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Sample records for caspase-2 mediated apoptotic

  1. Caspase-2 Mediated Apoptotic and Necrotic Murine Macrophage Cell Death Induced by Rough Brucella abortus

    PubMed Central

    Chen, Fang; He, Yongqun

    2009-01-01

    Brucella species are Gram-negative, facultative intracellular bacteria that cause zoonotic brucellosis. Survival and replication inside macrophages is critical for establishment of chronic Brucella infection. Virulent smooth B. abortus strain 2308 inhibits programmed macrophage cell death and replicates inside macrophages. Cattle B. abortus vaccine strain RB51 is an attenuated rough, lipopolysaccharide O antigen-deficient mutant derived from smooth strain 2308. B. abortus rough mutant RA1 contains a single wboA gene mutation in strain 2308. Our studies demonstrated that live RB51 and RA1, but not strain 2308 or heat-killed Brucella, induced both apoptotic and necrotic cell death in murine RAW264.7 macrophages and bone marrow derived macrophages. The same phenomenon was also observed in primary mouse peritoneal macrophages from mice immunized intraperitoneally with vaccine strain RB51 using the same dose as regularly performed in protection studies. Programmed macrophage cell death induced by RB51 and RA1 was inhibited by a caspase-2 inhibitor (Z-VDVAD-FMK). Caspase-2 enzyme activation and cleavage were observed at the early infection stage in macrophages infected with RB51 and RA1 but not strain 2308. The inhibition of macrophage cell death promoted the survival of rough Brucella cells inside macrophages. The critical role of caspase-2 in mediating rough B. abortus induced macrophage cell death was confirmed using caspase-2 specific shRNA. The mitochondrial apoptosis pathway was activated in macrophages infected with rough B. abortus as demonstrated by increase in mitochondrial membrane permeability and the release of cytochrome c to cytoplasm in macrophages infected with rough Brucella. These results demonstrate that rough B. abortus strains RB51 and RA1 induce apoptotic and necrotic murine macrophage cell death that is mediated by caspase-2. The biological relevance of Brucella O antigen and caspase-2-mediated macrophage cell death in Brucella pathogenesis and protective Brucella immunity is discussed. PMID:19714247

  2. Caspase-2 promotes cytoskeleton protein degradation during apoptotic cell death

    PubMed Central

    Vakifahmetoglu-Norberg, H; Norberg, E; Perdomo, A B; Olsson, M; Ciccosanti, F; Orrenius, S; Fimia, G M; Piacentini, M; Zhivotovsky, B

    2013-01-01

    The caspase family of proteases cleaves large number of proteins resulting in major morphological and biochemical changes during apoptosis. Yet, only a few of these proteins have been reported to selectively cleaved by caspase-2. Numerous observations link caspase-2 to the disruption of the cytoskeleton, although it remains elusive whether any of the cytoskeleton proteins serve as bona fide substrates for caspase-2. Here, we undertook an unbiased proteomic approach to address this question. By differential proteome analysis using two-dimensional gel electrophoresis, we identified four cytoskeleton proteins that were degraded upon treatment with active recombinant caspase-2 in vitro. These proteins were degraded in a caspase-2-dependent manner during apoptosis induced by DNA damage, cytoskeleton disruption or endoplasmic reticulum stress. Hence, degradation of these cytoskeleton proteins was blunted by siRNA targeting of caspase-2 and when caspase-2 activity was pharmacologically inhibited. However, none of these proteins was cleaved directly by caspase-2. Instead, we provide evidence that in cells exposed to apoptotic stimuli, caspase-2 probed these proteins for proteasomal degradation. Taken together, our results depict a new role for caspase-2 in the regulation of the level of cytoskeleton proteins during apoptosis. PMID:24309927

  3. Caspase-2 resides in the mitochondria and mediates apoptosis directly from the mitochondrial compartment

    PubMed Central

    Lopez-Cruzan, M; Sharma, R; Tiwari, M; Karbach, S; Holstein, D; Martin, CR; Lechleiter, JD; Herman, B

    2016-01-01

    Caspase-2 plays an important role in apoptosis induced by several stimuli, including oxidative stress. However, the subcellular localization of caspase-2, particularly its presence in the mitochondria, is unclear. It is also not known if cytosolic caspase-2 translocates to the mitochondria to trigger the intrinsic pathway of apoptosis or if caspase-2 is constitutively present in the mitochondria that then selectively mediates this apoptotic effect. Here, we demonstrate the presence of caspase-2 in purified mitochondrial fractions from in vitro-cultured cells and in liver hepatocytes using immunoblots and confocal microscopy. We show that mitochondrial caspase-2 is functionally active by performing fluorescence resonance energy transfer analyses using a mitochondrially targeted substrate flanked by donor and acceptor fluorophores. Cell-free apoptotic assays involving recombination of nuclear, cytosolic and mitochondrial fractions from the livers of wild type and Casp2−/− mice clearly point to a direct functional role for mitochondrial caspase-2 in apoptosis. Furthermore, cytochrome c release from Casp2−/− cells is decreased as compared with controls upon treatment with agents inducing mitochondrial dysfunction. Finally, we show that Casp2−/− primary skin fibroblasts are protected from oxidants that target the mitochondrial electron transport chain. Taken together, our results demonstrate that caspase-2 exists in the mitochondria and that it is essential for mitochondrial oxidative stress-induced apoptosis.

  4. Caspase 2-mediated tumor suppression involves survivin gene silencing.

    PubMed

    Guha, M; Xia, F; Raskett, C M; Altieri, D C

    2010-03-01

    One of the pivotal functions of endogenous tumor suppression is to oppose aberrant cell survival, but the molecular requirements of this process are not completely understood. Here, we show that caspase 2, a death effector with largely unknown functions, represses transcription of the survivin gene, a general regulator of cell division and cytoprotection in tumors. This pathway involves caspase 2 proteolytic cleavage of the nuclear factor kappaB (NFkappaB) activator, RIP1. In turn, loss of RIP1 abolishes transcription of NFkappaB target genes, including survivin, resulting in deregulated mitotic transitions, enhanced apoptosis and suppression of tumorigenicity in vivo. Therefore, caspase 2 functions as an endogenous inhibitor of NFkappaB-dependent cell survival and this mechanism may contribute to tumor suppression in humans. PMID:19935698

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

    PubMed Central

    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 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. PMID:24350060

  6. Caspase-2 mediates neuronal cell death induced by beta-amyloid.

    PubMed

    Troy, C M; Rabacchi, S A; Friedman, W J; Frappier, T F; Brown, K; Shelanski, M L

    2000-02-15

    beta-amyloid (Abeta) has been proposed to play a role in the pathogenesis of Alzheimer's disease (AD). Deposits of insoluble Abeta are found in the brains of patients with AD and are one of the pathological hallmarks of the disease. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. In our current study, treatment with nitric oxide generators protected against Abeta-induced death, whereas inhibition of nitric oxide synthase afforded no protection, suggesting that formation of peroxynitrite is not critical for Abeta-mediated death. Previous studies have shown that aggregated Abeta can induce caspase-dependent apoptosis in cultured neurons. In all of the neuronal populations studied here (hippocampal neurons, sympathetic neurons, and PC12 cells), cell death was blocked by the broad spectrum caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone and more specifically by the downregulation of caspase-2 with antisense oligonucleotides. In contrast, downregulation of caspase-1 or caspase-3 did not block Abeta(1-42)-induced death. Neurons from caspase-2 null mice were totally resistant to Abeta(1-42) toxicity, confirming the importance of this caspase in Abeta-induced death. The results indicate that caspase-2 is necessary for Abeta(1-42)-induced apoptosis in vitro. PMID:10662829

  7. Additive effects of nicotine and high-fat diet on hepatocellular apoptosis in mice: Involvement of caspase 2 and inducible nitric oxide synthase-mediated intrinsic pathway signaling

    PubMed Central

    Ivey, R.; Desai, M.; Green, K.; Sinha-Hikim, I.; Friedman, T. C.; Sinha-Hikim, A. P.

    2015-01-01

    Smoking is a major risk factor for diabetes and cardiovascular disease and may contribute to non-alcoholic fatty liver disease (NAFLD). The health risk associated with smoking is exaggerated by obesity and is the leading causes of morbidity and mortality worldwide. We recently demonstrated that combined treatment with nicotine and a high-fat diet (HFD) triggers greater oxidative stress, activates hepatocellular apoptosis, and exacerbates HFD-induced hepatic steatosis. Given that hepatocellular apoptosis plays a pivotal role in the pathogenesis of NAFLD, using this model of exacerbated hepatic steatosis, we elucidated the signal transduction pathways involved in HFD plus nicotine-induced liver cell death. Adult C57BL6 male mice were fed a normal chow diet or HFD with 60% of calories derived from fat and received twice daily IP injections of 0.75 mg/kg BW of nicotine or saline for 10 weeks. High resolution light microscopy revealed markedly higher lipid accumulation in hepatocytes from mice received HFD plus nicotine, compared to mice on HFD alone. Addition of nicotine to HFD further resulted in an increase in the incidence of hepatocellular apoptosis and was associated with activation of caspase 2, induction of inducible nitric oxide synthase (iNOS), and perturbation of the BAX/BCL-2 ratio. Together, our data indicate the involvement of caspase 2 and iNOS –mediated apoptotic signaling in nicotine plus HFD-induced hepatocellular apoptosis. Targeting the caspase 2-mediated death pathway may have a protective role in development and progression of NAFLD. PMID:24830635

  8. Additive effects of nicotine and high-fat diet on hepatocellular apoptosis in mice: involvement of caspase 2 and inducible nitric oxide synthase-mediated intrinsic pathway signaling.

    PubMed

    Ivey, R; Desai, M; Green, K; Sinha-Hikim, I; Friedman, T C; Sinha-Hikim, A P

    2014-07-01

    Smoking is a major risk factor for diabetes and cardiovascular disease and may contribute to nonalcoholic fatty liver disease (NAFLD). The health risk associated with smoking is exaggerated by obesity and is the leading causes of morbidity and mortality worldwide. We recently demonstrated that combined treatment with nicotine and a high-fat diet (HFD) triggers greater oxidative stress, activates hepatocellular apoptosis, and exacerbates HFD-induced hepatic steatosis. Given that hepatocellular apoptosis plays a pivotal role in the pathogenesis of NAFLD, using this model of exacerbated hepatic steatosis, we elucidated the signal transduction pathways involved in HFD plus nicotine-induced liver cell death. Adult C57BL6 male mice were fed a normal chow diet or HFD with 60% of calories derived from fat and received twice daily IP injections of 0.75?mg/kg BW of nicotine or saline for 10 weeks. High-resolution light microscopy revealed markedly higher lipid accumulation in hepatocytes from mice received HFD plus nicotine, compared to mice on HFD alone. Addition of nicotine to HFD further resulted in an increase in the incidence of hepatocellular apoptosis and was associated with activation of caspase 2, induction of inducible nitric oxide synthase (iNOS), and perturbation of the BAX/BCL-2 ratio. Together, our data indicate the involvement of caspase 2 and iNOS-mediated apoptotic signaling in nicotine plus HFD-induced hepatocellular apoptosis. Targeting the caspase 2-mediated death pathway may have a protective role in development and progression of NAFLD. PMID:24830635

  9. Proinflammatory Caspase-2-Mediated Macrophage Cell Death Induced by a Rough Attenuated Brucella suis Strain ▿ †

    PubMed Central

    Chen, Fang; Ding, Xicheng; Ding, Ying; Xiang, Zuoshuang; Li, Xinna; Ghosh, Debashis; Schurig, Gerhardt G.; Sriranganathan, Nammalwar; Boyle, Stephen M.; He, Yongqun

    2011-01-01

    Brucella spp. are intracellular bacteria that cause an infectious disease called brucellosis in humans and many domestic and wildlife animals. B. suis primarily infects pigs and is pathogenic to humans. The macrophage-Brucella interaction is critical for the establishment of a chronic Brucella infection. Our studies showed that smooth virulent B. suis strain 1330 (S1330) prevented programmed cell death of infected macrophages and rough attenuated B. suis strain VTRS1 (a vaccine candidate) induced strong macrophage cell death. To further investigate the mechanism of VTRS1-induced macrophage cell death, microarrays were used to analyze temporal transcriptional responses of murine macrophage-like J774.A1 cells infected with S1330 or VTRS1. In total 17,685 probe sets were significantly regulated based on the effects of strain, time and their interactions. A miniTUBA dynamic Bayesian network analysis predicted that VTRS1-induced macrophage cell death was mediated by a proinflammatory gene (the tumor necrosis factor alpha [TNF-α] gene), an NF-κB pathway gene (the IκB-α gene), the caspase-2 gene, and several other genes. VTRS1 induced significantly higher levels of transcription of 40 proinflammatory genes than S1330. A Mann-Whitney U test confirmed the proinflammatory response in VTRS1-infected macrophages. Increased production of TNF-α and interleukin 1β (IL-1β) were also detected in the supernatants in VTRS1-infected macrophage cell culture. Hyperphosphorylation of IκB-α was observed in macrophages infected with VTRS1 but not S1330. The important roles of TNF-α and IκB-α in VTRS1-induced macrophage cell death were further confirmed by individual inhibition studies. VTRS1-induced macrophage cell death was significantly inhibited by a caspase-2 inhibitor but not a caspase-1 inhibitor. The role of caspase-2 in regulating the programmed cell death of VTRS1-infected macrophages was confirmed in another study using caspase-2-knockout mice. In summary, VTRS1 induces a proinflammatory, caspase-2- and NF-κB-mediated macrophage cell death. This unique cell death differs from apoptosis, which is not proinflammatory. It is also different from classical pyroptosis, which is caspase-1 mediated. PMID:21464087

  10. Glycogen Synthase Kinase-3β and Caspase-2 Mediate Ceramide- and Etoposide-Induced Apoptosis by Regulating the Lysosomal-Mitochondrial Axis

    PubMed Central

    Lin, Chiou-Feng; Tsai, Cheng-Chieh; Huang, Wei-Ching; Wang, Yu-Chih; Tseng, Po-Chun; Tsai, Tsung-Ting; Chen, Chia-Ling

    2016-01-01

    Glycogen synthase kinase-3β (GSK-3β) regulates the sequential activation of caspase-2 and caspase-8 before mitochondrial apoptosis. Here, we report the regulation of Mcl-1 destabilization and cathepsin D-regulated caspase-8 activation by GSK-3β and caspase-2. Treatment with either the ceramide analogue C2-ceramide or the topoisomerase II inhibitor etoposide sequentially induced lysosomal membrane permeabilization (LMP), the reduction of mitochondrial transmembrane potential, and apoptosis. Following LMP, cathepsin D translocated from lysosomes to the cytoplasm, whereas inhibiting cathepsin D blocked mitochondrial apoptosis. Furthermore, cathepsin D caused the activation of caspase-8 but not caspase-2. Inhibiting GSK-3β and caspase-2 blocked Mcl-1 destabilization, LMP, cathepsin D re-localization, caspase-8 activation, and mitochondrial apoptosis. Expression of Mcl-1 was localized to the lysosomes, and forced expression of Mcl-1 prevented apoptotic signaling via the lysosomal-mitochondrial pathway. These results demonstrate the importance of GSK-3β and caspase-2 in ceramide- and etoposide-induced apoptosis through mechanisms involving Mcl-1 destabilization and the lysosomal-mitochondrial axis. PMID:26727221

  11. Metabolic Control of Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Caspase-2 Suppression by the B55?/Protein Phosphatase 2A (PP2A)*

    PubMed Central

    Huang, Bofu; Yang, Chih-Sheng; Wojton, Jeffrey; Huang, Nai-Jia; Chen, Chen; Soderblom, Erik J.; Zhang, Liguo; Kornbluth, Sally

    2014-01-01

    High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca2+/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr393/Ser395) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55? targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway. PMID:25378403

  12. Measurement of caspase-2 activation during different anti-tumor drugs induced apoptosis by FRET technique

    NASA Astrophysics Data System (ADS)

    Lin, Juqiang; Zeng, Shaoqun; Luo, Qingming; Rong, Chen; Zhang, Zhihong

    2007-11-01

    Caspase-2 is important for the engagement of the mitochondrial apoptotic pathway, in the presence of DNA-damaging agents, such as cisplatin; however, the mechanism by which caspase-2 executes apoptosis remains obscure. In this study, we carried out the measurements of the dynamics of caspase-2 activation in a single living cell by a FRET (fluorescence resonance energy transfer) probe. A FRET probe was constructed that encoded a CRS (caspase-2 recognition site) fused with a cyan fluorescent protein (CFP) and a red fluorescent protein (DsRed) (CFP-CRS-DsRed). Using this probe, we found that during TRAIL-induced apoptosis, caspase-2 was not activated, and caspase-2 activation occurred in etoposide and cisplatin treated cells. However, during cisplatin-induced apoptosis caspase-2 activation was initiated much earlier than that of etoposide. Cisplatin and etoposide is one of the most broadly used drugs in the Clinical applications of cancer chemotherapy, and TRAIL, which belongs to the TNF family proteins, can selectively induce apoptosis in many transformed cells but not in normal cells. Most of anticancer drugs can induce apoptosis mediated by the activation of caspase pathway. Thus, the perfect synergistic effect group of multi-drug can be selected by using our FRET probe.

  13. Caspase-2 impacts lung tumorigenesis and chemotherapy response in vivo.

    PubMed

    Terry, M R; Arya, R; Mukhopadhyay, A; Berrett, K C; Clair, P M; Witt, B; Salama, M E; Bhutkar, A; Oliver, T G

    2015-05-01

    Caspase-2 is an atypical caspase that regulates apoptosis, cell cycle arrest and genome maintenance, although the mechanisms are not well understood. Caspase-2 has also been implicated in chemotherapy response in lung cancer, but this function has not been addressed in vivo. Here we show that Caspase-2 functions as a tumor suppressor in Kras-driven lung cancer in vivo. Loss of Caspase-2 leads to enhanced tumor proliferation and progression. Despite being more histologically advanced, Caspase-2-deficient tumors are sensitive to chemotherapy and exhibit a significant reduction in tumor volume following repeated treatment. However, Caspase-2-deficient tumors rapidly rebound from chemotherapy with enhanced proliferation, ultimately hindering long-term therapeutic benefit. In response to DNA damage, Caspase-2 cleaves and inhibits Mdm2 and thereby promotes the stability of the tumor-suppressor p53. Caspase-2 expression levels are significantly reduced in human lung tumors with wild-type p53, in agreement with the model whereby Caspase-2 functions through Mdm2/p53 regulation. Consistently, p53 target genes including p21, cyclin G1 and Msh2 are reduced in Caspase-2-deficient tumors. Finally, we show that phosphorylation of p53-induced protein with a death domain 1 leads to Caspase-2-mediated cleavage of Mdm2, directly impacting p53 levels, activity and chemotherapy response. Together, these studies elucidate a Caspase-2-p53 signaling network that impacts lung tumorigenesis and chemotherapy response in vivo. PMID:25301067

  14. The enigma of caspase-2: the laymens view

    PubMed Central

    Krumschnabel, G; Sohm, B; Bock, F; Manzl, C; Villunger, A

    2012-01-01

    Proteolysis of cellular substrates by caspases (cysteine-dependent aspartate-specific proteases) is one of the hallmarks of apoptotic cell death. Although the activation of apoptotic caspases is considered a late-stage event in apoptosis signaling, past the commitment stage, one caspase family member, caspase-2, splits the cell death community into half those searching for evidence of an apical initiator function of this molecule and those considering it as an amplifier of the apoptotic caspase cascade, at best, if relevant for apoptosis at all. This review screens past and present biochemical as well as genetic evidence for caspase-2 function in cell death signaling and beyond. PMID:19023332

  15. The role of caspase-2 in stress-induced apoptosis

    PubMed Central

    Bouchier-Hayes, Lisa

    2010-01-01

    Abstract Caspase-2 is the most evolutionarily conserved of all the caspases, yet it has a poorly defined role in apoptotic pathways. This is mainly due to a dearth of techniques to determine the activation status of caspase-2 and the lack of an abnormal phenotype in caspase-2 deficient mice. Nevertheless, emerging evidence suggests that caspase-2 may have important functions in a number of stress-induced cell death pathways, in cell cycle maintenance and regulation of tumour progression. This review discusses recent advances that have been made to help elucidate the true role of this elusive caspase and the potential contribution of caspase-2 to the pathology of human diseases including cancer. PMID:20158568

  16. Natural indoles, indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM), attenuate staphylococcal enterotoxin B-mediated liver injury by downregulating miR-31 expression and promoting caspase-2-mediated apoptosis.

    PubMed

    Busbee, Philip B; Nagarkatti, Mitzi; Nagarkatti, Prakash S

    2015-01-01

    Staphylococcal enterotoxin B (SEB) is a potent superantigen capable of inducing inflammation characterized by robust immune cell activation and proinflammatory cytokine release. Exposure to SEB can result in food poisoning as well as fatal conditions such as toxic shock syndrome. In the current study, we investigated the effect of natural indoles including indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) on SEB-mediated liver injury. Injection of SEB into D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as indicated by an increase in enzyme aspartate transaminase (AST) levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. Administration of I3C and DIM (40 mg/kg), by intraperitonal injection, attenuated SEB-induced acute liver injury, as evidenced by decrease in AST levels, inflammatory cytokines and cellular infiltration in the liver. I3C and DIM triggered apoptosis in SEB-activated T cells primarily through activation of the intrinsic mitochondrial pathway. In addition, inhibitor studies involving caspases revealed that I3C and DIM-mediated apoptosis in these activated cells was dependent on caspase-2 but independent of caspase-8, 9 and 3. In addition, I3C and DIM caused a decrease in Bcl-2 expression. Both compounds also down-regulated miR-31, which directly targets caspase-2 and influences apoptosis in SEB-activated cells. Our data demonstrate for the first time that indoles can effectively suppress acute hepatic inflammation caused by SEB and that this may be mediated by decreased expression of miR-31 and consequent caspase-2-dependent apoptosis in T cells. PMID:25706292

  17. Natural Indoles, Indole-3-Carbinol (I3C) and 3,3’-Diindolylmethane (DIM), Attenuate Staphylococcal Enterotoxin B-Mediated Liver Injury by Downregulating miR-31 Expression and Promoting Caspase-2-Mediated Apoptosis

    PubMed Central

    Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

    2015-01-01

    Staphylococcal enterotoxin B (SEB) is a potent superantigen capable of inducing inflammation characterized by robust immune cell activation and proinflammatory cytokine release. Exposure to SEB can result in food poisoning as well as fatal conditions such as toxic shock syndrome. In the current study, we investigated the effect of natural indoles including indole-3-carbinol (I3C) and 3,3’-diindolylmethane (DIM) on SEB-mediated liver injury. Injection of SEB into D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as indicated by an increase in enzyme aspartate transaminase (AST) levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. Administration of I3C and DIM (40mg/kg), by intraperitonal injection, attenuated SEB-induced acute liver injury, as evidenced by decrease in AST levels, inflammatory cytokines and cellular infiltration in the liver. I3C and DIM triggered apoptosis in SEB-activated T cells primarily through activation of the intrinsic mitochondrial pathway. In addition, inhibitor studies involving caspases revealed that I3C and DIM-mediated apoptosis in these activated cells was dependent on caspase-2 but independent of caspase-8, 9 and 3. In addition, I3C and DIM caused a decrease in Bcl-2 expression. Both compounds also down-regulated miR-31, which directly targets caspase-2 and influences apoptosis in SEB-activated cells. Our data demonstrate for the first time that indoles can effectively suppress acute hepatic inflammation caused by SEB and that this may be mediated by decreased expression of miR-31 and consequent caspase-2-dependent apoptosis in T cells. PMID:25706292

  18. A nonapoptotic role for CASP2/caspase 2

    PubMed Central

    Tiwari, Meenakshi; Sharma, Lokendra K; Vanegas, Difernando; Callaway, Danielle A; Bai, Yidong; Lechleiter, James D; Herman, Brian

    2014-01-01

    CASP2/caspase 2 plays a role in aging, neurodegeneration, and cancer. The contributions of CASP2 have been attributed to its regulatory role in apoptotic and nonapoptotic processes including the cell cycle, DNA repair, lipid biosynthesis, and regulation of oxidant levels in the cells. Previously, our lab demonstrated CASP2-mediated modulation of autophagy during oxidative stress. Here we report the novel finding that CASP2 is an endogenous repressor of autophagy. Knockout or knockdown of CASP2 resulted in upregulation of autophagy in a variety of cell types and tissues. Reinsertion of Caspase-2 gene (Casp2) in mouse embryonic fibroblast (MEFs) lacking Casp2 (casp2−/−) suppresses autophagy, suggesting its role as a negative regulator of autophagy. Loss of CASP2-mediated autophagy involved AMP-activated protein kinase, mechanistic target of rapamycin, mitogen-activated protein kinase, and autophagy-related proteins, indicating the involvement of the canonical pathway of autophagy. The present study also demonstrates an important role for loss of CASP2-induced enhanced reactive oxygen species production as an upstream event in autophagy induction. Additionally, in response to a variety of stressors that induce CASP2-mediated apoptosis, casp2−/− cells demonstrate a further upregulation of autophagy compared with wild-type MEFs, and upregulated autophagy provides a survival advantage. In conclusion, we document a novel role for CASP2 as a negative regulator of autophagy, which may provide important insight into the role of CASP2 in various processes including aging, neurodegeneration, and cancer. PMID:24879153

  19. Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate

    PubMed Central

    Kitevska, Tanja; Roberts, SarahJ.; Pantaki-Eimany, Delara; Boyd, SarahE.; Scott, FionaL.; Hawkins, ChristineJ.

    2014-01-01

    Caspase-2 is an evolutionarily conserved but enigmatic protease whose biological role remains poorly understood. To date, research into the functions of caspase-2 has been hampered by an absence of reagents that can distinguish its activity from that of the downstream apoptotic caspase, caspase-3. Identification of protein substrates of caspase-2 that are efficiently cleaved within cells may also provide clues to the role of this protease. We used a yeast-based transcriptional reporter system to define the minimal substrate specificity of caspase-2. The resulting profile enabled the identification of candidate novel caspase-2 substrates. Caspase-2 cleaved one of these proteins, the cancer-associated transcription factor Runx1, although with relatively low efficiency. A fluorogenic peptide was derived from the sequence most efficiently cleaved in the context of the transcriptional reporter. This peptide, Ac-VDTTD-AFC, was efficiently cleaved by purified caspase-2 and auto-activating caspase-2in mammalian cells, and exhibited better selectivity for caspase-2 relative to caspase-3 than reagents that are currently available. We suggest that this reagent, used in parallel with the traditional caspase-3 substrate Ac-DEVD-AFC, will enable researchers to monitor caspase-2 activity in cell lysates and may assist in the determination of stimuli that activate caspase-2 invivo. PMID:24527765

  20. Bcl-2-regulated apoptosis and cytochrome c release can occur independently of both caspase-2 and caspase-9.

    PubMed

    Marsden, Vanessa S; Ekert, Paul G; Van Delft, Mark; Vaux, David L; Adams, Jerry M; Strasser, Andreas

    2004-06-21

    Apoptosis in response to developmental cues and stress stimuli is mediated by caspases that are regulated by the Bcl-2 protein family. Although caspases 2 and 9 have each been proposed as the apical caspase in that pathway, neither is indispensable for the apoptosis of leukocytes or fibroblasts. To investigate whether these caspases share a redundant role in apoptosis initiation, we generated caspase-2(-/-)9(-/-) mice. Their overt phenotype, embryonic brain malformation and perinatal lethality mirrored that of caspase-9(-/-) mice but were not exacerbated. Analysis of adult mice reconstituted with caspase-2(-/-)9(-/-) hematopoietic cells revealed that the absence of both caspases did not influence hematopoietic development. Furthermore, lymphocytes and fibroblasts lacking both remained sensitive to diverse apoptotic stimuli. Dying caspase-2(-/-)9(-/-) lymphocytes displayed multiple hallmarks of caspase-dependent apoptosis, including the release of cytochrome c from mitochondria, and their demise was antagonized by several caspase inhibitors. These findings suggest that caspases other than caspases 2 and 9 can promote cytochrome c release and initiate Bcl-2-regulated apoptosis. PMID:15210727

  1. Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2.

    PubMed

    Andersen, Joshua L; Johnson, Carrie E; Freel, Christopher D; Parrish, Amanda B; Day, Jennifer L; Buchakjian, Marisa R; Nutt, Leta K; Thompson, J Will; Moseley, M Arthur; Kornbluth, Sally

    2009-10-21

    The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1-cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1-cyclin B1 activity must be overcome for apoptosis to occur. PMID:19730412

  2. Endoplasmic Reticulum Stress Activates the Inflammasome via NLRP3- and Caspase-2-Driven Mitochondrial Damage.

    PubMed

    Bronner, Denise N; Abuaita, Basel H; Chen, Xiaoyun; Fitzgerald, Katherine A; Nuez, Gabriel; He, Yongqun; Yin, Xiao-Ming; O'Riordan, Mary X D

    2015-09-15

    Endoplasmic reticulum (ER) stress is observed in many human diseases, often associated with inflammation. ER stress can trigger inflammation through nucleotide-binding domain and leucine-rich repeat containing (NLRP3) inflammasome, which might stimulate inflammasome formation by association with damaged mitochondria. How ER stress triggers mitochondrial dysfunction and inflammasome activation is ill defined. Here we have used an infection model to show that the IRE1? ER stress sensor regulates regulated mitochondrial dysfunction through an NLRP3-mediated feed-forward loop, independently of ASC. IRE1? activation increased mitochondrial reactive oxygen species, promoting NLRP3 association with mitochondria. NLRP3 was required for ER stress-induced cleavage of caspase-2 and the pro-apoptotic factor, Bid, leading to subsequent release of mitochondrial contents. Caspase-2 and Bid were necessary for activation of the canonical inflammasome by infection-associated or general ER stress. These data identify an NLRP3-caspase-2-dependent mechanism that relays ER stress to the mitochondria to promote inflammation, integrating cellular stress and innate immunity. PMID:26341399

  3. Endoplasmic Reticulum Stress-Mediated Activation of p38 MAPK, Caspase-2 and Caspase-8 Leads to Abrin-Induced Apoptosis

    PubMed Central

    Mishra, Ritu; Karande, Anjali A.

    2014-01-01

    Abrin from Abrus precatorius plant is a potent protein synthesis inhibitor and induces apoptosis in cells. However, the relationship between inhibition of protein synthesis and apoptosis is not well understood. Inhibition of protein synthesis by abrin can lead to accumulation of unfolded protein in the endoplasmic reticulum causing ER stress. The observation of phosphorylation of eukaryotic initiation factor 2? and upregulation of CHOP (CAAT/enhancer binding protein (C/EBP) homologous protein), important players involved in ER stress signaling by abrin, suggested activation of ER stress in the cells. ER stress is also known to induce apoptosis via stress kinases such as p38 MAPK and JNK. Activation of both the pathways was observed upon abrin treatment and found to be upstream of the activation of caspases. Moreover, abrin-induced apoptosis was found to be dependent on p38 MAPK but not JNK. We also observed that abrin induced the activation of caspase-2 and caspase-8 and triggered Bid cleavage leading to mitochondrial membrane potential loss and thus connecting the signaling events from ER stress to mitochondrial death machinery. PMID:24664279

  4. Antitumor triptycene bisquinones induce a caspase-independent release of mitochondrial cytochrome c and a caspase-2-mediated activation of initiator caspase-8 and -9 in HL-60 cells by a mechanism which does not involve Fas signaling.

    PubMed

    Perchellet, Elisabeth M; Wang, Yang; Weber, Rebeka L; Lou, Kaiyan; Hua, Duy H; Perchellet, Jean-Pierre H

    2004-11-01

    Synthetic triptycene analogs (TT code number) mimic the antitumor effects of daunorubicin (DAU) in vitro, but have the advantage of blocking nucleoside transport, inhibiting both DNA topoisomerase I and II activities, and retaining their efficacy in multidrug-resistant (MDR) tumor cells. Since TT bisquinones induce poly(ADP-ribose) polymerase-1 (PARP-1) cleavage at 6 h and internucleosomal DNA fragmentation at 24 h, which are, respectively, early and late markers of apoptosis, these antitumor drugs were tested for their ability to trigger the release of mitochondrial cytochrome c (Cyt c) and the caspase activation cascade in the HL-60 cell system. Based on their ability to reduce the viability of wild-type, drug-sensitive HL-60-S cells in the nanomolar range, six lead antitumor TT bisquinones have been identified so far: TT2, TT13, TT16, TT19, TT24 and TT26. In accord with the fact that effector caspase-3 is responsible for PARP-1 cleavage, 4 microM concentrations of DAU and these TT bisquinones all maximally induce caspase-3 activity at 6 h in HL-60-S cells, an effect which persists when the drugs are removed after a 1-h pulse treatment. Since caspase-3 may be activated by initiator caspase-9 and -8, it is significant to show that such caspase activation cascade is induced by 4 microM DAU and TT bisquinones at 6 h in HL-60-S cells. Although the relationship is not perfect, the ability of TT analogs to induce caspase-3, -8 and -9 activities may be linked to their quinone functionality and cytotoxicity. Interestingly, 4 microM concentrations of TT bisquinones retain their ability to induce caspase-3, -8 and -9 activities at 6 h in the MDR HL-60-RV cell line where 4 microM DAU becomes totally ineffective. The release of mitochondrial Cyt c is also detected within 6 h in HL-60-S cells treated with 4 microM DAU or TT bisquinones, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9. Caspase-2 and -8 may both act upstream of mitochondria to promote Cyt c release, but caspase-2 is already maximally activated 6 h after 4 microM DAU or TT13 treatments, whereas DAU- or TT-induced caspase-8 and -9 activities peak at 9 h. Pre-treatments with 15 microM of the caspase-2 inhibitor benzyloxycarbonyl (z)-Val-Asp-Val-Ala-Asp (VDVAD)-fluoromethyl ketone (fmk) totally block DAU- and TT13-induced caspase-2, -8 and -9 activities, whereas pre-treatments with 15 microM of the caspase-8 inhibitor z-Ile-Glu-Thr-Asp (IETD)-fmk prevent DAU and TT13 from inducing caspase-8 activities without affecting their caspase-2- and -9-inducing activities, suggesting that the induction of apical caspase-2 activity by these drugs may be a critical upstream event required for the activation of other downstream caspases, including caspase-9 and the mitochondrial amplification loop through caspase-8. However, the mechanisms by which DAU and TT13 induce the release of mitochondrial Cyt c appear to be caspase-independent since they are both insensitive to similar pre-treatments with 100 microM of these specific caspase-2 and -8 inhibitors. Moreover, pre-treatments with 10 microg/ml of the antagonistic anti-Fas DX2 and ZB4 monoclonal antibodies (mAbs), and the neutralizing anti-Fas ligand (FasL) NOK-1 mAb are all unable to prevent DAU and TT13 from inducing Cyt c release and caspase-2, -8 and -9 activities, suggesting that the Fas-FasL signaling pathway is not involved in the mechanism by which these quinone antitumor drugs trigger apoptosis in HL-60 cells. PMID:15514562

  5. Degradomics Reveals That Cleavage Specificity Profiles of Caspase-2 and Effector Caspases Are Alike*

    PubMed Central

    Wejda, Magdalena; Impens, Francis; Takahashi, Nozomi; Van Damme, Petra; Gevaert, Kris; Vandenabeele, Peter

    2012-01-01

    Caspase-2 is considered an initiator caspase because its long prodomain contains a CARD domain that allows its recruitment and activation in several complexes by homotypic death domain-fold interactions. Because little is known about the function and specificity of caspase-2 and its physiological substrates, we compared the cleavage specificity profile of recombinant human caspase-2 with those of caspase-3 and -7 by analyzing cell lysates using N-terminal COmbined FRActional DIagonal Chromatography (COFRADIC). Substrate analysis of the 68 cleavage sites identified in 61 proteins revealed that the protease specificities of human caspases-2, -3, and -7 largely overlap, revealing the DEVD?G consensus cleavage sequence. We confirmed that Asp563 in eukaryotic translation initiation factor 4B (eIF4B) is a cleavage site preferred by caspase-2 not only in COFRADIC setup but also upon co-expression in HEK 293T cells. These results demonstrate that activated human caspase-2 shares remarkably overlapping protease specificity with the prototype apoptotic executioner caspases-3 and -7, suggesting that caspase-2 could function as a proapoptotic caspase once released from the activating complex. PMID:22825847

  6. ER-? mediates 17 ?-estradiol attenuation of HIV-1 Tat-induced apoptotic signaling

    PubMed Central

    Adams, Sheila M.; Aksenova, Marina V.; Aksenov, Michael Y.; Mactutus, Charles F.; Booze, Rosemarie M.

    2010-01-01

    The protective actions of estrogen have been well evaluated in various models of neurodegeneration. These neuroprotective mechanisms may include a direct neuronal anti-apoptotic effect as estrogen modulates actions of key regulators of the mitochondrial/intrinsic apoptotic cascade. We tested the ability of estrogen to protect against apoptotic signaling in cortical cell cultures exposed to Tat 1-86 (50nM), and additionally, whether the beneficial actions of estrogen involved an estrogen receptor sensitive mechanism. We demonstrated that estrogen pretreatment significantly delayed Tat-induced cell death in primary cortical cultures. Pretreatment with 17?-estradiol (10nM) attenuated the increased expression of anti-apoptotic protein Bcl-2, pro-apoptotic protein Bax and activation of caspases linked to mitochondrial apoptotic pathway following Tat exposure. In addition, select components of apoptotic pathway signaling appear more sensitive to estrogen receptor (ER) activation, as the addition of ER antagonist ICI 182,780 reversed estrogen downregulation of Bax and caspase 3, while estrogen effects on Tat-induced Bcl-2 and caspase 9 expression were maintained. Moreover, the addition of preferential ER? and ER? antagonists (MPP dihydrochloride and PHTPP) indicated that estrogen effects on caspase 3 may be mediated by both receptor subtypes, while ER? was more involved in estrogen effects on Bax. Our data suggest that estrogen intervenes against HIV Tat-induced cortical neuronal dysfunction via intersecting mitochondrial apoptotic pathway signaling in an ER-sensitive manner. PMID:20340172

  7. Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells.

    PubMed

    Park, B; Lee, J; Moon, H; Lee, G; Lee, D-H; Cho, J Hoon; Park, D

    2015-01-01

    During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways. PMID:26018733

  8. Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells

    PubMed Central

    Park, B; Lee, J; Moon, H; Lee, G; Lee, D-H; Hoon Cho, J; Park, D

    2015-01-01

    During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways. PMID:26018733

  9. Caspase-2: killer, savior and safeguardemerging versatile roles for an ill-defined caspase

    PubMed Central

    Krumschnabel, G; Manzl, C; Villunger, A

    2012-01-01

    Despite the early discovery of caspase-2, its physiological function has long remained an enigma. A number of recent publications now suggest not just one, but multiple functions, including roles in apoptosis, DNA repair and tumor suppression. How can one enzyme have so many talents? Considering the diversity of interaction partners and the specific mode of pro-apoptotic action proposed in these studies, caspase-2 might in fact represent a primordial protease serving numerous pathways, established before the advent of a more elaborate functionally diversified caspases system. PMID:19581929

  10. BAD-mediated apoptotic pathway is associated with human cancer development.

    PubMed

    Stickles, Xiaomang B; Marchion, Douglas C; Bicaku, Elona; Al Sawah, Entidhar; Abbasi, Forough; Xiong, Yin; Bou Zgheib, Nadim; Boac, Bernadette M; Orr, Brian C; Judson, Patricia L; Berry, Amy; Hakam, Ardeshir; Wenham, Robert M; Apte, Sachin M; Berglund, Anders E; Lancaster, Johnathan M

    2015-04-01

    The malignant transformation of normal cells is caused in part by aberrant gene expression disrupting the regulation of cell proliferation, apoptosis, senescence and DNA repair. Evidence suggests that the Bcl-2 antagonist of cell death (BAD)-mediated apoptotic pathway influences cancer chemoresistance. In the present study, we explored the role of the BAD-mediated apoptotic pathway in the development and progression of cancer. Using principal component analysis to derive a numeric score representing pathway expression, we evaluated clinico-genomic datasets (n=427) from corresponding normal, pre-invasive and invasive cancers of different types, such as ovarian, endometrial, breast and colon cancers in order to determine the associations between the BAD-mediated apoptotic pathway and cancer development. Immunofluorescence was used to compare the expression levels of phosphorylated BAD [pBAD (serine-112, -136 and -155)] in immortalized normal and invasive ovarian, colon and breast cancer cells. The expression of the BAD-mediated apoptotic pathway phosphatase, PP2C, was evaluated by RT-qPCR in the normal and ovarian cancer tissue samples. The growth-promoting effects of pBAD protein levels in the immortalized normal and cancer cells were assessed using siRNA depletion experiments with MTS assays. The expression of the BAD-mediated apoptotic pathway was associated with the development and/or progression of ovarian (n=106, p<0.001), breast (n=185, p<0.0008; n=61, p=0.04), colon (n=22, p<0.001) and endometrial (n=33, p<0.001) cancers, as well as with ovarian endometriosis (n=20, p<0.001). Higher pBAD protein levels were observed in the cancer cells compared to the immortalized normal cells, whereas PP2C gene expression was lower in the cancer compared to the ovarian tumor tissue samples (n=76, p<0.001). The increased pBAD protein levels after the depletion of PP2C conferred a growth advantage to the immortalized normal and cancer cells. The BAD-mediated apoptotic pathway is thus associated with the development of human cancers likely influenced by the protein levels of pBAD. PMID:25653146

  11. BAD-mediated apoptotic pathway is associated with human cancer development

    PubMed Central

    STICKLES, XIAOMANG B; MARCHION, DOUGLAS C; BICAKU, ELONA; SAWAH, ENTIDHAR AL; ABBASI, FOROUGH; XIONG, YIN; ZGHEIB, NADIM BOU; BOAC, BERNADETTE M; ORR, BRIAN C; JUDSON, PATRICIA L; BERRY, AMY; HAKAM, ARDESHIR; WENHAM, ROBERT M; APTE, SACHIN M; BERGLUND, ANDERS E; LANCASTER, JOHNATHAN M

    2015-01-01

    The malignant transformation of normal cells is caused in part by aberrant gene expression disrupting the regulation of cell proliferation, apoptosis, senescence and DNA repair. Evidence suggests that the Bcl-2 antagonist of cell death (BAD)-mediated apoptotic pathway influences cancer chemoresistance. In the present study, we explored the role of the BAD-mediated apoptotic pathway in the development and progression of cancer. Using principal component analysis to derive a numeric score representing pathway expression, we evaluated clinico-genomic datasets (n=427) from corresponding normal, pre-invasive and invasive cancers of different types, such as ovarian, endometrial, breast and colon cancers in order to determine the associations between the BAD-mediated apoptotic pathway and cancer development. Immunofluorescence was used to compare the expression levels of phosphorylated BAD [pBAD (serine-112, -136 and -155)] in immortalized normal and invasive ovarian, colon and breast cancer cells. The expression of the BAD-mediated apoptotic pathway phosphatase, PP2C, was evaluated by RT-qPCR in the normal and ovarian cancer tissue samples. The growth-promoting effects of pBAD protein levels in the immortalized normal and cancer cells were assessed using siRNA depletion experiments with MTS assays. The expression of the BAD-mediated apoptotic pathway was associated with the development and/or progression of ovarian (n=106, p<0.001), breast (n=185, p<0.0008; n=61, p=0.04), colon (n=22, p<0.001) and endometrial (n=33, p<0.001) cancers, as well as with ovarian endometriosis (n=20, p<0.001). Higher pBAD protein levels were observed in the cancer cells compared to the immortalized normal cells, whereas PP2C gene expression was lower in the cancer compared to the ovarian tumor tissue samples (n=76, p<0.001). The increased pBAD protein levels after the depletion of PP2C conferred a growth advantage to the immortalized normal and cancer cells. The BAD-mediated apoptotic pathway is thus associated with the development of human cancers likely influenced by the protein levels of pBAD. PMID:25653146

  12. Hydrogen sulfide-linked sulfhydration of NF-κB mediates its anti-apoptotic actions

    PubMed Central

    Sen, Nilkantha; Paul, Bindu D.; Gadalla, Moataz M.; Mustafa, Asif K.; Sen, Tanusree; Xu, Risheng; Kim, Seyun; Snyder, Solomon H.

    2011-01-01

    Summary Nuclear factor κB (NF-κB) is an anti-apoptotic transcription factor. We show that the anti-apoptotic actions of NF-κB are mediated by hydrogen sulfide (H2S) synthesized by cystathionine gamma-lyase (CSE). TNFα treatment triples H2S generation by stimulating binding of SP1 to the CSE promoter. H2S generated by CSE stimulates DNA binding and gene activation of NF-κB, processes that are abolished in CSE deleted mice. As CSE deletion leads to decreased glutathione levels, resultant oxidative stress may contribute to alterations in CSE mutant mice. H2S acts by sulfhydrating the p65 subunit of NF-κB at cysteine-38, which promotes its binding to the co-activator ribosomal protein S3 (RPS3). Sulfhydration of p65 predominates early following TNFα treatment, then declines and is succeeded by a reciprocal enhancement of p65 nitrosylation. Anti-apoptotic influences of NF-κB, which are markedly diminished in CSE mutant mice. Thus, sulfhydration of NF-κB appears to be a physiologic determinant of its anti-apoptotic transcriptional activity. PMID:22244329

  13. C. elegans transthyretin-like protein TTR-52 mediates recognition of apoptotic cells by the CED-1 phagocyte receptor

    PubMed Central

    Wang, Xiaochen; Li, Weida; Zhao, Dongfeng; Liu, Bin; Shi, Yong; Chen, Baohui; Yang, Hengwen; Guo, Pengfei; Geng, Xin; Shang, Zhihong; Peden, Erin; Kage-Nakadai, Eriko; Mitani, Shohei; Xue, Ding

    2010-01-01

    During apoptosis, dying cells are swiftly removed by phagocytes. How apoptotic cells are recognized by phagocytes is not fully understood. Here we report the identification and characterization of the C. elegans ttr-52 gene, which is required for efficient cell corpse engulfment and encodes a transthyretin-like protein. The TTR-52 protein is expressed in and secreted from C. elegans endoderm and clusters around apoptotic cells. Genetic analysis indicates that TTR-52 acts in the cell corpse engulfment pathway mediated by CED-1, CED-6, and CED-7 and affects clustering of the phagocyte receptor CED-1 around apoptotic cells. Interestingly, TTR-52 recognizes surface exposed phosphatidylserine (PS) in vivo and binds to both PS and the extracellular domain of CED-1 in vitro. Therefore, TTR-52 is the first bridging molecule identified in C. elegans that mediates recognition of apoptotic cells by cross-linking the PS “eat me” signal with the phagocyte receptor CED-1. PMID:20526330

  14. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD)

    PubMed Central

    Okada, Yasunobu; Maeno, Emi; Shimizu, Takahiro; Dezaki, Katsuya; Wang, Jun; Morishima, Shigeru

    2001-01-01

    A fundamental property of animal cells is the ability to regulate their own cell volume. Even under hypotonic stress imposed by either decreased extracellular or increased intracellular osmolarity, the cells can re-adjust their volume after transient osmotic swelling by a mechanism known as regulatory volume decrease (RVD). In most cell types, RVD is accomplished mainly by KCl efflux induced by parallel activation of K+ and Cl− channels. We have studied the molecular mechanism of RVD in a human epithelial cell line (Intestine 407). Osmotic swelling results in a significant increase in the cytosolic Ca2+ concentration and thereby activates intermediate-conductance Ca2+-dependent K+ (IK) channels. Osmotic swelling also induces ATP release from the cells to the extracellular compartment. Released ATP stimulates purinergic ATP (P2Y2) receptors, thereby inducing phospholipase C-mediated Ca2+ mobilization. Thus, RVD is facilitated by stimulation of P2Y2 receptors due to augmentation of IK channels. In contrast, stimulation of another G protein-coupled Ca2+-sensing receptor (CaR) enhances the activity of volume-sensitive outwardly rectifying Cl− channels, thereby facilitating RVD. Therefore, it is possible that Ca2+ efflux stimulated by swelling-induced and P2Y2 receptor-mediated intracellular Ca2+ mobilization activates the CaR, thereby secondarily upregulating the volume-regulatory Cl− conductance. On the other hand, the initial process towards apoptotic cell death is coupled to normotonic cell shrinkage, called apoptotic volume decrease (AVD). Stimulation of death receptors, such as TNFα receptor and Fas, induces AVD and thereafter biochemical apoptotic events in human lymphoid (U937), human epithelial (HeLa), mouse neuroblastoma × rat glioma hybrid (NG108-15) and rat phaeochromocytoma (PC12) cells. In those cells exhibiting AVD, facilitation of RVD is always observed. Both AVD induction and RVD facilitation as well as succeeding apoptotic events can be abolished by prior treatment with a blocker of volume-regulatory K+ or Cl− channels, suggesting that AVD is caused by normotonic activation of ion channels that are normally involved in RVD under hypotonic conditions. Therefore, it is likely that G protein-coupled receptors involved in RVD regulation and death receptors triggering AVD may share common downstream signals which should give us key clues to the detailed mechanisms of volume regulation and survival of animal cells. In this Topical Review, we look at the physiological ionic mechanisms of cell volume regulation and cell death-associated volume changes from the facet of receptor-mediated cellular processes. PMID:11283221

  15. SATB1 Mediates Long-Range Chromatin Interactions: A Dual Regulator of Anti-Apoptotic BCL2 and Pro-Apoptotic NOXA Genes

    PubMed Central

    Yang, Yin; Wang, Zongdan; Sun, Luan; Shao, Lipei; Yang, Nan; Yu, Dawei; Zhang, Xin; Han, Xiao; Sun, Yujie

    2015-01-01

    Aberrant expression of special AT-rich binding protein 1 (SATB1), a global genomic organizer, has been associated with various cancers, which raises the question of how higher-order chromatin structure contributes to carcinogenesis. Disruption of apoptosis is one of the hallmarks of cancer. We previously demonstrated that SATB1 mediated specific long-range chromosomal interactions between the mbr enhancer located within 3-UTR of the BCL2 gene and the promoter to regulate BCL2 expression during early apoptosis. In the present study, we used chromosome conformation capture (3C) assays and molecular analyses to further investigate the function of the SATB1-mediated higher-order chromatin structure in co-regulation of the anti-apoptotic BCL2 gene and the pro-apoptotic NOXA gene located 3.4Mb downstream on Chromosome 18. We demonstrated that the mbr enhancer spatially juxtaposed the promoters of BCL2 and NOXA genes through SATB1-mediated chromatin-loop in Jurkat cells. Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes. The SATB1-mediated dynamic switch of the chromatin loop structures was essential for the cooperative expression of the BCL2 and NOXA genes in apoptosis. Notably, the role of SATB1 was specific, since inhibition of SATB1 degradation by caspase-6 inhibitor or caspase-6-resistant SATB1 mutant reversed expression of BCL-2 and NOXA in response to apoptotic stimulation. This study reveals the critical role of SATB1-organized higher-order chromatin structure in regulating the dynamic equilibrium of apoptosis-controlling genes with antagonistic functions and suggests that aberrant SATB1 expression might contribute to cancer development by disrupting the co-regulated genes in apoptosis pathways. PMID:26422397

  16. Fas-Mediated Apoptotic Signaling in the Mouse Brain following Reovirus Infection?

    PubMed Central

    Clarke, Penny; Beckham, J. David; Leser, J. Smith; Hoyt, Cristen C.; Tyler, Kenneth L.

    2009-01-01

    Type 3 (T3) reovirus strains induce apoptotic neuronal cell death and lethal encephalitis in infected mice. T3 strain Dearing (T3D)-induced apoptosis in primary neuronal cultures occurs by a Fas-mediated mechanism and requires the activation of caspase 8. We now show that Fas mRNA is upregulated in the brains of mice infected with encephalitic reovirus T3D and T3 strain Abney (T3A) but not following infection with nonencephalitic reovirus type 1 strain Lang. Fas is upregulated in regions of the brain that are injured during infection with T3 reovirus strains and colocalizes with virus antigen in individual neurons. In contrast, levels of FasL mRNA induced by encephalitic and nonencephalitic reovirus strains do not differ significantly. Caspase 8, the initiator caspase associated with Fas-mediated apoptosis, is activated in the cortex and hippocampal regions of both T3D- and T3A-infected mice. Furthermore, Bid cleavage and the activation of caspase 9 in the brains of T3D-infected mice suggest that the caspase 8-dependent activation of mitochondrial apoptotic signaling contributes to virus-induced apoptosis. We have previously shown that the inhibition of c-Jun N-terminal kinase (JNK) signaling blocks T3D-induced apoptosis and improves the outcome of virus-induced encephalitis. We now show that the reovirus-induced upregulation of Fas requires JNK signaling, thereby providing a link between reovirus-induced death receptor signaling and mitogen-activated protein kinase pathways and a potential mechanism for the therapeutic action of JNK inhibition. PMID:19321603

  17. Characterization and expression analysis of a caspase-2 in an invertebrate echinoderm sea cumber Apostichopus japonicus.

    PubMed

    Ye, Shigen; Gao, Yang; Wang, Shengnan; Li, Qiang; Li, Ruijun; Li, Hua

    2016-01-01

    Caspase-2 is the most evolutionarily conserved member of the caspase family which mediates the programmed cell death and plays crucial roles in key cellular processes. In this study, a caspase-2 homolog was identified and functionally characterized in sea cucumber Apostichopus japonicus, which we named AjCASP. The full-length cDNA consists of 2100 bp with an ORF encoding a protein of 378 amino acids. The deduced amino acid sequence shows that AjCASP consists of a conserved CARD-CASP2 domain and a CASs domain containing two active residues, two proteolytic cleavage residues, a substrate pocket and a dimer interface as well. In addition, a p20 large subunit with a characteristic five-peptide motif (QACRG) and a p10 small subunit in C-terminal were identified in CASs domain. Above data demonstrated that AjCASP is similar to CED-3 (the caspase-2 homolog of nematode Caenorhabditis elegans), which is further confirmed by phylogenetic tree analysis. AjCASP was ubiquitously expressed in sea cucumber and the obviously higher expression level was observed in coelomocyte, respiratory tree and intestine. Real-time PCR analyses further demonstrated that AjCASP was significantly induced by LPS. Taken together, these results strongly suggest that AjCASP is a caspase-2 homolog and it may be involved in invertebrate immune response, especially in eliminating and degrading invading pathogens. PMID:26687532

  18. Caspase 2 Activation and ER Stress Drive Rapid Jurkat Cell Apoptosis by Clofibrate

    PubMed Central

    Penna, Fabio; Pin, Fabrizio; Costamagna, Domiziana; Reffo, Patrizia; Baccino, Francesco Maria; Bonelli, Gabriella; Costelli, Paola

    2012-01-01

    Differently from the antiapoptotic action most commonly assigned to peroxisome proliferators (PPs), we demonstrated that some of them, clofibrate (CF) in particular, display clearcut apoptogenic properties on rat hepatoma cell lines. We and others could confirm that CF as well as various other PPs can induce apoptosis in a variety of cells, including human liver, breast and lung cancer cell lines. The present study was aimed at investigating the cytotoxic action of CF on a neoplastic line of different origin, the human T leukemia Jurkat cells. We observed that CF rapidly triggers an extensive and morphologically typical apoptotic process on Jurkat cells, though not in primary T cells, which is completely prevented by the polycaspase inhibitor zVADfmk. Gene silencing studies demonstrated that CF-induced apoptosis in Jurkat cells is partially dependent on activation of caspase 2. Looking for a possible trigger of caspase 2 activation, we observed increased levels of phosphorylated eIF2? and JNK in CF-treated cells. Moreover, intracellular Ca2+ homeostasis was perturbed. Together, these findings are suggestive for the occurrence of ER stress, an event that is known to have the potential to activate caspase 2. The present observations demonstrate that CF induces in Jurkat cells a very fast and extensive apoptosis, that involves induction of ER stress and activation of caspases 2 and 3. Since apoptosis in Jurkat cells occurs at pharmacologically relevant concentrations of CF, the present findings encourage further in depth analysis in order to work out the potential implications of CF cytotoxcity on leukemic cells. PMID:23028936

  19. Involvement of caspase-2 and caspase-9 in endoplasmic reticulum stress-induced apoptosis: A role for the IAPs

    SciTech Connect

    Cheung, Herman H.; Lynn Kelly, N.; Liston, Peter; Korneluk, Robert G. . E-mail: bob@mgcheo.med.uottawa.ca

    2006-07-15

    Dysregulation of apoptosis is involved in a wide spectrum of disease ranging from proliferative to degenerative disorders. An emerging area of study in apoptosis is the critical contribution of the endoplasmic reticulum (ER) in both mitochondrial and ER specific apoptosis pathways. Here we show that brefeldin A and tunicamycin-mediated ER stress lead to caspase-dependent apoptosis involving caspase-2. Confocal microscopy and subcellular fractionation indicate that caspase-2 is localized to the ER, and following ER stress, the processing of caspase-2 and -9 is an early event preceding the activation of caspase-3 and -7 and the cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP). Inhibition and silencing of either caspase-2 or caspase-9 suppress ER stress-induced apoptosis, as demonstrated by annexin V binding. Similarly, transduction with an adenovirus encoding either Inhibitors of Apoptosis (IAP) protein HIAP1/c-IAP2 or HIAP2/c-IAP1 also suppresses ER stress-induced apoptosis. However, among HIAP1, HIAP2 and XIAP, only HIAP2 binds and inhibits caspase-2. Our results thus indicate a novel mechanism by which HIAP2 can regulate ER-initiated apoptosis by modulating the activity of caspase-2.

  20. Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells

    PubMed Central

    Dransfield, I; Zagórska, A; Lew, E D; Michail, K; Lemke, G

    2015-01-01

    Billions of inflammatory leukocytes die and are phagocytically cleared each day. This regular renewal facilitates the normal termination of inflammatory responses, suppressing pro-inflammatory mediators and inducing their anti-inflammatory counterparts. Here we investigate the role of the receptor tyrosine kinase (RTK) Mer and its ligands Protein S and Gas6 in the initial recognition and capture of apoptotic cells (ACs) by macrophages. We demonstrate extremely rapid binding kinetics of both ligands to phosphatidylserine (PtdSer)-displaying ACs, and show that ACs can be co-opsonized with multiple PtdSer opsonins. We further show that macrophage phagocytosis of ACs opsonized with Mer ligands can occur independently of a requirement for αV integrins. Finally, we demonstrate a novel role for Mer in the tethering of ACs to the macrophage surface, and show that Mer-mediated tethering and subsequent AC engulfment can be distinguished by their requirement for Mer kinase activity. Our results identify Mer as a receptor uniquely capable of both tethering ACs to the macrophage surface and driving their subsequent internalization. PMID:25695599

  1. Activation of p53-regulated pro-apoptotic signaling pathways in PrP-mediated myopathy

    PubMed Central

    Liang, Jingjing; Parchaliuk, Debra; Medina, Sarah; Sorensen, Garrett; Landry, Laura; Huang, Shenghai; Wang, Meiling; Kong, Qingzhong; Booth, Stephanie A

    2009-01-01

    Background We have reported that doxycycline-induced over-expression of wild type prion protein (PrP) in skeletal muscles of Tg(HQK) mice is sufficient to cause a primary myopathy with no signs of peripheral neuropathy. The preferential accumulation of the truncated PrP C1 fragment was closely correlated with these myopathic changes. In this study we use gene expression profiling to explore the temporal program of molecular changes underlying the PrP-mediated myopathy. Results We used DNA microarrays, and confirmatory real-time PCR and Western blot analysis to demonstrate deregulation of a large number of genes in the course of the progressive myopathy in the skeletal muscles of doxycycline-treated Tg(HQK) mice. These include the down-regulation of genes coding for the myofibrillar proteins and transcription factor MEF2c, and up-regulation of genes for lysosomal proteins that is concomitant with increased lysosomal activity in the skeletal muscles. Significantly, there was prominent up-regulation of p53 and p53-regulated genes involved in cell cycle arrest and promotion of apoptosis that paralleled the initiation and progression of the muscle pathology. Conclusion The data provides the first in vivo evidence that directly links p53 to a wild type PrP-mediated disease. It is evident that several mechanistic features contribute to the myopathy observed in PrP over-expressing mice and that p53-related apoptotic pathways appear to play a major role. PMID:19400950

  2. Polyglutamine-mediated dysfunction and apoptotic death of a Caenorhabditis elegans sensory neuron.

    PubMed

    Faber, P W; Alter, J R; MacDonald, M E; Hart, A C

    1999-01-01

    The effect of expressing human huntingtin fragments containing polyglutamine (polyQ) tracts of varying lengths was assessed in Caenorhabditis elegans ASH sensory neurons in young and old animals. Expression of a huntingtin fragment containing a polyQ tract of 150 residues (Htn-Q150) led to progressive ASH neurodegeneration but did not cause cell death. Progressive cell death and enhanced neurodegeneration were observed in ASH neurons that coexpressed Htn-Q150 and a subthreshold dose of a toxic OSM-10::green fluorescent protein (OSM-10::GFP) fusion protein. Htn-Q150 huntingtin protein fragments formed protein aggregates in ASH neurons, and the number of ASH neurons containing aggregates increased as animals aged. ASH neuronal cell death required ced-3 caspase function, indicating that the observed cell death is apoptotic. Of interest, ced-3 played a critical role in Htn-Q150-mediated neurodegeneration but not in OSM10::GFP-mediated ASH neurodegeneration. ced-3 function was important but not essential for the formation of protein aggregates. Finally, behavioral assays indicated that ASH neurons, coexpressing Htn-Q150 and OSM10::GFP, were functionally impaired at 3 days before the detection of neurodegeneration, cell death, and protein aggregates. PMID:9874792

  3. Caspase-2 modulates osteoclastogenesis through down-regulating oxidative stress.

    PubMed

    Callaway, Danielle A; Riquelme, Manuel A; Sharma, Ramaswamy; Lopez-Cruzan, Marisa; Herman, Brian A; Jiang, Jean X

    2015-07-01

    The loss of caspase-2 (Casp-2) in mice results in an osteopenic phenotype associated with increased numbers of osteoclasts in vivo. In this study, we show that Casp-2 is involved in osteoclastogenesis. Protein levels of Casp-2 decrease during the differentiation of macrophages to osteoclasts. Furthermore, siRNA-mediated Casp-2 knockdown in osteoclast precursors or differentiation of bone marrow macrophage (BMM) precursors from Casp2(-/-) mice results in increased osteoclast numbers and tartrate-resistant acid phosphatase (TRAP) activity. Casp2(-/-) osteoclasts are larger in size compared to wild-type osteoclasts and exhibited increased numbers of nuclei, perhaps due to increased precursor fusion. The loss of Casp-2 did not alter earlier stages of differentiation, but had a greater consequence on later stages involving NFATc1 auto-amplification and pre-osteoclast fusion. We have previously shown that the loss of Casp-2 results in increased oxidative stress in the bone. Reactive oxygen species (ROS) is known to play a critical role in late osteoclast differentiation and we show that total ROS and specifically, mitochondrial ROS, significantly increased in Casp2(-/-) BMM precursors after RANKL administration, with a concomitant reduction in FoxO3a and its target antioxidant enzymes, catalase and superoxide 2 (SOD2). Because mitochondrial ROS has been identified as a putative regulator of the later stages of differentiation, the heightened ROS levels in Casp2(-/-) cells likely promote precursor fusion and increased osteoclast numbers. In conclusion, our results indicate a novel role of Casp-2 in the osteoclast as a modulator of total and mitochondrial ROS and osteoclast differentiation. PMID:25796569

  4. Role of Apoptotic Proteins in REC-2006 Mediated Radiation Protection in Hepatoma Cell Lines

    PubMed Central

    Singh, Pankaj Kumar; Kumar, Raj; Sharma, Ashok; Arora, Rajesh; Chawla, Raman; Jain, Swatantra Kumar; Tripathi, Rajendra Prasad; Sharma, Rakesh Kumar

    2011-01-01

    The present study was carried out to evaluate the role of apoptotic proteins in REC-2006-mediated radiation protection in hepatoma cell lines. REC-2006 treatment 2?h before irradiation strongly inhibited the cleavage of ATM and PARP-1 in HepG2 cells. The expression of nuclear apoptosis inducing factor (AIF) was found to be more inhibited (~17%) in HepG2 cells in REC-2006 + radiation-treated group. More inhibition (~33%) of cytochrome c was observed in HepG2 cells upon REC-2006 treatment 2?h prior irradiation. Similarly, significantly more (P<.05) inhibition of Apaf-1, caspase-9 and caspase-3 was observed in REC-2006 + radition-treated group in HepG2 cells. REC-2006 treatment restored the expression of ICAD in HepG2 cells; however, no restoration was observed in Hep3B cells. Lower nuclear to cytoplasmic CAD ratio was observed in HepG2 cells (~0.6) as compared with Hep3B cells (~1.2) in REC-2006 + radiation-treated group. In conclusion, REC-2006 rendered higher protection in HepG2 cells by inhibiting the expression and translocation of AIF, inhibiting the cleavage of ATM and PARP-1, restoring the expression of ICAD, inhibiting the release of cytochrome c and thus modulating the expression of Apaf-1 caspase-9 and activity of caspase-3. PMID:21799693

  5. KAISO, a critical regulator of p53-mediated transcription of CDKN1A and apoptotic genes

    PubMed Central

    Koh, Dong-In; Han, Dohyun; Ryu, Hoon; Choi, Won-Il; Jeon, Bu-Nam; Kim, Min-Kyeong; Kim, Youngsoo; Kim, Jin Young; Parry, Lee; Clarke, Alan R.; Reynolds, Albert B.; Hur, Man-Wook

    2014-01-01

    An unresolved issue in genotoxic stress response is identification of induced regulatory proteins and how these activate tumor suppressor p53 to determine appropriate cell responses. Transcription factor KAISO was previously described to repress transcription following binding to methylated DNA. In this study, we show that KAISO is induced by DNA damage in p53-expressing cells and then interacts with the p53–p300 complex to increase acetylation of p53 K320 and K382 residues, although decreasing K381 acetylation. Moreover, the p53 with this particular acetylation pattern shows increased DNA binding and potently induces cell cycle arrest and apoptosis by activating transcription of CDKN1A (cyclin-dependent kinase inhibitor 1) and various apoptotic genes. Analogously, in Kaiso KO mouse embryonic fibroblast cells, p53-to-promoter binding and up-regulation of p21 and apoptosis gene expression is significantly compromised. KAISO may therefore be a critical regulator of p53-mediated cell cycle arrest and apoptosis in response to various genotoxic stresses in mammalian cells. PMID:25288747

  6. KAISO, a critical regulator of p53-mediated transcription of CDKN1A and apoptotic genes.

    PubMed

    Koh, Dong-In; Han, Dohyun; Ryu, Hoon; Choi, Won-Il; Jeon, Bu-Nam; Kim, Min-Kyeong; Kim, Youngsoo; Kim, Jin Young; Parry, Lee; Clarke, Alan R; Reynolds, Albert B; Hur, Man-Wook

    2014-10-21

    An unresolved issue in genotoxic stress response is identification of induced regulatory proteins and how these activate tumor suppressor p53 to determine appropriate cell responses. Transcription factor KAISO was previously described to repress transcription following binding to methylated DNA. In this study, we show that KAISO is induced by DNA damage in p53-expressing cells and then interacts with the p53-p300 complex to increase acetylation of p53 K320 and K382 residues, although decreasing K381 acetylation. Moreover, the p53 with this particular acetylation pattern shows increased DNA binding and potently induces cell cycle arrest and apoptosis by activating transcription of CDKN1A (cyclin-dependent kinase inhibitor 1) and various apoptotic genes. Analogously, in Kaiso KO mouse embryonic fibroblast cells, p53-to-promoter binding and up-regulation of p21 and apoptosis gene expression is significantly compromised. KAISO may therefore be a critical regulator of p53-mediated cell cycle arrest and apoptosis in response to various genotoxic stresses in mammalian cells. PMID:25288747

  7. Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster

    PubMed Central

    Xing, Yalan; Su, Tin Tin; Ruohola-Baker, Hannele

    2015-01-01

    Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. Here we show that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis and dissect the mechanism for this protection. We find that upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIA-BLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, Pvf1, a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer. PMID:25959206

  8. Lipid rafts and raft-mediated supramolecular entities in the regulation of CD95 death receptor apoptotic signaling.

    PubMed

    Gajate, Consuelo; Mollinedo, Faustino

    2015-05-01

    Membrane lipid rafts are highly ordered membrane domains enriched in cholesterol, sphingolipids and gangliosides that have the property to segregate and concentrate proteins. Lipid and protein composition of lipid rafts differs from that of the surrounding membrane, thus providing sorting platforms and hubs for signal transduction molecules, including CD95 death receptor-mediated signaling. CD95 can be recruited to rafts in a reversible way through S-palmitoylation following activation of cells with its physiological cognate ligand as well as with a wide variety of inducers, including several antitumor drugs through ligand-independent intracellular mechanisms. CD95 translocation to rafts can be modulated pharmacologically, thus becoming a target for the treatment of apoptosis-defective diseases, such as cancer. CD95-mediated signaling largely depends on protein-protein interactions, and the recruitment and concentration of CD95 and distinct downstream apoptotic molecules in membrane raft domains, forming raft-based supramolecular entities that act as hubs for apoptotic signaling molecules, favors the generation and amplification of apoptotic signals. Efficient CD95-mediated apoptosis involves CD95 and raft internalization, as well as the involvement of different subcellular organelles. In this review, we briefly summarize and discuss the involvement of lipid rafts in the regulation of CD95-mediated apoptosis that may provide a new avenue for cancer therapy. PMID:25702154

  9. Fatty acid synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death.

    PubMed

    Yang, C-S; Matsuura, K; Huang, N-J; Robeson, A C; Huang, B; Zhang, L; Kornbluth, S

    2015-06-01

    Blockade of fatty acid synthase (FASN), a key enzyme involved in de novo lipogenesis, results in robust death of ovarian cancer cells. However, known FASN inhibitors have proven to be poor therapeutic agents due to their ability to induce cachexia. Therefore, we sought to identify additional targets in the pathway linking FASN inhibition and cell death whose modulation might kill ovarian cancer cells without the attendant side effects. Here, we show that the initiator caspase-2 is required for robust death of ovarian cancer cells induced by FASN inhibitors. REDD1 (also known as Rtp801 or DDIT4), a known mTOR inhibitor previously implicated in the response to FASN inhibition, is a novel caspase-2 regulator in this pathway. REDD1 induction is compromised in ovarian cancer cells that do not respond to FASN inhibition. Inhibition of FASN induced an ATF4-dependent transcriptional induction of REDD1; downregulation of REDD1 prevented orlistat-induced activation of caspase-2, as monitored by its cleavage, proteolytic activity and dimerization. Abrogation of REDD1-mediated suppression of mTOR by TSC2 RNAi protected FASN inhibitor-sensitive ovarian cancer cells (OVCA420 cells) from orlistat-induced death. Conversely, suppression of mTOR with the chemical inhibitors PP242 or rapamycin-sensitized DOV13, an ovarian cancer cell line incapable of inducing REDD1, to orlistat-induced cell death through caspase-2. These findings indicate that REDD1 positively controls caspase-2-dependent cell death of ovarian cancer cells by inhibiting mTOR, placing mTOR as a novel upstream regulator of caspase-2 and supporting the possibility of manipulating mTOR to enhance caspase-2 activation in ovarian cancer. PMID:25151963

  10. Adenovirus type 12 E1B 55-kilodalton oncoprotein promotes p53-mediated apoptotic response of ovarian cancer to cisplatin.

    PubMed

    Wang, Junnai; Gao, Qinglei; Li, Qiang

    2015-08-01

    The tumor suppressor p53-mediated apoptotic response plays an important role in cisplatin resistant in ovarian cancer. The adenovirus (Ad) type 12 E1B 55-kDa protein binds to p53 and inactivates its transcriptional transactivation function. In this study, we test the hypothesis that Ad12 E1B 55-kDa oncoprotein promotes p53-mediated apoptotic response of ovarian cancer to cisplatin. First, we observed the upregulation protein level of p53 target genes in cisplatin-resistant or cisplatin-sensitive ovarian cancer by Western blotting. Second, after transfection of Ad12 E1b 55-kDa expression plasmid, the expressions of p53 target genes in A2780 cells were further enhanced. Co-IP experiment demonstrated Ad12 E1b 55 kDa associated with p53. MTT assay confirmed that the cell proliferation was enhanced after transfection, as well as the enhanced cell inhibitory rate in the presence of cisplatin. Using flow cytometry, transfection of Ad12 E1B 55-kDa protein induced apoptosis and promoted S-phase transition in proliferation. Finally, results showed that all these changes promoted by Ad12 E1b 55 kDa were attenuated by the exposure of specific inhibitor of p53 signaling, pifithrin-?. Taken together, we concluded that Ad E1B 55-kDa oncoprotein promotes p53-mediated apoptotic response of ovarian cancer to cisplatin. PMID:25820823

  11. Pro-apoptotic gene knockdown mediated by nanocomplexed siRNA reduces radiation damage in primary salivary gland cultures

    PubMed Central

    Arany, Szilvia; Xu, Qingfu; Hernady, Eric; Benoit, Danielle S.W.; Dewhurst, Steve; Ovitt, Catherine E.

    2012-01-01

    A critical issue in the management of head and neck tumors is radioprotection of the salivary glands. We have investigated whether siRNA-mediated gene knock down of pro-apoptotic mediators can reduce radiation-induced cellular apoptosis in salivary gland cells in vitro. We used novel, pH-responsive nanoparticles to deliver functionally active siRNAs into cultures of salivary gland cells. The nanoparticle molecules are comprised of cationic micelles that electrostatically interact with the siRNA, protecting it from nuclease attack, and also include pH-responsive endosomolytic constituents that promote release of the siRNA into the target cell cytoplasm. Transfection controls with Cy3-tagged siRNA/nanoparticle complexes showed efficiently internalized siRNAs in more than 70% of the submandibular gland cells. We found that introduction of siRNAs specifically targeting the Pkc? or Bax genes significantly blocked the induction of these pro-apoptotic proteins that normally occurs after radiation in cultured salivary gland cells. Furthermore, the level of cell death from subsequent radiation, as measured by caspase-3, TUNEL, and mitochondrial disruption assays, was significantly decreased. Thus, we have successfully demonstrated that the siRNA/ nanoparticle-mediated knock down of pro-apoptotic genes can prevent radiation-induced damage in submandibular gland primary cell cultures. PMID:22253051

  12. The BCL-2 family: key mediators of the apoptotic response to targeted anti-cancer therapeutics

    PubMed Central

    Hata, Aaron N.; Engelman, Jeffrey A.; Faber, Anthony C.

    2016-01-01

    The ability of cancer cells to suppress apoptosis is critical for carcinogenesis. The BCL-2 family proteins comprise the sentinel network that regulates the mitochondrial or intrinsic apoptotic response. Recent advances in our understanding of apoptotic signaling pathways have enabled methods to identify cancers that are primed to undergo apoptosis, and have revealed potential biomarkers that may predict which cancers will undergo apoptosis in response to specific therapies. Complementary efforts have focused on developing novel drugs that directly target anti-apoptotic BCL-2 family proteins. In this review, we summarize the current knowledge of the role of BCL-2 family members in cancer development and response to therapy, focusing on targeted therapeutics, recent progress in the development of apoptotic biomarkers, and therapeutic strategies designed to overcome deficiencies in apoptosis. PMID:25895919

  13. A novel anti-proliferative role of HMGA2in induction of apoptosis through caspase 2in primary human fibroblast cells

    PubMed Central

    Shi, Xi; Tian, Baoqing; Ma, Wenlong; Zhang, Na; Qiao, Yuehua; Li, Xiaoxue; Zhang, Yu; Huang, Baiqu; Lu, Jun

    2014-01-01

    The HMGA2 (high-mobility group AT-hook) protein has previously been shown as an oncoprotein, whereas ectopic expression of HMGA2 is found to induce growth arrest in primary cells. The precise mechanisms underlying this phenomenon remain to be unravelled. In the present study, we determined that HMGA2 was able to induce apoptosis in WI38 primary human cells. We show that WI38 cells expressing high level of HMGA2 were arrested at G2/M phase and exhibited apoptotic nuclear phenotypes. Meanwhile, the cleaved caspase 3 (cysteine aspartic acid-specific protease 3) was detected 8days after HMGA2 overexpression. Flow cytometric analysis confirmed that the ratio of cells undergoing apoptosis increased dramatically. Concurrently, other major apoptotic markers were also detected, including the up-regulation of p53, Bax and cleaved caspase 9, down-regulation of Bcl-2; as well as release of cytochrome c from the mitochondria. We further demonstrate that the shRNA (small-hairpin RNA)-mediated Apaf1 (apoptotic protease activating factor 1) silencing partially rescued the HMGA2-induced apoptosis, which was accompanied by the decrease of cleaved caspase-3 level and a decline of cell death ratio. Our results also reveal that ?H2A was accumulated in nuclei during the HMGA2-induced apoptosis along with the up-regulation of cleaved caspase 2, suggesting that the HMGA2-induced apoptosis was dependent on the pathway of DNA damage. Overall, the present study unravelled a novel function of HMGA2 in induction of apoptosis in human primary cell lines, and provided clues for clarification of the mechanistic action of HMGA2 in addition to its function as an oncoprotein. PMID:25300915

  14. Critical role for Death-Receptor Mediated Apoptotic Signaling in Viral Myocarditis

    PubMed Central

    DeBiasi, Roberta L.; Robinson, Bridget A.; Leser, J. Smith; Brown, R. Dale; Long, Carlin S.; Clarke, Penny

    2010-01-01

    Background Apoptosis of cardiac myocytes plays a key role in the pathogenesis of many cardiac diseases including viral myocarditis. The apoptotic signaling pathways that are activated during viral myocarditis and the role that these pathways play in disease pathogenesis have not been clearly delineated. Methods and Results We investigated the role of apoptotic signaling pathways following virus infection of primary cardiac myocytes. The death receptor associated initiator caspase, caspase 8, and the effector caspase, caspase 3, were significantly activated following infection of primary cardiac myocytes with myocarditic, but not non-myocarditic, reovirus strains. Furthermore, reovirus-induced cardiac myocyte apoptosis was significantly inhibited by soluble death receptors. In contrast, the mitochondrial membrane potential remained unaltered and caspase 9, the initiator caspase associated with mitochondrial apoptotic signaling, was only weakly activated in cardiac myocytes following infection with myocarditic reovirus strains. Inhibition of mitochondrial apoptotic signaling had no effect on reovirus-induced cardiac myocyte apoptosis. In accordance with our in vitro data, caspase 8, but not caspase 9, was significantly activated in the hearts of reovirus-infected mice. Conclusions Death receptor, but not mitochondrial, apoptotic signaling plays a key role in apoptosis following infection of cardiac myocytes with myocarditic reovirus strains. PMID:21055654

  15. Integrin ?V?5-mediated Removal of Apoptotic Cell Debris by the Eye Lens and Its Inhibition by UV Light Exposure.

    PubMed

    Chauss, Daniel; Brennan, Lisa A; Bakina, Olga; Kantorow, Marc

    2015-12-18

    Accumulation of apoptotic material is toxic and associated with cataract and other disease states. Identification of mechanisms that prevent accumulation of apoptotic debris is important for establishing the etiology of these diseases. The ocular lens is routinely assaulted by UV light that causes lens cell apoptosis and is associated with cataract formation. To date, no molecular mechanism for removal of toxic apoptotic debris has been identified in the lens. Vesicular debris within lens cells exposed to UV light has been observed raising speculation that lens cells themselves could act as phagocytes to remove toxic apoptotic debris. However, phagocytosis has not been confirmed as a function of the intact eye lens, and no mechanism for lens phagocytosis has been established. Here, we demonstrate that the eye lens is capable of phagocytizing extracellular lens cell debris. Using high throughput RNA sequencing and bioinformatics analysis, we establish that lens epithelial cells express members of the integrin ?V?5-mediated phagocytosis pathway and that internalized cell debris co-localizes with ?V?5 and with RAB7 and Rab-interacting lysosomal protein that are required for phagosome maturation and fusion with lysosomes. We demonstrate that the ?V?5 receptor is required for lens epithelial cell phagocytosis and that UV light treatment of lens epithelial cells results in damage to the ?V?5 receptor with concomitant loss of phagocytosis. These data suggest that loss of ?V?5-mediated phagocytosis by the eye lens could result in accumulation of toxic cell debris that could contribute to UV light-induced cataract formation. PMID:26527683

  16. Mucin 1 gene silencing inhibits the growth of SMMC-7721 human hepatoma cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways.

