Science.gov

Sample records for bak mediated apoptosis

  1. Resveratrol induces apoptosis via a Bak-mediated intrinsic pathway in human lung adenocarcinoma cells.

    PubMed

    Zhang, Weiwei; Wang, Xiaoping; Chen, Tongsheng

    2012-05-01

    Our recent study have shown that resveratrol (RV), a natural plant polyphenol found in red grape skins as well as other food product, induced apoptosis via the downstream factors, caspase-independent AIF and to lesser extent caspase-9, of intrinsic apoptosis pathway in human lung adenocarcinoma (ASTC-a-1) cells. This report is designed to explore the roles of the upstream mediators of the intrinsic pathway, such as Bak/Bax, Bim, Puma and Noxa, during RV-induced apoptosis in human lung adenocarcinoma (ASTC-a-1 and A549) cell lines. RV treatment remarkably induced the activation of Bak but not Bax, and silencing Bak but not Bax by shRNA almost completely prevented RV-induced cell death, mitochondrial dysfunction and also largely prevented RV-induced AIF release, demonstrating the preferential engagement of Bak but not Bax during RV-induced apoptosis. In addition, although RV treatment induced a significant degradation of Mcl-1, knockdown of Mcl-1 by shRNA only modestly increased RV-induced Bak activation. Interestingly, silencing Bim but not Puma and Noxa remarkably attenuated RV-induced cell death, loss of mitochondrial membrane potential, and Bak activation, suggesting the important roles of Bim. Collectively, our findings for the first time demonstrate that RV induces apoptosis dominantly via a Bak- but not Bax-mediated AIF-dependent mitochondrial apoptotic signaling pathway in which Bim but not Puma and Noxa may supply the force to trigger Bak activation and subsequent apoptosis in both ASTC-a-1 and A549 cell lines. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. TRIM22-Mediated Apoptosis is Associated with Bak Oligomerization in Monocytes

    PubMed Central

    Chen, Chi; Zhao, DongYan; Fang, Shu; Chen, QiXing; Cheng, BaoLi; Fang, XiangMing; Shu, Qiang

    2017-01-01

    Monocyte apoptosis is a key mechanism that orchestrates host immune responses during sepsis. TRIM22 is constitutively expressed at high levels in monocytes and plays important roles in the antiviral response and inflammation. Overexpression of TRIM22 interferes with the clonogenic growth of monocytic cells, suggesting that TRIM22 may regulate monocyte survival. However, the effect of TRIM22 on monocyte apoptosis remains unknown. In the present report, lipopolysaccharides (LPS)-primed human peripheral blood monocytes expressing higher levels of TRIM22 were more sensitive to apoptosis. This phenomenon was also observed in TRIM22-overexpressing THP-1 monocytes and was associated with the activation of caspase-9 and caspase-3, as well as the increased expression and oligomerization of the pro-apoptotic protein Bak. Similar expression patterns of TRIM22 and Bak were also observed in LPS-primed, apoptotic human peripheral blood monocytes. In addition, the deletion of either the RING domain or the SPRY domain of TRIM22 significantly attenuated TRIM22-mediated monocyte apoptosis and decreased Bak expression and oligomerization. Furthermore, in monocytes from septic patients, TRIM22 levels were down-regulated and positively correlated with Bak levels. Taken together, these results indicate that TRIM22 plays a critical role in monocyte apoptosis by regulating Bak oligomerization and may have a potential function in the pathogenesis of sepsis. PMID:28079123

  3. Bax, Bak and beyond - mitochondrial performance in apoptosis.

    PubMed

    Peña-Blanco, Aida; García-Sáez, Ana J

    2017-07-29

    Bax and Bak are members of the Bcl-2 family and core regulators of the intrinsic pathway of apoptosis. Upon apoptotic stimuli, they are activated and oligomerize at the mitochondrial outer membrane (MOM) to mediate its permeabilization, which is considered a key step in apoptosis. However, the molecular mechanism underlying Bax and Bak function has remained a key question in the field. Here, we review recent structural and biophysical evidence that has changed our understanding of how Bax and Bak promote MOM permeabilization. We also discuss how the spatial regulation of Bcl-2 family preference for binding partners contributes to regulate Bax and Bak activation. Finally, we consider the contribution of mitochondrial composition, dynamics and interaction with other organelles to apoptosis commitment. A new perspective is emerging, in which the control of apoptosis by Bax and Bak goes beyond them and is highly influenced by additional mitochondrial components. © 2017 Federation of European Biochemical Societies.

  4. Cytomegalovirus cell death suppressor vMIA blocks Bax- but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria.

    PubMed

    Arnoult, Damien; Bartle, Laura M; Skaletskaya, Anna; Poncet, Delphine; Zamzami, Naoufal; Park, Peter U; Sharpe, Juanita; Youle, Richard J; Goldmacher, Victor S

    2004-05-25

    We report that the cytomegalovirus-encoded cell death suppressor vMIA binds Bax and prevents Bax-mediated mitochondrial membrane permeabilization by sequestering Bax at mitochondria in the form of a vMIA-Bax complex. vMIA mutants with a defective mitochondria-targeting domain retain their Bax-binding function but not their ability to suppress mitochondrial membrane permeabilization or cell death. vMIA does not seem to either specifically associate with Bak or suppress Bak-mediated mitochondrial membrane permeabilization. Recent evidence suggests that the contribution of Bax and Bak in the mitochondrial apoptotic signaling pathway depends on the distinct phenotypes of cells, and it appears from our data that vMIA is capable of suppressing apoptosis in cells in which this pathway is dominated by Bax, but not in cells where Bak also plays a role.

  5. Bax and Bak Do Not Exhibit Functional Redundancy in Mediating Radiation-Induced Endothelial Apoptosis in the Intestinal Mucosa

    SciTech Connect

    Rotolo, Jimmy A.; Maj, Jerzy G.; Feldman, Regina; Ren, Decheng; Haimovitz-Friedman, Adriana; Cordon-Cardo, Carlos; Cheng, Emily H.-Y.; Kolesnick, Richard; Fuks, Zvi

    2008-03-01

    Purpose: To address in vivo the issue of whether Bax and Bak are functionally redundant in signaling apoptosis, capable of substituting for each other. Methods and Materials: Mice were exposed to whole-body radiation, and endothelial cell apoptosis was quantified using double immunostaining with TUNEL and anti-CD31 antibody. Crypt survival was determined at 3.5 days after whole-body radiation by the microcolony survival assay. Actuarial animal survival was calculated by the product-limit Kaplan-Meier method, and autopsies were performed to establish cause of death. Results: Radiation exposure of Bax- and Bak-deficient mice, both expressing a wild-type acid sphingomyelinase (ASMase) phenotype, indicated that Bax and Bak are both mandatory, though mutually independent, for the intestinal endothelial apoptotic response. However, neither affected epithelial apoptosis at crypt positions 4-5, indicating specificity toward endothelium. Furthermore, Bax deficiency and Bak deficiency each individually mimicked ASMase deficiency in inhibiting crypt lethality in the microcolony assay and in rescuing mice from the lethal gastrointestinal syndrome. Conclusions: The data indicate that Bax and Bak have nonredundant functional roles in the apoptotic response of the irradiated intestinal endothelium. The observation that Bax deficiency and Bak deficiency also protect crypts in the microcolony assay provides strong evidence that the microvascular apoptotic component is germane to the mechanism of radiation-induced damage to mouse intestines, regulating reproductive cell death of crypt stem cell clonogens.

