Outer Mitochondrial Membrane Localization of Apoptosis-Inducing Factor: Mechanistic Implications for Release

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Seong-Woon Yu

2009-10-01

Full Text Available Poly(ADP-ribose polymerase-1-dependent cell death (known as parthanatos plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor, but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.

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

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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.

Ginsenoside Rb1 Attenuates Oxygen-Glucose Deprivation-Induced Apoptosis in SH-SY5Y Cells via Protection of Mitochondria and Inhibition of AIF and Cytochrome c Release

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Pengfei Ge

2013-10-01

Full Text Available To investigate the role of mitochondria in the protective effects of ginsenoside Rb1 on cellular apoptosis caused by oxygen-glucose deprivation, in this study, MTT assay, TUNEL staining, flow cytometry, immunocytochemistry and western blotting were used to examine the cellular viability, apoptosis, ROS level, mitochondrial membrane potential, and the distribution of apoptosis inducing factor, cytochrome c, Bax and Bcl-2 in nucleus, mitochondria and cytoplasm. We found that pretreatment with GRb1 improved the cellular viability damaged by OGD. Moreover, GRb1 inhibited apoptosis in SH-SY5Y cells induced by OGD. Further studies showed that the elevation of cellular reactive oxygen species levels and the reduction of mitochondrial membrane potential caused by OGD were both counteracted by GRb1. Additionally, GRb1 not only suppressed the translocation of apoptosis inducing factor into nucleus and cytochrome c into cytoplasm, but also inhibited the increase of Bax within mitochondria and alleviated the decrease of mitochondrial Bcl-2. Our study indicates that the protection of GRb1 on OGD-induced apoptosis in SH-SY5Y cells is associated with its protection on mitochondrial function and inhibition of release of AIF and cytochrome c.

Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism.

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Han, Bing; Wang, Tong-Dan; Shen, Shao-Ming; Yu, Yun; Mao, Chan; Yao, Zhu-Jun; Wang, Li-Shun

2015-03-18

Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear. We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and related proteins were characterized by western blot. Immunofluorescence and subcellular fractionation were used to evaluate AIF nuclear translocation. Reactive oxygen species were assessed by using redox-sensitive dye DCFDA. AA005 induces a unique type of cell death in colorectal adenocarcinoma cells, characterized by lack of caspase-3 activation or apoptotic body formation, sensitivity to poly (ADP-ribose) polymerase inhibitor Olaparib (AZD2281) but not pan-caspase inhibitor Z-VAD.fmk, and dependence on apoptosis-inducing factor (AIF). AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage. Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells. Moreover, blocking activation of RIP-1 by necroptosis inhibitor necrotatin-1 inhibits AIF translocation and partially suppresses AA005-induced cell death in SW620 cells demonstrating that RIP-1 protein may be essential for cell death. AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway. Our work provided new mechanisms for AA005-induced cancer cell death and novel clues for cancer treatment via AIF dependent cell death.

Cardioprotection by modulation of mitochondrial respiration during ischemia–reperfusion: Role of apoptosis-inducing factor

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Xu, Aijun [Department of Internal Medicine (Division of Cardiology), Virginia Commonwealth University, Richmond, VA 23298 (United States); Department of Anesthesiology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 430030 (China); Szczepanek, Karol; Hu, Ying [Department of Internal Medicine (Division of Cardiology), Virginia Commonwealth University, Richmond, VA 23298 (United States); Lesnefsky, Edward J. [Department of Internal Medicine (Division of Cardiology), Virginia Commonwealth University, Richmond, VA 23298 (United States); Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298 (United States); Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA 23298 (United States); McGuire Department of Veterans Affairs Medical Center, Richmond, VA 23249 (United States); Chen, Qun, E-mail: qchen8@vcu.edu [Department of Internal Medicine (Division of Cardiology), Virginia Commonwealth University, Richmond, VA 23298 (United States)

2013-06-14

Highlights: •Blockade of electron transport prevents the loss of AIF from mitochondria during IR. •Blockade of electron transport decreases caspase-independent cell death during IR. •Mitochondrial AIF content is down-regulated in Harlequin mice. •Blockade of electron transport protects Harlequin mouse hearts during IR. •Amobarbital protection is partially dependent on mitochondrial AIF content. -- Abstract: The transient, reversible blockade of electron transport (BET) during ischemia or at the onset of reperfusion protects mitochondria and decreases cardiac injury. Apoptosis inducing factor (AIF) is located within the mitochondrial intermembrane space. A release of AIF from mitochondria into cytosol and nucleus triggers caspase-independent cell death. We asked if BET prevents the loss of AIF from mitochondria as a mechanism of protection in the buffer perfused heart. BET during ischemia with amobarbital, a rapidly reversible inhibitor of mitochondrial complex I, attenuated a release of AIF from mitochondria into cytosol, in turn decreasing the formation of cleaved and activated PARP-1. These results suggest that BET-mediated protection may occur through prevention of the loss of AIF from mitochondria during ischemia–reperfusion. In order to further clarify the role of mitochondrial AIF in BET-mediated protection, Harlequin (Hq) mice, a genetic model with mitochondrial AIF deficiency, were used to test whether BET could still decrease cell injury in Hq mouse hearts during reperfusion. BET during ischemia protected Hq mouse hearts against ischemia–reperfusion injury and improved mitochondrial function in these hearts during reperfusion. Thus, cardiac injury can still be decreased in the presence of down-regulated mitochondrial AIF content. Taken together, BET during ischemia protects both hearts with normal mitochondrial AIF content and hearts with mitochondrial AIF deficiency. Although preservation of mitochondrial AIF content plays a key role in

Cardioprotection by modulation of mitochondrial respiration during ischemia–reperfusion: Role of apoptosis-inducing factor

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Xu, Aijun; Szczepanek, Karol; Hu, Ying; Lesnefsky, Edward J.; Chen, Qun

2013-01-01

Highlights: •Blockade of electron transport prevents the loss of AIF from mitochondria during IR. •Blockade of electron transport decreases caspase-independent cell death during IR. •Mitochondrial AIF content is down-regulated in Harlequin mice. •Blockade of electron transport protects Harlequin mouse hearts during IR. •Amobarbital protection is partially dependent on mitochondrial AIF content. -- Abstract: The transient, reversible blockade of electron transport (BET) during ischemia or at the onset of reperfusion protects mitochondria and decreases cardiac injury. Apoptosis inducing factor (AIF) is located within the mitochondrial intermembrane space. A release of AIF from mitochondria into cytosol and nucleus triggers caspase-independent cell death. We asked if BET prevents the loss of AIF from mitochondria as a mechanism of protection in the buffer perfused heart. BET during ischemia with amobarbital, a rapidly reversible inhibitor of mitochondrial complex I, attenuated a release of AIF from mitochondria into cytosol, in turn decreasing the formation of cleaved and activated PARP-1. These results suggest that BET-mediated protection may occur through prevention of the loss of AIF from mitochondria during ischemia–reperfusion. In order to further clarify the role of mitochondrial AIF in BET-mediated protection, Harlequin (Hq) mice, a genetic model with mitochondrial AIF deficiency, were used to test whether BET could still decrease cell injury in Hq mouse hearts during reperfusion. BET during ischemia protected Hq mouse hearts against ischemia–reperfusion injury and improved mitochondrial function in these hearts during reperfusion. Thus, cardiac injury can still be decreased in the presence of down-regulated mitochondrial AIF content. Taken together, BET during ischemia protects both hearts with normal mitochondrial AIF content and hearts with mitochondrial AIF deficiency. Although preservation of mitochondrial AIF content plays a key role in

Neem oil limonoids induces p53-independent apoptosis and autophagy.

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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.

Neem oil limonoids induces p53-independent apoptosis and autophagy

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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

Silibinin induces apoptosis via calpain-dependent AIF nuclear translocation in U87MG human glioma cell death

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Kim Yong K

2011-04-01

Full Text Available Abstract Background Silibinin, a natural polyphenolic flavonoid, has been reported to induce cell death in various cancer cell types. However, the molecular mechanism is not clearly defined. Our previous study showed that silibinin induces glioma cell death and its effect was effectively prevented by calpain inhibitor. The present study was therefore undertaken to examine the role of calpain in the silibinin-induced glioma cell death. Methods U87MG cells were grown on well tissue culture plates and cell viability was measured by MTT assay. ROS generation and △ψm were estimated using the fluorescence dyes. PKC activation and Bax expression were measured by Western blot analysis. AIF nuclear translocation was determined by Western blot and immunocytochemistry. Results Silibinin induced activation of calpain, which was blocked by EGTA and the calpain inhibitor Z-Leu-Leu-CHO. Silibinin caused ROS generation and its effect was inhibited by calpain inhibitor, the general PKC inhibitor GF 109203X, the specific PKCδ inhibitor rottlerin, and catalase. Silibinin-induce cell death was blocked by calpain inhibitor and PKC inhibitors. Silibinin-induced PKCδ activation and disruption of △ψm were prevented by the calpain inhibitor. Silibinin induced AIF nuclear translocation and its effect was prevented by calpain inhibitor. Transfection of vector expressing microRNA of AIF prevented the silibinin-induced cell death. Conclusions Silibinin induces apoptotic cell death through a calpain-dependent mechanism involving PKC, ROS, and AIF nuclear translocation in U87MG human glioma cells.

Synergistic apoptosis of CML cells by buthionine sulfoximine and hydroxychavicol correlates with activation of AIF and GSH-ROS-JNK-ERK-iNOS pathway.

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Avik Acharya Chowdhury

Full Text Available BACKGROUND: Hydroxychavicol (HCH, a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS. The aim of the study is to optimize the oxidative stress -induced chronic myeloid leukemic (CML cell death by combining glutathione synthesis inhibitor, buthionine sulfoximine (BSO with HCH and studying the underlying mechanism. MATERIALS AND METHODS: Anti-proliferative activity of BSO and HCH alone or in combination against a number of leukemic (K562, KCL22, KU812, U937, Molt4, non-leukemic (A549, MIA-PaCa2, PC-3, HepG2 cancer cell lines and normal cell lines (NIH3T3, Vero was measured by MTT assay. Apoptotic activity in CML cell line K562 was detected by flow cytometry (FCM after staining with annexin V-FITC/propidium iodide (PI, detection of reduced mitochondrial membrane potential after staining with JC-1, cleavage of caspase- 3 and poly (ADP-ribose polymerase proteins by western blot analysis and translocation of apoptosis inducing factor (AIF by confocal microscopy. Intracellular reduced glutathione (GSH was measured by colorimetric assay using GSH assay kit. 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA and 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM were used as probes to measure intracellular increase in ROS and nitric oxide (NO levels respectively. Multiple techniques like siRNA transfection and pharmacological inhibition were used to understand the mechanisms of action. RESULTS: Non-apoptotic concentrations of BSO significantly potentiated HCH-induced apoptosis in K562 cells. BSO potentiated apoptosis-inducing activity of HCH in CML cells by caspase-dependent as well as caspase-independent but apoptosis inducing factor (AIF-dependent manner. Enhanced depletion of intracellular GSH induced by combined treatment correlated with induction of ROS. Activation of ROS- dependent JNK played a crucial role in ERK1/2 activation which subsequently induced the

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

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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.

Brassinosteroid-Induced Transcriptional Repression and Dephosphorylation-Dependent Protein Degradation Negatively Regulate BIN2-Interacting AIF2 (a BR Signaling-Negative Regulator) bHLH Transcription Factor.

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Kim, Yoon; Song, Ji-Hye; Park, Seon-U; Jeong, You-Seung; Kim, Soo-Hwan

2017-02-01

Brassinosteroids (BRs) are plant polyhydroxy-steroids that play important roles in plant growth and development via extensive signal integration through direct interactions between regulatory components of different signaling pathways. Recent studies have shown that diverse helix-loop-helix/basic helix-loop-helix (HLH/bHLH) family proteins are actively involved in control of BR signaling pathways and interact with other signaling pathways. In this study, we show that ATBS1-INTERACTING FACTOR 2 (AIF2), a nuclear-localized atypical bHLH transcription factor, specifically interacts with BRASSINOSTEROID-INSENSITIVE 2 (BIN2) among other BR signaling molecules. Overexpression of AIF2 down-regulated transcript expression of growth-promoting genes, thus resulting in retardation of growth. AIF2 renders plants hyposensitive to BR-induced root growth inhibition, but shows little effects on BR-promoted hypocotyl elongation. Notably, AIF2 was dephosphorylated by BR, and the dephosphorylated AIF2 was subject to proteasome-mediated degradation. AIF2 degradation was greatly induced by BR and ABA, but relatively slightly by other hormones such as auxin, gibberellin, cytokinin and ethylene. Moreover, AIF2 transcription was significantly suppressed by a BRI1/BZR1-mediated BR signaling pathway through a direct binding of BRASSINAZOLE RESISTANT 1 (BZR1) to the BR response element (BRRE) region of the AIF2 promoter. In conclusion, our study suggests that BIN2-driven AIF2 phosphorylation could augment the BIN2/AIF2-mediated negative circuit of BR signaling pathways, and the BR-induced transcriptional repression and protein degradation negatively regulate AIF2 transcription factor, reinforcing the BZR1/BES1-mediated positive BR signaling pathway. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Cigarette smoke causes caspase-independent apoptosis of bronchial epithelial cells from asthmatic donors.

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Fabio Bucchieri

Full Text Available Epidemiologic studies have demonstrated important links between air pollution and asthma. Amongst these pollutants, environmental cigarette smoke is a risk factor both for asthma pathogenesis and exacerbation. As the barrier to the inhaled environment, the bronchial epithelium is a key structure that is exposed to cigarette smoke.Since primary bronchial epithelial cells (PBECs from asthmatic donors are more susceptible to oxidant-induced apoptosis, we hypothesized that they would be susceptible to cigarette smoke-induced cell death.PBECs from normal and asthmatic donors were exposed to cigarette smoke extract (CSE; cell survival and apoptosis were assessed by fluorescence-activated cell sorting, and protective effects of antioxidants evaluated. The mechanism of cell death was evaluated using caspase inhibitors and immunofluorescent staining for apoptosis-inducing factor (AIF.Exposure of PBEC cultures to CSE resulted in a dose-dependent increase in cell death. At 20% CSE, PBECs from asthmatic donors exhibited significantly more apoptosis than cells from non-asthmatic controls. Reduced glutathione (GSH, but not ascorbic acid (AA, protected against CSE-induced apoptosis. To investigate mechanisms of CSE-induced apoptosis, caspase-3 or -9 inhibitors were tested, but these failed to prevent apoptosis; in contrast, CSE promoted nuclear translocation of AIF from the mitochondria. GSH reduced the number of nuclear-AIF positive cells whereas AA was ineffective.Our results show that PBECs from asthmatic donors are more susceptible to CSE-induced apoptosis. This response involves AIF, which has been implicated in DNA damage and ROS-mediated cell-death. Epithelial susceptibility to CSE may contribute to the impact of environmental tobacco smoke in asthma.

Water-Soluble Coenzyme Q10 Inhibits Nuclear Translocation of Apoptosis Inducing Factor and Cell Death Caused by Mitochondrial Complex I Inhibition

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Haining Li

2014-07-01

Full Text Available The objectives of the study were to explore the mechanism of rotenone-induced cell damage and to examine the protective effects of water-soluble Coenzyme Q10 (CoQ10 on the toxic effects of rotenone. Murine hippocampal HT22 cells were cultured with mitochondrial complex I inhibitor rotenone. Water-soluble CoQ10 was added to the culture media 3 h prior to the rotenone incubation. Cell viability was determined by alamar blue, reactive oxygen species (ROS production by dihydroethidine (DHE and mitochondrial membrane potential by tetramethyl rhodamine methyl ester (TMRM. Cytochrome c, caspase-9 and apoptosis-inducing factor (AIF were measured using Western blotting after 24 h rotenone incubation. Rotenone caused more than 50% of cell death, increased ROS production, AIF nuclear translocation and reduction in mitochondrial membrane potential, but failed to cause mitochondrial cytochrome c release and caspase-9 activation. Pretreatment with water-soluble CoQ10 enhanced cell viability, decreased ROS production, maintained mitochondrial membrane potential and prevented AIF nuclear translocation. The results suggest that rotenone activates a mitochondria-initiated, caspase-independent cell death pathway. Water-soluble CoQ10 reduces ROS accumulation, prevents the fall of mitochondrial membrane potential, and inhibits AIF translocation and subsequent cell death.

Basal metabolic state governs AIF-dependent growth support in pancreatic cancer cells

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Scott, Andrew J.; Wilkinson, Amanda S.; Wilkinson, John C.

2016-01-01

Apoptosis-inducing factor (AIF), named for its involvement in cell death pathways, is a mitochondrial protein that regulates metabolic homeostasis. In addition to supporting the survival of healthy cells, AIF also plays a contributory role to the development of cancer through its enzymatic activity, and we have previously shown that AIF preferentially supports advanced-stage prostate cancer cells. Here we further evaluated the role of AIF in tumorigenesis by exploring its function in pancreatic cancer, a disease setting that most often presents at an advanced stage by the time of diagnosis. A bioinformatics approach was first employed to investigate AIF mRNA transcript levels in pancreatic tumor specimens vs. normal tissues. AIF-deficient pancreatic cancer cell lines were then established via lentiviral infection. Immunoblot analysis was used to determine relative protein quantities within cells. Cell viability was measured by flow cytometry; in vitro and Matrigel™ growth/survival using Coulter™ counting and phase contrast microscopy; and glucose consumption in the absence and presence of Matrigel™ using spectrophotometric methods. Archival gene expression data revealed a modest elevation of AIF transcript levels in subsets of pancreatic tumor specimens, suggesting a possible role in disease progression. AIF expression was then suppressed in a panel of five pancreatic cancer cell lines that display diverse metabolic phenotypes. AIF ablation selectively crippled the growth of cells in vitro in a manner that directly correlated with the loss of mitochondrial respiratory chain subunits and altered glucose metabolism, and these effects were exacerbated in the presence of Matrigel™ substrate. This suggests a critical metabolic role for AIF to pancreatic tumorigenesis, while the spectrum of sensitivities to AIF ablation depends on basal cellular metabolic phenotypes. Altogether these data indicate that AIF supports the growth and survival of metabolically defined

Methadone induces CAD degradation and AIF-mediated necrotic-like cell death in neuroblastoma cells.

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Perez-Alvarez, Sergio; Iglesias-Guimarais, Victoria; Solesio, María E; Melero-Fernandez de Mera, Raquel María; Yuste, Víctor J; Galindo, María F; Jordán, Joaquín

2011-04-01

Methadone (d,l-methadone hydrochloride) is a full-opioid agonist, originally developed as a substitution for heroin or other opiates abusers. Nowadays methadone is also being applied as long-lasting analgesics in cancer, and it is proposed as a promising agent for leukemia therapy. Previously, we have demonstrated that high concentrations of methadone (0.5mM) induced necrotic-like cell death in SH-SY5Y cells. The pathway involved is caspase-independent but involves impairment of mitochondrial ATP synthesis and mitochondrial cytochrome c release. However, the downstream mitochondrial pathways remained unclear. Here, we studied the participation of apoptosis inducing factor (AIF) in methadone-induced cell death. Methadone resulted in a translocation of AIF from mitochondria to the nucleus. Translocation was inhibited by cyclosporine A, but not by lack of Bax protein. Therefore the effect seems mediated by the formation of the mitochondrial transition pore, but is apparently independent of Bax. Furthermore, methadone-treated SH-SY5Y nuclei show characteristics that are typical for stage I nuclear condensation. Methadone did not induce degradation of DNA into oligonucleosomal fragments or into high molecular weight DNA fragments. Absence of DNA fragmentation coincided with a considerable decrease in the levels of the caspase-actived endonuclase DNase and its chaperone-inhibitor ICAD. In conclusion, our results provide mechanistic insights into the molecular mechanisms that underlie methadone-induced cell death. This knowledge may prove useful to develop novel strategies to prevent toxic side-effects of methadone thereby sustaining its use as therapeutical agent against tumors. Copyright © 2010 Elsevier Ltd. All rights reserved.

Caspase-1 Deficiency Alleviates Dopaminergic Neuronal Death via Inhibiting Caspase-7/AIF Pathway in MPTP/p Mouse Model of Parkinson's Disease.

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Qiao, Chen; Zhang, Lin-Xia; Sun, Xi-Yang; Ding, Jian-Hua; Lu, Ming; Hu, Gang

2017-08-01

Caspase family has been recognized to be involved in dopaminergic (DA) neuronal death and to exert an unfavorable role in Parkinson's disease (PD) pathology. Our previous study has revealed that caspase-1, as an important component of NLRP3 inflammasome, induces microglia-mediated neuroinflammation in the pathogenesis of PD. However, the role of caspase-1 in DA neuronal degeneration in the onset of PD remains unclear. Here, we showed that caspase-1 knockout ameliorated DA neuronal loss and dyskinesia in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine/probenecid (MPTP/p)-induced PD model mice. We further found that caspase-1 knockout decreased MPTP/p-induced caspase-7 cleavage, subsequently inhibited nuclear translocation of poly (ADP-ribose) polymerase 1 (PARP1), and reduced the release of apoptosis-inducing factor (AIF). Consistently, we demonstrated that caspase-1 inhibitor suppressed caspase-7/PARP1/AIF-mediated apoptosis pathway by 1-methyl-4-phenylpyridinium ion (MPP + ) stimulation in SH-SY5Y cells. Caspase-7 overexpression reduced the protective effects of caspase-1 inhibitor on SH-SY5Y cell apoptosis. Collectively, our results have revealed that caspase-1 regulates DA neuronal death in the pathogenesis of PD in mice via caspase-7/PARP1/AIF pathway. These findings will shed new insight into the potential of caspase-1 as a target for PD therapy.

High level of Bcl-2 counteracts apoptosis mediated by a live rabies virus vaccine strain and induces long-term infection

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Thoulouze, Maria-Isabel; Lafage, Mireille; Yuste, Victor J.; Baloul, Leiela; Edelman, Lena; Kroemer, Guido; Israel, Nicole; Susin, Santos A.; Lafon, Monique

2003-01-01

We report here that rabies virus strains, currently used to immunize wildlife against rabies, induce not only caspase-dependent apoptosis in the human lymphoblastoid Jurkat T cell line (Jurkat-vect), but also a caspase-independent pathway involving the apoptosis-inducing factor (AIF). In contrast, a strain of neurotropic RV that does not induce apoptosis did not activate caspases or induce AIF translocation. Bcl-2 overproduction in Jurkat T cells (Jurkat-Bcl-2) abolished both pathways. ERA infection and production were similar in Jurkat-vect and Jurkat-Bcl-2 cells, indicating Bcl-2 has no direct antiviral effects. Bcl-2 production is naturally upregulated by day 3 in ERA-infected Jurkat-vect cultures. The increase in Bcl-2 levels seems to be controlled by the virus infection itself and results in the establishment of long-term, persistently infected cultures that continue to produce virus. Thus, in infections with live RV vaccine strains, infected cells may be productive reservoirs of virus in the long term. This may account for the high efficacy of live rabies vaccines

Mycotoxin zearalenone induces AIF- and ROS-mediated cell death through p53- and MAPK-dependent signaling pathways in RAW264.7 macrophages.

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Yu, Ji-Yeon; Zheng, Zhong-Hua; Son, Young-Ok; Shi, Xianglin; Jang, Young-Oh; Lee, Jeong-Chae

2011-12-01

Zearalenone (ZEN) is commonly found in many food commodities and is known to cause reproductive disorders and genotoxic effects. However, the mode of ZEN-induced cell death of macrophages and the mechanisms by which ZEN causes cytotoxicity remain unclear. The present study shows that ZEN treatment reduces viability of RAW264.7 cells in a dose-dependent manner. ZEN causes predominantly necrotic and late apoptotic cell death. ZEN treatment also results in the loss of mitochondrial membrane potential (MMP), mitochondrial changes in Bcl-2 and Bax proteins, and cytoplasmic release of cytochrome c and apoptosis-inducing factor (AIF). Pre-treatment of the cells with either z-VAD-fmk or z-IETD-fmk does not attenuate ZEN-mediated cell death, whereas catalase suppresses the ZEN-induced decrease in viability in RAW264.7 cells. Treating the cells with c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), or p53 inhibitor prevented ZEN-mediated changes, such as MMP loss, cellular reactive oxygen species (ROS) increase, and cell death. JNK or p38 MAPK inhibitor inhibited mitochondrial alterations of Bcl-2 and Bax proteins with attendant decreases in cellular ROS levels. Knockdown of AIF via siRNA transfection also diminished ZEN-induced cell death. Further, adenosine triphosphate was markedly depleted in the ZEN-exposed cells. Collectively, these results suggest that ZEN induces cytotoxicity in RAW264.7 cells via AIF- and ROS-mediated signaling, in which the activations of p53 and JNK/p38 play a key role. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

6-OHDA-induced apoptosis and mitochondrial dysfunction are mediated by early modulation of intracellular signals and interaction of Nrf2 and NF-κB factors

International Nuclear Information System (INIS)

Tobón-Velasco, Julio C.; Limón-Pacheco, Jorge H.; Orozco-Ibarra, Marisol; Macías-Silva, Marina; Vázquez-Victorio, Genaro; Cuevas, Elvis; Ali, Syed F.

2013-01-01

6-Hydroxydopamine (6-OHDA) is a neurotoxin that generates an experimental model of Parkinson's disease in rodents and is commonly employed to induce a lesion in dopaminergic pathways. The characterization of those molecular mechanisms linked to 6-OHDA-induced early toxicity is needed to better understand the cellular events further leading to neurodegeneration. The present work explored how 6-OHDA triggers early downstream signaling pathways that activate neurotoxicity in the rat striatum. Mitochondrial function, caspases-dependent apoptosis, kinases signaling (Akt, ERK 1/2, SAP/JNK and p38) and crosstalk between nuclear factor kappa B (NF-κB) and nuclear factor-erythroid-2-related factor 2 (Nrf2) were evaluated at early times post-lesion. We found that 6-OHDA initiates cell damage via mitochondrial complex I inhibition, cytochrome c and apoptosis-inducing factor (AIF) release, as well as activation of caspases 9 and 3 to induce apoptosis, kinase signaling modulation and NF-κB-mediated inflammatory responses, accompanied by inhibition of antioxidant systems regulated by the Nrf2 pathway. Our results suggest that kinases SAP/JNK and p38 up-regulation may play a role in the early stages of 6-OHDA toxicity to trigger intrinsic pathways for apoptosis and enhanced NF-κB activation. In turn, these cellular events inhibit the activation of cytoprotective mechanisms, thereby leading to a condition of general damage

Pulsed electromagnetic field affects intrinsic and endoplasmatic reticulum apoptosis induction pathways in MonoMac6 cell line culture.

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Kaszuba-Zwoinska, J; Chorobik, P; Juszczak, K; Zaraska, W; Thor, P J

2012-10-01

Current studies were aimed to elucidate influence of pulsed electromagnetic field stimulation on cell viability and apoptosis induction pathways. For the experimental model we have chosen monocytic cell line MonoMac6 and several apoptosis inducers with different mechanism of death induction like puromycin, colchicine, cyclophosphamide, minocycline and hydrogen peroxide. MonoMac6 cell line was grown at density 1x10(5) cells/well in 96-well culture plates. To induce cell death cell cultures were treated with different apoptosis inducers like puromycin, colchicine, cyclophosphamide, minocycline, hydrogen peroxide and at the same time with pulsed electromagnetic field 50 Hz, 45±5 mT (PEMF) for 4 hour per each stimulation, three times, in 24 hours intervals. Afterwards, cells were harvested for flow cytometry analysis of cell viability measured by annexin V-APC labeled and propidium iodide staining. Expression of apoptosis related genes was evaluated by semi quantitative reverse transcription (RT)-PCR assay. NuPAGE Novex Western blot analysis was carried out for apoptosis inducing factor (AIF) abundance in cytosolic and nuclear extracts of MonoMac6 cells. Puromycin, colchicine and minocycline activated cells and simultaneously treated with PEMF have shown out diminished percentage of annexinV positive (AnV+) cells comparing to controls without PEMF stimulation. MonaMac6 cells puromycin/colchicyne and PEMF treated were to a higher extent double stained (AnV+,PI+), which means increased late apoptotic as well as necrotic (PI+) cells, than non-stimulated controls. On the other hand, minocycline activated cells prior to PEMF treatment showed diminished amount of apoptotic and necrotic (annexin V, annexin V and propidium iodide, propidium iodide positive staining) cells. The opposite effect of PEMF on the percentage of annexin V positively stained cells has been achieved after treatment of MonoMac6 culture with cyclophoshamide and hydrogen peroxide. PEMF enhanced early

Daintain/AIF-1 (Allograft Inflammatory Factor-1) accelerates type 1 diabetes in NOD mice

Energy Technology Data Exchange (ETDEWEB)

Zhao, Yan-Ying, E-mail: biozyy@163.com [College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041 (China); Huang, Xin-Yuan [College of Life Science and Technology, Hubei Engineering University, Xiaogan 432000 (China); Chen, Zheng-Wang [Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074 (China)

2012-10-26

Highlights: Black-Right-Pointing-Pointer Daintain/AIF-1 is over-expressed in the blood of NOD mice suffering from insulitis. Black-Right-Pointing-Pointer Daintain/AIF-1 stimulates white blood cell proliferation in NOD mice. Black-Right-Pointing-Pointer Daintain/AIF-1 increases blood glucose levels and triggers type 1 diabetes. Black-Right-Pointing-Pointer Daintain/AIF-1 accelerates insulitis, while its antibody prevents insulitis. Black-Right-Pointing-Pointer Daintain/AIF-1 enhances the levels of nitric oxide in the pancreases of NOD mice. -- Abstract: A large body of experimental evidence suggests that cytokines trigger pancreatic {beta}-cell death in type 1 diabetes mellitus. Daintain/AIF-1 (Allograft Inflammatory Factor-1), a specific marker for activated macrophages, is accumulated in the pancreatic islets of pre-diabetic BB rats. In the present study, we demonstrate that daintain/AIF-1 is released into blood and the levels of daintain/AIF-1 in the blood of type 1 diabetes-prone non-obese diabetic (NOD) mice suffering from insulitis are significantly higher than that in healthy NOD mice. When injected intravenously into NOD mice, daintain/AIF-1 stimulates white blood cell proliferation, increases the concentrations of blood glucose, impairs insulin expression, up-regulates nitric oxide (NO) production in pancreases and accelerates diabetes in NOD mice, while the antibody against daintain/AIF-1 delays or prevents insulitis in NOD mice. These results imply daintain/AIF-1 triggers type 1 diabetes probably via arousing immune cells activation and induction of NO production in pancreas of NOD mice.

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

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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.

Perfluorononanoic acid-induced apoptosis in rat spleen involves oxidative stress and the activation of caspase-independent death pathway

International Nuclear Information System (INIS)

Fang, Xuemei; Feng, Yixing; Wang, Jianshe; Dai, Jiayin

2010-01-01

Perfluoroalkyl acid (PFAA)-induced apoptosis has been reported in many cell types. However, minimal information on its mode of action is available. This study explored the possible involvement of apoptotic signaling pathways in a nine-carbon-chain length PFAA-perfluorononanoic acid (PFNA)-induced splenocyte apoptosis. After a 14-day exposure to PFNA, rat spleens showed dose-dependent levels of apoptosis. The production of pro-inflammatory and anti-inflammatory cytokines was significantly increased and decreased, respectively. However, protein levels of tumor necrosis factor receptor 1 (TNFR1), fas-associated protein with death domain (FADD), caspase 8 and caspase 3, which are involved in inflammation-related and caspase-dependent apoptosis, were discordant. Peroxisome proliferator-activated receptors alpha (PPARα) and PPARγ genes expression was up-regulated in rats treated with 3 or 5 mg/kg/day of PFNA, and the level of hydrogen peroxide (H 2 O 2 ) increased concurrently in rats treated with the highest dose. Moreover, superoxide dismutase (SOD) activity and Bcl-2 protein levels were dramatically decreased in spleens after treatment with 3 and 5 mg/kg/day of PFNA. However, protein levels of Bax were unchanged. Apoptosis-inducing factor (AIF), an initiator of caspase-independent apoptosis, was significantly increased in all PFNA-dosed rats. Thus, oxidative stress and the activation of a caspase-independent apoptotic signaling pathway contributed to PFNA-induced apoptosis in rat splenocytes.

