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Sample records for ask-1 mediates apoptosis

  1. Pharmacological Inhibition of NOS Activates ASK1/JNK Pathway Augmenting Docetaxel-Mediated Apoptosis in Triple-Negative Breast Cancer.

    Science.gov (United States)

    Dávila-González, Daniel; Choi, Dong Soon; Rosato, Roberto R; Granados-Principal, Sergio M; Kuhn, John G; Li, Wen-Feng; Qian, Wei; Chen, Wen; Kozielski, Anthony J; Wong, Helen; Dave, Bhuvanesh; Chang, Jenny C

    2018-03-01

    Purpose: Chemoresistance in triple-negative breast cancer (TNBC) is associated with the activation of a survival mechanism orchestrated by the endoplasmic reticulum (EnR) stress response and by inducible nitric oxide synthase (iNOS). Our aim was to determine the effects of pharmacologic NOS inhibition on TNBC. Experimental Design: TNBC cell lines, SUM-159PT, MDA-MB-436, and MDA-MB-468, were treated with docetaxel and NOS inhibitor (L-NMMA) for 24, 48, and 72 hours. Apoptosis was assessed by flow cytometry using Annexin-V and propidium iodide. Western blot was used to assess ER stress and apoptosis, and rtPCR was used to evaluate s-XBP1. TNBC patient-derived xenografts (PDX) were treated either with vehicle, docetaxel, or combination therapy (NOS inhibition + docetaxel). Mouse weight and tumor volumes were recorded twice weekly. Docetaxel concentration was determined using mass spectrometry. To quantify proliferation and apoptosis, PDX tumor samples were stained using Ki67 and TUNEL assay. Results: In vitro, L-NMMA ameliorated the iNOS upregulation associated with docetaxel. Apoptosis increased when TNBC cells were treated with combination therapy. In TNBC PDXs, combination therapy significantly reduced tumor volume growth and increased survival proportions. In the BCM-5998 PDX model, intratumoral docetaxel concentration was higher in mice receiving combination therapy. Coupling docetaxel with NOS inhibition increased EnR-stress response via coactivation of ATF4 and CHOP, which triggered the pASK1/JNK proapoptotic pathway, promoting cleavage of caspases 3 and 9. Conclusions: iNOS is a critical target for docetaxel resistance in TNBC. Pharmacologic inhibition of NOS enhanced chemotherapy response in TNBC PDX models. Combination therapy may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy. Clin Cancer Res; 24(5); 1152-62. ©2018 AACR . ©2018 American Association for Cancer Research.

  2. MicroRNA-Mediated Down-Regulation of Apoptosis Signal-Regulating Kinase 1 (ASK1 Attenuates the Apoptosis of Human Mesenchymal Stem Cells (MSCs Transplanted into Infarcted Heart

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    Chang Youn Lee

    2016-10-01

    Full Text Available Stem cell therapy using adult stem cells, such as mesenchymal stem cells (MSCs has produced some promising results in treating the damaged heart. However, the low survival rate of MSCs after transplantation is still one of the crucial factors that limit the therapeutic effect of stem cells. In the damaged heart, oxidative stress due to reactive oxygen species (ROS production can cause the death of transplanted MSCs. Apoptosis signal-regulating kinase 1 (ASK1 has been implicated in the development of oxidative stress-related pathologic conditions. Thus, we hypothesized that down-regulation of ASK1 in human MSCs (hMSCs might attenuate the post-transplantation death of MSCs. To test this hypothesis, we screened microRNAs (miRNAs based on a miRNA-target prediction database and empirical data and investigated the anti-apoptotic effect of selected miRNAs on human adipose-derived stem cells (hASCs and on rat myocardial infarction (MI models. Our data indicated that miRNA-301a most significantly suppressed ASK1 expression in hASCs. Apoptosis-related genes were significantly down-regulated in miRNA-301a-enriched hASCs exposed to hypoxic conditions. Taken together, these data show that miRNA-mediated down-regulation of ASK1 protects MSCs during post-transplantation, leading to an increase in the efficacy of MSC-based cell therapy.

  3. 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to inhibit hepatocellular carcinoma cells

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    Fu, Meili; Wan, Fuqiang; Li, Zhengling; Zhang, Fenghua

    2016-01-01

    The aim of the present study is to investigate the potential anti-hepatocellular carcinoma (HCC) cell activity by 4SC-202, a novel class I HDAC inhibitor (HDACi). The associated signaling mechanisms were also analyzed. We showed that 4SC-202 treatment induced potent cytotoxic and proliferation–inhibitory activities against established HCC cell lines (HepG2, HepB3, SMMC-7721) and patient-derived primary HCC cells. Further, adding 4SC-202 in HCC cells activated mitochondrial apoptosis pathway, which was evidenced by mitochondrial permeability transition pore (mPTP) opening, cytochrome C cytosol release and caspase-3/-9 activation. Inhibition of this apoptosis pathway, by caspase-3/-9 inhibitors, mPTP blockers, or by shRNA-mediated knockdown of cyclophilin-D (Cyp-D, a key component of mPTP), significantly attenuated 4SC-202-induced HCC cell death and apoptosis. Reversely, over-expression of Cyp-D enhanced 4SC-202's sensitivity in HCC cells. Further studies showed that 4SC-202 induced apoptosis signal-regulating kinase 1 (ASK1) activation, causing it translocation to mitochondria and physical association with Cyp-D. This mitochondrial ASK1-Cyp-D complexation appeared required for mediating 4SC-202-induced apoptosis activation. ASK1 stable knockdown by targeted-shRNAs largely inhibited 4SC-202-induced mPTP opening, cytochrome C release, and following HCC cell apoptotic death. Together, we suggest that 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to potently inhibit human HCC cells. - Highlights: • 4SC-202 exerts potent anti-proliferative and cytotoxic activity against established/primary HCC cells. • SC-202-induced anti-HCC cell activity relies on caspase-dependent apoptosis activation. • 4SC-202 activates Cyp-D-dependent mitochondrial apoptosis pathway in HCC cells. • 4SC-202 activates ASK1 in HCC cells, causing it translocation to mitochondria. • Mitochondrial ASK1-Cyp-D complexation mediates 4SC-202's activity in HCC cells.

  4. 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to inhibit hepatocellular carcinoma cells

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    Fu, Meili, E-mail: fumeilidrlinyi@tom.com [Department of Infectious Disease, Linyi People' s Hospital, Linyi 276000 (China); Wan, Fuqiang [Department of Head and Neck Surgery, Linyi Tumor Hospital, Linyi 276000 (China); Li, Zhengling [Department of Nursing, Tengzhou Central People' s Hospital, Tengzhou 277500 (China); Zhang, Fenghua [Department of Operating Room, Linyi People' s Hospital, Linyi 276000 (China)

    2016-03-04

    The aim of the present study is to investigate the potential anti-hepatocellular carcinoma (HCC) cell activity by 4SC-202, a novel class I HDAC inhibitor (HDACi). The associated signaling mechanisms were also analyzed. We showed that 4SC-202 treatment induced potent cytotoxic and proliferation–inhibitory activities against established HCC cell lines (HepG2, HepB3, SMMC-7721) and patient-derived primary HCC cells. Further, adding 4SC-202 in HCC cells activated mitochondrial apoptosis pathway, which was evidenced by mitochondrial permeability transition pore (mPTP) opening, cytochrome C cytosol release and caspase-3/-9 activation. Inhibition of this apoptosis pathway, by caspase-3/-9 inhibitors, mPTP blockers, or by shRNA-mediated knockdown of cyclophilin-D (Cyp-D, a key component of mPTP), significantly attenuated 4SC-202-induced HCC cell death and apoptosis. Reversely, over-expression of Cyp-D enhanced 4SC-202's sensitivity in HCC cells. Further studies showed that 4SC-202 induced apoptosis signal-regulating kinase 1 (ASK1) activation, causing it translocation to mitochondria and physical association with Cyp-D. This mitochondrial ASK1-Cyp-D complexation appeared required for mediating 4SC-202-induced apoptosis activation. ASK1 stable knockdown by targeted-shRNAs largely inhibited 4SC-202-induced mPTP opening, cytochrome C release, and following HCC cell apoptotic death. Together, we suggest that 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to potently inhibit human HCC cells. - Highlights: • 4SC-202 exerts potent anti-proliferative and cytotoxic activity against established/primary HCC cells. • SC-202-induced anti-HCC cell activity relies on caspase-dependent apoptosis activation. • 4SC-202 activates Cyp-D-dependent mitochondrial apoptosis pathway in HCC cells. • 4SC-202 activates ASK1 in HCC cells, causing it translocation to mitochondria. • Mitochondrial ASK1-Cyp-D complexation mediates 4SC-202's activity in HCC cells.

  5. Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway.

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    Tsuchiya, Ayako; Kaku, Yoshiko; Nakano, Takashi; Nishizaki, Tomoyuki

    2015-11-01

    1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE) induces both necrosis/necroptosis and apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells. The present study was conducted to understand the mechanism for DAPE-induced apoptosis of NCI-H28 cells. DAPE induced caspase-independent apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells, and the effect of DAPE was prevented by antioxidants or an inhibitor of NADPH oxidase (NOX). DAPE generated reactive oxygen species (ROS) and inhibited activity of thioredoxin (Trx) reductase (TrxR). DAPE decreased an association of apoptosis signal-regulating kinase 1 (ASK1) with thioredoxin (Trx), thereby releasing ASK1. DAPE activated p38 mitogen-activated protein kinase (MAPK), which was inhibited by an antioxidant or knocking-down ASK1. In addition, DAPE-induced NCI-H28 cell death was also prevented by knocking-down ASK1. Taken together, the results of the present study indicate that DAPE stimulates NOX-mediated ROS production and suppresses TrxR activity, resulting in the decrease of reduced Trx and the dissociation of ASK1 from a complex with Trx, allowing sequential activation of ASK1 and p38 MAPK, to induce apoptosis of NCI-H28 MPM cells. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  6. Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway

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    Ayako Tsuchiya

    2015-11-01

    Full Text Available 1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE induces both necrosis/necroptosis and apoptosis of NCI-H28 malignant pleural mesothelioma (MPM cells. The present study was conducted to understand the mechanism for DAPE-induced apoptosis of NCI-H28 cells. DAPE induced caspase-independent apoptosis of NCI-H28 malignant pleural mesothelioma (MPM cells, and the effect of DAPE was prevented by antioxidants or an inhibitor of NADPH oxidase (NOX. DAPE generated reactive oxygen species (ROS and inhibited activity of thioredoxin (Trx reductase (TrxR. DAPE decreased an association of apoptosis signal-regulating kinase 1 (ASK1 with thioredoxin (Trx, thereby releasing ASK1. DAPE activated p38 mitogen-activated protein kinase (MAPK, which was inhibited by an antioxidant or knocking-down ASK1. In addition, DAPE-induced NCI-H28 cell death was also prevented by knocking-down ASK1. Taken together, the results of the present study indicate that DAPE stimulates NOX-mediated ROS production and suppresses TrxR activity, resulting in the decrease of reduced Trx and the dissociation of ASK1 from a complex with Trx, allowing sequential activation of ASK1 and p38 MAPK, to induce apoptosis of NCI-H28 MPM cells.

  7. Psoralen Inhibited Apoptosis of Osteoporotic Osteoblasts by Modulating IRE1-ASK1-JNK Pathway

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    Chen, Shuqing; Wang, Yongqian; Yang, Yubin; Xiang, Ting; Liu, Jiahui

    2017-01-01

    Osteoporosis is a common disease causing fracture in older populations. Abnormal apoptosis of osteoblasts contributes to the genesis of osteoporosis. Inhibiting apoptosis of osteoblasts provides a promising strategy to prevent osteoporosis. The proliferation of osteoblasts isolated from osteoporotic patients or healthy subjects was determined by MTT assay. Apoptosis was determined by Annexin V/PI assay. Protein expression was measured by western blot. The proliferation of osteoblasts isolated from osteoporotic patients was inhibited and the apoptosis level of these cells was higher than the osteoblasts from healthy subjects. Incubation with psoralen or estradiol significantly enhanced the proliferation and decreased the apoptosis level of osteoporotic osteoblasts. Western blot demonstrated that psoralen or estradiol treatment downregulated the expression of IRE1, p-ASK, p-JNK, and Bax. Meanwhile, expression of Bcl-2 was upregulated. Pretreatment by IRE1 agonist tunicamycin or JNK agonist anisomycin attenuated the effect of psoralen on osteoporotic osteoblasts. Psoralen inhibited apoptosis of osteoporotic osteoblasts by regulating IRE1-ASK1-JNK pathway. PMID:28349059

  8. A novel synthetic analogue of ω-3 17,18-epoxyeicosatetraenoic acid activates TNF receptor-1/ASK1/JNK signaling to promote apoptosis in human breast cancer cells.

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    Dyari, Herryawan Ryadi Eziwar; Rawling, Tristan; Chen, Yongjuan; Sudarmana, William; Bourget, Kirsi; Dwyer, Julie M; Allison, Sarah E; Murray, Michael

    2017-12-01

    A saturated analog of the cytochrome P450-mediated ω-3-17,18-epoxide of ω-3-eicosapentaenoic acid (C20E) activated apoptosis in human triple-negative MDA-MB-231 breast cancer cells. This study evaluated the apoptotic mechanism of C20E. Increased cytosolic cytochrome c expression and altered expression of pro- and antiapoptotic B-cell lymphoma-2 proteins indicated activation of the mitochondrial pathway. Caspase-3 activation by C20E was prevented by pharmacological inhibition and silencing of the JNK and p38 MAP kinases (MAPK), upstream MAPK kinases MKK4 and MKK7, and the upstream MAPK kinase kinase apoptosis signal-regulating kinase 1 (ASK1). Silencing of the death receptor TNF receptor 1 (TNFR1), but not Fas, DR4, or DR5, and the adapters TRADD and TNF receptor-associated factor 2, but not Fas-associated death domain, prevented C20E-mediated apoptosis. B-cell lymphoma-2 homology 3-interacting domain death agonist (Bid) cleavage by JNK/p38 MAPK linked the extrinsic and mitochondrial pathways of apoptosis. In further studies, an antibody against the extracellular domain of TNFR1 prevented apoptosis by TNF-α but not C20E. These findings suggest that C20E acts intracellularly at TNFR1 to activate ASK1-MKK4/7-JNK/p38 MAPK signaling and to promote Bid-dependent mitochondrial disruption and apoptosis. In in vivo studies, tumors isolated from C20E-treated nu/nu mice carrying MDA-MB-231 xenografts showed increased TUNEL staining and decreased Ki67 staining, reflecting increased apoptosis and decreased proliferation, respectively. ω-3-Epoxy fatty acids like C20E could be incorporated into treatments for triple-negative breast cancers.-Dyari, H. R. E., Rawling, T., Chen, Y., Sudarmana, W., Bourget, K., Dwyer, J. M., Allison, S. E., Murray, M. A novel synthetic analogue of ω-3 17,18-epoxyeicosatetraenoic acid activates TNF receptor-1/ASK1/JNK signaling to promote apoptosis in human breast cancer cells. © FASEB.

  9. Deletion of apoptosis signal-regulating kinase 1 (ASK1 protects pancreatic beta-cells from stress-induced death but not from glucose homeostasis alterations under pro-inflammatory conditions.

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    Emilie Pepin

    Full Text Available Type 2 diabetes is characterized by pancreatic beta-cell dysfunction and is associated with low-grade inflammation. Recent observations suggest that apoptosis signal-regulating kinase 1 (ASK1 is involved in beta-cell death in response to different stressors. In this study, we tested whether ASK1 deficiency protects beta-cells from glucolipotoxic conditions and cytokines treatment or from glucose homeostasis alteration induced by endotoxemia.Insulin secretion was neither affected upon shRNA-mediated downregulation of ASK1 in MIN6 cells nor in islets from ASK1-deficient mice. ASK1 silencing in MIN6 cells and deletion in islets did not prevent the deleterious effect of glucolipotoxic conditions or cytokines on insulin secretion. However, it protected MIN6 cells from death induced by ER stress or palmitate and islets from short term caspase activation in response to cytokines. Moreover, endotoxemia induced by LPS infusion increased insulin secretion during hyperglycemic clamps but the response was similar in wild-type and ASK1-deficient mice. Finally, insulin sensitivity in the presence of LPS was not affected by ASK1-deficiency.Our study demonstrates that ASK1 is not involved in beta-cell function and dysfunction but controls stress-induced beta-cell death.

  10. Cysteine residues mediate high‐affinity binding of thioredoxin to ASK1

    Czech Academy of Sciences Publication Activity Database

    Kylarová, Salome; Košek, Dalibor; Petrvalská, Olivia; Pšenáková, Katarína; Man, Petr; Večeř, J.; Herman, P.; Obšilová, Veronika; Obšil, Tomáš

    2016-01-01

    Roč. 283, č. 20 (2016), s. 3821-3838 ISSN 1742-464X R&D Projects: GA ČR(CZ) GA14-10061S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 ; RVO:61388971 Keywords : ASK 1 * cysteine * disulfide bond * mass spectrometry * TRX Subject RIV: CE - Biochemistry; EE - Microbiology, Virology (MBU-M) Impact factor: 3.902, year: 2016

  11. Structural Insight into the 14-3-3 Protein-dependent Inhibition of Protein Kinase ASK1 (Apoptosis Signal-regulating kinase 1)

    Czech Academy of Sciences Publication Activity Database

    Petrvalská, Olivia; Košek, Dalibor; Kukačka, Zdeněk; Tošner, Z.; Man, Petr; Večeř, J.; Herman, P.; Obšilová, Veronika; Obšil, Tomáš

    2016-01-01

    Roč. 291, č. 39 (2016), s. 20753-20765 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA14-10061S Institutional support: RVO:67985823 ; RVO:61388971 Keywords : 14-3-3 protein * apoptosis signal-regulating kinase 1 (ASK1) * fluorescence * nuclear magnetic resonance (NMR) * protein cross-linking * small-angle x-ray scattering (SAXS) Subject RIV: CE - Biochemistry Impact factor: 4.125, year: 2016

  12. IRE1α-TRAF2-ASK1 pathway is involved in CSTMP-induced apoptosis and ER stress in human non-small cell lung cancer A549 cells.

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    Zhang, Jiexia; Liang, Ying; Lin, Yongbin; Liu, Yuanbin; YouYou; Yin, Weiqiang

    2016-08-01

    CSTMP, a Tetramethylpyrazine (TMP) analogue, is designed and synthesized based on the pharmacophores of TMP and resveratrol. Recent studies showed that CSTMP had strong protective effects in endothelial cells apoptosis by its anti-oxidant activity. However, the pharmacological function of CSTMP in cancer have not been elucidated to date. The objective of this study was to investigate the anti-cancer effect of CSTMP against human non-small cell lung cancer (NSCLC) A549 cells and the underlying mechanisms. The cell proliferation and apoptosis were detected by MTT assay and flow cytometry. Caspases activity was determined spectrophotometricaly at 405nm using a microtiter plate reader. Western blot and real-time PCR was used to assess the protein and mRNA expression. Immunoprecipitation was used to examine the protein-protein interactions. CSTMP inhibited the proliferation and induced cell cycle arrest and apoptosis of A549 cells. Caspase3, 8, 9 and PARP-1 activation, and Bax/Bcl-2 ratio analyses demonstrated that the anti-cancer effect of CSTMP in A549 cells was mediated by promoting caspase- and mitochondria-dependent apoptosis. Furthermore, CSTMP induced ER stress in A549 cells as evidenced by elevated levels of GRP78, GRP94, CHOP, IRE1α, TRAF2, p-ASK1 and p-JNK, activation of caspase12 and 4, and enhanced formation of an IRE1α-TRAF2-ASK1 complex. Knockdown of IRE1α by siRNA suppressed activation of IRE1α, TRAF2, p-ASK1 and p-JNK in CSTMP treated A549 cells. In addition, the effects of CSTMP on the formation of an IRE1α-TRAF2-ASK1 complex, caspase- and mitochondria-dependent apoptosis were also reversed by IRE1α siRNA in A549 cells. Collectively, we showed that CSTMP induced apoptosis of A549 cells were through IRE1α-TRAF2-ASK1 complex-mediated ER stress, JNK activation, and mitochondrial dysfunction. These insights on this novel compound CSTMP may provide a novel anti-cancer candidate for the treatment of NSCLC. Copyright © 2016 Elsevier Masson SAS. All

  13. Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway

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    Ayako Tsuchiya; Yoshiko Kaku; Takashi Nakano; Tomoyuki Nishizaki

    2015-01-01

    1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE) induces both necrosis/necroptosis and apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells. The present study was conducted to understand the mechanism for DAPE-induced apoptosis of NCI-H28 cells. DAPE induced caspase-independent apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells, and the effect of DAPE was prevented by antioxidants or an inhibitor of NADPH oxidase (NOX). DAPE generated reactive oxygen species ...

  14. Alpha-mangostin induces apoptosis through activation of reactive oxygen species and ASK1/p38 signaling pathway in cervical cancer cells.

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    Lee, Chien-Hsing; Ying, Tsung-Ho; Chiou, Hui-Ling; Hsieh, Shu-Ching; Wen, Shiua-Hua; Chou, Ruey-Hwang; Hsieh, Yi-Hsien

    2017-07-18

    Alpha-mangostin, a natural xanthonoid, has been reported to possess the anti-cancer property in various types of human cancer. However, its effects and mechanism of α-mangostin in cervical cancer remain unclear. We found that α-mangostin effectively inhibited cell viability, resulted in loss of mitochondrial membrane potential (MMP), release of cytochrome C, increase of Bax, decrease of Bcl-2, and activation of caspase-9/caspase-3 cascade in cervical cancer cells. Alpha-mangostin elevated the contents of reactive oxygen species (ROS) to activate p38. Disrupting ASK1/p38 signaling pathway by a specific inhibitor of p38, or by the siRNAs against ASK1, MKK3/6, or p38, significantly abolished α-mangostin-induced cell death and apoptotic responses. Moreover, α-mangostin also repressed tumor growth in accordance with increased levels of p-ASK1, p-p38, cleaved-PARP and cleaved-caspase-3 in the tumor mass from the mouse xenograft model of cervical cancer. In the current study, we provided first evidence to demonstrate that dietary antioxidant α-mangostin could inhibit the tumor growth of cervical cancer cells through enhancing ROS amounts to activate ASK1/p38 signaling pathway and damage the integrity of mitochondria and thereby induction of apoptosis in cervical cancer cells.

  15. Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

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    Chan, Vera S. F.; Chung, Nancy P. Y.; Wang, Shu-Rong; Li, Zhongye; Ma, Jing; Lin, Chia-Wei; Hsieh, Ya-Ju; Chang, Kao-Ping; Kung, Sui-Sum; Wu, Yi-Chia; Chu, Cheng-Wei; Tai, Hsiao-Ting; Gao, George F.; Zheng, Bojian; Yokoyama, Kazunari K.; Austyn, Jonathan M.; Lin, Chen-Lung S.

    2013-01-01

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

  16. 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.

  17. Caspases: An apoptosis mediator

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    Tapan Kumar Palai

    2015-03-01

    Full Text Available The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy - dependent biochemical mechanisms. Apoptosis is a widely conserved phenomenon helping many processes, including normal cell turnover, proper development and functioning of the immune system, hormone dependent atrophy etc. Inappropriate apoptosis (either low level or high level leads to many developmental abnormalities like, neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer. To use cells for therapeutic purposes through generating cell lines, it is critical to study the cell cycle machinery and signalling pathways that controls cell death and apoptosis. Apoptotic pathways provide a fundamental protective mechanism that decreases cellular sensitivity to damaging events and allow proper developmental process in multi-cellular organisms. Major mediator of apoptosis is a family of proteins known as caspases. There are mainly fourteen types of caspases but out of them only ten caspasese have got essential role in controlling the process of apoptosis. These ten caspases have been categorized into either initiator caspases (caspase 2, 8, 9, 10 or executioner caspases (caspase 3, 6, 7. Although various types of caspases have been identified so far, the exact mechanisms of action of these groups of proteins is still to be fully understood. The aim of this review is to provide a detail overview of role of different caspases in regulating the process of apoptosis.

  18. Dissociation of Akt1 from its negative regulator JIP1 is mediated through the ASK1-MEK-JNK signal transduction pathway during metabolic oxidative stress: a negative feedback loop.

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    Song, Jae J; Lee, Yong J

    2005-07-04

    We have previously observed that metabolic oxidative stress-induced death domain-associated protein (Daxx) trafficking is mediated by the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. The relocalized Daxx from the nucleus to the cytoplasm during glucose deprivation participates in a positive regulatory feedback loop by binding to apoptosis signal-regulating kinase (ASK) 1. In this study, we report that Akt1 is involved in a negative regulatory feedback loop during glucose deprivation. Akt1 interacts with c-Jun NH(2)-terminal kinase (JNK)-interacting protein (JIP) 1, and Akt1 catalytic activity is inhibited. The JNK2-mediated phosphorylation of JIP1 results in the dissociation of Akt1 from JIP1 and subsequently restores Akt1 enzyme activity. Concomitantly, Akt1 interacts with stress-activated protein kinase/extracellular signal-regulated kinase (SEK) 1 (also known as MKK4) and inhibits SEK1 activity. Knockdown of SEK1 leads to the inhibition of JNK activation, JIP1-JNK2 binding, and the dissociation of Akt1 from JIP1 during glucose deprivation. Knockdown of JIP1 also leads to the inhibition of JNK activation, whereas the knockdown of Akt1 promotes JNK activation during glucose deprivation. Altogether, our data demonstrate that Akt1 participates in a negative regulatory feedback loop by interacting with the JIP1 scaffold protein.

  19. Ameliorating effect of TI-1-162, a hydroxyindenone derivative, against TNBS-induced rat colitis is mediated through suppression of RIP/ASK-1/MAPK signaling.

    Science.gov (United States)

    Gurung, Pallavi; Banskota, Suhrid; Katila, Nikita; Gautam, Jaya; Kadayat, Tara Man; Choi, Dong-Young; Lee, Eung Seok; Jeong, Tae Cheon; Kim, Jung-Ae

    2018-05-15

    The pathogenesis of inflammatory bowel disease (IBD) is associated with production of immense pro-inflammatory cytokines including TNF-α. Once generated, TNF-α stimulates production of various pro-inflammatory cytokines and disrupts mucosal barrier by inducing inflamed mucosal epithelial cell death. In the present study, we investigated inhibitory effects of TI-1-162, a hydroxyindenone derivative, against TNF-α-induced and TNBS-induced colon inflammation. TI-1-162 showed inhibitory effect on the TNF-α-induced adhesion of U937 monocytic cells to HT-29 colonic epithelial cells (IC 50 = 0.83 ± 0.12 μM), which is an in vitro model representing the initial step of colitis. In addition, TI-1-162 suppressed TNF-α-stimulated caspase-3 activation and HT-29 cell apoptosis. These in vitro inhibitory activities of TI-1-162 correlated to recovery changes in in vivo colon tissues, such as downregulation of adhesion molecules (ICAM-1, VCAM-1) and chemokines (CCL11, CXCL1, CXCL2, CXCL3, CX3CL1) revealed by gene expression array and Western blot analyses. Such molecular recovery of colon epithelium from TNBS-treated rats corresponded to the recovery in body weight, colon weight/length, and myeloperoxidase level by TI-1-162 (10 and 30 mg/kg/day, orally). In relation to action mechanism, TI-1-162 did not disturb TNF-α binding to its receptor, but suppressed phosphorylation of RIP-1, ASK-1, JNK and p38, and nuclear translocation of NF-kB and AP-1, which corresponded to down regulation of inflammatory cytokines in TNF-α-treated cells (HT-29 and U937) and TNBS-treated rat colon tissues. Taken together, the results indicate that the protective effects of TI-1-162 against colon inflammation and epithelial cell death are associated with its inhibitory action in RIP/ASK-1/MAPK signaling pathway downstream to TNF receptor 1. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Deletion of ASK1 Protects against Hyperoxia-Induced Acute Lung Injury.

    Directory of Open Access Journals (Sweden)

    Jutaro Fukumoto

    Full Text Available Apoptosis signal-regulating kinase 1 (ASK1, a member of the MAPK kinase kinase kinase (MAP3K family, is activated by various stimuli, which include oxidative stress, endoplasmic reticulum (ER stress, calcium influx, DNA damage-inducing agents and receptor-mediated signaling through tumor necrosis factor receptor (TNFR. Inspiration of a high concentration of oxygen is a palliative therapy which counteracts hypoxemia caused by acute lung injury (ALI-induced pulmonary edema. However, animal experiments so far have shown that hyperoxia itself could exacerbate ALI through reactive oxygen species (ROS. Our previous data indicates that ASK1 plays a pivotal role in hyperoxia-induced acute lung injury (HALI. However, it is unclear whether or not deletion of ASK1 in vivo protects against HALI. In this study, we investigated whether ASK1 deletion would lead to attenuation of HALI. Our results show that ASK1 deletion in vivo significantly suppresses hyperoxia-induced elevation of inflammatory cytokines (i.e. IL-1β and TNF-α, cell apoptosis in the lung, and recruitment of immune cells. In summary, the results from the study suggest that deletion of ASK1 in mice significantly inhibits hyperoxic lung injury.

  1. Stress-induced nuclear import of apoptosis signal-regulating kinase 1 is mediated by karyopherin α2/β1 heterodimer.

    Science.gov (United States)

    Sturchler, Emmanuel; Feurstein, Daniel; Chen, Weimin; McDonald, Patricia; Duckett, Derek

    2013-03-01

    The apoptosis signal-regulating kinase 1 (ASK1) is activated in response to a wide variety of extracellular stressors. Consequently, dysregulation of ASK1 is associated with multiple pathologies. Here, we show that ASK1 translocates from the cytoplasm to the nucleus in HEK293 cells and human cardiomyocytes in response to hydrogen peroxide (H(2)O(2)) or angiotensin respectively. Immunoprecipitation and mass spectrometry experiments reveal that ASK1 physically interacts with the karyopherin α2/β1 heterodimer in response to stress and genetic knockdown experiments confirm that this association mediates H(2)O(2)-induced ASK1 nuclear translocation. In addition, we have identified a nuclear localization signal (NLS)-like motif within the primary amino acid sequence of ASK1 composed of two clusters of basic amino acids separated by an intervening 16 amino acid spacer, KR[ACANDLLVDEFLKVSS]KKKK. Mutation of the downstream lysine cluster markedly reduces the H(2)O(2)-induced ASK1-karyopherin α2/β1 interaction and inhibits ASK1 nuclear translocation. Furthermore, we demonstrate that nuclear ASK1 is active and participates in H(2)O(2)-induced ASK1-mediated cell death. Collectively, our findings have identified a functional interaction between ASK1 and the karyopherin α2/β1 heterodimer and have also revealed a novel mechanism by which nuclear trafficking regulates the apoptotic function of ASK1 in response to stress. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Structural aspects of protein kinase ASK1 regulation

    Czech Academy of Sciences Publication Activity Database

    Obšil, Tomáš; Obšilová, Veronika

    2017-01-01

    Roč. 66, 1 Dec (2017), s. 31-36 ISSN 2212-4926 R&D Projects: GA ČR(CZ) GA16-02739S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : ASK1 kinase * apoptosis * thioredoxin * 14-3-3 protein Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology

  3. Apigenin promotes TRAIL-mediated apoptosis regardless of ROS generation.

    Science.gov (United States)

    Kang, Chang-Hee; Molagoda, Ilandarage Menu Neelaka; Choi, Yung Hyun; Park, Cheol; Moon, Dong-Oh; Kim, Gi-Young

    2018-01-01

    Apigenin is a bioactive flavone in several herbs including parsley, thyme, and peppermint. Apigenin possesses anti-cancer and anti-inflammatory properties; however, whether apigenin enhances TRAIL-mediated apoptosis in cancer cells is unknown. In the current study, we found that apigenin enhanced TRAIL-induced apoptosis by promoting caspase activation and death receptor 5 (DR5) expression and a chimeric antibody against DR5 completely blocked the apoptosis. Apigenin also upregulated reactive oxygen species (ROS) generation; however, intriguingly, ROS inhibitors, glutathione (GSH) or N-acetyl-l-cysteine (NAC), moderately increased apigenin/TRAIL-induced apoptosis. Additional results showed that an autophagy inducer, rapamycin, enhanced apigenin/TRAIL-mediated apoptosis by a slight increase of ROS generation. Accordingly, NAC and GSH rather decreased apigenin-induced autophagy formation, suggesting that apigenin-induced ROS generation increased autophagy formation. However, autophagy inhibitors, bafilomycin (BAF) and 3-methyladenine (3-MA), showed different result in apigenin/TRAIL-mediated apoptosis without ROS generation. 3-MA upregulated the apoptosis but remained ROS levels; however, no changes on apoptosis and ROS generation were observed by BAF treatment. Taken together, these findings reveal that apigenin enhances TRAIL-induced apoptosis by activating apoptotic caspases by upregulating DR5 expression regardless of ROS generation, which may be a promising strategy for an adjuvant of TRAIL. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation

    International Nuclear Information System (INIS)

    Umar, Sadiq; Hedaya, Omar; Singh, Anil K.; Ahmed, Salahuddin

    2015-01-01

    Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1–5 μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p < 0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p < 0.05; n = 4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p < 0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA. - Highlights: • Evolving evidence suggests that ASK1 plays a central role in rheumatic arthritis (RA). • TNF-α activates ASK1, which regulate downstream signaling through JNK/p38 activation in RA-FLS. • ASK1 may be used as a potential therapeutic target in RA. • Thymoquinone was able to selectively inhibit TNF-α-induced phosphorylation of ASK1 in RA-FLS. • Thymoquinone might serve as a potential small

  5. Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation

    Energy Technology Data Exchange (ETDEWEB)

    Umar, Sadiq; Hedaya, Omar; Singh, Anil K.; Ahmed, Salahuddin, E-mail: salah.ahmed@wsu.edu

    2015-09-15

    Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1–5 μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p < 0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p < 0.05; n = 4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p < 0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA. - Highlights: • Evolving evidence suggests that ASK1 plays a central role in rheumatic arthritis (RA). • TNF-α activates ASK1, which regulate downstream signaling through JNK/p38 activation in RA-FLS. • ASK1 may be used as a potential therapeutic target in RA. • Thymoquinone was able to selectively inhibit TNF-α-induced phosphorylation of ASK1 in RA-FLS. • Thymoquinone might serve as a potential small

  6. The effect of ASK1 on vascular permeability and edema formation in cerebral ischemia.

    Science.gov (United States)

    Song, Juhyun; Cheon, So Yeong; Lee, Won Taek; Park, Kyung Ah; Lee, Jong Eun

    2015-01-21

    Apoptosis signal-regulating kinase-1 (ASK1) is the mitogen-activated protein kinase kinase kinase (MAPKKK) and participates in the various central nervous system (CNS) signaling pathways. In cerebral ischemia, vascular permeability in the brain is an important issue because regulation failure of it results in edema formation and blood-brain barrier (BBB) disruption. To determine the role of ASK1 on vascular permeability and edema formation following cerebral ischemia, we first investigated ASK1-related gene expression using microarray analyses of ischemic brain tissue. We then measured protein levels of ASK1 and vascular endothelial growth factor (VEGF) in brain endothelial cells after hypoxia injury. We also examined protein expression of ASK1 and VEGF, edema formation, and morphological alteration through cresyl violet staining in ischemic brain tissue using ASK1-small interference RNA (ASK1-siRNA). Finally, immunohistochemistry was performed to examine VEGF and aquaporin-1 (AQP-1) expression in ischemic brain injury. Based on our findings, we propose that ASK1 is a regulating factor of vascular permeability and edema formation in cerebral ischemia. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Genetic Signatures of HIV-1 Envelope-mediated Bystander Apoptosis

    Science.gov (United States)

    Joshi, Anjali; Lee, Raphael T. C.; Mohl, Jonathan; Sedano, Melina; Khong, Wei Xin; Ng, Oon Tek; Maurer-Stroh, Sebastian; Garg, Himanshu

    2014-01-01

    The envelope (Env) glycoprotein of HIV is an important determinant of viral pathogenesis. Several lines of evidence support the role of HIV-1 Env in inducing bystander apoptosis that may be a contributing factor in CD4+ T cell loss. However, most of the studies testing this phenomenon have been conducted with laboratory-adapted HIV-1 isolates. This raises the question of whether primary Envs derived from HIV-infected patients are capable of inducing bystander apoptosis and whether specific Env signatures are associated with this phenomenon. We developed a high throughput assay to determine the bystander apoptosis inducing activity of a panel of primary Envs. We tested 38 different Envs for bystander apoptosis, virion infectivity, neutralizing antibody sensitivity, and putative N-linked glycosylation sites along with a comprehensive sequence analysis to determine if specific sequence signatures within the viral Env are associated with bystander apoptosis. Our studies show that primary Envs vary considerably in their bystander apoptosis-inducing potential, a phenomenon that correlates inversely with putative N-linked glycosylation sites and positively with virion infectivity. By use of a novel phylogenetic analysis that avoids subtype bias coupled with structural considerations, we found specific residues like Arg-476 and Asn-425 that were associated with differences in bystander apoptosis induction. A specific role of these residues was also confirmed experimentally. These data demonstrate for the first time the potential of primary R5 Envs to mediate bystander apoptosis in CD4+ T cells. Furthermore, we identify specific genetic signatures within the Env that may be associated with the bystander apoptosis-inducing phenotype. PMID:24265318

  8. SDF-1 induces TNF-mediated apoptosis in cardiac myocytes.

    Science.gov (United States)

    Jarrah, Andrew A; Schwarskopf, Martina; Wang, Edward R; LaRocca, Thomas; Dhume, Ashwini; Zhang, Shihong; Hadri, Lahouria; Hajjar, Roger J; Schecter, Alison D; Tarzami, Sima T

    2018-01-01

    Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation. One such chemokine, Stromal cell-derived factor-1 (SDF-1) also known as CXCL12, and its receptor, CXCR4, are expressed and functional in cardiac myocytes. SDF-1 both stimulates and enhances the cellular signal which attracts potentially beneficial stem cells for tissue repair within the ischemic heart. Paradoxically however, this chemokine is known to act in concert with the inflammatory cytokines of the innate immune response which contributes to cellular injury through the recruitment of inflammatory cells during ischemia. In the present study, we have demonstrated that SDF-1 has dose dependent effects on freshly isolated cardiomyocytes. Using Tunnel and caspase 3-activation assays, we have demonstrated that the treatment of isolated adult rat cardiac myocyte with SDF-1 at higher concentrations (pathological concentrations) induced apoptosis. Furthermore, ELISA data demonstrated that the treatment of isolated adult rat cardiac myocyte with SDF-1 at higher concentrations upregulated TNF-α protein expression which directly correlated with subsequent apoptosis. There was a significant reduction in SDF-1 mediated apoptosis when TNF-α expression was neutralized which suggests that SDF-1 mediated apoptosis is TNF-α-dependent. The fact that certain stimuli are capable of driving cardiomyocytes into apoptosis indicates that these cells are susceptible to clinically relevant apoptotic triggers. Our findings suggest that the elevated SDF-1 levels seen in a variety of clinical conditions, including ischemic myocardial infarction, may either directly or indirectly contribute to cardiac cell death via a TNF-α mediated pathway. This highlights the importance of this receptor/ligand in regulating the cardiomyocyte response to stress conditions.

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

    Science.gov (United States)

    Muzaffar, Suhail; Chattoo, Bharat B

    2017-03-01

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

  10. Involvement of Bim in Photofrin-mediated photodynamically induced apoptosis.

    Science.gov (United States)

    Wang, Xianwang; He, Xiaobing; Hu, Shujuan; Sun, Anbang; Lu, Chengbiao

    2015-01-01

    Photodynamic therapy (PDT) is a promising noninvasive technique, which has been successfully applied to the treatment of human cancers. Studies have shown that the Bcl-2 family proteins play important roles in PDT-induced apoptosis. However, whether Bcl-2-interacting mediator of cell death (Bim) is involved in photodynamic treatment remains unknown. In this study, we attempt to determine the effect of Bim on Photofrin photodynamic treatment (PPT)-induced apoptosis in human lung adenocarcinoma ASTC-a-1 cells. The translocation of Bim/Bax of the cells were monitored by laser confocal scanning microscope. The levels of Bim protein and activated caspase-3 in cells were detected by western blot assay. Caspase-3 activities were measured by Caspase-3 Fluorogenic Substrate (Ac-DEVD-AFC) analysis. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. The effect of Bim on PPT-induced apoptosis was determined by RNAi. BimL translocated to mitochondria in response to PPT, similar to the downstream pro-apoptotic protein Bax activation. PPT increased the level of Bim and activated caspase-3 in cells and that knockdown of Bim by RNAi significantly protected against caspase-3 activity. PPT-induced apoptosis were suppressed in cells transfected with shRNA-Bim. We demonstrated the involvement of Bim in PPT-induced apoptosis in human ASTC-a-1 lung adenocarcinoma cells and suggested that enhancing Bim activity might be a potential strategy for treating human cancers. © 2015 S. Karger AG, Basel.

  11. PARL mediates Smac proteolytic maturation in mitochondria to promote apoptosis.

    Science.gov (United States)

    Saita, Shotaro; Nolte, Hendrik; Fiedler, Kai Uwe; Kashkar, Hamid; Venne, A Saskia; Zahedi, René P; Krüger, Marcus; Langer, Thomas

    2017-04-01

    Mitochondria drive apoptosis by releasing pro-apoptotic proteins that promote caspase activation in the cytosol. The rhomboid protease PARL, an intramembrane cleaving peptidase in the inner membrane, regulates mitophagy and plays an ill-defined role in apoptosis. Here, we employed PARL-based proteomics to define its substrate spectrum. Our data identified the mitochondrial pro-apoptotic protein Smac (also known as DIABLO) as a PARL substrate. In apoptotic cells, Smac is released into the cytosol and promotes caspase activity by inhibiting inhibitors of apoptosis (IAPs). Intramembrane cleavage of Smac by PARL generates an amino-terminal IAP-binding motif, which is required for its apoptotic activity. Loss of PARL impairs proteolytic maturation of Smac, which fails to bind XIAP. Smac peptidomimetics, downregulation of XIAP or cytosolic expression of cleaved Smac restores apoptosis in PARL-deficient cells. Our results reveal a pro-apoptotic function of PARL and identify PARL-mediated Smac processing and cytochrome c release facilitated by OPA1-dependent cristae remodelling as two independent pro-apoptotic pathways in mitochondria.

  12. Regorafenib inhibits colorectal tumor growth through PUMA-mediated apoptosis

    Science.gov (United States)

    Chen, Dongshi; Wei, Liang; Yu, Jian; Zhang, Lin

    2014-01-01

    Purpose Regorafenib, a multi-kinase inhibitor targeting the Ras/Raf/MEK/ERK pathway, has recently been approved for the treatment of metastatic colorectal cancer (CRC). However, the mechanisms of action of regorafenib in CRC cells have been unclear. We investigated how regorafenib suppresses CRC cell growth and potentiates effects of other chemotherapeutic drugs. Experimental Design We determined whether and how regorafenib induces the expression of PUMA, a p53 target and a critical mediator of apoptosis in CRC cells. We also investigated whether PUMA is necessary for the killing and chemosensitization effects of regorafenib in CRC cells. Furthermore, xenograft tumors were used to test if PUMA mediates the in vivo antitumor, antiangiogenic and chemosensitization effects of regorafenib. Results We found that regorafenib treatment induces PUMA in CRC cells irrespective of p53 status through the NF-κB pathway following ERK inhibition and glycogen synthase kinase 3β (GSK3β) activation. Upregulation of PUMA is correlated with apoptosis induction in different CRC cell lines. PUMA is necessary for regorafenib-induced apoptosis in CRC cells. Chemosensitization by regorafenib is mediated by enhanced PUMA induction through different pathways. Furthermore, deficiency in PUMA abrogates the in vivo antitumor, antiangiogenic and chemosensitization effects of regorafenib. Conclusions Our results demonstrate a key role of PUMA in mediating the anticancer effects of regorafenib in CRC cells. They suggest that PUMA induction can be used as an indicator of regorafenib sensitivity, and also provide a rationale for manipulating the apoptotic machinery to improve the therapeutic efficacy of regorafenib and other targeted drugs. PMID:24763611

  13. Bystander apoptosis in human cells mediated by irradiated blood plasma

    Energy Technology Data Exchange (ETDEWEB)

    Vinnikov, Volodymyr, E-mail: vlad.vinnikov@mail.ru [Grigoriev Institute for Medical Radiology of the National Academy of Medical Science of Ukraine (Ukraine); Lloyd, David; Finnon, Paul [Centre for Radiation, Chemical and Environmental Hazards of the Health Protection Agency of the United Kingdom (United Kingdom)

    2012-03-01

    Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G{sub 0}-stage lymphocytes. Plasma was collected from healthy donors' blood irradiated in vitro to 0-40 Gy acute {gamma}-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24 h at 37 Degree-Sign C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4 Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2 {+-} 1.8% in plasma-free cultures, 21.6 {+-} 1.1% in cultures treated with plasma from unirradiated blood, 20.2 {+-} 1.4% in cultures with plasma from blood given 2-4 Gy and 16.7 {+-} 3.2% in cultures with plasma from blood given 6-10 Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

  14. Bystander apoptosis in human cells mediated by irradiated blood plasma

    International Nuclear Information System (INIS)

    Vinnikov, Volodymyr; Lloyd, David; Finnon, Paul

    2012-01-01

    Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G 0 -stage lymphocytes. Plasma was collected from healthy donors’ blood irradiated in vitro to 0–40 Gy acute γ-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24 h at 37 °C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4 Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2 ± 1.8% in plasma-free cultures, 21.6 ± 1.1% in cultures treated with plasma from unirradiated blood, 20.2 ± 1.4% in cultures with plasma from blood given 2–4 Gy and 16.7 ± 3.2% in cultures with plasma from blood given 6–10 Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

  15. PUMA mediates ER stress-induced apoptosis in portal hypertensive gastropathy

    OpenAIRE

    Tan, S; Wei, X; Song, M; Tao, J; Yang, Y; Khatoon, S; Liu, H; Jiang, J; Wu, B

    2014-01-01

    Mucosal apoptosis has been demonstrated to be an essential pathological feature in portal hypertensive gastropathy (PHG). p53-upregulated modulator of apoptosis (PUMA) was identified as a BH3-only Bcl-2 family protein that has an essential role in apoptosis induced by a variety of stimuli, including endoplasmic reticulum (ER) stress. However, whether PUMA is involved in mucosal apoptosis in PHG remains unclear, and whether PUMA induces PHG by mediating ER stress remains unknown. The aim of th...

  16. PUMA mediates ER stress-induced apoptosis in portal hypertensive gastropathy

    Science.gov (United States)

    Tan, S; Wei, X; Song, M; Tao, J; Yang, Y; Khatoon, S; Liu, H; Jiang, J; Wu, B

    2014-01-01

    Mucosal apoptosis has been demonstrated to be an essential pathological feature in portal hypertensive gastropathy (PHG). p53-upregulated modulator of apoptosis (PUMA) was identified as a BH3-only Bcl-2 family protein that has an essential role in apoptosis induced by a variety of stimuli, including endoplasmic reticulum (ER) stress. However, whether PUMA is involved in mucosal apoptosis in PHG remains unclear, and whether PUMA induces PHG by mediating ER stress remains unknown. The aim of the study is to investigate whether PUMA is involved in PHG by mediating ER stress apoptotic signaling. To identify whether PUMA is involved in PHG by mediating ER stress, gastric mucosal injury and apoptosis were studied in both PHG patients and PHG animal models using PUMA knockout (PUMA-KO) and PUMA wild-type (PUMA-WT) mice. The induction of PUMA expression and ER stress signaling were investigated, and the mechanisms of PUMA-mediated apoptosis were analyzed. GES-1 and SGC7901 cell lines were used to further identify whether PUMA-mediated apoptosis was induced by ER stress in vitro. Epithelial apoptosis and PUMA were markedly induced in the gastric mucosa of PHG patients and mouse PHG models. ER stress had a potent role in the induction of PUMA and apoptosis in PHG models, and the apoptosis was obviously attenuated in PUMA-KO mice. Although the targeted deletion of PUMA did not affect ER stress, mitochondrial apoptotic signaling was downregulated in mice. Meanwhile, PUMA knockdown significantly ameliorated ER stress-induced mitochondria-dependent apoptosis in vitro. These results indicate that PUMA mediates ER stress-induced mucosal epithelial apoptosis through the mitochondrial apoptotic pathway in PHG, and that PUMA is a potentially therapeutic target for PHG. PMID:24625987

  17. Helicobacter pylori enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in human gastric epithelial cells

    Science.gov (United States)

    Wu, Yi-Ying; Tsai, Hwei-Fang; Lin, We-Cheng; Chou, Ai-Hsiang; Chen, Hui-Ting; Yang, Jyh-Chin; Hsu, Ping-I; Hsu, Ping-Ning

    2004-01-01

    AIM: To investigate the relations between tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Helicobacter pylori (H pylori) infection in apoptosis of gastric epithelial cells and to assess the expression of TRAIL on the surface of infiltrating T-cells in H pylori-infected gastric mucosa. METHODS: Human gastric epithelial cell lines and primary gastric epithelial cells were co-cultured with H pylori in vitro, then recombinant TRAIL proteins were added to the culture. Apoptosis of gastric epithelial cells was determined by a specific ELISA for cell death. Infiltrating lymphocytes were isolated from H pylori-infected gastric mucosa, and expression of TRAIL in T cells was analyzed by flow cytometry. RESULTS: The apoptosis of gastric epithelial cell lines and primary human gastric epithelial cells was mildly increased by interaction with either TRAIL or H pylori alone. Interestingly, the apoptotic indices were markedly elevated when gastric epithelial cells were incubated with both TRAIL and H pylori (Control vs TRAIL and H pylori: 0.51 ± 0.06 vs 2.29 ± 0.27, P = 0.018). A soluble TRAIL receptor (DR4-Fc) could specifically block the TRAIL-mediated apoptosis. Further studies demonstrated that infiltrating T-cells in gastric mucosa expressed TRAIL on their surfaces, and the induction of TRAIL sensitivity by H pylori was dependent upon direct cell contact of viable bacteria, but not CagA and VacA of H pylori. CONCLUSION: H pylori can sensitize human gastric epithelial cells and enhance susceptibility to TRAIL-mediated apoptosis. Modulation of host cell sensitivity to apoptosis by bacterial interaction adds a new dimension to the immunopathogenesis of H pylori infection. PMID:15285015

  18. Modulating TRAIL-Mediated Apoptosis in Prostate Cancer Using Synthetic Triterpenoids

    National Research Council Canada - National Science Library

    Hyer, Marc L

    2005-01-01

    .... Concomitant treatment with CDDO or CDDO-Im reverses the TRAIL-resistant phenotype, leading to rapid induction of TRAIL-mediated apoptosis, while having no adverse effects on normal human mammary epithelial cells (HMEC...

  19. Perfluorooctanesulfonate Mediates Renal Tubular Cell Apoptosis through PPARgamma Inactivation.

    Directory of Open Access Journals (Sweden)

    Li-Li Wen

    Full Text Available Perfluorinated chemicals (PFCs are ubiquitously distributed in the environments including stainless pan-coating, raincoat, fire extinguisher, and semiconductor products. The PPAR family has been shown to contribute to the toxic effects of PFCs in thymus, immune and excretory systems. Herein, we demonstrated that perfluorooctanesulfonate (PFOS caused cell apoptosis through increasing ratio of Bcl-xS/xL, cytosolic cytochrome C, and caspase 3 activation in renal tubular cells (RTCs. In addition, PFOS increased transcription of inflammatory cytokines (i.e., TNFα, ICAM1, and MCP1 by NFκB activation. Conversely, PFOS reduced the mRNA levels of antioxidative enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase, as a result of reduced PPARγ transactivational activity by using reporter and chromatin immuoprecipitation (ChIP assays. PFOS reduced the protein interaction between PPARγ and PPARγ coactivator-1 alpha (PGC1α by PPARγ deacetylation through Sirt1 upregulation, of which the binding of PPARγ and PGC1α to a peroxisome proliferator response element (PPRE in the promoter regions of these antioxidative enzymes was alleviated in the ChIP assay. Furthermore, Sirt1 also deacetylated p53 and then increased the binding of p53 to Bax, resulting in increased cytosolic cytochrome C. The effect of PPARγ inactivation by PFOS was validated using the PPARγ antagonist GW9662, whereas the adverse effects of PFOS were prevented by PPARγ overexpression and activators, rosiglitozone and L-carnitine, in RTCs. The in vitro finding of protective effect of L-carnitine was substantiated in vivo using Balb/c mice model subjected to PFOS challenge. Altogether, we provide in vivo and in vitro evidence for the protective mechanism of L-carnitine in eliminating PFOS-mediated renal injury, at least partially, through PPARγ activation.

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

    Science.gov (United States)

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

    2015-08-01

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

  1. The ASK1 gene regulates B function gene expression in cooperation with UFO and LEAFY in Arabidopsis.

    Science.gov (United States)

    Zhao, D; Yu, Q; Chen, M; Ma, H

    2001-07-01

    The Arabidopsis floral regulatory genes APETALA3 (AP3) and PISTILLATA (PI) are required for the B function according to the ABC model for floral organ identity. AP3 and PI expression are positively regulated by the LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO) genes. UFO encodes an F-box protein, and we have shown previously that UFO genetically interacts with the ASK1 gene encoding a SKP1 homologue; both the F-box containing protein and SKP1 are subunits of ubiquitin ligases. We show here that the ask1-1 mutation can enhance the floral phenotypes of weak lfy and ap3 mutants; therefore, like UFO, ASK1 also interacts with LFY and AP3 genetically. Furthermore, our results from RNA in situ hybridizations indicate that ASK1 regulates early AP3 and PI expression. These results support the idea that UFO and ASK1 together positively regulate AP3 and PI expression. We propose that the UFO and ASK1 proteins are components of a ubiquitin ligase that mediates the proteolysis of a repressor of AP3 and PI expression. Our genetic studies also indicate that ASK1 and UFO play a role in regulating the number of floral organ primordia, and we discuss possible mechanisms for such a regulation.

  2. Ulinastatin Alone Does Not Reduce Caspase 3-mediated Apoptosis in Protease-positive Aeromonas hydrophilia-induced Sepsis

    Directory of Open Access Journals (Sweden)

    Bai-Horng Su

    2007-01-01

    Conclusion: PPAH-induced sepsis has a high mortality that is related to lymphocyte apoptosis. Ulinastatin alone does not significantly reduce caspase 3-mediated lymphocyte apoptosis. [J Formos Med Assoc 2007; 106(2:97-104

  3. VEGF masks BNIP3-mediated apoptosis of hypoxic endothelial cells.

    Science.gov (United States)

    Jurasz, Paul; Yurkova, Natasha; Kirshenbaum, Lorrie; Stewart, Duncan J

    2011-05-01

    Hypoxia results in the apoptotic death of myocytes, neurons, and epithelial cells, through the actions of Bcl-2 and Nineteen kilodalton Interacting Protein-3 (BNIP3). On the contrary, endothelial cells are especially adept at surviving conditions of oxygen deprivation via up-regulation of vascular endothelial growth factor (VEGF) the most potent endothelial survival factor. Both VEGF and BNIP3 expression are transcriptionally regulated by hypoxia inducible factor and may antagonize each other's affects in endothelial cells (ECs). Since factors that promote and inhibit apoptosis may be expressed at the same time in endothelial cells under hypoxic conditions, we decided to investigate whether VEGF and BNIP3 have opposing actions in endothelial cells. Human microvascular endothelial cells were exposed to hypoxic conditions in a Billups-Rothenburg chamber. Under hypoxic conditions BNIP3 expression by endothelial cells increased as measured by real-time PCR and immunoblot. After 48 h of hypoxia, EC apoptosis was assessed by flow cytometry and was lower than in corresponding normoxia serum starved controls. The increase in EC survival under hypoxic conditions corresponded with an increase in the expression of VEGF. Under normoxic conditions adenoviral BNIP3 over-expression promoted apoptosis of ECs; however, recombinant VEGF (100 pg/ml) antagonized the BNIP3 apoptosis promoting affects. SiRNA knockdown of VEGF expression by hypoxic ECs resulted in increased apoptosis with a concomitant increase in BNIP3 expression. SiRNA knockdown of BNIP3 expression by hypoxic ECs reduced the increase in EC apoptosis as a result of VEGF knockdown. We conclude that under hypoxic conditions VEGF counteracts and masks the apoptosis promoting affects of BNIP3.

  4. Hypercholesterolemia aggravates myocardial ischemia reperfusion injury via activating endoplasmic reticulum stress-mediated apoptosis.

    Science.gov (United States)

    Wu, Nan; Zhang, Xiaowen; Jia, Pengyu; Jia, Dalin

    2015-12-01

    The effect of hypercholesterolemia on myocardial ischemia reperfusion injury (MIRI) is in controversy and the underlying mechanism is still not well understood. In the present study, we firstly detected the effects of hypercholesterolemia on MIRI and the role of endoplasmic reticulum (ER) stress-mediated apoptosis pathway in this process. The infarct size was determined by TTC staining, and apoptosis was measured by the TUNEL method. The marker proteins of ER stress response and ER stress-mediated apoptosis pathway were detected by Western blot. The results showed that high cholesterol diet-induced hypercholesterolemia significantly increased the myocardial infarct size, the release of myocardium enzyme and the ratio of apoptosis, but did not affect the recovery of cardiac function. Moreover, hypercholesterolemia also remarkably up-regulated the expressions of ER stress markers (glucose-regulated protein 78 and calreticulin) and critical molecules in ER stress-mediated apoptosis pathway (CHOP, caspase 12, phospho-JNK). In conclusion, our study demonstrated that hypercholesterolemia enhanced myocardial vulnerability/sensitivity to ischemia reperfusion injury involved in aggravation the ER stress and activation of ER stress-mediated apoptosis pathway and it gave us a new insight into the underlying mechanisms associated with hypercholesterolemia-induced exaggerated MIRI and also provided a novel target for preventing MIRI in the presence of hypercholesterolemia. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. VDAC regulates AAC-mediated apoptosis and cytochromecrelease in yeast.

    Science.gov (United States)

    Trindade, Dário; Pereira, Clara; Chaves, Susana R; Manon, Stéphen; Côrte-Real, Manuela; Sousa, Maria J

    2016-08-25

    Mitochondrial outer membrane permeabilization is a key event in apoptosis processes leading to the release of lethal factors. We have previously shown that absence of the ADP/ATP carrier (AAC) proteins (yeast orthologues of mammalian ANT proteins) increased the resistance of yeast cells to acetic acid, preventing MOMP and the release of cytochrome c from mitochondria during acetic acid - induced apoptosis. On the other hand, deletion of POR1 (yeast voltage-dependent anion channel - VDAC) increased the sensitivity of yeast cells to acetic acid. In the present work, we aimed to further characterize the role of yeast VDAC in acetic acid - induced apoptosis and assess if it functionally interacts with AAC proteins. We found that the sensitivity to acetic acid resulting from POR1 deletion is completely abrogated by the absence of AAC proteins, and propose that Por1p acts as a negative regulator of acetic acid - induced cell death by a mechanism dependent of AAC proteins, by acting on AAC - dependent cytochrome c release. Moreover, we show that Por1p has a role in mitochondrial fusion that, contrary to its role in apoptosis, is not affected by the absence of AAC, and demonstrate that mitochondrial network fragmentation is not sufficient to induce release of cytochrome c or sensitivity to acetic acid - induced apoptosis. This work enhances our understanding on cytochrome c release during cell death, which may be relevant in pathological scenarios where MOMP is compromised.

  6. Aβ induces PUMA activation: a new mechanism for Aβ-mediated neuronal apoptosis.

    Science.gov (United States)

    Feng, Jie; Meng, Chengbo; Xing, Da

    2015-02-01

    p53 upregulated modulator of apoptosis (PUMA) is a promising tumor therapy target because it elicits apoptosis and profound sensitivity to radiation and chemotherapy. However, inhibition of PUMA may be beneficial for curbing excessive apoptosis associated with neurodegenerative disorders. Alzheimer's disease (AD) is a representative neurodegenerative disease in which amyloid-β (Aβ) deposition causes neurotoxicity. The regulation of PUMA during Aβ-induced neuronal apoptosis remains poorly understood. Here, we reported that PUMA expression was significantly increased in the hippocampus of transgenic mice models of AD and hippocampal neurons in response to Aβ. PUMA knockdown protected the neurons against Aβ-induced apoptosis. Furthermore, besides p53, PUMA transactivation was also regulated by forkhead box O3a through p53-independent manner following Aβ treatment. Notably, PUMA contributed to neuronal apoptosis through competitive binding of apoptosis repressor with caspase recruitment domain to activate caspase-8 that cleaved Bid into tBid to accelerate Bax mitochondrial translocation, revealing a novel pathway of Bax activation by PUMA to mediate Aβ-induced neuronal apoptosis. Together, we demonstrated that PUMA activation involved in Aβ-induced apoptosis, representing a drug target to antagonize AD progression. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2016-04-01

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

  8. Klotho Regulates 14-3-3ζ Monomerization and Binding to the ASK1 Signaling Complex in Response to Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Reynolds K Brobey

    Full Text Available The reactive oxygen species (ROS-sensitive apoptosis signal-regulating kinase 1 (ASK1 signaling complex is a key regulator of p38 MAPK activity, a major modulator of stress-associated with aging disorders. We recently reported that the ratio of free ASK1 to the complex-bound ASK1 is significantly decreased in Klotho-responsive manner and that Klotho-deficient tissues have elevated levels of free ASK1 which coincides with increased oxidative stress. Here, we tested the hypothesis that: 1 covalent interactions exist among three identified proteins constituting the ASK1 signaling complex; 2 in normal unstressed cells the ASK1, 14-3-3ζ and thioredoxin (Trx proteins simultaneously engage in a tripartite complex formation; 3 Klotho's stabilizing effect on the complex relied solely on 14-3-3ζ expression and its apparent phosphorylation and dimerization changes. To verify the hypothesis, we performed 14-3-3ζ siRNA knock-down experiments in conjunction with cell-based assays to measure ASK1-client protein interactions in the presence and absence of Klotho, and with or without an oxidant such as rotenone. Our results show that Klotho activity induces posttranslational modifications in the complex targeting 14-3-3ζ monomer/dimer changes to effectively protect against ASK1 oxidation and dissociation. This is the first observation implicating all three proteins constituting the ASK1 signaling complex in close proximity.

  9. Fas-Induced Apoptosis of Renal Cell Carcinoma is Mediated by Apoptosis Signal-Regulating Kinase 1 via Mitochondrial Damage-Dependent Caspase-8 Activation

    Directory of Open Access Journals (Sweden)

    Mohamed Hassan

    2009-01-01

    Full Text Available Renal cell carcinoma (RCC is a prototype of a chemo refractory tumour. It remains the most lethal of the common urologic cancers and is highly resistant to conventional therapy. Here, we confirmed the efficiency of anti-Fas monoclonal antibody (CH11 as alternative therapeutic approach for the treatment of RCC and investigated the molecular mechanism(s, whereby CH11 induces apoptosis of RCC cells. The present study shows an essential role for apoptosis signal-regulating kinase 1 (ASK1, together with both c-jun-N-terminal kinase (JNK and p38 pathways, and caspase-8 in this process. Furthermore, CH11-dependent induction of the ASK1–JNK/p38 pathways was found to activate the transcription factors AP-1 and ATF-2, and FADD-caspase-8-Bid signalling, resulting in the translocation of both Bax and Bak proteins, and subsequently mitochondrial dysregulation that is characterized by the loss of mitochondrial membrane potential (ΔΨm, cytochrome c release and cleavage of caspase-9, caspase-3 and PARP. Thus, the described molecular mechanisms of CH11-induced apoptosis suggest the reliability of Fas activation as an alternative therapeutic approach for the treatment of patients with advanced renal cell carcinoma.

  10. Fas receptor-mediated apoptosis : a clinical application?

    NARCIS (Netherlands)

    Timmer, T; de Vries, EGE; de Jong, S

    Fas is a membrane protein belonging to the death receptor family. Cross-linking of Fas by its ligand, FasL, or agonistic anti-Fas antibodies, induces apoptosis of cells expressing Fas on the membrane by triggering a cascade of caspases. Since many different tumours express Fas on their membrane,

  11. Oxidative Stress Mediates Radiation Lung Injury by Inducing Apoptosis

    International Nuclear Information System (INIS)

    Zhang Yu; Zhang Xiuwu; Rabbani, Zahid N.; Jackson, Isabel L.; Vujaskovic, Zeljko

    2012-01-01

    Purpose: Apoptosis in irradiated normal lung tissue has been observed several weeks after radiation. However, the signaling pathway propagating cell death after radiation remains unknown. Methods and Materials: C57BL/6J mice were irradiated with 15 Gy to the whole thorax. Pro-apoptotic signaling was evaluated 6 weeks after radiation with or without administration of AEOL10150, a potent catalytic scavenger of reactive oxygen and nitrogen species. Results: Apoptosis was observed primarily in type I and type II pneumocytes and endothelium. Apoptosis correlated with increased PTEN expression, inhibition of downstream PI3K/AKT signaling, and increased p53 and Bax protein levels. Transforming growth factor-β1, Nox4, and oxidative stress were also increased 6 weeks after radiation. Therapeutic administration of AEOL10150 suppressed pro-apoptotic signaling and dramatically reduced the number of apoptotic cells. Conclusion: Increased PTEN signaling after radiation results in apoptosis of lung parenchymal cells. We hypothesize that upregulation of PTEN is influenced by Nox4-derived oxidative stress. To our knowledge, this is the first study to highlight the role of PTEN in radiation-induced pulmonary toxicity.

  12. Oxidative Stress Mediates Radiation Lung Injury by Inducing Apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Yu; Zhang Xiuwu; Rabbani, Zahid N. [Department of Radiation Oncology, Duke University Medical Center, Durham, NC (United States); Jackson, Isabel L. [Department of Pathology, Duke University Medical Center, Durham, NC (United States); Vujaskovic, Zeljko, E-mail: vujas@radonc.duke.edu [Department of Radiation Oncology, Duke University Medical Center, Durham, NC (United States); Department of Pathology, Duke University Medical Center, Durham, NC (United States)

    2012-06-01

    Purpose: Apoptosis in irradiated normal lung tissue has been observed several weeks after radiation. However, the signaling pathway propagating cell death after radiation remains unknown. Methods and Materials: C57BL/6J mice were irradiated with 15 Gy to the whole thorax. Pro-apoptotic signaling was evaluated 6 weeks after radiation with or without administration of AEOL10150, a potent catalytic scavenger of reactive oxygen and nitrogen species. Results: Apoptosis was observed primarily in type I and type II pneumocytes and endothelium. Apoptosis correlated with increased PTEN expression, inhibition of downstream PI3K/AKT signaling, and increased p53 and Bax protein levels. Transforming growth factor-{beta}1, Nox4, and oxidative stress were also increased 6 weeks after radiation. Therapeutic administration of AEOL10150 suppressed pro-apoptotic signaling and dramatically reduced the number of apoptotic cells. Conclusion: Increased PTEN signaling after radiation results in apoptosis of lung parenchymal cells. We hypothesize that upregulation of PTEN is influenced by Nox4-derived oxidative stress. To our knowledge, this is the first study to highlight the role of PTEN in radiation-induced pulmonary toxicity.

  13. Cord blood stem cell-mediated induction of apoptosis in glioma downregulates X-linked inhibitor of apoptosis protein (XIAP.

    Directory of Open Access Journals (Sweden)

    Venkata Ramesh Dasari

    2010-07-01

    Full Text Available XIAP (X-linked inhibitor of apoptosis protein is one of the most important members of the apoptosis inhibitor family. XIAP is upregulated in various malignancies, including human glioblastoma. It promotes invasion, metastasis, growth and survival of malignant cells. We hypothesized that downregulation of XIAP by human umbilical cord blood mesenchymal stem cells (hUCBSC in glioma cells would cause them to undergo apoptotic death.We observed the effect of hUCBSC on two malignant glioma cell lines (SNB19 and U251 and two glioma xenograft cell lines (4910 and 5310. In co-cultures of glioma cells with hUCBSC, proliferation of glioma cells was significantly inhibited. This is associated with increased cytotoxicity of glioma cells, which led to glioma cell death. Stem cells induced apoptosis in glioma cells, which was evaluated by TUNEL assay, FACS analyses and immunoblotting. The induction of apoptosis is associated with inhibition of XIAP in co-cultures of hUCBSC. Similar results were obtained by the treatment of glioma cells with shRNA to downregulate XIAP (siXIAP. Downregulation of XIAP resulted in activation of caspase-3 and caspase-9 to trigger apoptosis in glioma cells. Apoptosis is characterized by the loss of mitochondrial membrane potential and upregulation of mitochondrial apoptotic proteins Bax and Bad. Cell death of glioma cells was marked by downregulation of Akt and phospho-Akt molecules. We observed similar results under in vivo conditions in U251- and 5310-injected nude mice brains, which were treated with hUCBSC. Under in vivo conditions, Smac/DIABLO was found to be colocalized in the nucleus, showing that hUCBSC induced apoptosis is mediated by inhibition of XIAP and activation of Smac/DIABLO.Our results indicate that downregulation of XIAP by hUCBSC treatment induces apoptosis, which led to the death of the glioma cells and xenograft cells. This study demonstrates the therapeutic potential of XIAP and hUCBSC to treat malignant

  14. Kaempferol Attenuates Cardiac Hypertrophy via Regulation of ASK1/MAPK Signaling Pathway and Oxidative Stress.

    Science.gov (United States)

    Feng, Hong; Cao, Jianlei; Zhang, Guangyu; Wang, Yanggan

    2017-07-01

    Kaempferol has been demonstrated to provide benefits for the treatment of atherosclerosis, coronary heart disease, hyperlipidemia, and diabetes through its antioxidant and anti-inflammatory properties. However, its role in cardiac hypertrophy remains to be elucidated. The aim of our study was to investigate the effects of kaempferol on cardiac hypertrophy and the underlying mechanism. Mice subjected to aorta banding were treated with or without kaempferol (100 mg/kg/d, p. o.) for 6 weeks. Echocardiography was performed to evaluate cardiac function. Mice hearts were collected for pathological observation and molecular mechanism investigation. H9c2 cardiomyocytes were stimulated with or without phenylephrine for in vitro study. Kaempferol significantly attenuated cardiac hypertrophy induced by aorta banding as evidenced by decreased cardiomyocyte areas and interstitial fibrosis, accompanied with improved cardiac functions and decreased apoptosis. The ASK1/MAPK signaling pathways (JNK1/2 and p38) were markedly activated in the aorta banding mouse heart but inhibited by kaempferol treatment. In in vitro experiments, kaempferol also inhibited the activity of ASK1/JNK1/2/p38 signaling pathway and the enlargement of H9c2 cardiomyocytes. Furthermore, our study revealed that kaempferol could protect the mouse heart and H9c2 cells from pathological oxidative stress. Our investigation indicated that treatment with kaempferol protects against cardiac hypertrophy, and its cardioprotection may be partially explained by the inhibition of the ASK1/MAPK signaling pathway and the regulation of oxidative stress. Georg Thieme Verlag KG Stuttgart · New York.

  15. Surfactant protein D delays Fas- and TRAIL-mediated extrinsic pathway of apoptosis in T cells.

    Science.gov (United States)

    Djiadeu, Pascal; Kotra, Lakshmi P; Sweezey, Neil; Palaniyar, Nades

    2017-05-01

    Only a few extracellular soluble proteins are known to modulate apoptosis. We considered that surfactant-associated protein D (SP-D), an innate immune collectin present on many mucosal surfaces, could regulate apoptosis. Although SP-D is known to be important for immune cell homeostasis, whether SP-D affects apoptosis is unknown. In this study we aimed to determine the effects of SP-D on Jurkat T cells and human T cells dying by apoptosis. Here we show that SP-D binds to Jurkat T cells and delays the progression of Fas (CD95)-Fas ligand and TRAIL-TRAIL receptor induced, but not TNF-TNF receptor-mediated apoptosis. SP-D exerts its effects by reducing the activation of initiator caspase-8 and executioner caspase-3. SP-D also delays the surface exposure of phosphatidylserine. The effect of SP-D was ablated by the presence of caspase-8 inhibitor, but not by intrinsic pathway inhibitors. The binding ability of SP-D to dying cells decreases during the early stages of apoptosis, suggesting the release of apoptotic cell surface targets during apoptosis. SP-D also delays FasL-induced death of primary human T cells. SP-D delaying the progression of the extrinsic pathway of apoptosis could have important implications in regulating immune cell homeostasis at mucosal surfaces.