  6. Caspase-dependent apoptosis induction by phenethyl isothiocyanate, a cruciferous vegetable-derived cancer chemopreventive agent, is mediated by Bak and Bax.

    PubMed

    Xiao, Dong; Zeng, Yan; Choi, Sunga; Lew, Karen L; Nelson, Joel B; Singh, Shivendra V

    2005-04-01

    The present study was undertaken to gain insights into the molecular mechanism of apoptosis induction by phenethyl isothiocyanate (PEITC) using prostate cancer cell lines derived from transgenic adenocarcinoma mouse prostate (TRAMP) mice (TRAMP-C1 and TRAMP-C2). The viability of TRAMP-C1 and TRAMP-C2 cells was reduced significantly in the presence of PEITC in a concentration-dependent manner as determined by sulforhodamine B and trypan blue dye exclusion assays. Treatment of TRAMP-derived cells with PEITC revealed features characteristic of apoptosis induction, including appearance of subdiploid cells (determined by flow cytometry), cytoplasmic histone-associated DNA fragmentation (determined by an ELISA assay), and cleavage of caspase-3 (determined by immunoblotting). The PEITC-induced apoptosis in TRAMP-derived cells was associated with a marked increase in the level of proapoptotic protein Bak and/or a decrease in the levels of antiapoptotic protein Mcl-1 or Bcl-xL and disruption of mitochondrial membrane potential. The SV40 immortalized mouse embryonic fibroblasts derived from Bak and Bax double knockout mice were significantly more resistant to PEITC-induced DNA fragmentation compared with wild-type or Bak-/- mouse embryonic fibroblasts. The PEITC-induced apoptosis in both cell lines was significantly attenuated in the presence of caspase inhibitors zVAD-fmk, zLEHD-fmk, and zIETD-fmk. Oral administration of PEITC (9 or 12 micromol PEITC/d, Monday-Friday) significantly retarded growth of TRAMP-C1 xenografts in nude mice without causing weight loss or any other side effects. The results of the present study indicate that caspase-dependent apoptosis by PEITC is mediated by Bak and Bax proteins.

  7. Bak and Bax Function To Limit Adenovirus Replication through Apoptosis Induction

    PubMed Central

    Cuconati, Andrea; Degenhardt, Kurt; Sundararajan, Ramya; Anschel, Alan; White, Eileen

    2002-01-01

    Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B 19K. The mechanism of apoptosis induction and the role that it plays in productive infection are not known. Unlike apoptosis mediated by death receptors, infection with proapoptotic E1B 19K mutant viruses did not induce cleavage of Bid but nonetheless induced changes in Bak and Bax conformation, Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B 19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax conformation. Infection with E1B 19K mutant viruses induced apoptosis in wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically increased E1A expression and virus replication. Thus, Bax- and Bak-mediated apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak function as an antiviral response at the cellular level. PMID:11932420

  8. Bak and Bax function to limit adenovirus replication through apoptosis induction.

    PubMed

    Cuconati, Andrea; Degenhardt, Kurt; Sundararajan, Ramya; Anschel, Alan; White, Eileen

    2002-05-01

    Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B 19K. The mechanism of apoptosis induction and the role that it plays in productive infection are not known. Unlike apoptosis mediated by death receptors, infection with proapoptotic E1B 19K mutant viruses did not induce cleavage of Bid but nonetheless induced changes in Bak and Bax conformation, Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B 19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax conformation. Infection with E1B 19K mutant viruses induced apoptosis in wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically increased E1A expression and virus replication. Thus, Bax- and Bak-mediated apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak function as an antiviral response at the cellular level.

  9. JNK-Bcl-2/Bcl-xL-Bax/Bak Pathway Mediates the Crosstalk between Matrine-Induced Autophagy and Apoptosis via Interplay with Beclin 1.

    PubMed

    Yang, Jiong; Yao, Shukun

    2015-10-27

    Autophagy is associated with drug resistance which has been a threat in chemotherapy of hepatocellular carcinoma (HCC). The interconnected molecular regulators between autophagy and apoptosis serve as switching points critical to the ultimate outcome of the cell. Our study was performed to investigate the crosstalk between autophagy and apoptosis in HCC after the treatment of matrine. Flow cytometry and TUNEL (terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling) assay were used to detect apoptosis in vitro and in vivo, respectively. Bax oligomerization and Cytochrome c release assay were performed. Immunoprecipitation and siRNA transfection were used to detect the interplay between Bcl-2/Bcl-xL,Bax, and Beclin 1. Our results showed that: (1) matrine not only activated caspase and PARP (poly ADP-ribose polymerase) cleavage, but also triggered autophagy as shown by the increased levels of LC3II, Beclin 1, and PI3KC3, and the decreased level of p62; (2) matrine treatment promoted the JNK-Bcl-2/ Bcl-xL-Bax/Bak pathway; (3) Bax was oligomerized, the mitochondrial membrane potential altered, and Cytochrome c was released subsequently; (4) Bax interacts with Beclin 1 and inhibits autophagy, which may be a new crosstalk point; and (5) finally, we showed that matrine suppressed the growth of a MHCC97L xenograft in vivo for the first time. In conclusion, the JNK-Bcl-2/Bcl-xL-Bax/Bak pathway mediates the crosstalk between matrine-induced autophagy and apoptosis via interplay with Beclin 1.

  10. JNK-Bcl-2/Bcl-xL-Bax/Bak Pathway Mediates the Crosstalk between Matrine-Induced Autophagy and Apoptosis via Interplay with Beclin 1

    PubMed Central

    Yang, Jiong; Yao, Shukun

    2015-01-01

    Autophagy is associated with drug resistance which has been a threat in chemotherapy of hepatocellular carcinoma (HCC). The interconnected molecular regulators between autophagy and apoptosis serve as switching points critical to the ultimate outcome of the cell. Our study was performed to investigate the crosstalk between autophagy and apoptosis in HCC after the treatment of matrine. Flow cytometry and TUNEL (terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling) assay were used to detect apoptosis in vitro and in vivo, respectively. Bax oligomerization and Cytochrome c release assay were performed. Immunoprecipitation and siRNA transfection were used to detect the interplay between Bcl-2/Bcl-xL,Bax, and Beclin 1. Our results showed that: (1) matrine not only activated caspase and PARP (poly ADP-ribose polymerase) cleavage, but also triggered autophagy as shown by the increased levels of LC3II, Beclin 1, and PI3KC3, and the decreased level of p62; (2) matrine treatment promoted the JNK-Bcl-2/Bcl-xL-Bax/Bak pathway; (3) Bax was oligomerized, the mitochondrial membrane potential altered, and Cytochrome c was released subsequently; (4) Bax interacts with Beclin 1 and inhibits autophagy, which may be a new crosstalk point; and (5) finally, we showed that matrine suppressed the growth of a MHCC97L xenograft in vivo for the first time. In conclusion, the JNK-Bcl-2/Bcl-xL-Bax/Bak pathway mediates the crosstalk between matrine-induced autophagy and apoptosis via interplay with Beclin 1. PMID:26516844

