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Sample records for sirna induces apoptosis

  1. Novel targets for sensitizing breast cancer cells to TRAIL-induced apoptosis with siRNA delivery.

    Science.gov (United States)

    Thapa, Bindu; Bahadur Kc, Remant; Uludağ, Hasan

    2018-02-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in variety of cancer cells without affecting most normal cells, which makes it a promising agent for cancer therapy. However, TRAIL therapy is clinically not effective due to resistance induction. To identify novel regulators of TRAIL that can aid in therapy, protein targets whose silencing sensitized breast cancer cells against TRAIL were screened with an siRNA library against 446 human apoptosis-related proteins in MDA-231 cells. Using a cationic lipopolymer (PEI-αLA) for delivery of library members, 16 siRNAs were identified that sensitized the TRAIL-induced death in MDA-231 cells. The siRNAs targeting BCL2L12 and SOD1 were further evaluated based on the novelty and their ability to sensitize TRAIL induced cell death. Silencing both targets sensitized TRAIL-mediated cell death in MDA-231 cells as well as TRAIL resistant breast cancer cells, MCF-7. Combination of TRAIL and siRNA silencing BCL2L12 had no effect in normal human umbilical vein cells and human bone marrow stromal cell. The silencing of BCL2L12 and SOD1 enhanced TRAIL-mediated apoptosis in MDA-231 cells via synergistically activating capsase-3 activity. Hence, here we report siRNAs targeting BCL2L12 and SOD1 as a novel regulator of TRAIL-induced cell death in breast cancer cells, providing a new approach for enhancing TRAIL therapy for breast cancer. The combination of siRNA targeting BCL2L12 and TRAIL can be a highly effective synergistic pair in breast cancer cells with minimal effect on the non-transformed cells. © 2017 UICC.

  2. Multifunctional selenium nanoparticles as carriers of HSP70 siRNA to induce apoptosis of HepG2 cells

    Directory of Open Access Journals (Sweden)

    Li Y

    2016-07-01

    Full Text Available Yinghua Li,1 Zhengfang Lin,1 Mingqi Zhao,1 Tiantian Xu,1 Changbing Wang,1 Huimin Xia,1,* Hanzhong Wang,2,* Bing Zhu1,* 1Guangzhou Women and Children’s Medical Center, Guangzhou, Guangdong, 2State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People’s Republic of China *These authors contributed equally to this work Abstract: Small interfering RNA (siRNA as a new therapeutic modality holds promise for cancer treatment, but it is unable to cross cell membrane. To overcome this limitation, nanotechnology has been proposed for mediation of siRNA transfection. Selenium (Se is a vital dietary trace element for mammalian life and plays an essential role in the growth and functioning of humans. As a novel Se species, Se nanoparticles have attracted more and more attention for their higher anticancer efficacy. In the present study, siRNAs with polyethylenimine (PEI-modified Se nanoparticles (Se@PEI@siRNA have been demonstrated to enhance the apoptosis of HepG2 cells. Heat shock protein (HSP-70 is overexpressed in many types of human cancer and plays a significant role in several biological processes including the regulation of apoptosis. The objective of this study was to silence inducible HSP70 and promote the apoptosis of Se-induced HepG2 cells. Se@PEI@siRNA were successfully prepared and characterized by various microscopic methods. Se@PEI@siRNA showed satisfactory size distribution, high stability, and selectivity between cancer and normal cells. The cytotoxicity of Se@PEI@siRNA was lower for normal cells than tumor cells, indicating that these compounds may have fewer side effects. The gene-silencing efficiency of Se@PEI@siRNA was significantly much higher than Lipofectamine 2000@siRNA and resulted in a significantly reduced HSP70 mRNA and protein expression in cancer cells. When the expression of HSP70 was diminished, the function of cell protection was also removed and cancer cells became more

  3. siRNA targeting PLK-1 induces apoptosis of synoviocytes in rheumatoid arthritis

    International Nuclear Information System (INIS)

    Wada, Makoto; Kawahito, Yutaka; Kimura, Shinya; Kohno, Masataka; Ishino, Hidetaka; Kimura, Mizuho; Omoto, Atsushi; Yamamoto, Aihiro; Hamaguchi, Masahide; Tsubouchi, Yasunori; Tokunaga, Daisaku; Hojo, Tatsuya; Ashihara, Eishi; Maekawa, Taira; Yoshikawa, Toshikazu

    2007-01-01

    Polo-like kinase-1 (PLK-1) is a member of the PLK family and participates in the control of cell mitosis. Here, we show that immunoreactive PLK-1 is strongly expressed in synoviocytes and some infiltrative mononuclear cells in synovial tissues from patients with rheumatoid arthritis (RA), while patients with osteoarthritis and injury show little or no expression of PLK-1 in synovial tissues. Western blot analysis shows that PLK is expressed and its expression is enhanced by IL-1β in RA synoviocytes. IL-1β also enhanced the cell growth of RA synoviocytes. Moreover, siRNA targeted against PLK-1 significantly decreases the expression of PLK-1 of RA synoviocytes stimulated by IL-1β and suppresses the proliferation of these synoviocytes through apoptosis. These findings suggest that PLK-1 plays a critical role in the proliferation of RA synoviocytes leading to bone destruction, and siRNA against PLK-1 is potentially useful for the treatment of RA

  4. IL13Rα2 siRNA inhibited cell proliferation, induced cell apoptosis, and suppressed cell invasion in papillary thyroid carcinoma cells

    Directory of Open Access Journals (Sweden)

    Gu MJ

    2018-03-01

    Full Text Available Mingjun Gu Department of Endocrinology, Shanghai Gongli Hospital, The Second Military Medical University, Shanghai, People’s Republic of China Aim: Papillary thyroid carcinoma (PTC is the most common type of thyroid cancer. Infiltrative growth and metastasis are the two most intractable characteristics of PTC. Interleukin-13 receptor α2 (IL13Rα2 with high affinity for Th2-derived cytokine IL-13 has been reported to be overexpressed in several tumors. In this study, an analysis of IL13Rα2 expression in PTC and matched paracancerous tissues was undertaken, and its biologic functions in PTC were assessed. Methods: IL13Rα2 and vascular endothelial growth factor (VEGF expression were detected by using real-time polymerase chain reaction and immunohistochemistry analyses. Cell proliferation, invasion, apoptosis, and caspase activity were measured with the Cell Counting Kit-8, Transwell, flow cytometry analyses, and biochemistry assay, respectively. Results: Upregulation of IL13Rα2 and VEGF was observed in PTC tissues compared with matched paracancerous tissues. Pearson’s correlation analysis indicated that IL13Rα2 mRNA level in the tested PTC tissues was positively correlated with VEGF mRNA level. Besides, inhibited cell proliferation, induced cell apoptosis, and suppressed cell invasion were detected in IL13Rα2-silenced TPC-1 cells. Increased activity of Caspase 3 and Caspase 9, along with elevated cleaved Caspase 3 and poly (ADP-ribose polymerase indicated the signal pathway of cell apoptosis induced by IL13Rα2 siRNA. In addition, downregulated metastasis- and angiogenesis-related proteins VEGF, VEGFR2, MMP2, and MMP9 indicated the decreased number of invading cells after knockdown of IL13Rα2. Conclusion: The results demonstrate that IL13Rα2 plays an important role in the progress of PTC. IL13Rα2 knockdown in PTC cells inhibited cell proliferation, induced cell apoptosis, and suppressed cell invasion. These data suggest that IL13Rα2

  5. Inhibitory Effect of Lycopene on Amyloid-β-Induced Apoptosis in Neuronal Cells.

    Science.gov (United States)

    Hwang, Sinwoo; Lim, Joo Weon; Kim, Hyeyoung

    2017-08-16

    Alzheimer's disease (AD) is a fatal neurodegenerative disease. Brain amyloid-β deposition is a crucial feature of AD, causing neuronal cell death by inducing oxidative damage. Reactive oxygen species (ROS) activate NF-κB, which induces expression of Nucling. Nucling is a pro-apoptotic factor recruiting the apoptosome complex. Lycopene is an antioxidant protecting from oxidative stress-induced cell damage. We investigated whether lycopene inhibits amyloid-β-stimulated apoptosis through reducing ROS and inhibiting mitochondrial dysfunction and NF-κB-mediated Nucling expression in neuronal SH-SY5Y cells. We prepared cells transfected with siRNA for Nucling or nontargeting control siRNA to determine the role of Nucling in amyloid-β-induced apoptosis. The amyloid-β increased intracellular and mitochondrial ROS levels, apoptotic indices (p53, Bax/Bcl-2 ratio, caspase-3 cleavage), NF-kB activation and Nucling expression, while cell viability, mitochondrial membrane potential, and oxygen consumption rate decreased in SH-SY5Y cells. Lycopene inhibited these amyloid-β-induced alterations. However, amyloid-β did not induce apoptosis, determined by cell viability and apoptotic indices (p53, Bax/Bcl-2 ratio, caspase-3 cleavage), in the cells transfected with siRNA for Nucling. Lycopene inhibited apoptosis by reducing ROS, and by inhibiting mitochondrial dysfunction and NF-κB-target gene Nucling expression in neuronal cells. Lycopene may be beneficial for preventing oxidative stress-mediated neuronal death in patients with neurodegeneration.

  6. Suberoyl bis-hydroxamic acid induces p53-dependent apoptosis of MCF-7 breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Zhi-gang ZHUANG; Fei FEI; Ying CHEN; Wei JIN

    2008-01-01

    Aim: To study the effects of suberoyl bis-hydroxamic acid (SBHA), an inhibitor of histone deacetylases, on the apoptosis of MCF-7 breast cancer cells. Meth-ods: Apoptosis in MCF-7 cells induced by SBHA was demonstrated by flow cytometric analysis, morphological observation, and DNA ladder. Mitochondrial membrane potential (△ψm) was measured using the fluorescent probe JC-1. The expressions of p53, p21, Bax, and PUMA were determined using RT-PCR or Western blotting analysis after the MCF-7 cells were treated with SBHA or p53 siRNA. Results: SBHA induced apoptosis in MCF-7 cells. The expressions of p53, p21, Bax, and PUMA were induced, and △ψm collapsed after treatment with SBHA. p53 siRNA abrogated the SBHA-induced apoptosis and the expressions of p53, p21, Bax, and PUMA. Conclusion: The activation of the p53 pathway is involved in SBHA-induced apoptosis in MCF-7 cells.

  7. Glucocorticoid Induced Leucine Zipper inhibits apoptosis of cardiomyocytes by doxorubicin

    International Nuclear Information System (INIS)

    Aguilar, David; Strom, Joshua; Chen, Qin M.

    2014-01-01

    Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found Glucocorticoid-Induced Leucine Zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes. - Highlights: • Corticosteroids act as a cytoprotective agent in cardiomyocytes • Corticosteroids induce GILZ expression in cardiomyocytes • Elevated GILZ results in resistance against apoptosis induced by doxorubicin • GILZ induces Bcl-xL protein without inducing Bcl-xL mRNA

  8. Downregulation of Lysyl Oxidase Protects Retinal Endothelial Cells From High Glucose-Induced Apoptosis.

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    Kim, Dongjoon; Mecham, Robert P; Trackman, Philip C; Roy, Sayon

    2017-05-01

    To investigate the effect of reducing high glucose (HG)-induced lysyl oxidase (LOX) overexpression and increased activity on retinal endothelial cell apoptosis. Rat retinal endothelial cells (RRECs) were grown in normal (N) or HG (30 mM glucose) medium for 7 days. In parallel, RRECs were grown in HG medium and transfected with LOX small interfering RNA (siRNA), scrambled siRNA as control, or exposed to β-aminopropionitrile (BAPN), a LOX inhibitor. LOX expression, AKT activation, and caspase-3 activity were determined by Western blot (WB) analysis and apoptosis by differential dye staining assay. Moreover, to determine whether diabetes-induced LOX overexpression alters AKT activation and promotes apoptosis, changes in LOX expression, AKT phosphorylation, caspase-3 activation, and Bax expression were assessed in retinas of streptozotocin (STZ)-induced diabetic mice and LOX heterozygous knockout (LOX+/-) mice. WB analysis indicated significant LOX overexpression and reduced AKT activation under HG condition in RRECs. Interestingly, when cells grown in HG were transfected with LOX siRNA or exposed to BAPN, the number of apoptotic cells was significantly decreased concomitant with increased AKT phosphorylation. Diabetic mouse retinas exhibited LOX overexpression, decreased AKT phosphorylation, and increased Bax and caspase-3 activation compared to values in nondiabetic mice. In LOX+/- mice, reduced LOX levels were observed with increased AKT activity, and reduced Bax and caspase-3 activity. Furthermore, decreased levels of LOX in the LOX+/- mice was protective against diabetes-induced apoptosis. Findings from this study indicate that preventing LOX overexpression may be protective against HG-induced apoptosis in retinal vascular cells associated with diabetic retinopathy.

  9. Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells.

    Science.gov (United States)

    Min, Kyoung-Jin; Nam, Ju-Ock; Kwon, Taeg Kyu

    2017-08-02

    Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki) cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose) polymerase (PARP), which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk) inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5) expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.

  10. Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells

    Directory of Open Access Journals (Sweden)

    Kyoung-jin Min

    2017-08-01

    Full Text Available Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose polymerase (PARP, which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5 expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.

  11. Cancer-targeting siRNA delivery from porous silicon nanoparticles.

    Science.gov (United States)

    Wan, Yuan; Apostolou, Sinoula; Dronov, Roman; Kuss, Bryone; Voelcker, Nicolas H

    2014-10-01

    Porous silicon nanoparticles (pSiNPs) with tunable pore size are biocompatible and biodegradable, suggesting that they are suitable biomaterials as vehicles for drug delivery. Loading of small interfering RNA (siRNA) into the pores of pSiNPs can protect siRNA from degradation as well as improve the cellular uptake. We aimed to deliver MRP1 siRNA loaded into pSiNPs to glioblastoma cells, and to demonstrate downregulation of MRP1 at the mRNA and protein levels. 50-220 nm pSiNPs with an average pore size of 26 nm were prepared, followed by electrostatic adsorption of siRNA into pores. Oligonucleotide loading and release profiles were investigated; MRP1 mRNA and protein expression, cell viability and cell apoptosis were studied. Approximately 7.7 µg of siRNA was loaded per mg of pSiNPs. Cells readily took up nanoparticles after 30 min incubation. siRNA-loaded pSiNPs were able to effectively downregulate target mRNA (~40%) and protein expression (31%), and induced cell apoptosis and necrosis (33%). siRNA loaded pSiNPs downregulated mRNA and protein expression and induced cell death. This novel siRNA delivery system may pave the way towards developing more effective tumor therapies.

  12. Targeted delivery of siRNA to macrophages for anti-inflammatory treatment.

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    Kim, Sang-Soo; Ye, Chunting; Kumar, Priti; Chiu, Isaac; Subramanya, Sandesh; Wu, Haoquan; Shankar, Premlata; Manjunath, N

    2010-05-01

    Inflammation mediated by tumor necrosis factor-alpha (TNF-alpha) and the associated neuronal apoptosis characterizes a number of neurologic disorders. Macrophages and microglial cells are believed to be the major source of TNF-alpha in the central nervous system (CNS). Here, we show that suppression of TNF-alpha by targeted delivery of small interfering RNA (siRNA) to macrophage/microglial cells dramatically reduces lipopolysaccharide (LPS)-induced neuroinflammation and neuronal apoptosis in vivo. Because macrophage/microglia express the nicotinic acetylcholine receptor (AchR) on their surface, we used a short AchR-binding peptide derived from the rabies virus glycoprotein (RVG) as a targeting ligand. This peptide was fused to nona-D-arginine residues (RVG-9dR) to enable siRNA binding. RVG-9dR was able to deliver siRNA to induce gene silencing in macrophages and microglia cells from wild type, but not AchR-deficient mice, confirming targeting specificity. Treatment with anti-TNF-alpha siRNA complexed to RVG-9dR achieved efficient silencing of LPS-induced TNF-alpha production by primary macrophages and microglia cells in vitro. Moreover, intravenous injection with RVG-9dR-complexed siRNA in mice reduced the LPS-induced TNF-alpha levels in blood as well as in the brain, leading to a significant reduction in neuronal apoptosis. These results demonstrate that RVG-9dR provides a tool for siRNA delivery to macrophages and microglia and that suppression of TNF-alpha can potentially be used to suppress neuroinflammation in vivo.

  13. Troglitazone induced apoptosis via PPARγ activated POX-induced ROS formation in HT29 cells.

    Science.gov (United States)

    Wang, Jing; Lv, XiaoWen; Shi, JiePing; Hu, XiaoSong; DU, YuGuo

    2011-08-01

    In order to investigate the potential mechanisms in troglitazone-induced apoptosis in HT29 cells, the effects of PPARγ and POX-induced ROS were explored. [3- (4, 5)-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, Annexin V and PI staining using FACS, plasmid transfection, ROS formation detected by DCFH staining, RNA interference, RT-PCR & RT-QPCR, and Western blotting analyses were employed to investigate the apoptotic effect of troglitazone and the potential role of PPARγ pathway and POX-induced ROS formation in HT29 cells. Troglitazone was found to inhibit the growth of HT29 cells by induction of apoptosis. During this process, mitochondria related pathways including ROS formation, POX expression and cytochrome c release increased, which were inhibited by pretreatment with GW9662, a specific antagonist of PPARγ. These results illustrated that POX upregulation and ROS formation in apoptosis induced by troglitazone was modulated in PPARγ-dependent pattern. Furthermore, the inhibition of ROS and apoptosis after POX siRNA used in troglitazone-treated HT29 cells indicated that POX be essential in the ROS formation and PPARγ-dependent apoptosis induced by troglitazone. The findings from this study showed that troglitazone-induced apoptosis was mediated by POX-induced ROS formation, at least partly, via PPARγ activation. Copyright © 2011 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.

  14. Andrographolide Analogue Induces Apoptosis and Autophagy Mediated Cell Death in U937 Cells by Inhibition of PI3K/Akt/mTOR Pathway.

    Directory of Open Access Journals (Sweden)

    Deepak Kumar

    Full Text Available Current chemotherapeutic agents based on apoptosis induction are lacking in desired efficacy. Therefore, there is continuous effort to bring about new dimension in control and gradual eradication of cancer by means of ever evolving therapeutic strategies. Various forms of PCD are being increasingly implicated in anti-cancer therapy and the complex interplay among them is vital for the ultimate fate of proliferating cells. We elaborated and illustrated the underlying mechanism of the most potent Andrographolide analogue (AG-4 mediated action that involved the induction of dual modes of cell death-apoptosis and autophagy in human leukemic U937 cells.AG-4 induced cytotoxicity was associated with redox imbalance and apoptosis which involved mitochondrial depolarisation, altered apoptotic protein expressions, activation of the caspase cascade leading to cell cycle arrest. Incubation with caspase inhibitor Z-VAD-fmk or Bax siRNA decreased cytotoxic efficacy of AG-4 emphasising critical roles of caspase and Bax. In addition, AG-4 induced autophagy as evident from LC3-II accumulation, increased Atg protein expressions and autophagosome formation. Pre-treatment with 3-MA or Atg 5 siRNA suppressed the cytotoxic effect of AG-4 implying the pro-death role of autophagy. Furthermore, incubation with Z-VAD-fmk or Bax siRNA subdued AG-4 induced autophagy and pre-treatment with 3-MA or Atg 5 siRNA curbed AG-4 induced apoptosis-implying that apoptosis and autophagy acted as partners in the context of AG-4 mediated action. AG-4 also inhibited PI3K/Akt/mTOR pathway. Inhibition of mTOR or Akt augmented AG-4 induced apoptosis and autophagy signifying its crucial role in its mechanism of action.Thus, these findings prove the dual ability of AG-4 to induce apoptosis and autophagy which provide a new perspective to it as a potential molecule targeting PCD for future cancer therapeutics.

  15. Gallic Acid Induces Apoptosis in Human Gastric Adenocarcinoma Cells.

    Science.gov (United States)

    Tsai, Chung-Lin; Chiu, Ying-Ming; Ho, Tin-Yun; Hsieh, Chin-Tung; Shieh, Dong-Chen; Lee, Yi-Ju; Tsay, Gregory J; Wu, Yi-Ying

    2018-04-01

    Gastric cancer is one of the most common malignant cancers with a poor prognosis and high mortality rate worldwide. Current treatment of gastric cancer includes surgery and chemotherapy as the main modalities, but the potentially severe side-effects of chemotherapy present a considerable challenge. Gallic acid is a trihydroxybenzoic acid found to exert an anticancer effect against a variety of cancer cells. The purpose of this study was to determine the anti-cancer activity of Galla chinensis and its main component gallic acid on human gastric adenocarcinoma cells. MTT assay and cell death ELISA were used to determine the apoptotic effect of Gallic Chinensis and gallic acid on human gastric adenocarcinoma cells. To determine the pathway and relevant components by which gallic acid-induced apoptosis is mediated through, cells were transfected with siRNA (Fas, FasL, DR5, p53) using Lipofectamine 2000. Reults: Gallic Chinensis and gallic acid induced apoptosis of human gastric adenocarcinoma cells. Gallic acid induced up-regulation of Fas, FasL, and DR5 expression in AGS cells. Transfection of cells with Fas, FasL, or DR5 siRNA reduced gallic acid-induced cell death. In addition, p53 was shown to be involved in gallic acid-mediated Fas, FasL, and DR5 expression as well as cell apoptosis in AGS cells. These results suggest that gallic acid has a potential role in the treatment of gastric cancer. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  16. Heme oxygenase-1 enhances autophagy in podocytes as a protective mechanism against high glucose-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Chenglong [Department of Endocrinology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing (China); Zheng, Haining [Department of Hyperbaric Oxygen, Nanjing General Hospital of Nanjing Military Command, Nanjing (China); Huang, Shanshan; You, Na; Xu, Jiarong; Ye, Xiaolong; Zhu, Qun; Feng, Yamin; You, Qiang; Miao, Heng [Department of Endocrinology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing (China); Ding, Dafa, E-mail: dingdafa2004@aliyun.com [Department of Endocrinology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing (China); Lu, Yibing, E-mail: luyibing2004@126.com [Department of Endocrinology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing (China)

    2015-10-01

    Injury and loss of podocytes play vital roles in diabetic nephropathy progression. Emerging evidence suggests autophagy, which is induced by multiple stressors including hyperglycemia, plays a protective role. Meanwhile, heme oxygenase-1 (HO-1) possesses powerful anti-apoptotic properties. Therefore, we investigated the impact of autophagy on podocyte apoptosis under diabetic conditions and its association with HO-1. Mouse podocytes were cultured in vitro; apoptosis was detected by flow cytometry. Transmission electron microscopy and biochemical autophagic flux assays were used to measure the autophagy markers microtubule-associated protein 1 light chain 3-II (LC3-II) and beclin-1. LC3-II and beclin-1 expression peaked 12–24 h after exposing podocytes to high glucose. Inhibition of autophagy with 3-methyladenine or Beclin-1 siRNAs or Atg 5 siRNAs sensitized cells to apoptosis, suggesting autophagy is a survival mechanism. HO-1 inactivation inhibited autophagy, which aggravated podocyte injury in vitro. Hemin-induced autophagy also protected podocytes from hyperglycemia in vitro and was abrogated by HO-1 siRNA. Adenosine monophosphate-activated protein kinase phosphorylation was higher in hemin-treated and lower in HO-1 siRNA-treated podocytes. Suppression of AMPK activity reversed HO-1-mediated Beclin-1 upregulation and autophagy, indicating HO-1-mediated autophagy is AMPK dependent. These findings suggest HO-1 induction and regulation of autophagy are potential therapeutic targets for diabetic nephropathy. - Highlights: • High glucose leads to increased autophagy in podocytes at an early stage. • The early autophagic response protects against high glucose-induced apoptosis. • Heme oxygenase-1 enhances autophagy and decreases high glucose -mediated apoptosis. • Heme oxygenase-1 induces autophagy through the activation of AMPK.

  17. Heme oxygenase-1 enhances autophagy in podocytes as a protective mechanism against high glucose-induced apoptosis

    International Nuclear Information System (INIS)

    Dong, Chenglong; Zheng, Haining; Huang, Shanshan; You, Na; Xu, Jiarong; Ye, Xiaolong; Zhu, Qun; Feng, Yamin; You, Qiang; Miao, Heng; Ding, Dafa; Lu, Yibing

    2015-01-01

    Injury and loss of podocytes play vital roles in diabetic nephropathy progression. Emerging evidence suggests autophagy, which is induced by multiple stressors including hyperglycemia, plays a protective role. Meanwhile, heme oxygenase-1 (HO-1) possesses powerful anti-apoptotic properties. Therefore, we investigated the impact of autophagy on podocyte apoptosis under diabetic conditions and its association with HO-1. Mouse podocytes were cultured in vitro; apoptosis was detected by flow cytometry. Transmission electron microscopy and biochemical autophagic flux assays were used to measure the autophagy markers microtubule-associated protein 1 light chain 3-II (LC3-II) and beclin-1. LC3-II and beclin-1 expression peaked 12–24 h after exposing podocytes to high glucose. Inhibition of autophagy with 3-methyladenine or Beclin-1 siRNAs or Atg 5 siRNAs sensitized cells to apoptosis, suggesting autophagy is a survival mechanism. HO-1 inactivation inhibited autophagy, which aggravated podocyte injury in vitro. Hemin-induced autophagy also protected podocytes from hyperglycemia in vitro and was abrogated by HO-1 siRNA. Adenosine monophosphate-activated protein kinase phosphorylation was higher in hemin-treated and lower in HO-1 siRNA-treated podocytes. Suppression of AMPK activity reversed HO-1-mediated Beclin-1 upregulation and autophagy, indicating HO-1-mediated autophagy is AMPK dependent. These findings suggest HO-1 induction and regulation of autophagy are potential therapeutic targets for diabetic nephropathy. - Highlights: • High glucose leads to increased autophagy in podocytes at an early stage. • The early autophagic response protects against high glucose-induced apoptosis. • Heme oxygenase-1 enhances autophagy and decreases high glucose -mediated apoptosis. • Heme oxygenase-1 induces autophagy through the activation of AMPK

  18. SET mediates TCE-induced liver cell apoptosis through dephosphorylation and upregulation of nucleolin.

    Science.gov (United States)

    Ren, Xiaohu; Huang, Xinfeng; Yang, Xifei; Liu, Yungang; Liu, Wei; Huang, Haiyan; Wu, Desheng; Zou, Fei; Liu, Jianjun

    2017-06-20

    Trichloroethylene (TCE) is an occupational and environmental chemical that can cause severe hepatotoxicity. While our previous studies showed that the phosphatase inhibitor SET is a key mediator of TCE-induced liver cell apoptosis, the molecular mechanisms remain elusive. Using quantitative phosphoproteomic analysis, we report here that nucleolin is a SET-regulated phosphoprotein in human liver HL-7702 cells. Functional analysis suggested that SET promoted dephosphorylation of nucleolin, decreased its binding to its transcriptional activator, c-myc, and upregulated nucleolin expression in TCE-treated cells. Importantly, TCE-induced hepatocyte apoptosis was significantly attenuated when nucleolin was downregulated with specific siRNAs. These findings indicate that TCE may induce hepatocyte apoptosis via SET-mediated dephosphorylation and overexpression of nucleolin.

  19. Ellipticine induces apoptosis in T-cell lymphoma via oxidative DNA damage

    DEFF Research Database (Denmark)

    Savorani, Cecilia; Manfé, Valentina; Biskup, Edyta

    2015-01-01

    (CTCL), a disease that is progressive, chemoresistant and refractory to treatment. We tested the effect of ellipticine in three cell lines with different p53 status: MyLa2000 (p53(wt/wt)), SeAx ((G245S)p53) and Hut-78 ((R196Stop)p53). Ellipticine caused apoptosis in MyLa2000 and SeAx and restored...... the transcriptional activity of (G245S)p53 in SeAx. However, p53 siRNA knockdown experiments revealed that p53 was not required for ellipticine-induced apoptosis in CTCL. The lipophilic antioxidant α-tocopherol inhibited ellipticine-dependent apoptosis and we linked the apoptotic response to the oxidative DNA damage....... Our results provide evidence that ellipticine-induced apoptosis is exerted through DNA damage and does not require p53 activation in T-cell lymphoma....

  20. Bim is a crucial regulator of apoptosis induced by Mycobacterium tuberculosis

    Science.gov (United States)

    Aguiló, N; Uranga, S; Marinova, D; Martín, C; Pardo, J

    2014-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, induces apoptosis in infected macrophages in vitro and in vivo. However, the molecular mechanism controlling this process is not known. In order to study the involvement of the mitochondrial apoptotic pathway in M. tuberculosis-induced apoptosis, we analysed cell death in M. tuberculosis-infected embryonic fibroblasts (MEFs) derived from different knockout mice for genes involved in this route. We found that apoptosis induced by M. tuberculosis is abrogated in the absence of Bak and Bax, caspase 9 or the executioner caspases 3 and 7. Notably, we show that MEF deficient in the BH3-only BCL-2-interacting mediator of cell death (Bim) protein were also resistant to this process. The relevance of these results has been confirmed in the mouse macrophage cell line J774, where cell transfection with siRNA targeting Bim impaired apoptosis induced by virulent mycobacteria. Notably, only infection with a virulent strain, but not with attenuated ESX-1-defective strains, such as Bacillus Calmette-Guerin and live-attenuated M. tuberculosis vaccine strain MTBVAC, induced Bim upregulation and apoptosis, probably implicating virulence factor early secreted antigenic target 6-kDa protein in this process. Our results suggest that Bim upregulation and apoptosis is mediated by the p38MAPK-dependent pathway. Our findings show that Bim is a master regulator of apoptosis induced by M. tuberculosis. PMID:25032866

  1. A preliminary study on action mechanisms of surviving expression in cell apoptosis induced by high-LET radiation

    International Nuclear Information System (INIS)

    Jin Xiaodong; Li Qiang; Gong Li; Wu Qingfeng; Li Ping; Dai Zhongying; Liu Xinguo; Tao Jiajun

    2010-01-01

    It has been proven that over-expression of surviving in cancerous cell lines is related to the radioresistance of cells to high-LET radiation in previous work. In this study, action mechanisms of surviving gene in apoptosis induced by high-LET radiation were investigated. We found that inhibiting surviving by siRNA had no notable influence on Bcl-2 and Bax expressions induced by carbon ions. Surviving depressed cell apoptosis through the inhibition of the activities of caspase-3 and -9 possibly in cell apoptosis induced by high-LET radiation. (authors)

  2. Peroxiredoxin IV Protects Cells From Radiation-Induced Apoptosis in Head-and-Neck Squamous Cell Carcinoma

    International Nuclear Information System (INIS)

    Park, Jung Je; Chang, Hyo Won; Jeong, Eun-Jeong; Roh, Jong-Lyel; Choi, Seung-Ho; Jeon, Sea-Yuong; Ko, Gyung Hyuck; Kim, Sang Yoon

    2009-01-01

    Purpose: Human peroxiredoxins (Prxs) are known as a family of thiol-specific antioxidant enzymes, among which Prx-I and -II play an important role in protecting cells from irradiation-induced cell death. It is not known whether Prx-IV also protects cells from ionizing radiation (IR). Methods and Materials: To evaluate the protective role of Prx-IV in IR, we transfected full-length Prx-IV cDNA into AMC-HN3 cells, which weakly express endogenous Prx-IV, and knocked down the expression of Prx-IV with siRNA methods using AMC-HN7 cells, which express high levels of endogenous Prx-IV. Radiosensitivity profiles in these cells were evaluated using clonogenic assay, FACS analysis, cell viability, and TUNEL assay. Results: Three Prx-IV expressing clones were isolated. Prx-IV regulated intracellular reactive oxygen species (ROS) levels and made cells more resistant to IR-induced apoptosis. Furthermore, the knockdown of Prx-IV with siRNA made cells more sensitive to IR-induced apoptosis. Conclusion: The results of these studies suggest that Prx-IV may play an important role in protecting cells from IR-induced apoptosis in head-and-neck squamous cell carcinoma

  3. Systematic analysis of off-target effects in an RNAi screen reveals microRNAs affecting sensitivity to TRAIL-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Enright Anton J

    2010-03-01

    Full Text Available Abstract Background RNA inhibition by siRNAs is a frequently used approach to identify genes required for specific biological processes. However RNAi screening using siRNAs is hampered by non-specific or off target effects of the siRNAs, making it difficult to separate genuine hits from false positives. It is thought that many of the off-target effects seen in RNAi experiments are due to siRNAs acting as microRNAs (miRNAs, causing a reduction in gene expression of unintended targets via matches to the 6 or 7 nt 'seed' sequence. We have conducted a careful examination of off-target effects during an siRNA screen for novel regulators of the TRAIL apoptosis induction pathway(s. Results We identified 3 hexamers and 3 heptamer seed sequences that appeared multiple times in the top twenty siRNAs in the TRAIL apoptosis screen. Using a novel statistical enrichment approach, we systematically identified a further 17 hexamer and 13 heptamer seed sequences enriched in high scoring siRNAs. The presence of one of these seeds sequences (which could explain 6 of 8 confirmed off-target effects is sufficient to elicit a phenotype. Three of these seed sequences appear in the human miRNAs miR-26a, miR-145 and miR-384. Transfection of mimics of these miRNAs protects several cell types from TRAIL-induced cell death. Conclusions We have demonstrated a role for miR-26a, miR-145 and miR-26a in TRAIL-induced apoptosis. Further these results show that RNAi screening enriches for siRNAs with relevant off-target effects. Some of these effects can be identified by the over-representation of certain seed sequences in high-scoring siRNAs and we demonstrate the usefulness of such systematic analysis of enriched seed sequences.

  4. Functional role of CCCTC binding factor (CTCF) in stress-induced apoptosis

    International Nuclear Information System (INIS)

    Li Tie; Lu Luo

    2007-01-01

    CTCF, a nuclear transcriptional factor, is a multifunctional protein and involves regulation of growth factor- and cytokine-induced cell proliferation/differentiation. In the present study, we investigated the role of CTCF in protecting stress-induced apoptosis in various human cell types. We found that UV irradiation and hyper-osmotic stress induced human corneal epithelial (HCE) and hematopoietic myeloid cell apoptosis detected by significantly increased caspase 3 activity and decreased cell viability. The stress-induced apoptotic response in these cells requires down-regulation of CTCF at both mRNA and protein levels, suggesting that CTCF may play an important role in downstream events of stress-induced signaling pathways. Inhibition of NFκB activity prevented stress-induced down-regulation of CTCF and increased cell viability against stress-induced apoptosis. The anti-apoptotic effect of CTCF was further studied by manipulating CTCF activities in HCE and hematopoietic cells. Transient transfection of cDNAs encoding full-length human CTCF markedly suppressed stress-induced apoptosis in these cells. In contrast, knocking down of CTCF mRNA using siRNA specific to CTCF significantly promoted stress-induced apoptosis. Thus, our results reveal that CTCF is a down stream target of stress-induced signaling cascades and it plays a significant anti-apoptotic role in regulation of stress-induced cellular responses in HCE and hematopoietic myeloid cells

  5. Egr-1 Upregulates Siva-1 Expression and Induces Cardiac Fibroblast Apoptosis

    Directory of Open Access Journals (Sweden)

    Karin Zins

    2014-01-01

    Full Text Available The early growth response transcription factor Egr-1 controls cell specific responses to proliferation, differentiation and apoptosis. Expression of Egr-1 and downstream transcription is closely controlled and cell specific upregulation induced by processes such as hypoxia and ischemia has been previously linked to multiple aspects of cardiovascular injury. In this study, we showed constitutive expression of Egr-1 in cultured human ventricular cardiac fibroblasts, used adenoviral mediated gene transfer to study the effects of continuous Egr-1 overexpression and studied downstream transcription by Western blotting, immunohistochemistry and siRNA transfection. Apoptosis was assessed by fluorescence microscopy and flow cytometry in the presence of caspase inhibitors. Overexpression of Egr-1 directly induced apoptosis associated with caspase activation in human cardiac fibroblast cultures in vitro assessed by fluorescence microscopy and flow cytometry. Apoptotic induction was associated with a caspase activation associated loss of mitochondrial membrane potential and transient downstream transcriptional up-regulation of the pro-apoptotic gene product Siva-1. Suppression of Siva-1 induction by siRNA partially reversed Egr-1 mediated loss of cell viability. These findings suggest a previously unknown role for Egr-1 and transcriptional regulation of Siva-1 in the control of cardiac accessory cell death.

  6. Noxa/Mcl-1 Balance Regulates Susceptibility of Cells to Camptothecin-Induced Apoptosis

    Directory of Open Access Journals (Sweden)

    Yide Mei

    2007-10-01

    Full Text Available Although camptothecin (CPT has been reported to induce apoptosis in various cancer cells, the molecular details of this regulation remain largely unknown. In this study, we demonstrate that 131-113-only protein Noxa is upregulated during CPT-induced apoptosis, which is independent of p53. In addition, we show that phosphatidylinositol 3-kinase (PI3K/Akt signaling pathway is responsible for Noxa's induction. Luciferase assay, cAMP response element binding protein (CREB knockdown experiments further demonstrate that CREB is involved in the transcriptional upregulation of Noxa. Moreover, blocking Noxa expression using specific small interfering ribonucleic acid (siRNA significantly reduces the apoptosis in response to CPT, indicating that Noxa is an essential mediator for CPT-induced apoptosis. Interestingly, antiapoptotic Mcl-1 was also upregulated through PI3K/Akt signaling pathway upon CPT treatment. Using immunoprecipitation assay, Noxa was found to interact with Mcl-1 in the presence or absence of CPT. Knockdown of Mcl-1 expression by short hairpin ribonucleic acid (shRNA was shown to potentiate CPT-induced apoptosis. Consistently, ectopic overexpression of Mcl-1 rescued cells from apoptosis induced by CPT. Cells coexpressing Noxa, Mcl-1 at different ratio correlates well with the extent of apoptosis, suggesting that the balance between Noxa, Mcl-1 may determine the susceptibility of HeLa cells to CPT-induced apoptosis.

  7. Dioscin induces caspase-independent apoptosis through activation of apoptosis-inducing factor in breast cancer cells.

    Science.gov (United States)

    Kim, Eun-Ae; Jang, Ji-Hoon; Lee, Yun-Han; Sung, Eon-Gi; Song, In-Hwan; Kim, Joo-Young; Kim, Suji; Sohn, Ho-Yong; Lee, Tae-Jin

    2014-07-01

    Dioscin, a saponin extracted from the roots of Polygonatum zanlanscianense, shows several bioactivities such as antitumor, antifungal, and antiviral properties. Although, dioscin is already known to induce cell death in variety cancer cells, the molecular basis for dioscin-induced cell death was not definitely known in cancer cells. In this study, we found that dioscin treatment induced cell death in dose-dependent manner in breast cancer cells such as MDA-MB-231, MDA-MB-453, and T47D cells. Dioscin decreased expressions of Bcl-2 and cIAP-1 proteins, which were down-regulated at the transcriptional level. Conversely, Mcl-1 protein level was down-regulated by facilitating ubiquitin/proteasome-mediated Mcl-1 degradation in dioscin-treated cells. Pretreatment with z-VAD fails to attenuate dioscin-induced cell death as well as caspase-mediated events such as cleavages of procaspase-3 and PARP. In addition, dioscin treatment increased the population of annexin V positive cells and induced DNA fragmentation in a dose-dependent manner in MDA-MB-231 cells. Furthermore, apoptosis inducing factor (AIF) was released from the mitochondria and translocated to the nucleus. Suppression in AIF expression by siRNA reduced dioscin-induced apoptosis in MDA-MB-231 cells. Taken together, our results demonstrate that dioscin-induced cell death was mediated via AIF-facilitating caspase-independent pathway as well as down-regulating anti-apoptotic proteins such as Bcl-2, cIAP-1, and Mcl-1 in breast cancer cells.

  8. Blockade of Inflammation and Apoptosis Pathways by siRNA Prolongs Cold Preservation Time and Protects Donor Hearts in a Porcine Model

    Directory of Open Access Journals (Sweden)

    Jia Wei

    2017-12-01

    Full Text Available In donor hearts from mini pigs, overtime cold preservation and ischemia-reperfusion injury cause poor graft quality and impaired heart function. Blockage of complement, apoptosis, and inflammation is considered a strategy for attenuating ischemia-reperfusion injury and protecting cardiac function. Minipig donor hearts were perfused and preserved in Celsior solution or transfection reagent containing Celsior solution with scramble siRNA or siRNAs targeting complement 3, caspase-8, caspase-3, and nuclear factor κB-p65 genes at 4°C and subsequently hemo-reperfused ex vivo (38°C or transplanted into recipients. The protective effect of the siRNA solution was evaluated by measuring cell apoptosis, structural alteration, protein markers for tissue damage and oxidative stress, and cardiac function. We found a reduction in cell apoptosis, myocardial damage, and tissue inflammation by reduced biochemistry and markers and protein expression of proinflammatory cytokines and improvement in cardiac function, as shown by the improved hemodynamic indices in 12-hr-preserved siRNA-treated hearts of both ex vivo and orthotopic transplantation models. These findings demonstrate that blockade of inflammation and apoptosis pathways using siRNA can prolong cold preservation time and better protect donor heart function in cardiac transplantation of large animals, which may be beneficial for human heart preservation.

  9. Fatty acid synthase regulates the chemosensitivity of breast cancer cells to cisplatin-induced apoptosis.

    Science.gov (United States)

    Al-Bahlani, Shadia; Al-Lawati, Hanaa; Al-Adawi, Moza; Al-Abri, Nadia; Al-Dhahli, Buthaina; Al-Adawi, Kawther

    2017-06-01

    Fatty acid synthase (FASN) is a key enzyme in fat biosynthesis that is over-expressed in advanced breast cancer stages. Cisplatin (CDDP) is a platinum-based drug used in the treatment of certain types of this disease. Although it was shown that FASN inhibition induced apoptosis by enhancing the cytotoxicity of certain drugs in breast cancer, its role in regulating the chemosensitivity of different types of breast cancer cells to CDDP-induced apoptosis is not established yet. Therefore, two different breast cancer cell lines; triple negative breast cancer (TNBC; MDA-MB-231) and triple positive breast cancer (TPBC; BT-474) cells were used to examine such role. We show that TNBC cells had naturally less fat content than TPBC cells. Subsequently, the fat content increased in both cells when treated with Palmitate rather than Oleate, whereas both fatty acids produced apoptotic ultra-structural effects and attenuated FASN expression. However, Oleate increased FASN expression in TPBC cells. CDDP decreased FASN expression and increased apoptosis in TNBC cells. These effects were further enhanced by combining CDDP with fatty acids. We also illustrate that the inhibition of FASN by either siRNA or exogenous inhibitor decreased CDDP-induced apoptosis in TPBC cells suggesting its role as an apoptotic factor, while an opposite finding was observed in TNBC cells when siRNA and fatty acids were used, suggesting its role as a survival factor. To our knowledge, we are the first to demonstrate a dual role of FASN in CDDP-induced apoptosis in breast cancer cells and how it can modulate their chemosensitivity.

  10. Hypoxia-inducible transcription factor-1α promotes hypoxia-induced A549 apoptosis via a mechanism that involves the glycolysis pathway

    International Nuclear Information System (INIS)

    Luo, FengMing; Liu, XiaoJing; Yan, NaiHong; Li, ShuangQing; Cao, GuiQun; Cheng, QingYing; Xia, QingJie; Wang, HongJing

    2006-01-01

    Hypoxia-inducible transcription factor-1α (HIF-1α), which plays an important role in controlling the hypoxia-induced glycolysis pathway, is a 'master' gene in the tissue hypoxia response during tumor development. However, its role in the apoptosis of non-small cell lung cancer remains unknown. Here, we have studied the effects of HIF-1α on apoptosis by modulating HIF-1α gene expression in A549 cells through both siRNA knock-down and over-expression. A549 cells were transfected with a HIF-1α siRNA plasmid or a HIF-1α expression vector. Transfected cells were exposed to a normoxic or hypoxic environment in the presence or absence of 25 mM HEPES and 2-deoxyglucose (2-DG) (5 mM). The expression of three key genes of the glycolysis pathway, glucose transporter type 1(GLUT1), phosphoglycerate kinase 1(PGK1), and hexokinase 1(HK1), were measured using real-time RT-PCR. Glycolysis was monitored by measuring changes of pH and lactate concentration in the culture medium. Apoptosis was detected by TUNEL assay and flow cytometry. Knocking down expression of HIF-1α inhibited the glycolysis pathway, increased the pH of the culture medium, and protected the cells from hypoxia-induced apoptosis. In contrast, over-expression of HIF-1α accelerated glycolysis in A549 cells, decreased the pH of the culture medium, and enhanced hypoxia-induced apoptosis. These effects of HIF-1α on glycolysis, pH of the medium, and apoptosis were reversed by treatment with the glycolytic inhibitor, 2-DG. Apoptosis induced by HIF-1α over-expression was partially inhibited by increasing the buffering capacity of the culture medium by adding HEPES. During hypoxia in A549 cells, HIF-1α promotes activity of the glycolysis pathway and decreases the pH of the culture medium, resulting in increased cellular apoptosis

  11. Silencing of Tumor Necrosis Factor Receptor 1 by siRNA in EC109 Cells Affects Cell Proliferation and Apoptosis

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    Ma Changhui

    2009-01-01

    Full Text Available Tumor necrosis factor receptor 1 (TNFR1 is a membrane receptor able to bind TNF-α or TNF-β. TNFR1 can suppress apoptosis by activating the NF-κB or JNK/SAPK signal transduction pathway, or it can induce apoptosis through a series of caspase cascade reactions; the particular effect may depend on the cell line. In the present study, we first showed that TNFR1 is expressed at both the gene and protein levels in the esophageal carcinoma cell line EC109. Then, by applying a specific siRNA, we silenced the expression of TNFR1; this resulted in a significant time-dependent promotion of cell proliferation and downregulation of the apoptotic rate. These results suggest that TNFR1 is strongly expressed in the EC109 cell line and that it may play an apoptosis-mediating role, which may be suppressed by highly activated NF-κB.

  12. Kaempferol Sensitizes Human Ovarian Cancer Cells-OVCAR-3 and SKOV-3 to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis via JNK/ERK-CHOP Pathway and Up-Regulation of Death Receptors 4 and 5.

    Science.gov (United States)

    Zhao, Yingmei; Tian, Binqiang; Wang, Yong; Ding, Haiying

    2017-10-26

    BACKGROUND Ovarian cancer is the most common gynecological malignancies in women, with high mortality rates worldwide. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) superfamily which preferentially induces apoptosis of cancer cells. However, acquired resistance to TRAIL hampers its therapeutic application. Identification of compounds that sensitize cancer cells to TRAIL is vital in combating resistance to TRAIL. The effect of kaempferol, a flavonoid enhancing TRAIL-induced apoptosis in ovarian cancer cells, was investigated in this study. MATERIAL AND METHODS The cytotoxic effects of TRAIL (25 ng/mL) and kaempferol (20-100 µM) on human ovarian cancer cells OVCAR-3 and SKOV-3 were assessed. Effect of kaempferol on the expression patterns of cell survival proteins (Bcl-xL, Bcl-2, survivin, XIAP, c-FLIP) and apoptotic proteins (caspase-3, caspase-8, caspase-9, Bax) were studied. The influence of kaempferol on expression of DR4 and DR5 death receptors on the cell surface and protein and mRNA levels was also analyzed. Apoptosis following silencing of DR5 and CHOP by small interfering RNA (siRNA), and activation of MAP kinases were analyzed as well. RESULTS Kaempferol enhanced apoptosis and drastically up-regulated DR4, DR5, CHOP, JNK, ERK1/2, p38 and apoptotic protein expression with decline in the expression of anti-apoptotic proteins. Further transfection with siRNA specific to CHOP and DR5 indicated the involvement of CHOP in DR5 up-regulation and also the contribution of DR5 in kaempferol-enhanced TRAIL-induced apoptosis. CONCLUSIONS Kaempferol sensitized ovarian cancer cells to TRAIL-induced apoptosis via up-regulation of DR4 and DR5 through ERK/JNK/CHOP pathways.

  13. Noxa/Mcl-1 Balance Regulates Susceptibility of Cells to Camptothecin-Induced Apoptosis1

    Science.gov (United States)

    Mei, Yide; Xie, Chongwei; Xie, Wei; Tian, Xu; Li, Mei; Wu, Mian

    2007-01-01

    Although camptothecin (CPT) has been reported to induce apoptosis in various cancer cells, the molecular details of this regulation remain largely unknown. In this study, we demonstrate that BH3-only protein Noxa is upregulated during CPT-induced apoptosis, which is independent of p53. In addition, we show that phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is responsible for Noxa's induction. Luciferase assay and cAMP response element binding protein (CREB) knockdown experiments further demonstrate that CREB is involved in the transcriptional upregulation of Noxa. Moreover, blocking Noxa expression using specific small interfering ribonucleic acid (siRNA) significantly reduces the apoptosis in response to CPT, indicating that Noxa is an essential mediator for CPT-induced apoptosis. Interestingly, antiapoptotic Mcl-1 was also upregulated through PI3K/Akt signaling pathway upon CPT treatment. Using immunoprecipitation assay, Noxa was found to interact with Mcl-1 in the presence or absence of CPT. Knockdown of Mcl-1 expression by short hairpin ribonucleic acid (shRNA) was shown to potentiate CPT-induced apoptosis. Consistently, ectopic overexpression of Mcl-1 rescued cells from apoptosis induced by CPT. Cells coexpressing Noxa and Mcl-1 at different ratio correlates well with the extent of apoptosis, suggesting that the balance between Noxa and Mcl-1 may determine the susceptibility of HeLa cells to CPT-induced apoptosis. PMID:17971907

  14. The combinational effect of E6/E7 siRNA and anti-miR-182 on apoptosis induction in HPV16-positive cervical cells.

    Science.gov (United States)

    Javadi, Hamidreza; Lotfi, Abbas Sahebghadam; Hosseinkhani, Saman; Mehrani, Hossein; Amani, Jafar; Soheili, Zahra Soheila; Hojati, Zahra; Kamali, Mehdi

    2018-06-06

    In the present research, we assumed that reducing the amounts of E6 and E7 oncoproteins by a specific siRNA sequence and recovering p53 and RB proteins, along with the recovery of the FOXO1 protein by applying anti-miR-182, would increase apoptosis and reduce proliferation rate in cancer cells. The HPV16-positive CaSki cervical cancer cell line was used. 48 hours after transfection of siRNA for targeting E6 and E7 oncoproteins and anti-miR-182, expression of its cellular targets p53, p21 and FOXO1 was assessed by real-time PCR, western blot analysis and immunocytofluorescence staining. In all treatments, apoptosis rate and viability were evaluated using Annexin-V-FITC apoptosis detection kits and MTT assays, respectively. Among the designed siRNAs, E6-1 and E7-2 proved the most effective in reducing E6 and E7 expressions by increasing the apoptotic rates to 12.4% and 16%, respectively, after 48 hours. Also, using anti-miR-182 increased apoptotic rate to 12.7% 48 hours after transfection of cervical cancer cells. The combinational use of either E6-1 or E7-2 siRNAs with anti-miR-182 resulted in a rise in apoptosis to 19.3% and 26%, respectively, higher than those obtained from the individual application of either without anti-miR-182. The simultaneous use of siRNA E6-1 and siRNA E7-2 with cisplatin increased sensitivity to cisplatin and reduced the viability of the cancer cells as compared to the use of cisplatin alone. The simultaneous use of cisplatin and anti-miR-182 had no considerable effect on viability or apoptosis rate compared to cisplatin alone.

  15. Polo-like kinase 1 siRNA-607 induces mitotic arrest and apoptosis in ...

    African Journals Online (AJOL)

    Polo-like kinase (Plk) 1 is overexpressed in many human malignancies including nasopharyngeal carcinoma, indicating its potential as a therapeutic target. Recently, using a simple cellular morphologybased strategy, we have identified several novel effective siRNAs against Plk1 including Plk1 siRNA- 607. In this study, we ...

  16. Intrinsic mechanism of estradiol-induced apoptosis in breast cancer cells resistant to estrogen deprivation.

    Science.gov (United States)

    Lewis, Joan S; Meeke, Kathleen; Osipo, Clodia; Ross, Eric A; Kidawi, Noman; Li, Tianyu; Bell, Eric; Chandel, Navdeep S; Jordan, V Craig

    2005-12-07

    We previously developed an estrogen receptor (ER)-positive breast cancer cell line (MCF-7:5C) that is resistant to long-term estrogen deprivation and undergoes rapid and complete apoptosis in the presence of physiologic concentrations of 17beta-estradiol. Here, we investigated the role of the mitochondrial apoptotic pathway in this process. Apoptosis in MCF-7:5C cells treated with estradiol, fulvestrant, or vehicle (control) was investigated by annexin V-propidium iodide double staining and 4',6-diamidino-2-phenylindole (DAPI) staining. Apoptosis was also analyzed in MCF-7:5C cells transiently transfected with small interfering RNAs (siRNAs) to apoptotic pathway components. Expression of apoptotic pathway intermediates was measured by western blot analysis. Mitochondrial transmembrane potential (psim) was determined by rhodamine-123 retention assay. Mitochondrial pathway activity was determined by cytochrome c release and cleavage of poly(ADP-ribose) polymerase (PARP) protein. Tumorigenesis was studied in ovariectomized athymic mice that were injected with MCF-7:5C cells. Differences between the treatment groups and control group were determined by two-sample t test or one-factor analysis of variance. All statistical tests were two-sided. MCF-7:5C cells treated with estradiol underwent apoptosis and showed increased expression of proapoptotic proteins, decreased psim, enhanced cytochrome c release, and PARP cleavage compared with cells treated with fulvestrant or vehicle. Blockade of Bax, Bim, and p53 mRNA expression by siRNA reduced estradiol-induced apoptosis relative to control by 76% [95% confidence interval (CI) = 73% to 79%, P estradiol-induced apoptosis in long-term estrogen-deprived breast cancer cells. Physiologic concentrations of estradiol could potentially be used to induce apoptosis and tumor regression in tumors that have developed resistance to aromatase inhibitors.

  17. Knockdown of long noncoding antisense RNA brain-derived neurotrophic factor attenuates hypoxia/reoxygenation-induced nerve cell apoptosis through the BDNF-TrkB-PI3K/Akt signaling pathway.

    Science.gov (United States)

    Zhong, Jian-Bin; Li, Xie; Zhong, Si-Ming; Liu, Jiu-Di; Chen, Chi-Bang; Wu, Xiao-Yan

    2017-09-27

    Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal cell apoptosis. The antisense RNA of brain-derived neurotrophic factor (BDNF-AS) is a natural antisense transcript that is transcribed opposite the gene that encodes BDNF. The aim of this study was to determine whether knockdown of BDNF-AS can suppress hypoxia/reoxygenation (H/R)-induced neuronal cell apoptosis and whether this is mediated by the BDNF-TrkB-PI3K/Akt pathway. We detected the expression of BDNF and BDNF-AS in brain tissue from 20 patients with cerebral infarction and five patients with other diseases (but no cerebral ischemia). We found that BDNF expression was significantly downregulated in patients with cerebral infarction, whereas the expression of BDNF-AS was significantly upregulated. In both human cortical neurons (HCN2) and human astrocytes, H/R significantly induced the expression of BDNF-AS, but significantly decreased BDNF expression. H/R also significantly induced apoptosis and reduced the mitochondrial membrane potential in these cells. Following downregulation of BDNF-AS by siRNA in human cortical neurons and human astrocyte cells, BDNF expression was significantly upregulated and the H/R-induced upregulation of BDNF-AS was significantly attenuated. BDNF-AS siRNA inhibited H/R-induced cell apoptosis and ameliorated the H/R-induced suppression of mitochondrial membrane potential. H/R inhibited the expression of BDNF, p-AKT/AKT, and TrKB, and this inhibition was recovered by BDNF-AS siRNA. In summary, this study indicates that BDNF-AS siRNA induces activation of the BDNF-TrkB-PI3K/Akt pathway following H/R-induced neurotoxicity. These findings will be useful toward the application of BDNF-AS siRNA for the treatment of neurodegenerative diseases.

  18. The role of cPLA2 in Methylglyoxal-induced cell apoptosis of HUVECs

    International Nuclear Information System (INIS)

    Yuan, Jie; Zhu, Chao; Hong, Yali; Sun, Zongxing; Fang, Xianjun; Wu, Biao; Li, Shengnan

    2017-01-01

    Methylglyoxal (MGO), a highly reactive dicarbonyl compound, is mainly formed as a byproduct of glycolysis. Elevated MGO level is known to induce apoptosis of vascular endothelial cells, which is implicated with progression of atherosclerosis and diabetic complications. However, the underlying mechanisms have not been exhaustively investigated yet. Here, we further characterized the mechanisms how MGO induced apoptosis in human umbilical vein endothelial cells (HUVECs). Our data revealed that cytosolic phospholipase A2 (cPLA2) played an important role in MGO-induced cell apoptosis. It was found that MGO could increase both the activity and expression of cPLA2. Inhibition of cPLA2 by Pyrrophenone (PYR) or siRNA significantly attenuated the MGO-induced apoptosis. Additionally, MGO time-dependently decreased the phosphorylation of nuclear factor κB (NF-κB). Pretreatment of the cells with NF-κB inhibitor, BAY11-7082, further increased MGO-induced apoptosis of HUVECs, indicating that NF-κB played a survival role in this MGO-induced apoptosis. Furthermore, in the presence of si-cPLA2 or PYR, MGO no longer decreased NF-κB phosphorylation. Beyond that, the antioxidant N-acetyl cysteine (NAC) could reverse the changes of both cPLA2 and NF-κB caused by MGO. p38, the upstream of cPLA2, was also significantly phosphorylated by MGO. However, p38 inhibitor failed to reverse the apoptosis induced by MGO. This study gives an important insight into the downstream signaling mechanisms of MGO, cPLA2-NF-κB, in endothelial apoptosis. - Highlights: • cPLA2 participated in MGO-induced HUVECs apoptosis. • Inhibition of NF-κB was involved in MGO-cPLA2-mediated cell apoptosis. • Antioxidant NAC attenuated MGO-induced cPLA2 activation and cell apoptosis.

  19. The role of cPLA2 in Methylglyoxal-induced cell apoptosis of HUVECs

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Jie; Zhu, Chao; Hong, Yali; Sun, Zongxing; Fang, Xianjun [Jiangsu Provincial Key Lab of Cardiovascular Diseases and Molecular intervention, Department of Pharmacology, Nanjing Medical University, Nanjing 210029 (China); Wu, Biao, E-mail: wubiao@ncu.edu.cn [Department of Surgery, The First Affiliated Hospital, Nanchang University (China); Li, Shengnan, E-mail: snli@njmu.edu.cn [Jiangsu Provincial Key Lab of Cardiovascular Diseases and Molecular intervention, Department of Pharmacology, Nanjing Medical University, Nanjing 210029 (China)

    2017-05-15

    Methylglyoxal (MGO), a highly reactive dicarbonyl compound, is mainly formed as a byproduct of glycolysis. Elevated MGO level is known to induce apoptosis of vascular endothelial cells, which is implicated with progression of atherosclerosis and diabetic complications. However, the underlying mechanisms have not been exhaustively investigated yet. Here, we further characterized the mechanisms how MGO induced apoptosis in human umbilical vein endothelial cells (HUVECs). Our data revealed that cytosolic phospholipase A2 (cPLA2) played an important role in MGO-induced cell apoptosis. It was found that MGO could increase both the activity and expression of cPLA2. Inhibition of cPLA2 by Pyrrophenone (PYR) or siRNA significantly attenuated the MGO-induced apoptosis. Additionally, MGO time-dependently decreased the phosphorylation of nuclear factor κB (NF-κB). Pretreatment of the cells with NF-κB inhibitor, BAY11-7082, further increased MGO-induced apoptosis of HUVECs, indicating that NF-κB played a survival role in this MGO-induced apoptosis. Furthermore, in the presence of si-cPLA2 or PYR, MGO no longer decreased NF-κB phosphorylation. Beyond that, the antioxidant N-acetyl cysteine (NAC) could reverse the changes of both cPLA2 and NF-κB caused by MGO. p38, the upstream of cPLA2, was also significantly phosphorylated by MGO. However, p38 inhibitor failed to reverse the apoptosis induced by MGO. This study gives an important insight into the downstream signaling mechanisms of MGO, cPLA2-NF-κB, in endothelial apoptosis. - Highlights: • cPLA2 participated in MGO-induced HUVECs apoptosis. • Inhibition of NF-κB was involved in MGO-cPLA2-mediated cell apoptosis. • Antioxidant NAC attenuated MGO-induced cPLA2 activation and cell apoptosis.

  20. Morphine Protects Spinal Cord Astrocytes from Glutamate-Induced Apoptosis via Reducing Endoplasmic Reticulum Stress

    Directory of Open Access Journals (Sweden)

    Chao Zhang

    2016-10-01

    Full Text Available Glutamate is not only a neurotransmitter but also an important neurotoxin in central nervous system (CNS. Chronic elevation of glutamate induces both neuronal and glial cell apoptosis. However, its effect on astrocytes is complex and still remains unclear. In this study, we investigated whether morphine, a common opioid ligand, could affect glutamate-induced apoptosis in astrocytes. Primary cultured astrocytes were incubated with glutamate in the presence/absence of morphine. It was found that morphine could reduce glutamate-induced apoptosis of astrocytes. Furthermore, glutamate activated Ca2+ release, thereby inducing endoplasmic reticulum (ER stress in astrocytes, while morphine attenuated this deleterious effect. Using siRNA to reduce the expression of κ-opioid receptor, morphine could not effectively inhibit glutamate-stimulated Ca2+ release in astrocytes, the protective effect of morphine on glutamate-injured astrocytes was also suppressed. These results suggested that morphine could protect astrocytes from glutamate-induced apoptosis via reducing Ca2+ overload and ER stress pathways. In conclusion, this study indicated that excitotoxicity participated in the glutamate mediated apoptosis in astrocytes, while morphine attenuated this deleterious effect via regulating Ca2+ release and ER stress.

  1. Activating transcription factor 6 mediates oxidized LDL-induced cholesterol accumulation and apoptosis in macrophages by up-regulating CHOP expression.

    Science.gov (United States)

    Yao, Shutong; Zong, Chuanlong; Zhang, Ying; Sang, Hui; Yang, Mingfeng; Jiao, Peng; Fang, Yongqi; Yang, Nana; Song, Guohua; Qin, Shucun

    2013-01-01

    This study was to explore whether activating transcription factor 6 (ATF6), an important sensor to endoplasmic reticulum (ER) stress, would mediate oxidized low-density lipoprotein (ox-LDL)- induced cholesterol accumulation and apoptosis in cultured macrophages and the underlying molecular mechanisms. Intracellular lipid droplets and total cholesterol levels were assayed by oil red O staining and enzymatic colorimetry, respectively. Cell viability and apoptosis were determined using MTT assay and AnnexinV-FITC apoptosis detection kit, respectively. The nuclear translocation of ATF6 in cells was detected by immunofluorescence analysis. Protein and mRNA levels were examined by Western blot analysis and real time-PCR, respectively. ATF6 siRNA was transfected to RAW264.7 cells by lipofectamin. Exposure of cells to ox-LDL induced glucose-regulated protein 78 (GRP78). C/EBP homologous protein (CHOP), a key-signaling component of ER stress-induced apoptosis, was up-regulated in ox-LDL-treated cells. ATF6, a factor that positively regulates CHOP expression, was activated by ox-LDL in a concentration- and time- dependent manner. The role of the ATF6-mediated ER stress pathway was further confirmed through the siRNA-mediated knockdown of ATF6, which attenuated ox-LDL-induced upregulation of CHOP, cholesterol accumulation and apoptosis in macrophages. In addition, the phosphorylation of double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK), another factor that positively regulates CHOP expression, was induced in the presence of ox-LDL, and PERK-specific siRNA also inhibited the ox-LDL-induced upregulation of CHOP and apoptosis in RAW264.7 cells. These results demonstrate that ER stress-related proteins, particularly ATF6 and its downstream molecule CHOP, are involved in ox-LDL-induced cholesterol accumulation and apoptosis in macrophages.

  2. SiRNA Crosslinked Nanoparticles for the Treatment of Inflammation-induced Liver Injury.

    Science.gov (United States)

    Tang, Yaqin; Zeng, Ziying; He, Xiao; Wang, Tingting; Ning, Xinghai; Feng, Xuli

    2017-02-01

    RNA interference mediated by small interfering RNA (siRNA) provides a powerful tool for gene regulation, and has a broad potential as a promising therapeutic strategy. However, therapeutics based on siRNA have had limited clinical success due to their undesirable pharmacokinetic properties. This study presents pH-sensitive nanoparticles-based siRNA delivery systems (PNSDS), which are positive-charge-free nanocarriers, composed of siRNA chemically crosslinked with multi-armed poly(ethylene glycol) carriers via acid-labile acetal linkers. The unique siRNA crosslinked structure of PNSDS allows it to have minimal cytotoxicity, high siRNA loading efficiency, and a stimulus-responsive property that enables the selective intracellular release of siRNA in response to pH conditions. This study demonstrates that PNSDS can deliver tumor necrosis factor alpha (TNF-α) siRNA into macrophages and induce the efficient down regulation of the targeted gene in complete cell culture media. Moreover, PNSDS with mannose targeting moieties can selectively accumulate in mice liver, induce specific inhibition of macrophage TNF-α expression in vivo, and consequently protect mice from inflammation-induced liver damages. Therefore, this novel siRNA delivering platform would greatly improve the therapeutic potential of RNAi based therapies.

  3. Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress

    International Nuclear Information System (INIS)

    Tang, C.-H.; Chiu, Y.-C.; Huang, C.-F.; Chen, Y.-W.; Chen, P.-C.

    2009-01-01

    Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromal cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis.

  4. Involvement of caspase-dependent and -independent apoptotic pathways in cisplatin-induced apoptosis

    Science.gov (United States)

    Liu, Lei; Zhang, Yingjie; Wang, Xianwang

    2009-02-01

    Cisplatin, an efficient anticancer agent, can trigger multiple apoptotic pathways in cancer cells. However, the signal transduction pathways in response to cisplatin-based chemotherapy are complicated, and the mechanism is not fully understood. In current study, we showed that, during cisplatin-induced apoptosis of human lung adenocarcinoma cells, both the caspase-dependent and -independent pathways were activated. Herein, we reported that after cisplatin treatment, the activities of caspase-9/-3 were sharply increased; pre-treatment with Z-LEHD-fmk (inhibitor of caspase-9), Z-DEVD-fmk (inhibitor of caspase-3), and Z-VAD-fmk (a pan-caspase inhibitor) increased cell viability and decreased apoptosis, suggesting that caspase-mediated apoptotic pathway was activated following cisplatin treatment. Confocal imaging of the cells transfected with AIF-GFP demonstrated that AIF release occurred about 9 h after cisplatin treatment. The event proceeded progressively over time, coinciding with a nuclear translocation and lasting for more than 2 hours. Down-regulation of AIF by siRNA also significantly increased cell viability and decreased apoptosis, these results suggested that AIF-mediated caspase-independent apoptotic pathway was involved in cispatin-induced apoptosis. In conclusion, the current study demonstrated that both caspase-dependent and -independent apoptotic pathways were involved in cisplatin-induced apoptosis in human lung adenocarcinoma cells.

  5. DNA damage-inducible transcript 4 (DDIT4) mediates methamphetamine-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes

    International Nuclear Information System (INIS)

    Chen, Rui; Wang, Bin; Chen, Ling; Cai, Dunpeng; Li, Bing; Chen, Chuanxiang; Huang, Enping; Liu, Chao; Lin, Zhoumeng; Xie, Wei-Bing; Wang, Huijun

    2016-01-01

    Methamphetamine (METH) is an amphetamine-like psychostimulant that is commonly abused. Previous studies have shown that METH can induce damages to the nervous system and recent studies suggest that METH can also cause adverse and potentially lethal effects on the cardiovascular system. Recently, we demonstrated that DNA damage-inducible transcript 4 (DDIT4) regulates METH-induced neurotoxicity. However, the role of DDIT4 in METH-induced cardiotoxicity remains unknown. We hypothesized that DDIT4 may mediate METH-induced autophagy and apoptosis in cardiomyocytes. To test the hypothesis, we examined DDIT4 protein expression in cardiomyocytes and in heart tissues of rats exposed to METH with Western blotting. We also determined the effects on METH-induced autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes using Western blot analysis, fluorescence microscopy and TUNEL staining. Our results showed that METH exposure increased DDIT4 expression and decreased phosphorylation of mTOR that was accompanied with increased autophagy and apoptosis both in vitro and in vivo. These effects were normalized after silencing DDIT4. On the other hand, rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes. These results suggest that DDIT4 mediates METH-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes. - Highlights: • METH exposure increases DDIT4 expression in cardiomyocytes. • DDIT4 mediates METH-induced autophagy and apoptosis in cardiomyocytes. • DDIT4 silencing protects cardiomyocytes against METH-caused autophagy and apoptosis.

  6. DNA damage-inducible transcript 4 (DDIT4) mediates methamphetamine-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Rui [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Department of Forensic Medicine, Guangdong Medical University, Dongguan 523808 (China); Wang, Bin; Chen, Ling; Cai, Dunpeng; Li, Bing; Chen, Chuanxiang; Huang, Enping [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Liu, Chao [Guangzhou Forensic Science Institute, Guangzhou 510030 (China); Lin, Zhoumeng [Institute of Computational Comparative Medicine and Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506 (United States); Xie, Wei-Bing, E-mail: xieweib@126.com [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Wang, Huijun, E-mail: hjwang711@yahoo.cn [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China)

    2016-03-15

    Methamphetamine (METH) is an amphetamine-like psychostimulant that is commonly abused. Previous studies have shown that METH can induce damages to the nervous system and recent studies suggest that METH can also cause adverse and potentially lethal effects on the cardiovascular system. Recently, we demonstrated that DNA damage-inducible transcript 4 (DDIT4) regulates METH-induced neurotoxicity. However, the role of DDIT4 in METH-induced cardiotoxicity remains unknown. We hypothesized that DDIT4 may mediate METH-induced autophagy and apoptosis in cardiomyocytes. To test the hypothesis, we examined DDIT4 protein expression in cardiomyocytes and in heart tissues of rats exposed to METH with Western blotting. We also determined the effects on METH-induced autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes using Western blot analysis, fluorescence microscopy and TUNEL staining. Our results showed that METH exposure increased DDIT4 expression and decreased phosphorylation of mTOR that was accompanied with increased autophagy and apoptosis both in vitro and in vivo. These effects were normalized after silencing DDIT4. On the other hand, rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes. These results suggest that DDIT4 mediates METH-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes. - Highlights: • METH exposure increases DDIT4 expression in cardiomyocytes. • DDIT4 mediates METH-induced autophagy and apoptosis in cardiomyocytes. • DDIT4 silencing protects cardiomyocytes against METH-caused autophagy and apoptosis.

  7. Andrographolide induces vascular smooth muscle cell apoptosis through a SHP-1-PP2A-p38MAPK-p53 cascade.

    Science.gov (United States)

    Chen, Yu-Ying; Hsieh, Cheng-Ying; Jayakumar, Thanasekaran; Lin, Kuan-Hung; Chou, Duen-Suey; Lu, Wan-Jung; Hsu, Ming-Jen; Sheu, Joen-Rong

    2014-07-10

    The abnormal growth of vascular smooth muscle cells (VSMCs) is considered a critical pathogenic process in inflammatory vascular diseases. We have previously demonstrated that protein phosphatase 2 A (PP2A)-mediated NF-κB dephosphorylation contributes to the anti-inflammatory properties of andrographolide, a novel NF-κB inhibitor. In this study, we investigated whether andrographolide causes apoptosis, and characterized its apoptotic mechanisms in rat VSMCs. Andrographolide activated the p38 mitogen-activated protein kinase (p38MAPK), leading to p53 phosphorylation. Phosphorylated p53 subsequently transactivated the expression of Bax, a pro-apoptotic protein. Transfection with pp2a small interfering RNA (siRNA) suppressed andrographolide-induced p38MAPK activation, p53 phosphorylation, and caspase 3 activation. Andrographolide also activated the Src homology 1 domain-containing protein tyrosine phosphatase (SHP-1), and induced PP2A dephosphorylation, both of which were inhibited by the SHP-1 inhibitor sodium stibogluconate (SSG) or shp-1 siRNA. SSG or shp-1 siRNA prevented andrographolide-induced apoptosis. These results suggest that andrographolide activates the PP2A-p38MAPK-p53-Bax cascade, causing mitochondrial dysfunction and VSMC death through an SHP-1-dependent mechanism.

  8. Formoxanthone C, isolated from Cratoxylum formosum ssp. pruniflorum, reverses anticancer drug resistance by inducing both apoptosis and autophagy in human A549 lung cancer cells.

    Science.gov (United States)

    Kaewpiboon, Chutima; Boonnak, Nawong; Kaowinn, Sirichat; Chung, Young-Hwa

    2018-02-15

    Multidrug resistance (MDR) cancer toward cancer chemotherapy is one of the obstacles in cancer therapy. Therefore, it is of interested to use formoxanthone C (1,3,5,6-tetraoxygenated xanthone; XanX), a natural compound, which showed cytotoxicity against MDR human A549 lung cancer (A549RT-eto). The treatment with XanX induced not only apoptosis- in A549RT-eto cells, but also autophagy-cell death. Inhibition of apoptosis did not block XanX-induced autophagy in A549RT-eto cells. Furthermore, suppression of autophagy by beclin-1 small interfering RNAs (siRNAs) did not interrupt XanX-induced apoptosis, indicating that XanX can separately induce apoptosis and autophagy. Of interest, XanX treatment reduced levels of histone deacetylase 4 (HDAC4) protein overexpressed in A549RT-etocells. The co-treatment with XanX and HDAC4 siRNA accelerated both autophagy and apoptosis more than that by XanX treatment alone, suggesting survival of HDAC4 in A549RT-eto cells. XanX reverses etoposide resistance in A549RT-eto cells by induction of both autophagy and apoptosis, and confers cytotoxicity through down-regulation of HDAC4. Copyright © 2017. Published by Elsevier Ltd.

  9. Targeted Delivery of siRNA to Macrophages for Anti-inflammatory Treatment

    OpenAIRE

    Kim, Sang-Soo; Ye, Chunting; Kumar, Priti; Chiu, Isaac; Subramanya, Sandesh; Wu, Haoquan; Shankar, Premlata; Manjunath, N

    2010-01-01

    Inflammation mediated by tumor necrosis factor-α (TNF-α) and the associated neuronal apoptosis characterizes a number of neurologic disorders. Macrophages and microglial cells are believed to be the major source of TNF-α in the central nervous system (CNS). Here, we show that suppression of TNF-α by targeted delivery of small interfering RNA (siRNA) to macrophage/microglial cells dramatically reduces lipopolysaccharide (LPS)-induced neuroinflammation and neuronal apoptosis in vivo. Because ma...

  10. Radiation-induced apoptosis

    International Nuclear Information System (INIS)

    Ohyama, Harumi

    1995-01-01

    Apoptosis is an active process of gene-directed cellular self-destruction that can be induced in many cell types via numerous physiological and pathological stimuli. We found that interphasedeath of thymocytes is a typical apoptosis showing the characteristic features of apoptosis including cell shrinkage, chromatin condensation and DNA degradation. Moderate dose of radiation induces extensive apoptosis in rapidly proliferating cell population such as the epithelium of intestinal crypt. Recent reports indicate that the ultimate form of radiation-induced mitotic death in several cells is also apoptosis. One of the hallmarks of apoptosis is the enzymatic internucleosomal degradation of chromatin DNA. We identified an endonuclease responsible for the radiation-induced DNA degradation in rat thymocytes. The death-sparing effects of interrupting RNA and protein synthesis suggested a cell genetic program for apoptosis. Apoptosis of thymocytes initiated by DNA damage, such as radiation and radio mimetic substance, absolutely requires the protein of p53 cancer suppresser gene. The cell death induced by glucocorticoid, or aging, has no such requirement. Expression of oncogene bcl-2 rescues cells from the apoptosis. Massive apoptosis in radiosensitive cells induced by higher dose radiation may be fatal. It is suggested that selective apoptotic elimination of cells would play an important role for protection against carcinogenesis and malformation through removal of cells with unrepaired radiation-induced DNA damages. Data to evaluate the significance of apoptosis in the radiation risk are still poor. Further research should be done in order to clarify the roles of the cell death on the acute and late effects of irradiation. (author)

  11. Peroxisome proliferator-activated receptor alpha acts as a mediator of endoplasmic reticulum stress-induced hepatocyte apoptosis in acute liver failure

    Directory of Open Access Journals (Sweden)

    Li Zhang

    2016-07-01

    Full Text Available Peroxisome proliferator-activated receptor α (PPARα is a key regulator to ameliorate liver injury in cases of acute liver failure (ALF. However, its regulatory mechanisms remain largely undetermined. Endoplasmic reticulum stress (ER stress plays an important role in a number of liver diseases. This study aimed to investigate whether PPARα activation inhibits ER stress-induced hepatocyte apoptosis, thereby protecting against ALF. In a murine model of D-galactosamine (D-GalN- and lipopolysaccharide (LPS-induced ALF, Wy-14643 was administered to activate PPARα, and 4-phenylbutyric acid (4-PBA was administered to attenuate ER stress. PPARα activation ameliorated liver injury, because pre-administration of its specific inducer, Wy-14643, reduced the serum aminotransferase levels and preserved liver architecture compared with that of controls. The protective effect of PPARα activation resulted from the suppression of ER stress-induced hepatocyte apoptosis. Indeed, (1 PPARα activation decreased the expression of glucose-regulated protein 78 (Grp78, Grp94 and C/EBP-homologous protein (CHOP in vivo; (2 the liver protection by 4-PBA resulted from the induction of PPARα expression, as 4-PBA pre-treatment promoted upregulation of PPARα, and inhibition of PPARα by small interfering RNA (siRNA treatment reversed liver protection and increased hepatocyte apoptosis; (3 in vitro PPARα activation by Wy-14643 decreased hepatocyte apoptosis induced by severe ER stress, and PPARα inhibition by siRNA treatment decreased the hepatocyte survival induced by mild ER stress. Here, we demonstrate that PPARα activation contributes to liver protection and decreases hepatocyte apoptosis in ALF, particularly through regulating ER stress. Therefore, targeting PPARα could be a potential therapeutic strategy to ameliorate ALF.

  12. Bupivacaine-induced apoptosis independently of WDR35 expression in mouse neuroblastoma Neuro2a cells

    Science.gov (United States)

    2012-01-01

    Background Bupivacaine-induced neurotoxicity has been shown to occur through apoptosis. Recently, bupivacaine was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in a human neuroblastoma cell line. We have reported that WDR35, a WD40-repeat protein, may mediate apoptosis through caspase-3 activation. The present study was undertaken to test whether bupivacaine induces apoptosis in mouse neuroblastoma Neuro2a cells and to determine whether ROS, p38 MAPK, and WDR35 are involved. Results Our results showed that bupivacaine induced ROS generation and p38 MAPK activation in Neuro2a cells, resulting in apoptosis. Bupivacaine also increased WDR35 expression in a dose- and time-dependent manner. Hydrogen peroxide (H2O2) also increased WDR35 expression in Neuro2a cells. Antioxidant (EUK-8) and p38 MAPK inhibitor (SB202190) treatment attenuated the increase in caspase-3 activity, cell death and WDR35 expression induced by bupivacaine or H2O2. Although transfection of Neuro2a cells with WDR35 siRNA attenuated the bupivacaine- or H2O2-induced increase in expression of WDR35 mRNA and protein, in contrast to our previous studies, it did not inhibit the increase in caspase-3 activity in bupivacaine- or H2O2-treated cells. Conclusions In summary, our results indicated that bupivacaine induced apoptosis in Neuro2a cells. Bupivacaine induced ROS generation and p38 MAPK activation, resulting in an increase in WDR35 expression, in these cells. However, the increase in WDR35 expression may not be essential for the bupivacaine-induced apoptosis in Neuro2a cells. These results may suggest the existence of another mechanism of bupivacaine-induced apoptosis independent from WDR35 expression in Neuro2a cells. PMID:23227925

  13. The ganglioside GM3 is associated with cisplatin-induced apoptosis in human colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Tae-Wook Chung

    Full Text Available Cisplatin (cis-diamminedichloroplatinum, CDDP is a well-known chemotherapeutic agent for the treatment of several cancers. However, the precise mechanism underlying apoptosis of cancer cells induced by CDDP remains unclear. In this study, we show mechanistically that CDDP induces GM3-mediated apoptosis of HCT116 cells by inhibiting cell proliferation, and increasing DNA fragmentation and mitochondria-dependent apoptosis signals. CDDP induced apoptosis within cells through the generation of reactive oxygen species (ROS, regulated the ROS-mediated expression of Bax, Bcl-2, and p53, and induced the degradation of the poly (ADP-ribosyl polymerase (PARP. We also checked expression levels of different gangliosides in HCT116 cells in the presence or absence of CDDP. Interestingly, among the gangliosides, CDDP augmented the expression of only GM3 synthase and its product GM3. Reduction of the GM3 synthase level through ectopic expression of GM3 small interfering RNA (siRNA rescued HCT116 cells from CDDP-induced apoptosis. This was evidenced by inhibition of apoptotic signals by reducing ROS production through the regulation of 12-lipoxigenase activity. Furthermore, the apoptotic sensitivity to CDDP was remarkably increased in GM3 synthase-transfected HCT116 cells compared to that in controls. In addition, GM3 synthase-transfected cells treated with CDDP exhibited an increased accumulation of intracellular ROS. These results suggest the CDDP-induced oxidative apoptosis of HCT116 cells is mediated by GM3.

  14. Combination of siRNA-directed Kras oncogene silencing and arsenic-induced apoptosis using a nanomedicine strategy for the effective treatment of pancreatic cancer.

    Science.gov (United States)

    Zeng, Linjuan; Li, Jingguo; Wang, Yong; Qian, Chenchen; Chen, Yinting; Zhang, Qiubo; Wu, Wei; Lin, Zhong; Liang, Jianzhong; Shuai, Xintao; Huang, Kaihong

    2014-02-01

    The synergetic inhibitory effects on human pancreatic cancer by nanoparticle-mediated siRNA and arsenic therapy were investigated both in vitro and in vivo. Poly(ethylene glycol)-block-poly(L-lysine) were prepared to form siRNA-complexed polyplex and poly(ethylene glycol)-block-poly(DL-lactide) were prepared to form arsenic-encapsulated vesicle, respectively. Down-regulation of the mutant Kras gene by siRNA caused defective abilities of proliferation, clonal formation, migration, and invasion of pancreatic cancer cells, as well as cell cycle arrest at the G0/G1 phase, which substantially enhanced the apoptosis-inducing effect of arsenic administration. Consequently, co-administration of the two nanomedicines encapsulating siRNA or arsenic showed ideal tumor growth inhibition both in vitro and in vivo as a result of synergistic effect of the siRNA-directed Kras oncogene silencing and arsenic-induced cell apoptosis. These results suggest that the combination of mutant Kras gene silencing and arsenic therapy using nanoparticle-mediated delivery strategy is promising for pancreatic cancer treatment. Treatment of pancreatic cancer remains a major challenge. These authors demonstrate a method that combines a siRNA-based Kras silencing with arsenic delivery to pancreatic cancer cells using nanoparticles, resulting in enhanced apoptosis induction in the treated cells. © 2013.

  15. High glucose induced oxidative stress and apoptosis in cardiac microvascular endothelial cells are regulated by FoxO3a.

    Directory of Open Access Journals (Sweden)

    Chaoming Peng

    Full Text Available Cardiac microvascular endothelial cells (CMECs dysfunction contributes to cardiovascular complications in diabetes, whereas, the underlying mechanism is not fully clarified. FoxO transcription factors are involved in apoptosis and reactive oxygen species (ROS production. Therefore, the present study was designed to elucidate the potential role of FoxO3a on the CMECs injury induced by high glucose.CMECs were isolated from hearts of adult rats and cultured in normal or high glucose medium for 6 h, 12 h and 24 h respectively. To down-regulate FoxO3a expression, CMECs were transfected with FoxO3a siRNA. ROS accumulation and apoptosis in CMECs were assessed by dihydroethidine (DHE staining and TUNEL assay respectively. Moreover, the expressions of Akt, FoxO3a, Bim and BclxL in CMECs were assessed by Western blotting assay.ROS accumulation in CMECs was significantly increased after high glucose incubation for 6 to 24 h. Meanwhile, high glucose also increased apoptosis in CMECs, correlated with decreased the phosphorylation expressions of Akt and FoxO3a. Moreover, high glucose incubation increased the expression of Bim, whereas increased anti-apoptotic protein BclxL. Furthermore, siRNA target FoxO3a silencing enhanced the ROS accumulation, whereas suppressed apoptosis in CMECs. FoxO3a silencing also abolished the disturbance of Bcl-2 proteins induced by high glucose in CMECs.Our data provide evidence that high glucose induced FoxO3a activation which suppressed ROS accumulation, and in parallel, resulted in apoptosis of CMECs.

  16. Downregulation of survivin by siRNA inhibits invasion and promotes apoptosis in neuroblastoma SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, L.; Liang, H. [Department of Pediatrics, Qilu Hospital, Shandong University, Jinan (China); Cao, W. [Department of Obstetrics, Qingdao Central Hospital, Qingdao (China); Xu, R.; Ju, X.L. [Department of Pediatrics, Qilu Hospital, Shandong University, Jinan (China)

    2014-05-23

    Neuroblastoma is a solid tumor that occurs mainly in children. Malignant neuroblastomas have a poor prognosis because conventional chemotherapeutic agents are not very effective. Survivin, a member of the inhibitor of the apoptosis protein family, plays a significant role in cell division, inhibition of apoptosis, and promotion of cell proliferation and invasion. Previous studies found that survivin is highly expressed in some malignant neuroblastomas and is correlated with poor prognosis. The aim of this study was to investigate whether survivin could serve as a potential therapeutic target of human neuroblastoma. We employed RNA interference to reduce survivin expression in the human neuroblastoma SH-SY5Y cell line and analyzed the effect of RNA interference on cell proliferation and invasion in vitro and in vivo. RNA interference of survivin led to a significant decrease in invasiveness and proliferation and increased apoptosis in SH-SY5Y cells in vitro. RNA interference of survivin inhibited tumor growth in vivo by 68±13% (P=0.002) and increased the number of apoptotic cells by 9.8±1.2% (P=0.001) compared with negative small interfering RNA (siRNA) treatment controls. Moreover, RNA interference of survivin inhibited the formation of lung metastases by 92% (P=0.002) and reduced microvascular density by 60% (P=0.0003). Survivin siRNA resulted in significant downregulation of survivin mRNA and protein expression both in vitro and in vivo compared with negative siRNA treatment controls. RNA interference of survivin was found to be a potent inhibitor of SH-SY5Y tumor growth and metastasis formation. These results support further clinical development of RNA interference of survivin as a treatment of neuroblastoma and other cancer types.

  17. Membrane type 1-matrix metalloproteinase/Akt signaling axis modulates TNF-α-induced procoagulant activity and apoptosis in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Hiroshi Ohkawara

    Full Text Available Membrane type 1-matrix metalloproteinase (MT1-MMP functions as a signaling molecule in addition to a proteolytic enzyme. Our hypothesis was that MT1-MMP cooperates with protein kinase B (Akt in tumor necrosis factor (TNF-α-induced signaling pathways of vascular responses, including tissue factor (TF procoagulant activity and endothelial apoptosis, in cultured human aortic endothelial cells (ECs. TNF-α (10 ng/mL induced a decrease in Akt phosphorylation within 60 minutes in ECs. A chemical inhibitor of MMP, TIMP-2 and selective small interfering RNA (siRNA-mediated suppression of MT1-MMP reversed TNF-α-triggered transient decrease of Akt phosphorylation within 60 minutes, suggesting that MT1-MMP may be a key regulator of Akt phosphorylation in TNF-α-stimulated ECs. In the downstream events, TNF-α increased TF antigen and activity, and suppressed the expression of thrombomodulin (TM antigen. Inhibition of Akt markedly enhanced TNF-α-induced expression of TF antigen and activity, and further reduced the expression of TM antigen. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-α. Moreover, TNF-α induced apoptosis of ECs through Akt- and forkhead box protein O1 (FoxO1-dependent signaling pathway and nuclear factor-kB (NF-kB activation. Knockdown of MT1-MMP by siRNA reversed apoptosis of ECs by inhibiting TNF-α-induced Akt-dependent regulation of FoxO1 in TNF-α-stimulated ECs. Immunoprecipitation demonstrated that TNF-α induced the changes in the associations between the cytoplasmic fraction of MT1-MMP and Akt in ECs. In conclusion, we show new evidence that MT1-MMP/Akt signaling axis is a key modifier for TNF-α-induced signaling pathways for modulation of procoagulant activity and apoptosis of ECs.