  16. Simultaneous modulation of the intrinsic and extrinsic pathways by simvastatin in mediating prostate cancer cell apoptosis

    International Nuclear Information System (INIS)

    Goc, Anna; Kochuparambil, Samith T; Al-Husein, Belal; Al-Azayzih, Ahmad; Mohammad, Shuaib; Somanath, Payaningal R

    2012-01-01

    Recent studies suggest the potential benefits of statins as anti-cancer agents. Mechanisms by which statins induce apoptosis in cancer cells are not clear. We previously showed that simvastatin inhibit prostate cancer cell functions and tumor growth. Molecular mechanisms by which simvastatin induce apoptosis in prostate cancer cells is not completely understood. Effect of simvastatin on PC3 cell apoptosis was compared with docetaxel using apoptosis, TUNEL and trypan blue viability assays. Protein expression of major candidates of the intrinsic pathway downstream of simvastatin-mediated Akt inactivation was analyzed. Gene arrays and western analysis of PC3 cells and tumor lysates were performed to identify the candidate genes mediating extrinsic apoptosis pathway by simvastatin. Data indicated that simvastatin inhibited intrinsic cell survival pathway in PC3 cells by enhancing phosphorylation of Bad, reducing the protein expression of Bcl-2, Bcl-xL and cleaved caspases 9/3. Over-expression of PC3 cells with Bcl-2 or DN-caspase 9 did not rescue the simvastatin-induced apoptosis. Simvastatin treatment resulted in increased mRNA and protein expression of molecules such as TNF, Fas-L, Traf1 and cleaved caspase 8, major mediators of intrinsic apoptosis pathway and reduced protein levels of pro-survival genes Lhx4 and Nme5. Our study provides the first report that simvastatin simultaneously modulates intrinsic and extrinsic pathways in the regulation of prostate cancer cell apoptosis in vitro and in vivo, and render reasonable optimism that statins could become an attractive anti-cancer agent

  17. Caspase-mediated apoptosis induction in zebrafish cerebellar Purkinje neurons.

    Science.gov (United States)

    Weber, Thomas; Namikawa, Kazuhiko; Winter, Barbara; Müller-Brown, Karina; Kühn, Ralf; Wurst, Wolfgang; Köster, Reinhard W

    2016-11-15

    The zebrafish is a well-established model organism in which to study in vivo mechanisms of cell communication, differentiation and function. Existing cell ablation methods are either invasive or they rely on the cellular expression of prokaryotic enzymes and the use of antibiotic drugs as cell death-inducing compounds. We have recently established a novel inducible genetic cell ablation system based on tamoxifen-inducible Caspase 8 activity, thereby exploiting mechanisms of cell death intrinsic to most cell types. Here, we prove its suitability in vivo by monitoring the ablation of cerebellar Purkinje cells (PCs) in transgenic zebrafish that co-express the inducible caspase and a fluorescent reporter. Incubation of larvae in tamoxifen for 8 h activated endogenous Caspase 3 and cell death, whereas incubation for 16 h led to the near-complete loss of PCs by apoptosis. We observed synchronous cell death autonomous to the PC population and phagocytosing microglia in the cerebellum, reminiscent of developmental apoptosis in the forebrain. Thus, induction of apoptosis through targeted activation of caspase by tamoxifen (ATTAC TM ) further expands the repertoire of genetic tools for conditional interrogation of cellular functions. © 2016. Published by The Company of Biologists Ltd.

  18. Role of CD137 signaling in dengue virus-mediated apoptosis

    International Nuclear Information System (INIS)

    Nagila, Amar; Netsawang, Janjuree; Srisawat, Chatchawan; Noisakran, Sansanee; Morchang, Atthapan; Yasamut, Umpa; Puttikhunt, Chunya; Kasinrerk, Watchara

    2011-01-01

    Highlights: → For the first time the role of CD137 in dengue virus (DENV) infection. → Induction of DENV-mediated apoptosis by CD137 signaling. → Sensitization to CD137-mediated apoptosis by dengue virus capsid protein (DENV C). → Nuclear localization of DENV C is required for CD137-mediated apoptosis. -- Abstract: Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. A double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.

  19. Role of CD137 signaling in dengue virus-mediated apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Nagila, Amar [Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Netsawang, Janjuree [Faculty of Medical Technology, Rangsit University, Bangkok (Thailand); Srisawat, Chatchawan [Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Noisakran, Sansanee [Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok (Thailand); Morchang, Atthapan; Yasamut, Umpa [Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Puttikhunt, Chunya [Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok (Thailand); Kasinrerk, Watchara [Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai (Thailand); Biomedical Technology Research Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency at Chiang Mai University, Chiang Mai (Thailand); and others

    2011-07-08

    Highlights: {yields} For the first time the role of CD137 in dengue virus (DENV) infection. {yields} Induction of DENV-mediated apoptosis by CD137 signaling. {yields} Sensitization to CD137-mediated apoptosis by dengue virus capsid protein (DENV C). {yields} Nuclear localization of DENV C is required for CD137-mediated apoptosis. -- Abstract: Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. A double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.

  20. 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

  1. Transmissible gastroenteritis virus infection induces apoptosis through FasL- and mitochondria-mediated pathways.

    Science.gov (United States)

    Ding, Li; Xu, Xingang; Huang, Yong; Li, Zhaocai; Zhang, Kuan; Chen, Guangda; Yu, Gaoshui; Wang, Zhisheng; Li, Wei; Tong, Dewen

    2012-07-06

    Transmissible gastroenteritis virus (TGEV) has been reported to induce apoptosis in swine testis (ST) cells. However, the mechanisms underlying TGEV-induced apoptosis are still unclear. In this study we observed that TGEV infection induced apoptosis in porcine kidney (PK-15) cells in a time- and dose-dependent manner. TGEV infection up-regulated FasL, activated FasL-mediated apoptotic pathway, leading to activation of caspase-8 and cleavage of Bid. In addition, TGEV infection down-regulated Bcl-2, up-regulated Bax expression, promoted translocation of Bax to mitochondria, activated mitochondria-mediated apoptotic pathway, which in turn caused the release of cytochrome c and the activation of caspase-9. Both extrinsic and intrinsic pathways activated downstream effector caspase-3, followed by the cleavage of PARP, resulting in cell apoptosis. Moreover, TGEV infection did not induce significant DNA fragmentation in ammonium chloride (NH(4)Cl) pretreated PK-15 cells or cells infected with UV-inactivated TGEV. In turn, block of caspases activation also did not affect TGEV replication. Taken together, this study demonstrates that TGEV-induced apoptosis is dependent on viral replication in PK-15 cells and occurs through activation of FasL- and mitochondria-mediated apoptotic pathways. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. The emphysematous lung is abnormally sensitive to TRAIL-mediated apoptosis

    Directory of Open Access Journals (Sweden)

    Milot Julie

    2011-08-01

    Full Text Available Abstract Background Alveolar apoptosis is increased in the emphysematous lung. However, mechanisms involved are not fully understood. Recently, we demonstrated that levels of TRAIL receptor 1 and 2, levels of p53, and Bax/Bcl-xL ratio were elevated in the lung of subjects with emphysema, despite smoking cessation. Thus, we postulate that due to chronic pulmonary oxidative stress, the emphysematous lung would be abnormally sensitive to TRAIL-mediated apoptosis. Methodology A549 cells were exposed to rTRAIL, cigarette smoke extract, and/or H2O2 prior to caspase-3 activity measurement and annexin V staining assessment. In addition, freshly resected lung samples were obtained from non-emphysematous and emphysematous subjects and exposed ex vivo to rTRAIL for up to 18 hours. Lung samples were harvested and levels of active caspase-3 and caspase-8 were measured from tissue lysates. Results Both cigarette smoke extract and H2O2 were able to sensitize A549 cells to TRAIL-mediated apoptosis. Moreover, following exposure to rTRAIL, caspase-3 and -8 were activated in lung explants from emphysematous subjects while being decreased in lung explants from non-emphysematous subjects. Significance of the study Alveolar sensitivity to TRAIL-mediated apoptosis is strongly increased in the emphysematous lung due to the presence of oxidative stress. This might be a new mechanism leading to increased alveolar apoptosis and persistent alveolar destruction following smoking cessation.

  3. CD40-mediated apoptosis in murine B-lymphoma lines containing mutated p53

    DEFF Research Database (Denmark)

    Hollmann, Annette C; Gong, Qiaoke; Owens, Trevor

    2002-01-01

    Crosslinking CD40 induces normal B-cells to proliferate and differentiate but causes many tumor cell lines to undergo apoptosis. As p53 is required for many apoptotic pathways, we analyzed the effects of CD40 ligation and their correlation with p53 function in four murine B-lymphoma lines. A20...... of detectable p21 mRNA in A20 and M12 cells. P21 mRNA was increased to detectable levels in M12 cells upon CD40 ligation; however, blocking this effect with the p53 inhibitor pifithrin had no effect on CD40-mediated apoptosis. Sequencing showed that p53 in A20 and M12 cells contained point mutations leading...... to amino acid substitutions in DNA binding regions, but was unmutated in WEHI231 and WEHI 279. These results suggest that CD40-mediated apoptosis can occur in the absence of functional p53....

  4. Combinatorial action of Grainyhead, Extradenticle and Notch in regulating Hox mediated apoptosis in Drosophila larval CNS.

    Science.gov (United States)

    Khandelwal, Risha; Sipani, Rashmi; Govinda Rajan, Sriivatsan; Kumar, Raviranjan; Joshi, Rohit

    2017-10-01

    Hox mediated neuroblast apoptosis is a prevalent way to pattern larval central nervous system (CNS) by different Hox genes, but the mechanism of this apoptosis is not understood. Our studies with Abdominal-A (Abd-A) mediated larval neuroblast (pNB) apoptosis suggests that AbdA, its cofactor Extradenticle (Exd), a helix-loop-helix transcription factor Grainyhead (Grh), and Notch signaling transcriptionally contribute to expression of RHG family of apoptotic genes. We find that Grh, AbdA, and Exd function together at multiple motifs on the apoptotic enhancer. In vivo mutagenesis of these motifs suggest that they are important for the maintenance of the activity of the enhancer rather than its initiation. We also find that Exd function is independent of its known partner homothorax in this apoptosis. We extend some of our findings to Deformed expressing region of sub-esophageal ganglia where pNBs undergo a similar Hox dependent apoptosis. We propose a mechanism where common players like Exd-Grh-Notch work with different Hox genes through region specific enhancers to pattern respective segments of larval central nervous system.

  5. Combinatorial action of Grainyhead, Extradenticle and Notch in regulating Hox mediated apoptosis in Drosophila larval CNS.

    Directory of Open Access Journals (Sweden)

    Risha Khandelwal

    2017-10-01

    Full Text Available Hox mediated neuroblast apoptosis is a prevalent way to pattern larval central nervous system (CNS by different Hox genes, but the mechanism of this apoptosis is not understood. Our studies with Abdominal-A (Abd-A mediated larval neuroblast (pNB apoptosis suggests that AbdA, its cofactor Extradenticle (Exd, a helix-loop-helix transcription factor Grainyhead (Grh, and Notch signaling transcriptionally contribute to expression of RHG family of apoptotic genes. We find that Grh, AbdA, and Exd function together at multiple motifs on the apoptotic enhancer. In vivo mutagenesis of these motifs suggest that they are important for the maintenance of the activity of the enhancer rather than its initiation. We also find that Exd function is independent of its known partner homothorax in this apoptosis. We extend some of our findings to Deformed expressing region of sub-esophageal ganglia where pNBs undergo a similar Hox dependent apoptosis. We propose a mechanism where common players like Exd-Grh-Notch work with different Hox genes through region specific enhancers to pattern respective segments of larval central nervous system.

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

    Directory of Open Access Journals (Sweden)

    Wei Hua

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

  7. Elevated pressure, a novel cancer therapeutic tool for sensitizing cisplatin-mediated apoptosis in A549

    International Nuclear Information System (INIS)

    Oh, Sangnam; Kim, Yanghee; Kim, Joonhee; Kwon, Daeho; Lee, Eunil

    2010-01-01

    Research highlights: → Sensitized apoptosis in cancer cells stimulated by EP precondition with p53 dependence. → EP attenuates several CDDP-resistance mechanisms. → No harmful effect of EP on normal fibroblasts. -- Abstract: Intensive cancer therapy strategies have thus far focused on sensitizing cancer cells to anticancer drug-mediated apoptosis to overcome drug resistance, and this strategy has led to more effective cancer therapeutics. Cisplatin (cis-diamminedichloroplatinum(II), CDDP) is an effective anticancer drug used to treat many types of cancer, including non-small cell lung carcinoma (NSCLC), and can be used in combination with various chemicals to enhance cancer cell apoptosis. Here, we introduce the use of elevated pressure (EP) in combination with CDDP for cancer treatment and explore the effects of EP on CDDP-mediated apoptosis in NSCLC cells. Our findings demonstrate that preconditioning NSCLC cells with EP sensitizes cells for CDDP-induced apoptosis. Enhanced apoptosis was dependent on p53 and HO-1 expression, and was associated with increased DNA damage and down-regulation of genes involved in nucleotide excision repair. The transcriptional levels of transporter proteins indicated that the mechanism by which EP-induced CDDP sensitization was intracellular drug accumulation. The protein levels of some antioxidants, such as hemeoxygenase-1 (HO-1), glutathione (GSH) and glutathione peroxidase (Gpx), were decreased in A549 cells exposed to EP via the down-regulation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf-2). Furthermore, normal human fibroblasts were resistant to EP treatment, with no elevated DNA damage or apoptosis. Collectively, these data show that administration of EP is a potential adjuvant tool for CDDP-based chemosensitivity of lung cancer cells that may reduce drug resistance.

  8. Curcumin enhances the antitumor effect of ABT-737 via activation of the ROS-ASK1-JNK pathway in hepatocellular carcinoma cells.

    Science.gov (United States)

    Zheng, Ruinian; You, Zhijian; Jia, Jun; Lin, Shunhuan; Han, Shuai; Liu, Aixue; Long, Huidong; Wang, Senming

    2016-02-01

    At present, the therapeutic treatment strategies for patients with hepatocellular carcinoma (HCC) remain unsatisfactory, and novel methods are urgently required to treat this disease. Members of the B cell lymphoma (Bcl)-2 family are anti‑apoptotic proteins, which are commonly expressed at high levels in certain HCC tissues and positively correlate with the treatment resistance of patients with HCC. ABT-737, an inhibitor of Bcl-2 anti-apoptotic proteins, has been demonstrated to exhibit potent antitumor effects in several types of tumor, including HCC. However, treatment with ABT-737 alone also activates certain pro-survival signaling pathways, which attenuate the antitumor validity of ABT-737. Curcumin, which is obtained from Curcuma longa, is also an antitumor potentiator in multiple types of cancer. In the present study, the synergistic effect of curcumin and ABT-737 on HCC cells was investigated for the first time, to the best of our knowledge. It was found that curcumin markedly enhanced the antitumor effects of ABT-737 on HepG2 cells, which was partially dependent on the induction of apoptosis, according to western blot analysis and flow cytometric apoptosis analysis. In addition, the sustained activation of the ROS-ASK1-c-Jun N-terminal kinase pathway may be an important mediator of the synergistic effect of curcumin and ABT-737. Collectively, these results indicated that the combination of curcumin and ABT-737 can efficaciously induce the death of HCC cells, and may offer a potential treatment strategy for patients with HCC.

  9. Inositol Hexakisphosphate Mediates Apoptosis in Human Breast Adenocarcinoma MCF-7 Cell Line via Intrinsic Pathway

    Science.gov (United States)

    Agarwal, Rakhee; Ali, Nawab

    2010-04-01

    Inositol polyphosphates (InsPs) are naturally occurring compounds ubiquitously present in plants and animals. Inositol hexakisphosphate (InsP6) is the most abundant among all InsPs and constitutes the major portion of dietary fiber in most cereals, legumes and nuts. Certain derivatives of InsPs also regulate cellular signaling mechanisms. InsPs have also been shown to reduce tumor formation and induce apoptosis in cancerous cells. Therefore, in this study, the effects of InsPs on apoptosis were studied in an attempt to investigate their potential anti-cancer therapeutic application and understand their mechanism of action. Acridine orange and ethidium bromide staining suggested that InsP6 dose dependently induced apoptosis in human breast adenocarcinoma MCF-7 cells. Among InsPs tested (InsP3, InsP4, InsP5, and InsP6), InsP6 was found to be the most effective in inducing apoptosis. Furthermore, effects of InsP6 were found most potent inducing apoptosis. Etoposide, the drug known to induce apoptosis in both in vivo and in vitro, was used as a positive control. Western blotting experiments using specific antibodies against known apoptotic markers suggested that InsP6 induced apoptotic changes were mediated via an intrinsic apoptotic pathway.

  10. Low dose gamma irradiation enhances defined signaling components of intercellular reactive oxygen-mediated apoptosis induction

    Energy Technology Data Exchange (ETDEWEB)

    Bauer, G, E-mail: georg.bauer@uniklinik-freiburg.de [Abteilung Virologie, Institut fuer Medizinische Mikrobiologie und Hygiene, Universitaet Freiburg, Freiburg (Germany)

    2011-01-01

    Transformed cells are selectively removed by intercellular ROS-mediated induction of apoptosis. Signaling is based on the HOCl and the NO/peroxynitrite pathway (major pathways) and the nitryl chloride and the metal-catalyzed Haber-Weiss pathway (minor pathways). During tumor progression, resistance against intercellular induction of apoptosis is acquired through expression of membrane-associated catalase. Low dose radiation of nontransformed cells has been shown to enhance intercellular induction of apoptosis. The present study was performed to define the signaling components which are modulated by low dose gamma irradiation. Low dose radiation induced the release of peroxidase from nontransformed, transformed and tumor cells. Extracellular superoxide anion generation was strongly enhanced in the case of transformed cells and tumor cells, but not in nontransformed cells. Enhancement of peroxidase release and superoxide anion generation either increased intercellular induction of apoptosis of transformed cells, or caused a partial protection under specific signaling conditions. In tumor cells, low dose radiation enhanced the production of major signaling components, but this had no effect on apoptosis induction, due to the strong resistance mechanism of tumor cells. Our data specify the nature of low dose radiation-induced effects on specific signaling components of intercellular induction of apoptosis at defined stages of multistep carcinogenesis.

  11. INOSITOL HEXAKISPHOSPHATE MEDIATES APOPTOSIS IN HUMAN BREAST ADENOCARCINOMA MCF-7 CELL LINE VIA INTRINSIC PATHWAY

    International Nuclear Information System (INIS)

    Agarwal, Rakhee; Ali, Nawab

    2010-01-01

    Inositol polyphosphates (InsP s ) are naturally occurring compounds ubiquitously present in plants and animals. Inositol hexakisphosphate (InsP 6 ) is the most abundant among all InsP s and constitutes the major portion of dietary fiber in most cereals, legumes and nuts. Certain derivatives of InsP s also regulate cellular signaling mechanisms. InsP s have also been shown to reduce tumor formation and induce apoptosis in cancerous cells. Therefore, in this study, the effects of InsPs on apoptosis were studied in an attempt to investigate their potential anti-cancer therapeutic application and understand their mechanism of action. Acridine orange and ethidium bromide staining suggested that InsP 6 dose dependently induced apoptosis in human breast adenocarcinoma MCF-7 cells. Among InsP s tested (InsP 3 , InsP 4 , InsP 5 , and InsP 6 ), InsP 6 was found to be the most effective in inducing apoptosis. Furthermore, effects of InsP 6 were found most potent inducing apoptosis. Etoposide, the drug known to induce apoptosis in both in vivo and in vitro, was used as a positive control. Western blotting experiments using specific antibodies against known apoptotic markers suggested that InsP 6 induced apoptotic changes were mediated via an intrinsic apoptotic pathway.

  12. Silver Nanoparticles Induce HePG-2 Cells Apoptosis Through ROS-Mediated Signaling Pathways

    Science.gov (United States)

    Zhu, Bing; Li, Yinghua; Lin, Zhengfang; Zhao, Mingqi; Xu, Tiantian; Wang, Changbing; Deng, Ning

    2016-04-01

    Recently, silver nanoparticles (AgNPs) have been shown to provide a novel approach to overcome tumors, especially those of hepatocarcinoma. However, the anticancer mechanism of silver nanoparticles is unclear. Thus, the purpose of this study was to estimate the effect of AgNPs on proliferation and activation of ROS-mediated signaling pathway on human hepatocellular carcinoma HePG-2 cells. A simple chemical method for preparing AgNPs with superior anticancer activity has been showed in this study. AgNPs were detected by transmission electronic microscopy (TEM) and energy dispersive X-ray (EDX). The size distribution and zeta potential of silver nanoparticles were detected by Zetasizer Nano. The average size of AgNPs (2 nm) observably increased the cellular uptake by endocytosis. AgNPs markedly inhibited the proliferation of HePG-2 cells through induction of apoptosis with caspase-3 activation and PARP cleavage. AgNPs with dose-dependent manner significantly increased the apoptotic cell population (sub-G1). Furthermore, AgNP-induced apoptosis was found dependent on the overproduction of reactive oxygen species (ROS) and affecting of MAPKs and AKT signaling and DNA damage-mediated p53 phosphorylation to advance HePG-2 cells apoptosis. Therefore, our results show that the mechanism of ROS-mediated signaling pathways may provide useful information in AgNP-induced HePG-2 cell apoptosis.

  13. Estradiol-mediated ERK phosphorylation and apoptosis in vascular smooth muscle cells requires GPR 30.

    Science.gov (United States)

    Ding, Qingming; Gros, Robert; Limbird, Lee E; Chorazyczewski, Jozef; Feldman, Ross D

    2009-11-01

    Recent studies suggest that the rapid and nongenomic effects of estradiol may be mediated through the G protein-coupled receptor dubbed GPR30 receptor. The present study examines the role of GPR30 versus a classical estrogen receptor (ERalpha) in mediating the growth regulatory effects of estradiol. GPR30 is readily detectable in freshly isolated vascular tissue but barely detectable in cultured vascular smooth muscle cells (VSMC). In freshly isolated aortic tissue, estradiol stimulated extracellular signal-regulated kinases (ERK) phosphorylation. In contrast, in cultured VSMC, where GPR30 expression is significantly reduced, estradiol inhibits ERK phosphorylation. Transfer of the genes encoding GPR30 led to estradiol stimulation of ERK phosphorylation, which is opposite the effects of estradiol in the primary culture of VSMCs. Transduction of the mineralocorticoid receptor (MR) had no effect on estradiol effects on ERK. Estradiol-mediated stimulation of ERK subsequent to heterologous GPR30 expression was pertussis toxin sensitive and phosphoinositide 3-kinase (PI3 kinase) dependent; under these conditions, estradiol also inhibited protein kinase A (PKA). In contrast, in the absence of GPR30 expression in cultured VSMC, estradiol stimulated PKA activity and inhibited ERK phosphorylation. To determine the functional effect of GPR30 (vs. estrogen receptor expression), we assessed estradiol-mediated apoptosis. In the absence of GPR30 expression, estradiol inhibited apoptosis. This effect was enhanced with ERalpha expression. In contrast, with GPR30 expression, estradiol stimulated apoptosis in an ERK-dependent manner. Thus the effect of estradiol on vascular smooth muscle cell apoptosis is likely dependent on the balance between ER-mediated PKA activation and GPR30-mediated PKA inhibition and PI3 kinase activation. Taken together, we postulate that modulation of GPR30 expression or activity may be an important determinant of the effects of estradiol in the vasculature.

  14. Voltage dependent anion channel-1 regulates death receptor mediated apoptosis by enabling cleavage of caspase-8

    International Nuclear Information System (INIS)

    Chacko, Alex D; Liberante, Fabio; Paul, Ian; Longley, Daniel B; Fennell, Dean A

    2010-01-01

    Activation of the extrinsic apoptosis pathway by tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a novel therapeutic strategy for treating cancer that is currently under clinical evaluation. Identification of molecular biomarkers of resistance is likely to play an important role in predicting clinical anti tumour activity. The involvement of the mitochondrial type 1 voltage dependent anion channel (VDAC1) in regulating apoptosis has been highly debated. To date, a functional role in regulating the extrinsic apoptosis pathway has not been formally excluded. We carried out stable and transient RNAi knockdowns of VDAC1 in non-small cell lung cancer cells, and stimulated the extrinsic apoptotic pathway principally by incubating cells with the death ligand TRAIL. We used in-vitro apoptotic and cell viability assays, as well as western blot for markers of apoptosis, to demonstrate that TRAIL-induced toxicity is VDAC1 dependant. Confocal microscopy and mitochondrial fractionation were used to determine the importance of mitochondria for caspase-8 activation. Here we show that either stable or transient knockdown of VDAC1 is sufficient to antagonize TRAIL mediated apoptosis in non-small cell lung cancer (NSCLC) cells. Specifically, VDAC1 is required for processing of procaspase-8 to its fully active p18 form at the mitochondria. Loss of VDAC1 does not alter mitochondrial sensitivity to exogenous caspase-8-cleaved BID induced mitochondrial depolarization, even though VDAC1 expression is essential for TRAIL dependent activation of the intrinsic apoptosis pathway. Furthermore, expression of exogenous VDAC1 restores the apoptotic response to TRAIL in cells in which endogenous VDAC1 has been selectively silenced. Expression of VDAC1 is required for full processing and activation of caspase-8 and supports a role for mitochondria in regulating apoptosis signaling via the death receptor pathway

  15. STAT3 mediates regorafenib-induced apoptosis in hepatocellular carcinoma.

    Science.gov (United States)

    Tai, Wei-Tien; Chu, Pei-Yi; Shiau, Chung-Wai; Chen, Yao-Li; Li, Yong-Shi; Hung, Man-Hsin; Chen, Li-Ju; Chen, Pei-Lung; Su, Jung-Chen; Lin, Ping-Yi; Yu, Hui-Chuan; Chen, Kuen-Feng

    2014-11-15

    Here, we aim to investigate the molecular mechanism of regorafenib and verify the potential druggable target for the treatment of hepatocellular carcinoma (HCC). HCC cell lines (PLC5, HepG2, Hep3B, SK-Hep1, and HA59T) were used to investigate the in vitro effect of regorafenib. Phosphatase activity was analyzed in HCC cells and purified SHP-1 proteins. PLC5-bearing mice were used to test the therapeutic efficiency of 20 and 40 mg/kg/d treatment with regorafenib ([Formula: see text] mice). The clinical relevance of STAT3 signaling was investigated with 142 tumor samples from different patients with HCC. Descriptive statistical analysis was used to compare the baseline characteristics of patients and the expression of p-STAT3. Regorafenib inhibited STAT3-related signaling in a dose-dependent manner and was a more potent inhibitor of STAT3 than sorafenib. Regorafenib increased SHP-1 phosphatase activity in purified SHP-1 protein directly. N-SH2 domain deletion and D61A mutants mimicking open-form SHP-1 partially abolished regorafenib-induced STAT3 inhibition and apoptosis. Importantly, a higher level of expression of STAT3 was found in patients with advanced clinical stages (P = 0.009) and poorly differentiated tumors (P = 0.035). Regorafenib induced significant tumor inhibition by relieving the autoinhibited N-SH2 domain of SHP-1 directly and inhibiting p-STAT3 signals. STAT3 may be suitable as a prognostic marker of HCC development, and may be a druggable target for HCC-targeted therapy using regorafenib. ©2014 American Association for Cancer Research.

  16. Apoptosis-mediated seasonal testicular regression in the Japanese Jungle Crow (Corvus macrorhynchos).

    Science.gov (United States)

    Islam, M Nazrul; Tsukahara, N; Sugita, S

    2012-06-01

    The present study investigated effects of apoptosis observed during seasonal testicular regression in Japanese Jungle Crows. The study was conducted during January to June 2008, 2009. Testes from adults captured during non-breeding (January), prebreeding (February to mid-March), main-breeding (late March to early May), transition (mid-May to late May), and post-breeding (June) seasons were analyzed. Apoptosis was assessed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Paired-testis volume increased 95-fold from the non-breeding to the main-breeding season (P Jungle Crows; however, testis function was terminated rapidly after the breeding season. Furthermore, we concluded, similar to other avian species, Sertoli cell apoptosis followed by massive germ cell death was responsible for rapid testicular regression in Jungle Crows. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. 17-AAG sensitized malignant glioma cells to death-receptor mediated apoptosis.

    Science.gov (United States)

    Siegelin, Markus David; Habel, Antje; Gaiser, Timo

    2009-02-01

    17-AAG is a selective HSP90-inhibitor that exhibited therapeutic activity in cancer. In this study three glioblastoma cell lines (U87, LN229 and U251) were treated with 17-AAG, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Treatment with subtoxic doses of 17-AAG in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rapid apoptosis in TRAIL-resistant glioma cells, suggesting that this combined treatment may offer an attractive strategy for treating gliomas. 17-AAG treatment down-regulated survivin through proteasomal degradation. In addition, over-expression of survivin attenuated cytotoxicity induced by the combination of 17-AAG and TRAIL. In summary, survivin is a key regulator of TRAIL-17-AAG mediated cell death in malignant glioma.

  18. Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species.

    Directory of Open Access Journals (Sweden)

    Eun-Ryeong Hahm

    Full Text Available Withaferin A (WA, a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC. The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.

  19. Structural and Functional Insight into Canarypox Virus CNP058 Mediated Regulation of Apoptosis

    Directory of Open Access Journals (Sweden)

    Mohd Ishtiaq Anasir

    2017-10-01

    Full Text Available Programmed cell death or apoptosis is an important component of host defense systems against viral infection. The B-cell lymphoma 2 (Bcl-2 proteins family is the main arbiter of mitochondrially mediated apoptosis, and viruses have evolved sequence and structural mimics of Bcl-2 to subvert premature host cell apoptosis in response to viral infection. The sequencing of the canarypox virus genome identified a putative pro-survival Bcl-2 protein, CNP058. However, a role in apoptosis inhibition for CNP058 has not been identified to date. Here, we report that CNP058 is able to bind several host cell pro-death Bcl-2 proteins, including Bak and Bax, as well as several BH3 only-proteins including Bim, Bid, Bmf, Noxa, Puma, and Hrk with high to moderate affinities. We then defined the structural basis for CNP058 binding to pro-death Bcl-2 proteins by determining the crystal structure of CNP058 bound to Bim BH3. CNP058 adopts the conserved Bcl-2 like fold observed in cellular pro-survival Bcl-2 proteins, and utilizes the canonical ligand binding groove to bind Bim BH3. We then demonstrate that CNP058 is a potent inhibitor of ultraviolet (UV induced apoptosis in a cell culture model. Our findings suggest that CNP058 is a potent inhibitor of apoptosis that is able to bind to BH3 domain peptides from a broad range of pro-death Bcl-2 proteins, and may play a key role in countering premature host apoptosis.

  20. JNK1 protects against glucolipotoxicity-mediated beta-cell apoptosis

    DEFF Research Database (Denmark)

    Prause, Michala; Christensen, Dan Ploug; Billestrup, Nils

    2014-01-01

    Pancreatic β-cell dysfunction is central to type 2 diabetes pathogenesis. Prolonged elevated levels of circulating free-fatty acids and hyperglycemia, also termed glucolipotoxicity, mediate β-cell dysfunction and apoptosis associated with increased c-Jun N-terminal Kinase (JNK) activity. Endoplas......Pancreatic β-cell dysfunction is central to type 2 diabetes pathogenesis. Prolonged elevated levels of circulating free-fatty acids and hyperglycemia, also termed glucolipotoxicity, mediate β-cell dysfunction and apoptosis associated with increased c-Jun N-terminal Kinase (JNK) activity....... Endoplasmic reticulum (ER) and oxidative stress are elicited by palmitate and high glucose concentrations further potentiating JNK activity. Our aim was to determine the role of the JNK subtypes JNK1, JNK2 and JNK3 in palmitate and high glucose-induced β-cell apoptosis. We established insulin-producing INS1...... INS1 cells showed increased apoptosis and cleaved caspase 9 and 3 compared to non-sense shRNA expressing control INS1 cells when exposed to palmitate and high glucose associated with increased CHOP expression, ROS formation and Puma mRNA expression. JNK2 shRNA expressing INS1 cells did not affect...

  1. CD4+ lymphocytes control gut epithelial apoptosis and mediate survival in sepsis.

    Science.gov (United States)

    Stromberg, Paul E; Woolsey, Cheryl A; Clark, Andrew T; Clark, Jessica A; Turnbull, Isaiah R; McConnell, Kevin W; Chang, Katherine C; Chung, Chun-Shiang; Ayala, Alfred; Buchman, Timothy G; Hotchkiss, Richard S; Coopersmith, Craig M

    2009-06-01

    Lymphocytes help determine whether gut epithelial cells proliferate or differentiate but are not known to affect whether they live or die. Here, we report that lymphocytes play a controlling role in mediating gut epithelial apoptosis in sepsis but not under basal conditions. Gut epithelial apoptosis is similar in unmanipulated Rag-1(-/-) and wild-type (WT) mice. However, Rag-1(-/-) animals have a 5-fold augmentation in gut epithelial apoptosis following cecal ligation and puncture (CLP) compared to septic WT mice. Reconstitution of lymphocytes in Rag-1(-/-) mice via adoptive transfer decreases intestinal apoptosis to levels seen in WT animals. Subset analysis indicates that CD4(+) but not CD8(+), gammadelta, or B cells are responsible for the antiapoptotic effect of lymphocytes on the gut epithelium. Gut-specific overexpression of Bcl-2 in transgenic mice decreases mortality following CLP. This survival benefit is lymphocyte dependent since gut-specific overexpression of Bcl-2 fails to alter survival when the transgene is overexpressed in Rag-1(-/-) mice. Further, adoptively transferring lymphocytes to Rag-1(-/-) mice that simultaneously overexpress gut-specific Bcl-2 results in improved mortality following sepsis. Thus, sepsis unmasks CD4(+) lymphocyte control of gut apoptosis that is not present under homeostatic conditions, which acts as a key determinant of both cellular survival and host mortality.

  2. Glutamate-mediated protection of crayfish glial cells from PDT-induced apoptosis

    Science.gov (United States)

    Rudkovskii, M. V.; Romanenko, N. P.; Berezhnaya, E. V.; Kovaleva, V. D.; Uzdensky, A. B.

    2011-03-01

    Photodynamic treatment that causes intense oxidative stress and kills cells is currently used in neurooncology. However, along with tumor it damages surrounding healthy neurons and glial cells. In order to study the possible role of glutamate-related signaling pathways in photodynamic injury of neurons and glia, we investigated photodynamic effect of alumophthalocyanine Photosens on isolated crayfish stretch receptor that consists of a single neuron surrounded by glial cells. The laser diode (670 nm, 0.4 W/cm2) was used for dye photoexcitation. Application of glutamate increased photodynamically induced necrosis of neurons and glial cells but significantly decreased glial apoptosis. The natural neuroglial mediator N-acetylaspartylglutamate, which releases glutamate after cleavage in the extracellular space by glutamate carboxypeptidase II, also inhibited photoinduced apoptosis. Inhibition of glutamate carboxypeptidase II, oppositely, enhanced apoptosis of glial cells. These data confirm the anti-apoptotic activity of glutamate. Application of NMDA or inhibition of NMDA receptors by MK801 did not influence photodynamic death of neurons and glial cells that indicated nonparticipation of NMDA receptors in these processes. Inhibition of metabotropic glutamate receptors by AP-3 decreased PDT-induced apoptosis. One can suggest that crayfish neurons naturally secrete NAAG, which being cleaved by GCOP produces glutamate. Glutamate prevents photoinduced apoptosis of glial cells possibly through metabotropic but not ionotropic glutamate receptors.

  3. Taraxerol Induces Cell Apoptosis through A Mitochondria-Mediated Pathway in HeLa Cells.

    Science.gov (United States)

    Yaoi, Xiangyang; Lu, Binyu; Lü, Chaotian; Bai, Qin; Yan, Dazhong; Xu, Hui

    2017-10-01

    Taraxerol acetate has potent anti-cancer effects via the induction of apoptosis, autophagy, cell cycle arrest, and inhibition of cell migration. However, whether taraxerol induced apoptosis and its underlying mechanisms of action is not clear. In the present study, we assess the effects of taraxerol on the mitochondrial apoptotic pathway and determine the release of cytochrome c to the cytosol and activation of caspases. In this experimental study, we mainly investigated the effect of taraxerol on HeLa cells. We tested cell viability by the MTT assay and morphologic changes, analyzed apoptosis by DAPI staining and flow cytometry. We also determined reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) using a Microplate Reader. In addition, the apoptotic proteins were tested by Western blot. Taraxerol enhanced ROS levels and attenuated the MMP (Δψm) in HeLa cells. Taraxerol induced apoptosis mainly via the mitochondrial pathway including the release of cytochrome c to the cytosol and activation of caspases 9 and 3, and anti-poly (ADPribose) polymerase (PARP). Taraxerol could induce the down-regulation of the anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax. It suppressed the PI3K/ Akt signaling pathway. These results demonstrated that taraxerol induced cell apoptosis through a mitochondria-mediated pathway in HeLa cells. Thus, taraxerol might be a potential anticervical cancer candidate.

  4. Chk2 regulates transcription-independent p53-mediated apoptosis in response to DNA damage

    International Nuclear Information System (INIS)

    Chen Chen; Shimizu, Shigeomi; Tsujimoto, Yoshihide; Motoyama, Noboru

    2005-01-01

    The tumor suppressor protein p53 plays a central role in the induction of apoptosis in response to genotoxic stress. The protein kinase Chk2 is an important regulator of p53 function in mammalian cells exposed to ionizing radiation (IR). Cells derived from Chk2-deficient mice are resistant to the induction of apoptosis by IR, and this resistance has been thought to be a result of the defective transcriptional activation of p53 target genes. It was recently shown, however, that p53 itself and histone H1.2 translocate to mitochondria and thereby induces apoptosis in a transcription-independent manner in response to IR. We have now examined whether Chk2 also regulates the transcription-independent induction of apoptosis by p53 and histone H1.2. The reduced ability of IR to induce p53 stabilization in Chk2-deficient thymocytes was associated with a marked impairment of p53 and histone H1 translocation to mitochondria. These results suggest that Chk2 regulates the transcription-independent mechanism of p53-mediated apoptosis by inducing stabilization of p53 in response to IR

  5. ROS accumulation by PEITC selectively kills ovarian cancer cells via UPR-mediated apoptosis

    Directory of Open Access Journals (Sweden)

    Yoon-hee eHong

    2015-07-01

    Full Text Available Unfolded protein response (UPR is crucial for both survival and death of mammalian cells, which is regulated by reactive oxygen species (ROS and nutrient depletion. In this study, we demonstrated the effect of ROS-accumulation, induced by β-phenethyl isothiocyanate (PEITC, on UPR mediated apoptosis in ovarian cancer cells. We used ovarian cancer cell lines, PA-1 and SKOV-3, with different p53 status (wild- and null- type, respectively. PEITC caused increased ROS-accumulation and inhibited proliferation selectively in ovarian cancer cells, and glutathione (GSH depletion in SKOV-3. However, PEITC did not cause any effect in normal ovarian epithelial cells and peripheral blood mononuclear cells. After 48 h of PEITC treatment (5 µM, apoptotic cell death was shown to increase significantly in the ovarian cancer cells and not in the normal cells. The key regulator of UPR-mediated apoptosis, CHOP/GADD153 and ER resident chaperone BiP/GRP78 were parallely up-regulated with activation of two major sensors of the UPR (PERK and ATF-6 in PA-1; PERK, and IRE1α in SKOV-3 in response to ROS accumulation induced by PEITC (5 µM. ROS scavenger, N-acetyl-cysteine (NAC, attenuated the effect of PEITC on UPR signatures (P-PERK, IRE1α, CHOP/GADD153, and BiP/GRP78, suggesting the involvement of ROS in UPR-mediated apoptosis. Altogether, PEITC induces UPR-mediated apoptosis in ovarian cancer cells via accumulation of ROS in a cancer-specific manner.

  6. Growth Hormone Mediates Its Protective Effect in Hepatic Apoptosis through Hnf6

    OpenAIRE

    Wang, Kewei; Wang, Minhua; Gannon, Maureen; Holterman, AiXuan

    2016-01-01

    Background and Aims Growth hormone (GH) not only supports hepatic metabolism but also protects against hepatocyte cell death. Hnf6 (or Oc1) belonging to the Onecut family of hepatocyte transcription factors known to regulate differentiated hepatic function, is a GH-responsive gene. We evaluate if GH mediates Hnf6 activity to attenuate hepatic apoptotic injury. Methods We used an animal model of hepatic apoptosis by bile duct ligation (BDL) with Hnf6 -/- (KO) mice in which hepatic Hnf6 was con...

  7. Role of NgR mediated oxidative stress in glucose induced RGC apoptosis

    Directory of Open Access Journals (Sweden)

    Yu-Bo Wang

    2017-02-01

    Full Text Available AIM: To investigate the effect of NgR mediated oxidative stress on the apoptosis of retinal ganglion cells(RGCsinduced by glucose. METHODS: RGC-5 cell were divided into 3 groups: control group(DMEM high glucose medium+10% fetal calf serum, high glucose group(DMEM high glucose medium+10% fetal calf serum+30mmol/L glucose,NEP1-40 group(DMEM high glucose medium+10% fetal calf serum+30mmol/L glucose+1μmol/L NEP1-40. Detections were performed after 3d culture: the state of cell growth was observed by microscopy. Cell viability was detected by CCK-8 kit. The apoptosis rate of RGC cells was detected by flow cytometry(FCM. The intensity of ROS of the cells were detected by fluorescence microscopy. Intracellular MDA levels and SOD activity were measured by related kits. Western blot was used to detect the expressions of Bcl-2 and Bax proteins. RESULTS: Compared with control group, high glucose group had a poor state and cell viability decreased, cell apoptosis rate significantly increased, ROS and MDA levels were significantly enhanced, SOD activity decreased, and the expression of anti-apoptotic protein Bcl-2 was decreased and the expression of pro apoptotic protein Bax was up-regulated. Compared with glucose group, after NgR expression was inhibited by NEP1-40, the oxidative stress reaction was reduced, Bcl-2/Bax was increased, the cell status was improved, the cell viability was increased, and the apoptosis rate was decreased in the NEP1-40 group(PCONCLUSION: High concentration of glucose can induce apoptosis of RGC-5 cells by NgR mediated oxidative stress reaction.

  8. Copper oxide nanoparticles induced mitochondria mediated apoptosis in human hepatocarcinoma cells.

    Science.gov (United States)

    Siddiqui, Maqsood A; Alhadlaq, Hisham A; Ahmad, Javed; Al-Khedhairy, Abdulaziz A; Musarrat, Javed; Ahamed, Maqusood

    2013-01-01

    Copper oxide nanoparticles (CuO NPs) are heavily utilized in semiconductor devices, gas sensor, batteries, solar energy converter, microelectronics and heat transfer fluids. It has been reported that liver is one of the target organs for nanoparticles after they gain entry into the body through any of the possible routes. Recent studies have shown cytotoxic response of CuO NPs in liver cells. However, the underlying mechanism of apoptosis in liver cells due to CuO NPs exposure is largely lacking. We explored the possible mechanisms of apoptosis induced by CuO NPs in human hepatocellular carcinoma HepG2 cells. Prepared CuO NPs were spherical in shape with a smooth surface and had an average diameter of 22 nm. CuO NPs (concentration range 2-50 µg/ml) were found to induce cytotoxicity in HepG2 cells in dose-dependent manner, which was likely to be mediated through reactive oxygen species generation and oxidative stress. Tumor suppressor gene p53 and apoptotic gene caspase-3 were up-regulated due to CuO NPs exposure. Decrease in mitochondrial membrane potential with a concomitant increase in the gene expression of bax/bcl2 ratio suggested that mitochondria mediated pathway involved in CuO NPs induced apoptosis. This study has provided valuable insights into the possible mechanism of apoptosis caused by CuO NPs at in vitro level. Underlying mechanism(s) of apoptosis due to CuO NPs exposure should be further invested at in vivo level.

  9. Copper oxide nanoparticles induced mitochondria mediated apoptosis in human hepatocarcinoma cells.

    Directory of Open Access Journals (Sweden)

    Maqsood A Siddiqui

    Full Text Available Copper oxide nanoparticles (CuO NPs are heavily utilized in semiconductor devices, gas sensor, batteries, solar energy converter, microelectronics and heat transfer fluids. It has been reported that liver is one of the target organs for nanoparticles after they gain entry into the body through any of the possible routes. Recent studies have shown cytotoxic response of CuO NPs in liver cells. However, the underlying mechanism of apoptosis in liver cells due to CuO NPs exposure is largely lacking. We explored the possible mechanisms of apoptosis induced by CuO NPs in human hepatocellular carcinoma HepG2 cells. Prepared CuO NPs were spherical in shape with a smooth surface and had an average diameter of 22 nm. CuO NPs (concentration range 2-50 µg/ml were found to induce cytotoxicity in HepG2 cells in dose-dependent manner, which was likely to be mediated through reactive oxygen species generation and oxidative stress. Tumor suppressor gene p53 and apoptotic gene caspase-3 were up-regulated due to CuO NPs exposure. Decrease in mitochondrial membrane potential with a concomitant increase in the gene expression of bax/bcl2 ratio suggested that mitochondria mediated pathway involved in CuO NPs induced apoptosis. This study has provided valuable insights into the possible mechanism of apoptosis caused by CuO NPs at in vitro level. Underlying mechanism(s of apoptosis due to CuO NPs exposure should be further invested at in vivo level.

  10. Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-09-01

    Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity as well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease. -- Highlights: ► Chlorpyrifos induces apoptosis. ► Chlorpyrifos inhibits mitochondrial complex I activity. ► ROS is involved in chlorpyrifos-induced apoptosis. ► Chlorpyrifos affects cellular antioxidant systems. ► Chlorpyrifos-induced apoptosis mediates activation of MAPK.

  11. Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

    International Nuclear Information System (INIS)

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

    2012-01-01

    Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity as well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease. -- Highlights: ► Chlorpyrifos induces apoptosis. ► Chlorpyrifos inhibits mitochondrial complex I activity. ► ROS is involved in chlorpyrifos-induced apoptosis. ► Chlorpyrifos affects cellular antioxidant systems. ► Chlorpyrifos-induced apoptosis mediates activation of MAPK.

  12. Helichrysetin Induces DNA Damage that Triggers JNK-Mediated Apoptosis in Ca Ski Cells.

    Science.gov (United States)

    Fong, Ho Yen; Abd Malek, Sri Nurestri; Yee, Hui Shin; Karsani, Saiful Anuar

    2017-01-01

    Cervical cancer has become one of the most common cancers in women and currently available treatment options for cervical cancer are very limited. Naturally occurring chalcones and its derivatives have been studied extensively as a potential anticancer agent in different types of cancer and helichrysetin is naturally occurring chalcone that possess potent antiproliferative activity toward human cancer cells. Inhibitory activity of helichrysetin was evaluated at different concentrations. Ability of helichrysetin to induce apoptosis and its relation with c-Jun N-terminal kinase (JNK)-mediated mechanism of apoptosis was assessed using flow cytometry and Western blotting. Helichrysetin inhibited Ca Ski cells at half maximal inhibitory concentration 30.62 ± 0.38 μM. This compound has the ability to induce DNA damage, mitochondrial membrane disruption, and loss of cell membrane integrity. We have shown that apoptosis was induced through the activation of JNK-mediated apoptosis by DNA damage in the cells then triggering p53-downstream apoptotic pathway with increased expression of pro-apoptotic proteins, Bax and caspase 3, and suppression of Bcl-2 anti-apoptotic protein. DNA damage in the cells also caused phosphorylation of protein ataxia-telangiectasia mutated, an activator of DNA damage response. We conclude that helichrysetin can inhibit Ca Ski cells through DNA damage-induced JNK-mediated apoptotic pathway highlighting the potential of this compound as anticancer agent for cervical cancer. Helichrysetin induced DNA damage in Ca Ski cellsDNA damage caused JNK-mediated phosphorylation of p53 resulting in p53-mediated apoptosisHelichrysetin is a potential DNA damage inducing agent through JNK activation to kill human cervical carcinoma cells. Abbreviations used: ATM: Ataxia-telangiectasia mutated, DAPI: 4',6-diamidino-2-phenylindole, DMSO: Dimethyl sulfoxide, FITC: Fluorescein isothiocyanate, IC 50 : Half maximal inhibitory concentration, JC1-5,5',6,6'-Tetrachloro: 1

  13. Par-4-mediated recruitment of Amida to the actin cytoskeleton leads to the induction of apoptosis

    International Nuclear Information System (INIS)

    Boosen, Meike; Vetterkind, Susanne; Koplin, Ansgar; Illenberger, Susanne; Preuss, Ute

    2005-01-01

    Par-4 (prostate apoptosis response-4) sensitizes cells to apoptotic stimuli, but the exact mechanisms are still poorly understood. Using Par-4 as bait in a yeast two-hybrid screen, we identified Amida as a novel interaction partner, a ubiquitously expressed protein which has been suggested to be involved in apoptotic processes. Complex formation of Par-4 and Amida occurs in vitro and in vivo and is mediated via the C-termini of both proteins, involving the leucine zipper of Par-4. Amida resides mainly in the nucleus but displays nucleo-cytoplasmic shuttling in heterokaryons. Upon coexpression with Par-4 in REF52.2 cells, Amida translocates to the cytoplasm and is recruited to actin filaments by Par-4, resulting in enhanced induction of apoptosis. The synergistic effect of Amida/Par-4 complexes on the induction of apoptosis is abrogated when either Amida/Par-4 complex formation or association of these complexes with the actin cytoskeleton is impaired, indicating that the Par-4-mediated relocation of Amida to the actin cytoskeleton is crucial for the pro-apoptotic function of Par-4/Amida complexes in REF52.2 cells. The latter results in enhanced phosphorylation of the regulatory light chain of myosin II (MLC) as has previously been shown for Par-4-mediated recruitment of DAP-like kinase (Dlk), suggesting that the recruitment of nuclear proteins involved in the regulation of apoptotic processes to the actin filament system by Par-4 represents a potent mechanism how Par-4 can trigger apoptosis

  14. 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.

  15. BH3-only protein BIM mediates heat shock-induced apoptosis.

    Science.gov (United States)

    Mahajan, Indra M; Chen, Miao-Der; Muro, Israel; Robertson, John D; Wright, Casey W; Bratton, Shawn B

    2014-01-01

    Acute heat shock can induce apoptosis through a canonical pathway involving the upstream activation of caspase-2, followed by BID cleavage and stimulation of the intrinsic pathway. Herein, we report that the BH3-only protein BIM, rather than BID, is essential to heat shock-induced cell death. We observed that BIM-deficient cells were highly resistant to heat shock, exhibiting short and long-term survival equivalent to Bax(-/-)Bak(-/-) cells and better than either Bid(-/-) or dominant-negative caspase-9-expressing cells. Only Bim(-/-) and Bax(-/-)Bak(-/-) cells exhibited resistance to mitochondrial outer membrane permeabilization and loss of mitochondrial inner membrane potential. Moreover, while dimerized caspase-2 failed to induce apoptosis in Bid(-/-) cells, it readily did so in Bim(-/-) cells, implying that caspase-2 kills exclusively through BID, not BIM. Finally, BIM reportedly associates with MCL-1 following heat shock, and Mcl-1(-/-) cells were indeed sensitized to heat shock-induced apoptosis. However, pharmacological inhibition of BCL-2 and BCL-X(L) with ABT-737 also sensitized cells to heat shock, most likely through liberation of BIM. Thus, BIM mediates heat shock-induced apoptosis through a BAX/BAK-dependent pathway that is antagonized by antiapoptotic BCL-2 family members.

  16. Sensitization of cancer cells to cyclophosphamide therapy by an organoselenium compound through ROS-mediated apoptosis.

    Science.gov (United States)

    Chakraborty, Pramita; Roy, Somnath Singha; Basu, Abhishek; Bhattacharya, Sudin

    2016-12-01

    Induction of apoptosis has been recognized as an excellent therapeutic approach in cancer. Selenium based compounds are well known for their antitumor and synergistic chemotherapeutic efficacy when combined with a standard antineoplastic drug. Previously, we have reported that an organoselenium compound, diphenylmethyl selenocyanate (DMSE) could effectively protect normal organs and tissues from the toxicity induced by a standard chemotherapeutic drug cyclophosphamide in a tumor bearing mouse model. In this study, as a further step, we have evaluated the effect of DMSE in sensitization of tumor cells to cyclophosphamide-induced cell death. We found that DMSE alone or in combination with cyclophosphamide could induce cell death mainly through apoptosis. Generation of reactive oxygen species (ROS) followed by down-regulation of antioxidant defense system in the tumor cells was hypothesized as the crucial cellular events occurred following DMSE treatment. In addition, DMSE in combination with cyclophosphamide also caused DNA damage in tumor cells which might be due to the consequence of oxidative stress induced by the combined therapy. Moreover, production of ROS subsequently activated p53, which in turn initiated release of mitochondrial cytochrome c via up-regulation of Bax and down-regulation of Bcl-2. Ultimately, the activation of caspase-3 played the major role to cleave PARP that finally led to apoptosis. All the above results together proposed that, DMSE sensitized tumor cells to cyclophosphamide therapy through ROS-induced p53 activation and mitochondria-mediated caspase dependent apoptosis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  17. Nuclear thioredoxin-1 is required to suppress cisplatin-mediated apoptosis of MCF-7 cells

    International Nuclear Information System (INIS)

    Chen, Xiao-Ping; Liu, Shou; Tang, Wen-Xin; Chen, Zheng-Wang

    2007-01-01

    Different cell line with increased thioredoxin-1 (Trx-1) showed a decreased or increased sensitivity to cell killing by cisplatin. Recently, several studies found that the subcellular localization of Trx-1 is closely associated with its functions. In this study, we explored the association of the nuclear Trx-1 with the cisplatin-mediated apoptosis of breast cancer cells MCF-7. Firstly, we found that higher total Trx-1 accompanied by no change of nuclear Trx-1 can not influence apoptosis induced by cisplatin in MCF-7 cells transferred with Trx-1 cDNA. Secondly, higher nuclear Trx-1 accompanied by no change of total Trx-1 can protect cells from apoptosis induced by cisplatin. Thirdly, high nuclear Trx-1 involves in the cisplatin-resistance in cisplatin-resistive cells. Meanwhile, we found that the mRNA level of p53 is closely correlated with the level of nuclear Trx-1. In summary, we concluded that the nuclear Trx-1 is required to resist apoptosis of MCF-7 cells induced by cisplatin, probably through up-regulating the anti-apoptotic gene, p53

  18. Nano Copper Induces Apoptosis in PK-15 Cells via a Mitochondria-Mediated Pathway.

    Science.gov (United States)

    Zhang, Hui; Chang, Zhenyu; Mehmood, Khalid; Abbas, Rao Zahid; Nabi, Fazul; Rehman, Mujeeb Ur; Wu, Xiaoxing; Tian, Xinxin; Yuan, Xiaodan; Li, Zhaoyang; Zhou, Donghai

    2018-01-01

    Nano-sized copper particles are widely used in various chemical, physical, and biological fields. However, earlier studies have shown that nano copper particles (40-100 μg/mL) can induce cell toxicity and apoptosis. Therefore, this study was conducted to investigate the role of nano copper in mitochondrion-mediated apoptosis in PK-15 cells. The cells were treated with different doses of nano copper (20, 40, 60, and 80 μg/mL) to determine the effects of apoptosis using acridine orange/ethidium bromide (AO/EB) fluorescence staining and a flow cytometry assay. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in the PK-15 cells were examined using commercially available kits. Moreover, the mRNA levels of the Bax, Bid, Caspase-3, and CYCS genes were assessed by real-time PCR. The results revealed that nano copper exposure induced apoptosis and changed the mitochondrial membrane potential. In addition, nano copper significantly altered the levels of the Bax, Bid, Caspase-3, and CYCS genes at a concentration of 40 μg/mL. To summarize, nano copper significantly (P nano copper can play an important role in inducing the apoptotic pathway in PK-15 cells, which may be the mechanism by which nano copper induces nephrotoxicity.

  19. Verapamil inhibits L-type calcium channel mediated apoptosis in human colon cancer cells.

    Science.gov (United States)

    Zawadzki, Antoni; Liu, Qing; Wang, Yusheng; Melander, Arne; Jeppsson, Bengt; Thorlacius, Henrik

    2008-11-01

    Treatment with calcium channel blockers have been associated with increased colon cancer mortality in epidemiologic studies. We examined the potential expression and function of calcium channels in two human colon cancer cell lines. Both primary (collected at operation) and commercially-available human colon cancer cell lines were used. The colon cancer cells were incubated with a calcium channel blocker (verapamil) and a calcium channel agonist (BayK 8644) at clinically relevant concentrations. L-type calcium channel mRNA was determined by reverse-transcription polymerase chain reaction. Intracellular calcium ion levels were measured with fluorometry and apoptosis with flow cytometry. Both types of cells expressed L-type calcium channel mRNA, comprising an alpha-1D and a beta-3 subunit, whereas the cells were negative for N-type and P-type channels. The selective calcium channel agonist (BayK 8644), dose-dependently increased intracellular calcium ion levels and the level of apoptosis in primary human colon cancer cells. Pretreatment with verapamil completely abolished both calcium channel agonist-induced influx of calcium and apoptosis in these cells. These data demonstrate that human colon cancer cells express L-type calcium channels that mediate calcium influx and apoptosis, which warrants further studies to determine whether calcium channel blockers may promote colon cancer growth.

  20. Chalcones and Dihydrochalcones Augment TRAIL-Mediated Apoptosis in Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Wojciech Krol

    2010-08-01

    Full Text Available Chalcones and dihydrochalcones exhibit chemopreventive and antitumor activity. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand is a natural endogenous anticancer agent. We examined the cytotoxic and apoptotic effect of chalcones and dihydrochalcones on TRAIL-mediated apoptosis in LNCaP prostate cancer cells. The cytotoxicity was evaluated by the MTT and LDH assays. The apoptosis was detected using annexin V-FITC by flow cytometry and fluorescence microscopy. The ΔΨm was evaluated using DePsipher staining by fluorescence microscopy. Our study showed that two tested chalcones (chalcone and 2’,6’dihydroxy-4’-methoxychalcone and three dihydrochalcones (2’,6’-dihydroxy-4’4-dimethoxydihydrochalcone, 2’,6’-dihydroxy-4’-methoxydihydro- chalcone,  and 2’,4’,6’-trihydroxydihydrochalcone, called phloretin markedly augmented TRAIL-induced apoptosis and cytotoxicity in LNCaP cells and confirmed the significant role of chalcones in chemoprevention of prostate cancer.

  1. Clitocine potentiates TRAIL-mediated apoptosis in human colon cancer cells by promoting Mcl-1 degradation.

    Science.gov (United States)

    Sun, Jian-Guo; Ruan, Feng; Zeng, Xue-Li; Xiang, Jun; Li, Xia; Wu, Ping; Fung, Kwok Pui; Liu, Fei-Yan

    2016-10-01

    Among anti-cancer candidate drugs, TRAIL might be the most specific agent against cancer cells due to its low toxicity to normal cells. Unfortunately, cancer cells usually develop drug resistance to TRAIL, which is a major obstacle for its clinical application. One promising strategy is co-administrating with sensitizer to overcome cancer cells resistance to TRAIL. Clitocine, a natural amino nucleoside purified from wild mushroom, is recently demonstrated that can induce apoptosis in multidrug-resistant human cancer cells by targeting Mcl-1. In the present study,we found that pretreatment with clitocine dramatically enhances TRAIL lethality in its resistant human colon cancer cells by inducing apoptosis. More importantly, combination of clitocine and TRAIL also effectively inhibits xenograft growth and induces tumor cells apoptosis in athymic mice. The disruption of the binding between Mcl-1 and Bak as well as mitochondrial translocation of Bax mediated by clitocine are identified as the key underlying mechanisms, which leading to mitochondrial membrane permeabilization. Enforced exogenous Mcl-1 can effectively attenuate clitocine/TRAIL-induced apoptosis by suppressing the activation of intrinsic apoptotic pathway. Furthermore, clitocine regulates Mcl-1 expression at the posttranslational level as no obvious change is observed on mRNA level and proteasome inhibitor MG132 almost blocks the Mcl-1 suppression by clitocine. In fact, more ubiquitinated Mcl-1 was detected under clitocine treatment. Our findings indicate that clitocine is potentially an effective adjuvant agent in TRAIL-based cancer therapy.

  2. Modulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by Helicobacter pylori in immune pathogenesis of gastric mucosal damage.

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    Tsai, Hwei-Fang; Hsu, Ping-Ning

    2017-02-01

    Helicobacter pylori infection is associated with chronic gastritis, peptic ulcer, gastric carcinoma, and gastric mucosa-associated lymphoid tissue lymphomas. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Enhanced gastric epithelial cell apoptosis during H. pylori infection was suggested to play an important role in the pathogenesis of chronic gastritis and gastric pathology. In addition to directly triggering apoptosis, H. pylori induces sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in gastric epithelial cells. Human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death-receptor signaling. The induction of TRAIL sensitivity by H. pylori is dependent upon the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex through downregulation of cellular FLICE-inhibitory protein. Moreover, H. pylori infection induces infiltration of T lymphocytes and triggers inflammation to augment apoptosis. In H. pylori infection, significant increases in CCR6 + CD3 + T cell infiltration in the gastric mucosa was observed, and the CCR6 ligand, CCL20 chemokine, was selectively expressed in inflamed gastric tissues. These mechanisms initiate chemokine-mediated T lymphocyte trafficking into inflamed epithelium and induce mucosal injury during Helicobacter infection. This article will review recent findings on the interactions of H. pylori with host-epithelial signaling pathways and events involved in the initiation of gastric pathology, including gastric inflammation and mucosal damage. Copyright © 2016. Published by Elsevier B.V.

  3. PAR-1 mediated apoptosis of breast cancer cells by V. cholerae hemagglutinin protease.

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    Ray, Tanusree; Pal, Amit

    2016-05-01

    Bacterial toxins have emerged as promising agents in cancer treatment strategy. Hemagglutinin (HAP) protease secreted by Vibrio cholerae induced apoptosis in breast cancer cells and regresses tumor growth in mice model. The success of novel cancer therapies depends on their selectivity for cancer cells with limited toxicity for normal tissues. Increased expression of Protease Activated Receptor-1 (PAR-1) has been reported in different malignant cells. In this study we report that HAP induced activation and over expression of PAR-1 in breast cancer cells (EAC). Immunoprecipitation studies have shown that HAP specifically binds with PAR-1. HAP mediated activation of PAR-1 caused nuclear translocation of p50-p65 and the phosphorylation of p38 which triggered the activation of NFκB and MAP kinase signaling pathways. These signaling pathways enhanced the cellular ROS level in malignant cells that induced the intrinsic pathway of cell apoptosis. PAR-1 mediated apoptosis by HAP of malignant breast cells without effecting normal healthy cells in the same environment makes it a good therapeutic agent for treatment of cancer.

  4. HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis.

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    Deng, Lin; Chen, Ming; Tanaka, Motofumi; Ku, Yonson; Itoh, Tomoo; Shoji, Ikuo; Hotta, Hak

    2015-09-01

    We previously reported that hepatitis C virus (HCV) infection induces Bax-triggered, mitochondrion-mediated apoptosis by using the HCV J6/JFH1 strain and Huh-7.5 cells. However, it was still unclear how HCV-induced Bax activation. In this study, we showed that the HCV-induced activation and mitochondrial accumulation of Bax were significantly attenuated by treatment with a general antioxidant, N-acetyl cysteine (NAC), or a specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, with the result suggesting that the reactive oxygen species (ROS)/JNK signalling pathway is upstream of Bax activation in HCV-induced apoptosis. We also demonstrated that HCV infection transcriptionally activated the gene for the pro-apoptotic protein Bim and the protein expression of three major splice variants of Bim (BimEL, BimL and BimS). The HCV-induced increase in the Bim mRNA and protein levels was significantly counteracted by treatment with NAC or SP600125, suggesting that the ROS/JNK signalling pathway is involved in Bim upregulation. Moreover, HCV infection led to a marked accumulation of Bim on the mitochondria to facilitate its interaction with Bax. On the other hand, downregulation of Bim by siRNA (small interfering RNA) significantly prevented HCV-mediated activation of Bax and caspase 3. Taken together, these observations suggest that HCV-induced ROS/JNK signalling transcriptionally activates Bim expression, which leads to Bax activation and apoptosis induction.

  5. Sevoflurane induces endoplasmic reticulum stress mediated apoptosis in hippocampal neurons of aging rats.

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    Gang Chen

    Full Text Available Elderly patients are more likely to suffer from postoperative memory impairment for volatile anesthetics could induce aging neurons degeneration and apoptosis while the mechanism was still elusive. Therefore we hypothesized that ER stress mediated hippocampal neurons apoptosis might play an important role in the mechanism of sevoflurane-induced cognitive impairment in aged rats. Thirty 18-month-old male Sprague-Dawley rats were divided into two groups: the sham anesthesia group (exposure to simply humidified 30-50% O2 balanced by N2 in an acrylic anesthetizing chamber for 5 hours and the sevoflurane anesthesia group (received 2% sevoflurane in the same humidified mixed air in an identical chamber for the same time. Spatial memory of rats was assayed by the Morris water maze test. The ultrastructure of the hippocampus was observed by transmission electron microscopy (TEM. The expressions of C/EBP homologous protein (CHOP and caspase-12 in the hippocampus were observed by immunohistochemistry and real-time PCR analysis. The apoptosis neurons were also assessed by TUNEL assay. The Morris water maze test showed that sevoflurane anesthesia induced spatial memory impairment in aging rats (P<0.05. The apoptotic neurons were condensed and had clumped chromatin with fragmentation of the nuclear membrane, verifying apoptotic degeneration in the sevoflurane group rats by TEM observation. The expressions of CHOP and caspase-12 increased, and the number of TUNEL positive cells of the hippocampus also increased in the sevoflurane group rats (P<0.05. The present results suggested that the long time exposure of sevoflurane could induce neuronal degeneration and cognitive impairment in aging rats. The ER stress mediated neurons apoptosis may play a role in the sevoflurane-induced memory impairment in aging rats.

  6. Proteomics informed by transcriptomics reveals Hendra virus sensitizes bat cells to TRAIL-mediated apoptosis.

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    Wynne, James W; Shiell, Brian J; Marsh, Glenn A; Boyd, Victoria; Harper, Jennifer A; Heesom, Kate; Monaghan, Paul; Zhou, Peng; Payne, Jean; Klein, Reuben; Todd, Shawn; Mok, Lawrence; Green, Diane; Bingham, John; Tachedjian, Mary; Baker, Michelle L; Matthews, David; Wang, Lin-Fa

    2014-01-01

    Bats are a major reservoir of emerging infectious viruses. Many of these viruses are highly pathogenic to humans however bats remain asymptomatic. The mechanism by which bats control viral replication is unknown. Here we utilize an integrated approach of proteomics informed by transcriptomics to compare the response of immortalized bat and human cells following infection with the highly pathogenic bat-borne Hendra virus (HeV). The host response between the cell lines was significantly different at both the mRNA and protein levels. Human cells demonstrated minimal response eight hours post infection, followed by a global suppression of mRNA and protein abundance. Bat cells demonstrated a robust immune response eight hours post infection, which led to the up-regulation of apoptosis pathways, mediated through the tumor necrosis factor-related apoptosis inducing ligand (TRAIL). HeV sensitized bat cells to TRAIL-mediated apoptosis, by up-regulating death receptor transcripts. At 48 and 72 hours post infection, bat cells demonstrated a significant increase in apoptotic cell death. This is the first study to comprehensively compare the response of bat and human cells to a highly pathogenic zoonotic virus. An early induction of innate immune processes followed by apoptosis of virally infected bat cells highlights the possible involvement of programmed cell death in the host response. Our study shows for the first time a side-by-side high-throughput analysis of a dangerous zoonotic virus in cell lines derived from humans and the natural bat host. This enables a way to search for divergent mechanisms at a molecular level that may influence host pathogenesis.

  7. The p53-induced Siva-1 plays a significant role in cisplatin-mediated apoptosis

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    Barkinge John

    2009-01-01

    Full Text Available Background: The pro-apoptotic protein Siva-1 functions in both extrinsic and intrinsic cell death signaling; however, the exact contribution of the endogenous Siva-1 to DNA damage-induced apoptosis is unclear. Using cisplatin, a chemotherapeutic drug, to induce DNA damage and cell death, we determined the role of Siva-1. Methods: Cisplatin treated HCT116 colorectal carcinoma cells (p53+/+ and -/- were used in the study. With the help of recombinant lentivirus that can express siSiva (siRNA that specifically targets Siva-1, we also generated Siva-1 knockdown HCT116 cells. Apoptosis was determined by tetramethyl rhodamine methyl ester (TMRM staining and propidium iodide (PI staining. Results: Treatment with cisplatin induced Siva-1 expression in a p53 dependent manner. In Siva-1 knockdown p53+/+ HCT116 colorectal carcinoma cells, loss of Siva-1 expression conferred significant resistance to cisplatin-induced apoptosis. Although Siva-1 levels were positively regulated by p53, Siva-1-induced apoptosis did not require p53. Despite the fact that Siva-1 lacks even a minimal BH3 domain, similar to other proapoptotic Bcl2 family members induced by p53, we showed that Siva-1 mediated apoptosis is characterized by Bax oligomerization and cytochrome c leakage from mitochondria. The putative amphipathic helical region in Siva-1 (SAH appeared to function analogously to a BH3 domain. Conclusion: The p53 induced Siva-1 is one of the effector molecules, which plays a significant role in DNA damage-induced cell death.

  8. 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.

  9. Vacuole-mitochondrial cross-talk during apoptosis in yeast: a model for understanding lysosome-mitochondria-mediated apoptosis in mammals.

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    Sousa, Maria João; Azevedo, Flávio; Azevedo, Flávìa; Pedras, Andreia; Pedras, Ana; Marques, Carolina; Coutinho, Olga P; Preto, Ana; Gerós, Hernâni; Chaves, Susana R; Côrte-Real, Manuela

    2011-10-01

    The yeast apoptosis field emerged with the finding that key components of the apoptotic machinery are conserved in these simple eukaryotes. Thus it became possible to exploit these genetically tractable organisms to improve our understanding of the intricate mechanisms of cell death in higher eukaryotes and of severe human diseases associated with apoptosis dysfunctions. Early on, it was recognized that a mitochondria-mediated apoptotic pathway showing similarities to the mammalian intrinsic pathway was conserved in yeast. Recently, lysosomes have also emerged as central players in mammalian apoptosis. Following LMP (lysosomal membrane permeabilization), lysosomal proteases such as cathepsins B, D and L are released into the cytosol and can trigger a mitochondrial apoptotic cascade. CatD (cathepsin D) can also have anti-apoptotic effects in some cellular types and specific contexts. Nonetheless, the mechanisms underlying LMP and the specific role of cathepsins after their release into the cytosol remain poorly understood. We have recently shown that yeast vacuoles, membrane-bound acidic organelles, which share many similarities to plant vacuoles and mammalian lysosomes, are also involved in the regulation of apoptosis and that the vacuolar protease Pep4p, orthologue of the human CatD, is released from the vacuole into the cytosol in response to acetic acid. Here, we discuss how the conservation of cell-death regulation mechanisms in yeast by the lysosome-like organelle and mitochondria may provide new insights into the understanding of the complex interplay between the mitochondria and lysosome-mediated signalling routes during mammalian apoptosis.

  10. Multiple metabolic hits converge on CD36 as novel mediator of tubular epithelial apoptosis in diabetic nephropathy.

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    Katalin Susztak

    2005-02-01

    Full Text Available Diabetic nephropathy (DNP is a common complication of type 1 and type 2 diabetes mellitus and the most common cause of kidney failure. While DNP manifests with albuminuria and diabetic glomerulopathy, its progression correlates best with tubular epithelial degeneration (TED and interstitial fibrosis. However, mechanisms leading to TED in DNP remain poorly understood.We found that expression of scavenger receptor CD36 coincided with proximal tubular epithelial cell (PTEC apoptosis and TED specifically in human DNP. High glucose stimulated cell surface expression of CD36 in PTECs. CD36 expression was necessary and sufficient to mediate PTEC apoptosis induced by glycated albumins (AGE-BSA and CML-BSA and free fatty acid palmitate through sequential activation of src kinase, and proapoptotic p38 MAPK and caspase 3. In contrast, paucity of expression of CD36 in PTECs in diabetic mice with diabetic glomerulopathy was associated with normal tubular epithelium and the absence of tubular apoptosis. Mouse PTECs lacked CD36 and were resistant to AGE-BSA-induced apoptosis. Recombinant expression of CD36 in mouse PTECs conferred susceptibility to AGE-BSA-induced apoptosis.Our findings suggest a novel role for CD36 as an essential mediator of proximal tubular apoptosis in human DNP. Because CD36 expression was induced by glucose in PTECs, and because increased CD36 mediated AGE-BSA-, CML-BSA-, and palmitate-induced PTEC apoptosis, we propose a two-step metabolic hit model for TED, a hallmark of progression in DNP.

