Sample records for caspase-2 mediated apoptotic

  1. Lovastatin-induced cardiac toxicity involves both oncotic and apoptotic cell death with the apoptotic component blunted by both caspase-2 and caspase-3 inhibitors

    Microsoft Academic Search

    Simon W Rabkin; Jennifer Y Kong

    2003-01-01

    The objective of this study was to evaluate the cardiac toxicity of the HMG–CoA reductase inhibitors by testing the hypothesis that lovastatin induces apoptotic and\\/or oncotic cell death in the myocyte element of the heart and further that cell death is mediated through interruption of the mevalonate pathway and that apoptosis is induced through activation of caspase-2 and caspase-3. Cardiomyocytes

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

    PubMed

    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 TNF? production, both of which are known to activate caspase-3 and caspase-8, respectively. In addition to TNF?, RB51-induced caspase-1 and IL-1? 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

  3. TNF receptors regulate vascular homeostasis in zebrafish through a caspase-8, caspase-2 and P53 apoptotic program that bypasses caspase-3

    PubMed Central

    Espín, Raquel; Roca, Francisco J.; Candel, Sergio; Sepulcre, María P.; González-Rosa, Juan M.; Alcaraz-Pérez, Francisca; Meseguer, José; Cayuela, María L.; Mercader, Nadia; Mulero, Victoriano

    2013-01-01

    SUMMARY Although it is known that tumor necrosis factor receptor (TNFR) signaling plays a crucial role in vascular integrity and homeostasis, the contribution of each receptor to these processes and the signaling pathway involved are still largely unknown. Here, we show that targeted gene knockdown of TNFRSF1B in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of TNFRSF1A or the activation of NF-?B rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. In endothelial cells, TNFRSF1A signals apoptosis through caspase-8, whereas TNFRSF1B signals survival via NF-?B. Similarly, TNF? promotes the apoptosis of human endothelial cells through TNFRSF1A and triggers caspase-2 and P53 activation. We have identified an evolutionarily conserved apoptotic pathway involved in vascular homeostasis that provides new therapeutic targets for the control of inflammation- and tumor-driven angiogenesis. PMID:22956347

  4. Coenzyme Q10 Rescues Ethanol-induced Corneal Fibroblast Apoptosis through the Inhibition of Caspase-2 Activation*

    PubMed Central

    Chen, Chun-Chen; Liou, Shiow-Wen; Chen, Chi-Chih; Chen, Wen-Chung; Hu, Fung-Rong; Wang, I-Jong; Lin, Shing-Jong

    2013-01-01

    Recent studies indicate that caspase-2 is involved in the early stages of apoptosis, particularly before the occurrence of mitochondrial damage. Here we report the important role of the coenzyme Q10 (CoQ10) on the activity of caspase-2 upstream of mitochondria in ethanol (EtOH)-treated corneal fibroblasts. After EtOH exposure, cells produce excessive reactive oxygen species formation, p53 expression, and most importantly, caspase-2 activation. After the activation of the caspase-2, the cells exhibited hallmarks of apoptotic pathway, such as mitochondrial damage and translocation of Bax and cytochrome c, which were then followed by caspase-3 activation. By pretreating the cells with a cell-permeable, biotinylated pan-caspase inhibitor, we identified caspase-2 as an initiator caspase in EtOH-treated corneal fibroblasts. Loss of caspase-2 inhibited EtOH-induced apoptosis. We further found that caspase-2 acts upstream of mitochondria to mediate EtOH-induced apoptosis. The loss of caspase-2 significantly inhibited EtOH-induced mitochondrial dysfunction, Bax translocation, and cytochrome c release from mitochondria. The pretreatment of CoQ10 prevented EtOH-induced caspase-2 activation and mitochondria-mediated apoptosis. Our data demonstrated that by blocking caspase-2 activity, CoQ10 can protect the cells from mitochondrial membrane change, apoptotic protein translocation, and apoptosis. Taken together, EtOH-induced mitochondria-mediated apoptosis is initiated by caspase-2 activation, which is regulated by CoQ10. PMID:23430247

  5. HOXA5Induced Apoptosis in Breast Cancer Cells Is Mediated by Caspases 2 and 8

    Microsoft Academic Search

    Hexin Chen; Seung Chung; Saraswati Sukumar

    2004-01-01

    HOXA5 is a transcriptional factor whose expression is lost in more than 60% of breast carcinomas. Our previous work demonstrated that the overexpression of HOXA5 in MCF7 cells resulted in cell death through a p53-dependent apoptotic pathway. To determine whether p53-independent apoptotic pathways are involved in HOXA5-induced cell death, we engineered a p53-mutant breast cancer cell line, Hs578T, to inducibly

  6. Anti-apoptotic effect of quercetin: Intervention in the JNK and ERK-mediated apoptotic pathways

    Microsoft Academic Search

    Yoshihisa Ishikawa; Masanori Kitamura

    2000-01-01

    Anti-apoptotic effect of quercetin: Intervention in the JNK- and ERK-mediated apoptotic pathways.BackgroundBioflavonoid quercetin inhibits hydrogen peroxide (H2O2)-induced apoptosis via intervention in the activator protein 1 (AP-1)-mediated apoptotic pathway. In this report, we investigated molecular events involved in the anti-apoptotic effect of quercetin, focusing especially on its effects on the family of mitogen-activated protein (MAP) kinases.MethodsCultured mesangial cells were exposed to

  7. 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.

  8. Caspase-2 regulates oncogene-induced senescence

    PubMed Central

    Gitenay, Delphine; Lallet-Daher, Hélène; Bernard, David

    2014-01-01

    Cellular senescence is activated by numerous cellular insults, in particular those driving cancer formation, resulting in stable proliferation arrest and acquisition of specific features. By self-opposing to oncogenic stimulation, senescence is considered as a failsafe program, allowing, when functional, to inhibit cancers occurrence. Compelling evidences suggest a tumor suppressive activity of caspase-2, eventually independently of its effect on cell death. The original results described here demonstrate that this tumor suppressive activity of caspase-2 is mediated, at least in part, by its pro-senescing activity. Indeed, we have demonstrated in vitro and in vivo that loss of function of caspase-2 allows to escape oncogenic stress induced senescence. These results are discussed in the context of known tumor suppressive activity of caspase-2. PMID:25114039

  9. 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

  10. 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

  11. 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

  12. PUMA-mediated mitochondrial apoptotic disruption by hypoxic postconditioning.

    PubMed

    Li, YuZhen; Guo, Qi; Liu, XiuHua; Wang, Chen; Song, DanDan

    2015-08-01

    Postconditioning can reduce ischemia-reperfusion (I/R)-induced cardiomyocyte apoptosis by targeting mitochondria. p53 upregulated modulator of apoptosis (PUMA) is involved in lethal I/R injury. Here, we hypothesized that postconditioning might inhibit mitochondrial pathway-mediated cardiomyocyte apoptosis by controlling PUMA expression. The cultured neonatal rat cardiomyocytes underwent 3 h of hypoxia and 3 h of reoxygenation. Postconditioning consisted of three cycles of 5 min reoxygenation and 5 min hypoxia after prolonged hypoxia. Hypoxic postconditioning reduced the levels of PUMA mRNA and protein. Concomitantly, the loss of mitochondrial membrane potential, cytochrome c release and caspase-3 activation were decreased significantly by postconditioning. Overexpression of PUMA increased greatly not only the number of apoptotic cardiomyocytes, but also the collapse of mitochondrial membrane potential, cytochrome c release and caspase-3 activation under postconditioning condition. The data suggest that reduction of PUMA expression mediates the endogenous cardioprotective mechanisms of postconditioning by disrupting mitochondrial apoptotic pathway. PMID:26043893

  13. An unexpected role for caspase-2 in neuroblastoma

    PubMed Central

    Dorstyn, L; Puccini, J; Nikolic, A; Shalini, S; Wilson, C H; Norris, M D; Haber, M; Kumar, S

    2014-01-01

    Caspase-2 has been implicated in various cellular functions, including cell death by apoptosis, oxidative stress response, maintenance of genomic stability and tumor suppression. The loss of the caspase-2 gene (Casp2) enhances oncogene-mediated tumorigenesis induced by E1A/Ras in athymic nude mice, and also in the E?-Myc lymphoma and MMTV/c-neu mammary tumor mouse models. To further investigate the function of caspase-2 in oncogene-mediated tumorigenesis, we extended our studies in the TH-MYCN transgenic mouse model of neuroblastoma. Surprisingly, we found that loss of caspase-2 delayed tumorigenesis in the TH-MYCN neuroblastoma model. In addition, tumors from TH-MYCN/Casp2?/? mice were predominantly thoracic paraspinal tumors and were less vascularized compared with tumors from their TH-MYCN/Casp2+/+ counterparts. We did not detect any differences in the expression of neuroblastoma-associated genes in TH-MYCN/Casp2?/? tumors, or in the activation of Ras/MAPK signaling pathway that is involved in neuroblastoma progression. Analysis of expression array data from human neuroblastoma samples showed a correlation between low caspase-2 levels and increased survival. However, caspase-2 levels correlated with clinical outcome only in the subset of MYCN-non-amplified human neuroblastoma. These observations indicate that caspase-2 is not a suppressor in MYCN-induced neuroblastoma and suggest a tissue and context-specific role for caspase-2 in tumorigenesis. PMID:25144718

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

    PubMed

    Kitevska, Tanja; Roberts, Sarah J; Pantaki-Eimany, Delara; Boyd, Sarah E; Scott, Fiona L; Hawkins, Christine J

    2014-02-17

    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-2 in 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 in vivo. PMID:24527765

  15. The enigma of caspase-2: the laymen's view

    Microsoft Academic Search

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

    2009-01-01

    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

  16. Caspase-2: Vestigial Remnant or Master Regulator?

    NSDL National Science Digital Library

    Carol M. Troy (Departments of Pathology and Neurology; Columbia University College of Physicians and Surgeons REV)

    2008-09-23

    Caspase-2, the second mammalian caspase to be identified and the most evolutionarily conserved caspase, has eluded classification. The lack of a profound phenotype in the caspase-2–deficient mouse resulted in decreased interest in caspase-2 for many years. However, advances in the field, including the identification of a potential activation complex and the development of methods to detect active caspase-2, now illuminate our understanding of the function of this caspase. These studies suggest that caspase-2 induces death through two pathways. First, caspase-2 induces cell death independently of the mitochondrial pathway, in a manner similar to that of ced-3, a caspase in Caenorhabditis elegans. Second, caspase-2 also induces cell death upstream of the mitochondrial pathway. The choice of pathway may depend on the type of death stimulus. The placing of caspase-2 upstream and independent of mitochondrial dysfunction provides a potentially new therapeutic target for aberrant cell death.

  17. Increased caspase-2 activity is associated with induction of apoptosis in IFN-beta sensitive melanoma cell lines.

    PubMed

    Kamiya, Takafumi; Okabayashi, Tamaki; Yokota, Shin-Ichi; Kan, Yuji; Ogino, Jiro; Yamashita, Toshiharu; Fujii, Nobuhiro; Jimbow, Kowichi

    2010-05-01

    Interferon (IFN) is believed to be one of the most effective anti-melanoma agents. Specifically, IFN-beta has the ability to induce apoptosis of melanoma cells. Induction of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has also been suggested to have a critical role in IFN-beta-induced apoptosis. To characterize the signaling pathway involved in IFN-beta-induced apoptosis, we analyzed the biological effects of IFN-beta on the cell death and caspase activation of melanoma cells. IFN-sensitive cell lines, MM418, SK-mel-23, and SK-mel-118, showed increased apoptotic populations correlated with the activation of caspase-2 and caspase-3 by IFN-beta. IFN-beta-induced apoptosis was significantly suppressed by inhibitors for caspase-2 or caspase-3, but not by inhibitors for caspase-8 or caspase-9 in these cell lines. TRAIL expression was observed in IFN-beta-treated cells of SK-mel-23 and SK-mel-118, but not in those cells of MM418, which showed massive IFN-beta-induced apoptosis and resistance to exogenous TRAIL-mediated apoptosis. G361 was resistant to IFN-beta-induced apoptosis but sensitive to exogenous TRAIL-mediated apoptosis. Furthermore, IFN-beta pretreatment significantly increased the sensitivity against exogenous TRAIL-mediated apoptosis and activation of caspase-2 in G361. These results suggested that caspase-2 activation is commonly associated with induction of IFN-beta-induced apoptosis in IFN-beta-sensitive melanoma cells. PMID:20187765

  18. 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

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

    PubMed

    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

  20. 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

  1. Pharmacological Inhibition of Caspase-2 Protects Axotomised Retinal Ganglion Cells from Apoptosis in Adult Rats

    PubMed Central

    Vigneswara, Vasanthy; Berry, Martin; Logan, Ann; Ahmed, Zubair

    2012-01-01

    Severing the axons of retinal ganglion cells (RGC) by crushing the optic nerve (ONC) causes the majority of RGC to degenerate and die, primarily by apoptosis. We showed recently that after ONC in adult rats, caspase-2 activation occurred specifically in RGC while no localisation of caspase-3 was observed in ganglion cells but in cells of the inner nuclear layer. We further showed that inhibition of caspase-2 using a single injection of stably modified siRNA to caspase-2 protected almost all RGC from death at 7 days, offering significant protection for up to 1 month after ONC. In the present study, we confirmed that cleaved caspase-2 was localised and activated in RGC (and occasional neurons in the inner nuclear layer), while TUNEL+ RGC were also observed after ONC. We then investigated if suppression of caspase-2 using serial intravitreal injections of the pharmacological inhibitor z-VDVAD-fmk (z-VDVAD) protected RGC from death for 15 days after ONC. Treatment of eyes with z-VDVAD suppressed cleaved caspase-2 activation by >85% at 3–4 days after ONC. Increasing concentrations of z-VDVAD protected greater numbers of RGC from death at 15 days after ONC, up to a maximum of 60% using 4000 ng/ml of z-VDVAD, compared to PBS treated controls. The 15-day treatment with 4000 ng/ml of z-VDVAD after ONC suppressed levels of cleaved caspase-2 but no significant changes in levels of cleaved caspase-3, -6, -7 or -8 were detected. Although suppression of caspase-2 protected 60% of RGC from death, RGC axon regeneration was not promoted. These results suggest that caspase-2 specifically mediates death of RGC after ONC and that suppression of caspase-2 may be a useful therapeutic strategy to enhance RGC survival not only after axotomy but also in diseases where RGC death occurs such as glaucoma and optic neuritis. PMID:23285297

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

    PubMed

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

    2010-11-01

    The protective actions of estrogen have been well evaluated in various models of neurodegeneration. These neuroprotective mechanisms may include a direct neuronal antiapoptotic 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 (50 nM), 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 (10 nM) attenuated the increased expression of antiapoptotic protein Bcl-2, proapoptotic 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 182780 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, whereas, was more involved in estrogen effects on Bax. Our data suggest that estrogen intervenes against HIV-1 Tat-induced cortical neuronal dysfunction via intersecting mitochondrial apoptotic pathway signaling in an ER-sensitive manner. PMID:20340172

  3. Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids.

    PubMed

    Lauber, K; Keppeler, H; Munoz, L E; Koppe, U; Schröder, K; Yamaguchi, H; Krönke, G; Uderhardt, S; Wesselborg, S; Belka, C; Nagata, S; Herrmann, M

    2013-09-01

    The phagocytic clearance of apoptotic cells is essential to prevent chronic inflammation and autoimmunity. The phosphatidylserine-binding protein milk fat globule-EGF factor 8 (MFG-E8) is a major opsonin for apoptotic cells, and MFG-E8(-/-) mice spontaneously develop a lupus-like disease. Similar to human systemic lupus erythematosus (SLE), the murine disease is associated with an impaired clearance of apoptotic cells. SLE is routinely treated with glucocorticoids (GCs), whose anti-inflammatory effects are consentaneously attributed to the transrepression of pro-inflammatory cytokines. Here, we show that the GC-mediated transactivation of MFG-E8 expression and the concomitantly enhanced elimination of apoptotic cells constitute a novel aspect in this context. Patients with chronic inflammation receiving high-dose prednisone therapy displayed substantially increased MFG-E8 mRNA levels in circulating monocytes. MFG-E8 induction was dependent on the GC receptor and several GC response elements within the MFG-E8 promoter. Most intriguingly, the inhibition of MFG-E8 induction by RNA interference or genetic knockout strongly reduced or completely abolished the phagocytosis-enhancing effect of GCs in vitro and in vivo. Thus, MFG-E8-dependent promotion of apoptotic cell clearance is a novel anti-inflammatory facet of GC treatment and renders MFG-E8 a prospective target for future therapeutic interventions in SLE. PMID:23832117

  4. Modulation of Orphan Nuclear Receptor Nur77-mediated Apoptotic Pathway by Acetylshikonin and Analogs

    PubMed Central

    Liu, Jie; Zhou, Wen; Li, Shao-Shun; Sun, Zhe; Lin, Bingzhen; Lang, Yuan-Yuan; He, Jia-You; Cao, Xihua; Yan, Tingdong; Wang, Li; Lu, Jiongming; Han, Young-Hoon; Cao, Yu; Zhang, Xiao-kun; Zeng, Jin-Zhang

    2008-01-01

    Shikonin derivatives, which are the active components of the medicinal plant Lithospermum erythrorhizon, exhibit many biological effects including apoptosis induction through undefined mechanisms. We recently discovered that orphan nuclear receptor Nur77 migrates from the nucleus to mitochondria, where it binds to Bcl-2 to induce apoptosis. Here, we report that certain shikonin derivatives could modulate the Nur77-Bcl-2 apoptotic pathway by increasing levels of Nur77 protein and promoting its mitochondrial targeting in cancer cells. Structural modification of acetylshikonin resulted in identification of a derivative 5,8-diacetoxyl-6-(1'-Acetoxyl-4'-methyl-3'-pentenyl)-1,4-naphthaquinones (SK07) that exhibited improved efficacy and specificity in activating the pathway. Unlike other Nur77 modulators, shikonins increased levels of Nur77 protein through their posttranscriptional regulation. The apoptotic effect of SK07 was impaired in Nur77 knockout cells and suppressed by co-treatment with leptomycin B (LMB) that inhibited Nur77 cytoplasmic localization. Furthermore, SK07 induced apoptosis in cells expressing the C-terminal half of Nur77 protein but not its N-terminal region. Our data also showed that SK07-induced apoptosis was associated with a Bcl-2 conformational change and Bax activation. Together, our results demonstrate that certain shikonin derivatives act as modulators of the Nur77-mediated apoptotic pathway and identify new shikonin-based lead that targets Nur77 for apoptosis induction. PMID:18974131

  5. Caspase-mediated pro-apoptotic interaction of panaxadiol and irinotecan in human colorectal cancer cells

    PubMed Central

    Du, Guang-Jian; Wang, Chong-Zhi; Zhang, Zhi-Yu; Wen, Xiao-Dong; Somogyi, Jacqueline; Calway, Tyler; He, Tong-Chuan; Du, Wei; Yuan, Chun-Su

    2012-01-01

    Objectives Panaxadiol (PD) is a purified sapogenin of ginseng saponins that exhibits anticancer activity. Irinotecan (IRN) is a second line anticancer drug, but clinical treatment with IRN is limited due to side effects. In this study, we investigated the possible synergistic anticancer effects of PD and IRN on human colorectal cancer cells and explored the potential role of apoptosis in the synergistic activities. Key findings The combination of PD and IRN significantly enhanced antiproliferative effects in HCT-116 cells (P < 0.05). Cell cycle analysis demonstrated that combining IRN treatment with PD significantly increased the G1-phase fractions of cells, compared with IRN treatment alone. In apoptotic assays, the combination of PD and IRN significantly increased the percentage of apoptotic cells compared with IRN alone (P < 0.01). Increased caspase 3 and caspase 9 activities were observed after treating with PD and IRN. The synergistic apoptotic effects were also supported by docking analysis, which demonstrated that PD and IRN bound two different chains of the caspase 3 protein. Conclusions Data from this study suggested that caspase 3- and caspase 9-mediated apoptosis may play an important role in the PD enhanced antiproliferative effects of IRN on human colorectal cancer cells. PMID:22471369

  6. Integrin ? PAT-2/CDC-42 Signaling Is Required for Muscle-Mediated Clearance of Apoptotic Cells in Caenorhabditis elegans

    PubMed Central

    Jiang, Hang-Shiang; Wu, Yi-Chun

    2012-01-01

    Clearance of apoptotic cells by engulfment plays an important role in the homeostasis and development of multicellular organisms. Despite the fact that the recognition of apoptotic cells by engulfment receptors is critical in inducing the engulfment process, the molecular mechanisms are still poorly understood. Here, we characterize a novel cell corpse engulfment pathway mediated by the integrin ? subunit PAT-2 in Caenorhabditis elegans and show that it specifically functions in muscle-mediated engulfment during embryogenesis. Inactivation of pat-2 results in a defect in apoptotic cell internalization. The PAT-2 extracellular region binds to the surface of apoptotic cells in vivo, and the intracellular region may mediate signaling for engulfment. We identify essential roles of small GTPase CDC-42 and its activator UIG-1, a guanine-nucleotide exchange factor, in PAT-2–mediated cell corpse removal. PAT-2 and CDC-42 both function in muscle cells for apoptotic cell removal and are co-localized in growing muscle pseudopods around apoptotic cells. Our data suggest that PAT-2 functions through UIG-1 for CDC-42 activation, which in turn leads to cytoskeletal rearrangement and apoptotic cell internalization by muscle cells. Moreover, in contrast to PAT-2, the other integrin ? subunit INA-1 and the engulfment receptor CED-1, which signal through the conserved signaling molecules CED-5 (DOCK180)/CED-12 (ELMO) or CED-6 (GULP) respectively, preferentially act in epithelial cells to mediate cell corpse removal during mid-embryogenesis. Our results show that different engulfing cells utilize distinct repertoires of receptors for engulfment at the whole organism level. PMID:22615577

  7. Integrin ? PAT-2/CDC-42 signaling is required for muscle-mediated clearance of apoptotic cells in Caenorhabditis elegans.

    PubMed

    Hsieh, Hsiao-Han; Hsu, Tsung-Yuan; Jiang, Hang-Shiang; Wu, Yi-Chun

    2012-01-01

    Clearance of apoptotic cells by engulfment plays an important role in the homeostasis and development of multicellular organisms. Despite the fact that the recognition of apoptotic cells by engulfment receptors is critical in inducing the engulfment process, the molecular mechanisms are still poorly understood. Here, we characterize a novel cell corpse engulfment pathway mediated by the integrin ? subunit PAT-2 in Caenorhabditis elegans and show that it specifically functions in muscle-mediated engulfment during embryogenesis. Inactivation of pat-2 results in a defect in apoptotic cell internalization. The PAT-2 extracellular region binds to the surface of apoptotic cells in vivo, and the intracellular region may mediate signaling for engulfment. We identify essential roles of small GTPase CDC-42 and its activator UIG-1, a guanine-nucleotide exchange factor, in PAT-2-mediated cell corpse removal. PAT-2 and CDC-42 both function in muscle cells for apoptotic cell removal and are co-localized in growing muscle pseudopods around apoptotic cells. Our data suggest that PAT-2 functions through UIG-1 for CDC-42 activation, which in turn leads to cytoskeletal rearrangement and apoptotic cell internalization by muscle cells. Moreover, in contrast to PAT-2, the other integrin ? subunit INA-1 and the engulfment receptor CED-1, which signal through the conserved signaling molecules CED-5 (DOCK180)/CED-12 (ELMO) or CED-6 (GULP) respectively, preferentially act in epithelial cells to mediate cell corpse removal during mid-embryogenesis. Our results show that different engulfing cells utilize distinct repertoires of receptors for engulfment at the whole organism level. PMID:22615577

  8. Orai1 and STIM1 mediate SOCE and contribute to apoptotic resistance of pancreatic adenocarcinoma.

    PubMed

    Kondratska, Kateryna; Kondratskyi, Artem; Yassine, Maya; Lemonnier, Loic; Lepage, Gilbert; Morabito, Angela; Skryma, Roman; Prevarskaya, Natalia

    2014-10-01

    The store-operated calcium channels (SOCs) represent one of the major calcium-entry pathways in non-excitable cells. SOCs and in particular their major components ORAI1 and STIM1 have been shown to be implicated in a number of physiological and pathological processes such as apoptosis, proliferation and invasion. Here we demonstrate that ORAI1 and STIM1 mediate store-operated calcium entry (SOCE) in pancreatic adenocarcinoma cell lines. We show that both ORAI1 and STIM1 play pro-survival anti-apoptotic role in pancreatic adenocarcinoma cell lines, as siRNA-mediated knockdown of ORAI1 and/or STIM1 increases apoptosis induced by chemotherapy drugs 5-fluorouracil (5-FU) or gemcitabine. We also demonstrate that both 5-FU and gemcitabine treatments increase SOCE in Panc1 pancreatic adenocarcinoma cell line via upregulation of ORAI1 and STIM1. Altogether our results reveal the novel calcium-dependent mechanism of action of the chemotherapy drugs 5-FU and gemcitabine and emphasize the anti-apoptotic role of ORAI1 and STIM1 in pancreatic adenocarcinoma cells. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau. PMID:24583265

  9. Abnormal Sperm Parameters in Humans Are Indicative of an Abortive Apoptotic Mechanism Linked to the Fas-Mediated Pathway

    Microsoft Academic Search

    Denny Sakkas; Ewa Mariethoz; Justin C. St. John

    1999-01-01

    The life cycle of many cell types can hinge on the presence of death factors that can control programmed cell death. The Fas-mediated apoptotic pathway has been implicated in controlling apoptosis during spermatogenesis in a number of mammalian species. In the human, the presence of nuclear DNA damage in ejaculated spermatozoa has pointed to a possible role for apoptosis during

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

    PubMed

    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 ?V 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

  11. DIEPOXYBUTANE ACTIVATES THE MITOCHONDRIAL APOPTOTIC PATHWAY AND MEDIATES APOPTOSIS IN HUMAN LYMPHOBLASTS THROUGH OXIDATIVE STRESS

    PubMed Central

    Yadavilli, Sridevi; Martinez-Ceballos, Eduardo; Snowden-Aikens, Janana; Hurst, Angela; Joseph, Tranole; Albrecht, Thomas; Muganda, Perpetua M.

    2007-01-01

    Diepoxybutane (DEB) is the most potent metabolite of the environmental chemical 1, 3-butadiene (BD), which is prevalent in petrochemical industrial areas. BD is a known mutagen and human carcinogen, and possesses multi-systems organ toxicity. We recently reported that DEB-induced cell death in TK6 lymphoblasts was due to the occurrence of apoptosis, and not necrosis. In this study, we investigated the molecular mechanisms responsible for DEB-induced apoptosis in these cells. Bax and Bak were found to be over-expressed and activated, and the mitochondrial trans-membrane potential was attenuated in cells undergoing DEB-induced apoptosis. Cytochrome c was depleted from the mitochondria of TK6 cells undergoing apoptosis, and was released into the cytosol in Jurkat-T lymphoblasts exposed to the same concentrations of DEB. Executioner caspase 3 was deduced to be activated by initiator caspase 9. DEB induced reactive oxygen species (ROS) formation, and the ROS scavenger N-acetyl-L-cysteine effectively blocked DEB-induced apoptosis in TK6 cells. Collectively, these results demonstrate that the mitochondrial apoptotic pathway is activated to mediate DEB-induced apoptosis in human TK6 lymphoblasts. These results further demonstrate that DEB-induced apoptosis is also mediated by the DEB-induced generation of ROS. This is the first report to examine the mechanism of DEB-induced apoptosis in human lymphoblasts. PMID:17693053

  12. Thai Acanthamoeba isolate (T4) induced apoptotic death in neuroblastoma cells via the Bax-mediated pathway.

    PubMed

    Chusattayanond, Araya Dharmkrong-at; Boonsilp, Siriphan; Kasisit, Jitra; Boonmee, Atsadang; Warit, Saradee

    2010-12-01

    A Thai Acanthamoeba isolate named AS recovered from a corneal scraping of a keratitis patient was genotypically determined as T4. AS trophozoites were used for studying Acanthamoeba-induced apoptosis in mouse neuroblastoma NA cells during in vitro co-cultivation. The Acanthamoeba-exposed NA cells showed signs of apoptosis including cell shrinkage, nuclear condensation and DNA fragmentation. The effect was confirmed by DNA laddering electrophoresis. Involvement of caspase enzymes and mitochondrial pro- and anti-apoptotic proteins (Bax and Bcl-2) in AS-induced apoptosis was determined. The use of Z-VAD-FMK, a pan-caspase inhibitor, significantly reduced the apoptotic effect, while Bax/Bcl-2 ratio analysis showed a significant increase in the expression of apoptotic proteins in AS-exposed NA cells. These results strongly indicated that apoptosis induced by AS trophozoites is caspase-dependent and is mediated by over-expression of pro-apoptotic proteins in the mitochondrial pathway. This is the first report on the role of Bax in mediating apoptosis induced by Acanthamoeba. PMID:20601106

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

    SciTech Connect

    Cheung, Herman H. [Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute II, Ottawa, Ontario, K1H 8L1 (Canada); Lynn Kelly, N. [Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute II, Ottawa, Ontario, K1H 8L1 (Canada); Liston, Peter [Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute II, Ottawa, Ontario, K1H 8L1 (Canada); Department of Paediatrics, University of Ottawa, Ottawa, Ontario, K1H 8L1 (Canada); Korneluk, Robert G. [Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute II, Ottawa, Ontario, K1H 8L1 (Canada) and Department of Paediatrics, University of Ottawa, Ottawa, Ontario, K1H 8L1 (Canada) and Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5 (Canada)]. 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.

  14. 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

  15. 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

  16. The scavenger receptor SCARF1 mediates apoptotic cell clearance and prevents autoimmunity

    PubMed Central

    Ramirez-Ortiz, Zaida G.; Pendergraft, William F.; Prasad, Amit; Byrne, Michael H.; Iram, Tal; Blanchette, Christopher J.; Luster, Andrew D.; Hacohen, Nir; Khoury, Joseph El; Means, Terry K.

    2013-01-01

    Clearance of apoptotic cells is critical for control of tissue homeostasis however the full range of receptor(s) on phagocytes responsible for recognition of apoptotic cells remains to be identified. Here we show that dendritic cells (DCs), macrophages and endothelial cells use scavenger receptor type F family member 1 (SCARF1) to recognize and engulf apoptotic cells via C1q. Loss of SCARF1 impairs uptake of apoptotic cells. Consequently, in SCARF1-deficient mice, dying cells accumulate in tissues leading to a lupus-like disease with the spontaneous generation of autoantibodies to DNA-containing antigens, immune cell activation, dermatitis and nephritis. The discovery of SCARF1 interactions with C1q and apoptotic cells provides insights into molecular mechanisms involved in maintenance of tolerance and prevention of autoimmune disease. PMID:23892722

  17. Bcl-2 family-mediated apoptotic effects of 3,3'-diindolylmethane (DIM) in human breast cancer cells.

    PubMed

    Hong, Chibo; Firestone, Gary L; Bjeldanes, Leonard F

    2002-03-15

    3,3'-Diindolylmethane (DIM) is a major in vivo derivative of the putative anticancer agent indole-3-carbinol (I3C), which is present in vegetables of the Brassica genus. At concentrations above 10 microM, DIM inhibited DNA synthesis and cell proliferation in both estrogen receptor replete (MCF-7) and deficient (MDA-MB-231) human breast cancer cells in a concentration- and time-dependent manner. These antiproliferative effects were accompanied by characteristic indications of programmed cell death in both cell lines, including externalization of phosphatidylserine, chromatin condensation, and DNA fragmentation. Furthermore, Western and Northern blot analyses, as well as coimmunoprecipitation assays, revealed that in both MCF-7 and MDA-MB-231 cells, DIM treatment decreased total transcript and protein levels of the apoptosis inhibitory protein Bcl-2, and the amount of Bcl-2 bound to the pro-apoptotic protein Bax. DIM treatment also caused an increase in Bax protein levels, but did not affect the level of Bax that was bound to Bcl-2. As a functional test of the role of Bcl-2 down-regulation in the DIM-induced apoptotic response, ectopic expression of Bcl-2 in MCF-7 cells was shown to attenuate the apoptotic effect of DIM. These results demonstrate that DIM can induce apoptosis in breast cancer cells independent of estrogen receptor status by a process that is mediated by the modulated expression of the Bax/Bcl-2 family of apoptotic regulatory factors. PMID:11931841

  18. EGF-Like Ligands Mediate Progesterone's Anti-Apoptotic Action on Macaque Granulosa Cells1

    PubMed Central

    Puttabyatappa, Muraly; Brogan, Rebecca S.; VandeVoort, Catherine A.; Chaffin, Charles L.

    2012-01-01

    ABSTRACT A local autocrine/paracrine role for progesterone is an absolute requirement for corpus luteum formation in primates. Despite this, the mechanism(s) remain obscure, although existing data suggest an anti-apoptotic action to be central. There are a limited number of progestin-regulated gene targets identified in the luteinizing primate follicle, suggesting that a small number of important genes may mediate progesterone action. Possible gene targets could be the epidermal growth factor (EGF) family members amphiregulin (AREG) and epiregulin (EREG). Using macaques undergoing controlled ovarian stimulation cycles, we show that the phosphorylation of EGF receptor (EGFR), ERK 1/2, and AKT increases 6 h after an ovulatory human chorionic gonadotropin (hCG) stimulus and remains activate through 24 h. Immunoreactive EREG and AREG ligands in the follicular fluid both increased in a time frame commensurate with EGFR phosphorylation. The mRNA expression of AREG and EREG in nonluteinized granulosa cells (NLGC) was induced in culture with hCG, an effect blocked by progesterone receptor (PGR) antagonists. Overexpression of PGR B in NLGC and treatment with a nonmetabolizable progestin did not increase either gene, indicating both progesterone and luteinizing hormone/CG are necessary. Addition of EGF and EGF-like ligands did not promote steroidogenesis in vitro by granulosa cells in the presence of gonadotropin, but were able to partially reverse RU486-induced cell death. These data suggest that progesterone promotes the expression of AREG and EREG, which in turn maintain viability of luteinizing granulosa cells, representing one possible mechanism whereby progesterone promotes corpus luteum formation in the primate. PMID:23136296

  19. Titanium dioxide induces apoptotic cell death through reactive oxygen species-mediated Fas upregulation and Bax activation

    PubMed Central

    Yoo, Ki-Chun; Yoon, Chang-Hwan; Kwon, Dongwook; Hyun, Kyung-Hwan; Woo, Soo Jung; Kim, Rae-Kwon; Lim, Eun-Jung; Suh, Yongjoon; Kim, Min-Jung; Yoon, Tae Hyun; Lee, Su-Jae

    2012-01-01

    Background Titanium dioxide (TiO2) has been widely used in many areas, including biomedicine, cosmetics, and environmental engineering. Recently, it has become evident that some TiO2 particles have a considerable cytotoxic effect in normal human cells. However, the molecular basis for the cytotoxicity of TiO2 has yet to be defined. Methods and results In this study, we demonstrated that combined treatment with TiO2 nanoparticles sized less than 100 nm and ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-dependent upregulation of Fas and conformational activation of Bax in normal human cells. Treatment with P25 TiO2 nanoparticles with a hydrodynamic size distribution centered around 70 nm (TiO2P25–70) together with ultraviolet A irradiation-induced caspase-dependent apoptotic cell death, accompanied by transcriptional upregulation of the death receptor, Fas, and conformational activation of Bax. In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO2-induced apoptotic cell death. Moreover, inhibition of reactive oxygen species with an antioxidant, N-acetyl-L-cysteine, clearly suppressed upregulation of Fas, conformational activation of Bax, and subsequent apoptotic cell death in response to combination treatment using TiO2P25–70 and ultraviolet A irradiation. Conclusion These results indicate that sub-100 nm sized TiO2 treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein, Bax. Elucidating the molecular mechanisms by which nanosized particles induce activation of cell death signaling pathways would be critical for the development of prevention strategies to minimize the cytotoxicity of nanomaterials. PMID:22419868

  20. 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

  1. Clathrin and AP2 are required for phagocytic receptor-mediated apoptotic cell clearance in Caenorhabditis elegans.

    PubMed

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

    2013-05-01

    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 ? 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

  2. 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 ? 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

  3. Beneficial Effects of Astragaloside IV for Hair Loss via Inhibition of Fas/Fas L-Mediated Apoptotic Signaling

    PubMed Central

    Kim, Mi Hye; Kim, Sung-Hoon; Yang, Woong Mo

    2014-01-01

    Apoptosis with premature termination of hair follicle growth induces several types of hair loss and is one of the crucial factors of hair loss. Astragaloside IV, which is a major component of Astragalus membranaceus, is a cycloartane triterpene saponin. Although an anti-apoptotic effect of Astragaloside IV has been reported, its effects against hair loss have not been investigated. To explore the underlying mechanisms of Astragaloside IV on apoptotic signaling in hair follicle, the dorsal skin of depilated C57BL/6 mice was topically treated with 1 and 100 ?M Astragaloside IV for 14 days. In Astragaloside IV-treated group, TUNEL-positive cells were reduced. We found that Astragaloside IV blocked the procaspase-8, resulting in the inhibition of caspase-3 and procaspase-9 activities. The changes were accompanied with down-regulation of Bax and p53, and up-regulation of Bcl-2 and Bcl-xL by Astragaloside IV treatment. In addition, activation of NF-?B and phosphorylation of I?B-? were inhibited, along with decreases in three MAPKs: ERK, SAPK/JNK and p38 by Astragaloside IV. The expressions of KGF, p21, TNF-? and IL-1?, which are keratinocyte terminal differentiation markers associated with catagen, were modulated by treatment with Astragaloside IV. These results demonstrated that Astragaloside IV is concerned with blocking the Fas/Fas L-mediated apoptotic pathway, which would be an alternative therapy for hair loss. PMID:24676213

  4. Apoptotic Mediators are Upregulated in the Skeletal Muscle of Chronic/Progressive Mouse Model of Parkinson's Disease.

    PubMed

    Erekat, Nour S

    2015-08-01

    Apoptosis has been implicated in the pathogenesis of Parkinson disease (PD). Parkinson disease is characterized by skeletal muscle abnormalities. The aim of this study is to illustrate the impact of PD induction on the expression of apoptotic mediators. Twenty normal albino mice were randomly selected and equally divided in control and PD groups. Chronic Parkinsonism was induced in the PD group using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTP/p). After that, samples from gastrocnemius muscles were evaluated by immunohistochemistry to examine the expression of p53 and active caspase-3 in the two groups of animals. P53 and active caspase-3 expression was significantly higher in gastrocnemius skeletal muscle in PD mice compared with that in the control mice (P value <0.01). Furthermore, we show PD gastrocnemius muscle atrophy measured by significant reduction (P < 0.01) in the muscle fiber cross-sectional area. Thus, our present data suggest that PD induction increased the expression of the apoptotic mediators p53 and active caspase-3 in gastrocnemius muscle, indicating the induction of apoptosis, which was correlative with gastrocnemius muscle atrophy subsequent to the induction of PD. Anat Rec, 298:1472-1478, 2015. © 2015 Wiley Periodicals, Inc. PMID:25704481

  5. Mizoribine-mediated apoptotic signaling pathway in human T-Cell line

    Microsoft Academic Search

    K. W. Seo; H. K. Lee; S. J. N. Choi; B. J. So; S. K. Kim; S. Y. Chung

    2005-01-01

    Mizoribine (MZR), an inhibitor of inosine monophosphate dehydrogenase, which depletes cellular guanadine triphosphate, is an immunosuppressive drug. The aim of this study was to evaluate the mechanism by which MZR exerts cytotoxic effects on human Jurkat T cells. Our study showed that MZR-induced apoptotic death of human Jurkat T cells is dose-dependent and time-dependent, as revealed by chromatin condensation and

  6. 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

  7. Phagocytic receptor signaling regulates clathrin and epsin-mediated cytoskeletal remodeling during apoptotic cell engulfment in C. elegans

    PubMed Central

    Shen, Qian; He, Bin; Lu, Nan; Conradt, Barbara; Grant, Barth D.; Zhou, Zheng

    2013-01-01

    The engulfment and subsequent degradation of apoptotic cells by phagocytes is an evolutionarily conserved process that efficiently removes dying cells from animal bodies during development. Here, we report that clathrin heavy chain (CHC-1), a membrane coat protein well known for its role in receptor-mediated endocytosis, and its adaptor epsin (EPN-1) play crucial roles in removing apoptotic cells in Caenorhabditis elegans. Inactivating epn-1 or chc-1 disrupts engulfment by impairing actin polymerization. This defect is partially suppressed by inactivating UNC-60, a cofilin ortholog and actin server/depolymerization protein, further indicating that EPN-1 and CHC-1 regulate actin assembly during pseudopod extension. CHC-1 is enriched on extending pseudopods together with EPN-1, in an EPN-1-dependent manner. Epistasis analysis places epn-1 and chc-1 in the same cell-corpse engulfment pathway as ced-1, ced-6 and dyn-1. CED-1 signaling is necessary for the pseudopod enrichment of EPN-1 and CHC-1. CED-1, CED-6 and DYN-1, like EPN-1 and CHC-1, are essential for the assembly and stability of F-actin underneath pseudopods. We propose that in response to CED-1 signaling, CHC-1 is recruited to the phagocytic cup through EPN-1 and acts as a scaffold protein to organize actin remodeling. Our work reveals novel roles of clathrin and epsin in apoptotic-cell internalization, suggests a Hip1/R-independent mechanism linking clathrin to actin assembly, and ties the CED-1 pathway to cytoskeleton remodeling. PMID:23861060

  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. 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 con¬centrations 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

  10. 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; Hébert, Marie-Josée; 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

  11. CHOP/GADD153 is a mediator of apoptotic death in substantia nigra dopamine neurons in an in vivo neurotoxin model of

    E-print Network

    Burke, Robert E

    CHOP/GADD153 is a mediator of apoptotic death in substantia nigra dopamine neurons in an in vivo- tive stress, Parkinson's disease, programmed cell death, substantia nigra. J. Neurochem. (2005) 95, 974-lesion day; PND, post- natal day; SSC, saline sodium citrate; SN, substantia nigra; TBS, Tris- buffered

  12. p85? 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 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. PMID:24477292

  13. Computational Modeling of Signaling Pathways Mediating Cell Cycle Checkpoint Control and Apoptotic Responses to Ionizing Radiation-Induced DNA Damage

    PubMed Central

    Zhao, Yuchao; Lou, In Chio; Conolly, Rory B.

    2012-01-01

    The shape of dose response of ionizing radiation (IR) induced cancer at low dose region, either linear non-threshold or J-shaped, has been a debate for a long time. This dose response relationship can be influenced by built-in capabilities of cells that minimize the fixation of IR-mediated DNA damage as pro-carcinogenic mutations. Key capabilities include sensing of damage, activation of cell cycle checkpoint arrests that provide time needed for repair of the damage as well as apoptosis. Here we describe computational modeling of the signaling pathways that link sensing of DNA damage and checkpoint arrest activation/apoptosis to investigate how these molecular-level interactions influence the dose response relationship for IR induced cancer. The model provides qualitatively accurate descriptions of the IR-mediated activation of cell cycle checkpoints and the apoptotic pathway, and of time-course activities and dose response of relevant regulatory proteins (e.g. p53 and p21). Linking to a two-stage clonal growth cancer model, the model described here successfully captured a monotonically increasing to a J-shaped dose response curve and identified one potential mechanism leading to the J-shape: the cell cycle checkpoint arrest time saturates with the increase of the dose. PMID:22740786

  14. 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.

  15. 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?–TRAF2–ASK1 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

  16. The role of lysosomes in BDE 47-mediated activation of mitochondrial apoptotic pathway in HepG2 cells.

    PubMed

    Liu, Xiaohui; Wang, Jian; Lu, Chengquan; Zhu, Chunyan; Qian, Bo; Li, Zhenwei; Liu, Chang; Shao, Jing; Yan, Jinsong

    2015-04-01

    Polybrominated diphenyl ethers (PBDEs) are a group of widely used flame retardants. The rising presence of PBDEs in human tissues has received considerable concerns with regard to potential health risks. While the mitochondrial-apoptotic pathway has been suggested in PBDEs-induced apoptosis, the role of lysosomes is yet to be understood. In the present study, HepG2 cells were exposed to BDE 47 at various concentrations and durations to establish the causal and temporal relationships among various cellular events, such as cell viability, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis, and expression of cytochrome C and caspase 3. The involvement of lysosomes was simultaneously studied by evaluating lysosomal membrane permeability (LMP) and changes in the expression of cathepsin B, a lysosome hydrolase. In addition, a cathepsin B inhibitor (10 ?M CA-074) was used to determine the involvement of lysosomes and potential interactions between lysosomes and mitochondria. Our results showed that ROS production was an initial response of HepG2 to BDE 47 exposure, followed by a decreased MMP; a loss of MMP caused additional ROS generation which acted to induce LMP; an increased LMP resulted in a release of cathepsin B which aggravated the loss of MMP leading to release of cytochrome C and caspase 3 and subsequent apoptosis. Pretreatment with CA-074 did not abolish the initial ROS generation, however, all downstream events were dramatically alleviated. Taken together, our data indicate that lysosomes might be involved in BDE 47-mediated mitochondrial-apoptotic pathway in HepG2 cells, possibly through feedback interactions between mitochondria and lysosomes. PMID:25479806

  17. 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 Central

    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.

    2014-01-01

    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.—Das, S., Sarkar, A., Ryan, K. A., Fox, S., Berger, A. H., Juncadella, I. J., Bimczok, D., Smythies, L. E., Harris, P. R., Ravichandran, K. S., Crowe, S. E., Smith, P. D., Ernst, P. B. 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. PMID:24509909

  18. 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

  19. Soy Isoflavones Genistein and Daidzein Exert Anti-Apoptotic Actions via a Selective ER-mediated Mechanism in Neurons following HIV-1 Tat1–86 Exposure

    PubMed Central

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

    2012-01-01

    Background HIV-1 viral protein Tat partially mediates the neural dysfunction and neuronal cell death associated with HIV-1 induced neurodegeneration and neurocognitive disorders. Soy isoflavones provide protection against various neurotoxic insults to maintain neuronal function and thus help preserve neurocognitive capacity. Methodology/Principal Findings We demonstrate in primary cortical cell cultures that 17?-estradiol or isoflavones (genistein or daidzein) attenuate Tat1–86-induced expression of apoptotic proteins and subsequent cell death. Exposure of cultured neurons to the estrogen receptor antagonist ICI 182,780 abolished the anti-apoptotic actions of isoflavones. Use of ER? or ER? specific antagonists determined the involvement of both ER isoforms in genistein and daidzein inhibition of caspase activity; ER? selectively mediated downregulation of mitochondrial pro-apoptotic protein Bax. The findings suggest soy isoflavones effectively diminished HIV-1 Tat-induced apoptotic signaling. Conclusions/Significance Collectively, our results suggest that soy isoflavones represent an adjunctive therapeutic option with combination anti-retroviral therapy (cART) to preserve neuronal functioning and sustain neurocognitive abilities of HIV-1 infected persons. PMID:22629415

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

    PubMed

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

    2012-05-01

    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-6-null 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

  1. 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-6–null 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

  2. 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

  3. 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

  4. Swainsonine Activates Mitochondria-mediated Apoptotic Pathway in Human Lung Cancer A549 Cells and Retards the Growth of Lung Cancer Xenografts

    PubMed Central

    Li, Zhaocai; Xu, Xingang; Huang, Yong; Ding, Li; Wang, Zhisheng; Yu, Gaoshui; Xu, Dan; Li, Wei; Tong, Dewen

    2012-01-01

    Swainsonine (1, 2, 8-trihyroxyindolizidine, SW), a natural alkaloid, has been reported to exhibit anti-cancer activity on several mouse models of human cancer and human cancers in vivo. However, the mechanisms of SW-mediated tumor regression are not clear. In this study, we investigated the effects of SW on several human lung cancer cell lines in vitro. The results showed that SW significantly inhibited these cells growth through induction of apoptosis in different extent in vitro. Further studies showed that SW treatment up-regulated Bax, down-regulated Bcl-2 expression, promoted Bax translocation to mitochondria, activated mitochondria-mediated apoptotic pathway, which in turn caused the release of cytochrome c, the activation of caspase-9 and caspase-3, and the cleavage of poly (ADP-ribose) polymerase (PARP), resulting in A549 cell apoptosis. However, the expression of Fas, Fas ligand (FasL) or caspase-8 activity did not appear significant changes in the process of SW-induced apoptosis. Moreover, SW treatment inhibited Bcl-2 expression, promoted Bax translocation, cytochrome c release and caspase-3 activity in xenograft tumor cells, resulting in a significant decrease of tumor volume and tumor weight in the SW-treated xenograft mice groups in comparison to the control group. Taken together, this study demonstrated for the first time that SW inhibited A549 cancer cells growth through a mitochondria-mediated, caspase-dependent apoptotic pathway in vitro and in vivo. PMID:22393311

  5. Apoptotic-like Leishmania exploit the host's autophagy machinery to reduce T-cell-mediated parasite elimination.

    PubMed

    Crauwels, Peter; Bohn, Rebecca; Thomas, Meike; Gottwalt, Stefan; Jäckel, Florian; Krämer, 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 showed--in contrast to viable parasites--that 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

  6. Apoptotic Cell Death Induced by Resveratrol Is Partially Mediated by the Autophagy Pathway in Human Ovarian Cancer Cells

    PubMed Central

    Lang, Fangfang; Qin, Zhaoyang; Li, Fang; Zhang, Huilin; Fang, Zhenghui; Hao, Enkui

    2015-01-01

    Resveratrol (trans-3,4,5’ –trihydroxystilbene) is an active compound in food, such as red grapes, peanuts, and berries. Resveratrol exhibits an anticancer effect on various human cancer cells. However, the mechanism of resveratrol-induced anti-cancer effect at the molecular level remains to be elucidated. In this study, the mechanism underlying the anti-cancer effect of resveratrol in human ovarian cancer cells (OVCAR-3 and Caov-3) was investigated using various molecular biology techniques, such as flow cytometry, western blotting, and RNA interference, with a major focus on the potential role of autophagy in resveratrol-induced apoptotic cell death. We demonstrated that resveratrol induced reactive oxygen species (ROS) generation, which triggers autophagy and subsequent apoptotic cell death. Resveratrol induced ATG5 expression and promoted LC3 cleavage. The apoptotic cell death induced by resveratrol was attenuated by both pharmacological and genetic inhibition of autophagy. The autophagy inhibitor chloroquine, which functions at the late stage of autophagy, significantly reduced resveratrol-induced cell death and caspase 3 activity in human ovarian cancer cells. We also demonstrated that targeting ATG5 by siRNA also suppressed resveratrol-induced apoptotic cell death. Thus, we concluded that a common pathway between autophagy and apoptosis exists in resveratrol-induced cell death in OVCAR-3 human ovarian cancer cells. PMID:26067645

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

    SciTech Connect

    Song, Shasha [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Wang, Shuang [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Biopharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China); Ma, Jun; Yao, Lan; Xing, Hao; Zhang, Lei; Liao, Lin [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Zhu, Daling, E-mail: dalingz@yahoo.com [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Biopharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China)

    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.

  8. Carbon monoxide protects hepatocytes from TNF-?\\/Actinomycin D by inhibition of the caspase-8-mediated apoptotic pathway

    Microsoft Academic Search

    Hoe Suk Kim; Patricia A. Loughran; Peter K. Kim; Timothy R. Billiar; Brian S. Zuckerbraun

    2006-01-01

    We have previously shown that carbon monoxide (CO) (250ppm) prevented tumor necrosis factor-? (TNF?)-induced apoptosis and activated the transcription factor NF-?B in hepatocytes both in vivo and in vitro. These studies were conducted to further determine the mechanisms by which CO suppresses apoptotic signaling in TNF? (10ng\\/ml) and Actinomycin D (ActD, 200ng\\/ml)-treated hepatocytes. Consistent with our previous findings, CO protected

  9. Mesenchymal Stem Cells from Rat Bone Marrow Downregulate Caspase3-mediated Apoptotic Pathway After Spinal Cord Injury in Rats

    Microsoft Academic Search

    Venkata Ramesh Dasari; Daniel G. Spomar; Craig Cady; Meena Gujrati; Jasti S. Rao; Dzung H. Dinh

    2007-01-01

    Mesenchymal stem cells have been intensively studied for their potential use in reparative strategies for neurodegenerative\\u000a diseases and traumatic injuries. We used mesenchymal stem cells (rMSC) from rat bone marrow to evaluate the therapeutic potential\\u000a after spinal cord injury (SCI). Immunohistochemistry confirmed a large number of apoptotic neurons and oligodendrocytes in\\u000a caudal segments 2 mm away from the lesion site. Expression

  10. Regulation of CRADD-caspase 2 cascade by histone deacetylase 1 in gastric cancer

    PubMed Central

    Shen, Qi; Tang, Wanfen; Sun, Jie; Feng, Lifeng; Jin, Hongchuan; Wang, Xian

    2014-01-01

    CRADD, also referred as RAIDD, is an adaptor protein that could interact with both caspase 2 and RIP that can promote apoptosis once activated. HDAC inhibitors are promising anti-cancer agents by inducing apoptosis of various cancer cells. In this study, we found that CRADD was induced by TSA (trichostatin A) to activate caspase 2-dependent apoptosis. CRADD was downregulated in gastric cancer and the restoration of its expression suppressed the viability of gastric cancer cells. HDAC1 was responsible for its downregulation in gastric cancer since HDAC1 siRNA upregulated CRADD expression and HDAC1 directly bound to the promoter of CRADD. Therefore, the high expression of HDAC1 can downregulate CRADD to confer gastric cancer cells the resistance to caspase 2-dependent apoptosis. HDAC inhibitors, potential anti-cancer drugs under investigation, can promote caspase 2-dependent apoptosis by inducing the expression of CRADD. PMID:25360218

  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. 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 (2 mM). 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

  13. The p53-inducible gene 3 involved in flavonoid-induced cytotoxicity through the reactive oxygen species-mediated mitochondrial apoptotic pathway in human hepatoma cells.

    PubMed

    Zhang, Qiang; Cheng, Guangdong; Qiu, Hongbin; Zhu, Liling; Ren, Zhongjuan; Zhao, Wei; Zhang, Tao; Liu, Lei

    2015-05-13

    Flavonoids have been reported to exhibit prooxidant cytotoxicity against cancer cells, but the underlying mechanism is still poorly understood. Here we investigated the potential mechanism that p53-inducible gene 3 (PIG3), a NADPH:quinone oxidoreductase, mediated the prooxidant cytotoxicity of flavonoids on human hepatoma HepG2 cells. The results showed that flavonoids (apigenin, luteolin, kaempferol, and quercetin) inhibited the growth of HepG2 cells in a dosage- and time-dependent manner, and induced the morphological changes characteristic of apoptosis in HepG2 cells. We also found that expression of PIG3 was increased markedly in HepG2 cells treated with flavonoids at both mRNA and protein levels, which was accompanied by increased intracellular ROS production and a decreased mitochondrial membrane potential (??m). All these effects were largely reversed through knockdown of the PIG3 gene in HepG2 cells. Western blotting indicated that flavonoids increased cytochrome c release, upregulated the ratio of Bax/Bcl-2, and activated the caspases-9 and -3. Moreover, knockdown of PIG3 could reverse the changes of these apoptotic-related proteins. These results suggest that PIG3 plays an important role in regulating the prooxidant activity and apoptosis-inducing action of flavonoids on HepG2 cells though the ROS-triggered mitochondrial apoptotic pathway. PMID:25820747

  14. Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3?-OH Single-strand DNA Breaks*

    PubMed Central

    Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Sánchez-Osuna, María; Casanelles, Elisenda; García-Belinchón, Mercè; Comella, Joan X.; Yuste, Victor J.

    2013-01-01

    Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD?/? cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3?-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3?-OH ends in single-strand rather than double-strand DNA nicks/breaks. PMID:23430749

  15. Necrotic cell-derived high mobility group box 1 attracts antigen-presenting cells but inhibits hepatocyte growth factor-mediated tropism of mesenchymal stem cells for apoptotic cell death.

    PubMed

    Vogel, S; Börger, V; Peters, C; Förster, M; Liebfried, P; Metzger, K; Meisel, R; Däubener, W; Trapp, T; Fischer, J C; Gawaz, M; Sorg, R V

    2015-07-01

    Tissue damage due to apoptotic or necrotic cell death typically initiates distinct cellular responses, leading either directly to tissue repair and regeneration or to immunological processes first, to clear the site, for example, of potentially damage-inducing agents. Mesenchymal stem cells (MSC) as well as immature dendritic cells (iDC) and monocytes migrate to injured tissues. MSC have regenerative capacity, whereas monocytes and iDC have a critical role in inflammation and induction of immune responses, including autoimmunity after tissue damage. Here, we investigated the influence of apoptotic and necrotic cell death on recruitment of MSC, monocytes and iDC, and identified hepatocyte growth factor (HGF) and the alarmin high mobility group box 1 (HMGB1) as key factors differentially regulating these migratory responses. MSC, but not monocytes or iDC, were attracted by apoptotic cardiomyocytic and neuronal cells, whereas necrosis induced migration of monocytes and iDC, but not of MSC. Only apoptotic cell death resulted in HGF production and HGF-mediated migration of MSC towards the apoptotic targets. In contrast, HMGB1 was predominantly released by the necrotic cells and mediated recruitment of monocytes and iDC via the receptor of advanced glycation end products. Moreover, necrotic cardiomyocytic and neuronal cells caused an HMGB1/toll-like receptor-4-dependent inhibition of MSC migration towards apoptosis or HGF, while recruitment of monocytes and iDC by necrosis or HMGB1 was not affected by apoptotic cells or HGF. Thus, the type of cell death differentially regulates recruitment of either MSC or monocytes and iDC through HGF and HMGB1, respectively, with a dominant, HMGB1-mediated role of necrosis in determining tropism after tissue injury. PMID:25571972

  16. Requirement of apoptotic protease-activating factor-1 for bortezomib-induced apoptosis but not for Fas-mediated apoptosis in human leukemic cells.

    PubMed

    Ottosson-Wadlund, Astrid; Ceder, Rebecca; Preta, Giulio; Pokrovskaja, Katja; Grafström, Roland C; Heyman, Mats; Söderhäll, Stefan; Grandér, Dan; Hedenfalk, Ingrid; Robertson, John D; Fadeel, Bengt

    2013-01-01

    Bortezomib is a highly selective inhibitor of the 26S proteasome and has been approved for clinical use in the treatment of relapsing and refractory multiple myeloma and mantle cell lymphoma. Clinical trials are also underway to assess the role of bortezomib in several other human malignancies, including leukemia. However, the mechanism(s) by which bortezomib acts remain to be fully understood. Here, we studied the molecular requirements of bortezomib-induced apoptosis using the human T-cell leukemic Jurkat cells stably transfected with or without shRNA against apoptotic protease-activating factor-1 (Apaf-1). The Apaf-1-deficient Jurkat T cells were resistant to bortezomib-induced apoptosis, as assessed by caspase-3 activity, poly(ADP-ribose) polymerase cleavage, phosphatidylserine externalization, and hypodiploid DNA content. In contrast, Apaf-1-deficient cells were sensitive to Fas-induced apoptosis. Bortezomib induced an upregulation of the pro-apoptotic protein Noxa, loss of mitochondrial transmembrane potential, and release of cytochrome c in cells expressing or not expressing Apaf-1. Transient silencing of Apaf-1 expression in RPMI 8402 T-cell leukemic cells also diminished bortezomib-induced apoptosis. Fas-associated death domain (FADD)-deficient Jurkat cells were resistant to Fas-mediated apoptosis yet remained sensitive to bortezomib. Our results show that bortezomib induces apoptosis by regulating pathways that are mechanistically different from those activated upon death receptor ligation. Furthermore, in silico analyses of public transcriptomics databases indicated elevated Apaf-1 expression in several hematologic malignancies, including acute lymphoblastic and myeloid leukemia. We also noted variable Apaf-1 expression in a panel of samples from patients with acute lymphoblastic leukemia. Our results suggest that the expression of Apaf-1 may be predictive of the response to proteasome inhibition. PMID:23093495

  17. 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

  18. Mesenchymal stem cells from rat bone marrow down regulate Caspase-3 mediated apoptotic pathway after spinal cord injury in rats

    PubMed Central

    Dasari, Venkata Ramesh; Spomar, Daniel G.; Cady, Craig; Gujrati, Meena; Rao, Jasti S.; Dinh, Dzung H.