Construction of Egr1-mediated human truncated apoptosis inducing factor expression vector and its expression regularity induced by radiation in breast cancer MCF-7 cells

International Nuclear Information System (INIS)

Wang Jianfeng; Gong Shouliang; Wang Zhicheng; Fang Fang; Liu Yang; Wu Jiahui

2012-01-01

Objective: To clone human truncated apoptosis inducing factor (AIF) cDNA sequence, and to construct early growth response 1 (Egr1)-mediated recombinant expression vector pcDNA 3.1-Egr1-AIF Δ1-480 (pEgr1-AIFΔ 1-480 ), and to observe its regularity induced by radiation in human breast cancer MCF-7 cells. Methods: The total mRNA extracted from human leukemia Jurkat cells used as template, and the human AIFΔ 1-480 was acquired by RT-PCR, and it was linked to pMD18T vector for sequencing. Egr1 fragment was digested from pMD19T-Egr1 by restrictive enzyme, and the Egr1-mediated expression plasmid pEgr1-AIFΔ 1-480 was constructed by gene recombination. There were control group, pcDNA3.1 group, pAIFΔ 1-480 group and pEgr1-AIFΔ 1-480 group in the experiment. After the plasmids in various groups were transfected into human breast cancer MCF-7 cells, the AIF and AIFΔ 1-480 protein expression time-effect (0, 2, 4, 12, 24 and 48 h after 2.0 Gy irradiation) and dose-effect (24 h after 0, 0.2, 0.5, 1.0, 2.0 and 5.0 Gy irradiation) regularity were measured by Western blotting method. Results: The sequencing results showed that the AIFΔ 1-480 acquired by RT-PCR was consistent with the sequence expected, the pEgr-AIFΔ 1-480 was confirmed by PCR and restrictive enzyme digestion. After 0-48 h the MCF-7 cells were irradiated by 2.0 Gy, and the AIF protein expressed in the cells in each group, and it increased significantly from 4 h and the AIF expressions in 4, 12, 24 and 48 h groups were higher than that in 0 h group (P<0.05), and it reached to maximum value at 48 h. But the AIFΔ 1-480 protein expressed in the cells in pAIFΔ 1-480 and pEgr1-AIFΔ 1-480 groups from 2 h (P<0.05), and it reached to peak value at 24 h. The AIFΔ 1-480 expressions in pEgr1-AIFΔ 1-480 group were higher than those in pAIFΔ 1-480 group at and 48 h (P<0.05). After the MCF-7 cells were irradiated by 0-5 Gy for 24 h, the AIF protein expressed in the cells in each group, but the AIFΔ 1-480 protein

Multiple signal transduction pathways in okadaic acid induced apoptosis in HeLa cells

International Nuclear Information System (INIS)

Jayaraj, R.; Gupta, Nimesh; Rao, P.V. Lakshmana

2009-01-01

Okadaic acid (OA) is the major component of diarrhetic shell fish poisoning toxins and a potent inhibitor of protein phosphatase 1 and 2A. We investigated the signal transduction pathways involved in OA induced cell death in HeLa cells. OA induced cytotoxicity and apoptosis at IC50 of 100 nM. OA treatment resulted in time dependent increase in reactive oxygen species and depleted intracellular glutathione levels. Loss of mitochondrial membrane permeability led to translocation of bax, cytochrome-c and AIF from mitochondria to cytosol. The cells under fluorescence microscope showed typical apoptotic morphology with condensed chromatin, and nuclear fragmentation. We investigated the mitochondrial-mediated caspase cascade. The time dependent activation and cleavage of of bax, caspases-8, 10, 9, 3 and 7 was observed in Western blot analysis. In addition to caspase-dependent pathway AIF mediated caspase-independent pathway was involved in OA mediated cell death. OA also caused time dependent inhibition of protein phosphatase 2A activity and phosphorylation of p38 and p42/44 MAP kinases. Inhibitor studies with Ac-DEVO-CHO and Z-VAD-FMK could not prevent the phosphorylation of p38 and p42/44 MAP kinases. Our experiments with caspase inhibitors Ac-DEVD-CHO, Z-IETD-FMK and Z-VAD-FMK inhibited capsase-3, 8 cleavages but did not prevent OA-induced apoptosis and DNA fragmentation. Similarly, pretreatment with cyclosporin-A and N-acetylcysteine could not prevent the DNA fragmentation. In summary, the results of our study show that OA induces multiple signal transduction pathways acting either independently or simultaneously leading to apoptosis

Calpain: a molecule to induce AIF-mediated necroptosis in RGC-5 following elevated hydrostatic pressure

Science.gov (United States)

2014-01-01

Background RIP3 (Receptor-interacting protein 3) pathway was mainly described as the molecular mechanism of necroptosis (programmed necrosis). But recently, non-RIP3 pathways were found to mediate necroptosis. We deliberate to investigate the effect of calpain, a molecule to induce necroptosis as reported (Cell Death Differ 19:245–256, 2012), in RGC-5 following elevated hydrostatic pressure. Results First, we identified the existence of necroptosis of RGC-5 after insult by using necrostatin-1 (Nec-1, necroptosis inhibitor) detected by flow cytometry. Immunofluorescence staining and western blot were used to detect the expression of calpain. Western blot analysis was carried out to describe the truncated AIF (tAIF) expression with or without pretreatment of ALLN (calpain activity inhibitor). Following elevated hydrostatic pressure, necroptotic cells pretreated with or without ALLN was stained by Annexin V/PI, The activity of calpain was also examined to confirm the inhibition effect of ALLN. The results showed that after cell injury there was an upregulation of calpain expression. Upon adding ALLN, the calpain activity was inhibited, and tAIF production was reduced upon injury along with the decreased number of necroptosis cells. Conclusion Our study found that calpain may induce necroptosis via tAIF-modulation in RGC-5 following elevated hydrostatic pressure. PMID:24884644

Involvement of VDAC, Bax and ceramides in the efflux of AIF from mitochondria during curcumin-induced apoptosis.

NARCIS (Netherlands)

Scharstuhl, A.; Mutsaers, H.A.M.; Pennings, S.W.C.; Russel, F.G.M.; Wagener, F.A.D.T.G.

2009-01-01

BACKGROUND: We previously identified curcumin as a potent inducer of fibroblast apoptosis, which could be used to treat hypertrophic scar formation. Here we investigated the underlying mechanism of this process. PRINCIPAL FINDINGS: Curcumin-induced apoptosis could not be blocked by

Fucoidan extract induces apoptosis in MCF-7 cells via a mechanism involving the ROS-dependent JNK activation and mitochondria-mediated pathways.

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Zhongyuan Zhang

Full Text Available BACKGROUND: Fucoidan extract (FE, an enzymatically digested compound with a low molecular weight, is extracted from brown seaweed. As a natural compound with various actions, FE is attractive, especially in Asian countries, for improving the therapeutic efficacy and safety of cancer treatment. The present study was carried out to investigate the anti-tumor properties of FE in human carcinoma cells and further examine the underlying mechanisms of its activities. METHODOLOGY/PRINCIPAL FINDING: FE inhibits the growth of MCF-7, MDA-MB-231, HeLa, and HT1080 cells. FE-mediated apoptosis in MCF-7 cancer cells is accompanied by DNA fragmentation, nuclear condensation, and phosphatidylserine exposure. FE induces mitochondrial membrane permeabilization (MMP through loss of mitochondrial membrane potential (ΔΨm and regulation of the expression of Bcl-2 family members. Release of apoptosis-inducing factor (AIF and cytochrome c precedes MMP. AIF release causes DNA fragmentation, the final stage of apoptosis, via a caspase-independent mitochondrial pathway. Additionally, FE was found to induce phosphorylation of c-Jun N-terminal kinase (JNK, p38, and extracellular signal-regulated kinase (ERK 1/2, and apoptosis was found to be attenuated by inhibition of JNK. Furthermore, FE-mediated apoptosis was found to involve the generation of reactive oxygen species (ROS, which are responsible for the decrease of ΔΨm and phosphorylation of JNK, p38, and ERK1/2 kinases. CONCLUSIONS/SIGNIFICANCE: These data suggest that FE activates a caspase-independent apoptotic pathway in MCF-7 cancer cells through activation of ROS-mediated MAP kinases and regulation of the Bcl-2 family protein-mediated mitochondrial pathway. They also provide evidence that FE deserves further investigation as a natural anticancer and cancer preventive agent.

Protective effect of nicotinamide adenine dinucleotide (NAD+) against spinal cord ischemia-reperfusion injury via reducing oxidative stress-induced neuronal apoptosis.

Science.gov (United States)

Xie, Lei; Wang, Zhenfei; Li, Changwei; Yang, Kai; Liang, Yu

2017-02-01

As previous studies demonstrate that oxidative stress and apoptosis play crucial roles in ischemic pathogenesis and nicotinamide adenine dinucleotide (NAD + ) treatment attenuates oxidative stress-induced cell death among primary neurons and astrocytes as well as significantly reduce cerebral ischemic injury in rats. We used a spinal cord ischemia injury (SCII) model in rats to verify our hypothesis that NAD + could ameliorate oxidative stress-induced neuronal apoptosis. Adult male rats were subjected to transient spinal cord ischemia for 60min, and different doses of NAD + were administered intraperitoneally immediately after the start of reperfusion. Neurological function was determined by Basso, Beattie, Bresnahan (BBB) scores. The oxidative stress level was assessed by superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. The degree of apoptosis was analyzed by deoxyuridinetriphosphate nick-end labeling (TUNEL) staining and protein levels of cleaved caspase-3 and AIF (apoptosis inducing factor). The results showed that NAD + at 50 or 100mg/kg significantly decreased the oxidative stress level and neuronal apoptosis in the spinal cord of ischemia-reperfusion rats compared with saline, as accompanied with the decreased oxidative stress, NAD + administration significantly restrained the neuronal apoptosis after ischemia injury while improved the neurological and motor function. These findings suggested that NAD + might protect against spinal cord ischemia-reperfusion via reducing oxidative stress-induced neuronal apoptosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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

Cambogin Induces Caspase-Independent Apoptosis through the ROS/JNK Pathway and Epigenetic Regulation in Breast Cancer Cells.

Science.gov (United States)

Shen, Kaikai; Xie, Jianling; Wang, Hua; Zhang, Hong; Yu, Mengyuan; Lu, Fangfang; Tan, Hongsheng; Xu, Hongxi

2015-07-01

Cambogin is a polycyclic polyprenylated acylphoroglucinol (PPAP) from the Garcinia genus, which has been used traditionally for cancer treatment across Southeastern Asia. In this study, we found that cambogin inhibited breast cancer cell proliferation and induced cell apoptosis in vitro. Cambogin induced the activation of the caspase-independent mitochondrial apoptotic pathway, as indicated by an increase in the ratio of Bax/Bcl-2 and the nuclear translocation of apoptosis inducing factor (AIF). Two-dimensional gel electrophoresis and mass spectrometry revealed that the expression of proteins involving in the radical oxygen species (ROS) pathway was among the most affected upon cambogin treatment. Cambogin enhanced cellular ROS production, and induced the activation of the ASK1-MKK4/MKK7-JNK/SAPK signaling pathway. Pretreatment with ROS scavenger N-acetylcysteine (NAC), an antioxidant, or the JNK inhibitor SP600125 was able to restore cell viability in the presence of cambogin. Importantly, cambogin treatment led to the activation of activating transcription factor-2 (ATF-2) and the trimethylation of histone H3K9 in the activator protein 1 (AP-1) binding region of the Bcl-2 gene promoter. Finally, cambogin exhibited a potential antitumor effect in MCF-7 breast cancer xenografts without apparent toxicity. Taken in conjunction, the present study indicates that cambogin can induce breast adenocarcinoma cell apoptosis and therefore represents therapeutic potential for cancer treatment. ©2015 American Association for Cancer Research.

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...

H2O2 INDUCES APOPTOSIS OF RABBIT CHONDROCYTES VIA BOTH THE EXTRINSIC AND THE CASPASE-INDEPENDENT INTRINSIC PATHWAYS

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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.

Suppression of postmitochondrial signaling and delayed response to UV-induced nuclear apoptosis in HeLa cells

International Nuclear Information System (INIS)

Sasai, Kaori; Yajima, Hirohiko; Suzuki, Fumio

2002-01-01

Activation of postmitochondrial pathways by UV irradiation was examined using mouse lymphoma 3SB and human leukemic Jurkat cells and two human carcinoma cell lines (HeLa and MCF-7). Exposure of 3SB and Jurkat cells resulted in large amounts of cytochrome c and apoptosis-inducing factor (AIF) being released into the cytosol, and a clear laddering pattern of DNA fragments was observed within 3 h of incubation after irradiation. Simultaneously, activation of caspase-9 and its downstream caspases was detected. HeLa and MCF-7 cells also showed extensive release of mitochondrial factors and caspase-9 activation at 4 to 6 h after exposure, but apoptotic nuclear changes appeared much later. Compared with 3SB and Jurkat cells, these carcinoma cell lines exhibited reduced activation of caspase-9-like proteolytic activity by UV radiation, and levels of caspase-3-like activity in HeLa cells were extremely low, similar to those in caspase-3-deficient MCF-7 cells. These results suggest that the delayed response to UV-induced nuclear apoptosis in HeLa cells is due to a reduced activation of the caspase cascade downstream of cytochrome c release and suppression of caspase-3 activity. (author)

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

The ability of AIF-1 to activate human vascular smooth muscle cells is lost by mutations in the EF-hand calcium-binding region

International Nuclear Information System (INIS)

Autieri, Michael V.; Chen Xing

2005-01-01

Allograft Inflammatory Factor-1 (AIF-1) is a cytoplasmic calcium-binding protein expressed in vascular smooth muscle cells (VSMC) in response to injury or cytokine stimulation. AIF-1 contains a partially conserved EF-hand calcium-binding domain, and participates in VSMC activation by activation of Rac1 and induction of Granulocyte-Colony Stimulating Factor (G-CSF) expression; however, the mechanism whereby AIF-1 mediates these effects is presently uncharacterized. To determine if calcium binding plays a functional role in AIF-1 activity, a single site-specific mutation was made in the EF-hand calcium-binding domain to abrogate binding of calcium (AIF-1ΔA), which was confirmed by calcium overlay. Functionally, similar to wild-type AIF-1, AIF-1ΔA was able to polymerize F-actin in vitro. However, in contrast to wild-type AIF-1, over-expression of AIF-1ΔA was unable to increase migration or proliferation of primary human VSMC. Further, it was unable to activate Rac1, or induce G-CSF expression to the degree as wild-type AIF-1. Taken together, modification of the wild-type EF-hand domain and native calcium-binding activity results in a loss of AIF-1 function. We conclude that appropriate calcium-binding potential is critical in AIF-1-mediated effects on VSMC pathophysiology, and that AIF-1 activity is mediated by Rac1 activation and G-CSF expression

Lebein, a Snake Venom Disintegrin, Induces Apoptosis in Human Melanoma Cells

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Manel B. Hammouda

2016-07-01

Full Text Available Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterized by its very invasive and chemoresistant properties. Despite the recent promising news from the field of immunotherapy, there is an urgent need for new therapeutic approaches that are free of resistance mechanisms and side effects. Anti-neoplasic properties have been highlighted for different disintegrins from snake venom including Lebein; however, the exact effect of Lebein on melanoma has not yet been defined. In this study, we showed that Lebein blocks melanoma cell proliferation and induces a more differentiated phenotype with inhibition of extracellular signal-regulated kinase (ERK phosphorylation and microphthalmia-associated transcription factor (MITF overexpression. Melanoma cells became detached but were less invasive with upregulation of E-cadherin after Lebein exposure. Lebein induced a caspase-independent apoptotic program with apoptosis inducing factor (AIF, BCL-2-associated X protein (BAX and Bim overexpression together with downregulation of B-cell lymphoma-2 (BCL-2. It generated a distinct response in reactive oxygen species (ROS generation and p53 levels depending on the p53 cell line status (wild type or mutant. Therefore, we propose Lebein as a new candidate for development of potential therapies for melanoma.

Protective role of morin, a flavonoid, against high glucose induced oxidative stress mediated apoptosis in primary rat hepatocytes.

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Radhika Kapoor

Full Text Available Apoptosis is an early event of liver damage in diabetes and oxidative stress has been linked to accelerate the apoptosis in hepatocytes. Therefore, the compounds that can scavenge ROS may confer regulatory effects on high-glucose induced apoptosis. In the present study, primary rat hepatocytes were exposed to high concentration (40 mM of glucose. At this concentration decreased cell viability and enhanced ROS generation was observed. Depleted antioxidant status of hepatocytes under high glucose stress was also observed as evident from transcriptional level and activities of antioxidant enzymes. Further, mitochondrial depolarisation was accompanied by the loss of mitochondrial integrity and altered expression of Bax and Bcl-2. Increased translocation of apoptotic proteins like AIF (Apoptosis inducing factor & Endo-G (endonuclease-G from its resident place mitochondria to nucleus was also observed. Cyt-c residing in the inter-membrane space of mitochondria also translocated to cytoplasm. These apoptotic proteins initiated caspase activation, DNA fragmentation, chromatin condensation, increased apoptotic DNA content in glucose treated hepatocytes, suggesting mitochondria mediated apoptotic mode of cell death. Morin, a dietary flavonoid from Psidium guajava was effective in increasing the cell viability and decreasing the ROS level. It maintained mitochondrial integrity, inhibited release of apoptotic proteins from mitochondria, prevented DNA fragmentation, chromatin condensation and hypodiploid DNA upon exposure to high glucose. This study confirms the capacity of dietary flavonoid Morin in regulating apoptosis induced by high glucose via mitochondrial mediated pathway through intervention of oxidative stress.

Is sub-chronic exercise in Combination with medicinal nanoparticles a protective strategy against Doxorubicin-induced Hepatic oxidative stress and apoptosis in aging model rats?

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Saied Kamal Sadat-Hoseini

2017-10-01

Full Text Available Objective(s: Oxidative stress and apoptosis are the major side effects of doxorubicin (DOX and the advantages accruing fromexercise and some medicinal herbs in mitigation of these toxic side effects is well documented. But so far, the effects of exercise in combination with medicinal nanoparticles on oxidative stress and apoptosis signaling simultaneously, in liver tissue are unknown. Hence, we investigated whether Treadmill Runningin combination with Nanocurcumin protects the liver tissue against these toxic side effects (oxidative stress and apoptosis simultaneously of DOX treatment in aging rats induced by D-galactose. Materials and Methods: Fifty-six Wistar male rats received a daily injection of D-galactose (100 mg/kg/day, i.p. then randomly assigned to 7 sub-groups. The training protocol included treadmill running progressively between 25 to 54 min/day and 15 to 20m/min, 5 days/week for six weeks. DOX (1 mg⋅mL−1⋅kg−1⋅day−1 was administrated intraperitoneally for 15 days and Nanocurcumin was administrated orally for 2 weeks (100 mg/kg/day. Results: Nanocurcumin Consumptionled to insignificant increase in SOD, MDA and insignificant decrease in AIF levels. Treadmill runningled to insignificant increase in SOD and insignificant decrease in AIF and MDA levels. The combination of Treadmill runningand Nanocurcumin led to significant decrease in MDA and insignificant increase in SOD and insignificant decrease in AIF levels. Conclusion: In conclusion, Treadmill runningexerciseand Nanocurcumin partly mitigates the toxic side effects of DOX treatment. But this amount of treatment does not play a required role against DOX-induced hepatic damage.

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

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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.

Alpha-tocopheryl succinate inhibits autophagic survival of prostate cancer cells induced by vitamin K3 and ascorbate to trigger cell death.

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Tomasetti, Marco; Nocchi, Linda; Neuzil, Jiri; Goodwin, Jacob; Nguyen, Maria; Dong, Lanfeng; Manzella, Nicola; Staffolani, Sara; Milanese, Claudio; Garrone, Beatrice; Alleva, Renata; Borghi, Battista; Santarelli, Lory; Guerrieri, Roberto

2012-01-01

The redox-silent vitamin E analog α-tocopheryl succinate (α-TOS) was found to synergistically cooperate with vitamin K3 (VK3) plus ascorbic acid (AA) in the induction of cancer cell-selective apoptosis via a caspase-independent pathway. Here we investigated the molecular mechanism(s) underlying cell death induced in prostate cancer cells by α-TOS, VK3 and AA, and the potential use of targeted drug combination in the treatment of prostate cancer. The generation of ROS, cellular response to oxidative stress, and autophagy were investigated in PC3 prostate cancer cells by using drugs at sub-toxic doses. We evaluated whether PARP1-mediated apoptosis-inducing factor (AIF) release plays a role in apoptosis induced by the combination of the agents. Next, the effect of the combination of α-TOS, VK3 and AA on tumor growth was examined in nude mice. VK3 plus AA induced early ROS formation associated with induction of autophagy in response to oxidative stress, which was reduced by α-TOS, preventing the formation of autophagosomes. α-TOS induced mitochondrial destabilization leading to the release of AIF. Translocation of AIF from mitochondria to the nucleus, a result of the combinatorial treatment, was mediated by PARP1 activation. The inhibition of AIF as well as of PARP1 efficiently attenuated apoptosis triggered by the drug combination. Using a mouse model of prostate cancer, the combination of α-TOS, VK3 and AA was more efficient in tumor suppression than when the drugs were given separately, without deleterious side effects. α-TOS, a mitochondria-targeting apoptotic agent, switches at sub-apoptotic doses from autophagy-dependent survival of cancer cells to their demise by promoting the induction of apoptosis. Given the grim prognosis for cancer patients, this finding is of potential clinical relevance.

Alpha-tocopheryl succinate inhibits autophagic survival of prostate cancer cells induced by vitamin K3 and ascorbate to trigger cell death.

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Marco Tomasetti

Full Text Available BACKGROUND: The redox-silent vitamin E analog α-tocopheryl succinate (α-TOS was found to synergistically cooperate with vitamin K3 (VK3 plus ascorbic acid (AA in the induction of cancer cell-selective apoptosis via a caspase-independent pathway. Here we investigated the molecular mechanism(s underlying cell death induced in prostate cancer cells by α-TOS, VK3 and AA, and the potential use of targeted drug combination in the treatment of prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: The generation of ROS, cellular response to oxidative stress, and autophagy were investigated in PC3 prostate cancer cells by using drugs at sub-toxic doses. We evaluated whether PARP1-mediated apoptosis-inducing factor (AIF release plays a role in apoptosis induced by the combination of the agents. Next, the effect of the combination of α-TOS, VK3 and AA on tumor growth was examined in nude mice. VK3 plus AA induced early ROS formation associated with induction of autophagy in response to oxidative stress, which was reduced by α-TOS, preventing the formation of autophagosomes. α-TOS induced mitochondrial destabilization leading to the release of AIF. Translocation of AIF from mitochondria to the nucleus, a result of the combinatorial treatment, was mediated by PARP1 activation. The inhibition of AIF as well as of PARP1 efficiently attenuated apoptosis triggered by the drug combination. Using a mouse model of prostate cancer, the combination of α-TOS, VK3 and AA was more efficient in tumor suppression than when the drugs were given separately, without deleterious side effects. CONCLUSIONS/SIGNIFICANCE: α-TOS, a mitochondria-targeting apoptotic agent, switches at sub-apoptotic doses from autophagy-dependent survival of cancer cells to their demise by promoting the induction of apoptosis. Given the grim prognosis for cancer patients, this finding is of potential clinical relevance.

Pharmacological targeting of HSP90 with 17-AAG induces apoptosis of myogenic cells through activation of the intrinsic pathway.

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Wagatsuma, Akira; Takayama, Yuzo; Hoshino, Takayuki; Shiozuka, Masataka; Yamada, Shigeru; Matsuda, Ryoichi; Mabuchi, Kunihiko

2017-12-16

We have shown that pharmacological inhibition of HSP90 ATPase activity induces apoptosis of myoblasts during their differentiation. However, the signaling pathways remain not fully characterized. We report that pharmacological targeting of HSP90 with 17-AAG activates the intrinsic pathway including caspase-dependent and caspase-independent pathways. 17-AAG induces the typical apoptotic phenotypes including PARP cleavage, chromatin condensation, and nuclear fragmentation with mitochondrial release of cytochrome c, Smac/DIABLO, procaspase-9 processing, and caspase-3 activation. AIF and EndoG redistribute from the mitochondria into the cytosol and are partially translocated to the nucleus in 17-AAG-treated cells. These results suggest that caspase-dependent and caspase-independent pathways should be considered in apoptosis of myogenic cells induced by inhibition of HSP90 ATPase activity.

LW-214, a newly synthesized flavonoid, induces intrinsic apoptosis pathway by down-regulating Trx-1 in MCF-7 human breast cells.

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Pan, Di; Li, Wei; Miao, Hanchi; Yao, Jing; Li, Zhiyu; Wei, Libin; Zhao, Li; Guo, Qinglong

2014-02-15

In this study, the anticancer effect of LW-214, a newly synthesized flavonoid, against MCF-7 human breast cancer cells and the underlying mechanisms were investigated. LW-214 triggered the mitochondrial apoptotic pathway by increasing Bax/Bcl-2 ratio, loss of mitochondrial membrane potential (ΔΨm) and caspase-9 activation, degradation of poly (ADP-ribose) polymerase (PARP), cytochrome c (Cyt c) release and apoptosis-inducing factor (AIF) transposition. Further research revealed that both the reactive oxygen species (ROS) generation and the apoptosis signal regulating kinase 1 (ASK1) activation by LW-214 were induced by down-regulating the thioredoxin-1 (Trx-1) expression. The ROS elevation and ASK1 activation induced a sustained phosphorylation of c-Jun N-terminal kinase (JNK), while SP600125, as known as JNK inhibitor, almost reversed LW-214-induced apoptosis in MCF-7 cells. Overexpression of Trx-1 in MCF-7 cells attenuated LW-214-mediated apoptosis as well as the JNK activation and reversed the expression of mitochondrial apoptosis-related protein. Accordingly, the in vivo study showed that LW-214 exhibited a potential antitumor effect in BALB/c species mice inoculated MCF-7 tumor with low systemic toxicity, and the mechanism was the same as in vitro study. Taken together, these findings indicated that LW-214 may down-regulated Trx-1 function, causing intracellular ROS generation and releasing the ASK1, and lead to JNK activation, which consequently induced the mitochondrial apoptosis in vitro and in vivo. Copyright © 2013 Elsevier Inc. All rights reserved.

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

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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)....

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

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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.

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

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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.

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

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Georgina P. Coló

2007-10-01

Full Text Available RAC3 pertenece a la familia de coactivadores de receptores nucleares p160, y se encuentra sobreexpresado en varios tumores. Demostramos previamente que RAC3 es coactivador del factor de transcripción anti-apoptótico NF-kapa;B. En este trabajo investigamos su rol en la apoptosis inducida por H2O2 en una línea celular no tumoral derivada de riñón embrionario humano (HEK293, y por el ligando inductor de apoptosis relacionado a TNF (TRAIL en una línea de leucemia mieloide crónica humana (K562, naturalmente resistente a la muerte por este estímulo. Observamos que las células tumorales K562 poseen niveles altos de RAC3 comparados con las células no tumorales HEK293. La sobreexpresión normal de coactivador o por transfección, inhibe la apoptosis mediante una disminución de la activación de caspasas, translocación del factor inductor de apoptosis (AIF al núcleo, aumento de la actividad de NF-kapa;B y las quinasas AKT y p38 y disminución de la quinasa ERK. Lo opuesto fue observado por disminución de RAC3 mediante la técnica de ARN interferente (RNAi en K562, aumentando así la apoptosis inducida por TRAIL. Estas evidencias sugieren que una sobreexpresión de RAC3 contribuye al desarrollo de tumores, participando en las cascadas que controlan la muerte celular por mecanismos no estrictamente dependientes de hormonas esteroideas y/o de acetilación, constituyendo esto un posible blanco de ataque para el tratamiento de tumores.RAC3 belongs to the family of p160 nuclear receptors coactivators and it is over-expressed in several tumors. We have previously shown that RAC3 is a NF-kappa;B coactivator. In this paper, we investigated the role of RAC3 in cell-sensitivity to apoptosis, using H2O2 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

Human herpesvirus 6A induces apoptosis of primary human fetal astrocytes via both caspase-dependent and -independent pathways

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Gu Bin

2011-12-01

Full Text Available Abstract Background Human herpesvirus 6 (HHV-6 is a T-lymphtropic and neurotropic virus that can infect various types of cells. Sequential studies reported that apoptosis of glia and neurons induced by HHV-6 might act a potential trigger for some central nervous system (CNS diseases. HHV-6 is involved in the pathogenesis of encephalitis, multiple sclerosis (MS and fatigue syndrome. However, the mechanisms responsible for the apoptosis of infected CNS cells induced by HHV-6 are poorly understood. In this study, we investigated the cell death processes of primary human fetal astrocytes (PHFAs during productive HHV-6A infection and the underlying mechanisms. Results HHV-6A can cause productive infection in primary human fetal astrocytes. Annexin V-PI staining and electron microscopic analysis indicated that HHV-6A was an inducer of apoptosis. The cell death was associated with activation of caspase-3 and cleavage of poly (ADP-ribose polymerase (PARP, which is known to be an important substrate for activated caspase-3. Caspase-8 and -9 were also significantly activated in HHV-6A-infected cells. Moreover, HHV-6A infection led to Bax up-regulation and Bcl-2 down-regulation. HHV-6A infection increased the release of Smac/Diablo, AIF and cytochrome c from mitochondria to cytosol, which induced apoptosis via the caspase-dependent and -independent pathways. In addition, we also found that anti-apoptotic factors such as IAPs and NF-κB decreased in HHV-6A infected PHFAs. Conclusion This is the first demonstration of caspase-dependent and -independent apoptosis in HHV-6A-infected glial cells. These findings would be helpful in understanding the mechanisms of CNS diseases caused by HHV-6.

Relative sensitivities of DCE-MRI pharmacokinetic parameters to arterial input function (AIF) scaling.

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Li, Xin; Cai, Yu; Moloney, Brendan; Chen, Yiyi; Huang, Wei; Woods, Mark; Coakley, Fergus V; Rooney, William D; Garzotto, Mark G; Springer, Charles S

2016-08-01

Dynamic-Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI) has been used widely for clinical applications. Pharmacokinetic modeling of DCE-MRI data that extracts quantitative contrast reagent/tissue-specific model parameters is the most investigated method. One of the primary challenges in pharmacokinetic analysis of DCE-MRI data is accurate and reliable measurement of the arterial input function (AIF), which is the driving force behind all pharmacokinetics. Because of effects such as inflow and partial volume averaging, AIF measured from individual arteries sometimes require amplitude scaling for better representation of the blood contrast reagent (CR) concentration time-courses. Empirical approaches like blinded AIF estimation or reference tissue AIF derivation can be useful and practical, especially when there is no clearly visible blood vessel within the imaging field-of-view (FOV). Similarly, these approaches generally also require magnitude scaling of the derived AIF time-courses. Since the AIF varies among individuals even with the same CR injection protocol and the perfect scaling factor for reconstructing the ground truth AIF often remains unknown, variations in estimated pharmacokinetic parameters due to varying AIF scaling factors are of special interest. In this work, using simulated and real prostate cancer DCE-MRI data, we examined parameter variations associated with AIF scaling. Our results show that, for both the fast-exchange-limit (FXL) Tofts model and the water exchange sensitized fast-exchange-regime (FXR) model, the commonly fitted CR transfer constant (K(trans)) and the extravascular, extracellular volume fraction (ve) scale nearly proportionally with the AIF, whereas the FXR-specific unidirectional cellular water efflux rate constant, kio, and the CR intravasation rate constant, kep, are both AIF scaling insensitive. This indicates that, for DCE-MRI of prostate cancer and possibly other cancers, kio and kep may be more suitable imaging

Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

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Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

2013-10-01

Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

Caspase dependent and independent mechanisms of apoptosis across gestation in a sheep model of placental insufficiency and intrauterine growth restriction.