    PubMed

    Yuan, Hongyan; Wang, Juan; Wang, Fengli; Zhang, Nannan; Li, Qiongshu; Xie, Fei; Chen, Tanxiu; Zhai, Ruiping; Wang, Fang; Guo, Yingying; Ni, Weihua; Tai, Guixiang

    2015-11-01

    Mucin 1 (MUC1) is an oncogene that has a crucial role in the pathogenesis and progression of the majority of epithelial malignant tumors. Our previous study demonstrated that MUC1 gene silencing inhibited the growth of SMMC‑7721 cells in vitro and in vivo, however, whether this growth inhibition is associated with apoptotic cell death remains to be elucidated. In the present study, it was found that MUC1 gene silencing not only resulted in the inhibition of SMMC‑7721 cell growth, determined using a clone formation assay in vitro and a tumor xenograft mouse model with an in vivo imaging system, but also induced apoptotic alterations in SMMC‑7721 cells, determined using Hoechst 33342 staining, flow cytometry with an Annexin V-PE staining and a DNA ladder assay. Further investigation using western blotting revealed that cytochrome c was released from the mitochondria into the cytoplasm, and caspase‑8 and caspase‑9 were activated in MUC1 gene‑silenced SMMC‑7721 cells. The pro‑apoptotic protein Bcl‑2‑associated X protein (Bax) and the tumor suppressor p53 were increased, while the anti‑apoptotic protein B‑cell lymphoma 2 was decreased in MUC1 gene‑silenced cells. In addition, results from the co‑immunoprecipitation experiments demonstrated that the MUC1 cytoplasmic tail can bind directly to Bax or caspase‑8 and these interactions were reduced upon MUC1 gene silencing in SMMC‑7721 cells. The above results indicate that MUC1 gene silencing induces growth inhibition in SMMC‑7721 cells through Bax‑mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways. PMID:26398332

  17. Mucin 1 gene silencing inhibits the growth of SMMC-7721 human hepatoma cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways

    PubMed Central

    YUAN, HONGYAN; WANG, JUAN; WANG, FENGLI; ZHANG, NANNAN; LI, QIONGSHU; XIE, FEI; CHEN, TANXIU; ZHAI, RUIPING; WANG, FANG; GUO, YINGYING; NI, WEIHUA; TAI, GUIXIANG

    2015-01-01

    Mucin 1 (MUC1) is an oncogene that has a crucial role in the pathogenesis and progression of the majority of epithelial malignant tumors. Our previous study demonstrated that MUC1 gene silencing inhibited the growth of SMMC-7721 cells in vitro and in vivo, however, whether this growth inhibition is associated with apoptotic cell death remains to be elucidated. In the present study, it was found that MUC1 gene silencing not only resulted in the inhibition of SMMC-7721 cell growth, determined using a clone formation assay in vitro and a tumor xenograft mouse model with an in vivo imaging system, but also induced apoptotic alterations in SMMC-7721 cells, determined using Hoechst 33342 staining, flow cytometry with an Annexin V-PE staining and a DNA ladder assay. Further investigation using western blotting revealed that cytochrome c was released from the mitochondria into the cytoplasm, and caspase-8 and caspase-9 were activated in MUC1 gene-silenced SMMC-7721 cells. The pro-apoptotic protein Bcl-2-associated X protein (Bax) and the tumor suppressor p53 were increased, while the anti-apoptotic protein B-cell lymphoma 2 was decreased in MUC1 gene-silenced cells. In addition, results from the co-immunoprecipitation experiments demonstrated that the MUC1 cytoplasmic tail can bind directly to Bax or caspase-8 and these interactions were reduced upon MUC1 gene silencing in SMMC-7721 cells. The above results indicate that MUC1 gene silencing induces growth inhibition in SMMC-7721 cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways. PMID:26398332

  18. Clathrin and AP2 Are Required for Phagocytic Receptor-Mediated Apoptotic Cell Clearance in Caenorhabditis elegans

    PubMed Central

    Liu, Xuezhao; Zhang, Yuanya; Liang, Jingjing; Qi, Xiaying; Du, Hongwei; Zou, Wei; Chen, Lianwan; Chai, Yongping; Ou, Guangshuo; Miao, Long; Wang, Yingchun; Yang, Chonglin

    2013-01-01

    Clathrin and the multi-subunit adaptor protein complex AP2 are central players in clathrin-mediated endocytosis by which the cell selectively internalizes surface materials. Here, we report the essential role of clathrin and AP2 in phagocytosis of apoptotic cells. In Caenorhabditis elegans, depletion of the clathrin heavy chain CHC-1 and individual components of AP2 led to a significant accumulation of germ cell corpses, which resulted from defects in both cell corpse engulfment and phagosome maturation required for corpse removal. CHC-1 and AP2 components associate with phagosomes in an inter-dependent manner. Importantly, we found that the phagocytic receptor CED-1 interacts with the α subunit of AP2, while the CED-6/Gulp adaptor forms a complex with both CHC-1 and the AP2 complex, which likely mediates the rearrangement of the actin cytoskeleton required for cell corpse engulfment triggered by the CED-1 signaling pathway. In addition, CHC-1 and AP2 promote the phagosomal association of LST-4/Snx9/18/33 and DYN-1/dynamin by forming a complex with them, thereby facilitating the maturation of phagosomes necessary for corpse degradation. These findings reveal a non-classical role of clathrin and AP2 and establish them as indispensable regulators in phagocytic receptor-mediated apoptotic cell clearance. PMID:23696751

  19. Lipid raft-mediated Fas/CD95 apoptotic signaling in leukemic cells and normal leukocytes and therapeutic implications.

    PubMed

    Gajate, Consuelo; Mollinedo, Faustino

    2015-11-01

    Plasma membrane is now recognized to contain tightly packed cholesterol/sphingolipid-rich domains, known as lipid or membrane rafts, which are more ordered than the surrounding lipid bilayer. Lipid rafts are crucial for the compartmentalization of signaling processes in the membrane, mostly involved in cell survival and immune response. However, in the last 15 years, a large body of evidence has also identified raft platforms as scaffolds for the recruitment and clustering of death receptor Fas/CD95 and downstream signaling molecules, leading to the concept of death-promoting lipid rafts. This raft-Fas/CD95 coclustering was first described at the early 2000s as the underlying mechanism for the proapoptotic action of the alkylphospholipid analog edelfosine in leukemic cells, hence facilitating protein-protein interactions and conveying apoptotic signals independently of Fas/CD95 ligand. Edelfosine induces apoptosis in hematologic cancer cells and activated T-lymphocytes. Fas/CD95 raft coclustering is also promoted by Fas/CD95 ligand, agonistic Fas/CD95 antibodies, and additional antitumor drugs. Thus, death receptor recruitment in rafts is a physiologic process leading to cell demise that can be pharmacologically modulated. This redistribution and local accumulation of apoptotic molecules in membrane rafts, which are usually accompanied by displacement of survival signaling molecules, highlight how alterations in the apoptosis/survival signaling balance in specialized membrane regions modulate cell fate. Membrane rafts might also modulate apoptotic and nonapoptotic death receptor signaling. Here, we discuss the role of lipid rafts in Fas/CD95-mediated apoptotic cell signaling in hematologic cancer cells and normal leukocytes, with a special emphasis on their involvement as putative therapeutic targets in cancer and autoimmune diseases. PMID:26246489

  20. Engulfment of Activated Apoptotic Cells Abolishes TGF-?Mediated Immunoregulation via the Induction of IL-6

    PubMed Central

    Notley, Clare A.; Brown, Mark A.; McGovern, Jenny L.; Jordan, Christine K.

    2015-01-01

    Phagocytosis of apoptotic cells (ACs) is usually a potent immunoregulatory signal but can also promote inflammation. In this article, we show that administration of apoptotic dendritic cells (DCs) inhibited inflammation in vivo through increasing production of TGF-? from intrinsic DCs and B cells. However, ACs derived from LPS-activated DCs failed to restrain inflammation because of a short-lived but marked IL-6 response, which abolished the increase in TGF-?. Inhibition of IL-6 restored the protective anti-inflammatory properties of aACs and the TGF-? response. DCs isolated from mice that had received resting but not activated ACs could transfer the suppression of inflammation to recipient mice. These transferred DCs stimulated B cell TGF-? production and relied on an intact B cell compartment to limit inflammation. These results highlight how the activation state of AC governs their ability to control inflammation through reciprocal regulation of IL-6 and TGF-?. PMID:25601923

  1. Suppression of macrophage-mediated phagocytosis of apoptotic cells by soluble ?-glucan due to a failure of PKC-?II translocation.

    PubMed

    Sekiguchi, Suzuno; Tomisawa, Yui; Ohki, Tomomi; Tsuboi, Kumiko; Nagata, Kisaburo; Kobayashi, Yoshiro

    2016-02-01

    If apoptotic cells are not removed efficiently, they may proceed to the stage of secondary necrosis, which would cause inflammation. Therefore, identification of cause(s) and agent(s) for down-modulating phagocytosis of apoptotic cells would help understand the pathologies. In this study we found that macrophage-mediated phagocytosis of apoptotic cells was suppressed by both soluble and particulate ?-glucan. This suppression was not observed when secondary necrotic cells were used. The adhesion of apoptotic cells to macrophages was not suppressed by soluble ?-glucan, suggesting that soluble ?-glucan suppresses phagocytosis at a post-adhesion step. Experiments involving PKC inhibitors suggested that PKC-?II is required for phagocytosis of apoptotic cells but not secondary necrotic ones by macrophages. Translocation of GFP-PKC-?II from the cytoplasm to membranes occurred upon interaction with apoptotic cells but not secondary necrotic ones. Such translocation was inhibited by soluble ?-glucan. Overall, this study suggests that suppression of macrophage-mediated phagocytosis of apoptotic cells by soluble ?-glucan is due to a failure of PKC-?II translocation. PMID:26745713

  2. Integrin ?PS3/??-mediated phagocytosis of apoptotic cells and bacteria in Drosophila.

    PubMed

    Nonaka, Saori; Nagaosa, Kaz; Mori, Toshinobu; Shiratsuchi, Akiko; Nakanishi, Yoshinobu

    2013-04-12

    Integrins exert a variety of cellular functions as heterodimers of two transmembrane subunits named ? and ?. Integrin ??, a ?-subunit of Drosophila integrin, is involved in the phagocytosis of apoptotic cells and bacteria. Here, we searched for an ?-subunit that forms a complex and cooperates with ??. Examinations of RNAi-treated animals suggested that ?PS3, but not any of four other ?-subunits, is required for the effective phagocytosis of apoptotic cells in Drosophila embryos. The mutation of ?PS3-encoding scb, deficiency, insertion of P-element, or alteration of nucleotide sequences, brought about a reduction in the level of phagocytosis. The defect in phagocytosis by deficiency was reverted by the forced expression of scb. Furthermore, flies in which the expression of both ?PS3 and ?? was inhibited by RNAi showed a level of phagocytosis almost equal to that observed in flies with RNAi for either subunit alone. A loss of ?PS3 also decreased the activity of larval hemocytes in the phagocytosis of Staphylococcus aureus. Finally, a co-immunoprecipitation analysis using a Drosophila cell line treated with a chemical cross-linker suggested a physical association between ?PS3 and ??. These results collectively indicated that integrin ?PS3/?? serves as a receptor in the phagocytosis of apoptotic cells and bacteria by Drosophila phagocytes. PMID:23426364

  3. Integrin ?PS3/??-mediated Phagocytosis of Apoptotic Cells and Bacteria in Drosophila*

    PubMed Central

    Nonaka, Saori; Nagaosa, Kaz; Mori, Toshinobu; Shiratsuchi, Akiko; Nakanishi, Yoshinobu

    2013-01-01

    Integrins exert a variety of cellular functions as heterodimers of two transmembrane subunits named ? and ?. Integrin ??, a ?-subunit of Drosophila integrin, is involved in the phagocytosis of apoptotic cells and bacteria. Here, we searched for an ?-subunit that forms a complex and cooperates with ??. Examinations of RNAi-treated animals suggested that ?PS3, but not any of four other ?-subunits, is required for the effective phagocytosis of apoptotic cells in Drosophila embryos. The mutation of ?PS3-encoding scb, deficiency, insertion of P-element, or alteration of nucleotide sequences, brought about a reduction in the level of phagocytosis. The defect in phagocytosis by deficiency was reverted by the forced expression of scb. Furthermore, flies in which the expression of both ?PS3 and ?? was inhibited by RNAi showed a level of phagocytosis almost equal to that observed in flies with RNAi for either subunit alone. A loss of ?PS3 also decreased the activity of larval hemocytes in the phagocytosis of Staphylococcus aureus. Finally, a co-immunoprecipitation analysis using a Drosophila cell line treated with a chemical cross-linker suggested a physical association between ?PS3 and ??. These results collectively indicated that integrin ?PS3/?? serves as a receptor in the phagocytosis of apoptotic cells and bacteria by Drosophila phagocytes. PMID:23426364

  4. Argon Mediates Anti-Apoptotic Signaling and Neuroprotection via Inhibition of Toll-Like Receptor 2 and 4

    PubMed Central

    Ulbrich, Felix; Kaufmann, Kai; Roesslein, Martin; Wellner, Franziska; Auwärter, Volker; Kempf, Jürgen; Loop, Torsten; Buerkle, Hartmut; Goebel, Ulrich

    2015-01-01

    Purpose Recently, the noble gas argon attracted significant attention due to its neuroprotective properties. However, the underlying molecular mechanism is still poorly understood. There is growing evidence that the extracellular regulated kinase 1/2 (ERK1/2) is involved in Argon´s protective effect. We hypothesized that argon mediates its protective effects via the upstream located toll-like receptors (TLRs) 2 and 4. Methods Apoptosis in a human neuroblastoma cell line (SH-SY5Y) was induced using rotenone. Argon treatment was performed after induction of apoptosis with different concentrations (25, 50 and 75 Vol% in oxygen 21 Vol%, carbon dioxide and nitrogen) for 2 or 4 hours respectively. Apoptosis was analyzed using flow cytometry (annexin-V (AV)/propidiumiodide (PI)) staining, caspase-3 activity and caspase cleavage. TLR density on the cells’ surface was analyzed using FACS and immunohistochemistry. Inhibition of TLR signaling and extracellular regulated kinase 1/2 (ERK1/2) were assessed by western blot, activity assays and FACS analysis. Results Argon 75 Vol% treatment abolished rotenone-induced apoptosis. This effect was attenuated dose- and time-dependently. Argon treatment was accompanied with a significant reduction of TLR2 and TLR4 receptor density and protein expression. Moreover, argon mediated increase in ERK1/2 phosphorylation was attenuated after inhibition of TLR signaling. ERK1/2 and TLR signaling inhibitors abolished the anti-apoptotic and cytoprotective effects of argon. Immunohistochemistry results strengthened these findings. Conclusion These findings suggest that argon-mediated anti-apoptotic and neuroprotective effects are mediated via inhibition of TLR2 and TLR4. PMID:26624894

  5. HSP70 mediates survival in apoptotic cellsBoolean network prediction and experimental validation

    PubMed Central

    Vasaikar, Suhas V.; Ghosh, Sourish; Narain, Priyam; Basu, Anirban; Gomes, James

    2015-01-01

    Neuronal stress or injury results in the activation of proteins, which regulate the balance between survival and apoptosis. However, the complex mechanism of cell signaling involving cell death and survival, activated in response to cellular stress is not yet completely understood. To bring more clarity about these mechanisms, a Boolean network was constructed that represented the apoptotic pathway in neuronal cells. FasL and neurotrophic growth factor (NGF) were considered as inputs in the absence and presence of heat shock proteins known to shift the balance toward survival by rescuing pro-apoptotic cells. The probabilities of survival, DNA repair and apoptosis as cellular fates, in the presence of either the growth factor or FasL, revealed a survival bias encoded in the network. Boolean predictions tested by measuring the mRNA level of caspase-3, caspase-8, and BAX in neuronal Neuro2a (N2a) cell line with NGF and FasL as external input, showed positive correlation with the observed experimental results for survival and apoptotic states. It was observed that HSP70 contributed more toward rescuing cells from apoptosis in comparison to HSP27, HSP40, and HSP90. Overexpression of HSP70 in N2a transfected cells showed reversal of cellular fate from FasL-induced apoptosis to survival. Further, the pro-survival role of the proteins BCL2, IAP, cFLIP, and NF?B determined by vertex perturbation analysis was experimentally validated through protein inhibition experiments using EM20-25, Embelin and Wedelolactone, which resulted in 1.27-, 1.26-, and 1.46-fold increase in apoptosis of N2a cells. The existence of a one-to-one correspondence between cellular fates and attractor states shows that Boolean networks may be employed with confidence in qualitative analytical studies of biological networks. PMID:26379495

  6. Regulation of Apoptotic Mediators Reveals Dynamic Responses to Thermal Stress in the Reef Building Coral Acropora millepora

    PubMed Central

    Pernice, Mathieu; Dunn, Simon R.; Miard, Thomas; Dufour, Sylvie; Dove, Sophie; Hoegh-Guldberg, Ove

    2011-01-01

    Background Mass coral bleaching is increasing in scale and frequency across the world's coral reefs and is being driven primarily by increased levels of thermal stress arising from global warming. In order to understand the impacts of projected climate change upon corals reefs, it is important to elucidate the underlying cellular mechanisms that operate during coral bleaching and subsequent mortality. In this respect, increased apoptotic cell death activity is an important cellular process that is associated with the breakdown of the mutualistic symbiosis between the cnidarian host and their dinoflagellate symbionts. Methodology/Principal Findings The present study reports the impacts of different stressors (colchicine and heat stress) on three phases of apoptosis: (i) the potential initiation by differential expression of Bcl-2 members, (ii) the execution of apoptotic events by activation of caspase 3-like proteases and (iii) and finally, the cell disposal indicated by DNA fragmentation in the reef building coral Acropora millepora. In corals incubated with colchicine, an increase in caspase 3-like activity and DNA fragmentation was associated with a relative down-regulation of Bcl-2, suggesting that the initiation of apoptosis may be mediated by the suppression of an anti-apoptotic mechanism. In contrast, in the early steps of heat stress, the induction of caspase-dependent apoptosis was related to a relative up-regulation of Bcl-2 consecutively followed by a delayed decrease in apoptosis activity. Conclusions/Significance In the light of these results, we propose a model of heat stress in coral hosts whereby increasing temperatures engage activation of caspase 3-dependent apoptosis in cells designated for termination, but also the onset of a delayed protective response involving overexpression of Bcl-2 in surviving cells. This mitigating response to thermal stress could conceivably be an important regulatory mechanism for cell survival in corals exposed to sudden environmental changes. PMID:21283671

  7. Cracking the Cytotoxicity Code: Apoptotic Induction of 10-Acetylirciformonin B is Mediated through ROS Generation and Mitochondrial Dysfunction

    PubMed Central

    Shih, Huei-Chuan; El-Shazly, Mohamed; Juan, Yung-Shun; Chang, Chao-Yuan; Su, Jui-Hsin; Chen, Yu-Cheng; Shih, Shou-Ping; Chen, Huei-Mei; Wu, Yang-Chang; Lu, Mei-Chin

    2014-01-01

    A marine furanoterpenoid derivative, 10-acetylirciformonin B (10AB), was found to inhibit the proliferation of leukemia, hepatoma, and colon cancer cell lines, with selective and significant potency against leukemia cells. It induced DNA damage and apoptosis in leukemia HL 60 cells. To fully understand the mechanism behind the 10AB apoptotic induction against HL 60 cells, we extended our previous findings and further explored the precise molecular targets of 10AB. We found that the use of 10AB increased apoptosis by 8.9%–87.6% and caused disruption of mitochondrial membrane potential (MMP) by 15.2%–95.2% in a dose-dependent manner, as demonstrated by annexin-V/PI and JC-1 staining assays, respectively. Moreover, our findings indicated that the pretreatment of HL 60 cells with N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger, diminished MMP disruption and apoptosis induced by 10AB, suggesting that ROS overproduction plays a crucial rule in the cytotoxic activity of 10AB. The results of a cell-free system assay indicated that 10AB could act as a topoisomerase catalytic inhibitor through the inhibition of topoisomerase IIα. On the protein level, the expression of the anti-apoptotic proteins Bcl-xL and Bcl-2, caspase inhibitors XIAP and survivin, as well as hexokinase II were inhibited by the use of 10AB. On the other hand, the expression of the pro-apoptotic protein Bax was increased after 10AB treatment. Taken together, our results suggest that 10AB-induced apoptosis is mediated through the overproduction of ROS and the disruption of mitochondrial metabolism. PMID:24857964

  8. Cracking the cytotoxicity code: apoptotic induction of 10-acetylirciformonin B is mediated through ROS generation and mitochondrial dysfunction.

    PubMed

    Shih, Huei-Chuan; El-Shazly, Mohamed; Juan, Yung-Shun; Chang, Chao-Yuan; Su, Jui-Hsin; Chen, Yu-Cheng; Shih, Shou-Ping; Chen, Huei-Mei; Wu, Yang-Chang; Lu, Mei-Chin

    2014-05-01

    A marine furanoterpenoid derivative, 10-acetylirciformonin B (10AB), was found to inhibit the proliferation of leukemia, hepatoma, and colon cancer cell lines, with selective and significant potency against leukemia cells. It induced DNA damage and apoptosis in leukemia HL 60 cells. To fully understand the mechanism behind the 10AB apoptotic induction against HL 60 cells, we extended our previous findings and further explored the precise molecular targets of 10AB. We found that the use of 10AB increased apoptosis by 8.9%-87.6% and caused disruption of mitochondrial membrane potential (MMP) by 15.2%-95.2% in a dose-dependent manner, as demonstrated by annexin-V/PI and JC-1 staining assays, respectively. Moreover, our findings indicated that the pretreatment of HL 60 cells with N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger, diminished MMP disruption and apoptosis induced by 10AB, suggesting that ROS overproduction plays a crucial rule in the cytotoxic activity of 10AB. The results of a cell-free system assay indicated that 10AB could act as a topoisomerase catalytic inhibitor through the inhibition of topoisomerase II?. On the protein level, the expression of the anti-apoptotic proteins Bcl-xL and Bcl-2, caspase inhibitors XIAP and survivin, as well as hexokinase II were inhibited by the use of 10AB. On the other hand, the expression of the pro-apoptotic protein Bax was increased after 10AB treatment. Taken together, our results suggest that 10AB-induced apoptosis is mediated through the overproduction of ROS and the disruption of mitochondrial metabolism. PMID:24857964

  9. Local Augmented Angiotensinogen Secreted from Apoptotic Vascular Endothelial Cells Is a Vital Mediator of Vascular Remodelling

    PubMed Central

    Wu, Shyh-Jong; Chu, Chih-Sheng; Shih, Shih-Chuan; Hbert, Marie-Jose; Kuo, Mei-Chuan; Hsieh, Ya-Ju

    2015-01-01

    Vascular remodelling is a critical vasculopathy found in atheromatous diseases and allograft failures. The local renin angiotensin system (RAS) has been implicated in vascular remodelling. However, the mechanisms by which the augmented local RAS is associated with the initial event of endothelial cell apoptosis in injured vasculature remain undefined. We induced the apoptosis of human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs) through serum starvation (SS). After the cells were subjected to SS, we found that the mRNA expression of angiotensinogen (AGT) was increased by >3-fold in HUVECs and by approximately 2.5-fold in VSMCs. In addition, the expression of angiotensin-converting enzyme (ACE) mRNA was increased in VSMCs but decreased to 50% in HUVECs during the same apoptotic process. Increases in the expression of AGT protein and angiotensin II (Ang II) were found in a serum-free medium conditioned by HUVECs (SSC). The increased Ang II was suppressed using lisinopril (an ACE inhibitor) treatment. Moreover, the activation of ERK1/2 induced by the SSC in VSMCs was also suppressed by losartan. In conclusion, we first demonstrated that the augmented AGT released from apoptotic endothelial cells acts as a vital progenitor of Ang II to accelerate vascular remodelling, and we suggest that blocking local augmented Ang II might be an effective strategy for restraining intimal hyperplasia. PMID:26147666

  10. MiR-125a-5p decreases after long non-coding RNA HOTAIR knockdown to promote cancer cell apoptosis by releasing caspase 2.

    PubMed

    Tang, L; Shen, H; Li, X; Li, Z; Liu, Z; Xu, J; Ma, S; Zhao, X; Bai, X; Li, M; Wang, Q; Ji, J

    2016-01-01

    HOTAIR (homeobox transcript antisense RNA), one of the prototypical long non-coding RNAs, has been verified overexpressed in multiple carcinomas and has emerged as a promising novel anticancer target. Its well-established role is acting as a predictor of poor prognosis and promoting cancer cell metastasis. Recently, another important mission of HOTAIR was uncovered that targeting HOTAIR caused cancer cell apoptosis. Nevertheless, so far there is no published data elaborating the mechanism. Here, we report that microRNA miR-125a-5p decreases and releases caspase 2 to promote cancer cell apoptosis after HOTAIR knockdown. We applied siRNAs targeting HOTAIR to various cancer cells, and observed apoptosis in all of these cell lines. RNA sequencing detected that miR-125a-5p was decreased after HOTAIR knockdown and miR-125a-5p mimics could rescue the apoptosis induced by HOTAIR deficiency. Luciferase assays identified caspase 2, an initiator caspase, to be a new target of miR-125a-5p. Elevated expression and subsequent cleavage of caspase 2 was observed after HOTAIR knockdown or inhibition of miR-125a-5p. RNAi of caspase 2 could attenuate the apoptosis induced by HOTAIR knockdown. In 80 clinical colon cancer tissues, HOTAIR and miR-125a-5p levels were higher than adjacent tissues, whereas caspase 2 was lower. MiR-125a-5p expression level was significantly correlated with colon tumor size, lymph node metastasis and clinical stage. These findings indicate that miR-125a-5p decreases after HOTAIR knockdown to promote cancer cell apoptosis by releasing caspase 2. Our work reveals a previously unidentified apoptotic mechanism, which might be exploitable in anticancer drug development. PMID:26962687

  11. Association of a Methanol Extract of Rheum undulatum L. Mediated Cell Death in AGS Cells with an Intrinsic Apoptotic Pathway

    PubMed Central

    Hong, Noo Ri; Park, Hyun Soo; Ahn, Tae Seok; Jung, Myeong Ho; Kim, Byung Joo

    2015-01-01

    Objectives: Rheum undulatum L. has traditionally been used for the treatment of many diseases in Asia. However, its anti-proliferative activity in cancer has still not been studied. In the present study, we investigated the anti-cancer effects of methanol extract of Rheum undulatum L. (MERL) on human adenocarcinoma gastric cell lines (AGS). Methods: To investigate the anti-cancer effect of MERL on AGS cells, we treated the AGS cells with varying concentrations of MERL and performed 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Cell cycle analyses, measurements of the mitochondrial membrane potential (MMP), caspase activity assays and Western blots were conducted to determine whether AGS cell death occurred by apoptosis. Results: Treatment with MERL significantly inhibited growth of AGS cells in a concentration dependent manner. MERL treatment in AGS cells leaded to increased accumulation of apoptotic sub G1 phase cells in a concentration dependent manner. In control cultures, 5.38% of the cells were in the sub G1 phase. In MERL treated cells, however, this percentage was significantly increased (9.95% at 70 ?g/mL, 15.94% at 140 ?g/mL, 26.56% at 210 ?g/mL and 38.08% at 280 ?g/mL). MERL treatment induced the decreased expression of pro-caspase-8 and -9 in a concentration dependent manner, whereas the expression of the active form of caspase-3 was increased. A subsequent Western blot analysis revealed increased cleaved levels of poly (ADP-ribose) polymerase (PARP) protein. Also, treatment with MERL increased the activities of caspase-3 and -9 compared with the control. MERL treatment increased the levels of the pro-apoptotic truncated Bid (tBid) and Bcl2 Antagonist X (Bax) proteins and decreased the levels of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein, whose is the stabilization of mitochondria. However, inhibitions of p38, extracellular signal regulated kinases (ERKs) and C-Jun N-terminal kinases (JNK) by MERL treatment did not affect cell death. Conclusion: These results suggest that MERL mediated cell death is associated with an intrinsic apoptotic pathway in AGS cells. PMID:26120485

  12. p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa.

    PubMed

    Villunger, Andreas; Michalak, Ewa M; Coultas, Leigh; Mllauer, Franziska; Bck, Gunther; Ausserlechner, Michael J; Adams, Jerry M; Strasser, Andreas

    2003-11-01

    Apoptosis provoked by DNA damage requires the p53 tumor suppressor, but which of the many p53-regulated genes are required has remained unknown. Two genes induced by this transcription factor, noxa and puma (bbc3), stand out, because they encode BH3-only proteins, proapoptotic members of the Bcl-2 family required to initiate apoptosis. In mice with either noxa or puma disrupted, we observed decreased DNA damage-induced apoptosis in fibroblasts, although only loss of Puma protected lymphocytes from cell death. Puma deficiency also protected cells against diverse p53-independent cytotoxic insults, including cytokine deprivation and exposure to glucocorticoids, the kinase inhibitor staurosporine, or phorbol ester. Hence, Puma and Noxa are critical mediators of the apoptotic responses induced by p53 and other agents. PMID:14500851

  13. Apoptotic effects of Antrodia cinnamomea fruiting bodies extract are mediated through calcium and calpain-dependent pathways in Hep 3B cells.

    PubMed

    Kuo, Po-Lin; Hsu, Ya-Ling; Cho, Chien-Yu; Ng, Lean-Teik; Kuo, Yueh-Hsiung; Lin, Chun-Ching

    2006-08-01

    Antrodia cinnamomea is well known in Taiwan as a traditional medicine for treating cancer and inflammation. The purpose of this study was to evaluate the apoptotic effects of ethylacetate extract from A. cinnamomea (EAC) fruiting bodies in Hep 3B, a liver cancer cell line. EAC decreased cell proliferation of Hep 3B cells by inducing apoptotic cell death. EAC treatment increased the level of calcium (Ca2+) in the cytoplasm and triggered the subsequent activation of calpain and caspase-12. EAC also initiated the mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 in Hep 3B cells. Furthermore, the mitochondrial apoptotic pathway amplified the calpain pathway by Bid and Bax interaction and Ca2+ translocation. We have therefore concluded that the molecular mechanisms during EAC-mediated proliferation inhibition in Hep 3B cells were due to: (1) apoptosis induction, (2) triggering of Ca2+/calpain pathway, (3) disruption of mitochondrial function, and (4) apoptotic signaling being amplified by cross-talk between the calpain/Bid/Bax and Ca2+/mitochondrial apoptotic pathways. PMID:16600460

  14. p85α recruitment by the CD300f phosphatidylserine receptor mediates apoptotic cell clearance required for autoimmunity suppression

    PubMed Central

    Tian, Linjie; Choi, Seung-Chul; Murakami, Yousuke; Allen, Joselyn; Morse, Herbert C.; Qi, Chen-Feng; Krzewski, Konrad; Coligan, John E

    2014-01-01

    Apoptotic cell (AC) clearance is essential for immune homeostasis. Here we show that mouse CD300f (CLM-1) recognizes outer membrane-exposed phosphatidylserine, and regulates the phagocytosis of ACs. CD300f accumulates in phagocytic cups at AC contact sites. Phosphorylation within CD300f cytoplasmic tail tyrosine-based motifs initiates signals that positively or negatively regulate AC phagocytosis. Y276 phosphorylation is necessary for enhanced CD300f-mediated phagocytosis through the recruitment of the p85α regulatory subunit of phosphatidylinositol-3-kinase (PI3K). CD300f-PI3K association leads to activation of downstream Rac/Cdc42 GTPase and mediates changes of F-actin that drive AC engulfment. Importantly, primary macrophages from CD300f-deficient mice have impaired phagocytosis of ACs. The biological consequence of CD300f deficiency is predisposition to autoimmune disease development, as FcγRIIB-deficient mice develop a systemic lupus erythematosus-like disease at a markedly accelerated rate if CD300f is absent. In this report we identify the mechanism and role of CD300f in AC phagocytosis and maintenance of immune homeostasis. PMID:24477292

  15. p85? recruitment by the CD300f phosphatidylserine receptor mediates apoptotic cell clearance required for autoimmunity suppression

    NASA Astrophysics Data System (ADS)

    Tian, Linjie; Choi, Seung-Chul; Murakami, Yousuke; Allen, Joselyn; Morse, Herbert C., III; Qi, Chen-Feng; Krzewski, Konrad; Coligan, John E.

    2014-01-01

    Apoptotic cell (AC) clearance is essential for immune homeostasis. Here we show that mouse CD300f (CLM-1) recognizes outer membrane-exposed phosphatidylserine, and regulates the phagocytosis of ACs. CD300f accumulates in phagocytic cups at AC contact sites. Phosphorylation within CD300f cytoplasmic tail tyrosine-based motifs initiates signals that positively or negatively regulate AC phagocytosis. Y276 phosphorylation is necessary for enhanced CD300f-mediated phagocytosis through the recruitment of the p85? regulatory subunit of phosphatidylinositol-3-kinase (PI3K). CD300f-PI3K association leads to activation of downstream Rac/Cdc42 GTPase and mediates changes of F-actin that drive AC engulfment. Importantly, primary macrophages from CD300f-deficient mice have impaired phagocytosis of ACs. The biological consequence of CD300f deficiency is predisposition to autoimmune disease development, as Fc?RIIB-deficient mice develop a systemic lupus erythematosus-like disease at a markedly accelerated rate if CD300f is absent. In this report we identify the mechanism and role of CD300f in AC phagocytosis and maintenance of immune homeostasis.

  16. Targeted Apoptotic Effects of Thymoquinone and Tamoxifen on XIAP Mediated Akt Regulation in Breast Cancer

    PubMed Central

    Rajput, Shashi; Kumar, B. N. Prashanth; Sarkar, Siddik; Das, Subhasis; Azab, Belal; Santhekadur, Prasanna K.; Das, Swadesh K.; Emdad, Luni; Sarkar, Devanand; Fisher, Paul B.; Mandal, Mahitosh

    2013-01-01

    X-linked inhibitor of apoptosis protein (XIAP) is constitutively expressed endogenous inhibitor of apoptosis, exhibit its antiapoptotic effect by inactivating key caspases such as caspase-3, caspase-7 and caspase-9 and also play pivotal role in rendering cancer chemoresistance. Our studies showed the coadministration of TQ and TAM resulting in a substantial increase in breast cancer cell apoptosis and marked inhibition of cell growth both in vitro and in vivo. Anti-angiogenic and anti-invasive potential of TQ and TAM was assessed through in vitro studies. This novel combinatorial regimen leads to regulation of multiple cell signaling targets including inactivation of Akt and XIAP degradation. At molecular level, TQ and TAM synergistically lowers XIAP expression resulting in binding and activation of caspase-9 in apoptotic cascade, and interfere with cell survival through PI3-K/Akt pathway by inhibiting Akt phosphorylation. Cleaved caspase-9 further processes other intracellular death substrates such as PARP thereby shifting the balance from survival to apoptosis, indicated by rise in the sub-G1 cell population. This combination also downregulates the expression of Akt-regulated downstream effectors such as Bcl-xL, Bcl-2 and induce expression of Bax, AIF, cytochrome C and p-27. Consistent with these results, overexpression studies further confirmed the involvement of XIAP and its regulatory action on Akt phosphorylation along with procaspase-9 and PARP cleavage in TQ-TAM coadministrated induced apoptosis. The ability of TQ and TAM in inhibiting XIAP was confirmed through siRNA-XIAP cotransfection studies. This novel modality may be a promising tool in breast cancer treatment. PMID:23613836

  17. Fibril growth and seeding capacity play key roles in ?-synuclein-mediated apoptotic cell death.

    PubMed

    Mahul-Mellier, A-L; Vercruysse, F; Maco, B; Ait-Bouziad, N; De Roo, M; Muller, D; Lashuel, H A

    2015-12-01

    The role of extracellular ?-synuclein (?-syn) in the initiation and the spreading of neurodegeneration in Parkinson's disease (PD) has been studied extensively over the past 10 years. However, the nature of the ?-syn toxic species and the molecular mechanisms by which they may contribute to neuronal cell loss remain controversial. In this study, we show that fully characterized recombinant monomeric, fibrillar or stabilized forms of oligomeric ?-syn do not trigger significant cell death when added individually to neuroblastoma cell lines. However, a mixture of preformed fibrils (PFFs) with monomeric ?-syn becomes toxic under conditions that promote their growth and amyloid formation. In hippocampal primary neurons and ex vivo hippocampal slice cultures, ?-syn PFFs are capable of inducing a moderate toxicity over time that is greatly exacerbated upon promoting fibril growth by addition of monomeric ?-syn. The causal relationship between ?-syn aggregation and cellular toxicity was further investigated by assessing the effect of inhibiting fibrillization on ?-syn-induced cell death. Remarkably, our data show that blocking fibril growth by treatment with known pharmacological inhibitor of ?-syn fibrillization (Tolcapone) or replacing monomeric ?-syn by monomeric ?-synuclein in ?-syn mixture composition prevent ?-syn-induced toxicity in both neuroblastoma cell lines and hippocampal primary neurons. We demonstrate that exogenously added ?-syn fibrils bind to the plasma membrane and serve as nucleation sites for the formation of ?-syn fibrils and promote the accumulation and internalization of these aggregates that in turn activate both the extrinsic and intrinsic apoptotic cell death pathways in our cellular models. Our results support the hypothesis that ongoing aggregation and fibrillization of extracellular ?-syn play central roles in ?-syn extracellular toxicity, and suggest that inhibiting fibril growth and seeding capacity constitute a viable strategy for protecting against ?-syn-induced toxicity and slowing the progression of neurodegeneration in PD and other synucleinopathies. PMID:26138444

  18. The ER Stress-Mediated Mitochondrial Apoptotic Pathway and MAPKs Modulate Tachypacing-Induced Apoptosis in HL-1 Atrial Myocytes

    PubMed Central

    Shi, Jiaojiao; Jiang, Qi; Ding, Xiangwei; Xu, Wenhua; Wang, Dao W.; Chen, Minglong

    2015-01-01

    Background and Object Cell apoptosis is a contributing factor in the initiation, progression and relapse of atrial fibrillation (AF), a life-threatening illness accompanied with stroke and heart failure. However, the regulatory cascade of apoptosis is intricate and remains unidentified, especially in the setting of AF. The aim of this study was to explore the roles of endoplasmic reticulum (ER) stress, mitochondrial apoptotic pathway (MAP), mitogen-activated protein kinases (MAPKs), and their cross-talking in tachypacing-induced apoptosis. Methods and Results HL-1 cells were cultured in the presence of tachypacing for 24 h to simulate atrial tachycardia remodeling. Results showed that tachypacing reduced cell viability measured by the cell counting kit-8, dissipated mitochondrial membrane potential detected by JC-1 staining and resulted in approximately 50% apoptosis examined by Hoechst staining and annexin V/propidium iodide staining. In addition, the proteins involved in ER stress, MAP and MAPKs were universally up-regulated or activated via phosphorylation, as confirmed by western blotting; and reversely silencing of ER stress, caspase-3 (the ultimate executor of MAP) and MAPKs with specific inhibitors prior to pacing partially alleviated apoptosis. An inhibitor of ER stress was applied to further investigate the responses of mitochondria and MAPKs to ER stress, and results indicated that suppression of ER stress comprehensively but incompletely attenuated the activation of MAP and MAPKs aroused by tachypacing, with the exception of ERK1/2, one branch of MAPKs. Conclusions Our study suggested tachypacing-induced apoptosis is regulated by ER stress-mediated MAP and MAPKs. Thus, the above three components are all promising anti-apoptotic targets in AF patients and ER stress appears to play a dominant role due to its comprehensive effects. PMID:25689866

  19. Reduced IRE1? mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor

    PubMed Central

    Son, S M; Byun, J; Roh, S-E; Kim, S J; Mook-Jung, I

    2014-01-01

    The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca2+. Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer's and Parkinson diseases. One key regulator that underlies cell survival and Ca2+ homeostasis during ER stress responses is inositol-requiring enzyme 1? (IRE1?). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca2+ dysregulation via the IRE1?-dependent signaling pathway. In this study, we show that inactivation of IRE1? by RNA interference increases cytosolic Ca2+ concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca2+ through the InsP3 receptor (InsP3R). The Ca2+ efflux in IRE1?-deficient cells correlates with dissociation of the Ca2+-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1?TRAF2ASK1 complex. The increased cytosolic concentration of Ca2+ induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca2+ dysregulation-induced mitochondrial abnormalities and cell death in IRE1?-deficient cells can be blocked by depleting ROS or inhibiting Ca2+ influx into the mitochondria. These results demonstrate the importance of IRE1? in Ca2+ homeostasis and cell survival during ER stress and reveal a previously unknown Ca2+-mediated cell death signaling between the IRE1?InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion. PMID:24743743

  20. Heat Stress Induces Apoptosis through a Ca2+-Mediated Mitochondrial Apoptotic Pathway in Human Umbilical Vein Endothelial Cells

    PubMed Central

    Liu, Zhifeng; Geng, Yan; Liu, Yunsong; Tong, Huasheng; Tang, Youqing; Qiu, Junmin; Su, Lei

    2014-01-01

    Background Heat stress can be acutely cytotoxic, and heat stress-induced apoptosis is a prominent pathological feature of heat-related illnesses, although the precise mechanisms by which heat stress triggers apoptosis are poorly defined. Methods The percentages of viability and cell death were assessed by WST-1 and LDH release assays. Apoptosis was assayed by DNA fragmentation and caspase activity. Expression of cleaved PARP, Apaf-1, phospho-PERK, Phospho-eIF2a, ATF4, XBP-1s, ATF6, GRP78, phospho-IP3R, RYR and SERCA was estimated by Western blot. The effect of calcium overload was determined using flow cytometric analysis with the fluorescent probe Fluo-3/AM. The generation of ROS (O2?, H2O2, NO) was labeled by confocal laser scanning microscopy images of fluorescently and flow cytometry. Results In this study, we found that heat stress in HUVEC cells activated initiators of three major unfolded protein response (UPR) signaling transduction pathways: PERK-eIF2a-ATF4, IRE1-XBP-1S and ATF6 to protect against ER stress, although activation declined over time following cessation of heat stress. Furthermore, we show that intense heat stress may induce apoptosis in HUVEC cells through the calcium-mediated mitochondrial apoptotic pathway, as indicated by elevation of cytoplasmic Ca2+, expression of Apaf-1, activation of caspase-9 and caspase-3, PARP cleavage, and ultimately nucleosomal DNA fragmentation; Reactive oxygen species (ROS) appear to act upstream in this process. In addition, we provide evidence that IP3R upregulation may promote influx of Ca2+ into the cytoplasm after heat stress. Conclusion Our findings describe a novel mechanism for heat stress-induced apoptosis in HUVEC cells: following elevation of cytoplasm Ca2+, activation of the mitochondrial apoptotic pathway via the IP3R upregulation, with ROS acting as an upstream regulator of the process. PMID:25549352

  1. Tetraspanin CD37 Directly Mediates Transduction of Survival and Apoptotic Signals

    PubMed Central

    Lapalombella, Rosa; Yeh, Yuh-Ying; Wang, Liwen; Ramanunni, Asha; Rafiq, Sarwish; Jha, Shruti; Staubli, Justin; Lucas, David M.; Mani, Rajeswaran; Herman, Sarah E. M.; Johnson, Amy J.; Lozanski, Arletta; Andritsos, Leslie; Jones, Jeffrey; Flynn, Joseph M.; Lannutti, Brian; Thompson, Peter; Algate, Paul; Stromatt, Scott; Jarjoura, David; Mo, Xiaokui; Wang, Dasheng; Chen, Ching-Shih; Lozanski, Gerard; Heerema, Nyla A.; Tridandapani, Susheela; Freitas, Michael A.; Muthusamy, Natarajan; Byrd, John C.

    2012-01-01

    SUMMARY Tetraspanins are commonly believed to act only as molecular facilitators, with no direct role in signal transduction. We herein demonstrate that upon ligation, CD37, a tetraspanin molecule expressed on mature normal and transformed B-cells, becomes tyrosine phosphorylated, associates with proximal signaling molecules, and initiates a cascade of events leading to apoptosis. Moreover, we have identified two tyrosine residues with opposing regulatory functions, one lies in the N-terminal domain of CD37 in a predicted ITIM-like motif and mediates SHP1-dependent death whereas the second lies in a predicted ITAM motif in the C-terminal domain of CD37 and counteracts death signals by mediating phosphatidylinositol 3-kinase-dependent survival. PMID:22624718

  2. Chandipura Virus Induces Neuronal Death through Fas-Mediated Extrinsic Apoptotic Pathway

    PubMed Central

    Ghosh, Sourish; Dutta, Kallol

    2013-01-01

    Chandipura virus (CHPV; genus Vesiculovirus, family Rhabdoviridae) is an emerging tropical pathogen with a case fatality rate of 55 to 75% that predominantly affects children in the age group of 2 to 16 years. Although it has been established as a neurotropic virus causing encephalitis, the molecular pathology leading to neuronal death is unknown. The present study elucidates for the first time the mechanism of cell death in neurons after CHPV infection that answers the basic cause of CHPV-mediated neurodegeneration. Through various cell death assays in vitro and in vivo, a relationship between viral replication within neuron and neuronal apoptosis has been established. We report that expression of CHPV phosphoprotein increases up to 6 h postinfection and diminishes thereafter in neuronal cell lines, signifying the replicative phase of CHPV. Various analyses conducted during the investigation established that CHPV-infected neurons are undergoing apoptosis through an extrinsic pathway mediated through the Fas-associated death domain (FADD) following activation of caspase-8 and -3 and prominent cleavage of poly(ADP-ribose) polymerase (PARP). Knocking down the expression of caspase-3, the final executioner of apoptosis, in a neuronal cell line by endoribonuclease-prepared small interfering RNA (siRNA) validated its pivotal role in CHPV-mediated neurodegeneration by showing reduction in apoptosis after CHPV infection. PMID:24027318

  3. Protection of cells from nitric oxide-mediated apoptotic death by glutathione C?? derivative.

    PubMed

    Hu, Zhen; Zhang, Chunhua; Tang, Peiyi; Li, Cuiyun; Yao, Yuhuan; Sun, Shaofan; Zhang, Li; Huang, Yudong

    2012-07-01

    The influence of the glutathione C?? derivative on the cytotoxicity of a highly reactive free radical NO (nitric oxide) has been investigated. Consistent with its cytoprotective abilities, the derivative scavenges ROS (reactive oxygen species) and RNS (reactive nitrogen species) both in vitro and under cell-free conditions. Moreover, the glutathione C?? derivative protected PC12 cells from the cytotoxic effect of the NO-releasing compound, SNP (sodium nitroprusside). Addition of glutathione C?? derivative alone did not induce apoptosis and necrosis. The results suggest that the glutathione C?? derivative has the potential to prevent NO-mediated cell death without evident toxicity. PMID:22439806

  4. Paracrine apoptotic effect of p53 mediated by tumor suppressor Par-4.

    PubMed

    Burikhanov, Ravshan; Shrestha-Bhattarai, Tripti; Hebbar, Nikhil; Qiu, Shirley; Zhao, Yanming; Zambetti, Gerard P; Rangnekar, Vivek M

    2014-01-30

    The guardian of the genome, p53, is often mutated in cancer and may contribute to therapeutic resistance. Given that p53 is intact and functional in normal tissues, we harnessed its potential to inhibit the growth of p53-deficient cancer cells. Specific activation of p53 in normal fibroblasts selectively induced apoptosis in p53-deficient cancer cells. This paracrine effect was mediated by p53-dependent secretion of the tumor suppressor Par-4. Accordingly, the activation of p53 in normal mice, but not p53(-)/(-) or Par-4(-)/(-) mice, caused systemic elevation of Par-4, which induced apoptosis of p53-deficient tumor cells. Mechanistically, p53 induced Par-4 secretion by suppressing the expression of its binding partner, UACA, which sequesters Par-4. Thus, normal cells can be empowered by p53 activation to induce Par-4 secretion for the inhibition of therapy-resistant tumors. PMID:24412360

  5. Paracrine Apoptotic Effect of p53 Mediated by Tumor Suppressor Par-4

    PubMed Central

    Burikhanov, Ravshan; Shrestha-Bhattarai, Tripti; Hebbar, Nikhil; Qiu, Shirley; Zhao, Yanming; Zambetti, Gerard P.; Rangnekar, Vivek M.

    2014-01-01

    Summary The guardian of the genome, p53, is often mutated in cancer and may contribute to therapeutic resistance. As p53 is intact and functional in normal tissues, we harnessed its potential to inhibit the growth of p53-deficient cancer cells. Specific activation of p53 in normal fibroblasts selectively induced apoptosis in p53-deficient cancer cells. This paracrine effect was mediated by p53-dependent secretion of the tumor suppressor Par-4. Accordingly, activation of p53 in normal mice, but not in p53?/? or Par-4?/? mice, caused systemic elevation of Par-4 that induced apoptosis of p53-deficient tumor cells. Mechanistically, p53 induced Par-4 secretion by suppressing the expression of UACA, which sequesters Par-4. Thus, normal cells can be empowered by p53 activation to induce Par-4 secretion for inhibition of therapy-resistant tumors. PMID:24412360

  6. Inflammasome-mediated pyroptotic and apoptotic cell death, and defense against infection

    PubMed Central

    Aachoui, Youssef; Sagulenko, Vitaliya; Miao, Edward A; Stacey, Katryn J.

    2013-01-01

    Cell death is an effective strategy to limit intracellular infections. Canonical inflammasomes, including NLRP3, NLRC4, and AIM2, recruit and activate caspase-1 in response to a range of microbial stimuli and endogenous danger signals. Caspase-1 then promotes the secretion of IL-1? and IL-18 and a rapid form of lytic programmed cell death termed pyroptosis. A second inflammatory caspase, mouse caspase-11, mediates pyroptotic death through an unknown non-canonical inflammasome system in response to cytosolic bacteria. In addition, recent work shows that inflammasomes can also recruit procaspase-8, initiating apoptosis. The induction of multiple pathways of cell death has probably evolved to counteract microbial evasion of cell death pathways. PMID:23707339

  7. Caspase-2 promotes obesity, the metabolic syndrome and nonalcoholic fatty liver disease.

    PubMed

    Machado, M V; Michelotti, G A; Jewell, M L; Pereira, T A; Xie, G; Premont, R T; Diehl, A M

    2016-01-01

    Obesity and its resulting metabolic disturbances are major health threats. In response to energy surplus, overtaxed adipocytes release fatty acids and pro-inflammatory factors into the circulation, promoting organ fat accumulation (including nonalcoholic fatty liver disease), insulin resistance and the metabolic syndrome. Recently, caspase-2 was linked to lipoapoptosis, so we hypothesized that caspase-2 might be a critical determinant of metabolic syndrome pathogenesis. Caspase-2-deficient and wild-type mice were fed a Western diet (high-fat diet, enriched with saturated fatty acids and 0.2% cholesterol, supplemented with fructose and glucose in the drinking water) for 16 weeks. Metabolic and hepatic outcomes were evaluated. In vitro studies assessed the role of caspase-2 in adipose tissue proliferative properties and susceptibility for lipoapoptosis. Caspase-2-deficient mice fed a Western diet were protected from abdominal fat deposition, diabetes mellitus, dyslipidemia and hepatic steatosis. Adipose tissue in caspase-2-deficient mice was more proliferative, upregulated mitochondrial uncoupling proteins consistent with browning, and was resistant to cell hypertrophy and cell death. The liver was protected from steatohepatitis through a decrease in circulating fatty acids and more efficient hepatic fat metabolism, and from fibrosis as a consequence of reduced fibrogenic stimuli from fewer lipotoxic hepatocytes. Caspase-2 deficiency protected mice from diet-induced obesity, metabolic syndrome and nonalcoholic fatty liver disease. Further studies are necessary to assess caspase-2 as a therapeutic target for those conditions. PMID:26890135

  8. The protection of cells from nitric oxide-mediated apoptotic death by mechanochemically synthesized fullerene (C(60)) nanoparticles.

    PubMed

    Misirkic, Maja S; Todorovic-Markovic, Biljana M; Vucicevic, Ljubica M; Janjetovic, Kristina D; Jokanovic, Vukoman R; Dramicanin, Miroslav D; Markovic, Zoran M; Trajkovic, Vladimir S

    2009-04-01

    The influence of fullerene (C(60)) nanoparticles on the cytotoxicity of a highly reactive free radical nitric oxide (NO) was investigated. Fullerene nanoparticles were prepared by mechanochemically assisted complexation with anionic surfactant sodium dodecyl sulfate, macrocyclic oligosaccharide gamma-cyclodextrin or the copolymer ethylene vinyl acetate-ethylene vinyl versatate. C(60) nanoparticles were characterized by UV-vis and atomic force microscopy. While readily internalized by mouse L929 fibroblasts, C(60) nanoparticles were not cytotoxic. Moreover, they partially protected L929 cells from the cytotoxic effect of NO-releasing compounds sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO) and 3-morpholino-sydnonimine (SIN-1). C(60) nanoparticles reduced SNP-induced apoptotic cell death by preventing mitochondrial depolarization, caspase activation, cell membrane phosphatidylserine exposure and DNA fragmentation. The protective action of C(60) nanoparticles was not exerted via direct interaction with NO, but through neutralization of mitochondria-produced superoxide radical in NO-treated cells, as demonstrated by using different redox-sensitive reporter fluorochromes. These data suggest that C(60) complexes with appropriate host molecules might be plausible candidates for preventing NO-mediated cell injury in inflammatory/autoimmune disorders. PMID:19195698

  9. F-box protein Fbxl18 mediates polyubiquitylation and proteasomal degradation of the pro-apoptotic SCF subunit Fbxl7

    PubMed Central

    Liu, Y; Lear, T; Zhao, Y; Zhao, J; Zou, C; Chen, B B; Mallampalli, R K

    2015-01-01

    Fbxl7, a subunit of the SCF (Skp-Cul1-F-box protein) complex induces mitotic arrest in cells; however, molecular factors that control its cellular abundance remain largely unknown. Here, we identified that an orphan F-box protein, Fbxl18, targets Fbxl7 for its polyubiquitylation and proteasomal degradation. Lys 109 within Fbxl7 is an essential acceptor site for ubiquitin conjugation by Fbxl18. An FQ motif within Fbxl7 serves as a molecular recognition site for Fbxl18 interaction. Ectopically expressed Fbxl7 induces apoptosis in Hela cells, an effect profoundly accentuated after cellular depletion of Fbxl18 protein or expression of Fbxl7 plasmids encoding mutations at either Lys 109 or within the FQ motif. Ectopic expression of Fbxl18 plasmid-limited apoptosis caused by overexpressed Fbxl7 plasmid. Thus, Fbxl18 regulates apoptosis by mediating ubiquitin-dependent proteasomal degradation of the pro-apoptotic protein Fbxl7 that may impact cellular processes involved in cell cycle progression. PMID:25654763

  10. The apoptotic engulfment protein Ced-6 participates in clathrin-mediated yolk uptake in Drosophila egg chambers

    PubMed Central

    Jha, Anupma; Watkins, Simon C.; Traub, Linton M.

    2012-01-01

    Clathrin-mediated endocytosis and phagocytosis are both selective surface internalization processes but have little known mechanistic similarity or interdependence. Here we show that the phosphotyrosine-binding (PTB) domain protein Ced-6, a well-established phagocytosis component that operates as a transducer of so-called eat-me signals during engulfment of apoptotic cells and microorganisms, is expressed in the female Drosophila germline and that Ced-6 expression correlates with ovarian follicle development. Ced-6 exhibits all the known biochemical properties of a clathrin-associated sorting protein, yet ced-6null flies are semifertile despite massive accumulation of soluble yolk precursors in the hemolymph. This is because redundant sorting signals within the cytosolic domain of the Drosophila vitellogenin receptor Yolkless, a low density lipoprotein receptor superfamily member, occur; a functional atypical dileucine signal binds to the endocytic AP-2 clathrin adaptor directly. Nonetheless, the Ced-6 PTB domain specifically recognizes the noncanonical Yolkless FXNPXA sorting sequence and in HeLa cells promotes the rapid, clathrin-dependent uptake of a Yolkless chimera lacking the distal dileucine signal. Ced-6 thus operates in vivo as a clathrin adaptor. Because the human Ced-6 orthologue GULP similarly binds to clathrin machinery, localizes to cell surface clathrin-coated structures, and is enriched in placental clathrin-coated vesicles, new possibilities for Ced-6/Gulp operation during phagocytosis must be considered. PMID:22398720

  11. Curcumin induces apoptosis through the mitochondria-mediated apoptotic pathway in HT-29 cells*

    PubMed Central

    Wang, Jin-bo; Qi, Li-li; Zheng, Shui-di; Wu, Tian-xing

    2009-01-01

    Objective: To investigate the effects of curcumin on release of cytochrome c and expressions of Bcl-2, Bax, Bad, Bcl-xL, caspase-3, poly ADP-ribose polymerase (PARP), and survivin of HT-29 cells. Methods: HT-29 cells were treated with curcumin (0~80 ?mol/L) for 24 h. The release of cytochrome c from the mitochondria and the apoptosis-related proteins Bax, Bcl-2, Bcl-xL, Bad, caspase-3, PARP, and survivin were determined by Western blot analysis and their mRNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR). Results: Curcumin significantly induced the growth inhibition and apoptosis of HT-29 cells. A decrease in expressions of Bcl-2, Bcl-xL and survivin was observed after exposure to 10~80 ?mol/L curcumin, while the levels of Bax and Bad increased in the curcumin-treated cells. Curcumin also induced the release of cytochrome c, the activation of caspase-3, and the cleavage of PARP in a dose-dependent manner. Conclusion: These data suggest that curcumin induced the HT-29 cell apoptosis possibly via the mitochondria-mediated pathway. PMID:19235267

  12. IRES-mediated translation of the pro-apoptotic Bcl2 family member PUMA

    PubMed Central

    Shaltouki, Atossa; Harford, Terri J.; Komar, Anton A.; Weyman, Crystal M.

    2013-01-01

    The proapoptotic Bcl-2 family member PUMA is a critical regulator of apoptosis. We have previously shown that PUMA plays a pivotal role in the apoptosis associated with skeletal myoblast differentiation and that a MyoD-dependent mechanism is responsible for the increased expression of PUMA in these cells. Herein, we report that the increased expression of PUMA under these conditions involves regulation at the level of translation. Specifically, we have found that the increase in PUMA protein levels occurs under conditions of decreased total protein synthesis, eIF2-alpha phosphorylation and hypophosphorylation of eIF4E-BP, suggesting that PUMA translation is proceeding via an alternative initiation mechanism. Polyribosome analysis of PUMA mRNA further corroborated this suggestion. A combination of in vitro and ex vivo (cellular) approaches has provided evidence suggesting that PUMA mRNA 5'UTR harbors an Internal Ribosome Entry Site (IRES) element. Using mono- and bi-cistronic reporter constructs, we have delineated an mRNA fragment that allows for cap-independent translation in vitro and ex vivo (in skeletal myoblasts) in response to culture in differentiation media (DM), or in response to treatment with the DNA-damaging agent, etoposide. This mRNA fragment also supports translation in HeLa and 293T cells. Thus, our data has revealed a novel IRES-mediated regulation of PUMA expression in several cell types and in response to several stimuli. These findings contribute to our understanding and potential manipulation of any developmental or therapeutic scenario involving PUMA.

  13. Efficacious gene silencing in serum and significant apoptotic activity induction by survivin downregulation mediated by new cationic gemini tocopheryl lipids.

    PubMed

    Kumar, Krishan; Maiti, Bappa; Kondaiah, Paturu; Bhattacharya, Santanu

    2015-02-01

    Nonviral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We present here six new tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient coliposomal formulations derived from each of these geminis and a helper lipid, dioleoylphosphatidylethanolamine (DOPE), were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized coliposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz., HEK 293T, HeLa, and Caco-2, significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and in the presence of serum (FBS). Notably, the knockdown activity of coliposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR, and Western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin, significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines. PMID:25438085

  14. Apoptotic-like Leishmania exploit the hosts autophagy machinery to reduce T-cell-mediated parasite elimination

    PubMed Central

    Crauwels, Peter; Bohn, Rebecca; Thomas, Meike; Gottwalt, Stefan; Jckel, Florian; Krmer, Susi; Bank, Elena; Tenzer, Stefan; Walther, Paul; Bastian, Max; van Zandbergen, Ger

    2015-01-01

    Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showedin contrast to viable parasitesthat apoptotic-like parasites enter an LC3+, autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4+ T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferation-reducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis. PMID:25801301

  15. Estrogen receptor-alpha 36 mediates the anti-apoptotic effect of estradiol in triple negative breast cancer cells via a membrane-associated mechanism.

    PubMed

    Chaudhri, Reyhaan A; Hadadi, Agreen; Lobachev, Kirill S; Schwartz, Zvi; Boyan, Barbara D

    2014-11-01

    17?-Estradiol can promote the growth and development of several estrogen receptor (ER)-negative breast cancers. The effects are rapid and non-genomic, suggesting that a membrane-associated ER is involved. ER?36 has been shown to mediate rapid, non-genomic, membrane-associated effects of 17?-estradiol in several cancer cell lines, including triple negative HCC38 breast cancer cells. Moreover, the effect is anti-apoptotic. The aim of this study was to determine if ER?36 mediates this anti-apoptotic effect, and to elucidate the mechanism involved. Taxol was used to induce apoptosis in HCC38 cells, and the effect of 17?-estradiol pre-treatment was determined. Antibodies to ER?36, signal pathway inhibitors, ER?36 deletion mutants, and ER?36-silencing were used prior to these treatments to determine the role of ER?36 in these effects and to determine which signaling molecules were involved. We found that the anti-apoptotic effect of 17?-estradiol in HCC38 breast cancer cells is in fact mediated by membrane-associated ER?36. We also showed that this signaling occurs through a pathway that requires PLD, LPA, and PI3K; G?s and calcium signaling may also be involved. In addition, dynamic palmitoylation is required for the membrane-associated effect of 17?-estradiol. Exon 9 of ER?36, a unique exon to ER?36 not found in other identified splice variants of ER? with previously unknown function, is necessary for these effects. This study provides a working model for a mechanism by which estradiol promotes anti-apoptosis through membrane-associated ER?36, suggesting that ER?36 may be a potential membrane target for drug design against breast cancer, particularly triple negative breast cancer. PMID:25108195

  16. Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network.

    PubMed

    Pietkiewicz, Sabine; Eils, Roland; Krammer, Peter H; Giese, Natalia; Lavrik, Inna N

    2015-11-15

    Combination therapy of cancer is based on the cumulative effects mediated by several drugs. Although molecular mechanisms of action of each particular drug are partially elucidated, understanding of the dynamic cross-talk between different cell death pathways at the quantitative level induced by combination therapy is still missing. Here, we exemplified this question for the death receptor (DR) networks in pancreatic cancer cells. We demonstrate that the combined action of CD95L and gemcitabine in pancreatic cancer cells leads to the simultaneous induction of caspase-dependent and caspase-independent cell death. The pro-apoptotic effects are mediated through down-regulation of the anti-apoptotic proteins c-FLIP and Mcl-1, while caspase-independent cell death was blocked by inhibition of the kinase activity of RIP1. Furthermore, gemcitabine co-treatment strongly increased the amount of cells undergoing CD95-induced RIP1-regulated necrosis. Imaging flow cytometry has enabled us to get the quantitative insights into the apoptosis-necroptosis network and reveal that the majority of the cells upon the CD95L/gemcitabine co-treatment undergoes necroptosis. Our data underlie the importance of the quantitative understanding of the interplay between different cell death modalities, which is essential for the development of anti-cancer therapies. Taken together, our results are important for combination therapy of pancreatic cancer comprising chemotherapeutics and DR-agonists and offer a possibility to sensitize cells with defects in the apoptotic machinery towards necroptosis-type-mediated death. PMID:26453936

  17. Impaired Clearance of Early Apoptotic Cells Mediated by Inhibitory IgG Antibodies in Patients with Primary Sjgren's Syndrome

    PubMed Central

    Manoussakis, Menelaos N.; Fragoulis, George E.; Vakrakou, Aigli G.; Moutsopoulos, Haralampos M.

    2014-01-01

    Objectives Deficient efferocytosis (i.e. phagocytic clearance of apoptotic cells) has been frequently reported in systemic lupus erythematosus (SLE). Todate, patients with primary Sjgren's syndrome (SS) have not been assessed for phagocytosis of apoptotic cells (ApoCell-phagocytosis) and of particulate targets (microbeads, MB-phagocytosis). Design ApoCell-phagocytosis and MB-phagocytosis were comparatively assessed by flow cytometry in peripheral blood specimens and monocyte-derived macrophage (MDM) preparations from healthy blood donors (HBD) and consecutive SS, SLE and rheumatoid arthritis (RA) patients. Cross-admixture ApoCell-phagocytosis experiments were also performed using phagocytes from HBD or patients, and apoptotic cells pretreated with whole sera or purified serum IgG derived from patients or HBD. Results Compared to HBD, approximately half of SS and SLE patients studied (but not RA) manifested significantly reduced ApoCell-phagocytosis (p<0.001) and MB-phagocytosis (p<0.003) by blood-borne phagocytes that correlated inversely with disease activity (p?0.004). In cross-admixture assays, healthy monocytes showed significantly reduced ApoCell-phagocytosis when fed with apoptotic cells that were pretreated with sera or purified serum IgG preparations from SS and SLE patients (p<0.0001, compared to those from HBD or RA). Such aberrant effect of the SS and SLE sera and IgG preparations correlated linearly with their content of IgG antibodies against apoptotic cells (p?0.0001). Phagocytic dysfunction maybe also present in certain SS and SLE patients, as supported by deficient capacity of MDM for ApoCell-phagocytosis and MB-phagocytosis under patients' serum-free conditions. Conclusion Similarly to SLE, efferocytosis is frequently impaired in SS and is primarily due to the presence of inhibitory IgG anti-ApoCell antibodies and secondarily to phagocytes' dysfunction. PMID:25396412

  18. Death receptor-mediated apoptotic signaling is activated in the brain following infection with West Nile virus in the absence of a peripheral immune response.

    PubMed

    Clarke, Penny; Leser, J Smith; Quick, Eamon D; Dionne, Kalen R; Beckham, J David; Tyler, Kenneth L

    2014-01-01

    Apoptosis is an important mechanism of West Nile virus (WNV) pathogenesis within the central nervous system (CNS). The signaling pathways that result in WNV-induced apoptotic neuronal death within the CNS have not been established. In this study, we identified death receptor (DR)-induced apoptosis as a pathway that may be important in WNV pathogenesis, based on the pattern of differential gene expression in WNV-infected, compared to uninfected, brains. Reverse transcription-PCR (RT-PCR) and Western blotting confirmed that genes involved in DR-induced apoptotic signaling are upregulated in the brain following WNV infection. Activity of the DR-associated initiator caspase, caspase 8, was also increased in the brains of WNV-infected mice and occurred in association with cleavage of Bid and activation of caspase 9. These results demonstrate that DR-induced apoptotic signaling is activated in the brain following WNV infection and suggest that the caspase 8-dependent cleavage of Bid promotes intrinsic apoptotic signaling within the brains of infected animals. Utilization of a novel ex vivo brain slice culture (BSC) model of WNV encephalitis revealed that inhibition of caspase 8 decreases virus-induced activation of caspase 3 and tissue injury. The BSC model allows us to examine WNV-induced pathogenesis in the absence of a peripheral immune response. Thus, our results indicate that WNV-induced neuronal injury in the brain is mediated by DR-induced apoptosis signaling and can occur in the absence of infiltrating immune cells. However, astrocytes and microglia were activated in WNV-infected BSC, suggesting that local immune responses influence WNV pathogenesis. PMID:24198425

  19. Death Receptor-Mediated Apoptotic Signaling Is Activated in the Brain following Infection with West Nile Virus in the Absence of a Peripheral Immune Response

    PubMed Central

    Leser, J. Smith; Quick, Eamon D.; Dionne, Kalen R.; Beckham, J. David; Tyler, Kenneth L.

    2014-01-01

    Apoptosis is an important mechanism of West Nile virus (WNV) pathogenesis within the central nervous system (CNS). The signaling pathways that result in WNV-induced apoptotic neuronal death within the CNS have not been established. In this study, we identified death receptor (DR)-induced apoptosis as a pathway that may be important in WNV pathogenesis, based on the pattern of differential gene expression in WNV-infected, compared to uninfected, brains. Reverse transcription-PCR (RT-PCR) and Western blotting confirmed that genes involved in DR-induced apoptotic signaling are upregulated in the brain following WNV infection. Activity of the DR-associated initiator caspase, caspase 8, was also increased in the brains of WNV-infected mice and occurred in association with cleavage of Bid and activation of caspase 9. These results demonstrate that DR-induced apoptotic signaling is activated in the brain following WNV infection and suggest that the caspase 8-dependent cleavage of Bid promotes intrinsic apoptotic signaling within the brains of infected animals. Utilization of a novel ex vivo brain slice culture (BSC) model of WNV encephalitis revealed that inhibition of caspase 8 decreases virus-induced activation of caspase 3 and tissue injury. The BSC model allows us to examine WNV-induced pathogenesis in the absence of a peripheral immune response. Thus, our results indicate that WNV-induced neuronal injury in the brain is mediated by DR-induced apoptosis signaling and can occur in the absence of infiltrating immune cells. However, astrocytes and microglia were activated in WNV-infected BSC, suggesting that local immune responses influence WNV pathogenesis. PMID:24198425

  20. The BH4 domain of anti-apoptotic Bcl-XL, but not that of the related Bcl-2, limits the voltage-dependent anion channel 1 (VDAC1)-mediated transfer of pro-apoptotic Ca2+ signals to mitochondria.

    PubMed

    Monaco, Giovanni; Decrock, Elke; Arbel, Nir; van Vliet, Alexander R; La Rovere, Rita M; De Smedt, Humbert; Parys, Jan B; Agostinis, Patrizia; Leybaert, Luc; Shoshan-Barmatz, Varda; Bultynck, Geert

    2015-04-01

    Excessive Ca(2+) fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca(2+)-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP3Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca(2+)-dependent apoptosis by binding and inhibiting IP3Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP3Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca(2+) uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1(-/-) cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca(2+) uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca(2+) release in the cytosol or to prevent apoptosis, as done instead by an IP3R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca(2+) uptake into the mitochondria. PMID:25681439

  1. Two Programmed Cell Death Systems in Escherichia coli: An Apoptotic-Like Death Is Inhibited by the mazEF-Mediated Death Pathway

    PubMed Central

    Erental, Ariel; Sharon, Idith; Engelberg-Kulka, Hanna

    2012-01-01

    In eukaryotes, the classical form of programmed cell death (PCD) is apoptosis, which has as its specific characteristics DNA fragmentation and membrane depolarization. In Escherichia coli a different PCD system has been reported. It is mediated by the toxinantitoxin system module mazEF. The E. coli mazEF module is one of the most thoroughly studied toxinantitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. mazEF-mediated cell death is a population phenomenon requiring the quorum-sensing pentapeptide NNWNN designated Extracellular Death Factor (EDF). mazEF is triggered by several stressful conditions, including severe damage to the DNA. Here, using confocal microscopy and FACS analysis, we show that under conditions of severe DNA damage, the triggered mazEF-mediated cell death pathway leads to the inhibition of a second cell death pathway. The latter is an apoptotic-like death (ALD); ALD is mediated by recA and lexA. The mazEF-mediated pathway reduces recA mRNA levels. Based on these results, we offer a molecular model for the maintenance of an altruistic characteristic in cell populations. In our model, the ALD pathway is inhibited by the altruistic EDF-mazEF-mediated death pathway. PMID:22412352

  2. Methylantcinate A induces tumor specific growth inhibition in oral cancer cells via Bax-mediated mitochondrial apoptotic pathway.

    PubMed

    Tsai, Wan-Chi; Rao, Yerra Koteswara; Lin, Shih-Shen; Chou, Ming-Yung; Shen, Yi-Ting; Wu, Chih-Hao; Geethangili, Madamanchi; Yang, Chi-Chiang; Tzeng, Yew-Min

    2010-10-15

    An ergostane type triterpenoid methylantcinate A (MAA) isolated from the fruiting bodies of Antrodia camphorata inhibited the growth of oral cancer cell lines OEC-M1 and OC-2 in a dose-dependent manner, without cytotoxic to normal oral gingival fibroblast cells. The major mechanism of growth inhibition was apoptosis induction, as shown by flow cytometric analysis of annexin V-FITC and propidium iodide staining, caspase-3 activation and DNA fragmentation. The increased expression of pro-apoptotic Bax, poly-(ADP-ribose) polymerase cleavage, and activated caspase-3 and decreased expression of anti-apoptotic Bcl-2 and Bcl-xL were also observed. These results provide the first evidence that the anti-oral cancer effects of MAA may involve a mechanism through the mitochondrial dependent pathway. Thus, results reported here may offer further impulse to the development of MAA analogues as potential chemotherapeutic targets for oral cancer complications. PMID:20817519

  3. Tip60 HAT Activity Mediates APP Induced Lethality and Apoptotic Cell Death in the CNS of a Drosophila Alzheimer's Disease Model

    PubMed Central

    Pirooznia, Sheila K.; Chiu, Kellie; Koduri, Sravanthi; Elefant, Felice

    2012-01-01

    Histone acetylation of chromatin promotes dynamic transcriptional responses in neurons that influence neuroplasticity critical for cognitive ability. It has been demonstrated that Tip60 histone acetyltransferase (HAT) activity is involved in the transcriptional regulation of genes enriched for neuronal function as well as the control of synaptic plasticity. Accordingly, Tip60 has been implicated in the neurodegenerative disorder Alzheimer's disease (AD) via transcriptional regulatory complex formation with the AD linked amyloid precursor protein (APP) intracellular domain (AICD). As such, inappropriate complex formation may contribute to AD-linked neurodegeneration by misregulation of target genes involved in neurogenesis; however, a direct and causative epigenetic based role for Tip60 HAT activity in this process during neuronal development in vivo remains unclear. Here, we demonstrate that nervous system specific loss of Tip60 HAT activity enhances APP mediated lethality and neuronal apoptotic cell death in the central nervous system (CNS) of a transgenic AD fly model while remarkably, overexpression of Tip60 diminishes these defects. Notably, all of these effects are dependent upon the C-terminus of APP that is required for transcriptional regulatory complex formation with Tip60. Importantly, we show that the expression of certain AD linked Tip60 gene targets critical for regulating apoptotic pathways are modified in the presence of APP. Our results are the first to demonstrate a functional interaction between Tip60 and APP in mediating nervous system development and apoptotic neuronal cell death in the CNS of an AD fly model in vivo, and support a novel neuroprotective role for Tip60 HAT activity in AD neurodegenerative pathology. PMID:22848598

  4. Tip60 HAT activity mediates APP induced lethality and apoptotic cell death in the CNS of a Drosophila Alzheimer's disease model.

    PubMed

    Pirooznia, Sheila K; Sarthi, Jessica; Johnson, Ashley A; Toth, Meridith S; Chiu, Kellie; Koduri, Sravanthi; Elefant, Felice

    2012-01-01

    Histone acetylation of chromatin promotes dynamic transcriptional responses in neurons that influence neuroplasticity critical for cognitive ability. It has been demonstrated that Tip60 histone acetyltransferase (HAT) activity is involved in the transcriptional regulation of genes enriched for neuronal function as well as the control of synaptic plasticity. Accordingly, Tip60 has been implicated in the neurodegenerative disorder Alzheimer's disease (AD) via transcriptional regulatory complex formation with the AD linked amyloid precursor protein (APP) intracellular domain (AICD). As such, inappropriate complex formation may contribute to AD-linked neurodegeneration by misregulation of target genes involved in neurogenesis; however, a direct and causative epigenetic based role for Tip60 HAT activity in this process during neuronal development in vivo remains unclear. Here, we demonstrate that nervous system specific loss of Tip60 HAT activity enhances APP mediated lethality and neuronal apoptotic cell death in the central nervous system (CNS) of a transgenic AD fly model while remarkably, overexpression of Tip60 diminishes these defects. Notably, all of these effects are dependent upon the C-terminus of APP that is required for transcriptional regulatory complex formation with Tip60. Importantly, we show that the expression of certain AD linked Tip60 gene targets critical for regulating apoptotic pathways are modified in the presence of APP. Our results are the first to demonstrate a functional interaction between Tip60 and APP in mediating nervous system development and apoptotic neuronal cell death in the CNS of an AD fly model in vivo, and support a novel neuroprotective role for Tip60 HAT activity in AD neurodegenerative pathology. PMID:22848598

  5. Autocrine secretion of 15d-PGJ2 mediates simvastatin-induced apoptotic burst in human metastatic melanoma cells

    PubMed Central

    Wasinger, Christine; Knzl, Martin; Minichsdorfer, Christoph; Hller, Christoph; Zellner, Maria; Hohenegger, Martin

    2014-01-01

    Background and Purpose Despite new therapeutic approaches, metastatic melanomas still have a poor prognosis. Statins reduce low-density lipoprotein cholesterol and exert anti-inflammatory and anti-proliferative actions. We have recently shown that simvastatin triggers an apoptotic burst in human metastatic melanoma cells by the synthesis of an autocrine factor. Experimental Approach The current in vitro study was performed in human metastatic melanoma cell lines (A375, 518a2) and primary human melanocytes and melanoma cells. The secretome of simvastatin-stressed cells was analysed with two-dimensional difference gel electrophoresis and MS. The signalling pathways involved were analysed at the protein and mRNA level using pharmacological approaches and siRNA technology. Key Results Simvastatin was shown to activate a stress cascade, leading to the synthesis of 15-deoxy-12,14-PGJ2 (15d-PGJ2), in a p38- and COX-2-dependent manner. Significant concentrations of 15d-PGJ2 were reached in the medium of melanoma cells, which were sufficient to activate caspase 8 and the mitochondrial pathway of apoptosis. Inhibition of lipocalin-type PGD synthase, a key enzyme for 15d-PGJ2 synthesis, abolished the apoptotic effect of simvastatin. Moreover, 15d-PGJ2 was shown to bind to the fatty acid-binding protein 5 (FABP5), which was up-regulated and predominantly detected in the secretome of simvastatin-stressed cells. Knockdown of FABP5 abolished simvastatin-induced activation of PPAR-? and amplified the apoptotic response. Conclusions and Implications We characterized simvastatin-induced activation of the 15d-PGJ2/FABP5 signalling cascades, which triggered an apoptotic burst in melanoma cells but did not affect primary human melanocytes. These data support the rationale for the pharmacological targeting of 15d-PGJ2 in metastatic melanoma. PMID:25091578

  6. Icariin displays anticancer activity against human esophageal cancer cells via regulating endoplasmic reticulum stress-mediated apoptotic signaling

    PubMed Central

    Fan, Chongxi; Yang, Yang; Liu, Yong; Jiang, Shuai; Di, Shouyin; Hu, Wei; Ma, Zhiqiang; Li, Tian; Zhu, Yifang; Xin, Zhenlong; Wu, Guiling; Han, Jing; Li, Xiaofei; Yan, Xiaolong

    2016-01-01

    In this study, we investigated the antitumor activity of icariin (ICA) in human esophageal squamous cell carcinoma (ESCC) in vitro and in vivo and explored the role of endoplasmic reticulum stress (ERS) signaling in this activity. ICA treatment resulted in a dose- and time-dependent decrease in the viability of human EC109 and TE1 ESCCs. Additionally, ICA exhibited strong antitumor activity, as evidenced by reductions in cell migration, adhesion, and intracellular glutathione (GSH) levels and by increases in the EC109 and TE1 cell apoptotic index, Caspase 9 activity, reactive oxygen species (ROS) level, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Furthermore, ICA treatments upregulated the levels of ERS-related molecules (p-PERK, GRP78, ATF4, p-eIF2α, and CHOP) and a pro-apoptotic protein (PUMA) and simultaneously downregulated an anti-apoptotic protein (Bcl2) in the two ESCC cell lines. The downregulation of ERS signaling using eIF2α siRNA desensitized EC109 and TE1 cells to ICA treatment, and the upregulation of ERS signaling using thapsigargin sensitized EC109 and TE1 cells to ICA treatment. In summary, ERS activation may represent a mechanism of action for the anticancer activity of ICA in ESCCs, and the activation of ERS signaling may represent a novel therapeutic intervention for human esophageal cancer. PMID:26892033

  7. Icariin displays anticancer activity against human esophageal cancer cells via regulating endoplasmic reticulum stress-mediated apoptotic signaling.

    PubMed

    Fan, Chongxi; Yang, Yang; Liu, Yong; Jiang, Shuai; Di, Shouyin; Hu, Wei; Ma, Zhiqiang; Li, Tian; Zhu, Yifang; Xin, Zhenlong; Wu, Guiling; Han, Jing; Li, Xiaofei; Yan, Xiaolong

    2016-01-01

    In this study, we investigated the antitumor activity of icariin (ICA) in human esophageal squamous cell carcinoma (ESCC) in vitro and in vivo and explored the role of endoplasmic reticulum stress (ERS) signaling in this activity. ICA treatment resulted in a dose- and time-dependent decrease in the viability of human EC109 and TE1 ESCCs. Additionally, ICA exhibited strong antitumor activity, as evidenced by reductions in cell migration, adhesion, and intracellular glutathione (GSH) levels and by increases in the EC109 and TE1 cell apoptotic index, Caspase 9 activity, reactive oxygen species (ROS) level, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Furthermore, ICA treatments upregulated the levels of ERS-related molecules (p-PERK, GRP78, ATF4, p-eIF2α, and CHOP) and a pro-apoptotic protein (PUMA) and simultaneously downregulated an anti-apoptotic protein (Bcl2) in the two ESCC cell lines. The downregulation of ERS signaling using eIF2α siRNA desensitized EC109 and TE1 cells to ICA treatment, and the upregulation of ERS signaling using thapsigargin sensitized EC109 and TE1 cells to ICA treatment. In summary, ERS activation may represent a mechanism of action for the anticancer activity of ICA in ESCCs, and the activation of ERS signaling may represent a novel therapeutic intervention for human esophageal cancer. PMID:26892033

  8. P38 MAP kinase mediates apoptosis after genipin treatment in non-small-cell lung cancer H1299 cells via a mitochondrial apoptotic cascade.

    PubMed

    Yang, Xue; Yao, Jie; Luo, Yue; Han, Yongguang; Wang, Zuobai; Du, Linfang

    2013-01-01

    Genipin, an active constituent of Gardenia fruit, has been reported to show an anti-tumor effect in several cancer cell systems. Here, we demonstrate how genipin exhibits a strong apoptotic cell death effect in human non-small-cell lung cancer H1299 cells. Genipin-mediated decrease in cell viability was observed through apoptosis as demonstrated by induction of a sub-G1 peak through flow cytometry, DNA fragmentation measured by TUNEL assay, and cleavage of poly ADP-ribose-polymerase. During genipin-induced apoptosis, the mitochondrial execution pathway was activated by caspase-9 and -3 activation as examined by a kinetic study, cytochrome c release, and a dose-dependent increase in Bax/Bcl-2 ratio. A search for the downstream pathway reveals that genipin-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2. SB203580, a p38MAPK inhibitor, markedly blocked the formation of TUNEL-positive apoptotic cells in genipin-treated cells. Besides, the interference of p38MAPK inhibited Bax expression and cytochrome c release. Altogether, our observations imply that genipin causes increased levels of Bax in response to p38MAPK signaling, which results in the initiation of mitochondrial death cascade, and therefore it holds promise as a potential chemotherapeutic agent for the treatment of H1299 cells. PMID:23603895

  9. Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway

    SciTech Connect

    Song, Shasha; Wang, Shuang; Ma, Jun; Yao, Lan; Xing, Hao; Zhang, Lei; Liao, Lin; Zhu, Daling

    2013-08-01

    Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxia in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway. Highlights: • BVR expresses in PASMC and is up-regulated by hypoxia in protein and mRNA levels. • BVR/bilirubin contribute to the inhibitive process of hypoxia on PASMC apoptosis. • Bilirubin protects PASMC from apoptosis under hypoxia via ERK1/2 pathway.

  10. Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells.

    PubMed

    Urresti, Jorge; Ruiz-Meana, Marisol; Coccia, Elena; Arvalo, Juan Carlos; Castellano, Jos; Fernndez-Sanz, Celia; Galenkamp, Koen M O; Planells-Ferrer, Laura; Moubarak, Rana S; Llecha-Cano, Nria; Reix, Stphanie; Garca-Dorado, David; Barneda-Zahonero, Bruna; Comella, Joan X

    2016-01-15

    Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG. PMID:26582200

  11. Pronounced transcriptional regulation of apoptotic and TNF-NF-kappa-B signaling genes during the course of thymoquinone mediated apoptosis in HeLa cells.