  11. In vitro effects of Panax ginseng in aristolochic acid-mediated renal tubulotoxicity: apoptosis versus regeneration.

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    Bunel, Valérian; Antoine, Marie-Hélène; Nortier, Joëlle; Duez, Pierre; Stévigny, Caroline

    2015-03-01

    This in vitro study aimed to determine the effects of a Panax ginseng extract on aristolochic acid-mediated toxicity in HK-2 cells. A methanolic extract of ginseng (50 µg/mL) was able to reduce cell survival after treatment with 50 µM aristolochic acid for 24, 48, and 72 h, as evidenced by a resazurin reduction assay. This result was confirmed by a flow cytometric evaluation of apoptosis using annexin V-PI staining, and indicated higher apoptosis rates in cells treated with aristolochic acid and P. ginseng extract compared with aristolochic acid alone. However, P. ginseng extract by itself (5 and 50 µg/mL) increased the Ki-67 index, indicating an enhancement in cellular proliferation. Cell cycle analysis excluded a P. ginseng extract-mediated induction of G2/M cell cycle arrest such as the one typically observed with aristolochic acid. Finally, β-catenin acquisition was found to be accelerated when cells were treated with both doses of ginseng, suggesting that the epithelial phenotype of renal proximal tubular epithelial cells was maintained. Also, ginseng treatment (5 and 50 µg/mL) reduced the oxidative stress activity induced by aristolochic acid after 24 and 48 h. These results indicate that the ginseng extract has a protective activity towards the generation of cytotoxic reactive oxygen species induced by aristolochic acid. However, the ginseng-mediated alleviation of oxidative stress did not correlate with a decrease but rather with an increase in aristolochic acid-induced apoptosis and death. This deleterious herb-herb interaction could worsen aristolochic acid tubulotoxicity and reinforce the severity and duration of the injury. Nevertheless, increased cellular proliferation and migration, along with the improvement in the epithelial phenotype maintenance, indicate that ginseng could be useful for improving tubular regeneration and the recovery following drug-induced kidney injury. Such dual activities of ginseng certainly warrant further in vivo

  12. Apoptosis-mediated inhibition of human breast cancer cell proliferation by lemon citrus extract.

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    Alshatwi, Ali A; Shafi, Gowhar; Hasan, Tarique N; Al-Hazzani, Amal A; Alsaif, Mohammed A; Alfawaz, Mohammed A; Lei, K Y; Munshi, Anjana

    2011-01-01

    Dietary phytochemicals have a variety of antitumor properties. In the present study, the antitumor activity of methanolic extract of lemon fruit (lemon extract; LE) (LE) on the MCF-7 breast cancer cell line was investigated in vitro. Apoptotic cell death was analyzed using the TUNEL assay. In addition, the apoptosis mediated by LE extract in the MCF-7 cells was associated with the increased expression of the tumor suppressor p53 and caspase-3. Additionally, the expression of a pro-apoptotic gene, bax, was increased, and the expression of an anti-apoptotic gene, bcl-2, was decreased by LE extract treatment, resulting in a shift in the Bax:Bcl-2 ratio to one that favored apoptosis. The expression of a major apoptotic gene, caspase-3, was increased by LE extract treatment. In light of the above results, we concluded that LE extract can induce the apoptosis of MCF-7 breast cancer cells via Bax-related caspase-3 activation. This study provides experimental data that are relevant to the possible future clinical use of LE to treat breast cancer.

  13. Heat shock protein 70-mediated sensitization of cells to apoptosis by Carboxyl-Terminal Modulator Protein

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    Sack Ragna

    2009-07-01

    Full Text Available Abstract Background The serine/threonine protein kinase B (PKB/Akt is involved in insulin signaling, cellular survival, and transformation. Carboxyl-terminal modulator protein (CTMP has been identified as a novel PKB binding partner in a yeast two-hybrid screen, and appears to be a negative PKB regulator with tumor suppressor-like properties. In the present study we investigate novel mechanisms by which CTMP plays a role in apoptosis process. Results CTMP is localized to mitochondria. Furthermore, CTMP becomes phosphorylated following the treatment of cells with pervanadate, an insulin-mimetic. Two serine residues (Ser37 and Ser38 were identified as novel in vivo phosphorylation sites of CTMP. Association of CTMP and heat shock protein 70 (Hsp70 inhibits the formation of complexes containing apoptotic protease activating factor 1 and Hsp70. Overexpression of CTMP increased the sensitivity of cells to apoptosis, most likely due to the inhibition of Hsp70 function. Conclusion Our data suggest that phosphorylation on Ser37/Ser38 of CTMP is important for the prevention of mitochondrial localization of CTMP, eventually leading to cell death by binding to Hsp70. In addition to its role in PKB inhibition, CTMP may therefore play a key role in mitochondria-mediated apoptosis by localizing to mitochondria.

  14. Macrophage Akt1 Kinase-Mediated Mitophagy Modulates Apoptosis Resistance and Pulmonary Fibrosis.

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    Larson-Casey, Jennifer L; Deshane, Jessy S; Ryan, Alan J; Thannickal, Victor J; Carter, A Brent

    2016-03-15

    Idiopathic pulmonary fibrosis (IPF) is a devastating lung disorder with increasing incidence. Mitochondrial oxidative stress in alveolar macrophages is directly linked to pulmonary fibrosis. Mitophagy, the selective engulfment of dysfunctional mitochondria by autophagasomes, is important for cellular homeostasis and can be induced by mitochondrial oxidative stress. Here, we show Akt1 induced macrophage mitochondrial reactive oxygen species (ROS) and mitophagy. Mice harboring a conditional deletion of Akt1 in macrophages (Akt1(-/-)Lyz2-cre) and Park2(-/-) mice had impaired mitophagy and reduced active transforming growth factor-β1 (TGF-β1). Although Akt1 increased TGF-β1 expression, mitophagy inhibition in Akt1-overexpressing macrophages abrogated TGF-β1 expression and fibroblast differentiation. Importantly, conditional Akt1(-/-)Lyz2-cre mice and Park2(-/-) mice had increased macrophage apoptosis and were protected from pulmonary fibrosis. Moreover, IPF alveolar macrophages had evidence of increased mitophagy and displayed apoptosis resistance. These observations suggest that Akt1-mediated mitophagy contributes to alveolar macrophage apoptosis resistance and is required for pulmonary fibrosis development. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. FOXO3-mediated up-regulation of Bim contributes to rhein-induced cancer cell apoptosis.

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    Wang, Jiao; Liu, Shu; Yin, Yancun; Li, Mingjin; Wang, Bo; Yang, Li; Jiang, Yangfu

    2015-03-01

    The anthraquinone compound rhein is a natural agent in the traditional Chinese medicine rhubarb. Preclinical studies demonstrate that rhein has anticancer activity. Treatment of a variety of cancer cells with rhein may induce apoptosis. Here, we report that rhein induces atypical unfolded protein response in breast cancer MCF-7 cells and hepatoma HepG2 cells. Rhein induces CHOP expression, eIF2α phosphorylation and caspase cleavage, while it does not induce glucose-regulated protein 78 (GRP78) expression in both MCF-7 and HepG2 cells. Meanwhile, rhein inhibits thapsigargin-induced GRP78 expression and X box-binding protein 1 splicing. In addition, rhein inhibits Akt phosphorylation and stimulates FOXO transactivation activity. Rhein induces Bim expression in MCF-7 and HepG2 cells, which can be abrogated by FOXO3a knockdown. Knockdown of FOXO3a or Bim abrogates rhein-induced caspase cleavage and apoptosis. The chemical chaperone 4-phenylbutyrate acid antagonizes the induction of FOXO activation, Bim expression and caspase cleavage by rhein, indicating that protein misfolding may be involved in triggering these deleterious effects. We conclude that FOXO3a-mediated up-regulation of Bim is a key mechanism underlying rhein-induced cancer cells apoptosis.

  16. Hyperoxia accelerates Fas-mediated signaling and apoptosis in the lungs of Legionella pneumophila pneumonia

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    Tanabe Yoshinari

    2011-04-01

    Full Text Available Abstract Background Oxygen supplementation is commonly given to the patients with severe pneumonia including Legionella disease. Recent data suggested that apoptosis may play an important role, not only in the pathogenesis of Legionella pneumonia, but also in oxygen-induced tissue damage. In the present study, the lethal sensitivity to Legionella pneumonia were compared in the setting of hyperoxia between wild-type and Fas-deficient mice. Findings C57BL/6 mice and B6.MRL-Faslpr mice characterized with Fas-deficiency were used in this study. After intratracheal administration of L. pneumophila, mice were kept in hyperoxic conditions (85-90% O2 conc. in an airtight chamber for 3 days. Bone-marrow derived macrophages infected with L. pneumophila were also kept in hyperoxic conditions. Caspase activity and cytokine production were determined by using commercially available kits. Smaller increases of several apoptosis markers, such as caspase-3 and -8, were demonstrated in Fas-deficient mice, even though the bacterial burdens in Fas-deficient and wild type mice were similar. Bone-marrow derived macrophages from Fas-deficient mice were shown to be more resistant to Legionella-induced cytotoxicity than those from wild-type mice under hyperoxia. Conclusions These results demonstrated that Fas-mediated signaling and apoptosis may be a crucial factor in the pathogenesis of Legionella pneumonia in the setting of hyperoxia.

  17. Pw1/Peg3 is a potential cell death mediator and cooperates with Siah1a in p53-mediated apoptosis

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    Relaix, Frédéric; Wei, Xiao-jun; Li, Wei; Pan, Jianjing; Lin, Yahong; Bowtell, David D.; Sassoon, David A.; Wu, Xiangwei

    2000-01-01

    Induction of wild-type p53 in mouse fibroblasts causes cell cycle arrest at the G1 phase, whereas coexpression of p53 and the protooncogene c-myc induces apoptosis. Although p53 transcriptional activity generally is required for both pathways, the molecular components mediating p53-dependent apoptosis are not well understood. To identify factors that could mediate p53-induced cell death, we used a comparative RNA differential display procedure. We have identified Pw1/Peg3 as a gene product induced during p53/c-myc-mediated apoptosis. Pw1/Peg3 is not induced during p53-mediated G1 growth arrest nor by c-myc alone. Although it is not clear whether the induction of Pw1/Peg3 depends on p53 activity, we show that Pw1/Peg3 interacts with a p53-inducible gene product Siah1a. We demonstrate that coexpression of Pw1/Peg3 with Siah1a induces apoptosis independently of p53 whereas expression of Pw1/Peg3 or Siah1a separately has no effect on cell death. These data suggest that Siah1a and Pw1/Peg3 cooperate in the p53-mediated cell death pathway. Furthermore, we show that inhibiting Pw1/Peg3 activity blocks p53-induced apoptosis. The observation that Pw1/Peg3 is necessary for the p53 apoptotic response suggests a pivotal role for this gene in determining cell death versus survival. PMID:10681424

  18. Tannic Acid Induces Endoplasmic Reticulum Stress-Mediated Apoptosis in Prostate Cancer.

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    Nagesh, Prashanth K B; Hatami, Elham; Chowdhury, Pallabita; Kashyap, Vivek K; Khan, Sheema; Hafeez, Bilal B; Chauhan, Subhash C; Jaggi, Meena; Yallapu, Murali M

    2018-03-07

    Endoplasmic reticulum (ER) stress is an intriguing target with significant clinical importance in chemotherapy. Interference with ER functions can lead to the accumulation of unfolded proteins, as detected by transmembrane sensors that instigate the unfolded protein response (UPR). Therefore, controlling induced UPR via ER stress with natural compounds could be a novel therapeutic strategy for the management of prostate cancer. Tannic acid (a naturally occurring polyphenol) was used to examine the ER stress mediated UPR pathway in prostate cancer cells. Tannic acid treatment inhibited the growth, clonogenic, invasive, and migratory potential of prostate cancer cells. Tannic acid demonstrated activation of ER stress response (Protein kinase R-like endoplasmic reticulum kinase (PERK) and inositol requiring enzyme 1 (IRE1)) and altered its regulatory proteins (ATF4, Bip, and PDI) expression. Tannic acid treatment affirmed upregulation of apoptosis-associated markers (Bak, Bim, cleaved caspase 3, and cleaved PARP), while downregulation of pro-survival proteins (Bcl-2 and Bcl-xL). Tannic acid exhibited elevated G₁ population, due to increase in p18 INK4C and p21 WAF1/CIP1 expression, while cyclin D1 expression was inhibited. Reduction of MMP2 and MMP9, and reinstated E-cadherin signifies the anti-metastatic potential of this compound. Altogether, these results demonstrate that tannic acid can promote apoptosis via the ER stress mediated UPR pathway, indicating a potential candidate for cancer treatment.

  19. Sildenafil (Viagra) sensitizes prostate cancer cells to doxorubicin-mediated apoptosis through CD95

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    Das, Anindita; Durrant, David; Mitchell, Clint; Dent, Paul; Batra, Surinder K.; Kukreja, Rakesh C.

    2016-01-01

    We previously reported that Sildenafil enhances apoptosis and antitumor efficacy of doxorubicin (DOX) while attenuating its cardiotoxic effect in prostate cancer. In the present study, we investigated the mechanism by which sildenafil sensitizes DOX in killing of prostate cancer (PCa) cells, DU145. The death receptor Fas (APO-1 or CD95) induces apoptosis in many carcinoma cells, which is negatively regulated by anti-apoptotic molecules such as FLIP (Fas-associated death domain (FADD) interleukin-1-converting enzyme (FLICE)-like inhibitory protein). Co-treatment of PCa cells with sildenafil and DOX for 48 hours showed reduced expression of both long and short forms of FLIP (FLIP-L and -S) as compared to individual drug treatment. Over-expression of FLIP-s with an adenoviral vector attentuated the enhanced cell-killing effect of DOX and sildenafil. Colony formation assays also confirmed that FLIP-S over-expression inhibited the DOX and sildenafil-induced synergistic killing effect as compared to the cells infected with an empty vector. Moreover, siRNA knock-down of CD95 abolished the effect of sildenafil in enhancing DOX lethality in cells, but had no effect on cell killing after treatment with a single agent. Sildenafil co-treatment with DOX inhibited DOX-induced NF-κB activity by reducing phosphorylation of IκB and nuclear translocation of the p65 subunit, in addition to down regulation of FAP-1 (Fas associated phosphatase-1, a known inhibitor of CD95-mediated apoptosis) expression. This data provides evidence that the CD95 is a key regulator of sildenafil and DOX mediated enhanced cell death in prostate cancer. PMID:26716643

  20. 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

    2016-04-01

    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 pro-apoptotic 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.

  1. Ectopic expression of Bcl-2, but not Bcl-xL rescues Ramos B cells from Fas-mediated apoptosis.

    Science.gov (United States)

    Alam, M K; Davison, S; Siddiqui, N; Norton, J D; Murphy, J J

    1997-12-01

    The human Burkitt lymphoma Ramos B cell line can be induced to undergo apoptosis in response to a variety of different agents, including calcium ionophores, anti-immunoglobulin (Ig) and macromolecular synthesis inhibitors. In addition, following up-regulation of the Fas (CD95) surface receptor by CD40 ligation, these cells also become susceptible to apoptosis induction by Fas ligation. We have previously shown that protection from calcium ionophore- and macromolecular synthesis inhibitor-induced apoptosis by CD40 ligation is associated with a rapid up-regulation of Bcl-xL followed by a more moderate and delayed up-regulation of Bcl-2. We show here that overexpression of Bcl-xL, like Bcl-2, protects Ramos cells from apoptosis induction in response to calcium ionophore, anti-Ig and macromolecular synthesis inhibition. However, in contrast to Bcl-2, ectopic overexpression of Bcl-xL does not rescue from Fas-mediated apoptosis. Thus, in Ramos B cells, the Fas apoptotic pathway exhibits differential sensitivity to inhibition by Bcl-2 family members. These findings also suggest that CD40 signaling provides a switch which renders the cells susceptible to Fas-ligand mediated apoptosis through up-regulation of Fas whilst affording protection from anti-Ig-induced apoptosis through up-regulation of Bcl-xL.

  2. MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Teng, E-mail: tengyu33@yahoo.com [Department of Dermatology, Shandong Ji-ning No. 1 People’s Hospital, Shandong Province 272011 (China); Ji, Jiang [Department of Dermatology, The Second Hospital Affiliated of Soochow University, SuZhou, Jiangsu Province 215000 (China); Guo, Yong-li [Department of Oncology, Shandong Ji-ning No. 1 People’s Hospital, Shandong Province 272011 (China)

    2013-11-08

    Highlights: •Curcumin activates MST1 in melanoma cells. •MST1 mediates curcumin-induced apoptosis of melanoma cells. •ROS production is involved in curcumin-induced MST1 activation. •MST1 mediates curcumin-induced JNK activation in melanoma cells. •MST1 mediates curcumin-induced Foxo3a nuclear translocation and Bim expression. -- Abstract: Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen species (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.

  3. MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

    International Nuclear Information System (INIS)

    Yu, Teng; Ji, Jiang; Guo, Yong-li

    2013-01-01

    Highlights: •Curcumin activates MST1 in melanoma cells. •MST1 mediates curcumin-induced apoptosis of melanoma cells. •ROS production is involved in curcumin-induced MST1 activation. •MST1 mediates curcumin-induced JNK activation in melanoma cells. •MST1 mediates curcumin-induced Foxo3a nuclear translocation and Bim expression. -- Abstract: Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen species (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

  4. Identification of DR5 as a critical, NF-κB-regulated mediator of Smac-induced apoptosis.

    Science.gov (United States)

    Eckhardt, I; Roesler, S; Fulda, S

    2013-11-28

    Smac mimetic promotes apoptosis by neutralizing inhibitor of apoptosis (IAP) proteins and is considered as a promising cancer therapeutic. Although an autocrine/paracrine tumor necrosis factor-α (TNFα) loop has been implicated in Smac mimetic-induced cell death, little is yet known about additional factors that determine sensitivity to Smac mimetic. Using genome-wide gene expression analysis, we identify death receptor 5 (DR5) as a novel key mediator of Smac mimetic-induced apoptosis. Although several cell lines that are sensitive to the Smac mimetic BV6 die in a TNFα-dependent manner, A172 glioblastoma cells undergo BV6-induced apoptosis largely independently of TNFα/TNFR1, as the TNFα-blocking antibody Enbrel or TNFR1 knockdown provide little protection. Yet, BV6-stimulated nuclear factor-κB (NF-κB) activation is critically required for apoptosis, as inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) blocks BV6-induced apoptosis. Unbiased genome-wide gene expression studies in IκBα-SR-overexpressing cells versus vector control cells reveal that BV6 increases DR5 expression in a NF-κB-dependent manner. Importantly, this BV6-stimulated upregulation of DR5 is critically required for apoptosis, as transient or stable knockdown of DR5 significantly inhibits BV6-triggered apoptosis. In addition, DR5 silencing attenuates formation of a RIP1/FADD/caspase-8 cytosolic cell death complex and activation of caspase-8, -3 and -9. By identifying DR5 as a critical mediator of Smac mimetic-induced apoptosis, our findings provide novel insights into the determinants that control susceptibility of cancer cells to Smac mimetic.

  5. Differential activation of CD95-mediated apoptosis related proteins in proximal and distal tubules during rat renal development.

    Science.gov (United States)

    Song, Xiao-Feng; Tian, He; Zhang, Zhen-Xing

    2016-10-01

    The CD95-mediated apoptotic pathway is the best characterized of the death receptor-mediated apoptotic pathways. The present study characterized localization and expression of proteins involved in CD95-mediated apoptosis during rat renal development. Kidneys were obtained from embryonic (E) 18 and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups. Immunohistochemical characterization revealed that CD95, FasL and cleaved caspase-3 were strongly expressed in proximal tubules and weakly expressed in distal tubules, but that expression of caspase-8 in distal tubules was stronger than that in proximal tubules. Results from terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that levels of apoptosis in proximal tubules slowly increased after E18, while those of distal tubules slowly decreased after P5. Western blotting demonstrated that expression of CD95, FasL and FADD was very weak during embryonic development, but rapidly increased at P14. Expression of cleaved caspase-3 was maintained at high levels after P1, while caspase-8 expression gradually reached a peak at P7. Results from this study reveal that the CD95-mediated apoptotic pathway is a key driver of apoptosis in proximal tubules during late postnatal kidney development in rats and suggest that apoptosis in distal tubules is mediated by a different apoptotic pathway. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Piroxicam and C-phycocyanin mediated apoptosis in 1,2-dimethylhydrazine dihydrochloride induced colon carcinogenesis: exploring the mitochondrial pathway.

    Science.gov (United States)

    Saini, Manpreet Kaur; Sanyal, Sankar Nath; Vaiphei, Kim

    2012-04-01

    Apoptosis is a synchronized procedure of cell death that is regulated by caspases and proapoptotic proteins. During apoptosis, translocation of cytochrome c, an electron carrier, from mitochondria into the cytosol is regulated by Bcl-2 family members. Cytochrome c in association with an apoptotic protease activating factor (Apaf), a proapoptotic protein essential for cell differentiation and procaspase-9 form the apoptosome complex, which consecutively activates effector caspase, caspase-3, and coordinate the implementation of apoptosis. In the current study, an attempt has been made to gain insight into piroxicam, a traditional nonsteroidal antiinflammatory drug and c-phycocyanin, a biliprotein from Spirulina platensis (cyanobacterium) mediated apoptosis in DMH-induced colon cancer. Male Sprague-Dawley rats were segregated into 5 groups: control, DMH, DMH + piroxicam, DMH + c-phycocyanin, and DMH + piroxicam + c-phycocyanin. Results illustrated that piroxicam and c-phycocyanin treatments stimulate cytochrome c release by downregulating the Bcl-2 (an antiapoptotic protein) expression significantly, while promoting the level of Bax (a proapoptotic protein), thereby activating caspases (caspases-9 and -3) and Apaf-1. The outcomes of the present study clearly signify that piroxicam and c-phycocyanin may mediate mitochondrial-dependent apoptosis in DMH-induced colon cancer. Moreover, apoptosis induction was more apparent in the combination regimen of piroxicam and c-phycocyanin than the individual drugs alone.

  7. Receptor-mediated endocytosis is not required for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis.

    Science.gov (United States)

    Kohlhaas, Susan L; Craxton, Andrew; Sun, Xiao-Ming; Pinkoski, Michael J; Cohen, Gerald M

    2007-04-27

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 degrees C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells.

  8. Withaferin A Induces Oxidative Stress-Mediated Apoptosis and DNA Damage in Oral Cancer Cells

    Science.gov (United States)

    Chang, Hsueh-Wei; Li, Ruei-Nian; Wang, Hui-Ru; Liu, Jing-Ru; Tang, Jen-Yang; Huang, Hurng-Wern; Chan, Yu-Hsuan; Yen, Ching-Yu

    2017-01-01

    Withaferin A (WFA) is one of the most active steroidal lactones with reactive oxygen species (ROS) modulating effects against several types of cancer. ROS regulation involves selective killing. However, the anticancer and selective killing effects of WFA against oral cancer cells remain unclear. We evaluated whether the killing ability of WFA is selective, and we explored its mechanism against oral cancer cells. An MTS tetrazolium cell proliferation assay confirmed that WFA selectively killed two oral cancer cells (Ca9-22 and CAL 27) rather than normal oral cells (HGF-1). WFA also induced apoptosis of Ca9-22 cells, which was measured by flow cytometry for subG1 percentage, annexin V expression, and pan-caspase activity, as well as western blotting for caspases 1, 8, and 9 activations. Flow cytometry analysis shows that WFA-treated Ca9-22 oral cancer cells induced G2/M cell cycle arrest, ROS production, mitochondrial membrane depolarization, and phosphorylated histone H2A.X (γH2AX)-based DNA damage. Moreover, pretreating Ca9-22 cells with N-acetylcysteine (NAC) rescued WFA-induced selective killing, apoptosis, G2/M arrest, oxidative stress, and DNA damage. We conclude that WFA induced oxidative stress-mediated selective killing of oral cancer cells. PMID:28936177

  9. TNF-α- mediated-p38-dependent signaling pathway contributes to myocyte apoptosis in rats subjected to surgical trauma.

    Science.gov (United States)

    Wu, Huaxing; Wang, Guonian; Li, Shuai; Zhang, Mingyue; Li, Hulun; Wang, Kun

    2015-01-01

    The accumulation of cytokines in the plasma after trauma can induce myocyte apoptosis. We aimed to identify which cytokine(s) present in the plasma responsible for myocyte apoptosis, and delineated the signal transduction mechanism in rats subjected to surgical trauma. Rats were randomized into two groups: control and trauma groups, which was divided into five subgroups: posttraumatic 0, 3, 6, 12, and 24 h subgroups. Cardiomyocytes isolated from traumatized rats were incubated with one of the factors for 12 h (normal plasma; Cytomix; TNF-α; IL-1β; IFN-γ; trauma plasma; anti-TNF-α antibody; SB203580). Myocyte apoptosis, cytokine levels, and MAPKs activation, as the primary experimental outcomes, were measured by TUNEL, flow cytometry, ELISA and Western blot, respectively. Myocyte apoptosis was induced by surgical trauma during the early stage after trauma. Accompanying this change, plasma TNF-α, IL-1β, and IFN-γ levels were elevated in traumatized rats. Incubation of traumatized cardiomyocytes with cytomix or TNF-α alone induced myocyte apoptosis, and increased the activation of p38 and ERK1/2. Myocyte apoptosis and p38 activation were elevated in traumatized cardiomyocytes with trauma plasma, and these increases were partly abolished by anti-TNF-α antibody or SB203580. Our study demonstrated that there exists the TNF-α-mediated-p38-dependent signaling pathway that contributed to posttraumatic myocyte apoptosis of rats undergoing surgical trauma. © 2015 S. Karger AG, Basel.

  10. TNF-α- Mediated-p38-Dependent Signaling Pathway Contributes to Myocyte Apoptosis in Rats Subjected to Surgical Trauma

    Directory of Open Access Journals (Sweden)

    Huaxing Wu

    2015-03-01

    Full Text Available Background: The accumulation of cytokines in the plasma after trauma can induce myocyte apoptosis. We aimed to identify which cytokine(s present in the plasma responsible for myocyte apoptosis, and delineated the signal transduction mechanism in rats subjected to surgical trauma. Methods: Rats were randomized into two groups: control and trauma groups, which was divided into five subgroups: posttraumatic 0, 3, 6, 12, and 24 h subgroups. Cardiomyocytes isolated from traumatized rats were incubated with one of the factors for 12 h (normal plasma; Cytomix; TNF-α; IL-1β; IFN-γ; trauma plasma; anti-TNF-α antibody; SB203580. Myocyte apoptosis, cytokine levels, and MAPKs activation, as the primary experimental outcomes, were measured by TUNEL, flow cytometry, ELISA and Western blot, respectively. Results: Myocyte apoptosis was induced by surgical trauma during the early stage after trauma. Accompanying this change, plasma TNF-α, IL-1β, and IFN-γ levels were elevated in traumatized rats. Incubation of traumatized cardiomyocytes with cytomix or TNF-α alone induced myocyte apoptosis, and increased the activation of p38 and ERK1/2. Myocyte apoptosis and p38 activation were elevated in traumatized cardiomyocytes with trauma plasma, and these increases were partly abolished by anti-TNF-α antibody or SB203580. Conclusion: Our study demonstrated that there exists the TNF-α-mediated-p38-dependent signaling pathway that contributed to posttraumatic myocyte apoptosis of rats undergoing surgical trauma.

  11. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ding, L.; Wu, J.P. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China); Xu, G. [Fudan University, Jinshan Hospital, Center Laboratory, Shanghai, China, Center Laboratory, Jinshan Hospital, Fudan University, Shanghai (China); Zhu, B.; Zeng, Q.M.; Li, D.F.; Lu, W. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China)

    2014-05-09

    Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

  12. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    Directory of Open Access Journals (Sweden)

    L. Ding

    2014-06-01

    Full Text Available Current studies find that degenerated cartilage endplates (CEP of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

  13. Apoptosis and autophagy induced by pyropheophorbide-α methyl ester-mediated photodynamic therapy in human osteosarcoma MG-63 cells.

    Science.gov (United States)

    Huang, Qiu; Ou, Yun-Sheng; Tao, Yong; Yin, Hang; Tu, Ping-Hua

    2016-06-01

    Pyropheophorbide-α methyl ester (MPPa) was a second-generation photosensitizer with many potential applications. Here, we explored the impact of MPPa-mediated photodynamic therapy (MPPa-PDT) on the apoptosis and autophagy of human osteosarcoma (MG-63) cells as well as the relationships between apoptosis and autophagy of the cells, and investigated the related molecular mechanisms. We found that MPPa-PDT demonstrated the ability to inhibit MG-63 cell viability in an MPPa concentration- and light dose-dependent manner, and to induce apoptosis via the mitochondrial apoptosis pathway. Additionally, MPPa-PDT could also induce autophagy of MG-63 cell. Meanwhile, the ROS scavenger N-acetyl-L-cysteine (NAC) and the Jnk inhibitor SP600125 were found to inhibit the MPPa-PDT-induced autophagy, and NAC could also inhibit Jnk phosphorylation. Furthermore, pretreatment with the autophagy inhibitor 3-methyladenine or chloroquine showed the potential in reducing the apoptosis rate induced by MPPa-PDT in MG-63 cells. Our results indicated that the mitochondrial pathway was involved in MPPa-PDT-induced apoptosis of MG-63 cells. Meanwhile the ROS-Jnk signaling pathway was involved in MPPa-PDT-induced autophagy, which further promoted the apoptosis in MG-63 cells.

  14. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    International Nuclear Information System (INIS)

    Ding, L.; Wu, J.P.; Xu, G.; Zhu, B.; Zeng, Q.M.; Li, D.F.; Lu, W.

    2014-01-01

    Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis

  15. Singly protonated dehydronorcantharidin silver coordination polymer induces apoptosis of lung cancer cells via reactive oxygen species-mediated mitochondrial pathway.

    Science.gov (United States)

    Li, Senpeng; Zhang, Shuo; Jin, Xing; Tan, Xuejie; Lou, Jianfang; Zhang, Xiumei; Zhao, Yunxue

    2014-10-30

    Silver complexes have been shown to possess antimicrobial and anticancer properties. Ag-SP-DNC, a novel silver and singly protonated dehydronorcantharidin complex, was synthesized in our previous study. In this study, we offer evidence that Ag-SP-DNC elicits a reactive oxygen species (ROS)-mediated mitochondrial apoptosis in lung cancer cells. Ag-SP-DNC inhibited the growth of A549 cells by inducing G2/M phase cell cycle arrest and apoptosis. Ag-SP-DNC induced apoptosis was associated with the levels of intracellular ROS. The further study revealed that Ag-SP-DNC disrupted the mitochondrial membrane potential, induced the caspase-3 activation and led to the translocation of apoptosis inducing factor and endonucleaseG to the nucleus. These findings have important implications for the development of silver complexes for anticancer applications. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  16. Naringin prevents ovariectomy-induced osteoporosis and promotes osteoclasts apoptosis through the mitochondria-mediated apoptosis pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Fengbo [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Graduate School of Tianjin Medical University, No. 22, Qixiangtai Street, Heping District, Tianjin 300070 (China); Sun, Xiaolei; Ma, Jianxiong [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Ma, Xinlong, E-mail: gengxiao502@163.com [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Zhao, Bin; Zhang, Yang; Tian, Peng [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Li, Yanjun [Graduate School of Tianjin Medical University, No. 22, Qixiangtai Street, Heping District, Tianjin 300070 (China); Han, Zhe [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China)

    2014-09-26

    Highlights: • Naringin possesses many pharmacological activities, promotes the proliferation of osteoblast. • Undecalcified histological obtain dynamic parameters of callus formation and remodeling. • Naringin regulate osteoclast apoptosis by mitochondrial pathway. - Abstract: Naringin, the primary active compound of the traditional Chinese medicine Rhizoma drynariae, possesses many pharmacological activities. The present study is an effort to explore the anti-osteoporosis potential of naringin in vivo and in vitro. In vivo, we used ovariectomized rats to clarify the mechanisms by which naringin anti-osteoporosis. In vitro, we used osteoclasts to investigate naringin promotes osteoclasts apoptosis. Naringin was effective at enhancing BMD, trabecular thickness, bone mineralization, and mechanical strength in a dose-dependent manner. The result of RT-PCR analysis revealed that naringin down-regulated the mRNA expression levels of BCL-2 and up-regulated BAX, caspase-3 and cytochrome C. In addition, naringin significantly reduced the bone resorption area in vitro. These findings suggest that naringin promotes the apoptosis of osteoclasts by regulating the activity of the mitochondrial apoptosis pathway and prevents OVX-induced osteoporosis in rats.

  17. Naringin prevents ovariectomy-induced osteoporosis and promotes osteoclasts apoptosis through the mitochondria-mediated apoptosis pathway

    International Nuclear Information System (INIS)

    Li, Fengbo; Sun, Xiaolei; Ma, Jianxiong; Ma, Xinlong; Zhao, Bin; Zhang, Yang; Tian, Peng; Li, Yanjun; Han, Zhe

    2014-01-01

    Highlights: • Naringin possesses many pharmacological activities, promotes the proliferation of osteoblast. • Undecalcified histological obtain dynamic parameters of callus formation and remodeling. • Naringin regulate osteoclast apoptosis by mitochondrial pathway. - Abstract: Naringin, the primary active compound of the traditional Chinese medicine Rhizoma drynariae, possesses many pharmacological activities. The present study is an effort to explore the anti-osteoporosis potential of naringin in vivo and in vitro. In vivo, we used ovariectomized rats to clarify the mechanisms by which naringin anti-osteoporosis. In vitro, we used osteoclasts to investigate naringin promotes osteoclasts apoptosis. Naringin was effective at enhancing BMD, trabecular thickness, bone mineralization, and mechanical strength in a dose-dependent manner. The result of RT-PCR analysis revealed that naringin down-regulated the mRNA expression levels of BCL-2 and up-regulated BAX, caspase-3 and cytochrome C. In addition, naringin significantly reduced the bone resorption area in vitro. These findings suggest that naringin promotes the apoptosis of osteoclasts by regulating the activity of the mitochondrial apoptosis pathway and prevents OVX-induced osteoporosis in rats

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    Atthapan Morchang

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

  20. Mouse Lung Fibroblast Resistance to Fas-Mediated Apoptosis Is Dependent on the Baculoviral Inhibitor of Apoptosis Protein 4 and the Cellular FLICE-Inhibitory Protein

    Science.gov (United States)

    Predescu, Sanda A.; Zhang, Jian; Bardita, Cristina; Patel, Monal; Godbole, Varun; Predescu, Dan N.

    2017-01-01

    A characteristic feature of idiopathic pulmonary fibrosis (IPF) is accumulation of apoptotic resistant fibroblasts/myofibroblasts in the fibroblastic foci. As caveolin (Cav)-null mice develop pulmonary fibrosis (PF), we hypothesized that the participating fibroblasts display an apoptosis-resistant phenotype. To test this hypothesis and identify the molecular mechanisms involved we isolated lung fibroblasts from Cav-null mice and examined the expression of several inhibitors of apoptosis (IAPs), of c-FLIP, of Bcl-2 proteins and of the death receptor CD95/Fas. We found significant increase in XIAP and c-FLIP constitutive protein expression with no alteration of Bcl-2 and lower levels of CD95/Fas. The isolated fibroblasts were then treated with the CD95/Fas ligand (FasL) to induce apoptosis. While the morphological and biochemical alterations induced by FasL were similar in wild-type (wt) and Cav-null mouse lung fibroblasts, the time course and the extent of the alterations were greater in the Cav-null fibroblasts. Several salient features of Cav-null fibroblasts response such as loss of membrane potential, fragmentation of the mitochondrial continuum concurrent with caspase-8 activation, and subsequent Bid cleavage, prior to caspase-3 activation were detected. Furthermore, M30 antigen formation, phosphatidylserine expression and DNA fragmentation were caspase-3 dependent. SiRNA-mediated silencing of XIAP and c-FLIP, individually or combined, enhanced the sensitivity of lung fibroblasts to FasL-induced apoptosis. Pharmacological inhibition of Bcl-2 had no effect. Together our findings support a mechanism in which CD95/Fas engagement activates caspase-8, inducing mitochondrial apoptosis through Bid cleavage. XIAP and c-FLIP fine tune this process in a cell-type specific manner. PMID:28352235

  1. Intense picosecond pulsed electric fields induce apoptosis through a mitochondrial-mediated pathway in HeLa cells

    Science.gov (United States)

    HUA, YUAN-YUAN; WANG, XIAO-SHU; ZHANG, YU; YAO, CHEN-GUO; ZHANG, XI-MING; XIONG, ZHENG-AI

    2012-01-01

    The application of pulsed electric fields (PEF) is emerging as a new technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely, but the research of the biological effects of psPEF on cells is limited. Electric theory predicts that intense psPEF will target mitochondria and lead to changes in transmembrane potential, therefore, it is hypothesized that it can induce mitochondrial-mediated apoptosis. HeLa cells were exposed to psPEF in this study to investigate this hypothesis. MTT assay demonstrated that intense psPEF significantly inhibited the proliferation of HeLa cells in a dose-dependent manner. Typical characteristics of apoptosis in HeLa cells were observed, using transmission electron microscopy. Loss of mitochondrial transmembrane potential was explored using laser scanning confocal microscopy with Rhodamine-123 (Rh123) staining. Furthermore, the mitochondrial apoptotic events were also confirmed by western blot analysis for the release of cytochrome C and apoptosis-inducing factor from mitochondria into the cytosol. In addition, activation of caspase-3, caspase-9, upregulation of Bax, p53 and downregulation of Bcl-2 were observed in HeLa cells also indicating apoptosis. Taken together, these results demonstrate that intense psPEF induce cell apoptosis through a mitochondrial-mediated pathway. PMID:22307872

  2. 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.

  3. The induction of autophagy against mitochondria-mediated apoptosis in lung cancer cells by a ruthenium (II) imidazole complex

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    Peng, Fa; Jie, Xinming; Dongye, Guangzhi; Cai, Kangrong; Feng, Ruibing; Li, Baojun; Zeng, Qingwang; Lun, Kaiyi; Chen, Jincan; Xu, Bilian

    2016-01-01

    In the present study, it was found that the ruthenium (II) imidazole complex [Ru(Im)4(dppz)]2+ (Ru1) could induce significant growth inhibition and apoptosis in A549 and NCI-H460 cells. Apart from the induction of apoptosis, it was reported for the first time that Ru1 induced an autophagic response in A549 and NCI-H460 cells as evidenced by the formation of autophagosomes, acidic vesicular organelles (AVOs), and the up-regulation of LC3-II. Furthermore, scavenging of reactive oxygen species (ROS) by antioxidant NAC or Tiron inhibited the release of cytochrome c, caspase-3 activity, and eventually rescued cancer cells from Ru1-mediated apoptosis, suggesting that Ru1 inducing apoptosis was partially caspase 3-dependent by triggering ROS-mediated mitochondrial dysfunction in A549 and NCI-H460 cells. Further study indicated that the extracellular signal-regulated kinase (ERK) signaling pathway was involved in Ru1-induced autophagy in A549 and NCI-H460 cells. Moreover, blocking autophagy using pharmacological inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) enhanced Ru1-induced apoptosis, indicating the cytoprotective role of autophagy in Ru1-treated A549 and NCI-H460 cells. Finally, the in vivo mice bearing A549 xenografts, Ru1 dosed at 10 or 20 mg/kg significantly inhibited tumor growth. PMID:27811372

  4. Downregulation of β1,4-galactosyltransferase 1 inhibits CDK11p58-mediated apoptosis induced by cycloheximide

    International Nuclear Information System (INIS)

    Li Zejuan; Wang Hanzhou; Zong Hongliang; Sun Qing; Kong Xiangfei; Jiang Jianhai; Gu Jianxin

    2005-01-01

    Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34 cdc2 -related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11 p58 induces apoptosis is not clear. Some evidences suggested β1,4-galactosyltransferase 1 (β1,4-GT 1) might participate in apoptosis induced by CDK11 p58 . In this study, we demonstrated that ectopically expressed β1,4-GT 1 increased CDK11 p58 -mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of β1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11 p58 -overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of β1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11 p58 by caspase-3 was reduced. We proposed that β1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11 p58 . This may represent a new mechanism of β1,4-GT 1 in CHX-induced apoptosis of CDK11 p58 -overexpressing cells

  5. Microglia are mediators of Borrelia burgdorferi-induced apoptosis in SH-SY5Y neuronal cells.

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    Tereance A Myers

    2009-11-01

    Full Text Available Inflammation has long been implicated as a contributor to pathogenesis in many CNS illnesses, including Lyme neuroborreliosis. Borrelia burgdorferi is the spirochete that causes Lyme disease and it is known to potently induce the production of inflammatory mediators in a variety of cells. In experiments where B. burgdorferi was co-cultured in vitro with primary microglia, we observed robust expression and release of IL-6 and IL-8, CCL2 (MCP-1, CCL3 (MIP-1alpha, CCL4 (MIP-1beta and CCL5 (RANTES, but we detected no induction of microglial apoptosis. In contrast, SH-SY5Y (SY neuroblastoma cells co-cultured with B. burgdorferi expressed negligible amounts of inflammatory mediators and also remained resistant to apoptosis. When SY cells were co-cultured with microglia and B. burgdorferi, significant neuronal apoptosis consistently occurred. Confocal microscopy imaging of these cell cultures stained for apoptosis and with cell type-specific markers confirmed that it was predominantly the SY cells that were dying. Microarray analysis demonstrated an intense microglia-mediated inflammatory response to B. burgdorferi including up-regulation in gene transcripts for TLR-2 and NFkappabeta. Surprisingly, a pathway that exhibited profound changes in regard to inflammatory signaling was triggering receptor expressed on myeloid cells-1 (TREM1. Significant transcript alterations in essential p53 pathway genes also occurred in SY cells cultured in the presence of microglia and B. burgdorferi, which indicated a shift from cell survival to preparation for apoptosis when compared to SY cells cultured in the presence of B. burgdorferi alone. Taken together, these findings indicate that B. burgdorferi is not directly toxic to SY cells; rather, these cells become distressed and die in the inflammatory surroundings generated by microglia through a bystander effect. If, as we hypothesized, neuronal apoptosis is the key pathogenic event in Lyme neuroborreliosis, then

  6. Epidermal growth factor prevents thallium(I)- and thallium(III)-mediated rat pheochromocytoma (PC12) cell apoptosis.

    Science.gov (United States)

    Pino, María Teresa Luján; Marotte, Clarisa; Verstraeten, Sandra Viviana

    2017-03-01

    We have reported recently that the proliferation of PC12 cells exposed to micromolar concentrations of Tl(I) or Tl(III) has different outcomes, depending on the absence (EGF - cells) or the presence (EGF + cells) of epidermal growth factor (EGF) added to the media. In the current work, we investigated whether EGF supplementation could also modulate the extent of Tl(I)- or Tl(III)-induced cell apoptosis. Tl(I) and Tl(III) (25-100 μM) decreased cell viability in EGF - but not in EGF + cells. In EGF - cells, Tl(I) decreased mitochondrial potential, enhanced H 2 O 2 generation, and activated mitochondrial-dependent apoptosis. In addition, Tl(III) increased nitric oxide production and caused a misbalance between the anti- and pro-apoptotic members of Bcl-2 family. Tl(I) increased ERK1/2, JNK, p38, and p53 phosphorylation in EGF - cells. In these cells, Tl(III) did not affect ERK1/2 and JNK phosphorylation but increased p53 phosphorylation that was related to the promotion of cell senescence. In addition, this cation significantly activated p38 in both EGF - and EGF + cells. The specific inhibition of ERK1/2, JNK, p38, or p53 abolished Tl(I)-mediated EGF - cell apoptosis. Only when p38 activity was inhibited, Tl(III)-mediated apoptosis was prevented in EGF - and EGF + cells. Together, current results indicate that EGF partially prevents the noxious effects of Tl by preventing the sustained activation of MAPKs signaling cascade that lead cells to apoptosis and point to p38 as a key mediator of Tl(III)-induced PC12 cell apoptosis.

  7. Synergistic property of cordycepin in cultivated Cordyceps militaris-mediated apoptosis in human leukemia cells.

    Science.gov (United States)

    Chou, Shang-Min; Lai, Wan-Jung; Hong, Tzu-Wen; Lai, Jui-Ya; Tsai, Sheng-Hong; Chen, Yen-Hsun; Yu, Sz-Hsien; Kao, Cheng-Hsiang; Chu, Richard; Ding, Shih-Torng; Li, Tsai-Kun; Shen, Tang-Long

    2014-10-15

    Cordyceps militaris is a well-known Chinese traditional medicinal mushroom frequently used for tonics and recently of a potential interest for cancer intervention. Here, we explored the cancer cell killing activity of the hot water extracts of C. militaris cultured mycelia (CM(MY)) and cultivated fruiting bodies (CM(FB)). We found that CM(FB) exhibited a greater cytotoxic effect against various cancer cells over CM(MY). Apoptotic phenotypes including apoptotic body formation, DNA laddering, caspase 3 activation and cleavage of PARP proteins were induced by CM(FB) treatment but only slightly induced by same concentration of CM(MY) treatment in human HL-60 leukemia cells. Cordycepin in CM(FB) (10.47 mg/g) is significantly higher (∼ 15.2 times) than that of CM(MY) (0.69 mg/g). Using isobolographic analysis, the synergy of cytotoxicity was observed across different combined concentrations of CM(MY) and cordycepin. By complementing cordycepin into CM(MY) to the level comparable with CM(FB), we observed that CM(MY) (500 μg/ml) with cordycepin (4.8 μg/ml) induced apoptosis to a level similar to that induced by CM(FB) (500 μg/ml). Together, our results suggest that cordycepin possesses a synergistic cytotoxic effect with Cordyceps militaris-mediated apoptosis in human leukemia cells and therefore explaining a better anti-proliferating activity of CM(FB) over CM(MY). Copyright © 2014 Elsevier GmbH. All rights reserved.

  8. Fas- and Mitochondria-Mediated Signaling Pathway Involved in Osteoblast Apoptosis Induced by AlCl3.

    Science.gov (United States)

    Xu, Feibo; Ren, Limin; Song, Miao; Shao, Bing; Han, Yanfei; Cao, Zheng; Li, Yanfei

    2017-10-12

    Aluminum (Al) is known to induce apoptosis of osteoblasts (OBs). However, the mechanism is not yet established. To investigate the apoptotic mechanism of OBs induced by aluminum trichloride (AlCl 3 ), the primary OBs from the craniums of fetal Wistar rats were exposed to 0 mg/mL (control group, CG), 0.06 mg/mL (low-dose group, LG), 0.12 mg/mL (mid-dose group, MG), and 0.24 mg/mL (high-dose group, HG) AlCl 3 for 24 h, respectively. We observed that AlCl 3 induced OB apoptosis with the appearance of apoptotic morphology and increase of apoptosis rate. Additionally, AlCl 3 treatment activated mitochondrial-mediated signaling pathway, accompanied by mitochondrial membrane potential (ΔΨm) depolarization, release of cytochrome c from the mitochondria to the cytoplasm, as well as survival signal-related factor caspase-9 and caspase-3 activation. AlCl 3 exposure also activated Fas/Fas ligand signaling pathway, presented as Fas, Fas ligand, and Fas-associated death domain expression enhancement and caspase-8 activation, as well as the hydrolysis of Bid to truncated Bid, suggesting that the Fas-mediated signaling pathway might aggravate mitochondria-mediated OB apoptosis through hydrolyzing Bid. Furthermore, AlCl 3 exposure inhibited Bcl-2 protein expression and increased the expressions of Bax, Bak, and Bim in varying degrees. These results indicated that AlCl 3 exposure induced OB apoptosis through activating Fas- and mitochondria-mediated signaling pathway and disrupted B-cell lymphoma-2 family proteins.

  9. Knockdown of apoptosis signal-regulating kinase 1 affects ischaemia-induced astrocyte activation and glial scar formation.

    Science.gov (United States)

    Cheon, So Yeong; Cho, Kyoung Joo; Song, Juhyun; Kim, Gyung Whan

    2016-04-01

    Reactive astrocytes play an essential role in determining the tissue response to ischaemia. Formation of a glial scar can block the neuronal outgrowth that is required for restoration of damaged tissue. Therefore, regulation of astrocyte activation is important; however, the mediator of this process has not been fully elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is an early responder to oxidative stress, and plays a pivotal role in the intracellular signalling pathway of apoptosis, inflammation, and differentiation. To confirm whether ASK1 mediates astrocyte activation and leads to glial scar formation after cerebral ischaemia, we conducted in vivo and in vitro experiments. C57BL/6 mice were subjected to occlusion of the middle cerebral artery, and astrocyte cultures were exposed to oxygen-glucose deprivation. After silencing of ASK1 , astrocyte-associated genes were downregulated, as seen with the use of microarrays. The glial fibrillary acidic protein (GFAP) level was decreased, and correlated with the reduction in the ASK1 level. In astrocytes, reduction in the ASK1 level decreased the activity of the p38 pathway, and the levels of transcription factors for GFAP and GFAP transcripts after hypoxia. In the chronic phase, ASK1 depletion reduced glial scar formation and conserved neuronal structure, which may lead to better functional recovery. These data suggest that ASK1 may be an important mediator of ischaemia-induced astrocyte activation and scar formation, and could provide a potential therapeutic target for treatment after ischaemic stroke. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  10. Inhibition of Drp1 protects against senecionine-induced mitochondria-mediated apoptosis in primary hepatocytes and in mice

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    Xiao Yang

    2017-08-01

    Full Text Available Pyrrolizidine alkaloids (PAs are a group of compounds found in various plants and some of them are widely consumed in the world as herbal medicines and food supplements. PAs are potent hepatotoxins that cause irreversible liver injury in animals and humans. However, the mechanisms by which PAs induce liver injury are not clear. In the present study, we determined the hepatotoxicity and molecular mechanisms of senecionine, one of the most common toxic PAs, in primary cultured mouse and human hepatocytes as well as in mice. We found that senecionine administration increased serum alanine aminotransferase levels in mice. H&E and TUNEL staining of liver tissues revealed increased hemorrhage and hepatocyte apoptosis in liver zone 2 areas. Mechanistically, senecionine induced loss of mitochondrial membrane potential, release of mitochondrial cytochrome c as well as mitochondrial JNK translocation and activation prior to the increased DNA fragmentation and caspase-3 activation in primary cultured mouse and human hepatocytes. SP600125, a specific JNK inhibitor, and ZVAD-fmk, a general caspase inhibitor, alleviated senecionine-induced apoptosis in primary hepatocytes. Interestingly, senecionine also caused marked mitochondria fragmentation in hepatocytes. Pharmacological inhibition of dynamin-related protein1 (Drp1, a protein that is critical to regulate mitochondrial fission, blocked senecionine-induced mitochondrial fragmentation and mitochondrial release of cytochrome c and apoptosis. More importantly, hepatocyte-specific Drp1 knockout mice were resistant to senecionine-induced liver injury due to decreased mitochondrial damage and apoptosis. In conclusion, our results uncovered a novel mechanism of Drp1-mediated mitochondrial fragmentation in senecionine-induced liver injury. Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by PAs. Keywords: Senecionine, Drp1

  11. Knockdown of c‑Myc activates Fas-mediated apoptosis and sensitizes A549 cells to radiation.

    Science.gov (United States)

    Zhang, Jing; Zhou, Ling; Nan, Zhaodi; Yuan, Qing; Wen, Jie; Xu, Maolei; Li, Youjie; Li, Baosheng; Wang, Pingyu; Liu, Changmin; Ma, Ying; Chen, Shaoshui; Xie, Shuyang

    2017-10-01

    Several studies have demonstrated that cancer radiosensitivity is associated with the deregulation of c‑Myc, but the relationship between c‑Myc and Fas in radioresistance of lung adenocarcinoma remains unclear. In this study, we established radiation-resistant A549 cell model (A549/R), and investigated the roles of c‑Myc and Fas in radiation-induced cytotoxicity of A549 cells. Apoptosis detection showed that there were fewer apoptotic cells in A549/R cells treated with radiation than in A549 cells. Western blotting results demonstrated the inverse expression pattern of c‑Myc and Fas in A549 and A549/R cells. Suppression of c‑Myc expression by small interfering RNA (siRNA) displayed enhancement of Fas-mediated apoptosis in A549/R cells, accompanying a significant decrease of Bid, Bcl‑2, pro‑caspase‑8, -9 and -3 and increase of Bax. In contrast, Fas-mediated apoptosis was attenuated while Fas expression was suppressed by ectopic expression of c‑Myc in A549 cells. Moreover, decreased cell viability and increased induction of apoptosis were observed in A549/R cells followed by combinational treatment of c‑Myc siRNA and irradiation, whereas, upregulation of c‑Myc reduced the sensitivity of A549 cells to irradiation. These results indicated that c‑Myc and Fas regulated the sensitivity of A549 cells to irradiation by regulating caspase‑8-mediated Bid activation and the subsequent association with the mitochondrial pathway of apoptosis.

  12. Hepatitis C virus infection induces apoptosis through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway.

    Science.gov (United States)

    Deng, Lin; Adachi, Tetsuya; Kitayama, Kikumi; Bungyoku, Yasuaki; Kitazawa, Sohei; Ishido, Satoshi; Shoji, Ikuo; Hotta, Hak

    2008-11-01

    We previously reported that cells harboring the hepatitis C virus (HCV) RNA replicon as well as those expressing HCV NS3/4A exhibited increased sensitivity to suboptimal doses of apoptotic stimuli to undergo mitochondrion-mediated apoptosis (Y. Nomura-Takigawa, et al., J. Gen. Virol. 87:1935-1945, 2006). Little is known, however, about whether or not HCV infection induces apoptosis of the virus-infected cells. In this study, by using the chimeric J6/JFH1 strain of HCV genotype 2a, we demonstrated that HCV infection induced cell death in Huh7.5 cells. The cell death was associated with activation of caspase 3, nuclear translocation of activated caspase 3, and cleavage of DNA repair enzyme poly(ADP-ribose) polymerase, which is known to be an important substrate for activated caspase 3. These results suggest that HCV-induced cell death is, in fact, apoptosis. Moreover, HCV infection activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change and its increased accumulation on mitochondrial membranes. Concomitantly, HCV infection induced disruption of mitochondrial transmembrane potential, followed by mitochondrial swelling and release of cytochrome c from mitochondria. HCV infection also caused oxidative stress via increased production of mitochondrial superoxide. On the other hand, HCV infection did not mediate increased expression of glucose-regulated protein 78 (GRP78) or GRP94, which are known as endoplasmic reticulum (ER) stress-induced proteins; this result suggests that ER stress is not primarily involved in HCV-induced apoptosis in our experimental system. Taken together, our present results suggest that HCV infection induces apoptosis of the host cell through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway(s).

  13. Mechanical ventilation-induced apoptosis in newborn rat lung is mediated via FasL/Fas pathway.

    Science.gov (United States)

    Kroon, Andreas A; Delriccio, Veronica; Tseu, Irene; Kavanagh, Brian P; Post, Martin

    2013-12-01

    Mechanical ventilation induces pulmonary apoptosis and inhibits alveolar development in preterm infants, but the molecular basis for the apoptotic injury is unknown. The objective was to determine the signaling mechanism(s) of ventilation (stretch)-induced apoptosis in newborn rat lung. Seven-day-old rats were ventilated with room air for 24 h using moderate tidal volumes (8.5 ml/kg). Isolated fetal rat lung epithelial and fibroblast cells were subjected to continuous cyclic stretch (5, 10, or 17% elongation) for up to 12 h. Prolonged ventilation significantly increased the number of apoptotic alveolar type II cells (i.e., terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling and anti-cleaved caspase-3 immunochemistry) and was associated with increased expression of the apoptotic mediator Fas ligand (FasL). Fetal lung epithelial cells, but not fibroblasts, subjected to maximal (i.e., 17%, but not lesser elongation) cyclic stretch exhibited increased apoptosis (i.e., nuclear fragmentation and DNA laddering), which appeared to be mediated via the extrinsic pathway (increased expression of FasL and cleaved caspase-3, -7, and -8). The intrinsic pathway appeared not to be involved [minimal mitochondrial membrane depolarization (JC-1 flow analysis) and no activation of caspase-9]. Universal caspases inhibition and neutralization of FasL abrogated the stretch-induced apoptosis. Prolonged mechanical ventilation induces apoptosis of alveolar type II cells in newborn rats and the mechanism appears to involve activation of the extrinsic death pathway via the FasL/Fas system.

  14. A ROS-dependent and Caspase-3-mediated apoptosis in sheep bronchial epithelial cells in response to Mycoplasma Ovipneumoniae infections.

    Science.gov (United States)

    Xue, Di; Li, Yanan; Jiang, Zhongjia; Deng, Guangcun; Li, Min; Liu, Xiaoming; Wang, Yujiong

    2017-05-01

    Mycoplasma Ovipneumoniae (M. ovipneumoniae) is a primary etiological agent of enzootic pneumonia in sheep and goats. It can enter and colonize ovine respiratory epithelial cells to establish an infection, which leads a serious cell death of epithelial cells. However, the nature of the interaction between pathogen of M. ovipneumoniae and host cells in the cell injury is currently not well understood. In this study, we investigated the epithelial cell apoptosis caused by an infection of M. ovipneumoniae in sheep primary air-liquid interface (ALI) epithelial cultures. The results showed that M. ovipneumoniae could specifically bind to ciliated cells at early stage of infection. Flow cytometric analysis demonstrated that an infection of M. ovipneumoniae induced a time-dependent cell apoptotic cell death, accompanied with an increased production of extracellular nitric oxide (NO), intracellular reactive oxygen species (ROS) production and activation of caspase-3 signaling in sheep bronchial epithelial cells. The induced cell apoptosis was further confirmed by a transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay. Interestingly, the M. ovipneumoniae-induced apoptosis and activation of caspase-3 were correlated with the production of ROS but not NO. Mechanistically, M. ovipneumoniae-induced cell apoptosis was mediated by a mechanism by increasing the expression of phosphorylation of p38 and pro-apoptotic proteins, and activating caspase-3, caspase-8 and poly ADP-ribose polymerase (PARP) cleavage. These results suggest a ROS-dependent and caspase-3-mediated cell apoptosis in sheep bronchial epithelial cells in response to M. ovipneumoniae infections. Copyright © 2017. Published by Elsevier B.V.

  15. Heme Oxygenase-1 Mediates Oxidative Stress and Apoptosis in Coxsackievirus B3-Induced Myocarditis

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    Oana N. Ursu

    2014-01-01

    Full Text Available Background: Heme oxygenase-1 (HO-1, which is suggested to play a role in defending the organism against oxidative stress-mediated injuries, can be induced by diverse factors including viruses and iron. As coxsackievirus B3 (CVB3-infected SWR/J mice susceptible for chronic myocarditis were found to have a significant iron incorporation and HO-1 upregulation in the myocardium, we aimed to investigate the molecular interplay between HO-1 expression and iron homeostasis in the outcome of viral myocarditis. Methods and Results: In susceptible SWR/J mice, but not in resistant C57BL/6 mice, we observed at later stages of CVB3 myocarditis significant iron deposits in macrophages and also in cardiomyocytes, which were spatially associated with oxidative stress, upregulation of HO-1 and caspase-3 activation. HO-1, which is also expressed in cultivated RAW 264.7 macrophages upon incubation with iron and/or CVB3, could be downregulated by inhibition of NO/iNOS using L-NAME. Moreover, specific inhibition of HO-1 by tin mesoporphyrin revealed a suppression of superoxide production in iron and/or CVB3-treated macrophages. The molecular relationship of HO-1 and caspase-3 activation was proven by downregulation with HO-1 siRNA in iron- and/or CVB3-treated cultivated cells. Importantly, iron was found to increase viral replication in vitro. Conclusion: These results indicate that HO-1 induces a paracrine signalling in macrophages via reactive oxygen species production, mediating apoptosis of heart muscle cells at later stages of myocarditis. Notably, in genetically susceptible mice iron potentiates the detrimental effects of CVB3 by the NO/HO-1 pathway, thus increasing cardiac pathogenicity.

  16. Cadmium induces Ca2+ mediated, calpain-1/caspase-3-dependent apoptosis in primary cultured rat proximal tubular cells.

    Science.gov (United States)

    Wang, Hong; Zhai, Nianhui; Chen, Ying; Xu, Haibin; Huang, Kehe

    2017-07-01

    Calcium, as a ubiquitous second messenger, governs a large array of cellular processes and is necessary for cell survival. More recently, it was observed that the cytosolic Ca 2+ concentration ([Ca 2+ ] c ) elevation could induce apoptosis in primary cultured rat proximal tubular (rPT) cells exposed to cadmium (Cd), but the concrete mechanism is still unclear. This study was designed to investigate the signal pathway involved in [Ca 2+ ] c elevation-mediated apoptosis. The results confirmed the elevation of [Ca 2+ ] c by confocal microscopy and enhancement of the apoptosis by Hoechst 33258 staining and flow cytometer when rPT cells were exposed to Cd for 12h. Then we demonstrated that Cd enhanced the protein levels of active calpain-1 and caspase-3 in rPT cells. Pretreatment with a cytosolic Ca 2+ chelator, 1,2-Bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), markedly blocked the up-regulation of active calpain-1 and caspase-3 and inhibited the apoptosis induced by Cd. Further, rPT cells were pretreated with a cell-permeable selective calpain-1 inhibitor, 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) and caspase-3 inhibitor, N-Acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO), respectively. PD150606 significantly attenuated the up-regulation of active caspase-3 and the apoptosis induced by Cd. As expected, inhibition of active caspase-3 by Ac-DEVD-CHO decreased the apoptosis induced by Cd. Taken together, it could be concluded that [Ca 2+ ] c elevation did act as a pro-apoptotic signal in Cd-induced cytotoxicity of rPT cells, triggered calpain-1 and caspase-3 activation in turn, and induced apoptosis of rPT cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Piroxicam, a traditional non-steroidal anti-inflammatory drug (NSAID) causes apoptosis by ROS mediated Akt activation.

    Science.gov (United States)

    Rai, Neha; Sarkar, Munna; Raha, Sanghamitra

    2015-12-01

    Piroxicam (Px) belongs to the oxicam group of the non-steroidal anti-inflammatory drugs (NSAIDs) and have been shown to exert chemopreventive and chemotherapeutic effects in animal models and cultured animal cells. However, little is known about the mode of action of Px and its cellular targets. We explored the role of Px, in triggering apoptosis and examined the involvement of upstream cellular mechanisms in apoptosis induction by Px. Our studies with human breast cancer cells MCF-7 show that Px induces reactive oxygen species (ROS) generation along with apoptotic cell death. ROS release lead to Akt activation. On evaluation it became evident that ROS mediated apoptosis induction was due to Akt activation (hyper phosphorylation). Silencing the expression of Akt using siRNA and a specific Akt inhibitor, triciribine further confirmed the findings. However Px failed to cause ROS generation, cell death or Akt phosphorylation in another human breast cancer cells MDA-MB-231 which is estrogen receptor negative and more aggressive compared to MCF-7 cells. This suggests that Px has cell type specific effects. Thus we revealed for the first time that Px can induce apoptosis by ROS mediated Akt hyperphosphorylation/activation. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  18. Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration

    International Nuclear Information System (INIS)

    Braun, Ralf J.

    2012-01-01

    Mitochondrial damage and dysfunction are common hallmarks for neurodegenerative disorders, including Alzheimer, Parkinson, Huntington diseases, and the motor neuron disorder amyotrophic lateral sclerosis. Damaged mitochondria pivotally contribute to neurotoxicity and neuronal cell death in these disorders, e.g., due to their inability to provide the high energy requirements for neurons, their generation of reactive oxygen species (ROS), and their induction of mitochondrion-mediated cell death pathways. Therefore, in-depth analyses of the underlying molecular pathways, including cellular mechanisms controlling the maintenance of mitochondrial function, is a prerequisite for a better understanding of neurodegenerative disorders. The yeast Saccharomyces cerevisiae is an established model for deciphering mitochondrial quality control mechanisms and the distinct mitochondrial roles during apoptosis and programmed cell death. Cell death upon expression of various human neurotoxic proteins has been characterized in yeast, revealing neurotoxic protein-specific differences. This review summarizes how mitochondria are affected in these neurotoxic yeast models, and how they are involved in the execution and prevention of cell death. I will discuss to which extent this mimics the situation in other neurotoxic model systems, and how this may contribute to a better understanding of the mitochondrial roles in the human disorders.

  19. The Proteasome Inhibitor Bortezomib Sensitizes AML with Myelomonocytic Differentiation to TRAIL Mediated Apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Dijk, Marianne van; Murphy, Eoin [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Natural Sciences, National University of Ireland, University Road, Galway (Ireland); Morrell, Ruth [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Natural Sciences, National University of Ireland, University Road, Galway (Ireland); School of Medicine, National University of Ireland, University Road, Galway (Ireland); Knapper, Steven [Department of Haematology, School of Medicine, Cardiff University, Heath Park, CF14 4XN Cardiff (United Kingdom); O' Dwyer, Michael [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Medicine, National University of Ireland, University Road, Galway (Ireland); Samali, Afshin; Szegezdi, Eva, E-mail: eva.szegezdi@nuigalway.ie [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Natural Sciences, National University of Ireland, University Road, Galway (Ireland)

    2011-03-15

    Acute myeloid leukemia (AML) is an aggressive stem cell malignancy that is difficult to treat. There are limitations to the current treatment regimes especially after disease relapse, and therefore new therapeutic agents are urgently required which can overcome drug resistance whilst avoiding unnecessary toxicity. Among newer targeted agents, both tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and proteasome inhibitors show particular promise. In this report we show that a combination of the proteasome inhibitor bortezomib and TRAIL is effective against AML cell lines, in particular, AML cell lines displaying myelomonocytic/monocytic phenotype (M4/M5 AML based on FAB classification), which account for 20-30% of AML cases. We show that the underlying mechanism of sensitization is at least in part due to bortezomib mediated downregulation of c-FLIP and XIAP, which is likely to be regulated by NF-κB. Blockage of NF-κB activation with BMS-345541 equally sensitized myelomonocytic AML cell lines and primary AML blasts to TRAIL.

  20. The Proteasome Inhibitor Bortezomib Sensitizes AML with Myelomonocytic Differentiation to TRAIL Mediated Apoptosis

    Directory of Open Access Journals (Sweden)

    Eva Szegezdi

    2011-03-01

    Full Text Available Acute myeloid leukemia (AML is an aggressive stem cell malignancy that is difficult to treat. There are limitations to the current treatment regimes especially after disease relapse, and therefore new therapeutic agents are urgently required which can overcome drug resistance whilst avoiding unnecessary toxicity. Among newer targeted agents, both tumor necrosis factor (TNF-related apoptosis-inducing ligand (TRAIL and proteasome inhibitors show particular promise. In this report we show that a combination of the proteasome inhibitor bortezomib and TRAIL is effective against AML cell lines, in particular, AML cell lines displaying myelomonocytic/monocytic phenotype (M4/M5 AML based on FAB classification, which account for 20-30% of AML cases. We show that the underlying mechanism of sensitization is at least in part due to bortezomib mediated downregulation of c-FLIP and XIAP, which is likely to be regulated by NF-κB. Blockage of NF-κB activation with BMS-345541 equally sensitized myelomonocytic AML cell lines and primary AML blasts to TRAIL.

  1. Apoptosis-mediated in vivo toxicity of hydroxylated fullerene nanoparticles in soil nematode Caenorhabditis elegans.

    Science.gov (United States)

    Cha, Yun Jeong; Lee, Jaesang; Choi, Shin Sik

    2012-03-01

    Although a number of manufactured nanoparticles are applied for the medical and clinical purposes, the understanding of interaction between nanomaterials and biological systems are still insufficient. Using nematode Caenorhabditis elegans model organism, we here investigated the in vivo toxicity or safety of hydroxylated fullerene nanoparticles known to detoxify anti-cancer drug-induced oxidative damages in mammals. The survival ratio of C. elegans rapidly decreased by the uptake of nanoparticles from their L4 larval stage with resulting in shortened lifespan (20 d). Both reproduction rate and body size of C. elegans were also reduced after exposure to 100 μg mL(-1) of fullerol. We found ectopic cell corpses caused by apoptotic cell death in the adult worms grown with fullerol nanoparticles. By the mutation of core pro-apoptotic regulator genes, ced-3 and ced-4, these nanoparticle-induced cell death were significantly suppressed, and the viability of animals consequently increased despite of nanoparticle uptake. The apoptosis-mediated toxicity of nanoparticles particularly led to the disorder of digestion system in the animals containing a large number of undigested foods in their intestine. These results demonstrated that the water-soluble fullerol nanoparticles widely used in medicinal applications have a potential for inducing apoptotic cell death in multicellular organisms despite of their antioxidative detoxifying property. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    International Nuclear Information System (INIS)

    Wang, Yi-Fen; Shyu, Huey-Wen; Chang, Yi-Chuang; Tseng, Wei-Chang; Huang, Yeou-Lih; Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling; Chen, Chang-Yu

    2012-01-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  3. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi-Fen; Shyu, Huey-Wen [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chang, Yi-Chuang [Department of Nursing, Fooyin University, Kaohsiung, Taiwan (China); Tseng, Wei-Chang [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Huang, Yeou-Lih [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chen, Chang-Yu, E-mail: mt037@mail.fy.edu.tw [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China)

    2012-03-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  4. Sulforaphane induces reactive oxygen species-mediated mitotic arrest and subsequent apoptosis in human bladder cancer 5637 cells.

    Science.gov (United States)

    Park, Hyun Soo; Han, Min Ho; Kim, Gi-Young; Moon, Sung-Kwon; Kim, Wun-Jae; Hwang, Hye Jin; Park, Kun Young; Choi, Yung Hyun

    2014-02-01

    The present study was undertaken to determine whether sulforaphane-derived reactive oxygen species (ROS) might cause growth arrest and apoptosis in human bladder cancer 5637 cells. Our results show that the reduced viability of 5637 cells by sulforaphane is due to mitotic arrest, but not the G2 phase. The sulforaphane-induced mitotic arrest correlated with an induction of cyclin B1 and phosphorylation of Cdk1, as well as a concomitant increased complex between cyclin B1 and Cdk1. Sulforaphane-induced apoptosis was associated with the activation of caspase-8 and -9, the initiators caspases of the extrinsic and intrinsic apoptotic pathways, respectively, and activation of effector caspase-3 and cleavage of poly (ADP-ribose) polymerase. However, blockage of caspase activation inhibited apoptosis and abrogated growth inhibition in sulforaphane-treated 5637 cells. This study further investigated the roles of ROS with respect to mitotic arrest and the apoptotic effect of sulforaphane, and the maximum level of ROS accumulation was observed 3h after sulforaphane treatment. However, a ROS scavenger, N-acetyl-L-cysteine, notably attenuated sulforaphane-mediated apoptosis as well as mitotic arrest. Overall, these results suggest that sulforaphane induces mitotic arrest and apoptosis of 5637 cells via a ROS-dependent pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen Ngoc, Tam Dan [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Son, Young-Ok [Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Lim, Shin-Saeng [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Shi, Xianglin [Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Kim, Jong-Ghee [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Heo, Jung Sun [Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Choe, Youngji [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Jeon, Young-Mi, E-mail: young@jbnu.ac.kr [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Lee, Jeong-Chae, E-mail: leejc88@jbnu.ac.kr [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of)

    2012-03-15

    Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G{sub 2}/M phase. The addition of NaF induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53 in mESCs.

  6. Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

    International Nuclear Information System (INIS)

    Nguyen Ngoc, Tam Dan; Son, Young-Ok; Lim, Shin-Saeng; Shi, Xianglin; Kim, Jong-Ghee; Heo, Jung Sun; Choe, Youngji; Jeon, Young-Mi; Lee, Jeong-Chae

    2012-01-01

    Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G 2 /M phase. The addition of NaF induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53 in mESCs.

  7. E2F1-Mediated Induction of NFYB Attenuates Apoptosis via Joint Regulation of a Pro-Survival Transcriptional Program.

    Directory of Open Access Journals (Sweden)

    Xiaolei Jiang

    Full Text Available The E2F1 transcription factor regulates cell proliferation and apoptosis through the control of a considerable variety of target genes. Previous work has detailed the role of other transcription factors in mediating the specificity of E2F function. Here we identify the NF-YB transcription factor as a novel direct E2F1 target. Genome-wide expression analysis of the effects of NFYB knockdown on E2F1-mediated transcription identified a large group of genes that are co-regulated by E2F1 and NFYB. We also provide evidence that knockdown of NFYB enhances E2F1-induced apoptosis, suggesting a pro-survival function of the NFYB/E2F1 joint transcriptional program. Bioinformatic analysis suggests that deregulation of these NFY-dependent E2F1 target genes might play a role in sarcomagenesis as well as drug resistance.

  8. Caspase-2 mediates a Brucella abortus RB51-induced hybrid cell death having features of apoptosis and pyroptosis

    Directory of Open Access Journals (Sweden)

    Denise Nicole Bronner

    2013-11-01

    Full Text Available Programmed cell death (PCD can play a crucial role in tuning the immune response to microbial infection. Although PCD can occur in different forms, all are mediated by a family of proteases called caspases. Caspase-2 is the most conserved caspase; however its function in cell death is ill-defined. Previously we demonstrated that live attenuated cattle vaccine strain Brucella abortus RB51 induces caspase-2-mediated PCD of infected macrophages. However, the mechanism of caspase-2-mediated cell death pathway remained unclear. In this study, we found that caspase-2 mediated proinflammatory cell death of RB51-infected macrophages and regulated many genes in different PCD pathways. We show that the activation of proapoptotic caspases-3 and -8 was dependent upon caspase-2. Caspase-2 regulated mitochondrial cytochrome c release and TNFα production, both of which are known to activate caspase-3 and caspase-8, respectively. In addition to TNFα, RB51-induced caspase-1 and IL-1β production was also driven by caspase-2-mediated mitochondrial dysfunction. Interestingly, pore formation, a phenomenon commonly associated with caspase-1-mediated pyroptosis, occurred; however it did not contribute to RB51-induced proinflammatory cell death. Our data suggest that caspase-2 acts as an initiator caspase that mediates a novel RB51-induced hybrid cell death that simulates but differs from typical apoptosis and pyroptosis. The initiator role of the caspase-2-mediated cell death was also conserved in cellular stress-induced cell death of macrophages treated with etoposide, naphthalene, or anti-Fas. Caspase-2 also regulated caspase-3 and -8 activation, as well as cell death in macrophages treated with each of the three reagents. Taken together, our data has demonstrated that caspase-2 can play an important role in mediating a proinflammatory response and a hybrid cell death that demonstrates features of both apoptosis and pyroptosis.