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Monson, Troy; Wright, Tanner; Galan, Henry L; Reynolds, Paul R; Arroyo, Juan A

2017-05-01

Increased placental apoptosis is a hallmark of intrauterine growth restricted (IUGR). Several molecules have been shown to be involved in the control of apoptosis during this disease. Our objective was to determine the expression of Bcl2, Bax, phospho XIAP, AIF, caspase 3 and 9, and telomerase activity across gestation in an ovine hyperthermia-induced model of IUGR. Pregnant sheep were placed in hyperthermic (HT) conditions to induce IUGR along with age-matched controls. Placental tissues were collected at 55 (early), 95 (mid-gestation) and 130 (near-term) days of gestational age (dGA) to determine the expression of apoptotic molecules during the development of IUGR. Compared to the control placenta, IGUR pregnancies showed: significantly reduced placental Bcl2 in early gestation (55 dGA) with a significant increase observed at mid gestation (95 dGA); decreased placental pXIAP at both mid and near term gestational days (95 and 130 dGA); placental AIF increased only at 55 dGA (early gestation); active caspase 3 increased at both mid and near term gestational days (95 and 130 dGA); caspase 9 only increased at mid gestation (95 dGA) and decreased Telomerase activity near term. Placental apoptosis, mediated in part by the apoptosis related molecule, participates in the development of IUGR. Findings from this study suggest a caspase-independent apoptotic pathway during early gestation and caspase-dependent apoptosis at mid and near term gestation. The data also implicate decreased activation of XIAP as a plausible factor involved in the control of placental apoptosis during IUGR.

JS-K, a GST-activated nitric oxide generator, induces DNA double-strand breaks, activates DNA damage response pathways, and induces apoptosis in vitro and in vivo in human multiple myeloma cells.

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Kiziltepe, Tanyel; Hideshima, Teru; Ishitsuka, Kenji; Ocio, Enrique M; Raje, Noopur; Catley, Laurence; Li, Chun-Qi; Trudel, Laura J; Yasui, Hiroshi; Vallet, Sonia; Kutok, Jeffery L; Chauhan, Dharminder; Mitsiades, Constantine S; Saavedra, Joseph E; Wogan, Gerald N; Keefer, Larry K; Shami, Paul J; Anderson, Kenneth C

2007-07-15

Here we investigated the cytotoxicity of JS-K, a prodrug designed to release nitric oxide (NO(*)) following reaction with glutathione S-transferases, in multiple myeloma (MM). JS-K showed significant cytotoxicity in both conventional therapy-sensitive and -resistant MM cell lines, as well as patient-derived MM cells. JS-K induced apoptosis in MM cells, which was associated with PARP, caspase-8, and caspase-9 cleavage; increased Fas/CD95 expression; Mcl-1 cleavage; and Bcl-2 phosphorylation, as well as cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (EndoG) release. Moreover, JS-K overcame the survival advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells. Mechanistic studies revealed that JS-K-induced cytotoxicity was mediated via NO(*) in MM cells. Furthermore, JS-K induced DNA double-strand breaks (DSBs) and activated DNA damage responses, as evidenced by neutral comet assay, as well as H2AX, Chk2 and p53 phosphorylation. JS-K also activated c-Jun NH(2)-terminal kinase (JNK) in MM cells; conversely, inhibition of JNK markedly decreased JS-K-induced cytotoxicity. Importantly, bortezomib significantly enhanced JS-K-induced cytotoxicity. Finally, JS-K is well tolerated, inhibits tumor growth, and prolongs survival in a human MM xenograft mouse model. Taken together, these data provide the preclinical rationale for the clinical evaluation of JS-K to improve patient outcome in MM.

Aspartame-induced apoptosis in PC12 cells.

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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.

Induction and regulation of tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand-mediated apoptosis in renal cell carcinoma.

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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.

An Involvement of PI3-K/Akt Activation and Inhibition of AIF Translocation in Neuroprotective Effects of Undecylenic Acid (UDA) Against Pro-Apoptotic Factors-Induced Cell Death in Human Neuroblastoma SH-SY5Y Cells.

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Jantas, Danuta; Piotrowski, Marek; Lason, Wladyslaw

2015-12-01

Undecylenic acid (UDA), a naturally occurring 11-carbon unsaturated fatty acid, has been used for several years as an economical antifungal agent and a nutritional supplement. Recently, the potential usefulness of UDA as a neuroprotective drug has been suggested based on the ability of this agent to inhibit μ-calpain activity. In order to verify neuroprotective potential of UDA, we tested protective efficacy of this compound against cell damage evoked by pro-apoptotic factors (staurosporine and doxorubicin) and oxidative stress (hydrogen peroxide) in human neuroblastoma SH-SY5Y cells. We showed that UDA partially protected SH-SY5Y cells against the staurosporine- and doxorubicin-evoked cell death; however, this effect was not connected with its influence on caspase-3 activity. UDA decreased the St-induced changes in mitochondrial and cytosolic AIF level, whereas in Dox-model it affected only the cytosolic AIF content. Moreover, UDA (1-40 μM) decreased the hydrogen peroxide-induced cell damage which was connected with attenuation of hydrogen peroxide-mediated necrotic (PI staining, ADP/ATP ratio) and apoptotic (mitochondrial membrane potential, caspase-3 activation, AIF translocation) changes. Finally, we demonstrated that an inhibitor of PI3-K/Akt (LY294002) but not MAPK/ERK1/2 (U0126) pathway blocked the protection mediated by UDA in all tested models of SH-SY5Y cell injury. These in vitro data point to UDA as potentially effective neuroprotectant the utility of which should be further validated in animal studies. © 2015 Wiley Periodicals, Inc.

Arctigenin, a dietary phytoestrogen, induces apoptosis of estrogen receptor-negative breast cancer cells through the ROS/p38 MAPK pathway and epigenetic regulation.

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Hsieh, Chia-Jung; Kuo, Po-Lin; Hsu, Ying-Chan; Huang, Ya-Fang; Tsai, Eing-Mei; Hsu, Ya-Ling

2014-02-01

This study investigates the anticancer effect of arctigenin (ATG), a natural lignan product of Arctium lappa L., in human breast cancer MDA-MB-231 cells. Results indicate that ATG inhibits MDA-MB-231 cell growth by inducing apoptosis in vitro and in vivo. ATG triggers the mitochondrial caspase-independent pathways, as indicated by changes in Bax/Bcl-2 ratio, resulting in AIF and EndoG nuclear translocation. ATG increased cellular reactive oxygen species (ROS) production by increasing p22(phox)/NADPH oxidase 1 interaction and decreasing glutathione level. ATG clearly increases the activation of p38 MAPK, but not JNK and ERK1/2. Antioxidant EUK-8, a synthetic catalytic superoxide and hydrogen peroxide scavenger, significantly decreases ATG-mediated p38 activation and apoptosis. Blocking p38 with a specific inhibitor suppresses ATG-mediated Bcl-2 downregulation and apoptosis. Moreover, ATG activates ATF-2, a transcription factor activated by p38, and then upregulates histone H3K9 trimethylation in the Bcl-2 gene promoter region, resulting in Bcl-2 downregulation. Taken together, the results demonstrate that ATG induces apoptosis of MDA-MB-231 cells via the ROS/p38 MAPK pathway and epigenetic regulation of Bcl-2 by upregulation of histone H3K9 trimethylation. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Effects of Ginkgo biloba extract on the apoptosis of oxygen and glucose-deprived SH-SY5Y cells and its mechanism.

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Ba, Xiao-Hong; Min, Lian-Qiu

2015-01-01

The aim was to observe the effects of the extract of Ginkgo biloba (EGb761) on the apoptosis of oxygen and glucose-deprived (OGD) human neuroblastoma cells (SH-SY5Y) cells and explore its mechanism. SH-SY5Y cells were divided into normal control group, OGD group, OGD for 4 h and EGb761-pretreated groups including very low-concentration (20 μg/ml), low-concentration group (25 μg/ml), moderate-concentration group (50 μg/ml) and high-concentration group (100 μg/ml). Twenty four hours after reoxygenation, cell viability was determined with 3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide assay, apoptosis rate was detected with annexin V-fluorescein isothiocyanate/propidium iodide double staining flow cytometry and the protein level of apoptosis-inducing factor (AIF) was observed with immunofluorescence technique in each group. Cell viability was significantly lower in OGD group than in EGb761-pretreated groups, especially in moderate-concentration group (50 μg/ml) (P cells probably through inhibiting AIF nuclear translocation. This study provides a theoretical basis for the application of EGb761 in clinical practice.

Prediction of localization and interactions of apoptotic proteins

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Matula Pavel

2009-07-01

Full Text Available Abstract During apoptosis several mitochondrial proteins are released. Some of them participate in caspase-independent nuclear DNA degradation, especially apoptosis-inducing factor (AIF and endonuclease G (endoG. Another interesting protein, which was expected to act similarly as AIF due to the high sequence homology with AIF is AIF-homologous mitochondrion-associated inducer of death (AMID. We studied the structure, cellular localization, and interactions of several proteins in silico and also in cells using fluorescent microscopy. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the lipid membranes. Bioinformatic predictions were conducted to analyze the interactions of the studied proteins with each other and with other possible partners. We conducted molecular modeling of proteins with unknown 3D structures. These models were then refined by MolProbity server and employed in molecular docking simulations of interactions. Our results show data acquired using a combination of modern in silico methods and image analysis to understand the localization, interactions and functions of proteins AMID, AIF, endonuclease G, and other apoptosis-related proteins.

Lebein, a snake venom disintegrin, suppresses human colon cancer cells proliferation and tumor-induced angiogenesis through cell cycle arrest, apoptosis induction and inhibition of VEGF expression.

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Zakraoui, Ons; Marcinkiewicz, Cezary; Aloui, Zohra; Othman, Houcemeddine; Grépin, Renaud; Haoues, Meriam; Essafi, Makram; Srairi-Abid, Najet; Gasmi, Ammar; Karoui, Habib; Pagès, Gilles; Essafi-Benkhadir, Khadija

2017-01-01

Lebein, is an heterodimeric disintegrin isolated from Macrovipera lebetina snake venom that was previously characterized as an inhibitor of ADP-induced platelet aggregation. In this study, we investigated the effect of Lebein on the p53-dependent growth of human colon adenocarcinoma cell lines. We found that Lebein significantly inhibited LS174 (p53wt), HCT116 (p53wt), and HT29 (p53mut) colon cancer cell viability by inducing cell cycle arrest through the modulation of expression levels of the tumor suppression factor p53, cell cycle regulating proteins cyclin D1, CDK2, CDK4, retinoblastoma (Rb), CDK1, and cyclin-dependent kinase inhibitors p21 and p27. Interestingly, Lebein-induced apoptosis of colon cancer cells was dependent on their p53 status. Thus, in LS174 cells, cell death was associated with PARP cleavage and the activation of caspases 3 and 8 while in HCT116 cells, Lebein induced caspase-independent apoptosis through increased expression of apoptosis inducing factor (AIF). In LS174 cells, Lebein triggers the activation of the MAPK ERK1/2 pathway through induction of reactive oxygen species (ROS). It also decreased cell adhesion and migration to fibronectin through down regulation of α5β1 integrin. Moreover, Lebein significantly reduced the expression of two angiogenesis stimulators, Vascular Endothelial Growth Factor (VEGF) and Neuropilin 1 (NRP1). It inhibited the VEGF-induced neovascularization process in the quail embryonic CAM system and blocked the development of human colon adenocarcinoma in nude mice. Overall, our work indicates that Lebein may be useful to design a new therapy against colon cancer. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Resveratrol induces growth arrest and apoptosis through activation of FOXO transcription factors in prostate cancer cells.

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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

Peroxynitrite induces apoptosis of mouse cochlear hair cells via a Caspase-independent pathway in vitro.

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Cao, Zhixin; Yang, Qianqian; Yin, Haiyan; Qi, Qi; Li, Hongrui; Sun, Gaoying; Wang, Hongliang; Liu, Wenwen; Li, Jianfeng

2017-11-01

Peroxynitrite (ONOO - ) is a potent and versatile oxidant implicated in a number of pathophysiological processes. The present study was designed to investigate the effect of ONOO - on the cultured cochlear hair cells (HCs) of C57BL/6 mice in vitro as well as the possible mechanism underlying the action of such an oxidative stress. The in vitro primary cultured cochlear HCs were subjected to different concentrations of ONOO - , then, the cell survival and morphological changes were examined by immunofluorescence and transmission electron microscopy (TEM), the apoptosis was determined by Terminal deoxynucleotidyl transferase dUNT nick end labeling (TUNEL) assay, the mRNA expressions of Caspase-3, Caspase-8, Caspase-9, Apaf1, Bcl-2, and Bax were analyzed by RT-PCR, and the protein expressions of Caspase-3 and AIF were assessed by immunofluorescence. This work demonstrated that direct exposure of primary cultured cochlear HCs to ONOO - could result in a base-to-apex gradient injury of HCs in a concentration-dependent manner. Furthermore, ONOO - led to much more losses of outer hair cells than inner hair cells mainly through the induction of apoptosis of HCs as evidenced by TEM and TUNEL assays. The mRNA expressions of Caspase-8, Caspase-9, Apaf1, and Bax were increased and, meanwhile, the mRNA expression of Bcl-2 was decreased in response to ONOO - treatment. Of interesting, the expression of Caspase-3 had no significant change, whereas, the expression alteration of AIF was observed. These results suggested that ONOO - can effectively damage the survival of cochlear HCs via triggering the apoptotic pathway. The findings from this work suggest that ONOO - -induced apoptosis is mediated, at least in part, via a Caspase-independent pathway in cochlear HCs.

Fisetin Induces Apoptosis of HSC3 Human Oral Cancer Cells Through Endoplasmic Reticulum Stress and Dysfunction of Mitochondria-mediated Signaling Pathways.

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Shih, Yung-Luen; Hung, Fang-Ming; Lee, Ching-Hsiao; Yeh, Ming-Yang; Lee, Mei-Hui; Lu, Hsu-Feng; Chen, Yung-Liang; Liu, Jia-You; Chung, Jing-Gung

2017-01-01

Oral cancer has been reported to be one of the major cancer-related diseases in human populations and the treatment of oral cancer is still unsatisfied. Fisetin, is a flavonoid from plants and has several biological activities such as antioxidant, anti-inflammatory and anticancer function, but its cytotoxicity in human oral cancer cells is unknown. In the present study, we investigated fisetin-induced cytotoxic effects on HSC3 human oral cancer cells in vitro. Materials and Methods/Results: We used flow cytometric assay to show fisetin induced apoptotic cell death through increased reactive oxygen species and Ca 2+ , but reduced the mitochondrial membrane potential and increased caspase-8, -9 and -3 activities in HSC3 cells. Furthermore, we also used 4' 6-diamidino-2-phenylindole staining to show that fisetin induced chromatin condensation (apoptotic cell death), and Comet assay to show that fisetin induced DNA damage in HSC3 cells. Western blotting was used to examine the levels of apoptotic-associated protein and results indicated that fisetin increased expression of pro-apoptotic proteins such as B-cell lymphoma 2 (BCL2) antagonist/killer (BAK) and BCL2-associated X (BAX) but reduced that of anti-apoptotic protein such as BCL2 and BCL-x, and increased the cleaved forms of caspase-3, -8 and -9, and cytochrome c, apoptosis-inducing factor (AIF) and endonuclease G (ENDO G) in HSC3 cells. Confocal microscopy showed that fisetin increased the release of cytochrome c, AIF and ENDO G from mitochondria into the cytoplasm. Based on these observations, we suggest that fisetin induces apoptotic cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

A Taiwanese Propolis Derivative Induces Apoptosis through Inducing Endoplasmic Reticular Stress and Activating Transcription Factor-3 in Human Hepatoma Cells

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Fat-Moon Suk

2013-01-01

Full Text Available Activating transcription factor-(ATF- 3, a stress-inducible transcription factor, is rapidly upregulated under various stress conditions and plays an important role in inducing cancer cell apoptosis. NBM-TP-007-GS-002 (GS-002 is a Taiwanese propolin G (PPG derivative. In this study, we examined the antitumor effects of GS-002 in human hepatoma Hep3B and HepG2 cells in vitro. First, we found that GS-002 significantly inhibited cell proliferation and induced cell apoptosis in dose-dependent manners. Several main apoptotic indicators were found in GS-002-treated cells, such as the cleaved forms of caspase-3, caspase-9, and poly(ADP-ribose polymerase (PARP. GS-002 also induced endoplasmic reticular (ER stress as evidenced by increases in ER stress-responsive proteins including glucose-regulated protein 78 (GRP78, growth arrest- and DNA damage-inducible gene 153 (GADD153, phosphorylated eukaryotic initiation factor 2α (eIF2α, phosphorylated protein endoplasmic-reticular-resident kinase (PERK, and ATF-3. The induction of ATF-3 expression was mediated by mitogen-activated protein kinase (MAPK signaling pathways in GS-002-treated cells. Furthermore, we found that GS-002 induced more cell apoptosis in ATF-3-overexpressing cells. These results suggest that the induction of apoptosis by the propolis derivative, GS-002, is partially mediated through ER stress and ATF-3-dependent pathways, and GS-002 has the potential for development as an antitumor drug.

1-Benzyl-2-Phenylbenzimidazole (BPB, a Benzimidazole Derivative, Induces Cell Apoptosis in Human Chondrosarcoma through Intrinsic and Extrinsic Pathways

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Ju-Fang Liu

2012-12-01

Full Text Available In this study, we investigated the anticancer effects of a new benzimidazole derivative, 1-benzyl-2-phenyl -benzimidazole (BPB, in human chondrosarcoma cells. BPB-mediated apoptosis was assessed by the MTT assay and flow cytometry analysis. The in vivo efficacy was examined in a JJ012 xenograft model. Here we found that BPB induced apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 but not in primary chondrocytes. BPB induced upregulation of Bax, Bad and Bak, downregulation of Bcl-2, Bid and Bcl-XL and dysfunction of mitochondria in chondrosarcoma. In addition, BPB also promoted cytosolic releases AIF and Endo G. Furthermore, it triggered extrinsic death receptor-dependent pathway, which was characterized by activating Fas, FADD and caspase-8. Most importantly, animal studies revealed a dramatic 40% reduction in tumor volume after 21 days of treatment. Thus, BPB may be a novel anticancer agent for the treatment of chondrosarcoma.

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)

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

Cantharidin Induced Oral Squamous Cell Carcinoma Cell Apoptosis via the JNK-Regulated Mitochondria and Endoplasmic Reticulum Stress-Related Signaling Pathways.

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Chin-Chuan Su

Full Text Available Oral cancer is a subtype of head and neck cancer which represents 2.65% of all human malignancies. Most of oral cancer is histopathologically diagnosed as oral squamous cell carcinoma (OSCC. OSCC is characterized by a high degree of local invasion and a high rate of metastasis to the cervical lymph nodes. How to prevention and treatment of OSCC is important and imperative. Here, we investigated the therapeutic effect and molecular mechanism of cantharidin, an active compound isolated from blister beetles, on OSCC in vitro. Results showed that cantharidin significantly decreased cell viability in human tongue squamous carcinoma-derived SAS, CAL-27, and SCC-4 cell lines. The further mechanistic studies were carried out in SAS cells. Cantharidin also significantly increased apoptosis-related signals, including caspase-9, caspase-7 and caspase-3 proteins. Besides, cantharidin decreased mitochondrial transmembrane potential (MMP and induced cytochrome c and apoptosis inducing factor (AIF release. Cantharidin also increased Bax, Bid, and Bak protein expressions and decreased Bcl-2 protein expression. Cantharidin could also increase the endoplasmic reticulum (ER stress signals, including the expressions of phosphorylated eIF-2α and CHOP, but not Grp78 and Grp94. Furthermore, cantharidin reduced pro-caspase-12 protein expression. In signals of mitogen-activated protein kinases, cantharidin increased the phosphorylation of JNK, but not ERK and p38. Transfection of shRNA-JNK to OSCC cells effectively reversed the cantharidin-induced cell apoptotic signals, including the mitochondrial and ER stress-related signaling molecules. Taken together, these findings suggest that cantharidin induces apoptosis in OSCC cells via the JNK-regulated mitochondria and ER stress-related signaling pathways.

Comparison of first pass bolus AIFs extracted from sequential 18F-FDG PET and DSC-MRI of mice

International Nuclear Information System (INIS)

Evans, Eleanor; Sawiak, Stephen J.; Ward, Alexander O.; Buonincontri, Guido; Hawkes, Robert C.; Adrian Carpenter, T.

2014-01-01

Accurate kinetic modelling of in vivo physiological function using positron emission tomography (PET) requires determination of the tracer time–activity curve in plasma, known as the arterial input function (AIF). The AIF is usually determined by invasive blood sampling methods, which are prohibitive in murine studies due to low total blood volumes. Extracting AIFs from PET images is also challenging due to large partial volume effects (PVE). We hypothesise that in combined PET with magnetic resonance imaging (PET/MR), a co-injected bolus of MR contrast agent and PET ligand can be tracked using fast MR acquisitions. This protocol would allow extraction of a MR AIF from MR contrast agent concentration–time curves, at higher spatial and temporal resolution than an image-derived PET AIF. A conversion factor could then be applied to the MR AIF for use in PET kinetic analysis. This work has compared AIFs obtained from sequential DSC-MRI and PET with separate injections of gadolinium contrast agent and 18 F-FDG respectively to ascertain the technique′s validity. An automated voxel selection algorithm was employed to improve MR AIF reproducibility. We found that MR and PET AIFs displayed similar character in the first pass, confirmed by gamma variate fits (p<0.02). MR AIFs displayed reduced PVE compared to PET AIFs, indicating their potential use in PET/MR studies

Platelet-derived growth factor (PDGF)-signaling mediates radiation-induced apoptosis in human prostate cancer cells with loss of p53 function

International Nuclear Information System (INIS)

Kim, Harold E.; Han, Sue J.; Kasza, Thomas; Han, Richard; Choi, Hyeong-Seon; Palmer, Kenneth C.; Kim, Hyeong-Reh C.

1997-01-01

Platelet-derived growth factor (PDGF) signals a diversity of cellular responses in vitro, including cell proliferation, survival, transformation, and chemotaxis. PDGF functions as a 'competence factor' to induce a set of early response genes expressed in G 1 including p21 WAF1/CIP1 , a functional mediator of the tumor suppressor gene p53 in G 1 /S checkpoint. For PDGF-stimulated cells to progress beyond G 1 and transit the cell cycle completely, progression factors in serum such as insulin and IGF-1 are required. We have recently shown a novel role of PDGF in inducing apoptosis in growth-arrested murine fibroblasts. The PDGF-induced apoptosis is rescued by insulin, suggesting that G 1 /S checkpoint is a critical determinant for PDGF-induced apoptosis. Because recent studies suggest that radiation-induced signal transduction pathways interact with growth factor-mediated signaling pathways, we have investigated whether activation of the PDGF-signaling facilitates the radiation-induced apoptosis in the absence of functional p53. For this study we have used the 125-IL cell line, a mutant p53-containing, highly metastatic, and hormone-unresponsive human prostate carcinoma cell line. PDGF signaling is constitutively activated by transfection with a p28 v-sis expression vector, which was previously shown to activate PDGF α- and β- receptors. Although the basal level of p21 WAF1/CIP1 expression and radiation-induced apoptosis were not detectable in control 125-IL cells as would be predicted in mutant p53-containing cells, activation of PDGF-signaling induced expression of p21 WAF1/CIP1 and radiation-induced apoptosis. Our study suggests that the level of 'competence' growth factors including PDGF may be one of the critical determinants for radiation-induced apoptosis, especially in cells with loss of p53 function at the site of radiotherapy in vivo

Rituximab enhances radiation-triggered apoptosis in non-Hodgkin's lymphoma cells via caspase-dependent and - independent mechanisms

International Nuclear Information System (INIS)

Skvortsova, I.; Skvortsov, S.; Popper, B.A.; Haidenberger, A.; Saurer, M.; Gunkel, A.R.; Zwierzina, H.; Lukas, P.

2006-01-01

Rituximab (RTX), a chimeric human anti-CD20 monoclonal antibody, is currently employed in the treatment of malignant non-Hodgkin's lymphoma (NHL) either alone or in combination with other cytotoxic approaches. The present study examines the effects of ionizing radiation in combination with RTX on proliferation and apoptosis development in B-lymphoma RL and Raji cells. RTX was used at a concentration of 10 μg/mL 24 hours prior to irradiation at a single dose of 9 Gy. CD20 expression, cell viability, apoptosis, mitochondrial membrane potential and apoptosis-related proteins were evaluated in the treated B cells. The constitutive level of CD20 expression in RL and Raji lymphoma cells did not play an essential role in RTX-induced cell growth delay. Both lymphoma cells showed similar inhibition of cell proliferation without apoptosis development in response to RTX treatment. Exposure to ionizing radiation induced cell growth delay and apoptosis in RL cells, whereas Raji cells showed moderate radio-resistance and activation of cell growth at 24 hours after irradiation, which was accompanied by increased radiation-triggered CD20 expression. The simultaneous exposure of lymphoma cells to ionizing radiation and RTX abrogated radioresistance of Raji cells and significantly enhanced cell growth delay and apoptosis in RL cells. X-linked inhibitor of apoptosis protein (XIAP) and the inducible form of heat shock protein 70 (Hsp70) were positively modulated by RTX in combination with ionizing radiation in order to induce apoptosis. Furthermore, it was demonstrated that mitochondrial membrane potential dissipation is not an essential component to induce apoptosis-inducing factor (AIF) maturation and apoptosis. Our results show that RTX-triggered enhancement of radiation-induced apoptosis and cell growth delay is achieved by modulation of proteins involved in programmed cell death. (author)

Lycopene protects human SH-SY5Y neuroblastoma cells against hydrogen peroxide-induced death via inhibition of oxidative stress and mitochondria-associated apoptotic pathways

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FENG, CHUNSHENG; LUO, TIANFEI; ZHANG, SHUYAN; LIU, KAI; ZHANG, YANHONG; LUO, YINAN; GE, PENGFEI

2016-01-01

Oxidative stress, which is characterized by excessive production of reactive oxygen species (ROS), is a common pathway that results in neuronal injury or death due to various types of pathological stress. Although lycopene has been identified as a potent antioxidant, its effect on hydrogen peroxide (H2O2)-induced neuronal damage remains unclear. In the present study, pretreatment with lycopene was observed to protect SH-SY5Y neuroblastoma cells against H2O2-induced death via inhibition of apoptosis resulting from activation of caspase-3 and translocation of apoptosis inducing factor (AIF) to the nucleus. Furthermore, the over-produced ROS, as well as the reduced activities of anti-oxidative enzymes, superoxide dismutase and catalase, were demonstrated to be alleviated by lycopene. Additionally, lycopene counteracted H2O2-induced mitochondrial dysfunction, which was evidenced by suppression of mitochondrial permeability transition pore opening, attenuation of the decline of the mitochondrial membrane potential, and inhibition of the increase of Bax and decrease of Bcl-2 levels within the mitochondria. The release of cytochrome c and AIF from the mitochondria was also reduced. These results indicate that lycopene is a potent neuroprotectant against apoptosis, oxidative stress and mitochondrial dysfunction, and could be administered to prevent neuronal injury or death. PMID:27035331

MicroRNA-195 induced apoptosis in hypoxic chondrocytes by targeting hypoxia-inducible factor 1 alpha.

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Bai, R; Zhao, A-Q; Zhao, Z-Q; Liu, W-L; Jian, D-M

2015-02-01

The chondrocytes, the resident cells of cartilage, are maintained and take effects in the whole life upon chronic hypoxic exposure, which hypoxia-inducible factor 1 alpha (HIF-1α) play pivotal roles in response to. Dysregulation of some microRNA (miRNAs) have also been identified to be involved in hypoxia-related physiologic and pathophysiologic responses in some tissues or cell lines. However, the mechanism of miRNAs reponse to hypoxia remain largely unknown in chondrocytes, including the microRNA-195 (miR-195). AIM To investigate the effects of microRNAs (miRNAs) and hypoxia-inducible factor 1 alpha (HIF-1α) on chondrocytes in physiologic environment. We compared the expression of miR-195 and HIF-1α mRNA on hypoxia with that on normoxia in ATDC 5 cells by qRT-PCR. Further experiments was performed to confirmed the relationships of miR-195 and HIF-1α by bioinformatics analysis and dual reporter gene assay. we also assessed the effect of miR-195 on apoptosis in hypoxic ATDC 5 cells by transfect with miR-195 mimics. It was found the downregulated miR-195 and upregulated HIF-1α were present in hypoxic ATDC 5 cells. miR-195 negatively regulated HIF-1α by targeting its 3'-untranslated region. Moreover, the founding indicated miR-195 greatly increased apoptosis and downregulated HIF-1α mRNA occurred simultaneously in hypoxic chondrocytes. We concluded that miR-195 induced apoptosis in hypoxic chondrocytes by directly targeting HIF-1α.

Brain-derived neurotrophic factor exerts neuroprotective actions against amyloid β-induced apoptosis in neuroblastoma cells

OpenAIRE

KIM, JIN HEE

2014-01-01

Alzheimer’s disease (AD) brains demonstrate decreased levels of brain-derived neurotrophic factor (BDNF) and increased levels of β-amyloid peptide (Aβ), which is neurotoxic. The present study assessed the impact of BDNF on the toxic effects of Aβ25–35-induced apoptosis and the effects on BDNF-mediated signaling using the MTT assay, western blotting and reverse transcription quantitative polymerase chain reaction. Aβ25–35 was found to induce an apoptosis, dose-dependent effect on SH-SY5Y neuro...

Daintain/AIF-1 Plays Roles in Coronary Heart Disease via Affecting the Blood Composition and Promoting Macrophage Uptake and Foam Cell Formation

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Junhan Wang

2013-07-01

Full Text Available Background: Daintain/AIF-1 is an inflammatory polypeptide factor/allograft inflammatory factor 1 derived from macrophages. It is characterized in APOE-/- mice as a novel inflammatory factor associated with atherosclerosis. The purpose of this study was to characterize its function in human atherosclerosis. Methods: Immunohistochemistry was used to identify the expression of daintain/AIF-1 in vessel segments within and far from atherosclerotic plaques; High-performance liquid chromatography (HPLC was used to display the effects of daintain/AIF-1 on C-reactive protein (CRP, oxidative capacity and superoxide dismutase (SOD in vivo; Oil Red O Staining was used to show the effects of daintain/AIF-1 on uptake of oxidized low density lipoprotein (ox-LDL into U937 cells, a macrophage line; Western Blot was used to test scavenger receptor A (SRA expression. Results: A high density of daintain/AIF-1 was observed in the tunica intima and media of coronary artery with atherosclerotic plaque, and fewer daintain/AIF-1 in the vessels without atherosclerotic plaque; Daintain/AIF-1 injected intravenously into BALB/c mice boosted oxidative capacity, significantly impaired SOD activities and augmented the CRP level in blood. According to the oil red O test, daintain/AIF-1 profoundly facilitated the uptake of ox-LDL in U937 macrophages and formation of foam cells in the endothelium. We also found that the molecular mechanisms are effective by promoting overexpression of SRA on macrophages. Conclusion: These findings implicate that the inflammatory factor daintain/AIF-1 is closely associated with atherogenesis, and could be further characterized as a novel risk factor for atherosclerosis

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.