    PubMed

    Sakalar, Cagri; Yuruk, Merve; Kaya, Tugba; Aytekin, Metin; Kuk, Salih; Canatan, Halit

    2013-11-01

    Thymoquinone (TQ) is the active ingredient extracted from the essential oil of Nigella sativa. A number of studies implicated TQ as an antitumor agent. In this study, cytotoxic effects of the oil of N. sativa and TQ were evaluated on human cervical cancer cell line, HeLa cells. IC50 value was ~0.125 ?l/ml for N. sativa oil preparations and 12.5 ?M for TQ. TQ strongly inhibited wound healing at all concentrations ranging from 12.5 to 100 ?M in a scratch wound healing assay. Additionally, induction of apoptosis by TQ was assessed by Giemsa staining and TQ was found to induce apoptosis in cancer cells especially at concentrations of 50 and 100 ?M. TQ-mediated transcriptional regulation of 84 genes involved in apoptosis was studied using a PCR array. At low dose (12.5 ?M), TQ was found to induce expression of four pro-apoptotic genes: BIK (~22.7-fold), FASL (~2.9-fold), BCL2L10 (~2.1-fold), and CASP1 (~2-fold). TQ was also found to reduce the expression of an anti-apoptotic gene implicated in NF-kappa-B signaling and cancer: RELA (~8-fold). At high dose (100 ?M), TQ mediated the expression of 21 genes implicated directly in apoptosis (6 genes), TNF signaling (10 genes), and NF-kappa-B signaling (3 genes) such as BIK, BID, TNFRSF10A, TNFRSF10B, TNF, TRAF3, RELA, and RELB. In conclusion, this study implicates the role of TQ in the inhibition of cancer cell proliferation and migration. At the same time, our results strongly suggest that TQ intervenes with TNF and NF-kappa-B signaling during TQ-mediated induction of apoptosis in cancer cells. PMID:23943306

  12. γ-Secretase Activity Is Required for Regulated Intramembrane Proteolysis of Tumor Necrosis Factor (TNF) Receptor 1 and TNF-mediated Pro-apoptotic Signaling.

    PubMed

    Chhibber-Goel, Jyoti; Coleman-Vaughan, Caroline; Agrawal, Vishal; Sawhney, Neha; Hickey, Emer; Powell, James C; McCarthy, Justin V

    2016-03-11

    The γ-secretase protease and associated regulated intramembrane proteolysis play an important role in controlling receptor-mediated intracellular signaling events, which have a central role in Alzheimer disease, cancer progression, and immune surveillance. An increasing number of γ-secretase substrates have a role in cytokine signaling, including the IL-6 receptor, IL-1 receptor type I, and IL-1 receptor type II. In this study, we show that following TNF-converting enzyme-mediated ectodomain shedding of TNF type I receptor (TNFR1), the membrane-bound TNFR1 C-terminal fragment is subsequently cleaved by γ-secretase to generate a cytosolic TNFR1 intracellular domain. We also show that clathrin-mediated internalization of TNFR1 C-terminal fragment is a prerequisite for efficient γ-secretase cleavage of TNFR1. Furthermore, using in vitro and in vivo model systems, we show that in the absence of presenilin expression and γ-secretase activity, TNF-mediated JNK activation was prevented, assembly of the TNFR1 pro-apoptotic complex II was reduced, and TNF-induced apoptosis was inhibited. These observations demonstrate that TNFR1 is a γ-secretase substrate and suggest that γ-secretase cleavage of TNFR1 represents a new layer of regulation that links the presenilins and the γ-secretase protease to pro-inflammatory cytokine signaling. PMID:26755728

  13. Do plants mediate their anti-diabetic effects through anti-oxidant and anti-apoptotic actions? an in vitro assay of 3 Indian medicinal plants

    PubMed Central

    2013-01-01

    Background Both experimental and clinical studies suggest that oxidative stress plays a major role in the pathogenesis of both types of diabetes mellitus. This oxidative stress leads to ?-cell destruction by apoptosis. Hence exploring agents modulating oxidative stress is an effective strategy in the treatment of both Type I and Type II diabetes. Plants are a major source of anti-oxidants and exert protective effects against oxidative stress in biological systems. Phyllanthus emblica, Curcuma longa and Tinospora cordifolia are three such plants widely used in Ayurveda for their anti-hyperglycemic activity. Additionally their anti-oxidant properties have been scientifically validated in various experimental in vitro and in vivo models. Hence the present in vitro study was planned to assess whether the anti-hyperglycemic effects of the hydro-alcoholic extracts of Phyllanthus emblica (Pe) and Curcuma longa (Cl) and aqueous extract of Tinospora cordifolia (Tc) are mediated through their antioxidant and/or anti-apoptotic property in a streptozotocin induced stress model. Methods RINm5F cell line was used as a model of pancreatic ?-cells against stress induced by streptozotocin (2mM). Non-toxic concentrations of the plant extracts were identified using MTT assay. Lipid peroxidation through MDA release, modulation of apoptosis and insulin release were the variables measured to assess streptozotocin induced damage and protection afforded by the plant extracts. Results All 3 plants extracts significantly inhibited MDA release from RIN cells indicating protective effect against STZ induced oxidative damage. They also exhibited a dose dependent anti-apoptotic effect as seen by a decrease in the sub G0 population in response to STZ. None of the plant extracts affected insulin secretion from the cells to a great extent. Conclusion The present study thus demonstrated that the protective effect of the selected medicinal plants against oxidative stress induced by STZ in vitro, which was exerted through their anti-oxidant and anti-apoptotic actions. PMID:24093976

  14. Biomechanical insult switches PEA-15 activity to uncouple its anti-apoptotic function and promote erk mediated tissue remodeling.

    PubMed

    Exler, Rachel E; Guo, Xiaoxin; Chan, Darren; Livne-Bar, Izhar; Vicic, Nevena; Flanagan, John G; Sivak, Jeremy M

    2016-01-15

    Biomechanical insult contributes to many chronic pathological processes, yet the resulting influences on signal transduction mechanisms are poorly understood. The retina presents an excellent mechanotransduction model, as mechanical strain on sensitive astrocytes of the optic nerve head (ONH) is intimately linked to chronic tissue remodeling and excavation by matrix metalloproteinases (MMPs), and apoptotic cell death. However, the mechanism by which these effects are induced by biomechanical strain is unclear. We previously identified the small adapter protein, PEA-15 (phosphoprotein enriched in astrocytes), through proteomic analyses of human ONH astrocytes subjected to pathologically relevant biomechanical insult. Under resting conditions PEA-15 is regulated through phosphorylation of two key serine residues to inhibit extrinsic apoptosis and ERK1/2 signaling. However, we surprisingly observed that biomechanical insult dramatically switches PEA-15 phosphorylation and function to uncouple its anti-apoptotic activity, and promote ERK1/2-dependent MMP-2 and MMP-9 secretion. These results reveal a novel cell autonomous mechanism by which biomechanical strain rapidly modifies this signaling pathway to generate altered tissue injury responses. PMID:26615958

  15. PRKACA mediates resistance to HER2-targeted therapy in breast cancer cells and restores anti-apoptotic signaling.

    PubMed

    Moody, S E; Schinzel, A C; Singh, S; Izzo, F; Strickland, M R; Luo, L; Thomas, S R; Boehm, J S; Kim, S Y; Wang, Z C; Hahn, W C

    2015-04-16

    Targeting HER2 with antibodies or small molecule inhibitors in HER2-positive breast cancer leads to improved survival, but resistance is a common clinical problem. To uncover novel mechanisms of resistance to anti-HER2 therapy in breast cancer, we performed a kinase open reading frame screen to identify genes that rescue HER2-amplified breast cancer cells from HER2 inhibition or suppression. In addition to multiple members of the MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) signaling pathways, we discovered that expression of the survival kinases PRKACA and PIM1 rescued cells from anti-HER2 therapy. Furthermore, we observed elevated PRKACA expression in trastuzumab-resistant breast cancer samples, indicating that this pathway is activated in breast cancers that are clinically resistant to trastuzumab-containing therapy. We found that neither PRKACA nor PIM1 restored MAPK or PI3K activation after lapatinib or trastuzumab treatment, but rather inactivated the pro-apoptotic protein BAD, the BCl-2-associated death promoter, thereby permitting survival signaling through BCL-XL. Pharmacological blockade of BCL-XL/BCL-2 partially abrogated the rescue effects conferred by PRKACA and PIM1, and sensitized cells to lapatinib treatment. These observations suggest that combined targeting of HER2 and the BCL-XL/BCL-2 anti-apoptotic pathway may increase responses to anti-HER2 therapy in breast cancer and decrease the emergence of resistant disease. PMID:24909179

  16. PRKACA Mediates Resistance to HER2-Targeted Therapy in Breast Cancer Cells and Restores Anti-Apoptotic Signaling

    PubMed Central

    Moody, Susan E.; Schinzel, Anna C.; Singh, Shambhavi; Izzo, Francesca; Strickland, Matthew R.; Luo, Leo; Thomas, Sapana R.; Boehm, Jesse S.; Kim, So Young; Wang, Zhigang C.; Hahn, William C.

    2014-01-01

    Targeting HER2 with antibodies or small molecule inhibitors in HER2-positive breast cancer leads to improved survival, but resistance is a common clinical problem. To uncover novel mechanisms of resistance to anti-HER2 therapy in breast cancer, we performed a kinase open reading frame (ORF) screen to identify genes that rescue HER2-amplified breast cancer cells from HER2 inhibition or suppression. In addition to multiple members of the MAPK and PI3K signaling pathways, we discovered that expression of the survival kinases PRKACA and PIM1 rescued cells from anti-HER2 therapy. Furthermore, we observed elevated PRKACA expression in trastuzumab-resistant breast cancer samples, indicating that this pathway is activated in breast cancers that are clinically resistant to trastuzumab-containing therapy. We found that neither PRKACA nor PIM1 restored MAPK or PI3K activation after lapatinib or trastuzumab treatment, but rather inactivated the pro-apoptotic protein BAD, thereby permitting survival signaling through BCL-XL. Pharmacological blockade of BCL-XL/BCL-2 partially abrogated the rescue effects conferred by PRKACA and PIM1, and sensitized cells to lapatinib treatment. These observations suggest that combined targeting of HER2 and the BCL-XL/BCL-2 anti-apoptotic pathway may increase responses to anti-HER2 therapy in breast cancer and decrease the emergence of resistant disease. PMID:24909179

  17. RXR?, PXR and CAR xenobiotic receptors mediate the apoptotic and neurotoxic actions of nonylphenol in mouse hippocampal cells.

    PubMed

    Litwa, E; Rzemieniec, J; Wnuk, A; Lason, W; Krzeptowski, W; Kajta, M

    2016-02-01

    In the present study, we investigated the role of the retinoid X receptor (RXR), the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), in the apoptotic and toxic effects of nonylphenol in mouse primary neuronal cell cultures. Our study demonstrated that nonylphenol activated caspase-3 and induced lactate dehydrogenase (LDH) release in hippocampal cells, which was accompanied by an increase in the mRNA expression and protein levels of RXR?, PXR and CAR. Nonylphenol stimulated Rxra, Pxr, and Car mRNA expression. These effects were followed by increase in the protein levels of particular receptors. Immunofluorescence labeling revealed the cellular distribution of RXR?, PXR and CAR in hippocampal neurons in response to nonylphenol, shortening of neurites and cytoplasmic shrinking, as indicated by MAP2 staining. It also showed NP-induced translocation of receptor-specific immunofluorescence from cytoplasm to the nucleus. The use of specific siRNAs demonstrated that Rxra-, Pxr-, and Car-siRNA-transfected cells were less vulnerable to nonylphenol-induced activation of caspase-3 and LDH, thus confirming the key involvement of RXR?/PXR/CAR signaling pathways in the apoptotic and neurotoxic actions of nonylphenol. These new data give prospects for the targeting xenobiotic nuclear receptors to protect the developing nervous system against endocrine disrupting chemicals. PMID:26643981

  18. Correlation of glucocorticoid-mediated E4BP4 upregulation with altered expression of pro- and anti-apoptotic genes in CEM human lymphoblastic leukemia cells.

    PubMed

    Beach, Jessica A; Nary, Laura J; Hovanessian, Rebeka; Medh, Rheem D

    2014-08-29

    In Caenorhabditiselegans, motorneuron apoptosis is regulated via a ces-2-ces-1-egl-1 pathway. We tested whether human CEM lymphoblastic leukemia cells undergo apoptosis via an analogous pathway. We have previously shown that E4BP4, a ces-2 ortholog, mediates glucocorticoid (GC)-dependent upregulation of BIM, an egl-1 ortholog, in GC-sensitive CEM C7-14 cells and in CEM C1-15mE#3 cells, which are sensitized to GCs by ectopic expression of E4BP4. In the present study, we demonstrate that the human ces-1 orthologs, SLUG and SNAIL, are not significantly repressed in correlation with E4BP4 expression. Expression of E4BP4 homologs, the PAR family genes, especially HLF, encoding a known anti-apoptotic factor, was inverse to that of E4BP4 and BIM. Expression of pro- and anti-apoptotic genes in CEM cells was analyzed via an apoptosis PCR Array. We identified BIRC3 and BIM as genes whose expression paralleled that of E4BP4, while FASLG, TRAF4, BCL2A1, BCL2L1, BCL2L2 and CD40LG as genes whose expression was opposite to that of E4BP4. PMID:25101525

  19. LBH589, a deacetylase inhibitor, induces apoptosis in adult T-cell leukemia/lymphoma cells via activation of a novel RAIDD-caspase-2 pathway

    PubMed Central

    Hasegawa, H; Yamada, Y; Tsukasaki, K; Mori, N; Tsuruda, K; Sasaki, D; Usui, T; Osaka, A; Atogami, S; Ishikawa, C; Machijima, Y; Sawada, S; Hayashi, T; Miyazaki, Y; Kamihira, S

    2011-01-01

    Adult T-cell leukemia/lymphoma (ATLL), an aggressive neoplasm etiologically associated with human T-lymphotropic virus type-1 (HTLV-1), is resistant to treatment. In this study, we examined the effects of a new inhibitor of deacetylase enzymes, LBH589, on ATLL cells. LBH589 effectively induced apoptosis in ATLL-related cell lines and primary ATLL cells and reduced the size of tumors inoculated in SCID mice. Analyses, including with a DNA microarray, revealed that neither death receptors nor p53 pathways contributed to the apoptosis. Instead, LBH589 activated an intrinsic pathway through the activation of caspase-2. Furthermore, small interfering RNA experiments targeting caspase-2, caspase-9, RAIDD, p53-induced protein with a death domain (PIDD) and RIPK1 (RIP) indicated that activation of RAIDD is crucial and an event initiating this pathway. In addition, LBH589 caused a marked decrease in levels of factors involved in ATLL cell proliferation and invasion such as CCR4, IL-2R and HTLV-1 HBZ-SI, a spliced form of the HTLV-1 basic zipper factor HBZ. In conclusion, we showed that LBH589 is a strong inducer of apoptosis in ATLL cells and uncovered a novel apoptotic pathway initiated by activation of RAIDD. PMID:21242994

  20. Enhanced tolerance against early and late apoptotic oxidative stress in mammalian neurons through nicotinamidase and sirtuin mediated pathways.

    PubMed

    Chong, Zhao Zhong; Maiese, Kenneth

    2008-08-01

    Focus upon therapeutic strategies that intersect between pathways that govern cellular metabolism and cellular survival may offer the greatest impact for the treatment of a number of neurodegenerative and metabolic disorders, such as diabetes mellitus. In this regard, we investigated the role of a Drosophila nicotinamidase (DN) in mammalian SH-SY5Y neuronal cells during oxidative stress. We demonstrate that during free radical exposure to nitric oxide generators DN neuronal expression significantly increased cell survival and blocked cellular membrane injury. Furthermore, DN neuronal expression prevented both apoptotic late DNA degradation and early phosphatidylserine exposure that may serve to modulate inflammatory cell activation in vivo. Nicotinamidase activity that limited nicotinamide cellular concentrations appeared to be necessary for DN neuroprotection, since application of progressive nicotinamide concentrations could abrogate the benefits of DN expression during oxidative stress. Pathways that involved sirtuin activation and SIRT1 were suggested to be vital, at least in part, for DN to confer protection through a series of studies. First, application of resveratrol increased cell survival during oxidative stress either alone or in conjunction with the expression of DN to a similar degree, suggesting that DN may rely upon SIRT1 activation to foster neuronal protection. Second, the overexpression of either SIRT1 or DN in neurons prevented apoptotic injury specifically in neurons expressing these proteins during oxidative stress, advancing the premise that DN and SIRT1 may employ similar pathways for neuronal protection. Third, inhibition of sirtuin activity with sirtinol was detrimental to neuronal survival during oxidative stress and prevented neuronal protection during overexpression of DN or SIRT1, further supporting that SIRT1 activity may be necessary for DN neuroprotection during oxidative stress. Implementation of further work to elucidate the cellular mechanisms that govern nicotinamidase activity in mammalian cells may offer novel avenues for the treatment of disorders tied to oxidative stress and cellular metabolic dysfunction. PMID:18691073

  1. Par6 is an essential mediator of apoptotic response to transforming growth factor beta in NMuMG immortalized mammary cells

    PubMed Central

    2014-01-01

    Background We previously observed that the TGFbeta-Par6 pathway mediates loss of polarity and apoptosis in NMuMG cells. Here we investigate the contribution of Par6 versus TGFbeta receptor I activation to TGFbeta-induced apoptosis in association with changes in apico-basal polarity. We focus on the effect of Par6 activation on alpha6beta4 integrin expression and localization, and Nuclear Factor-kappaB (p65/RelA) activation, previously shown to mediate polarity-dependent cell survival. Methods Using immunoblotting and/or immunofluorescence we investigated the effect of TGFbeta1 on apoptosis, alpha6, beta4 and beta1 integrin expression/localization, and p65/RelA phosphorylation/localization in monolayer and three-dimensional (3D) cultures of NMuMG cells with an overactive or inactive Par6 pathway. Results were quantified by band densitometry or as percent of 3D structures displaying a phenotype. Differences among means were compared by two-way ANOVA. Results Blocking Par6 activation inhibits TGFbeta-induced apoptosis. Par6 overactivation enhances TGFbeta-induced apoptosis, notably after 6-day exposure to TGFbeta (p?apoptotic stimuli. 48-hour TGFbeta treatment reduced beta4 integrin levels in NMuMG monolayers and significantly reduced the basal localization of alpha6 (p?apoptotic response. After 6-day exposure to TGFbeta, Par6-dependent changes to beta4 integrin were no longer apparent, but there was reduced phosphorylation of p65/RelA (p?apoptotic or pro-survival factor in breast cancer, and potentially aid in predicting patients prognosis and therapy response. PMID:24581220

  2. SCAR/WAVE-mediated processing of engulfed apoptotic corpses is essential for effective macrophage migration in Drosophila

    PubMed Central

    Evans, I R; Ghai, P A; Urban?i?, V; Tan, K-L; Wood, W

    2013-01-01

    In vitro studies have shown that SCAR/WAVE activates the Arp2/3 complex to generate actin filaments, which in many cell types are organised into lamellipodia that are thought to have an important role in cell migration. Here we demonstrate that SCAR is utilised by Drosophila macrophages to drive their developmental and inflammatory migrations and that it is regulated via the Hem/Kette/Nap1-containing SCAR/WAVE complex. SCAR is also important in protecting against bacterial pathogens and in wound repair as SCAR mutant embryos succumb more readily to both sterile and infected wounds. However, in addition to driving the formation of lamellipodia in macrophages, SCAR is required cell autonomously for the correct processing of phagocytosed apoptotic corpses by these professional phagocytes. Removal of this phagocytic burden by preventing apoptosis rescues macrophage lamellipodia formation and partially restores motility. Our results show that efficient processing of phagosomes is critical for effective macrophage migration in vivo. These findings have important implications for the resolution of macrophages from chronic wounds and the behaviour of those associated with tumours, because phagocytosis of debris may serve to prolong the presence of these cells at these sites of pathology. PMID:23328632

  3. Antiapoptotic and Antioxidant Properties of Orthosiphon stamineus Benth (Cat's Whiskers): Intervention in the Bcl-2-Mediated Apoptotic Pathway

    PubMed Central

    Abdelwahab, Siddig Ibrahim; Mohan, Syam; Mohamed Elhassan, Manal; Al-Mekhlafi, Nabil; Mariod, Abdelbasit Adam; Abdul, Ahmad Bustamam; Abdulla, Mahmood Ameen; Alkharfy, Khalid M.

    2011-01-01

    Antiapoptotic and antioxidant activities of aqueous-methanolic extract (CAME) of Orthosiphonstamineus Benth(OS), and its hexane (HF), chloroform (CF), n-butanol (NBF), ethyl acetate (EAF) and water (WF) fractions were investigated. Antioxidant properties were evaluated using the assays of Folin-Ciocalteu, aluminiumtrichloride, ?-carotene bleaching and DPPH. The role of OS against hydrogen peroxide induced apoptosis on MDA-M231 epithelial cells was examined using MTT assay, phase contrast microscope, colorimetric assay of caspase-3, western blot and quantitative real-time PCR. Results showed that EAF showed the highest total phenolic content followed by CAME, NBF, WF, CF and HF, respectively. Flavonoid content was in the order of the CF > EAF > HF > CAME > NBF > WF. The IC50 values on DPPH assay for different extract/fractions were 126.2 23, 31.25 1.2, 15.25 2.3, 13.56 1.9, 23.0 3.2, and 16.66 1.5??g/ml for HF, CF, EAF, NBF, WF and CAME, respectively. OSreduced the oxidation of ?-carotene by hydroperoxides. Cell death was dose-dependently inhibited by pretreatment with OS. Caspase-3 and distinct morphological features suggest the anti-apoptotic activities of OS. This plant not only increased the expression of Bcl-2, but also decreased Bax expression, and ultimately reduced H2O2-induced apoptosis. The current results showed that phenolics may provide health and nutritional benefits. PMID:21234328

  4. Repression of mammary stem/progenitor cells by p53 is mediated by notch and separable from apoptotic activity

    PubMed Central

    Tao, Luwei; Roberts, Amy L.; Dunphy, Karen A.; Bigelow, Carol; Yan, Haoheng; Jerry, D. Joseph

    2012-01-01

    Breast cancer is the most common tumor among women with inherited mutations in the p53 gene (Li-Fraumeni syndrome). The tumors represent the basal-like subtype which has been suggested to originate from mammary stem/progenitor cells. In mouse mammary epithelium, mammosphere-forming potential was increased with decreased dosage of the gene encoding the p53 tumor suppressor protein (Trp53). Limiting dilution transplantation also showed a 3.3-fold increase in the frequency of long-term regenerative mammary stem cells in Trp53?/? mice. The repression of mammospheres by p53 was apparent despite the absence of apoptotic responses to radiation indicating a dissociation of these two activities of p53. The effects of p53 on progenitor cells were also observed in TM40A cells using both mammosphere-forming assays and the DsRed-let7c-sensor. The frequency of long-term label-retaining epithelial cells (LRECs) was decreased in Trp53?/? mammary glands indicating that asymmetric segregation of DNA is diminished and contributes to the expansion of the mammary stem cells. Treatment with an inhibitor of ?-secretase (DAPT) reduced the number of Trp53?/? mammospheres to the level found in Trp53+/+ cells. These results demonstrate that basal levels of p53 restrict mammary stem/progenitor cells through Notch and that the Notch pathway is a therapeutic target to prevent expansion of this vulnerable pool of cells. PMID:21280161

  5. Charge profile analysis reveals that activation of pro-apoptotic regulators Bax and Bak relies on charge transfer mediated allosteric regulation.

    PubMed

    Ionescu, Crina-Maria; Svobodov Va?ekov, Radka; Prehn, Jochen H M; Huber, Heinrich J; Ko?a, Jaroslav

    2012-01-01

    The pro-apoptotic proteins Bax and Bak are essential for executing programmed cell death (apoptosis), yet the mechanism of their activation is not properly understood at the structural level. For the first time in cell death research, we calculated intra-protein charge transfer in order to study the structural alterations and their functional consequences during Bax activation. Using an electronegativity equalization model, we investigated the changes in the Bax charge profile upon activation by a functional peptide of its natural activator protein, Bim. We found that charge reorganizations upon activator binding mediate the exposure of the functional sites of Bax, rendering Bax active. The affinity of the Bax C-domain for its binding groove is decreased due to the Arg94-mediated abrogation of the Ser184-Asp98 interaction. We further identified a network of charge reorganizations that confirms previous speculations of allosteric sensing, whereby the activation information is conveyed from the activation site, through the hydrophobic core of Bax, to the well-distanced functional sites of Bax. The network was mediated by a hub of three residues on helix 5 of the hydrophobic core of Bax. Sequence and structural alignment revealed that this hub was conserved in the Bak amino acid sequence, and in the 3D structure of folded Bak. Our results suggest that allostery mediated by charge transfer is responsible for the activation of both Bax and Bak, and that this might be a prototypical mechanism for a fast activation of proteins during signal transduction. Our method can be applied to any protein or protein complex in order to map the progress of allosteric changes through the proteins' structure. PMID:22719244

  6. Differential regulation of caspase-2 in MPP?-induced apoptosis in primary cortical neurons.

    PubMed

    Hu, Hsin-I; Chang, Hsin-Hou; Sun, Der-Shan

    2015-03-01

    Parkinson's disease (PD), among the most common neurodegenerative diseases worldwide for which there is no cure, is characterized as progressive dopaminergic neuron loss in the substantia nigra through an unknown mechanism. Administering 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) causes neuronal cell death and Parkinsonism in humans. Commonly used in animal models of PD, MPTP can metabolize to 1-methyl-4-phenylpyridinium (MPP(+)); however, the detailed mechanism through which MPP(+) causes neuronal cell death remains undetermined. Previous reports have indicated those knockout mice with Bcl-2 associated protein X (Bax) or caspase-2, two mitochondrial outer membrane permeabilization inducers, are resistant to MPTP administration, suggesting that mitochondria are involved in MPP(+)-triggered apoptosis. Our previous study showed that MPP(+)-triggered apoptosis can be distinguished from spontaneous apoptosis of primary cortical neurons. In the present study, we verified the involvement of mitochondria in MPP(+)-induced and spontaneous apoptosis in cortical neurons through confocal microscope analysis. We demonstrated that caspase-2 activation is specific to MPP(+)-induced apoptosis and occurs before Bax translocation to the mitochondria. Caspase-2 activation is one of the few early molecular events identified in PD models. PMID:25645943

  7. ERK-Mediated Activation of Fas Apoptotic Inhibitory Molecule 2 (Faim2) Prevents Apoptosis of 661W Cells in a Model of Detachment-Induced Photoreceptor Cell Death

    PubMed Central

    Besirli, Cagri G.; Zheng, Qiong-Duon; Reed, David M.; Zacks, David N.

    2012-01-01

    In this study, we examined the role of Fas apoptotic inhibitory molecule 2 (Faim2), an inhibitor of the Fas signaling pathway, and its regulation by stress kinase signaling during Fas-mediated apoptosis of 661W cells, an immortalized photoreceptor-like cell line Treatment of 661W cells with a Fas-activating antibody led to increased levels of Faim2. Both ERK and JNK stress kinase pathways were activated in Fas-treated 661W cells, but only the inhibition of the ERK pathway reduced the levels of Faim2. Blocking the ERK pathway using a pharmacological inhibitor increased the susceptibility of 661W cells to Fas-induced caspase activation and apoptosis. When the levels of Faim2 were reduced in 661W cells by siRNA knockdown, Fas activating antibody treatment resulted in earlier and more robust caspase activation, and increased cell death. These results demonstrate that Faim2 acts as a neuroprotectant during Fas-mediated apoptosis of 661W cells. The expression of Faim2 is triggered, at least in part, by Fas-receptor activation and subsequent ERK signaling. Our findings identify a novel protective pathway that auto-regulates Fas-induced photoreceptor apoptosis in vitro. Modulation of this pathway to increase Faim2 expression may be a potential therapeutic option to prevent photoreceptor death. PMID:23029562

  8. Nitric oxide mediates coral bleaching through an apoptotic-like cell death pathway: evidence from a model sea anemone-dinoflagellate symbiosis.

    PubMed

    Hawkins, Thomas D; Bradley, Benjamin J; Davy, Simon K

    2013-12-01

    Coral bleaching (involving the loss of symbiotic algae from the cnidarian host) is a major threat to coral reefs and appears to be mediated at the cellular level by nitric oxide (NO). In this study, we examined the specific role of NO in bleaching using the sea anemone Aiptasia pulchella, a model system for the study of corals. Exposure of A. pulchella to high-temperature shock (26-33C over <1 h) or an NO donor (S-nitrosoglutathione) resulted in significant increases in host caspase-like enzyme activity. These responses were reflected in the intensities of bleaching, which were significantly higher in heat- or NO-treated specimens than in controls maintained in seawater at 26C. Notably, the inhibition of caspase-like activity prevented bleaching even in the presence of an NO donor or at elevated temperature. The additional use of an NO scavenger controlled for effects mediated by agents other than NO. We also exposed A. pulchella to a more ecologically relevant treatment (an increase from 26 to 33C over 6-7 d). Again, host NO synthesis correlated with the activation of caspase-like enzyme activity. Therefore, we conclude that NO's involvement in cnidarian bleaching arises through the regulation of host apoptotic pathways. PMID:23934282

  9. Activation of NF-?B is required for mediating proliferative and anti-apoptotic effects of progastrin on proximal colonic crypts of mice, in vivo

    PubMed Central

    Umar, Shahid; Sarkar, Shubhashish; Cowey, Stephanie; Singh, Pomila

    2010-01-01

    Mice over-expressing progastrin in intestinal mucosa (Fabp-PG mice) are at an increased risk of proximal colon carcinogenesis in response to azoxymethane. In here we report a significant increase in the length of proximal colonic crypts in Fabp-PG mice, associated with potent anti-apoptotic effects of progastrin, which likely contributed to the previously reported increase in colon carcinogenesis in Fabp-PG mice. Phosphorylation of IKK?/?, I?B? and p65NF-?B was significantly elevated in proximal colonic crypts of Fabp-PG versus wild-type mice, which was associated with degradation of I?B? and nuclear translocation/activation of p65. Surprisingly, distal colonic crypt cells were not as responsive to elevated levels of progastrin in Fabp-PG mice. Annexin II, recently described as a high affinity receptor for progastrin, strongly co-localized with progastrin intracellularly and on basolateral membranes of proximal crypt cells, providing evidence that annexin-II binds progastrin in situ in colonic crypt cells. Proliferative and anti-apoptotic effects of progastrin on proximal crypts of Fabp-PG mice were attenuated to wild type levels, on treatment with NEMO peptide (an inhibitor of NF-?B activation), demonstrating for the first time a critical role of NF-?B in mediating hyperproliferative affects of progastrin on colonic crypts of Fabp-PG mice, in vivo. Thus, down regulation of NF-?B may significantly reduce the increased risk of colon carcinogenesis in response to progastrin. PMID:18521082

  10. Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

    SciTech Connect

    Ji Lili; Chen Ying; Liu Tianyu; Wang Zhengtao

    2008-09-15

    Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 {mu}M)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 {mu}M clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 {mu}M) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway.

  11. The apoptotic effect of brucine from the seed of Strychnos nux-vomica on human hepatoma cells is mediated via Bcl-2 and Ca2+ involved mitochondrial pathway.

    PubMed

    Deng, Xukun; Yin, Fangzhou; Lu, Xiaoyu; Cai, Baochang; Yin, Wu

    2006-05-01

    In an attempt to dissect the mechanism of Strychnos nux-vomica, a commonly used Chinese folk medicine in the therapy of liver cancer, the cytotoxic effects of four alkaloids in Strychnos nux-vomica, brucine, brucine N-oxide, strychnine, and isostrychnine, on human hepatoma cells (HepG2) were screened by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrasolium bromide (MTT) assay. Brucine, among the four alkaloids, exhibited the strongest toxic effect, the mechanism of which was found to cause HepG2 cell apoptosis, since brucine caused HepG2 cell shrinkage, the formation of apoptotic bodies, DNA fragmentation, cell cycle arrest, as well as phosphatidylserine externalization, all of which are typical characteristics of apoptotic programmed cell death. Brucine-induced HepG2 cell apoptosis was caspase dependent, with caspase-3 activated by caspase-9. Brucine also caused the proteolytic processing of caspase-9. In addition, brucine caused depolarization of the mitochondrial membrane of HepG2 cells, the inhibition of which by cyclosporine A completely abrogated the activation of casapses and release of cytochrome c in brucine-treated HepG2 cells. These findings suggested a pivotal role of mitochondrial membrane depolarization in HepG2 cell apoptosis elicited by brucine. Furthermore, brucine induced a rapid and sustained elevation of intracellular [Ca2+], which compromised the mitochondrial membrane potential and triggered the process of HepG2 cell apoptosis. Finally, Bcl-2 was found to predominately control the whole event of cell apoptosis induced by brucine. The elevation of [Ca2+]i caused by brucine was also suppressed by overexpression of Bcl-2 protein in HepG2 cells. From the facts given above, Ca2+ and Bcl-2 mediated mitochondrial pathway were found to be involved in brucine-induced HepG2 cell apoptosis. PMID:16443926

  12. Non-conventional apoptotic response to ionising radiation mediated by N-methyl D-aspartate receptors in immature neuronal cells

    PubMed Central

    SAMARI, NADA; DE SAINT-GEORGES, LOUIS; PANI, GIUSEPPE; BAATOUT, SARAH; LEYNS, LUC; BENOTMANE, MOHAMMED ABDERRAFI

    2013-01-01

    During cortical development, N-methyl D-aspartate (NMDA) receptors are highly involved in neuronal maturation and synapse establishment. Their implication in the phenomenon of excitotoxicity has been extensively described in several neurodegenerative diseases due to the permissive entry of Ca2+ ions and massive accumulation in the intracellular compartment, which is highly toxic to cells. Ionising radiation is also a source of stress to the cells, particularly immature neurons. Their capacity to induce cell death has been described for various cell types either by directly damaging the DNA or indirectly through the generation of reactive oxygen species responsible for the activation of a battery of stress response effectors leading in certain cases, to cell death. In this study, in order to determine whether a link exists between NMDA receptors-mediated excitotoxicity and radiation-induced cell death, we evaluated radiation-induced cell death in vitro and in vivo in maturing neurons during the fetal period. Cell death induction was assessed by TUNEL, caspase-3 activity and DNA ladder assays, with or without the administration of dizocilpine (MK-801), a non-competitive NMDA receptor antagonist which blocks neuronal Ca2+ influx. To further investigate the possible involvement of Ca2+-dependent enzyme activation, known to occur at high Ca2+ concentrations, we examined the protective effect of a calpain inhibitor on cell death induced by radiation. Doses ranging from 0.2 to 0.6 Gy of X-rays elicited a clear apoptotic response that was prevented by the injection of dizocilpine (MK-801) or calpain inhibitor. These data demonstrate the involvement of NMDA receptors in radiation-induced neuronal death by the activation of downstream effectors, including calpain-related pathways. An increased apoptotic process elicited by radiation, occurring independently of the normal developmental scheme, may eliminate post-mitotic but immature neuronal cells and deeply impair the establishment of the neuronal network, which in the case of cortical development is critical for cognitive capacities. PMID:23338045

  13. Mangiferin Attenuates Diabetic Nephropathy by Inhibiting Oxidative Stress Mediated Signaling Cascade, TNFα Related and Mitochondrial Dependent Apoptotic Pathways in Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Pal, Pabitra Bikash; Sinha, Krishnendu; Sil, Parames C.

    2014-01-01

    Oxidative stress plays a crucial role in the progression of diabetic nephropathy in hyperglycemic conditions. It has already been reported that mangiferin, a natural C-glucosyl xanthone and polyhydroxy polyphenol compound protects kidneys from diabetic nephropathy. However, little is known about the mechanism of its beneficial action in this pathophysiology. The present study, therefore, examines the detailed mechanism of the beneficial action of mangiferin on STZ-induced diabetic nephropathy in Wister rats as the working model. A significant increase in plasma glucose level, kidney to body weight ratio, glomerular hypertrophy and hydropic changes as well as enhanced nephrotoxicity related markers (BUN, plasma creatinine, uric acid and urinary albumin) were observed in the experimental animals. Furthermore, increased oxidative stress related parameters, increased ROS production and decreased the intracellular antioxidant defenses were detected in the kidney. Studies on the oxidative stress mediated signaling cascades in diabetic nephropathy demonstrated that PKC isoforms (PKCα, PKCβ and PKCε), MAPKs (p38, JNK and ERK1/2), transcription factor (NF-κB) and TGF-β1 pathways were involved in this pathophysiology. Besides, TNFα was released in this hyperglycemic condition, which in turn activated caspase 8, cleaved Bid to tBid and finally the mitochorndia-dependent apoptotic pathway. In addition, oxidative stress also disturbed the proapoptotic-antiapoptotic (Bax and Bcl-2) balance and activated mitochorndia-dependent apoptosis via caspase 9, caspase 3 and PARP cleavage. Mangiferin treatment, post to hyperglycemia, successfully inhibited all of these changes and protected the cells from apoptotic death. PMID:25233093

  14. Temozolomide-mediated DNA methylation in human myeloid precursor cells:differential involvement of intrinsic and extrinsic apoptotic pathways

    PubMed Central

    Wang, Haiyan; Cai, Shanbao; Ernstberger, Aaron; Bailey, Barbara J.; Wang, Michael Z.; Cai, Wenjing; Goebel, W. Scott; Czader, Magdalena B.; Crean, Colin; Suvannasankhah, Attaya; Shokolenkoc, Inna; Wilson, Glenn L.; Baluyut, Arthur R.; Mayo, Lindsey D.; Pollok, Karen E.

    2013-01-01

    Purpose An understanding of how hematopoietic cells respond to therapy that causes myelosuppression will help develop approaches to prevent this potentially life-threatening toxicity. The goal of this study was to determine how human myeloid precursor cells (MP) respond to temozolomide (TMZ)-induced DNA damage. Experimental Design We developed an ex vivo primary human MP cells model system to investigate the involvement of cell-death pathways using a known myelosuppressive regimen of O6-benzylguanine (6BG) and TMZ. Results Exposure to 6BG/TMZ led to increases in p53, p21, ?-H2AX, and mitochondrial DNA damage. Increases in mitochondrial membrane depolarization correlated with increased caspase-9 and caspase-3 activities following 6BG/TMZ treatment. These events correlated with decreases in activated AKT, downregulation of the DNA repair protein O6methylguanine-DNA methyltransferase (MGMT), and increased cell death. During MP cell expansion, FAS/CD95/APO1(FAS) expression increased over time and was present on ~100% of the cells following exposure to 6BG/TMZ. While c-flipshort, an endogenous inhibitor of FAS-mediated signaling, was decreased in 6BG/TMZ-treated versus control, 6BG-, or TMZ alone-treated cells, there were no changes in caspase-8 activity. Additionally, there were no changes in the extent of cell death in MP cells exposed to 6BG/TMZ in the presence of neutralizing or agonistic anti-FAS antibodies, indicating that FAS-mediated signaling was not operative. Conclusions In human MP cells, 6BG/TMZ-initiated apoptosis occurred by intrinsic, mitochondrial-mediated and not extrinsic, FAS-mediated apoptosis. Human MP cells represent a clinically relevant model system for gaining insight into how hematopoietic cells respond to chemotherapeutics and offer an approach for selecting effective chemotherapeutic regimens with limited hematopoietic toxicity. PMID:23536437

  15. Regulation of anti-apoptotic signaling by Kruppel-like factors 4 and 5 mediates lapatinib resistance in breast cancer.

    PubMed

    Farrugia, M K; Sharma, S B; Lin, C-C; McLaughlin, S L; Vanderbilt, D B; Ammer, A G; Salkeni, M A; Stoilov, P; Agazie, Y M; Creighton, C J; Ruppert, J M

    2015-01-01

    The Kruppel-like transcription factors (KLFs) 4 and 5 (KLF4/5) are coexpressed in mouse embryonic stem cells, where they function redundantly to maintain pluripotency. In mammary carcinoma, KLF4/5 can each impact the malignant phenotype, but potential linkages to drug resistance remain unclear. In primary human breast cancers, we observed a positive correlation between KLF4/5 transcript abundance, particularly in the human epidermal growth factor receptor 2 (HER2)-enriched subtype. Furthermore, KLF4/5 protein was rapidly upregulated in human breast cancer cells following treatment with the HER2/epidermal growth factor receptor inhibitor, lapatinib. In addition, we observed a positive correlation between these factors in the primary tumors of genetically engineered mouse models (GEMMs). In particular, the levels of both factors were enriched in the basal-like tumors of the C3(1) TAg (SV40 large T antigen transgenic mice under control of the C3(1)/prostatein promoter) GEMM. Using tumor cells derived from this model as well as human breast cancer cells, suppression of KLF4 and/or KLF5 sensitized HER2-overexpressing cells to lapatinib. Indicating cooperativity, greater effects were observed when both genes were depleted. KLF4/5-deficient cells had reduced basal mRNA and protein levels of the anti-apoptotic factors myeloid cell leukemia 1 (MCL1) and B-cell lymphoma-extra large (BCL-XL). Moreover, MCL1 was upregulated by lapatinib in a KLF4/5-dependent manner, and enforced expression of MCL1 in KLF4/5-deficient cells restored drug resistance. In addition, combined suppression of KLF4/5 in cultured tumor cells additively inhibited anchorage-independent growth, resistance to anoikis and tumor formation in immunocompromised mice. Consistent with their cooperative role in drug resistance and other malignant properties, KLF4/5 levels selectively stratified human HER2-enriched breast cancer by distant metastasis-free survival. These results identify KLF4 and KLF5 as cooperating protumorigenic factors and critical participants in resistance to lapatinib, furthering the rationale for combining anti-MCL1/BCL-XL inhibitors with conventional HER2-targeted therapies. PMID:25789974

  16. Regulation of anti-apoptotic signaling by Kruppel-like factors 4 and 5 mediates lapatinib resistance in breast cancer

    PubMed Central

    Farrugia, M K; Sharma, S B; Lin, C-C; McLaughlin, S L; Vanderbilt, D B; Ammer, A G; Salkeni, M A; Stoilov, P; Agazie, Y M; Creighton, C J; Ruppert, J M

    2015-01-01

    The Kruppel-like transcription factors (KLFs) 4 and 5 (KLF4/5) are coexpressed in mouse embryonic stem cells, where they function redundantly to maintain pluripotency. In mammary carcinoma, KLF4/5 can each impact the malignant phenotype, but potential linkages to drug resistance remain unclear. In primary human breast cancers, we observed a positive correlation between KLF4/5 transcript abundance, particularly in the human epidermal growth factor receptor 2 (HER2)-enriched subtype. Furthermore, KLF4/5 protein was rapidly upregulated in human breast cancer cells following treatment with the HER2/epidermal growth factor receptor inhibitor, lapatinib. In addition, we observed a positive correlation between these factors in the primary tumors of genetically engineered mouse models (GEMMs). In particular, the levels of both factors were enriched in the basal-like tumors of the C3(1) TAg (SV40 large T antigen transgenic mice under control of the C3(1)/prostatein promoter) GEMM. Using tumor cells derived from this model as well as human breast cancer cells, suppression of KLF4 and/or KLF5 sensitized HER2-overexpressing cells to lapatinib. Indicating cooperativity, greater effects were observed when both genes were depleted. KLF4/5-deficient cells had reduced basal mRNA and protein levels of the anti-apoptotic factors myeloid cell leukemia 1 (MCL1) and B-cell lymphoma-extra large (BCL-XL). Moreover, MCL1 was upregulated by lapatinib in a KLF4/5-dependent manner, and enforced expression of MCL1 in KLF4/5-deficient cells restored drug resistance. In addition, combined suppression of KLF4/5 in cultured tumor cells additively inhibited anchorage-independent growth, resistance to anoikis and tumor formation in immunocompromised mice. Consistent with their cooperative role in drug resistance and other malignant properties, KLF4/5 levels selectively stratified human HER2-enriched breast cancer by distant metastasis-free survival. These results identify KLF4 and KLF5 as cooperating protumorigenic factors and critical participants in resistance to lapatinib, furthering the rationale for combining anti-MCL1/BCL-XL inhibitors with conventional HER2-targeted therapies. PMID:25789974

  17. Cinobufagin exerts anti-proliferative and pro-apoptotic effects through the modulation ROS-mediated MAPKs signaling pathway.

    PubMed

    Baek, Seung Ho; Kim, Chulwon; Lee, Jong Hyun; Nam, Dongwoo; Lee, Junhee; Lee, Seok-Geun; Chung, Won-Seok; Jang, Hyeung-Jin; Kim, Sung-Hoon; Ahn, Kwang Seok

    2015-06-01

    Cinobufagin (CBG) is a cardiotoxic bufanolide steroid secreted by the skin and parotid venom glands of the Asiatic toad Bufo bufo gargarizans (called Chan-Su). Although CBG is known to exhibit anti-cancer activities, very little is known about its potential mechanism(s) of action. In this study, we investigated whether CBG mediates its effect through the modulation of the mitogen-activated protein kinases (MAPKs) signaling pathway in human multiple myeloma (MM) U266 cells. We found that CBG caused the significant activation of ERK, JNK and p38 MAPK in U266 cells. CBG showed much higher cytotoxicity against U266 cells as compared to peripheral blood mononuclear cells (PBMC). Induction of CBG increased reactive oxygen species (ROS) generation from mitochondria, which is associated with the induction of apoptosis as characterized by increased sub-G1 DNA contents of cell cycle, positive Annexin V binding, activation of caspase-3 and cleavage of PARP. Inhibition of ROS generation by N-acetyl-l-cysteine (NAC) significantly prevented CBG-induced ERK, JNK and p38 MAPK activation and apoptosis. CBG also down-regulated the expression of various downstream gene products that mediate cell proliferation, survival, angiogenesis and metastasis. Interestingly, ERK, JNK and p38MAPK pharmacological inhibitors blocked CBG-induced MAPKs activation and ERK inhibitor (PD98059) also prevented the CBG-induced caspase-3 activation and PARP cleavage in U266 cells. Taken together, these findings suggest that CBG can act as a potent anticancer agent against MM and possibly exerts its effects through the ROS-mediated activation of ERK, JNK and p38 MAPK leading to the activation of caspase-3 in U266 cells. PMID:25982794

  18. A Novel Form of DAP5 Protein Accumulates in Apoptotic Cells as a Result of Caspase Cleavage and Internal Ribosome Entry Site-Mediated Translation

    PubMed Central

    Henis-Korenblit, Sivan; Strumpf, Naomi Levy; Goldstaub, Dan; Kimchi, Adi

    2000-01-01

    Death-associated protein 5 (DAP5) (also named p97 and NAT1) is a member of the translation initiation factor 4G (eIF4G) family that lacks the eIF4E binding site. It was previously implicated in apoptosis, based on the finding that a dominant negative fragment of the protein protected against cell death. Here we address its function and two distinct levels of regulation during apoptosis that affect the protein both at translational and posttranslational levels. DAP5 protein was found to be cleaved at a single caspase cleavage site at position 790, in response to activated Fas or p53, yielding a C-terminal truncated protein of 86 kDa that is capable of generating complexes with eIF4A and eIF3. Interestingly, while the overall translation rate in apoptotic cells was reduced by 60 to 70%, in accordance with the simultaneous degradation of the two major mediators of cap-dependent translation, eIF4GI and eIF4GII, the translation rate of DAP5 protein was selectively maintained. An internal ribosome entry site (IRES) element capable of directing the translation of a reporter gene when subcloned into a bicistronic vector was identified in the 5? untranslated region of DAP5 mRNA. While cap-dependent translation from this transfected vector was reduced during Fas-induced apoptosis, the translation via the DAP5 IRES was selectively maintained. Addition of recombinant DAP5/p97 or DAP5/p86 to cell-free systems enhanced preferentially the translation through the DAP5 IRES, whereas neutralization of the endogenous DAP5 in reticulocyte lysates by adding a dominant negative DAP5 fragment interfered with this translation. The DAP5/p86 apoptotic form was more potent than DAP5/p97 in these functional assays. Altogether, the data suggest that DAP5 is a caspase-activated translation factor which mediates cap-independent translation at least from its own IRES, thus generating a positive feedback loop responsible for the continuous translation of DAP5 during apoptosis. PMID:10611228

  19. Suppression of ATAD2 inhibits hepatocellular carcinoma progression through activation of p53- and p38-mediated apoptotic signaling

    PubMed Central

    Lu, Wen-Jing; Chua, Mei-Sze; So, Samuel K.

    2015-01-01

    The ATPase family, AAA domain containing 2 (ATAD2) is highly expressed in multiple cancers. We aim to understand the clinical and biological significance of ATAD2 over-expression in hepatocellular carcinoma (HCC), as a means to validate it as a therapeutic target in HCC. We demonstrated that ATAD2 was over-expressed in HCC patients, where high ATAD2 levels were significantly correlated with aggressive phenotypes such as high AFP levels, advanced tumor stages, and vascular invasion. Using RNA interference, suppression of ATAD2 in HCC cell lines decreased cell viability, migration, and invasion, and induced apoptosis in vitro. Furthermore, we identified p53 and p38 as key proteins that mediate apoptosis induced by ATAD2 suppression. In HCC cells, we demonstrated that ATAD2 directly interacted with MKK3/6, which prevented p38 activation and therefore inhibited p38-mediated apoptosis. In vivo, suppression of ATAD2 impaired the growth of HepG2 and Hep3B subcutaneous xenografts, accompanied by enhanced apoptosis and p-p53 and p-p38 levels. Our results validate that ATAD2 is an important negative regulator of apoptosis, and that neutralizing its activity has promising anti-tumor effects in HCC cells. PMID:26497681

  20. RNAi-mediated Downregulation of MMP-2 Activates the Extrinsic Apoptotic Pathway in Human Glioma Xenograft Cells

    PubMed Central

    Gondi, Christopher S.; Dinh, Dzung H.; Gujrati, Meena; Rao, Jasti S.

    2009-01-01

    Malignant gliomas are characterized by invasive and infiltrative behavior that generally involves the destruction of normal brain tissue. Strategies to treat infiltrating gliomas, such as chemotherapy and gene therapy, have remained largely unsuccessful. The infiltrative nature of gliomas can be attributed largely to proteases, which include serine, metallo and cysteine proteases. Our previous work and that of others strongly suggest a relationship between the expression of uPAR, MMP-9, and MMP-2; this relationship is generally indicative of the infiltrative phenotype of gliomas. In the present study, we have demonstrated that the RNAi-mediated downregulation of MMP-2 induces apoptosis in the 4910 human glioma xenograft cell line. Using western blot analysis, we observed that caspase-8 levels increased in MMP-2-downregulated cells whereas TRADD and TRAF-2 levels decreased. Further, NIK levels increased in MMP-2-downregulated cells. To determine the nuclear localization of AIF and I?B-?, we analyzed the levels of AIF, I?B-? and p-I?B-? in the cytosolic and nuclear fractions of MMP-2-downregulated cells. Western blot analysis revealed that MMP-2 downregulation resulted in the translocation of AIF to the nucleus and also inhibited the nuclear localization of p-I?B-?. To confirm the involvement of AIF, we performed FACS analysis to determine the integrity of the mitochondrial membrane using the Mito-PT method. FACS analysis showed that the downregulation of MMP-2 caused a collapse in the mitochondrial cell membrane. Immunolocalization of AIF revealed that in MMP-2-downregulated cells, AIF translocates to the nucleus, thereby enabling the induction of apoptosis. RT-PCR analysis revealed that caspase-8 was overexpressed 57-fold, whereas p73 was downregulated 28-fold. Evidence of apoptosis was determined by TUNEL assay and visualization of nuclear fragmentation by DAPI staining. In summary, it is evident from our results that MMP-2 downregulation induces caspase-8 and AIF-mediated apoptosis and, as such, shows potential for glioma therapy. PMID:19724922

  1. Ablation of axial structures activates apoptotic pathways in somite cells of the chick embryo.

    PubMed

    Sanders, E J; Parker, E

    2001-11-01

    It has been known for some time that ablation of the neural tube and/or the notochord in the chick embryo leads to a massive wave of cell death in the adjacent somites. It is postulated that in the normal embryo, survival signals emanate from the neural tube and/or notochord that suppress apoptosis in the cells of the somites, except for a small population of sclerotome cells that are programmed to die naturally. In this study we show that axial ablation results in the death of sclerotome and not somitic neural crest cells, and we have examined the apoptotic response of these cells to the ablation. We show that several elements of the apoptotic cascade become detectable in somite cells in response to the withdrawal of survival signals. We demonstrate the down-regulation of bcl-2 protein in the somites adjacent to, and caudal to, the site of ablation, corresponding to the region that displays an elevated level of cell death. Although caspase-9 appeared to be activated in somites at all levels of the trunk, caspase-2 showed a clear response to the ablation of the axial structures. Removal of the neural tube and notochord produced an up-regulation of caspase-2 activity in somites in the region of the operation. Cleavage of two down-stream substrates of these caspases was examined. The cleavage of poly (ADP-ribose) polymerase (PARP) was apparent in somites at all levels of the trunk, and showed only a modest up-regulation after ablation. By contrast, the cleavage of DNA fragmentation factor (DFF45) showed a marked up-regulation in response to ablation, suggesting that this is a primary substrate for a caspase-dependent apoptotic mechanism. Evidence was also found for a caspase-independent mechanism, since the expression of apoptosis-inducing factor (AIF) was found to be very sensitive to, and up-regulated in somites by, axial ablation. Because the wave of apoptosis that is precipitated in somites by removal of the axial structures may be mediated by BMP-4, we examined the levels of BMP-4 in somites in response to axial ablation. BMP-4 expression was clearly up-regulated in somites adjacent to, or close to, the site of operation. PMID:11789986

  2. Efficacy of PLGA-loaded apigenin nanoparticles in Benzo[a]pyrene and ultraviolet-B induced skin cancer of mice: mitochondria mediated apoptotic signalling cascades.

    PubMed

    Das, Sreemanti; Das, Jayeeta; Samadder, Asmita; Paul, Avijit; Khuda-Bukhsh, Anisur Rahman

    2013-12-01

    Skin cancer is increasing at an alarming rate and becoming resistant to conventional chemotherapy necessitating improved drug delivery system. We loaded apigenin (Ap), a dietary flavonoid having anti-cancer property, with poly (lactic-co-glycolide) (PLGA) nanoparticles (NAp) to explore if nano-encapsulation could enhance anti-carcinogenic effect against ultra-violet B (UVB) and Benzo(a)pyrene (BaP) induced skin tumor and mitochondrial dysfunction in mice. Particle size, morphology and zeta potential of NAp were determined using dynamic light scattering and atomic force microscopy. Tumor incidence and multiplicity in UVB-BaP induced mice with/without NAp treatment were ascertained and their histolopathological sections and chromosomal aberrations were studied. ROS accumulation and mitochondrial functioning through relevant markers like mitochondrial transmembrane potential were analyzed. Mitochondrial volume changes/swelling, cytochrome c (cyt c) release, mRNA and protein expressions of Apaf-1, bax, bcl-2, cyt c, cleaved caspase-9 and 3 were studied. Results showed that NAp produced better effects than Ap, due to their smaller size, and faster mobility. NAp reduced tissue damage and frequency of chromosomal aberrations, increased ROS accumulation to mediate mitochondrial-apoptosis through modulation of several apoptotic markers and mitochondrial matrix swelling. NAp showed ameliorative potentials in combating skin cancer and therefore has greater prospect of use in therapeutic management of skin cancer. PMID:24120900

  3. Apoptotic induction with bifunctional E.coli cytosine deaminase-uracil phosphoribosyltransferase mediated suicide gene therapy is synergized by curcumin treatment in vitro.

    PubMed

    Gopinath, P; Ghosh, Siddhartha Sankar

    2008-05-01

    Development of novel suicide gene therapy vector with potential application in cancer treatment has a great impact on human health. Investigation to understand molecular mechanism of cell death is necessary to evaluate the therapeutic application of suicide vectors. For example, the bifunctional E.coli cytosine deaminase & uracil phosphoribosyltransferase fusion (CD-UPRT) gene expression is known to sensitize a wide range of cells toward nontoxic prodrug 5-flurocytosine (5-FC) by converting it to toxic compounds, but the exact pathway of cell death is yet to be defined. Herein, we investigated the mechanism of cell death by 5-FC/CD-UPRT suicide system in both cancer and non-cancer cells and found that the optimum 5-FC concentration led to programmed cell death in vitro. The CD-UPRT expression of transfected cells was measured by the RT-PCR analysis. Biochemical assays, such as mitochondrial activity (MTS) and lactate dehydrogenase (LDH) measurements exhibited cell death. Microscopic experiments showed characteristic onset of apoptosis which was further supported by internucleosomal DNA cleavage of BrdU labeled cellular DNA, appearance of characteristic laddering of chromosomal DNA and involvement of caspase pathway. Furthermore, the 5-FC/CD-UPRT-mediated apoptosis was potentiated with addition of a known anticancer agent curcumin. Our in vitro studies confirmed synergistic induction of apoptotic pathway in the combination treatment. Therefore, combination of 5-FC/CD-UPRT with curcumin could be a potential chemosensitization strategy for cancer treatment. PMID:18092145

  4. Puerarin attenuates glucocorticoid-induced apoptosis of hFOB1.19 cells through the JNK-and Akt-mediated mitochondrial apoptotic pathways

    PubMed Central

    YU, DONGDONG; MU, SHUAI; ZHAO, DANYANG; WANG, GUANGBIN; CHEN, ZHIGUANG; REN, HONGFEI; FU, QIN

    2015-01-01

    Puerarin is an active component of Pueraria lobata, which is a commonly used Chinese herbal medicine for the treatment of osteoporosis. The present study aimed to evaluate the osteoprotective effect of puerarin on glucocorticoid (GC)-induced apoptosis of osteoblasts in vitro. The effects of puerarin on dexamethasone (DEX)-induced cell apoptosis were assessed using enzyme-linked immunosorbent assay and a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and found that the viability of hFOB1.19 cells was significantly increased following exposure to between 10−6 and 10−10 M puerarin, with a maximal anti-apoptotic effect at a concentration of 10−8 M. In addition, compared with the control group, puerarin upregulated the transcription and protein levels of B-cell lymphoma-2 and downregulated B-cell-associated X protein in the hFOB1.19 cells. Puerarin attenuated the DEX-induced release of cytochrome c and cleavage of caspase-3, and treatment with puerarin inhibited the c-Jun N-terminal kinase (JNK) pathway and activated the phosphoinositide 3-kinase (PI3K)/Akt pathway in the hFOB1.19 cells. Furthermore, the Akt inhibitor, LY294002, partly eliminated the protective effect of puerarin on DEX-induced apoptosis, and puerarin combined with the JNK inhibitor, SP600125, suppressed DEX-induced apoptosis to a lesser extent than in the cells treated with SP600125 alone. These results suggested that the JNK and PI3K/Akt signaling pathways mediate the inhibitory effects of puerarin on apoptosis in the hFOB1.19 cells. In conclusion, puerarin prevented DEX-induced apoptosis of hFOB1.19 cells via inhibition of the JNK pathway and activation of the PI3K/Akt signaling pathway in the cells, dependent on the mitochondrial apoptotic pathway. These results support puerarin as a promising target in the treatment of GC-induced osteoporosis. PMID:26101183

  5. Puerarin attenuates glucocorticoid-induced apoptosis of hFOB1.19 cells through the JNK- and Akt-mediated mitochondrial apoptotic pathways.

    PubMed

    Yu, Dongdong; Mu, Shuai; Zhao, Danyang; Wang, Guangbin; Chen, Zhiguang; Ren, Hongfei; Fu, Qin

    2015-08-01

    Puerarin is an active component of Pueraria lobata, which is a commonly used Chinese herbal medicine for the treatment of osteoporosis. The present study aimed to evaluate the osteoprotective effect of puerarin on glucocorticoid (GC)-induced apoptosis of osteoblasts in vitro. The effects of puerarin on dexamethasone (DEX)-induced cell apoptosis were assessed using enzyme-linked immunosorbent assay and a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and found that the viability of hFOB1.19 cells was significantly increased following exposure to between 10(-6) and 10(-10) M puerarin, with a maximal anti-apoptotic effect at a concentration of 10(-8) M. In addition, compared with the control group, puerarin upregulated the transcription and protein levels of B-cell lymphoma-2 and downregulated B-cell-associated X protein in the hFOB1.19 cells. Puerarin attenuated the DEX-induced release of cytochrome c and cleavage of caspase-3, and treatment with puerarin inhibited the c-Jun N-terminal kinase (JNK) pathway and activated the phosphoinositide 3-kinase (PI3K)/Akt pathway in the hFOB1.19 cells. Furthermore, the Akt inhibitor, LY294002, partly eliminated the protective effect of puerarin on DEX-induced apoptosis, and puerarin combined with the JNK inhibitor, SP600125, suppressed DEX-induced apoptosis to a lesser extent than in the cells treated with SP600125 alone. These results suggested that the JNK and PI3K/Akt signaling pathways mediate the inhibitory effects of puerarin on apoptosis in the hFOB1.19 cells. In conclusion, puerarin prevented DEX-induced apoptosis of hFOB1.19 cells via inhibition of the JNK pathway and activation of the PI3K/Akt signaling pathway in the cells, dependent on the mitochondrial apoptotic pathway. These results support puerarin as a promising target in the treatment of GC-induced osteoporosis. PMID:26101183

  6. Polypyrimidine Track-binding Protein Binding Downstream of Caspase-2 Alternative Exon 9 Represses Its Inclusion*

    PubMed Central

    Ct, Jocelyn; Dupuis, Sophie; Wu, Jane Y.

    2007-01-01

    We have been using the caspase-2 pre-mRNA as a model system to study the importance of alternative splicing in the regulation of programmed cell death. Inclusion or skipping of a cassette-type exon in the 3? portion of this pre-mRNA leads to the production of isoforms with antagonistic activity in apoptosis. We previously identified a negative regulatory element (In100) located in the intron downstream of alternative exon 9. The upstream portion of this element harbors a decoy 3? acceptor site that engages in nonproductive commitment complex interactions with the 5? splice site of exon 9. This in turn confers a competitive advantage to the exon-skipping splicing pattern. Further characterization of the In100 element reveals a second, functionally distinct, domain located downstream from the decoy 3? acceptor site. This downstream domain harbors several polypyrimidine track-binding protein (PTB)-binding sites. We show that PTB binding to these sites correlates with the negative effect on exon 9 inclusion. Finally, we show that both domains of the In100 element can function independently to repress exon 9 inclusion, although PTB binding in the vicinity of the decoy 3? splice site can modulate its activity. Our results thus reveal a complex composite element that regulates caspase-2 exon 9 alternative splicing through a novel mechanism. PMID:11116151

  7. Immunosuppressive effects of apoptotic cells

    NASA Astrophysics Data System (ADS)

    Voll, Reinhard E.; Herrmann, Martin; Roth, Edith A.; Stach, Christian; Kalden, Joachim R.; Girkontaite, Irute

    1997-11-01

    Apoptotic cell death is important in the development and homeostasis of multicellular organisms and is a highly controlled means of eliminating dangerous, damaged or unnecessary cells without causing an inflammatory response or tissue damage,. We now show that the presence of apoptotic cells during monocyte activation increases their secretion of the anti-inflammatory and immunoregulatory cytokine interleukin 10 (IL-10) and decreases secretion of the proinflammatory cytokines tumour necrosis factor-? (TNF-?), IL-1 and IL-12. This may inhibit inflammation and contribute to impaired cell-mediated immunity in conditions associated with increased apoptosis, such as viral infections, pregnancy, cancer and exposure to radiation.

  8. Apoptotic Response through a High Mobility Box 1 Protein-Dependent Mechanism in LPS/GalN-Induced Mouse Liver Failure and Glycyrrhizin-Mediated Inhibition

    PubMed Central

    Kuroda, Noriyuki; Inoue, Kouji; Ikeda, Tadayuki; Hara, Yaiko; Wake, Kenjiro; Sato, Tetsuji

    2014-01-01

    HMGB1 is a nuclear component involved in nucleosome stabilization and transcription regulation, but extracellularly it is able to serve as a potential late mediator of lethality. In the present study, we explored inflammation-promoting activity of HMGB1 and blockade of extracellular release of HMGB1 by glycyrrhizin (GL) in LPS/GalN-triggered mouse liver injury. At 1 to 10 h after LPS/GalN-treatment, mice were anesthetized to collect blood from heart puncture, and serum transaminase and HMGB1 were evaluated. Administration of LPS/GalN precipitated tissue injury associated with time-dependent alteration in HMGB1 serum levels. At 8 h nuclear immunoreactive products were remarkably reduced and extracellular HMGB1 expression was found exclusively in the pericentral foci. The treatment with GL significantly down-regulated the serum levels of ALT, AST, and HMGB1 in addition to the strong inhibition of tissue injury and extracellular immunoreactivity to HMGB1 and to acetylated-lysine. Furthermore, GL brought about a significant decrease in the number of apoptotic hepatocytes labeled with TUNEL-method. On the basis of these results, three apoptosis-associated genes were identified with microarray analysis and real-time PCR. The ChIP-assay revealed the binding of HMGB1 protein to Gsto1 promoter sequence in LPS/GalN-treated mice and the remarkable decrease in combined HMGB1 protein by GL. The current findings claim that a single injection of LPS/GalN might stimulate apoptosis of hepatocytes through the binding of HMGB1 protein to Gsto1 promoter region and that GL-treatment might prevent the apoptosis and inflammatory infiltrates caused with LPS/GalN-injection by disturbing the binding of HMGB1 protein to Gsto1 promoter sequence. PMID:24690901

  9. Oridonin triggers apoptosis in colorectal carcinoma cells and suppression of microRNA-32 expression augments oridonin-mediated apoptotic effects.

    PubMed

    Yang, Jie; Jiang, Hai; Wang, Chunyu; Yang, Bo; Zhao, Lijun; Hu, Dongling; Qiu, Guihua; Dong, Xiaolin; Xiao, Bin

    2015-05-01

    Oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, has been found to exhibit various anti-tumor effects. In this work, to investigate its pharmacological effects on human colorectal carcinoma HCT-116 and LoVo cells, cell proliferation and apoptosis were respectively evaluated by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, annexin V-FITC, and propidium iodide (PI) staining. Western blotting was used to detect the expression levels of Bim, Bax, Bcl-2, cytosolic cytochrome c, procaspase-9, cleaved caspase-9, procaspase-3, and caspase-3 proteins. Caspase-Glo-9 and Caspase-Glo-3 assays were applied to determine caspase-9 and caspase-3 activity. MicroRNA-32 (miR-32) expression level was detected by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The in vivo anti-tumor effects of oridonin were evaluated using cell lines HCT-116 and LoVo xenograft model. The results indicated that oridonin effectively inhibited cell proliferation and induced apoptosis in HCT-116 and LoVo cells in a concentration-dependent manner. Oridonin treatment upregulated the expression levels of Bim, Bax, cytosolic cytochrome c, cleaved caspase-9 and cleaved caspase-3 proteins, downregulated the expression levels of Bcl-2, procaspase-9 and procaspase-3 proteins, and meanwhile obviously activated caspase-9 and caspase-3 in a dose-dependent manner in HCT-116 and LoVo cells. The results of qRT-PCR demonstrated that oridonin treatment significantly decreased miR-32 expression, and furthermore, suppression of miR-32 expression by miR-32 inhibitors augmented oridonin-mediated inhibitory and apoptotic effects in HCT-116 and LoVo cells. In vivo results indicated that oridonin administration through intraperitoneal injection suppressed tumor growth in nude mice. Therefore, these findings suggest that oridonin maybe is a potential candidate for colorectal cancer treatment. PMID:26054686

  10. Di-O-demethylcurcumin protects SK-N-SH cells against mitochondrial and endoplasmic reticulum-mediated apoptotic cell death induced by Aβ25-35.

    PubMed

    Pinkaew, Decha; Changtam, Chatchawan; Tocharus, Chainarong; Thummayot, Sarinthorn; Suksamrarn, Apichart; Tocharus, Jiraporn

    2015-01-01

    Alzheimer's disease (AD) is a neurodegenerative and progressive disorder. The hallmark of pathological AD is amyloid plaque which is the accumulation of amyloid β (Aβ) in extracellular neuronal cells and neurofibrillary tangles (NFT) in neuronal cells, which lead to neurotoxicity via reactive oxygen species (ROS) generation related apoptosis. Loss of synapses and synaptic damage are the best correlates of cognitive decline in AD. Neuronal cell death is the main cause of brain dysfunction and cognitive impairment. Aβ activates neuronal death via endoplasmic reticulum (ER) stress and mitochondria apoptosis pathway. This study investigated the underlying mechanisms and effects of di-O-demethylcurcumin in preventing Aβ-induced apoptosis. Pretreatment with di-O-demethylcurcumin for 2 h, which was followed by Aβ25-35 (10 µM) in human neuroblastoma SK-N-SH cells improved cell viability by using MTS assay and decreased neuronal cell apoptosis. Pretreatment with di-O-demethylcurcumin attenuated the number of nuclear condensations and number of apoptotic cells in Aβ25-35-induced group in a concentration-dependent manner by using transmission electron microscope (TEM) and flow cytometry, respectively. Di-O-demethylcurcumin also increased the ratio of Bcl-XL/Bax protein, and reduced intracellular ROS level, cytochrome c protein expression, cleaved caspase-9 protein expression, and cleaved caspase-3 protein expression. Additionally, di-O-demethylcurcumin treatment also reduced the expression of ER stress protein markers, including protein kinase RNA like endoplasmic reticulum kinase (PERK) phosphorylation, eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation, inositol-requiring enzyme 1 (IRE1) phosphorylation, X-box-binding protein-1 (XBP-1), activating transcription factor (ATF6), C/EBP homologous protein (CHOP), and cleaved caspase-12 protein. CHOP and cleaved caspase-12 protein are the key mediators of apoptosis. Our data suggest that di-O-demethylcurcumin is a candidate protectant against neuronal death through its suppression of the apoptosis mediated by mitochondrial death and ER stress pathway. PMID:25451798

  11. Brain angiogenesis inhibitor 1 is expressed by gastric phagocytes during infection with Helicobacter pylori and mediates the recognition and engulfment of human apoptotic gastric epithelial cells.

    PubMed

    Das, Soumita; Sarkar, Arup; Ryan, Kieran A; Fox, Sarah; Berger, Alice H; Juncadella, Ignacio J; Bimczok, Diane; Smythies, Lesley E; Harris, Paul R; Ravichandran, Kodi S; Crowe, Sheila E; Smith, Phillip D; Ernst, Peter B

    2014-05-01

    After Helicobacter pylori infection in humans, gastric epithelial cells (GECs) undergo apoptosis due to stimulation by the bacteria or inflammatory cytokines. In this study, we assessed the expression and function of brain angiogenesis inhibitor 1 (BAI1) in the engulfment of apoptotic GECs using human tissue and cells. After induction of apoptosis by H. pylori or camptothecin, there was a 5-fold increase in the binding of apoptotic GECs to THP-1 cells or peripheral blood monocyte-derived macrophages as assayed by confocal microscopy or conventional and imaging flow cytometry. Binding was impaired 95% by pretreating apoptotic cells with annexin V, underscoring the requirement for phosphatidylserine recognition. The phosphatidylserine receptor BAI1 was expressed in human gastric biopsy specimens and gastric phagocytes. To confirm the role of BAI1 in apoptotic cell clearance, the functional domain of BAI1 was used as a competitive inhibitor or BAI1 expression was inhibited by small interfering RNA. Both approaches decreased binding and engulfment >40%. Exposing THP-1 cells to apoptotic cells inhibited IL-6 production from 1340 to <364 pg/ml; however, this decrease was independent of phagocytosis. We conclude that recognition of apoptotic cells by BAI1 contributes to their clearance in the human gastric mucosa and this is associated with anti-inflammatory effects. PMID:24509909

  12. Maslinic Acid, a Natural Triterpene, Induces a Death Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma Cells.

    PubMed

    Reyes-Zurita, Fernando J; Rufino-Palomares, Eva E; García-Salguero, Leticia; Peragón, Juan; Medina, Pedro P; Parra, Andrés; Cascante, Marta; Lupiáñez, José A

    2016-01-01

    Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin. PMID:26751572

  13. Maslinic Acid, a Natural Triterpene, Induces a Death Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma Cells

    PubMed Central

    Reyes-Zurita, Fernando J.; Rufino-Palomares, Eva E.; García-Salguero, Leticia; Peragón, Juan; Medina, Pedro P.; Parra, Andrés; Cascante, Marta; Lupiáñez, José A.

    2016-01-01

    Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin. PMID:26751572

  14. A putative role of Drep1 in apoptotic DNA fragmentation system in fly is mediated by direct interaction with Drep2 and Drep4.

    PubMed

    Park, Ok Kyung; Park, Hyun Ho

    2013-04-01

    DNA fragmentation is common phenomenon for apoptotic cell death. DNA fragmentation factor, called DFF40 (CAD: mouse homologue), is a main nuclease for apoptotic DNA fragmentation. Nuclease activity of DFF40 is normally inhibited by DFF45 by tight interaction via CIDE domain without apoptotic stimuli. Once effector caspase is activated during apoptosis signaling, it cleave DFF45, allowing DFF40 to enter the nucleus and cleave chromosomal DNA. Unlike mammalian system, apoptotic DNA fragmentation in the fly might be controlled by four DFF-related proteins, known as Drep1, Drep2, Drep3 and Drep4. Although the function of Drep1 and Drep4 is well known as DFF45 and DFF40 homologues, respectively, the function of Drep2 and Drep3 is still unclear. DFF-related proteins contain a conserved CIDE domain of ~90 amino acid residues that is involved in protein-protein interaction. Here, we showed that Drep1 directly bind to Drep2 as well as Drep4 via CIDE domain. In addition, we found that the interaction of Drep2 and Drep4 to Drep1 was not competitive indicating that Drep2 and Drep4 bind different place of Drep1. All together, we suggest that Drep1 might be involved in apoptotic DNA fragmentation of fly system by direct interaction with Drep2 as well as Drep4. PMID:23417746

  15. Human chorionic gonadotropin suppresses human breast cancer cell growth directly via p53-mediated mitochondrial apoptotic pathway and indirectly via ovarian steroid secretion.

    PubMed

    Yuri, Takashi; Kinoshita, Yuichi; Emoto, Yuko; Yoshizawa, Katsuhiko; Tsubura, Airo

    2014-03-01

    The tumor-suppressive effects of human chorionic gonadotropin (hCG) against human breast cancer cells were examined. In cell viability assays, hCG inhibited the growth of three human breast cancer cell lines (estrogen receptor (ER)-positive KPL-1 and MCF-7, and ER-negative MKL-F cells), and the growth inhibition activity of hCG was most pronounced against KPL-1 cells (luteinizing hormone/chorionic gonadotropin receptor (LHCGR)-positive and luminal-A subtype). In hCG-treated KPL-1 cells, immunoblotting analysis revealed the expression of tumor suppressor protein p53 peaking at 12 h following treatment, followed by cleavage of caspase-9 and caspase-3 at 24 h and 48 h, respectively. KPL-1-transplanted athymic mice were divided into 3 groups: a sham-treated group that received an inoculation of KPL-1 cells at 6 weeks of age followed by daily intraperitoneal (i.p.) injection of saline; an in vitro hCG-treated KPL-1 group that received an inoculation of KPL-1 cells pre-treated with 100 IU/ml hCG in vitro for 48 h at 6 weeks of age, followed by daily i.p. injection of saline; and an in vivo hCG-treated group that received an KPL-1 cell inoculation at 6 weeks of age, followed by daily i.p. injection of 100 IU hCG. The daily injections of saline or hCG continued until the end of the experiment when mice reached 11 weeks of age. KPL-1 tumor growth was retarded in in vitro and in vivo hCG-treated mice compared to sham-treated controls, and the final tumor volume and tumor weight tended to be suppressed in the in vitro hCG-treated group and were significantly suppressed in the in vivo hCG-treated group. In vivo 100-IU hCG injections for 5 weeks elevated serum estradiol levels (35.7 vs. 23.5 pg/ml); thus, the mechanisms of hCG action may be directly coordinated via the p53-mediated mitochondrial apoptotic pathway and indirectly through ovarian steroid secretion that elevates estrogen levels. It is thus concluded that hCG may be an attractive agent for treating human breast cancer expressing both LHCGR and ER. PMID:24596382

  16. A Novel Herbal Medicine, KIOM-C, Induces Autophagic and Apoptotic Cell Death Mediated by Activation of JNK and Reactive Oxygen Species in HT1080 Human Fibrosarcoma Cells

    PubMed Central

    Kim, Aeyung; Im, Minju; Yim, Nam-Hui; Kim, Taesoo; Ma, Jin Yeul

    2014-01-01

    KIOM-C was recently demonstrated to have anti-metastatic activity in highly malignant cancer cells via suppression of NF-?B-mediated MMP-9 activity. In addition, it was reported to be effective for clearance of the influenza virus by increasing production of anti-viral cytokines, such as TNF-? and IFN-?, and efficacious in the treatment of pigs suffering from porcine circovirus-associated disease (PCVAD). In this study, we investigated whether KIOM-C induces cancer cell death and elucidated the underlying anti-cancer mechanisms. In addition, we examined whether KIOM-C oral administration suppresses in vivo tumor growth of HT1080 cells in athymic nude mice. We initially found that KIOM-C at concentrations of 500 and 1000 g/ml caused dose- and time-dependent cell death in cancer cells, but not normal hepatocytes, to approximately 50% of control levels. At the early stage of KIOM-C treatment (12 h), cells were arrested in G1 phase, which was accompanied by up-regulation of p21 and p27, down-regulation of cyclin D1, and subsequent increases in apoptotic and autophagic cells. Following KIOM-C treatment, the extent of caspase-3 activation, PARP cleavage, Beclin-1 expression, and LC3-II conversion was remarkably up-regulated, but p62 expression was down-regulated. Phosphorylation of AMPK, ULK, JNK, c-jun, and p53 was increased significantly in response to KIOM-C treatment. The levels of intracellular ROS and CHOP expression were also increased. In particular, the JNK-specific inhibitor SP600125 blocked KIOM-C-induced ROS generation and CHOP expression almost completely, which consequently almost completely rescued cell death, indicating that JNK activation plays a critical role in KIOM-C-induced cell death. Furthermore, daily oral administration of 85 and 170 mg/kg KIOM-C efficiently suppressed the tumorigenic growth of HT1080 cells, without systemic toxicity. These results collectively suggest that KIOM-C efficiently induces cancer cell death by both autophagy and apoptosis via activation of JNK signaling pathways, and KIOM-C represents a safe and potent herbal therapy for treating malignancies. PMID:24878898

  17. A novel herbal medicine, KIOM-C, induces autophagic and apoptotic cell death mediated by activation of JNK and reactive oxygen species in HT1080 human fibrosarcoma cells.

    PubMed

    Kim, Aeyung; Im, Minju; Yim, Nam-Hui; Kim, Taesoo; Ma, Jin Yeul

    2014-01-01

    KIOM-C was recently demonstrated to have anti-metastatic activity in highly malignant cancer cells via suppression of NF-?B-mediated MMP-9 activity. In addition, it was reported to be effective for clearance of the influenza virus by increasing production of anti-viral cytokines, such as TNF-? and IFN-?, and efficacious in the treatment of pigs suffering from porcine circovirus-associated disease (PCVAD). In this study, we investigated whether KIOM-C induces cancer cell death and elucidated the underlying anti-cancer mechanisms. In addition, we examined whether KIOM-C oral administration suppresses in vivo tumor growth of HT1080 cells in athymic nude mice. We initially found that KIOM-C at concentrations of 500 and 1000 g/ml caused dose- and time-dependent cell death in cancer cells, but not normal hepatocytes, to approximately 50% of control levels. At the early stage of KIOM-C treatment (12 h), cells were arrested in G1 phase, which was accompanied by up-regulation of p21 and p27, down-regulation of cyclin D1, and subsequent increases in apoptotic and autophagic cells. Following KIOM-C treatment, the extent of caspase-3 activation, PARP cleavage, Beclin-1 expression, and LC3-II conversion was remarkably up-regulated, but p62 expression was down-regulated. Phosphorylation of AMPK, ULK, JNK, c-jun, and p53 was increased significantly in response to KIOM-C treatment. The levels of intracellular ROS and CHOP expression were also increased. In particular, the JNK-specific inhibitor SP600125 blocked KIOM-C-induced ROS generation and CHOP expression almost completely, which consequently almost completely rescued cell death, indicating that JNK activation plays a critical role in KIOM-C-induced cell death. Furthermore, daily oral administration of 85 and 170 mg/kg KIOM-C efficiently suppressed the tumorigenic growth of HT1080 cells, without systemic toxicity. These results collectively suggest that KIOM-C efficiently induces cancer cell death by both autophagy and apoptosis via activation of JNK signaling pathways, and KIOM-C represents a safe and potent herbal therapy for treating malignancies. PMID:24878898

  18. Assessment of in-utero venlafaxine induced, ROS-mediated, apoptotic neurodegeneration in fetal neocortex and neurobehavioral sequelae in rat offspring.