  9. Qualitative and Quantitative Analysis of ROS-Mediated Oridonin-Induced Oesophageal Cancer KYSE-150 Cell Apoptosis by Atomic Force Microscopy.

    Directory of Open Access Journals (Sweden)

    Jiang Pi

    Full Text Available High levels of intracellular reactive oxygen species (ROS in cells is recognized as one of the major causes of cancer cell apoptosis and has been developed into a promising therapeutic strategy for cancer therapy. However, whether apoptosis associated biophysical properties of cancer cells are related to intracellular ROS functions is still unclear. Here, for the first time, we determined the changes of biophysical properties associated with the ROS-mediated oesophageal cancer KYSE-150 cell apoptosis using high resolution atomic force microscopy (AFM. Oridonin was proved to induce ROS-mediated KYSE-150 cell apoptosis in a dose dependent manner, which could be reversed by N-acetylcysteine (NAC pretreatment. Based on AFM imaging, the morphological damage and ultrastructural changes of KYSE-150 cells were found to be closely associated with ROS-mediated oridonin-induced KYSE-150 cell apoptosis. The changes of cell stiffness determined by AFM force measurement also demonstrated ROS-dependent changes in oridonin induced KYSE-150 cell apoptosis. Our findings not only provided new insights into the anticancer effects of oridonin, but also highlighted the use of AFM as a qualitative and quantitative nanotool to detect ROS-mediated cancer cell apoptosis based on cell biophysical properties, providing novel information of the roles of ROS in cancer cell apoptosis at nanoscale.

  10. Post-operative infection and sepsis in humans is associated with deficient gene expression of γc cytokines and their apoptosis mediators.

    LENUS (Irish Health Repository)

    White, Mary

    2011-06-01

    Lymphocyte homeostasis is dependent on the γc cytokines. We hypothesised that sepsis in humans is associated with differential gene expression of the γc cytokines and their associated apoptosis mediators.

  11. High Glucose-Mediated Tyrosine Nitration of PI3-Kinase: A Molecular Switch of Survival and Apoptosis in Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Sally L. Elshaer

    2018-03-01

    Full Text Available Diabetes and hyperglycemia are associated with increased retinal oxidative and nitrative stress and vascular cell death. Paradoxically, high glucose stimulates expression of survival and angiogenic growth factors. Therefore, we examined the hypothesis that high glucose-mediated tyrosine nitration causes inhibition of the survival protein PI3-kinase, and in particular, its regulatory p85 subunit in retinal endothelial cell (EC cultures. Retinal EC were cultured in high glucose (HG, 25 mM for 3 days or peroxynitrite (PN, 100 µM overnight in the presence or absence of a peroxynitrite decomposition catalyst (FeTPPs, 2.5 µM, or the selective nitration inhibitor epicatechin (100 µM. Apoptosis of ECs was assessed using TUNEL assay and caspase-3 activity. Immunoprecipitation and Western blot were used to assess protein expression and tyrosine nitration of p85 subunit and its interaction with the p110 subunit. HG or PN accelerated apoptosis of retinal ECs compared to normal glucose (NG, 5 mM controls. HG- or PN-treated cells also showed significant increases in tyrosine nitration on the p85 subunit of PI3-kinase that inhibited its association with the catalytic p110 subunit and impaired PI3-kinase/Akt kinase activity. Decomposing peroxynitrite or blocking tyrosine nitration of p85 restored the activity of PI3-kinase, and prevented apoptosis and activation of p38 MAPK. Inhibiting p38 MAPK or overexpression of the constitutively activated Myr-Akt construct prevented HG- or peroxynitrite-mediated apoptosis. In conclusion, HG impairs pro-survival signals and causes accelerated EC apoptosis, at least in part via tyrosine nitration and inhibition of PI3-kinase. Inhibitors of nitration can be used in adjuvant therapy to delay diabetic retinopathy and microvascular complication.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Insights on the involvement of (-)-epigallocatechin gallate in ER stress-mediated apoptosis in age-related macular degeneration.

    Science.gov (United States)

    Karthikeyan, Bose; Harini, Lakshminarasimhan; Krishnakumar, Vaithilingam; Kannan, Velu Rajesh; Sundar, Krishnan; Kathiresan, Thandavarayan

    2017-01-01

    Endoplasmic reticulum (ER) stress-mediated apoptosis is a well-known factor in the pathogenesis of age-related macular degeneration (AMD). ER stress leads to accumulation of misfolded proteins, which in turn activates unfolded protein response (UPR) of the cell for its survival. The prolonged UPR of ER stress promotes cell death; however, the transition between adaptation and ER stress-induced apoptosis has not been clearly understood. Hence, the present study investigates the regulatory effect of (-)-epigallocatechin gallate (EGCG) on ER stress-induced by hydrogen peroxide (H 2 O 2 ) and disturbance of calcium homeostasis by thapsigargin (TG) in mouse retinal pigment epithelial (MRPE) cells. The oxidant molecules influenced MRPE cells showed an increased level of intracellular calcium [Ca 2+ ] i in ER and transferred to mitochondria through ER-mitochondrial tether site then increased ROS production. EGCG restores [Ca 2+ ] i homeostasis by decreasing ROS production through inhibition of prohibitin1 which regulate ER-mitochondrial tether site and inhibit apoptosis. Effect of EGCG on ER stress-mediated apoptosis was elucidated by exploring the UPR signalling pathways. EGCG downregulated GRP78, CHOP, PERK, ERO1α, IRE1α, cleaved PARP, cleaved caspase 3, caspase 12 and upregulated expression of calnexinin MRPE cells. In addition to this, inhibition of apoptosis by EGCG was also confirmed with expression of proteins Akt, PTEN and GSK3β. MRPE cells with EGCG upregulates phosphorylation of Akt at ser473 and phospho ser380 of PTEN, but phosphorylation at ser9 of GSK3β was inhibited. Further, constitutively active (myristoylated) CA-Akt transfected in MRPE cells had an increased Akt activity in EGCG influenced cells. These findings strongly suggest that antioxidant molecules inhibit cell death through the proper balancing of [Ca 2+ ] i and ROS production in order to maintain UPR of ER in MRPE cells. Thus, modulation of UPR signalling may provide a potential target for

  14. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hongling; Huang, Yong; Du, Qian; Luo, Xiaomao; Zhang, Liang; Zhao, Xiaomin; Tong, Dewen, E-mail: dwtong@nwsuaf.edu.cn

    2015-01-09

    Highlights: • PPV reduces PK-15 cells viability by inducing apoptosis. • PPV infection induces apoptosis through mitochondria-mediated pathway. • PPV infection activates p53 to regulate the mitochondria apoptotic signaling. - Abstract: Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection.

  15. Off-Target Function of the Sonic-Hedgehog Inhibitor Cyclopamine in Mediating Apoptosis via Nitric Oxide-Dependent Neutral Sphingomyelinase 2/Ceramide Induction

    OpenAIRE

    Meyers-Needham, Marisa; Lewis, Jocelyn A.; Gencer, Salih; Sentelle, R. David; Saddoughi, Sahar A.; Clarke, Christopher J.; Hannun, Yusuf A.; Norell, Haakan; da Palma, Telma Martins; Nishimura, Michael; Kraveka, Jacqueline M.; Khavandgar, Zohreh; Murshed, Monzur; Cevik, M. Ozgur; Ogretmen, Besim

    2012-01-01

    Sonic hedgehog (SHh) signaling is important in the pathogenesis of various human cancers, such as medulloblastomas, and it has been identified as a valid target for anti-cancer therapeutics. The SHh inhibitor cyclopamine induces apoptosis. The bioactive sphingolipid ceramide mediates cell death in response to various chemotherapeutic agents; however, ceramide’s roles/mechanisms in cyclopamine-induced apoptosis are unknown. Here, we report that cyclopamine mediates ceramide generation selectiv...

  16. ARF and ATM/ATR cooperate in p53-mediated apoptosis upon oncogenic stress

    International Nuclear Information System (INIS)

    Pauklin, Siim; Kristjuhan, Arnold; Maimets, Toivo; Jaks, Viljar

    2005-01-01

    Induction of apoptosis is pivotal for eliminating cells with damaged DNA or deregulated proliferation. We show that tumor suppressor ARF and ATM/ATR kinase pathways cooperate in the induction of apoptosis in response to elevated expression of c-myc, β-catenin or human papilloma virus E7 oncogenes. Overexpression of oncogenes leads to the formation of phosphorylated H2AX foci, induction of Rad51 protein levels and ATM/ATR-dependent phosphorylation of p53. Inhibition of ATM/ATR kinases abolishes both induction of Rad51 and phosphorylation of p53, and remarkably reduces the level of apoptosis induced by co-expression of oncogenes and ARF. However, the induction of apoptosis is downregulated in p53-/- cells and does not depend on activities of ATM/ATR kinases, indicating that efficient induction of apoptosis by oncogene activation depends on coordinated action of ARF and ATM/ATR pathways in the regulation of p53

  17. Ex vivo malignant glioma cells are sensitive to Fas (CD95/APO-1) ligand-mediated apoptosis.

    Science.gov (United States)

    Frei, K; Ambar, B; Adachi, N; Yonekawa, Y; Fontana, A

    1998-07-01

    Fas (also known as CD95/APO-1) is a cell surface receptor and member of the tumor necrosis factor receptor superfamily which mediates apoptosis in sensitive cells upon oligomerization by specific antibodies or by its ligand (FasL). Recently, human glioma cell lines were found to be susceptible to Fas-mediated apoptosis triggered by alpha-Fas antibodies. However, whether the Fas system can also be targeted in ex vivo high grade gliomas is at present unknown. In the present investigation, alpha-Fas antibodies and FasL were tested in short-term monolayer cultures or in colony forming assays established from freshly resected tumors of patients with anaplastic astrocytomas (WHO grade III) and glioblastoma multiforme (WHO grade IV). Anti-Fas antibodies induced only moderate apoptosis in four of the 19 tested glioma cell cultures. This contrasts FasL which induced apoptosis in all of the 19 tumor cell cultures analyzed. Mean cytotoxicity of glioma cell cultures treated for 48 h with alpha-Fas antibodies or FasL was 9.6% and 44.3%, respectively. Irrespective of whether alpha-Fas antibodies or FasL were used, pretreatment with recombinant hu (rhu) IFN-gamma and rhuTNF-alpha for 48 h did not sensitize glioma cells to Fas-mediated cytotoxicity. The long-term effect by FasL on tumor colony formation was more striking. FasL treatment resulted in more than 90% inhibition of clonal tumor cell growth of all the eight high grade gliomas analyzed. These results suggest that Fas targeting by FasL but not by alpha-Fas antibodies may provide a promising approach for locoregional glioma treatment.

  18. Curcumin protects microglia and primary rat cortical neurons against HIV-1 gp120-mediated inflammation and apoptosis.

    Directory of Open Access Journals (Sweden)

    Luyan Guo

    Full Text Available Curcumin is a molecule found in turmeric root that has anti-inflammatory, antioxidant, and anti-tumor properties and has been widely used as both an herbal drug and a food additive to treat or prevent neurodegenerative diseases. To explore whether curcumin is able to ameliorate HIV-1-associated neurotoxicity, we treated a murine microglial cell line (N9 and primary rat cortical neurons with curcumin in the presence or absence of neurotoxic HIV-1 gp120 (V3 loop protein. We found that HIV-1 gp120 profoundly induced N9 cells to produce reactive oxygen species (ROS, tumor necrosis factor-α (TNF-α and monocyte chemoattractant protein-1 (MCP-1. HIV-1 gp120 also induced apoptosis of primary rat cortical neurons. Curcumin exerted a powerful inhibitory effect against HIV-1 gp120-induced neuronal damage, reducing the production of ROS, TNF-α and MCP-1 by N9 cells and inhibiting apoptosis of primary rat cortical neurons. Curcumin may exert its biological activities through inhibition of the delayed rectification and transient outward potassium (K(+ current, as curcumin effectively reduced HIV-1 gp120-mediated elevation of the delayed rectification and transient outward K(+ channel current in neurons. We conclude that HIV-1 gp120 increases ROS, TNF-α and MCP-1 production in microglia, and induces cortical neuron apoptosis by affecting the delayed rectification and transient outward K(+ channel current. Curcumin reduces production of ROS and inflammatory mediators in HIV-1-gp120-stimulated microglia, and protects cortical neurons against HIV-1-mediated apoptosis, most likely through inhibition of HIV-1 gp120-induced elevation of the delayed rectification and transient outward K(+ current.

  19. Differential induction of p53-mediated apoptosis in medulloblastomas and gliomas correlates with their ability to induce bax

    International Nuclear Information System (INIS)

    Shu, H.-K.G.; Furman, Felix; Dee, Suzanne; Israel, Mark A.

    1997-01-01

    Purpose/Objective: Medulloblastoma cell lines readily undergo a p53-mediated apoptosis following exposure to ionizing radiation, while glioma cell lines do not undergo significant levels of apoptosis following irradiation. This study attempts to define some of the molecular events that characterize the differential ability medulloblastomas and gliomas to undergo radiation-induced apoptosis. Materials and Methods: The medulloblastoma cell lines D283 and D341 and the glioma cell lines U87, U343 and U563 were used in this study. All five cell lines were confirmed to have a wild type p53 by their ability to induce p21 protein levels and to undergo a cell-cycle arrest in G1 following treatment with ionizing radiation. Also, 3 clonal derivatives of D283 were used. Two of the clones were derived following transfection with an expression plasmid containing a dominant negative mutant p53 (Arg175 --> His) expressed from the CMV promoter (D283/53.6 and D283/53.7), while the remaining clone was derived following transfection with that same expression plasmid without mutant p53 (D283/vec). All irradiation experiments were performed on Phillips RT-250 X-ray unit using 250 Kvp X-rays. In each case, 5 Gy of ionizing radiation was given at a dose rate of 250 cGy/minute. Apoptosis was quantitated by staining fixed cells with propidium iodide and determining the percentage of cells with subdiploid DNA content by flow cytometry. Northern blot analysis was performed using standard methods. Results: The D283 and D341 cell lines exhibited a significant induction of apoptosis when assayed 2 days following treatment with radiation while the U87, U343 and U563 cell lines displayed only minimal induction of apoptosis when assayed following treatment at that time. RNA was prepared from the different cell lines that were unirradiated, 6 hours or 24 hours post-irradiation. Northern blots were made of the total RNAs and probed for bax, bcl-2 and bcl-x mRNA. This analysis detected no significant

  20. Ganoderma atrum polysaccharide ameliorates anoxia/reoxygenation-mediated oxidative stress and apoptosis in human umbilical vein endothelial cells.

    Science.gov (United States)

    Zhang, Yan-Song; Li, Wen-Juan; Zhang, Xian-Yi; Yan, Yu-Xin; Nie, Shao-Ping; Gong, De-Ming; Tang, Xiao-Fang; He, Ming; Xie, Ming-Yong

    2017-05-01

    Ganoderma atrum polysaccharide (PSG-1), a main polysaccharide from Ganoderma atrum, possesses potent antioxidant capacity and cardiovascular benefits. The aim of this study was to investigate the role of PSG-1 in oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs) under anoxia/reoxygenation (A/R) injury conditions. The results showed that exposure of HUVECs to A/R triggered cell death and apoptosis. Administration of PSG-1 significantly inhibited A/R-induced cell death and apoptosis in HUVECs. PSG-1-reduced A/R injury was mediated via mitochondrial apoptotic pathway, as evidenced by elevation of mitochondrial Bcl-2 protein and mitochondrial membrane potential, and attenuation of Bax translocation, cytochrome c release and caspases activation. Furthermore, PSG-1 enhanced the activities of superoxide dismutase, catalase and glutathione peroxidase and glutathione content, and concomitantly attenuated reactive oxygen species generation, lipid peroxidation and glutathione disulfide content. The antioxidant, N-acetyl-l-cysteine, significantly ameliorated all of these endothelial injuries caused by A/R, suggesting that antioxidant activities might play a key role in PSG-1-induced endothelial protection. Taken together, these findings suggested that PSG-1 could be as a promising adjuvant against endothelial dysfunction through ameliorating oxidative stress and apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Mir143-BBC3 cascade reduces microglial survival via interplay between apoptosis and autophagy: Implications for methamphetamine-mediated neurotoxicity

    Science.gov (United States)

    Zhang, Yuan; Shen, Kai; Bai, Ying; Lv, Xuan; Huang, Rongrong; Zhang, Wei; Chao, Jie; Nguyen, Lan K.; Hua, Jun; Gan, Guangming; Hu, Gang; Yao, Honghong

    2016-01-01

    ABSTRACT BBC3 (BCL2 binding component 3) is a known apoptosis inducer; however, its role in microglial survival remains poorly understood. In addition to the classical transcription factor TRP53, Mir143 is involved in BBC3 expression at the post-transcriptional level. Here, we identify unique roles of Mir143-BBC3 in mediating microglial survival via the regulation of the interplay between apoptosis and autophagy. Autophagy inhibition accelerated methamphetamine-induced apoptosis, whereas autophagy induction attenuated the decrease in microglial survival. Moreover, anti-Mir143-dependent BBC3 upregulation reversed the methamphetamine-induced decrease in microglial survival via the regulation of apoptosis and autophagy. The in vivo relevance of these findings was confirmed in mouse models, which demonstrated that the microinjection of anti-Mir143 into the hippocampus ameliorated the methamphetamine-induced decrease in microglia as well as that observed in heterozygous Mir143+/− mice. These findings provide new insight regarding the specific contributions of Mir143-BBC3 to microglial survival in the context of drug abuse. PMID:27464000

  2. Hsp90 inhibitor NMS-E973 exerts the anticancer effect against glioblastoma via induction of PUMA-mediated apoptosis.

    Science.gov (United States)

    Sun, Libo; Yang, Shoujun; Chi, Guonan; Jin, Xingyi

    2018-01-01

    Glioblastoma is one of the most aggressive and common malignancies of the central nervous system in humans. Owing to the correlation of high Hsp90 expression with prognosis and clinical pathology features of diverse types of cancer, targeting Hsp90 with small-molecule inhibitors has become a promising anticancer strategy. In this study, we aimed to explore the possibility of anticancer effect of NMS-E973 in giloblastoma and elucidate the mechanism. Cell based MTT assay and colony formation assay were used to detect cell viability. Apoptosis was analyzed by nuclear staining with Hoechst 33258 and Annexin V/propidium iodide staining followed by flow cytometry. Western-blot and RT-PCR were used to detect gene expression. Xenograft assay was used to explore the anticancer effect of NMS-E973 in vivo. We found that NMS-E973 induces apoptosis and inhibits cell growth in glioblastoma cells in cell culture and xenograft models. As a proapoptotic Bcl-2 member, PUMA was induced by NMS-E973 in a p53-dependent manner in glioblastoma in cell culture, thereby inducing apoptosis in glioblastoma cells. Furthermore, PUMA was induced by NMS-E973 treatment in xenograft tumors, and deficiency in PUMA significantly suppressed the antitumor effects of NMS-E973. Our study suggests that PUMA-mediated apoptosis is important for the therapeutic responses of NMS-E973. Induction of PUMA might be a potential biomarker for predicting NMS-E973 responses.

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

    Science.gov (United States)

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

    2015-04-01

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

  4. Farnesiferol C induces cell cycle arrest and apoptosis mediated by oxidative stress in MCF-7 cell line.

    Science.gov (United States)

    Hasanzadeh, Davoud; Mahdavi, Majid; Dehghan, Gholamreza; Charoudeh, Hojjatollah Nozad

    2017-01-01

    Farnesiferol C is one of the major compounds, isolated from Ferula asafoetida (a type of coumarins) and used for cancer treatment as a folk remedy. Treatment of many cancers depends on oxidative stress situation. In this study, we sought the hypothesis that oxidative stress induced by Farnesiferol C contribute to anticancer property and induce apoptosis in MCF-7, human breast cancer cell line. We investigated the effect of Farnesiferol C on oxidative stress by measurement of some enzymes activity including catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), as well as some parameters such as total thiol and ROS levels. Also we evaluated Farnesiferol C effects on the cell cycle and apoptosis induction by using flow cytometry analysis. Our findings demonstrated that Farnesiferol C significantly induced apoptosis mediated by increasing in the cellular ROS levels. This compound increased cellular SOD and CAT activities in 24 and 48 h and reduced activity of these enzymes after 72 h exposure. Furthermore, MDA and total thiol levels were increased and decreased, respectively in the cells treated with Farnesiferol C after 24-72 h. G0/G1 phase cell cycle arrest followed by induction of apoptosis was also observed in MCF-7 cells after treatment with Farnesiferol C. According to these data, Farnesiferol C has a therapeutic effect on MCF-7 cells and can be suitable candidate for breast cancer treatment; however it is necessary for further experiments.

  5. Ursolic acid-mediated changes in glycolytic pathway promote cytotoxic autophagy and apoptosis in phenotypically different breast cancer cells.

    Science.gov (United States)

    Lewinska, Anna; Adamczyk-Grochala, Jagoda; Kwasniewicz, Ewa; Deregowska, Anna; Wnuk, Maciej

    2017-06-01

    Plant-derived pentacyclic triterpenotids with multiple biological activities are considered as promising candidates for cancer therapy and prevention. However, their mechanisms of action are not fully understood. In the present study, we have analyzed the effects of low dose treatment (5-20 µM) of ursolic acid (UA) and betulinic acid (BA) on breast cancer cells of different receptor status, namely MCF-7 (ER + , PR +/- , HER2 - ), MDA-MB-231 (ER - , PR - , HER2 - ) and SK-BR-3 (ER - , PR - , HER2 + ). UA-mediated response was more potent than BA-mediated response. Triterpenotids (5-10 µM) caused G0/G1 cell cycle arrest, an increase in p21 levels and SA-beta-galactosidase staining that was accompanied by oxidative stress and DNA damage. UA (20 µM) also diminished AKT signaling that affected glycolysis as judged by decreased levels of HK2, PKM2, ATP and lactate. UA-induced energy stress activated AMPK that resulted in cytotoxic autophagy and apoptosis. UA-mediated elevation in nitric oxide levels and ATM activation may also account for AMPK activation-mediated cytotoxic response. Moreover, UA-promoted apoptosis was associated with decreased pERK1/2 signals and the depolarization of mitochondrial membrane potential. Taken together, we have shown for the first time that UA at low micromolar range may promote its anticancer action by targeting glycolysis in phenotypically distinct breast cancer cells.

  6. Caspase-2 mediates a Brucella abortus RB51-induced hybrid cell death having features of apoptosis and pyroptosis.

    Science.gov (United States)

    Bronner, Denise N; O'Riordan, Mary X D; He, Yongqun

    2013-01-01

    Programmed cell death (PCD) can play a crucial role in tuning the immune response to microbial infection. Although PCD can occur in different forms, all are mediated by a family of proteases called caspases. Caspase-2 is the most conserved caspase, however, its function in cell death is ill-defined. Previously we demonstrated that live attenuated cattle vaccine strain Brucella abortus RB51 induces caspase-2-mediated and caspase-1-independent PCD of infected macrophages. We also discovered that rough attenuated B. suis strain VTRS1 induces a caspase-2-mediated and caspase-1-independent proinflammatory cell death in infected macrophages, which was tentatively coined "caspase-2-mediated pyroptosis". However, the mechanism of caspase-2-mediated cell death pathway remained unclear. In this study, we found that caspase-2 mediated proinflammatory cell death of RB51-infected macrophages and regulated many genes in different PCD pathways. We show that the activation of proapoptotic caspases-3 and -8 was dependent upon caspase-2. Caspase-2 regulated mitochondrial cytochrome c release and TNFα production, both of which are known to activate caspase-3 and caspase-8, respectively. In addition to TNFα, RB51-induced caspase-1 and IL-1β production was also driven by caspase-2-mediated mitochondrial dysfunction. Interestingly, pore formation, a phenomenon commonly associated with caspase-1-mediated pyroptosis, occurred; however, unlike its role in S. typhimurium-induced pyroptosis, pore formation did not contribute to RB51-induced proinflammatory cell death. Our data suggest that caspase-2 acts as an initiator caspase that mediates a novel RB51-induced hybrid cell death that simulates but differs from typical non-proinflammatory apoptosis and caspase-1-mediated proinflammatory pyroptosis. The initiator role of the caspase-2-mediated cell death was also conserved in cellular stress-induced cell death of macrophages treated with etoposide, naphthalene, or anti-Fas. Caspase-2 also

  7. Effect of modified Wuzi Yanzong Pill () on Tip60-mediated apoptosis in testis of male rats after microwave radiation.

    Science.gov (United States)

    Hu, Hai-Xiang; Sun, Jing; Gao, Ya-Jing; Fang, Hong; Xu, Shao-Qiang; Dong, Jing; Wei, Li-Zhao; Luo, Shao-Bo; Shen, Chuan-Yun; Zhang, Qi-Long; Xie, Ya-Lan

    2017-10-24

    To investigate the effect of a modified Wuzi Yanzong Pill (, WZYZP) on the male rats' testis after microwave radiation, as well as its potential mechanism. Forty-five male rats were randomly assigned to three groups: the control group, the radiation group, and the WZYZP group. The rats in the radiation group and WZYZP group were exposed to microwave radiation for 15 min once, while the rats in the control group were not exposed to any radiation. The rats in the WZYZP group were given a modified of WZYZP by gavage daily for 7 days. Apoptosis in the testis was evaluated using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. Histopathological alterations of the testis were observed by haematoxylin-eosin (HE) staining. Tat-interactive protein, 60kD (Tip60) and p53 expressions were determined by Western blotting. The apoptosis index (AI) in the radiation group was higher than that of the WZYZP group and control group on day 1 (D1), day 7 (D7) day 14 (D14) after radiation (Pradiation group, the partial seminiferous tubules were collapsed, basement membranes of the seminiferous epithelia became detached. WZYZP could restore the morphological changes. There was no expression of Tip60 among the three groups on D7 and D14. The expression of p53 was higher in the radiation group than in the control group (Pradiation on D7 and D14 (Peffects may partly result from its ability to intervene in Tip60 mediated apoptosis.

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

    Directory of Open Access Journals (Sweden)

    Tamaki Iwai

    2016-11-01

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

  9. Nanoparticle Delivery of Artesunate Enhances the Anti-tumor Efficiency by Activating Mitochondria-Mediated Cell Apoptosis

    Science.gov (United States)

    Liu, Rui; Yu, Xiwei; Su, Chang; Shi, Yijie; Zhao, Liang

    2017-06-01

    Artemisinin and its derivatives were considered to exert a broad spectrum of anti-cancer activities, and they induced significant anti-cancer effects in tumor cells. Artemisinin and its derivatives could be absorbed quickly, and they were widely distributed, selectively killing tumor cells. Since low concentrations of artesunate primarily depended on oncosis to induce cell death in tumor cells, its anti-tumor effects were undesirable and limited. To obtain better anti-tumor effects, in this study, we took advantage of a new nanotechnology to design novel artesunate-loaded bovine serum albumin nanoparticles to achieve the mitochondrial accumulation of artesunate and induce mitochondrial-mediated apoptosis. The results showed that when compared with free artesunate's reliance on oncotic death, artesunate-loaded bovine serum albumin nanoparticles showed higher cytotoxicity and their significant apoptotic effects were induced through the distribution of artesunate in the mitochondria. This finding indicated that artesunate-loaded bovine serum albumin nanoparticles damaged the mitochondrial integrity and activated mitochondrial-mediated cell apoptosis by upregulating apoptosis-related proteins and facilitating the rapid release of cytochrome C.

  10. Butyrate suppresses colonic inflammation through HDAC1-dependent Fas upregulation and Fas-mediated apoptosis of T cells

    Science.gov (United States)

    Zimmerman, Mary A.; Singh, Nagendra; Martin, Pamela M.; Thangaraju, Muthusamy; Ganapathy, Vadivel; Waller, Jennifer L.; Shi, Huidong; Robertson, Keith D.; Munn, David H.

    2012-01-01

    Butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit protective effects toward inflammatory diseases such as ulcerative colitis (UC) and inflammation-mediated colorectal cancer. Recent studies have shown that chronic IFN-γ signaling plays an essential role in inflammation-mediated colorectal cancer development in vivo, whereas genome-wide association studies have linked human UC risk loci to IFNG, the gene that encodes IFN-γ. However, the molecular mechanisms underlying the butyrate-IFN-γ-colonic inflammation axis are not well defined. Here we showed that colonic mucosa from patients with UC exhibit increased signal transducer and activator of transcription 1 (STAT1) activation, and this STAT1 hyperactivation is correlated with increased T cell infiltration. Butyrate treatment-induced apoptosis of wild-type T cells but not Fas-deficient (Faslpr) or FasL-deficient (Fasgld) T cells, revealing a potential role of Fas-mediated apoptosis of T cells as a mechanism of butyrate function. Histone deacetylase 1 (HDAC1) was found to bind to the Fas promoter in T cells, and butyrate inhibits HDAC1 activity to induce Fas promoter hyperacetylation and Fas upregulation in T cells. Knocking down gpr109a or slc5a8, the genes that encode for receptor and transporter of butyrate, respectively, resulted in altered expression of genes related to multiple inflammatory signaling pathways, including inducible nitric oxide synthase (iNOS), in mouse colonic epithelial cells in vivo. Butyrate effectively inhibited IFN-γ-induced STAT1 activation, resulting in inhibition of iNOS upregulation in human colon epithelial and carcinoma cells in vitro. Our data thus suggest that butyrate delivers a double-hit: induction of T cell apoptosis to eliminate the source of inflammation and suppression of IFN-γ-mediated inflammation in colonic epithelial cells, to suppress colonic inflammation. PMID:22517765

  11. Urokinase mediates endothelial cell survival via induction of the X-linked inhibitor of apoptosis protein

    DEFF Research Database (Denmark)

    Prager, Gerald W; Mihaly, Judit; Brunner, Patrick M

    2008-01-01

    Urokinase-type plasminogen activator (uPA) additionally elicits a whole array of pro-angiogenic responses, such as differentiation, proliferation, and migration. In this study, we demonstrate that in endothelial cells uPA also protects against apoptosis by transcriptional up-regulation and partia......Urokinase-type plasminogen activator (uPA) additionally elicits a whole array of pro-angiogenic responses, such as differentiation, proliferation, and migration. In this study, we demonstrate that in endothelial cells uPA also protects against apoptosis by transcriptional up......-regulation and partially by mRNA stabilization of inhibitor of apoptosis proteins, most prominently the X-linked inhibitor of apoptosis protein (XIAP). The antiapoptotic activity of uPA was dependent on its protease activity, the presence of uPA receptor (uPAR) and low-density lipoprotein receptor-related protein (LRP...

  12. Iodinated contrast media induce neutrophil apoptosis through a mitochondrial and caspase mediated pathway.

    LENUS (Irish Health Repository)

    Fanning, N F

    2012-02-03

    Iodinated contrast media (ICM) can induce apoptosis (programmed cell death) in renal, myocardial and endothelial cells. Following intravascular injection, circulating immune cells are exposed to high concentrations of ICM. As neutrophils constitutively undergo apoptosis we hypothesized that ICM may adversely affect neutrophil survival. Our aim was to investigate the effect of ICM on neutrophil apoptosis. Neutrophils were isolated from healthy subjects and cultured in vitro with ionic (diatrizoate and ioxaglate) and non-ionic (iohexol and iotrolan) ICM. The effect of ICM on neutrophil apoptosis in both unstimulated and lipopolysaccharide-stimulated neutrophils was determined by annexin V flow cytometry. The influence of physicochemical properties of the different ICM on apoptosis of neutrophils was also studied. We further investigated the effects of ICM on key intracellular signal pathways, including p38 mitogen-activated protein kinase (MAPK) by Western blotting, and mitochondrial depolarization and caspase activity by flow cytometry. Isoiodine concentrations (20 mg ml(-1)) of ionic (diatrizoate 69.6+\\/-2.9%; ioxaglate 58.9+\\/-2.0%) and non-ionic (iohexol 57.3+\\/-2.9%; iotrolan 57.1+\\/-2.6%) ICM significantly induced neutrophil apoptosis over control levels (47.7+\\/-1.4%). The apoptotic effect of ICM was influenced by their chemical structure, with ionic ICM having a more significant (p<0.01) apoptotic effect than non-ionic ICM (p<0.05). Furthermore, ICM reversed the anti-apoptotic effect of lipopolysaccharide (1000 ng ml(-1)) treated neutrophils to control levels (23.0+\\/-3.5% to 61.2+\\/-5.3%; n=4; p<0.05). These agents induce apoptosis through a p38 MAPK independent pathway that results in mitochondrial depolarization, and is dependent on caspase activation. As neutrophils play a central role in host response to infection and injury, ICM, through induction of neutrophil apoptosis, could have a significant deleterious effect on host immune defence and

  13. Expression of Cyt-c-Mediated Mitochondrial Apoptosis-Related Proteins in Rat Renal Proximal Tubules during Development.

    Science.gov (United States)

    Song, Xiao-Feng; Tian, He; Zhang, Ping; Zhang, Zhen-Xing

    2017-01-01

    Apoptosis regulates embryogenesis, organ metamorphosis and tissue homeostasis. Mitochondrial signaling is an apoptotic pathway, in which Cyt-c and Apaf-1 are transformed into an apoptosome, which activates procaspase-9 and triggers apoptosis. This study evaluated Cyt-c, Apaf-1 and caspase-9 expression during renal development. Kidneys from embryonic (E) 16-, 18-, and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups were obtained. Immunohistochemical analysis, dual-labeled immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique assay and Western blot were performed in addition to histological analysis. Immunohistochemistry showed that Cyt-c was strongly expressed in proximal and distal tubules (DTs) at all time points. Caspase-9 and Apaf-1 were strongly expressed in proximal tubules (PTs) but only weakly expressed in DTs. Dual-labeled immunofluorescence showed that most tubules expressed both Cyt-c and Apaf-1, except for some tubules that only expressed Cyt-c. The TUNEL assay showed a greater percentage of apoptotic cells in PTs compared to DTs. Apaf-1 and cleaved caspase-9 protein expression gradually increased during the embryonic period and peaked during the early postnatal period but apparently declined from P7. Cyt-c protein expression was weak during the embryonic period but obviously increased after P1. This study showed that PTs are more sensitive to apoptosis than DTs during rat renal development, even though both tubule segments contain a large number of mitochondria. Furthermore, Cyt-c-mediated mitochondrial apoptosis-related proteins play an important role in PTs during the early postnatal kidney development. © 2016 S. Karger AG, Basel.

  14. Lymphotropic Virions Affect Chemokine Receptor-Mediated Neural Signaling and Apoptosis: Implications for Human Immunodeficiency Virus Type 1-Associated Dementia

    Science.gov (United States)

    Zheng, Jialin; Ghorpade, Anuja; Niemann, Douglas; Cotter, Robin L.; Thylin, Michael R.; Epstein, Leon; Swartz, Jennifer M.; Shepard, Robin B.; Liu, Xiaojuan; Nukuna, Adeline; Gendelman, Howard E.

    1999-01-01

    Chemokine receptors pivotal for human immunodeficiency virus type 1 (HIV-1) infection in lymphocytes and macrophages (CCR3, CCR5, and CXCR4) are expressed on neural cells (microglia, astrocytes, and/or neurons). It is these cells which are damaged during progressive HIV-1 infection of the central nervous system. We theorize that viral coreceptors could effect neural cell damage during HIV-1-associated dementia (HAD) without simultaneously affecting viral replication. To these ends, we studied the ability of diverse viral strains to affect intracellular signaling and apoptosis of neurons, astrocytes, and monocyte-derived macrophages. Inhibition of cyclic AMP, activation of inositol 1,4,5-trisphosphate, and apoptosis were induced by diverse HIV-1 strains, principally in neurons. Virions from T-cell-tropic (T-tropic) strains (MN, IIIB, and Lai) produced the most significant alterations in signaling of neurons and astrocytes. The HIV-1 envelope glycoprotein, gp120, induced markedly less neural damage than purified virions. Macrophage-tropic (M-tropic) strains (ADA, JR-FL, Bal, MS-CSF, and DJV) produced the least neural damage, while 89.6, a dual-tropic HIV-1 strain, elicited intermediate neural cell damage. All T-tropic strain-mediated neuronal impairments were blocked by the CXCR4 antibody, 12G5. In contrast, the M-tropic strains were only partially blocked by 12G5. CXCR4-mediated neuronal apoptosis was confirmed in pure populations of rat cerebellar granule neurons and was blocked by HA1004, an inhibitor of calcium/calmodulin-dependent protein kinase II, protein kinase A, and protein kinase C. Taken together, these results suggest that progeny HIV-1 virions can influence neuronal signal transduction and apoptosis. This process occurs, in part, through CXCR4 and is independent of CD4 binding. T-tropic viruses that traffic in and out of the brain during progressive HIV-1 disease may play an important role in HAD neuropathogenesis. PMID:10482576

  15. Heat Killed Attenuated Leishmania Induces Apoptosis of HepG2 Cells Through ROS Mediated p53 Dependent Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Dipayan Bose

    2016-03-01

    Full Text Available Background/Aims: Cytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. Methods: Spectrophotometric study to analyze cell death and levels of different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different apoptotic markers. Results: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. Conclusion: It has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therapy.

  16. Mitochondrial-mediated apoptosis in lymphoma cells by the diterpenoid lactone Andrographolide, the active component of Andrographis paniculata

    Science.gov (United States)

    Yang, Shuo; Evens, Andrew M.; Prachand, Sheila; Singh, Amareshwar T.K; Bhalla, Savita; David, Kevin; Gordon, Leo I.

    2010-01-01

    Purpose Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an herbal medicine used in Asia. It has been reported to have anti-inflammatory, antihypertensive, anti-viral and immune-stimulant properties. Furthermore, it has been shown to inhibit cancer cell proliferation and induce apoptosis in leukemia and solid tumor cell lines. Experimental Design We studied the Burkitt p53 mutated Ramos cell line, the mantle-cell lymphoma (MCL) line Granta, the follicular lymphoma (FL) cell line HF-1 and the diffuse large B-cell lymphoma (DLBCL) cell line SUDHL4, as well as primary cells from patients with FL, DLBCL, and MCL. Results We found that andrographolide resulted in dose- and time-dependent cell death as measured by MTT. Andrographolide significantly increased reactive oxygen species (ROS) production in all cell lines. To determine mechanism of cell death, we measured apoptosis by Annexin-V/propidium iodide (PI) in the presence and absence of the antioxidant N-acetyl-L-cysteine (NAC), the glutathione-depleting agent buthionine sulfoxamine (BSO), or caspase inhibitors. We found that apoptosis was greatly enhanced by BSO, blocked by NAC, and accompanied by PARP cleavage and activation of caspases 3, 8 and 9. We measured BAX conformational change, and mitochondrial membrane potential, and using mouse embryonic fibroblast (MEF) Bax/Bak double knockouts (MEFBax−/−/Bak−/−), we found that apoptosis was mediated through mitochondrial pathways, but dependent on caspases in both cell lines and in patient samples. Conclusions Andrographolide caused ROS-dependent apoptosis in lymphoma cell lines and in primary tumor samples, which was enhanced by depletion of GSH and inhibited by NAC or the pan-caspase inhibitor Z-VAD-FMK. Further studies of diterpenoid lactones in lymphoma are warranted. PMID:20798229

  17. Mitochondrial-mediated apoptosis in lymphoma cells by the diterpenoid lactone andrographolide, the active component of Andrographis paniculata.

    Science.gov (United States)

    Yang, Shuo; Evens, Andrew M; Prachand, Sheila; Singh, Amareshwar T K; Bhalla, Savita; David, Kevin; Gordon, Leo I

    2010-10-01

    Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an herbal medicine used in Asia. It has been reported to have anti-inflammatory, antihypertensive, antiviral, and immune-stimulant properties. Furthermore, it has been shown to inhibit cancer cell proliferation and induce apoptosis in leukemia and solid tumor cell lines. We studied the Burkitt p53-mutated Ramos cell line, the mantle cell lymphoma (MCL) line Granta, the follicular lymphoma (FL) cell line HF-1, and the diffuse large B-cell lymphoma (DLBCL) cell line SUDHL4, as well as primary cells from patients with FL, DLBCL, and MCL. We found that andrographolide resulted in dose- and time-dependent cell death as measured by MTT. Andrographolide significantly increased reactive oxygen species (ROS) production in all cell lines. To determine mechanism of cell death, we measured apoptosis by Annexin V/propidium iodide in the presence and absence of the antioxidant N-acetyl-l-cysteine (NAC), the glutathione (GSH)-depleting agent buthionine sulfoxamine (BSO), or caspase inhibitors. We found that apoptosis was greatly enhanced by BSO, blocked by NAC, and accompanied by poly(ADP-ribose) polymerase cleavage and activation of caspase-3, caspase-8, and caspase-9. We measured BAX conformational change and mitochondrial membrane potential, and using mouse embryonic fibroblast (MEF) Bax/Bak double knockouts (MEF(Bax-/-/Bak-/-)), we found that apoptosis was mediated through mitochondrial pathways, but dependent on caspases in both cell lines and patient samples. Andrographolide caused ROS-dependent apoptosis in lymphoma cell lines and in primary tumor samples, which was enhanced by depletion of GSH and inhibited by NAC or the pan-caspase inhibitor Z-VAD-FMK. Further studies of diterpenoid lactones in lymphoma are warranted. ©2010 AACR.

  18. Hsp90 inhibitor NMS-E973 exerts the anticancer effect against glioblastoma via induction of PUMA-mediated apoptosis

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    Sun L

    2018-03-01

    Full Text Available Libo Sun,1 Shoujun Yang,2 Guonan Chi,1 Xingyi Jin1 1First Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changhun, Jilin, People’s Republic of China; 2Department of Rehabilitation, China-Japan Union Hospital of Jilin University, Changhun, Jilin, People’s Republic of China Background: Glioblastoma is one of the most aggressive and common malignancies of the central nervous system in humans. Owing to the correlation of high Hsp90 expression with prognosis and clinical pathology features of diverse types of cancer, targeting Hsp90 with small-molecule inhibitors has become a promising anticancer strategy.Purpose: In this study, we aimed to explore the possibility of anticancer effect of NMS-E973 in giloblastoma and elucidate the mechanism.Methods: Cell based MTT assay and colony formation assay were used to detect cell viability. Apoptosis was analyzed by nuclear staining with Hoechst 33258 and Annexin V/propidium iodide staining followed by flow cytometry. Western-blot and RT-PCR were used to detect gene expression. Xenograft assay was used to explore the anticancer effect of NMS-E973 in vivo.Results: We found that NMS-E973 induces apoptosis and inhibits cell growth in glioblastoma cells in cell culture and xenograft models. As a proapoptotic Bcl-2 member, PUMA was induced by NMS-E973 in a p53-dependent manner in glioblastoma in cell culture, thereby inducing apoptosis in glioblastoma cells. Furthermore, PUMA was induced by NMS-E973 treatment in xenograft tumors, and deficiency in PUMA significantly suppressed the antitumor effects of NMS-E973.Conclusion: Our study suggests that PUMA-mediated apoptosis is important for the therapeutic responses of NMS-E973. Induction of PUMA might be a potential biomarker for predicting NMS-E973 responses. Keywords: NMS-E973, PUMA, glioblastoma, apoptosis

  19. Valsartan reduces AT1-AA-induced apoptosis through suppression oxidative stress mediated ER stress in endothelial progenitor cells.

    Science.gov (United States)

    Wang, Z-C; Qi, J; Liu, L-M; Li, J; Xu, H-Y; Liang, B; Li, B

    2017-03-01

    Valsartan has been reported to have the function of treating hypertension and improving the prognosis of patients. Many studies indicated that valsartan can also increase angiotensin II, andosterone and plasma renin activity (PRA). Autoantibodies against the angiotensin II type 1 receptor (AT1-AA) have been showed to increase reactive oxygen species (ROS) and calcium (Ca2+) and result in apoptosis in vascular smooth muscle cells. In this study, we attempted to explore the effect of valsartan on AT1-AA-induced apoptosis in endothelial progenitor cells. Endothelial progenitor cells (EPCs) were cultured. The cytotoxicity was determined by MTT assay. EPCs apoptosis was determined by DAPI staining and flow cytometry. Reactive oxygen species, intracellular calcium concentration and calpain activity were measured using Fluostar Omega Spectrofluorimeter. The expression of p-ERK, p-eIF-2a, CHOP, Bcl-2 and caspase-3 were detected by Western blot. MTT assays showed valsartan significantly inhibited AT1-AA- induced decline of the viability of EPCs. DAPI staining and flow cytometry results indicated valsartan inhibited AT1-AA-induced decline of the viability of EPCs via inhibiting AT1-AA-induced apoptosis. Furthermore, the increasing of reactive oxygen species, intracellular calcium and calpain activity induced by AT1-AA in EPCs were also recovered after pre-treated with valsartan. Meanwhile, the upregulation of p-ERK, p-eIF-2a and CHOP, downregulation of Bcl-2, and activation of Caspase-3 caused by AT1-AA were reversed after pre-incubated with valsartan. Valsartan could inhibit AT1-AA-induced apoptosis through inhibiting oxidative stress mediated ER stress in EPCs.

  20. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro.

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    Fuqiang Xing

    Full Text Available Recent studies have reported that exposure of mammalian cells to microwave radiation may have adverse effects such as induction of cell apoptosis. However, the molecular mechanisms underlying microwave induced mammalian cell apoptosis are not fully understood. Here, we report a novel mechanism: exposure to 1800MHz microwave radiation induces p53-dependent cell apoptosis through cytochrome c-mediated caspase-3 activation pathway. We first measured intensity of microwave radiation from several electronic devices with an irradiation detector. Mouse NIH/3T3 and human U-87 MG cells were then used as receivers of 1800MHz electromagnetic radiation (EMR at a power density of 1209 mW/m2. Following EMR exposure, cells were analyzed for viability, intracellular reactive oxygen species (ROS generation, DNA damage, p53 expression, and caspase-3 activity. Our analysis revealed that EMR exposure significantly decreased viability of NIH/3T3 and U-87 MG cells, and increased caspase-3 activity. ROS burst was observed at 6 h and 48 h in NIH/3T3 cells, while at 3 h in U-87 MG cells. Hoechst 33258 staining and in situ TUNEL assay detected that EMR exposure increased DNA damage, which was significantly restrained in the presence of N-acetyl-L-cysteine (NAC, an antioxidant. Moreover, EMR exposure increased the levels of p53 protein and p53 target gene expression, promoted cytochrome c release from mitochondrion, and increased caspase-3 activity. These events were inhibited by pretreatment with NAC, pifithrin-α (a p53 inhibitor and caspase inhibitor. Collectively, our findings demonstrate, for the first time, that 1800MHz EMR induces apoptosis-related events such as ROS burst and more oxidative DNA damage, which in turn promote p53-dependent caspase-3 activation through release of cytochrome c from mitochondrion. These findings thus provide new insights into physiological mechanisms underlying microwave-induced cell apoptosis.

  1. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro

    Science.gov (United States)

    Xing, Fuqiang; Zhan, Qiuqiang; He, Yiduo; Cui, Jiesheng; He, Sailing; Wang, Guanyu

    2016-01-01

    Recent studies have reported that exposure of mammalian cells to microwave radiation may have adverse effects such as induction of cell apoptosis. However, the molecular mechanisms underlying microwave induced mammalian cell apoptosis are not fully understood. Here, we report a novel mechanism: exposure to 1800MHz microwave radiation induces p53-dependent cell apoptosis through cytochrome c-mediated caspase-3 activation pathway. We first measured intensity of microwave radiation from several electronic devices with an irradiation detector. Mouse NIH/3T3 and human U-87 MG cells were then used as receivers of 1800MHz electromagnetic radiation (EMR) at a power density of 1209 mW/m2. Following EMR exposure, cells were analyzed for viability, intracellular reactive oxygen species (ROS) generation, DNA damage, p53 expression, and caspase-3 activity. Our analysis revealed that EMR exposure significantly decreased viability of NIH/3T3 and U-87 MG cells, and increased caspase-3 activity. ROS burst was observed at 6 h and 48 h in NIH/3T3 cells, while at 3 h in U-87 MG cells. Hoechst 33258 staining and in situ TUNEL assay detected that EMR exposure increased DNA damage, which was significantly restrained in the presence of N-acetyl-L-cysteine (NAC, an antioxidant). Moreover, EMR exposure increased the levels of p53 protein and p53 target gene expression, promoted cytochrome c release from mitochondrion, and increased caspase-3 activity. These events were inhibited by pretreatment with NAC, pifithrin-α (a p53 inhibitor) and caspase inhibitor. Collectively, our findings demonstrate, for the first time, that 1800MHz EMR induces apoptosis-related events such as ROS burst and more oxidative DNA damage, which in turn promote p53-dependent caspase-3 activation through release of cytochrome c from mitochondrion. These findings thus provide new insights into physiological mechanisms underlying microwave-induced cell apoptosis. PMID:27689798

  2. USP22 Induces Cisplatin Resistance in Lung Adenocarcinoma by Regulating γH2AX-Mediated DNA Damage Repair and Ku70/Bax-Mediated Apoptosis

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    Aman Wang

    2017-05-01

    Full Text Available Resistance to platinum-based chemotherapy is one of the most important reasons for treatment failure in advanced non-small cell lung cancer, but the underlying mechanism is extremely complex and unclear. The present study aimed to investigate the correlation of ubiquitin-specific peptidase 22 (USP22 with acquired resistance to cisplatin in lung adenocarcinoma. In this study, we found that overexpression of USP22 could lead to cisplatin resistance in A549 cells. USP22 and its downstream proteins γH2AX and Sirt1 levels are upregulated in the cisplatin- resistant A549/CDDP cell line. USP22 enhances DNA damage repair and induce cisplatin resistance by promoting the phosphorylation of histone H2AX via deubiquitinating histone H2A. In addition, USP22 decreases the acetylation of Ku70 by stabilizing Sirt1, thus inhibiting Bax-mediated apoptosis and inducing cisplatin resistance. The cisplatin sensitivity in cisplatin-resistant A549/CDDP cells was restored by USP22 inhibition in vivo and vitro. In summary, our findings reveal the dual mechanism of USP22 involvement in cisplatin resistance that USP22 can regulate γH2AX-mediated DNA damage repair and Ku70/Bax-mediated apoptosis. USP22 is a potential target in cisplatin-resistant lung adenocarcinoma and should be considered in future therapeutic practice.

  3. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

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    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province (China); Dong, Wei-Guo, E-mail: dongwg1966@yahoo.com.cn [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province (China)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. Black-Right-Pointing-Pointer G{sub 2}/M phase arrest and chromatin condensation and nuclear fragmentation were induced. Black-Right-Pointing-Pointer Noscapine promoted apoptosis via mitochondrial pathways. Black-Right-Pointing-Pointer Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC{sub 50} = 75 {mu}M). This cytotoxicity was reflected by cell cycle arrest at G{sub 2}/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  4. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

    International Nuclear Information System (INIS)

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang; Dong, Wei-Guo

    2012-01-01

    Highlights: ► Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. ► G 2 /M phase arrest and chromatin condensation and nuclear fragmentation were induced. ► Noscapine promoted apoptosis via mitochondrial pathways. ► Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC 50 = 75 μM). This cytotoxicity was reflected by cell cycle arrest at G 2 /M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  5. Endosomes and lysosomes are involved in early steps of Tl(III)-mediated apoptosis in rat pheochromocytoma (PC12) cells.

    Science.gov (United States)

    Hanzel, Cecilia E; Almeira Gubiani, María F; Verstraeten, Sandra V

    2012-11-01

    The mechanisms that mediate thallium (Tl) toxicity are still not completely understood. The exposure of rat pheochromocytoma (PC12) cells to Tl(I) or Tl(III) activates both mitochondrial (Tl(I) and Tl(III)) and extrinsic (Tl(III)) pathways of apoptosis. In this work we evaluated the hypothesis that the effects of Tl(III) may be mediated by the damage to lysosomes, where it might be incorporated following the route of iron uptake. PC12 cells exposed for 3 h to 100 μM Tl(III) presented marked endosomal acidification, effect that was absent when cells were incubated in a serum-free medium and that was fully recovered when the latter was supplemented with transferrin. After 6 h of incubation the colocalization of cathepsins D and B with the lysosomal marker Lamp-1 was decreased together with an increase in the total activity of the enzymes. A permanent damage to lysosomes after 18 h of exposure was evidenced from the impairment of acridine orange uptake. Cathepsin D caused the cleavage of pro-apoptotic protein BID that is involved in the activation of the intrinsic pathway of apoptosis. Supporting that, BID cleavage and the activation of caspase 3 by Tl(III) were fully prevented when cells were preincubated with cathepsin D inhibitor (pepstatin A) and only partially prevented when cathepsin B inhibitor (E64d) was used. None of these inhibitors affected BID cleavage or caspase 3 activation in Tl(I)-treated cells. Together, experimental results support the role of Tl(III) uptake by the acidic cell compartments and their involvement in the early steps of Tl(III)-mediated PC12 cells apoptosis.

  6. Curcumin induces ER stress-mediated apoptosis through selective generation of reactive oxygen species in cervical cancer cells.

    Science.gov (United States)

    Kim, Boyun; Kim, Hee Seung; Jung, Eun-Ji; Lee, Jung Yun; K Tsang, Benjamin; Lim, Jeong Mook; Song, Yong Sang

    2016-05-01

    Prolonged accumulation of misfolded or unfolded proteins caused by cellular stress, including oxidative stress, induces endoplasmic reticulum stress, which then activates an unfolded protein response (UPR). ER stress is usually maintained at higher levels in cancer cells as compared to normal cells due to altered metabolism in cancer. Here, we investigated whether curcumin is ER stress-mediated apoptosis in cervical cancer cells, and ROS increased by curcumin are involved in the process as an upstream contributor. Curcumin inhibited proliferation of cervical cancer cells (C33A, CaSki, HeLa, and ME180) and induced apoptotic cell death. Curcumin activated ER-resident UPR sensors, such as PERK, IRE-1α, and ATF6, and their downstream-signaling proteins in cervical cancer cells, but not in normal epithelial cells and peripheral blood mononuclear cells (PBMCs). CHOP, a key factor involved in ER stress-mediated apoptosis, was also activated by curcumin. CHOP decreased the ratio of anti-apoptotic protein Bcl-2 to pro-apoptotic protein Bax expression, and subsequently increased the apoptotic population of cervical cancer cells. Furthermore, curcumin elevated levels of intracellular reactive oxygen species (ROS) in cervical cancer cells, but not in normal epithelial cells. Scavenging ROS resulted in inhibition of ER stress and partially restored cell viability in curcumin-treated cancer cells. Collectively, these observations show that curcumin promotes ER stress-mediated apoptosis in cervical cancer cells through increase of cell type-specific ROS generation. Therefore, modulation of these differential responses to curcumin between normal and cervical cancer cells could be an effective therapeutic strategy without adverse effects on normal cells. © 2015 Wiley Periodicals, Inc.

  7. Effects of Baicalin on inflammatory mediators and pancreatic acinar cell apoptosis in rats with sever acute pancreatitis

    Directory of Open Access Journals (Sweden)

    zhang xiping

    2009-02-01

    Full Text Available

    • BACKGROUND: To investigate the effects of Baicalin and Octreotide on inflammatory mediators and pancreatic acinar cells apoptosis of rats with severe acute pancreatitis (SAP.
    • METHODS: SD rats were randomly divided into sham operated group (I group, model control group (II group, Baicalin treated group (III group and Octreotide treated group (IV group. Each group was also divided into subgroup of 3, 6 and 12 h (n = 15. The mortality rate, ascites/body weight ratio as well as the level of endotoxin, NO and ET-1 in blood were measured. The pathological severity score of pancreas, apoptotic indexes, and expression levels of Bax and Bcl-2 proteins in each group were investigated.
    • RESULTS: The survival rate of III and IV group has a significant difference compared with II group (P12 h < 0.05. The ascites volume, contents of inflammatory mediators in blood and pathological severity score of pancreas of III and IV group declined at different degrees compared to II group (P < 0.05, P < 0.01 or P < 0.001. Apoptotic index in III group was significantly higher than that in II group at 3 and 6 h (P3, 6 h < 0.05. Apoptotic index in IV group was significantly higher than that in II group at pancreatic tail at 6 h (P6 h < 0.05. Expression level of Bax in III group was significantly higher than that in II group (pancreatic head P3 h,6 h < 0.01, pancreatic tail P3 h < 0.001.
    • CONCLUSIONS: Compared with Octreotide in the treatment of SAP, the protective mechanisms of Baicalin include reducing the excessive inflammatory mediators’ release, inducing the pancreatic acinar cells apoptosis.
    • KEY WORDS: Severe acute pancreatitis, baicalin, octreotide, inflammatory mediators, apoptosis, tissue microarrays.

  8. The molecular mechanism of dopamine-induced apoptosis: identification and characterization of genes that mediate dopamine toxicity.

    Science.gov (United States)

    Barzilai, A; Zilkha-Falb, R; Daily, D; Stern, N; Offen, D; Ziv, I; Melamed, E; Shirvan, A

    2000-01-01

    Parkinson's disease (PD) is a progressive neurological disorder caused by rather selective degeneration of the dopaminergic (DA) neurons in the substantia nigra. Though subject to intensive research, the etiology of this nigral neuronal loss is still enigmatic and treatment is basically symptomatic. The current major hypothesis suggests that nigral neuronal death in PD is due to excessive oxidative stress generated by auto- and enzymatic oxidation of the endogenous neurotransmitter dopamine (DA), the formation of neuromelanin and presence of high concentrations of iron. We have found that DA toxicity is mediated through its oxidative metabolites. Whereas thiol-containing antioxidants provided marked protection against DA toxicity, ascorbic acid accelerated DA-induced death. Using the differential display approach, we sought to isolate and characterize genes whose expression is altered in response to DA toxicity. We found an upregulation of the collapsin response mediator protein (CRM) and TCP-1delta in sympathetic neurons, which undergo dopamine-induced apoptosis. The isolation of these genes led us to examine the expression and activity of CRM and TCP-1delta related genes. Indeed, we found a significant induction of mRNAs of the secreted collapsin-1 and the mitochondrial stress protein HSP60. Antibodies directed against collapsin-1 provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. In a parallel study, using antisense technology, we found that inhibition of TCP-1delta expression significantly reduced DA-induced neuronal death. These findings suggest a functional role for collapsin-1 and TCP-1delta as positive mediators of DA-induced neuronal apoptosis.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  10. Immunoregulatory mechanisms in Chagas disease: modulation of apoptosis in T-cell mediated immune responses.

    Science.gov (United States)

    Chaves, Ana Thereza; de Assis Silva Gomes Estanislau, Juliana; Fiuza, Jacqueline Araújo; Carvalho, Andréa Teixeira; Ferreira, Karine Silvestre; Fares, Rafaelle Christine Gomes; Guimarães, Pedro Henrique Gazzinelli; de Souza Fagundes, Elaine Maria; Morato, Maria José; Fujiwara, Ricardo Toshio; da Costa Rocha, Manoel Otávio; Correa-Oliveira, Rodrigo

    2016-04-30

    Chronic Chagas disease presents different clinical manifestations ranging from asymptomatic (namely indeterminate) to severe cardiac and/or digestive. Previous results have shown that the immune response plays an important role, although no all mechanisms are understood. Immunoregulatory mechanisms such as apoptosis are important for the control of Chagas disease, possibly affecting the morbidity in chronic clinical forms. Apoptosis has been suggested to be an important mechanism of cellular response during T. cruzi infection. We aimed to further understand the putative role of apoptosis in Chagas disease and its relation to the clinical forms of the disease. Apoptosis of lymphocytes, under antigenic stimuli (soluble T. cruzi antigens - TcAg) where compared to that of non-stimulated cells. Apoptosis was evaluated using the expression of annexin and caspase 3(+) by T cells and the percentage of cells positive evaluated by flow cytometry. In addition activation and T cell markers were used for the identification of TCD4(+) and TCD8(+) subpopulations. The presence of intracellular and plasma cytokines were also evaluated. Analysis of the activation status of the peripheral blood cells showed that patients with Chagas disease presented higher levels of activation determined by the expression of activation markers, after TcAg stimulation. PCR array were used to evaluate the contribution of this mechanism in specific cell populations from patients with different clinical forms of human Chagas disease. Our results showed a reduced proliferative response associated a high expression of T CD4(+)CD62L(-) cells in CARD patients when compared with IND group and NI individuals. We also observed that both groups of patients presented a significant increase of CD4(+) and CD8(+) T cell subsets in undergoing apoptosis after in vitro stimulation with T. cruzi antigens. In CARD patients, both CD4(+) and CD8(+) T cells expressing TNF-α were highly susceptible to undergo apoptosis

  11. Betulinic Acid Induces Apoptosis in Differentiated PC12 Cells Via ROS-Mediated Mitochondrial Pathway.

    Science.gov (United States)

    Wang, Xi; Lu, Xiaocheng; Zhu, Ronglan; Zhang, Kaixin; Li, Shuai; Chen, Zhongjun; Li, Lixin

    2017-04-01

    Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.

  12. Alteration of Regulation of Fas/FasL Mediated Apoptosis in Gastric Cancer

    Directory of Open Access Journals (Sweden)

    Reggie García Robles

    2009-04-01

    Full Text Available Gastric cancer is an important neoplasticdisease in all around the world because its highincidence and mortality. Otherwise, apoptosis isa key process of programmed cell death duringembryogenesis, regulation of immune system,and holding the tissue homeostasis. Besides,the escape of apoptosis by different ways is anessential molecular aspect for the developmentof cancer. In this article we present an exhaustivereview of the current evidence of the roleof apoptosis through Fas/FasL pathway in thedevelopment of gastric carcinogenesis, includingsince early stages like in appearance of preneoplasticlesions. Finally, we think that a bettercomprehension of the signaling pathway Fas/FasL role in the different stages of gastric carcinogenesiscould let us know more about the implicatedmolecular ways and the physiopathologicalchanges in the appearance of this disease.

  13. Splice variants of the forkhead box protein AFX exhibit dominant negative activity and inhibit AFXalpha-mediated tumor cell apoptosis.

    Directory of Open Access Journals (Sweden)

    Eun Jig Lee

    2008-07-01

    Full Text Available Loss-of-function in the apoptosis-inducing genes is known to facilitate tumorigenesis. AFX (FOXO4, a member of forkhead transcription factors functions as a tumor suppressor and has 2 isoforms, AFXalpha (505 a.a. and AFXzeta (450 a.a.. In human cancer cells, we identified an N-terminally deleted form of AFXalpha (alpha198-505, translated from a downstream start and 2 short N-terminal AFX proteins (90, and 101 a.a. produced by aberrant splicing.We investigated the expression and role of these AFX variants. Cell transduction study revealed that short N-terminal AFX proteins were not stable. Though alpha(198-505 protein expression was detected in the cytoplasm and nucleus, alpha(198-505 expressing cells did not show a nucleocytoplasmic shuttling mediated by PI3 kinase signaling. Whereas, we observed this shuttling in cells expressing either AFXalpha or AFXzeta protein. AFXzeta and alpha(198-505 lost the ability to transactivate BCL6 or suppress cyclin D2 gene expression. These variants did not induce cancer cell death whereas AFXalpha resulted in apoptosis. We found that AFXzeta and alpha(198-505 suppress the AFXalpha stimulation of BCL6 promoter in a dose dependent manner, indicating dominant negative activity. These variants also inhibited AFXalpha induction of apoptosis.Loss of function by aberrant splicing and the dominant negative activity of AFX variants may provide a mechanism for enhanced survival of neoplastic cells.

  14. MOAP-1 Mediates Fas-Induced Apoptosis in Liver by Facilitating tBid Recruitment to Mitochondria.

    Science.gov (United States)

    Tan, Chong Teik; Zhou, Qi-Ling; Su, Yu-Chin; Fu, Nai Yang; Chang, Hao-Chun; Tao, Ran N; Sukumaran, Sunil K; Baksh, Shairaz; Tan, Yee-Joo; Sabapathy, Kanaga; Yu, Chun-Dong; Yu, Victor C

    2016-06-28

    Fas apoptotic signaling regulates diverse physiological processes. Acute activation of Fas signaling triggers massive apoptosis in liver. Upon Fas receptor stimulation, the BH3-only protein Bid is cleaved into the active form, tBid. Subsequent tBid recruitment to mitochondria, which is facilitated by its receptor MTCH2 at the outer mitochondrial membrane (OMM), is a critical step for commitment to apoptosis via the effector proteins Bax or Bak. MOAP-1 is a Bax-binding protein enriched at the OMM. Here, we show that MOAP-1-deficient mice are resistant to Fas-induced hepatocellular apoptosis and lethality. In the absence of MOAP-1, mitochondrial accumulation of tBid is markedly impaired. MOAP-1 binds to MTCH2, and this interaction appears necessary for MTCH2 to engage tBid. These findings reveal a role for MOAP-1 in Fas signaling in the liver by promoting MTCH2-mediated tBid recruitment to mitochondria. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  15. MOAP-1 Mediates Fas-Induced Apoptosis in Liver by Facilitating tBid Recruitment to Mitochondria

    Directory of Open Access Journals (Sweden)

    Chong Teik Tan

    2016-06-01

    Full Text Available Fas apoptotic signaling regulates diverse physiological processes. Acute activation of Fas signaling triggers massive apoptosis in liver. Upon Fas receptor stimulation, the BH3-only protein Bid is cleaved into the active form, tBid. Subsequent tBid recruitment to mitochondria, which is facilitated by its receptor MTCH2 at the outer mitochondrial membrane (OMM, is a critical step for commitment to apoptosis via the effector proteins Bax or Bak. MOAP-1 is a Bax-binding protein enriched at the OMM. Here, we show that MOAP-1-deficient mice are resistant to Fas-induced hepatocellular apoptosis and lethality. In the absence of MOAP-1, mitochondrial accumulation of tBid is markedly impaired. MOAP-1 binds to MTCH2, and this interaction appears necessary for MTCH2 to engage tBid. These findings reveal a role for MOAP-1 in Fas signaling in the liver by promoting MTCH2-mediated tBid recruitment to mitochondria.

  16. Andrographolide sensitizes cancer cells to TRAIL-induced apoptosis via p53-mediated death receptor 4 up-regulation.

    Science.gov (United States)

    Zhou, Jing; Lu, Guo-Dong; Ong, Chye-Sun; Ong, Choon-Nam; Shen, Han-Ming

    2008-07-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an important member of the tumor necrosis factor subfamily with great potential in cancer therapy. Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess potent anti-inflammatory and anticancer activities. Here, we showed that pretreatment with Andro significantly enhances TRAIL-induced apoptosis in various human cancer cell lines, including those TRAIL-resistant cells. Such sensitization is achieved through transcriptional up-regulation of death receptor 4 (DR4), a death receptor of TRAIL. In search of the molecular mechanisms responsible for DR4 up-regulation, we found that the tumor suppressor p53 plays an essential role in DR4 transcriptional activation. Andro is capable of activating p53 via increased p53 phosphorylation and protein stabilization, a process mediated by enhanced reactive oxygen species production and subsequent c-Jun NH(2)-terminal kinase activation. Pretreatment with an antioxidant (N-acetylcysteine) or a c-Jun NH(2)-terminal kinase inhibitor (SP600125) effectively prevented Andro-induced p53 activation and DR4 up-regulation and eventually blocked the Andro-induced sensitization on TRAIL-induced apoptosis. Taken together, these results present a novel anticancer effect of Andro and support its potential application in cancer therapy to overcome TRAIL resistance.

  17. Targeting GRP75 improves HSP90 inhibitor efficacy by enhancing p53-mediated apoptosis in hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Weiwei Guo

    Full Text Available Heat shock protein 90 (HSP90 inhibitors are potential drugs for cancer therapy. The inhibition of HSP90 on cancer cell growth largely through degrading client proteins, like Akt and p53, therefore, triggering cancer cell apoptosis. Here, we show that the HSP90 inhibitor 17-AAG can induce the expression of GRP75, a member of heat shock protein 70 (HSP70 family, which, in turn, attenuates the anti-growth effect of HSP90 inhibition on cancer cells. Additionally, 17-AAG enhanced binding of GRP75 and p53, resulting in the retention of p53 in the cytoplasm. Blocking GRP75 with its inhibitor MKT-077 potentiated the anti-tumor effects of 17-AAG by disrupting the formation of GRP75-p53 complexes, thereby facilitating translocation of p53 into the nuclei and leading to the induction of apoptosis-related genes. Finally, dual inhibition of HSP90 and GRP75 was found to significantly inhibit tumor growth in a liver cancer xenograft model. In conclusion, the GRP75 inhibitor MKT-077 enhances 17-AAG-induced apoptosis in HCCs and increases p53-mediated inhibition of tumor growth in vivo. Dual targeting of GRP75 and HSP90 may be a useful strategy for the treatment of HCCs.

  18. Ruthenium complexes with phenylterpyridine derivatives target cell membrane and trigger death receptors-mediated apoptosis in cancer cells.

    Science.gov (United States)

    Deng, Zhiqin; Gao, Pan; Yu, Lianling; Ma, Bin; You, Yuanyuan; Chan, Leung; Mei, Chaoming; Chen, Tianfeng

    2017-06-01

    Elucidation of the communication between metal complexes and cell membrane may provide useful information for rational design of metal-based anticancer drugs. Herein we synthesized a novel class of ruthenium (Ru) complexes containing phtpy derivatives (phtpy = phenylterpyridine), analyzed their structure-activity relationship and revealed their action mechanisms. The result showed that, the increase in the planarity of hydrophobic Ru complexes significantly enhanced their lipophilicity and cellular uptake. Meanwhile, the introduction of nitro group effectively improved their anticancer efficacy. Further mechanism studies revealed that, complex (2c), firstly accumulated on cell membrane and interacted with death receptors to activate extrinsic apoptosis signaling pathway. The complex was then transported into cell cytoplasm through transferrin receptor-mediated endocytosis. Most of the intracellular 2c accumulated in cell plasma, decreasing the level of cellular ROS, inducing the activation of caspase-9 and thus intensifying the apoptosis. At the same time, the residual 2c can translocate into cell nucleus to interact with DNA, induce DNA damage, activate p53 pathway and enhance apoptosis. Comparing with cisplatin, 2c possesses prolonged circulation time in blood, comparable antitumor ability and importantly, much lower toxicity in vivo. Taken together, this study uncovers the role of membrane receptors in the anticancer actions of Ru complexes, and provides fundamental information for rational design of membrane receptor targeting anticancer drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Up-Regulation of P21 Inhibits TRAIL-Mediated Extrinsic Apoptosis, Contributing Resistance to SAHA in Acute Myeloid Leukemia Cells

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    Xing Wu

    2014-08-01

    Full Text Available Background/Aim: P21, a multifunctional cell cycle-regulatory molecule, regulates apoptotic cell death. In this study we examined the effect of altered p21 expression on the sensitivity of acute myeloid leukemia cells in response to HDAC inhibitor SAHA treatment and investigated the underlying mechanism. Methods: Stably transfected HL60 cell lines were established in RPMI-1640 with supplementation of G-418. Cell viability was measured by MTT assay. Western blot was applied to assess the protein expression levels of target genes. Cell apoptosis was monitored by AnnexinV-PE/7AAD assay. Results: We showed HL60 cells that that didn't up-regulate p21 expression were more sensitive to SAHA-mediated apoptosis than NB4 and U937 cells that had increased p21 level. Enforced expression of p21 in HL60 cells reduced sensitivity to SAHA and blocked TRAIL-mediated apoptosis. Conversely, p21 silencing in NB4 cells enhanced SAHA-mediated apoptosis and lethality. Finally, we found that combined treatment with SAHA and rapamycin down-regulated p21 and enhanced apoptosis in AML cells. Conclusion: We conclude that up-regulated p21 expression mediates resistance to SAHA via inhibition of TRAIL apoptotic pathway. P21 may serve as a candidate biomarker to predict responsiveness or resistance to SAHA-based therapy in AML patients. In addition, rapamycin may be an effective agent to override p21-mediated resistance to SAHA in AML patients.

  20. Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway.