  18. Perivascular delivery of Notch 1 siRNA inhibits injury-induced arterial remodeling.

    Directory of Open Access Journals (Sweden)

    Eileen M Redmond

    Full Text Available To determine the efficacy of perivascular delivery of Notch 1 siRNA in preventing injury-induced arterial remodeling.Carotid artery ligation was performed to induce arterial remodeling. After 14 days, morphometric analysis confirmed increased vSMC growth and subsequent media thickening and neointimal formation. Laser capture microdissection, quantitative qRT-PCR and immunoblot analysis of medial tissue revealed a significant increase in Notch1 receptor and notch target gene, Hrt 1 and 2 expression in the injured vessels. Perivascular delivery of Notch 1 siRNA by pluronic gel inhibited the injury-induced increase in Notch 1 receptor and target gene expression when compared to scrambled siRNA controls while concomitantly reducing media thickening and neointimal formation to pre-injury, sham-operated levels. Selective Notch 1 knockdown also reversed the injury-induced inhibition of pro-apoptotic Bax expression while decreasing injury-induced anti-apoptotic Bcl-XL expression to sham-operated control levels. In parallel experiments, proliferative cyclin levels, as measured by PCNA expression, were reversed to sham-operated control levels following selective Notch 1 knockdown.These results suggest that injury-induced arterial remodeling can be successfully inhibited by localized perivascular delivery of Notch 1 siRNA.

  19. Role of annexin A5 in cisplatin-induced toxicity in renal cells: molecular mechanism of apoptosis.

    Science.gov (United States)

    Jeong, Jin-Joo; Park, Nahee; Kwon, Yeo-Jung; Ye, Dong-Jin; Moon, Aree; Chun, Young-Jin

    2014-01-24

    Annexin A5 belongs to a large family of calcium-binding and phospholipid-binding proteins and may act as an endogenous regulator of various pathophysiological processes. There is increasing evidence that annexin A5 is related to cytotoxicity, but the precise function of this protein has yet to be elucidated. In this study, we aimed to verify the function of annexin A5 in the apoptosis of renal epithelial cells. Real-time PCR and Western blot analysis, together with immunofluorescence analysis, showed that the expression of annexin A5 significantly increased in the presence of cisplatin in both human and rat renal epithelial cells. With regard to the mechanism of cisplatin-induced apoptosis, apoptosis-inducing factor (AIF) release into the cytosol was observed, and the underlying mechanism was identified as voltage-dependent anion channel (VDAC) oligomerization. Mitochondrial membrane potential (Δψm) was found to be greatly disrupted in cisplatin-treated cells. Moreover, cisplatin strongly induced translocation of annexin A5 into mitochondria. To understand the functional significance of annexin A5 in renal cell death, we used a siRNA-mediated approach to knock down annexin A5. Annexin A5 depletion by siRNA led to decreased annexin A5 translocation into mitochondria and significantly reduced VDAC oligomerization and AIF release. Annexin A5 siRNA also increased cell viability compared with the control. Moreover, expression of annexin A5 was induced by other nephrotoxicants such as CdCl2 and bacitracin. Taken together, our data suggest that annexin A5 may play a crucial role in cisplatin-induced toxicity by mediating the mitochondrial apoptotic pathway via the induction and oligomerization of VDAC.

  20. ClC-3 deficiency protects preadipocytes against apoptosis induced by palmitate in vitro and in type 2 diabetes mice.

    Science.gov (United States)

    Huang, Yun-Ying; Huang, Xiong-Qin; Zhao, Li-Yan; Sun, Fang-Yun; Chen, Wen-Liang; Du, Jie-Yi; Yuan, Feng; Li, Jie; Huang, Xue-Lian; Liu, Jie; Lv, Xiao-Fei; Guan, Yong-Yuan; Chen, Jian-Wen; Wang, Guan-Lei

    2014-11-01

    Palmitate, a common saturated free fatty acid (FFA), has been demonstrated to induce preadipocyte apoptosis in the absence of adipogenic stimuli, suggesting that preadipocytes may be prone to apoptosis under adipogenic insufficient conditions, like type 2 diabetes mellitus (T2DM). ClC-3, encoding Cl(-) channel or Cl(-)/H(+) antiporter, is critical for cell fate choices of proliferation versus apoptosis under diseased conditions. However, it is unknown whether ClC-3 is related with preadipocyte apoptosis induced by palmitate or T2DM. Palmitate, but not oleate, induced apoptosis and increase in ClC-3 protein expression and endoplasmic reticulum (ER) stress in 3T3-L1 preadipocyte. ClC-3 specific siRNA attenuated palmitate-induced apoptosis and increased protein levels of Grp78, ATF4, CHOP and phosphorylation of JNK1/2, whereas had no effects on increased phospho-PERK and phospho-eIF2α protein expression. Moreover, the enhanced apoptosis was shown in preadipocytes from high-sucrose/fat, low-dose STZ induced T2DM mouse model with hyperglycemia, hyperlipidemia (elevated serum TG and FFA levels) and insulin resistance. ClC-3 knockout significantly attenuated preadipocyte apoptosis and the above metabolic disorders in T2DM mice. These data demonstrated that ClC-3 deficiency prevent preadipocytes against palmitate-induced apoptosis via suppressing ER stress, and also suggested that ClC-3 may play a role in regulating cellular apoptosis and disorders of glucose and lipid metabolism during T2DM.

  1. Random small interfering RNA library screen identifies siRNAs that induce human erythroleukemia cell differentiation.

    Science.gov (United States)

    Fan, Cuiqing; Xiong, Yuan; Zhu, Ning; Lu, Yabin; Zhang, Jiewen; Wang, Song; Liang, Zicai; Shen, Yan; Chen, Meihong

    2011-03-01

    Cancers are characterized by poor differentiation. Differentiation therapy is a strategy to alleviate malignant phenotypes by inducing cancer cell differentiation. Here we carried out a combinatorial high-throughput screen with a random siRNA library on human erythroleukemia K-562 cell differentiation. Two siRNAs screened from the library were validated to be able to induce erythroid differentiation to varying degrees, determined by CD235 and globin up-regulation, GATA-2 down-regulation, and cell growth inhibition. The screen we performed here is the first trial of screening cancer differentiation-inducing agents from a random siRNA library, demonstrating that a random siRNA library can be considered as a new resource in efforts to seek new therapeutic agents for cancers. As a random siRNA library has a broad coverage for the entire genome, including known/unknown genes and protein coding/non-coding sequences, screening using a random siRNA library can be expected to greatly augment the repertoire of therapeutic siRNAs for cancers.

  2. Membrane Type 1–Matrix Metalloproteinase/Akt Signaling Axis Modulates TNF-α-Induced Procoagulant Activity and Apoptosis in Endothelial Cells

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    Ohkawara, Hiroshi; Ishibashi, Toshiyuki; Sugimoto, Koichi; Ikeda, Kazuhiko; Ogawa, Kazuei; Takeishi, Yasuchika

    2014-01-01

    Membrane type 1–matrix metalloproteinase (MT1-MMP) functions as a signaling molecule in addition to a proteolytic enzyme. Our hypothesis was that MT1-MMP cooperates with protein kinase B (Akt) in tumor necrosis factor (TNF)-α-induced signaling pathways of vascular responses, including tissue factor (TF) procoagulant activity and endothelial apoptosis, in cultured human aortic endothelial cells (ECs). TNF-α (10 ng/mL) induced a decrease in Akt phosphorylation within 60 minutes in ECs. A chemical inhibitor of MMP, TIMP-2 and selective small interfering RNA (siRNA)-mediated suppression of MT1-MMP reversed TNF-α-triggered transient decrease of Akt phosphorylation within 60 minutes, suggesting that MT1-MMP may be a key regulator of Akt phosphorylation in TNF-α-stimulated ECs. In the downstream events, TNF-α increased TF antigen and activity, and suppressed the expression of thrombomodulin (TM) antigen. Inhibition of Akt markedly enhanced TNF-α-induced expression of TF antigen and activity, and further reduced the expression of TM antigen. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-α. Moreover, TNF-α induced apoptosis of ECs through Akt- and forkhead box protein O1 (FoxO1)-dependent signaling pathway and nuclear factor-kB (NF-kB) activation. Knockdown of MT1-MMP by siRNA reversed apoptosis of ECs by inhibiting TNF-α-induced Akt-dependent regulation of FoxO1 in TNF-α-stimulated ECs. Immunoprecipitation demonstrated that TNF-α induced the changes in the associations between the cytoplasmic fraction of MT1-MMP and Akt in ECs. In conclusion, we show new evidence that MT1-MMP/Akt signaling axis is a key modifier for TNF-α-induced signaling pathways for modulation of procoagulant activity and apoptosis of ECs. PMID:25162582

  3. Effect of perilipin-5 on apoptosis of cardiac microvascular endothelial cells induced by high fat and high glucose in mice

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    Jin DU

    2017-12-01

    Full Text Available Objective To investigate the effects and mechanisms of perilipin-5 (Plin5 on the apoptosis of mouse cardiac microvascular endothelial cells induced by high fat and high glucose. Methods The mouse cardiac microvascular endothelial cells (MCMECs cultured with high glucose medium were respectively given 0, 100, 300 and 500μmol/L palmitic acid for 24 hours. In order to explore the effects and mechanisms of Plin5 on MCMECs injuries induced by high fat and high glucose, MCMECs exposed to 300μmol/L palmitic acid for 24 hours were divided into control group, Scra siRNA group and Plin5 siRNA group. The control group was only treated with transfection reagent, the Scra siRNA group was given treatment of transfection reagent and garbled RNA, the Plin5 siRNA group was given treatment of transfection reagent and Plin5 specific siRNA. In order to further confirm the specific mechanism of Plin5 in high fat/glucose inducing MCMECs injury, MCMECs in Plin5 siRNA group were divided into vehicle group and N-acetyl cysteine (NAC group, and given the same intervention of high fat. The apoptotic rate was detected by flow cytometry, qRT-PCR and Western blotting were respectively used to detect the mRNA and protein expression of Plin5, and the intracellular reactive oxygen species (ROS level was tested by DHE staining and ELISA kit. Results The apoptotic rate of MCMECs was increased in a fat concentration-dependent manner (P<0.05. Compared with 0μmol/L palmitic acid group, the intracellular ROS content and the expression of Plin5 increased significantly in 300μmol/L palmitic acid group (P<0.05. Compared with the control group and the Scra siRNA group, the intracellular ROS content and apoptotic rate increased significantly in Plin5 siRNA group under the action of 300μmol/L palmitic acid (P<0.05. Compared with the vehicle group, the intracellular ROS content and apoptotic rate decreased remarkably in NAC group (P<0.05. Conclusion With inhibition of oxidative stress

  4. A Monoclonal Antibody against Wnt-1 Induces Apoptosis in Human Cancer Cells

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    Biao He

    2004-01-01

    Full Text Available Aberrant activation of the Wingless-type (Wnt/β-catenin signaling pathway is associated with a variety of human cancers. Little is known regarding the role that Wnt ligands play in human carcinogenesis. To test whether a Wnt-1 signal is a survival factor in human cancer cells and thus may serve as a potential cancer therapeutic target, we investigated the effect of inhibition of Wnt-1 signaling in a variety of human cancer cell lines, including non small cell lung cancer, breast cancer, mesothelioma, and sarcoma. Both monoclonal antibody and RNA interference (RNAi were used to inhibit Wnt-1 signaling. We found that incubation of a monoclonal anti-Wnt-1 antibody induced apoptosis and caused downstream protein changes in cancer cells overexpressing Wnt-1. In contrast, apoptosis was not detected in cells lacking or having minimal Wnt-1 expression after the antibody incubation. RNAi targeting of Wnt-1 in cancer cells overexpressing Wnt-1 demonstrated similar downstream protein changes and induction of apoptosis. The antibody also suppressed tumor growth in vivo. Our results indicate that both monoclonal anti-Wnt-1 antibody and Wnt-1 siRNA inhibit Wnt-1 signaling and can induce apoptosis in human cancer cells. These findings hold promise as a novel therapeutic strategy for cancer.

  5. siRNA inhibition of telomerase enhances the anti-cancer effect of doxorubicin in breast cancer cells

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    Dong, Xuejun; Liu, Anding; Zer, Cindy; Feng, Jianguo; Zhen, Zhuan; Yang, Mingfeng; Zhong, Li

    2009-01-01

    Doxorubicin is an effective breast cancer drug but is hampered by a severe, dose-dependent toxicity. Concomitant administration of doxorubicin and another cancer drug may be able to sensitize tumor cells to the cytotoxicity of doxorubicin and lowers the therapeutic dosage. In this study, we examined the combined effect of low-dose doxorubicin and siRNA inhibition of telomerase on breast cancer cells. We found that when used individually, both treatments were rapid and potent apoptosis inducers; and when the two treatments were combined, we observed an enhanced and sustained apoptosis induction in breast cancer cells. siRNA targeting the mRNA of the protein component of telomerase, the telomerase reverse transcriptase (hTERT), was transfected into two breast cancer cell lines. The siRNA inhibition was confirmed by RT-PCR and western blot on hTERT mRNA and protein levels, respectively, and by measuring the activity level of telomerase using the TRAP assay. The effect of the hTERT siRNA on the tumorigenicity of the breast cancer cells was also studied in vivo by injection of the siRNA-transfected breast cancer cells into nude mice. The effects on cell viability, apoptosis and senescence of cells treated with hTERT siRNA, doxorubicin, and the combined treatment of doxorubicin and hTERT siRNA, were examined in vitro by MTT assay, FACS and SA-β-galactosidase staining. The hTERT siRNA effectively knocked down the mRNA and protein levels of hTERT, and reduced the telomerase activity to 30% of the untreated control. In vivo, the tumors induced by the hTERT siRNA-transfected cells were of reduced sizes, indicating that the hTERT siRNA also reduced the tumorigenic potential of the breast cancer cells. The siRNA treatment reduced cell viability by 50% in breast cancer cells within two days after transfection, while 0.5 μM doxorubicin treatment had a comparable effect but with a slower kinetics. The combination of hTERT siRNA and 0.5 μM doxorubicin killed twice as many

  6. Decreased expression of MUC1 induces apoptosis and inhibits migration in pancreatic cancer PANC-1 cells via regulation of Slug pathway.

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    Zhao, Ping; Meng, Meng; Xu, Bin; Dong, Aiping; Ni, Guangzhen; Lu, Lianfang

    2017-12-06

    MUC1, a membrane tethered mucin glycoprotein, is overexpressed in > 60% of human pancreatic cancers (PCs), and is associated with poor prognosis and enhanced metastasis. Here, we report the effect of silencing MUC1 expression on the growth, migration and invasive ability of pancreatic cancer cells, and explored its mechanisms. We observed that siRNA mediated suppression of the MUC1 expression significantly reduced invasive and migrative capability and induced apoptosis of the pancreatic cancer PANC-1 cells. We found that Slug was inhibited in the MUC1 siRNA transfected PANC-1 cells (MUC1 siRNA/PANC-1 cells). Expression of PUMA and E-cadherin was increased in the MUC1 siRNA/PANC-1 cells. PANC-1 cells overexpressing full long Slug gene (when transfected with Slug cDNA plasmid) significantly inhibited PUMA and E-cadherin expression in the MUC1 siRNA/PANC-1 cells. Silencing PUMA expression inhibited apoptosis in the MUC1 siRNA transfected PANC-1 cells (MUC1 siRNA/PANC-1 cells). Silencing E-cadherin expression restored the invasion and migration ability in the MUC1 siRNA/PANC-1 cells. We therefore concluded that silencing MUC1 expression inhibited migration and invasion, and induced apoptosis of PANC-1 cells via downregulation of Slug and upregulation of Slug dependent PUMA and E-cadherin expression. MUC1 could serve as a potential therapeutic target in pancreatic cancer.

  7. Axin1 up-regulated 1 accelerates stress-induced cardiomyocytes apoptosis through activating Wnt/β-catenin signaling.

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    Ye, Xing; Lin, Junyi; Lin, Zebin; Xue, Aimin; Li, Liliang; Zhao, Ziqin; Liu, Li; Shen, Yiwen; Cong, Bin

    2017-10-15

    Stress-induced cardiomyocyte apoptosis contributes to the pathogenesis of a variety of cardiovascular diseases, but how stress induces cardiomyocyte apoptosis remains largely unclear. The present study aims to investigate the effects of Axin1 up-regulated 1 (Axud1), a novel pro-apoptotic protein, on the cardiomyocyte survival and the underlying mechanisms. To this end, a rat model under restraint stress (RS) was established and in vitro stress-induced cardiomyocytes culture was achieved. Our data showed that Axud1 was upregulated in the rat myocardia after exposure to RS. Anti-apoptotic Bcl-2 was decreased, whereas pro-apoptotic Bax and Cleaved caspase-3 (Cc3) were increased in a time-dependent manner. The Wnt/β-catenin signaling was observed to be interestingly activated in heart undergoing RS. In addition, the treatment of norepinephrine (NE) to in vitro cardiomyocytes increased Axud1 level and induced cell apoptosis. Wnt/β-catenin signaling was consistently activated. Knockdown of Axud1 using specific siRNA blunted NE-induced cardiomyocytes apoptosis and also inactivated the Wnt/β-catenin signaling. XAV-939, an inhibitor of Wnt/β-catenin signaling, partially reversed the pro-apoptotic effect of NE. In conclusion, Axud1 accelerated stress-induced cardiomyocytes apoptosis through activation of Wnt/β-catenin signaling pathway. Our data provided novel evidence that therapeutic strategies against Axud1 or Wnt/β-catenin signaling might be promising in relation to RS-induced myocardial injury. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Holotoxin A1 Induces Apoptosis by Activating Acid Sphingomyelinase and Neutral Sphingomyelinase in K562 and Human Primary Leukemia Cells

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    Seong-Hoon Yun

    2018-04-01

    Full Text Available Marine triterpene glycosides are attractive candidates for the development of anticancer agents. Holotoxin A1 is a triterpene glycoside found in the edible sea cucumber, Apostichopus (Stichopus japonicus. We previously showed that cladoloside C2, the 25(26-dihydro derivative of holotoxin A1, induced apoptosis in human leukemia cells by activating ceramide synthase 6. Thus, we hypothesized that holotoxin A1, which is structurally similar to cladoloside C2, might induce apoptosis in human leukemia cells through the same molecular mechanism. In this paper, we compared holotoxin A1 and cladoloside C2 for killing potency and mechanism of action. We found that holotoxin A1 induced apoptosis more potently than cladoloside C2. Moreover, holotoxin A1 induced apoptosis in K562 cells by activating caspase-8 and caspase-3, but not by activating caspase-9. During holotoxin A1-induced apoptosis, acid sphingomyelinase (SMase and neutral SMase were activated in both K562 cells and human primary leukemia cells. Specifically inhibiting acid SMase and neutral SMаse with chemical inhibitors or siRNAs significantly inhibited holotoxin A1–induced apoptosis. These results indicated that holotoxin A1 might induce apoptosis by activating acid SMase and neutral SMase. In conclusion, holotoxin A1 represents a potential anticancer agent for treating leukemia. Moreover, the aglycone structure of marine triterpene glycosides might affect the mechanism involved in inducing apoptosis.

  9. Down-regulation of cellular FLICE-inhibitory protein (Long Form contributes to apoptosis induced by Hsp90 inhibition in human lung cancer cells

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    Wang Qilin

    2012-12-01

    Full Text Available Abstract Background Cellular FLICE-Inhibitory Protein (long form, c-FLIPL is a critical negative regulator of death receptor-mediated apoptosis. Overexpression of c-FLIPL has been reported in many cancer cell lines and is associated with chemoresistance. In contrast, down-regulation of c-FLIP may drive cancer cells into cellular apoptosis. This study aims to demonstrate that inhibition of the heat shock protein 90 (Hsp90 either by inhibitors geldanamycin/17-N-Allylamino-17-demethoxygeldanamycin (GA/17-AAG or siRNA technique in human lung cancer cells induces c-FLIPL degradation and cellular apoptosis through C-terminus of Hsp70-interacting protein (CHIP-mediated mechanisms. Methods Calu-1 and H157 cell lines (including H157-c-FLIPL overexpressing c-FLIPL and control cell H157-lacZ were treated with 17-AAG and the cell lysates were prepared to detect the given proteins by Western Blot and the cell survival was assayed by SRB assay. CHIP and Hsp90 α/β proteins were knocked down by siRNA technique. CHIP and c-FLIPL plasmids were transfected into cells and immunoprecipitation experiments were performed to testify the interactions between c-FLIPL, CHIP and Hsp90. Results c-FLIPL down-regulation induced by 17-AAG can be reversed with the proteasome inhibitor MG132, which suggested that c-FLIPL degradation is mediated by a ubiquitin-proteasome system. Inhibition of Hsp90α/β reduced c-FLIPL level, whereas knocking down CHIP expression with siRNA technique inhibited c-FLIPL degradation. Furthermore, c-FLIPL and CHIP were co-precipitated in the IP complexes. In addition, overexpression of c-FLIPL can rescue cancer cells from apoptosis. When 17-AAG was combined with an anti-cancer agent celecoxib(CCB, c-FLIPL level declined further and there was a higher degree of caspase activation. Conclusion We have elucidated c-FLIPL degradation contributes to apoptosis induced by Hsp90 inhibition, suggesting c-FLIP and Hsp90 may be the promising combined targets

  10. Molybdenum induces pancreatic β-cell dysfunction and apoptosis via interdependent of JNK and AMPK activation-regulated mitochondria-dependent and ER stress-triggered pathways

    International Nuclear Information System (INIS)

    Yang, Tsung-Yuan; Yen, Cheng-Chieh; Lee, Kuan-I; Su, Chin-Chuan; Yang, Ching-Yao; Wu, Chin-Ching; Hsieh, Shang-Shu; Ueng, Kwo-Chang; Huang, Chun-Fa

    2016-01-01

    Molybdenum (Mo), a well-known toxic environmental and industrial pollutant, causes adverse health effects and diseases in humans and has received attention as a potential risk factor for DM. However, the roles of Mo in the mechanisms of the toxicological effects in pancreatic β-cells are mostly unclear. In this study, the results revealed dysfunction of insulin secretion and apoptosis in the pancreatic β-cell-derived RIN-m5F cells and the isolated mouse islets in response to Mo. These effects were accompanied by a mitochondria-dependent apoptotic signals including a decreased in the MMP, an increase in cytochrome c release, and the activation of caspase cascades and PARP. In addition, ER stress was triggered as indicated by several key molecules of the UPR. Furthermore, exposure to Mo induced the activation of ERK1/2, JNK, AMPKα, and GSK3-α/β. Pretreatment with specific pharmacological inhibitors (in RIN-m5F cells and isolated mouse islets) of JNK (SP600125) and AMPK (Compound C) or transfection with si-RNAs (in RIN-m5F cells) specific to JNK and AMPKα effectively prevented the Mo-induced apoptosis and related signals, but inhibitors of ERK1/2 and GSK3-α/β (PD98059 and LiCl, respectively) did not reverse the Mo-induced effects. Additionally, both the inhibitors and specific si-RNAs could suppress the Mo-induced phosphorylation of JNK and AMPKα each other. Taken together, these results suggest that Mo exerts its cytotoxicity on pancreatic β-cells by inducing dysfunction and apoptosis via interdependent JNK and AMPK activation downstream-regulated mitochondrial-dependent and ER stress-triggered apoptosis pathways. - Highlights: • Molybdenum (Mo) induces pancreatic β-cell dysfunction and apoptosis. • Mo causes β-cell death via mitochondria-dependent caspase cascades signals. • ER stress-triggered apoptotic pathway also regulates Mo-induced β-cell death. • Interdependent of JNK and AMPK activation involves in Mo-induced β-cell apoptosis.

  11. Molybdenum induces pancreatic β-cell dysfunction and apoptosis via interdependent of JNK and AMPK activation-regulated mitochondria-dependent and ER stress-triggered pathways

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Tsung-Yuan [Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China); Yen, Cheng-Chieh [Department of Occupational Safety and Health, College of Health Care and Management, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Occupational Medicine, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China); Lee, Kuan-I [Department of Emergency, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taichung 427, Taiwan (China); Su, Chin-Chuan [Department of Otorhinolaryngology, Head and Neck Surgery, Changhua Christian Hospital, Changhua County 500, Taiwan (China); Graduate Institute of Basic Medical Science, School of Medicine, College of Medicine, China Medical University, Taichung 404, Taiwan (China); Yang, Ching-Yao [Department of Surgery, National Taiwan University Hospital, Taipei 100, Taiwan (China); Department of Surgery, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Wu, Chin-Ching [Department of Public Health, China Medical University, Taichung 404, Taiwan (China); Hsieh, Shang-Shu, E-mail: gile1123@yahoo.com.tw [Department of Emergency, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taichung 427, Taiwan (China); Ueng, Kwo-Chang, E-mail: kcueng@gmail.com [Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China); School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan (China); Huang, Chun-Fa, E-mail: cfhuang@mail.cmu.edu.tw [School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan (China)

    2016-03-01

    Molybdenum (Mo), a well-known toxic environmental and industrial pollutant, causes adverse health effects and diseases in humans and has received attention as a potential risk factor for DM. However, the roles of Mo in the mechanisms of the toxicological effects in pancreatic β-cells are mostly unclear. In this study, the results revealed dysfunction of insulin secretion and apoptosis in the pancreatic β-cell-derived RIN-m5F cells and the isolated mouse islets in response to Mo. These effects were accompanied by a mitochondria-dependent apoptotic signals including a decreased in the MMP, an increase in cytochrome c release, and the activation of caspase cascades and PARP. In addition, ER stress was triggered as indicated by several key molecules of the UPR. Furthermore, exposure to Mo induced the activation of ERK1/2, JNK, AMPKα, and GSK3-α/β. Pretreatment with specific pharmacological inhibitors (in RIN-m5F cells and isolated mouse islets) of JNK (SP600125) and AMPK (Compound C) or transfection with si-RNAs (in RIN-m5F cells) specific to JNK and AMPKα effectively prevented the Mo-induced apoptosis and related signals, but inhibitors of ERK1/2 and GSK3-α/β (PD98059 and LiCl, respectively) did not reverse the Mo-induced effects. Additionally, both the inhibitors and specific si-RNAs could suppress the Mo-induced phosphorylation of JNK and AMPKα each other. Taken together, these results suggest that Mo exerts its cytotoxicity on pancreatic β-cells by inducing dysfunction and apoptosis via interdependent JNK and AMPK activation downstream-regulated mitochondrial-dependent and ER stress-triggered apoptosis pathways. - Highlights: • Molybdenum (Mo) induces pancreatic β-cell dysfunction and apoptosis. • Mo causes β-cell death via mitochondria-dependent caspase cascades signals. • ER stress-triggered apoptotic pathway also regulates Mo-induced β-cell death. • Interdependent of JNK and AMPK activation involves in Mo-induced β-cell apoptosis.

  12. 2'-Hydroxycinnamaldehyde induces apoptosis through HSF1-mediated BAG3 expression.

    Science.gov (United States)

    Nguyen, Hai-Anh; Kim, Soo-A

    2017-01-01

    BAG3, a member of BAG co-chaperone family, is induced by stressful stimuli such as heat shock and heavy metals. Through interaction with various binding partners, BAG3 is thought to play a role in cellular adaptive responses against stressful conditions in normal and neoplastic cells. 2'-Hydroxycinnamaldehyde (HCA) is a natural derivative of cinnamaldehyde and has antitumor activity in various cancer cells. In the present study, for the first time, we identified that HCA induced BAG3 expression and BAG3-mediated apoptosis in cancer cells. The apoptotic cell death induced by HCA was demonstrated by caspase-7, -9 and PARP activation, and confirmed by Annexin V staining in both SW480 and SW620 colon cancer cells. Notably, both the mRNA and protein levels of BAG3 were largely induced by HCA in a dose- and time-dependent manner. By showing transcription factor HSF1 activation, we demonstrated that HCA induces the expression of BAG3 through HSF1 activation. More importantly, knockdown of BAG3 expression using siRNA largely inhibited HCA-induced apoptosis, suggesting that BAG3 is actively involved in HCA-induced cancer cell death. Considering the importance of the stress response mechanism in cancer progression, our results strongly suggest that BAG3 could be a potential target for anticancer therapy.

  13. Silencing of the HER2/neu Gene by siRNA Inhibits Proliferation and Induces Apoptosis in HER2/neu-Overexpressing Breast Cancer Cells

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    Timo Faltus

    2004-11-01

    Full Text Available In eukaryotes, double-stranded (ds RNA induces sequence-specific inhibition of gene expression referred to as RNA interference (RNAi. We exploited RNAi to define the role of HER2/neu in the neoplastic proliferation of human breast cancer cells. We transfected SK-BR-3, BT-474, MCF-7, and MDA-MB-468 breast cancer cells with short interfering RNA (siRNA targeted against human HER2/neu and analyzed the specific inhibition of HER2/neu expression by Northern and Western blots. Transfection with HER2/neu-specific siRNA resulted in a sequence-specific decrease in HER2/neu mRNA and protein levels. Moreover, transfection with HER2/neu siRNA caused cell cycle arrest at G0/G1 in the breast cancer cell lines SKBR-3 and BT-474, consistent with a powerful RNA silencing effect. siRNA treatment resulted in an antiproliferative and apoptotic response in cells overexpressing HER2/neu, but had no influence in cells with almost no expression of HER2/neu proteins like MDA-MB-468 cells. These data indicate that HER2/neu function is essential for the proliferation of HER2/neuoverexpressing breast cancer cells. Our observations suggest that siRNA targeted against human HER2/neu may be valuable tools as anti proliferative agents that display activity against neoplastic cells at very low doses.

  14. Fluid shear stress suppresses TNF-α-induced apoptosis in MC3T3-E1 cells: Involvement of ERK5-AKT-FoxO3a-Bim/FasL signaling pathways

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    Bin, Geng; Bo, Zhang; Jing, Wang; Jin, Jiang; Xiaoyi, Tan; Cong, Chen; Liping, An; Jinglin, Ma; Cuifang, Wang; Yonggang, Chen [The Second Hospital of Lanzhou University, #82 Cuiyingmen, Lanzhou, 730000 Gansu (China); Orthopaedics Key Laboratory of Gansu Province, Lanzhou, 730000 Gansu (China); Yayi, Xia, E-mail: xiayayildey@163.com [The Second Hospital of Lanzhou University, #82 Cuiyingmen, Lanzhou, 730000 Gansu (China); Orthopaedics Key Laboratory of Gansu Province, Lanzhou, 730000 Gansu (China)

    2016-05-01

    TNF-α is known to induce osteoblasts apoptosis, whereas mechanical stimulation has been shown to enhance osteoblast survival. In the present study, we found that mechanical stimulation in the form of fluid shear stress (FSS) suppresses TNF-α induced apoptosis in MC3T3-E1 cells. Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family that has been implicated in cell survival. We also demonstrated that FSS imposed by flow chamber in vitro leads to a markedly activation of ERK5, which was shown to be protective against TNF-α-induced apoptosis, whereas the transfection of siRNA against ERK5 (ERK5-siRNA) reversed the FSS-medicated anti-apoptotic effects. An initial FSS-mediated activation of ERK5 that phosphorylates AKT to increase its activity, and a following forkhead box O 3a (FoxO3a) was phosphorylated by activated AKT. Phosphorylated FoxO3a is sequestered in the cytoplasm, and prevents it from translocating to nucleus where it can increase the expression of FasL and Bim. The inhibition of AKT-FoxO3a signalings by a PI3K (PI3-kinase)/AKT inhibitor (LY294002) or the transfection of ERK5-siRNA led to the nuclear translocation of non-phosphorylated FoxO3a, and increased the protein expression of FasL and Bim. In addition, the activation of caspase-3 by TNF-α was significantly inhibited by aforementioned FSS-medicated mechanisms. In brief, the activation of ERK5-AKT-FoxO3a signaling pathways by FSS resulted in a decreased expression of FasL and Bim and an inhibition of caspase-3 activation, which exerts a protective effect that prevents osteoblasts from apoptosis. - Highlights: • Fluid shear stress inhibits osteoblast apoptosis induced by TNF-α. • Inhibition of ERK5 activity by transfection of ERK5 siRNA blocks FSS-mediated anti-apoptotic effect in osteoblast. • Activated ERK5-AKT-FoxO3a-Bim/FasL signaling pathways by FSS is required to protect osteoblast from apoptosis.

  15. Resveratrol attenuates methylglyoxal-induced mitochondrial dysfunction and apoptosis by Sestrin2 induction

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    Seo, Kyuhwa; Seo, Suho; Han, Jae Yun; Ki, Sung Hwan; Shin, Sang Mi, E-mail: smshin@chosun.ac.kr

    2014-10-15

    Methylglyoxal is found in high levels in the blood and other tissues of diabetic patients and exerts deleterious effects on cells and tissues. Previously, we reported that resveratrol, a polyphenol in grapes, induced the expression of Sestrin2 (SESN2), a novel antioxidant protein, and inhibited hepatic lipogenesis. This study investigated whether resveratrol protects cells from the methylglyoxal-induced toxicity via SESN2 induction. Methylglyoxal significantly induced cell death in HepG2 cells. However, cells pretreated with resveratrol were rescued from methylglyoxal-induced apoptosis. Resveratrol attenuated glutathione (GSH) depletion and ROS production promoted by methylglyoxal. Moreover, mitochondrial damage was observed by methylglyoxal treatment, but resveratrol restored mitochondrial function, as evidenced by the observed lack of mitochondrial permeability transition and increased ADP/ATP ratio. Resveratrol treatment inhibited SESN2 depletion elicited by methylglyoxal. SESN2 overexpression repressed methylglyoxal-induced mitochondrial dysfunction and apoptosis. Likewise, rotenone-induced cytotoxicity was not observed in SESN2 overexpressed cells. Furthermore, siRNA knockdown of SESN2 reduced the ability of resveratrol to prevent methylglyoxal-induced mitochondrial permeability transition. In addition, when mice were exposed to methylglyoxal after infection of Ad-SESN2, the plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and GSH depletion by methylglyoxal in liver was reduced in Ad-SESN2 infected mice. Our results demonstrated that resveratrol is capable of protecting cells from methylglyoxal-induced mitochondrial dysfunction and oxidative stress via SESN2 induction. - Highlights: • Resveratrol decreased methylglyoxal-induced apoptosis. • Resveratrol attenuated GSH depletion and ROS production promoted by methylglyoxal. • Resveratrol restored the mitochondrial function by Sestrin2 induction. • Induction of Sestrin2

  16. Resveratrol attenuates methylglyoxal-induced mitochondrial dysfunction and apoptosis by Sestrin2 induction

    International Nuclear Information System (INIS)

    Seo, Kyuhwa; Seo, Suho; Han, Jae Yun; Ki, Sung Hwan; Shin, Sang Mi

    2014-01-01

    Methylglyoxal is found in high levels in the blood and other tissues of diabetic patients and exerts deleterious effects on cells and tissues. Previously, we reported that resveratrol, a polyphenol in grapes, induced the expression of Sestrin2 (SESN2), a novel antioxidant protein, and inhibited hepatic lipogenesis. This study investigated whether resveratrol protects cells from the methylglyoxal-induced toxicity via SESN2 induction. Methylglyoxal significantly induced cell death in HepG2 cells. However, cells pretreated with resveratrol were rescued from methylglyoxal-induced apoptosis. Resveratrol attenuated glutathione (GSH) depletion and ROS production promoted by methylglyoxal. Moreover, mitochondrial damage was observed by methylglyoxal treatment, but resveratrol restored mitochondrial function, as evidenced by the observed lack of mitochondrial permeability transition and increased ADP/ATP ratio. Resveratrol treatment inhibited SESN2 depletion elicited by methylglyoxal. SESN2 overexpression repressed methylglyoxal-induced mitochondrial dysfunction and apoptosis. Likewise, rotenone-induced cytotoxicity was not observed in SESN2 overexpressed cells. Furthermore, siRNA knockdown of SESN2 reduced the ability of resveratrol to prevent methylglyoxal-induced mitochondrial permeability transition. In addition, when mice were exposed to methylglyoxal after infection of Ad-SESN2, the plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and GSH depletion by methylglyoxal in liver was reduced in Ad-SESN2 infected mice. Our results demonstrated that resveratrol is capable of protecting cells from methylglyoxal-induced mitochondrial dysfunction and oxidative stress via SESN2 induction. - Highlights: • Resveratrol decreased methylglyoxal-induced apoptosis. • Resveratrol attenuated GSH depletion and ROS production promoted by methylglyoxal. • Resveratrol restored the mitochondrial function by Sestrin2 induction. • Induction of Sestrin2

  17. Interferon beta induces apoptosis in nasopharyngeal carcinoma cells via the TRAIL-signaling pathway.

    Science.gov (United States)

    Makowska, Anna; Wahab, Lora; Braunschweig, Till; Kapetanakis, Nikiforos-Ioannis; Vokuhl, Christian; Denecke, Bernd; Shen, Lian; Busson, Pierre; Kontny, Udo

    2018-03-06

    The combination of neoadjuvant chemotherapy, radiochemotherapy, and maintenance therapy with interferon beta (IFNβ) has led to superior results in the treatment of children and adolescents with nasopharyngeal carcinoma (NPC). However, nothing is known about the mechanism of the antitumor activity of IFNβ in NPC. Here, we investigate the role of IFNβ on apoptosis in NPC cells. Six NPC cell lines, one patient-derived NPC xenograft (PDX) and one SV40-transformed nasoepithelial cell line were used. Induction of apoptosis by IFNβ was measured by flow cytometric analysis of subG1-DNA-content, Hoechst 33258 staining and activation of caspase-3. Dissection of death ligand signaling pathways included measuring surface expression of its components by flow cytometry, activation by death ligands and neutralization with specific antibodies and siRNA. IFNβ induced apoptosis at concentrations achievable in humans in five of six NPC cell lines and in PDX cells but not in nasoepithelial cells. Inhibition of caspases-3 and -8 abrogated this effect suggesting IFNβ promoted apoptosis through the extrinsic pathway. IFNβ induced surface expression of TRAIL and TRAIL-R2 and the addition of an anti-TRAIL-antibody or transfection with TRAIL-siRNA blocked IFNβ-induced apoptosis. No induction of TRAIL-expression was noted in the IFNβ-resistant cell line. In conclusion, IFNβ leads to apoptosis in NPC cells in an autocrine way via the induction of TRAIL expression and subsequent activation of the TRAIL-signaling pathway. The mechanism described could at least partly explain the clinical benefit of IFNβ in the treatment of NPC. Further studies in a mouse-xenograft model are warranted to substantiate this effect in vivo .

  18. Involvement of Endoplasmic Reticulum Stress in Capsaicin-Induced Apoptosis of Human Pancreatic Cancer Cells

    Directory of Open Access Journals (Sweden)

    Shengzhang Lin

    2013-01-01

    Full Text Available Capsaicin, main pungent ingredient of hot chilli peppers, has been shown to have anticarcinogenic effect on various cancer cells through multiple mechanisms. In this study, we investigated the apoptotic effect of capsaicin on human pancreatic cancer cells in both in vitro and in vivo systems, as well as the possible mechanisms involved. In vitro, treatment of both the pancreatic cancer cells (PANC-1 and SW1990 with capsaicin resulted in cells growth inhibition, G0/G1 phase arrest, and apoptosis in a dose-dependent manner. Knockdown of growth arrest- and DNA damage-inducible gene 153 (GADD153, a marker of the endoplasmic-reticulum-stress- (ERS- mediated apoptosis pathway, by specific siRNA attenuated capsaicin-induced apoptosis both in PANC-1 and SW1990 cells. Moreover, in vivo studies capsaicin effectively inhibited the growth and metabolism of pancreatic cancer and prolonged the survival time of pancreatic cancer xenograft tumor-induced mice. Furthermore, capsaicin increased the expression of some key ERS markers, including glucose-regulated protein 78 (GRP78, phosphoprotein kinase-like endoplasmic reticulum kinase (phosphoPERK, and phosphoeukaryotic initiation factor-2α (phospho-eIF2α, activating transcription factor 4 (ATF4 and GADD153 in tumor tissues. In conclusion, we for the first time provide important evidence to support the involvement of ERS in the induction of apoptosis in pancreatic cancer cells by capsaicin.

  19. Diclofenac inhibits tumor necrosis factor-α-induced nuclear factor-κB activation causing synergistic hepatocyte apoptosis.

    Science.gov (United States)

    Fredriksson, Lisa; Herpers, Bram; Benedetti, Giulia; Matadin, Quraisha; Puigvert, Jordi C; de Bont, Hans; Dragovic, Sanja; Vermeulen, Nico P E; Commandeur, Jan N M; Danen, Erik; de Graauw, Marjo; van de Water, Bob

    2011-06-01

    Drug-induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor α (TNF-α). HepG2 cells were treated with diclofenac followed by TNF-α challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF-α. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF-α-mediated enhancement involved activation of caspase-3 through a caspase-8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c-Jun N-terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF-α-induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF-α-mediated nuclear factor kappaB (NF-κB) translocation oscillation in association with reduced NF-κB transcriptional activity. This was associated with inhibition by diclofenac of the TNF-α-induced phosphorylation of the inhibitor of NF-κB alpha (IκBα). Finally, inhibition of IκB kinase β (IKKβ) with BMS-345541 as well as stable lentiviral short hairpin RNA (shRNA)-based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNF-α-induced cytotoxicity. Together, our data suggest a model whereby diclofenac-mediated stress signaling suppresses TNF-α-induced survival signaling routes and sensitizes cells to apoptosis. Copyright © 2011 American Association for the Study of Liver Diseases.

  20. PEGylated carboxymethyl chitosan/calcium phosphate hybrid anionic nanoparticles mediated hTERT siRNA delivery for anticancer therapy.

    Science.gov (United States)

    Xie, Ying; Qiao, Hongzhi; Su, Zhigui; Chen, Minglei; Ping, Qineng; Sun, Minjie

    2014-09-01

    Lack of safe and effective delivery vehicle is the main obstacle for siRNA mediated cancer therapy. In this study, we synthesized a pH-sensitive polymer of PEG grafted carboxymethyl chitosan (PEG-CMCS) and developed anionic-charged hybrid nanoparticles of PEG-CMCS and calcium phosphate (CaP) for siRNA delivery through a single-step self-assembly method in aqueous condition. The formed nanoparticles with charge of around -8.25 mv and average diameter of 102.1 nm exhibited efficient siRNA encapsulation and enhanced colloidal and serum stability. The test in vitro indicated that the nanoparticles entered into HepG2 cells by endocytosis, and achieved endosomal escape of siRNA effectively due to the pH-responsive disassembly of nanoparticles and dissolution of CaP in the endosome. Reporter gene silencing assay showed that luciferase siRNA delivered by the anionic nanoparticles could achieve gene silencing efficacy comparable to that of conventional Lipofectamine 2000. Additionally, dramatic hTERT knockdown mediated by the anionic nanoparticles transfection induced significant apoptosis of HepG2 cells in vitro. After intravenous injection in tumor-bearing BALB/c nude mice, the nanoparticles specifically accumulated into tumor regions by EPR effect, leading to efficient and specific gene silencing sequentially. Most importantly, the nanoparticles carrying hTERT siRNA inhibited tumor growth significantly via silencing hTERT expression and inducing cells apoptosis in HepG2 tumor xenograft. Moreover, comprehensive safety studies of the nanoparticles confirmed their superior safety both in vitro and in vivo. We concluded that the PEG-CMCS/CaP hybrid anionic nanoparticles possessed potential as a safe and effective siRNA delivery system for anticancer therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Apoptosis and reduced cell proliferation of HL-60 cell line caused by human telomerase reverse transcriptase inhibition by siRNA.

    Science.gov (United States)

    Miri-Moghaddam, Ebrahim; Deezagi, Abdolkhaleg; Soheili, Zahra Sohaila; Shariati, Parvin

    2010-01-01

    The close correlation between telomerase activity and human telomerase reverse transcriptase (hTERT) expression has made hTERT to be considered as a selective molecular target for human cancer therapy. In this study, the ability of short-interfering RNA (siRNA) to downregulate hTERT expression and its correlation with cell growth and apoptosis in the promyelocytic cell line HL-60 was evaluated. hTERT siRNA was designed and transfected to HL-60. hTERT mRNA expression, cell proliferation and apoptotic cells were measured. The results indicated that hTERT siRNA resulted in 97.2 ± 0.6% downregulation of the hTERT mRNA content; inhibition of the cell proliferation rate was about 52.8 ± 2.3% and the apoptotic index of cells was 30.5 ± 1.5%. hTERT plays an essential role in cell proliferation and control of the viability of leukemic cells, thus promising the development of drugs for leukemia. Copyright © 2010 S. Karger AG, Basel.

  2. TNF/TNFR1 pathway and endoplasmic reticulum stress are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes

    International Nuclear Information System (INIS)

    Zhang, Fu-Tao; Ding, Yi; Shah, Zahir; Xing, Dan; Gao, Yuan; Liu, Dong Ming; Ding, Ming-Xing

    2014-01-01

    Background and purpose: Quinolones cause obvious cartilaginous lesions in juvenile animals by chondrocyte apoptosis, which results in the restriction of their use in pediatric and adolescent patients. Studies showed that chondrocytes can be induced to produce TNFα, and the cisternae of the endoplasmic reticulum in quinolone-treated chondrocytes become dilated. We investigated whether TNF/TNFR 1 pathway and endoplasmic reticulum stress (ERs) are involved in ofloxacin (a typical quinolone)-induced apoptosis of juvenile canine chondrocytes. Experimental approach: Canine juvenile chondrocytes were treated with ofloxacin. Cell survival and apoptosis rates were determined with MTT method and flow cytometry, respectively. The gene expression levels of the related signaling molecules (TNFα, TNFR 1 , TRADD, FADD and caspase-8) in death receptor pathways and main apoptosis-related molecules (calpain, caspase-12, GADD153 and GRP78) in ERs were measured by qRT-PCR. The gene expression of TNFR 1 was suppressed with its siRNA. The protein levels of TNFα, TNFR 1 and caspase-12 were assayed using Western blotting. Key results: The survival rates decreased while apoptosis rates increased after the chondrocytes were treated with ofloxacin. The mRNA levels of the measured apoptosis-related molecules in death receptor pathways and ERs, and the protein levels of TNFα, TNFR 1 and caspase-12 increased after the chondrocytes were exposed to ofloxacin. The downregulated mRNA expressions of TNFR 1 , Caspase-8 and TRADD, and the decreased apoptosis rates of the ofloxacin-treated chondrocytes occurred after TNFR 1 –siRNA interference. Conclusions and implications: Ofloxacin-induced chondrocyte apoptosis in a time- and concentration-dependent fashion. TNF/TNFR 1 pathway and ERs are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes in the early stage. - Highlights: • Chondrocyte apoptosis is induced by ofloxacin in a time- and concentration-dependent manners.

  3. Gallic Acid Induces a Reactive Oxygen Species-Provoked c-Jun NH2-Terminal Kinase-Dependent Apoptosis in Lung Fibroblasts

    Science.gov (United States)

    Chen, Chiu-Yuan; Chen, Kun-Chieh; Yang, Tsung-Ying; Liu, Hsiang-Chun; Hsu, Shih-Lan

    2013-01-01

    Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by fibroblasts proliferation and extracellular matrix accumulation. Induction of fibroblast apoptosis therefore plays a crucial role in the resolution of this disease. Gallic acid (3,4,5-trihydroxybenzoic acid), a common botanic phenolic compound, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in lung fibroblasts apoptosis induced by gallic acid. We found that treatment with gallic acid resulted in activation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB, Akt), but not p38MAPK, in mouse lung fibroblasts. Inhibition of JNK using pharmacologic inhibitor (SP600125) and genetic knockdown (JNK specific siRNA) significantly inhibited p53 accumulation, reduced PUMA and Fas expression, and abolished apoptosis induced by gallic acid. Moreover, treatment with antioxidants (vitamin C, N-acetyl cysteine, and catalase) effectively diminished gallic acid-induced hydrogen peroxide production, JNK and p53 activation, and cell death. These observations imply that gallic acid-mediated hydrogen peroxide formation acts as an initiator of JNK signaling pathways, leading to p53 activation and apoptosis in mouse lung fibroblasts. PMID:23533505

  4. Blockade of store-operated calcium entry alleviates high glucose-induced neurotoxicity via inhibiting apoptosis in rat neurons.

    Science.gov (United States)

    Xu, Zhenkuan; Xu, Wenzhe; Song, Yan; Zhang, Bin; Li, Feng; Liu, Yuguang

    2016-07-25

    Altered store-operated calcium entry (SOCE) has been suggested to be involved in many diabetic complications. However, the association of altered SOCE and diabetic neuronal damage remains unclear. This study aimed to investigate the effects of altered SOCE on primary cultured rat neuron injury induced by high glucose. Our data demonstrated that high glucose increased rat neuron injury and upregulated the expression of store-operated calcium channel (SOC). Inhibition of SOCE by a pharmacological inhibitor and siRNA knockdown of stromal interaction molecule 1 weakened the intracellular calcium overload, restored mitochondrial membrane potential, downregulated cytochrome C release and inhibited cell apoptosis. As well, treatment with the calcium chelator BAPTA-AM prevented cell apoptosis by ameliorating the high glucose-increased intracellular calcium level. These findings suggest that SOCE blockade may alleviate high glucose-induced neuronal damage by inhibiting apoptosis. SOCE might be a promising therapeutic target in diabetic neurotoxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

    Science.gov (United States)

    Safaralizadeh, Reza; Soheili, Zahra-Soheila; Deezagi, Abdolkhaleg; Pourpak, Zahra; Samiei, Shahram; Moin, Mostafa

    2009-12-01

    FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast

  6. Apoptosis-inducing factor (Aif1) mediates anacardic acid-induced apoptosis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Muzaffar, Suhail; Chattoo, Bharat B

    2017-03-01

    Anacardic acid is a medicinal phytochemical that inhibits proliferation of fungal as well as several types of cancer cells. It induces apoptotic cell death in various cell types, but very little is known about the mechanism involved in the process. Here, we used budding yeast Saccharomyces cerevisiae as a model to study the involvement of some key elements of apoptosis in the anacardic acid-induced cell death. Plasma membrane constriction, chromatin condensation, DNA degradation, and externalization of phosphatidylserine (PS) indicated that anacardic acid induces apoptotic cell death in S. cerevisiae. However, the exogenous addition of broad-spectrum caspase inhibitor Z-VAD-FMK or deletion of the yeast caspase Yca1 showed that the anacardic acid-induced cell death is caspase independent. Apoptosis-inducing factor (AIF1) deletion mutant was resistant to the anacardic acid-induced cell death, suggesting a key role of Aif1. Overexpression of Aif1 made cells highly susceptible to anacardic acid, further confirming that Aif1 mediates anacardic acid-induced apoptosis. Interestingly, instead of the increase in the intracellular reactive oxygen species (ROS) normally observed during apoptosis, anacardic acid caused a decrease in the intracellular ROS levels. Quantitative real-time PCR analysis showed downregulation of the BIR1 survivin mRNA expression during the anacardic acid-induced apoptosis.

  7. Chemotherapy-Induced Apoptosis in a Transgenic Model of Neuroblastoma Proceeds Through p53 Induction

    Directory of Open Access Journals (Sweden)

    Louis Chesler

    2008-11-01

    Full Text Available Chemoresistance in neuroblastoma is a significant issue complicating treatment of this common pediatric solid tumor. MYCN-amplified neuroblastomas are infrequently mutated at p53 and are chemosensitive at diagnosis but acquire p53 mutations and chemoresistance with relapse. Paradoxically, Myc-driven transformation is thought to require apoptotic blockade. We used the TH-MYCN transgenic murine model to examine the role of p53-driven apoptosis on neuroblastoma tumorigenesis and the response to chemotherapy. Tumors formed with high penetrance and low latency in p53-haploinsufficient TH-MYCN mice. Cyclophosphamide (CPM induced a complete remission in p53 wild type TH-MYCN tumors, mirroring the sensitivity of childhood neuroblastoma to this agent. Treated tumors showed a prominent proliferation block, induction of p53 protein, and massive apoptosis proceeding through induction of the Bcl-2 homology domain-3-only proteins PUMA and Bim, leading to the activation of Bax and cleavage of caspase-3 and -9. Apoptosis induced by CPM was reduced in p53-haploinsufficient tumors. Treatment of MYCN-expressing human neuroblastoma cell lines with CPM induced apoptosis that was suppressible by siRNA to p53. Taken together, the results indicate that the p53 pathway plays a significant role in opposing MYCN-driven oncogenesis in a mouse model of neuroblastoma and that basal inactivation of the pathway is achieved in progressing tumors. This, in part, explains the striking sensitivity of such tumors to chemotoxic agents that induce p53-dependent apoptosis and is consistent with clinical observations that therapy-associated mutations in p53 are a likely contributor to the biology of tumors at relapse and secondarily mediate resistance to therapy.

  8. TNF/TNFR{sub 1} pathway and endoplasmic reticulum stress are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Fu-Tao; Ding, Yi; Shah, Zahir; Xing, Dan; Gao, Yuan; Liu, Dong Ming; Ding, Ming-Xing, E-mail: dmx@mail.hzau.edu.cn

    2014-04-15

    Background and purpose: Quinolones cause obvious cartilaginous lesions in juvenile animals by chondrocyte apoptosis, which results in the restriction of their use in pediatric and adolescent patients. Studies showed that chondrocytes can be induced to produce TNFα, and the cisternae of the endoplasmic reticulum in quinolone-treated chondrocytes become dilated. We investigated whether TNF/TNFR{sub 1} pathway and endoplasmic reticulum stress (ERs) are involved in ofloxacin (a typical quinolone)-induced apoptosis of juvenile canine chondrocytes. Experimental approach: Canine juvenile chondrocytes were treated with ofloxacin. Cell survival and apoptosis rates were determined with MTT method and flow cytometry, respectively. The gene expression levels of the related signaling molecules (TNFα, TNFR{sub 1}, TRADD, FADD and caspase-8) in death receptor pathways and main apoptosis-related molecules (calpain, caspase-12, GADD153 and GRP78) in ERs were measured by qRT-PCR. The gene expression of TNFR{sub 1} was suppressed with its siRNA. The protein levels of TNFα, TNFR{sub 1} and caspase-12 were assayed using Western blotting. Key results: The survival rates decreased while apoptosis rates increased after the chondrocytes were treated with ofloxacin. The mRNA levels of the measured apoptosis-related molecules in death receptor pathways and ERs, and the protein levels of TNFα, TNFR{sub 1} and caspase-12 increased after the chondrocytes were exposed to ofloxacin. The downregulated mRNA expressions of TNFR{sub 1}, Caspase-8 and TRADD, and the decreased apoptosis rates of the ofloxacin-treated chondrocytes occurred after TNFR{sub 1}–siRNA interference. Conclusions and implications: Ofloxacin-induced chondrocyte apoptosis in a time- and concentration-dependent fashion. TNF/TNFR{sub 1} pathway and ERs are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes in the early stage. - Highlights: • Chondrocyte apoptosis is induced by ofloxacin in a time- and

  9. Tissue factor/FVIIa activates Bcl-2 and prevents doxorubicin-induced apoptosis in neuroblastoma cells

    International Nuclear Information System (INIS)

    Fang, Jun; Gu, Lubing; Zhu, Ningxi; Tang, Hao; Alvarado, Carlos S; Zhou, Muxiang

    2008-01-01

    Tissue factor (TF) is a transmembrane protein that acts as a receptor for activated coagulation factor VII (FVIIa), initiating the coagulation cascade. Recent studies demonstrate that expression of tumor-derived TF also mediates intracellular signaling relevant to tumor growth and apoptosis. Our present study investigates the possible mechanism by which the interaction between TF and FVIIa regulates chemotherapy resistance in neuroblastoma cell lines. Gene and siRNA transfection was used to enforce TF expression in a TF-negative neuroblastoma cell line and to silence endogenous TF expression in a TF-overexpressing neuroblastoma line, respectively. The expression of TF, Bcl-2, STAT5, and Akt as well as the phosphorylation of STAT5 and Akt in gene transfected cells or cells treated with JAK inhibitor and LY294002 were determined by Western blot assay. Tumor cell growth was determined by a clonogenic assay. Cytotoxic and apoptotic effect of doxorubicin on neuroblastoma cell lines was analyzed by WST assay and annexin-V staining (by flow cytometry) respectively. Enforced expression of TF in a TF-negative neuroblastoma cell line in the presence of FVIIa induced upregulation of Bcl-2, leading to resistance to doxorubicin. Conversely, inhibition of endogenous TF expression in a TF-overexpressing neuroblastoma cell line using siRNA resulted in down-regulation of Bcl-2 and sensitization to doxorubicin-induced apoptosis. Additionally, neuroblastoma cells expressing high levels of either endogenous or transfected TF treated with FVIIa readily phosphorylated STAT5 and Akt. Using selective pharmacologic inhibitors, we demonstrated that JAK inhibitor I, but not the PI3K inhibitor LY294002, blocked the TF/FVIIa-induced upregulation of Bcl-2. This study shows that in neuroblastoma cell lines overexpressed TF ligated with FVIIa produced upregulation of Bcl-2 expression through the JAK/STAT5 signaling pathway, resulting in resistance to apoptosis. We surmise that this TF

  10. Oxidative stress by monosodium urate crystals promotes renal cell apoptosis through mitochondrial caspase-dependent pathway in human embryonic kidney 293 cells: mechanism for urate-induced nephropathy.

    Science.gov (United States)

    Choe, Jung-Yoon; Park, Ki-Yeun; Kim, Seong-Kyu

    2015-01-01

    The aim of this study is to clarify the effect of oxidative stress on monosodium urate (MSU)-mediated apoptosis of renal cells. Quantitative real-time polymerase chain reaction and immunoblotting for Bcl-2, caspase-9, caspase-3, iNOS, cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), IL-18, TNF receptor-associated factor-6 (TRAF-6), and mitogen-activated protein kinases were performed on human embryonic kidney 293 (HEK293) cells, which were stimulated by MSU crystals. Fluorescence-activated cell sorting was performed using annexin V for assessment of apoptosis. Reactive oxygen species (ROS) were measured. IL-1β siRNA was used for blocking IL-1β expression. MSU crystals promoted ROS, iNOS, and COX-2 expression and also increased TRAF-6 and IL-1β expression in HEK293 cells, which was inhibited by an antioxidant ascorbic acid. Caspase-dependent renal cell apoptosis was induced through attenuation of Bcl-2 and enhanced caspase-3 and caspase-9 expression by MSU crystals, which was significantly reversed by ascorbic acid and transfection of IL-1β siRNA to HEK293 cells. Ascorbic acid inhibited phosphorylation of extracellular signal-regulated kinase and Jun N-terminal protein kinase stimulated by MSU crystals. ROS accumulation and iNOS and COX-2 mRNA expression by MSU crystals was also suppressed by transfection with IL-1β siRNA. Oxidative stress generated by MSU crystals promotes renal apoptosis through the mitochondrial caspase-dependent apoptosis pathway.

  11. Interference of silibinin with IGF-1R signalling pathways protects human epidermoid carcinoma A431 cells from UVB-induced apoptosis

    International Nuclear Information System (INIS)

    Liu, Weiwei; Otkur, Wuxiyar; Li, Lingzhi; Wang, Qiong; He, Hao; Zang, Linghe; Hayashi, Toshihiko; Tashiro, Shin-ichi; Onodera, Satoshi; Xia, Mingyu; Ikejima, Takashi

    2013-01-01

    Highlights: ► Silibinin protects A431 cells from UVB irradiation-induced apoptosis. ► Up-regulation of the IGF-1R-JNK/ERK pathways by UVB induces cell apoptosis. ► Silibinin inhibits IGF-1R pathways to repress caspase-8-mediated apoptosis. -- Abstract: Ultraviolet B (UVB) from sunlight is a major cause of cutaneous lesion. Silibinin, a traditional hepatic protectant, elicits protective effects against UVB-induced cellular damage. In A431 cells, the insulin-like growth factor-1 receptor (IGF-1R) was markedly up-regulated by UVB irradiation. The activation of the IGF-1R signalling pathways contributed to apoptosis of the cells rather than rescuing the cells from death. Up-regulated IGF-1R stimulated downstream mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinases (JNK) and extracellular signal-regulated protein kinases 1/2 (ERK1/2). The subsequent activation of caspase-8 and caspase-3 led to apoptosis. The activation of IGF-1R signalling pathways is the cause of A431 cell death. The pharmacological inhibitors and the small interfering RNA (siRNA) targeting IGF-1R suppressed the downstream activation of JNK/ERK-caspases to help the survival of the UVB-irradiated A431 cells. Indeed, silibinin treatment suppressed the IGF-1R-JNK/ERK pathways and thus protected the cells from UVB-induced apoptosis

  12. RAC3 nuclear receptor co-activator has a protective role in the apoptosis induced by different stimuli

    International Nuclear Information System (INIS)

    Colo, Georgina P.; Rubio, Maria F.; Alvarado, Cecilia V.; Costas, Monica A.

    2007-01-01

    RAC3 belongs to the family of p160 nuclear receptors co activators and it is over-expressed in several tumors. We have previously shown that RAC3 is a NF-κB co activator. In this paper, we investigated the role of RAC3 in cell-sensitivity to apoptosis, using H 2 O 2 in the human embryonic kidney cell line (HEK293), and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) in a human chronic myeloid leukemia cell line (K562) naturally resistant to TRAIL. We observed that the tumoral K562 cells have high levels of RAC3 if compared with the non-tumoral HEK293 cells. The normal or transfected co activator over-expression inhibits apoptosis through a diminished caspase activity and AIF nuclear translocation, increased NF--κB, AKT and p38, and decreased ERK activities. In contrast, inhibition of RAC3 by siRNA induced sensitivity of K562 to TRAIL-induced apoptosis. Such results suggest that over-expression of RAC3 contributes to tumor development through molecular mechanisms that do not depend strictly on acetylation and/or steroid hormones, which control cell death. This could be a possible target for future tumor therapies. (author) [es

  13. TFF1 inhibits proliferation and induces apoptosis of gastric cancer cells in vitro

    Directory of Open Access Journals (Sweden)

    Yanli Ge

    2012-05-01

    Full Text Available Trefoil Factor Family (TFF plays an essential role in the intestinal epithelial restitution, but the relationship between TFF1 and gastric cancer (GC is still unclear. The present study aimed to determine the role of TFF1 in repairing gastric mucosa and in the pathogenesis of GC.The TFF1 expression in different gastric mucosas was measured with immunohistochemistry. Then, siRNA targeting TFF1 or plasmids expressing TFF1 gene were transfected into BGC823 cells, SGC7901 cells and GES-1 cells. The cell proliferation was detected with MTT assay and apoptosis and cell cycle measured by flow cytometry.From normal gastric mucosa to mucosa with dysplasia and to gastric cancer, the TFF1 expression had a decreasing trend. Down-regulation of TFF1 expression significantly reduced the apoptosis of three cell lines and markedly facilitated their proliferation but had no significant effect on cell cycle. Over-expression of TFF1 could promote apoptosis of three cell lines and inhibit proliferation but had no pronounced effect on cell cycle. TFF1 can inhibit proliferation and induce apoptosis of GC cells in vitro.

  14. Suppression of c-Myc induces apoptosis via an AMPK/mTOR-dependent pathway by 4-O-methyl-ascochlorin in leukemia cells.

    Science.gov (United States)

    Shin, Jae-Moon; Jeong, Yun-Jeong; Cho, Hyun-Ji; Magae, Junji; Bae, Young-Seuk; Chang, Young-Chae

    2016-05-01

    4-O-Methyl-ascochlorin (MAC) is a methylated derivative of the prenyl-phenol antibiotic ascochlorin, which was isolated from an incomplete fungus, Ascochyta viciae. Although the effects of MAC on apoptosis have been reported, the underlying mechanisms remain unknown. Here, we show that MAC promoted apoptotic cell death and downregulated c-Myc expression in K562 human leukemia cells. The effect of MAC on apoptosis was similar to that of 10058-F4 (a c-Myc inhibitor) or c-Myc siRNA, suggesting that the downregulation of c-Myc expression plays a role in the apoptotic effect of MAC. Further investigation showed that MAC downregulated c-Myc by inhibiting protein synthesis. MAC promoted the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and its target proteins, including p70S6 K and 4E-BP-1. Treatment of cells with AICAR (an AMPK activator), rapamycin (an mTOR inhibitor), or mTOR siRNA downregulated c-Myc expression and induced apoptosis to a similar extent to that of MAC. These results suggest that the effect of MAC on apoptosis induction in human leukemia cells is mediated by the suppression of c-Myc protein synthesis via an AMPK/mTOR-dependent mechanism.

  15. Inhibition of PKR protects against H2O2-induced injury on neonatal cardiac myocytes by attenuating apoptosis and inflammation.

    Science.gov (United States)

    Wang, Yongyi; Men, Min; Xie, Bo; Shan, Jianggui; Wang, Chengxi; Liu, Jidong; Zheng, Hui; Yang, Wengang; Xue, Song; Guo, Changfa

    2016-12-08

    Reactive oxygenation species (ROS) generated from reperfusion results in cardiac injury through apoptosis and inflammation, while PKR has the ability to promote apoptosis and inflammation. The aim of the study was to investigate whether PKR is involved in hydrogen peroxide (H 2 O 2 ) induced neonatal cardiac myocytes (NCM) injury. In our study, NCM, when exposed to H 2 O 2 , resulted in persistent activation of PKR due to NCM endogenous RNA. Inhibition of PKR by 2-aminopurine (2-AP) or siRNA protected against H 2 O 2 induced apoptosis and injury. To elucidate the mechanism, we revealed that inhibition of PKR alleviated H 2 O 2 induced apoptosis companied by decreased caspase3/7 activity, BAX and caspase-3 expression. We also revealed that inhibition of PKR suppressed H 2 O 2 induced NFκB pathway and NLRP3 activation. Finally, we found ADAR1 mRNA and protein expression were both induced after H 2 O 2 treatment through STAT-2 dependent pathway. By gain and loss of ADAR1 expression, we confirmed ADAR1 modulated PKR activity. Therefore, we concluded inhibition of PKR protected against H 2 O 2 -induced injury by attenuating apoptosis and inflammation. A self-preservation mechanism existed in NCM that ADAR1 expression is induced by H 2 O 2 to limit PKR activation simultaneously. These findings identify a novel role for PKR/ADAR1 in myocardial reperfusion injury.

  16. Avian reovirus S1133-induced apoptosis is associated with Bip/GRP79-mediated Bim translocation to the endoplasmic reticulum.

    Science.gov (United States)

    Lin, Ping-Yuan; Liu, Hung-Jen; Chang, Ching-Dong; Chen, Yo-Chia; Chang, Chi-I; Shih, Wen-Ling

    2015-04-01

    In this study the mechanism of avian reovirus (ARV) S1133-induced pathogenesis was investigated, with a focus on the contribution of ER stress to apoptosis. Our results showed that upregulation of the ER stress response protein, as well as caspase-3 activation, occurred in ARV S1133-infected cultured cells and in SPF White Leghorn chicks organs. Upon infection, Bim was translocated specifically to the ER, but not mitochondria, in the middle to late infectious stages. In addition, ARV S1133 induced JNK phosphorylation and promoted JNK-Bim complex formation, which correlated with the Bim translocation and apoptosis induction that was observed at the same time point. Knockdown of BiP/GRP78 by siRNA and inhibition of BiP/GRP78 using EGCG both abolished the formation of the JNK-Bim complex, caspase-3 activation, and subsequent apoptosis induction by ARV S1133 efficiently. These results suggest that BiP/GRP78 played critical roles and works upstream of JNK-Bim in response to the ARV S1133-mediated apoptosis process.

  17. Baicalein Induces Apoptosis and Autophagy via Endoplasmic Reticulum Stress in Hepatocellular Carcinoma Cells

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    Zhongxia Wang

    2014-01-01

    Full Text Available Background. Hepatocellular carcinoma (HCC remains a disastrous disease and the treatment for HCC is rather limited. Separation and identification of active compounds from traditionally used herbs in HCC treatment may shed light on novel therapeutic drugs for HCC. Methods. Cell viability and colony forming assay were conducted to determine anti-HCC activity. Morphology of cells and activity of caspases were analyzed. Antiapoptotic Bcl-2 family proteins and JNK were also examined. Levels of unfolded protein response (UPR markers were determined and intracellular calcium was assayed. Small interfering RNAs (siRNAs were used to investigate the role of UPR and autophagy in baicalein-induced cell death. Results. Among four studied flavonoids, only baicalein exhibited satisfactory inhibition of viability and colony formation of HCC cells within water-soluble concentration. Baicalein induced apoptosis via endoplasmic reticulum (ER stress, possibly by downregulating prosurvival Bcl-2 family, increasing intracellular calcium, and activating JNK. CHOP was the executor of cell death during baicalein-induced ER stress while eIF2α and IRE1α played protective roles. Protective autophagy was also triggered by baicalein in HCC cells. Conclusion. Baicalein exhibits prominent anti-HCC activity. This flavonoid induces apoptosis and protective autophagy via ER stress. Combination of baicalein and autophagy inhibitors may represent a promising therapy against HCC.

  18. Therapeutic Effects of Myeloid Cell Leukemia-1 siRNA on Human Acute Myeloid Leukemia Cells

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    Hadi Karami

    2014-05-01

    Full Text Available Purpose: Up-regulation of Mcl-1, a known anti-apoptotic protein, is associated with the survival and progression of various malignancies including leukemia. The aim of this study was to explore the effect of Mcl-1 small interference RNA (siRNA on the proliferation and apoptosis of HL-60 acute myeloid leukemia (AML cells. Methods: siRNA transfection was performed using Lipofectamine™2000 reagent. Relative mRNA and protein expressions were quantified by quantitative real-time PCR and Western blotting, respectively. Trypan blue assay was performed to assess tumor cell proliferation after siRNA transfection. The cytotoxic effect of Mcl-1 siRNA on leukemic cells was measured using MTT assay. Apoptosis was detected using ELISA cell death assay. Results: Mcl-1 siRNA clearly lowered both Mcl-1 mRNA and protein levels in a time-dependent manner, leading to marked inhibition of cell survival and proliferation. Furthermore, Mcl-1 down-regulation significantly enhanced the extent of HL-60 apoptotic cells. Conclusion: Our results suggest that the down-regulation of Mcl-1 by siRNA can effectively trigger apoptosis and inhibit the proliferation of leukemic cells. Therefore, Mcl-1 siRNA may be a potent adjuvant in AML therapy.

  19. Andrographolide Induces Apoptosis of C6 Glioma Cells via the ERK-p53-Caspase 7-PARP Pathway

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    Shih-Hung Yang

    2014-01-01

    Full Text Available Background. Glioma is the most malignant tumor of the central nervous system. Efforts on the development of new chemotherapy are mandatory. Andrographolide (AND, a diterpenoid lactone isolated from the Andrographis paniculata, has been shown to have antitumor activities in several types of cancer cells. Whether AND can exert its antitumor activity in glioblastoma cells remains unknown. This study examined the anticancer effects of AND, both in vitro and in vivo. Methods. Cell apoptosis was assayed by flow cytometry and nuclear staining. The signaling pathway for AND was determined by western blotting. The effects of AND on tumor growth was evaluated in a mouse model. Results and Conclusion. In vitro, with application of specific inhibitors and siRNA, AND-induced apoptosis was proven through ROS-ERK-P53-caspase 7-PARP signaling pathway. In vivo, AND significantly retarded tumor growth and caused regression of well-formed tumors in vivo. Furthermore, AND did not induce apoptosis or activate ERK and p53 in primary cultured astrocyte cells, and it may serve as a potential therapeutic candidate for the treatment of glioma.

  20. Down-regulation of procaspase-8 expression by focal adhesion kinase protects HL-60 cells from TRAIL-induced apoptosis

    International Nuclear Information System (INIS)

    Tamagiku, Yuji; Sonoda, Yoshiko; Kunisawa, Mari; Ichikawa, Daiju; Murakami, Yayoi; Aizu-Yokota, Eriko; Kasahara, Tadashi

    2004-01-01

    We have demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as hydrogen peroxide, etoposide, and ionizing radiation compared with the vector-transfected (HL-60/Vect) cells. HL-60/FAK cells are highly resistant to TRAIL-induced apoptosis, while original HL-60 or HL-60/Vect cells were sensitive. TRAIL at 500 ng/ml induced significant DNA fragmentation, activation of caspase-8 and 3, the processing of a proapoptotic BID, and mitochondrial release of cytochrome c in HL-60/Vect cells, whereas no such events were observed in the HL-60/FAK cells. In particular, the expression of procaspase-8 gene and subsequent cleavage of caspase-8 were markedly reduced in HL-60/FAK cells, while expression of TRAIL-receptor 2 and 3, TRADD, and FADD was equivalent in both types of cells. In HL-60/FAK cells, the phosphoinositide 3 (PI3)-kinase/Akt survival pathway was constitutively activated, accompanied by significant induction of inhibitor-of-apoptosis proteins, XIAP, RIP, and Bcl-XL. The introduction of FAK siRNA in HL-60/FAK cells sensitized them against TRAIL-induced apoptosis, confirming that overexpressed FAK downregulates procaspase-8 expression, which subsequently inhibits downstream apoptosis pathway in the HL-60/FAK cells

  1. A novel benzofuran derivative, ACDB, induces apoptosis of human chondrosarcoma cells through mitochondrial dysfunction and endoplasmic reticulum stress.

    Science.gov (United States)

    Su, Chen-Ming; Chen, Chien-Yu; Lu, Tingting; Sun, Yi; Li, Weimin; Huang, Yuan-Li; Tsai, Chun-Hao; Chang, Chih-Shiang; Tang, Chih-Hsin

    2016-12-13

    Chondrosarcoma is one of the bone tumor with high mortality in respond to poor radiation and chemotherapy treatment. Here, we analyze the antitumor activity of a novel benzofuran derivative, 2-amino-3-(2-chlorophenyl)-6-(4-dimethylaminophenyl)benzofuran-4-yl acetate (ACDB), in human chondrosarcoma cells. ACDB increased the cell apoptosis of human chondrosarcomas without harm in chondrocytes. ACDB also enhanced endoplasmic reticulum (ER) stress, which was characterized by varieties in the cytosolic calcium levels and induced the expression of glucose-regulated protein (GRP) and calpain. Furthermore, the ACDB-induced chondrosarcoma apoptosis was associated with the upregulation of the B cell lymphoma-2 (Bcl-2) family members including pro- and anti-apoptotic proteins, downregulation of dysfunctional mitochondria that released cytochrome C, and subsequent activation of caspases-3. In addition, the ACDB-mediated cellular apoptosis was suppressed by transfecting cells with glucose-regulated protein (GRP) and calpain siRNA or treating cells with ER stress chelators and caspase inhibitors. Interestingly, animal experiments illustrated a reduction in the tumor volume following ACDB treatment. Together, these results suggest that ACDB may be a novel tumor suppressor of chondrosarcoma, and this study demonstrates that the novel antitumor agent, ACDB, induced apoptosis by mitochondrial dysfunction and ER stress in human chondrosarcoma cells in vitro and in vivo.

  2. Regulation of apoptosis-inducing factor-mediated, cisplatin-induced apoptosis by Akt

    OpenAIRE

    Yang, X; Fraser, M; Abedini, M R; Bai, T; Tsang, B K

    2008-01-01

    Cisplatin is a first-line chemotherapeutic for ovarian cancer, although chemoresistance limits treatment success. Apoptosis, an important determinant of cisplatin sensitivity, occurs via caspase-dependent and -independent mechanisms. Activation of the protein kinase Akt, commonly observed in ovarian tumours, confers resistance to ovarian cancer cells via inhibition of caspase-dependent apoptosis. However, the effect of Akt on cisplatin-induced, caspase-independent apoptosis remains unclear. W...

  3. Hyperthermia-induced apoptosis

    NARCIS (Netherlands)

    Nijhuis, E.H.A.

    2008-01-01

    This thesis describes a number of studies that investigated several aspects of heat-induced apoptosis in human lymphoid malignancies. Cells harbour both pro- and anti-apoptotic proteins and the balance between these proteins determines whether a cell is susceptible to undergo apoptosis. In this

  4. Bim and Bmf synergize to induce apoptosis in Neisseria gonorrhoeae infection.

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    Oliver Kepp

    2009-03-01

    Full Text Available Bcl-2 family proteins including the pro-apoptotic BH3-only proteins are central regulators of apoptotic cell death. Here we show by a focused siRNA miniscreen that the synergistic action of the BH3-only proteins Bim and Bmf is required for apoptosis induced by infection with Neisseria gonorrhoeae (Ngo. While Bim and Bmf were associated with the cytoskeleton of healthy cells, they both were released upon Ngo infection. Loss of Bim and Bmf from the cytoskeleton fraction required the activation of Jun-N-terminal kinase-1 (JNK-1, which in turn depended on Rac-1. Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases. Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-X(L, respectively. Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells.

  5. Degradation of Mcl-1 through GSK-3β Activation Regulates Apoptosis Induced by Bufalin in Non-Small Cell Lung Cancer H1975 Cells

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    Xiao-hong Kang

    2017-04-01

    Full Text Available Background/Aims: Mcl-1, an anti-apoptotic Bcl-2 family member, is often overexpressed in non-small cell lung cancer (NSCLC. Bufalin has been reported to induce apoptosis in various tumor cells. However, there is no report showing that bufalin could downregulate Mcl-1 expression in NSCLC. Methods: Cell proliferation was analyzed by cell counting kit-8 (CCK-8 assay in H1975 cells. Cell apoptosis was detected by flow cytometry. Mcl-1 mRNA was detected by RT-PCR. The expression of apoptosis-associated proteins in H1975 cells was detected by western blotting. The levels of Mcl-1 ubiquitination and NOXA were analyzed by Immunoprecipitation assay. Results: Cell growth was inhibited by bufalin in a time and dose-dependent manner. Bufalin induced apoptosis in NSCLC cells by activating caspase cascades and downregulating Mcl-1 expression. However, overexpression of Mcl-1 diminished bufalin-induced apoptosis. Furthermore, bufalin did not reduce Mcl-1 mRNA expression in H1975 cells, but strongly promoted Mcl-1 protein degradation. Proteasome inhibitor MG132 markedly prevented the degradation of Mcl-1 and blocked bufalin-induced Mcl-1 reduction. Bufalin did not significantly affect NOXA protein levels, but downregulated the expression of p-GSK-3β. GSK-3 inhibitor and GSK-3β siRNA resulted in increased levels of Mcl-1 and reversed the bufalin-induced Mcl-1 degradation. Conclusion: Bufalin induced cell apoptosis in H1975 cells may be through downregulation of Mcl-1. Proteasomal degradation of Mcl-1 via GSK-3β activation was involved in bufalin-induced apoptosis.

  6. Indomethacin promotes apoptosis in gastric cancer cells through concomitant degradation of Survivin and Aurora B kinase proteins.

    Science.gov (United States)

    Chiou, Shiun-Kwei; Hoa, Neil; Hodges, Amy; Ge, Lishen; Jadus, Martin R

    2014-09-01

    Regular usage of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with reduced incidence of a variety of cancers. The molecular mechanisms underlying these chemopreventive effects remain poorly understood. This current investigation showed that in gastric cancer cells: (1) Indomethacin treatment enhanced the degradation of chromosomal passenger proteins, Survivin and Aurora B kinase; (2) Indomethacin treatment down-regulated Aurora B kinase activity in a cell cycle-independent fashion; (3) siRNA knockdown of Survivin level promoted Aurora B kinase protein degradation, and vice versa; (4) ectopic overexpression of Survivin blocked reduction of Aurora B kinase level and activity by indomethacin treatment, and vice versa; (5) siRNA knockdown of Aurora B kinase level and AZD1152 inhibition of its activity induced apoptosis, and overexpression of Aurora B kinase inhibited indomethacin-induced apoptosis; (6) indomethacin treatment reduced Aurora B kinase level, coinciding with reduction of Survivin level and induction of apoptosis, in KATO III and HT-29 cells, and in mouse gastric mucosa. A role for Aurora B kinase function in NSAID-induced apoptosis was not previously explored. Thus this report provides better understanding of the molecular mechanisms underlying the anti-cancer effect of NSAIDs by elucidating a significant role for Aurora B kinase in indomethacin-induced apoptosis.

  7. Cyproterone acetate enhances TRAIL-induced androgen-independent prostate cancer cell apoptosis via up-regulation of death receptor 5.

    Science.gov (United States)

    Chen, Linjie; Wolff, Dennis W; Xie, Yan; Lin, Ming-Fong; Tu, Yaping

    2017-03-07

    cyproterone acetate-induced CHOP and DR5 up-regulation. More importantly, siRNA silencing of CHOP significantly reduced cyproterone acetate-induced DR5 up-regulation and TRAIL sensitivity in prostate cancer cells. Our study shows a novel effect of cyproterone acetate on apoptosis pathways in prostate cancer cells and raises the possibility that a combination of TRAIL with cyproterone acetate could be a promising strategy for treating castration-resistant prostate cancer.

  8. p53-Induced Apoptosis Occurs in the Absence of p14ARF in Malignant Pleural Mesothelioma

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    Sally Hopkins-Donaldson

    2006-07-01

    Full Text Available Malignant pleural mesotheliomas (MPMs are usually wild type for the p53 gene but contain homozygous deletions in the INK4A locus that encodes p14ARF, an inhibitor of p53-MDM2 interaction. Previous findings suggest that lack of p14ARF expression and the presence of SV40 large T antigen (L-Tag result in p53 inactivation in MPM. We did not detect SV40 L-Tag mRNA in either MPM cell lines or primary cultures, treatment of p14ARF-deficient cells with cisplatin (CDDP increased both total and phosphorylated p53 and enhanced p53 DNA-binding activity. On incubation with CDDP, levels of positively regulated p53 transcriptional targets p21WAF, PIG3, MDM2, Bax, PUMA increased in p14ARF-deficient cells, whereas negatively regulated survivin decreased. Significantly, p53-induced apoptosis was activated by CDDP in p14ARF-deficient cells, treatment with p53-specific siRNA rendered them more CDDP-resistant. p53 was also activated by: 1 inhibition of MDM2 (using nutlin-3; 2 transient overexpression of p14ARF; and 3 targeting of survivin using antisense oligonucleotides. However, it is noteworthy that only survivin downregulation sensitized cells to CDDP-induced apoptosis. These results suggest that p53 is functional in the absence of p14ARF in MPM and that targeting of the downstream apoptosis inhibitor survivin can sensitize to CDDP-induced apoptosis.

  9. Efficient Inhibition of wear debris-induced inflammation by locally delivered siRNA

    International Nuclear Information System (INIS)

    Peng Xiaochun; Tao Kun; Cheng Tao; Zhu Junfeng; Zhang Xianlong

    2008-01-01

    Aseptic loosening is the most common long-term complication of total joint replacement, which is associated with the generation of wear debris. The purpose of this study was to investigate the inhibitory effect of small interfering RNA (siRNA) targeting tumor necrosis factor-α (TNF-α) on wear debris-induced inflammation. A local delivery of lentivirus-mediated TNF-α siRNA into the modified murine air pouch, which was stimulated by polymethylmethacrylate (PMMA) particles, resulted in significant blockage of TNF-α both in mRNA and protein levels for up to 4 weeks. In addition, significant down-regulation of interleukin-1 (IL-1) and interleukin-6 (IL-6) was observed in TNF-α siRNA-treated pouches. The safety profile of gene therapy was proven by Bioluminescent assay and quantitative fluorescent flux. Histological analysis revealed less inflammatory responses (thinner pouch membrane and decreased cellular infiltration) in TNF-α siRNA-treated pouches. These findings suggest that local delivery of TNF-α siRNA might be an excellent therapeutic candidate to inhibit particle-induced inflammation.

  10. Inhibitory heterotrimeric GTP-binding proteins inhibit hydrogen peroxide-induced apoptosis by up-regulation of Bcl-2 via NF-κB in H1299 human lung cancer cells

    International Nuclear Information System (INIS)

    Seo, Mi Ran; Nam, Hyo-Jung; Kim, So-Young; Juhnn, Yong-Sung

    2009-01-01

    Inhibitory heterotrimeric GTP-binding proteins (Gi proteins) mediate a variety of signaling pathways by coupling receptors and effectors to regulate cellular proliferation, differentiation, and apoptosis. However, the role of Gi proteins in the modulation of hydrogen peroxide-induced apoptosis is not clearly understood. Thus, we investigated the effect of Gi proteins on hydrogen peroxide-induced apoptosis and the underlying mechanisms in H1299 human lung cancer cells. The stable expression of constitutively active alpha subunits of Gi1 (Gαi1QL), Gi2, or Gi3 inhibited hydrogen peroxide-induced apoptosis. The expression of Gαi1QL up-regulated Bcl-2 expression, and the knockdown of Bcl-2 with siRNA abolished the anti-apoptotic effect of Gαi1QL. Gαi1 induced the transcription of Bcl-2 by activation of NF-κB, which resulted from an increase in NF-κB p50 protein. We conclude that Gαi1 inhibits hydrogen peroxide-induced apoptosis of H1299 lung cancer cells by up-regulating the transcription of Bcl-2 through a p50-mediated NF-κB activation.

  11. Knockdown of hypoxia-inducible factor-1 alpha reduces proliferation, induces apoptosis and attenuates the aggressive phenotype of retinoblastoma WERI-Rb-1 cells under hypoxic conditions.

    Science.gov (United States)

    Xia, Tian; Cheng, Hao; Zhu, Yu

    2014-01-01

    Hypoxia-inducible factor-1 alpha (HIF-1α) plays a critical role in tumor cell adaption to hypoxia by inducing the transcription of numerous genes. The role of HIF-1α in malignant retinoblastoma remains unclear. We analyzed the role of HIF-1α in WERI-Rb-1 retinoblastoma cells under hypoxic conditions. CoCl2 (125 mmol/L) was added to the culture media to mimic hypoxia. HIF-1α was silenced using siRNA. Gene and protein expression were measured by semi-quantitative RT-PCR and Western blotting. Cell cycle and apoptosis were analyzed by flow cytometry. Cell proliferation, adhesion and invasion were assayed using MTT, Transwell invasion, and cell adhesion assays respectively. Hypoxia significantly upregulated HIF-1α protein expression and the HIF-1α target genes VEGF, GLUT1, and Survivin mRNA. HIF-1α mRNA expression was not affected by hypoxia. Transfection of the siRNA expression plasmid pRNAT-CMV3.2/Neo-HIF-1α silenced HIF-1α by approximately 80% in hypoxic WERI-Rb-1 cells. The knockdown of HIF-1α under hypoxic conditions downregulated VEGF, GLUT1, and Survivin mRNA. It also inhibited proliferation, promoted apoptosis, induced the G0/G1 phase cell cycle arrest, and reduced the adhesion and invasion of WERI-Rb-1 cells. HIF-1α plays a major role in the survival and aggressive phenotype of retinoblastoma cells under hypoxic conditions. Targeting HIF-1α may be a promising therapeutic strategy for human malignant retinoblastoma.

  12. TRAIL-induced cleavage and inactivation of SPAK sensitizes cells to apoptosis

    International Nuclear Information System (INIS)

    Polek, Tara C.; Talpaz, Moshe; Spivak-Kroizman, Taly R.

    2006-01-01

    Ste20-related proline-alanine-rich kinase (SPAK) has been linked to various cellular processes, including proliferation, differentiation, and ion transport regulation. Recently, we showed that SPAK mediates signaling by the TNF receptor, RELT. The presence of a caspase cleavage site in SPAK prompted us to study its involvement in apoptotic signaling induced by another TNF member, TRAIL. We show that TRAIL stimulated caspase 3-like proteases that cleaved SPAK at two distinct sites. Cleavage had little effect on the activity of SPAK but removed its substrate-binding domain. In addition, TRAIL reduced the activity of SPAK in HeLa cells in a caspase-independent manner. Thus, TRAIL inhibited SPAK by two mechanisms: activation of caspases, which removed its substrate-binding domain, and caspase-independent down-regulation of SPAK activity. Furthermore, reducing the amount of SPAK by siRNA increased the sensitivity of HeLa cells to TRAIL-induced apoptosis. Thus, TRAIL down-regulation of SPAK is an important event that enhances its apoptotic effects

  13. Anti-RhoC siRNAs inhibit the proliferation and invasiveness of breast cancer cells via modulating the KAI1, MMP9, and CXCR4 expression

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    Xu X

    2017-03-01

    Full Text Available Xu-Dong Xu,1 Han-Bin Shen,1 Li Zhu,2 Jian-Qin Lu,2 Lin Zhang,3 Zhi-Yong Luo,3 Ya-Qun Wu3 1Department of Thyroid and Breast Surgery, The Fifth Hospital of Wuhan, Hanyang District, 2Department of Oncology, 3Department of Thyroid and Breast Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China Abstract: Overexpression of RhoC in breast cancer cells indicates poor prognosis. In the present study, we aim to investigate the possible antitumor effects of anti-RhoC small-interfering RNA (siRNA in inflammatory breast cancer cells. In this study, a specific anti-RhoC siRNA was used to inhibit RhoC synthesis. Transfection of anti-RhoC siRNA into two IBC cells SUM149 and SUM190 induced extensive degradation of target mRNA and led to significant decrease in the synthesis of protein. Anti-RhoC siRNA inhibited cell proliferation and invasion, increased cell apoptosis, and induced cell cycle arrest in vitro. Moreover, the transfection of siRNA increased the expression of KAI1 and decreased the expression of MMP9 and CXCR4 in both mRNA and protein levels. Furthermore, transplantation tumor experiments in BALB/c-nu mice showed that intratumoral injection of anti-RhoC siRNA inhibited tumor growth and increased survival rate. Our results suggested that RhoC gene silencing with specific anti-RhoC siRNA would be a potential therapeutic method for metastatic breast cancer. Keywords: gene silencing, proliferation, apoptosis, cell cycle arrest

  14. Lentivirus-Mediated Knockdown of Astrocyte Elevated Gene-1 Inhibits Growth and Induces Apoptosis through MAPK Pathways in Human Retinoblastoma Cells.