    PubMed

    Singh, Manish; Singh, K P; Shukla, Shubha; Dikshit, Madhu

    2015-02-01

    Venlafaxine (VEN), a serotonin and noradrenaline reuptake inhibitor is being used as a drug of choice for treating clinical depression even during pregnancy. It is an important therapeutic option in the treatment of perinatal depression, but the effects of VEN on fetus and the newborn are uncertain. Therefore, present study was undertaken to investigate the safety of in-utero exposure to VEN in terms of developmental neurotoxicity and neurodegenerative potential by using prenatal rat model. The selected doses of VEN (25, 40 and 50mg/kg) were administered to pregnant rats from GD 5 to 19 through oral gavage. The fetal brains were dissected and processed for histopathological measurements of neocortical thickness that showed significant reduction. Considering vulnerability of immature brain to free radical injury, VEN exposed neocortices were tested for reactive oxygen species (ROS) levels which were significantly increased. As ROS play important role in the initiation of apoptotic mechanisms, we explored for in situ detection of apoptosis by confocal microscopy that showed enhanced apoptosis including chromatin condensation which was further reconfirmed by electron microscopy. Substantially increased levels of pro-apoptotic protein Bax and decreased levels of anti-apoptotic protein Bcl2 as shown by western blotting also supported the increased neuro-apoptotic degeneration. For further correlation of these findings, prenatally VEN exposed young-adult rat offspring were assessed for open field exploratory behavior that showed increased anxiety-like and stereotypic responses indicating disturbed neurobehavioral pattern. The study concludes that prenatal VEN exposure may primarily enhance ROS generation that plays a key role in regulating release of proapoptotic factors from mitochondria and thereby enhancing apoptotic neurodegeneration that affect proliferation, migration and differentiation of cells, resulting in neuronal deficits manifested as long term neurobehavioral impairments. PMID:25450524

  19. Taurine protects HK-2 cells from oxidized LDL-induced cytotoxicity via the ROS-mediated mitochondrial and p53-related apoptotic pathways

    SciTech Connect

    Chang, Chun-Yu; Shen, Chao-Yu; Kang, Chao-Kai; Sher, Yuh-Pyng; Sheu, Wayne H.-H.; Chang, Chia-Che; Lee, Tsung-Han

    2014-09-15

    Oxidized LDL (oxLDL) induces a pro-oxidative environment and promotes apoptosis, causing the progression of renal diseases in humans. Taurine is a semi-essential amino acid in mammals and has been shown to be a potent endogenous antioxidant. The kidney plays a pivotal role in maintaining the balance of taurine. However, the mechanisms underlying the protective effects of taurine against oxLDL-induced injury in renal epithelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effects of taurine on human proximal tubular epithelial (HK-2) cells exposed to oxLDL and explored the related mechanisms. We observed that oxLDL increased the contents of ROS and of malondialdehyde (MDA), which is a lipid peroxidation by-product that acts as an indicator of the cellular oxidation status. In addition, oxLDL induced cell death and apoptosis in HK-2 cells. Pretreatment with taurine at 100 μM significantly attenuated the oxLDL-induced cytotoxicity. We determined that oxLDL triggered the phosphorylation of ERK and, in turn, the activation of p53 and other apoptosis-related events, including calcium accumulation, destabilization of the mitochondrial permeability and disruption of the balance between pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins. The malfunctions induced by oxLDL were effectively blocked by taurine. Thus, our results suggested that taurine exhibits potential therapeutic activity by preventing oxLDL-induced nephrotoxicity. The inhibition of oxLDL-induced epithelial apoptosis by taurine was at least partially due to its anti-oxidant activity and its ability to modulate the ERK and p53 apoptotic pathways. - Highlights: • Oxidized LDL induced cytotoxicity and apoptosis in HK-2 cells. • Pretreatment with taurine attenuated oxLDL-induced nephrotoxicity. • Taurine protected against renal damages through inhibition of ROS generation. • Taurine prevented apoptosis through modulation of the p53 phosphorylation.

  20. The tumor-modulatory effects of Caspase-2 and Pidd1 do not require the scaffold protein Raidd.

    PubMed

    Peintner, L; Dorstyn, L; Kumar, S; Aneichyk, T; Villunger, A; Manzl, C

    2015-11-01

    The receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD/CRADD) functions as a dual adaptor and is a constituent of different multi-protein complexes implicated in the regulation of inflammation and cell death. Within the PIDDosome complex, RAIDD connects the cell death-related protease, Caspase-2, with the p53-induced protein with a death domain 1 (PIDD1). As such, RAIDD has been implicated in DNA-damage-induced apoptosis as well as in tumorigenesis. As loss of Caspase-2 leads to an acceleration of tumor onset in the Eμ-Myc mouse lymphoma model, whereas loss of Pidd1 actually delays onset of this disease, we set out to interrogate the role of Raidd in cancer in more detail. Our data obtained analyzing Eμ-Myc/Raidd(-/-) mice indicate that Raidd is unable to protect from c-Myc-driven lymphomagenesis. Similarly, we failed to observe a modulatory effect of Raidd deficiency on DNA-damage-driven cancer. The role of Caspase-2 as a tumor suppressor and that of Pidd1 as a tumor promoter can therefore be uncoupled from their ability to interact with the Raidd scaffold, pointing toward the existence of alternative signaling modules engaging these two proteins in this context. PMID:25857265

  1. The tumor-modulatory effects of Caspase-2 and Pidd1 do not require the scaffold protein Raidd

    PubMed Central

    Peintner, L; Dorstyn, L; Kumar, S; Aneichyk, T; Villunger, A; Manzl, C

    2015-01-01

    The receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD/CRADD) functions as a dual adaptor and is a constituent of different multi-protein complexes implicated in the regulation of inflammation and cell death. Within the PIDDosome complex, RAIDD connects the cell death-related protease, Caspase-2, with the p53-induced protein with a death domain 1 (PIDD1). As such, RAIDD has been implicated in DNA-damage-induced apoptosis as well as in tumorigenesis. As loss of Caspase-2 leads to an acceleration of tumor onset in the E?-Myc mouse lymphoma model, whereas loss of Pidd1 actually delays onset of this disease, we set out to interrogate the role of Raidd in cancer in more detail. Our data obtained analyzing E?-Myc/Raidd?/? mice indicate that Raidd is unable to protect from c-Myc-driven lymphomagenesis. Similarly, we failed to observe a modulatory effect of Raidd deficiency on DNA-damage-driven cancer. The role of Caspase-2 as a tumor suppressor and that of Pidd1 as a tumor promoter can therefore be uncoupled from their ability to interact with the Raidd scaffold, pointing toward the existence of alternative signaling modules engaging these two proteins in this context. PMID:25857265

  2. Oldenlandia diffusa extracts exert antiproliferative and apoptotic effects on human breast cancer cells through ERα/Sp1-mediated p53 activation.

    PubMed

    Gu, Guowei; Barone, Ines; Gelsomino, Luca; Giordano, Cinzia; Bonofiglio, Daniela; Statti, Giancarlo; Menichini, Francesco; Catalano, Stefania; Andò, Sebastiano

    2012-10-01

    Breast cancer is the most frequent tumor and a major cause of death among women. Estrogens play a crucial role in breast tumor growth, which is the rationale for the use of hormonal antiestrogen therapies. Unfortunately, not all therapeutic modalities are efficacious and it is imperative to develop new effective antitumoral drugs. Oldenlandia diffusa (OD) is a well-known medicinal plant used to prevent and treat many disorders, especially cancers. The aim of this study was to investigate the effects of OD extracts on breast cancer cell proliferation. We observed that OD extracts strongly inhibited anchorage-dependent and -independent cell growth and induced apoptosis in estrogen receptor alpha (ERα)-positive breast cancer cells, whereas proliferation and apoptotic responses of MCF-10A normal breast epithelial cells were unaffected. Mechanistically, OD extracts enhance the tumor suppressor p53 expression as a result of an increased binding of ERα/Sp1 complex to the p53 promoter region. Finally, we isolated ursolic and oleanolic acids as the bioactive compounds able to upregulate p53 expression and inhibit breast cancer cell growth. These acids were greatly effective in reducing tamoxifen-resistant growth of a derivative MCF-7 breast cancer cell line resistant to the antiestrogen treatment. Our results evidence how OD, and its bioactive compounds, exert antiproliferative and apoptotic effects selectively in ERα-positive breast cancer cells, highlighting the potential use of these herbal extracts as breast cancer preventive and/or therapeutic agents. PMID:22213398

  3. Estrogen receptor ? mediates the effects of notoginsenoside R1 on endotoxin-induced inflammatory and apoptotic responses in H9c2 cardiomyocytes.

    PubMed

    Zhong, Lei; Zhou, Xing-Lu; Liu, Yan-Song; Wang, Yi-Min; Ma, Fei; Guo, Bao-Liang; Yan, Zhao-Qi; Zhang, Qing-Yuan

    2015-07-01

    Estrogen receptors (ERs) are important for preventing endotoxin-induced myocardial dysfunction. Therefore, plant-derived phytoestrogens, which target ERs may also affect endotoxin-induced toxicity in cardiomyocytes. Our previous study revealed that notoginsenoside-R1 (NG-R1), a predominant phytoestrogen from Panax notoginseng, protects against cardiac dysfunction. However, the effects of NG-R1 on cardiomyocytes and the precise cellular/molecular mechanisms underlying its action remain to be elucidated. In the present study, pretreatment with NG-R1 suppressed the lipopolysaccharide (LPS)-induced degradation of inhibitor of nuclear factor-?B (NF-?B) ?, the activation of NF-?B and caspase-3, and the subsequent myocardial inflammatory and apoptotic responses in H9c2 cardiomyocytes. An increase in the mRNA and protein expression of ER? was also observed in the NG-R1-treated cardiomyocytes. However, the expression pattern of ER? remained unaltered. Furthermore, the cardioprotective properties of NG-R1 against LPS-induced apoptosis and the inflammatory response in cardiomyocytes were attenuated by ICI 182780, a non-selective ER? antagonist, and methyl-piperidino-pyrazole, a selective ER? antagonist. These findings suggested that NG-R1 reduced endotoxin-induced cardiomyocyte apoptosis and the inflammatory response via the activation of ER?. Therefore, NG-R1 exerted direct anti-inflammatory and anti-apoptotic effects on the cardiomyocytes, representing a potent agent for the treatment of myocardial inflammation during septic shock. PMID:25738436

  4. Human and Simian Immunodeficiency Virus-Mediated Upregulation of the Apoptotic Factor TRAIL Occurs in Antigen-Presenting Cells from AIDS-Susceptible but Not from AIDS-Resistant Species?

    PubMed Central

    Kim, Nayoung; Dabrowska, Alicja; Jenner, Richard G.; Aldovini, Anna

    2007-01-01

    Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections lead to AIDS in humans and rhesus macaques (RM), while they are asymptomatic in species naturally infected with SIV, such as chimpanzees, sooty mangabeys (SM), and African green monkeys (AGM). Differential CD4+ T-cell apoptosis may be responsible for these species-specific differences in susceptibility to disease. To identify factors that influence the different apoptotic responses of these species, we analyzed virus-infected human and nonhuman primate peripheral blood mononuclear cells (PBMC). We found that the apoptotic factor TRAIL was present at higher levels in human and RM PBMC cultures and was mediating, at least in part, CD4+ T-cell apoptosis in these cultures. The species-specific increase in TRAIL and death receptor expression observed with cultures also occurred in vivo in SIV-infected RM but not in SIV-infected SM. In human and RM myeloid immature dendritic cells and macrophages, the virus-induced expression of TRAIL and other interferon-inducible genes, which did not occur in the same cells from chimpanzee, SM, and AGM, was Tat dependent. Our results link the differential induction of TRAIL in human and nonhuman primate cells to species-specific differences in disease susceptibility. PMID:17494085

  5. Carboxylation of multiwalled carbon nanotube attenuated the cytotoxicity by limiting the oxidative stress initiated cell membrane integrity damage, cell cycle arrestment, and death receptor mediated apoptotic pathway.

    PubMed

    Liu, Zhenbao; Liu, Yanfei; Peng, Dongming

    2015-08-01

    In this study, the effects of carboxylated multiwalled carbon nanotubes (MWCNTs-COOH) on human normal liver cell line L02 was compared with that of pristine multiwalled carbon nanotubes (p-MWCNTs). It was shown that compared with MWCNTs-COOH, p-MWCNTs induced apoptosis, reduced the level of intracellular antioxidant glutathione more significantly, and caused severer cell membrane damage as demonstrated by lactate dehydrogenase leakage. Cell cycles were arrested by both MWCNTs, while p-MWCNTs induced higher ratio of G0/G1 phase arrestment as compared with MWCNTs-COOH. Caspase-8 was also activated after both MWCNTs exposure, indicating extrinsic apoptotic pathway was involved in the apoptosis induced by MWCNTs exposure, more importantly, MWCNTs-COOH significantly reduced the activation of caspase-8 as compared with p-MWCNTs. All these results suggested that MWCNTs-COOH might be safer for in vivo application as compared with p-MWCNTs. PMID:25684371

  6. Follicle-stimulating Hormone Regulates Pro-apoptotic Protein Bcl-2-interacting Mediator of Cell Death-Extra Long (BimEL)-induced Porcine Granulosa Cell Apoptosis*

    PubMed Central

    Wang, Xian-Long; Wu, Yi; Tan, Lu-Bin; Tian, Zhen; Liu, Jing-Hao; Zhu, De-Sheng; Zeng, Shen-Ming

    2012-01-01

    The pro-apoptotic protein Bim (B-cell lymphoma-2 (Bcl-2)-interacting modulator of cell death) has recently been identified and shown to promote cell death in response to several stimuli. In this report, we investigated the role of Bim in porcine follicular atresia. Initially, Bim cDNA was cloned and characterized from porcine ovarian tissue. Porcine Bim had three alternative splicing variants (Bim-extra long, Bim-long, and Bim-short), all containing the consensus Bcl-2 homology 3 domain. We then found the Bim-extra long (BimEL) protein, the most abundant isoform of Bim, was strongly expressed and co-localized with apoptotic (TUNEL-positive) granulosa cells from porcine atretic follicles. Furthermore, overexpression of BimEL triggered apoptosis in granulosa cells. In primary granulosa cell cultures under basal conditions, we observed that BimEL expression was dampened by treatment with follicle-stimulating hormone (FSH). The role of the PI3K/Akt pathway in the regulation of repression was clarified by the use of the PI3K inhibitor, LY294002, and by transfection with Akt siRNA. Forkhead Box Protein O3a (FoxO3a), a well defined transcriptional activator of Bim, was phosphorylated at Ser-253 and inactivated after FSH stimulation. Also, FSH abolished FoxO3a nuclear accumulation in response to LY294002. Finally, chromatin immunoprecipitation assays demonstrated that FoxO3a directly bound and activated the bim promoter. Taken together, we conclude that BimEL induces porcine granulosa cell apoptosis during follicular atresia, and its expression is regulated by FSH via the PI3K/Akt/FoxO3a pathway. PMID:22235114

  7. dLin52 is crucial for dE2F and dRBF mediated transcriptional regulation of pro-apoptotic gene hid.

    PubMed

    Bhaskar, Pradeep Kumar; Surabhi, Satya; Tripathi, Bipin Kumar; Mukherjee, Ashim; Mutsuddi, Mousumi

    2014-09-01

    Drosophila lin52 (dlin52) is a member of Myb transcription regulator complex and it shows a dynamic pattern of expression in all Drosophila tissues. Myb complex functions to activate or repress transcription in a site-specific manner; however, the detailed mechanism is yet to be clearly understood. Members of the Drosophila melanogaster Myb-MuvB/dREAM complex have been known to regulate expression of a wide range of genes including those involved in regulating apoptosis. E2F and its corepressor RBF also belong to this complex and together they regulate expression of genes involved in cell cycle progression, apoptosis, differentiation, and development. In the present study, we examined whether the depletion of dlin52 in developing photoreceptor neurons results in enhanced apoptosis and disorganisation of the ommatidia. Strikingly, we found that dLin52 is essential for transcriptional repression of the pro-apoptotic gene, hid; decrease in dlin52 levels led to dramatic induction of hid and apoptosis in eye-antennal discs. Reduction of Rpd3 (HDAC1), another member of the dREAM complex, also led to marginal upregulation of Hid. In addition, we also demonstrated that an optimum level of dLin52 is needed for dE2F1/2 activity on the hid promoter. dlin52 cooperates with dRBF and dE2F1/2 for recruitment of repressor complex on the hid promoter. Preliminary data indicate that Rpd3/HDAC1 also contributes to hid repression. Based on the findings, we conclude that dLin52 functions as a co-factor and modulates activity of members of dMyb/dREAM complex at hid promoter, thus regulating apoptosis by repressing this pro-apoptotic gene in the developing Drosophila eye. PMID:24863159

  8. Anticancer and apoptotic activities of oleanolic acid are mediated through cell cycle arrest and disruption of mitochondrial membrane potential in HepG2 human hepatocellular carcinoma cells

    PubMed Central

    ZHU, YUE-YONG; HUANG, HONG-YAN; WU, YIN-LIAN

    2015-01-01

    Hepatocellular carcinoma (HCC) is an aggressive form of cancer, with high rates of morbidity and mortality, a poor prognosis and limited therapeutic options. The objective of the present study was to demonstrate the anticancer activity of oleanolic acid in HepG2 human HCC cells. Cell viability was evaluated using an MTT assay, following administration of various doses of oleanolic acid. The effect of oleanolic acid on cell cycle phase distribution and mitochondrial membrane potential was evaluated using flow cytometry with propidium iodide and rhodamine-123 DNA-binding cationic fluorescent dyes. Fluorescence microscopy was employed to detect morphological changes in HepG2 cells following oleanolic acid treatment. The results revealed that oleanolic acid induced a dose-dependent, as well as time-dependent inhibition in the growth of HepG2 cancer cells. Following acridine orange and ethidium bromide staining, treatment with various doses (0, 5, 25 and 50 M) of oleanolic acid induced typical morphological changes associated with apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation. Cell cycle analysis revealed that oleanolic acid induced cell cycle arrest in HepG2 cells at the sub-G1 (apoptotic) phase of the cell cycle, in a dose-dependent manner. Staining with Annexin V-fluorescein isothiocyanate and propidium iodide revealed that apoptosis occurred early in these cells. Oleanolic acid treatment also resulted in fragmentation of nuclear DNA in a dose-dependent manner, producing the typical features of DNA laddering on an agarose gel. The results also demonstrated that oleanolic acid treatment resulted in a potent loss of mitochondrial membrane potential, which also occurred in a dose-dependent manner. Therefore, oleanolic acid may be used as a therapeutic agent in the treatment of human HCC. PMID:26151733

  9. Anticancer and apoptotic activities of oleanolic acid are mediated through cell cycle arrest and disruption of mitochondrial membrane potential in HepG2 human hepatocellular carcinoma cells.

    PubMed

    Zhu, Yue-Yong; Huang, Hong-Yan; Wu, Yin-Lian

    2015-10-01

    Hepatocellular carcinoma (HCC) is an aggressive form of cancer, with high rates of morbidity and mortality, a poor prognosis and limited therapeutic options. The objective of the present study was to demonstrate the anticancer activity of oleanolic acid in HepG2 human HCC cells. Cell viability was evaluated using an MTT assay, following administration of various doses of oleanolic acid. The effect of oleanolic acid on cell cycle phase distribution and mitochondrial membrane potential was evaluated using flow cytometry with propidium iodide and rhodamine?123 DNA?binding cationic fluorescent dyes. Fluorescence microscopy was employed to detect morphological changes in HepG2 cells following oleanolic acid treatment. The results revealed that oleanolic acid induced a dose?dependent, as well as time?dependent inhibition in the growth of HepG2 cancer cells. Following acridine orange and ethidium bromide staining, treatment with various doses (0, 5, 25 and 50 M) of oleanolic acid induced typical morphological changes associated with apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation. Cell cycle analysis revealed that oleanolic acid induced cell cycle arrest in HepG2 cells at the sub?G1 (apoptotic) phase of the cell cycle, in a dose?dependent manner. Staining with Annexin V?fluorescein isothiocyanate and propidium iodide revealed that apoptosis occurred early in these cells. Oleanolic acid treatment also resulted in fragmentation of nuclear DNA in a dose?dependent manner, producing the typical features of DNA laddering on an agarose gel. The results also demonstrated that oleanolic acid treatment resulted in a potent loss of mitochondrial membrane potential, which also occurred in a dose?dependent manner. Therefore, oleanolic acid may be used as a therapeutic agent in the treatment of human HCC. PMID:26151733

  10. Pheophorbidea-mediated photodynamic therapy induces apoptotic cell death in murine oral squamous cell carcinoma in vitro and in vivo.

    PubMed

    Ahn, Mee-Young; Kwon, Seong-Min; Kim, Yong-Chul; Ahn, Sang-Gun; Yoon, Jung-Hoon

    2012-06-01

    Photodynamic therapy (PDT) with several photosensitizers is a promising modality for the treatment of cancer. In this study, the therapeutic effect of PDT using the synthetic photosensitizer pheophorbide a (Pa-PDT) was examined in AT-84 murine oral squamous cell carcinoma (OSCC) cells. The MTT assay revealed that Pa-PDT induced cell growth inhibition in a dose- and time-dependent manner. Pa-PDT treatment significantly induced intracellular ROS generation, which is critical for cell death induced by Pa-PDT. Cell cycle analysis showed the increased sub-G1 proportion of cells in Pa-PDT-treated cells. Induction of apoptotic cell death was confirmed by DAPI staining and the reduction of mitochondrial membrane potential (??m) on Pa-PDT-treated cells. The changes in apoptosis-related molecules were next examined using western blotting. Cytochrome c release and cleavage of caspase-3 and PAPR were observed in AT-84 cells, whereas Bcl-2 protein levels were decreased. To determine the therapeutic effect of Pa-PDT in vivo, a murine OSCC animal model was used. Treatment of mice with Pa-PDT significantly inhibited tumor growth, especially PDT with Pa intravenous administration (i.v. Pa-PDT), and increased proliferative cell nuclear antigen (PCNA) levels and TUNEL-stained apoptotic cells compared to vehicle-treated controls. The data demonstrate that the in vitro effects of Pa-PDT on the inhibition of tumor cell proliferation and induction of apoptosis correlate to the anticancer activity of Pa-PDT in vivo. Our findings suggest the therapeutic potential of Pa-PDT in OSCC. PMID:22470106

  11. Translocation of a Bak C-Terminus Mutant from Cytosol to Mitochondria to Mediate Cytochrome c Release: Implications for Bak and Bax Apoptotic Function

    PubMed Central

    Ferrer, Pedro Eitz; Frederick, Paul; Gulbis, Jacqueline M.

    2012-01-01

    Background One of two proapoptotic Bcl-2 proteins, Bak or Bax, is required to permeabilize the mitochondrial outer membrane during apoptosis. While Bax is mostly cytosolic and translocates to mitochondria following an apoptotic stimulus, Bak is constitutively integrated within the outer membrane. Membrane anchorage occurs via a C-terminal transmembrane domain that has been studied in Bax but not in Bak, therefore what governs their distinct subcellular distribution is uncertain. In addition, whether the distinct subcellular distributions of Bak and Bax contributes to their differential regulation during apoptosis remains unclear. Methodology/Principal Findings To gain insight into Bak and Bax targeting to mitochondria, elements of the Bak C-terminus were mutated, or swapped with those of Bax. Truncation of the C-terminal six residues (C-segment) or substitution of three basic residues within the C-segment destabilized Bak. Replacing the Bak C-segment with that from Bax rescued stability and function, but unexpectedly resulted in a semi-cytosolic protein, termed Bak/BaxCS. When in the cytosol, both Bax and Bak/BaxCS sequestered their hydrophobic transmembrane domains in their hydrophobic surface groove. Upon apoptotic signalling, Bak/BaxCS translocated to the mitochondrial outer membrane, inserted its transmembrane domain, oligomerized, and released cytochrome c. Despite this Bax-like subcellular distribution, Bak/BaxCS retained Bak-like regulation following targeting of Mcl-1. Conclusions/Significance Residues in the C-segment of Bak and of Bax contribute to their distinct subcellular localizations. That a semi-cytosolic form of Bak, Bak/BaxCS, could translocate to mitochondria and release cytochrome c indicates that Bak and Bax share a conserved mode of activation. In addition, the differential regulation of Bak and Bax by Mcl-1 is predominantly independent of the initial subcellular localizations of Bak and Bax. PMID:22442658

  12. Non-apoptotic and extracellular activity of Granzyme B mediates resistance to Treg suppression by HLA-DRnegCD25hiCD127lo Tregs in multiple sclerosis and in response to IL-6

    PubMed Central

    Bhela, Siddheshvar; Kempsell, Christine; Manohar, Monali; Dominguez-Villar, Margarita; Griffin, Russell; Bhatt, Pooja; Kivisakk-Webb, Pia; Fuhlbrigge, Robert; Kupper, Thomas; Weiner, Howard; Baecher-Allan, Clare

    2015-01-01

    In autoimmune patients, regulatory T cells are increasingly found to be unable to suppress patient-derived T cells, an outcome referred to as Treg resistance. Here we show that CD4 T cells from patients with MS resist suppression by patient derived- or healthy donor derived- ex vivo Tregs. Importantly, we report that Granzyme B (GzmB) contributes to this Treg resistance via a novel, apoptosis-independent mechanism. We show that memory CD4+CD127loFoxP3+ Treg subsets do not express GzmB, while activated, non-regulatory CD4 T cells isolated from patients with MS express higher levels of GzmB than cells from healthy donors. In contrast to the intracellular GzmB that mediates apoptosis, GzmB can be found in extracellular fluids where it is hypothesized to regulate other cellular processes. Here we show that providing extracellular GzmB strongly inhibits Treg suppression, without altering Treg viability. However, if GzmB and GzmB-specific inhibitor are both provided to the co-cultures, Treg suppression occurs. Thus, these data suggest that a novel activity of extracellular GzmB is to regulate Treg suppression. In addition, we find that the suppression-abrogating cytokine, IL-6, augments GzmB expression by human CD4 T cells, and inhibits Treg suppression via this non-apoptotic GzmB-mediated mechanism. Lastly, in examining the mechanism whereby GzmB inhibits Treg function, we show that extracellular GzmB reduces Treg expression of CD39 and PD-L1. Altogether, these data indicate that extracellular GzmB plays an unexpected, non-apoptotic role in regulating Treg suppression and suggest that inactivation of specifically the extracellular activity of GzmB may be an efficacious therapeutic in autoimmunity. PMID:25637022

  13. Apoptotic cell removal.

    PubMed

    Henson, P M; Bratton, D L; Fadok, V A

    2001-10-01

    Ingestion by professional or amateur phagocytes is the fate of most cells that undergo apoptosis. Studies in both Caenorhabditis elegans and mammals are now converging to reveal some of the key mechanisms and consequences of this removal process. At least seven corpse removal genes in nematodes have mammalian equivalents, and represent elements of signaling pathways involved in uptake. In mammals, a wide variety of apoptotic cell recognition receptors has been implicated and appears to be divided into two categories, involved in tethering the apoptotic cell or triggering an uptake mechanism related to macropinocytosis. Apoptotic cell removal is normally efficient and non-inflammatory. By contrast, the process may become subverted by parasites to yield a more favorable growth environment, or in other cases lead to fibrosis. Removal may also clinch the apoptotic process itself in cells not yet completely committed to death. PMID:11591341

  14. Cytoprotective and anti-apoptotic effects of Satureja khuzestanica essential oil against busulfan-mediated sperm damage and seminiferous tubules destruction in adult male mice.

    PubMed

    Nasimi, P; Vahdati, A; Tabandeh, M R; Khatamsaz, S

    2016-02-01

    We studied the protective effect of Satureja khuzestanica essential oil (SKEO) against damage caused by busulfan on testis in male mice. The NMRI mice (n=40) were assigned to four groups including: G1: control, G2: treated with busulfan for 4days (3.2mgkg(-1) ), G3: receive busulfan (4days, 3.2mgkg(-1) ) and SKEO (28days, 225mgkg(-1) ) at the same time, G4: pre-treated with SKEO (7days, 225mgkg(-1) ) and subsequently cotreated with busulfan (4days, 3.2mgkg(-1) ) and SKEO (28days, 225mgkg(-1) ). The histological changes of testis were analysed using H&E staining. Sperm parameters, cytotoxic and apoptotic factors were also studied by computer-aided sperm analyzer, MTT and TUNEL assays respectively. Our results showed that SKEO pre-administration significantly improved all parameters of epididymal spermatozoa and decreased germinal epithelium destruction following busulfan chemotherapy. We also found lower MTT levels and TUNEL-positive cells in SKEO pre-treated groups. In conclusion, SKEO possesses beneficial effects on sperm parameters when taken before chemotherapy and continued during and after chemotherapy for a long time, than when used short-term coinciding with the chemotherapy. Our results support valuable data about the application of SKEO for protection against adverse effects of busulfan on male genital system in patients under chemotherapy. PMID:26011020

  15. Bag-1 promotes cell survival through c-Myc-mediated ODC upregulation that is not preferred under apoptotic stimuli in MCF-7 cells.

    PubMed

    Ozfiliz, Pelin; Kizilboga, Tugba; Demir, Salih; Alkurt, Gizem; Palavan-Unsal, Narin; Arisan, Elif Damla; Dinler-Doganay, Gizem

    2015-07-01

    Bag-1, Bcl-2 associated athanogene-1, is a multifunctional protein that can regulate a wide variety of cellular processes: proliferation, cell survival, transcription, apoptosis and motility. Bag-1 interacts with various targets in the modulation of these pathways; yet molecular details of Bag-1's involvement in each cellular event are still unclear. We first showed that forced Bag-1 expression promotes cell survival and prevents drug-induced apoptosis in MCF-7 breast cancer cells. Increased mRNA expressions of c-myc protooncogene and ornithine decarboxylase (ODC), biosynthetic enzyme of polyamines, were detected in Bag-1L+ cells, and western blots against the protein product of c-Myc and ODC confirmed these findings. Once ODC, a c-Myc target, gets activated, polyamine biosynthesis increases. We observed enhanced polyamine content in the Bag-1L+ cells. On the contrary, when polyamine catabolic mechanisms were investigated, Bag-1 silencing suppressed biosynthesis of polyamines because of the downregulation of ODC and upregulation of PAO. Exposure of cells to apoptotic inducers enhances the cell death mechanism by producing toxic products such as H2 O2 and aldehydes. Bag-1L+ cells prevented drug-induced PAO activation leading to a decrease in H2 O2 production following cisplatin or paclitaxel treatment. In this line, our results suggested that Bag-1 indirectly affects cell survival through c-Myc activated signalling that causes elevation of ODC levels, leading to an increase of the polyamine content. PMID:26178413

  16. Evodiamine induces apoptosis and enhances apoptotic effects of erlotinib in wild-type EGFR NSCLC cells via S6K1-mediated Mcl-1 inhibition.

    PubMed

    Li, Yang-Ling; Pan, Yi-Ni; Wu, Wen-Jue; Mao, Shi-Ying; Sun, Jiao; Zhao, Yi-Ming; Dong, Jing-Yin; Zhang, Da-Yong; Pan, Jian-Ping; Zhang, Chong; Lin, Neng-Ming

    2016-02-01

    Erlotinib is effective in NSCLC patients with known drug-sensitizing EGFR mutations, but its clinical efficacy in patients with wild-type EGFR or acquired resistance to erlotinib remains modest. Evodiamine is a chemical extracted from the Evodia rutaecarpa (Juss.) Benth, we showed that evodiamine could induce anti-proliferation and apoptosis in four wild-type EGFR NSCLC cell lines, and combining evodiamine with erlotinib might successfully inhibit cell proliferation and survival in wild-type EGFR NSCLC cells, characterized as erlotinib-resistant. In addition, evodiamine plus erlotinib significantly increased the apoptotic rate of NSCLC cells, as compared to single agent treatment alone. Further investigation of the mechanism underlying these effects revealed that evodiamine plus erlotinib might downregulate Mcl-1 expression through the mTOR/S6K1 control of its translation. Thus, our study has revealed evodiamine as a pertinent sensitizer to erlotinib and the strategy of combining erlotinib with evodiamine appears to be an attractive option for reversing resistance to erlotinib. PMID:26757927

  17. The anti-apoptotic effect of IGF-1 on tissue resident stem cells is mediated via PI3-kinase dependent secreted frizzled related protein 2 (Sfrp2) release

    SciTech Connect

    Gehmert, Sebastian; Sadat, Sanga; Song Yaohua; Yan Yasheng; Alt, Eckhard

    2008-07-11

    Previous studies suggest that IGF-1 may be used as an adjuvant to stem cell transfer in order to improve cell engraftment in ischemic tissue. In the current study, we investigated the effect of IGF-1 on serum deprivation and hypoxia induced stem cell apoptosis and the possible mechanisms involved. Exposure of adipose tissue derived stem cells (ASCs) to serum deprivation and hypoxia resulted in significant apoptosis in ASC which is partially prevented by IGF-1. IGF-1's anti-apoptotic effect was abolished in ASCs transfected with Sfrp2 siRNA but not by the control siRNA. Using Western blot analysis, we demonstrated that serum deprivation and hypoxia reduced the expression of nuclear {beta}-catenin, which is reversed by IGF-1. IGF-1's effect on {beta}-catenin expression was abolished by the presence of PI3-kinase inhibitor LY294002 or in ASCs transfected with Sfrp2 siRNA. These results suggest that IGF-1, through the release of the Sfrp2, contributes to cell survival by stabilizing {beta}-catenin.

  18. Mediation of endogenous antioxidant enzymes and apoptotic signaling by resveratrol following muscle disuse in the gastrocnemius muscles of young and old rats

    PubMed Central

    Jackson, Janna R.; Ryan, Michael J.; Hao, Yanlei

    2010-01-01

    Hindlimb suspension (HLS) elicits muscle atrophy, oxidative stress, and apoptosis in skeletal muscle. Increases in oxidative stress can have detrimental effects on muscle mass and function, and it can potentially lead to myonuclear apoptosis. Resveratrol is a naturally occurring polyphenol possessing both antioxidant and antiaging properties. To analyze the capacity of resveratrol to attenuate oxidative stress, apoptosis and muscle force loss were measured following 14 days of HLS. Young (6 mo) and old (34 mo) rats were administered either 12.5 mgkg?1day?1 of trans-resveratrol, or 0.1% carboxymethylcellulose for 21 days, including 14 days of HLS. HLS induced a significant decrease in plantarflexor isometric force, but resveratrol blunted this loss in old animals. Resveratrol increased gastrocnemius catalase activity, MnSOD activity, and MnSOD protein content following HLS. Resveratrol reduced hydrogen peroxide and lipid peroxidation levels in muscles from old animals after HLS. Caspase 9 abundance was reduced and Bcl-2 was increased, but other apoptotic markers were not affected by resveratrol in the gastrocnemius muscle after HLS. The data indicate that resveratrol has a protective effect against oxidative stress and muscle force loss in old HLS animals; however, resveratrol was unable to attenuate apoptosis following HLS. These results suggest that resveratrol has the potential to be an effective therapeutic agent to treat muscle functional decrements via improving the redox status associated with disuse. PMID:20861279

  19. In the absence of cellular poly (A) binding protein, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53

    SciTech Connect

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B.; Bag, Jnanankur

    2011-06-03

    Highlights: {yields} PABP knock down and cell apoptosis. {yields} Nuclear translocation of GAPDH in PABP depleted cells. {yields} Role of p53 in apoptosis of PABP depleted cells. {yields} Bax translocation and cytochrome C release and caspase 3 activation following PABP depletion. {yields} Association of p53 with Bcl2 and Bax. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) interacts with 3' poly (A) tract of eukaryotic mRNA and is important for both translation and stability of mRNA. Previously, we have shown that depletion of PABP by siRNA prevents protein synthesis and consequently leads to cell death through apoptosis. In the present investigation, we studied the mechanism of cell apoptosis. We show that in the absence of PABP, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53. As a result, p53 translocated to the mitochondria to initiate Bax mediated apoptosis.

  20. Anti Proliferative and Pro Apoptotic Effects of Flavonoid Quercetin Are Mediated by CB1 Receptor in Human Colon Cancer Cell Lines.

    PubMed

    Refolo, Maria Grazia; D'Alessandro, Rosalba; Malerba, Natascia; Laezza, Chiara; Bifulco, Maurizio; Messa, Caterina; Caruso, Maria Gabriella; Notarnicola, Maria; Tutino, Valeria

    2015-12-01

    Quercetin, the major constituent of flavonoid and widely present in fruits and vegetables, is an attractive compound for cancer prevention due to its beneficial anti proliferative effects, showing a crucial role in the regulation of apoptosis and cell cycle signaling. In vitro studies have demonstrated that quercetin specifically influences colon cancer cell proliferation. Our experiments, using human colon adenocarcinoma cells, confirmed the anti proliferative effect of quercetin and gave intriguing new insight in to the knowledge of the mechanisms involved. We observed a significant increase in the expression of the endocannabinoids receptor (CB1-R) after quercetin treatment. CB1-R can be considered an estrogen responsive receptor and quercetin, having a structure similar to that of the estrogens, can interact with CB1-R leading to the regulation of cell growth. In order to clarify the contribution of the CB1-R to the quercetin action, we investigated some of the principal molecular pathways that are inhibited or activated by this natural compound. In particular we detected the inhibition of the major survival signals like the PI3K/Akt/mTOR and an induction of the pro apoptotic JNK/JUN pathways. Interestingly, the metabolism of β-catenin was modified by flavonoid both directly and through activated CB1-R. In all the experiments done, the quercetin action has proven to be reinforced by anandamide (Met-F-AEA), a CB1-R agonist, and partially counteracted by SR141716, a CB1-R antagonist. These findings open new perspectives for anticancer therapeutic strategies. PMID:25893829

  1. Apoptotic cells subjected to cold/warming exposure disorganize apoptotic microtubule network and undergo secondary necrosis.

    PubMed

    Oropesa-vila, Manuel; Fernndez-Vega, Alejandro; de la Mata, Mario; Garrido-Maraver, Juan; Cotn, David; Paz, Marina Villanueva; Pavn, Ana Delgado; Cordero, Mario D; Alcocer-Gmez, Elizabet; de Lavera, Isabel; Lema, Rafael; Zaderenko, Ana Paula; Snchez-Alczar, Jos A

    2014-09-01

    Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath the plasma membrane which plays a critical role in preserving cell morphology and plasma membrane integrity. The aim of this study was to examine the effect of cold/warming exposure on apoptotic microtubules and plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptotic H460 cells that cold/warming exposure disorganized apoptotic microtubules and allowed the access of active caspases to the cellular cortex and the cleavage of essential proteins in the preservation of plasma membrane permeability. Cleavage of cellular cortex and plasma membrane proteins, such as ?-spectrin, paxilin, focal adhesion kinase and calcium ATPase pump (PMCA-4) involved in cell calcium extrusion resulted in increased plasma permeability and calcium overload leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the addition of the pan-caspase inhibitor z-VAD during cold/warming exposure that induces AMN depolymerization avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Likewise, apoptotic microtubules stabilization by taxol during cold/warming exposure also prevented cellular cortex and plasma membrane protein cleavage and secondary necrosis. Furthermore, microtubules stabilization or caspase inhibition during cold/warming exposure was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that cold/warming exposure of apoptotic cells induces secondary necrosis which can be prevented by both, microtubule stabilization or caspase inhibition. PMID:25027509

  2. Folate deficiency triggers an oxidative-nitrosative stress-mediated apoptotic cell death and impedes insulin biosynthesis in RINm5F pancreatic islet ?-cells: relevant to the pathogenesis of diabetes.

    PubMed

    Hsu, Hung-Chih; Chiou, Jeng-Fong; Wang, Yu-Huei; Chen, Chia-Hui; Mau, Shin-Yi; Ho, Chun-Te; Chang, Pey-Jium; Liu, Tsan-Zon; Chen, Ching-Hsein

    2013-01-01

    It has been postulated that folic acid (folate) deficiency (FD) may be a risk factor for the pathogenesis of a variety of oxidative stress-triggered chronic degenerative diseases including diabetes, however, the direct evidence to lend support to this hypothesis is scanty. For this reason, we set out to study if FD can trigger the apoptotic events in an insulin-producing pancreatic RINm5F islet ? cells. When these cells were cultivated under FD condition, a time-dependent growth impediment was observed and the demise of these cells was demonstrated to be apoptotic in nature proceeding through a mitochondria-dependent pathway. In addition to evoke oxidative stress, FD condition could also trigger nitrosative stress through a NF-?B-dependent iNOS-mediated overproduction of nitric oxide (NO). The latter compound could then trigger depletion of endoplasmic reticulum (ER) calcium (Ca(2+)) store leading to cytosolic Ca(2+) overload and caused ER stress as evidence by the activation of CHOP expression. Furthermore, FD-induced apoptosis of RINm5F cells was found to be correlated with a time-dependent depletion of intracellular glutathione (GSH) and a severe down-regulation of Bcl-2 expression. Along the same vein, we also demonstrated that FD could severely impede RINm5F cells to synthesize insulin and their abilities to secret insulin in response to glucose stimulation were appreciably hampered. Even more importantly, we found that folate replenishment could not restore the ability of RINm5F cells to resynthesize insulin. Taken together, our data provide strong evidence to support the hypothesis that FD is a legitimate risk factor for the pathogenesis of diabetes. PMID:24223745

  3. TRAF2 mediates JNK and STAT3 activation in response to IL-1? and IFN? and facilitates apoptotic death of insulin-producing ?-cells.

    PubMed

    Prause, Michala; Berchtold, Lukas Adrian; Urizar, Adriana Ibarra; Hyldgaard Trauelsen, Mette; Billestrup, Nils; Mandrup-Poulsen, Thomas; Strling, Joachim

    2016-01-15

    Interleukin-1? (IL-1?) and interferon-? (IFN?) contribute to type 1 diabetes (T1D) by inducing ?-cell death. Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins are adaptors that transduce signaling from a variety of membrane receptors including cytokine receptors. We show here that IL-1? and IFN? upregulate the expression of TRAF2 in insulin-producing INS-1E cells and isolated rat pancreatic islets. siRNA-mediated knockdown (KD) of TRAF2 in INS-1E cells reduced IL-1?-induced phosphorylation of JNK1/2, but not of p38 or ERK1/2 mitogen-activated protein kinases. TRAF2 KD did not modulate NF?B activation by cytokines, but reduced cytokine-induced inducible nitric oxide synthase (iNOS) promotor activity and expression. We further observed that IFN?-stimulated phosphorylation of STAT3 required TRAF2. KD of TRAF2 or STAT3 reduced cytokine-induced caspase 3/7 activation, but, intriguingly, potentiated cytokine-mediated loss of plasma membrane integrity and augmented the number of propidium iodide-positive cells. Finally, we found that TRAF2 KD increased cytokine-induced production of reactive oxygen species (ROS). In summary, our data suggest that TRAF2 is an important mediator of IL-1? and IFN? signaling in pancreatic ?-cells. PMID:26610752

  4. Synthetic 1,4-anthracenedione analogs induce cytochrome c release, caspase-9, -3, and -8 activities, poly(ADP-ribose) polymerase-1 cleavage and internucleosomal DNA fragmentation in HL-60 cells by a mechanism which involves caspase-2 activation but not Fas signaling.

    PubMed

    Perchellet, Elisabeth M; Wang, Yang; Weber, Rebeka L; Sperfslage, Bonnie J; Lou, Kaiyan; Crossland, Justin; Hua, Duy H; Perchellet, Jean-Pierre

    2004-02-01

    Synthetic analogs of 1,4-anthraquinone (AQ code number), a compound that mimics the antiproliferative effects of daunorubicin (daunomycin) in the nanomolar range in vitro but has the advantage of blocking nucleoside transport and retaining its efficacy in multidrug-resistant tumor cells, were tested for their ability to induce apoptosis in the HL-60 cell system. AQ10 and, especially, the new lead antiproliferative compounds AQ8 and AQ9 reduce the growth and integrity of wild-type, drug-sensitive, HL-60-S cells more effectively than AQ1, suggesting that various methyl group substituents at C6 may enhance the bioactivity of the parent compound. Internucleosomal DNA fragmentation, a late marker of apoptosis, is similarly induced in a biphasic manner by increasing concentrations of AQ8 and AQ9 at 24 hr. Poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, an early event required for cells committed to apoptosis, is detected within 3-6 hr in HL-60-S cells treated with AQ9. In accord with the fact that the caspases 9 and 3 cascade is responsible for PARP-1 cleavage, the activities of initiator caspase-9 and effector caspase-3 are induced by AQ9 in the same time- and concentration-dependent manners and to the same maximal degrees in both the HL-60-S and multidrug-resistant HL-60-RV cell lines. Interestingly, a 1-hr pulse treatment is sufficient for AQ8 and AQ9 to maximally induce caspase-9 and -3 activities at 6 hr. The release of mitochondrial cytochrome c (Cyt c) is also detected within 3-6hr in HL-60-S cells treated with AQ9, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9. Moreover, AQ analogs induce Cyt c release, caspase-9 and -3 activities and PARP-1 cleavage in relation with their abilities to decrease tumor cell growth and integrity, AQ8 and AQ9 being consistently the most effective. Since apical caspases 2 and 8 may both act upstream of mitochondria to promote Cyt c release, it is significant to show that AQ9 maximally induces caspase-2 and -8 activities at 6 and 9 hr, respectively. During AQ8 treatment, the caspase-2 inhibitor benzyloxycarbonyl (z)-Val-Asp-Val-Ala-Asp (VDVAD)-fluoromethyl ketone (fmk) totally blocks caspase-9, -3, and -8 activations, whereas the caspase-8 inhibitor z-Ile-Glu-Thr-Asp-(IETD)-fmk does not prevent caspase-2, -9, and -3 activations, suggesting that AQ-induced caspase-2 activity is an upstream event critical for the activation of the downstream caspases 9 and 3 cascade, including the mitochondrial amplification loop through caspase-8. However, these caspase-2 and -8 inhibitors fail to alter AQ8-induced Cyt c release, suggesting that AQs might also target mitochondria independently from caspase activation. Furthermore, the antagonistic anti-Fas DX2 and ZB4 monoclonal antibodies (mAbs), which block the induction of Cyt c release and caspase-2, -8, and -9 activities by the agonistic anti-Fas CH11 mAb, and the neutralizing anti-Fas ligand (FasL) NOK-1 mAb all fail to inhibit AQ9-induced Cyt c release and caspase-2, -8, and -9 activities, suggesting that the FasL/Fas signaling pathway is not involved in the mechanism by which antiproliferative AQ analogs trigger apoptosis in HL-60 cells. PMID:15037204

  5. (−)-Epigallocatechin-3-Gallate Induces Non-Apoptotic Cell Death in Human Cancer Cells via ROS-Mediated Lysosomal Membrane Permeabilization

    PubMed Central

    Zhang, Yin; Yang, Nai-Di; Zhou, Fan; Shen, Ting; Duan, Ting; Zhou, Jing; Shi, Yin; Zhu, Xin-Qiang; Shen, Han-Ming

    2012-01-01

    (−)-Epigallocatechin-3-gallate (EGCG) is the most extensive studied tea polyphenol for its anti-cancer function. In this study, we report a novel mechanism of action for EGCG-mediated cell death by identifying the critical role of lysosomal membrane permeabilization (LMP). First, EGCG-induced cell death in human cancer cells (both HepG2 and HeLa) was found to be caspase-independent and accompanied by evident cytosolic vacuolization, only observable when cells were treated in serum-free medium. The cytosolic vacuolization observed in EGCG-treated cells was most probably caused by lysosomal dilation. Interestingly, EGCG was able to disrupt autophagic flux at the degradation stage by impairment of lysosomal function, and EGCG-induced cell death was independent of Atg5 or autophagy. The key finding of this study is that EGCG is able to trigger LMP, as evidenced by Lyso-Tracker Red staining, cathepsin D cytosolic translocation and cytosolic acidification. Consistently, a lysosomotropic agent, chloroquine, effectively rescues the cell death via suppressing LMP-caused cytosolic acidification. Lastly, we found that EGCG promotes production of intracellular ROS upstream of LMP and cell death, as evidenced by increased level of ROS in cells treated with EGCG and the protective effects of antioxidant N-acetylcysteine (NAC) against EGCG-mediated LMP and cell death. Taken together, data from our study reveal a novel mechanism underlying EGCG-induced cell death involving ROS and LMP. Therefore, understanding this lysosome-associated cell death pathway shed new lights on the anti-cancer effects of EGCG. PMID:23056433

  6. Transcriptomic Analysis Unveils Correlations between Regulative Apoptotic Caspases and Genes of Cholesterol Homeostasis in Human Brain

    PubMed Central

    Picco, Raffaella; Tomasella, Andrea; Fogolari, Federico; Brancolini, Claudio

    2014-01-01

    Regulative circuits controlling expression of genes involved in the same biological processes are frequently interconnected. These circuits operate to coordinate the expression of multiple genes and also to compensate dysfunctions in specific elements of the network. Caspases are cysteine-proteases with key roles in the execution phase of apoptosis. Silencing of caspase-2 expression in cultured glioblastoma cells allows the up-regulation of a limited number of genes, among which some are related to cholesterol homeostasis. Lysosomal Acid Lipase A (LIPA) was up-regulated in two different cell lines in response to caspase-2 down-regulation and cells silenced for caspase-2 exhibit reduced cholesterol staining in the lipid droplets. We expanded this observation by large-scale analysis of mRNA expression. All caspases were analyzed in terms of co-expression in comparison with 166 genes involved in cholesterol homeostasis. In the brain, hierarchical clustering has revealed that the expression of regulative apoptotic caspases (CASP2, CASP8 CASP9, CASP10) and of the inflammatory CASP1 is linked to several genes involved in cholesterol homeostasis. These correlations resulted in altered GBM (Glioblastoma Multiforme), in particular for CASP1. We have also demonstrated that these correlations are tissue specific being reduced (CASP9 and CASP10) or different (CASP2) in the liver. For some caspases (CASP1, CASP6 and CASP7) these correlations could be related to brain aging. PMID:25330190

  7. HLA-DR Alpha 2 Mediates Negative Signalling via Binding to Tirc7 Leading to Anti-Inflammatory and Apoptotic Effects in Lymphocytes In Vitro and In Vivo

    PubMed Central

    Schlawinsky, Mirko; Heinemann, Thomas; Schulze, Anke; Höhne, Wolfgang; Krause, Gerd; Kalka-Moll, Wiltrud; Fraser, Patricia; Volk, Hans-Dieter; Löhler, Jürgen; Milford, Edgar L.; Utku, Nalân

    2008-01-01

    Classically, HLA-DR expressed on antigen presenting cells (APC) initiates lymphocyte activation via presentation of peptides to TCR bearing CD4+ T-Cells. Here we demonstrate that HLA-DR alpha 2 domain (sHLA-DRα2) also induces negative signals by engaging TIRC7 on lymphocytes. This interaction inhibits proliferation and induces apoptosis in CD4+ and CD8+ T-cells via activation of the intrinsic pathway. Proliferation inhibition is associated with SHP-1 recruitment by TIRC7, decreased phosphorylation of STAT4, TCR-ζ chain & ZAP70, and inhibition of IFN-γ and FasL expression. HLA-DRα2 and TIRC7 co-localize at the APC-T cell interaction site. Triggering HLA-DR - TIRC7 pathway demonstrates that sHLA-DRα2 treatment inhibits proinflammatory-inflammatory cytokine expression in APC & T cells after lipopolysaccaride (LPS) stimulation in vitro and induces apoptosis in vivo. These results suggest a novel antiproliferative role for HLA-DR mediated via TIRC7, revise the notion of an exclusive stimulatory interaction of HLA-DR with CD4+ T cells and highlights a novel physiologically relevant regulatory pathway. PMID:18270567

  8. Loss of the Birt–Hogg–Dubé tumor suppressor results in apoptotic resistance due to aberrant TGFβ-mediated transcription

    PubMed Central

    Cash, T P; Gruber, J J; Hartman, T R; Henske, E P; Simon, M C

    2011-01-01

    Birt–Hogg–Dubé (BHD) syndrome is an inherited cancer susceptibility disease characterized by skin and kidney tumors, as well as cystic lung disease, which results from loss-of-function mutations in the BHD gene. BHD is also inactivated in a significant fraction of patients with sporadic renal cancers and idiopathic cystic lung disease, and little is known about its mode of action. To investigate the molecular and cellular basis of BHD tumor suppressor activity, we generated mutant Bhd mice and embryonic stem cell lines. BHD-deficient cells exhibited defects in cell-intrinsic apoptosis that correlated with reduced expression of the BH3-only protein Bim, which was similarly observed in all human and murine BHD-related tumors examined. We further demonstrate that Bim deficiency in Bhd−/− cells is not a consequence of elevated mTOR or ERK activity, but results instead from reduced Bim transcription associated with a general loss of TGFβ-mediated transcription and chromatin modifications. In aggregate, this work identifies a specific tumor suppressive mechanism for BHD in regulating TGFβ-dependent transcription and apoptosis, which has implications for the development of targeted therapies. PMID:21258407

  9. An investigation on the cytotoxicity and caspase-mediated apoptotic effect of biologically synthesized silver nanoparticles using Podophyllum hexandrum on human cervical carcinoma cells.

    PubMed

    Jeyaraj, Murugaraj; Rajesh, Manoharan; Arun, Renganathan; MubarakAli, Davoodbasha; Sathishkumar, Gnanasekar; Sivanandhan, Ganeshan; Dev, Gnanajothi Kapil; Manickavasagam, Markandan; Premkumar, Kumpati; Thajuddin, Nooruddin; Ganapathi, Andy

    2013-02-01

    Now-a-days synthesis and characterization of silver nanoparticles (AgNPs) through biological entity is quite interesting to employ AgNPs for various biomedical applications in general and treatment of cancer in particular. This paper presents the green synthesis of AgNPs using leaf extract of Podophyllum hexandrum Royle and optimized with various parameters such as pH, temperature, reaction time, volume of extract and metal ion concentration for synthesis of AgNPs. TEM, XRD and FTIR were adopted for characterization. The synthesized nanoparticles were found to be spherical shaped with average size of 14 nm. Effects of AgNPs were analyzed against human cervical carcinoma cells by MTT Assay, quantification of ROS, RT-PCR and western blotting techniques. The overall result indicates that AgNPs can selectively inhibit the cellular mechanism of HeLa by DNA damage and caspase mediated cell death. This biological procedure for synthesis of AgNPs and selective inhibition of cancerous cells gives an alternative avenue to treat human cancer effectively. PMID:23117153

  10. Rapamycin-enhanced mitomycin C-induced apoptotic death is mediated through the S6K1BadBak pathway in peritoneal carcinomatosis

    PubMed Central

    Song, X; Dilly, A-K; Kim, S-Y; Choudry, H A; Lee, Y J

    2014-01-01

    Peritoneal carcinomatosis (PC) is the most common secondary cancerous disease, and more effective novel regimens are needed. In this study, we identified a novel combination treatment for PC, chemotherapeutic agent mitomycin C in combination with mTOR (mammalian target of rapamycin) inhibitor rapamycin. We observed that the combination of mitomycin C and rapamycin induced synergistic cytotoxicity and apoptosis, which was mediated through an increase in caspase activation. The combination of mitomycin C and rapamycin inactivated p70 S6 ribosomal kinase (S6K1) and dephosphorylated Bad, leading to dissociation of Bcl-xL from Bak, which resulted in Bak oligomerization, mitochondria dysfunction and cytochrome c release. PF-4708671, a S6K1-specific inhibitor, enhanced the combination treatment-induced apoptosis, whereas S6K1 E389 DeltaCT-HA (S6K1 active form) dramatically decreased the induction of apoptosis. In addition, the combination treatment significantly inhibited LS174T intraperitoneal tumor growth in vivo. This study provides a preclinical rationale for apoptosis induction linked with the mTOR pathway through a combination of chemotherapeutic agents and mTOR inhibitor, and will support this combinatorial strategy to PC patients. PMID:24901052

  11. Combination of Protoporphyrin IX-mediated Sonodynamic Treatment with Doxorubicin Synergistically Induced Apoptotic Cell Death of a Multidrug-Resistant Leukemia K562/DOX Cell Line.

    PubMed

    Wang, Xiaobing; Jia, Yali; Su, Xiaomin; Wang, Pan; Zhang, Kun; Feng, Xiaolan; Liu, Quanhong

    2015-10-01

    The main objective of this study was to evaluate the efficacy of administration of doxorubicin (DOX) in combination with protoporphyrin IX (PpIX)-assisted low-level therapeutic ultrasound (US) in K562/DOX cells as a potential strategy in cancer therapy. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the cytotoxicity of different treatments. Apoptosis was analyzed using annexin V-PE/7-amino-actinomycin D staining. Changes in DNA fragmentation, intracellular reactive oxygen species production, cellular membrane permeability, P-glycoprotein expression and DOX uptake were analyzed with flow cytometry. Under optimal conditions, PpIX-US significantly aggravated DOX-induced K562/DOX cell death, compared with either monotherapy. Synergistic potentiation of DNA damage, generation of reactive oxygen species and P-glycoprotein inhibition were observed. Plasma membrane integrity changed slightly after US exposure, and DOX uptake was notably improved after PpIX-US exposure. The results indicate that PpIX-US could increase the susceptibility of tumors to antineoplastic drugs, suggesting a clinical potential method for sonodynamic therapy-mediated tumor chemotherapy. PMID:26166458

  12. Flow cytometry enumeration of apoptotic cancer cells by apoptotic rate.

    PubMed

    Diaz, David; Prieto, Alfredo; Reyes, Eduardo; Barcenilla, Hugo; Monserrat, Jorge; Alvarez-Mon, Melchor

    2015-01-01

    Most authors currently quantify the frequency of apoptotic cells in a given phenotypically defined population after calculating the apoptotic index (AI), i.e., the percentage of apoptotic cells displaying a specific linage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. Firstly, apoptotic cells fragment into apoptotic bodies that later disintegrate. Secondly, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. This chapter describes a flow cytometry method to calculate the apoptotic rate (AR) that takes into account both cell fragmentation and loss of lineage antigen expression on measurement of apoptosis using flow cytometry ratiometric cell enumeration that emerges as a more accurate method of measurement of the occurrence of apoptosis in normal and tumoral cell cultures. PMID:25308258

  13. Flow cytometry enumeration of apoptotic cancer cells by apoptotic rate.

    PubMed

    Diaz, David; Prieto, Alfredo; Reyes, Eduardo; Barcenilla, Hugo; Monserrat, Jorge; Alvarez-Mon, Melchor

    2008-01-01

    Most authors currently quantify the frequency of apoptotic cells in a given phenotypically defined population after calculating the apoptotic index (AI), that is, the percentage of apoptotic cells displaying a specific lineage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. First, apoptotic cells fragment into apoptotic bodies that later disintegrate. Second, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. This chapter will describe a flow cytometry method to calculate the apoptotic rate (AR) that takes into account both cell fragmentation and loss of LAg expression on measurement of apoptosis using flow cytometry ratiometric cell enumeration that emerges as a more accurate method of measurement of the occurrence of apoptosis in normal and tumoral cell cultures. PMID:18175809

  14. Cyclic AMP is both a pro-apoptotic and anti-apoptotic second messenger

    PubMed Central

    Insel, Paul A.; Zhang, Lingzhi; Murray, Fiona; Yokouchi, Hiroshi; Zambon, Alexander C.

    2011-01-01

    The second messenger cyclic AMP (cAMP) can either stimulate or inhibit programmed cell death (apoptosis). Here, we review examples of cell types that show pro-apoptotic or anti-apoptotic responses to increases in cAMP. We also show that cells can have both such responses, although predominantly having one or the other. Protein kinase A (PKA)-promoted changes in phosphoylation and gene expression can mediate pro-apoptotic responses, such as in murine S49 lymphoma cells, based on evidence that mutants lacking PKA fail to undergo cAMP-promoted, mitochondria-dependent apoptosis. Mechanisms for the anti-apoptotic response to cAMP likely involve Epac (Exchange protein activated by cAMP), a cAMP-regulated effector that is a guanine nucleotide exchange factor (GEF) for the low molecular weight G-protein, Rap1. Therapeutic approaches that activate PKA-mediated pro-apoptosis or that block Epac-mediated anti-apoptotisis may provide a means to enhance cell killing, such as in certain cancers. By contrast, efforts to block PKA or stimulate Epac have the potential to be useful in diseases settings (such as heart failure) associated with cAMP-promoted apoptosis. PMID:21385327

  15. The pro-apoptotic and anti-invasive effects of hypericin-mediated photodynamic therapy are enhanced by hyperforin or aristoforin in HT-29 colon adenocarcinoma cells.

    PubMed

    Šemeláková, Martina; Mikeš, Jaromír; Jendželovský, Rastislav; Fedoročko, Peter

    2012-12-01

    Photodynamic therapy is a rapidly-developing anti-cancer approach for the treatment of various types of malignant as well as non-malignant diseases. In this study, hypericin-mediated photodynamic therapy (HY-PDT) in sub-optimal dose was combined with hyperforin (HP) or its stable derivative aristoforin (AR) in an effort to improve efficacy on the cellular level. The logic of this combination is based on the fact that both bioactive compounds naturally occur in plants of Hypericum sp. At relatively low concentrations up to 5 μM, hyperforin and aristoforin were able to stimulate onset of apoptosis in HT-29 colon adenocarcinoma cells exposed to HY-PDT, inhibit cell cycle progression, suppress expression of matrixmetalloproteinases-2/-9 together with cell adhesivity, thereby affecting the clonogenic potential of the cells. As the action of aristoforin was more pronounced, in line with our assumption, these changes were also linked in this case with hypericin accumulation and increased ROS generation leading to dissipation of mitochondrial membrane potential in a significant portion of the cells, as well as activation of caspase-3. Comparison of HT-29 cells to another colon adenocarcinoma-derived cell line HCT-116 demonstrated significant differences in sensitivity of different cell lines to PDT, however, accumulated effect of HY-PDT with HP/AR proved similar in both tested cell lines. The presented data may help to elucidate the mechanisms of action for different bioactive constituents of St. John's wort, which are increasingly recognized as being able to regulate a variety of pathobiological processes, thus possessing potential therapeutic properties. PMID:23099482

  16. Chondroitin Sulfate as a Molecular Portal That Preferentially Mediates the Apoptotic Killing of Tumor Cells by Penetratin-directed Mitochondria-disrupting Peptides*

    PubMed Central

    Yang, Hao; Liu, Shan; Cai, Huawei; Wan, Lin; Li, Shengfu; Li, Youping; Cheng, Jingqiu; Lu, Xiaofeng

    2010-01-01

    The use of cell-penetrating peptides (CPPs) as drug carriers for targeted therapy is limited by the unrestricted cellular translocation of CPPs. The preferential induction of tumor cell death by penetratin (Antp)-directed peptides (PNC27 and PNC28), however, suggests that the CPP Antp may contribute to the preferential cytotoxicity of these peptides. Using PNC27 as a molecular model, we constructed three novel peptides (PT, PR9, and PD3) by replacing the leader peptide Antp with one of three distinct CPPs (TAT, R9, or DPV3), respectively. The IC50 values of PNC27 in tumor cells were 23 times lower than in normal cells. However, all three engineered peptides demonstrated similar cytotoxic effects in tumor and normal cells. Another three chimeric peptides containing the leader peptide Antp with different mitochondria-disrupting peptides (KLA-Antp (KGA), B27-Antp (BA27), and B28-Antp (BA28)), preferentially induced apoptosis in tumor cells. The IC50 values of these peptides (310 ?m) were 36 times lower in tumor cells than in normal cells. In contrast, TAT-directed peptides (TAT-KLA (TK), TAT-B27 (TB27), and TAT-B28 (TB28)), were cytotoxic to both tumor and normal cells. These data demonstrate that the leader peptide Antp contributes to the preferential cytotoxicity of Antp-directed peptides. Furthermore, Antp-directed peptides bind chondroitin sulfate (CS), and the removal of endogenous CS reduces the cytotoxic effects of Antp-directed peptides in tumor cells. The overexpression of CS in tumor cells is positively correlated to the cell entry and cytotoxicity of Antp- directed peptides. These results suggest that CS overexpression in tumor cells is an important molecular portal that mediates the preferential cytotoxicity of Antp-directed peptides. PMID:20484051

  17. p66Shc mediates high-glucose and angiotensin II-induced oxidative stress renal tubular injury via mitochondrial-dependent apoptotic pathway

    PubMed Central

    Xiao, Li; Nie, Jing; Liu, Fu-you; Ling, Guang-hui; Zhu, Xue-jing; Tang, Wen-bin; Chen, Wen-cui; Xia, Yun-cheng; Zhan, Ming; Ma, Ming-ming; Peng, You-ming; Liu, Hong; Liu, Ying-hong; Kanwar, Yashpal S.

    2010-01-01

    p66Shc, a promoter of apoptosis, modulates oxidative stress response and cellular survival, but its role in the progression of diabetic nephropathy is relatively unknown. In this study, mechanisms by which p66Shc modulates high-glucose (HG)- or angiotensin (ANG) II-induced mitochondrial dysfunction were investigated in renal proximal tubular cells (HK-2 cells). Expression of p66Shc and its phosphorylated form (p-p66Shc, serine residue 36) and apoptosis were notably increased in renal tubules of diabetic mice, suggesting an increased reactive oxygen species production. In vitro, HG and ANG II led to an increased expression of total and p-p66Shc in HK-2 cells. These changes were accompanied with increased production of mitochondrial H2O2, reduced mitochondrial membrane potential, increased translocation of mitochondrial cytochrome c from mitochondria into cytosol, upregulation of the expression of caspase-9, and ultimately reduced cell survival. Overexpression of a dominant-negative Ser36 mutant p66Shc (p66ShcS36A) or treatment of p66Shc- or PKC-β-short interfering RNAs partially reversed these changes. Treatment of HK-2 cells with HG and ANG II also increased the protein-protein association between p-p66Shc and Pin1, an isomerase, in the cytosol, and with cytochrome c in the mitochondria. These interactions were partially disrupted with the treatment of PKC-β inhibitor or Pin1-short interfering RNA. These data suggest that p66Shc mediates HG- and ANG II-induced mitochondrial dysfunctions via PKC-β and Pin1-dependent pathways in renal tubular cells. PMID:20739391

  18. Topological Transitions in Mitochondrial Membranes controlled by Apoptotic Proteins

    NASA Astrophysics Data System (ADS)

    Hwee Lai, Ghee; Sanders, Lori K.; Mishra, Abhijit; Schmidt, Nathan W.; Wong, Gerard C. L.; Ivashyna, Olena; Schlesinger, Paul H.

    2010-03-01

    The Bcl-2 family comprises pro-apoptotic proteins, capable of permeabilizing the mitochondrial membrane, and anti-apoptotic members interacting in an antagonistic fashion to regulate programmed cell death (apoptosis). They offer potential therapeutic targets to re-engage cellular suicide in tumor cells but the extensive network of implicated protein-protein interactions has impeded full understanding of the decision pathway. We show, using synchrotron x-ray diffraction, that pro-apoptotic proteins interact with mitochondrial-like model membranes to generate saddle-splay (negative Gaussian) curvature topologically required for pore formation, while anti-apoptotic proteins can deactivate curvature generation by molecules drastically different from Bcl-2 family members and offer evidence for membrane-curvature mediated interactions general enough to affect very disparate systems.

  19. Complementary proteomic tools for the dissection of apoptotic proteolysis events.

    PubMed

    Pham, Victoria C; Pitti, Robert; Anania, Veronica G; Bakalarski, Corey E; Bustos, Daisy; Jhunjhunwala, Suchit; Phung, Qui T; Yu, Kebing; Forrest, William F; Kirkpatrick, Donald S; Ashkenazi, Avi; Lill, Jennie R

    2012-05-01

    Proteolysis is a key regulatory event that controls intracellular and extracellular signaling through irreversible changes in a protein's structure that greatly alters its function. Here we describe a platform for profiling caspase substrates which encompasses two highly complementary proteomic techniques--the first is a differential gel based approach termed Global Analyzer of SILAC-derived Substrates of Proteolysis (GASSP) and the second involves affinity enrichment of peptides containing a C-terminal aspartic acid residue. In combination, these techniques have enabled the profiling of a large cellular pool of apoptotic-mediated proteolytic events across a wide dynamic range. By applying this integrated proteomic work flow to analyze proteolytic events resulting from the induction of intrinsic apoptosis in Jurkat cells via etoposide treatment, 3346 proteins were quantified, of which 360 proteins were identified as etoposide-induced proteolytic substrates, including 160 previously assigned caspase substrates. In addition to global profiling, a targeted approach using BAX HCT116 isogenic cell lines was utilized to dissect pre- and post-mitochondrial extrinsic apoptotic cleavage events. By employing apoptotic activation with a pro-apoptotic receptor agonist (PARA), a limited set of apoptotic substrates including known caspase substrates such as BH3 interacting-domain death agonist (BID) and Poly (ADP-ribose) polymerase (PARP)-1, and novel substrates such as Basic Transcription Factor 3, TRK-fused gene protein (TFG), and p62/Sequestosome were also identified. PMID:22432722

  20. Anti-C1q autoantibodies from active lupus nephritis patients could inhibit the clearance of apoptotic cells and complement classical pathway activation mediated by C1q in vitro.

    PubMed

    Pang, Yun; Yang, Xiao-Wei; Song, Yan; Yu, Feng; Zhao, Ming-Hui

    2014-12-01

    Anti-C1q antibodies are prevalent in patients with active lupus nephritis and were found to be closely associated with renal involvement and predictive for a flare of nephritis. However, the pathogenesis of anti-C1q antibodies involved in human lupus nephritis remains unclear. C1q, which plays a key role in apoptotic cell and immune complex removal, is a very important functional molecule in the pathogenesis of SLE. The aim of this study was to investigate the influence of anti-C1q autoantibodies from active lupus nephritis patients on the bio-functions of C1q in vitro. We purified IgG autoantibodies against C1q from lupus nephritis patients, and found that they could recognize C1q bound on early apoptotic cells at 30 ?g/ml, and could significantly decrease the phagocytosis by macrophages of early apoptotic cells opsonized by 50 ?g/ml C1q in comparison with normal IgG. Levels of circulating immune complexes of the ten patients were measured by a circulating immune complexes (CIC)-C1q Enzyme Immunoassay Kit. Anti-C1q autoantibodies affinity purified by microtiter plates could significantly inhibit the deposition of C3c on CIC-C1q in a dose dependent manner in comparison with IgG from 10 healthy blood donors. The binding of opsonized immune complexes to RBCs was significantly inhibited by anti-C1q autoantibodies purified by microtiter plates in a dose dependent manner. Our observations suggest that serum anti-C1q autoantibodies from active lupus nephritis patients could interfere with some biological function of C1q in vitro. PMID:25092568

  1. Induction of Noxa-mediated apoptosis by modified vaccinia virus Ankara depends on viral recognition by cytosolic helicases, leading to IRF-3/IFN-?-dependent induction of pro-apoptotic Noxa.

    PubMed

    Eitz Ferrer, Pedro; Potthoff, Stephanie; Kirschnek, Susanne; Gasteiger, Georg; Kastenmller, Wolfgang; Ludwig, Holger; Paschen, Stefan A; Villunger, Andreas; Sutter, Gerd; Drexler, Ingo; Hcker, Georg

    2011-06-01

    Viral infection is a stimulus for apoptosis, and in order to sustain viral replication many viruses are known to carry genes encoding apoptosis inhibitors. F1L, encoded by the orthopoxvirus modified vaccinia virus Ankara (MVA) has a Bcl-2-like structure. An MVA mutant lacking F1L (MVA?F1L) induces apoptosis, indicating that MVA infection activates and F1L functions to inhibit the apoptotic pathway. In this study we investigated the events leading to apoptosis upon infection by MVA?F1L. Apoptosis largely proceeded through the pro-apoptotic Bcl-2 family protein Bak with some contribution from Bax. Of the family of pro-apoptotic BH3-only proteins, only the loss of Noxa provided substantial protection, while the loss of Bim had a minor effect. In mice, MVA preferentially infected macrophages and DCs in vivo. In both cell types wt MVA induced apoptosis albeit more weakly than MVA?F1L. The loss of Noxa had a significant protective effect in macrophages, DC and primary lymphocytes, and the combined loss of Bim and Noxa provided strong protection. Noxa protein was induced during infection, and the induction of Noxa protein and apoptosis induction required transcription factor IRF3 and type I interferon signalling. We further observed that helicases RIG-I and MDA5 and their signalling adapter MAVS contribute to Noxa induction and apoptosis in response to MVA infection. RNA isolated from MVA-infected cells induced Noxa expression and apoptosis when transfected in the absence of viral infection. We thus here describe a pathway leading from the detection of viral RNA during MVA infection by the cytosolic helicase-pathway, to the up-regulation of Noxa and apoptosis via IRF3 and type I IFN signalling. PMID:21698224

  2. Apoptosis-inducing active components from Corbicula fluminea through activation of caspase-2 and production of reactive oxygen species in human leukemia HL-60 cells.

    PubMed

    Huang, Ying-Tang; Huang, Yi-Hsuan; Hour, Tzhy-Chyuan; Pan, Bonnie Sun; Liu, Yeuk-Chuen; Pan, Min-Hsiung

    2006-08-01

    The anti-cancer effects and possible mechanisms of the freshwater clam (Corbicula fluminea Muller) and its active compounds (FME) on cell viability in human leukemia HL-60 cells were investigated. This study demonstrated that FME was able to inhibit cell proliferation in a concentration- and time-dependent manner. Treatment with FME caused induction of caspase-2, caspase-3, caspase-6, caspase-8, and caspase-9 activity in a time-dependent manner, but not affect caspase-1 activity; it induced the proteolysis of DNA fragmentation factor (DFF-45) and poly(ADP-ribose) polymerase (PARP). Induction of cell death by FME was completely prevented by a pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) and a caspase-2 inhibitor, Z-Val-Asp-Val-Ala-Asp-FMK (Z-VDVAD-FMK). Furthermore, treatment with FME caused a rapid loss of mitochondrial transmembrane potential, stimulation of generation of reactive oxygen species (ROS), release of mitochondrial cytochrome c into cytosol, and GSH depletion. Anti-oxidants such as N-acetylcysteine, catalase, superoxide dismutase, allopurinol, and pyrrolidine dithiocarbamate, but not diphenylene iodonium, significantly inhibited FME-induced cell death. In addition, the results showed that FME-induced apoptosis was accompanied by up-regulation of Bax and Bad, and down-regulation of Bcl-2 and Bcl-XL. Taken together, induction of apoptosis on HL-60 cells by FME was mainly associated with ROS production, GSH depletion, mitochondrial dysfunction, and caspase activation. PMID:16545898

  3. TNF? inhibits apoptotic cell clearance in the lung, exacerbating acute inflammation

    PubMed Central

    Borges, Valeria M.; Vandivier, R. William; McPhillips, Kathleen A.; Kench, Jennifer A.; Morimoto, Konosuke; Groshong, Steve D.; Richens, Tiffany R.; Graham, Brian B.; Muldrow, Alaina M.; Heule, Lea Van; Henson, Peter M.

    2009-01-01

    Efficient removal of apoptotic cells is essential for resolution of inflammation. Failure to clear dying cells can exacerbate lung injury and lead to persistent inflammation and autoimmunity. Here we show that TNF? blocks apoptotic cell clearance by alveolar macrophages and leads to proinflammatory responses in the lung. Compared with mice treated with intratracheal TNF? or exogenous apoptotic cells, mice treated with the combination of TNF? plus apoptotic cells demonstrated reduced apoptotic cell clearance from the lungs and increased recruitment of inflammatory leukocytes to the air spaces. Treatment with intratracheal TNF? had no effect on the removal of exogenous apoptotic cells from the lungs of TNF? receptor-1 (p55) and -2 (p75) double mutant mice and no effect on leukocyte recruitment. Bronchoalveolar lavage from mice treated with TNF? plus apoptotic cells contained increased levels of proinflammatory cytokines IL-6, KC, and MCP-1, but exhibited no change in levels of anti-inflammatory cytokines IL-10 and TGF-?. Administration of TNF? plus apoptotic cells during LPS-induced lung injury augmented neutrophil accumulation and proinflammatory cytokine production. These findings suggest that the presence of TNF? in the lung can alter the response of phagocytes to apoptotic cells leading to inflammatory cell recruitment and proinflammatory mediator production. PMID:19648283

  4. TNFalpha inhibits apoptotic cell clearance in the lung, exacerbating acute inflammation.

    PubMed

    Borges, Valeria M; Vandivier, R William; McPhillips, Kathleen A; Kench, Jennifer A; Morimoto, Konosuke; Groshong, Steve D; Richens, Tiffany R; Graham, Brian B; Muldrow, Alaina M; Van Heule, Lea; Henson, Peter M; Janssen, William J

    2009-10-01

    Efficient removal of apoptotic cells is essential for resolution of inflammation. Failure to clear dying cells can exacerbate lung injury and lead to persistent inflammation and autoimmunity. Here we show that TNFalpha blocks apoptotic cell clearance by alveolar macrophages and leads to proinflammatory responses in the lung. Compared with mice treated with intratracheal TNFalpha or exogenous apoptotic cells, mice treated with the combination of TNFalpha plus apoptotic cells demonstrated reduced apoptotic cell clearance from the lungs and increased recruitment of inflammatory leukocytes to the air spaces. Treatment with intratracheal TNFalpha had no effect on the removal of exogenous apoptotic cells from the lungs of TNFalpha receptor-1 (p55) and -2 (p75) double mutant mice and no effect on leukocyte recruitment. Bronchoalveolar lavage from mice treated with TNFalpha plus apoptotic cells contained increased levels of proinflammatory cytokines IL-6, KC, and MCP-1, but exhibited no change in levels of anti-inflammatory cytokines IL-10 and TGF-beta. Administration of TNFalpha plus apoptotic cells during LPS-induced lung injury augmented neutrophil accumulation and proinflammatory cytokine production. These findings suggest that the presence of TNFalpha in the lung can alter the response of phagocytes to apoptotic cells leading to inflammatory cell recruitment and proinflammatory mediator production. PMID:19648283

  5. Apoptotic effect of novel Schiff Based CdCl2(C14H21N3O2) complex is mediated via activation of the mitochondrial pathway in colon cancer cells

    PubMed Central

    Hajrezaie, Maryam; Paydar, Mohammadjavad; Looi, Chung Yeng; Moghadamtousi, Soheil Zorofchian; Hassandarvish, Pouya; Salga, Muhammad Saleh; Karimian, Hamed; Shams, Keivan; Zahedifard, Maryam; Majid, Nazia Abdul; Ali, Hapipah Mohd; Abdulla, Mahmood Ameen

    2015-01-01

    The development of metal-based agents has had a tremendous role in the present progress in cancer chemotherapy. One well-known example of metal-based agents is Schiff based metal complexes, which hold great promise for cancer therapy. Based on the potential of Schiff based complexes for the induction of apoptosis, this study aimed to examine the cytotoxic and apoptotic activity of a CdCl2(C14H21N3O2) complex on HT-29 cells. The complex exerted a potent suppressive effect on HT-29 cells with an IC50 value of 2.57 ± 0.39 after 72 h of treatment. The collapse of the mitochondrial membrane potential and the elevated release of cytochrome c from the mitochondria to the cytosol indicate the involvement of the intrinsic pathway in the induction of apoptosis. The role of the mitochondria-dependent apoptotic pathway was further proved by the significant activation of the initiator caspase-9 and the executioner caspases-3 and -7. In addition, the activation of caspase-8, which is associated with the suppression of NF-κB translocation to the nucleus, also revealed the involvement of the extrinsic pathway in the induced apoptosis. The results suggest that the CdCl2(C14H21N3O2) complex is able to induce the apoptosis of colon cancer cells and is a potential candidate for future cancer studies. PMID:25764970

  6. Acriflavine enhances radiosensitivity of colon cancer cells through endoplasmic reticulum stress-mediated apoptosis.

    PubMed

    Lim, Min-Jin; Ahn, Ji-Yeon; Han, Youngsoo; Yu, Chi-Ho; Kim, Mi-Hyoung; Lee, Sae-Lo-Oom; Lim, Dae-Seog; Song, Jie-Young

    2012-08-01

    Radiotherapy (RT) is one of the most effective tools in the clinical treatment of cancer. Because the tumor suppressor p53 plays a central role in radiation-mediated responses, including cell cycle-arrest and apoptosis, a number of studies have suggested that p53 could be a useful therapeutic target of anti-cancer agents. Accordingly, we sought to discover a new agent capable of increasing p53 activity. HCT116 colon cancer cells, containing wild-type p53, were stably transfected with a p53 responsive-luciferase (p53-Luc) reporter gene. A cell-based high-throughput screen of 7920 synthetic small molecules was performed in duplicate. Of the screened compounds, acriflavine (ACF) significantly increased p53-Luc activity in a concentration-dependent manner without causing toxicity. Pretreatment with ACF enhanced the induction of p53 protein expression and phosphorylation on serine 15 by ?-irradiation. Clonogenic assays showed that ACF pretreatment also potentiated radiation-induced cell death. The combination of irradiation and ACF treatment induced mitochondrial release of cytochrome c and significant activation of caspase-3 with PARP cleavage in colon cancer cells, demonstrating typical apoptotic cell death. Combined treatment with ACF and radiation increased the expression of Bax and Bad, while decreasing expression of Bcl-2. In addition, the ACF/radiation treatment combination induced endoplasmic reticulum (ER) stress responses mediated by IRE1? (inositol-requiring transmembrane kinase and endonuclease 1?), eIF-2? (eukaryotic initiation factor 2?), caspase-2/12, and CHOP (C/EBP homologous protein). The knockdown of IRE1? by siRNA inhibited the apoptotic cell death induced by ACF/radiation treatment. In vivo studies showed that combined treatment with ACF and radiation significantly inhibited the growth of tumors in colorectal cancer xenografted mice. These results indicate that ACF acts through p53-dependent mitochondrial pathways and ER stress signals, and could be a promising radiosensitizer. PMID:22564437

  7. Pro-apoptotic and pro-autophagic effects of the Aurora kinase A inhibitor alisertib (MLN8237) on human osteosarcoma U-2 OS and MG-63 cells through the activation of mitochondria-mediated pathway and inhibition of p38 MAPK/PI3K/Akt/mTOR signaling pathway

    PubMed Central

    Niu, Ning-Kui; Wang, Zi-Li; Pan, Shu-Ting; Ding, Hui-Qiang; Au, Giang HT; He, Zhi-Xu; Zhou, Zhi-Wei; Xiao, Guozhi; Yang, Yin-Xue; Zhang, Xueji; Yang, Tianxin; Chen, Xiao-Wu; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Osteosarcoma (OS) is the most common malignant bone tumor occurring mostly in children and adolescents between 10 and 20 years of age with poor response to current therapeutics. Alisertib (ALS, MLN8237) is a selective Aurora kinase A inhibitor that displays anticancer effects on several types of cancer. However, the role of ALS in the treatment of OS remains unknown. This study aimed to investigate the effects of ALS on the cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT) and the underlying mechanisms in two human OS cell lines U-2 OS and MG-63. The results showed that ALS had potent growth inhibitory, pro-apoptotic, pro-autophagic, and EMT inhibitory effects on U-2 OS and MG-63 cells. ALS remarkably induced G2/M arrest and down-regulated the expression levels of cyclin-dependent kinases 1 and 2 and cyclin B1 in both U-2 OS and MG-63 cells. ALS markedly induced mitochondria-mediated apoptosis with a significant increase in the expression of key pro-apoptotic proteins and a decrease in main anti-apoptotic proteins. Furthermore, ALS promoted autophagic cell death via the inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways, and activation of 5′-AMP-dependent kinase (AMPK) signaling pathway. Inducers or inhibitors of apoptosis or autophagy simultaneously altered ALS-induced apoptotic and autophagic death in both U-2 OS and MG-63 cells, suggesting a crosstalk between these two primary modes of programmed cell death. Moreover, ALS suppressed EMT-like phenotypes with a marked increase in the expression of E-cadherin but a decrease in N-cadherin in U-2 OS and MG-63 cells. ALS treatment also induced reactive oxygen species (ROS) generation but inhibited the expression levels of sirtuin 1 and nuclear factor-erythroid-2-related factor 2 (Nrf2) in both cell lines. Taken together, these findings show that ALS promotes apoptosis and autophagy but inhibits EMT via PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways with involvement of ROS- and sirtuin 1-associated pathways in U-2 OS and MG-63 cells. ALS is a promising anticancer agent in OS treatment and further studies are needed to confirm its efficacy and safety in OS chemotherapy. PMID:25792811

  8. Apoptotic markers in protozoan parasites.

    PubMed

    Jimnez-Ruiz, Antonio; Alzate, Juan Fernando; Macleod, Ewan Thomas; Lder, Carsten Gnter Kurt; Fasel, Nicolas; Hurd, Hilary

    2010-01-01

    The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms.In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities. PMID:21062457

  9. Apoptotic markers in protozoan parasites

    PubMed Central

    2010-01-01

    The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms. In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities. PMID:21062457

  10. p38alpha, but not p38beta, inhibits the phosphorylation and presence of c-FLIPS in DISC to potentiate Fas-mediated caspase-8 activation and type I apoptotic signaling.