    2007-01-01

    Mesenchymal stem cells have been intensively studied for their potential use in reparative strategies for neurodegenerative diseases and traumatic injuries. We used mesenchymal stem cells (rMSC) from rat bone marrow to evaluate the therapeutic potential after spinal cord injury (SCI). Immunohistochemistry confirmed a large number of apoptotic neurons and oligodendrocytes in caudal segments 2mm away from the lesion site. Expression of caspase-3 on both neurons and oligodendrocytes after SCI was significantly downregulated by rMSC. Caspase-3 downregulation by rMSC involves increased expression of FLIP and XIAP in the cytosol and inhibition of PARP cleavage in the nucleus. Animals treated with rMSC had higher BBB scores and better recovery of hind limb sensitivity. Treatment with rMSC had a positive effect on behavioral outcome and histopathological assessment after SCI. The ability of rMSC to incorporate into the spinal cord, differentiate and to improve locomotor recovery hold promise for a potential cure after SCI. PMID:17564836

  19. 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

  20. Caspase-2 Maintains Bone Homeostasis by Inducing Apoptosis of Oxidatively-Damaged Osteoclasts

    PubMed Central

    Sharma, Ramaswamy; Callaway, Danielle; Vanegas, Difernando; Bendele, Michelle; Lopez-Cruzan, Marisa; Horn, Diane; Guda, Teja; Fajardo, Roberto; Abboud-Werner, Sherry; Herman, Brian

    2014-01-01

    Osteoporosis is a silent disease, characterized by a porous bone micro-structure that enhances risk for fractures and associated disabilities. Senile, or age-related osteoporosis (SO), affects both men and women, resulting in increased morbidity and mortality. However, cellular and molecular mechanisms underlying senile osteoporosis are not fully known. Recent studies implicate the accumulation of reactive oxygen species (ROS) and increased oxidative stress as key factors in SO. Herein, we show that loss of caspase-2, a cysteine aspartate protease involved in oxidative stress-induced apoptosis, results in total body and femoral bone loss in aged mice (20% decrease in bone mineral density), and an increase in bone fragility (30% decrease in fracture strength). Importantly, we demonstrate that genetic ablation or selective inhibition of caspase-2 using zVDVAD-fmk results in increased numbers of bone-resorbing osteoclasts and enhanced tartrate-resistant acid phosphatase (TRAP) activity. Conversely, transfection of osteoclast precursors with wild type caspase-2 but not an enzymatic mutant, results in a decrease in TRAP activity. We demonstrate that caspase-2 expression is induced in osteoclasts treated with oxidants such as hydrogen peroxide and that loss of caspase-2 enhances resistance to oxidants, as measured by TRAP activity, and decreases oxidative stress-induced apoptosis of osteoclasts. Moreover, oxidative stress, quantified by assessment of the lipid peroxidation marker, 4-HNE, is increased in Casp2-/- bone, perhaps due to a decrease in antioxidant enzymes such as SOD2. Taken together, our data point to a critical and novel role for caspase-2 in maintaining bone homeostasis by modulating ROS levels and osteoclast apoptosis during conditions of enhanced oxidative stress that occur during aging. PMID:24691516

  1. Identification and characterization of ABCB1-mediated and non-apoptotic sebum secretion in differentiated hamster sebocytes.

    PubMed

    Kurihara, Hirokazu; Sato, Takashi; Akimoto, Noriko; Ogura, Takayuki; Ito, Akira

    2011-12-01

    Sebaceous glands secrete sebum onto the skin surface in a holocrine manner and as such a thin lipid layer is formed as a physiological barrier. In the present study, extracellular level of triacylglycerols (TG), a major sebum component, as well as intracellular TG accumulation was augmented in insulin-differentiated hamster sebocytes (DHS). The DHS exhibited phosphatidylserine exposure in an apoptosis-independent manner. In addition, intracellular ATP level and membrane-transporter activity using a substrate, Rhodamine 123, were highly detectable in the DHS rather than in the undifferentiated hamster sebocytes. A membrane-transporter activating reagent, 2'(3')-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP), enhanced transporter activity, extracellular TG level, and phosphatidylserine exposure in the DHS. Both transporter activity and TG secretion were suppressed by R-verapamil, a potent membrane-transporter inhibitor, in the BzATP-treated and untreated DHS. Furthermore, the gene expression and production of ATP-binding cassette subfamily B member 1 (ABCB1) were augmented in the DHS. ABCB1 was also detectable in sebaceous glands in the skin of hamsters. Moreover, the cell-differentiation- and BzATP-augmented transporter activity and TG secretion were dose-dependently inhibited by adding not only an ABCB1 antibody but also a selective inhibitor of ABCB1, PSC833. Thus, these results provide novel evidence that ABCB1 is involved in sebum secretion in the DHS, which is associated with non-apoptotic phosphatidylserine exposure and the increased level of intracellular ATP. These findings should accelerate the understanding of sebum secretion occurring in a holocrine-independent manner in sebaceous glands, and may contribute to the development of therapies for sebaceous gland disorders such as acne, seborrhea, and xerosis. PMID:21889999

  2. 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

  3. Anti-proliferative and pro-apoptotic effects of lichen-derived compound protolichesterinic acid are not mediated by its lipoxygenase-inhibitory activity.

    PubMed

    Bessadóttir, M; Eiríksson, F F; Becker, S; Ögmundsdóttir, M H; Ómarsdóttir, S; Thorsteinsdóttir, M; Ögmundsdóttir, H M

    2015-07-01

    Lipoxygenases (LOXs) and their products are involved in several biological functions and have been associated with carcinogenesis. Protolichesterinic acid (PA), a lichen metabolite, inhibits 5- and 12-LOX and has anti-proliferative effects on various cancer cell lines. Here, PA was shown to inhibit proliferation of multiple myeloma cells, RPMI 8226 and U266, and pancreatic cancer cells AsPC-1. Apoptosis was induced only in multiple myeloma cells. Cell-cycle associated changes in expression and sub-cellular localization of 5- and 12-LOX were not affected by PA but increased cytoplasmic localisation was found to accompany morphological changes at later stages. Assessment by mass spectrometry showed that PA entered the pancreatic cancer cells. However, effects on LOX metabolites were only evident after treatment with concentrations exceeding those having anti-proliferative effects and no effects were measurable in the myeloma cells. We conclude that the anti-proliferative and pro-apoptotic effects of PA are not mediated directly through inhibition of LOX activity. PMID:25964147

  4. (?)-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

  5. Effect of cilostazol pretreatment on the PARP/AIF-mediated apoptotic pathway in rat cerebral ischemia-reperfusion models.

    PubMed

    Ba, Xiao-Hong; Cai, Li-Ping; Han, Wei

    2014-05-01

    The aim of this study was to observe the expression of poly ADP-ribose polymerase (PARP) and apoptosis-inducing factor (AIF) in the CA1 region of the hippocampus and to explore whether cilostazol pretreatment exerts a protective effect on the brain through the PARP/AIF-mediated pathway in a rat model of cerebral ischemia-reperfusion. Rats were randomly divided into three groups: Sham-surgery, ischemia-reperfusion and cilostazol (n=45 rats/group). Rat models of middle cerebral artery occlusion were prepared using a thread occlusion method. Rats in the cilostazol group were administered 30 mg/kg intragastric cilostazol 6 and 2 h before brain ischemia, respectively. Following reperfusion, samples were collected at different time-points (6, 24 and 72 h) and each group was further subdivided into three subgroups (n=15 rats/subgroup). Apoptosis was measured using the terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling method. The protein expression levels of AIF and PARP were detected using western blot analysis and the expression levels of AIF mRNA were determined using the reverse transcription-polymerase chain reaction. AIF nuclear translocation occurred following local cerebral ischemia-reperfusion injury. Apoptosis, levels of AIF and PARP protein expression and levels of AIF mRNA expression were significantly increased in the ischemia-reperfusion group compared with the sham-surgery group (P<0.05). However, apoptosis and the expression levels of AIF protein, PARP protein and AIF mRNA at different time-points were significantly decreased in the cilostazol group compared with the ischemia-reperfusion group (P<0.05). In conclusion, cilostazol has a protective effect on rat cerebral ischemia-reperfusion injury, and acts by inhibiting nerve cell apoptosis by preventing the excessive activation of PARP and AIF nuclear translocation. PMID:24940413

  6. 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

  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. 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

  9. Selective growth regulatory and pro-apoptotic effects of DIM is mediated by AKT and NF-kappaB pathways in prostate cancer cells.

    PubMed

    Li, Yiwei; Chinni, Sreenivasa R; Sarkar, Fazlul H

    2005-01-01

    Prostate cancer is the second leading cause of cancer related deaths in men in the United States. I3C and its in vivo dimeric product, DIM, have been found to inhibit the growth of prostate cancer cells. However, the molecular mechanism(s) by which DIM elicits its effects on prostate cancer cells has not been fully elucidated. We have previously shown that I3C induces apoptosis and inhibits the activation of NF-kappaB pathway, which could be mediated via Akt signaling pathway. In this study, we investigated whether there is any cross-talk between Akt and NF-kappaB during DIM-induced apoptosis in PC-3 prostate cancer cells. We found that DIM inhibited cell growth and induced apoptosis in PC-3 prostate cancer cells but not in non-tumorigenic CRL2221 human prostate epithelial cells. DIM also inhibited EGFR expression, PI3K kinase activity, and Akt activation, and abrogated the EGF-induced activation of PI3K in prostate cancer cells. NF-kappaB DNA-binding analysis and transfection studies with Akt cDNA constructs revealed that Akt transfection resulted in the induction of NF-kappaB activity and this was inhibited by DIM treatment. DIM treatment also showed significant induction of apoptosis in non-transfected cells compared to Akt and Akt-Myr transfected prostate cancer cells. From these results, we conclude that the inhibition of Akt and NF-kappaB activity and their cross-talk is a novel mechanism by which DIM inhibits cell growth and induces apoptotic processes in prostate cancer cells but not in non-tumorigenic prostate epithelial cells. PMID:15574364

  10. Increased expression of caspase 2 in experimental and human temporal lobe epilepsy

    Microsoft Academic Search

    Susanna Narkilahti; Leena Jutila; Irina Alafuzoff; Kari Karkola; Leo Paljärvi; Arto Immonen; Matti Vapalahti; Esa Mervaala; Reetta Kälviäinen; Asla Pitkänen

    2007-01-01

    Temporal lobe epilepsy (TLE) is often caused by a neurodegenerative brain insult that triggers epileptogenesis, and eventually\\u000a results in spontaneous seizures, i.e., epilepsy. Understanding the mechanisms of cell death is a key for designing new drug\\u000a therapies for preventing the neurodegeneration associated with TLE. Here, we investigated the expression of caspase 2, a protein\\u000a involved in programmed cell death, during

  11. 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

  12. Berberine-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species generation and mitochondrial-related apoptotic pathway.

    PubMed

    Xie, Juan; Xu, Yinyan; Huang, Xinyan; Chen, Yanni; Fu, Jing; Xi, Mingming; Wang, Li

    2015-02-01

    Berberine has drawn extensive attention toward their wide range of biochemical and pharmacological effects, including antineoplastic effect in recent years, but the precise mechanisms remain unclear. Treatment of human breast cancer cells (MCF-7 and MDA-MB-231 cells) with berberine induced inhibition of cell viability in concentration- and time-dependent manner irrespective of their estrogen receptor (ER) expression. Hoechst33342 staining confirmed berberine induced breast cancer cell apoptosis in time-dependent manner. Because apoptosis induction is considered to be a crucial strategy for cancer prevention and therapy, berberine may be an effective chemotherapeutic agent against breast cancer. To explore the precise mechanism, berberine-induced oxidative stress and mitochondrial-related apoptotic pathway in human breast cancer cells were investigated in this study. In both MCF-7 and MDA-MB-231 cells, berberine increased the production of reactive oxygen species (ROS), which activated the pro-apoptotic JNK signaling. Phosphorylated JNK triggered mitochondria membrane potential (??m) depolarization and downregulation expression of anti-apoptotic protein Bcl-2 concomitant with the upregulation expression of pro-apoptotic protein Bax. Downregulation of anti-apoptotic Bcl-2 family protein in parallel with loss of ??m, leading to increased the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, and eventually triggered the caspase-dependent and caspase-independent apoptosis. Taken together, our study reveled that berberine exerted an antitumor activity in breast cancer cells by reactive oxygen species generation and mitochondrial-related apoptotic pathway. These finding provide an insight into the potential of berberine for breast cancer therapy. PMID:25352028

  13. 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

  14. Triggering of death receptor apoptotic signaling by human papillomavirus 16 E2 protein in cervical cancer cell lines is mediated by interaction with c-FLIP

    Microsoft Academic Search

    Wei Wang; Yong Fang; Ni Sima; Yan Li; Wei Li; Li Li; Linfei Han; Shujie Liao; Zhiqiang Han; Qinglei Gao; Kezhen Li; Dongrui Deng; Li Meng; Jianfeng Zhou; Shixuan Wang; Ding Ma

    2011-01-01

    Human papillomavirus (HPV) E2 gene disruption is one of the key features of HPV-induced cervical malignant transformation. Though it is thought to prevent\\u000a progression of carcinogenesis, the pro-apoptotic function of E2 protein remains poorly understood. This study shows that expression\\u000a of HPV16 E2 induces apoptosis both in HPV-positive and -negative cervical cancer cell lines and leads to hyperactivation of\\u000a caspase-8

  15. Macrophage Response to Apoptotic Cells Varies with the Apoptotic Trigger and Is Not Altered by a Deficiency in LRP Expression

    Microsoft Academic Search

    Ana Kozmar; Mallary C. Greenlee-Wacker; Suzanne S. Bohlson

    2010-01-01

    Rapid engulfment of apoptotic cells in the absence of inflammation is required for maintenance of normal tissue homeostasis. The low-density lipoprotein receptor-related protein-1 (LRP\\/CD91) is a receptor mediating interactions between macrophages and apoptotic cells, but recent reports have challenged the requirement of this surface protein in this process. To explore the role of LRP in the recognition of apoptotic cells,

  16. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine co-expressing pro-apoptotic caspase-3.

    PubMed

    Gartner, Tatiana; Romano, Marta; Suin, Vanessa; Kalai, Michaël; Korf, Hannelie; De Baetselier, Patrick; Huygen, Kris

    2008-03-10

    DNA vaccination is a potent means for inducing strong cell-mediated immune responses and protective immunity against viral, bacterial and parasite pathogens in rodents. In an attempt to increase cross-presentation through apoptosis, the DNA-encoding caspase-2 prodomain followed by wild-type or catalytically inactive mutated caspase-3 was inserted into a plasmid encoding the 32 kDa mycolyl transferase (Ag85A) from Mycobacterium tuberculosis. Transient transfection showed that the mutated caspase induced slow apoptosis, normal protein expression and NF-kappaB activation while wild-type caspase induced rapid apoptosis, lower protein expression and no NF-kappaB activation. Ag85A specific antibody production was increased by co-expressing the mutated and decreased by co-expressing the wild-type caspase. Vaccination with pro-apoptotic plasmids triggered more Ag85A specific IFN-gamma producing spleen cells, and more efficient IL-2 and IFN-gamma producing memory cells in spleen and lungs after M. tuberculosis challenge. Compared to DNA-encoding secreted Ag85A, vaccination with DNA co-expressing wild-type caspase increased protection after infection with M. tuberculosis, while vaccination with plasmid co-expressing mutated caspase was not protective, possibly due to the stimulation of IL-6, IL-10 and IL-17A production. PMID:18280621

  17. Multi-level disruption of the extrinsic apoptotic pathway mediates resistance of leukemia cells to TNF-related apoptosis-inducing ligand (TRAIL).

    PubMed

    Leahomschi, S; Molinsky, J; Klanova, M; Andera, L; Peterka, M; Gasova, Z; Klener, P; Trneny, M; Necas, E; Simonova, T; Zivny, J; Klener, P

    2013-01-01

    Disruption of apoptotic pathways belongs to commonly reported molecular mechanisms that underlie cancer drug resistance. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, Apo2L) is a cytokine of the TNF family with selective anti-tumor activity and minimal toxicity toward healthy tissues. Primary leukemia cells are, however, largely intrinsically resistant to TRAIL-induced apoptosis. In this study we analyzed molecular differences between TRAIL-resistant K562 cell line and TRAIL-sensitive K562 clones. We demonstrate that TRAIL-sensitive K562 cells differ from the TRAIL-resistant cell line by cell surface downregulation of TRAIL decoy receptor 1, upregulation of both TRAIL death receptors, enhanced assembly and improved functioning of the death-inducing signaling complex, and increased cytoplasmic protein expression of CASP8 and key proapoptotic BCL2 members BID, BIM, BAD and BAK. The molecular basis of the intrinsic leukemia cell TRAIL resistance thus appears a consequence of the multi-level disruption of the extrinsic apoptotic pathway. The results of this study also suggest that the leukemia TRAIL-resistance is functional, leaving a possibility of overcoming the resistance by preexposure of the leukemia cells to potent TRAIL sensitizers, e.g. BH3-mimetics. PMID:23259793

  18. Multi-level disruption of the extrinsic apoptotic pathway mediates resistance of leukemia cells to TNF-related apoptosis-inducing ligand (TRAIL).

    PubMed

    Leahomschi, S; Molinsky, J; Klanova, M; Andera, L; Peterka, M; Gasova, Z; Sr, P Klener; Trneny, M; Necas, E; Simonova, T; Zivny, J; Jr, P Klener

    2012-11-25

    Disruption of apoptotic pathways belongs to commonly reported molecular mechanisms that underlie cancer drug resistance. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, Apo2L) is a?cytokine of the TNF family with selective anti-tumor activity and minimal toxicity toward healthy tissues. Primary leukemia cells are, however, largely intrinsically resistant to TRAIL-induced apoptosis. In this study we analyzed molecular differences between TRAIL-resistant K562 cell line and TRAIL-sensitive K562 clones. We demonstrate that TRAIL-sensitive K562 cells differ from the TRAIL-resistant cell line by cell surface downregulation of TRAIL decoy receptor 1, upregulation of both TRAIL death receptors, enhanced assembly and improved functioning of the death-inducing signaling complex, and increased cytoplasmic protein expression of CASP8 and key proapoptotic BCL2 members BID, BIM, BAD and BAK. The molecular basis of the intrinsic leukemia cell TRAIL resistance thus appears a?consequence of the multi-level disruption of the extrinsic apoptotic pathway. The results of this study also suggest that the leukemia TRAIL-resistance is functional, leaving a?possibility of overcoming the resistance by preexposure of the leukemia cells to potent TRAIL sensitizers, e.g. BH3-mimetics. Keywords: leukemia, drug-resistance, TRAIL, apoptosis, BCL2 family. PMID:23176284

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

    PubMed Central

    ZHONG, LEI; ZHOU, XING-LU; LIU, YAN-SONG; WANG, YI-MIN; MA, FEI; GUO, BAO-LIANG; YAN, ZHAO-QI; ZHANG, QING-YUAN

    2015-01-01

    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

  20. Adaptation to ER Stress Is Mediated by Differential Stabilities of Pro-Survival and Pro-Apoptotic mRNAs and Proteins

    PubMed Central

    Rutkowski, D. Thomas; Arnold, Stacey M; Miller, Corey N; Wu, Jun; Li, Jack; Gunnison, Kathryn M; Mori, Kazutoshi; Sadighi Akha, Amir A.; Raden, David; Kaufman, Randal J

    2006-01-01

    The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a signaling cascade known as the unfolded protein response (UPR). Although activation of the UPR is well described, there is little sense of how the response, which initiates both apoptotic and adaptive pathways, can selectively allow for adaptation. Here we describe the reconstitution of an adaptive ER stress response in a cell culture system. Monitoring the activation and maintenance of representative UPR gene expression pathways that facilitate either adaptation or apoptosis, we demonstrate that mild ER stress activates all UPR sensors. However, survival is favored during mild stress as a consequence of the intrinsic instabilities of mRNAs and proteins that promote apoptosis compared to those that facilitate protein folding and adaptation. As a consequence, the expression of apoptotic proteins is short-lived as cells adapt to stress. We provide evidence that the selective persistence of ER chaperone expression is also applicable to at least one instance of genetic ER stress. This work provides new insight into how a stress response pathway can be structured to allow cells to avert death as they adapt. It underscores the contribution of posttranscriptional and posttranslational mechanisms in influencing this outcome. PMID:17090218

  1. Viral apoptotic mimicry.

    PubMed

    Amara, Ali; Mercer, Jason

    2015-08-01

    As opportunistic pathogens, viruses have evolved many elegant strategies to manipulate host cells for infectious entry and replication. Viral apoptotic mimicry, defined by the exposure of phosphatidylserine - a marker for apoptosis - on the pathogen surface, is emerging as a common theme used by enveloped viruses to promote infection. Focusing on the four best described examples (vaccinia virus, dengue virus, Ebola virus and pseudotyped lentivirus), we summarize our current understanding of apoptotic mimicry as a mechanism for virus entry, binding and immune evasion. We also describe recent examples of non-enveloped viruses that use this mimicry strategy, and discuss future directions and how viral apoptotic mimicry could be targeted therapeutically. PMID:26052667

  2. 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

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

    PubMed

    Oropesa-Ávila, Manuel; Fernández-Vega, Alejandro; de la Mata, Mario; Garrido-Maraver, Juan; Cotán, David; Paz, Marina Villanueva; Pavón, Ana Delgado; Cordero, Mario D; Alcocer-Gómez, Elizabet; de Lavera, Isabel; Lema, Rafael; Zaderenko, Ana Paula; Sánchez-Alcázar, José 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

  4. 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. [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (Canada)] [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (Canada); Bag, Jnanankur, E-mail: jbag@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (Canada)] [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (Canada)

    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.

  5. ROS generation mediates the anti-cancer effects of WZ35 via activating JNK and ER stress apoptotic pathways in gastric cancer

    PubMed Central

    Zou, Peng; Zhang, Junru; Xia, Yiqun; Kanchana, Karvannan; Guo, Guilong; Chen, Wenbo; Huang, Yi; Wang, Zhe; Yang, Shulin; Liang, Guang

    2015-01-01

    Gastric cancer is one of the leading causes of cancer mortality in the world, and finding novel agents and strategies for the treatment of advanced gastric cancer is of urgent need. Curcumin is a well-known natural product with anti-cancer ability, but is limited by its poor chemical stability. In this study, an analog of curcumin with high chemical stability, WZ35, was designed and evaluated for its anti-cancer effects and underlying mechanisms against human gastric cancer. WZ35 showed much stronger anti-proliferative effects than curcumin, accompanied by dose-dependent induction of cell cycle arrest and apoptosis in gastric cancer cells. Mechanistically, our data showed that WZ35 induced reactive oxygen species (ROS) production, resulting in the activation of both JNK-mitochondrial and ER stress apoptotic pathways and eventually cell apoptosis in SGC-7901 cells. Blockage of ROS production totally reversed WZ35-induced JNK and ER stress activation as well as cancer cell apoptosis. In vivo, WZ35 showed a significant reduction in SGC-7901 xenograft tumor size in a dose-dependent manner. Taken together, this work provides a novel anticancer candidate for the treatment of gastric cancer, and importantly, reveals that increased ROS generation might be an effective strategy in human gastric cancer treatment. PMID:25714022

  6. ROS generation mediates the anti-cancer effects of WZ35 via activating JNK and ER stress apoptotic pathways in gastric cancer.

    PubMed

    Zou, Peng; Zhang, Junru; Xia, Yiqun; Kanchana, Karvannan; Guo, Guilong; Chen, Wenbo; Huang, Yi; Wang, Zhe; Yang, Shulin; Liang, Guang

    2015-03-20

    Gastric cancer is one of the leading causes of cancer mortality in the world, and finding novel agents and strategies for the treatment of advanced gastric cancer is of urgent need. Curcumin is a well-known natural product with anti-cancer ability, but is limited by its poor chemical stability. In this study, an analog of curcumin with high chemical stability, WZ35, was designed and evaluated for its anti-cancer effects and underlying mechanisms against human gastric cancer. WZ35 showed much stronger anti-proliferative effects than curcumin, accompanied by dose-dependent induction of cell cycle arrest and apoptosis in gastric cancer cells. Mechanistically, our data showed that WZ35 induced reactive oxygen species (ROS) production, resulting in the activation of both JNK-mitochondrial and ER stress apoptotic pathways and eventually cell apoptosis in SGC-7901 cells. Blockage of ROS production totally reversed WZ35-induced JNK and ER stress activation as well as cancer cell apoptosis. In vivo, WZ35 showed a significant reduction in SGC-7901 xenograft tumor size in a dose-dependent manner. Taken together, this work provides a novel anticancer candidate for the treatment of gastric cancer, and importantly, reveals that increased ROS generation might be an effective strategy in human gastric cancer treatment. PMID:25714022

  7. 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 [Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, SCRB2, Box 951, 7435 Fannin Street, Houston, TX 77030 (United States); Alt, Eckhard [Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, SCRB2, Box 951, 7435 Fannin Street, Houston, TX 77030 (United States)], E-mail: ealt@mdanderson.org

    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.

  8. 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. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 2770-2777, 2015. PMID:25684371

  9. Terminal Deoxynucleotidyl Transferase (TdT)-Mediated dUTP Nick-End Labeling (TUNEL) for Detection of Apoptotic Cells in Drosophila.

    PubMed

    Denton, Donna; Kumar, Sharad

    2015-01-01

    A characteristic feature of apoptosis is DNA fragmentation. This fragmentation can be detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) of DNA in dying cells. Here, we present a protocol for TUNEL detection of apoptosis in Drosophila larval tissue, but these techniques can be adapted for other tissues and developmental stages. PMID:26034307

  10. AlphaII-spectrin is an in vitro target for caspase-2, and its cleavage is regulated by calmodulin binding.

    PubMed Central

    Rotter, Björn; Kroviarski, Yolande; Nicolas, Gaël; Dhermy, Didier; Lecomte, Marie-Christine

    2004-01-01

    The spectrin-actin scaffold underlying the lipid bilayer is considered to participate in cell-shape stabilization and in the organization of specialized membrane subdomains. These structures are dynamic and likely to undergo frequent remodelling during changes in cell shape. Proteolysis of spectrin, which occurs during apoptosis, leads to destabilization of the scaffold. It is also one of the major processes involved in membrane remodelling. Spectrins, the main components of the membrane skeleton, are the targets for two important protease systems: m- and micro-calpains (Ca2+-activated proteases) and caspase-3 (activated during apoptosis). In this paper, we show that caspase-2 also targets spectrin in vitro, and we characterize Ca2+/calmodulin-dependent regulation of spectrin cleavage by caspases. Yeast two-hybrid screening reveals that the large isoform (1/L) of procaspase-2 specifically binds to alphaII-spectrin, while the short isoform does not. Like caspase-3, caspase-2 cleaves alphaII-spectrin in vitro at residue Asp-1185. This study emphasizes a role of executioner caspase for caspase-2. We also demonstrated that the executioner caspase-7 but not caspase-6 cleaves spectrin at residue Asp-1185 in vitro. This spectrin cleavage by caspases 2, 3 and 7 is inhibited by the Ca2+-dependent binding of calmodulin to spectrin. In contrast, calmodulin binding enhances spectrin cleavage by calpain at residue Tyr-1176. These results indicate that alphaII-spectrin cleavage is highly influenced by Ca2+ homoeostasis and calmodulin, which therefore represent potential regulators of the stability and the plasticity of the spectrin-based skeleton. PMID:14599290

  11. Apoptosis signaling triggered by the marine alkaloid ascididemin is routed via caspase-2 and JNK to mitochondria

    Microsoft Academic Search

    Verena M Dirsch; Stephanie O Kirschke; Michael Estermeier; Bert Steffan; Angelika M Vollmar

    2004-01-01

    The marine alkaloid ascididemin (ASC) was shown to exert cytotoxicity even against multidrug-resistant cancer cells. Here, we address the signaling pathways utilized by ASC to trigger apoptosis in Jurkat leukemia T cells. We show that ASC (0.5–20 ?M) induces a mitochondrial pathway that requires the activation of the initiator caspase-2 upstream of mitochondria. ASC-triggered apoptosis occurred independent of CD95, but

  12. 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

  13. Induction of apoptosis and apoptotic mediators in balb\\/C splenic lymphocytes by dietary n?3 and n?6 fatty acids

    Microsoft Academic Search

    C. P. Reddy Avula; A. K. Zaman; R. Lawrence; G. Fernandes

    1999-01-01

    The present study was designed to investigate the effect of diatery n?6 and n?3 polyunsaturated fatty acids (PUFA) on anti-CD3\\u000a and anti-Fas antibody-induced apoptosis and its mediators in mouse spleen cells. Nutritionally adequate semipurified diets\\u000a containing either 5% w\\/w corn oil (n?6 PUFA) or fish oil (n?3 PUFA) were fed to weanling female Balb\\/C mice, and 24 wk later\\u000a mice

  14. Caspase-3 dependent proteolytic activation of protein kinase C delta mediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration.

    PubMed

    Kaul, Siddharth; Kanthasamy, Arthi; Kitazawa, Masashi; Anantharam, Vellareddy; Kanthasamy, Anumantha G

    2003-09-01

    1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), induces apoptosis in dopaminergic neurons; however, the cellular mechanisms underlying the degenerative process are not well understood. In the present study, we demonstrate that caspase-3 mediated proteolytic activation of protein kinase C delta (PKC delta) is critical in MPP+-induced oxidative stress and apoptosis. MPP+ exposure in rat dopaminergic neuronal cells resulted in time-dependent increases in reactive oxygen species generation, cytochrome c release, and caspase-9 and caspase-3 activation. Interestingly, MPP+ induced proteolytic cleavage of PKC delta (72-74 kDa) into a 41-kDa catalytic and a 38-kDa regulatory subunit, resulting in persistently increased kinase activity. The caspase-3 inhibitor Z-DEVD-fmk effectively blocked MPP+-induced PKC delta cleavage and kinase activity, suggesting that the proteolytic activation is caspase-3 mediated. Similar results were seen in MPP+-treated rat midbrain slices. Z-DEVD-fmk and the PKC delta specific inhibitor rottlerin almost completely blocked MPP+-induced DNA fragmentation. The superoxide dismutase mimetic, MnTBAP also effectively attenuated MPP+-induced caspase-3 activation, PKC delta cleavage, and DNA fragmentation. Furthermore, rottlerin attenuated MPP+-induced caspase-3 activity without affecting basal activity, suggesting positive feedback activation of caspase-3 by PKC delta. Intracellular delivery of catalytically active recombinant PKC delta significantly increased caspase-3 activity, further indicating that PKC delta regulates caspase-3 activity. Finally, over-expression of a kinase inactive PKC delta K376R mutant prevented MPP+-induced caspase activation and DNA fragmentation, confirming the pro-apoptotic function of PKC delta in dopaminergic cell death. Together, we demonstrate for the first time that MPP+-induced oxidative stress proteolytically activates PKC delta in a caspase-3-dependent manner to induce apoptosis and up-regulate the caspase cascade in dopaminergic neuronal cells. PMID:14511319

  15. 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 2–3 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 (3–10 ?m) were 3–6 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

  16. A sustained deficiency of mitochondrial respiratory complex III induces an apoptotic cell death through the p53-mediated inhibition of pro-survival activities of the activating transcription factor 4

    PubMed Central

    Evstafieva, A G; Garaeva, A A; Khutornenko, A A; Klepikova, A V; Logacheva, M D; Penin, A A; Novakovsky, G E; Kovaleva, I E; Chumakov, P M

    2014-01-01

    Generation of energy in mitochondria is subjected to physiological regulation at many levels, and its malfunction may result in mitochondrial diseases. Mitochondrial dysfunction is associated with different environmental influences or certain genetic conditions, and can be artificially induced by inhibitors acting at different steps of the mitochondrial electron transport chain (ETC). We found that a short-term (5?h) inhibition of ETC complex III with myxothiazol results in the phosphorylation of translation initiation factor eIF2? and upregulation of mRNA for the activating transcription factor 4 (ATF4) and several ATF4-regulated genes. The changes are characteristic for the adaptive integrated stress response (ISR), which is known to be triggered by unfolded proteins, nutrient and metabolic deficiency, and mitochondrial dysfunctions. However, after a prolonged incubation with myxothiazol (13–17?h), levels of ATF4 mRNA and ATF4-regulated transcripts were found substantially suppressed. The suppression was dependent on the p53 response, which is triggered by the impairment of the complex III-dependent de novo biosynthesis of pyrimidines by mitochondrial dihydroorotate dehydrogenase. The initial adaptive induction of ATF4/ISR acted to promote viability of cells by attenuating apoptosis. In contrast, the induction of p53 upon a sustained inhibition of ETC complex III produced a pro-apoptotic effect, which was additionally stimulated by the p53-mediated abrogation of the pro-survival activities of the ISR. Interestingly, a sustained inhibition of ETC complex I by piericidine did not induce the p53 response and stably maintained the pro-survival activation of ATF4/ISR. We conclude that a downregulation of mitochondrial ETC generally induces adaptive pro-survival responses, which are specifically abrogated by the suicidal p53 response triggered by the genetic risks of the pyrimidine nucleotide deficiency. PMID:25375376

  17. The Anti-Apoptotic and Cardioprotective Effects of Salvianolic Acid A on Rat Cardiomyocytes following Ischemia/Reperfusion by DUSP-Mediated Regulation of the ERK1/2/JNK Pathway

    PubMed Central

    Chen, Qiuping; Zhu, Shasha; Liu, Yang; Pan, Defeng; Chen, Xiaohu; Li, Dongye

    2014-01-01

    The purpose of this study was to observe the effects of salvianolic acid A (SAA) pretreatment on the myocardium during ischemia/reperfusion (I/R) and to illuminate the interrelationships among dual specificity protein phosphatase (DUSP) 2/4/16, ERK1/2 and JNK pathways during myocardial I/R, with the ultimate goal of elucidating how SAA exerts cardioprotection against I/R injury (IRI). Wistar rats were divided into the following six groups: control group (CON), I/R group, SAA+I/R group, ERK1/2 inhibitor PD098059+I/R group (PD+I/R), PD+SAA+I/R group, and JNK inhibitor SP600125+I/R group (SP+I/R). The cardioprotective effects of SAA on the myocardium during I/R were investigated with a Langendorff device. Heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximum rate of ventricular pressure rise and fall (±dp/dtmax), myocardial infarction areas (MIA), lactate dehydrogenase (LDH), and cardiomyocytes apoptosis were monitored. To determine the crosstalk betwee JNK and ERK1/2 via DUSP2/4/16 with SAA pretreatment, siRNA-DUSP2/4/16 were performed. The expression levels of Bcl-2, Bax, caspase 3, p-JNK, p-ERK1/2 and DUSP2/4/16 in cardiomyocytes were assayed by Western blot. Our results showed that LDH, MIA and cell apoptosis were decreased, and various parameters of heart function were improved by SAA pretreatment and SP application. In the I/R group, the expression levels of p-ERK1/2 and DUSP4/16 were not significantly different compared with the CON group, however, the protein expression levels of p-ERK1/2, Bcl-2 and DUSP4/16 were higher, while p-JNK, Bax, caspase 3 and DUSP2 levels were reduced among the SAA+I/R, PD+SAA+I/R and SP+I/R groups. The above indices were not significantly different between the SAA+I/R and SP+I/R groups. Compared with the SAA+I/R group, p-ERK1/2 was increased and p-JNK was decreased in the SAA+si-DUSP2+I/R, however, p-ERK was downregulated and p-JNK was upregulated in SAA+si-DUSP4+I/R group. SAA exerts an anti-apoptotic role against myocardial IRI by inhibiting DUSP2-mediated JNK dephosphorylation and activating DUSP4/16-mediated ERK1/2 phosphorylation. PMID:25019380

  18. Goniothalamin-induced oxidative stress, DNA damage and apoptosis via caspase-2 independent and Bcl-2 independent pathways in Jurkat T-cells

    PubMed Central

    Inayat-Hussain, S.H.; Chan, K.M.; Rajab, N.F.; Din, L.B.; Chow, S.C.; Kizilors, A.; Farzaneh, F.; Williams, G.T.

    2010-01-01

    Goniothalamin (GTN) isolated from Goniothalamus sp. has been demonstrated to induce apoptosis in a variety of cancer cell lines including Jurkat T leukemia cells. However, the mechanism of GTN-induced apoptosis upstream of mitochondria is still poorly defined. In this study, GTN caused a decrease in GSH with an elevation of reactive oxygen species as early as 30 min and DNA damage as assessed by Comet assay. Analysis using topoisomerase II processing of supercoiled pBR 322 DNA showed that GTN caused DNA damage via a topoisomerase II-independent pathway suggesting that cellular oxidative stress may contribute to genotoxicity. A 12-fold increase of caspase-2 activity was observed in GTN-treated Jurkat cells after 4 h treatment and this was confirmed using Western blotting. Although the caspase-2 inhibitor Z-VDVAD-FMK inhibited the proteolytic activity of caspase-2, apoptosis ensued confirming that caspase-2 activity was not crucial for GTN-induced apoptosis. However, GTN-induced apoptosis was completely abrogated by N-acetylcysteine further confirming the role of oxidative stress. Since cytochrome c release was observed as early as 1 h without any appreciable change in Bcl-2 protein expression, we further investigated whether overexpression of Bcl-2 confers resistance in GTN-induced cytotoxicity. Using a panel of Jurkat Bcl-2 transfectants, GTN cytotoxicity was not abrogated in these cells. In conclusion, GTN induces DNA damage and oxidative stress resulting in apoptosis which is independent of both caspase-2 and Bcl-2. PMID:20026395

  19. Temporal regulation of apoptotic and anti-apoptotic molecules after middle cerebral artery occlusion followed by reperfusion.

    PubMed

    Chelluboina, Bharath; Klopfenstein, Jeffrey D; Gujrati, Meena; Rao, Jasti S; Veeravalli, Krishna Kumar

    2014-02-01

    A tremendous effort has been expended to elucidate the role of apoptotic molecules in ischemia. However, many agents that target apoptosis, despite their proven efficacy in animal models, have failed to translate that efficacy and specificity in clinical settings. Therefore, comprehensive knowledge of apoptotic mechanisms involving key apoptotic regulatory molecules and the temporal expression profiles of various apoptotic molecules after cerebral ischemia may provide insight for the development of better therapeutic strategies aimed at cerebral ischemia. The present study investigates the extent of apoptosis and the regulation of apoptotic molecules both at mRNA and protein levels at various time points after focal cerebral ischemia in a rat model of middle cerebral artery occlusion. In this study, we performed various techniques, such as TTC (2,3,5-triphenyltetrazolium chloride), H&E (hematoxylin and eosin), and TUNEL (terminal deoxy nucleotidyl transferase-mediated nick-end labeling) staining, along with polymerase chain reaction (PCR) microarray, antibody microarray, reverse transcription (RT)-PCR, immunofluorescence, and immunoblot analyses. Our research provided a large list of pro-apoptotic and anti-apoptotic molecules and their temporal expression profiles both at the mRNA and protein levels. This information could be very useful for designing future stroke therapies and aid in targeting the right molecules at critical time to obtain maximum therapeutic benefit. PMID:23813097

  20. P2X7 receptor-mediated scavenger activity of mononuclear phagocytes toward non-opsonized particles and apoptotic cells is inhibited by serum glycoproteins but remains active in cerebrospinal fluid.

    PubMed

    Gu, Ben J; Duce, James A; Valova, Valentina A; Wong, Bruce; Bush, Ashley I; Petrou, Steven; Wiley, James S

    2012-05-18

    Rapid phagocytosis of non-opsonized particles including apoptotic cells is an important process that involves direct recognition of the target by multiple scavenger receptors including P2X7 on the phagocyte surface. Using a real-time phagocytosis assay, we studied the effect of serum proteins on this phagocytic process. Inclusion of 1-5% serum completely abolished phagocytosis of non-opsonized YG beads by human monocytes. Inhibition was reversed by pretreatment of serum with 1-10 mM tetraethylenepentamine, a copper/zinc chelator. Inhibitory proteins from the serum were determined as negatively charged glycoproteins (pI < 6) with molecular masses between 100 and 300 kDa. A glycoprotein-rich inhibitory fraction of serum not only abolished YG bead uptake but also inhibited phagocytosis of apoptotic lymphocytes or neuronal cells by human monocyte-derived macrophages. Three copper- and/or zinc-containing serum glycoproteins, ceruloplasmin, serum amyloid P-component, and amyloid precursor protein, were identified, and the purified proteins were shown to inhibit the phagocytosis of beads by monocytes as well as phagocytosis of apoptotic neuronal cells by macrophages. Human adult cerebrospinal fluid, which contains very little glycoprotein, had no inhibitory effect on phagocytosis of either beads or apoptotic cells. These data suggest for the first time that metal-interacting glycoproteins present within serum are able to inhibit the scavenger activity of mononuclear phagocytes toward insoluble debris and apoptotic cells. PMID:22461619

  1. P2X7 Receptor-mediated Scavenger Activity of Mononuclear Phagocytes toward Non-opsonized Particles and Apoptotic Cells Is Inhibited by Serum Glycoproteins but Remains Active in Cerebrospinal Fluid*

    PubMed Central

    Gu, Ben J.; Duce, James A.; Valova, Valentina A.; Wong, Bruce; Bush, Ashley I.; Petrou, Steven; Wiley, James S.

    2012-01-01

    Rapid phagocytosis of non-opsonized particles including apoptotic cells is an important process that involves direct recognition of the target by multiple scavenger receptors including P2X7 on the phagocyte surface. Using a real-time phagocytosis assay, we studied the effect of serum proteins on this phagocytic process. Inclusion of 1–5% serum completely abolished phagocytosis of non-opsonized YG beads by human monocytes. Inhibition was reversed by pretreatment of serum with 1–10 mm tetraethylenepentamine, a copper/zinc chelator. Inhibitory proteins from the serum were determined as negatively charged glycoproteins (pI < 6) with molecular masses between 100 and 300 kDa. A glycoprotein-rich inhibitory fraction of serum not only abolished YG bead uptake but also inhibited phagocytosis of apoptotic lymphocytes or neuronal cells by human monocyte-derived macrophages. Three copper- and/or zinc-containing serum glycoproteins, ceruloplasmin, serum amyloid P-component, and amyloid precursor protein, were identified, and the purified proteins were shown to inhibit the phagocytosis of beads by monocytes as well as phagocytosis of apoptotic neuronal cells by macrophages. Human adult cerebrospinal fluid, which contains very little glycoprotein, had no inhibitory effect on phagocytosis of either beads or apoptotic cells. These data suggest for the first time that metal-interacting glycoproteins present within serum are able to inhibit the scavenger activity of mononuclear phagocytes toward insoluble debris and apoptotic cells. PMID:22461619

  2. Annexin-1 and Peptide Derivatives Are Released by Apoptotic Cells and Stimulate Phagocytosis of Apoptotic Neutrophils by Macrophages1

    Microsoft Academic Search

    Michael Scannell; Michelle B. Flanagan; Kieran J. Wynne; Gerard Cagney; Catherine Godson; Paola Maderna

    The resolution of inflammation is a dynamically regulated process that may be subverted in many pathological conditions. Macrophage (M) phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation as this process prevents the exposure of tissues at the inflammatory site to the noxious contents of lytic cells. It is increasingly appreciated that endogenously produced mediators,

  3. The anti-apoptotic role of interleukin-6 in human cervical cancer is mediated by up-regulation of Mcl-1 through a PI 3-K\\/Akt pathway

    Microsoft Academic Search

    Lin-Hung Wei; Min-Liang Kuo; Chi-An Chen; Chia-Hung Chou; Wen-Fang Cheng; Ming-Cheng Chang; Jen-Liang Su; Chang-Yao Hsieh

    2001-01-01

    Interleukin-6 (IL-6), a multifunctional cytokine, has recently been implicated in human cervical cancer, though the mechanism remains elusive. This study demonstrates that the anti-apoptotic protein Mcl-1 and IL-6 was concomitantly expressed in human cervical cancer tissues and cell lines, but not in normal cervix tissues. Upon IL-6 treatment, Mcl-1, but not other Bcl-2 family members, was rapidly up-regulated peaking at

  4. Autologous dendritic cells or cells expressing both B7-1 and MUC1 can rescue tumor-specific cytotoxic T lymphocytes from MUC1-mediated apoptotic cell death

    Microsoft Academic Search

    Keiichi Kontani; Osamu Taguchi; Tatsuhiko Narita; Nozomu Hiraiwa; Satoru Sawai; Jun Hanaoka; Masutaro Ichinose; Noriaki Tezuka; Shuhei Inoue; Shozo Fujino; Reiji Kannagi

    We attempted to induce MUC1-specific cytotoxic T lymphocytes (CTLs) by mixed-lympho- cyte tumor cell culture (MLTC) using two alloge- neic MUC1-positive cancer cell lines, T-47D and MCF7. The induced CTLs exhibited MUC1-specific cytotoxicity 16 days after the initial stimulation. However, these CTLs underwent apoptotic death within 16 days. To examine whether the B7-1 mol- ecule is required for the expansion

  5. H. pylori Infection Inhibits Phagocyte Clearance of Apoptotic Gastric Epithelial Cells

    PubMed Central

    Bimczok, Diane; Smythies, Lesley E.; Waites, Ken B.; Grams, Jayleen M.; Stahl, Richard D.; Mannon, Peter J.; Peter, Shajan; Wilcox, C. Mel; Harris, Paul R.; Das, Soumita; Ernst, Peter B.; Smith, Phillip D.

    2013-01-01

    Increased apoptotic death of gastric epithelial cells is a hallmark of H. pylori infection, and altered epithelial cell turnover is an important contributor to gastric carcinogenesis. To address the fate of apoptotic gastric epithelial cells and their role in H. pylori mucosal disease, we investigated phagocyte clearance of apoptotic gastric epithelial cells in H. pylori infection. Human gastric mononuclear phagocytes were analyzed for their ability to take up apoptotic epithelial cells in vivo using immunofluorescence analysis. We then used primary human gastric epithelial cells induced to undergo apoptosis by exposure to live H. pylori to study apoptotic cell uptake by autologous monocyte-derived macrophages. We show that HLA-DR+ mononuclear phagocytes in human gastric mucosa contain cytokeratin-positive and TUNEL-positive apoptotic epithelial cell material, indicating that gastric phagocytes are involved in apoptotic epithelial cell clearance. We further show that H. pylori both increased apoptosis in primary gastric epithelial cells and decreased phagocytosis of the apoptotic epithelial cells by autologous monocyte-derived macrophages. Reduced macrophage clearance of apoptotic cells was mediated in part by H. pylori-induced macrophage TNF-?, which was expressed at higher levels in H. pylori-infected, compared to uninfected, gastric mucosa. Importantly, we show that H. pylori-infected gastric mucosa contained significantly higher numbers of apoptotic epithelial cells and higher levels of non-phagocytosed TUNEL-positive apoptotic material, consistent with a defect in apoptotic cell clearance. Thus, as shown in other autoimmune and chronic inflammatory diseases, insufficient phagocyte clearance may contribute to the chronic and self-perpetuating inflammation in human H. pylori infection. PMID:23686492

  6. 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 Central

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

    2011-01-01

    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

  7. ERK mediates anti-apoptotic effect through phosphorylation and cytoplasmic localization of p21 Waf1\\/Cip1\\/Sdi in response to DNA damage in normal human embryonic fibroblast (HEF) cells

    Microsoft Academic Search

    Jee-In Heo; Soo-Jin Oh; Yoon-Jung Kho; Jeong-Hyeon Kim; Hong-Joon Kang; Seong-Hoon Park; Hyun-Seok Kim; Jong-Yeon Shin; Min-Ju Kim; Sung Chan Kim; Jae-Bong Park; Jaebong Kim; Jae-Yong Lee

    2011-01-01

    Since anti-apoptotic effect of ERK has not been elucidated clearly in DNA-damage-induced cell death, the role of ERK was examined\\u000a in normal HEF cells treated with mild DNA damage using etoposide or camptothecin. ERK was activated by DNA damage in HEF cells.\\u000a PD98059 increased apoptosis and reduced DNA-damage-induced p21Waf1\\/Cip1\\/Sdi level. Depletion of p21Waf1\\/Cip1\\/Sdi induced cell death and PD98059 induced additional

  8. 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

  9. Interaction of Apoptotic Cells with Macrophages Upregulates COX-2/PGE2 and HGF Expression via a Positive Feedback Loop

    PubMed Central

    Byun, Ji Yeon; Youn, Young-So; Lee, Ye-Ji; Choi, Youn-Hee; Woo, So-Yeon; Kang, Jihee Lee

    2014-01-01

    Recognition of apoptotic cells by macrophages is crucial for resolution of inflammation, immune tolerance, and tissue repair. Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) and hepatocyte growth factor (HGF) play important roles in the tissue repair process. We investigated the characteristics of macrophage COX-2 and PGE2 expression mediated by apoptotic cells and then determined how macrophages exposed to apoptotic cells in vitro and in vivo orchestrate the interaction between COX-2/PGE2 and HGF signaling pathways. Exposure of RAW 264.7 cells and primary peritoneal macrophages to apoptotic cells resulted in induction of COX-2 and PGE2. The COX-2 inhibitor NS-398 suppressed apoptotic cell-induced PGE2 production. Both NS-398 and COX-2-siRNA, as well as the PGE2 receptor EP2 antagonist, blocked HGF expression in response to apoptotic cells. In addition, the HGF receptor antagonist suppressed increases in COX-2 and PGE2 induction. The in vivo relevance of the interaction between the COX-2/PGE2 and HGF pathways through a positive feedback loop was shown in cultured alveolar macrophages following in vivo exposure of bleomycin-stimulated lungs to apoptotic cells. Our results demonstrate that upregulation of the COX-2/PGE2 and HGF in macrophages following exposure to apoptotic cells represents a mechanism for mediating the anti-inflammatory and antifibrotic consequences of apoptotic cell recognition. PMID:24959005

  10. Isoobtusilactone A-induced apoptosis in human hepatoma Hep G2 cells is mediated via increased NADPH oxidase-derived reactive oxygen species (ROS) production and the mitochondria-associated apoptotic mechanisms.

    PubMed

    Chen, Chung-Yi; Liu, Tsan-Zon; Chen, Ching-Hsein; Wu, Chih-Chung; Cheng, Jiin-Tsuey; Yiin, Shuenn-Jiun; Shih, Ming-Kuei; Wu, Mei-Jem; Chern, Chi-Liang

    2007-07-01

    Chemoprevention by the use of naturally occurring substances is becoming a promising strategy to prevent cancer. In this study, the effects of isoobtusilactone A, a novel constituent isolated from the leaves of Cinnamomum kotoense, on the proliferation of human hepatoma Hep G2 cells were studied. Under our experimental conditions, isoobtusilactone A was found to elicit a concentration-dependent growth impediment (IC(50)=37.5 microM). The demise of these cells induced by isoobtusilactone A was apoptotic in nature, exhibiting a concentration-dependent increase in sub-G(1) fraction and DNA fragmentation. Subcellular fractionation analysis further revealed that Bax translocation to mitochondria resulted in a rapid release of cytochrome c, followed by activation of caspase 3 and PARP cleavage, and finally cell death. Isoobtusilactone A-treated cells also displayed transient increase of ROS during the earlier stage of the experiment, followed by the disruption of mitochondrial transmembrane potential (DeltaPsi(m)). The presence of a ROS scavenger (N-acetyl-L-cysteine) and an inhibitor of NADPH oxidase (diphenyleneiodonium chloride) blocked ROS production and the subsequent apoptotic cell death. In addition, in order to investigate the acute toxicity of isoobtusilactone A, groups of 5-6-week old Sprague-Dawley rats were subjected to oral administration of 350, or 700 mg/kg bw isoobtusilactone A four times each week for two weeks. There was no significant difference between control animals and treated animals with respect to the body weight gain, the body weight ratio of liver, spleen and kidney, haematological and clinical chemistry parameters. Taken together, our data suggest that ROS generated through the activation of NADPH oxidase plays an essential role in apoptosis induced by isoobtusilactone A, and the dosages of isoobtusilactone A tested in this study did not cause animal toxicity. PMID:17321026

  11. Protective role of D-saccharic acid-1,4-lactone in alloxan induced oxidative stress in the spleen tissue of diabetic rats is mediated by suppressing mitochondria dependent apoptotic pathway.

    PubMed

    Rashid, Kahkashan; Bhattacharya, Semantee; Sil, Parames C

    2012-03-01

    The present study investigated the role of D-saccharic acid 1,4-lactone (DSL) in the spleen tissue of alloxan (ALX) induced diabetic rats. Diabetes was induced in rats by injecting ALX (at a dose of 120 mg/kg body weight) intraperitoneally in sterile normal saline. Elevated levels of blood glucose, glycosylated Hb and TNF? decreased levels of plasma insulin and disturbed intra-cellular antioxidant machineries were detected in ALX exposed animals. Oral administration of DSL at a dose of 80 mg/kg body weight, however, restored these alterations in diabetic rats. Studies on the mechanism of ALX-induced diabetes showed that hyperglycemia caused disruption of mitochondrial membrane potential in the spleen, released cytochrome C in the cytosol, activated caspase 3 and ultimately led to apoptotic cell death. Results suggest that DSL possesses the ability of protecting the spleen tissue from ALX-induced hyperglycemia and thus could act as an anti-diabetic agent in lessening diabetes associated spleen dysfunction. PMID:22239106

  12. EBNA3C-Mediated Regulation of Aurora Kinase B Contributes to Epstein-Barr Virus-Induced B-Cell Proliferation through Modulation of the Activities of the Retinoblastoma Protein and Apoptotic Caspases

    PubMed Central

    Jha, Hem Chandra; Lu, Jie; Saha, Abhik; Cai, Qiliang; Banerjee, Shuvomoy; Prasad, Mahadesh A. J.

    2013-01-01

    Epstein-Barr virus (EBV) is an oncogenic gammaherpesvirus that is implicated in several human malignancies, including Burkitt's lymphoma (BL), posttransplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma (NPC), and AIDS-associated lymphomas. Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential EBV latent antigens, can induce mammalian cell cycle progression through its interaction with cell cycle regulators. Aurora kinase B (AK-B) is important for cell division, and deregulation of AK-B is associated with aneuploidy, incomplete mitotic exit, and cell death. Our present study shows that EBNA3C contributes to upregulation of AK-B transcript levels by enhancing the activity of its promoter. Further, EBNA3C also increased the stability of the AK-B protein, and the presence of EBNA3C leads to reduced ubiquitination of AK-B. Importantly, EBNA3C in association with wild-type AK-B but not with its kinase-dead mutant led to enhanced cell proliferation, and AK-B knockdown can induce nuclear blebbing and cell death. This phenomenon was rescued in the presence of EBNA3C. Knockdown of AK-B resulted in activation of caspase 3 and caspase 9, along with poly(ADP-ribose) polymerase 1 (PARP1) cleavage, which is known to be an important contributor to apoptotic signaling. Importantly, EBNA3C failed to stabilize the kinase-dead mutant of AK-B compared to wild-type AK-B, which suggests a role for the kinase domain in AK-B stabilization and downstream phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). This study demonstrates the functional relevance of AK-B kinase activity in EBNA3C-regulated B-cell proliferation and apoptosis. PMID:23986604

  13. EBNA3C-mediated regulation of aurora kinase B contributes to Epstein-Barr virus-induced B-cell proliferation through modulation of the activities of the retinoblastoma protein and apoptotic caspases.

    PubMed

    Jha, Hem Chandra; Lu, Jie; Saha, Abhik; Cai, Qiliang; Banerjee, Shuvomoy; Prasad, Mahadesh A J; Robertson, Erle S

    2013-11-01

    Epstein-Barr virus (EBV) is an oncogenic gammaherpesvirus that is implicated in several human malignancies, including Burkitt's lymphoma (BL), posttransplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma (NPC), and AIDS-associated lymphomas. Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential EBV latent antigens, can induce mammalian cell cycle progression through its interaction with cell cycle regulators. Aurora kinase B (AK-B) is important for cell division, and deregulation of AK-B is associated with aneuploidy, incomplete mitotic exit, and cell death. Our present study shows that EBNA3C contributes to upregulation of AK-B transcript levels by enhancing the activity of its promoter. Further, EBNA3C also increased the stability of the AK-B protein, and the presence of EBNA3C leads to reduced ubiquitination of AK-B. Importantly, EBNA3C in association with wild-type AK-B but not with its kinase-dead mutant led to enhanced cell proliferation, and AK-B knockdown can induce nuclear blebbing and cell death. This phenomenon was rescued in the presence of EBNA3C. Knockdown of AK-B resulted in activation of caspase 3 and caspase 9, along with poly(ADP-ribose) polymerase 1 (PARP1) cleavage, which is known to be an important contributor to apoptotic signaling. Importantly, EBNA3C failed to stabilize the kinase-dead mutant of AK-B compared to wild-type AK-B, which suggests a role for the kinase domain in AK-B stabilization and downstream phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). This study demonstrates the functional relevance of AK-B kinase activity in EBNA3C-regulated B-cell proliferation and apoptosis. PMID:23986604

  14. PDT-treated apoptotic cells induce macrophage synthesis NO

    NASA Astrophysics Data System (ADS)

    Song, S.; Xing, D.; Zhou, F. F.; Chen, W. R.

    2009-11-01

    Nitric oxide (NO) is a biologically active molecule which has multi-functional in different species. As a second messenger and neurotransmitter, NO is not only an important regulatory factor between cells' information transmission, but also an important messenger in cell-mediated immunity and cytotoxicity. On the other side, NO is involving in some diseases' pathological process. In pathological conditions, the macrophages are activated to produce a large quantity of nitric oxide synthase (iNOS), which can use L-arginine to produce an excessive amount of NO, thereby killing bacteria, viruses, parasites, fungi, tumor cells, as well as in other series of the immune process. In this paper, photofrin-based photodynamic therapy (PDT) was used to treat EMT6 mammary tumors in vitro to induce apoptotic cells, and then co-incubation both apoptotic cells and macrophages, which could activate macrophage to induce a series of cytotoxic factors, especially NO. This, in turn, utilizes macrophages to activate a cytotoxic response towards neighboring tumor cells. These results provided a new idea for us to further study the immunological mechanism involved in damaging effects of PDT, also revealed the important function of the immune effect of apoptotic cells in PDT.

  15. Clearance of apoptotic cells by phagocytes

    Microsoft Academic Search

    L-P Erwig; P M Henson

    2008-01-01

    Phagocytic clearance of apoptotic cells may be considered to consist of four distinct steps: accumulation of phagocytes at the site where apoptotic cells are located; recognition of dying cells through a number of bridge molecules and receptors; engulfment by a unique uptake process; and processing of engulfed cells within phagocytes. Here, we will discuss these individual steps that collectively are

  16. Tolerance to apoptotic cells is regulated by indoleamine 2,3-dioxygenase

    PubMed Central

    Ravishankar, Buvana; Liu, Haiyun; Shinde, Rahul; Chandler, Phillip; Baban, Babak; Tanaka, Masato; Munn, David H.; Mellor, Andrew L.; Karlsson, Mikael C. I.; McGaha, Tracy L.

    2012-01-01

    Tolerance to self-antigens present in apoptotic cells is critical to maintain immune-homeostasis and prevent systemic autoimmunity. However, mechanisms that sustain self-tolerance are poorly understood. Here we show that systemic administration of apoptotic cells to mice induced splenic expression of the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). IDO expression was confined to the splenic marginal zone and was abrogated by depletion of CD169+ cells. Pharmacologic inhibition of IDO skewed the immune response to apoptotic cells, resulting in increased proinflammatory cytokine production and increased effector T-cell responses toward apoptotic cell-associated antigens. Presymptomatic lupus-prone MRLlpr/lpr mice exhibited abnormal elevated IDO expression in the marginal zone and red pulp and inhibition of IDO markedly accelerated disease progression. Moreover, chronic exposure of IDO-deficient mice to apoptotic cells induced a lupus-like disease with serum autoreactivity to double-stranded DNA associated with renal pathology and increased mortality. Thus, IDO limits innate and adaptive immunity to apoptotic self-antigens and IDO-mediated regulation inhibits inflammatory pathology caused by systemic autoimmune disease. PMID:22355111

  17. Modulation of caspases and their non-apoptotic functions by Legionella pneumophila.

    PubMed

    Amer, Amal O

    2010-02-01

    Legionella pneumophila has become a model system to decipher the non-apoptotic functions of caspases and their role in immunity. In permissive cells, the L. pneumophila-containing vacuole evades endosomal traffic and is remodelled by the endoplasmic reticulum. Evasion of the endosomes is mediated by the Dot/Icm type IV secretion system. Upon L. pneumophila infection of genetically restrictive cells such as wild-type (WT) C57Bl/6J murine macrophages, flagellin is sensed by the NOD-like receptor Nlrc4 leading to caspase-1 activation by the inflammasome complex. Then, caspase-7 is activated downstream of the Nlrc4 inflammasome, promoting non-apoptotic functions such as L. pneumophila-containing phagosome maturation and bacterial degradation. Interestingly, caspase-3 is activated in permissive cells during early stages of infection. However, caspase-3 activation does not lead to apoptosis until late stages of infection because it is associated with potent Dot/Icm-mediated anti-apoptotic stimuli that render the infected cells resistant to external apoptotic inducers. Therefore, the role of caspase-1 and non-apoptotic functions of executioner caspases are temporally and spatially modulated during infection by L. pneumophila, which determine permissiveness to intracellular bacterial proliferation. This review will examine the novel activation pathways of caspases by L. pneumophila and discuss their role in genetic restriction and permissiveness to infection. PMID:19863553

  18. Age-related activation of mitochondrial caspase-independent apoptotic signaling in rat gastrocnemius muscle

    PubMed Central

    Marzetti, Emanuele; Wohlgemuth, Stephanie Eva; Lees, Hazel Anne; Chung, Hae-Young; Giovannini, Silvia; Leeuwenburgh, Christiaan

    2008-01-01

    Mitochondria-mediated apoptosis represents a central process driving age-related muscle loss. However, the temporal relation between mitochondrial apoptotic signaling and sarcopenia as well as the regulation of release of pro-apoptotic factors from the mitochondria has not been elucidated. In this study, we investigated mitochondrial apoptotic signaling in skeletal muscle of rats across a wide age range. We also investigated whether mitochondrial-driven apoptosis was accompanied by changes in the expression of Bcl-2 proteins and components of the mitochondrial permeability transition pore (mPTP). Analyses were performed on gastrocnemius muscle of 8-, 18-, 29- and 37- month-old male Fischer344×Brown Norway rats (9 per group). Muscle weight declined progressively with advancing age, concomitant with increased apoptotic DNA fragmentation. Cytosolic and nuclear levels of apoptosis inducing factor (AIF) and endonuclease G (EndoG) increased in old and senescent animals. In contrast, cytosolic levels of cytochrome c were unchanged with age. Mitochondrial Bcl-2, Bax and Bid increased dramatically in 37-month-old rats, with no changes in the Bax/Bcl-2 ratio in any of the age groups. Finally, expression of cyclophilin D was enhanced at very old age. Our findings indicate that the mitochondrial caspase-independent apoptotic pathway may play a more prominent role in skeletal muscle loss than caspase-mediated apoptosis. PMID:18579179

  19. Rg1 exhibits neuroprotective effects by inhibiting the endoplasmic reticulum stress?mediated c?Jun N?terminal protein kinase apoptotic pathway in a rat model of Alzheimer's disease.