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    Yan Yuan

    Full Text Available Cadmium (Cd is an extremely toxic metal, capable of severely damaging several organs, including the brain. Studies have shown that Cd disrupts intracellular free calcium ([Ca(2+]i homeostasis, leading to apoptosis in a variety of cells including primary murine neurons. Calcium is a ubiquitous intracellular ion which acts as a signaling mediator in numerous cellular processes including cell proliferation, differentiation, and survival/death. However, little is known about the role of calcium signaling in Cd-induced apoptosis in neuronal cells. Thus we investigated the role of calcium signaling in Cd-induced apoptosis in primary rat cerebral cortical neurons. Consistent with known toxic properties of Cd, exposure of cerebral cortical neurons to Cd caused morphological changes indicative of apoptosis and cell death. It also induced elevation of [Ca(2+]i and inhibition of Na(+/K(+-ATPase and Ca(2+/Mg(2+-ATPase activities. This Cd-induced elevation of [Ca(2+]i was suppressed by an IP3R inhibitor, 2-APB, suggesting that ER-regulated Ca(2+ is involved. In addition, we observed elevation of reactive oxygen species (ROS levels, dysfunction of cytochrome oxidase subunits (COX-I/II/III, depletion of mitochondrial membrane potential (ΔΨm, and cleavage of caspase-9, caspase-3 and poly (ADP-ribose polymerase (PARP during Cd exposure. Z-VAD-fmk, a pan caspase inhibitor, partially prevented Cd-induced apoptosis and cell death. Interestingly, apoptosis, cell death and these cellular events induced by Cd were blocked by BAPTA-AM, a specific intracellular Ca(2+ chelator. Furthermore, western blot analysis revealed an up-regulated expression of Bcl-2 and down-regulated expression of Bax. However, these were not blocked by BAPTA-AM. Thus Cd toxicity is in part due to its disruption of intracellular Ca(2+ homeostasis, by compromising ATPases activities and ER-regulated Ca(2+, and this elevation in Ca(2+ triggers the activation of the Ca(2+-mitochondria apoptotic

  1. Autophagy Inhibition Enhances the Mitochondrial-Mediated Apoptosis Induced by Mangrove (Avicennia marina) Extract in Human Breast Cancer Cells

    KAUST Repository

    Esau, Luke

    2015-01-10

    %. Conclusion: Our data provide evidence that AM extract triggers ROS-mediated autophagy as well as caspase-independent apoptosis. The results also strengthen the view that concurrent targeting of apoptotic and autophagic pathways may provide effective therapeutic strategy against cancer.

  2. Role of endoplasmic reticulum stress in alpha-TEA mediated TRAIL/DR5 death receptor dependent apoptosis.

    Directory of Open Access Journals (Sweden)

    Richa Tiwary

    2010-07-01

    Full Text Available Alpha-TEA (RRR-alpha-tocopherol ether-linked acetic acid analog, a derivative of RRR-alpha-tocopherol (vitamin E exhibits anticancer actions in vitro and in vivo in variety of cancer types. The objective of this study was to obtain additional insights into the mechanisms involved in alpha-TEA induced apoptosis in human breast cancer cells.alpha-TEA induces endoplasmic reticulum (ER stress as indicated by increased expression of CCAAT/enhancer binding protein homologous protein (CHOP as well as by enhanced expression or activation of specific markers of ER stress such as glucose regulated protein (GRP78, phosphorylated alpha subunit of eukaryotic initiation factor 2 (peIF-2alpha, and spliced XBP-1 mRNA. Knockdown studies using siRNAs to TRAIL, DR5, JNK and CHOP as well as chemical inhibitors of ER stress and caspase-8 showed that: i alpha-TEA activation of DR5/caspase-8 induces an ER stress mediated JNK/CHOP/DR5 positive amplification loop; ii alpha-TEA downregulation of c-FLIP (L protein levels is mediated by JNK/CHOP/DR5 loop via a JNK dependent Itch E3 ligase ubiquitination that further serves to enhance the JNK/CHOP/DR5 amplification loop by preventing c-FLIP's inhibition of caspase-8; and (iii alpha-TEA downregulation of Bcl-2 is mediated by the ER stress dependent JNK/CHOP/DR5 signaling.Taken together, ER stress plays an important role in alpha-TEA induced apoptosis by enhancing DR5/caspase-8 pro-apoptotic signaling and suppressing anti-apoptotic factors c-FLIP and Bcl-2 via ER stress mediated JNK/CHOP/DR5/caspase-8 signaling.

  3. LACE1 interacts with p53 and mediates its mitochondrial translocation and apoptosis

    Czech Academy of Sciences Publication Activity Database

    Česneková, J.; Spáčilová, J.; Hansíková, H.; Houštěk, Josef; Zeman, J.; Stibůrek, L.

    2016-01-01

    Roč. 7, č. 30 (2016), s. 47687-47698 ISSN 1949-2553 R&D Projects: GA ČR(CZ) GA13-07223S Institutional support: RVO:67985823 Keywords : p53 * LACE1 * mitochondria * apoptosis * translocation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.168, year: 2016

  4. Coenzyme Q blocks biochemical but not receptor-mediated apoptosis by increasing mitochondrial antioxidant protection

    Czech Academy of Sciences Publication Activity Database

    Alleva, R.; Tomasetti, M.; Anděra, Ladislav; Gellert, N.; Borghi, B.; Weber, C.; Murphy, M. P.; Neužil, J.

    2001-01-01

    Roč. 503, č. 1 (2001), s. 46-50 ISSN 0014-5793 R&D Projects: GA ČR GA301/99/0350 Institutional research plan: CEZ:AV0Z5052915 Keywords : coenzyme Q * apoptosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.644, year: 2001

  5. Carnosol induces ROS-mediated beclin1-independent autophagy and apoptosis in triple negative breast cancer.

    Directory of Open Access Journals (Sweden)

    Yusra Al Dhaheri

    Full Text Available In this study we investigated the in vitro and in vivo anticancer effect of carnosol, a naturally occurring polyphenol, in triple negative breast cancer.We found that carnosol significantly inhibited the viability and colony growth induced G2 arrest in the triple negative MDA-MB-231. Blockade of the cell cycle was associated with increased p21/WAF1 expression and downregulation of p27. Interestingly, carnosol was found to induce beclin1-independent autophagy and apoptosis in MDA-MB-231 cells. The coexistence of both events, autophagy and apoptosis, was confirmed by electron micrography. Induction of autophagy was found to be an early event, detected within 3 h post-treatment, which subsequently led to apoptosis. Carnosol treatment also caused a dose-dependent increase in the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2. Moreover, we show that carnosol induced DNA damage, reduced the mitochondrial potential and triggered the activation of the intrinsic and extrinsic apoptotic pathway. Furthermore, we found that carnosol induced a dose-dependent generation of reactive oxygen species (ROS and inhibition of ROS by tiron, a ROS scavenger, blocked the induction of autophagy and apoptosis and attenuated DNA damage. To our knowledge, this is the first report to identify the induction of autophagy by carnosol.In conclusion our findings provide strong evidence that carnosol may be an alternative therapeutic candidate against the aggressive form of breast cancer and hence deserves more exploration.

  6. Honokiol induces reactive oxygen species-mediated apoptosis in Candida albicans through mitochondrial dysfunction.

    Science.gov (United States)

    Sun, Lingmei; Liao, Kai; Hang, Chengcheng; Wang, Dayong

    2017-01-01

    To investigate the effects of honokiol on induction of reactive oxygen species (ROS), antioxidant defense systems, mitochondrial dysfunction, and apoptosis in Candida albicans. To measure ROS accumulation, 2',7'-dichlorofluorescein diacetate fluorescence was used. Lipid peroxidation was assessed using both fluorescence staining and a thiobarbituric acid reactive substances (TBARS) assay. Protein oxidation was determined using dinitrophenylhydrazine derivatization. Antioxidant enzymatic activities were measured using commercially available detection kits. Superoxide dismutase (SOD) genes expression was measured using real time RT-PCR. To assess its antifungal abilities and effectiveness on ROS accumulation, honokiol and the SOD inhibitor N,N'-diethyldithiocarbamate (DDC) were used simultaneously. Mitochondrial dysfunction was assessed by measuring the mitochondrial membrane potential (mtΔψ). Honokiol-induced apoptosis was assessed using an Annexin V-FITC apoptosis detection kit. ROS, lipid peroxidation, and protein oxidation occurred in a dose-dependent manner in C. albicans after honokiol treatment. Honokiol caused an increase in antioxidant enzymatic activity. In addition, honokiol treatment induced SOD genes expression in C. albicans cells. Moreover, addition of DDC resulted in increased endogenous ROS levels and potentiated the antifungal activity of honokiol. Mitochondrial dysfunction was confirmed by measured changes to mtΔψ. The level of apoptosis increased in a dose-dependent manner after honokiol treatment. Collectively, these results indicate that honokiol acts as a pro-oxidant in C. albicans. Furthermore, the SOD inhibitor DDC can be used to potentiate the activity of honokiol against C. albicans.

  7. Thymoquinone Induces Mitochondria-Mediated Apoptosis in Acute Lymphoblastic Leukaemia in Vitro

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    Bassem Y. Sheikh

    2013-09-01

    Full Text Available There has been a growing interest in naturally occurring compounds from traditional medicine with anti-cancer potential. Nigella sativa (black seed is one of the most widely studied plants. This annual herb grows in countries bordering the Mediterranean Sea and India. Thymoquinone (TQ is an active ingredient isolated from Nigella sativa. The anti-cancer effect of TQ, via the induction of apoptosis resulting from mitochondrial dysfunction, was assessed in an acute lymphocyte leukemic cell line (CEMss with an IC50 of 1.5 µg/mL. A significant increase in chromatin condensation in the cell nucleus was observed using fluorescence analysis. The apoptosis was then confirmed by Annexin V and an increased number of cellular DNA breaks in treated cells were observed as a DNA ladder. Treatment of CEMss cells with TQ encouraged apoptosis with cell death-transducing signals by a down-regulation of Bcl-2 and up-regulation of Bax. Moreover, the significant generation of cellular ROS, HSP70 and activation of caspases 3 and 8 were also observed in the treated cells. The mitochondrial apoptosis was clearly associated with the S phase cell cycle arrest. In conclusion, the results from the current study indicated that TQ could be a promising agent for the treatment of leukemia.

  8. Honokiol induces reactive oxygen species-mediated apoptosis in Candida albicans through mitochondrial dysfunction.

    Directory of Open Access Journals (Sweden)

    Lingmei Sun

    Full Text Available To investigate the effects of honokiol on induction of reactive oxygen species (ROS, antioxidant defense systems, mitochondrial dysfunction, and apoptosis in Candida albicans.To measure ROS accumulation, 2',7'-dichlorofluorescein diacetate fluorescence was used. Lipid peroxidation was assessed using both fluorescence staining and a thiobarbituric acid reactive substances (TBARS assay. Protein oxidation was determined using dinitrophenylhydrazine derivatization. Antioxidant enzymatic activities were measured using commercially available detection kits. Superoxide dismutase (SOD genes expression was measured using real time RT-PCR. To assess its antifungal abilities and effectiveness on ROS accumulation, honokiol and the SOD inhibitor N,N'-diethyldithiocarbamate (DDC were used simultaneously. Mitochondrial dysfunction was assessed by measuring the mitochondrial membrane potential (mtΔψ. Honokiol-induced apoptosis was assessed using an Annexin V-FITC apoptosis detection kit.ROS, lipid peroxidation, and protein oxidation occurred in a dose-dependent manner in C. albicans after honokiol treatment. Honokiol caused an increase in antioxidant enzymatic activity. In addition, honokiol treatment induced SOD genes expression in C. albicans cells. Moreover, addition of DDC resulted in increased endogenous ROS levels and potentiated the antifungal activity of honokiol. Mitochondrial dysfunction was confirmed by measured changes to mtΔψ. The level of apoptosis increased in a dose-dependent manner after honokiol treatment.Collectively, these results indicate that honokiol acts as a pro-oxidant in C. albicans. Furthermore, the SOD inhibitor DDC can be used to potentiate the activity of honokiol against C. albicans.

  9. Carnosol Induces ROS-Mediated Beclin1-Independent Autophagy and Apoptosis in Triple Negative Breast Cancer

    Science.gov (United States)

    Al Dhaheri, Yusra; Attoub, Samir; Ramadan, Gaber; Arafat, Kholoud; Bajbouj, Khuloud; Karuvantevida, Noushad; AbuQamar, Synan; Eid, Ali; Iratni, Rabah

    2014-01-01

    Background In this study we investigated the in vitro and in vivo anticancer effect of carnosol, a naturally occurring polyphenol, in triple negative breast cancer. Results We found that carnosol significantly inhibited the viability and colony growth induced G2 arrest in the triple negative MDA-MB-231. Blockade of the cell cycle was associated with increased p21/WAF1 expression and downregulation of p27. Interestingly, carnosol was found to induce beclin1-independent autophagy and apoptosis in MDA-MB-231 cells. The coexistence of both events, autophagy and apoptosis, was confirmed by electron micrography. Induction of autophagy was found to be an early event, detected within 3 h post-treatment, which subsequently led to apoptosis. Carnosol treatment also caused a dose-dependent increase in the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2). Moreover, we show that carnosol induced DNA damage, reduced the mitochondrial potential and triggered the activation of the intrinsic and extrinsic apoptotic pathway. Furthermore, we found that carnosol induced a dose-dependent generation of reactive oxygen species (ROS) and inhibition of ROS by tiron, a ROS scavenger, blocked the induction of autophagy and apoptosis and attenuated DNA damage. To our knowledge, this is the first report to identify the induction of autophagy by carnosol. Conclusion In conclusion our findings provide strong evidence that carnosol may be an alternative therapeutic candidate against the aggressive form of breast cancer and hence deserves more exploration. PMID:25299698

  10. Interaction of caveolin-1 with Ku70 inhibits Bax-mediated apoptosis.

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    Huafei Zou

    Full Text Available Caveolin-1, the structural protein component of caveolae, acts as a scaffolding protein that functionally regulates signaling molecules. We show that knockdown of caveolin-1 protein expression enhances chemotherapeutic drug-induced apoptosis and inhibits long-term survival of colon cancer cells. In vitro studies demonstrate that caveolin-1 is a novel Ku70-binding protein, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82-101 to the caveolin-binding domain (CBD of Ku70 (amino acids 471-478. Cell culture data show that caveolin-1 binds Ku70 after treatment with chemotherapeutic drugs. Mechanistically, we found that binding of caveolin-1 to Ku70 inhibits the chemotherapeutic drug-induced release of Bax from Ku70, activation of Bax, translocation of Bax to mitochondria and apoptosis. Potentiation of apoptosis by knockdown of caveolin-1 protein expression is greatly reduced in the absence of Bax expression. Finally, we found that overexpression of wild type Ku70, but not a mutant form of Ku70 that cannot bind to caveolin-1 (Ku70 Φ→A, limits the chemotherapeutic drug-induced Ku70/Bax dissociation and apoptosis. Thus, caveolin-1 acts as an anti-apoptotic protein in colon cancer cells by binding to Ku70 and inhibiting Bax-dependent cell death.

  11. The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis

    Science.gov (United States)

    Kim, Seok-Jo; Cheresh, Paul; Jablonski, Renea P.; Williams, David B.; Kamp, David W.

    2015-01-01

    Convincing evidence has emerged demonstrating that impairment of mitochondrial function is critically important in regulating alveolar epithelial cell (AEC) programmed cell death (apoptosis) that may contribute to aging-related lung diseases, such as idiopathic pulmonary fibrosis (IPF) and asbestosis (pulmonary fibrosis following asbestos exposure). The mammalian mitochondrial DNA (mtDNA) encodes for 13 proteins, including several essential for oxidative phosphorylation. We review the evidence implicating that oxidative stress-induced mtDNA damage promotes AEC apoptosis and pulmonary fibrosis. We focus on the emerging role for AEC mtDNA damage repair by 8-oxoguanine DNA glycosylase (OGG1) and mitochondrial aconitase (ACO-2) in maintaining mtDNA integrity which is important in preventing AEC apoptosis and asbestos-induced pulmonary fibrosis in a murine model. We then review recent studies linking the sirtuin (SIRT) family members, especially SIRT3, to mitochondrial integrity and mtDNA damage repair and aging. We present a conceptual model of how SIRTs modulate reactive oxygen species (ROS)-driven mitochondrial metabolism that may be important for their tumor suppressor function. The emerging insights into the pathobiology underlying AEC mtDNA damage and apoptosis is suggesting novel therapeutic targets that may prove useful for the management of age-related diseases, including pulmonary fibrosis and lung cancer. PMID:26370974

  12. Protective Role of Complement C3 Against Cytokine-Mediated beta-Cell Apoptosis

    DEFF Research Database (Denmark)

    Dos Santos, Reinaldo S.; Marroqui, Laura; Grieco, Fabio A.

    2017-01-01

    silencing exacerbates apoptosis under both basal condition and following exposure to cytokines, and it increases chemokine expression upon cytokine treatment. C3 exerts its prosurvival effects via AKT activation and c-Jun N-terminal kinase inhibition. Exogenously added C3 also protects against cytokine...

  13. Homocysteine induces PUMA-mediated mitochondrial apoptosis in SH-SY5Y cells.

    Science.gov (United States)

    Jang, Yumi; Kim, Juhae; Ko, Je Won; Kwon, Young Hye

    2016-11-01

    Previous studies have reported that homocysteine induced endoplasmic reticulum (ER) stress in neuronal cells, proposing the underlying mechanism by which it could induce neurotoxicity. Induction of pro-apoptotic transcription factor C/EBP homologous protein (CHOP) and activation of caspase-4 by calpain have been suggested to be an important route in inducing apoptosis in response to ER stress. In this study, we investigated the molecular pathway of homocysteine-induced apoptosis in caspase-4 deficient SH-SY5Y human neuroblastoma cells. Homocysteine significantly increased mRNA levels of CHOP and p53, resulting in the upregulation of their downstream target gene, p53 up-regulated modulator of apoptosis (PUMA). In cells treated with homocysteine, Bcl-2-associated X protein (BAX) protein levels, cytochrome c release from the mitochondria, and caspase-9 activation were significantly increased. Consistently, a caspase-9 inhibitor significantly alleviated homocysteine-induced cytotoxicity. Significantly lower BAX mRNA levels and caspase-9 activation were observed in cells transfected with siRNA for PUMA. Taken together, our findings suggest that PUMA would be involved in the possible crosstalk between the ER and the mitochondria in the homocysteine-induced apoptosis of caspase-4 deficient SH-SY5Y cells.

  14. Iron-Mediated Lysosomal Membrane Permeabilization in Ethanol-Induced Hepatic Oxidative Damage and Apoptosis: Protective Effects of Quercetin

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    Yanyan Li

    2016-01-01

    Full Text Available Iron, in its free ferrous states, can catalyze Fenton reaction to produce OH∙, which is recognized as a crucial role in the pathogenesis of alcoholic liver diseases (ALD. As a result of continuous decomposition of iron-containing compounds, lysosomes contain a pool of redox-active iron. To investigate the important role of intralysosomal iron in alcoholic liver injury and the potential protection of quercetin, male C57BL/6J mice fed by Lieber De Carli diets containing ethanol (30% of total calories were cotreated by quercetin or deferoxamine (DFO for 15 weeks and ethanol-incubated mice primary hepatocytes were pretreated with FeCl3, DFO, and bafilomycin A1 at their optimal concentrations and exposure times. Chronic ethanol consumption caused an evident increase in lysosomal redox-active iron accompanying sustained oxidative damage. Iron-mediated ROS could trigger lysosomal membrane permeabilization (LMP and subsequent mitochondria apoptosis. The hepatotoxicity was attenuated by reducing lysosomal iron while being exacerbated by escalating lysosomal iron. Quercetin substantially alleviated the alcoholic liver oxidative damage and apoptosis by decreasing lysosome iron and ameliorating iron-mediated LMP, which provided a new prospective of the use of quercetin against ALD.

  15. Pazopanib, a novel multi-kinase inhibitor, shows potent antitumor activity in colon cancer through PUMA-mediated apoptosis.

    Science.gov (United States)

    Zhang, Lingling; Wang, Huanan; Li, Wei; Zhong, Juchang; Yu, Rongcheng; Huang, Xinfeng; Wang, Honghui; Tan, Zhikai; Wang, Jiangang; Zhang, Yingjie

    2017-01-10

    Colon cancer is still the third most common cancer which has a high mortality but low five-year survival rate. Novel tyrosine kinase inhibitors (TKI) such as pazopanib become effective antineoplastic agents that show promising clinical activity in a variety of carcinoma, including colon cancer. However, the precise underlying mechanism against tumor is unclear. Here, we demonstrated that pazopanib promoted colon cancer cell apoptosis through inducing PUMA expression. Pazopanib induced p53-independent PUMA activation by inhibiting PI3K/Akt signaling pathway, thereby activating Foxo3a, which subsequently bound to the promoter of PUMA to activate its transcription. After induction, PUMA activated Bax and triggered the intrinsic mitochondrial apoptosis pathway. Furthermore, administration of pazopanib highly suppressed tumor growth in a xenograft model. PUMA deletion in cells and tumors led to resistance of pazopanib, indicating PUMA-mediated pro-apoptotic and anti-tumor effects in vitro and in vivo. Combing pazopanib with some conventional or novel drugs, produced heightened and synergistic antitumor effects that were associated with potentiated PUMA induction via different pathways. Taken together, these results establish a critical role of PUMA in mediating the anticancer effects of pazopanib in colon cancer cells and provide the rationale for clinical evaluation.

  16. Requirement of Nuclear Factor κB for Smac Mimetic–Mediated Sensitization of Pancreatic Carcinoma Cells for Gemcitabine-Induced Apoptosis

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    Dominic Stadel

    2011-12-01

    Full Text Available Defects in apoptosis contribute to treatment resistance and poor outcome of pancreatic cancer, calling for novel therapeutic strategies. Here, we provide the first evidence that nuclear factor (NF κB is required for Smac mimetic– mediated sensitization of pancreatic carcinoma cells for gemcitabine-induced apoptosis. The Smac mimetic BV6 cooperates with gemcitabine to reduce cell viability and to induce apoptosis. In addition, BV6 significantly enhances the cytotoxicity of several anticancer drugs against pancreatic carcinoma cells, including doxorubicin, cisplatin, and 5-fluorouracil. Molecular studies reveal that BV6 stimulates NF-κB activation, which is further increased in the presence of gemcitabine. Importantly, inhibition of NF-κB by overexpression of the dominant-negative IκBα superrepressor significantly decreases BV6- and gemcitabine-induced apoptosis, demonstrating that NF-κB exerts a proapoptotic function in this model of apoptosis. In support of this notion, inhibition of tumor necrosis factor α (TNFα by the TNFα blocking antibody Enbrel reduces BV6- and gemcitabine-induced activation of caspase 8 and 3, loss of mitochondrial membrane potential, and apoptosis. By demonstrating that BV6 and gemcitabine trigger a NF-κB–dependent, TNFα-mediated loop to activate apoptosis signaling pathways and caspase-dependent apoptotic cell death, our findings have important implications for the development of Smac mimetic–based combination protocols in the treatment of pancreatic cancer.

  17. Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1a cells

    Energy Technology Data Exchange (ETDEWEB)

    Fuchs, Dominik [Research Group Molecular Neuro-Oncology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg (Germany); Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg (Germany); Daniel, Volker; Sadeghi, Mahmoud; Opelz, Gerhard [Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg (Germany); Naujokat, Cord, E-mail: cord.naujokat@med.uni-heidelberg.de [Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg (Germany)

    2010-04-16

    Leukemia stem cells are known to exhibit multidrug resistance by expression of ATP-binding cassette (ABC) transporters which constitute transmembrane proteins capable of exporting a wide variety of chemotherapeutic drugs from the cytosol. We show here that human promyeloblastic leukemia KG-1a cells exposed to the histone deacetylase inhibitor phenylbutyrate resemble many characteristics of leukemia stem cells, including expression of functional ABC transporters such as P-glycoprotein, BCRP and MRP8. Consequently, KG-1a cells display resistance to the induction of apoptosis by various chemotherapeutic drugs. Resistance to apoptosis induction by chemotherapeutic drugs can be reversed by cyclosporine A, which effectively inhibits the activity of P-glycoprotein and BCRP, thus demonstrating ABC transporter-mediated drug resistance in KG-1a cells. However, KG-1a are highly sensitive to apoptosis induction by salinomycin, a polyether ionophore antibiotic that has recently been shown to kill human breast cancer stem cell-like cells and to induce apoptosis in human cancer cells displaying multiple mechanisms of drug and apoptosis resistance. Whereas KG-1a cells can be adapted to proliferate in the presence of apoptosis-inducing concentrations of bortezomib and doxorubicin, salinomycin does not permit long-term adaptation of the cells to apoptosis-inducing concentrations. Thus, salinomycin should be regarded as a novel and effective agent for the elimination of leukemia stem cells and other tumor cells exhibiting ABC transporter-mediated multidrug resistance.

  18. Artesunate induces ROS-mediated apoptosis in doxorubicin-resistant T leukemia cells.

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    Thomas Efferth

    Full Text Available BACKGROUND: A major obstacle for successful cancer treatment often is the development of drug resistance in cancer cells during chemotherapy. Therefore, there is an urgent need for novel drugs with improved efficacy against tumor cells and with less toxicity on normal cells. Artesunate (ART, a powerful anti-malarial herbal compound, has been shown to inhibit growth of various tumor cell lines in vitro and of xenografted Kaposi's sarcoma in mice in vivo. However, the molecular mechanisms by which ART exerts its cytotoxicity have not been elucidated. The ART-class of anti-malarial compounds is attractive due to their activity against multidrug-resistant Plasmodium falciparum and Plasmodium vivax strains. Another salient feature of these compounds is the lack of severe side effects in malaria patients. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we used T-cell leukemias as a model system to study the molecular mechanisms of ART-induced apoptosis. The most typical anticancer drugs are DNA intercalators such as Doxorubicin. To investigate drug sensitivity and resistance, we chose a Doxorubicin-resistant leukemia cell line and investigated the killing effect of ART on these cells. We show that ART induces apoptosis in leukemic T cells mainly through the mitochondrial pathway via generation of reactive oxygen species (ROS, a mechanism different from Doxorubicin. This is confirmed by the fact that the antioxidant N-Acetyle-Cysteine (NAC could completely block ROS generation and, consequently, inhibited ART-induced apoptosis. Therefore, ART can overcome the Doxorubicin-resistance and induce the Doxorubicin-resistant leukemia cells to undergo apoptosis. We also show that ART can synergize with Doxorubicin to enhance apoptotic cell death in leukemic T cells. This synergistic effect can be largely explained by the fact that ART and Doxorubicin use different killing mechanisms. CONCLUSIONS: Our studies raise the possibility to develop ART in

  19. Xylopine Induces Oxidative Stress and Causes G2/M Phase Arrest, Triggering Caspase-Mediated Apoptosis by p53-Independent Pathway in HCT116 Cells

    Directory of Open Access Journals (Sweden)

    Luciano de Souza Santos

    2017-01-01

    Full Text Available Xylopine is an aporphine alkaloid that has cytotoxic activity to cancer cells. In this study, the underlying mechanism of xylopine cytotoxicity was assessed in human colon carcinoma HCT116 cells. Xylopine displayed potent cytotoxicity in different cancer cell lines in monolayer cultures and in a 3D model of cancer multicellular spheroids formed from HCT116 cells. Typical morphology of apoptosis, cell cycle arrest in the G2/M phase, increased internucleosomal DNA fragmentation, loss of the mitochondrial transmembrane potential, and increased phosphatidylserine externalization and caspase-3 activation were observed in xylopine-treated HCT116 cells. Moreover, pretreatment with a caspase-3 inhibitor (Z-DEVD-FMK, but not with a p53 inhibitor (cyclic pifithrin-α, reduced xylopine-induced apoptosis, indicating induction of caspase-mediated apoptosis by the p53-independent pathway. Treatment with xylopine also caused an increase in the production of reactive oxygen/nitrogen species (ROS/RNS, including hydrogen peroxide and nitric oxide, but not superoxide anion, and reduced glutathione levels were decreased in xylopine-treated HCT116 cells. Application of the antioxidant N-acetylcysteine reduced the ROS levels and xylopine-induced apoptosis, indicating activation of ROS-mediated apoptosis pathway. In conclusion, xylopine has potent cytotoxicity to different cancer cell lines and is able to induce oxidative stress and G2/M phase arrest, triggering caspase-mediated apoptosis by the p53-independent pathway in HCT116 cells.

  20. CYP3A-mediated apoptosis of dauricine in cultured human bronchial epithelial cells and in lungs of CD-1 mice

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hua; Shen, Shuijie [Center for Developmental Therapeutics, Seattle Children' s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, WA 98101 (United States); Chen, Xiaoyan; Zhong, Dafang [Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203 (China); Zheng, Jiang, E-mail: jiang.zheng@seattlechildrens.org [Center for Developmental Therapeutics, Seattle Children' s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, WA 98101 (United States)

    2012-06-15

    Dauricine is the major bioactive component isolated from the root of Menispermum dauricum DC and has shown promising pharmacologic activities with a great potential for clinical use. Recently, we found that intraperitoneal exposure of dauricine produced selective pulmonary injury in mice. A quinone methide metabolite of dauricine was identified and is suggested to be associated with the pulmonary toxicity of dauricine. The present study evaluated the apoptotic effect of dauricine in cultured cells and mice, determined the change in cellular glutathione (GSH) contents after exposure to dauricine, investigated the role of GSH depletion in dauricine-induced cytotoxicity and apoptosis, and examined the role of CYP3A in dauricine-induced GSH depletion and apoptosis. Dauricine was found to induce apoptosis in NL-20 cells. Additionally, intraperitoneal administration of dauricine caused GSH depletion and apoptosis in lungs of mice. Treatment with ketoconazole, an inhibitor of CYP3A, reversed cellular GSH depletion in lungs of mice given dauricine and showed protective effect on dauricine-induced apoptosis in lungs of mice. This indicates that metabolic activation is involved in dauricine-induced GSH-depletion, cytotoxicity and apoptosis. The glutathione depletor L-buthionine sulfoximine showed potentiating effect on cytotoxicity and apoptosis induced by dauricine. We propose that dauricine is metabolized to a quinone methide intermediate which depletes cellular GSH, and the depletion of GSH may trigger and/or intensify the cytotoxicity and apoptosis induced by dauricine. -- Highlights: ► Dauricine induced apoptosis in lungs in mice and in cultured human pulmonary cells. ► Dauricine depleted cellular GSH in lungs of mice and in the human pulmonary cells. ► CYP3A subfamily mediated GSH depletion and apoptosis induced by dauricine. ► L-Buthionine sulfoximine potentiated dauricine-induced GSH depletion and apoptosis.

  1. Exogenous estradiol enhances apoptosis in regressing post-partum rat corpora lutea possibly mediated by prolactin

    Directory of Open Access Journals (Sweden)

    Telleria Carlos M

    2005-08-01

    Full Text Available Abstract Background In pregnant rats, structural luteal regression takes place after parturition and is associated with cell death by apoptosis. We have recently shown that the hormonal environment is responsible for the fate of the corpora lutea (CL. Changing the levels of circulating hormones in post-partum rats, either by injecting androgen, progesterone, or by allowing dams to suckle, was coupled with a delay in the onset of apoptosis in the CL. The objectives of the present investigation were: i to examine the effect of exogenous estradiol on apoptosis of the rat CL during post-partum luteal regression; and ii to evaluate the post-partum luteal expression of the estrogen receptor (ER genes. Methods In a first experiment, rats after parturition were separated from their pups and injected daily with vehicle or estradiol benzoate for 4 days. On day 4 post-partum, animals were sacrificed, blood samples were taken to determine serum concentrations of hormones, and the ovaries were isolated to study apoptosis in situ. In a second experiment, non-lactating rats after parturition received vehicle, estradiol benzoate or estradiol benzoate plus bromoergocryptine for 4 days, and their CL were isolated and used to study apoptosis ex vivo. In a third experiment, we obtained CL from rats on day 15 of pregnancy and from non-lactating rats on day 4 post-partum, and studied the expression of the messenger RNAs (mRNAs encoding the ERalpha and ERbeta genes. Results Exogenous administration of estradiol benzoate induced an increase in the number of apoptotic cells within the CL on day 4 post-partum when compared with animals receiving vehicle alone. Animals treated with the estrogen had higher serum prolactin and progesterone concentrations, with no changes in serum androstenedione. Administration of bromoergocryptine blocked the increase in serum prolactin and progesterone concentrations, and DNA fragmentation induced by the estrogen treatment. ERalpha and

  2. Spirulina platensis prevents high glucose-induced oxidative stress mitochondrial damage mediated apoptosis in cardiomyoblasts.

    Science.gov (United States)

    Jadaun, Pratiksha; Yadav, Dhananjay; Bisen, Prakash Singh

    2018-04-01

    The current study was undertaken to study the effect of Spirulina platensis (Spirulina) extract on enhanced oxidative stress during high glucose induced cell death in H9c2 cells. H9c2 cultured under high glucose (33 mM) conditions resulted in a noteworthy increase in oxidative stress (free radical species) accompanied by loss of mitochondrial membrane potential, release of cytochrome c, increase in caspase activity and pro-apoptotic protein (Bax). Spirulina extract (1 μg/mL), considerably inhibited increased ROS and RNS levels, reduction in cytochrome c release, raise in mitochondrial membrane potential, decreased the over expression of proapoptotic protein Bax and suppressed the Bax/Bcl2 ratio with induced apoptosis without affecting cell viability. Overall results suggest that Spirulina extract plays preventing role against enhanced oxidative stress during high glucose induced apoptosis in cardiomyoblasts as well as related dysfunction in H9c2 cells.

  3. L-carnitine reduces doxorubicin-induced apoptosis through a prostacyclin-mediated pathway in neonatal rat cardiomyocytes.

    Science.gov (United States)

    Chao, Hung-Hsin; Liu, Ju-Chi; Hong, Hong-Jye; Lin, Jia-wei; Chen, Cheng-Hsien; Cheng, Tzu-Hurng

    2011-01-21

    Clinical use of doxorubicin is greatly limited by its severe cardiotoxic side effects. L-carnitine is a vitamin-like substance which has been successfully used in many cardiomyopathies, however, the intracellular mechanism(s) remain unclear. The objective of this study was set to evaluate the protective effect of L-carnitine on doxorubicin-induced cardiomyocyte apoptosis, and to explore its intracellular mechanism(s). Primary cultured neonatal rat cardiomyocytes were treated with doxorubicin (1 µM) with or without pretreatment with L-carnitine (1-30 mM). Lactate dehydrogenase assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining, and flow cytometry measurement were used to assess cytotoxicity and apoptosis. Fluorescent probes 2',7'-dichlorofluorescein diacetate and chemiluminescence assay of superoxide production were used to detect the production of reactive oxygen species. Western blotting was used to evaluate the quantity of cleaved caspase-3, cytosol cytochrome c, and Bcl-x(L) expression. L-carnitine inhibited doxorubicin-induced reactive oxygen species generation and NADPH oxidase activation, reduced the quantity of cleaved caspase-3 and cytosol cytochrome c, and increased Bcl-x(L) expression, resulting in protecting cardiomyocytes from doxorubicin-induced apoptosis. In addition, L-carnitine was found to increase the prostacyclin (PGI(2)) generation in cardiomyocytes. The siRNA transfection for PGI(2) synthase significantly reduced L-carnitine-induced PGI(2) and L-carnitine's protective effect. Furthermore, blockade the potential PGI(2) receptors, including PGI(2) receptors (IP receptors), and peroxisome proliferator-activated receptors alpha and delta (PPARα and PPARδ), revealed that the siRNA-mediated blockage of PPARα considerably reduced the anti-apoptotic effect of L-carnitine. These findings suggest that L-carnitine protects cardiomyocytes from doxorubicin-induced apoptosis in part through PGI(2

  4. Interferon-α and cyclooxygenase-2 inhibitor cooperatively mediates TRAIL-induced apoptosis in hepatocellular carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zuo, Chaohui, E-mail: zuochaohui@vip.sina.com [Department of Gastroduodenal and Pancreatic Surgery, Translation Medicine Research Center of Liver Cancer, Hunan Province Tumor Hospital & Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha, Hunan Province (China); Department of Pathology, Immunology and Laboratory Medicine and Shands Cancer Center, University of Florida, Gainesville, FL (United States); Qiu, Xiaoxin [Department of Gastroduodenal and Pancreatic Surgery, Translation Medicine Research Center of Liver Cancer, Hunan Province Tumor Hospital & Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha, Hunan Province (China); Cancer Research Institute, University of South China, Hengyang, Hunan Province (China); Liu, Nianli; Yang, Darong [Cancer Research Institute, University of South China, Hengyang, Hunan Province (China); Xia, Man [Department of Gastroduodenal and Pancreatic Surgery, Translation Medicine Research Center of Liver Cancer, Hunan Province Tumor Hospital & Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha, Hunan Province (China); Department of Pathology, Immunology and Laboratory Medicine and Shands Cancer Center, University of Florida, Gainesville, FL (United States); Liu, Jingshi [Department of Gastroduodenal and Pancreatic Surgery, Translation Medicine Research Center of Liver Cancer, Hunan Province Tumor Hospital & Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha, Hunan Province (China); Wang, Xiaohong [Cancer Research Institute, University of South China, Hengyang, Hunan Province (China); and others

    2015-05-01

    Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Interferon-alpha (IFN-α) has recently been recognized to harbor therapeutic potential in the prevention and treatment of HCC, but it remains controversial as to whether IFN-α exerts direct cytotoxicity against HCC. Cyclooxygenase-2 (COX-2) is overexpressed in HCC and is considered to play a role in hepatocarcinogenesis. Therefore, we aimed to elucidate the combined effect of a COX-2 inhibitor, celecoxib, and IFN-α on in vitro growth suppression of HCC using the hepatoma cell line HLCZ01 and the in vivo nude mouse xenotransplantation model using HLCZ01 cells. Treatment with celecoxib and IFN-α synergistically inhibited cell proliferation in a dose- and time-dependent manner. Apoptosis was identified by 4',6-diamidino-2-phenylindole dihydrochloride and fluorescent staining. IFN-α upregulated the expression of TRAIL, while celecoxib increased the expression of TRAIL receptors. The combined regimen with celecoxib and IFN-α reduced the growth of xenotransplanted HCCs in nude mice. The regulation of IFN-α- and COX-2 inhibitor-induced cell death is impaired in a subset of TRAIL-resistant cells. The molecular mechanisms of HCC cells resistant to TRAIL-induced apoptosis were explored using molecular biological and immunological methods. Interferon-α and the COX-2 inhibitor celecoxib synergistically increased TRAIL-induced apoptosis in hepatocellular carcinoma. These data suggest that IFN-α and celecoxib may offer a novel role with important implications in designing new therapeutics for TRAIL-resistant tumors. - Highlights: ●The cytotoxic effect of TRAIL on a developed HCC HLCZ01 cells infected with HBV. ●IFN-α and celecoxib induced apoptosis in HLCZ01 cells infected with HBV. ●The combined regime reduced the growth of xenotransplanted HCCs in nude mice model.

  5. Slug promotes survival during metastasis through suppression of Puma-mediated apoptosis

    OpenAIRE

    Kim, Seaho; Yao, Jiahong; Suyama, Kimita; Qian, Xia; Qian, Bin-Zhi; Bandyopadhyay, Sanmay; Loudig, Olivier; De Leon-Rodriguez, Carlos; Zhou, Zhen Ni; Segall, Jeffrey; Macian, Fernando; Norton, Larry; Hazan, Rachel. B.

    2014-01-01

    Tumor cells must overcome apoptosis to survive throughout metastatic dissemination and distal organ colonization. Here we show in the Polyoma Middle T mammary tumor model that N-cadherin expression causes Slug upregulation, which in turn promotes carcinoma cell survival. Slug was dramatically upregulated in metastases relative to primary tumors. Consistent with a role in metastasis, Slug knockdown in carcinoma cells suppressed lung colonization by decreasing cell survival at metastatic sites,...

  6. Decreased apoptosis during CAR-mediated hepatoprotection against lithocholic acid-induced liver injury in mice.

    Science.gov (United States)

    Beilke, Lisa D; Aleksunes, Lauren M; Olson, Erik R; Besselsen, David G; Klaassen, Curtis D; Dvorak, Katerina; Cherrington, Nathan J

    2009-07-10

    Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein that is regulated by the constitutive androstane receptor (CAR). Activation of CAR can protect the liver against bile acid-induced toxicity and it may have a role in cell death via apoptosis by altering expression of Bcl-2 family proteins such as myeloid cell leukemia-1 (Mcl-1). Our aim was to determine if activation of CAR reduces hepatocellular apoptosis during cholestasis as a mechanism of hepatoprotection. CAR(+/+) (WT) and CAR(-/-) (CAR-null) mice were pre-treated with compounds known to activate CAR prior to induction of intrahepatic cholestasis using the secondary bile acid lithocholic acid (LCA). Pre-treatment with the CAR activators phenobarbital (PB) and TCPOBOP (TC), as well as the non-CAR activator pregnenolone 16alpha-carbontrile (PCN), protected against LCA-induced liver injury in WT mice, whereas liver injury was more extensive without CAR (CAR-null). Unexpectedly, expression of anti-apoptotic Mcl-1 and Bcl-x(L) was not increased in hepatoprotected mice. Compared to unprotected groups, apoptosis was decreased in hepatoprotected mice as evidenced by the absence of cleaved caspase 3 (cCasp3). In contrast to the cytoplasmic localization in the injured livers (LCA and oltipraz), Mcl-1 protein was localized in the nucleus of hepatoprotected livers to potentially promote cell survival. This study demonstrates that although apoptosis is reduced in hepatoprotected mice pre-treated with CAR and non-CAR activators; hepatoprotection is not directly a result of CAR-induced Mcl-1 expression.

  7. Increased sensitivity of African American triple negative breast cancer cells to nitric oxide-induced mitochondria-mediated apoptosis

    International Nuclear Information System (INIS)

    Martinez, Luis; Thames, Easter; Kim, Jinna; Chaudhuri, Gautam; Singh, Rajan; Pervin, Shehla

    2016-01-01

    Breast cancer is a complex heterogeneous disease where many distinct subtypes are found. Younger African American (AA) women often present themselves with aggressive form of breast cancer with unique biology which is very difficult to treat. Better understanding the biology of AA breast tumors could lead to development of effective treatment strategies. Our previous studies indicate that AA but not Caucasian (CA) triple negative (TN) breast cancer cells were sensitive to nitrosative stress-induced cell death. In this study, we elucidate possible mechanisms that contribute to nitric oxide (NO)-induced apoptosis in AA TN breast cancer cells. Breast cancer cells were treated with various concentrations of long-acting NO donor, DETA-NONOate and cell viability was determined by trypan blue exclusion assay. Apoptosis was determined by TUNEL and caspase 3 activity as well as changes in mitochondrial membrane potential. Caspase 3 and Bax cleavage, levels of Cu/Zn superoxide dismutase (SOD) and Mn SOD was assessed by immunoblot analysis. Inhibition of Bax cleavage by Calpain inhibitor, and levels of reactive oxygen species (ROS) as well as SOD activity was measured in NO-induced apoptosis. In vitro and in vivo effect of NO treatment on mammary cancer stem cells (MCSCs) was assessed. NO induced mitocondria-mediated apoptosis in all AA but not in CA TN breast cancer cells. We found significant TUNEL-positive cells, cleavage of Bax and caspase-3 activation as well as depolarization mitochondrial membrane potential only in AA TN breast cancer cells exposed to NO. Inhibition of Bax cleavage and quenching of ROS partially inhibited NO-induced apoptosis in AA TN cells. Increase in ROS coincided with reduction in SOD activity in AA TN breast cancer cells. Furthermore, NO treatment of AA TN breast cancer cells dramatically reduced aldehyde dehydrogenase1 (ALDH1) expressing MCSCs and xenograft formation but not in breast cancer cells from CA origin. Ethnic differences in breast

  8. Aromadendrene oxide 2, induces apoptosis in skin epidermoid cancer cells through ROS mediated mitochondrial pathway.

    Science.gov (United States)

    Pavithra, P S; Mehta, Alka; Verma, Rama S

    2018-03-15

    Aromadendrene oxide 2 (AO-(2)) is an oxygenated sesquiterpene naturally found as a chemical component of essential oils. In the present study anticancer activity of AO-(2) has been investigated on A431 human epidermoid cancer and precancerous HaCaT cells. Cell viability was used to detect cytotoxic activity. Mechanism of cell death induced by AO-(2) treatments was studied using Annexin V-FITC/PI binding, cell cycle analysis, measurement of MMP and ROS generation by flow cytometry. Expression of apoptosis related proteins was investigated by western blot. AO-(2) inhibited the growth and colony formation ability of A431 and HaCaT cells in concentration dependent manner. It induced cell cycle arrest at G0/G1 phase and apoptosis through intracellular ROS accumulation. Inhibition of intracellular ROS by ascorbic acid and N-acetyl cysteine treatment completely blocked apoptotic effect. N-acetyl cysteine treatment significantly reversed G0/G1 arrest induced by AO-(2). AO-(2) treatment caused loss of mitochondrial membrane potential (ΔΨm), increase in Bax/Bcl-2 ratios, cytochrome c release, activation of caspases (cleaved caspase-3 and caspase-9) and PARP cleavage. AO-(2) also significantly inhibited the growth of multicellular tumor spheroids of A431 and HaCaT cells. The results of the present study reveals that AO-(2) a chemical component of essential oils induces apoptosis in A431 and HaCaT cells. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. MicroRNA 196B regulates FAS-mediated apoptosis in colorectal cancer cells

    Science.gov (United States)

    Kang, In-Hong; Park, Won Cheol; Seo, Geom-Seog; Choi, Suck-Chei; Kim, Hun-Soo; Moon, Hyung-Bae; Yun, Ki-Jung; Chae, Soo-Cheon

    2015-01-01

    Using miRNA microarray analysis, we identified 31 miRNAs that were significantly up-regulated or down-regulated in colon cancer tissues. We chose MIR196B, which was specifically up-regulated in colon cancer, for further study. We identified 18 putative MIR196B target genes by comparing between the mRNAs down-regulated in MIR196B-overexpressed cells and the assumed MIR196B target genes predicted by public bioinformatics tools. The association between MIR196B and FAS was verified in this study. FAS expression was constitutively elevated in normal human colorectal tissues. However, its expression was often reduced in human colorectal cancer. The decrease in FAS expression could be responsible for the reduction of apoptosis in colorectal cancer cells. In colorectal cancer tissue, we showed that MIR196B up-regulation was mutually followed by down regulation of FAS expression. We also showed that MIR196B directly repressed FAS expression in colorectal cells. Furthermore, anti-MIR196B up-regulated FAS expression and increased apoptosis in colorectal cancer cell lines. Our results suggest that the up-regulation of MIR196B modulates apoptosis in colorectal cancer cells by partially repressing FAS expression and that anti-MIR196B could be a potential candidate as an anti-cancer drug in colorectal cancer therapy. PMID:25605245

  10. The ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis.

    Science.gov (United States)

    Zhao, D; Yang, M; Solava, J; Ma, H

    1999-09-01

    Normal flower development likely requires both specific and general regulators. We have isolated an Arabidopsis mutant ask1-1 (for -Arabidopsis skp1-like1-1), which exhibits defects in both vegetative and reproductive development. In the ask1-1mutant, rosette leaf growth is reduced, resulting in smaller than normal rosette leaves, and internodes in the floral stem are shorter than normal. Examination of cell sizes in these organs indicates that cell expansion is normal in the mutant, but cell number is reduced. In the mutant, the numbers of petals and stamens are reduced, and many flowers have one or more petals with a reduced size. In addition, all mutant flowers have short stamen filaments. Furthermore, petal/stamen chimeric organs are found in many flowers. These results indicate that the ASK1 gene affects the size of vegetative and floral organs. The ask1 floral phenotype resembles somewhat that of the Arabidopsis ufo mutants in that both genes affect whorls 2 and 3. We therefore tested for possible interactions between ASK1 and UFO by analyzing the phenotypes of ufo-2 ask1-1 double mutant plants. In these plants, vegetative development is similar to that of the ask1-1 single mutant, whereas the floral defects are more severe than those in either single mutant. Interior to the first whorl, the double mutant flowers have more sepals or sepal-like organs than are found in ufo-2, and less petals than ask1-1. Our results suggest that ASK1 interacts with UFO to control floral organ identity in whorls 2 and 3. This is very intriguing because ASK1 is very similar in sequence to the yeast SKP1 protein and UFO contains an F-box, a motif known to interact with SKP1 in yeast. Although the precise mechanism of ASK1 and UFO action is unknown, our results support the hypothesis that these two proteins physically interact in vivo. Copyright 1999 Wiley-Liss, Inc.

  11. Nicotine-induced embryonic malformations mediated by apoptosis from increasing intracellular calcium and oxidative stress.

    Science.gov (United States)

    Zhao, Zhiyong; Reece, E Albert

    2005-10-01

    Tobacco smoking by women during pregnancy increases the risk of congenital birth defects in the infants. Among the smoke products, nicotine is believed to be the major teratogenic factor that perturbs embryonic development. However, the role of nicotine in embryonic malformations has not been addressed, and the mechanisms by which nicotine affects embryonic development remain to be delineated. To investigate the effects of nicotine on early embryogenesis, murine embryos at embryonic day (E) 8.5 were dissected out of the uteri, cultured in a roller bottle system, and treated with nicotine (0.6-6 microM) or vehicle. Embryonic morphogenesis and growth were examined in terms of structural morphology and crown/rump length, respectively. Programmed cell death (apoptosis) was assessed using LysoTracker Red staining of whole mount embryos and TUNEL assay of tissue sections. Changes in intracellular calcium concentration ([Ca2+]i) and reactive oxygen species (ROS) production were assessed using fluorescent dyes (Flu-4, AM; H2DCFDA, respectively) under a confocal microscope. To further investigate the role of intracellular calcium and ROS in nicotine-induced embryopathy, embryos were treated with BAPTA-AM (2 microM) to inhibit [Ca2+]i elevation and ascorbic acid (vitamin C; 100 microg/ml) to scavenge ROS in presence of nicotine (6 microM). The embryos treated with nicotine in 3-6 microM were smaller than those treated with vehicle. Most of the embryos had open neural tube in the anterior (brain) regions. The embryos treated with 6 microM nicotine also exhibited severe defects in the posterior trunk, resembling caudal dysplasia. Excessive apoptosis was observed in the deformed structures. Significant increases in [Ca2+]i and ROS were seen in the tissues that had higher levels of apoptosis. Furthermore, inhibition of [Ca2+]i and scavenging of ROS significantly reduced embryonic malformation and apoptotic rates in the embryos. Nicotine affects embryonic development in a

  12. Nitric oxide-induced eosinophil apoptosis is dependent on mitochondrial permeability transition (mPT, JNK and oxidative stress: apoptosis is preceded but not mediated by early mPT-dependent JNK activation

    Directory of Open Access Journals (Sweden)

    Ilmarinen-Salo Pinja

    2012-08-01

    Full Text Available Abstract Background Eosinophils are critically involved in the pathogenesis of asthma. Nitric oxide (NO is produced in high amounts in asthmatic lungs and has an important role as a regulator of lung inflammation. NO was previously shown to induce eosinophil apoptosis mediated via c-jun N-terminal kinase (JNK and caspases. Our aim was to clarify the cascade of events leading to NO-induced apoptosis in granulocyte macrophage-colony stimulating factor (GM-CSF-treated human eosinophils concentrating on the role of mitochondria, reactive oxygen species (ROS and JNK. Methods Apoptosis was determined by flow cytometric analysis of relative DNA content, by Annexin-V labelling and/or morphological analysis. Immunoblotting was used to study phospho-JNK (pJNK expression. Mitochondrial membrane potential was assessed by JC-1-staining and mitochondrial permeability transition (mPT by loading cells with calcein acetoxymethyl ester (AM and CoCl2 after which flow cytometric analysis was conducted. Statistical significance was calculated by repeated measures analysis of variance (ANOVA or paired t-test. Results NO-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP induced late apoptosis in GM-CSF-treated eosinophils. SNAP-induced apoptosis was suppressed by inhibitor of mPT bongkrekic acid (BA, inhibitor of JNK SP600125 and superoxide dismutase-mimetic AEOL 10150. Treatment with SNAP led to late loss of mitochondrial membrane potential. Additionally, we found that SNAP induces early partial mPT (1 h that was followed by a strong increase in pJNK levels (2 h. Both events were prevented by BA. However, these events were not related to apoptosis because SNAP-induced apoptosis was prevented as efficiently when BA was added 16 h after SNAP. In addition to the early and strong rise, pJNK levels were less prominently increased at 20–30 h. Conclusions Here we demonstrated that NO-induced eosinophil apoptosis is mediated via ROS, JNK and late mPT. Additionally

  13. Regulation of Microglia and Macrophage Polarization via Apoptosis Signal-Regulating Kinase 1 Silencing after Ischemic/Hypoxic Injury

    Directory of Open Access Journals (Sweden)

    So Yeong Cheon

    2017-08-01

    Full Text Available Inflammation is implicated in ischemic stroke and is involved in abnormal homeostasis. Activation of the immune system leads to breakdown of the blood–brain barrier and, thereby, infiltration of immune cells into the brain. Upon cerebral ischemia, infiltrated macrophages and microglia (resident CNS immune cell are activated, change their phenotype to M1 or M2 based on the microenvironment, migrate toward damaged tissue, and are involved in repair or damage. Those of M1 phenotype release pro-inflammatory mediators, which are associated with tissue damage, while those of M2 phenotype release anti-inflammatory mediators, which are related to tissue recovery. Moreover, late inflammation continually stimulates immune cell infiltration and leads to brain infarction. Therefore, regulation of M1/M2 phenotypes under persistent inflammatory conditions after cerebral ischemia is important for brain repair. Herein, we focus on apoptosis signal-regulating kinase 1 (ASK1, which is involved in apoptotic cell death, brain infarction, and production of inflammatory mediators after cerebral ischemia. We hypothesized that ASK1 is involved in the polarization of M1/M2 phenotype and the function of microglia and macrophage during the late stage of ischemia/hypoxia. We investigated the effects of ASK1 in mice subjected to middle cerebral artery occlusion and on BV2 microglia and RAW264.7 macrophage cell lines subjected to oxygen-glucose deprivation. Our results showed that ASK1 silencing effectively reduced Iba-1 or CD11b-positive cells in ischemic areas, suppressed pro-inflammatory cytokines, and increased anti-inflammatory mediator levels at 7 days after cerebral ischemia. In cultured microglia and macrophages, ASK1 inhibition, induced by NQDI-1 drug, decreased the expression and release of M1-associated factors and increased those of M2-associated factors after hypoxia/reperfusion (H/R. At the gene level, ASK1 inhibition suppressed M1-associated genes and

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

    Science.gov (United States)

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

    2016-05-01

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

  15. Reactive oxygen species-mediated apoptosis contributes to chemosensitization effect of saikosaponins on cisplatin-induced cytotoxicity in cancer cells

    Directory of Open Access Journals (Sweden)

    He Fan

    2010-12-01

    Full Text Available Abstract Background Saikosaponin-a and -d, two naturally occurring compounds derived from Bupleurum radix, have been shown to exert anti-cancer activity in several cancer cell lines. However, the effect of combination of saikosaponins with chemotherapeutic drugs has never been addressed. Thus, we investigated whether these two saikosaponins have chemosensitization effect on cisplatin-induced cancer cell cytotoxicity. Methods Two cervical cancer cell lines, HeLa and Siha, an ovarian cancer cell line, SKOV3, and a non-small cell lung cancer cell line, A549, were treated with saikosaponins or cisplatin individually or in combination. Cell death was quantitatively detected by the release of lactate dehydrogenase (LDH using a cytotoxicity detection kit. Cellular ROS was analyzed by flow cytometry. Apoptosis was evaluated by AO/EB staining, flow cytometry after Anexin V and PI staining, and Western blot for caspase activation. ROS scavengers and caspase inhibitor were used to determine the roles of ROS and apoptosis in the effects of saikosaponins on cisplatin-induced cell death. Results Both saikosaponin-a and -d sensitized cancer cells to cisplatin-induced cell death in a dose-dependent manner, which was accompanied with induction of reactive oxygen species (ROS accumulation. The dead cells showed typical apoptotic morphologies. Both early apoptotic and late apoptotic cells detected by flow cytometry were increased in saikosaponins and cisplatin cotreated cells, accompanied by activation of the caspase pathway. The pan-caspase inhibitor z-VAD and ROS scanvengers butylated hydroxyanisole (BHA and N-acetyl-L-cysteine (NAC dramatically suppressed the potentiated cytotoxicity achieved by combination of saikosaponin-a or -d and cisplatin. Conclusions These results suggest that saikosaponins sensitize cancer cells to cisplatin through ROS-mediated apoptosis, and the combination of saikosaponins with cisplatin could be an effective therapeutic strategy.

  16. PI3K/Akt signaling mediated Hexokinase-2 expression inhibits cell apoptosis and promotes tumor growth in pediatric osteosarcoma

    Energy Technology Data Exchange (ETDEWEB)

    Zhuo, Baobiao; Li, Yuan; Li, Zhengwei; Qin, Haihui; Sun, Qingzeng; Zhang, Fengfei; Shen, Yang; Shi, Yingchun [Department of Surgery, The Children' s Hospital of Xuzhou, Xuzhou, Jiangsu Province 221006 (China); Wang, Rong, E-mail: wangrong2008163@163.com [Department of Ultrasonography, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu Province 221006 (China)

    2015-08-21

    Accumulating evidence has shown that PI3K/Akt pathway is frequently hyperactivated in osteosarcoma (OS) and contributes to tumor initiation and progression. Altered phenotype of glucose metabolism is a key hallmark of cancer cells including OS. However, the relationship between PI3K/Akt pathway and glucose metabolism in OS remains largely unexplored. In this study, we showed that elevated Hexokinase-2 (HK2) expression, which catalyzes the first essential step of glucose metabolism by conversion of glucose into glucose-6-phosphate, was induced by activated PI3K/Akt signaling. Immunohistochemical analysis showed that HK2 was overexpressed in 83.3% (25/30) specimens detected and was closely correlated with Ki67, a cell proliferation index. Silencing of endogenous HK2 resulted in decreased aerobic glycolysis as demonstrated by reduced glucose consumption and lactate production. Inhibition of PI3K/Akt signaling also suppressed aerobic glycolysis and this effect can be reversed by reintroduction of HK2. Furthermore, knockdown of HK2 led to increased cell apoptosis and reduced ability of colony formation; meanwhile, these effects were blocked by 2-Deoxy-D-glucose (2-DG), a glycolysis inhibitor through its actions on hexokinase, indicating that HK2 functions in cell apoptosis and growth were mediated by altered aerobic glycolysis. Taken together, our study reveals a novel relationship between PI3K/Akt signaling and aerobic glycolysis and indicates that PI3K/Akt/HK2 might be potential therapeutic approaches for OS. - Highlights: • PI3K/Akt signaling contributes to elevated expression of HK2 in osteosarcoma. • HK2 inhibits cell apoptosis and promotes tumor growth through enhanced Warburg effect. • Inhibition of glycolysis blocks the oncogenic activity of HK2.

  17. PI3K/Akt signaling mediated Hexokinase-2 expression inhibits cell apoptosis and promotes tumor growth in pediatric osteosarcoma

    International Nuclear Information System (INIS)

    Zhuo, Baobiao; Li, Yuan; Li, Zhengwei; Qin, Haihui; Sun, Qingzeng; Zhang, Fengfei; Shen, Yang; Shi, Yingchun; Wang, Rong

    2015-01-01

    Accumulating evidence has shown that PI3K/Akt pathway is frequently hyperactivated in osteosarcoma (OS) and contributes to tumor initiation and progression. Altered phenotype of glucose metabolism is a key hallmark of cancer cells including OS. However, the relationship between PI3K/Akt pathway and glucose metabolism in OS remains largely unexplored. In this study, we showed that elevated Hexokinase-2 (HK2) expression, which catalyzes the first essential step of glucose metabolism by conversion of glucose into glucose-6-phosphate, was induced by activated PI3K/Akt signaling. Immunohistochemical analysis showed that HK2 was overexpressed in 83.3% (25/30) specimens detected and was closely correlated with Ki67, a cell proliferation index. Silencing of endogenous HK2 resulted in decreased aerobic glycolysis as demonstrated by reduced glucose consumption and lactate production. Inhibition of PI3K/Akt signaling also suppressed aerobic glycolysis and this effect can be reversed by reintroduction of HK2. Furthermore, knockdown of HK2 led to increased cell apoptosis and reduced ability of colony formation; meanwhile, these effects were blocked by 2-Deoxy-D-glucose (2-DG), a glycolysis inhibitor through its actions on hexokinase, indicating that HK2 functions in cell apoptosis and growth were mediated by altered aerobic glycolysis. Taken together, our study reveals a novel relationship between PI3K/Akt signaling and aerobic glycolysis and indicates that PI3K/Akt/HK2 might be potential therapeutic approaches for OS. - Highlights: • PI3K/Akt signaling contributes to elevated expression of HK2 in osteosarcoma. • HK2 inhibits cell apoptosis and promotes tumor growth through enhanced Warburg effect. • Inhibition of glycolysis blocks the oncogenic activity of HK2

  18. Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

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    Yu-Qin Zhang

    Full Text Available The role of Pokemon (POK erythroid myeloid ontogenic actor, a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy.

  19. Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

    Science.gov (United States)

    Zhang, Yu-Qin; Xiao, Chuan-Xing; Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

    2013-01-01

    The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy.

  20. Thymoquinone inhibits the CXCL12-induced chemotaxis of multiple myeloma cells and increases their susceptibility to Fas-mediated apoptosis.

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    Gamal Badr

    Full Text Available In multiple myeloma (MM, malignant plasma cells reside in the bone marrow, where they accumulate in close contact with stromal cells. The mechanisms responsible for the chemotaxis of malignant plasma cells are still poorly understood. Thus, we investigated the mechanisms involved in the chemotaxis of MDN and XG2 MM cell lines. Both cell lines strongly expressed CCR9, CXCR3 and CXCR4 chemokine receptors but only migrated toward CXCL12. Activation of CXCR4 by CXCL12 resulted in the association of CXCR4 with CD45 and activation of PLCβ3, AKT, RhoA, IκBα and ERK1/2. Using siRNA-silencing techniques, we showed CD45/CXCR4 association is essential for CXCL12-induced migration of MM cells. Thymoquinone (TQ, the major active component of the medicinal herb Nigella sativa Linn, has been described as a chemopreventive and chemotherapeutic compound. TQ treatment strongly inhibited CXCL12-mediated chemotaxis in MM cell lines as well as primary cells isolated from MM patients, but not normal PBMCs. Moreover, TQ significantly down-regulated CXCR4 expression and CXCL12-mediated CXCR4/CD45 association in MM cells. Finally, TQ also induced the relocalization of cytoplasmic Fas/CD95 to the membrane of MM cells and increased CD95-mediated apoptosis by 80%. In conclusion, we demonstrate the potent anti-myeloma activity of TQ, providing a rationale for further clinical evaluation.

  1. Tetrapeptide DEVD-aldehyde or YVAD-chloromethylketone inhibits Fas/Apo-1(CD95)-mediated apoptosis in renal-cell-cancer cells.

    Science.gov (United States)

    Tomita, Y; Kawasaki, T; Bilim, V; Takeda, M; Takahashi, K

    1996-09-27

    The apoptotic machinery has been intensively investigated, and interleukin-1-beta-converting enzyme (ICE) and its homologs directly mediate apoptosis by means of their unique protease activity. Fas/Apo1 (CD95), a member of the TNF-receptor family, mediates apoptosis by binding to its ligand, which is mainly expressed on lymphocytes. Here, we investigated the expression and function of both molecules in renal-cell cancer (RCC). The expression of Fas was examined in 6 RCC cell lines by immunoblotting and all of them expressed Fas. ICE and CPP32/YAMA were also identified among the cell lines. We earlier examined ACHN cells expressing low levels of BCL-2, as well as KRC/Y cells with high levels of BCL-2. Here, we found that the anti-Fas monoclonal antibody, CH-11, induced apoptosis in a dose-dependent fashion more remarkably in ACHN cells. Pre-incubation with the tetrapeptide YVAD-chloromethyl-ketone or DEVD-aldehyde inhibited Fas-mediated apoptosis. These findings suggest that, in RCC, apoptosis is induced by lymphocytes bearing Fas-L, and that it is achieved through the proteolytic action of CPP32/YAMA and/or ICE, or another member of the ICE/ced-3 protease family.

  2. RNAi mediated gene silencing of ITPA using a targeted nanocarrier: Apoptosis induction in SKBR3 cancer cells.

    Science.gov (United States)

    Charbgoo, Fahimeh; Behmanesh, Mehrdad; Nikkhah, Maryam; Kane, Eric G

    2017-08-01

    A pure nucleotide pool is required for high-fidelity DNA replication and prevention of carcinogenesis in living cells. Human inosine triphosphatase (ITPase), encoded by the ITPA gene, plays a critical role in maintaining the purity of the cellular nucleotide pool by excluding nucleotides that enhance mutagenesis. ITPase is a nucleoside triphosphate pyrophosphatase that hydrolyzes the non-canonical nucleotides inosine triphosphate (ITP) and xanthine triphosphate (XTP). The monophosphate products of ITPase reactions are subsequently excluded from the nucleotide pool and the improper substitution of ITP and XTP into DNA and RNA is prevented. Previous studies show that deficiency in ITPA can suppress cellular growth and enhance DNA instability. In this study, we evaluated the influence of effective ITPA down-regulation on the induction of apoptosis in a human cancer cell line using folate-single wall nanotubes (SWNT) as a targeted nanocarrier. We assessed whether SWNT enhances IPTA-siRNA transfection efficiency in cancer cells using folate as a homing device. Since folate receptor is considerably overexpressed in cancer cells, conjugation of SWNTs to folate could enhance their cancer-specific penetrance. We found that nanocarrier mediated ITPA-siRNA transfection into SKBR3 cells caused significant reduction of ITPA mRNA expression level and complete down-regulation of the ITPase protein product. The silencing of ITPA led to promotion of apoptosis in SWNT-treated SKBR3 cancer cells. © 2017 John Wiley & Sons Australia, Ltd.

  3. Protective effect of bone marrow mesenchymal stem cells on PC12 cells apoptosis mediated by TAG1.

    Science.gov (United States)

    Zhang, Yu-Zhen; Lou, Ji-Yu; Bai, Hong-Ying; Wang, Yun-Liang; Li, Jin-Feng; Yin, Hong-Lei

    2015-01-01

    This study aims to explore the protection effect of bone marrow mesenchymal stem cells (BMSCs) on PC12 cells apoptosis mediated by transient axonal glycoprotein 1 (TAG1). PC12 cells were divided into control group, Aβ25-35 group and BMSCs + Aβ25-35 group. The effects of BMSCs on PC12 cells treated by Aβ25-35 were detected using MTT, Hoechst 33258 and Annexin V-FITC/PI staining methods. The expression levels of TAG1, β-amyloid precursor protein (APP), AICD and p53 were determined by RT-PCR and Western blotting methods. The expression levels of Bax and Bcl-2 were determined by Western blotting method. The activity of Caspase 3 was detected by spectrophotometric method. MTT results showed that cell activity decreased after the treatment of 20 μM Aβ25-35 for 48 h (PPC12 cells while the apoptosis of PC12 cells was inhibited in BMSCs + Aβ25-35 group. RT-PCR and Western blotting methods showed that 20 μM Aβ25-35 could increase the expression levels of TAG1, APP, AICD and p53 (PPC12 cells, which maybe related with TAG1/APP/AICD signal pathway.

  4. Rhein Elicits In Vitro Cytotoxicity in Primary Human Liver HL-7702 Cells by Inducing Apoptosis through Mitochondria-Mediated Pathway

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    Guy-Armel Bounda

    2015-01-01

    Full Text Available Objective. To study rhein-induced apoptosis signaling pathway and to investigate its molecular mechanisms in primary human hepatic cells. Results. Cell viability of HL-7702 cells treated with rhein showed significant decrease in dose-dependent manner. Following rhein treatment (25 μM, 50 μM, and 100 μM for 12 h, the detection of apoptotic cells was significantly analyzed by flow cytometry and nuclear morphological changes by Hoechst 33258, respectively. Fatty degeneration studies showed upregulation level of the relevant hepatic markers (P < 0.01. Caspase activities expressed significant upregulation of caspase-3, caspase-9, and caspase-8. Moreover, apoptotic cells by rhein were significantly inhibited by Z-LEHD-FMK and Z-DEVD-FMK, caspase-9 inhibitor, and caspase-3 inhibitor, respectively. Overproduction of reactive oxygen species, lipid peroxidation, and loss of mitochondrial membrane potential were detected by fluorometry. Additionally, NAC, a ROS scavenger, significantly attenuated rhein-induced oxidative damage in HL-7702 cells. Furthermore, real-time qPCR results showed significant upregulation of p53, PUMA, Apaf-1, and Casp-9 and Casp-3 mRNA, with no significant changes of Fas and Cytochrome-c. Immunoblotting revealed significant Cytochrome-c release from mitochondria into cytosol and no change in Fas expression. Conclusion. Taken together, these observations suggested that rhein could induce apoptosis in HL-7702 cells via mitochondria-mediated signal pathway with involvement of oxidative stress mechanism.

  5. EZH2-mediated Puma gene repression regulates non-small cell lung cancer cell proliferation and cisplatin-induced apoptosis.

    Science.gov (United States)

    Liu, Haidan; Li, Wei; Yu, Xinfang; Gao, Feng; Duan, Zhi; Ma, Xiaolong; Tan, Shiming; Yuan, Yunchang; Liu, Lijun; Wang, Jian; Zhou, Xinmin; Yang, Yifeng

    2016-08-30

    Polycomb group (PcG) proteins are highly conserved epigenetic effectors that maintain the silenced state of genes. EZH2 is the catalytic core and one of the most important components of the polycomb repressive complex 2 (PRC2). In non-small cell lung cancer (NSCLC) cells and primary lung tumors, we found that PRC2 components, including EZH2, are overexpressed. High levels of EZH2 protein were associated with worse overall survival rate in NSCLC patients. RNA interference mediated attenuation of EZH2 expression blunted the malignant phenotype in this setting, exerting inhibitory effects on cell proliferation, anchorage-independent growth, and tumor development in a xenograft mouse model. Unexpectedly, we discovered that, in the suppression of EZH2, p53 upregulated modulator of apoptosis (PUMA) expression was concomitantly induced. This is achieved through EZH2 directly binds to the Puma promoter thus epigenetic repression of PUMA expression. Furthermore, cisplatin-induced apoptosis of EZH2-knocking down NSCLC cells was elevated as a consequence of increased PUMA expression. Our work reveals a novel epigenetic regulatory mechanism controlling PUMA expression and suggests that EZH2 offers a candidate molecular target for NSCLC therapy and EZH2-regulated PUMA induction would synergistically increase the sensitivity to platinum agents in non-small cell lung cancers.

  6. A Lentinus edodes polysaccharide induces mitochondrial-mediated apoptosis in human cervical carcinoma HeLa cells.

    Science.gov (United States)

    Ya, Guowei

    2017-10-01

    In this study, a homogeneous polysaccharide (LEP1) with an average molecular weight of 53kDa was successfully purified from the fruiting bodies of Lentinus edodes and its anticancer efficacy on human cervical carcinoma HeLa cells in vitro and associated possible molecular mechanism were also evaluated. MTT assay showed that LEP1 exhibited a dose-dependent inhibitory effect on the proliferation of HeLa cells and caused apoptotic death. Our present findings provided the first evidence that LEP1 induced the apoptosis of HeLa cells via a mitochondria dependent pathway, as indicated by an increase in Bax/Bcl-2 ratio, a loss of mitochondrial membrane potential (Δym), the release of cytochrome c from the mitochondria to the cytosol, activation of caspase-9 and caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP) in HeLa cells. These combined results unequivocally indicated that the involvement of mitochondria-mediated signaling pathway in LEP1-induced apoptosis and strongly provided experimental evidence for the use of LEP1 as a potential therapeutic agent in the prevention and treatment of human cervical carcinoma. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. HSPB1 facilitates ERK-mediated phosphorylation and degradation of BIM to attenuate endoplasmic reticulum stress-induced apoptosis.

    Science.gov (United States)

    Kennedy, Donna; Mnich, Katarzyna; Oommen, Deepu; Chakravarthy, Reka; Almeida-Souza, Leonardo; Krols, Michiel; Saveljeva, Svetlana; Doyle, Karen; Gupta, Sanjeev; Timmerman, Vincent; Janssens, Sophie; Gorman, Adrienne M; Samali, Afshin

    2017-08-31

    BIM, a pro-apoptotic BH3-only protein, is a key regulator of the intrinsic (or mitochondrial) apoptosis pathway. Here, we show that BIM induction by endoplasmic reticulum (ER) stress is suppressed in rat PC12 cells overexpressing heat shock protein B1 (HSPB1 or HSP27) and that this is due to enhanced proteasomal degradation of BIM. HSPB1 and BIM form a complex that immunoprecipitates with p-ERK1/2. We found that HSPB1-mediated proteasomal degradation of BIM is dependent on MEK-ERK signaling. Other studies have shown that several missense mutations in HSPB1 cause the peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease, which is associated with nerve degeneration. Here we show that cells overexpressing CMT-related HSPB1 mutants exhibited increased susceptibility to ER stress-induced cell death and high levels of BIM. These findings identify a novel function for HSPB1 as a negative regulator of BIM protein stability leading to protection against ER stress-induced apoptosis, a function that is absent in CMT-associated HSPB1 mutants.