Comparison of first pass bolus AIFs extracted from sequential {sup 18}F-FDG PET and DSC-MRI of mice

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Evans, Eleanor, E-mail: ee244@cam.ac.uk [Wolfson Brain Imaging Centre, Department of Clinical Neurosciences, School of Clinical Medicine, University of Cambridge, Cambridge Biomedical Campus, Cambridge, CB2 0QQ (United Kingdom); Sawiak, Stephen J. [Wolfson Brain Imaging Centre, Department of Clinical Neurosciences, School of Clinical Medicine, University of Cambridge, Cambridge Biomedical Campus, Cambridge, CB2 0QQ (United Kingdom); Behavioural and Clinical Neuroscience Institute, Department of Experimental Psychology, University of Cambridge, Cambridge, CB2 3EB (United Kingdom); Ward, Alexander O.; Buonincontri, Guido; Hawkes, Robert C.; Adrian Carpenter, T. [Wolfson Brain Imaging Centre, Department of Clinical Neurosciences, School of Clinical Medicine, University of Cambridge, Cambridge Biomedical Campus, Cambridge, CB2 0QQ (United Kingdom)

2014-01-11

Accurate kinetic modelling of in vivo physiological function using positron emission tomography (PET) requires determination of the tracer time–activity curve in plasma, known as the arterial input function (AIF). The AIF is usually determined by invasive blood sampling methods, which are prohibitive in murine studies due to low total blood volumes. Extracting AIFs from PET images is also challenging due to large partial volume effects (PVE). We hypothesise that in combined PET with magnetic resonance imaging (PET/MR), a co-injected bolus of MR contrast agent and PET ligand can be tracked using fast MR acquisitions. This protocol would allow extraction of a MR AIF from MR contrast agent concentration–time curves, at higher spatial and temporal resolution than an image-derived PET AIF. A conversion factor could then be applied to the MR AIF for use in PET kinetic analysis. This work has compared AIFs obtained from sequential DSC-MRI and PET with separate injections of gadolinium contrast agent and {sup 18}F-FDG respectively to ascertain the technique′s validity. An automated voxel selection algorithm was employed to improve MR AIF reproducibility. We found that MR and PET AIFs displayed similar character in the first pass, confirmed by gamma variate fits (p<0.02). MR AIFs displayed reduced PVE compared to PET AIFs, indicating their potential use in PET/MR studies.

AIF programs supporting industry self-improvement

International Nuclear Information System (INIS)

Szalay, R.A.; Bivens, A.C.

1985-01-01

The Atomic Industrial Forum (AIF) report ''Nuclear Power in America's Future'' identifies those actions that must be taken by both government and industry if the nuclear power option is to be preserved. This includes initiatives that: 1) reduce construction costs and lead times; 2) establish a national energy policy that reflects the role of electricity and the nuclear power component; 3) provide better financial arrangements; 4) reform licensing and regulation; 5) enhance the light water reactor design; 6) inform the public on the benefits and contributions of nuclear power; and 7) complete the fuel cycle. In attempting to solve nuclear power's problems, the AIF has programs that support and push these needed actions. Much of the initiative must be taken by industry to enhance the nuclear power option. This paper discusses some AIF initiatives and programs that support industry self-improvement

Brazilian Red Propolis Induces Apoptosis-Like Cell Death and Decreases Migration Potential in Bladder Cancer Cells

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Karine Rech Begnini

2014-01-01

Full Text Available Natural products continue to be an invaluable resource of anticancer drug discovery in recent years. Propolis is known for its biological activities such as antimicrobial and antitumor effects. This study assessed the effects of Brazilian red propolis (BRP on apoptosis and migration potential in human bladder cancer cells. The effect of BRP ethanolic extract (25, 50, and 100 μg/mL on 5637 cells was determined by MTT, LIVE/DEAD, and migration (scratch assay assays. Apoptosis induction was investigated through flow cytometry and gene expression profile was investigated by qRT-PCR. Results showed cytotoxicity on MTT and LIVE/DEAD assays, with IC50 values of 95 μg/mL in 24 h of treatment. Cellular migration of 5637 cells was significantly inhibited through lower doses of BRP ethanolic extract (25 and 50 μg/mL. Flow cytometry analyses showed that BRP induced cytotoxicity through apoptosis-like mechanisms in 5637 cells and qRT-PCR revealed increased levels of Bax/Bcl-2 ratio, p53, AIF, and antioxidant enzymes genes. Data suggest that BRP may be a potential source of drugs to bladder cancer treatment.

Brazilian red propolis induces apoptosis-like cell death and decreases migration potential in bladder cancer cells.

Science.gov (United States)

Begnini, Karine Rech; Moura de Leon, Priscila Marques; Thurow, Helena; Schultze, Eduarda; Campos, Vinicius Farias; Martins Rodrigues, Fernanda; Borsuk, Sibele; Dellagostin, Odir Antônio; Savegnago, Lucielli; Roesch-Ely, Mariana; Moura, Sidnei; Padilha, Francine F; Collares, Tiago; Pêgas Henriques, João Antonio; Seixas, Fabiana Kömmling

2014-01-01

Natural products continue to be an invaluable resource of anticancer drug discovery in recent years. Propolis is known for its biological activities such as antimicrobial and antitumor effects. This study assessed the effects of Brazilian red propolis (BRP) on apoptosis and migration potential in human bladder cancer cells. The effect of BRP ethanolic extract (25, 50, and 100 μg/mL) on 5637 cells was determined by MTT, LIVE/DEAD, and migration (scratch assay) assays. Apoptosis induction was investigated through flow cytometry and gene expression profile was investigated by qRT-PCR. Results showed cytotoxicity on MTT and LIVE/DEAD assays, with IC50 values of 95 μg/mL in 24 h of treatment. Cellular migration of 5637 cells was significantly inhibited through lower doses of BRP ethanolic extract (25 and 50 μg/mL). Flow cytometry analyses showed that BRP induced cytotoxicity through apoptosis-like mechanisms in 5637 cells and qRT-PCR revealed increased levels of Bax/Bcl-2 ratio, p53, AIF, and antioxidant enzymes genes. Data suggest that BRP may be a potential source of drugs to bladder cancer treatment.

Infection of Human Fallopian Tube Epithelial Cells with Neisseria gonorrhoeae Protects Cells from Tumor Necrosis Factor Alpha-Induced Apoptosis

Science.gov (United States)

Morales, Priscilla; Reyes, Paz; Vargas, Macarena; Rios, Miguel; Imarai, Mónica; Cardenas, Hugo; Croxatto, Horacio; Orihuela, Pedro; Vargas, Renato; Fuhrer, Juan; Heckels, John E.; Christodoulides, Myron; Velasquez, Luis

2006-01-01

Following infection with Neisseria gonorrhoeae, bacteria may ascend into the Fallopian tubes (FT) and induce salpingitis, a major cause of infertility. In the FT, interactions between mucosal epithelial cells and gonococci are pivotal events in the pathogen's infection cycle and the inflammatory response. In the current study, primary FT epithelial cells were infected in vitro with different multiplicities of infection (MOI) of Pil+ Opa+ gonococci. Bacteria showed a dose-dependent association with cells and induced the secretion of tumor necrosis factor alpha (TNF-α). A significant finding was that gonococcal infection (MOI = 1) induced apoptosis in approximately 30% of cells, whereas increasing numbers of bacteria (MOI = 10 to 100) did not induce apoptosis. Apoptosis was observed in only 11% of cells with associated bacteria, whereas >84% of cells with no adherent bacteria were apoptotic. TNF-α was a key contributor to apoptosis, since (i) culture supernatants from cells infected with gonococci (MOI = 1) induced apoptosis in naïve cultures, suggesting that a soluble factor was responsible; (ii) gonococcal infection-induced apoptosis was inhibited with anti-TNF-α antibodies; and (iii) the addition of exogenous TNF-α induced apoptosis, which was inhibited by the presence of increasing numbers of bacteria (MOI = 10 to 100). These data suggest that TNF-α-mediated apoptosis of FT epithelial cells is likely a primary host defense mechanism to prevent pathogen colonization. However, epithelial cell-associated gonococci have evolved a mechanism to protect the cells from undergoing TNF-α-mediated apoptosis, and this modulation of the host innate response may contribute to establishment of infection. Understanding the antiapoptotic mechanisms used by Neisseria gonorrhoeae will inform the pathogenesis of salpingitis and could suggest new intervention strategies for prevention and treatment of the disease. PMID:16714596

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...

Expression of AIF-1 and RANTES in Unexplained Spontaneous Abortion and Possible Association with Alloimmune Abortion

Institute of Scientific and Technical Information of China (English)

Yong-hong LI; Hai-lin WANG; Ya-juan ZHANG

2007-01-01

Objective To investigate the effects of allograft inflammatory factor-1(AIF-1)and (RANTES) in sera and deciduas on unexplained early spontaneous abortion.Methods AIF-1 and RANTES were examined in sera and deciduas/endometria of 43 unexplained early spontaneous abortion women (group A),40 healthy women with early pregnancy(group B)and 20 healthy women with no pregnancy (group C). Immunohistochemistry and enzyme linked immunosorbent assay (ELISA) were used in this study. Results AIF-1 protein was expressed both in deciduas of group A and in endometria of group C.In group A, H scores in the recurrent abortion deciduas specimens were significantly greater than those in the first abortion;in endometrium,expression of AIF-1 was greater in the secretory than in proliferative phase of group C.In group B,concentrations of RANTES in sera were higher in 7th-8th week of pregnancy than in 6th-7th and ＞8th week of pregnancy;expression of AIF-1 protein showed a negative correlation with RASNTES concentration;a significant increase of the RANTES levels in sera and tissue was observed in group B. Conclusion These results demonstrate, for the first time,that AIF-1 are expressed in deciduas of unexplained spontaneous abortion suggesting that AIF-1 involve in alloimmune abortion; RANTES might act as a novel blocking antibody;AIF-1 and RANTES might act as reliable markers for diagnosis of early alloimmune abortion.

Hepatocyte growth factor enhances death receptor-induced apoptosis by up-regulating DR5

International Nuclear Information System (INIS)

Li, Yang; Fan, Xing; Goodwin, C Rory; Laterra, John; Xia, Shuli

2008-01-01

Hepatocyte growth factor (HGF) and its receptor c-MET are commonly expressed in malignant gliomas and embryonic neuroectodermal tumors including medulloblastoma and appear to play an important role in the growth and dissemination of these malignancies. Dependent on cell context and the involvement of specific downstream effectors, both pro- and anti-apoptotic effects of HGF have been reported. Human medulloblastoma cells were treated with HGF for 24–72 hours followed by death receptor ligand TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) for 24 hours. Cell death was measured by MTT and Annexin-V/PI flow cytometric analysis. Changes in expression levels of targets of interest were measured by Northern blot analysis, quantitative reverse transcription-PCR, Western blot analysis as well as immunoprecipitation. In this study, we show that HGF promotes medulloblastoma cell death induced by TRAIL. TRAIL alone triggered apoptosis in DAOY cells and death was enhanced by pre-treating the cells with HGF for 24–72 h prior to the addition of TRAIL. HGF (100 ng/ml) enhanced TRAIL (10 ng/ml) induced cell death by 36% (P < 0.001). No cell death was associated with HGF alone. Treating cells with PHA-665752, a specific c-Met receptor tyrosine kinase inhibitor, significantly abrogated the enhancement of TRAIL-induced cell death by HGF, indicating that its death promoting effect requires activation of its canonical receptor tyrosine kinase. Cell death induced by TRAIL+HGF was predominately apoptotic involving both extrinsic and intrinsic pathways as evidenced by the increased activation of caspase-3, 8, 9. Promotion of apoptosis by HGF occurred via the increased expression of the death receptor DR5 and enhanced formation of death-inducing signal complexes (DISC). Taken together, these and previous findings indicate that HGF:c-Met pathway either promotes or inhibits medulloblastoma cell death via pathway and context specific mechanisms

Low Oxygen Modulates Multiple Signaling Pathways, Increasing Self-Renewal, While Decreasing Differentiation, Senescence, and Apoptosis in Stromal MIAMI Cells

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Rios, Carmen; D'Ippolito, Gianluca; Curtis, Kevin M.; Delcroix, Gaëtan J.-R.; Gomez, Lourdes A.; El Hokayem, Jimmy; Rieger, Megan; Parrondo, Ricardo; de las Pozas, Alicia; Perez-Stable, Carlos; Howard, Guy A.

2016-01-01

Human bone marrow multipotent mesenchymal stromal cell (hMSC) number decreases with aging. Subpopulations of hMSCs can differentiate into cells found in bone, vasculature, cartilage, gut, and other tissues and participate in their repair. Maintaining throughout adult life such cell subpopulations should help prevent or delay the onset of age-related degenerative conditions. Low oxygen tension, the physiological environment in progenitor cell-rich regions of the bone marrow microarchitecture, stimulates the self-renewal of marrow-isolated adult multilineage inducible (MIAMI) cells and expression of Sox2, Nanog, Oct4a nuclear accumulation, Notch intracellular domain, notch target genes, neuronal transcriptional repressor element 1 (RE1)-silencing transcription factor (REST), and hypoxia-inducible factor-1 alpha (HIF-1α), and additionally, by decreasing the expression of (i) the proapoptotic proteins, apoptosis-inducing factor (AIF) and Bak, and (ii) senescence-associated p53 expression and β-galactosidase activity. Furthermore, low oxygen increases canonical Wnt pathway signaling coreceptor Lrp5 expression, and PI3K/Akt pathway activation. Lrp5 inhibition decreases self-renewal marker Sox2 mRNA, Oct4a nuclear accumulation, and cell numbers. Wortmannin-mediated PI3K/Akt pathway inhibition leads to increased osteoblastic differentiation at both low and high oxygen tension. We demonstrate that low oxygen stimulates a complex signaling network involving PI3K/Akt, Notch, and canonical Wnt pathways, which mediate the observed increase in nuclear Oct4a and REST, with simultaneous decrease in p53, AIF, and Bak. Collectively, these pathway activations contribute to increased self-renewal with concomitant decreased differentiation, cell cycle arrest, apoptosis, and/or senescence in MIAMI cells. Importantly, the PI3K/Akt pathway plays a central mechanistic role in the oxygen tension-regulated self-renewal versus osteoblastic differentiation of progenitor cells. PMID:27059084

Prohibitin (PHB) acts as a potent survival factor against ceramide induced apoptosis in rat granulosa cells.

Science.gov (United States)

Chowdhury, Indrajit; Branch, Alicia; Olatinwo, Moshood; Thomas, Kelwyn; Matthews, Roland; Thompson, Winston E

2011-08-29

Ceramide is a key factor in inducing germ cell apoptosis by translocating from cumulus cells into the adjacent oocyte and lipid rafts through gap junctions. Therefore studies designed to elucidate the mechanistic pathways in ceramide induced granulosa cell (GC) apoptosis and follicular atresia may potentially lead to the development of novel lipid-based therapeutic strategies that will prevent infertility and premature menopause associated with chemo and/or radiation therapy in female cancer patients. Our previous studies have shown that Prohibitin (PHB) is intimately involved in GCs differentiation, atresia, and luteolysis. In the present study, we have examined the functional effects of loss-/gain-of-function of PHB using adenoviral technology in delaying apoptosis induced by the physiological ligand ceramide in rat GCs. Under these experimental conditions, exogenous ceramide C-8 (50 μM) augmented the expression of mitochondrial PHB and subsequently cause the physical destruction of GC by the release of mitochondrial cytochrome c and activation of caspase-3. In further studies, silencing of PHB expression by adenoviral small interfering RNA (shRNA) sensitized GCs to ceramide C8-induce apoptosis. In contrast, adenovirus (Ad) directed overexpression of PHB in GCs resulted in increased PHB content in mitochondria and delayed the onset of ceramide induced apoptosis in the infected GCs. Taken together, these results provide novel evidences that a critical level of PHB expression within the mitochondria plays a key intra-molecular role in GC fate by mediating the inhibition of apoptosis and may therefore, contribute significantly to ceramide induced follicular atresia. Copyright © 2011 Elsevier Inc. All rights reserved.

An Ingenol Derived from Euphorbia kansui Induces Hepatocyte Cytotoxicity by Triggering G0/G1 Cell Cycle Arrest and Regulating the Mitochondrial Apoptosis Pathway in Vitro

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Xiaojing Yan

2016-06-01

Full Text Available Natural product lingenol, a purified diterpenoid compound derived from the root of Euphorbia kansui, exerts serious hepatotoxicity; however, the molecular mechanisms remain to be defined. In the present study, cell counting Kit-8 (CCK-8, inverted phase contrast microscope and flow cytometry were used to demonstrate that lingenol significantly inhibited L-O2 cells proliferation, and induced cell cycle arrest and apoptosis. Moreover, the results investigated that lingenol markedly disrupted mitochondrial functions by high content screening (HCS. In addition, the up-regulation of cytochrome c, AIF and Apaf-1 and activation of caspases were found in L-O2 cells detected by Western blotting and ELISA assay, which was required for lingenol activation of cytochrome c-mediated caspase cascades and AIF-mediated DNA damage. Mechanistic investigations revealed that lingenol significantly down-regulated the Bcl-2/Bax ratio and enhanced the reactive oxygen species (ROS in L-O2 cells. These data collectively indicated that lingenol modulation of ROS and Bcl-2/Bax ratio led to cell cycle arrest and mitochondrial-mediated apoptosis in L-O2 cells in vitro. All of these results will be helpful to reveal the hepatotoxicity mechanism of Euphorbia kansui and to effectively guide safer and better clinical application of this herb.

Casticin impairs cell growth and induces cell apoptosis via cell cycle arrest in human oral cancer SCC-4 cells.

Science.gov (United States)

Chou, Guan-Ling; Peng, Shu-Fen; Liao, Ching-Lung; Ho, Heng-Chien; Lu, Kung-Wen; Lien, Jin-Cherng; Fan, Ming-Jen; La, Kuang-Chi; Chung, Jing-Gung

2018-02-01

Casticin, a polymethoxyflavone, present in natural plants, has been shown to have biological activities including anti-cancer activities. Herein, we investigated the anti-oral cancer activity of casticin on SCC-4 cells in vitro. Viable cells, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS) production, and Ca 2+ production, levels of ΔΨ m and caspase activity were measured by flow cytometric assay. Cell apoptosis associated protein expressions were examined by Western blotting and confocal laser microscopy. Results indicated that casticin induced cell morphological changes, DNA condensation and damage, decreased the total viable cells, induced G 2 /M phase arrest in SCC-4 cells. Casticin promoted ROS and Ca 2+ productions, decreases the levels of ΔΨ m , promoted caspase-3, -8, and -9 activities in SCC-4 cells. Western blotting assay demonstrated that casticin affect protein level associated with G2/M phase arrest and apoptosis. Confocal laser microscopy also confirmed that casticin increased the translocation of AIF and cytochrome c in SCC-4 cells. In conclusion, casticin decreased cell number through G 2 /M phase arrest and the induction of cell apoptosis through caspase- and mitochondria-dependent pathways in SCC-4 cells. © 2017 Wiley Periodicals, Inc.

Oxidative stress induction by T-2 toxin causes DNA damage and triggers apoptosis via caspase pathway in human cervical cancer cells

International Nuclear Information System (INIS)

Chaudhari, Manjari; Jayaraj, R.; Bhaskar, A.S.B.; Lakshmana Rao, P.V.

2009-01-01

T-2 toxin is the most toxic trichothecene and both humans and animals suffer from several pathological conditions after consumption of foodstuffs contaminated with trichothecenes. We investigated the molecular mechanism of T-2 toxin induced cytotoxicity and cell death in HeLa cells. T-2 toxin at LC50 of 10 ng/ml caused time dependent increase in cytotoxicity as assessed by dye uptake, lactatedehydrogenase leakage and MTT assay. The toxin caused generation of reactive oxygen species as early as 30 min followed by significant depletion of glutathione levels and increased lipid peroxidation. The results indicate oxidative stress as underlying mechanism of cytotoxicity. Single stranded DNA damage after T-2 treatment was observed as early as 2 and 4 h by DNA diffusion assay. The cells exhibited apoptotic morphology like condensed chromatin and nuclear fragmentation after 4 h of treatment. Downstream of T-2 induced oxidative stress and DNA damage a time dependent increase in expression level of p53 protein was observed. The increase in Bax/Bcl2 ratio indicated shift in response, in favour of apoptotic process in T-2 toxin treated cells. Western blot analysis showed increase in levels of mitochondrial apoptogenic factors Bax, Bcl-2, cytochrome-c followed by activation of caspases-9, -3 and -7 leading to DNA fragmentation and apoptosis. In addition to caspase-dependent pathway, our results showed involvement of caspase-independent AIF pathway in T-2 induced apoptosis. Broad spectrum caspase inhibitor z-VAD-fmk could partially protect the cells from DNA damage but could not inhibit AIF induced oligonucleosomal DNA fragmentation beyond 4 h. Results of the study clearly show that oxidative stress is the underlying mechanism by which T-2 toxin causes DNA damage and apoptosis.

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 ...

TATA-binding protein-associated factor 7 regulates polyamine transport activity and polyamine analog-induced apoptosis.

Science.gov (United States)

Fukuchi, Junichi; Hiipakka, Richard A; Kokontis, John M; Nishimura, Kazuhiro; Igarashi, Kazuei; Liao, Shutsung

2004-07-16

Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.

Connective tissue growth factor mediates TGF-β1-induced low-grade serous ovarian tumor cell apoptosis.

Science.gov (United States)

Cheng, Jung-Chien; Chang, Hsun-Ming; Leung, Peter C K

2017-10-17

Ovarian low-grade serous carcinoma (LGSC) is a rare disease and is now considered to be a distinct entity from high-grade serous carcinoma (HGSC), which is the most common and malignant form of epithelial ovarian cancer. Connective tissue growth factor (CTGF) is a secreted matricellular protein that has been shown to modulate many biological functions by interacting with multiple molecules in the microenvironment. Increasing evidence indicates that aberrant expression of CTGF is associated with cancer development and progression. Transforming growth factor-β1 (TGF-β1) is a well-known molecule that can strongly up-regulate CTGF expression in different types of normal and cancer cells. Our previous study demonstrated that TGF-β1 induces apoptosis of LGSC cells. However, the effect of TGF-β1 on CTGF expression in LGSC needs to be defined. In addition, whether CTGF mediates TGF-β1-induced LGSC cell apoptosis remains unknown. In the present study, we show that TGF-β1 treatment up-regulates CTGF expression by activating SMAD3 signaling in two human LGSC cell lines. Additionally, siRNA-mediated CTGF knockdown attenuates TGF-β1-induced cell apoptosis. Moreover, our results show that the inhibitory effect of the CTGF knockdown on TGF-β1-induced cell apoptosis is mediated by down-regulating SMAD3 expression. This study demonstrates an important role for CTGF in mediating the pro-apoptotic effects of TGF-β1 on LGCS.

Oxaliplatin triggers necrosis as well as apoptosis in gastric cancer SGC-7901 cells

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Wu, Ping; Zhu, Xueping [Department of Immunology, Anhui Medical University, Hefei 230032 (China); Jin, Wei [Department of Otolaryngology, Chaohu Hospital of Anhui Medical University, Chaohu 238000 (China); Hao, Shumei; Liu, Qi [Department of Immunology, Anhui Medical University, Hefei 230032 (China); Zhang, Linjie, E-mail: zlj33@ahmu.edu.cn [Department of Immunology, Anhui Medical University, Hefei 230032 (China)

2015-05-01

Intrinsic apoptotic pathway is considered to be responsible for cell death induced by platinum anticancer drugs. While in this study, we found that, necrosis is an indispensable pathway besides apoptosis in oxaliplatin-treated gastric cancer SGC-7901 cells. Upon exposure to oxaliplatin, both apoptotic and necrotic features were observed. The majority of dead cells were double positive for Annexin V and propidium iodide (PI). Moreover, mitochondrial membrane potential collapsed and caspase cascades were activated. However, ultrastructural changes under transmission electron microscope, coupled with the release of cellular contents, demonstrated the rupture of the plasma membrane. Oxaliplatin administration did not stimulate reactive oxygen species (ROS) production and autophagy, but elevated the protein level of Bmf. In addition, receptor interacting protein 1 (RIP1), but not receptor interacting protein 3 (RIP3) and its downstream components participated in this death process. Necrostatin-1 (Nec-1) blocked oxaliplatin-induced cell death nearly completely, whereas z-VAD-fmk could partially suppress cell death. Oxaliplatin treatment resulted in poly(ADP-ribose) polymerase-1 (PARP-1) overactivation, as indicated by the increase of poly(ADP-ribose) (PAR), which led to NAD{sup +} and ATP depletion. PARP-1 inhibitor, olaparib, could significantly block oxaliplatin-induced cell death, thus confirming that PARP-1 activation is mainly responsible for the cytotoxicity of oxaliplatin. Phosphorylation of H2AX at Ser139 and translocalization of apoptosis-inducing factor (AIF) are critical for this death process. Taken together, these results indicate that oxaliplatin can bypass canonical cell death pathways to kill gastric cancer cells, which may be of therapeutic advantage in the treatment of gastric cancer. - Highlights: • Oxaliplatin induces apoptotic and necrotic cell death. • Nec-1 can inhibit oxaliplatin-induced cell death nearly completely. • RIP3 and its

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

Poly (ADP-ribose polymerase plays an important role in intermittent hypoxia-induced cell death in rat cerebellar granule cells

Directory of Open Access Journals (Sweden)

Chiu Sheng-Chun

2012-03-01

Full Text Available Abstract Background Episodic cessation of airflow during sleep in patients with sleep apnea syndrome results in intermittent hypoxia (IH. Our aim was to investigate the effects of IH on cerebellar granule cells and to identify the mechanism of IH-induced cell death. Methods Cerebellar granule cells were freshly prepared from neonatal Sprague-Dawley rats. IH was created by culturing the cerebellar granule cells in the incubators with oscillating O2 concentration at 20% and 5% every 30 min for 1-4 days. The results of this study are based on image analysis using a confocal microscope and associated software. Cellular oxidative stress increased with increase in IH. In addition, the occurrence of cell death (apoptosis and necrosis increased as the duration of IH increased, but decreased in the presence of an iron chelator (phenanthroline or poly (ADP-ribose polymerase (PARP inhibitors [3-aminobenzamide (3-AB and DPQ]. The fluorescence of caspase-3 remained the same regardless of the duration of IH, and Western blots did not detect activation of caspase-3. However, IH increased the ratio of apoptosis-inducing factor (AIF translocation to the nucleus, while PARP inhibitors (3-AB reduced this ratio. Results According to our findings, IH increased oxidative stress and subsequently leading to cell death. This effect was at least partially mediated by PARP activation, resulting in ATP depletion, calpain activation leading to AIF translocation to the nucleus. Conclusions We suggest that IH induces cell death in rat primary cerebellar granule cells by stimulating oxidative stress PARP-mediated calpain and AIF activation.

Generation of reactive oxygen species by a novel berberine–bile acid analog mediates apoptosis in hepatocarcinoma SMMC-7721 cells

Energy Technology Data Exchange (ETDEWEB)

Li, Qingyong, E-mail: li_qingyong@126.com [Key Laboratory of Forest Plant Ecology (Northeast Forestry University), Ministry of Education (China); Zhang, Li; Zu, Yuangang; Liu, Tianyu; Zhang, Baoyou; He, Wuna [Key Laboratory of Forest Plant Ecology (Northeast Forestry University), Ministry of Education (China)

2013-04-19

Graphical abstract: - Highlights: • Anticancer effects of B4, a novel berberine–bile acid analog, were tested. • B4 inhibited cell proliferation in hepatocellular carcinoma cells. • It also stimulated mitochondrial ROS production and membrane depolarization. • Effects of B4 were inhibited by a non-specific ROS scavenger. • Regulation of ROS generation may be a strategy for treating hepatic carcinoma. - Abstract: 2,3-Methenedioxy-9-O-(3′α,7′α-dihydroxy-5′β-cholan-24′-propy-lester) berberine (B4) is a novel berberine–bile acid analog synthesized in our laboratory. Previously, we showed that B4 exerted greater cytotoxicity than berberine in several human cancer cell lines. Therefore, we further evaluated the mechanism governing its anticancer actions in hepatocellular carcinoma SMMC-7721 cells. B4 inhibited the proliferation of SMMC-7721 cells, and stimulated reactive oxygen species (ROS) production and mitochondrial membrane depolarization; anti-oxidant capacity was reduced. B4 also induced the release of cytochrome c from the mitochondria to the cytosol and an increase in poly ADP-ribose polymerase (PARP) cleavage products, reflective of caspase-3 activation. Moreover, B4 induced the nuclear translocation of apoptosis-inducing factor (AIF) and a rise in DNA fragmentation. Pretreatment with the anti-oxidant N-acetylcysteine (NAC) inhibited B4-mediated effects, including cytotoxicity, ROS production, mitochondrial membrane depolarization increase in intracellular Ca{sup 2+}, cytochrome c release, PARP cleavage, and AIF translocation. Our data suggest that B4 induces ROS-triggered caspase-dependent and caspase-independent apoptosis pathways in SMMC-7721 cells and that ROS production may be a specific potential strategy for treating hepatic carcinoma.

Verification Based on Set-Abstraction Using the AIF Framework

DEFF Research Database (Denmark)

Mödersheim, Sebastian Alexander

The AIF framework is a novel method for analyzing advanced security protocols, web services, and APIs, based a new abstract interpretation method. It consists of the specification language AIF and a translation/abstraction processes that produces a set of first-order Horn clauses. These can...

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

17-beta estradiol inhibits oxidative stress-induced accumulation of AIF into nucleolus and PARP1-dependent cell death via estrogen receptor alpha.

Science.gov (United States)

Batnasan, Enkhzaya; Wang, Ruoxi; Wen, Jitao; Ke, Yueshuang; Li, Xiaoxue; Bohio, Ameer Ali; Zeng, Xianlu; Huo, Hongliang; Han, Liping; Boldogh, Istvan; Ba, Xueqing

2015-01-05

Oxidative stress-induced DNA damage results in over-activation of poly(ADP-ribose) polymerase 1 (PARP1), leading to parthanatos, a newly discovered cell elimination pathway. Inhibition of PARP1-dependent cell death has shown to improve the outcome of diseases, including stroke, heart ischemia, and neurodegenerative diseases. In the present study we aimed to detect whether estrogen plays a protective role in inhibiting parthanatos. We utilized human mammary adenocarcinoma cells (MCF7) that abundantly express the estrogen receptor alpha and beta (ERα and ERβ). Parthanatos was induced by challenging the cells with hydrogen peroxide (H2O2). Microscopic imaging and molecular biological techniques, such as Western blot analysis and RNA interference, were performed. The results showed 17β estradiol (E2) protected MCF7 cells from PARP1-dependent cell death by decreasing protein PARylation, and AIF translocation into nuclei/nucleoli. Down-regulation of ERα expression by siRNA before E2 addition resulted in the failure of the E2-mediated inhibition of H2O2-induced protein PARylation and AIF nucleolar translocation. Together these data suggest that estrogen via its alpha-type receptor inhibits oxidative stress-induced, PARP1-dependent cell death. The present study provided us insight into how to apply hormone therapy in intervention of parthanatos-implicated ischemic and degenerative diseases. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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.

Tumor Necrosis Factor-α Induced Apoptosis in U937 Cells Promotes Cathepsin D-Independent Stefin B Degradation.