  11. A New Fungal Diterpene Induces VDAC1-dependent Apoptosis in Bax/Bak-deficient Cells.

    PubMed

    Huang, Li; Han, Junjie; Ben-Hail, Danya; He, Luwei; Li, Baowei; Chen, Ziheng; Wang, Yueying; Yang, Yanlei; Liu, Lei; Zhu, Yushan; Shoshan-Barmatz, Varda; Liu, Hongwei; Chen, Quan

    2015-09-25

    The pro-apoptotic Bax and Bak proteins are considered central to apoptosis, yet apoptosis occurs in their absence. Here, we asked whether the mitochondrial protein VDAC1 mediates apoptosis independently of Bax/Bak. Upon screening a fungal secondary metabolite library for compounds inducing apoptosis in Bax/Bak-deficient mouse embryonic fibroblasts, we identified cyathin-R, a new cyathane diterpenoid compound able to activate apoptosis in the absence of Bax/Bak via promotion of the VDAC1 oligomerization that mediates cytochrome c release. Diphenylamine-2-carboxilic acid, an inhibitor of VDAC1 conductance and oligomerization, inhibited cyathin-R-induced VDAC1 oligomerization and apoptosis. Similarly, Bcl-2 overexpression conferred resistance to cyathin-R-induced apoptosis and VDAC1 oligomerization. Silencing of VDAC1 expression prevented cyathin-R-induced apoptosis. Finally, cyathin-R effectively attenuated tumor growth and induced apoptosis in Bax/Bak-deficient cells implanted into a xenograft mouse model. Hence, this study identified a new compound promoting VDAC1-dependent apoptosis as a potential therapeutic option for cancerous cells lacking or presenting inactivated Bax/Bak. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Spontaneous and drug-induced apoptosis is mediated by conformational changes of Bax and Bak in B-cell chronic lymphocytic leukemia.

    PubMed

    Bellosillo, Beatriz; Villamor, Neus; López-Guillermo, Armando; Marcé, Silvia; Bosch, Francesc; Campo, Elias; Montserrat, Emili; Colomer, Dolors

    2002-09-01

    The role of Bax and Bak, 2 proapoptotic proteins of the Bcl-2 family, was analyzed in primary B-cell chronic lymphocytic leukemia (CLL) cells following in vitro treatment with fludarabine, dexamethasone, or the combination of fludarabine with cyclophosphamide and mitoxantrone (FCM). A strong correlation was found between the number of apoptotic cells and the percentage of cells stained with antibodies recognizing conformational changes of Bax (n = 33; r = 0.836; P <.001) or Bak (n = 10; r = 0.948; P <.001). Preincubation of CLL cells with Z-VAD.fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone), a broad caspase inhibitor, abolished caspase-3 activation, exposure of phosphatidylserine residues, and reactive oxygen species generation; partially reversed the loss of transmembrane mitochondrial potential (DeltaPsim); but did not affect Bax or Bak conformational changes. These results indicate that the conformational changes of Bax and Bak occur upstream of caspase activation or are caspase independent. Following drug-induced apoptosis, Bax integrates into mitochondria, as demonstrated by fluorescence microscopy and Western blot, without changes in the total amount of Bax or Bak protein. Fludarabine and FCM induce p53 stabilization, but do not seem to be essential in inducing Bax and Bak conformational changes, as they are also observed in dexamethasone-treated CLL cells. These results demonstrate that, in CLL cells, the change in the intracellular localization of Bax from cytosol to mitochondria and the conformational changes of Bax and Bak are among the early steps in the induction of cell death.

  13. Artemisinin induces caspase-8/9-mediated and Bax/Bak-independent apoptosis in human lung adenocarcinoma (ASTC-a-1) cells.

    PubMed

    Xiao, Feng-Lian; Gao, Wei-Jie; Liu, Cheng-Yi; Wang, Xiao-Ping; Chen, Tong-Sheng

    2011-01-01

    Artemisinin (ARTE), an antimalarial phytochemical component from the sweet wormwood plant, has been shown a potential anticancer activity by inducing cell apoptosis. The aim of this report is to explore the mechanism of ARTE-induced human lung adenocarcinoma (ASTC-a-1) cell apoptosis. Cell counting kit (CCK-8) assay showed that ARTE induced cytotoxcity in a dose- and time-dependent manner. Confocal microscopy fluorescence imaging of cells stained with Hoechst 33258 and flow cytometry (FCM) analysis of cells stained with Annexin V-FITC/propidium iodide (PI) showed that ARTE induced reactive oxygen species (ROS)-dependent apoptosis. Confocal fluorescence resonance energy transfer (FRET) imaging of single living cells expressing SCAT3, SCAT9 or CFP-Bid-YFP and fluorometic substrate assay showed that ARTE induced the activation of caspase-3, -8 and -9. Moreover, inhibition of caspase-8 or -9 completely blocked ARTE-induced apoptosis which was only partially attenuated by caspase-3 inhibitor. Interestingly, silencing Bax and Bak by RNA interference (RNAi) did not attenuate ARTE-induced apoptosis. Collectively, ARTE induces caspase-dependent but Bax/Bak-independent apoptosis in ASTC-a-1 cells.

  14. Sevoflurane post-conditioning protects primary rat cortical neurons against oxygen-glucose deprivation/resuscitation via down-regulation in mitochondrial apoptosis axis of Bid, Bim, Puma-Bax and Bak mediated by Erk1/2.

    PubMed

    Zhang, Li-Min; Zhao, Xiao-Chun; Sun, Wen-Bo; Li, Rui; Jiang, Xiao-Jing

    2015-10-15

    Temporal post-conditioning helps provide neuroprotection against brain injury secondary to ischemia-reperfusion and is considered an effective intervention, but the exact mechanism of sevoflurane post-conditioning is unclear. The essential axis involves activator Bid, Bim, Puma (BH3s), Bax, and Bak; activates the mitochondrial death program; and might be involved in a cell death signal. Extracellular signal-related kinases 1/2 (Erk1/2) play a pivotal role in cell growth and proliferation. We hypothesized that sevoflurane post-conditioning might inhibit Bid, Bim, Puma, Bax, and Bak expression and is activated by phosphor-Erk1/2 to decrease neuronal death. To test this hypothesis, we exposed primary cortical neuron cultures to oxygen-glucose deprivation for 1h, along with resuscitation for 24h (OGD/R). MTT assays, propidium iodide uptake (PI), JC-1 fluorescence, and Western blot indicated the following: decreased cell viability (P<0.05); increased cell death (P<0.05); decreased mitochondrial membrane potential (P<0.05); and decreased Bid, Bim, Puma, Bax, and Bak expression with OGD/R exposure. Inhibition of Erk1/2 phosphorylation could attenuate sevoflurane post-conditioning that mediated an increase in neuronal viability and mitochondrial membrane potential, as well as a decrease in cell death and Bid, Bim, Puma, Bax, and Bak expression after OGD/R treatment. The results demonstrated that sevoflurane post-conditioning caused a marked decrease in cortical neuronal death secondary to OGD/R exposure through the downregulation of the mitochondrial apoptosis axis involving Bid, Bim, Puma, Bax, and Bak that was mediated by the phosphorylation/activation of Erk1/2.