    PubMed

    Tourian, Leon; Zhao, Hong; Srikant, Coimbatore B

    2004-12-15

    Pharmacological inhibitors of JNK (SP600125) and p38 (PD169316) sensitize tumor cells to Fas-mediated apoptosis. PD169316 is less potent than SP600125 and diminishes its effect when present together. Because the p38 isoforms that promote (p38alpha) or inhibit (p38beta) apoptosis are both suppressed by PD169316, we investigated their regulatory involvement in Fas-signaling. We report here, that p38alpha, but not p38beta, exerts its proapoptotic effect by inhibiting the phosphorylation and presence of c-FLIPS, but not c-FLIPL, in the DISC to promote caspase-8 activation and type I signaling in Fas-activated Jurkat cells. Its effect was enhanced by enforced expression of Flag-tagged p38alpha and was attenuated by its inactive mutant (p38alpha-AGF) or by translational silencing. By contrast, type II signaling was facilitated by p38alpha-dependent mitochondrial presence of tBid and inhibition of Bcl-2 (Ser70) phosphorylation as well as by p38alpha/beta-dependent mitochondrial localization of Bax and inhibition of phosphorylation of Bad (Ser112/Ser155). Potentiation of Fas-mediated apoptosis by the inhibition of JNK1/2 correlated with the loss of Bad (Ser136) phosphorylation and was dependent on the stimulatory effect of p38alpha on DISC and the downstream effects of both p38alpha and p38beta. These data underscore the need to reassess the findings obtained with pan-p38 inhibitors and suggest that activation of p38alpha coupled with targeted inhibition of p38beta and JNK1/2 should optimally sensitize tumor cells to Fas-mediated apoptosis. PMID:15572410

  11. Calpain Inhibition Protects against Virus-Induced Apoptotic Myocardial Injury

    PubMed Central

    DeBiasi, Roberta L.; Edelstein, Charles L.; Sherry, Barbara; Tyler, Kenneth L.

    2001-01-01

    Viral myocarditis is an important cause of human morbidity and mortality for which reliable and effective therapy is lacking. Using reovirus strain 8B infection of neonatal mice, a well-characterized experimental model of direct virus-induced myocarditis, we now demonstrate that myocardial injury results from apoptosis. Proteases play a critical role as effectors of apoptosis. The activity of the cysteine protease calpain increases in reovirus-infected myocardiocytes and can be inhibited by the dipeptide alpha-ketoamide calpain inhibitor Z-Leu-aminobutyric acid-CONH(CH2)3-morpholine (CX295). Treatment of reovirus-infected neonatal mice with CX295 protects them against reovirus myocarditis as documented by (i) a dramatic reduction in histopathologic evidence of myocardial injury, (ii) complete inhibition of apoptotic myocardial cell death as identified by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, (iii) a reduction in serum creatine phosphokinase, and (iv) improved weight gain. These findings are the first evidence for the importance of a calpain-associated pathway of apoptotic cell death in viral disease. Inhibition of apoptotic signaling pathways may be an effective strategy for the treatment of viral disease in general and viral myocarditis in particular. PMID:11119604

  12. EPO Mediates Neurotrophic, Neuroprotective, Anti-Oxidant, and Anti-Apoptotic Effects via Downregulation of miR-451 and miR-885-5p in SH-SY5Y Neuron-Like Cells

    PubMed Central

    Alural, Begum; Duran, Gizem Ayna; Tufekci, Kemal Ugur; Allmer, Jens; Onkal, Zeynep; Tunali, Dogan; Genc, Kursad; Genc, Sermin

    2014-01-01

    Erythropoietin (EPO) is a neuroprotective cytokine, which has been applied in several animal models presenting neurological disorders. One of the proposed modes of action resulting in neuroprotection is post-transcriptional gene expression regulation. This directly brings to mind microRNAs (miRNAs), which are small non-coding RNAs that regulate gene expression at the post-transcriptional level. It has not yet been evaluated whether miRNAs participate in the biological effects of EPO or whether it, inversely, modulates specific miRNAs in neuronal cells. In this study, we employed miRNA and mRNA arrays to identify how EPO exerts its biological function. Notably, miR-451 and miR-885-5p are downregulated in EPO-treated SH-SY5Y neuronal-like cells. Accordingly, target prediction and transcriptome analysis of cells treated with EPO revealed an alteration of the expression of genes involved in apoptosis, cell survival, proliferation, and migration. Low expression of miRNAs in SH-SY5Y was correlated with high expression of their target genes, vascular endothelial growth factor A, matrix metallo peptidase 9 (MMP9), cyclin-dependent kinase 2 (CDK2), erythropoietin receptor, Mini chromosome maintenance complex 5 (MCM5), B-cell lymphoma 2 (BCL2), and Galanin (GAL). Cell viability, apoptosis, proliferation, and migration assays were carried out for functional analysis after transfection with miRNA mimics, which inhibited some biological actions of EPO such as neuroprotection, anti-oxidation, anti-apoptosis, and migratory effects. In this study, we report for the first time that EPO downregulates the expression of miRNAs (miR-451 and miR-885-5p) in SH-SY5Y neuronal-like cells. The correlation between the over-expression of miRNAs and the decrease in EPO-mediated biological effects suggests that miR-451 and miR-885-5p may play a key role in the mediation of biological function. PMID:25324845

  13. A Novel Anticancer Agent, 8-Methoxypyrimido[4?,5?:4,5]thieno(2,3-b) Quinoline-4(3H)-One Induces Neuro 2a Neuroblastoma Cell Death through p53-Dependent, Caspase-Dependent and -Independent Apoptotic Pathways

    PubMed Central

    Sahu, Upasana; Sidhar, Himakshi; Ghate, Pankaj S.; Advirao, Gopal M.; Raghavan, Sathees C.; Giri, Ranjit K.

    2013-01-01

    Neuroblastoma is the most common cancer in infants and fourth most common cancer in children. Despite recent advances in cancer treatments, the prognosis of stage-IV neuroblastoma patients continues to be dismal which warrant new pharmacotherapy. A novel tetracyclic condensed quinoline compound, 8-methoxypyrimido [4?,5?:4,5]thieno(2,3-b) quinoline-4(3H)-one (MPTQ) is a structural analogue of an anticancer drug ellipticine and has been reported to posses anticancer property. Study on MPTQ on neuroblastoma cells is very limited and mechanisms related to its cytotoxicity on neuroblastoma cells are completely unknown. Here, we evaluated the anticancer property of MPTQ on mouse neuro 2a and human SH-SY5Y neuroblastoma cells and investigated the mechanisms underlying MPTQ-mediated neuro 2a cell death. MPTQ-mediated neuro 2a and SH-SY5Y cell deaths were found to be dose and time dependent. Moreover, MPTQ induced cell death reached approximately 99.8% and 90% in neuro 2a and SH-SY5Y cells respectively. Nuclear oligonucleosomal DNA fragmentation and Terminal dUTP Nick End Labelling assays indicated MPTQ-mediated neuro 2a cell death involved apoptosis. MPTQ-mediated apoptosis is associated with increased phosphorylation of p53 at Ser15 and Ser20 which correlates with the hyperphosphorylation of Ataxia-Telangiectasia mutated protein (ATM). Immunocytochemical analysis demonstrated the increased level of Bax protein in MPTQ treated neuro 2a cells. MPTQ-mediated apoptosis is also associated with increased activation of caspase-9, -3 and -7 but not caspase-2 and -8. Furthermore, increased level of caspase-3 and cleaved Poly (ADP Ribose) polymerase were observed in the nucleus of MPTQ treated neuro 2a cells, suggesting the involvement of caspase-dependent intrinsic but not extrinsic apoptotic pathway. Increased nuclear translocation of apoptosis inducing factor suggests additional involvement of caspase-independent apoptosis pathway in MPTQ treated neuro 2a cells. Collectively, MPTQ-induced neuro 2a cell death is mediated by ATM and p53 activation, and Bax-mediated activation of caspase-dependent and caspase-independent mitochondrial apoptosis pathways. PMID:23824039

  14. Intercellular transfer of apoptotic signals via electrofusion

    SciTech Connect

    Park, Jin Suk; Lee, Wilson; McCulloch, Christopher A.

    2012-05-01

    We determined whether cells that are induced to undergo anoikis by matrix detachment can initiate apoptosis in healthy cells following electroporation-induced fusion. Separate populations of MDCK cells undergoing anoikis and stained with FITC-annexin or viable MDCK cells that were labeled with spectrally discrete fluorescent beads were electroporated. Cells were analyzed by flow cytometry for enumeration of viable cells with beads, apoptotic cells or fused cells. Electroporation promoted a 49-fold increase of the percentage of viable cells that had fused with apoptotic cells. Apoptotic cell-viable cell fusions were 8-fold more likely to not attach to cell culture plastic and 2.3-fold less likely to proliferate after 24 hr incubation than viable cell fusion controls. These data demonstrate that apoptotic signals can be transferred between cells by electrofusion, possibly suggesting a novel investigative approach for optimizing targeted cell deletion in cancer treatment.

  15. Role of microglia in ethanol’s apoptotic action on hypothalamic neuronal cells in primary cultures

    PubMed Central

    Boyadjieva, Nadka I.; Sarkar, Dipak K.

    2010-01-01

    Background Microglia are the major inflammatory cells in the central nervous system and play a role in brain injuries as well as brain diseases. In this study, we determined the role of microglia in ethanol’s apoptotic action on neuronal cells obtained from the mediobasal hypothalamus and maintained in primary cultures. We also tested the effect of cAMP, a signaling molecule critically involved in hypothalamic neuronal survival, on microglia-mediated ethanol’s neurotoxic action. Methods Ethanol’s neurotoxic action was determined on enriched fetal mediobasal hypothalamic neuronal cells with or without microglia cells or ethanol-activated microglia conditioned media. Ethanol’s apoptotic action was determined using nucleosome assay. Microglia activation was determined using OX6 histochemistry and by measuring inflammatory cytokines secretion from microglia in cultures using enzyme-linked immunosorbent assay (ELISA). An immunoneutralization study was conducted to identify the role of a cytokine involved in ethanol’s apoptotic action. Results We show here that ethanol at a dose range of 50 and 100 mM induces neuronal death by an apoptotic process. Ethanol’s ability to induce an apoptotic death of neurons is increased by the presence of ethanol-activated microglia conditioned media. In the presence of ethanol, microglia showed elevated secretion of various inflammatory cytokines, of which TNF-α shows significant apoptotic action on mediobasal hypothalamic neuronal cells. Ethanol’s neurotoxic action was completely prevented by cAMP. The cell-signaling molecule also prevented ethanol-activated microglial production of TNF-α. Immunoneutralization of TNF-α prevented microglia-derived media’s ability to induce neuronal death. Conclusions These results suggest that ethanol’s apoptotic action on hypothalamic neuronal cells might be mediated via microglia, possibly via increased production of TNF-α. Furthermore, cAMP reduces TNF-α production from microglia to prevent ethanol’s neurotoxic action. PMID:20662807

  16. Serum-dependent processing of late apoptotic cells for enhanced efferocytosis.

    PubMed

    Liang, Y Y; Arnold, T; Michlmayr, A; Rainprecht, D; Perticevic, B; Spittler, A; Oehler, R

    2014-01-01

    Binding of the serum protein complement component C1q to the surface of dying cells facilitates their clearance by phagocytes in a process termed efferocytosis. Here, we investigate during which phase of apoptotic cell death progression C1q binding takes place. Purified C1q was found to bind to all dying cells and, albeit weaker, also to viable cells. The presence of serum abrogated completely the binding to viable cells. In addition, C1q binding to dying cells was limited to a specific subpopulation of late apoptotic/secondary necrotic cells. Co-culturing serum-treated apoptotic cells with human monocytes revealed a much higher phagocytosis of C1q-positive than of C1q-negative late apoptotic/secondary necrotic cells. But this phagocytosis-promoting activity could not be observed with purified C1q. Serum-treated C1q-positive late apoptotic/secondary necrotic cells exhibited a similar volume, a similar degraded protein composition, but a much lower DNA content in comparison with the remaining late apoptotic/secondary necrotic cells. This was mediated by a serum-bound nuclease activity that could be abrogated by G-actin, which is a specific inhibitor of serum DNase I. These results show that serum factors are involved in the prevention of C1q binding to viable cells and in the processing of late apoptotic/secondary necrotic cells promoting cell death progression toward apoptotic bodies. This process leads to the exposure of C1q-binding structures and facilitates efferocytosis. PMID:24874736

  17. Ras Homolog Enriched in Brain (Rheb) Enhances Apoptotic Signaling*

    PubMed Central

    Karassek, Sascha; Berghaus, Carsten; Schwarten, Melanie; Goemans, Christoph G.; Ohse, Nadine; Kock, Gerd; Jockers, Katharina; Neumann, Sebastian; Gottfried, Sebastian; Herrmann, Christian; Heumann, Rolf; Stoll, Raphael

    2010-01-01

    Rheb is a homolog of Ras GTPase that regulates cell growth, proliferation, and regeneration via mammalian target of rapamycin (mTOR). Because of the well established potential of activated Ras to promote survival, we sought to investigate the ability of Rheb signaling to phenocopy Ras. We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNF?, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner. Knocking down endogenous Rheb or applying rapamycin led to partial protection, identifying Rheb as a mediator of cell death. Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNF? but did not prevent Rheb-mediated apoptosis. To gain structural insight into the signaling mechanisms, we determined the structure of Rheb-GDP by NMR. The complex adopts the typical canonical fold of RasGTPases and displays the characteristic GDP-dependent picosecond to nanosecond backbone dynamics of the switch I and switch II regions. NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNF?-induced apoptosis and suppressed enhancement by Rheb overexpression. In conclusion, Rheb-mTOR activation not only promotes normal cell growth but also enhances apoptosis in response to diverse toxic stimuli via an ASK-1-mediated mechanism. Pharmacological regulation of the Rheb/mTORC1 pathway using rapamycin should take the presence of cellular stress into consideration, as this may have clinical implications. PMID:20685651

  18. The Paradoxical Pro- and Anti-apoptotic Actions of GSK3 in the Intrinsic and Extrinsic Apoptosis Signaling Pathways

    PubMed Central

    Beurel, Eléonore; Jope, Richard S.

    2006-01-01

    Few things can be considered to be more important to a cell than its threshold for apoptotic cell death, which can be modulated up or down, but rarely in both directions, by a single enzyme. Therefore, it came as quite a surprise to find that one enzyme, glycogen synthase kinase-3 (GSK3), has the perplexing capacity to either increase or decrease the apoptotic threshold. These apparently paradoxical effects now are known to be due to GSK3 oppositely regulating the two major apoptotic signaling pathways. GSK3 promotes cell death caused by the mitochondrial intrinsic apoptotic pathway, but inhibits the death receptor-mediated extrinsic apoptotic signaling pathway. Intrinsic apoptotic signaling, activated by cell damage, is promoted by GSK3 by facilitation of signals that cause disruption of mitochondria and by regulation of transcription factors that control the expression of anti- or pro-apoptotic proteins. The extrinsic apoptotic pathway entails extracellular ligands stimulating cell-surface death receptors that initiate apoptosis by activating caspase-8, and this early step in extrinsic apoptotic signaling is inhibited by GSK3. Thus, GSK3 modulates key steps in each of the two major pathways of apoptosis, but in opposite directions. Consequently, inhibitors of GSK3 provide protection from intrinsic apoptosis signaling but potentiate extrinsic apoptosis signaling. Studies of this eccentric ability of GSK3 to oppositely influence two types of apoptotic signaling have shed light on important regulatory mechanisms in apoptosis and provide the foundation for designing the rational use of GSK3 inhibitors for therapeutic interventions. PMID:16935409

  19. Genetic dissection of c-myc apoptotic pathways.

    PubMed

    Nesbit, C E; Tersak, J M; Grove, L E; Drzal, A; Choi, H; Prochownik, E V

    2000-06-29

    All biological functions mediated by the c-myc oncoprotein require an intact transactivation domain (TAD). We compared TAD mutants for their ability to promote apoptosis of 32D myeloid cells in response to interleukin-3 (IL-3) deprivation and exposure to chemotherapeutic drugs, and to activate ornithine decarboxylase, an endogenous c-myc target. Different sub-regions of the TAD were required to mediate each function. cDNA microarrays were then used to identify multiple c-myc-regulated transcripts, some of which were also modulated by IL-3 or cytotoxic drugs, as well as by specific sub-regions of the TAD. Several of the c-myc-regulated transcripts had also been previously identified as targets for IFN-gamma. The functional consequences of their deregulation were manifested by a marked sensitivity of c-myc-overexpressing cells to IFN-gamma-mediated apoptosis. Our results establish that several well-characterized functions of c-myc are separable and correlate with the expression of a novel group of target genes, some of which also mediate the apoptotic action of IFN-gamma. PMID:10918575

  20. Ulinastatin inhibits oxidant-induced endothelial hyperpermeability and apoptotic signaling

    PubMed Central

    Li, Guicheng; Li, Tao; Li, Yunfeng; Cai, Shumin; Zhang, Zhiming; Zeng, Zhenhua; Wang, Xingmin; Gao, Youguang; Li, Yunfeng; Chen, Zhongqing

    2014-01-01

    Oxidants are important signaling molecules known to increase endothelial permeability. Studies implicate reactive oxygen species (ROS) and the intrinsic apoptotic signaling cascades as mediators of vascular hyperpermeability. Here we report the protective effects of ulinastatin, a serine protease inhibitor with antiapoptotic properties, against oxidant-induced endothelial monolayer hyperpermeability. HUVECs were respectively pretreated with 10,000 and 50,000 u/l ulinastatin, followed by stimulation of 0.6 mM H2O2. Monolayer permeability was determined by transendothelial electrical resistance (TER); Mitochondrial release of cytochrome c was determined by enzyme-linked immunosorbent assay; Caspase-3 activity was measured by fluorometric assay; Adherens junction protein β-catenin was detected by immunofluorescense staining; Ratio of cell apoptosis was evaluated by Annexin-V/PI double stain assay; Mitochondrial membrane potential (Δψm) was determined with JC-1; Intracellular ATP content was assayed by a commercial kit; Bax and Bcl-2 expression were estimated by western blotting; Intracellular reactive oxygen species (ROS) level was measured by DCFH-DA. H2O2 exposure resulted in endothelial hyperpermeability and ROS formation (P < 0.05). The activation of mitochondrial intrinsic apoptotic signaling pathway was evidenced from BAX up-regulation, Bcl-2 down-regulation, mitochondrial depolarization, an increase in cytochrome c release, and activation of caspase-3 (P < 0.05). UTI (50,000 u/l) attenuated endothelial hyperpermeability, ROS formation, mitochondrial dysfunction, cytochrome c release, activation of caspase-3, and disruption of cell adherens junctions (P < 0.05). Together, these results demonstrate that UTI provides protection against vascular hyperpermeability by modulating the intrinsic apoptotic signaling. PMID:25550770

  1. Therapeutic targets in the mitochondrial apoptotic pathway.

    PubMed

    Häcker, Georg; Paschen, Stefan A

    2007-04-01

    Every cell in the human body has most of the components of the apoptotic apparatus and is thus principally equipped to die by apoptosis. Situations of increased or decreased apoptosis contribute to many forms of human disease, making this pathway an attractive target of therapeutic intervention. The past few years have seen an enormous refinement in the understanding how apoptosis works on a molecular level and the role of mitochondria as a central element in apoptotic signal transduction has become obvious. Here, the authors consider the events that are critical in this mitochondrial pathway, in particular at mitochondria but also upstream and downstream. The authors' opinion is presented on the merits and feasibility of approaches that aim at treating disease by interfering with the mitochondrial apoptotic pathway. PMID:17373881

  2. Functional, morphological, and apoptotic alterations in skeletal muscle of ARC deficient mice.

    PubMed

    Mitchell, Andrew S; Smith, Ian C; Gamu, Daniel; Donath, Stefan; Tupling, A Russell; Quadrilatero, Joe

    2015-03-01

    Apoptotic signaling plays an important role in the development and maintenance of healthy skeletal muscle. However, dysregulation of apoptotic signals in skeletal muscle is associated with atrophy and loss of function. Apoptosis repressor with caspase recruitment domain (ARC) is a potent anti-apoptotic protein that is highly expressed in skeletal muscle; however, its role in this tissue has yet to be elucidated. To investigate whether ARC deficiency has morphological, functional, and apoptotic consequences, skeletal muscle from 18 week-old wild-type and ARC knockout (KO) mice was studied. In red muscle (soleus), we found lower maximum tetanic force, as well as a shift towards a greater proportion of type II fibers in ARC KO mice. Furthermore, the soleus of ARC KO mice exhibited lower total, as well as fiber type-specific cross sectional area in type I and IIA fibers. Interestingly, these changes in ARC KO mice corresponded with increased DNA fragmentation, albeit independent of caspase or calpain activation. However, cytosolic fractions of red muscle from ARC KO mice had higher apoptosis inducing factor content, suggesting increased mitochondrial-mediated, caspase-independent apoptotic signaling. This was confirmed in isolated mitochondrial preparations, as mitochondria from skeletal muscle of ARC KO mice were more susceptible to calcium stress. Interestingly, white muscle from ARC KO mice showed no signs of altered apoptotic signaling or detrimental morphological differences. Results from this study suggest that even under basal conditions ARC influences muscle apoptotic signaling, phenotype, and function, particularly in slow and/or oxidative muscle. PMID:25596718

  3. Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells

    PubMed Central

    Yamaguchi, Hiroshi; Maruyama, Toshihiko; Urade, Yoshihiro; Nagata, Shigekazu

    2014-01-01

    Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a calm down signal. DOI: http://dx.doi.org/10.7554/eLife.02172.001 PMID:24668173

  4. Pro-apoptotic Bid is required for the resolution of the effector phase of inflammatory arthritis

    PubMed Central

    Scatizzi, John C; Hutcheson, Jack; Bickel, Emily; Haines, G Kenneth; Perlman, Harris

    2007-01-01

    Rheumatoid arthritis is an autoimmune disease characterized by hyperplasia of the synovial lining and destruction of cartilage and bone. Recent studies have suggested that a lack of apoptosis contributes to the hyperplasia of the synovial lining and to the failure in eliminating autoreactive cells. Mice lacking Fas or Bim, two pro-apoptotic proteins that mediate the extrinsic and intrinsic death cascades, respectively, develop enhanced K/BxN serum transfer-induced arthritis. Since the pro-apoptotic protein Bid functions as an intermediate between the extrinsic and intrinsic apoptotic pathways, we examined the role that it plays in inflammatory arthritis. Mice deficient in Bid (Bid-/-) show a delay in the resolution of K/BxN serum transfer-induced arthritis. Bid-/- mice display increased inflammation, bone destruction, and pannus formation compared to wild-type mice. Furthermore, Bid-/- mice have elevated levels of CXC chemokine and IL-1? in serum, which are associated with more inflammatory cells throughout the arthritic joint. In addition, there are fewer apoptotic cells in the synovium of Bid-/- compared to Wt mice. These data suggest that extrinsic and intrinsic apoptotic pathways cooperate through Bid to limit development of inflammatory arthritis. PMID:17509138

  5. Human intrahepatic biliary epithelial cells engulf blebs from their apoptotic peers

    PubMed Central

    Rong, G-H; Yang, G-X; Ando, Y; Zhang, W; He, X-S; Leung, P S C; Coppel, R L; Ansari, A A; Zhong, R; Gershwin, M E

    2013-01-01

    The phagocytic clearance of apoptotic cells is critical for tissue homeostasis; a number of non-professional phagocytic cells, including epithelial cells, can both take up and process apoptotic bodies, including the release of anti-inflammatory mediators. These observations are particularly important in the case of human intrahepatic biliary cells (HiBEC), because such cells are themselves a target of destruction in primary biliary cirrhosis, the human autoimmune disease. To address the apoptotic ability of HiBECs, we have focused on their ability to phagocytize apoptotic blebs from autologous HiBECs. In this study we report that HiBEC cells demonstrate phagocytic function from autologous HiBEC peers accompanied by up-regulation of the chemokines CCL2 [monocyte chemotactic protein-1 (MCP-1)] and CXCL8 [interleukin (IL)-8]. In particular, HiBEC cells express the phagocytosis-related receptor phosphatidylserine receptors (PSR), implying that HiBECs function through the eat-me signal phosphatidylserine expressed by apoptotic cells. Indeed, although HiBEC cells acquire antigen-presenting cell (APC) function, they do not change the expression of classic APC function surface markers after engulfment of blebs, both with and without the presence of Toll-like receptor (TLR) stimulation. These results are important not only for understanding of the normal physiological function of HiBECs, but also explain the inflammatory potential and reduced clearance of HiBEC cells following the inflammatory cascade in primary biliary cirrhosis. PMID:23480189

  6. Degradation of nuclear matrix and DNA cleavage in apoptotic thymocytes.

    PubMed

    Weaver, V M; Carson, C E; Walker, P R; Chaly, N; Lach, B; Raymond, Y; Brown, D L; Sikorska, M

    1996-01-01

    In dexamethasone-treated thymocyte cultures an increase in nuclear proteolytic activity paralleled chromatin fragmentation and the appearance of small apoptotic cells. The elevation of nuclear proteolytic activity was accompanied by site-specific degradation of nuclear mitotic apparatus protein and lamin B, two essential components of the nuclear matrix. Nuclear mitotic apparatus protein phosphorylation and cleavage into 200 and 48 kDa fragments occurred within 30 minutes of dexamethasone treatment. Cleavage of lamin B, which generated a fragment of 46 kDa consistent with the central rod domain of the protein, was also detected after 30 minutes of exposure to the steroid hormone. The level of lamin B phosphorylation did not change as a result of the dexamethasone treatment and the lamina did not solubilize until the later stages of apoptosis. Initial DNA breaks, detected by the terminal transferase-mediated dUTP-biotin nick end labeling assay, occurred throughout the nuclei and solubilization of lamina was not required for this process to commence. The data presented in this paper support a model of apoptotic nuclear destruction brought about by the site-specific proteolysis of key structural proteins. Both the nuclear mitotic apparatus protein and lamin B were specifically targeted by protease(s) at early stages of the cell death pathway, which possibly initiate the cascade of degradative events in apoptosis. PMID:8834789

  7. Cisplatin-induced apoptotic cell death in Mongolian gerbil cochlea.

    PubMed

    Alam, S A; Ikeda, K; Oshima, T; Suzuki, M; Kawase, T; Kikuchi, T; Takasaka, T

    2000-03-01

    Cisplatin is well known to cause cochleotoxicity. In order to determine the underlying mechanisms of cisplatin-induced cell death in the cochlea, we investigated the apoptotic changes and the expression of bcl-2 family proteins controlling apoptosis. Mongolian gerbils were administered 4 mg/kg/day cisplatin consecutively for 5 days. The cisplatin-treated animals showed a significant deterioration in the responses of both distortion product otoacoustic emissions and the endocochlear potential as compared with those of the age-matched controls, suggesting outer hair cell and stria vascularis dysfunction. The presence of DNA fragmentation revealed by a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling method was recognized in the organ of Corti, the spiral ganglion, and the stria vascularis in the cisplatin-treated animals whereas almost negative results were obtained in the control animals. The nuclear morphology obtained by Hoechst 33342 staining revealed pyknotic and condensed nuclei, confirming the presence of the characteristic features of apoptosis. A significant increase and reduction in the number of bax- and bcl-2-positive cells, respectively, following cisplatin treatment was observed in the cells of the organ of Corti, the spiral ganglion, and the lateral wall. These findings suggest a critical role for bcl-2 family proteins in the regulation of apoptotic cell death induced by cisplatin. The underlying mechanisms of the cisplatin-induced cell death are discussed. PMID:10713493

  8. SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

    SciTech Connect

    Herbert, Katharine J.; Cook, Anthony L. Snow, Elizabeth T.

    2014-06-15

    Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment. - Highlights: • Impaired microRNA biogenesis promotes apoptotic resistance in HaCaT keratinocytes. • TP53 mutations suppress miR-34a-mediated regulation of SIRT1 expression. • SIRT1 inhibition increases p53 acetylation in HaCaTs, restoring apoptosis.

  9. Apoptotic mimicry by an obligate intracellular parasite downregulates macrophage microbicidal activity.

    PubMed

    de Freitas Balanco, J M; Moreira, M E; Bonomo, A; Bozza, P T; Amarante-Mendes, G; Pirmez, C; Barcinski, M A

    2001-11-27

    Programmed cell death by apoptosis of unnecessary or potentially harmful cells is clearly beneficial to multicellular organisms. Proper functioning of such a program demands that the removal of dying cells proceed without an inflammatory reaction. Phosphatidylserine (PS) is one of the ligands displayed by apoptotic cells that participates in their noninflammatory removal when recognized by neighboring phagocytes. PS ligation induces the release of transforming growth factor-beta (TGF-beta), an antiinflammatory cytokine that mediates the suppression of macrophage-mediated inflammation. In Hydra vulgaris, an organism that stands at the base of metazoan evolution, the selective advantage provided by apoptosis lies in the fact that Hydra can survive recycling apoptotic cells by phagocytosis. In unicellular organisms, it has been proposed that altruistic death benefits clonal populations of yeasts and trypanosomatids. Now we show that advantageous features of the apoptotic process can operate without death as the necessary outcome. Leishmania spp are able to evade the killing activity of phagocytes and establish themselves as obligate intracellular parasites. Amastigotes, responsible for disease propagation, similar to apoptotic cells, inhibit macrophage activity by exposing PS. Exposed PS participates in amastigote internalization. Recognition of this moiety by macrophages induces TGF-beta secretion and IL-10 synthesis, inhibits NO production, and increases susceptibility to intracellular leishmanial growth. PMID:11728310

  10. Delivery of Intracellular-acting Biologics in Pro-Apoptotic Therapies

    PubMed Central

    Li, Hongmei; Nelson, Chris E.; Evans, Brian C.; Duvall, Craig L.

    2013-01-01

    The recent elucidation of molecular regulators of apoptosis and their roles in cellular oncogenesis has motivated the development of biomacromolecular anticancer therapeutics that can activate intracellular apoptotic signaling pathways. Pharmaceutical scientists have employed a variety of classes of biologics toward this goal, including antisense oligodeoxynucleotides, small interfering RNA, proteins, antibodies, and peptides. However, stability in the in vivo environment, tumor-specific biodistribution, cell internalization, and localization to the intracellular microenvironment where the targeted molecule is localized pose significant challenges that limit the ability to directly apply intracellular-acting, pro-apoptotic biologics for therapeutic use. Thus, approaches to improve the pharmaceutical properties of therapeutic biomacromolecules are of great significance and have included chemically modifying the bioactive molecule itself or formulation with auxiliary compounds. Recently, promising advances in delivery of pro-apoptotic biomacromolecular agents have been made using tools such as peptide stapling, cell penetrating peptides, fusogenic peptides, liposomes, nanoparticles, smart polymers, and synergistic combinations of these components. This review will discuss the molecular mediators of cellular apoptosis, the respective mechanisms by which these mediators are dysregulated in cellular oncogenesis, the history and development of both nucleic-acid and amino-acid based drugs, and techniques to achieve intracellular delivery of these biologics. Finally, recent applications where pro-apoptotic functionality has been achieved through delivery of intracellular-acting biomacromolecular drugs will be highlighted. PMID:21348831

  11. Skeletal muscle stem cells express anti-apoptotic ErbB receptors during activation from quiescence

    SciTech Connect

    Golding, Jon P. . E-mail: j.p.golding@open.ac.uk; Calderbank, Emma; Partridge, Terence A.; Beauchamp, Jonathan R.

    2007-01-15

    To be effective for tissue repair, satellite cells (the stem cells of adult muscle) must survive the initial activation from quiescence. Using an in vitro model of satellite cell activation, we show that erbB1, erbB2 and erbB3, members of the EGF receptor tyrosine kinase family, appear on satellite cells within 6 h of activation. We show that signalling via erbB2 provides an anti-apoptotic survival mechanism for satellite cells during the first 24 h, as they progress to a proliferative state. Inhibition of erbB2 signalling with AG825 reduced satellite cell numbers, concomitant with elevated caspase-8 activation and TUNEL labelling of apoptotic satellite cells. In serum-free conditions, satellite cell apoptosis could be largely prevented by a mixture of erbB1, erbB3 and erbB4 ligand growth factors, but not by neuregulin alone (erbB3/erbB4 ligand). Furthermore, using inhibitors specific to discrete intracellular signalling pathways, we identify MEK as a pro-apoptotic mediator, and the erbB-regulated factor STAT3 as an anti-apoptotic mediator during satellite cell activation. These results implicate erbB2 signalling in the preservation of a full compliment of satellite cells as they activate in the context of a damaged muscle.

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

    NASA Astrophysics Data System (ADS)

    Liu, Lei; Zhang, Yingjie; Wang, Xianwang

    2009-02-01

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

  13. Apoptotic hair cell death after transient cochlear ischemia in gerbils.

    PubMed

    Taniguchi, Masafumi; Hakuba, Nobuhiro; Koga, Kenichiro; Watanabe, Futoshi; Hyodo, Jun; Gyo, Kiyofumi

    2002-12-20

    The mechanisms of cochlear hair cell death following exposure to transient inner ear ischemia were investigated in gerbils histologically. The animals were subjected to ischemic insult by occluding both vertebral arteries for 15 min. Hoechst 33342 nuclear staining showed that inner hair cells (IHCs) underwent sporadic degeneration via nuclear condensation, which peaked 12 hours after the ischemia. Furthermore, nuclear DNA fragmentation was noted by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling method. Transmission electron microscopy revealed morphological changes in the IHCs characteristic of apoptosis, including karyopyknosis, chromatin condensation. These findings suggest that apoptotic cell death is the major process in hair cell degeneration in this animal model. PMID:12499849

  14. Apoptotic osteocytes and the control of targeted bone resorption.

    PubMed

    Plotkin, Lilian I

    2014-03-01

    Studies from the 1950s and 1960s already recognize the fact that osteocytes, although long living cells, die, as evidenced by accumulation of osteocytic lacunae devoid of cells. More recently, it was demonstrated that these cells die by apoptosis. The rate of osteocyte apoptosis is regulated by the age of the bone, as well as by systemic hormones, local growth factors, cytokines, pharmacological agents, and mechanical forces. Apoptotic osteocytes, in turn, recruit osteoclasts to initiate targeted bone resorption. This results in the removal of "dead" bone and may improve the mechanical properties of the skeleton. However, the molecular regulators of osteocyte survival and targeted bone remodeling are not completely known. In this review, the current knowledge on the molecular mechanism that lead to osteocyte death or survival, and the signals that mediate targeted bone resorption is discussed. PMID:24470254

  15. The N-Terminus of CD14 Acts to Bind Apoptotic Cells and Confers Rapid-Tethering Capabilities on Non-Myeloid Cells

    PubMed Central

    Thomas, Leanne; Bielemeier, Anne; Lambert, Peter A.; Darveau, Richard P.; Marshall, Lindsay J.; Devitt, Andrew

    2013-01-01

    Cell death and removal of cell corpses in a timely manner is a key event in both physiological and pathological situations including tissue homeostasis and the resolution of inflammation. Phagocytic clearance of cells dying by apoptosis is a complex sequential process comprising attraction, recognition, tethering, signalling and ultimately phagocytosis and degradation of cell corpses. A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within this process. The role of myeloid cell CD14 in mediating apoptotic cell interactions with macrophages has long been known though key molecules and residues involved have not been defined. Here we sought to further dissect the function of CD14 in apoptotic cell clearance. A novel panel of THP-1 cell-derived phagocytes was employed to demonstrate that CD14 mediates effective apoptotic cell interactions with macrophages in the absence of detectable TLR4 whilst binding and responsiveness to LPS requires TLR4. Using a targeted series of CD14 point mutants expressed in non-myeloid cells we reveal CD14 residue 11 as key in the binding of apoptotic cells whilst other residues are reported as key for LPS binding. Importantly we note that expression of CD14 in non-myeloid cells confers the ability to bind rapidly to apoptotic cells. Analysis of a panel of epithelial cells reveals that a number naturally express CD14 and that this is competent to mediate apoptotic cell clearance. Taken together these data suggest that CD14 relies on residue 11 for apoptotic cell tethering and it may be an important tethering molecule on so called non-professional phagocytes thus contributing to apoptotic cell clearance in a non-myeloid setting. Furthermore these data establish CD14 as a rapid-acting tethering molecule, expressed in monocytes, which may thus confer responsiveness of circulating monocytes to apoptotic cell derived material. PMID:23936239

  16. Macrophage ADAM17 Deficiency Augments CD36-Dependent Apoptotic Cell Uptake and the Linked Anti-Inflammatory Phenotype

    PubMed Central

    Driscoll, Will S.; Vaisar, Tomas; Tang, Jingjing; Wilson, Carole L.; Raines, Elaine W.

    2013-01-01

    Rationale Apoptotic cell phagocytosis (efferocytosis) is mediated by specific receptors and is essential for resolution of inflammation. In chronic inflammation, apoptotic cell clearance is dysfunctional and soluble levels of several apoptotic cell receptors are elevated. Reports have identified proteolytic cleavage as a mechanism capable of releasing soluble apoptotic cell receptors, but the functional implications of their proteolysis are unclear. Objective To test the hypothesis that ADAM17-mediated cleavage of apoptotic cell receptors limits efferocytosis in vivo. Methods and Results In vivo comparison of macrophage efferocytosis in wildtype and Adam17-null hematopoietic chimeras demonstrates that ADAM17 deficiency leads to a 60% increase in efferocytosis and an enhanced anti-inflammatory phenotype in a model of peritonitis. In vitro uptake of phosphatidylserine liposomes identifies the dual-pass apoptotic cell receptor CD36 as a major contributor to enhanced efferocytosis, and CD36 surface levels are elevated on macrophages from Adam17-null mice. Further, temporal elevation of CD36 expression with inflammation may also contribute to its impact. Soluble CD36 from macrophage-conditioned media is comprised of two species based on Western blotting, and mass spectrometry identifies three N-terminal peptides, which represent probable cleavage sites. Levels of soluble CD36 are decreased in Adam17-null conditioned media, providing evidence for involvement of ADAM17 in CD36 cleavage. Importantly, enhanced efferocytosis in vivo by macrophages lacking ADAM17 is CD36 dependent and accelerates macrophage clearance from the peritoneum, thus promoting resolution of inflammation and highlighting the impact of increased apoptotic cell uptake. Conclusions Our studies demonstrate the importance of ADAM17-mediated proteolysis for in vivo efferocytosis regulation, and suggest a possible mechanistic link between chronic inflammation and defective efferocytosis. PMID:23584255

  17. Stabilization of apoptotic cells: generation of zombie cells

    PubMed Central

    Oropesa-Ávila, M; Andrade-Talavera, Y; Garrido-Maraver, J; Cordero, M D; de la Mata, M; Cotán, D; Paz, M V; Pavón, A D; Alcocer-Gómez, E; de Lavera, I; Lema, R; Zaderenko, A P; Rodríguez-Moreno, A; Sánchez-Alcázar, J A

    2014-01-01

    Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn2+ (a caspase inhibitor) and coenzyme Q10 (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy. PMID:25118929

  18. Stabilization of apoptotic cells: generation of zombie cells.

    PubMed

    Oropesa-Ávila, M; Andrade-Talavera, Y; Garrido-Maraver, J; Cordero, M D; de la Mata, M; Cotán, D; Paz, M V; Pavón, A D; Alcocer-Gómez, E; de Lavera, I; Lema, R; Zaderenko, A P; Rodríguez-Moreno, A; Sánchez-Alcázar, J A

    2014-01-01

    Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn(2+) (a caspase inhibitor) and coenzyme Q10 (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy. PMID:25118929

  19. Apoptotic Sphingolipid Ceramide in Cancer Therapy

    PubMed Central

    Huang, Wei-Ching; Chen, Chia-Ling; Lin, Yee-Shin; Lin, Chiou-Feng

    2011-01-01

    Apoptosis, also called programmed cell death, is physiologically and pathologically involved in cellular homeostasis. Escape of apoptotic signaling is a critical strategy commonly used for cancer tumorigenesis. Ceramide, a derivative of sphingolipid breakdown products, acts as second messenger for multiple extracellular stimuli including growth factors, chemical agents, and environmental stresses, such as hypoxia, and heat stress as well as irradiation. Also, ceramide acts as tumor-suppressor lipid because a variety of stress stimuli cause apoptosis by increasing intracellular ceramide to initiate apoptotic signaling. Defects on ceramide generation and sphingolipid metabolism are developed for cancer cell survival and cancer therapy resistance. Alternatively, targeting ceramide metabolism to correct these defects might provide opportunities to overcome cancer therapy resistance. PMID:21490804

  20. Apoptotic Cells Can Deliver Chemotherapeutics to Engulfing Macrophages and Suppress Inflammatory Cytokine Production*

    PubMed Central

    Perez, Beatriz; Paquette, Nicholas; Padassi, Helena; Zhai, Bo; White, Kristin; Skvirsky, Rachel; Lacy-Hulbert, Adam; Stuart, Lynda M.

    2012-01-01

    Immunosuppression via cell-cell contact with apoptotic cells is a well studied immunological phenomenon. Although the original studies of immune repression used primary cells, which undergo spontaneous cell death or apoptosis in response to irradiation, more recent studies have relied on chemotherapeutic agents to induce apoptosis in cell lines. In this work, we demonstrate that Jurkat cells induced to die with actinomycin D suppressed inflammatory cytokine production by macrophages, whereas cells treated with etoposide did not. This immune repression mediated by actinomycin D-treated cells did not require phagocytosis or cell-cell contact and thus occurs through a different mechanism from that seen with primary apoptotic neutrophils. Moreover, cells induced to die with etoposide and then treated for a short time with actinomycin D also suppressed macrophage responses, indicating that suppression was mediated by actinomycin D independent of the mechanism of cell death. Finally, phagocytosis of actinomycin D-treated cells caused apoptosis in macrophages, and suppression could be blocked by inhibition of caspase activity in the target macrophage. Together, these data indicate that apoptotic cells act as Trojan horses, delivering actinomycin D to engulfing macrophages. Suppression of cytokine production by macrophages is therefore due to exposure to actinomycin D from apoptotic cells and is not the result of cell-receptor interactions. These data suggest that drug-induced death may not be an appropriate surrogate for the immunosuppressive activity of apoptotic cells. Furthermore, these effects of cytotoxic drugs on infiltrating immune phagocytes may have clinical ramifications for their use as antitumor therapies. PMID:22433861

  1. Variability in apoptotic response to poliovirus infection.

    PubMed

    Romanova, Lyudmila I; Belov, George A; Lidsky, Peter V; Tolskaya, Elena A; Kolesnikova, Marina S; Evstafieva, Alexandra G; Vartapetian, Andrey B; Egger, Denise; Bienz, Kurt; Agol, Vadim I

    2005-01-20

    In several cell types, poliovirus activates the apoptotic program, implementation of which is suppressed by viral antiapoptotic functions. In such cells, productive infection leads to a necrotic cytopathic effect (CPE), while abortive reproduction, associated with inadequate viral antiapoptotic functions, results in apoptosis. Here, we describe two other types of cell response to poliovirus infection. Murine L20B cells expressing human poliovirus receptor responded to the infection by both CPE and apoptosis concurrently. Interruption of productive infection decreased rather than increased the proportion of apoptotic cells. Productive infection was accompanied by the early efflux of cytochrome c from the mitochondria in a proportion of cells and by activation of DEVD-specific caspases. Inactivation of caspase-9 resulted in a marked, but incomplete, prevention of the apoptotic response of these cells to viral infection. Thus, the poliovirus-triggered apoptotic program in L20B cells was not completely suppressed by the viral antiapoptotic functions. In contrast, human rhabdomyosarcoma RD cells did not develop appreciable apoptosis during productive or abortive infection, exhibiting inefficient efflux of cytochrome c from mitochondria and no marked activation of DEVD-specific caspases. The cells were also refractory to several nonviral apoptosis inducers. Nevertheless, typical caspase-dependent signs of apoptosis in a proportion of RD cells were observed after cessation of viral reproduction. Such "late" apoptosis was also observed in productively infected HeLa cells. In addition, a tiny proportion of all studied cells were TUNEL positive even in the presence of a caspase inhibitor. Degradation of DNA in such cells appeared to be a postmortem phenomenon. Biological relevance of variable host responses to viral infection is discussed. PMID:15629772

  2. PPAR? activation following apoptotic cell instillation promotes resolution of lung inflammation and fibrosis via regulation of efferocytosis and proresolving cytokines.

    PubMed

    Yoon, Y-S; Kim, S-Y; Kim, M-J; Lim, J-H; Cho, M-S; Kang, J L

    2015-09-01

    Changes in macrophage phenotype have been implicated in apoptotic cell-mediated immune modulation via induction of peroxisome proliferator-activated receptor-? (PPAR?). In this study, we characterized PPAR? induction by apoptotic cell instillation over the course of bleomycin-induced lung injury in C57BL/6 mice. Next, the role of PPAR? activation in resolving lung inflammation and fibrosis was investigated. Our data demonstrate that apoptotic cell instillation after bleomycin results in immediate and prolonged enhancement of PPAR? mRNA and protein in alveolar macrophages and lung. Moreover, PPAR? activity and expression of its target molecules, including CD36, macrophage mannose receptor, and arginase 1, were persistently enhanced following apoptotic cell instillation. Coadministration of the PPAR? antagonist, GW9662, reversed the enhanced efferocytosis, and the reduced proinflammatory cytokine expression, neutrophil recruitment, myeloperoxidase activity, hydroxyproline contents, and fibrosis markers, including type 1 collagen ?2, fibronectin and ?-smooth muscle actin (?-SMA), in the lung by apoptotic cell instillation. In addition, inhibition of PPAR? activity reversed the expression of transforming growth factor-? (TGF-?), interleukin (IL)-10, and hepatocyte growth factor (HGF). These findings indicate that one-time apoptotic cell instillation contributes to anti-inflammatory and antifibrotic responses via upregulation of PPAR? expression and subsequent activation, leading to regulation of efferocytosis and production of proresolving cytokines. PMID:25586556

  3. PPARγ activation following apoptotic cell instillation promotes resolution of lung inflammation and fibrosis via regulation of efferocytosis and proresolving cytokines

    PubMed Central

    Yoon, Y-S; Kim, S-Y; Kim, M-J; Lim, J-H; Cho, M-S; Kang, J L

    2015-01-01

    Changes in macrophage phenotype have been implicated in apoptotic cell-mediated immune modulation via induction of peroxisome proliferator-activated receptor-γ (PPARγ). In this study, we characterized PPARγ induction by apoptotic cell instillation over the course of bleomycin-induced lung injury in C57BL/6 mice. Next, the role of PPARγ activation in resolving lung inflammation and fibrosis was investigated. Our data demonstrate that apoptotic cell instillation after bleomycin results in immediate and prolonged enhancement of PPARγ mRNA and protein in alveolar macrophages and lung. Moreover, PPARγ activity and expression of its target molecules, including CD36, macrophage mannose receptor, and arginase 1, were persistently enhanced following apoptotic cell instillation. Coadministration of the PPARγ antagonist, GW9662, reversed the enhanced efferocytosis, and the reduced proinflammatory cytokine expression, neutrophil recruitment, myeloperoxidase activity, hydroxyproline contents, and fibrosis markers, including type 1 collagen α2, fibronectin and α-smooth muscle actin (α-SMA), in the lung by apoptotic cell instillation. In addition, inhibition of PPARγ activity reversed the expression of transforming growth factor-β (TGF-β), interleukin (IL)-10, and hepatocyte growth factor (HGF). These findings indicate that one-time apoptotic cell instillation contributes to anti-inflammatory and antifibrotic responses via upregulation of PPARγ expression and subsequent activation, leading to regulation of efferocytosis and production of proresolving cytokines. PMID:25586556

  4. Bcl-2 Inhibitors: Targeting Mitochondrial Apoptotic Pathways in Cancer Therapy

    PubMed Central

    Kang, Min H.; Reynolds, C. Patrick

    2010-01-01

    Defects in apoptotic pathways can promote cancer cell survival and also confer resistance to antineoplastic drugs. One pathway being targeted for antineoplastic therapy is the anti-apoptotic B-cell lymphoma-2 (Bcl-2) family of proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl1/A-1, and Bcl-B) that bind to and inactivate BH3-domain pro-apoptotic proteins. Signals transmitted by cellular damage (including antineoplastic drugs) or cytokine deprivation can initiate apoptosis via the intrinsic apoptotic pathway. It is controversial whether some BH3-domain proteins (Bim or tBid) directly activate multidomain pro-apoptotic proteins (e.g., Bax and Bak) or act via inhibition of those anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl1/A-1, and Bcl-B) that stabilize pro-apoptotic proteins. Overexpression of anti-apoptotic Bcl-2 family members has been associated with chemotherapy resistance in various human cancers, and preclinical studies have shown that agents targeting anti-apoptotic Bcl-2 family members have preclinical activity as single agents and in combination with other antineoplastic agents. Clinical trials of several investigational drugs targeting the Bcl-2 family (oblimersen sodium, AT-101, ABT-263, GX15-070) are ongoing. Here, we review the role of the Bcl-2 family in apoptotic pathways and those agents that are known and/or designed to inhibit the anti-apoptotic Bcl-2 family of proteins. PMID:19228717

  5. Improvement of DC vaccine with ALA-PDT induced immunogenic apoptotic cells for skin squamous cell carcinoma

    PubMed Central

    Zhou, Feifan; Wang, Xiaojie; Shi, Lei; Zhang, Haiyan; Wang, Peiru; Yang, Degang; Zhang, Linglin; Chen, Wei R.; Wang, Xiuli

    2015-01-01

    Dendritic cell (DC) based vaccines have emerged as a promising immunotherapy for cancers. However, most DC vaccines so far have achieved only limited success in cancer treatment. Photodynamic therapy (PDT), an established cancer treatment strategy, can cause immunogenic apoptosis to induce an effective antitumor immune response. In this study, we developed a DC-based cancer vaccine using immunogenic apoptotic tumor cells induced by 5-aminolevulinic acid (ALA) mediated PDT. The maturation of DCs induced by PDT-treated apoptotic cells was evaluated using electron microscopy, FACS, and ELISA. The anti-tumor immunity of ALA-PDT-DC vaccine was tested with a mouse model. We observed the maturations of DCs potentiated by ALA-PDT treated tumor cells, including morphology maturation (enlargement of dendrites and increase of lysosomes), phenotypic maturation (upregulation of surface expression of MHC-II, DC80, and CD86), and functional maturation (enhanced capability to secrete IFN-γ and IL-12, and to induce T cell proliferation). Most interestingly, PDT-induced apoptotic tumor cells are more capable of potentiating maturation of DCs than PDT-treated or freeze/thaw treated necrotic tumor cells. ALA-PDT-DC vaccine mediated by apoptotic cells provided protection against tumors in mice, far stronger than that of DC vaccine obtained from freeze/thaw treated tumor cells. Our results indicate that immunogenic apoptotic tumor cells can be more effective in enhancing a DC-based cancer vaccine, which could improve the clinical application of PDT-DC vaccines. PMID:25915530

  6. Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis

    PubMed Central

    Oropesa-vila, M; Fernndez-Vega, A; de la Mata, M; Maraver, J G; Cordero, M D; Cotn, D; de Miguel, M; Calero, C P; Paz, M V; Pavn, A D; Snchez, M A; Zaderenko, A P; Ybot-Gonzlez, P; Snchez-Alczar, J A

    2013-01-01

    Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as ?-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit ?4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na+/Ca2+ exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na+/K+ pump subunit ? was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis. PMID:23470534

  7. Angiotensin II-induced p53-dependent cardiac apoptotic cell death: its prevention by metallothionein.

    PubMed

    Liu, Qiuju; Wang, Guanjun; Zhou, Guihua; Tan, Yi; Wang, Xiuli; Wei, Wei; Liu, Lucheng; Xue, Wanli; Feng, Wenke; Cai, Lu

    2009-12-15

    Apoptotic cell death was found to play a critical role in the development of diabetic cardiomyopathy. As one of pathogenic components of diabetes angiotensin II (Ang II) induced cardiac cell death in vitro and in vivo through induction of reactive oxygen and nitrogen species. However, Ang II-induced cell death signaling in the heart remains unclear. The present study was to investigate whether Ang II induces p53 expression and activation and if so, whether Ang II-induced cardiac cell death is p53-dependent, and whether a potent antioxidant metallothionein (MT) prevents Ang II-induced p53 expression, and associate apoptotic cell death signaling. A cardiac cell line (H9c2) was exposed to Ang II. We found that exposure of H9c2 cells to Ang II at 10, 50 and 100 nM for 24 h induced a significant apoptotic effect, measured by DNA fragmentation and cleaved caspase-3. Induction of apoptotic cell death by Ang II can be completely blocked by p53 inhibitor Pitithrin-alpha. Exposure of H9c2 cells to Ang II also significantly increased p53 phosphorylation, DNA double strand breaks and Bax/Bcl-2 ratio. All these effects were not observed in H9c2MT7 cells that forcedly overexpresses human MT-IIA gene, suggesting the preventive effect of antioxidant MT against Ang II-induced p53 activation and its apoptotic death signaling. Furthermore, the in vitro finding was validated in animal models by supplying Ang II to wild-type mice (WT) and MT-TG mice that has cardiac-specifically overexpressed MT gene. Ang II-induced significant up-regulation of p53 expression along with an increase in Bax/Bcl-2 ratio in the hearts of WT mice, but not MT-TG mice. These results suggest that Ang II-induced cardiac apoptotic cell death is mediated by p53 apoptotic signaling pathway, which is related to oxidative stress. Antioxidant MT can completely prevent Ang II-induced p53 activation and associated apoptotic effect in the heart. PMID:19808082

  8. The Apoptotic Pathway as a Therapeutic Target in Sepsis

    PubMed Central

    Wesche-Soldato, Doreen E.; Swan, Ryan Z.; Chung, Chun-Shiang; Ayala, Alfred

    2006-01-01

    Recent research has yielded many interesting and potentially important therapeutic targets in sepsis. Specifically, the effects of antagonistic anti-cytokine therapies (tumor necrosis factor-alpha [TNF-α], interleukin-1 [IL-1]) and anti-endotoxin strategies utilizing antibodies against endotoxin or endotoxin receptor/carrier molecules (anti-CD14 or anti-LPS-binding protein) have been studied. Unfortunately, these approaches often failed clinically, and in many cases, the efficacy of these treatments was dependent on the severity of sepsis. Recently, clinical trials using insulin to lock blood glucose levels and activated protein C treatment have showed that while they provided some survival benefit, their efficacy does not appear to be predicated solely upon anti-inflammatory effects. Here, we will review work done in animal models of polymicrobial sepsis and clinical findings that support the hypothesis that apoptosis in the immune system is a pathologic event in sepsis that can be a therapeutic target. In this respect, experimental studies looking at the septic animal suggest that loss of lymphocytes during sepsis may be due to dysregulated apoptosis and that this appears to be brought on by a variety of mediators effecting ‘intrinsic’ as well as ‘extrinsic’ cell death pathways. From a therapeutic perspective this has provided a number of novel targets for clinically successful current, as well as future therapies, such as caspases (caspase inhibition/protease inhibition), pro-apoptotic protein-expression (via administration and/or over-expression of Bcl-2) and the death receptor family Fas-FasL (via. FasFP [fas fusion protein] and the application of siRNA against a number pro-apoptotic factors). PMID:17430119

  9. Multiple pathways regulating the anti-apoptotic protein clusterin in breast cancer

    PubMed Central

    Ranney, Melissa K.; Ahmed, Ikhlas S.A.; Potts, Kelly R.; Craven, Rolf J.

    2012-01-01

    Cancer chemotherapy inhibits tumor growth, in part, by triggering apoptosis, and anti-apoptotic proteins reduce the effectiveness of chemotherapy. Clusterin, a chaperone-like protein that binds to apoptotic and DNA repair proteins, is induced by chemotherapy and promotes tumor cell survival. Histone deacetylase inhibitors (HDIs) such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA) are pharmacological agents that induce differentiation and apoptosis in cancer cells by altering chromatin structure, and we have found that combinations of chemotherapeutic drugs such as doxorubicin and HDIs efficiently induce apoptosis, even though they paradoxically induce high levels of clusterin. The hyper-expressed form of clusterin localizes to mitochondria, inhibits cytochrome c release, and is inhibited by the proteasome. When HDIs are used as single agents, clusterin suppresses cytochrome c release and apoptosis. However, doxorubicin/HDI-induced apoptosis is not inhibited by clusterin, and clusterin-resistant apoptosis corresponds with markers of the extrinsic/receptor-mediated apoptotic pathway. Thus, chemotherapy-HDI combinations are capable of overcoming an innate anti-apoptotic pathway of tumor cells, suggesting that chemotherapy-HDI combinations have potential for treating advanced stage breast cancer. PMID:17689225

  10. Boolean Model of Yeast Apoptosis as a Tool to Study Yeast and Human Apoptotic Regulations

    PubMed Central

    Kazemzadeh, Laleh; Cvijovic, Marija; Petranovic, Dina

    2012-01-01

    Programmed cell death (PCD) is an essential cellular mechanism that is evolutionary conserved, mediated through various pathways and acts by integrating different stimuli. Many diseases such as neurodegenerative diseases and cancers are found to be caused by, or associated with, regulations in the cell death pathways. Yeast Saccharomyces cerevisiae, is a unicellular eukaryotic organism that shares with human cells components and pathways of the PCD and is therefore used as a model organism. Boolean modeling is becoming promising approach to capture qualitative behavior and describe essential properties of such complex networks. Here we present large literature-based and to our knowledge first Boolean model that combines pathways leading to apoptosis (a type of PCD) in yeast. Analysis of the yeast model confirmed experimental findings of anti-apoptotic role of Bir1p and pro-apoptotic role of Stm1p and revealed activation of the stress protein kinase Hog proposing the maximal level of activation upon heat stress. In addition we extended the yeast model and created an in silico humanized yeast in which human pro- and anti-apoptotic regulators Bcl-2 family and Valosin-contain protein (VCP) are included in the model. We showed that accumulation of Bax in silico humanized yeast shows apoptotic markers and that VCP is essential target of Akt Signaling. The presented Boolean model provides comprehensive description of yeast apoptosis network behavior. Extended model of humanized yeast gives new insights of how complex human disease like neurodegeneration can initially be tested. PMID:23233838

  11. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

    SciTech Connect

    Lee, Dae-Hee; Kim, Dong-Wook; Jung, Chang-Hwa; Lee, Yong J.; Park, Daeho

    2014-09-15

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway.

  12. Biophysical investigation of the apoptotic force

    NASA Astrophysics Data System (ADS)

    Toyama, Yusuke; Peralta, Xomalin; Wells, Adrienne; Kiehart, Daniel; Edwards, Glenn

    2009-03-01

    Understanding tissue dynamics during development requires knowledge of how cells produce and respond to forces. We have experimentally shown that apoptosis (programmed cell death, which remodels tissue by eliminating cells) also contributes a significant tissue force that promotes cell sheet fusion during dorsal closure in Drosophila development [Science, 321, 1683 (2008)]. By genetically suppressing (enhancing) apoptosis, we slow (increase) the rate of dorsal closure. These changes correlate with the forces produced by the amnioserosa tissue and the rate of seam formation (zipping) for two advancing sheets of lateral epidermis. This apoptotic force is used to drive cell sheet movements during development, a role not classically attributed to apoptosis.

  13. GSIV serine/threonine kinase can induce apoptotic cell death via p53 and pro-apoptotic gene Bax upregulation in fish cells.

    PubMed

    Reshi, Latif; Wu, Horng-Cherng; Wu, Jen-Leih; Wang, Hao-Ven; Hong, Jiann-Ruey

    2016-04-01

    Previous studies have shown that GSIV induces apoptotic cell death through upregulation of the pro-apoptotic genes Bax and Bak in Grouper fin cells (GF-1 cells). However, the role of viral genome-encoded protein(s) in this death process remains unknown. In this study, we demonstrated that the Giant seaperch iridovirus (GSIV) genome encoded a serine/threonine kinase (ST kinase) protein, and induced apoptotic cell death via a p53-mediated Bax upregulation approach and a downregulation of Bcl-2 in fish cells. The ST kinase expression profile was identified through Western blot analyses, which indicated that expression started at day 1 h post-infection (PI), increased up to day 3, and then decreased by day 5 PI. This profile indicated the role of ST kinase expression during the early and middle phases of viral replication. We then cloned the ST kinase gene and tested its function in fish cells. The ST kinase was transiently expressed and used to investigate possible novel protein functions. The transient expression of ST kinase in GF-1 cells resulted in apoptotic cell features, as revealed with Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) assays and Hoechst 33258 staining at 24 h (37 %) and 48 h post-transfection (PT) (49 %). Then, through studies on the mechanism of cell death, we found that ST kinase overexpression could upregulate the anti-stress gene p53 and the pro-apoptotic gene Bax at 48 h PT. Interestingly, this upregulation of p53 and Bax also correlated to alterations in the mitochondria function that induced loss of mitochondrial membrane potential (MMP) and activated the initiator caspase-9 and the effector caspase-3 in the downstream. Moreover, when the p53-dependent transcriptional downstream gene was blocked by a specific transcriptional inhibitor, it was found that pifithrin-α not only reduced Bax expression, but also averted cell death in GF-1 cells during the ST kinase overexpression. Taken altogether, these results suggested that aquatic GSIV ST kinase could induce apoptosis via upregulation of p53 and Bax expression, resulting in mitochondrial disruption, which activated a downstream caspases-mediated cell death pathway. PMID:26833308

  14. Increased membrane permeability of apoptotic thymocytes: a flow cytometric study.

    PubMed

    Ormerod, M G; Sun, X M; Snowden, R T; Davies, R; Fearnhead, H; Cohen, G M

    1993-01-01

    We have recently developed a method for the separation and quantification of viable apoptotic cells without the need for permeabilisation or fixation of the cells. The method is based on the observation that apoptotic rat thymocytes fluoresce more brightly than normal cells after a brief incubation with the DNA binding dye, Hoechst 33342. In order to understand these differences, we have investigated the uptake of Hoechst 33342 by normal and apoptotic thymocytes. By staining with fluorescein diacetate, we have shown that the efflux of fluorescein from apoptotic cells is more rapid than that from normal thymocytes. This result demonstrated an increase in the permeability of the plasma membrane of the apoptotic thymocytes and it is this change which probably results in the more rapid uptake of Hoechst 33342. The data also revealed two populations of apoptotic thymocytes. PMID:8404365

  15. Organization of the Mitochondrial Apoptotic BAK Pore

    PubMed Central

    Aluvila, Sreevidya; Mandal, Tirtha; Hustedt, Eric; Fajer, Peter; Choe, Jun Yong; Oh, Kyoung Joon

    2014-01-01

    The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices ?1 and ?6 disengage from the rest of the domain, leaving helices ?2-?5 as a folded unit. Helices ?2-?5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX BH3-in-groove homodimer. Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of ?3 and ?5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a worm hole. PMID:24337568

  16. Pro-apoptotic NOXA is implicated in atmospheric-pressure plasma-induced melanoma cell death

    NASA Astrophysics Data System (ADS)

    Ishaq, M.; Bazaka, K.; Ostrikov, K.

    2015-11-01

    Atmospheric-pressure plasma (APP) has been successfully used to treat several types of cancers in vivo and in vitro, with the effect being primarily attributed to the generation of reactive oxygen species (ROS). However, the mechanisms by which APP induces apoptosis in cancer cells require further elucidation. In this study, the effects of APP on the expression of 500 genes in melanoma Mel007 cancer cells were examined. Pro-apoptotic phorbol-12-myristate-13-acetate-induced protein (PMAIP1), also known as NOXA, was highly expressed as a result of APP treatment in a dose-dependent manner. Blocking of ROS using scavenger NAC or silencing of NOXA gene by RNA interference inhibited the APP-induced NOXA genes upregulation and impaired caspases 3/7 mediated apoptosis, confirming the important role plasma-generated ROS species and pro-apoptotic NOXA play in APP-induced cancer cell death.

  17. Pseudorabies virus glycoprotein gE triggers ERK1/2 phosphorylation and degradation of the pro-apoptotic protein Bim in epithelial cells.

    PubMed

    Pontes, Maria S; Van Waesberghe, Cliff; Nauwynck, Hans; Verhasselt, Bruno; Favoreel, Herman W

    2016-02-01

    ERK1/2 (Extracellular signal Regulated Kinase 1/2) signaling is a key cellular signaling axis controlling many cellular events, including cell survival. Activation of ERK 1/2 may trigger an anti-apoptotic response, and different viruses have been shown to benefit from this process. We have described recently that the viral glycoprotein gE mediates pseudorabies virus (PRV)-induced activation of ERK 1/2 in T lymphocytes. In the present study, we report that PRV gE-mediated ERK 1/2 phosphorylation also occurs in epithelial cells and that in these cells, gE-mediated ERK 1/2 signaling is associated with degradation of the pro-apoptotic protein Bim. Our results for the first time link the viral glycoprotein gE, an important alphaherpesvirus virulence factor, with the apoptotic signaling pathway. PMID:26721325

  18. Leptin is an anti-apoptotic effector in placental cells involving p53 downregulation.

    PubMed

    Toro, Ayelén Rayen; Maymó, Julieta Lorena; Ibarbalz, Federico Matías; Pérez-Pérez, Antonio; Maskin, Bernardo; Faletti, Alicia Graciela; Sánchez-Margalet, Víctor; Varone, Cecilia Laura

    2014-01-01

    Leptin, a peripheral signal synthetized by the adipocyte to regulate energy metabolism, can also be produced by placenta, where it may work as an autocrine hormone. We have previously demonstrated that leptin promotes proliferation and survival of trophoblastic cells. In the present work, we aimed to study the molecular mechanisms that mediate the survival effect of leptin in placenta. We used the human placenta choriocarcinoma BeWo and first trimester Swan-71 cell lines, as well as human placental explants. We tested the late phase of apoptosis, triggered by serum deprivation, by studying the activation of Caspase-3 and DNA fragmentation. Recombinant human leptin added to BeWo cell line and human placental explants, showed a decrease on Caspase-3 activation. These effects were dose dependent. Maximal effect was achieved at 250 ng leptin/ml. Moreover, inhibition of endogenous leptin expression with 2 µM of an antisense oligonucleotide, reversed Caspase-3 diminution. We also found that the cleavage of Poly [ADP-ribose] polymerase-1 (PARP-1) was diminished in the presence of leptin. We analyzed the presence of low DNA fragments, products from apoptotic DNA cleavage. Placental explants cultivated in the absence of serum in the culture media increased the apoptotic cleavage of DNA and this effect was prevented by the addition of 100 ng leptin/ml. Taken together these results reinforce the survival effect exerted by leptin on placental cells. To improve the understanding of leptin mechanism in regulating the process of apoptosis we determined the expression of different intermediaries in the apoptosis cascade. We found that under serum deprivation conditions, leptin increased the anti-apoptotic BCL-2 protein expression, while downregulated the pro-apoptotic BAX and BID proteins expression in Swan-71 cells and placental explants. In both models leptin augmented BCL-2/BAX ratio. Moreover we have demonstrated that p53, one of the key cell cycle-signaling proteins, is downregulated in the presence of leptin under serum deprivation. On the other hand, we determined that leptin reduced the phosphorylation of Ser-46 p53 that plays a pivotal role for apoptotic signaling by p53. Our data suggest that the observed anti-apoptotic effect of leptin in placenta is in part mediated by the p53 pathway. In conclusion, we provide evidence that demonstrates that leptin is a trophic factor for trophoblastic cells. PMID:24922063

  19. Antcin B and its ester derivative from Antrodia camphorata induce apoptosis in hepatocellular carcinoma cells involves enhancing oxidative stress coincident with activation of intrinsic and extrinsic apoptotic pathway.

    PubMed

    Hsieh, Yun-Chih; Rao, Yerra Koteswara; Whang-Peng, Jacqueline; Huang, Chi-Ying F; Shyue, Song-Kun; Hsu, Shih-Lan; Tzeng, Yew-Min

    2011-10-26

    The triterpenoids methylantcinate B (MAB) and antcin B (AB), isolated from the medicinal mushroom Antrodia camphorata , have been identified as strong cytotoxic agents against various type of cancer cells; however, the mechanisms of MAB- and AB-induced cytotoxicity have not been adequately explored. This study investigated the roles of caspase cascades, reactive oxygen species (ROS), DNA damage, mitochondrial disruption, and Bax and Bcl-2 proteins in MAB- and AB-induced apoptosis of hepatocellular carcinoma (HCC) HepG2 cells. Here, we showed that MAB and AB induced apoptosis in HepG2 cells, as characterized by increased DNA fragmentation, cleavage of PARP, sub-G1 population, chromatin condensation, loss of mitochondrial membrane potential, and release of cytochrome c. Increasing the levels of caspase-2, -3, -8, and -9 activities was involved in MAB- and AB-induced apoptosis, and they could be attenuated by inhibitors of specific caspases, indicating that MAB and AB triggered the caspase-dependent apoptotic pathway. Additionally, the enhanced apoptotic effect correlates with high expression of Fas, Fas ligand, as well as Bax and decreased protein levels of Bcl-(XL) and Bcl-2, suggesting that both the extrinsic and intrinsic apoptosis pathways were involved in the apoptotic processes. Incubation of HepG2 cells with antioxidant enzymes superoxide dismutase and catalase and antioxidants N-acetylcysteine and ascorbic acid attenuated the ROS generation and apoptosis induced by MAB and AB, which indicate that ROS plays a pivotal role in cell death. NADPH oxidase activation was observed in MAB- and AB-stimulated HepG2 cells; however, inhibition of such activation by diphenylamine significantly blocked MAB- and AB-induced ROS production and increased cell viability. Taken together, our results provide the first evidence that triterpenoids MAB and AB induced a NADPH oxidase-provoked oxidative stress and extrinsic and intrinsic apoptosis as a critical mechanism of cause cell death in HCC cells. PMID:21916504

  20. Proteomic analysis for the anti-apoptotic effects of cystamine on apoptosis-prone macrophage.

    PubMed

    Kao, Shao-Hsuan; Hsu, Tsai-Ching; Yu, Jau-Song; Chen, Jeng-Ting; Li, Sin-Lun; Lai, Wen-Xian; Tzang, Bor-Show

    2010-06-01

    Increased macrophage vulnerability is associated with progression of systemic lupus erythematosus. Our previous studies have shown that cystamine, an inhibitor of transglutaminase 2 (TG2), alleviated the apoptosis of hepatocyte and brain cell in lupus-prone mice NZB/W-F1. In present study, we further investigated the effects of cystamine on apoptosis-prone macrophages (APMs) in the lupus mice. Using two-dimensional gel electrophoresis (2-DE) analysis, we found that cystamine induced a differential protein expression pattern of APM as comparing to the PBS control. The protein spots presenting differential level between cystamine and PBS treatment were then identified by peptide-mass fingerprinting (PMF). After bioinformatic analysis, these identified proteins were found involved in mitochondrial apoptotic pathway, oxidative stress, and mitogen-activated protein (MAP) kinase-mediated pathway. Further investigation revealed that cystamine significantly decreased the levels of apoptotic Bax and Apaf-1 and the activity of caspase-3, and increased the levels of anti-apoptotic Bcl-2 in APM. We also found that these apoptotic mediators were up-regulated in a correlation with the progression of lupus severity in NZB/W-F1, which were little affected in BALB/c mice. We also found that the reduced serum glutathione was restored by cystamine in NZB/W-F1. Interestingly, the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in APM and the phagocytic ability was diminished in presence of cystamine. In conclusion, our findings indicate that cystamine significantly inhibited mitochondrial pathway, induced antioxidant proteins, and diminished phosphorylation of extracellular ERK1/2, which may alleviate the apoptosis and the phagocytic ability of APM. PMID:20512926

  1. Abnormal Production of Pro- and Anti-Inflammatory Cytokines by Lupus Monocytes in Response to Apoptotic Cells

    PubMed Central

    Sule, Sangeeta; Rosen, Antony; Petri, Michelle; Akhter, Ehtisham; Andrade, Felipe

    2011-01-01

    Monocytes are a key component of the innate immune system involved in the regulation of the adaptive immune response. Previous studies have focused on apoptotic cell clearance abnormalities in systemic lupus erythematosus (SLE) monocytes. However, whether SLE monocytes might express unique patterns of cytokine secretion in response to apoptotic cells is still unknown. Here, we used monocytes from healthy controls and SLE patients to evaluate the production of TNF-? and TGF-? in response to apoptotic cells. Upon recognition of apoptotic material, monocytes from healthy controls showed prominent TGF-? secretion (mean SD: 824.6144.3 pg/ml) and minimal TNF-? production (mean SD: 32.62.1 pg/ml). In contrast, monocytes from SLE patients had prominent TNF-? production (mean SD: 302.2337.5 pg/ml) and diminished TGF-? secretion (mean SD: 685.9615.9 pg/ml), a difference that was statistically significant compared to normal monocytes (p?10?6 for TNF-? secretion, and p?=?0.0031 for TGF-?, respectively). Interestingly, the unique cytokine response by SLE monocytes was independent of their phagocytic clearance efficiency, opsonizing autoantibodies and disease activity. We further showed that nucleic acids from apoptotic cells play important role in the induction of TNF-? by lupus monocytes. Together, these observations suggest that, in addition to potential clearance defects, monocytes from SLE patients have an abnormal balance in the secretion of anti- and pro-inflammatory cytokines in response to apoptotic cells. Since the abnormal cytokine response to apoptotic material in SLE is not related to disease activity and opsonizing autoantibodies, it is possible that this response might be an intrinsic property of lupus monocytes. The studies focus attention on toll-like receptors (TLRs) and their downstream pathways as mediators of this response. PMID:21423726

  2. Protective effects of melittin on transforming growth factor-{beta}1 injury to hepatocytes via anti-apoptotic mechanism

    SciTech Connect

    Lee, Woo-Ram; Park, Ji-Hyun; Kim, Kyung-Hyun; Park, Yoon-Yub; Han, Sang-Mi; Park, Kwan-kyu

    2011-10-15

    Melittin is a cationic, hemolytic peptide that is the main toxic component in the venom of the honey bee (Apis mellifera). Melittin has multiple effects, including anti-bacterial, anti-viral and anti-inflammatory, in various cell types. However, the anti-apoptotic mechanisms of melittin have not been fully elucidated in hepatocytes. Apoptosis contributes to liver inflammation and fibrosis. Knowledge of the apoptotic mechanisms is important to develop new and effective therapies for treatment of cirrhosis, portal hypertension, liver cancer, and other liver diseases. In the present study, we investigated the anti-apoptotic effect of melittin on transforming growth factor (TGF)-{beta}1-induced apoptosis in hepatocytes. TGF-{beta}1-treated hepatocytes were exposed to low doses (0.5 and 1 {mu}g/mL) and high dose (2 {mu}g/mL) of melittin. The low doses significantly protected these cells from DNA damage in TGF-{beta}1-induced apoptosis compared to the high dose. Also, melittin suppressed TGF-{beta}1-induced apoptotic activation of the Bcl-2 family and caspase family of proteins, which resulted in the inhibition of poly-ADP-ribose polymerase (PARP) cleavage. These results demonstrate that TGF-{beta}1 induces hepatocyte apoptosis and that an optimal dose of melittin exerts anti-apoptotic effects against TGF-{beta}1-induced injury to hepatocytes via the mitochondrial pathway. These results suggest that an optimal dose of melittin can serve to protect cells against TGF-{beta}1-mediated injury. - Highlights: > We investigated the anti-apoptotic effect of melittin on TGF-{beta}1-induced hepatocyte. > TGF-{beta}1 induces hepatocyte apoptosis. > TGF-{beta}1-treated hepatocytes were exposed to low doses and high dose of melittin. > Optimal dose of melittin exerts anti-apoptotic effects to hepatocytes.

  3. Poncirin Induces Apoptosis in AGS Human Gastric Cancer Cells through Extrinsic Apoptotic Pathway by up-Regulation of Fas Ligand

    PubMed Central

    Venkatarame Gowda Saralamma, Venu; Nagappan, Arulkumar; Hong, Gyeong Eun; Lee, Ho Jeong; Yumnam, Silvia; Raha, Suchismita; Heo, Jeong Doo; Lee, Sang Joon; Lee, Won Sup; Kim, Eun Hee; Kim, Gon Sup

    2015-01-01

    Poncirin, a natural bitter flavanone glycoside abundantly present in many species of citrus fruits, has various biological benefits such as anti-oxidant, anti-microbial, anti-inflammatory and anti-cancer activities. The anti-cancer mechanism of Poncirin remains elusive to date. In this study, we investigated the anti-cancer effects of Poncirin in AGS human gastric cancer cells (gastric adenocarcinoma). The results revealed that Poncirin could inhibit the proliferation of AGS cells in a dose-dependent manner. It was observed Poncirin induced accumulation of sub-G1 DNA content, apoptotic cell population, apoptotic bodies, chromatin condensation, and DNA fragmentation in a dose-dependent manner in AGS cells. The expression of Fas Ligand (FasL) protein was up-regulated dose dependently in Poncirin-treated AGS cells Moreover, Poncirin in AGS cells induced activation of Caspase-8 and -3, and subsequent cleavage of poly(ADP-ribose) polymerase (PARP). Inhibitor studies’ results confirm that the induction of caspase-dependent apoptotic cell death in Poncirin-treated AGS cells was led by the Fas death receptor. Interestingly, Poncirin did not show any effect on mitochondrial membrane potential (ΔΨm), pro-apoptotic proteins (Bax and Bak) and anti-apoptotic protein (Bcl-xL) in AGS-treated cells followed by no activation in the mitochondrial apoptotic protein caspase-9. This result suggests that the mitochondrial-mediated pathway is not involved in Poncirin-induced cell death in gastric cancer. These findings suggest that Poncirin has a potential anti-cancer effect via extrinsic pathway-mediated apoptosis, possibly making it a strong therapeutic agent for human gastric cancer. PMID:26393583

  4. Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells.