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Bidovec, Katja; Božič, Janja; Dolenc, Iztok; Turk, Boris; Turk, Vito; Stoka, Veronika

2017-12-01

Lysosomal cathepsins were previously found to be involved in tumor necrosis factor-α (TNFα)-induced apoptosis. However, there are opposing views regarding their role as either initiators or amplifiers of the signaling cascade as well as the order of molecular events during this process. In this study, we investigated the role of cathepsin D (catD) in TNFα/cycloheximide-induced apoptosis in U937 human monocytic cells. TNFα-induced apoptosis proceeds through caspase-8 activation, processing of the pro-apoptotic molecule Bid, mitochondrial membrane permeabilization, and caspase-3 activation. The translocation of lysosomal catD into the cytosol was a late event, suggesting that lysosomal membrane permeabilization and the release of cathepsins are not required for the induction of apoptosis, but rather amplifies the process through the generation of reactive oxygen species. For the first time, we show that apoptosis is accompanied by degradation of the cysteine cathepsin inhibitor stefin B (StfB). CatD did not exhibit a crucial role in this step. However, this degradation was partially prevented through pre-incubation with the antioxidant N-acetyl cysteine, although it did not prevent apoptosis and its progression. These results suggest that the degradation of StfB, as a response to TNFα, could induce a cell death amplification effect as a result of progressive damage to lysosomes during TNFα treatment. J. Cell. Biochem. 118: 4813-4820, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

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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.

Soluble Prokaryotic Expression and Purification of Bioactive Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand.

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Do, Bich Hang; Nguyen, Minh Tan; Song, Jung-A; Park, Sangsu; Yoo, Jiwon; Jang, Jaepyeong; Lee, Sunju; So, Seoungjun; Yoon, Yejin; Kim, Inki; Lee, Kyungjin; Jang, Yeon Jin; Choe, Han

2017-12-28

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as an antitumor agent owing to its ability to induce apoptosis of cancer cells without imparting toxicity toward most normal cells. TRAIL is produced in poor yield because of its insoluble expression in the cytoplasm of E. coli . In this study, we achieved soluble expression of TRAIL by fusing maltose-binding protein (MBP), b'a' domain of protein disulfide isomerase (PDIb'a'), or protein disulfide isomerase at the N-terminus of TRAIL. The TRAIL was purified using subsequent immobilized metal affinity chromatography and amylose-binding chromatography, with the tag removal using tobacco etch virus protease. Approximately 4.5 mg of pure TRAIL was produced from 125 ml flask culture with a purification yield of 71.6%. The endotoxin level of the final product was 0.4 EU/μg, as measured by the Limulus amebocyte lysate endotoxin assay. The purified TRAIL was validated and shown to cause apoptosis of HeLa cells with an EC₅₀ and Hill coefficient of 0.6 ± 0.03 nM and 2.41 ± 0.15, respectively. The high level of apoptosis in HeLa cells following administration of purified TRAIL indicates the significance and novelty of this method for producing high-grade and high-yield TRAIL.

The phytopathogenic virulent effector protein RipI induces apoptosis in budding yeast Saccharomyces cerevisiae.

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Deng, Meng-Ying; Sun, Yun-Hao; Li, Pai; Fu, Bei; Shen, Dong; Lu, Yong-Jun

2016-10-01

Virulent protein toxins secreted by the bacterial pathogens can cause cytotoxicity by various molecular mechanisms to combat host cell defense. On the other hand, these proteins can also be used as probes to investigate the defense pathway of host innate immunity. Ralstonia solanacearum, one of the most virulent bacterial phytopathogens, translocates more than 70 effector proteins via type III secretion system during infection. Here, we characterized the cytotoxicity of effector RipI in budding yeast Saccharomyce scerevisiae, an alternative host model. We found that over-expression of RipI resulted in severe growth defect and arginine (R) 117 within the predicted integrase motif was required for inhibition of yeast growth. The phenotype of death manifested the hallmarks of apoptosis. Our data also revealed that RipI-induced apoptosis was independent of Yca1 and mitochondria-mediated apoptotic pathways because Δyca1 and Δaif1 were both sensitive to RipI as compared with the wild type. We further demonstrated that RipI was localized in the yeast nucleus and the N-terminal 1-174aa was required for the localization. High-throughput RNA sequencing analysis showed that upon RipI over-expression, 101 unigenes of yeast ribosome presented lower expression level, and 42 GO classes related to the nucleus or recombination were enriched with differential expression levels. Taken together, our data showed that a nuclear-targeting effector RipI triggers yeast apoptosis, potentially dependent on its integrase function. Our results also provided an alternative strategy to dissect the signaling pathway of cytotoxicity induced by the protein toxins. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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.

Evidence that tumor necrosis factor-related apoptosis inducing ligand (TRAIL) inhibits angiogenesis by inducing vascular endothelial cell apoptosis

International Nuclear Information System (INIS)

Chen, Pei-Lin; Easton, Alexander S.

2010-01-01

Tumor necrosis factor (TNF) and its related ligands TNF-related apoptosis inducing ligand (TRAIL) and Fas ligand (FasL) play roles in the regulation of vascular responses, but their effect on the formation of new blood vessels (angiogenesis) is unclear. Therefore, we have examined the effects of these ligands on angiogenesis modeled with primary cultures of human umbilical vein endothelial cells (HUVEC). To examine angiogenesis in the context of the central nervous system, we have also modeled cerebral angiogenesis with the human brain endothelial cell line hCMEC/D3. Parameters studied were bromodeoxyuridine (BrdU) incorporation and cell number (MTT) assay (to assess endothelial proliferation), scratch assay (migration) and networks on Matrigel (tube formation). In our hands, neither TRAIL nor FasL (1, 10, and 100 ng/ml) had an effect on parameters of angiogenesis in the HUVEC model. In hCMEC/D3 cells by contrast, TRAIL inhibited all parameters (10-100 ng/ml, 24 h). This was due to apoptosis, since its action was blocked by the pan-caspase inhibitor zVADfmk (5 x 10 -5 mol/l) and TRAIL increased caspase-3 activity 1 h after application. However FasL (100 ng/ml) increased BrdU uptake without other effects. We conclude that TRAIL has different effects on in vitro angiogenesis depending on which model is used, but that FasL is generally ineffective when applied in vitro. The data suggest that TRAIL primarily influences angiogenesis by the induction of vascular endothelial apoptosis, leading to vessel regression.

Sugammadex, a Neuromuscular Blockade Reversal Agent, Causes Neuronal Apoptosis in Primary Cultures

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Palanca, José M.; Aguirre-Rueda, Diana; Granell, Manuel V.; Aldasoro, Martin; Garcia, Alma; Iradi, Antonio; Obrador, Elena; Mauricio, Maria Dolores; Vila, Jose; Gil-Bisquert, Anna; Valles, Soraya L.

2013-01-01

Sugammadex, a γ-cyclodextrin that encapsulates selectively steroidal neuromuscular blocking agents, such as rocuronium or vecuronium, has changed the face of clinical neuromuscular pharmacology. Sugammadex allows a rapid reversal of muscle paralysis. Sugammadex appears to be safe and well tolerated. Its blood-brain barrier penetration is poor (Sugammadex in neurons in primary culture. Here we show that clinically relevant sugammadex concentrations cause apoptotic/necrosis neuron death in primary cultures. Studies on the underlying mechanism revealed that sugammadex-induced activation of mitochondria-dependent apoptosis associates with depletion of neuronal cholesterol levels. Furthermore SUG increase CytC, AIF, Smac/Diablo and CASP-3 protein expression in cells in culture. Potential association of SUG-induced alteration in cholesterol homeostasis with oxidative stress and apoptosis activation occurs. Furthermore, resistance/sensitivity to oxidative stress differs between neuronal cell types. PMID:23983586

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

The Role of the E2F Transcription Factor Family in UV-Induced Apoptosis

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Orla Gannon

2011-12-01

Full Text Available The E2F transcription factor family is traditionally associated with cell cycle control. However, recent data has shown that activating E2Fs (E2F1-3a are potent activators of apoptosis. In contrast, the recently cloned inhibitory E2Fs (E2F7 and 8 appear to antagonize E2F-induced cell death. In this review we will discuss (i the potential role of E2Fs in UV-induced cell death and (ii the implications of this to the development of UV-induced cutaneous malignancies.

Modeling Dynamic Contrast-Enhanced MRI Data with a Constrained Local AIF

DEFF Research Database (Denmark)

Duan, Chong; Kallehauge, Jesper F.; Pérez-Torres, Carlos J

2018-01-01

PURPOSE: This study aims to develop a constrained local arterial input function (cL-AIF) to improve quantitative analysis of dynamic contrast-enhanced (DCE)-magnetic resonance imaging (MRI) data by accounting for the contrast-agent bolus amplitude error in the voxel-specific AIF. PROCEDURES....... RESULTS: When the data model included the cL-AIF, tracer kinetic parameters were correctly estimated from in silico data under contrast-to-noise conditions typical of clinical DCE-MRI experiments. Considering the clinical cervical cancer data, Bayesian model selection was performed for all tumor voxels...

Role of mitochondria-associated hexokinase II in cancer cell death induced by 3-Bromopyruvate

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Chen, Zhao; Zhang, Hui; Lu, Weiqin; Huang, Peng

2009-01-01

Summary It has long been observed that cancer cells rely more on glycolysis to generate ATP and actively use certain glycolytic metabolic intermediates for biosynthesis. Hexokinase II (HKII) is a key glycolytic enzyme that plays a role in the regulation of the mitochondria-initiated apoptotic cell death. As a potent inhibitor of hexokinase, 3-bromopyruvate (3-BrPA) is known to inhibit cancer cell energy metabolism and trigger cell death, supposedly through depletion of cellular ATP. The current study showed that 3-BrPA caused a covalent modification of HKII protein and directly triggered its dissociation from mitochondria, leading to a specific release of apoptosis-inducing factor (AIF) from the mitochondria to cytosol and eventual cell death. Co-immunoprecipitation revealed a physical interaction between HKII and AIF. Using a competitive peptide of HKII, we showed that the dissociation of hexokinase II from mitochondria alone could cause apoptotic cell death, especially in the mitochondria-deficient ρ0 cells that highly express HKII. Interestingly, the dissociation of HKII itself did no directly affect the mitochondrial membrane potential, ROS generation, and oxidative phosphorylation. Our study suggests that the physical association between HKII and AIF is important for the normal localization of AIF in the mitochondria, and disruption of this protein complex by 3-BrPA leads to their release from the mitochondria and eventual cell death. PMID:19285479

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

The 19?kDa Mycobacterium tuberculosis Lipoprotein (LpqH) Induces Macrophage Apoptosis through Extrinsic and Intrinsic Pathways: A Role for the Mitochondrial Apoptosis-Inducing Factor

OpenAIRE

S?nchez, Alejandro; Espinosa, Patricia; Garc?a, Teresa; Mancilla, Ra?l

2012-01-01

We describe the association of caspase-dependent and caspase-independent mechanisms in macrophage apoptosis induced by LpqH, a 19 kDa Mycobacterium tuberculosis lipoprotein. LpqH triggered TLR2 activation, with upregulation of death receptors and ligands, which was followed by a death receptor signaling cascade with activation of initiator caspase 8 and executioner caspase 3. In this caspase-mediated phase, mitochondrial factors were involved in loss of mitochondrial transmembrane potential (...

Host and Viral Factors in HIV-Mediated Bystander Apoptosis

Science.gov (United States)

Garg, Himanshu; Joshi, Anjali

2017-01-01

Human immunodeficiency virus (HIV) infections lead to a progressive loss of CD4 T cells primarily via the process of apoptosis. With a limited number of infected cells and vastly disproportionate apoptosis in HIV infected patients, it is believed that apoptosis of uninfected bystander cells plays a significant role in this process. Disease progression in HIV infected individuals is highly variable suggesting that both host and viral factors may influence HIV mediated apoptosis. Amongst the viral factors, the role of Envelope (Env) glycoprotein in bystander apoptosis is well documented. Recent evidence on the variability in apoptosis induction by primary patient derived Envs underscores the role of Env glycoprotein in HIV disease. Amongst the host factors, the role of C-C Chemokine Receptor type 5 (CCR5), a coreceptor for HIV Env, is also becoming increasingly evident. Polymorphisms in the CCR5 gene and promoter affect CCR5 cell surface expression and correlate with both apoptosis and CD4 loss. Finally, chronic immune activation in HIV infections induces multiple defects in the immune system and has recently been shown to accelerate HIV Env mediated CD4 apoptosis. Consequently, those factors that affect CCR5 expression and/or immune activation in turn indirectly regulate HIV mediated apoptosis making this phenomenon both complex and multifactorial. This review explores the complex role of various host and viral factors in determining HIV mediated bystander apoptosis. PMID:28829402

Fisetin-induced apoptosis of human oral cancer SCC-4 cells through reactive oxygen species production, endoplasmic reticulum stress, caspase-, and mitochondria-dependent signaling pathways.

Science.gov (United States)

Su, Chen-Hsuan; Kuo, Chao-Lin; Lu, Kung-Wen; Yu, Fu-Shun; Ma, Yi-Shih; Yang, Jiun-Long; Chu, Yung-Lin; Chueh, Fu-Shin; Liu, Kuo-Ching; Chung, Jing-Gung

2017-06-01

Oral cancer is one of the cancer-related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin-induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin-induced cell death and associated signal pathways on human oral cancer SCC-4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca 2+ , mitochondria membrane potential (ΔΨ m ), and caspase-8, -9, and -3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca 2+ production, and decreased the level of ΔΨ m and increased caspase-3, -8, and -9 activities in SCC-4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin-induced cell apoptosis in SCC-4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl-2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC-4 cells. We also used ATF-6α, ATF-6β, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria-, and caspase-dependent pathways. © 2017 Wiley Periodicals, Inc.

Small kinetochore associated protein (SKAP promotes UV-induced cell apoptosis through negatively regulating pre-mRNA processing factor 19 (Prp19.

Directory of Open Access Journals (Sweden)

Shan Lu

Full Text Available Apoptosis is a regulated cellular suicide program that is critical for the development and maintenance of healthy tissues. Previous studies have shown that small kinetochore associated protein (SKAP cooperates with kinetochore and mitotic spindle proteins to regulate mitosis. However, the role of SKAP in apoptosis has not been investigated. We have identified a new interaction involving SKAP, and we propose a mechanism through which SKAP regulates cell apoptosis. Our experiments demonstrate that both overexpression and knockdown of SKAP sensitize cells to UV-induced apoptosis. Further study has revealed that SKAP interacts with Pre-mRNA processing Factor 19 (Prp19. We find that UV-induced apoptosis can be inhibited by ectopic expression of Prp19, whereas silencing Prp19 has the opposite effect. Additionally, SKAP negatively regulates the protein levels of Prp19, whereas Prp19 does not alter SKAP expression. Finally, rescue experiments demonstrate that the pro-apoptotic role of SKAP is executed through Prp19. Taken together, these findings suggest that SKAP promotes UV-induced cell apoptosis by negatively regulating the anti-apoptotic protein Prp19.

Differential modulatory effects of GSK-3β and HDM2 on sorafenib-induced AIF nuclear translocation (programmed necrosis in melanoma

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Mier James W

2011-09-01

Full Text Available Abstract Background GSK-3β phosphorylates numerous substrates that govern cell survival. It phosphorylates p53, for example, and induces its nuclear export, HDM2-dependent ubiquitination, and proteasomal degradation. GSK-3β can either enhance or inhibit programmed cell death, depending on the nature of the pro-apoptotic stimulus. We previously showed that the multikinase inhibitor sorafenib activated GSK-3β and that this activation attenuated the cytotoxic effects of the drug in various BRAF-mutant melanoma cell lines. In this report, we describe the results of studies exploring the effects of GSK-3β on the cytotoxicity and antitumor activity of sorafenib combined with the HDM2 antagonist MI-319. Results MI-319 alone increased p53 levels and p53-dependent gene expression in melanoma cells but did not induce programmed cell death. Its cytotoxicity, however, was augmented in some melanoma cell lines by the addition of sorafenib. In responsive cell lines, the MI-319/sorafenib combination induced the disappearance of p53 from the nucleus, the down modulation of Bcl-2 and Bcl-xL, the translocation of p53 to the mitochondria and that of AIF to the nuclei. These events were all GSK-3β-dependent in that they were blocked with a GSK-3β shRNA and facilitated in otherwise unresponsive melanoma cell lines by the introduction of a constitutively active form of the kinase (GSK-3β-S9A. These modulatory effects of GSK-3β on the activities of the sorafenib/MI-319 combination were the exact reverse of its effects on the activities of sorafenib alone, which induced the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF only in cells in which GSK-3β activity was either down modulated or constitutively low. In A375 xenografts, the antitumor effects of sorafenib and MI-319 were additive and associated with the down modulation of Bcl-2 and Bcl-xL, the nuclear translocation of AIF, and increased suppression of tumor angiogenesis

Degradation of Epidermal Growth Factor Receptor Mediates Dasatinib-Induced Apoptosis in Head and Neck Squamous Cell Carcinoma Cells

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Yu-Chin Lin

2012-06-01

Full Text Available Epidermal growth factor receptor (EGFR is an important oncoprotein that promotes cell growth and proliferation. Dasatinib, a bcr-abl inhibitor, has been approved clinically for the treatment of chronic myeloid leukemia and demonstrated to be effective against solid tumors in vitro through Src inhibition. Here, we disclose that EGFR degradation mediated dasatinib-induced apoptosis in head and neck squamous cell carcinoma (HNSCC cells. HNSCC cells, including Ca9-22, FaDu, HSC3, SAS, SCC-25, and UMSCC1, were treated with dasatinib, and cell viability, apoptosis, and underlying signal transduction were evaluated. Dasatinib exhibited differential sensitivities against HNSCC cells. Growth inhibition and apoptosis were correlated with its inhibition on Akt, Erk, and Bcl-2, irrespective of Src inhibition. Accordingly, we found that down-regulation of EGFR was a determinant of dasatinib sensitivity. Lysosome inhibitor reversed dasatinib-induced EGFR down-regulation, and c-cbl activity was increased by dasatinib, indicating that dasatinib-induced EGFR down-regulation might be through c-cbl-mediated lysosome degradation. Increased EGFR activation by ligand administration rescued cells from dasatinib-induced apoptosis, whereas inhibition of EGFR enhanced its apoptotic effect. Estrogen receptor α (ERα was demonstrated to play a role in Bcl-2 expression, and dasatinib inhibited ERα at the pretranslational level. ERα was associated with EGFR in dasatinib-treated HNSCC cells. Furthermore, the xenograft model showed that dasatinib inhibited HSC3 tumor growth through in vivo down-regulation of EGFR and ERα. In conclusion, degradation of EGFR is a novel mechanism responsible for dasatinib-induced apoptosis in HNSCC cells.

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.

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

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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.

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

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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.

Quantifying dynamic contrast-enhanced MRI of the knee in children with juvenile rheumatoid arthritis using an arterial input function (AIF) extracted from popliteal artery enhancement, and the effect of the choice of the AIF on the kinetic parameters.

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Workie, Dagnachew W; Dardzinski, Bernard J

2005-09-01

Quantification of dynamic contrast-enhanced (DCE) MRI based on pharmacokinetic modeling requires specification of the arterial input function (AIF). A full representation of the plasma concentration data, including the initial rise and decay parts, considering the delay and dispersion of the bolus contrast is important. This work deals with modeling of DCE-MRI data from the knees of children with a history of juvenile rheumatoid arthritis (JRA) by using an AIF extracted from the signal enhancement data from the nearby popliteal artery. Three models for the AIFs were considered: a triexponential (AIF1), a gamma-variate plus a biexponential (AIF2), and a biexponential (AIF3). The pharmacokinetic parameters obtained from the model were Ktrans', kep, and V'p. The results from AIF1 and AIF2 showed no statistically significant difference. However, some statistically significant differences were seen with AIF3, particularly for parameters Ktrans' and V'p in the synovium (SNVM). These results suggest the importance of obtaining an appropriate AIF representation in pharmacokinetic modeling of JRA. Specifically, the initial rising part of the AIF should be incorporated for optimal pharmacokinetic modeling results. The pharmacokinetic parameters (mean+/-SD) derived from AIF1, using the average plasma concentration data, were as follows: SNVM Ktrans'(min-1)=0.52+/-0.34, kep(min-1)=0.71+/-0.39, and V'p=0.33+/-0.16, and for the distal femoral physis (DFP) Ktrans'(min-1)=1.83+/-1.78, kep(min-1)=2.65+/-1.80, and V'p=0.46+/-0.31. The pharmacokinetic parameters in the SNVM may be useful for investigating activity and therapeutic efficacy in studies of JRA. Longitudinal studies are necessary to find or demonstrate the parameter that is more sensitive to disease activity. Copyright (c) 2005 Wiley-Liss, Inc.

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

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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.

Roscovitine sensitizes leukemia and lymphoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis

Czech Academy of Sciences Publication Activity Database

Molinsky, J.; Klánová, M.; Koc, Michal; Beranová, Lenka; Anděra, Ladislav; Ludvíková, Z.; Bohmova, M.; Gasova, Z.; Strnad, Miroslav; Ivánek, R.; Trněný, M.; Nečas, E.; Živný, J.; Klener, P.

2013-01-01

Roč. 54, č. 2 (2013), s. 372-380 ISSN 1042-8194 R&D Projects: GA MZd NS10287 Institutional research plan: CEZ:AV0Z50380511 Institutional support: RVO:68378050 Keywords : roscovitine * TRAIL * synergism * apoptosis * leukemia * lymphoma Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.605, year: 2013

Andrographolide sensitizes prostate cancer cells to TRAIL-induced apoptosis

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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.

DNA Damage and Cell Cycle Arrest Induced by Protoporphyrin IX in Sarcoma 180 Cells

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Qing Li

2013-09-01

Full Text Available Background: Porphyrin derivatives have been widely used in photodynamic therapy as effective sensitizers. Protoporphyrin IX (PpIX, a well-known hematoporphyrin derivative component, shows great potential to enhance light induced tumor cell damage. However, PpIX alone could also exert anti-tumor effects. The mechanisms underlying those direct effects are incompletely understood. This study thus investigated the putative mechanisms underlying the anti-tumor effects of PpIX on sarcoma 180 (S180 cells. Methods: S180 cells were treated with different concentrations of PpIX. Following the treatment, cell viability was evaluated by the 3-(4, 5- dimethylthiazol-2-yl-2, 5-diphenyltetrazoliumbromide (MTT assay; Disruption of mitochondrial membrane potential was measured by flow cytometry; The trans-location of apoptosis inducer factor (AIF from mitochondria to nucleus was visualized by confocal laser scanning microscopy; DNA damage was detected by single cell gel electrophoresis; Cell cycle distribution was analyzed by DNA content with flow cytometry; Cell cycle associated proteins were detected by western blotting. Results: PpIX (≥ 1 µg/ml significantly inhibited proliferation and reduced viability of S180 cells in a dose-dependent manner. PpIX rapidly and significantly triggered mitochondrial membrane depolarization, AIF (apoptosis inducer factor translocation from mitochondria to nucleus and DNA damage, effects partially relieved by the specific inhibitor of MPTP (mitochondrial permeability transition pore. Furthermore, S phase arrest and upregulation of the related proteins of P53 and P21 were observed following 12 and 24 h PpIX exposure. Conclusion: PpIX could inhibit tumor cell proliferation by induction of DNA damage and cell cycle arrest in the S phase.

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

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.

TG-interacting factor transcriptionally induced by AKT/FOXO3A is a negative regulator that antagonizes arsenic trioxide-induced cancer cell apoptosis

International Nuclear Information System (INIS)

Liu, Zi-Miao; Tseng, Hong-Yu; Cheng, Ya-Ling; Yeh, Bi-Wen; Wu, Wen-Jeng; Huang, Huei-Sheng

2015-01-01

Arsenic trioxide (ATO) is a multi-target drug approved by the Food and Drug Administration as the first-line chemotherapeutic agent for the treatment of acute promyelocytic leukemia. In addition, several clinical trials are being conducted with arsenic-based drugs for the treatment of other hematological malignancies and solid tumors. However, ATO's modest clinical efficacy on some cancers, and potential toxic effects on humans have been reported. Determining how best to reduce these adverse effects while increasing its therapeutic efficacy is obviously a critical issue. Previously, we demonstrated that the JNK-induced complex formation of phosphorylated c-Jun and TG-interacting factor (TGIF) antagonizes ERK-induced cyclin-dependent kinase inhibitor CDKN1A (p21 WAF1/CIP1 ) expression and resultant apoptosis in response to ATO in A431 cells. Surprisingly, at low-concentrations (0.1–0.2 μM), ATO increased cellular proliferation, migration and invasion, involving TGIF expression, however, at high-concentrations (5–20 μM), ATO induced cell apoptosis. Using a promoter analysis, TGIF was transcriptionally regulated by ATO at the FOXO3A binding site (− 1486 to − 1479 bp) via the c-Src/EGFR/AKT pathway. Stable overexpression of TGIF promoted advancing the cell cycle into the S phase, and attenuated 20 μM ATO-induced apoptosis. Furthermore, blockage of the AKT pathway enhanced ATO-induced CDKN1A expression and resultant apoptosis in cancer cells, but overexpression of AKT1 inhibited CDKN1A expression. Therefore, we suggest that TGIF is transcriptionally regulated by the c-Src/EGFR/AKT pathway, which plays a role as a negative regulator in antagonizing ATO-induced CDKN1A expression and resultant apoptosis. Suppression of these antagonistic effects might be a promising therapeutic strategy toward improving clinical efficacy of ATO. - Highlights: • ATO-induced biphasic survival responses of cancer cells depend on low- or high-concentrations. • TGIF mediates

TG-interacting factor transcriptionally induced by AKT/FOXO3A is a negative regulator that antagonizes arsenic trioxide-induced cancer cell apoptosis

Energy Technology Data Exchange (ETDEWEB)

Liu, Zi-Miao; Tseng, Hong-Yu; Cheng, Ya-Ling [Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan (China); Yeh, Bi-Wen [Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan (China); Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Department of Urology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Wu, Wen-Jeng [Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Department of Urology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Huang, Huei-Sheng, E-mail: huanghs@mail.ncku.edu.tw [Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan (China)

2015-05-15

Arsenic trioxide (ATO) is a multi-target drug approved by the Food and Drug Administration as the first-line chemotherapeutic agent for the treatment of acute promyelocytic leukemia. In addition, several clinical trials are being conducted with arsenic-based drugs for the treatment of other hematological malignancies and solid tumors. However, ATO's modest clinical efficacy on some cancers, and potential toxic effects on humans have been reported. Determining how best to reduce these adverse effects while increasing its therapeutic efficacy is obviously a critical issue. Previously, we demonstrated that the JNK-induced complex formation of phosphorylated c-Jun and TG-interacting factor (TGIF) antagonizes ERK-induced cyclin-dependent kinase inhibitor CDKN1A (p21{sup WAF1/CIP1}) expression and resultant apoptosis in response to ATO in A431 cells. Surprisingly, at low-concentrations (0.1–0.2 μM), ATO increased cellular proliferation, migration and invasion, involving TGIF expression, however, at high-concentrations (5–20 μM), ATO induced cell apoptosis. Using a promoter analysis, TGIF was transcriptionally regulated by ATO at the FOXO3A binding site (− 1486 to − 1479 bp) via the c-Src/EGFR/AKT pathway. Stable overexpression of TGIF promoted advancing the cell cycle into the S phase, and attenuated 20 μM ATO-induced apoptosis. Furthermore, blockage of the AKT pathway enhanced ATO-induced CDKN1A expression and resultant apoptosis in cancer cells, but overexpression of AKT1 inhibited CDKN1A expression. Therefore, we suggest that TGIF is transcriptionally regulated by the c-Src/EGFR/AKT pathway, which plays a role as a negative regulator in antagonizing ATO-induced CDKN1A expression and resultant apoptosis. Suppression of these antagonistic effects might be a promising therapeutic strategy toward improving clinical efficacy of ATO. - Highlights: • ATO-induced biphasic survival responses of cancer cells depend on low- or high-concentrations. • TGIF

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

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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.

Mitotic catastrophe occurs in the absence of apoptosis in p53-null cells with a defective G1 checkpoint.

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Michalis Fragkos

Full Text Available Cell death occurring during mitosis, or mitotic catastrophe, often takes place in conjunction with apoptosis, but the conditions in which mitotic catastrophe may exhibit features of programmed cell death are still unclear. In the work presented here, we studied mitotic cell death by making use of a UV-inactivated parvovirus (adeno-associated virus; AAV that has been shown to induce a DNA damage response and subsequent death of p53-defective cells in mitosis, without affecting the integrity of the host genome. Osteosarcoma cells (U2OSp53DD that are deficient in p53 and lack the G1 cell cycle checkpoint respond to AAV infection through a transient G2 arrest. We found that the infected U2OSp53DD cells died through mitotic catastrophe with no signs of chromosome condensation or DNA fragmentation. Moreover, cell death was independent of caspases, apoptosis-inducing factor (AIF, autophagy and necroptosis. These findings were confirmed by time-lapse microscopy of cellular morphology following AAV infection. The assays used readily revealed apoptosis in other cell types when it was indeed occurring. Taken together the results indicate that in the absence of the G1 checkpoint, mitotic catastrophe occurs in these p53-null cells predominantly as a result of mechanical disruption induced by centrosome overduplication, and not as a consequence of a suicide signal.

Bim is a crucial regulator of apoptosis induced by Mycobacterium tuberculosis

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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

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)

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

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

Oleuropein isolated from Fraxinus rhynchophylla inhibits glutamate-induced neuronal cell death by attenuating mitochondrial dysfunction.

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Kim, Mi Hye; Min, Ju-Sik; Lee, Joon Yeop; Chae, Unbin; Yang, Eun-Ju; Song, Kyung-Sik; Lee, Hyun-Shik; Lee, Hong Jun; Lee, Sang-Rae; Lee, Dong-Seok

2017-04-27

Glutamate-induced neurotoxicity is related to excessive oxidative stress accumulation and results in the increase of neuronal cell death. In addition, glutamate has been reported to lead to neurodegenerative diseases, including Parkinson's and Alzheimer's diseases.It is well known that Fraxinus rhynchophylla contains a significant level of oleuropein (Ole), which exerts various pharmacological effects. However, the mechanism of neuroprotective effects of Ole is still poorly defined. In this study, we aimed to investigate whether Ole prevents glutamate-induced toxicity in HT-22 hippocampal neuronal cells. The exposure of the glutamate treatment caused neuronal cell death through an alteration of Bax/Bcl-2 expression and translocation of mitochondrial apoptosis-inducing factor (AIF) to the cytoplasm of HT-22 cells. In addition, glutamate induced an increase in dephosphorylation of dynamin-related protein 1 (Drp1), mitochondrial fragmentation, and mitochondrial dysfunction. The pretreatment of Ole decreased Bax expression, increased Bcl-2 expression, and inhibited the translocation of mitochondrial AIF to the cytoplasm. Furthermore, Ole amended a glutamate-induced mitochondrial dynamic imbalance and reduced the number of cells with fragmented mitochondria, regulating the phosphorylation of Drp1 at amino acid residue serine 637. In conclusion, our results show that Ole has a preventive effect against glutamate-induced toxicity in HT-22 hippocampal neuronal cells. Therefore, these data imply that Ole may be an efficient approach for the treatment of neurodegenerative diseases.

Apoptotic cells can induce non-autonomous apoptosis through the TNF pathway

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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

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

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.

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

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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.

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...

Nutrient Availability Alters the Effect of Autophagy on Sulindac Sulfide-Induced Colon Cancer Cell Apoptosis

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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.

Mitochondrial dysfunction in lyssavirus-induced apoptosis.

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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.