  15. Disordered clusters of Bak dimers rupture mitochondria during apoptosis

    PubMed Central

    Uren, Rachel T; O’Hely, Martin; Iyer, Sweta; Bartolo, Ray; Shi, Melissa X; Brouwer, Jason M; Alsop, Amber E; Dewson, Grant; Kluck, Ruth M

    2017-01-01

    During apoptosis, Bak and Bax undergo major conformational change and form symmetric dimers that coalesce to perforate the mitochondrial outer membrane via an unknown mechanism. We have employed cysteine labelling and linkage analysis to the full length of Bak in mitochondria. This comprehensive survey showed that in each Bak dimer the N-termini are fully solvent-exposed and mobile, the core is highly structured, and the C-termini are flexible but restrained by their contact with the membrane. Dimer-dimer interactions were more labile than the BH3:groove interaction within dimers, suggesting there is no extensive protein interface between dimers. In addition, linkage in the mobile Bak N-terminus (V61C) specifically quantified association between dimers, allowing mathematical simulations of dimer arrangement. Together, our data show that Bak dimers form disordered clusters to generate lipidic pores. These findings provide a molecular explanation for the observed structural heterogeneity of the apoptotic pore. DOI: http://dx.doi.org/10.7554/eLife.19944.001 PMID:28182867

  16. Synthetic Bichalcone TSWU-BR23 Induces Apoptosis of Human Colon Cancer HT-29 Cells by p53-Mediated Mitochondrial Oligomerization of BAX/BAK and Lipid Raft Localization of CD95/FADD.

    PubMed

    Lin, Meng-Liang; Chen, Shih-Shun; Wu, Tian-Shung

    2015-10-01

    A synthetic bichalcone analog, (E)-1-(3-((4-(4-acetylphenyl)piperazin-1-yl)methyl)-4-hydroxy-5-methoxyphenyl)-3-(pyridin-3-yl)prop-2-en-1-one (TSWU-BR23), has been shown to induce apoptosis in human colon cancer HT-29 cells involving the induction of CD95 and FAS-associated protein death domain (FADD), but its precise mechanism of action has not been fully elucidated. Using cell-surface biotinylation and sucrose density-gradient-based membrane flotation techniques, we showed that the disruption of TSWU-BR23-induced lipid raft localization of CD95/FADD by cholesterol-depleting agent (methyl-β-cyclodextrin) was reversed by cholesterol replenishment. Blockade of p53 expression by short-hairpin RNA (shRNA) suppressed oligomeric Bcl-2-associated x protein (BAX)/Bcl-2 antagonist killer 1 (BAK)-mediated mitochondrial apoptosis but did not inhibit lipid raft localization of CD95/FADD and pro-caspase-8 cleavage induced by TSWU-BR23. Co-expression of p53 shRNA and dominant-negative mutant of FADD completely inhibited TSWU-BR32-induced mitochondrial apoptotic cell death. Collectively, these data demonstrate that TSWU-BR23 leads to HT-29 cell apoptosis by inducing p53-mediated mitochondrial oligomerization of BAX/BAK and the localization of CD95/FADD with lipid rafts at the cell surface. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  17. mRNA for N-Bak, a neuron-specific BH3-only splice isoform of Bak, escapes nonsense-mediated decay and is translationally repressed in the neurons

    PubMed Central

    Jakobson, M; Lintulahti, A; Arumäe, U

    2012-01-01

    mRNA for neuronal Bak (N-Bak), a splice variant of pro-apoptotic Bcl-2 family member Bak is expressed in the neurons. Surprisingly the endogeneous N-Bak protein cannot be demonstrated in the neurons, although the antibodies recognize N-Bak protein from in vitro translation or transiently transfected cells. As N-Bak mRNA contains premature termination codon (PTC) at 89 nucleotides upstream from the last exon–exon junction, it could be degraded by nonsense-mediated decay (NMD) during the pioneer round of translation thus explaining the absence of the protein. We show here that the endogeneous neuronal N-Bak mRNA is not the NMD substrate, as it is not accumulating by cycloheximide treatment, it has a long lifetime, and even prevention of PTC by interfering with the alternative splicing did not lead to translation of the Bak mRNA. N-Bak protein is also not revealed by proteasome inhibitors. Our data suggest strong translational arrest of N-Bak mRNA in the neurons. We show that this arrest is partially mediated by 5′-untranslated region of Bak mRNA and it is not released during mitochondrial apoptosis. PMID:22297299

  18. mRNA for N-Bak, a neuron-specific BH3-only splice isoform of Bak, escapes nonsense-mediated decay and is translationally repressed in the neurons.

    PubMed

    Jakobson, M; Lintulahti, A; Arumäe, U

    2012-02-02

    mRNA for neuronal Bak (N-Bak), a splice variant of pro-apoptotic Bcl-2 family member Bak is expressed in the neurons. Surprisingly the endogeneous N-Bak protein cannot be demonstrated in the neurons, although the antibodies recognize N-Bak protein from in vitro translation or transiently transfected cells. As N-Bak mRNA contains premature termination codon (PTC) at 89 nucleotides upstream from the last exon-exon junction, it could be degraded by nonsense-mediated decay (NMD) during the pioneer round of translation thus explaining the absence of the protein. We show here that the endogeneous neuronal N-Bak mRNA is not the NMD substrate, as it is not accumulating by cycloheximide treatment, it has a long lifetime, and even prevention of PTC by interfering with the alternative splicing did not lead to translation of the Bak mRNA. N-Bak protein is also not revealed by proteasome inhibitors. Our data suggest strong translational arrest of N-Bak mRNA in the neurons. We show that this arrest is partially mediated by 5'-untranslated region of Bak mRNA and it is not released during mitochondrial apoptosis.

  19. Paclitaxel-Induced Apoptosis Is BAK-Dependent, but BAX and BIM-Independent in Breast Tumor

    PubMed Central

    Nakajima, Wataru; Richardson, Amanda C.; Windle, Jolene J.; Harada, Hisashi

    2013-01-01

    Paclitaxel (Taxol)-induced cell death requires the intrinsic cell death pathway, but the specific participants and the precise mechanisms are poorly understood. Previous studies indicate that a BH3-only protein BIM (BCL-2 Interacting Mediator of cell death) plays a role in paclitaxel-induced apoptosis. We show here that BIM is dispensable in apoptosis with paclitaxel treatment using bim−/− MEFs (mouse embryonic fibroblasts), the bim−/− mouse breast tumor model, and shRNA-mediated down-regulation of BIM in human breast cancer cells. In contrast, both bak−/− MEFs and human breast cancer cells in which BAK was down-regulated by shRNA were more resistant to paclitaxel. However, paclitaxel sensitivity was not affected in bax−/− MEFs or in human breast cancer cells in which BAX was down-regulated, suggesting that paclitaxel-induced apoptosis is BAK-dependent, but BAX-independent. In human breast cancer cells, paclitaxel treatment resulted in MCL-1 degradation which was prevented by a proteasome inhibitor, MG132. A Cdk inhibitor, roscovitine, blocked paclitaxel-induced MCL-1 degradation and apoptosis, suggesting that Cdk activation at mitotic arrest could induce subsequent MCL-1 degradation in a proteasome-dependent manner. BAK was associated with MCL-1 in untreated cells and became activated in concert with loss of MCL-1 expression and its release from the complex. Our data suggest that BAK is the mediator of paclitaxel-induced apoptosis and could be an alternative target for overcoming paclitaxel resistance. PMID:23577147

  20. Phylogenetically Distant Viruses Use the Same BH3-Only Protein Puma to Trigger Bax/Bak-Dependent Apoptosis of Infected Mouse and Human Cells.