    PubMed

    Mu, Jun-Shan; Lin, Hang; Ye, Jian-Xin; Lin, Min; Cui, Xiao-Ping

    2015-09-01

    The neuroprotective agents currently used to treat Alzheimer's disease (AD) often only target one aspect of the disease process. Therefore, identifying effective drug targets associated with the pathogenesis of AD is critical for the production of novel AD therapeutic strategies. The present study aimed to investigate the underlying mechanisms of the neuroprotective effects of Rg1 on a rat model of AD. A double transgenic ??amyloid (A?) precursor protein/PS1 rat model was established, which co?expressed mutations associated with AD. A? plaques and neurofibrillary tangles (NFTs) were detected by immunohistochemistry. The detection of the protein expression levels of caspase?3 and terminal deoxynucleotidyl?transferase?mediated dUTP nick end labeling (TUNEL) staining were used to determine the level of apoptosis in the brain tissue. The expression levels of the endoplasmic reticulum (ER) stress biomarker, glucose?regulated protein 78 (Grp78), and the mitochondrial apoptosis biomarkers, B?cell lymphoma 2 (Bcl?2) and Bcl?2?associated X protein (Bax), were analyzed by western blotting. Furthermore, the expression of the proteins associated with the ER stress unfolded protein response (UPR) was determined, in order to examine the levels of ER stress. The mRNA expression of downstream genes of UPR were also detected by reverse transcription?polymerase chain reaction. The protein expression levels of the apoptosis?associated phosphorylated?c?Jun N?terminal protein kinase (p?JNK), caspase?12 and cAMP response element?binding transcription factor homologous protein were determined by western blotting. The results of the present study indicated that the accumulation of NFTs and A? plaques was significantly decreased in the Rg1?treated AD rats, compared with untreated AD rats. The expression of caspase?3 and the number of TUNEL?positive cells were also significantly decreased in the Rg1?treated rats, as compared with the AD rats. Furthermore, treatment with Rg1 significantly reduced the expression of Grp78, and triggered inositol?requiring enzyme?1 (IRE?1) and phosphorylated protein kinase RNA?like ER kinase?associated ER stress. The IRE?1 UPR pathway downstream gene, tumor necrosis factor receptor?associated factor 2, was significantly decreased in rats treated with Rg1, compared with untreated AD rats. Furthermore, the activation of p?JNK was also inhibited when AD rats were treated with Rg1. In conclusion, Rg1 was shown to function as an important factor that inhibits the accumulation of NFTs and A? via inhibition of the ER stress?mediated pathway. Blocking of this pathway was triggered by the IRE?1 and TRAF2 pathway, as a result of inhibition of the expression of p?JNK. PMID:26016457

  20. PARP Inhibition Restores Extrinsic Apoptotic Sensitivity in Glioblastoma

    PubMed Central

    Karpel-Massler, Georg; Pareja, Fresia; Aimé, Pascaline; Shu, Chang; Chau, Lily; Westhoff, Mike-Andrew; Halatsch, Marc-Eric; Crary, John F.; Canoll, Peter; Siegelin, Markus D.

    2014-01-01

    Background Resistance to apoptosis is a paramount issue in the treatment of Glioblastoma (GBM). We show that targeting PARP by the small molecule inhibitors, Olaparib (AZD-2281) or PJ34, reduces proliferation and lowers the apoptotic threshold of GBM cells in vitro and in vivo. Methods The sensitizing effects of PARP inhibition on TRAIL-mediated apoptosis and potential toxicity were analyzed using viability assays and flow cytometry in established GBM cell lines, low-passage neurospheres and astrocytes in vitro. Molecular analyses included western blots and gene silencing. In vivo, effects on tumor growth were examined in a murine subcutaneous xenograft model. Results The combination treatment of PARP inhibitors and TRAIL led to an increased cell death with activation of caspases and inhibition of formation of neurospheres when compared to single-agent treatment. Mechanistically, pharmacological PARP inhibition elicited a nuclear stress response with up-regulation of down-stream DNA-stress response proteins, e.g., CCAAT enhancer binding protein (C/EBP) homology protein (CHOP). Furthermore, Olaparib and PJ34 increased protein levels of DR5 in a concentration and time-dependent manner. In turn, siRNA-mediated suppression of DR5 mitigated the effects of TRAIL/PARP inhibitor-mediated apoptosis. In addition, suppression of PARP-1 levels enhanced TRAIL-mediated apoptosis in malignant glioma cells. Treatment of human astrocytes with the combination of TRAIL/PARP inhibitors did not cause toxicity. Finally, the combination treatment of TRAIL and PJ34 significantly reduced tumor growth in vivo when compared to treatment with each agent alone. Conclusions PARP inhibition represents a promising avenue to overcome apoptotic resistance in GBM. PMID:25531448

  1. Identification of apoptotic bodies in equine semen.

    PubMed

    Caselles, A B; Miro-Moran, A; Morillo Rodriguez, A; Gallardo Bolaños, J M; Ortega-Ferrusola, C; Salido, G M; Peña, F J; Tapia, J A; Aparicio, I M

    2014-04-01

    Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and to study the relationship between the quantity of these bodies and cell concentration, the volume of ejaculate, viability and motility. Moreover, we also studied whether the presence apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa to being incubated at 37 °C or being frozen and thawed. Fresh equine semen was stained with fluorescent dyes such as M540 and Annexin-V. Active Caspase 3 was studied in fresh semen through Western blotting and immunofluorescence with a specific antibody. Sperm kinematics was assessed in fresh, incubated and thawed samples using computer-assisted semen analysis, and viability was evaluated with the LIVE/DEAD Sperm Viability Kit. Overall, our results demonstrate for the first time the presence of apoptotic bodies in equine semen. The quantity of apoptotic bodies was highly variable among stallions and was positively correlated with Caspase 3 activity in fresh samples and negatively correlated with the viability and motility of stallion spermatozoa after the cryopreservation process. PMID:24467598

  2. Honeybee venom induces calcium-dependent but caspase-independent apoptotic cell death in human melanoma A2058 cells.

    PubMed

    Tu, Wu-Chun; Wu, Chun-Chi; Hsieh, Hui-Lien; Chen, Chiu-Yuan; Hsu, Shih-Lan

    2008-08-01

    Honeybee (Apis mellifera) venom (BV) has been reported to exhibit anticancer effects, but its mode of action at the cellular and molecular levels remains largely unknown. We found that honeybee venom induced apoptosis in human melanoma A2058 cells but not in normal skin fibroblast Detroit 551 cells. The BV-induced apoptosis was accompanied by generation of reactive oxygen species and alteration of mitochondrial membrane potential transition. Treatment with antioxidants significantly attenuated BV-induced apoptosis. Although caspase-2 and -3 were slightly activated by BV, inhibitors of caspase-2 and -3 could not block BV-induced apoptosis in A2058 cells. Data from immunostaining indicated that EndoG and AIF were translocated from mitochondria to the cytosol or nucleus, suggesting that BV induces apoptosis in A2058 cells via a caspase-independent pathway. In addition, cJun N-terminal kinases (JNK) and ERK were rapidly activated after a 5 min incubation with BV, while p38 and AKT were inactivated after 30 min administration of BV. Inhibition of JNK significantly attenuated BV-triggered apoptotic death. Moreover, BV induced a rapid and marked increase in cytosolic calcium ion. Incubation of cells under calcium-free conditions effectively diminished BV-induced apoptosis. Furthermore, when the calcium-free treatment was combined with ouabain, the recovery of cellular calcium fluctuation protected A2058 cells against BV-induced apoptosis. Finally, treatment of A2058 cells with melittin, the major component of BV, resulted in similar elevation of calcium levels and cell killing effects, suggesting that melittin is the major determinant in BV-triggered cell death. These observations provide a molecular explanation for the antiproliferative properties of BV, and suggest that this agent may be useful in treating melanoma. PMID:18602939

  3. Expression of ?B-crystallin overrides the anti-apoptotic activity of XIAP

    PubMed Central

    Lee, Jee Suk; Kim, Hye Young; Jeong, Na Young; Lee, Sang Yeob; Yoon, Young Geol; Choi, Yung Hyun; Yan, Chunlan; Chu, In-Sun; Koh, Hyungjong; Park, Hwan Tae; Yoo, Young Hyun

    2012-01-01

    Although crystallins are major structural proteins in the lens, ?-crystallins perform non-lens functions, and ?B-crystallin has been shown to act as an anti-apoptotic mediator in various cells. The present study was undertaken to examine whether ?B-crystallin expressed in human malignant glioma cells exerts anti-apoptotic acitivity. In addition, we sought to elucidate the mechanism underlying any observed anti-apoptotic function of ?B-crystallin in these cells. Three glioma cell lines, U373MG, U118MG, and T98G, were used. We observed that only the U373MG cell line expresses ?B-crystallin, whereas the other 2 glioma cell lines, U118MG and T98G, demonstrated no endogenous expression of ?B-crystallin. We next observed that the silencing of ?B-crystallin sensitized U373MG cells to suberoylanilide hydroxamic acid (SAHA)–induced apoptosis and that ?B-crystallin associates with caspase-3 and XIAP. Because XIAP is the most potent suppressor of mammalian apoptosis through the direct binding with caspases, we assessed whether XIAP also plays an anti-apoptotic role in SAHA-induced apoptosis in ?B-crystallin-expressing U373MG cells. Of note, the silencing of XIAP did not alter the amount of cell death induced by SAHA, indicating that XIAP does not exert an anti-apoptotic activity in U373MG cells. We then determined whether the ectopic expression of ?B-crystallin in glioma cells caused a loss of the anti-apoptotic activity of XIAP. Accordingly, we established 2 ?B-crystallin over-expressing glioma cell lines, U118MG and T98G, and found that the silencing of XIAP did not sensitize these cells to SAHA-induced apoptosis. These findings suggest that ?B-crystallin expressed in glioma cells overrides the anti-apoptotic activity exerted by XIAP. PMID:23074197

  4. 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

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

    PubMed

    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 H?O?. 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 ?-catenin was detected by immunofluorescense staining; Ratio of cell apoptosis was evaluated by Annexin-V/PI double stain assay; Mitochondrial membrane potential (??m) 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. H?O? 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

  6. IRAS is an anti-apoptotic protein.

    PubMed

    Dontenwill, Monique; Piletz, John E; Chen, Michael; Baldwin, James; Pascal, Geraldine; Ronde, Philippe; Dupuy, Laurence; Greney, Hugues; Takeda, Ken; Bousquetd, Pascal

    2003-12-01

    Active cell death, also known as apoptosis, has been implicated in the pathophysiology of diseases such as cancer, heart failure and neurodegenerative disorders. We report the anti-apoptotic function of IRAS, which was previously shown to bind imidazoline ligands. The amino acid sequence of human IRAS (hIRAS) is unrelated to known proteins, except for rat IRAS and a mouse homologue named nischarin, which binds the alpha5 integrin subunit of the fibronectin receptor. When stably transfected into PC12 cells, hIRAS localizes to the cytosol as a 167 kDa immunoreactive protein. Clonal PC12 cell lines expressing hIRAS displayed normal serum growth responses. However, hIRAS expression led to prolonged cell survival against known apoptotic stimuli: serum starvation or thapsigargin or staurosporine treatments. The apoptotic population of hIRAS-expressing cells was significantly reduced, and this protection was achieved by a decrease in caspase-3 activity, phosphatidylserine translocation, and nuclear fragmentation. Similar protective effect was obtained in COS7 cells transiently transfected with hIRAS. A partial activation of the PI3 kinase pathway is possibly implicated in the anti-apoptotic effect of IRAS. Thus, IRAS appears to represent a previously unknown anti-apoptotic protein involved in the regulation of cell survival. PMID:15028619

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

    SciTech Connect

    Golding, Jon P. [Department of Biological Sciences, Open University, Walton Hall, Milton Keynes, MK7 6AA (United Kingdom)]. E-mail: j.p.golding@open.ac.uk; Calderbank, Emma [Muscle Cell Biology Group, Medical Research Council Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN (United Kingdom); Partridge, Terence A. [Muscle Cell Biology Group, Medical Research Council Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN (United Kingdom); Beauchamp, Jonathan R. [Muscle Cell Biology Group, Medical Research Council Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN (United Kingdom)

    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.

  8. Fetal bovine serum requirement for pyrrolidine dithiocarbamate-induced apoptotic cell death of MCF7 breast tumor cells

    Microsoft Academic Search

    Da Hee Oh; Jun Soo Bang; Hyun Mi Choi; Hyung-In Yang; Myung Chul Yoo; Kyoung Soo Kim

    2010-01-01

    Pyrrolidine dithiocarbamate (PDTC) can form a complex with metal ions and then act as a proteasome inhibitor, which leads to tumor cell apoptosis, and could therefore be developed as an anticancer agent. In our efforts to find factors that induce PDTC-mediated apoptosis of tumor cells, the effect of serum concentration on the apoptotic activity of PDTC was investigated. PDTC could

  9. Apoptotic response of malignant rhabdoid tumor cells

    Microsoft Academic Search

    Silvano Nocentini

    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

  10. 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

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

    PubMed Central

    Oropesa-Ávila, M; Fernández-Vega, A; de la Mata, M; Maraver, J G; Cordero, M D; Cotán, D; de Miguel, M; Calero, C P; Paz, M V; Pavón, A D; Sánchez, M A; Zaderenko, A P; Ybot-González, P; Sánchez-Alcázar, 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

  12. Complement-dependent Clearance of Apoptotic Cells by Human Macrophages

    Microsoft Academic Search

    Dror Mevorach; John O. Mascarenhas; Debra Gershov; Keith B. Elkon

    1998-01-01

    Summary Apoptotic cells are rapidly engulfed by phagocytes, but the receptors and ligands responsible for this phenomenon are incompletely characterized. Previously described receptors on blood- derived macrophages have been characterized in the absence of serum and show a relatively low uptake of apoptotic cells. Addition of serum to the phagocytosis assays increased the uptake of apoptotic cells by more than

  13. Identification of a factor that links apoptotic cells to phagocytes

    Microsoft Academic Search

    Rikinari Hanayama; Masato Tanaka; Keiko Miwa; Azusa Shinohara; Akihiro Iwamatsu; Shigekazu Nagata

    2002-01-01

    Apoptotic cells are rapidly engulfed by phagocytes to prevent the release of potentially noxious or immunogenic intracellular materials from the dying cells, thereby preserving the integrity and function of the surrounding tissue. Phagocytes engulf apoptotic but not healthy cells, indicating that the apoptotic cells present a signal to the phagocytes, and the phagocytes recognize the signal using a specific receptor.

  14. Modulation of Apoptotic Pathways of Macrophages by Surface-Functionalized Multi-Walled Carbon Nanotubes

    PubMed Central

    Jiang, Yuanqin; Zhang, Honggang; Wang, Yange; Chen, Min; Ye, Shefang; Hou, Zhenqing; Ren, Lei

    2013-01-01

    Biomedical applications of carbon nanotubes (CNTs) often involve improving their hydrophilicity and dispersion in biological media by modifying them through noncovalent or covalent functionalization. However, the potential adverse effects of surface-functionalized CNTs have not been well characterized. In this study, we functionalized multi-walled CNTs (MWCNTs) via carboxylation, to produce MWCNTs-COOH, and via poly (ethylene glycol) linking, to produce MWCNTs-PEG. We used these functionalized MWCNTs to study the effect of surface functionalization on MWCNTs-induced toxicity to macrophages, and elucidate the underlying mechanisms of action. Our results revealed that MWCNTs-PEG were less cytotoxic and were associated with less apoptotic cell death of macrophages than MWCNTs-COOH. Additionally, MWCNTs-PEG induced less generation of reactive oxygen species (ROS) involving less activation of NADPH oxidase compared with MWCNTs-COOH, as evidenced by membrane translocation of p47phox and p67phox in macrophages. The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-?B. These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways. Our study thus provides new insights into the molecular basis for the surface properties affecting CNTs toxicity. PMID:23755279

  15. Pre-Transplant IgG Reactivity to Apoptotic Cells Correlates with Late kidney Allograft Loss

    PubMed Central

    Gao, Baoshan; Moore, Carolina; Porcheray, Fabrice; Rong, Chunshu; Abidoglu, Cem; DeVito, Julie; Paine, Rosemary; Girouard, Timothy C.; Saidman, Susan L.; Schoenfeld, David; Levin, Bruce; Wong, Waichi; Elias, Nahel; Schuetz, Christian; Rosales, Ivy A.; Fu, Yaowen; Zorn, Emmanuel

    2014-01-01

    Pre-existing serum antibodies have long been associated with graft loss in transplant candidates. While most studies have focused on HLA-specific antibodies, the contribution of non-HLA-reactive antibodies has been largely overlooked. We have recently characterized monoclonal antibodies secreted by B cell clones derived from kidney allograft recipients with rejection that selectively bind to apoptotic cells. Here, we assessed the presence of such antibodies in pre-transplant serum from 300 kidney transplant recipients and examined their contribution to the graft outcomes. Kaplan-Meier survival analysis revealed that patients with high pre-transplant IgG reactivity to apoptotic cells had a significantly increased rate of late graft loss. The effect was only apparent after approximately 1 year post-transplant. Moreover, the association between pre-transplant IgG reactivity to apoptotic cells and graft loss was still significant after excluding patients with high reactivity to HLA. This reactivity was almost exclusively mediated by IgG1 and IgG3 with complement fixing and activating properties. Overall, our findings support the view that IgG reactivity to apoptotic cells contribute to pre-sensitization. Taking these antibodies into consideration alongside anti-HLA antibodies during candidate evaluation would likely improve the transplant risk assessment. PMID:24935695

  16. Pregnenolone, a cholesterol metabolite, induces glioma cell apoptosis via activating extrinsic and intrinsic apoptotic pathways

    PubMed Central

    XIAO, XIAO; CHEN, LIJUN; OUYANG, YING; ZHU, WENBO; QIU, PENGXIN; SU, XINWEN; DOU, YUNLING; TANG, LIPENG; YAN, MIN; ZHANG, HAIPENG; YANG, XIAOXIAO; XU, DONG; YAN, GUANGMEI

    2014-01-01

    Gliomas are one of the most common types of malignant tumors worldwide, however, an effective therapeutic strategy not yet been fully determined. The present study aimed to investigate the anti-glioma activity and underlying mechanisms of pregnenolone, which originates from cholesterol and is metabolized into important steroid hormones in the body. The results demonstrated that 100 ?M pregnenolone induced a significant loss of cell viability in various malignant glioma cell lines. In the U-87 MG, LN-18 and C6 cell lines, the loss of cell viability resulted from cell apoptosis, which was evidenced by apoptotic nuclear morphology changes and caspase 3 activation. Moreover, the increased activities of caspase 8 and 9 strongly indicated that pregnenolone activated the extrinsic and intrinsic pathways of apoptosis. Additionally, glioma cell apoptosis was prevented by the general caspase inhibitor, Z-VAD-FMK. In the C6 cells, upregulation of Fas and Fas ligand triggered the activation of the extrinsic pathway, whereas knockdown of Fas significantly abrogated the cell apoptosis that was induced by pregnenolone. Furthermore, downregulation of the anti-apoptotic protein, B-cell lymphoma 2 and upregulation of pro-apoptotic proteins, such as Bax and Bak, activated the intrinsic pathway. In conclusion, pregnenolone induced glioma cell apoptosis in a caspase-dependent manner, which was mediated by activation of the extrinsic and intrinsic apoptotic pathways. PMID:25013479

  17. Apoptotic Membrane Blebbing Is Regulated by Myosin Light Chain Phosphorylation

    PubMed Central

    Mills, Jason C.; Stone, Nicole L.; Erhardt, Joseph; Pittman, Randall N.

    1998-01-01

    The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis. PMID:9456322

  18. Gene polymorphisms, apoptotic capacity and cancer risk

    Microsoft Academic Search

    Evgeny N. Imyanitov

    2009-01-01

    Programmed cell death has been implicated in various aspects of cancer development. Apoptotic capacity is a subject of significant\\u000a interindividual variations, which are largely attributed to hereditary traits. Single nucleotide polymorphisms (SNPs) located\\u000a within cell death genes may influence cancer risk in various ways. Low activity of apoptosis may favor cancer development\\u000a because of the failure to eliminate cellular clones

  19. Current Understanding of the Mechanisms for Clearance of Apoptotic Cells—A Fine Balance

    PubMed Central

    Hawkins, Lois A; Devitt, Andrew

    2013-01-01

    Apoptosis is an important cell death mechanism by which multicellular organisms remove unwanted cells. It culminates in a rapid, controlled removal of cell corpses by neighboring or recruited viable cells. Whilst many of the molecular mechanisms that mediate corpse clearance are components of the innate immune system, clearance of apoptotic cells is an anti-inflammatory process. Control of cell death is dependent on competing pro-apoptotic and anti-apoptotic signals. Evidence now suggests a similar balance of competing signals is central to the effective removal of cells, through so called ‘eat me’ and ‘don’t eat me’ signals. Competing signals are also important for the controlled recruitment of phagocytes to sites of cell death. Consequently recruitment of phagocytes to and from sites of cell death can underlie the resolution or inappropriate propagation of cell death and inflammation. This article highlights our understanding of mechanisms mediating clearance of dying cells and discusses those mechanisms controlling phagocyte migration and how inappropriate control may promote important pathologies. PMID:25278779

  20. MicroRNAs control the apoptotic threshold in primed pluripotent stem cells through regulation of BIM

    PubMed Central

    Pernaute, Barbara; Spruce, Thomas; Smith, Kimberley M.; Sánchez-Nieto, Juan Miguel; Manzanares, Miguel; Cobb, Bradley

    2014-01-01

    Mammalian primed pluripotent stem cells have been shown to be highly susceptible to cell death stimuli due to their low apoptotic threshold, but how this threshold is regulated remains largely unknown. Here we identify microRNA (miRNA)-mediated regulation as a key mechanism controlling apoptosis in the post-implantation epiblast. Moreover, we found that three miRNA families, miR-20, miR-92, and miR-302, control the mitochondrial apoptotic machinery by fine-tuning the levels of expression of the proapoptotic protein BIM. These families therefore represent an essential buffer needed to maintain cell survival in stem cells that are primed for not only differentiation but also cell death. PMID:25184675

  1. Mangiferin Has an Additive Effect on the Apoptotic Properties of Hesperidin in Cyclopia sp. Tea Extracts

    PubMed Central

    Bartoszewski, Rafal; Hering, Anna; Marsza??, Marcin; Stefanowicz Hajduk, Justyna; Bartoszewska, Sylwia; Kapoor, Niren; Kochan, Kinga; Ochocka, Renata

    2014-01-01

    A variety of biological pro-health activities have been reported for mangiferin and hesperidin, two major phenolic compounds of Honeybush (Cyclopia sp.) tea extracts. Given their increasing popularity, there is a need for understanding the mechanisms underlying the biological effects of these compounds. In this study, we used real-time cytotoxicity cellular analysis of the Cyclopia sp. extracts on HeLa cells and found that the higher hesperidin content in non-fermented "green" extracts correlated with their higher cytotoxicity compared to the fermented extracts. We also found that mangiferin had a modulatory effect on the apoptotic effects of hesperidin. Quantitative PCR analysis of hesperidin-induced changes in apoptotic gene expression profile indicated that two death receptor pathway members, TRADD and TRAMP, were up regulated. The results of this study suggest that hesperidin mediates apoptosis in HeLa cells through extrinsic pathway for programmed cell death. PMID:24633329

  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 [Department of Pathology, College of Medicine, Catholic University of Daegu, Daegu (Korea, Republic of); Park, Yoon-Yub [Department of Physiology, College of Medicine, Catholic University of Daegu, Daegu (Korea, Republic of); Han, Sang-Mi [Department of Agricultural Biology, National Institute of Agricultural Science and Technology, Suwon (Korea, Republic of); Park, Kwan-kyu, E-mail: kkpark@cu.ac.kr [Department of Pathology, College of Medicine, Catholic University of Daegu, Daegu (Korea, Republic of)

    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. Leptin Is an Anti-Apoptotic Effector in Placental Cells Involving p53 Downregulation

    PubMed Central

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

  4. Effect of Transient Maternal Hypotension on Apoptotic Cell Death in Foetal Rat Brain

    PubMed Central

    Özyürek, Hamit; Bayrak, Sibel; Pehlivano?lu, Bilge; Atilla, Pergin; Balkanc?, Zeynep Dicle; Çakar, Nur; Anlar, Banu

    2014-01-01

    Background: Intrauterine perfusion insufficiency induced by transient maternal hypotension has been reported to be associated with foetal brain malformations. However, the effects of maternal hypotension on apoptotic processes in the foetal brain have not been investigated experimentally during the intrauterine period. Aims: The aim of this study was to investigate the effects of transient maternal hypotension on apoptotic cell death in the intrauterine foetal brain. Study Design: Animal experimentation. Methods: Three-month-old female Wistar albino rats were allocated into four groups (n=5 each). The impact of hypoxic/ischemic injury induced by transient maternal hypotension on the 15th day of pregnancy (late gestation) in rats was investigated at 48 (H17 group) or 96 hours (H19 group) after the insult. Control groups underwent the same procedure except for induction of hypotension (C17 and H17 groups). Brain sections of one randomly selected foetus from each pregnant rat were histopathologically evaluated for hypoxic/ischemic injury in the metencephalon, diencephalon, and telencephalon by terminal transferase-mediated dUTP nick end labelling and active cysteine-dependent aspartate-directed protease-3 (caspase-3) positivity for cell death. Results: The number of terminal transferase-mediated dUTP nick end labelling (+) cells in all the areas examined was comparable in both hypotension and control groups. The H17 group had active caspase-3 (+) cells in the metencephalon and telencephalon, sparing diencephalon, whereas the C19 and H19 groups had active caspase-3 (+) cells in all three regions. The number of active caspase-3 (+) cells in the telencephalon in the H19 group was higher compared with the metencephalon and diencephalon and compared with H17 group (p<0.05). Conclusion: Our results suggest that prenatal hypoxic/ischemic injury triggers apoptotic mechanisms. Therefore, blockade of apoptotic pathways, considering the time pattern of the insult, may constitute a potential neuroprotective approach for the detrimental effects of prenatal hypoperfusion. PMID:25207175

  5. Cell membrane oxidative damage induced by gamma-radiation and apoptotic sensitivity.

    PubMed

    Mishra, Kaushala P

    2004-01-01

    Ionizing radiation-generated reactive oxygen species (ROS) resulting in oxidative damage to the cell membrane and its consequent role in the mechanism of apoptotic cell death have been receiving growing attention in cellular radiobiology. In recent years, evidence has accumulated to suggest that it is the damage to the cell membrane that contributes to the radiation cell killing. It has been demonstrated that degradation of membrane-bound sphingomyelinase (SMase) after irradiation of bovine endothelial cell produces ceramide, which initiates an apoptotic cascade, suggesting membrane-triggered events in the mechanism of cellular apoptosis. Fluorescence and electron spin resonance (ESR) studies from gamma-irradiation ofliposomal vesicles have shown that radiation-mediated lipid damage was modified by the inclusion of structure-modulating agents (e.g., cholesterol) and antioxidants (e.g., tocopherol, eugenol). The magnitude of the modification of the damage was found to be dependent on the concentration of these modifiers. Moreover, experiments on dipalmitoyl phosphatidyl choline (DPPC) unilamellar liposomes demonstrated a biphasic behavior of radiation damage, which was remarkably modified by ascorbic acid and alpha-tocopherol in a concentration-dependent fashion. The comparison of their protective effects showed that ascorbic acid was less effective than tocopherol against radiation damage to liposomes. Studies on irradiated mouse thymocytes employing FDA fluorescence probe have suggested post-irradiation time- and dose-dependent changes in membrane permeability. The determination of induction of apoptosis in irradiated thymocytes showed a time-dependent DNA fragmentation, suggesting that radiation-induced permeability changes and occurrence of apoptotic death in thymocytes were closely correlated. These results are discussed, with an emphasis on membrane-damage-mediated apoptotic death with relevance to improvement of cancer radiotherapy. PMID:14994996

  6. Protective effects of melittin on transforming growth factor-?1 injury to hepatocytes via anti-apoptotic mechanism.

    PubMed

    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)-?1-induced apoptosis in hepatocytes. TGF-?1-treated hepatocytes were exposed to low doses (0.5 and 1 ?g/mL) and high dose (2 ?g/mL) of melittin. The low doses significantly protected these cells from DNA damage in TGF-?1-induced apoptosis compared to the high dose. Also, melittin suppressed TGF-?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-?1 induces hepatocyte apoptosis and that an optimal dose of melittin exerts anti-apoptotic effects against TGF-?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-?1-mediated injury. PMID:21871910

  7. Apoptotic cell clearance: basic biology and therapeutic potential

    PubMed Central

    Poon, Ivan K. H.; Lucas, Christopher D.

    2014-01-01

    Prompt removal of apoptotic cells by phagocytes is important for maintaining tissue homeostasis. The molecular and cellular events that underpin apoptotic cell recognition and uptake, and the subsequent biological responses are increasingly better defined. The detection and disposal of apoptotic cells generally promote an anti-inflammatory response at the tissue level, as well as immunological tolerance. Consequently, defects in apoptotic cell clearance have been linked with a variety of inflammatory diseases and autoimmunity. Conversely, under certain conditions such as killing tumour cells by specific cell death inducers, the recognition of apoptotic tumour cells can promote an immunogenic response and anti-tumour immunity. Here, we review the current understanding of the complex process of apoptotic cell clearance in physiology and pathology, and discuss how this knowledge could be harnessed for new therapeutic strategies. PMID:24481336

  8. Neuroprotective Effect of Resveratrol Against Methamphetamine-Induced Dopaminergic Apoptotic Cell Death in a Cell Culture Model of Neurotoxicity

    PubMed Central

    Kanthasamy, Kavin; Gordon, Richard; Jin, Huajun; Anantharam, Vellareddy; Ali, Syed; Kanthasamy, Anumantha G; Kanthasamy, Arthi

    2011-01-01

    A growing body of evidence suggests that oxidative stress-mediated cell death signaling mechanisms may exert neurotoxic effects of methamphetamine (MA)-induced dopaminergic neuronal loss. However, the means by which oxidative stress induced by MA causes neurodegeneration remains unclear. In recent years, resveratrol has garnered considerable attention owing to its antioxidant, anti-inflammatory, anti-aging, and neuroprotective properties. In the present study, we sought to investigate the neuroprotective effects of resveratrol against apoptotic cell death in a mesencephalic dopaminergic neuronal cell culture model of MA neurotoxicity. MA treatment in the N27 dopaminergic neuronal cell model produced a time-dependent activation of the apoptotic cascade involving caspase-3 and DNA fragmentation. We found that the caspase-3 activation preceded DNA fragmentation. Notably, treatment with resveratrol almost completely attenuated MA-induced caspase-3 activity, but only partially reduced apoptotic cell death. We conclude that the neuroprotective effect of resveratrol is at least in part mediated by suppression of caspase-3 dependent cell death pathways. Collectively, our results demonstrate that resveratrol can attenuate MA-induced apoptotic cell death and suggest that resveratrol or its analogs may have therapeutic benefits in mitigating MA-induced dopaminergic neurodegeneration. PMID:21886561

  9. Synergistic apoptotic response between valproic acid and fludarabine in chronic lymphocytic leukaemia (CLL) cells involves the lysosomal protease cathepsin B

    PubMed Central

    Yoon, J-Y; Szwajcer, D; Ishdorj, G; Benjaminson, P; Xiao, W; Kumar, R; Johnston, J B; Gibson, S B

    2013-01-01

    Fludarabine, a nucleoside analogue, is commonly used in combination with other agents for the treatment of chronic lymphocytic leukaemia (CLL). In previous studies, valproic acid (VPA), an inhibitor of histone deacetylases, combined with fludarabine to synergistically increase apoptotic cell death in CLL cells. In the present study, we found that the combination of fludarabine and VPA decreases the level of the anti-apoptotic proteins Mcl-1 and XIAP in primary CLL cells. Treatment with fludarabine alone, or in combination with VPA, led to the loss of lysosome integrity, and chemical inhibition of the lysosomal protease cathepsin B, using CA074-Me, was sufficient to reduce apoptosis. VPA treatment increased cathepsin B levels and activities in primary CLL cells, thereby priming CLL cells for lysosome-mediated cell death. Six previously treated patients with relapsed CLL were treated with VPA, followed by VPA/fludarabine combination. The combined therapy resulted in reduced lymphocyte count in five out of six and reduced lymph node sizes in four out of six patients. In vivo VPA treatment increased histone-3 acetylation and cathepsin B expression levels. Thus, the synergistic apoptotic response with VPA and fludarabine in CLL is mediated by cathepsin B activation leading to a decrease in the anti-apoptotic proteins. PMID:24141622

  10. The anti-apoptotic activity of melatonin in neurodegenerative diseases

    PubMed Central

    Wang, Xin

    2009-01-01

    Melatonin plays a neuroprotective role in models of neurodegenerative diseases. However, the molecular mechanisms underlying neuroprotection by melatonin are not well-understood. Apoptotic cell death in the central nervous system is a feature of neurodegenerative diseases. The intrinsic and extrinsic apoptotic pathways and the anti-apoptotic survival signal pathways play critical roles in neurodegeneration. This review summarizes the reports to date showing inhibition by melatonin of the intrinsic apoptotic pathways in neurodegenerative diseases including stroke, Alzheimer’s disease, Parkinson's disease, Huntington’s disease, and Amyotrophic Lateral Sclerosis. Furthermore, the activation of survival signal pathways by melatonin in neurodegenerative diseases is discussed. PMID:19818070

  11. 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

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

    SciTech Connect

    Zeilstra, Jurrit; Joosten, Sander P.J. [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands)] [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Wensveen, Felix M. [Department of Experimental Immunology, Academic Medical Center, Amsterdam (Netherlands)] [Department of Experimental Immunology, Academic Medical Center, Amsterdam (Netherlands); Dessing, Mark C.; Schuetze, Denise M. [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands)] [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Eldering, Eric [Department of Experimental Immunology, Academic Medical Center, Amsterdam (Netherlands)] [Department of Experimental Immunology, Academic Medical Center, Amsterdam (Netherlands); Spaargaren, Marcel [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands)] [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Pals, Steven T., E-mail: s.t.pals@amc.uva.nl [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands)

    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.

  13. Apoptotic effect of red wine polyphenols on human colon cancer SNU-C4 cells

    Microsoft Academic Search

    M.-J. Kim; Y.-J. Kim; H.-J. Park; J.-H. Chung; K.-H. Leem; H.-K. Kim

    2006-01-01

    Polyphenols in fruits, soybean, vegetables, herbs, roots and leaves act as bioactive components related with prevention of cancer, heart diseases and diabetes. We investigated the apoptotic effects of polyphenols from red wine on human colon cancer cells SNU-C4 using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, reverse transcription-polymerase chain reaction

  14. The Arf GAP CNT-2 regulates the apoptotic fate in C. elegans asymmetric neuroblast divisions.

    PubMed

    Singhvi, Aakanksha; Teuliere, Jerome; Talavera, Karla; Cordes, Shaun; Ou, Guangshuo; Vale, Ronald D; Prasad, Brinda C; Clark, Scott G; Garriga, Gian

    2011-06-01

    During development, all cells make the decision to live or die. Although 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 GTPase-activating protein (GAP) of the AGAP family, as a novel regulator of this type of neuroblast division. Loss of CNT-2 alters daughter cell size and causes the apoptotic cell to adopt the fate of its sister cell, resulting in extra neurons. CNT-2's Arf GAP activity is 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

  15. 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

  16. 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

  17. Hypochlorite-modified low-density lipoprotein induces the apoptotic machinery in Jurkat T-cell lines

    Microsoft Academic Search

    Ulrike Resch; Michaela Semlitsch; Astrid Hammer; Heidrun Susani-Etzerodt; Henning Walczak; Wolfgang Sattler; Ernst Malle

    2011-01-01

    Myeloperoxidase is abundantly present in inflammatory diseases where activation of monocytes\\/macrophages and T-cell-mediated immune response occurs. The potent oxidant hypochlorous acid (HOCl), generated by the myeloperoxidase–H2O2–chloride system of activated phagocytes, converts low-density lipoprotein (LDL) into a proinflammatory lipoprotein particle. Here, we investigated the apoptotic effect of HOCl–LDL, an in vivo occurring LDL modification, on human T-cell lymphoblast-like Jurkat cells. Experiments

  18. Apoptotic mechanism of MCF7 breast cells in vivo and in vitro induced by photodynamic therapy with C-phycocyanin

    Microsoft Academic Search

    Bing Li; Xianming Chu; Meihua Gao; Wuxiu Li

    2010-01-01

    The aim of this study was to investigate the pro-apoptotic mechanism of C-phycocyanin (C-PC)-mediated photody- namic therapy (PDT) in a murine tumor model and cul- tured MCF-7 cells. The mice were divided into four groups: control, He - Ne laser radiation, C-PC treatment, and C-PC treatment 1 He - Ne laser radiation. The effects of C-PC and\\/or laser on immune

  19. Macrophage Recognition of ICAM-3 on Apoptotic Leukocytes1

    Microsoft Academic Search

    Odette D. Moffatt; Andrew Devitt; Elaine D. Bell; David L. Simmons; Christopher D. Gregory

    Cells undergoing apoptosis are cleared rapidly by phagocytes, thus preventing tissue damage caused by loss of plasma membrane integrity. In this study, we show that the surface of leukocytes is altered during apoptosis such that the first Ig-like domain of ICAM-3 (CD50) can participate in the recognition and phagocytosis of the apoptotic cells by macrophages. Macrophage recog- nition of apoptotic

  20. Exposure of Phosphatidylethanolamine on the Surface of Apoptotic Cells

    Microsoft Academic Search

    Kazuo Emoto; Noriko Toyama-Sorimachi; Hajime Karasuyama; Keizo Inoue; Masato Umeda

    1997-01-01

    In the early stages of apoptosis, phosphatidylserine (PS) is translocated from the inner side of the plasma membrane to the outer layer, which allows phagocytes to recognize and engulf the apoptotic cells. In this study we have analyzed the cell surface exposure of phosphatidylethanolamine (PE) in apoptotic CTLL-2 cells, a cytotoxic T cell line, using a tetracyclic polypeptide of 19

  1. 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

  2. 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...

  3. Characterization of the Effects of Cross-Linking of Macrophage CD44 Associated with Increased Phagocytosis of Apoptotic PMN

    PubMed Central

    Hart, Simon P.; Rossi, Adriano G.; Haslett, Christopher; Dransfield, Ian

    2012-01-01

    Control of macrophage capacity for apoptotic cell clearance by soluble mediators such as cytokines, prostaglandins and lipoxins, serum proteins, and glucocorticoids may critically determine the rate at which inflammation resolves. Previous studies suggested that macrophage capacity for clearance of apoptotic neutrophils was profoundly altered following binding of CD44 antibodies. We have used a number of different approaches to further define the mechanism by which CD44 rapidly and specifically augment phagocytosis of apoptotic neutrophils. Use of Fab' fragments unequivocally demonstrated a requirement for cross-linking of macrophage surface CD44. The molecular mechanism of CD44-augmented phagocytosis was shown to be opsonin-independent and to be distinct from the Mer/protein S pathway induced by glucocorticoids and was not functional for clearance of apoptotic eosinophils. CD44-cross-linking also altered macrophage migration and induced cytoskeletal re-organisation together with phosphorylation of paxillin and activation of Rac2. Investigation of signal transduction pathways that might be critical for CD44 augmentation of phagocytosis revealed that Ca2+ signalling, PI-3 kinase pathways and altered cAMP signalling were not involved, but did implicate a key role for tyrosine phosphorylation events. Finally, although CD44 antibodies were able to augment phagocytosis of apoptotic neutrophils by murine peritoneal and bone marrow-derived macrophages, we did not observe a difference in the clearance of neutrophils following induction of peritonitis with thioglycollate in CD44-deficient animals. Together, these data demonstrate that CD44 cross-linking induces a serum opsonin-independent mechanism of macrophage phagocytosis of apoptotic neutrophils that is associated with reduced macrophage migration and cytoskeletal reorganisation. PMID:22427969

  4. Identification of MMP-15 as an anti-apoptotic factor in cancer cells.

    PubMed

    Abraham, Reimar; Schäfer, Juliane; Rothe, Mike; Bange, Johannes; Knyazev, Pjotr; Ullrich, Axel

    2005-10-01

    We have performed an in vitro selection for an anti-apoptotic phenotype that resembles the selection process that pre-malignant cells undergo in the initial phase of carcinogenesis in vivo. Using the cervical carcinoma cell line HeLa S3 as a model system, the selection procedure yielded cell clones that displayed increased resistance to apoptosis induced by Fas, tumor necrosis factor-related apoptosis-inducing ligand, and serum starvation. Gene expression profiling using gene family focused cDNA arrays revealed numerous genes that are differentially expressed in HeLa S3 and the resistant subclones and therefore are potentially involved in the definition of sensitivity to apoptotic stimuli. From the genes identified in this functional genomics approach we validated the anti-apoptotic activity of the membrane-anchored matrix metalloproteinase 15 (MMP-15) by means of small interfering RNA-mediated knock-down and ectopic expression in parental HeLa S3 cells and, to confirm a more general significance of our findings, in other cancer cell lines. The in vivo relevance of these findings is supported by the overexpression of MMP-15 in human lung adenocarcinoma compared with normal lung. Because MMP-15 is known to promote invasion, our results suggest that this protease connects metastasis and apoptosis resistance by an unknown regulatory mechanism. Our findings therefore strongly suggest that cancer characteristics such as metastatic potential, which are thought to evolve late in cancer progression, could be manifested early on by selection for an anti-apoptotic phenotype. PMID:16093241

  5. C1q and Mannose Binding Lectin Engagement of Cell Surface Calreticulin and Cd91 Initiates Macropinocytosis and Uptake of Apoptotic Cells

    PubMed Central

    Ogden, Carol Anne; deCathelineau, Aimee; Hoffmann, Peter R.; Bratton, Donna; Ghebrehiwet, Berhane; Fadok, Valerie A.; Henson, Peter M.

    2001-01-01

    Removal of apoptotic cells is essential for maintenance of tissue homeostasis, organogenesis, remodeling, development, and maintenance of the immune system, protection against neoplasia, and resolution of inflammation. The mechanisms of this removal involve recognition of the apoptotic cell surface and initiation of phagocytic uptake into a variety of cell types. Here we provide evidence that C1q and mannose binding lectin (MBL), a member of the collectin family of proteins, bind to apoptotic cells and stimulate ingestion of these by ligation on the phagocyte surface of the multifunctional protein, calreticulin (also known as the cC1qR), which in turn is bound to the endocytic receptor protein CD91, also known as the ?-2-macroglobulin receptor. Use of these proteins provides another example of apoptotic cell clearance mediated by pattern recognition molecules of the innate immune system. Ingestion of the apoptotic cells through calreticulin/CD91 stimulation is further shown to involve the process of macropinocytosis, implicated as a primitive and relatively nonselective uptake mechanism for C1q- and MBL-enhanced engulfment of whole, intact apoptotic cells, as well as cell debris and foreign organisms to which these molecules may bind. PMID:11560994

  6. Fetal bovine serum requirement for pyrrolidine dithiocarbamate-induced apoptotic cell death of MCF-7 breast tumor cells.

    PubMed

    Oh, Da Hee; Bang, Jun Soo; Choi, Hyun Mi; Yang, Hyung-In; Yoo, Myung Chul; Kim, Kyoung Soo

    2010-12-15

    Pyrrolidine dithiocarbamate (PDTC) can form a complex with metal ions and then act as a proteasome inhibitor, which leads to tumor cell apoptosis, and could therefore be developed as an anticancer agent. In our efforts to find factors that induce PDTC-mediated apoptosis of tumor cells, the effect of serum concentration on the apoptotic activity of PDTC was investigated. PDTC could not induce MCF-7 breast tumor cell death in serum-free media but significantly induced cell death in a dose-dependent manner at concentrations of ?25 ?M in media containing 10% fetal bovine serum. PDTC-mediated cell death was also dependent on serum concentration. PDTC-mediated cell death occurred through apoptosis. Similar to that in normal FBS, PDTC-mediated apoptotic cell death was also induced in media containing dialyzed FBS, indicating that PDTC-mediated apoptosis does not require metal ions or salts, but rather proteins in fetal bovine serum. In addition, differential apoptotic effects of PDTC were not observed with inhibitors of NF-?B activation such as N-acetylcysteine (NAC), Fenofibrate and carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal (MG132) or with the metal-binding agent, 5-chloro-7-iodo-8-hydroxyquinoline (Clioquinol). These results indicate that serum is required for PDTC-mediated apoptosis and that zinc-binding compounds such as PDTC, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and Clioquinol may each have their own mechanisms by which they induce tumor cell death, even though they are all classified as zinc-binding compounds. PMID:20868670

  7. Human RIF1 encodes an anti-apoptotic factor required for DNA repair

    PubMed Central

    Wang, Haibo; Zhao, Ailian; Chen, Lin; Zhong, Xueyan; Liao, Ji; Gao, Min; Cai, Minghua; Lee, Dong-Hyun; Li, Jing; Chowdhury, Dipanjan; Yang, Yun-gui; Pfeifer, Gerd P.; Yen, Yun; Xu, Xingzhi

    2009-01-01

    Human Rap1-interacting protein 1 (RIF1) contributes to the ataxia telangiectasia, mutated-mediated DNA damage response against the dexterous effect of DNA lesions and plays a critical role in the S-phase checkpoint. However, the molecular mechanisms by which human RIF1 conquers DNA aberrations remain largely unknown. We here showed that inhibition of RIF1 expression by small interfering RNA led to defective homologous recombination-mediated DNA double-strand break repair and sensitized cancer cells to camptothecin or staurosporine treatment. RIF1 underwent caspase-dependent cleavage upon apoptosis. We further found that RIF1 was highly expressed in human breast tumors, and its expression status was positively correlated with differentiation degrees of invasive ductal carcinoma of the breast. Our results suggest that RIF1 encodes an anti-apoptotic factor required for DNA repair and is a potential target for cancer treatment. PMID:19483192

  8. 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

  9. 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

  10. Survival and apoptotic pathways initiated by TNF-alpha: modeling and predictions.

    PubMed

    Rangamani, Padmini; Sirovich, Lawrence

    2007-08-01

    We present a mathematical model which includes TNF-alpha initiated survival and apoptotic cascades, as well as nuclear transcription of IkappaB. These pathways play a crucial role in deciding cell fate in response to inflammation and infection. Our model incorporates known specific protein-protein interactions as identified by experiments. Using these biochemical interactions, we develop a mathematical model of the NF-kappaB-mediated survival and caspase-mediated apoptosis pathways. Using mass action kinetics, we follow the formation of the survival and late complexes as well as the dynamics of DNA fragmentation. The effect of TNF-alpha concentration on DNA fragmentation is modeled and compares well with experiment. Nuclear transcription is also modeled phenomenologically by means of time lagged cytosolic concentrations. This results in transcription related concentrations undergoing under-damped oscillations, in qualitative and quantitative agreement with experiment. Using a tumor cell as a hypothetical model, we explore the interplay between the components of the survival and apoptotic pathways. Results are presented which make predictions on the limits of cellular oscillations in terms of time delay, initial concentration ratios and other features of the model. The model also makes clear predictions on cell viability in terms of DNA damage within the framework of TNF-alpha stimulus duration. PMID:17171720

  11. Apoptosis in immune-mediated diseases.

    PubMed

    Sankari, S Leena; Babu, N Aravindha; Rajesh, E; Kasthuri, M

    2015-04-01

    Apoptosis plays a significant role in both the physiological and pathological process. A dysfunctional apoptotic system can lead to either excessive removal or prolonged survival of cells. Therefore, dysregulation is involved in the pathogenesis of a variety of immunological diseases. The present review aims to provide an overview regarding role of apoptosis in immune-mediated disease. PMID:26015710

  12. Proteases in Fas-mediated apoptosis.

    PubMed

    Zhivotovsky, B; Burgess, D H; Schlegel, J; Pörn, 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

  13. Mechanisms of AH receptor-mediated responses 

    E-print Network

    Dong, Lian

    1996-01-01

    regulator in cell apoptotic pathways, and bcl-2 gene expression by estrogen in ER' breast cancer cell lines. The effects of TCDD on E2-mediated bcl-2 gene expression were investigated in this study. The E2-induced bcl-2 niRNA levels were inhibited by TCDD...

  14. In vitro senescence and apoptotic cell death of human megakaryocytes.

    PubMed

    Zauli, G; Vitale, M; Falcieri, E; Gibellini, D; Bassini, A; Celeghini, C; Columbaro, M; Capitani, S

    1997-09-15

    To investigate the fate of human megakaryocytes, CD34+ hematopoietic progenitor cells were purified from the peripheral blood or bone marrow of healthy donors and seeded in serum-free chemically defined suspension cultures. In the presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived cells showed an eightfold numerical expansion and a progressive maturation along the megakaryocytic lineage. Megakaryocyte maturation was characterized ultrastructurally by the presence of a demarcation membrane system and phenotypically by a high surface expression of alpha(IIb)beta3 integrin. The number of mature megakaryocytes peaked at days 12 to 15 of culture. On the other hand, the number of platelets released in the culture supernatant by CD34-derived megakaryocytes peaked at days 18 to 21, when a high percentage of megakaryocytes showed the characteristic features of apoptosis, as evaluated by electron microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end-labeling technique (TUNEL) and uptake of propidium iodide. In other experiments, primary alpha(IIb)beta3+ megakaryocytic cells were directly purified from the bone marrow aspirates of normal donors and seeded in serum-free suspension cultures. In the absence of cytokines, alpha(IIb)beta3+ megakaryocytes progressively underwent apoptotic cell death. The addition of TPO but not interleukin-3 or erythropoietin showed some protection of alpha(IIb)beta3+ cells from apoptosis at early culture times (days 2 to 4), but it did not show any significant effect at later time points. These findings suggest that the terminal phase of the megakaryocyte life span is characterized by the onset of apoptosis, which can be modulated only to a certain extent by TPO. PMID:9310474

  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. 3-O-(E)-p-coumaroyl tormentic acid from Eriobotrya japonica leaves induces caspase-dependent apoptotic cell death in human leukemia cell line.

    PubMed

    Kikuchi, Takashi; Akazawa, Hiroyuki; Tabata, Keiichi; Manosroi, Aranya; Manosroi, Jiradej; Suzuki, Takashi; Akihisa, Toshihiro

    2011-01-01

    Eleven triterpene acids, 1-11, isolated from the leaves of Eriobotrya japonica, were evaluated for inhibition of DNA topoisomerase (Topo) I and cytotoxicity against human leukemia (HL60) and melanoma cell lines (CRL1579). Among the compounds tested, four compounds, ?-oleanolic acid (4), ursolic acid (5), 3-O-(E)-p-coumaroyl tormentic acid (8), and betulinic acid (10), exhibited potent Topo I inhibitory activity (IC(50) 20.3-36.5 µM) and cytotoxicity against HL60 (EC(50) 5.0-8.1 µM). Upon assessing the apoptosis-inducing activity in HL60 cells, compound 8 exhibited induction of apoptosis detected by the observation of DNA fragmentation and membrane phospholipid exposure in flow cytometry. Western blot analysis showed that compound 8 markedly reduced the levels of procaspases-3 and 9, while being increased the levels of cleaved caspases-3 and 9. On the other hand, compound 8 exerted almost no influence on the expression of caspase-8. In addition, compound 8 increased significantly Bax/Bcl-2 ratio and activated caspase-2. These results suggested that compound 8 induced apoptotic cell death in HL60 via mainly mitochondrial pathway by, at least in part, Topo I inhibition. Therefore, compound 8 may be promising lead compound for developing an effective drug for treatment of leukemia. PMID:21372421

  17. BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

    PubMed Central

    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-XL 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

  18. A novel compound modified from tanshinone inhibits tumor growth in vivo via activation of the intrinsic apoptotic pathway.

    PubMed

    Tian, Hong-Lei; Yu, Ting; Xu, Nai-Ning; Feng, Chao; Zhou, Li-Ying; Luo, Hou-Wei; Chang, Donald C; Le, Xiao-Feng; Luo, Kathy Qian

    2010-11-01

    A novel compound, acetyltanshinone IIA (ATA) was obtained from chemical modifications of tanshinone TIIA (TIIA) isolated from a medicinal plant, Salvia miltiorrhiza. ATA exhibited increased water solubility and stronger apoptotic activity on multiple cancer cell lines than TIIA. ATA displayed a higher growth inhibition ability on breast cancer especially HER2 positive cells than normal cells and it inhibited xenografted tumor growth in mice. Mechanistic studies showed that ATA could induce significant reactive oxygen species (ROS) generation, Bax translocation to mitochondria, resulting in mitochondria damage, cytochrome c release, caspase-3 activation and apoptotic cell death. ATA-mediated ROS production and its downstream apoptotic events could be blocked by an antioxidant agent, propyl gallate, indicating the prominent role of ROS in ATA-induced apoptosis. Overexpression of Bcl-2 protein reduced ATA-induced cell death. In conclusion, ATA is a novel anticancer agent with potent in vitro and in vivo anticancer ability. ROS-mediated Bax activation should be the mechanism by which ATA induces apoptosis and inhibits tumor growth. PMID:20494511

  19. Gamma tocotrienol, a potent radioprotector, preferentially upregulates expression of anti-apoptotic genes to promote intestinal cell survival.

    PubMed

    Suman, Shubhankar; Datta, Kamal; Chakraborty, Kushal; Kulkarni, Shilpa S; Doiron, Kathryn; Fornace, Albert J; Sree Kumar, K; Hauer-Jensen, Martin; Ghosh, Sanchita P

    2013-10-01

    Gamma tocotrienol (GT3) has been reported as a potent ameliorator of radiation-induced gastrointestinal (GI) toxicity when administered prophylactically. This study aimed to evaluate the role of GT3 mediated pro- and anti-apoptotic gene regulation in protecting mice from radiation-induced GI damage. Male 10- to 12-weeks-old CD2F1 mice were administered with a single dose of 200 mg/kg of GT3 or equal volume of vehicle (5% Tween-80) 24 h before exposure to 11 Gy of whole-body ?-radiation. Mouse jejunum was surgically removed 4 and 24h after radiation exposure, and was used for PCR array, histology, immunohistochemistry, and immunoblot analysis. Results were compared among vehicle pre-treated no radiation, vehicle pre-treated irradiated, and GT3 pre-treated irradiated groups. GT3 pretreated irradiated groups, both 4h and 24h after radiation, showed greater upregulation of anti-apoptotic gene expression than vehicle pretreated irradiated groups. TUNEL staining and intestinal crypt analysis showed protection of jejunum after GT3 pre-treatment and immunoblot results were supportive of PCR data. Our study demonstrated that GT3-mediated protection of intestinal cells from a GI-toxic dose of radiation occurred via upregulation of antiapoptotic and downregulation of pro-apoptotic factors, both at the transcript as well as at the protein levels. PMID:23941772

  20. Disturbances of apoptotic cell clearance in systemic lupus erythematosus

    Microsoft Academic Search

    Wen-Hai Shao; Philip L Cohen

    2011-01-01

    Systemic lupus erythematosus is a multifactorial autoimmune disease with an as yet unknown etiopathogenesis. It is widely\\u000a thought that self-immunization in systemic lupus is driven by defective clearance of dead and dying cells. In lupus patients,\\u000a large numbers of apoptotic cells accumulate in various tissues including germinal centers. In the present review, we discuss\\u000a the danger signals released by apoptotic

  1. Clearance of apoptotic and necrotic cells and its immunological consequences

    Microsoft Academic Search

    Dmitri V. Krysko; Katharina D’Herde; Peter Vandenabeele

    2006-01-01

    The ultimate and most favorable fate of almost all dying cells is engulfment by neighboring or specialized cells. Efficient\\u000a clearance of cells undergoing apoptotic death is crucial for normal tissue homeostasis and for the modulation of immune responses.\\u000a Engulfment of apoptotic cells is finely regulated by a highly redundant system of receptors and bridging molecules on phagocytic\\u000a cells that detect

  2. Apoptotic cell clearance in chronic inflammation of lateral neck cysts

    Microsoft Academic Search

    Wieslaw Dobros; Karolina Burda; Krzysztof Guzik; Joanna Koziel; Jan Potempa

    The mechanism driving accumulation of large numbers of apoptotic and necrotic neutrophils in inflamed lateral neck cysts (LNC),\\u000a in the absence of infection, remains obscure. The cellular content of cysts obtained from 17 patients was co-cultured with\\u000a human macrophages. Phagocytosis levels of cyst-derived neutrophils were determined and compared to the uptake of spontaneously\\u000a apoptotic neutrophils. Simultaneously, the expression of cytokines

  3. Apoptotic markers in human blood platelets treated with peroxynitrite.

    PubMed

    Wachowicz, Barbara; Rywaniak, Joanna Zofia; Nowak, Pawe?

    2008-12-01

    Platelets are anucleated cells that upon activation by agonists or during storage may develop apoptotic events. The role of peroxynitrite and its reactive intermediates in apoptotic process in blood platelets is unknown. In order to study the appearance of biomarkers of apoptosis in platelets after treatment with peroxynitrite and with thrombin different markers were chosen: annexin V binding (phosphatidylserine exposure), platelet microparticle formation, mitochondrial membrane depolarization, caspase-3 activation and P-selectin expression. In gel-filtrated platelets treated with different concentrations of peroxynitrite (0.01, 0.1, 1.0 mM, 10 minute, 37 degrees C) a significant increase of phosphatidylserine exposure (about 36% at the highest concentration, p < 0.01) and the platelet microparticle formation were observed. Peroxynitrite caused a dose-dependent caspase-3 activation and depolarization of mitochondrial potential. The same apoptotic markers were appeared in thrombin-activated platelets. Dose-dependent tyrosine nitration in platelet proteins caused by peroxynitrite was reduced in the presence of (-)-epicatechin. Moreover, (-)-epicatechin distinctly reduced the level of apoptotic markers. The obtained results indicate that peroxynitrite responsible for oxidative/nitrative stress and changes in platelet function may promote in vitro apoptotic events in human gel-filtrated platelets via intrinsic pathway. Nitration of tyrosine seems to be partly associated with the appearance of apoptotic markers in platelets. PMID:19012180

  4. Clearance of apoptotic and necrotic cells and its immunological consequences.

    PubMed

    Krysko, Dmitri V; D'Herde, Katharina; Vandenabeele, Peter

    2006-10-01

    The ultimate and most favorable fate of almost all dying cells is engulfment by neighboring or specialized cells. Efficient clearance of cells undergoing apoptotic death is crucial for normal tissue homeostasis and for the modulation of immune responses. Engulfment of apoptotic cells is finely regulated by a highly redundant system of receptors and bridging molecules on phagocytic cells that detect molecules specific for dying cells. Recognition of necrotic cells by phagocytes is less well understood than recognition of apoptotic cells, but an increasing number of recent studies, which are discussed here, are highlighting its importance. New observations indicate that the interaction of macrophages with dying cells initiates internalization of the apoptotic or necrotic targets, and that internalization can be preceded by "zipper"-like and macropinocytotic mechanisms, respectively. We emphasize that clearance of dying cells is an important fundamental process serving multiple functions in the regulation of normal tissue turnover and homeostasis, and is not just simple anti- or pro-inflammatory responses. Here we review recent findings on genetic pathways participating in apoptotic cell clearance, mechanisms of internalization, and molecules involved in engulfment of apoptotic versus necrotic cells, as well as their immunological consequences and relationships to disease pathogenesis. PMID:16951923

  5. Obesity impairs apoptotic cell clearance in asthma

    PubMed Central

    Fernandez-Boyanapalli, Ruby; Goleva, Elena; Kolakowski, Christena; Min, Elysia; Day, Brian; Leung, Donald Y. M.; Riches, David W. H.; Bratton, Donna L.; Sutherland, E. Rand

    2014-01-01

    Background Asthma in obese adults is typically more severe and less responsive to glucocorticoids than asthma in nonobese adults. Objective We sought to determine whether the clearance of apoptotic inflammatory cells (efferocytosis) by airway macrophages was associated with altered inflammation and reduced glucocorticoid sensitivity in obese asthmatic patients. Methods We investigated the relationship of efferocytosis by airway (induced sputum) macrophages and blood monocytes to markers of monocyte programming, in vitro glucocorticoid response, and systemic oxidative stress in a cohort of adults with persistent asthma. Results Efferocytosis by airway macrophages was assessed in obese (n = 14) and nonobese (n = 19) asthmatic patients. Efferocytosis by macrophages was 40% lower in obese than nonobese subjects, with a mean efferocytic index of 1.77 (SD, 1.07) versus 3.00 (SD, 1.25; P < .01). A similar reduction of efferocytic function was observed in blood monocytes of obese participants. In these monocytes there was also a relative decrease in expression of markers of alternative (M2) programming associated with efferocytosis, including peroxisome proliferator-activated receptor ? and CX3 chemokine receptor 1. Macrophage efferocytic index was significantly correlated with dexamethasone-induced mitogen-activated protein kinase phosphatase 1 expression (? = 0.46, P < .02) and baseline glucocorticoid receptor ? expression (? = 0.44, P < .02) in PBMCs. Plasma 4-hydroxynonenal levels were increased in obese asthmatic patients at 0.33 ng/mL (SD, 0.15 ng/mL) versus 0.16 ng/mL (SD, 0.08 ng/mL) in nonobese patients (P = .006) and was inversely correlated with macrophage efferocytic index (? = ?0.67, P = .02). Conclusions Asthma in obese adults is associated with impaired macrophage/monocyte efferocytosis. Impairment of this anti-inflammatory process is associated with altered monocyte/macrophage programming, reduced glucocorticoid responsiveness, and systemic oxidative stress. PMID:23154082

  6. Rnd3 haploinsufficient mice are predisposed to hemodynamic stress and develop apoptotic cardiomyopathy with heart failure.