Myostatin induces mitochondrial metabolic alteration and typical apoptosis in cancer cells

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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

Crystal structure of the regulatory subunit of archaeal initiation factor 2B (aIF2B) from hyperthermophilic archaeon Pyrococcus horikoshii OT3: a proposed structure of the regulatory subcomplex of eukaryotic IF2B

International Nuclear Information System (INIS)

Kakuta, Yoshimitsu; Tahara, Maino; Maetani, Shigehiro; Yao, Min; Tanaka, Isao; Kimura, Makoto

2004-01-01

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine-nucleotide exchange factor for eukaryotic initiation factor 2 (eIF2). eIF2B is a heteropentameric protein composed of α-ε subunits. The α, β, and δ subunits form a regulatory subcomplex, while the γ and ε form a catalytic subcomplex. Archaea possess homologues of α, β, and δ subunits of eIF2B. Here, we report the three-dimensional structure of an archaeal regulatory subunit (aIF2Bα) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 determined by X-ray crystallography at 2.2 A resolution. aIF2Bα consists of two subdomains, an N-domain (residues 1-95) and a C-domain (residues 96-276), connected by a long α-helix (α5: 78-106). The N-domain contains a five helix bundle structure, while the C-domain folds into the α/β structure, thus showing similarity to D-ribose-5-phosphate isomerase structure. The presence of two molecules in the crystallographic asymmetric unit and the gel filtration analysis suggest a dimeric structure of aIF2Bα in solution, interacting with each other by C-domains. Furthermore, the crystallographic 3-fold symmetry generates a homohexameric structure of aIF2Bα; the interaction is primarily mediated by the long α-helix at the N-domains. This structure suggests an architecture of the three subunits, α, β, and δ, in the regulatory subcomplex within eIF2B

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

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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.

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.

Influenza virus induces apoptosis via BAD-mediated mitochondrial dysregulation.

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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.

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

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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.

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.

Characterization of apoptosis-related oxidoreductases from Neurospora crassa.

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Patrícia Carneiro

Full Text Available The genome from Neurospora crassa presented three open reading frames homologous to the genes coding for human AIF and AMID proteins, which are flavoproteins with oxidoreductase activities implicated in caspase-independent apoptosis. To investigate the role of these proteins, namely within the mitochondrial respiratory chain, we studied their cellular localization and characterized the respective null mutant strains. Efficiency of the respiratory chain was analyzed by oxygen consumption studies and supramolecular organization of the OXPHOS system was assessed through BN-PAGE analysis in the respective null mutant strains. The results demonstrate that, unlike in mammalian systems, disruption of AIF in Neurospora does not affect either complex I assembly or function. Furthermore, the mitochondrial respiratory chain complexes of the mutant strains display a similar supramolecular organization to that observed in the wild type strain. Further characterization revealed that N. crassa AIF appears localized to both the mitochondria and the cytoplasm, whereas AMID was found exclusively in the cytoplasm. AMID2 was detected in both mitochondria and cytoplasm of the amid mutant strain, but was barely discernible in wild type extracts, suggesting overlapping functions for the two proteins.

Evidence for a Proangiogenic Activity of TNF-Related Apoptosis-Inducing Ligand

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Paola Secchiero

2004-07-01

Full Text Available Starting from the observation that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/ Apo-2L protein is expressed in both malignant and inflammatory cells in some highly vascularized soft tissue sarcomas, the angiogenic potential of TRAIL was investigated in a series of in vitro assays. Recombinant soluble TRAIL induced endothelial cell migration and vessel tube formation to a degree comparable to vascular endothelial growth factor (VEGF, one of the best-characterized angiogenic factors. However, the proangiogenic activity of TRAIL was not mediated by endogenous expression of VEGF. Although TRAIL potentiated VEGF-induced extracellular signal-regulated kinase (ERK phosphorylation and endothelial cell proliferation, the combination of TRAIL + VEGF did not show additive effects with respect to VEGF alone in inducing vessel tube formation. Thus, although TRAIL has gained attention as a potential anticancer therapeutic for its ability to induce apoptosis in a variety of cancer cells, our present data suggest that TRAIL might also play an unexpected role in promoting angiogenesis, which might have therapeutic implications.

Protective role of benfotiamine, a fat soluble vitamin B1 analogue, in the lipopolysaccharide–induced cytotoxic signals in murine macrophages

Science.gov (United States)

Yadav, Umesh C S; Kalariya, Nilesh M; Srivastava, Satish K; Ramana, Kota V

2010-01-01

The study has been designed to investigate the molecular mechanisms by which benfotiamine, a lipid-soluble analogue of Vitamin B1 effects lipopolysaccharide (LPS) – induced inflammatory signals leading to cytotoxicity in mouse macrophage cell line RAW264.7. Benfotiamine prevented LPS-induced apoptosis, expression of Bcl-2 family of pro-apoptotic proteins, caspase-3 activation and PARP cleavage, altered mitochondrial membrane potential and release of cytochrome-c and apoptosis inducing factor (AIF), phosphorylation and subsequent activation of p38-MAPK, stress activated kinases (SAPK/JNK), Protein kinase C, and cytoplasmic-phospholipase A2 in RAW cells. Further, phosphorylation and degradation of inhibitory kappa B (IκB) and consequent activation and nuclear translocation of redox-sensitive transcription factor NF-κB was significantly prevented by benfotiamine. The LPS-induced increased expression of cytokines and chemokines and other inflammatory marker proteins iNOS and COX-2 and their metabolic products NO and PGE2 were also blocked significantly. Thus, our results elucidate the molecular mechanism of anti-inflammatory action of benfotiamine in LPS-induced inflammation in murine macrophage. Benfotiamine suppresses oxidative stress-induced NF-κB activation and prevents the bacterial endotoxin-induced inflammation indicating that vitamin B1 supplementation could be beneficial in the treatment of inflammatory diseases. PMID:20219672

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.

Research Advances on Pathways of Nickel-Induced Apoptosis

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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

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

The chalcone flavokawain B induces G2/M cell-cycle arrest and apoptosis in human oral carcinoma HSC-3 cells through the intracellular ROS generation and downregulation of the Akt/p38 MAPK signaling pathway.

Science.gov (United States)

Hseu, You-Cheng; Lee, Meng-Shiou; Wu, Chi-Rei; Cho, Hsin-Ju; Lin, Kai-Yuan; Lai, Guan-Hua; Wang, Sheng-Yang; Kuo, Yueh-Hsiung; Kumar, K J Senthil; Yang, Hsin-Ling

2012-03-07

Chalcones have been described to represent cancer chemopreventive food components that are rich in fruits and vegetables. In this study, we examined the anti-oral cancer effect of flavokawain B (FKB), a naturally occurring chalcone isolated from Alpinia pricei (shell gingers), and revealed its molecular mechanism of action. Treatment of human oral carcinoma (HSC-3) cells with FKB (1.25-10 μg/mL; 4.4-35.2 μM) inhibited cell viability and caused G(2)/M arrest through reductions in cyclin A/B1, Cdc2, and Cdc25C levels. Moreover, FKB treatment resulted in the induction of apoptosis, which was associated with DNA fragmentation, mitochondria dysfunction, cytochrome c and AIF release, caspase-3 and caspase-9 activation, and Bcl-2/Bax dysregulation. Furthermore, increased Fas activity and procaspase-8, procaspase-4, and procaspase-12 cleavages were accompanied by death receptor and ER-stress, indicating the involvement of mitochondria, death-receptor, and ER-stress signaling pathways. FKB induces apoptosis through ROS generation as evidenced by the upregulation of oxidative-stress markers HO-1/Nrf2. This mechanism was further confirmed by the finding that the antioxidant N-acetylcysteine (NAC) significantly blocked ROS generation and consequently inhibited FKB-induced apoptosis. Moreover, FKB downregulated the phosphorylation of Akt and p38 MAPK, while their inhibitors LY294002 and SB203580, respectively, induced G(2)/M arrest and apoptosis. The profound reduction in cell number was observed in combination treatment with FKB and Akt/p38 MAPK inhibitors, indicating that the disruption of Akt and p38 MAPK cascades plays a functional role in FKB-induced G(2)/M arrest and apoptosis in HSC-3 cells.

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.

Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis

International Nuclear Information System (INIS)

Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting

2012-01-01

Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K 3 ) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ∼ 12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. -- Highlights: ► Menadione causes mitochondrial superoxide accumulation and injury. ► Menadione-induced cell death is caspase-independent, due to rapid depletion of ATP

Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis

Energy Technology Data Exchange (ETDEWEB)

Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting, E-mail: BTZhu@kumc.edu

2012-07-15

Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K{sub 3}) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ∼ 12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. -- Highlights: ► Menadione causes mitochondrial superoxide accumulation and injury. ► Menadione-induced cell death is caspase-independent, due to rapid depletion of

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

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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

Sulforaphane Induces Cell Death Through G2/M Phase Arrest and Triggers Apoptosis in HCT 116 Human Colon Cancer Cells.

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Liu, Kuo-Ching; Shih, Ting-Ying; Kuo, Chao-Lin; Ma, Yi-Shih; Yang, Jiun-Long; Wu, Ping-Ping; Huang, Yi-Ping; Lai, Kuang-Chi; Chung, Jing-Gung

2016-01-01

Sulforaphane (SFN), an isothiocyanate, exists exclusively in cruciferous vegetables, and has been shown to possess potent antitumor and chemopreventive activity. However, there is no available information that shows SFN affecting human colon cancer HCT 116 cells. In the present study, we found that SFN induced cell morphological changes, which were photographed by contrast-phase microscopy, and decreased viability. SFN also induced G2/M phase arrest and cell apoptosis in HCT 116 cells, which were measured with flow cytometric assays. Western blotting indicated that SFN increased Cyclin A, cdk 2, Cyclin B and WEE1, but decreased Cdc 25C, cdk1 protein expressions that led to G2/M phase arrest. Apoptotic cell death was also confirmed by Annexin V/PI and DAPI staining and DNA gel electrophoresis in HCT 116 cells after exposure to SFN. The flow cytometric assay also showed that SFN induced the generation of reactive oxygen species (ROS) and Ca[Formula: see text] and decreased mitochondria membrane potential and increased caspase-8, -9 and -3 activities in HCT 116 cell. Western blotting also showed that SFN induced the release of cytochrome c, and AIF, which was confirmed by confocal microscopy examination. SFN induced ER stress-associated protein expression. Based on those observations, we suggest that SFN may be used as a novel anticancer agent for the treatment of human colon cancer in the future.

15,16-Dihydrotanshinone I, a Compound of Salvia miltiorrhiza Bunge, Induces Apoptosis through Inducing Endoplasmic Reticular Stress in Human Prostate Carcinoma Cells

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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.

Toll-like Receptor 3 Regulates Angiogenesis and Apoptosis in Prostate Cancer Cell Lines through Hypoxia-Inducible Factor 1α

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Alessio Paone

2010-07-01

Full Text Available Toll-like receptors (TLRs recognize microbial/viral-derived components that trigger innate immune response and conflicting data implicate TLR agonists in cancer, either as protumor or antitumor agents. We previously demonstrated that TLR3 activation mediated by its agonist poly(I:C induces antitumor signaling, leading to apoptosis of prostate cancer cells LNCaP and PC3 with much more efficiency in the former than in the second more aggressive line. The transcription factor hypoxia-induciblefactor 1 (HIF-1regulates several cellular processes, includingapoptosis, in response to hypoxia and to other stimuli also in normoxic conditions. Here we describe a novel protumor machinery triggered by TLR3 activation in PC3 cells consisting of increased expression of the specific 1.3 isoform of HIF-1α and nuclear accumulation of HIF-1 complex in normoxia, resulting in reduced apoptosis and in secretion of functional vascular endothelial growth factor (VEGF. Moreover, we report that, in the less aggressive LNCaP cells, TLR3 activation fails to induce nuclear accumulation of HIF-1α. However, the transfection of 1.3 isoform of hif-1α in LNCaP cells allows poly(I:CI-induced HIF-1 activation, resulting in apoptosis protection and VEGF secretion. Altogether, our findings demonstrate that differences in the basal level of HIF-1α expression in different prostate cancer cell lines underlie their differential response to TLR3 activation, suggesting a correlation between different stages of malignancy, hypoxic gene expression, and beneficial responsiveness to TLR agonists.

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

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

Tumor necrosis factor-α attenuates starvation-induced apoptosis through upregulation of ferritin heavy chain in hepatocellular carcinoma cells

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Kou, Xingrui; Zhao, Qiudong; Zhao, Xue; Li, Rong; Wei, Lixin; Wu, Mengchao; Jing, Yingying; Deng, Weijie; Sun, Kai; Han, Zhipeng; Ye, Fei; Yu, Guofeng; Fan, Qingmin; Gao, Lu

2013-01-01

Tumor microenviroment is characteristic of inflammation, ischemia and starvation of nutrient. TNF-α, which is an extraordinarily pleiotropic cytokine, could be an endogenous tumor promoter in some tumor types. The basic objective of this study was to investigate the effects of TNF-α on the cell viability and apoptosis of hepatocellular carcinoma cells under serum starvation, and to identify the molecular mechanisms involved. For this purpose, five different concentrations of TNF-α and two different serum settings (serum-cultured and serum-deprived) were used to investigate the effects of TNF-α on the cell viability and apoptosis of Hep3B and SMMC-7721 cells. TNF-α (10 ng/ml) attenuated serum starvation-induced apoptosis of hepatocellular carcinoma cells, and autophagy conferred this process. BAY11-7082, a specific inhibitor of NF-κB, reversed the suppression of serum starvation-induced apoptosis by TNF-α. Moreover, TNF-α-induced NF-κB transactivation was suppressed by autophagy inhibitor 3-MA. In addition, TNF-α up-regulated Ferritin heavy chain (FHC) transiently by NF-κB activation and FHC levels were correlated with the TNF-α-induced protection against serum starvation-mediated apoptosis of hepatocellular carcinoma cells. Furthermore, FHC-mediated inhibition of apoptosis depended on suppressing ROS accumulation. Our findings suggested that autophagy conferred the TNF-α protection against serum starvation-mediated apoptosis of hepatocellular carcinoma cells, the mechanism involved with the activation of the TNF-α/ NF-κB /FHC signaling pathway

LW6, a hypoxia-inducible factor 1 inhibitor, selectively induces apoptosis in hypoxic cells through depolarization of mitochondria in A549 human lung cancer cells.

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Sato, Mariko; Hirose, Katsumi; Kashiwakura, Ikuo; Aoki, Masahiko; Kawaguchi, Hideo; Hatayama, Yoshiomi; Akimoto, Hiroyoshi; Narita, Yuichiro; Takai, Yoshihiro

2015-09-01

Hypoxia‑inducible factor 1 (HIF‑1) activates the transcription of genes that act upon the adaptation of cancer cells to hypoxia. LW6, an HIF‑1 inhibitor, was hypothesized to improve resistance to cancer therapy in hypoxic tumors by inhibiting the accumulation of HIF‑1α. A clear anti‑tumor effect under low oxygen conditions would indicate that LW6 may be an improved treatment strategy for cancer in hypoxia. In the present study, the HIF‑1 inhibition potential of LW6 on the growth and apoptosis of A549 lung cancer cells in association with oxygen availability was evaluated. LW6 was observed to inhibit the expression of HIF‑1α induced by hypoxia in A549 cells at 20 mM, independently of the von Hippel‑Lindau protein. In addition, at this concentration, LW6 induced hypoxia‑selective apoptosis together with a reduction in the mitochondrial membrane potential. The intracellular reactive oxygen species levels increased in LW6‑treated hypoxic A549 cells and LW6 induced a hypoxia‑selective increase of mitochondrial O2•‑. In conclusion, LW6 inhibited the growth of hypoxic A549 cells by affecting the mitochondria. The inhibition of the mitochondrial respiratory chain is suggested as a potentially effective strategy to target apoptosis in cancer cells.

Primary bovine skeletal muscle cells enters apoptosis rapidly via the intrinsic pathway when available oxygen is removed.

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Sissel Beate Rønning

Full Text Available Muscle cells undergo changes post-mortem during the process of converting muscle into meat, and this complex process is far from revealed. Recent reports have suggested programmed cell death (apoptosis to be important in the very early period of converting muscle into meat. The dynamic balance that occurs between anti-apoptotic members, such as Bcl-2, and pro-apoptotic members (Bid, Bim helps determine whether the cell initiates apoptosis. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to investigate if apoptosis is induced when oxygen is removed from the growth medium. Primary bovine muscle cells were differentiated to form myotubes, and anoxia was induced for 6h. The anoxic conditions significantly increased (P<0.05 the relative gene expression of anti- and pro-apoptotic markers (Aif, Bcl-2, Bid and Bim, and the PARK7 (P<0.05 and Grp75 (Hsp70 protein expressions were transiently increased. The anoxic conditions also led to a loss of mitochondrial membrane potential, which is an early apoptotic event, as well as cytochrome c release from the mitochondria. Finally, reorganization and degradation of cytoskeletal filaments occurred. These results suggest that muscle cells enters apoptosis via the intrinsic pathway rapidly when available oxygen in the muscle diminishes post-mortem.

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

Diclofenac inhibits tumor necrosis factor-a-induced nuclear factor-kB activation causing synergic hepatocyte apoptosis

NARCIS (Netherlands)

Frederiksson, L; Herpers, B; Benedetti, G; Matadin, Q; Puigvert, J.C.; de Bont, H; Dragovic, S.; Vermeulen, N.P.E.; Commandeur, J.N.M.; Danen, E; de Graauw, M; van de Water, B.

2011-01-01

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

Microcystin-LR Induces Apoptosis via NF-κB /iNOS Pathway in INS-1 Cells

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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.

Hypoxia promotes apoptosis of neuronal cells through hypoxia-inducible factor-1α-microRNA-204-B-cell lymphoma-2 pathway.

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Wang, Xiuwen; Li, Ji; Wu, Dongjin; Bu, Xiangpeng; Qiao, Yong

2016-01-01

Neuronal cells are highly sensitive to hypoxia and may be subjected to apoptosis when exposed to hypoxia. Several apoptosis-related genes and miRNAs involve in hypoxia-induced apoptosis. This study aimed to examine the role of HIF1α-miR-204-BCL-2 pathway in hypoxia-induced apoptosis in neuronal cells. Annexin V/propidium iodide assay was performed to analyze cell apoptosis in AGE1.HN and PC12 cells under hypoxic or normoxic conditions. The expression of BCL-2 and miR-204 were determined by Western blot and qRT-PCR. The effects of miR-204 overexpression or knockdown on the expression of BCL-2 were evaluated by luciferase assay and Western blot under hypoxic or normoxic conditions. HIF-1α inhibitor YC-1 and siHIF-1α were employed to determine the effect of HIF-1α on the up-regulation of miR-204 and down-regulation of BCL-2 induced by hypoxia. Apoptosis assay showed the presence of apoptosis induced by hypoxia in neuronal cells. Moreover, we found that hypoxia significantly down-regulated the expression of BCL-2, and increased the mRNA level of miR-204 in neuronal cells than that in control. Bioinformatic analysis and luciferase reporter assay demonstrated that miR-204 directly targeted and regulated the expression of BCL-2. Specifically, the expression of BCL-2 was inhibited by miR-204 mimic and enhanced by miR-204 inhibitor. Furthermore, we detected that hypoxia induced cell apoptosis via HIF-1α/miR-204/BCL-2 in neuronal cells. This study demonstrated that HIF-1α-miR-204-BCL-2 pathway contributed to apoptosis of neuronal cells induced by hypoxia, which could potentially be exploited to prevent spinal cord ischemia-reperfusion injury. © 2015 by the Society for Experimental Biology and Medicine.

Distributed capillary adiabatic tissue homogeneity model in parametric multi-channel blind AIF estimation using DCE-MRI.

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Kratochvíla, Jiří; Jiřík, Radovan; Bartoš, Michal; Standara, Michal; Starčuk, Zenon; Taxt, Torfinn

2016-03-01

One of the main challenges in quantitative dynamic contrast-enhanced (DCE) MRI is estimation of the arterial input function (AIF). Usually, the signal from a single artery (ignoring contrast dispersion, partial volume effects and flow artifacts) or a population average of such signals (also ignoring variability between patients) is used. Multi-channel blind deconvolution is an alternative approach avoiding most of these problems. The AIF is estimated directly from the measured tracer concentration curves in several tissues. This contribution extends the published methods of multi-channel blind deconvolution by applying a more realistic model of the impulse residue function, the distributed capillary adiabatic tissue homogeneity model (DCATH). In addition, an alternative AIF model is used and several AIF-scaling methods are tested. The proposed method is evaluated on synthetic data with respect to the number of tissue regions and to the signal-to-noise ratio. Evaluation on clinical data (renal cell carcinoma patients before and after the beginning of the treatment) gave consistent results. An initial evaluation on clinical data indicates more reliable and less noise sensitive perfusion parameter estimates. Blind multi-channel deconvolution using the DCATH model might be a method of choice for AIF estimation in a clinical setup. © 2015 Wiley Periodicals, Inc.

Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner

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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

The role of tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) in mediating autophagy in myositis skeletal muscle: A potential non-immune mechanism of muscle damage

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Alger, Heather M.; Raben, Nina; Pistilli, Emidio; Francia, Dwight; Rawat, Rashmi; Getnet, Derese; Ghimbovschi, Svetlana; Chen, Yi-Wen; Lundberg, Ingrid E.; Nagaraju, Kanneboyina

2011-01-01

Objective Multinucleated cells are relatively resistant to classical apoptosis, and the factors initiating cell-death and damage in myositis are not well defined. We hypothesized that non-immune autophagic cell death may play a role in muscle fiber damage. Recent literature indicates that tumor necrosis factor-alpha-related apoptosis inducing ligand (TRAIL) may induce both NFκB (nuclear factor kappa-light chain enhancer of activated B cells) activation and autophagic cell death in other systems. Here, we have investigated its role in cell death and pathogenesis in vitro and in vivo using myositis (human and mouse) muscle tissues. Methods Gene expression profiling indicated that expression of TRAIL and several autophagy markers was specifically upregulated in myositis muscle tissue; these results were confirmed by immunohistochemistry and immunoblotting. We also analyzed TRAIL-induced cell death (apoptosis and autophagy) and NFκB activation in vitro in cultured cells. Results TRAIL was expressed predominantly in muscle fibers of myositis, but not in biopsies from normal or other dystrophic-diseased muscle. Autophagy markers were upregulated in human and mouse models of myositis. TRAIL expression was restricted to regenerating/atrophic areas of muscle fascicles, blood vessels, and infiltrating lymphocytes. TRAIL induced NFκB activation and IκB degradation in cultured cells that are resistant to TRAIL-induced apoptosis but undergo autophagic cell death. Conclusion Our data demonstrate that TRAIL is expressed in myositis muscle and may mediate both activation of NFκB and autophagic cell death in myositis. Thus, this non-immune pathway may be an attractive target for therapeutic intervention in myositis. PMID:21769834

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

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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.

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.

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

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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.

SCGB3A2 Inhibits Acrolein-Induced Apoptosis through Decreased p53 Phosphorylation.

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Kurotani, Reiko; Shima, Reika; Miyano, Yuki; Sakahara, Satoshi; Matsumoto, Yoshie; Shibata, Yoko; Abe, Hiroyuki; Kimura, Shioko

2015-04-28

Chronic obstructive pulmonary disease (COPD), a major global health problem with increasing morbidity and mortality rates, is anticipated to become the third leading cause of death worldwide by 2020. COPD arises from exposure to cigarette smoke. Acrolein, which is contained in cigarette smoke, is the most important risk factor for COPD. It causes lung injury through altering apoptosis and causes inflammation by augmenting p53 phosphorylation and producing reactive oxygen species (ROS). Secretoglobin (SCGB) 3A2, a secretory protein predominantly present in the epithelial cells of the lungs and trachea, is a cytokine-like small molecule having anti-inflammatory, antifibrotic, and growth factor activities. In this study, the effect of SCGB3A2 on acrolein-related apoptosis was investigated using the mouse fibroblast cell line MLg as the first step in determining the possible therapeutic value of SCGB3A2 in COPD. Acrolein increased the production of ROS and phosphorylation of p53 and induced apoptosis in MLg cells. While the extent of ROS production induced by acrolein was not affected by SCGB3A2, p53 phosphorylation was significantly decreased by SCGB3A2. These results demonstrate that SCGB3A2 inhibited acrolein-induced apoptosis through decreased p53 phosphorylation, not altered ROS levels.

SCGB3A2 Inhibits Acrolein-Induced Apoptosis through Decreased p53 Phosphorylation

International Nuclear Information System (INIS)

Kurotani, Reiko; Shima, Reika; Miyano, Yuki; Sakahara, Satoshi; Matsumoto, Yoshie; Shibata, Yoko; Abe, Hiroyuki; Kimura, Shioko

2015-01-01

Chronic obstructive pulmonary disease (COPD), a major global health problem with increasing morbidity and mortality rates, is anticipated to become the third leading cause of death worldwide by 2020. COPD arises from exposure to cigarette smoke. Acrolein, which is contained in cigarette smoke, is the most important risk factor for COPD. It causes lung injury through altering apoptosis and causes inflammation by augmenting p53 phosphorylation and producing reactive oxygen species (ROS). Secretoglobin (SCGB) 3A2, a secretory protein predominantly present in the epithelial cells of the lungs and trachea, is a cytokine-like small molecule having anti-inflammatory, antifibrotic, and growth factor activities. In this study, the effect of SCGB3A2 on acrolein-related apoptosis was investigated using the mouse fibroblast cell line MLg as the first step in determining the possible therapeutic value of SCGB3A2 in COPD. Acrolein increased the production of ROS and phosphorylation of p53 and induced apoptosis in MLg cells. While the extent of ROS production induced by acrolein was not affected by SCGB3A2, p53 phosphorylation was significantly decreased by SCGB3A2. These results demonstrate that SCGB3A2 inhibited acrolein-induced apoptosis through decreased p53 phosphorylation, not altered ROS levels

Valsartan protects HK-2 cells from contrast media-induced apoptosis by inhibiting endoplasmic reticulum stress.

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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.

A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells

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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. �

Sulforaphane reverses glucocorticoid-induced apoptosis in osteoblastic cells through regulation of the Nrf2 pathway

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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

The protective effect of resveratrol on human lens epithelial cells against ultraviolet-induced apoptosis

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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.

Correlation of the microculture-kinetic drug-induced apoptosis assay with patient outcomes in initial treatment of adult acute myelocytic leukemia.

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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.

Heart tissue of harlequin (hq)/Big Blue mice has elevated reactive oxygen species without significant impact on the frequency and nature of point mutations in nuclear DNA

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Crabbe, Rory A. [Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7 (Canada); Hill, Kathleen A., E-mail: khill22@uwo.ca [Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7 (Canada)

2010-09-10

Age is a major risk factor for heart disease, and cardiac aging is characterized by elevated mitochondrial reactive oxygen species (ROS) with compromised mitochondrial and nuclear DNA integrity. To assess links between increased ROS levels and mutations, we examined in situ levels of ROS and cII mutation frequency, pattern and spectrum in the heart of harlequin (hq)/Big Blue mice. The hq mouse is a model of premature aging with mitochondrial dysfunction and increased risk of oxidative stress-induced heart disease with the means for in vivo mutation detection. The hq mutation produces a significant downregulation in the X-linked apoptosis-inducing factor gene (Aif) impairing both the antioxidant and oxidative phosphorylation functions of AIF. Brain and skin of hq disease mice have elevated frequencies of point mutations in nuclear DNA and histopathology characterized by cell loss. Reports of associated elevations in ROS in brain and skin have mixed results. Herein, heart in situ ROS levels were elevated in hq disease compared to AIF-proficient mice (p < 0.0001) yet, mutation frequency and pattern were similar in hq disease, hq carrier and AIF-proficient mice. Heart cII mutations were also assessed 15 days following an acute exposure to an exogenous ROS inducer (10 mg paraquat/kg). Acute paraquat exposure with a short mutant manifestation period was insufficient to elevate mutation frequency or alter mutation pattern in the post-mitotic heart tissue of AIF-proficient mice. Paraquat induction of ROS requires mitochondrial complex I and thus is likely compromised in hq mice. Results of this preliminary survey and the context of recent literature suggest that determining causal links between AIF deficiency and the premature aging phenotypes of specific tissues is better addressed with assay of mitochondrial ROS and large-scale changes in mitochondrial DNA in specific cell types.

Heart tissue of harlequin (hq)/Big Blue mice has elevated reactive oxygen species without significant impact on the frequency and nature of point mutations in nuclear DNA

International Nuclear Information System (INIS)

Crabbe, Rory A.; Hill, Kathleen A.

2010-01-01

Age is a major risk factor for heart disease, and cardiac aging is characterized by elevated mitochondrial reactive oxygen species (ROS) with compromised mitochondrial and nuclear DNA integrity. To assess links between increased ROS levels and mutations, we examined in situ levels of ROS and cII mutation frequency, pattern and spectrum in the heart of harlequin (hq)/Big Blue mice. The hq mouse is a model of premature aging with mitochondrial dysfunction and increased risk of oxidative stress-induced heart disease with the means for in vivo mutation detection. The hq mutation produces a significant downregulation in the X-linked apoptosis-inducing factor gene (Aif) impairing both the antioxidant and oxidative phosphorylation functions of AIF. Brain and skin of hq disease mice have elevated frequencies of point mutations in nuclear DNA and histopathology characterized by cell loss. Reports of associated elevations in ROS in brain and skin have mixed results. Herein, heart in situ ROS levels were elevated in hq disease compared to AIF-proficient mice (p < 0.0001) yet, mutation frequency and pattern were similar in hq disease, hq carrier and AIF-proficient mice. Heart cII mutations were also assessed 15 days following an acute exposure to an exogenous ROS inducer (10 mg paraquat/kg). Acute paraquat exposure with a short mutant manifestation period was insufficient to elevate mutation frequency or alter mutation pattern in the post-mitotic heart tissue of AIF-proficient mice. Paraquat induction of ROS requires mitochondrial complex I and thus is likely compromised in hq mice. Results of this preliminary survey and the context of recent literature suggest that determining causal links between AIF deficiency and the premature aging phenotypes of specific tissues is better addressed with assay of mitochondrial ROS and large-scale changes in mitochondrial DNA in specific cell types.

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

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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.

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

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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.

Effect of aging on UVC-induced apoptosis of rat splenocytes

International Nuclear Information System (INIS)

Radziszewska, E.; Piwocka, K.; Bielak-Zmijewska, A.; Sikora, E.; Skierski, J.

2000-01-01

UVC-induced apoptotic symptoms such as morphological changes, DNA fragmentation, Bcl-2 and Bax protein expression were examined in primary splenocyte cultures from young (3 months) and old (24 months) rats. The activities of AP-1 and CRE transcription factors in UVC-irradiated splenocytes were also assessed. At 24 h after UVC irradiation 40% of cells derived from young rats were found to be apoptotic, which was twice as much as in splenocytes from old rats. Apoptosis in cells from old rats did not give typical symptoms like a ''DNA ladder'' and Bcl-2 protein downregulation, in contrast to splenocytes from young rats. No AP-1 transcription factor activity was found in UVC-irradiated splenocytes from old animals and only a trace activity in splenocytes from young animals. This indicates that, UVC-induced apoptosis in rat splenocytes is practically AP-1 independent and that cells from old rats are less sensitive to UVC irradiation than splenocytes from young rats. (author)

Propolis augments apoptosis induced by butyrate via targeting cell survival pathways.