    PubMed

    Papaianni, Emanuela; El Maadidi, Souhayla; Schejtman, Andrea; Neumann, Simon; Maurer, Ulrich; Marino-Merlo, Francesca; Mastino, Antonio; Borner, Christoph

    2015-01-01

    Viruses can trigger apoptosis of infected host cells if not counteracted by cellular or viral anti-apoptotic proteins. These protective proteins either inhibit the activation of caspases or they act as Bcl-2 homologs to prevent Bax/Bak-mediated outer mitochondrial membrane permeabilization (MOMP). The exact mechanism by which viruses trigger MOMP has however remained enigmatic. Here we use two distinct types of viruses, a double stranded DNA virus, herpes simplex virus-1 (HSV-1) and a positive sense, single stranded RNA virus, Semliki Forest virus (SFV) to show that the BH3-only protein Puma is the major mediator of virus-induced Bax/Bak activation and MOMP induction. Indeed, when Puma was genetically deleted or downregulated by shRNA, mouse embryonic fibroblasts and IL-3-dependent monocytes as well as human colon carcinoma cells were as resistant to virus-induced apoptosis as their Bax/Bak double deficient counterparts (Bax/Bak-/-). Puma protein expression started to augment after 2 h postinfection with both viruses. Puma mRNA levels increased as well, but this occurred after apoptosis initiation (MOMP) because it was blocked in cells lacking Bax/Bak or overexpressing Bcl-xL. Moreover, none of the classical Puma transcription factors such as p53, p73 or p65 NFκB were involved in HSV-1-induced apoptosis. Our data suggest that viruses use a Puma protein-dependent mechanism to trigger MOMP and apoptosis in host cells.

  1. Phylogenetically Distant Viruses Use the Same BH3-Only Protein Puma to Trigger Bax/Bak-Dependent Apoptosis of Infected Mouse and Human Cells

    PubMed Central

    Papaianni, Emanuela; El Maadidi, Souhayla; Schejtman, Andrea; Neumann, Simon; Maurer, Ulrich; Marino-Merlo, Francesca; Mastino, Antonio; Borner, Christoph

    2015-01-01

    Viruses can trigger apoptosis of infected host cells if not counteracted by cellular or viral anti-apoptotic proteins. These protective proteins either inhibit the activation of caspases or they act as Bcl-2 homologs to prevent Bax/Bak-mediated outer mitochondrial membrane permeabilization (MOMP). The exact mechanism by which viruses trigger MOMP has however remained enigmatic. Here we use two distinct types of viruses, a double stranded DNA virus, herpes simplex virus-1 (HSV-1) and a positive sense, single stranded RNA virus, Semliki Forest virus (SFV) to show that the BH3-only protein Puma is the major mediator of virus-induced Bax/Bak activation and MOMP induction. Indeed, when Puma was genetically deleted or downregulated by shRNA, mouse embryonic fibroblasts and IL-3-dependent monocytes as well as human colon carcinoma cells were as resistant to virus-induced apoptosis as their Bax/Bak double deficient counterparts (Bax/Bak-/-). Puma protein expression started to augment after 2 h postinfection with both viruses. Puma mRNA levels increased as well, but this occurred after apoptosis initiation (MOMP) because it was blocked in cells lacking Bax/Bak or overexpressing Bcl-xL. Moreover, none of the classical Puma transcription factors such as p53, p73 or p65 NFκB were involved in HSV-1-induced apoptosis. Our data suggest that viruses use a Puma protein-dependent mechanism to trigger MOMP and apoptosis in host cells. PMID:26030884

  2. MCMV-mediated Inhibition of the Pro-apoptotic Bak Protein Is Required for Optimal In Vivo Replication

    PubMed Central

    Fleming, Peter; Kvansakul, Marc; Voigt, Valentina; Kile, Benjamin T.; Kluck, Ruth M.; Huang, David C. S.; Degli-Esposti, Mariapia A.; Andoniou, Christopher E.

    2013-01-01

    Successful replication and transmission of large DNA viruses such as the cytomegaloviruses (CMV) family of viruses depends on the ability to interfere with multiple aspects of the host immune response. Apoptosis functions as a host innate defence mechanism against viral infection, and the capacity to interfere with this process is essential for the replication of many viruses. The Bcl-2 family of proteins are the principle regulators of apoptosis, with two pro-apoptotic members, Bax and Bak, essential for apoptosis to proceed. The m38.5 protein encoded by murine CMV (MCMV) has been identified as Bax-specific inhibitor of apoptosis. Recently, m41.1, a protein product encoded by the m41 open reading frame (ORF) of MCMV, has been shown to inhibit Bak activity in vitro. Here we show that m41.1 is critical for optimal MCMV replication in vivo. Growth of a m41.1 mutant was attenuated in multiple organs, a defect that was not apparent in Bak−/− mice. Thus, m41.1 promotes MCMV replication by inhibiting Bak-dependent apoptosis during in vivo infection. The results show that Bax and Bak mediate non-redundant functions during MCMV infection and that the virus produces distinct inhibitors for each protein to counter the activity of these proteins. PMID:23468630

  3. Epigenetic control of mitochondrial cell death through PACS1-mediated regulation of BAX/BAK oligomerization.

    PubMed

    Brasacchio, Daniella; Alsop, Amber E; Noori, Tahereh; Lufti, Mariam; Iyer, Sweta; Simpson, Kaylene J; Bird, Phillip I; Kluck, Ruth M; Johnstone, Ricky W; Trapani, Joseph A

    2017-06-01

    PCAF and ADA3 associate within the same macromolecular complexes to control the transcription of many genes, including some that regulate apoptosis. Here we show that PCAF and ADA3 regulate the expression of PACS1, whose protein product is a key component of the machinery that sorts proteins among the trans-Golgi network and the endosomal compartment. We describe a novel role for PACS1 as a regulator of the intrinsic pathway of apoptosis and mitochondrial outer membrane permeabilization. Cells with decreased PACS1 expression were refractory to cell death mediated by a variety of stimuli that operate through the mitochondrial pathway, including human granzyme B, staurosporine, ultraviolet radiation and etoposide, but remained sensitive to TRAIL receptor ligation. The mitochondria of protected cells failed to release cytochrome c as a result of perturbed oligomerization of BAX and BAK. We conclude that PCAF and ADA3 transcriptionally regulate PACS1 and that PACS1 is a key regulator of BAX/BAK oligomerization and the intrinsic (mitochondrial) pathway to apoptosis.