    PubMed

    Tth, Beta; Garabuczi, Eva; Sarang, Zsolt; Vereb, Gyrgy; Vmosi, Gyrgy; Aeschlimann, Daniel; Blask, Bernadett; Bcsi, Blint; Erddi, Ferenc; Lacy-Hulbert, Adam; Zhang, Ailiang; Falasca, Laura; Birge, Raymond B; Balajthy, Zoltn; Melino, Gerry; Fss, Lszl; Szondy, Zsuzsa

    2009-02-15

    Transglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin beta(3). We have previously shown that TG2(-/-) mice develop an age-dependent autoimmunity due to defective in vivo clearance of apoptotic cells. Here we report that TG2 on the cell surface and in guanine nucleotide-bound form promotes phagocytosis. Besides being a binding partner for integrin beta(3), a receptor known to mediate the uptake of apoptotic cells via activating Rac1, we also show that TG2 binds MFG-E8 (milk fat globulin EGF factor 8), a protein known to bridge integrin beta(3) to apoptotic cells. Finally, we report that in wild-type macrophages one or two engulfing portals are formed during phagocytosis of apoptotic cells that are characterized by accumulation of integrin beta(3) and Rac1. In the absence of TG2, integrin beta(3) cannot properly recognize the apoptotic cells, is not accumulated in the phagocytic cup, and its signaling is impaired. As a result, the formation of the engulfing portals, as well as the portals formed, is much less efficient. We propose that TG2 has a novel function to stabilize efficient phagocytic portals. PMID:19201861

  5. Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

    PubMed Central

    Hesketh, Emily E.; Dransfield, Ian; Kluth, David C.; Hughes, Jeremy

    2015-01-01

    Autoimmunity is associated with defective phagocytic clearance of apoptotic cells. IgM deficient mice exhibit an autoimmune phenotype consistent with a role for circulating IgM antibodies in apoptotic cell clearance. We have extensively characterised IgM binding to non-apoptotic and apoptotic mouse thymocytes and human Jurkat cells using flow cytometry, confocal imaging and electron microscopy. We demonstrate strong specific IgM binding to a subset of Annexin-V (AnnV)+PI (Propidium Iodide)+ apoptotic cells with disrupted cell membranes. Electron microscopy studies indicated that IgM+AnnV+PI+ apoptotic cells exhibited morphologically advanced apoptosis with marked plasma membrane disruption compared to IgM-AnnV+PI+ apoptotic cells, suggesting that access to intracellular epitopes is required for IgM to bind. Strong and comparable binding of IgM to permeabilised non-apoptotic and apoptotic cells suggests that IgM bound epitopes are 'apoptosis independent' such that IgM may bind any cell with profound disruption of cell plasma membrane integrity. In addition, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognize and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption. The role of IgM in vivo in conditions characterized by severe cell damage such as ischemic injury, sepsis and thrombotic microangiopathies merits further exploration. PMID:26121639

  6. Genes of the mitochondrial apoptotic pathway in Mytilus galloprovincialis.

    PubMed

    Estévez-Calvar, Noelia; Romero, Alejandro; Figueras, Antonio; Novoa, Beatriz

    2013-01-01

    Bivalves play vital roles in marine, brackish, freshwater and terrestrial habitats. In recent years, these ecosystems have become affected through anthropogenic activities. The ecological success of marine bivalves is based on the ability to modify their physiological functions in response to environmental changes. One of the most important mechanisms involved in adaptive responses to environmental and biological stresses is apoptosis, which has been scarcely studied in mollusks, although the final consequence of this process, DNA fragmentation, has been frequently used for pollution monitoring. Environmental stressors induce apoptosis in molluscan cells via an intrinsic pathway. Many of the proteins involved in vertebrate apoptosis have been recognized in model invertebrates; however, this process might not be universally conserved. Mytilus galloprovincialis is presented here as a new model to study the linkage between molecular mechanisms that mediate apoptosis and marine bivalve ecological adaptations. Therefore, it is strictly necessary to identify the key elements involved in bivalve apoptosis. In the present study, six mitochondrial apoptotic-related genes were characterized, and their gene expression profiles following UV irradiation were evaluated. This is the first step for the development of potential biomarkers to assess the biological responses of marine organisms to stress. The results confirmed that apoptosis and, more specifically, the expression of the genes involved in this process can be used to assess the biological responses of marine organisms to stress. PMID:23626691

  7. Genes of the Mitochondrial Apoptotic Pathway in Mytilus galloprovincialis

    PubMed Central

    Figueras, Antonio; Novoa, Beatriz

    2013-01-01

    Bivalves play vital roles in marine, brackish, freshwater and terrestrial habitats. In recent years, these ecosystems have become affected through anthropogenic activities. The ecological success of marine bivalves is based on the ability to modify their physiological functions in response to environmental changes. One of the most important mechanisms involved in adaptive responses to environmental and biological stresses is apoptosis, which has been scarcely studied in mollusks, although the final consequence of this process, DNA fragmentation, has been frequently used for pollution monitoring. Environmental stressors induce apoptosis in molluscan cells via an intrinsic pathway. Many of the proteins involved in vertebrate apoptosis have been recognized in model invertebrates; however, this process might not be universally conserved. Mytilus galloprovincialis is presented here as a new model to study the linkage between molecular mechanisms that mediate apoptosis and marine bivalve ecological adaptations. Therefore, it is strictly necessary to identify the key elements involved in bivalve apoptosis. In the present study, six mitochondrial apoptotic-related genes were characterized, and their gene expression profiles following UV irradiation were evaluated. This is the first step for the development of potential biomarkers to assess the biological responses of marine organisms to stress. The results confirmed that apoptosis and, more specifically, the expression of the genes involved in this process can be used to assess the biological responses of marine organisms to stress. PMID:23626691

  8. Transglutaminase 2 Promotes Both Caspase-dependent and Caspase-independent Apoptotic Cell Death via the Calpain/Bax Protein Signaling Pathway*

    PubMed Central

    Yoo, Je-Ok; Lim, Young-Cheol; Kim, Young-Myeong; Ha, Kwon-Soo

    2012-01-01

    Transglutaminase 2 (TG2) is a versatile protein that is implicated in significant biological processes, including cell death and degenerative diseases. A possible role of TG2 in the apoptotic death of cancer cells induced by photodynamic therapy (PDT) was suggested recently; however, the mechanism by which TG2 regulates apoptotic responses to PDT remains to be elucidated. In this study, we investigated the key signaling pathways stimulated during apoptotic cell death following PDT and whether inhibition of TG2 activation using pharmacological approaches and siRNAs affects the signaling pathways. PDT caused the release of both cytochrome c and apoptosis-inducing factor (AIF) by damaging mitochondria, which resulted in caspase-dependent and caspase-independent apoptotic cell death, respectively. Released AIF translocated to the nucleus and, synergistically with the caspase-dependent pathway, led to apoptotic cell death. Both the caspase cascade and the activation of AIF following PDT were mediated by TG2 activation. In addition, PDT-activated calpain was responsible for the sequential events of Bax translocation, the collapse of ??m, caspase-3 activation, and AIF translocation, all of which were provoked by TG2 activation. Together, these results demonstrate that PDT with a chlorin-based photosensitizer targets TG2 by activating calpain-induced Bax translocation, which induces apoptotic cell death through both caspase-dependent and AIF-mediated pathways. Moreover, these results indicate that TG2 may be a possible therapeutic target for PDT treatment of cancer. PMID:22418443

  9. The regulation of apoptotic cell death.

    PubMed

    Amarante-Mendes, G P; Green, D R

    1999-09-01

    Apoptosis is a fundamental biological phenomenon in which the death of a cell is genetically and biochemically regulated. Different molecules are involved in the regulation of the apoptotic process. Death receptors, coupled to distinct members of the caspases as well as other adapter molecules, are involved in the initiation of the stress signals (The Indictment). Members of the Bcl-2 family control at the mitochondrial level the decision between life and death (The Judgement). The effector caspases are responsible for all morphological and biochemical changes related to apoptosis including the "eat-me" signals perceived by phagocytes and neighboring cells (The Execution). Finally, apoptosis would have little biological significance without the recognition and removal of the dying cells (The Burial). PMID:10464379

  10. The Anti-Apoptotic Role of Neuroglobin

    PubMed Central

    Brittain, Thomas

    2012-01-01

    The small heme-protein neuroglobin is expressed at high concentrations in certain brain neurons and in the rod cells of the retina. This paper reviews the many studies which have recently identified a protective role for neuroglobin, in a wide range of situations involving apoptotic cell death. The origins of this protective mechanism are discussed in terms of both experimental results and computational modeling of the intrinsic pathway of apoptosis, which shows that neuroglobin can intervene in this process by a reaction with released mitochondrial cytochrome c. An integrated model, based on the various molecular actions of both neuroglobin and cytochrome c, is developed, which accounts for the cellular distribution of neuroglobin. PMID:24710547

  11. Structure and apoptotic function of p73

    PubMed Central

    Yoon, Mi-Kyung; Ha, Ji-Hyang; Lee, Min-Sung; Chi, Seung-Wook

    2015-01-01

    p73 is a structural and functional homologue of the p53 tumor suppressor protein. Like p53, p73 induces apoptosis and cell cycle arrest and transactivates p53-responsive genes, conferring its tumor suppressive activity. In addition, p73 has unique roles in neuronal development and differentiation. The importance of p73-induced apoptosis lies in its capability to substitute the pro-apoptotic activity of p53 in various human cancer cells in which p53 is mutated or inactive. Despite the great importance of p73-induced apoptosis in cancer therapy, little is known about the molecular basis of p73-induced apoptosis. In this review, we discuss the p73 structures reported to date, detailed structural comparisons between p73 and p53, and current understanding of the transcription-dependent and -independent mechanisms of p73-induced apoptosis. [BMB Reports 2015; 48(2): 81-90] PMID:25441426

  12. Apoptotic cell-treated dendritic cells induce immune tolerance by specifically inhibiting development of CD4(+) effector memory T cells.

    PubMed

    Zhou, Fang; Zhang, Guang-Xian; Rostami, Abdolmohamad

    2016-02-01

    CD4(+) memory T cells play an important role in induction of autoimmunity and chronic inflammatory responses; however, regulatory mechanisms of CD4(+) memory T cell-mediated inflammatory responses are poorly understood. Here we show that apoptotic cell-treated dendritic cells inhibit development and differentiation of CD4(+) effector memory T cells in vitro and in vivo. Simultaneously, intravenous transfer of apoptotic T cell-induced tolerogenic dendritic cells can block development of experimental autoimmune encephalomyelitis (EAE), an inflammatory disease of the central nervous system in C57 BL/6J mouse. Our results imply that it is effector memory CD4(+) T cells, not central memory CD4(+) T cells, which play a major role in chronic inflammatory responses in mice with EAE. Intravenous transfer of tolerogenic dendritic cells induced by apoptotic T cells leads to immune tolerance by specifically blocking development of CD4(+) effector memory T cells compared with results of EAE control mice. These results reveal a new mechanism of apoptotic cell-treated dendritic cell-mediated immune tolerance in vivo. PMID:26111522

  13. Human Cells Display Reduced Apoptotic Function Relative to Chimpanzee Cells

    PubMed Central

    McDonald, John F.

    2012-01-01

    Previously published gene expression analyses suggested that apoptotic function may be reduced in humans relative to chimpanzees and led to the hypothesis that this difference may contribute to the relatively larger size of the human brain and the increased propensity of humans to develop cancer. In this study, we sought to further test the hypothesis that humans maintain a reduced apoptotic function relative to chimpanzees by conducting a series of apoptotic function assays on human, chimpanzee and macaque primary fibroblastic cells. Human cells consistently displayed significantly reduced apoptotic function relative to the chimpanzee and macaque cells. These results are consistent with earlier findings indicating that apoptotic function is reduced in humans relative to chimpanzees. PMID:23029431

  14. WNT signaling controls expression of pro-apoptotic BOK and BAX in intestinal cancer

    SciTech Connect

    Zeilstra, Jurrit; Joosten, Sander P.J.; Wensveen, Felix M.; Dessing, Mark C.; Schuetze, Denise M.; Eldering, Eric; Spaargaren, Marcel; Pals, Steven T.

    2011-03-04

    Research highlights: {yields} Intestinal adenomas initiated by aberrant activation of the WNT pathway displayed an increased sensitivity to apoptosis. {yields} Expression profiling of apoptosis-related genes in Apc{sup Min/+} mice revealed the differential expression of pro-apoptotic Bok and Bax. {yields} APC-mutant adenomatous crypts in FAP patients showed strongly increased BAX immunoreactivity. {yields} Blocking of {beta}-catenin/TCF-4-mediated signaling in colon cancer cells reduced the expression of BOK and BAX. -- Abstract: In a majority of cases, colorectal cancer is initiated by aberrant activation of the WNT signaling pathway. Mutation of the genes encoding the WNT signaling components adenomatous polyposis coli or {beta}-catenin causes constitutively active {beta}-catenin/TCF-mediated transcription, driving the transformation of intestinal crypts to cancer precursor lesions, called dysplastic aberrant crypt foci. Deregulated apoptosis is a hallmark of adenomatous colon tissue. However, the contribution of WNT signaling to this process is not fully understood. We addressed this role by analyzing the rate of epithelial apoptosis in aberrant crypts and adenomas of the Apc{sup Min/+} mouse model. In comparison with normal crypts and adenomas, aberrant crypts displayed a dramatically increased rate of apoptotic cell death. Expression profiling of apoptosis-related genes along the crypt-villus axis and in Apc mutant adenomas revealed increased expression of two pro-apoptotic Bcl-2 family members in intestinal adenomas, Bok and Bax. Analysis of the colon of familial adenomatous polyposis (FAP) patients along the crypt-to-surface axis, and of dysplastic crypts, corroborated this expression pattern. Disruption of {beta}-catenin/TCF-4-mediated signaling in the colorectal cancer cell line Ls174T significantly decreased BOK and BAX expression, confirming WNT-dependent regulation in intestinal epithelial cells. Our results suggest a feedback mechanism by which uncontrolled epithelial cell proliferation in the stem cell compartment can be counterbalanced by an increased propensity to undergo cell death.

  15. Unc93b Induces Apoptotic Cell Death and Is Cleaved by Host and Enteroviral Proteases

    PubMed Central

    Harris, Katharine G.; Coyne, Carolyn B.

    2015-01-01

    Unc93b is an endoplasmic reticulum (ER)-resident transmembrane protein that serves to bind and traffic toll-like receptors (TLRs) from the ER to their appropriate subcellular locations for ligand sensing. Because of its role in TLR trafficking, Unc93b is necessary for an effective innate immune response to coxsackievirus B3 (CVB), a positive-sense single stranded RNA virus belonging to the enterovirus family. Here, we show that Unc93b is cleaved by a CVB-encoded cysteine protease (3Cpro) during viral replication. Further, we define a role for Unc93b in the induction of apoptotic cell death and show that expression of wild-type Unc93b, but not a mutant incapable of binding TLRs or exiting the ER (H412R), induces apoptosis. Furthermore, we show that cellular caspases activated during apoptosis directly cleave Unc93b. Interestingly, we show that the 3Cpro- and caspase-mediated cleavage of Unc93b both occur within ten amino acids in the distal N-terminus of Unc93b. Mechanistically, neither caspase-mediated nor 3Cpro-mediated cleavage of Unc93b altered its trafficking function, inhibited its role in facilitating TLR3 or TLR8 signaling, or altered its apoptosis-inducing effects. Taken together, our studies show that Unc93b is targeted by both viral- and host cell-specific proteases and identify a function of Unc93b in the induction of apoptotic cell death. PMID:26509685

  16. Ceramide metabolism regulates autophagy and apoptotic cell death induced by melatonin in liver cancer cells.

    PubMed

    Ordoez, Raquel; Fernndez, Anna; Prieto-Domnguez, Nstor; Martnez, Laura; Garca-Ruiz, Carmen; Fernndez-Checa, Jos C; Mauriz, Jos L; Gonzlez-Gallego, Javier

    2015-09-01

    Autophagy is a process that maintains homeostasis during stress, although it also contributes to cell death under specific contexts. Ceramides have emerged as important effectors in the regulation of autophagy, mediating the crosstalk with apoptosis. Melatonin induces apoptosis of cancer cells; however, its role in autophagy and ceramide metabolism has yet to be clearly elucidated. This study was aimed to evaluate the effect of melatonin administration on autophagy and ceramide metabolism and its possible link with melatonin-induced apoptotic cell death in hepatocarcinoma (HCC) cells. Melatonin (2 mm) transiently induced autophagy in HepG2 cells through JNK phosphorylation, characterized by increased Beclin-1 expression, p62 degradation, and LC3II and LAMP-2 colocalization, which translated in decreased cell viability. Moreover, ATG5 silencing sensitized HepG2 cells to melatonin-induced apoptosis, suggesting a dual role of autophagy in cell death. Melatonin enhanced ceramide levels through both de novo synthesis and acid sphingomyelinase (ASMase) stimulation. Serine palmitoyltransferase (SPT) inhibition with myriocin prevented melatonin-induced autophagy and ASMase inhibition with imipramine-impaired autophagy flux. However, ASMase inhibition partially protected HepG2 cells against melatonin, while SPT inhibition significantly enhanced cell death. Findings suggest a crosstalk between SPT-mediated ceramide generation and autophagy in protecting against melatonin, while specific ASMase-induced ceramide production participates in melatonin-mediated cell death. Thus, dual blocking of SPT and autophagy emerges as a potential strategy to potentiate the apoptotic effects of melatonin in liver cancer cells. PMID:25975536

  17. Apoptotic cell responses in the splenic marginal zone: a paradigm for immunologic reactions to apoptotic antigens with implications for autoimmunity.

    PubMed

    McGaha, Tracy L; Karlsson, Mikael C I

    2016-01-01

    Apoptotic cells drive innate regulatory responses that result in tolerogenic immunity. This is a critical aspect of cell physiology as apoptotic cells expose potentially dangerous nuclear antigens on the surface in apoptotic blebs, and failure in their recognition, phagocytosis, or destruction can cause dramatic autoimmunity in experimental models and is linked to development and progression of systemic pathology in human. The marginal zone is a specialized splenic environment that serves as a transitional site from circulation to peripheral lymphoid structures. The marginal zone serves a key role in trapping of particulates and initiation of innate responses against systemic microbial pathogens. However in recent years, it has become clear the marginal zone is also important for initiation of immune tolerance to apoptotic cells, driving a coordinated response involving multiple phagocyte and lymphocyte subsets. Recent reports linking defects in splenic macrophage function to systemic lupus erythematosus in a manner analogous to marginal zone macrophages in lupus-prone mice provide an impetus to better understand the mechanistic basis of the apoptotic cell response in the marginal zone and its general applicability to apoptotic cell-driven tolerance at other tissue sites. In this review, we discuss immune responses to apoptotic cells in the spleen in general and the marginal zone in particular, the relationship of these responses to autoimmune disease, and comparisons to apoptotic cell immunity in humans. PMID:26683143

  18. Autophagy exacerbates caspase-dependent apoptotic cell death after short times of starvation.

    PubMed

    Mattiolo, Paolo; Yuste, Victor J; Boix, Jacint; Ribas, Judit

    2015-12-15

    Autophagy is generally regarded as a mechanism to promote cell survival. However, autophagy can occasionally be the mechanism responsible of cell demise. We have found that a concomitant depletion of glucose, nutrients and growth factors provoked cell death in a variety of cell lines. This death process was contingent upon caspase activation and was mediated by BAX/BAK proteins, thus indicating its apoptotic nature and the engagement of an intrinsic pathway. In order to abrogate autophagy, 3-methyladenine (3-MA), BECLIN-1 siRNA and Atg5 knock-out (Tet-Off type) approaches were alternatively employed. Irrespective of the procedure, at short times of starvation, we found that the ongoing autophagy was sensitizing cells to the permeabilization of the mitochondrial outer membrane (MOMP), caspase activation and, therefore, apoptosis. On the contrary, at longer times of starvation, autophagy displayed its characteristic pro-survival effect on cells. As far as we know, we provide the first experimental paradigm where time is the only variable determining the final outcome of autophagy. In other words, we have circumscribed in time the shift transforming autophagy from a cell death to a protection mechanism. Moreover, at short times, starvation-driven autophagy exacerbated the apoptotic cell death caused by several antitumor agents. In agreement with this fact, their apoptotic effects were greatly diminished by autophagy inhibition. The implications of these facts in tumor biology will be discussed. PMID:26441250

  19. Apoptotic-cell-derived membrane microparticles and IFN-? induce an inflammatory immune response.

    PubMed

    Niessen, Anna; Heyder, Petra; Krienke, Stefan; Blank, Norbert; Tykocinski, Lars-Oliver; Lorenz, Hanns-Martin; Schiller, Martin

    2015-07-15

    A dysregulation in the clearance of apoptotic material is considered a major pathogenetic factor for the emergence of autoimmune diseases. Apoptotic-cell-derived membrane microparticles (AdMPs), which are released from the cell surface during apoptosis, have been implicated in the pathogenesis of autoimmunity. Also of importance are cytokines, such as interferon-? (IFN-?), which is known to be a major player in patients with systemic lupus erythematosus (SLE). This study investigates the combined effect of AdMPs and IFN-? on professional phagocytes. In the presence of IFN-?, phagocytosis of AdMPs by human monocytes was significantly increased in a dose-dependent manner. The combination of AdMPs and raised IFN-? concentrations resulted in an increase in the secretion of pro-inflammatory cytokines and an upregulation of surface molecule expression involved in antigen uptake. In addition, macrophage polarisation was shifted towards a more inflammatory type of cell. The synergism between IFN-? and AdMPs seemed to be mediated by an upregulation of phosphorylated STAT1. Our results indicate that IFN-?, together with AdMPs, amplify the initiation and maintenance of inflammation. This mechanism might especially play a crucial role in disorders with a defective clearance of apoptotic material. PMID:26034070

  20. Anti-apoptotic Bcl-2 Family Proteins Disassemble Ceramide Channels*s

    PubMed Central

    Siskind, Leah J.; Feinstein, Laurence; Yu, Tingxi; Davis, Joseph S.; Jones, David; Choi, Jinna; Zuckerman, Jonathan E.; Tan, Wenzhi; Hill, R. Blake; Hardwick, J. Marie; Colombini, Marco

    2009-01-01

    Early in mitochondria-mediated apoptosis, the mitochondrial outer membrane becomes permeable to proteins that, when released into the cytosol, initiate the execution phase of apoptosis. Proteins in the Bcl-2 family regulate this permeabilization, but the molecular composition of the mitochondrial outer membrane pore is under debate. We reported previously that at physiologically relevant levels, ceramides form stable channels in mitochondrial outer membranes capable of passing the largest proteins known to exit mitochondria during apoptosis (Siskind, L. J., Kolesnick, R. N., and Colombini, M. (2006) Mitochondrion 6, 118125). Here we show that Bcl-2 proteins are not required for ceramide to form protein-permeable channels in mitochondrial outer membranes. However, both recombinant human Bcl-xL and CED-9, the Caenorhabditis elegans Bcl-2 homologue, disassemble ceramide channels in the mitochondrial outer membranes of isolated mitochondria from rat liver and yeast. Importantly, Bcl-xL and CED-9 disassemble ceramide channels in the defined system of solvent-free planar phospholipid membranes. Thus, ceramide channel disassembly likely results from direct interaction with these anti-apoptotic proteins. Mutants of Bcl-xL act on ceramide channels as expected from their ability to be anti-apoptotic. Thus, ceramide channels may be one mechanism for releasing pro-apoptotic proteins from mitochondria during the induction phase of apoptosis. PMID:18171672

  1. Bcl-xL promotes metastasis independent of its anti-apoptotic activity.

    PubMed

    Choi, Soyoung; Chen, Zhengming; Tang, Laura H; Fang, Yuanzhang; Shin, Sandra J; Panarelli, Nicole C; Chen, Yao-Tseng; Li, Yi; Jiang, Xuejun; Du, Yi-Chieh Nancy

    2016-01-01

    Bcl-xL suppresses mitochondria-mediated apoptosis and is frequently overexpressed in cancer to promote cancer cell survival. Bcl-xL also promotes metastasis. However, it is unclear whether this metastatic function is dependent on its anti-apoptotic activity in the mitochondria. Here we demonstrate that Bcl-xL promotes metastasis independent of its anti-apoptotic activity. We show that apoptosis-defective Bcl-xL mutants and an engineered Bcl-xL targeted to the nucleus promote epithelial-mesenchymal transition, migration, invasion and stemness in pancreatic neuroendocrine tumour (panNET) and breast cancer cell lines. However, Bcl-xL proteins targeted to the mitochondria or outside of the nucleus do not have these functions. We confirm our findings in spontaneous and xenograft mouse models. Furthermore, Bcl-xL exerts metastatic function through epigenetic modification of the TGF? promoter to increase TGF? signalling. Consistent with these findings, we detect nuclear Bcl-xL in human metastatic panNETs. Taken together, the metastatic function of Bcl-xL is independent of its anti-apoptotic activity and its residence in the mitochondria. PMID:26785948

  2. Analysis of apoptotic pathways by multiparametric flow cytometry: application to HIV infection.

    PubMed

    Lecoeur, Hervé; Melki, Marie-Thérèse; Saïdi, Héla; Gougeon, Marie-Lise

    2008-01-01

    Flow cytometry analysis of apoptosis allows the detection, at the single cell level, of essential features of apoptotic cells. They include alterations in plasma membrane integrity, detected with the 7-aminoactinomycin D assay, translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane analyzed with the annexin-V/PI assay, DNA strand breaks in apoptotic nuclei measured with the in situ nick translation and terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling assays, and morphological modifications evidenced with FSC/SSC criteria. In addition, mitochondrial events such as the drop in transmembrane potential DeltaPsi(m) can be detected with the cationic lipophilic dye 3,3'-dihexyloxacarbocyanine iodide and downregulation of the Bcl-2 molecule by specific intracellular staining. Multiparametric flow cytometry combines all these approaches for a thorough sequential analysis of apoptosis, especially for heterogenous populations such as human peripheral mononuclear cells. Several examples of combined staining of apoptotic cells are shown on peripheral blood lymphocytes from chronically HIV-infected patients, prone to undergo premature apoptosis. PMID:18662564

  3. Bcl-xL promotes metastasis independent of its anti-apoptotic activity

    PubMed Central

    Choi, Soyoung; Chen, Zhengming; Tang, Laura H.; Fang, Yuanzhang; Shin, Sandra J.; Panarelli, Nicole C.; Chen, Yao-Tseng; Li, Yi; Jiang, Xuejun; Du, Yi-Chieh Nancy

    2016-01-01

    Bcl-xL suppresses mitochondria-mediated apoptosis and is frequently overexpressed in cancer to promote cancer cell survival. Bcl-xL also promotes metastasis. However, it is unclear whether this metastatic function is dependent on its anti-apoptotic activity in the mitochondria. Here we demonstrate that Bcl-xL promotes metastasis independent of its anti-apoptotic activity. We show that apoptosis-defective Bcl-xL mutants and an engineered Bcl-xL targeted to the nucleus promote epithelial–mesenchymal transition, migration, invasion and stemness in pancreatic neuroendocrine tumour (panNET) and breast cancer cell lines. However, Bcl-xL proteins targeted to the mitochondria or outside of the nucleus do not have these functions. We confirm our findings in spontaneous and xenograft mouse models. Furthermore, Bcl-xL exerts metastatic function through epigenetic modification of the TGFβ promoter to increase TGFβ signalling. Consistent with these findings, we detect nuclear Bcl-xL in human metastatic panNETs. Taken together, the metastatic function of Bcl-xL is independent of its anti-apoptotic activity and its residence in the mitochondria. PMID:26785948

  4. Anti-apoptotic effects of suppressor of cytokine signaling 3 and 1 in psoriasis

    PubMed Central

    Madonna, S; Scarponi, C; Pallotta, S; Cavani, A; Albanesi, C

    2012-01-01

    Because of their genetically determined capacity to respond to pro-inflammatory stimuli, keratinocytes have a crucial role in the pathogenesis of psoriasis. Upon IFN-? and TNF-? exposure, psoriatic keratinocytes express exaggerated levels of inflammatory mediators, and show aberrant hyperproliferation and terminal differentiation. The thickening of psoriasic skin also results from a peculiar resistance of keratinocytes to cytokine-induced apoptosis. In this study, we investigated on the molecular mechanisms concurring to the resistance of psoriatic keratinocytes to cell death, focusing on the role having suppressor of cytokine signaling (SOCS)1 and SOCS3, two molecules abundantly expressed in IFN-?/TNF-?-activated psoriatic keratinocytes, in sustaining anti-apoptotic pathways. We found that SOCS1 and SOCS3 suppress cytokine-induced apoptosis by sustaining the activation of the PI3K/AKT pathway in keratinocytes. The latter determines the activation of the anti-apoptotic NF-?B cascade and, in parallel, the inhibition of the pro-apoptotic BAD function in keratinocytes. For the first time, we report that phosphorylated AKT and phosphorylated BAD are strongly expressed in lesional psoriatic skin, compared with healthy or not lesional skin, and they strictly correlate to the high expression of SOCS1 and SOCS3 molecules in the psoriatic epidermis. Finally, the depletion of SOCS1 and SOCS3, as well as the chemical inactivation of PI3K activity in psoriatic keratinocytes, definitively unveils the role of PI3K/AKT cascade on the resistance of diseased keratinocytes to apoptosis. PMID:22739986

  5. The Arf GAP CNT-2 regulates the apoptotic fate in C. elegans asymmetric neuroblast divisions

    PubMed Central

    Singhvi, Aakanksha; Teuliere, Jerome; Talavera, Karla; Cordes, Shaun; Ou, Guangshuo; Vale, Ronald D.; Prasad, Brinda C.; Clark, Scott G.; Garriga, Gian

    2011-01-01

    Summary During development, all cells make the decision to live or die. While the molecular mechanisms that execute the apoptotic program are well defined, less is known about how cells decide whether to live or die. In C. elegans, this decision is linked to how cells divide asymmetrically [1, 2]. Several classes of molecules are known to regulate asymmetric cell divisions in metazoans, yet these molecules do not appear to control C. elegans divisions that produce apoptotic cells [3]. We identified CNT-2, an Arf GAP protein of the AGAP family, as a novel regulator of this type of neuroblast division. Loss of CNT-2 altered daughter cell size and caused the apoptotic cell to adopt the fate of its sister cell, resulting in extra neurons. CNT-2’s Arf GAP activity was essential for its function in these divisions. The N-terminus of CNT-2, which contains a GTPase-like domain that defines the AGAP class of Arf GAPs, negatively regulates CNT-2’s function. We provide evidence that CNT-2 regulates receptor-mediated endocytosis and consider the implications of its role in asymmetric cell divisions. PMID:21596567

  6. Combined effects of fine particulate matter and lipopolysaccharide on apoptotic responses in NR8383 macrophages.

    PubMed

    Xiong, Qi; Ru, Qin; Chen, Lin; Yue, Kai; Tian, Xiang; Ma, Baomiao; Liu, Lu; Wu, Rihui; Xu, Congyue; Pi, Mingshan; Li, Chaoying

    2015-01-01

    Alveolar macrophages (AM) are the predominant lung cells responsible for both ingestion and clearance of inhaled particulate matter (PM). The aims of this study were (1) to examine effects of fine PM on rat NR8383 cell line apoptosis, and (2) to determine whether NR8383 cell functions are further affected when exposed to fine PM in the presence of inflammation induced by lipopolysaccharide (LPS). Standard Reference Material 2786 (SRM 2786) for fine PM was used to measure the following parameters: cytotoxicity, apoptotic rate, Bax/Bcl-2 expression, nitric oxide (NO) production, and reactive oxygen species (ROS) generation in NR8383 cells. Data showed that SRM 2786 alone induced damage and apoptosis in NR8383 cells in a concentration-dependent manner as demonstrated by significant decrease in expression of Bcl-2 and increase in expression of Bax, suggesting fine PM might trigger apoptosis involving a mitochondria-mediated apoptotic pathway. In addition, there was elevated production of free radicals, such as NO and ROS, suggesting oxidative stress plays a role in the observed apoptotic responses. Further, LPS pretreatment enhanced apoptosis of NR8383 cells induced by SRM 2786. Consequently, data indicate that SRM 2786 triggered cell apoptosis in NR8383 cells, probably by mechanisms involving oxidative stress, as evidenced by elevated NO and ROS levels, while the degree of apoptosis was further aggravated by inflammation. PMID:25785558

  7. Parkin Promotes Degradation of the Mitochondrial Pro-Apoptotic ARTS Protein

    PubMed Central

    Kemeny, Stav; Dery, Dikla; Loboda, Yelena; Rovner, Marshall; Lev, Tali; Zuri, Dotan; Finberg, John P. M.; Larisch, Sarit

    2012-01-01

    Parkinson’s disease (PD) is associated with excessive cell death causing selective loss of dopaminergic neurons. Dysfunction of the Ubiquitin Proteasome System (UPS) is associated with the pathophysiology of PD. Mutations in Parkin which impair its E3-ligase activity play a major role in the pathogenesis of inherited PD. ARTS (Sept4_i2) is a mitochondrial protein, which initiates caspase activation upstream of cytochrome c release in the mitochondrial apoptotic pathway. Here we show that Parkin serves as an E3-ubiquitin ligase to restrict the levels of ARTS through UPS-mediated degradation. Though Parkin binds equally to ARTS and Sept4_i1 (H5/PNUTL2), the non-apoptotic splice variant of Sept4, Parkin ubiquitinates and degrades only ARTS. Thus, the effect of Parkin on ARTS is specific and probably related to its pro-apoptotic function. High levels of ARTS are sufficient to promote apoptosis in cultured neuronal cells, and rat brains treated with 6-OHDA reveal high levels of ARTS. However, over-expression of Parkin can protect cells from ARTS-induced apoptosis. Furthermore, Parkin loss-of-function experiments reveal that reduction of Parkin causes increased levels of ARTS and apoptosis. We propose that in brain cells in which the E3-ligase activity of Parkin is compromised, ARTS levels increase and facilitate apoptosis. Thus, ARTS is a novel substrate of Parkin. These observations link Parkin directly to a pro-apoptotic protein and reveal a novel connection between Parkin, apoptosis, and PD. PMID:22792159

  8. Apoptosis and apoptotic mimicry in Leishmania: an evolutionary perspective

    PubMed Central

    El-Hani, Charbel N.; Borges, Valria M.; Wanderley, Joo L. M.; Barcinski, Marcello A.

    2012-01-01

    Apoptotic death and apoptotic mimicry are defined respectively as a non-accidental death and as the mimicking of an apoptotic-cell phenotype, usually by phosphatidylserine (PS) exposure. In the case of the murine infection by Leishmania spp, apoptotic death has been described in promastigotes and apoptotic mimicry in amastigotes. In both situations they are important events of the experimental murine infection by this parasite. In the present review we discuss what features we need to consider if we want to establish if a behavior shown by Leishmania is altruistic or not: does the behavior increases the fitness of organisms other than the one showing it? Does this behavior have a cost for the actor? If we manage to show that a given behavior is costly for the actor and beneficial for the recipient of the action, we will be able to establish it as altruistic. From this perspective, we can argue that apoptotic-like death and apoptotic mimicry are both altruistic with the latter representing a weaker altruistic behavior than the former. PMID:22912937

  9. Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

    PubMed Central

    Shklyar, Boris; Shklover, Jeny; Kurant, Estee

    2013-01-01

    The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance. PMID:23979068

  10. Engineered apoptotic nucleases for chromatin research.

    PubMed

    Xiao, Fei; Widlak, Piotr; Garrard, William T

    2007-01-01

    We have created new genomics tools for chromatin research by genetically engineering the human and mouse major apoptotic nucleases that are responsible for internucleosomal DNA cleavage, DNA fragmentation factor (DFF). Normally, in its inactive form, DFF is a heterodimer composed of a 45-kDa chaperone inhibitor subunit (DFF45 or ICAD), and a 40-kDa latent endonuclease subunit (DFF40 or CAD). Upon caspase-3 cleavage of DFF45, DFF40 forms active endonuclease homo-oligomers. Although Saccharomyces cerevisiae lacks DFF, expression of caspase-3 is lethal in this organism, but expression of the highly sequence-specific tobacco etch virus protease (TEVP) is harmless. Therefore, we inserted TEVP cleavage sites immediately downstream of the two caspase-3 cleavage sites within DFF45, generating a novel form of DFF (DFF-T) whose nuclease activity proved to be exclusively under the control of TEVP. We demonstrate that co-expression of TEVP and DFF-T under galactose control results in nucleosomal DNA laddering and cell death in S. cerevisiae. We also created synthetic DFF genes with optimized codons for high-level expression in Eschericia coli or S. cerevisiae. We further demonstrate the excellence of the synthetic gene products for in vitro mapping of the nucleosome positions and hypersensitive sites in specific genes such as the yeast PHO5. PMID:17626049

  11. Disturbances of apoptotic cell clearance in systemic lupus erythematosus

    PubMed Central

    2011-01-01

    Systemic lupus erythematosus is a multifactorial autoimmune disease with an as yet unknown etiopathogenesis. It is widely thought that self-immunization in systemic lupus is driven by defective clearance of dead and dying cells. In lupus patients, large numbers of apoptotic cells accumulate in various tissues including germinal centers. In the present review, we discuss the danger signals released by apoptotic cells, their triggering of inflammatory responses, and the breakdown of B-cell tolerance. We also review the pathogenic role of apoptotic cell clearance in systemic lupus erythematosus. PMID:21371352

  12. Screening of pro-apoptotic genes upregulated in an experimental street rabies virus-infected neonatal mouse brain.

    PubMed

    Ubol, Sukathida; Kasisith, Jitra; Pitidhammabhorn, Dhanesh; Tepsumethanol, Veera

    2005-01-01

    Rabies virus (RABV) is able to induce apoptotic death of target cells. The molecular pathway of RABV-induced cell death is partially known. In the present study, cDNA array analysis was used as a tool to screen for pro-apoptotic genes that may be involved in RABV induction. RNA was extracted from the infected CNS and from mock-infected controls. When the mean gene expression was compared between the infected group and controls, 21 potential apoptotic genes were identified that exhibited more than 2.5-fold difference in their expression levels. These 21 genes can be grouped into two groups, those genes that participate in the commitment phase and those that play a role as executioners. Examples of genes in commitment phase were death receptors (Fas-L receptor, TNF-receptor), lysosomal proteases, calpain, caspase-1, signaling molecules (ERK, p38MAPK) and bcl-2 family members. Cytochrome c and caspase-3 were representatives of executioners. Based on types of genes activated during the commitment phase, two independent apoptotic mechanisms may be activated in response to the RV infection. The first is immune-mediated death which may operate through the receptor-ligand pathway activated by caspase-1 and the pro-inflammatory cytokine, IL-1beta. The other mechanism is a protease-mediated process which involves lysosomal proteases and calcium-dependent neutral proteases. These two stimulating pathways were followed by Bad, Bak, Bid activation and subsequently the upregulation of cytochrome c and caspase-3. In addition, mobilization of K+ ion and other accessory apoptotic genes such as annexins and clusterin were also upregulated. PMID:15905604

  13. Cardiorenal Syndrome Type 1: Activation of Dual Apoptotic Pathways.

    PubMed

    Pastori, Silvia; Virz, Grazia Maria; Brocca, Alessandra; de Cal, Massimo; Cantaluppi, Vincenzo; Castellani, Chiara; Fedrigo, Marny; Thiene, Gaetano; Valente, Maria Luisa; Angelini, Annalisa; Vescovo, Giorgio; Ronco, Claudio

    2015-10-01

    Cardiorenal syndrome type 1 (CRS1) pathophysiology is complex, and immune-mediated damage, including alterations in the immune response with monocyte apoptosis and cytokine release, has been reported as a potential mechanism. In this study, we examined the putative role of renal tubular epithelial cell (RTC) apoptosis as a pathogenic mechanism in CRS1. In particular, we investigated the caspase pathways involved in induced apoptosis. We enrolled 29 patients with acute heart failure (AHF), 11 patients with CRS1, and 15 controls (CTR) without AHF or acute kidney injury (AKI). Patients who had AKI prior to the episode of AHF or who had any other potential causes of AKI were excluded. Plasma from different groups was incubated with RTCs for 24 h. Subsequently, cell apoptosis, DNA fragmentation, and caspase-3, -8, and -9 activities were investigated in RTCs incubated with AHF, CRS1, and CTR plasma. A p value <0.5 was considered statistically significant. A quantitative analysis of apoptosis showed significantly higher apoptosis rates in CRS1 patients compared to AHF patients and CTR (p < 0.01). This increase in apoptosis was strongly confirmed by caspase-3 levels (? = 0.73). Caspase-8 and -9 were significantly higher in CRS1 patients compared to AHF patients and CTR (p < 0.01). Furthermore, caspase-3 levels showed a significantly positive correlation with caspase-8 (? = 0.57) and -9 (? = 0.47; p < 0.001). This study demonstrated the significantly heightened presence of dual apoptotic disequilibrium in CRS1. Our findings indicated that apoptosis may have a central role in the mechanism of CRS1, and it could be a potential therapeutic target in this syndrome. PMID:26648947

  14. Cardiorenal Syndrome Type 1: Activation of Dual Apoptotic Pathways

    PubMed Central

    Pastori, Silvia; Virz, Grazia Maria; Brocca, Alessandra; de Cal, Massimo; Cantaluppi, Vincenzo; Castellani, Chiara; Fedrigo, Marny; Thiene, Gaetano; Valente, Maria Luisa; Angelini, Annalisa; Vescovo, Giorgio; Ronco, Claudio

    2015-01-01

    Cardiorenal syndrome type 1 (CRS1) pathophysiology is complex, and immune-mediated damage, including alterations in the immune response with monocyte apoptosis and cytokine release, has been reported as a potential mechanism. In this study, we examined the putative role of renal tubular epithelial cell (RTC) apoptosis as a pathogenic mechanism in CRS1. In particular, we investigated the caspase pathways involved in induced apoptosis. We enrolled 29 patients with acute heart failure (AHF), 11 patients with CRS1, and 15 controls (CTR) without AHF or acute kidney injury (AKI). Patients who had AKI prior to the episode of AHF or who had any other potential causes of AKI were excluded. Plasma from different groups was incubated with RTCs for 24 h. Subsequently, cell apoptosis, DNA fragmentation, and caspase-3, ?8, and ?9 activities were investigated in RTCs incubated with AHF, CRS1, and CTR plasma. A p value <0.5 was considered statistically significant. A quantitative analysis of apoptosis showed significantly higher apoptosis rates in CRS1 patients compared to AHF patients and CTR (p < 0.01). This increase in apoptosis was strongly confirmed by caspase-3 levels (? = 0.73). Caspase-8 and ?9 were significantly higher in CRS1 patients compared to AHF patients and CTR (p < 0.01). Furthermore, caspase-3 levels showed a significantly positive correlation with caspase-8 (? = 0.57) and ?9 (? = 0.47; p < 0.001). This study demonstrated the significantly heightened presence of dual apoptotic disequilibrium in CRS1. Our findings indicated that apoptosis may have a central role in the mechanism of CRS1, and it could be a potential therapeutic target in this syndrome. PMID:26648947

  15. The pro-apoptotic role of autophagy in breast cancer

    PubMed Central

    Suman, S; Das, T P; Reddy, R; Nyakeriga, A M; Luevano, J E; Konwar, D; Pahari, P; Damodaran, C

    2014-01-01

    Background: Autophagy is a catabolic process that has a vital role in cancer progression and treatment. Current chemotherapeutic agents, which target autophagy, result in growth inhibition in many cancer types. In this study, we examined the role of autophagy in breast cancer (BCa) patients as well as BCa cell lines. Methods: Tissue microarray was used to detect the expression of an autophagy marker, LC3B in BCa patients (normal/hyperplasia=8; grade-I=15, grade-II=84, and grade-III=27) and BCa cell lines. To modulate the activation of autophagy, we used novel herbal compound nimocinol acetate (NA) in BCa cell lines and the anticancer activity was measured by phenotypic and molecular analysis. Results: LC3B is highly expressed in tumours as compared with normal tissues. Activation of LC3B in NA-treated BCa (MCF-7 and MDA-MB-231) cells was evident as compared with other autophagy makers. Further, our results confirmed that NA-transcriptionally regulates LC3B (as confirmed by mRNA levels and reporter assay), which resulted in the formation of acidic autophagy vesicles and autolysosomes in BCa cells. Nimocinol acetate inhibited mTOR-mediated pro-survival signalling that resulted in inhibition of growth in BCa cells without affecting normal breast epithelial cells. Downregulation of LC3B expression by siRNA significantly inhibited the anticancer effects of NA in BCa cells. Conclusions: Together, our results suggest that LC3B is highly expressed in BCa tissues and increasing the threshold of LC3B activation dictates the pro-apoptotic function, which in turn, suppresses the growth of BCa cells. Nimocinol acetate could be a potential agent for treatment of BCa. PMID:24945999

  16. Expression of animal CED-9 anti-apoptotic gene in tobacco modifies plasma membrane ion fluxes in response to salinity and oxidative stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Apoptosis, one form of programmed cell death (PCD), plays an important role in mediating plant adaptive responses to the environment. Recent studies suggest that expression of animal anti-apoptotic genes in transgenic plants may be an efficient way of enhancing stress resistance in economically impo...

  17. Cobra venom cytotoxins; apoptotic or necrotic agents?

    PubMed

    Ebrahim, Karim; Shirazi, Farshad H; Mirakabadi, Abbas Zare; Vatanpour, Hossein

    2015-12-15

    Organs homeostasis is controlled by a dynamic balance between cell proliferation and apoptosis. Failure to induction of apoptosis has been implicated in tumor development. Cytotoxin-I (CTX-I) and cytotoxin-II (CTX-II) are two physiologically active polypeptides found in Caspian cobra venom. Anticancer activity and mechanism of cell death induced by these toxins have been studied. The toxins were purified by different chromatographic steps and their cytotoxicity and pattern of cell death were determined by MTT, LDH release, acridine orange/ethidium bromide (AO/EtBr) double staining, flow cytometric analysis, caspase-3 activity and neutral red assays. The IC50 of CTX-II in MCF-7, HepG2, DU-145 and HL-60 was 4.11.3, 21.24.4, 9.41.8?g/mL and 16.31.9 respectively while the IC50 of this toxin in normal MDCK cell line was 54.53.9?g/mL. LDH release suddenly increase after a specific toxins concentrations in all cell lines. AO/EtBr double staining, flow cytometric analysis and caspase-3 activity assay confirm dose and time-dependent induction of apoptosis by both toxins. CTX-I and CTX-II treated cells lost their lysosomal membrane integrity and couldn't uptake neutral red day. CTX-I and CTX-II showed significant anticancer activity with minimum effects on normal cells and better IC50 compared to current anticancer drug; cisplatin. They induce their apoptotic effect via lysosomal pathways and release of cathepsins to cytosol. These effects were seen in limited rage of toxins concentrations and pattern of cell death rapidly changes to necrosis by increase in toxin's concentration. In conclusion, significant apoptogenic effects of these toxins candidate them as a possible anticancer agent. PMID:26482932

  18. Apoptotic cell clearance in chronic inflammation of lateral neck cysts.

    PubMed

    Dobros, Wieslaw; Burda, Karolina; Guzik, Krzysztof; Koziel, Joanna; Potempa, Jan

    2012-03-01

    The mechanism driving accumulation of large numbers of apoptotic and necrotic neutrophils in inflamed lateral neck cysts (LNC), in the absence of infection, remains obscure. The cellular content of cysts obtained from 17 patients was co-cultured with human macrophages. Phagocytosis levels of cyst-derived neutrophils were determined and compared to the uptake of spontaneously apoptotic neutrophils. Simultaneously, the expression of cytokines in macrophages exposed to cyst contents was measured. In comparison to spontaneously apoptotic neutrophils, the phagocytosis of LNC-derived neutrophils by macrophages was inefficient. An inverse correlation between neutrophil content in LNC and their uptake was observed. Macrophages co-cultured with cyst contents responded with variable expression of IL-6, TNF-? and IL-10. The hindered clearance of apoptotic neutrophils in LNC may lead to secondary necrosis of these cells and stimulation of the inflammatory reaction. Together with local production of anti-inflammatory cytokines, this may fuel chronic inflammation in the cysts. PMID:21755330

  19. Regulation of mammalian horizontal gene transfer by apoptotic DNA fragmentation.

    PubMed

    Yan, B; Wang, H; Li, F; Li, C-Y

    2006-12-18

    Previously it was shown that horizontal DNA transfer between mammalian cells can occur through the uptake of apoptotic bodies, where genes from the apoptotic cells were transferred to neighbouring cells phagocytosing the apoptotic bodies. The regulation of this process is poorly understood. It was shown that the ability of cells as recipient of horizontally transferred DNA was enhanced by deficiency of p53 or p21. However, little is known with regard to the regulation of DNA from donor apoptotic cells. Here we report that the DNA fragmentation factor/caspase-activated DNase (DFF/CAD), which is the endonuclease responsible for DNA fragmentation during apoptosis, plays a significant role in regulation of horizontal DNA transfer. Cells with inhibited DFF/CAD function are poor donors for horizontal gene transfer (HGT) while their ability of being recipients of HGT is not affected. PMID:17146478

  20. Severe apoptotic enteropathy caused by methotrexate treatment for rheumatoid arthritis.

    PubMed

    Toquet, Ségolène; Nguyen, Yohan; Sabbagh, Adel; Djerada, Zoubir; Boulagnon, Camille; Bani-Sadr, Firouzé

    2016-03-01

    The folic acid antagonist methotrexate is a cornerstone treatment of rheumatoid arthritis. Its use is limited chiefly by gastrointestinal toxicity, which is among the main reasons for methotrexate discontinuation. Here, we report the case of a 40-year-old man on chronic methotrexate therapy in whom life-threatening apoptotic enteropathy with watery diarrhea and hypovolemic shock developed after he was switched from the oral to the intramuscular route, with no change in dosage. Colonic biopsies suggested drug-induced colitis, showing a nonspecific, mildly inflammatory infiltrate of lymphocytes and plasma cells, dilated damaged crypts, and a marked increase in basal crypt apoptosis (>20 apoptotic bodies/100 crypts). Clinicians should be aware that methotrexate can cause life-threatening apoptotic enteropathy. Increased basal crypt apoptosis in colonic biopsies with more than 5 apoptotic bodies/100 crypts should routinely suggest drug-induced enteropathy. PMID:26494588

  1. Cannabidiol induced a contrasting pro-apoptotic effect between freshly isolated and precultured human monocytes.

    PubMed

    Wu, Hsin-Ying; Chang, An-Chi; Wang, Chia-Chi; Kuo, Fu-Hua; Lee, Chi-Ya; Liu, Der-Zen; Jan, Tong-Rong

    2010-08-01

    It has been documented that cannabidiol (CBD) induced apoptosis in a variety of transformed cells, including lymphocytic and monocytic leukemias. In contrast, a differential sensitivity between normal lymphocytes and monocytes to CBD-mediated apoptosis has been reported. The present study investigated the pro-apoptotic effect of CBD on human peripheral monocytes that were either freshly isolated or precultured for 72h. CBD markedly enhanced apoptosis of freshly isolated monocytes in a time- and concentration-dependent manner, whereas precultured monocytes were insensitive. By comparison, both cells were sensitive to doxorubicin-induced apoptosis. CBD significantly diminished the cellular thiols and glutathione in freshly isolated monocytes. The apoptosis induced by CBD was abrogated in the presence of N-acetyl-L-cysteine, a precursor of glutathione. In addition, precultured monocytes contained a significantly greater level of glutathione and heme oxygenase-1 (HO-1) compared to the freshly isolated cells. The HO-1 competitive inhibitor zinc protoporphyrin partially but significantly restored the sensitivity of precultured monocytes to CBD-mediated apoptosis. Collectively, our results demonstrated a contrasting pro-apoptotic effect of CBD between precultured and freshly isolated monocytes, which was closely associated with the cellular level of glutathione and the antioxidative capability of the cells. PMID:20471992

  2. PKC? is an anti-apoptotic kinase that predicts poor prognosis in breast and lung cancer.

    PubMed

    Zurgil, Udi; Ben-Ari, Assaf; Rotem-Dai, Noa; Karp, Galia; Krasnitsky, Ella; Frost, Sigal A; Livneh, Etta

    2014-12-01

    The successful treatment of cancer in a disseminated stage using chemotherapy is limited by the occurrence of drug resistance, often mediated by anti-apoptotic mechanisms. Thus the challenge is to pinpoint the underlying key factors and to develop therapies for their direct targeting. Protein kinase C (PKC) enzymes are promising candidates, as some PKCs were shown to be involved in regulation of apoptosis. Our studies and others have shown that PKC? is an anti-apoptotic kinase, able to confer protection on tumour cells against stress and chemotherapy. We have demonstrated that PKC? shuttles between the cytoplasm and the nucleus and that upon DNA damage is tethered at the nuclear membrane. The C1b domain mediates translocation of PKC? to the nuclear envelope and, similar to the full-length protein, could also confer protection against cell death. Furthermore, its localization in cell and nuclear membranes in breast cancer biopsies of neoadjuvant-treated breast cancer patients was an indicator for poor survival and a predictor for the effectiveness of treatment. PKC? is also a novel biomarker for poor prognosis in non-small-cell lung cancer (NSCLC). Thus PKC? presents a potential target for therapy where inhibition of its activity and/or translocation to membranes could interfere with the resistance to chemotherapy. PMID:25399563

  3. Cannabidiol induced a contrasting pro-apoptotic effect between freshly isolated and precultured human monocytes

    SciTech Connect

    Wu, Hsin-Ying; Chang, An-Chi; Wang, Chia-Chi; Kuo, Fu-Hua; Lee, Chi-Ya; Liu, Der-Zen; Jan, Tong-Rong

    2010-08-01

    It has been documented that cannabidiol (CBD) induced apoptosis in a variety of transformed cells, including lymphocytic and monocytic leukemias. In contrast, a differential sensitivity between normal lymphocytes and monocytes to CBD-mediated apoptosis has been reported. The present study investigated the pro-apoptotic effect of CBD on human peripheral monocytes that were either freshly isolated or precultured for 72 h. CBD markedly enhanced apoptosis of freshly isolated monocytes in a time- and concentration-dependent manner, whereas precultured monocytes were insensitive. By comparison, both cells were sensitive to doxorubicin-induced apoptosis. CBD significantly diminished the cellular thiols and glutathione in freshly isolated monocytes. The apoptosis induced by CBD was abrogated in the presence of N-acetyl-{sub L}-cysteine, a precursor of glutathione. In addition, precultured monocytes contained a significantly greater level of glutathione and heme oxygenase-1 (HO-1) compared to the freshly isolated cells. The HO-1 competitive inhibitor zinc protoporphyrin partially but significantly restored the sensitivity of precultured monocytes to CBD-mediated apoptosis. Collectively, our results demonstrated a contrasting pro-apoptotic effect of CBD between precultured and freshly isolated monocytes, which was closely associated with the cellular level of glutathione and the antioxidative capability of the cells.

  4. ANTI-APOPTOTIC ACTIONS OF VASOPRESSIN IN H32 NEURONS INVOLVE MAP KINASE TRANSACTIVATION AND BAD PHOSPHORYLATION

    PubMed Central

    Chen, Jun; Volpi, Simona; Aguilera, Greti

    2008-01-01

    Vasopressin (VP) secreted within the brain modulates neuronal function acting as a neurotransmitter. Based on the observation that VP prevented serum deprivation-induced cell death in the neuronal cell line, H32, which expresses endogenous V1 receptors, we tested the hypothesis that VP has anti-apoptotic properties. Flow cytometry experiments showed that 10nM VP prevented serum deprivation-induced cell death and annexin V binding. Serum deprivation increased caspase-3 activity in a time and serum concentration dependent manner, and VP prevented these effects through interaction with receptors of V1 subtype. The signaling pathways mediating the anti-apoptotic effect of VP involve mitogen activated protein (MAP) kinase and extracellular signal-regulated kinases (ERK), Ca2+/calmodulin dependent kinase (CaMK) and protein kinase C (PKC). Western blot analyses revealed time-dependent decreases of Bad phosphorylation and increases in cytosolic levels of cytochrome c following serum deprivation, effects which were prevented by 10nM VP. These data demonstrate that activation of endogenous V1 VP receptors prevents serum deprivation-induced apoptosis, through phosphorylation-inactivation of the pro-apoptotic protein, Bad, and consequent decreases in cytosolic cytochome c and caspase-3 activation. The data suggest that VP has anti-apoptotic activity in neurons and that VP may act as a neuroprotective agent in the brain. PMID:18402937

  5. Phosphatidylserine receptor is required for the engulfment of dead apoptotic cells and for normal embryonic development in zebrafish.

    PubMed

    Hong, Jiann-Ruey; Lin, Gen-Hwa; Lin, Cliff Ji-Fan; Wang, Wan-Ping; Lee, Chien-Chung; Lin, Tai-Lang; Wu, Jen-Leih

    2004-11-01

    During development, the role of the phosphatidylserine receptor (PSR) in the removal of apoptotic cells that have died is poorly understood. We have investigated this role of PSR in developing zebrafish. Programmed cell death began during the shield stage, with dead cells being engulfed by a neighboring cell that showed a normal-looking nucleus and the nuclear condensation multi-micronuclei of an apoptotic cell. The zebrafish PSR engulfing receptor was cloned (zfpsr), and its nucleotide sequence was compared with corresponding sequences in Drosophila melanogaster (76% identity), human (74%), mouse (72%) and Caenorhabditis elegans (60%). The PSR receptor contained a jmjC domain (residues 143-206) that is a member of the cupin metalloenzyme superfamily, but in this case serves an as yet unknown function(s). psr knockdown by a PSR morpholino oligonucleotide led to accumulation of a large number of dead apoptotic cells in whole early embryo. These cells interfered with embryonic cell migration. In addition, normal development of the somite, brain, heart and notochord was sequentially disrupted up to 24 hours post-fertilization. Development could be rescued in defective embryos by injecting psr mRNA. These results are consistent with a PSR-dependent system in zebrafish embryos that engulfs apoptotic cells mediated by PSR-phagocytes during development, with the system assuming an important role in the normal development of tissues such as the brain, heart, notochord and somite. PMID:15469976

  6. Comparison of automated haematology analysers for detection of apoptotic lymphocytes.

    PubMed

    Taga, K; Sawaya, M; Yoshida, M; Kaneko, M; Okada, M; Taniho, M

    2002-06-01

    Automated haematology analysers can rapidly provide accurate blood cell counts and white blood cell differentials. In this study, we evaluated four different haematology analysers for the detection of apoptotic lymphocytes in peripheral blood: MAXM A/L Retic, H*2, Cell-Dyn 3500 and NE-8000. With the MAXM A/L Retic haematology analyser, the apoptotic lymphocyte cluster appeared below the original lymphocyte cluster on the volume/DF1, and to the right under the original lymphocyte cluster on the volume/DF2 scattergrams. With the H*2 haematology analyser, the apoptotic polymorphonuclear lymphocytes produced a higher lobularity index on the BASO channel. With the Cell-Dyn 3500 haematology analyser, the apoptotic lymphocyte cluster appeared to the right side of the original lymphocyte cluster on the 0D/10D scattergram and to the left side of the polymorphonuclear cluster on the 90D/10D scattergram. With the NE-8000 haematology analyser, the apoptotic lymphocyte cluster was not distinguishable. Thus, apoptotic lymphocytes are readily detected on scattergrams generated by selected haematology analysers. PMID:12067276

  7. Apoptotic response of malignant rhabdoid tumor cells

    PubMed Central

    Nocentini, Silvano

    2003-01-01

    Background Malignant rhabdoid tumors (MRTs) are extremely aggressive and resist current radio- and chemotherapic treatments. To gain insight into the dysfunctions of MRT cells, the apoptotic response of a model cell line, MON, was analyzed after exposure to several genotoxic and non-genotoxic agents employed separately or in association. Results Fluorescence microscopy of chromatin morphology and electrophoretic analysis of internucleosomal DNA fragmentation revealed that MON cells were, comparatively to HeLa cells, resistant to apoptosis after treatment with etoposide, cisplatin (CisPt) or X-rays, but underwent some degree of apoptosis after ultraviolet (UV) C irradiation. Concomitant treatment of MON cells with X-rays or vinblastine and the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin resulted in synergistic induction of apoptosis. Western blot analysis showed that the p53 protein was upregulated in MON cells after exposure to all the different agents tested, singly or in combination. In treated cells, the p53 downstream effectors p21WAF1/CIP1, Mdm2 and Bax were induced with some inconsistency with regard to the accumulation of p53. Poly ADP-ribose polymerase (PARP) cleavage, indicative of ongoing apoptosis, occurred in UVC-irradiated cells and, especially, in cells treated with combinations of X-rays or vinblastine with wortmannin. However, there was moderate or no PARP cleavage in cells treated with CisPt, X-rays, vinblastine or wortmannin singly or with the combinations X-rays plus CisPt or vinblastine and CisPt plus vinblastine or wortmannin. The synergistic effect on the induction of apoptosis exerted by some agent combinations corresponded with synergy in respect of MON cell growth inhibition. Conclusion These results suggest abnormalities in the p53 pathway and apoptosis control in MRT cells. The Ras/PI3-K/AKT signaling pathway might also be deregulated in these cells by generating an excess of survival factors. These dysfunctions might contribute to the resistance of MRTs to current antineoplastic treatments and could warrant consideration in the search of new therapeutic approaches. PMID:12904267

  8. Low expression of pro-apoptotic Bcl-2 family proteins sets the apoptotic threshold in Waldenström macroglobulinemia.

    PubMed

    Gaudette, B T; Dwivedi, B; Chitta, K S; Poulain, S; Powell, D; Vertino, P; Leleu, X; Lonial, S; Chanan-Khan, A A; Kowalski, J; Boise, L H

    2016-01-28

    Waldenström macroglobulinemia (WM) is a proliferative disorder of IgM-secreting, lymphoplasmacytoid cells that inhabit the lymph nodes and bone marrow. The disease carries a high prevalence of activating mutations in MyD88 (91%) and CXCR4 (28%). Because signaling through these pathways leads to Bcl-xL induction, we examined Bcl-2 family expression in WM patients and cell lines. Unlike other B-lymphocyte-derived malignancies, which become dependent on expression of anti-apoptotic proteins to counter expression of pro-apoptotic proteins, WM samples expressed both pro- and anti-apoptotic Bcl-2 proteins at low levels similar to their normal B-cell and plasma cell counterparts. Three WM cell lines expressed pro-apoptotic Bcl-2 family members Bim or Bax and Bak at low levels, which determined their sensitivity to inducers of intrinsic apoptosis. In two cell lines, miR-155 upregulation, which is common in WM, was responsible for the inhibition of FOXO3a and Bim expression. Both antagonizing miR-155 to induce Bim and proteasome inhibition increased the sensitivity to ABT-737 in these lines indicating a lowering of the apoptotic threshold. In this manner, treatments that increase pro-apoptotic protein expression increase the efficacy of agents treated in combination in addition to direct killing. PMID:25893290

  9. Surface codebiophysical signals for apoptotic cell clearance

    NASA Astrophysics Data System (ADS)

    Biermann, Mona; Mauerder, Christian; Brauner, Jan M.; Chaurio, Ricardo; Janko, Christina; Herrmann, Martin; Muoz, Luis E.

    2013-12-01

    Apoptotic cell death and the clearance of dying cells play an important and physiological role in embryonic development and normal tissue turnover. In contrast to necrosis, apoptosis proceeds in an anti-inflammatory manner. It is orchestrated by the timed release and/or exposure of so-called find-me, eat me and tolerate me signals. Mononuclear phagocytes are attracted by various find-me signals, including proteins, nucleotides, and phospholipids released by the dying cell, whereas the involvement of granulocytes is prevented via stay away signals. The exposure of anionic phospholipids like phosphatidylserine (PS) by apoptotic cells on the outer leaflet of the plasma membrane is one of the main eat me signals. PS is recognized by a number of innate receptors as well as by soluble bridging molecules on the surface of phagocytes. Importantly, phagocytes are able to discriminate between viable and apoptotic cells both exposing PS. Due to cytoskeleton remodeling PS has a higher lateral mobility on the surfaces of apoptotic cells thereby promoting receptor clustering on the phagocyte. PS not only plays an important role in the engulfment process, but also acts as tolerate me signal inducing the release of anti-inflammatory cytokines by phagocytes. An efficient and fast clearance of apoptotic cells is required to prevent secondary necrosis and leakage of intracellular danger signals into the surrounding tissue. Failure or prolongation of the clearance process leads to the release of intracellular antigens into the periphery provoking inflammation and development of systemic inflammatory autoimmune disease like systemic lupus erythematosus. Here we review the current findings concerning apoptosis-inducing pathways, important players of apoptotic cell recognition and clearance as well as the role of membrane remodeling in the engulfment of apoptotic cells by phagocytes.

  10. Three sorting nexins drive the degradation of apoptotic cells in response to PtdIns(3)P signaling

    PubMed Central

    Lu, Nan; Shen, Qian; Mahoney, Timothy R.; Liu, Xianghua; Zhou, Zheng

    2011-01-01

    Apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Phagosome maturation requires phosphatidylinositol 3-phosphate [PtdIns(3)P], yet how PtdIns(3)P triggers phagosome maturation remains largely unknown. Through a genome-wide PtdIns(3)P effector screen in the nematode Caenorhabditis elegans, we identified LST-4/SNX9, SNX-1, and SNX-6, three BAR domain-containing sorting nexins, that act in two parallel pathways to drive PtdIns(3)P-mediated degradation of apoptotic cells. We found that these proteins were enriched on phagosomal surfaces through association with PtdIns(3)P and through specific proteinprotein interaction, and they promoted the fusion of early endosomes and lysosomes to phagosomes, events essential for phagosome maturation. Specifically, LST-4 interacts with DYN-1 (dynamin), an essential phagosome maturation initiator, to strengthen DYN-1s association to phagosomal surfaces, and facilitates the maintenance of the RAB-7 GTPase on phagosomal surfaces. Furthermore, both LST-4 and SNX-1 promote the extension of phagosomal tubules to facilitate the docking and fusion of intracellular vesicles. Our findings identify the critical and differential functions of two groups of sorting nexins in phagosome maturation and reveal a signaling cascade initiated by phagocytic receptor CED-1, mediated by PtdIns(3)P, and executed through these sorting nexins to degrade apoptotic cells. PMID:21148288

  11. CD3-specific antibody-induced immune tolerance involves transforming growth factor-beta from phagocytes digesting apoptotic T cells.

    PubMed

    Perruche, Sylvain; Zhang, Pin; Liu, Yongzhong; Saas, Philippe; Bluestone, Jeffrey A; Chen, Wanjun

    2008-05-01

    Intact CD3-specific antibody transiently depletes large numbers of T cells and subsequently induces long-term immune tolerance. The underlying mechanisms for the systemic tolerance, however, remain unclear. We show here that treatment of normal mice with intact antibody to CD3 increases systemic transforming growth factor-beta (TGF-beta) produced by phagocytes exposed to apoptotic T cells. Among the phagocytes, macrophages and immature dendritic cells (iDCs) secrete TGF-beta upon ingestion of apoptotic T cells, which induces CD4+Foxp3+ regulatory T cells in culture and contributes to immune tolerance mediated by CD3-specific antibody in vivo. In accordance with these results, depletion of macrophages and iDCs not only abrogates CD3-specific antibody-mediated prevention of myelin oligodendrocyte glycoprotein-induced acute experimental autoimmune encephalomyelitis (EAE), but also reverses the therapeutic effects of antibody to CD3 on established disease in a model of relapsing-remitting EAE. Thus, CD3-specific antibody-induced immune tolerance is associated with TGF-beta production in phagocytes involved in clearing apoptotic T cells, which suggests that apoptosis is linked to active suppression in immune tolerance. PMID:18438416

  12. Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells.

    PubMed

    Kauntz, Henriette; Bousserouel, Souad; Goss, Francine; Raul, Francis

    2011-10-01

    Silibinin, a flavonolignan isolated from the milk thistle plant (Silybum marianum), possesses anti-neoplastic properties. In vitro and in vivo studies have recently shown that silibinin inhibits the growth of colorectal cancer (CRC). The present study investigates the mechanisms of silibinin-induced cell death using an in vitro model of human colon cancer progression, consisting of primary tumor cells (SW480) and their derived metastatic cells (SW620) isolated from a metastasis of the same patient. Silibinin induced apoptotic cell death evidenced by DNA fragmentation and activation of caspase-3 in both cell lines. Silibinin enhanced the expression (protein and mRNA) of TNF-related apoptosis-inducing ligand (TRAIL) death receptors (DR4/DR5) at the cell surface in SW480 cells, and induced their expression in TRAIL-resistant SW620 cells normally not expressing DR4/DR5. Caspase-8 and -10 were activated demonstrating the involvement of the extrinsic apoptotic pathway in silibinin-treated SW480 and SW620 cells. The protein Bid was cleaved in SW480 cells indicating a cross-talk between extrinsic and intrinsic apoptotic pathway. We demonstrated that silibinin activated also the intrinsic apoptotic pathway in both cell lines, including the perturbation of the mitochondrial membrane potential, the release of cytochrome c into the cytosol and the activation of caspase-9. Simultaneously to apoptosis, silibinin triggered an autophagic response. The inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in silibinin-treated cells. Taken together, our data show that silibinin initiated in SW480 and SW620 cells an autophagic-mediated survival response overwhelmed by the activation of both the extrinsic and intrinsic apoptotic pathways. PMID:21779837

  13. Proteases in Fas-mediated apoptosis.

    PubMed

    Zhivotovsky, B; Burgess, D H; Schlegel, J; Prn, M I; Vanags, D; Orrenius, S

    1997-01-01

    Involvement of a unique family of cysteine proteases in the multistep apoptotic process has been documented. Cloning of several mammalian genes identifies some components of this cellular response. However, it is currently unclear which protease plays a role as a signal and/or effector of apoptosis. We summarize contributions to the data concerning proteases in Fas-mediated apoptosis. PMID:9015753

  14. Rho kinase regulates fragmentation and phagocytosis of apoptotic cells

    SciTech Connect

    Orlando, Kelly A.; Stone, Nicole L.; Pittman, Randall N. . E-mail: pittman@pharm.med.upenn.edu

    2006-01-01

    During the execution phase of apoptosis, a cell undergoes cytoplasmic and nuclear changes that prepare it for death and phagocytosis. The end-point of the execution phase is condensation into a single apoptotic body or fragmentation into multiple apoptotic bodies. Fragmentation is thought to facilitate phagocytosis; however, mechanisms regulating fragmentation are unknown. An isoform of Rho kinase, ROCK-I, drives membrane blebbing through its activation of actin-myosin contraction; this raises the possibility that ROCK-I may regulate other execution phase events, such as cellular fragmentation. Here, we show that COS-7 cells fragment into a number of small apoptotic bodies during apoptosis; treating with ROCK inhibitors (Y-27632 or H-1152) prevents fragmentation. Latrunculin B and blebbistatin, drugs that interfere with actin-myosin contraction, also inhibit fragmentation. During apoptosis, ROCK-I is cleaved and activated by caspases, while ROCK-II is not activated, but rather translocates to a cytoskeletal fraction. siRNA knock-down of ROCK-I but not ROCK-II inhibits fragmentation of dying cells, consistent with ROCK-I being required for apoptotic fragmentation. Finally, cells dying in the presence of the ROCK inhibitor Y-27632 are not efficiently phagocytized. These data show that ROCK plays an essential role in fragmentation and phagocytosis of apoptotic cells.

  15. Carbocisteine promotes phagocytosis of apoptotic cells by alveolar macrophages.

    PubMed

    Inoue, Masako; Ishibashi, Yuji; Nogawa, Hisashi; Yasue, Tokutaro

    2012-02-29

    Clearance of apoptotic cells, so-called efferocytosis, by alveolar macrophages (AMs) is important for lung homeostasis and is impaired in pulmonary inflammatory diseases, such as chronic obstructive pulmonary disease and asthma. Carbocisteine, a mucoregulatory drug, corrects the contents of fucose in airway mucus and has anti-inflammatory properties in airway inflammation. Thus, we conducted the present study to better understand the anti-inflammatory properties of carbocisteine. First, we induced airway inflammation in mice with lipopolysaccharide intratracheally. Carbocisteine significantly decreased neutrophil numbers in bronchoalveolar lavage fluid at the resolution phase of inflammation, implying the promotion of neutrophil clearance. Then, we investigated whether carbocisteine would enhance the efferocytosis by AMs isolated from mice and found that this drug promoted not only the phagocytosis but also the binding of apoptotic cells to AMs in vitro. Furthermore, carbocisteine decreased the fucose residues stained with fluorescent fucose-binding lectin, Lens culinaris agglutinin, on the cell surface of AMs. We found here that removing fucose residues from cell surfaces of AMs by fucosidase markedly enhanced both the binding and phagocytosis of apoptotic cells. Finally, AMs from mice orally given carbocisteine also promoted both the binding and phagocytosis ex vivo similarly to in vitro. These results suggest that carbocisteine could promote the clearance of apoptotic cells by AMs in airway. In addition, the present findings suggest that the binding and phagocytosis of apoptotic cells may be modulated by fucose residues on the cell surface of AMs. PMID:22222820

  16. Rnd3 haploinsufficient mice are predisposed to hemodynamic stress and develop apoptotic cardiomyopathy with heart failure

    PubMed Central

    Yue, X; Yang, X; Lin, X; Yang, T; Yi, X; Dai, Y; Guo, J; Li, T; Shi, J; Wei, L; Fan, G-C; Chen, C; Chang, J

    2014-01-01

    Rho family guanosine triphosphatase (GTPase) 3 (Rnd3), a member of the small Rho GTPase family, has been suggested to regulate cell actin cytoskeleton dynamics, cell migration, and apoptosis through the Rho kinase-dependent signaling pathway. The biological function of Rnd3 in the heart is unknown. The downregulation of small GTPase Rnd3 transcripts was found in patients with end-stage heart failure. The pathological significance of Rnd3 loss in the transition to heart failure remains unexplored. To investigate the functional consequence of Rnd3 downregulation and the associated molecular mechanism, we generated Rnd3+/? haploinsufficient mice to mimic the downregulation of Rnd3 observed in the failing human heart. Rnd3+/? mice were viable; however, the mice developed heart failure after pressure overload by transverse aortic constriction (TAC). Remarkable apoptosis, increased caspase-3 activity, and elevated Rho kinase activity were detected in the Rnd3+/? haploinsufficient animal hearts. Pharmacological inhibition of Rho kinase by fasudil treatment partially improved Rnd3+/? mouse cardiac functions and attenuated myocardial apoptosis. To determine if Rho-associated coiled-coil kinase 1 (ROCK1) was responsible for Rnd3 deficiency-mediated apoptotic cardiomyopathy, we established a double-knockout mouse line, the Rnd3 haploinsufficient mice with ROCK1-null background (Rnd3+/?/ROCK1?/?). Again, genetic deletion of ROCK1 partially but not completely rescued Rnd3 deficiency-mediated heart failure phenotype. These data suggest that downregulation of Rnd3 correlates with cardiac loss of function as in heart failure patients. Animals with Rnd3 haploinsufficiency are predisposed to hemodynamic stress. Hyperactivation of Rho kinase activity is responsible in part for the apoptotic cardiomyopathy development. Further investigation of ROCK1-independent mechanisms in Rnd3-mediated cardiac remodeling should be the focus for future study. PMID:24901055

  17. Apoptotic activities of thymoquinone, an active ingredient of black seed (Nigella sativa), in cervical cancer cell lines.

    PubMed

    Ichwan, Solachuddin J A; Al-Ani, Imad M; Bilal, Hakim G; Suriyah, Wastuti H; Taher, Muhammad; Ikeda, Masa A

    2014-10-31

    Thymoquinone (TQ) is the main constituent of black seed (Nigella sativa, spp) essential oil which shows promising in vitro and in vivo anti-neoplastic activities in different tumor cell lines. However, to date there are only a few reports regarding the apoptotic effects of TQ on cervical cancer cells. Here, we report that TQ stimulated distinct apoptotic pathways in two human cervical cell lines, Siha and C33A. TQ markedly induced apoptosis as demonstrated by cell cycle analysis in both cell lines. Moreover, quantitative PCR revealed that TQ induced apoptosis in Siha cells through p53-dependent pathway as shown by elevated level of p53-mediated apoptosis target genes, whereas apoptosis in C33A cells was mainly associated with the activation of caspase-3. These results support previous findings on TQ as a potential therapeutic agent for human cervical cancer. PMID:25241984

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

    PubMed

    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. PMID:24427286

  19. Effects of vitamin E on the cinnamaldehyde-induced apoptotic mechanism in human PLC/PRF/5 cells.

    PubMed

    Wu, Shu-Jing; Ng, Lean-Teik; Lin, Chun-Ching

    2004-11-01

    1. Cinnamaldehyde has been shown to be effective in inducing cell apoptosis in a number of human cancer cells. The aim of the present study was to investigate the effect of vitamin E on the apoptotic signalling mechanism induced by cinnamaldehyde in human hepatoma PLC/PRF/5 cells. 2. Using the XTT assay, cinnamaldehyde exhibited a powerful antiproliferative effect on PLC/PRF/5 cells. Apoptosis was elicited when cells were treated with 1 micromol/L cinnamaldehyde, as characterized by the appearance of phosphatidylserine on the outer surface of the plasma membrane. 3. The apoptotic effect induced by cinnamaldehyde could be further supported by the release of cytochrome c, Smac/Diablo and Omi/HtrA2 from mitochondria to the cytosol and activation of caspase 3. Cinnamaldehyde also upregulated the expression of pro-apoptotic protein (Bax) and down-regulated the levels of anti-apoptotic proteins, such as Bcl-2 and the inhibitor of apoptosis protein family (X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein (cIAP)-1 and cIAP-2). 4. Cinnamaldehyde induces the generation of reactive oxygen species (ROS) in cells. Following the pre-incubation of PLC/PRF/5 cells with anti-oxidants, it was found that 100 micromol/L vitamin E significantly diminished the effect of cinnamaldehyde-induced apoptosis, whereas a lesser effect was seen with on 100 micromol/L N-acetyl-L-cysteine. Vitamin E effectively blocked the release of cytochrome c, Smac/Diablo and Omi/HtrA2 from mitochondria to the cytosol in cells treated with cinnamaldehyde. Vitamin E also markedly suppressed caspase 3 activation. The expression of apoptotic inhibitors (XIAP, cIAP-1, cIAP-2) and anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) proteins was affected by vitamin E pretreatment. 5. Taken together, the results suggest that cinnamaldehyde triggers apoptosis possibly through the mitochondrial pathway. Pretreatment with vitamin E markedly prevented cinnamaldehyde-mediated apoptosis, which was associated with the modulation of XIAP, cIAP-1, cIAP-2, Bcl-2 and Bax protein activity. PMID:15566391

  20. Dual apoptotic DNA fragmentation system in the fly: Drep2 is a novel nuclease of which activity is inhibited by Drep3.

    PubMed

    Park, Ok Kyeung; Park, Hyun Ho

    2012-09-21

    DNA fragmentation is the hallmark of apoptotic cells and mainly mediated by the DNA fragmentation factor DFF40(CAD)/DFF45(ICAD). DFF40 is a novel nuclease, whereas DFF45 is an inhibitor that can suppress the nuclease activity. Apoptotic DNA fragmentation in the fly is controlled by four DFF-related proteins, known as Drep1, 2, 3 and 4. However, the functions of Drep2 and Drep3 are totally unknown. Here, we found that Drep2 is a novel nuclease whose activity is inhibited by Drep3 through a tight interaction with the CIDE domain. Our results suggest that the fly has dual apoptotic DNA fragmentation systems: Drep1: Drep4 and Drep2: Drep3 complexes. PMID:22850116

  1. Neuroprotection with metformin and thymoquinone against ethanol-induced apoptotic neurodegeneration in prenatal rat cortical neurons

    PubMed Central

    2012-01-01

    Background Exposure to ethanol during early development triggers severe neuronal death by activating multiple stress pathways and causes neurological disorders, such as fetal alcohol effects or fetal alcohol syndrome. This study investigated the effect of ethanol on intracellular events that predispose developing neurons for apoptosis via calcium-mediated signaling. Although the underlying molecular mechanisms of ethanol neurotoxicity are not completely determined, mitochondrial dysfunction, altered calcium homeostasis and apoptosis-related proteins have been implicated in ethanol neurotoxicity. The present study was designed to evaluate the neuroprotective mechanisms of metformin (Met) and thymoquinone (TQ) during ethanol toxicity in rat prenatal cortical neurons at gestational day (GD) 17.5. Results We found that Met and TQ, separately and synergistically, increased cell viability after ethanol (100 mM) exposure for 12 hours and attenuated the elevation of cytosolic free calcium [Ca2+]c. Furthermore, Met and TQ maintained normal physiological mitochondrial transmembrane potential (??M), which is typically lowered by ethanol exposure. Increased cytosolic free [Ca2+]c and lowered mitochondrial transmembrane potential after ethanol exposure significantly decreased the expression of a key anti-apoptotic protein (Bcl-2), increased expression of Bax, and stimulated the release of cytochrome-c from mitochondria. Met and TQ treatment inhibited the apoptotic cascade by increasing Bcl-2 expression. These compounds also repressed the activation of caspase-9 and caspase-3 and reduced the cleavage of PARP-1. Morphological conformation of cell death was assessed by TUNEL, Fluoro-Jade-B, and PI staining. These staining methods demonstrated more cell death after ethanol treatment, while Met, TQ or Met plus TQ prevented ethanol-induced apoptotic cell death. Conclusion These findings suggested that Met and TQ are strong protective agents against ethanol-induced neuronal apoptosis in primary rat cortical neurons. The collective data demonstrated that Met and TQ have the potential to ameliorate ethanol neurotoxicity and revealed a possible protective target mechanism for the damaging effects of ethanol during early brain development. PMID:22260211

  2. Induction of discrete apoptotic pathways by bromo-substituted indirubin derivatives in invasive breast cancer cells

    SciTech Connect

    Nicolaou, Katerina A.; Liapis, Vasilis; Evdokiou, Andreas; Constantinou, Constantina; Magiatis, Prokopios; Skaltsounis, Alex L.; Koumas, Laura; Costeas, Paul A.; Constantinou, Andreas I.

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer The effects of 6BIO and 7BIO are evaluated against five breast cancer cell lines. Black-Right-Pointing-Pointer 6BIO induces a caspase dependent apoptotic effect via the intrinsic pathway. Black-Right-Pointing-Pointer 7BIO promotes G{sub 2}/M cells cycle arrest. Black-Right-Pointing-Pointer 7BIO triggers a caspase-8 mediated apoptotic pathway. Black-Right-Pointing-Pointer 7BIO triggers and a caspase independent pathway. -- Abstract: Indirubin derivatives gained interest in recent years for their anticancer and antimetastatic properties. The objective of the present study was to evaluate and compare the anticancer properties of the two novel bromo-substituted derivatives 6-bromoindirubin-3 Prime -oxime (6BIO) and 7-bromoindirubin-3 Prime -oxime (7BIO) in five different breast cancer cell lines. Cell viability assays identified that 6BIO and 7BIO are most effective in preventing the proliferation of the MDA-MB-231-TXSA breast cancer cell line from a total of five breast cancer cell lined examined. In addition it was found that the two compounds induce apoptosis via different mechanisms. 6BIO induces caspase-dependent programmed cell death through the intrinsic (mitochondrial) caspase-9 pathway. 7BIO up-regulates p21 and promotes G{sub 2}/M cell cycle arrest which is subsequently followed by the activation of two different apoptotic pathways: (a) a pathway that involves the upregulation of DR4/DR5 and activation of caspase-8 and (b) a caspase independent pathway. In conclusion, this study provides important insights regarding the molecular pathways leading to cell cycle arrest and apoptosis by two indirubin derivatives that can find clinical applications in targeted cancer therapeutics.

  3. Dual-functional bio-derived nanoparticulates for apoptotic antitumor therapy.

    PubMed

    Ding, Yang; Wang, Yazhe; Opoku-Damoah, Yaw; Wang, Cheng; Shen, Lingjia; Yin, Lifang; Zhou, Jianping

    2015-12-01

    The application of bio-derived nanoparticulates has gained a remarkable degree of interest as a promising sustained-release, site-targeted and completely biodegradable delivery system for chemotherapeutics. We hereby introduce a dual-functionalized biomimetic nanovector, cell-penetrating peptide (CPP)-anchored recombinant high density lipoproteins (cp-rHDL), which affords high payload and improved targeting of gambogic acid (GA), a therapeutic agent for apoptotic antitumor therapy. GA-loaded cp-rHDL nanoparticles (cp-rHDL/GA) consisted of hydrophobic core modulating GA, apolipoprotein A-I (apo A-I) for attractive integrating and tumor-homing, and lipophilic anchored R6H4 (RRRRRRHHHH, a pH-responsive CPP) offering a pH-controlled penetrating potential. Upon stepwise incubation with apo A-I and R6H4, cp-rHDL/GA presented several merits, including desirable physicochemical properties, superior biostability, and favorable buffering capacity resulting in proton sponge effect. Synergistic intracellular mechanism for scavenger receptor class B type I (SR-BI)-mediated direct transmembrane delivery, and pH-responsive R6H4 associated endocytotic pathway with rapid endo-lysosomal escape was also observed. This tailored cp-rHDL/GA displayed remarkable cytotoxicity and apoptotic effect via triggering p53 pathway, and provided approximately 5-fold increase in IC50 compared to free GA. Moreover, this rational biomimetic therapeutic strategy attained superior tumor accumulation and significant inhibition of tumor growth in HepG2 xenograft tumor animal models without measurable adverse effect. Results of this study demonstrated that bio-derived cp-rHDL/GA presents pH-responsive penetrating potential and efficient cellular internalization. This dual-functionalization model will open an avenue for exploration of multi-functional bio-derived drug delivery, thereby rendering potential broad applications in apoptotic anticancer therapy. PMID:26344366

  4. β-Amyloid protein (Aβ) and human amylin regulation of apoptotic genes occurs through the amylin receptor.