    PubMed

    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

  7. Surface code—biophysical signals for apoptotic cell clearance

    NASA Astrophysics Data System (ADS)

    Biermann, Mona; Maueröder, Christian; Brauner, Jan M.; Chaurio, Ricardo; Janko, Christina; Herrmann, Martin; Muñoz, 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.

  8. SIGN-R1 and complement factors are involved in the systemic clearance of radiation-induced apoptotic cells in whole-body irradiated mice.

    PubMed

    Park, Jin-Yeon; Loh, SoHee; Cho, Eun-Hee; Choi, Hyeong-Jwa; Na, Tae-Young; Nemeno, Judee Grace E; Lee, Jeong Ik; Yoon, Taek Joon; Choi, In-Soo; Lee, Minyoung; Lee, Jae-Seon; Kang, Young-Sun

    2015-08-01

    Although SIGN-R1-mediated complement activation pathway has been shown to enhance the systemic clearance of apoptotic cells, the role of SIGN-R1 in the clearance of radiation-induced apoptotic cells has not been characterized and was investigated in this study. Our data indicated that whole-body ?-irradiation of mice increased caspase-3(+) apoptotic lymphocyte numbers in secondary lymphoid organs. Following ?-irradiation, SIGN-R1 and complements (C4 and C3) were simultaneously increased only in the mice spleen tissue among the assessed tissues. In particular, C3 was exclusively activated in the spleen. The delayed clearance of apoptotic cells was markedly prevalent in the spleen and liver of SIGN-R1 KO mice, followed by a significant increase of CD11b(+) cells. These results indicate that SIGN-R1 and complement factors play an important role in the systemic clearance of radiation-induced apoptotic innate immune cells to maintain tissue homeostasis after ?-irradiation. PMID:26079881

  9. 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

  10. Apoptotic and Autophagic Effects of Sesbania grandiflora Flowers in Human Leukemic Cells

    PubMed Central

    Chakraborty, Biswajit; Chowdhury, Chinmay; Das, Padma

    2013-01-01

    Background Identification of cytotoxic compounds that induce apoptosis has been the mainstay of anti-cancer therapeutics for several decades. In recent years, focus has shifted to inducing multiple modes of cell death coupled with reduced systemic toxicity. The plant Sesbania grandiflora is widely used in Indian traditional medicine for the treatment of a broad spectrum of diseases. This encouraged us to investigate into the anti-proliferative effect of a fraction (F2) isolated from S. grandiflora flowers in cancer cells and delineate the underlying involvement of apoptotic and autophagic pathways. Principal Findings Using MTT based cell viability assay, we evaluated the cytotoxic potential of fraction F2. It was the most effective on U937 cells (IC50?18.6 µg/ml). Inhibition of growth involved enhancement of Annexin V positivity. This was associated with elevated reactive oxygen species generation, measured by flow cytometry and reduced oxygen consumption – both effects being abrogated by anti-oxidant NAC. This caused stimulation of pro-apoptotic proteins and concomitant inhibition of anti-apoptotic protein expressions inducing mitochondrial depolarization, as measured by flow cytometry and release of cytochrome c. Interestingly, even with these molecular features of apoptosis, F2 was able to alter Atg protein levels and induce LC3 processing. This was accompanied by formation of autophagic vacuoles as revealed by fluorescence and transmission electron microscopy – confirming the occurrence of autophagy. Eventually, F2 triggered caspase cascade – executioners of programmed cell death and AIF translocation to nuclei. This culminated in cleavage of the DNA repair enzyme, poly (ADP-ribose) polymerase that caused DNA damage as proved by staining with Hoechst 33258 leading to cell death. Conclusions The findings suggest fraction F2 triggers pro-oxidant activity and mediates its cytotoxicity in leukemic cells via apoptosis and autophagy. Thus, it merits consideration and further investigation as a therapeutic option for the treatment of leukemia. PMID:23967233

  11. Treadmill exercise ameliorates apoptotic cell death in the retinas of diabetic rats.

    PubMed

    Kim, Dae-Young; Jung, Sun-Young; Kim, Chang-Ju; Sung, Yun-Hee; Kim, Jae-Deung

    2013-06-01

    Apoptotic neuronal cell death in the retina is a hallmark of diabetic retinopathy. Exercise has been recommended for the alleviation of symptoms in patients with diabetes. In the present study, the effect of treadmill exercise on apoptosis in the retinas of diabetic rats was investigated. Diabetes was induced by intraperitoneal injection of streptozotocin. The rats in the exercise groups ran on a treadmill for 30 min/day, 5 times a week, over the course of 6 weeks. In this study, the terminal deoxynucleotidyl transferase?mediated dUTP nick?end labeling (TUNEL) assay, immunohistochemistry staining of caspase?3 and western blot analysis for Bax, Bcl?2 and phosphorylated protein kinase B (p?Akt) in the retinas of diabetic rats were performed. The results demonstrated that the number of TUNEL? and caspase?3?positive cells was increased in the retinas of diabetic rats, whereas treadmill exercise decreased these numbers. In addition, the expression of the pro?apoptotic protein Bax and the anti?apoptotic protein Bcl?2 was enhanced in the retinas of diabetic rats. Treadmill exercise suppressed Bax and enhanced Bcl?2 levels. The expression of the cell survival factor, p?Akt, was decreased in the retinas of diabetic rats and treadmill exercise increased the expression of p?Akt. The results of the present study demonstrated that treadmill exercise ameliorated diabetes?induced apoptosis in retinal cells by enhancing p?Akt levels in the retina. Treadmill exercise represents an effective strategy to delay or prevent the onset of ocular complications in diabetic patients. PMID:23620139

  12. Induction of discrete apoptotic pathways by bromo-substituted indirubin derivatives in invasive breast cancer cells

    SciTech Connect

    Nicolaou, Katerina A. [Department of Biological Sciences, University of Cyprus, Nicosia (Cyprus)] [Department of Biological Sciences, University of Cyprus, Nicosia (Cyprus); Liapis, Vasilis; Evdokiou, Andreas [Department of Surgery, Basil Hetzel Institute, Adelaide University, Adelaide (Australia)] [Department of Surgery, Basil Hetzel Institute, Adelaide University, Adelaide (Australia); Constantinou, Constantina [St. George's University of London Medical School at the University of Nicosia, Nicosia (Cyprus)] [St. George's University of London Medical School at the University of Nicosia, Nicosia (Cyprus); Magiatis, Prokopios; Skaltsounis, Alex L. [Faculty of Pharmacy, University of Athens, Athens (Greece)] [Faculty of Pharmacy, University of Athens, Athens (Greece); Koumas, Laura; Costeas, Paul A. [Center for Study of Hematological Malignancies, Nicosia (Cyprus)] [Center for Study of Hematological Malignancies, Nicosia (Cyprus); Constantinou, Andreas I., E-mail: andreasc@ucy.ac.cy [Department of Biological Sciences, University of Cyprus, Nicosia (Cyprus)

    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.

  13. 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

  14. 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

  15. Horizontal transfer of DNA by the uptake of apoptotic bodies.

    PubMed

    Holmgren, L; Szeles, A; Rajnavölgyi, E; Folkman, J; Klein, G; Ernberg, I; Falk, K I

    1999-06-01

    In this study we have raised the question of whether DNA can be transferred from one cell to another by phagocytosis of apoptotic bodies. We have used integrated copies of the Epstein-Barr virus (EBV) as a marker to follow the fate and expression pattern of apoptotic DNA in the phagocytotic host. Apoptosis was induced in EBV-carrying cell lines by irradiation before cultivation with either human fibroblasts, macrophages, or bovine aortic endothelial cells. Analysis of the expression pattern of EBV-encoded genes was performed by immunofluorescent staining as well as in situ hybridization. Cocultivation of apoptotic bodies from lymphoid cell lines containing integrated but not episomal copies of EBV resulted in expression of the EBV-encoded genes EBER and EBNA1 in the recipient cells at a high frequency. Fluorescence in situ hybridization analysis showed uptake of human chromatin as well as integrated EBV-DNA into the nuclei of bovine aortic endothelial cells. These data show that DNA may be rescued and reused from apoptotic bodies by somatic cells. In addition, our findings suggest that apoptotic bodies derived from EBV-carrying B lymphocytes may serve as the source of viral transfer to cells that lack receptors for the EBV virus in vivo. PMID:10339505

  16. Nicotinamide Inhibits Alkylating Agent-Induced Apoptotic Neurodegeneration in the Developing Rat Brain

    PubMed Central

    Naseer, Muhammad Imran; Ullah, Ikram; Suh, Joo Won; Kim, Myeong Ok

    2011-01-01

    Background Exposure to the chemotherapeutic alkylating agent thiotepa during brain development leads to neurological complications arising from neurodegeneration and irreversible damage to the developing central nerve system (CNS). Administration of single dose of thiotepa in 7-d postnatal (P7) rat triggers activation of apoptotic cascade and widespread neuronal death. The present study was aimed to elucidate whether nicotinamide may prevent thiotepa-induced neurodegeneration in the developing rat brain. Methodology/Principal Findings Neuronal cell death induced by thiotepa was associated with the induction of Bax, release of cytochrome-c from mitochondria into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1). Post-treatment of developing rats with nicotinamide suppressed thiotepa-induced upregulation of Bax, reduced cytochrome-c release into the cytosol and reduced expression of activated caspase-3 and cleavage of PARP-1. Cresyl violet staining showed numerous dead cells in the cortex hippocampus and thalamus; post-treatment with nicotinamide reduced the number of dead cells in these brain regions. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) and immunohistochemical analysis of caspase-3 show that thiotepa-induced cell death is apoptotic and that it is inhibited by nicotinamide treatment. Conclusion Nicotinamide (Nic) treatment with thiotepa significantly improved neuronal survival and alleviated neuronal cell death in the developing rat. These data demonstrate that nicotinamide shows promise as a therapeutic and neuroprotective agent for the treatment of neurodegenerative disorders in newborns and infants. PMID:22164206

  17. Advanced Glycation Endproducts Stimulate Osteoblast Apoptosis Via the MAP Kinase and Cytosolic Apoptotic Pathways

    PubMed Central

    Alikhani, Mani; Alikhani, Zoubin; Boyd, Coy; MacLellan, Christine M.; Raptis, Markos; Liu, Rongkun; Pischon, Nicole; Trackman, Philip C.; Gerstenfeld, Louis; Graves, Dana T.

    2007-01-01

    We have previously shown that diabetes significantly enhances apoptosis of osteoblastic cells in vivo and that the enhanced apoptosis contributes to diabetes impaired new bone formation. A potential mechanism is enhanced apoptosis stimulated by advanced glycation endproducts (AGEs). To investigate this further, an advanced glycation product, carboxymethyl lysine modified collagen (CML-collagen) was injected in vivo and stimulated a 5 fold increase in calvarial periosteal cell apoptosis compared to unmodified collagen. It also induced apoptosis in primary cultures of human or neonatal rat osteoblastic cells or MC-3T3-E1 cells in vitro. Moreover, the apoptotic effect was largely mediated through RAGE receptor. CML-collagen increased p38 and JNK activity 3.2 and 4.4 fold, respectively. Inhibition of p38 and JNK reduced CML-collagen stimulated apoptosis by 45% and 59% and by 90% when used together (P<0.05). The predominant apoptotic pathway induced by CML-collagen involved caspase-8 activation of caspase-3 and was independent of NF-?B activation. When osteoblastic cells were exposed to a long-term low dose incubation with CML-collagen there was a higher degree of apoptosis compared to short term incubation. In more differentiated osteoblastic cultures apoptosis was enhanced even further. These results indicate that advanced glycation endproducts, which accumulate in diabetic and aged individuals may promote apoptosis of osteoblastic cells and contribute to deficient bone formation. PMID:17064973

  18. Molecular mechanism of PDT-induced apoptotic cells stimulation NO production in macrophages

    NASA Astrophysics Data System (ADS)

    Song, Sheng; Zhou, Fei-fan; Yang, Si-hua; Chen, Wei R.

    2011-03-01

    It is well known that apoptotic cells (AC) participate in immune response. The immune response induced by AC, either immunostimulatory or immunosuppressive, have been extensively studied. However, the molecular mechanisms of the immunostimulatory effects induced by PDT-treated AC remain unclear. Nitric oxide (NO) is an important signal transduction molecule and has been implicated in a variety of functions. It has also been found to play an important role not only as a cytotoxic effector but an immune regulatory mediator. In this study, we demonstrate that the PDT-induced apoptotic tumor cells stimulate the production of NO in macrophages by up-regulating expression of inducible nitric oxide synthase (iNOS). In addition, we show that AC, through toll-like receptors (TLRs), can activate myeloid differentiation factor-88 (MyD88), indicating that AC serves as an intercellular signal to induce iNOS expression in immune cells after PDT treatment. This study provided more details for understanding the molecular mechanism of the immune response induced by PDT-treated AC.

  19. Resveratrol disrupts peroxynitrite-triggered mitochondrial apoptotic pathway: a role for Bcl-2.

    PubMed

    Brito, Paula M; Simões, Núria F; Almeida, Leonor M; Dinis, Teresa C P

    2008-08-01

    Resveratrol (3,4',5-trihydroxystilbene) is a phytochemical believed to be partly responsible for the cardioprotective effects of red wine due to its numerous biological activities. Here, we studied biochemical pathways underlying peroxynitrite-mediated apoptosis in endothelial cells and potential mechanisms responsible for resveratrol cytoprotective action. Peroxynitrite triggered endothelial cell apoptosis through caspases-8, -9 and -3 activation implying both mitochondrial and death receptor apoptotic pathways. Resveratrol was able to prevent peroxynitrite-induced caspases-3 and -9 activation, but not caspase-8 activation. Additionally, peroxynitrite increased intracellular levels of Bax without affecting those of Bcl-2, increasing consequently the Bax/Bcl-2 ratio. This ratio decreased when cells where pre-incubated with 10 and 50 muM resveratrol, mainly due to resveratrol ability per se to increase Bcl-2 intracellular levels without affecting Bax intracellular levels. These results propose an additional mechanism whereby resveratrol may exert its cardioprotective effects and suggest a key role for Bcl-2 in the resveratrol anti-apoptotic action, especially in disrupting peroxynitrite-triggered mitochondrial pathway. PMID:18584328

  20. Apoptotic and anti-proliferative effects of all-trans retinoic acid

    SciTech Connect

    Zamora, Monica [Departament de Bioquimica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona Diagonal 645, E-08028-Barcelona (Spain); Ortega, Juan Alberto [Departament de Bioquimica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona Diagonal 645, E-08028-Barcelona (Spain); Alana, Lide [Departament de Bioquimica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona Diagonal 645, E-08028-Barcelona (Spain); Vinas, Octavi [Departament de Bioquimica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona Diagonal 645, E-08028-Barcelona (Spain); Mampel, Teresa [Departament de Bioquimica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona Diagonal 645, E-08028-Barcelona (Spain)]. 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.

  1. Scythe: a novel reaper-binding apoptotic regulator.

    PubMed Central

    Thress, K; Henzel, W; Shillinglaw, W; Kornbluth, S

    1998-01-01

    Reaper is a central regulator of apoptosis in Drosophila melanogaster. With no obvious catalytic activity or homology to other known apoptotic regulators, reaper's mechanism of action has been obscure. We recently reported that recombinant Drosophila reaper protein induced rapid mitochondrial cytochrome c release, caspase activation and apoptotic nuclear fragmentation in extracts of Xenopus eggs. We now report the purification of a 150 kDa reaper-interacting protein from Xenopus egg extracts, which we have named Scythe. Scythe is highly conserved among vertebrates and contains a ubiquitin-like domain near its N-terminus. Immunodepletion of Scythe from extracts completely prevented reaper-induced apoptosis without affecting apoptosis triggered by activated caspases. Moreover, a truncated variant of Scythe lacking the N-terminal domain induced apoptosis even in the absence of reaper. These data suggest that Scythe is a novel apoptotic regulator that is an essential component in the pathway of reaper-induced apoptosis. PMID:9799223

  2. 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

  3. Die another way – non-apoptotic mechanisms of cell death

    PubMed Central

    Tait, Stephen W. G.; Ichim, Gabriel; Green, Douglas R.

    2014-01-01

    ABSTRACT Regulated, programmed cell death is crucial for all multicellular organisms. Cell death is essential in many processes, including tissue sculpting during embryogenesis, development of the immune system and destruction of damaged cells. The best-studied form of programmed cell death is apoptosis, a process that requires activation of caspase proteases. Recently it has been appreciated that various non-apoptotic forms of cell death also exist, such as necroptosis and pyroptosis. These non-apoptotic cell death modalities can be either triggered independently of apoptosis or are engaged should apoptosis fail to execute. In this Commentary, we discuss several regulated non-apoptotic forms of cell death including necroptosis, autophagic cell death, pyroptosis and caspase-independent cell death. We outline what we know about their mechanism, potential roles in vivo and define outstanding questions. Finally, we review data arguing that the means by which a cell dies actually matters, focusing our discussion on inflammatory aspects of cell death. PMID:24833670

  4. Differential modulation of endotoxin responsiveness by human caspase-12 polymorphisms

    Microsoft Academic Search

    Maya Saleh; John P. Vaillancourt; Rona K. Graham; Matthew Huyck; Srinivasa M. Srinivasula; Emad S. Alnemri; Martin H. Steinberg; Vikki Nolan; Clinton T. Baldwin; Richard S. Hotchkiss; Timothy G. Buchman; Barbara A. Zehnbauer; Michael R. Hayden; Lindsay A. Farrer; Sophie Roy; Donald W. Nicholson

    2004-01-01

    Caspases mediate essential key proteolytic events in inflammatory cascades and the apoptotic cell death pathway. Human caspases functionally segregate into two distinct subfamilies: those involved in cytokine maturation (caspase-1, -4 and -5) and those involved in cellular apoptosis (caspase-2, -3, -6, -7, -8, -9 and -10). Although caspase-12 is phylogenetically related to the cytokine maturation caspases, in mice it has

  5. Distinct lipid effects on tBid and Bim activation of membrane permeabilization by pro-apoptotic Bax.

    PubMed

    Shamas-Din, Aisha; Bindner, Scott; Chi, Xiaoke; Leber, Brian; Andrews, David W; Fradin, Cécile

    2015-05-01

    After exposure to stressful stimuli, apoptotic signals can be relayed to mitochondria by pro-apoptotic activator proteins, tBid (truncated Bid/p15) and Bim (Bcl-2 interacting mediator), which activate Bax (Bcl-2 associated X protein) and or Bak (Bcl-2 antagonist/killer) to induce mitochondrial outer membrane (MOM) permeabilization (MOMP). These protein-protein and protein-membrane interactions are critical for apoptosis regulation, since MOMP irreversibly leads to cell death. Whereas the distinct roles of tBid and Bim as sensors of different types of stress are well recognized, it is not known whether the molecular mechanisms whereby they initiate MOMP are the same. In the present study, we compare membrane permeabilization by Bax activated by either cBid [cleaved Bid (p7 and p15)] or Bim and we examine the role of membrane lipids in the recruitment and activation of these three Bcl-2 (B-cell lymphoma 2) pro-apoptotic proteins. We employ fluorescently-labelled proteins and liposomes to quantify the effects of specific lipids on each of the well-characterized steps in Bax-mediated membrane permeabilization. We show that high levels of cholesterol in the membrane inhibit permeabilization by categorically identifying the recruitment of Bax by the activators and Bax insertion in the membrane as the steps being hindered by cholesterol. Furthermore, we show that binding of both cBid and Bim to membranes is facilitated by electrostatic interactions with anionic phospholipids. However, whereas Bim does not require any particular anionic lipids, the conformational change in tBid depends on cardiolipin (CL). This suggests that CL can activate tBid in a similar manner to Mtch2 (mitochondrial carrier homologue 2). Thus, lipids modify multiple aspects of Bax-mediated membrane permeabilization. PMID:25714678

  6. Novel oncolytic adenovirus sensitizes renal cell carcinoma cells to radiotherapy via mitochondrial apoptotic cell death.

    PubMed

    Chen, Ren-Fu; Li, Yue-Yan; Li, Lian-Tao; Cheng, Qian; Jiang, Guan; Zheng, Jun-Nian

    2015-03-01

    Renal cell carcinoma is the most frequent kidney malignancy and patients with metastatic disease have a poor prognosis. Suppressed apoptosis and marked invasiveness are distinctive features of renal cell carcinoma. In the present study, a dual?regulated oncolytic adenovirus expressing the interluekin (IL)?24 gene (Ki67?ZD55?IL?24) was constructed utilizing the Ki67 promoter to replace the native viral promoter of the E1A gene. Whether the combination of Ki67?ZD55?IL?24?mediated gene virotherapy and radiotherapy produced increased cytotoxicity in renal cell carcinoma cells via mitochondrial apoptotic cell death was investigated. The data indicated that this novel strategy has the potential to be further developed into an effective approach to treat renal cell carcinoma. The results showed that the combination of Ki67?ZD55?IL?24 and radiotherapy significantly enhanced anti?tumour activity via increasing the induction of apoptosis in melanoma cells compared with the other agents. PMID:25411768

  7. PNUTS knockdown potentiates the apoptotic effect of Roscovitine in breast and colon cancer cells

    PubMed Central

    De Leon, Gabriel; Cavino, Margaret; D’Angelo, Mikilyn; Krucher, Nancy A

    2013-01-01

    The phosphorylation state of Retinoblastoma protein (Rb) plays a role in cell proliferation and apoptosis. Within cells, cyclin dependent kinases (cdks) phosphorylate Rb in response to growth stimulatory signals, whereas protein phosphatase 1 (PP1) dephosphorylates Rb when cells stop proliferating or undergo apoptosis in response to anti-proliferative or stress signals. Stimulation of PP1 activity via siRNA mediated knockdown of its interacting protein PNUTS (Phosphatase Nuclear Targeting Subunit) leads to Rb dephosphorylation and apoptosis in cancer cells. Here we utilize two separate methods to modulate the phosphorylation state of Rb in cancer cells. Kinase activity toward Rb is inhibited by the clinically relevant cdk inhibitor, Roscovitine. In addition, siRNA mediated PNUTS knockdown stimulates phosphatase activity toward Rb. Either of these treatments in cancer cells causes a two-fold stimulation of apoptosis. When activation of phosphatase activity is combined with inhibition of cdk activity toward Rb, however, cells exhibit a 4-fold increase in apoptosis. The mechanism by which PNUTS knockdown mediated PP1 activation leads to apoptosis was determined to be dependent on the activity of the transcription factor E2F1. The Rb phosphorylation profiles resulting from each treatment were analyzed and found to be similar but not identical. In addition, the two treatments differentially effect the expression of bcl-2 family proteins. Thus inhibition of cdk activity and activation of PP1 activity toward pRb are functionally distinct processes that together increase the apoptotic effect in cells. PMID:20372802

  8. PNUTS knockdown potentiates the apoptotic effect of Roscovitine in breast and colon cancer cells.

    PubMed

    De Leon, Gabriel; Cavino, Margaret; D'Angelo, Mikilyn; Krucher, Nancy A

    2010-05-01

    The phosphorylation state of Retinoblastoma protein (Rb) plays a role in cell proliferation and apoptosis. Within cells, cyclin dependent kinases (cdks) phosphorylate Rb in response to growth stimulatory signals, whereas protein phosphatase 1 (PP1) dephosphorylates Rb when cells stop proliferating or undergo apoptosis in response to anti-proliferative or stress signals. Stimulation of PP1 activity via siRNA mediated knockdown of its interacting protein PNUTS (Phosphatase Nuclear Targeting Subunit) leads to Rb dephosphorylation and apoptosis in cancer cells. We utilized two separate methods to modulate the phosphorylation state of Rb in cancer cells. Kinase activity toward Rb is inhibited by the clinically relevant cdk inhibitor, Roscovitine. In addition, siRNA mediated PNUTS knockdown stimulates phosphatase activity toward Rb. Either of these treatments in cancer cells causes a 2-fold stimulation of apoptosis. When activation of phosphatase activity is combined with inhibition of cdk activity toward Rb, however, cells exhibit a 4-fold increase in apoptosis. The mechanism by which PNUTS knockdown mediated PP1 activation leads to apoptosis was determined to be dependent on the activity of the transcription factor E2F1. The Rb phosphorylation profiles resulting from each treatment were analyzed and found to be similar but not identical. In addition, the two treatments differentially effect the expression of bcl-2 family proteins. Thus inhibition of cdk activity and activation of PP1 activity toward pRb are functionally distinct processes that together increase the apoptotic effect in cells. PMID:20372802

  9. 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

  10. Small GTPase CDC-42 promotes apoptotic cell corpse clearance in response to PAT-2 and CED-1 in C. elegans

    PubMed Central

    Neukomm, L J; Zeng, S; Frei, A P; Huegli, P A; Hengartner, M O

    2014-01-01

    The rapid clearance of dying cells is important for the well-being of multicellular organisms. In C. elegans, cell corpse removal is mainly mediated by three parallel engulfment signaling cascades. These pathways include two small GTPases, MIG-2/RhoG and CED-10/Rac1. Here we present the identification and characterization of CDC-42 as a third GTPase involved in the regulation of cell corpse clearance. Genetic analyses performed by both loss of cdc-42 function and cdc-42 overexpression place cdc-42 in parallel to the ced-2/5/12 signaling module, in parallel to or upstream of the ced-10 module, and downstream of the ced-1/6/7 module. CDC-42 accumulates in engulfing cells at membranes surrounding apoptotic corpses. The formation of such halos depends on the integrins PAT-2/PAT-3, UNC-112 and the GEF protein UIG-1, but not on the canonical ced-1/6/7 or ced-2/5/12 signaling modules. Together, our results suggest that the small GTPase CDC-42 regulates apoptotic cell engulfment possibly upstream of the canonical Rac GTPase CED-10, by polarizing the engulfing cell toward the apoptotic corpse in response to integrin signaling and ced-1/6/7 signaling in C. elegans. PMID:24632947

  11. Secondary necrosis of apoptotic neutrophils induced by the human cathelicidin LL-37 is not proinflammatory to phagocytosing macrophages

    PubMed Central

    Li, Hsin-Ni; Barlow, Peter G.; Bylund, Johan; Mackellar, Annie; Björstad, Åse; Conlon, James; Hiemstra, Pieter S.; Haslett, Chris; Gray, Mohini; Simpson, A. John; Rossi, Adriano G.; Davidson, Donald J.

    2009-01-01

    Cathelicidins are CHDP with essential roles in innate host defense but also more recently associated with the pathogenesis of certain chronic diseases. These peptides have microbicidal potential and the capacity to modulate innate immunity and inflammatory processes. PMN are key innate immune effector cells with pivotal roles in defense against infection. The appropriate regulation of PMN function, death, and clearance is critical to innate immunity, and dysregulation is implicated in disease pathogenesis. The efferocytosis of apoptotic PMN, in contrast to necrotic cells, is proposed to promote the resolution of inflammation. We demonstrate that the human cathelicidin LL-37 induced rapid secondary necrosis of apoptotic human PMN and identify an essential minimal region of LL-37 required for this activity. Using these LL-37-induced secondary necrotic PMN, we characterize the consequence for macrophage inflammatory responses. LL-37-induced secondary necrosis did not inhibit PMN ingestion by monocyte-derived macrophages and in contrast to expectation, was not proinflammatory. Furthermore, the anti-inflammatory effects of apoptotic PMN on activated macrophages were retained and even potentiated after LL-37-induced secondary necrosis. However, this process of secondary necrosis did induce the release of potentially harmful PMN granule contents. Thus, we suggest that LL-37 can be a potent inducer of PMN secondary necrosis during inflammation without promoting macrophage inflammation but may mediate host damage through PMN granule content release under chronic or dysregulated conditions. PMID:19581375

  12. Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization but partially affects its apoptotic activity

    SciTech Connect

    Lee, Y.-H. [Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan (China); Cheng, C.-M. [Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan (China); Chang, Y.-F. [Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan (China); Wang, T.-Y. [Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan (China); Yuo, C.-Y. [Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan (China); Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan (China); 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.

  13. Engulfment of cerebral apoptotic bodies controls the course of prion disease in a mouse strain-dependent manner.

    PubMed

    Kranich, Jan; Krautler, Nike Julia; Falsig, Jeppe; Ballmer, Boris; Li, Shulei; Hutter, Gregor; Schwarz, Petra; Moos, Rita; Julius, Christian; Miele, Gino; Aguzzi, Adriano

    2010-09-27

    Progressive accumulation of PrP(Sc), a hallmark of prion diseases, occurs when conversion of PrP(C) into PrP(Sc) is faster than PrP(Sc) clearance. Engulfment of apoptotic bodies by phagocytes is mediated by Mfge8 (milk fat globule epidermal growth factor 8). In this study, we show that brain Mfge8 is primarily produced by astrocytes. Mfge8 ablation induced accelerated prion disease and reduced clearance of cerebellar apoptotic bodies in vivo, as well as excessive PrP(Sc) accumulation and increased prion titers in prion-infected C57BL/6 × 129Sv mice and organotypic cerebellar slices derived therefrom. These phenotypes correlated with the presence of 129Sv genomic markers in hybrid mice and were not observed in inbred C57BL/6 Mfge8(-/-) mice, suggesting the existence of additional strain-specific genetic modifiers. Because Mfge8 receptors are expressed by microglia and depletion of microglia increases PrP(Sc) accumulation in organotypic cerebellar slices, we conclude that engulfment of apoptotic bodies by microglia may be an important pathway of prion clearance controlled by astrocyte-borne Mfge8. PMID:20837697

  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. Lipoxins, Aspirin-Triggered Epi-Lipoxins, Lipoxin Stable Analogues, and the Resolution of Inflammation: Stimulation of Macrophage Phagocytosis of Apoptotic Neutrophils In Vivo

    Microsoft Academic Search

    SIOBHAN MITCHELL; GRAHAM THOMAS; KILLEEN HARVEY; DAVID COTTELL; KEIRA REVILLE; GIOVANNI BERLASCONI; NICOS A. PETASIS; LARS ERWIG; ANDREW J. REES; JOHN SAVILL; HUGH R. BRADY; CATHERINE GODSON

    Lipoxins (LX) are eicosanoids with antiinflamma- tory activity in glomerulonephritis (GN) and inflammatory diseases, hypersensitivity, and ischemia reperfusion injury. It has been demonstrated that LXA4 stimulates non-phlogis- tic phagocytosis of apoptotic polymorphonuclear neutro- phils (PMN) by monocyte-derived macrophages (M) in vitro, suggesting a role for LX as endogenous pro-resolution lipid mediators. It is here reported that LXA4, LXB4, the aspirin-triggered

  16. NDK-1, the Homolog of NM23-H1/H2 Regulates Cell Migration and Apoptotic Engulfment in C. elegans

    PubMed Central

    Fancsalszky, Luca; Monostori, Eszter; Farkas, Zsolt; Pourkarimi, Ehsan; Masoudi, Neda; Hargitai, Balázs; Bosnar, Maja Herak; Deželjin, Martina; Zsákai, Annamária; Vellai, Tibor; Mehta, Anil; Takács-Vellai, Krisztina

    2014-01-01

    Abnormal regulation of cell migration and altered rearrangement of cytoskeleton are characteristic of metastatic cells. The first described suppressor of metastatic processes is NM23-H1, which displays NDPK (nucleoside-diphosphate kinase) activity. To better understand the role of nm23 genes in cell migration, we investigated the function of NDK-1, the sole Caenorhabditis elegans homolog of group I NDPKs in distal tip cell (DTC) migration. Dorsal phase of DTC migration is regulated by integrin mediated signaling. We find that ndk-1 loss of function mutants show defects in this phase. Epistasis analysis using mutants of the ?-integrin ina-1 and the downstream functioning motility-promoting signaling module (referred to as CED-10 pathway) placed NDK-1 downstream of CED-10/Rac. As DTC migration and engulfment of apoptotic corpses are analogous processes, both partially regulated by the CED-10 pathway, we investigated defects of apoptosis in ndk-1 mutants. Embryos and germ cells defective for NDK-1 showed an accumulation of apoptotic cell corpses. Furthermore, NDK-1::GFP is expressed in gonadal sheath cells, specialized cells for engulfment and clearence of apoptotic corpses in germ line, which indicates a role for NDK-1 in apoptotic corpse removal. In addition to the CED-10 pathway, engulfment in the worm is also mediated by the CED-1 pathway. abl-1/Abl and abi-1/Abi, which function in parallel to both CED-10/CED-1 pathways, also regulate engulfment and DTC migration. ndk-1(-);abi-1(-) double mutant embryos display an additive phenotype (e. g. enhanced number of apoptotic corpses) which suggests that ndk-1 acts in parallel to abi-1. Corpse number in ndk-1(-);ced-10(-) double mutants, however, is similar to ced-10(-) single mutants, suggesting that ndk-1 acts downstream of ced-10 during engulfment. In addition, NDK-1 shows a genetic interaction with DYN-1/dynamin, a downstream component of the CED-1 pathway. In summary, we propose that NDK-1/NDPK might represent a converging point of CED-10 and CED-1 pathways in the process of cytoskeleton rearrangement. PMID:24658123

  17. Cardioprotective activity of urocortin by preventing caspase-independent, non-apoptotic death in cultured neonatal rat cardiomyocytes exposed to ischemia

    SciTech Connect

    Takatani-Nakase, Tomoka, E-mail: nakase@mukogawa-u.ac.jp [Department of Pharmaceutics, School of Pharmaceutical Sciences, Mukogawa Women's University, 11-68, Koshien, Nishinomiya, Hyogo 663-8179 (Japan)] [Department of Pharmaceutics, School of Pharmaceutical Sciences, Mukogawa Women's University, 11-68, Koshien, Nishinomiya, Hyogo 663-8179 (Japan); Takahashi, Koichi, E-mail: koichi@mukogawa-u.ac.jp [Department of Pharmaceutics, School of Pharmaceutical Sciences, Mukogawa Women's University, 11-68, Koshien, Nishinomiya, Hyogo 663-8179 (Japan)] [Department of Pharmaceutics, School of Pharmaceutical Sciences, Mukogawa Women's University, 11-68, Koshien, Nishinomiya, Hyogo 663-8179 (Japan)

    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.

  18. Prostaglandin D2 synthase: Apoptotic factor in alzheimer plasma, inducer of reactive oxygen species, inflammatory cytokines and dialysis dementia

    PubMed Central

    Maesaka, John K.; Sodam, Bali; Palaia, Thomas; Ragolia, Louis; Batuman, Vecihi; Miyawaki, Nobuyuki; Shastry, Shubha; Youmans, Steven; El-Sabban, Marwan

    2013-01-01

    Background: Apoptosis, reactive oxygen species (ROS) and inflammatory cytokines have all been implicated in the development of Alzheimer’s disease (AD). Objectives: The present study identifies the apoptotic factor that was responsible for the fourfold increase in apoptotic rates that we previously noted when pig proximal tubule, LLC-PK1, cells were exposed to AD plasma as compared to plasma from normal controls and multi-infarct dementia. Patients and Methods: The apoptotic factor was isolated from AD urine and identified as lipocalin-type prostaglandin D2 synthase (L-PGDS). L-PGDS was found to be the major apoptotic factor in AD plasma as determined by inhibition of apoptosis approximating control levels by the cyclo-oxygenase (COX) 2 inhibitor, NS398, and the antibody to L-PGDS. Blood levels of L-PGDS, however, were not elevated in AD. We now demonstrate a receptor-mediated uptake of L-PGDS in PC12 neuronal cells that was time, dose and temperature-dependent and was saturable by competition with cold L-PGDS and albumin. Further proof of this endocytosis was provided by an electron microscopic study of gold labeled L-PGDS and immunofluorescence with Alexa-labeled L-PGDS. Results: The recombinant L-PGDS and wild type (WT) L-PGDS increased ROS but only the WTL-PGDS increased IL6 and TNF?, suggesting that differences in glycosylation of L-PGDS in AD was responsible for this discrepancy. Conclusions: These data collectively suggest that L-PGDS might play an important role in the development of dementia in patients on dialysis and of AD. PMID:24475446

  19. Estrogen Negatively Regulates the Pro-apoptotic Function of Mixed Lineage Kinase 3 in Estrogen Receptor Positive Breast Cancer

    PubMed Central

    Rangasamy, Velusamy; Mishra, Rajakishore; Mehrotra, Suneet; Sondarva, Gautam; Ray, Rajarshi S.; Rao, Arundhati; Chatterjee, Malay; Rana, Basabi; Rana, Ajay

    2009-01-01

    Estrogen stimulates growth and inhibits apoptosis of breast cancer cells via genomic and non-genomic actions. However, the detailed mechanism by which estrogen inhibits the pro-apoptotic pathways that might impede the normal homeostasis and action of chemotherapeutic drugs in breast cancer cells is not well understood. Here, we report a negative regulation of a pro-apoptotic kinase, Mixed Lineage Kinase 3 (MLK3) by 17?-estradiol (E2) that hinders cytotoxic drug-induced cell death in estrogen receptor positive (ER+) breast cancer cells. MLK3 kinase activities were significantly higher in estrogen receptor negative (ER?), progesterone receptor negative (PR?) primary human breast tumors, suggesting that E2 might have a negative role in regulating MLK3 kinase activity. The kinase activities of MLK3 and its downstream target, JNK were rapidly inhibited by E2 in ER+ but not in ER? breast cancer cells. The inhibition of MLK3 kinase activity by E2 was mediated via activation of protein kinase B (PKB/AKT) because specific knockdown of AKT1/2 prevented the E2-induced inhibition of MLK3. Furthermore, E2-induced inhibition of MLK3 kinase activity involved a direct phosphorylation of MLK3 at Ser 674 site by AKT, which resulted in an attenuation of the pro-apoptotic function of MLK3. In addition, a pan-MLK inhibitor (CEP-11004) significantly attenuated Taxol-induced cell death, which was further synergized by E2. Thus, our data suggest that E2 negatively regulates the pro-apoptotic function of MLK3 during breast cancer pathogenesis and therefore MLK3 and other MLK family members might play an important role in cytotoxic drug-induced cell death in ER+ breast cancer cells. PMID:20145118

  20. No death without life: vital functions of apoptotic effectors

    Microsoft Academic Search

    L Galluzzi; N Joza; E Tasdemir; M C Maiuri; M Hengartner; J M Abrams; N Tavernarakis; J Penninger; F Madeo; G Kroemer

    2008-01-01

    As a result of the genetic experiments performed in Caenorhabditis elegans, it has been tacitly assumed that the core proteins of the ‘apoptotic machinery’ (CED-3, -4, -9 and EGL-1) would be solely involved in cell death regulation\\/execution and would not exert any functions outside of the cell death realm. However, multiple studies indicate that the mammalian orthologs of these C.

  1. Near death experiences: Poxvirus regulation of apoptotic death

    Microsoft Academic Search

    John M. Taylor; Michele Barry

    2006-01-01

    Apoptosis, or programmed cell death, plays a critical role in the elimination of virus-infected cells. As a result, a growing number of viruses encode numerous potent anti-apoptotic proteins to counteract apoptosis in an effort to prolong their own survival. This review describes the numerous mechanisms by which poxviruses inhibit apoptosis thereby modulating life and death of the cell.

  2. Molecular mechanisms of late apoptotic\\/necrotic cell clearance

    Microsoft Academic Search

    I K H Poon; M D Hulett; C R Parish

    2010-01-01

    Phagocytosis serves as one of the key processes involved in development, maintenance of tissue homeostasis, as well as in eliminating pathogens from an organism. Under normal physiological conditions, dying cells (e.g., apoptotic and necrotic cells) and pathogens (e.g., bacteria and fungi) are rapidly detected and removed by professional phagocytes such as macrophages and dendritic cells (DCs). In most cases, specific

  3. Phenotype of apoptotic lymphocytes in children with Down syndrome

    Microsoft Academic Search

    Solaf M Elsayed; Ghada M Elsayed

    2009-01-01

    BACKGROUND: Down syndrome (DS) is the most common and best-known chromosomal disorder and is associated with several other pathologic conditions including immunodeficiency which makes a significant contribution to morbidity and mortality. Various immunological theories and observations to explain the predisposition of individuals with DS to various infections have been published, one of which is increased apoptotic cells. AIM: The aim

  4. Cytostatic and apoptotic actions of TGF-? in homeostasis and cancer

    Microsoft Academic Search

    Peter M. Siegel; Joan Massagué

    2003-01-01

    The cytostatic and apoptotic functions of transforming growth factor-? (TGF-?) help restrain the growth of mammalian tissues; loss of these effects leads to hyperproliferative disorders and is common in cancer. However, tumour cells that are relieved from TGF-? growth constraints might then overproduce this cytokine to create a local immunosuppressive environment that fosters tumour growth and exacerbates the invasive and

  5. Apoptotic versus proliferative activities in human benign prostatic hyperplasia

    Microsoft Academic Search

    Natasha Kyprianou; Huacheng Tu; Stephen C Jacobs

    1996-01-01

    Cell growth in the normal prostate is regulated by a delicate balance between cell death and cell proliferation (ie, apoptotic v proliferative activity). Disruption of the molecular mechanisms that regulate these two processes may underline the abnormal growth of the gland leading to benign prostatic hyperplasia (BPH). In this study, the incidence of programmed cell death (apoptosis) and cell proliferation

  6. Morphometric Quantification of Apoptotic Stages in Cell Culture

    Microsoft Academic Search

    Maurizio Sabbatini; Chiarella Bozzo; Mario Castellucci; Mario Cannas

    2004-01-01

    Apoptosis is an active process of self-destruction, whereby cells undergo physiological cell death. It occurs during development and regulation of tissue homeostasis or as a result of changes in environmental stimuli. Chromatin condensation and nuclear fragmentation, which are typical features of apoptotic nuclei, are usually quantified by fluorescent DNA dyes. The present study reports a reliable method to analyze morphological

  7. Apoptotic Osteocytes Regulate Osteoclast Precursor Recruitment and Differentiation In Vitro

    E-print Network

    You, Lidan

    Apoptotic Osteocytes Regulate Osteoclast Precursor Recruitment and Differentiation In Vitro Saja A, Toronto, ON, Canada ABSTRACT Fatigue loading causes a spatial distribution of osteocyte apoptosis co-localized with bone resorption spaces peaking around microdamage sites. Since osteocytes have been shown to regulate

  8. Sorafenib sensitizes hepatocellular carcinoma cells to physiological apoptotic stimuli.

    PubMed

    Fernando, Joan; Sancho, Patricia; Fernández-Rodriguez, Conrado M; Lledó, José L; Caja, Laia; Campbell, Jean S; Fausto, Nelson; Fabregat, Isabel

    2012-04-01

    Sorafenib increases survival rate of patients with advanced hepatocellular carcinoma (HCC). The mechanism underlying this effect is not completely understood. In this work we have analyzed the effects of sorafenib on autocrine proliferation and survival of different human HCC cell lines. Our results indicate that sorafenib in vitro counteracts autocrine growth of different tumor cells (Hep3B, HepG2, PLC-PRF-5, SK-Hep1). Arrest in S/G2/M cell cycle phases were observed coincident with cyclin D1 down-regulation. However, sorafenib's main anti-tumor activity seems to occur through cell death induction which correlated with caspase activation, increase in the percentage of hypodiploid cells, activation of BAX and BAK and cytochrome c release from mitochondria to cytosol. In addition, we observed a rise in mRNA and protein levels of the pro-apoptotic "BH3-domain only" PUMA and BIM, as well as decreased protein levels of the anti-apoptotic MCL1 and survivin. PUMA targeting knock-down, by using specific siRNAs, inhibited sorafenib-induced apoptotic features. Moreover, we obtained evidence suggesting that sorafenib also sensitizes HCC cells to the apoptotic activity of transforming growth factor-? (TGF-?) through the intrinsic pathway and to tumor necrosis factor-? (TNF) through the extrinsic pathway. Interestingly, sensitization to sorafenib-induced apoptosis is characteristic of liver tumor cells, since untransformed hepatocytes did not respond to sorafenib inducing apoptosis, either alone or in combination with TGF-? or TNF. Indeed, sorafenib effectiveness in delaying HCC late progression might be partly related to a selectively sensitization of HCC cells to apoptosis by disrupting autocrine signals that protect them from adverse conditions and pro-apoptotic physiological cytokines. PMID:21604268

  9. Differentiation of monocytes to macrophages induced by influenza virus-infected apoptotic cells

    Microsoft Academic Search

    Noboru Uchide; Kunio Ohyama; Bo Yuan; Toshio Bessho; Toshio Yamakawa

    The effect of the culture supernatant of influenza virus (IV)-infected apoptotic and non-apoptotic cells on the differentiation of monocytes to macro- phages was investigated. IV infection induced apoptotic DNA fragmentation in cultured chorion cells but not in amnion cells prepared from human foetal membrane tissue. To examine the differ- entiation of monocytes to macrophages, an ad- hesion assay was employed

  10. Preemptive donor apoptotic cell infusions induce IFN-?-producing myeloid derived suppressor cells for cardiac allograft protection1

    PubMed Central

    Bryant, Jane; Lerret, Nadine M.; Wang, Jiao-jing; Kang, Hee-Kap; Tasch, James; Zhang, Zheng; Luo, Xunrong

    2014-01-01

    We have previously shown that preemptive infusion of apoptotic donor splenocytes treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) induces long-term allograft survival in full MHC-mismatched models of allogeneic islet and cardiac transplantation. The role of myeloid derived suppressor cells (MDSCs) in the graft protection provided by ECDI-SPs is unclear. In this study, we demonstrate that infusions of ECDI-SPs increase two populations of CD11b+ cells in the spleen that phenotypically resemble monocytic-like (CD11b+Ly6CHI) and granulocytic-like (CD11b+Gr1HI) MDSCs. Both populations suppress T cell proliferation in vitro, and traffic to the cardiac allografts in vivo to mediate their protection via inhibition of local CD8 T cell accumulation and potentially also via induction and homing of regulatory T cells. Importantly, repeated treatments with ECDI-SPs induce the CD11b+Gr1HI cells to produce a high level of IFN-? and to exhibit an enhanced responsiveness to IFN-? by expressing higher levels of downstream effector molecules ido and nos2. Consequently, neutralization of IFN-? completely abolishes the suppressive capacity of this population. We conclude that donor ECDI-SPs induce the expansion of two populations of MDSCs important for allograft protection mediated in part by intrinsic IFN-? dependent mechanisms. This form of preemptive donor apoptotic cell infusions has significant potential for the therapeutic manipulation of MDSCs for transplant tolerance induction. PMID:24808363

  11. 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

  12. Rybp interacts with Hippi and enhances Hippi-mediated apoptosis

    Microsoft Academic Search

    Sasha E. Stanton; Jennifer K. Blanck; Joseph Locker; Nicole Schreiber-Agus

    2007-01-01

    Rybp (DEDAF) has been shown to interact with DED-containing proteins and to encode pro-apoptotic functions. Here we characterize\\u000a a novel interaction between Rybp and Hippi, a protein implicated in neuronal apoptosis as well as in the pathogenesis of Huntington’s\\u000a disease. Rybp can synergize with Hippi to enhance Caspase 8-mediated apoptosis and also appears to be essential for Hippi-mediated\\u000a apoptosis. Moreover,

  13. Depletion of Hepatic Glutathione Prevents Death Receptor-Dependent Apoptotic and Necrotic Liver Injury in Mice

    PubMed Central

    Hentze, Hannes; Gantner, Florian; Kolb, Stefan A.; Wendel, Albrecht

    2000-01-01

    The activation of the death receptors, tumor necrosis factor-receptor-1 (TNF-R1) or CD95, is a hallmark of inflammatory or viral liver disease. In different murine in vivo models, we found that livers depleted of ?-glutamyl-cysteinyl-glycine (GSH) by endogenous enzymatic conjugation after phorone treatment were resistant against death receptor-elicited injury as assessed by transaminase release and histopathology. In apoptotic models initiated by engagement of CD95, or by injection of TNF or lipopolysaccharide into galactosamine-sensitized mice, hepatic caspase-3-like proteases were not activated in the GSH-depleted state. Under GSH depletion, also caspase-independent, TNF-R1-mediated injury (high-dose actinomycin D or ?-amanitin), as well as necrotic hepatotoxicity (high-dose lipopolysaccharide) were entirely blocked. In the T-cell-dependent model of concanavalin A-induced hepatotoxicity, GSH depletion resulted in a suppression of interferon-? release, delay of systemic TNF release, hepatic nuclear factor-?B activation, and an abrogation of sinusoidal endothelial cell detachment as assessed by electron microscopy. When GSH depletion was initiated 3 hours after concanavalin A injection, ie, after the peak of early pro-inflammatory cytokines, livers were still protected. We conclude that sufficient hepatic GSH levels are a prerequisite for the execution of death receptor-mediated hepatocyte demise. PMID:10854226

  14. 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

  15. Clearing the Dead: Apoptotic Cell Sensing, Recognition, Engulfment, and Digestion

    PubMed Central

    Hochreiter-Hufford, Amelia; Ravichandran, Kodi S.

    2013-01-01

    Clearance of apoptotic cells is the final stage of programmed cell death. Uncleared corpses can become secondarily necrotic, promoting inflammation and autoimmunity. Remarkably, even in tissues with high cellular turnover, apoptotic cells are rarely seen because of efficient clearance mechanisms in healthy individuals. Recently, significant progress has been made in understanding the steps involved in prompt cell clearance in vivo. These include the sensing of corpses via “find me” signals, the recognition of corpses via “eat me” signals and their cognate receptors, the signaling pathways that regulate cytoskeletal rearrangement necessary for engulfment, and the responses of the phagocyte that keep cell clearance events “immunologically silent.” This study focuses on our understanding of these steps. PMID:23284042

  16. 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

  17. Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response

    PubMed Central

    Nantajit, Danupon; Fan, Ming; Duru, Nadire; Wen, Yunfei; Reed, John C.; Li, Jian Jian

    2010-01-01

    The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53?/? cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53?/? cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53. PMID:20808790

  18. Usage of whey protein may cause liver damage via inflammatory and apoptotic responses.

    PubMed

    Gürgen, S G; Yücel, A T; Karaku?, Aç; Çeçen, D; Özen, G; Koçtürk, S

    2015-07-01

    The purpose of this study was to investigate the long- and short-term inflammatory and apoptotic effects of whey protein on the livers of non-exercising rats. Thirty rats were divided into three groups namely (1) control group, (2) short-term whey (WS) protein diet (252 g/kg for 5 days), and (3) long-term whey (WL) protein diet (252 g/kg for 4 weeks). Interleukin 1? (IL-1?), IL-6, tumor necrosis factor ? (TNF-?), and cytokeratin 18 (CK-18-M30) were assessed using enzyme-linked immunosorbent assay and immunohistochemical methods. Apoptosis was evaluated using the terminal transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method. Hepatotoxicity was evaluated by quanitation of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Based on the biochemical levels and immunohistochemical results, the highest level of IL-1? was identified in the WL group (p < 0.01). The IL-6 and TNF-? results were slightly lower in the WS group than in the control group and were highest in the WL group (p < 0.01). The CK-18-M30 and TUNEL results were highest in the WS group and exhibited medium intensity in the WL group (p < 0.01). AST results were statistically significant for all groups, while our ALT groups were particularly significant between the WL and control groups (p < 0.01). The results showed that when whey protein is used in an uninformed manner and without exercising, adverse effects on the liver may occur by increasing the apoptotic signal in the short term and increasing inflammatory markers and hepatotoxicity in the long term. PMID:25352651

  19. Pro-apoptotic function of GABA-related transcripts following stroke.

    PubMed

    Jaenisch, Nadine; Popp, Anke; Guenther, Madlen; Schnabel, Juliane; Witte, Otto W; Frahm, Christiane

    2014-10-01

    Following cerebral injuries such as stroke, a structural and functional reorganization of the impaired tissue occurs, which is often accompanied by a re-expression of developmental genes. During brain development, embryonic splice variants of the GABA-synthesizing GAD67 gene (collectively termed EGAD) participate in cell proliferation, migration, and neuronal differentiation. We thus hypothesized an involvement of EGAD in post-ischemic plasticity. EGAD transcripts were up-regulated at early reperfusion times in the injured area following transient middle cerebral artery occlusion (with a peak expression of 4.5-fold at 6h in C57BL/6 mice). Cell-specific analysis by a combination of radioactive in situ hybridization and immunolabeling revealed EGAD up-regulation in TUNEL-positive neurons. This unexpected cell death-associated expression of EGAD was confirmed in cell culture models of ischemia (combined oxygen-glucose deprivation) and apoptosis (staurosporine). Staurosporine-mediated cell death led to cleaved Caspase-3 activation, a key regulator of apoptosis following stroke. Blocking of staurosporine-associated EGAD expression via antisense RNA treatment reduced cleaved Caspase-3 activation by ~30%. In addition to the involvement of EGAD in proliferative processes during brain development, we found here that EGAD participates in cell death under pathophysiological conditions in the adult brain. Re-expression of EGAD in neurons following stroke may play a role in aberrant cell cycle activation, consequently being pro-apoptotic. Our observation of a new GABA related pro-apoptotic mechanism and its successful modification might be of significant clinical relevance. PMID:24983209

  20. Altered mitochondrial apoptotic pathway in placentas from undernourished rat gestations

    PubMed Central

    Desai, Mina; Michael Nelson, D.; Ross, Michael G.

    2011-01-01

    Maternal undernutrition (MUN) during pregnancy results in intrauterine growth-restricted (IUGR) fetuses and small placentas. Although reduced fetal nutrient supply has been presumed to be etiologic in IUGR, MUN-induced placental dysfunction may occur prior to detectable fetal growth restriction. Placental growth impairment may result from apoptosis signaled by mitochondria in response to reduced energy substrate. Therefore, we sought to determine the presence of mitochondrial-induced apoptosis under MUN and ad libitum diet (AdLib) pregnancies. Pregnant rats were fed an AdLib or a 50% MUN diet from embryonic day 10 (E10) to E20. At E20, fetuses and placentas from proximal- and mid-horns (extremes of nutrient/oxygen supply) were collected. Right-horn placentas were used to quantify apoptosis. Corresponding left-horn placentas were separated into basal (hormone production) and labyrinth (feto-maternal exchange) zones, and protein expression of the mitochondrial pathway was determined. Our results show that the MUN placentas had significantly increased apoptosis, with lower expression of cytosolic and mitochondrial anti-apoptotic Bcl2 and Bcl-XL, and significantly higher expression of pro-apoptotic Bax and Bak especially in the labyrinth zone. This was paralleled by higher coimmunostaining with the mitochondrial marker manganese superoxide dismutase (MnSOD), indicating transition of pro-apoptotic factors to the mitochondrial membrane. Also, cytosolic cytochrome c and activated caspases-9 and -3 were significantly higher in all MUN. Conversely, peroxisome proliferator-activator receptor-? (PPAR?), a member of the nuclear receptor family with anti-apoptotic properties, was significantly downregulated in both zones and horns. Our results suggest that MUN during rat pregnancy enhances mitochondria-dependent apoptosis in the placenta, probably due to the downregulation of PPAR? expression. PMID:21918224

  1. Apoptotic lymphocytes induce progenitor cell mobilization after exercise.

    PubMed

    Mooren, Frank C; Krüger, 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

  2. 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

  3. Effect of PDT-treated apoptotic cells on macrophages

    NASA Astrophysics Data System (ADS)

    Song, Sheng; Xing, Da; Zhou, Fei-fan; Chen, Wei R.

    2009-02-01

    Recently, the long-term immunological effects of photodynamic therapy have attracted much attention. PDT induced immune response was mainly initiated through necrotic cells and apoptotic cells, as well as immune cells such as macrophages. Nitric oxide (NO) as an important regulatory factor in signal transfer between cells has been wildly studied for generation, development, and metastasis of tumors. NO synthase is a key enzyme in nitric oxide synthesis. However, inducible nitric oxide synthase (iNOS) is usually activated under pathological conditions, such as stress and cancer, which can produce high levels of nitric oxide and contribute to tumor cytotoxicity. In addition, increased NO production by iNOS has been associated with the host immune response and cell apoptosis, which play an important role in many carcinogenesis and anti-carcinoma mechanisms. This study focuses on the NO production in macrophages, induced by mouse breast carcinoma apoptotic cells treated by PDT in vitro, and on the effects of immune response induced by apoptotic cells in tumor cells growth.

  4. Membrane Protected Apoptotic Trophoblast Microparticles Contain Nucleic Acids

    PubMed Central

    Orozco, Aaron F.; Jorgez, Carolina J.; Horne, Cassandra; Marquez-Do, Deborah A.; Chapman, Matthew R.; Rodgers, John R.; Bischoff, Farideh Z.; Lewis, Dorothy E.