  4. Expression levels of the BAK1 and BCL2 genes highlight the role of apoptosis in age-related hearing impairment

    PubMed Central

    Falah, Masoumeh; Najafi, Mohammad; Houshmand, Massoud; Farhadi, Mohammad

    2016-01-01

    Age-related hearing impairment (ARHI) is a progressive and a common sensory disorder in the elderly and will become an increasingly important clinical problem given the growing elderly population. Apoptosis of cochlear cells is an important factor in animal models of ARHI. As these cells cannot regenerate, their loss leads to irreversible hearing impairment. Identification of molecular mechanisms can facilitate disease prevention and effective treatment. In this study, we compared the expression of the genes BAK1 and BCL2 as two arms of the intrinsic apoptosis pathway between patients with ARHI and healthy subjects. ARHI and healthy subjects were selected after an ear nose throat examination, otoscopic investigation, and pure tone audiometry. RNA was extracted from peripheral blood samples, and relative gene expression levels were measured using quantitative real-time polymerase chain reaction. BAK1 and the BAK1/BCL2 ratio were statistically significantly upregulated in the ARHI subjects. The BAK1/BCL2 ratio was positively correlated with the results of the audiometric tests. Our results indicate that BAK-mediated apoptosis may be a core mechanism in the progression of ARHI in humans, similar to finding in animal models. Moreover, the gene expression changes in peripheral blood samples could be used as a rapid and simple biomarker for early detection of ARHI. PMID:27555755

  5. Stepwise activation of BAX and BAK by tBID, BIM, and PUMA initiates mitochondrial apoptosis.

    PubMed

    Kim, Hyungjin; Tu, Ho-Chou; Ren, Decheng; Takeuchi, Osamu; Jeffers, John R; Zambetti, Gerard P; Hsieh, James J-D; Cheng, Emily H-Y

    2009-11-13

    While activation of BAX/BAK by BH3-only molecules (BH3s) is essential for mitochondrial apoptosis, the underlying mechanisms remain unsettled. Here we demonstrate that BAX undergoes stepwise structural reorganization leading to mitochondrial targeting and homo-oligomerization. The alpha1 helix of BAX keeps the alpha9 helix engaged in the dimerization pocket, rendering BAX as a monomer in cytosol. The activator BH3s, tBID/BIM/PUMA, attack and expose the alpha1 helix of BAX, resulting in secondary disengagement of the alpha9 helix and thereby mitochondrial insertion. Activator BH3s remain associated with the N-terminally exposed BAX through the BH1 domain to drive homo-oligomerization. BAK, an integral mitochondrial membrane protein, has bypassed the first activation step, explaining why its killing kinetics are faster than those of BAX. Furthermore, death signals initiated at ER induce BIM and PUMA to activate mitochondrial apoptosis. Accordingly, deficiency of Bim/Puma impedes ER stress-induced BAX/BAK activation and apoptosis. Our study provides mechanistic insights regarding the spatiotemporal execution of BAX/BAK-governed cell death.

  6. Prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim reduces the association of Bcl-2 with Bak or Bim, provoking Bak activation and mitochondrial apoptosis in nocodazole-treated Jurkat T cells.

    PubMed

    Han, Cho Rong; Jun, Do Youn; Lee, Ji Young; Kim, Young Ho

    2014-01-01

    Treatment of Jurkat T cells with the microtubule-depolymerizing agent nocodazole (NOC) caused prometaphase arrest and apoptosis. NOC-induced mitochondrial apoptotic events including Bak activation, Δψm loss, cytochrome c release, and caspase cascade activation were blocked by Bcl-2 overexpression. However, mitotic arrest, Cdc25C activation, upregulation of cyclin B1 levels, Cdk1 activation, Bcl-2 phosphorylation at Thr-56 and Ser-70, and Bim phosphorylation were retained. The treatment of Jurkat T cells concomitantly with NOC and the G1/S-blocking agent hydroxyurea resulted in G1/S arrest and complete abrogation of all apoptotic events. The association of Bcl-2 with Bim or Bak declined after the prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim, whereas the association of Bcl-2 with Bax remained relatively constant. Although Bax was redistributed from the cytosol to the mitochondria, resulting in an increase in the mitochondrial level of Bax following NOC treatment, the subcellular localization of Bcl-2, Bim, Bak and apoptosis-inducing factor was confined to the mitochondrial fraction irrespective of NOC treatment. Experiments using selective caspase inhibitors showed that mitochondria-dependent activation of caspase-9 and -3 was crucial for NOC-induced apoptosis. NOC-induced phosphorylation of Bcl-2 and Bim, Δψm loss, and mitochondria-dependent apoptotic events were significantly suppressed by a Cdk1 inhibitor roscovitine, but not by the JNK inhibitor SP600125 or the p38 MAPK inhibitor SB203580. These results show that the prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim, which was mediated by Cdk1, could reduce the association of Bcl-2 with Bak or Bim to allow Bak activation and mitochondrial apoptotic events in Jurkat T cells exposed to NOC.

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

    PubMed

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

    2015-08-01

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

  8. Motifs of VDAC2 required for mitochondrial Bak import and tBid-induced apoptosis

    PubMed Central

    Naghdi, Shamim; Várnai, Péter; Hajnóczky, György

    2015-01-01

    Voltage-dependent anion channel (VDAC) proteins are major components of the outer mitochondrial membrane. VDAC has three isoforms with >70% sequence similarity and redundant roles in metabolite and ion transport. However, only Vdac2−/− (V2−/−) mice are embryonic lethal, indicating a unique and fundamental function of VDAC2 (V2). Recently, a specific V2 requirement was demonstrated for mitochondrial Bak import and truncated Bid (tBid)-induced apoptosis. To determine the relevant domain(s) of V2 involved, VDAC1 (V1) and V2 chimeric constructs were created and used to rescue V2−/− fibroblasts. Surprisingly, the commonly cited V2-specific N-terminal extension and cysteines were found to be dispensable for Bak import and high tBid sensitivity. In gain-of-function studies, V2 (123–179) was the minimal sequence sufficient to render V1 competent to support Bak insertion. Furthermore, in loss-of-function experiments, T168 and D170 were identified as critical residues. These motifs are conserved in zebrafish V2 (zfV2) that also rescued V2-deficient fibroblasts. Because high-resolution structures of zfV2 and mammalian V1 have become available, we could superimpose these structures and recognized that the critical V2-specific residues help to create a distinctive open “pocket” on the cytoplasmic surface that could facilitate Bak recruitment. PMID:26417093

  9. Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis

    PubMed Central

    Westphal, D; Kluck, R M; Dewson, G

    2014-01-01

    The central role of the Bcl-2 family in regulating apoptotic cell death was first identified in the 1980s. Since then, significant in-roads have been made in identifying the multiple members of this family, characterizing their form and function and understanding how their interactions determine whether a cell lives or dies. In this review we focus on the recent progress made in characterizing the proapoptotic Bcl-2 family members, Bax and Bak. This progress has resolved longstanding controversies, but has also challenged established theories in the apoptosis field. We will discuss different models of how these two proteins become activated and different ‘modes' by which they are inhibited by other Bcl-2 family members. We will also discuss novel conformation changes leading to Bak and Bax oligomerization and speculate how these oligomers might permeabilize the mitochondrial outer membrane. PMID:24162660

  10. Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis.

    PubMed

    Westphal, D; Kluck, R M; Dewson, G

    2014-02-01

    The central role of the Bcl-2 family in regulating apoptotic cell death was first identified in the 1980s. Since then, significant in-roads have been made in identifying the multiple members of this family, characterizing their form and function and understanding how their interactions determine whether a cell lives or dies. In this review we focus on the recent progress made in characterizing the proapoptotic Bcl-2 family members, Bax and Bak. This progress has resolved longstanding controversies, but has also challenged established theories in the apoptosis field. We will discuss different models of how these two proteins become activated and different 'modes' by which they are inhibited by other Bcl-2 family members. We will also discuss novel conformation changes leading to Bak and Bax oligomerization and speculate how these oligomers might permeabilize the mitochondrial outer membrane.