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    Ying Chang

    Full Text Available To explore expression and function of astrocyte elevated gene-1 (AEG-1 in human retinoblastoma (RB.The expression of AEG-1 in histological sections of human RBs and in RB cell lines was examined using immunohistochemical staining and RT-PCR and Western blotting respectively. We knocked down AEG-1 gene levels by AEG-1-siRNA lentivirus transfection of human RB cell lines SO-RB50 and Y79, and using an MTT assay, we assessed the role of AEG-1 on RB cell proliferation. The biological significance of lentivirus transfection induced AEG-1 down-regulation was examined by assessing the apoptosis rate in the transfected RB cells by Annexin V-APC staining and flow cytometry. We additionally measured the expression of Bcl-2, Bax, cleaved-caspase-3 and caspase-3, and the phosphorylation and non-phosphorylation alternation of MAPKs.AEG-1 expression was detected to be strongly positive in the histological slides of 35 out of 54 (65% patients with RB. AEG-1 expression increased significantly (P<0.05 with tumor stage. In the RB cell lines SO-RB50, Y79 and WERI-RB1 as compared with retinal pigment epithelium cells, expression of AEG-1 mRNA and AEG-1 protein was significantly higher. In AEG-1-siRNA lentivirus transfected cell cultures as compared with negative control lentivirus transfected cell cultures, levels of AEG-1 mRNA and of AEG-1 protein (P<0.05 and cell growth rates (P<0.01 were significantly lower, and apoptosis rate (P<0.001, Bax/Bcl-2 ratio and cleaved-caspase-3 protein level were significantly increased. The P-ERK/ERK ratio was significantly decreased in the AEG-1-siRNA lentivirus transfected cell lines.Expression of AEG-1 was associated with RB, in histological slides of patients and in cell culture experiments. Lentivirus transfection induced knockdown of AEG-1 had a tumor suppressive effect, potentially by tumor cell apoptosis induction through inhibition of ERK.

  15. Ubiquitin-dependent system controls radiation induced apoptosis

    International Nuclear Information System (INIS)

    Delic, J.; Magdelenat, H.; Glaisner, S.; Magdelenat, H.; Maciorowski, Z.

    1997-01-01

    The selective proteolytic pathway, dependent upon 'N-end rule' protein recognition/ubiquitination and on the subsequent proteasome dependent processing of ubiquitin conjugates, operates in apoptosis induced by γ-irradiation. The proteasome inhibitor peptide aldehyde, MG132, efficiently induced apoptosis and was also able (at doses lower than those required for apoptosis induction) to potentiate apoptosis induced by DNA damage. Its specificity is suggested by the induction of the ubiquitin (UbB and UbC) and E1 (ubiquitin activating enzyme) genes and by an altered ubiquitination pattern. More selectively, a di-peptide competitor of the 'N-end rule' of ubiquitin dependent protein processing inhibited radiation induced apoptosis. This inhibition is also followed by an altered ubiquitination pattern and by activation of Poly (ADP-ribose) polymerase (PARP). These data strongly suggest that early apoptosis radiation induced events are controlled by ubiquitin-dependent proteolytic processing. (author)

  16. Thimerosal-induced apoptosis in mouse C2C12 myoblast cells occurs through suppression of the PI3K/Akt/survivin pathway.

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    Wen-Xue Li

    Full Text Available BACKGROUND: Thimerosal, a mercury-containing preservative, is one of the most widely used preservatives and found in a variety of biological products. Concerns over its possible toxicity have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be markedly cytotoxic to neural tissue. However, little is known regarding thimerosal-induced toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells. METHODOLOGY/PRINCIPAL FINDINGS: The study showed that C2C12 myoblast cells underwent inhibition of proliferation and apoptosis after exposure to thimerosal (125-500 nM for 24, 48 and 72 h. Thimerosal caused S phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining and immunoblotting. The data revealed that thimerosal could trigger the leakage of cytochrome c from mitochondria, followed by cleavage of caspase-9 and caspase-3, and that an inhibitor of caspase could suppress thimerosal-induced apoptosis. Thimerosal inhibited the phosphorylation of Akt(ser473 and survivin expression. Wortmannin, a PI3K inhibitor, inhibited Akt activity and decreased survivin expression, resulting in increased thimerosal-induced apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF-I (50 ng/ml increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of survivin expression by siRNA enhanced thimerosal-induced cell apoptosis, while overexpression of survivin prevented thimerosal-induced apoptosis. Taken together, the data show that the PI3K/Akt/survivin pathway plays an important role in the thimerosal-induced apoptosis in C2C12 cells. CONCLUSIONS/SIGNIFICANCE: Our results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and finally causes apoptosis via inhibition of PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic

  17. Resveratrol suppresses IGF-1 induced human colon cancer cell proliferation and elevates apoptosis via suppression of IGF-1R/Wnt and activation of p53 signaling pathways

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    Radhakrishnan Sridhar

    2010-05-01

    Full Text Available Abstract Background Obesity is a global phenomenon and is associated with various types of cancer, including colon cancer. There is a growing interest for safe and effective bioactive compounds that suppress the risk for obesity-promoted colon cancer. Resveratrol (trans-3, 4', 5,-trihydroxystilbene, a stilbenoid found in the skin of red grapes and peanuts suppresses many types of cancers by regulating cell proliferation and apoptosis through a variety of mechanisms, however, resveratrol effects on obesity-promoted colon cancer are not clearly established. Methods We investigated the anti-proliferative effects of resveratrol on HT-29 and SW480 human colon cancer cells in the presence and absence of insulin like growth factor-1 (IGF-1; elevated during obesity and elucidated the mechanisms of action using IGF-1R siRNA in HT-29 cells which represents advanced colon carcinogenesis. Results Resveratrol (100-150 μM exhibited anti-proliferative properties in HT-29 cells even after IGF-1 exposure by arresting G0/G1-S phase cell cycle progression through p27 stimulation and cyclin D1 suppression. Treatment with resveratrol suppressed IGF-1R protein levels and concurrently attenuated the downstream Akt/Wnt signaling pathways that play a critical role in cell proliferation. Targeted suppression of IGF-1R using IGF-1R siRNA also affected these signaling pathways in a similar manner. Resveratrol treatment induced apoptosis by activating tumor suppressor p53 protein, whereas IGF-1R siRNA treatment did not affect apoptosis. Our data suggests that resveratrol not only suppresses cell proliferation by inhibiting IGF-1R and its downstream signaling pathways similar to that of IGF-1R siRNA but also enhances apoptosis via activation of the p53 pathway. Conclusions For the first time, we report that resveratrol suppresses colon cancer cell proliferation and elevates apoptosis even in the presence of IGF-1 via suppression of IGF-1R/Akt/Wnt signaling pathways and

  18. Resveratrol suppresses IGF-1 induced human colon cancer cell proliferation and elevates apoptosis via suppression of IGF-1R/Wnt and activation of p53 signaling pathways

    International Nuclear Information System (INIS)

    Vanamala, Jairam; Reddivari, Lavanya; Radhakrishnan, Sridhar; Tarver, Chris

    2010-01-01

    Obesity is a global phenomenon and is associated with various types of cancer, including colon cancer. There is a growing interest for safe and effective bioactive compounds that suppress the risk for obesity-promoted colon cancer. Resveratrol (trans-3, 4', 5,-trihydroxystilbene), a stilbenoid found in the skin of red grapes and peanuts suppresses many types of cancers by regulating cell proliferation and apoptosis through a variety of mechanisms, however, resveratrol effects on obesity-promoted colon cancer are not clearly established. We investigated the anti-proliferative effects of resveratrol on HT-29 and SW480 human colon cancer cells in the presence and absence of insulin like growth factor-1 (IGF-1; elevated during obesity) and elucidated the mechanisms of action using IGF-1R siRNA in HT-29 cells which represents advanced colon carcinogenesis. Resveratrol (100-150 μM) exhibited anti-proliferative properties in HT-29 cells even after IGF-1 exposure by arresting G 0 /G 1 -S phase cell cycle progression through p27 stimulation and cyclin D1 suppression. Treatment with resveratrol suppressed IGF-1R protein levels and concurrently attenuated the downstream Akt/Wnt signaling pathways that play a critical role in cell proliferation. Targeted suppression of IGF-1R using IGF-1R siRNA also affected these signaling pathways in a similar manner. Resveratrol treatment induced apoptosis by activating tumor suppressor p53 protein, whereas IGF-1R siRNA treatment did not affect apoptosis. Our data suggests that resveratrol not only suppresses cell proliferation by inhibiting IGF-1R and its downstream signaling pathways similar to that of IGF-1R siRNA but also enhances apoptosis via activation of the p53 pathway. For the first time, we report that resveratrol suppresses colon cancer cell proliferation and elevates apoptosis even in the presence of IGF-1 via suppression of IGF-1R/Akt/Wnt signaling pathways and activation of p53, suggesting its potential role as a

  19. c-Abl is an upstream regulator of acid sphingomyelinase in apoptosis induced by inhibition of integrins αvβ3 and αvβ5.

    Directory of Open Access Journals (Sweden)

    Xiuhai Ren

    Full Text Available Inhibition of integrins αvβ3/αvβ5 by the cyclic function-blocking peptide, RGDfV (Arg-Gly-Asp-Phe-Val can induce apoptosis in both normal cells and tumor cells. We show that RGDfV induced apoptosis in ECV-304 carcinoma cells, increased activity and mRNA expression of acid sphingomyelinase (ASM, and increased ceramides C(16, C(18:0, C(24:0 and C(24:1 while decreasing the corresponding sphingomyelins. siRNA to ASM decreased RGDfV-induced apoptosis as measured by TUNEL, PARP cleavage, mitochondrial depolarization, and caspase-3 and caspase-8 activities, as well as by annexinV in a 3D collagen model. These findings indicate a causal role for ASM in RGDfV-induced apoptosis in ECV-304. We have shown that c-Abl, a non-receptor tyrosine kinase, also mediates RGDfV-induced apoptosis. However, c-Abl, has not been previously linked to ASM in any system. Here we show that STI-571 (imatinib, inhibitor of c-Abl inhibited RGDfV-induced ASM activity. Furthermore, STI-571 and c-Abl-siRNA both inhibited RGDfV-induced increase in ASM mRNA, but ASM-siRNA did not affect c-Abl phosphorylation or expression, supporting that c-Abl regulates the RGDfV-induced increase in ASM expression. These studies implicate ASM as a mediator of apoptosis induced by inhibition of integrins αvβ3/αvβ5, and for the first time place c-Abl as an upstream regulator of ASM expression and activity.

  20. Therapeutic Silencing of Bcl-2 by Systemically Administered siRNA Nanotherapeutics Inhibits Tumor Growth by Autophagy and Apoptosis and Enhances the Efficacy of Chemotherapy in Orthotopic Xenograft Models of ER (− and ER (+ Breast Cancer

    Directory of Open Access Journals (Sweden)

    Ibrahim Tekedereli

    2013-01-01

    Full Text Available Bcl-2 is overexpressed in about a half of human cancers and 50–70% of breast cancer patients, thereby conferring resistance to conventional therapies and making it an excellent therapeutic target. Small interfering RNA (siRNA offers novel and powerful tools for specific gene silencing and molecularly targeted therapy. Here, we show that therapeutic silencing of Bcl-2 by systemically administered nanoliposomal (NL-Bcl-2 siRNA (0.15 mg siRNA/kg, intravenous twice a week leads to significant antitumor activity and suppression of growth in both estrogen receptor-negative (ER(− MDA-MB-231 and ER-positive (+ MCF7 breast tumors in orthotopic xenograft models (P < 0.05. A single intravenous injection of NL-Bcl-2-siRNA provided robust and persistent silencing of the target gene expression in xenograft tumors. NL-Bcl-2-siRNA treatment significantly increased the efficacy of chemotherapy when combined with doxorubicin in both MDA-MB-231 and MCF-7 animal models (P < 0.05. NL-Bcl-2-siRNA treatment-induced apoptosis and autophagic cell death, and inhibited cyclin D1, HIF1α and Src/Fak signaling in tumors. In conclusion, our data provide the first evidence that in vivo therapeutic targeting Bcl-2 by systemically administered nanoliposomal-siRNA significantly inhibits growth of both ER(− and ER(+ breast tumors and enhances the efficacy of chemotherapy, suggesting that therapeutic silencing of Bcl-2 by siRNA is a viable approach in breast cancers.

  1. Protective effect of Sestrin2 on apoptosis of dendritic cells induced by high mobility group box-1 protein

    Directory of Open Access Journals (Sweden)

    Li-xue WANG

    2018-04-01

    Full Text Available Objective To investigate the potential role of Sestrin2 (SESN2 in regulating the apoptosis of dendritic cells (DCs induced by high mobility group box-1 protein (HMGB1. Methods DCs (the murine DC cell line DC2.4 were cultured with or without HMGB1 stimulation (cultured with 10ng/ml HMGB1 for 8, 24 and 48 hours, or cultured with HMGB1 for 48 hours at different concentrations of 1, 10 and 100ng/ml, respectively, n=4. The protein level of SESN2, cleaved-caspase-3 and Bcl-2 were analyzed with Western blotting. Localization of SESN2 in cells was observed under confocal laser microscope (LSCM. Cell apoptosis was analyzed with flow cytometry. In addition, DC2.4 cells were transfected with lentivirus containing SESN2 LV-RNA, SESN2 siRNA sequence expressing plasmids or blank vector (NC, NS, n=4. These cells were then stimulated with HMGB1 (100ng/ml for 48 hours, and the apoptosis was accessed as mentioned above. Results Compared with the control group, the expression of SESN2 was obviously up-regulated after HMGB1 (10ng/ml stimulation for 24 and 48 hours (P<0.05. In a dose-dependent response, the expression of SESN2 was markedly enhanced in treatment with 1, 10, 100ng/ml HMGB1 for 48 hours (P<0.05. Compared with the control group (7.35%±1.33%, the percentage of apoptosis was significantly increased with 10, 100ng/ml HMGB1 for 48 hours [(17.02%±4.85%, 17.48%±4.04%, respectively, P<0.05 or P<0.01]. After transfection, compared with blank vector group, the apoptosis of SESN2 siRNA group obviously elevated [(65.96%±2.50% vs. (50.01%±2.07%, P<0.05], and cleaved-caspase-3 expression significantly increased while Bcl-2 expression obviously decreased. In SESN2 LV-RNA group, the apoptosis significantly decreased [(35.57%±1.69% vs. (49.04%±4.87%, P<0.05], and cleaved-caspase-3 expression decreased and Bcl-2 expression obviously increased compared with blank vector group (P<0.05. Conclusion SESN2 has a protective effect against HMGB1 induced apoptosis of DC2

  2. Opposing effects of PI3K/Akt and Smad-dependent signaling pathways in NAG-1-induced glioblastoma cell apoptosis.

    Directory of Open Access Journals (Sweden)

    Zhiguo Zhang

    Full Text Available Nonsteroidal anti-inflammatory drug (NSAID activated gene-1 (NAG-1 is a divergent member of the transforming growth factor-beta (TGF-β superfamily. NAG-1 plays remarkable multifunctional roles in controlling diverse physiological and pathological processes including cancer. Like other TGF-β family members, NAG-1 can play dual roles during cancer development and progression by negatively or positively modulating cancer cell behaviors. In glioblastoma brain tumors, NAG-1 appears to act as a tumor suppressor gene; however, the precise underlying mechanisms have not been well elucidated. In the present study, we discovered that overexpression of NAG-1 induced apoptosis in U87 MG, U118 MG, U251 MG, and T98G cell lines via the intrinsic mitochondrial pathway, but not in A172 and LN-229 cell lines. NAG-1 could induce the phosphorylation of PI3K/Akt and Smad2/3 in all six tested glioblastoma cell lines, except Smad3 phosphorylation in A172 and LN-229 cell lines. In fact, Smad3 expression and its phosphorylation were almost undetectable in A172 and LN-229 cells. The PI3K inhibitors promoted NAG-1-induced glioblastoma cell apoptosis, while siRNAs to Smad2 and Smad3 decreased the apoptosis rate. NAG-1 also stimulated the direct interaction between Akt and Smad3 in glioblastoma cells. Elevating the level of Smad3 restored the sensitivity to NAG-1-induced apoptosis in A172 and LN-229 cells. In conclusion, our results suggest that PI3K/Akt and Smad-dependent signaling pathways display opposing effects in NAG-1-induced glioblastoma cell apoptosis.

  3. Sensitization of human colon cancer cells to sodium butyrate-induced apoptosis by modulation of sphingosine kinase 2 and protein kinase D

    International Nuclear Information System (INIS)

    Xiao, Min; Liu, Yungang; Zou, Fei

    2012-01-01

    Sphingosine kinases (SphKs) have been recognized as important proteins regulating cell proliferation and apoptosis. Of the two isoforms of SphK (SphK1 and SphK2), little is known about the functions of SphK2. Sodium butyrate (NaBT) has been established as a promising chemotherapeutic agent, but the precise mechanism for its effects is unknown. In this study, we investigated the role of SphK2 in NaBT-induced apoptosis of HCT116 colon cancer cells. The results indicated that following NaBT treatment SphK2 was translocated from the nucleus to the cytoplasm, leading to its accumulation in the cytoplasm; in the meantime, only mild apoptosis occurred. However, downregulation of SphK2 resulted in sensitized apoptosis, and overexpression of SphK2 led to even lighter apoptosis; these strongly indicate an inhibitory role of SphK2 in cell apoptosis induced by NaBT. After knocking down protein kinase D (PKD), another protein reported to be critical in cell proliferation/apoptosis process, by using siRNA, blockage of cytoplasmic accumulation of SphK2 and sensitized apoptosis following NaBT treatment were observed. The present study suggests that PKD and SphK2 may form a mechanism for the resistance of cancer cells to tumor chemotherapies, such as HCT116 colon cancer cells to NaBT, and these two proteins may become molecular targets for designation of new tumor-therapeutic drugs. -- Highlights: ► In the present study sodium butyrate (10 mM) induced mild apoptosis of cancer cells. ► The apoptosis was negatively regulated by cytoplasmic Sphingosine Kinase 2 (SphK2). ► Translocation of SphK2 from nucleus to cytoplasm was mediated by protein kinase D. ► Downregulation of SphK2 or protein kinase D leads to sensitized cell apoptosis.

  4. microRNA-340 induces apoptosis by downregulation of BAG3 in ovarian cancer SKOV3 cells.

    Science.gov (United States)

    Qu, Fei; Wang, Xiufen

    2017-08-01

    Aberrant expression of miR-340 has been found in several kinds of cancers including ovarian cancer. Pro-apoptotic and anti-metastasis roles of miR-340 in ovarian cancer have also been reported; however, the underling molecular mechanisms by which miR-340 suppresses ovarian cancer are still unclear. This study focused on the role and molecular mechanism of miR-340 in ovarian cancer. Human ovarian carcinoma SKOV3 cells were used and transfected with miR-340 mimic, miR-340 inhibitor and their correspondingly negative controls (mimic control and inhibitor control). Thereafter, cell viability, apoptosis, and the expressions of apoptosis-associated factors and BAG3 were respectively assessed by MTT assay, flow cytometry, qRT-PCR and Western blotting. SKOV3 cells were then co-transfected with miR-340 inhibitor and BAG3 targeted siRNA, then cell viability, apoptosis and the expression of apoptosis-associated factors were retested. Besides, the expressions of main factors in PI3K/AKT pathway were detected. Overexpression of miR-340 suppressed BAG3 cells viability (P BAG3 was negatively regulated by miR-340 (P BAG3 silence significantly induced cell apoptosis (P BAG3 silence abolished miR-340 suppression-induced activation of PI3K and AKT. This study revealed the tumor suppressive role of miR-340 in SKOV3 cells by negative regulation of BAG3. PI3K/AKT pathway might be involved in the regulation of miR-340 and BAG3.

  5. Aspartame-induced apoptosis in PC12 cells.

    Science.gov (United States)

    Horio, Yukari; Sun, Yongkun; Liu, Chuang; Saito, Takeshi; Kurasaki, Masaaki

    2014-01-01

    Aspartame is an artificial sweetner added to many low-calorie foods. The safety of aspartame remains controversial even though there are many studies on its risks. In this study, to understand the physiological effects of trace amounts of artificial sweetners on cells, the effects of aspartame on apoptosis were investigated using a PC12 cell system. In addition, the mechanism of apoptosis induced by aspartame in PC12 cells and effects on apoptotic factors such as cytochrome c, apoptosis-inducing factor, and caspase family proteins were studied by Western blotting and RT-PCR. Aspartame-induced apoptosis in PC12 cells in a dose-dependent manner. In addition, aspartame exposure increased the expressions of caspases 8 and 9, and cytochrome c. These results indicate that aspartame induces apoptosis mainly via mitochondrial pathway involved in apoptosis due to oxigen toxicity. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. 14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis

    International Nuclear Information System (INIS)

    Wang, Lai; Chen, Man; Yuan, Lin; Xiang, Yuting; Zheng, Ruimao; Zhu, Shigong

    2014-01-01

    Highlights: • 14,15-EET inhibits OGD-induced apoptosis in cortical neurons. • Mitochondrial biogenesis of cortical neurons is promoted by 14,15-EET. • 14,15-EET preserves mitochondrial function of cortical neurons under OGD. • CREB mediates effect of 14,15-EET on mitochondrial biogenesis and function. - Abstract: 14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen–glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1

  7. 14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Lai; Chen, Man; Yuan, Lin; Xiang, Yuting [Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing (China); Zheng, Ruimao, E-mail: rmzheng@pku.edu.cn [Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing (China); Zhu, Shigong, E-mail: sgzhu@bjmu.edu.cn [Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing (China)

    2014-07-18

    Highlights: • 14,15-EET inhibits OGD-induced apoptosis in cortical neurons. • Mitochondrial biogenesis of cortical neurons is promoted by 14,15-EET. • 14,15-EET preserves mitochondrial function of cortical neurons under OGD. • CREB mediates effect of 14,15-EET on mitochondrial biogenesis and function. - Abstract: 14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen–glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1.

  8. Molecular mechanism of apoptosis and characterization of apoptosis induced by radiation

    International Nuclear Information System (INIS)

    Li Yumin; Zhang Yuguang; Li Yukun

    1999-01-01

    The major discoveries of apoptosis research in recent years were reviewed briefly. The mechanisms of caspases/ICE gene family and bcl-2 gene family on apoptosis were analyzed. And the signal transduction pathway of apoptosis found currently has been summarized. The characterizations of apoptosis induced by radiation such as time-effects, dose-effects and the radiosensibility were summed up

  9. Upregulation of LYAR induces neuroblastoma cell proliferation and survival.

    Science.gov (United States)

    Sun, Yuting; Atmadibrata, Bernard; Yu, Denise; Wong, Matthew; Liu, Bing; Ho, Nicholas; Ling, Dora; Tee, Andrew E; Wang, Jenny; Mungrue, Imran N; Liu, Pei Y; Liu, Tao

    2017-09-01

    The N-Myc oncoprotein induces neuroblastoma by regulating gene transcription and consequently causing cell proliferation. Paradoxically, N-Myc is well known to induce apoptosis by upregulating pro-apoptosis genes, and it is not clear how N-Myc overexpressing neuroblastoma cells escape N-Myc-mediated apoptosis. The nuclear zinc finger protein LYAR has recently been shown to modulate gene expression by forming a protein complex with the protein arginine methyltransferase PRMT5. Here we showed that N-Myc upregulated LYAR gene expression by binding to its gene promoter. Genome-wide differential gene expression studies revealed that knocking down LYAR considerably upregulated the expression of oxidative stress genes including CHAC1, which depletes intracellular glutathione and induces oxidative stress. Although knocking down LYAR expression with siRNAs induced oxidative stress, neuroblastoma cell growth inhibition and apoptosis, co-treatment with the glutathione supplement N-acetyl-l-cysteine or co-transfection with CHAC1 siRNAs blocked the effect of LYAR siRNAs. Importantly, high levels of LYAR gene expression in human neuroblastoma tissues predicted poor event-free and overall survival in neuroblastoma patients, independent of the best current markers for poor prognosis. Taken together, our data suggest that LYAR induces proliferation and promotes survival of neuroblastoma cells by repressing the expression of oxidative stress genes such as CHAC1 and suppressing oxidative stress, and identify LYAR as a novel co-factor in N-Myc oncogenesis.

  10. A hybrid of coumarin and phenylsulfonylfuroxan induces caspase-dependent apoptosis and cytoprotective autophagy in lung adenocarcinoma cells.

    Science.gov (United States)

    Wang, Qian; Guo, Yalan; Jiang, Shanshan; Dong, Mengxue; Kuerban, Kudelaidi; Li, Jiyang; Feng, Meiqing; Chen, Ying; Ye, Li

    2018-01-15

    Lung adenocarcinoma is the most primary histologic subtype of non-small cell lung cancer (NSCLC). Compound 8b, a novel coumarin derivative with phenylsulfonylfuroxan group, shows significant antiproliferation activity against lung adenocarcinoma cell with low toxicity. This study aims to uncover the potential of compound 8b in relation to apoptosis as well as autophagy induction in lung adenocarcinoma cells. The cytotoxicity and apoptosis of A549 and H1299 cells induced by compound 8b were detected by MTT, microscope and western blot analysis. Autophagy was determined by TEM, confocal microscopy and western blot analysis. Akt/mTOR and Erk signaling pathway were also examined by western blot analysis. First, significant growth inhibition and caspase-dependent apoptosis were observed in compound 8b-treated A549 and H1299 cells. Then, we confirmed compound 8b-induced autophagy by autophagosomes formation, upregulated expression of autophagy-related protein LC3-II and autophagic flux. Importantly, abolishing autophagy using inhibitors and ATG5 siRNA enhanced the cytotoxicity of compound 8b, indicating the cytoprotective role of autophagy in lung adenocarcinoma. Further mechanistic investigations suggested that Akt/mTOR and Erk signaling pathways contributed to autophagy induction by compound 8b. This results demonstrate that compound 8b induces caspase-dependent apoptosis as well as cytoprotective autophagy in lung adenocarcinoma cells, which may provide scientific evidence for developing this furoxan-based NO-releasing coumarin derivative as a potential anti-lung adenocarcinoma therapeutic agents. Copyright © 2017 Elsevier GmbH. All rights reserved.

  11. DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells.

    Science.gov (United States)

    Fukuhara, Shinichiro; Chang, Inik; Mitsui, Yozo; Chiyomaru, Takeshi; Yamamura, Soichiro; Majid, Shahana; Saini, Sharanjot; Hirata, Hiroshi; Deng, Guoren; Gill, Ankurpreet; Wong, Darryn K; Shiina, Hiroaki; Nonomura, Norio; Dahiya, Rajvir; Tanaka, Yuichiro

    2014-11-30

    Mismatch repair (MMR) enzymes have been shown to be deficient in prostate cancer (PCa). MMR can influence the regulation of tumor development in various cancers but their role on PCa has not been investigated. The aim of the present study was to determine the functional effects of the mutL-homolog 1 (MLH1) gene on growth of PCa cells. The DU145 cell line has been established as MLH1-deficient and thus, this cell line was utilized to determine effects of MLH1 by gene expression. Lack of MLH1 protein expression was confirmed by Western blotting in DU145 cells whereas levels were high in normal PWR-1E and RWPE-1 prostatic cells. MLH1-expressing stable transfectant DU145 cells were then created to characterize the effects this MMR gene has on various growth properties. Expression of MLH1 resulted in decreased cell proliferation, migration and invasion properties. Lack of cell growth in vivo also indicated a tumor suppressive effect by MLH1. Interestingly, MLH1 caused an increase in apoptosis along with phosphorylated c-Abl, and treatment with MLH1 siRNAs countered this effect. Furthermore, inhibition of c-Abl with STI571 also abrogated the effect on apoptosis caused by MLH1. These results demonstrate MLH1 protects against PCa development by inducing c-Abl-mediated apoptosis.

  12. Mitochondrial dysfunction in lyssavirus-induced apoptosis.

    Science.gov (United States)

    Gholami, Alireza; Kassis, Raïd; Real, Eléonore; Delmas, Olivier; Guadagnini, Stéphanie; Larrous, Florence; Obach, Dorothée; Prevost, Marie-Christine; Jacob, Yves; Bourhy, Hervé

    2008-05-01

    Lyssaviruses are highly neurotropic viruses associated with neuronal apoptosis. Previous observations have indicated that the matrix proteins (M) of some lyssaviruses induce strong neuronal apoptosis. However, the molecular mechanism(s) involved in this phenomenon is still unknown. We show that for Mokola virus (MOK), a lyssavirus of low pathogenicity, the M (M-MOK) targets mitochondria, disrupts the mitochondrial morphology, and induces apoptosis. Our analysis of truncated M-MOK mutants suggests that the information required for efficient mitochondrial targeting and dysfunction, as well as caspase-9 activation and apoptosis, is held between residues 46 and 110 of M-MOK. We used a yeast two-hybrid approach, a coimmunoprecipitation assay, and confocal microscopy to demonstrate that M-MOK physically associates with the subunit I of the cytochrome c (cyt-c) oxidase (CcO) of the mitochondrial respiratory chain; this is in contrast to the M of the highly pathogenic Thailand lyssavirus (M-THA). M-MOK expression induces a significant decrease in CcO activity, which is not the case with M-THA. M-MOK mutations (K77R and N81E) resulting in a similar sequence to M-THA at positions 77 and 81 annul cyt-c release and apoptosis and restore CcO activity. As expected, the reverse mutations, R77K and E81N, introduced in M-THA induce a phenotype similar to that due to M-MOK. These features indicate a novel mechanism for energy depletion during lyssavirus-induced apoptosis.

  13. Therapeutic potential of inhibiting ABCE1 and eRF3 genes via siRNA strategy using chitosan nanoparticles in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cengiz, Bagdat Burcu; Asik, Mehmet Dogan [Hacettepe University, Nanotechnology and Nanomedicine Division (Turkey); Kara, Goknur [Hacettepe University, Biochemistry Division, Chemistry Department (Turkey); Turk, Mustafa [Kirikkale University, Bioengineering Department (Turkey); Denkbas, Emir Baki, E-mail: denkbas@hacettepe.edu.tr [Hacettepe University, Biochemistry Division, Chemistry Department (Turkey)

    2015-04-15

    In recent years, targeted cancer therapy strategies have begun to take the place of the conventional treatments. Inhibition of the specific genes, involved in cancer progress, via small interfering RNA (siRNA) has become one of the promising therapeutic approaches for cancer therapy. However, due to rapid nuclease degradation and poor cellular uptake of siRNA, a suitable carrier for siRNA penetration inside the cells is required. We used chitosan nanoparticles (CS-NPs) to efficiently deliver ATP-binding casette E1 (ABCE1) and eukaryotic release factor 3 (eRF3)-targeting siRNAs, individually and together, to reduce the proliferation and induce the apoptosis of breast cancer cells. The CS-NPs were generated by ionic gelation method using tripolyphosphate (TPP) as a crosslinker. Nanoparticles (NPs) were obtained with diameters ranging between 110 and 230 nm and the zeta potential of approximately 27 mV optimizing the solution pH to 4.5 and CS/TPP mass ratio to 3:1. Loading efficiencies of 98.69 % ± 0.051 and 98.83 % ± 0.047 were achieved when ABCE1 siRNA and eRF3 siRNA were entrapped into the NPs, respectively. Cell proliferation assay demonstrated that siRNA-loaded CS-NPs were more effective on cancer cells when compared to siRNAs without CS-NPs. Parallel results were also obtained by apoptosis/necrosis, double-staining analysis. Within our study, the potency of ABCE1 and eRF3 siRNAs were shown for the first time with this kind of polymeric delivery system. The results also indicated that ABCE1 and eRF3, important molecules in protein synthesis, could serve as effective targets to inhibit the cancer cells.

  14. Inhibition of PDGFR by CP-673451 induces apoptosis and increases cisplatin cytotoxicity in NSCLC cells via inhibiting the Nrf2-mediated defense mechanism.

    Science.gov (United States)

    Yang, Yang; Deng, Yanchao; Chen, Xiangcui; Zhang, Jiahao; Chen, Yueming; Li, Huachao; Wu, Qipeng; Yang, Zhicheng; Zhang, Luyong; Liu, Bing

    2018-05-29

    Platelet-derived growth factor receptors (PDGFRs) are abundantly expressed by stromal cells in the non-small cell lung cancer (NSCLC) microenvironment, and in a subset of cancer cells, usually with their overexpression and/or activating mutation. However, the effect of PDGFR inhibition on lung cancer cells themselves has been largely neglected. In this study, we investigated the anticancer activity of CP-673451, a potent and selective inhibitor of PDGFRβ, on NSCLC cell lines (A549 and H358) and the potential mechanism. The results showed that inhibition of PDGFRβ by CP-673451 induced a significant increase in cell apoptosis, accompanied by ROS accumulation. However, CP-673451 exerted less cytotoxicity in normal lung epithelial cell line BEAS-2B cells determined by MTT and apoptosis assay. Elimination of ROS by NAC reversed the CP-673451-induced apoptosis in NSCLC cells. Furthermore, CP-673451 down-regulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) probably through inhibition of PI3K/Akt pathway. Rescue of Nrf2 activity counteracted the effects of CP-673451 on cell apoptosis and ROS accumulation. Silencing PDGFRβ expression by PDGFRβ siRNA exerted similar effects with CP-673451 in A549 cells, and when PDGFRβ was knockdowned by PDGFRβ siRNA, CP-673451 produced no additional effects on cell viability, ROS and GSH production, Nrf2 expression as well as PI3K/Akt pathway activity. Specifically, Nrf2 plays an indispensable role in NSCLC cell sensitivity to platinum-based treatments and we found that combination of CP-673451 and cisplatin produced a synergistic anticancer effect and substantial ROS production in vitro. Therefore, these results clearly demonstrate the effectiveness of inhibition of PDGFRβ against NSCLC cells and strongly suggest that CP-673451 may be a promising adjuvant chemotherapeutic drug. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Delivery of small interfering RNA for inhibition of endothelial cell apoptosis by hypoxia and serum deprivation

    International Nuclear Information System (INIS)

    Cho, Seung-Woo; Hartle, Lauren; Son, Sun Mi; Yang, Fan; Goldberg, Michael; Xu, Qiaobing; Langer, Robert; Anderson, Daniel G.

    2008-01-01

    RNA interference (RNAi) for anti-angiogenic or pro-apoptotic factors in endothelial cells (ECs) has great potential for the treatment of ischemic diseases by promoting angiogenesis or inhibiting apoptosis. Here, we report the utility of small interfering RNA (siRNA) in inhibiting EC apoptosis induced by tumor necrosis factor-α (TNF-α). siRNA was designed and synthesized targeting tumor necrosis factor-α receptor-1 (TNFR-1) and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were cultured under in vitro hypoxic and serum-deprived conditions to simulate in vivo ischemic conditions. Two days after liposomal delivery of siRNA targeting TNFR-1 and SHP-1, significant silencing of each target (TNFR-1; 76.5% and SHP-1; 97.2%) was detected. Under serum-deprived hypoxic (1% oxygen) conditions, TNF-α expression in HUVECs increased relative to normoxic (20% oxygen) and serum-containing conditions. Despite enhanced TNF-α expression, suppression of TNFR-1 or SHP-1 by siRNA delivery not only enhanced expression of angiogenic factors (KDR/Flk-1 and eNOS) and anti-apoptotic factor (Bcl-xL) but also reduced expression of a pro-apoptotic factor (Bax). Transfection of TNFR-1 or SHP-1 siRNA significantly decreased the HUVEC apoptosis while significantly enhancing HUVEC proliferation and capillary formation. The present study demonstrates that TNFR-1 and SHP-1 may be useful targets for the treatment of myocardial or hindlimb ischemia

  16. Polydatin inhibits cell proliferation and induces apoptosis in laryngeal cancer and HeLa cells via suppression of the PDGF/AKT signaling pathway.

    Science.gov (United States)

    Li, Haixia; Shi, Baoyuan; Li, Yanyun; Yin, Fengfang

    2017-07-01

    Polydatin (PD), a stilbene compound extracted from Polygonum cuspidatum, is suggested to possess anti-cancer activities, including inhibition of cell proliferation, cell cycle arrest, and induction of apoptosis. The platelet-derived growth factor (PDGF)/AKT signaling pathway plays complex roles in tumor suppression. However, the effect of PD on the PDGF/AKT signaling pathway in laryngeal cancer and HeLa cells has not been explored. MTT assay and flow cytometry showed that PD inhibited cell proliferation and induced apoptosis in Hep-2 and AMC-HN-8 cells. Western blot analysis indicated that PD inhibited the expression levels of PDGF-B and phosphorylated AKT (p-AKT) in both cells. Treatment of PDGF-B siRNA or PDGFR inhibitor found that after the PDGF signaling was inactivated, p-AKT expression was significantly decreased in Hep-2 cells. Tumor xenograft experiment in nude mice indicated PD significantly inhibited the growth of Hep-2 cells in vivo. In conclusion, PD inhibited cell proliferation and induced apoptosis in laryngeal cancer and HeLa cells via inactivation of the PDGF/AKT signaling pathway. © 2017 Wiley Periodicals, Inc.

  17. SIRT1 protects cardiac cells against apoptosis induced by zearalenone or its metabolites α- and β-zearalenol through an autophagy-dependent pathway

    International Nuclear Information System (INIS)

    Ben Salem, Intidhar; Boussabbeh, Manel; Da Silva, Julie Pires; Guilbert, Arnaud; Bacha, Hassen; Abid-Essefi, Salwa; Lemaire, Christophe

    2017-01-01

    Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium in cereals and agricultural products. The major ZEN metabolites are α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL). In the present study, we investigated the underlying mechanism of the toxicity induced by ZEN, α-ZOL and β-ZOL in cardiac cells (H9c2). We show that treatment with ZEN or its metabolites induces the activation of the mitochondrial pathway of apoptosis as characterized by an increase in ROS generation, a loss of mitochondrial transmembrane potential (ΔΨm) and an activation of caspases. Besides, we demonstrate that these mycotoxins promote the activation of autophagy before the onset of apoptosis. Indeed, we observed that a short-time (6 h) treatment with ZEN, α-ZOL or β-ZOL, increased the level of Beclin-1 and LC3-II and induced the accumulation of the CytoID® autophagy detection probe. Moreover, the inhibition of autophagy by Chloroquine significantly increased cell death induced by ZEN, α-ZOL or β-ZOL, suggesting that the activation of autophagy serves as a cardioprotective mechanism against these mycotoxins. In addition, we found that the inhibition (EX527) or the knockdown of SIRT1 (siRNA) significantly increased apoptosis induced by ZEN or its derivatives, whereas SIRT1 activation with RSV greatly prevents the cytotoxic effects of these mycotoxins. By contrast, when autophagy was inhibited by CQ, the activation of SIRT1 by RSV had no protection against the cardiotoxicity of ZEN or its metabolites, suggesting that SIRT1 protects cardiac cells by an autophagy-dependent pathway. - Highlights: • ZEN, α- and β-ZOL induce the mitochondrial pathway of apoptosis in cardiac cells. • Inhibition of autophagy enhanced ZEN-, α-ZOL- and β-ZOL-induced apoptosis. • SIRT1 activates autophagy to protect cells from ZEN, α- and β-ZOL-induced toxicity.

  18. The proapoptotic activity of C-terminal domain of apoptosis-inducing factor (AIF is separated from its N-terminal

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    YONG ZHANG

    2009-01-01

    Full Text Available Apoptosis-inducing factor (AIF is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFΔ1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFΔ1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFΔ1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFΔ1-480. Therefore, AIFΔ 1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFΔ 1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFΔ 1-480. Human Jurkat cells transfected with the immuno-AIFΔl-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFΔl-480 gene as a novel approach to treating HER2-overexpressing cancers.

  19. The dependence receptor Ret induces apoptosis in somatotrophs through a Pit-1/p53 pathway, preventing tumor growth.

    Science.gov (United States)

    Cañibano, Carmen; Rodriguez, Noela L; Saez, Carmen; Tovar, Sulay; Garcia-Lavandeira, Montse; Borrello, Maria Grazia; Vidal, Anxo; Costantini, Frank; Japon, Miguel; Dieguez, Carlos; Alvarez, Clara V

    2007-04-18

    Somatotrophs are the only pituitary cells that express Ret, GFRalpha1 and GDNF. This study investigated the effects of Ret in a somatotroph cell line, in primary pituitary cultures and in Ret KO mice. Ret regulates somatotroph numbers by inducing Pit-1 overexpression, leading to increased p53 expression and apoptosis, both of which can be prevented with Ret or Pit-1 siRNA. The Pit-1 overexpression is mediated by sustained activation of PKCdelta, JNK, c/EBPalpha and CREB induced by a complex of Ret, caspase 3 and PKCdelta. In the presence of GDNF, Akt is activated, and the Pit-1 overexpression and resulting apoptosis are blocked. The adenopituitary of Ret KO mice is larger than normal, showing Pit-1 and somatotroph hyperplasia. In normal animals, activation of the Ret/Pit-1/p53 pathway by retroviral introduction of Ret blocked tumor growth in vivo. Thus, somatotrophs have an intrinsic mechanism for controlling Pit-1/GH production through an apoptotic/survival pathway. Ret might be of value for treatment of pituitary adenomas.

  20. Chk2 mediates RITA-induced apoptosis.

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    de Lange, J; Verlaan-de Vries, M; Teunisse, A F A S; Jochemsen, A G

    2012-06-01

    Reactivation of the p53 tumor-suppressor protein by small molecules like Nutlin-3 and RITA (reactivation of p53 and induction of tumor cell apoptosis) is a promising strategy for cancer therapy. The molecular mechanisms involved in the responses to RITA remain enigmatic. Several groups reported the induction of a p53-dependent DNA damage response. Furthermore, the existence of a p53-dependent S-phase checkpoint has been suggested, involving the checkpoint kinase Chk1. We have recently shown synergistic induction of apoptosis by RITA in combination with Nutlin-3, and we observed concomitant Chk2 phosphorylation. Therefore, we investigated whether Chk2 contributes to the cellular responses to RITA. Strikingly, the induction of apoptosis seemed entirely Chk2 dependent. Transcriptional activity of p53 in response to RITA required the presence of Chk2. A partial rescue of apoptosis observed in Noxa knockdown cells emphasized the relevance of p53 transcriptional activity for RITA-induced apoptosis. In addition, we observed an early p53- and Chk2-dependent block of DNA replication upon RITA treatment. Replicating cells seemed more prone to entering RITA-induced apoptosis. Furthermore, the RITA-induced DNA damage response, which was not a secondary effect of apoptosis induction, was strongly attenuated in cells lacking p53 or Chk2. In conclusion, we identified Chk2 as an essential mediator of the cellular responses to RITA.

  1. 14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis.

    Science.gov (United States)

    Wang, Lai; Chen, Man; Yuan, Lin; Xiang, Yuting; Zheng, Ruimao; Zhu, Shigong

    2014-07-18

    14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen-glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Csk regulates angiotensin II-induced podocyte apoptosis.

    Science.gov (United States)

    Zhang, Lu; Ren, Zhilong; Yang, Qian; Ding, Guohua

    2016-07-01

    Increasing data have shown that angiotensin II (Ang II) perpetuates podocyte injury and promotes progression to end-stage kidney disease. The mechanism underlying Ang II-induced podocyte apoptosis has not been established. C-terminal Src kinase (Csk) is a cytoplasmic kinase that interacts with scaffolding proteins involved in cell growth, adhesion, and polarization, and the role of Csk in regulating cellular apoptosis has gradually attracted attention. This study evaluates the role of Csk in Ang II-induced podocyte apoptosis. In vivo, Wistar rats were randomly subjected to a normal saline or Ang II infusion. In vitro, we exposed differentiated mouse podocytes to Ang II. Ang II increased Csk expression and induced podocyte apoptosis, stimulated Csk translocation and binding to Caveolin-1, and stimulated decreased Fyn pY416, increased Fyn pY529, and nephrin dephosphorylation. Csk knockdown prevented Ang II-induced podocyte apoptosis, reduced Fyn kinase inactivation, and increased the interaction between nephrin and the activated form of Fyn, accompanied by a reduced interaction between Csk and Caveolin-1. These findings indicate that Ang II induces podocyte injury via a Csk-dependent pathway.

  3. Globular Adiponectin Inhibits the Apoptosis of Mesenchymal Stem Cells Induced by Hypoxia and Serum Deprivation via the AdipoR1-Mediated Pathway

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    Xia-Qiu Tian

    2016-02-01

    Full Text Available Background/Aims: Poor viability of transplanted mesenchymal stem cells (MSCs within the ischemic heart limits their therapeutic potential for cardiac repair. Globular adiponectin (gAPN exerts anti-apoptotic effects on several types of stem cells. Herein, we investigated the effect of gAPN on the MSCs against apoptosis induced by hypoxia and serum deprivation (H/SD. Methods: MSCs exposed to H/SD conditions were treated with different concentrations of gAPN. To identify the main type of receptor, MSCs were transfected with siRNA targeting adiponectin receptor 1 or 2 (AdipoR1 or AdipoR2. To elucidate the downstream pathway, MSCs were pre-incubated with AMPK inhibitor Compound C. Apoptosis, caspase-3 activity and mitochondrial membrane potential were evaluated. Results: H/SD-induced MSCs apoptosis and caspase-3 activation were attenuated by gAPN in a concentration-dependent manner. gAPN increased Bcl-2 and decreased Bax expressions. The loss of mitochondrial membrane potential induced by H/SD was also abolished by gAPN. The protective effect of gAPN was significantly attenuated after the knockdown of AdipoR1 rather than AdipoR2. Moreover, Compound C partly suppressed the anti-apoptotic effect of gAPN. Conclusions: gAPN inhibits H/SD-induced apoptosis in MSCs via AdipoR1-mediated pathway, possibly linked to the activation of AMPK. gAPN may be a novel survival factor for MSCs in the ischemic engraftment environment.

  4. Globular Adiponectin Inhibits the Apoptosis of Mesenchymal Stem Cells Induced by Hypoxia and Serum Deprivation via the AdipoR1-Mediated Pathway.

    Science.gov (United States)

    Tian, Xia-Qiu; Yang, Yue-Jin; Li, Qing; Huang, Pei-Sen; Li, Xiang-Dong; Jin, Chen; Qi, Kang; Jiang, Lei-Pei; Chen, Gui-Hao

    2016-01-01

    Poor viability of transplanted mesenchymal stem cells (MSCs) within the ischemic heart limits their therapeutic potential for cardiac repair. Globular adiponectin (gAPN) exerts anti-apoptotic effects on several types of stem cells. Herein, we investigated the effect of gAPN on the MSCs against apoptosis induced by hypoxia and serum deprivation (H/SD). MSCs exposed to H/SD conditions were treated with different concentrations of gAPN. To identify the main type of receptor, MSCs were transfected with siRNA targeting adiponectin receptor 1 or 2 (AdipoR1 or AdipoR2). To elucidate the downstream pathway, MSCs were pre-incubated with AMPK inhibitor Compound C. Apoptosis, caspase-3 activity and mitochondrial membrane potential were evaluated. H/SD-induced MSCs apoptosis and caspase-3 activation were attenuated by gAPN in a concentration-dependent manner. gAPN increased Bcl-2 and decreased Bax expressions. The loss of mitochondrial membrane potential induced by H/SD was also abolished by gAPN. The protective effect of gAPN was significantly attenuated after the knockdown of AdipoR1 rather than AdipoR2. Moreover, Compound C partly suppressed the anti-apoptotic effect of gAPN. gAPN inhibits H/SD-induced apoptosis in MSCs via AdipoR1-mediated pathway, possibly linked to the activation of AMPK. gAPN may be a novel survival factor for MSCs in the ischemic engraftment environment. © 2016 The Author(s) Published by S. Karger AG, Basel.

  5. RNAi screen reveals a role of SPHK2 in dengue virus-mediated apoptosis in hepatic cell lines.

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    Atthapan Morchang

    Full Text Available Hepatic dysfunction is a feature of dengue virus (DENV infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show apoptosis, which relates to the pathogenesis of DENV infection. However, how DENV induced liver injury is not fully understood. In this study, we aim to identify the factors that influence cell death by employing an apoptosis-related siRNA library screening. Our results show the effect of 558 gene silencing on caspase 3-mediated apoptosis in DENV-infected Huh7 cells. The majority of genes that contributed to apoptosis were the apoptosis-related kinase enzymes. Tumor necrosis factor superfamily member 12 (TNFSF12, and sphingosine kinase 2 (SPHK2, were selected as the candidate genes to further validate their influences on DENV-induced apoptosis. Transfection of siRNA targeting SPHK2 but not TNFSF12 genes reduced apoptosis determined by Annexin V/PI staining. Knockdown of SPHK2 did not reduce caspase 8 activity; however, did significantly reduce caspase 9 activity, suggesting its involvement of SPHK2 in the intrinsic pathway of apoptosis. Treatment of ABC294649, an inhibitor of SPHK2, reduced the caspase 3 activity, suggesting the involvement of its kinase activity in apoptosis. Knockdown of SPHK2 significantly reduced caspase 3 activity not only in DENV-infected Huh7 cells but also in DENV-infected HepG2 cells. Our results were consistent across all of the four serotypes of DENV infection, which supports the pro-apoptotic role of SPHK2 in DENV-infected liver cells.

  6. Nur77 inhibits oxLDL induced apoptosis of macrophages via the p38 MAPK signaling pathway

    International Nuclear Information System (INIS)

    Shao, Qin; Han, Fei; Peng, Shi; He, Ben

    2016-01-01

    The interaction between macrophages and oxLDL plays a crucial role in the initiation and progression of atherosclerosis. As a key initiator in a number of plaque promoting processes, oxLDL induces variable effects such as cell apoptosis or proliferation. Orphan nuclear receptor Nur77 is potently induced in macrophages by diverse stimuli, suggesting that it is of importance in vascular inflammation resulting in atherosclerosis, but whether Nur77 induction is detrimental or protective is unclear. In our study, we explore the role of Nur77 in the regulation of oxLDL-induced macrophage apoptosis and the signaling pathways that are involved. We found that oxLDL induced Nur77 expression in a dose and time dependent fashion, and cell viability was decreased in parallel. To determine whether Nur77 induction contributes to the loss of cell viability or is a protective mechanism, the effect of Nur77 overexpression was examined. Importantly, Nur77 overexpression inhibited the oxLDL-induced decrease of cell viability, inhibited the production of apoptotic bodies and restored DNA synthesis following oxLDL exposure. Furthermore, we found that Nur77 induction is mediated through the p38 MAPK signaling pathway. After pretreatment with SB203580, cell viability was decreased, the expression of CyclinA2 and PCNA was attenuated and the percentage of cell apoptosis was enhanced. Likewise, Nur77 overexpression increased the expression of the cell cycle genes PCNA and p21, and attenuated the increase in caspase-3. On the other hand, knockdown of Nur77 expression by specific siRNA resulted in the increased expression of caspase 3. The results demonstrate that Nur77 is induced by oxLDL via the p38 MAPK signaling pathway, which is involved in the regulation of cell survival. Nur77 enhanced cell survival via suppressing apoptosis, without affecting cell proliferation of activated macrophages, which may be beneficial in patients with atherosclerosis. - Highlights: • oxLDL could induce Nur77

  7. Nur77 inhibits oxLDL induced apoptosis of macrophages via the p38 MAPK signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Qin; Han, Fei; Peng, Shi; He, Ben, E-mail: heben@medmail.com.cn

    2016-03-18

    The interaction between macrophages and oxLDL plays a crucial role in the initiation and progression of atherosclerosis. As a key initiator in a number of plaque promoting processes, oxLDL induces variable effects such as cell apoptosis or proliferation. Orphan nuclear receptor Nur77 is potently induced in macrophages by diverse stimuli, suggesting that it is of importance in vascular inflammation resulting in atherosclerosis, but whether Nur77 induction is detrimental or protective is unclear. In our study, we explore the role of Nur77 in the regulation of oxLDL-induced macrophage apoptosis and the signaling pathways that are involved. We found that oxLDL induced Nur77 expression in a dose and time dependent fashion, and cell viability was decreased in parallel. To determine whether Nur77 induction contributes to the loss of cell viability or is a protective mechanism, the effect of Nur77 overexpression was examined. Importantly, Nur77 overexpression inhibited the oxLDL-induced decrease of cell viability, inhibited the production of apoptotic bodies and restored DNA synthesis following oxLDL exposure. Furthermore, we found that Nur77 induction is mediated through the p38 MAPK signaling pathway. After pretreatment with SB203580, cell viability was decreased, the expression of CyclinA2 and PCNA was attenuated and the percentage of cell apoptosis was enhanced. Likewise, Nur77 overexpression increased the expression of the cell cycle genes PCNA and p21, and attenuated the increase in caspase-3. On the other hand, knockdown of Nur77 expression by specific siRNA resulted in the increased expression of caspase 3. The results demonstrate that Nur77 is induced by oxLDL via the p38 MAPK signaling pathway, which is involved in the regulation of cell survival. Nur77 enhanced cell survival via suppressing apoptosis, without affecting cell proliferation of activated macrophages, which may be beneficial in patients with atherosclerosis. - Highlights: • oxLDL could induce Nur77

  8. Research Advances on Pathways of Nickel-Induced Apoptosis

    Science.gov (United States)

    Guo, Hongrui; Chen, Lian; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Xun; Wu, Bangyuan

    2015-01-01

    High concentrations of nickel (Ni) are harmful to humans and animals. Ni targets a number of organs and produces multiple toxic effects. Apoptosis is important in Ni-induced toxicity of the kidneys, liver, nerves, and immune system. Apoptotic pathways mediated by reactive oxygen species (ROS), mitochondria, endoplasmic reticulum (ER), Fas, and c-Myc participate in Ni-induced cell apoptosis. However, the exact mechanism of apoptosis caused by Ni is still unclear. Understanding the mechanism of Ni-induced apoptosis may help in designing measures to prevent Ni toxicity. PMID:26703593

  9. Visualizing Vpr-induced G2 arrest and apoptosis.

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    Tomoyuki Murakami

    Full Text Available Vpr is an accessory protein of human immunodeficiency virus type 1 (HIV-1 with multiple functions. The induction of G2 arrest by Vpr plays a particularly important role in efficient viral replication because the transcriptional activity of the HIV-1 long terminal repeat is most active in G2 phase. The regulation of apoptosis by Vpr is also important for immune suppression and pathogenesis during HIV infection. However, it is not known whether Vpr-induced apoptosis depends on the ability of Vpr to induce G2 arrest, and the dynamics of Vpr-induced G2 arrest and apoptosis have not been visualized. We performed time-lapse imaging to examine the temporal relationship between Vpr-induced G2 arrest and apoptosis using HeLa cells containing the fluorescent ubiquitination-based cell cycle indicator2 (Fucci2. The dynamics of G2 arrest and subsequent long-term mitotic cell rounding in cells transfected with the Vpr-expression vector were visualized. These cells underwent nuclear mis-segregation after prolonged mitotic processes and then entered G1 phase. Some cells subsequently displayed evidence of apoptosis after prolonged mitotic processes and nuclear mis-segregation. Interestingly, Vpr-induced apoptosis was seldom observed in S or G2 phase. Likewise, visualization of synchronized HeLa/Fucci2 cells infected with an adenoviral vector expressing Vpr clearly showed that Vpr arrests the cell cycle at G2 phase, but does not induce apoptosis at S or G2 phase. Furthermore, time-lapse imaging of HeLa/Fucci2 cells expressing SCAT3.1, a caspase-3-sensitive fusion protein, clearly demonstrated that Vpr induces caspase-3-dependent apoptosis. Finally, to examine whether the effects of Vpr on G2 arrest and apoptosis were reversible, we performed live-cell imaging of a destabilizing domain fusion Vpr, which enabled rapid stabilization and destabilization by Shield1. The effects of Vpr on G2 arrest and subsequent apoptosis were reversible. This study is the first to

  10. Effective gene silencing activity of prodrug-type 2'-O-methyldithiomethyl siRNA compared with non-prodrug-type 2'-O-methyl siRNA.

    Science.gov (United States)

    Hayashi, Junsuke; Nishigaki, Misa; Ochi, Yosuke; Wada, Shun-Ichi; Wada, Fumito; Nakagawa, Osamu; Obika, Satoshi; Harada-Shiba, Mariko; Urata, Hidehito

    2018-07-01

    Small interfering RNAs (siRNAs) are an active agent to induce gene silencing and they have been studied for becoming a biological and therapeutic tool. Various 2'-O-modified RNAs have been extensively studied to improve the nuclease resistance. However, the 2'-O-modified siRNA activities were often decreased by modification, since the bulky 2'-O-modifications inhibit to form a RNA-induced silencing complex (RISC). We developed novel prodrug-type 2'-O-methyldithiomethyl (MDTM) siRNA, which is converted into natural siRNA in an intracellular reducing environment. Prodrug-type 2'-O-MDTM siRNAs modified at the 5'-end side including 5'-end nucleotide and the seed region of the antisense strand exhibited much stronger gene silencing effect than non-prodrug-type 2'-O-methyl (2'-O-Me) siRNAs. Furthermore, the resistances for nuclease digestion of siRNAs were actually enhanced by 2'-O-MDTM modifications. Our results indicate that 2'-O-MDTM modifications improve the stability of siRNA in serum and they are able to be introduced at any positions of siRNA. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Chaetocin induces endoplasmic reticulum stress response and leads to death receptor 5-dependent apoptosis in human non-small cell lung cancer cells.

    Science.gov (United States)

    Liu, Xianfang; Guo, Sen; Liu, Xiangguo; Su, Ling

    2015-11-01

    Epigenetic abnormalities are associated with non-small cell lung cancer (NSCLC) initiation and progression. Epigenetic drugs are being studied and in clinical trials. However, the molecular mechanism underlying the apoptosis by the epigenetic agents remains unclear. SUV39H1 is an important methyl-transferase for lysine 9 on histone H3 and usually related to gene transcriptional suppression, and chaetocin acts as the inhibitor of SUV39H1. We demonstrated here that chaetocin effectively suppressed the growth of multiple lung cancer cells through inducing apoptosis in a death receptor 5 (DR5)-dependent manner. Chaetocin treatment activated endoplasmic reticulum (ER) stress which gave rise to the up-regulation of ATF3 and CHOP. Furthermore, ATF3 and CHOP contributed to the induction of DR5 and subsequent apoptosis. When SUV39H1 was silenced with siRNA, the expression of ATF3, CHOP and DR5 was elevated. Thereafter, knockdown of SUV39H1 induced apoptosis in NSCLC cells. In summary, chaetocin pharmacologically inhibits the activity of SUV39H1 which provokes ER stress and results in up-regulation of ATF3 and CHOP, leading to DR5-dependent apoptosis eventually. These findings provide a novel interpretation on the anti-neoplastic activity of epigenetic drugs as a new therapeutic approach in NSCLC.

  12. Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation.

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    Jiateng Zhong

    Full Text Available CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER, nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.

  13. 18α-Glycyrrhetinic acid lethality for neuroblastoma cells via de-regulating the Beclin-1/Bcl-2 complex and inducing apoptosis.

    Science.gov (United States)

    Rahman, Md Ataur; Bishayee, Kausik; Habib, Khadija; Sadra, Ali; Huh, Sung-Oh

    2016-10-01

    18α-Glycyrrhetinic acid (18-GA) is a known gap-junction inhibitor with demonstrated anticancer effects. However, the different modes of cell cytotoxicity for 18-GA remain to be characterized. In this study, 18-GA reduced the expression of cell-cell interaction proteins (N- and VE-cadherin), and led to a dose-dependent increase in cytotoxicity of the neuroblastoma cells tested, but was less toxic toward actively dividing human embryonic kidney cells. We found that 18-GA could induce both autophagy and apoptosis. 18-GA mediated autophagy was due to accumulation of Atg5, Atg7 and LC3II and degradation of p62. Individual siRNAs against Atg5 and Atg7 prevented autophagy and resulted in a further loss of viability with 18-GA. In addition, combination of 18-GA with autophagy inhibitor chloroquine produced a more significant cell death. This implied a pro-survival function for autophagy induction with 18-GA. 18-GA also led to the destabilization of Bcl-2/Beclin-1 interaction and cleavage of Beclin-1, a protein known to play role in apoptosis and autophagy induction. Treatment of cells by a pan-caspase inhibitor or a caspase-3 siRNA prevented a large portion of 18-GA mediated cytotoxicity, demonstrating that caspase-dependent apoptosis induction was responsible for most of the observed cytotoxicity. In terms of signaling, 18-GA led to reduced phosphorylation of all three classes of MAP kinases. Taken together, 18-GA or its pathways may lead to more effective, targeted therapeutics against neuroblastoma. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Ghrelin protects against depleted uranium-induced apoptosis of MC3T3-E1 cells through oxidative stress-mediated p38-mitogen-activated protein kinase pathway

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    Hao, Yuhui; Liu, Cong; Huang, Jiawei; Gu, Ying; Li, Hong; Yang, Zhangyou; Liu, Jing [State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, No. 30 Gaotanyan Street, Shapingba District, Chongqing 400038 (China); Wang, Weidong, E-mail: wwdwyl@sina.com [Department of Radiation Oncology, Shanghai Jiao Tong University Affiliated Sixth People Hospital, Shanghai 200233 (China); Li, Rong, E-mail: yuhui_hao@126.com [State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, No. 30 Gaotanyan Street, Shapingba District, Chongqing 400038 (China)

    2016-01-01

    Depleted uranium (DU) mainly accumulates in the bone over the long term. Osteoblast cells are responsible for the formation of bone, and they are sensitive to DU damage. However, studies investigating methods of reducing DU damage in osteoblasts are rarely reported. Ghrelin is a stomach hormone that stimulates growth hormones released from the hypothalamic–pituitary axis, and it is believed to play an important physiological role in bone metabolism. This study evaluates the impact of ghrelin on DU-induced apoptosis of the osteoblast MC3T3-E1 and investigates its underlying mechanisms. The results show that ghrelin relieved the intracellular oxidative stress induced by DU, eliminated reactive oxygen species (ROS) and reduced lipid peroxidation by increasing intracellular GSH levels; in addition, ghrelin effectively suppressed apoptosis, enhanced mitochondrial membrane potential, and inhibited cytochrome c release and caspase-3 activation after DU exposure. Moreover, ghrelin significantly reduced the expression of DU-induced phosphorylated p38-mitogen-activated protein kinase (MAPK). A specific inhibitor (SB203580) or specific siRNA of p38-MAPK could significantly suppress DU-induced apoptosis and related signals, whereas ROS production was not affected. In addition, ghrelin receptor inhibition could reduce the anti-apoptosis effect of ghrelin on DU and reverse the effect of ghrelin on intracellular ROS and p38-MAPK after DU exposure. These results suggest that ghrelin can suppress DU-induced apoptosis of MC3T3-E1 cells, reduce DU-induced oxidative stress by interacting with its receptor, and inhibit downstream p38-MAPK activation, thereby suppressing the mitochondrial-dependent apoptosis pathway. - Highlights: • Ghrelin suppressed DU-induced apoptosis of MC3T3-E1 cells. • Ghrelin inhibited DU-induced oxidative stress and further p38-MAPK activation. • Ghrelin further suppressed mitochondrial-dependent apoptosis pathway. • The anti-oxidation effect of

  15. Autotaxin is induced by TSA through HDAC3 and HDAC7 inhibition and antagonizes the TSA-induced cell apoptosis.

    Science.gov (United States)

    Li, Song; Wang, Baolu; Xu, Yan; Zhang, Junjie

    2011-02-12

    Autotaxin (ATX) is a secreted glycoprotein with the lysophospholipase D (lysoPLD) activity to convert lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a bioactive lysophospholipid involved in diverse biological actions. ATX is highly expressed in some cancer cells and contributes to their tumorigenesis, invasion, and metastases, while in other cancer cells ATX is silenced or expressed at low level. The mechanism of ATX expression regulation in cancer cells remains largely unknown. In the present study, we demonstrated that trichostatin A (TSA), a well-known HDAC inhibitor (HDACi), significantly induced ATX expression in SW480 and several other cancer cells with low or undetectable endogenous ATX expression. ATX induction could be observed when HDAC3 and HDAC7 were down-regulated by their siRNAs. It was found that HDAC7 expression levels were low in the cancer cells with high endogenous ATX expression. Exogenous over-expression of HDAC7 inhibited ATX expression in these cells in a HDAC3-dependent manner. These data indicate that HDAC3 and HDAC7 collaboratively suppress ATX expression in cancer cells, and suggest that TSA induce ATX expression by inhibiting HDAC3 and HDAC7. The biological significance of this regulation mechanism was revealed by demonstrating that TSA-induced ATX protected cancer cells against TSA-induced apoptosis by producing LPA through its lysoPLD activity, which could be reversed by BrP-LPA and S32826, the inhibitors of the ATX-LPA axis. We have demonstrated that ATX expression is repressed by HDAC3 and HDAC7 in cancer cells. During TSA treatment, ATX is induced due to the HDAC3 and HDAC7 inhibition and functionally antagonizes the TSA-induced apoptosis. These results reveal an internal HDACi-resistant mechanism in cancer cells, and suggest that the inhibition of ATX-LPA axis would be helpful to improve the efficacy of HDACi-based therapeutics against cancer.

  16. Characterization of radiation-induced Apoptosis in rodent cell lines

    International Nuclear Information System (INIS)

    Guo, Min; Chen, Changhu; Ling, C.C.

    1997-01-01

    For REC:myc(ch1), Rat1 and Rat1:myc b cells, we determined the events in the development of radiation-induced apoptosis to be in the following order: cell division followed by chromatin condensation, membrane blebbing, loss of adhesion and the uptake of vital dye. Experimental data which were obtained using 4 He ions of well defined energies and which compared the dependence of apoptosis and clonogenic survival on 4 He range strongly suggested that in our cells both apoptosis and loss of clonogenic survival resulted from radiation damage to the cell nucleus. Corroboratory evidence was that BrdU incorporation sensitized these cells to radiation-induced apoptosis. Comparing the dose response for apoptosis and the clonogenic survival curves for Rat1 and Rat1:myc b cells, we concluded that radiation-induced cell inactivation as assayed by clonogenic survival, and that a modified linear-quadratic model, proposed previously, modeled such a contribution effectively. In the same context, the selective increase in radiation-induced apoptosis. Comparing the dose response for apoptosis and the clonogenic survival curves for Rat1 and Rat1:myc b cells, we concluded that radiation-induced apoptosis contributed to the overall radiation-induced cell inactivation as assayed by clonogenic survival, and that a modified linear-quadratic model, proposed previously, modeled such a contribution effectively. In the same context, the selective increase in radiation-induced apoptosis during late S and G 2 phases reduced the relative radioresistance observed for clonogenic survival during late S and G 2 phases. 30 refs., 8 figs

  17. Effect of pH on radiation-induced apoptosis

    International Nuclear Information System (INIS)

    Chang, W. Song; Park, Heon J.; Lyons, John C.; Auger, Elizabeth A.; Lee, Hyung-Sik

    1996-01-01

    Purpose/Objective: The effect of environmental pH on the radiation-induced apoptosis in tumor cells in vitro was investigated. Materials and Methods: SCK mammary adenocarcinoma cells of A/J mice were irradiated with γ-rays using a 137 Cs irradiator and incubated in media of different pHs. After incubation at 37 degree sign C for 24-120 hrs., the extent of apoptosis was determined using agarose gel electrophoresis of DNA, in situ TUNEL staining, flow cytometry, and release of 3 H from 3 H-thymidine labeled cells. The membrane integrity, using the trypan blue exclusion method, and the clonogenicity of the cells were also determined. Results: Irradiation with 2-12 Gy of γ-rays induced apoptosis in pH 7.5 medium within 48 hrs. The radiation-induced apoptosis progressively declined as the medium pH was lowered so that little apoptosis occurred in 48 hrs. after irradiation with 12 Gy in pH 6.6 medium. However, when the cells were irradiated and incubated for 48 hrs. in pH 6.6 medium and then medium was replaced with pH 7.5 medium, apoptosis promptly occurred. Apoptosis also occurred even in pH 6.6 medium when the cells were irradiated and maintained in pH 7.5 medium for 8 hrs. or longer post-irradiation before incubation in pH 6.6 medium. Conclusion: An acidic environment markedly suppresses radiation-induced apoptosis probably by suppressing the expression of initial signals responsible for irradiation-induced apoptosis. Indications are that the signals persist in an acidic environment and trigger apoptosis when the environmental acidity is eased. Our results suggest that the acidic environment in human tumors may inhibit the apoptosis after irradiation. However, apoptosis may be triggered when reoxygenation occurs after irradiation, and thus, the intratumor environment becomes less acidic after irradiation. Not only the change in pO 2 but the change in pH during the course of fractionated radiotherapy may greatly influence the outcome of the treatment

  18. COX-2 and PPAR-γ confer cannabidiol-induced apoptosis of human lung cancer cells.

    Science.gov (United States)

    Ramer, Robert; Heinemann, Katharina; Merkord, Jutta; Rohde, Helga; Salamon, Achim; Linnebacher, Michael; Hinz, Burkhard

    2013-01-01

    The antitumorigenic mechanism of cannabidiol is still controversial. This study investigates the role of COX-2 and PPAR-γ in cannabidiol's proapoptotic and tumor-regressive action. In lung cancer cell lines (A549, H460) and primary cells from a patient with lung cancer, cannabidiol elicited decreased viability associated with apoptosis. Apoptotic cell death by cannabidiol was suppressed by NS-398 (COX-2 inhibitor), GW9662 (PPAR-γ antagonist), and siRNA targeting COX-2 and PPAR-γ. Cannabidiol-induced apoptosis was paralleled by upregulation of COX-2 and PPAR-γ mRNA and protein expression with a maximum induction of COX-2 mRNA after 8 hours and continuous increases of PPAR-γ mRNA when compared with vehicle. In response to cannabidiol, tumor cell lines exhibited increased levels of COX-2-dependent prostaglandins (PG) among which PGD(2) and 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) caused a translocation of PPAR-γ to the nucleus and induced a PPAR-γ-dependent apoptotic cell death. Moreover, in A549-xenografted nude mice, cannabidiol caused upregulation of COX-2 and PPAR-γ in tumor tissue and tumor regression that was reversible by GW9662. Together, our data show a novel proapoptotic mechanism of cannabidiol involving initial upregulation of COX-2 and PPAR-γ and a subsequent nuclear translocation of PPAR-γ by COX-2-dependent PGs.

  19. MCPIP1 Regulates Alveolar Macrophage Apoptosis and Pulmonary Fibroblast Activation After in vitro Exposure to Silica.

    Science.gov (United States)

    Wang, Xingang; Zhang, Yuxia; Zhang, Wei; Liu, Haijun; Zhou, Zewei; Dai, Xiaoniu; Cheng, Yusi; Fang, Shencun; Zhang, Yingming; Yao, Honghong; Chao, Jie

    2016-05-01

    Silicosis is a fatal and fibrotic pulmonary disease caused by the inhalation of silica. After arriving at the alveoli, silica is ingested by alveolar macrophages (AMOs), in which monocyte chemotactic protein-induced protein 1 (MCPIP1) plays an essential role in controlling macrophage-mediated inflammatory responses. However, the mechanism of action of MCPIP1 in silicosis is poorly understood. Primary rat AMOs were isolated and treated with SiO2 (50 µg/cm(2)). MCPIP1 and AMO activation/apoptosis markers were detected by immunoblotting. MCPIP1 was down-regulated using siRNA in AMOs. The effects of AMOs on fibroblast activation and migration were evaluated using a gel contraction assay, a scratch assay, and a nested collagen matrix migration model. After exposure to SiO2, MCPIP1 was significantly increased in rat AMOs. Activation and apoptosis markers in AMOs were up-regulated after exposure to SiO2 Following siRNA-mediated silencing of MCPIP1 mRNA, the markers of AMO activation and apoptosis were significantly decreased. Rat pulmonary fibroblasts (PFBs) cultured in conditional medium from AMOs treated with MCPIP1 siRNA and SiO2 showed significantly less activation and migration compared with those cultured in conditional medium from AMOs treated with control siRNA and SiO2 CONCLUSION: Our data suggest a vital role for MCPIP1 in AMO apoptosis and PFB activation/migration induced by SiO2. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. Aspartame-induced apoptosis in PC12 cells

    OpenAIRE

    Horio, Yukari; Sun, Yongkun; Liu, Chuang; Saito, Takeshi; Kurasaki, Masaaki

    2014-01-01

    Aspartame is an artificial sweetner added to many low-calorie foods. The safety of aspartame remains controversial even though there are many studies on its risks. In this study, to understand the physiological effects of trace amounts of artificial sweetners on cells, the effects of aspartame on apoptosis were investigated using a PC12 cell system. In addition, the mechanism of apoptosis induced by aspartame in PC12 cells and effects on apoptotic factors such as cytochrome c, apoptosis-induc...

  1. siRNA and innate immunity.

    Science.gov (United States)

    Robbins, Marjorie; Judge, Adam; MacLachlan, Ian

    2009-06-01

    Canonical small interfering RNA (siRNA) duplexes are potent activators of the mammalian innate immune system. The induction of innate immunity by siRNA is dependent on siRNA structure and sequence, method of delivery, and cell type. Synthetic siRNA in delivery vehicles that facilitate cellular uptake can induce high levels of inflammatory cytokines and interferons after systemic administration in mammals and in primary human blood cell cultures. This activation is predominantly mediated by immune cells, normally via a Toll-like receptor (TLR) pathway. The siRNA sequence dependency of these pathways varies with the type and location of the TLR involved. Alternatively nonimmune cell activation may also occur, typically resulting from siRNA interaction with cytoplasmic RNA sensors such as RIG1. As immune activation by siRNA-based drugs represents an undesirable side effect due to the considerable toxicities associated with excessive cytokine release in humans, understanding and abrogating this activity will be a critical component in the development of safe and effective therapeutics. This review describes the intracellular mechanisms of innate immune activation by siRNA, the design of appropriate sequences and chemical modification approaches, and suitable experimental methods for studying their effects, with a view toward reducing siRNA-mediated off-target effects.

  2. Lithium protects ethanol-induced neuronal apoptosis

    International Nuclear Information System (INIS)

    Zhong Jin; Yang Xianlin; Yao Weiguo; Lee Weihua

    2006-01-01

    Lithium is widely used for the treatment of bipolar disorder. Recent studies have demonstrated its neuroprotective effect. Ethanol is a potent neurotoxin that is particularly harmful to the developing nervous system. In this study, we evaluated lithium's neuroprotection against ethanol-induced apoptosis. Transient exposure of infant mice to ethanol caused apoptotic cell death in brain, which was prevented significantly by administering a low dose of lithium 15 min later. In cultured cerebellar granule neurons, ethanol-induced apoptosis and activation of caspase-3/9, both of which were prevented by lithium. However, lithium's protection is not mediated by its commonly known inhibition of glycogen synthase3β, because neither ethanol nor lithium has significant effects on the phosphorylation of Akt (ser473) or GSK3β (ser9). In addition, the selective GSK-3β inhibitor SB-415286 was unable to prevent ethanol-induced apoptosis. These data suggest lithium may be used as a potential preventive measure for ethanol-induced neurological deficits

  3. Paricalcitol attenuates lipopolysaccharide-induced inflammation and apoptosis in proximal tubular cells through the prostaglandin E₂ receptor EP4

    Directory of Open Access Journals (Sweden)

    Yu Ah Hong

    2017-06-01

    Full Text Available Background: Vitamin D is considered to exert a protective effect on various renal diseases but its underlying molecular mechanism remains poorly understood. This study aimed to determine whether paricalcitol attenuates inflammation and apoptosis during lipopolysaccharide (LPS-induced renal proximal tubular cell injury through the prostaglandin E₂ (PGE₂ receptor EP4. Methods: Human renal tubular epithelial (HK-2 cells were pretreated with paricalcitol (2 ng/mL for 1 hour and exposed to LPS (1 μg/mL. The effects of paricalcitol pretreatment in relation to an EP4 blockade using AH-23848 or EP4 small interfering RNA (siRNA were investigated. Results: The expression of cyclooxygenase-2, PGE₂, and EP4 were significantly increased in LPS-exposed HK-2 cells treated with paricalcitol compared with cells exposed to LPS only. Paricalcitol prevented cell death induced by LPS exposure, and the cotreatment of AH-23848 or EP4 siRNA offset these cell-protective effects. The phosphorylation and nuclear translocation of p65 nuclear factor-kappaB (NF-κB were decreased and the phosphorylation of Akt was increased in LPS-exposed cells with paricalcitol treatment. AH-23848 or EP4 siRNA inhibited the suppressive effects of paricalcitol on p65 NF-κB nuclear translocation and the activation of Akt. The production of proinflammatory cytokines and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were attenuated by paricalcitol in LPS exposed HK-2 cells. The cotreatment with an EP4 antagonist abolished these anti-inflammatory and antiapoptotic effects. Conclusion: EP4 plays a pivotal role in anti-inflammatory and antiapoptotic effects through Akt and NF-κB signaling after paricalcitol pretreatment in LPS-induced renal proximal tubule cell injury.

  4. The ROS/NF-κB/NR4A2 pathway is involved in H2O2 induced apoptosis of resident cardiac stem cells via autophagy.

    Science.gov (United States)

    Shi, Xingxing; Li, Wenjing; Liu, Honghong; Yin, Deling; Zhao, Jing

    2017-09-29

    Cardiac stem cells (CSCs)-based therapy provides a promising avenue for the management of ischemic heart diseases. However, engrafted CSCs are subjected to acute cell apoptosis in the ischemic microenvironment. Here, stem cell antigen 1 positive (Sca-1 + ) CSCs proved to own therapy potential were cultured and treated with H 2 O 2 to mimic the ischemia situation. As autophagy inhibitor, 3-methyladenine (3MA), inhibited H 2 O 2 -induced CSCs apoptosis, thus we demonstrated that H 2 O 2 induced autophagy-dependent apoptosis in CSCs, and continued to find key proteins responsible for the crosstalk between autophagy and apoptosis. Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2), increased upon cardiomyocyte injury with unknown functions in CSCs, was increased by H 2 O 2 . NR4A2 siRNA attenuated H 2 O 2 induced autophagy and apoptosis in CSCs, which suggested an important role of NR4A2 in CSCs survival in ischemia conditions. Reactive oxygen species (ROS) and NF-κB (P65) subunit were both increased by H 2 O 2 . Either the ROS scavenger, N-acetyl-l-cysteine (NAC) or NF-κB signaling inhibitor, bay11-7082 could attenuate H 2 O 2 -induced autophagy and apoptosis in CSCs, which suggested they were involved in this process. Furthermore, NAC inhibited NF-κB activities, while bay11-7082 inhibited NR4A2 expression, which revealed a ROS/NF-κB/NR4A2 pathway responsible for H 2 O 2 -induced autophagy and apoptosis in CSCs. Our study supports a new clue enhancing the survival rate of CSCs in the infarcted myocardium for cell therapy in ischemic cardiomyopathy.

  5. MDR1 siRNA loaded hyaluronic acid-based CD44 targeted nanoparticle systems circumvent paclitaxel resistance in ovarian cancer

    Science.gov (United States)

    Yang, Xiaoqian; Lyer, Arun K.; Singh, Amit; Choy, Edwin; Hornicek, Francis J.; Amiji, Mansoor M.; Duan, Zhenfeng

    2015-02-01

    Development of multidrug resistance (MDR) is an almost universal phenomenon in patients with ovarian cancer, and this severely limits the ultimate success of chemotherapy in the clinic. Overexpression of the MDR1 gene and corresponding P-glycoprotein (Pgp) is one of the best known MDR mechanisms. MDR1 siRNA based strategies were proposed to circumvent MDR, however, systemic, safe, and effective targeted delivery is still a major challenge. Cluster of differentiation 44 (CD44) targeted hyaluronic acid (HA) based nanoparticle has been shown to successfully deliver chemotherapy agents or siRNAs into tumor cells. The goal of this study is to evaluate the ability of HA-PEI/HA-PEG to deliver MDR1 siRNA and the efficacy of the combination of HA-PEI/HA-PEG/MDR1 siRNA with paclitaxel to suppress growth of ovarian cancer. We observed that HA-PEI/HA-PEG nanoparticles can efficiently deliver MDR1 siRNA into MDR ovarian cancer cells, resulting in down-regulation of MDR1 and Pgp expression. Administration of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles followed by paclitaxel treatment induced a significant inhibitory effect on the tumor growth, decreased Pgp expression and increased apoptosis in MDR ovarian cancer mice model. Our findings suggest that CD44 targeted HA-PEI/HA-PEG/MDR1 siRNA nanoparticles can serve as a therapeutic tool with great potentials to circumvent MDR in ovarian cancer.

  6. Involvement of Nrf2-Mediated Upregulation of Heme Oxygenase-1 in Mollugin-Induced Growth Inhibition and Apoptosis in Human Oral Cancer Cells

    Directory of Open Access Journals (Sweden)

    Young-Man Lee

    2013-01-01

    Full Text Available Although previous studies have shown that mollugin, a bioactive phytochemical isolated from Rubia cordifolia L. (Rubiaceae, exhibits antitumor effects, its biological activity in oral cancer has not been reported. We thus investigated the effects and putative mechanism of apoptosis induced by mollugin in human oral squamous cell carcinoma cells (OSCCs. Results show that mollugin induces cell death in a dose-dependent manner in primary and metastatic OSCCs. Mollugin-induced cell death involved apoptosis, characterized by the appearance of nuclear shrinkage, flow cytometric analysis of sub-G1 phase arrest, and annexin V-FITC and propidium iodide staining. Western blot analysis and RT-PCR revealed that mollugin suppressed activation of NF-κB and NF-κB-dependent gene products involved in antiapoptosis (Bcl-2 and Bcl-xl, invasion (MMP-9 and ICAM-1, and angiogenesis (FGF-2 and VEGF. Furthermore, mollugin induced the activation of p38, ERK, and JNK and the expression of heme oxygenase-1 (HO-1 and nuclear factor E2–related factor 2 (Nrf2. Mollugin-induced growth inhibition and apoptosis of HO-1 were reversed by an HO-1 inhibitor and Nrf2 siRNA. Collectively, this is the first report to demonstrate the effectiveness of mollugin as a candidate for a chemotherapeutic agent in OSCCs via the upregulation of the HO-1 and Nrf2 pathways and the downregulation of NF-κB.

  7. Gene silencing of HPV16 E6/E7 induced by promoter-targeting siRNA in SiHa cells

    OpenAIRE

    Hong, D; Lu, W; Ye, F; Hu, Y; Xie, X

    2009-01-01

    Background: Recently, transcriptional gene silencing induced by small interfering RNA (siRNA) was found in mammalian and human cells. However, previous studies focused on endogenous genes. Methods: In this study, we designed siRNA targeting the promoter of human papillomavirus 16 E6/E7 and transfected it into the cervical cancer cell line, SiHa. E6 and E7 mRNA and protein expression were detected in cells treated by promoter-targeting siRNA. Futhermore, cellular growth, proliferation, apoptos...

  8. Tetramethylpyrazine suppresses transient oxygen-glucose deprivation-induced connexin32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathway in cultured hippocampal neurons.

    Science.gov (United States)

    Gong, Gu; Yuan, Libang; Cai, Lin; Ran, Maorong; Zhang, Yulan; Gong, Huaqu; Dai, Xuemei; Wu, Wei; Dong, Hailong

    2014-01-01

    Tetramethylpyrazine (TMP) has been widely used in China as a drug for the treatment of various diseases. Recent studies have suggested that TMP has a protective effect on ischemic neuronal damage. However, the exact mechanism is still unclear. This study aims to investigate the mechanism of TMP mediated ischemic hippocampal neurons injury induced by oxygen-glucose deprivation (OGD). The effect of TMP on hippocampal neurons viability was detected by MTT assay, LDH release assay and apoptosis rate was measured by flow cytometry. TMP significantly suppressed neuron apoptosis in a concentration-dependent manner. TMP could significantly reduce the elevated levels of connexin32 (Cx32) induced by OGD. Knockdown of Cx32 by siRNA attenuated OGD injury. Moreover, our study showed that viability was increased in siRNA-Cx32-treated-neurons, and neuron apoptosis was suppressed by activating Bcl-2 expression and inhibiting Bax expression. Over expression of Cx32 could decrease neurons viability and increase LDH release. Furthermore, OGD increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the neuron injury and Cx32 up-regulation. Taken together, TMP can reverse the OGD-induced Cx32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathways.

  9. Tetramethylpyrazine suppresses transient oxygen-glucose deprivation-induced connexin32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathway in cultured hippocampal neurons.

    Directory of Open Access Journals (Sweden)

    Gu Gong

    Full Text Available Tetramethylpyrazine (TMP has been widely used in China as a drug for the treatment of various diseases. Recent studies have suggested that TMP has a protective effect on ischemic neuronal damage. However, the exact mechanism is still unclear. This study aims to investigate the mechanism of TMP mediated ischemic hippocampal neurons injury induced by oxygen-glucose deprivation (OGD. The effect of TMP on hippocampal neurons viability was detected by MTT assay, LDH release assay and apoptosis rate was measured by flow cytometry. TMP significantly suppressed neuron apoptosis in a concentration-dependent manner. TMP could significantly reduce the elevated levels of connexin32 (Cx32 induced by OGD. Knockdown of Cx32 by siRNA attenuated OGD injury. Moreover, our study showed that viability was increased in siRNA-Cx32-treated-neurons, and neuron apoptosis was suppressed by activating Bcl-2 expression and inhibiting Bax expression. Over expression of Cx32 could decrease neurons viability and increase LDH release. Furthermore, OGD increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the neuron injury and Cx32 up-regulation. Taken together, TMP can reverse the OGD-induced Cx32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathways.

  10. siRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases.

    Science.gov (United States)

    Rebelo, Thalia M; Vania, Leila; Ferreira, Eloise; Weiss, Stefan F T

    2018-07-01

    The 37 kDa/67 kDa laminin receptor (LRP/LR) is over-expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell-surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down-regulated via siRNA technology. MTT assays revealed that LRP knock-down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin-V FITC/PI assays confirmed this observation. Additionally, caspase-3 activity assays revealed that apoptosis was induced in both cell lines after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knock-down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway. siRNAs mediated LRP knock-down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis. Copyright © 2018. Published by Elsevier Inc.

  11. Hyperthermia: an effective strategy to induce apoptosis in cancer cells.

    Science.gov (United States)

    Ahmed, Kanwal; Tabuchi, Yoshiaki; Kondo, Takashi

    2015-11-01

    Heat has been used as a medicinal and healing modality throughout human history. The combination of hyperthermia (HT) with radiation and anticancer agents has been used clinically and has shown positive results to a certain extent. However, the clinical results of HT treatment alone have been only partially satisfactory. Cell death following HT treatment is a function of both temperature and treatment duration. HT induces cancer cell death through apoptosis; the degree of apoptosis and the apoptotic pathway vary in different cancer cell types. HT-induced reactive oxygen species production are responsible for apoptosis in various cell types. However, the underlying mechanism of signal transduction and the genes related to this process still need to be elucidated. In this review, we summarize the molecular mechanism of apoptosis induced by HT, enhancement of heat-induced apoptosis, and the genetic network involved in HT-induced apoptosis.

  12. Gene expression profiling associated with angiotensin II type 2 receptor-induced apoptosis in human prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Nana Pei

    Full Text Available Increased expression of angiotensin II type 2 receptor (AT2R induces apoptosis in numerous tumor cell lines, with either Angiotensin II-dependent or Angiotensin II-independent regulation, but its molecular mechanism remains poorly understood. Here, we used PCR Array analysis to determine the gene and microRNA expression profiles in human prostate cancer cell lines transduced with AT2R recombinant adenovirus. Our results demonstrated that AT2R over expression leads to up-regulation of 6 apoptosis-related genes (TRAIL-R2, BAG3, BNIPI, HRK, Gadd45a, TP53BP2, 2 cytokine genes (IL6 and IL8 and 1 microRNA, and down-regulation of 1 apoptosis-related gene TNFSF10 and 2 cytokine genes (BMP6, BMP7 in transduced DU145 cells. HRK was identified as an up-regulated gene in AT2R-transduced PC-3 cells by real-time RT-PCR. Next, we utilized siRNAs to silence the up-regulated genes to further determine their roles on AT2R overexpression mediated apoptosis. The results showed downregulation of Gadd45a reduced the apoptotic effect by ∼30% in DU145 cells, downregulation of HRK reduced AT2R-mediated apoptosis by more than 50% in PC-3 cells, while downregulation of TRAIL-R2 enhanced AT2R-mediated apoptosis more than 4 times in DU145 cells. We also found that the effects on AT2R-mediated apoptosis caused by downregulation of Gadd45a, TRAIL-R2 and HRK were independent in activation of p38 MAPK, p44/42 MAPK and p53. Taken together, our results demonstrated that TRAIL-R2, Gadd45a and HRK may be novel target genes for further study of the mechanism of AT2R-mediated apoptosis in prostate cancer cells.

  13. 3,3'-diindolylmethane potentiates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of gastric cancer cells.