  8. Early-life exposure to bisphenol a induces liver injury in rats involvement of mitochondria-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Wei Xia

    Full Text Available Exposure to bisphenol A (BPA, a monomer widely used to manufacture polycarbonate plastics, has been reported to be associated with abnormalities of liver function and hepatic damage. However, the molecular mechanism under the pathogenesis of hepatic injury is unclear. In this study, the effect of perinatal exposure to BPA at the reference dose of 50 µg/kg/day on the apoptotic index in the liver of rat offspring was investigated. Increased levels of ALT and enhanced cell apoptosis were observed in the liver of rat offspring at 15 and 21 weeks, and significantly increased activity of caspase-3 and caspase-9 and elevated levels of cytochrome c were also confirmed. In addition, significant change in the expression levels of Bcl-2 and Bax were found in BPA-treated offspring at 21 weeks. For in vitro experiments, liver mitochondria were isolated from neonatal rats and were treated with BPA. BPA treatment led to a significant increase in mitochondrial permeability transition. Moreover, the supernatant from BPA-treated mitochondria significantly increased apoptotic changes in nuclei isolated from liver tissue. In conclusion, the study demonstrates that BPA induces mitochondria-mediated apoptosis in hepatic cells, which may contribute to long-term hepatotoxicity induced by early-life exposure to BPA.

  9. Small molecule obatoclax (GX15-070) antagonizes MCL-1 and overcomes MCL-1-mediated resistance to apoptosis

    Science.gov (United States)

    Nguyen, Mai; Marcellus, Richard C.; Roulston, Anne; Watson, Mark; Serfass, Lucile; Murthy Madiraju, S. R.; Goulet, Daniel; Viallet, Jean; Bélec, Laurent; Billot, Xavier; Acoca, Stephane; Purisima, Enrico; Wiegmans, Adrian; Cluse, Leonie; Johnstone, Ricky W.; Beauparlant, Pierre; Shore, Gordon C.

    2007-01-01

    Elevated expression of members of the BCL-2 pro-survival family of proteins can confer resistance to apoptosis in cancer cells. Small molecule obatoclax (GX15-070), which is predicted to occupy a hydrophobic pocket within the BH3 binding groove of BCL-2, antagonizes these members and induces apoptosis, dependent on BAX and BAK. Reconstitution in yeast confirmed that obatoclax acts on the pathway and overcomes BCL-2-, BCL-XL-, BCL-w-, and MCL-1-mediated resistance to BAX or BAK. The compound potently interfered with the direct interaction between MCL-1 and BAK in intact mitochondrial outer membrane and inhibited the association between MCL-1 and BAK in intact cells. MCL-1 has been shown to confer resistance to the BCL-2/BCL-XL/BCL-w-selective antagonist ABT-737 and to the proteasome inhibitor bortezomib. In both cases, this resistance was overcome by obatoclax. These findings support a rational clinical development opportunity for the compound in cancer indications or treatments where MCL-1 contributes to resistance to cell killing. PMID:18040043

  10. Phorbol Esters Isolated from Jatropha Meal Induced Apoptosis-Mediated Inhibition in Proliferation of Chang and Vero Cell Lines

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    Syahida Ahmad

    2012-10-01

    Full Text Available The direct feeding of Jatropha meal containing phorbol esters (PEs indicated mild to severe toxicity symptoms in various organs of different animals. However, limited information is available on cellular and molecular mechanism of toxicity caused by PEs present in Jatropha meal. Thus, the present study was conducted to determine the cytotoxic and mode of action of PEs isolated from Jatropha meal using human hepatocyte (Chang and African green monkey kidney (Vero cell lines. The results showed that isolated PEs inhibited cell proliferation in a dose-dependent manner in both cell lines with the CC50 of 125.9 and 110.3 μg/mL, respectively. These values were compatible to that of phorbol 12-myristate 13-acetate (PMA values as positive control i.e., 124.5 and 106.3 μg/mL respectively. Microscopic examination, flow cytometry and DNA fragmentation results confirmed cell death due to apoptosis upon treatment with PEs and PMA at CC50 concentration for 24 h in both cell lines. The Western blot analysis revealed the overexpression of PKC-δ and activation of caspase-3 proteins which could be involved in the mechanism of action of PEs and PMA. Consequently, the PEs isolated form Jatropha meal caused toxicity and induced apoptosis-mediated proliferation inhibition toward Chang and Vero cell lines involving over-expression of PKC-δ and caspase-3 as their mode of actions.

  11. Copper Oxide Nanoparticles Induced Mitochondria Mediated Apoptosis in Human Hepatocarcinoma Cells

    OpenAIRE

    Siddiqui, Maqsood A.; Alhadlaq, Hisham A.; Ahmad, Javed; Al-Khedhairy, Abdulaziz A.; Musarrat, Javed; Ahamed, Maqusood

    2013-01-01

    Copper oxide nanoparticles (CuO NPs) are heavily utilized in semiconductor devices, gas sensor, batteries, solar energy converter, microelectronics and heat transfer fluids. It has been reported that liver is one of the target organs for nanoparticles after they gain entry into the body through any of the possible routes. Recent studies have shown cytotoxic response of CuO NPs in liver cells. However, the underlying mechanism of apoptosis in liver cells due to CuO NPs exposure is largely lack...

  12. BIM-Mediated AKT Phosphorylation Is a Key Modulator of Arsenic Trioxide-Induced Apoptosis in Cisplatin-Sensitive and -Resistant Ovarian Cancer Cells

    Science.gov (United States)

    Yuan, Zhu; Wang, Fang; Zhao, Zhiwei; Zhao, Xinyu; Qiu, Ji; Nie, Chunlai; Wei, Yuquan

    2011-01-01

    Background Chemo-resistance to cisplatin-centered cancer therapy is a major obstacle to the effective treatment of human ovarian cancer. Previous reports indicated that arsenic trioxide (ATO) induces cell apoptosis in both drug-sensitive and -resistant ovarian cancer cells. Principal Findings In this study, we determined the molecular mechanism of ATO-induced apoptosis in ovarian cancer cells. Our data demonstrated that ATO induced cell apoptosis by decreasing levels of phosphorylated AKT (p-AKT) and activating caspase-3 and caspase-9. Importantly, BIM played a critical role in ATO-induced apoptosis. The inhibition of BIM expression prevented AKT dephosphorylation and inhibited caspase-3 activation during cell apoptosis. However, surprisingly, gene silencing of AKT or FOXO3A had little effect on BIM expression and phosphorylation. Moreover, the activation of caspase-3 by ATO treatment improved AKT dephosphorylation, not only by cleaving the regulatory A subunit of protein phosphatase 2A (PP2A), but also by increasing its activation. Furthermore, our data indicated that the c-Jun N-terminal kinases (JNK) pathway is involved in the regulation of BIM expression. Conclusions We demonstrated the roles of BIM in ATO-induced apoptosis and the molecular mechanisms of BIM expression regulated by ATO during ovarian cancer cell apoptosis. Our findings suggest that BIM plays an important role in regulating p-AKT by activating caspase-3 and that BIM mediates the level of AKT phosphorylation to determine the threshold for overcoming cisplatin resistance in ovarian cancer cells. PMID:21655183

  13. Magnetic nanoparticle-mediated hyperthermia therapy induces tumour growth inhibition by apoptosis and Hsp90/AKT modulation.

    Science.gov (United States)

    Shetake, Neena G; Kumar, Amit; Gaikwad, Snehal; Ray, Pritha; Desai, Sejal; Ningthoujam, Raghumani Singh; Vatsa, Rajesh Kumar; Pandey, Badri N

    2015-01-01

    We have evaluated the hyperthermia efficacy of oleic acid-functionalised Fe(3)O(4) magnetic nanoparticles (MN-OA) under in vivo conditions and elucidated the underlying mechanism of tumour growth inhibition. The efficacy and mechanism of tumour growth inhibition by MN-OA-mediated magnetic hyperthermia therapy (MHT) was evaluated in a murine fibrosarcoma tumour model (WEHI-164) using techniques such as TUNEL assay, Western blotting (WB), immunofluorescence (IF) staining and histopathological examination. In addition, bio-distribution of MN-OA in tumour/other target organs and its effect on normal organ function were studied by Prussian blue staining and serum biochemical analysis, respectively. MN-OA-induced MHT resulted in significant inhibition of tumour growth as determined by measurement of tumour volume, as well as by in vivo imaging of tumour derived from luciferase-transfected WEHI-164 cells. Histopathology analysis showed presence of severe apoptosis and reduced tumour cells proliferation, which was further confirmed by TUNEL assay, reduced expression of Ki-67 and enhanced level of cleaved caspase-3, in tumours treated with MHT. Moreover, expression of heat stress marker, Hsp90 and its client protein, AKT/PKB was reduced by ∼50 and 80%, respectively, in tumours treated with MHT as studied by WB and IF staining. Serum analysis suggested insignificant toxicity of MN-OA (in terms of liver and kidney function), which was further correlated with minimal accumulation of MN-OA in target organs. These results suggest the involvement of apoptosis and Hsp90/AKT modulation in MN-OA-mediated MHT-induced tumour growth inhibition.

  14. Death receptor and mitochondria-mediated hepatocyte apoptosis underlies liver dysfunction in rats exposed to organic pollutants from drinking water.

    Science.gov (United States)

    Yang, Guanghong; Zhou, Zhiwei; Cen, Yanli; Gui, Xiaolin; Zeng, Qibing; Ao, Yunxia; Li, Qian; Wang, Shiran; Li, Jun; Zhang, Aihua

    2015-01-01

    Persistent organic pollutants in drinking water impose a substantial risk to the health of human beings, but the evidence for liver toxic effect and the underlying mechanism is scarce. This study aimed to examine the liver toxicity and elucidate the molecular mechanism of organic pollutants in drinking water in normal human liver cell line L02 cells and rats. The data showed that organic extraction from drinking water remarkably impaired rat liver function, evident from the increase in the serum level of alanine aminotransferase, aspartate aminotransferase, and cholinesterase, and decrease in the serum level of total protein and albumin. Organic extraction dose-dependently induced apoptotic cell death in rat liver and L02 cells. Administration of rats with organic extraction promoted death receptor signaling pathway through the increase in gene and protein expression level of Fas and FasL. Treatment of rats with organic extraction also induced mitochondria-mediated apoptosis via increasing the expression level of proapoptotic protein, Bax, but decreasing the expression level of antiapoptotic protein, Bcl-2, resulting in an upregulation of cytochrome c and activation of caspase cascade at both transcriptional and post-transcriptional levels. Moreover, organic extraction enhanced rat liver glutathione S-transferases activity and reactive oxygen species generation, and upregulated aryl hydrocarbon receptor and glutathione S-transferase A1 at both transcriptional and translational levels. Collectively, the results indicate that organic extraction from drinking water impairs liver function, with the involvement of death receptor and mitochondria-mediated apoptosis in rats. The results provide evidence and molecular mechanisms for organic pollutants in drinking water-induced liver dysfunction, which may help prevent and treat organic extraction-induced liver injury.

  15. Genomic analysis reveals a potential role for cell cycle perturbation in HCV-mediated apoptosis of cultured hepatocytes.

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    Kathie-Anne Walters

    2009-01-01

    Full Text Available The mechanisms of liver injury associated with chronic HCV infection, as well as the individual roles of both viral and host factors, are not clearly defined. However, it is becoming increasingly clear that direct cytopathic effects, in addition to immune-mediated processes, play an important role in liver injury. Gene expression profiling during multiple time-points of acute HCV infection of cultured Huh-7.5 cells was performed to gain insight into the cellular mechanism of HCV-associated cytopathic effect. Maximal induction of cell-death-related genes and appearance of activated caspase-3 in HCV-infected cells coincided with peak viral replication, suggesting a link between viral load and apoptosis. Gene ontology analysis revealed that many of the cell-death genes function to induce apoptosis in response to cell cycle arrest. Labeling of dividing cells in culture followed by flow cytometry also demonstrated the presence of significantly fewer cells in S-phase in HCV-infected relative to mock cultures, suggesting HCV infection is associated with delayed cell cycle progression. Regulation of numerous genes involved in anti-oxidative stress response and TGF-beta1 signaling suggest these as possible causes of delayed cell cycle progression. Significantly, a subset of cell-death genes regulated during in vitro HCV infection was similarly regulated specifically in liver tissue from a cohort of HCV-infected liver transplant patients with rapidly progressive fibrosis. Collectively, these data suggest that HCV mediates direct cytopathic effects through deregulation of the cell cycle and that this process may contribute to liver disease progression. This in vitro system could be utilized to further define the cellular mechanism of this perturbation.

  16. 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.

  17. Adenosine monophosphate activated protein kinase (AMPK), a mediator of estradiol-induced apoptosis in long-term estrogen deprived breast cancer cells.

    Science.gov (United States)

    Chen, Haiyan; Wang, Ji-Ping; Santen, Richard J; Yue, Wei

    2015-06-01

    Estrogens stimulate growth of hormone-dependent breast cancer but paradoxically induce tumor regress under certain circumstances. We have shown that long-term estrogen deprivation (LTED) enhances the sensitivity of hormone dependent breast cancer cells to estradiol (E2) so that physiological concentrations of estradiol induce apoptosis in these cells. E2-induced apoptosis involve both intrinsic and extrinsic pathways but precise mechanisms remain unclear. We found that exposure of LTED MCF-7 cells to E2 activated AMP activated protein kinase (AMPK). In contrast, E2 inhibited AMPK activation in wild type MCF-7 cells where E2 prevents apoptosis. As a result of AMPK activation, the transcriptional activity of FoxO3, a downstream factor of AMPK, was up-regulated in E2 treatment of LTED. Increased activity of FoxO3 was demonstrated by up-regulation of three FoxO3 target genes, Bim, Fas ligand (FasL), and Gadd45α. Among them, Bim and FasL mediate intrinsic and extrinsic apoptosis respectively and Gadd45α causes cell cycle arrest at the G2/M phase. To further confirm the role of AMPK in apoptosis, we used AMPK activator AICAR in wild type MCF-7 cells and examined apoptosis, proliferation and expression of Bim, FasL, and Gadd45α. The effects of AICAR on these parameters recapitulated those observed in E2-treated LTED cells. Activation of AMPK by AICAR also increased expression of Bax in MCF-7 cells and its localization to mitochondria, which is a required process for apoptosis. These results reveal that AMPK is an important factor mediating E2-induced apoptosis in LTED cells, which is implicative of therapeutic potential for relapsing breast cancer after hormone therapy.

  18. Slug promotes survival during metastasis through suppression of Puma-mediated apoptosis.

    Science.gov (United States)

    Kim, Seaho; Yao, Jiahong; Suyama, Kimita; Qian, Xia; Qian, Bin-Zhi; Bandyopadhyay, Sanmay; Loudig, Olivier; De Leon-Rodriguez, Carlos; Zhou, Zhen Ni; Segall, Jeffrey; Macian, Fernando; Norton, Larry; Hazan, Rachel B

    2014-07-15

    Tumor cells must overcome apoptosis to survive throughout metastatic dissemination and distal organ colonization. Here, we show in the Polyoma Middle T mammary tumor model that N-cadherin (Cdh2) expression causes Slug (Snai2) upregulation, which in turn promotes carcinoma cell survival. Slug was dramatically upregulated in metastases relative to primary tumors. Consistent with a role in metastasis, Slug knockdown in carcinoma cells suppressed lung colonization by decreasing cell survival at metastatic sites, but had no effect on tumor cell invasion or extravasation. In support of this idea, Slug inhibition by shRNA sensitized tumor cells to apoptosis by DNA damage, resulting in caspase-3 and PARP cleavage. The prosurvival effect of Slug was found to be caused by direct repression of the proapoptotic gene, Puma (Bbc3), by Slug. Consistent with a pivotal role for a Slug-Puma axis in metastasis, inhibition of Puma by RNA interference in Slug-knockdown cells rescued lung colonization, whereas Puma overexpression in control tumor cells suppressed lung metastasis. The survival function of the Slug-Puma axis was confirmed in human breast cancer cells, where Slug knockdown increased Puma expression and inhibited lung colonization. This study demonstrates a pivotal role for Slug in carcinoma cell survival, implying that disruption of the Slug-Puma axis may impinge on the survival of metastatic cells. ©2014 American Association for Cancer Research.

  19. ER stress-mediated apoptosis in a new mouse model of osteogenesis imperfecta.

    Directory of Open Access Journals (Sweden)

    Thomas S Lisse

    2008-02-01

    Full Text Available Osteogenesis imperfecta is an inherited disorder characterized by increased bone fragility, fractures, and osteoporosis, and most cases are caused by mutations affecting the type I collagen genes. Here, we describe a new mouse model for Osteogenesis imperfecta termed Aga2 (abnormal gait 2 that was isolated from the Munich N-ethyl-N-nitrosourea mutagenesis program and exhibited phenotypic variability, including reduced bone mass, multiple fractures, and early lethality. The causal gene was mapped to Chromosome 11 by linkage analysis, and a C-terminal frameshift mutation was identified in the Col1a1 (procollagen type I, alpha 1 gene as the cause of the disorder. Aga2 heterozygous animals had markedly increased bone turnover and a disrupted native collagen network. Further studies showed that abnormal proalpha1(I chains accumulated intracellularly in Aga2/+ dermal fibroblasts and were poorly secreted extracellularly. This was associated with the induction of an endoplasmic reticulum stress-specific unfolded protein response involving upregulation of BiP, Hsp47, and Gadd153 with caspases-12 and -3 activation and apoptosis of osteoblasts both in vitro and in vivo. These studies resulted in the identification of a new model for Osteogenesis imperfecta, and identified a role for intracellular modulation of the endoplasmic reticulum stress-associated unfolded protein response machinery toward osteoblast apoptosis during the pathogenesis of disease.

  20. The signalling axis mediating neuronal apoptosis in response to [Pt(O,O'-acac)(γ-acac)(DMS)].

    Science.gov (United States)

    Muscella, Antonella; Calabriso, Nadia; Vetrugno, Carla; Fanizzi, Francesco Paolo; De Pascali, Sandra Angelica; Marsigliante, Santo

    2011-06-01

    It was previously shown that [Pt(O,O'-acac)(γ-acac)(DMS)] induces apoptosis in various cancer cells and exerts antimetastatic responses in vitro. In rats, [Pt(O,O'-acac)(γ-acac)(DMS)] reaches the central nervous system in quantities higher than cisplatin causing less excitotoxicity. The aim of the present paper was to investigate whether [Pt(O,O'-acac)(γ-acac)(DMS)] is able to exert cytotoxic effects on SH-SY5Y human neuroblastoma cell line, and to study the intracellular transduction mechanisms underlying these effects. Here we have demonstrated that [Pt(O,O'-acac)(γ-acac)(DMS)] was more effective than cisplatin in provoking apoptosis characterized by: (a) mitochondria depolarization, (b) decrease of Bcl-2 expression and increase of BAX expressions with cytosol-to-mitochondria translocation, (c) activation of caspase-7 and -9 and (d) generation of reactive oxygen species (ROS). [Pt(O,O'-acac)(γ-acac)(DMS)] provoked the activation of the following signalling kinases that were interacting with each other: PKC-δ and -ɛ, ERK1/2, p38MAPK, JNK1/2, NF-κB, c-src and FAK. We found that ROS generated by NADPH oxidase was responsible for the [Pt(O,O'-acac)(γ-acac)(DMS)]-mediated PKC-δ and -ɛ activation and consequential phosphorylation of all MAPKs. [Pt(O,O'-acac)(γ-acac)(DMS)]-induced mitochondrial apoptosis was blocked when p38MAPK and JNK1/2 were inhibited, whilst the effects on Bax/Bcl-2 mRNA and protein levels were blocked inhibiting NF-κB. NF-κB nuclear translocation was blocked inhibiting MEK1/2 activity. In addition to the induction of apoptosis [Pt(O,O'-acac)(γ-acac)(DMS)] downregulated pro-survival pathway. Survival inhibition started from mitochondrial ROS generation which induced c-src, FAK and Akt activation. In conclusion, our results suggest that [Pt(O,O'-acac)(γ-acac)(DMS)] may be considered a promising compound for the treatment of neuroblastoma. Further studies are warranted to explore in detail the therapeutic potential of this compound

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

    Science.gov (United States)

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

    2016-04-01

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

  2. Glucocorticoid-Mediated Repression of the Oncogenic microRNA Cluster miR-17∼92 Contributes to the Induction of Bim and Initiation of Apoptosis

    Science.gov (United States)

    Molitoris, Jason K.; McColl, Karen S.

    2011-01-01

    Synthetic glucocorticoids were one of the first effective treatments for lymphoid malignancies because of their ability to induce apoptosis and are still used in combination with other chemotherapeutic agents. Up-regulation of Bim, a proapoptotic member of the B-cell lymphoma-2 family, is an important mediator of glucocorticoid-induced apoptosis. Although glucocorticoids are known to elevate Bim mRNA and protein, little is known about the mechanism. Here, we report that glucocorticoids repress the expression of the microRNA cluster miR-17∼92, which results in elevated Bim protein expression as a mechanism by which glucocorticoids induce Bim. Using a luciferase-Bim 3′ untranslated region construct, we demonstrate that glucocorticoids mediate Bim induction posttranscriptionally after miR-17∼92 repression, resulting in increased Bim protein expression. Overexpression of miR-17∼92 microRNAs decreases Bim induction and attenuates glucocorticoid-mediated apoptosis. Conversely, knockdown of miR-17∼92 increases Bim protein expression and glucocorticoid-mediated apoptosis. These findings indicate that endogenous levels of miR-17∼92 repress Bim expression in T-cell lymphoid malignancies and that glucocorticoids induce Bim expression via down-regulation of the miR-17∼92 microRNA cluster. Our findings present a novel mechanism that contributes to the up-regulation of Bim and induction of apoptosis in lymphocytes after glucocorticoid treatment. Furthermore, our work demonstrating that inhibition of miR-17∼92 increases glucocorticoid-induced apoptosis highlights the potential importance of miR-17∼92 as a therapeutic target in leukemias and lymphomas. PMID:21239610

  3. The efficacy and mechanism of apoptosis induction by hypericin-mediated sonodynamic therapy in THP-1 macrophages

    Directory of Open Access Journals (Sweden)

    Li XS

    2015-01-01

    sonodynamic therapy (SDT group. Both ROS generation and MDA levels were significantly reduced by the ROS scavenger N-acetyl cysteine (NAC and the singlet oxygen scavenger sodium azide. Most of the loss of ΔΨm was inhibited by pretreatment with NAC, sodium azide, and the mPTP inhibitor cyclosporin A (CsA. mPTP opening was induced upon SDT but was reduced by pretreatment with bongkrekic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium, CsA, and NAC. Western blot analyses revealed translocation of BAX and cytochrome C, downregulated expression of Bcl-2, and upregulated expression of cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose polymerase in the SDT group, which were reversed by NAC.Conclusion: HY mediated SDT-induced apoptosis in THP-1 macrophages via ROS generation. Then, the proapoptotic factor BAX translocated from the cytosol to the mitochondria, increasing the ratio of BAX/Bcl-2, and the mPTP opened to release cytochrome C. This study demonstrated the great potential of HY-mediated SDT for treating atherosclerosis. Keywords: apoptosis, hypericin, sonodynamic therapy, mitochondria–caspase pathway, atherosclerosis

  4. Caspase-mediated cleavage of Beclin1 inhibits autophagy and promotes apoptosis induced by S1 in human ovarian cancer SKOV3 cells.

    Science.gov (United States)

    Li, Xiaoning; Su, Jing; Xia, Meihui; Li, Hongyan; Xu, Ye; Ma, Chunhui; Ma, Liwei; Kang, Jingsong; Yu, Huimei; Zhang, Zhichao; Sun, Liankun

    2016-02-01

    S1, a novel BH3 mimetic, can induce apoptosis dependent on Bax/Bak through inhibition of Bcl-2 in various tumors. S1 also induces autophagy through interrupting the interaction of Bcl-2 and Beclin1. Our results showed that S1 induces apoptosis in human ovarian cancer SKOV3 cells in a time- and dose-dependent manner. Autophagy precedes apoptosis, in SKOV3 cells treated with S1 (6 μmol/L), autophagy reached the maximum peak at 12 h after treatment and decreased to 24 h. In SKOV3 cells treated with different concentrations of S1 for 24 h, the highest level of autophagy was observed with 5 μmol/L and decreased to 10 μmol/L. Autophagy inhibitors 3-MA and CQ enhanced apoptosis induced by S1 in SKOV3 cells. However, overactivation of caspases in apoptosis induced by S1 may inhibit the autophagy-inducing function of Beclin1. Because the pan-caspase inhibitor Z-VAD recovered the autophagy-inducing function of Beclin1 through reduction of activated caspase-mediated cleavage of Beclin1. Furthermore, the Beclin1 cleavage products could further increase apoptosis induced by S1 in SKOV3 cells. This indicates that apoptosis induced by high doses and long exposure of S1 causes the overactivation of caspases and subsequent cleavage of Beclin1, and inhibits the protection of autophagy. Moreover, the cleaved product of Beclin1 further promotes apoptosis induced by S1 in SKOV3 cells. Our results suggest this may be a molecular mechanism for enhancing the sensitivity of cancer cells to apoptosis induced by small molecular compound targeting Bcl-2.

  5. RGS6 Suppresses Ras-induced Cellular Transformation by Facilitating Tip60-mediated Dnmt1 Degradation and Promoting Apoptosis

    Science.gov (United States)

    Huang, Jie; Stewart, Adele; Maity, Biswanath; Hagen, Jussara; Fagan, Rebecca L.; Yang, Jianqi; Quelle, Dawn E.; Brenner, Charles; Fisher, Rory A.

    2014-01-01

    The RAS protooncogene plays a central role in regulation of cell proliferation, and point mutations leading to oncogenic activation of Ras occur in a large number of human cancers. Silencing of tumor suppressor genes by DNA methyltransferase 1 (Dnmt1) is essential for oncogenic cellular transformation by Ras, and Dnmt1 is over-expressed in numerous human cancers. Here we provide new evidence that the pleiotropic Regulator of G protein Signaling (RGS) family member RGS6 suppresses Ras-induced cellular transformation by facilitating Tip60-mediated degradation of Dmnt1 and promoting apoptosis. Employing mouse embryonic fibroblasts (MEFs) from wild type (WT) and RGS6−/− mice, we found that oncogenic Ras induced up-regulation of RGS6, which in turn blocked Ras-induced cellular transformation. RGS6 functions to suppress cellular transformation in response to oncogenic Ras by down regulating Dnmt1 protein expression leading to inhibition of Dnmt1-mediated anti-apoptotic activity. Further experiments showed that RGS6 functions as a scaffolding protein for both Dnmt1 and Tip60 and is required for Tip60-mediated acetylation of Dnmt1 and subsequent Dnmt1 ubiquitylation and degradation. The RGS domain of RGS6, known only for its GAP activity toward Gα subunits, was sufficient to mediate Tip60 association with RGS6. This work demonstrates a novel signaling action for RGS6 in negative regulation of oncogene-induced transformation and provides new insights into our understanding of the mechanisms underlying Ras-induced oncogenic transformation and regulation of Dnmt1 expression. Importantly, these findings identify RGS6 as an essential cellular defender against oncogenic stress and a potential therapeutic target for developing new cancer treatments. PMID:23995786

  6. The effects of benzene exposure on apoptosis in epithelial lung cells: localization by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and the immunocytochemical localization of apoptosis-related gene products.

    Science.gov (United States)

    Weaver, C V; Liu, S-P; Lu, J-F; Lin, B-S

    2007-05-01

    Although benzene, a well-known human carcinogen, has been shown to induce apoptosis in vitro, no studies have been carried out to confirm and characterize its role in activating apoptosis in vivo. The present study investigated the effects of benzene inhalation on the epithelial cells lining the respiratory tract including bronchioles, terminal bronchioles, respiratory bronchioles and alveoli of male Sprague-Dawley rats. Inhalation of benzene 300 ppm for 7 days induced apoptotic changes in the parenchymal components in the lung that significantly exceeded the events of programmed cell death in normal control tissues. Apoptosis was confirmed by the electrophoretic analysis of internucleosomal DNA fragmentation of benzene-exposed lung tissues, which exhibited 180-200 bp laddering subunits indicative of genomic DNA degradation. Furthermore, semi-quantitative analysis of intracellular localization of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling TUNEL) showed a significant (p respiratory bronchiolar 60.8% segmental epithelial components as well as alveolar (55%) epithelia. Analysis of immunohistochemical expression of apoptosis-related gene products also supported the hypothesis that benzene can induce apoptosis in chemosensitive target cells in the lung parenchyma. Quantitative immunhistochemistry showed a statistically significant increase p respiratory system that demonstrates that benzene inhalation induces lung cell apoptosis as confirmed by DNA electrophoresis, in situ nick end labeling, and the upregulation of apoptosis-related gene products that facilitate caspase-cleaved enzymes which lead to cell degradation via programmed cell death. These responses may represent an important defense mechanism within the parenchymal cells of the respiratory system that reduce mutational hazard and the potential carcinogenic effects of benzene-initiated pathogenesis.

  7. Solanine Induces Mitochondria-Mediated Apoptosis in Human Pancreatic Cancer Cells

    Directory of Open Access Journals (Sweden)

    Hongwei Sun

    2014-01-01

    Full Text Available Steroid alkaloids have been suggested as potential anticancer compounds. However, the underlying mechanisms of how steroid alkaloids inhibit the tumor growth are largely unknown. Here, we reported that solanine, a substance of steroid alkaloids, has a positive effect on the inhibition of pancreatic cancer cell growth in vitro and in vivo. In pancreatic cancer cells and nu/nu nude mice model, we found that solanine inhibited cancer cells growth through caspase-3 dependent mitochondrial apoptosis. Mechanically, solanine promotes the opening of mitochondrial membrane permeability transition pore (MPTP by downregulating the Bcl-2/Bax ratio; thereafter, Cytochrome c and Smac are released from mitochondria into cytosol to process the caspase-3 zymogen into an activated form. Moreover, we found that the expression of tumor metastasis related proteins, MMP-2 and MMP-9, was also decreased in the cells treated with solanine. Therefore, our results suggested that solanine was an effective compound for the treatment of pancreatic cancer.

  8. COX-2 mediates PM2.5-induced apoptosis and inflammation in vascular endothelial cells.

    Science.gov (United States)

    Yin, Jie; Xia, Weiwei; Li, Yuanyuan; Guo, Chuchu; Zhang, Yue; Huang, Songming; Jia, Zhanjun; Zhang, Aihua

    2017-01-01

    Emerging evidence demonstrated that particulate matter 2.5 (PM2.5) exposure served as an important risk factor of cardiovascular diseases. Some studies also reported that COX-2/mPGES-1/PGE2 cascade played a pathogenic role in vascular injury. However, the relationship between the PM2.5 exposure and the activation of COX-2/mPGES-1/PGE2 cascade in endothelial cells is still unknown. In the present study, mouse aorta endothelial cells were exposed to PM2.5. Strikingly, following the PM2.5 treatment, we observed dose- and time-dependent upregulation of COX-2 at both protein and mRNA levels as determined by Western blotting and qRT-PCR, respectively. However, COX-1 mRNA expression was not affected by PM2.5 treatment. Next, we examined mPGES-1 expression. As expected, mPGES-1 protein was markedly increased by PM2.5 exposure in line with a significant increment of PGE2 release in medium. At the same time, we observed a dose-dependent upregulation of another two PGE2 synthases of mPGES-2 and cPGES determined by qRT-PCR. Inhibition of COX-2 by using a specific COX-2 inhibitor NS-398 markedly blocked cell apoptosis, inflammation, and PGE2 secretion. Taken together, these results suggested that PM2.5 could activate inflammatory axis of COX-2/PGES/PGE2 in vascular endothelial cells to promote cell apoptosis and inflammatory response.

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

    OpenAIRE

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

    2017-01-01

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

  10. Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway

    Science.gov (United States)

    Mandal, Abhishek; Das, Sushmita; Roy, Saptarshi; Ghosh, Ayan Kumar; Sardar, Abul Hasan; Verma, Sudha; Saini, Savita; Singh, Ruby; Abhishek, Kumar; Kumar, Ajay; Mandal, Chitra; Das, Pradeep

    2016-01-01

    The growth and survival of intracellular parasites depends on the availability of extracellular nutrients. Deprivation of nutrients viz glucose or amino acid alters redox balance in mammalian cells as well as some lower organisms. To further understand the relationship, the mechanistic role of L-arginine in regulation of redox mediated survival of Leishmania donovani promastigotes was investigated. L-arginine deprivation from the culture medium was found to inhibit cell growth, reduce proliferation and increase L-arginine uptake. Relative expression of enzymes, involved in L-arginine metabolism, which leads to polyamine and trypanothione biosynthesis, were downregulated causing decreased production of polyamines in L-arginine deprived parasites and cell death. The resultant increase in reactive oxygen species (ROS), due to L-arginine deprivation, correlated with increased NADP+/NADPH ratio, decreased superoxide dismutase (SOD) level, increased lipid peroxidation and reduced thiol content. A deficiency of L-arginine triggered phosphatidyl serine externalization, a change in mitochondrial membrane potential, release of intracellular calcium and cytochrome-c. This finally led to DNA damage in Leishmania promastigotes. In summary, the growth and survival of Leishmania depends on the availability of extracellular L-arginine. In its absence the parasite undergoes ROS mediated, caspase-independent apoptosis-like cell death. Therefore, L-arginine metabolism pathway could be a probable target for controlling the growth of Leishmania parasites and disease pathogenesis. PMID:26808657

  11. Protective role of complement C3 against cytokine-mediated beta cell apoptosis

    DEFF Research Database (Denmark)

    Dos Santos, R. S.; Marroqui, L.; Grieco, F. A.

    2017-01-01

    Background and aims: Type 1 diabetes is a chronic autoimmune disease characterized by pancreatic islet inflammation and β-cell destruction by pro-inflammatory cytokines and other mediators. The complement system, a major component of the immune system, has been recently shown to also act in metab...

  12. Proapoptotic protein Smac mediates apoptosis in cisplatin-resistant ovarian cancer cells when treated with the anti-tumor agent AT101.

    Science.gov (United States)

    Hu, Wenbin; Wang, Fang; Tang, Jingsheng; Liu, Xinyu; Yuan, Zhu; Nie, Chunlai; Wei, Yuquan

    2012-01-02

    Chemoresistance of ovarian cancer has been previously attributed to the expression and activation of Bcl-2 family proteins. BH3-mimetic molecules possessing potential anticancer activity are able to inhibit antiapoptotic Bcl-2 family proteins. AT101 (R-(-)-gossypol), a natural BH3-mimetic molecule, has shown anti-tumor activity as a single agent and in combination with standard anticancer therapies in a variety of tumor models. Here, we report the effect of AT101 on apoptosis in cisplatin-resistant ovarian cancer cells and identify the major molecular events that determine sensitivity. AT101 induced cell apoptosis by activating Bax through a conformational change, translocation, and oligomerization. The inhibition of Bax expression only partially prevented caspase-3 cleavage. However, the gene silencing of Bax had no effect on mitochondrial Smac release. Further experiments demonstrated that Smac reduction inhibited caspase-3 activation and attenuated cell apoptosis. More importantly, the inhibition of Smac or overexpression of XIAP attenuated Bax activation in ovarian cells. Furthermore, our data indicate that the Akt-p53 pathway is involved in the regulation of Smac release. Taken together, our data demonstrate the role of Smac and the molecular mechanisms of AT101-induced apoptosis of chemoresistant ovarian cancer cells. Our findings suggest that AT101 not only triggers Bax activation but also induces mitochondrial Smac release. Activated Smac can enhance Bax-mediated cellular apoptosis. Therefore, Smac mediates Bax activation to determine the threshold for overcoming cisplatin resistance in ovarian cancer cells.

  13. Proapoptotic Protein Smac Mediates Apoptosis in Cisplatin-resistant Ovarian Cancer Cells When Treated with the Anti-tumor Agent AT101*

    Science.gov (United States)

    Hu, Wenbin; Wang, Fang; Tang, Jingsheng; Liu, Xinyu; Yuan, Zhu; Nie, Chunlai; Wei, Yuquan

    2012-01-01

    Chemoresistance of ovarian cancer has been previously attributed to the expression and activation of Bcl-2 family proteins. BH3-mimetic molecules possessing potential anticancer activity are able to inhibit antiapoptotic Bcl-2 family proteins. AT101 (R-(−)-gossypol), a natural BH3-mimetic molecule, has shown anti-tumor activity as a single agent and in combination with standard anticancer therapies in a variety of tumor models. Here, we report the effect of AT101 on apoptosis in cisplatin-resistant ovarian cancer cells and identify the major molecular events that determine sensitivity. AT101 induced cell apoptosis by activating Bax through a conformational change, translocation, and oligomerization. The inhibition of Bax expression only partially prevented caspase-3 cleavage. However, the gene silencing of Bax had no effect on mitochondrial Smac release. Further experiments demonstrated that Smac reduction inhibited caspase-3 activation and attenuated cell apoptosis. More importantly, the inhibition of Smac or overexpression of XIAP attenuated Bax activation in ovarian cells. Furthermore, our data indicate that the Akt-p53 pathway is involved in the regulation of Smac release. Taken together, our data demonstrate the role of Smac and the molecular mechanisms of AT101-induced apoptosis of chemoresistant ovarian cancer cells. Our findings suggest that AT101 not only triggers Bax activation but also induces mitochondrial Smac release. Activated Smac can enhance Bax-mediated cellular apoptosis. Therefore, Smac mediates Bax activation to determine the threshold for overcoming cisplatin resistance in ovarian cancer cells. PMID:22052903

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

    Directory of Open Access Journals (Sweden)

    Deepak Kumar

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

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

    Science.gov (United States)

    Kumar, Deepak; Das, Bimolendu; Sen, Rupashree; Kundu, Priyanka; Manna, Alak; Sarkar, Avijit; Chowdhury, Chinmay; Chatterjee, Mitali; Das, Padma

    2015-01-01

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

  16. Small molecule XIAP inhibitors cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells and overcome Bcl-2-mediated resistance.

    Science.gov (United States)

    Fakler, Melanie; Loeder, Sandra; Vogler, Meike; Schneider, Katja; Jeremias, Irmela; Debatin, Klaus-Michael; Fulda, Simone

    2009-02-19

    Defects in apoptosis contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL), calling for novel strategies that counter apoptosis resistance. Here, we demonstrate for the first time that small molecule inhibitors of the antiapoptotic protein XIAP cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells. XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, synergize with TRAIL to trigger apoptosis and to inhibit clonogenic survival of acute leukemia cells, whereas they do not affect viability of normal peripheral blood lymphocytes, suggesting some tumor selectivity. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases, loss of mitochondrial membrane potential, and cytochrome c release in a caspase-dependent manner, indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Of note, XIAP inhibitors even overcome Bcl-2-mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Importantly, XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In vivo, they significantly reduce leukemic burden in a mouse model of pediatric ALL engrafted in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus, XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood ALL.

  17. [TRPM8 mediates PC-12 neuronal cell apoptosis induced by oxygen-glucose deprivation through cAMP-PKA/UCP4 signaling].

    Science.gov (United States)

    Li, Hong-Wei; Zhou, Bin; Zhang, Hai-Hong

    2016-08-20

    To explore the molecular mechanism responsible for apoptosis of PC-12 neuronal cells induced by oxygen-glucose deprivation (OGD). PC12 cells were exposed to OGD for 24 h to simulate ischemia-reperfusion injury. Flow cytometry was employed detect the cell apoptosis, and the expresions of TRPM8, UCP4, cAMP and PKA in the exposed cells were detected with RT-PCR and Western blotting. The changes in the expressions of Bax, Bcl-2, cAMP, PKA and UCP4 proteins were detected in the exposed cells in resposne to inhibition of TRPM8 and cAMP-PKA signal or over-expression of UCP4. OGD for 24 induced obvious apoptosis in PC-12 cells and caused TRPM8 over-expression and inhibition of UCP4 and cAMP-PKA signaling. Inhibiting TRPM8 expression reduced the cell apoptosis and up-regulated cAMP, p-PKA and UCP4 in the cells exposed to OGD. In cells exposed to OGD, inhibition of TRPM8 and cAMP-PKA signaling suppressed the expressio of UCP4 and increased the cell apoptosis. TRPM8 mediates OGD-induced PC12 cell apoptosis through cAMP-PKA/UCP4 signaling.

  18. A cardinal role for cathepsin d in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci.

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    Martin A Bewley

    2011-01-01

    Full Text Available The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D(-/- hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function.

  19. A role for the non-receptor tyrosine kinase ACK1 in TNF-alpha-mediated apoptosis and proliferation in human intestinal epithelial caco-2 cells.

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    Zhao, Xinmei; Lv, Chaolan; Chen, Shengbo; Zhi, Fachao

    2017-09-16

    The roles of tumor necrosis factor alpha (TNF-alpha) and its mediators in cellular processes related to intestinal diseases remain elusive. In this study, we aimed to determine the biological role of activated Cdc42-associated kinase 1 (ACK1) in TNF-alpha-mediated apoptosis and proliferation in Caco-2 cells. ACK1 expression was knocked down using ACK1-specific siRNAs, and ACK1 activity was disrupted using a small molecule ACK1 inhibitor. The Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) and the BrdU incorporation assays were used to measure apoptosis and cell proliferation, respectively. ACK1-specific siRNA and the pharmacological ACK1 inhibitor significantly abrogated the TNF-alpha-mediated anti-apoptotic effects and proliferation of Caco-2 cells. Interestingly, TNF-alpha activated ACK1 at tyrosine 284 (Tyr284), and the ErbB family of proteins was implicated in ACK1 activation in Caco-2 cells. ACK1-Tyr284 was required for protein kinase B (AKT) activation, and ACK1 signaling was mediated through recruiting and phosphorylating the down-stream adaptor protein AKT, which likely promoted cell proliferation in response to TNF-alpha. Moreover, ACK1 activated AKT and Src enhanced nuclear factor-кB (NF-кB) activity, suggesting a correlation between NF-кB signaling and TNF-alpha-mediated apoptosis in Caco-2 cells. Our results demonstrate that ACK1 plays an important role in modulating TNF-alpha-induced aberrant cell proliferation and apoptosis, mediated in part by ACK1 activation. ACK1 and its down-stream effectors may hold promise as therapeutic targets in the prevention and treatment of gastrointestinal cancers, in particular, those induced by chronic intestinal inflammation. © 2017 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

  20. Human T-Cell Leukemia Virus Type 1 Tax-Deregulated Autophagy Pathway and c-FLIP Expression Contribute to Resistance against Death Receptor-Mediated Apoptosis

    Science.gov (United States)

    Wang, Weimin; Zhou, Jiansuo; Shi, Juan; Zhang, Yaxi; Liu, Shilian

    2014-01-01

    ABSTRACT The human T-cell leukemia virus type 1 (HTLV-1) Tax protein is considered to play a central role in the process that leads to adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 Tax-expressing cells show resistance to apoptosis induced by Fas ligand (FasL) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). The regulation of Tax on the autophagy pathway in HeLa cells and peripheral T cells was recently reported, but the function and underlying molecular mechanism of the Tax-regulated autophagy are not yet well defined. Here, we report that HTLV-1 Tax deregulates the autophagy pathway, which plays a protective role during the death receptor (DR)-mediated apoptosis of human U251 astroglioma cells. The cellular FLICE-inhibitory protein (c-FLIP), which is upregulated by Tax, also contributes to the resistance against DR-mediated apoptosis. Both Tax-induced autophagy and Tax-induced c-FLIP expression require Tax-induced activation of IκB kinases (IKK). Furthermore, Tax-induced c-FLIP expression is regulated through the Tax-IKK-NF-κB signaling pathway, whereas Tax-triggered autophagy depends on the activation of IKK but not the activation of NF-κB. In addition, DR-mediated apoptosis is correlated with the degradation of Tax, which can be facilitated by the inhibitors of autophagy. IMPORTANCE Our study reveals that Tax-deregulated autophagy is a protective mechanism for DR-mediated apoptosis. The molecular mechanism of Tax-induced autophagy is also illuminated, which is different from Tax-increased c-FLIP. Tax can be degraded via manipulation of autophagy and TRAIL-induced apoptosis. These results outline a complex regulatory network between and among apoptosis, autophagy, and Tax and also present evidence that autophagy represents a new possible target for therapeutic intervention for the HTVL-1 related diseases. PMID:24352466

  1. Association of reactive oxygen species-mediated signal transduction with in vitro apoptosis sensitivity in chronic lymphocytic leukemia B cells.

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    Adam L Palazzo

    Full Text Available BACKGROUND: Chronic lymphocytic leukemia (CLL is a B cell malignancy with a variable clinical course and unpredictable response to therapeutic agents. Single cell network profiling (SCNP utilizing flow cytometry measures alterations in signaling biology in the context of molecular changes occurring in malignancies. In this study SCNP was used to identify proteomic profiles associated with in vitro apoptotic responsiveness of CLL B cells to fludarabine, as a basis for ultimately linking these with clinical outcome. METHODOLOGY/PRINCIPAL FINDING: SCNP was used to quantify modulated-signaling of B cell receptor (BCR network proteins and in vitro F-ara-A mediated apoptosis in 23 CLL samples. Of the modulators studied the reactive oxygen species, hydrogen peroxide (H₂O₂, a known intracellular second messenger and a general tyrosine phosphatase inhibitor stratified CLL samples into two sub-groups based on the percentage of B cells in a CLL sample with increased phosphorylation of BCR network proteins. Separately, in the same patient samples, in vitro exposure to F-ara-A also identified two sub-groups with B cells showing competence or refractoriness to apoptotic induction. Statistical analysis showed that in vitro F-ara-A apoptotic proficiency was highly associated with the proficiency of CLL B cells to undergo H₂O₂-augmented signaling. CONCLUSIONS/SIGNIFICANCE: This linkage in CLL B cells among the mechanisms governing chemotherapy-induced apoptosis increased signaling of BCR network proteins and a likely role of phosphatase activity suggests a means of stratifying patients for their response to F-ara-A based regimens. Future studies will examine the clinical applicability of these findings and also the utility of this approach in relating mechanism to function of therapeutic agents.

  2. Embelin-Induced Apoptosis of Human Prostate Cancer Cells Is Mediated through Modulation of Akt and β-Catenin Signaling.

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    Nahee Park

    Full Text Available There is increasing evidence that embelin, an active component of Embelia ribes, induces apoptosis in human cancer cells, but the detailed mechanisms are still unclear. Here, we have investigated the effect of embelin on the growth of human prostate cancer cells. Embelin strongly inhibited cell growth especially in human prostate cancer cell lines, including PC3, DU145, LNCaP-LN3 and normal prostate epithelial cell, RWPE-1 compared to breast cancer (MDA-MB-231, MCF-7, and T47D, hepatoma (HepG2, Hep3B, and HuH-7, or choriocarcinoma (JEG-3. We observed that embelin induced apoptosis of PC3 cells in a time-dependent manner correlated with decreased expression of Bcl-2, Bcl-xL, and Mcl-1, increased translocation of Bax into mitochondria, and a reduction in the mitochondrial membrane potential. Furthermore, embelin induced voltage-dependent anion channel (VDAC 1 expression and oligomerization, which may promote cytochrome c and AIF release. Because embelin was able to inhibit Akt activation and cyclooxygenase-2 expression, the effects on Wnt/ β-catenin signaling were determined. Embelin activated glycogen synthase kinase (GSK-3β by preventing phosphorylation and suppressed β-catenin expression. Attenuation of β-catenin-mediated TCF transcriptional activity and gene transcription, such as cyclin D1, c-myc, and matrix metalloproteinase (MMP-7, were shown in embelin-treated cells. The changes in β-catenin levels in response to embelin were blocked by lithium chloride, a GSK-3 inhibitor, indicating that embelin may decrease β-catenin expression via GSK-3β activation. Furthermore, exposure of PC3 cells to embelin resulted in a significant decrease in cell migration and invasion. In conclusion, these findings suggest that inhibition of Akt signaling and activation of GSK-3β partially contributes to the pro-apoptotic effect of embelin in prostate cancer cells.

  3. 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.

  4. Neferine Potentiates the Antitumor Effect of Cisplatin in Human Lung Adenocarcinoma Cells Via a Mitochondria-Mediated Apoptosis Pathway.

    Science.gov (United States)

    Sivalingam, Kalai Selvi; Paramasivan, Poornima; Weng, Ching Feng; Viswanadha, Vijaya Padma

    2017-09-01

    Cisplatin is one of the most potent chemotherapeutic agents for the treatment of many types of solid tumors but its efficacy is often limited by the development of resistance and dose limiting toxicity. Neferine is an alkaloid isolated from seed embryo of Nelumbo nucifera, it has recently been shown to have anticancer effects in various human cancer cell lines. The present investigation is designed to study the chemosensitizing ability of neferine with cisplatin in A549 cells. Neferine potentiates the cisplatin induced apoptosis through the exploration of characteristic apoptotic morphological changes, induced sub-G1 cell cycle arrest, ROS hypergeneration, significant loss of cellular antioxidant enzymes, as well as loss of mitochondrial membrane potential (ΔΨM). Furthermore our results revealed that neferine combined with cisplatin down regulate the expression of Bcl-2 and up regulate the expression of Bax, Bad, Bak, release of cytochrome c, p53 levels, then activated cleavage forms of caspase-9, caspase-3, and PARP. Moreover neferine and cisplatin combination significantly down regulated the protein levels of FAK and VEGF. In addition, we observed the activity of MMP-2 and MMP-9. Thus this study provides molecular evidence for the ROS mediated apoptosis of the combinatorial regimen of cisplatin and neferine in lung cancer cells. Thus these results suggest that using neferine with cisplatin combinatorial regimen could be potentiating the effect of cisplatin and neferine reduces the cisplatin dose requirement in cancer chemotherapy. J. Cell. Biochem. 118: 2865-2876, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  5. Reversal of drug-induced gingival overgrowth by UV-mediated apoptosis of gingival fibroblasts - an in vitro study.

    Science.gov (United States)

    Ritchhart, Casey; Joy, Anita

    2018-02-07

    Gingival overgrowth (GO) is an undesirable result of certain drugs like Cyclosporine A (CsA). Histopathology of GO shows hyperplasia of gingival epithelium, expansion of connective tissue with increased collagen, or a combination. Factors such as age, gender, oral hygiene, duration, and dosage also influence onset and severity of GO. One of the mechanisms behind uncontrolled cell proliferation in drug-induced GO is inhibition of apoptotic pathways, with a consequent effect on normal cell turnover. Our objective was to determine if UV photo-treatment would activate apoptosis in the gingival fibroblast component. Human gingival fibroblast cells (HGF-1) were exposed to 200ng/ml or 400ng/ml CsA and maintained for 3, 6, and 9 days, followed by UV radiation for 2, 5, or 10min (N=6). Naïve (no CsA or UV), negative (UV, no CsA), and positive controls (CsA, no UV) were designated. Prior to UV treatment, growth media was replaced with 1M PBS to prevent absorption of UV radiation by serum proteins, and cells were incubated in growth media for 24h post-UV before processing for TUNEL assay, cell proliferation assays, or immunofluorescence. Data showed a temporal increase in proliferation of HGF-1 cells under the influence of CsA. The 200ng/ml dose was more effective in causing over-proliferation. UV treatment for 10min resulted in significant reduction in cell numbers, as evidenced by counts and proliferation assays. Our study is a first step to further evaluate UV-mediated apoptosis as a mechanism to control certain forms of GO. Copyright © 2018 Elsevier GmbH. All rights reserved.

  6. Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation.

    Science.gov (United States)

    Galleu, Antonio; Riffo-Vasquez, Yanira; Trento, Cristina; Lomas, Cara; Dolcetti, Luigi; Cheung, Tik Shing; von Bonin, Malte; Barbieri, Laura; Halai, Krishma; Ward, Sophie; Weng, Ling; Chakraverty, Ronjon; Lombardi, Giovanna; Watt, Fiona M; Orchard, Kim; Marks, David I; Apperley, Jane; Bornhauser, Martin; Walczak, Henning; Bennett, Clare; Dazzi, Francesco

    2017-11-15

    The immunosuppressive activity of mesenchymal stromal cells (MSCs) is well documented. However, the therapeutic benefit is completely unpredictable, thus raising concerns about MSC efficacy. One of the affecting factors is the unresolved conundrum that, despite being immunosuppressive, MSCs are undetectable after administration. Therefore, understanding the fate of infused MSCs could help predict clinical responses. Using a murine model of graft-versus-host disease (GvHD), we demonstrate that MSCs are actively induced to undergo perforin-dependent apoptosis by recipient cytotoxic cells and that this process is essential to initiate MSC-induced immunosuppression. When examining patients with GvHD who received MSCs, we found a striking parallel, whereby only those with high cytotoxic activity against MSCs responded to MSC infusion, whereas those with low activity did not. The need for recipient cytotoxic cell activity could be replaced by the infusion of apoptotic MSCs generated ex vivo. After infusion, recipient phagocytes engulf apoptotic MSCs and produce indoleamine 2,3-dioxygenase, which is ultimately necessary for effecting immunosuppression. Therefore, we propose the innovative concept that patients should be stratified for MSC treatment according to their ability to kill MSCs or that all patients could be treated with ex vivo apoptotic MSCs. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  7. Active hexose-correlated compound enhances extrinsic-pathway-mediated apoptosis of Acute Myeloid Leukemic cells.

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    Kavin Fatehchand

    Full Text Available Active Hexose Correlated Compound (AHCC has been shown to have many immunostimulatory and anti-cancer activities in mice and in humans. As a natural product, AHCC has potential to create safer adjuvant therapies in cancer patients. Acute Myeloid Leukemia (AML is the least curable and second-most common leukemia in adults. AML is especially terminal to those over 60 years old, where median survival is only 5 to 10 months, due to inability to receive intensive chemotherapy. Hence, the purpose of this study was to investigate the effects of AHCC on AML cells both in vitro and in vivo. Results showed that AHCC induced Caspase-3-dependent apoptosis in AML cell lines as well as in primary AML leukopheresis samples. Additionally, AHCC induced Caspase-8 cleavage as well as Fas and TRAIL upregulation, suggesting involvement of the extrinsic apoptotic pathway. In contrast, monocytes from healthy donors showed suppressed Caspase-3 cleavage and lower cell death. When tested in a murine engraftment model of AML, AHCC led to significantly increased survival time and decreased blast counts. These results uncover a mechanism by which AHCC leads to AML-cell specific death, and also lend support for the further investigation of AHCC as a potential adjuvant for the treatment of AML.

  8. Montelukast Induces Apoptosis-Inducing Factor-Mediated Cell Death of Lung Cancer Cells

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    Ming-Ju Tsai

    2017-06-01

    Full Text Available Developing novel chemo-prevention techniques and advancing treatment are key elements to beating lung cancer, the most common cause of cancer mortality worldwide. Our previous cohort study showed that cysteinyl leukotriene receptor antagonists, mainly montelukast, decreased the lung cancer risk in asthma patients. In the current study, we conducted in vivo and in vitro experiments to demonstrate the inhibiting effect of montelukast on lung cancer and to investigate the underlying mechanisms. Using Lewis lung carcinoma-bearing mice, we showed that feeding montelukast significantly delayed the tumor growth in mice (p < 0.0001. Montelukast inhibited cell proliferation and colony formation and induced the cell death of lung cancer cells. Further investigation showed the down-regulation of B-cell lymphoma 2 (Bcl-2, up-regulation of Bcl-2 homologous antagonist/killer (Bak, and nuclear translocation of apoptosis-inducing factor (AIF in montelukast-treated lung cancer cells. Montelukast also markedly decreased the phosphorylation of several proteins, such as with no lysine 1 (WNK1, protein kinase B (Akt, extracellular signal-regulated kinase 1/2 (Erk1/2, MAPK/Erk kinase (MEK, and proline-rich Akt substrate of 40-kDa (PRAS40, which might contribute to cell death. In conclusion, montelukast induced lung cancer cell death via the nuclear translocation of AIF. This study confirmed the chemo-preventive effect of montelukast shown in our previous cohort study. The utility of montelukast in cancer prevention and treatment thus deserves further studies.

  9. PTEN counteracts FBXL2 to promote IP3R3- and Ca2+-mediated apoptosis limiting tumour growth.

    Science.gov (United States)

    Kuchay, Shafi; Giorgi, Carlotta; Simoneschi, Daniele; Pagan, Julia; Missiroli, Sonia; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Collazo-Lorduy, Ana; Castillo-Martin, Mireia; Cordon-Cardo, Carlos; Sebti, Said M; Pinton, Paolo; Pagano, Michele

    2017-06-22

    In response to environmental cues that promote IP3 (inositol 1,4,5-trisphosphate) generation, IP3 receptors (IP3Rs) located on the endoplasmic reticulum allow the 'quasisynaptical' feeding of calcium to the mitochondria to promote oxidative phosphorylation. However, persistent Ca 2+ release results in mitochondrial Ca 2+ overload and consequent apoptosis. Among the three mammalian IP3Rs, IP3R3 appears to be the major player in Ca 2+ -dependent apoptosis. Here we show that the F-box protein FBXL2 (the receptor subunit of one of 69 human SCF (SKP1, CUL1, F-box protein) ubiquitin ligase complexes) binds IP3R3 and targets it for ubiquitin-, p97- and proteasome-mediated degradation to limit Ca 2+ influx into mitochondria. FBXL2-knockdown cells and FBXL2-insensitive IP3R3 mutant knock-in clones display increased cytosolic Ca 2+ release from the endoplasmic reticulum and sensitization to Ca 2+ -dependent apoptotic stimuli. The phosphatase and tensin homologue (PTEN) gene is frequently mutated or lost in human tumours and syndromes that predispose individuals to cancer. We found that PTEN competes with FBXL2 for IP3R3 binding, and the FBXL2-dependent degradation of IP3R3 is accelerated in Pten -/- mouse embryonic fibroblasts and PTEN-null cancer cells. Reconstitution of PTEN-null cells with either wild-type PTEN or a catalytically dead mutant stabilizes IP3R3 and induces persistent Ca 2+ mobilization and apoptosis. IP3R3 and PTEN protein levels directly correlate in human prostate cancer. Both in cell culture and xenograft models, a non-degradable IP3R3 mutant sensitizes tumour cells with low or no PTEN expression to photodynamic therapy, which is based on the ability of photosensitizer drugs to cause Ca 2+ -dependent cytotoxicity after irradiation with visible light. Similarly, disruption of FBXL2 localization with GGTi-2418, a geranylgeranyl transferase inhibitor, sensitizes xenotransplanted tumours to photodynamic therapy. In summary, we identify a novel molecular

  10. 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.

  11. DC-CIK cells derived from ovarian cancer patient menstrual blood activate the TNFR1-ASK1-AIP1 pathway to kill autologous ovarian cancer stem cells.

    Science.gov (United States)

    Qin, Wenxing; Xiong, Ying; Chen, Juan; Huang, Yongyi; Liu, Te

    2018-03-22

    Ovarian cancer stem cells (OCSCs) are highly carcinogenic and have very strong resistance to traditional chemotherapeutic drugs; therefore, they are an important factor in ovarian cancer metastasis and recurrence. It has been reported that dendritic cell (DC)-cytokine-induced killer (CIK) cells have significant killing effects on all cancer cells across many systems including the blood, digestive, respiratory, urinary and reproductive systems. However, whether DC-CIK cells can selectively kill OCSCs is currently unclear. In this study, we collected ovarian cancer patient menstrual blood (OCPMB) samples to acquire mononuclear cells and isolated DC-CIK cells in vitro. In addition, autologous CD44+/CD133+ OCSCs were isolated and used as target cells. The experimental results showed that when DC-CIK cells and OCSCs were mixed and cultured in vitro at ratios of 5:1, 10:1 and 50:1, the DC-CIK cells killed significant amounts of OCSCs, inhibited their invasion in vitro and promoted their apoptosis. The qPCR and Western blot results showed that DC-CIK cells stimulated high expression levels and phosphorylation of TNFR1, ASK1, AIP1 and JNK in OCSCs through the release of TNF-α. After the endogenous TNFR1 gene was knocked out in OCSCs using the CRISPR/Cas9 technology, the killing function of DC-CIK cells on target OCSCs was significantly attenuated. The results of the analyses of clinical samples suggested that the TNFR1 expression level was negatively correlated with ovarian cancer stage and prognosis. Therefore, we innovatively confirmed that DC-CIK cells derived from OCPMB could secret TNF-α to activate the expression of the TNFR1-ASK1-AIP1-JNK pathway in OCSCs and kill autologous OCSCs. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  12. NuMA and nuclear lamins behave differently in Fas-mediated apoptosis.

    Science.gov (United States)

    Taimen, Pekka; Kallajoki, Markku

    2003-02-01

    NuMA is a nuclear matrix protein that has an essential function in the organization of the mitotic spindle. Here we have studied the fate of NuMA in Fas-treated apoptotic Jurkat T and HeLa cells. We show that in both cell lines NuMA is an early target protein for caspases and that NuMA is cleaved coincidently with poly(ADP-ribose) polymerase-1 (PARP-1) and nuclear lamin B. NuMA is cleaved differently in Jurkat T and HeLa cells, suggesting that different sets of caspases are activated in these cell lines. The normal diffuse intranuclear distribution of NuMA changed during apoptosis: first NuMA condensed, then concentrated in the center of the nucleus and finally encircled the nuclear fragments within the apoptotic bodies. NuMA seems to be preferentially cleaved by caspase-3 in vivo since it was not cleaved in staurosporine-treated caspase-3-null MCF-7 breast cancer cells. The cleavage of NuMA, lamin B and PARP-1 was inhibited in the presence of three different caspase inhibitors: z-DEVD-FMK, z-VEID-FMK and z-IETD-FMK. Furthermore, in the presence of caspase inhibitors approximately 5-10% of the cells showed atypical apoptotic morphology. These cells had convoluted nuclei, altered chromatin structure and additionally, they were negative for NuMA and lamins. Since caspase-8, -3 and -7 were not activated and PARP was not cleaved in these cells as judged by western blotting and immunofluorescence studies, it is likely that this is an atypical form of programmed cell death owing to a proteinase(s) independent of caspases. These results characterize the role of NuMA in programmed cell death and suggest that cleavage of NuMA plays a role in apoptotic nuclear breakdown.

  13. Protective role of complement C3 against cytokine-mediated beta cell apoptosis

    DEFF Research Database (Denmark)

    Dos Santos, R. S.; Marroqui, L.; Grieco, F. A.

    2017-01-01

    Background and aims: Type 1 diabetes is a chronic autoimmune disease characterized by pancreatic islet inflammation and β-cell destruction by pro-inflammatory cytokines and other mediators. The complement system, a major component of the immune system, has been recently shown to also act...... in metabolic organs, such as liver, adipose tissue, and pancreas. In the present study we identified complement C3 as an important hub of a cytokine-modified complement network in human islets and characterized the role of C3 in β-cell survival....

  14. Blocking RhoA/ROCK inhibits the pathogenesis of pemphigus vulgaris by suppressing oxidative stress and apoptosis through TAK1/NOD2-mediated NF-κB pathway.

    Science.gov (United States)

    Liang, Junqin; Zeng, Xuewen; Halifu, Yilinuer; Chen, Wenjing; Hu, Fengxia; Wang, Peng; Zhang, Huan; Kang, Xiaojing

    2017-12-01

    Oxidative stress and apoptosis play critical roles in pemphigus vulgaris (PV). The main aim of the present study was to investigate the effects of RhoA/ROCK signaling on UVB-induced oxidative damage, and to delineate the molecular mechanisms involved in the UVB-mediated inflammatory and apoptotic response. In HaCaT cells, we observed that blockage of RhoA/ROCK signaling with the inhibitor CT04 or Y27632 greatly inhibited the UVB-mediated increase in intracellular reactive oxygen species (ROS). Additionally, inhibition of RhoA/ROCK signaling reduced UVB-induced apoptosis, as exemplified by a reduction in DNA fragmentation, and also elevated anti-apoptotic Bcl-2 protein, concomitant with reduced levels of pro-apoptotic protein Bax, caspase-3 cleavage and decreased PARP-1 protein. The release of inflammatory mediators TNF-α, IL-1β, and IL-6 was also attenuated. Mechanically, we observed that blockage of RhoA/ROCK repressed the TAK1/NOD2-mediated NF-κB pathway in HaCaT cells exposed to UVB. Taken together, these data reveal that RhoA/ROCK signaling is one of the regulators contributing to oxidative damage and apoptosis in human keratinocytes, suggesting that RhoA/ROCK signaling has strong potential to be used as a useful therapeutic target in skin diseases including PV.

  15. DNA damage-induced primordial follicle oocyte apoptosis and loss of fertility require TAp63-mediated induction of Puma and Noxa

    Science.gov (United States)

    Kerr, Jeffrey B.; Hutt, Karla J.; Michalak, Ewa M.; Cook, Michele; Vandenberg, Cassandra J.; Liew, Seng H.; Bouillet, Philippe; Mills, Alea; Scott, Clare L.; Findlay, Jock K.; Strasser, Andreas

    2012-01-01

    SUMMARY Trp63, a transcription factor related to the tumor suppressor p53, is activated by diverse stimuli and can initiate a range of cellular responses. TAp63 is the predominant Trp53 family member in primordial follicle oocytes and essential for their apoptosis triggered by DNA damage in vivo. Following γ-irradiation, induction of the pro-apoptotic BH3-only members Puma and Noxa was observed in primordial follicle oocytes from wt and Trp53−/− mice but not in those from TAp63 deficient mice. Primordial follicle oocytes from mice lacking Puma or both Puma and Noxa were protected from γ-irradiation-induced apoptosis and, remarkably, could produce healthy offspring. Hence, PUMA and NOXA are critical for DNA damage-induced, TAp63-mediated primordial follicle oocyte apoptosis. Thus, blockade of PUMA may protect fertility during cancer therapy and prevent premature menopause, improving women’s health. PMID:23000175

  16. Upregulation of Fas-Fas-L (CD95/CD95L)-mediated epithelial apoptosis--a putative role in pouchitis?

    LENUS (Irish Health Repository)

    Coffey, J C

    2012-02-03

    INTRODUCTION: Ileal pouch-anal anastomosis (IPAA) remains the gold standard for patients with refractory ulcerative colitis. Pouchitis causes considerable morbidity in 40% of patients with IPAA. This study examined the role of increased epithelial apoptosis in the etiology of pouchitis. METHODS: Following ethical approval pouch biopsies taken from patients with a history of pouchitis were compared with age-matched controls from patients who were pouchitis free. Apoptosis was detected immunohistochemically using a monoclonal antibody (M30) and terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin end labeling (TUNEL). Villous atrophy was assessed histologically and correlated with levels of apoptosis. Epithelial Fas-ligand (L) was also assessed immunohistochemically. RESULTS: A significant increase in TUNEL staining was seen at the epithelial but not at the lamina propria level for known pouchitis patients versus controls (0.091 vs 0.035; P < 0.01). Similarly, epithelial M30 immunoreactivity (0.225 vs 0.082; P < 0.05) and villous atrophy (0.035 vs 0.10; P < 0.05) were significantly increased in pouches with previous pouchitis when compared with normal pouches. Upregulation of Fas-L expression was characteristic of this epithelium. Mononuclear cells were strongly positive for Fas-L. Increased epithelial levels of apoptosis correlated with increased levels of villous atrophy. CONCLUSIONS: Our data suggest a role for elevated Fas-Fas-L (CD95-CD95L)-mediated epithelial apoptosis in the etiology of pouchitis. Increased levels of villous atrophy may result from increased apoptosis and thereby predispose to infection by otherwise apathogenic organisms.

  17. Effect of Ca2EDTA on zinc mediated inflammation and neuronal apoptosis in hippocampus of an in vivo mouse model of hypobaric hypoxia.

    Directory of Open Access Journals (Sweden)

    Udayabanu Malairaman

    Full Text Available Calcium overload has been implicated as a critical event in glutamate excitotoxicity associated neurodegeneration. Recently, zinc accumulation and its neurotoxic role similar to calcium has been proposed. Earlier, we reported that free chelatable zinc released during hypobaric hypoxia mediates neuronal damage and memory impairment. The molecular mechanism behind hypobaric hypoxia mediated neuronal damage is obscure. The role of free zinc in such neuropathological condition has not been elucidated. In the present study, we investigated the underlying role of free chelatable zinc in hypobaric hypoxia-induced neuronal inflammation and apoptosis resulting in hippocampal damage.Adult male Balb/c mice were exposed to hypobaric hypoxia and treated with saline or Ca2EDTA (1.25 mM/kg i.p daily for four days. The effects of Ca2EDTA on apoptosis (caspases activity and DNA fragmentation, pro-inflammatory markers (iNOS, TNF-α and COX-2, NADPH oxidase activity, poly(ADP ribose polymerase (PARP activity and expressions of Bax, Bcl-2, HIF-1α, metallothionein-3, ZnT-1 and ZIP-6 were examined in the hippocampal region of brain.Hypobaric hypoxia resulted in increased expression of metallothionein-3 and zinc transporters (ZnT-1 and ZIP-6. Hypobaric hypoxia elicited an oxidative stress and inflammatory response characterized by elevated NADPH oxidase activity and up-regulation of iNOS, COX-2 and TNF-α. Furthermore, hypobaric hypoxia induced HIF-1α protein expression, PARP activation and apoptosis in the hippocampus. Administration of Ca2EDTA significantly attenuated the hypobaric hypoxia induced oxidative stress, inflammation and apoptosis in the hippocampus.We propose that hypobaric hypoxia/reperfusion instigates free chelatable zinc imbalance in brain associated with neuroinflammation and neuronal apoptosis. Therefore, zinc chelating strategies which block zinc mediated neuronal damage linked with cerebral hypoxia and other neurodegenerative conditions can be

  18. Effect of Ca2EDTA on Zinc Mediated Inflammation and Neuronal Apoptosis in Hippocampus of an In Vivo Mouse Model of Hypobaric Hypoxia

    Science.gov (United States)

    Malairaman, Udayabanu; Dandapani, Kumaran; Katyal, Anju

    2014-01-01

    Background Calcium overload has been implicated as a critical event in glutamate excitotoxicity associated neurodegeneration. Recently, zinc accumulation and its neurotoxic role similar to calcium has been proposed. Earlier, we reported that free chelatable zinc released during hypobaric hypoxia mediates neuronal damage and memory impairment. The molecular mechanism behind hypobaric hypoxia mediated neuronal damage is obscure. The role of free zinc in such neuropathological condition has not been elucidated. In the present study, we investigated the underlying role of free chelatable zinc in hypobaric hypoxia-induced neuronal inflammation and apoptosis resulting in hippocampal damage. Methods Adult male Balb/c mice were exposed to hypobaric hypoxia and treated with saline or Ca2EDTA (1.25 mM/kg i.p) daily for four days. The effects of Ca2EDTA on apoptosis (caspases activity and DNA fragmentation), pro-inflammatory markers (iNOS, TNF-α and COX-2), NADPH oxidase activity, poly(ADP ribose) polymerase (PARP) activity and expressions of Bax, Bcl-2, HIF-1α, metallothionein-3, ZnT-1 and ZIP-6 were examined in the hippocampal region of brain. Results Hypobaric hypoxia resulted in increased expression of metallothionein-3 and zinc transporters (ZnT-1 and ZIP-6). Hypobaric hypoxia elicited an oxidative stress and inflammatory response characterized by elevated NADPH oxidase activity and up-regulation of iNOS, COX-2 and TNF-α. Furthermore, hypobaric hypoxia induced HIF-1α protein expression, PARP activation and apoptosis in the hippocampus. Administration of Ca2EDTA significantly attenuated the hypobaric hypoxia induced oxidative stress, inflammation and apoptosis in the hippocampus. Conclusion We propose that hypobaric hypoxia/reperfusion instigates free chelatable zinc imbalance in brain associated with neuroinflammation and neuronal apoptosis. Therefore, zinc chelating strategies which block zinc mediated neuronal damage linked with cerebral hypoxia and other

  19. Raf-1/CK2 and RhoA/ROCK signaling promote TNF-α-mediated endothelial apoptosis via regulating vimentin cytoskeleton.

    Science.gov (United States)

    Yang, Lifeng; Tang, Lian; Dai, Fan; Meng, Guoliang; Yin, Runting; Xu, Xiaole; Yao, Wenjuan

    2017-08-15

    Both RhoA/ROCK and Raf-1/CK2 pathway play essential roles in cell proliferation, apoptosis, differentiation, and multiple other common cellular functions. We previously reported that vimentin is responsible for TNF-α-induced cell apoptosis. Herein, we investigated the regulation of RhoA/ROCK and Raf-1/CK2 signaling on vimentin filaments and endothelial apoptosis mediated by TNF-α. Treatment with TNF-α significantly induced the activation of RhoA and ROCK, and the expression of ROCK1. RhoA deficiency could obviously inhibit ROCK activation and ROCK1 expression induced by TNF-α. Both RhoA deficiency and ROCK activity inhibition (Y-27632) greatly inhibited endothelial apoptosis and preserved cell viability in TNF-α-induced human umbilical vein endothelial cells (HUVECs). Also vimentin phosphorylation and the remodeling of vimentin or phospho-vimentin induced by TNF-α were obviously attenuated by RhoA suppression and ROCK inhibition. TNF-α-mediated vimentin cleavage was significantly inhibited by RhoA suppression and ROCK inhibition through decreasing the activation of caspase3 and 8. Furthermore, TNF-α treatment greatly enhanced the activation of Raf-1. Suppression of Raf-1 or CK2 by its inhibitor (GW5074 or TBB) blocked vimentin phosphorylation, remodeling and endothelial apoptosis, and preserved cell viability in TNF-α-induced HUVECs. However, Raf-1 inhibition showed no significant effect on TNF-α-induced ROCK expression and activation, suggesting that the regulation of Raf-1/CK2 signaling on vimentin was independent of ROCK. Taken together, these results indicate that both RhoA/ROCK and Raf-1/CK2 pathway are responsible for TNF-α-mediated endothelial cytotoxicity via regulating vimentin cytoskeleton. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. N-methyl-d-aspartate receptor-mediated calcium overload and endoplasmic reticulum stress are involved in interleukin-1beta-induced neuronal apoptosis in rat hippocampus.