    PubMed

    Jhamandas, Jack H; Mactavish, David

    2012-01-01

    Deposition of amyloid-beta (Aβ) protein, a 39-43 amino acid peptide, in the brain is a major pathological feature of Alzheimer's disease (AD). We have previously provided evidence that in primary cultures of rat basal forebrain and human fetal neurons (HFNs), neurotoxic effects of oligomeric Aβ are expressed through the amylin receptor. In this study, we utilized RT-PCR arrays to compare RNA expression levels of 84 markers for pro and anti- apoptotic signalling pathways following exposure of HFNs to either Aβ(1-42) (20 μM) or human amylin (2 μM). Oligomeric Aβ(1-42) or human amylin was applied to HFNs alone or after pre-treatment of cultures with the amylin receptor antagonist, AC253. Changes in RNA levels were then quantified and compared to each other in order to identify increases or decreases in gene expression of apoptotic markers. Applications of Aβ(1-42) or human amylin, but not the inactive inverse sequence Aβ(42-1) or rat amylin, resulted in a time-dependent marked increase in mediators of apoptosis including a 10- to 30-fold elevations in caspases 3, 6, 9, BID and XIAP levels. Amylin receptor antagonists, AC253 (10 μM) or AC187 (10 μM), significantly attenuated the induction of several pro-apoptotic mediators up-regulated following exposure to Aβ(1-42) or human amylin and increased the expression of several anti-apoptotic markers. These data allow us to identify key elements in the Aβ-induced apoptosis that are blocked by antagonism of the amylin receptor and further support the potential for amylin receptor blockade as a potential therapeutic avenue in AD. PMID:21947943

  5. VDAC1 selectively transfers apoptotic Ca2+ signals to mitochondria

    PubMed Central

    De Stefani, D; Bononi, A; Romagnoli, A; Messina, A; De Pinto, V; Pinton, P; Rizzuto, R

    2012-01-01

    Voltage-dependent anion channels (VDACs) are expressed in three isoforms, with common channeling properties and different roles in cell survival. We show that VDAC1 silencing potentiates apoptotic challenges, whereas VDAC2 has the opposite effect. Although all three VDAC isoforms are equivalent in allowing mitochondrial Ca2+ loading upon agonist stimulation, VDAC1 silencing selectively impairs the transfer of the low-amplitude apoptotic Ca2+ signals. Co-immunoprecipitation experiments show that VDAC1, but not VDAC2 and VDAC3, forms complexes with IP3 receptors, an interaction that is further strengthened by apoptotic stimuli. These data highlight a non-redundant molecular route for transferring Ca2+ signals to mitochondria in apoptosis. PMID:21720385

  6. Novel functions of viral anti-apoptotic factors

    PubMed Central

    Liang, Chengyu; Oh, Byung-Ha; Jung, Jae U.

    2015-01-01

    Cellular apoptosis is of major importance in the struggle between virus and host. Although many viruses use various strategies to control the cell death machinery by encoding anti-apoptotic virulence factors, it is now becoming clear that, in addition to their role in inhibiting apoptosis, these factors function in multiple immune and metabolic pathways to promote fitness and pathogenesis. In this Progress article, we discuss novel functions of viral anti-apoptotic factors in the regulation of autophagy in the nuclear factor-?B (NF-?B) pathway and in interferon signalling, with a focus on persistent and oncogenic gammaherpesviruses. If viral anti-apoptotic proteins are to be properly exploited as targets for antiviral drugs, their diverse and complex roles should be considered. PMID:25363821

  7. Macrophages programmed by apoptotic cells inhibit epithelial-mesenchymal transition in lung alveolar epithelial cells via PGE2, PGD2, and HGF.

    PubMed

    Yoon, Young-So; Lee, Ye-Ji; Choi, Youn-Hee; Park, Young Mi; Kang, Jihee Lee

    2016-01-01

    Apoptotic cell clearance results in the release of growth factors and the action of signaling molecules involved in tissue homeostasis maintenance. Here, we investigated whether and how macrophages programmed by apoptotic cells inhibit the TGF-?1-induced Epithelial-mesenchymal transition (EMT) process in lung alveolar epithelial cells. Treatment with conditioned medium derived from macrophages exposed to apoptotic cells, but not viable or necrotic cells, inhibited TGF-?1-induced EMT, including loss of E-cadherin, synthesis of N-cadherin and ?-smooth muscle actin, and induction of EMT-activating transcription factors, such as Snail1/2, Zeb1/2, and Twist1. Exposure of macrophages to cyclooxygenase (COX-2) inhibitors (NS-398 and COX-2 siRNA) or RhoA/Rho kinase inhibitors (Y-27632 and RhoA siRNA) and LA-4 cells to antagonists of prostaglandin E2 (PGE2) receptor (EP4 [AH-23848]), PGD2 receptors (DP1 [BW-A868C] and DP2 [BAY-u3405]), or the hepatocyte growth factor (HGF) receptor c-Met (PHA-665752), reversed EMT inhibition by the conditioned medium. Additionally, we found that apoptotic cell instillation inhibited bleomycin-mediated EMT in primary mouse alveolar type II epithelial cells in vivo. Our data suggest a new model for epithelial cell homeostasis, by which the anti-EMT programming of macrophages by apoptotic cells may control the progressive fibrotic reaction via the production of potent paracrine EMT inhibitors. PMID:26875548

  8. Binding, degradation and apoptotic activity of stearoylethanolamide in rat C6 glioma cells.

    PubMed Central

    Maccarrone, Mauro; Pauselli, Riccardo; Di Rienzo, Marianna; Finazzi-Agr, Alessandro

    2002-01-01

    Stearoylethanolamide (SEA) is present in human, rat and mouse brain in amounts comparable with those of the endocannabinoid anandamide (arachidonoylethanolamide; AEA). Yet, the biological activity of SEA has never been investigated. We synthesized unlabelled and radiolabelled SEA to investigate its binding, degradation and biological activity in rat C6 glioma cells. We report that SEA binds to a specific site distinct from known cannabinoid or vanilloid receptors, and that AEA and capsazepine partly (approx. 50%) antagonized this binding. Treatment of C6 cells with SEA inhibits cellular nitric oxide synthase and does not affect adenylate cyclase, whereas treatment with cannabinoid type 1 agonist 2-arachidonoylglycerol activates the former enzyme and inhibits the latter. C6 cells also have a specific SEA membrane transporter, which is inhibited by NO, and a fatty acid amide hydrolase capable of cleaving SEA. In these cells, SEA shows pro-apoptotic activity, due to elevation of intracellular calcium, activation of the arachidonate cascade and mitochondrial uncoupling. NO further enhances SEA-induced apoptosis. Moreover, the cannabinoid type 1 receptor-mediated decrease in cAMP induced by AEA in C6 cells is potentiated by SEA, suggesting that this compound also has an 'entourage' effect. Taken together, this study shows that SEA is an endocannabinoid-like compound which binds to and is transported by new components of the endocannabinoid system. It seems noteworthy that degradation and pro-apoptotic activity of SEA are regulated by NO in a way opposite to that reported for AEA. PMID:12010121

  9. Apoptotic and anti-proliferative effects of all-trans retinoic acid

    SciTech Connect

    Zamora, Monica; Ortega, Juan Alberto; Alana, Lide; Vinas, Octavi; Mampel, Teresa . E-mail: tmampel@ub.edu

    2006-06-10

    We examined the apoptotic and anti-proliferative effects of all-trans retinoic acid (atRA) in HeLa cells. Our results demonstrated that HeLa cells were more sensitive to the anti-proliferative effects of atRA than to its apoptotic effects. Furthermore, we demonstrated that caspase inhibition attenuates cell death but does not alter the atRA-dependent reduction in cell proliferation, which suggests that atRA-induced apoptosis is independent of the arrest in cell proliferation. To check whether ANT proteins mediated these atRA effects, we transiently transfected cells with expression vectors encoding for individual ANT (adenine nucleotide translocase 1-3). Our results revealed that ANT1 and ANT3 over-expressing HeLa cells increased their atRA sensitivity. Thus, our results not only demonstrate the different functional activities of ANT isoforms, but also contribute to a better understanding of the properties of atRA as an anti-tumoral agent used in cancer therapy.

  10. Cell death stages in single apoptotic and necrotic cells monitored by Raman microspectroscopy.

    PubMed

    Brauchle, Eva; Thude, Sibylle; Brucker, Sara Y; Schenke-Layland, Katja

    2014-01-01

    Although apoptosis and necrosis have distinct features, the identification and discrimination of apoptotic and necrotic cell death in vitro is challenging. Immunocytological and biochemical assays represent the current gold standard for monitoring cell death pathways; however, these standard assays are invasive, render large numbers of cells and impede continuous monitoring experiments. In this study, both room temperature (RT)-induced apoptosis and heat-triggered necrosis were analyzed in individual Saos-2 and SW-1353 cells by utilizing Raman microspectroscopy. A targeted analysis of defined cell death modalities, including early and late apoptosis as well as necrosis, was facilitated based on the combination of Raman spectroscopy with fluorescence microscopy. Spectral shifts were identified in the two cell lines that reflect biochemical changes specific for either RT-induced apoptosis or heat-mediated necrosis. A supervised classification model specified apoptotic and necrotic cell death based on single cell Raman spectra. To conclude, Raman spectroscopy allows a non-invasive, continuous monitoring of cell death, which may help shedding new light on complex pathophysiological or drug-induced cell death processes. PMID:24732136

  11. Cell death stages in single apoptotic and necrotic cells monitored by Raman microspectroscopy

    PubMed Central

    Brauchle, Eva; Thude, Sibylle; Brucker, Sara Y.; Schenke-Layland, Katja

    2014-01-01

    Although apoptosis and necrosis have distinct features, the identification and discrimination of apoptotic and necrotic cell death in vitro is challenging. Immunocytological and biochemical assays represent the current gold standard for monitoring cell death pathways; however, these standard assays are invasive, render large numbers of cells and impede continuous monitoring experiments. In this study, both room temperature (RT)-induced apoptosis and heat-triggered necrosis were analyzed in individual Saos-2 and SW-1353 cells by utilizing Raman microspectroscopy. A targeted analysis of defined cell death modalities, including early and late apoptosis as well as necrosis, was facilitated based on the combination of Raman spectroscopy with fluorescence microscopy. Spectral shifts were identified in the two cell lines that reflect biochemical changes specific for either RT-induced apoptosis or heat-mediated necrosis. A supervised classification model specified apoptotic and necrotic cell death based on single cell Raman spectra. To conclude, Raman spectroscopy allows a non-invasive, continuous monitoring of cell death, which may help shedding new light on complex pathophysiological or drug-induced cell death processes. PMID:24732136

  12. The anti-apoptotic members of the Bcl-2 family are attractive tumor-associated antigens

    PubMed Central

    Andersen, Mads Hald

    2010-01-01

    Anti-apoptotic members of the Bcl-2 family (Bcl-2, Bcl-X(L) and Mcl-2) are pivotal regulators of apoptotic cell death. They are all highly overexpressed in cancers of different origin in which they enhance the survival of the cancer cells. Consequently, they represent prime candidates for anti-cancer therapy and specific antisense oligonucleotides or small molecule inhibitors have shown broad anti-cancer activities in pre-clinical models and are currently tested in clinical trials. In addition, immune-mediated tumor destruction is emerging as an interesting modality to treat cancer patients. Notably, spontaneous cellular immune responses against the Bcl-2 family proteins have been identified as frequent features in cancer patients underscoring that these proteins are natural targets for the immune system. Thus, Bcl-2 family may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies, alone or in the combination with conventional therapy. Here, we summarize the current knowledge of Bcl-2 family proteins as T-cell antigens, which has set the stage for the first explorative trial using these antigens in therapeutic vaccinations against cancer, and discuss future opportunities. PMID:21304176

  13. Cadmium-Induced Oxidative Stress and Apoptotic Changes in the Testis of Freshwater Crab, Sinopotamon henanense

    PubMed Central

    Wang, Lan; Xu, Tuan; Lei, Wen-wen; Liu, Dong-mei; Li, Ying-jun; Xuan, Rui-jing; Ma, Jing-jin

    2011-01-01

    Cadmium (Cd), one of the most toxic environmental and industrial pollutants, is known to exert gonadotoxic and spermiotoxic effects. In the present study, we examined the toxic effect of Cd on the testis of freshwater crab, Sinopotamon henanense. Crabs were exposed to different Cd concentrations (from 0 to 116.00 mgL?1) for 7 d. Oxidative stress and apoptotic changes in the testes were detected. The activities of SOD, GPx and CAT initially increased and subsequently decreased with increasing Cd concentrations, which was accompanied with the increase in malondialdehyde (MDA) and H2O2 content in a concentration-dependent manner. Typical morphological characteristic and physiological changes of apoptosis were observed using a variety of methods (HE staining, AO/EB double fluorescent staining, Transmission Electron Microscope observation and DNA fragmentation analysis), and the activities of caspase-3 and caspase-9 were increased in a concentration-dependent manner after Cd exposure. These results led to the conclusion that Cd could induced oxidative damage as well as apoptosis in the testis, and the apoptotic processes may be mediated via mitochondria-dependent apoptosis pathway by regulating the activities of caspase-3 and caspase-9. PMID:22132153

  14. Levels of pro-apoptotic regulator Bad and anti-apoptotic regulator Bcl-xL determine the type of the apoptotic logic gate

    PubMed Central

    2013-01-01

    Background Apoptosis is a tightly regulated process: cellular survive-or-die decisions cannot be accidental and must be unambiguous. Since the suicide program may be initiated in response to numerous stress stimuli, signals transmitted through a number of checkpoints have to be eventually integrated. Results In order to analyze possible mechanisms of the integration of multiple pro-apoptotic signals, we constructed a simple model of the Bcl-2 family regulatory module. The module collects upstream signals and processes them into life-or-death decisions by employing interactions between proteins from three subgroups of the Bcl-2 family: pro-apoptotic multidomain effectors, pro-survival multidomain restrainers, and pro-apoptotic single domain BH3-only proteins. Although the model is based on ordinary differential equations (ODEs), it demonstrates that the Bcl-2 family module behaves akin to a Boolean logic gate of the type dependent on levels of BH3-only proteins (represented by Bad) and restrainers (represented by Bcl-xL). A low level of pro-apoptotic Bad or a high level of pro-survival Bcl-xL implies gate AND, which allows for the initiation of apoptosis only when two stress stimuli are simultaneously present: the rise of the p53 killer level and dephosphorylation of kinase Akt. In turn, a high level of Bad or a low level of Bcl-xL implies gate OR, for which any of these stimuli suffices for apoptosis. Conclusions Our study sheds light on possible signal integration mechanisms in cells, and spans a bridge between modeling approaches based on ODEs and on Boolean logic. In the proposed scheme, logic gates switching results from the change of relative abundances of interacting proteins in response to signals and involves system bistability. Consequently, the regulatory system may process two analogous inputs into a digital survive-or-die decision. PMID:23883471

  15. Pro-apoptotic Sorafenib signaling in murine hepatocytes depends on malignancy and is associated with PUMA expression in vitro and in vivo

    PubMed Central

    Sonntag, R; Gassler, N; Bangen, J-M; Trautwein, C; Liedtke, C

    2014-01-01

    The multi-kinase inhibitor Sorafenib increases the survival of patients with advanced hepatocellular carcinoma (HCC). Current data suggest that Sorafenib inhibits cellular proliferation and angiogenesis and promotes apoptosis. However, the underlying pro-apoptotic molecular mechanisms are incompletely understood. Here we compared the pro-apoptotic and anti-proliferative properties of Sorafenib in murine hepatoma cells and syngeneic healthy hepatocytes in vitro and in animal models of HCC and liver regeneration in vivo. In vitro, we demonstrate that cell cycle activity and expression of anti-apoptotic Bcl-2 like proteins are similarly downregulated by Sorafenib in Hepa1-6 hepatoma cells and in syngeneic primary hepatocytes. However, Sorafenib-mediated activation of caspase-3 and induction of apoptosis were exclusively found in hepatoma cells, but not in matching primary hepatocytes. We validated these findings in vivo by applying an isograft HCC transplantation model and partial hepatectomy (PH) in C57BL/6 mice. Sorafenib treatment activated caspase-3 and thus apoptosis selectively in small tumor foci that originated from implanted Hepa1-6 cells but not in surrounding healthy hepatocytes. Similarly, Sorafenib did not induce apoptosis after PH. However, Sorafenib treatment transiently inhibited cell cycle progression and resulted in mitotic catastrophe and enhanced non-apoptotic liver injury during regeneration. Importantly, Sorafenib-mediated apoptosis in hepatoma cells was associated with the expression of p53-upregulated-modulator-of-apoptosis (PUMA). In contrast, regenerating livers after PH revealed downregulation of PUMA and were completely protected from Sorafenib-mediated apoptosis. We conclude that Sorafenib induces apoptosis selectively in hepatoma cells but not in healthy hepatocytes and can additionally increase non-apoptotic hepatocyte injury in the regenerating liver. PMID:24481444

  16. AICAR Enhances the Phagocytic Ability of Macrophages towards Apoptotic Cells through P38 Mitogen Activated Protein Kinase Activation Independent of AMP-Activated Protein Kinase

    PubMed Central

    Lee, Hyun-Jung; Lee, Seong-Heon; Choi, Jeong-Il; Bae, Hong-Beom

    2015-01-01

    Recent studies have suggested that 5-aminoimidazole-4-carboxamide-1-?-D-ribofuranoside (AICAR) increases macrophage phagocytosis through adenosine monophosphate-activated protein kinase (AMPK). However, little information is available on the effects of AICAR on the clearance of apoptotic cells by macrophages, known as efferocytosis, which is essential in maintaining tissue homeostasis and resolving inflammation. AICAR increased p38 MAPK activation and the phagocytosis of apoptotic cells by macrophages, which were inhibited by the p38 MAPK inhibitor, SB203580, the TGF-beta-activated kinase 1 (TAK1) inhibitor, (5Z)-7-oxozeaenol, and siRNA-mediated knock-down of p38?. AICAR increased phosphorylation of Akt, but the inhibition of PI3K/Akt activity using LY294002 did not affect the AICAR-induced changes in efferocytosis in macrophages. CGS15943, a non-selective adenosine receptor antagonist, did not affect AICAR-induced changes in efferocytosis, but dipyridamole, an adenosine transporter inhibitor, diminished the AICAR-mediated increases in efferocytosis. AICAR-induced p38 MAPK phosphorylation was not inhibited by the AMPK inhibitor, compound C, or siRNA-mediated knock-down of AMPK?1. Inhibition of AMPK using compound C or 5-iodotubercidin did not completely block AICAR-mediated increases in efferocytosis. Furthermore, AICAR also increased the removal of apoptotic neutrophils or thymocytes in mouse lungs. These results reveal a novel mechanism by which AICAR increases macrophage-mediated phagocytosis of apoptotic cells and suggest that AICAR may be used to treat efferocytosis-related inflammatory conditions. PMID:26020972

  17. AICAR Enhances the Phagocytic Ability of Macrophages towards Apoptotic Cells through P38 Mitogen Activated Protein Kinase Activation Independent of AMP-Activated Protein Kinase.

    PubMed

    Quan, Hui; Kim, Joung-Min; Lee, Hyun-Jung; Lee, Seong-Heon; Choi, Jeong-Il; Bae, Hong-Beom

    2015-01-01

    Recent studies have suggested that 5-aminoimidazole-4-carboxamide-1-?-D-ribofuranoside (AICAR) increases macrophage phagocytosis through adenosine monophosphate-activated protein kinase (AMPK). However, little information is available on the effects of AICAR on the clearance of apoptotic cells by macrophages, known as efferocytosis, which is essential in maintaining tissue homeostasis and resolving inflammation. AICAR increased p38 MAPK activation and the phagocytosis of apoptotic cells by macrophages, which were inhibited by the p38 MAPK inhibitor, SB203580, the TGF-beta-activated kinase 1 (TAK1) inhibitor, (5Z)-7-oxozeaenol, and siRNA-mediated knock-down of p38?. AICAR increased phosphorylation of Akt, but the inhibition of PI3K/Akt activity using LY294002 did not affect the AICAR-induced changes in efferocytosis in macrophages. CGS15943, a non-selective adenosine receptor antagonist, did not affect AICAR-induced changes in efferocytosis, but dipyridamole, an adenosine transporter inhibitor, diminished the AICAR-mediated increases in efferocytosis. AICAR-induced p38 MAPK phosphorylation was not inhibited by the AMPK inhibitor, compound C, or siRNA-mediated knock-down of AMPK?1. Inhibition of AMPK using compound C or 5'-iodotubercidin did not completely block AICAR-mediated increases in efferocytosis. Furthermore, AICAR also increased the removal of apoptotic neutrophils or thymocytes in mouse lungs. These results reveal a novel mechanism by which AICAR increases macrophage-mediated phagocytosis of apoptotic cells and suggest that AICAR may be used to treat efferocytosis-related inflammatory conditions. PMID:26020972

  18. Macrophage migration inhibitory factor interacts with HBx and inhibits its apoptotic activity

    SciTech Connect

    Zhang Shimeng; Lin Ruxian; Zhou Zhe; Wen Siyuan; Lin Li; Chen Suhong; Shan Yajun; Cong Yuwen; Wang Shengqi . E-mail: sqwang@nic.bmi.ac.cn

    2006-04-07

    HBx, a transcriptional transactivating protein of hepatitis B virus (HBV), is required for viral infection and has been implicated in virus-mediated liver oncogenesis. However, the precise molecular mechanism remains largely elusive. We used the yeast two-hybrid system to identify that HBx interacts with MIF directly. Macrophage migration inhibitory factor (MIF) is implicated in the regulation of inflammation, cell growth, and even tumor formation. The interaction between HBx and MIF was verified with co-immunoprecipitation, GST pull-down, and cellular colocalization. The expression of MIF was up-regulated in HBV particle producing cell 2.2.15 compared with HepG2 cell. Both HBx and MIF cause HepG2 cell G /G{sub 1} phase arrest, proliferation inhibition, and apoptosis. However, MIF can counteract the apoptotic effect of HBx. These results may provide evidence to explain the link between HBV infection and hepatocellular carcinoma.

  19. Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores.

    PubMed

    Salvador-Gallego, Raquel; Mund, Markus; Cosentino, Katia; Schneider, Jale; Unsay, Joseph; Schraermeyer, Ulrich; Engelhardt, Johann; Ries, Jonas; Garca-Sez, Ana J

    2016-02-15

    Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual-color single-molecule localization-based super-resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc-shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane. PMID:26783362

  20. Baicalin Reverses Depressive-Like Behaviours and Regulates Apoptotic Signalling Induced by Olfactory Bulbectomy.

    PubMed

    Yu, Hai-Yang; Yin, Zhu-Jun; Yang, Shui-Jin; Ma, Shi-Ping; Qu, Rong

    2016-03-01

    Apoptosis is thought to be involved in neurological disorders including major depression. In this study, we examined whether the polyphenolic compound baicalin could decrease apoptosis in the olfactory bulbectomy (OBX) depression rat model. OBX rats exhibited decreased performance in depression-like behavioural tests and showed evidence of increased oxidative stress, decreased synaptophysin expression, and hippocampal apoptosis. Treatment with baicalin (20 and 40 mg/kg) significantly reversed all of these changes. Baicalin modulated the levels or activity of malondialdehyde, superoxide dismutase, and glutathione peroxidase and prevented apoptotic protease-activating factor-1 expression, effectively suppressing caspase-mediated apoptosis signalling cascades. Our results demonstrate that baicalin has potent antidepressant activity, likely because of its ability to suppress apoptosis. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26681067

  1. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases.

    PubMed

    Dlamini, Zodwa; Tshidino, Shonisani C; Hull, Rodney

    2015-01-01

    Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets. PMID:26580598

  2. The apoptotic thanatotranscriptome associated with the liver of cadavers.

    PubMed

    Javan, Gulnaz T; Can, Ismail; Finley, Sheree J; Soni, Shivani

    2015-12-01

    Gene expression investigations are well-established components of ante mortem studies with broad applications ranging from elucidating basic mechanisms responsible for normal physiological processes to discovering therapeutic targets in pathophysiological conditions. However, gene expression studies and their application in the medico-legal field are still in their infancy. Therefore, the present study focuses on RNA using PCR array in the analysis of gene expression associated with tissues taken from actual criminal cases. RNA was extracted from the liver tissues of bodies with PMIs between 6 and 48h. The results demonstrated that mRNA was stable up to 48h postmortem. Further, as cell death is an indispensable and necessary part of the biological life cycle, apoptotic gene expression profiles were investigated. The gene expression related to the programmed cell death found in body tissues after death is defined as the apoptotic thanatotranscriptome (thanatos-, Greek for death). On comparison of control and decaying tissues, the results show that with time, pro-apoptotic genes such as caspases are up-regulated and the expression of genes responsible for anti-apoptosis such as BCL2 and BAG3 were down-regulated. Thus, this current work gives a unique perspective of the apoptotic thanatotranscriptome that is affected after death. Up to the present time, gene expression in bodies from criminal cases has not been reported in literature using PCR array techniques. Thus, this thanatotranscriptome study provides insight into postmortem gene activity with potential applications in medico-legal investigations. PMID:26318598

  3. Regulation of Apoptotic Endonucleases by EndoG.

    PubMed

    Zhdanov, Dmitry D; Fahmi, Tariq; Wang, Xiaoying; Apostolov, Eugene O; Sokolov, Nikolai N; Javadov, Sabzali; Basnakian, Alexei G

    2015-05-01

    Cells contain several apoptotic endonucleases, which appear to act simultaneously before and after cell death by destroying the host cell DNA. It is largely unknown how the endonucleases are being induced and whether they can regulate each other. This study was performed to determine whether apoptotic mitochondrial endonuclease G (EndoG) can regulate expression of other apoptotic endonucleases. The study showed that overexpression of mature EndoG in kidney tubular epithelial NRK-52E cells can increase expression of caspase-activated DNase (CAD) and four endonucleases that belong to DNase I group including DNase I, DNase X, DNase IL2, and DNase ?, but not endonucleases of the DNase 2 group. The induction of DNase I-type endonucleases was associated with DNA degradation in promoter/exon 1 regions of the endonuclease genes. These results together with findings on colocalization of immunostained endonucleases and TUNEL suggest that DNA fragmentation after EndoG overexpression was caused by DNase I endonucleases and CAD in addition to EndoG itself. Overall, these data provide first evidence for the existence of the integral network of apoptotic endonucleases regulated by EndoG. PMID:25849439

  4. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    PubMed Central

    Dlamini, Zodwa; Tshidino, Shonisani C.; Hull, Rodney

    2015-01-01

    Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets. PMID:26580598

  5. Monitoring circulating apoptotic cells by in-vivo flow cytometry

    NASA Astrophysics Data System (ADS)

    Wei, Xunbin; Tan, Yuan; Chen, Yun; Zhang, Li; Li, Yan; Liu, Guangda; Wu, Bin; Wang, Chen

    2008-02-01

    Chemotherapies currently constitute one main venue of cancer treatment. For a large number of adult and elderly patients, however, treatment options are poor. These patients may suffer from disease that is resistant to conventional chemotherapy or may not be candidates for curative therapies because of advanced age or poor medical conditions. To control disease in these patients, new therapies must be developed that are selectively targeted to unique characteristics of tumor cell growth and metastasis. A reliable early evaluation and prediction of response to the chemotherapy is critical to its success. Chemotherapies induce apoptosis in tumor cells and a portion of such apoptotic cancer cells may be present in the circulation. However, the fate of circulating tumor cells is difficult to assess with conventional methods that require blood sampling. We report the in situ measurement of circulating apoptotic cells in live animals using in vivo flow cytometry, a novel method that enables real-time detection and quantification of circulating cells without blood extraction. Apoptotic cells are rapidly cleared from the circulation with a half-life of ~10 minutes. Real-time monitoring of circulating apoptotic cells can be useful for detecting early changes in disease processes, as well as for monitoring response to therapeutic intervention.

  6. The peculiar apoptotic behavior of skeletal muscle cells.

    PubMed

    Salucci, Sara; Burattini, Sabrina; Baldassarri, Valentina; Battistelli, Michela; Canonico, Barbara; Valmori, Aurelio; Papa, Stefano; Falcieri, Elisabetta

    2013-08-01

    Apoptosis plays an active role in maintaining skeletal muscle homeostasis. Its deregulation is involved in several skeletal muscle disorders such as dystrophies, myopathies, disuse and sarcopenia. The aim of this work was to study in vitro the apoptotic behavior induced by etoposide, staurosporine and hydrogen peroxide in the C2C12 skeletal muscle cell line, comparing myoblast vs myotube sensitivity, investigated by means of morphological and cytofluorimetric analyses. Myotubes appeared more resistant than myoblasts to apoptotic induction. In myoblasts treated with etoposide, nuclei with chromatin condensation were observed, in the presence of a diffuse DNA fragmentation, as shown by confocal microscopy. The latter also appeared in myotubes, where apoptotic and normal nuclei coexisted inside the same syncytium. After staurosporine treatment, myobalsts evidenced late apoptotic features and a high number of TUNEL-positive nuclei. Secondary necrosis appeared in myotubes, where myonuclei with cleaved DNA again coexisted with normal myonuclei. After H?O? exposure, myotubes, differently from myoblasts, showed a poor sensitivity to cell death. Intriguingly, autophagic granules appeared abundantly in myotubes after each treatment. In myotubes, mitochondria were better preserved than in myoblasts since those which were damaged were probably degraded through autophagic processes. These findings demonstrate a scarce sensitivity of myotubes to apoptotic stimuli due to acquisition of an apoptosis-resistant phenotype during differentiation. The presence of nuclear-dependent "territorial" death domains in the syncytium could explain a slower death of myotubes compared to mononucleated cells. In addition, autophagy could preserve and protect muscle cell integrity against chemical stimuli, making C2C12 cells, in particular myotubes, more resistant to apoptosis induction. PMID:23400589

  7. Follicular dendritic cells control engulfment of apoptotic bodies by secreting Mfge8

    PubMed Central

    Kranich, Jan; Krautler, Nike Julia; Heinen, Ernst; Polymenidou, Magdalini; Bridel, Claire; Schildknecht, Anita; Huber, Christoph; Kosco-Vilbois, Marie H.; Zinkernagel, Rolf; Miele, Gino; Aguzzi, Adriano

    2008-01-01

    The secreted phosphatidylserine-binding protein milk fat globule epidermal growth factor 8 (Mfge8) mediates engulfment of apoptotic germinal center B cells by tingible-body macrophages (TBM?s). Impairment of this process can contribute to autoimmunity. We show that Mfge8 is identical to the mouse follicular dendritic cell (FDC) marker FDC-M1. In bone-marrow chimeras between wild-type and Mfge8?/? mice, all splenic Mfge8 was derived from FDCs rather than TBM?s. However, Mfge8?/? TBM?s acquired and displayed Mfge8 only when embedded in Mfge8+/+ stroma, or when situated in lymph nodes draining exogenous recombinant Mfge8. These findings indicate a licensing role for FDCs in TBM?-mediated removal of excess B cells. Lymphotoxin-deficient mice lacked FDCs and splenic Mfge8, and suffer from autoimmunity similar to Mfge8?/? mice. Hence, FDCs facilitate TBM?-mediated corpse removal, and their malfunction may be involved in autoimmunity. PMID:18490487

  8. Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization but partially affects its apoptotic activity

    SciTech Connect

    Lee, Y.-H.; Cheng, C.-M.; Chang, Y.-F.; Wang, T.-Y.; Yuo, C.-Y.; E-mail: m815006@kmu.edu.tw

    2007-03-09

    Apoptin, a chicken anemia virus-encoded protein, induces apoptosis in human tumor cells but not in normal cells. In addition, Apoptin also exhibits tumor-specific nuclear localization and tumor-specific phosphorylation on threonine 108 (T108). Here, we studied the effects of T108 phosphorylation on the tumor-specific nuclear localization and apoptotic activity of Apoptin. We first showed that a hemagglutinin (HA)-tagged Apoptin, but not the green fluorescent protein-fused Apoptin used in many previous studies, exhibited the same intracellular distribution pattern as native Apoptin. We then made and analyzed an HA-Apoptin mutant with its T108 phosphorylation site abolished. We found that Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization and abolishing the T108 phosphorylation of Apoptin does affect its apoptotic activity in tumor cells but only partially. Our results support the previous finding that Apoptin contains two distinct apoptosis domains located separately at the N- and C-terminal regions and suggest that the T108 phosphorylation may only be required for the apoptotic activity mediated through the C-terminal apoptosis domain.

  9. Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains.

    PubMed

    Iyer, S; Bell, F; Westphal, D; Anwari, K; Gulbis, J; Smith, B J; Dewson, G; Kluck, R M

    2015-10-01

    Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak ?9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that ?9 does not line a pore. Bak (and Bax) activation allowed linkage of ?9 to neighboring ?9 segments, identifying an ?9:?9 interface in Bak (and Bax) oligomers. Although the linkage pattern along ?9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The ?9:?9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when ?9:?9 linkage in the membrane was combined with ?6:?6 linkage on the membrane surface, the ?6-?9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore. PMID:25744027

  10. Externalization and recognition by macrophages of large subunit of eukaryotic translation initiation factor 3 in apoptotic cells

    SciTech Connect

    Nakai, Yuji; Shiratsuchi, Akiko; Manaka, Junko; Nakayama, Hiroshi; Takio, Koji; Zhang Jianting; Suganuma, Tatsuo; Nakanishi, Yoshinobu . E-mail: nakanaka@kenroku.kanazawa-u.ac.jp

    2005-09-10

    We previously isolated a monoclonal antibody named PH2 that inhibits phosphatidylserine-mediated phagocytosis of apoptotic cells by macrophages [C. Fujii, A. Shiratsuchi, J. Manaka, S. Yonehara, Y. Nakanishi. Cell Death Differ. 8 (2001) 1113-1122]. We report here the identification of the cognate antigen. A protein bound by PH2 in Western blotting was identified as the 170-kDa subunit of eukaryotic translation initiation factor 3 (eIF3 p170/eIF3a). When eIF3a was expressed in a culture cell line as a protein fused to green fluorescence protein, the fusion protein was detected at the cell surface only after the induction of apoptosis. The same phenomenon was seen when the localization of endogenous eIF3a was determined using anti-eIF3a antibody, and eIF3a seemed to be partially degraded during apoptosis. Furthermore, bacterially expressed N-terminal half of eIF3a fused to glutathione S-transferase bound to the surface of macrophages and inhibited phagocytosis of apoptotic cells by macrophages when it was added to phagocytosis reactions. These results collectively suggest that eIF3a translocates to the cell surface upon apoptosis, probably after partial degradation, and bridges apoptotic cells and macrophages to enhance phagocytosis.

  11. Functional antagonism between pro-apoptotic BIM and anti-apoptotic BCL-XL in MYC-induced lymphomagenesis.

    PubMed

    Delbridge, A R D; Grabow, S; Bouillet, P; Adams, J M; Strasser, A

    2015-04-01

    Genomic analyses revealed that many cancers have acquired abnormalities in their expression of pro- or anti-apoptotic members of the BCL-2 protein family. It is, however, unknown whether changes in pro- or anti-apoptotic BCL-2 family members have similar impact on tumorigenesis or whether changes in one subgroup have disproportionate impact. We compared the consequences of concomitant loss of anti-apoptotic Bclx and pro-apoptotic Bim on MYC-induced lymphomagenesis. Whereas only loss of both Bclx alleles markedly forestalled tumorigenesis, loss of a single Bim allele overcame this blockade. Conversely, loss of even a single Bim allele sufficed to substantially accelerate lymphomagenesis, and only loss of both but not loss of a single allele of Bclx could attenuate this acceleration. The evidence that modest (two-fold) monoallelic changes in the expression of at least some BH3-only proteins can profoundly impact tumorigenesis suggests that such aberrations, imposed by epigenetic or genetic changes, may expedite tumorigenesis more effectively than elevated expression of pro-survival BCL-2 family members. These findings further our understanding of the mechanisms of lymphomagenesis and possibly also cancer therapy. PMID:24858047

  12. Apoptotic cell death and its relationship to gastric carcinogenesis

    PubMed Central

    Bir, Ferda; Calli-Demirkan, Nese; Tufan, A Cevik; Akbulut, Metin; Satiroglu-Tufan, N Lale

    2007-01-01

    AIM: To investigate the apoptotic process of cells within the intestinal metaplasia areas co-localizing with chronic gastritis and gastric carcinomas and to analyze the involvement of proteins regulating apoptosis in the process of intestinal metaplasia related gastric carcinogenesis. METHODS: Forty-two gastric carcinoma and seventeen chronic gastritis cases were included in this study. All cases were examined for the existence of intestinal metaplasia. Ten cases randomly selected from each group were processed for TUNEL assay. TUNEL positive cells within the intestinal metaplasia areas, co-localizing either to gastric carcinoma or chronic gastritis, were counted and converted to apoptotic indices. In addition, p53, bcl-2 and bax expression patterns within these tissues were analyzed on the basis of immunohistochemistry. RESULTS: Twenty-eight of the cases were intestinal and 14 of the cases were diffuse type adenocarcinomas. 64% (27/42) of the gastric carcinoma cases had intestinal metaplasia. Intestinal metaplasia co-localized more with intestinal type carcinomas compared with diffuse type carcinomas [75% (21/28) vs 42% (6/14), respectively; P ? 0.05]. The mean apoptotic index in tumor cells was 0.70 0.08. The mean apoptotic index in intestinal metaplasias co-localizing to tumors was significantly higher than that of intestinal metaplasias co-localizing to chronic gastritis (0.70 0.03 vs 0.09 0.01, respectively; P ? 0.05). p53 positivity was not observed in areas of intestinal metaplasia adjacent to tumors or chronic gastritis. Intestinal metaplasia areas adjacent to tumors showed lower cytoplasmic bcl-2 positivity compared to intestinal metaplasia areas adjacent to chronic gastritis [55.5% (15/27) vs 70.5% (12/17), respectively]. On the other hand, intestinal metaplasia areas adjacent to tumors showed significantly higher cytoplasmic bax positivity compared to intestinal metaplasia areas adjacent to chronic gastritis [44.4% (12/27) vs 11.7% (2/17), respectively; P ? 0.05]. CONCLUSION: Existence of apoptotic cells on the basis of TUNEL positivity is shown in intestinal metaplasias co-localizing to both diffuse and intestinal type gastric cancers in this study. Our results also suggested bax expression dependent induction of apoptosis especially in intestinal metaplasia areas adjacent to tumors. These findings strongly support the involvement of apoptotic mechanisms in the process of gastric carcinogenesis especially in the transition from intestinal metaplasia to gastric cancer. It may be suggested that induction of apoptosis in intestinal metaplasia areas adjacent to tumors may involve different mechanisms than induction by chronic inflammation. PMID:17589896

  13. Cardioprotective activity of urocortin by preventing caspase-independent, non-apoptotic death in cultured neonatal rat cardiomyocytes exposed to ischemia

    SciTech Connect

    Takatani-Nakase, Tomoka; Takahashi, Koichi

    2010-11-12

    Research highlights: {yields} Ischemia induces high level of iPLA{sub 2} resulting in caspase-independent myocyte death. {yields} Urocortin causes iPLA{sub 2} down-regulation leading to avoidance of non-apoptotic death. {yields} The survival-promoting effect of urocortin is abrogated by CRH receptor antagonist. -- Abstract: Caspase-independent, non-apoptotic cell death in ischemic heart disease is considered to be one of the important therapeutic targets, however, the detailed mechanisms of this cell death process are not clear. In this study, we investigated the mechanisms of non-apoptotic cell death in cultured neonatal rat cardiomyocytes during ischemia, and the cardioprotection by preventing the mechanisms. We found that ischemia caused elevation of the phospholipase A{sub 2} (iPLA{sub 2}) expression in the myocytes, leading to distinctive non-apoptotic nuclear shrinkage, and cell death. Moreover, we investigated whether the potent cardioprotective corticotropin-releasing hormone (CRH), urocortin, which had been less focused on non-apoptotic cell death, inhibits the ischemic myocyte death. Ischemia-augmented nuclear shrinkage of the myocytes was suppressed by the pretreatment of {approx}10 nM urocortin before the cells were exposed to ischemia. Urocortin could significantly suppress the expression and activity of iPLA{sub 2}, resulting in preventing the ischemia-induced cell death. The survival-promoting effect of urocortin was abrogated by the CRH receptor antagonist astressin. These findings provide the first evidence linking the targets of the urocortin-mediated cardioprotection to the suppression of the caspase-independent, non-apoptotic death in cardiac myocytes exposed to ischemia.

  14. Manipulating the apoptotic pathway: potential therapeutics for cancer patients

    PubMed Central

    Bates, Darcy J P; Lewis, Lionel D

    2013-01-01

    This review summarizes the current state of scientific understanding of the apoptosis pathway, with a focus on the proteins involved in the pathway, their interactions and functions. This forms the rationale for detailing the preclinical and clinical pharmacology of drugs that modulate the pivotal proteins in this pathway, with emphasis on drugs that are furthest advanced in clinical development as anticancer agents. There is a focus on describing drugs that modulate three of the most promising targets in the apoptosis pathway, namely antibodies that bind and activate the death receptors, small molecules that inhibit the anti-apoptotic Bcl-2 family proteins, and small molecules and antisense oligonucleotides that inactivate the inhibitors of apoptosis, all of which drive the equilibrium of the apoptotic pathway towards apoptosis. These structurally different yet functionally related groups of drugs represent a promising novel approach to anticancer therapeutics whether used as monotherapy or in combination with either classical cytotoxic or other molecularly targeted anticancer agents. PMID:23782006

  15. Apoptotic Death of Cancer Stem Cells for Cancer Therapy

    PubMed Central

    He, Ying-Chun; Zhou, Fang-Liang; Shen, Yi; Liao, Duan-Fang; Cao, Deliang

    2014-01-01

    Cancer stem cells (CSCs) play crucial roles in tumor progression, chemo- and radiotherapy resistance, and recurrence. Recent studies on CSCs have advanced understanding of molecular oncology and development of novel therapeutic strategies. This review article updates the hypothesis and paradigm of CSCs with a focus on major signaling pathways and effectors that regulate CSC apoptosis. Selective CSC apoptotic inducers are introduced and their therapeutic potentials are discussed. These include synthetic and natural compounds, antibodies and recombinant proteins, and oligonucleotides. PMID:24823879

  16. The major apoptotic pathway activated and suppressed by poliovirus.

    PubMed

    Belov, George A; Romanova, Lyudmila I; Tolskaya, Elena A; Kolesnikova, Marina S; Lazebnik, Yuri A; Agol, Vadim I

    2003-01-01

    Cells respond to poliovirus infection by switching on the apoptotic program, implementation of which is usually suppressed by viral antiapoptotic functions. We show here that poliovirus infection of HeLa cells or derivatives of MCF-7 cells was accompanied by the efflux of cytochrome c from mitochondria. This efflux occurred during both abortive infection (e.g., interrupted by guanidine-HCl and ending with apoptosis) and productive infection (leading to cytopathic effect). The former type of infection, but not the latter, was accompanied by truncation of the proapoptotic protein Bid. The virus-triggered cytochrome c efflux was suppressed by overexpression of Bcl-2. Both abortive and productive infections also resulted in a decreased level of procaspase-9, as revealed by Western blotting. In the former case, this decrease was accompanied by the accumulation of a protein with the electrophoretic mobility of active caspase-9. In contrast, in the productively infected cells, the latter protein was absent but caspase-9-related polypeptides with altered mobility could be detected. Both caspase-9 and caspase-3 were shown to be essential for the development of such hallmarks of virus-induced apoptosis as chromatin condensation, DNA degradation, and nuclear fragmentation. These and some other results suggest the following scenario. Poliovirus infection activates the apoptotic pathway, involving mitochondrial damage, cytochrome c efflux, and consecutive activation of caspase-9 and caspase-3. The apoptotic signal appears to be amplified by a loop which includes secondary processing of Bid. The implementation of the apoptotic program in productively infected cells may be suppressed, however, by the viral antiapoptotic functions, which act at a step(s) downstream of the cytochrome c efflux. The suppression appears to be caused, at least in part, by aberrant processing and degradation of procaspase-9. PMID:12477809

  17. A mathematical model for apoptotic switch in Drosophila

    NASA Astrophysics Data System (ADS)

    Ziraldo, Riccardo; Ma, Lan

    2015-10-01

    Apoptosis is an evolutionarily-conserved process of autonomous cell death. The molecular switch mechanism underlying the fate decision of apoptosis in mammalian cells has been intensively studied by mathematical modeling. In contrast, the apoptotic switch in invertebrates, with highly conserved signaling proteins and pathway, remains poorly understood mechanistically and calls for theoretical elucidation. In this study, we develop a mathematical model of the apoptosis pathway in Drosophila and compare the switch mechanism to that in mammals. Enumeration of the elementary reactions for the model demonstrates that the molecular interactions among the signaling components are considerably different from their mammalian counterparts. A notable distinction in network organization is that the direct positive feedback from the effector caspase (EC) to the initiator caspase in mammalian pathway is replaced by a double-negative regulation in Drosophila. The model is calibrated by experimental input-output relationship and the simulated trajectories exhibit all-or-none bimodal behavior. Bifurcation diagrams confirm that the model of Drosophila apoptotic switch possesses bistability, a well-recognized feature for an apoptosis system. Since the apoptotic protease activating factor-1 (APAF1) induced irreversible activation of caspase is an essential and beneficial property for the mammalian apoptotic switch, we perform analysis of the bistable caspase activation with respect to the input of DARK protein, the Drosophila homolog of APAF1. Interestingly, this bistable behavior in Drosophila is predicted to be reversible. Further analysis suggests that the mechanism underlying the systems property of reversibility is the double-negative feedback from the EC to the initiator caspase. Using theoretical modeling, our study proposes plausible evolution of the switch mechanism for apoptosis between organisms.

  18. Apoptotic lymphocytes induce progenitor cell mobilization after exercise.

    PubMed

    Mooren, Frank C; Krger, Karsten

    2015-07-15

    There is evidence that apoptotic cells and their components have immunmodulatory properties and signaling function. The present study investigated first whether exercise-induced apoptosis and exercise-induced mobilization of progenitor cells are similarly affected by subjects' training status and, second, whether the appearance of dying cells in the circulation might mobilize progenitor cells. CD1 SWISS mice were subjected to a 10-wk endurance training using free wheel running or served as untrained controls. Mice of both groups performed an intensive exercise test after the training period at a velocity corresponding to 80% maximal oxygen uptake for 30 min. Cells from blood and bone marrow were analyzed, and apoptosis and number of progenitor cells determined via flow cytometry. In a second experiment, apoptotic cells were transferred into recipient mice, and mobilization of progenitor cells was analyzed while vital cells served as controls. In untrained animals, the exhaustive exercise was followed by an enhanced rate of annexin V positive CD3(+) cells in blood and bone marrow (P < 0.05), whereas no increase was found in trained mice. Similarly, exercise mobilized Sca-1(+)/c-kit(+) and Sca-1(+)/Flk(+) cells in untrained (P < 0.05) but not trained mice. Furthermore, application of apoptotic cells and their supernatant mobilized Sca-1(+)/c-kit(+) cells into the blood (P < 0.05), whereas Sca-1(+)/Flk(+) cells were not affected. The present study demonstrated that both lymphocyte apoptosis, as well as mobilization of progenitor cells are similarly related to training status. Furthermore, apoptotic cells seem to induce signals that effectively mobilize hematopoietic progenitor cells. The relevance of this effect for the adaptation to exercise stimuli remains to be shown. PMID:26023229

  19. The complexity of apoptotic cell death in mollusks: An update.

    PubMed

    Romero, A; Novoa, B; Figueras, A

    2015-09-01

    Apoptosis is a type of programmed cell death that produces changes in cell morphology and in biochemical intracellular processes without inflammatory reactions. The components of the apoptotic pathways are conserved throughout evolution. Caspases are key molecules involved in the transduction of the death signal and are responsible for many of the biochemical and morphological changes associated with apoptosis. Nowadays, It is known that caspases are activated through two major apoptotic pathways (the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway), but there are also evidences of at least other alternative pathway (the perforin/granzyme pathway). Apoptosis in mollusks seems to be similar in complexity to apoptosis in vertebrates but also has unique features maybe related to their recurrent exposure to environmental changes, pollutants, pathogens and also related to the sedentary nature of some stages in the life cycle of mollusks bivalves and gastropods. As in other animals, apoptotic process is involved in the maintenance of tissue homeostasis and also constitutes an important immune response that can be triggered by a variety of stimuli, including cytokines, hormones, toxic insults, viruses, and protozoan parasites. The main goal of this work is to present the current knowledge of the molecular mechanisms of apoptosis in mollusks and to highlight those steps that need further study. PMID:25862972

  20. PDT-apoptotic tumor cells induce macrophage immune response

    NASA Astrophysics Data System (ADS)

    Zhou, Fei-fan; Xing, Da; Chen, Wei R.

    2008-02-01

    Photodynamic therapy (PDT) functions as a cancer therapy through two major cell death mechanisms: apoptosis and necrosis. Immunological responses induced by PDT has been mainly associated with necrosis while apoptosis associated immune responses have not fully investigated. Heat shock proteins (HSPs) play an important role in regulating immune responses. In present study, we studied whether apoptotic tumor cells could induce immune response and how the HSP70 regulates immune response. The endocytosis of tumor cells by the activated macrophages was observed at single cell level by LSM. The TNF-? release of macrophages induced by co-incubated with PDT-apoptotic tumor cells was detected by ELISA. We found that apoptotic tumor cells treated by PDT could activate the macrophages, and the immune effect decreased evidently when HSP70 was blocked. These findings not only show that apoptosis can induce immunological responses, but also show HSP70 may serves as a danger signal for immune cells and induce immune responses to regulate the efficacy of PDT.

  1. Apoptotic cell signaling in cancer progression and therapy

    PubMed Central

    Plati, Jessica; Bucur, Octavian; Khosravi-Far, Roya

    2011-01-01

    Apoptosis is a tightly regulated cell suicide program that plays an essential role in the development and maintenance of tissue homeostasis by eliminating unnecessary or harmful cells. Impairment of this native defense mechanism promotes aberrant cellular proliferation and the accumulation of genetic defects, ultimately resulting in tumorigenesis, and frequently confers drug resistance to cancer cells. The regulation of apoptosis at several levels is essential to maintain the delicate balance between cellular survival and death signaling that is required to prevent disease. Complex networks of signaling pathways act to promote or inhibit apoptosis in response to various cues. Apoptosis can be triggered by signals from within the cell, such as genotoxic stress, or by extrinsic signals, such as the binding of ligands to cell surface death receptors. Various upstream signaling pathways can modulate apoptosis by converging on, and thereby altering the activity of, common central control points within the apoptotic signaling pathways, which involve the BCL-2 family proteins, inhibitor of apoptosis (IAP) proteins, and FLICE-inhibitory protein (c-FLIP). This review highlights the role of these fundamental regulators of apoptosis in the context of both normal apoptotic signaling mechanisms and dysregulated apoptotic pathways that can render cancer cells resistant to cell death. In addition, therapeutic strategies aimed at modulating the activity of BCL-2 family proteins, IAPs, and c-FLIP for the targeted induction of apoptosis are briefly discussed. PMID:21340093

  2. In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins.

    PubMed

    Senft, D; Weber, A; Saathoff, F; Berking, C; Heppt, M V; Kammerbauer, C; Rothenfusser, S; Kellner, S; Kurgyis, Z; Besch, R; Hcker, G

    2015-01-01

    Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak. PMID:26610208

  3. In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins

    PubMed Central

    Senft, D; Weber, A; Saathoff, F; Berking, C; Heppt, M V; Kammerbauer, C; Rothenfusser, S; Kellner, S; Kurgyis, Z; Besch, R; Hcker, G

    2015-01-01

    Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak. PMID:26610208

  4. Apoptotic neurodegeneration in the context of traumatic injury to the developing brain.

    TOXLINE Toxicology Bibliographic Information

    Bittigau P; Sifringer M; Felderhoff-Mueser U; Ikonomidou C

    2004-10-01

    Head trauma is the leading cause of death and disability in the pediatric population. Some recent studies on neuropathological and biochemical features of traumatic injury to the developing brain revealed interesting aspects and potential targets for future research. Trauma triggers both excitotoxic and apoptotic neurodegeneration in the developing rat brain. Apoptotic neurodegeneration occurs in a delayed fashion over several days and contributes in an age-dependent fashion to neuropathologic outcome following head trauma, with the immature brain being exceedingly sensitive. Biochemical studies indicate that both the extrinsic and the intrinsic apoptotic pathways are involved in pathogenesis of apoptotic cell death following trauma in the developing brain and that caspase inhibition ameliorates apoptotic neurodegeneration in an infant head trauma model. Given the major contribution of apoptotic neurodegeneration to neuropathologic outcome following trauma to the developing brain, interference with apoptotic pathways may comprise a potential therapeutic target in pediatric traumatic brain injury.

  5. Apoptotic neurodegeneration in the context of traumatic injury to the developing brain.

    PubMed

    Bittigau, Petra; Sifringer, Marco; Felderhoff-Mueser, Ursula; Ikonomidou, Chrysanthy

    2004-10-01

    Head trauma is the leading cause of death and disability in the pediatric population. Some recent studies on neuropathological and biochemical features of traumatic injury to the developing brain revealed interesting aspects and potential targets for future research. Trauma triggers both excitotoxic and apoptotic neurodegeneration in the developing rat brain. Apoptotic neurodegeneration occurs in a delayed fashion over several days and contributes in an age-dependent fashion to neuropathologic outcome following head trauma, with the immature brain being exceedingly sensitive. Biochemical studies indicate that both the extrinsic and the intrinsic apoptotic pathways are involved in pathogenesis of apoptotic cell death following trauma in the developing brain and that caspase inhibition ameliorates apoptotic neurodegeneration in an infant head trauma model. Given the major contribution of apoptotic neurodegeneration to neuropathologic outcome following trauma to the developing brain, interference with apoptotic pathways may comprise a potential therapeutic target in pediatric traumatic brain injury. PMID:15581279

  6. SorCS2 regulates dopaminergic wiring and is processed into an apoptotic two-chain receptor in peripheral glia.

    PubMed

    Glerup, Simon; Olsen, Ditte; Vaegter, Christian B; Gustafsen, Camilla; Sjoegaard, Susanne S; Hermey, Guido; Kjolby, Mads; Molgaard, Simon; Ulrichsen, Maj; Boggild, Simon; Skeldal, Sune; Fjorback, Anja N; Nyengaard, Jens R; Jacobsen, Jan; Bender, Dirk; Bjarkam, Carsten R; Srensen, Esben S; Fchtbauer, Ernst-Martin; Eichele, Gregor; Madsen, Peder; Willnow, Thomas E; Petersen, Claus M; Nykjaer, Anders

    2014-06-01

    Balancing trophic and apoptotic cues is critical for development and regeneration of neuronal circuits. Here we identify SorCS2 as a proneurotrophin (proNT) receptor, mediating both trophic and apoptotic signals in conjunction with p75(NTR). CNS neurons, but not glia, express SorCS2 as a single-chain protein that is essential for proBDNF-induced growth cone collapse in developing dopaminergic processes. SorCS2- or p75(NTR)-deficient in mice caused reduced dopamine levels and metabolism and dopaminergic hyperinnervation of the frontal cortex. Accordingly, both knockout models displayed a paradoxical behavioral response to amphetamine reminiscent of ADHD. Contrary, in PNS glia, but not in neurons, proteolytic processing produced a two-chain SorCS2 isoform that mediated proNT-dependent Schwann cell apoptosis. Sciatic nerve injury triggered generation of two-chain SorCS2 in p75(NTR)-positive dying Schwann cells, with apoptosis being profoundly attenuated in Sorcs2(-/-) mice. In conclusion, we have demonstrated that two-chain processing of SorCS2 enables neurons and glia to respond differently to proneurotrophins. PMID:24908487

  7. Extracellular heat shock proteins protect U937 cells from H2O2-induced apoptotic cell death.

    PubMed

    Franco, Lourdes; Terrinca, Jorge; Rodrguez, Ana B; Espino, Javier; Pariente, Jos A

    2016-01-01

    The cytoprotective role of heat shock proteins (HSPs) has been demonstrated in various cell types however, only few studies have investigated the role of extracellular exposure to HSPs in the survival of human lymphoma cell line U937. In the present study, we investigated the effect of extracellular exposure to four HSPs (HSP90, HSP70, HSP60, and HSP47) on apoptotic cell death induced by either oxidative stress (hydrogen peroxide) or endoplasmic reticulum stress-mediated intracellular calcium overload. It was found that extracellular exposure to HSPs reduced the cytotoxicity induced by hydrogen peroxide, but not that evoked by thapsigargin (a specific inhibitor of cytosolic calcium reuptake which is able to induce endoplasmic reticulum stress with subsequent intracellular calcium overload). Similarly, it was observed that exogenous HSPs were able to suppress the caspase-3 activation induced by hydrogen peroxide. These findings indicate that extracellular HSPs increase the resistance of human lymphoma cell line U937 to apoptotic cell death induced by hydrogen peroxide and diminish oxidative stress-mediated injures. PMID:26530166

  8. Diphenyleneiodonium Inhibits Apoptotic Cell Death of Gastric Epithelial Cells Infected with Helicobacter pylori in a Korean Isolate

    PubMed Central

    Cho, Soon Ok; Lim, Joo Weon

    2015-01-01

    NADPH oxidase produces a large amount of reactive oxygen species (ROS) in Helicobacter pylori (H. pylori)-induced gastric epithelial cells. Even though ROS mediate apoptotic cell death, direct involvement of NADPH oxidase on H. pylori-induced apoptosis remains unclear. Besides, H. pylori isolates show a high degree of genetic variability. The predominant genotype of H. pylori in Korea has been reported as cagA+, vacA s1b, m2, iceA genotype. Present study aims to investigate whether NADPH oxidase-generated ROS mediate apoptosis in human gastric epithelial AGS cells infected with H. pylori in a Korean isolate. AGS cells were pretreated with or without an NADPH oxidase inhibitor diphenyleneiodonium (DPI) and cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1. Cell viability, hydrogen peroxide level, DNA fragmentation, and protein levels of p53, Bcl-2, and Bax were determined. Results showed that H. pylori inhibited cell viability with the density of H. pylori added to the cells. Inhibition of NADPH oxidase by DPI suppressed H. pylori-induced cell death, increased hydrogen peroxide, DNA fragmentation, and the ratio of Bax/Bcl-2, and p53 induction in AGS cells dose-dependently. The results suggest that targeting NADPH oxidase may prevent the development of gastric inflammation associated with H. pylori infection by suppressing abnormal apoptotic cell death of gastric epithelial cells. PMID:26069142

  9. The Extrathyronine Actions of Iodine as Antioxidant, Apoptotic, and Differentiation Factor in Various Tissues

    PubMed Central

    Anguiano, Brenda; Delgado, Guadalupe

    2013-01-01

    Background Seaweed is an important dietary component and a rich source of iodine in several chemical forms in Asian communities. Their high consumption of this element (25 times higher than in Western countries) has been associated with the low incidence of benign and cancerous breast and prostate disease in Japanese people. Summary We review evidence showing that, in addition to being a component of the thyroid hormone, iodine can be an antioxidant as well as an antiproliferative and differentiation agent that helps to maintain the integrity of several organs with the ability to take up iodine. In animal and human studies, molecular iodine (I2) supplementation exerts a suppressive effect on the development and size of both benign and cancerous neoplasias. Investigations by several groups have demonstrated that these effects can be mediated by a variety of mechanisms and pathways, including direct actions, in which the oxidized iodine dissipates the mitochondrial membrane potential, thereby triggering mitochondrion-mediated apoptosis, and indirect effects through iodolipid formation and the activation of peroxisome proliferator–activated receptors type gamma, which, in turn, trigger apoptotic or differentiation pathways. Conclusions We propose that the International Council for the Control of Iodine Deficient Disorders recommend that iodine intake be increased to at least 3 mg/day of I2 in specific pathologies to obtain the potential extrathyroidal benefits described in the present review. PMID:23607319

  10. Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells.

    PubMed

    Wang, X; Gerdes, H-H

    2015-07-01

    Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context. PMID:25571977

  11. Linking Metabolic Abnormalities to Apoptotic Pathways in Beta Cells in Type 2 Diabetes

    PubMed Central

    Wali, Jibran A.; Masters, Seth L.; Thomas, Helen E.

    2013-01-01

    Pancreatic beta-cell apoptosis is an important feature of islets in type 2 diabetes. Apoptosis can occur through two major pathways, the extrinsic or death receptor mediated pathway, and the intrinsic or Bcl-2-regulated pathway. Hyperglycaemia, hyperlipidaemia and islet amyloid poly-peptide (IAPP) represent important possible causes of increased beta-cell apoptosis. Hyperglycaemia induces islet-cell apoptosis by the intrinsic pathway involving molecules of the Bcl-2 family. High concentrations of palmitate also activate intrinsic apoptosis in islets cells. IAPP oligomers can induce apoptosis by both intrinsic and extrinsic pathways. IL-1? produced through NLRP3 inflammasome activation can also induce islet cell death. Activation of the NLRP3 inflammasome may not be important for glucose or palmitate induced apoptosis in islets but may be important for IAPP mediated cell death. Endoplasmic reticulum (ER) and oxidative stress have been observed in beta cells in type 2 diabetes, and these could be the link between upstream metabolic abnormalities and downstream apoptotic machinery. PMID:24709700

  12. Effects of postnatal nicotine exposure on apoptotic markers in the developing piglet brain.

    PubMed

    Machaalani, R; Waters, K A; Tinworth, K D

    2005-01-01

    Exposure to cigarette smoke is a risk factor for the sudden infant death syndrome (SIDS), but the ability to distinguish between the neuropathological effects of pre- versus postnatal exposure is limited in the clinical setting. To test whether postnatal nicotine exposure could contribute to the increased neuronal expression of apoptotic markers that we have previously observed in SIDS infants, as well as including study of gender influences, we developed a piglet model to mimic passive smoking in the early postnatal period. Piglets were exposed to nicotine (2 mg/kg/day infused via an implanted osmotic minipump) within 48 h of birth until the age of 13-14 days, when the brain was collected for study. Four piglet groups included: control females (n=7), control males (n=7), nicotine females (n=7), and nicotine males (n=7). Apoptotic markers included immunohistochemistry for activated caspase-3, and for DNA fragmentation or terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) in seven nuclei of the brainstem caudal medulla and two subregions of the hippocampus (CA4 and dentate gyrus). Among control females compared with males, there was less active caspase-3 and less TUNEL in the dorsal motor nucleus of vagus (DMNV), and there was less TUNEL in the nucleus of the spinal trigeminal tract (NSTT). Compared with controls, nicotine-exposed male piglets had increased TUNEL staining in the cuneate nucleus (P=0.05), and increased active caspase-3 in the hypoglossal, gracile and dentate gyrus (P<0.05 for each). Nicotine-exposed females showed no change in TUNEL staining in any of the nuclei studied, but increased active caspase-3 in the hypoglossal, DMNV and NSTT (P<0.05 for each). These results show for the first time that postnatal nicotine exposure can lead to an increase in apoptotic markers in the brain. In piglets, these effects showed regional and gender-specific differences, suggesting that passive, postnatal nicotine exposure may be responsible for some neuropathological changes observed in infants dying from SIDS. PMID:15802186

  13. Counteraction of Apoptotic and Inflammatory Effects of Adriamycin in the Liver Cell Culture by Clinopitolite.

    PubMed

    Yapislar, Hande; Taskin, Eylem; Ozdas, Sule; Akin, Demet; Sonmez, Emine

    2016-04-01

    Growing evidence has been reported on adriamycin (ADR) hepatotoxicity in literature. Hepatotoxicity caused by the use of drugs has a serious undesirable effect in the cure of cancer patients that needs to be eliminated. The exact mechanism of ADR on non-cancerous tissue still remains to be a mystery. The zeolite (clinoptilolite) minerals form a complex group of aluminosilicates that often occur as accessory minerals in intermediate and basic rocks. In light of this information, we investigated the possible anti-inflammatory and anti-apoptotic effects of clinoptilolite in ADR that is inducing the toxicity in primary liver cell culture. Primary liver cell culture from rat was used in the study. We had three experiment groups including the following: (1) cells treated only with 50 μM ADR for 24 h, (2) cells treated with the 50 μM ADR for 24 h and then treated with 10(-4) M zeolite for 1 h, and (3) cells were incubated with 50 μM ADR for 24 h and then incubated with 10(-4) M zeolite for 24 h to test its long-term effects. After that, western blotting was performed in order to evaluate protein expression levels of several inflammation markers including IL-1β, tumor necrosis factor (TNF)-α, and nuclear factor kappa B (NF-κB), and immunohistochemistry was carried out to detect apoptosis in liver cell culture. Also, TdT-dUTP Terminal Nick-End Labeling (TUNEL) method was used for detecting apoptosis. We found elevated levels of inflammatory protein and apoptotic markers in ADR-administered cells (p < 0.05). Inflammatory and apoptotic markers decreased significantly after treated with zeolite (p < 0.05). The present study was pointed out that ADR causes hepatotoxicity via apoptosis and/or inflammation processes resulting from initiator NF-κB and TNF which causes proinflammatory mediators such as IL-1β. Elevation of inflammation might give rise to trigger apoptosis. Clinoptilolite counteracted the apoptosis and inflammation induced by ADR arising from the decrease in NF-κB, TNF-α, and IL-1β protein levels. PMID:26306587

  14. Regulation of Apoptotic Pathways by Stylophora pistillata (Anthozoa, Pocilloporidae) to Survive Thermal Stress and Bleaching

    PubMed Central

    Kvitt, Hagit; Rosenfeld, Hanna; Zandbank, Keren; Tchernov, Dan

    2011-01-01

    Elevated seawater temperatures are associated with coral bleaching events and related mortality. Nevertheless, some coral species are able to survive bleaching and recover. The apoptotic responses associated to this ability were studied over 3 years in the coral Stylophora pistillata from the Gulf of Eilat subjected to long term thermal stress. These include caspase activity and the expression profiles of the S. pistillata caspase and Bcl-2 genes (StyCasp and StyBcl-2-like) cloned in this study. In corals exposed to thermal stress (32 or 34C), caspase activity and the expression levels of the StyBcl-2-like gene increased over time (648 h) and declined to basal levels within 72 h of thermal stress. Distinct transcript levels were obtained for the StyCasp gene, with stimulated expression from 6 to 48 h of 34C thermal stress, coinciding with the onset of bleaching. Increased cell death was detected in situ only between 6 to 48 h of stress and was limited to the gastroderm. The bleached corals survived up to one month at 32C, and recovered back symbionts when placed at 24C. These results point to a two-stage response in corals that withstand thermal stress: (i) the onset of apoptosis, accompanied by rapid activation of anti-oxidant/anti-apoptotic mediators that block the progression of apoptosis to other cells and (ii) acclimatization of the coral to the chronic thermal stress alongside the completion of symbiosis breakdown. Accordingly, the coral's ability to rapidly curb apoptosis appears to be the most important trait affecting the coral's thermotolerance and survival. PMID:22194880

  15. Localization of dynein light chains 1 and 2 and their pro-apoptotic ligands.

    PubMed

    Day, Catherine L; Puthalakath, Hamsa; Skea, Gretchen; Strasser, Andreas; Barsukov, Igor; Lian, Lu-Yun; Huang, David C S; Hinds, Mark G

    2004-02-01

    The dynein and myosin V motor complexes are multi-protein structures that function to transport molecules and organelles within the cell. DLC (dynein light-chain) proteins, found as components of both dynein and myosin V motor complexes, connect the complexes to their cargoes. One of the roles of these motor complexes is to selectively sequester the pro-apoptotic 'BH3-only' (Bcl-2 homology 3-only) proteins, Bim (Bcl-2-interacting mediator of cell death) and Bmf (Bcl-2-modifying factor), and so regulate their cell death-inducing function. In vivo DLC2 is found exclusively as a component of the myosin V motor complex and Bmf binds DLC2 selectively. On the other hand, Bim interacts with DLC1 (LC8), an integral component of the dynein motor complex. The two DLCs share 93% sequence identity yet show unambiguous in vivo specificity for their respective BH3-only ligands. To investigate this specificity the three-dimensional solution structure of DLC2 was elucidated using NMR spectroscopy. In vitro structural and mutagenesis studies show that Bmf and Bim have identical binding characteristics to recombinant DLC2 or DLC1. Thus the selectivity shown by Bmf and Bim for binding DLC1 or DLC2, respectively, does not reside in their DLC-binding domains. Remarkably, mutational analysis of DLC1 and DLC2 indicates that a single surface residue (residue 41) determines the specific localization of DLCs with their respective motor complexes. These results suggest a molecular mechanism for the specific compartmentalization of DLCs and their pro-apoptotic cargoes and implicate other protein(s) in defining the specificity between the cargoes and the DLC proteins. PMID:14561217

  16. Localization of dynein light chains 1 and 2 and their pro-apoptotic ligands.

    PubMed Central

    Day, Catherine L; Puthalakath, Hamsa; Skea, Gretchen; Strasser, Andreas; Barsukov, Igor; Lian, Lu-Yun; Huang, David C S; Hinds, Mark G

    2004-01-01

    The dynein and myosin V motor complexes are multi-protein structures that function to transport molecules and organelles within the cell. DLC (dynein light-chain) proteins, found as components of both dynein and myosin V motor complexes, connect the complexes to their cargoes. One of the roles of these motor complexes is to selectively sequester the pro-apoptotic 'BH3-only' (Bcl-2 homology 3-only) proteins, Bim (Bcl-2-interacting mediator of cell death) and Bmf (Bcl-2-modifying factor), and so regulate their cell death-inducing function. In vivo DLC2 is found exclusively as a component of the myosin V motor complex and Bmf binds DLC2 selectively. On the other hand, Bim interacts with DLC1 (LC8), an integral component of the dynein motor complex. The two DLCs share 93% sequence identity yet show unambiguous in vivo specificity for their respective BH3-only ligands. To investigate this specificity the three-dimensional solution structure of DLC2 was elucidated using NMR spectroscopy. In vitro structural and mutagenesis studies show that Bmf and Bim have identical binding characteristics to recombinant DLC2 or DLC1. Thus the selectivity shown by Bmf and Bim for binding DLC1 or DLC2, respectively, does not reside in their DLC-binding domains. Remarkably, mutational analysis of DLC1 and DLC2 indicates that a single surface residue (residue 41) determines the specific localization of DLCs with their respective motor complexes. These results suggest a molecular mechanism for the specific compartmentalization of DLCs and their pro-apoptotic cargoes and implicate other protein(s) in defining the specificity between the cargoes and the DLC proteins. PMID:14561217

  17. Autoantibodies from Sjgren's syndrome induce activation of both the intrinsic and extrinsic apoptotic pathways in human salivary gland cell line A-253.

    PubMed

    Sisto, M; Lisi, S; Castellana, D; Scagliusi, P; D'Amore, M; Caprio, S; Scagliusi, A; Acquafredda, A; Panaro, M A; Mitolo, V

    2006-08-01

    Sjgren's syndrome (SS) is an autoimmune rheumatic disease that targets salivary and lachrymal glands, characterized by a high concentration of serum autoantibodies directed against nuclear and cytoplasmic antigens. It is known that autoantibodies can enter viable cells and this phenomenon has functional consequences including activation of apoptotic process. The objective of this work was to explore whether autoantibodies contained in IgG purified from Sjgren sera trigger apoptotic process in an experimental model represented by the human salivary gland cell line A-253. To define if the intrinsic or extrinsic pathways are activated, we examined which caspases are critical for inducing cell death. The results have demonstrated that morphological changes and DNA laddering, consistent with apoptotic cell death, occurred in A-253 cells treated with IgG from Sjgren sera. Sjgren IgG induced cleavage and activation of the effector caspase-3 and degradation of the caspase-3 substrate poly(ADP-ribose)polymerase. Both the intrinsic and extrinsic apoptotic pathways were activated, since both caspase-8 and caspase-9 cleavages occurred. In conclusion, autoantibodies contained in IgG purified from Sjgren sera mediate apoptosis of the A-253 cell line in a caspase-dependent manner. PMID:16797160

  18. STAT1, STAT3 and p38MAPK are involved in the apoptotic effect induced by a chimeric cyclic interferon-{alpha}2b peptide

    SciTech Connect

    Blank, Viviana C.; Pena, Clara; Roguin, Leonor P.

    2010-02-15

    In the search of mimetic peptides of the interferon-{alpha}2b molecule (IFN-{alpha}2b), we have previously designed and synthesized a chimeric cyclic peptide of the IFN-{alpha}2b that inhibits WISH cell proliferation by inducing an apoptotic response. Here, we first studied the ability of this peptide to activate intracellular signaling pathways and then evaluated the participation of some signals in the induction of apoptosis. Stimulation of WISH cells with the cyclic peptide showed tyrosine phosphorylation of Jak1 and Tyk2 kinases, tyrosine and serine phosphorylation of STAT1 and STAT3 transcription factors and activation of p38 MAPK pathway, although phosphorylation levels or kinetics were in some conditions different to those obtained under IFN-{alpha}2b stimulus. JNK and p44/42 pathways were not activated by the peptide in WISH cells. We also showed that STAT1 and STAT3 downregulation by RNA interference decreased the antiproliferative activity and the amount of apoptotic cells induced by the peptide. Pharmacological inhibition of p38 MAPK also reduced the peptide growth inhibitory activity and the apoptotic effect. Thus, we demonstrated that the cyclic peptide regulates WISH cell proliferation through the activation of Jak/STAT signaling pathway. In addition, our results indicate that p38 MAPK may also be involved in cell growth regulation. This study suggests that STAT1, STAT3 and p38 MAPK would be mediating the antitumor and apoptotic response triggered by the cyclic peptide in WISH cells.

  19. PEGylated apoptotic protein-loaded PLGA microspheres for cancer therapy

    PubMed Central

    Byeon, Hyeong Jun; Kim, Insoo; Choi, Ji Su; Lee, Eun Seong; Shin, Beom Soo; Youn, Yu Seok

    2015-01-01

    The aim of the current study was to investigate the antitumor potential of poly (D,L-lactic-co-glycolic acid) microspheres (PLGA MSs) containing polyethylene glycol (PEG)-conjugated (PEGylated) tumor necrosis factor–related apoptosis-inducing ligand (PEG-TRAIL). PEG-TRAIL PLGA MSs were prepared by using a water-in-oil-in-water double-emulsion method, and the apoptotic activities of supernatants released from the PLGA MSs at days 1, 3, and 7 were examined. The antitumor effect caused by PEG-TRAIL PLGA MSs was evaluated in pancreatic Mia Paca-2 cell-xenografted mice. PEG-TRAIL PLGA MS was found to be spherical and 14.4±1.06 μm in size, and its encapsulation efficiency was significantly greater than that of TRAIL MS (85.7%±4.1% vs 43.3%±10.9%, respectively). The PLGA MS gradually released PEG-TRAIL for 14 days, and the released PEG-TRAIL was shown to have clear apoptotic activity in Mia Paca-2 cells, whereas TRAIL released after 1 day had a negligible activity. Finally, PEG-TRAIL PLGA MS displayed remarkably greater antitumor efficacy than blank or TRAIL PLGA MS in Mia Paca-2 cell-xenografted mice in terms of tumor volume and weight, apparently due to increased stability and well-retained apoptotic activity of PEG-TRAIL in PLGA MS. We believe that this PLGA MS system, combined with PEG-TRAIL, should be considered a promising candidate for treating pancreatic cancer. PMID:25632232

  20. Quercetin sensitizes fluconazole-resistant candida albicans to induce apoptotic cell death by modulating quorum sensing.

    PubMed

    Singh, B N; Upreti, D K; Singh, B R; Pandey, G; Verma, S; Roy, S; Naqvi, A H; Rawat, A K S

    2015-04-01

    Quorum sensing (QS) regulates group behaviors of Candida albicans such as biofilm, hyphal growth, and virulence factors. The sesquiterpene alcohol farnesol, a QS molecule produced by C. albicans, is known to regulate the expression of virulence weapons of this fungus. Fluconazole (FCZ) is a broad-spectrum antifungal drug that is used for the treatment of C. albicans infections. While FCZ can be cytotoxic at high concentrations, our results show that at much lower concentrations, quercetin (QC), a dietary flavonoid isolated from an edible lichen (Usnea longissima), can be implemented as a sensitizing agent for FCZ-resistant C. albicans NBC099, enhancing the efficacy of FCZ. QC enhanced FCZ-mediated cell killing of NBC099 and also induced cell death. These experiments indicated that the combined application of both drugs was FCZ dose dependent rather than QC dose dependent. In addition, we found that QC strongly suppressed the production of virulence weapons-biofilm formation, hyphal development, phospholipase, proteinase, esterase, and hemolytic activity. Treatment with QC also increased FCZ-mediated cell death in NBC099 biofilms. Interestingly, we also found that QC enhances the anticandidal activity of FCZ by inducing apoptotic cell death. We have also established that this sensitization is reliant on the farnesol response generated by QC. Molecular docking studies also support this conclusion and suggest that QC can form hydrogen bonds with Gln969, Thr1105, Ser1108, Arg1109, Asn1110, and Gly1061 in the ATP binding pocket of adenylate cyclase. Thus, this QS-mediated combined sensitizer (QC)-anticandidal agent (FCZ) strategy may be a novel way to enhance the efficacy of FCZ-based therapy of C. albicans infections. PMID:25645848

  1. Quercetin Sensitizes Fluconazole-Resistant Candida albicans To Induce Apoptotic Cell Death by Modulating Quorum Sensing

    PubMed Central

    Singh, B. R.; Pandey, G.; Verma, S.; Roy, S.; Naqvi, A. H.

    2015-01-01

    Quorum sensing (QS) regulates group behaviors of Candida albicans such as biofilm, hyphal growth, and virulence factors. The sesquiterpene alcohol farnesol, a QS molecule produced by C. albicans, is known to regulate the expression of virulence weapons of this fungus. Fluconazole (FCZ) is a broad-spectrum antifungal drug that is used for the treatment of C. albicans infections. While FCZ can be cytotoxic at high concentrations, our results show that at much lower concentrations, quercetin (QC), a dietary flavonoid isolated from an edible lichen (Usnea longissima), can be implemented as a sensitizing agent for FCZ-resistant C. albicans NBC099, enhancing the efficacy of FCZ. QC enhanced FCZ-mediated cell killing of NBC099 and also induced cell death. These experiments indicated that the combined application of both drugs was FCZ dose dependent rather than QC dose dependent. In addition, we found that QC strongly suppressed the production of virulence weapons—biofilm formation, hyphal development, phospholipase, proteinase, esterase, and hemolytic activity. Treatment with QC also increased FCZ-mediated cell death in NBC099 biofilms. Interestingly, we also found that QC enhances the anticandidal activity of FCZ by inducing apoptotic cell death. We have also established that this sensitization is reliant on the farnesol response generated by QC. Molecular docking studies also support this conclusion and suggest that QC can form hydrogen bonds with Gln969, Thr1105, Ser1108, Arg1109, Asn1110, and Gly1061 in the ATP binding pocket of adenylate cyclase. Thus, this QS-mediated combined sensitizer (QC)-anticandidal agent (FCZ) strategy may be a novel way to enhance the efficacy of FCZ-based therapy of C. albicans infections. PMID:25645848

  2. Metabolic regulation of CaMKII protein and caspases in Xenopus laevis egg extracts.

    PubMed

    McCoy, Francis; Darbandi, Rashid; Chen, Si-Ing; Eckard, Laura; Dodd, Keela; Jones, Kelly; Baucum, Anthony J; Gibbons, Jennifer A; Lin, Sue-Hwa; Colbran, Roger J; Nutt, Leta K

    2013-03-29

    The metabolism of the Xenopus laevis egg provides a cell survival signal. We found previously that increased carbon flux from glucose-6-phosphate (G6P) through the pentose phosphate pathway in egg extracts maintains NADPH levels and calcium/calmodulin regulated protein kinase II (CaMKII) activity to phosphorylate caspase 2 and suppress cell death pathways. Here we show that the addition of G6P to oocyte extracts inhibits the dephosphorylation/inactivation of CaMKII bound to caspase 2 by protein phosphatase 1. Thus, G6P sustains the phosphorylation of caspase 2 by CaMKII at Ser-135, preventing the induction of caspase 2-mediated apoptotic pathways. These findings expand our understanding of oocyte biology and clarify mechanisms underlying the metabolic regulation of CaMKII and apoptosis. Furthermore, these findings suggest novel approaches to disrupt the suppressive effects of the abnormal metabolism on cell death pathways. PMID:23400775

  3. Glucocorticoids inhibit the apoptotic actions of UV-C but not Fas ligand in hepatoma cells: direct evidence for a critical role of Bcl-xL.