    Science.gov (United States)

    Ye, Yang; Miao, Shuhan; Wang, Yan; Zhou, Jianwei; Lu, Rongzhu

    2015-05-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) specifically kills cancer cells without destroying the majority of healthy cells. However, numerous types of cancer cell, including gastric cancer cells, tend to be resistant to TRAIL. The bioactive product 3,3'-diindolylmethane (DIM), which is derived from cruciferous vegetables, is also currently recognized as a candidate anticancer agent. In the present study, a Cell Counting Kit 8 cell growth assay and an Annexin V-fluorescein isothiocyanate apoptosis assay were performed to investigate the potentiating effect of DIM on TRAIL-induced apoptosis in gastric cancer cells, and the possible mechanisms of this potentiation. The results obtained demonstrated that, compared with TRAIL or DIM treatment alone, co-treatment with TRAIL (25 or 50 ng/ml) and DIM (10 µmol/l) induced cytotoxic and apoptotic effects in BGC-823 and SGC-7901 gastric cancer cells. Furthermore, western blot analysis revealed that the protein expression levels of death receptor 5 (DR5), CCAAT/enhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) were upregulated in the co-treated gastric cancer cells. To the best of our knowledge, the present study is the first to provide evidence that DIM sensitizes TRAIL-induced inhibition of proliferation and apoptosis in gastric cancer cells, accompanied by the upregulated expression of DR5, CHOP and GRP78 proteins, which may be involved in endoplasmic reticulum stress mechanisms.

  14. Evaluation of a curcumin analog as an anti-cancer agent inducing ER stress-mediated apoptosis in non-small cell lung cancer cells

    International Nuclear Information System (INIS)

    Liu, Zhiguo; Wang, Yi; Sun, Yusheng; Ren, Luqing; Huang, Yi; Cai, Yuepiao; Weng, Qiaoyou; Shen, Xueqian; Li, Xiaokun; Liang, Guang

    2013-01-01

    Recent advances have highlighted the importance of the endoplasmic reticulum (ER) in cell death processes. Pharmacological interventions that effectively enhance tumor cell death through activating ER stress have attracted a great deal of attention for anti-cancer therapy. A bio-evaluation on 113 curcumin analogs against four cancer cell lines was performed through MTT assay. Furthermore, real time cell assay and flow cytometer were used to evaluate the apoptotic induction of (1E,4E)-1,5-bis(5-bromo-2-ethoxyphenyl)penta-1,4-dien-3-one (B82). Western blot, RT-qPCR, and siRNA were then utilized to confirm whether B82-induced apoptosis is mediated through activating ER stress pathway. Finally, the in vivo anti-tumor effect of B82 was evaluated. B82 exhibited strong anti-tumor activity in non-small cell lung cancer (NSCLC) H460 cells. Treatment with B82 significantly induced apoptosis in H460 cells in vitro and inhibited H460 tumor growth in vivo. Further studies demonstrated that the B82-induced apoptosis is mediated by activating ER stress both in vitro and in vivo. A new monocarbonyl analog of curcumin, B82, exhibited anti-tumor effects on H460 cells via an ER stress-mediated mechanism. B82 could be further explored as a potential anticancer agent for the treatment of NSCLC

  15. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Pan Shiow-Lin

    2009-05-01

    Full Text Available Abstract In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1 in denbinobin-induced apoptosis in human lung adenocarcinoma (A549 cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN, two antioxidants (N-acetyl-L-cysteine (NAC and glutathione (GSH, a c-Jun N-terminal kinase (JNK inhibitor (SP600125, and an activator protein-1 (AP-1 inhibitor (curcumin. Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

  16. Potentiation of apoptosis by histone deacetylase inhibitors and doxorubicin combination: cytoplasmic cathepsin B as a mediator of apoptosis in multiple myeloma.

    Science.gov (United States)

    Cheriyath, V; Kuhns, M A; Kalaycio, M E; Borden, E C

    2011-03-15

    Although inhibitors of histone deacetylase inhibitors (HDACis) in combination with genotoxins potentiate apoptosis, the role of proteases other than caspases in this process remained elusive. Therefore, we examined the potentiation of apoptosis and related mechanisms of HDACis and doxorubicin combination in a panel of myeloma cell lines and in 25 primary myelomas. At IC(50) concentrations, sodium butyrate (an HDACi) or doxorubicin alone caused little apoptosis. However, their combination potentiated apoptosis and synergistically reduced the viability of myeloma cells independent of p53 and caspase 3-7 activation. Potentiated apoptosis correlated with nuclear translocation of apoptosis-inducing factor, suggesting the induction of caspase 3- and 7-independent pathways. Consistent with this, butyrate and doxorubicin combination significantly increased the activity of cytoplasmic cathepsin B. Inhibition of cathepsin B either with a small-molecule inhibitor or downregulation with a siRNA reversed butyrate- and doxorubicin-potentiated apoptosis. Finally, ex vivo, clinically relevant concentrations of butyrate or SAHA (suberoylanilide hydroxamic acid, vorinostat, an HDACi in clinical testing) in combination with doxorubicin significantly (Pmediating apoptosis potentiated by HDACi and doxorubicin combinations in myeloma. Our results support a molecular model of lysosomal-mitochondrial crosstalk in HDACi- and doxorubicin-potentiated apoptosis through the activation of cathepsin B.

  17. Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme.

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    Ruth Villalonga-Planells

    2011-04-01

    Full Text Available Glioblastoma multiforme (GBM is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.

  18. Efficient inhibition of fibroblast proliferation and collagen expression by ERK2 siRNAs

    International Nuclear Information System (INIS)

    Li, Fengfeng; Fan, Cunyi; Cheng, Tao; Jiang, Chaoyin; Zeng, Bingfang

    2009-01-01

    Transforming growth factor-β1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which ERK2 is supposed to be crucial. Based on these assumptions, lentivirus (LV)-mediated small interfering RNAs (siRNAs) targeting ERK2 were used to suppress the proliferation and collagen expression of rat joint adhesion tissue fibroblasts (RJATFs). Among four siRNAs examined, siRNA1 caused an 84% reduction in ERK2 expression (p < 0.01) and was selected as the most efficient siRNA for use in this study. In subsequent experiments, significant downregulation of types I and III collagen were observed by quantitative RT-PCR and Western blot analyses. MTT assays and flow cytometry revealed marked inhibition of RJATF proliferation, but no apoptosis. In conclusion, LV-mediated ERK2 siRNAs may represent novel therapies or drug targets for preventing joint adhesion formation.

  19. Protein phosphatase 2A mediates JS-K-induced apoptosis by affecting Bcl-2 family proteins in human hepatocellular carcinoma HepG2 cells.

    Science.gov (United States)

    Liu, Ling; Huang, Zile; Chen, Jingjing; Wang, Jiangang; Wang, Shuying

    2018-04-25

    Protein phosphatase 2A (PP2A) is an important enzyme within various signal transduction pathways. The present study was investigated PP2A mediates JS-K-induced apoptosis by affecting Bcl-2 family protein. JS-K showed diverse inhibitory effects in five HCC cell lines, especially HepG2 cells. JS-K caused a dose- and time-dependent reduction in cell viability and increased in levels of LDH release. Meanwhile, JS-K- induced apoptosis was characterized by mitochondrial membrane potential reduction, Hoechst 33342 + /PI + dual staining, release of cytochrome c (Cyt c), and activation of cleaved caspase-9/3. Moreover, JS-K-treatment could lead to the activation of protein phosphatase 2A-C (PP2A-C), decrease of anti-apoptotic Bcl-2 family-protein expression including p-Bcl-2 (Ser70), Bcl-2, Bcl-xL, and Mcl-1 as well as the increase of pro-apoptosis Bcl-2 family-protein including Bim, Bad, Bax, and Bak. Furthermore, JS-K caused a marked increase of intracellular NO levels while pre-treatment with Carboxy-PTIO (a NO scavenger) reduced the cytotoxicity effects and the apoptosis rate. Meanwhile, pre-treatment with Carboxy-PTIO attenuated the JS-K-induced up-regulation of PP2A, Cyt c, and cleaved-caspase-9/3 activation. The silencing PP2A-C by siRNA could abolish the activation of PP2A-C, down-regulation of anti-apoptotic Bcl-2 family-protein (p-Bcl-2, Bcl-2, Bcl-xL, and Mcl-1), increase of pro-apoptosis Bcl-2 family-protein (Bim, Bad, Bax, and Bak) and apoptotic-related protein (Cyt c, cleaved caspase-9/3) that were caused by JS-K in HepG2 cells. In addition, pre-treatment with OA (a PP2A inhibitor) also attenuated the above effects induced by JS-K. In summary, NO release from JS-K induces apoptosis through PP2A activation, which contributed to the regulation of Bcl-2 family proteins. © 2018 Wiley Periodicals, Inc.

  20. The mitochondria-mediate apoptosis of Lepidopteran cells induced by azadirachtin.

    Directory of Open Access Journals (Sweden)

    Jingfei Huang

    Full Text Available Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS generation, activation of mitochondrial permeability transition pores (MPTPs and loss of mitochondrial membrane potential (MMP were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP inhibitor cyclosporin A (CsA, which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis.

  1. The mitochondria-mediate apoptosis of Lepidopteran cells induced by azadirachtin.

    Science.gov (United States)

    Huang, Jingfei; Lv, Chaojun; Hu, Meiying; Zhong, Guohua

    2013-01-01

    Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue) was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS) generation, activation of mitochondrial permeability transition pores (MPTPs) and loss of mitochondrial membrane potential (MMP) were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP) inhibitor cyclosporin A (CsA), which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis.

  2. Andrographolide induces apoptotic and non-apoptotic death and enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in gastric cancer cells.

    Science.gov (United States)

    Lim, Sung-Chul; Jeon, Ho Jong; Kee, Keun Hong; Lee, Mi Ja; Hong, Ran; Han, Song Iy

    2017-05-01

    Andrographolide, a natural compound isolated from Andrographis paniculata , has been reported to possess antitumor activity. In the present study, the effect of andrographolide in human gastric cancer (GC) cells was investigated. Andrographolide induced cell death with apoptotic and non-apoptotic features. At a low concentration, andrographolide potentiated apoptosis and reduction of clonogenicity triggered by recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL). Exposure of GC cells to andrographolide altered the expression level of several growth-inhibiting and apoptosis-regulating proteins, including death receptors. It was demonstrated that activity of the TRAIL-R2 (DR5) pathway was critical in the development of andrographolide-mediated rhTRAIL sensitization, since its inhibition significantly reduced the extent of apoptosis induced by the combination of rhTRAIL and andrographolide. In addition, andrographolide increased reactive oxygen species (ROS) generation in a dose-dependent manner. N-acetyl cysteine prevented andrographolide-mediated DR5 induction and the apoptotic effect induced by the combination of rhTRAIL and andrographolide. Collectively, the present study demonstrated that andrographolide enhances TRAIL-induced apoptosis through induction of DR5 expression. This effect appears to involve ROS generation in GCs.

  3. Overexpression of BAG3 Attenuates Hypoxia-Induced Cardiomyocyte Apoptosis by Inducing Autophagy

    Directory of Open Access Journals (Sweden)

    Jiankai Zhang

    2016-07-01

    Full Text Available Background: Hypoxia is a well-known factor in the promotion of apoptosis, which contributes to the development of numerous cardiac diseases, such as heart failure and myocardial infarction. Inhibiting apoptosis is an important therapeutic strategy for the treatment of related heart diseases caused by ischemia/hypoxic injury. Previous studies have demonstrated that BAG3 plays an important role in cardiomyocyte apoptosis and survival. However, the role of BAG3 in hypoxia-induced cardiomyocyte apoptosis remains to be clarified. Here, we demonstrate that BAG3 is induced by hypoxia stimuli in cultured cardiomyocytes. Methods: BAG3 expression level was measured in H9c2 cells treated with hypoxia for 48 h. Cell proliferation and apoptosis were tested using MTT assay and Annexin V FITC-PI staining assay, respectively. The mRNA or protein expression level of BAG3, LC3-I, LC3-II, Atg5, NF-κB p65 and phosphorylated NF-κB p65 were assessed by qRT-PCR and western blot assay, respectively. Resluts: Overexpression of BAG3 inhibited cell apoptosis and promoted proliferation in hypoxia-injured H9c2 cells. Furthermore, autophagy and NF-κB were activated by BAG3 overexpression, and the NF-κB inhibitor PDTC could inhibit the activation of autophagy induced by BAG3 overexpression. In addition, the autophagy inhibitor 3-MA partly impeded the inhibitory effect of BAG3 on hypoxia-induced cardiomyocyte apoptosis. Conclusion: these results suggested that overexpression of BAG3 promoted cell proliferation and inhibited apoptosis by activating autophagy though the NF-κB signaling pathway in hypoxia-injured cardiomyocytes.

  4. Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

    Science.gov (United States)

    Pijpers, A H; van Setten, P A; van den Heuvel, L P; Assmann, K J; Dijkman, H B; Pennings, A H; Monnens, L A; van Hinsbergh, V W

    2001-04-01

    The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

  5. Hydroxychloroquine-conjugated gold nanoparticles for improved siRNA activity.

    Science.gov (United States)

    Perche, F; Yi, Y; Hespel, L; Mi, P; Dirisala, A; Cabral, H; Miyata, K; Kataoka, K

    2016-06-01

    Current technology of siRNA delivery relies on pharmaceutical dosage forms to route maximal doses of siRNA to the tumor. However, this rationale does not address intracellular bottlenecks governing silencing activity. Here, we tested the impact of hydroxychloroquine conjugation on the intracellular fate and silencing activity of siRNA conjugated PEGylated gold nanoparticles. Addition of hydroxychloroquine improved endosomal escape and increased siRNA guide strand distribution to the RNA induced silencing complex (RISC), both crucial obstacles to the potency of siRNA. This modification significantly improved gene downregulation in cellulo. Altogether, our data suggest the benefit of this modification for the design of improved siRNA delivery systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Fas-induced apoptosis in malnourished infants

    African Journals Online (AJOL)

    EL-HAKIM

    deprivation in animals, including man11. Factor of apoptosis signal (Fas) induces apoptosis in activated T cells when they are repeatedly stimulated by antigen and functions to maintain T cell tolerance by deleting auto reactive cells12. The functional role of Fas (CD95) in the immune system has been examined in a variety ...

  7. Ketamine-induced apoptosis in cultured rat cortical neurons

    International Nuclear Information System (INIS)

    Takadera, Tsuneo; Ishida, Akira; Ohyashiki, Takao

    2006-01-01

    Recent data suggest that anesthetic drugs cause neurodegeneration during development. Ketamine is frequently used in infants and toddlers for elective surgeries. The purpose of this study is to determine whether glycogen synthase kinase-3 (GSK-3) is involved in ketamine-induced apoptosis. Ketamine increased apoptotic cell death with morphological changes which were characterized by cell shrinkage, nuclear condensation or fragmentation. In addition, insulin growth factor-1 completely blocked the ketamine-induced apoptotic cell death. Ketamine decreased Akt phosphorylation. GSK-3 is known as a downstream target of Akt. The selective inhibitors of GSK-3 prevented the ketamine-induced apoptosis. Moreover, caspase-3 activation was accompanied by the ketamine-induced cell death and inhibited by the GSK-3 inhibitors. These results suggest that activation of GSK-3 is involved in ketamine-induced apoptosis in rat cortical neurons

  8. [Advances in Parvovirus Non-structural Protein NS1 Induced Apoptosis].

    Science.gov (United States)

    Tu, Mengyu; Liu, Fei; Chen, Shun; Wang, Mingshu; Cheng, Anchun

    2015-11-01

    Until now, more than seventeen parvovirus have been reported which can infect mammals and poultries. The infected cells appeared different properties of apoptosis and death, present a typical cytopathic effect. NS1 is a major nonstructural protein of parvovirus, with a conservative structure and function, which plays an important role in the viral life cycle. In addition to the influence on viral replication, the NS1 also participates in apoptosis induced by viruses. Parvovirus induced apoptosis which is mainly mediated by mitochondrial pathway, this review summarized the latest research progresses of parvovirus induced apoptosis.

  9. Eosinophil Resistance to Glucocorticoid-Induced Apoptosis is Mediated by the Transcription Factor NFIL3

    Science.gov (United States)

    Pazdrak, Konrad; Moon, Young; Straub, Christof; Stafford, Susan; Kurosky, Alexander

    2016-01-01

    The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs' effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired prop-aptoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils' response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apoptotic response regardless of IL-5 stimulation. Proteomic analysis revealed that both Nuclear Factor IL-3 (NFIL3) and Map Kinase Phosphatase 1 (MKP1) were inducible by IL-5 and enhanced by Dex; however, CpdA had no effect on NFIL3 and MKP1 expression. We found that inhibiting NFIL3 with specific siRNA or by blocking the IL-5-inducible Pim-1 kinase abrogated the protective effect of IL-5 on Dex-induced apoptosis, indicating crosstalk between IL-5 anti-apoptotic pathways and GR-mediated TA signaling occurring via the NFIL3 molecule. Collectively, these results indicate that 1) GCs' TA pathway may support eosinophil viability in IL-5-stimulated cells through synergistic upregulation of NFIL3; and 2) functional inhibition of IL-5 signaling (anti-Pim1) or the use of selective GR agonists that don't upregulate NFIL3 may be effective strategies for the restoring pro-apoptotic effect of GCs on IL-5-activated eosinophils. PMID:26880402

  10. Deregulation of Mitochondria-Shaping Proteins Opa-1 and Drp-1 in Manganese-Induced Apoptosis

    Science.gov (United States)

    Alaimo, Agustina; Gorojod, Roxana M.; Beauquis, Juan; Muñoz, Manuel J.; Saravia, Flavia; Kotler, Mónica L.

    2014-01-01

    Mitochondria are dynamic organelles that undergo fusion and fission processes. These events are regulated by mitochondria-shaping proteins. Changes in the expression and/or localization of these proteins lead to a mitochondrial dynamics impairment and may promote apoptosis. Increasing evidence correlates the mitochondrial dynamics disruption with the occurrence of neurodegenerative diseases. Therefore, we focused on this topic in Manganese (Mn)-induced Parkinsonism, a disorder associated with Mn accumulation preferentially in the basal ganglia where mitochondria from astrocytes represent an early target. Using MitoTracker Red staining we observed increased mitochondrial network fission in Mn-exposed rat astrocytoma C6 cells. Moreover, Mn induced a marked decrease in fusion protein Opa-1 levels as well as a dramatic increase in the expression of fission protein Drp-1. Additionally, Mn provoked a significant release of high MW Opa-1 isoforms from the mitochondria to the cytosol as well as an increased Drp-1 translocation to the mitochondria. Both Mdivi-1, a pharmacological Drp-1 inhibitor, and rat Drp-1 siRNA reduced the number of apoptotic nuclei, preserved the mitochondrial network integrity and prevented cell death. CsA, an MPTP opening inhibitor, prevented mitochondrial Δψm disruption, Opa-1 processing and Drp-1 translocation to the mitochondria therefore protecting Mn-exposed cells from mitochondrial disruption and apoptosis. The histological analysis and Hoechst 33258 staining of brain sections of Mn-injected rats in the striatum showed a decrease in cellular mass paralleled with an increase in the occurrence of apoptotic nuclei. Opa-1 and Drp-1 expression levels were also changed by Mn-treatment. Our results demonstrate for the first time that abnormal mitochondrial dynamics is implicated in both in vitro and in vivo Mn toxicity. In addition we show that the imbalance in fusion/fission equilibrium might be involved in Mn-induced apoptosis. This knowledge may

  11. Deregulation of mitochondria-shaping proteins Opa-1 and Drp-1 in manganese-induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Agustina Alaimo

    Full Text Available Mitochondria are dynamic organelles that undergo fusion and fission processes. These events are regulated by mitochondria-shaping proteins. Changes in the expression and/or localization of these proteins lead to a mitochondrial dynamics impairment and may promote apoptosis. Increasing evidence correlates the mitochondrial dynamics disruption with the occurrence of neurodegenerative diseases. Therefore, we focused on this topic in Manganese (Mn-induced Parkinsonism, a disorder associated with Mn accumulation preferentially in the basal ganglia where mitochondria from astrocytes represent an early target. Using MitoTracker Red staining we observed increased mitochondrial network fission in Mn-exposed rat astrocytoma C6 cells. Moreover, Mn induced a marked decrease in fusion protein Opa-1 levels as well as a dramatic increase in the expression of fission protein Drp-1. Additionally, Mn provoked a significant release of high MW Opa-1 isoforms from the mitochondria to the cytosol as well as an increased Drp-1 translocation to the mitochondria. Both Mdivi-1, a pharmacological Drp-1 inhibitor, and rat Drp-1 siRNA reduced the number of apoptotic nuclei, preserved the mitochondrial network integrity and prevented cell death. CsA, an MPTP opening inhibitor, prevented mitochondrial Δψm disruption, Opa-1 processing and Drp-1 translocation to the mitochondria therefore protecting Mn-exposed cells from mitochondrial disruption and apoptosis. The histological analysis and Hoechst 33258 staining of brain sections of Mn-injected rats in the striatum showed a decrease in cellular mass paralleled with an increase in the occurrence of apoptotic nuclei. Opa-1 and Drp-1 expression levels were also changed by Mn-treatment. Our results demonstrate for the first time that abnormal mitochondrial dynamics is implicated in both in vitro and in vivo Mn toxicity. In addition we show that the imbalance in fusion/fission equilibrium might be involved in Mn-induced apoptosis

  12. Deregulation of mitochondria-shaping proteins Opa-1 and Drp-1 in manganese-induced apoptosis.

    Science.gov (United States)

    Alaimo, Agustina; Gorojod, Roxana M; Beauquis, Juan; Muñoz, Manuel J; Saravia, Flavia; Kotler, Mónica L

    2014-01-01

    Mitochondria are dynamic organelles that undergo fusion and fission processes. These events are regulated by mitochondria-shaping proteins. Changes in the expression and/or localization of these proteins lead to a mitochondrial dynamics impairment and may promote apoptosis. Increasing evidence correlates the mitochondrial dynamics disruption with the occurrence of neurodegenerative diseases. Therefore, we focused on this topic in Manganese (Mn)-induced Parkinsonism, a disorder associated with Mn accumulation preferentially in the basal ganglia where mitochondria from astrocytes represent an early target. Using MitoTracker Red staining we observed increased mitochondrial network fission in Mn-exposed rat astrocytoma C6 cells. Moreover, Mn induced a marked decrease in fusion protein Opa-1 levels as well as a dramatic increase in the expression of fission protein Drp-1. Additionally, Mn provoked a significant release of high MW Opa-1 isoforms from the mitochondria to the cytosol as well as an increased Drp-1 translocation to the mitochondria. Both Mdivi-1, a pharmacological Drp-1 inhibitor, and rat Drp-1 siRNA reduced the number of apoptotic nuclei, preserved the mitochondrial network integrity and prevented cell death. CsA, an MPTP opening inhibitor, prevented mitochondrial Δψm disruption, Opa-1 processing and Drp-1 translocation to the mitochondria therefore protecting Mn-exposed cells from mitochondrial disruption and apoptosis. The histological analysis and Hoechst 33258 staining of brain sections of Mn-injected rats in the striatum showed a decrease in cellular mass paralleled with an increase in the occurrence of apoptotic nuclei. Opa-1 and Drp-1 expression levels were also changed by Mn-treatment. Our results demonstrate for the first time that abnormal mitochondrial dynamics is implicated in both in vitro and in vivo Mn toxicity. In addition we show that the imbalance in fusion/fission equilibrium might be involved in Mn-induced apoptosis. This knowledge may

  13. Cytoprotection by fructose and other ketohexoses during bile salt-induced apoptosis of hepatocytes.

    Science.gov (United States)

    Zeid, I M; Bronk, S F; Fesmier, P J; Gores, G J

    1997-01-01

    Toxic bile salts cause hepatocyte necrosis at high concentrations and apoptosis at lower concentrations. Although fructose prevents bile salt-induced necrosis, the effect of fructose on bile salt-induced apoptosis is unclear. Our aim was to determine if fructose also protects against bile salt-induced apoptosis. Fructose inhibited glycochenodeoxycholate (GCDC)-induced apoptosis in a concentration-dependent manner with a maximum inhibition of 72% +/- 10% at 10 mmol/L. First, we determined if fructose inhibited apoptosis by decreasing adenosine triphosphate (ATP) and intracellular pH (pHi). Although fructose decreased ATP to effects, alterations in the expression of bcl-2, or metal chelation, we next determined if the poorly metabolized ketohexoses, tagatose and sorbose, also inhibited apoptosis; unexpectedly, both ketohexoses inhibited apoptosis. Because bile salt-induced apoptosis and necrosis are inhibited by fructose, these data suggest that similar processes initiate bile salt-induced hepatocyte necrosis and apoptosis. In contrast, acidosis, which inhibits necrosis, potentiates apoptosis. Thus, ketohexose-sensitive pathways appear to initiate both bile salt-induced cell apoptosis and necrosis, whereas dissimilar, pH-sensitive, effector mechanisms execute these two different cell death processes.

  14. Data on cell growth inhibition induced by anti-VEGF siRNA delivered by Stealth liposomes incorporating G2 PAMAM-cholesterol versus Metafectene® as a function of exposure time and siRNA concentration

    Directory of Open Access Journals (Sweden)

    Nasim Golkar

    2016-09-01

    Full Text Available In this data article, carboxyfluorescein-loaded liposomes were prepared and purified from free carboxyfluorescein using gel filtration chromatography in the first part. In the next part, following preparation of anti-VEGF siRNA loaded liposomes incorporating hydrophobically modified G2 PAMAM dendrimer (G2-Chol40% (Golkar et al., 2016 [1], the cell growth inhibition induced by the formulations (siRNA/Metafectene complexes and siRNA loaded liposomes incorporating hydrophobic G2 was evaluated at two exposure times through MTT assay in a breast cancer cell (SKBR-3 and compared by two-way ANOVA. Keywords: Anti-VEGF siRNA, Cell growth inhibition, Polyamidoaminedendrimer, Liposome

  15. Snake venom toxin from vipera lebetina turanica induces apoptosis of colon cancer cells via upregulation of ROS- and JNK-mediated death receptor expression

    International Nuclear Information System (INIS)

    Park, Mi Hee; Jo, MiRan; Won, Dohee; Song, Ho Sueb; Han, Sang Bae; Song, Min Jong; Hong, Jin Tae

    2012-01-01

    Abundant research suggested that the cancer cells avoid destruction by the immune system through down-regulation or mutation of death receptors. Therefore, it is very important that finding the agents that increase the death receptors of cancer cells. In this study, we demonstrated that the snake venom toxin from Vipera lebetina turanica induce the apoptosis of colon cancer cells through reactive oxygen species (ROS) and c-Jun N-terminal kinases (JNK) dependent death receptor (DR4 and DR5) expression. We used cell viability assays, DAPI/TUNEL assays, as well as western blot for detection of apoptosis related proteins and DRs to demonstrate that snake venom toxin-induced apoptosis is DR4 and DR5 dependent. We carried out transient siRNA knockdowns of DR4 and DR5 in colon cancer cells. We showed that snake venom toxin inhibited growth of colon cancer cells through induction of apoptosis. We also showed that the expression of DR4 and DR5 was increased by treatment of snake venom toxin. Moreover, knockdown of DR4 or DR5 reversed the effect of snake venom toxin. Snake venom toxin also induced JNK phosphorylation and ROS generation, however, pretreatment of JNK inhibitor and ROS scavenger reversed the inhibitory effect of snake venom toxin on cancer cell proliferation, and reduced the snake venom toxin-induced upregulation of DR4 and DR5 expression. Our results indicated that snake venom toxin could inhibit human colon cancer cell growth, and these effects may be related to ROS and JNK mediated activation of death receptor (DR4 and DR5) signals

  16. Overexpression of BAG3 Attenuates Hypoxia-Induced Cardiomyocyte Apoptosis by Inducing Autophagy.

    Science.gov (United States)

    Zhang, Jiankai; He, Zhangyou; Xiao, Wenjian; Na, Qingqing; Wu, Tianxiu; Su, Kaixin; Cui, Xiaojun

    2016-01-01

    Hypoxia is a well-known factor in the promotion of apoptosis, which contributes to the development of numerous cardiac diseases, such as heart failure and myocardial infarction. Inhibiting apoptosis is an important therapeutic strategy for the treatment of related heart diseases caused by ischemia/hypoxic injury. Previous studies have demonstrated that BAG3 plays an important role in cardiomyocyte apoptosis and survival. However, the role of BAG3 in hypoxia-induced cardiomyocyte apoptosis remains to be clarified. Here, we demonstrate that BAG3 is induced by hypoxia stimuli in cultured cardiomyocytes. BAG3 expression level was measured in H9c2 cells treated with hypoxia for 48 h. Cell proliferation and apoptosis were tested using MTT assay and Annexin V FITC-PI staining assay, respectively. The mRNA or protein expression level of BAG3, LC3-I, LC3-II, Atg5, NF-x03BA;B p65 and phosphorylated NF-x03BA;B p65 were assessed by qRT-PCR and western blot assay, respectively. Resluts: Overexpression of BAG3 inhibited cell apoptosis and promoted proliferation in hypoxia-injured H9c2 cells. Furthermore, autophagy and NF-x03BA;B were activated by BAG3 overexpression, and the NF-x03BA;B inhibitor PDTC could inhibit the activation of autophagy induced by BAG3 overexpression. In addition, the autophagy inhibitor 3-MA partly impeded the inhibitory effect of BAG3 on hypoxia-induced cardiomyocyte apoptosis. these results suggested that overexpression of BAG3 promoted cell proliferation and inhibited apoptosis by activating autophagy though the NF-x03BA;B signaling pathway in hypoxia-injured cardiomyocytes. © 2016 The Author(s) Published by S. Karger AG, Basel.

  17. Andrographolide sensitizes prostate cancer cells to TRAIL-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Ruo-Jing Wei

    2018-01-01

    Full Text Available Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL is a promising agent for anticancer therapy. The identification of small molecules that can establish the sensitivity of prostate cancer (PCa cells to TRAIL-induced apoptosis is crucial for the targeted treatment of PCa. PC3, DU145, JAC-1, TsuPr1, and LNCaP cells were treated with Andrographolide (Andro and TRAIL, and the apoptosis was measured using the Annexin V/PI double staining method. Real time-polymerase chain reaction (PCR and Western blot analysis were performed to measure the expression levels of target molecules. RNA interference technique was used to down-regulate the expression of the target protein. We established a nude mouse xenograft model of PCa, which was used to measure the caspase-3 activity in the tumor cells using flow cytometry. In this research study, our results demonstrated that Andro preferentially increased the sensitivity of PCa cells to TRAIL-induced apoptosis at subtoxic concentrations, and the regulation mechanism was related to the up-regulation of DR4. In addition, it also increased the p53 expression and led to the generation of reactive oxygen species (ROS in the cells. Further research revealed that the DR4 inhibition, p53 expression, and ROS generation can significantly reduce the apoptosis induced by the combination of TRAIL and Andro in PCa cells. In conclusion, Andro increases the sensitivity of PCa cells to TRAIL-induced apoptosis through the generation of ROS and up-regulation of p53 and then promotes PCa cell apoptosis associated with the activation of DR4.

  18. Hypoxia-induced p53 modulates both apoptosis and radiosensitivity via AKT

    Science.gov (United States)

    Leszczynska, Katarzyna B.; Foskolou, Iosifina P.; Abraham, Aswin G.; Anbalagan, Selvakumar; Tellier, Céline; Haider, Syed; Span, Paul N.; O’Neill, Eric E.; Buffa, Francesca M.; Hammond, Ester M.

    2015-01-01

    Restoration of hypoxia-induced apoptosis in tumors harboring p53 mutations has been proposed as a potential therapeutic strategy; however, the transcriptional targets that mediate hypoxia-induced p53-dependent apoptosis remain elusive. Here, we demonstrated that hypoxia-induced p53-dependent apoptosis is reliant on the DNA-binding and transactivation domains of p53 but not on the acetylation sites K120 and K164, which, in contrast, are essential for DNA damage–induced, p53-dependent apoptosis. Evaluation of hypoxia-induced transcripts in multiple cell lines identified a group of genes that are hypoxia-inducible proapoptotic targets of p53, including inositol polyphosphate-5-phosphatase (INPP5D), pleckstrin domain–containing A3 (PHLDA3), sulfatase 2 (SULF2), B cell translocation gene 2 (BTG2), cytoplasmic FMR1-interacting protein 2 (CYFIP2), and KN motif and ankyrin repeat domains 3 (KANK3). These targets were also regulated by p53 in human cancers, including breast, brain, colorectal, kidney, bladder, and melanoma cancers. Downregulation of these hypoxia-inducible targets associated with poor prognosis, suggesting that hypoxia-induced apoptosis contributes to p53-mediated tumor suppression and treatment response. Induction of p53 targets, PHLDA3, and a specific INPP5D transcript mediated apoptosis in response to hypoxia through AKT inhibition. Moreover, pharmacological inhibition of AKT led to apoptosis in the hypoxic regions of p53-deficient tumors and consequently increased radiosensitivity. Together, these results identify mediators of hypoxia-induced p53-dependent apoptosis and suggest AKT inhibition may improve radiotherapy response in p53-deficient tumors. PMID:25961455

  19. Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells.

    Science.gov (United States)

    Kim, Tae Hwan; Kim, Ju Sung; Kim, Zoo Haye; Huang, Ren Bin; Chae, Young Lye; Wang, Ren Sheng

    2014-07-10

    Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an

  20. Cr(VI) induces mitochondrial-mediated and caspase-dependent apoptosis through reactive oxygen species-mediated p53 activation in JB6 Cl41 cells

    International Nuclear Information System (INIS)

    Son, Young-Ok; Hitron, J. Andrew; Wang Xin; Chang Qingshan; Pan Jingju; Zhang Zhuo; Liu Jiankang; Wang Shuxia; Lee, Jeong-Chae; Shi Xianglin

    2010-01-01

    Cr(VI) compounds are known to cause serious toxic and carcinogenic effects. Cr(VI) exposure can lead to a severe damage to the skin, but the mechanisms involved in the Cr(VI)-mediated toxicity in the skin are unclear. The present study examined whether Cr(VI) induces cell death by apoptosis or necrosis using mouse skin epidermal cell line, JB6 Cl41 cells. We also investigated the cellular mechanisms of Cr(VI)-induced cell death. This study showed that Cr(VI) induced apoptotic cell death in a dose-dependent manner, as demonstrated by the appearance of cell shrinkage, the migration of cells into the sub-G1 phase, the increase of Annexin V positively stained cells, and the formation of nuclear DNA ladders. Cr(VI) treatment resulted in the increases of mitochondrial membrane depolarization and caspases activation. Electron spin resonance (ESR) and fluorescence analysis revealed that Cr(VI) increased intracellular levels of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion radical in dose-dependent manner. Blockage of p53 by si-RNA transfection suppressed mitochondrial changes of Bcl-2 family composition, mitochondrial membrane depolarization, caspase activation and PARP cleavage, leading to the inhibition of Cr(VI)-induced apoptosis. Further, catalase treatment prevented p53 phosphorylation stimulated by Cr(VI) with the concomitant inhibition of caspase activation. These results suggest that Cr(VI) induced a mitochondrial-mediated and caspase-dependent apoptosis in skin epidermal cells through activation of p53, which are mainly mediated by reactive oxidants generated by the chemical.

  1. Inhibition of VDAC1 prevents Ca²⁺-mediated oxidative stress and apoptosis induced by 5-aminolevulinic acid mediated sonodynamic therapy in THP-1 macrophages.

    Science.gov (United States)

    Chen, Haibo; Gao, Weiwei; Yang, Yang; Guo, Shuyuan; Wang, Huan; Wang, Wei; Zhang, Shuisheng; Zhou, Qi; Xu, Haobo; Yao, Jianting; Tian, Zhen; Li, Bicheng; Cao, Wenwu; Zhang, Zhiguo; Tian, Ye

    2014-12-01

    Ultrasound combined with endogenous protoporphyrin IX derived from 5-aminolevulinic acid (ALA-SDT) is known to induce apoptosis in multiple cancer cells and macrophages. Persistent retention of macrophages in the plaque has been implicated in the pathophysiology and progression of atherosclerosis. Here we investigated the effects of inhibition of voltage-dependent anion channel 1 (VDAC1) on ALA-SDT-induced THP-1 macrophages apoptosis. Cells were pre-treated with VDAC1 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) disodium salt for 1 h or downregulated VDAC1 expression by small interfering RNA and exposed to ultrasound. Cell viability was assessed by MTT assay, and cell apoptosis along with necrosis was evaluated by Hoechst 33342/propidium iodide staining and flow cytometry. Levels of cytochrome c release was assessed by confocal microscope and Western blot. The levels of full length caspases, caspase activation, and VDAC isoforms were analyzed by Western blot. Intracellular reactive oxygen species generation, mitochondrial membrane permeability, and intracellular Ca(2+) [Ca(2+)]i levels were measured with fluorescent probes. We confirmed that the pharmacological inhibition of VDAC1 by DIDS notably prevented ALA-SDT-induced cell apoptosis in THP-1 macrophages. Additionally, DIDS significantly inhibited intracellular ROS generation and apoptotic biochemical changes such as inner mitochondrial membrane permeabilization, loss of mitochondrial membrane potential, cytochrome c release and activation of caspase-3 and caspase-9. Moreover, ALA-SDT elevated the [Ca(2+)]i levels and it was also notably reduced by DIDS. Furthermore, both of intracellular ROS generation and cell apoptosis were predominately inhibited by Ca(2+) chelating reagent BAPTA-AM. Intriguingly, ALA-treatment markedly augmented VDAC1 protein levels exclusively, and the downregulation of VDAC1 expression by specific siRNA also significantly abolished cell apoptosis. Altogether, these

  2. Oxidative Stress-Responsive Apoptosis Inducing Protein (ORAIP) Plays a Critical Role in High Glucose-Induced Apoptosis in Rat Cardiac Myocytes and Murine Pancreatic β-Cells.

    Science.gov (United States)

    Yao, Takako; Fujimura, Tsutomu; Murayama, Kimie; Okumura, Ko; Seko, Yoshinori

    2017-10-18

    We previously identified a novel apoptosis-inducing humoral factor in the conditioned medium of hypoxic/reoxygenated-cardiac myocytes. We named this novel post-translationally-modified secreted-form of eukaryotic translation initiation factor 5A Oxidative stress-Responsive Apoptosis-Inducing Protein (ORAIP). We confirmed that myocardial ischemia/reperfusion markedly increased plasma ORAIP levels and rat myocardial ischemia/reperfusion injury was clearly suppressed by neutralizing anti-ORAIP monoclonal antibodies (mAbs) in vivo. In this study, to investigate the mechanism of cell injury of cardiac myocytes and pancreatic β-cells involved in diabetes mellitus (DM), we analyzed plasma ORAIP levels in DM model rats and the role of ORAIP in high glucose-induced apoptosis of cardiac myocytes in vitro. We also examined whether recombinant-ORAIP induces apoptosis in pancreatic β-cells. Plasma ORAIP levels in DM rats during diabetic phase were about 18 times elevated as compared with non-diabetic phase. High glucose induced massive apoptosis in cardiac myocytes (66.2 ± 2.2%), which was 78% suppressed by neutralizing anti-ORAIP mAb in vitro. Furthermore, recombinant-ORAIP clearly induced apoptosis in pancreatic β-cells in vitro. These findings strongly suggested that ORAIP plays a pivotal role in hyperglycemia-induced myocardial injury and pancreatic β-cell injury in DM. ORAIP will be a biomarker and a critical therapeutic target for cardiac injury and progression of DM itself.

  3. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  4. Radiation-induced apoptosis in F9 teratocarcinoma cells

    International Nuclear Information System (INIS)

    Langley, R.E.; Palayoor, S.T.; Coleman, C.N.; Bump, E.A.

    1994-01-01

    We have found that F9 murine teratocarcinoma cells undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation. We studied the time course, radiation dose-response, and the effects of protein and RNA synthesis inhibitors on this process. The response is dose dependent in the range 2-12 Gy. Internucleosomal DNA fragmentation can be detected as early as 6 h postirradiation and is maximal by 48 h. Cycloheximide, a protein synthesis inhibitor, and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, an RNA synthesis inhibitor, both induced internucleosomal DNA fragmentation in the unirradiated cells and enhanced radiation-induced DNA fragmentation. F9 cells can be induced to differentiate into cells resembling endoderm with retinoic acid. After irradiation, differentiated F9 cells exhibit less DNA fragmentation than stem cells. This indicates that ionizing radiation can induce apoptosis in non-lymphoid tumours. We suggest that embryonic tumour cells may be particularly susceptible to agents that induce apoptosis. (Author)

  5. Radiation-induced apoptosis in F9 teratocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Langley, R E; Palayoor, S T; Coleman, C N; Bump, E A [Joint Center for Radiation Therapy and Dana Farber Cancer Inst., Boston (United States)

    1994-05-01

    We have found that F9 murine teratocarcinoma cells undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation. We studied the time course, radiation dose-response, and the effects of protein and RNA synthesis inhibitors on this process. The response is dose dependent in the range 2-12 Gy. Internucleosomal DNA fragmentation can be detected as early as 6 h postirradiation and is maximal by 48 h. Cycloheximide, a protein synthesis inhibitor, and 5,6-dichloro-1-[beta]-D-ribofuranosylbenzimidazole, an RNA synthesis inhibitor, both induced internucleosomal DNA fragmentation in the unirradiated cells and enhanced radiation-induced DNA fragmentation. F9 cells can be induced to differentiate into cells resembling endoderm with retinoic acid. After irradiation, differentiated F9 cells exhibit less DNA fragmentation than stem cells. This indicates that ionizing radiation can induce apoptosis in non-lymphoid tumours. We suggest that embryonic tumour cells may be particularly susceptible to agents that induce apoptosis. (Author).

  6. Icariside II induces apoptosis in U937 acute myeloid leukemia cells: role of inactivation of STAT3-related signaling.

    Directory of Open Access Journals (Sweden)

    Sang-Hun Kang

    Full Text Available BACKGROUND: The aim of this study is to determine anti-cancer effect of Icariside II purified from the root of Epimedium koreanum Nakai on human acute myeloid leukemia (AML cell line U937. METHODOLOGY/PRINCIPAL FINDINGS: Icariside II blocked the growth U937 cells in a dose- and time-dependent manner. In this anti-proliferation process, this herb compound rendered the cells susceptible to apoptosis, manifested by enhanced accumulation of sub-G1 cell population and increased the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL-positive cells. Icariside II was able to activate caspase-3 and cleaved poly (ADP-ribose polymerase (PARP in a time-dependent manner. Concurrently, the anti-apoptotic proteins, such as bcl-x(L and survivin in U937 cells, were downregulated by Icariside II. In addition, Icariside II could inhibit STAT3 phosphorylation and function and subsequently suppress the activation of Janus activated kinase 2 (JAK2, the upstream activators of STAT3, in a dose- and time-dependent manner. Icariside II also enhanced the expression of protein tyrosine phosphatase (PTP SH2 domain-containing phosphatase (SHP-1, and the addition of sodium pervanadate (a PTP inhibitor prevented Icariside II-induced apoptosis as well as STAT3 inactivation in STAT3 positive U937 cells. Furthermore, silencing SHP-1 using its specific siRNA significantly blocked STAT3 inactivation and apoptosis induced by Icariside II in U937 cells. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated that via targeting STAT3-related signaling, Icariside II sensitizes U937 cells to apoptosis and perhaps serves as a potent chemotherapeutic agent for AML.

  7. Beta-irradiation used for systemic radioimmunotherapy induces apoptosis and activates apoptosis pathways in leukaemia cells

    International Nuclear Information System (INIS)

    Friesen, Claudia; Lubatschofski, Annelie; Debatin, Klaus-Michael; Kotzerke, Joerg; Buchmann, Inga; Reske, Sven N.

    2003-01-01

    Beta-irradiation used for systemic radioimmunotherapy (RIT) is a promising treatment approach for high-risk leukaemia and lymphoma. In bone marrow-selective radioimmunotherapy, beta-irradiation is applied using iodine-131, yttrium-90 or rhenium-188 labelled radioimmunoconjugates. However, the mechanisms by which beta-irradiation induces cell death are not understood at the molecular level. Here, we report that beta-irradiation induced apoptosis and activated apoptosis pathways in leukaemia cells depending on doses, time points and dose rates. After beta-irradiation, upregulation of CD95 ligand and CD95 receptor was detected and activation of caspases resulting in apoptosis was found. These effects were completely blocked by the broad-range caspase inhibitor zVAD-fmk. In addition, irradiation-mediated mitochondrial damage resulted in perturbation of mitochondrial membrane potential, caspase-9 activation and cytochrome c release. Bax, a death-promoting protein, was upregulated and Bcl-x L , a death-inhibiting protein, was downregulated. We also found higher apoptosis rates and earlier activation of apoptosis pathways after gamma-irradiation in comparison to beta-irradiation at the same dose rate. Furthermore, irradiation-resistant cells were cross-resistant to CD95 and CD95-resistant cells were cross-resistant to irradiation, indicating that CD95 and irradiation used, at least in part, identical effector pathways. These findings demonstrate that beta-irradiation induces apoptosis and activates apoptosis pathways in leukaemia cells using both mitochondrial and death receptor pathways. Understanding the timing, sequence and molecular pathways of beta-irradiation-mediated apoptosis may allow rational adjustment of chemo- and radiotherapeutic strategies. (orig.)

  8. The apoptosis of CHO cells induced by X-rays

    International Nuclear Information System (INIS)

    Lu Zhaohong; Zhao Jingyong; Zhu Mingqing; Shi Xijin; Wang Chunlei

    2004-01-01

    The work is to study the mechanism of toxic effects on reproductive system and apoptosis of Chinese hamster ovary (CHO) cells induced by X-rays. CHO cell was exposed to X-rays 2 to 20 Gy. Apoptosis and morphological changes of the cells were observed by fluorescent microscopy and flow cytometry analyzer with double staining with Annexin V/PI. The apoptosis could be observed at 24, 48 and 72h after the exposure, but it was more obvious 48 and 72 h after the exposure. Rate of the apoptosis increased along with radiation dose were elevated. Some morphological changes, such as irregular agglomerate of chromatins, pycnosis and periphery distribution of nuclei, crescent-moon-like cells, small apoptosis body, were observed. Radiation results DNA damage in the CHO cells, and the damage cannot be repaired, hence the induced cell apoptosis. (authors)

  9. Sequential activation of proteases in radiation induced apoptosis

    International Nuclear Information System (INIS)

    Watters, D.; Waterhouse, N.

    1997-01-01

    Full text: Significant advances have been made in recent years in unraveling the molecular mechanisms of apoptosis particularly in relation to Fas- and TNF-mediated cell death, however there are considerable gaps in our knowledge of the processes involved in apoptosis induced by ionizing radiation. We have used the degradation of specific proteolytic targets in a pair of isogenic Burkitt's Iymphoma cells lines (BL30A, sensitive and BL30K resistant) to study the sequence of events in the execution of radiation-induced apoptosis. Fodrin can be cleaved to fragments of 150 kDa and 120 kDa. In the case of Fas-mediated apoptosis both cleavages are inhibited by the caspase inhibitor zVAD-fmk at 10 μM, a concentration which inhibits all the hallmarks of apoptosis. However in radiation-induced apoptosis, inhibition of the clevage of fodrin to the 150 kDa fragment requires 100 μM zVAD-fink while apoptosis itself is inhibited at 10 μM. This suggests that different enzymes are responsible for the generation of the 150 kDa fragment in the two models of apoptosis. Fodrin has been reported to be cleaved by μ-calpain to a 150 kDa fragment however, the involvement of μ-calpain in apoptosis has not yet been established. In murine fodrin there is a caspase cleavage site within 1 kDa of the calpain cleavage site. In vitro studies using purified enzymes showed that only caspase-3 and μ-calpain could cleave fodrin in untreated cell extracts to the same sized fragments as seen during apoptosis in vivo. We provide evidence for the early activation of μ-calpain after ionizing radiation in the sensitive BL30A cell line, and show that the time course of μ-calpain activation parallels that of the appearance of the 150 kDa fragment. Caspase-3 is activated much later and is likely to be responsible for the generation of the 120 kDa fragment. μ-Calpain was not activated in the resistant cell line. Based on these results we propose a model for the proteolytic cascade in radiation-induced

  10. Neuroprotective effects of ganoderma lucidum polysaccharides against oxidative stress-induced neuronal apoptosis

    Science.gov (United States)

    Sun, Xin-zhi; Liao, Ying; Li, Wei; Guo, Li-mei

    2017-01-01

    Ganoderma lucidum polysaccharides have protective effects against apoptosis in neurons exposed to ischemia/reperfusion injury, but the mechanisms are unclear. The goal of this study was to investigate the underlying mechanisms of the effects of ganoderma lucidum polysaccharides against oxidative stress-induced neuronal apoptosis. Hydrogen peroxide (H2O2) was used to induce apoptosis in cultured cerebellar granule cells. In these cells, ganoderma lucidum polysaccharides remarkably suppressed H2O2-induced apoptosis, decreased expression of caspase-3, Bax and Bim and increased that of Bcl-2. These findings suggested that ganoderma lucidum polysaccharides regulate expression of apoptosis-associated proteins, inhibit oxidative stress-induced neuronal apoptosis and, therefore, have significant neuroprotective effects. PMID:28761429

  11. The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis

    NARCIS (Netherlands)

    Greijer, A.E.; Wall, E. van der

    2004-01-01

    Apoptosis can be induced in response to hypoxia. The severity of hypoxia determines whether cells become apoptotic or adapt to hypoxia and survive. A hypoxic environment devoid of nutrients prevents the cell undergoing energy dependent apoptosis and cells become necrotic. Apoptosis regulatory

  12. Zinc finger protein 598 inhibits cell survival by promoting UV-induced apoptosis.

    Science.gov (United States)

    Yang, Qiaohong; Gupta, Romi

    2018-01-19

    UV is one of the major causes of DNA damage induced apoptosis. However, cancer cells adopt alternative mechanisms to evade UV-induced apoptosis. To identify factors that protect cancer cells from UV-induced apoptosis, we performed a genome wide short-hairpin RNA (shRNA) screen, which identified Zinc finger protein 598 (ZNF598) as a key regulator of UV-induced apoptosis. Here, we show that UV irradiation transcriptionally upregulates ZNF598 expression. Additionally, ZNF598 knockdown in cancer cells inhibited UV-induced apoptosis. In our study, we observe that ELK1 mRNA level as well as phosphorylated ELK1 levels was up regulated upon UV irradiation, which was necessary for UV irradiation induced upregulation of ZNF598. Cells expressing ELK1 shRNA were also resistant to UV-induced apoptosis, and phenocopy ZNF598 knockdown. Upon further investigation, we found that ZNF598 knockdown inhibits UV-induced apoptotic gene expression, which matches with decrease in percentage of annexin V positive cell. Similarly, ectopic expression of ZNF598 promoted apoptotic gene expression and also increased annexin V positive cells. Collectively, these results demonstrate that ZNF598 is a UV irradiation regulated gene and its loss results in resistance to UV-induced apoptosis.

  13. The effects of cysteamine on the radiation-induced apoptosis

    International Nuclear Information System (INIS)

    Choi, Young Min; Cho, Heung Lae; Park, Chang Gyo; Lee, Hyung Sik; Hur, Won Joo

    2000-01-01

    To investigate the pathways of radiation induced apoptosis and the effect of cysteamine (β-mercaptoethylamine), as a radioprotector, on it. HL-60 cells were assigned to control, irradiated, and cysteamine (1 mM, 10 mM) pretreated groups. Irradiation was given in a single fraction of 10 Gy (6 MV x-ray) and cysteamine was administered 1 hour before irradiation. The activities of caspase-8 were measured in control and irradiated group to evaiuate its relation to the radiation induced apoptosis. To evaluate the role of cysteamine in radiation induced apoptosis, the number of viable cells, the expression and activity or caspase-3, and the expression of poly (ADP-ribose) polymerase (PARP) were measured and compared after irradiating the HL cells with cysteamine pretreatment or not. The intracellular caspase-8 activity, known to be related to the death receptor induced apoptosis, was not affected by irradiation( p>0.05). The number of viable cells began to decrease from 6 hours after irradiation (p>0.05), but the number of viable cells in 1 mM cysteamine pretreated group was not decreased after irradiation and was similar to those in the control group. In caspase-3 analyses, known as apoptosis executioner, its expression was not different but its activity was increased by irradialion(p>0.05). However, this increase of activity was suppressed by the pretreatment of 1 mM cysteamine. The cleavage of PARP, thought to be resulted from caspase-3 activation, occurred, after irradiation, which was attenuated by the pretreatment of 1 mM cysteamine. These results show that radiation induced apoptotic process is somewhat different from death receptor induced one and the pretreatment of 1 mM cysteamine has a tendency to decrease the radiation-induced apoptosis in HL-60 cells

  14. MicroRNAs regulate B-cell receptor signaling-induced apoptosis

    NARCIS (Netherlands)

    Kluiver, J. L.; Chen, C-Z

    Apoptosis induced by B-cell receptor (BCR) signaling is critical for antigen-driven selection, a process critical to tolerance and immunity. Here, we examined the roles of microRNAs (miRNAs) in BCR signaling-induced apoptosis using the widely applied WEHI-231 model. Comparison of miRNA levels in

  15. The characteristics and mechanism of apoptosis induced by internal irradiation

    International Nuclear Information System (INIS)

    Hong Chengjiao; Zhang Junning; Zhu Shoupeng

    2001-01-01

    Apoptosis in tumor cells induced by radionuclides is likely the most effective way to cure cancer. In order to explore the possibility in clinic application, the characteristics and mechanism of apoptosis induced by internal irradiation were investigated. The apoptosis and expressions of bcl-2mRNA, bcl-2 and bax of K 562 cells following internal exposure with different accumulated absorbed doses of strontium-89 were studied. 6 h after irradiation, the characteristics of apoptosis and necrosis appeared in K 562 cells. The apoptosis and necrosis enhanced with the prolongation of internally contaminated time at 6 h, 9 h, 12 h, 24 h and 48 h. The expressions of bcl-2mRNA decreased at 12 h, most remarkably at 24 h. The expressions of bcl-2 decreased after irradiation whereas bax had no obvious changes. The results suggest that the apoptosis induced by internal exposure may be regulated by lower expressions of bcl-2mRNA and bcl-2, lower bcl-2/bax value

  16. Apoptosis-induced lymphopenia in sepsis and other severe injuries.

    Science.gov (United States)

    Girardot, Thibaut; Rimmelé, Thomas; Venet, Fabienne; Monneret, Guillaume

    2017-02-01

    Sepsis and other acute injuries such as severe trauma, extensive burns, or major surgeries, are usually followed by a period of marked immunosuppression. In particular, while lymphocytes play a pivotal role in immune response, their functions and numbers are profoundly altered after severe injuries. Apoptosis plays a central role in this process by affecting immune response at various levels. Indeed, apoptosis-induced lymphopenia duration and depth have been associated with higher risk of infection and mortality in various clinical settings. Therapies modulating apoptosis represent an interesting approach to restore immune competence after acute injury, although their use in clinical practice still presents several limitations. After briefly describing the apoptosis process in physiology and during severe injuries, we will explore the immunological consequences of injury-induced lymphocyte apoptosis, and describe associations with clinically relevant outcomes in patients. Therapeutic perspectives targeting apoptosis will also be discussed.

  17. Angiotensin II protects primary rat hepatocytes against bile salt-induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Golnar Karimian

    Full Text Available UNLABELLED: Angiotensin II (AT-II is a pro-fibrotic compound that acts via membrane-bound receptors (AT-1R/AT-2R and thereby activates hepatic stellate cells (HSCs. AT-II receptor blockers (ARBs are thus important candidates in the treatment of liver fibrosis. However, multiple case reports suggest that AT-1R blockers may induce hepatocyte injury. Therefore, we investigated the effect of AT-II and its receptor blockers on cytokine-, oxidative stress- and bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to TNF-α/Actinomycin D, the ROS-generating agent menadione or the bile salts: glycochenodeoxycholic acid (GCDCA and tauro-lithocholic acid-3 sulfate (TLCS, to induce apoptosis. AT-II (100 nmol/L was added 10 minutes prior to the cell death-inducing agent. AT-1R antagonists (Sartans and the AT-2R antagonist PD123319 were used at 1 µmol/L. Apoptosis (caspase-3 activity, acridine orange staining and necrosis (Sytox green staining were quantified. Expression of CHOP (marker for ER stress and AT-II receptor mRNAs were quantified by Q-PCR. AT-II dose-dependently reduced GCDCA-induced apoptosis of hepatocytes (-50%, p<0.05 without inducing necrosis. In addition, AT-II reduced TLCS-induced apoptosis of hepatocytes (-50%, p<0.05. However, AT-II did not suppress TNF/Act-D and menadione-induced apoptosis. Only the AT-1R antagonists abolished the protective effect of AT-II against GCDCA-induced apoptosis. AT-II increased phosphorylation of ERK and a significant reversal of the protective effect of AT-II was observed when signaling kinases, including ERK, were inhibited. Moreover, AT-II prevented the GCDCA-induced expression of CHOP (the marker of the ER-mediated apoptosis. CONCLUSION: Angiotensin II protects hepatocytes from bile salt-induced apoptosis through a combined activation of PI3-kinase, MAPKs, PKC pathways and inhibition of bile salt-induced ER stress. Our results suggest a mechanism for the observed hepatocyte

  18. Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

    Directory of Open Access Journals (Sweden)

    Motoharu Ono

    Full Text Available We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

  19. Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

    Science.gov (United States)

    Ono, Motoharu; Yamada, Kayo; Avolio, Fabio; Afzal, Vackar; Bensaddek, Dalila; Lamond, Angus I

    2015-01-01

    We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

  20. Apoptosis-inducing factor plays a critical role in caspase-independent, pyknotic cell death in hydrogen peroxide-exposed cells.

    Science.gov (United States)

    Son, Young-Ok; Jang, Yong-Suk; Heo, Jung-Sun; Chung, Wan-Tae; Choi, Ki-Choon; Lee, Jeong-Chae

    2009-06-01

    It has been proposed that continuously generated hydrogen peroxide (H(2)O(2)) inhibits typical apoptosis and instead initiates an alternate, apoptosis-inducing factor (AIF)-dependent process. Aside from the role of AIF, however, the detailed morphological characterization of H(2)O(2)-induced cell death is not complete. This study examined the cellular mechanism(s) by which the continuous presence of H(2)O(2) induces cell death. We also further analyzed the precise role of AIF by inhibiting its expression with siRNA. Exposure of cells to H(2)O(2) generated by glucose oxidase caused mitochondrion-mediated, caspase-independent cell death. In addition, H(2)O(2) exposure resulted in cell shrinkage and chromatin condensation without nuclear fragmentation, indicating that H(2)O(2) stimulates a pyknotic cell death. Further analysis of AIF-transfected cells clearly demonstrated that nuclear translocation of AIF is the most important event required for nuclear condensation, phosphatidyl serine translocation, and ultimately cell death in H(2)O(2)-exposed cells. Furthermore, ATP was rapidly and severely depleted in cells exposed to H(2)O(2) generated by glucose oxidase but not by H(2)O(2) added as a bolus. Suppression of the H(2)O(2)-mediated ATP depletion by 3-aminobenzamide led to a significant increase of nuclear fragmentation in glucose oxidase-exposed cells. Collectively, these findings suggest that an acute energy reduction by H(2)O(2) causes caspase-independent and AIF-dependent cell death.

  1. Spironolactone induces apoptosis in human mononuclear cells. Association between apoptosis and cytokine suppression

    DEFF Research Database (Denmark)

    Mikkelsen, Martin; Sønder, S U; Nersting, J

    2006-01-01

    Spironolactone (SPIR) has been described to suppress accumulation of pro-inflammatory cytokines. Here, the suppression of TNF-alpha in lipopolysaccharide (LPS)-stimulated mononuclear cell cultures was confirmed. However, SPIR was also found to induce apoptosis, prompting the investigations...... of a possible association between the two effects: The apoptosis-inducing and the cytokine-suppressive effects of SPIR correlated with regard to the effective concentration range. Also, pre-incubation experiments demonstrated a temporal separation of the two effects of ... preceding apoptosis. An association between the two effects was also seen when testing several SPIR analogues. Contrary to TNF-alpha, the levels of IL-1beta increased in SPIR-treated cultures. However, the amount of IL-1beta in the supernatants depended upon the order of SPIR and LPS addition, as IL-1beta...

  2. Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

    Directory of Open Access Journals (Sweden)

    Annika Sommerfeld

    2015-05-01

    Full Text Available Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP signal with induction of the dual specificity mitogen-activated protein (MAP kinase phosphatase 1 (MKP-1, which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4 and c-jun-NH2-terminal kinase (JNK activation. Furthermore, TUDC induced a protein kinase A (PKA-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

  3. Bisphenol A induces spermatocyte apoptosis in rare minnow Gobiocypris rarus

    International Nuclear Information System (INIS)

    Zhang, Yingying; Cheng, Mengqian; Wu, Lang; Zhang, Guo; Wang, Zaizhao

    2016-01-01

    Highlights: • Adult male G. rarus were exposed to 225 μg/L BPA for 7, 21 and 63 days. • BPA could induce spermatocyte apoptosis in rare minnow testis. • The mitochondrial apoptotic pathway participated in the germ cell apoptosis. • The spermatocyte apoptosis was likely initiated by BPA induced meiosis arrest. - Abstract: Bisphenol A (BPA) is an endocrine disruptor, and could induce germ cells apoptosis in the testis of mammals. But whether it could affect fish in the same mechanism has not’ been studied till now. In the present study, to investigate the influence of BPA on testis germ cells in fish, adult male rare minnow Gobiocypris rarus were exposed to 225 μg L"−"1 (0.99 μM) BPA for 1, 3 and 9 weeks. Through TdT-mediated dUTP nick end labeling (TUNEL) and transmission electron microscope (TEM) analysis, we found that the amount of apoptotic spermatocytes significantly increased in a time dependent manner following BPA exposure. Western Blot results showed that the ratio of Bcl2/Bax, the important apoptosis regulators in intrinsic mitochondrial apoptotic pathway, was significantly decreased. qPCR showed that mRNA expression of several genes in mitochondrial apoptotic pathway including bcl2, bax, casp9, cytc and mcl1b were significantly changed following BPA exposure. In addition, mRNA expression of meiosis regulation genes (kpna7 and wee2), and genes involved in both apoptosis and meiosis (birc5, ccna1, and gsa1a) were also affected by BPA. Taken together, the present study demonstrated that BPA could induce spermatocytes apoptosis in rare minnow testis, and the apoptosis was probably under regulation of intrinsic mitochondrial apoptotic pathway. Moreover, the spermatocyte apoptosis was likely initiated by BPA induced meiosis arrest.

  4. Bisphenol A induces spermatocyte apoptosis in rare minnow Gobiocypris rarus

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yingying; Cheng, Mengqian; Wu, Lang; Zhang, Guo; Wang, Zaizhao, E-mail: zzwang@nwsuaf.edu.cn

    2016-10-15

    Highlights: • Adult male G. rarus were exposed to 225 μg/L BPA for 7, 21 and 63 days. • BPA could induce spermatocyte apoptosis in rare minnow testis. • The mitochondrial apoptotic pathway participated in the germ cell apoptosis. • The spermatocyte apoptosis was likely initiated by BPA induced meiosis arrest. - Abstract: Bisphenol A (BPA) is an endocrine disruptor, and could induce germ cells apoptosis in the testis of mammals. But whether it could affect fish in the same mechanism has not’ been studied till now. In the present study, to investigate the influence of BPA on testis germ cells in fish, adult male rare minnow Gobiocypris rarus were exposed to 225 μg L{sup −1} (0.99 μM) BPA for 1, 3 and 9 weeks. Through TdT-mediated dUTP nick end labeling (TUNEL) and transmission electron microscope (TEM) analysis, we found that the amount of apoptotic spermatocytes significantly increased in a time dependent manner following BPA exposure. Western Blot results showed that the ratio of Bcl2/Bax, the important apoptosis regulators in intrinsic mitochondrial apoptotic pathway, was significantly decreased. qPCR showed that mRNA expression of several genes in mitochondrial apoptotic pathway including bcl2, bax, casp9, cytc and mcl1b were significantly changed following BPA exposure. In addition, mRNA expression of meiosis regulation genes (kpna7 and wee2), and genes involved in both apoptosis and meiosis (birc5, ccna1, and gsa1a) were also affected by BPA. Taken together, the present study demonstrated that BPA could induce spermatocytes apoptosis in rare minnow testis, and the apoptosis was probably under regulation of intrinsic mitochondrial apoptotic pathway. Moreover, the spermatocyte apoptosis was likely initiated by BPA induced meiosis arrest.

  5. Lysophosphatidic acid rescues bone mesenchymal stem cells from hydrogen peroxide-induced apoptosis.

    Science.gov (United States)

    Wang, Xian-Yun; Fan, Xue-Song; Cai, Lin; Liu, Si; Cong, Xiang-Feng; Chen, Xi

    2015-03-01

    The increase of reactive oxygen species in infracted heart significantly reduces the survival of donor mesenchymal stem cells, thereby attenuating the therapeutic efficacy for myocardial infarction. In our previous study, we demonstrated that lysophosphatidic acid (LPA) protects bone marrow-derived mesenchymal stem cells (BMSCs) against hypoxia and serum deprivation-induced apoptosis. However, whether LPA protects BMSCs from H2O2-induced apoptosis was not examined. In this study, we report that H2O2 induces rat BMSC apoptosis whereas LPA pre-treatment effectively protects BMSCs from H2O2-induced apoptosis. LPA protection of BMSC from the induced apoptosis is mediated mostly through LPA3 receptor. Furthermore, we found that membrane G protein Gi2 and Gi3 are involved in LPA-elicited anti-apoptotic effects through activation of ERK1/2- and PI3 K-pathways. Additionally, H2O2 increases levels of type II of light chain 3B (LC3B II), an autophagy marker, and H2O2-induced autophagy thus protected BMSCs from apoptosis. LPA further increases the expression of LC3B II in the presence of H2O2. In contrast, autophagy flux inhibitor bafilomycin A1 has no effect on LPA's protection of BMSC from H2O2-induced apoptosis. Taken together, our data suggest that LPA rescues H2O2-induced apoptosis mainly by interacting with Gi-coupled LPA3, resulting activation of the ERK1/2- and PI3 K/AKT-pathways and inhibition caspase-3 cleavage, and LPA protection of BMSCs against the apoptosis is independent of it induced autophagy.

  6. Kaempferol induces ATM/p53-mediated death receptor and mitochondrial apoptosis in human umbilical vein endothelial cells.

    Science.gov (United States)

    Lee, Chiu-Fang; Yang, Jai-Sing; Tsai, Fuu-Jen; Chiang, Ni-Na; Lu, Chi-Cheng; Huang, Yu-Syuan; Chen, Chun; Chen, Fu-An

    2016-05-01

    Kaempferol is a member of the flavonoid compounds found in vegetables and fruits. It is shown to exhibit biological impact and anticancer activity, but no report exists on the angiogenic effect of kaempferol and induction of cell apoptosis in vitro. In this study, we investigated the role of kaempferol on anti-angiogenic property and the apoptotic mechanism of human umbilical vein endothelial cells (HUVECs). Our results demonstrated that kaempferol decreased HUVEC viability in a time- and concentration-dependent manner. Kaempferol also induced morphological changes and sub-G1 phase cell population (apoptotic cells). Kaempferol triggered apoptosis of HUVECs as detecting by DNA fragmentation, comet assay and immunofluorescent staining for activated caspase-3. The caspase signals, including caspase-8, -9 and -3, were time-dependently activated in HUVECs after kaempferol exposure. Furthermore, pre-treatment with a specific inhibitor of caspase-8 (Z-IETD-FMK) significantly reduced the activity of caspase-8, -9 and -3, indicating that extrinsic pathway is a major signaling pathway in kaempferol-treated HUVECs. Importantly, kaempferol promoted reactive oxygen species (ROS) evaluated using flow cytometric assay in HUVECs. We further investigated the upstream extrinsic pathway and showed that kaempferol stimulated death receptor signals [Fas/CD95, death receptor 4 (DR4) and DR5] through increasing the levels of phosphorylated p53 and phosphorylated ATM pathways in HUVECs, which can be individually confirmed by N-acetylcysteine (NAC), ATM specific inhibitor (caffeine) and p53 siRNA. Based on these results, kaempferol-induced HUVEC apoptosis was involved in an ROS-mediated p53/ATM/death receptor signaling. Kaempferol might possess therapeutic effects on cancer treatment in anti-vascular targeting.

  7. Fisetin induces apoptosis through mitochondrial apoptosis pathway in human uveal melanoma cells.

    Science.gov (United States)

    Wang, Kai; Hu, Dan-Ning; Lin, Hui-Wen; Yang, Wei-En; Hsieh, Yi-Hsien; Chien, Hsiang-Wen; Yang, Shun-Fa

    2018-05-01

    Fisetin, a diatery flavonoid, been reported that possess anticancer effects in various cancers. The purpose of the study was to investigate the antitumor effects of fisetin in cultured uveal melanoma cell lines and compared with normal retinal pigment epithelial (RPE) cells. MTT assay was used for evaluating cytotoxic effects of fisetin. Flow cytometry study was used for the determination of apoptosis. JC-1 fluorescent reader was used to determine mitochondrial transmembrane potential changes. The results shown that fisetin dose-dependently decreased the cell viability of uveal melanoma cells but not influenced the cell viability of RPE cells. Apoptosis of uveal melanoma cells was induced by fisetin efficiently. Fisetin inhibited antiapoptotic Bcl-2 family proteins and damaged the mitochondrial transmembrane potential. The levels of proapoptotic Bcl-2 proteins, cytochrome c, and various caspase activities were increased by fisetin. In conclusion, fisetin induces apoptosis of uveal melanoma cells selectively and may be a promising agent to be explored for the treatment of uveal melanoma. © 2018 Wiley Periodicals, Inc.

  8. Dose-effect relationship of apoptosis induced by fission-neutron in murine thymocytes

    International Nuclear Information System (INIS)

    Yuan Bin; Li Liang; Xue Wencheng; Sun Jianmin; Wang Baoqin

    2000-01-01

    Objective: To investigate the effectiveness of high LET fission-neutron to induce apoptosis in murine thymocytes and to compare it with that of low LET 60 Co γ-ray. Methods: Apoptosis induction was studied qualitatively by light and transmission electron microscopy and DNA gel electrophoresis,also quantitatively by flow cytometry(FCM) and diphenylamine (DPA)methods. Results: DNA ladders of murine thymocytes were detectable, the typical apoptosis of thymocytes could be observed morphologically by means of light and electron microscopy at 6 h after fission-neutron irradiation with doses ranging from 0.5 to 5.0 Gy, meanwhile the percentages of apoptosis increased with increasing doses. After exposure to γ-rays with doses ranging from 1.0 to 30 Gy, the experimental results were similar to those from neutron radiation. The incidence of apoptosis peaked at about 20 Gy, the percentages did not increase further when doses increased. Conclusion: Apoptosis of murine thymocytes can be induced when mice are exposed to either fission-neutron (0.5-5.0 Gy) or to γ-ray (1-30 Gy). Although the relationship between apoptosis and radiation doses is similar, the percentage of apoptosis induced by neutron irradiation is higher than that induced by γ-irradiation. The RBE values of fission-neutron for inducing apoptosis murine thymocytes are 2.09 (by FCM method) and 2.37 (by DPA method), respectively. These results also suggest that fission-neutron-induced murine immune tissue is more severe than that induced by γ-rays at several hours post-irradiation and this might be the basis for heavy damage to immune tissues induced by fission-neutron-irradiation in later period

  9. Ursodeoxycholic Acid Induces Death Receptor-mediated Apoptosis in Prostate Cancer Cells

    Science.gov (United States)

    Lee, Won Sup; Jung, Ji Hyun; Panchanathan, Radha; Yun, Jeong Won; Kim, Dong Hoon; Kim, Hye Jung; Kim, Gon Sup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

    2017-01-01

    Background Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. Methods Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. Results UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. Conclusions UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer. PMID:28382282

  10. Heat Shock Protein 70 Neutralizes Apoptosis-Inducing Factor

    Directory of Open Access Journals (Sweden)

    Guido Kroemer

    2001-01-01

    Full Text Available Programmed cell death (apoptosis is the physiological process responsible for the demise of superfluous, aged, damaged, mutated, and ectopic cells. Its normal function is essential both for embryonic development and for maintenance of adult tissue homeostasis. Deficient apoptosis participates in cancerogenesis, whereas excessive apoptosis leads to unwarranted cell loss accounting for disparate diseases including neurodegeneration and AIDS. One critical step in the process of apoptosis consists in the permeabilization of mitochondrial membranes, leading to the release of proteins which normally are secluded behind the outer mitochondrial membrane[1]. For example, cytochrome c, which is normally confined to the mitochondrial intermembrane space, is liberated from mitochondria and interacts with a cytosolic protein, Apaf-1, causing its oligomerization and constitution of the so-called apoptosome, a protein complex which activates a specific class of cysteine proteases, the caspases[2]. Another example concerns the so-called apoptosis-inducing factor (AIF, another mitochondrial intermembrane protein which can translocate to the nucleus where it induces chromatin condensation and DNA fragmentation[3].

  11. mPGES-1-derived prostaglandin E2 stimulates Stat3 to promote podocyte apoptosis.

    Science.gov (United States)

    Yu, Jing; Wu, Yimei; Wang, Lu; Zhang, Wen; Xu, Man; Song, Jiayu; Fu, Yu; Cui, Yiyun; Gong, Wei; Li, Shuzhen; Xia, Weiwei; Huang, Songming; Zhang, Aihua; Jia, Zhanjun

    2017-11-01

    We previously reported that microsomal prostaglandin E synthase-1 (mPGES-1) contributed to adriamycin (Adr)-induced podocyte apoptosis. However, the molecular mechanism remains unclear. Here we studied the role of mPGES-1/PGE2 cascade in activating Stat3 signaling and the contribution of Stat3 in PGE2- and Adr-induced podocyte apoptosis. In murine podocytes, PGE2 dose- and time-dependently increased the phosphorylation of Stat3 in line with the enhanced cell apoptosis and reduced podocyte protein podocin. In agreement with the increased Stat3 phosphorylation, Stat3-derived cytokines including IL-6, IL-17, MCP-1, and ICAM-1 were significantly upregulated following PGE2 treatment. By application of a specific Stat3 inhibitor S3I-201, PGE2-induced podocyte apoptosis was largely abolished in parallel with a blockade of podocin reduction. Next, we observed that Adr treatment also enhanced p-Stat3 and activated mPGES-1/PGE2 cascade. Blockade of Stat3 by S3I-201 significantly ameliorated Adr-induced cell apoptosis and podocin reduction. More interestingly, silencing mPGES-1 in podocytes by mPGES-1 siRNA blocked Adr-induced increments of Stat-3 phosphorylation, PGE2 production, and Stat3-derived inflammatory cytokines. Taken together, this study suggested that mPGES-1-derived PGE2 could activate Stat3 signaling to promote podocyte apoptosis. Targeting mPGES-1/PGE2/Stat3 signaling might be a potential strategy for the treatment of podocytopathy.

  12. Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis

    International Nuclear Information System (INIS)

    Graaf, Aniek O. de; Heuvel, Lambert P. van den; Dijkman, Henry B.P.M.; Abreu, Ronney A. de; Birkenkamp, Kim U.; Witte, Theo de; Reijden, Bert A. van der; Smeitink, Jan A.M.; Jansen, Joop H.

    2004-01-01

    Bcl-2 family proteins regulate apoptosis at the level of mitochondria. To examine the mechanism of Bcl-2 function, we investigated the effects of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on two hematopoietic cell lines and Bcl-2 overexpressing transfectants. CCCP directly interferes with mitochondrial function and induces apoptosis. We show that Bcl-2 inhibits apoptosis and that the antiapoptotic effect of Bcl-2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Bcl-2 does not prevent the decrease in mitochondrial membrane potential nor the alterations in cellular ATP content induced by CCCP in FL5.12 and Jurkat cells. A higher number of mitochondria was observed in untreated Bcl-2 transfected cells compared to parental cells, as shown by electron microscopy. Exposure to CCCP induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, with apparent swelling and loss of cristae in parental cells. Bcl-2 clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. These data suggest that CCCP induces apoptosis by structural disruption of mitochondria and that Bcl-2 prevents apoptosis and mitochondrial degeneration by preserving mitochondrial integrity

  13. Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis.

    Science.gov (United States)

    de Graaf, Aniek O; van den Heuvel, Lambert P; Dijkman, Henry B P M; de Abreu, Ronney A; Birkenkamp, Kim U; de Witte, Theo; van der Reijden, Bert A; Smeitink, Jan A M; Jansen, Joop H

    2004-10-01

    Bcl-2 family proteins regulate apoptosis at the level of mitochondria. To examine the mechanism of Bcl-2 function, we investigated the effects of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on two hematopoietic cell lines and Bcl-2 overexpressing transfectants. CCCP directly interferes with mitochondrial function and induces apoptosis. We show that Bcl-2 inhibits apoptosis and that the antiapoptotic effect of Bcl-2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Bcl-2 does not prevent the decrease in mitochondrial membrane potential nor the alterations in cellular ATP content induced by CCCP in FL5.12 and Jurkat cells. A higher number of mitochondria was observed in untreated Bcl-2 transfected cells compared to parental cells, as shown by electron microscopy. Exposure to CCCP induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, with apparent swelling and loss of cristae in parental cells. Bcl-2 clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. These data suggest that CCCP induces apoptosis by structural disruption of mitochondria and that Bcl-2 prevents apoptosis and mitochondrial degeneration by preserving mitochondrial integrity.

  14. Effect of bFGF on radiation-induced apoptosis of vascular endothelial cells

    International Nuclear Information System (INIS)

    Gu Qingyang; Wang Dewen; Li Yuejuan; Peng Ruiyun; Dong Bo; Wang Zhaohai; Liu Jie; Deng Hua; Jiang Tao

    2003-01-01

    Objective: To study the effect of bFGF on radiation-induced apoptosis vascular endothelial cells. Methods: A cell line PAE (porcine aortic endothelial cells) and primary cultured HUVEC (human umbilical vein endothelial cells) were irradiated with 60 Co γ-rays to establish cell apoptosis models. Flow cytometry with annexin-V-FITC + PI labeling was used to evaluate cell apoptosis. Different amounts of bFGF were used to study their effects on radiation-induced endothelial cell apoptosis. Results and Conclusions: It is found that bFGF could inhibit radiation-induced endothelial cell apoptosis in a considerable degree

  15. Neem oil limonoids induces p53-independent apoptosis and autophagy.

    Science.gov (United States)

    Srivastava, Pragya; Yadav, Neelu; Lella, Ravi; Schneider, Andrea; Jones, Anthony; Marlowe, Timothy; Lovett, Gabrielle; O'Loughlin, Kieran; Minderman, Hans; Gogada, Raghu; Chandra, Dhyan

    2012-11-01

    Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.

  16. Neem oil limonoids induces p53-independent apoptosis and autophagy

    Science.gov (United States)

    Chandra, Dhyan

    2012-01-01

    Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells. PMID:22915764

  17. Hypoxia-induced p53 modulates both apoptosis and radiosensitivity via AKT

    NARCIS (Netherlands)

    Leszczynska, K.B.; Foskolou, I.P.; Abraham, A.G.; Anbalagan, S.; Tellier, C.; Haider, S.; Span, P.N.; O'Neill, E.E.; Buffa, F.M.; Hammond, E.M.

    2015-01-01

    Restoration of hypoxia-induced apoptosis in tumors harboring p53 mutations has been proposed as a potential therapeutic strategy; however, the transcriptional targets that mediate hypoxia-induced p53-dependent apoptosis remain elusive. Here, we demonstrated that hypoxia-induced p53-dependent

  18. Andrographolide induces apoptotic and non-apoptotic death and enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in gastric cancer cells

    OpenAIRE

    Lim, Sung-Chul; Jeon, Ho Jong; Kee, Keun Hong; Lee, Mi Ja; Hong, Ran; Han, Song Iy

    2017-01-01

    Andrographolide, a natural compound isolated from Andrographis paniculata, has been reported to possess antitumor activity. In the present study, the effect of andrographolide in human gastric cancer (GC) cells was investigated. Andrographolide induced cell death with apoptotic and non-apoptotic features. At a low concentration, andrographolide potentiated apoptosis and reduction of clonogenicity triggered by recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL)....

  19. related apoptosis-inducing ligand in transplastomic tobacco

    African Journals Online (AJOL)

    -inducing ligand (sTRAIL) can, as the whole length TRAIL protein, bind with its receptors and specifically induce the apoptosis of cancer cells; therefore, it has been developed as a potential therapeutic agent for various cancer treatments.

  20. Interdependence of Bad and Puma during ionizing-radiation-induced apoptosis.

    Science.gov (United States)

    Toruno, Cristhian; Carbonneau, Seth; Stewart, Rodney A; Jette, Cicely

    2014-01-01

    Ionizing radiation (IR)-induced DNA double-strand breaks trigger an extensive cellular signaling response that involves the coordination of hundreds of proteins to regulate DNA repair, cell cycle arrest and apoptotic pathways. The cellular outcome often depends on the level of DNA damage as well as the particular cell type. Proliferating zebrafish embryonic neurons are highly sensitive to IR-induced apoptosis, and both p53 and its transcriptional target puma are essential mediators of the response. The BH3-only protein Puma has previously been reported to activate mitochondrial apoptosis through direct interaction with the pro-apoptotic Bcl-2 family proteins Bax and Bak, thus constituting the role of an "activator" BH3-only protein. This distinguishes it from BH3-only proteins like Bad that are thought to indirectly promote apoptosis through binding to anti-apoptotic Bcl-2 family members, thereby preventing the sequestration of activator BH3-only proteins and allowing them to directly interact with and activate Bax and Bak. We have shown previously that overexpression of the BH3-only protein Bad in zebrafish embryos supports normal embryonic development but greatly sensitizes developing neurons to IR-induced apoptosis. While Bad has previously been shown to play only a minor role in promoting IR-induced apoptosis of T cells in mice, we demonstrate that Bad is essential for robust IR-induced apoptosis in zebrafish embryonic neural tissue. Moreover, we found that both p53 and Puma are required for Bad-mediated radiosensitization in vivo. Our findings show the existence of a hierarchical interdependence between Bad and Puma whereby Bad functions as an essential sensitizer and Puma as an essential activator of IR-induced mitochondrial apoptosis specifically in embryonic neural tissue.

  1. Cytosolic NADP(+)-dependent isocitrate dehydrogenase regulates cadmium-induced apoptosis.

    Science.gov (United States)

    Shin, Seoung Woo; Kil, In Sup; Park, Jeen-Woo

    2010-04-01

    Cadmium ions have a high affinity for thiol groups. Therefore, they may disturb many cellular functions. We recently reported that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme to supply NADPH, a major source of reducing equivalents to the cytosol. Cadmium decreased the activity of IDPc both as a purified enzyme and in cultured cells. In the present study, we demonstrate that the knockdown of IDPc expression in HEK293 cells greatly enhances apoptosis induced by cadmium. Transfection of HEK293 cells with an IDPc small interfering RNA significantly decreased the activity of IDPc and enhanced cellular susceptibility to cadmium-induced apoptosis as indicated by the morphological evidence of apoptosis, DNA fragmentation and condensation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. Taken together, our results suggest that suppressing the expression of IDPc enhances cadmium-induced apoptosis of HEK293 cells by increasing disruption of the cellular redox status. Copyright 2009 Elsevier Inc. All rights reserved.

  2. Nrdp1 Increases Ischemia Induced Primary Rat Cerebral Cortical Neurons and Pheochromocytoma Cells Apoptosis Via Downregulation of HIF-1α Protein

    Directory of Open Access Journals (Sweden)

    Yuan Zhang

    2017-09-01

    Full Text Available Neuregulin receptor degradation protein-1 (Nrdp1 is an E3 ubiquitin ligase that targets proteins for degradation and regulates cell growth, apoptosis and oxidative stress in various cell types. We have previously shown that Nrdp1 is implicated in ischemic cardiomyocyte death. In this study, we investigated the change of Nrdp1 expression in ischemic neurons and its role in ischemic neuronal injury. Primary rat cerebral cortical neurons and pheochromocytoma (PC12 cells were infected with adenoviral constructs expressing Nrdp1 gene or its siRNA before exposing to oxygen-glucose deprivation (OGD treatment. Our data showed that Nrdp1 was upregulated in ischemic brain tissue 3 h after middle cerebral artery occlusion (MCAO and in OGD-treated neurons. Of note, Nrdp1 overexpression by Ad-Nrdp1 enhanced OGD-induced neuron apoptosis, while knockdown of Nrdp1 with siRNA attenuated this effect, implicating a role of Nrdp1 in ischemic neuron injury. Moreover, Nrdp1 upregulation is accompanied by increased protein ubiquitylation and decreased protein levels of ubiquitin-specific protease 8 (USP8 in OGD-treated neurons, which led to a suppressed interaction between USP8 and HIF-1α and subsequently a reduction in HIF-1α protein accumulation in neurons under OGD conditions. In conclusion, our data support an important role of Nrdp1 upregulation in ischemic neuronal death, and suppressing the interaction between USP8 and HIF-1α and consequently the hypoxic adaptive response of neurons may account for this detrimental effect.

  3. Tumor necrosis factor related apoptosis inducing ligand triggers apoptosis in dividing but not in differentiating human epidermal keratinocytes

    NARCIS (Netherlands)

    Jansen, Bastiaan J. H.; van Ruissen, Fred; Cerneus, Stefanie; Cloin, Wendy; Bergers, Mieke; van Erp, Piet E. J.; Schalkwijk, Joost

    2003-01-01

    Using serial analysis of gene expression we have previously identified the expression of several pro-apoptotic and anti-apoptotic genes in cultured human primary epidermal keratinocytes, including tumor necrosis factor related apoptosis inducing ligand (TRAIL). TRAIL is a potent inducer of apoptosis

  4. Tempo enhances heat-induced apoptosis by mitochondrial targeting of Bax

    International Nuclear Information System (INIS)

    Zhao, Q.-L.; Fujiwara, Y.; Kondo, T.

    2003-01-01

    A stable membrane-permeable nitroxide, Tempo, exerts an SOD-like antioxidant activity against ROS. Reportedly, Tempo inhibits ROS-induced thymocyte apoptosis, while 10 mM Tempo activates JNK1 to induce apoptosis in breast cancer cells. We have observed that nontoxic 5 mM Tempo enhances suboptimal hyperthermia (44 deg C/10 min)-induced apoptosis in U937 cells. Here we report the enhancing mechanism, focusing on activation and targeting of Bax to mitochondria and cytochrome c release. Methods: U937 cells were treated with either Tempo (5 mM, 37 deg C/10 min), heating (44 deg C/10 min), or Tempo-plus-heating, washed and incubated for various times up to 6 h, until assessing apoptosis, mitochondrial potential (ΔΨ>), and amount of superoxide by flow cytometry using Annexin V-FITC/PI, TMRM, and dihydroethidium, respectively. Bax, Bcl-2 and Bcl-XL, and cytochrome c were detected by western blotting, activated Bax was by immunoprecipitation, and ATP was by a luciferase assay. Bax targeting to and cytochrome c release from mitochorndria were also detected immunocytochemically under fluorescent microscopy. Results and Discussion: Treatment of U937 cells with 5 mM Tempo for 10 min at 37 deg C or suboptimal heating (44 deg C/ 10 min) alone did not induce apoptosis. The combined treatment with 5 mM Tempo and 44 deg C for 10 min dramatically induced ∼90% apoptosis in 6 h, as did the 44 deg C/30 min heating. During the enhanced apoptosis, cytochrome c release progressed. Although signals of Bcl-2, Bcl-XL and Bax in cell lysates were not altered, Bax was specifically activated and translocated to mitochondria after the combined treatment. Further, loss of ΔΨ>and decreases in superoxide and ATP progressed after the combined treatment, suggesting that the treatment may disturb mitochondrial electron transport. Thus, Tempo sensitizes the heat-induced apoptosis through (1) targeting of Bax to mitochondria and releasing cytochrome c, and (2) mitochondrial dysfunction

  5. Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis.

    Directory of Open Access Journals (Sweden)

    Jintao Zhang

    (siRNA targeting BIP and CHOP methods, the induction of BIP, PDI, IRE1a, and LC3-II was blocked, but PARP cleavage was markedly enhanced.Taken together, these results suggested that sodium butyrate-induced autophagy was mediated by endoplasmic reticulum stress, and that preventing autophagy by blocking the endoplasmic reticulum stress response enhanced sodium butyrate-induced apoptosis. These results provide novel insights into the anti-tumor mechanisms of butyric acid.

  6. Ionizing radiation induces apoptosis in hematopoietic stem and progenitor cells

    International Nuclear Information System (INIS)

    Meng, A.; Zhou, D.; Geiger, H.; Zant, G.V.