    2008-01-01

    Microparticles (MPs) that circulate in blood may be a source of DNA for molecular analyses, including prenatal genetic diagnoses. Because MPs are heterogeneous in nature, however, further characterization is important before use in clinical settings. One key question is whether DNA is either bound to aggregates of blood proteins and lipid micelles or intrinsically associated with MPs from dying cells. To test the latter hypothesis, we asked whether MPs derived in vitro from dying cells were similar to those in maternal plasma. JEG-3 cells model extravillous trophoblasts, which predominate during the first trimester of pregnancy when prenatal diagnosis is most relevant. MPs were derived from apoptosis and increased over 48 hours. Compared with necrotic MPs, DNA in apoptotic MPs was more fragmented and resistant to plasma DNases. Membrane-specific dyes indicated that apoptotic MPs had more membranous material, which protects nucleic acids, including RNA. Flow cytometry showed that MPs derived from dying cells displayed light scatter and DNA staining similar to MPs found in maternal plasma. Quantification of maternal MPs using characteristics defined by MPs generated in vitro revealed a significant increase of DNA+ MPs in the plasma of women with preeclampsia compared with plasma from women with normal pregnancies. Apoptotic MPs are therefore a likely source of stable DNA that could be enriched for both early genetic diagnosis and monitoring of pathological pregnancies. PMID:18974299

  5. Role of group A p21-activated kinases in the anti-apoptotic activity of the pseudorabies virus US3 protein kinase.

    PubMed

    Van den Broeke, C; Radu, M; Nauwynck, H J; Chernoff, J; Favoreel, H W

    2011-01-01

    The alphaherpesvirus US3 kinase is a conserved multifunctional serine/threonine kinase that plays a role in several processes, including modulation of the actin cytoskeleton, egress of virus particles from the nucleus and inhibition of apoptosis. However, the mechanisms used by the US3 protein to exert its functions remain poorly understood. Recently, we identified the group A p21-activated kinases PAK1 and PAK2 as important effectors in the US3-mediated cytoskeletal rearrangements. Here, we investigated if group A PAKs are also involved in the anti-apoptotic properties of US3. Infection experiments using a group A PAK inhibitor pointed at a moderate role for group A PAKs in the anti-apoptotic properties of US3. Furthermore, infection assays using wild type and US3null PRV in wild type MEF, PAK1(-/-) MEF and PAK2(-/-) MEF indicated that PAK2 does not play a role in US3-mediated inhibition of apoptosis during infection, whereas PAK1 plays a significant, yet limited role. Experiments in US3-transfected MEF using staurosporine as apoptosis trigger confirmed these observations. These results show that PAK1 plays a significant, yet limited, role in the anti-apoptotic activity of US3. PMID:21093504

  6. Treatment with annexin V increases immunogenicity of apoptotic human T-cells in Balb\\/c mice

    Microsoft Academic Search

    C M Stach; X Turnay; R E Voll; P M Kern; W Kolowos; T D Beyer; J R Kalden; M Herrmann

    2000-01-01

    Exposure of phosphatidylserine on the outer leaflet of the cytoplasmic membrane is an early event during apoptotic cell death and serves as a recognition signal for phagocytes. Usually the clearance of apoptotic cells does not initiate inflammation or immune response. We investigated the immune response in Balb\\/c mice towards apoptotic human T-cells. Animals injected with apoptotic cells showed significantly reduced

  7. The expression patterns of pro-apoptotic and anti-apoptotic factors in human fetal and adult ovary.

    PubMed

    Poljicanin, Ana; Vukusic Pusic, Tanja; Vukojevic, Katarina; Caric, Ana; Vilovic, Katarina; Tomic, Snjezana; Soljic, Violeta; Saraga-Babic, Mirna

    2013-07-01

    The influence of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins on the cell death (caspase-3, TUNEL) of different ovarian cell lineages was immunohistochemically analyzed in six fetal and five adult human ovaries in order to disclose possible mechanisms of cell number control. Mild to moderate expression of Bcl-2 characterized ovarian surface epithelium, follicular cells and oocytes of 15 and 22 week human ovaries, while expression of Bax and caspase-3 gradually increased in all ovarian cell populations, except caspase-3 in the ovarian surface epithelium. Different levels of Bax and Bcl-2 proteins co-expression characterized fetal ovarian cells, while TUNEL and caspase-3 co-expression was found only in some of them. In adult ovaries, Bcl-2 was moderately and Bax strongly expressed in the surface ovarian epithelium and stroma. Bcl-2 and Bax expression in granulosa and theca interna cells varied depending on the stage of follicular atresia. Caspase-3 apoptotic cells characterized granulosa cells of adult atretic follicles. Our results indicate that intracellular levels of Bcl-2 and Bax protein might regulate the final destiny of developing germ cells. Caspase-3 dependent apoptosis seems to be the most important, but not the only cell death pathway in ovaries. In adult ovaries, caspase-dependent cell death characterized granulosa cells, but not the germ cells. PMID:23295106

  8. 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; Nör, 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

  9. 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

  10. Resveratrol protects astrocytes against traumatic brain injury through inhibiting apoptotic and autophagic cell death

    PubMed Central

    Lin, C-J; Chen, T-H; Yang, L-Y; Shih, C-M

    2014-01-01

    Traumatic brain injury (TBI) is often caused by accidents that damage the brain. TBI can induce glutamate excitotoxicity and lead to neuronal and glial cell death. In this study, we investigated the mechanism of cell death during the secondary damage caused by TBI in vivo and in vitro, as well as the protective effect of resveratrol (RV). Here we report that glycogen synthase kinase-3? (GSK-3?) activation and microtubule-associated protein light chain 3 processing were induced in rat brains exposed to TBI. In the in vitro TBI model, apoptotic and autophagic cell death were induced through glutamate-mediated GSK-3? activation in normal CTX TNA2 astrocytes. The GSK-3? inhibitor SB216763 or transfection of GSK-3? small-interfering RNA increases cell survival. By contrast, overexpression of GSK-3? enhanced glutamate excitotoxicity. Administration of RV reduced cell death in CTX TNA2 astrocytes by suppressing reactive oxygen species (ROS)-mediated GSK-3? activation, the mechanism by which RV also exerted protective effects in vivo. Mitochondrial damages, including the opening of mitochondrial permeability transition pore (MPTP) and mitochondrial depolarization, were induced by glutamate through the ROS/GSK-3? pathway. Moreover, cyclosporine A, an MPTP inhibitor, suppressed mitochondrial damage and the percentages of cells undergoing autophagy and apoptosis and thereby increased cell survival. Taken together, our results demonstrated that cell death occurring after TBI is induced through the ROS/GSK-3?/mitochondria signaling pathway and that administration of RV can increase cell survival by suppressing GSK-3?-mediated autophagy and apoptosis. Therefore, the results indicated that resveratrol may serve as a potential therapeutic agent in the treatment of TBI. PMID:24675465

  11. Effect of Reactive Oxygen Species Generation in Rabbit Corneal Epithelial Cells on Inflammatory and Apoptotic Signaling Pathways in the Presence of High Osmotic Pressure

    PubMed Central

    Li, Bing; Wang, Weifang; Lin, Anjuan; Sheng, Minjie

    2013-01-01

    It is generally accepted that high osmotic pressure (HOP) of lacrimal fluid is the core mechanism causing ocular inflammation and injury. However, the association between HOP and the regulation of cell inflammatory response and apoptotic pathways remains unclear. In the present study, we used HOP to interfere with in vitro cultured rabbit corneal epithelial cells, and found that HOP increased the generation of reactive oxygen species (ROS) in rabbit corneal epithelial cells, and increased ROS in turn induced the activation of JNK inflammatory signaling pathway, which further promoted the expression of pro-inflammatory factor NF-?? and induced the generation of inflammatory factor IL-1? and TNF-?. In addition, HOP-induced ROS in rabbit corneal epithelial cells regulated the CD95/CD95L-mediated cell apoptotic signaling pathway by activating JNK inflammatory signaling pathway. These findings may serve as new theoretical basis and a new way of thinking about the treatment of ocular diseases, especially dry eye. PMID:23977369

  12. Blockade of PKCdelta proteolytic activation by loss of function mutants rescues mesencephalic dopaminergic neurons from methylcyclopentadienyl manganese tricarbonyl (MMT)-induced apoptotic cell death.

    PubMed

    Anantharam, V; Kitazawa, M; Latchoumycandane, C; Kanthasamy, A; Kanthasamy, A G

    2004-12-01

    The use of methylcyclopentadienyl manganese tricarbonyl (MMT) as a gasoline additive has raised health concerns and increased interest in understanding the neurotoxic effects of manganese. Chronic exposure to inorganic manganese causes Manganism, a neurological disorder somewhat similar to Parkinson's disease. However, the cellular mechanism by which MMT, an organic manganese compound, induces neurotoxicity in dopaminergic neuronal cells remains unclear. Therefore, we systematically investigated apoptotic cell-signaling events following exposure to 3-200 microM MMT in mesencephalic dopaminergic neuronal (N27) cells. MMT treatment resulted in a time- and dose-dependent increase in reactive oxygen species generation and cell death in N27 cells. The cell death was preceded by sequential activation of mitochondrial-dependent proapoptotic events including cytochrome c release, caspase-3 activation, and DNA fragmentation, indicating that the mitochondrial-dependent apoptotic cascade primarily triggers MMT-induced apoptotic cell death. Importantly, MMT induced proteolytic cleavage of protein kinase Cdelta (PKCdelta), resulting in persistently increased kinase activity. The proteolytic activation of PKCdelta was suppressed by treatment with 100 microM Z-VAD-FMK and 100 microM Z-DEVD-FMK, suggesting that caspase-3 mediates the proteolytic activation of PKCdelta. Pretreatment with 100 microM Z-DEVD-FMK and 5 microM rottlerin (a PKCdelta inhibitor) also significantly attenuated MMT-induced DNA fragmentation. Furthermore, overexpression of either the kinase inactive dominant negative PKCdelta(K376R) mutant or the caspase cleavage resistant PKCdelta(D327A) mutant rescued N27 cells from MMT-induced DNA fragmentation. Collectively, these results demonstrate that the mitochondrial-dependent apoptotic cascade mediates apoptosis via proteolytic activation of PKCdelta in MMT-induced dopaminergic degeneration and suggest that PKCdelta may serve as an attractive therapeutic target in Parkinson-related neurological diseases. PMID:15681813

  13. Adaptor protein GULP is involved in stabilin-1-mediated phagocytosis.

    PubMed

    Park, Seung-Yoon; Kim, Sang-Yeob; Kang, Kae-Bok; Kim, In-San

    2010-07-30

    The clearance of apoptotic cells is critical during cellular homeostasis as well as inflammation resolution. Recently, we found that stabilin-1 is a phagocytic receptor that is involved in the clearance of apoptotic cells. However, the downstream signaling pathway of stabilin-1-mediated phagocytosis remains to be investigated. Here we identify that GULP is able to specifically interact with the NPxF/Y motif of stabilin-1 cytoplasmic region. The PTB domain of GULP is necessary for interaction with stabilin-1. GULP is enriched around PS-coated beads for phagocytosis and co-localized with stabilin-1. Downregulation of endogenous GULP expression decreased stabilin-1-mediated phagocytosis. Thus, these results indicate that GULP functions as an adaptor protein for stabilin-1-mediated phagocytosis. PMID:20599701

  14. 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 ?m 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

  15. Effects of Cyclooxygenase Inhibitors on Apoptotic Neuroretinal Cells

    PubMed Central

    Brust, Anja-Kristina; Ulbrich, Holger K.; Seigel, Gail M.; Pfeiffer, Norbert; Grus, Franz H.

    2008-01-01

    Glaucoma is characterized by a loss of retinal ganglion cells (RGC) which is associated with a decrease of visual function. Neuroprotective agents as a new therapeutic strategy could prevent the remaining neurons from apoptotic cell death. Previous studies have shown the involvement of the Cyclooxygenase (COX)-2 signalling in the apoptotic death of neurons. Herein we investigated the neuroprotective effect of COX-1/COX-2- and selective COX-2- inhibitors on apoptotic. R28, a neuroretinal cell line and determined the PGE2 levels by ELISA. Furthermore we investigated differences in protein expression in the cells after exposure to elevated pressure compared to untreated cells by ProteinChip analysis. In addition, a protein profiling study of the cells after exposure to elevated pressure was performed. The protein expression profiles were measured by SELDI-TOF (Surface Enhanced Laser Desorption/Ionization-time of flight) Protein Chips. The protein identification was performed by mass spectrometry (MS). It could be shown that COX-2 inhibition significantly prevented the cells from apoptosis and reduced the PGE2 concentrations. Selective COX-2 inhibitors were significant more potent than non-selective inhibitors or COX-1 inhibitors. We found differently expressed protein patterns in neuroretinal cells cultured at atmospheric pressure compared to those cells exposed to elevated pressure with or without celecoxib respectively. We identified three biomarkers, ubiquitin, HSP10 and NDKB, which were differently expressed in the groups. However, our data indicates a distinct neuroprotective effect of COX-2 inhibition. The local treatment with selective COX-2 inhibitors might provide an innovative strategy of therapeutic intervention for glaucoma. PMID:19578520

  16. Autoimmune Disease and Impaired Uptake of Apoptotic Cells in MFG-E8Deficient Mice

    Microsoft Academic Search

    Rikinari Hanayama; Masato Tanaka; Kay Miyasaka; Katsuyuki Aozasa; Masato Koike; Yasuo Uchiyama; Shigekazu Nagata

    2004-01-01

    Apoptotic cells expose phosphatidylserine and are swiftly engulfed by macrophages. Milk fat globule epidermal growth factor (EGF) factor 8 (MFG-E8) is a protein that binds to apoptotic cells by recognizing phosphatidylserine and that enhances the engulfment of apoptotic cells by macrophages. We report that tingible body macrophages in the germinal centers of the spleen and lymph nodes strongly express MFG-E8.

  17. 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

  18. PIAS3 activates the intrinsic apoptotic pathway in non-small cell lung cancer cells independent of p53 status

    PubMed Central

    Dabir, Snehal; Kluge, Amy; McColl, Karen; Liu, Yu; Lam, Minh; Halmos, Balazs; Wildey, Gary; Dowlati, Afshin

    2014-01-01

    Protein inhibitor of activated STAT3 (PIAS3) is an endogenous inhibitor of STAT3 that negatively regulates STAT3 transcriptional activity and cell growth and demonstrates limited expression in the majority of human squamous cell carcinomas of the lung. In the present study we sought to determine if PIAS3 inhibits cell growth in non-small cell lung cancer (NSCLC) cell lines by inducing apoptosis. Our results demonstrate that over-expression of PIAS3 promotes mitochondrial depolarization, leading to cytochrome c release, caspase 9 and 3 activation and PARP cleavage. This intrinsic pathway activation was associated with decreased Bcl-xL expression and increased Noxa expression and was independent of p53 status. Furthermore, PIAS3 inhibition of STAT3 activity was also p53 independent. Microarray experiments were performed to discover STAT3-independent mediators of PIAS3-induced apoptosis by comparing the apoptotic gene expression signature induced by PIAS3 over-expression with that induced by STAT3 siRNA. The results showed that a subset of apoptotic genes was uniquely expressed only after PIAS3 expression. Thus, PIAS3 may represent a promising lung cancer therapeutic target because of its p53-independent efficacy as well as its potential to synergize with Bcl-2 targeted inhibitors. PMID:23959540

  19. A novel SOD-mimetic permeability transition inhibitor agent protects ischemic heart by inhibiting both apoptotic and necrotic cell death.

    PubMed

    Bognar, Zita; Kalai, Tamas; Palfi, Anita; Hanto, Katalin; Bognar, Balazs; Mark, Laszlo; Szabo, Zoltan; Tapodi, Antal; Radnai, Balazs; Sarszegi, Zsolt; Szanto, Arpad; Gallyas, Ferenc; Hideg, Kalman; Sumegi, Balazs; Varbiro, Gabor

    2006-09-01

    In ischemia-reperfusion injuries, elevated calcium and reactive oxygen species (ROS) induce mitochondrial permeability transition (mPT), which plays a pivotal role in mediating damages and cell death. Inhibition of mPT decreases necrotic cell death; however, during reperfusion, the continuous production of ROS may contribute to the temporary opening of the pore and thus the onset of the delayed apoptotic cell death. Based on amiodarone structure, we developed the first SOD-mimetic mPT inhibitor (HO-3538) that can eliminate ROS in the microenvironment of the permeability pore. In isolated mitochondria, HO-3538 inhibited mPT and the release of proapoptotic mitochondrial proteins. It had a ROS scavenging effect and antiapoptotic effect in a cardiomyocyte line and it diminished release of mitochondrial proapoptotic proteins. Furthermore, HO-3538 significantly enhanced the recovery of mitochondrial energy metabolism and functional cardiac parameters; decreased infarct size, lipid peroxidation, and protein oxidation; and suppressed necrotic as well as apoptotic cell death pathways in Langendorff-perfused hearts. In these respects it was somewhat superior to its two constituents, amiodarone and a pyrrol-derivative free radical scavenger. These data suggest that the SOD-mimetic mPT inhibitors are ideal candidates for drug development for the alleviation of postinfarct myocardial injuries. PMID:16895804

  20. Senescence may mediate conversion of tau phosphorylation-induced apoptotic escape to neurodegeneration.

    PubMed

    Wang, Jian-Zhi; Wang, Zhi-Hao

    2015-08-01

    Neurodegeneration is the characteristic pathology in the brains of Alzheimer's disease (AD). However, the nature and molecular mechanism leading to the degeneration are not clarified. Given that only the neurons filled with neurofibrillary tangles survive to the end stage of the disease and the major component of the tangles is the hyperphosphorylated tau proteins, it is conceivable that tau hyperphosphorylation must play a crucial role in AD neurodegeneration. We have demonstrated that tau hyperphosphorylation renders the cells more resistant to the acute apoptosis. The molecular mechanisms involve substrate competition of tau and ?-catenin for glycogen synthase kinase 3? (GSK-3?); activation of Akt; preservation of Bcl-2 and suppression of Bax, cytosolic cytochrome-c, and caspase-3 activity; and upregulation of unfolded protein response (UPR), i.e., up-regulating phosphorylation of PERK, eIF2 and IRE1 with an increased cleavage of ATF6 and ATF4. On the other hand, tau hyperphosphorylation promotes its intracellular accumulation and disrupts axonal transport; hyperphosphorylated tau also impairs cholinergic function and inhibits proteasome activity. These findings indicate that tau hyperphosphorylation and its intracellular accumulation play dual role in the evolution of AD. We speculate that transient tau phosphorylation helps cells abort from an acute apoptosis, while persistent tau hyperphosphorylation/accumulation may trigger cell senescence that eventually causes a chronic neurodegeneration. Therefore, the nature of "AD neurodegeneration" may represent a new type of tau-regulated chronic neuron death; and the stage of cell senescence may provide a broad window for the intervention of AD. PMID:25777063

  1. Short term apoptotic activity of intravitreal bevacizumab on rabbit retina

    PubMed Central

    Türkcü, Fatih Mehmet; Alp, Mehmet Numan; Türkcü, Gül; Kulaço?lu, Sezer; Kural, Gülcan

    2013-01-01

    AIM To evaluate the safety and the short term apoptotic activity of intravitreal bevacizumab in rabbit eyes by histopathological analysis. METHODS Twenty-eight eyes of 14 rabbits were divided into three groups: 8 rabbits in group 1 and 3 rabbits in each of group 2 and group 3. Intravitreal bevacizumab (1.25mg/0.05mL) was applied to the right eyes of each subject in group 1 and group 2 (11 eyes) and the same volume of saline was applied to the left eyes of each subject in group 1 and group 3 (11 eyes). The left eyes in group 2 and the right eyes in group 3 were left untreated and used as control. Enucleated eyes were used for histopathologic analyses. RESULTS After immunohistochemical staining with caspase-3 and p53, there was no histological evidence of toxicity to the retina and the optic nerve in any of the sections that were analyzed in all three groups. In addition, vascular endothelial cells located at the retina and the optic nerve tissues in all groups showed a similar staining pattern with caspase-3 and p53. CONCLUSION Our study showed that intravitreal bevacizumab with the dose of 1.25mg/0.05mL caused no histological signs of toxicity or apoptotic activity on the rabbit retina. PMID:24392325

  2. A simple method for accurate estimation of apoptotic cells.

    PubMed

    Singh, N P

    2000-04-10

    A simple, sensitive, and reliable "DNA diffusion" assay for the quantification of apoptosis is described. Human lymphocytes and human lymphoblastoid cells, MOLT-4, were exposed to 0, 12.5, 25, 50, or 100 rad of X-rays. After 24 h of incubation, cells were mixed with agarose, microgels were made, and cells were lysed in high salt and detergents. DNA was precipitated in microgels by ethanol. Staining of DNA was done with an intense fluorescent dye, YOYO-1. Apoptotic cells show a halo of granular DNA with a hazy outer boundary. Necrotic cells, resulting from hyperthermia treatment, on the other hand, show an unusually large homogeneous nucleus with a clearly defined boundary. The number of cells with apoptotic and necrotic appearance can be scored and quantified by using a fluorescent microscope. Results were compared with other methods of apoptosis measurement: morphological estimations of apoptosis and DNA ladder pattern formation in regular agarose gel electrophoresis. Validation of the technique was done using some known inducers of apoptosis and necrosis (hyperthermia, hydrogen peroxide, mitoxantrone, novobiocin, and sodium ascorbate). PMID:10739681

  3. Heat-Induced Fibrillation of BclXL Apoptotic Repressor

    PubMed Central

    Bhat, Vikas; Olenick, Max B.; Schuchardt, Brett J.; Mikles, David C.; Deegan, Brian J.; McDonald, Caleb B.; Seldeen, Kenneth L.; Kurouski, Dmitry; Faridi, Mohd Hafeez; Shareef, Mohammed M.; Gupta, Vineet; Lednev, Igor K.; Farooq, Amjad

    2013-01-01

    The BclXL apoptotic repressor bears the propensity to associate into megadalton oligomers in solution, particularly under acidic pH. Herein, using various biophysical methods, we analyze the effect of temperature on the oligomerization of BclXL. Our data show that BclXL undergoes irreversible aggregation and assembles into highly-ordered rope-like homogeneous fibrils with length in the order of mm and a diameter in the ?m-range under elevated temperatures. Remarkably, the formation of such fibrils correlates with the decay of a largely ?-helical fold into a predominantly ?-sheet architecture of BclXL in a manner akin to the formation of amyloid fibrils. Further interrogation reveals that while BclXL fibrils formed under elevated temperatures show no observable affinity toward BH3 ligands, they appear to be optimally primed for insertion into cardiolipin bicelles. This salient observation strongly argues that BclXL fibrils likely represent an on-pathway intermediate for insertion into mitochondrial outer membrane during the onset of apoptosis. Collectively, our study sheds light on the propensity of BclXL to form amyloid-like fibrils with important consequences on its mechanism of action in gauging the apoptotic fate of cells in health and disease. PMID:23714425

  4. 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

  5. Jedi-1 and MEGF10 signal engulfment of apoptotic neurons through the tyrosine kinase Syk

    PubMed Central

    Scheib, Jami L; Sullivan, Chelsea S; Carter, Bruce D

    2012-01-01

    During the development of the peripheral nervous system there is extensive apoptosis and these neuronal corpses need to be cleared to prevent an inflammatory response. Recently, Jedi-1 and MEGF10, both expressed in glial precursor cells, were identified in mouse as having an essential role in this phagocytosis (Wu et al., 2009); however, the mechanisms by which they promote engulfment remained unknown. Both Jedi-1 and MEGF10 are homologous to the Drosophila melanogaster receptor Draper, which mediates engulfment through activation of the tyrosine kinase Shark. Here, we identify Syk, the mammalian homolog of Shark, as a signal transducer for both Jedi-1 and MEGF10. Syk interacted with each receptor independently through the Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) in their intracellular domains. The interaction was enhanced by phosphorylation of the tyrosines in the ITAMs by Src family kinases (SFKs). Jedi association with Syk and activation of the kinase was also induced by exposure to dead cells. Expression of either Jedi-1 or MEGF10 in HeLa cells facilitated engulfment of carboxylated microspheres to a similar extent and there was no additive effect when they were co-expressed. Mutation of the ITAM tyrosines of Jedi-1 and MEGF10 prevented engulfment. The SFK inhibitor PP2 or a selective Syk inhibitor (BAY 61-3606) also blocked engulfment. Similarly, in co-cultures of glial precursors and dying sensory neurons from embryonic mice, addition of PP2 or knock down of endogenous Syk decreased the phagocytosis of apoptotic neurons. These results indicate that both Jedi-1 and MEGF10 can mediate phagocytosis independently through the recruitment of Syk. PMID:22993420

  6. Death ligand concentration and the membrane proximal signaling module regulate the type 1/ type 2 choice in apoptotic death signaling

    E-print Network

    Raychaudhuri, Subhadip

    2015-01-01

    Apoptotic death pathways are frequently activated by death ligand induction and subsequent activation of the membrane proximal signaling module. Death receptors cluster upon binding to death ligands, leading to formation of a membrane proximal death-inducing-signaling-complex (DISC). In this membrane proximal signalosome, initiator caspases (caspase 8) are processed resulting in activation of both type 1 and type 2 pathways of apoptosis signaling. How the type 1/type 2 choice is made is an important question in the systems biology of apoptosis signaling. In this study, we utilize a Monte Carlo based in silico approach to elucidate the role of membrane proximal signaling module in the type 1/type 2 choice of apoptosis signaling. Our results provide crucial mechanistic insights into the formation of DISC signalosome and caspase 8 activation. Increased concentration of death ligands was shown to correlate with increased type 1 activation. We also study the caspase 6 mediated system level feedback activation of a...

  7. 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...

  8. CDK4 inhibition and doxorubicin mediate breast cancer cell apoptosis through Smad3 and survivin.

    PubMed

    Tarasewicz, Elizabeth; Hamdan, Randala; Straehla, Joelle; Hardy, Ashley; Nunez, Omar; Zelivianski, Stanislav; Dokic, Danijela; Jeruss, Jacqueline S

    2014-10-01

    Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGF? superfamily member Smad3. We sought to determine if CDK4 inhibition and doxorubicin chemotherapy could impact Smad3-mediated cell/colony growth and apoptosis in breast cancer cells. Parental and cyclin D1-overexpressing MCF7 cells were treated with CDK4 inhibitor, doxorubicin, or combination therapy and cell proliferation, apoptosis, colony formation, and expression of apoptotic proteins were evaluated using an MTS assay, TUNEL staining, 3D Matrigel assay, and apoptosis array/immunoblotting. Study cells were also transduced with WT Smad3 or a Smad3 construct resistant to CDK4 phosphorylation (5M) and colony formation and expression of apoptotic proteins were assessed. Treatment with CDK4 inhibitor/doxorubicin combination therapy, or transduction with 5M Smad3, resulted in a similar decrease in colony formation. Treating cyclin D overexpressing breast cancer cells with combination therapy also resulted in the greatest increase in apoptosis, resulted in decreased expression of anti-apoptotic proteins survivin and XIAP, and impacted subcellular localization of pro-apoptotic Smac/DIABLO. Additionally, transduction of 5M Smad3 and doxorubicin treatment resulted in the greatest change in apoptotic protein expression. Collectively, this work showed the impact of CDK4 inhibitor-mediated, Smad3-regulated tumor suppression, which was augmented in doxorubicin-treated cyclin D-overexpressing study cells. PMID:25006666

  9. 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 ?-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

  10. 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 ?-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

  11. The phosphatidylserine receptor TIM-4 does not mediate direct signaling.

    PubMed

    Park, Daeho; Hochreiter-Hufford, Amelia; Ravichandran, Kodi S

    2009-02-24

    Engulfment of apoptotic cells is an active process coordinated by receptors on phagocytes and ligands on apoptotic cells [1]. Phosphatidylserine (PtdSer) is a key ligand on apoptotic cells, and recently three PtdSer recognition receptors have been identified, namely, TIM-4, BAI1, and Stabilin-2 [1-6]. Whereas BAI1 is dependent on the ELMO1/Dock180/Rac signaling module, and Stablilin-2 appears to use the intracellular adaptor GULP [2, 3, 7], little is known about how TIM-4 transduces signals downstream of PtdSer recognition [8]. To test the role of known engulfment signaling pathways in TIM-4-mediated engulfment, we used a combination of dominant-negative mutants, knockdown of specific signaling proteins, and knockout cell lines. TIM-4 appears to be largely independent of the two known engulfment signaling pathways [7, 9-17], yet the TIM-4-mediated uptake is inhibited by cytoskeleton disrupting drugs. Remarkably, a version of TIM-4 lacking its cytoplasmic tail promoted corpse uptake via PtdSer recognition. Moreover, replacement of the transmembrane region of TIM-4 with a glycophosphatidylinositol anchor still promoted engulfment comparable to wild-type TIM-4. Thus, the transmembrane region and cytoplasmic tail of TIM-4 are dispensable for apoptotic cell engulfment, and we propose that TIM-4 is a PtdSer tethering receptor without any direct signaling of its own. PMID:19217291

  12. 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

  13. Caenorhabditis elegans Myotubularin MTM-1 Negatively Regulates the Engulfment of Apoptotic Cells

    Microsoft Academic Search

    Wei Zou; Qun Lu; Dongfeng Zhao; Weida Li; James Mapes; Yuting Xie; Xiaochen Wang

    2009-01-01

    During programmed cell death, apoptotic cells are recognized and rapidly engulfed by phagocytes. Although a number of genes have been identified that promote cell corpse engulfment, it is not well understood how phagocytosis of apoptotic cells is negatively regulated. Here we have identified Caenorhabditis elegans myotubularin MTM-1 as a negative regulator of cell corpse engulfment. Myotubularins (MTMs) constitute a large,

  14. Phagocytic Receptor CED1 Initiates a Signaling Pathway for Degrading Engulfed Apoptotic Cells

    Microsoft Academic Search

    Xiaomeng Yu; Nan Lu; Zheng Zhou

    2008-01-01

    Apoptotic cells in animals are engulfed by phagocytic cells and subsequently degraded inside phagosomes. To study the mechanisms controlling the degradation of apoptotic cells, we developed time-lapse imaging protocols in developing Caenorhabditis elegans embryos and established the temporal order of multiple events during engulfment and phagosome maturation. These include sequential enrichment on phagocytic membranes of phagocytic receptor cell death abnormal

  15. Autoantigens as substrates for apoptotic proteases: implications for the pathogenesis of systemic autoimmune disease

    Microsoft Academic Search

    Antony Rosen; Livia Casciola-Rosen

    1999-01-01

    Systemic autoimmune diseases are a genetically complex, heterogeneous group of diseases in which the immune system targets a diverse, but highly specific group of intracellular autoantigens. The clustering and marked concentration of these molecules in the surface blebs of apoptotic cells, and their modification by apoptosis-specific proteolytic cleavage and\\/or phosphorylation at these sites, has focused attention on a unique apoptotic

  16. Fitness Landscapes of Evolved Apoptotic Cellular Automata Daniel Ashlock and Sharon McNicholas

    E-print Network

    Ashlock, Dan

    Fitness Landscapes of Evolved Apoptotic Cellular Automata Daniel Ashlock and Sharon McNicholas Abstract--This paper examines the fitness landscape for evo- lutionary algorithms evolving cellular automata rules to satisfy an apoptotic fitness function. This fitness function requires the automata

  17. Spatiotemporal evolution of apoptotic neurodegeneration following traumatic injury to the developing rat brain

    Microsoft Academic Search

    Philip V. Bayly; Krikor T. Dikranian; Erin E. Black; Chainllie Young; Yue-Qin Qin; Joann Labruyere; John W. Olney

    2006-01-01

    Closed head injury to the developing rat brain causes an acute excitotoxic lesion and axonal disruption at the impact site followed by a delayed pattern of apoptotic damage at various distant sites. Using an electromagnetic impact device to deliver a precisely controlled degree of mechanical deformation to the P7 infant rat skull, we studied the distribution of distant apoptotic lesions

  18. Autoantigens are translocated into small apoptotic bodies during early stages of apoptosis

    Microsoft Academic Search

    M Schiller; I Bekeredjian-Ding; P Heyder; N Blank; A D Ho; H-M Lorenz

    2008-01-01

    A dysregulation of apoptosis or an ineffective clearance of apoptotic material is suspected to be involved in the pathogenesis of systemic lupus erythematodes. Subcellular fragments such as apoptotic bodies (ABs) have been recognized as modulators of intercellular communication and immune function. In this context, we have been interested whether nuclear and cytoplasmic antigens are relocated into ABs. In the present

  19. Cholesterol-derived novel anti-apoptotic agents on the structural basis of ginsenoside Rk1

    E-print Network

    Suh, Young-Ger

    that ginsenoside Rg3 isolated from the root of genus Panax (ginseng), which exerts many pharmacological activitiesCholesterol-derived novel anti-apoptotic agents on the structural basis of ginsenoside Rk1 Sujin Cholesterol Ginsenoside Rk1 a b s t r a c t Design and synthesis of cholesterol-derived anti-apoptotic agents

  20. Pro-apoptotic gene regulation in the Caribbean fruit fly, Anastrepha suspensa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcriptional activation of pro-apoptotic genes in response to cytotoxic stimuli is a conserved feature of the cell death pathway proposed for metazoans. However, understanding the extent of this conservation in insects, as well as other organisms, has been limited by the lack of known pro-apoptot...

  1. Cooperative binding of Annexin A5 to phosphatidylserine on apoptotic cell membranes

    NASA Astrophysics Data System (ADS)

    Janko, Christina; Jeremic, Ivica; Biermann, Mona; Chaurio, Ricardo; Schorn, Christine; Muñoz, Luis E.; Herrmann, Martin

    2013-12-01

    Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major ‘eat-me’ signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

  2. 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. [Department of Pathology and Laboratory Medicine, 422 Curie Blvd., 505 Stellar-Chance Laboratories, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 (United States); Chambers, Jerome A. [Department of Pathology and Laboratory Medicine, 422 Curie Blvd., 505 Stellar-Chance Laboratories, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 (United States); Pankhong, Panyupa [Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Bangkok 10700 (Thailand); Chattergoon, Michael [Department of Pathology and Laboratory Medicine, 422 Curie Blvd., 505 Stellar-Chance Laboratories, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 (United States); Attatippaholkun, Watcharee [Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Bangkok 10700 (Thailand); Dang, Kesen [Department of Pathology and Laboratory Medicine, 422 Curie Blvd., 505 Stellar-Chance Laboratories, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 (United States); Shah, Neelima [Department of Pathology and Laboratory Medicine, 422 Curie Blvd., 505 Stellar-Chance Laboratories, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 (United States); Weiner, David B. [Department of Pathology and Laboratory Medicine, 422 Curie Blvd., 505 Stellar-Chance Laboratories, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 (United States)]. 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-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV.

  3. Repeated low-dose 17?-estradiol treatment prevents activation of apoptotic signaling both in the synaptosomal and cellular fraction in rat prefrontal cortex following cerebral ischemia.

    PubMed

    Stanojlovi?, Miloš; Zlatkovi?, Jelena; Guševac, Ivana; Grkovi?, Ivana; Mitrovi?, Nataša; Zari?, Marina; Horvat, Anica; Drakuli?, Dunja

    2015-01-01

    Disturbance in blood circulation is associated with numerous pathological conditions characterized by cognitive decline and neurodegeneration. Activation of pro-apoptotic signaling previously detected in the synaptosomal fraction may underlie neurodegeneration in the prefrontal cortex of rats submitted to permanent bilateral common carotid arteries occlusion (two-vessel occlusion, 2VO). 17?-Estradiol (E) exerts potent neuroprotective effects in the brain affecting, among other, ischemia-induced pathological changes. As most significant changes in rats submitted to 2VO were observed on 7th day following the insult, of interest was to examine whether 7 day treatment with low dose of E (33.3?µg/kg/day) prevents formerly reported neurodegeneration and may represent additional therapy during the early post-ischemic period. Role of E treatment on apoptotic pathway was monitored on Bcl-2 family members, cytochrome c, caspase 3 and PARP protein level in the synaptosomal (P2) fraction of the prefrontal cortex. Furthermore, changes of these proteins were examined in the cytosolic, mitochondrial and nuclear fraction, with the emphasis on potential involvement of extracellular signal-regulated kinases (ERK) and protein kinase B (Akt) activation and their role in nuclear translocation of transcriptional nuclear factor kappa B (NF-kB) associated with alteration of Bax and Bcl-2 gene expression. The extent of cellular damage was determined using DNA fragmentation and Fluoro-Jade B staining. The absence of activation of apoptotic cascade both in the P2 and cell accompanied with decreased DNA fragmentation and number of degenerating neurons clearly indicates that E treatment ensures the efficient protection against ischemic insult. Moreover, E-mediated modulation of pro-apoptotic signaling in the cortical cellular fractions involves cooperative activation of ERK and Akt, which may be implicated in the observed prevention of neurodegenerative changes. PMID:25777481

  4. DEFICIENT IN VITRO AND IN VIVO PHAGOCYTOSIS OF APOPTOTIC T CELLS BY RESIDENT MURINE ALVEOLAR MACROPHAGES

    PubMed Central

    Hu, Bin; Sonstein, Joanne; Christensen, Paul J.; Punturieri, Antonello; Curtis, Jeffrey L.

    2015-01-01

    Apoptotic lymphocytes are readily identified in murine lungs, both during the response to particulate antigen and in normal mice. Because apoptotic lymphocytes are seldom detected in other organs, we hypothesized that alveolar macrophages (AMø) clear apoptotic lymphocytes poorly. To test this hypothesis, we compared in vitro phagocytosis of apoptotic thymocytes by resident AMø and peritoneal Mø (PMø) from normal C57BL/6 mice. AMø were deficient relative to PMø both in percentage containing apoptotic thymocytes (19.1 ± 1.0% versus 96.0 ± 2.6% positive) and in phagocytic index (0.23 ± 0.02 versus 4.2 ± 0.67). This deficiency was not due to kinetic differences, was seen with six other inbred mouse strains, and was not observed using carboxylate-modified polystyrene microbeads. Annexin V blockade indicated that both Mø types cleared apoptotic T cells by a mechanism involving phosphatidylserine expression. By contrast, neither mAb blockade of a variety of receptors (CD11b, CD29, CD51, and CD61) known to be involved in clearance of apoptotic cells, nor the tetrapeptide RGDS, blocked ingestion by either type of Mø. To confirm these studies, apoptotic thymocytes were given intratracheally or intraperitoneally to normal mice and then AMø or PMø were recovered 30–240 min later. Ingestion of apoptotic thymocytes by AMø in vivo was significantly decreased at all times. Defective ingestion of apoptotic lymphocytes may preserve AMø capacity to produce proinflammatory cytokines in host defense, but could contribute to development of autoimmunity by failing to eliminate nucleosomes. PMID:10925298

  5. Neuronal activity-dependent protection against apoptotic and oxidative insults 

    E-print Network

    Baxter, Paul Stuart

    2012-06-22

    Patterns of physiological electrical activity in the central nervous system (CNS) cause longlasting changes in gene expression that promote neuronal survival. These changes can be mediated by signalling pathways activated ...

  6. Reduced expression of CD44 on monocytes and neutrophils in systemic lupus erythematosus: relations with apoptotic neutrophils and disease activity

    Microsoft Academic Search

    A P Cairns; A D Crockard; J R McConnell; P A Courtney; A L Bell

    2001-01-01

    BACKGROUNDIncreased numbers of apoptotic neutrophils, and impaired monocyte\\/macrophage clearance of apoptotic cells, have been demonstrated in systemic lupus erythematosus (SLE). CD44 is implicated in the clearance of apoptotic neutrophils.OBJECTIVETo determine the expression of CD44 on peripheral blood monocytes and neutrophils in SLE, and examine the relations with disease activity and numbers of circulating apoptotic neutrophils.METHODSPeripheral blood was sampled from 31

  7. MBP1 mediated apoptosis involves cytochrome c release from mitochondria

    Microsoft Academic Search

    Asish K Ghosh; Mainak Majumder; Robert Steele; Ta-Jen Liu; Ratna B Ray

    2002-01-01

    MBP-1, a cellular factor, appears to be involved in multiple functions, including transcriptional modulation, apoptosis and cell growth regulation. In this study, we have investigated the signaling pathway involved in MBP-1 mediated apoptotic cell death. Human carcinoma cells infected with a replication deficient adenovirus expressing MBP-1 (AdMBP-1) induced apoptosis, when compared with cells infected by replication-defective adenovirus (dl312) as a

  8. Mitochondria mediated cell death in diabetes.

    PubMed

    Szabadkai, Gyorgy; Duchen, Michael R

    2009-12-01

    Mitochondrial dysfunction plays a role in the pathogenesis of a wide range of diseases that involve disordered cellular fuel metabolism and survival/death pathways, including neurodegenerative diseases, cancer and diabetes. Cytokine, virus recognition and cellular stress pathways converging on mitochondria cause apoptotic and/or necrotic cell death of beta-cells in type-1 diabetes. Moreover, since mitochondria generate crucial metabolic signals for glucose stimulated insulin secretion (GSIS), mitochondrial dysfunction underlies both the functional derangement of GSIS and (over-nutrition) stress-induced apoptotic/necrotic beta-cell death, hallmarks of type-2 diabetes. The apparently distinct mechanisms governing beta-cell life/death decisions during the development of diabetes provide a remarkable example where remote metabolic, immune and stress signalling meet with mitochondria mediated apoptotic/necrotic death pathways to determine the fate of the beta-cell. We summarize the main findings supporting such a pivotal role of mitochondria in beta-cell death in the context of current trends in diabetes research. PMID:19466549

  9. Role of factors downstream of caspases in nuclear disassembly during apoptotic execution.

    PubMed Central

    Samejima, K; Villa, P; Earnshaw, W C

    1999-01-01

    We used cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway to analyse the events of apoptotic execution. So-called S/M extracts from morphologically normal 'committed-stage' cells induce apoptotic morphology and DNA cleavage in substrate nuclei. These apoptotic changes appear to require the function of multiple caspases (cysteine aspartases, a specialized class of proteases) acting in parallel. Extracts from 'execution-stage' apoptotic cells induce apoptotic events in added nuclei in a caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. 'Execution-stage' extracts contain an ICAD/DFF45-inhibitable nuclease resembling CAD, plus another activity that is required for the apoptotic chromatin condensation. 'Committed-stage' S/M extracts lack these downstream activities. These observations reveal that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves. They also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to the execution phase of apoptosis. PMID:10582245

  10. O Death Where Is Thy Sting? Immunologic Tolerance To Apoptotic Self

    PubMed Central

    Ravishankar, Buvana; McGaha, Tracy L.

    2013-01-01

    In higher organisms, innate scavenging cells maintain physiologic homeostasis by removal of the billions of apoptotic cells generated on a daily basis. Apoptotic cell removal requires efficient recognition and uptake by professional and non-professional phagocytic cells, which are governed by an array of soluble and apoptotic cell-integral signals resulting in immunologically silent clearance. While apoptosis is associated with profound suppression of adaptive and innate inflammatory immunity, we have only begun to scratch the surface in understanding how immunologic tolerance to apoptotic self manifest at either the molecular or cellular level. In the last 10 years data has emerged implicating professional phagocytes, most notably stromal macrophages and CD8?+CD103+ dendritic cells, as critical in initiation of the regulatory cascade that will ultimately lead to long-term whole animal immune tolerance. Importantly, recent work by our lab and others has shown that alterations in apoptotic cell perception by the innate immune system either by removal of critical phagocytic sentinels in secondary lymphoid organs or blockage of immunosuppressive pathways leads to pronounced inflammation with a breakdown of tolerance towards self. This challenges the paradigm that apoptotic cells are inherently immunosuppressive suggesting that apoptotic cell tolerance is a “context dependent” event. PMID:23377225

  11. Marginal zone CD169+ macrophages coordinate apoptotic cell-driven cellular recruitment and tolerance

    PubMed Central

    Ravishankar, Buvana; Shinde, Rahul; Liu, Haiyun; Chaudhary, Kapil; Bradley, Jillian; Lemos, Henrique P.; Chandler, Phillip; Tanaka, Masato; Munn, David H.; Mellor, Andrew L.; McGaha, Tracy L.

    2014-01-01

    Tolerance to apoptotic cells is essential to prevent inflammatory pathology. Though innate responses are critical for immune suppression, our understanding of early innate immunity driven by apoptosis is lacking. Herein we report apoptotic cells induce expression of the chemokine CCL22 in splenic metallophillic macrophages, which is critical for tolerance. Systemic challenge with apoptotic cells induced rapid production of CCL22 in CD169+ (metallophillic) macrophages, resulting in accumulation and activation of FoxP3+ Tregs and CD11c+ dendritic cells, an effect that could be inhibited by antagonizing CCL22-driven chemotaxis. This mechanism was essential for suppression after apoptotic cell challenge, because neutralizing CCL22 or its receptor, reducing Treg numbers, or blocking effector mechanisms abrogated splenic TGF-? and IL-10 induction; this promoted a shift to proinflammatory cytokines associated with a failure to suppress T cells. Similarly, CCR4 inhibition blocked long-term, apoptotic cell-induced tolerance to allografts. Finally, CCR4 inhibition resulted in a systemic breakdown of tolerance to self after apoptotic cell injection with rapid increases in anti-dsDNA IgG and immune complex deposition. Thus, the data demonstrate CCL22-dependent chemotaxis is a key early innate response required for apoptotic cell-induced suppression, implicating a previously unknown mechanism of macrophage-dependent coordination of early events leading to stable tolerance. PMID:24591636

  12. ARK, the Apaf-1 related killer in Drosophila, requires diverse domains for its apoptotic activity.

    PubMed

    Srivastava, M; Scherr, H; Lackey, M; Xu, D; Chen, Z; Lu, J; Bergmann, A

    2007-01-01

    In mammals and Drosophila, apoptotic caspases are under positive control of the CED-4-like proteins Apaf-1 and ARK, respectively. In an EMS-mutagenesis screen, we isolated 33 ark mutants as recessive suppressors of hid-induced apoptosis. The ark mutants are loss-of-function alleles characterized by reduced developmental apoptosis. Using the phenotypic series of these alleles, we identified helical domain I in the nucleotide oligomerization domain as critical for ARK's apoptotic activity. Interestingly, the WD40 region may also have an unanticipated positive requirement for the apoptotic activity of ARK. Considering structural information, we discuss the roles of these domains for assembly and activity of the ARK apoptosome, and propose that the WD40 region is anti-apoptotic in the absence of apoptotic signals, and pro-apoptotic in the presence of such signals. Furthermore, a defined null allele reveals that ark is required for most, but not all apoptosis suggesting the existence of an ARK-independent apoptotic pathway. PMID:16645639

  13. Environmental mediation

    SciTech Connect

    Watson, J.L. (Kirkland and Ellis, Denver, CO); Danielson, L.J.

    1983-01-01

    Stemming from a 1975 Homestake Mining Company plan for developing uranium mining and milling in Saguache County, Colorado, protesters and the company agreed to a site-specific mediation process while an appealed decision was pending. The authors describe the mediation process which took nearly a year to reach settlement agreements, discussing the potential benefits of mediation in resolving such disputes and shedding some light on the dynamics of successful resolutions in environmental cases. Copies of the Statement of Understanding, the Mediation Agreement, and the Covenant Not to Sue are appended.

  14. Heat stress downregulates FLIP and sensitizes cells to Fas receptor-mediated apoptosis

    Microsoft Academic Search

    S E F Tran; A Meinander; T H Holmström; A Rivero-Müller; K M Heiskanen; E K Linnau; M J Courtney; D D Mosser; L Sistonen; J E Eriksson

    2003-01-01

    The heat shock response and death receptor-mediated apoptosis are both key physiological determinants of cell survival. We found that exposure to a mild heat stress rapidly sensitized Jurkat and HeLa cells to Fas-mediated apoptosis. We further demonstrate that Hsp70 and the mitogen-activated protein kinases, critical molecules involved in both stress-associated and apoptotic responses, are not responsible for the sensitization. Instead,

  15. Cell cycle regulation and apoptotic cell death in experimental colon carcinogenesis: intervening with cyclooxygenase-2 inhibitors.

    PubMed

    Saini, Manpreet Kaur; Sanyal, Sankar Nath

    2015-01-01

    Relative imbalance in the pathways regulating cell cycle, cell proliferation, or cell death marks a prerequisite for neoplasm. C-phycocyanin, a biliprotein from Spirulina platensis and a selective COX-2 inhibitor along with piroxicam, a traditional nonsteroidal antiinflammatory drug was used to investigate the role of cell cycle regulatory proteins and proinflammatory transcription factor NF?B in 1,2-dimethylhydrazine dihydrochloride (DMH)-induced rat colon carcinogenesis. Cell cycle regulators [cyclin D1, cyclin E, cyclin dependent kinase 2 (CDK2), CDK4, and p53], NF?B (p65) pathway, and proliferating cell nuclear antigen (PCNA) were evaluated by gene and protein expression, whereas apoptosis was studied by terminal deoxynucleotidyl transferase dUTP nick end labeling and apoptotic bleb assay. Molecular docking of ligand protein interaction was done to validate the in vivo results. Cyclin D1, cyclin E, CDK2, and CDK4 were overexpressed in DMH, whereas piroxicam and c-phycocyanin promoted the cell cycle arrest by downregulating them. Both drugs mediated apoptosis through p53 activation. Piroxicam and c-phycocyanin also stimulated antiproliferation by restraining PCNA expression and reduced cell survival via inhibiting NF?B (p65) pathway. Molecular docking revealed that phycocyanobilin (a chromophore of c-phycocyanin) interact with DNA binding site of NF?B. Inhibition of cyclin/CDK complex by piroxicam and c-phycocyanin affects the expression of p53 in colon cancer followed by downregulation of NF?B and PCNA levels, thus substantiating the antineoplastic role of these agents. PMID:25825916

  16. Proteins Associated with the Exon Junction Complex Also Control the Alternative Splicing of Apoptotic Regulators

    PubMed Central

    Michelle, Laetitia; Cloutier, Alexandre; Toutant, Johanne; Shkreta, Lulzim; Thibault, Philippe; Durand, Mathieu; Garneau, Daniel; Gendron, Daniel; Lapointe, Elvy; Couture, Sonia; Le Hir, Hervé; Klinck, Roscoe; Elela, Sherif Abou; Prinos, Panagiotis

    2012-01-01

    Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-xS splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level. PMID:22203037

  17. From embryo to adult: persistent neurogenesis and apoptotic cell death shape the lobster deutocerebrum.

    PubMed

    Harzsch, S; Miller, J; Benton, J; Beltz, B

    1999-05-01

    Neuronal plasticity and synaptic remodeling play important roles during the development of the invertebrate nervous system. In addition, structural neuroplasticity as a result of long-term environmental changes, behavioral modifications, age, and experience have been demonstrated in the brains of sexually mature insects. In adult vertebrates, persistent neurogenesis is found in the granule cell layer of the mammalian hippocampus and the subventricular zone, as well as in the telencephalon of songbirds, indicating that persistent neurogenesis, which is presumably related to plasticity and learning, may be an integral part of the normal biology of the mature brain. In decapod crustaceans, persistent neurogenesis among olfactory projection neurons is a common principle that shapes the adult brain, indicating a remarkable degree of life-long structural plasticity. The present study closes a gap in our knowledge of this phenomenon by describing the continuous cell proliferation and gradual displacement of proliferation domains in the central olfactory pathway of the American lobster Homarus americanus from early embryonic through larval and juvenile stages into adult life. Neurogenesis in the deutocerebrum was examined by the in vivo incorporation of bromodeoxyuridine, and development and structural maturation of the deutocerebral neuropils were studied using immunohistochemistry against Drosophila synapsin. The role of apoptotic cell death in shaping the developing deutocerebrum was studied using the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling method, combined with immunolabeling using an antiphospho histone H3 mitosis marker. Our results indicate that, in juvenile and adult lobsters, birth and death of olfactory interneurons occur in parallel, suggesting a turnover of these cells. When the persistent neurogenesis and concurrent death of interneurons in the central olfactory pathway of the crustacean brain are taken into account with the life-long turnover of olfactory receptor cells in crustacean antennules, a new, highly dynamic picture of olfaction in crustaceans emerges. PMID:10212307

  18. Tula hantavirus triggers pro-apoptotic signals of ER stress in Vero E6 cells.

    PubMed

    Li, Xiao-Dong; Lankinen, Hilkka; Putkuri, Niina; Vapalahti, Olli; Vaheri, Antti

    2005-03-01

    Tula virus is a member of the Hantavirus genus of the family Bunyaviridae. Viruses of this family have an unusual pattern of intracellular maturation at the ER-Golgi compartment. We recently found that Tula virus, similar to several other hantaviruses, is able to induce apoptosis in cultured cells [Li, X.D., Kukkonen, S., Vapalahti, O., Plyusnin, A., Lankinen, H., Vaheri, A., 2004. Tula hantavirus infection of Vero E6 cells induces apoptosis involving caspase 8 activation. J. Gen. Virol. 85, 3261-3268.]. However, the cellular mechanisms remain to be clarified. In this study, we demonstrate that the progressive replication of Tula virus in Vero E6 cells initiates several death programs that are intimately associated with ER stress: (1) early activation of ER-resident caspase-12; (2) phosphorylation of Jun NH2-terminal kinase (JNK) and its downstream target transcriptional factor, c-jun; (3) induction of the pro-apoptotic transcriptional factor, growth arrest- and DNA damage-inducible gene 153, or C/EBP homologous protein (Gadd153/chop); and (4) changes in the ER-membrane protein BAP31 implying cross-talk with the mitochondrial apoptosis pathway. Furthermore, we confirmed that a sustained ER stress was induced marked by an increased expression of an ER chaperone Grp78/BiP. Taken together, we have identified involvement of ER stress-mediated death program in Tula virus-infected Vero E6 cells which provides a new approach to understand the mechanisms in hantavirus-induced apoptosis. PMID:15708603

  19. Manganese Nanoparticle Activates Mitochondrial Dependent Apoptotic Signaling and Autophagy in Dopaminergic Neuronal Cells

    PubMed Central

    Ngwa, Hilary Afeseh; Kanthasamy, Arthi; Gu, Yan; Fang, Ning; Anantharam, Vellareddy; Kanthasamy, Anumantha G.

    2011-01-01

    The production of man-made nanoparticles for various modern applications has increased exponentially in recent years, but the potential health effects of most nanoparticles are not well characterized. Unfortunately, in vitro nanoparticle toxicity studies are extremely limited by yet unresolved problems relating to dosimetry. In the present study, we systematically characterized manganese (Mn) nanoparticle sizes and examined the nanoparticle-induced oxidative signaling in dopaminergic neuronal cells. Differential interference contrast (DIC) microscopy and transmission electron microscopy (TEM) studies revealed that Mn nanoparticles range in size from single nanoparticles (~25 nM) to larger agglomerates when in treatment media. Manganese nanoparticles were effectively internalized in N27 dopaminergic neuronal cells, and they induced a time-dependent upregulation of the transporter protein transferrin. Exposure to 25–400 µg/mL Mn nanoparticles induced cell death in a time- and dose-dependent manner. Mn nanoparticles also significantly increased ROS, accompanied by a caspase-mediated proteolytic cleavage of proapoptotic protein kinase C? (PKC?), as well as activation loop phosphorylation. Blocking Mn nanoparticle-induced ROS failed to protect against the neurotoxic effects, suggesting the involvement of other pathways. Further mechanistic studies revealed changes in Beclin1 and LC3, indicating that Mn nanoparticles induce autophagy. Primary mesencephalic neuron exposure to Mn nanoparticles induced loss of TH positive dopaminergic neurons and neuronal processes. Collectively, our results suggest that Mn nanoparticles effectively enter dopaminergic neuronal cells and exert neurotoxic effects by activating an apoptotic signaling pathway and autophagy, emphasizing the need for assessing possible health risks associated with an increased use of Mn nanoparticles in modern applications. PMID:21856324

  20. Asymmetric neuroblast divisions producing apoptotic cells require the cytohesin GRP-1 in Caenorhabditis elegans.

    PubMed

    Teuliere, Jerome; Cordes, Shaun; Singhvi, Aakanksha; Talavera, Karla; Garriga, Gian

    2014-09-01

    Cytohesins are Arf guanine nucleotide exchange factors (GEFs) that regulate membrane trafficking and actin cytoskeletal dynamics. We report here that GRP-1, the sole Caenorhabditis elegans cytohesin, controls the asymmetric divisions of certain neuroblasts that divide to produce a larger neuronal precursor or neuron and a smaller cell fated to die. In the Q neuroblast lineage, loss of GRP-1 led to the production of daughter cells that are more similar in size and to the transformation of the normally apoptotic daughter into its sister, resulting in the production of extra neurons. Genetic interactions suggest that GRP-1 functions with the previously described Arf GAP CNT-2 and two other Arf GEFs, EFA-6 and BRIS-1, to regulate the activity of Arf GTPases. In agreement with this model, we show that GRP-1's GEF activity, mediated by its SEC7 domain, is necessary for the posterior Q cell (Q.p) neuroblast division and that both GRP-1 and CNT-2 function in the Q.posterior Q daughter cell (Q.p) to promote its asymmetry. Although functional GFP-tagged GRP-1 proteins localized to the nucleus, the extra cell defects were rescued by targeting the Arf GEF activity of GRP-1 to the plasma membrane, suggesting that GRP-1 acts at the plasma membrane. The detection of endogenous GRP-1 protein at cytokinesis remnants, or midbodies, is consistent with GRP-1 functioning at the plasma membrane and perhaps at the cytokinetic furrow to promote the asymmetry of the divisions that require its function. PMID:25053664

  1. Straw blood cell count, growth, inhibition and comparison to apoptotic bodies

    PubMed Central

    Wu, Yonnie; Henry, David C; Heim, Kyle; Tomkins, Jeffrey P; Kuan, Cheng-Yi

    2008-01-01

    Background Mammalian cells transform into individual tubular straw cells naturally in tissues and in response to desiccation related stress in vitro. The transformation event is characterized by a dramatic cellular deformation process which includes: condensation of certain cellular materials into a much smaller tubular structure, synthesis of a tubular wall and growth of filamentous extensions. This study continues the characterization of straw cells in blood, as well as the mechanisms of tubular transformation in response to stress; with specific emphasis placed on investigating whether tubular transformation shares the same signaling pathway as apoptosis. Results There are approximately 100 billion, unconventional, tubular straw cells in human blood at any given time. The straw blood cell count (SBC) is 45 million/ml, which accounts for 6.9% of the bloods dry weight. Straw cells originating from the lungs, liver and lymphocytes have varying nodules, hairiness and dimensions. Lipid profiling reveals severe disruption of the plasma membrane in CACO cells during transformation. The growth rates for the elongation of filaments and enlargement of rabbit straw cells is 0.6~1.1 (?m/hr) and 3.8 (?m3/hr), respectively. Studies using apoptosis inhibitors and a tubular transformation inhibitor in CACO2 cells and in mice suggested apoptosis produced apoptotic bodies are mediated differently than tubular transformation produced straw cells. A single dose of 0.01 mg/kg/day of p38 MAPK inhibitor in wild type mice results in a 30% reduction in the SBC. In 9 domestic animals SBC appears to correlate inversely with an animal's average lifespan (R2 = 0.7). Conclusion Straw cells are observed residing in the mammalian blood with large quantities. Production of SBC appears to be constant for a given animal and may involve a stress-inducible protein kinase (P38 MAPK). Tubular transformation is a programmed cell survival process that diverges from apoptosis. SBCs may be an important indicator of intrinsic aging-related stress. PMID:18492269

  2. Antiproliferative property and apoptotic effect of xanthorrhizol on MDA-MB-231 breast cancer cells.

    PubMed

    Cheah, Yew Hoong; Nordin, Fariza Juliana; Tee, Thiam Tsui; Azimahtol, Hawariah Lope Pihie; Abdullah, Noor Rain; Ismail, Zakiah

    2008-01-01

    Xanthorrhizol is a natural sesquiterpenoid compound isolated from the rhizome of Curcuma xanthorrhizza Roxb (Zingerberaceae). Recent studies of xanthorrhizol in cell cultures strongly support the role of xanthorrhizol as an antiproliferative agent. In our study, we tested the antiproliferative effect of xanthorrhizol using different breast cancer cell lines. The invasive breast cancer cell line, MDA-MB-231, was then selected for further investigations. Treatment with xanthorrhizol caused 50% growth inhibition on MDA-MB-231 cells at 8.67 +/- 0.79 microg/ml as determined by sulforhodamine B (SRB) assay. Hoechst 33258 nuclear staining assay showed the rate of apoptosis of MDA-MB-231 cells to increase in response to xanthorrhizol treatment. Immunofluorescence staining using antibody MitoCapture and fluorescein isothiocyanate (FITC)-labeled cytochrome c revealed the possibility of altered mitochondrial transmembrane potential and the release of cytochrome c respectively. This was further confirmed by Western-blotting, where cytochrome c was showed to migrate from mitochondrial fraction to the cytosol fraction of treated MDA-MB-231 cells. Caspase activity assay showed the involvement of caspase-3 and caspase-9, but not caspase-6 or caspase-8 in MDA-MB-231 apoptotic cell death. Subsequently, cleavage of PARP-1 protein is suggested. These data suggest treatment with xanthorrhizol modulates MDA-MB-231 cell apoptosis through the mitochondria-mediated pathway subsequent to the disruption of mitochondrial transmembrane potential, release of cytochrome c, activation of caspase-3 and caspase-9, and the modulation of PARP-1 protein. PMID:19189649

  3. Signalling pathway involving GULP, MAPK and Rac1 for SR-BI-induced phagocytosis of apoptotic cells.

    PubMed

    Osada, Yoichi; Sunatani, Toshiro; Kim, In-San; Nakanishi, Yoshinobu; Shiratsuchi, Akiko

    2009-03-01

    Class B scavenger receptor type I (SR-BI) is a phosphatidylserine (PS)-recognizing receptor of testicular Sertoli cells responsible for the phagocytosis of spermatogenic cells undergoing apoptosis. Here, we determined signal mediators that compose a signalling pathway for SR-BI-induced phagocytosis. Results of a yeast two-hybrid analysis and a cell-free binding assay indicated that SR-BI binds to engulfment adapter protein (GULP) using the C-terminal intracellular domain. A co-immunoprecipitation analysis showed the existence of a complex of GULP and SR-BI in cells prior to the activation of SR-BI by PS. A reduction of GULP expression in phagocytes decreased the SR-BI-mediated phagocytosis of apoptotic cells. Administration to phagocytes of PS-containing liposomes increased the levels of the GTP-bound form of Rac1 and the phosphorylated forms of mitogen-activated protein kinases (MAPK) p38 and extracellular signal-related kinase 1 and 2. Finally, lowering the expression of GULP abrogated MAPK phosphorylation, and the presence of MAPK inhibitors reduced the level of GTP-bound Rac1 in PS-activated phagocytes. These results collectively suggested the following signalling pathway for the SR-BI-induced phagocytosis: (i) PS-recognizing SR-BI activates associated GULP; (ii) activated GULP induces MAPK phosphorylation; (iii) activated MAPK increases GTP-bound Rac1; and (iv) activated Rac1 induces a rearrangement of the actin cytoskeleton. PMID:19122200

  4. Effect of thrombopoietin receptor agonists on the apoptotic profile of platelets in patients with chronic immune thrombocytopenia.