    Science.gov (United States)

    Dong, Yilong; Kalueff, Allan V; Song, Cai

    2017-06-15

    Increased levels of interleukin (IL)-1β and its gene expression are implicated in the etiology of Alzheimer's disease (AD). IL-1β activates microglia and stimulates glutamatergic N-methyl-d-aspartate receptor NMDA receptor expression, thereby disturbing intracellular Ca 2+ homeostasis. Ca 2+ disequilibrium, in turn, may trigger endoplasmic reticulum (ER) stress, contributing to overall excitotoxicity and neuronal death that evoke AD. However, it is unclear whether IL-1β-induced neuronal apoptosis is mediated by the glutamatergic system, ER stress and/or Ca 2+ dysfunction. The present study investigated the role of NMDA receptor (NMDAR) in ER stress and IL-1β-evoked neuronal death by assessing NMDAR-induced Ca 2+ overload and NMDA-mediated ER stress. Male Long Evans rats were treated with IL-1β (with or without NMDAR antagonist MK801) injected intracerebroventricularly for 8days. Glutamate concentration was measured by HPLC, and mRNA and protein expression of microglial biomarkers and NMDAR, as well as markers of Ca 2+ overload (caplain2) and ER stress (glucose-regulated protein 78, GRP78, and C/EBP homologous protein-10, CHOP), were assessed by real-time PCR and western blot. Apoptosis was also evaluated in the hippocampal neurons using TUNEL. Overall, IL-1β induced robust neuronal apoptosis, accompanied by upregulated NMDAR, caplain2, GRP78 and CHOP. MK801 pretreatment significantly attenuated neuronal apoptosis and NMDA up-regulation, also reducing GRP78 and CHOP expression. In summary, these results suggest that IL-1β may disturb intracellular Ca 2+ homeostasis via NMDAR-mediated mechanism, thereby triggering neuronal apoptosis by enhancing ER stress. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Opposed arsenite-mediated regulation of p53-survivin is involved in neoplastic transformation, DNA damage, or apoptosis in human keratinocytes

    International Nuclear Information System (INIS)

    Li, Yuan; Jiang, Rongrong; Zhao, Yue; Xu, Yuan; Ling, Min; Pang, Ying; Shen, Lu; Zhou, Yun; Zhang, Jianping; Zhou, Jianwei; Wang, Xinru; Liu, Qizhan

    2012-01-01

    Highlights: ► Different concentrations of arsenite cause biphasic effects in HaCaT cells. ► p53-survivin signal pathway plays a role in arsenite-induced biphasic effects. ► ERKs inactivate p53, but improve survivin expression by NF-κB/mot-2. ► JNKs block survivin expression by preventing p53 from mdm2-mediated degradation. ► ERKs and JNKs play roles in arsenite-induced biphasic effects. -- Abstract: Biphasic dose–response relationship induced by environmental agents is often characterized with the effect of low-dose stimulation and high dose inhibition. Some studies showed that arsenite may induce cell proliferation and apoptosis via biphasic dose–response relationship in human cells; however, mechanisms underlying this phenomenon are not well understood. Our present study shows that, for human keratinocytes (HaCaT) cells, a low concentration of arsenite activates extracellular signal-regulated kinases (ERKs), which leads to up-regulation of nuclear factor κB (NF-κB) binding to DNA and to elevated, NF-κB-dependent expression of mot-2 (a p53 inhibitor) and survivin (an inhibitor of apoptosis). Activation of p53 is blocked, and neoplastic transformation is enhanced. Inhibition of ERKs reduces cell proliferation and neoplastic transformation. In contrast, a high concentration of arsenite activates c-Jun N-terminal kinases (JNKs), positive regulators of p53, by binding to p53 and preventing its murine double minute 2 (mdm2)-mediated degradation. The elevated levels of p53 lead to repair of DNA damage and apoptosis. Inhibition of JNKs increases DNA damage but decreases apoptosis. By identifying a mechanism whereby ERKs and JNKs-mediated regulation of the p53-survivin signal pathway is involved in the biphasic effects of arsenite on human keratinocytes, our data expand understanding of arsenite-induced cell proliferation, neoplastic transformation, DNA damage, and apoptosis.

  2. Melatonin Reverses Fas, E2F-1 and Endoplasmic Reticulum Stress Mediated Apoptosis and Dysregulation of Autophagy Induced by the Herbicide Atrazine in Murine Splenocytes.

    Directory of Open Access Journals (Sweden)

    Shweta Sharma

    Full Text Available Exposure to the herbicide Atrazine (ATR can cause immunotoxicity, apart from other adverse consequences for animal and human health. We aimed at elucidating the apoptotic mechanisms involved in immunotoxicity of ATR and their attenuation by Melatonin (MEL. Young Swiss mice were divided into control, ATR and MEL+ATR groups based on daily (x14 intraperitoneal administration of the vehicle (normal saline, ATR (100 mg/kg body weight and MEL (20 mg/kg body weight with ATR. Isolated splenocytes were processed for detection of apoptosis by Annexin V-FITC and TUNEL assays, and endoplasmic reticulum (ER stress by immunostaining. Key proteins involved in apoptosis, ER stress and autophagy were quantified by immunoblotting. ATR treatment resulted in Fas-mediated activation of caspases 8 and 3 and inactivation of PARP1 which were inhibited significantly by co-treatment with MEL. MEL also attenuated the ATR-induced, p53 independent mitochondrial apoptosis through upregulation of E2F-1 and PUMA and suppression of their downstream target Bax. An excessive ER stress triggered by ATR through overexpression of ATF-6α, spliced XBP-1, CREB-2 and GADD153 signals was reversed by MEL. MEL also reversed the ATR-induced impairment of autophagy which was indicated by a decline in BECN-1, along with significant enhancement in LC3B-II and p62 expressions. Induction of mitochondrial apoptosis, ER stress and autophagy dysregulation provide a new insight into the mechanism of ATR immunotoxicity. The cytoprotective role of MEL, on the other hand, was defined by attenuation of ER stress, Fas-mediated and p53 independent mitochondria-mediated apoptosis as well as autophagy signals.

  3. Antiproliferative activity of Alisol B in MDA-MB-231 cells is mediated by apoptosis, dysregulation of mitochondrial functions, cell cycle arrest and generation of reactive oxygen species.

    Science.gov (United States)

    Zhang, Aifeng; Sheng, Yuqing; Zou, Mingchang

    2017-03-01

    Previous studies have demonstrated that Alisol B has inhibitory activity in cancer cells. However, the exact mechanism through which inhibition is achieved is still poorly understood. In the present study, the authors examined the effects of Alisol B in human breast cancer cells. Alisol B showed significant anticancer activity in MDA-MB-231 cells. The results demonstrated that the cytotoxicity induced by Alisol B was mediated by induction of apoptosis, decrease in mitochondrial membrane potential, cell cycle arrest, activation of caspases and accumulation of ROS (reactive oxygen species) level. Interestingly, pretreatment of cells with the general caspase inhibitor z-VAD-FMK significantly prevented Alisol B-induced apoptosis. Furthermore, western blot analysis revealed the upregulation of p-p38 and downregulation of p-AKT, p-p65 and p-mTOR. Taken together, the above results suggest that Alisol B suppresses the growth of MDA-MB-231 cells mainly through induction of apoptosis; this outcome may represent the major mechanism of Alisol B-mediated apoptosis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  4. Interferon-α mediates human beta cell HLA class I overexpression, endoplasmic reticulum stress and apoptosis, three hallmarks of early human type 1 diabetes.

    Science.gov (United States)

    Marroqui, Laura; Dos Santos, Reinaldo S; Op de Beeck, Anne; Coomans de Brachène, Alexandra; Marselli, Lorella; Marchetti, Piero; Eizirik, Decio L

    2017-04-01

    Three hallmarks of the pancreatic islets in early human type 1 diabetes are overexpression of HLA class I, endoplasmic reticulum (ER) stress and beta cell apoptosis. The mediators of these phenomena remain to be defined. The type I interferon IFNα is expressed in human islets from type 1 diabetes patients and mediates HLA class I overexpression. We presently evaluated the mechanisms involved in IFNα-induced HLA class I expression in human beta cells and determined whether this cytokine contributes to ER stress and apoptosis. IFNα-induced inflammation, ER stress and apoptosis were evaluated by RT-PCR, western blot, immunofluorescence and nuclear dyes, and proteins involved in type I interferon signalling were inhibited by small interfering RNAs. All experiments were performed in human islets or human EndoC-βH1 cells. IFNα upregulates HLA class I, inflammation and ER stress markers in human beta cells via activation of the candidate gene TYK2, and the transcription factors signal transducer and activator of transcription 2 and IFN regulatory factor 9. Furthermore, it acts synergistically with IL-1β to induce beta cell apoptosis. The innate immune effects induced by IFNα may induce and amplify the adaptive immune response against human beta cells, indicating that IFNα has a central role in the early phases of diabetes.

  5. Induction of apoptosis and reversal of permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM by ginsenoside Rh2

    Science.gov (United States)

    Zhang, Hui; Gong, Jian; Zhang, Huilai; Kong, Di

    2015-01-01

    Multidrug resistance is a phenomenon that cancer cells develop a cross-resistant phenotype against several unrelated drugs, and permeability glycoprotein derived from the overexpression of multidrug resistance gene 1 has been taken as the most significant cause of multidrug resistance. In the present study, ginsenoside Rh2 was used to reverse permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM cell line. Effects of ginsenoside Rh2 on the apoptotic process and caspase-3 activity of MCF-7 and MCF-7/ADM cell lines were determined using flow cytometry and microplate reader. Methyl thiazolyl tetrazolium test was conducted to assess the IC50 values of ginsenoside Rh2 and adriamycin on MCF-7 and MCF-7/ADM cultures; Rhodamin 123 assay was used to assess the retention of permeability glycoprotein after ginsenoside Rh2 treatment; flow cytometry and real time polymerase chain reaction were used to determine the expression levels of permeability glycoprotein and multidrug resistance gene 1 in drug-resistant cells and their parental cells after exposure to ginsenoside Rh2. The results showed that ginsenoside Rh2, except for inducing apoptosis, had the ability to reverse multidrug resistance in MCF-7/ADM cell line without changing the expression levels of permeability glycoprotein and multidrug resistance gene 1. Our findings provided some valuable information for the application of ginsenoside Rh2 in cancer therapy, especially for multidrug resistance reversal in clinic. PMID:26191135

  6. Seleno-short-chain chitosan induces apoptosis in human non-small-cell lung cancer A549 cells through ROS-mediated mitochondrial pathway.

    Science.gov (United States)

    Zhao, Yana; Zhang, Shaojing; Wang, Pengfei; Fu, Shengnan; Wu, Di; Liu, Anjun

    2017-12-01

    Seleno-short-chain chitosan (SSCC) is a synthesized chitosan derivative. In this study, antitumor activity and underlying mechanism of SSCC on human non-small-cell lung cancer A549 cells were investigated in vitro. The MTT assay showed that SSCC could inhibit cell viability in a dose- and time-dependent manner, and 200 μg/ml SSCC exhibited significantly toxic effects on A549 cells. The cell cycle assay showed that SSCC triggered S phase cell cycle arrest in a dose- and time-dependent manner, which was related to a downregulation of S phase associated cyclin A. The DAPI staining and Annexin V-FITC/PI double staining identified that the SSCC could induce A549 cells apoptosis. Further studies found that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) by DCFH-DA and Rhodamin 123 staining, respectively. Meanwhile, free radical scavengers N-acetyl-L-cysteine (NAC) pretreatment confirmed that SSCC-induced A549 cells apoptosis was associated with ROS generation. Furthermore, real-time PCR and western blot assay showed that SSCC up-regulated Bax and down-regulated Bcl-2, subsequently incited the release of cytochrome c from mitochondria to cytoplasm, activated the increase of cleaved-caspase 3 and finally induced A549 cells apoptosis in vitro. In general, the present study demonstrated that SSCC induced A549 cells apoptosis via ROS-mediated mitochondrial apoptosis pathway.

  7. Study on the apoptosis mediated by cytochrome c and factors that affect the activation of bovine longissimus muscle during postmortem aging.

    Science.gov (United States)

    Zhang, Jiaying; Yu, Qunli; Han, Ling; Chen, Cheng; Li, Hang; Han, Guangxing

    2017-06-01

    This study investigates whether bovine longissimus muscle cell apoptosis occurs during postmortem aging and whether apoptosis is dependent on the mitochondria pathway. This study also determines the apoptosis process mediated by cytochrome c after its release from mitochondria and the factors that affect the activation processes. Results indicate that apoptotic nuclei were detected at 12 h postmortem. Cytochrome c release from the mitochondria to the cytoplasm activated the caspase-9 and caspase-3 at early postmortem aging and the activation of caspase-9 occurs before the activation of caspase-3. The pH level decreased during the first 48 h postmortem, whereas the mitochondria membrane permeability increased from 6 to 12 h. Results demonstrate that an apoptosis process of bovine muscle occurred during postmortem aging. Apoptosis was dependent on the mitochondria pathway and occurred at early postmortem aging. Increased mitochondria membrane permeability and low pH are necessary conditions for the release of cytochrome c during postmortem aging.

  8. TNF-α Mediates the Intrinsic and Extrinsic Pathway in Propofol-Induced Neuronal Apoptosis Via PI3K/Akt Signaling Pathway in Rat Prefrontal Cortical Neurons.

    Science.gov (United States)

    Deng, Xiaoyuan; Chen, Bo; Wang, Bin; Zhang, Junfang; Liu, Hongliang

    2017-10-01

    Propofol can cause developing neuronal apoptosis in both in vivo and in vitro studies, and the mechanism is unclear till now. Our previous study has demonstrated that propofol can increase the TNF-α expression in the prefrontal cortex in rat developing brain, the TNF-α antagonist, etanercept, can inhibit propofol-induced neuronal apoptosis, but little is known about how TNF-α mediates that process. This study reveals that propofol at clinically relevant concentrations increases the TNF-α synthesis and release in neurons, and induces neuronal apoptosis; etanercept significantly reduces neuronal apoptosis, the elevation of cleaved caspase-8 and cleaved caspase-9, or the Akt phosphorylation induced by propofol, while the selective PI3K antagonist blocks the neuroprotection of etanercept. Propofol does not change the expression of P2X7 receptor in neurons, and the P2X7 receptor antagonist cannot affect the TNF-α synthesis or release after propofol treatment. These results suggest that propofol can increase the synthesis and release of TNF-α in the primary cultured prefrontal cortical neurons, TNF-α contributes to the intrinsic and extrinsic pathway in propofol-induced neuronal apoptosis via PI3K/Akt signaling pathway, and P2X7R is not involved in the synthesis and release of TNF-α induced by propofol.

  9. Antisense oligonucleotide mediated knockdown of HOXC13 affects cell growth and induces apoptosis in tumor cells and over expression of HOXC13 induces 3D-colony formation.

    Science.gov (United States)

    Kasiri, Sahba; Ansari, Khairul I; Hussain, Imran; Bhan, Arunoday; Mandal, Subhrangsu S

    2013-01-01

    HOXC13 is a homeobox containing gene that plays crucial roles in hair development and origin of replication. Herein, we investigated the biochemical functions of HOXC13 and explored its potential roles in tumor cell viability. We have designed a phosphorothioate based antisense-oligonucleotide that specifically knockdown HOXC13 in cultured cells. Cell viability and cytotoxicity assays demonstrated that HOXC13 is essential for cell growth and viability. Antisense-mediated knockdown of HOXC13 affected the cell viability and induced apoptosis in cultured tumor cells. HOXC13 regulates the expression of cyclins and antisense-mediated knockdown of HOXC13 resulted in cell cycle arrest and apoptosis in colon cancer cells. Finally over expression of HOXC13 resulted in 3D-colony formation in soft-agar assay indicating its potential roles in cell proliferation and tumorigenesis.

  10. Depletion of TFAP2E attenuates adriamycin-mediated apoptosis in human neuroblastoma cells.

    Science.gov (United States)

    Hoshi, Reina; Watanabe, Yosuke; Ishizuka, Yoshiaki; Hirano, Takayuki; Nagasaki-Maeoka, Eri; Yoshizawa, Shinsuke; Uekusa, Shota; Kawashima, Hiroyuki; Ohashi, Kensuke; Sugito, Kiminobu; Fukuda, Noboru; Nagase, Hiroki; Soma, Masayoshi; Ozaki, Toshinori; Koshinaga, Tsugumichi; Fujiwara, Kyoko

    2017-04-01

    Neuroblastoma is a childhood malignancy originating from the sympathetic nervous system and accounts for approximately 15% of all pediatric cancer-related deaths. To newly identify gene(s) implicated in the progression of neuroblastoma, we investigated aberrantly methylated genomic regions in mouse skin tumors. Previously, we reported that TFAP2E, a member of activator protein-2 transcription factor family, is highly methylated within its intron and its expression is strongly suppressed in mouse skin tumors compared with the normal skin. In the present study, we analyzed public data of neuroblastoma patients and found that lower expression levels of TFAP2E are significantly associated with a shorter survival. The data indicate that TFAP2E acts as a tumor suppressor of neuroblastoma. Consistent with this notion, TFAP2E-depleted neuroblastoma NB1 and NB9 cells displayed a substantial resistance to DNA damage arising from adriamycin (ADR), cisplatin (CDDP) and ionizing radiation (IR). Silencing of TFAP2E caused a reduced ADR-induced proteolytic cleavage of caspase-3 and PARP. Of note, compared with the untransfected control cells, ADR-mediated stimulation of CDK inhibitor p21WAF1 was markedly upregulated in TFAP2E‑knocked down cells. Therefore, our present findings strongly suggest that TFAP2E has a pivotal role in the regulation of DNA damage response in NB cells through the induction of p21WAF1.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  12. Artemisinin induces A549 cell apoptosis dominantly via a reactive oxygen species-mediated amplification activation loop among caspase-9, -8 and -3.

    Science.gov (United States)

    Gao, Weijie; Xiao, Fenglian; Wang, Xiaoping; Chen, Tongsheng

    2013-10-01

    This report is designed to explore the roles of caspase-8, -9 and -3 in artemisinin (ARTE)-induced apoptosis in non-small cell lung cancer cells (A549 cells). ARTE induced reactive oxygen species (ROS)-mediated apoptosis in dose- and time-dependent fashion. Although ARTE treatment did not induce Bid cleavage and significant loss of mitochondrial membrane potential, it induced release of Smac and AIF but not cytochrome c from mitochondria, and silencing of Bak but not Bax significantly prevented ARTE-induced cytotoxicity. Moreover, ARTE treatment induced ROS-dependent activation of caspase-9, -8 and -3. Of the utmost importance, silencing or inhibiting any one of caspase-8, -9 and -3 almost completely prevented ARTE-induced activation of all the three caspases and remarkably abrogated the cytotoxicity of ARTE, suggesting that ARTE triggered an amplification activation loop among caspase-9, -8 and -3. Collectively, our data demonstrate that ARTE induces a ROS-mediated amplification activation loop among caspase-9, -8 and -3 to dominantly mediate the apoptosis of A549 cells.

  13. Aloe-emodin-mediated photodynamic therapy induces autophagy and apoptosis in human osteosarcoma cell line MG‑63 through the ROS/JNK signaling pathway.

    Science.gov (United States)

    Tu, Pinghua; Huang, Qiu; Ou, Yunsheng; Du, Xing; Li, Kaiting; Tao, Yong; Yin, Hang

    2016-06-01

    The present study was carried out to investigate the effect and mechanisms of aloe‑emodin (AE)-mediated photodynamic therapy (AE-PDT) on the human osteosarcoma cell line MG-63. After treatment with AE-PDT, the human osteosarcoma cell line MG-63 was tested for levels of viability, autophagy, reactive oxygen species (ROS) and apoptosis and changes in cell morphology with the Cell Counting Kit-8 (CCK‑8), monodansylcadaverine (MDC) and Hoechst staining and transmission electron microscopy. The expression of proteins including LC-3, cleaved caspase-3, Beclin-1, Bcl-2, p-JNK, t-JNK and β-actin was examined with western blotting. AE-PDT significantly inhibited the viability of the MG-63 cells in an AE-concentration- and PDT energy density-dependent manner. Autophagy and apoptosis of MG-63 cells was substantially promoted in the AE-PDT group compared to the control group, the AE alone group and the light emitting diode (LED) alone group. Inhibition of autophagy by 3-methyladenine (3-MA) (5 mM) and chloroquine (CQ) (15 µM) significantly promoted the apoptosis rate and improved the sensitivity of the MG-63 cells to AE-PDT. AE-PDT was found to induce the expression of ROS and p-JNK. ROS scavenger, N-acetyl-L-cysteine (NAC, 5 mM), was able to hinder the autophagy, apoptosis and phosphorylation of JNK, and JNK inhibitor (SP600125, 10 µM) significantly inhibited the autophagy and apoptosis, and attenuated the sensitivity of MG63 cells to AE-PDT. In conclusion, AE-PDT induced the autophagy and apoptosis of human osteosarcoma cell line MG-63 through the activation of the ROS-JNK signaling pathway. Autophagy may play a protective role during the early stage following treatment of AE-PDT.

  14. Off-target function of the Sonic hedgehog inhibitor cyclopamine in mediating apoptosis via nitric oxide-dependent neutral sphingomyelinase 2/ceramide induction.

    Science.gov (United States)

    Meyers-Needham, Marisa; Lewis, Jocelyn A; Gencer, Salih; Sentelle, R David; Saddoughi, Sahar A; Clarke, Christopher J; Hannun, Yusuf A; Norell, Haakan; da Palma, Telma Martins; Nishimura, Michael; Kraveka, Jacqueline M; Khavandgar, Zohreh; Murshed, Monzur; Cevik, M Ozgur; Ogretmen, Besim

    2012-05-01

    Sonic hedgehog (SHh) signaling is important in the pathogenesis of various human cancers, such as medulloblastomas, and it has been identified as a valid target for anticancer therapeutics. The SHh inhibitor cyclopamine induces apoptosis. The bioactive sphingolipid ceramide mediates cell death in response to various chemotherapeutic agents; however, ceramide's roles/mechanisms in cyclopamine-induced apoptosis are unknown. Here, we report that cyclopamine mediates ceramide generation selectively via induction of neutral sphingomyelin phosphodiesterase 3, SMPD3 (nSMase2) in Daoy human medulloblastoma cells. Importantly, short interfering RNA-mediated knockdown of nSMase2 prevented cyclopamine-induced ceramide generation and protected Daoy cells from drug-induced apoptosis. Accordingly, ectopic wild-type N-SMase2 caused cell death, compared with controls, which express the catalytically inactive N-SMase2 mutant. Interestingly, knockdown of smoothened (Smo), a target protein for cyclopamine, or Gli1, a downstream signaling transcription factor of Smo, did not affect nSMase2. Mechanistically, our data showed that cyclopamine induced nSMase2 and cell death selectively via increased nitric oxide (NO) generation by neuronal-nitric oxide synthase (n-NOS) induction, in Daoy medulloblastoma, and multiple other human cancer cell lines. Knockdown of n-NOS prevented nSMase2 induction and cell death in response to cyclopamine. Accordingly, N-SMase2 activity-deficient skin fibroblasts isolated from homozygous fro/fro (fragilitas ossium) mice exhibited resistance to NO-induced cell death. Thus, our data suggest a novel off-target function of cyclopamine in inducing apoptosis, at least in part, by n-NOS/NO-dependent induction of N-SMase2/ceramide axis, independent of Smo/Gli inhibition. ©2012 AACR

  15. Upconversion nanoparticle-mediated photodynamic therapy induces THP-1 macrophage apoptosis via ROS bursts and activation of the mitochondrial caspase pathway.

    Science.gov (United States)

    Zhu, Xing; Wang, Hao; Zheng, Longbin; Zhong, Zhaoyu; Li, Xuesong; Zhao, Jing; Kou, Jiayuan; Jiang, Yueqing; Zheng, Xiufeng; Liu, Zhongni; Li, Hongxia; Cao, Wenwu; Tian, Ye; Wang, You; Yang, Liming

    2015-01-01

    Atherosclerosis (AS) is the most vital cardiovascular disease, which poses a great threat to human health. Macrophages play an important role in the progression of AS. Photodynamic therapy (PDT) has emerged as a useful therapeutic modality not only in the treatment of cancer but also in the treatment of AS. The purpose of this study was to determine the molecular mechanisms underlying the activity of PDT, using mesoporous-silica-coated upconversion fluorescent nanoparticles encapsulating chlorin e6 (UCNPs-Ce6) in the induction of apoptosis in THP-1 macrophages. Here, we investigated the ability of UCNPs-Ce6-mediated PDT to induce THP-1 macrophage apoptosis by facilitating the induction of reactive oxygen species (ROS) and regulation of mitochondrial permeability transition pore (MPTP) to depolarize mitochondrial membrane potential (MMP). Both Bax translocation and the release of cytochrome C were examined using immunofluorescence and Western blotting. Our results indicated that the levels of ROS were significantly increased in the PDT group, resulting in both MPTP opening and MMP depolarization, which led to apoptosis. In addition, immunofluorescence and Western blotting revealed that PDT induced both Bax translocation and the release of cytochrome C, as well as upregulation of cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase. Therefore, we demonstrated that UCNPs-Ce6-mediated PDT induces apoptosis in THP-1 macrophages via ROS bursts. The proapoptotic factor Bax subsequently translocates from the cytosol to the mitochondria, resulting in the MPTP opening and cytochrome C release. This study demonstrated the great potential of UCNPs-Ce6-mediated PDT in the treatment of AS.

  16. Bifidobacterial recombinant thymidine kinase-ganciclovir gene therapy system induces FasL and TNFR2 mediated antitumor apoptosis in solid tumors

    International Nuclear Information System (INIS)

    Wang, Changdong; Ma, Yongping; Hu, Qiongwen; Xie, Tingting; Wu, Jiayan; Zeng, Fan; Song, Fangzhou

    2016-01-01

    Directly targeting therapeutic suicide gene to a solid tumor is a hopeful approach for cancer gene therapy. Treatment of a solid tumor by an effective vector for a suicide gene remains a challenge. Given the lack of effective treatments, we constructed a bifidobacterial recombinant thymidine kinase (BF-rTK) -ganciclovir (GCV) targeting system (BKV) to meet this requirement and to explore antitumor mechanisms. Bifidobacterium (BF) or BF-rTK was injected intratumorally with or without ganciclovir in a human colo320 intestinal xenograft tumor model. The tumor tissues were analyzed using apoptosis antibody arrays, real time PCR and western blot. The colo320 cell was analyzed by the gene silencing method. Autophagy and necroptosis were also detected in colo320 cell. Meanwhile, three human digestive system xenograft tumor models (colorectal cancer colo320, gastric cancer MKN-45 and liver cancer SSMC-7721) and a breast cancer (MDA-MB-231) model were employed to validate the universality of BF-rTK + GCV in solid tumor gene therapy. The survival rate was evaluated in three human cancer models after the BF-rTK + GCV intratumor treatment. The analysis of inflammatory markers (TNF-α) in tumor indicated that BF-rTK + GCV significantly inhibited TNF-α expression. The results suggested that BF-rTK + GCV induced tumor apoptosis without autophagy and necroptosis occurrence. The apoptosis was transduced by multiple signaling pathways mediated by FasL and TNFR2 and mainly activated the mitochondrial control of apoptosis via Bid and Bim, which was rescued by silencing Bid or/and Bim. However, BF + GCV only induced apoptosis via Fas/FasL signal pathway accompanied with increased P53 expression. We further found that BF-rTK + GCV inhibited the expression of the inflammatory maker of TNF-α. However, BF-rTK + GCV did not result in necroptosis and autophagy. BF-rTK + GCV induced tumor apoptosis mediated by FasL and TNFR2 through the mitochondrial control of apoptosis via Bid and Bim

  17. N-Cadherin-Mediated Activation of PI3K/Akt-GSK-3β Signaling Attenuates Nucleus Pulposus Cell Apoptosis Under High-Magnitude Compression

    Directory of Open Access Journals (Sweden)

    Pei Li

    2017-11-01

    Full Text Available Background/Aims: Mechanical overloading-induced nucleus pulposus (NP apoptosis plays an important role in the pathogenesis of intervertebral disc degeneration. N-cadherin (N-CDH-mediated signaling preserves normal NP cell phenotype. This study aims to investigate the effects of N-CDH on NP cell apoptosis under high-magnitude compression and the underlying mechanism behind this process. Methods: Rat NP cells seeded on scaffold were perfusion-cultured using a self-developed perfusion bioreactor for 5 days and experienced different magnitudes (2% and 20% compressive deformation, respectively of compression at a frequency of 1.0 Hz for 4 hours once per day. The un-loaded NP cells were used as controls. Lentivirus-mediated N-CDH overexpression and inhibitor LY294002 were used to further investigate the role of N-CDH and PI3K/Akt pathway under high-magnitude compression, respectively. NP cell apoptosis was evaluated by caspase-3 activity measured using a commercial kit, flow cytometry, and expression of apoptosis-related molecules analyzed by real-time PCR and western blotting assays. Results: High-magnitude compression significantly increased apoptotic NP cells, caspase-3 activity and expression of pro-apoptotic molecules (Bax and caspase-3/cleaved caspase-3, but decreased expression of anti-apoptotic molecule (Bcl-2. High-magnitude compression decreased expression of N-CDH, p-Akt and p-GSK-3β. However, N-CDH overexpression attenuated NP cell apoptosis and increased expression of p-Akt and p-GSK-3β under high-magnitude compression. Further analysis showed that inhibition of the PI3K/Akt pathway suppressed NP cell apoptosis and decreased expression of p-GSK-3β, but had no significant effects on N-CDH expression under high-magnitude compression. Conclusion: N-CDH can attenuate NP cell apoptosis through activating the PI3K/Akt-GSK-3β signaling under high-magnitude compression.

  18. The selective target of capsaicin on FASN expression and de novo fatty acid synthesis mediated through ROS generation triggers apoptosis in HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Hathaichanok Impheng

    Full Text Available The inhibition of the mammalian de novo synthesis of long-chain saturated fatty acids (LCFAs by blocking the fatty acid synthase (FASN enzyme activity in tumor cells that overexpress FASN can promote apoptosis, without apparent cytotoxic to non-tumor cells. The present study aimed to focus on the potent inhibitory effect of capsaicin on the fatty acid synthesis pathway inducing apoptosis of capsaicin in HepG2 cells. The use of capsaicin as a source for a new FASN inhibitor will provide new insight into its possible application as a selective anti-cancer therapy. The present findings showed that capsaicin promoted apoptosis as well as cell cycle arrest in the G0/G1 phase. The onset of apoptosis was correlated with a dissipation of mitochondrial membrane potential (ΔΨm. Apoptotic induction by capsaicin was mediated by inhibition of FASN protein expression which was accompanied by decreasing its activity on the de novo fatty acid synthesis. The expression of FASN was higher in HepG2 cells than in normal hepatocytes that were resistant to undergoing apoptosis following capsaicin administration. Moreover, the inhibitory effect of capsaicin on FASN expression and activity was found to be mediated by an increase of intracellular reactive oxygen species (ROS generation. Treatment of HepG2 cells with capsaicin failed to alter ACC and ACLY protein expression, suggesting ACC and ACLY might not be the specific targets of capsaicin to induce apoptosis. An accumulation of malonyl-CoA level following FASN inhibition represented a major cause of mitochondrial-dependent apoptotic induction instead of deprivation of fatty acid per se. Here, we also obtained similar results with C75 that exhibited apoptosis induction by reducing the levels of fatty acid without any change in the abundance of FASN expression along with increasing ROS production. Collectively, our results provide novel evidence that capsaicin exhibits a potent anti-cancer property by targeting

  19. Early apoptosis and cell death induced by ATX-S10Na (II)-mediated photodynamic therapy are Bax- and p53-dependent in human colon cancer cells.

    Science.gov (United States)

    Mitsunaga, Makoto; Tsubota, Akihito; Nariai, Kohichi; Namiki, Yoshihisa; Sumi, Makoto; Yoshikawa, Tetsuya; Fujise, Kiyotaka

    2007-02-07

    To investigate the roles of Bax and p53 proteins in photosensitivity of human colon cancer cells by using lysosome-localizing photosensitizer, ATX-S10Na (II). HCT116 human colon cancer cells and Bax-null or p53-null isogenic derivatives were irradiated with a diode laser. Early apoptosis and cell death in response to photodynamic therapy were determined by MTT assays, annexin V assays, transmission electron microscopy assays, caspase assays and western blotting. Induction of early apoptosis and cell death was Bax- and p53-dependent. Bax and p53 were required for caspase-dependent apoptosis. The levels of anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-x(L), were decreased in Bax- and p53-independent manner. Our results indicate that early apoptosis and cell death of human colon cancer cells induced by photodynamic therapy with lysosome-localizing photosensitizer ATX-S10Na (II) are mediated by p53-Bax network and low levels of Bcl-2 and Bcl-x(L) proteins. Our results might help in formulating new therapeutic approaches in photodynamic therapy.

  20. FLASH knockdown sensitizes cells to Fas-mediated apoptosis via down-regulation of the anti-apoptotic proteins, MCL-1 and Cflip short.

    Directory of Open Access Journals (Sweden)

    Song Chen

    Full Text Available FLASH (FLICE-associated huge protein or CASP8AP2 is a large multifunctional protein that is involved in many cellular processes associated with cell death and survival. It has been reported to promote apoptosis, but we show here that depletion of FLASH in HT1080 cells by siRNA interference can also accelerate the process. As shown previously, depletion of FLASH halts growth by down-regulating histone biosynthesis and arrests the cell cycle in S-phase. FLASH knockdown followed by stimulating the cells with Fas ligand or anti-Fas antibodies was found to be associated with a more rapid cleavage of PARP, accelerated activation of caspase-8 and the executioner caspase-3 and rapid progression to cellular disintegration. As is the case for most anti-apoptotic proteins, FLASH was degraded soon after the onset of apoptosis. Depletion of FLASH also resulted in the reduced intracellular levels of the anti-apoptotic proteins, MCL-1 and the short isoform of cFLIP. FLASH knockdown in HT1080 mutant cells defective in p53 did not significantly accelerate Fas mediated apoptosis indicating that the effect was dependent on functional p53. Collectively, these results suggest that under some circumstances, FLASH suppresses apoptosis.

  1. Esculetin induces apoptosis in human gastric cancer cells through a cyclophilin D-mediated mitochondrial permeability transition pore associated with ROS.

    Science.gov (United States)

    Pan, Hui; Wang, Bao-Hui; Lv, Wang; Jiang, Yan; He, Lei

    2015-12-05

    Esculetin is a coumarin derivative from natural plants that has been commonly used as a folk medicine and has been reported to have beneficial pharmacological and biochemical activities; however, the mechanism by which esculetin prevents human gastric cancer cell growth is still largely unknown. In this study, we investigated the effect of esculetin on human gastric cancer cells and explored the cell death mechanism. Our data indicated that esculetin inhibited the growth of human gastric cancer cells in a dose- and time-dependent manner and apoptosis was the main cause of decreased cell viability in esculetin-treated cells. Additionally, esculetin treatment increased the activity of caspase-9 and caspase-3, and resulted in the appearance of the PARP cleavage product; and esculetin-induced cell death and apoptosis was decreased by pretreatment with CsA and NAC, but not BA; these results demonstrate that esculetin induced apoptosis via the caspase-dependent mitochondrial pathway in human gastric cancer cells in which cyclophilin D mediated the cytotoxic action by triggering the opening of the mitochondrial permeability transition pore; and the generation of ROS not only was a consequence of mitochondrial dysfunction, but also triggered esculetin-induced apoptosis. These results reveal a novel mechanism of esculetin on gastric cancer cells and suggest that esculetin could be a novel agent in the treatment of gastric cancer. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  2. Fluorochloridone induces primary cultured Sertoli cells apoptosis: Involvement of ROS and intracellular calcium ions-mediated ERK1/2 activation.

    Science.gov (United States)

    Liu, Luqing; Chang, Xiuli; Zhang, Yubin; Wu, Chunhua; Li, Rui; Tang, Liming; Zhou, Zhijun

    2018-03-01

    Fluorochloridone (FLC) is a widely used pyrrolidone selective herbicide and reported to induce testis injuries in male rats, but the underlying mechanism is largely unknown. In the present study, primary-cultured Sertoli cells were exposed to FLC at the concentration of 0-10.00μM to study the mechanism of FLC-induced apoptosis. The roles of ROS, intracellular calcium, endoplasmic reticulum (ER), and ERK1/2 were looked at with ROS scavenger N-acetyl-cysteine (NAC), intracellular calcium chelator BAPTA-AM, ER calcium depleting agent thapsigargin (TG), and ERK1/2 inhibitor U0126, respectively. FLC induced dose-dependent apoptosis increase as well as the elevation in levels of ROS, intracellular calcium, and ERK1/2 activation. FLC treatment led to constantly increasing apoptotic rates and ERK1/2 activation over time, while inversed-V shaped change tendencies of ROS and intracellular calcium levels were observed. FLC-induced ROS generation disrupted the intracellular calcium homeostasis by attacking the ER, and the elevated intracellular calcium levels resulted in ERK1/2 over-phosphorylation and consequently promoted Sertoli cell apoptosis. Taken together, ROS and intracellular calcium-mediated ERK1/2 activation led to FLC-induced Sertoli cell apoptosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. 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

  4. Chinese Herbal Formula, Modified Danggui Buxue Tang, Attenuates Apoptosis of Hematopoietic Stem Cells in Immune-Mediated Aplastic Anemia Mouse Model

    Directory of Open Access Journals (Sweden)

    Jingwei Zhou

    2017-01-01

    Full Text Available A derivative formula, DGBX, which is composed of three herbs (Radix astragali, Radix Angelicae sinensis, and Coptis chinensis Franch, is derived from a famous Chinese herbal formula, Danggui Buxue Tang (DBT (Radix astragali and Radix Angelicae sinensis. We aimed to investigate the effects of DGBX on the regulation of the balance between proliferation and apoptosis of hematopoietic stem cells (HSCs due to the aberrant immune response in a mouse model of aplastic anemia (AA. Cyclosporine (CsA, an immunosuppressor, was used as the positive control. Our results indicated that DGBX could downregulate the production of IFNγ in bone marrow cells by interfering with the binding between SLAM and SAP and the expressions of Fyn and T-bet. This herbal formula can also inhibit the activation of Fas-mediated apoptosis, interferon regulatory factor-1-induced JAK/Stat, and eukaryotic initiation factor 2 signaling pathways and thereby induce proliferation and attenuate apoptosis of HSCs. In conclusion, DGBX can relieve the immune-mediated destruction of HSCs, repair hematopoietic failure, and recover the hematopoietic function of HSCs in hematogenesis. Therefore, DGBX can be used in traditional medicine against AA as a complementary and alternative immunosuppressive therapeutic formula.

  5. Estrogen-Related Receptor Alpha Confers Methotrexate Resistance via Attenuation of Reactive Oxygen Species Production and P53 Mediated Apoptosis in Osteosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Peng Chen

    2014-01-01

    Full Text Available Osteosarcoma (OS is a malignant tumor mainly occurring in children and adolescents. Methotrexate (MTX, a chemotherapy agent, is widely used in treating OS. However, treatment failures are common due to acquired chemoresistance, for which the underlying molecular mechanisms are still unclear. In this study, we report that overexpression of estrogen-related receptor alpha (ERRα, an orphan nuclear receptor, promoted cell survival and blocked MTX-induced cell death in U2OS cells. We showed that MTX induced ROS production in MTX-sensitive U2OS cells while ERRα effectively blocked the ROS production and ROS associated cell apoptosis. Our further studies demonstrated that ERRα suppressed ROS induction of tumor suppressor P53 and its target genes NOXA and XAF1 which are mediators of P53-dependent apoptosis. In conclusion, this study demonstrated that ERRα plays an important role in the development of MTX resistance through blocking MTX-induced ROS production and attenuating the activation of p53 mediated apoptosis signaling pathway, and points to ERRα as a novel target for improving osteosarcoma therapy.

  6. Cisplatin Induces Apoptosis Through the Endoplasmic Reticulum-mediated, Calpain 1 Pathway in Triple-negative Breast Cancer Cells.

    Science.gov (United States)

    Al-Bahlani, Shadia M; Al-Bulushi, Khadija H; Al-Alawi, Zaina M; Al-Abri, Nadia Y; Al-Hadidi, Zuweina R; Al-Rawahi, Shaikha S

    2017-06-01

    Breast cancer is the most common cancer in women worldwide. Triple-negative breast cancer (TNBC) is an aggressive type that can be treated using platinum-based chemotherapy such as cisplatin (cis-diamminedichloroplatinum II). Although the calpain protein is essential in many cellular processes, including apoptosis, cell signaling, and proliferation, its role in cisplatin-induced apoptosis in TNBC cells is not fully understood. The present study assessed calpain 1-dependent, cisplatin-induced apoptosis in TNBC cells. MDA-MB231 cells were treated with different concentrations of cisplatin (0, 20, and 40 μM). The cisplatin deposit and its effect on endoplasmic reticulum and, subsequently, calcium release were detected using transmission electron microscopy and Von Koss staining, respectively. Calpain 1 messenger RNA, protein content, and apoptosis was measured using reverse transcriptase-polymerase chain reaction, Western blotting, and Hoechst stain, respectively. In addition, calpain modulation, by either activation or inhibition, and its effect on cisplatin-induced apoptosis were assessed. Our results showed that cisplatin induced endoplasmic reticulum stress, indicated by an increase in calcium staining and protein expression of glucose-regulated protein 78 and calmodulin, followed by cleavage of α-fodrin and caspase-12 and, eventually, apoptosis. Cyclopiazonic acid showed a similar effect and enhanced the sensitivity of these cells to cisplatin treatment. In contrast, calpain 1 inhibition by both specific small interfering RNA and exogenous inhibitor (calpeptin) attenuated cisplatin-induced apoptosis in these cells. Altogether, these findings suggest, for the first time, that calpain 1 activation by endoplasmic reticulum plays an essential role in sensitizing TNBC cells to cisplatin-induced apoptosis. This finding will allow exploration of new insights for the treatment of TNBC by overcoming its resistance to apoptosis. Copyright © 2016 Elsevier Inc. All

  7. Ubiquitination in apoptosis signaling

    NARCIS (Netherlands)

    van de Kooij, L.W.

    2014-01-01

    The work described in this thesis focuses on ubiquitination and protein degradation, with an emphasis on how these processes regulate apoptosis signaling. More specifically, our aims were: 1. To increase the understanding of ubiquitin-mediated regulation of apoptosis signaling. 2. To identify the E3

  8. Siderocalin/Lcn2/NGAL/24p3 does not drive apoptosis through gentisic acid mediated iron withdrawal in hematopoietic cell lines.

    Directory of Open Access Journals (Sweden)

    Colin Correnti

    Full Text Available Siderocalin (also lipocalin 2, NGAL or 24p3 binds iron as complexes with specific siderophores, which are low molecular weight, ferric ion-specific chelators. In innate immunity, siderocalin slows the growth of infecting bacteria by sequestering bacterial ferric siderophores. Siderocalin also binds simple catechols, which can serve as siderophores in the damaged urinary tract. Siderocalin has also been proposed to alter cellular iron trafficking, for instance, driving apoptosis through iron efflux via BOCT. An endogenous siderophore composed of gentisic acid (2,5-dihydroxybenzoic acid substituents was proposed to mediate cellular efflux. However, binding studies reported herein contradict the proposal that gentisic acid forms high-affinity ternary complexes with siderocalin and iron, or that gentisic acid can serve as an endogenous siderophore at neutral pH. We also demonstrate that siderocalin does not induce cellular iron efflux or stimulate apoptosis, questioning the role siderocalin plays in modulating iron metabolism.

  9. SIRT2-mediated FOXO3a deacetylation drives its nuclear translocation triggering FasL-induced cell apoptosis during renal ischemia reperfusion.

    Science.gov (United States)

    Wang, Yan; Mu, Yu; Zhou, Xiaorui; Ji, Huaixue; Gao, Xing; Cai, Wen Wen; Guan, Qiuhua; Xu, Tie

    2017-04-01

    We have found that Fas/FasL-mediated "extrinsic" pathway promoted cell apoptosis induced by renal ischemic injury. This study is to elucidate the upstream mechanism regulating FasL-induced extrinsic pathway during renal ischemia/reperfusion. Results demonstrated that when SIRT2 was activated by renal ischemia/reperfusion, activated SIRT2 could bind to and deacetylate FOXO3a, promoting FOXO3a nuclear translocation which resulted in an increase of nuclear FOXO3a along with FasL expression and activation of caspase8 and caspase3, triggering cell apoptosis during renal ischemia/reperfusion. The administration of SIRT2 inhibitor AGK2 prior to renal ischemia decreased significantly the number of apoptotic renal tubular cells and alleviated ultrastructure injury. These results indicate that inhibition of FOXO3a deacetylation might be a promising therapeutic approach for renal ischemia /reperfusion injury.

  10. High CDK6 protects cells from fulvestrant-mediated apoptosis and is a predictor of resistance to fulvestrant in estrogen receptor-positive metastatic breast cancer

    DEFF Research Database (Denmark)

    Alves, Carla Maria Lourenco; Elias, Daniel; Lyng, Maria B

    2016-01-01

    in cell proliferation, apoptosis and kinase activity. Furthermore, we evaluated CDK6 expression in metastatic samples from breast cancer patients treated or not with fulvestrant. RESULTS: We found increased expression of CDK6 in two fulvestrant-resistant cell models vs. sensitive cells. Reduction of CDK6...... expression impaired fulvestrant-resistant cell growth and induced apoptosis. Treatment with palbociclib re-sensitized fulvestrant-resistant cells to fulvestrant through alteration of retinoblastoma protein phosphorylation. High CDK6 levels in metastatic samples from two independent cohorts of breast cancer.......511, respectively). CONCLUSIONS: Our results indicate that upregulation of CDK6 may be an important mechanism in overcoming fulvestrant-mediated growth inhibition in breast cancer cells. Patients with advanced ER+ breast cancer exhibiting high CDK6 expression in the metastatic lesions show shorter PFS upon...

  11. Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells

    DEFF Research Database (Denmark)

    Zheng, Lin; Terman, Alexei; Hallbeck, Martin

    2011-01-01

    and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production......, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase...... and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration....

  12. MOAP-1 Mediates Fas-Induced Apoptosis in Liver by Facilitating tBid Recruitment to Mitochondria

    OpenAIRE

    Chong Teik Tan; Qi-Ling Zhou; Yu-Chin Su; Nai Yang Fu; Hao-Chun Chang; Ran N. Tao; Sunil K. Sukumaran; Shairaz Baksh; Yee-Joo Tan; Kanaga Sabapathy; Chun-Dong Yu; Victor C. Yu

    2016-01-01

    Fas apoptotic signaling regulates diverse physiological processes. Acute activation of Fas signaling triggers massive apoptosis in liver. Upon Fas receptor stimulation, the BH3-only protein Bid is cleaved into the active form, tBid. Subsequent tBid recruitment to mitochondria, which is facilitated by its receptor MTCH2 at the outer mitochondrial membrane (OMM), is a critical step for commitment to apoptosis via the effector proteins Bax or Bak. MOAP-1 is a Bax-binding protein enriched at the ...

  13. Chronic nandrolone administration promotes oxidative stress, induction of pro-inflammatory cytokine and TNF-α mediated apoptosis in the kidneys of CD1 treated mice

    Energy Technology Data Exchange (ETDEWEB)

    Riezzo, Irene; Turillazzi, Emanuela; Bello, Stefania; Cantatore, Santina [Department of Forensic Pathology, University of Foggia, Foggia (Italy); Cerretani, Daniela [Pharmacology Unit, Department of Medicine, Surgery and Neuroscience, University of Siena, Siena (Italy); Di Paolo, Marco [Department of Forensic Pathology, University of Pisa, Pisa (Italy); Fiaschi, Anna Ida [Pharmacology Unit, Department of Medicine, Surgery and Neuroscience, University of Siena, Siena (Italy); Frati, Paola [Department of Anatomical, Histological, Forensic and Orthopaedic Sciences, University of Rome Sapienza, Viale Regina Elena 336, 00161 Rome (Italy); Neri, Margherita [Department of Forensic Pathology, University of Foggia, Foggia (Italy); Pedretti, Monica [Department of Forensic Pathology, University of Pisa, Pisa (Italy); Fineschi, Vittorio, E-mail: vfinesc@tin.it [Department of Anatomical, Histological, Forensic and Orthopaedic Sciences, University of Rome Sapienza, Viale Regina Elena 336, 00161 Rome (Italy)

    2014-10-01

    Nandrolone decanoate administration and strenuous exercise increase the extent of renal damage in response to renal toxic injury. We studied the role played by oxidative stress in the apoptotic response caused by nandrolone decanoate in the kidneys of strength-trained male CD1 mice. To measure cytosolic enzyme activity, glutathione peroxidase (GPx), glutathione reductase (GR) and malondialdehyde (MDA) were determined after nandrolone treatment. An immunohistochemical study and Western blot analysis were performed to evaluate cell apoptosis and to measure the effects of renal expression of inflammatory mediators (IL-1β, TNF-α) on the induction of apoptosis (HSP90, TUNEL). Dose-related oxidative damage in the kidneys of treated mice is shown by an increase in MDA levels and by a reduction of antioxidant enzyme GR and GPx activities, resulting in the kidney's reduced radical scavenging ability. Renal specimens of the treated group showed relevant glomeruli alterations and increased immunostaining and protein expressions, which manifested significant focal segmental glomerulosclerosis. The induction of proinflammatory cytokine expression levels was confirmed by Western blot analysis. Long-term administration of nandrolone promotes oxidative injury in the mouse kidneys. TNF-α mediated injury due to nandrolone in renal cells appears to play a role in the activation of both the intrinsic and extrinsic apoptosis pathways. - Highlights: • We analyze abuse of nandrolone decanoate in strength-trained male CD1 mice. • Nandrolone decanoate administration increases oxidative stress. • Increased cytokine expressions were observed. • Renal apoptosis was described. • Long-term administration of nandrolone promotes oxidative injury in mice kidney.

  14. Taurine Pretreatment Prevents Isoflurane-Induced Cognitive Impairment by Inhibiting ER Stress-Mediated Activation of Apoptosis Pathways in the Hippocampus in Aged Rats.

    Science.gov (United States)

    Zhang, Yanan; Li, Dongliang; Li, Haiou; Hou, Dailiang; Hou, Jingdong

    2016-10-01

    Isoflurane, a commonly used inhalation anesthetic, may induce neurocognitive deficits, especially in elderly patients after surgery. Recent study demonstrated that isoflurane caused endoplasmic reticulum (ER) stress and subsequent neuronal apoptosis in the brain, contributing to cognitive deficits. Taurine, a major intracellular free amino acid, has been shown to inhibit ER stress and neuronal apoptosis in several neurological disorders. Here, we examined whether taurine can prevent isoflurane-induced ER stress and cognitive impairment in aged rats. Thirty minutes prior to a 4-h 1.3 % isoflurane exposure, aged rats were treated with vehicle or taurine at low, middle and high doses. Aged rats without any treatment served as control. The brains were harvested 6 h after isoflurane exposure for molecular measurements, and behavioral study was performed 2 weeks later. Compared with control, isoflurane increased expression of hippocampal ER stress biomarkers including glucose-regulated protein 78, phosphorylated (P-) inositol-requiring enzyme 1, P-eukaryotic initiation factor 2-α (EIF2α), activating transcription factor 4 (ATF-4), cleaved ATF-6 and C/EBP homologous protein, along with activation of apoptosis pathways as indicated by decreased B cell lymphoma 2 (BCL-2)/BCL2-associated X protein, increased expressions of cytochrome-c and cleaved caspase-3. Taurine pretreatment dose-dependently inhibited isoflurane-induced increase in expression of ER stress biomarkers except for P-EIF2α and ATF-4, and reversed isoflurane-induced changes in apoptosis-related proteins. Moreover, isoflurane caused spatial working memory deficits in aged rats, which were prevented by taurine pretreatment. The results indicate that taurine pretreatment prevents anesthetic isoflurane-induced cognitive impairment by inhibiting ER stress-mediated activation of apoptosis pathways in the hippocampus in aged rats.

  15. Myricetin inhibits Akt survival signaling and induces Bad-mediated apoptosis in a low dose ultraviolet (UV)-B-irradiated HaCaT human immortalized keratinocytes.

    Science.gov (United States)

    Kim, Wanyeon; Yang, Hee Jung; Youn, HyeSook; Yun, Young Ju; Seong, Ki Moon; Youn, BuHyun

    2010-01-01

    Deregulation of cell survival pathways and resistance to apoptosis are generally accepted as crucial aspects of tumorigenesis. As in many tumors, increasing occurrence of human skin cancer and other conflicting effects of solar ultraviolet (UV) radiation enhance the demand for novel chemoprevention agents. Myricetin, a naturally occurring phytochemical, is potent in anti-cancer promoting activity and affords to the chemopreventive potential of several healthy-foods, including fruits and vegetables. We demonstrate here that myricetin inhibits Akt activity to induce apoptosis in a low dose ('repairable dose') UVB-irradiated keratinocytes. Treatment of UVB-irradiated HaCaT cells with an apoptosis-inducing concentration of myricetin (20 microM) resulted in a decrease in phosphorylation of Akt leading to inhibition of its kinase activity. Myricetin treatment also caused a decrease in phosphorylation of Bad (a pro-apoptotic protein), a direct target of Akt in signaling pathway. Interaction between Bad and 14-3-3beta was reduced markedly in UVB-irradiated cells upon a treatment with myricetin. Comparable to these results, myricetin treatment promoted mitochondrial translocation of Bad, loss of the mitochondrial membrane potential, and release of the mitochondrial apoptotic proteins including cytochrome c, Smac, and AIF. Ectopic expression of constitutively active Akt granted statistically significant protection against myricetin-induced apoptosis. In addition, myricetin-induced apoptosis in UVB-irradiated cells was notably attenuated in the presence of caspase inhibitors. Together, these results indicate that myricetin might take on potent chemopreventive activity by inhibiting the Akt-mediated survival signaling axis in UVB-induced skin carcinogenesis.

  16. TNFα cooperates with IFN-γ to repress Bcl-xL expression to sensitize metastatic colon carcinoma cells to TRAIL-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Feiyan Liu

    Full Text Available BACKGROUND: TNF-related apoptosis-inducing ligand (TRAIL is an immune effector molecule that functions as a selective anti-tumor agent. However, tumor cells, especially metastatic tumor cells often exhibit a TRAIL-resistant phenotype, which is currently a major impediment in TRAIL therapy. The aim of this study is to investigate the synergistic effect of TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: The efficacy and underlying molecular mechanism of cooperation between TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis were examined. The functional significance of TNFα- and IFN-γ-producing T lymphocyte immunotherapy in combination with TRAIL therapy in suppression of colon carcinoma metastasis was determined in an experimental metastasis mouse model. We observed that TNFα or IFN-γ alone exhibits minimal sensitization effects, but effectively sensitized metastatic colon carcinoma cells to TRAIL-induced apoptosis when used in combination. TNFα and IFN-γ cooperate to repress Bcl-xL expression, whereas TNFα represses Survivin expression in the metastatic colon carcinoma cells. Silencing Bcl-xL expression significantly increased the metastatic colon carcinoma cell sensitivity to TRAIL-induced apoptosis. Conversely, overexpression of Bcl-xL significantly decreased the tumor cell sensitivity to TRAIL-induced apoptosis. Furthermore, TNFα and IFN-γ also synergistically enhanced TRAIL-induced caspase-8 activation. TNFα and IFN-γ was up-regulated in activated primary and tumor-specific T cells. TRAIL was expressed in tumor-infiltrating immune cells in vivo, and in tumor-specific cytotoxic T lymphocytes (CTL ex vivo. Consequently, TRAIL therapy in combination with TNFα/IFN-γ-producing CTL adoptive transfer immunotherapy effectively suppressed colon carcinoma metastasis in vivo. CONCLUSIONS/SIGNIFICANCE: TNFα and IFN

  17. Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

    International Nuclear Information System (INIS)

    Fu, Wen; Gorodeski, George I; McCormick, Tom; Qi, Xiaoping; Luo, Liping; Zhou, Lingyin; Li, Xin; Wang, Bing-Cheng; Gibbons, Heidi E; Abdul-Karim, Fadi W

    2009-01-01

    augmented calcium influx were depended on the expression of the P2X 7 receptor, while the BzATP-induced apoptosis depended on calcium influx. (h) The BzATP-induced apoptosis could be blocked by co-treatment with inhibitors of caspase-9 and caspase-3, but not of caspase-8. (a) P2X 7 -dependent apoptosis is an important mechanism that controls the development and progression of epidermal neoplasia in the mouse. (b) The P2X 7 -dependent apoptosis is mediated by calcium influx via P2X 7 pores, and involves the caspase-9 (mitochondrial) pathway. (c) The diminished pro-apoptotic effect of BzATP in mouse cancer keratinocytes is possibly the result of low expression of the P2X 7 receptor. (d) Activation of P2X 7 -dependent apoptosis, e.g. with BzATP could be a novel chemotherapeutic growth-preventive modality for papillomas and epithelial cancers in vivo

  18. Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

    Directory of Open Access Journals (Sweden)

    Fu Wen

    2009-04-01

    BzATP. (g Pore formation and the augmented calcium influx were depended on the expression of the P2X7 receptor, while the BzATP-induced apoptosis depended on calcium influx. (h The BzATP-induced apoptosis could be blocked by co-treatment with inhibitors of caspase-9 and caspase-3, but not of caspase-8. Conclusion (a P2X7-dependent apoptosis is an important mechanism that controls the development and progression of epidermal neoplasia in the mouse. (b The P2X7-dependent apoptosis is mediated by calcium influx via P2X7 pores, and involves the caspase-9 (mitochondrial pathway. (c The diminished pro-apoptotic effect of BzATP in mouse cancer keratinocytes is possibly the result of low expression of the P2X7 receptor. (d Activation of P2X7-dependent apoptosis, e.g. with BzATP could be a novel chemotherapeutic growth-preventive modality for papillomas and epithelial cancers in vivo.

  19. Post operative infection and sepsis in humans is associated with deficient gene expression of gammac cytokines and their apoptosis mediators.

    LENUS (Irish Health Repository)

    White, Mary

    2011-06-28

    Abstract Introduction Lymphocyte homeostasis is dependent on the γc cytokines. We hypothesised that sepsis in humans is associated with differential gene expression of the γc cytokines and their associated apoptosis mediators. Methods The study population consisted of a total of 60 patients with severe sepsis, 15 with gram negative bacteraemia, 10 healthy controls and 60 patients undergoing elective lung resection surgery. Pneumonia was diagnosed by CDC NNIC criteria. Gene expression in peripheral blood leukocytes (PBLs) of interleukin (IL)-2, 7, 15 and interferon (IFN)-γ, Bax, Bim, Bcl-2 was determined by qRT-PCR and IL-2 and IL-7 serum protein levels by ELISA. Gene expression of IL-2, 7 and IFN-γ was measured in peripheral blood leukocytes (PBL), cultured in the presence of lipopolysacharide (LPS) and CD3 binding antibody (CD3ab) Results IL-2 gene expression was lower in the bacteraemia group compared with controls, and lower still in the sepsis group (P < 0.0001). IL-7 gene expression was similar in controls and bacteraemia, but lower in sepsis (P < 0.0001). IL-15 gene expression was similar in the three groups. Bcl-2 gene expression was less (P < 0.0001) and Bim gene expression was greater (P = 0.0003) in severe sepsis compared to bacteraemic and healthy controls. Bax gene expression was similar in the three groups. In lung resection surgery patients, post-operative pneumonia was associated with a perioperative decrease in IL-2 mRNA (P < 0.0001) and IL-7 mRNA (P = 0.003). IL-2 protein levels were reduced in sepsis and bacteraemia compared to controls (P = 0.02) but similar in pneumonia and non-pneumonia groups. IL-7 protein levels were similar in all groups. In cultured PBLs, IFN-γ gene expression was decreased in response to LPS and increased in response to CD3ab with sepsis: IL-7 gene expression increased in response to LPS in controls and to CD3ab with sepsis; Bcl-2 gene expression decreased in response to combined CD3ab and IL-2 with sepsis

  20. Study on the effect of reactive oxygen species-mediated oxidative stress on the activation of mitochondrial apoptosis and the tenderness of yak meat.

    Science.gov (United States)

    Wang, Lin-Lin; Yu, Qun-Li; Han, Ling; Ma, Xiu-Li; Song, Ren-De; Zhao, Suo-Nan; Zhang, Wen-Hua

    2018-04-01

    This study investigated the effect of reactive oxygen species-mediated oxidative stress on activation of mitochondrial apoptosis and tenderness of yak meat during postmortem ageing. Oxidative stress degree, Ca 2+ levels, membrane permeability transition pore opening, mitochondrial membrane potential, apoptotic factors and the shear force were examined. Results showed that the ROS generated by H 2 O 2 significantly increased mitochondrial oxidative stress by decreasing the activities of superoxide dismutase, catalase and glutathione peroxidase, and increasing lipid peroxidation. Furthermore, oxidative stress enhanced Ca 2+ production and cytochrome c release, changed the levels of Bcl-2 family proteins and activated caspase-9 and -3 activities. Ultimately, oxidative stress increased the apoptosis rate and tenderness of yak meat. These observations confirmed that ROS-mediated oxidative stress participates in the activation of the apoptotic cascade reaction involving Ca 2+ and Bcl-2 family proteins. The results further suggested that ROS-mediated oxidative stress plays a significant role in meat tenderization through the mitochondrial apoptotic pathway. Copyright © 2017. Published by Elsevier Ltd.

  1. p21(WAF1/CIP1 upregulation through the stress granule-associated protein CUGBP1 confers resistance to bortezomib-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Cristina Gareau

    Full Text Available p21(WAF1/CIP1 is a well known cyclin-dependent kinase inhibitor induced by various stress stimuli. Depending on the stress applied, p21 upregulation can either promote apoptosis or prevent against apoptotic injury. The stress-mediated induction of p21 involves not only its transcriptional activation but also its posttranscriptional regulation, mainly through stabilization of p21 mRNA levels. We have previously reported that the proteasome inhibitor MG132 induces the stabilization of p21 mRNA, which correlates with the formation of cytoplasmic RNA stress granules. The mechanism underlying p21 mRNA stabilization, however, remains unknown.We identified the stress granules component CUGBP1 as a factor required for p21 mRNA stabilization following treatment with bortezomib ( =  PS-341/Velcade. This peptide boronate inhibitor of the 26S proteasome is very efficient for the treatment of myelomas and other hematological tumors. However, solid tumors are sometimes refractory to bortezomib treatment. We found that depleting CUGBP1 in cancer cells prevents bortezomib-mediated p21 upregulation. FISH experiments combined to mRNA stability assays show that this effect is largely due to a mistargeting of p21 mRNA in stress granules leading to its degradation. Altering the expression of p21 itself, either by depleting CUGBP1 or p21, promotes bortezomib-mediated apoptosis.We propose that one key mechanism by which apoptosis is inhibited upon treatment with chemotherapeutic drugs might involve upregulation of the p21 protein through CUGBP1.

  2. Dietary flavonoid fisetin regulates aluminium chloride-induced neuronal apoptosis in cortex and hippocampus of mice brain.

    Science.gov (United States)

    Prakash, Dharmalingam; Sudhandiran, Ganapasam

    2015-12-01

    Dietary flavonoids have been suggested to promote brain health by protecting brain parenchymal cells. Recently, understanding the possible mechanism underlying neuroprotective efficacy of flavonoids is of great interest. Given that fisetin exerts neuroprotection, we have examined the mechanisms underlying fisetin in regulating Aβ aggregation and neuronal apoptosis induced by aluminium chloride (AlCl3) administration in vivo. Male Swiss albino mice were induced orally with AlCl3 (200 mg/kg. b.wt./day/8 weeks). Fisetin (15 mg/Kg. b.wt. orally) was administered for 4 weeks before AlCl3-induction and administered simultaneously for 8 weeks during AlCl3-induction. We found aggregation of Amyloid beta (Aβ 40-42), elevated expressions of Apoptosis stimulating kinase (ASK-1), p-JNK (c-Jun N-terminal Kinase), p53, cytochrome c, caspases-9 and 3, with altered Bax/Bcl-2 ratio in favour of apoptosis in cortex and hippocampus of AlCl3-administered mice. Furthermore, TUNEL and fluoro-jade C staining demonstrate neurodegeneration in cortex and hippocampus. Notably, treatment with fisetin significantly (P<0.05) reduced Aβ aggregation, ASK-1, p-JNK, p53, cytochrome c, caspase-9 and 3 protein expressions and modulated Bax/Bcl-2 ratio. TUNEL-positive and fluoro-jade C stained cells were also significantly reduced upon fisetin treatment. We have identified the involvement of fisetin in regulating ASK-1 and p-JNK as possible mediator of Aβ aggregation and subsequent neuronal apoptosis during AlCl3-induced neurodegeneration. These findings define the possibility that fisetin may slow or prevent neurodegneration and can be utilised as neuroprotective agent against Alzheimer's and Parkinson's disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. The majority of lamina propria CD4(+) T-cells from scid mice with colitis undergo Fas-mediated apoptosis in vivo

    DEFF Research Database (Denmark)

    Bregenholt, S; Petersen, T R; Claesson, Mogens Helweg

    2001-01-01

    We have previously shown that adoptively transferred CD4(+) T-cells mediate an chronic colitis in severe combined immune deficient (scid) mice. Colitis is accompanied by activation and apoptosis of Fas ligand and TNF-alpha expressing CD4(+) T-cells in the diseased colonic lamina propria (Eur. J...... CD4(+) T-cells in this model of murine chronic colitis, and suggest an important role for the Fas-FasL system in the maintenance of homeostasis of locally proliferating T-cells....

  4. Signaling Rho-kinase mediates inflammation and apoptosis in T cells and renal tubules in cisplatin nephrotoxicity.

    Science.gov (United States)

    Nozaki, Yuji; Kinoshita, Koji; Hino, Shoichi; Yano, Tomohiro; Niki, Kaoru; Hirooka, Yasuaki; Kishimoto, Kazuya; Funauchi, Masanori; Matsumura, Itaru

    2015-04-15

    Nephrotoxicity is a frequent complication of cisplatin-induced chemotherapy, in which T cells are known to promote acute kidney injury (AKI). Apoptosis and necrosis of tubules and inflammatory events also contribute to the nephrotoxicity. A delineation of the mechanisms that underlie the inappropriate renal and tubular inflammation can thus provide important insights into potential therapies for cisplatin-induced AKI. Rho-kinases are known to act as molecular switches controlling several critical cellular functions, including cell migration, cytokine production, and apoptosis. Here, we show that the Rho-kinase inhibitor fasudil attenuated cisplatin nephrotoxicity, resulting in less histological damage, improved renal function, and the infiltration of fewer leukocytes into the kidney. Renal nuclear factor-κB activation and apoptosis were reduced, and the expressions of proinflammatory renal cytokine and chemokine mRNA were decreased. Urinary and renal kidney injury molecule-1 (Kim-1) expression was also reduced, a finding that is consistent with diminished kidney injury. In the current study, we also showed that fasudil could be protective of the impaired tubules. In vitro, fasudil reduced the apoptosis (annexin-V+PI cells) and cytokine production (tumor necrosis factor+ cells) in T cells and the apoptosis (annexin-V+PI cells) and tubular damage (Kim-1+ cells) in proximal tubular cells by flow cytometric analysis. As Rho-kinase plays an important role in promoting cisplatin nephrotoxicity, inhibiting Rho-kinase may be a therapeutic strategy for preventing cisplatin-induced AKI. Copyright © 2015 the American Physiological Society.

  5. Propensity of crocin to offset Vipera russelli venom induced oxidative stress mediated neutrophil apoptosis: a biochemical insight.

    Science.gov (United States)

    Santhosh, M Sebastin; Sundaram, M Shanmuga; Sunitha, K; Jnaneshwari, S; Devaraja, S; Kemparaju, K; Girish, K S

    2016-01-01

    Viper envenomation results in inflammation at the bitten site as well as target organs. Neutrophils and other polymorphonuclear leukocytes execute inflammation resolving mechanism and will undergo apoptosis after completing the task. However, the target specific toxins induce neutrophil apoptosis at the bitten site and in circulation prior to their function, thus reducing their number. Circulating activated neutrophils are major source of inflammatory cytokines and leakage of reactive oxygen species (ROS)/other toxic intermediates resulting in aggravation of inflammatory response at the bitten/target site. Therefore, neutralization of venom induced neutrophil apoptosis reduces inflammation besides increasing the functional neutrophil population. Therefore, the present study investigates the venom induced perturbances in isolated human neutrophils and its neutralization by crocin (Crocus sativus) a potent antioxidant carotenoid. Human neutrophils on treatment with venom resulted in altered ROS generation, intracellular Ca(2+) mobilization, mitochondrial membrane depolarization, cyt-c translocation, caspase activation, phosphatidylserine externalization and DNA damage. On the other hand significant protection against oxidative stress and apoptosis were evidenced in crocin pre-treated groups. In conclusion the viper venom induces neutrophil apoptosis and results in aggravation of inflammation and tissue damage. The present study demands the necessity of an auxiliary therapy in addition to antivenin therapy to treat secondary/overlooked complications of envenomation.

  6. BH3-only protein Bim is upregulated and mediates the apoptosis of cardiomyocytes under glucose and oxygen-deprivation conditions.

    Science.gov (United States)

    Huang, Chahua; Li, Juxiang; Hong, Kui; Xia, Zhen; Xu, Yan; Cheng, Xiaoshu

    2015-03-01

    Bim is a potent pro-apoptotic BH3-only Bcl-2 member. However, the expression of Bim and its role in cardiac injury induced by ischemia remain unclear. H9c2 cells were subjected to a glucose and oxygen-deprived (GOD) condition in vitro, mimicking ischemia environment in vivo. GOD treatment augmented the expression of Bim and induced the apoptosis of H9c2 cells. Silencing of Bim by RNAi significantly attenuated GOD-induced cytotoxicity, suppressed mitochondrial membrane potential △Ψm loss, inhibited caspase 3 activation and reduced apoptosis. The data demonstrate that Bim is upregulated by GOD in a time-dependent manner in H9c2 cells, and enhances mitochondrial apoptosis dependent on the activation of caspase 3. Silencing of Bim may be a promising therapeutic strategy in ischemia related heart diseases. © 2014 International Federation for Cell Biology.

  7. Cypripedin, a phenanthrenequinone from Dendrobium densiflorum, sensitizes non-small cell lung cancer H460 cells to cisplatin-mediated apoptosis.

    Science.gov (United States)

    Wattanathamsan, Onsurang; Treesuwan, Surassawadee; Sritularak, Boonchoo; Pongrakhananon, Varisa

    2018-03-01

    The life-threatening potential of lung cancer has increased over the years due to its acquisition of chemotherapeutic resistance, especially to cisplatin, a first-line therapy. In response to this development, researchers have turned their attention to several compounds derived from natural origins, including cypripedin (CYP), a phenanthrenequinone substance extracted from Dendrobium densiflorum. The aim of the present study was to investigate the ability of CYP to induce apoptosis and enhance cisplatin-mediated death of human lung cancer NCI-H460 cells using cell viability and apoptosis assays. The induction of apoptosis by CYP was observed at a concentration of > 50 μM with the appearance of morphological changes, including DNA condensation and chromatin fragmentation. Together with, CYP was able to activate caspase-3 and downregulate the anti-apoptotic proteins Bcl-2 and Bcl-xL. Also, a non-cytotoxic dose of CYP synergistically potentiated the effect of cisplatin in non-small cell lung cancer line H460 cells, which clearly exhibited the apoptotic phenotype. Western blot analysis revealed that the underlying mechanism involved the downregulation of anti-apoptotic Bcl-xL, whereas the levels of other apoptotic regulatory proteins were not altered. This study provides interesting information on the potent effect of CYP as a chemotherapeutic sensitizer that could be further developed to improve the clinical outcomes of lung cancer patients.

  8. Addition of exogenous NAD+ prevents mefloquine-induced neuroaxonal and hair cell degeneration through reduction of caspase-3-mediated apoptosis in cochlear organotypic cultures.