    2003-01-01

    The aims of this study was to determine if ionizing radiation (IR) induces apoptosis in hematopoietic stem (HSC) and progenitor cells. Lin-cells were isolated from mouse bone marrow (BM) and pretreated with vehicle or 100 μM z-VAD 1 h prior to exposure to 4 Gy IR. The apoptotic and/or necrotic responses of these cells to IR were analyzed by measuring the annexin V and/or 7-AAD staining in HSC and progenitor populations using flow cytometry, and hematopoietic function of these cells was determined by CAFC assay. Exposure of Lin-cells to IR selectively decreased the numbers of HSC and progenitors in association with an increase in apoptosis in a time-dependent manner. Pretreatment of Lin- cells with z-VAD significantly inhibited IR-induced apoptosis and the decrease in the numbers of HSC and progenitors. However, IR alone or in combination with z-VAD did not lead to a significant increase in necrotic cell death in either HSC or progenitors. In addition, pretreatment of BM cells with z-VAD significantly attenuated IR-induced reduction in the frequencies of day-7, -28 and -35 CAFC. Exposure of HSC and progenitors to IR induces apoptosis. The induction of HSC and progenitor apoptosis contributes to IR-induced suppression of their hematopoietic function

  7. Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells

    International Nuclear Information System (INIS)

    Park, Jae Hyeon; Lee, Jeong Eun; Shin, In Chul; Koh, Hyun Chul

    2013-01-01

    Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. - Highlights: ► Chlorpyrifos (CPF) is cytotoxic to SH-SY5Y cells ► CPF-induced cytotoxicity is mediated by

  8. Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jae Hyeon [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); Lee, Jeong Eun [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Shin, In Chul [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Koh, Hyun Chul, E-mail: hckoh@hanyang.ac.kr [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of)

    2013-04-01

    Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. - Highlights: ► Chlorpyrifos (CPF) is cytotoxic to SH-SY5Y cells ► CPF-induced cytotoxicity is mediated by

  9. Modulation of iridovirus-induced apoptosis by endocytosis, early expression, JNK, and apical caspase

    International Nuclear Information System (INIS)

    Chitnis, Nilesh S.; D'Costa, Susan M.; Paul, Eric R.; Bilimoria, Shaen L.

    2008-01-01

    Chilo iridescent virus (CIV) is the type species for the family Iridoviridae, which are large, isometric, cytoplasmic dsDNA viruses. We examined the mechanism of apoptosis induction by CIV. High CIV doses (CIV XS ; 400 μg/ml), UV-irradiated virus (CIV UV ; 10 μg/ml) and CVPE (CIV protein extract; 10 μg/ml) induced apoptosis in 60% of treated Choristoneura fumiferana (IPRI-CF-124T) cells. Normal doses of infectious CIV (10 μg/ml) induced apoptosis in only 10% of C. fumiferana (CF) cells. Apoptosis was inhibited by Z-IETD-FMK, an apical caspase inhibitor, indicating that CIV-induced apoptosis requires caspase activity. The putative caspase in CF cells was designated Cf-caspase-i. CIV UV or CVPE enhanced Cf-caspase-i activity by 80% at 24 h relative to mock-treated cells. Since the MAP kinase pathway induces or inhibits apoptosis depending on the context, we used JNK inhibitor SP600125 and demonstrated drastic suppression of CVPE-induced apoptosis. Thus, the JNK signaling pathway is significant for apoptosis in this system. Virus interaction with the cell surface was not sufficient for apoptosis since CIV UV particles bound to polysterene beads failed to induce apoptosis. Endocytosis inhibitors (bafilomycin or ammonium chloride) negated apoptosis induction by CIV UV , CIV XS or CVPE indicating that entry through this mode is required. Given the weak apoptotic response to infectious CIV, we postulated that viral gene expression inhibited apoptosis. CIV infection of cells pretreated with cycloheximide induced apoptosis in 69% of the cells compared to 10% in normal infections. Furthermore, blocking viral DNA replication with aphidicolin or phosphonoacetic acid suppressed apoptosis and Cf-caspase-i activity, indicating that early viral expression is necessary for inhibition of apoptosis, and de novo synthesis of viral proteins is not required for induction. We show for the first time that, in a member of the family Iridoviridae, apoptosis: (i) requires entry and

  10. The role of apoptosis in MCLR-induced developmental toxicity in zebrafish embryos

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Cheng [College of Fisheries, Huazhong Agricultural University, Wuhan 430070 (China); Sun, Hong [Hubei Maternal and Child Health Hospital, Wuhan 430070 (China); Xie, Ping [Donghu Experimental Station of Lake Ecosystems, State Key Laboratory for Freshwater Ecology and Biotechnology of China, Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan 430072 (China); Wang, Jianghua; Zhang, Guirong; Chen, Nan [College of Fisheries, Huazhong Agricultural University, Wuhan 430070 (China); Yan, Wei, E-mail: Yanwei75126@163.com [Institute of Agricultural Quality Standards and Testing Technology, Hubei Academy of Agricultural Sciences, Wuhan 430064 (China); Li, Guangyu, E-mail: ligy2001@163.com [College of Fisheries, Huazhong Agricultural University, Wuhan 430070 (China); Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070 (China)

    2014-04-01

    Highlights: • MCLR-induced apoptosis in the heart of developing embryos leads to the growth delay in zebrafish. • MCLR-triggered apoptosis might be induced by ROS. • P53–Bax–Bcl-2 and caspase-dependent apoptotic pathway contribute greatly to MCLR-induced apoptosis. Abstract: We previously demonstrated that cyanobacteria-derived microcystin–leucine–arginine (MCLR) is able to induce developing toxicity, such as malformation, growth delay and also decreased heart rates in zebrafish embryos. However, the molecular mechanisms by which MCLR induces its toxicity during the development of zebrafish remain largely unknown. Here, we evaluate the role of apoptosis in MCLR-induced developmental toxicity. Zebrafish embryos were exposed to various concentrations of MCLR (0, 0.2, 0.5, 2, and 5.0 mg L⁻¹ for 96 h, at which time reactive oxygen species (ROS) was significantly induced in the 2 and 5.0 mg L⁻¹ MCLR exposure groups. Acridine orange (AO) staining and terminal deoxynucleotide transferase-mediated deoxy-UTP nick end labelling (TUNEL) assay showed that MCLR exposure resulted in cell apoptosis. To test the apoptotic pathway, the expression pattern of several apoptotic-related genes was examined for the level of enzyme activity, gene and protein expression, respectively. The overall results demonstrate that MCLR induced ROS which consequently triggered apoptosis in the heart of developing zebrafish embryos. Our results also indicate that the p53–Bax–Bcl-2 pathway and the caspase-dependent apoptotic pathway play major roles in MCLR-induced apoptosis in the developing embryos.

  11. Efficient construction of an inverted minimal H1 promoter driven siRNA expression cassette: facilitation of promoter and siRNA sequence exchange.

    Directory of Open Access Journals (Sweden)

    Hoorig Nassanian

    2007-08-01

    Full Text Available RNA interference (RNAi, mediated by small interfering RNA (siRNA, is an effective method used to silence gene expression at the post-transcriptional level. Upon introduction into target cells, siRNAs incorporate into the RNA-induced silencing complex (RISC. The antisense strand of the siRNA duplex then "guides" the RISC to the homologous mRNA, leading to target degradation and gene silencing. In recent years, various vector-based siRNA expression systems have been developed which utilize opposing polymerase III promoters to independently drive expression of the sense and antisense strands of the siRNA duplex from the same template.We show here the use of a ligase chain reaction (LCR to develop a new vector system called pInv-H1 in which a DNA sequence encoding a specific siRNA is placed between two inverted minimal human H1 promoters (approximately 100 bp each. Expression of functional siRNAs from this construct has led to efficient silencing of both reporter and endogenous genes. Furthermore, the inverted H1 promoter-siRNA expression cassette was used to generate a retrovirus vector capable of transducing and silencing expression of the targeted protein by>80% in target cells.The unique design of this construct allows for the efficient exchange of siRNA sequences by the directional cloning of short oligonucleotides via asymmetric restriction sites. This provides a convenient way to test the functionality of different siRNA sequences. Delivery of the siRNA cassette by retroviral transduction suggests that a single copy of the siRNA expression cassette efficiently knocks down gene expression at the protein level. We note that this vector system can potentially be used to generate a random siRNA library. The flexibility of the ligase chain reaction suggests that additional control elements can easily be introduced into this siRNA expression cassette.

  12. Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress.

    Science.gov (United States)

    Zhang, Qiang; Liu, Jianing; Chen, Shulan; Liu, Jing; Liu, Lijuan; Liu, Guirong; Wang, Fang; Jiang, Wenxin; Zhang, Caixia; Wang, Shuangyu; Yuan, Xiao

    2016-04-01

    It is well recognized that mandibular growth, which is caused by a variety of functional appliances, is considered to be the result of both neuromuscular and skeletal adaptations. Accumulating evidence has demonstrated that apoptosis plays an important role in the adaptation of skeletal muscle function. However, the underlying mechanism of apoptosis that is induced by stretch continues to be incompletely understood. Endoplasmic reticulum stress (ERS), a newly defined signaling pathway, initiates apoptosis. This study seeks to determine if caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress in myoblast and its underlying mechanism. Apoptosis was assessed by Hochest staining, DAPI staining and annexin V binding and PI staining. ER chaperones, such as GRP78, CHOP and caspase-12, were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Furthermore, caspase-12 inhibitor was used to value the mechanism of the caspase-12 pathway. Apoptosis of myoblast, which is subjected to cyclic stretch, was observed in a time-dependent manner. We found that GRP78 mRNA and protein were significantly increased and CHOP and caspase-12 were activated in myoblast that was exposed to cyclic stretch. Caspase-12 inhibition reduced stretch-induced apoptosis, and caspase-12 activated caspase-3 to induce apoptosis. We concluded that caspase-12 played an important role in stretch-induced apoptosis that is associated by endoplasmic reticulum stress by activating caspase-3.

  13. Inhibition of autophagy exerts anti-colon cancer effects via apoptosis induced by p53 activation and ER stress

    International Nuclear Information System (INIS)

    Sakitani, Kosuke; Hirata, Yoshihiro; Hikiba, Yohko; Hayakawa, Yoku; Ihara, Sozaburo; Suzuki, Hirobumi; Suzuki, Nobumi; Serizawa, Takako; Kinoshita, Hiroto; Sakamoto, Kei; Nakagawa, Hayato; Tateishi, Keisuke; Maeda, Shin; Ikenoue, Tsuneo; Kawazu, Shoji; Koike, Kazuhiko

    2015-01-01

    Although some molecularly targeted drugs for colorectal cancer are used clinically and contribute to a better prognosis, the current median survival of advanced colorectal cancer patients is not sufficient. Autophagy, a basic cell survival mechanism mediated by recycling of cellular amino acids, plays an important role in cancer. Recently, autophagy has been highlighted as a promising new molecular target. The unfolded protein response (UPR) reportedly act in complementary fashion with autophagy in intestinal homeostasis. However, the roles of UPR in colon cancer under autophagic inhibition remain to be elucidated. We aim to clarify the inhibitory effect of autophagy on colon cancer. We crossed K19 CreERT and Atg5 flox/flox mice to generate Atg5 flox/flox /K19 CreERT mice. Atg5 flox/flox /K19 CreERT mice were first treated with azoxymethane/dextran sodium sulfate and then injected with tamoxifen to inhibit autophagy in CK19-positive epithelial cells. To examine the anti-cancer mechanisms of autophagic inhibition, we used colon cancer cell lines harboring different p53 gene statuses, as well as small interfering RNAs (siRNAs) targeting Atg5 and immunoglobulin heavy-chain binding protein (BiP), a chaperone to aid folding of unfolded proteins. Colon tumors in Atg5 flox/flox /K19 CreERT mice showed loss of autophagic activity and decreased tumor size (the total tumor diameter was 28.1 mm in the control and 20.7 mm in Atg5 flox/flox /K19 CreERT mice, p = 0.036). We found that p53 and UPR/endoplasmic reticulum (ER) stress-related proteins, such as cleaved caspase 3, and CAAT/enhancer-binding protein homologous protein, are up-regulated in colon tumors of Atg5 flox/flox /K19 CreERT mice. Although Atg5 and BiP silencing, respectively, increased apoptosis in p53 wild type cells, Atg5 silencing alone did not show the same effect on apoptosis in p53 mutant cells. However, co-transfection of Atg5 and BiP siRNAs led to increased apoptosis in p53 mutant cells. Blocking autophagy

  14. Dihydroartemisinin induces apoptosis preferentially via a Bim-mediated intrinsic pathway in hepatocarcinoma cells.

    Science.gov (United States)

    Qin, Guiqi; Zhao, ChuBiao; Zhang, Lili; Liu, Hongyu; Quan, Yingyao; Chai, Liuying; Wu, Shengnan; Wang, Xiaoping; Chen, Tongsheng

    2015-08-01

    This report is designed to dissect the detail molecular mechanism by which dihydroartemisinin (DHA), a derivative of artemisinin, induces apoptosis in human hepatocellular carcinoma (HCC) cells. DHA induced a loss of the mitochondrial transmemberane potential (ΔΨm), release of cytochrome c, activation of caspases, and externalization of phosphatidylserine indicative of apoptosis induction. Compared with the modest inhibitory effects of silencing Bax, silencing Bak largely prevented DHA-induced ΔΨm collapse and apoptosis though DHA induced a commensurable activation of Bax and Bak, demonstrating a key role of the Bak-mediated intrinsic apoptosis pathway. DHA did not induce Bid cleavage and translocation from cytoplasm to mitochondria and had little effects on the expressions of Puma and Noxa, but did increase Bim and Bak expressions and decrease Mcl-1 expression. Furthermore, the cytotoxicity of DHA was remarkably reduced by silencing Bim, and modestly but significantly reduced by silencing Puma or Noxa. Silencing Bim or Noxa preferentially reduced DHA-induced Bak activation, while silencing Puma preferentially reduced DHA-induced Bax activation, demonstrating that Bim and to a lesser extent Noxa act as upstream mediators to trigger the Bak-mediated intrinsic apoptosis pathway. In addition, silencing Mcl-1 enhanced DHA-induced Bak activation and apoptosis. Taken together, our data demonstrate a crucial role of Bim in preferentially regulating the Bak/Mcl-1 rheostat to mediate DHA-induced apoptosis in HCC cells.

  15. Molecular mechanism of X-ray-induced p53-dependent apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Nakano, Hisako [Tokyo Metropolitan Inst. of Medical Center (Japan)

    1999-03-01

    Radiation-induced cell death has been classified into the interphase- and mitotic-ones, both of which apoptosis involving. This review described the molecular mechanism of the apoptosis, focusing on its p53-dependent process. It is known that there are genes regulating cell death either negatively or positively and the latter is involved in apoptosis. As an important factor in the apoptosis, p53 has become remarkable since it was shown that X-ray-induced apoptosis required RNA and protein syntheses in thymocytes and those cells of p53 gene-depleted mouse were shown to be resistant to gamma-ray-induced apoptosis. Radiation sensitivity of MOLT-4 cells derived from human T cell leukemia, exhibiting the typical X-ray-induced p53-dependent apoptosis, depends on the levels of p53 mRNA and protein. p53 is a gene suppressing tumor and also a transcription factor. Consequently, mutation of p53 conceivably leads to the failure of cell cycle regulation, which allows damaged cells to divide without both repair and exclusion due to loss of the apoptotic mechanism, and finally results in carcinogenesis. The radiation effect occurs in the order of the cell damage, inhibition of p53-Mdm2 binding, accumulation of p53, activation of mdm2 transcription, Mdm2 accumulation, p53-protein degradation and recovery to the steady state level. Here, the cystein protease (caspases) plays an important role as a disposing mechanism for cells scheduled to die. However, many are unknown to be solved in future. (K.H.) 119 refs.

  16. Osteoblasts Protect AML Cells from SDF-1-Induced Apoptosis

    Science.gov (United States)

    Kremer, Kimberly N.; Dudakovic, Amel; McGee-Lawrence, Meghan E.; Philips, Rachael L.; Hess, Allan D.; Smith, B. Douglas; van Wijnen, Andre J.; Karp, Judith E.; Kaufmann, Scott H.; Westendorf, Jennifer J.; Hedin, Karen E.

    2014-01-01

    The bone marrow provides a protective environment for acute myeloid leukemia (AML) cells that often allows leukemic stem cells to survive standard chemotherapeutic regimens. Targeting these leukemic stem cells within the bone marrow is critical for preventing relapse. We recently demonstrated that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis in AML cell lines and in patient samples expressing high levels of its receptor, CXCR4. Here we show that a subset of osteoblast lineage cells within the bone marrow can protect AML cells from undergoing apoptosis in response to the SDF-1 naturally present in that location. In co-culture systems, osteoblasts at various stages of differentiation protected AML cell lines and patient isolates from SDF-1-induced apoptosis. The differentiation of the osteoblast cell lines, MC3T3 and W-20-17, mediated this protection via a cell contact-independent mechanism. In contrast, bone marrow-derived mesenchymal cells, the precursors of osteoblasts, induced apoptosis in AML cells via a CXCR4-dependent mechanism and failed to protect AML cells from exogenously added SDF-1. These results indicate that osteoblasts in the process of differentiation potently inhibit the SDF-1-driven apoptotic pathway of CXCR4-expressing AML cells residing in the bone marrow. Drugs targeting this protective mechanism could potentially provide a new approach to treating AML by enhancing the SDF-1-induced apoptosis of AML cells residing within the bone marrow microenvironment. PMID:24851270

  17. Increased radiosensitivity and radiation-induced apoptosis in SRC-3 knockout mice

    International Nuclear Information System (INIS)

    Jin Jie; Wang Yu; Xu Yang; Chen Shilei; Wang Junping; Ran Xinze; Su Yongping; Wang Jin

    2014-01-01

    Steroid receptor coactivator-3 (SRC-3), a multifunctional transcriptional coactivator, plays an important role in regulation of cell apoptosis in chemoresistant cancer cells. However, its role in radiation-induced apoptosis in hematopoietic cells is still unclear. In this study, we used SRC-3 knockout (SRC-3 -/- ) mice to assess the role of SRC-3 in radiation-induced hematopoietic injury in vivo. After a range of doses of irradiation, SRC-3 -/- mice exhibited lower counts of peripheral blood cells and bone marrow (BM) mononuclear cells and excessive BM depression, which resulted in a significantly higher mortality compared with wildtype mice. Moreover, BM mononuclear cells obtained from SRC-3 -/- mice showed a remarkable increase in radiation-induced apoptosis. Collectively, our data demonstrate that SRC-3 plays a role in radiation-induced apoptosis of BM hematopoietic cells. Regulation of SRC-3 might influence the radiosensitivity of hematopoietic cells, which highlights a potential therapeutic target for radiation-induced hematopoietic injury. (author)

  18. Thermal Stability of siRNA Modulates Aptamer- conjugated siRNA Inhibition

    Directory of Open Access Journals (Sweden)

    Alexey Berezhnoy

    2012-01-01

    Full Text Available Oligonucleotide aptamer-mediated in vivo cell targeting of small interfering RNAs (siRNAs is emerging as a useful approach to enhance the efficacy and reduce the adverse effects resulting from siRNA-mediated genetic interference. A current main impediment in aptamer-mediated siRNA targeting is that the activity of the siRNA is often compromised when conjugated to an aptamer, often requiring labor intensive and time consuming design and testing of multiple configurations to identify a conjugate in which the siRNA activity has not been significantly reduced. Here, we show that the thermal stability of the siRNA is an important parameter of siRNA activity in its conjugated form, and that siRNAs with lower melting temperature (Tm are not or are minimally affected when conjugated to the 3′ end of 2′F-pyrimidine-modified aptamers. In addition, the configuration of the aptamer-siRNA conjugate retains activity comparable with the free siRNA duplex when the passenger strand is co-transcribed with the aptamer and 3′ overhangs on the passenger strand are removed. The approach described in this paper significantly reduces the time and effort necessary to screening siRNA sequences that retain biological activity upon aptamer conjugation, facilitating the process of identifying candidate aptamer-siRNA conjugates suitable for in vivo testing.

  19. Protection of betulin against cadmium-induced apoptosis in hepatoma cells

    International Nuclear Information System (INIS)

    Oh, Seon-Hee; Choi, Jeong-Eun; Lim, Sung-Chul

    2006-01-01

    The protective effects of betulin (BT) against cadmium (Cd)-induced cytotoxicity have been previously reported. However, the mechanisms responsible for these protective effects are unclear. Therefore, this study investigated the mechanisms responsible for the protection of BT against Cd-induced cytotoxicity in human hepatoma cell lines. The protection of BT against Cd cytotoxicity was more effective in the HepG2 than in the Hep3B cells. The protection of BT on Cd-induced cytotoxicity in the HepG2 cells appeared to be related to the inhibition of apoptosis, as determined by PI staining and DNA fragmentation analysis. The anti-apoptosis exerted by BT involved the blocking of Cd-induced reactive oxygen species (ROS) generation, the abrogation of the Cd-induced Fas upregulation, the blocking of caspase-8-dependent Bid activation, and subsequent inhibition of mitochondrial pathway. The BT pretreatment did not affect the p21 and p53 expression levels, when compared with those of the treated cells with Cd alone. BT induced the transient S phase arrest at an early stage and the G /G 1 arrest at a relatively late stage, but it did not observe the sub-G1 apoptotic peak. In the Hep3B cells, Cd did not induce ROS generation. The BT pretreatment partially inhibited the Cd-induced apoptosis, which was related with the incomplete blockage in caspase-9 or -3 activation, as well as in Bax activation. Taken together, it was found that Cd can induce apoptosis via the Fas-dependent and -independent apoptosis pathways. However, the observed protective effects of BT were clearly more sensitive to Fas-expressing HepG2 cells than to Fas-deficient Hep3B cells

  20. JS-K promotes apoptosis by inducing ROS production in human prostate cancer cells.

    Science.gov (United States)

    Qiu, Mingning; Chen, Lieqian; Tan, Guobin; Ke, Longzhi; Zhang, Sai; Chen, Hege; Liu, Jianjun

    2017-03-01

    Reactive oxygen species (ROS) are chemical species that alter redox status, and are responsible for inducing carcinogenesis. The purpose of the present study was to assess the effects of the glutathione S transferase-activated nitric oxide donor prodrug, JS-K, on ROS accumulation and on proliferation and apoptosis in human prostate cancer cells. Cell proliferation and apoptosis, ROS accumulation and the activation of the mitochondrial signaling pathway were measured. The results demonstrated that JS-K may inhibit prostate cancer cell growth in a dose- and time-dependent manner, and induce ROS accumulation and apoptosis in a dose-dependent manner. With increasing concentrations of JS-K, expression of pro-apoptotic proteins increased, but Bcl-2 expression decreased. Additionally, the antioxidant N-acetylcysteine reversed JS-K-induced cell apoptosis; conversely, the pro-oxidant glutathione disulfide exacerbated JS-K-induced apoptosis. In conclusion, the data suggest that JS-K induces prostate cancer cell apoptosis by increasing ROS levels.

  1. Ceramide-Induced Apoptosis in Renal Tubular Cells: A Role of Mitochondria and Sphingosine-1-Phoshate

    Science.gov (United States)

    Ueda, Norishi

    2015-01-01

    Ceramide is synthesized upon stimuli, and induces apoptosis in renal tubular cells (RTCs). Sphingosine-1 phosphate (S1P) functions as a survival factor. Thus, the balance of ceramide/S1P determines ceramide-induced apoptosis. Mitochondria play a key role for ceramide-induced apoptosis by altered mitochondrial outer membrane permeability (MOMP). Ceramide enhances oligomerization of pro-apoptotic Bcl-2 family proteins, ceramide channel, and reduces anti-apoptotic Bcl-2 proteins in the MOM. This process alters MOMP, resulting in generation of reactive oxygen species (ROS), cytochrome C release into the cytosol, caspase activation, and apoptosis. Ceramide regulates apoptosis through mitogen-activated protein kinases (MAPKs)-dependent and -independent pathways. Conversely, MAPKs alter ceramide generation by regulating the enzymes involving ceramide metabolism, affecting ceramide-induced apoptosis. Crosstalk between Bcl-2 family proteins, ROS, and many signaling pathways regulates ceramide-induced apoptosis. Growth factors rescue ceramide-induced apoptosis by regulating the enzymes involving ceramide metabolism, S1P, and signaling pathways including MAPKs. This article reviews evidence supporting a role of ceramide for apoptosis and discusses a role of mitochondria, including MOMP, Bcl-2 family proteins, ROS, and signaling pathways, and crosstalk between these factors in the regulation of ceramide-induced apoptosis of RTCs. A balancing role between ceramide and S1P and the strategy for preventing ceramide-induced apoptosis by growth factors are also discussed. PMID:25751724

  2. O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling.

    Science.gov (United States)

    Shi, Jianhua; Gu, Jin-hua; Dai, Chun-ling; Gu, Jianlan; Jin, Xiaoxia; Sun, Jianming; Iqbal, Khalid; Liu, Fei; Gong, Cheng-Xin

    2015-09-28

    Apoptosis plays an important role in neural development and neurological disorders. In this study, we found that O-GlcNAcylation, a unique protein posttranslational modification with O-linked β-N-acetylglucosamine (GlcNAc), promoted apoptosis through attenuating phosphorylation/activation of AKT and Bad. By using co-immunoprecipitation and mutagenesis techniques, we identified O-GlcNAc modification at both Thr308 and Ser473 of AKT. O-GlcNAcylation-induced apoptosis was attenuated by over-expression of AKT. We also found a dynamic elevation of protein O-GlcNAcylation during the first four hours of cerebral ischemia, followed by continuous decline after middle cerebral artery occlusion (MCAO) in the mouse brain. The elevation of O-GlcNAcylation coincided with activation of cell apoptosis. Finally, we found a negative correlation between AKT phosphorylation and O-GlcNAcylation in ischemic brain tissue. These results indicate that cerebral ischemia induces a rapid increase of O-GlcNAcylation that promotes apoptosis through down-regulation of AKT activity. These findings provide a novel mechanism through which O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling.

  3. Knockdown of HIF-1α and IL-8 induced apoptosis of hepatocellular carcinoma triggers apoptosis of vascular endothelial cells.

    Science.gov (United States)

    Choi, Sung Hoon; Park, Jun Yong; Kang, Wonseok; Kim, Seung Up; Kim, Do Young; Ahn, Sang Hoon; Ro, Simon Wonsang; Han, Kwang-Hyub

    2016-01-01

    A local hypoxic microenvironment is one of the most important characteristics of solid tumors. Hypoxia inducible factor-1α (HIF-1α) and Interleukin-8 (IL-8) activate tumor survival from hypoxic-induced apoptosis in each pathway. This study aimed to evaluate whether knockdown of HIF-1α and IL-8 induced apoptosis of the hepatocellular carcinoma (HCC) and endothelial cell lines. HCC cell lines were infected with adenovirus-expressing shRNA for HIF-1α and IL-8 and maintained under hypoxic conditions (1% O2, 24 h). The expression levels of HIF-1α and both apoptotic and growth factors were examined by real-time quantitative PCR and western blot. We also investigated apoptosis by TUNEL assay (FACS and Immunofluorescence) and measured the concentration of cytochrome C. Inhibition of HIF-1α and IL-8 up-regulated the expression of apoptotic factors while downregulating anti-apoptotic factors simultaneously. Knockdown of HIF-1α and IL-8 increased the concentration of cytochrome C and enhanced DNA fragmentation in HCC cell lines. Moreover, culture supernatant collected from the knockdown of HIF-1α and IL-8 in HCC cell lines induced apoptosis in human umbilical vein endothelial cells under hypoxia, and the expression of variable apoptotic ligand increased from HCC cell lines, time-dependently. These data suggest that adenovirus-mediated knockdown of HIF-1α and IL-8 induced apoptosis in HCC cells and triggered apoptosis of vascular endothelial cells.

  4. Binding of HIV-1 gp120 to DC-SIGN promotes ASK-1-dependent activation-induced apoptosis of human dendritic cells.

    Directory of Open Access Journals (Sweden)

    Yongxiong Chen

    2013-01-01

    Full Text Available During disease progression to AIDS, HIV-1 infected individuals become increasingly immunosuppressed and susceptible to opportunistic infections. It has also been demonstrated that multiple subsets of dendritic cells (DC, including DC-SIGN⁺ cells, become significantly depleted in the blood and lymphoid tissues of AIDS patients, which may contribute to the failure in initiating effective host immune responses. The mechanism for DC depletion, however, is unclear. It is also known that vast quantities of viral envelope protein gp120 are shed from maturing HIV-1 virions and form circulating immune complexes in the serum of HIV-1-infected individuals, but the pathological role of gp120 in HIV-1 pathogenesis remains elusive. Here we describe a previously unrecognized mechanism of DC death in chronic HIV-1 infection, in which ligation of DC-SIGN by gp120 sensitizes DC to undergo accelerated apoptosis in response to a variety of activation stimuli. The cultured monocyte-derived DC and also freshly-isolated DC-SIGN⁺ blood DC that were exposed to either cross-linked recombinant gp120 or immune-complex gp120 in HIV⁺ serum underwent considerable apoptosis after CD40 ligation or exposure to bacterial lipopolysaccharide (LPS or pro-inflammatory cytokines such as TNFα and IL-1β. Furthermore, circulating DC-SIGN⁺ DC that were isolated directly from HIV-1⁺ individuals had actually been pre-sensitized by serum gp120 for activation-induced exorbitant apoptosis. In all cases the DC apoptosis was substantially inhibited by DC-SIGN blockade. Finally, we showed that accelerated DC apoptosis was a direct consequence of excessive activation of the pro-apoptotic molecule ASK-1 and transfection of siRNA against ASK-1 significantly prevented the activation-induced excessive DC death. Our study discloses a previously unknown mechanism of immune modulation by envelope protein gp120, provides new insights into HIV immunopathogenesis, and suggests potential

  5. Apoptosis induced by high- and low-LET radiations

    International Nuclear Information System (INIS)

    Hendry, J.H.; Potten, C.S.; Merritt, A.

    1995-01-01

    Cell death after irradiation occurs by apoptosis in certain cell populations in tissues. The phenomenon also occurs after high linear energy transfer (LET) irradiation, and the relative biological effectiveness (RBE) is 3 to 4 (with respect to low-LET radiation and apoptosis in intestinal crypts) for neutrons with energies of 14 MeV and up to 600 MeV. It is thought that p53 plays a role in the phenomenon, as radiation-induced apoptosis is not observed in p53-null animals. (orig.)

  6. Mechanisms of methicillin-resistant Staphylococcus aureus pneumonia-induced intestinal epithelial apoptosis.

    Science.gov (United States)

    Perrone, Erin E; Jung, Enjae; Breed, Elise; Dominguez, Jessica A; Liang, Zhe; Clark, Andrew T; Dunne, W Michael; Burd, Eileen M; Coopersmith, Craig M

    2012-07-01

    Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia-induced sepsis is a common cause of morbidity in the intensive care unit. Although pneumonia is initiated in the lungs, extrapulmonary manifestations occur commonly. In light of the key role the intestine plays in the pathophysiology of sepsis, we sought to determine whether MRSA pneumonia induces intestinal injury. FVB/N mice were subjected to MRSA or sham pneumonia and killed 24 h later. Septic animals had a marked increase in intestinal epithelial apoptosis by both hematoxylin-eosin and active caspase 3 staining. Methicillin-resistant S. aureus-induced intestinal apoptosis was associated with an increase in the expression of the proapoptotic proteins Bid and Bax and the antiapoptotic protein Bcl-xL in the mitochondrial pathway. In the receptor-mediated pathway, MRSA pneumonia induced an increase in Fas ligand but decreased protein levels of Fas, FADD, pFADD, TNF-R1, and TRADD. To assess the functional significance of these changes, MRSA pneumonia was induced in mice with genetic manipulations in proteins in either the mitochondrial or receptor-mediated pathways. Both Bid-/- mice and animals with intestine-specific overexpression of Bcl-2 had decreased intestinal apoptosis compared with wild-type animals. In contrast, Fas ligand-/- mice had no alterations in apoptosis. To determine if these findings were organism-specific, similar experiments were performed in mice subjected to Pseudomonas aeruginosa pneumonia. Pseudomonas aeruginosa induced gut apoptosis, but unlike MRSA, this was associated with increased Bcl-2 and TNF-R1 and decreased Fas. Methicillin-resistant S. aureus pneumonia thus induces organism-specific changes in intestinal apoptosis via changes in both the mitochondrial and receptor-mediated pathways, although the former may be more functionally significant.

  7. Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

    International Nuclear Information System (INIS)

    Yang, Wei; Sun, Ting; Cao, Jianping; Liu, Fenju; Tian, Ye; Zhu, Wei

    2012-01-01

    Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1α (HIF-1α) and miR-210 expression and cell arrest in the G 0 /G 1 phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G 0 /G 1 phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: ► miR-210 downregulation radiosensitized hypoxic hepatoma. ► AIFM3 was identified as a direct target gene of miR-210. ► miR-210 might be a therapeutic target to hypoxic hepatoma.

  8. Cadmium induces apoptosis in pancreatic β-cells through a mitochondria-dependent pathway: the role of oxidative stress-mediated c-Jun N-terminal kinase activation.

    Directory of Open Access Journals (Sweden)

    Kai-Chih Chang

    Full Text Available Cadmium (Cd, one of well-known highly toxic environmental and industrial pollutants, causes a number of adverse health effects and diseases in humans. The growing epidemiological studies have suggested a possible link between Cd exposure and diabetes mellitus (DM. However, the toxicological effects and underlying mechanisms of Cd-induced pancreatic β-cell injury are still unknown. In this study, we found that Cd significantly decreased cell viability, and increased sub-G1 hypodiploid cells and annexin V-Cy3 binding in pancreatic β-cell-derived RIN-m5F cells. Cd also increased intracellular reactive oxygen species (ROS generation and malondialdehyde (MDA production and induced mitochondrial dysfunction (the loss of mitochondrial membrane potential (MMP and the increase of cytosolic cytochrome c release, the decreased Bcl-2 expression, increased p53 expression, poly (ADP-ribose polymerase (PARP cleavage, and caspase cascades, which accompanied with intracellular Cd accumulation. Pretreatment with the antioxidant N-acetylcysteine (NAC effectively reversed these Cd-induced events. Furthermore, exposure to Cd induced the phosphorylations of c-jun N-terminal kinases (JNK, extracellular signal-regulated kinases (ERK1/2, and p38-mitogen-activated protein kinase (MAPK, which was prevented by NAC. Additionally, the specific JNK inhibitor SP600125 or JNK-specific small interference RNA (si-RNA transfection suppressed Cd-induced β-cell apoptosis and related signals, but not ERK1/2 and p38-MAPK inhibitors (PD98059 and SB203580 did not. However, the JNK inhibitor or JNK-specific si-RNA did not suppress ROS generation in Cd-treated cells. These results indicate that Cd induces pancreatic β-cell death via an oxidative stress downstream-mediated JNK activation-triggered mitochondria-regulated apoptotic pathway.

  9. Endoplasmic reticulum stress-induced apoptosis accompanies enhanced expression of multiple inositol polyphosphate phosphatase 1 (Minpp1): a possible role for Minpp1 in cellular stress response.

    Science.gov (United States)

    Kilaparty, Surya P; Agarwal, Rakhee; Singh, Pooja; Kannan, Krishnaswamy; Ali, Nawab

    2016-07-01

    Inositol polyphosphates represent a group of differentially phosphorylated inositol metabolites, many of which are implicated to regulate diverse cellular processes such as calcium mobilization, vesicular trafficking, differentiation, apoptosis, etc. The metabolic network of these compounds is complex and tightly regulated by various kinases and phosphatases present predominantly in the cytosol. Multiple inositol polyphosphate phosphatase 1 (Minpp1) is the only known endoplasmic reticulum (ER) luminal enzyme that hydrolyzes various inositol polyphosphates in vitro as well as in vivo conditions. However, access of the Minpp1 to cytosolic substrates has not yet been demonstrated clearly and hence its physiological function. In this study, we examined a potential role for Minpp1 in ER stress-induced apoptosis. We generated a custom antibody and characterized its specificity to study the expression of Minpp1 protein in multiple mammalian cells under experimentally induced cellular stress conditions. Our results demonstrate a significant increase in the expression of Minpp1 in response to a variety of cellular stress conditions. The protein expression was corroborated with the expression of its mRNA and enzymatic activity. Further, in an attempt to link the role of Minpp1 to apoptotic stress, we studied the effect of Minpp1 expression on apoptosis following silencing of the Minpp1 gene by its specific siRNA. Our results suggest an attenuation of apoptotic parameters following knockdown of Minpp1. Thus, in addition to its known role in inositol polyphosphate metabolism, we have identified a novel role for Minpp1 as a stress-responsive protein. In summary, our results provide, for the first time, a probable link between ER stress-induced apoptosis and Minpp1 expression.

  10. Evaluation of the neuronal apoptotic pathways involved in cytoskeletal disruption-induced apoptosis.

    Science.gov (United States)

    Jordà, Elvira G; Verdaguer, Ester; Jimenez, Andrés; Arriba, S Garcia de; Allgaier, Clemens; Pallàs, Mercè; Camins, Antoni

    2005-08-01

    The cytoskeleton is critical to neuronal functioning and survival. Cytoskeletal alterations are involved in several neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. We studied the possible pathways involved in colchicine-induced apoptosis in cerebellar granule neurons (CGNs). Although colchicine evoked an increase in caspase-3, caspase-6 and caspase-9 activation, selective caspase inhibitors did not attenuate apoptosis. Inhibitors of other cysteine proteases such as PD150606 (a calpain-specific inhibitor), Z-Phe-Ala fluoromethyl ketone (a cathepsins-inhibitors) and N(alpha)-p-tosyl-l-lysine chloromethyl ketone (serine-proteases inhibitor) also had no effect on cell death/apoptosis induced by colchicine. However, BAPTA-AM 10 microM (intracellular calcium chelator) prevented apoptosis mediated by cytoskeletal alteration. These data indicate that calcium modulates colchicine-induced apoptosis in CGNs. PARP-1 inhibitors did not prevent apoptosis mediated by colchicine. Finally, colchicine-induced apoptosis in CGNs was attenuated by kenpaullone, a cdk5 inhibitor. Kenpaullone and indirubin also prevented cdk5/p25 activation mediated by colchicine. These findings indicate that cytoskeletal alteration can compromise cdk5 activation, regulating p25 formation and suggest that cdk5 inhibitors attenuate apoptosis mediated by cytoskeletal alteration. The present data indicate the potential therapeutic value of drugs that prevent the formation of p25 for the treatment of neurodegenerative disorders.

  11. [Study on thaspine in inducing apoptosis of A549 cell].

    Science.gov (United States)

    Zhang, Yan-min; He, Lang-chong

    2007-04-01

    To investigate the effect of thaspine on the cellular proliferation, apoptosis and cell cycle in A549 cell line. A549 cell was cultured with different concentrations of thaspine. Cellular proliferation was detected with MTT, apoptosis and cell cycle were checked with Flow Cytometer, and change of microstructure was observed by transmission electron microscope. Thaspine could inhibit the proliferation and induce apoptosis of A549 cell in a time-dose dependent manner. Cell cycle was significantly stopped at the S phase by thaspine with FCM technology. Under electronic microscope, the morphology of A549 cell showed nuclear karyopycnosis, chromatin agglutination and typical apoptotic body when the cell was treated with thaspine. Thaspine has the effects of anti-tumor and inducing apoptosis.

  12. Wnt1 inhibits hydrogen peroxide-induced apoptosis in mouse cardiac stem cells.

    Directory of Open Access Journals (Sweden)

    Jingjin Liu

    Full Text Available BACKGROUND: Because of their regenerative and paracrine abilities, cardiac stem cells (CSCs are the most appropriate, optimal and promising candidates for the development of cardiac regenerative medicine strategies. However, native and exogenous CSCs in ischemic hearts are exposed to various pro-apoptotic or cytotoxic factors preventing their regenerative and paracrine abilities. METHODS AND RESULTS: We examined the effects of H2O2 on mouse CSCs (mCSCs, and observed that hydrogen peroxide (H2O2 treatment induces mCSCs apoptosis via the caspase 3 pathway, in a dose-dependent manner. We then examined the effects of Wnt1 over-expression on H2O2-induced apoptosis in mCSCs and observed that Wnt1 significantly decreased H2O2-induced apoptosis in mCSCs. On the other hand, inhibition of the canonical Wnt pathway by the secreted frizzled related protein 2 (SFRP2 or knockdown of β-catenin in mCSCs reduced cells resistance to H2O2-induced apoptosis, suggesting that Wnt1 predominantly prevents H2O2-induced apoptosis through the canonical Wnt pathway. CONCLUSIONS: Our results provide the first evidences that Wnt1 plays an important role in CSCs' defenses against H2O2-induced apoptosis through the canonical Wnt1/GSK3β/β-catenin signaling pathway.

  13. Influenza virus induces apoptosis via BAD-mediated mitochondrial dysregulation.

    Science.gov (United States)

    Tran, Anh T; Cortens, John P; Du, Qiujiang; Wilkins, John A; Coombs, Kevin M

    2013-01-01

    Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication.

  14. Enhanced 15-HPETE production during oxidant stress induces apoptosis of endothelial cells.

    Science.gov (United States)

    Sordillo, Lorraine M; Weaver, James A; Cao, Yu-Zhang; Corl, Chris; Sylte, Matt J; Mullarky, Isis K

    2005-05-01

    Oxidant stress plays an important role in the etiology of vascular diseases by increasing rates of endothelial cell apoptosis, but few data exist on the mechanisms involved. Using a unique model of oxidative stress based on selenium deficiency (-Se), the effects of altered eicosanoid production on bovine aortic endothelial cells (BAEC) apoptosis was evaluated. Oxidant stress significantly increased the immediate oxygenation product of arachidonic acid metabolized by the 15-lipoxygenase pathway, 15-hydroxyperoxyeicosatetraenoic acid (15-HPETE). Treatment of -Se BAEC with TNFalpha/cyclohexamide (CHX) exhibited elevated levels of apoptosis, which was significantly reduced by the addition of a specific 15-lipoxygenase inhibitor PD146176. Furthermore, the addition of 15-HPETE to PD146176-treated BAEC, partially restored TNF/CHX-induced apoptosis. Increased exposure to 15-HPETE induced apoptosis, as determined by internucleosomal DNA fragmentation, chromatin condensation, caspase-3 activation, and caspase-9 activation, which suggests mitochondrial dysfunction. The expression of Bcl-2 protein also was decreased in -Se BAEC. Addition of a caspase-9 inhibitor (LEHD-fmk) completely blocked 15-HPETE-induced chromatin condensation in -Se BAEC, suggesting that 15-HPETE-induced apoptosis is caspase-9 dependent. Increased apoptosis of BAEC as a result of oxidant stress and subsequent production of 15-HPETE may play a critical role in a variety of inflammatory based diseases.

  15. Endoplasmic reticulum stress and IRE-1 signaling cause apoptosis in colon cancer cells in response to andrographolide treatment.

    Science.gov (United States)

    Banerjee, Aditi; Ahmed, Hafiz; Yang, Peixin; Czinn, Steven J; Blanchard, Thomas G

    2016-07-05

    The plant metabolite andrographolide induces cell cycle arrest and apoptosis in cancer cells. The mechanism(s) by which andrographolide induces apoptosis however, have not been elucidated. The present study was performed to determine the molecular events that promote apoptosis in andrographolide treated cells using T84, HCT116 and COLO 205 colon cancer cell lines. Andrographolide was determined to limit colony formation and Ki67 expression, alter nuclear morphology, increase cytoplasmic histone-associated-DNA-fragments, and increase cleaved caspase-3 levels. Andrographolide also induced significantly higher expression of endoplasmic reticulum (ER) stress proteins GRP-78 and IRE-1 by 48 h but not PERK or ATF6. Apoptosis signaling molecules BAX, spliced XBP-1 and CHOP were also significantly increased. Moreover, chemical inhibition of ER stress or IRE-1 depletion with siRNA in andrographolide treated cells significantly limited expression of IRE-1 and CHOP as determined by immunofluorescence staining, real time PCR, or immunobloting. This was accompanied by a decreased BAX/Bcl-2 ratio. Andrographolide significantly promotes cancer cell death compared to normal cells. These data demonstrate that andrographolide associated ER stress contributes to apoptosis through the activation of a pro-apoptotic GRP-78/IRE-1/XBP-1/CHOP signaling pathway.

  16. Myostatin induces mitochondrial metabolic alteration and typical apoptosis in cancer cells

    Science.gov (United States)

    Liu, Y; Cheng, H; Zhou, Y; Zhu, Y; Bian, R; Chen, Y; Li, C; Ma, Q; Zheng, Q; Zhang, Y; Jin, H; Wang, X; Chen, Q; Zhu, D

    2013-01-01

    Myostatin, a member of the transforming growth factor-β superfamily, regulates the glucose metabolism of muscle cells, while dysregulated myostatin activity is associated with a number of metabolic disorders, including muscle cachexia, obesity and type II diabetes. We observed that myostatin induced significant mitochondrial metabolic alterations and prolonged exposure of myostatin induced mitochondria-dependent apoptosis in cancer cells addicted to glycolysis. To address the underlying mechanism, we found that the protein levels of Hexokinase II (HKII) and voltage-dependent anion channel 1 (VDAC1), two key regulators of glucose metabolisms as well as metabolic stress-induced apoptosis, were negatively correlated. In particular, VDAC1 was dramatically upregulated in cells that are sensitive to myostatin treatment whereas HKII was downregulated and dissociated from mitochondria. Myostatin promoted the translocation of Bax from cytosol to mitochondria, and knockdown of VDAC1 inhibited myostatin-induced Bax translocation and apoptosis. These apoptotic changes can be partially rescued by repletion of ATP, or by ectopic expression of HKII, suggesting that perturbation of mitochondrial metabolism is causally linked with subsequent apoptosis. Our findings reveal novel function of myostatin in regulating mitochondrial metabolism and apoptosis in cancer cells. PMID:23412387

  17. Myeloperoxidase serves as a redox switch that regulates apoptosis in epithelial ovarian cancer.

    Science.gov (United States)

    Saed, Ghassan M; Ali-Fehmi, Rouba; Jiang, Zhong L; Fletcher, Nicole M; Diamond, Michael P; Abu-Soud, Husam M; Munkarah, Adnan R

    2010-02-01

    Resistance to apoptosis is a key feature of cancer cells and is believed to be regulated by nitrosonium ion (NO(+))-induced S-nitrosylation of key enzymes. Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), is utilized by MPO to generated NO(+). We sought to investigate the expression of myeloperoxidase (MPO) and iNOS in epithelial ovarian cancer (EOC) and determine their effect on S-nitrosylation of caspase-3 and its activity as well as apoptosis. MPO and iNOS expression were determined using immunofluorescence in SKOV-3 and MDAH-2774 and EOC tissue sections. S-nitrosylation of caspase-3 and its activity, levels of MPO and iNOS, as well as apoptosis, were evaluated in the EOC cells before and after silencing MPO or iNOS genes with specific siRNA probes utilizing real-time RT-PCR, ELISA, and TUNEL assays. MPO and iNOS are expressed in EOC cell lines and in over 60% of invasive EOC cases with no expression in normal ovarian epithelium. Indeed, silencing of MPO or iNOS gene expression resulted in decreased S-nitrosylation of caspase-3, increased caspase-3 activity, and increased apoptosis but with a more significant effect when silencing MPO. MPO and iNOS are colocalized to the same cells in EOC but not in the normal ovarian epithelium. Silencing of either MPO or iNOS significantly induced apoptosis, highlighting their role as a redox switch that regulates apoptosis in EOC. Understanding the mechanisms by which MPO functions as a redox switch in regulating apoptosis in EOC may lead to future diagnostic tools and therapeutic interventions. Copyright 2009 Elsevier Inc. All rights reserved.

  18. Metformin protects rat hepatocytes against bile acid-induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Titia E Woudenberg-Vrenken

    Full Text Available BACKGROUND: Metformin is used in the treatment of Diabetes Mellitus type II and improves liver function in patients with non-alcoholic fatty liver disease (NAFLD. Metformin activates AMP-activated protein kinase (AMPK, the cellular energy sensor that is sensitive to changes in the AMP/ATP-ratio. AMPK is an inhibitor of mammalian target of rapamycin (mTOR. Both AMPK and mTOR are able to modulate cell death. AIM: To evaluate the effects of metformin on hepatocyte cell death. METHODS: Apoptotic cell death was induced in primary rat hepatocytes using either the bile acid glycochenodeoxycholic acid (GCDCA or TNFα in combination with actinomycin D (actD. AMPK, mTOR and phosphoinositide-3 kinase (PI3K/Akt were inhibited using pharmacological inhibitors. Apoptosis and necrosis were quantified by caspase activation, acridine orange staining and Sytox green staining respectively. RESULTS: Metformin dose-dependently reduces GCDCA-induced apoptosis, even when added 2 hours after GCDCA, without increasing necrotic cell death. Metformin does not protect against TNFα/ActD-induced apoptosis. The protective effect of metformin is dependent on an intact PI3-kinase/Akt pathway, but does not require AMPK/mTOR-signaling. Metformin does not inhibit NF-κB activation. CONCLUSION: Metformin protects against bile acid-induced apoptosis and could be considered in the treatment of chronic liver diseases accompanied by inflammation.

  19. The small molecule survivin inhibitor YM155 may be an effective treatment modality for colon cancer through increasing apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Li, Wan Lu, E-mail: lvvlchina@msn.cn [Department of Pathology, Yonsei University Wonju College of Medicine, Wonju (Korea, Republic of); Lee, Mi-Ra, E-mail: mira1125@yonsei.ac.kr [Department of Pathology, Yonsei University Wonju College of Medicine, Wonju (Korea, Republic of); Cho, Mee-Yon, E-mail: meeyon@yonsei.ac.kr [Department of Pathology, Yonsei University Wonju College of Medicine, Wonju (Korea, Republic of); Institute of Genomic Cohort, Yonsei University Wonju College of Medicine, Wonju (Korea, Republic of)

    2016-03-04

    Survivin has a known beneficial role in the survival of both cancer cells and normal cells. Therapies targeting survivin have been proposed as an alternative treatment modality for various tumors; however, finding the proper indication for this toxic therapy is critical for reducing unavoidable side effects. We recently observed that high survivin expression in CD133{sup +} cells is related to chemoresistance in Caco-2 colon cancer cells. However, the effect of survivin-targeted therapy on CD133{sup +} colon cancer is unknown. In this study, we investigated the roles of CD133 and survivin expression in colon cancer biology in vitro and comparatively analyzed the anticancer effects of survivin inhibitor on CD133{sup +} cells (ctrl-siRNA group) and small interfering RNA (siRNA)-induced CD133{sup −} cells (CD133-siRNA group) obtained from a single colon cancer cell line. CD133 knockdown via siRNA transfection did not change the tumorigenicity of cells, although in vitro survivin expression levels in CD133{sup +} cells were higher than those in siRNA-induced CD133{sup −} cells. The transfection procedure seemed to induce survivin expression. Notably, a significant number of CD133{sup −} cells (33.8%) was found in the cell colonies of the CD133-siRNA group. In the cell proliferation assay after treatment, YM155 and a combination of YM155 and 5-fluorouracil (5-FU) proved to be far more effective than 5-FU alone. A significantly increased level of apoptosis was observed with increasing doses of YM155 in all groups. However, significant differences in therapeutic effect and apoptosis among the mock, ctrl-siRNA, and CD133-siRNA groups were not detected. Survivin inhibitor is an effective treatment modality for colon cancer; however, the role of CD133 and the use of survivin expression as a biomarker for this targeted therapy must be verified.

  20. The small molecule survivin inhibitor YM155 may be an effective treatment modality for colon cancer through increasing apoptosis

    International Nuclear Information System (INIS)

    Li, Wan Lu; Lee, Mi-Ra; Cho, Mee-Yon

    2016-01-01

    Survivin has a known beneficial role in the survival of both cancer cells and normal cells. Therapies targeting survivin have been proposed as an alternative treatment modality for various tumors; however, finding the proper indication for this toxic therapy is critical for reducing unavoidable side effects. We recently observed that high survivin expression in CD133"+ cells is related to chemoresistance in Caco-2 colon cancer cells. However, the effect of survivin-targeted therapy on CD133"+ colon cancer is unknown. In this study, we investigated the roles of CD133 and survivin expression in colon cancer biology in vitro and comparatively analyzed the anticancer effects of survivin inhibitor on CD133"+ cells (ctrl-siRNA group) and small interfering RNA (siRNA)-induced CD133"− cells (CD133-siRNA group) obtained from a single colon cancer cell line. CD133 knockdown via siRNA transfection did not change the tumorigenicity of cells, although in vitro survivin expression levels in CD133"+ cells were higher than those in siRNA-induced CD133"− cells. The transfection procedure seemed to induce survivin expression. Notably, a significant number of CD133"− cells (33.8%) was found in the cell colonies of the CD133-siRNA group. In the cell proliferation assay after treatment, YM155 and a combination of YM155 and 5-fluorouracil (5-FU) proved to be far more effective than 5-FU alone. A significantly increased level of apoptosis was observed with increasing doses of YM155 in all groups. However, significant differences in therapeutic effect and apoptosis among the mock, ctrl-siRNA, and CD133-siRNA groups were not detected. Survivin inhibitor is an effective treatment modality for colon cancer; however, the role of CD133 and the use of survivin expression as a biomarker for this targeted therapy must be verified.

  1. Physcion induces mitochondria-driven apoptosis in colorectal cancer cells via downregulating EMMPRIN.

    Science.gov (United States)

    Chen, Xuehong; Gao, Hui; Han, Yantao; Ye, Junli; Xie, Jing; Wang, Chunbo

    2015-10-05

    Physcion, an anthraquinone derivative widely isolated and characterized from both terrestrial and marine sources, has anti-tumor effects on a variety of carcinoma cells, mainly through inhibition of cell proliferation, apoptosis induction and cell cycle arrest. However, little is known about the mechanisms underlying its role in tumor progression. In the present study, we investigated the molecular mechanisms involved in physcion-induced apoptosis in human colorectal cancer (CRC) lines HCT116. Our results showed that physcion inhibited tumor cell viability in a dose- and time-dependent manner, and induced cell apoptosis via intrinsic mitochondrial pathway. Our results also revealed that physcion treatment significantly inhibited extracelluar matrix metalloproteinase inducer (EMMPRIN) expression in HCT116 cells in a dose-dependent manner and overexpression of EMMPRIN protein markedly reduced physcion-induced cell apoptosis. Furthermore, our results strongly indicated the modulating effect of physcion on EMMPRIN is correlated with AMP-activated protein kinase (AMPK)/Hypoxia-inducible factor 1α (HIF-1α) signaling pathway. Our data provide the first experimental evidence that physcion induces mitochondrial apoptosis in CRC cells by downregulating of EMMPRIN via AMPK/HIF-1α signaling pathway and suggest a new mechanism to explain its anti-tumor effects. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. PLK-1 Silencing in Bladder Cancer by siRNA Delivered With Exosomes.

    Science.gov (United States)

    Greco, Kristin A; Franzen, Carrie A; Foreman, Kimberly E; Flanigan, Robert C; Kuo, Paul C; Gupta, Gopal N

    2016-05-01

    To use exosomes as a vector to deliver small interfering ribonucleic acid (siRNA) to silence the polo-like kinase 1 (PLK-1) gene in bladder cancer cells. Exosomes were isolated from both human embryonic kidney 293 (HEK293) cell and mesenchymal stem cell (MSC) conditioned media. Fluorescently labeled exosomes were co-cultured with bladder cancer and normal epithelial cells and uptake was quantified by image cytometry. PLK-1 siRNA and negative control siRNA were loaded into HEK293 and MSC exosomes using electroporation. An invasive bladder cancer cell line (UMUC3) was co-cultured with the electroporated exosomes. Quantitative reverse transcriptase polymerase chain reaction was performed. Protein analysis was performed by Western blot. Annexin V staining and MTT assays were used to investigate effects on apoptosis and viability. Bladder cancer cell lines internalize an increased percentage of HEK293 exosomes when compared to normal bladder epithelial cells. Treatment of UMUC3 cells with exosomes electroporated with PLK-1 siRNA achieved successful knockdown of PLK-1 mRNA and protein when compared to cells treated with negative control exosomes. HEK293 and MSC exosomes were effectively used as a delivery vector to transport PLK-1 siRNA to bladder cancer cells in vitro, resulting in selective gene silencing of PLK-1. The use of exosomes as a delivery vector for potential intravesical therapy is attractive. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Arsenic-induced alteration in intracellular calcium homeostasis induces head kidney macrophage apoptosis involving the activation of calpain-2 and ERK in Clarias batrachus

    International Nuclear Information System (INIS)

    Banerjee, Chaitali; Goswami, Ramansu; Datta, Soma; Rajagopal, R.; Mazumder, Shibnath

    2011-01-01

    We had earlier shown that exposure to arsenic (0.50 μM) caused caspase-3 mediated head kidney macrophage (HKM) apoptosis involving the p38-JNK pathway in Clarias batrachus. Here we examined the roles of calcium (Ca 2+ ) and extra-cellular signal-regulated protein kinase (ERK), the other member of MAPK-pathway on arsenic-induced HKM apoptosis. Arsenic-induced HKM apoptosis involved increased expression of ERK and calpain-2. Nifedipine, verapamil and EGTA pre-treatment inhibited the activation of calpain-2, ERK and reduced arsenic-induced HKM apoptosis as evidenced from reduced caspase-3 activity, Annexin V-FITC-propidium iodide and Hoechst 33342 staining. Pre-incubation with ERK inhibitor U 0126 inhibited the activation of calpain-2 and interfered with arsenic-induced HKM apoptosis. Additionally, pre-incubation with calpain-2 inhibitor also interfered with the activation of ERK and inhibited arsenic-induced HKM apoptosis. The NADPH oxidase inhibitor apocynin and diphenyleneiodonium chloride also inhibited ERK activation indicating activation of ERK in arsenic-exposed HKM also depends on signals from NADPH oxidase pathway. Our study demonstrates the critical role of Ca 2+ homeostasis on arsenic-induced HKM apoptosis. We suggest that arsenic-induced alteration in intracellular Ca 2+ levels initiates pro-apoptotic ERK and calpain-2; the two pathways influence each other positively and induce caspase-3 mediated HKM apoptosis. Besides, our study also indicates the role of ROS in the activation of ERK pathway in arsenic-induced HKM apoptosis in C. batrachus. - Highlights: → Altered Ca 2+ homeostasis leads to arsenic-induced HKM apoptosis. → Calpain-2 plays a critical role in the process. → ERK is pro-apoptotic in arsenic-induced HKM apoptosis. → Arsenic-induced HKM apoptosis involves cross talk between calpain-2 and ERK.

  4. Radiation-induced apoptosis in different pH environments in vitro

    International Nuclear Information System (INIS)

    Lee, Hyung-Sik; Park, Heon J.; Lyons, John C.; Griffin, Robert J.; Auger, Elizabeth A.; Song, Chang W.

    1997-01-01

    Purpose: The effect of environmental pH on the radiation-induced apoptosis in tumor cells in vitro was investigated. Methods and Materials: Mammary adenocarcinoma cells of A/J mice (SCK cells) were irradiated with γ-rays using a 137 Cs irradiator and incubated in media of different pHs. After incubation at 37 deg. C for 24-120 h the extent of apoptosis was determined using agarose gel electrophoresis, TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining, flow cytometry, and release of 3 H from 3 H-thymidine labeled cells. The clonogenicity of the cells irradiated in different pH medium was determined, and the progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry. Results: Irradiation with 2-12 Gy of γ-rays induced apoptosis in SCK cells in pH 7.5 medium within 48 h as judged from the results of four different assays mentioned. Radiation-induced apoptosis declined as the medium pH was lowered from 7.5 to 6.4. Specifically, the radiation-induced degradation of DNA including the early DNA breaks, as determined with the TUNEL method, progressively declined as the medium pH was lowered so that little DNA fragmentation occurred 48 h after irradiation with 12 Gy in pH 6.6 medium. When the cells were irradiated and incubated for 48 h in pH 6.6 medium and the medium was replaced with pH 7.5 medium, DNA fragmentation promptly occurred. DNA fragmentation also occurred even in pH 6.6 medium when the cells were irradiated and maintained in pH 7.5 medium for 8 h or longer post-irradiation before incubation in pH 6.6 medium. The radiation-induced G 2 arrest in pH 6.6 medium lasted markedly longer than that in pH 7.5 medium. Conclusion: Radiation-induced apoptosis in SCK cells in vitro is reversibly suppressed in an acidic environment. Taking the results of four different assays together, it was concluded that early step(s) in the apoptotic pathway, probably the DNA break or upstream of DNA break, is

  5. Comparative study on 4 quantitative detection methods of apoptosis induced by radiation

    International Nuclear Information System (INIS)

    Yang Yepeng; Chen Guanying; Zhou Mei; Shen Qinjian; Shen Lei; Zhu Yingbao

    2004-01-01

    Objective: To reveal the capability of 4 apoptosis-detecting methods to discriminate between apoptosis and necrosis and show their respective advantages and shortcomings through comparison of detected results and analysis of detection mechanism. Methods: Four methods, PI staining-flow cytometric detection (P-F method), TUNEL labeling-flow cytometric detection (T-F method), annexing V-FITC/PI vital staining-flow cytometric detection (A-F method) and Hoechst/PI vital staining-fluorescence microscopic observation (H-O method), were used to determine apoptosis and necrosis in human breast cancer MCF-7 cell line induced by γ-rays. Hydroxycamptothecine and sodium azide were used to induce positive controls of apoptosis and necrosis respectively. Results: All 4 methods showed good time-dependent and dose dependent respondence to apoptosis induced by γ-rays and hydroxycamptothecine. Apoptotic cell ratios and curve slopes obtained from P-F method were minimum and, on the contrary, those from T-F method were maximum among these 4 methods. With A-F method and H-O method, two sets of data, apoptosis and necrosis, could be gained respectively and the data gained from these two methods were close to equal. A-F method and H-O method could distinguish necrosis induced by sodium azide from apoptosis while P-F method and T-F method presented false increase of apoptosis. Conclusions: P-F method and T-F method can not discriminate between apoptosis and necrosis. P-F method is less sensitive but more simple, convenient and economical than T-F method. A-F method and H-O method can distinguish necrosis from apoptosis. A-F method is more costly but more quick and reliable than H-O method. H-O method is economical, practical and morphological changes of cells and nucleus can be observed simultaneously with it. (authors)

  6. Transarterial chemoembolization of hepatocellular carcinoma in a rat model: the effect of additional injection of survivin siRNA to the treatment protocol

    International Nuclear Information System (INIS)

    Vogl, Thomas J.; Oppermann, Elsie; Qian, Jun; Imlau, Ulli; Tran, Andreas; Hamidavi, Yousef; Korkusuz, Huedayi; Bechstein, Wolf Otto; Nour-Eldin, Nour-Eldin Abdel-Rehim; Gruber-Rouh, Tatjana; Hammerstingl, Renate; Naguib, Nagy Naguib Naeem

    2016-01-01

    Transarterial chemoembolization is one of the most widely accepted interventional treatment options for treatment of hepatocellular carcinoma. Still there is a lack of a standard protocol regarding the injected chemotherapeutics. Survivin is an inhibitor of Apoptosis protein that functions to inhibit apoptosis, promote proliferation, and enhance invasion. Survivin is selectively up-regulated in many human tumors. Small interfering RNA (siRNA) can trigger an RNA interference response in mammalian cells and induce strong inhibition of specific gene expression including Survivin. The aim of the study is to assess the effectiveness of the additional injection of Survivin siRNA to the routine protocol of Transarterial Chemoembolization (TACE) for the treatment of hepatocellular carcinoma in a rat model. The study was performed on 20 male ACI rats. On day 0 a solid Morris Hepatoma 3924A was subcapsullary implanted in the liver. On day 12 MRI measurement of the initial tumor volume (V1) was performed. TACE was performed on day 13. The rats were divided into 2 groups; Group (A, n = 10) in which 0.1 mg mitomycin, 0.1 ml lipiodol and 5.0 mg degradable starch microspheres were injected in addition 2.5 nmol survivin siRNA were injected. The same agents were injected in Group (B,=10) without Survivin siRNA. MRI was repeated on day 25 to assess the tumor volume (V2). The tumor growth ratio (V2/V1) was calculated. Western blot and immunohistochemical analysis were performed. For group A the mean tumor growth ratio (V2/V1) was 1.1313 +/− 0.1381, and was 3.1911 +/− 0.1393 in group B. A statistically significant difference between both groups was observed regarding the inhibition of tumor growth (P < 0.0001) where Group A showed more inhibition compared to Group B. Similarly immunohistochemical analysis showed significantly lower (p < 0.002) VEGF staining in group A compared to group B. Western Blot analysis showed a similar difference in VEGF expression (P < 0.0001). The

  7. Aspirin Induces Apoptosis through Release of Cytochrome c from Mitochondria

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    Katja C. Zimmermann

    2000-01-01

    Full Text Available Nonsteroidal anti-inflammatory drugs (NSAID reduce the risk for cancer, due to their anti proliferative and apoptosis-inducing effects. A critical pathway for apoptosis involves the release of cytochrome c from mitochondria, which then interacts with Apaf-1 to activate caspase proteases that orchestrate cell death. In this study we found that treatment of a human cancer cell line with aspirin induced caspase activation and the apoptotic cell morphology, which was blocked by the caspase inhibitor zVAD-fmk. Further analysis of the mechanism underlying this apoptotic event showed that aspirin induces translocation of Bax to the mitochondria and triggers release of cytochrome c into the cytosol. The release of cytochrome c from mitochondria was inhibited by overexpression of the antiapoptotic protein Bcl-2 and cells that lack Apaf-1 were resistant to aspirin-induced apoptosis. These data provide evidence that the release of cytochrome c is an important part of the apoptotic mechanism of aspirin.

  8. Effects of ceramide inhibition on radiation-induced apoptosis in human leukemia MOLT-4 cells

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Eriko; Inanami, Osamu; Asanuma, Taketoshi; Kuwabara, Mikinori [Hokkaido Univ., Graduate School of Veterinary Medicine, Sapporo, Hokkaido (Japan)

    2006-03-15

    In the present study, using inhibitors of ceramide synthase (fumonisin B{sub 1}), ketosphinganine synthetase (L-cycloserine), acid sphingomyelinase (D609 and desipramine) and neutral sphingomyelinase (GW4869), the role of ceramide in X-ray-induced apoptosis was investigated in MOLT-4 cells. The diacylglycerol kinase (DGK) assay showed that the intracellular concentration of ceramide increased time-dependently after X irradiation of cells, and this radiation-induced accumulation of ceramide did not occur prior to the appearance of apoptotic cells. Treatment with D609 significantly inhibited radiation-induced apoptosis, but did not inhibit the increase of intracellular ceramide. Treatment with desipramine or GW4869 prevented neither radiation-induced apoptosis nor the induced increase of ceramide. On the other hand, fumonisin B{sub 1} and L-cycloserine had no effect on the radiation-induced induction of apoptosis, in spite of significant inhibition of the radiation-induced ceramide. From these results, it was suggested that the increase of the intracellular concentration of ceramide was not essential for radiation-induced apoptosis in MOLT-4 cells. (author)

  9. Effects of ceramide inhibition on radiation-induced apoptosis in human leukemia MOLT-4 cells

    International Nuclear Information System (INIS)

    Takahashi, Eriko; Inanami, Osamu; Asanuma, Taketoshi; Kuwabara, Mikinori

    2006-01-01

    In the present study, using inhibitors of ceramide synthase (fumonisin B 1 ), ketosphinganine synthetase (L-cycloserine), acid sphingomyelinase (D609 and desipramine) and neutral sphingomyelinase (GW4869), the role of ceramide in X-ray-induced apoptosis was investigated in MOLT-4 cells. The diacylglycerol kinase (DGK) assay showed that the intracellular concentration of ceramide increased time-dependently after X irradiation of cells, and this radiation-induced accumulation of ceramide did not occur prior to the appearance of apoptotic cells. Treatment with D609 significantly inhibited radiation-induced apoptosis, but did not inhibit the increase of intracellular ceramide. Treatment with desipramine or GW4869 prevented neither radiation-induced apoptosis nor the induced increase of ceramide. On the other hand, fumonisin B 1 and L-cycloserine had no effect on the radiation-induced induction of apoptosis, in spite of significant inhibition of the radiation-induced ceramide. From these results, it was suggested that the increase of the intracellular concentration of ceramide was not essential for radiation-induced apoptosis in MOLT-4 cells. (author)

  10. Arsenic induced apoptosis in rat liver following repeated 60 days exposure

    International Nuclear Information System (INIS)

    Bashir, Somia; Sharma, Yukti; Irshad, M.; Nag, T.C.; Tiwari, Monica; Kabra, M.; Dogra, T.D.

    2006-01-01

    Background: Accumulation of the wide spread environmental toxin arsenic in liver results in hepatotoxcity. Exposure to arsenite and other arsenicals has been previously shown to induce apoptosis in certain tumor cell lines at low (1-3 μM) concentration. Aim: The present study was focused to elucidate the role of free radicals in arsenic toxicity and to investigate the nature of in vivo sodium arsenite induced cell death in liver. Methods: Male wistar rats were exposed to arsenite at three different doses of 0.05, 2.5 and 5 mg/l for 60 days. Oxidative stress in liver was measured by estimating pro-oxidant and antioxidant activity in liver. Histopathological examination of liver was carried out by light and transmission electron microscopy. Analysis of DNA fragmentation by gel electrophoresis was used to identify apoptosis after the exposure. Terminal deoxy-nucleotidyl transferase mediated dUTP Nick end-labeling (TUNEL) assay was used to qualify and quantify apoptosis. Results: A significant increase in cytochrome-P450 and lipid peroxidation accompanied with a significant alteration in the activity of many of the antioxidants was observed, all suggestive of arsenic induced oxidative stress. Histopathological examination under light and transmission electron microscope suggested a combination of ongoing necrosis and apoptosis. DNA-TUNEL showed an increase in apoptotic cells in liver. Agarose gel electrophoresis of DNA of hepatocytes resulted in a characteristic ladder pattern. Conclusion: Chronic arsenic administration induces a specific pattern of apoptosis called post-mitotic apoptosis

  11. Decoy receptor 3 suppresses FasL-induced apoptosis via ERK1/2 activation in pancreatic cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yi; Li, Dechun; Zhao, Xin; Song, Shiduo; Zhang, Lifeng; Zhu, Dongming [Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Wang, Zhenxin [Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Chen, Xiaochen [Department of Pathology, The Obstetrics & Gynecology Hospital of Fudan University, Shanghai 200090 (China); Zhou, Jian, E-mail: zhoujian20150602@126.com [Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China)

    2015-08-07

    Resistance to Fas Ligand (FasL) mediated apoptosis plays an important role in tumorigenesis. Decoy receptor 3 (DcR3) is reported to interact with FasL and is overexpressed in some malignant tumors. We sought to investigate the role of DcR3 in resistance to FasL in pancreatic cancer. We compared expression of apoptosis related genes between FasL-resistant SW1990 and FasL-sensitive Patu8988 pancreatic cell lines by microarray analysis. We explored the impact of siRNA knockdown of, or exogenous supplementation with, DcR3 on FasL-induced cell growth inhibition in pancreatic cancer cell lines and expression of proteins involved in apoptotic signaling. We assessed the level of DcR3 protein and ERK1/2 phosphorylation in tumor and non-tumor tissue samples of 66 patients with pancreatic carcinoma. RNAi knockdown of DcR3 expression in SW1990 cells reduced resistance to FasL-induced apoptosis, and supplementation of Patu8988 with rDcR3 had the opposite effect. RNAi knockdown of DcR3 in SW1990 cells elevated expression of caspase 3, 8 and 9, and reduced ERK1/2 phosphorylation (P < 0.05), but did not alter phosphorylated-Akt expression. 47 tumor tissue specimens, but only 15 matched non-tumor specimens stained for DcR3 (χ{sup 2} = 31.1447, P < 0.001). The proliferation index of DcR3 positive specimens (14.26  ±  2.67%) was significantly higher than that of DcR3 negative specimens (43.58  ±  7.88%, P < 0.01). DcR3 expression positively correlated with p-ERK1/2 expression in pancreatic cancer tissues (r = 0.607, P < 0.001). DcR3 enhances ERK1/2 phosphorylation and opposes FasL signaling in pancreatic cancer cells. - Highlights: • We investigated the role of DcR3 in FasL resistance in pancreatic cancer. • Knockdown of DcR3 in SW1990 cells reduced resistance to FasL-induced apoptosis. • DcR3 knockdown also elevated caspase expression, and reduced ERK1/2 phosphorylation. • Tumor and non-tumor tissues were collected from 66 pancreatic carcinoma patients

  12. Stearoyl-CoA Desaturase-1 Protects Cells against Lipotoxicity-Mediated Apoptosis in Proximal Tubular Cells

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    Tamaki Iwai

    2016-11-01

    Full Text Available Saturated fatty acid (SFA-related lipotoxicity is a pathogenesis of diabetes-related renal proximal tubular epithelial cell (PTEC damage, closely associated with a progressive decline in renal function. This study was designed to identify a free fatty acid (FFA metabolism-related enzyme that can protect PTECs from SFA-related lipotoxicity. Among several enzymes involved in FFA metabolism, we identified stearoyl-CoA desaturase-1 (SCD1, whose expression level significantly decreased in the kidneys of high-fat diet (HFD-induced diabetic mice, compared with non-diabetic mice. SCD1 is an enzyme that desaturates SFAs, converting them to monounsaturated fatty acids (MUFAs, leading to the formation of neutral lipid droplets. In culture, retrovirus-mediated overexpression of SCD1 or MUFA treatment significantly ameliorated SFA-induced apoptosis in PTECs by enhancing intracellular lipid droplet formation. In contrast, siRNA against SCD1 exacerbated the apoptosis. Both overexpression of SCD1 and MUFA treatment reduced SFA-induced apoptosis via reducing endoplasmic reticulum stress in cultured PTECs. Thus, HFD-induced decrease in renal SCD1 expression may play a pathogenic role in lipotoxicity-induced renal injury, and enhancing SCD1-mediated desaturation of SFA and subsequent formation of neutral lipid droplets may become a promising therapeutic target to reduce SFA-induced lipotoxicity. The present study provides a novel insight into lipotoxicity in the pathogenesis of diabetic nephropathy.

  13. Study of progesterone mechanisms in radio-induced apoptosis prevention

    International Nuclear Information System (INIS)

    Vares, G.

    2004-10-01

    The purpose of this work was to study the modulation of radiation-induced cell death of human mammary tumoral cells by progesterone. On the one hand, we observed that progesterone protects cells against radiation-induced apoptosis and increases the proportion of surviving and proliferating damaged cells. On the other hand, a transcriptome analysis was undertaken in irradiated cells treated by progesterone, using DNA micro-arrays. This let us highlight several transcriptional dis-regulations that are likely to explain the protective effect of the hormone; in particular, we showed that progesterone regulates the expression of genes implicated in apoptosis signaling by death receptors. Knowing the crucial role of hormonal control and of apoptosis regulation in breast cancer initiation, our results may help to achieve a better understanding of the implication of progesterone in mammary carcinogenesis. (author)

  14. Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70.

    Science.gov (United States)

    Hsu, Shu-Fen; Hsu, Chuan-Chih; Cheng, Bor-Chih; Lin, Cheng-Hsien

    2014-11-01

    Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.

  15. Isolation and identification of gene mediating radiation-induced apoptosis in human leukemia U937 cells

    International Nuclear Information System (INIS)

    Tong Xin; Luo Ying; Dong Yan; Sun Zhixian

    1998-01-01

    Objective: Increasing evidences suggest that Caspase family proteases play an important role in the effector mechanism of apoptotic cell death. Radiation (IR) can induce apoptosis in tumor cells, so it is very important to isolate and identify the member of the Caspase family proteases involved in IR-induced apoptosis, and this would contribute to the understanding of the mechanism responsible for apoptosis execution. Methods: A PCR approach to isolate genes for IR-induced apoptosis was developed. The approach used degenerated oligonucleotide encoding the highly conserved peptides that were present in all known Caspases. Results: Protease inhibitors special for Caspases could block the apoptotic cell death caused by IR, and Caspase-3 was isolated from irradiated human leukemia U937 cells. Conclusion: Caspases involve in IR-induced apoptosis, and Caspase-3 is the pivotal element of IR-induced apoptosis

  16. CD36 Mediated Fatty Acid-Induced Podocyte Apoptosis via Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Wei Hua

    Full Text Available Hyperlipidemia-induced apoptosis mediated by fatty acid translocase CD36 is associated with increased uptake of ox-LDL or fatty acid in macrophages, hepatocytes and proximal tubular epithelial cells, leading to atherosclerosis, liver damage and fibrosis in obese patients, and diabetic nephropathy (DN, respectively. However, the specific role of CD36 in podocyte apoptosis in DN with hyperlipidemia remains poorly investigated.The expression of CD36 was measured in paraffin-embedded kidney tissue samples (Ctr = 18, DN = 20 by immunohistochemistry and immunofluorescence staining. We cultured conditionally immortalized mouse podocytes (MPC5 and treated cells with palmitic acid, and measured CD36 expression by real-time PCR, Western blot analysis and immunofluorescence; lipid uptake by Oil red O staining and BODIPY staining; apoptosis by flow cytometry assay, TUNEL assay and Western blot analysis; and ROS production by DCFH-DA fluorescence staining. All statistical analyses were performed using SPSS 21.0 statistical software.CD36 expression was increased in kidney tissue from DN patients with hyperlipidemia. Palmitic acid upregulated CD36 expression and promoted its translocation from cytoplasm to plasma membrane in podocytes. Furthermore, palmitic acid increased lipid uptake, ROS production and apoptosis in podocytes, Sulfo-N-succinimidyloleate (SSO, the specific inhibitor of the fatty acid binding site on CD36, decreased palmitic acid-induced fatty acid accumulation, ROS production, and apoptosis in podocytes. Antioxidant 4-hydroxy-2,2,6,6- tetramethylpiperidine -1-oxyl (tempol inhibited the overproduction of ROS and apoptosis in podocytes induced by palmitic acid.CD36 mediated fatty acid-induced podocyte apoptosis via oxidative stress might participate in the process of DN.

  17. Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wei, E-mail: detachedy@yahoo.com.cn [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Sun, Ting [Brain and Nerve Research Laboratory, The First Affiliated Hospital, Soochow University, Suzhou (China); Cao, Jianping; Liu, Fenju [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Tian, Ye [Department of Radiotherapy and Oncology, The Second Affiliated Hospital, Soochow University, Suzhou (China); Zhu, Wei [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China)

    2012-05-01

    Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1{alpha} (HIF-1{alpha}) and miR-210 expression and cell arrest in the G{sub 0}/G{sub 1} phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G{sub 0}/G{sub 1} phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: Black-Right-Pointing-Pointer miR-210 downregulation radiosensitized hypoxic hepatoma. Black-Right-Pointing-Pointer AIFM3 was identified as a direct target gene of miR-210. Black-Right-Pointing-Pointer miR-210 might be a therapeutic target to hypoxic hepatoma.

  18. Fast neutrons-induced apoptosis is Fas-independent in lymphoblastoid cells

    International Nuclear Information System (INIS)

    Fischer, Barbara; Benzina, Sami; Jeannequin, Pierre; Dufour, Patrick; Bergerat, Jean-Pierre; Denis, Jean-Marc; Gueulette, John; Bischoff, Pierre L.

    2005-01-01

    We have previously shown that ionizing radiation-induced apoptosis in human lymphoblastoid cells differs according to their p53 status, and that caspase 8-mediated cleavage of BID is involved in the p53-dependent pathway. In the present study, we investigated the role of Fas signaling in caspase 8 activation induced by fast neutrons irradiation in these cells. Fas and FasL expression was assessed by flow cytometry and by immunoblot. We also measured Fas aggregation after irradiation by fluorescence microscopy. We found a decrease of Fas expression after irradiation, but no change in Fas ligand expression. We also showed that, in contrast to the stimulation of Fas by an agonistic antibody, Fas aggregation did not occur after irradiation. Altogether, our data strongly suggest that fast neutrons induced-apoptosis is Fas-independent, even in p53-dependent apoptosis

  19. Atorvastatin Inhibits Myocardial Apoptosis in a Swine Model of Coronary Microembolization by Regulating PTEN/PI3K/Akt Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Jiangyou Wang

    2016-01-01

    Full Text Available Background/Aims: Phosphatase and tensin homolog deleted on chromosome ten (PTEN has been recognized as a promoter of apoptosis in various tissues, and revealed to be up-regulated in circumstances of coronary microembolization (CME. However, whether this functional protein could be modified by pretreatment of atorvastatin in models of CME has not been disclosed yet. Methods: Swine CME was induced by intra-coronary injection of inertia plastic microspheres (diameter 42 μm into left anterior descending coronary, with or without pretreatment of atorvastatin or PTEN siRNA. Echocardiologic measurements, pathologic examination, TUNEL staining and western blotting were applied to assess their functional, morphological and molecular effects in CME. Results: PTEN were aberrantly up-regulated in cardiomyocytes following CME, with both the mRNA and protein levels increased after CME modeling. Pretreatment with atorvastatin could attenuate the induction of PTEN. Furthermore, down-regulation of PTEN in vivo via siRNA was associated with an improved cardiac function, attenuated myocardial apoptosis, and concomitantly inhibited expressions of key proapoptotic proteins such as Bax, cleaved-caspase-3. Interestingly, atorvastatin could markedly attenuate PTEN expression and therefore partially reverse cardiac dysfunction and attenuate the apoptosis of the myocardium following CME. Conclusion: Modulation of PTEN was probably as a potential mechanism involved in the beneficial effects of pretreatment of atorvastatin to cardiac function and apoptosis in large animal models of CME.

  20. Curcumin induces apoptosis of upper aerodigestive tract cancer cells by targeting multiple pathways.

    Directory of Open Access Journals (Sweden)

    A R M Ruhul Amin

    Full Text Available Curcumin, a natural compound isolated from the Indian spice "Haldi" or "curry powder", has been used for centuries as a traditional remedy for many ailments. Recently, the potential use of curcumin in cancer prevention and therapy urges studies to uncover the molecular mechanisms associated with its anti-tumor effects. In the current manuscript, we investigated the mechanism of curcumin-induced apoptosis in upper aerodigestive tract cancer cell lines and showed that curcumin-induced apoptosis is mediated by the modulation of multiple pathways such as induction of p73, and inhibition of p-AKT and Bcl-2. Treatment of cells with curcumin induced both p53 and the related protein p73 in head and neck and lung cancer cell lines. Inactivation of p73 by dominant negative p73 significantly protected cells from curcumin-induced apoptosis, whereas ablation of p53 by shRNA had no effect. Curcumin treatment also strongly inhibited p-AKT and Bcl-2 and overexpression of constitutively active AKT or Bcl-2 significantly inhibited curcumin-induced apoptosis. Taken together, our findings suggest that curcumin-induced apoptosis is mediated via activating tumor suppressor p73 and inhibiting p-AKT and Bcl-2.

  1. Effect of cycloheximide and actinomycin D on radionuclide 235U-induced apoptosis

    International Nuclear Information System (INIS)

    Fu Qiang; Zhang Lansheng; Zhu Shoupeng

    1999-01-01

    Objective: The mechanism of apoptosis induced by radionuclide 235 U was studied. Methods: MTT and JAM assay were used to analyse the cell viability and quantification of fragmented DNA. Results: The inhibitor of protein cycloheximide (CHX), and the inhibitor of RNA synthesis, actinomycin D. cannot inhibit the apoptosis induced by 235 U, but CHX can partly inhibit apoptotic cells DNA fragmentation. Conclusion: The pathway of apoptosis induced by radionuclide 235 U is different from X-and γ-ray external irradiation, protein synthesis is not essential for it, but synthetic endonuclease is necessary for DNA fragmentation of apoptotic cells

  2. Protein Kinase C-{delta} mediates down-regulation of heterogeneous nuclear ribonucleoprotein K protein: involvement in apoptosis induction

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Feng-Hou [NO.3 People' s Hospital affiliated to Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 201900 (China); The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Wu, Ying-Li [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Zhao, Meng [Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China); Liu, Chuan-Xu; Wang, Li-Shun [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Chen, Guo-Qiang, E-mail: chengq@shsmu.edu.cn [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China)

    2009-11-15

    We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C delta ({Delta}PKC-{delta}). By subcellular proteome analysis, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as being significantly down-regulated in NSC606985-treated leukemic NB4 cells. HnRNP K, a docking protein for DNA, RNA, and transcriptional or translational molecules, is implicated in a host of processes involving the regulation of gene expression. However, the molecular mechanisms of hnRNP K reduction and its roles during apoptosis are still not understood. In the present study, we found that, following the appearance of the {Delta}PKC-{delta}, hnRNP K protein was significantly down-regulated in NSC606985, doxorubicin, arsenic trioxide and ultraviolet-induced apoptosis. We further provided evidence that {Delta}PKC-{delta} mediated the down-regulation of hnRNP K protein during apoptosis: PKC-{delta} inhibitor could rescue the reduction of hnRNP K; hnRNP K failed to be decreased in PKC-{delta}-deficient apoptotic KG1a cells; conditional induction of {Delta}PKC-{delta} in U937T cells directly down-regulated hnRNP K protein. Moreover, the proteasome inhibitor also inhibited the down-regulation of hnRNP K protein by apoptosis inducer and the conditional expression of {Delta}PKC-{delta}. More intriguingly, the suppression of hnRNP K with siRNA transfection significantly induced apoptosis. To our knowledge, this is the first demonstration that proteolytically activated PKC-{delta} down-regulates hnRNP K protein in a proteasome-dependent manner, which plays an important role in apoptosis induction.

  3. Atrazine-induced apoptosis of splenocytes in BALB/C mice

    Directory of Open Access Journals (Sweden)

    Zheng Jing

    2011-10-01

    Full Text Available Abstract Background Atrazine (2-chloro-4-ethytlamino-6-isopropylamine-1,3,5-triazine; ATR, is the most commonly applied broad-spectrum herbicide in the world. Unintentional overspray of ATR poses an immune function health hazard. The biomolecular mechanisms responsible for ATR-induced immunotoxicity, however, are little understood. This study presents on our investigation into the apoptosis of splenocytes in mice exposed to ATR as we explore possible immunotoxic mechanisms. Methods Oral doses of ATR were administered to BALB/C mice for 21 days. The histopathology, lymphocyte apoptosis and the expression of apoptosis-related proteins from the Fas/Fas ligand (FasL apoptotic pathway were examined from spleen samples. Results Mice administered ATR exhibited a significant decrease in spleen and thymus weight. Electron microscope histology of ultrathin sections of spleen revealed degenerative micromorphology indicative of apoptosis of splenocytes. Flow cytometry revealed that the percentage of apoptotic lymphocytes increased in a dose-dependent manner after ATR treatment. Western blots identified increased expression of Fas, FasL and active caspase-3 proteins in the treatment groups. Conclusions ATR is capable of inducing splenocytic apoptosis mediated by the Fas/FasL pathway in mice, which could be the potential mechanism underlying the immunotoxicity of ATR.

  4. Statins induce apoptosis in rat and human myotube cultures by inhibiting protein geranylgeranylation but not ubiquinone

    International Nuclear Information System (INIS)

    Johnson, Timothy E.; Zhang, Xiaohua; Bleicher, Kimberly B.; Dysart, Gary; Loughlin, Amy F.; Schaefer, William H.; Umbenhauer, Diane R.

    2004-01-01

    Statins are widely used to treat lipid disorders. These drugs are safe and well tolerated; however, in <1% of patients, myopathy and/or rhabdomyolysis can develop. To better understand the mechanism of statin-induced myopathy, we examined the ability of structurally distinct statins to induce apoptosis in an optimized rat myotube model. Compound A (a lactone) and Cerivastatin (an open acid) induced apoptosis, as measured by TUNEL and active caspase 3 staining, in a concentration- and time-dependent manner. In contrast, an epimer of Compound A (Compound B) exhibited a much weaker apoptotic response. Statin-induced apoptosis was completely prevented by mevalonate or geranylgeraniol, but not by farnesol. Zaragozic acid A, a squalene synthase inhibitor, caused no apoptosis on its own and had no effect on Compound-A-induced myotoxicity, suggesting the apoptosis was not a result of cholesterol synthesis inhibition. The geranylgeranyl transferase inhibitors GGTI-2133 and GGTI-2147 caused apoptosis in myotubes; the farnesyl transferase inhibitor FTI-277 exhibited a much weaker effect. In addition, the prenylation of rap1a, a geranylgeranylated protein, was inhibited by Compound A in myotubes at concentrations that induced apoptosis. A similar statin-induced apoptosis profile was seen in human myotube cultures but primary rat hepatocytes were about 200-fold more resistant to statin-induced apoptosis. Although the statin-induced hepatotoxicity could be attenuated with mevalonate, no effect was found with either geranylgeraniol or farnesol. In studies assessing ubiquinone levels after statin treatment in rat and human myotubes, there was no correlation between ubiquinone levels and apoptosis. Taken together, these observations suggest that statins cause apoptosis in myotube cultures in part by inhibiting the geranylgeranylation of proteins, but not by suppressing ubiquinone concentration. Furthermore, the data from primary hepatocytes suggests a cell-type differential

  5. Mycobacterium avium MAV2052 protein induces apoptosis in murine macrophage cells through Toll-like receptor 4.

    Science.gov (United States)

    Lee, Kang-In; Choi, Han-Gyu; Son, Yeo-Jin; Whang, Jake; Kim, Kwangwook; Jeon, Heat Sal; Park, Hye-Soo; Back, Yong Woo; Choi, Seunga; Kim, Seong-Woo; Choi, Chul Hee; Kim, Hwa-Jung

    2016-04-01

    Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨm), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨm loss, and apoptosis were all significantly reduced in TLR4(-/-) macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.