    PubMed

    Mitchell, William Beau; Pinheiro, Mariana P; Boulad, Nayla; Kaplan, David; Edison, Michele N; Psaila, Bethan; Karpoff, Marissa; White, Michael J; Josefsson, Emma C; Kile, Benjamin T; Bussel, James B

    2014-12-01

    Platelet survival depends upon mediators of apoptosis e.g., Bcl-xL, Bax, and Bak, which are regulated by thrombopoietin (TPO)-mediated AKT signaling. Thrombopoietin receptor (TPO-R) signaling might decrease platelet and/or megakaryocyte apoptosis and increase the platelet count. This study therefore explored anti-apoptotic effects of TPO-R-agonists in vivo on platelets of patients with immune thrombocytopenia. Patients received eltrombopag or romiplostim for two weeks. Total, immature, and large platelet counts were assessed as were Bcl-xL inhibitor assay; Bcl-xL Western blot; and flow cytometric (FACS) analysis of the AKT-signaling pathway. Eight/ten patients had platelet responses to eltrombopag and all three to romiplostim. Platelet sensitivity to apoptosis by Bcl-xL inhibition was greater in pretreatment patients than controls. This sensitivity normalized after one week of therapy, but surprisingly returned to pretreatment levels at week two. FACS analysis revealed increased AKT-pathway signaling after one week, followed by a decrease at week two. Platelet counts correlated with the Bcl-xL /Bak ratio. Platelet survival may be enhanced by TPO-R-agonists as a transient decrease in platelet sensitivity to apoptosis was accompanied by transient activation of AKT. However, this mechanism has only a short-lived effect. Megakaryocytes and platelets already present at the start of TPO-R-agonist treatment appear to respond differently than those generated de novo. PMID:25132654

  5. Mediating connectors

    Microsoft Academic Search

    R. M. Balzer; N. M. Goldman

    1999-01-01

    We have developed a technology for mediating all shared library calls. These mediators can be dynamically installed and removed during execution. They can be used as an instrument for these interfaces, to monitor their behavior, to integrate legacy components together, or to encapsulate potentially harmful or unreliable components. Since modern commercial operating systems promote packaging third party functionality as shared

  6. Non-apoptotic functions of caspase-7 during osteogenesis

    PubMed Central

    Svandova, E; Lesot, H; Vanden Berghe, T; Tucker, A S; Sharpe, P T; Vandenabeele, P; Matalova, E

    2014-01-01

    Caspase-3 and -7 are generally known for their central role in the execution of apoptosis. However, their function is not limited to apoptosis and under specific conditions activation has been linked to proliferation or differentiation of specialised cell types. In the present study, we followed the localisation of the activated form of caspase-7 during intramembranous (alveolar and mandibular bones) and endochondral (long bones of limbs) ossification in mice. In both bone types, the activated form of caspase-7 was detected from the beginning of ossification during embryonic development and persisted postnatally. The bone status was investigated by microCT in both wild-type and caspase-7-deficient adult mice. Intramembranous bone in mutant mice displayed a statistically significant decrease in volume while the mineral density was not altered. Conversely, endochondral bone showed constant volume but a significant decrease in mineral density in caspase-7 knock-out mice. Cleaved caspase-7 was present in a number of cells that did not show signs of apoptosis. PCR array analysis of the mandibular bone of caspase-7-deficient versus wild-type mice pointed to a significant decrease in mRNA levels for Msx1 and Smad1 in early bone formation. These observations might explain the decrease in the alveolar bone volume of adult knock-out mice. In conclusion, this study is the first to report a non-apoptotic function of caspase-7 in osteogenesis and also demonstrates further specificities in endochondral versus intramembranous ossification. PMID:25118926

  7. Neuropeptide Y receptors activation protects rat retinal neural cells against necrotic and apoptotic cell death induced by glutamate

    PubMed Central

    Santos-Carvalho, A; Elvas, F; Álvaro, A R; Ambrósio, A F; Cavadas, C

    2013-01-01

    It has been claimed that glutamate excitotoxicity might have a role in the pathogenesis of several retinal degenerative diseases, including glaucoma and diabetic retinopathy. Neuropeptide Y (NPY) has neuroprotective properties against excitotoxicity in the hippocampus, through the activation of Y1, Y2 and/or Y5 receptors. The principal objective of this study is to investigate the potential protective role of NPY against glutamate-induced toxicity in rat retinal cells (in vitro and in an animal model), unraveling the NPY receptors and intracellular mechanisms involved. Rat retinal neural cell cultures were prepared from newborn Wistar rats (P3-P5) and exposed to glutamate (500??M) for 24?h. Necrotic cell death was evaluated by propidium iodide (PI) assay and apoptotic cell death using TUNEL and caspase-3 assays. The cell types present in culture were identified by immunocytochemistry. The involvement of NPY receptors was assessed using selective agonists and antagonists. Pre-treatment of cells with NPY (100?nM) inhibited both necrotic cell death (PI-positive cells) and apoptotic cell death (TUNEL-positive cells and caspase 3-positive cells) triggered by glutamate, with the neurons being the cells most strongly affected. The activation of NPY Y2, Y4 and Y5 receptors inhibited necrotic cell death, while apoptotic cell death was only prevented by the activation of NPY Y5 receptor. Moreover, NPY neuroprotective effect was mediated by the activation of PKA and p38K. In the animal model, NPY (2.35?nmol) was intravitreally injected 2?h before glutamate (500?nmol) injection into the vitreous. The protective role of NPY was assessed 24?h after glutamate (or saline) injection by TUNEL assay and Brn3a (marker of ganglion cells) immunohistochemistry. NPY inhibited the increase in the number of TUNEL-positive cells and the decrease in the number of Brn3a-positive cells induced by glutamate. In conclusion, NPY and NPY receptors can be considered potential targets to treat retinal degenerative diseases, such as glaucoma and diabetic retinopathy. PMID:23681231

  8. Antitumour effect of polyoxomolybdates: induction of apoptotic cell death and autophagy in in vitro and in vivo models

    PubMed Central

    Ogata, A; Yanagie, H; Ishikawa, E; Morishita, Y; Mitsui, S; Yamashita, A; Hasumi, K; Takamoto, S; Yamase, T; Eriguchi, M

    2007-01-01

    Polyoxomolybdates (PMs) as discrete molybdenum-oxide cluster anions have been investigated in the course of study of their medical applications. Here, we show the significant antitumour potency of the polyoxomolybdate [Me3NH]6[H2MoV12O28(OH)12(MoVIO3)4]·2H2O (PM-17), which is a photo-reduced compound of [NH3Pri]6[Mo7O24]·3H2O. The effect of PM-17 on the growth of cancer cell lines and xenografts was assessed by a cell viability test and analysis of tumour expansion rate. Morphological analysis was carried out by Hoechst staining, flow-cytometric analysis of Annexin V staining, terminal deoxynucleotidyl transferase-mediated ‘nick-end' labelling staining, and electron-microscopic analysis. Activation of autophagy was detected by western blotting and fluorescence-microscopic analysis of the localisation of GFP-LC3 in transfected tumour cells. PM-17 inhibited the growth of human pancreatic cancer (AsPC-1) xenografts in a nude mice model, and induced morphological alterations in tumour cells. Correspondingly, PM-17 repressed the proliferation of AsPC-1 cells and human gastric cancer cells (MKN45) depending on the dose in vitro. We observed apoptotic patterns as the formation of apoptotic small bodies and translocation of phosphatidylserine by Hoechst staining and flow-cytometric analysis following Annexin V staining, and in parallel, autophagic conformation by the formulation of autophagosomes and localisation of GFP-LC3 by electron- and fluorescence-microscopic analysis. PMID:18087283

  9. Investigating Migration Inhibition and Apoptotic Effects of Fomitopsis pinicola Chloroform Extract on Human Colorectal Cancer SW-480 Cells

    PubMed Central

    Wang, Pan; Wang, Lu; Fan, Jianping; Wang, Xiaobing; Liu, Quanhong

    2014-01-01

    Background Fomitopsis pinicola (Sw. Ex Fr.m) Karst (FPK) which belongs to the Basidiomycota fungal class is one of the most popular medical fungi in China. It has been used for many diseases: cancer, heart diseases, diabetes and so on. However, little study on the pro-apoptotic effect and migration inhibition of FPK chloroform extract (FPKc) has been reported and the possible involved mechanism has not been illuminated. Methodology/Principal Findings Chemical analysis was performed by HPLC which showed ergosterol (ES) concentration was 105 µg/mg. MTT assay revealed that FPKc could selectively inhibit SW-480 cells viability with the IC50 of 190.28 µg/ml. Wound healing and transwell assay indicated that FPKc could inhibit the migration of SW-480 cells obviously, FPKc could also dramatically decreased the matrix metalloproteinases-2, 9 (MMP-2 and MMP-9) expression. Annexin V–FITC/PI staining, nuclear Hoechst 33342 staining and DNA fragmentation analysis revealed that FPKc and ES could induce SW-480 cells apoptosis. The apoptosis process closely involved in ROS accumulation and depletion of GSH, activation of caspase 3, poly (ADP-ribose) polymerase (PARP) degradation. FPKc could also up-regulate P53 expression and thus lead to G1 phase arrest. When SW-480 cells were pretreated with N-acetylcysteine (NAC), the ROS generation, cell viability and apoptotic ratio were partially declined, which indicated that ROS was vertical in the pro-apoptosis process induced by FPKc. Moreover, in the whole process, ES which has been previously found in FPKc had the similar effect to FPKc. Thus we could conclude that ES, as one of the highest abundant components in FPKc, might also be one of the active constituents. Conclusion/Significance FPKc could inhibit the migration of SW-480 cells, induce SW-480 cells G1 phase arrest and cause ROS-mediated apoptosis effect. And ES might be one of the effective constituents in the whole process. PMID:24992193

  10. The Chinese herbal formula Liuwei dihuang protects dopaminergic neurons against Parkinson's toxin through enhancing antioxidative defense and preventing apoptotic death.

    PubMed

    Tseng, Yu-Ting; Chang, Fang-Rong; Lo, Yi-Ching

    2014-04-15

    Liuwei dihuang (LWDH), a widely used traditional Chinese medicine (TCM), has been employed as an anti-aging prescription to improve declined function. Parkinson's disease (PD) is a common adult-onset neurodegenerative disorder characterized by the degeneration of dopaminergic nigrostriatal neurons with complex pathological mechanisms, including oxidative stress. Increasing evidence indicate that TCM has the potential to be neuroprotective drugs because of their antioxidant characteristics. The aim of this study is to investigate the mechanisms of LWDH-mediated protection in Parkinson's toxin-induced dopaminergic neurodegeneration by evaluating water extract of LWDH (LWDH-WE) in 1-methyl-4-phenylpyridinium (MPP(+))-treated primary mesencephalic neurons and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated C57BL/6 mice. In the present study, chemical profiling and quantitative analysis of LWDH-WE were revealed using 3D-HPLC technique, and were confirmed by the data of three batches of LWDH-WE. In primary mesencephalic neuronal cultures, LWDH-WE decreased MPP(+)-induced loss of tyrosine hydroxylase (TH)-positive neurons and increase of Annexin V-positive neurons. LWDH-WE reduced MPP(+)-induced oxidative damage via increasing antioxidant defense (SOD, GSH), decreasing ROS production, and down-regulating NADPH oxidases (Nox2 and Nox4). Also, LWDH-WE inhibited neuronal apoptosis by improving mitochondrial membrane potential, increasing antiapoptotic protein Bcl-2 expression, and down-regulating apoptotic signaling (Bax, cytochrome c, cleaved-caspase-3) in MPP(+)-treated neurons. In MPTP-treated C57BL/6 mice, LWDH-WE attenuated TH-positive neuronal loss in substantia nigra pars compacta (SNpc), and improved locomotor activity of mice. In conclusion, the present results reveal that LWDH-WE possesses protection on dopaminergic neurons through enhancing antioxidant defense and decreasing apoptotic death, suggesting the potential benefits of LWDH-WE for PD treatment. PMID:24411708

  11. In situ detection of apoptotic cells by TUNEL in the gill epithelium of the developing brown trout (Salmo trutta)

    PubMed Central

    ROJO, M. C.; GONZALEZ, M. E.

    1998-01-01

    Apoptosis is a form of naturally occurring cell death during development and it is characterised by extensive DNA fragmentation. Apoptosis is easily detected in the gill epithelium of brown trout embryos in ultrathin sections (Rojo et al. 1997). Here we provide the first biochemical evidence for apoptosis in the gill epithelium of brown trout embryos, using in situ end-labelling of DNA breaks (Gavrieli et al. 1992). Embryos at d 57 of development as well as those at hatching, were processed to analyse the distribution of apoptotic cells in the gills. The extent of apoptosis revealed by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling method technique is considerably greater than apoptosis detected by nuclear morphology. This method revealed that apoptosis was frequent at hatching, although it was also present during embryonic development. The presence and distribution of stained nuclei were different depending on the developmental stage. In embryos of 57 d, apoptotic flattened nuclei were dispersed in the gill epithelium, whereas at hatching, they were mainly grouped in the tips of the filaments and in the gill arches. TUNEL also revealed a distinct pattern of nuclear staining: at hatching, the intense staining covered the entire cell, but in embryos it was restricted to the nucleus. These results show the functional relevance of apoptosis at hatching, when apoptosis seems to be the unique process by which cell numbers in the gill epithelium are adjusted, in order to prepare for the new extrinsic conditions affecting the free-living life of alevins. PMID:9877294

  12. Recombinant osteopontin attenuates experimental cerebral vasospasm following subarachnoid hemorrhage in rats through an anti-apoptotic mechanism.

    PubMed

    He, Junchi; Liu, Mindi; Liu, Zhen; Luo, Liangsheng

    2015-06-22

    Cerebral vasospasm (CVS) is an important pathological process following subarachnoid hemorrhage (SAH). Osteopontin (OPN), a pleiotropic extracellular glycoprotein, has been reported to be able to induce MKP-1 in the spastic cerebral arteries and prevent vasospasm after SAH. The purpose of this study was to investigate the protective effects of recombinant OPN (r-OPN) on CVS following SAH and the underlying mechanisms associated with its anti-apoptotic effect. Eighty male Sprague Dawley rats (weighing 300-375g) were randomly assigned to four groups: (1) sham+vehicle (n=20), (2) SAH+vehicle (n=20), (3) SAH+OPN0.03 (0.03?g) (n=20), (4) SAH+OPN0.1 (0.1?g) (n=20). The double injection model of cisterna magna was performed on day 0 and 48h after the first induction. r-OPN was administered intraventricularly nearly 30min after the first SAH. After neurological score assessment, rats were sacrificed 72h after the first SAH. The cross-sectional area and thickness of basilar arteries (BA) were measured under Hematoxylin-eosin (H&E) staining. Endothelial cell apoptosis was identified by terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining. Immunohistochemistry was used to assess the expression of p-Akt and cleaved caspase-3 in BA. Western blot analysis was applied to evaluate the expression of p-Akt, cleaved caspase-3, Bax and Bcl-2 in BA. r-OPN improved neurological scores and attenuated vasospasm. r-OPN significantly reduced expression of cleaved caspase-3 and Bax in BA following SAH, and increased the level of p-Akt and Bcl-2, coupled with reduced apoptosis of endothelial cell in BA. These results demonstrate that r-OPN can attenuate vasospasm after SAH through a suppressed apoptotic response, which may provide a novel therapeutic target for cerebral vasospasm. PMID:25779039

  13. Fish oil supplementation reverses the effect of cholesterol on apoptotic gene expression in smooth muscle cells

    PubMed Central

    2010-01-01

    Background Nutritional control of gene regulation guides the transformation of smooth muscle cells (SMC) into foam cells in atherosclerosis. Oxidative stress has been reported in areas of lipid accumulation, activating proliferation genes. Suppression of oxidative stress by antioxidant administration reduces this activation and the progression of lesions. We hypothesized that fish oil consumption may protect against atherosclerotic vascular disease. The study objective was to determine the effects of dietary cholesterol and fish-oil intake on the apoptotic pathways induced by 25-hydroxycholesterol (25-HC) in SMC cultures. Methods An in vivo/in vitro cell model was used, culturing SMC isolated from chicks exposed to an atherogenic cholesterol-rich diet with 5% of cholesterol (SMC-Ch) alone or followed by an anti-atherogenic fish oil-rich diet with 10% of menhaden oil (SMC-Ch-FO) and from chicks on standard diet (SMC-C). Cells were exposed to 25-HC, studying apoptosis levels by flow cytometry (Annexin V) and expressions of caspase-3, c-myc, and p53 genes by quantitative real-time reverse transcriptase-polymerase chain reaction. Results: Exposure to 25-HC produced apoptosis in all three SMC cultures, which was mediated by increases in caspase-3, c-myc, and p53 gene expression. Changes were more marked in SMC-Ch than in SMC-C, indicating that dietary cholesterol makes SMC more susceptible to 25-HC-mediated apoptosis. Expression of p53 gene was elevated in SMC-Ch-FO. This supports the proposition that endogenous levels of p53 protect SMC against apoptosis and possibly against the development of atherosclerosis. Fish oil attenuated the increase in c-myc levels observed in SMC-C and SMC-Ch, possibly through its influence on the expression of antioxidant genes. Conclusion Replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of the cholesterol-induced changes, increasing the resistance of SMC to apoptosis. PMID:20630092

  14. Caspase-dependant activation of chymotrypsin-like proteases mediates nuclear events during Jurkat T cell apoptosis

    SciTech Connect

    O'Connell, A.R. [National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland); Lee, B.W. [Immunochemistry Technologies LLC., 9401 James Avenue South Suite 155, Bloomington, MN 55431 (United States); Stenson-Cox, C. [National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland)]. E-mail: catherine.stenson@nuigalway.ie

    2006-06-30

    Apoptosis involves a cascade of biochemical and morphological changes resulting in the systematic disintegration of the cell. Caspases are central mediators of this process. Supporting and primary roles for serine proteases as pro-apoptotic mediators have also been highlighted. Evidence for such roles comes largely from the use of pharmacological inhibitors; as a consequence information regarding their apoptotic function and biochemical properties has been limited. Here, we circumvented limitations associated with traditional serine protease inhibitors through use of a fluorescently labelled inhibitor of serine proteases (FLISP) that allowed for analysis of the specificity, regulation and positioning of apoptotic serine proteases within a classical apoptotic cascade. We demonstrate that staurosporine triggers a caspase-dependant induction of chymotrypsin-like activity in the nucleus of apoptotic Jurkat T cells. We show that serine protease activity is required for the generation of late stage nuclear events including condensation, fragmentation and DNA degradation. Furthermore, we reveal caspase-dependant activation of two chymotrypsin-like protein species that we hypothesize mediate cell death-associated nuclear events.

  15. Quantitative analysis of apoptotic decisions in single cells and cell populations

    E-print Network

    Albeck, John G

    2007-01-01

    Apoptosis is a form of programmed cell death that is essential for the elimination of damaged or unneeded cells in multicellular organisms. Inactivation of apoptotic cell death is a necessary step in the development of ...

  16. Golgi Anti-apoptotic Proteins Are Highly Conserved Ion Channels That Affect Apoptosis and Cell Migration

    E-print Network

    Carrara, Guia; Saraiva, Nuno; Parsons, Maddy; Byrne, Bernadette; Prole, David L.; Taylor, Colin W.; Smith, Geoffrey L.

    2015-02-24

    Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses...

  17. Leukemia . Author manuscript Sirolimus enhances the effect of apoptotic cell infusion on hematopoietic

    E-print Network

    Paris-Sud XI, Université de

    Leukemia . Author manuscript Page /1 6 Sirolimus enhances the effect of apoptotic cell infusion not candidate for standard myeloablative AHCT and diseases other than acute leukemia. However, and despite some

  18. Evolutionary analyses of caspase-8 and its paralogs: Deep origins of the apoptotic signaling pathways.

    PubMed

    Sakamaki, Kazuhiro; Imai, Kenichiro; Tomii, Kentaro; Miller, David J

    2015-07-01

    Although Caenorhabditis and Drosophila proved invaluable in unraveling the molecular mechanisms of apoptosis, it is now clear that these animals are of limited value for understanding the evolution of apoptotic systems. Whereas data from these invertebrates led to the assumption that the extrinsic apoptotic pathway is restricted to vertebrates, recent data from cnidarians and sponges indicate that this pathway predates bilaterian origins. Here we review the phylogenetic distribution of caspase-8, the initiator caspase of the extrinsic apoptotic pathway, its paralogs and other components of the network. The ancestral caspase-8 gave rise to four paralogs early in vertebrate evolution, and these have been maintained in many tetrapods. However, eutherians have lost caspase-18 and myomorph rodents have lost caspase-10, these losses suggesting functional redundancy amongst caspase-8 paralogs. The apoptotic network of the eumetazoan ancestor appears to have been complex and vertebrate like, and is only now being revealed by studying simple animals. PMID:26010168

  19. Abl Kinase Inhibits the Engulfment of Apoptotic [corrected] Cells in Caenorhabditis elegans

    E-print Network

    Hurwitz, Michael Eliezer

    The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One ...

  20. COMPARISON OF CELLULAR AND NUCLEAR FLOW CYTOMETRIC TECHNIQUES FOR DISCRIMINATING APOPTOTIC SUBPOPULATIONS

    EPA Science Inventory

    We compared cellular flow cytometric methods employing carboxyfluorescein (CF), Hoechst 33342, and Hoechst 33258 with a nuclear method in their ability to discriminate apoptotic subpopulations in rat thymocyte cultures exposed to dexamethasone r tributyltin. n the nuclear techniq...

  1. Apoptotic signal molecules in skin biopsies of cutaneous lupus erythematosus: analysis using tissue microarray.

    PubMed

    Toberer, Ferdinand; Sykora, Jaromir; Göttel, Daniel; Hartschuh, Wolfgang; Werchau, Siegfried; Enk, Alexander; Joos, Stefan; Krammer, Peter H; Kuhn, Annegret

    2013-10-01

    Cutaneous lupus erythematosus (CLE) is a heterogeneous autoimmune disease. Different pathogenetic mechanisms, including the accumulation of apoptotic keratinocytes in CLE, have been reported. Therefore, we investigated whether CLE and other inflammatory skin diseases differ with regard to the epidermal expression of molecules that are crucial for the initiation and regulation of apoptosis. In this study, 241 skin biopsies from patients with CLE, psoriasis (PSO), lichen planus (LP) and healthy controls (HCs) were analysed immunohistochemically using the tissue microarray (TMA) technique. The TUNEL assay and anti-activated caspase-3 antibodies revealed a significant increase of apoptotic keratinocytes in CLE lesions compared with HCs. Furthermore, we detected a significant increase in the epidermal expression of CD95 in CLE specimens compared with PSO, LP and HCs. These data suggest that the accumulation of apoptotic keratinocytes in CLE might be due to the increased epidermal expression of CD95, resulting in increased activity of the extrinsic apoptotic pathway in the disease. PMID:24079735

  2. Apoptotic Signaling through CD95 (Fas\\/Apo1) Activates an Acidic Sphlngomyellnase

    Microsoft Academic Search

    Maria Grazia Cifone; Paola Roncaioli; S Maria Rita; Miyuki Azuma; Lewis L. Lanier; Angela Santoni; Roberto Testi

    2010-01-01

    Summary Intraceilular pathways leading from membrane receptor engagement to apoptotic cell death are still poorly characterized. We investigated the intracellular signaling generated after cross-linking of CD95 (Fas\\/Apo-1 antigen), a broadly expressed ceil surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 monodonal antibody was produced by immunizing mice with human CD95-transfected L ceils.

  3. Role of Ionic Fluxes in the Apoptotic Cell Death of Cultured Cerebellar Granule Neurons

    Microsoft Academic Search

    A. Franco-Cea; A. Valencia; S. Sánchez-Armass; G. Domínguez; J. Morán

    2004-01-01

    Cultured cerebellar granule neurons (CGC) increase survival in a medium containing 25 mM KCl (K25), and they die apoptotically when cultures are treated with staurosporine (St) or are transferred to a 5-mM KCl containing medium (K5). Apoptotic CGC show nuclear condensation and caspase-3 activation. Cell death induced by these conditions was partially prevented when cultures were maintained under alkaline conditions,

  4. CD14-dependent clearance of apoptotic cells by human macrophages: the role of phosphatidylserine

    Microsoft Academic Search

    A Devitt; S Pierce; C Oldreive; W H Shingler; C D Gregory

    2003-01-01

    Apoptotic-cell clearance is dependent on several macrophage surface molecules, including CD14. Phosphatidylserine (PS) becomes externalised during apoptosis and participates in the clearance process through its ability to bind to a novel receptor, PS-R. CD14 has the proven ability to bind phospholipids and may function as an alternative receptor for the externalised PS of apoptotic cells. Here we demonstrate that CD14

  5. CpG-ODN enhances ingestion of apoptotic neutrophils by macrophages

    Microsoft Academic Search

    Jiong Wang; Wei-Lin Huang; Rong-Yu Liu

    2009-01-01

    The clearance of apoptotic neutrophils by macrophages plays an important role in the process of inflammatory response. In\\u000a the present study, we examined the ability of macrophages to ingest apoptotic neutrophils after activated by synthetic oligodeoxynucleotides\\u000a containing CpG motifs (CpG-ODN) in vitro. The results showed that, while CpG-ODN at the experimental concentration had no\\u000a cytotoxic effect on the viability of

  6. The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis

    Microsoft Academic Search

    José A. Sánchez-Alcázar; Ángeles Rodríguez-Hernández; Mario D. Cordero; Daniel J. M. Fernández-Ayala; Gloria Brea-Calvo; Katherina Garcia; Plácido Navas

    2007-01-01

    It has recently been shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis. We demonstrate\\u000a that this microtubule reformation occurs in many cell types and under different apoptotic stimuli. We confirm that the apoptotic\\u000a microtubule network possesses a novel organization, whose nucleation appears independent of conventional ?-tubulin ring complex\\u000a containing structures. Our analysis suggests that microtubules

  7. Photodynamic treatment induces an apoptotic pathway involving calcium, nitric oxide, p53, p21-activated kinase 2, and c-Jun N-terminal kinase and inactivates survival signal in human umbilical vein endothelial cells.

    PubMed

    Chan, Wen-Hsiung

    2011-01-01

    Photodynamic treatment (PDT) elicits a diverse range of cellular responses, including apoptosis. Previously, we showed that PDT stimulates caspase-3 activity, and subsequent cleavage and activation of p21-activated kinase 2 (PAK2) in human epidermal carcinoma A431 cells. In the current study, pretreatment with nitric oxide (NO) scavengers inhibited PDT-induced mitochondrial membrane potential (MMP) changes, activation of caspase-9, caspase-3, p21-activated protein kinase 2 (PAK2) and c-Jun N-terminal kinase (JNK), and gene expression of p53 and p21 involved in apoptotic signaling. Moreover, PAK2 activity was required for PDT-induced JNK activation and apoptosis. Inhibition of p53 mRNA expression using small interfering RNA (siRNA) additionally blocked activation of PAK2 and apoptosis induced by PDT. Importantly, our data also show that PDT triggers cell death via inactivation of ERK-mediated anti-apoptotic pathway. PDT triggers cell death via inactivation of the HSP90/multi-chaperone complex and subsequent degradation of Ras, further inhibiting anti-apoptotic processes, such as the Ras?ERK signal transduction pathway. Furthermore, we did not observe two-stage JNK activation for regulation of PAK2 activity in the PDT-induced apoptotic pathway in HUVECs, which was reported earlier in A431 cells. Based on the collective results, we have proposed a model for the PDT-triggered inactivation of the survival signal and apoptotic signaling cascade with Rose Bengal (RB), which sequentially involves singlet oxygen, Ca(2+), NO, p53, caspase-9, caspase-3, PAK2, and JNK. PMID:21541041

  8. Extracellular matrix proteins modulate antimigratory and apoptotic effects of Doxorubicin.

    PubMed

    Said, Georges; Guilbert, Marie; Morjani, Hamid; Garnotel, Roselyne; Jeannesson, Pierre; El Btaouri, Hassan

    2012-01-01

    Anticancer drug resistance is a multifactorial process that includes acquired and de novo drug resistances. Acquired resistance develops during treatment, while de novo resistance is the primary way for tumor cells to escape chemotherapy. Tumor microenvironment has been recently shown to be one of the important factors contributing to de novo resistance and called environment-mediated drug resistance (EMDR). Two forms of EMDR have been described: soluble factor-mediated drug resistance (SFM-DR) and cell adhesion-mediated drug resistance (CAM-DR). Anthracyclines, among the most potent chemotherapeutic agents, are widely used in clinics against hematopoietic and solid tumors. Their main mechanism of action relies on the inhibition of topoisomerase I and/or II and the induction of apoptosis. Beyond this well-known antitumor activity, it has been recently demonstrated that anthracyclines may display potent anti-invasive effects when used at subtoxic concentrations. In this paper, we will describe two particular modes of EMDR by which microenvironment may influence tumor-cell response to one of these anthracyclines, doxorubicin. The first one considers the influence of type I collagen on the antimigratory effect of doxorubicin (CAM-DR). The second considers the protection of tumor cells by thrombospondin-I against doxorubicin-induced apoptosis (SFM-DR). PMID:22811904

  9. SAFB re-distribution marks steps of the apoptotic process

    SciTech Connect

    Lee, Youn-Bok; Colley, Shane; Norman, Michel; Biamonti, Giuseppe [Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology (LINE), Bristol University, Dorothy Hodgkin Building, Whitson Street Bristol BS1 3NY (United Kingdom); Uney, James B. [Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology (LINE), Bristol University, Dorothy Hodgkin Building, Whitson Street Bristol BS1 3NY (United Kingdom)], E-mail: James.uney@bris.ac.uk

    2007-11-01

    We have found novel functions of scaffold attachment factor-B1 (SAFB) during apoptosis. The experiments showed that SAFB moved into the nucleolus 15 min after the induction of apoptosis and before the release of cytochrome c into the cytoplasm. Two hours later SAFB formed a peri-nucleolar ring-like structure and this occurred after cytochrome c release and before PARP cleavage. Digestion with RNase suggested that the peri-nucleolar ring structure was dependent on RNA integrity and a RNA moiety formed part of this structure. Studies using SAFB deletion mutants showed that the formation of the peri-nucleolar structure was not mediated by the DNA binding (SAP) or the RNA binding (RRM) domain of SAFB but was instead dependent on the S/K and R/E coiled-coil regions: a result suggesting that the structure is formed via protein interactions. In addition, SAFB cleavage was shown to be mediated by caspase-3 and occurred after the formation of the peri-nucleolar ring and after cleavage of PARP (characteristic of proteins having a direct role in apoptosis). A determinant for this cleavage is located in the DNA binding domain and we hypothesize that SAFB may direct the reorganization and segregation of nuclear RNA and DNA prior to endonuclease-mediated DNA cleavage.

  10. Tumor suppressor gene RBM5 delivered by attenuated Salmonella inhibits lung adenocarcinoma through diverse apoptotic signaling pathways

    PubMed Central

    2013-01-01

    Background RBM5 (RNA-binding motif protein 5, also named H37/LUCA-15) gene from chromosome 3p21.3 has been demonstrated to be a tumor suppressor. Current researches in vitro confirm that RBM5 can suppress the growth of lung adenocarcinoma cells by inducing apoptosis. There is still no effective model in vivo, however, that thoroughly investigates the effect and molecular mechanism of RBM5 on lung adenocarcinoma. Method We established the transplanted tumor model on BALB/c nude mice using the A549 cell line. The mice were treated with the recombinant plasmids carried by attenuated Salmonella to induce the overexpression of RBM5 in tumor tissues. RBM5 overexpression was confirmed by immunohistochemistry staining. H&E staining was performed to observe the histological performance on plasmids-treated A549 xenografts. Apoptosis was assessed by TUNEL staining with a TUNEL detection kit. Apoptosis-regulated genes were detected by Western blot. Results We successful established the lung adenocarcinoma animal model in vivo. The growth of tumor xenografts was significantly retarded on the mice treated with pcDNA3.1-RBM5 carried by attenuated Salmonella compared to that on mice treated with pcDNA3.1. Overexpression of RBM5 enhanced the apoptosis in tumor xenografts. Furthermore, the expression of Bcl-2 protein was decreased significantly, while the expression of BAX, TNF-?, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and cleaved PARP proteins was significantly increased in the pcDNA3.1-RBM5-treated mice as compared to that in the control mice. Conclusions In this study, we established a novel animal model to determine RBM5 function in vivo, and concluded that RBM5 inhibited tumor growth in mice by inducing apoptosis. The study suggests that although RBM5’s involvement in the death receptor-mediated apoptotic pathway is still to be investigated, RBM5-mediated growth suppression, at least in part, employs regulation of the mitochondrial apoptotic pathways. PMID:23721095

  11. Glucocorticoids induce protein S-dependent phagocytosis of apoptotic neutrophils by human macrophages.

    PubMed

    McColl, Aisleen; Bournazos, Stylianos; Franz, Sandra; Perretti, Mauro; Morgan, B Paul; Haslett, Christopher; Dransfield, Ian

    2009-08-01

    During resolution of an inflammatory response, recruited neutrophil granulocytes undergo apoptosis and are removed by tissue phagocytes before induction of secondary necrosis without provoking proinflammatory cytokine production and release. Promotion of physiological neutrophil clearance mechanisms may represent a viable therapeutic strategy for the treatment of inflammatory or autoimmune diseases in which removal of apoptotic cells is impaired. The mechanism underlying enhancement of macrophage capacity for phagocytosis of apoptotic cells by the powerful anti-inflammatory drugs of the glucocorticoid family has remained elusive. In this study, we report that human monocyte-derived macrophages cultured in the presence of dexamethasone exhibit augmented capacity for phagocytosis of membrane-intact, early apoptotic cells only in the presence of a serum factor. Our results eliminate a role for a number of potential opsonins, including complement, pentraxin-3, and fibronectin. Using ion-exchange and gel filtration chromatography, we identified a high molecular mass serum fraction containing C4-binding protein and protein S responsible for the augmentation of phagocytosis of apoptotic neutrophils. Because the apoptotic neutrophils used in this study specifically bind protein S, we suggest that glucocorticoid treatment of macrophages induces a switch to a protein S-dependent apoptotic cell recognition mechanism. Consistent with this suggestion, pretreatment of macrophages with Abs to Mer tyrosine kinase, a member of the Tyro3/Axl/Mer family of receptor tyrosine kinases, prevented glucocorticoid augmentation of phagocytosis. Induction of a protein S/Mer tyrosine kinase-dependent apoptotic cell clearance pathway may contribute to the potent anti-inflammatory effects of glucocorticoids, representing a potential target for promoting resolution of inflammatory responses. PMID:19597001

  12. Interaction of CED-6/GULP, an adapter protein involved in engulfment of apoptotic cells with CED-1 and CD91/low density lipoprotein receptor-related protein (LRP).

    PubMed

    Su, Hua Poo; Nakada-Tsukui, Kumiko; Tosello-Trampont, Annie-Carole; Li, Yonghe; Bu, Guojun; Henson, Peter M; Ravichandran, Kodimangalam S

    2002-04-01

    The prompt clearance of cells undergoing apoptosis is critical during embryonic development, normal tissue turnover, as well as inflammation and autoimmunity. The molecular details of the engulfment of apoptotic cells are not fully understood. ced-6 and its human homologue gulp, encode an adapter protein, whose function in engulfment is highly evolutionarily conserved; however, the upstream and downstream components of CED-6 mediated signaling are not known. Recently, ced-1 has been shown to encode a transmembrane protein on phagocytic cells, with two functional sequence motifs in its cytoplasmic tail that are important for engulfment. In this study, using a combination of biochemical approaches and yeast two-hybrid analysis, we present evidence for a physical interaction between GULP/CED-6 and one of the two motifs (NPXY motif) in the cytoplasmic tail of CED-1. The phosphotyrosine binding domain of GULP was necessary and sufficient for this interaction. Since the precise mammalian homologue of CED-1 is not known, we undertook a database search for human proteins that contain the motifs shown to be important for CED-1 function and identified CD91/LRP (low density lipoprotein receptor-related protein) as one candidate. Interestingly, recent studies have also identified CD91/LRP as a receptor involved in the phagocytosis of apoptotic cells in mammals. The GULP phosphotyrosine binding domain was able to specifically interact with one specific NPXY motif in the CD91 cytoplasmic tail. During these studies we have also identified the mouse GULP sequence. These studies suggest a physical link between CED-1 or CD91/LRP and the adapter protein CED-6/GULP during engulfment of apoptotic cells and further elucidate the pathway suggested by the genetic studies. PMID:11729193

  13. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

    SciTech Connect

    Morotomi-Yano, Keiko; Akiyama, Hidenori [Institute of Pulsed Power Science, Kumamoto University, Kumamoto 860-8555 (Japan)] [Institute of Pulsed Power Science, Kumamoto University, Kumamoto 860-8555 (Japan); Yano, Ken-ichi, E-mail: yanoken@kumamoto-u.ac.jp [Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto 860-8555 (Japan)] [Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto 860-8555 (Japan)

    2013-08-30

    Highlights: •Nanosecond pulsed electric field (nsPEF) is a new and unique means for life sciences. •Apoptosis was induced by nsPEF exposure in Jurkat cells. •No signs of apoptosis were detected in HeLa S3 cells exposed to nsPEFs. •Formation of poly(ADP-ribose) was induced in nsPEF-exposed HeLa S3 cells. •Two distinct modes of cell death were activated by nsPEF in a cell-dependent manner. -- Abstract: 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.

  14. Reactive oxygen species (ROS) mediates the mitochondrial-dependent apoptosis induced by transforming growth factor b in fetal hepatocytes

    Microsoft Academic Search

    BLANCA HERRERA; M. ALVAREZ; ARANZAZU SANCHEZ; MARGARITA FERNANDEZ; CESAR RONCERO; MANUEL BENITO; ISABEL FABREGAT

    Treatment of fetal rat hepatocytes with transforming growth factor beta (TGF-b) is followed by apoptotic cell death. Analysis of radical oxygen species (ROS) content and mitochondrial transmembrane po- tential (Dcm), using specific fluorescent probes in FACScan and confocal microscopy, showed that TGF-b mediates ROS production that precedes the loss of Dcm, the release of cytochrome c, and the activation of

  15. Cell, Vol. 104, 4356, January 12, 2001, Copyright 2001 by Cell Press CED-1 Is a Transmembrane Receptor that Mediates

    E-print Network

    Horvitz, H. Robert

    Receptor that Mediates Cell Corpse Engulfment in C. elegans serine receptor (PSR) (Fadok et al., 2000 for the engulfment of apoptotic corpses by macrophages (Franc et al., 1999). During the development; Sulston et al., 1983; Gu-grammed cell death. ced-1 encodes a transmembrane protein similar to human SREC

  16. Suppression of Fas-mediated apoptosis via steric shielding by filovirus glycoproteins.

    PubMed

    Noyori, Osamu; Nakayama, Eri; Maruyama, Junki; Yoshida, Reiko; Takada, Ayato

    2013-11-29

    Apoptotic death of virus-infected cells is generally thought to be a defense mechanism to limit the spread of infectious virions by eliminating virus-producing cells in host animals. On the other hand, several viruses have been shown to have anti-apoptotic mechanisms to facilitate efficient viral replication and transmission. In this study, we found that the filovirus glycoprotein (GP) expressed on cell surfaces formed a steric shield over the Fas molecule and that GP-expressing cells showed resistance to cell death induced by a Fas agonistic antibody. These results suggest that filovirus GP-mediated steric shielding may interfere with the Fas-induced apoptotic signal transduction in infected cells and serve as an immune evasion mechanism for filoviruses. PMID:24239546

  17. Homocysteine and its thiolactone may promote apoptotic events in blood platelets in vitro.

    PubMed

    Olas, Beata; Malinowska, Joanna; Rywaniak, Joanna

    2010-01-01

    The actions of homocysteine and its major metabolite, cyclic thioester, homocysteine thiolactone on endothelial cells, blood platelets, plasmatic fibrinogen and plasminogen--the important major components of haemostasis, regulating the flowing properties of blood--are complex and sometimes controversial. Homocysteine (Hcys) can promote apoptosis in endothelial cells, but the role of Hcys and its thiolactone in the apoptotic process in blood platelets is unknown. In order to study the appearance of apoptosis in platelets after treatment with the reduced form of Hcys or its thiolactone different markers were chosen: annexin V binding (phosphatidylserine exposure), platelet microparticle formation, mitochondrial membrane depolarization and ?IIb?3 expression in vitro. Apoptotic events and platelet activation were measured by a flow cytometer. In gel-filtered platelets treated with different concentrations of the reduced form of Hcys (25, 50 and 100 µM, 10 min) a significant increase of phosphatidylserine exposure (about 37% at the highest concentration, p < 0.001) and platelet microparticle formation were observed. Homocysteine caused also a dose-dependent depolarization of mitochondrial potential. The same apoptotic markers appeared in HTL-treated platelets (0.2 and 1 µM). Moreover, resveratrol (25 µM), a well known antioxidant, distinctly reduced the level of apoptotic markers. The obtained results indicate that Hcys and its thiolactone may promote in vitro apoptotic events in human gel-filtered platelets. PMID:20701459

  18. GRK6 deficiency in mice causes autoimmune disease due to impaired apoptotic cell clearance

    PubMed Central

    Nakaya, Michio; Tajima, Mitsuru; Kosako, Hidetaka; Nakaya, Takeo; Hashimoto, Akiko; Watari, Kenji; Nishihara, Hiroaki; Ohba, Mina; Komiya, Shiori; Tani, Naoki; Nishida, Motohiro; Taniguchi, Hisaaki; Sato, Yoji; Matsumoto, Mitsuru; Tsuda, Makoto; Kuroda, Masahiko; Inoue, Kazuhide; Kurose, Hitoshi

    2013-01-01

    Efficient engulfment of apoptotic cells is critical for maintaining tissue homoeostasis. When phagocytes recognize ‘eat me’ signals presented on the surface of apoptotic cells, this subsequently induces cytoskeletal rearrangement of phagocytes for the engulfment through Rac1 activation. However, the intracellular signalling cascades that result in Rac1 activation remain largely unknown. Here we show that G-protein-coupled receptor kinase 6 (GRK6) is involved in apoptotic cell clearance. GRK6 cooperates with GIT1 to activate Rac1, which promotes apoptotic engulfment independently from the two known DOCK180/ELMO/Rac1 and GULP1/Rac1 engulfment pathways. As a consequence, GRK6-deficient mice develop an autoimmune disease. GRK6-deficient mice also have increased iron stores in splenic red pulp in which F4/80+ macrophages are responsible for senescent red blood cell clearance. Our results reveal previously unrecognized roles for GRK6 in regulating apoptotic engulfment and its fundamental importance in immune and iron homoeostasis. PMID:23443560

  19. Capture of cell-derived microvesicles (exosomes and apoptotic bodies) by human plasmacytoid dendritic cells.

    PubMed

    Bastos-Amador, Patricia; Pérez-Cabezas, Begoña; Izquierdo-Useros, Nuria; Puertas, Maria C; Martinez-Picado, Javier; Pujol-Borrell, Ricardo; Naranjo-Gómez, Mar; Borràs, Francesc E

    2012-05-01

    cDCs and pDCs differ in multiple aspects. Among those, antigen capture is a recognized feature of cDCs, whereas pDCs display poor capacity to capture cell-derived antigens. However, animal models of organ transplantation suggested a role for pDCs in tolerance induction via phagocytosis of donor antigens. In a transplantation setting, microvesicles, such as apoptotic bodies and exosomes secreted by the graft, may be potential sources of alloantigen. Here, we tested the capacity of human pDCs to capture exosomes and apoptotic bodies from Jurkat T cells. Exosomes and apoptotic bodies were indeed captured by pDCs, although required longer times of incubation when compared with the highly endocytic cDCs. In cDCs and pDCs, exosome capture was more efficient than apoptotic bodies. Endocytosis inhibitors clearly impaired exosome capture by cDCs, although this could not be verified in pDCs as a result of cellular toxicity. Functionally, capture of Jurkat-derived exosomes did not induce nor prevent pDC maturation, and exosome-loaded pDCs induced T cell proliferation, suggesting a link between capture and presentation. Thus, exosomes and apoptotic bodies may be sources of antigen for human pDCs. PMID:22319103

  20. Number of apoptotic cells as a prognostic marker in invasive breast cancer

    PubMed Central

    Jong, J S de; Diest, P J van; Baak, J P A

    2000-01-01

    Apoptosis plays an important role in tumorigenesis. Tumour growth is determined by the rate of cell proliferation and cell death. We counted the number of apoptotic cells in haematoxylin and eosin (H&E)-stained tumour sections in series of 172 grade I and II invasive breast cancers with long-term follow-up. The number of apoptotic cells in ten high-power fields were converted to the number of apoptotic cells per mm2to obtain the apoptotic index (AI). The AI showed a positive correlation to the mitotic activity index (MAI) (P = 0.0001), histological grade (P< 0.0001) and worse tumour differentiation. Patients with high AI showed shorter overall survival than patients with low AI in the total group as well as in the lymph node-positive group. Tumour size, MAI, lymph node status and AI were independent prognostic indicators in multivariate analysis. The AI was shown to be of additional prognostic value to the MAI in the total patients group as well as in the lymph node-positive group. The correlation between the AI and the MAI points to linked mechanisms of apoptosis and proliferation. Since apoptotic cells can be counted with good reproducibility in H&E-stained tumour sections, the AI may be used as an additional prognostic indicator in invasive breast cancer. © 2000 Cancer Research Campaign PMID:10646890

  1. Apico-basal forces exerted by apoptotic cells drive epithelium folding.

    PubMed

    Monier, Bruno; Gettings, Melanie; Gay, Guillaume; Mangeat, Thomas; Schott, Sonia; Guarner, Ana; Suzanne, Magali

    2015-02-12

    Epithelium folding is a basic morphogenetic event that is essential in transforming simple two-dimensional epithelial sheets into three-dimensional structures in both vertebrates and invertebrates. Folding has been shown to rely on apical constriction. The resulting cell-shape changes depend either on adherens junction basal shift or on a redistribution of myosin II, which could be driven by mechanical signals. Yet the initial cellular mechanisms that trigger and coordinate cell remodelling remain largely unknown. Here we unravel the active role of apoptotic cells in initiating morphogenesis, thus revealing a novel mechanism of epithelium folding. We show that, in a live developing tissue, apoptotic cells exert a transient pulling force upon the apical surface of the epithelium through a highly dynamic apico-basal myosin II cable. The apoptotic cells then induce a non-autonomous increase in tissue tension together with cortical myosin II apical stabilization in the surrounding tissue, eventually resulting in epithelium folding. Together our results, supported by a theoretical biophysical three-dimensional model, identify an apoptotic myosin-II-dependent signal as the initial signal leading to cell reorganization and tissue folding. This work further reveals that, far from being passively eliminated as generally assumed (for example, during digit individualization), apoptotic cells actively influence their surroundings and trigger tissue remodelling through regulation of tissue tension. PMID:25607361

  2. DdrO is an essential protein that regulates the radiation desiccation response and the apoptotic-like cell death in the radioresistant Deinococcus radiodurans bacterium.

    PubMed

    Devigne, Alice; Ithurbide, Solenne; Bouthier de la Tour, Claire; Passot, Fanny; Mathieu, Martine; Sommer, Suzanne; Servant, Pascale

    2015-06-01

    Deinococcus radiodurans is known for its extreme radioresistance. Comparative genomics identified a radiation-desiccation response (RDR) regulon comprising genes that are highly induced after DNA damage and containing a conserved motif (RDRM) upstream of their coding region. We demonstrated that the RDRM sequence is involved in cis-regulation of the RDR gene ddrB?in vivo. Using a transposon mutagenesis approach, we showed that, in addition to ddrO encoding a predicted RDR repressor and irrE encoding a positive regulator recently shown to cleave DdrO in Deinococcus?deserti, two genes encoding ?-keto-glutarate dehydrogenase subunits are involved in ddrB regulation. In wild-type cells, the DdrO cell concentration decreased transiently in an IrrE-dependent manner at early times after irradiation. Using a conditional gene inactivation system, we showed that DdrO depletion enhanced expression of three RDR proteins, consistent with the hypothesis that DdrO acts as a repressor of the RDR regulon. DdrO-depleted cells loose viability and showed morphological changes evocative of an apoptotic-like response, including membrane blebbing, defects in cell division and DNA fragmentation. We propose that DNA repair and apoptotic-like death might be two responses mediated by the same regulators, IrrE and DdrO, but differently activated depending on the persistence of IrrE-dependent DdrO cleavage. PMID:25754115

  3. Phosphorylation Drives an Apoptotic Protein to Activate Antiapoptotic Genes

    PubMed Central

    Halder, Umesh Chandra; Bhowmick, Rahul; Roy Mukherjee, Tapasi; Nayak, Mukti Kant; Chawla-Sarkar, Mamta

    2013-01-01

    During infection, viral proteins target cellular pathways that regulate cellular innate immune responses and cell death. We demonstrate that influenza A virus matrix 1 protein (M1), an established proapoptotic protein, activates nuclear factor-?B member RelB-mediated survival genes (cIAP1, cIAP2, and cFLIP), a function that is linked with its nuclear translocation during early infection. Death domain-associated protein 6 (Daxx) is a transcription co-repressor of the RelB-responsive gene promoters. During influenza virus infection M1 binds to and stabilizes Daxx protein by preventing its ubiquitination and proteasomal degradation. Binding of M1 with Daxx through its Daxx binding motif prevents binding of RelB and Daxx, resulting in up-regulation of survival genes. This interaction also prevents promoter recruitment of DNA methyltransferases (Dnmt1 and Dnmt3a) and lowers CpG methylation of the survival gene promoters, leading to the activation of these genes. Thus, M1 prevents repressional function of Daxx during infection, thereby exerting a survival role. In addition to its nuclear localization signal, translocation of M1 to the nucleus depends on cellular kinase-mediated phosphorylation as the protein kinase C inhibitor calphostin C effectively down-regulates virus replication. The study reconciles the ambiguity of dual antagonistic function of viral protein and potentiates a possible target to limit virus infection. PMID:23548901

  4. CARDIOPROTECTIVE AND ANTI-APOPTOTIC EFFECTS OF HEME OXYGENASE-1 IN THE FAILING HEART

    PubMed Central

    Wang, Guangwu; Hamid, Tariq; Keith, Rachel J.; Zhou, Guihua; Partridge, Charles R.; Xiang, Xilin; Kingery, Justin R.; Lewis, Robert K.; Li, Qianhong; Rokosh, D. Gregg; Ford, Rachael; Spinale, Francis G.; Riggs, Daniel W.; Srivastava, Sanjay; Bhatnagar, Aruni; Bolli, Roberto; Prabhu, Sumanth D.

    2010-01-01

    Background Heme oxygenase-1 (HO-1) is an inducible stress-response protein that imparts antioxidant and anti-apoptotic effects. However, its pathophysiological role in cardiac remodeling and chronic heart failure (HF) is unknown. We hypothesized that induction of HO-1 in HF ameliorates pathological remodeling. Methods and Results Adult male non-transgenic (NTG) and myocyte-restricted HO-1 transgenic (TG) mice underwent either sham operation or coronary ligation to induce HF. Four weeks after ligation, NTG HF mice exhibited post-infarction LV remodeling and dysfunction, hypertrophy, fibrosis, oxidative stress, apoptosis, and reduced capillary density, associated with a two-fold increase in HO-1 expression in noninfarcted myocardium. Compared with NTG, HO-1 TG HF mice exhibited significantly (p < 0.05) improved post-infarction survival (94% vs. 57%), and less LV dilatation (end-diastolic volume 46 ± 8 vs. 85 ± 32 µL), mechanical dysfunction (ejection fraction 65 ± 9 vs. 49 ± 16 %), hypertrophy (LV/tibia length 4.4 ± 0.4 vs. 5.2 ± 0.6 mg/mm), interstitial fibrosis (11.2 ± 3.1 vs. 18.5 ± 3.5 % ), and oxidative stress (3-fold reduction in tissue malondialdehyde). Moreover, myocyte-specific HO-1 overexpression in HF promoted tissue neovascularization and ameliorated myocardial p53 expression (2-fold reduction) and apoptosis. Inisolated mitochondria, mitochondrial permeability transition (MPT) was inhibited by HO-1 in a carbon monoxide (CO)-dependent manner, and was recapitulated by the CO donor CORM-3. HO-1-derived CO also prevented H2O2-induced cardiomyocyte apoptosis and cell death. Finally, in vivo treatment with CORM-3 alleviated post-infarction LV remodeling, p53 expression, and apoptosis. Conclusions HO-1 induction in the failing heart is an important cardioprotective adaptation that opposes pathological LV remodeling, and this effect is mediated, at least in part, by CO-dependent inhibition of MPT and apoptosis. Augmentation of HO-1 or its product, CO, may represent a novel therapeutic strategy for ameliorating HF. PMID:20404253

  5. Protective Role of Morin, a Flavonoid, against High Glucose Induced Oxidative Stress Mediated Apoptosis in Primary Rat Hepatocytes

    PubMed Central

    Kapoor, Radhika; Kakkar, Poonam

    2012-01-01

    Apoptosis is an early event of liver damage in diabetes and oxidative stress has been linked to accelerate the apoptosis in hepatocytes. Therefore, the compounds that can scavenge ROS may confer regulatory effects on high-glucose induced apoptosis. In the present study, primary rat hepatocytes were exposed to high concentration (40 mM) of glucose. At this concentration decreased cell viability and enhanced ROS generation was observed. Depleted antioxidant status of hepatocytes under high glucose stress was also observed as evident from transcriptional level and activities of antioxidant enzymes. Further, mitochondrial depolarisation was accompanied by the loss of mitochondrial integrity and altered expression of Bax and Bcl-2. Increased translocation of apoptotic proteins like AIF (Apoptosis inducing factor) & Endo-G (endonuclease-G) from its resident place mitochondria to nucleus was also observed. Cyt-c residing in the inter-membrane space of mitochondria also translocated to cytoplasm. These apoptotic proteins initiated caspase activation, DNA fragmentation, chromatin condensation, increased apoptotic DNA content in glucose treated hepatocytes, suggesting mitochondria mediated apoptotic mode of cell death. Morin, a dietary flavonoid from Psidium guajava was effective in increasing the cell viability and decreasing the ROS level. It maintained mitochondrial integrity, inhibited release of apoptotic proteins from mitochondria, prevented DNA fragmentation, chromatin condensation and hypodiploid DNA upon exposure to high glucose. This study confirms the capacity of dietary flavonoid Morin in regulating apoptosis induced by high glucose via mitochondrial mediated pathway through intervention of oxidative stress. PMID:22899998

  6. Complex changes in the apoptotic and cell differentiation programs during initiation of the hair follicle response to chemotherapy

    PubMed Central

    Sharova, Tatyana Y.; Poterlowicz, Krzysztof; Botchkareva, Natalia V.; Kondratiev, Nikita A.; Aziz, Ahmar; Spiegel, Jeffrey H.; Botchkarev, Vladimir A.; Sharov, Andrey A.

    2014-01-01

    Chemotherapy has severe side-effects for normal rapidly proliferating organs, such as hair follicle, and causes massive apoptosis in hair matrix keratinocytes followed by hair loss. To define the molecular signature of hair follicle response to chemotherapy, human scalp hair follicles cultured ex vivo were treated with doxorubicin and global microarray analysis was performed 3 hours after treatment. Microarray data revealed changes in expression of 504 genes in doxorubicin-treated hair follicles versus the controls. Among these genes, upregulations of several tumor necrosis factor family of apoptotic receptors (FAS, TRAIL receptors 1/2), as well as of a large number of the keratin-associated protein genes were seen after doxorubicin treatment. Hair follicle apoptosis induced by doxorubicin was significantly inhibited by either TRAIL neutralizing antibody or caspase 8 inhibitor, thus suggesting a novel role for TRAIL receptor signaling in mediating doxorubicin-induced hair loss. These data demonstrate that the early phase of the hair follicle response to doxorubicin includes upregulation of apoptosis-associated markers, as well as substantial re-organization of the terminal differentiation programs in hair follicle keratinocytes. These data provide an important platform for further studies towards the design of novel approaches for management of chemotherapy-induced hair loss. PMID:24999588

  7. Low Density Lipoprotein Receptor-related Protein 1 Promotes Anti-apoptotic Signaling in Neurons by Activating Akt Survival Pathway*

    PubMed Central

    Fuentealba, Rodrigo A.; Liu, Qiang; Kanekiyo, Takahisa; Zhang, Juan; Bu, Guojun

    2009-01-01

    The low density lipoprotein receptor-related protein 1 (LRP1) is a multi-ligand receptor abundantly expressed in neurons. Previous work has shown that brain LRP1 levels are decreased during aging and in Alzheimer disease. Although mounting evidence has demonstrated a role for LRP1 in the metabolism of apolipoprotein E/lipoprotein and amyloid-? peptide, whether LRP1 also plays a direct role in neuronal survival is not clear. Here, we show that LRP1 expression is critical for the survival of primary neurons under stress conditions including trophic withdrawal, the presence of apoptosis inducers, or amyloid-?-induced neurotoxicity. Using lentiviral short hairpin RNA to knock down endogenous LRP1 expression, we showed that a depletion of LRP1 leads to an activation of caspase-3 and increased neuronal apoptosis, an effect that was rescued by a caspase-3 inhibitor. A correlation between decreased Akt phosphorylation and the activation of caspase-3 was demonstrated in LRP1 knocked down neurons. Notably, LRP1 knockdown decreased insulin receptor levels in primary neurons, suggesting that decreased neuronal survival might be a consequence of an impaired insulin receptor signaling pathway. Correspondingly, both insulin receptor and phospho-Akt levels were decreased in LRP1 forebrain knock-out mice. These results demonstrate that LRP1 mediates anti-apoptotic function in neurons by regulating insulin receptor and the Akt survival pathway and suggest that restoring LRP1 expression in Alzheimer disease brain might be beneficial to inhibiting neurodegeneration. PMID:19815552

  8. Futile caspase-8 activation during the apoptotic cell death induced by DNA damaging agents in human B-lymphoblasts.

    PubMed

    Vit, J P; Guillouf, C; Rosselli, F

    2001-09-10

    Caspase-8 plays an essential role in apoptosis induced by Fas activation. Moreover, caspase-8 can be processed also in response to exposure to genotoxic agents. To decipher the role of caspase-8 in DNA damaging agent (DDA)-induced apoptosis as well as the pathway(s) leading to its activation in response to genotoxic stress, we investigated caspase-8 processing induced by ionizing radiation (IR) or mitomycin C (MMC) treatment in human B-lymphoblasts. Altogether, our observations establish that caspase-8 is actively processed in both receptor-mediated and DDA-induced cell death. However, while Fas-dependent apoptosis absolutely required caspase-8 activity, it is not necessary for completion of the apoptotic program induced by IR and MMC. Experiments performed to understand the molecular pathway(s) of the caspase-8 activation after DDA demonstrated that for both IR and MMC, the Fas/Fas-L interaction is dispensable. Data obtained from caspase inhibitors and from lymphoblasts carrying mutations in ATM and FANCC proteins, involved in DDA response, clearly showed that distinct mechanisms are responsible for caspase-8 activation by IR and MMC in B-lymphoblasts. IR-dependent processing of caspase-8 involves ATM, mitochondrial collapse, FANCC, and caspase-3 activation. Caspase-8 activation by MMC evokes the mitochondrial pathways involving FANCC but not ATM. Collectively, our data indicate that caspase-8 activation is essentially a bystander effect and not a major determinant of the behavior of DDA-exposed cells. PMID:11525634

  9. The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling

    PubMed Central

    Mukherjee, Amitava; Morosky, Stefanie A.; Delorme-Axford, Elizabeth; Dybdahl-Sissoko, Naomi; Oberste, M. Steven; Wang, Tianyi; Coyne, Carolyn B.

    2011-01-01

    The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor (PRR) pathways that converge on the induction of type I interferons (IFNs). Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation. Here we show that the 3Cpro cysteine protease of coxsackievirus B3 (CVB3) cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) as a mechanism to escape host immunity. We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture. CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3Cpro, occurred at specific sites and within specialized domains of each molecule, and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors. 3Cpro-mediated MAVS cleavage occurred within its proline-rich region, led to its relocalization from the mitochondrial membrane, and ablated its downstream signaling. We further show that 3Cpro cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm. Taken together, these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition. PMID:21436888

  10. Impaired Clearance of Apoptotic Cells in Chronic Inflammatory Diseases: Therapeutic Implications

    PubMed Central

    Szondy, Zsuzsa; Garabuczi, Éva; Joós, Gergely; Tsay, Gregory J.; Sarang, Zsolt

    2014-01-01

    In healthy individuals, billions of cells die by apoptosis every day. Removal of the dead cells by phagocytosis (a process called efferocytosis) must be efficient to prevent secondary necrosis and the consequent release of pro-inflammatory cell contents that damages the tissue environment and provokes autoimmunity. In addition, detection and removal of apoptotic cells generally induces an anti-inflammatory response. As a consequence improper clearance of apoptotic cells, being the result of either genetic anomalies and/or a persistent disease state, contributes to the establishment and progression of a number of human chronic inflammatory diseases such as autoimmune and neurological disorders, inflammatory lung diseases, obesity, type 2 diabetes, or atherosclerosis. During the past decade, our knowledge about the mechanism of efferocytosis has significantly increased, providing therapeutic targets through which impaired phagocytosis of apoptotic cells and the consequent inflammation could be influenced in these diseases. PMID:25136342

  11. Salvia x jamensis J. Compton: Trichomes, essential oil constituents and cytotoxic-apoptotic activity.

    PubMed

    Fraternale, Daniele; Albertini, Maria Cristina; Rudov, Alexander; Flamini, Guido; Ricci, Donata; Bisio, Angela; Battistelli, Michela; Accorsi, Augusto

    2013-01-01

    Salvia x jamensis J. Compton is a hybrid between Salvia greggii A. Gray and Salvia microphylla Kunt. In this study, we describe three hair types identified by Scanning Electron Microscopy. In the essential oil of the aerial parts of S. jamensis 56 different compounds were identified. The two main constituents were ?-caryophyllene (14.8%) and ?-pinene (6.8%). Cytotoxic-apoptotic activity of S. x jamensis essential oil has been investigated by using U937 cell line. The essential oil EC50 for cell number and for cell apoptosis have been shown to be 360 and 320?µg?mL(-1), respectively. Among the constituents of the oil examined, only ?-caryophyllene, ?-pinene and ?-pinene displayed cytotoxic and apoptotic activities. For the first time, it has been demonstrated that some of the pure constituents identified within S. x jamensis essential oil are responsible for its cytotoxic-apoptotic activity when properly combined. PMID:23030520

  12. Mitochondrial staining allows robust elimination of apoptotic and damaged cells during cell sorting.

    PubMed

    Barteneva, Natasha S; Ponomarev, Eugeny D; Tsytsykova, Alla; Armant, Myriam; Vorobjev, Ivan A

    2014-04-01

    High-speed fluorescence-activated cell sorting is relevant for a plethora of applications, such as PCR-based techniques, microarrays, cloning, and propagation of selected cell populations. We suggest a simple cell-sorting technique to eliminate early and late apoptotic and necrotic cells, with good signal-to-noise ratio and a high-purity yield. The mitochondrial potential dye, TMRE (tetramethylrhodamine ethyl ester perchlorate), was used to separate viable and non-apoptotic cells from the cell sorting samples. TMRE staining is reversible and does not affect cell proliferation and viability. Sorted TMRE(+) cells contained a negligible percentage of apoptotic and damaged cells and had a higher proliferative potential as compared with their counterpart cells, sorted on the basis of staining with DNA viability dye. This novel sorting technique using TMRE does not interfere with subsequent functional assays and is a method of choice for the enrichment of functionally active, unbiased cell populations. PMID:24394470

  13. Anti-apoptotic BCL-2 family proteins in acute neural injury.

    PubMed

    Anilkumar, Ujval; Prehn, Jochen H M

    2014-01-01

    Cells under stress activate cell survival and cell death signaling pathways. Cell death signaling frequently converges on mitochondria, a process that is controlled by the activities of pro- and anti-apoptotic B-cell lymphoma 2 (BCL-2) proteins. In this review, we summarize current knowledge on the control of neuronal survival, development and injury by anti-apoptotic BCL-2 family proteins. We discuss overlapping and differential effects of the individual family members BCL-2, BCL-extra long (BCL-XL), myeloid cell leukemia 1 (MCL-1), and BCL2-like 2 (BCL-W) in the control of survival during development and pathophysiological processes such as trophic factor withdrawal, ischemic injury, excitotoxicity, oxidative stress and energy stress. Finally we discuss recent evidence that several anti-apoptotic BCL-2 proteins influence mitochondrial bioenergetics and control neuronal Ca(2+) homeostasis independent of their classical role in cell death signaling. PMID:25324720

  14. Anti-apoptotic BCL-2 family proteins in acute neural injury

    PubMed Central

    Anilkumar, Ujval; Prehn, Jochen H. M.