    PubMed

    Scoltock, A B; Heimlich, G; Cidlowski, J A

    2007-04-01

    Our laboratory has shown that glucocorticoids can inhibit apoptosis in rat hepatoma cells; however, the mechanisms are incompletely understood. To address this issue we sought to determine if glucocorticoid inhibition is effective when death is induced by stimuli that more selectively activate either the intrinsic (UV-C) or extrinsic (FasL) apoptotic pathways. Using flow cytometric analysis, we show that pretreatment of HTC cells with dexamethasone (Dex) inhibits UV-C- but not FasL-induced apoptosis. This inhibition requires Dex pretreatment and can be abrogated by the glucocorticoid antagonist RU486 indicating glucocorticoid receptor-mediated action. Dex increases anti-apoptotic Bcl-x(L) at both mRNA and protein levels. The Bcl-x(L) protein level remains elevated even after apoptosis induction with either UV-C or FasL although only UV-C-induced cell death is inhibited. Repression of Bcl-x(L) protein with siRNA abrogates the anti-apoptotic effect of glucocorticoids. Together these data provide direct evidence that Bcl-x(L) mediates glucocorticoid inhibition of UV-C induced apoptosis. PMID:17170751

  4. Akt attenuates apoptotic death through phosphorylation of H2A under hydrogen peroxide-induced oxidative stress in PC12 cells and hippocampal neurons

    PubMed Central

    Park, Ji Hye; Kim, Chung Kwon; Lee, Sang Bae; Lee, Kyung-Hoon; Cho, Sung-Woo; Ahn, Jee-Yin

    2016-01-01

    Although the essential role of protein kinase B (PKB)/Akt in cell survival signaling has been clearly established, the mechanism by which Akt mediates the cellular response to hydrogen peroxide (H2O2)-induced oxidative stress remains unclear. We demonstrated that Akt attenuated neuronal apoptosis through direct association with histone 2A (H2A) and phosphorylation of H2A at threonine 17. At early time points during H2O2 exposure of PC12 cells and primary hippocampal neurons, when the cells can tolerate the level of DNA damage, Akt was activated and phosphorylated H2A, leading to inhibition of apoptotic death. At later time points, Akt delivered the NAD+-dependent protein deacetylase Sirtuin 2 (Sirt 2) to the vicinity of phosphorylated H2A in response to irreversible DNA damage, thereby inducing H2A deacetylation and subsequently leading to apoptotic death. Ectopically expressed T17A-substituted H2A minimally interacted with Akt and failed to prevent apoptosis under oxidative stress. Thus Akt-mediated H2A phosphorylation has an anti-apoptotic function in conditions of H2O2-induced oxidative stress in neurons and PC12 cells. PMID:26899247

  5. Induction of apoptotic cell death in hen granulosa cells by ceramide.

    PubMed

    Witty, J P; Bridgham, J T; Johnson, A L

    1996-12-01

    Recent studies have demonstrated that ovarian follicle atresia occurs extensively before follicle selection into the avian preovulatory hierarchy, and that this process is mediated via granulosa cell apoptosis. Subsequent to follicle selection, granulosa cells are inherently resistant to apoptosis, and such resistance is correlated with increased expression of death suppressor genes such as bcl-xlong. In the present studies we used this avian ovary model system to 1) identify cellular characteristics and mechanisms related to apoptotic cell death of granulosa cells in vitro, and 2) further characterize functional differences between apoptosis-susceptible (4- to 8-mm follicle) and apoptosis-resistant (preovulatory follicle) granulosa cells. Treatment of granulosa cells from the largest preovulatory follicle with N-octanoylsphingosine (C8-ceramide) results in pronounced oligonucleosome formation, a hallmark of apoptosis. That this is indicative of programmed cell death is supported by an increased incidence of pyknotic nuclei and apoptotic bodies in C8-ceramide-treated samples compared to that in control cultured cells. Tumor necrosis factor-alpha, a stimulator of ceramide production, actively promotes oligonucleosome formation in apoptosis-susceptible, but not in apoptosis-resistant, granulosa cells. Induction of apoptosis is also observed after exposure of apoptosis-resistant granulosa cells to sphingomyelinase treatment and UV irradiation, which are known to stimulate endogenous ceramide production, and to the anticancer drug, daunorubicin, which initiates de novo ceramide biosynthesis via activation of ceramide synthase. Although treatment of granulosa cells with fumonisin B1, a specific ceramide synthase inhibitor, blocks daunorubicin-stimulated oligonucleosome formation, UV-induced cell death is unaffected. Taken together, these results demonstrate that pharmacological factors known to mimic the actions of ceramide or stimulate ceramide production can induce oligonucleosome formation and programmed cell death in granulosa cells. More importantly, however, the ability of a physiologically relevant initiator of ceramide biosynthesis, tumor necrosis factor-alpha, to promote cell death is evident only in apoptosis-susceptible granulosa cells collected from atresia-prone prehierarchal follicles. These data provide support for ceramide as an important intracellular signaling mechanism, mediating granulosa cell apoptosis and follicle atresia. PMID:8940345

  6. Tumor-targeting novel manganese complex induces ROS-mediated apoptotic and autophagic cancer cell death

    PubMed Central

    LIU, JIA; GUO, WENJIE; LI, JING; LI, XIANG; GENG, JI; CHEN, QIUYUN; GAO, JING

    2015-01-01

    In this study, the antitumor activity of the novel manganese (II) compound, Adpa-Mn {[(Adpa)Mn(Cl)(H2O)] (Adpa=bis(2-pyridylmethyl)amino-2-propionic acid)}, and its possible mechanisms of action were investigated. In vitro, the growth inhibitory effects of Adpa-Mn (with IC50 values lower than 15 ?M) on tumor cell lines were examined by MTT assay. We found that this compound was more selective against cancer cells than the popular chemotherapeutic reagent, cisplatin. We then found that Adpa-Mn achieved its selectivity against cancer cells through the transferrin (Tf)-transferrin receptor (TfR) system, which is highly expressed in tumor cells. Furthermore, Adpa-Mn induced both apoptosis and autophagy, as indicated by chromatin condensation, the activation of poly(ADP-ribose) polymerase (PARP), Annexin V/prop-idium iodide staining, an enhanced fluorescence intensity of monodansylcadaverine (MDC), as well as the elevated expression of the autophagy-related protein, microtubule-associated protein 1 light chain 3 (LC3). In addition, Adpa-Mn induced the generation of intracellular reactive oxygen species (ROS) and its anticancer effects were significantly reduced following pre-treatment with the antioxidant, N-acetyl cysteine, indicating that ROS triggered cell death. In vivo, the induction of apoptosis and autophagy in tumor tissue was confirmed following treatment with Adpa-Mn, which contributed to its significant antitumor activity against hepatocellular carcinoma (Hep-A cell) xenografts at 10 mg/kg. Taken together, these data suggest the possible use of Adpa-Mn as a novel anticancer drug. PMID:25604962

  7. Adiponectin mediates antiproliferative and apoptotic responses in human MCF7 breast cancer cells

    SciTech Connect

    Dieudonne, Marie-Noelle; Bussiere, Marianne; Dos Santos, Esther; Leneveu, Marie-Christine; Giudicelli, Yves . E-mail: biochip@wanadoo.fr; Pecquery, Rene

    2006-06-23

    It is well established that obesity is a risk factor for breast cancer and that blood levels of adiponectin, a hormone mainly secreted by white adipocytes, are inversely correlated with the body fat mass. As adiponectin elicits anti-proliferative effects in some cell types, we tested the hypothesis that adiponectin could influence human breast cancer MCF-7 cell growth. Here we show that MCF-7 cells express adiponectin receptors and respond to human recombinant adiponectin by reducing their growth, AMPkinase activation, and p42/p44 MAPkinase inactivation. Further, we demonstrate that the anti-proliferative effect of adiponectin involves activation of cell apoptosis and inhibition of cell cycle. These findings suggest that adiponectin could act in vivo as a paracrine/endocrine growth inhibitor towards mammary epithelial cells. Moreover, adipose adiponectin production being strongly reduced in obesity, this study may help to explain why obesity is a risk factor of developing breast cancers.

  8. IL-10 restricts dendritic cell (DC) growth at the monocyte-to-monocyte-derived DC interface by disrupting anti-apoptotic and cytoprotective autophagic molecular machinery.

    PubMed

    Martin, Carla; Espaillat, Mel Pilar; Santiago-Schwarz, Frances

    2015-12-01

    An evolving premise is that cytoprotective autophagy responses are essential to monocyte-macrophage differentiation. Whether autophagy functions similarly during the monocyte-to-dendritic cell (DC) transition is unclear. IL-10, which induces apoptosis in maturing human DCs, has been shown to inhibit starvation-induced autophagy in murine macrophage cell lines. Based on the strict requirement that Bcl-2-mediated anti-apoptotic processes are implemented during the monocyte-to-DC transition, we hypothesized that cytoprotective autophagy responses also operate at the monocyte-DC interface and that IL-10 inhibits both anti-apoptotic and cytoprotective autophagy responses at this critical juncture. In support of our premise, we show that levels of anti-apoptotic Bcl-2 and autophagy-associated LC3 and Beclin-1 proteins are coincidentally upregulated during the monocyte-to-DC transition. Autophagy was substantiated by increased autophagosome visualization after bafilomycin treatment. Moreover, the autophagy inhibitor 3-MA restricted DC differentiation by prompting apoptosis. IL-10 implemented apoptosis that was coincidentally associated with reduced levels of Bcl-2 and widespread disruption of the autophagic flux. During peak apoptosis, IL-10 produced the death of newly committed DCs. However, cells surviving the IL-10 apoptotic schedule were highly phagocytic macrophage-like cells displaying reduced capacity to stimulate allogeneic nave T cells in a mixed leukocyte reaction, increased levels of LC3, and mature autophagosomes. Thus, IL-10's negative control of DC-driven adaptive immunity at the monocyte-DC interface includes disruption of coordinately regulated molecular networks involved in pro-survival autophagy and anti-apoptotic responses. PMID:26395023

  9. Synergistic Combination of Small Molecule Inhibitor and RNA interference Against Anti-apoptotic Bcl-2 Protein in Head and Neck Cancer Cells

    PubMed Central

    Lin, Yen-Ling; Durmaz, Yasemin Yuksel; Nr, Jacques E.; ElSayed, Mohamed E. H.

    2014-01-01

    B-cell lymphoma 2 (Bcl-2) is an anti-apoptotic protein that is over-expressed in head and neck squamous cell carcinomas, which has been implicated in development of radio- and chemo-resistance. Small molecule inhibitors such as AT-101 (a BH3-mimetic drug) have been developed to inhibit the anti-apoptotic activity of Bcl-2 proteins, which proved effective in restoring radio- and chemo-sensitivity in head and neck cancer cells. However, high doses of AT-101 are associated with gastrointestinal, hepatic, and fertility side effects, which prompted the search for other Bcl-2 inhibitors. Short interfering RNA (siRNA) proved to inhibit anti-apoptotic Bcl-2 protein expression and trigger cancer cell death. However, transforming siRNA molecules into a viable therapy remains a challenge due to the lack of efficient and biocompatible carriers. We report the development of degradable star-shaped polymers that proved to condense anti-Bcl-2 siRNA into smart pH-sensitive and membrane-destabilizing particles that shuttle their cargo past the endosomal membrane and into the cytoplasm of head and neck cancer cells. Results show that smart anti-Bcl-2 particles reduced the mRNA and protein levels of anti-apoptotic Bcl-2 protein in UM-SCC-17B cancer cells by 50-60% and 65-75%, respectively. Results also show that combining smart anti-Bcl-2 particles with the IC25 of AT-101 (inhibitory concentration responsible for killing 25% of the cells) synergistically inhibit cancer cell proliferation and increase cell apoptosis, which reduced the survival of UM-SCC-17B cancer cells compared to treatment with AT-101 alone. Results indicate the therapeutic benefit of combining siRNA-mediated knockdown of anti-apoptotic Bcl-2 protein expression with low doses of AT-101 for inhibiting the growth of head and neck cancer cells. PMID:23734725

  10. Binding of Anti-SSA Antibodies to Apoptotic Fetal Cardiocytes Stimulates uPA/uPAR-Dependent Activation of TGF beta and Potentiates Fibrosis

    PubMed Central

    Briassouli, Paraskevi; Rifkin, Daniel; Clancy, Robert M.; Buyon, Jill P.

    2011-01-01

    In congenital heart block (CHB), binding of maternal anti-SSA/Ro antibodies to fetal apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases uPA/uPAR-dependent plasmin activation. Since the uPA/uPAR system plays a role in TGF beta activation, we evaluated whether anti-Ro binding to apoptotic cardiocytes enhances plasmin-mediated activation of TGF beta thereby promoting a profibrosing phenotype. Supernatants from co-cultures of healthy cardiocytes and apoptotic cardiocytes bound by IgG from a mother whose child had CHB (apo-CHB-IgG) exhibited significantly increased levels of active TGF beta compared to supernatants from co-cultures of healthy cardiocytes and apoptotic cardiocytes preincubated with IgG (apo-nl-IgG) from a healthy donor. Treatment of the culture medium with anti-TGF beta antibody or TGF beta inhibitor (SB431542) abrogated the luciferase response thereby confirming TGF beta dependency. Increased uPA levels and activity were present in supernatants generated from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes compared to healthy cardiocytes and and apo nl-IgG cardiocytes, respectively. Treatment of apo-CHB-IgG cardiocytes with anti-uPAR or anti-uPA antibodies or plasmin inhibitor aprotinin prior to coculturing with healthy cardiocytes attenuated TGF beta activation. Supernatants derived from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes promoted Smad2 phosphorylation and fibroblast transdifferentiation as evidenced by increased SMAc and collagen expression, which decreased when fibroblasts were treated with supernatants from cocultures pretreated with uPAR antibodies. These data suggest that binding of anti-Ro antibodies to apoptotic cardiocytes triggers TGF beta activation, by virtue of increasing uPAR-dependent uPA activity, thus initiating and amplifying a cascade of events that promote myofibroblast transdifferentiation and scar. PMID:22013113

  11. Macrophages programmed by apoptotic cells inhibit epithelial-mesenchymal transition in lung alveolar epithelial cells via PGE2, PGD2, and HGF

    PubMed Central

    Yoon, Young-So; Lee, Ye-Ji; Choi, Youn-Hee; Park, Young Mi; Kang, Jihee Lee

    2016-01-01

    Apoptotic cell clearance results in the release of growth factors and the action of signaling molecules involved in tissue homeostasis maintenance. Here, we investigated whether and how macrophages programmed by apoptotic cells inhibit the TGF-β1-induced Epithelial-mesenchymal transition (EMT) process in lung alveolar epithelial cells. Treatment with conditioned medium derived from macrophages exposed to apoptotic cells, but not viable or necrotic cells, inhibited TGF-β1-induced EMT, including loss of E-cadherin, synthesis of N-cadherin and α-smooth muscle actin, and induction of EMT-activating transcription factors, such as Snail1/2, Zeb1/2, and Twist1. Exposure of macrophages to cyclooxygenase (COX-2) inhibitors (NS-398 and COX-2 siRNA) or RhoA/Rho kinase inhibitors (Y-27632 and RhoA siRNA) and LA-4 cells to antagonists of prostaglandin E2 (PGE2) receptor (EP4 [AH-23848]), PGD2 receptors (DP1 [BW-A868C] and DP2 [BAY-u3405]), or the hepatocyte growth factor (HGF) receptor c-Met (PHA-665752), reversed EMT inhibition by the conditioned medium. Additionally, we found that apoptotic cell instillation inhibited bleomycin-mediated EMT in primary mouse alveolar type II epithelial cells in vivo. Our data suggest a new model for epithelial cell homeostasis, by which the anti-EMT programming of macrophages by apoptotic cells may control the progressive fibrotic reaction via the production of potent paracrine EMT inhibitors. PMID:26875548

  12. Allograft tolerance induced by donor apoptotic lymphocytes requires phagocytosis in the recipient

    NASA Technical Reports Server (NTRS)

    Sun, E.; Gao, Y.; Chen, J.; Roberts, A. I.; Wang, X.; Chen, Z.; Shi, Y.

    2004-01-01

    Cell death through apoptosis plays a critical role in regulating cellular homeostasis. Whether the disposal of apoptotic cells through phagocytosis can actively induce immune tolerance in vivo, however, remains controversial. Here, we report in a rat model that without using immunosuppressants, transfusion of apoptotic splenocytes from the donor strain prior to transplant dramatically prolonged survival of heart allografts. Histological analysis verified that rejection signs were significantly ameliorated. Splenocytes from rats transfused with donor apoptotic cells showed a dramatically decreased response to donor lymphocyte stimulation. Most importantly, blockade of phagocytosis in vivo, either with gadolinium chloride to disrupt phagocyte function or with annexin V to block binding of exposed phosphotidylserine to its receptor on phagocytes, abolished the beneficial effect of transfused apoptotic cells on heart allograft survival. Our results demonstrate that donor apoptotic cells promote specific allograft acceptance and that phagocytosis of apoptotic cells in vivo plays a crucial role in maintaining immune tolerance.

  13. Allograft tolerance induced by donor apoptotic lymphocytes requires phagocytosis in the recipient.

    PubMed

    Sun, E; Gao, Y; Chen, J; Roberts, A I; Wang, X; Chen, Z; Shi, Y

    2004-12-01

    Cell death through apoptosis plays a critical role in regulating cellular homeostasis. Whether the disposal of apoptotic cells through phagocytosis can actively induce immune tolerance in vivo, however, remains controversial. Here, we report in a rat model that without using immunosuppressants, transfusion of apoptotic splenocytes from the donor strain prior to transplant dramatically prolonged survival of heart allografts. Histological analysis verified that rejection signs were significantly ameliorated. Splenocytes from rats transfused with donor apoptotic cells showed a dramatically decreased response to donor lymphocyte stimulation. Most importantly, blockade of phagocytosis in vivo, either with gadolinium chloride to disrupt phagocyte function or with annexin V to block binding of exposed phosphotidylserine to its receptor on phagocytes, abolished the beneficial effect of transfused apoptotic cells on heart allograft survival. Our results demonstrate that donor apoptotic cells promote specific allograft acceptance and that phagocytosis of apoptotic cells in vivo plays a crucial role in maintaining immune tolerance. PMID:15375386

  14. In vitro apoptotic and DNA damaging potential of nanobarium oxide

    PubMed Central

    Alarifi, Saud; Ali, Daoud; Al-Bishri, Widad

    2016-01-01

    Barium oxide nanoparticles (BaONPs) are an important industrial compound and are widely used in polymers and paints. In this study, apoptotic and genotoxic effects of BaONPs in mouse embryonic fibroblast (L929) cells were determined by using single-cell gel test. In vitro cytotoxicity assays were performed to assess BaONPs’ toxicity in L929 cells. Mild cytotoxicity was observed in L929 cells due to BaONPs. BaONPs increased lipid peroxidation, catalase, and superoxide dismutase levels and lowered glutathione levels in L929 cells. This was accompanied by concomitant generation of reactive oxygen species and activation of caspase-3 in BaONPs-treated L929 cells. On the other hand, when we exposed L929 cells to BaONPs for 24 and 48 hours (comet assay), there was a duration- and dose-dependent increase in DNA impairment detected in the single-cell gel test. Thus, BaONPs exhibit genotoxic and apoptotic effects in L929 cells, most likely due to initiation of oxidative damage. PMID:26834473

  15. In vitro apoptotic and DNA damaging potential of nanobarium oxide.

    PubMed

    Alarifi, Saud; Ali, Daoud; Al-Bishri, Widad

    2016-01-01

    Barium oxide nanoparticles (BaONPs) are an important industrial compound and are widely used in polymers and paints. In this study, apoptotic and genotoxic effects of BaONPs in mouse embryonic fibroblast (L929) cells were determined by using single-cell gel test. In vitro cytotoxicity assays were performed to assess BaONPs' toxicity in L929 cells. Mild cytotoxicity was observed in L929 cells due to BaONPs. BaONPs increased lipid peroxidation, catalase, and superoxide dismutase levels and lowered glutathione levels in L929 cells. This was accompanied by concomitant generation of reactive oxygen species and activation of caspase-3 in BaONPs-treated L929 cells. On the other hand, when we exposed L929 cells to BaONPs for 24 and 48 hours (comet assay), there was a duration- and dose-dependent increase in DNA impairment detected in the single-cell gel test. Thus, BaONPs exhibit genotoxic and apoptotic effects in L929 cells, most likely due to initiation of oxidative damage. PMID:26834473

  16. Protopine, a novel microtubule-stabilizing agent, causes mitotic arrest and apoptotic cell death in human hormone-refractory prostate cancer cell lines.

    PubMed

    Chen, Chun-Han; Liao, Cho-Hwa; Chang, Ya-Ling; Guh, Jih-Hwa; Pan, Shiow-Lin; Teng, Che-Ming

    2012-02-01

    In this study, we investigated the anticancer effect of protopine on human hormone-refractory prostate cancer (HRPC) cells. Protopine exhibited an anti-proliferative effect by induction of tubulin polymerization and mitotic arrest, which ultimately led to apoptotic cell death. The data suggest that protopine increased the activity of cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex and that contributed to cell apoptosis by modulating mitochondria-mediated signaling pathways, such as Bcl-2 phosphorylation and Mcl-1 down-regulation. In conclusion, the data suggest that protopine is a novel microtubule stabilizer with anticancer activity in HRPC cells through apoptotic pathway by modulating Cdk1 activity and Bcl-2 family of proteins. PMID:22033245

  17. c-Jun regulates the stability of anti-apoptotic ?Np63 in amyloid-?-induced apoptosis.

    PubMed

    Fonseca, Maria B; Nunes, Ana F; Rodrigues, Ceclia M P

    2012-01-01

    p63, the structural and functional homologue of p53, is expressed either as a full-length isoform, containing a transactivation (TA) domain (TAp63), or as a truncated isoform, which lacks TA (?Np63). Amyloid-? (A?) incubation of neuronal cells results in stress-induced cell death through poorly understood mechanisms. We investigated the role of p63 in A?-induced stress. Our results show that A?-induced apoptosis of rat PC12 neuronal-like cells and primary cortical neurons was associated with stabilization of pro-apoptotic TAp63 and, most importantly, degradation of anti-apoptotic ?Np63 through a MAPK- and proteasome-dependent mechanism. This was associated with increased c-Jun, and partially modulated by tauroursodeoxycholic acid. As expected, classic genotoxic insults resulted in c-Jun upregulation and concomitant ?Np63 reduction. Endogenous and ectopic ?Np63 expression was also markedly reduced by c-Jun overexpression. Further, A?-mediated ?Np63 degradation occurred in a c-Jun-dependent manner. Downregulation of c-Jun expression by specific c-Jun siRNA abrogated the reduction of ?Np63 levels following A? insult, whereas overexpression of c-Jun led to its degradation. c-Jun significantly decreased ?Np63 half-life. Together, these findings demonstrate that the abundance of anti-apoptotic ?Np63 in response to A?-induced cell stress is regulated by a c-Jun-dependent mechanism, and highlight the importance of finding novel targets for potential therapeutic intervention. PMID:22045494

  18. Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival

    PubMed Central

    Eichhorn, Joshua M.; Alford, Sarah E.; Sakurikar, Nandini; Chambers, Timothy C.

    2014-01-01

    Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins. PMID:24556425

  19. Bis(ethyl)norspermine potentiates the apoptotic activity of the pure antiestrogen ICI 182780 in breast cancer cells.

    PubMed

    Balabhadrapathruni, Srivani; Santhakumaran, Latha M; Thomas, T J; Shirahata, Akira; Gallo, Michael A; Thomas, Thresia

    2005-01-01

    We studied the effects of ICI 182780 and bis(ethyl)norspermine (BE-3-3-3) on cell growth and apoptosis of estrogen receptor-positive MCF-7 breast cancer cells. Combination treatment with 100 nM ICI 182780 and 5 microM BE-3-3-3 for 6 days inhibited cell growth by 74.3+/-8.4% in MCF-7 cells, compared to that of 25.4+/-5.8 and 45.8+/-12.2%, respectively, when ICI 182780 and BE-3-3-3 were used as single agents. Treatment with 100 nM ICI 182780 and 5 microM BE-3-3-3 as single agents resulted in 9.1+/-1.0% and 35.1+/-4.5% apoptosis, respectively, as measured by APO-BRDU assay. When ICI 182780 and BE-3-3-3 were used in combination, the percentage of apoptosis was 60.6+/-3.8%. Improved efficacy of ICI 182780 and BE-3-3-3 combination on growth inhibition was observed for T-47D cells also. Western blot analysis showed that combinations of ICI 182780 and BE-3-3-3 caused down-regulation of the anti-apoptotic Bcl-2 and Bcl-XL proteins and increased the level of the pro-apoptotic Bax protein. Combination treatment also increased caspase-8 activation. Analysis of polyamine levels 48 h after combination treatment showed that spermidine and spermine levels were down regulated significantly. These studies indicate a potentially effective combination strategy for breast cancer treatment. Our results also link the down-regulation of polyamine pathway to apoptotic cell death and regulation of mediators of cell death. PMID:15583809

  20. Memantine blocks mitochondrial OPA1 and cytochrome c release, and subsequent apoptotic cell death in glaucomatous retina

    PubMed Central

    Ju, Won-Kyu; Kim, Keun-Young; Angert, Mila; Duong-Polk, Karen X.; Lindsey, James D.; Ellisman, Mark H.; Weinreb, Robert N.

    2009-01-01

    Purpose To determine whether intraocular pressure (IOP) elevation alters OPA1 expression and triggers OPA1 release, as well as whether the uncompetitive N-methyl-D-aspartate (NMDA) glutamate receptor antagonist memantine blocks OPA1 release and subsequent apoptotic cell death in glaucomatous DBA/2J mouse retina. Methods Preglaucomatous DBA/2J mice received memantine (5 mg/kg, i.p. injection, twice a day for 3 months) and IOP in the eyes was measured monthly. RGC loss was counted following Fluoro-Gold labeling. OPA1, Dnm1, Bcl-2 and Bax mRNA were measured by Taqman qPCR. OPA1 protein was assessed by immunohistochemistry and Western blot. Apoptotic cell death was assessed by TUNEL staining. Results Memantine treatment significantly increased RGC survival in glaucomatous DBA/2J mice. Memantine treatment increased the 75 kDa OPA1 isoform but did not alter the 80 and 90 kDa isoforms. The isoforms of OPA1 were significantly increased in the cytosol of the vehicle-treated glaucomatous retinas but were significantly decreased in memantine-treated glaucomatous retinas. OPA1 immunoreactivity was decreased in the photoreceptors of both vehicle- and memantine-treated glaucomatous retinas but was increased in the outer plexiform layer of only the memantine-treated glaucomatous retinas. Memantine blocked apoptotic cell death in the GCL, increased Bcl-2 gene expression, and decreased Bax gene expression. Conclusions OPA1 release from mitochondria in glaucomatous mouse retina is inhibited by blockade of glutamate receptor activation. Because this OPA1 effect was accompanied by increased Bcl-2 expression, decreased Bax expression and apoptosis blockade, glutamate receptor activation in the glaucomatous retina may involve a distinct mitochondria-mediated cell death pathway. PMID:18936150

  1. Improved bioavailability of targeted Curcumin delivery efficiently regressed cardiac hypertrophy by modulating apoptotic load within cardiac microenvironment.

    PubMed

    Ray, Aramita; Rana, Santanu; Banerjee, Durba; Mitra, Arkadeep; Datta, Ritwik; Naskar, Shaon; Sarkar, Sagartirtha

    2016-01-01

    Cardiomyocyte apoptosis acts as a prime modulator of cardiac hypertrophy leading to heart failure, a major cause of human mortality worldwide. Recent therapeutic interventions have focussed on translational applications of diverse pharmaceutical regimes among which, Curcumin (from Curcuma longa) is known to have an anti-hypertrophic potential but with limited pharmacological efficacies due to low aqueous solubility and poor bioavailability. In this study, Curcumin encapsulated by carboxymethyl chitosan (CMC) nanoparticle conjugated to a myocyte specific homing peptide was successfully delivered in bioactive form to pathological myocardium for effective regression of cardiac hypertrophy in a rat (Rattus norvegicus) model. Targeted nanotization showed higher cardiac bioavailability of Curcumin at a low dose of 5mg/kg body weight compared to free Curcumin at 35mg/kg body weight. Moreover, Curcumin/CMC-peptide treatment during hypertrophy significantly improved cardiac function by downregulating expression of hypertrophy marker genes (ANF, β-MHC), apoptotic mediators (Bax, Cytochrome-c) and activity of apoptotic markers (Caspase 3 and PARP); whereas free Curcumin in much higher dose showed minimal improvement during compromised cardiac function. Targeted Curcumin treatment significantly lowered p53 expression and activation in diseased myocardium via inhibited interaction of p53 with p300-HAT. Thus attenuated acetylation of p53 facilitated p53 ubiquitination and reduced the apoptotic load in hypertrophied cardiomyocytes; thereby limiting cardiomyocytes' need to enter the regeneration cycle during hypertrophy. This study elucidates for the first time an efficient targeted delivery regimen for Curcumin and also attributes towards probable mechanistic insight into its therapeutic potential as a cardio-protective agent for regression of cardiac hypertrophy. PMID:26612707

  2. COMPUTATIONAL MODELING OF SIGNALING PATHWAYS MEDIATING CELL CYCLE AND APOPTOTIC RESPONSES TO IONIZING RADIATION MEDIATED DNA DAMAGE

    EPA Science Inventory

    Demonstrated of the use of a computational systems biology approach to model dose response relationships. Also discussed how the biologically motivated dose response models have only limited reference to the underlying molecular level. Discussed the integration of Computational S...

  3. High-Content Analysis of Pro-Apoptotic EphA4 Dependence Receptor Functions using Small Molecule Libraries

    PubMed Central

    Nelersa, Claudiu M.; Barreras, Henry; Runko, Erik; Ricard, Jerome; Shi, Yan; Bixby, John L.; Lemmon, Vance P.; Liebl, Daniel J.

    2015-01-01

    Small molecule compounds (SMCs) can provide an inexpensive and selective approach to modifying biological responses. High-content analysis (HCA) of SMC libraries can help identify candidate molecules that inhibit or activate cellular responses. In particular, regulation of cell death has important implications for many pathological conditions. Dependence receptors are a new classification of pro-apoptotic membrane receptors that, unlike classic death receptors, initiate apoptotic signals in the absence of their ligands. EphA4 has recently been identified as a dependence receptor that may have important functions in conditions as disparate as cancer biology and CNS injury and disease. To screen potential candidate SMCs that inhibit or activate EphA4-induced cell death, HCA of a SMC library was performed using stable EphA4-expressing NIH3T3 cells. Our results describe a high-content method for screening dependence receptor-signaling pathways, and demonstrate that several candidate SMCs can inhibit EphA4-mediated cell death. PMID:22492230

  4. X-linked Inhibitor of Apoptosis Protein promotes the degradation of its antagonist, the pro-apoptotic ARTS protein.

    PubMed

    Bornstein, Bavat; Edison, Natalia; Gottfried, Yossi; Lev, Tali; Shekhtman, Anna; Gonen, Hedva; Rajalingam, Krishnaraj; Larisch, Sarit

    2012-03-01

    ARTS (Sept4_i2) is a mitochondrial pro-apoptotic tumor suppressor protein. In response to apoptotic signals, ARTS translocates to the cytosol where it promotes caspase activation through caspase de-repression and proteasome mediated degradation of X-linked Inhibitor of Apoptosis Protein (XIAP). Here we show that XIAP regulates the levels of ARTS by serving as its ubiquitin ligase, thereby providing a potential feedback mechanism to protect against unwanted apoptosis. Using both in vitro and in vivo ubiquitination assays we found that ARTS is directly ubiquitinated by XIAP. Moreover, we found that XIAP-induced ubiquitination and degradation is prevented by removal of the first four amino acids in the N-terminus of ARTS, which contains a single lysine residue at position 3. Thus, this lysine at position 3 is a likely target for ubiquitination by XIAP. Importantly, although the stabilized ARTS lacking its first 4 residues binds XIAP as well as the full length ARTS, it is more potent in promoting apoptosis than the full length ARTS. This suggests that increased stability of ARTS has a significant effect on its ability to induce apoptosis. Collectively, our data reveal a mutual regulatory mechanism by which ARTS and XIAP control each other's levels through the ubiquitin proteasome system. PMID:22185822

  5. Molecular analysis of neutrophil spontaneous apoptosis reveals a strong role for the pro-apoptotic BH3-only protein Noxa.

    PubMed

    Kirschnek, S; Vier, J; Gautam, S; Frankenberg, T; Rangelova, S; Eitz-Ferrer, P; Grespi, F; Ottina, E; Villunger, A; Hcker, H; Hcker, G

    2011-11-01

    Neutrophils enter the peripheral blood from the bone marrow and die after a short time. Molecular analysis of spontaneous neutrophil apoptosis is difficult as these cells die rapidly and cannot be easily manipulated. We use conditional Hoxb8 expression to generate mouse neutrophils and test the regulation of apoptosis by extensive manipulation of B-cell lymphoma protein 2 (Bcl-2)-family proteins. Spontaneous apoptosis was preceded by downregulation of anti-apoptotic Bcl-2 proteins. Loss of the pro-apoptotic Bcl-2 homology domain (BH3)-only protein Bcl-2-interacting mediator of cell death (Bim) gave some protection, but only neutrophils deficient in both BH3-only proteins, Bim and Noxa, were strongly protected against apoptosis. Function of Noxa was at least in part neutralization of induced myeloid leukemia cell differentiation protein (Mcl-1) in neutrophils and progenitors. Loss of Bim and Noxa preserved neutrophil function in culture, and apoptosis-resistant cells remained in circulation in mice. Apoptosis regulated by Bim- and Noxa-driven loss of Mcl-1 is thus the final step in neutrophil differentiation, required for the termination of neutrophil function and neutrophil-dependent inflammation. PMID:21660046

  6. Tolerance strategies employing antigen-coupled apoptotic cells and carboxylated PLG nanoparticles for the treatment of type 1 diabetes.

    PubMed

    Prasad, Suchitra; Xu, Dan; Miller, Stephen D

    2012-01-01

    The development of therapies that specifically target autoreactive immune cells for the prevention and treatment of type 1 diabetes (T1D) without inducing generalized immunosuppression that often compromises the host's ability to clear non-self antigen is highly desired. This review discusses the mechanisms and potential therapeutic applications of antigen-specific T cell tolerance techniques using syngeneic apoptotic cellular carriers and synthetic nanoparticles that are covalently cross-linked to diabetogenic peptides or proteins through ethylene carbodiimide (ECDI) to prevent and treat T1D. Experimental models have demonstrated that intravenous injection of autoantigen decorated splenocytes and biodegradable nanoparticles through ECDI fixation effectively induce and maintain antigen-specific T cell abortive activation and anergy by T cell intrinsic and extrinsic mechanisms. The putative mechanisms include, but are not limited to, the uptake and processing of antigen-coupled nanoparticles or apoptotic cellular carriers for tolerogenic presentation by host splenic antigen-presenting cells, the induction of regulatory T cells, and the secretion of immune-suppressive cytokines, such as IL-10 and TGF-β. The safety profile and efficacy of this approach in preclinical animal models of T1D, including non-obese diabetic (NOD), BDC2.5 transgenic, and humanized mice, have been extensively investigated, and will be the focus of this review. Translation of this approach to clinical trials of T1D and other T cell-mediated autoimmune diseases will also be reviewed in this chapter. PMID:23804269

  7. Pro-apoptotic Bim suppresses breast tumor cell metastasis and is a target gene of SNAI2.

    PubMed

    Merino, D; Best, S A; Asselin-Labat, M-L; Vaillant, F; Pal, B; Dickins, R A; Anderson, R L; Strasser, A; Bouillet, P; Lindeman, G J; Visvader, J E

    2015-07-23

    Evasion of cell death is fundamental to the development of cancer and its metastasis. The role of the BCL-2-mediated (intrinsic) apoptotic program in these processes remains poorly understood. Here we have investigated the relevance of the pro-apoptotic protein BIM to breast cancer progression using the MMTV-Polyoma middle-T (PyMT) transgenic model. BIM deficiency in PyMT females did not affect primary tumor growth, but substantially increased the survival of metastatic cells within the lung. These data reveal a role for BIM in the suppression of breast cancer metastasis. Intriguingly, we observed a striking correlation between the expression of BIM and the epithelial to mesenchymal transition transcription factor SNAI2 at the proliferative edge of the tumors. Overexpression and knockdown studies confirmed that these two genes were coordinately expressed, and chromatin immunoprecipitation analysis further revealed that Bim is a target of SNAI2. Taken together, our findings suggest that SNAI2-driven BIM-induced apoptosis may temper metastasis by governing the survival of disseminating breast tumor cells. PMID:25263453

  8. Krabbe disease: involvement of connexin43 in the apoptotic effects of sphingolipid psychosine on mouse oligodendrocyte precursors.

    PubMed

    Graziano, A C E; Parenti, R; Avola, R; Cardile, V

    2016-01-01

    Krabbe disease is a genetic demyelinating syndrome characterized by deficiency of the enzyme ?-galactosylceramidase, lysosomal psychosine accumulation, and loss of myelin-forming cells. In this study, some apoptotic markers such as apoptotic index (AI), DNA fragmentation, caspase-3, PTEN, Bad, and PI3K were determined in oligodendrocyte precursors from wild type or twitcher mice untreated or treated with psychosine. Twitcher is a natural mouse model of Krabbe disease containing a premature stop codon (W339X) in the ?-galactosylceramidase gene. Moreover, a possible involvement of connexin (Cx)43 in cell death of oligodendrocyte precursors induced by psychosine was investigated with the final aim to provide a contribution to the knowledge of the molecular mechanisms and pathophysiological events that occur in Krabbe disease. Connexins are a multigene family of structurally related trans-membrane proteins able to modulate essential cellular processes such as proliferation, differentiation and migration. Among these, Cx43 is the predominant isoform in many cell types, including neural progenitor cells. Our results showed an increase of AI, DNA fragmentation, caspase-3, PTEN, Bad, and Cx43 associated to a decrease of PI3K, pAKT and pBad. Taken together, these findings suggest an involvement of Cx43 in the psychosine-mediated apoptosis of primary oligodendrocyte progenitors from wild type or twitcher mice, used for the first time as cell models in comparison. It could open unexplored perspective also for other demyelinating diseases. PMID:26459425

  9. Genotoxic stress/p53-induced DNAJB9 inhibits the pro-apoptotic function of p53

    PubMed Central

    Lee, H J; Kim, J M; Kim, K H; Heo, J I; Kwak, S J; Han, J A

    2015-01-01

    DNAJB9 is a recently isolated member of the molecular chaperone gene family, whose precise function is largely unknown. In the present study, we have identified DNAJB9 as an inducible gene of the tumor suppressor p53. DNAJB9 expression was induced by p53 or genotoxic stress in a p53-dependent manner, which was mediated by the Ras/Raf/ERK pathway. In addition, depletion of DNAJB9 by using siRNAs greatly increased genotoxic stress/p53-induced apoptosis, suggesting that DNAJB9 inhibits the pro-apoptotic function of p53. We also found that DNAJB9 physically interacts with p53 through its J domain, through which it inhibits the pro-apoptotic function of p53. Moreover, DNAJB9 colocalized with p53 in both cytoplasm and nucleus under genotoxic conditions. Together, these results demonstrate that DNAJB9 is a downstream target of p53 that belongs to the group of negative feedback regulators of p53. PMID:25146923

  10. Artocarpus communis Induces Autophagic Instead of Apoptotic Cell Death in Human Hepatocellular Carcinoma Cells.

    PubMed

    Tzeng, Cheng-Wei; Tzeng, Wen-Sheng; Lin, Liang-Tzung; Lee, Chiang-Wen; Yen, Ming-Hong; Yen, Feng-Lin; Lin, Chun-Ching

    2015-01-01

    For centuries, natural plant extracts have played an important role in traditional medicine for curing and preventing diseases. Studies have revealed that Artocarpus communis possess various bioactivities, such as anti-inflammation, anti-oxidant, and anticancer activities. A. communis offers economic value as a source of edible fruit, yields timber, and is widely used in folk medicines. However, little is known about its molecular mechanisms of anticancer activity. Here, we demonstrate the antiproliferative activity of A. communis methanol extract (AM) and its dichloromethane fraction (AD) in two human hepatocellular carcinoma (HCC) cell lines, HepG2 and PLC/PRF/5. Colony assay showed the long-term inhibitory effect of both extracts on cell growth. DNA laddering and immunoblotting analyses revealed that both extracts did not induce apoptosis in the hepatoma cell lines. AM and AD-treated cells demonstrated different cell cycle distribution compared to UV-treated cells, which presented apoptotic cell death with high sub-G1 ratio. Instead, acridine orange staining revealed that AM and AD triggered autophagosome accumulation. Immunoblotting showed a significant expression of autophagy-related proteins, which indicated the autophagic cell death (ACD) of hepatoma cell lines. This study therefore demonstrates that A. communis AM and its dichloromethane fraction can induce ACD in HCC cells and elucidates the potential of A. communis extracts for development as anti tumor therapeutic agents that utilize autophagy as mechanism in mediating cancer cell death. PMID:25967668

  11. Apoptotic effects of ?-mangostin from the fruit hull of Garcinia mangostana on human malignant glioma cells.

    PubMed

    Chang, Hui-Fang; Huang, Wen-Tsung; Chen, Hui-Ju; Yang, Ling-Ling

    2010-01-01

    Gliomas are a common type of primary brain tumor with glioblastoma multiforme accounting for the majority of human brain tumors. In this paper, high grade human malignant glioblastomas (MGs) including U87 MG and GBM 8401 were used to evaluate the antitumor effects of ?-mangostin, a xanthone derivative isolated and purified from the hull of the tropical fruit Garcinia mangostana. The ?-mangostin showed potent antiproliferative activity toward MGs in dose- and time-dependent manners. In addition, flow cytometric analysis of cell morphology in the apoptotic cells revealed an increase in hypodiploid cells in ?-mangostin treated U87 MG and GBM 8401 cells, while significant enhancement of intracellular peroxide production was detected in the same ?-mangostin treated cells by DCHDA assay and DiOC(6)(3) stain. g-Mangostin induced apoptosis, which in turn mediates cytotoxicity in human MG cells was prevented by the addition of catalase. Naturally derived medicines and herbal therapies are drawing increasing attention in regard to the treatment of many health issues, and this includes the testing of new phytochemicals or nutrients for brain tumor patients. This has led to ?-mangostin being identified as a potential leading compound for the development of an anti-brain tumor agent. PMID:21139533

  12. An unfractionated fucoidan from Ascophyllum nodosum: extraction, characterization, and apoptotic effects in vitro.

    PubMed

    Foley, Sarah A; Szegezdi, Eva; Mulloy, Barbara; Samali, Afshin; Tuohy, Maria G

    2011-09-23

    An unfractionated fucoidan was extracted from the brown alga Ascophyllum nodosum. Extraction of fucoidan from seaweed was carried out using an innovative low-chemical process. A combinational approach involving compositional analysis, HPAEC, IR analysis, GPC, and NMR was employed to elucidate the composition and structure of an unfractionated fucoidan from A. nodosum. This fucoidan is composed mainly of fucose (52.1%), and also galactose (6.1%), glucose (21.3%), and xylose (16.5%). Sulfate content was determined to be 19%. GPC data indicated a polydisperse fucoidan containing two main size fractions (47 and 420 kDa). NMR analyses revealed a fucoidan displaying broad, complex signals as expected for such a high molecular weight and heterogeneous polymer with resonances consistent with a fucoidan isolated previously from A. nodosum. The effects of fucoidan on the apoptosis of human colon carcinoma cells and fucoidan-mediated signaling pathways were also investigated. Fucoidan decreased cell viability and induced apoptosis of HCT116 colon carcinoma cells. Fucoidan treatment of HCT116 cells induced activation of caspases-9 and -3 and the cleavage of PARP, led to apoptotic morphological changes, and altered mitochondrial membrane permeability. These results detail the structure and biological activity of an unfractionated fucoidan from A. nodosum. PMID:21875034

  13. Apoptotic effects of Photofrin-Diomed 630-PDT on SHEEC human esophageal squamous cancer cells

    PubMed Central

    Gao, Shegan; Zhang, Mengxi; Zhu, Xiaojuan; Qu, Zhifeng; Shan, Tanyou; Xie, Xuanhu; Wang, Ying; Feng, Xiaoshan

    2015-01-01

    Photodynamic therapy (PDT) using photofrin-II is a clinically effective treatment for both non-neoplastic and neoplastic diseases. Herein, we performed an in vitro experiment to study the anti-tumor effect and mechanisms of photofrin-II mediated PDT for esophageal squamous cell carcinoma (ESCC) cell line, SHEEC. In this study, human ESCC cell line SHEEC and parental normal cell line SHEE were used. The anti-tumor effect of PDT was determined by evaluating cell viability using CCK-8 assay, apoptosis and generation of reactive oxygen species (ROS). PDT induced significant apoptosis in SHEEC and SHEE cells in a time- and photofrin-II dose-dependent manner. Furthermore, PDT treatment induced significant death of SHEEC, instead of SHEE cells. The apoptotic outcome was accompanied by concurrent generation of ROS. In summary, PDT shed light on therapy of ESCC, functioning as a useful tool for ESCC clinical treatment, providing a better understanding of Photofrin-Diomed 630-PDT in SHEEC cells. PMID:26628993

  14. A novel prohibitin-binding compound induces the mitochondrial apoptotic pathway through NOXA and BIM upregulation.

    PubMed

    Moncunill-Massaguer, Cristina; Saura-Esteller, Jos; Prez-Perarnau, Alba; Palmeri, Claudia Mariela; Nez-Vzquez, Sonia; Cosialls, Ana M; Gonzlez-Girons, Diana M; Pomares, Helena; Korwitz, Anne; Preciado, Sara; Albericio, Fernando; Lavilla, Rodolfo; Pons, Gabriel; Langer, Thomas; Iglesias-Serret, Daniel; Gil, Joan

    2015-12-01

    We previously described diaryl trifluorothiazoline compound 1a (hereafter referred to as fluorizoline) as a first-in-class small molecule that induces p53-independent apoptosis in a wide range of tumor cell lines. Fluorizoline directly binds to prohibitin 1 and 2 (PHBs), two proteins involved in the regulation of several cellular processes, including apoptosis. Here we demonstrate that fluorizoline-induced apoptosis is mediated by PHBs, as cells depleted of these proteins are highly resistant to fluorizoline treatment. In addition, BAX and BAK are necessary for fluorizoline-induced cytotoxic effects, thereby proving that apoptosis occurs through the intrinsic pathway. Expression analysis revealed that fluorizoline induced the upregulation of Noxa and Bim mRNA levels, which was not observed in PHB-depleted MEFs. Finally, Noxa-/-/Bim-/- MEFs and NOXA-downregulated HeLa cells were resistant to fluorizoline-induced apoptosis. All together, these findings show that fluorizoline requires PHBs to execute the mitochondrial apoptotic pathway. PMID:26497683

  15. A novel prohibitin-binding compound induces the mitochondrial apoptotic pathway through NOXA and BIM upregulation

    PubMed Central

    Moncunill-Massaguer, Cristina; Saura-Esteller, José; Pérez-Perarnau, Alba; Palmeri, Claudia Mariela; Núñez-Vázquez, Sonia; Cosialls, Ana M.; González-Gironès, Diana M.; Pomares, Helena; Korwitz, Anne; Preciado, Sara; Albericio, Fernando; Lavilla, Rodolfo; Pons, Gabriel; Langer, Thomas; Iglesias-Serret, Daniel; Gil, Joan

    2015-01-01

    We previously described diaryl trifluorothiazoline compound 1a (hereafter referred to as fluorizoline) as a first-in-class small molecule that induces p53-independent apoptosis in a wide range of tumor cell lines. Fluorizoline directly binds to prohibitin 1 and 2 (PHBs), two proteins involved in the regulation of several cellular processes, including apoptosis. Here we demonstrate that fluorizoline-induced apoptosis is mediated by PHBs, as cells depleted of these proteins are highly resistant to fluorizoline treatment. In addition, BAX and BAK are necessary for fluorizoline-induced cytotoxic effects, thereby proving that apoptosis occurs through the intrinsic pathway. Expression analysis revealed that fluorizoline induced the upregulation of Noxa and Bim mRNA levels, which was not observed in PHB-depleted MEFs. Finally, Noxa−/−/Bim−/− MEFs and NOXA-downregulated HeLa cells were resistant to fluorizoline-induced apoptosis. All together, these findings show that fluorizoline requires PHBs to execute the mitochondrial apoptotic pathway. PMID:26497683

  16. Apoptotic Mechanisms of Peroxisome ProliferatorActivated Receptor-? Activation in Acinar Cells During Acute Pancreatitis

    PubMed Central

    Xu, Ping; Lou, Xiao-Li; Chen, Cheng

    2016-01-01

    Objective The objective of this study was to determine the mechanism by which activation of peroxisome proliferatoractivated receptor-? promotes apoptosis of acinar cells in pancreatitis. Methods AR42j cells pretreated with the peroxisome proliferatoractivated receptor-? agonist pioglitazone were activated by cerulein as an in vitro model of acute pancreatitis. Inflammatory cytokines and amylase were detected by enzyme-linked immunosorbent assay. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was measured by flow cytometry and terminal deoxynucleotidyl transferasemediated dUTP nick end labeling staining. Activity of caspases was determined. Bax and Bcl-2 levels were assayed by Western blot. Results Cytokines, amylase, and cellular proliferation decreased in pioglitazone-pretreated cells. Pioglitazone increased the activity of caspases 3, 8, and 9 in cerulein-activated AR42j cells as well as in the pancreas of rats 3 hours after induction of severe acute pancreatitis. Acinar cell apoptosis was induced by reducing the mitochondrial membrane potential in the pioglitazone group. Pioglitazone increased expression of proapoptotic Bax proteins and decreased antiapoptotic Bcl-2 in cerulein-induced AR42j cells and decreased Bcl-2 levels in pancreatic tissue of severe acute pancreatitis rats 1 and 3 hours after induction. Conclusion Pioglitazone may promote apoptosis of acinar cells through both intrinsic and extrinsic apoptotic pathways in acute pancreatitis. PMID:26495791

  17. Selective apoptotic cell death effects of oral cancer cells treated with destruxin B

    PubMed Central

    2014-01-01

    Background Recent studies have revealed that destruxins (Dtx) have potent cytotoxic activities on individual cancer cells, however, data on oral cancer cells especial human are absent. Methods Destruxin B (DB) was isolated and used to evaluate the selective cytotoxicity with human oral cancer cell lines, GNM (Neck metastasis of gingival carcinoma) and TSCCa (Tongue squamous cell carcinoma) cells, and normal gingival fibroblasts (GF) were also included as controls. Cells were tested with different concentrations of DB for 24, 48, and 72 h by MTT assay. Moreover, the mechanism of cytotoxicity was investigated using caspase-3 Immunofluorescence, annexin V/PI staining, and the expression of caspase-3, Bax, and Bcl-2 by western blotting after treated with different concentrations of DB for 72 h as parameters for apoptosis analyses. Results The results show that DB exhibited significant (p < 0.01) and selective time- and dose-dependent inhibitory effects on GNM and TSCCa cells viability but not on GF cells. The data suggested that DB is capable to induce tumor specific growth inhibition in oral GNM and TSCCa cancer cells via Bax/Bcl-2-mediated intrinsic mitochondrial apoptotic pathway in time- and dose-dependent manners. Conclusions This is the first report on the anti-proliferation effect of DB in oral cancer cells. The results reported here may offer further evidences to the development of DB as a potential complementary chemotherapeutic target for oral cancer complications. PMID:24972848

  18. Molecular analysis of the apoptotic effects of BPA in acute myeloid leukemia cells

    PubMed Central

    Bontempo, Paola; Mita, Luigi; Doto, Antonella; Miceli, Marco; Nebbioso, Angela; Lepore, Ilaria; Franci, GianLuigi; Menafra, Roberta; Carafa, Vincenzo; Conte, Mariarosaria; De Bellis, Floriana; Manzo, Fabio; Di Cerbo, Vincenzo; Benedetti, Rosaria; D'Amato, Loredana; Marino, Maria; Bolli, Alessandro; Del Pozzo, Giovanna; Diano, Nadia; Portaccio, Marianna; Mita, Gustavo D; Vietri, Maria Teresa; Cioffi, Michele; Nola, Ernesto; Dell'Aversana, Carmela; Sica, Vincenzo; Molinari, Anna Maria; Altucci, Lucia

    2009-01-01

    Background: BPA (bisphenol A or 2,2-bis(4-hydroxy-phenol)propane) is present in the manufacture of polycarbonate plastic and epoxy resins, which can be used in impact-resistant safety equipment and baby bottles, as protective coatings inside metal food containers, and as composites and sealants in dentistry. Recently, attention has focused on the estrogen-like and carcinogenic adverse effects of BPA. Thus, it is necessary to investigate the cytotoxicity and apoptosis-inducing activity of this compound. Methods: Cell cycle, apoptosis and differentiation analyses; western blots. Results: BPA is able to induce cell cycle arrest and apoptosis in three different acute myeloid leukemias. Although some granulocytic differentiation concomitantly occurred in NB4 cells upon BPA treatment, the major action was the induction of apoptosis. BPA mediated apoptosis was caspase dependent and occurred by activation of extrinsic and intrinsic cell death pathways modulating both FAS and TRAIL and by inducing BAD phosphorylation in NB4 cells. Finally, also non genomic actions such as the early decrease of both ERK and AKT phosphorylation were induced by BPA thus indicating that a complex intersection of regulations occur for the apoptotic action of BPA. Conclusion: BPA is able to induce apoptosis in leukemia cells via caspase activation and involvement of both intrinsic and extrinsic pathways of apoptosis. PMID:19538739

  19. Interferon-induced mechanosensing defects impede apoptotic cell clearance in lupus

    PubMed Central

    Li, Hao; Fu, Yang-Xin; Wu, Qi; Zhou, Yong; Crossman, David K.; Yang, PingAr; Li, Jun; Luo, Bao; Morel, Laurence M.; Kabarowski, Janusz H.; Yagita, Hideo; Ware, Carl F.; Hsu, Hui-Chen; Mountz, John D.

    2015-01-01

    Systemic lupus erythematosus (SLE) is a severe autoimmune disease that is associated with increased circulating apoptotic cell autoantigens (AC-Ags) as well as increased type I IFN signaling. Here, we describe a pathogenic mechanism in which follicular translocation of marginal zone (MZ) B cells in the spleens of BXD2 lupus mice disrupts marginal zone macrophages (MZMs), which normally clear AC debris and prevent follicular entry of AC-Ags. Phagocytosis of ACs by splenic MZMs required the megakaryoblastic leukemia 1 (MKL1) transcriptional coactivatormediated mechanosensing pathway, which was maintained by MZ B cells through expression of membrane lymphotoxin-?1?2 (mLT). Specifically, type I IFNinduced follicular shuttling of mLT-expressing MZ B cells disengaged interactions between these MZ B cells and LT? receptorexpressing MZMs, thereby downregulating MKL1 in MZMs. Loss of MKL1 expression in MZMs led to defective F-actin polymerization, inability to clear ACs, and, eventually, MZM dissipation. Aggregation of plasmacytoid DCs in the splenic perifollicular region, follicular translocation of MZ B cells, and loss of MKL1 and MZMs were also observed in an additional murine lupus model and in the spleens of patients with SLE. Collectively, the results suggest that lupus might be interrupted by strategies that maintain or enhance mechanosensing signaling in the MZM barrier to prevent follicular entry of AC-Ags. PMID:26098211

  20. Roles of microRNA-1 in hypoxia-induced apoptotic insults to neuronal cells.

    PubMed

    Chang, Chia-Yu; Lui, Tai-Ngar; Lin, Jia-Wei; Lin, Yi-Ling; Hsing, Chung-Hsi; Wang, Jhi-Joung; Chen, Ruei-Ming

    2016-01-01

    Hypoxia is a common occurrence in brain tumors and traumatic brain injury. microRNA (miR)-1 participates in the regulation of brain development and neuronal function. Interestingly, miR-1 can mediate ischemia-induced injury to cardiomyocytes. This study was designed to evaluate the roles of miR-1 in hypoxia-induced insults to neurons and the possible mechanisms. Exposure of neuro-2a cells to oxygen/glucose deprivation (OGD) or cobalt chloride decreased cell viability and induced cell apoptosis in time-dependent manners. In parallel, OGD caused augmentation of cellular Bax and cytochrome c levels, a reduction in the mitochondrial membrane potential (MMP), activation of caspase-3, and fragmentation of DNA. miR-1 was induced in neuro-2a cells by OGD. Knocking down miR-1 expression using specific antisense inhibitors significantly alleviated OGD-induced neuronal death. Administration of OGD to neuro-2a cells induced heat-shock protein (HSP)-70 messenger (m)RNA and protein expressions. A bioinformatic search revealed that miR-1-specific binding elements exist in the 3'-untranslated region of HSP-70 mRNA. Overexpression of miR-1 simultaneously attenuated OGD-induced HSP-70 mRNA and protein expressions. In comparison, knocking down miR-1 expression synergistically enhanced OGD-induced HSP-70 mRNA. As to the mechanism, reducing miR-1 expression lowered OGD-induced alterations in the MMP, caspase-3 activation, DNA fragmentation, and cell apoptosis. Taken together, this study shows that miR-1 can target HSP-70 expression and consequently mediate hypoxia-induced apoptotic insults to neuro-2a cells via an intrinsic Bax-mitochondrion-caspase protease pathway. PMID:25238743

  1. Ochratoxin A Inhibits Mouse Embryonic Development by Activating a Mitochondrion-Dependent Apoptotic Signaling Pathway

    PubMed Central

    Hsuuw, Yan-Der; Chan, Wen-Hsiung; Yu, Jau-Song

    2013-01-01

    Ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, both in vitro and in vivo. In the present study, we explored the cytotoxic effects exerted by OTA on the blastocyst stage of mouse embryos, on subsequent embryonic attachment, on outgrowth in vitro, and following in vivo implantation via embryo transfer. Mouse blastocysts were incubated with or without OTA (1, 5, or 10 ?M) for 24 h. Cell proliferation and growth were investigated using dual differential staining; apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay; and embryo implantation and post-implantation development were assessed by examination of in vitro growth and the outcome of in vivo embryo transfer, respectively. Blastocysts treated with 10 ?M OTA displayed a significantly increased level of apoptosis and a reduction in total cell number. Interestingly, we observed no marked difference in implantation success rate between OTA-pretreated and control blastocysts either during in vitro embryonic development (following implantation in a fibronectin-coated culture dish) or after in vivo embryo transfer. However, in vitro treatment with 10 ?M OTA was associated with increased resorption of post-implantation embryos by the mouse uterus, and decreased fetal weight upon embryo transfer. Our results collectively indicate that in vitro exposure to OTA triggers apoptosis and retards early post-implantation development after transfer of embryos to host mice. In addition, OTA induces apoptosis-mediated injury of mouse blastocysts, via reactive oxygen species (ROS) generation, and promotes mitochondrion-dependent apoptotic signaling processes that impair subsequent embryonic development. PMID:23296271

  2. Mechanism of dichotomy between CD8+ responses elicited by apoptotic and necrotic cells

    PubMed Central

    Buckwalter, Matthew R.; Srivastava, Pramod K.

    2013-01-01

    Apoptotic cells are significantly more immunogenic than necrotic cells, even though both forms are identical in antigenic content. When a combination of apoptotic and necrotic cells are used to immunize, the phenotype conferred by apoptotic cells, i.e., high immunogenicity, is dominant. However, necrotic cells are not immunosuppressive or tolerogenic. Apoptotic and necrotic cells are taken up by antigen-presenting cells in an equivalent manner. The priming of nave T cell response is also equivalent. However, the CD8+ T cells elicited by apoptotic cells expand, accumulate, and express effector function, while those primed by the necrotic cells do not. This dichotomy does not extend to CD4+ cells. Apoptotic and necrotic cells elicit equivalent CD4+ T cell priming, accumulation, and function. The deficit in CD8+ T cell function elicited by necrotic cells can be overcome to varying degrees by anti-CD40 antibody and ligands for TLR4 and TLR9; conversely, the immunogenicity of apoptotic cells can be abrogated by blocking anti-CD154 antibody. Our results indicate that immunization with apoptotic cells leads to engagement of CD40 on antigen-presenting cells; this is essential for their ability to elicit mature functional CD8+ cells. The necrotic cells fail to engage CD40, and this failure is the basis of their lack of immunogenicity. These differences have consequences for the understanding of mechanisms of cross-presentation and for efforts toward immunotherapy of cancers and autoimmune pathologies. PMID:23390373

  3. Cigarette Smoke Inhibits Engulfment of Apoptotic Cells by Macrophages through Inhibition of Actin Rearrangement

    PubMed Central

    Minematsu, Naoto; Blumental-Perry, Anna; Shapiro, Steven D.

    2011-01-01

    Exposure to cigarette smoke (CS) was shown to impair the capacity of macrophages to clear bacteria and apoptotic cells. Here, we show that both the exposure of macrophages to cigarette smoke extract (CSE) in vitro and an acute single exposure to CS in vivo impair the macrophage clearance of apoptotic polymorphonuclear leukocytes (PMNs). Upon longer periods of exposure to smoke in vivo (412 weeks), the impaired capacity of macrophages to clear apoptotic cells persisted after the cessation of smoking, with slow recovery to normality observed 4 weeks later. With respect to the mechanism by which CS impairs the macrophage uptake of apoptotic PMNs, we did not detect altered surface expression of receptors associated with apoptotic cell clearance. We did observe the impaired phosphorylation of the guanine nucleotide exchange factor Vav1 and the downstream inhibition of Ras-related C3 botulinum toxin substrate 1 (Rac1) activation. Consistent with these findings, CS impaired the macrophage cytoskeletal changes observed after stimulation with apoptotic cells. A loss of actin occurred at the leading edge, manifested as impaired ruffling of the cell membrane and a decreased capacity to engulf apoptotic cells. The inability to clear PMNs would lead to a greater release of destructive PMN products, and would diminish the reparative phenotype induced by the macrophage clearance of apoptotic cells. PMID:20525804

  4. Gamma irradiation induced apoptotic changes in the chromatin structure of human erythroleukemia K562 cells.

    PubMed

    Banfalvi, Gaspar; Klaisz, Mariann; Ujvarosi, Kinga; Trencsenyi, Gyorgy; Rozsa, David; Nagy, Gabor

    2007-12-01

    Exponentially growing human erythroleukemia K562 cells were synchronized by centrifugal elutriation prior to and after Co60 gamma-irradiation (4 Gy). Forward scatter flow cytometry used for size analysis revealed the increase of an early apoptotic cell population ranging from lower (0.05 C-value) to higher DNA content (approximately 1 C) as the cells progressed through the S phase. The increase in cellular DNA content expressed in C-values correlated with apoptotic chromatin changes manifested as many small apoptotic bodies in early S phase and larger but less numerous disintegrated apoptotic bodies in late S phase. Most significant changes after exposure to gamma-irradiation took place in early S phase resulting in an increase of nuclear size by more than 50%. Cell fractions containing irradiated cells showed enhanced growth arrest at 2.4 C-value, which was accompanied by apoptosis. Apoptotic cell cycle arrest near to the G1/G0 checkpoint and apoptotic changes indicate that the radiation resistance of K562 cells is related to the bypass of the early stage of the p53 apoptotic pathway. Apoptotic changes in chromatin structure induced by gamma-irradiation indicate that these injury-specific changes can be identified and distinguished from chromatin changes induced by UV radiation or heavy metals. PMID:17924194

  5. Penicillium antifungal protein (PAF) is involved in the apoptotic and autophagic processes of the producer Penicillium chrysogenum.

    PubMed

    Kovács, Barbara; Hegedűs, Nikoletta; Bálint, Mihály; Szabó, Zsuzsa; Emri, Tamás; Kiss, Gréta; Antal, Miklós; Pócsi, István; Leiter, Eva

    2014-09-01

    PAF, which is produced by the filamentous fungus Pencicillium chrysogenum, is a small antifungal protein, triggering ROS-mediated apoptotic cell death in Aspergillus nidulans. In this work, we provide information on the function of PAF in the host P. chrysogenum considering that carbon-starving cultures of the Δpaf mutant strain showed significantly reduced apoptosis rates in comparison to the wild-type (wt) strain. Moreover, the addition of PAF to the Δpaf strain resulted in a twofold increase in the apoptosis rate. PAF was also involved in the regulation of the autophagy machinery of this fungus, since several Saccharomyces cerevisiae autophagy-related ortholog genes, e.g. those of atg7, atg22 and tipA, were repressed in the deletion strain. This phenomenon was accompanied by the absence of autophagosomes in the Δpaf strain, even in old hyphae. PMID:25261948

  6. Apoptotic and anti-metastatic effects of the whole skin of Venenum bufonis in A549 human lung cancer cells

    PubMed Central

    PARK, JEONG-SEOK; SHIN, DONG YEOK; LEE, YEON-WEOL; CHO, CHONG-KWAN; KIM, GI YOUNG; KIM, WUN-JAE; YOO, HWA-SEUNG; CHOI, YUNG HYUN

    2012-01-01

    In the present study, the effects of the whole skin of Venenum bufonis on apoptotic and anti-invasive activity in A549 human lung cancer cells were investigated. Treatment with extract of the whole skin of V. bufonis (SVB) resulted in a significant decrease in cell growth of A549 cells, depending on dosage, which was associated with apoptosis induction, as proved by chromatin condensation and accumulation of apoptotic fraction. SVB treatment induced expression of death receptor-related proteins, such as death receptor 4, which further triggered activation of caspase-8 and cleavage of Bid. In addition, the increase in apoptosis by SVB treatment was correlated with dysfunction of mitochondria, activation of caspase-9 and -3, downregulation of IAP family proteins, such as XIAP, cIAP-1 and cIAP-2, and concomitant degradation of activated caspase-3-specific target proteins, such as poly (ADP-ribose) polymerase and β-catenin proteins. However, z-DEVD-fmk, a caspase-3-specific inhibitor, blocked SVB-induced apoptosis and increased the survival rate of SVB-treated cells, indicating that activation of caspase-3 plays a key role in SVB-induced apoptosis. In addition, within concentrations that were not cytotoxic to A549 cells, SVB induced marked inhibition of cell motility and invasiveness. Activities of matrix metalloproteinase (MMP)-2 and MMP-9 in AGS cells were dose-dependently inhibited by treatment with SVB, and this was also correlated with a decrease in expression of their mRNA and proteins, and upregulation of tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 mRNA expression. Further studies are needed; however, the results indicated that SVB induces apoptosis of A549 cells through a signaling cascade of death receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways. Our data also demonstrated that MMPs are critical targets of SVB-induced anti-invasiveness in A549 cells. PMID:22200726

  7. Gastroprotective effect of nymphayol isolated from Nymphaea stellata (Willd.) flowers: contribution of antioxidant, anti-inflammatory and anti-apoptotic activities.

    PubMed

    Antonisamy, Paulrayer; Subash-Babu, Pandurangan; Alshatwi, Ali A; Aravinthan, Adithan; Ignacimuthu, Savarimuthu; Choi, Ki Choon; Kim, Jong-Hoon

    2014-12-01

    Gastric ulcer is an illness that affects a great number of people worldwide. The goal of the present research was to assess the anti-ulcerogenic activity of nymphayol (NYM), isolated from Nymphaea stellata, against an ethanol-induced ulcer model in rats. Administration of ethanol elevates the levels of the ulcer index (UI) along with causing tremendous increases in lipid peroxidation and myeloperoxidase (MPO) and significant decreases in gastric mucus, catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), and prostaglandin E2 (PGE2). However, the NYM- (45 mg/kg) pretreated animals showed considerable increases in antioxidants, gastric mucus, and PGE2 level and significant decreases in UI, lipid peroxidation, and MPO level. Pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) were increased and the level of interleukin-10 (IL-10), an anti-inflammatory cytokine, was decreased in ethanol-induced ulcerated animals, and these inequalities were amended by NYM pretreatment. Pro-apoptotic markers including caspase-8, caspase-9, and caspase-3 were decreased and Bcl-2, an anti-apoptotic marker, was increased through NYM pretreatment, as compared with the ethanol-induced ulcer group. Pretreatment with indomethacin, SC560, rofecoxib, and Nω-Nitro-L-arginine methyl ester (L-NAME) considerably prevented the ulcer protective activity of NYM (45 mg/kg), indicating the involvement of cyclooxygenase (COX) and nitric oxide synthase (NOS) in NYM-mediated gastroprotection against ethanol-induced ulcer. These outcomes suggest that the gastroprotective effect of NYM might be mediated by adjustment of inflammatory mediators and apoptotic markers and increasing antioxidants. PMID:25289771

  8. Delisheng, a Chinese medicinal compound, exerts anti-proliferative and pro-apoptotic effects on HepG2 cells through extrinsic and intrinsic pathways.

    PubMed

    Lu, Chuang-xin; Nan, Ke-jun; Nie, Yan-li; Hai, Ya-nan; Jiao, Min

    2010-10-01

    The anti-proliferative, cytotoxic and apoptogenic activities of delisheng, a Chinese medicinal compound, has been investigated. In this study, the hepatocarcinoma cell line (HepG2) and the liver cell line (L-02) were exposed to delisheng (6.25, 50 and 100 ?l/ml). Delisheng suppressed the proliferation and viability of normal liver L-02 cells slightly, but strongly inhibited the proliferation and viability of hepatocarcinoma HepG2 cells. The flow cytometric analysis of HepG2 cells demonstrated that delisheng primarily arrested the HepG2 cells at the G1 phase of the cell cycle. Annexin V-FITC/PI staining corroborates the apoptogenic nature of delisheng on HepG2 cells. The anti-proliferative and pro-apoptotic effect of delisheng in HepG2 cells was associated with changes in the Bcl-2/Bax ratio and the induction of caspase-mediated apoptosis. Upregulation of DR5 expression was observed in HepG2 cells after treatment with delisheng. The findings from the present study suggest that delisheng has selective cytotoxic activities against HepG2 cells. Delisheng triggered time- and dose-dependent apoptosis in HepG2 cells by activating the mitochondria-mediated and death receptor-mediated apoptotic pathways. PMID:20012371

  9. Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes.

    PubMed

    Erenpreisa, J; Freivalds, T; Roach, H; Alston, R

    1997-07-01

    Apoptotic cell nuclei are known to stain hyperchromatically with absorption dyes and dimly with many DNA fluorochromes. We hypothesised that both optical phenomena have the same cause--the ability of apoptotic chromatin to aggregate cationic dyes. This hypothesis was tested using prednisolone-primed rat thymus, which is known to contain apoptotic cells. The apoptotic cells were classified as early and late, based on their morphology, in thin and semithin sections and in thymus imprints on slides. Direct reaction for DNA strand breaks (TUNEL) indicated the presence of breaks in both categories of cells, with more intense labelling in late apoptosis. The chromatin ultrastructure of early apoptotic cells initially retained the supranucleosomal order of packaging which characterises control cells, whereas the dense chromatin of late apoptotic cells possessed the degraded structure. Absorption spectra of the toluidine blue-stained early apoptotic cell chromatin revealed a metachromatic shift, indicating a change of DNA conformation and polymerisation of the dye. When the staining was performed by acridine orange (preceded by a short acid treatment), a paradoxical several-fold increase of fluorescence intensity at a several-fold dilution of the dye was found. The simultaneous reduction of the ratio of red to green components of fluorescence confirmed that the concentration-dependent fluorescence quenching was due to aggregation of the dye. The results suggest that the enhanced affinity of the chromatin of early apoptotic cells for cationic dyes is associated with conformational relaxation rather than degradation of DNA. In late apoptotic cells, the very dense packaging of degraded DNA promotes further aggregation of dyes. The results suggest alternative methods for detection and discrimination of early and late apoptotic cells. PMID:9377226

  10. ERK1/2 acts as a switch between necrotic and apoptotic cell death in ether phospholipid edelfosine-treated glioblastoma cells.

    PubMed

    Melo-Lima, Sara; Lopes, Maria C; Mollinedo, Faustino

    2015-01-01

    Glioblastoma is characterized by constitutive apoptosis resistance and survival signaling expression, but paradoxically is a necrosis-prone neoplasm. Incubation of human U118 glioblastoma cells with the antitumor alkylphospholipid analog edelfosine induced a potent necrotic cell death, whereas apoptosis was scarce. Preincubation of U118 cells with the selective MEK1/2 inhibitor U0126, which inhibits MEK1/2-mediated activation of ERK1/2, led to a switch from necrosis to caspase-dependent apoptosis following edelfosine treatment. Combined treatment of U0126 and edelfosine totally inhibited ERK1/2 phosphorylation, and led to RIPK1 and RelA/NF-?B degradation, together with a strong activation of caspase-3 and -8. This apoptotic response was accompanied by the activation of the intrinsic apoptotic pathway with mitochondrial transmembrane potential loss, Bcl-xL degradation and caspase-9 activation. Inhibition of ERK phosphorylation also led to a dramatic increase in edelfosine-induced apoptosis when the alkylphospholipid analog was used at a low micromolar range, suggesting that ERK phosphorylation acts as a potent regulator of apoptotic cell death in edelfosine-treated U118 cells. These data show that inhibition of MEK1/2-ERK1/2 signaling pathway highly potentiates edelfosine-induced apoptosis in glioblastoma U118 cells and switches the type of edelfosine-induced cell death from necrosis to apoptosis. PMID:25749008

  11. Establishment of a specific cell death induction system in Bombyx mori by a transgene with the conserved apoptotic regulator, mouse Bcl-2-associated X protein (mouse Bax).

    PubMed

    Sumitani, M; Sakurai, T; Kasashima, K; Kobayashi, S; Uchino, K; Kanzaki, R; Tamura, T; Sezutsu, H

    2015-12-01

    The induction of apoptosis in vivo is a useful tool for investigating the functions and importance of particular tissues. B-cell leukaemia/lymphoma 2-associated X protein (Bax) functions as a pro-apoptotic factor and induces apoptosis in several organisms. The Bax-mediated apoptotic system is widely conserved from Caenorhabditis elegans to humans. In order to establish a tissue-specific cell death system in the domestic silkworm, Bombyx mori, we constructed a transgenic silkworm that overexpressed mouse Bax (mBax) in particular tissues by the Gal4-upstream activation sequence system. We found that the expression of mBax induced specific cell death in the silk gland, fat body and sensory cells. Fragmentation of genomic DNA was observed in the fat body, which expressed mBax, thereby supporting apoptotic cell death in this tissue. Using this system, we also demonstrated that specific cell death in sensory cells attenuated the response to the sex pheromone bombykol. These results show that we successfully established a tissue-specific cell death system in vivo that enabled specific deficiencies in particular tissues. The inducible cell death system may provide useful means for industrial applications of the silkworm and possible utilization for other species. PMID:26426866

  12. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells.

    PubMed

    Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi

    2013-08-30

    Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs. PMID:23899527

  13. Norepinephrine-induced apoptotic and hypertrophic responses in H9c2 cardiac myoblasts are characterized by different repertoire of reactive oxygen species generation

    PubMed Central

    Thakur, Anita; Alam, Md. Jahangir; Ajayakumar, MR; Ghaskadbi, Saroj; Sharma, Manish; Goswami, Shyamal K.

    2015-01-01

    Despite recent advances, the role of ROS in mediating hypertrophic and apoptotic responses in cardiac myocytes elicited by norepinephrine (NE) is rather poorly understood. We demonstrate through our experiments that H9c2 cardiac myoblasts treated with 2 µM NE (hypertrophic dose) generate DCFH-DA positive ROS only for 2 h; while those treated with 100 µM NE (apoptotic dose) sustains generation for 48 h, followed by apoptosis. Though the levels of DCFH fluorescence were comparable at early time points in the two treatment sets, its quenching by DPI, catalase and MnTmPyP suggested the existence of a different repertoire of ROS. Both doses of NE also induced moderate levels of H2O2 but with different kinetics. Sustained but intermittent generation of highly reactive species detectable by HPF was seen in both treatment sets but no peroxynitrite was generated in either conditions. Sustained generation of hydroxyl radicals with no appreciable differences were noticed in both treatment sets. Nevertheless, despite similar profile of ROS generation between the two conditions, extensive DNA damage as evident from the increase in 8-OH-dG content, formation of γ-H2AX and PARP cleavage was seen only in cells treated with the higher dose of NE. We therefore conclude that hypertrophic and apoptotic doses of NE generate distinct but comparable repertoire of ROS/RNS leading to two very distinct downstream responses. PMID:26070033

  14. Diallyl disulfide enhances carbon ion beams-induced apoptotic cell death in cervical cancer cells through regulating Tap73 /?Np73.

    PubMed

    Di, Cuixia; Sun, Chao; Li, Hongyan; Si, Jing; Zhang, Hong; Han, Lu; Zhao, Qiuyue; Liu, Yang; Liu, Bin; Miao, Guoying; Gan, Lu; Liu, Yuanyuan

    2015-12-01

    Diallyl disulfide (DADS), extracted from crushed garlic by steam-distillation, has been reported to provide the anticancer activity in severalcancertypes. However,the effect of DADS on high-LET carbon beams - induced cell death remains unknown. Therefore, we used human cervical cancer cells to elucidate the molecular effects ofthis dallylsulfide. Radiotherapy remains the mainstay of treatment, especially in advanced cervical cancer and there is still space to improve the radiosensitivity to reduce radiation dosage. In this study, we found that radiation effects evoked by high-LET carbon beam was marked by inhibition of cell viability, cell cycle arrest, significant rise of apoptotic cells, regulation of transcription factor, such as p73, as well as alterations of crucial mediator of theapoptosispathway. We further demonstratedthat pretreatment of 10 M DADS in HeLa cells exposed to radiation resulted in decrease in cell viability and increased radiosensitivity. Additionally, cells pretreated with DADS obviously inhibited the radiation-induced G2/M phase arrest, but promoted radiation-induced apoptosis. Moreover, combination DADS and the radiation exacerbated the activation of apoptosis pathways through up-regulated ration of pro-apoptotic Tap73 to anti-apoptotic ?Np73, and its downstream proteins, such as FASLG, and APAF1. Taken together, these results suggest thatDADS is a potentialcandidate as radio sensitive agent for cervical cancer. PMID:26505313

  15. Protective Role of Malvidin-3-Glucoside on Peroxynitrite-Induced Damage in Endothelial Cells by Counteracting Reactive Species Formation and Apoptotic Mitochondrial Pathway

    PubMed Central

    Paixão, Joana; Dinis, Teresa C. P.; Almeida, Leonor M.

    2012-01-01

    The health-promoted benefits of anthocyanins, including vascular protective effects and antiatherogenic properties, have now been recognized, but the involved molecular mechanisms have not been well elucidated. Following our previous work on cytoprotective mechanisms of some anthocyanins against apoptosis triggered by peroxynitrite in endothelial cells, here we investigated the protective role of malvidin-3-glucoside, a major dietary anthocyanin, on such deleterious process, by exploring the interference on cellular reactive species formation and on apoptotic mitochondrial pathway. Preincubation of cells with 25 μM malvidin-3-glucoside protected efficiently endothelial cells from peroxynitrite-promoted apoptotic death, an effect which may be partially mediated by its ability to decrease the formation of reactive species after cell aggression, as assessed by the dichlorodihydrofluorescein diacetate assay and by carbonyl groups formation. Moreover, malvidin-3-glucoside inhibited mitochondrial apoptotic signaling pathways induced by peroxynitrite, by counteracting mitochondrial membrane depolarization, the activation of caspase-3 and -9, and the increase in the expression of the proapoptotic Bax protein. Altogether, our data expands our knowledge about the molecular mechanisms underlying the vascular protection afforded by malvidin-3-glucoside, and anthocyanins in general, in the context of prevention of endothelial dysfunction and atherosclerosis. PMID:22792413

  16. Multisite phosphorylation of c-Jun at threonine 91/93/95 triggers the onset of c-Jun pro-apoptotic activity in cerebellar granule neurons

    PubMed Central

    Reddy, C E; Albanito, L; De Marco, P; Aiello, D; Maggiolini, M; Napoli, A; Musti, A M

    2013-01-01

    Cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation is a model of election to study the interplay of pro-apoptotic and pro-survival signaling pathways in neuronal cell death. In this model, the c-Jun N-terminal kinase (JNK) induces pro-apoptotic genes through the c-Jun/activator protein 1 (AP-1) transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/AP-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. Here, we used this model system to study the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK sites in neuronal cell death. We found that TK-deprivation led to c-Jun multiphosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of CGCs. Conversely, lithium prevented T91T93 phosphorylation and cell death without affecting the S63 site, suggesting that T91T93 phosphorylation triggers c-Jun pro-apoptotic activity. Accordingly, a c-Jun mutant lacking the T95 priming site for T91/93 phosphorylation protected CGCs from apoptosis, whereas it was able to induce neurite outgrowth in PC12 cells. Vice versa, a c-Jun mutant bearing aspartate substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass spectrometry analysis confirmed multiphosphorylation of c-Jun at T91/T93/T95 in cells. Moreover, JNK phosphorylated recombinant c-Jun at T91/T93 in a T95-dependent manner. On the basis of our results, we propose that T91/T93/T95 multiphosphorylation of c-Jun functions as a sensitivity amplifier of the JNK cascade, setting the threshold for c-Jun pro-apoptotic activity in neuronal cells. PMID:24113186

  17. Drug-induced apoptotic neurodegeneration in the developing brain.

    PubMed

    Olney, John W; Wozniak, David F; Jevtovic-Todorovic, Vesna; Farber, Nuri B; Bittigau, Petra; Ikonomidou, Chysanthy

    2002-10-01

    Physiological cell death (PCD), a process by which redundant or unsuccessful neurons are deleted by apoptosis (cell suicide) from the developing central nervous system, has been recognized as a natural phenomenon for many years. Whether environmental factors can interact with PCD mechanisms to increase the number of neurons undergoing PCD, thereby converting this natural phenomenon into a pathological process, is an interesting question for which new answers are just now becoming available. In a series of recent studies we have shown that 2 major classes of drugs (those that block NMDA glutamate receptors and those that promote GABAA receptor activation), when administered to immature rodents during the period of synaptogenesis, trigger widespread apoptotic neurodegeneration throughout the developing brain. In addition, we have found that ethanol, which has both NMDA antagonist and GABAmimetic properties, triggers a robust pattern of apoptotic neurodegeneration, thereby deleting large numbers of neurons from many different regions of the developing brain. These findings provide a more likely explanation than has heretofore been available for the reduced brain mass and lifelong neurobehavioral disturbances associated with the human fetal alcohol syndrome (FAS). The period of synaptogenesis, also known as the brain growth spurt period, occurs in different species at different times relative to birth. In rats and mice it is a postnatal event, but in humans it extends from the sixth month of gestation to several years after birth. Thus, there is a period in pre- and postnatal human development, lasting for several years, during which immature CNS neurons are prone to commit suicide if exposed to intoxicating concentrations of drugs with NMDA antagonist or GABAmimetic properties. These findings are important, not only because of their relevance to the FAS, but because there are many agents in the human environment, other than ethanol, that have NMDA antagonist or GABAmimetic properties. Such agents include drugs that may be abused by pregnant mothers (ethanol, phencyclidine [angel dust], ketamine [Special K], nitrous oxide [laughing gas], barbiturates, benzodiazepines), and many medicinals used in obstetric and pediatric neurology (anticonvulsants), and anesthesiology (all general anesthetics are either NMDA antagonists or GABAmimetics). PMID:12408236

  18. Cardio Protective Effects of Lumbrokinase and Dilong on Second-Hand Smoke-Induced Apoptotic Signaling in the Heart of a Rat Model.

    PubMed

    Liao, Hung-En; Lai, Chao-Hung; Ho, Tsung-Jung; Yeh, Yu-Lan; Jong, Gwo-Ping; Kuo, Wu-Hsien; Chung, Li-Chin; Pai, Pei-ying; Wen, Su-Ying; Huang, Chih-Yang

    2015-06-30

    Exposure to second-hand tobacco smoke (SHS) has been epidemiologically linked to heart disease among non-smokers. However, the molecular mechanism behind SHS-induced cardiac disease is not well known. This study found that SD rats exposed to cigarette smoke at a dose of 10 cigarettes for 30 min twice a day for 1 month had a reduced left ventricle-to-tibia length ratio (mg/mm), increased cardiomyocyte apoptosis by TUNEL assay and a wider interstitial space by H&E staining. However, lumbrokinase and dilong both reversed the effects of SHS. Western blotting demonstrated significantly increased expression of the pro-apoptotic protein caspase-3 in the hearts of the rats exposed to SHS. Elevated protein expression levels of Fas, FADD and the apoptotic initiator activated caspase-8, a molecule in the death-receptor-dependent pathway, coupled with increased t-Bid and apoptotic initiator activated caspase-9 were found. Molecules in the mitochondria-dependent pathway, which disrupts mitochondrial membrane potential, were also found in rats exposed to SHS. These factors indicate myocardial apoptosis. However, treatment with lumbrokinase and dilong inhibited SHS-induced apoptosis. Regarding regulation of the survival pathway, we found in western blot analysis that cardiac protein expression of pAkt, Bcl2, and Bcl-xL was significantly down-regulated in rats exposed to SHS. These effects were reversed with lumbrokinase and dilong treatment. The effects of SHS on cardiomyocytes were also found to be mediated by the Fas death receptor-dependent apoptotic pathway, an unbalanced mitochondria membrane potential and decreased survival signaling. However, treatment with both lumbrokinase and dilong inhibited the effects of SHS. Our data suggest that lumbrokinase and dilong may prevent heart disease in SHS-exposed non-smokers. PMID:26014124

  19. Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway

    SciTech Connect

    Ramanathan, Mathura P.; Chambers, Jerome A.; Pankhong, Panyupa; Chattergoon, Michael; Attatippaholkun, Watcharee; Dang, Kesen; Shah, Neelima; Weiner, David B. . E-mail: dbweiner@mail.med.upenn.edu

    2006-02-05

    The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-