  6. Studying arsenic trioxide-induced apoptosis of Colo-16 cells with ...

    African Journals Online (AJOL)

    Jane

    2011-10-05

    Oct 5, 2011 ... induced apoptosis at the single cell level. Key words: Two-photon laser scanning microscopy, confocal laser scanning microscopy, human skin squamous carcinoma cells (Colo-16 cells), arsenic trioxide, apoptosis. INTRODUCTION. Although arsenic is poisonous and chronic arsenic exposure from ...

  7. Role of Bax in resveratrol-induced apoptosis of colorectal carcinoma cells

    International Nuclear Information System (INIS)

    Mahyar-Roemer, Mojgan; Köhler, Hans; Roemer, Klaus

    2002-01-01

    The natural plant polyphenol resveratrol present in some foods including grapes, wine, and peanuts, has been implicated in the inhibition, delay, and reversion of cellular events associated with heart diseases and tumorigenesis. Recent work has suggested that the cancer chemoprotective effect of the compound is primarily linked to its ability to induce cell division cycle arrest and apoptosis, the latter possibly through the activation of pro-apoptotic proteins such as Bax. The expression, subcellular localization, and importance of Bax for resveratrol-provoked apoptosis were assessed in human HCT116 colon carcinoma cells and derivatives with both bax alleles inactivated. Low to moderate concentrations of resveratrol induced co-localization of cellular Bax protein with mitochondria, collapse of the mitochondrial membrane potential, activation of caspases 3 and 9, and finally, apoptosis. In the absence of Bax, membrane potential collapse was delayed, and apoptosis was reduced but not absent. Resveratrol inhibited the formation of colonies by both HCT116 and HCT116 bax -/- cells. Resveratrol at physiological doses can induce a Bax-mediated and a Bax-independent mitochondrial apoptosis. Both can limit the ability of the cells to form colonies

  8. Susceptibility of different subsets of immature thymocytes to apoptosis induced by anti-TCRmAb

    International Nuclear Information System (INIS)

    Li Hongmei; Zhong Renqian; Yu Jiaping; Kong Xiantao; Chen Weifeng

    2003-01-01

    To analysis the susceptibility of different subsets of immature mice thymocytes to apoptosis induced by anti-TCRmAbs in vitro apoptosis was induced in unfractionated mice thymocytes by anti-TCRmAb. In Vivo apoptosis was induced in BALB/c mice by anti-TCR mAb, and thymocytes were examined by FACS. Results showed that CD4 + CD8 + DP thymocytes and CD4 - CD8 + CD3 - thymocytes were equally sensitive to apoptosis after treatment with the anti-TCR mAb. In sharp contrast, the early migrants or precursor containing thymocytes which are CD4 - CD8 - CD3 - TN have a lower spontaneous apoptosis rate and were relatively resistant to the anti-TCR mAb. The findings showed a breakpoint in thymocyte sensitivity to apoptosis which occurs after the onset of CD4 - CD8 + CD3 expression, suggesting that susceptibility of thymocytes to apoptosis is developmentally regulated

  9. Gonadal steroids modulate Fas-induced apoptosis of lactotropes and somatotropes.

    Science.gov (United States)

    Jaita, Gabriela; Zárate, Sandra; Ferrari, Luciana; Radl, Daniela; Ferraris, Jimena; Eijo, Guadalupe; Zaldivar, Verónica; Pisera, Daniel; Seilicovich, Adriana

    2011-02-01

    We have previously reported that Fas activation induces apoptosis of anterior pituitary cells from rats at proestrus but not at diestrus and in an estrogen-dependent manner. In this study, we evaluated the effect of Fas activation on apoptosis of lactotropes and somatotropes during the estrous cycle and explored the action of gonadal steroids on Fas-induced apoptosis. Also, we studied whether changes in Fas expression are involved in the apoptotic response of anterior pituitary cells. Fas activation increased the percentage of TUNEL-positive lactotropes and somatotropes at proestrus but not at diestrus. FasL triggered apoptosis of somatotropes only when cells from ovariectomized rats were cultured in the presence of 17 β-estradiol (E2). Progesterone (P4) blocked the apoptotic action of the Fas/FasL system in lactotropes and somatotropes incubated with E2. Both E2 and P4 increased the percentage of cells expressing Fas at the cell membrane. Our results show that Fas activation induces apoptosis of lactotropes and somatotropes at proestrus but not at diestrus. Gonadal steroids may be involved in the apoptotic response of lactotropes and somatotropes, suggesting that Fas activation is implicated in the renewal of these pituitary subpopulations during the estrous cycle. The effect of gonadal steroids on Fas expression may be only partially involved in regulation of the Fas/FasL apoptotic pathway in the anterior pituitary gland.

  10. Norcantharidin (NCTD) induces mitochondria mediated apoptosis in ...

    African Journals Online (AJOL)

    Administrator

    2011-06-15

    Jun 15, 2011 ... cancer deaths for both sexes being attributable to hepatoma. However ..... Resveratrol induces apoptosis and cell cycle arrest of human T24 bladder cancer cells in ... involvement of the CD95 receptor/ligand. J. Cancer. Res.

  11. Apoptosis of nasopharyngeal carcinoma cell line (CNE-2) induced by neutron irradiation

    International Nuclear Information System (INIS)

    Liang Ke; He Shaoqin; Feng Yan; Tang Jinhua; Feng Qinfu; Shen Yu; Yin Weibo; Xu Guozhen; Liu Xinfan; Wang Luhua; Gao Li

    1999-01-01

    Objective: To study the apoptotic response of the nasopharyngeal carcinoma cell line (CNE-2) induced by neutron irradiation. Methods: CNE-2 cells were cultured as usual. Using the techniques of DNA agarose gel electrophoresis and DNA special fluorescent staining, the status of apoptosis in CNE-2 cells after neutron irradiation was detected. Results: It was shown that the apoptosis can be induced in CNE-2 cell after neutron radiation. Six hrs, after different doses of neutron (0/0.667/1.333/2.000/2.667/3.333 Gy) and X-ray 0/2/4/6/8/10 Gy) irradiation the apoptotic rates were 2.4%, 6.3%, 7.1%, 9.5%, 13.5%, 14.6% and 2.4%, 3.8%, 5.7%, 7.8%, 10.4%, 11.7%, respectively; at 48 hrs they were 18.3%, 21.5%, 22.8%, 29.3%, 34.2% and 13.7%, 17.6%, 21.3%, 25.6%, 28.9%, respectively. At 10 hrs after neutron irradiation the DNA ladder of apoptosis could be detected between 0.667-3.333 Gy doses in CNE-2 cells by DNA agarose gel electrophoresis. Conclusion: Neutron radiation can induce apoptosis in tumor cells. Compared with the X-ray, neutron induces apoptosis in larger extent than X-ray in the same condition; meanwhile, apoptosis after irradiation is dose and time dependent

  12. Kinetics of apoptotic markers in exogeneously induced apoptosis of EL4 cells.

    Science.gov (United States)

    Jessel, Robert; Haertel, Steffen; Socaciu, Carmen; Tykhonova, Svetlana; Diehl, Horst A

    2002-01-01

    We investigated the time-dependence of apoptotic events in EL4 cells by monitoring plasma membrane changes in correlation to DNA fragmentation and cell shrinkage. We applied three apoptosis inducers (staurosporine, tubericidine and X-rays) and we looked at various markers to follow the early-to-late apoptotic events: phospholipid translocation (identified through annexin V-fluorescein assay and propidium iodide), lipid package (via merocyanine assay), membrane fluidity and anisotropy (via fluorescent measurements), DNA fragmentation by the fluorescence-labeling test and cell size measurements. The different apoptotic inducers caused different reactions of the cells: staurosporine induced apoptosis most rapidly in a high number of cells, tubercidine triggered apoptosis only in the S phase cells, while X-rays caused a G2/M arrest and subsequently apoptosis. Loss of lipid asymmetry is promptly detectable after one hour of incubation time. The phosphatidylserine translocation, decrease of lipid package and anisotropy, and the increase of membrane fluidity appeared to be based on the same process of lipid asymmetry loss. Therefore, the DNA fragmentation and the cell shrinkage appear to be parallel and independent processes running on different time scales but which are kinetically inter-related. The results indicate different signal steps to apoptosis dependent on inducer characteristics but the kinetics of "early-to-late" apoptosis appears to be a fixed program.

  13. Canine distemper virus induces apoptosis in cervical tumor derived cell lines

    Directory of Open Access Journals (Sweden)

    Rajão Daniela S

    2011-06-01

    Full Text Available Abstract Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi, by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control.

  14. Suppression of heat shock protein 70 by siRNA enhances the antitumor effects of cisplatin in cultured human osteosarcoma cells.

    Science.gov (United States)

    Mori, Yuki; Terauchi, Ryu; Shirai, Toshiharu; Tsuchida, Shinji; Mizoshiri, Naoki; Arai, Yuji; Kishida, Tsunao; Fujiwara, Hiroyoshi; Mazda, Osam; Kubo, Toshikazu

    2017-09-01

    Although advances in chemotherapy have improved the prognosis for osteosarcoma, some patients do not respond sufficiently to treatment. Heat shock protein 70 (Hsp70) is expressed at high levels in cancer cells and attenuates the therapeutic efficacy of anticancer agents, resulting in a poorer prognosis. This study investigated whether small interfering RNA (siRNA)-mediated inhibition of Hsp70 expression in an osteosarcoma cell line would enhance sensitivity to cisplatin. The expression of Hsp70 with cisplatin treatment was observed by using Western blotting and real-time reverse transcription polymerase chain reaction (RT-PCR). Changes in the IC 50 of cisplatin when Hsp70 was inhibited by siRNA were evaluated. Cisplatin's effectiveness in inducing apoptosis was assessed by assay of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), caspase-3 activity, and mitochondrial membrane potential. Up-regulation of Hsp70 expression was dependent on the concentration of cisplatin. Inhibition of Hsp70 expression significantly reduced the IC 50 of cisplatin. When cisplatin was added to osteosarcoma cells with Hsp70 expression inhibited, a significant increase in apoptosis was demonstrated in TUNEL, caspase-3, and mitochondrial membrane potential assays. Inhibition of Hsp70 expression induced apoptosis in cultured osteosarcoma cells, indicating that Hsp70 inhibition enhanced sensitivity to cisplatin. Inhibition of Hsp70 expression may provide a new adjuvant therapy for osteosarcoma.

  15. Butyrate down regulates BCL-XL and sensitizes human fibroblasts to radiation and chemotherapy induced apoptosis

    International Nuclear Information System (INIS)

    Chung, Diana H.; Ljungman, Mats; Zhang Fenfen; Chen Feng; McLaughlin, William P.

    1997-01-01

    Purpose/Objective: Butyrate is a short chain fatty acid that has been implicated in the induction of cell cycle arrest, cell differentiation and apoptosis. The purpose of this study was to determine if butyrate treatment sensitizes cells to radiation or chemotherapy induced apoptosis. Materials and Methods: Normal neonatal human diploid fibroblasts were used throughout this study. Apoptosis was scored and quantified using three different methods. First, cell morphology using propidium iodide and fluorescence microscopy was used to qualitatively determine apoptosis and to quantify the percentage of cells undergoing apoptosis. Second, apoptosis induced DNA degradation was scored by quantifying the amount of cells appearing in a sub-G1 peak using fixed and PI-stained cells and flow cytometry. Third, apoptosis-induced DNA degradation was examined by using an assay involving direct lysis of cells in the wells of agarose gels followed by conventional gel electrophoresis. Western blotting was used to quantify the cellular levels of the apoptosis regulators, Bcl-2, Bcl-XL and Bax. Results: Human diploid fibroblasts, which were resistant to radiation induced apoptosis, were found to undergo massive apoptosis when radiation was combined with butyrate treatment. Sensitization was obtained when butyrate was added before or after radiation although the combination of both pre and post-treatment was the most effective. Butyrate was also found to enhance UV light and cisplatin-induced apoptosis. These findings correlated with a reduction of the apoptosis antagonist Bcl-XL. Bcl-XL levels significantly dropped in a time and dose dependent manner. In addition, butyrate effectively blocked UV-induced accumulation of p53. Conclusion: Our results suggest that butyrate may be an attractive agent to use in combination with radiation or chemotherapy to lower the apoptotic threshold of tumor cells, regardless of the p53 status of the tumor cells

  16. A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Ren-Jie [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan (China); Lin, Su-Shuan [Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan (China); Wu, Wen-Ren [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Chen, Lih-Ren [Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan (China); Division of Physiology, Livestock Research Institute, Council of Agriculture, Taiwan (China); Li, Chien-Feng [Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan (China); Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan (China); National Institute of Cancer Research, National Health Research Institute, Tainan, Taiwan (China); Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Chen, Han-De; Chou, Chien-Ting; Chen, Ya-Chun [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Liang, Shih-Shin [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Chien, Shang-Tao [Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan (China); Shiue, Yow-Ling, E-mail: ylshiue@mail.nsysu.edu.tw [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung, Taiwan (China)

    2016-11-15

    The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G{sub 2}/M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G{sub 2}/M cell cycle regulators, ABT-751 induced G{sub 2}/M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. - Highlights: • An anti-microtubule agent, ABT-751, induces autophagy and apoptosis in Huh-7 cells.

  17. Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3

    International Nuclear Information System (INIS)

    Ozeki, Nobuaki; Hase, Naoko; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2015-01-01

    A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ) and IL-1β-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CM treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells. - Highlights: • Pro-inflammatory cytokines induce MMP-3 activity in human odontoblast-like cells. • Increased MMP-3 activity can promote cell proliferation in odontoblasts. • Specific loss of MMP-3 increases apoptosis in odontoblasts. • MMP-3 has potential as a promising new target for pupal repair and regeneration

  18. Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Hase, Naoko; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464-8651, Aichi (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan); Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464-8651, Aichi (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan)

    2015-02-01

    A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ) and IL-1β-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CM treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells. - Highlights: • Pro-inflammatory cytokines induce MMP-3 activity in human odontoblast-like cells. • Increased MMP-3 activity can promote cell proliferation in odontoblasts. • Specific loss of MMP-3 increases apoptosis in odontoblasts. • MMP-3 has potential as a promising new target for pupal repair and regeneration.

  19. Ultraviolet B irradiation of human leukaemia HL-60 cells in vitro induces apoptosis

    International Nuclear Information System (INIS)

    Martin, S.J.; Cotter, T.G.

    1991-01-01

    UV radiation is known to be a potent agent for the induction of programmed cell death (apoptosis) in human skin. However, the mechanistic aspects of UV-induced apoptosis remain ill-defined. In this study the effects of varying periods of UV-irradiation on the human leukaemia HL-60 cell line and on five other human cell lines were investigated.HL-60 cells were found to rapidly undergo apoptosis en masse after short periods of UV-irradiation whereas prolonged exposure of these cells to this form of radiation induced a more rapid form of cell death which was suggestive of necrosis, the pathological mode of cell death. UV-induced apoptosis in cell lines was characterized by morphological changes as well as DNA fragmentation into unit multiples of ∼ 200 bp, which was indicative of endogenous endonuclease activation. This DNA fragmentation pattern was not detected in cells immediately after UV-irradiation, and was therefore not the result of direct UV-induced DNA damage. UV-induced apoptosis of the HL-60 cell line was found to require extracellular calcium and to be inhibited in a dose-dependent way by zinc added to the culture medium. (author)

  20. SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity

    Energy Technology Data Exchange (ETDEWEB)

    Kubo, Takanori, E-mail: kubo-t@yasuda-u.ac.jp [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan); Yanagihara, Kazuyoshi [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan); Division of Genetics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Takei, Yoshifumi [Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumi-cho, Showa-ku, Nagoya 466-8550 (Japan); Mihara, Keichiro [Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553 (Japan); Sato, Yuichiro; Seyama, Toshio [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan)

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer SiRNAs conjugated with aromatic compounds (Ar-siRNAs) at 5 Prime -sense strand were synthesized. Black-Right-Pointing-Pointer Ar-siRNAs increased resistance against nuclease degradation. Black-Right-Pointing-Pointer Ar-siRNAs were thermodynamically stable compared with the unmodified siRNA. Black-Right-Pointing-Pointer High levels of cellular uptake and cytoplasmic localization were found. Black-Right-Pointing-Pointer Strong gene-silencing efficacy was exhibited in the Ar-siRNAs. -- Abstract: Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, such as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.

  1. Ultrasound-guided delivery of siRNA and a chemotherapeutic drug by using microbubble complexes: In vitro and in vivo evaluations in a prostate cancer model

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Yun Jung; Yoon, Young Il; Lee, Hak Jong [Dept. of Radiology, Seoul National University Bundang Hospital, Seongnam (Korea, Republic of); Yoon, Tae Jong [Dept. of Applied Bioscience, College of Life Science, CHA University, Pocheon (Korea, Republic of)

    2016-07-15

    To evaluate the effectiveness of ultrasound and microbubble-liposome complex (MLC)-mediated delivery of siRNA and doxorubicin into prostate cancer cells and its therapeutic capabilities both in vitro and in vivo. Microbubble-liposome complexes conjugated with anti-human epidermal growth factor receptor type 2 (Her2) antibodies were developed to target human prostate cancer cell lines PC-3 and LNCaP. Intracellular delivery of MLC was observed by confocal microscopy. We loaded MLC with survivin-targeted small interfering RNA (siRNA) and doxorubicin, and delivered it into prostate cancer cells. The release of these agents was facilitated by ultrasound application. Cell viability was analyzed by MTT assay after the delivery of siRNA and doxorubicin. Survivin-targeted siRNA loaded MLC was delivered into the xenograft mouse tumor model. Western blotting was performed to quantify the expression of survivin in vivo. Confocal microscopy demonstrated substantial intracellular uptake of MLCs in LNCaP, which expresses higher levels of Her2 than PC-3. The viability of LNCaP cells was significantly reduced after the delivery of MLCs loaded with siRNA and doxorubicin (85.0 ± 2.9%), which was further potentiated by application of ultrasound (55.0 ± 3.5%, p = 0.009). Survivin expression was suppressed in vivo in LNCaP tumor xenograft model following the ultrasound and MLC-guided delivery of siRNA (77.4 ± 4.90% to 36.7 ± 1.34%, p = 0.027). Microbubble-liposome complex can effectively target prostate cancer cells, enabling intracellular delivery of the treatment agents with the use of ultrasound. Ultrasound and MLC-mediated delivery of survivin-targeted siRNA and doxorubicin can induce prostate cell apoptosis and block survivin expression in vitro and in vivo.

  2. Ultrasound-Guided Delivery of siRNA and a Chemotherapeutic Drug by Using Microbubble Complexes: In Vitro and In Vivo Evaluations in a Prostate Cancer Model

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Yun Jung [Department of Radiology, Seoul National University Bundang Hospital, Seongnam 13620 (Korea, Republic of); Department of Radiology, Seoul National University College of Medicine, Seoul 03080 (Korea, Republic of); Yoon, Young Il [Department of Radiology, Seoul National University Bundang Hospital, Seongnam 13620 (Korea, Republic of); Department of Radiology, Seoul National University College of Medicine, Seoul 03080 (Korea, Republic of); Program in Nano Science and Technology, Department of Transdisciplinary Studies, Seoul National University Graduate School of Convergence Science and Technology, Suwon 16229 (Korea, Republic of); Yoon, Tae-Jong [Department of Applied Bioscience, College of Life Science, CHA University, Pocheon 11160 (Korea, Republic of); College of Pharmacy, Ajou University, Suwon 16499 (Korea, Republic of); Lee, Hak Jong [Department of Radiology, Seoul National University Bundang Hospital, Seongnam 13620 (Korea, Republic of); Department of Radiology, Seoul National University College of Medicine, Seoul 03080 (Korea, Republic of); Program in Nano Science and Technology, Department of Transdisciplinary Studies, Seoul National University Graduate School of Convergence Science and Technology, Suwon 16229 (Korea, Republic of)

    2016-11-01

    To evaluate the effectiveness of ultrasound and microbubble-liposome complex (MLC)-mediated delivery of siRNA and doxorubicin into prostate cancer cells and its therapeutic capabilities both in vitro and in vivo. Microbubble-liposome complexes conjugated with anti-human epidermal growth factor receptor type 2 (Her2) antibodies were developed to target human prostate cancer cell lines PC-3 and LNCaP. Intracellular delivery of MLC was observed by confocal microscopy. We loaded MLC with survivin-targeted small interfering RNA (siRNA) and doxorubicin, and delivered it into prostate cancer cells. The release of these agents was facilitated by ultrasound application. Cell viability was analyzed by MTT assay after the delivery of siRNA and doxorubicin. Survivin-targeted siRNA loaded MLC was delivered into the xenograft mouse tumor model. Western blotting was performed to quantify the expression of survivin in vivo. Confocal microscopy demonstrated substantial intracellular uptake of MLCs in LNCaP, which expresses higher levels of Her2 than PC-3. The viability of LNCaP cells was significantly reduced after the delivery of MLCs loaded with siRNA and doxorubicin (85.0 ± 2.9%), which was further potentiated by application of ultrasound (55.0 ± 3.5%, p = 0.009). Survivin expression was suppressed in vivo in LNCaP tumor xenograft model following the ultrasound and MLC-guided delivery of siRNA (77.4 ± 4.90% to 36.7 ± 1.34%, p = 0.027). Microbubble-liposome complex can effectively target prostate cancer cells, enabling intracellular delivery of the treatment agents with the use of ultrasound. Ultrasound and MLC-mediated delivery of survivin-targeted siRNA and doxorubicin can induce prostate cell apoptosis and block survivin expression in vitro and in vivo.

  3. Ultrasound-Guided Delivery of siRNA and a Chemotherapeutic Drug by Using Microbubble Complexes: In Vitro and In Vivo Evaluations in a Prostate Cancer Model

    International Nuclear Information System (INIS)

    Bae, Yun Jung; Yoon, Young Il; Yoon, Tae-Jong; Lee, Hak Jong

    2016-01-01

    To evaluate the effectiveness of ultrasound and microbubble-liposome complex (MLC)-mediated delivery of siRNA and doxorubicin into prostate cancer cells and its therapeutic capabilities both in vitro and in vivo. Microbubble-liposome complexes conjugated with anti-human epidermal growth factor receptor type 2 (Her2) antibodies were developed to target human prostate cancer cell lines PC-3 and LNCaP. Intracellular delivery of MLC was observed by confocal microscopy. We loaded MLC with survivin-targeted small interfering RNA (siRNA) and doxorubicin, and delivered it into prostate cancer cells. The release of these agents was facilitated by ultrasound application. Cell viability was analyzed by MTT assay after the delivery of siRNA and doxorubicin. Survivin-targeted siRNA loaded MLC was delivered into the xenograft mouse tumor model. Western blotting was performed to quantify the expression of survivin in vivo. Confocal microscopy demonstrated substantial intracellular uptake of MLCs in LNCaP, which expresses higher levels of Her2 than PC-3. The viability of LNCaP cells was significantly reduced after the delivery of MLCs loaded with siRNA and doxorubicin (85.0 ± 2.9%), which was further potentiated by application of ultrasound (55.0 ± 3.5%, p = 0.009). Survivin expression was suppressed in vivo in LNCaP tumor xenograft model following the ultrasound and MLC-guided delivery of siRNA (77.4 ± 4.90% to 36.7 ± 1.34%, p = 0.027). Microbubble-liposome complex can effectively target prostate cancer cells, enabling intracellular delivery of the treatment agents with the use of ultrasound. Ultrasound and MLC-mediated delivery of survivin-targeted siRNA and doxorubicin can induce prostate cell apoptosis and block survivin expression in vitro and in vivo

  4. Dissection of pathways leading to antigen receptor-induced and Fas/CD95-induced apoptosis in human B cells

    NARCIS (Netherlands)

    Lens, S. M.; den Drijver, B. F.; Pötgens, A. J.; Tesselaar, K.; van Oers, M. H.; van Lier, R. A.

    1998-01-01

    To dissect intracellular pathways involved in B cell Ag receptor (BCR)-mediated and Fas-induced human B cell death, we isolated clones of the Burkitt lymphoma cell line Ramos with different apoptosis sensitivities. Selection for sensitivity to Fas-induced apoptosis also selected for clones with

  5. Regulation of radiation-induced protein kinase Cδ activation in radiation-induced apoptosis differs between radiosensitive and radioresistant mouse thymic lymphoma cell lines

    International Nuclear Information System (INIS)

    Nakajima, Tetsuo; Yukawa, Osami; Tsuji, Hideo; Ohyama, Harumi; Wang, Bing; Tatsumi, Kouichi; Hayata, Isamu; Hama-Inaba, Hiroko

    2006-01-01

    Protein kinase Cδ (PKCδ) has an important role in radiation-induced apoptosis. The expression and function of PKCδ in radiation-induced apoptosis were assessed in a radiation-sensitive mouse thymic lymphoma cell line, 3SBH5, and its radioresistant variant, XR223. Rottlerin, a PKCδ-specific inhibitor, completely abolished radiation-induced apoptosis in 3SBH5. Radiation-induced PKCδ activation correlated with the degradation of PKCδ, indicating that PKCδ activation through degradation is involved in radiation-induced apoptosis in radiosensitive 3SBH5. In radioresistant XR223, radiation-induced PKCδ activation was lower than that in radiosensitive 3SBH5. Cytosol PKCδ levels in 3SBH5 decreased markedly after irradiation, while those in XR223 did not. There was no apparent change after irradiation in the membrane fractions of either cell type. In addition, basal cytosol PKCδ levels in XR223 were higher than those in 3SBH5. These results suggest that the radioresistance in XR223 to radiation-induced apoptosis is due to a difference in the regulation of radiation-induced PKCδ activation compared to that of 3SBH5. On the other hand, Atm -/- mouse thymic lymphoma cells were more radioresistant to radiation-induced apoptosis than wild-type mouse thymic lymphoma cells. Irradiated wild-type cells, but not Atm -/- cells, had decreased PKCδ levels, indicating that the Atm protein is involved in radiation-induced apoptosis through the induction of PKCδ degradation. The decreased Atm protein levels induced by treatment with Atm small interfering RNA had no effect on radiation-induced apoptosis in 3SBH5 cells. These results suggest that the regulation of radiation-induced PKCδ activation, which is distinct from the Atm-mediated cascade, determines radiation sensitivity in radiosensitive 3SBH5 cells

  6. BIM is the primary mediator of MYC-induced apoptosis in multiple solid tissues.

    Science.gov (United States)

    Muthalagu, Nathiya; Junttila, Melissa R; Wiese, Katrin E; Wolf, Elmar; Morton, Jennifer; Bauer, Barbara; Evan, Gerard I; Eilers, Martin; Murphy, Daniel J

    2014-09-11

    MYC is one of the most frequently overexpressed oncogenes in human cancer, and even modestly deregulated MYC can initiate ectopic proliferation in many postmitotic cell types in vivo. Sensitization of cells to apoptosis limits MYC's oncogenic potential. However, the mechanism through which MYC induces apoptosis is controversial. Some studies implicate p19ARF-mediated stabilization of p53, followed by induction of proapoptotic BH3 proteins NOXA and PUMA, whereas others argue for direct regulation of BH3 proteins, especially BIM. Here, we use a single experimental system to systematically evaluate the roles of p19ARF and BIM during MYC-induced apoptosis, in vitro, in vivo, and in combination with a widely used chemotherapeutic, doxorubicin. We find a common specific requirement for BIM during MYC-induced apoptosis in multiple settings, which does not extend to the p53-responsive BH3 family member PUMA, and find no evidence of a role for p19ARF during MYC-induced apoptosis in the tissues examined. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Killing Effect of Ad5/F35-APE1 siRNA Recombinant Adenovirus in Combination with Hematoporphrphyrin Derivative-Mediated Photodynamic Therapy on Human Nonsmall Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Lei Xia

    2013-01-01

    Full Text Available The main goal of this work is to investigate the killing effects and molecular mechanism of photodynamic therapy (PDT mediated by the Ad5/F35-APE1 siRNA recombinant adenovirus in combination with a hematoporphrphyrin derivative (HpD in the A549 human lung adenocarcinoma cell line in vitro to provide a theoretical reference for treating lung cancer by HpD-PDT. By using the technologies of MTT, flow cytometry, ELISA, and western blot, we observed that the proliferation inhibition and apoptosis of the A549 cells were significantly higher than the control group ( after HpD-PDT was performed. The inhibitory efficiency is dependent on the HpD concentration and laser intensity dose. The inhibitory effect on the proliferation of A549 cells of Ad5/F35-APE1 siRNA is more significant after combining with PDT, as indicated by a significant elevation of the intracellular ROS level and the expression of inflammatory factors (. The HpD-PDT-induced expression of the APE1 protein reached the peak after 24 h in A549 cells. The inhibition of APE1 expression in A549 cells was most significant after 48 hours of infection by Ad5/F35-APE1 siRNA recombinant adenovirus (10 MOI. In conclusion, the Ad5/F35-APE1 siRNA recombinant adenovirus could efficiently inhibit the HpD-PDT-induced APE1 expression hence could significantly enhance the killing effect of HpD-PDT in lung cancer cells.

  8. Apoptosis-promoted tumorigenesis: γ-irradiation-induced thymic lymphomagenesis requires Puma-driven leukocyte death

    OpenAIRE

    Michalak, Ewa M.; Vandenberg, Cassandra J.; Delbridge, Alex R.D.; Wu, Li; Scott, Clare L.; Adams, Jerry M.; Strasser, Andreas

    2010-01-01

    Although tumor development requires impaired apoptosis, we describe a novel paradigm of apoptosis-dependent tumorigenesis. Because DNA damage triggers apoptosis through p53-mediated induction of BH3-only proteins Puma and Noxa, we explored their roles in γ-radiation-induced thymic lymphomagenesis. Surprisingly, whereas Noxa loss accelerated it, Puma loss ablated tumorigenesis. Tumor suppression by Puma deficiency reflected its protection of leukocytes from γ-irradiation-induced death, because...

  9. Proteasomal Dysfunction Induced By Diclofenac Engenders Apoptosis Through Mitochondrial Pathway.

    Science.gov (United States)

    Amanullah, Ayeman; Upadhyay, Arun; Chhangani, Deepak; Joshi, Vibhuti; Mishra, Ribhav; Yamanaka, Koji; Mishra, Amit

    2017-05-01

    Diclofenac is the most commonly used phenylacetic acid derivative non-steroidal anti-inflammatory drug (NSAID) that demonstrates significant analgesic, antipyretic, and anti-inflammatory effects. Several epidemiological studies have demonstrated anti-proliferative activity of NSAIDs and examined their apoptotic induction effects in different cancer cell lines. However, the precise molecular mechanisms by which these pharmacological agents induce apoptosis and exert anti-carcinogenic properties are not well known. Here, we have observed that diclofenac treatment induces proteasome malfunction and promotes accumulation of different critical proteasome substrates, including few pro-apoptotic proteins in cells. Exposure of diclofenac consequently elevates aggregation of various ubiquitylated misfolded proteins. Finally, we have shown that diclofenac treatment promotes apoptosis in cells, which could be because of mitochondrial membrane depolarization and cytochrome c release into cytosol. This study suggests possible beneficial insights of NSAIDs-induced apoptosis that may improve our existing knowledge in anti-proliferative interspecific strategies development. J. Cell. Biochem. 118: 1014-1027, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. Hydroxyoctadecadienoic acids regulate apoptosis in human THP-1 cells in a PPARγ-dependent manner.

    Science.gov (United States)

    Vangaveti, Venkat N; Shashidhar, Venkatesh M; Rush, Catherine; Malabu, Usman H; Rasalam, Roy R; Collier, Fiona; Baune, Bernhard T; Kennedy, Richard L

    2014-12-01

    Macrophage apoptosis, a key process in atherogenesis, is regulated by oxidation products, including hydroxyoctadecadienoic acids (HODEs). These stable oxidation products of linoleic acid (LA) are abundant in atherosclerotic plaque and activate PPARγ and GPR132. We investigated the mechanisms through which HODEs regulate apoptosis. The effect of HODEs on THP-1 monocytes and adherent THP-1 cells were compared with other C18 fatty acids, LA and α-linolenic acid (ALA). The number of cells was reduced within 24 hours following treatment with 9-HODE (p labelling of cells (p blocked by the caspase inhibitor DEVD-CHO. The PPARγ antagonist T0070907 further increased apoptosis, suggestive of the PPARγ-regulated apoptotic effects induced by 9-HODE. The use of siRNA for GPR132 showed no evidence that the effect of HODEs was mediated through this receptor. 9-HODE and 13-HODE are potent--and specific--regulators of apoptosis in THP-1 cells. Their action is PPARγ-dependent and independent of GPR132. Further studies to identify the signalling pathways through which HODEs increase apoptosis in macrophages may reveal novel therapeutic targets for atherosclerosis.

  11. NFAT2 mediates high glucose-induced glomerular podocyte apoptosis through increased Bax expression

    International Nuclear Information System (INIS)

    Li, Ruizhao; Zhang, Li; Shi, Wei; Zhang, Bin; Liang, Xinling; Liu, Shuangxin; Wang, Wenjian

    2013-01-01

    Background: Hyperglycemia promotes podocyte apoptosis and plays a key role in the pathogenesis of diabetic nephropathy. However, the mechanisms that mediate hyperglycemia-induced podocyte apoptosis is still far from being fully understood. Recent studies reported that high glucose activate nuclear factor of activated T cells (NFAT) in vascular smooth muscle or pancreatic β-cells. Here, we sought to determine if hyperglycemia activates NFAT2 in cultured podocyte and whether this leads to podocyte apoptosis. Meanwhile, we also further explore the mechanisms of NFAT2 activation and NFAT2 mediates high glucose-induced podocyte apoptosis. Methods: Immortalized mouse podocytes were cultured in media containing normal glucose (NG), or high glucose (HG) or HG plus cyclosporine A (a pharmacological inhibitor of calcinerin) or 11R-VIVIT (a special inhibitor of NFAT2). The activation of NFAT2 in podocytes was detected by western blotting and immunofluorescence assay. The role of NFAT2 in hyperglycemia-induced podocyte apoptosis was further evaluated by observing the inhibition of NFAT2 activation by 11R-VIVIT using flow cytometer. Intracellular Ca 2+ was monitored in HG-treated podcocytes using Fluo-3/AM. The mRNA and protein expression of apoptosis gene Bax were measured by real time-qPCR and western blotting. Results: HG stimulation activated NFAT2 in a time- and dose-dependent manner in cultured podocytes. Pretreatment with cyclosporine A (500 nM) or 11R-VIVIT (100 nM) completely blocked NFAT2 nuclear accumulation. Meanwhile, the apoptosis effects induced by HG were also abrogated by concomitant treatment with 11R-VIVIT in cultured podocytes. We further found that HG also increased [Ca 2+ ]i, leading to activation of calcineurin, and subsequent increased nuclear accumulation of NFAT2 and Bax expression in cultured podocytes. Conclusion: Our results identify a new finding that HG-induced podocyte apoptosis is mediated by calcineurin/NFAT2/Bax signaling pathway, which may

  12. NFAT2 mediates high glucose-induced glomerular podocyte apoptosis through increased Bax expression

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ruizhao, E-mail: liruizhao1979@126.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Zhang, Li, E-mail: Zhanglichangde@163.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Southern Medical University, Guangzhou, Guangdong (China); Shi, Wei, E-mail: shiwei.gd@139.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Zhang, Bin, E-mail: zhangbinyes@yahoo.com.cn [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Liang, Xinling, E-mail: xinlingliang@yahoo.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Liu, Shuangxin, E-mail: mplsxi@yahoo.com.cn [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Wang, Wenjian, E-mail: wwjph@yahoo.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China)

    2013-04-15

    Background: Hyperglycemia promotes podocyte apoptosis and plays a key role in the pathogenesis of diabetic nephropathy. However, the mechanisms that mediate hyperglycemia-induced podocyte apoptosis is still far from being fully understood. Recent studies reported that high glucose activate nuclear factor of activated T cells (NFAT) in vascular smooth muscle or pancreatic β-cells. Here, we sought to determine if hyperglycemia activates NFAT2 in cultured podocyte and whether this leads to podocyte apoptosis. Meanwhile, we also further explore the mechanisms of NFAT2 activation and NFAT2 mediates high glucose-induced podocyte apoptosis. Methods: Immortalized mouse podocytes were cultured in media containing normal glucose (NG), or high glucose (HG) or HG plus cyclosporine A (a pharmacological inhibitor of calcinerin) or 11R-VIVIT (a special inhibitor of NFAT2). The activation of NFAT2 in podocytes was detected by western blotting and immunofluorescence assay. The role of NFAT2 in hyperglycemia-induced podocyte apoptosis was further evaluated by observing the inhibition of NFAT2 activation by 11R-VIVIT using flow cytometer. Intracellular Ca{sup 2+} was monitored in HG-treated podcocytes using Fluo-3/AM. The mRNA and protein expression of apoptosis gene Bax were measured by real time-qPCR and western blotting. Results: HG stimulation activated NFAT2 in a time- and dose-dependent manner in cultured podocytes. Pretreatment with cyclosporine A (500 nM) or 11R-VIVIT (100 nM) completely blocked NFAT2 nuclear accumulation. Meanwhile, the apoptosis effects induced by HG were also abrogated by concomitant treatment with 11R-VIVIT in cultured podocytes. We further found that HG also increased [Ca{sup 2+}]i, leading to activation of calcineurin, and subsequent increased nuclear accumulation of NFAT2 and Bax expression in cultured podocytes. Conclusion: Our results identify a new finding that HG-induced podocyte apoptosis is mediated by calcineurin/NFAT2/Bax signaling pathway

  13. The BH3 α-Helical Mimic BH3-M6 Disrupts Bcl-XL, Bcl-2, and MCL-1 Protein-Protein Interactions with Bax, Bak, Bad, or Bim and Induces Apoptosis in a Bax- and Bim-dependent Manner*

    Science.gov (United States)

    Kazi, Aslamuzzaman; Sun, Jiazhi; Doi, Kenichiro; Sung, Shen-Shu; Takahashi, Yoshinori; Yin, Hang; Rodriguez, Johanna M.; Becerril, Jorge; Berndt, Norbert; Hamilton, Andrew D.; Wang, Hong-Gang; Sebti, Saïd M.

    2011-01-01

    A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-XL, and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-XL and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-XL, Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-XL/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-XL, Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612. PMID:21148306

  14. Palmitate induces VSMC apoptosis via toll like receptor (TLR)4/ROS/p53 pathway.

    Science.gov (United States)

    Zhang, Yuanjun; Xia, Guanghao; Zhang, Yaqiong; Liu, Juxiang; Liu, Xiaowei; Li, Weihua; Lv, Yaya; Wei, Suhong; Liu, Jing; Quan, Jinxing

    2017-08-01

    Toll-like receptor 4 (TLR4) has been implicated in vascular inflammation, as well as in the pathogenesis of atherosclerosis and diabetes. Vascular smooth muscle cell (VSMC) apoptosis has been shown to induce plaque vulnerability in atherosclerosis. Previous studies reported that palmitate induced apoptosis in VSMCs; however, the role of TLR4 in palmitate-induced apoptosis in VSMCs has not yet been defined. In this study, we investigated whether or not palmitate-induced apoptosis depended on the activation of the TLR4 pathway. VSMCs were treated with or without palmitate, CRISPR/Cas9z-mediated genome editing methods were used to deplete TLR4 expression, while NADPH oxidase inhibitors were used to inhibit reactive oxygen species (ROS) generation. Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, ROS was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) method, the mRNA and protein expression levels of caspase 3, caspase 9, BCL-2 and p53 were studied by real-time polymerase chain reaction (RT-PCR) and ELISA. Palmitate significantly promotes VSMC apoptosis, ROS generation, and expression of caspase 3, caspase 9 and p53; while NADPH oxidase inhibitor pretreatment markedly attenuated these effects. Moreover, knockdown of TLR4 significantly blocked palmitate-induced ROS generation and VSMC apoptosis accompanied by inhibition of caspase 3, caspase 9, p53 expression and restoration of BCL-2 expression. Our results suggest that palmitate-induced apoptosis depends on the activation of the TLR4/ROS/p53 signaling pathway, and that TLR4 may be a potential therapeutic target for the prevention and treatment of atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Down-regulation of long non-coding RNA TUG1 inhibits osteosarcoma cell proliferation and promotes apoptosis.

    Science.gov (United States)

    Zhang, Qiang; Geng, Pei-Liang; Yin, Pei; Wang, Xiao-Lin; Jia, Jin-Peng; Yao, Jie

    2013-01-01

    To investigate the expression level of TUG1 and one of its transcript variants (n377360) in osteosarcoma cells and assess the role of TUG1 in proliferation and apoptosis in the U2OS cell line. TUG1 and n377360 expression levels in patients with osteosarcomas and the U2OS human osteosarcoma cell line were evaluated using real-time quantitative PCR. U2OS cells were transected with TUG1 and n377360 siRNA or non-targeting siRNA. MTS was performed to assess the cell proliferation and flow cytometry was applied to analyze apoptosis. We found significantly higher TUG1 and n377360 expression levels in osteosarcoma tissues compared with matched non-tumorous tissues. In line with this, suppression of TUG1 and n377360 expression by siRNA significantly impaired the cell proliferation potential of osteosarcoma cells. Furthermore, inhibition of TUG1 expression significantly promoted osteosarcoma cell apoptosis. The overexpression of TUG1 and n377360 in osteosarcoma specimens and the functional role of TUG1 and n377360 regarding cell proliferation and apoptosis in an osteosarcoma cell line provided evidence that the use of TUG1 or n377360 may be a viable but an as yet unexplored therapeutic strategy in tumors that over express these factors.

  16. Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis.

    Science.gov (United States)

    Shaw, Catherine A; Webb, David J; Rossi, Adriano G; Megson, Ian L

    2009-05-07

    Nitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-). In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMvarphi), and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP) is able to limit apoptosis in this cell type. Characterisation of the NO-related species generated by (Z)-1- [2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-, chloride (GEA-3162) was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR) spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMvarphi. Resultant MDMvarphi were treated for 24 h with DETA/NO (100 - 1000 muM) or GEA-3162 (10 - 300 muM) in the presence or absence of BAY 41-2272 (1 muM), isobutylmethylxanthine (IBMX; 1 muM), 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 muM) or 8-bromo-cGMP (1 mM). Apoptosis in MDMvarphi was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO) had no effect on cell viability, but ONOO- (GEA-3162) caused a concentration-dependent induction of apoptosis in MDMvarphi. Preconditioning of MDMvarphi with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX), or the NO-independent stimulator of soluble guanylate cyclase, BAY 41-2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner. These results

  17. Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Rossi Adriano G

    2009-05-01

    Full Text Available Abstract Background Nitric oxide (NO can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-. In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMϕ, and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP is able to limit apoptosis in this cell type. Methods Characterisation of the NO-related species generated by (Z-1- [2-(2-aminoethyl-N-(2-ammonioethylamino]diazen-1-ium-1,2-diolate (DETA/NO and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl-, chloride (GEA-3162 was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMϕ. Resultant MDMϕ were treated for 24 h with DETA/NO (100 – 1000 μM or GEA-3162 (10 – 300 μM in the presence or absence of BAY 41–2272 (1 μM, isobutylmethylxanthine (IBMX; 1 μM, 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 μM or 8-bromo-cGMP (1 mM. Apoptosis in MDMϕ was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Results Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO had no effect on cell viability, but ONOO- (GEA-3162 caused a concentration-dependent induction of apoptosis in MDMϕ. Preconditioning of MDMϕ with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX, or the NO-independent stimulator of soluble guanylate cyclase, BAY 41–2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner

  18. Apoptosis and radiosensitivity induced by N-acety1 phytosphingosine, in human cancer cell line

    International Nuclear Information System (INIS)

    Kim, Y. H.; Kim, K. S.; Han, Y. S.; Jeon, S. J.; Song, J. Y.; Jung, I. S.; Hong, S. H.; Yun, Y. S.; Park, J. S.

    2004-01-01

    Ceramide is a key lipid molecule in signal transduction with a role in various regulatory pathways including differentiation, proliferation and especially apoptosis. Ionizing radiation-induced apoptosis is associated with accumulation of ceramide, and the sphingomyelinase deficiency results in radioresistance. We investigated the exogenous treatment of N-acetyl-phytosphingosine (NAPS), an analogue of N-acetyl-sphingosine (C 2 -Ceramide), and C 2 -ceramide exert apoptotic effect on human T cell lymphoma Jurkat cells and breast cancer cell line MDA-MB-231. NAPS and C 2 -Ceramide has cytotoxic effect in time- and dose-dependent manner, and increased caspase-3, 8 activity. However, NAPS induced apoptosis more effectively, and increased caspase activity induced by NAPS is more higher than C 2 -ceramide. Moreover, NAPS decreased clonogenicity of irradiated cells and increased radiation-induced apoptosis significantly. Increased cell death by irradiation in the presence of NAPS is owing to the increase of caspase activity. These data suggest that NAPS might be used for lead as a new type of radiosensitizing agent increasing radiation-induced apoptosis

  19. Cloning and Characterization of Genes that Inhibit TRAIL-Induced Apoptosis of Breast Cancer Cells

    National Research Council Canada - National Science Library

    Shu, Hong-Bing

    2003-01-01

    ...). However, some cancer cells are resistant to TRAIL-induced apoptosis (3, 4, 6-13). The purpose of this proposed study is to clone and characterize such inhibitory genes of TRAIL-induced apoptosis...

  20. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    PURPOSE: The immune privilege of the eye has been thought to be dependent on physical barriers and absence of lymphatic vessels. However, the immune privilege may also involve active immunologic processes, as recent studies have indicated. The purpose of the present study was to investigate whether...... human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...

  1. Roles of acid sphingomyelinase activation in neuronal cells apoptosis induced by microwave irradiation

    International Nuclear Information System (INIS)

    Zhang Lei; Xu Shangcheng; Zhang Guangbin; Yu Zhengping

    2009-01-01

    The present study is to examine the effect of microwave on acid sphingomyelinase (ASM) activity and expression, and to explore the role of ASM activation in neuronal cells apoptosis induced by microwave irradiation. Primary cultured hippocampal neurons were irradiated by 30 W/cm 2 microwave for 10 min, and ASM activity assay was used to investigate ASM activity alteration. RT-PCR and western blot were used to detect ASM mRNA and protein expression respectively. Apoptosis was observed by Hoechst 33342 fluorescence staining. ASM specific inhibitor imipramine was applied to inhibit ASM activation. It has been found that apoptosis rate of primary cultured hippocampal neurons increased significantly after microwave irradiation. ASM was activated while ASM mRNA and protein expression were upregulated in neurons after microwave irradiation. Pretreatment with imipramine could reverse neuronal apoptosis induced by microwave irradiation. Results show that microwave irradiation causes increment of ASM activation and expression and ASM activation is involved in microwave induced neuronal apoptosis. (authors)

  2. Induction and regulation of tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand-mediated apoptosis in renal cell carcinoma.

    Science.gov (United States)

    Griffith, Thomas S; Fialkov, Jonathan M; Scott, David L; Azuhata, Takeo; Williams, Richard D; Wall, Nathan R; Altieri, Dario C; Sandler, Anthony D

    2002-06-01

    The lack of effective therapy for disseminated renal cell carcinoma (RCC) has stimulated the search for novel treatments including immunotherapeutic strategies. However, poor therapeutic responses and marked toxicity associated with immunological agents has limited their use. The tumor necrosis factor family member tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2 ligand induces apoptosis in a variety of tumor cell types, while having little cytotoxic activity against normal cells. In this study the activation and regulation of TRAIL-induced apoptosis and TRAIL receptor expression in human RCC cell lines and pathologic specimens was examined. TRAIL induced caspase-mediated apoptotic death of RCC cells with variable sensitivities among the cell lines tested. Compared with TRAIL-sensitive RCC cell lines (A-498, ACHN, and 769-P), the TRAIL-resistant RCC cell line (786-O) expressed lesser amounts of the death-inducing TRAIL receptors, and greater amounts of survivin, an inhibitor of apoptosis. Incubation of 786-O with actinomycin D increased the expression of the death-inducing TRAIL receptors and, concomitantly, decreased the intracellular levels of survivin, resulting in TRAIL-induced apoptotic death. The link between survivin and TRAIL regulation was confirmed when an increase in TRAIL resistance was observed after overexpression of survivin in the TRAIL-sensitive, survivin-negative RCC line A-498. These findings, along with our observation that TRAIL receptors are expressed in RCC tumor tissue, suggest that TRAIL may be useful as a therapeutic agent for RCC and that survivin may partially regulate TRAIL-induced cell death.

  3. Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes

    Directory of Open Access Journals (Sweden)

    Yue Wang

    2016-11-01

    Full Text Available The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC. Levels of reactive oxygen species (ROS, malondialdehyde (MDA, and glutathione (GSH, activities of superoxide dismutase (SOD and catalase (CAT, mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

  4. Molecular Mechanisms of Particle Ration Induced Apoptosis in Lymphocyte

    Science.gov (United States)

    Shi, Yufang

    Space radiation, composed of high-energy charged nuclei (HZE particles) and protons, has been previously shown to severely impact immune homeostasis in mice. To determine the molecular mechanisms that mediate acute lymphocyte depletion following exposure to HZE particle radiation mice were exposed to particle radiation beams at Brookhaven National Laboratory. We found that mice given whole body 5 6Fe particle irradiation (1GeV /n) had dose-dependent losses in total lymphocyte numbers in the spleen and thymus (using 200, 100 and 50 cGy), with thymocytes being more sensitive than splenocytes. All phenotypic subsets were reduced in number. In general, T cells and B cells were equally sensitive, while CD8+ T cells were more senstive than CD4+ T cells. In the thymus, immature CD4+CD8+ double-positive thymocytes were exquisitely sensitive to radiation-induced losses, single-positive CD4 or CD8 cells were less sensitive, and the least mature double negative cells were resistant. Irradiation of mice deficient in genes encoding essential apoptosis-inducing proteins revealed that the mechanism of lymphocyte depletion is independent of Fas ligand and TRAIL (TNF-ralated apoptosis-inducing ligand), in contrast to γ-radiation-induced lymphocyte losses which require the Fas-FasL pathway. Using inhibitors in vitro, lymphocyte apoptosis induced by HZE particle radiation was found to be caspase dependent, and not involve nitric oxide or oxygen free radicals.

  5. Radiation-induced apoptosis in differentially modulated by PTK inhibitora in K562 cells

    International Nuclear Information System (INIS)

    Lee, Hyung Sik; Moon, Chang Woo; Hur, Won Joo; Jeong, Su Jin; Jeong Min Ho; Lee, Jeong Hyeon; Lim, Young Jin; Park, Heon Joo

    2000-01-01

    The effect of PTK inhibitors (herbimycin A and genistein) on the induction of radiation-induced apoptosis in Ph-positive K562 leukemia cell line was investigated. K562 cells in exponential growth phase were irradiated with a linear accelerator at room temperature. For 6 MV X-ray irradiation and drug treatment, cultures were initiated at 2x10 6 cells/ml. The cells were irradiated with 10Gy. Stock solutions of herbimycin A and genistein were prepared in dimethyl sulphoxide (DMSO). After incubation at 37 .deg. for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and TUNEL assay. The progression of cells through the cell cycle after irradiation and drug treatment was also determined with flow cytometry. Western blot analysis was used to monitor bcl-2, bcl-X-L and bax protein levels. Treatment with 10 Gy X-irradiation did not result in the induction of apoptosis. The HMA alone (500 nM) also failed to induce apoptosis. By contrast, incubation of K562 cells with HMA after irradiation resulted in a substantial induction of nuclear condensation and fragmentation by agarose gel electrophoresis and TUNEL assay. Genistein failed to enhance the ability of X-irradiation to induce DNA fragmentation. Enhancement of apoptosis by HMA was not attributable to downregulation of the bcl-2 or bcl-X-L anti-apoptotic proteins. When the cells were irradiated and maintained with HMA, the percentage of cells in G2/M phase decreased to 30-40% at 48 h. On the other hand, cells exposed to 10 Gy X-irradiation alone or maintained with genistein did not show marked cell cycle redistribution. We have shown that nanomolar concentrations of the PTK inhibitor HMA synergize with X-irradiation in inducing the apoptosis in Ph (+) K562 leukemia cell line. While, genistein, a PTK inhibitor which is not selective for p210 bcr/abl failed to enhance the radiation induced apoptosis in K562 cells. It is unlikely that the ability of HMA to enhance apoptosis in K562 cells is

  6. The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis in a Bax- and Bim-dependent manner.

    Science.gov (United States)

    Kazi, Aslamuzzaman; Sun, Jiazhi; Doi, Kenichiro; Sung, Shen-Shu; Takahashi, Yoshinori; Yin, Hang; Rodriguez, Johanna M; Becerril, Jorge; Berndt, Norbert; Hamilton, Andrew D; Wang, Hong-Gang; Sebti, Saïd M

    2011-03-18

    A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.

  7. Regulation of singlet oxygen-induced apoptosis by cytosolic NADP+-dependent isocitrate dehydrogenase.

    Science.gov (United States)

    Kim, Sun Yee; Lee, Su Min; Tak, Jean Kyoung; Choi, Kyeong Sook; Kwon, Taeg Kyu; Park, Jeen-Woo

    2007-08-01

    Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. Recently, we demonstrated that the control of redox balance and the cellular defense against oxidative damage are the primary functions of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) through supplying NADPH for antioxidant systems. In this report, we demonstrate that modulation of IDPc activity in HL-60 cells regulates singlet oxygen-induced apoptosis. When we examined the protective role of IDPc against singlet oxygen-induced apoptosis with HL-60 cells transfected with the cDNA for mouse IDPc in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc expressed in target cells and their susceptibility to apoptosis. The results suggest that IDPc plays an important protective role in apoptosis of HL-60 cells induced by singlet oxygen.

  8. Autophagy inhibition enhances apigenin-induced apoptosis in human breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Xuchen Cao; Bowen Liu; Wenfeng Cao; Weiran Zhang; Fei Zhang; Hongmeng Zhao; Ran Meng

    2013-01-01

    Apigenin (4',5,7-trihydroxyflavone) is a member of the flavone subclass of flavonoids present in fruits and vegetables.The involvement of autophagy in the apigenin-induced apoptotic death of human breast cancer cells was investigated.Cell proliferation and viability were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays.Flow cytometry,fluorescent staining and Western blot analysis were employed to detect apoptosis and autophagy,and the role of autophagy was assessed using autophagy inhibitors.Apigenin dose-and time-dependently repressed the proliferation and clonogenic survival of the human breast cancer T47D and MDA-MB-231 cell lines.The death of T47D and MDA-MB-231 cells was due to apoptosis associated with increased levels of Caspase3,PARP cleavage and Bax/Bcl-2 ratios.The results from flow cytometry and fluorescent staining also verified the occurrence of apoptosis.In addition,the apigenin-treated cells exhibited autophagy,as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles (AVOs)by flow cytometry.Furthermore,the results of the Western blot analysis revealed that the level of LC3-Ⅱ,the processed form of LC3-Ⅰ,was increased.Treatment with the autophagy inhibitor,3-methyladenine (3-MA),significantly enhanced the apoptosis induced by apigenin,which was accompanied by an increase in the level of PARP cleavage.Similar results were also confirmed by flow cytometry and fluorescence microscopy.These results indicate that apigenin has apoptosis-and autophagy-inducing effects in breast cancer cells.Autophagy plays a cyto-protective role in apigenin-induced apoptosis,and the combination of apigenin and an autophagy inhibitor may be a promising strategy for breast cancer control.

  9. Apoptotic cells can induce non-autonomous apoptosis through the TNF pathway

    Science.gov (United States)

    Pérez-Garijo, Ainhoa; Fuchs, Yaron; Steller, Hermann

    2013-01-01

    Apoptotic cells can produce signals to instruct cells in their local environment, including ones that stimulate engulfment and proliferation. We identified a novel mode of communication by which apoptotic cells induce additional apoptosis in the same tissue. Strong induction of apoptosis in one compartment of the Drosophila wing disc causes apoptosis of cells in the other compartment, indicating that dying cells can release long-range death factors. We identified Eiger, the Drosophila tumor necrosis factor (TNF) homolog, as the signal responsible for apoptosis-induced apoptosis (AiA). Eiger is produced in apoptotic cells and, through activation of the c-Jun N-terminal kinase (JNK) pathway, is able to propagate the initial apoptotic stimulus. We also show that during coordinated cell death of hair follicle cells in mice, TNF-α is expressed in apoptotic cells and is required for normal cell death. AiA provides a mechanism to explain cohort behavior of dying cells that is seen both in normal development and under pathological conditions. DOI: http://dx.doi.org/10.7554/eLife.01004.001 PMID:24066226

  10. DMPD: Pathogen-induced apoptosis of macrophages: a common end for different pathogenicstrategies. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 11207583 Pathogen-induced apoptosis of macrophages: a common end for different path...ml) Show Pathogen-induced apoptosis of macrophages: a common end for different pathogenicstrategies. PubmedI...D 11207583 Title Pathogen-induced apoptosis of macrophages: a common end for diff

  11. Novel targeted therapy for neuroblastoma: silencing the MXD3 gene using siRNA.

    Science.gov (United States)

    Duong, Connie; Yoshida, Sakiko; Chen, Cathy; Barisone, Gustavo; Diaz, Elva; Li, Yueju; Beckett, Laurel; Chung, Jong; Antony, Reuben; Nolta, Jan; Nitin, Nitin; Satake, Noriko

    2017-09-01

    BackgroundNeuroblastoma is the second most common extracranial cancer in children. Current therapies for neuroblastoma, which use a combination of chemotherapy drugs, have limitations for high-risk subtypes and can cause significant long-term adverse effects in young patients. Therefore, a new therapy is needed. In this study, we investigated the transcription factor MXD3 as a potential therapeutic target in neuroblastoma.MethodsMXD3 expression was analyzed in five neuroblastoma cell lines by immunocytochemistry and quantitative real-time reverse transcription PCR, and in 18 primary patient tumor samples by immunohistochemistry. We developed nanocomplexes using siRNA and superparamagnetic iron oxide nanoparticles to target MXD3 in neuroblastoma cell lines in vitro as a single-agent therapeutic and in combination with doxorubicin, vincristine, cisplatin, or maphosphamide-common drugs used in current neuroblastoma treatment.ResultsMXD3 was highly expressed in neuroblastoma cell lines and in patient tumors that had high-risk features. Neuroblastoma cells treated in vitro with the MXD3 siRNA nanocomplexes showed MXD3 protein knockdown and resulted in cell apoptosis. Furthermore, on combining MXD3 siRNA nanocomplexes with each of the four drugs, all showed additive efficacy.ConclusionThese results indicate that MXD3 is a potential new target and that the use of MXD3 siRNA nanocomplexes is a novel therapeutic approach for neuroblastoma.

  12. Drosophila MOF regulates DIAP1 and induces apoptosis in a JNK dependent pathway.

    Science.gov (United States)

    Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Koteswara Rao, G; Bag, Indira; Bhadra, Utpal; Pal-Bhadra, Manika

    2016-03-01

    Histone modulations have been implicated in various cellular and developmental processes where in Drosophila Mof is involved in acetylation of H4K16. Reduction in the size of larval imaginal discs is observed in the null mutants of mof with increased apoptosis. Deficiency involving Hid, Reaper and Grim [H99] alleviated mof (RNAi) induced apoptosis in the eye discs. mof (RNAi) induced apoptosis leads to activation of caspases which is suppressed by over expression of caspase inhibitors like P35 and Diap1 clearly depicting the role of caspases in programmed cell death. Also apoptosis induced by knockdown of mof is rescued by JNK mutants of bsk and tak1 indicating the role of JNK in mof (RNAi) induced apoptosis. The adult eye ablation phenotype produced by ectopic expression of Hid, Rpr and Grim, was restored by over expression of Mof. Accumulation of Mof at the Diap1 promoter 800 bp upstream of the transcription start site in wild type larvae is significantly higher (up to twofolds) compared to mof (1) mutants. This enrichment coincides with modification of histone H4K16Ac indicating an induction of direct transcriptional up regulation of Diap1 by Mof. Based on these results we propose that apoptosis triggered by mof (RNAi) proceeds through a caspase-dependent and JNK mediated pathway.

  13. LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells

    Science.gov (United States)

    Figarola, James L.; Singhal, Jyotsana; Rahbar, Samuel; Awasthi, Sanjay

    2014-01-01

    Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis. PMID:24615331

  14. Toll-like receptor 9 is required for opioid-induced microglia apoptosis.

    Directory of Open Access Journals (Sweden)

    Lei He

    2011-04-01

    Full Text Available Opioids have been widely applied in clinics as one of the most potent pain relievers for centuries, but their abuse has deleterious physiological effects beyond addiction. However, the underlying mechanism by which microglia in response to opioids remains largely unknown. Here we show that morphine induces the expression of Toll-like receptor 9 (TLR9, a key mediator of innate immunity and inflammation. Interestingly, TLR9 deficiency significantly inhibited morphine-induced apoptosis in microglia. Similar results were obtained when endogenous TLR9 expression was suppressed by the TLR9 inhibitor CpGODN. Inhibition of p38 MAPK by its specific inhibitor SB203580 attenuated morphine-induced microglia apoptosis in wild type microglia. Morphine caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type microglia, but not in TLR9 deficient microglia. In addition, morphine treatment failed to induce an increased levels of phosphorylated p38 MAPK and MAP kinase kinase 3/6 (MKK3/6, the upstream MAPK kinase of p38 MAPK, in either TLR9 deficient or µ-opioid receptor (µOR deficient primary microglia, suggesting an involvement of MAPK and µOR in morphine-mediated TLR9 signaling. Moreover, morphine-induced TLR9 expression and microglia apoptosis appears to require μOR. Collectively, these results reveal that opioids prime microglia to undergo apoptosis through TLR9 and µOR as well. Taken together, our data suggest that inhibition of TLR9 and/or blockage of µOR is capable of preventing opioid-induced brain damage.

  15. The correlation between spontaneous and radiation-induced apoptosis in T3B bladder cancer (histological grade G3), and the precedence between the two kinds of apoptosis for predicting clinical prognosis

    International Nuclear Information System (INIS)

    Harada, Satoshi; Sato, Ryuichi; Nakamura, Ryuji; Oikawa, Hiroshi; Oikawa, Hirobumi; Ohgi, Shie; Tamakawa, Yoshiharu; Yanagisawa, Toru

    2000-01-01

    Purpose: The correlation between the frequency of spontaneous and radiation-induced apoptosis, and the precedence between those for predicting prognosis were studied at clinical level. Methods and Materials: Twenty-one patients (mean age, 65.8 years; 16 men and 5 women) with bladder cancer (transitional cell carcinoma Grade 3, T3bN0M0, Stage IIIb) underwent intraoperative radiotherapy: single 30-Gy 12-MV electron beam irradiation to bladder, followed by total cystectomy 6 h after irradiation. The specimens of pretreatment and irradiated bladder cancer were assayed for apoptosis, using TUNEL staining with counter staining of hematoxylin. The apoptotic index (AI) was calculated by dividing the number of apoptotic cells by the total number of cells and multiplying by 100. The Pearson's linear fitting was used to test the correlation between spontaneous and radiation-induced apoptosis. The Kaplan-Meier product-limit estimation was used for overall survival (OS) and freedom from recurrence (FFR). The precedence between spontaneous and radiation-induced apoptosis for predicting the clinical prognosis was estimated using the proportional hazard regression. Results: The mean AI of spontaneous and radiation-induced apoptosis was 1.18 ± 0.16 and 2.63 ± 0.45, respectively, which was significantly different. There was strong correlation between spontaneous and radiation-induced apoptosis (r 2 = 0.864, adjusted r 2 = 0.857). Radiation-induced apoptosis was estimated by equitation: y (radiation-induced apoptosis) = 2.67 x (spontaneous apoptosis) -0.52. However, the proportional hazard regression test indicated that only spontaneous apoptosis was significant for predicting OS and FFR (vertical bar t vertical bar > 0.2), but radiation-induced apoptosis was not. Conclusion: Estimating AI in radiation-induced apoptosis from AI in spontaneous apoptosis is possible. However, spontaneous apoptosis is more accurate in predicting clinical prognosis

  16. Advanced glycation endproducts alter functions and promote apoptosis in endothelial progenitor cells through receptor for advanced glycation endproducts mediate overpression of cell oxidant stress.

    Science.gov (United States)

    Chen, Jianfei; Song, Minbao; Yu, Shiyong; Gao, Pan; Yu, Yang; Wang, Hong; Huang, Lan

    2010-02-01

    Endothelial progenitor cells (EPCs) play an important role in preventing atherosclerosis. The factors that regulate the function of EPCs are not completely clear. Increased formation of advanced glycation endproducts (AGEs) is generally regarded as one of the main mechanisms responsible for vascular damage in patients with diabetes and atherosclerosis. AGEs lead to the generation of reactive oxygen species (ROS) and part of the regenerative capacity of EPCs seems to be due to their low baseline ROS levels and reduced sensitivity to ROS-induced cell apoptosis. Therefore, we tested the hypothesis that AGEs can alter functions and promote apoptosis in EPCs through overpress cell oxidant stress. EPCs, isolated from bone marrow, were cultured in the absence or presence of AGEs (50, 100, and 200 microg/ml). A modified Boyden's chamber was used to assess the migration of EPCs and the number of recultured EPCs was counted to measure the adhesiveness function. MTT assay was used to determine the proliferation function. ROS were analyzed using the ROS assay kit. A spectrophotometer was used to assess superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity, and PCR was used to test mRNA expression of SOD and GSH-PX. SiRNA was used to block receptor for advanced glycation endproducts (RAGEs) expression. Apoptosis was evaluated by Annexin V immunostaining and TUNEL staining. Co-culturing with AGEs increases ROS production, decreases anti-oxidant defenses, overpresses oxidant stress, inhibits the proliferation, migration, and adhesion of EPCs, and induces EPCs apoptosis. In addition, these effects were attenuated during block RAGE protein expression by siRNA. AGEs may serve to impair EPCs functions through RAGE-mediate oxidant stress, and promote EPCs sensitivity toward oxidative-stress-mediated apoptosis, which indicates a new pathophysiological mechanism of disturbed vascular adaptation in atherosclerosis and suggests that lower levels of AGEs might improve the

  17. The antifibrotic effects of TGF-β1 siRNA on hepatic fibrosis in rats

    International Nuclear Information System (INIS)

    Lang, Qing; Liu, Qi; Xu, Ning; Qian, Ke-Li; Qi, Jing-Hu; Sun, Yin-Chun; Xiao, Lang; Shi, Xiao-Feng

    2011-01-01

    Highlights: → We constructed CCL4 induced liver fibrosis model successfully. → We proofed that the TGF-β1 siRNA had a definite therapy effect to CCL4 induced liver fibrosis. → The therapy effect of TGF-β1 siRNA had dose-dependent. -- Abstract: Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-β1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-β1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague-Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-β1 siRNA 0.125 mg/kg treatment group, TGF-β1 siRNA 0.25 mg/kg treatment group and TGF-β1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin-Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-β1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-β1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-β1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-β1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-β1 siRNA negative control group and the TGF-β1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the mRNA expression of TGF-β1, type I collagen and type III collagen (P < 0

  18. The antifibrotic effects of TGF-{beta}1 siRNA on hepatic fibrosis in rats

    Energy Technology Data Exchange (ETDEWEB)

    Lang, Qing; Liu, Qi [Key Laboratory of Molecular Biology for Infectious Diseases, Ministry of Education, Instituted for Virus Hepatitis and Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing (China); Xu, Ning [The Second Hospital of YuLin, Shanxi Province (China); Qian, Ke-Li; Qi, Jing-Hu; Sun, Yin-Chun; Xiao, Lang [Key Laboratory of Molecular Biology for Infectious Diseases, Ministry of Education, Instituted for Virus Hepatitis and Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing (China); Shi, Xiao-Feng, E-mail: sxff2003@yahoo.com.cn [Key Laboratory of Molecular Biology for Infectious Diseases, Ministry of Education, Instituted for Virus Hepatitis and Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing (China)

    2011-06-10

    Highlights: {yields} We constructed CCL4 induced liver fibrosis model successfully. {yields} We proofed that the TGF-{beta}1 siRNA had a definite therapy effect to CCL4 induced liver fibrosis. {yields} The therapy effect of TGF-{beta}1 siRNA had dose-dependent. -- Abstract: Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-{beta}1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-{beta}1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague-Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-{beta}1 siRNA 0.125 mg/kg treatment group, TGF-{beta}1 siRNA 0.25 mg/kg treatment group and TGF-{beta}1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin-Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-{beta}1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-{beta}1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-{beta}1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-{beta}1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-{beta}1 siRNA negative control group and the TGF-{beta}1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the m

  19. Nutrient Availability Alters the Effect of Autophagy on Sulindac Sulfide-Induced Colon Cancer Cell Apoptosis

    Directory of Open Access Journals (Sweden)

    Shiun-Kwei Chiou

    2012-01-01

    Full Text Available Autophagy is a catabolic process by which a cell degrades its intracellular materials to replenish itself. Induction of autophagy under various cellular stress stimuli can lead to either cell survival or cell death via apoptotic and/or autophagic (nonapoptotic pathways. The NSAID sulindac sulfide induces apoptosis in colon cancer cells. Here, we show that inhibition of autophagy under serum-deprived conditions resulted in significant reductions of sulindac sulfide-induced apoptosis in HT-29 colon cancer cells. In contrast, inhibition of autophagy under conditions where serum is available significantly increased sulindac sulfide-induced apoptosis in HT-29 cells. We previously showed that the apoptosis inhibitor, survivin, plays a role in regulating NSAID-induced apoptosis and autophagic cell death. Here, we show that survivin protein half-life is increased in the presence of autophagy inhibitors under serum-deprived conditions, but not under conditions when serum is available. Thus, the increased levels of survivin may be a factor contributing to inhibition of sulindac sulfide-induced apoptosis under serum-deprived conditions. These results suggest that whether a cell lives or dies due to autophagy induction depends on the balance of factors that regulate both autophagic and apoptotic processes.

  20. Herbal medicine as inducers of apoptosis in cancer treatment.

    Science.gov (United States)

    Safarzadeh, Elham; Sandoghchian Shotorbani, Siamak; Baradaran, Behzad

    2014-10-01

    Cancer is uncontrolled growth of abnormal cells in the body. Nowadays, cancer is considered as a human tragedy and one of the most prevalent diseases in the wide, and its mortality resulting from cancer is being increased. It seems necessary to identify new strategies to prevent and treat such a deadly disease. Control survival and death of cancerous cell are important strategies in the management and therapy of cancer. Anticancer agents should kill the cancerous cell with the minimal side effect on normal cells that is possible through the induction of apoptosis. Apoptosis is known as programmed cell death in both normal and damaged tissues. This process includes some morphologically changes in cells such as rapid condensation and budding of the cell, formation of membrane-enclosed apoptotic bodies with well-preserved organelles. Induction of apoptosis is one of the most important markers of cytotoxic antitumor agents. Some natural compounds including plants induce apoptotic pathways that are blocked in cancer cells through various mechanisms in cancer cells. Multiple surveys reported that people with cancer commonly use herbs or herbal products. Vinca Alkaloids, Texans, podo phyllotoxin, Camptothecins have been clinically used as Plant derived anticancer agents. The present review summarizes the literature published so far regarding herbal medicine used as inducers of apoptosis in cancer.

  1. Valsartan protects HK-2 cells from contrast media-induced apoptosis by inhibiting endoplasmic reticulum stress.

    Science.gov (United States)

    Peng, Ping-An; Wang, Le; Ma, Qian; Xin, Yi; Zhang, Ou; Han, Hong-Ya; Liu, Xiao-Li; Ji, Qing-Wei; Zhou, Yu-Jie; Zhao, Ying-Xin

    2015-12-01

    Contrast-induced acute kidney injury (CI-AKI) is associated with increasing in-hospital and long-term adverse clinical outcomes in high-risk patients undergoing percutaneous coronary intervention (PCI). Contrast media (CM)-induced renal tubular cell apoptosis is reported to participate in this process by activating endoplasmic reticulum (ER) stress. An angiotensin II type 1 receptor (AT1R) antagonist can alleviate ER stress-induced renal apoptosis in streptozotocin (STZ)-induced diabetic mice and can reduce CM-induced renal apoptosis by reducing oxidative stress and reversing the enhancement of bax mRNA and the reduction of bcl-2 mRNA, but the effect of the AT1R blocker on ER stress in the pathogenesis of CI-AKI is still unknown. In this study, we explored the effect of valsartan on meglumine diatrizoate-induced human renal tubular cell apoptosis by measuring changes in ER stress-related biomarkers. The results showed that meglumine diatrizoate caused significant cell apoptosis by up-regulating the expression of ER stress markers, including glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), CCAAT/enhancer-binding protein-homologous protein (CHOP) and caspase 12, in a time- and dose-dependent manner, which could be alleviated by preincubation with valsartan. In conclusion, valsartan had a potential nephroprotective effect on meglumine diatrizoate-induced renal cell apoptosis by inhibiting ER stress. © 2015 International Federation for Cell Biology.

  2. Radiation-induced apoptosis of lymphocytes in peripheral blood

    International Nuclear Information System (INIS)

    Oh, Yoon Kyeong; Lee, Tae Bum; Nam, Taek Keun; Kee, Keun Hong; Choi, Cheol Hee

    2003-01-01

    This study quantitatively evaluated the apoptosis in human peripheral blood lymphocytes using flow cytometry, and investigated the possibility of using this method, with a small amount of blood, and the time and dose dependence of radiation-induced apoptosis. Peripheral blood lymphocytes were isolated from the heparinized venous blood of 11 healthy volunteers, 8 men and 3 women, with each 10 ml of blood being divided into 15 samples. The blood lymphocytes were irradiated using a linear accelerator at a dose rate of 2.4 Gy/min, to deliver doses of 0.5, 1, 2 and 5 Gy. The control samples, and irradiated cells, were maintained in culture medium for 24, 48 and 72 hours following the irradiation. The number of apoptotic cells after the in vitro X-irradiation was measured by flow cytometry after incubation periods of 24, 48 and 72 hours. We also observed the apoptotic cells using a DNA fragmentation assay and electron microscopy. The rate of spontaneous apoptosis increased in relation to the time interval following irradiation (1.761±0.161, 3.563±0.564, 11.098±2.849, at 24, 48, and 72 hours). The apoptotic cells also increased in the samples irradiated with 0.5, 1, 2 and 5 Gy, in a radiation dose and time interval after irradiation manner, with the apoptosis being too great at 72 hours after irradiation. The dose-response curves were characterized by an initial steep increase in the number of apoptotic cells for irradiation doses below 2 Gy, with a flattening of the curves as the dose approached towards 5 Gy. The flow cytometric assay technique yielded adequate data, and required less than 1 mL of blood. The time and dose dependence of the radiation-induced apoptosis, was also shown. It is suggested that the adequate time interval required for the evaluation of apoptosis would be 24 to 48 hours after blood sampling

  3. Aminomethylphosphonic Acid and Methoxyacetic Acid Induce Apoptosis in Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Keshab R. Parajuli

    2015-05-01

    Full Text Available Aminomethylphosphonic acid (AMPA and its parent compound herbicide glyphosate are analogs to glycine, which have been reported to inhibit proliferation and promote apoptosis of cancer cells, but not normal cells. Methoxyacetic acid (MAA is the active metabolite of ester phthalates widely used in industry as gelling, viscosity and stabilizer; its exposure is associated with developmental and reproductive toxicities in both rodents and humans. MAA has been reported to suppress prostate cancer cell growth by inducing growth arrest and apoptosis. However, it is unknown whether AMPA and MAA can inhibit cancer cell growth. In this study, we found that AMPA and MAA inhibited cell growth in prostate cancer cell lines (LNCaP, C4-2B, PC-3 and DU-145 through induction of apoptosis and cell cycle arrest at the G1 phase. Importantly, the AMPA-induced apoptosis was potentiated with the addition of MAA, which was due to downregulation of the anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, leading to activation of caspases 7 and 3. These results demonstrate that the combination of AMPA and MAA can promote the apoptosis of prostate cancer cells, suggesting that they can be used as potential therapeutic drugs in the treatment of prostate cancer.

  4. Aminomethylphosphonic acid and methoxyacetic acid induce apoptosis in prostate cancer cells.

    Science.gov (United States)

    Parajuli, Keshab R; Zhang, Qiuyang; Liu, Sen; You, Zongbing

    2015-05-22

    Aminomethylphosphonic acid (AMPA) and its parent compound herbicide glyphosate are analogs to glycine, which have been reported to inhibit proliferation and promote apoptosis of cancer cells, but not normal cells. Methoxyacetic acid (MAA) is the active metabolite of ester phthalates widely used in industry as gelling, viscosity and stabilizer; its exposure is associated with developmental and reproductive toxicities in both rodents and humans. MAA has been reported to suppress prostate cancer cell growth by inducing growth arrest and apoptosis. However, it is unknown whether AMPA and MAA can inhibit cancer cell growth. In this study, we found that AMPA and MAA inhibited cell growth in prostate cancer cell lines (LNCaP, C4-2B, PC-3 and DU-145) through induction of apoptosis and cell cycle arrest at the G1 phase. Importantly, the AMPA-induced apoptosis was potentiated with the addition of MAA, which was due to downregulation of the anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2), leading to activation of caspases 7 and 3. These results demonstrate that the combination of AMPA and MAA can promote the apoptosis of prostate cancer cells, suggesting that they can be used as potential therapeutic drugs in the treatment of prostate cancer.

  5. Gallic acid induces apoptosis in EGFR-mutant non-small cell lung cancers by accelerating EGFR turnover.

    Science.gov (United States)

    Nam, Boas; Rho, Jin Kyung; Shin, Dong-Myung; Son, Jaekyoung

    2016-10-01

    Gallic acid is a common botanic phenolic compound, which is present in plants and foods worldwide. Gallic acid is implicated in various biological processes such as cell growth and apoptosis. Indeed, gallic acid has been shown to induce apoptosis in many cancer types. However, the molecular mechanisms of gallic acid-induced apoptosis in cancer, particularly lung cancer, are still unclear. Here, we report that gallic acid induces apoptosis in EGFR-mutant non-small cell lung cancer (NSCLC) cells, but not in EGFR-WT NSCLC cells. Treatment with gallic acid resulted in a significant reduction in proliferation and induction of apoptosis, only in EGFR-mutant NSCLC cells. Interestingly, treatment with gallic acid led to a robust decrease in EGFR levels, which is critical for NSCLC survival. Treatment with gallic acid had no significant effect on transcription, but induced EGFR turnover. Indeed, treatment with a proteasome inhibitor dramatically reversed gallic acid-induced EGFR downregulation. Moreover, treatment with gallic acid induced EGFR turnover leading to apoptosis in EGFR-TKI (tyrosine kinase inhibitor)-resistant cell lines, which are dependent on EGFR signaling for survival. Thus, these studies suggest that gallic acid can induce apoptosis in EGFR-dependent lung cancers that are dependent on EGFR for growth and survival via acceleration of EGFR turnover. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Relationship between radiation induced activation of DNA repair genes and radiation induced apoptosis in human cell line A431

    International Nuclear Information System (INIS)

    Bom, Hee Seung; Min, Jung Jun; Kim, Kyung Keun; Choi, Keun Hee

    2000-01-01

    The purpose of this study was to evaluate the relationship between radiation-induced acivation of DNA repair genes and radiation induced apoptosis in A431 cell line. Five and 25 Gys of gamma radiation were given to A431 cells by a Cs-137 cell irradiator. Apoptosis was evaluated by flow cytometry using annexin V-fluorescein isothiocyanate and propidium iodide staining. The expression of DNA repair genes was evaluated by both Northern and Western blot analyses. The number of apoptotic cells increased with the increased radiation dose. It increased most significantly at 12 hours after irradiation. Expression of p53, p21, and ℎRAD50 reached the highest level at 12 hours after 5 Gy irradiation. In response to 25 Gy irradiation, ℎRAD50 and p21 were expressed maximally at 12 hours, but p53 and GADD45 genes showed the highest expression level after 12 hours. Induction of apoptosis and DNA repair by ionizing radiation were closely correlated. The peak time of inducing apoptosis and DNA repair was 12 hours in this study model. ℎRAD50, a recently discovered DNA repair gene, was also associated with radiation-induced apoptosis.=20

  7. [RNA interference of HERC4 inhibits proliferation, apoptosis and migration of cervical cancer Hela cells].

    Science.gov (United States)

    Wei, Min; Zhang, Yan-Ling; Chen, Lan; Cai, Cui-Xia; Wang, Han-Duo

    2016-02-20

    To explore the effects of silencing HERC4 on the proliferation, apoptosis, and migration of cervical cancer cell line Hela and the possible molecular mechanisms. Three HERC4-specific small interfering RNAs (siRNAs) were transfected into Hela cells, and HERC4 expression in the cells was examined with Western blotting. CCK-8 assay, annexin V-FITC/PI assay, and wound healing assay were used to assess the effect of HERC4 silencing on the proliferation, apoptosis and migration ability of Hela cells. The expression levels of cyclin D1 and Bcl-2 in the cells were detected using Western blotting. Transfection of siRNA-3 resulted in significantly decreased HERC4 protein expression (PHela cells, increased the apoptosis rate (PHela cells in vitro, and the underlying mechanisms may involve the down-regulation of cyclin D1 and Bcl-2.

  8. RITA inhibits growth of human hepatocellular carcinoma through induction of apoptosis.

    Science.gov (United States)

    Wang, Haihe; Chen, Guofu; Wang, Hongzhi; Liu, Chunbo

    2013-01-01

    RBP-J-interacting and tubulin-associated (RITA) is a novel RBP-J-interacting protein that downregulates Notch-mediated transcription. The current study focuses on the antitumor effect of RITA in human hepatocellular carcinoma (HCC) and aims to explore its molecular mechanism. Thirty paired HCC and adjacent non-tumoral liver samples were analyzed by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RITA overexpression was induced by transfection of a pcDNA3.1-Flag-RITA plasmid into HepG2 cells. RITA knockdown was achieved by siRNA transfection. mRNA and protein expression of target genes were quantified by qRT-PCR and Western blotting, respectively. Cell proliferation and apoptosis were measured using MTT assay and flow cytometry. Our results demonstrate that adjacent nontumoral liver samples exhibited increased RITA expression compared to HCC tissues (p RITA levels were associated with tumor differentiation status. Overexpression of RITA suppressed cell proliferation and promoted early apoptosis, while its silencing promoted cell growth dramatically (p RITA overexpression upregulated p53 and reduced cyclin E levels, whereas silencing of RITA had the opposite effect on p53 and cyclin E expression. Our in vitro results represent the first evidence that RITA might suppress tumor growth and induce apoptosis in HCCs, and may be a potent antitumoral agent for HCC treatment that deserves further exploration.

  9. GSK-3beta inhibition enhances sorafenib-induced apoptosis in melanoma cell lines.

    Science.gov (United States)

    Panka, David J; Cho, Daniel C; Atkins, Michael B; Mier, James W

    2008-01-11

    Glycogen synthase kinase-3beta (GSK-3beta) can participate in the induction of apoptosis or, alternatively, provide a survival signal that minimizes cellular injury. We previously demonstrated that the multikinase inhibitor sorafenib induces apoptosis in melanoma cell lines. In this report, we show that sorafenib activates GSK-3beta in multiple subcellular compartments and that this activation undermines the lethality of the drug. Pharmacologic inhibition and/or down-modulation of the kinase enhances sorafenib-induced apoptosis as determined by propidium iodide staining and by assessing the mitochondrial release of apoptosis-inducing factor and Smac/DIABLO. Conversely, the forced expression of a constitutively active form of the enzyme (GSK-3beta(S9A)) protects the cells from the apoptotic effects of the drug. This protective effect is associated with a marked increase in basal levels of Bcl-2, Bcl-x(L), and survivin and a diminution in the degree to which these anti-apoptotic proteins are down-modulated by sorafenib exposure. Sorafenib down-modulates the pro-apoptotic Bcl-2 family member Noxa in cells with high constitutive GSK-3beta activity. Pharmacologic inhibition of GSK-3beta prevents the disappearance of Noxa induced by sorafenib and enhances the down-modulation of Mcl-1. Down-modulation of Noxa largely eliminates the enhancing effect of GSK-3 inhibition on sorafenib-induced apoptosis. These data provide a strong rationale for the use of GSK-3beta inhibitors as adjuncts to sorafenib treatment and suggest that preservation of Noxa may contribute to their efficacy.

  10. Knockdown of TFIIS by RNA silencing inhibits cancer cell proliferation and induces apoptosis

    International Nuclear Information System (INIS)

    Hubbard, Kyle; Catalano, Jennifer; Puri, Raj K; Gnatt, Averell

    2008-01-01

    A common element among cancer cells is the presence of improperly controlled transcription. In these cells, the degree of specific activation of some genes is abnormal, and altering the aberrant transcription may therefore directly target cancer. TFIIS is a transcription elongation factor, which directly binds the transcription motor, RNA Polymerase II and allows it to read through various transcription arrest sites. We report on RNA interference of TFIIS, a transcription elongation factor, and its affect on proliferation of cancer cells in culture. RNA interference was performed by transfecting siRNA to specifically knock down TFIIS expression in MCF7, MCF10A, PL45 and A549 cells. Levels of TFIIS expression were determined by the Quantigene method, and relative protein levels of TFIIS, c-myc and p53 were determined by C-ELISA. Induction of apoptosis was determined by an enzymatic Caspase 3/7 assay, as well as a non-enzymatic assay detecting cytoplasmic mono- and oligonucleosomes. A gene array analysis was conducted for effects of TFIIS siRNA on MCF7 and MCF10A cell lines. Knockdown of TFIIS reduced cancer cell proliferation in breast, lung and pancreatic cancer cell lines. More specifically, TFIIS knockdown in the MCF7 breast cancer cell line induced cancer cell death and increased c-myc and p53 expression whereas TFIIS knockdown in the non-cancerous breast cell line MCF10A was less affected. Differential effects of TFIIS knockdown in MCF7 and MCF10A cells included the estrogenic, c-myc and p53 pathways, as observed by C-ELISA and gene array, and were likely involved in MCF7 cell-death. Although transcription is a fundamental process, targeting select core transcription factors may provide for a new and potent avenue for cancer therapeutics. In the present study, knockdown of TFIIS inhibited cancer cell proliferation, suggesting that TFIIS could be studied as a potential cancer target within the transcription machinery

  11. UVC-induced apoptosis in Dubca cells is independent of JNK activation and p53Ser-15 phosphorylation

    International Nuclear Information System (INIS)

    Chathoth, Shahanas; Thayyullathil, Faisal; Hago, Abdulkader; Shahin, Allen; Patel, Mahendra; Galadari, Sehamuddin

    2009-01-01

    Ultraviolet C (UVC) irradiation in mammalian cell lines activates a complex signaling network that leads to apoptosis. By using Dubca cells as a model system, we report the presence of a UVC-induced apoptotic pathway that is independent of c-Jun N-terminal kinases (JNKs) activation and p53 phosphorylation at Ser 15 . Irradiation of Dubca cells with UVC results in a rapid JNK activation and phosphorylation of its downstream target c-Jun, as well as, phosphorylation of activating transcription factor 2 (ATF2). Pre-treatment with JNK inhibitor, SP600125, inhibited UVC-induced c-Jun phosphorylation without preventing UVC-induced apoptosis. Similarly, inhibition of UVC-induced p53 phosphorylation did not prevent Dubca cell apoptosis, suggesting that p53 Ser-15 phosphorylation is not associated with UVC-induced apoptosis signaling. The pan-caspase inhibitor z-VAD-fmk inhibited UVC-induced PARP cleavage, DNA fragmentation, and ultimately apoptosis of Dubca cells. Altogether, our study clearly indicates that UVC-induced apoptosis is independent of JNK and p53 activation in Dubca cells, rather, it is mediated through a caspase dependent pathway. Our findings are not in line with the ascribed critical role for JNKs activation, and downstream phosphorylation of targets such as c-Jun and ATF2 in UVC-induced apoptosis.

  12. Novel TRAIL sensitizer Taraxacum officinale F.H. Wigg enhances TRAIL-induced apoptosis in Huh7 cells.

    Science.gov (United States)

    Yoon, Ji-Yong; Cho, Hyun-Soo; Lee, Jeong-Ju; Lee, Hyo-Jung; Jun, Soo Young; Lee, Jae-Hye; Song, Hyuk-Hwan; Choi, SangHo; Saloura, Vassiliki; Park, Choon Gil; Kim, Cheol-Hee; Kim, Nam-Soon

    2016-04-01

    TRAIL (TNF-related apoptosis inducing ligand) is a promising anti-cancer drug target that selectively induces apoptosis in cancer cells. However, many cancer cells are resistant to TRAIL-induced apoptosis. Therefore, reversing TRAIL resistance is an important step for the development of effective TRAIL-based anti-cancer therapies. We previously reported that knockdown of the TOR signaling pathway regulator-like (TIPRL) protein caused TRAIL-induced apoptosis by activation of the MKK7-c-Jun N-terminal Kinase (JNK) pathway through disruption of the MKK7-TIPRL interaction. Here, we identified Taraxacum officinale F.H. Wigg (TO) as a novel TRAIL sensitizer from a set of 500 natural products using an ELISA system and validated its activity by GST pull-down analysis. Furthermore, combination treatment of Huh7 cells with TRAIL and TO resulted in TRAIL-induced apoptosis mediated through inhibition of the MKK7-TIPRL interaction and subsequent activation of MKK7-JNK phosphorylation. Interestingly, HPLC analysis identified chicoric acid as a major component of the TO extract, and combination treatment with chicoric acid and TRAIL induced TRAIL-induced cell apoptosis via JNK activation due to inhibition of the MKK7-TIPRL interaction. Our results suggest that TO plays an important role in TRAIL-induced apoptosis, and further functional studies are warranted to confirm the importance of TO as a novel TRAIL sensitizer for cancer therapy. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  13. Radiation-induced apoptosis in the neonatal and adult rat spinal cord.