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Eric Drago

Full Text Available Diet is one of the major lifestyle factors affecting incidence of colorectal cancer (CC, and despite accumulating evidence that numerous diet-derived compounds modulate CC incidence, definitive dietary recommendations are not available. We propose a strategy that could facilitate the design of dietary supplements with CC-preventive properties. Thus, nutrient combinations that are a source of apoptosis-inducers and inhibitors of compensatory cell proliferation pathways (e.g., AKT signaling may produce high levels of programmed death in CC cells. Here we report the combined effect of butyrate, an apoptosis inducer that is produced through fermentation of fiber in the colon, and propolis, a honeybee product, on CC cells. We established that propolis increases the apoptosis of CC cells exposed to butyrate through suppression of cell survival pathways such as the AKT signaling. The programmed death of CC cells by combined exposure to butyrate and propolis is further augmented by inhibition of the JNK signaling pathway. Analyses on the contribution of the downstream targets of JNK signaling, c-JUN and JAK/STAT, to the apoptosis of butyrate/propolis-treated CC cells ascertained that JAK/STAT signaling has an anti-apoptotic role; whereas, the role of cJUN might be dependent upon regulatory cell factors. Thus, our studies ascertained that propolis augments apoptosis of butyrate-sensitive CC cells and re-sensitizes butyrate-resistant CC cells to apoptosis by suppressing AKT signaling and downregulating the JAK/STAT pathway. Future in vivo studies should evaluate the CC-preventive potential of a dietary supplement that produces high levels of colonic butyrate, propolis, and diet-derived JAK/STAT inhibitors.

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.

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.

The role of autophagy in THP-1 macrophages resistance to HIV- vpr-induced apoptosis

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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.

The effects of humanin and its analogues on male germ cell apoptosis induced by chemotherapeutic drugs.

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Jia, Yue; Ohanyan, Aikoui; Lue, Yan-He; Swerdloff, Ronald S; Liu, Peter Y; Cohen, Pinchas; Wang, Christina

2015-04-01

Human (HN) prevents stress-induced apoptosis in many cells/tissues. In this study we showed that HN ameliorated chemotherapy [cyclophosphamide (CP) and Doxorubicin (DOX)]-induced male germ cell apoptosis both ex vivo in seminiferous tubule cultures and in vivo in the testis. HN acts by several putative mechanisms via binding to: an IL-12 like trimeric membrane receptor; BAX; or insulin-like growth factor binding protein-3 (IGFBP-3, a proapoptotic factor). To understand the mechanisms of HN on male germ cell apoptosis, we studied five HN analogues including: HNG (HN-S14G, a potent agonist), HNG-F6A (no binding to IGFBP-3), HN-S7A (no self-dimerization), HN-C8P (no binding to BAX), and HN-L12A (a HN antagonist) on CP-induced male germ cell apoptosis in mice. CP-induced germ cell apoptosis was inhibited by HN, HNG, HNG-F6A, HN-S7A, and HN-C8P (less effective); but not by HN-L12A. HN-L12A, but not HN-S7A or HN-C8P, blocked the protective effect of HN against CP-induced male germ cell apoptosis. HN, HN-S7A, and HN-C8P restored CP-suppressed STAT3 phosphorylation. These results suggest that HN: (1) decreases DOX (ex vivo) and CP (in vivo) induced male germ cell apoptosis; (2) action is mediated by the membrane receptor/STAT3 with minor contribution by BAX-binding pathway; (3) self-dimerization or binding to IGFBP-3 may not be involved in HN's effect in testis. HN is an important molecule in the regulation of germ cell homeostasis after injury and agonistic analogues may be developed for treating male infertility or protection against chemotherapy side effects.

GSK-3beta inhibition enhances sorafenib-induced apoptosis in melanoma cell lines.

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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.

Zoledronate induces apoptosis in cells from fibro-cellular membrane of unicameral bone cyst (UBC).

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Yu, John; Chang, Seong-Sil; Suratwala, Sanjeev; Chung, Woo-Sik; Abdelmessieh, Peter; Lee, Hahn-Jun; Yang, Jay; Lee, Francis Young-In

2005-09-01

Unicameral bone cyst (UBC) is a benign cystic lesion in children which is prone to fracture. Various treatments are available, but recurrence after different types of percutaneous injection therapy can cause bone destruction and pathologic fracture. The potential therapeutic effects of anti-resorptive agents, such as bisphosphonates, have not been investigated for UBC. The objective of this study was to characterize the cells from the fibro-cellular membrane of unicameral bone cyst (UBC cells) and to determine whether zoledronate, a nitrogen-containing bisphosphonate, could induce apoptosis in UBC cells. Flow cytometry and immunoblotting were performed in order to determine whether zoledronate induced apoptosis. Cells derived from normal human trabecular bones were used as controls against UBC cells to compare the effect of zoledronate in inducing apoptosis. Immunohisto/cytochemistry (IHC/ICC) and mini-array analyses were performed on tissues and cultured cells. Isolated peripheral blood mononuclear cells were incubated with conditioned media from the UBC cells to determine whether they are capable of inducing osteoclastogenesis. UBC membrane is composed of cells staining positively with CD68, SDF-1, STRO-1 and RANKL, but in vitro cells showed no staining with antibodies to CD68 and STRO-1, suggesting that there was a clonal selection of stromal cells during cell culture. UBC cells also express RUNX2 (runt-related transcription factor-2, core binding factor-1), a key transcription factor for osteoblastic differentiation. In addition, media collected from UBC cells induced a generation of multi-nucleated osteoclast-like cells of peripheral blood mononuclear cells. Zoledronate induced apoptosis of UBC cells in a dose-dependent manner. Apoptosis was evidenced by induction of the active cleaved form of caspase-3. The baseline apoptotic fractions were similar in UBC cells and trabecular bone cells. However, in the overall apoptotic fractions in this study, trabecular

Insulin like growth factor-1 prevents 1-mentyl-4-phenylphyridinium-induced apoptosis in PC12 cells through activation of glycogen synthase kinase-3beta

International Nuclear Information System (INIS)

Sun, Xin; Huang, Luqi; Zhang, Min; Sun, Shenggang; Wu, Yan

2010-01-01

Dopaminergic neurons are lost mainly through apoptosis in Parkinson's disease. Insulin like growth factor-1 (IGF-1) inhibits apoptosis in a wide variety of tissues. Here we have shown that IGF-1 protects PC12 cells from toxic effects of 1-methyl-4-phenylpyridiniumion (MPP + ). Treatment of PC12 cells with recombinant human IGF-1 significantly decreased apoptosis caused by MPP + as measured by acridine orange/ethidium bromide staining. IGF-1 treatment induced sustained phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) as shown by western blot analysis. The anti-apoptotic effect of IGF-1 was abrogated by LY294002, which indirectly inhibits phosphorylation of GSK-3beta. Lithium chloride (LiCl), a known inhibitor of GSK-3beta, also blocked MPP + -induced apoptosis. Finally, although IGF-1 enhanced phosphorylation of extracellular signal-regulated kinases ERK1 and 2 (ERK1/2), PD98059, a specific inhibitor of ERK1/2, did not alter the survival effect of IGF-1. Thus, our findings indicate that IGF-1 protects PC12 cells exposed to MPP + from apoptosis via the GSK-3beta signaling pathway.

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

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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.

[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.

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

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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.

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

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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.

Up-regulation of hypoxia inducible factor-1α by cobalt chloride correlates with proliferation and apoptosis in PC-2 cells

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Dai Zhi-Jun

2012-03-01

Full Text Available Abstract Background The exact mechanism of the effects of hypoxia on the proliferation and apoptosis in carcinoma cells is still conflicting. This study investigated the variation of hypoxia-inducible factor-1α(HIF-1α expression and the apoptosis effect of hypoxia stimulated by cobalt chloride (CoCl2 in pancreatic cancer PC-2 cells. Methods PC-2 cells were cultured with different concentration (50-200 μmol/L of CoCl2 after 24-120 hours to simulate hypoxia in vitro. The proliferation of PC-2 cells was examined by MTT assay. The cellular morphology of PC-2 cells were observed by light inverted microscope and transmission electron microscope(EM. The expression of HIF-1α on mRNA and protein level was measured by semi-quantitive RT-PCR and Western blot analysis. Apoptosis of PC-2 cells were demonstrated by flow cytometry with Annexin V-FITC/PI double staining. Results MTT assay showed that the proliferation of PC-2 cells were stimulated in the first 72 h, while after treated over 72 h, a dose- dependent inhibition of cell growth could be observed. By using transmission electron microscope, swollen chondrosomes, accumulated chromatin under the nuclear membrane and apoptosis bodies were observed. Flow cytometer(FCM analysis showed the apoptosis rate was correlated with the dosage of CoCl2. RT-PCR and Western blot analysis indicated that hypoxia could up-regulate the expression of HIF-1α on both mRNA and protein levels. Conclusion Hypoxic microenvironment stimulated by CoCl2 could effectively induce apoptosis and influence cell proliferation in PC-2 cells, the mechanism could be related to up-expression of HIF-1α.

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

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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.

Smac Mimetic Bypasses Apoptosis Resistance in FADD- or Caspase-8-Deficient Cells by Priming for Tumor Necrosis Factor α-Induced Necroptosis

Directory of Open Access Journals (Sweden)

Bram Laukens

2011-10-01

Full Text Available Searching for new strategies to bypass apoptosis resistance, we investigated the potential of the Smac mimetic BV6 in Jurkat leukemia cells deficient in key molecules of the death receptor pathway. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant, FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis in a synergistic manner. In contrast to TNFα, Smac mimetic significantly enhances CD95-induced apoptosis in wild-type but not in FADD-deficient cells. Interestingly, Smac mimetic- and TNFα-mediated cell death occurs without characteristic features of apoptosis (i.e., caspase activation, DNA fragmentation in FADD-deficient cells. By comparison, Smac mimetic and TNFα trigger activation of caspase-8, -9, and -3 and DNA fragmentation in wild-type cells. Consistently, the caspase inhibitor zVAD.fmk fails to block Smac mimetic- and TNFα-triggered cell death in FADD- or caspase-8-deficient cells, while it confers protection in wild-type cells. By comparison, necrostatin-1, an RIP1 kinase inhibitor, abolishes Smac mimetic- and TNFα-induced cell death in FADD- or caspase-8-deficient. Thus, Smac mimetic enhances TNFα-induced cell death in leukemia cells via two distinct pathways in a context-dependent manner: it primes apoptosis-resistant cells lacking FADD or caspase-8 to TNFα-induced, RIP1-dependent and caspase-independent necroptosis, whereas it sensitizes apoptosis-proficient cells to TNFα-mediated, caspase-dependent apoptosis. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance.

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

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

Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation

DEFF Research Database (Denmark)

Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid

2004-01-01

Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induc......Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation......, possibly because of conflicting growth control. Interestingly, E7/p21-induced cell death is associated with the activation of a newly identified mediator of apoptosis, namely cathepsin B. Activation of the cellular caspases is undetectable in cells undergoing E7/p21-induced apoptosis. To our knowledge...

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.

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

Iron dysregulation combined with aging prevents sepsis-induced apoptosis.

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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.

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.

Valsartan Protects Against Contrast-Induced Acute Kidney Injury in Rats by Inhibiting Endoplasmic Reticulum Stress-Induced Apoptosis.

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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.

Curcumin-induced fibroblast apoptosis and in vitro wound contraction are regulated by antioxidants and heme oxygenase: implications for scar formation.

NARCIS (Netherlands)

Scharstuhl, A.; Mutsaers, H.A.M.; Pennings, S.W.C.; Szarek, W.A.; Russel, F.G.M.; Wagener, F.A.D.T.G.

2009-01-01

Fibroblast apoptosis plays a crucial role in normal and pathological scar formation and therefore we studied whether the putative apoptosis-inducing factor curcumin affects fibroblast apoptosis and may function as a novel therapeutic. We show that 25-microM curcumin causes fibroblast apoptosis and

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.

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...

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.

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)

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).

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.)

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)

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

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

Sirt1 protects against oxidative stress-induced renal tubular cell apoptosis by the bidirectional regulation of catalase expression

International Nuclear Information System (INIS)

Hasegawa, Kazuhiro; Wakino, Shu; Yoshioka, Kyoko; Tatematsu, Satoru; Hara, Yoshikazu; Minakuchi, Hitoshi; Washida, Naoki; Tokuyama, Hirobumi; Hayashi, Koichi; Itoh, Hiroshi

2008-01-01

NAD + -dependent protein deacetylase Sirt1 regulates cellular apoptosis. We examined the role of Sirt1 in renal tubular cell apoptosis by using HK-2 cells, proximal tubular cell lines with or without reactive oxygen species (ROS), H 2 O 2 . Without any ROS, Sirt1 inhibitors enhanced apoptosis and the expression of ROS scavenger, catalase, and Sirt1 overexpression downregulated catalase. When apoptosis was induced with H 2 O 2 , Sirt1 was upregulated with the concomitant increase in catalase expression. Sirt1 overexpression rescued H 2 O 2 -induced apoptosis through the upregulation of catalase. H 2 O 2 induced the nuclear accumulation of forkhead transcription factor, FoxO3a and the gene silencing of FoxO3a enhanced H 2 O 2 -induced apoptosis. In conclusion, endogenous Sirt1 maintains cell survival by regulating catalase expression and by preventing the depletion of ROS required for cell survival. In contrast, excess ROS upregulates Sirt1, which activates FoxO3a and catalase leading to rescuing apoptosis. Thus, Sirt1 constitutes a determinant of renal tubular cell apoptosis by regulating cellular ROS levels

Diosgenin induces apoptosis in IGF-1-stimulated human thyrocytes through two caspase-dependent pathways

International Nuclear Information System (INIS)

Mu, Shumin; Tian, Xingsong; Ruan, Yongwei; Liu, Yuantao; Bian, Dezhi; Ma, Chunyan; Yu, Chunxiao; Feng, Mei; Wang, Furong; Gao, Ling; Zhao, Jia-jun

2012-01-01

Highlights: ► Diosgenin induces apoptosis in IGF-1-treated thyrocytes through two caspase pathways. ► Diosgenin inhibits FLIP and activates caspase-8 in FAS related-pathway. ► Diosgenin increases ROS, regulates the ratio of Bax/Bcl-2 in mitochondrial pathway. -- Abstract: Insulin-like growth factor-1 (IGF-1) is a growth factor of the thyroid that has been shown in our previous study to possess proliferative and antiapoptotic effects in FRTL-5 cell lines through the upregulation of cyclin D and Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). Diosgenin, a natural steroid sapogenin from plants, has been shown to induce apoptosis in many cell lines, with the exception of thyroid cells. In this report, we investigated the apoptotic effect and mechanism of diosgenin in IGF-1-stimulated primary human thyrocytes. Primary human thyrocytes were preincubated with or without IGF-1 for 24 h and subsequently exposed to varying concentrations of diosgenin for different times. We found that diosgenin induced apoptosis in human thyrocytes pretreated with IGF-1 in a dose-dependent manner through the activation of caspase cascades. Moreover, diosgenin inhibited FLIP and activated caspase-8 in the FAS-related apoptotic pathway. Diosgenin increased the production of ROS, regulated the balance of Bax and Bcl-2 and cleaved caspase-9 in the mitochondrial apoptotic pathway. These results indicate that diosgenin induces apoptosis in IGF-1-stimulated primary human thyrocytes through two caspase-dependent pathways.

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.

Ruxolitinib synergizes with DMF to kill via BIM+BAD-induced mitochondrial dysfunction and via reduced SOD2/TRX expression and ROS.

Science.gov (United States)

Tavallai, Mehrad; Booth, Laurence; Roberts, Jane L; McGuire, William P; Poklepovic, Andrew; Dent, Paul

2016-04-05

We determined whether the myelofibrosis drug ruxolitinib, an inhibitor of Janus kinases 1/2 (JAK1 and JAK2), could interact with the multiple sclerosis drug dimethyl-fumarate (DMF) to kill tumor cells; studies used the in vivo active form of the drug, mono-methyl fumarate (MMF). Ruxolitinib interacted with MMF to kill brain, breast, lung and ovarian cancer cells, and enhanced the lethality of standard of care therapies such as paclitaxel and temozolomide. MMF also interacted with other FDA approved drugs to kill tumor cells including Celebrex® and Gilenya®. The combination of [ruxolitinib + MMF] inactivated ERK1/2, AKT, STAT3 and STAT5; reduced expression of MCL-1, BCL-XL, SOD2 and TRX; increased BIM expression; decreased BAD S112 S136 phosphorylation; and enhanced pro-caspase 3 cleavage. Expression of activated forms of STAT3, MEK1 or AKT each significantly reduced drug combination lethality; prevented BAD S112 S136 dephosphorylation and decreased BIM expression; and preserved TRX, SOD2, MCL-1 and BCL-XL expression. The drug combination increased the levels of reactive oxygen species in cells, and over-expression of TRX or SOD2 prevented drug combination tumor cell killing. Over-expression of BCL-XL or knock down of BAX, BIM, BAD or apoptosis inducing factor (AIF) protected tumor cells. The drug combination increased AIF : HSP70 co-localization in the cytosol but this event did not prevent AIF : eIF3A association in the nucleus.

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

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

Apoptosis in fish: environmental factors and programmed cell death.

Science.gov (United States)

AnvariFar, Hossein; Amirkolaie, Abdolsamad Keramat; Miandare, Hamed Kolangi; Ouraji, Hossein; Jalali, M Ali; Üçüncü, Sema İşisağ

2017-06-01

Apoptosis, a form of programmed cell death, is a critical component in maintaining homeostasis and growth in all tissues and plays a significant role in immunity and cytotoxicity. In contrast to necrosis or traumatic cell death, apoptosis is a well-controlled and vital process characterized mainly by cytoplasmic shrinkage, chromatin condensation, DNA fragmentation, membrane blebbing and apoptotic bodies. Our understanding of apoptosis is partly based on observations in invertebrates but mainly in mammals. Despite the great advantages of fish models in studying vertebrate development and diseases and the tremendous interest observed in recent years, reports on apoptosis in fish are still limited. Although apoptotic machinery is well conserved between aquatic and terrestrial organisms throughout the history of evolution, some differences exist in key components of apoptotic pathways. Core parts of apoptotic machinery in fish are virtually expressed as equivalent to the mammalian models. Some differences are, however, evident, such as the extrinsic and intrinsic pathways of apoptosis including lack of a C-terminal region in the Fas-associated protein with a death domain in fish. Aquatic species inhabit a complex and highly fluctuating environment, making these species good examples to reveal features of apoptosis that may not be easily investigated in mammals. Therefore, in order to gain a wider view on programmed cell death in fish, interactions between the main environmental factors, chemicals and apoptosis are discussed in this review. It is indicated that apoptosis can be induced in fish by exposure to environmental stressors during different stages of the fish life cycle.

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

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.

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

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.

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...

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.

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.

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.

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

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.

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.

A benzoxazine derivative induces vascular endothelial cell apoptosis in the presence of fibroblast growth factor-2 by elevating NADPH oxidase activity and reactive oxygen species levels.

Science.gov (United States)

Zhao, Jing; He, Qiuxia; Cheng, Yizhe; Zhao, Baoxiang; Zhang, Yun; Zhang, Shangli; Miao, Junying

2009-09-01

Previously, we found that 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (DBO) promoted apoptosis of human umbilical vascular endothelial cells (HUVECs) deprived of growth factors. In this study, we aimed to investigate the effect of DBO and its mechanism of action on angiogenesis and apoptosis of HUVECs in the presence of fibroblast growth factor-2 (FGF-2), which promotes angiogenesis and inhibits apoptosis in vivo and in vitro. DBO significantly inhibited capillary-like tube formation by promoting apoptosis of HUVECs in the presence of FGF-2 in vitro. Furthermore, DBO elevated the levels of reactive oxygen species (ROS) and nitric oxide (NO) and increased the activity of NADPH oxidase and inducible nitric oxide synthase (iNOS) in promoting apoptosis under this condition. Moreover, when NADPH oxidase was inhibited by its specific inhibitor, dibenziodolium chloride (DPI), DBO could not elevate ROS and NO levels in HUVECs. The data suggest that DBO is a new modulator of apoptosis in vitro, and it might function by increasing the activity of NADPH oxidase and iNOS, subsequently elevating the levels of ROS and NO in HUVECs. The findings of this study provide a new small molecule for investigating the FGF-2/NADPH oxidase/iNOS signaling pathway in apoptosis.

Critical role of poly(ADP-ribose) polymerase-1 in modulating the mode of cell death caused by continuous oxidative stress.

Science.gov (United States)

Son, Young-Ok; Kook, Sung-Ho; Jang, Yong-Suk; Shi, Xianglin; Lee, Jeong-Chae

2009-11-01

Continuously generated hydrogen peroxide (H(2)O(2)) inhibits typical apoptosis and instead initiates a caspase-independent, apoptosis-inducing factor (AIF)-mediated pyknotic cell death. This may be related to H(2)O(2)-mediated DNA damage and subsequent ATP depletion, although the exact mechanisms by which the mode of cell death is decided after H(2)O(2) exposure are still unclear. Accumulated evidence and our previous data led us to hypothesize that continuously generated H(2)O(2), not an H(2)O(2) bolus, induces severe DNA damage, signaling poly(ADP-ribose) polymerase-1 (PARP-1) activation, ATP depletion, and eventually caspase-independent cell death. Results from the present study support that H(2)O(2) generated continuously by glucose oxidase causes excessive DNA damage and PARP-1 activation. Blockage of PARP-1 by a siRNA transfection or by pharmacological inhibitor resulted in the significant inhibition of ATP depletion, loss of mitochondrial membrane potential, nuclear translocation of AIF and endonuclease G, and eventually conversion to caspase-dependent apoptosis. Overall, the current study demonstrates the different roles of PARP-1 inhibition in modulation of cell death according to the method of H(2)O(2) exposure, that is, continuous generation versus a direct addition. (c) 2009 Wiley-Liss, Inc.

Akt interacts directly with Smad3 to regulate the sensitivity to TGF-beta induced apoptosis.

Science.gov (United States)

Conery, Andrew R; Cao, Yanna; Thompson, E Aubrey; Townsend, Courtney M; Ko, Tien C; Luo, Kunxin

2004-04-01

Transforming growth factor beta (TGF-beta) induces both apoptosis and cell-cycle arrest in some cell lines, but only growth arrest in others. It is not clear how this differential response to TGF-beta is specified. Smad proteins are critical mediators of TGF-beta signalling. After stimulation by TGF-beta, Smad2 and Smad3 become phosphorylated by the activated TGF-beta receptor kinases, oligomerize with Smad4, translocate to the nucleus and regulate the expression of TGF-beta target genes. Here we report that the sensitivity to TGF-beta induced apoptosis is regulated by crosstalk between the Akt/PKB serine/threonine kinase and Smad3 through a mechanism that is independent of Akt kinase activity. Akt interacts directly with unphosphorylated Smad3 to sequester it outside the nucleus, preventing its phosphorylation and nuclear translocation. This results in inhibition of Smad3-mediated transcription and apoptosis. Furthermore, the ratio of Smad3 to Akt correlates with the sensitivity of cells to TGF-beta induced apoptosis. Alteration of this ratio changes the apoptotic, but not the growth-inhibitory, responses of cells to TGF-beta. These findings identify an important determinant of sensitivity to TGF-beta-induced apoptosis that involves crosstalk between the TGF-beta and phosphatidylinositol-3-OH kinase (PI(3)K) pathways.

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.

[Roles of KLF5 in inhibition TNFα-induced SK-BR-3 breast cancer cell apoptosis].

Science.gov (United States)

Shi, Jianhong; Liu, Caiyun; Zhang, Anyi; Cui, Naipeng; Wang, Bing; Chen, Baoping; Ma, Zhenfeng

2014-07-08

To explore the expression levels and roles of Krüpple-like factor 5 (KLF5) in tumor necrosis factor α (TNFα)-induced SK-BR-3 breast cancer cells. SK-BR-3 breast cancer cells were stimulated by TNFα at different concentrations (0, 1, 5, 10, 20 µg/L) for specified durations (0, 6, 12, 24, 36 h). Western blot was performed to detect KLF5 protein levels. Then Western blot and quantitative real-time PCR (qRT-PCR) were used to detect the expression levels of apoptosis genes. Flow cytometry and qRT-PCR were used to observe the effects of exogenous KLF5 on TNFα-induced apoptosis of SK-BR-3 breast cancer cell. KLF5 expression levels significantly decreased in TNFα-stimulated SK-BR-3 breast cancer cells in a concentration- and time-dependent manner. Quantitative RT-PCR results showed that TNFα up-regulate apoptosis gene caspase 3, caspase 9 and bax expression levels and down-regulate bcl-1 level in SK-BR-3 cells. Adenovirus expression vectors of pAd-GFP and pAd-GFP-KLF5 were constructed and used to infect SK-BR-3 breast cancer cells. Over-expression of GFP-KLF5 inhibited apoptosis in TNFα-stimulated SK-BR-3 breast cancer cells. TNFα reduces KLF5 expression in SK-BR-3 breast cancer cells and KLF5 participates in TNFα-induced SK-BR-3 cell apoptosis.

Angiotensin II induces apoptosis in intestinal epithelial cells through the AT2 receptor, GATA-6 and the Bax pathway

International Nuclear Information System (INIS)

Sun, Lihua; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Yang, Yang; Yang, Hua

2012-01-01

Highlights: ► Ang II-induced apoptosis in intestinal epithelial cell through AT2 receptor. ► The apoptosis process involves in the Bax/Bcl-2 intrinsic pathway. ► GATA-6 short hairpin RNA reduced Bax expression, but not Bcl-2. ► GATA-6 may play a critical role in apoptosis in response to the Ang II challenge. -- Abstract: Angiotensin II (Ang II) has been shown to play an important role in cell apoptosis. However, the mechanisms of Ang-II-induced apoptosis in intestinal epithelial cells are not fully understood. GATA-6 is a zinc finger transcription factor expressed in the colorectal epithelium, which directs cell proliferation, differentiation and apoptosis. In the present study we investigated the underlying mechanism of which GATA-6 affects Ang-II induced apoptosis in intestinal epithelial cells. The in vitro intestinal epithelial cell apoptosis model was established by co-culturing Caco-2 cells with Ang II. Pretreatment with Angiotensin type 2 (AT2) receptor antagonist, PD123319, significantly reduced the expression of Bax and prevented the Caco-2 cells apoptosis induced by Ang II. In addition, Ang II up-regulated the expression of GATA-6. Interestingly, GATA-6 short hairpin RNA prevented Ang II-induced intestinal epithelial cells apoptosis and reduced the expression of Bax, but not Bcl-2. Taken together, the present study suggests that Angiotensin II promotes apoptosis in intestinal epithelial cells through GATA-6 and the Bax pathway in an AT2 receptor-dependent manner.

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

Eukaryotic translation initiation factor 5A induces apoptosis in colon cancer cells and associates with the nucleus in response to tumour necrosis factor α signalling

International Nuclear Information System (INIS)

Taylor, Catherine A.; Sun Zhong; Cliche, Dominic O.; Ming, Hong; Eshaque, Bithi; Jin Songmu; Hopkins, Marianne T.; Thai, Boun; Thompson, John E.

2007-01-01

Eukaryotic translation initiation factor 5A (eIF5A) is thought to function as a nucleocytoplasmic shuttle protein. There are reports of its involvement in cell proliferation, and more recently it has also been implicated in the regulation of apoptosis. In the present study, we examined the effects of eIF5A over-expression on apoptosis and of siRNA-mediated suppression of eIF5A on expression of the tumour suppressor protein, p53. Over-expression of either eIF5A or a mutant of eIF5A incapable of being hypusinated was found to induce apoptosis in colon carcinoma cells. Our results also indicate that eIF5A is required for expression of p53 following the induction of apoptosis by treatment with Actinomycin D. Depiction of eIF5A localization by indirect immunofluorescence has indicated, for the first time, that the protein is rapidly translocated from the cytoplasm to the nucleus by death receptor activation or following treatment with Actinomycin D. These findings collectively indicate that unhypusinated eIF5A may have pro-apoptotic functions and that eIF5A is rapidly translocated to the nucleus following the induction of apoptotic cell death

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

Diosgenin induces apoptosis in IGF-1-stimulated human thyrocytes through two caspase-dependent pathways

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Mu, Shumin [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Hospital Affiliated to Shandong Traditional Chinese Medicine University, Jinan 250011 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China); Tian, Xingsong; Ruan, Yongwei [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Liu, Yuantao [The Second Hospital of Shandong University, Jinan 250033 (China); Bian, Dezhi [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Jining Medical College, Jining 272013 (China); Ma, Chunyan [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Yu, Chunxiao [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China); Feng, Mei [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Wang, Furong [Shandong University of Traditional Chinese Medicine, Jinan 250011 (China); Gao, Ling [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China); Zhao, Jia-jun, E-mail: jjzhao@medmail.com.cn [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China)

2012-02-10

Highlights: Black-Right-Pointing-Pointer Diosgenin induces apoptosis in IGF-1-treated thyrocytes through two caspase pathways. Black-Right-Pointing-Pointer Diosgenin inhibits FLIP and activates caspase-8 in FAS related-pathway. Black-Right-Pointing-Pointer Diosgenin increases ROS, regulates the ratio of Bax/Bcl-2 in mitochondrial pathway. -- Abstract: Insulin-like growth factor-1 (IGF-1) is a growth factor of the thyroid that has been shown in our previous study to possess proliferative and antiapoptotic effects in FRTL-5 cell lines through the upregulation of cyclin D and Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). Diosgenin, a natural steroid sapogenin from plants, has been shown to induce apoptosis in many cell lines, with the exception of thyroid cells. In this report, we investigated the apoptotic effect and mechanism of diosgenin in IGF-1-stimulated primary human thyrocytes. Primary human thyrocytes were preincubated with or without IGF-1 for 24 h and subsequently exposed to varying concentrations of diosgenin for different times. We found that diosgenin induced apoptosis in human thyrocytes pretreated with IGF-1 in a dose-dependent manner through the activation of caspase cascades. Moreover, diosgenin inhibited FLIP and activated caspase-8 in the FAS-related apoptotic pathway. Diosgenin increased the production of ROS, regulated the balance of Bax and Bcl-2 and cleaved caspase-9 in the mitochondrial apoptotic pathway. These results indicate that diosgenin induces apoptosis in IGF-1-stimulated primary human thyrocytes through two caspase-dependent pathways.

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

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.

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

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

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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.

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

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.

Ethanol acts as a potent agent sensitizing colon cancer cells to the TRAIL-induced apoptosis

Czech Academy of Sciences Publication Activity Database

Vaculová, Alena; Hofmanová, Jiřina; Souček, Karel; Anděra, Ladislav; Kozubík, Alois

2004-01-01

Roč. 577, 1-2 (2004), s. 309-313 ISSN 0014-5793 R&D Projects: GA ČR GA524/04/0895; GA AV ČR KSK5011112; GA AV ČR IBS5004009 Institutional research plan: CEZ:AV0Z5004920 Keywords : TNF-related apoptosis-inducing ligand * ethanol * apoptosis Subject RIV: BO - Biophysics Impact factor: 3.843, year: 2004

Effect of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) combined with ionizing radiation on proliferation and apoptosis of breast cancer MCF-7 cell lines

International Nuclear Information System (INIS)

Zhang Yusong; Fu Jinxiang; Zhou Jianying; Zhou Liying; Guo Xiaokui; Zhuang Zhixiang

2007-01-01

Objective: To investigate the effect of Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) on breast cancer MCF-7 cell lines and the possibility of TRAIL combined with radiotherapy. Methods: 1 x 10 4 /ml MCF-7 cell suspension were added to each well of 96-well plates, MCF cell were treated with radiotherapy(RT), TRAIL at different concentration or RT combined with TRAIL. MTT working solution was added and calculated the inhibitory rates of MCF-7 cells. MCF-7 cell suspension was added to 6-well plates then treated with TRAIL(1 μg/ml), 8 Gy RT or TRAIL combined with 8 Gy RT. The rates of apoptosis were detected by flow cytometry after incubated 48 h. RT-PCR methods were employed to analyze the expression of apoptosis related gene in different treatment group. Results: MCF-7 cell lines were resistant to TRAIL, but the inhibitory rate was upregulated when MCF-7 cell was treated with TRAIL combined with RT, which had a significant difference compared with RT or TRAIL alone. The expression of Bcl-2 and Bcl-Xl gene were down-regulated when MCF-7 cell lines was treated with 8 Gy RT combined with TRAIL. Conclusions: In vitro, MCF-7 cell lines are resistant to TRAIL, but TRAIL combined with radiotherapy increased the cytotoxic effect. TRAIL has a promising prospect in clinical use. (authors)

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.