    2014-01-01

    Cells under stress activate cell survival and cell death signaling pathways. Cell death signaling frequently converges on mitochondria, a process that is controlled by the activities of pro- and anti-apoptotic B-cell lymphoma 2 (BCL-2) proteins. In this review, we summarize current knowledge on the control of neuronal survival, development and injury by anti-apoptotic BCL-2 family proteins. We discuss overlapping and differential effects of the individual family members BCL-2, BCL-extra long (BCL-XL), myeloid cell leukemia 1 (MCL-1), and BCL2-like 2 (BCL-W) in the control of survival during development and pathophysiological processes such as trophic factor withdrawal, ischemic injury, excitotoxicity, oxidative stress and energy stress. Finally we discuss recent evidence that several anti-apoptotic BCL-2 proteins influence mitochondrial bioenergetics and control neuronal Ca2+ homeostasis independent of their classical role in cell death signaling. PMID:25324720

  15. Mediated Faces

    Microsoft Academic Search

    Judith S. Donath

    2001-01-01

    Incorporating faces into mediated discussions is a complex design problem. The face conveys social and personal identity; it reports fleeting changes of emotion and the cumulative effects of often repeated expressions. The face both expresses and betrays: it shows what the person wishes to convey - and much more. We are highly attuned to recognizing and interpreting faces (though these

  16. Gamma-hydroxybutyrate, acting through an anti-apoptotic mechanism, protects native and amyloid-precursor-protein-transfected neuroblastoma cells against oxidative stress-induced death.

    PubMed

    Wendt, G; Kemmel, V; Patte-Mensah, C; Uring-Lambert, B; Eckert, A; Schmitt, M J; Mensah-Nyagan, A G

    2014-03-28

    Clinical observations suggested that gamma-hydroxybutyrate (GHB) protects nerve cells against death but the direct proofs are missing. Here, we combined several approaches to investigate GHB capacity to protect human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced death. To increase the patho-physiological relevancy of our study, we used native SH-SY5Y cells and SH-SY5Y cells stably transfected with the wild-type amyloid-precursor-protein (APPwt) or control-vector-pCEP4. Trypan Blue exclusion and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide) assays combined with pharmacological analyses showed that H2O2 reduced native and genetically modified cell viability and APPwt-transfected cells were the most vulnerable. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and activated caspase-3 staining assessed by flow cytometry revealed a basally elevated apoptotic signal in APPwt-transfected cells. Reverse-transcription, real-time quantitative polymerase chain reaction (qPCR) and Western blotting showed that mRNA and protein basal ratios of apoptotic modulators Bax/Bcl-2 were also high in APPwt-transfected cells. GHB efficiently and dose-dependently rescued native and genetically modified cells from H2O2-induced death. Interestingly, GHB, which strongly decreased elevated basal levels of TUNEL-staining, activated caspase 3-labeling and Bax/Bcl-2 in APPwt-transfected cells, also counteracted H2O2-evoked increased apoptotic markers in native and genetically modified SH-SY5Y cells. Since GHB did not promote cell proliferation, anti-apoptotic action through the down-regulation of Bax/Bcl-2 ratios and/or caspase 3 activity appears as a critical mechanism involved in GHB-induced protection of SH-SY5Y cells against APPwt-overexpression- or H2O2-evoked death. Altogether, these results, providing multi-parametric evidence for the existence of neuroprotective action of GHB, also open interesting perspectives for the development of GHB analog-based strategies against neurodegeneration or nerve cell death. PMID:24456637

  17. Characterization of Cyclin E Expression in Multiple Myeloma and Its Functional Role in Seliciclib-Induced Apoptotic Cell Death

    PubMed Central

    Shimoni, Avichai; Ostrovsky, Olga; Samookh, Michal; Peled, Amnon; Nagler, Arnon

    2012-01-01

    Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cell that eventually develops resistance to chemotherapy. Drug resistance, differentiation block and increased survival of the MM tumor cells result from high genomic instability. Chromosomal translocations, the most common genomic alterations in MM, lead to dysregulation of cyclin D, a regulatory protein that governs the activation of key cell cycle regulator – cyclin dependent kinase (CDK). Genomic instability was reported to be affected by over expression of another CDK regulator - cyclin E (CCNE). This occurs early in tumorigenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore sought to investigate the role of cyclin E in MM. CCNE1 expression was found to be heterogeneous in various MM cell lines (hMMCLs). Incubation of hMMCLs with seliciclib, a selective CDK-inhibitor, results in apoptosis which is accompanied by down regulation of MCL1 and p27. Ectopic over expression of CCNE1 resulted in reduced sensitivity of the MM tumor cells in comparison to the paternal cell line, whereas CCNE1 silencing with siRNA increased the cell sensitivity to seliciclib. Adhesion to FN of hMMCLs was prevented by seliciclib, eliminating adhesion–mediated drug resistance of MM cells. Combination of seliciclib with flavopiridol effectively reduced CCNE1 and CCND1 protein levels, increased subG1 apoptotic fraction and promoted MM cell death in BMSCs co-culture conditions, therefore over-coming stroma-mediated protection. We suggest that seliciclib may be considered as essential component of modern anti MM drug combination therapy. PMID:22558078

  18. Intrinsic and extrinsic apoptotic pathways are involved in rat testis by cold water immersion-induced acute and chronic stress.

    PubMed

    Juárez-Rojas, Adriana Lizbeth; García-Lorenzana, Mario; Aragón-Martínez, Andrés; Gómez-Quiroz, Luis Enrique; Del Socorro Retana-Márquez, María

    2015-08-01

    Testicular apoptosis is activated by stress, but it is not clear which signaling pathway is activated in response to stress. The aim of this study was to investigate whether intrinsic, extrinsic, or both apoptotic signaling pathways are activated by acute and chronic stress. Adult male rats were subjected to cold water immersion-induced stress for 1, 20, 40, and 50 consecutive days. The seminiferous tubules:apoptotic cell ratio was assayed on acute (1 day) and chronic (20, 40, 50 days) stress. Apoptotic markers, including cleaved-caspase 3 and 8, the pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins were also determined after acute and chronic stress induction. Additionally, epididymal sperm quality was evaluated, as well as corticosterone and testosterone levels. An increase in tubule apoptotic cell count percentage after an hour of acute stress and during chronic stress induction was observed. The apoptotic cells rate per tubule increment was only detected one hour after acute stress, but not with chronic stress. Accordingly, there was an increase in Bax, cleaved caspase-8 and caspase-3 pro-apoptotic proteins with a decrease of anti-apoptotic Bcl-2 in both acutely and chronically stressed male testes. In addition, sperm count, viability, as well as total and progressive motility were low in chronically stressed males. Finally, the levels of corticosterone increased whereas testosterone levels decreased in chronically stressed males. Activation of the extrinsic apoptotic pathway was shown by cleaved caspase-8 increase whereas the intrinsic apoptotic pathway activation was determined by the increase of Bax, along with Bcl-2 decrease, making evident a cross-talk between these two pathways with the activation of caspase-3. These results suggest that both acute and chronic stress can potentially activate the intrinsic/extrinsic apoptosis pathways in testes. Chronic stress also reduces the quality of epididymal spermatozoa, possibly due to a decrease in testosterone. PMID:25867867

  19. Anti-apoptotic signaling as a cytoprotective mechanism in mammalian hibernation

    PubMed Central

    Mamady, Hapsatou; Tessier, Shannon N.

    2013-01-01

    In the context of normal cell turnover, apoptosis is a natural phenomenon involved in making essential life and death decisions. Apoptotic pathways balance signals which promote cell death (pro-apoptotic pathways) or counteract these signals (anti-apoptotic pathways). We proposed that changes in anti-apoptotic proteins would occur during mammalian hibernation to aid cell preservation during prolonged torpor under cellular conditions that are highly injurious to most mammals (e.g. low body temperatures, ischemia). Immunoblotting was used to analyze the expression of proteins associated with pro-survival in six tissues of thirteen-lined ground squirrels, Ictidomys tridecemlineatus. The brain showed a concerted response to torpor with significant increases in the levels of all anti-apoptotic targets analyzed (Bcl-2, Bcl-xL, BI-1, Mcl-1, cIAP1/2, xIAP) as well as enhanced phosphorylation of Bcl-2 at S70 and T56. Heart responded similarly with most anti-apoptotic proteins elevated significantly during torpor except for Bcl-xL and xIAP that decreased and Mcl-1 that was unaltered. In liver, BI-1 increased whereas cIAP1/2 decreased. In kidney, there was an increase in BI-1, cIAP and xIAP but decreases in Bcl-xL and p-Bcl-2(T56) content. In brown adipose tissue, protein levels of BI-1, cIAP1/2, and xIAP decreased significantly during torpor (compared with euthermia) whereas Bcl-2, Bcl-xL, Mcl-1 were unaltered; however, Bcl-2 showed enhanced phosphorylation at Thr56 but not at Ser70. In skeletal muscle, only xIAP levels changed significantly during torpor (an increase). The data show that anti-apoptotic pathways have organ-specific responses in hibernators with a prominent potential role in heart and brain where coordinated enhancement of anti-apoptotic proteins occurred in response to torpor. PMID:23638364

  20. Anti-Apoptotic Machinery Protects the Necrotrophic Fungus Botrytis cinerea from Host-Induced Apoptotic-Like Cell Death during Plant Infection

    PubMed Central

    Shlezinger, Neta; Minz, Anna; Gur, Yonatan; Hatam, Ido; Dagdas, Yasin F.; Talbot, Nicholas J.; Sharon, Amir

    2011-01-01

    Necrotrophic fungi are unable to occupy living plant cells. How such pathogens survive first contact with living host tissue and initiate infection is therefore unclear. Here, we show that the necrotrophic grey mold fungus Botrytis cinerea undergoes massive apoptotic-like programmed cell death (PCD) following germination on the host plant. Manipulation of an anti-apoptotic gene BcBIR1 modified fungal response to PCD-inducing conditions. As a consequence, strains with reduced sensitivity to PCD were hyper virulent, while strains in which PCD was over-stimulated showed reduced pathogenicity. Similarly, reduced levels of PCD in the fungus were recorded following infection of Arabidopsis mutants that show enhanced susceptibility to B. cinerea. When considered together, these results suggest that Botrytis PCD machinery is targeted by plant defense molecules, and that the fungal anti-apoptotic machinery is essential for overcoming this host-induced PCD and hence, for establishment of infection. As such, fungal PCD machinery represents a novel target for fungicides and antifungal drugs. PMID:21876671

  1. ALA-PDT mediated DC vaccine for skin squamous cell carcinoma

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

    Dendritic cell (DC) based vaccine has emerged as a promising immunotherapy for cancers. However, most DC vaccines so far have only achieved 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. The anti-tumor immunity of ALA-PDT-DC vaccine was tested with mouse model. We observed the maturations of DCs potentiated by ALA-PDT treated tumor cells, including phenotypic maturation (upregulation of surface expression of MHC-II, DC80, and CD86), and functional maturation (enhanced capability to secret INF-? and IL-12). ALA-PDT-DC vaccine mediated by apoptotic cells provided protection against tumor 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 DC-based cancer vaccine, which could improve the clinical application of PDT- DC vaccines.

  2. Apoptotic cell death and altered calcium homeostasis caused by frataxin depletion in dorsal root ganglia neurons can be prevented by BH4 domain of Bcl-xL protein.

    PubMed

    Mincheva-Tasheva, Stefka; Obis, Elia; Tamarit, Jordi; Ros, Joaquim

    2014-04-01

    Friedreich ataxia (FRDA) is a neurodegenerative disease characterized by a decreased expression of the mitochondrial protein frataxin. Major neurological symptoms of the disease are due to degeneration of dorsal root ganglion (DRG) sensory neurons. In this study we have explored the neurodegenerative events occurring by frataxin depletion on primary cultures of neurons obtained from rat DRGs. Reduction of 80% of frataxin levels in these cells was achieved by transduction with lentivirus containing shRNA silencing sequences. Frataxin depletion caused mitochondrial membrane potential decrease, neurite degeneration and apoptotic cell death. A marked increase of free intracellular Ca(2+) levels and alteration in Ca(2+)-mediated signaling pathways was also observed, thus suggesting that altered calcium homeostasis can play a pivotal role in neurodegeneration caused by frataxin deficiency. These deleterious effects were reverted by the addition of a cell-penetrant TAT peptide coupled to the BH4, the anti-apoptotic domain of Bcl-x(L). Treatment of cultured frataxin-depleted neurons with TAT-BH4 was able to restore the free intracellular Ca(2+) levels and protect the neurons from degeneration. These observations open the possibility of new therapies of FRDA based on modulating the Ca(2+) signaling and prevent apoptotic process to protect DRG neurons from neurodegeneration. PMID:24242291

  3. AMP-Activated Protein Kinase ? 2 Isoform Suppression in Primary Breast Cancer Alters AMPK Growth Control and Apoptotic Signaling.

    PubMed

    Fox, Melissa M; Phoenix, Kathryn N; Kopsiaftis, Stavros G; Claffey, Kevin P

    2013-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is a metabolic regulator that promotes energy conservation and restoration when cells are exposed to nutrient stress. Given the high metabolic requirement of cancer cells, AMPK activation has been suggested as a potential preventative and therapeutic target. However, previous findings have shown that AMPK activity is diminished in some cancers. Expression of the 2 catalytic isoforms, AMPK?1 and AMPK?2, was evaluated in primary breast cancer and matched nontumor-adjacent tissue samples using immunohistochemistry. AMPK-dependent growth signaling events were examined in primary human mammary epithelial cells (HMECs) using RNAi to understand the importance of AMPK?2 in normal growth regulation. To test whether AMPK?2 would reinstate growth control and apoptotic mechanisms in breast cancer cells, metabolic stress assays and tumor xenografts were performed in MCF-7 cells, expressing low levels of AMPK?2, with stable transfection of either green fluorescent protein (GFP) or AMPK?2 expression constructs. AMPK?2 was found to be significantly suppressed in breast cancer tissue samples, whereas AMPK?1 was not. In normal HMECs, low glucose stress resulted in AMPK-driven growth inhibition. Interestingly, this response was ablated when AMPK?2 was silenced. Metabolic stress assays in MCF-7 cells indicated that AMPK?2 expression reduced both mTOR signaling and cyclin D1 expression, contributing to G1-phase cell cycle arrest. Cells expressing AMPK?2 underwent apoptosis more readily than GFP control cells. Xenograft studies demonstrated that MCF-7 tumors expressing AMPK?2 display reduced proliferation and increased apoptotic events. Furthermore, AMPK?2 xenografts exhibited diminished cyclin D1 levels along with an increased amount of nuclear p53, thereby implicating the AMPK?2-p53 signaling axis as a mediator of cell apoptosis. Together, these results highlight the significance of reduced AMPK activity contributing to human carcinogenesis and, specifically, the role of AMPK?2 with respect to its control of normal mammary epithelial cell growth and its reduced expression in breast cancer. PMID:23946867

  4. Distinct Modes of Macrophage Recognition for Apoptotic and Necrotic Cells Are Not Specified Exclusively by Phosphatidylserine Exposure

    Microsoft Academic Search

    Regina E. Cocco; David S. Ucker

    2001-01-01

    The distinction between physiological (apoptotic) and pathological (necrotic) cell deaths reflects mechanistic differences in cellular disintegration and is of functional significance with respect to the outcomes that are triggered by the cell corpses. Mechanistically, apoptotic cells die via an active and ordered pathway; necrotic deaths, conversely, are chaotic and passive. Macrophages and other phagocytic cells recognize and engulf these dead

  5. Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT, annexin-V and 7AAD cytofluorometric staining methods

    Microsoft Academic Search

    Hervé Lecoeur; Eric Ledru; Marie-Christine Prévost; Marie-Lise Gougeon

    1997-01-01

    The present article compares the reliability of four previously described cytofluorometric methods of apoptosis quantification for phenotyping apoptotic human lymphocytes. Each of these assays detects distinct cellular alterations of the apoptotic process. Alteration in plasma membrane integrity can be evaluated following 7-AAD incorporation and the translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane can

  6. A new insight into phagocytosis of apoptotic cells: proteolytic enzymes divert the recognition and clearance of polymorphonuclear leukocytes by macrophages

    Microsoft Academic Search

    K Guzik; M Bzowska; J Smagur; O Krupa; M Sieprawska; J Travis; J Potempa

    2007-01-01

    The recognition of phosphatidylserine (PS) on the surface of any apoptotic cell is considered to be a key event for its clearance. We challenge this concept by showing that pretreatment of neutrophils with either host or bacterial protease affects their uptake by human monocyte-derived macrophages without having an effect on cell-surface PS presentation. Specifically, whereas preincubation of apoptotic neutrophils with

  7. RECOGNITION AND PHAGOCYTOSIS OF APOPTOTIC T CELLS BY RESIDENT MURINE TISSUE MACROPHAGES REQUIRES MULTIPLE SIGNAL TRANSDUCTION EVENTS

    PubMed Central

    Hu, Bin; Punturieri, Antonello; Todt, Jill; Sonstein, Joanne; Polak, Timothy; Curtis, Jeffrey L.

    2015-01-01

    Macrophages (Mø) ingest apoptotic cells with unique effects on their cytokine production, but the signaling pathways involved are virtually unknown. Signal transduction in response to recognition of apoptotic thymocytes by resident murine alveolar (AMø) or peritoneal (PMø) Mø was studied by in vitro phagocytosis assay. Phagocytosis was decreased in a dose-dependent and non-toxic fashion by inhibiting phosphatidylinositol 3 kinase (wortmannin and LY294002), protein tyrosine phosphorylation (herbimycin A, genistein, piceatannol and, for AMø only, PP2), and protein kinase C (staurosporine, Gö 6976 and calphostin C). Exposure of Mø to apoptotic or heat-killed thymocytes, but not to viable thymocytes, rapidly activated ERK1/2, as detected by specific phosphorylation, but did not activate NF-?B or MAP kinases p38 or JNK. Mø phagocytosis of apoptotic T cells requires tyrosine, serine/threonine, and lipid phosphorylation. Mø recognition of apoptotic T cells triggers rapid but limited MAP kinase activation. PMID:11994514

  8. Photoluminescent graphene quantum dots for in vivo imaging of apoptotic cells.

    PubMed

    Roy, Prathik; Periasamy, Arun Prakash; Lin, Chiu-Ya; Her, Guor-Mour; Chiu, Wei-Jane; Li, Chi-Lin; Shu, Chia-Lun; Huang, Chih-Ching; Liang, Chi-Te; Chang, Huan-Tsung

    2015-02-14

    Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro; however, there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescence properties of plant leaf-derived graphene quantum dots (GQDs) modified with annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with the GQDs exhibiting high biocompatibility as they were excreted from the zebrafish's body without affecting its growth significantly at a concentration lower than 2 mg mL(-1) over a period of 4 to 72 hour post fertilization. The GQDs have further been used to image human breast adenocarcinoma cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), and normal human mammary epithelial cell line (MCF-10A). These results are indispensable to further the advance of graphene-based nanomaterials for biomedical applications. PMID:25569453

  9. Nitric Oxide Induces Cell Death by Regulating Anti-Apoptotic BCL2 Family Members

    Microsoft Academic Search

    Colleen M. Snyder; Emelyn H. Shroff; Jing Liu; Navdeep S. Chandel; Syed A. Aziz

    2009-01-01

    Nitric oxide (NO) activates the intrinsic apoptotic pathway to induce cell death. However, the mechanism by which this pathway is activated in cells exposed to NO is not known. Here we report that BAX and BAK are activated by NO and that cytochrome c is released from the mitochondria. Cells deficient in Bax and Bak or Caspase-9 are completely protected

  10. Anti-inflammatory response following uptake of apoptotic bodies by meningothelial cells

    PubMed Central

    2014-01-01

    Background Meningothelial cells (MECs) are the cellular components of the meninges. As such, they provide important barrier function for the central nervous system (CNS) building the interface between neuronal tissue and the cerebrospinal fluid (CSF), and are also part of the immune response of the CNS. Methods Human, immortalized MECs were analyzed by flow cytometry and confocal microscopy to study the uptake of apoptotic cells. Furthermore, cytokine and chemokine production by MECs was analyzed by cytokine array and ELISA. Results We found that MECs are highly active phagocytes able of ingesting and digesting large amounts of apoptotic cells. Furthermore, the uptake of apoptotic cells by MECs was immune suppressive via inhibiting the secretion of pro-inflammatory and chemoattractant cytokines and chemokines IL-6, IL-8, IL-16, MIF, and CXCL1, while increasing the secretion of anti-inflammatory IL-1 receptor antagonist by MECs. Conclusion MECs respond with the secretion of anti-inflammatory cytokines and chemokines following the uptake of apoptotic cells potentially connecting these cells to processes important for the shut-down of immune responses in the brain. PMID:24565420

  11. Reconstitution and Engineering of Apoptotic Protein Interactions on the Bacterial Cell Surface

    PubMed Central

    Sun, Jingjing; Abdeljabbar, Diya M.; Clarke, Nicole; Bellows, Meghan L.; Floudas, Christodoulos A.; Link, A. James

    2010-01-01

    The interactions between pro- and anti-apoptotic members of the Bcl-2 class of proteins control whether a cell lives or dies, and the study of these protein–protein interactions has been an area of intense research. In this report, we describe a new tool for the study and engineering of apoptotic protein interactions that is based on the flow cytometric detection of these interactions on the surface of Escherichia coli. After validation of the assay with the well-studied interaction between the Bak(72–87) peptide and the anti-apoptotic protein Bcl-xL, the effect of both increasing and decreasing Bak peptide length on Bcl-xL binding was investigated. Previous work demonstrated that the Bak(72–87) peptide also binds to the anti-apoptotic protein Bcl-2, albeit with lower binding affinity compared to Bcl-xL. Here, we demonstrate that a slightly longer Bak peptide corresponding to amino acids 72–89 of Bak binds Bcl-xL and Bcl-2 equally well. Approximate binding affinity calculations on these peptide–protein complexes confirm the experimental observations. The flow cytometric assay was also used to screen a saturation mutagenesis library of Bak(72–87) variants for improved affinity to Bcl-xL. The best variants obtained from this library exhibit an apparent Kd to Bcl-xL 4-fold lower than that of wild-type Bak(72–87). PMID:19766123

  12. A rapid and strong apoptotic process is triggered by hyperosmotic stress in cultured rat cardiac myocytes

    Microsoft Academic Search

    Anita Galvez; María Paz Morales; José Miguel Eltit; Paula Ocaranza; Loreto Carrasco; Ximena Campos; Mario Sapag-Hagar; Guillermo Díaz-Araya; Sergio Lavandero

    2001-01-01

    In all cell types, the maintenance of normal cell volume is an essential homeostatic function. Relatively little is known about the induction of apoptosis by hyperosmotic stress and its molecular mechanism in terminally differentiated cardiac myocytes. We compared the apoptotic response of cultured neonatal rat cardiomyoctes to hyperosmotic stress by sorbitol (SOR) with those induced by doxorubicin (Doxo) or angiotensin

  13. The Effects of NAD+ on Apoptotic Neuronal Death and Mitochondrial Biogenesis and Function after Glutamate Excitotoxicity

    PubMed Central

    Wang, Xiaowan; Li, Hailong; Ding, Shinghua

    2014-01-01

    NAD+ is an essential co-enzyme for cellular energy metabolism and is also involved as a substrate for many cellular enzymatic reactions. It has been shown that NAD+ has a beneficial effect on neuronal survival and brain injury in in vitro and in vivo ischemic models. However, the effect of NAD+ on mitochondrial biogenesis and function in ischemia has not been well investigated. In the present study, we used an in vitro glutamate excitotoxicity model of primary cultured cortical neurons to study the effect of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function. Our results show that supplementation of NAD+ could effectively reduce apoptotic neuronal death, and apoptotic inducing factor translocation after neurons were challenged with excitotoxic glutamate stimulation. Using different approaches including confocal imaging, mitochondrial DNA measurement and Western blot analysis of PGC-1 and NRF-1, we also found that NAD+ could significantly attenuate glutamate-induced mitochondrial fragmentation and the impairment of mitochondrial biogenesis. Furthermore, NAD+ treatment effectively inhibited mitochondrial membrane potential depolarization and NADH redistribution after excitotoxic glutamate stimulation. Taken together, our results demonstrated that NAD+ is capable of inhibiting apoptotic neuronal death after glutamate excitotoxicity via preserving mitochondrial biogenesis and integrity. Our findings provide insights into potential neuroprotective strategies in ischemic stroke. PMID:25387075

  14. Delayed mitochondrial dysfunction in apoptotic hair cells in chinchilla cochleae following exposure to impulse noise.

    PubMed

    Hu, Bo Hua

    2007-06-01

    Apoptotic death of hair cells (HCs) in the cochlea has been found following exposure to intense noise. The current study was designed to examine the mitochondrial energetic function of HCs during the course of noise-induced apoptosis. Two aspects of the mitochondrial energetic function, succinate dehydrogenase (SDH) activity and mitochondrial membrane potential (MMP), were examined in HCs of chinchilla cochleae following exposure to a series of 75 pairs of impulse noises at 155 dB pSPL. The results showed that nuclear condensation and uptake of propidium iodide or trypan blue appeared at 10 min after the noise exposure, indicating a rapid progression of HC apoptosis. However, SDH activity was preserved at this time point. As the time elapsed (1 hr or 24 hrs) after the noise exposure, all newly-generated apoptotic HCs showed strong SDH activity, indicating the preservation of SDH activity during the course of apoptosis. Examination of MMP with rhodamine 123 staining revealed that MMP was sustained in the apoptotic HCs having mild nuclear condensation, even after the occurrence of cell membrane leakage. MMP was reduced with further progression of nuclear condensation. These results suggest the presence of a delayed mitochondrial dysfunction in apoptotic HCs following exposure to intense noise. PMID:17268771

  15. Induction of mesangial interleukin-6 synthesis by apoptotic U937 cells and monocytes

    Microsoft Academic Search

    Stefan Heidenreich; Toshinobu Sato; Michael Schmidt; Christian August; Janneke J Timmerman; Leendert A van Es; Mohamed R Daha

    1997-01-01

    Induction of mesangial interleukin-6 synthesis by apoptotic U937 cells and monocytes. Infiltration of the glomerular mesangium by monocytes and macrophages is a central pathologic feature in various forms of glomerulonephritis. Dependent on the presence and activity of local survival factors, monocytes may undergo apoptosis. Therefore, we looked for the interaction between cultured human mesangial cells (HMC) and intact, necrotic or

  16. Phosphatidylserine expression and phagocytosis of apoptotic thymocytes during differentiation of monocytic cells

    Microsoft Academic Search

    Melissa K. Callahan; Margaret S. Halleck; Stephen Krahling; Andrew J. Henderson; Patrick Williamson; Robert A. Schlegel

    2003-01-01

    Expression of phosphatidylserine (PS) on the surface of both macrophages and their apo- ptotic targets is required for efficient phagocytosis. Monocytes, the precursors of macrophages, do not express PS on their surface and do not efficiently phagocytose apoptotic cells. We report here that PS appears on the surface of both human mono- cytic U937 cells and primary human monocytes as

  17. Signal transduction induced by apoptotic cells inhibits HIV transcription in monocytes\\/macrophages

    Microsoft Academic Search

    Bethsebah N. Gekonge; Gillian Schiralli; Robert A. Schlegel; Andrew J. Henderson

    2006-01-01

    The primary targets of HIV are CD4 T cells and macrophages. HIV infection is associ- ated with an increase in apoptosis of infected and uninfected CD4 T cells, and these infected cells undergo apoptosis and produce HIV virions with phosphatidylserine (PS) on their surface. During phagocytosis of apoptotic cells, macrophages, us- ing an array of receptors, are able to perceive

  18. Death-defying immunity: do apoptotic cells influence antigen processing and presentation?

    Microsoft Academic Search

    Matthew L. Albert

    2004-01-01

    The clearance of apoptotic cells has been paid much attention for its role not only in tissue homeostasis, but also as a source of antigen for immune tolerance and activation. The complexity of this process has been borne out by the many receptor families and signalling pathways involved; however, an important aspect of the biology has so far been overlooked.

  19. Fas ligand\\/Fas system in the brain: regulator of immune and apoptotic responses

    Microsoft Academic Search

    Chulhee Choi; Etty N. Benveniste

    2004-01-01

    Apoptosis, also known as programmed cell death, is the major type of cell death involved in normal development, regeneration, proliferation and pathologic degeneration in the central nervous system (CNS). The apoptotic process can be divided further into two pathways depending on the involvement of mitochondria and related biochemical cascades. The internal pathway of apoptosis is initiated by a variety of

  20. Modulation of apoptotic and inflammatory genes by bioflavonoids and angiotensin II inhibition in ureteral obstruction

    Microsoft Academic Search

    Eric A Jones; Asha Shahed; Daniel A Shoskes

    2000-01-01

    Objectives. Ureteral obstruction results in an injury response that can progress to irreversible renal fibrosis and tubular atrophy by apoptosis. The molecular events leading to apoptosis from obstruction are not well understood. We investigated the effect of bioflavonoids and angiotensin II inhibition on apoptotic and inflammatory gene expression in a model of unilateral ureteral obstruction (UUO).Methods. Complete UUO was produced

  1. MODULATION OF APOPTOTIC AND INFLAMMATORY GENES BY BIOFLAVONOIDS AND ANGIOTENSIN II INHIBITION IN URETERAL OBSTRUCTION

    Microsoft Academic Search

    ERIC A. JONES; ASHA SHAHED; DANIEL A. SHOSKES

    Objectives. Ureteral obstruction results in an injury response that can progress to irreversible renal fibrosis and tubular atrophy by apoptosis. The molecular events leading to apoptosis from obstruction are not well understood. We investigated the effect of bioflavonoids and angiotensin II inhibition on apoptotic and inflammatory gene expression in a model of unilateral ureteral obstruction (UUO). Methods. Complete UUO was

  2. Lanthanum chloride promotes mitochondrial apoptotic pathway in primary cultured rat astrocytes.

    PubMed

    Yang, Jinghua; Liu, Qiufang; Qi, Ming; Lu, Shuai; Wu, Shengwen; Xi, Qi; Cai, Yuan

    2013-09-01

    Population surveys and animal experiments have shown that rare earth elements (REEs) cause neurological defects. However, the detailed mechanisms underlying these effects are still unclear. Given that lanthanum is commonly used for investigating into REEs-induced neurological defects, this study chose lanthanum chloride (LaCl3 ) to show that LaCl3 promotes mitochondrial apoptotic pathway in primary cultured rat astrocytes by regulating expression of Bcl-2 family proteins. The main findings of this study are (1) LaCl3 treatment (0.25, 0.5, and 1.0 mM for 12-48 h) induced the astrocytes damages with a concentration-dependent manner, which were confirmed with methyl thiazolyl tetrazolium and lactate dehydrogenase release assays, and morphological examination. (2) A 24 h treatment of LaCl3 concentration-dependently decreased mitochondrial membrane potential, increased cytochrome c release from mitochondria into cytosol, elevated caspase 9 and 3 expression, and promoted astrocyte apoptosis. (3) LaCl3 treatment increased the ratio of pro-apoptotic Bax and antiapoptotic Bcl-2 proteins, which in turn broke the balance among pro-apoptotic and antiapoptotic Bcl-2 family proteins, leading to astrocyte apoptosis. Our results indicate that LaCl3 alters Bcl-2 family protein expressions, which in turn promote mitochondrial apoptotic pathway, and thus astrocytic damage. PMID:21793157

  3. Bax Affects Production of Reactive Oxygen by the Mitochondria of Non-apoptotic Neurons

    PubMed Central

    Kirkland, Rebecca A.; Franklin, James L.

    2007-01-01

    Depriving sympathetic neurons in cell culture of nerve growth factor (NGF) causes their apoptotic death. Bax-induced release of cytochrome c from mitochondria and the subsequent activation of cytosolic caspases are central to this death. A Bax-dependent increase of mitochondrial-derived reactive oxygen species (ROS) that is an important component of the apoptotic cascade in these cells begins soon after NGF withdrawal. Here we report that Bax can also influence mitochondrial production of ROS in non-apoptotic sympathetic neurons. We determined ROS levels by using confocal microscopy to monitor changes in the fluorescence intensity of a redox-sensitive dye loaded into single cells. ROS levels were similar in NGF-replete bax wild-type neurons and neurons from which bax had been deleted. To enhance any effects that Bax might have on ROS levels in NGF-replete cells we exposed cultures to the ATP synthase inhibitor, oligomycin. This treatment hyperpolarizes mitochondrial membrane potential (??m), an event that can favor increased ROS production. NGF-replete neurons from mice in which bax had been deleted had much higher levels of mitochondrial-derived ROS when treated with oligomycin than did bax wild-type cells. Oligomycin treatment also caused greater hyperpolarization of ??m in bax-deleted cells than in wild-type cells. These findings indicate that Bax can affect mitochondrial ROS production in non-apoptotic neurons and may do so by altering ??m. PMID:17097638

  4. Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria.

    PubMed

    Oropesa, Manuel; de la Mata, Mario; Maraver, Juan Garrido; Cordero, Mario D; Cotán, David; Rodríguez-Hernández, Angeles; Domínguez-Moñino, Irene; de Miguel, Manuel; Navas, Plácido; Sánchez-Alcázar, José A

    2011-04-01

    Microtubule cytoskeleton is reformed during apoptosis, forming a cortical structure beneath plasma membrane, which plays an important role in preserving cell morphology and plasma membrane integrity. However, the maintenance of the apoptotic microtubule network (AMN) during apoptosis is not understood. In the present study, we examined apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1? cells. We demonstrate that AMN was organized in apoptotic cells with high ATP levels and hyperpolarized mitochondria and, on the contrary, was dismantled in apoptotic cells with low ATP levels and mitochondrial depolarization. AMN disorganization after mitochondrial depolarization was associated with increased plasma membrane permeability assessed by enhancing LDH release and increased intracellular calcium levels. Living cell imaging monitoring of both, microtubule dynamics and mitochondrial membrane potential, showed that AMN persists during apoptosis coinciding with cycles of mitochondrial hyperpolarization. Eventually, AMN was disorganized when mitochondria suffered a large depolarization and cell underwent secondary necrosis. AMN stabilization by taxol prevented LDH release and calcium influx even though mitochondria were depolarized, suggesting that AMN is essential for plasma membrane integrity. Furthermore, high ATP levels and mitochondria polarization collapse after oligomycin treatment in apoptotic cells suggest that ATP synthase works in "reverse" mode during apoptosis. These data provide new explanations for the role of AMN and mitochondria during apoptosis. PMID:21311976

  5. Apoptotic Cells Deliver Processed Antigen to Dendritic Cells for Cross-Presentation

    E-print Network

    Paris-Sud XI, Université de

    . There is, however, growing evidence that apoptotic death need not be an endpoint, and that dying cells the early events of recognition and internalization of the dying cell have been characterized, the antigen that a second pathway exists in which transfer of processed antigen from the dying cell allows formation

  6. Vessel-associated myogenic precursors control macrophage activation and clearance of apoptotic cells.

    PubMed

    Bosurgi, L; Brunelli, S; Rigamonti, E; Monno, A; Manfredi, A A; Rovere-Querini, P

    2015-01-01

    Swift and regulated clearance of apoptotic cells prevents the accumulation of cell remnants in injured tissues and contributes to the shift of macrophages towards alternatively activated reparatory cells that sustain wound healing. Environmental signals, most of which are unknown, in turn control the efficiency of the clearance of apoptotic cells and as such determine whether tissues eventually heal. In this study we show that vessel-associated stem cells (mesoangioblasts) specifically modulate the expression of genes involved in the clearance of apoptotic cells and in macrophage alternative activation, including those of scavenger receptors and of molecules that bridge dying cells and phagocytes. Mesoangioblasts, but not immortalized myoblasts or neural precursor cells, enhance CD163 membrane expression in vitro as assessed by flow cytometry, indicating that the effect is specific. Mesoangioblasts transplanted in acutely or chronically injured skeletal muscles determine the expansion of the population of CD163(+) infiltrating macrophages and increase the extent of CD163 expression. Conversely, macrophages challenged with mesoangioblasts engulf significantly better apoptotic cells in vitro. Collectively, the data reveal a feed-forward loop between macrophages and vessel-associated stem cells, which has implications for the skeletal muscle homeostatic response to sterile injury and for diseases in which homeostasis is jeopardized, including muscle dystrophies and inflammatory myopathies. PMID:24749786

  7. The traditional Chinese medicine Cordyceps sinensis and its effects on apoptotic homeostasis

    Microsoft Academic Search

    E. J. Buenz; B. A. Bauer; T. W. Osmundson; T. J. Motley

    2005-01-01

    Cordyceps sinensis is a medicinal fungus of Traditional Chinese Medicine. While there are a wide range of reported uses of Cordyceps sinensis in the literature, the reports that extracts of this fungus may alter apoptotic homeostasis are most intriguing. However, there are significant challenges regarding research surrounding Cordyceps sinensis, such as the difficulty identifying the various species of Cordyceps and

  8. Pivotal Advance: Macrophages become resistant to cholesterol-induced death after phagocytosis of apoptotic cells

    Microsoft Academic Search

    Dongying Cui; Edward Thorp; Yankun Li; Nan Wang; Laurent Yvan-Charvet; Alan R. Tall; Ira Tabas

    2007-01-01

    One of the most important functions of macrophages is the phagocytosis of apoptotic cells (ACs). ACs deliver large amounts mem- brane-derived cholesterol to phagocytes, which, if not handled properly, can be cytotoxic. In atherosclerosis, where the ACs are cholesterol- loaded, this situation is exaggerated, because the ACs deliver both endogenous membrane choles- terol and stored lipoprotein-derived cholesterol. To examine how

  9. Comparative Study of Four Different Sperm Washing Methods Using Apoptotic Markers in Ram Spermatozoa

    Microsoft Academic Search

    ELENA MARTI; ROSAURA PEREZ-PE; TERESA MUINO-BLANCO; JOSEA. CEBRIAN-PEREZ

    The accurate measurement of semen fertilizing potential is of great importance in determining the acceptability of processed semen for breeding purposes. A good sperm preparation technique results in a sample with high viability and motility and also takes into account other parameters such as the capacitation and apoptotic state which could compromise the ability to fertilize an oocyte. In this

  10. Toward the early evaluation of therapeutic effects: an electrochemical platform for ultrasensitive detection of apoptotic cells.

    PubMed

    Zhang, Jing-Jing; Zheng, Ting-Ting; Cheng, Fang-Fang; Zhang, Jian-Rong; Zhu, Jun-Jie

    2011-10-15

    The ability for early evaluation of therapeutic effects is a significant challenge in leukemia research. To address this challenge, we developed a novel electrochemical platform for ultrasensitive and selective detection of apoptotic cells in response to therapy. In order to construct the platform, a novel three-dimensional (3-D) architecture was initially fabricated after combining nitrogen-doped carbon nanotubes and gold nanoparticles via a layer-by-layer method. The formed architecture provided an effective matrix for annexin V with high stability and bioactivity to enhance sensitivity. On the basis of the specific recognition between annexin V and phosphatidylserine on the apoptotic cell membrane, the annexin V/3-D architecture interface showed a predominant capability for apoptotic cell capture. Moreover, a lectin-based nanoprobe was designed by noncovalent assembly of concanavalin A on CdTe quantum dots (QDs)-labeled silica nanospheres with poly(allylamine hydrochloride) as a linker. This nanoprobe incorporated both the specific carbohydrate recognition and the multilabeled QDs-based signal amplification. By coupling with the QDs-based nanoprobe and electrochemical stripping analysis, the proposed sandwich-type cytosensor showed an excellent analytical performance for the ultrasensitive detection of apoptotic cells (as low as 48 cells), revealing great potential toward the early evaluation of therapeutic effects. PMID:21888423

  11. Macrophage Surface Expression of Annexins I and II in the Phagocytosis of Apoptotic Lymphocytes

    Microsoft Academic Search

    Xiaoxuan Fan; Stephen Krahling; Douglas Smith; Patrick Williamson; Robert A. Schlegel

    2004-01-01

    When cells undergo apoptosis, or programmed cell death, they expose phosphatidylserine (PS) on their surface. Macro- phages that efficiently phagocytose apoptotic cells also express PS on their surface, although at a lower level. The PS exposed on both cells is required for phagocytosis, because uptake is inhibited by masking PS on either cell with annexin V, a PS-binding protein. The

  12. DOI: 10.1002/cbic.200500149 Fluorescent Detection of Apoptotic Cells by

    E-print Network

    Smith, Bradley D.

    An important structural feature of healthy animal cells is the asymmetric distribution of phospholipids betweenDOI: 10.1002/cbic.200500149 Fluorescent Detection of Apoptotic Cells by Using Zinc Coordination the two leaflets of the cell plasma membrane.[1] In particular, phosphati- dylserine (PS), which usually

  13. Letter to the Editor Detection of apoptotic cells using a synthetic

    E-print Network

    Smith, Bradley D.

    lead to false-positive results because most animal cells have a Ca2 þ -dependent scramblase that canLetter to the Editor Detection of apoptotic cells using a synthetic fluorescent sensor for membrane surfaces that contain phosphatidylserine Cell Death and Differentiation (2003) 10, 1357­1359. doi:10

  14. Release of intact, monomeric cytochrome c from apoptotic and necrotic cells

    Microsoft Academic Search

    R Jemmerson; B LaPlante; A Treeful

    2002-01-01

    Cytochrome c (Cyt c) has been shown to translocate from mitochondria to the cytoplasm of cells early in apoptosis. In this study sandwich ELISAs for Cyt c were used to determine if Cyt c is ultimately released from apoptotic and necrotic cells. Gel-filtration and cation-exchange chromatographies, in conjunction with immunoreactivity in ELISA, and Western blotting were employed to examine the

  15. Apoptotic cells induce arginase II in macrophages, thereby attenuating NO production

    Microsoft Academic Search

    Axel M. Johann; Vera Barra; Anne-Marie Kuhn; Andreas Weigert; Andreas von Knethen; Bernhard Brune

    2007-01-01

    In recent years it has become apparent that removal of apoptotic cells (AC) by professional phagocytes alters the macrophage phenotype. This change is characterized by attenuated proinflammatory cytokine expression and NO production, which mech- anistically remained unexplained. With the intention to explore molecular mechanisms underlying reduced NO formation, we showed that NO production in IFN- stimulated murine RAW264.7 macrophages exposed

  16. Research Article Skeletal muscle stem cells express anti-apoptotic ErbB

    E-print Network

    Golding, Jon

    Research Article Skeletal muscle stem cells express anti-apoptotic ErbB receptors during activation a Department of Biological Sciences, Open University, Walton Hall, Milton Keynes, MK7 6AA, UK b Muscle Cell cells of adult muscle) must survive the initial activation from quiescence. Using an in vitro model

  17. Propofol inhibits burn injury-induced hyperpermeability through an apoptotic signal pathway in microvascular endothelial cells

    PubMed Central

    Tian, K.Y.; Liu, X.J.; Xu, J.D.; Deng, L.J.; Wang, G.

    2015-01-01

    Recent studies have revealed that an intrinsic apoptotic signaling cascade is involved in vascular hyperpermeability and endothelial barrier dysfunction. Propofol (2,6-diisopropylphenol) has also been reported to inhibit apoptotic signaling by regulating mitochondrial permeability transition pore (mPTP) opening and caspase-3 activation. Here, we investigated whether propofol could alleviate burn serum-induced endothelial hyperpermeability through the inhibition of the intrinsic apoptotic signaling cascade. Rat lung microvascular endothelial cells (RLMVECs) were pretreated with propofol at various concentrations, followed by stimulation with burn serum, obtained from burn-injury rats. Monolayer permeability was determined by transendothelial electrical resistance. Mitochondrial release of cytochrome C was measured by ELISA. Bax and Bcl-2 expression and mitochondrial release of second mitochondrial-derived activator of caspases (smac) were detected by Western blotting. Caspase-3 activity was assessed by fluorometric assay; mitochondrial membrane potential (??m) was determined with JC-1 (a potential-sensitive fluorescent dye). Intracellular ATP content was assayed using a commercial kit, and reactive oxygen species (ROS) were measured by dichlorodihydrofluorescein diacetate (DCFH-DA). Burn serum significantly increased monolayer permeability (P<0.05), and this effect could be inhibited by propofol (P<0.05). Compared with a sham treatment group, intrinsic apoptotic signaling activation - indicated by Bax overexpression, Bcl-2 downregulation, ??m reduction, decreased intracellular ATP level, increased cytosolic cytochrome C and smac, and caspase-3 activation - was observed in the vehicle group. Propofol not only attenuated these alterations (P<0.05 for all), but also significantly decreased burn-induced ROS production (P<0.05). Propofol attenuated burn-induced RLMVEC monolayer hyperpermeability by regulating the intrinsic apoptotic signaling pathway. PMID:25760023

  18. Propofol inhibits burn injury-induced hyperpermeability through an apoptotic signal pathway in microvascular endothelial cells.

    PubMed

    Tian, K Y; Liu, X J; Xu, J D; Deng, L J; Wang, G

    2015-05-01

    Recent studies have revealed that an intrinsic apoptotic signaling cascade is involved in vascular hyperpermeability and endothelial barrier dysfunction. Propofol (2,6-diisopropylphenol) has also been reported to inhibit apoptotic signaling by regulating mitochondrial permeability transition pore (mPTP) opening and caspase-3 activation. Here, we investigated whether propofol could alleviate burn serum-induced endothelial hyperpermeability through the inhibition of the intrinsic apoptotic signaling cascade. Rat lung microvascular endothelial cells (RLMVECs) were pretreated with propofol at various concentrations, followed by stimulation with burn serum, obtained from burn-injury rats. Monolayer permeability was determined by transendothelial electrical resistance. Mitochondrial release of cytochrome C was measured by ELISA. Bax and Bcl-2 expression and mitochondrial release of second mitochondrial-derived activator of caspases (smac) were detected by Western blotting. Caspase-3 activity was assessed by fluorometric assay; mitochondrial membrane potential (??m) was determined with JC-1 (a potential-sensitive fluorescent dye). Intracellular ATP content was assayed using a commercial kit, and reactive oxygen species (ROS) were measured by dichlorodihydrofluorescein diacetate (DCFH-DA). Burn serum significantly increased monolayer permeability (P<0.05), and this effect could be inhibited by propofol (P<0.05). Compared with a sham treatment group, intrinsic apoptotic signaling activation - indicated by Bax overexpression, Bcl-2 downregulation, ??m reduction, decreased intracellular ATP level, increased cytosolic cytochrome C and smac, and caspase-3 activation - was observed in the vehicle group. Propofol not only attenuated these alterations (P<0.05 for all), but also significantly decreased burn-induced ROS production (P<0.05). Propofol attenuated burn-induced RLMVEC monolayer hyperpermeability by regulating the intrinsic apoptotic signaling pathway. PMID:25760023

  19. Evaluation of intracellular signalling pathways in response to insulin-like growth factor I in apoptotic-resistant activated human hepatic stellate cells

    Microsoft Academic Search

    Alessandra Gentilini; Benedetta Lottini; Marco Brogi; Alessandra Caligiuri; Lorenzo Cosmi; Fabio Marra; Massimo Pinzani

    2009-01-01

    BACKGROUND: Human hepatic stellate cells have been shown to be resistant to apoptotic stimuli. This is likely dependent on the activation of anti-apoptotic pathways upon transition of these cells to myofibroblast-like cells. In particular, previous studies have demonstrated an increased expression of the anti-apoptotic protein Bcl-2 and a decreased expression of the pro-apoptotic protein Bax during the transition of the

  20. BAK overexpression mediates p53-independent apoptosis inducing effects on human gastric cancer cells

    Microsoft Academic Search

    Qiang-Song Tong; Li-Duan Zheng; Liang Wang; Jun Liu; Wei Qian

    2004-01-01

    BACKGROUND: BAK (Bcl-2 homologous antagonist\\/killer) is a novel pro-apoptotic gene of the Bcl-2 family. It has been reported that gastric tumors have reduced BAK levels when compared with the normal mucosa. Moreover, mutations of the BAK gene have been identified in human gastrointestinal cancers, suggesting that a perturbation of BAK-mediated apoptosis may contribute to the pathogenesis of gastric cancer. In

  1. Apoptosis overrides survival signals through a caspase-mediated dominant-negative NF-?B loop

    Microsoft Academic Search

    Bodo Levkau; Marta Scatena; Cecilia M. Giachelli; Russell Ross; Elaine W. Raines

    1999-01-01

    The transcription factor NF-?B is an important regulator of gene expression during immune and inflammatory responses, and can also protect against apoptosis. Here we show that endothelial cells undergo apoptosis when deprived of growth factors. Surviving viable cells exhibit increased activity of NF-?B, whereas apoptotic cells show caspase-mediated cleavage of the NF-?B p65\\/RelA subunit. This cleavage leads to loss of

  2. The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism

    PubMed Central

    Sullivan, Chelsea S.; Scheib, Jami L.; Ma, Zhong; Dang, Rajan P.; Schafer, Johanna M.; Hickman, Francis E.; Brodsky, Frances M.; Ravichandran, Kodi S.; Carter, Bruce D.

    2014-01-01

    During the development of the peripheral nervous system, the large number of apoptotic neurons generated are phagocytosed by glial precursor cells. This clearance is mediated, in part, through the mammalian engulfment receptor Jedi-1. However, the mechanisms by which Jedi-1 mediates phagocytosis are poorly understood. Here we demonstrate that Jedi-1 associates with GULP, the mammalian homologue of CED-6, an adaptor protein required for phagocytosis mediated by the nematode engulfment receptor CED-1. Silencing GULP or mutating the NPXY motif in Jedi-1, which is required for GULP binding, prevents Jedi-1–mediated phagocytosis. How GULP promotes engulfment is not known. Of interest, we find that Jedi-1–induced phagocytosis requires GULP binding to clathrin heavy chain (CHC). During engulfment, CHC is tyrosine phosphorylated, which is required for Jedi-mediated engulfment. Both phosphoclathrin and actin accumulate around engulfed microspheres. Furthermore, knockdown of CHC in HeLa cells prevents Jedi-1–mediated engulfment of microspheres, and knockdown in glial precursors prevents the engulfment of apoptotic neurons. Taken together, these results reveal that Jedi-1 signals through recruitment of GULP, which promotes phagocytosis through a noncanonical phosphoclathrin-dependent mechanism. PMID:24743597

  3. The adaptor protein GULP promotes Jedi-1-mediated phagocytosis through a clathrin-dependent mechanism.

    PubMed

    Sullivan, Chelsea S; Scheib, Jami L; Ma, Zhong; Dang, Rajan P; Schafer, Johanna M; Hickman, Francis E; Brodsky, Frances M; Ravichandran, Kodi S; Carter, Bruce D

    2014-06-15

    During the development of the peripheral nervous system, the large number of apoptotic neurons generated are phagocytosed by glial precursor cells. This clearance is mediated, in part, through the mammalian engulfment receptor Jedi-1. However, the mechanisms by which Jedi-1 mediates phagocytosis are poorly understood. Here we demonstrate that Jedi-1 associates with GULP, the mammalian homologue of CED-6, an adaptor protein required for phagocytosis mediated by the nematode engulfment receptor CED-1. Silencing GULP or mutating the NPXY motif in Jedi-1, which is required for GULP binding, prevents Jedi-1-mediated phagocytosis. How GULP promotes engulfment is not known. Of interest, we find that Jedi-1-induced phagocytosis requires GULP binding to clathrin heavy chain (CHC). During engulfment, CHC is tyrosine phosphorylated, which is required for Jedi-mediated engulfment. Both phosphoclathrin and actin accumulate around engulfed microspheres. Furthermore, knockdown of CHC in HeLa cells prevents Jedi-1-mediated engulfment of microspheres, and knockdown in glial precursors prevents the engulfment of apoptotic neurons. Taken together, these results reveal that Jedi-1 signals through recruitment of GULP, which promotes phagocytosis through a noncanonical phosphoclathrin-dependent mechanism. PMID:24743597

  4. Friendly fire against neutrophils: proteolytic enzymes confuse the recognition of apoptotic cells by macrophages.

    PubMed

    Guzik, Krzysztof; Potempa, Jan

    2008-02-01

    Physiologically the only acceptable fate for almost all damaged or unwanted cells is their apoptotic death, followed by engulfment of the corpses by healthy neighbors or professional phagocytes. Efficient clearance of cells that have succumbed to apoptosis is crucial for normal tissue homeostasis, and for the modulation of immune responses. The disposal of apoptotic cells is finely regulated by a highly redundant system of receptors, bridging molecules and 'eat me' signals. The complexity of the system is reflected by the term: 'engulfment synapse', used to describe the interaction between a phagocytic cell and its target. In healthy humans, dying neutrophils are the most abundant and important targets for such recognition and engulfment. In inflammation the scope and importance of this complicated task is further increased. Paradoxically, despite growing evidence highlighting the priority of neutrophils clearance, the recognition of these cells by phagocytes is not as well understood as the recognition of other apoptotic cell types. New findings indicate that the interaction of phosphatidylserine (PS) on apoptotic neutrophils with its receptor on macrophages is not as critical for the specific clearance of neutrophil corpses it was previously believed. In this review we focus on recent findings regarding alternative, PS-independent "eat me" signals expressed on neutrophils during cell death and activation. Based on our own research, we emphasize the clearance of dying neutrophils, especially at the focus of bacterial infection; and the associated inflammatory reaction, which occurs in a highly proteolytic milieu containing both host and bacteria-derived proteinases. In these environments, eat-me signals expressed by neutrophils are drastically modified; arguing against the phospholipid-based detection of apoptotic cells, but supporting the importance of proteinaceous ligand(s) for the recognition of neutrophils by macrophages. In this context we discuss the effect of the gingipain R (Rgp) proteinases from Porphyromonas gingivalis on neutrophils interactions with macrophages. Since the recognition of apoptotic neutrophils is an important fundamental process, serving multiple functions in the regulation of immunity and homeostasis, we hypothesize that many pathogenic bacteria may have developed similar strategies to confuse macrophage-neutrophil interaction as a common pathogenic strategy. PMID:17964056

  5. Pleiotrophin mediates hematopoietic regeneration via activation of RAS.

    PubMed

    Himburg, Heather A; Yan, Xiao; Doan, Phuong L; Quarmyne, Mamle; Micewicz, Eva; McBride, William; Chao, Nelson J; Slamon, Dennis J; Chute, John P

    2014-11-01

    Hematopoietic stem cells (HSCs) are highly susceptible to ionizing radiation-mediated death via induction of ROS, DNA double-strand breaks, and apoptotic pathways. The development of therapeutics capable of mitigating ionizing radiation-induced hematopoietic toxicity could benefit both victims of acute radiation sickness and patients undergoing hematopoietic cell transplantation. Unfortunately, therapies capable of accelerating hematopoietic reconstitution following lethal radiation exposure have remained elusive. Here, we found that systemic administration of pleiotrophin (PTN), a protein that is secreted by BM-derived endothelial cells, substantially increased the survival of mice following radiation exposure and after myeloablative BM transplantation. In both models, PTN increased survival by accelerating the recovery of BM hematopoietic stem and progenitor cells in vivo. PTN treatment promoted HSC regeneration via activation of the RAS pathway in mice that expressed protein tyrosine phosphatase receptor-zeta (PTPRZ), whereas PTN treatment did not induce RAS signaling in PTPRZ-deficient mice, suggesting that PTN-mediated activation of RAS was dependent upon signaling through PTPRZ. PTN strongly inhibited HSC cycling following irradiation, whereas RAS inhibition abrogated PTN-mediated induction of HSC quiescence, blocked PTN-mediated recovery of hematopoietic stem and progenitor cells, and abolished PTN-mediated survival of irradiated mice. These studies demonstrate the therapeutic potential of PTN to improve survival after myeloablation and suggest that PTN-mediated hematopoietic regeneration occurs in a RAS-dependent manner. PMID:25250571

  6. Low extracellular pH augments TRAIL-induced apoptotic death through the mitochondria-mediated caspase signal transduction pathway

    Microsoft Academic Search

    Yong J Lee; Jae J Song; Jin H Kim; Hyeong-Reh Choi Kim; Young K Song

    2004-01-01

    Tumor necrosis factor-related apoptosis inducing ligand (TRAIL\\/APO-2L), a member of the tumor necrosis factor (TNF) gene family, is considered as one of the most promising cancer therapeutic agents due to its ability to selectively kill tumor cells. Although microenvironments of solid tumors (hypoxia, nutrient deprivation, and low pH) often affect the effectiveness of chemotherapy, few studies have been reported on

  7. Anti-apoptotic effect of retinoic acid on retinal progenitor cells mediated by a protein kinase A-dependent mechanism

    Microsoft Academic Search

    Roman Kholodenko; Irina Kholodenko; Viktor Sorokin; Anna Tolmazova; Olga Sazonova; Anton Buzdin

    2007-01-01

    Retinal progenitor cells (RPCs) are neural stem cells able to differentiate into any normal adult retinal cell type, except for pigment epithelial cells. Retinoic acid (RA) is a powerful growth\\/differentiation factor that generally causes growth inhibition, differentiation and\\/or apoptosis. In this study, we demonstrate that RA not only affects mouse RPC differentiation but also improves cell survival by reducing spontaneous

  8. p85  Acts as a Novel Signal Transducer for Mediation of Cellular Apoptotic Response to UV Radiation

    Microsoft Academic Search

    Lun Song; Jingxia Li; Jianping Ye; Gang Yu; Jin Ding; Dongyun Zhang; Weiming Ouyang; Zigang Dong; Sung O. Kim; Chuanshu Huang

    2007-01-01

    Apoptosis is an important cellular response to UV radiation (UVR), but the corresponding mechanisms remain largely unknown. Here we report that the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI-3K) exerted a proapoptotic role in response to UVR through the induction of tumor necrosis factor alpha (TNF-) gene expression. This special effect of p85 was unrelated to the PI-3K-dependent signaling pathway.