    Science.gov (United States)

    Li, Y Q; Wong, C S

    2000-09-01

    This study was designed to characterize radiation-induced apoptosis in the spinal cord of the neonatal and young adult rat. Spinal cords (C2-T2) of 1-, 2- and 10-week-old rats were irradiated with a single dose of 8, 18 or 22 Gy. Apoptosis was assessed histologically according to its specific morphological features or by using the TUNEL assay. Cell proliferation was assessed immunohistochemically using BrdU. Identities of cell types undergoing apoptosis were assessed using immunohistochemistry or in situ hybridization using markers for neurons, glial progenitor cells, microglia, oligodendrocytes and astrocytes. The time course of radiation-induced apoptosis in 1- or 2-week-old rat spinal cord was similar to that in the young adult rat spinal cord. A peak response was observed at about 8 h after irradiation, and the apoptosis index returned to the levels in nonirradiated spinal cords at 24 h. The neonatal rat spinal cord demonstrated increased apoptosis compared to the adult. Values for total yield of apoptosis over 24 h induced by 8 Gy in the neonatal rat spinal cord were significantly greater than that in the adult. Immunohistochemistry studies using Leu7, galactocerebroside, Rip and adenomatous polyposis coli tumor suppressor protein indicated that most apoptotic cells were cells of the oligodendroglial lineage regardless of the age of the animal. No evidence of Gfap or factor VIII-related antigen-positive apoptotic cells was observed, and there was a small number of apoptotic microglial cells (lectin-Rca1 positive) in the neonatal and adult rat spinal cord. In the neonatal but not adult rat spinal cord, about 10% of the apoptotic cells appeared to be neurons and were immunoreactive for synaptophysin. Labeling indices (LI) for BrdU in nonirradiated 1- and 2-week-old rat spinal cord were 20.0 and 16.3%, respectively, significantly greater than the LI of 1.0% in the 10-week-old rat spinal cord. At 8 h after a single dose of 8 Gy, 13.4% of the apoptotic cells were

  14. TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

    International Nuclear Information System (INIS)

    Ding, Li; Huang, Yong; Du, Qian; Dong, Feng; Zhao, Xiaomin; Zhang, Wenlong; Xu, Xingang; Tong, Dewen

    2014-01-01

    Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence

  15. Study on apoptosis of prostate cancer cell induced by 125I seed irradiation

    International Nuclear Information System (INIS)

    Liao Anyan; Wang Junjie; Wang Jidong; Zhuang Hongqing; Zhao Yong

    2007-01-01

    Objective: To explore the mechanism of apoptosis induced by 125 I seed irradiation on PC3 cells. Methods: Human prostate cancer cell line PC3 was treated by irradiation of 125 I (2.77 cGy/h) with various dose. Agarose gel electrophoresis of DNA and flows cytometry were used to detect the apoptosis of PC3 cells and indirect immunofluorescence assay was used to detect the expression of Bcl-2. The activity of Caspase-3 was measured by Caspase Colorimetric Assay Kits. Results: Apoptosis of PC3 cells could be efficiently induced by 125 I seed irradiation. The apoptotic peaks were found by flow cytometry and DNA ladder appeared on 1.8% agarose gel. The activity of Caspase-3 on PC3 cells treated by 125 I seed irradiation was not changed significantly. Bcl-2 gene expression was down-regulated with the sample concentration increased. Conclusion: 125 I irradiation can induce the apoptosis of PC3 cells and the mechanism of apoptosis is related with down regulation of Bcl-2 gene expression and is not related with Caspase-3 activity. (authors)

  16. Chlorella vulgaris triggers apoptosis in hepatocarcinogenesis-induced rats*

    Science.gov (United States)

    Mohd Azamai, Emey Suhana; Sulaiman, Suhaniza; Mohd Habib, Shafina Hanim; Looi, Mee Lee; Das, Srijit; Abdul Hamid, Nor Aini; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum

    2009-01-01

    Chlorella vulgaris (CV) has been reported to have antioxidant and anticancer properties. We evaluated the effect of CV on apoptotic regulator protein expression in liver cancer-induced rats. Male Wistar rats (200~250 g) were divided into eight groups: control group (normal diet), CDE group (choline deficient diet supplemented with ethionine in drinking water to induce hepatocarcinogenesis), CV groups with three different doses of CV (50, 150, and 300 mg/kg body weight), and CDE groups treated with different doses of CV (50, 150, and 300 mg/kg body weight). Rats were sacrificed at various weeks and liver tissues were embedded in paraffin blocks for immunohistochemistry studies. CV, at increasing doses, decreased the expression of anti-apoptotic protein, Bcl-2, but increased the expression of pro-apoptotic protein, caspase 8, in CDE rats, which was correlated with decreased hepatoctyes proliferation and increased apoptosis as determined by bromodeoxy-uridine (BrdU) labeling and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay, respectively. Our study shows that CV has definite chemopreventive effect by inducing apoptosis via decreasing the expression of Bcl-2 and increasing the expression of caspase 8 in hepatocarcinogenesis-induced rats. PMID:19198018

  17. O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling

    OpenAIRE

    Shi, Jianhua; Gu, Jin-hua; Dai, Chun-ling; Gu, Jianlan; Jin, Xiaoxia; Sun, Jianming; Iqbal, Khalid; Liu, Fei; Gong, Cheng-Xin

    2015-01-01

    Apoptosis plays an important role in neural development and neurological disorders. In this study, we found that O-GlcNAcylation, a unique protein posttranslational modification with O-linked β-N-acetylglucosamine (GlcNAc), promoted apoptosis through attenuating phosphorylation/activation of AKT and Bad. By using co-immunoprecipitation and mutagenesis techniques, we identified O-GlcNAc modification at both Thr308 and Ser473 of AKT. O-GlcNAcylation-induced apoptosis was attenuated by over-expr...

  18. Influence of radiation-induced apoptosis on development brain in molecular regulation

    International Nuclear Information System (INIS)

    Gu Guixiong

    2000-01-01

    An outline of current status on the influence of radiation on the development brain was given. Some genes as immediate early gene, Bcl-2 family, p53, heat shock protein and AT gene play an important regulation role in ionizing radiation-induced development brain cells apoptosis. And such biological factor as nerve growth factor, interleukin-1, tumor necrosis factor, basic fibroblast growth factor, transforming growth factor and so on have a vital protection function against ionizing radiation-induced cells apoptosis

  19. Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7.

    Science.gov (United States)

    Bi, Yanzhao; Zhang, Yifan; Cui, Chunying; Ren, Lulu; Jiang, Xueyun

    Nanodiamond (ND) is a renowned material in nonviral small interfering RNA (siRNA) carrier field due to its unique physical, chemical, and biological properties. In our previous work, it was proven that ND could deliver siRNA into cells efficiently and downregulate the expression of desired protein. However, synthesizing a high-efficient tumor-targeting carrier using ND is still a challenge. In this study, a novel carrier, NDCONH(CH 2 ) 2 NH-VDGR, was synthesized for siRNA delivery, and its properties were characterized with methods including Fourier transform infrared spectrometry, transmission electron microscopy, scanning electron microscopy, gel retardation assay, differential scanning calorimetry, confocal microscopy, releasing test, real-time polymerase chain reaction (PCR) assay, enzyme-linked immunosorbent assay (ELISA), flow cytometry, cytotoxicity assay, and gene-silencing efficacy assay in vitro and in vivo. The mechanism of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA-induced tumor apoptosis was evaluated via flow cytometer assay using Annexin V-fluorescein isothiocyanate/propidium iodide staining method. The NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA nanoparticle with 60-110 nm diameter and 35.65±3.90 mV zeta potential was prepared. For real-time PCR assay, the results showed that the expression of survivin mRNA was reduced to 46.77%±6.3%. The expression of survivin protein was downregulated to 48.49%±2.25%, as evaluated by ELISA assay. MTT assay showed that NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA had an inhibitory effect on MCF-7 cell proliferation. According to these results, the survivin-siRNA could be delivered, transported, and released stably, which benefits in increasing the gene-silencing effect. Therefore, as an siRNA carrier, NDCONH(CH 2 ) 2 NH-VDGR was suggested to be used in siRNA delivery system and in cancer treatments.

  20. Recombinant Vaccinia Viruses Coding Transgenes of Apoptosis-Inducing Proteins Enhance Apoptosis But Not Immunogenicity of Infected Tumor Cells

    Science.gov (United States)

    Tkachenko, Anastasiya; Richter, Vladimir

    2017-01-01

    Genetic modifications of the oncolytic vaccinia virus (VV) improve selective tumor cell infection and death, as well as activation of antitumor immunity. We have engineered a double recombinant VV, coding human GM-CSF, and apoptosis-inducing protein apoptin (VV-GMCSF-Apo) for comparing with the earlier constructed double recombinant VV-GMCSF-Lact, coding another apoptosis-inducing protein, lactaptin, which activated different cell death pathways than apoptin. We showed that both these recombinant VVs more considerably activated a set of critical apoptosis markers in infected cells than the recombinant VV coding GM-CSF alone (VV-GMCSF-dGF): these were phosphatidylserine externalization, caspase-3 and caspase-7 activation, DNA fragmentation, and upregulation of proapoptotic protein BAX. However, only VV-GMCSF-Lact efficiently decreased the mitochondrial membrane potential of infected cancer cells. Investigating immunogenic cell death markers in cancer cells infected with recombinant VVs, we demonstrated that all tested recombinant VVs were efficient in calreticulin and HSP70 externalization, decrease of cellular HMGB1, and ATP secretion. The comparison of antitumor activity against advanced MDA-MB-231 tumor revealed that both recombinants VV-GMCSF-Lact and VV-GMCSF-Apo efficiently delay tumor growth. Our results demonstrate that the composition of GM-CSF and apoptosis-inducing proteins in the VV genome is very efficient tool for specific killing of cancer cells and for activation of antitumor immunity. PMID:28951871

  1. Paclitaxel Induces Apoptosis in Breast Cancer Cells through Different Calcium—Regulating Mechanisms Depending on External Calcium Conditions

    Science.gov (United States)

    Pan, Zhi; Avila, Andrew; Gollahon, Lauren

    2014-01-01

    Previously, we reported that endoplasmic reticulum calcium stores were a direct target for paclitaxel initiation of apoptosis. Furthermore, the actions of paclitaxel attenuated Bcl-2 resistance to apoptosis through endoplasmic reticulum-mediated calcium release. To better understand the calcium-regulated mechanisms of paclitaxel-induced apoptosis in breast cancer cells, we investigated the role of extracellular calcium, specifically; whether influx of extracellular calcium contributed to and/or was necessary for paclitaxel-induced apoptosis. Our results demonstrated that paclitaxel induced extracellular calcium influx. This mobilization of extracellular calcium contributed to subsequent cytosolic calcium elevation differently, depending on dosage. Under normal extracellular calcium conditions, high dose paclitaxel induced apoptosis-promoting calcium influx, which did not occur in calcium-free conditions. In the absence of extracellular calcium an “Enhanced Calcium Efflux” mechanism in which high dose paclitaxel stimulated calcium efflux immediately, leading to dramatic cytosolic calcium decrease, was observed. In the absence of extracellular calcium, high dose paclitaxel’s stimulatory effects on capacitative calcium entry and apoptosis could not be completely restored. Thus, normal extracellular calcium concentrations are critical for high dose paclitaxel-induced apoptosis. In contrast, low dose paclitaxel mirrored controls, indicating that it occurs independent of extracellular calcium. Thus, extracellular calcium conditions only affect efficacy of high dose paclitaxel-induced apoptosis. PMID:24549172

  2. The protective effect of resveratrol on human lens epithelial cells against ultraviolet-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Xue - Fang Chen

    2013-06-01

    Full Text Available AIM: To investigate the protective effect of resveratrol on human lens epithelial cells against ultraviolet-induced apoptosis. METHODS:Subcultured human lens epithelial cell line, ultraviolet induced cell apoptosis, 20μmol/L resveratrol pretreated cell, the indicators change was observed: rate of apoptosis was detected by flow cytometry and apoptosis-related factors of caspses-3 and caspase-9 were detected by colorimetric detection, ultrastructure changes were observed under transmission electron microscope. RESULTS: Flow cytometry instrument testing found that resveratrol can suppress the apoptosis induced by ultraviolet irradiation, caspses-3 and caspase-9 content in positive control group were significantly higher than that of the negative control group at the same time period, the difference was statistically significant(P<0.05; caspses-3 and caspase-9 content in experimental group were lower than that in the positive control group at the same time, the difference was statistically significant(P<0.05. In addition, the damage of human lens epithelial cells was alleviated with the incubation time of resveratrol elongated. CONCLUSION:Resveratrol may inhibit ultraviolet-induced apoptosis of human lens epithelial cells, it has preventive function against radioactive cataract, and it can provide reliable evidence for pursuing effective medicine to prevent and treat cataract.

  3. Correlation of the microculture-kinetic drug-induced apoptosis assay with patient outcomes in initial treatment of adult acute myelocytic leukemia.

    Science.gov (United States)

    Strickland, Stephen A; Raptis, Anastasios; Hallquist, Allan; Rutledge, James; Chernick, Michael; Perree, Mathieu; Talbott, Mahsa S; Presant, Cary A

    2013-03-01

    Overall survival (OS) with acute myeloid leukemia (AML) remains poor. Determining prognostic factors will help in selecting patients for appropriate treatments. Our aim was to determine whether the level of drug-induced apoptosis (chemosensitivity) demonstrated by the microculture-kinetic drug-induced apoptosis (MiCK) assay significantly predicted outcomes after standard AML induction therapy. A total of 109 patients with untreated AML had blood and/or bone marrow aspirate samples analyzed for anthracycline-induced apoptosis using the MiCK assay. The amount of apoptosis observed over 48 h was determined and expressed as kinetic units of apoptosis (KU). Complete remission (CR) was significantly higher (72%) in patients with high idarubicin-induced apoptosis >3 KU compared to patients with apoptosis ≤ 3 KU (p = 0.01). Multivariate analysis showed the only significant variables to be idarubicin-induced apoptosis and karyotype. Median overall survival of patients with idarubicin-induced apoptosis >3 KU was 16.1 months compared to 4.5 months in patients with apoptosis ≤ 3 KU (p = 0.004). Multivariate analysis showed the only significant variable to be idarubicin-induced apoptosis. Chemotherapy-induced apoptosis measured by the MiCK assay demonstrated significant correlation with outcomes and appears predictive of complete remission and overall survival for patients receiving standard induction chemotherapy.

  4. Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells.

    Science.gov (United States)

    Wang, Zheng; Cheng, Xingan; Meng, Qianqian; Wang, Peidan; Shu, Benshui; Hu, Qiongbo; Hu, Meiying; Zhong, Guohua

    2015-07-01

    Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells

    International Nuclear Information System (INIS)

    Ozeki, Nobuaki; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kinoshita, Katsue; Hase, Naoko; Nakata, Kazuhiko; Kondo, Ayami; Mogi, Makio; Nakamura, Hiroshi

    2014-01-01

    We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7 + hSMSC)-derived osteoblast-like (α7 + hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7 + hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7 + hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7 + hSMSC-OB cells are regulated by ADAM-28. - Highlights: • IL-1β induces the MMP-13 and ADAM-28 expression in human osteoblast-like cells. • IL-1β-induced MMP-13 expression increases proliferation and decreased apoptosis. • MMP-13 expression induced by IL-1β is regulated by ADAM-28. • proMMP-13 appears to be cleaved into its active form via ADAM-28

  6. IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kinoshita, Katsue; Hase, Naoko; Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya, Aichi 464-8650 (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya, Aichi 464-8650 (Japan); Nakamura, Hiroshi [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan)

    2014-04-15

    We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7{sup +}hSMSC)-derived osteoblast-like (α7{sup +}hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7{sup +}hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7{sup +}hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7{sup +}hSMSC-OB cells are regulated by ADAM-28. - Highlights: • IL-1β induces the MMP-13 and ADAM-28 expression in human osteoblast-like cells. • IL-1β-induced MMP-13 expression increases proliferation and decreased apoptosis. • MMP-13 expression induced by IL-1β is regulated by ADAM-28. • proMMP-13 appears to be cleaved into its active form via

  7. Advanced oxidation protein products induce chondrocyte apoptosis via receptor for advanced glycation end products-mediated, redox-dependent intrinsic apoptosis pathway.

    Science.gov (United States)

    Wu, Qian; Zhong, Zhao-Ming; Zhu, Si-Yuan; Liao, Cong-Rui; Pan, Ying; Zeng, Ji-Huan; Zheng, Shuai; Ding, Ruo-Ting; Lin, Qing-Song; Ye, Qing; Ye, Wen-Bin; Li, Wei; Chen, Jian-Ting

    2016-01-01

    Pro-inflammatory cytokine-induced chondrocyte apoptosis is a primary cause of cartilage destruction in the progression of rheumatoid arthritis (RA). Advanced oxidation protein products (AOPPs), a novel pro-inflammatory mediator, have been confirmed to accumulate in patients with RA. However, the effect of AOPPs accumulation on chondrocyte apoptosis and the associated cellular mechanisms remains unclear. The present study demonstrated that the plasma formation of AOPPs was enhanced in RA rats compared with normal. Then, chondrocyte were treated with AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. Exposure of chondrocyte to AOPPs activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and increased expression of NADPH oxidase subunits, which was mediated by receptor for advanced glycation end products (RAGE), but not scavenger receptor CD36. Moreover, AOPPs challenge triggered NADPH oxidase-dependent ROS generation which induced mitochondrial dysfunction and endoplasmic reticulum stress resulted in activation of caspase family that eventually lead to apoptosis. Lastly, blockade of RAGE, instead of CD36, largely attenuated these signals. Our study demonstrated first time that AOPPs induce chondrocyte apoptosis via RAGE-mediated and redox-dependent intrinsic apoptosis pathway in vitro. These data implicates that AOPPs may represent a novel pathogenic factor that contributes to RA progression. Targeting AOPPs-triggered cellular mechanisms might emerge as a promising therapeutic option for patients with RA.

  8. Caffeic Acid Induces Apoptosis in Human Cervical Cancer Cells Through the Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Wei-Chun Chang

    2010-12-01

    Conclusion: Caffeic acid induces apoptosis by inhibiting Bcl-2 activity, leading to release of cytochrome c and subsequent activation of caspase-3, indicating that caffeic acid induces apoptosis via the mitochondrial apoptotic pathway. This also suggests that caffeic acid has a strong anti-tumor effect and may be a promising chemopreventive or chemotherapeutic agent.

  9. El coactivador de receptores nucleares RAC3 tiene un rol protector de la Apoptosis inducida por distintos estímulos RAC3 nuclear receptor co-activator has a protective role in the apoptosis induced by different stimuli

    Directory of Open Access Journals (Sweden)

    Georgina P. Coló

    2007-10-01

    of RAC3 if compared with the non-tumoral HEK293 cells. The normal or transfected coactivator over-expression inhibits apoptosis through a diminished caspase activity and AIF nuclear translocation, increased NF-kappa;B, AKT and p38, and decreased ERK activities. In contrast, inhibition of RAC3 by siRNA induced sensitivity of K562 to TRAIL-induced apoptosis. Such results suggest that over-expression of RAC3 contributes to tumor development through molecular mechanisms that do not depend strictly on acetylation and/or steroid hormones, which control cell death. This could be a possible target for future tumor therapies.

  10. Globular Adiponectin Attenuated H2O2-Induced Apoptosis in Rat Chondrocytes by Inducing Autophagy Through the AMPK/ mTOR Pathway.

    Science.gov (United States)

    Hu, Junzheng; Cui, Weiding; Ding, Wenxiao; Gu, Yanqing; Wang, Zhen; Fan, Weimin

    2017-01-01

    Chondrocyte apoptosis is closely related to the development and progression of osteoarthritis. Global adiponectin (gAPN), secreted from adipose tissue, possesses potent anti-inflammatory and antiapoptotic properties in various cell types. This study aimed to investigate the role of autophagy induced by gAPN in the suppression of H2O2-induced apoptosis and the potential mechanism of gAPN-induced autophagy in chondrocytes. H2O2 was used to induce apoptotic injury in rat chondrocytes. CCK-8 assay was performed to determine the viability of cells treated with different concentrations of gAPN with or without H2O2. Cell apoptosis was detected by flow cytometry and TUNEL staining. Mitochondrial membrane potential was examined using JC-1 fluorescence staining assay. The autophagy inhibitors 3-MA and Bafilomycin A1 were used to treat cells and then evaluate the effect of gAPN-induced autophagy. To determine the downstream pathway, chondrocytes were preincubated with the AMPK inhibitor Compound C. Beclin-1, LC3B, P62 and apoptosis-related proteins were identified by Western blot analysis. H2O2 (400 µM)-induced chondrocytes apoptosis and caspase-3 activation were attenuated by gAPN (0.5 µg/mL). gAPN increased Bcl-2 expression and decreased Bax expression. The loss of mitochondrial membrane potential induced by H2O2 was also abolished by gAPN. Furthermore, the antiapoptotic effect of gAPN was related to gAPN-induced autophagy by increased formation of Beclin-1 and LC3B and P62 degradation. In particular, the inhibition of gAPN-induced autophagy by 3-MA prevented the protective effect of gAPN on apoptosis induced by H2O2. Moreover, gAPN increased p-AMPK expression and decreased p-mTOR expression. Compound C partly suppressed the expression of autophagy-related proteins and restored the expression of p-mTOR suppressed by gAPN. Thus, the AMPK/mTOR pathway played an important role in the induction of autophagy and protection of H2O2-induced chondrocytes apoptosis by gAPN. g

  11. Globular Adiponectin Attenuated H2O2-Induced Apoptosis in Rat Chondrocytes by Inducing Autophagy Through the AMPK/ mTOR Pathway

    Directory of Open Access Journals (Sweden)

    Junzheng Hu

    2017-08-01

    Full Text Available Background/Aims: Chondrocyte apoptosis is closely related to the development and progression of osteoarthritis. Global adiponectin (gAPN, secreted from adipose tissue, possesses potent anti-inflammatory and antiapoptotic properties in various cell types. This study aimed to investigate the role of autophagy induced by gAPN in the suppression of H2O2-induced apoptosis and the potential mechanism of gAPN-induced autophagy in chondrocytes. Methods: H2O2 was used to induce apoptotic injury in rat chondrocytes. CCK-8 assay was performed to determine the viability of cells treated with different concentrations of gAPN with or without H2O2. Cell apoptosis was detected by flow cytometry and TUNEL staining. Mitochondrial membrane potential was examined using JC-1 fluorescence staining assay. The autophagy inhibitors 3-MA and Bafilomycin A1 were used to treat cells and then evaluate the effect of gAPN-induced autophagy. To determine the downstream pathway, chondrocytes were preincubated with the AMPK inhibitor Compound C. Beclin-1, LC3B, P62 and apoptosis-related proteins were identified by Western blot analysis. Results: H2O2 (400 µM-induced chondrocytes apoptosis and caspase-3 activation were attenuated by gAPN (0.5 µg/mL. gAPN increased Bcl-2 expression and decreased Bax expression. The loss of mitochondrial membrane potential induced by H2O2 was also abolished by gAPN. Furthermore, the antiapoptotic effect of gAPN was related to gAPN-induced autophagy by increased formation of Beclin-1 and LC3B and P62 degradation. In particular, the inhibition of gAPN-induced autophagy by 3-MA prevented the protective effect of gAPN on apoptosis induced by H2O2. Moreover, gAPN increased p-AMPK expression and decreased p-mTOR expression. Compound C partly suppressed the expression of autophagy-related proteins and restored the expression of p-mTOR suppressed by gAPN. Thus, the AMPK/mTOR pathway played an important role in the induction of autophagy and protection of

  12. Biological dosimetry: the potential use of radiation-induced apoptosis in human T-lymphocytes

    International Nuclear Information System (INIS)

    Menz, R.; Andres, R.; Larsson, B.; Ozsahin, M.; Crompton, N.E.A.; Trott, K.

    1997-01-01

    An assay for biological dosimetry based on the induction of apoptosis in human T-lymphocytes is described. Radiation-induced apoptosis was assessed by flow cytometric identification of cells displaying apoptosis-associated DNA condensation. CD4 and CD8 T-lymphocytes were analysed. They were recognized on the basis of their cell-surface antigens. Four parameters were measured for both cell types: cell size, granularity, antigen immunofluorescence and DNA content. Apoptosis was quantified as the fraction of CD4-, or CD8-positive cells with a characteristic reduction of cell size and DNA content. At doses below 1 Gy, levels of radiation-induced apoptosis increased for up to 5 days after irradiation. Optimal dose discrimination was observed 4 days after irradiation, at which time the dose-response curves were linear, with a slope of 8% ± 0.5% per 0.1 Gy. In controlled, dose-response experiments the lowest dose level at which the radiation-induced apoptosis frequency was still significantly above control was 0.05 Gy. After 5 days post-irradiation incubation, intra- and interdonor variations were measured and found to be similar; thus, apoptotic levels depend more on the dose than on the donor. The results demonstrate the potential of this assay as a biological dosimeter. (orig.)

  13. Hot water extract of Chlorella vulgaris induced DNA damage and apoptosis

    Science.gov (United States)

    Yusof, Yasmin Anum Mohd; Md. Saad, Suhana; Makpol, Suzana; Shamaan, Nor Aripin; Ngah, Wan Zurinah Wan

    2010-01-01

    OBJECTIVES: The aim of this study was to determine the antiproliferative and apoptotic effects of hot water extracts of Chlorella vulgaris on hepatoma cell line HepG2. INTRODUCTION: The search for food and spices that can induce apoptosis in cancer cells has been a major study interest in the last decade. Chlorella vulgaris, a unicellular green algae, has been reported to have antioxidant and anti‐cancer properties. However, its chemopreventive effects in inhibiting the growth of cancer cells have not been studied in great detail. METHODS: HepG2 liver cancer cells and WRL68 normal liver cells were treated with various concentrations (0‐4 mg/ml) of hot water extract of C. vulgaris after 24 hours incubation. Apoptosis rate was evaluated by TUNEL assay while DNA damage was assessed by Comet assay. Apoptosis proteins were evaluated by Western blot analysis. RESULTS: Chlorella vulgaris decreased the number of viable HepG2 cells in a dose dependent manner (p Chlorella vulgaris tested. Evaluation of apoptosis by TUNEL assay showed that Chlorella vulgaris induced a higher apoptotic rate (70%) in HepG2 cells compared to normal liver cells, WRL68 (15%). Western blot analysis showed increased expression of pro‐ apoptotic proteins P53, Bax and caspase‐3 in the HepG2 cells compared to normal liver cells WRL68, and decreased expression of the anti‐apoptotic protein Bcl‐2. CONCLUSIONS: Chlorella vulgaris may have anti‐cancer effects by inducing apoptosis signaling cascades via an increased expression of P53, Bax and caspase‐3 proteins and through a reduction of Bcl‐2 protein, which subsequently lead to increased DNA damage and apoptosis. PMID:21340229

  14. Valsartan Protects Against Contrast-Induced Acute Kidney Injury in Rats by Inhibiting Endoplasmic Reticulum Stress-Induced Apoptosis.

    Science.gov (United States)

    Sun, Yan; Peng, Ping-An; Ma, Yue; Liu, Xiao-Li; Yu, Yi; Jia, Shuo; Xu, Xiao-Han; Wu, Si-Jing; Zhou, Yu-Jie

    2017-01-01

    Contrast-induced acute kidney injury (CI-AKI) is a serious complication of the administration of iodinated contrast media (CM) for diagnostic and interventional cardiovascular procedures and is associated with substantial morbidity and mortality. While the preventative measures can mitigate the risk of CI-AKI, there remains a need for novel and effective therapeutic approaches. The pathogenesis of CI-AKI is complex and not completely understood. CM-induced renal tubular cell apoptosis caused by the activation of endoplasmic reticulum (ER) stress is involved in CIAKI. We previously demonstrated that valsartan alleviated CM-induced human renal tubular cell apoptosis by inhibiting ER stress in vitro. However, the nephroprotective effect of valsartan on CI-AKI in vivo has not been investigated. Therefore, the aim of this study was to explore the protective effect of valsartan in a rat model of CI-AKI by measuring the amelioration of renal damage and the changes in ER stressrelated biomarkers. Our results showed that the radiocontrast agent meglumine diatrizoate caused significant renal insufficiency, renin-angiotensin system (RAS) activation, and renal tubular apoptosis by triggering ER stress through activation of glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), caspase 12, CCAAT/enhancer-binding protein-homologous protein (CHOP) and c-Jun N-terminal protein kinase (JNK) (Pvalsartan significantly alleviated renal dysfunction, pathological injury, and apoptosis along with the inhibition of ER stressrelated biomarkers (PValsartan could protect against meglumine diatrizoate-induced kidney injury in rats by inhibiting the ER stress-induced apoptosis, making it a promising strategy for preventing CI-AKI. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  15. Down-regulation of HSP27 sensitizes TRAIL-resistant tumor cell to TRAIL-induced apoptosis

    DEFF Research Database (Denmark)

    Zhuang, Hongqin; Jiang, Weiwei; Cheng, Wei

    2010-01-01

    Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) has recently emerged as a cancer therapeutic agent because it preferentially induces apoptosis in human cancer over normal cells. Most tumor cells, including lung cancer cell line A549, unfortunately, are resistant to TRAIL tre...

  16. Caspase-Independent Apoptosis Induced by Reperfusion Following Ischemia without Bile Duct Occlusion in Rat Liver.

    Science.gov (United States)

    Matsui, Nobuaki; Yoshioka, Rie; Nozawa, Asako; Kobayashi, Naonobu; Shichijo, Yukari; Yoshikawa, Tadatoshi; Akagi, Masaaki

    2017-01-01

    The contribution of caspases to hepatic ischemia/reperfusion (I/R)-induced apoptosis has not been completely understood yet. Several studies have demonstrated increased caspase activity during I/R and the protective effect of caspase inhibitors against I/R injuries. However, reports with opposing results also exist. Herein, we examined the contribution of caspases to the I/R-induced hepatic apoptosis in rats using caspase inhibitors and specific substrates of caspases. Hepatic I/R was induced via a 2-h occlusion of the portal vein and the hepatic artery, without conducting bile duct occlusion. DNA laddering and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL)-positive cells were increased at 3 h after reperfusion. Pretreatment with caspase inhibitors (Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-cmk) 2 or 10 mg/kg intravenously (i.v.), 20 mg/kg intraperitoneally (i.p.), Z-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-fmk) 3 mg/kg i.v.) failed to reduce apoptosis induced by I/R. Interestingly, apoptosis induced by the portal triad (hepatic artery, portal vein, and bile duct) occlusion/reperfusion could be marginally attenuated using Z-Asp-cmk (2 mg/kg i.v.). The cleavage activity for Ac-DEVD-α-(4-methylcoumaryl-7-amide) (MCA), a caspase-3/7/8/9 substrate, was significantly increased by I/R. Conversely, the cleavage activities for Ac-DNLD-MCA and MCA-VDQVDGW[K-DNP]-NH 2 , specific substrates for caspase-3 and -7 respectively, were decreased by I/R. Protein expression of the cellular inhibitor of apoptosis protein 2 (c-IAP2), an endogenous caspase inhibitor, was increased by ischemia. Nuclear translocation of the apoptosis-inducing factor (AIF), an initiator protein of caspase-independent apoptosis, was also increased during I/R. These results suggest that caspases are inhibited by c-IAP2 induced during ischemia and that AIF may be involved in initiation of apoptosis induced by hepatic I/R without

  17. Curcumin induces apoptosis and protective autophagy in castration-resistant prostate cancer cells through iron chelation

    Directory of Open Access Journals (Sweden)

    Yang C

    2017-02-01

    Full Text Available Chunguang Yang,1,* Xueyou Ma,1,* Zhihua Wang,1 Xing Zeng,1 Zhiquan Hu,1 Zhangqun Ye,1 Guanxin Shen2 1Department of Urology, Tongji Hospital, 2Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China *These authors contributed equally to this work Background: Curcumin induces apoptosis and autophagy in different cancer cells. Moreover, chemical and biological experiments have evidenced that curcumin is a biologically active iron chelator and induces cytotoxicity through iron chelation. We thus hypothesized that curcumin may induce apoptosis and autophagy in castration-resistant prostate cancer (CRPC cells through its iron-chelating properties.Materials and methods: CRPC cells were loaded with curcumin alone or in combination with ferric ammonium citrate (FAC. Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. Apoptosis was assessed by flow cytometry, terminal deoxynucleotidyl transferase nick end labeling (TUNEL assay and caspase activity. Autophagy status was analyzed by the detection of autophagosomes and light chain 3-II (LC3-II using transmission electron microscopy and Western blot. Iron-binding activity of curcumin was assessed by spectrophotometry and MTT assay. The expression levels of transferrin receptor 1 (TfR1 and iron regulatory protein 1 (IRP1 were examined by Western blot.Results: Curcumin induced apoptosis and autophagy in CRPC cells. Combining curcumin with autophagy inhibitors (3-methyladenine [3-MA] synergized the apoptotic effect of curcumin. Moreover, curcumin bound to FAC at a ratio of ~1:1, as assessed by spectrophotometry and MTT assay. Apoptosis and autophagy induced by curcumin were counteracted by equal amounts of FAC. At apoptosis- and autophagy-inducing concentrations, curcumin enhanced the expression levels of TfR1 and IRP1, indicative of iron deprivation induced by curcumin

  18. Calcium signals and caspase-12 participated in paraoxon-induced apoptosis in EL4 cells.

    Science.gov (United States)

    Li, Lan; Cao, Zhiheng; Jia, Pengfei; Wang, Ziren

    2010-04-01

    In order to investigate whether calcium signals participate in paraoxon (POX)-induced apoptosis in EL4 cells, real-time laser scanning confocal microscopy (LSCM) was used to detect Ca(2+) changes during the POX application. Apoptotic rates of EL4 cells and caspase-12 expression were also evaluated. POX (1-10nM) increased intracellular calcium concentration ([Ca(2+)]i) in EL4 cells in a dose-dependent manner at early stage (0-2h) of POX application, and apoptotic rates of EL4 cells after treatment with POX for 16h were also increased in a dose-dependent manner. Pre-treatment with EGTA, heparin or procaine attenuated POX-induced [Ca(2+)]i elevation and apoptosis. Additionally, POX up-regulated caspase-12 expression in a dose-dependent manner, and pre-treatment with EGTA, heparin or procaine significantly inhibited POX-induced increase of caspase-12 expression. Our results suggested that POX induced [Ca(2+)]i elevation in EL4 cells at the early stage of POX-induced apoptosis, which might involve Ca(2+) efflux from the endoplasmic reticulum (ER) and Ca(2+) influx from extracellular medium. Calcium signals and caspase-12 were important upstream messengers in POX-induced apoptosis in EL4 cells. The ER-associated pathway possibly operated in this apoptosis. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  19. Blockade of store-operated calcium entry alleviates ethanol-induced hepatotoxicity via inhibiting apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Cui, Ruibing [Department of Hepatology and Gastroenterology, Qilu Hospital of Shandong University, Jinan, Shandong Province 250012 (China); Yan, Lihui [Shandong Normal University, Jinan, Shandong Province 250012 (China); Luo, Zheng; Guo, Xiaolan [Department of Hepatology and Gastroenterology, Qilu Hospital of Shandong University, Jinan, Shandong Province 250012 (China); Yan, Ming, E-mail: ymylh@163.com [Department of Hepatology and Gastroenterology, Qilu Hospital of Shandong University, Jinan, Shandong Province 250012 (China)

    2015-08-15

    Extracellular Ca{sup 2+} influx has been suggested to play a role in ethanol-induced hepatocyte apoptosis and necrosis. Previous studies indicated that store-operated Ca{sup 2+} entry (SOCE) was involved in liver injury induced by ethanol in HepG2 cells. However, the mechanisms underlying liver injury caused by SOCE remain unclear. We aimed to investigate the effects and mechanism of SOCE inhibition on liver injury induced by ethanol in BRL cells and Sprague–Dawley rats. Our data demonstrated that ethanol (0–400 mM) dose-dependently increased hepatocyte injury and 100 mM ethanol significantly upregulated the mRNA and protein expression of SOC for at least 72 h in BRL cells. Blockade of SOCE by pharmacological inhibitors and sh-RNA knockdown of STIM1 and Orai1 attenuated intracellular Ca{sup 2+} overload, restored the mitochondrial membrane potential (MMP), decreased cytochrome C release and inhibited ethanol-induced apoptosis. STIM1 and Orai1 expression was greater in ethanol-treated than control rats, and the SOCE inhibitor corosolic acid ameliorated the histopathological findings and alanine transaminase and aspartate transaminase activity as well as decreased cytochrome C release and inhibited alcohol-induced cell apoptosis. These findings suggest that SOCE blockade could alleviate alcohol-induced hepatotoxicity via inhibiting apoptosis. SOCE might be a useful therapeutic target in alcoholic liver diseases. - Highlights: • Blockade of SOCE alleviated overload of Ca{sup 2+} and hepatotoxicity after ethanol application. • Blockade of SOCE inhibited mitochondrial apoptosis after ethanol application. • SOCE might be a useful therapeutic target in alcoholic liver diseases.

  20. Ghrelin ameliorates the human alveolar epithelial A549 cell apoptosis induced by lipopolysaccharide

    International Nuclear Information System (INIS)

    Huang, Chunrong; Zheng, Haichong; He, Wanmei; Lu, Guifang; Li, Xia; Deng, Yubin; Zeng, Mian

    2016-01-01

    Ghrelin is a gastric acyl-peptide that plays an inhibitory role in cell apoptosis. Herein we investigate the protective effects of ghrelin in LPS-induced apoptosis of human alveolar epithelial A549 cells, along with the possible molecular mechanisms. LPS exposure impaired cell viability and increased apoptosis of A549 cells significantly in concentration- and time-dependent manners embodied in increased Bax and cleaved caspase-3 production, coupled with decreased Bcl-2 levels. Simultaneously, LPS remarkably decreased the expression of phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated kinas (ERK) in A549 cells. However, ghrelin'pretreatment ameliorated LPS-caused alterations in the ratio of Bax/Bcl-2 and cleaved caspase-3 expression, whereas activated the PI3K/Akt and ERK signaling. These results demonstrate that ghrelin lightens LPS-induced apoptosis of human alveolar epithelial cells partly through activating the PI3K/Akt and ERK pathway and thereby might benefit alleviating septic ALI. -- Graphical abstract: Ghrelin ameliorates the human alveolar epithelial A549 cells apoptosis induced by lipopolysaccharide partly through activating the PI3K/Akt and ERK pathway. Display Omitted -- Highlights: •It has been observed that LPS insult significantly increased apoptosis in A549 cells. •Both Akt and ERK signaling are critical adapter molecules to mediate the ghrelin-mediated proliferative effect. •Ghrelin may have a therapeutic effect in the prevention of LPS-induced apoptosis.

  1. Ghrelin ameliorates the human alveolar epithelial A549 cell apoptosis induced by lipopolysaccharide

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chunrong; Zheng, Haichong; He, Wanmei; Lu, Guifang; Li, Xia [Department of Medical Intensive Care Unit, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China); Deng, Yubin, E-mail: dengyub@mail.sysu.edu.cn [Research Center of Translational Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China); Zeng, Mian, E-mail: zengmian2004@163.com [Department of Medical Intensive Care Unit, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China)

    2016-05-20

    Ghrelin is a gastric acyl-peptide that plays an inhibitory role in cell apoptosis. Herein we investigate the protective effects of ghrelin in LPS-induced apoptosis of human alveolar epithelial A549 cells, along with the possible molecular mechanisms. LPS exposure impaired cell viability and increased apoptosis of A549 cells significantly in concentration- and time-dependent manners embodied in increased Bax and cleaved caspase-3 production, coupled with decreased Bcl-2 levels. Simultaneously, LPS remarkably decreased the expression of phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated kinas (ERK) in A549 cells. However, ghrelin'pretreatment ameliorated LPS-caused alterations in the ratio of Bax/Bcl-2 and cleaved caspase-3 expression, whereas activated the PI3K/Akt and ERK signaling. These results demonstrate that ghrelin lightens LPS-induced apoptosis of human alveolar epithelial cells partly through activating the PI3K/Akt and ERK pathway and thereby might benefit alleviating septic ALI. -- Graphical abstract: Ghrelin ameliorates the human alveolar epithelial A549 cells apoptosis induced by lipopolysaccharide partly through activating the PI3K/Akt and ERK pathway. Display Omitted -- Highlights: •It has been observed that LPS insult significantly increased apoptosis in A549 cells. •Both Akt and ERK signaling are critical adapter molecules to mediate the ghrelin-mediated proliferative effect. •Ghrelin may have a therapeutic effect in the prevention of LPS-induced apoptosis.

  2. TIMP-1 gene deficiency increases tumour cell sensitivity to chemotherapy-induced apoptosis

    DEFF Research Database (Denmark)

    Davidsen, Marie Louise; Würts, S.Ø.; Rømer, Maria Unni Koefoed

    2006-01-01

    deficiency increases the response to chemotherapy considerably, confirming that TIMP-1 protects the cells from apoptosis. This is to our knowledge the first study investigating TIMP-1 and chemotherapy-induced apoptosis employing a powerful model system comprising TIMP-1 gene-deficient cells...... this hypothesis, we have established TIMP-1 gene-deficient and TIMP-1 wild-type fibrosarcoma cells from mouse lung tissue. We have characterised these cells with regard to TIMP-1 genotype, TIMP-1 expression, malignant transformation and sensitivity to chemotherapy-induced apoptosis. We show that TIMP-1 gene...... and their genetically identical wild-type controls. For future studies, this cell system can be used to uncover the mechanisms and signalling pathways involved in the TIMP-1-mediated inhibition of apoptosis as well as to investigate the possibility of using TIMP-1 inhibitors to optimise the effect of conventional...

  3. A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Shi, Junwei; Zhang, Huan; Fang, Liurong; Xi, Yongqiang; Zhou, Yanrong; Luo, Rui; Wang, Dang; Xiao, Shaobo; Chen, Huanchun

    2014-01-01

    Highlights: • We developed a novel firefly luciferase based biosensor to detect apoptosis. • The novel biosensor 233-DnaE-DEVDG was reliable, sensitive and convenient. • 233-DnaE-DEVDG faithfully indicated ESAT-6 family proteins of Mycobacterium tuberculosis induced apoptosis. • EsxA, esxT and esxL in ESAT-6 family proteins induced apoptosis. • Activation of nuclear factor-κB (NF-κB) participated in esxT-induced apoptosis. - Abstract: The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis

  4. A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Junwei; Zhang, Huan; Fang, Liurong; Xi, Yongqiang; Zhou, Yanrong; Luo, Rui; Wang, Dang, E-mail: wangdang511@126.com; Xiao, Shaobo; Chen, Huanchun

    2014-10-03

    Highlights: • We developed a novel firefly luciferase based biosensor to detect apoptosis. • The novel biosensor 233-DnaE-DEVDG was reliable, sensitive and convenient. • 233-DnaE-DEVDG faithfully indicated ESAT-6 family proteins of Mycobacterium tuberculosis induced apoptosis. • EsxA, esxT and esxL in ESAT-6 family proteins induced apoptosis. • Activation of nuclear factor-κB (NF-κB) participated in esxT-induced apoptosis. - Abstract: The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.

  5. Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    Directory of Open Access Journals (Sweden)

    Bi YZ

    2016-11-01

    Full Text Available Yanzhao Bi, Yifan Zhang, Chunying Cui, Lulu Ren, Xueyun Jiang School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China Abstract: Nanodiamond (ND is a renowned material in nonviral small interfering RNA (siRNA carrier field due to its unique physical, chemical, and biological properties. In our previous work, it was proven that ND could deliver siRNA into cells efficiently and downregulate the expression of desired protein. However, synthesizing a high-efficient tumor-targeting carrier using ND is still a challenge. In this study, a novel carrier, NDCONH(CH22NH-VDGR, was synthesized for siRNA delivery, and its properties were characterized with methods including Fourier transform infrared spectrometry, transmission electron microscopy, scanning electron microscopy, gel retardation assay, differential scanning calorimetry, confocal microscopy, releasing test, real-time polymerase chain reaction (PCR assay, enzyme-linked immunosorbent assay (ELISA, flow cytometry, cytotoxicity assay, and gene-silencing efficacy assay in vitro and in vivo. The mechanism of NDCONH(CH22NH-VDGR/survivin-siRNA-induced tumor apoptosis was evaluated via flow cytometer assay using Annexin V–fluorescein isothiocyanate/propidium iodide staining method. The NDCONH(CH22NH-VDGR/survivin-siRNA nanoparticle with 60–110 nm diameter and 35.65±3.90 mV zeta potential was prepared. For real-time PCR assay, the results showed that the expression of survivin mRNA was reduced to 46.77%±6.3%. The expression of survivin protein was downregulated to 48.49%±2.25%, as evaluated by ELISA assay. MTT assay showed that NDCONH(CH22NH-VDGR/survivin-siRNA had an inhibitory effect on MCF-7 cell proliferation. According to these results, the survivin-siRNA could be delivered, transported, and released stably, which benefits in increasing the gene-silencing effect. Therefore, as an siRNA carrier, NDCONH(CH22NH-VDGR was suggested

  6. FoxP3 inhibits proliferation and induces apoptosis of gastric cancer cells by activating the apoptotic signaling pathway

    International Nuclear Information System (INIS)

    Ma, Gui-Fen; Chen, Shi-Yao; Sun, Zhi-Rong; Miao, Qing; Liu, Yi-Mei; Zeng, Xiao-Qing; Luo, Tian-Cheng; Ma, Li-Li; Lian, Jing-Jing; Song, Dong-Li

    2013-01-01

    Highlights: ► The article revealed FoxP3 gene function in gastric cancer firstly. ► Present the novel roles of FoxP3 in inhibiting proliferation and promoting apoptosis in gastric cancer cells. ► Overexpression of FoxP3 increased proapoptotic molecules and repressed antiapoptotic molecules. ► Silencing of FoxP3 reduced the expression of proapoptotic genes, such as PARP, caspase-3 and caspase-9. ► FoxP3 is sufficient for activating the apoptotic signaling pathway. -- Abstract: Forkhead Box Protein 3 (FoxP3) was identified as a key transcription factor to the occurring and function of the regulatory T cells (Tregs). However, limited evidence indicated its function in tumor cells. To elucidate the precise roles and underlying molecular mechanism of FoxP3 in gastric cancer (GC), we examined the expression of FoxP3 and the consequences of interfering with FoxP3 gene in human GC cell lines, AGS and MKN45, by multiple cellular and molecular approaches, such as immunofluorescence, gene transfection, CCK-8 assay, clone formation assay, TUNEL assay, Flow cytometry, immunoassay and quantities polymerase chain reaction (PCR). As a result, FoxP3 was expressed both in nucleus and cytoplasm of GC cells. Up-regulation of FoxP3 inhibited cell proliferation and promoted cell apoptosis. Overexpression of FoxP3 increased the protein and mRNA levels of proapoptotic molecules, such as poly ADP-ribose polymerase1 (PARP), caspase-3 and caspase-9, and repressed the expression of antiapoptotic molecules, such as cellular inhibitor of apoptosis-1 (c-IAP1) and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2). Furthermore, silencing of FoxP3 by siRNA in GC cells reduced the expression of proapoptotic genes, such as PARP, caspase-3 and caspase-9. Collectively, our findings identify the novel roles of FoxP3 in inhibiting proliferation and inducing apoptosis in GC cells by regulating apoptotic signaling, which could be a promising therapeutic approach for gastric cancer.

  7. Iron dysregulation combined with aging prevents sepsis-induced apoptosis.

    Science.gov (United States)

    Javadi, Pardis; Buchman, Timothy G; Stromberg, Paul E; Turnbull, Isaiah R; Vyas, Dinesh; Hotchkiss, Richard S; Karl, Irene E; Coopersmith, Craig M

    2005-09-01

    Sepsis, iron loading, and aging cause independent increases in gut epithelial and splenic apoptosis. It is unknown how their combination will affect apoptosis and systemic cytokine levels. Hfe-/- mice (a murine homologue of hemochromatosis) abnormally accumulate iron in their tissues. Aged (24-26 months) or mature (16-18 months) Hfe-/- mice and wild type (WT) littermates were subjected to cecal ligation and puncture (CLP) or sham laparotomy. Intestine, spleen, and blood were harvested 24 h later and assessed for apoptosis and cytokine levels. Gut epithelial and splenic apoptosis were low in both aged septic and sham Hfe-/- mice, regardless of the amount of iron in their diet. Mature septic WT mice had increased apoptosis compared to age-matched sham WT mice. Mature septic Hfe-/- mice had similar levels of intestinal cell death to age-matched septic WT mice but higher levels of splenic apoptosis. Apoptosis was significantly lower in septic aged Hfe-/- mice than septic mature Hfe-/- animals. Interleukin-6 was elevated in septic aged Hfe-/- mice compared to sham mice. Although sepsis, chronic iron dysregulation, and aging each increase gut and splenic apoptosis, their combination yields cell death levels similar to sham animals despite the fact that aged Hfe-/- mice are able to mount an inflammatory response following CLP and mature Hfe-/- mice have elevated sepsis-induced apoptosis. Combining sepsis with two risk factors that ordinarily increase cell death and increase mortality in CLP yields an apoptotic response that could not have been predicted based upon each element in isolation.

  8. N-acetylcysteine protects against cadmium-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in testes.

    Science.gov (United States)

    Ji, Yan-Li; Wang, Hua; Zhang, Cheng; Zhang, Ying; Zhao, Mei; Chen, Yuan-Hua; Xu, De-Xiang

    2013-03-01

    Cadmium (Cd) is a reproductive toxicant that induces germ cell apoptosis in the testes. Previous studies have demonstrated that endoplasmic reticulum (ER) stress is involved in Cd-induced germ cell apoptosis. The aim of the present study was to investigate the effects of N-acetylcysteine (NAC), an antioxidant, on Cd-induced ER stress and germ cell apoptosis in the testes. Male CD-1 mice were intraperitoneally injected with CdCl2 (2.0 mg kg(-1)). As expected, acute Cd exposure induced germ cell apoptosis in the testes, as determined by terminal dUTP nick-end labelling (TUNEL). However, the administration of NAC alleviated Cd-induced germ cell apoptosis in the testes. Further analysis showed that NAC attenuated the Cd-induced upregulation of testicular glucose-regulated protein 78 (GRP78), an important ER molecular chaperone. Moreover, NAC inhibited the Cd-induced phosphorylation of testicular eukaryotic translation initiation factor 2α (eIF2α), a downstream target of the double-stranded RNA-activated kinase-like ER kinase (PERK) pathway. In addition, NAC blocked the Cd-induced activation of testicular X binding protein (XBP)-1, indicating that NAC attenuates the Cd-induced ER stress and the unfolded protein response (UPR). Interestingly, NAC almost completely prevented the Cd-induced elevation of C/EBP homologous protein (CHOP) and phosphorylation of c-Jun N-terminal kinase (JNK), two components of the ER stress-mediated apoptotic pathway. In conclusion, NAC protects against Cd-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in the testes.

  9. Roles of PTEN-induced putative kinase 1 and dynamin-related protein 1 in transient global ischemia-induced hippocampal neuronal injury

    International Nuclear Information System (INIS)

    Chen, Shang-Der; Lin, Tsu-Kung; Yang, Ding-I.; Lee, Su-Ying; Shaw, Fu-Zen; Liou, Chia-Wei; Chuang, Yao-Chung

    2015-01-01

    Recent studies showed that increased mitochondrial fission is an early event of cell death during cerebral ischemia and dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, which may be regulated by PTEN-induced putative kinase 1 (PINK1), a mitochondrial serine/threonine-protein kinase thought to protect cells from stress-induced mitochondrial dysfunction and regulate mitochondrial fission. However, the roles of PINK1 and Drp1 in hippocampal injury caused by transient global ischemia (TGI) remain unknown. We therefore tested the hypothesis that TGI may induce PINK1 causing downregulation of Drp1 phosphorylation to enhance hippocampal neuronal survival, thus functioning as an endogenous neuroprotective mechanism. We found progressively increased PINK1 expression in the hippocampal CA1 subfield1-48 h following TGI, reaching the maximal level at 4 h. Despite lack of changes in the expression level of total Drp1 and phosphor-Drp1 at Ser637, TGI induced a time-dependent increase of Drp1 phosphorlation at Ser616 that peaked after 24 h. Notably, PINK1-siRNA increased p-Drp1(Ser616) protein level in hippocampal CA1 subfield 24 h after TGI. The PINK1 siRNA also aggravated the TGI-induced oxidative DNA damage with an increased 8-hydroxy-deoxyguanosine (8-OHdG) content in hippocampal CA1 subfield. Furthermore, PINK1 siRNA also augmented TGI-induced apoptosis as evidenced by the increased numbers of TUNEL-positive staining and enhanced DNA fragmentation. These findings indicated that PINK1 is an endogenous protective mediator vital for neuronal survival under ischemic insult through regulating Drp1 phosphorylation at Ser616. - Highlights: • Transient global ischemia increases expression of PINK1 and p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA decreases PINK1 expression but increases p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA augments oxidative stress and neuronal damage in hippocampal CA1 subfield

  10. Roles of PTEN-induced putative kinase 1 and dynamin-related protein 1 in transient global ischemia-induced hippocampal neuronal injury

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Shang-Der, E-mail: chensd@adm.cgmh.org.tw [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Lin, Tsu-Kung [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Yang, Ding-I. [Institute of Brain Science and Brain Research Center, National Yang-Ming University, Taipei, Taiwan (China); Lee, Su-Ying [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Shaw, Fu-Zen [Department of Psychology, National Cheng Kung University, Tainan, Taiwan (China); Liou, Chia-Wei [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Chuang, Yao-Chung, E-mail: ycchuang@adm.cgmh.org.tw [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China)

    2015-05-01

    Recent studies showed that increased mitochondrial fission is an early event of cell death during cerebral ischemia and dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, which may be regulated by PTEN-induced putative kinase 1 (PINK1), a mitochondrial serine/threonine-protein kinase thought to protect cells from stress-induced mitochondrial dysfunction and regulate mitochondrial fission. However, the roles of PINK1 and Drp1 in hippocampal injury caused by transient global ischemia (TGI) remain unknown. We therefore tested the hypothesis that TGI may induce PINK1 causing downregulation of Drp1 phosphorylation to enhance hippocampal neuronal survival, thus functioning as an endogenous neuroprotective mechanism. We found progressively increased PINK1 expression in the hippocampal CA1 subfield1-48 h following TGI, reaching the maximal level at 4 h. Despite lack of changes in the expression level of total Drp1 and phosphor-Drp1 at Ser637, TGI induced a time-dependent increase of Drp1 phosphorlation at Ser616 that peaked after 24 h. Notably, PINK1-siRNA increased p-Drp1(Ser616) protein level in hippocampal CA1 subfield 24 h after TGI. The PINK1 siRNA also aggravated the TGI-induced oxidative DNA damage with an increased 8-hydroxy-deoxyguanosine (8-OHdG) content in hippocampal CA1 subfield. Furthermore, PINK1 siRNA also augmented TGI-induced apoptosis as evidenced by the increased numbers of TUNEL-positive staining and enhanced DNA fragmentation. These findings indicated that PINK1 is an endogenous protective mediator vital for neuronal survival under ischemic insult through regulating Drp1 phosphorylation at Ser616. - Highlights: • Transient global ischemia increases expression of PINK1 and p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA decreases PINK1 expression but increases p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA augments oxidative stress and neuronal damage in hippocampal CA1 subfield.

  11. Irigenin sensitizes TRAIL-induced apoptosis via enhancing pro-apoptotic molecules in gastric cancer cells.

    Science.gov (United States)

    Xu, Ying; Gao, Cheng-Cheng; Pan, Zhen-Guo; Zhou, Chuan-Wen

    2018-02-12

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) holds promising value for cancer therapy due to its capacity to induce apoptosis in cancer cells. Nevertheless, TRAIL therapy is greatly hampered by its resistance. Irigenin (Iri), isoflavonoids, can be isolated from the rhizome of Belamcanda chinensis, and has been shown anti-cancer properties. In this study, we explored if Iri could enhance TRAIL-regulated apoptosis in TRAIL resistant gastric cancer cells. Iri significantly potentiated TRAIL-triggered cytotoxicity. Iri alone and TRAIL alone showed no effective role in apoptosis induction, whereas combined treatment with Iri and TRAIL markedly induced apoptosis in cancer cells, as evidenced by the up-regulation of cleaved Caspase-8/-9/-3 and PARP. Additionally, the sensitization to TRAIL was along with the enhancement of pro-apoptotic proteins, including FAS-associated protein with death domain (FADD), death receptor 5 (DR5) and Bax. And suppressing FADD, DR5 and Bax by si RNA significantly reduced the apoptosis and enhanced the cell viability induced by the co-application of Iri and TRAIL. Moreover, the sensitization to TRAIL was accompanied by the decrease of Cellular-FLICE inhibitory protein (c-FLIP), Bcl-2 and Survivin. Additionally, Iri could sensitize TRAIL to produce reactive oxygen species (ROS). Pre-treatment of N-acetyl-cysteine (NAC), ROS scavenger, attenuated Iri plus TRAIL-induced apoptosis and improved cell viability. Finally, combination of Iri and TRAIL inhibited tumor growth in the xenograft model. Collectively, our present study gave new insights into the effects of Iri on potentiating TRAIL-sensitivity, and suggested that Iri could be a potential candidate for sensitizer of TRAIL-resistant cancer cell treatment. Copyright © 2018. Published by Elsevier Inc.

  12. Herbal Medicine as Inducers of Apoptosis in Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Elham Safarzadeh

    2014-12-01

    Full Text Available Cancer is uncontrolled growth of abnormal cells in the body. Nowadays, cancer is considered as a human tragedy and one of the most prevalent diseases in the wide, and its mortality resulting from cancer is being increased. It seems necessary to identify new strategies to prevent and treat such a deadly disease. Control survival and death of cancerous cell are important strategies in the management and therapy of cancer. Anticancer agents should kill the cancerous cell with the minimal side effect on normal cells that is possible through the induction of apoptosis. Apoptosis is known as programmed cell death in both normal and damaged tissues. This process includes some morphologically changes in cells such as rapid condensation and budding of the cell, formation of membrane-enclosed apoptotic bodies with well-preserved organelles. Induction of apoptosis is one of the most important markers of cytotoxic antitumor agents. Some natural compounds including plants induce apoptotic pathways that are blocked in cancer cells through various mechanisms in cancer cells. Multiple surveys reported that people with cancer commonly use herbs or herbal products. Vinca Alkaloids, Texans, podo phyllotoxin, Camptothecins have been clinically used as Plant derived anticancer agents. The present review summarizes the literature published so far regarding herbal medicine used as inducers of apoptosis in cancer.

  13. Decreases in Casz1 mRNA by an siRNA Complex Do not Alter Blood Pressure in Mice.

    Science.gov (United States)

    Ji, Su-Min; Shin, Young-Bin; Park, So-Yon; Lee, Hyeon-Ju; Oh, Bermseok

    2012-03-01

    Recent genomewide association studies of large samples have identified genes that are associated with blood pressure. The Global Blood Pressure Genetics (Global BPgen) and Cohorts for Heart and Aging Research in Genome Epidemiology (CHARGE) consortiums identified 14 loci that govern blood pressure on a genomewide significance level, one of which is CASZ1 confirmed in both Europeans and Asians. CASZ1 is a zinc finger transcription factor that controls apoptosis and cell fate and suppresses neuroblastoma tumor growth by reprogramming gene expression, like a tumor suppressor. To validate the function of CASZ1 in blood pressure, we decreased Casz1 mRNA levels in mice by siRNA. Casz1 siRNA reduced mRNA levels by 59% in a mouse cell line. A polyethylenimine-mixed siRNA complex was injected into mouse tail veins, reducing Casz1 mRNA expression to 45% in the kidney. However, blood pressure in the treated mice was unaffected, despite a 55% reduction in Casz1 mRNA levels in the kidney on multiple siRNA injections daily. Even though Casz1 siRNA-treated mice did not experience any significant change in blood pressure, our study demonstrates the value of in vivo siRNA injection in analyzing the function of candidate genes identified by genomewide association studies.

  14. Dopamine induces neutrophil apoptosis through a dopamine D-1 receptor-independent mechanism.

    LENUS (Irish Health Repository)

    Sookhai, S

    2012-02-03

    BACKGROUND: For the normal resolution of an acute inflammatory response, neutrophil (PMN) apoptosis is essential to maintain immune homeostasis and to limit inappropriate host tissue damage. A delay in PMN apoptosis has been implicated in the pathogenesis of the systemic inflammatory response syndrome (SIRS). Dopamine, a biogenic amine with known cardiovascular and neurotransmitter properties, is used in patients with SIRS to maintain hemodynamic stability. We sought to determine whether dopamine may also have immunoregulatory properties capable of influencing PMN apoptosis, function, and activation state in patients with SIRS. METHODS: PMNs were isolated from healthy volunteers and patients with SIRS and treated with varying doses of dopamine and a dopamine D-1 receptor agonist, fenoldopam. PMN apoptosis was assessed every 6 hours with use of propidium iodide DNA staining and PMN function was assessed with use of respiratory burst activity, phagocytosis ability, and CD11a, CD11b, and CD18 receptor expression as functional markers. RESULTS: There was a significant delay in PMN apotosis in patients with SIRS compared with controls. Treatment of isolated PMNs from both healthy controls and patients with SIRS with 10 and 100 mumol\\/L dopamine induced apoptosis. PMN ingestive and cytocidal capacity were both decreased in patients with SIRS compared with controls. Treatment with dopamine significantly increased phagocytic function. Fenoldopam did not induce PMN apoptosis. CONCLUSION: Our data demonstrate for the first time that dopamine induces PMN apoptosis and modulates PMN function both in healthy controls and in patients with SIRS. These results indicate that dopamine may be beneficial during SIRS through a nonhemodynamic PMN-dependent proapoptotic mechanism.

  15. A reactive oxygen species activation mechanism contributes to JS-K-induced apoptosis in human bladder cancer cells.

    Science.gov (United States)

    Qiu, Mingning; Chen, Lieqian; Tan, Guobin; Ke, Longzhi; Zhang, Sai; Chen, Hege; Liu, Jianjun

    2015-10-13

    Reactive oxygen species (ROS) and cellular oxidant stress are regulators of cancer cells. The alteration of redox status, which is induced by increased generation of ROS, results in increased vulnerability to oxidative stress. The aim of this study is to investigate the influence of O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, C13H16N6O8) on proliferation and apoptosis in bladder cancer cells and explored possible ROS-related mechanisms. Our results indicated that JS-K could suppress bladder cancer cell proliferation in a concentration- and time-dependent manner and induce apoptosis and ROS accumulation in a concentration-dependent manner. With increasing concentrations of JS-K, expression of proteins that are involved in cell apoptosis increased in a concentration-dependent manner. Additionally, the antioxidant N-acetylcysteine (NAC) reversed JS-K-induced cell apoptosis; conversely, the prooxidant oxidized glutathione (GSSG) exacerbated JS-K-induced cell apoptosis. Furthermore, we found that nitrites, which were generated from the oxidation of JS-K-released NO, induced apoptosis in bladder cancer cells to a lower extent through the ROS-related pathway. In addition, JS-K was shown to enhance the chemo-sensitivity of doxorubicin in bladder cancer cells. Taken together, the data suggest that JS-K-released NO induces bladder cancer cell apoptosis by increasing ROS levels, and nitrites resulting from oxidation of NO have a continuous apoptosis-inducing effect.

  16. siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

    Science.gov (United States)

    Vickers, Timothy A; Crooke, Stanley T

    2012-07-01

    While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

  17. Fungal secondary metabolites rasfonin induces autophagy, apoptosis and necroptosis in renal cancer cell line

    Directory of Open Access Journals (Sweden)

    Hui Sun

    2016-04-01

    Full Text Available Rasfonin (A304 is a fungal natural product isolated from the fermentation substrate of Talaromyces sp. 3656-A1, which was named according to its activity against the small G-protein Ras. In a former study, we demonstrated that it induced autophagy and apoptosis; however, whether rasfonin activated necroptosis remained unknown. Moreover, the interplay among different cell death processes induced by rasfonin was unexplored. In the present study, we revealed that, in addition of promoting autophagy and caspase-dependent apoptosis, rasfonin also activated necroptosis. Nectrostatin-1 (Nec-1, an inhibitor of necroptosis, affected rasfonin-induced autophagy in a time-dependent manner concurring with an increased caspase-dependent apoptosis. The aforementioned results were confirmed by knockdown of receptor-interacting protein 1 (RIP1, a crucial necrostatin-1-targeted adaptor kinase mediating cell death and survival. Taken together, the data presented indicate that rasfonin activates various cell death pathways, and RIP1 plays a critical role in rasfonin-induced autophagy and apoptosis.

  18. Kurarinol induces hepatocellular carcinoma cell apoptosis through suppressing cellular signal transducer and activator of transcription 3 signaling

    International Nuclear Information System (INIS)

    Shu, Guangwen; Yang, Jing; Zhao, Wenhao; Xu, Chan; Hong, Zongguo; Mei, Zhinan; Yang, Xinzhou

    2014-01-01

    Kurarinol is a flavonoid isolated from roots of the medical plant Sophora flavescens. However, its cytotoxic activity against hepatocellular carcinoma (HCC) cells and toxic effects on mammalians remain largely unexplored. Here, the pro-apoptotic activities of kurarinol on HCC cells and its toxic impacts on tumor-bearing mice were evaluated. The molecular mechanisms underlying kurarinol-induced HCC cell apoptosis were also investigated. We found that kurarinol dose-dependently provoked HepG2, Huh-7 and H22 HCC cell apoptosis. In addition, kurarinol gave rise to a considerable decrease in the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) in HCC cells. Suppression of STAT3 signaling is involved in kurarinol-induced HCC cell apoptosis. In vivo studies showed that kurarinol injection substantially induced transplanted H22 cell apoptosis with low toxic impacts on tumor-bearing mice. Similarly, the transcriptional activity of STAT3 in transplanted tumor tissues was significantly suppressed after kurarinol treatment. Collectively, our current research demonstrated that kurarinol has the capacity of inducing HCC cell apoptosis both in vitro and in vivo with undetectable toxic impacts on the host. Suppressing STAT3 signaling is implicated in kurarinol-mediated HCC cell apoptosis. - Highlights: • Kurarinol induces hepatocellular carcinoma (HCC) cell apoptosis. • Kurarinol induces HCC cell apoptosis via inhibiting STAT3. • Kurarinol exhibits low toxic effects on tumor-bearing animals

  19. Antimony trioxide-induced apoptosis is dependent on SEK1/JNK signaling.

    Science.gov (United States)

    Mann, Koren K; Davison, Kelly; Colombo, Myrian; Colosimo, April L; Diaz, Zuanel; Padovani, Alessandra M S; Guo, Qi; Scrivens, P James; Gao, Wenli; Mader, Sylvie; Miller, Wilson H

    2006-01-05

    Very little is known concerning the toxicity of antimony, despite its commercial use as a flame retardant and medical use as a treatment for parasitic infections. Our previous studies show that antimony trioxide (Sb(2)O(3)) induces growth inhibition in patient-derived acute promyelocytic leukemia (APL) cell lines, a disease in which a related metal, arsenic trioxide (As(2)O(3)), is used clinically. However, signaling pathways initiated by Sb(2)O(3) treatment remain undefined. Here, we show that Sb(2)O(3) treatment of APL cells is associated with increased apoptosis as well as differentiation markers. Sb(2)O(3)-induced reactive oxygen species (ROS) correlated with increased apoptosis. In addition, when we decreased the buffering capacity of the cell by depleting glutathione, ROS production and apoptosis was enhanced. Arsenic-resistant APL cells with increased glutathione levels exhibited increased cross-resistance to Sb(2)O(3). Based on studies implicating c-jun kinase (JNK) in the mediation of the response to As(2)O(3), we investigated the role for JNK in Sb(2)O(3)-induced apoptosis. Sb(2)O(3) activates JNK and its downstream target, AP-1. In fibroblasts with a genetic deletion in SEK1, an upstream regulator of JNK, Sb(2)O(3)-induced growth inhibition as well as JNK activation was decreased. These data suggest roles for ROS and the SEK1/JNK pathway in the cytotoxicity associated with Sb(2)O(3) exposure.

  20. Solid nano-in-nanoparticles for potential delivery of siRNA.

    Science.gov (United States)

    Amsalem, Orit; Nassar, Taher; Benhamron, Sandrine; Lazarovici, Philip; Benita, Simon; Yavin, Eylon

    2017-07-10

    siRNA-based therapeutics possess great potential to treat a wide variety of genetic disorders. However, they suffer from low cellular uptake and short half-lives in blood circulation; issues that remain to be addressed. This work is, to the best of our knowledge, the first to report the production of solid nano-in-nanoparticles, termed double nano carriers (DNCs) by means of the innovative technology of nano spray drying. DNCs (with a median size of 580-770nm) were produced by spraying at low temperatures (50°C) to prevent damage to heat-sensitive biomacromolecules like siRNA. DNCs consisting of Poly (d,l-lactide-co-glycolide) used as a wall material, encapsulating 20% human serum albumin primary nanoparticles (PNPs) loaded with siRNA, were obtained as a dry nanoparticulate powder with smooth spherical surfaces and a unique inner morphology. Incubation of pegylated or non-pegylated DNCs under sink conditions at 37°C, elicited a controlled release profile of the siRNA for up to 12 or 24h, respectively, with a minimal burst effect. Prolonged incubation of pegylated DNCs loaded with active siRNA (anti EGFR) in an A549 epithelial cell culture monolayer did not induce any apparent cytotoxicity. A slow degradation of the internalized DNCs by the cells was also observed resulting in the progressive release of the siRNA for up to 6days, as corroborated by laser confocal microscopy. The structural integrity and silencing activity of the double encapsulated siRNA were fully preserved, as demonstrated by HPLC, gel electrophoresis, and potent RNAi activity of siRNA extracted from DNCs. These results demonstrate the potential use of DNCs as a nano drug delivery system for systemic administration and controlled release of siRNA and potentially other sensitive bioactive macromolecules. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Resveratrol induces growth arrest and apoptosis through activation of FOXO transcription factors in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Qinghe Chen

    2010-12-01

    Full Text Available Resveratrol, a naturally occurring phytopolyphenol compound, has attracted extensive interest in recent years because of its diverse pharmacological characteristics. Although resveratrol possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. The present study was carried out to examine whether PI3K/AKT/FOXO pathway mediates the biological effects of resveratrol.Resveratrol inhibited the phosphorylation of PI3K, AKT and mTOR. Resveratrol, PI3K inhibitors (LY294002 and Wortmannin and AKT inhibitor alone slightly induced apoptosis in LNCaP cells. These inhibitors further enhanced the apoptosis-inducing potential of resveratrol. Overexpression of wild-type PTEN slightly induced apoptosis. Wild type PTEN and PTEN-G129E enhanced resveratrol-induced apoptosis, whereas PTEN-G129R had no effect on proapoptotic effects of resveratrol. Furthermore, apoptosis-inducing potential of resveratrol was enhanced by dominant negative AKT, and inhibited by wild-type AKT and constitutively active AKT. Resveratrol has no effect on the expression of FKHR, FKHRL1 and AFX genes. The inhibition of FOXO phosphorylation by resveratrol resulted in its nuclear translocation, DNA binding and transcriptional activity. The inhibition of PI3K/AKT pathway induced FOXO transcriptional activity resulting in induction of Bim, TRAIL, p27/KIP1, DR4 and DR5, and inhibition of cyclin D1. Similarly, resveratrol-induced FOXO transcriptional activity was further enhanced when activation of PI3K/AKT pathway was blocked. Over-expression of phosphorylation deficient mutants of FOXO proteins (FOXO1-TM, FOXO3A-TM and FOXO4-TM induced FOXO transcriptional activity, which was further enhanced by resveratrol. Inhibition of FOXO transcription factors by shRNA blocked resveratrol-induced upregulation of Bim, TRAIL, DR4, DR5, p27/KIP1 and apoptosis, and inhibition of cyclin D1 by

  2. VCC-1 over-expression inhibits cisplatin-induced apoptosis in HepG2 cells

    International Nuclear Information System (INIS)

    Zhou, Zhitao; Lu, Xiao; Zhu, Ping; Zhu, Wei; Mu, Xia; Qu, Rongmei; Li, Ming

    2012-01-01

    Highlights: ► VCC-1 is hypothesized to be associated with carcinogenesis. ► Levels of VCC-1 are increased significantly in HCC. ► Over-expression of VCC-1 could promotes cellular proliferation rate. ► Over-expression of VCC-1 inhibit the cisplatin-provoked apoptosis in HepG2 cells. ► VCC-1 plays an important role in control the tumor growth and apoptosis. -- Abstract: Vascular endothelial growth factor-correlated chemokine 1 (VCC-1), a recently described chemokine, is hypothesized to be associated with carcinogenesis. However, the molecular mechanisms by which aberrant VCC-1 expression determines poor outcomes of cancers are unknown. In this study, we found that VCC-1 was highly expressed in hepatocellular carcinoma (HCC) tissue. It was also associated with proliferation of HepG2 cells, and inhibition of cisplatin-induced apoptosis of HepG2 cells. Conversely, down-regulation of VCC-1 in HepG2 cells increased cisplatin-induced apoptosis of HepG2 cells. In summary, these results suggest that VCC-1 is involved in cisplatin-induced apoptosis of HepG2 cells, and also provides some evidence for VCC-1 as a potential cellular target for chemotherapy.

  3. Sensitization to UV-induced apoptosis by the histone deacetylase inhibitor trichostatin A (TSA)

    International Nuclear Information System (INIS)

    Kim, Myoung Sook; Baek, Jin Hyen; Chakravarty, Devulapalli; Sidransky, David; Carrier, France

    2005-01-01

    UV-induced apoptosis is a protective mechanism that is primarily caused by DNA damage. Cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts are the main DNA adducts triggered by UV radiation. Because the formation of DNA lesions in the chromatin is modulated by the structure of the nucleosomes, we postulated that modification of chromatin compaction could affect the formation of the lesions and consequently apoptosis. To verify this possibility we treated human colon carcinoma RKO cells with the histone deacetylase inhibitor trichostatin A (TSA) prior to exposure to UV radiation. Our data show that pre-treatment with TSA increased UV killing efficiency by more than threefold. This effect correlated with increased formation of CPDs and consequently apoptosis. On the other hand, TSA treatment after UV exposure rather than before had no more effect than UV radiation alone. This suggests that a primed (opened) chromatin status is required to sensitize the cells. Moreover, TSA sensitization to UV-induced apoptosis is p53 dependent. p53 and acetylation of the core histones may thus contribute to UV-induced apoptosis by modulating the formation of DNA lesions on chromatin

  4. Naringin prevents ovariectomy-induced osteoporosis and promotes osteoclasts apoptosis through the mitochondria-mediated apoptosis pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Fengbo [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Graduate School of Tianjin Medical University, No. 22, Qixiangtai Street, Heping District, Tianjin 300070 (China); Sun, Xiaolei; Ma, Jianxiong [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Ma, Xinlong, E-mail: gengxiao502@163.com [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Zhao, Bin; Zhang, Yang; Tian, Peng [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Li, Yanjun [Graduate School of Tianjin Medical University, No. 22, Qixiangtai Street, Heping District, Tianjin 300070 (China); Han, Zhe [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China)

    2014-09-26

    Highlights: • Naringin possesses many pharmacological activities, promotes the proliferation of osteoblast. • Undecalcified histological obtain dynamic parameters of callus formation and remodeling. • Naringin regulate osteoclast apoptosis by mitochondrial pathway. - Abstract: Naringin, the primary active compound of the traditional Chinese medicine Rhizoma drynariae, possesses many pharmacological activities. The present study is an effort to explore the anti-osteoporosis potential of naringin in vivo and in vitro. In vivo, we used ovariectomized rats to clarify the mechanisms by which naringin anti-osteoporosis. In vitro, we used osteoclasts to investigate naringin promotes osteoclasts apoptosis. Naringin was effective at enhancing BMD, trabecular thickness, bone mineralization, and mechanical strength in a dose-dependent manner. The result of RT-PCR analysis revealed that naringin down-regulated the mRNA expression levels of BCL-2 and up-regulated BAX, caspase-3 and cytochrome C. In addition, naringin significantly reduced the bone resorption area in vitro. These findings suggest that naringin promotes the apoptosis of osteoclasts by regulating the activity of the mitochondrial apoptosis pathway and prevents OVX-induced osteoporosis in rats.

  5. Naringin prevents ovariectomy-induced osteoporosis and promotes osteoclasts apoptosis through the mitochondria-mediated apoptosis pathway

    International Nuclear Information System (INIS)

    Li, Fengbo; Sun, Xiaolei; Ma, Jianxiong; Ma, Xinlong; Zhao, Bin; Zhang, Yang; Tian, Peng; Li, Yanjun; Han, Zhe

    2014-01-01

    Highlights: • Naringin possesses many pharmacological activities, promotes the proliferation of osteoblast. • Undecalcified histological obtain dynamic parameters of callus formation and remodeling. • Naringin regulate osteoclast apoptosis by mitochondrial pathway. - Abstract: Naringin, the primary active compound of the traditional Chinese medicine Rhizoma drynariae, possesses many pharmacological activities. The present study is an effort to explore the anti-osteoporosis potential of naringin in vivo and in vitro. In vivo, we used ovariectomized rats to clarify the mechanisms by which naringin anti-osteoporosis. In vitro, we used osteoclasts to investigate naringin promotes osteoclasts apoptosis. Naringin was effective at enhancing BMD, trabecular thickness, bone mineralization, and mechanical strength in a dose-dependent manner. The result of RT-PCR analysis revealed that naringin down-regulated the mRNA expression levels of BCL-2 and up-regulated BAX, caspase-3 and cytochrome C. In addition, naringin significantly reduced the bone resorption area in vitro. These findings suggest that naringin promotes the apoptosis of osteoclasts by regulating the activity of the mitochondrial apoptosis pathway and prevents OVX-induced osteoporosis in rats

  6. The role of autophagy in THP-1 macrophages resistance to HIV- vpr-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Hua-ying, E-mail: zhouhuaying_2004@126.com; Zheng, Yu-huang; He, Yan; Chen, Zi; He, Bo

    2017-02-01

    Macrophages are resistant to cell death and are one of HIV reservoirs. HIV viral protein Vpr has the potential to promote infection of and survival of macrophages, which could be a highly significant factor in the development and/or maintenance of macrophage viral reservoirs. However, the impact of vpr on macrophages resistance to apoptosis is yet to be comprehended. Autophagy is a cell survival mechanism under stress state. In this study, we investigated whether autophagy is involved in macrophages resistant to vpr-induced apoptosis. Using the THP1 macrophages, we studied the interconnection between macrophages resistance to apoptosis and autophagy. We found that vpr is able to trigger autophagy in transfected THP-1 macrophages confirmed by electron microscopy (EM) and western blot analysis, and inhibition of autophagy with 3MA increased vpr-induced apoptosis. The results indicate that autophagy may be responsible for maintenance of macrophage HIV reservoirs. - Highlights: • HIV Vpr is able to trigger autophagy in transfected THP-1 macrophages. • Autophagy inhibition increases vpr-transfected THP1-macrophages apoptosis. • Autophagy is involved in THP-1 macrophages resistant to vpr-induced apoptosis.

  7. The Common Inhalational Anesthetic Sevoflurane Induces Apoptosis and Increases β-Amyloid Protein Levels

    Science.gov (United States)

    Dong, Yuanlin; Zhang, Guohua; Zhang, Bin; Moir, Robert D.; Xia, Weiming; Marcantonio, Edward R.; Culley, Deborah J.; Crosby, Gregory; Tanzi, Rudolph E.; Xie, Zhongcong

    2009-01-01

    Objective: To assess the effects of sevoflurane, the most commonly used inhalation anesthetic, on apoptosis and β-amyloid protein (Aβ) levels in vitro and in vivo. Subjects: Naive mice, H4 human neuroglioma cells, and H4 human neuroglioma cells stably transfected to express full-length amyloid precursor protein. Interventions: Human H4 neuroglioma cells stably transfected to express full-length amyloid precursor protein were exposed to 4.1% sevoflurane for 6 hours. Mice received 2.5% sevoflurane for 2 hours. Caspase-3 activation, apoptosis, and Aβ levels were assessed. Results: Sevoflurane induced apoptosis and elevated levels of β-site amyloid precursor protein-cleaving enzyme and Aβ in vitro and in vivo. The caspase inhibitor Z-VAD decreased the effects of sevoflurane on apoptosis and Aβ. Sevoflurane-induced caspase-3 activation was attenuated by the γ-secretase inhibitor L-685,458 and was potentiated by Aβ. These results suggest that sevoflurane induces caspase activation which, in turn, enhances β-site amyloid precursor protein–cleaving enzyme and Aβ levels. Increased Aβ levels then induce further rounds of apoptosis. Conclusions: These results suggest that inhalational anesthetic sevoflurane may promote Alzheimer disease neuropathogenesis. If confirmed in human subjects, it may be prudent to caution against the use of sevoflurane as an anesthetic, especially in those suspected of possessing excessive levels of cerebral Aβ. PMID:19433662

  8. Protection of neuroblastoma Neuro2A cells from hypoxia-induced apoptosis by cyclic phosphatidic acid (cPA.

    Directory of Open Access Journals (Sweden)

    Mari Gotoh

    Full Text Available Cyclic phosphatidic acid (cPA is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We have previously shown that cPA significantly suppresses ischemia-induced delayed neuronal death and the accumulation of glial fibrillary acidic protein in the CA1 region of the rat hippocampus. These results indicated that the systemic administration of cPA can protect hippocampal neurons against ischemia-induced delayed neuronal cell death. In the current study, we investigated the effects of cPA on neuronal cell death caused by hypoxia in vitro and the molecular mechanisms underlying these effects. We used cobalt chloride (CoCl(2 to expose cells to hypoxic conditions in vitro. Treating mouse neuroblastoma (Neuro2A cells with CoCl(2 induced nuclear DNA condensation and phosphatidylserine exposure. However, adding cPA led to the suppression of CoCl(2-induced apoptosis in a cPA dose-dependent manner and attenuated the increase in the Bax/Bcl-2 ratio caused by CoCl(2. Quantitative PCR analysis showed that Neuro2A cells strongly express the LPA(1, LPA(2, and LPA(6, which are G-protein coupled receptors that can be activated by cPA. To date, LPA(1 and LPA(2 have been reported to exhibit antiapoptotic activity. Therefore, to assess the roles of LPA(1 and LPA(2 on cPA-induced neuroprotective functions, Ki16425, a selective LPA(1 and LPA(3 antagonist, was adopted to know the LPA(1 function and siRNA was used to knockdown the expression of LPA(2. On the basis of our results, we propose that cPA-induced protection of Neuro2A cells from CoCl(2-induced hypoxia damage is mediated via LPA(2.

  9. In vivo therapeutic potential of Dicer-hunting siRNAs targeting infectious hepatitis C virus.

    Science.gov (United States)

    Watanabe, Tsunamasa; Hatakeyama, Hiroto; Matsuda-Yasui, Chiho; Sato, Yusuke; Sudoh, Masayuki; Takagi, Asako; Hirata, Yuichi; Ohtsuki, Takahiro; Arai, Masaaki; Inoue, Kazuaki; Harashima, Hideyoshi; Kohara, Michinori

    2014-04-23

    The development of RNA interference (RNAi)-based therapy faces two major obstacles: selecting small interfering RNA (siRNA) sequences with strong activity, and identifying a carrier that allows efficient delivery to target organs. Additionally, conservative region at nucleotide level must be targeted for RNAi in applying to virus because hepatitis C virus (HCV) could escape from therapeutic pressure with genome mutations. In vitro preparation of Dicer-generated siRNAs targeting a conserved, highly ordered HCV 5' untranslated region are capable of inducing strong RNAi activity. By dissecting the 5'-end of an RNAi-mediated cleavage site in the HCV genome, we identified potent siRNA sequences, which we designate as Dicer-hunting siRNAs (dh-siRNAs). Furthermore, formulation of the dh-siRNAs in an optimized multifunctional envelope-type nano device inhibited ongoing infectious HCV replication in human hepatocytes in vivo. Our efforts using both identification of optimal siRNA sequences and delivery to human hepatocytes suggest therapeutic potential of siRNA for a virus.

  10. Lymphocytes from wasted mice express enhanced spontaneous and {gamma}-ray-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Woloschak, G.E. [Argonne National Lab., IL (United States)]|[Loyola Univ. Medical Center, Maywood, IL (United States); Chang-Liu, Chin-Mei [Argonne National Lab., IL (United States); Chung, Jen; Libertin, C.R. [Loyola Univ. Medical Center, Maywood, IL (United States)

    1993-09-01

    Mice bearing the autosomal recessive mutation wasted (wst/wst) display a disease pattern including faulty repair of DNA damage in lymphocytes after radiation exposure, neurologic abnormalities, and immunodeficiency. Many of the features of this mouse model have suggested a premature or increased spontaneous frequency of apoptosis in thymocytes; past work has shown an inability to establish cultured T cell lines, an abnormally high death rate of stimulated T cells in culture, and an increased sensitivity of T cells to the killing effects of ionizing radiations in wst/wst mice relative to controls. The experiments reported here were designed to examine splenic and thymic lymphocytes from wasted and control mice for signs of early apoptosis. Our results revealed enhanced expression of Rp-8 mRNA (associated with apoptosis) in thymic lymphocytes and reduced expression in splenic lymphocytes of wst/wst mice relative to controls; expression of Rp-2 and Td-30 mRNA (induced during apoptosis) were not detectable in spleen or thymus. Higher spontaneous DNA fragmentation was observed in wasted mice than in controls; however, {gamma}-ray-induced DNA fragmentation peaked at a lower dose and occurred to a greater extent in wasted mice relative to controls. These results provide evidence for high spontaneous and {gamma}-ray-induced apoptosis in T cells of wasted mice as a mechanism underlying the observed lymphocyte and DNA repair abnormalities.

  11. The effects of herbs on the radiation-induced apoptosis in intestinal crypt cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sung Ho; An, Mi Ra; Nah, Seung Yeol; Lee, Jong Hwan; Kim, Jae Ha; Shin, Dong Ho [Chonnam National Univ., Gwangju (Korea, Republic of); Jo, Sung Kee [KAERI, Daejeon (Korea, Republic of); Jang, Jong Sik [Sangju National Univ., Sangju (Korea, Republic of)

    2001-03-15

    This study was performed to determine the effect of several herbs on radiation-induced apoptosis in jejunal crypt cells. Longyanrou(Euphoris logana), Suanzaoren(Zizyphus vulgaris), Yuanzhi(Polygala tenuifolia), Rensan(Panax ginseng), Fuling(Poria cocos), Muxiang(Saussurea lappa), Chuanxiong(Cnidium offcinale), Baishaoyao(Paeonia lactifolia), Shengma(Cimicifuga heracleifolia), Chaihu(Bupleurum falcatum) and Dongchongxiacao(Paecilomyces japonica) reduced the frequency of radiation-induced apoptosis(p<0.05). Although the mechanisms of this effect remain to be elucidated, these results indicated that Longyanrou, Suanzaoren, Yuanzhi, Rensan, Fuling, Muxiang, Chuanxiong, Baishaoyao, Shengma, Chaihu and Dongchongxiacao might be useful inhibitors of apoptosis, especially since these are relative nontoxic natural products.

  12. Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway

    Directory of Open Access Journals (Sweden)

    Liang Chi-Ming

    2009-01-01

    Full Text Available Abstract Background Numerous proteins can be converted to amyloid-like fibrils to increase cytotoxicity and induce apoptosis, but the methods generally require a high concentration of protein, vigorous shaking, or fibril seed. As well, the detailed mechanism of the cytotoxic effects is not well characterized. In this study, we have developed a novel process to convert native proteins into the fibrillar form. We used globular bovine serum albumin (BSA as a model protein to verify the properties of the fibrillar protein, investigated its cellular effects and studied the signaling cascade induced by the fibrillar protein. Results We induced BSA, a non-cytotoxic globular protein, to become fibril by a novel process involving Superdex-200 column chromatography in the presence of anionic or zwittergenic detergent(s. The column pore size was more important than column matrix composite in fibril formation. The fibrillar BSA induced apoptosis in BHK-21 cell as well as breast cancer cell line T47D. Pre-treating cells with anti-integrin antibodies blocked the apoptotic effect. Fibrillar BSA, but not globular BSA, bound to integrin, dephosphorylated focal adhesion kinase (FAK, Akt and glycogen synthase kinase-3β (GSK-3β. Conclusion We report on a novel process for converting globular proteins into fibrillar form to cause apoptosis by modulating the integrin/FAK/Akt/GSK-3β/caspase-3 signaling pathway. Our findings may be useful for understanding the pathogenesis of amyloid-like fibrils and applicable for the development of better therapeutic agents that target the underlying mechanism(s of the etiologic agents.