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.

Analysis of TNF-related apoptosis-inducing ligand and receptors and implications in thymus biology and myasthenia gravis.

Science.gov (United States)

Kanatli, Irem; Akkaya, Bahar; Uysal, Hilmi; Kahraman, Sevim; Sanlioglu, Ahter Dilsad

2017-02-01

Myasthenia Gravis is an autoantibody-mediated, neuromuscular junction disease, and is usually associated with thymic abnormalities presented as thymic tumors (~10%) or hyperplastic thymus (~65%). The exact role of thymus in Myasthenia Gravis development is not clear, yet many patients benefit from thymectomy. The apoptotic ligand TNF-Related Apoptosis-Inducing Ligand is thought to be involved in the regulation of thymocyte counts, although conflicting results are reported. We investigated differential expression profiles of TNF-Related Apoptosis-Inducing Ligand and its transmembrane receptors, Nuclear Factor-kB activation status, and apoptotic cell counts in healthy thymic tissue and pathological thymus from Myasthenia Gravis patients. All tissues expressed TNF-Related Apoptosis-Inducing Ligand and its receptors, with hyperplastic tissue having the highest expression levels of death receptors DR4 and DR5. No detectable Nuclear Factor-kB activation, at least via the canonical Protein Kinase A-mediated p65 Ser276 phosphorylation, was evident in any of the tissues studied. Apoptotic cell counts were higher in MG-associated tissue compared to the normal thymus. Possible use of the TNF-Related Apoptosis-Inducing Ligand within the concept of an apoptotic ligand-mediated medical thymectomy in thymoma- or thymic hyperplasia-associated Myasthenia Gravis is also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins.

Science.gov (United States)

Tollefson, A E; Toth, K; Doronin, K; Kuppuswamy, M; Doronina, O A; Lichtenstein, D L; Hermiston, T W; Smith, C A; Wold, W S

2001-10-01

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus

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

Ionizing radiation induces apoptosis in hematopoietic stem and progenitor cells

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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

TanshinoneIIA and cryptotanshinone protect against hypoxia-induced mitochondrial apoptosis in H9c2 cells.

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Hyou-Ju Jin

Full Text Available Mitochondrial apoptosis pathway is an important target of cardioprotective signalling. Tanshinones, a group of major bioactive compounds isolated from Salvia miltiorrhiza, have been reported with actions against inflammation, oxidative stress, and myocardial ischemia reperfusion injury. However, the actions of these compounds on the chronic hypoxia-related mitochondrial apoptosis pathway have not been investigated. In this study, we examined the effects and molecular mechanisms of two major tanshonones, tanshinone IIA (TIIA and cryptotanshinone (CT on hypoxia induced apoptosis in H9c2 cells. Cultured H9c2 cells were treated with TIIA and CT (0.3 and 3 μΜ 2 hr before and during an 8 hr hypoxic period. Chronic hypoxia caused a significant increase in hypoxia inducible factor 1α expression and the cell late apoptosis rate, which was accompanied with an increase in caspase 3 activity, cytochrome c release, mitochondria membrane potential and expression of pro-apoptosis proteins (Bax and Bak. TIIA and CT (0.3 and 3 μΜ, in concentrations without affecting the cell viability, significantly inhibited the late apoptosis and the changes of caspase 3 activity, cytochrome c release, and mitochondria membrane potential induced by chronic hypoxia. These compounds also suppressed the overexpression of Bax and reduced the ratio of Bax/Bcl-2. The results indicate that TIIA and CT protect against chronic hypoxia induced cell apoptosis by regulating the mitochondrial apoptosis signaling pathway, involving inhibitions of mitochondria hyperpolarization, cytochrome c release and caspase 3 activity, and balancing anti- and pro-apoptotic proteins in Bcl-2 family proteins.

Mitochondrial control of cell death induced by hyperosmotic stress.

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Criollo, Alfredo; Galluzzi, Lorenzo; Maiuri, M Chiara; Tasdemir, Ezgi; Lavandero, Sergio; Kroemer, Guido

2007-01-01

HeLa and HCT116 cells respond differentially to sorbitol, an osmolyte able to induce hypertonic stress. In these models, sorbitol promoted the phenotypic manifestations of early apoptosis followed by complete loss of viability in a time-, dose-, and cell type-specific fashion, by eliciting distinct yet partially overlapping molecular pathways. In HCT116 but not in HeLa cells, sorbitol caused the mitochondrial release of the caspase-independent death effector AIF, whereas in both cell lines cytochrome c was retained in mitochondria. Despite cytochrome c retention, HeLa cells exhibited the progressive activation of caspase-3, presumably due to the prior activation of caspase-8. Accordingly, caspase inhibition prevented sorbitol-induced killing in HeLa, but only partially in HCT116 cells. Both the knock-out of Bax in HCT116 cells and the knock-down of Bax in A549 cells by RNA interference reduced the AIF release and/or the mitochondrial alterations. While the knock-down of Bcl-2/Bcl-X(L) sensitized to sorbitol-induced killing, overexpression of a Bcl-2 variant that specifically localizes to mitochondria (but not of the wild-type nor of a endoplasmic reticulum-targeted form) strongly inhibited sorbitol effects. Thus, hyperosmotic stress kills cells by triggering different molecular pathways, which converge at mitochondria where pro- and anti-apoptotic members of the Bcl-2 family exert their control.

The Roles of ROS and Caspases in TRAIL-Induced Apoptosis and Necroptosis in Human Pancreatic Cancer Cells.

Directory of Open Access Journals (Sweden)

Min Zhang

Full Text Available Death signaling provided by tumor necrosis factor (TNF-related apoptosis-inducing ligand (TRAIL can induce death in cancer cells with little cytotoxicity to normal cells; this cell death has been thought to involve caspase-dependent apoptosis. Reactive oxygen species (ROS are also mediators that induce cell death, but their roles in TRAIL-induced apoptosis have not been elucidated fully. In the current study, we investigated ROS and caspases in human pancreatic cancer cells undergoing two different types of TRAIL-induced cell death, apoptosis and necroptosis. TRAIL treatment increased ROS in two TRAIL-sensitive pancreatic cancer cell lines, MiaPaCa-2 and BxPC-3, but ROS were involved in TRAIL-induced apoptosis only in MiaPaCa-2 cells. Unexpectedly, inhibition of ROS by either N-acetyl-L-cysteine (NAC, a peroxide inhibitor, or Tempol, a superoxide inhibitor, increased the annexin V-/propidium iodide (PI+ early necrotic population in TRAIL-treated cells. Additionally, both necrostatin-1, an inhibitor of receptor-interacting protein kinase 1 (RIP1, and siRNA-mediated knockdown of RIP3 decreased the annexin V-/PI+ early necrotic population after TRAIL treatment. Furthermore, an increase in early apoptosis was induced in TRAIL-treated cancer cells under inhibition of either caspase-2 or -9. Caspase-2 worked upstream of caspase-9, and no crosstalk was observed between ROS and caspase-2/-9 in TRAIL-treated cells. Together, these results indicate that ROS contribute to TRAIL-induced apoptosis in MiaPaCa-2 cells, and that ROS play an inhibitory role in TRAIL-induced necroptosis of MiaPaCa-2 and BxPC-3 cells, with caspase-2 and -9 playing regulatory roles in this process.

Apoptosis of mouse hippocampal cells induced by Taenia crassiceps metacestode factor.

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Zepeda, N; Solano, S; Copitin, N; Chávez, J L; Fernández, A M; García, F; Tato, P; Molinari, J L

2017-03-01

Seizures, headache, depression and neurological deficits are the signs and symptoms most frequently reported in human neurocysticercosis. However, the cause of the associated learning and memory deficits is unknown. Here, we used Taenia crassiceps infection in mice as a model of human cysticercosis. The effects of T. crassiceps metacestode infection or T. crassiceps metacestode factor (MF) treatment on mouse hippocampal cells were studied; control mice were included. At 45 days after infection or treatment of the mice with MF, all mice were anaesthetized and perfused transcardially with saline followed by phosphate-buffered 10% formalin. Then the brains were carefully removed. Coronal sections stained using several techniques were analysed. Extensive and significant apoptosis was found in the experimental animals, mainly in the dentate gyrus, CA1, CA2, CA3 and neighbouring regions, in comparison with the apparently intact cells from control mice (P < 0.01). These results suggest that neurological deficits, especially the learning and memory deficits, may be generated by extensive apoptosis of hippocampal cells.

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

Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells

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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

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

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

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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.

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

Scopadulciol, Isolated from Scoparia dulcis, Induces β-Catenin Degradation and Overcomes Tumor Necrosis Factor-Related Apoptosis Ligand Resistance in AGS Human Gastric Adenocarcinoma Cells.

Science.gov (United States)

Fuentes, Rolly G; Toume, Kazufumi; Arai, Midori A; Sadhu, Samir K; Ahmed, Firoj; Ishibashi, Masami

2015-04-24

Scopadulciol (1), a scopadulan-type diterpenoid, was isolated from Scoparia dulcis along with three other compounds (2-4) by an activity-guided approach using the TCF reporter (TOP) luciferase-based assay system. A fluorometric microculture cytotoxicity assay (FMCA) revealed that compound 1 was cytotoxic to AGS human gastric adenocarcinoma cells. The treatment of AGS cells with 1 decreased β-catenin levels and also inhibited its nuclear localization. The pretreatment of AGS cells with a proteasome inhibitor, either MG132 or epoxomicin, protected against the degradation of β-catenin induced by 1. The 1-induced degradation of β-catenin was also abrogated in the presence of pifithrin-α, an inhibitor of p53 transcriptional activity. Compound 1 inhibited TOP activity in AGS cells and downregulated the protein levels of cyclin D1, c-myc, and survivin. Compound 1 also sensitized AGS cells to tumor necrosis factor-related apoptosis ligand (TRAIL)-induced apoptosis by increasing the levels of the death receptors, DR4 and DR5, and decreasing the level of the antiapoptotic protein Bcl-2. Collectively, our results demonstrated that 1 induced the p53- and proteasome-dependent degradation of β-catenin, which resulted in the inhibition of TCF/β-catenin transcription in AGS cells. Furthermore, 1 enhanced apoptosis in TRAIL-resistant AGS when combined with TRAIL.

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

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

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

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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.

Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis☆

Science.gov (United States)

Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting

2013-01-01

Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K3) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ~12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. PMID:22575170

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) enhances vascular and renal damage induced by hyperlipidemic diet in ApoE-knockout mice.

Science.gov (United States)

Muñoz-García, Begoña; Moreno, Juan Antonio; López-Franco, Oscar; Sanz, Ana Belén; Martín-Ventura, José Luis; Blanco, Julia; Jakubowski, Aniela; Burkly, Linda C; Ortiz, Alberto; Egido, Jesús; Blanco-Colio, Luis Miguel

2009-12-01

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily of cytokines. TWEAK binds and activates the Fn14 receptor, and may regulate apoptosis, inflammation, and angiogenesis, in different pathological conditions. We have evaluated the effect of exogenous TWEAK administration as well as the role of endogenous TWEAK on proinflammatory cytokine expression and vascular and renal injury severity in hyperlipidemic ApoE-knockout mice. ApoE(-/-) mice were fed with hyperlipidemic diet for 4 to 10 weeks, then randomized and treated with saline (controls), TWEAK (10 microg/kg/d), anti-TWEAK neutralizing mAb (1000 microg/kg/d), TWEAK plus anti-TWEAK antibody (10 microg TWEAK +1000 microg anti-TWEAK/kg/d), or nonspecific IgG (1000 microg/kg/d) daily for 9 days. In ApoE(-/-) mice, exogenous TWEAK administration in ApoE(-/-) mice induced activation of NF-kappaB, a key transcription factor implicated in the regulation of the inflammatory response, in vascular and renal lesions. Furthermore, TWEAK treatment increased chemokine expression (RANTES and MCP-1), as well as macrophage infiltration in atherosclerotic plaques and renal lesions. These effects were associated with exacerbation of vascular and renal damage. Conversely, treatment of ApoE(-/-) mice with an anti-TWEAK blocking mAb decreased NF-kappaB activation, proinflammatory cytokine expression, macrophage infiltration, and vascular and renal injury severity, indicating a pathological role for endogenous TWEAK. Finally, in murine vascular smooth muscle cells or tubular cells, either ox-LDL or TWEAK treatment increased expression and secretion of both RANTES and MCP-1. Furthermore, ox-LDL and TWEAK synergized for induction of MCP-1 and RANTES expression and secretion. Our results suggest that TWEAK exacerbates the inflammatory response associated with a high lipid-rich diet. TWEAK may be a novel therapeutic target to prevent vascular and renal damage associated with

Inhibition of NF-κB Pathway and Modulation of MAPK Signaling Pathways in Glioblastoma and Implications for Lovastatin and Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL Combination Therapy.

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Pi Chu Liu

Full Text Available Glioblastoma is a common malignant brain tumor and it is refractory to therapy because it usually contains a mixture of cell types. The tumor necrosis factor-related apoptosis inducing ligand (TRAIL has been shown to induce apoptosis in a range of tumor cell types. Previously, we found that two human glioblastoma cell lines are resistant to TRAIL, while lovastatin sensitizes these glioblastoma cells to TRAIL-induced cell death. In this study, we investigated the mechanisms underlying the TRAIL-induced apoptosis in human glioblastoma cell lines by lovastatin. Furthermore, we have confirmed the anti-tumor effect of combination therapy with lovastatin and TRAIL in the subcutaneous brain tumor model. We showed that lovastatin significantly up-regulated the expression of death receptor 5 (DR5 in glioblastoma cell lines as well as in tumor-bearing mice with peri-tumoral administration of lovastatin. Further study in glioblastoma cell lines suggested that lovastatin treatment could inhibit NF-κB and Erk/MAPK pathways but activates JNK pathway. These results suggest that lovastatin sensitizes TRAIL-induced apoptosis by up-regulation of DR5 level via NF-κB inactivation, but also directly induces apoptosis by dysregulation of MAPK pathway. Our in vivo study showed that local peri-tumoral co-injection of lovastatin and TRAIL substantially reduced tumor growth compared with single injection of lovastatin or TRAIL in subcutaneous nude mice model. This study suggests that combined treatment of lovastatin and TRAIL is a promising therapeutic strategy to TRAIL-resistant glioblastoma.

α-Hispanolol sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis via death receptor up-regulation

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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

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

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

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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.

Identification of RIP1 as a critical mediator of Smac mimetic-mediated sensitization of glioblastoma cells for Drozitumab-induced apoptosis.

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Cristofanon, S; Abhari, B A; Krueger, M; Tchoghandjian, A; Momma, S; Calaminus, C; Vucic, D; Pichler, B J; Fulda, S

2015-04-16

This study aims at evaluating the combination of the tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL)-receptor 2 (TRAIL-R2)-specific antibody Drozitumab and the Smac mimetic BV6 in preclinical glioblastoma models. To this end, the effect of BV6 and/or Drozitumab on apoptosis induction and signaling pathways was analyzed in glioblastoma cell lines, primary glioblastoma cultures and glioblastoma stem-like cells. Here, we report that BV6 and Drozitumab synergistically induce apoptosis and reduce colony formation in several glioblastoma cell lines (combination indextrigger the formation of a cytosolic receptor-interacting protein (RIP) 1/Fas-associated via death domain (FADD)/caspase-8-containing complex and subsequent activation of caspase-8 and -3. BV6- and Drozitumab-induced apoptosis is blocked by the caspase inhibitor zVAD.fmk, pointing to caspase-dependent apoptosis. RNA interference-mediated silencing of RIP1 almost completely abolishes the BV6-conferred sensitization to Drozitumab-induced apoptosis, indicating that the synergism critically depends on RIP1 expression. In contrast, both necrostatin-1, a RIP1 kinase inhibitor, and Enbrel, a TNFα-blocking antibody, do not interfere with BV6/Drozitumab-induced apoptosis, demonstrating that apoptosis occurs independently of RIP1 kinase activity or an autocrine TNFα loop. In conclusion, the rational combination of BV6 and Drozitumab presents a promising approach to trigger apoptosis in glioblastoma, which warrants further investigation.

Modulators of Response to Tumor Necrosis-Related Apoptosis-Inducing Ligand (TRAIL) Therapy in Ovarian Cancer

National Research Council Canada - National Science Library

Behbakht, Kian

2008-01-01

.... More effective therapies are urgently needed. One of the most promising therapies in development for ovarian cancer is the use of either the Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL...

Osteoblasts Protect AML Cells from SDF-1-Induced Apoptosis

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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

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)

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

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.

Nicotine prevents the apoptosis induced by menadione in human lung cancer cells

International Nuclear Information System (INIS)

Zhang Tao; Lu Heng; Shang Xuan; Tian Yihao; Zheng Congyi; Wang Shiwen; Cheng Hanhua; Zhou Rongjia

2006-01-01

Approximately 50% of long-term cigarette smokers die prematurely from the adverse effects of smoking, including on lung cancer and other illnesses. Nicotine is a main component in tobacco and has been implicated as a potential factor in the pathogenesis of human lung cancer. However, the mechanism of nicotine action in the development of lung cancer remains largely unknown. In the present study, we designed a nicotine-apoptosis system, by pre-treatment of nicotine making lung cancer cell A549 to be in a physiological nicotine environment, and observed that nicotine promoted cell proliferation and prevented the menadione-induced apoptosis, and exerts its role of anti-apoptosis by shift of apoptotic stage induced by menadione from late apoptotic stage to early apoptotic stage, in which NF-κB was up-regulated. Interference analysis of NF-κB in A549 cells showed that knock down of NF-κB resulted in apoptosis promotion and counteracted the protective effect of nicotine. The findings suggest that nicotine has potential effect in lung cancer genesis, especially in patients with undetectable early tumor development and development of specific NF-κB inhibitors would represent a potentially exciting new pharmacotherapy for tobacco-related lung cancer

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

Directory of Open Access Journals (Sweden)

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.

The microRNA-302b-inhibited insulin-like growth factor-binding protein 2 signaling pathway induces glioma cell apoptosis by targeting nuclear factor IA.

Directory of Open Access Journals (Sweden)

Chin-Cheng Lee

Full Text Available MicroRNAs are small noncoding RNAs that post-transcriptionally control the expression of genes involved in glioblastoma multiforme (GBM development. Although miR-302b functions as a tumor suppressor, its role in GBM is still unclear. Therefore, this study comprehensively explored the roles of miR-302b-mediated gene networks in GBM cell death. We found that miR-302b levels were significantly higher in primary astrocytes than in GBM cell lines. miR-302b overexpression dose dependently reduced U87-MG cell viability and induced apoptosis through caspase-3 activation and poly(ADP ribose polymerase degradation. A transcriptome microarray revealed 150 downregulated genes and 380 upregulated genes in miR-302b-overexpressing cells. Nuclear factor IA (NFIA, higher levels of which were significantly related to poor survival, was identified as a direct target gene of miR-302b and was involved in miR-302b-induced glioma cell death. Higher NFIA levels were observed in GBM cell lines and human tumor sections compared with astrocytes and non-tumor tissues, respectively. NFIA knockdown significantly enhanced apoptosis. We found high levels of insulin-like growth factor-binding protein 2 (IGFBP2, another miR-302b-downregulated gene, in patients with poor survival. We verified that NFIA binds to the IGFBP2 promoter and transcriptionally enhances IGFBP2 expression levels. We identified that NFIA-mediated IGFBP2 signaling pathways are involved in miR-302b-induced glioma cell death. The identification of a regulatory loop whereby miR-302b inhibits NFIA, leading to a decrease in expression of IGFBP-2, may provide novel directions for developing therapies to target glioblastoma tumorigenesis.

Bee venom induces apoptosis through intracellular Ca2+ -modulated intrinsic death pathway in human bladder cancer cells.

Science.gov (United States)

Ip, Siu-Wan; Chu, Yung-Lin; Yu, Chun-Shu; Chen, Po-Yuan; Ho, Heng-Chien; Yang, Jai-Sing; Huang, Hui-Ying; Chueh, Fu-Shin; Lai, Tung-Yuan; Chung, Jing-Gung

2012-01-01

To focus on bee venom-induced apoptosis in human bladder cancer TSGH-8301 cells and to investigate its signaling pathway to ascertain whether intracellular calcium iron (Ca(2+)) is involved in this effect. Bee venom-induced cytotoxic effects, productions of reactive oxygen species and Ca(2+) and the level of mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry. Apoptosis-associated proteins were examined by Western blot analysis and confocal laser microscopy. Bee venom-induced cell morphological changes and decreased cell viability through the induction of apoptosis in TSGH-8301 cell were found. Bee venom promoted the protein levels of Bax, caspase-9, caspase-3 and endonuclease G. The enhancements of endoplasmic reticulum stress-related protein levels were shown in bee venom-provoked apoptosis of TSGH-8301 cells. Bee venom promoted the activities of caspase-3, caspase-8, and caspase-9, increased Ca(2+) release and decreased the level of ΔΨm. Co-localization of immunofluorescence analysis showed the releases of endonuclease G and apoptosis-inducing factor trafficking to nuclei for bee venom-mediated apoptosis. The images revealed evidence of nuclear condensation and formation of apoptotic bodies by 4',6-diamidino-2-phenylindole staining and DNA gel electrophoresis showed the DNA fragmentation in TSGH-8301 cells. Bee venom treatment induces both caspase-dependent and caspase-independent apoptotic death through intracellular Ca(2+) -modulated intrinsic death pathway in TSGH-8301 cells. © 2011 The Japanese Urological Association.

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

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.

Protective role of Nrf2 against mechanical-stretch-induced apoptosis in mouse fibroblasts: a potential therapeutic target of mechanical-trauma-induced stress urinary incontinence.

Science.gov (United States)

Li, Qiannan; Li, Bingshu; Liu, Cheng; Wang, Linlin; Tang, Jianming; Hong, Li

2018-01-10

We investigated the protective effect and underlying molecular mechanism of nuclear factor-E2-related factor 2 (Nrf2) against mechanical-stretch-induced apoptosis in mouse fibroblasts. Normal cells, Nrf2 silencing cells, and Nrf2 overexpressing cells were respectively divided into two groups-nonintervention and cyclic mechanical strain (CMS)-subjected to CMS of 5333 μ (1.0 Hz for 4 h), six groups in total (control, CMS, shNfe212, shNfe212 + CMS, LV-shNfe212, and LV-shNfe212 + CMS). After treatment, cell apoptosis; cell-cycle distribution; expressions of Nrf2, Bax, Bcl-2, Cyt-C, caspase-3, caspase-9, cleaved-caspase-3, and cleaved-caspase-9; mitochondrial membrane potential (ΔΨm); reactive oxygen species (ROS); and malondialdehyde (MDA) levels were measured. Thirty virgin female C57BL/6 mice were divided into two groups: control (without intervention) and vaginal distension (VD) groups, which underwent VD for 1 h with an 8-mm dilator (0.3 ml saline). Leak-point pressure (LPP) was tested on day 7 after VD; Nrf2 expression, apoptosis, and MDA levels were then measured in urethra and anterior vaginal wall. Mechanical stretch decreased Nrf2 messenger RNA (mRNA) and protein expressions. Overexpression of Nrf2 alleviated mechanical-stretch-induced cell apoptosis; S-phase arrest of cell cycle; up-regulation of Bax, cytochrome C (Cyt-C), ROS, MDA, ratio of cleaved-caspase-3/caspase-3 and cleaved-caspase-9/caspase-9; and exacerbated the decrease of Bcl2 and ΔΨm in L929 cells. On the contrary, silencing of Nrf2 showed opposite effects. Besides, VD reduced LPP levels and Nrf2 expression and increased cell apoptosis and MDA generation in the urethra and anterior vaginal wall. Nrf2 exhibits a protective role against mechanical-stretch -induced apoptosis on mouse fibroblasts, which might indicate a potential therapeutic target of mechanical-trauma-induced stress urinary incontinence (SUI).

Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoposis

International Nuclear Information System (INIS)

Zhou Yijun; Wang Jiahe; Zhang Jin

2006-01-01

Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-κB, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-κB, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease

Belinostat-induced apoptosis and growth inhibition in pancreatic cancer cells involve activation of TAK1-AMPK signaling axis

International Nuclear Information System (INIS)

Wang, Bing; Wang, Xin-bao; Chen, Li-yu; Huang, Ling; Dong, Rui-zen

2013-01-01

Highlights: •Belinostat activates AMPK in cultured pancreatic cancer cells. •Activation of AMPK is important for belinostat-induced cytotoxic effects. •ROS and TAK1 are involved in belinostat-induced AMPK activation. •AMPK activation mediates mTOR inhibition by belinostat. -- Abstract: Pancreatic cancer accounts for more than 250,000 deaths worldwide each year. Recent studies have shown that belinostat, a novel pan histone deacetylases inhibitor (HDACi) induces apoptosis and growth inhibition in pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In the current study, we found that AMP-activated protein kinase (AMPK) activation was required for belinostat-induced apoptosis and anti-proliferation in PANC-1 pancreatic cancer cells. A significant AMPK activation was induced by belinostat in PANC-1 cells. Inhibition of AMPK by RNAi knockdown or dominant negative (DN) mutation significantly inhibited belinostat-induced apoptosis in PANC-1 cells. Reversely, AMPK activator AICAR and A-769662 exerted strong cytotoxicity in PANC-1 cells. Belinostat promoted reactive oxygen species (ROS) production in PANC-1 cells, increased ROS induced transforming growth factor-β-activating kinase 1 (TAK1)/AMPK association to activate AMPK. Meanwhile, anti-oxidants N-Acetyl-Cysteine (NAC) and MnTBAP as well as TAK1 shRNA knockdown suppressed belinostat-induced AMPK activation and PANC-1 cell apoptosis. In conclusion, we propose that belinostat-induced apoptosis and growth inhibition require the activation of ROS-TAK1-AMPK signaling axis in cultured pancreatic cancer cells

Belinostat-induced apoptosis and growth inhibition in pancreatic cancer cells involve activation of TAK1-AMPK signaling axis

Energy Technology Data Exchange (ETDEWEB)

Wang, Bing, E-mail: wangbin69@yahoo.com; Wang, Xin-bao; Chen, Li-yu; Huang, Ling; Dong, Rui-zen

2013-07-19

Highlights: •Belinostat activates AMPK in cultured pancreatic cancer cells. •Activation of AMPK is important for belinostat-induced cytotoxic effects. •ROS and TAK1 are involved in belinostat-induced AMPK activation. •AMPK activation mediates mTOR inhibition by belinostat. -- Abstract: Pancreatic cancer accounts for more than 250,000 deaths worldwide each year. Recent studies have shown that belinostat, a novel pan histone deacetylases inhibitor (HDACi) induces apoptosis and growth inhibition in pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In the current study, we found that AMP-activated protein kinase (AMPK) activation was required for belinostat-induced apoptosis and anti-proliferation in PANC-1 pancreatic cancer cells. A significant AMPK activation was induced by belinostat in PANC-1 cells. Inhibition of AMPK by RNAi knockdown or dominant negative (DN) mutation significantly inhibited belinostat-induced apoptosis in PANC-1 cells. Reversely, AMPK activator AICAR and A-769662 exerted strong cytotoxicity in PANC-1 cells. Belinostat promoted reactive oxygen species (ROS) production in PANC-1 cells, increased ROS induced transforming growth factor-β-activating kinase 1 (TAK1)/AMPK association to activate AMPK. Meanwhile, anti-oxidants N-Acetyl-Cysteine (NAC) and MnTBAP as well as TAK1 shRNA knockdown suppressed belinostat-induced AMPK activation and PANC-1 cell apoptosis. In conclusion, we propose that belinostat-induced apoptosis and growth inhibition require the activation of ROS-TAK1-AMPK signaling axis in cultured pancreatic cancer cells.

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.)

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.

Shikonin ameliorates isoproterenol (ISO)-induced myocardial damage through suppressing fibrosis, inflammation, apoptosis and ER stress.

Science.gov (United States)

Yang, Jun; Wang, Zhao; Chen, Dong-Lin

2017-09-01

Shikonin, isolated from the roots of herbal plant Lithospermum erythrorhizon, is a naphthoquinone. It has been reported to exert beneficial anti-inflammatory effects and anti-oxidant properties in various diseases. Isoproterenol (ISO) has been widely used to establish cardiac injury in vivo and in vitro. However, shikonin function in ISO-induced cardiac injury remains uncertain. In our study, we attempted to investigate the efficiency and possible molecular mechanism of shikonin in cardiac injury treatment induced by ISO. In vivo, C57BL6 mice were subcutaneously injected with 5mg/kg ISO to induce heart failure. And mice were given a gavage of shikonin (2 or 4mg/kg/d, for four weeks). Cardiac function, fibrosis indices, inflammation response, apoptosis and endoplasmic reticulum (ER) stress were calculated. Pathological alterations, fibrosis-, inflammation-, apoptosis- and ER stress-related molecules were examined. In ISO-induced cardiac injury, shikonin significantly ameliorated heart function, decreased myocardial fibrosis, suppressed inflammation, attenuated apoptosis and ER stress through impeding collagen accumulation, Toll like receptor 4/nuclear transcription factor κB (TLR4/NF-κB), Caspase-3 and glucose-regulated protein 78 (GRP78) signaling pathways activity, relieving heart failure in vivo. Also, in vitro, shikonin attenuated ISO-induced cardiac muscle cells by reducing fibrosis, inflammation, apoptosis and ER stress. Our findings indicated that shikonin treatment attenuated ISO-induced heart injury, providing an effective therapeutic strategy for heart failure treatment for future. Copyright © 2017. Published by Elsevier Masson SAS.

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.

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.

NF-kappaB and p53 are the dominant apoptosis-inducing transcription factors elicited by the HIV-1 envelope.

Science.gov (United States)

Perfettini, Jean-Luc; Roumier, Thomas; Castedo, Maria; Larochette, Nathanael; Boya, Patricia; Raynal, Brigitte; Lazar, Vladimir; Ciccosanti, Fabiola; Nardacci, Roberta; Penninger, Josef; Piacentini, Mauro; Kroemer, Guido

2004-03-01

The coculture of cells expressing the HIV-1 envelope glycoprotein complex (Env) with cells expressing CD4 results into cell fusion, deregulated mitosis, and subsequent cell death. Here, we show that NF-kappaB, p53, and AP1 are activated in Env-elicited apoptosis. The nuclear factor kappaB (NF-kappaB) super repressor had an antimitotic and antiapoptotic effect and prevented the Env-elicited phosphorylation of p53 on serine 15 and 46, as well as the activation of AP1. Transfection with dominant-negative p53 abolished apoptosis and AP1 activation. Signs of NF-kappaB and p53 activation were also detected in lymph node biopsies from HIV-1-infected individuals. Microarrays revealed that most (85%) of the transcriptional effects of HIV-1 Env were blocked by the p53 inhibitor pifithrin-alpha. Macroarrays led to the identification of several Env-elicited, p53-dependent proapoptotic transcripts, in particular Puma, a proapoptotic "BH3-only" protein from the Bcl-2 family known to activate Bax/Bak. Down modulation of Puma by antisense oligonucleotides, as well as RNA interference of Bax and Bak, prevented Env-induced apoptosis. HIV-1-infected primary lymphoblasts up-regulated Puma in vitro. Moreover, circulating CD4+ lymphocytes from untreated, HIV-1-infected donors contained enhanced amounts of Puma protein, and these elevated Puma levels dropped upon antiretroviral therapy. Altogether, these data indicate that NF-kappaB and p53 cooperate as the dominant proapoptotic transcription factors participating in HIV-1 infection.