  11. OCT4 promotes tumorigenesis and inhibits apoptosis of cervical cancer cells by miR-125b/BAK1 pathway

    PubMed Central

    Wang, Y-D; Cai, N; Wu, X-L; Cao, H-Z; Xie, L-L; Zheng, P-S

    2013-01-01

    Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene that maintains the pluripotency and self-renewal properties of embryonic stem cells. Although there is emerging evidence that it can function as oncogene in several cancers, the role in mediating cervical cancer remains unexplored. Here we found that OCT4 protein expression showed a pattern of gradual increase from normal cervix to cervical carcinoma in situ and then to invasive cervical cancer. Overexpression of OCT4 in two types of cervical cancer cells promotes the carcinogenesis, and inhibits cancer cell apoptosis. OCT4 induces upregulation of miR-125b through directly binding to the promoter of miR-125b-1 confirmed by chromatin immunoprecipitation analysis. MiRNA-125b overexpression suppressed apoptosis and expression of BAK1 protein. In contrast, miR-125b sponge impaired the anti-apoptotic effect of OCT4, along with the upregulated expression of BAK1. Significantly, Luciferase assay showed that the activity of the wild-type BAK1 3′-untranslated region reporter was suppressed and this suppression was diminished when the miR-125b response element was mutated or deleted. In addition, we observed negative correlation between levels of BAK1 and OCT4, and positive between OCT4 and miR-125b in primary cervical cancers. These findings suggest an undescribed regulatory pathway in cervical cancer, by which OCT4 directly induces expression of miR-125b, which inhibits its direct target BAK1, leading to suppression of cervical cancer cell apoptosis. PMID:23928699

  12. Bz-423 superoxide signals B cell apoptosis via Mcl-1, Bak, and Bax.

    PubMed

    Blatt, Neal B; Boitano, Anthony E; Lyssiotis, Costas A; Opipari, Anthony W; Glick, Gary D

    2009-10-15

    Bz-423 is a pro-apoptotic 1,4-benzodiazepine with therapeutic properties in murine models of lupus demonstrating selectivity for autoreactive lymphocytes. Bz-423 modulates the F(1)F(0)-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. In order to understand some of the features that contribute to the increased sensitivity of lymphocytes, we report the signaling pathway engaged by Bz-423 in a Burkitt lymphoma cell line (Ramos). Following the generation of superoxide, Bz-423-induced apoptosis requires the activation of Bax and Bak to induce mitochondrial outer membrane permeabilization and cytochrome c release. Knockdown of the BH3-only proteins Bad, Bim, Bik, and Puma inhibits Bz-423 apoptosis, suggesting that these proteins serve as upstream sensors of the oxidant stress induced by Bz-423. Treatment with Bz-423 results in superoxide-dependent Mcl-1 degradation, implicating this protein as the link between Bz-423-induced superoxide and Bax and Bak activation. In contrast to fibroblasts, B cell death induced by Bz-423 is independent of c-Jun N-terminal kinase. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a specific apoptotic response that differs across cell types.

  13. Double knockout of Bax and Bak from kidney proximal tubules reduces unilateral urethral obstruction associated apoptosis and renal interstitial fibrosis

    PubMed Central

    Mei, Shuqin; Li, Lin; Wei, Qingqing; Hao, Jielu; Su, Yunchao; Mei, Changlin; Dong, Zheng

    2017-01-01

    Interstitial fibrosis, a common pathological feature of chronic kidney diseases, is often associated with apoptosis in renal tissues. To determine the associated apoptotic pathway and its role in renal interstitial fibrosis, we established a mouse model in which Bax and Bak, two critical genes in the intrinsic pathway of apoptosis, were deleted specifically from kidney proximal tubules and used this model to examine renal apoptosis and interstitial fibrosis following unilateral urethral obstruction (UUO). It was shown that double knockout of Bax and Bak from proximal tubules attenuated renal tubular cell apoptosis and suppressed renal interstitial fibrosis in UUO. The results indicate that the intrinsic pathway of apoptosis contributes significantly to the tubular apoptosis and renal interstitial fibrosis in kidney diseases. PMID:28317867

  14. Ethanolic extract of Tulipa edulis Bak induces apoptosis in SGC-7901 human gastric carcinoma cells via the mitochondrial signaling pathway

    PubMed Central

    LIN, RUHUI; LI, ZUANFANG; LIN, JIUMAO; YE, JINXIA; CAI, QIAOYAN; CHEN, LIDIAN; PENG, JUN

    2015-01-01

    Tulipa edulis Bak (TEB) is an active ingredient in various traditional Chinese medicine compounds and is commonly used to treat swelling and redness, remove toxicity and eliminate stagnation, as well as to prevent and treat certain cancer types. However, the underlying molecular mechanism of the anticancer activity of TEB remains unclear. The aim of the current study was to investigate the effect and underlying mechanism of the ethanolic extract of TEB (EETEB) on SGC-7901 human gastric carcinoma cells. An MTT assay was performed to analyze cell viability. In addition, transmission electron microscopy, an Annexin V/fluorescein isothiocyanate assay, a JC-1 assay and laser scanning confocal microscopy with DAPI staining were used to determine the rate of apoptosis. Furthermore, reverse transcription-polymerase chain reaction and western blot analysis were used to detect the expression levels of the apoptosis gene and protein. EETEB was identified to inhibit the growth of SGC-7901 cells in a dose-dependent manner and induce changes in cell morphology. At the molecular level, EETEB induced SGC-7901 cell DNA fragmentation, loss of plasma membrane and asymmetrical collapse of the mitochondrial membrane potential, while it increased the expression of pro-apoptotic B-cell lymphoma-2 (Bcl-2)-associated X protein and reduced expression of anti-apoptotic Bcl-2. Thus, the results of the current study revealed that the application of EETEB may inhibit the growth of the SGC-7901 cells due to mitochondria-mediated apoptosis. PMID:26622854

  15. Ethanolic extract of Tulipa edulis Bak induces apoptosis in SGC-7901 human gastric carcinoma cells via the mitochondrial signaling pathway.

    PubMed

    Lin, Ruhui; Li, Zuanfang; Lin, Jiumao; Ye, Jinxia; Cai, Qiaoyan; Chen, Lidian; Peng, Jun

    2015-10-01

    Tulipa edulis Bak (TEB) is an active ingredient in various traditional Chinese medicine compounds and is commonly used to treat swelling and redness, remove toxicity and eliminate stagnation, as well as to prevent and treat certain cancer types. However, the underlying molecular mechanism of the anticancer activity of TEB remains unclear. The aim of the current study was to investigate the effect and underlying mechanism of the ethanolic extract of TEB (EETEB) on SGC-7901 human gastric carcinoma cells. An MTT assay was performed to analyze cell viability. In addition, transmission electron microscopy, an Annexin V/fluorescein isothiocyanate assay, a JC-1 assay and laser scanning confocal microscopy with DAPI staining were used to determine the rate of apoptosis. Furthermore, reverse transcription-polymerase chain reaction and western blot analysis were used to detect the expression levels of the apoptosis gene and protein. EETEB was identified to inhibit the growth of SGC-7901 cells in a dose-dependent manner and induce changes in cell morphology. At the molecular level, EETEB induced SGC-7901 cell DNA fragmentation, loss of plasma membrane and asymmetrical collapse of the mitochondrial membrane potential, while it increased the expression of pro-apoptotic B-cell lymphoma-2 (Bcl-2)-associated X protein and reduced expression of anti-apoptotic Bcl-2. Thus, the results of the current study revealed that the application of EETEB may inhibit the growth of the SGC-7901 cells due to mitochondria-mediated apoptosis.