  13. Radiation-induced apoptosis of chicken lymphocyte B-cell line DT40

    International Nuclear Information System (INIS)

    Furusawa, Y.; Aoki, M.; Takakura, K.

    2003-01-01

    Full text: Ionizing radiation causes lesions of DNA, cell cycle arrest, induced cell death, and apoptosis in the irradiated cells. Then it is easy to expect that those events would be increased in a cell line which is defective in DNA repair system. However, induction of apoptosis by irradiation takes so complicated process when the cells are defective of DNA repair system. Indeed by many recent studies it has been clarified that DNA repair gene is also concerned with apoptotic event and some study shows the contrary data. Thus, the relationship between the genetics of apoptosis and that of DNA repair is still unclear. In this study two kinds of DNA repair proteins, Rad54 and Ku70, were focused. Proteins of Rad54 and Ku70 have important role at two type of DNA repair systems called homologous recombination repair and non-homologous end joining repair, respectively. 4 phenotypes of DT40, parent type, ku70-/-, rad54-/- and ku70-/-/rad54-/- were used to study the radiation-induced apoptosis (Previous study shows that survival fraction of 4 phenotypes of DT40 is decreased in the cell line, in which DNA repair gene is defective). From the results in this study, two things are clarifies. One is that the dependence of apoptotic index on phenotypes is so different between at low dose and at high dose irradiation. The other is that Ku70 has effective role to induce apoptosis in DT40 irradiated with high dose X-rays

  14. Paclitaxel-induced apoptosis is BAK-dependent, but BAX and BIM-independent in breast tumor.

    Directory of Open Access Journals (Sweden)

    Anna V Miller

    Full Text Available Paclitaxel (Taxol-induced cell death requires the intrinsic cell death pathway, but the specific participants and the precise mechanisms are poorly understood. Previous studies indicate that a BH3-only protein BIM (BCL-2 Interacting Mediator of cell death plays a role in paclitaxel-induced apoptosis. We show here that BIM is dispensable in apoptosis with paclitaxel treatment using bim(-/- MEFs (mouse embryonic fibroblasts, the bim(-/- mouse breast tumor model, and shRNA-mediated down-regulation of BIM in human breast cancer cells. In contrast, both bak (-/- MEFs and human breast cancer cells in which BAK was down-regulated by shRNA were more resistant to paclitaxel. However, paclitaxel sensitivity was not affected in bax(-/- MEFs or in human breast cancer cells in which BAX was down-regulated, suggesting that paclitaxel-induced apoptosis is BAK-dependent, but BAX-independent. In human breast cancer cells, paclitaxel treatment resulted in MCL-1 degradation which was prevented by a proteasome inhibitor, MG132. A Cdk inhibitor, roscovitine, blocked paclitaxel-induced MCL-1 degradation and apoptosis, suggesting that Cdk activation at mitotic arrest could induce subsequent MCL-1 degradation in a proteasome-dependent manner. BAK was associated with MCL-1 in untreated cells and became activated in concert with loss of MCL-1 expression and its release from the complex. Our data suggest that BAK is the mediator of paclitaxel-induced apoptosis and could be an alternative target for overcoming paclitaxel resistance.

  15. Dihydrotestosterone (DHT) modulates the ability of NSAIDs to induce apoptosis of prostate cancer cells.

    Science.gov (United States)

    Andrews, Peter; Krygier, Scott; Djakiew, Daniel

    2002-03-01

    Recent evidence indicates that nonsteroidal antiinflammatory drugs (NSAIDs) are effective in the treatment and prevention of prostate cancer. In the study reported here, we investigated the ability of the steroid hormone dihydrotestosterone (DHT) to modulate NSAID-induced apoptosis of prostate cancer cells. Using in vitro models of androgen-sensitive and androgen-insensitive human prostate cancer cells, we evaluated the ability of a specific cyclooxygenase-2 inhibitor (NS-398) and a nonspecific cyclooxygenase inhibitor (indomethacin) to induce apoptosis in the presence of various concentrations of DHT. Apoptosis was quantified using the TUNEL method and verified by electron microscopy. We found that increasing concentrations of DHT significantly enhanced the ability of NS-398 and indomethacin to induce apoptosis of androgen-sensitive LNCaP cells. The ability of NSAIDs to induce apoptosis of androgen-insensitive PC-3 cells, however, was not affected by the presence of DHT. Higher levels of DHT in the incubation medium both before as well as following exposure to NSAIDs enhanced apoptosis of LNCaP cells. Another steroid hormone that interacts with the androgen receptor in LNCaP cells (progesterone) also promoted apoptosis of these cells. Increasing concentrations of DHT caused LNCaP cells to shift from the S and G(2)/M to the G(0)/G(1) stages of the cell cycle. These observations support the use of DHT in combination with NSAIDs in the treatment of prostate cancer, and indicate that DHT is an important issue to address in clinical trials of NSAIDs since androgen ablation is a common treatment for prostate cancer.

  16. SOX6 and PDCD4 enhance cardiomyocyte apoptosis through LPS-induced miR-499 inhibition.

    Science.gov (United States)

    Jia, Zhuqing; Wang, Jiaji; Shi, Qiong; Liu, Siyu; Wang, Weiping; Tian, Yuyao; Lu, Qin; Chen, Ping; Ma, Kangtao; Zhou, Chunyan

    2016-02-01

    Sepsis-induced cardiac apoptosis is one of the major pathogenic factors in myocardial dysfunction. As it enhances numerous proinflammatory factors, lipopolysaccharide (LPS) is considered the principal mediator in this pathological process. However, the detailed mechanisms involved are unclear. In this study, we attempted to explore the mechanisms involved in LPS-induced cardiomyocyte apoptosis. We found that LPS stimulation inhibited microRNA (miR)-499 expression and thereby upregulated the expression of SOX6 and PDCD4 in neonatal rat cardiomyocytes. We demonstrate that SOX6 and PDCD4 are target genes of miR-499, and they enhance LPS-induced cardiomyocyte apoptosis by activating the BCL-2 family pathway. The apoptosis process enhanced by overexpression of SOX6 or PDCD4, was rescued by the cardiac-abundant miR-499. Overexpression of miR-499 protected the cardiomyocytes against LPS-induced apoptosis. In brief, our results demonstrate the existence of a miR-499-SOX6/PDCD4-BCL-2 family pathway in cardiomyocytes in response to LPS stimulation.

  17. Piroxicam, a traditional non-steroidal anti-inflammatory drug (NSAID) causes apoptosis by ROS mediated Akt activation.

    Science.gov (United States)

    Rai, Neha; Sarkar, Munna; Raha, Sanghamitra

    2015-12-01

    Piroxicam (Px) belongs to the oxicam group of the non-steroidal anti-inflammatory drugs (NSAIDs) and have been shown to exert chemopreventive and chemotherapeutic effects in animal models and cultured animal cells. However, little is known about the mode of action of Px and its cellular targets. We explored the role of Px, in triggering apoptosis and examined the involvement of upstream cellular mechanisms in apoptosis induction by Px. Our studies with human breast cancer cells MCF-7 show that Px induces reactive oxygen species (ROS) generation along with apoptotic cell death. ROS release lead to Akt activation. On evaluation it became evident that ROS mediated apoptosis induction was due to Akt activation (hyper phosphorylation). Silencing the expression of Akt using siRNA and a specific Akt inhibitor, triciribine further confirmed the findings. However Px failed to cause ROS generation, cell death or Akt phosphorylation in another human breast cancer cells MDA-MB-231 which is estrogen receptor negative and more aggressive compared to MCF-7 cells. This suggests that Px has cell type specific effects. Thus we revealed for the first time that Px can induce apoptosis by ROS mediated Akt hyperphosphorylation/activation. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  18. H2O2 INDUCES APOPTOSIS OF RABBIT CHONDROCYTES VIA BOTH THE EXTRINSIC AND THE CASPASE-INDEPENDENT INTRINSIC PATHWAYS

    Directory of Open Access Journals (Sweden)

    CAIPING ZHUANG

    2013-07-01

    Full Text Available Osteoarthritis (OA, one of the most common joint diseases with unknown etiology, is characterized by the progressive destruction of articular cartilage and the apoptosis of chondrocytes. The purpose of this study is to elucidate the molecular mechanisms of H2O2-mediated rabbit chondrocytes apoptosis. CCK-8 assay showed that H2O2 treatment induced a remarkable reduction of cell viability, which was further verified by the remarkable phosphatidylserine externalization after H2O2 treatment for 1 h, the typical characteristics of apoptosis. H2O2 treatment induced a significant dysfunction of mitochondrial membrane potential (ΔΨm, but did not induce casapse-9 activation, indicating that H2O2 treatment induced caspase-independent intrinsic apoptosis that was further verified by the fact that silencing of AIF but not inhibiting caspase-9 potently prevented H2O2-induced apoptosis. H2O2 treatment induced a significant increase of caspase-8 and -3 activation, and inhibition of caspase-8 or -3 significantly prevented H2O2-induced apoptosis, suggesting that the extrinsic pathway played an important role. Collectively, our findings demonstrate that H2O2 induces apoptosis via both the casapse-8-mediated extrinsic and the caspase-independent intrinsic apoptosis pathways in rabbit chondrocytes.

  19. Thymocyte apoptosis induced by p53-dependent and independent pathways

    International Nuclear Information System (INIS)

    Clarke, A.R.; Purdie, C.A.; Harrison, D.J.; Morris, R.G.; Bird, C.C.; Hooper, M.L.; Wyllie, A.H.

    1993-01-01

    The authors studied the dependence of apoptosis on p53 expression in cells from the thymus cortex. Short-term thymocyte cultures were prepared from mice constitutively heterozygous or homozygous for a deletion in the p53 gene introduced into the germ line after gene targeting. Wild-type thymocytes readily undergo apoptosis after treatment with ionizing radiation, the glucocorticoid methylprednisolone, or etoposide (an inhibitor of topoisomerase II), or after Ca 2+ -dependent activation by phorbol ester and a calcium ionophore. In contrast, homozygous null p53 thymocytes are resistant to induction of apoptosis by radiation or etoposide, but retain normal sensitivity to glucocorticoid and calcium. The time-dependent apoptosis that occurs in untreated cultures is unaffected by p53 status. Cells heterozygous for p53 deletion are partially resistant to radiation and etoposide. Results show that p53 exerts a significant and dose-dependent effect in the initiation of apoptosis, but only when it is induced by agents that cause DNA-strand breakage. (Author)

  20. Melatonin resists oxidative stress-induced apoptosis in nucleus pulposus cells.

    Science.gov (United States)

    He, Ruijun; Cui, Min; Lin, Hui; Zhao, Lei; Wang, Jiayu; Chen, Songfeng; Shao, Zengwu

    2018-04-15

    Intervertebral disc degeneration (IVDD) is thought to be the major cause of low back pain (LBP), which is still in lack of effective etiological treatment. Oxidative stress has been demonstrated to participate in the impairment of nucleus pulposus cells (NPCs). As the most important neuroendocrine hormone in biological clock regulation, melatonin (MLT) is also featured by good antioxidant effect. In this study, we investigated the effect and mechanisms of melatonin on oxidative stress-induced damage in rat NPCs. Cytotoxicity of H 2 O 2 and protecting effect of melatonin were analyzed with Cell Counting kit-8 (CCK-8). Cell apoptosis rate was detected by Annexin V-FITC/PI staining. DCFH-DA probe was used for the reactive oxygen species (ROS) detection. The mitochondrial membrane potential (MMP) changes were analyzed with JC-1 probe. Intracellular oxidation product and reductants were measured through enzymatic reactions. Extracellular matrix (ECM) and apoptosis associated proteins were analyzed with Western blot assays. Melatonin preserved cell viability of NPCs under oxidative stress. The apoptosis rate, ROS level and malonaldehyde (MDA) declined with melatonin. MLT/H 2 O 2 group showed higher activities of GSH and SOD. The fall of MMP receded and the expression of ECM protein increased with treatment of melatonin. The mitochondrial pathway of apoptosis was inhibited by melatonin. Melatonin alleviated the oxidative stress-induced apoptosis of NPCs. Melatonin could be a promising alternative in treatment of IVDD. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Involvement of phorbol-12-myristate-13-acetate-induced protein 1 in goniothalamin-induced TP53-dependent and -independent apoptosis in hepatocellular carcinoma-derived cells

    International Nuclear Information System (INIS)

    Kuo, Kung-Kai; Chen, Yi-Ling; Chen, Lih-Ren; Li, Chien-Feng; Lan, Yu-Hsuan; Chang, Fang-Rong; Wu, Yang-Chang; Shiue, Yow-Ling

    2011-01-01

    The objective was to investigate the upstream apoptotic mechanisms that were triggered by a styrylpyrone derivative, goniothalamin (GTN), in tumor protein p53 (TP53)-positive and -negative hepatocellular carcinoma (HCC)-derived cells. Effects of GTN were evaluated by the flow cytometry, alkaline comet assay, immunocytochemistry, small-hairpin RNA interference, mitochondria/cytosol fractionation, quantitative reverse transcription-polymerase chain reaction, immunoblotting analysis and caspase 3 activity assays in two HCC-derived cell lines. Results indicated that GTN triggered phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, also known as NOXA)-mediated apoptosis via TP53-dependent and -independent pathways. In TP53-positive SK-Hep1 cells, GTN furthermore induced TP53 transcription-dependent and -independent apoptosis. After GTN treatment, accumulation of reactive oxygen species, formation of DNA double-strand breaks, transactivation of TP53 and/or PMAIP1 gene, translocation of TP53 and/or PMAIP1 proteins to mitochondria, release of cytochrome c from mitochondria, cleavage of caspases and induction of apoptosis in both cell lines were sustained. GTN might represent a novel class of anticancer drug that induces apoptosis in HCC-derived cells through PMAIP1 transactivation regardless of the status of TP53 gene. - Highlights: → Goniothalamin (GTN) induced apoptosis in hepatocellular carcinomas-derived cells. → The apoptosis induced by GTN is PMAIP1-dependent, regardless of TP53 status. → The apoptosis induced by GTN might be TP53 transcription-dependent or -independent. → GTN-induced apoptosis is mitochondria- and caspases-mediated.

  2. Cuprous oxide nanoparticles selectively induce apoptosis of tumor cells

    Science.gov (United States)

    Wang, Ye; Zi, Xiao-Yuan; Su, Juan; Zhang, Hong-Xia; Zhang, Xin-Rong; Zhu, Hai-Ying; Li, Jian-Xiu; Yin, Meng; Yang, Feng; Hu, Yi-Ping

    2012-01-01

    In the rapid development of nanoscience and nanotechnology, many researchers have discovered that metal oxide nanoparticles have very useful pharmacological effects. Cuprous oxide nanoparticles (CONPs) can selectively induce apoptosis and suppress the proliferation of tumor cells, showing great potential as a clinical cancer therapy. Treatment with CONPs caused a G1/G0 cell cycle arrest in tumor cells. Furthermore, CONPs enclosed in vesicles entered, or were taken up by mitochondria, which damaged their membranes, thereby inducing apoptosis. CONPs can also produce reactive oxygen species (ROS) and initiate lipid peroxidation of the liposomal membrane, thereby regulating many signaling pathways and influencing the vital movements of cells. Our results demonstrate that CONPs have selective cytotoxicity towards tumor cells, and indicate that CONPs might be a potential nanomedicine for cancer therapy. PMID:22679374

  3. Glutathione Depletion Induced by c-Myc Downregulation Triggers Apoptosis on Treatment with Alkylating Agents1

    Science.gov (United States)

    Biroccio, Annamaria; Benassi, Barbara; Fiorentino, Francesco; Zupi, Gabriella

    2004-01-01

    Abstract Here we investigate the mechanism(s) involved in the c-Myc-dependent drug response of melanoma cells. By using three M14-derived c-Myc low-expressing clones, we demonstrate that alkylating agents, cisplatin and melphalan, trigger apoptosis in the c-Myc antisense transfectants, but not in the parental line. On the contrary, topoisomerase inhibitors, adriamycin and camptothecin, induce apoptosis to the same extent regardless of c-Myc expression. Because we previously demonstrated that c-Myc downregulation decreases glutathione (GSH) content, we evaluated the role of GSH in the apoptosis induced by the different drugs. In control cells treated with one of the alkylating agents or the others, GSH depletion achieved by l-buthionine-sulfoximine preincubation opens the apoptotic pathway. The apoptosis proceeded through early Bax relocalization, cytochrome c release, and concomitant caspase-9 activation, whereas reactive oxygen species production and alteration of mitochondria membrane potential were late events. That GSH was determining in the c-Myc-dependent drug-induced apoptosis was demonstrated by altering the intracellular GSH content of the c-Myc low-expressing cells up to the level of controls. Indeed, GSH ethyl ester-mediated increase of GSH abrogated apoptosis induced by cisplatin and melphalan by inhibition of Bax/cytochrome c redistribution. The relationship among c-Myc, GSH content, and the response to alkylating agent has been also evaluated in the M14 Myc overexpressing clones as well as in the melanoma JR8 c-Myc antisense transfectants. All together, these results demonstrate that GSH plays a key role in governing c-Myc-dependent drug-induced apoptosis. PMID:15153331

  4. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

    International Nuclear Information System (INIS)

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang; Dong, Wei-Guo

    2012-01-01

    Highlights: ► Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. ► G 2 /M phase arrest and chromatin condensation and nuclear fragmentation were induced. ► Noscapine promoted apoptosis via mitochondrial pathways. ► Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC 50 = 75 μM). This cytotoxicity was reflected by cell cycle arrest at G 2 /M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  5. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner

    Directory of Open Access Journals (Sweden)

    Gretel G. Pellegrini

    2016-07-01

    Full Text Available Oats contain unique bioactive compounds known as avenanthramides (AVAs with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT and Nrf2 Knockout (KO osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast

  6. Tob1 induces apoptosis and inhibits proliferation, migration and invasion of gastric cancer cells by activating Smad4 and inhibiting β‑catenin signaling.

    Science.gov (United States)

    Kundu, Juthika; Wahab, S M Riajul; Kundu, Joydeb Kumar; Choi, Yoon-La; Erkin, Ozgur Cem; Lee, Hun Seok; Park, Sang Gyu; Shin, Young Kee

    2012-09-01

    Transducer of ErbB-2.1 (Tob1), a tumor suppressor protein, is inactivated in a variety of cancers including stomach cancer. However, the role of Tob1 in gastric carcinogenesis remains elusive. The present study aimed to investigate whether Tob1 could inhibit gastric cancer progression in vitro, and to elucidate its underlying molecular mechanisms. We found differential expression of Tob1 in human gastric cancer (MKN28, AGS and MKN1) cells. The overexpression of Tob1 induced apoptosis in MKN28 and AGS cells, which was associated with sub-G1 arrest, activation of caspase-3, induction of Bax, inhibition of Bcl-2 and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, Tob1 inhibited proliferation, migration and invasion, which were reversed in MKN1 and AGS cells transfected with Tob1 siRNA. Overexpression of Tob1 in MKN28 and AGS cells induced the expression of Smad4, leading to the increased expression and the promoter activity of p15, which was diminished by silencing of Tob1 using specific siRNA. Tob1 decreased the phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β) in MKN28 and AGS cells, resulting in the reduced protein expression and the transcriptional activity of β‑catenin, which in turn decreased the expression of cyclin D1, cyclin-dependent kinase-4 (CDK4), urokinase plasminogen activator receptor (uPAR) and peroxisome proliferator and activator receptor-δ (PPARδ). Conversely, silencing of Tob1 induced the phosphorylation of Akt and GSK-3β, and increased the expression of β‑catenin and its target genes. Collectively, our study demonstrates that the overexpression of Tob1 inhibits gastric cancer progression by activating Smad4- and inhibiting β‑catenin-mediated signaling pathways.

  7. 15,16-Dihydrotanshinone I, a Compound of Salvia miltiorrhiza Bunge, Induces Apoptosis through Inducing Endoplasmic Reticular Stress in Human Prostate Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Mao-Te Chuang

    2011-01-01

    Full Text Available 5,16-dihydrotanshinone I (DHTS is extracted from Salvia miltiorrhiza Bunge (tanshen root and was found to be the most effective compound of tanshen extracts against breast cancer cells in our previous studies. However, whether DHTS can induce apoptosis through an endoplasmic reticular (ER stress pathway was examined herein. In this study, we found that DHTS significantly inhibited the proliferation of human prostate DU145 carcinoma cells and induced apoptosis. DHTS was able to induce ER stress as evidenced by the upregulation of glucose regulation protein 78 (GRP78/Bip and CAAT/enhancer binding protein homologous protein/growth arrest- and DNA damage-inducible gene 153 (CHOP/GADD153, as well as increases in phosphorylated eukaryotic initiation factor 2α (eIF2α, c-jun N-terminal kinase (JNK, and X-box-binding protein 1 (XBP1 mRNA splicing forms. DHTS treatment also caused significant accumulation of polyubiquitinated proteins and hypoxia-inducible factor (HIF-1α, indicating that DHTS might be a proteasome inhibitor that is known to induce ER stress or enhance apoptosis caused by the classic ER stress-dependent mechanism. Moreover, DHTS-induced apoptosis was reversed by salubrinal, an ER stress inhibitor. Results suggest that DHTS can induce apoptosis of prostate carcinoma cells via induction of ER stress and/or inhibition of proteasome activity, and may have therapeutic potential for prostate cancer patients.

  8. Apoptosis and autophagy induced by pyropheophorbide-α methyl ester-mediated photodynamic therapy in human osteosarcoma MG-63 cells.

    Science.gov (United States)

    Huang, Qiu; Ou, Yun-Sheng; Tao, Yong; Yin, Hang; Tu, Ping-Hua

    2016-06-01

    Pyropheophorbide-α methyl ester (MPPa) was a second-generation photosensitizer with many potential applications. Here, we explored the impact of MPPa-mediated photodynamic therapy (MPPa-PDT) on the apoptosis and autophagy of human osteosarcoma (MG-63) cells as well as the relationships between apoptosis and autophagy of the cells, and investigated the related molecular mechanisms. We found that MPPa-PDT demonstrated the ability to inhibit MG-63 cell viability in an MPPa concentration- and light dose-dependent manner, and to induce apoptosis via the mitochondrial apoptosis pathway. Additionally, MPPa-PDT could also induce autophagy of MG-63 cell. Meanwhile, the ROS scavenger N-acetyl-L-cysteine (NAC) and the Jnk inhibitor SP600125 were found to inhibit the MPPa-PDT-induced autophagy, and NAC could also inhibit Jnk phosphorylation. Furthermore, pretreatment with the autophagy inhibitor 3-methyladenine or chloroquine showed the potential in reducing the apoptosis rate induced by MPPa-PDT in MG-63 cells. Our results indicated that the mitochondrial pathway was involved in MPPa-PDT-induced apoptosis of MG-63 cells. Meanwhile the ROS-Jnk signaling pathway was involved in MPPa-PDT-induced autophagy, which further promoted the apoptosis in MG-63 cells.

  9. Dopamine Attenuates Ketamine-Induced Neuronal Apoptosis in the Developing Rat Retina Independent of Early Synchronized Spontaneous Network Activity.

    Science.gov (United States)

    Dong, Jing; Gao, Lingqi; Han, Junde; Zhang, Junjie; Zheng, Jijian

    2017-07-01

    Deprivation of spontaneous rhythmic electrical activity in early development by anesthesia administration, among other interventions, induces neuronal apoptosis. However, it is unclear whether enhancement of neuronal electrical activity attenuates neuronal apoptosis in either normal development or after anesthesia exposure. The present study investigated the effects of dopamine, an enhancer of spontaneous rhythmic electrical activity, on ketamine-induced neuronal apoptosis in the developing rat retina. TUNEL and immunohistochemical assays indicated that ketamine time- and dose-dependently aggravated physiological and ketamine-induced apoptosis and inhibited early-synchronized spontaneous network activity. Dopamine administration reversed ketamine-induced neuronal apoptosis, but did not reverse the inhibitory effects of ketamine on early synchronized spontaneous network activity despite enhancing it in controls. Blockade of D1, D2, and A2A receptors and inhibition of cAMP/PKA signaling partially antagonized the protective effect of dopamine against ketamine-induced apoptosis. Together, these data indicate that dopamine attenuates ketamine-induced neuronal apoptosis in the developing rat retina by activating the D1, D2, and A2A receptors, and upregulating cAMP/PKA signaling, rather than through modulation of early synchronized spontaneous network activity.

  10. Sulforaphane reverses glucocorticoid-induced apoptosis in osteoblastic cells through regulation of the Nrf2 pathway

    Directory of Open Access Journals (Sweden)

    Lin H

    2014-07-01

    Full Text Available Hao Lin,1,* Bo Wei,1,* Guangsheng Li,1 Jinchang Zheng,1 Jiecong Sun,1 Jiaqi Chu,2 Rong Zeng,1 Yanru Niu21Department of Spinal Surgery, Affiliated Hospital of Guangdong Medical College, Zhanjiang, People’s Republic of China; 2Laboratory Institute of Minimally Invasive Orthopedic Surgery, Affiliated Hospital of Guangdong Medical College, Zhanjiang, People’s Republic of China *These authors contributed equally to this work Abstract: Apoptosis of osteoblasts triggered by high-dose glucocorticoids (GCs has been identified as a major cause of osteoporosis. However, the underlying molecular mechanisms accounting for this action remain elusive, which has impeded the prevention and cure of this side effect. Sulforaphane (SFP is a naturally occurring isothiocyanate that has huge health benefits for humans. In this study, by using osteoblastic MC3T3-E1 cells as a model, we demonstrate the protective effects of SFP against dexamethasone (Dex-induced apoptosis and elucidate the underlying molecular mechanisms. The results show that SFP could effectively inhibit the Dex-induced growth inhibition and release of lactate dehydrogenase in MC3T3-E1 cells. Treatment with Dex induced caspase-dependent apoptosis in MC3T3-E1 cells, as evidenced by an increase in the Sub-G1 phase, chromatin condensation, and deoxyribonucleic acid fragmentation, which were significantly suppressed by coincubation with SFP. Mitochondria-mediated apoptosis pathway contributed importantly to Dex-induced apoptosis, as revealed by the activation of caspase-3/-9 and subsequent cleavage of poly adenosine diphosphate ribose polymerase, which was also effectively blocked by SFP. Moreover, treatments of Dex strongly induced overproduction of reactive oxygen species and inhibited the expression of nuclear factor erythroid 2-related factor 2 (Nrf2 and the downstream effectors HO1 and NQO1. However, cotreatment with SFP effectively reversed this action of Dex. Furthermore, silencing of Nrf2 by

  11. Mechanisms of caffeine-induced inhibition of UVB carcinogenesis

    Directory of Open Access Journals (Sweden)

    Allan H Conney

    2013-06-01

    Full Text Available Sunlight-induced nonmelanoma skin cancer is the most prevalent cancer in the United States with more than 2 million cases per year. Several studies have shown an inhibitory effect of caffeine administration on UVB-induced skin cancer in mice, and these studies are paralleled by epidemiology studies that indicate an inhibitory effect of coffee drinking on nonmelanoma skin cancer in humans. Strikingly, decaffeinated coffee consumption had no such inhibitory effect.Mechanism studies indicate that caffeine has a sunscreen effect that inhibits UVB-induced formation of thymine dimers and sunburn lesions in the epidermis of mice. In addition, caffeine administration has a biological effect that enhances UVB-induced apoptosis thereby enhancing the elimination of damaged precancerous cells, and caffeine administration also enhances apoptosis in tumors. Caffeine administration enhances UVB-induced apoptosis by p53-dependent and p53-independent mechanisms. Exploration of the p53-independent effect indicated that caffeine administration enhanced UVB-induced apoptosis by inhibiting the UVB-induced increase in ATR-mediated formation of phospho-Chk1 (Ser345 and abolishing the UVB-induced decrease in cyclin B1 which resulted in caffeine-induced premature and lethal mitosis in mouse skin. In studies with cultured primary human keratinocytes, inhibition of ATR with siRNA against ATR inhibited Chk1 phosphorylation and enhanced UVB-induced apoptosis. Transgenic mice with decreased epidermal ATR function that were irradiated chronically with UVB had 69% fewer tumors at the end of the study compared with irradiated littermate controls with normal ATR function. These results, which indicate that genetic inhibition of ATR (like pharmacologic inhibition of ATR via caffeine inhibits UVB-induced carcinogenesis and supports the concept that ATR-mediated phosphorylation of Chk1 is an important target for caffeine’s inhibitory effect on UVB-induced carcinogenesis.

  12. Intranasal delivery of antiviral siRNA.

    Science.gov (United States)

    Barik, Sailen

    2011-01-01

    Intranasal administration of synthetic siRNA is an effective modality of RNAi delivery for the prevention and therapy of respiratory diseases, including pulmonary infections. Vehicles used for nasal siRNA delivery include established as well as novel reagents, many of which have been recently optimized. In general, they all promote significant uptake of siRNA into the lower respiratory tract, including the lung. When properly designed and optimized, these siRNAs offer significant protection against respiratory viruses such as influenza virus, parainfluenza virus and respiratory syncytial virus (RSV). Nasally administered siRNA remains within the lung and does not access systemic blood flow, as judged by its absence in other major organs such as liver, heart, kidney, and skeletal muscle. Adverse immune reaction is generally not encountered, especially when immunogenic and/or off-target siRNA sequences and toxic vehicles are avoided. In fact, siRNA against RSV has entered Phase II clinical trials in human with promising results. Here, we provide a standardized procedure for using the nose as a specific route for siRNA delivery into the lung of laboratory animals. It should be clear that this simple and efficient system has enormous potential for therapeutics.

  13. Dopamine-induced apoptosis of lactotropes is mediated by the short isoform of D2 receptor.

    Science.gov (United States)

    Radl, Daniela Betiana; Ferraris, Jimena; Boti, Valeria; Seilicovich, Adriana; Sarkar, Dipak Kumar; Pisera, Daniel

    2011-03-25

    Dopamine, through D2 receptor (D2R), is the major regulator of lactotrope function in the anterior pituitary gland. Both D2R isoforms, long (D2L) and short (D2S), are expressed in lactotropes. Although both isoforms can transduce dopamine signal, they differ in the mechanism that leads to cell response. The administration of D2R agonists, such as cabergoline, is the main pharmacological treatment for prolactinomas, but resistance to these drugs exists, which has been associated with alterations in D2R expression. We previously reported that dopamine and cabergoline induce apoptosis of lactotropes in primary culture in an estrogen-dependent manner. In this study we used an in vivo model to confirm the permissive action of estradiol in the apoptosis of anterior pituitary cells induced by D2R agonists. Administration of cabergoline to female rats induced apoptosis, measured by Annexin-V staining, in anterior pituitary gland from estradiol-treated rats but not from ovariectomized rats. To evaluate the participation of D2R isoforms in the apoptosis induced by dopamine we used lactotrope-derived PR1 cells stably transfected with expression vectors encoding D2L or D2S receptors. In the presence of estradiol, dopamine induced apoptosis, determined by ELISA and TUNEL assay, only in PR1-D2S cells. To study the role of p38 MAPK in apoptosis induced by D2R activation, anterior pituitary cells from primary culture or PR1-D2S were incubated with an inhibitor of the p38 MAPK pathway (SB203850). SB203580 blocked the apoptotic effect of D2R activation in lactotropes from primary cultures and PR1-D2S cells. Dopamine also induced p38 MAPK phosphorylation, determined by western blot, in PR1-D2S cells and estradiol enhanced this effect. These data suggest that, in the presence of estradiol, D2R agonists induce apoptosis of lactotropes by their interaction with D2S receptors and that p38 MAPK is involved in this process.

  14. The roles of DNA damage-dependent signals and MAPK cascades in tributyltin-induced germline apoptosis in Caenorhabditis elegans.

    Science.gov (United States)

    Wang, Yun; Wang, Shunchang; Luo, Xun; Yang, Yanan; Jian, Fenglei; Wang, Xuemin; Xie, Lucheng

    2014-08-01

    The induction of apoptosis is recognized to be a major mechanism of tributyltin (TBT) toxicity. However, the underlying signaling pathways for TBT-induced apoptosis remain unclear. In this study, using the nematode Caenorhabditis elegans, we examined whether DNA damage response (DDR) pathway and mitogen-activated protein kinase (MAPK) signaling cascades are involved in TBT-induced germline apoptosis and cell cycle arrest. Our results demonstrated that exposing worms to TBT at the dose of 10nM for 6h significantly increased germline apoptosis in N2 strain. Germline apoptosis was absent in strains that carried ced-3 or ced-4 loss-of-function alleles, indicating that both caspase protein CED-3 and Apaf-1 protein CED-4 were required for TBT-induced apoptosis. TBT-induced apoptosis was blocked in the Bcl-2 gain-of-function strain ced-9(n1950), whereas TBT induced a minor increase in the BH3-only protein EGL-1 mutated strain egl-1(n1084n3082). Checkpoint proteins HUS-1 and CLK-2 exerted proapoptotic effects, and the null mutation of cep-1, the homologue of tumor suppressor gene p53, significantly inhibited TBT-induced apoptosis. Apoptosis in the loss-of-function strains of ERK, JNK and p38 MAPK signaling pathways were completely or mildly suppressed under TBT stress. These results were supported by the results of mRNA expression levels of corresponding genes. The present study indicated that TBT-induced apoptosis required the core apoptotic machinery, and that DDR genes and MAPK pathways played essential roles in signaling the processes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Radiation induced apoptosis and initial DNA damage are inversely related in locally advanced breast cancer patients

    International Nuclear Information System (INIS)

    Pinar, Beatriz; Henríquez-Hernández, Luis Alberto; Lara, Pedro C; Bordon, Elisa; Rodriguez-Gallego, Carlos; Lloret, Marta; Nuñez, Maria Isabel; De Almodovar, Mariano Ruiz

    2010-01-01

    DNA-damage assays, quantifying the initial number of DNA double-strand breaks induced by radiation, have been proposed as a predictive test for radiation-induced toxicity. Determination of radiation-induced apoptosis in peripheral blood lymphocytes by flow cytometry analysis has also been proposed as an approach for predicting normal tissue responses following radiotherapy. The aim of the present study was to explore the association between initial DNA damage, estimated by the number of double-strand breaks induced by a given radiation dose, and the radio-induced apoptosis rates observed. Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radio-induced apoptosis at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Radiation-induced apoptosis increased in order to radiation dose and data fitted to a semi logarithmic mathematical model. A positive correlation was found among radio-induced apoptosis values at different radiation doses: 1, 2 and 8 Gy (p < 0.0001 in all cases). Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). A statistically significant inverse correlation was found between initial damage to DNA and radio-induced apoptosis at 1 Gy (p = 0.034). A trend toward 2 Gy (p = 0.057) and 8 Gy (p = 0.067) was observed after 24 hours of incubation. An inverse association was observed for the first time between these variables, both considered as predictive factors to radiation toxicity

  16. Apoptosis induced by radionuclide 153Sm and expression of relevant genes in three different cancer cells

    International Nuclear Information System (INIS)

    Zou Baomin; Duan Xiaoyi; Chen Wei; Hu Guoying

    2003-01-01

    To study apoptosis of PC-3, ER-75-30 and A549 cells induced by radionuclide 153 Sm and the expression of bcl-2, bax in apoptosis cells, MTT assay was used to detect the anti-tumor effect, light microscope, transmission electron microscope, flow cytometer were used to detect apoptosis, while image analysis was used to detect the expression of bcl-2 and bax. 153 Sm showed anti-tumor effect and could induce tumor cell apoptosis. Both bcl-2 and bax played an important role in apoptosis. Different kind of cells had different sensitivity to 153 Sm

  17. Functional analysis of molecular mechanisms of radiation induced apoptosis, that are not mediated by DNA damages

    International Nuclear Information System (INIS)

    Angermeier, Marita; Moertl, Simone

    2012-01-01

    The effects of low-dose irradiation pose new challenges on the radiation protection efforts. Enhanced cellular radiation sensitivity is displayed by disturbed cellular reactions and resulting damage like cell cycle arrest, DNA repair and apoptosis. Apoptosis serves as genetically determinate parameter for the individual radiation sensitivity. In the frame of the project the radiation-induced apoptosis was mechanistically investigated. Since ionizing radiation induced direct DNA damage and generates a reactive oxygen species, the main focus of the research was the differentiation and weighting of DNA damage mediated apoptosis and apoptosis caused by the reactive oxygen species (ROS).

  18. [NRH2 induces cell apoptosis of cerebral tissues around hematomas after intracerebral hemorrhage through up-regulating proNGF, sortilin and p75NTR expressions].

    Science.gov (United States)

    Zeng, Zhiqing; Liu, Hong; Jiang, Di

    2015-04-01

    between ≤ 6 hours group and control group. In comparison with control group and ≤ 6 hours group, the expression levels of NRH2 and sortilin mRNAs and proteins and Bax expression started to increase in 6-24 hours, reached the peak in 24-72 hours, and then stayed a higher level after 72 hours, whereas there were no significant differences in the above indicators between ≤ 6 hours group and control group. There was no obvious change in Bcl-2 expression level between ≤ 6 hours group and control group. The level of Bcl-2 decreased in all groups of intracerebral hemorrhage for over 6 hours, and reached the nadir in 24-72 hours. Astrocytes transfected with NRH2 siRNA displayed a significant decrease in proNGF, sortilin and p75NTR protein levels as compared with scramble siRNA or blank control groups. The expression of NRH2 would increase in the cerebral tissues around hematomas after intracerebral hemorrhage. NRH2 might enhance the ratio of Bax/Bcl-2 by promoting the expressions of proNGF, sortilin and p75NTR, thereby inducing brain cell apoptosis.

  19. A mechanism for suppression of the CDP-choline pathway during apoptosis

    OpenAIRE

    Morton, Craig C.; Aitchison, Adam J.; Gehrig, Karsten; Ridgway, Neale D.

    2013-01-01

    Inhibition of the CDP-choline pathway during apoptosis restricts the availability of phosphatidylcholine (PtdCho) for assembly of membranes and synthesis of signaling factors. The N-terminal nuclear localization signal (NLS) in CTP:phosphocholine cytidylyltransferase (CCT)α is removed during apoptosis but the caspase(s) involved and the contribution to suppression of the CDP-choline pathway is unresolved. In this study we utilized siRNA silencing of caspases in HEK293 cells and caspase 3-defi...

  20. LP-THAE induced tumor cell apoptosis of rabbit VX2 liver carcinoma

    International Nuclear Information System (INIS)

    Chen Shengli; Quan Yi; Huang Zicheng; Chen Guodong; Zhu Dongliang

    2007-01-01

    Objective: To research tumor cell apoptosis induced by Lp-THAE of rabbit VX2 liver implanted tumor. Methods: 27 New Zealand white rabbits implanted with VX2 tumor at left middle lobe of the liver divided into three groups: Group A(n= 9) Lp-THAE: treated through transhepatic artery catheterization; Group B(n=9) THAI and Group C(n=9) as control. The rabbits were executed at second to fifth day after treatment. HE dye microscopy was taken for counting the typical apoptosis cells and calculating apoptosis index (ApI). FITC-AnnexinV/PI assay was used for measuring apoptosis by flow cytometry. Results: The ApI of tumor central area and marginal area were (17.769±2.417)%, (4.129±1.172)%, P<0.01. The percentages of tumor cell apoptosis and tumor cell necrosis were (16.483±1.404)%, (9.478±0.964)%, P<0.01 and (43.559±5.053)%, (33.460±1.840)%, P=0.093. The total percentages of tumor cell apoptosis and necrosis were (60.042±13.979)%, (42.938±8.979)%, P< 0.01, at tumor center and marginal area in THAE group respectively. The ApI, percentages of tumor cell apoptosis and necrosis in THAE group were significantly higher than those of THAI group (P<0.01). The percentages of tumor cell apoptosis at tumor center area in THAE group were significantly higher than those of tumor marginal area(P<0.01). Conclusion: Induced tumor cell apoptosis and necrosis are two mechanisms of action for Lp-THAE treatment of liver carcinoma. (authors)

  1. p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Shi-Wei [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Wu, Chun-Ying [Division of Gastroenterology and Hepatology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Wang, Yen-Ting [Department of Medical Research and Education, Cheng Hsin General Hospital, Taipei, Taiwan (China); Kao, Jun-Kai [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Department of Pediatrics, Children' s Hospital, Changhua Christian Hospital, Changhua, Taiwan (China); Lin, Chi-Chen; Chang, Chia-Che; Mu, Szu-Wei; Chen, Yu-Yu [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Chiu, Husan-Wen [Institute of Biotechnology, National Cheng-Kung University, Tainan, Taiwan (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan (China); Chang, Chuan-Hsun [Department of Surgical Oncology, Cheng Hsin General Hospital, Taipei, Taiwan (China); Department of Nutrition Therapy, Cheng Hsin General Hospital, Taipei, Taiwan (China); School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan (China); Liang, Shu-Mei [Institute of Biotechnology, National Cheng-Kung University, Tainan, Taiwan (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan (China); Chen, Yi-Ju [Department of Dermatology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Huang, Jau-Ling [Department of Bioscience Technology, Chang Jung Christian University, Tainan, Taiwan (China); Shieh, Jeng-Jer, E-mail: shiehjj@vghtc.gov.tw [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan (China)

    2013-02-15

    Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status. - Highlights: ► Compound C caused more apoptosis in p53 wildtype than p53-mutant skin cancer cells. ► Compound C can upregulate p53 expression and induce p53 activation. ► Compound C induced p53 effects were dependent on ROS induced DNA damage pathway. ► p53-knockdown attenuated compound C-induced apoptosis but not autophagy. ► Compound C-induced apoptosis in skin cancer cells was dependent on p53 status.

  2. p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells

    International Nuclear Information System (INIS)

    Huang, Shi-Wei; Wu, Chun-Ying; Wang, Yen-Ting; Kao, Jun-Kai; Lin, Chi-Chen; Chang, Chia-Che; Mu, Szu-Wei; Chen, Yu-Yu; Chiu, Husan-Wen; Chang, Chuan-Hsun; Liang, Shu-Mei; Chen, Yi-Ju; Huang, Jau-Ling; Shieh, Jeng-Jer

    2013-01-01

    Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status. - Highlights: ► Compound C caused more apoptosis in p53 wildtype than p53-mutant skin cancer cells. ► Compound C can upregulate p53 expression and induce p53 activation. ► Compound C induced p53 effects were dependent on ROS induced DNA damage pathway. ► p53-knockdown attenuated compound C-induced apoptosis but not autophagy. ► Compound C-induced apoptosis in skin cancer cells was dependent on p53 status

  3. The pathway of estradiol-induced apoptosis in patients with systemic lupus erythematosus.

    Science.gov (United States)

    Rastin, Maryam; Hatef, Mohammad Reza; Tabasi, Nafisseh; Mahmoudi, Mahmoud

    2012-03-01

    Systemic lupus erythematosus (SLE) is a disease with unknown etiology. The pathologic role of sex hormones and apoptosis in SLE has often been discussed. We studied the effects of estradiol in the pathway of induced apoptosis in Iranian SLE patients. T lymphocytes from 35 SLE patients and 20 age-matched controls were isolated and cultured in the presence of 10(-8) M 17-β estradiol. The expression levels of Fas, Fas ligand (FasL), Bcl-2, caspase-8, and caspase-9 mRNAs were determined semiquantitatively in comparison to the expression level of beta actin RNA. Estradiol exposure did not have any significant effects on the expression levels of Fas, Bcl-2, and caspase-9 in SLE patients and controls. However, the expression levels of FasL and caspase-8 were significantly increased in SLE patients, but not in controls. This suggests the probable involvement of extrinsic apoptosis pathway in estradiol-induced apoptosis in SLE.

  4. Taurine inhibits 2,5-hexanedione-induced oxidative stress and mitochondria-dependent apoptosis in PC12 cells.

    Science.gov (United States)

    Li, Shuangyue; Guan, Huai; Qian, Zhiqiang; Sun, Yijie; Gao, Chenxue; Li, Guixin; Yang, Yi; Piao, Fengyuan; Hu, Shuhai

    2017-04-07

    2,5-hexanedione (HD) is the ultimate neurotoxic metabolite of hexane, causing the progression of nerve diseases in human. It was reported that HD induced apoptosis and oxidative stress. Taurine has been shown to be a potent antioxidant. In the present study, we investigated the protection of taurine against HD-induced apoptosis in PC12 cells and the underlying mechanism. Our results showed the decreased viability and increased apoptosis in HD-exposed PC12 cells. HD also induced the disturbance of Bax and Bcl-2 expression, the loss of MMP, the release of mitochondrial cytochrome c and caspase-3 activation in PC12 cells. Moreover, HD resulted in an increase in reactive oxygen species (ROS) level and a decline in the activities of superoxidedismutase and catalase in PC12 cells. However, taurine pretreatment ameliorated the increased apoptosis and the alterations in key regulators of mitochondria-dependent pathway in PC12 exposed to HD. The increased ROS level and the decreased activities of the antioxidant enzymes in HD group were attenuated by taurine. These results indicate that pretreatment of taurine may, at least partly, prevent HD-induced apoptosis via inhibiting mitochondria-dependent pathway. It is also suggested that the potential of taurine against HD-induced apoptosis may benefit from its anti-oxidative property.

  5. Microcystin-LR Induces Apoptosis via NF-κB /iNOS Pathway in INS-1 Cells

    Directory of Open Access Journals (Sweden)

    Kai Shen

    2011-07-01

    Full Text Available Cyanobacterial toxins, especially the microcystins, are found in eutrophied waters throughout the world, and their potential to impact on human and animal health is a cause for concern. Microcystin-LR (MC-LR is one of the common toxic microcystin congeners and occurs frequently in diverse water systems. Recent work suggested that apoptosis plays a major role in the toxic effects induced by MC-LR in hepatocytes. However, the roles of MC-LR in pancreatic beta cells have not been fully established. The aim of the present study was to assess possible in vitro effects of MC-LR on cell apoptosis in the rat insulinoma cell line, INS-1. Our results demonstrated that MC-LR promoted selectively activation of NF-κB (increasing nuclear p50/p65 translocation and increased the mRNA and protein levels of induced nitric oxide synthase (iNOS. The chronic treatment with MC-LR stimulated nitric oxide (NO production derived from iNOS and induced apoptosis in a dose dependent manner in INS-1 cells. Meanwhile, this effect was inhibited by the NF-κB inhibitor PDTC, which reversed the apoptosis induced by MC-LR. Our observations indicate that MC-LR induced cell apoptosis via an iNOS-dependent pathway. A well-known nuclear transcription factor, NF-κB, is activated and mediates intracellular nitric oxide synthesis. We suggest that the apoptosis induced by chronic MC-LR in vivo presents a possible cause of β-cell dysfunction, as a key environmental factor in the development of diabetes mellitus.

  6. LyGDI expression in HeLa cells increased its sensitivity to radiation-induced apoptosis

    International Nuclear Information System (INIS)

    Zhou Xinwen; Xu Yaxiang

    2006-01-01

    Objective: In order to confirm whether LyGDI has apoptotic signal transduction function and can increase the apoptotic rate of radiation-induced cell death, the lyGDI and mutant D19lyGDI gene, which constructed with the pCDNA3. 1 His A, were transfected into no-endogenous lyGDI HeLa cells. Methods Transient expressions of lyGDI and D19lyGDI in HeLa cells were analyzed by Western blot using anti-mono antibody of LyGDI and Xpress tag. Cell apoptosis was assayed with Annexin V-FITC apoptosis kit. To select stable clone, the transferred HeLa cells had been maintained in G418 medium for 3 weeks, then a cell line, which stably expressed LyGDI and mutant D19lyGDI, was selected. The selected cell line was irradiated with 12 Gy 60 Co y-rays. Caspase-3 activity of the cells was determined by Western blot and cell viability by clone-forming assay after 48 hours post-irradiation culture. Results: Western blot and Annexin V-FITC apoptotic analysis revealed that lyGDI and D19lyGDI transient expressions in HeLa cells induced apoptosis; Caspase-3 activity measurement and clone-forming assay showed that lyGDI increased sensitivity to radiation-induced cell apoptosis. Conclusions: lyGDI performs function in apoptosis signal transduction, its expression in HeLa cells can increase the sensitivity to radiation-induced cell apoptosis. (authors)

  7. Chloroquinone Inhibits Cell Proliferation and Induces Apoptosis in ...

    African Journals Online (AJOL)

    Purpose: To demonstrate the role of chloroquinone (CQ) in inducing apoptosis in HONE-1 and HNE-1, nasopharyngeal carcinoma (NPC) cell lines. Methods: Water-soluble tetrazolium salt (WST)-1 assay was used for the determination of cell proliferation while an inverted microscope was employed for the analysis of ...

  8. Mechanisms of chromium (VI)-induced apoptosis in anterior pituitary cells.

    Science.gov (United States)

    Quinteros, Fernanda A; Machiavelli, Leticia I; Miler, Eliana A; Cabilla, Jimena P; Duvilanski, Beatriz H

    2008-07-30

    Hexavalent chromium (Cr (VI)) is a highly toxic metal. Exposure to Cr (VI) compounds may affect reproductive functions. Due to the importance of anterior pituitary hormones on reproductive physiology we have studied the effects of Cr (VI) on anterior pituitary. We previously demonstrated that, after in vivo Cr (VI) administration, Cr accumulates in the pituitary gland and affects prolactin secretion. In vitro, Cr (VI) causes apoptosis in anterior pituitary cells due to oxidative stress generation. To better understand the mechanisms involved in Cr (VI)-induced apoptosis we studied: (a) whether Cr (VI) affects the intracellular antioxidant response and (b) which of the apoptotic factors participates in Cr (VI) effect. Our results show that Cr (VI) treatment induces a decrease in catalase and glutathione peroxidase (GPx) activity but does not modify glutathione reductase (GR) activity. Cr (VI) exposure causes an increase of GSH levels. p53 and Bax mRNA are also upregulated by the metal. Pifithrin alpha, a p53 transcriptional inhibitor, increases Cr (VI) cytotoxicity, suggesting a role of p53 as a survival molecule. The antioxidant N-acetyl-cysteine (NAC) could prevent Bax mRNA increase and caspase 3 activation, confirming that Cr (VI)-induced apoptosis involves oxidative stress generation.

  9. 20(S-Protopanaxadiol-Induced Apoptosis in MCF-7 Breast Cancer Cell Line through the Inhibition of PI3K/AKT/mTOR Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Hong Zhang

    2018-04-01

    Full Text Available 20(S-Protopanaxadiol (PPD is one of the major active metabolites of ginseng. It has been reported that 20(S-PPD shows a broad spectrum of antitumor effects. Our research study aims were to investigate whether apoptosis of human breast cancer MCF-7 cells could be induced by 20(S-PPD by targeting the Phosphatidylinositol 3-kinase/Protein kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR signal pathway in vitro and in vivo. Cell cycle analysis was performed by Propidium Iodide (PI staining. To overexpress and knock down the expression of mTOR, pcDNA3.1-mTOR and mTOR small interfering RNA (siRNA transient transfection assays were used, respectively. Cell viability and apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT-test and Annexin V /PI double-staining after transfection. The antitumor effect in vivo was determined by the nude mice xenograft assay. After 24 h of incubation, treatment with 20(S-PPD could upregulate phosphorylated-Phosphatase and tensin homologue deleted on chromosome 10 (p-PTEN expression and downregulate PI3K/AKT/mTOR-pathway protein expression. Moreover, G0/G1 cell cycle arrest in MCF-7 cells could be induced by 20(S-PPD treatment at high concentrations. Furthermore, overexpression or knockdown of mTOR could inhibit or promote the apoptotic effects of 20(S-PPD. In addition, tumor volumes were partially reduced by 20(S-PPD at 100 mg/kg in a MCF-7 xenograft model. Immunohistochemical staining indicated a close relationship between the inhibition of tumor growth and the PI3K/AKT/mTOR signal pathway. PI3K/AKT/mTOR pathway-mediated apoptosis may be one of the potential mechanisms of 20(S-PPD treatment.

  10. Iodinated contrast media induce neutrophil apoptosis through a mitochondrial and caspase mediated pathway.

    LENUS (Irish Health Repository)

    Fanning, N F

    2012-02-03

    Iodinated contrast media (ICM) can induce apoptosis (programmed cell death) in renal, myocardial and endothelial cells. Following intravascular injection, circulating immune cells are exposed to high concentrations of ICM. As neutrophils constitutively undergo apoptosis we hypothesized that ICM may adversely affect neutrophil survival. Our aim was to investigate the effect of ICM on neutrophil apoptosis. Neutrophils were isolated from healthy subjects and cultured in vitro with ionic (diatrizoate and ioxaglate) and non-ionic (iohexol and iotrolan) ICM. The effect of ICM on neutrophil apoptosis in both unstimulated and lipopolysaccharide-stimulated neutrophils was determined by annexin V flow cytometry. The influence of physicochemical properties of the different ICM on apoptosis of neutrophils was also studied. We further investigated the effects of ICM on key intracellular signal pathways, including p38 mitogen-activated protein kinase (MAPK) by Western blotting, and mitochondrial depolarization and caspase activity by flow cytometry. Isoiodine concentrations (20 mg ml(-1)) of ionic (diatrizoate 69.6+\\/-2.9%; ioxaglate 58.9+\\/-2.0%) and non-ionic (iohexol 57.3+\\/-2.9%; iotrolan 57.1+\\/-2.6%) ICM significantly induced neutrophil apoptosis over control levels (47.7+\\/-1.4%). The apoptotic effect of ICM was influenced by their chemical structure, with ionic ICM having a more significant (p<0.01) apoptotic effect than non-ionic ICM (p<0.05). Furthermore, ICM reversed the anti-apoptotic effect of lipopolysaccharide (1000 ng ml(-1)) treated neutrophils to control levels (23.0+\\/-3.5% to 61.2+\\/-5.3%; n=4; p<0.05). These agents induce apoptosis through a p38 MAPK independent pathway that results in mitochondrial depolarization, and is dependent on caspase activation. As neutrophils play a central role in host response to infection and injury, ICM, through induction of neutrophil apoptosis, could have a significant deleterious effect on host immune defence and

  11. The Bcl-2-Beclin 1 interaction in (-)-gossypol-induced autophagy versus apoptosis in prostate cancer cells.

    Science.gov (United States)

    Lian, Jiqin; Karnak, David; Xu, Liang

    2010-11-01

    Bcl-2 is a key dual regulator of autophagy and apoptosis, but how the level of Bcl-2 influences the cellular decision between autophagy and apoptosis is unclear. The natural BH3-mimetic (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, whereas apoptosis is preferentially induced in androgen-dependent or -independent cells with low Bcl-2. (-)-Gossypol induces autophagy via blocking Bcl-2-Beclin 1 interaction at the endoplasmic reticulum (ER), together with downregulating Bcl-2, upregulating Beclin 1 and activating the autophagic pathway. Furthermore, (-)-gossypol-induced autophagy is Beclin 1- and Atg5-dependent. These results provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which could facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.

  12. Ku70 inhibits gemcitabine-induced DNA damage and pancreatic cancer cell apoptosis

    International Nuclear Information System (INIS)

    Ma, Jiali; Hui, Pingping; Meng, Wenying; Wang, Na; Xiang, Shihao

    2017-01-01

    The current study focused on the role of Ku70, a DNA-dependent protein kinase (DNA-PK) complex protein, in pancreatic cancer cell resistance to gemcitabine. In both established cell lines (Mia-PaCa-2 and PANC-1) and primary human pancreatic cancer cells, shRNA/siRNA-mediated knockdown of Ku70 significantly sensitized gemcitabine-induced cell death and proliferation inhibition. Meanwhile, gemcitabine-induced DNA damage and subsequent pancreatic cancer cell apoptosis were also potentiated with Ku70 knockdown. On the other hand, exogenous overexpression of Ku70 in Mia-PaCa-2 cells suppressed gemcitabine-induced DNA damage and subsequent cell apoptosis. In a severe combined immune deficient (SCID) mice Mia-PaCa-2 xenograft model, gemcitabine-induced anti-tumor activity was remarkably pontificated when combined with Ku70 shRNA knockdown in the xenografts. The results of this preclinical study imply that Ku70 might be a primary resistance factor of gemcitabine, and Ku70 silence could significantly chemo-sensitize gemcitabine in pancreatic cancer cells. - Highlights: • Ku70 knockdown sensitizes gemcitabine-induced killing of pancreatic cancer cells. • Ku70 knockdown facilitates gemcitabine-induced DNA damage and cell apoptosis. • Ku70 overexpression deceases gemcitabine's sensitivity in pancreatic cancer cells. • Ku70 knockdown sensitizes gemcitabine-induced anti-tumor activity in vivo.

  13. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    International Nuclear Information System (INIS)

    Bhattacharjee, Rajesh; Xiang, Wenpei; Wang, Yinna; Zhang, Xiaoying; Billiar, Timothy R.

    2012-01-01

    Highlights: ► cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. ► cAMP blocks NF-κB activation induced by TNF and actinomycin D. ► cAMP blocks DISC formation following TNF and actinomycin D exposure. ► cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor α (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found that cAMP exerts its affect at the proximal level of TNF signaling by inhibiting the formation of the DISC

  14. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Rajesh [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Xiang, Wenpei [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People' s Republic of China (China); Wang, Yinna [Vascular Medicine Institute, University of Pittsburgh School of Medicine, 10051-5A BST 3, 3501 Fifth Avenue, Pittsburgh, PA 15261 (United States); Zhang, Xiaoying [Department of Medicine/Endocrinology Division, University of Pittsburgh Medical Center, 200 Lothrop St., Pittsburgh, PA 15213 (United States); Billiar, Timothy R., E-mail: billiartr@upmc.edu [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found

  15. Linear ubiquitin chain induces apoptosis and inhibits tumor growth.

    Science.gov (United States)

    Qin, Zhoushuai; Jiang, Wandong; Wang, Guifen; Sun, Ying; Xiao, Wei

    2018-01-01

    Ubiquitination of proliferating cell nuclear antigen (PCNA) plays an important role in DNA damage response. Ectopic expression of PCNA fused at either terminus with ubiquitin (Ub) lacking two C-terminal glycine residues induces translesion DNA synthesis which resembles synthesis mediated by PCNA monoubiquitination. PCNA fused with Ub containing the C-terminal Gly residues at the C-terminus can be further polyubiquitinated in a Gly-dependent manner, which inhibits cell proliferation and induces ATR-dependent replication checkpoint. In this study, we surprisingly found that PCNA fused to a head-to-tail linear Ub chain induces apoptosis in a Ub chain length-dependent manner. Further investigation revealed that the apoptotic effect is actually induced by the linear Ub chain independently from PCNA, as the Ub chain fused to GFP or an epitope tag still efficiently induces apoptosis. It is revealed that the artificial linear Ub chain differs from endogenously encoded linear Ub chains in that its Ubs contain a Ub-G76S substitution, making the Ub chain resistant to cleavage by deubiquitination enzymes. We demonstrated in this study that ectopic expression of the artificial Ub chain alone in cultured human cancer cells is sufficient to inhibit tumor growth in a xenograft mouse model, making the linear Ub chain a putative anti-cancer agent.

  16. VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

    Energy Technology Data Exchange (ETDEWEB)

    Qian, Qinyi [Department of Ultrasonograph, Changshu No. 2 People’s Hospital, Changshu (China); Zhou, Hao; Chen, Yan [Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou (China); Shen, Chenglong [Department of General Surgery, Changshu No. 2 People’s Hospital, Changshu (China); He, Songbing; Zhao, Hua; Wang, Liang [Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou (China); Wan, Daiwei, E-mail: 372710369@qq.com [Department of Hepatobiliary Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou (China); Gu, Wen, E-mail: 505339704@qq.com [Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou (China)

    2014-01-17

    Highlights: •This research confirmed VMP1 as a regulator of autophagy in colorectal cancer cell lines. •We proved the pro-survival role of VMP1-mediated autophagy in colorectal cancer cell lines. •We found the interaction between VMP1 and BECLIN1 also existing in colorectal cancer cell lines. -- Abstract: Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.

  17. Essential roles of caspases and their upstream regulators in rotenone-induced apoptosis

    International Nuclear Information System (INIS)

    Lee Jihjong; Huang, M.-S.; Yang, I-C.; Lai, T.-C.; Wang, J.-L.; Pang, V.F.; Hsiao, M.; Kuo, M.Y.P.

    2008-01-01

    In the present study, we examined whether caspases and their upstream regulators are involved in rotenone-induced cytotoxicity. Rotenone significantly inhibited the proliferation of oral cancer cell lines in a dose-dependent manner compared to normal oral mucosal fibroblasts. Flow cytometric analysis of DNA content showed that rotenone treatment induced apoptosis following G2/M arrest. Western blotting showed activation of both the caspase-8 and caspase-9 pathways, which differed from previous studies conducted in other cell types. Furthermore, p53 protein and its downstream pro-apoptotic target, Bax, were induced in SAS cells after treatment with rotenone. Rotenone-induced apoptosis was inhibited by antioxidants (glutathione, N-acetylcysteine, and tiron). In conclusion, our results demonstrate significant involvement of caspases and their upstream regulators in rotenone-induced cytotoxicity

  18. Insights into the therapeutic potential of hypoxia-inducible factor-1α small interfering RNA in malignant melanoma delivered via folate-decorated cationic liposomes

    Directory of Open Access Journals (Sweden)

    Chen Z

    2016-03-01

    Full Text Available Zhongjian Chen,1,* Tianpeng Zhang,2,* Baojian Wu,2 Xingwang Zhang2 1Department of Pharmaceutics, Shanghai Dermatology Hospital, 2Division of Pharmaceutics, College of Pharmacy, Jinan University, Gangzhou, People’s Republic of China *These authors contributed equally to this work Abstract: Malignant melanoma (MM represents the most dangerous form of skin cancer, and its incidence is expected to rise in the coming time. However, therapy for MM is limited by low topical drug concentration and multidrug resistance. This article aimed to develop folate-decorated cationic liposomes (fc-LPs for hypoxia-inducible factor-1α (HIF-1α small interfering (siRNA delivery, and to evaluate the potential of such siRNA/liposome complexes in MM therapy. HIF-1α siRNA-loaded fc-LPs (siRNA-fc-LPs were prepared by a film hydration method followed by siRNA incubation. Folate decoration of liposomes was achieved by incorporation of folate/oleic acid-diacylated oligochitosans. The resulting siRNA-fc-LPs were 95.3 nm in size with a ζ potential of 2.41 mV. The liposomal vectors exhibited excellent loading capacity and protective effect toward siRNA. The in vitro cell transfection efficiency was almost parallel to the commercially available Lipofectamine™ 2000. Moreover, the anti-melanoma activity of HIF-1α siRNA was significantly enhanced through fc-LPs. Western blot analysis and apoptosis test demonstrated that siRNA-fc-LPs substantially reduced the production of HIF-1α-associated protein and induced the apoptosis of hypoxia-tolerant melanoma cells. Our designed liposomal vectors might be applicable as siRNA delivery vehicle to systemically or topically treat MM. Keywords: malignant melanoma, HIF-1α siRNA, chitosan, cationic liposomes, gene therapy

  19. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province (China); Dong, Wei-Guo, E-mail: dongwg1966@yahoo.com.cn [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province (China)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. Black-Right-Pointing-Pointer G{sub 2}/M phase arrest and chromatin condensation and nuclear fragmentation were induced. Black-Right-Pointing-Pointer Noscapine promoted apoptosis via mitochondrial pathways. Black-Right-Pointing-Pointer Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC{sub 50} = 75 {mu}M). This cytotoxicity was reflected by cell cycle arrest at G{sub 2}/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  20. Fem1b, a proapoptotic protein, mediates proteasome inhibitor-induced apoptosis of human colon cancer cells.

    Science.gov (United States)

    Subauste, M Cecilia; Sansom, Owen J; Porecha, Nehal; Raich, Natacha; Du, Liqin; Maher, Joseph F

    2010-02-01

    In the treatment of colon cancer, the development of resistance to apoptosis is a major factor in resistance to therapy. New molecular approaches to overcome apoptosis resistance, such as selectively upregulating proapoptotic proteins, are needed in colon cancer therapy. In a mouse model with inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene, reflecting the pathogenesis of most human colon cancers, the gene encoding feminization-1 homolog b (Fem1b) is upregulated in intestinal epithelium following Apc inactivation. Fem1b is a proapoptotic protein that interacts with apoptosis-inducing proteins Fas, tumor necrosis factor receptor-1 (TNFR1), and apoptotic protease activating factor-1 (Apaf-1). Increasing Fem1b expression induces apoptosis of cancer cells, but effects on colon cancer cells have not been reported. Fem1b is a homolog of feminization-1 (FEM-1), a protein in Caenorhabditis elegans that is regulated by proteasomal degradation, but whether Fem1b is likewise regulated by proteasomal degradation is unknown. Herein, we found that Fem1b protein is expressed in primary human colon cancer specimens, and in malignant SW620, HCT-116, and DLD-1 colon cancer cells. Increasing Fem1b expression, by transfection of a Fem1b expression construct, induced apoptosis of these cells. We found that proteasome inhibitor treatment of SW620, HCT-116, and DLD-1 cells caused upregulation of Fem1b protein levels, associated with induction of apoptosis. Blockade of Fem1b upregulation with morpholino antisense oligonucleotide suppressed the proteasome inhibitor-induced apoptosis of these cells. In conclusion, the proapoptotic protein Fem1b is downregulated by the proteasome in malignant colon cancer cells and mediates proteasome inhibitor-induced apoptosis of these cells. Therefore, Fem1b could represent a novel molecular target to overcome apoptosis resistance in therapy of colon cancer.

  1. Capsaicin sensitizes TRAIL-induced apoptosis through Sp1-mediated DR5 up-regulation: Involvement of Ca2+ influx

    International Nuclear Information System (INIS)

    Moon, Dong-Oh; Kang, Chang-Hee; Kang, Sang-Hyuck; Choi, Yung-Hyun; Hyun, Jin-Won; Chang, Weon-Young; Kang, Hee-Kyoung; Koh, Young-Sang; Maeng, Young-Hee; Kim, Young-Ree; Kim, Gi-Young

    2012-01-01

    Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various malignant cells, several cancers including human hepatocellular carcinoma (HCC) exhibit potent resistance to TRAIL-induced cell death. The aim of this study is to evaluate the anti-cancer potential of capsaicin in TRAIL-induced cancer cell death. As indicated by assays that measure phosphatidylserine exposure, mitochondrial activity and activation of caspases, capsaicin potentiated TRAIL-resistant cells to lead to cell death. In addition, we found that capsaicin induces the cell surface expression of TRAIL receptor DR5, but not DR4 through the activation Sp1 on its promoter region. Furthermore, we investigated that capsaicin-induced DR5 expression and apoptosis are inhibited by calcium chelator or inhibitors for calmodulin-dependent protein kinase. Taken together, our data suggest that capsaicin sensitizes TRAIL-mediated HCC cell apoptosis by DR5 up-regulation via calcium influx-dependent Sp1 activation. Highlights: ► Capsaicin sensitizes TRAIL-induced apoptosis through activation of caspases. ► Capsaicin induces expression of DR5 through Sp1 activation. ► Capsaicin activates calcium signaling pathway.

  2. α-Hispanolol sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis via death receptor up-regulation

    Energy Technology Data Exchange (ETDEWEB)

    Mota, Alba, E-mail: amota@iib.uam.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain); Jiménez-Garcia, Lidia, E-mail: ljimenez@isciii.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain); Herránz, Sandra, E-mail: sherranz@isciii.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain); Heras, Beatriz de las, E-mail: lasheras@ucm.es [Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense de Madrid (UCM), Madrid (Spain); Hortelano, Sonsoles, E-mail: shortelano@isciii.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain)

    2015-08-01

    Hispanolone derivatives have been previously described as anti-inflammatory and antitumoral agents. However, their effects on overcoming Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance remain to be elucidated. In this study, we analyzed the cytotoxic effects of the synthetic hispanolone derivative α-hispanolol (α-H) in several tumor cell lines, and we evaluated the induction of apoptosis, as well as the TRAIL-sensitizing potential of α-H in the hepatocellular carcinoma cell line HepG2. Our data show that α-H decreased cell viability in a dose-dependent manner in HeLa, MDA-MB231, U87 and HepG2 cell lines, with a more prominent effect in HepG2 cells. Interestingly, α-H had no effect on non-tumoral cells. α-H induced activation of caspase-8 and caspase-9 and also increased levels of the proapoptotic protein Bax, decreasing antiapoptotic proteins (Bcl-2, X-IAP and IAP-1) in HepG2 cells. Specific inhibition of caspase-8 abrogated the cascade of caspase activation, suggesting that the extrinsic pathway has a critical role in the apoptotic events induced by α-H. Furthermore, combined treatment of α-H with TRAIL enhanced apoptosis in HepG2 cells, activating caspase-8 and caspase-9. This correlated with up-regulation of both the TRAIL death receptor DR4 and DR5. DR4 or DR5 neutralizing antibodies abolished the effect of α-H on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the death receptor pathway. Our results demonstrate that α-H induced apoptosis in the human hepatocellular carcinoma cell line HepG2 through activation of caspases and induction of the death receptor pathway. In addition, we describe a novel function of α-H as a sensitizer on TRAIL-induced apoptotic cell death in HepG2 cells. - Highlights: • α-Hispanolol induced apoptosis in the human hepatocellular carcinoma cell line HepG2. • α-Hispanolol induced activation of caspases and the death receptor pathway. • α-Hispanolol enhanced

  3. α-Hispanolol sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis via death receptor up-regulation

    International Nuclear Information System (INIS)

    Mota, Alba; Jiménez-Garcia, Lidia; Herránz, Sandra; Heras, Beatriz de las; Hortelano, Sonsoles

    2015-01-01

    Hispanolone derivatives have been previously described as anti-inflammatory and antitumoral agents. However, their effects on overcoming Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance remain to be elucidated. In this study, we analyzed the cytotoxic effects of the synthetic hispanolone derivative α-hispanolol (α-H) in several tumor cell lines, and we evaluated the induction of apoptosis, as well as the TRAIL-sensitizing potential of α-H in the hepatocellular carcinoma cell line HepG2. Our data show that α-H decreased cell viability in a dose-dependent manner in HeLa, MDA-MB231, U87 and HepG2 cell lines, with a more prominent effect in HepG2 cells. Interestingly, α-H had no effect on non-tumoral cells. α-H induced activation of caspase-8 and caspase-9 and also increased levels of the proapoptotic protein Bax, decreasing antiapoptotic proteins (Bcl-2, X-IAP and IAP-1) in HepG2 cells. Specific inhibition of caspase-8 abrogated the cascade of caspase activation, suggesting that the extrinsic pathway has a critical role in the apoptotic events induced by α-H. Furthermore, combined treatment of α-H with TRAIL enhanced apoptosis in HepG2 cells, activating caspase-8 and caspase-9. This correlated with up-regulation of both the TRAIL death receptor DR4 and DR5. DR4 or DR5 neutralizing antibodies abolished the effect of α-H on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the death receptor pathway. Our results demonstrate that α-H induced apoptosis in the human hepatocellular carcinoma cell line HepG2 through activation of caspases and induction of the death receptor pathway. In addition, we describe a novel function of α-H as a sensitizer on TRAIL-induced apoptotic cell death in HepG2 cells. - Highlights: • α-Hispanolol induced apoptosis in the human hepatocellular carcinoma cell line HepG2. • α-Hispanolol induced activation of caspases and the death receptor pathway. • α-Hispanolol enhanced

  4. Portulaca oleracea extracts protect human keratinocytes and fibroblasts from UV-induced apoptosis.

    Science.gov (United States)

    Lee, Suyeon; Kim, Ki Ho; Park, Changhoon; Lee, Jong-Suk; Kim, Young Heui

    2014-10-01

    Portulaca oleracea extracts, known as Ma Chi Hyun in the traditional Korean medicine, show a variety of biomedical efficacies including those in anti-inflammation and anti-allergy. In this study, we investigate the protective activity of the P. oleracea extracts against UVB-induced damage in human epithelial keratinocytes and fibroblasts by several apoptosis-related tests. The results suggest that P. oleracea extracts have protective effects from UVB-induced apoptosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Nano Copper Induces Apoptosis in PK-15 Cells via a Mitochondria-Mediated Pathway.

    Science.gov (United States)

    Zhang, Hui; Chang, Zhenyu; Mehmood, Khalid; Abbas, Rao Zahid; Nabi, Fazul; Rehman, Mujeeb Ur; Wu, Xiaoxing; Tian, Xinxin; Yuan, Xiaodan; Li, Zhaoyang; Zhou, Donghai

    2018-01-01

    Nano-sized copper particles are widely used in various chemical, physical, and biological fields. However, earlier studies have shown that nano copper particles (40-100 μg/mL) can induce cell toxicity and apoptosis. Therefore, this study was conducted to investigate the role of nano copper in mitochondrion-mediated apoptosis in PK-15 cells. The cells were treated with different doses of nano copper (20, 40, 60, and 80 μg/mL) to determine the effects of apoptosis using acridine orange/ethidium bromide (AO/EB) fluorescence staining and a flow cytometry assay. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in the PK-15 cells were examined using commercially available kits. Moreover, the mRNA levels of the Bax, Bid, Caspase-3, and CYCS genes were assessed by real-time PCR. The results revealed that nano copper exposure induced apoptosis and changed the mitochondrial membrane potential. In addition, nano copper significantly altered the levels of the Bax, Bid, Caspase-3, and CYCS genes at a concentration of 40 μg/mL. To summarize, nano copper significantly (P nano copper can play an important role in inducing the apoptotic pathway in PK-15 cells, which may be the mechanism by which nano copper induces nephrotoxicity.

  6. Pathway of 3-MCPD-induced apoptosis in human embryonic kidney cells.

    Science.gov (United States)

    Ji, Jian; Zhu, Pei; Sun, Chao; Sun, Jiadi; An, Lu; Zhang, Yinzhi; Sun, Xiulan

    2017-01-01

    3-Chloropropane-1,2-diol (3-MCPD) is a heat-produced contaminant formed during the preparation of soy sauce worldwide. The present investigation was conducted to determine the molecular aspects of 3-MCPD toxicity on human embryonic kidney cells (HEK293). Cell viability and apoptosis were assessed in response to exposure to 3-MCPD using the MTT assay and high-content screening (HCS). DNA damage, intracellular reactive oxygen species (ROS) and apoptosis-related proteins were evaluated. Genes related with apoptosis were detected by qPCR-array for further understanding the 3-MCPD induced cell apoptosis signaling pathway. Our results clearly showed that 3-MCPD treatment inhibits cell proliferation and reactive oxygen species generation. qPCR-array indicated that nine apoptotic genes were up-regulated more than 2-fold and six down-regulated more than 2-fold. Genes associated with the mitochondrial apoptotic pathway, especially BCL2 family genes, changed significantly, indicating that the mitochondrial apoptotic pathway is activated. Death receptor pathway-related genes, TNFRSF11B and TNFRSF1A, changed significantly, indicating that the death receptor pathway is also activated, resulting in the inhibition of cell growth and proliferation as well as induction of apoptosis. To sum up, the experiment results indicated that 3-MCPD induced HEK293 cell toxicity through the death receptor pathway and mitochondrial pathway.

  7. Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis.

    Science.gov (United States)

    Parra, Valentina; Eisner, Veronica; Chiong, Mario; Criollo, Alfredo; Moraga, Francisco; Garcia, Alejandra; Härtel, Steffen; Jaimovich, Enrique; Zorzano, Antonio; Hidalgo, Cecilia; Lavandero, Sergio

    2008-01-15

    In cells, mitochondria are organized as a network of interconnected organelles that fluctuate between fission and fusion events (mitochondrial dynamics). This process is associated with cell death. We investigated whether activation of apoptosis with ceramides affects mitochondrial dynamics and promotes mitochondrial fission in cardiomyocytes. Neonatal rat cardiomyocytes were incubated with C(2)-ceramide or the inactive analog dihydro-C(2)-ceramide for up to 6 h. Three-dimensional images of cells loaded with mitotracker green were obtained by confocal microscopy. Dynamin-related protein-1 (Drp-1) and mitochondrial fission protein 1 (Fis1) distribution and levels were studied by immunofluorescence and western blot. Mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c (cyt c) distribution were used as indexes of early activation of apoptosis. Cell viability and DNA fragmentation were determined by propidium iodide staining/flow cytometry, whereas cytotoxicity was evaluated by lactic dehydrogenase activity. To decrease the levels of the mitochondrial fusion protein mitofusin 2, we used an antisense adenovirus (AsMfn2). C(2)-ceramide, but not dihydro-C(2)-ceramide, promoted rapid fragmentation of the mitochondrial network in a concentration- and time-dependent manner. C(2)-ceramide also increased mitochondrial Drp-1 and Fis1 content, Drp-1 colocalization with Fis1, and caused early activation of apoptosis. AsMfn2 accentuated the decrease in DeltaPsi(m) and cyt c redistribution induced by C(2)-ceramide. Doxorubicin, which induces cardiomyopathy and apoptosis through ceramide generation, also stimulated mitochondrial fragmentation. Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.

  8. Curcumin protects neurons against oxygen-glucose deprivation/reoxygenation-induced injury through activation of peroxisome proliferator-activated receptor-γ function.

    Science.gov (United States)

    Liu, Zun-Jing; Liu, Hong-Qiang; Xiao, Cheng; Fan, Hui-Zhen; Huang, Qing; Liu, Yun-Hai; Wang, Yu

    2014-11-01

    The turmeric derivative curcumin protects against cerebral ischemic injury. We previously demonstrated that curcumin activates peroxisome proliferator-activated receptor-γ (PPARγ), a ligand-activated transcription factor involved in both neuroprotective and anti-inflammatory signaling pathways. This study tested whether the neuroprotective effects of curcumin against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury of rat cortical neurons are mediated (at least in part) by PPARγ. Curcumin (10 μM) potently enhanced PPARγ expression and transcriptional activity following OGD/R. In addition, curcumin markedly increased neuronal viability, as evidenced by decreased lactate dehydrogenase release and reduced nitric oxide production, caspase-3 activity, and apoptosis. These protective effects were suppressed by coadministration of the PPARγ antagonist 2-chloro-5-nitrobenzanilide (GW9662) and by prior transfection of a small-interfering RNA (siRNA) targeting PPARγ, treatments that had no toxic effects on healthy neurons. Curcumin reduced OGD/R-induced accumulation of reactive oxygen species and inhibited the mitochondrial apoptosis pathway, as indicated by reduced release of cytochrome c and apoptosis-inducing factor and maintenance of both the mitochondrial membrane potential and the Bax/Bcl-2 ratio. Again, GW9662 or PPARγ siRNA transfection mitigated the protective effects of curcumin on mitochondrial function. Curcumin suppressed IκB kinase phosphorylation and IκB degradation, thereby inhibiting nuclear factor-κ B (NF-κB) nuclear translocation, effects also blocked by GW9662 or PPARγ siRNA. Immunoprecipitation experiments revealed that PPARγ interacted with NF-κB p65 and inhibited NF-κB activation. The present study provides strong evidence that at least some of the neuroprotective effects of curcumin against OGD/R are mediated by PPARγ activation. Copyright © 2014 Wiley Periodicals, Inc.

  9. PI3K inhibition enhances doxorubicin-induced apoptosis in sarcoma cells.

    Directory of Open Access Journals (Sweden)

    Diana Marklein

    Full Text Available We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX. We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.

  10. Taurine Protects Lens Epithelial Cells Against Ultraviolet B-Induced Apoptosis.

    Science.gov (United States)

    Dayang, Wu; Dongbo, Pang

    2017-10-01

    The massive uptake of compatible osmolytes is a self-protective response shared by lens exposed to hypertonic stress and ultraviolet stress. This study aimed to investigate the protective effects of taurine against ultraviolet B-induced cytotoxicity in the lens epithelial cells. Real-time PCR was used to measure osmolytes transport. Radioimmunoassay was used to measure osmolytes uptake. Cell counting kit-8 assays were used to measure cellular viability. Flow cytometry analysis was used to measure apoptosis level. Compared with normotonic stress, hypertonic stress-induced osmolytes uptake into the lens epithelial cells such as betaine, myoinositol and taurine. UVB exposure increased osmolytes transporter mRNA expression together with osmolytes uptake. Moreover, taurine suppressed UVB-induced cell apoptosis in the lens epithelial cells significantly. The effect of compatible osmolyte taurine on cell survival rate may play an important role in cell resistance and adaption to UVB exposure.

  11. Acetyl-CoA Carboxylase-α Inhibitor TOFA Induces Human Cancer Cell Apoptosis

    Science.gov (United States)

    Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-fang; Cao, Deliang

    2009-01-01

    Acetyl-CoA carboxylase-α (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC50 at approximately 5.0, 5.0, and 4.5 μg/ml, respectively. TOFA at 1.0–20.0 μg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 μM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis. PMID:19450551

  12. Acetyl-CoA carboxylase-alpha inhibitor TOFA induces human cancer cell apoptosis.

    Science.gov (United States)

    Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-Fang; Cao, Deliang

    2009-07-31

    Acetyl-CoA carboxylase-alpha (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC(50) at approximately 5.0, 5.0, and 4.5 microg/ml, respectively. TOFA at 1.0-20.0 microg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 microM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis.

  13. Extremely Low Frequency Magnetic Fields Induce Spermatogenic Germ Cell Apoptosis: Possible Mechanism

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    Sang-Kon Lee

    2014-01-01

    Full Text Available The energy generated by an extremely low frequency electromagnetic field (ELF-EMF is too weak to directly induce genotoxicity. However, it is reported that an extremely low frequency magnetic field (ELF-MF is related to DNA strand breakage and apoptosis. The testes that conduct spermatogenesis through a dynamic cellular process involving meiosis and mitosis seem vulnerable to external stress such as heat, MF exposure, and chemical or physical agents. Nevertheless the results regarding adverse effects of ELF-EMF on human or animal reproductive functions are inconclusive. According to the guideline of the International Commission on Non-Ionizing Radiation Protection (ICNIRP; 2010 for limiting exposure to time-varying MF (1 Hz to 100 kHz, overall conclusion of epidemiologic studies has not consistently shown an association between human adverse reproductive outcomes and maternal or paternal exposure to low frequency fields. In animal studies there is no compelling evidence of causal relationship between prenatal development and ELF-MF exposure. However there is increasing evidence that EL-EMF exposure is involved with germ cell apoptosis in testes. Biophysical mechanism by which ELF-MF induces germ cell apoptosis has not been established. This review proposes the possible mechanism of germ cell apoptosis in testes induced by ELF-MF.

  14. Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes

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    Xu, Jiqian; Hu, Houxiang; Chen, Bin; Yue, Rongchuan; Zhou, Zhou; Liu, Yin; Zhang, Shuang; Xu, Lei; Wang, Huan; Yu, Zhengping

    2015-01-01

    Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes. PMID:26291709

  15. Apoptosis induced by knockdown of uPAR and MMP-9 is mediated by inactivation of EGFR/STAT3 signaling in medulloblastoma.

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    Ramaprasada Rao Kotipatruni

    Full Text Available Medulloblastoma is a highly invasive cancer of central nervous system diagnosed mainly in children. Matrix metalloproteinase-9 (MMP-9 and urokinase plasminogen activator receptor (uPAR are over expressed in several cancers and well established for their roles in tumor progression. The present study is aimed to determine the consequences of targeting these molecules on medulloblastoma progression.Radiation is one of the foremost methods applied for treating cancer and considerable evidence showed that radiation elevated uPAR and MMP-9 expression in medulloblastoma cell. Therefore efforts are made to target these molecules in non-irradiated and irradiated medulloblastoma cells. Our results showed that siRNA-mediated knockdown of uPAR and MMP-9, either alone or in combination with radiation modulated a series of events leading to apoptosis. Down regulation of uPAR and MMP-9 inhibited the expression of anti-apoptotic molecules like Bcl-2, Bcl-xL, survivin, XIAP and cIAPI; activated BID cleavage, enhanced the expression of Bak and translocated cyctochrome C to cytosol. Capsase-3 and -9 activities were also increased in uPAR- and MMP-9-downregulated cells. The apoptosis induced by targeting MMP-9 and uPAR was initiated by inhibiting epidermal growth factor receptor (EGFR mediated activation of STAT3 and NF-κB related signaling molecules. Silencing uPAR and MMP-9 inhibited DNA binding activity of STAT3 and also reduced the recruitment of STAT3 protein at the promoter region of Bcl-2 and survivin genes. Our results suggest that inhibiting uPAR and MMP-9 reduced the expression of anti-apoptotic molecules by inactivating the transcriptional activity of STAT3. In addition, treating pre-established medulloblastoma with siRNAs against uPAR and MMP-9 both alone or in combination with radiation suppressed uPAR, MMP-9, EGFR, STAT3 expression and induced Bak activation leading to apoptosis.Taken together, our results illustrated that RNAi mediated targeting of

  16. SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis

    International Nuclear Information System (INIS)

    Srisuttee, Ratakorn; Koh, Sang Seok; Malilas, Waraporn; Moon, Jeong; Cho, Il-Rae; Jhun, Byung Hak; Horio, Yoshiyuki; Chung, Young-Hwa

    2012-01-01

    Highlights: ► Up-regulation of SIRT1 protein and activity sensitizes Hep3B-HBX cells to oxidative stress-induced apoptosis. ► Nuclear localization of SIRT1 is not required for oxidation-induced apoptosis. ► Ectopic expression and enhanced activity of SIRT1 attenuate JNK phosphorylation. ► Inhibition of SIRT1 activity restores resistance to oxidation-induced apoptosis through JNK activation. -- Abstract: We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of β-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.

  17. SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Srisuttee, Ratakorn [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Koh, Sang Seok [Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology, Daejeon 305-333 (Korea, Republic of); Department of Functional Genomics, University of Science and Technology, Daejeon 305-333 (Korea, Republic of); Malilas, Waraporn; Moon, Jeong; Cho, Il-Rae [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Jhun, Byung Hak [Department of Applied Nanoscience, Pusan National University, Busan 609-735 (Korea, Republic of); Horio, Yoshiyuki [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Chung, Young-Hwa, E-mail: younghc@pusan.ac.kr [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer Up-regulation of SIRT1 protein and activity sensitizes Hep3B-HBX cells to oxidative stress-induced apoptosis. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for oxidation-induced apoptosis. Black-Right-Pointing-Pointer Ectopic expression and enhanced activity of SIRT1 attenuate JNK phosphorylation. Black-Right-Pointing-Pointer Inhibition of SIRT1 activity restores resistance to oxidation-induced apoptosis through JNK activation. -- Abstract: We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of {beta}-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.

  18. [Knock-down of BCL11A expression in breast cancer cells promotes MDA-MB-231 cell apoptosis].

    Science.gov (United States)

    Li, Hongli; Gui, Chen; Yan, Lijun

    2016-11-01

    Objective To detect the expression and pathological significance of B-cell CLL/lymphoma 11A (BCL11A) in breast cancer and investigate the effect of its silencing on the apoptosis of human MDA-MB-231 breast cancer cells. MethodsImmunohistochemistry was used to detect the expression of BCL11A in 62 cases of human breast cancer tissues and 8 cases of normal tissues. We synthesized siRNA targeting BCL11A, and then siRNA was transfected into MDA-MB-231 cells. Forty-eight hours later, the suppression effect of siRNA on BCL11A was determined by quantitative real-time PCR and Western blotting. The apoptosis of MDA-MB-231 cells was detected by flow cytometry. Results The BCL11A protein was mainly expressed in cytoplasm. The expression level of BCL11A in breast cancer tissues was higher than that in paracancerous tissues. The expression had correlations with tumor grade, tumor stage, while it had no correlations with the patients' age and tumor size. BCL11A-siRNA significantly suppressed the expression of BCL11A mRNA and protein as compared with the control group. MDA-MB-231 cells transfected with BCL11A-siRNA had higher apoptosis rate compared with the control group. Conclusion The BCL11A protein is highly expressed in breast cancer and knock-down of BCL11A promotes the apoptosis of MDA-MB-231 cells.

  19. A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells.

    Science.gov (United States)

    Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Hamana, Hiroshi; Nakagawa, Hidetoshi; Jin, Aishun; Lin, Zhezhu; Muraguchi, Atsushi

    2014-10-31

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Cyclooxygenase inhibitors induce apoptosis in oral cavity cancer cells by increased expression of nonsteroidal anti-inflammatory drug-activated gene

    International Nuclear Information System (INIS)

    Kim, Kyung-Su; Yoon, Joo-Heon; Kim, Jin Kook; Baek, Seung Joon; Eling, Thomas E.; Lee, Won Jae; Ryu, Ji-Hwan; Lee, Jeung Gweon; Lee, Joo-Hwan; Yoo, Jong-Bum

    2004-01-01

    We have investigated whether NAG-1 is induced in oral cavity cancer cells by various NSAIDs and if apoptosis induced by NSAIDs can be linked directly with the induction of NAG-1. NAG-1 expression was increased by diclofenac, aceclofenac, indomethacin, ibuprofen, and sulindac sulfide, in the order of NAG-1 induction, but not by acetaminophen, piroxicam or NS-398. Diclofenac was the most effective NAG-1 inducer. Incubation with diclofenac inhibited cell proliferation and induced apoptosis. The expression of NAG-1 was observed in advance of the induction of apoptosis. Conditioned medium from NAG-1-overexpressing Drosophila cells inhibited SCC 1483 cells proliferation and induced apoptosis. In summary, some NSAIDs induce NAG-1 expression in oral cavity cancer cells and the induced NAG-1 protein appears to mediate apoptosis. Therefore, NSAIDs may be considered as a possible chemopreventive agent against oral cavity cancer