  9. Apoptotic Regulation by the Crk Adapter Protein Mediated by Interactions with Wee1 and Crm1\\/Exportin

    Microsoft Academic Search

    Jesse J. Smith; D. Ashley Richardson; Jan Kopf; Minoru Yoshida; Robert E. Hollingsworth; Sally Kornbluth

    2002-01-01

    The adapter protein Crk contains an SH2 domain and two SH3 domains. Through binding of particular ligands to the SH2 domain and the N-terminal SH3 domain, Crk has been implicated in a number of signaling processes, including regulation of cell growth, cell motility, and apoptosis. We report here that the C-terminal SH3 domain, never shown to bind any specific signaling

  10. Bcl2 family-mediated apoptotic effects of 3,3?-diindolylmethane (DIM) in human breast cancer cells

    Microsoft Academic Search

    Chibo Hong; Gary L. Firestone; Leonard F. Bjeldanes

    2002-01-01

    3,3?-Diindolylmethane (DIM) is a major invivo derivative of the putative anticancer agent indole-3-carbinol (I3C), which is present in vegetables of the Brassica genus. At concentrations above 10?M, DIM inhibited DNA synthesis and cell proliferation in both estrogen receptor replete (MCF-7) and deficient (MDA-MB-231) human breast cancer cells in a concentration- and time-dependent manner. These antiproliferative effects were accompanied by characteristic

  11. Lutein Protects against Methotrexate-Induced and Reactive Oxygen Species-Mediated Apoptotic Cell Injury of IEC-6 Cells

    PubMed Central

    Chang, Chi-Jen; Lin, Ji-Fan; Chang, Hsun-Hsien; Lee, Gon-Ann; Hung, Chi-Feng

    2013-01-01

    Purpose High-dose chemotherapy using methotrexate (MTX) frequently induces side effects such as mucositis that leads to intestinal damage and diarrhea. Several natural compounds have been demonstrated of their effectiveness in protecting intestinal epithelial cells from these adverse effects. In this paper, we investigated the protection mechanism of lutein against MTX-induced damage in IEC-6 cells originating from the rat jejunum crypt. Methods The cell viability, induced-apoptosis, reactive oxygen species (ROS) generation, and mitochondrial membrane potential in IEC-6 cells under MTX treatment were examined in the presence or absence of lutein. Expression level of Bcl2, Bad and ROS scavenging enzymes (including SOD, catalase and Prdx1) were detected by quantitative RT-PCR. Results The cell viability of IEC-6 cells exposed to MTX was decreased in a dose- and time-dependent manner. MTX induces mitochondrial membrane potential loss, ROS generation and caspase 3 activation in IEC-6 cells. The cytotoxicity of MTX was reduced in IEC-6 cells by the 24 h pre-treatment of lutein. We found that pre-treatment of lutein significantly reduces MTX-induced ROS and apoptosis. The expression of SOD was up-regulated by the pre-treatment of lutein in the MTX-treated IEC-6 cells. These results indicated that lutein can protect IEC-6 cells from the chemo-drugs induced damage through increasing ROS scavenging ability. Conclusion The MTX-induced apoptosis of IEC-6 cells was shown to be repressed by the pre-treatment of lutein, which may represent a promising adjunct to conventional chemotherapy for preventing intestinal damages. PMID:24039779

  12. Methanolic extract of Origanum vulgare ameliorates type 1 diabetes through antioxidant, anti-inflammatory and anti-apoptotic activity.

    PubMed

    Vujicic, Milica; Nikolic, Ivana; Kontogianni, Vassiliki G; Saksida, Tamara; Charisiadis, Pantelis; Orescanin-Dusic, Zorana; Blagojevic, Dusko; Stosic-Grujicic, Stanislava; Tzakos, Andreas G; Stojanovic, Ivana

    2015-03-14

    Type 1 diabetes (T1D), an autoimmune inflammatory disorder, develops as a consequence of pancreatic ?-cell destruction and results in hyperglycaemia. Since current T1D therapy mainly involves insulin replacement, the aim of the present study was to evaluate the therapeutic potential of Origanum vulgare L. ssp. hirtum (Greek oregano) leaf extract rich in biophenols for the treatment of T1D. The phytochemical profile of methanolic oregano extract (MOE) and aqueous oregano extract (AOE) was determined by liquid chromatography/electrospray ion-trap tandem MS (LC/DAD/ESI-MSn), while their main compounds were quantified by HPLC with diode array detection. After establishing their potent in vitro antioxidant activity, the extracts were administered to C57BL/6 mice treated with multiple low doses of streptozotocin for diabetes induction. While prophylactic AOE therapy had no impact on diabetes induction, MOE reduced diabetes incidence and preserved normal insulin secretion. In addition, MOE scavenged reactive oxygen and nitrogen species and, therefore, alleviated the need for the up-regulation of antioxidant enzymes. MOE treatment specifically attenuated the pro-inflammatory response mediated by T helper 17 cells and enhanced anti-inflammatory T helper 2 and T regulatory cells through the impact on specific signalling pathways and transcription factors. Importantly, MOE preserved ?-cells from in vitro apoptosis via blockade of caspase 3. Finally, rosmarinic acid, a predominant compound in MOE, exhibited only partial protection from diabetes induction. In conclusion, acting as an antioxidant, immunomodulator and in an anti-apoptotic manner, MOE protected mice from diabetes development. Seemingly, there is more than one compound responsible for the beneficial effect of MOE. PMID:25671817

  13. 8-Chloro-cAMP induces apoptotic cell death in a human mammary carcinoma cell (MCF-7) line.

    PubMed Central

    Bøe, R.; Gjertsen, B. T.; Døskeland, S. O.; Vintermyr, O. K.

    1995-01-01

    8-Cl-cAMP and 8-NH2-cAMP induced MCF-7 cell death. The type(s) of cell death were studied in more detail and compared with the cell death type (apoptosis) induced by okadaic acid, an inhibitor of serine/threonine phosphatases. By morphological criteria dying cells showed loss of cell-cell interactions and microvilli, condensation of nuclear chromatin and segregation of cytoplasmic organelles. By in situ nick end-labelling, using digoxigenin-conjugated dUTP as probe, a large fraction of 8-Cl-cAMP, 8-NH2-cAMP and 8-Cl-adenosine-exposed cells stained positively in the advanced stages of death. In the early phase of chromatin condensation the cells stained negatively. Specific (internucleosomal) DNA fragmentation was not observed. The MCF-7 cell death induced by 8-Cl-cAMP and 8-NH2-cAMP was not mediated by activation of the cAMP kinase since more stable cAMP analogues (8-CPT-cAMP and N6-benzoyl-cAMP) or forskolin failed to induce death. Furthermore, 8-Cl-cAMP action was counteracted by adenosine deaminase and 3-isobutyl-1-methylxanthine, and mimicked by 8-Cl-adenosine, a major metabolite of 8-Cl-cAMP. It is concluded that 8-Cl- and 8-NH2-cAMP can induce morphological and biochemical effects resembling apoptotic cell death in MCF-7 cells through their conversion into potent cytotoxic metabolite(s). Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7577461

  14. Human acute promyelocytic leukemia NB4 cells are sensitive to esculetin through induction of an apoptotic mechanism.

    PubMed

    Rubio, Virginia; Calviño, Eva; García-Pérez, Ana; Herráez, Angel; Diez, José C

    2014-09-01

    Acute promyelocytic leukemia (APL) is a type of cancer, in which immature cells called promyelocytes proliferate abnormally. Human NB4 cell line appears to be a suitable in vitro model to express the characteristics of APL. In this work, we have investigated the effects of esculetin, a coumarin derivative with antioxidant properties, on the viability, the induction of apoptosis and the expression of apoptotic factors in NB4 cells. Cells treated with esculetin at several concentrations (20-500 ?M) and for different times (5-24 h) showed a concentration- and time-dependent viability decrease with increased subdiploid DNA production. Esculetin inhibited cell cycle progression and induced DNA fragmentation. Moreover, annexin-V-FITC cytometry assays suggested that increased toxicity is due to both early and late apoptosis. This apoptosis process is be mediated by activation of caspase-3 and caspase-9. Treatments with progressively increasing concentrations (from 100 ?M to 500 ?M) of esculetin produced a reduction of Bcl2/Bax ratio in NB4 cells at 19 h, without affecting p53 levels. Proapoptotic action of esculetin involves the ERK MAP kinase cascade since increased levels of phosphorylated ERK were observed after those treatments. Increments in the levels of phosphorylated-Akt were also observed. Additionally, esculetin induced the loss of mitochondrial membrane potential with a release of cytochrome c into the cytosol which starts at 6 h of treatment with esculetin and increases up to 24 h. Esculetin induced an increase in superoxide anion at long times of treatment and a reduction of peroxides at short times (1 h) with an observed increase at 2-4 h of treatment. No significant changes in NO production was observed. Esculetin reduced the GSH levels in a time-dependent manner. In summary, the present work shows the cytotoxic action of esculetin as an efficient tool to study apoptosis mechanism induction on NB4 cell line used as a relevant model of APL disease. PMID:24995577

  15. hMOB3 modulates MST1 apoptotic signaling and supports tumor growth in glioblastoma multiforme

    PubMed Central

    Tang, Fengyuan; Zhang, Lei; Xue, Gongda; Hynx, Debby; Wang, Yuhua; Cron, Peter D.; Hundsrucker, Christian; Hergovich, Alexander; Frank, Stephan; Hemmings, Brian A.; Schmitz-Rohmer, Debora

    2014-01-01

    New therapeutic targets are needed that circumvent inherent therapeutic resistance of glioblastoma multiforme (GBM). Here we report such a candidate target in the uncharacterized adaptor protein hMOB3, which we show is upregulated in GBM. In a search for its biochemical function, we found that hMOB3 specifically interacts with MST1 kinase in response to apoptotic stimuli and cell-cell contact. Moreover, hMOB3 negatively regulated apoptotic signaling by MST1 in GBM cells by inhibiting the MST1 cleavage-based activation process. Physical interaction between hMOB3 and MST1 was essential for this process. In vivo investigations established that hMOB3 sustains GBM cell growth at high cell density and promotes tumorigenesis. Our results suggest hMOB3 as a candidate therapeutic target for the treatment of malignant gliomas. PMID:24872389

  16. Die-hard survivors: heterogeneity in apoptotic thresholds may underlie chemoresistance

    PubMed Central

    Ogden, Angela; Rida, Padmashree CG; Reid, Michelle D.; Kucuk, Omer; Aneja, Ritu

    2015-01-01

    The unmatched efficacy of microtubule-targeting agents (MTAs) as chemotherapeutics was once assumed to originate from their impact on mitotic processes; however, this misconception is being eroded by amassing data that MTAs instead target interphase functions in patients’ tumors. What remains murky is how MTAs target malignant cells over non-malignant ones if proliferation rates do not distinguish them. In many instances, malignant cells are actually more ‘primed’ for apoptosis than non-malignant ones. Nevertheless, even if most cells within the tumor are more apoptosis-susceptible than those in healthy tissues, there likely exist small subpopulations of apoptosis-resistant clones that engender incomplete responses to MTAs and relapse. Therefore, intratumor heterogeneity in terms of proximity to the apoptotic threshold must be better understood to facilitate the design of chemotherapeutic regimens, which may benefit from including drugs like BH3 mimetics that help in lowering the apoptotic threshold of tumor cells within these chemoresistant subpopulations. PMID:25695344

  17. Monitoring the Clearance of Apoptotic and Necrotic Cells in the Nematode Caenorhabditis elegans

    PubMed Central

    Li, Zao; Lu, Nan; He, Xiangwei; Zhou, Zheng

    2014-01-01

    The nematode Caenorhabditis elegans is an excellent model organism for studying the mechanisms controlling cell death, including apoptosis, a cell suicide event, and necrosis, pathological cell deaths caused by environmental insults or genetic alterations. C. elegans has also been established as a model for understanding how dying cells are cleared from animal bodies. In particular, the transparent nature of worm bodies and eggshells make C. elegans particularly amenable for live-cell microscopy. Here we describe methods for identifying apoptotic and necrotic cells in living C. elegans embryos, larvae, and adults and for monitoring their clearance during development. We further discuss specific methods to distinguish engulfed from unengulfed apoptotic cells, and methods to monitor cellular and molecular events occurring during phagosome maturation. These methods are based on Differential Interference Contrast (DIC) microscopy or fluorescence microscopy using GFP-based reporters. PMID:23733578

  18. Photoluminescent graphene quantum dots for in vivo imaging of apoptotic cells

    NASA Astrophysics Data System (ADS)

    Roy, Prathik; Periasamy, Arun Prakash; Lin, Chiu-Ya; Her, Guor-Mour; Chiu, Wei-Jane; Li, Chi-Lin; Shu, Chia-Lun; Huang, Chih-Ching; Liang, Chi-Te; Chang, Huan-Tsung

    2015-01-01

    Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro; however, there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescence properties of plant leaf-derived graphene quantum dots (GQDs) modified with annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with the GQDs exhibiting high biocompatibility as they were excreted from the zebrafish's body without affecting its growth significantly at a concentration lower than 2 mg mL-1 over a period of 4 to 72 hour post fertilization. The GQDs have further been used to image human breast adenocarcinoma cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), and normal human mammary epithelial cell line (MCF-10A). These results are indispensable to further the advance of graphene-based nanomaterials for biomedical applications.Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro; however, there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescence properties of plant leaf-derived graphene quantum dots (GQDs) modified with annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with the GQDs exhibiting high biocompatibility as they were excreted from the zebrafish's body without affecting its growth significantly at a concentration lower than 2 mg mL-1 over a period of 4 to 72 hour post fertilization. The GQDs have further been used to image human breast adenocarcinoma cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), and normal human mammary epithelial cell line (MCF-10A). These results are indispensable to further the advance of graphene-based nanomaterials for biomedical applications. Electronic supplementary information (ESI) available: Experimental discussion on synthesis, characterization, cellular imaging, cytotoxicity of GQDs in addition to its effect on zebrafish embryos, preparation of annexin V (A5)-modified GQDs (AbA5-GQDs), staining procedures and imaging are given. Figures for XRD, UV-vis absorption, photoluminescence of GQDs, mortality of zebrafish, time course recording of morphology of zebrafish embryos and morphology of adult zebrafish exposed to GQDs are illustrated. See DOI: 10.1039/c4nr07005d

  19. Antitumor effects of traditional Chinese medicine targeting the cellular apoptotic pathway

    PubMed Central

    Xu, Huanli; Zhao, Xin; Liu, Xiaohui; Xu, Pingxiang; Zhang, Keming; Lin, Xiukun

    2015-01-01

    Defects in apoptosis are common phenomena in many types of cancer and are also a critical step in tumorigenesis. Targeting the apoptotic pathway has been considered an intriguing strategy for cancer therapy. Traditional Chinese medicine (TCM) has been used in the People’s Republic of China for thousands of years, and many of the medicines have been confirmed to be effective in the treatment of a number of tumors. With increasing cancer rates worldwide, the antitumor effects of TCMs have attracted more and more attention globally. Many of the TCMs have been shown to have antitumor activity through multiple targets, and apoptosis pathway-related targets have been extensively studied and defined to be promising. This review focuses on several antitumor TCMs, especially those with clinical efficacy, based on their effects on the apoptotic signaling pathway. The problems with and prospects of development of TCMs as anticancer agents are also presented.

  20. mediation: R package for causal mediation analysis

    E-print Network

    Tingley, Dustin

    In this paper, we describe the R package mediation for conducting causal mediation analysis in applied empirical research. In many scientific disciplines, the goal of researchers is not only estimating causal effects of a ...

  1. Epigallocatechin-3-gallate and zinc provide anti-apoptotic protection against hypoxia/reoxygenation injury in H9c2 rat cardiac myoblast cells

    PubMed Central

    ZENG, XING; TAN, XUERUI

    2015-01-01

    It has previously been demonstrated that phospha-tidylinositol-3-kinase (PI3K)/Akt and cleaved caspase-3 serve critical roles in the apoptosis of cardiac myocytes following ischemia/reperfusion injury. Epigallocatechin-3-gallate (EGCG), the predominant catechin component of green tea, has been reported to have potential cardioprotective effects in primary cultures of cardiac myocytes exposed to I/R injury, mediated through inhibition of signal transducers and activators of transcription-1 activity. In addition, it is also known that the biological behavior of EGCG may be influenced by metal ions, for example the hepatoprotective activity of EGCG has been reported to be enhanced by zinc. In the present study, the protective effects of EGCG with zinc were assessed on cultures of rat cardiac myoblasts exposed to hypoxia/reoxygenation (H/R) injury. H9c2 cells were subjected to 3-h hypoxia, followed by 1-h reperfusion. EGCG and/or zinc were perfused prior to induced hypoxic stress. It was demonstrated that when EGCG interacted with zinc, the anti-apoptotic activity was significantly enhanced. To the best of our knowledge, the current study was the first to demonstrate that EGCG + Zn2+ protects H9c2 cells against H/R injury through activation of the PI3K/Akt pathway, as determined by western blotting. Since EGCG + Zn2+ may, at least in part, protect cardiac myocytes against H/R-induced apoptotic cell death, the PI3K/Akt pathway of EGCG may be enhanced by its interactions with zinc during H/R injury. Furthermore, it was suggested that a similar procedure may be implemented in a clinical setting, in order to maximize PI3K/Akt activation levels in patients with acute coronary artery disease. EGCG and zinc may therefore represent effective agents for use in the prevention of I/R injury in clinical practice. PMID:25872640

  2. Epigallocatechin?3?gallate and zinc provide anti?apoptotic protection against hypoxia/reoxygenation injury in H9c2 rat cardiac myoblast cells.

    PubMed

    Zeng, Xing; Tan, Xuerui

    2015-08-01

    It has previously been demonstrated that phosphatidylinositol?3?kinase (PI3K)/Akt and cleaved caspase?3 serve critical roles in the apoptosis of cardiac myocytes following ischemia/reperfusion injury. Epigallocatechin?3?gallate (EGCG), the predominant catechin component of green tea, has been reported to have potential cardioprotective effects in primary cultures of cardiac myocytes exposed to I/R injury, mediated through inhibition of signal transducers and activators of transcription?1 activity. In addition, it is also known that the biological behavior of EGCG may be influenced by metal ions, for example the hepatoprotective activity of EGCG has been reported to be enhanced by zinc. In the present study, the protective effects of EGCG with zinc were assessed on cultures of rat cardiac myoblasts exposed to hypoxia/reoxygenation (H/R) injury. H9c2 cells were subjected to 3?h hypoxia, followed by 1?h reperfusion. EGCG and/or zinc were perfused prior to induced hypoxic stress. It was demonstrated that when EGCG interacted with zinc, the anti?apoptotic activity was significantly enhanced. To the best of our knowledge, the current study was the first to demonstrate that EGCG + Zn2+ protects H9c2 cells against H/R injury through activation of the PI3K/Akt pathway, as determined by western blotting. Since EGCG + Zn2+ may, at least in part, protect cardiac myocytes against H/R?induced apoptotic cell death, the PI3K/Akt pathway of EGCG may be enhanced by its interactions with zinc during H/R injury. Furthermore, it was suggested that a similar procedure may be implemented in a clinical setting, in order to maximize PI3K/Akt activation levels in patients with acute coronary artery disease. EGCG and zinc may therefore represent effective agents for use in the prevention of I/R injury in clinical practice. PMID:25872640

  3. Mch3, a Novel Human Apoptotic Cysteine Protease Highly Related to CPP321

    Microsoft Academic Search

    Teresa Fernandes-Alnemri; Robert Armstrong; Joseph Krebs; Ljjuan Wang; Zailin Yu; Carlo M. Croce; Guy Salveson; William C. Eamshaw; Gerald Litwack; Emad S. Alnemri; K. J. Ti

    Recent evidence suggests that mammalian cysteine pretenses related to Caenorhabditis elegans CED-3 are key components of mammalian pro grammed cell death or apoptosis. We have shown recently that the CPP32 and Mch2a cystelne proteases cleave the apoptotic markers poly(ADP. ribose) polymerase (PARP) and lamins, respectively. Here we report the cloning of a new Ced-3\\/interleukin 1@-convertlng enzyme-related gene, designated Mch3, that

  4. Mechanisms of failed apoptotic cell clearance by phagocyte subsets in cardiovascular disease

    Microsoft Academic Search

    Edward B. Thorp

    2010-01-01

    Recent evidence in humans indicate that defective phagocytic clearance of dying cells is linked to progression of advanced\\u000a atherosclerotic lesions, the precursor to atherothrombosis, ischemic heart disease, and leading cause of death in the industrialized\\u000a world. During atherogenesis, apoptotic cell turnover in the vascular wall is counterbalanced by neighboring phagocytes with\\u000a high clearance efficiency, thereby limiting cellularity and maintaining lesion

  5. Neonatal primary neuronal death induced by capsaicin and axotomy involves an apoptotic mechanism

    Microsoft Academic Search

    Tomosada Sugimoto; Chun Xiao; Hiroyuki Ichikawa

    1998-01-01

    To clarify the mechanism of capsaicin-induced primary neuronal cell death, newborn and adult rats were given a subcutaneous injection of capsaicin (50 mg\\/kg). Neonatal capsaicin injection induced neuronal apoptosis in the trigeminal ganglion. Apoptotic neurons had peripheral stacks of long parallel endoplasmic reticulum that are characteristic to primary neurons of the B-type, and exhibited nucleoplasmic condensation, nuclear shrinkage and cytoplasmic

  6. Apoptotic activities of ethanol extracts from some Apiaceae on human leukaemia cell lines

    Microsoft Academic Search

    A. Bogucka-Kocka; H. D. Smolarz; J. Kocki

    2008-01-01

    Apiaceae are a family of medicinal plants widely used in traditional medicine. The apoptotic activities of seven ethanol extracts from fruits of seven species of Apiaceae, Eryngium planum, Archangelica officinalis, Pastinaca sativa, Heracleum sibiricum, Carum carvi, Foeniculum vulgare, Levisticum officinale against ML-1—human acute myeloblastic leukaemia, J-45.01—human acute T cell leukaemia, EOL—human eosinophilic leukaemia, HL-60—human Caucasian promyelocytic leukaemia, 1301—human T cell

  7. Drosophila activated Cdc42 kinase has an anti-apoptotic function.

    PubMed

    Schoenherr, Jessica A; Drennan, J Michelle; Martinez, Juan S; Chikka, Madhusudana Rao; Hall, Mark C; Chang, Henry C; Clemens, James C

    2012-01-01

    Activated Cdc42 kinases (Acks) are evolutionarily conserved non-receptor tyrosine kinases. Activating somatic mutations and increased ACK1 protein levels have been found in many types of human cancers and correlate with a poor prognosis. ACK1 is activated by epidermal growth factor (EGF) receptor signaling and functions to regulate EGF receptor turnover. ACK1 has additionally been found to propagate downstream signals through the phosphorylation of cancer relevant substrates. Using Drosophila as a model organism, we have determined that Drosophila Ack possesses potent anti-apoptotic activity that is dependent on Ack kinase activity and is further activated by EGF receptor/Ras signaling. Ack anti-apoptotic signaling does not function through enhancement of EGF stimulated MAP kinase signaling, suggesting that it must function through phosphorylation of some unknown effector. We isolated several putative Drosophila Ack interacting proteins, many being orthologs of previously identified human ACK1 interacting proteins. Two of these interacting proteins, Drk and yorkie, were found to influence Ack signaling. Drk is the Drosophila homolog of GRB2, which is required to couple ACK1 binding to receptor tyrosine kinases. Drk knockdown blocks Ack survival activity, suggesting that Ack localization is important for its pro-survival activity. Yorkie is a transcriptional co-activator that is downstream of the Salvador-Hippo-Warts pathway and promotes transcription of proliferative and anti-apoptotic genes. We find that yorkie and Ack synergistically interact to produce tissue overgrowth and that yorkie loss of function interferes with Ack anti-apoptotic signaling. Our results demonstrate how increased Ack signaling could contribute to cancer when coupled to proliferative signals. PMID:22615583

  8. Malignant mixed Mullerian tumors of the uterus: histopathological evaluation of cell cycle and apoptotic regulatory proteins

    Microsoft Academic Search

    Rani Kanthan; Jenna-Lynn B Senger; Dana Diudea

    2010-01-01

    AIM: The aim of our study was to evaluate survival outcomes in malignant mixed Mullerian tumors (MMMT) of the uterus with respect to the role of cell cycle and apoptotic regulatory proteins in the carcinomatous and sarcomatous components. METHODS: 23 cases of uterine MMMT identified from the Saskatchewan Cancer Agency (1970-1999) were evaluated. Immunohistochemical expression of Bad, Mcl-1, bcl-x, bak,

  9. Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions.

    PubMed

    Deegan, Shane; Saveljeva, Svetlana; Logue, Susan E; Pakos-Zebrucka, Karolina; Gupta, Sanjeev; Vandenabeele, Peter; Bertrand, Mathieu J M; Samali, Afshin

    2014-01-01

    Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells. PMID:25470234

  10. The Gastroprotective Effect of Menthol: Involvement of Anti-Apoptotic, Antioxidant and Anti-Inflammatory Activities

    PubMed Central

    Rozza, Ariane Leite; Meira de Faria, Felipe; Souza Brito, Alba Regina; Pellizzon, Cláudia Helena

    2014-01-01

    The aim of this research was to investigate the anti-apoptotic, antioxidant and anti-inflammatory properties of menthol against ethanol-induced gastric ulcers in rats. Wistar rats were orally treated with vehicle, carbenoxolone (100 mg/kg) or menthol (50 mg/kg) and then treated with ethanol to induce gastric ulcers. After euthanasia, stomach samples were prepared for histological slides and biochemical analyses. Immunohistochemical analyses of the cytoprotective and anti-apoptotic heat-shock protein-70 (HSP-70) and the apoptotic Bax protein were performed. The neutrophils were manually counted. The activity of the myeloperoxidase (MPO) was measured. To determine the level of antioxidant functions, the levels of glutathione (GSH), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and superoxide dismutase (SOD) were measured using ELISA. The levels of the pro-inflammatory cytokines tumor necrosis factor-? (TNF-?) and interleukin-6 (IL-6) and the anti-inflammatory cytokine interleukin-10 (IL-10) were assessed using ELISA kits. The menthol treated group presented 92% gastroprotection compared to the vehicle-treated group. An increased immunolabeled area was observed for HSP-70, and a decreased immunolabeled area was observed for the Bax protein in the menthol treated group. Menthol treatment induced a decrease in the activity of MPO and SOD, and the protein levels of GSH, GSH-Px and GR were increased. There was also a decrease in the levels of TNF-? and IL-6 and an increase in the level of IL-10. In conclusion, oral treatment with menthol displayed a gastroprotective activity through anti-apoptotic, antixidant and anti-inflammatory mechanisms. PMID:24466200

  11. Death receptor-induced apoptotic and necrotic cell death: differential role of caspases and mitochondria

    Microsoft Academic Search

    G Denecker; D Vercammen; M Steemans; T Vanden Berghe; G Brouckaert; G Van Loo; B Zhivotovsky; W Fiers; J Grooten; W Declercq; P Vandenabeele

    2001-01-01

    In L929sAhFas cells, tumor necrosis factor (TNF) leads to necrotic cell death, whereas agonistic anti-Fas antibodies elicit apoptotic cell death. Apoptosis, but not necrosis, is correlated with a rapid externalization of phosphatidylserine and the appearance of a hypoploid population. During necrosis no cytosolic and organelle-associated active caspase-3 and -7 fragments are detectable. The necrotic process does not involve proteolytic generation

  12. Oncogenic Properties of Apoptotic Tumor Cells in Aggressive B Cell Lymphoma

    PubMed Central

    Ford, Catriona A.; Petrova, Sofia; Pound, John D.; Voss, Jorine J.L.P.; Melville, Lynsey; Paterson, Margaret; Farnworth, Sarah L.; Gallimore, Awen M.; Cuff, Simone; Wheadon, Helen; Dobbin, Edwina; Ogden, Carol Anne; Dumitriu, Ingrid E.; Dunbar, Donald R.; Murray, Paul G.; Ruckerl, Dominik; Allen, Judith E.; Hume, David A.; van Rooijen, Nico; Goodlad, John R.; Freeman, Tom C.; Gregory, Christopher D.

    2015-01-01

    Summary Background Cells undergoing apoptosis are known to modulate their tissue microenvironments. By acting on phagocytes, notably macrophages, apoptotic cells inhibit immunological and inflammatory responses and promote trophic signaling pathways. Paradoxically, because of their potential to cause death of tumor cells and thereby militate against malignant disease progression, both apoptosis and tumor-associated macrophages (TAMs) are often associated with poor prognosis in cancer. We hypothesized that, in progression of malignant disease, constitutive loss of a fraction of the tumor cell population through apoptosis could yield tumor-promoting effects. Results Here, we demonstrate that apoptotic tumor cells promote coordinated tumor growth, angiogenesis, and accumulation of TAMs in aggressive B cell lymphomas. Through unbiased “in situ transcriptomics” analysis—gene expression profiling of laser-captured TAMs to establish their activation signature in situ—we show that these cells are activated to signal via multiple tumor-promoting reparatory, trophic, angiogenic, tissue remodeling, and anti-inflammatory pathways. Our results also suggest that apoptotic lymphoma cells help drive this signature. Furthermore, we demonstrate that, upon induction of apoptosis, lymphoma cells not only activate expression of the tumor-promoting matrix metalloproteinases MMP2 and MMP12 in macrophages but also express and process these MMPs directly. Finally, using a model of malignant melanoma, we show that the oncogenic potential of apoptotic tumor cells extends beyond lymphoma. Conclusions In addition to its profound tumor-suppressive role, apoptosis can potentiate cancer progression. These results have important implications for understanding the fundamental biology of cell death, its roles in malignant disease, and the broader consequences of apoptosis-inducing anti-cancer therapy. PMID:25702581

  13. A mutation of human cytochrome c enhances the intrinsic apoptotic pathway but causes only thrombocytopenia.

    PubMed

    Morison, Ian M; Cramer Bordé, Elisabeth M; Cheesman, Emma J; Cheong, Pak Leng; Holyoake, Andrew J; Fichelson, Serge; Weeks, Robert J; Lo, Alexandra; Davies, Stefan M K; Wilbanks, Sigurd M; Fagerlund, Robert D; Ludgate, Mathew W; da Silva Tatley, Fernanda M; Coker, Melanie S A; Bockett, Nicholas A; Hughes, Gillian; Pippig, Diana A; Smith, Mark P; Capron, Claude; Ledgerwood, Elizabeth C

    2008-04-01

    We report the first identified mutation in the gene encoding human cytochrome c (CYCS). Glycine 41, invariant throughout eukaryotes, is substituted by serine in a family with autosomal dominant thrombocytopenia caused by dysregulated platelet formation. The mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro. Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome c activity is not a critical regulator of most physiological apoptosis. PMID:18345000

  14. Animal Anti-Apoptotic Genes Enhance Recovery from Drought Stress in Tobacco

    Microsoft Academic Search

    TALA AWADA; DAVID D. DUNIGAN; MARTIN B. DICKMAN

    The anti-apoptotic nematode CED-9 gene and a 3' non-coding mRNA region of the human Bcl-2 gene, referred to as 161-1, enhanced resistance to drought and recovery after re-watering in transgenic Nicotiana tabacum L. CV Glurk plants. The effect of drought on plant functions was investigated by withholding water from pots for 6, 8, 10 and 13 days. Results showed that

  15. ATR and Chk1 Suppress a Caspase3–Dependent Apoptotic Response Following DNA Replication Stress

    Microsoft Academic Search

    Katie Myers; Mary E. Gagou; Pedro Zuazua-Villar; Rene Rodriguez; Mark Meuth

    2009-01-01

    The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-related (ATR) play major roles in the regulation of cellular responses to DNA damage or replication stress. The pro-apoptotic role of ATM and p53 in response to ionizing radiation (IR) has been widely investigated. Much less is known about the control of apoptosis following DNA replication stress. Recent work indicates

  16. The green algae Ulva fasciata Delile extract induces apoptotic cell death in human colon cancer cells.

    PubMed

    Ryu, Min Ju; Kim, Areum Daseul; Kang, Kyoung Ah; Chung, Ha Sook; Kim, Hye Sun; Suh, In Soo; Chang, Weon Young; Hyun, Jin Won

    2013-01-01

    This study investigated the mechanisms underlying the cytotoxicity of the green algae Ulva fasciata Delile. U. fasciata extract (UFE) inhibited the growth of HCT 116 human colon cancer cells by 50% at a concentration of 200 ?g/ml. In addition, UFE stimulated the production of intracellular reactive oxygen species, an effect that was abolished by pretreatment with N-acetyl cysteine, which also inhibited the cytotoxic effects of UFE. UFE also induced morphological changes indicative of apoptosis, such as the formation of apoptotic bodies, DNA fragmentation, an increase in the population of apoptotic sub-G(1) phase cells, and mitochondrial membrane depolarization. Concomitant activation of the mitochondria-dependent apoptotic pathway occurred via modulation of Bax and Bcl-2 expression, resulting in disruption of the mitochondrial membrane potential and activation of caspase-9 and caspase-3. This is the first report to demonstrate the cytotoxic effect of U. fasciata on human colon cancer cells and to provide a possible mechanism for this activity. PMID:23299316

  17. BCDO2 acts as a carotenoid scavenger and gatekeeper for the mitochondrial apoptotic pathway

    PubMed Central

    Lobo, Glenn P.; Isken, Andrea; Hoff, Sylvia; Babino, Darwin; von Lintig, Johannes

    2012-01-01

    Carotenoids and their metabolites are widespread and exert key biological functions in living organisms. In vertebrates, the carotenoid oxygenase BCMO1 converts carotenoids such as ?,?-carotene to retinoids, which are required for embryonic pattern formation and cell differentiation. Vertebrate genomes encode a structurally related protein named BCDO2 but its physiological function remains undefined. Here, we show that BCDO2 is expressed as an oxidative stress-regulated protein during zebrafish development. Targeted knockdown of this mitochondrial enzyme resulted in anemia at larval stages. Marker gene analysis and staining for hemoglobin revealed that erythropoiesis was not impaired but that erythrocytes underwent apoptosis in BCDO2-deficient larvae. To define the mechanism of this defect, we have analyzed the role of BCDO2 in human cell lines. We found that carotenoids caused oxidative stress in mitochondria that eventually led to cytochrome c release, proteolytic activation of caspase 3 and PARP1, and execution of the apoptotic pathway. Moreover, BCDO2 prevented this induction of the apoptotic pathway by carotenoids. Thus, our study identifying BCDO2 as a crucial protective component against oxidative stress establishes this enzyme as mitochondrial carotenoid scavenger and a gatekeeper of the intrinsic apoptotic pathway. PMID:22764054

  18. Role of ionic fluxes in the apoptotic cell death of cultured cerebellar granule neurons.

    PubMed

    Franco-Cea, A; Valencia, A; Sánchez-Armass, S; Domínguez, G; Morán, J

    2004-01-01

    Cultured cerebellar granule neurons (CGC) increase survival in a medium containing 25 mM KCl (K25), and they die apoptotically when cultures are treated with staurosporine (St) or are transferred to a 5-mM KCl containing medium (K5). Apoptotic CGC show nuclear condensation and caspase-3 activation. Cell death induced by these conditions was partially prevented when cultures were maintained under alkaline conditions, which also induced a marked reduction of the caspase-3 activation. The acidification of the medium further increased cell death induced by both stimuli. Cultures transferred to K5 suffered an immediate intracellular alkalinization that remained constant during the time K5 was present. In contrast, St did not modify cytosolic pH at any of the evaluated times. On the other hand, DIDS, furosemide, and bumetanide prevented CGC death induced by K5 and St. Other drugs such as amiloride, EIPA, tamoxifen, NEM, or NPPB did not modify cell death induced by these conditions. Both DIDS and bumetanide markedly inhibited the processing and activation of caspase-3, and DIDS prevented the nuclear condensation induced by K5 and St. These findings suggest that pH is a condition that could contribute to the modulation of cell death induced by some stimuli and that other ions, such as potassium, could have a role in the initial phase of apoptotic death of CGC. PMID:14992282

  19. Anti-apoptotic and pro-survival effect of protocatechuic acid on hypertensive hearts.

    PubMed

    Deng, Jeng-Shyan; Lee, Shin-Da; Kuo, Wei-Wen; Fan, Ming-Jen; Lin, Yueh-Min; Hu, Wei-Syun; Huang, Yi-Ching; Velmurugan, Bharath Kumar; Tsai, Fuu-Jen; Tsai, Chang-Hai; Huang, Chih-Yang

    2014-02-25

    Cardiac apoptosis was found in hearts from hypertensive animals, therefore in this study we aimed to evaluate the anti-apoptotic and pro-survival effects of protocatechuic acid (PCA) on hypertensive hearts. At first we found that, sedentary group (SHR)-PCA group's decreased TUNEL-positive apoptotic cells than SHR group alone. Protein levels of Fas ligand, Fas death receptor, Fas-associated death domain (FADD), Bid, t-Bid, Bax, cytochrome c, activated caspase-8, activated caspase 9 and activated caspase-3 were decreased in SHR-PCA group compared with SHR group. Moreover, SHR-PCA groups increased pro-survival pathway proteins like IGF1, pIGF1R, pPI3K, p-Akt, Bcl-xL, and Bcl-2 than SHR and sedentary normotensive group (WKY). All these finding suggest us that, Protocatechuic acid prevented hypertension-enhanced cardiac Fas-dependent and mitochondria-dependent apoptotic pathways and enhanced cardiac pro-survival pathway in rat models. PMID:24355328

  20. Journey to the grave: signaling events regulating removal of apoptotic cells.

    PubMed

    Kinchen, Jason M; Ravichandran, Kodi S

    2007-07-01

    Programmed cell death is critical both for organ formation during development and during adult life, when billions of cells must be removed every day. The culmination of the apoptotic process is the specific recognition and engulfment of the apoptotic cell by a phagocyte. A number of recent studies have revealed a series of evolutionarily conserved proteins that link corpse recognition to membrane movement, facilitating the internalization of the target and its subsequent degradation. Two potential signaling modules have been identified: one involving the CED-12/ELMO and CED-5/Dock180 proteins, which function as a bipartite guanine nucleotide exchange factor (GEF) for Rac1, and a second involving CED-1/LRP1 (a potential engulfment receptor) and the adaptor protein CED-6/GULP. Recognition of the apoptotic cell modulates cytokine secretion by the phagocyte, resulting in an anti-inflammatory state distinct from that induced by necrotic cells. The recent molecular delineation of the phagocytic process and the identification of novel signaling proteins involved in engulfment have provided an exciting new platform for future studies into this biologically important process. PMID:17591687

  1. p53 Dependent Apoptotic Cell Death Induces Embryonic Malformation in Carassius auratus under Chronic Hypoxia

    PubMed Central

    Dasgupta, Subrata; Sawant, Bhawesh T.; Chadha, Narinder K.; Pal, Asim K.

    2014-01-01

    Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD), leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf) and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD), ultimately resulting in significant (p<0.05) embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos. PMID:25068954

  2. pknE, a serine/threonine kinase of Mycobacterium tuberculosis modulates multiple apoptotic paradigms.

    PubMed

    Kumar, Dinesh; Narayanan, Sujatha

    2012-06-01

    Mycobacterium tuberculosis, an intracellular pathogen that causes tuberculosis has developed multifactorial mechanisms to evade host signaling responses. Apoptosis, an important innate host immune response that clears the invading pathogen is suppressed by M. tuberculosis to gain persistence. Here, we examined the various apoptotic events suppressed by Protein Kinase E, (pknE) a Serine Threonine Protein Kinase (STPK) of M. tuberculosis in macrophages infected with ?pknE, a deletion mutant of pknE vs. the wild type strain H(37)Rv using microarray. The data showed increased expression of genes involved in apoptosis and chemokines with suppressed pro-inflammatory cytokines, co-stimulatory molecules, arginase1 and iNOS. The microarray data was validated using qRT-PCR, PCR array, oligoGE array, arginase assay and/or ELISA. Furthermore, we analyzed the phosphorylation of Akt that promotes cell survival using western blotting. ?pknE infected macrophages reduced the phosphorylation of Akt that correlates with the observed apoptotic responses. Experiments performed using exogenous nitrate donor, sodium nitro prusside to demonstrate the role of pknE during nitrate stress showed similar apoptotic responses to that of endogenous nitrate stress in ?pknE infected macrophages. Our data confirms the role of pknE in the intra cellular survival of M. tuberculosis by suppressing apoptosis during nitrate stress. PMID:21945589

  3. Gene expression of anti- and pro-apoptotic proteins in malignant and normal plasma cells

    PubMed Central

    Jourdan, Michel; Rème, Thierry; Goldschmidt, Harmut; Fiol, Geneviève; Pantesco, Véronique; De Vos, John; Rossi, Jean-François; Hose, Dirk; Klein, Bernard

    2009-01-01

    Summary Survival of malignant plasma cells is a key event in disease occurrence, progression and chemoresistance. Using DNA-microarrays, we analysed the expression of genes coding for 58 proteins linked with extrinsic and intrinsic apoptotic pathways, caspases and inhibitor of apoptosis proteins. We considered 6 memory B cells (MBC), 7 plasmablasts (PPC), 7 bone marrow plasma cells (BMPC) and purified myeloma cells (MMC) from 92 newly-diagnosed patients. 40 out of the 58 probe sets make it possible to separate MBC, PPC and BMPC in 3 homogeneous clusters, characterized by an elevated expression of TNFRSF10A, TNFRSF10B, BCL2A1, CASP8, CASP9 and PMAIP1 genes for MBC, of FAS, FADD, AIFM1, BIRC5, CASP2, CASP3 and CASP6 for PPC and of BCL2, MCL1, BID, BIRC3 and XIAP for BMPC. Thus, B cell differentiation is associated in change of expression of pro-apoptotic and anti-apoptotic genes. Regarding MMC, the major finding is TNFSF10 upregulation in MMC that might be counteracted by a high osteoprotegerin production by BM stromal cells and a decreased expression of FAS, APAF1 and BNIP3 compared to normal BMPC. Out of the 40 genes, CASP2 and BIRC5 expression in MMC had adverse prognosis in 2 independent series of previously-untreated patients. PMID:19183193

  4. Different Apoptotic Pathways Activated by Oxaliplatin in Primary Astrocytes vs. Colo-Rectal Cancer Cells

    PubMed Central

    Zanardelli, Matteo; Micheli, Laura; Nicolai, Raffaella; Failli, Paola; Ghelardini, Carla; Di Cesare Mannelli, Lorenzo

    2015-01-01

    Oxaliplatin-based chemotherapy improves the outcomes of metastatic colorectal cancer patients. Its most significant and dose-limiting side effect is the development of a neuropathic syndrome. The mechanism of the neurotoxicity is unclear. The limited knowledge about differences existing between neurotoxic and antitumor effects hinders the discovery of effective and safe adjuvant therapies. In vitro, we suggested cell-specific activation apoptotic pathways in normal nervous cells (astrocytes) vs. colon-cancer cells (HT-29). In the present research we compared the apoptotic signals evoked by oxaliplatin in astrocytes and HT-29 analyzing the intrinsic and extrinsic apoptotic pathways. In astrocytes, oxaliplatin induced a mitochondrial derangement measured as cytosolic release of cytochrome C, increase in superoxide anion levels and decreased expression of the antiapoptotic protein Bcl-2. Caspase-8, a main initiator of the extrinsic process remained unaltered. On the contrary, in HT-29 oxaliplatin increased caspase-8 activity and Bid expression, thus activating the extrinsic apoptosis, while the Bcl-2 increased expression blocked the mitochondrial damage. Data suggest the preferred activation of the intrinsic apoptosis as oxaliplatin damage signaling in normal nervous cells. The extrinsic pathway prevails in tumor cells indicating a possible strategy for planning new molecules to treat oxaliplatin-dependent neurotoxicity without negatively influence chemotherapy. PMID:25761243

  5. Tomato lycopene attenuates myocardial infarction induced by isoproterenol: Electrocardiographic, biochemical and anti-apoptotic study

    PubMed Central

    Aman, Upaganlawar; Vaibhav, Patel; Balaraman, R

    2012-01-01

    Objective To assess the protective effects of lycopene on electrocardiographic, hemodynamic, biochemical and apoptotic changes in isoproterenol induced myocardial infarction. Methods Myocardial infarction was induced in rats by subcutaneous injection of isoproterenol (200 mg/kg) for two consecutive days at an interval of 24 h. Rats were treated with lycopene (10 mg/kg/day, p.o.) for a period of 30 days and isoproterenol (ISO) was injected on the 29th and 30th day. At the end of experiment i.e. on the 31st day electrocardiographic, hemodynamic, biochemical and apoptotic changes were monitored from control and experimental groups. Results ISO injected rats showed a significant alteration in electrocardiograph pattern and hemodynamic changes (i.e. systolic, diastolic and mean arterial pressure). It also showed significant increase in C-reactive protein, myeloperoxidase, nitrite levels and Caspase-3 protease activity. In addition, it also exhibited alteration in the levels of electrolytes (Na+, K+ and Ca2+), vitamin E, uric acid and serum protein. Gel electrophoresis of ISO injected rats showed increase in DNA fragmentation. Triphenyl tetrazolium chloride staining of the heart section shows increase area of infarction in ISO injected rats. Pre-co-treatment with lycopene significantly prevented the ISO induced alteration in ECG, haemodynamic, biochemical and apoptotic changes. Conclusions The present result shows that treatment of lycopene in ISO injected rats significantly attenuates induced myocardial infarction. PMID:23569928

  6. In vivo CaspaseTracker biosensor system for detecting anastasis and non-apoptotic caspase activity

    PubMed Central

    Tang, Ho Lam; Tang, Ho Man; Fung, Ming Chiu; Hardwick, J. Marie

    2015-01-01

    The discovery that mammalian cells can survive late-stage apoptosis challenges the general assumption that active caspases are markers of impending death. However, tools have not been available to track healthy cells that have experienced caspase activity at any time in the past. Therefore, to determine if cells in whole animals can undergo reversal of apoptosis, known as anastasis, we developed a dual color CaspaseTracker system for Drosophila to identify cells with ongoing or past caspase activity. Transient exposure of healthy females to environmental stresses such as cold shock or starvation activated the CaspaseTracker coincident with caspase activity and apoptotic morphologies in multiple cell types of developing egg chambers. Importantly, when stressed flies were returned to normal conditions, morphologically healthy egg chambers and new progeny flies were labeled by the biosensor, suggesting functional recovery from apoptotic caspase activation. In striking contrast to developing egg chambers, which lack basal caspase biosensor activation under normal conditions, many adult tissues of normal healthy flies exhibit robust caspase biosensor activity in a portion of cells, including neurons. The widespread persistence of CaspaseTracker-positivity implies that healthy cells utilize active caspases for non-apoptotic physiological functions during and after normal development. PMID:25757939

  7. Acidic pH Promotes Oligomerization and Membrane Insertion of the BclXL Apoptotic Repressor

    PubMed Central

    Bhat, Vikas; Kurouski, Dmitry; Olenick, Max B.; McDonald, Caleb B.; Mikles, David C.; Deegan, Brian J.; Seldeen, Kenneth L.; Lednev, Igor K.; Farooq, Amjad

    2012-01-01

    Solution pH is believed to serve as an intricate regulatory switch in the induction of apoptosis central to embryonic development and cellular homeostasis. Herein, using an array of biophysical techniques, we provide evidence that acidic pH promotes the assembly of BclXL apoptotic repressor into a megaldalton oligomer with a plume-like appearance and harboring structural features characteristic of a molten globule. Strikingly, our data reveal that pH tightly modulates not only oligomerization but also ligand binding and membrane insertion of BclXL in a highly subtle manner. Thus, while oligomerization and the accompanying molten globular content of BclXL is least favorable at pH 6, both of these structural features become more pronounced under acidic and alkaline conditions. However, membrane insertion of BclXL appears to be predominantly favored under acidic conditions. In a remarkable contrast, while ligand binding to BclXL optimally occurs at pH 6, it is diminished by an order of magnitude at lower and higher pH. This reciprocal relationship between BclXL oligomerization and ligand binding lends new insights into how pH modulates functional versatility of a key apoptotic regulator and strongly argues that the molten globule may serve as an intermediate primed for membrane insertion in response to apoptotic cues. PMID:22960132

  8. In vitro assessment of oxidative stress and apoptotic mechanisms of garlic extract in the treatment of acute promyelocytic leukemia

    PubMed Central

    Yedjou, Clement G.; Tchounwou, Paul B.

    2012-01-01

    Introduction Garlic supplementation in diet has been shown to be beneficial to cancer patients. Recently, its pharmacological role in the prevention and treatment of cancer has received increasing attention. However, the mechanisms by which garlic extract (GE) induces cytotoxicity, oxidative stress, and apoptosis in cancer cells remain largely unknown. Objective The present study was designed to use HL-60 cells as a test model to evaluate whether or not GE-induced cytotoxicty and apoptosis in human leukemia (HL-60) cells is mediated through oxidative stress. Methods Human leukemia (HL-60) cells were treated with different concentrations of GE for 12 hr. Cell survival was determined by MTT assay. The extent of oxidative cell/tissue damage was determined by measuring malondialdehyde (lipid peroxidation biomarker) concentrations by spectrophotometry. Cell apoptosis was measured by flow cytometry assessment (Annexin-V and caspase-3 assays) and agarose gel electrophoresis (DNA laddering assay). Results Data obtained from the MTT assay indicated that GE significantly (p < 0.05) reduced the viability of HL-60 cells in a concentration-dependent manner. We detected a significant (p < 0.05) increase in malondialdehyde (MDA) concentrations in GE-treated HL-60 cells compared to the control. Flow cytometry data showed a strong concentration-response relationship between GE exposure and Annexin-V positive HL-60 cells. Similarly, a statistically significant and concentration-dependent increase (p <0.05) were recorded with regard to caspase-3 activity in HL-60 cells undergoing late apoptosis. These results were confirmed by data of DNA laddering assay showing a clear evidence of nucleosomal DNA fragmentation in GE-treated cells. Conclusion Our finding indicates that GE-induced cytotoxicity and apoptosis in HL-60 cells involve phosphatidylserine externalization, caspase-3 activation, and nucleosomal DNA fragmentation associated with the formation of MDA, a by-product of lipid peroxidation and biomarker of oxidative stress. At therapeutic concentrations, GE-induced cytotoxic and apoptotic effects in HL-60 cells is mediated by oxidative stress. PMID:23847719

  9. Upregulation of Hsp72 mediates anoxia/reoxygenation neuroprotection in the freshwater turtle via modulation of ROS.

    PubMed

    Kesaraju, Shailaja; Nayak, Gauri; Prentice, Howard M; Milton, Sarah L

    2014-09-25

    The neuroprotective role of Hsp72 has been demonstrated in several ischemic/stroke models to occur primarily through mediation of apoptotic pathways, and a number of heat shock proteins are upregulated in animal models capable of extended anoxic survival. In the present study, we investigated the role of Hsp72 on cell death and apoptotic regulators in one anoxia tolerant model system, the freshwater turtle Trachemys scripta. Since Hsp72 is known to regulate apoptosis through interactions with Bcl-2, we manipulated the levels of Hsp72 and Bcl-2 with siRNA in neuronally enriched primary cell cultures and examined downstream effects. The knockdown of either Hsp72 or Bcl-2 induced cell death during anoxia and reoxygenation. Knockdown of Bcl-2 resulted in increases in apoptotic markers and increased ROS levels 2-fold. However, significant knockdown of Hsp72 did not have any effect on the expression of key mitochondrial apoptotic regulators such as Cytochrome c and caspase-3. Hsp72 knockdown however significantly increased apoptosis inducing factor in both anoxia and reoxygenation and resulted in a six-fold induction of hydrogen peroxide levels. These findings suggest that the neuroprotection offered by Hsp72 in the anoxia/reoxygenation tolerant turtle is through the mediation of ROS levels and not through modulation of caspase-dependent pathways. PMID:25107858

  10. Apoptotic Signaling in Normal and Cancer Cell Biology: Monitoring Apoptosis Biomarkers to Help Understand development and Oncologic Processes

    NSDL National Science Digital Library

    Saul Rosenberg (Abbott Laboratories; )

    2009-09-30

    This webinar provides an overview of apoptosis, surveys some of the key processes controlled by apoptosis and the assays available for monitoring of these processes, and discusses options available for monitoring apoptotic induction in various model systems.

  11. Leptin suppresses non-apoptotic cell death in ischemic rat cardiomyocytes by reduction of iPLA2 activity.

    PubMed

    Takatani-Nakase, Tomoka; Takahashi, Koichi

    2015-07-17

    Caspase-independent, non-apoptotic cell death is an important therapeutic target in myocardial ischemia. Leptin, an adipose-derived hormone, is known to exhibit cytoprotective effects on the ischemic heart, but the mechanisms are poorly understood. In this research, we found that pretreatment of leptin strongly suppressed ischemic-augmented nuclear shrinkage and non-apoptotic cell death on cardiomyocytes. Leptin was also shown to significantly inhibit the activity of iPLA2, which is considered to play crucial roles in non-apoptotic cell death, resulting in effective prevention of ischemia-induced myocyte death. These findings provide the first evidence of a protective mechanism of leptin against ischemia-induced non-apoptotic cardiomyocyte death. PMID:25979360

  12. Natural Proteasome Inhibitor Celastrol Suppresses Androgen-Independent Prostate Cancer Progression by Modulating Apoptotic Proteins and NF-kappaB

    E-print Network

    Dai, Yao; Jeffrey, Tang; Wenhua, Xiaojie; Meng, Yang; Burstein, Ezra; Lawrence, Theodore S.; Xu, Liang

    2010-12-10

    the proteasome, celastrol suppresses proliferation, invasion and angiogenesis by inducing the apoptotic machinery and attenuating constitutive NF-?B activity in AIPC both in vitro and in vivo. Celastrol as an active ingredient of traditional herbal medicine could...

  13. Two pathways converge at CED-10 to mediate actin rearrangement and corpse removal in C. elegans.

    PubMed

    Kinchen, Jason M; Cabello, Juan; Klingele, Doris; Wong, Kelvin; Feichtinger, Richard; Schnabel, Heinke; Schnabel, Ralf; Hengartner, Michael O

    2005-03-01

    The removal of apoptotic cells is essential for the physiological well being of the organism. In Caenorhabditis elegans, two conserved, partially redundant genetic pathways regulate this process. In the first pathway, the proteins CED-2, CED-5 and CED-12 (mammalian homologues CrkII, Dock180 and ELMO, respectively) function to activate CED-10 (Rac1). In the second group, the candidate receptor CED-1 (CD91/LRP/SREC) probably recognizes an unknown ligand on the apoptotic cell and signals via its cytoplasmic tail to the adaptor protein CED-6 (hCED-6/GULP), whereas CED-7 (ABCA1) is thought to play a role in membrane dynamics. Molecular understanding of how the second pathway promotes engulfment of the apoptotic cell is lacking. Here, we show that CED-1, CED-6 and CED-7 are required for actin reorganization around the apoptotic cell corpse, and that CED-1 and CED-6 colocalize with each other and with actin around the dead cell. Furthermore, we find that the CED-10(Rac) GTPase acts genetically downstream of these proteins to mediate corpse removal, functionally linking the two engulfment pathways and identifying the CED-1, -6 and -7 signalling module as upstream regulators of Rac activation. PMID:15744306

  14. Hydrogen peroxide is a mediator of indole-3-acetic acid/horseradish peroxidase-induced apoptosis.

    PubMed

    Kim, Dong-Seok; Jeon, Sang-Eun; Jeong, Yun-Mi; Kim, So-Young; Kwon, Sun-Bang; Park, Kyoung-Chan

    2006-02-20

    Recently, we reported that a combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) induces apoptosis in G361 human melanoma cells. However, the apoptotic mechanism involved has been poorly studied. It is known that when IAA is oxidized by HRP, free radicals are produced, and since oxidative stress can induce apoptosis, we investigated whether reactive oxygen species (ROS) are involved in IAA/HRP-induced apoptosis. Our results show that IAA/HRP-induced free radical production is inhibited by catalase, but not by superoxide dismutase or sodium formate. Furthermore, catalase was found to prevent IAA/HRP-induced apoptotic cell death, indicating that IAA/HRP-produced hydrogen peroxide (H2O2) may be involved in the apoptotic process. Moreover, the antiapoptotic effect of catalase is potentiated by NADPH, which is known to protect catalase. On further investigating the IAA/HRP-mediated apoptotic pathway, we found that the IAA/HRP reaction leads to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, which was also blocked by catalase. Additionally, we found that IAA/HRP produces H2O2 and induces peroxiredoxin (Prx) sulfonylation. Consequently, our results suggest that H2O2 plays a major role in IAA/HRP-induced apoptosis. PMID:16460736

  15. Protective effects of melittin on transforming growth factor-?1 injury to hepatocytes via anti-apoptotic mechanism

    Microsoft Academic Search

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

    2011-01-01

    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

  16. Molecular Basis of Anti-Apoptotic Effect of Immunophilin Ligands on Hydrogen Peroxide–Induced Apoptosis in Human Glioma Cells

    Microsoft Academic Search

    Ken-ichi Tanaka; Masato Asanuma; Norio Ogawa

    2004-01-01

    To clarify the molecular basis of the cytoprotective properties of immunophilin ligands (IPLs), the anti-apoptotic effects of IPLs were determined in human glioma U251 cells. GPI1046 and V10367, non-immunosuppressive IPLs (NI-IPLs), as well as FK506, an immunosuppressive IPL (I-IPL), had cytoprotective effects against hydrogen peroxide (H2O2)–induced apoptotic cell death in U251 cells. H2O2 increased both the ratio of bax\\/bcl-2 and

  17. Delayed Apoptotic Cell Clearance and Lupus-like Autoimmunity in Mice Lacking the c-mer Membrane Tyrosine Kinase

    Microsoft Academic Search

    Philip L. Cohen; Roberto Caricchio; Valsamma Abraham; Todd D. Camenisch; J. Charles Jennette; Robert A. S. Roubey; H. Shelton Earp; Glenn Matsushima; Elizabeth A. Reap

    2002-01-01

    Mice lacking the membrane tyrosine kinase c-mer have been shown to have altered macro- phage cytokine production and defective phagocytosis of apoptotic cells despite normal phago- cytosis of other particles. We show here that c-mer-deficient mice have impaired clearance of infused apoptotic cells and that they develop progressive lupus-like autoimmunity, with anti- bodies to chromatin, DNA, and IgG. The autoimmunity

  18. Apoptotic effects of hydrogen peroxide and vitamin C on chicken embryonic fibroblasts: redox state and programmed cell death

    Microsoft Academic Search

    D. P. Jin; C. Y. Li; H. J. Yang; W. X. Zhang; C. L. Li; W. J. Guan; Y. H. Ma

    The pro-apoptotic effects of hydrogen peroxide and the purported anti-apoptotic effects of Vitamin C on chicken embryonic\\u000a fibroblasts were investigated. Hydrogen peroxide induced morphological changes in a dose dependent manner, and a myriad of\\u000a autophagosomes were observed using transmission electron microscopy. Doxorubicin elicited alterations were not inhibited by\\u000a co-incubation with Vitamin C except that mitochondrial structure was slightly improved. TUNEL

  19. Pim-1 Mediated Signaling during the Process of Cardiac Remodeling Following Myocardial Infarction in Ovine Hearts

    PubMed Central

    Gao, Yang; Li, Tieluo; Wu, Changfu; Bittle, Gregory J.; Chen, Shengxi; Wu, Zhongjun J.; Griffith, Bartley P.

    2013-01-01

    Objectives The serine/threonine kinase Pim-1 was recently identified as a cardiomyocyte survival regulator downstream of Akt. The present study aims to examine Pim-1 activity and its association with the post MI remodeling myocardium in a clinically relevant large animal model. Methods Apical myocardial infarction of approximately 25% left ventricular mass was created in an ovine model. Regional post-infarction deformation of the left ventricle was monitored by sonomicrometry and quantified using areal remodeling strain (i.e., areal expansion). Myocardial tissues were harvested at 12 weeks from the adjacent and remote regions of the infarct for analysis of Pim-1 mediated survival signaling proteins as well as apoptotic activity. Results The cDNA coding sequences of two ovine Pim-1 kinase isoforms, 44 and 33 kDa, were identified. Both isoforms were detected in heart tissue and the overall Pim-1 expression was found to be tightly controlled at multiple molecular levels. Pim-1 as well as the Pim-1 mediated survival signaling proteins Bcl-2, Bcl-xL, and phospho-Bad (Ser112), were upregulated in the adjacent region at 12 weeks post-infarction and their expression correlated positively with the degree of the remodeling, which was accompanied by significant upregulations of the PP2A/BAD mediated apoptotic signaling proteins. However these upregulations were imbalanced, such that p-BAD (Ser112)/BAD decreased in the adjacent region of the infarcted hearts. Apoptotic activity also increased with remodeling strain. Conclusions Despite an observed intrinsic upregulation of survival proteins, the imbalanced activation of apoptotic pathways resulted in evident apoptosis in the adjacent region. PMID:23899906

  20. Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway.

    PubMed

    Cai, Lu; Li, Wei; Wang, Guangwu; Guo, Luping; Jiang, Youchun; Kang, Y James

    2002-06-01

    Diabetic cardiomyopathy is related directly to hyperglycemia. Cell death such as apoptosis plays a critical role in cardiac pathogenesis. Whether hyperglycemia induces myocardial apoptosis, leading to diabetic cardiomyopathy, remains unclear. We tested the hypothesis that apoptotic cell death occurs in the diabetic myocardium through mitochondrial cytochrome c-mediated caspase-3 activation pathway. Diabetic mice produced by streptozotocin and H9c2 cardiac myoblast cells exposed to high levels of glucose were used. In the hearts of diabetic mice, apoptotic cell death occurred as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Correspondingly, caspase-3 activation as determined by enzymatic assay and mitochondrial cytochrome c release detected by Western blotting analysis were observed. Supplementation of insulin inhibited diabetes-induced myocardial apoptosis as well as suppressed hyperglycemia. To explore whether apoptosis in diabetic hearts is related directly to hyperglycemia, we exposed cardiac myoblast H9c2 cells to high levels of glucose (22 and 33 mmol/l) in cultures. Apoptotic cell death was detected by TUNEL assay and DAPI nuclear staining. Caspase-3 activation with a concomitant mitochondrial cytochrome c release was also observed. Apoptosis or activation of caspase-3 was not observed in the cultures exposed to the same concentrations of mannitol. Inhibition of caspase-3 with a specific inhibitor, Ac-DEVD-cmk, suppressed apoptosis induced by high levels of glucose. In addition, reactive oxygen species (ROS) generation was detected in the cells exposed to high levels of glucose. These results suggest that hyperglycemia directly induces ap