    Directory of Open Access Journals (Sweden)

    Dalian Ding

    Full Text Available Mefloquine is widely used for the treatment of malaria. However, this drug is known to induce neurological side effects including depression, anxiety, balance disorder, and sensorineural hearing loss. Yet, there is currently no treatment for these side effects.In this study, we show that the coenzyme NAD(+, known to play a critical role in maintaining the appropriate cellular redox environment, protects cochlear axons and sensory hair cells from mefloquine-induced degeneration in cultured rat cochleae. Mefloquine alone destroyed hair cells and nerve fiber axons in rat cochlear organotypics cultures in a dose-dependent manner, while treatment with NAD(+ protected axons and hair cells from mefloquine-induced degeneration. Furthermore, cochlear organs treated with mefloquine showed increased oxidative stress marker levels, including superoxide and protein carbonyl, and increased apoptosis marker levels, including TUNEL-positive nuclei and caspases-3. Treatment with NAD(+ reduced the levels of these oxidative stress and apoptosis markers.Taken together, our findings suggest that that mefloquine disrupts the cellular redox environment and induces oxidative stress in cochlear hair cells and nerve fibers leading to caspases-3-mediated apoptosis of these structures. Exogenous NAD(+ suppresses mefloquine-induced oxidative stress and prevents the degeneration of cochlear axons and sensory hair cells caused by mefloquine treatment.

  9. Addition of exogenous NAD+ prevents mefloquine-induced neuroaxonal and hair cell degeneration through reduction of caspase-3-mediated apoptosis in cochlear organotypic cultures.

    Science.gov (United States)

    Ding, Dalian; Qi, Weidong; Yu, Dongzhen; Jiang, Haiyan; Han, Chul; Kim, Mi-Jung; Katsuno, Kana; Hsieh, Yun Hua; Miyakawa, Takuya; Salvi, Richard; Tanokura, Masaru; Someya, Shinichi

    2013-01-01

    Mefloquine is widely used for the treatment of malaria. However, this drug is known to induce neurological side effects including depression, anxiety, balance disorder, and sensorineural hearing loss. Yet, there is currently no treatment for these side effects. In this study, we show that the coenzyme NAD(+), known to play a critical role in maintaining the appropriate cellular redox environment, protects cochlear axons and sensory hair cells from mefloquine-induced degeneration in cultured rat cochleae. Mefloquine alone destroyed hair cells and nerve fiber axons in rat cochlear organotypics cultures in a dose-dependent manner, while treatment with NAD(+) protected axons and hair cells from mefloquine-induced degeneration. Furthermore, cochlear organs treated with mefloquine showed increased oxidative stress marker levels, including superoxide and protein carbonyl, and increased apoptosis marker levels, including TUNEL-positive nuclei and caspases-3. Treatment with NAD(+) reduced the levels of these oxidative stress and apoptosis markers. Taken together, our findings suggest that that mefloquine disrupts the cellular redox environment and induces oxidative stress in cochlear hair cells and nerve fibers leading to caspases-3-mediated apoptosis of these structures. Exogenous NAD(+) suppresses mefloquine-induced oxidative stress and prevents the degeneration of cochlear axons and sensory hair cells caused by mefloquine treatment.

  10. Hemagglutinin protease secreted by V. cholerae induced apoptosis in breast cancer cells by ROS mediated intrinsic pathway and regresses tumor growth in mice model.

    Science.gov (United States)

    Ray, Tanusree; Chakrabarti, Monoj Kumar; Pal, Amit

    2016-02-01

    Conventional anticancer therapies are effective but have side effects, so alternative targets are being developed. Bacterial toxins that can kill cells or alter the cellular processes like proliferation, apoptosis and differentiation have been reported for cancer treatment. In this study we have shown antitumor activity of hemagglutinin protease (HAP) secreted by Vibrio cholerae. One µg of HAP showed potent antitumor activity when injected into Ehrlich ascites carcinoma (EAC) tumors in Swiss albino mice. Weekly administration of this dose is able to significantly diminish a large tumor volume within 3 weeks and increases the survival rates of cancerous mice. HAP showed apoptotic activity on EAC and other malignant cells. Increased level of pro-apoptotic p53 with increased ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2 signify that HAP induced apoptogenic signals lead to death of the tumor cells. In vivo and ex vivo studies suggest that mitochondrial dependent intrinsic pathway is responsible for this apoptosis. The level of ROS in malignant cells is reported to be higher than the normal healthy cells. HAP induces oxidative stress and increases the level of ROS in malignant cells which is significantly higher than the normal healthy cells. As a result the malignant cells cross the threshold level of ROS for cell survival faster than normal healthy cells. This mechanism causes HAP mediated apoptosis in malignant cells, but normal cells remain unaltered in the same environment. Our study suggests that HAP may be used as a new candidate drug for cancer therapy.

  11. TGFβ1 induces apoptosis in invasive prostate cancer and bladder cancer cells via Akt-independent, p38 MAPK and JNK/SAPK-mediated activation of caspases

    International Nuclear Information System (INIS)

    Al-Azayzih, Ahmad; Gao, Fei; Goc, Anna; Somanath, Payaningal R.

    2012-01-01

    Highlights: ► TGFβ induced apoptosis in invasive prostate cancer and bladder cancer cells. ► TGFβ inhibited prostate/bladder cancer cell proliferation and colony/foci formation. ► TGFβ induced prostate/bladder cancer cell apoptosis independent of Akt inhibition. ► TGFβ inhibited ERK1/2 phosphorylation in prostate/bladder cancer cells. ► TGFβ induced p38 MAPK and JNK-mediated activation of caspases-9, -8 and -3. -- Abstract: Recent findings indicate that advanced stage cancers shun the tumor suppressive actions of TGFβ and inexplicably utilize the cytokine as a tumor promoter. We investigated the effect of TGFβ1 on the survival and proliferation of invasive prostate (PC3) and bladder (T24) cancer cells. Our study indicated that TGFβ1 decreased cell viability and induced apoptosis in invasive human PC3 and T24 cells via activation of p38 MAPK-JNK-Caspase9/8/3 pathway. Surprisingly, no change in the phosphorylation of pro-survival Akt kinase was observed. We postulate that TGFβ1 pathway may be utilized for specifically targeting urological cancers without inflicting side effects on normal tissues.

  12. Mangosenone F, A Furanoxanthone from Garciana mangostana, Induces Reactive Oxygen Species-Mediated Apoptosis in Lung Cancer Cells and Decreases Xenograft Tumor Growth.

    Science.gov (United States)

    Seo, Kyung Hye; Ryu, Hyung Won; Park, Mi Jin; Park, Ki Hun; Kim, Jin Hyo; Lee, Mi-Ja; Kang, Hyeon Jung; Kim, Sun Lim; Lee, Jin Hwan; Seo, Woo Duck

    2015-11-01

    Mangosenone F (MSF), a natural xanthone, was isolated form Carcinia mangotana, and a few studies have reported its glycosidase inhibitor effect. In this study we investigated the anti lung cancer effect of MSF both in vitro and in vivo. MSF inhibited cancer cell cytotoxicity and induced and induced apoptosis via reactive oxygen species (ROS) generation in NCI-H460. MSF treatment also showed in pronounced release of apoptogenic cytochrome c from the mitochondria to the cytosol, downregulation of Bcl-2 and Bcl-xL, and upregulation of Bax, suggesting that caspase-mediated pathways were involved in MSF-induced apoptosis. ROS activation of the mitogen-activated protein kinase signaling pathway was shown to play a predominant role in the apoptosis mechanism of MSF. Compared with cisplatin treatment, MSF treatment showed significantly increased inhibition of the growth of NCI-H460 cells xenografted in nude mice. Together, these results indicate the potential of MSF as a candidate natural anticancer drug by promoting ROS production. Copyright © 2015 John Wiley & Sons, Ltd.

  13. The induction of apoptosis in HepG-2 cells by ruthenium(II) complexes through an intrinsic ROS-mediated mitochondrial dysfunction pathway.

    Science.gov (United States)

    Zeng, Chuan-Chuan; Lai, Shang-Hai; Yao, Jun-Hua; Zhang, Cheng; Yin, Hui; Li, Wei; Han, Bing-Jie; Liu, Yun-Jun

    2016-10-21

    Four new ruthenium(II) polypyridyl complexes [Ru(N-N)2(dhbn)](ClO4)2 (N-N = dmb: 4,4'-dimethyl-2,2'-bipyridine 1; bpy = 2,2'-bipyridine 2; phen = 1,10-phenanthroline 3; dmp = 2,9-dimethyl-1,10-phenanthroline 4) were synthesized and characterized. The cytotoxicity in vitro of the ligand and complexes toward HepG-2, HeLa, MG-63 and A549 were assayed by MTT method. The IC50 values of the complexes against the above cells range from 17.7 ± 1.1 to 45.1 ± 2.8 μM. The cytotoxic activity of the complexes against HepG-2 cells follows the order of 4 > 2 > 3 > 1. Ligand shows no cytotoxic activity against the selected cell lines. Cellular uptake, apoptosis, comet assay, reactive oxygen species, mitochondrial membrane potential, cell cycle arrest, and the expression of proteins involved in apoptosis pathway induced by the complexes were investigated. The results indicate that complexes 1-4 induce apoptosis in HepG-2 cells through an intrinsic ROS-mediated mitochondrial dysfunction pathway. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  14. Doxorubicin resistance mediated by cytoplasmic macrophage colony-stimulating factor is associated with switch from apoptosis to autophagic cell death in MCF-7 breast cancer cells

    Science.gov (United States)

    Zhang, Mengxia; Zhang, Hailiang; Tang, Fan; Wang, Yuhua; Mo, Zhongcheng; Lei, Xiaoyong

    2016-01-01

    Macrophage colony-stimulating factor is a vital factor in maintaining the biological function of monocyte–macrophage lineage. It is expressed in many tumor tissues and cancer cells. Recent findings indicate that macrophage colony-stimulating factor might contribute to chemoresistance, but the precise mechanisms are unclear. This study was to explore the effect of macrophage colony-stimulating factor on doxorubicin resistance in MCF-7 breast cancer cells and the possible mechanism. In the study, the human breast cancer cells, MCF-7, were transfected with macrophage colony-stimulating factor. We document that cytoplasmic macrophage colony-stimulating factor induces doxorubicin resistance and inhibits apoptosis in MCF-7 cells. Further studies demonstrated that cytoplasmic macrophage colony-stimulating factor-mediated apoptosis inhibition was dependent on the activation of PI3K/Akt/Survivin pathway. More importantly, we found that macrophage colony-stimulating factor-induced autophagic cell death in doxorubicin-treated MCF-7 cells. Taken together, we show for the first time that macrophage colony-stimulating factor-induced doxorubicin resistance is associated with the changes in cell death response with defective apoptosis and promotion of autophagic cell death. PMID:27439542

  15. Prospective clinical biomarkers of caspase-mediated apoptosis associated with neuronal and neurovascular damage following stroke and other severe brain injuries: Implications for chronic neurodegeneration

    Directory of Open Access Journals (Sweden)

    Olena Y Glushakova

    2017-01-01

    Full Text Available Acute brain injuries, including ischemic and hemorrhagic stroke, as well as traumatic brain injury (TBI, are major worldwide health concerns with very limited options for effective diagnosis and treatment. Stroke and TBI pose an increased risk for the development of chronic neurodegenerative diseases, notably chronic traumatic encephalopathy, Alzheimer's disease, and Parkinson's disease. The existence of premorbid neurodegenerative diseases can exacerbate the severity and prognosis of acute brain injuries. Apoptosis involving caspase-3 is one of the most common mechanisms involved in the etiopathology of both acute and chronic neurological and neurodegenerative diseases, suggesting a relationship between these disorders. Over the past two decades, several clinical biomarkers of apoptosis have been identified in cerebrospinal fluid and peripheral blood following ischemic stroke, intracerebral and subarachnoid hemorrhage, and TBI. These biomarkers include selected caspases, notably caspase-3 and its specific cleavage products such as caspase-cleaved cytokeratin-18, caspase-cleaved tau, and a caspase-specific 120 kDa αII-spectrin breakdown product. The levels of these biomarkers might be a valuable tool for the identification of pathological pathways such as apoptosis and inflammation involved in injury progression, assessment of injury severity, and prediction of clinical outcomes. This review focuses on clinical studies involving biomarkers of caspase-3-mediated pathways, following stroke and TBI. The review further examines their prospective diagnostic utility, as well as clinical utility for improved personalized treatment of stroke and TBI patients and the development of prophylactic treatment chronic neurodegenerative disease.

  16. Brazilian green propolis induced apoptosis in human lung cancer A549 cells through mitochondrial-mediated pathway.

    Science.gov (United States)

    Frión-Herrera, Yahima; Díaz-García, Alexis; Ruiz-Fuentes, Jenny; Rodríguez-Sánchez, Hermis; Sforcin, José Maurício

    2015-10-01

    Propolis effect on the growth and apoptosis of human lung adenocarcinoma (A549 cells) was investigated as well as its mechanisms. Cells were incubated with propolis for 72 h, and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were employed to assess cell viability and the inhibitory concentration (IC). Apoptosis was detected by Acridine Orange/Ethidium Bromide and 4',6-diamidino-2-phenylindole staining after 24 and 48 h of incubation with ¼ IC50 of propolis by testing the mitochondrial membrane potential (ΔΨm) and the expression of apoptosis-related genes (p53, Caspase-3, Bax, Bcl-2, Bcl-XL , Noxa, Puma and p21) by reverse transcription polymerase chain reaction. Propolis displayed antiproliferative and cytotoxic effects on A549 cells in a dose- and time-dependent manner, but it did not suppress the growth of normal Vero cells. An enhanced apoptosis was seen in A549 propolis-treated cells after 48 h compared with the control cells. Propolis decreased mitochondrial membrane potential by overexpression of pro-apoptotic genes (Bax and Noxa) and reduction of the antiapoptotic gene Bcl-XL . The expression level of other genes remained unchanged (p53, Caspse-3 and Bax), whereas p21 expression was increased. Propolis induced caspase-independent apoptosis through a p53-independent mitochondrial pathway, and cell cycle arrest by upregulation of p21. Although propolis induces apoptosis mainly by p53-independent manner, it may be induced by another pathway, and new insights may arise for preventing or treating lung cancer. © 2015 Royal Pharmaceutical Society.

  17. Nicotinamide induces mitochondrial-mediated apoptosis through oxidative stress in human cervical cancer HeLa cells.

    Science.gov (United States)

    Feng, Yi; Wang, Yonghua; Jiang, Chengrui; Fang, Zishui; Zhang, Zhiqiang; Lin, Xiaoying; Sun, Liwei; Jiang, Weiying

    2017-07-15

    Nicotinamide participates in energy metabolism and influences cellular redox status and modulates multiple pathways related with both cellular survival and death. Recent studies have shown that it induced proliferation inhibition and apoptosis in many cancer cells. However, little is known about the effects of nicotinamide on human cervical cancer cells. We aimed to evaluate the effects of the indicated concentrations nicotinamide on cell proliferation, apoptosis and redox-related parameters in HeLa cells and investigated the apoptotic mechanism. After the treatment of the indicated concentrations nicotinamide, HeLa cell proliferation was evaluated by the CCK-8 assay and the production of ROS (reactive oxygen species) was measured using 2',7'-Dichlorofluorescin diacetate. The apoptotic effect was confirmed by observing the cellular and nuclear morphologies with fluorescence microscope and apoptotic rate of HeLa cell apoptosis was measured by flow cytometry using Annexin-V method. Moreover, we examined the mitochondrial membrane potential by JC-1 method and measured the expression of apoptosis related genes using qRT-PCR and immunoblotting. Nicotinamide restrained the HeLa cell proliferation and significantly increased the accumulation of ROS and depletion of GSH at relatively high concentrations. Furthermore, nicotinamide promoted HeLa cell apoptosis via the intrinsic mitochondrial apoptotic pathway. Our study revealed that nicotinamide induced the apoptosis through oxidative stress and intrinsic mitochondrial apoptotic pathways in HeLa cell. The results emerge that nicotinamide may be an inexpensive, safe and promising therapeutic agent or a neoadjuvant chemotherapy for cervical cancer patients, as well useful to find new drugs for cervical cancer therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Platelet-Derived Growth Factor-BB Protects Mesenchymal Stem Cells (MSCs) Derived From Immune Thrombocytopenia Patients Against Apoptosis and Senescence and Maintains MSC-Mediated Immunosuppression.

    Science.gov (United States)

    Zhang, Jia-Min; Feng, Fei-Er; Wang, Qian-Ming; Zhu, Xiao-Lu; Fu, Hai-Xia; Xu, Lan-Ping; Liu, Kai-Yan; Huang, Xiao-Jun; Zhang, Xiao-Hui

    2016-12-01

    : Immune thrombocytopenia (ITP) is characterized by platelet destruction and megakaryocyte dysfunction. Mesenchymal stem cells (MSCs) from ITP patients (MSC-ITP) do not exhibit conventional proliferative abilities and thus exhibit defects in immunoregulation, suggesting that MSC impairment might be a mechanism involved in ITP. Platelet-derived growth factor (PDGF) improves growth and survival in various cell types. Moreover, PDGF promotes MSC proliferation. The aim of the present study was to analyze the effects of PDGF-BB on MSC-ITP. We showed that MSC-ITP expanded more slowly and appeared flattened and larger. MSC-ITP exhibited increased apoptosis and senescence compared with controls. Both the intrinsic and extrinsic pathways account for the enhanced apoptosis. P53 and p21 expression were upregulated in MSC-ITP, but inhibition of p53 with pifithrin-α markedly inhibited apoptosis and senescence. Furthermore, MSCs from ITP patients showed a lower capacity for inhibiting the proliferation of activated T cells inducing regulatory T cells (Tregs) and suppressing the synthesis of anti-glycoprotein (GP)IIb-IIIa antibodies. PDGF-BB treatment significantly decreased the expression of p53 and p21 and increased survivin expression in MSC-ITP. In addition, the apoptotic rate and number of senescent cells in ITP MSCs were reduced. Their impaired ability for inhibiting activated T cells, inducing Tregs, and suppressing the synthesis of anti-GPIIb-IIIa antibodies was restored after PDGF-BB treatment. In conclusion, we have demonstrated that PDGF-BB protects MSCs derived from ITP patients against apoptosis, senescence, and immunomodulatory defects. This protective effect of PDGF-BB is likely mediated via the p53/p21 pathway, thus potentially providing a new therapeutic approach for ITP. Immune thrombocytopenia (ITP) is characterized by platelet destruction and megakaryocyte dysfunction. Platelet-derived growth factor (PDGF) improves growth and survival in various cell types

  19. In vitro cytotoxicity of Artemisia vulgaris L. essential oil is mediated by a mitochondria-dependent apoptosis in HL-60 leukemic cell line.

    Science.gov (United States)

    Saleh, Ayman M; Aljada, Ahmad; Rizvi, Syed A A; Nasr, Amre; Alaskar, Ahmed S; Williams, Jack D

    2014-07-07

    The essential oil (EO) of Artemisia vulgaris L. has been traditionally used worldwide for treating a large number of diseases. Although major components in A. vulgaris EO have been shown to inhibit growth of different cancer cells, as pure compounds or part of other plants extracted oil, no information is known about its anti-proliferative activities. Therefore, the current investigation has evaluated the toxicity of the plant extracted oil from buds (AVO-b) and leaves (AVO-l) and characterized their growth inhibitory effects on cancer cells. AVO-b and AVO-l from A. vulgaris L. were extracted by hydrodistillation, and their effect on the viability of human HL-60 promyelocytic leukemia and various other cancer cell lines was tested using MTT assay. Flow cytometric analysis of apoptosis, DNA fragmentation assay, caspases enzymatic activities and Western blotting were used to determine the apoptotic pathway triggered by their action on HL-60 cells. Low concentrations of AVO-b and AVO-l inhibited the growth of HL-60 cells in a dose- and time-dependent manner. Employing flow cytometric, DNA fragmentation and caspase activation analyses, demonstrated that the cytotoxic effect of the oils is mediated by a caspase-dependent apoptosis. Kinetic studies in the presence and absence specific caspase inhibitors showed that activation of caspase-8 was dependent and subsequent to the activation of caspases-9 and -3. In addition, the essential oil caused a disruption of the mitochondrial transmembrane potential (ΔΨm), increased the release of cytochrome c to the cytosol, and altered the expression of certain members of Bcl-2 family (Bcl-2, Bax and Bid), Apaf-1 and XIAP. Interestingly, low doses of AVO-b and AVO-1 also induced apoptosis in various cancer cell lines, but not in noncancerous cells. The results demonstrate that the EO-induced apoptosis in HL-60 cells is mediated by caspase-dependent pathways, involving caspases-3, -9, and -8, which are initiated by Bcl-2/Bax

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

    Science.gov (United States)

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

    2014-12-01

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Xia-Qiu Tian

    2016-02-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  4. Identification of cytotoxic mediators and their putative role in the signaling pathways during docosahexaenoic acid (DHA)-induced apoptosis of cancer cells.

    Science.gov (United States)

    Das, Moitreyi; Das, Sumantra

    2016-12-01

    Docosahexaenoic acid (DHA), an important w-3 fatty acid exhibits differential behavior in cancer cells of neural origin when compared to that in normal healthy astrocytes. Treatment of C6 glioma and SH-SY5Y cell lines and primary astrocytes, representing the neoplastic cells and normal healthy cells respectively, with 100 µM DHA for 24 h showed significant loss of cell viability in the both the cancer cells as determined by MTT assay, whereas the primary astrocytes cultures were unaffected. Such loss of cell viability was due to apoptosis as confirmed by TUNEL staining and caspase-3 activation in cancer cells. Proteomic approach, employing 2-dimensional gel electrophoresis (2DE), difference gel electrophoresis (DIGE), and MALDI-TOF-TOF analysis identified six proteins which unlike in the astrocytes, were differently altered in the cancer cells upon exposure to DHA, suggesting their putative contribution in causing apoptosis in these cells. Of these, annexin A2, calumenin, pyruvate kinase M2 isoform, 14-3-3ζ were downregulated while aldo keto reductase-1B8 (AKR1B8) and glutathione-S-transferase P1 subunit (GSTP1) showed upregulation by DHA in the cancer cells. siRNA-mediated knockdown of AKR1B8 and GSTP1 inhibit DHA-induced apoptosis confirming their role in apoptotic process. Furthermore, western blot analysis identified upregulation of PPARα and the MAP kinases, JNK and p38 as well as increased ROS production selectively in the cell lines. Results suggest that DHA selectively induces apoptosis in the neural cell lines by regulating the expression of the above proteins to activate multiple apoptotic pathways which in association with excess ROS and activated MAPKs promote cell death.

  5. Hempseed oil induces reactive oxygen species- and C/EBP homologous protein-mediated apoptosis in MH7A human rheumatoid arthritis fibroblast-like synovial cells.

    Science.gov (United States)

    Jeong, Mini; Cho, Jaewook; Shin, Jong-Il; Jeon, Yong-Joon; Kim, Jin-Hyun; Lee, Sung-Joon; Kim, Eun-Soo; Lee, Kyungho

    2014-07-03

    The medicinal efficacy of hempseed (Cannabis sativa L.), which is rich in polyunsaturated fatty acids, in atopic dermatitis, inflammation, and rheumatoid arthritis (RA) has been suggested for centuries. Hempseed has been used as a treatment for these diseases in Korean and Chinese folk medicine. The aim of the study is to investigate the effects of hempseed oil (HO) on MH7A human RA fibroblast-like synovial cells. MH7A cells were used to study the anti-rheumatoid effects of hempseed (Cannabis sativa L., cv. Cheungsam/Cannabaceae) oil by investigating cell viability, apoptosis, lipid accumulation, oxidative stress, and endoplasmic reticulum (ER) stress-induced apoptosis. HO treatment reduced the survival rate of MH7A cells and promoted apoptotic cell death in a time- and dose-dependent manner. Both lipid accumulation and the level of intracellular reactive oxygen species (ROS) increased in HO-treated MH7A cells. Co-treatment with the antioxidant Tiron effectively abrogated the cytotoxic effects of HO; the ROS level was reduced, cell viability was recovered, and apoptotic cell death was significantly diminished. Moreover, HO-treated cells exhibited increased expression of the major ER stress markers, glucose-regulated protein 78 and C/EBP homologous protein (CHOP). The siRNA-mediated knockdown of CHOP prevented HO-induced apoptosis. Our results suggest that HO treatment induced lipid accumulation, ROS production, CHOP expression, and apoptosis in MH7A cells, and that CHOP functions as an anti-rheumatoid factor downstream of HO in MH7A cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Penta-acetyl geniposide-induced apoptosis involving transcription of NGF/p75 via MAPK-mediated AP-1 activation in C6 glioma cells

    International Nuclear Information System (INIS)

    Peng, C.-H.; Huang, C.-N.; Hsu, S.-P.; Wang, C.-J.

    2007-01-01

    We have demonstrated the herbal derivative penta-acetyl geniposide ((Ac) 5 GP) induces C6 glioma cell apoptosis through the critical sphingomyelinase (SMase)/nerve growth factor (NGF)/p75 and its downstream signals. It has been reported mitogen-activated protein kinase (MAPK) mediates NGF synthesis induced by SMase activation. In this study, ERK, p38 and JNK are shown to mediate (Ac) 5 GP-induced glioma cell apoptosis and elevation of NGF and p75. Treatment of PD98059 (ERK-specific inhibitor), SB203580 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) decreases the elevation of NGF and p75 mRNA induced by (Ac) 5 GP, indicating possible transcription regulation via MAPKs. The results of nuclear extract blotting and EMSA further confirm (Ac) 5 GP maximally increases AP-1 and NF-κB DNA binding at 6 h. Inhibition of ERK, p38 and JNK block the activation of AP-1 and NF-κB, suggesting these MAPKs are involved in (Ac) 5 GP-induced transcription regulation. We thereby used RT-PCR to analyze cells treated with (Ac) 5 GP, with or without AP-1 or NF-κB inhibitors. AP-1 inhibitor NDGA decreases NGF/p75 and expression of FasL and caspase 3 induced by (Ac) 5 GP, suggesting the importance of AP-1 in mediating NGF/p75 and their downstream apoptotic signals. However, FasL and caspase 3 do not change with the NF-κB inhibitor PDTC; NF-κB might be linked to other cellular events. Overall, we demonstrate that MAPK mediates (Ac) 5 GP-induced activation of AP-1, promoting the transcription of NGF/p75 and downstream apoptotic signals. These results further highlight the potential therapeutic effects of (Ac) 5 GP in chemoprevention or as an anti-tumor agent

  7. Resistance of activated human T(h)2 cells to NO-induced apoptosis is mediated by gamma-glutamyltranspeptidase

    NARCIS (Netherlands)

    Roozendaal, R; Vellenga, E; de Jong, MA; Traanberg, KF; Postma, DS; de Monchy, JGR; Kauffman, HF

    Activation-induced death of inflammatory cells (AICD) has an important function in immune maintenance, Type 1 T-h cells are known to be more susceptible to AICD than T(h)2 cells. In the current study we examined whether NO-induced apoptosis also preferentially eliminates T(h)1 cells over Th2 cells.

  8. Quercetin enhances TRAIL-mediated apoptosis in colon cancer cells by inducing the accumulation of death receptors in lipid rafts

    Czech Academy of Sciences Publication Activity Database

    Psahoulia, F.H.; Drosopoulos, K.G.; Doubravská, Lenka; Anděra, Ladislav; Pintzas, A.

    2007-01-01

    Roč. 6, č. 9 (2007), s. 2591-2599 ISSN 1535-7163 R&D Projects: GA MŠk 1M0506 Institutional research plan: CEZ:AV0Z50520514 Keywords : TRAIL * apoptosis * lipid rafts Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.800, year: 2007

  9. TGF-beta-induced apoptosis in endothelial cells mediated by M6P/IGFII-R and mini-plasminogen

    Czech Academy of Sciences Publication Activity Database

    Leksa, V.; Godar, S.; Schiller, H.B.; Fuertbauer, E.; Muhammad, A.; Slezakova, K.; Hořejší, Václav; Steinlein, P.; Weidle, U.H.; Binder, B.R.; Stockinger, H.

    2005-01-01

    Roč. 118, č. 19 (2005), s. 4577-4586 ISSN 0021-9533 Grant - others:Austrian Science Fund(AT) 005 Institutional research plan: CEZ:AV0Z5052915; CEZ:AV0Z50520514 Keywords : apoptosis * TGF-beta * mini-plasminogen Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.543, year: 2005

  10. Blueberry and malvidin inhibit cell cycle progression and induce mitochondrial-mediated apoptosis by abrogating the JAK/STAT-3 signalling pathway.

    Science.gov (United States)

    Baba, Abdul Basit; Nivetha, Ramesh; Chattopadhyay, Indranil; Nagini, Siddavaram

    2017-11-01

    Blueberries, a rich source of anthocyanins have attracted considerable attention as functional foods that confer immense health benefits including anticancer properties. Herein, we assessed the potential of blueberry and its major constituent malvidin to target STAT-3, a potentially druggable oncogenic transcription factor with high therapeutic index. We demonstrate that blueberry abrogates the JAK/STAT-3 pathway and modulates downstream targets that influence cell proliferation and apoptosis in a hamster model of oral oncogenesis. Further, we provide mechanistic evidence that blueberry and malvidin function as STAT-3 inhibitors in the oral cancer cell line SCC131. Blueberry and malvidin suppressed STAT-3 phosphorylation and nuclear translocation thereby inducing cell cycle arrest and mitochondrial-mediated apoptosis. However, the combination of blueberry and malvidin with the STAT-3 inhibitor S3I-201 was more efficacious in STAT-3 inhibition relative to single agents. The present study has provided leads for the development of novel combinations of compounds that can serve as inhibitors of STAT-mediated oncogenic signalling. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Cell surface-bound heat shock protein 70 (Hsp70) mediates perforin-independent apoptosis by specific binding and uptake of granzyme B.

    Science.gov (United States)

    Gross, Catharina; Koelch, Walter; DeMaio, Antonio; Arispe, Nelson; Multhoff, Gabriele

    2003-10-17

    Cell surface-bound heat shock protein 70 (Hsp70) renders tumor cells more sensitive to the cytolytic attack mediated by natural killer (NK) cells. A 14-amino acid Hsp70 sequence, termed TKD (TKDNNLLGRFELSG, aa450-463) could be identified as the extracellular localized recognition site for NK cells. Here, we show by affinity chromatography that both, full-length Hsp70-protein and Hsp70-peptide TKD, specifically bind a 32-kDa protein derived from NK cell lysates. The serine protease granzyme B was uncovered as the 32-kDa Hsp70-interacting protein using matrix-assisted laser desorption ionization time-of-flight mass peptide fingerprinting. Incubation of tumor cells with increasing concentrations of perforin-free, isolated granzyme B shows specific binding and uptake in a dose-dependent manner and results in initiation of apoptosis selectively in tumor cells presenting Hsp70 on the cell surface. Remarkably, Hsp70 cation channel activity was also determined selectively in purified phospholipid membranes of Hsp70 membrane-positive but not in membrane-negative tumor cells. The physiological role of our findings was demonstrated in primary NK cells showing elevated cytoplasmic granzyme B levels following contact with TKD. Furthermore, an increased lytic activity of Hsp70 membrane-positive tumor cells could be associated with granzyme B release by NK cells. Taken together we propose a novel perforin-independent, granzyme B-mediated apoptosis pathway for Hsp70 membrane-positive tumor cells.

  12. Green tea polyphenols induce cell death in breast cancer MCF-7 cells through induction of cell cycle arrest and mitochondrial-mediated apoptosis.

    Science.gov (United States)

    Liu, Shu-Min; Ou, Shi-Yi; Huang, Hui-Hua

    In order to study the molecular mechanisms of green tea polyphenols (GTPs) in treatment or prevention of breast cancer, the cytotoxic effects of GTPs on five human cell lines (MCF-7, A549, Hela, PC3, and HepG2 cells) were determined and the antitumor mechanisms of GTPs in MCF-7 cells were analyzed. The results showed that GTPs exhibited a broad spectrum of inhibition against the detected cancer cell lines, particularly the MCF-7 cells. Studies on the mechanisms revealed that the main modes of cell death induced by GTPs were cell cycle arrest and mitochondrial-mediated apoptosis. Flow cytometric analysis showed that GTPs mediated cell cycle arrest at both G1/M and G2/M transitions. GTP dose dependently led to apoptosis of MCF-7 cells via the mitochondrial pathways, as evidenced by induction of chromatin condensation, reduction of mitochondrial membrane potential (ΔΨ m ), improvement in the generation of reactive oxygen species (ROS), induction of DNA fragmentation, and activations of caspase-3 and caspase-9 in the present paper.

  13. Liver injury in acute hepatitis A is associated with decreased frequency of regulatory T cells caused by Fas-mediated apoptosis.

    Science.gov (United States)

    Choi, Yoon Seok; Lee, Jeewon; Lee, Hyun Woong; Chang, Dong-Yeop; Sung, Pil Soo; Jung, Min Kyung; Park, Jun Yong; Kim, Ja Kyung; Lee, Jung Il; Park, Hana; Cheong, Jae Youn; Suh, Kyung-Suk; Kim, Hyung Joon; Lee, June Sung; Kim, Kyung-Ah; Shin, Eui-Cheol

    2015-08-01

    Foxp3(+)CD4(+)CD25(+) regulatory T cells (Tregs) control immune responses, but their role in acute viral hepatitis remains elusive. Herein, we investigated alteration in the peripheral blood Treg population during acute hepatitis A (AHA) and its implication in the immune-mediated liver injury. The study included 71 patients with AHA, and peripheral blood mononuclear cells (PBMCs) were isolated. The suppressive activity of Treg population was determined by assessing anti-CD3/CD28-stimulated proliferation of Treg-depleted and reconstituted PBMCs. Treg cell frequency, phenotype and apoptosis in PBMCs were analysed by flow cytometry. The frequency of circulating Tregs was reduced during AHA. Moreover, the suppressive activity of the total Treg pool in the peripheral blood was attenuated during AHA. Treg frequency and suppressive activity of the Treg population inversely correlated with the serum alanine aminotransferase level. Fas was overexpressed on Tregs during AHA, suggesting their susceptibility to Fas-induced apoptosis. Indeed, increased apoptotic death was observed in Tregs of patients with AHA compared with healthy controls. In addition, agonistic anti-Fas treatment further increased apoptotic death of Tregs from patients with AHA. The decreased Treg frequency and Fas overexpression on Tregs were not observed in other acute liver diseases such as acute hepatitis B, acute hepatitis C and toxic/drug-induced hepatitis. The size of the Treg pool was contracted during AHA, resulting from apoptosis of Tregs induced by a Fas-mediated mechanism. Decrease in Treg numbers led to reduced suppressive activity of the Treg pool and consequently resulted in severe liver injury during AHA. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  14. A targeted proteomics approach to the quantitative analysis of ERK/Bcl-2-mediated anti-apoptosis and multi-drug resistance in breast cancer.

    Science.gov (United States)

    Yang, Ting; Xu, Feifei; Sheng, Yuan; Zhang, Wen; Chen, Yun

    2016-10-01

    Apoptosis suppression caused by overexpression of anti-apoptotic proteins is a central factor to the acquisition of multi-drug resistance (MDR) in breast cancer. As a highly conserved anti-apoptotic protein, Bcl-2 can initiate an anti-apoptosis response via an ERK1/2-mediated pathway. However, the details therein are still far from completely understood and a quantitative description of the associated proteins in the biological context may provide more insights into this process. Following our previous attempts in the quantitative analysis of MDR mechanisms, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics was continually employed here to describe ERK/Bcl-2-mediated anti-apoptosis. A targeted proteomics assay was developed and validated first for the simultaneous quantification of ERK1/2 and Bcl-2. In particular, ERK isoforms (i.e., ERK1 and ERK2) and their differential phosphorylated forms including isobaric ones were distinguished. Using this assay, differential protein levels and site-specific phosphorylation stoichiometry were observed in parental drug-sensitive MCF-7/WT cancer cells and drug-resistant MCF-7/ADR cancer cells and breast tissue samples from two groups of patients who were either suspected or diagnosed to have drug resistance. In addition, quantitative analysis of the time course of both ERK1/2 and Bcl-2 in doxorubicin (DOX)-treated MCF-7/WT cells confirmed these findings. Overall, we propose that targeted proteomics can be used generally to resolve more complex cellular events.

  15. Equol enhances tamoxifen’s anti-tumor activity by induction of caspase-mediated apoptosis in MCF-7 breast cancer cells

    International Nuclear Information System (INIS)

    Charalambous, Christiana; Pitta, Chara A; Constantinou, Andreas I

    2013-01-01

    Soy phytoestrogens, such as daidzein and its metabolite equol, have been proposed to be responsible for the low breast cancer rate in Asian women. Since the majority of estrogen receptor positive breast cancer patients are treated with tamoxifen, the basic objective of this study is to determine whether equol enhances tamoxifen’s anti-tumor effect, and to identify the molecular mechanisms involved. For this purpose, we examined the individual and combined effects of equol and tamoxifen on the estrogen-dependent MCF-7 breast cancer cells using viability assays, annexin-V/PI staining, cell cycle and western blot analysis. We found that equol (>50 μM) and 4-hydroxy-tamoxifen (4-OHT; >100 nM) significantly reduced the MCF-7 cell viability. Furthermore, the combination of equol (100 μM) and 4-OHT (10 μM) induced apoptosis more effectively than each compound alone. Subsequent treatment of MCF-7 cells with the pan-caspase inhibitor Z-VAD-FMK inhibited equol- and 4-OHT-mediated apoptosis, which was accompanied by PARP and α-fodrin cleavage, indicating that apoptosis is mainly caspase-mediated. These compounds also induced a marked reduction in the bcl-2:bax ratio, which was accompanied by caspase-9 and caspase-7 activation and cytochrome-c release to the cytosol. Taken together, these data support the notion that the combination of equol and tamoxifen activates the intrinsic apoptotic pathway more efficiently than each compound alone. Consequently, equol may be used therapeutically in combination treatments and clinical studies to enhance tamoxifen’s effect by providing additional protection against estrogen-responsive breast cancers

  16. Chlorella sorokiniana induces mitochondrial-mediated apoptosis in human non-small cell lung cancer cells and inhibits xenograft tumor growth in vivo.

    Science.gov (United States)

    Lin, Ping-Yi; Tsai, Ching-Tsan; Chuang, Wan-Ling; Chao, Ya-Hsuan; Pan, I-Horng; Chen, Yu-Kuo; Lin, Chi-Chen; Wang, Bing-Yen

    2017-02-01

    Lung cancer is one of the leading causes of cancer related deaths worldwide. Marine microalgae are a source of biologically active compounds and are widely consumed as a nutritional supplement in East Asian countries. It has been reported that Chlorella or Chlorella extracts have various beneficial pharmacological compounds that modulate immune responses; however, no studies have investigated the anti-cancer effects of Chlorella sorokiniana (CS) on non-small cell lung cancer (NSCLC). In this study, we evaluated the anti-cancer effects of CS in two human NSCLC cell lines (A549 and CL1-5 human lung adenocarcinoma cells), and its effects on tumor growth in a subcutaneous xenograft tumor model. We also investigated the possible molecular mechanisms governing the pharmacological function of CS. Our results showed that exposure of the two cell lines to CS resulted in a concentration-dependent reduction in cell viability. In addition, the percentage of apoptotic cells increased in a dose-dependent manner, suggesting that CS might induce apoptosis in human NSCLC cells. Western blot analysis revealed that exposure to CS resulted in increased protein expression of the cleaved/activated forms of caspase-3, caspase-9, and PARP, except caspase-8. ZDEVD (caspase-3 inhibitor) and Z-LEHD (caspase-9 inhibitor) were sufficient at preventing apoptosis in both A549 and CL1-5 cells, proving that CS induced cell death via the mitochondria-mediated apoptotic pathway. Exposure of A549 and CL1-5 cells to CS for 24 h resulted in decreased expression of Bcl-2 protein and increased expression of Bax protein as well as decreased expression of two IAP family proteins, survivin and XIAP. We demonstrated that CS induces mitochondrial-mediated apoptosis in NSCLC cells via downregulation of Bcl-2, XIAP and survivin. In addition, we also found that the tumors growth of subcutaneous xenograft in vivo was markedly inhibited after oral intake of CS.

  17. Effects of shRNA-Mediated Silencing of PKM2 Gene on Aerobic Glycolysis, Cell Migration, Cell Invasion, and Apoptosis in Colorectal Cancer Cells.

    Science.gov (United States)

    Yan, Xiao-Ling; Zhang, Xue-Bin; Ao, Ran; Guan, Lin

    2017-12-01

    This study aims to explore the effects of shRNA-mediated silencing on Pyruvate kinase type M2 (PKM2) gene during aerobic glycolysis in colorectal cancer (CRC) cells. CRC tissues and adjacent normal tissues were obtained from 136 patients diagnosed with qRT-PCR, Western blotting, and immunohistochemistry (IHC) were performed to detect mRNA and protein expressions of PKM2. CRC cells were divided into a blank, vector, and PKM2-shRNA groups. Hexokinase (HK) and PKM2 activity were both determined by glucose-6-phosphate dehydrogenase (G-6-PD) coupled colorimetric assay and enzyme coupling rate method. The extracellular lactate concentration was measured by ultraviolet spectrophotometer and caspase activity was measured using spectrophotometry. The proliferation, cell cycle, apoptosis, invasion, and migration of CRC cells were detected by cell counting kit-8 (CCK-8) assay, flow cytometry, transwell assay, and scratch test. Three groups of nude mice were injected with 0.2 mL single-cell suspension from the blank, vector, and PKM2-shRNA groups, respectively. PKM2 protein content in CRC tissues was higher than that in adjacent normal tissues. Results showed that the PKM2-shRNA group exhibited significantly lower mRNA and protein expressions of PKM2, decreased PKM2 activity, reduced lactate metabolism level, increased cell apoptosis rate, elevated caspase-3 and caspase-9 activity, weakened proliferation, and a reduction in cell invasion and migration ability compared to the vector and blank groups. The optical density (OD) value was lower in the PKM2-shRNA group than in the blank and vector groups. These findings indicate that shRNA-mediated silencing of PKM2 gene promotes apoptosis and inhibits aerobic glycolysis, proliferation, migration, and invasion in CRC cells. J. Cell. Biochem. 118: 4792-4803, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  18. Alpha-mangostin induces apoptosis through activation of reactive oxygen species and ASK1/p38 signaling pathway in cervical cancer cells

    OpenAIRE

    Lee, Chien-Hsing; Ying, Tsung-Ho; Chiou, Hui-Ling; Hsieh, Shu-Ching; Wen, Shiua-Hua; Chou, Ruey-Hwang; Hsieh, Yi-Hsien

    2017-01-01

    Alpha-mangostin, a natural xanthonoid, has been reported to possess the anti-cancer property in various types of human cancer. However, its effects and mechanism of ?-mangostin in cervical cancer remain unclear. We found that ?-mangostin effectively inhibited cell viability, resulted in loss of mitochondrial membrane potential (MMP), release of cytochrome C, increase of Bax, decrease of Bcl-2, and activation of caspase-9/caspase-3 cascade in cervical cancer cells. Alpha-mangostin elevated the...

  19. Phospho-Rb mediating cell cycle reentry induces early apoptosis following oxygen-glucose deprivation in rat cortical neurons.

    Science.gov (United States)

    Yu, Ying; Ren, Qing-Guo; Zhang, Zhao-Hui; Zhou, Ke; Yu, Zhi-Yuan; Luo, Xiang; Wang, Wei

    2012-03-01

    The aim of this study was to investigate the relationship between cell cycle reentry and apoptosis in cultured cortical neurons following oxygen-glucose deprivation (OGD). We found that the percentage of neurons with BrdU uptake, TUNEL staining, and colocalized BrdU uptake and TUNEL staining was increased relative to control 6, 12 and 24 h after 1 h of OGD. The number of neurons with colocalized BrdU and TUNEL staining was decreased relative to the number of TUNEL-positive neurons at 24 h. The expression of phosphorylated retinoblastoma protein (phospho-Rb) was significantly increased 6, 12 and 24 h after OGD, parallel with the changes in BrdU uptake. Phospho-Rb and TUNEL staining were colocalized in neurons 6 and 12 h after OGD. This colocalization was strikingly decreased 24 h after OGD. Treatment with the cyclin-dependent kinase inhibitor roscovitine (100 μM) decreased the expression of phospho-Rb and reduced neuronal apoptosis in vitro. These results demonstrated that attempted cell cycle reentry with phosphorylation of Rb induce early apoptosis in neurons after OGD and there must be other mechanisms involved in the later stages of neuronal apoptosis besides cell cycle reentry. Phosphoralated Rb may be an important factor which closely associates aberrant cell cycle reentry with the early stages of neuronal apoptosis following ischemia/hypoxia in vitro, and pharmacological interventions for neuroprotection may be useful directed at this keypoint.

  20. Hesperidin inhibits HeLa cell proliferation through apoptosis mediated by endoplasmic reticulum stress pathways and cell cycle arrest

    International Nuclear Information System (INIS)

    Wang, Yaoxian; Yu, Hui; Zhang, Jin; Gao, Jing; Ge, Xin; Lou, Ge

    2015-01-01

    Hesperidin (30, 5, 9-dihydroxy-40-methoxy-7-orutinosyl flavanone) is a flavanone that is found mainly in citrus fruits and has been shown to have some anti-neoplastic effects. The aim of the present study was to investigate the effect of hesperidin on apoptosis in human cervical cancer HeLa cells and to identify the mechanism involved. Cells were treated with hesperidin (0, 20, 40, 60, 80, and 100 μM) for 24, 48, or 72 h and relative cell viability was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Hesperidin inhibited the proliferation of HeLa cells in a concentration- and time-dependent manner. Hesperidin-induced apoptosis in HeLa cells was characterized by increased nuclear condensation and DNA fragmentation. Furthermore, increased levels of GADD153/CHOP and GRP78 indicated hesperidin-induced apoptosis in HeLa cells involved a caspase-dependent pathway, presumably downstream of the endoplasmic reticulum stress pathway. Both of these proteins are hallmarks of endoplasmic reticulum stress. Hesperidin also promoted the formation of reactive oxygen species, mobilization of intracellular Ca 2+ , loss of mitochondrial membrane potential (ΔΨm), increased release of cytochrome c and apoptosis-inducing factor from mitochondria, and promoted capase-3 activation. It also arrested HeLa cells in the G0/G1 phase in the cell cycle by downregulating the expression of cyclinD1, cyclinE1, and cyclin-dependent kinase 2 at the protein level. The effect of hesperidin was also verified on the human colon cancer cell HT-29 cells. We concluded that hesperidin inhibited HeLa cell proliferation through apoptosis involving endoplasmic reticulum stress pathways and cell cycle arrest

  1. Atherosclerosis-Associated Endothelial Cell Apoptosis by MiR-429-Mediated Down Regulation of Bcl-2

    Directory of Open Access Journals (Sweden)

    Tao Zhang

    2015-10-01

    Full Text Available Background/Aims: Endothelial cell injury and subsequent apoptosis play a key role in the development and pathogenesis of atherosclerosis, which is hallmarked by dysregulated lipid homeostasis, aberrant immunity and inflammation, and plaque-instability-associated coronary occlusion. Nevertheless, our understanding of the mechanisms underlying endothelial cell apoptosis is still limited. MicroRNA-429 (miR-29 is a known cancer suppressor that promotes cancer cell apoptosis. However, it is unknown whether miR-429 may be involved in the development of atherosclerosis through similar mechanisms. We addressed these questions in the current study. Methods: We examined the levels of endothelial cell apoptosis in ApoE (-/- mice suppled with high-fat diet (HFD, a mouse model for atherosclerosis (simplified as HFD mice. We analyzed the levels of anti-apoptotic protein Bcl-2 and the levels of miR-429 in the purified CD31+ endothelial cells from mouse aorta. Prediction of the binding between miR-429 and 3'-UTR of Bcl-2 mRNA was performed by bioinformatics analyses and confirmed by a dual luciferase reporter assay. The effects of miR-429 were further analyzed in an in vitro model using oxidized low-density lipoprotein (ox-LDL-treated human aortic endothelial cells (HAECs. Results: HFD mice developed atherosclerosis in 12 weeks, while the control ApoE (-/- mice that had received normal diet (simplified as NOR mice did not. HFD mice had significantly lower percentage of endothelial cells and significantly higher percentage of mesenchymal cells in the aorta than NOR mice. Significantly higher levels of endothelial cell apoptosis were detected in HFD mice, resulting from decreases in Bcl-2 protein, but not mRNA. The decreases in Bcl-2 in endothelial cells were due to increased levels of miR-429, which suppressed the translation of Bcl-2 mRNA via 3'-UTR binding. These in vivo findings were reproduced in vitro on ox-LDL-treated HAECs. Conclusion: Atherosclerosis

  2. Regulatory RNA Key Player in p53-Mediated Apoptosis in Embryonic Stem Cells | Center for Cancer Research

    Science.gov (United States)

    Embryonic stem cells (ESCs) must maintain the integrity of their genomes or risk passing potentially deleterious mutations on to numerous tissues. Thus, ESCs have a unique genome surveillance system and easily undergo apoptosis or differentiation when DNA damage is detected. The protein p53 is known to promote differentiation in mouse ESCs (mESCs), but its role in DNA damage-induced apoptosis (DIA) is unclear. p53 may have a pro-apoptotic function since it can regulate apoptotic genes in embryonal cells. Given that ESCs have a distinct transcriptional program, Jing Huang, Ph.D., of CCR’s Laboratory of Cancer Biology and Genetics, and his colleagues wondered whether p53 might regulate DIA in ESCs by utilizing the ESC-specific expression program.

  3. siRNA-mediated knockdown against CDCA1 and KNTC2, both frequently overexpressed in colorectal and gastric cancers, suppresses cell proliferation and induces apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Kaneko, Naoyuki [Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Miyagi 980-8575 (Japan); Department of Surgery, Tohoku University School of Medicine, Sendai, Miyagi 980-8575 (Japan); Miura, Koh [Department of Surgery, Tohoku University School of Medicine, Sendai, Miyagi 980-8575 (Japan); Gu, Zhaodi [Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Miyagi 980-8575 (Japan); Karasawa, Hideaki; Ohnuma, Shinobu; Sasaki, Hiroyuki [Department of Surgery, Tohoku University School of Medicine, Sendai, Miyagi 980-8575 (Japan); Tsukamoto, Nobukazu; Yokoyama, Satoru; Yamamura, Akihiro [Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Miyagi 980-8575 (Japan); Department of Surgery, Tohoku University School of Medicine, Sendai, Miyagi 980-8575 (Japan); Nagase, Hiroki [Division of Cancer Genetics, Department of Advanced Medical Science, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610 (Japan); Shibata, Chikashi; Sasaki, Iwao [Department of Surgery, Tohoku University School of Medicine, Sendai, Miyagi 980-8575 (Japan); Horii, Akira, E-mail: horii@m.tains.tohoku.ac.jp [Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Miyagi 980-8575 (Japan)

    2009-12-25

    Ndc80 has been shown to play an important role in stable microtubule-kinetochore attachment, chromosome alignment, and spindle checkpoint activation in mitosis. It is composed of two heterodimers, CDCA1-KNTC2 and SPC24-SPC25. Overexpression of CDCA1 and KNTC2 is reported to be associated with poor prognosis in non-small cell lung cancers (NSCLC), and siRNA-mediated knockdown against CDCA1 or KNTC2 has been found to inhibit cell proliferation and induction of apoptosis in NSCLC, ovarian cancer, cervical cancer and glioma. Therefore, CDCA1 and KNTC2 can be considered good candidates for molecular target therapy as well as diagnosis in some cancers. However, the role of the Ndc80 complex in colorectal and gastric cancers (CRC and GC) still remains unclear. In the present study, we used qRT-PCR to evaluate the expression levels of CDCA1, KNTC2, SPC24 and SPC25 in CRC and GC and employed siRNA-mediated knockdown to examine cell proliferation and apoptosis. mRNA overexpression of these four genes was observed in CRCs and GCs when compared with the corresponding normal mucosae. Additionally, the expression levels of tumor/normal ratios of CDCA1, KNTC2, SPC24 and SPC25 correlated with each other in CRCs. MTT assays revealed that cell growths after the siRNA-mediated knockdown of either CDCA1 or KNTC2 were significantly suppressed, and flow cytometry analyses revealed significant increases of the subG1 fractions after knockdown against both genes. Our present results suggest that expressional control of component molecules of Ndc80 can be utilized for molecular target therapy of patients with CRC and GC.

  4. Adenovirus-mediated truncated Bid overexpression induced by the Cre/LoxP system promotes the cell apoptosis of CD133+ ovarian cancer stem cells.

    Science.gov (United States)

    Long, Qifang; Yang, Ru; Lu, Weixian; Zhu, Weipei; Zhou, Jundong; Zheng, Cui; Zhou, Dongmei; Yu, Ling; Wu, Jinchang

    2017-01-01

    Cancer stem cells are a small subset of cancer cells that contribute to cancer progression, metastasis, chemoresistance and recurrence. CD133-positive (CD133+) ovarian cancer cells have been identified as ovarian cancer stem cells. Adenovirus-mediated gene therapy is an innovative therapeutic method for cancer treatment. In the present study, we aimed to develop a new gene therapy to specifically eliminate CD133+ ovarian cancer stem cells by targeting CD133. We used the Cre/LoxP system to augment the selective expression of the truncated Bid (tBid) gene as suicide gene therapy in CD133+ ovarian cancer stem cells. The adenovirus (Ad)-CD133-Cre expressing Cre recombinase under the control of the CD133 promoter and Ad-CMV-LoxP-Neo-LoxP-tBid expressing tBid under the control of the CMV promoter were successfully constructed using the Cre/LoxP switching system. The co-infection of Ad-CMV-LoxP-Neo-LoxP-tBid and Ad-CD133-Cre selectively induced tBid overexpression, which inhibited cell growth and triggered the cell apoptosis of CD133+ ovarian cancer stem cells. The Cre/LoxP system-mediated tBid overexpression activated the pro-apoptotic signaling pathway and augmented the cytotoxic effect of cisplatin in CD133+ ovarian cancer stem cells. Furthermore, in xenograft experiments, co-infection with the two recombinant adenoviruses markedly suppressed tumor growth in vivo and promoted cell apoptosis in tumor tissues. Taken together, the present study provides evidence that the adenovirus-mediated tBid overexpression induced by the Cre/LoxP system can effectively eliminate CD133+ ovarian cancer stem cells, representing a novel therapeutic strategy for the treatment of ovarian cancer.

  5. siRNA-mediated knockdown against CDCA1 and KNTC2, both frequently overexpressed in colorectal and gastric cancers, suppresses cell proliferation and induces apoptosis

    International Nuclear Information System (INIS)

    Kaneko, Naoyuki; Miura, Koh; Gu, Zhaodi; Karasawa, Hideaki; Ohnuma, Shinobu; Sasaki, Hiroyuki; Tsukamoto, Nobukazu; Yokoyama, Satoru; Yamamura, Akihiro; Nagase, Hiroki; Shibata, Chikashi; Sasaki, Iwao; Horii, Akira

    2009-01-01

    Ndc80 has been shown to play an important role in stable microtubule-kinetochore attachment, chromosome alignment, and spindle checkpoint activation in mitosis. It is composed of two heterodimers, CDCA1-KNTC2 and SPC24-SPC25. Overexpression of CDCA1 and KNTC2 is reported to be associated with poor prognosis in non-small cell lung cancers (NSCLC), and siRNA-mediated knockdown against CDCA1 or KNTC2 has been found to inhibit cell proliferation and induction of apoptosis in NSCLC, ovarian cancer, cervical cancer and glioma. Therefore, CDCA1 and KNTC2 can be considered good candidates for molecular target therapy as well as diagnosis in some cancers. However, the role of the Ndc80 complex in colorectal and gastric cancers (CRC and GC) still remains unclear. In the present study, we used qRT-PCR to evaluate the expression levels of CDCA1, KNTC2, SPC24 and SPC25 in CRC and GC and employed siRNA-mediated knockdown to examine cell proliferation and apoptosis. mRNA overexpression of these four genes was observed in CRCs and GCs when compared with the corresponding normal mucosae. Additionally, the expression levels of tumor/normal ratios of CDCA1, KNTC2, SPC24 and SPC25 correlated with each other in CRCs. MTT assays revealed that cell growths after the siRNA-mediated knockdown of either CDCA1 or KNTC2 were significantly suppressed, and flow cytometry analyses revealed significant increases of the subG1 fractions after knockdown against both genes. Our present results suggest that expressional control of component molecules of Ndc80 can be utilized for molecular target therapy of patients with CRC and GC.

  6. Csseverin inhibits apoptosis through mitochondria-mediated pathways triggered by Ca2 + dyshomeostasis in hepatocarcinoma PLC cells.

    Science.gov (United States)

    Shi, Mengchen; Zhou, Lina; Zhao, Lu; Shang, Mei; He, Tongtong; Tang, Zeli; Sun, Hengchang; Ren, Pengli; Lin, Zhipeng; Chen, Tingjin; Yu, Jinyun; Xu, Jin; Yu, Xinbing; Huang, Yan

    2017-11-01

    Numerous experimental and epidemiological studies have demonstrated a link between Clonorchis sinensis (C. sinensis) infestation and cholangiocarcinoma (CCA) as well as hepatocellular carcinoma (HCC). The underlying molecular mechanism involved in the malignancy of CCA and HCC has not yet been addressed. Csseverin, a component of the excretory/secretory products of C. sinensis (CsESPs), was confirmed to cause obvious apoptotic inhibition in the human HCC cell line PLC. However, the antiapoptotic mechanism is unclear. In the present study, we investigated the cellular features of the antiapoptotic mechanism upon transfection of the Csseverin gene. In the present study, we evaluated the effects of Csseverin gene overexpression on the apoptosis of PLC cells using an Annexin PE/7-AAD assay. Western blotting was applied to quantify the activation of caspase-3 and caspase-9, the mitochondrial translocation of Bax and the release of Cyt c upon Csseverin overexpression in PLC cells. Laser scanning confocal microscopy was used to analyze the changes of intracellular calcium. Fluorescence assay and immunofluorescence assays were performed to observe the changes of the mitochondrial permeability transition pore (MPTP). The overexpression of Csseverin in PLC cells showed apoptosis resistance after the induction of apoptosis. Additionally, the activation of caspase-3 and caspase-9 was specifically weakened in Csseverin overexpression PLC cells. The overexpression of Csseverin reduced the increase in intracellular free Ca2+, thereby inhibiting MPTP opening in PLC cells. Moreover, Bax mitochondrial translocation and the subsequent release of Cyt c were downregulated in apoptotic Csseverin overexpression PLC cells. The present findings suggest that Csseverin, a component of CsESPs, confers protection from human HCC cell apoptosis via the inactivation of membranous Ca2+ channels. Csseverin might be involved in the process of HCC through C. sinensis infestation in affected patients.

  7. LDH-A silencing suppresses breast cancer tumorigenicity through induction of oxidative stress mediated mitochondrial pathway apoptosis.

    Science.gov (United States)

    Wang, Zhi-Yu; Loo, Tjing Yung; Shen, Jian-Gang; Wang, Neng; Wang, Dong-Mei; Yang, De-Po; Mo, Sui-Lin; Guan, Xin-Yuan; Chen, Jian-Ping

    2012-02-01

    LDH-A, as the critical enzyme accounting for the transformation from pyruvate into lactate, has been demonstrated to be highly expressed in various cancer cells and its silencing has also been approved relating to increased apoptosis in lymphoma cells. In this study, we intend to investigate the correlation between LDH-A and other clinicopathological factors of breast cancer and whether LDH-A silencing could suppress breast cancer growth, and if so the potential mechanisms. 46 breast cancer specimens were collected to study the relation between LDH-A expression and clinicopathological characteristics including menopause, tumor size, node involvement, differentiation, and pathological subtypes classified by ER, PR, and Her-2. shRNAs were designed and applied to silence LDH-A expression in breast cancer cell lines MCF-7 and MDA-MB-231. The effects of LDH-A reduction on cancer cells were studied by a series of in vitro and in vivo experiments, including cell growth assay, apoptosis evaluation, oxidative stress detection, transmission electron microscopy observation, and tumor formation assay on nude mice. LDH-A expression was found to correlate significantly with tumor size and to be independent for other clinicopathological factors. LDH-A reduction resulted in an inhibited cancer cell proliferation, elevated intracellular oxidative stress, and induction of mitochondrial pathway apoptosis. Meanwhile, the tumorigenic ability of LDH-A deficient cancer cells was significantly limited in both breast cancer xenografts. The Ki67 positive cancer cells were significantly reduced in LDH-A deficiency tumor samples, while the apoptosis ratio was enhanced. Our results suggested that LDH-A inhibition might offer a promising therapeutic strategy for breast cancer.

  8. Distinct functional domains of PNMA5 mediate protein-protein interaction, nuclear localization, and apoptosis signaling in human cancer cells.

    Science.gov (United States)

    Lee, Yong Hoi; Pang, Siew Wai; Poh, Chit Laa; Tan, Kuan Onn

    2016-09-01

    Members of paraneoplastic Ma (PNMA) family have been identified as onconeuronal antigens, which aberrant expressions in cancer cells of patients with paraneoplastic disorder (PND) are closely linked to manifestation of auto-immunity, neuro-degeneration, and cancer. The purpose of present study was to determine the role of PNMA5 and its functional relationship to MOAP-1 (PNMA4) in human cancer cells. PNMA5 mutants were generated through deletion or site-directed mutagenesis and transiently expressed in human cancer cell lines to investigate their role in apoptosis, subcellular localization, and potential interaction with MOAP-1 through apoptosis assays, fluorescence microscopy, and co-immunoprecipitation studies, respectively. Over-expressed human PNMA5 exhibited nuclear localization pattern in both MCF-7 and HeLa cells. Deletion mapping and mutagenesis studies showed that C-terminus of PNMA5 is responsible for nuclear localization, while the amino acid residues (391KRRR) within the C-terminus of PNMA5 are required for nuclear targeting. Deletion mapping and co-immunoprecipitation studies showed that PNMA5 interacts with MOAP-1 and N-terminal domain of PNMA5 is required for interaction with MOAP-1. Furthermore, co-expression of PNMA5 and MOAP-1 in MCF-7 cells significantly enhanced chemo-sensitivity of MCF-7 to Etoposide treatment, indicating that PNMA5 and MOAP-1 interact synergistically to promote apoptotic signaling in MCF-7 cells. Our results show that PNMA5 promotes apoptosis signaling in HeLa and MCF-7 cells and interacts synergistically with MOAP-1 through its N-terminal domain to promote apoptosis and chemo-sensitivity in human cancer cells. The C-terminal domain of PNMA5 is required for nuclear localization; however, both N-and C-terminal domains of PNMA5 appear to be required for pro-apoptotic function.

  9. Saponins isolated from Asparagus induce apoptosis in human hepatoma cell line HepG2 through a mitochondrial-mediated pathway

    Science.gov (United States)

    Ji, Y.; Ji, C.; Yue, L.; Xu, H.

    2012-01-01

    Objective Many scientific studies have shown that Asparagus officinalis has an antitumour effect and enhances human immunity, but the active components and the antitumour mechanisms are unclear. We investigated the effects of saponins isolated from Asparagus on proliferation and apoptosis in the human hepatoma cell line HepG2. Methods HepG2 cells were treated with varying concentrations of Asparagus saponins at various times. Using mtt and flow cytometry assays, we evaluated the effects of Asparagus saponins on the growth and apoptosis of HepG2 cells. Transmission electron microscopy was used to observe the morphology of cell apoptosis. Confocal laser scanning microscopy was used to analyze intracellular calcium ion concentration, mitochondrial permeability transition pore (mptp), and mitochondrial membrane potential (mmp). Spectrophotometry was applied to quantify the activity of caspase-9 and caspase-3. Flow cytometry was used to investigate the levels of reactive oxygen species (ros) and pH, and the expressions of Bcl2, Bax, CytC, and caspase-3, in HepG2 cells. Results Asparagus saponins inhibited the growth of HepG2 cells in a dose-dependent manner. The median inhibitory concentration (IC50) was 101.15 mg/L at 72 hours. The apoptosis morphology at 72 hours of treatment was obvious, showing cell protuberance, concentrated cytoplasm, and apoptotic bodies. The apoptotic rates at 72 hours were 30.9%, 51.7%, and 62.1% (for saponin concentrations of 50 mg/L, 100 mg/L, 200 mg/L). Treatment with Asparagus saponins for 24 hours increased the intracellular level of ros and Ca2+, lowered the pH, activated intracellular mptp, and decreased mmp in a dose-dependent manner. Treatment also increased the activity of caspase-9 and caspase-3, downregulated the expression of Bcl2, upregulated the expression of Bax, and induced release of CytC and activation of caspase-3. Conclusions Asparagus saponins induce apoptosis in HepG2 cells through a mitochondrial-mediated and caspase

  10. Artonin E induces p53-independent G1 cell cycle arrest and apoptosis through ROS-mediated mitochondrial pathway and livin suppression in MCF-7 cells

    Directory of Open Access Journals (Sweden)

    Etti IC

    2017-03-01

    Full Text Available Imaobong Christopher Etti,1,2 Abdullah Rasedee,3 Najihah Mohd Hashim,4 Ahmad Bustamam Abdul,5 Arifah Kadir,6 Swee Keong Yeap,7 Peter Waziri,5 Ibrahim Malami,5 Kian Lam Lim,8 Christopher J Etti9 1Pharmacology and Toxicology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Malaysia; 2Department of Pharmacology and Toxicology, University of Uyo, Uyo, Nigeria; 3Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Malaysia; 4Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 5MAKNA-Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Malaysia; 6Department of Veterinary Preclinical Science, Universiti Putra Malaysia, Serdang, Malaysia; 7Laboratory of Vaccine and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Malaysia; 8Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Cheras, Selangor, Malaysia; 9Department of Agricultural and Food Engineering, University of Uyo, Uyo, Nigeria Abstract: Artonin E is a prenylated flavonoid compound isolated from the stem bark of Artocarpus elasticus. This phytochemical has been previously reported to be drug-like with full compliance to Lipinski’s rule of five and good physicochemical properties when compared with 95% of orally available drugs. It has also been shown to possess unique medicinal properties that can be utilized in view of alleviating most human disease conditions. In this study, we investigated the cytotoxic mechanism of Artonin E in MCF-7 breast cancer cells, which has so far not been reported. In this context, Artonin E significantly suppressed the breast cancer cell’s viability while inducing apoptosis in a dose-dependent manner. This apoptosis induction was caspase dependent, and it is mediated mainly through the intrinsic pathway with the elevation of total reactive oxygen species

  11. Quercetin enhances hypoxia-mediated apoptosis via direct inhibition of AMPK activity in HCT116 colon cancer.

    Science.gov (United States)

    Kim, Hak-Su; Wannatung, Tirawat; Lee, Sooho; Yang, Woo Kyeom; Chung, Sung Hyun; Lim, Jong-Seok; Choe, Wonchae; Kang, Insug; Kim, Sung-Soo; Ha, Joohun

    2012-09-01

    Tumor hypoxia is considered the best validated target in clinical oncology because of its significant contribution to chemotherapy failure and drug resistance. As an approach to target hypoxia, we assessed the potential of quercetin, a flavonoid widely distributed in plants, as a anticancer agent under hypoxic conditions and examined its pharmacological mechanisms by primarily focusing on the role of AMP-activated protein kinase (AMPK). Quercetin significantly attenuated tumor growth in an HCT116 cancer xenograft in vivo model with a substantial reduction of AMPK activity. In a cell culture system, quercetin more dramatically induced apoptosis of HCT116 cancer cells under hypoxic conditions than normoxic conditions, and this was tightly associated with inhibition of hypoxia-induced AMPK activity. An in vitro kinase assay demonstrated that quercetin directly inhibits AMPK activity. Inhibition of AMPK by expressing a dominant-negative form resulted in an increase of apoptosis under hypoxia, and a constitutively active form of AMPK effectively blocked quercetin-induced apoptosis under hypoxia. Collectively, our data suggest that quercetin directly inhibits hypoxia-induced AMPK, which plays a protective role against hypoxia. Quercetin also reduced the activity of hypoxia-inducible factor-1 (HIF-1), a major transcription factor for adaptive cellular response to hypoxia. Moreover, quercetin sensitized HCT116 cancer cells to the anticancer drugs cisplatin and etoposide under hypoxic conditions. Our findings suggest that AMPK may serve as a novel target for overcoming tumor hypoxia-associated negative aspects.

  12. Caspase-10 Is the Key Initiator Caspase Involved in Tributyltin-Mediated Apoptosis in Human Immune Cells

    Directory of Open Access Journals (Sweden)

    Harald F. Krug

    2012-01-01

    Full Text Available Tributyltin (TBT is one of the most toxic compounds produced by man and distributed in the environment. A multitude of toxic activities have been described, for example, immunotoxic, neurotoxic, and endocrine disruptive effects. Moreover, it has been shown for many cell types that they undergo apoptosis after treatment with TBT and the cell death of immune cells could be the molecular background of its immunotoxic effect. As low as 200 nM up to 1 μM of TBT induces all signs of apoptosis in Jurkat T cells within 1 to 24 hrs of treatment. When compared to Fas-ligand control stimulation, the same sequence of events occurs: membrane blebbing, phosphatidylserine externalisation, the activation of the “death-inducing signalling complex,” and the following sequence of cleavage processes. In genetically modified caspase-8-deficient Jurkat cells, the apoptotic effects are only slightly reduced, whereas, in FADD-negative Jurkat cells, the TBT effect is significantly diminished. We could show that caspase-10 is recruited by the TRAIL-R2 receptor and apoptosis is totally prevented when caspase-10 is specifically inhibited in all three cell lines.

  13. Grape seed proanthocyanidins (GSPs inhibit the growth of cervical cancer by inducing apoptosis mediated by the mitochondrial pathway.

    Directory of Open Access Journals (Sweden)

    Qing Chen

    Full Text Available Grape seed proanthocyanidins (GSPs, a biologically active component of grape seeds, have been reported to possess a wide array of pharmacological and biochemical properties. Recently, the inhibitory effects of GSPs on various cancers have been reported, but their effects on cervical cancer remain unclear. Here, we explored the effect of GSPs on cervical cancer using in vitro and in vivo models. In vitro, the treatment of HeLa and SiHa cells with GSPs resulted in a significant inhibition of cell viability. Further investigation indicated that GSPs led to the dose-dependent induction of apoptosis in cancer cells. The underlying mechanism was associated with increased expression of the pro-apoptotic protein Bak-1, decreased expression of the anti-apoptotic protein Bcl-2, the loss of mitochondrial membrane potential, and the activation of caspase-3, suggesting that GSPs induced cervical cancer cell apoptosis through the mitochondrial pathway. In addition, the administration of GSPs (0.1%, 0.2%, and 0.4%, w/v as a supplement in drinking water significantly inhibited the tumor growth of HeLa and SiHa cells in athymic nude mice, and the number of apoptotic cells in those tumors was also increased significantly. Taken together, our studies demonstrated that GSPs could inhibit the growth of cervical cancer by inducing apoptosis through the mitochondrial pathway, which provides evidence indicating that GSPs may be a potential chemopreventive and/or chemotherapeutic agent for cervical cancer.

  14. Andrographolide isolated from Andrographis paniculata induces cell cycle arrest and mitochondrial-mediated apoptosis in human leukemic HL-60 cells.

    Science.gov (United States)

    Cheung, Hon-Yeung; Cheung, Sau-Ha; Li, Jieliang; Cheung, Chui-Shan; Lai, Wai-Ping; Fong, Wang-Fun; Leung, Fo-Man

    2005-12-01

    The in vitro cytotoxicities of the ethanol extract of Andrographis paniculata (APE) and its main diterpenoid components were evaluated in various cancer cells. APE was found to be significantly growth inhibitory to human acute myeloid leukemic HL-60 cells with an IC (50) value of 14.01 microg/mL after 24 h of treatment. Among the three main diterpenoids in A. paniculata, andrographolide exhibited the highest degree of cytotoxicity followed by deoxyandrographolide while neoandrographolide was the least effective. Laser confocal microscopy and gel electrophoresis studies revealed chromosomal DNA fragmentations suggesting the occurrence of apoptosis. An increase of G (0)/G (1) phase cells from 51.88 % to 78.69 % was noted after andrographolide treatment for 36 h. The G (0)/G (1) phase arrest and apoptosis were associated with disappearance of mitochondrial cytochrome c and increased expression of Bax but decreased expression of Bcl-2 proteins in the inhibited cells. Although the order of all these events has not been determined, it is concluded that APE and andrographolide induce cell cycle arrest and affect an intrinsic mitochondria-dependent pathway of apoptosis by regulating the expression of some pro-apoptotic markers in HL-60 cells.

  15. Andrographolide sodium bisulfate-induced apoptosis and autophagy in human proximal tubular endothelial cells is a ROS-mediated pathway.

    Science.gov (United States)

    Lu, Hong; Zhang, Xin-Yue; Wang, Yi-Qi; Zheng, Xiao-Liang; Yin-Zhao; Xing, Wen-Min; Zhang, Qin

    2014-03-01

    The nephrotoxic mechanisms of andrographolide sodium bisulfate (ASB) remain largely unknown. This study attempted to explore the mechanism of ASB-induced nephrotoxicity using human proximal tubular endothelial cells (HK-2). For this study HK-2 cells were treated with rising concentrations of ASB. Their survival rate was detected using MTT assay and ultrastructure was observed with electron microscopy. L-Lactate dehydrogenase (LDH) assay was followed by examination