  16. CXCR4 Chemokine Receptor Signaling Induces Apoptosis in Acute Myeloid Leukemia Cells via Regulation of the Bcl-2 Family Members Bcl-XL, Noxa, and Bak*

    PubMed Central

    Kremer, Kimberly N.; Peterson, Kevin L.; Schneider, Paula A.; Meng, X. Wei; Dai, Haiming; Hess, Allan D.; Smith, B. Douglas; Rodriguez-Ramirez, Christie; Karp, Judith E.; Kaufmann, Scott H.; Hedin, Karen E.

    2013-01-01

    The CXCR4 chemokine receptor promotes survival of many different cell types. Here, we describe a previously unsuspected role for CXCR4 as a potent inducer of apoptosis in acute myeloid leukemia (AML) cell lines and a subset of clinical AML samples. We show that SDF-1, the sole ligand for CXCR4, induces the expected migration and ERK activation in the KG1a AML cell line transiently overexpressing CXCR4, but ERK activation did not lead to survival. Instead, SDF-1 treatment led via a CXCR4-dependent mechanism to apoptosis, as evidenced by increased annexin V staining, condensation of chromatin, and cleavage of both procaspase-3 and PARP. This SDF-1-induced death pathway was partially inhibited by hypoxia, which is often found in the bone marrow of AML patients. SDF-1-induced apoptosis was inhibited by dominant negative procaspase-9 but not by inhibition of caspase-8 activation, implicating the intrinsic apoptotic pathway. Further analysis showed that this pathway was activated by multiple mechanisms, including up-regulation of Bak at the level of mRNA and protein, stabilization of the Bak activator Noxa, and down-regulation of antiapoptotic Bcl-XL. Furthermore, adjusting expression levels of Bak, Bcl-XL, or Noxa individually altered the level of apoptosis in AML cells, suggesting that the combined modulation of these family members by SDF-1 coordinates their interplay to produce apoptosis. Thus, rather than mediating survival, SDF-1 may be a means to induce apoptosis of CXCR4-expressing AML cells directly in the SDF-1-rich bone marrow microenvironment if the survival cues of the bone marrow are disrupted. PMID:23798675

  17. Bz-423 superoxide signals apoptosis via selective activation of JNK, Bak, and Bax.

    PubMed

    Blatt, Neal B; Boitano, Anthony E; Lyssiotis, Costas A; Opipari, Anthony W; Glick, Gary D

    2008-11-01

    Bz-423 is a proapoptotic 1,4-benzodiazepine with potent therapeutic properties in murine models of lupus and psoriasis. Bz-423 modulates the F(1)F(0)-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. Herein, we report the signaling pathway activated by Bz-423 in mouse embryonic fibroblasts containing knockouts of key apoptotic proteins. Bz-423-induced superoxide activates cytosolic ASK1 and its release from thioredoxin. A mitogen-activated protein kinase cascade follows, leading to the specific phosphorylation of JNK. JNK signals activation of Bax and Bak which then induces mitochondrial outer membrane permeabilization to cause the release of cytochrome c and a commitment to apoptosis. The response of these cells to Bz-423 is critically dependent on both superoxide and JNK activation as antioxidants and the JNK inhibitor SP600125 prevents Bax translocation, cytochrome c release, and cell death. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a sequential and specific apoptotic response. Collectively, these data suggest that the selectivity of Bz-423 observed in vivo results from cell-type specific differences in redox balance and signaling by ASK1 and Bcl-2 proteins.

  18. Metaxins 1 and 2, two proteins of the mitochondrial protein sorting and assembly machinery, are essential for Bak activation during TNF alpha triggered apoptosis.

    PubMed

    Cartron, Pierre-François; Petit, Elise; Bellot, Grégory; Oliver, Lisa; Vallette, François M

    2014-09-01

    The proteins Bax and Bak are central in the execution phase of apoptosis; however, little is known about the partners involved in the control of this complex process. Here, we show that mitochondrial Bak is incorporated into a VDAC2/Mtx1/Mtx2 multi-protein complex in both resting and dying cells. VDAC2 is a porin that has previously been described as a partner of Bak while Mtx1 and Mtx2 are two proteins of the mitochondrial sorting and assembly machinery (SAM) that have been implicated in TNF-induced apoptosis. We show that, after the induction of apoptosis, Bak switches from its association with Mtx2 and VDAC2 to interact with Mtx1. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. AICAR induces Bax/Bak-dependent apoptosis through upregulation of the BH3-only proteins Bim and Noxa in mouse embryonic fibroblasts.

    PubMed

    González-Gironès, Diana M; Moncunill-Massaguer, Cristina; Iglesias-Serret, Daniel; Cosialls, Ana M; Pérez-Perarnau, Alba; Palmeri, Claudia M; Rubio-Patiño, Camila; Villunger, Andreas; Pons, Gabriel; Gil, Joan

    2013-08-01

    5-Aminoimidazole-4-carboxamide (AICA) riboside (AICAR) is a nucleoside analogue that is phosphorylated to 5-amino-4-imidazolecarboxamide ribotide (ZMP), which acts as an AMP mimetic and activates AMP-activated protein kinase (AMPK). It has been recently described that AICAR triggers apoptosis in chronic lymphocytic leukemia (CLL) cells, and its mechanism of action is independent of AMPK as well as p53. AICAR-mediated upregulation of the BH3-only proteins BIM and NOXA correlates with apoptosis induction in CLL cells. Here we propose mouse embryonic fibroblasts (MEFs) as a useful model to analyze the mechanism of AICAR-induced apoptosis. ZMP formation was required for AICAR-induced apoptosis, though direct Ampk activation with A-769662 failed to induce apoptosis in MEFs. AICAR potently induced apoptosis in Ampkα1 (-/-) /α2 (-/-) MEFs, demonstrating an Ampk-independent mechanism of cell death activation. In addition, AICAR acts independently of p53, as MEFs lacking p53 also underwent apoptosis normally. Notably, MEFs lacking Bax and Bak were completely resistant to AICAR-induced apoptosis, confirming the involvement of the mitochondrial pathway in its mechanism of action. Apoptosis was preceded by ZMP-dependent but Ampk-independent modulation of the mRNA levels of different Bcl-2 family members, including Noxa, Bim and Bcl-2. Bim protein levels were accumulated upon AICAR treatment of MEFs, suggesting its role in the apoptotic process. Strikingly, MEFs lacking both Bim and Noxa displayed high resistance to AICAR. These findings support the notion that MEFs are a useful system to further dissect the mechanism of AICAR-induced apoptosis.

  20. Cancer-selective death of human breast cancer cells by leelamine is mediated by bax and bak activation.

    PubMed

    Sehrawat, Anuradha; Kim, Su-Hyeong; Hahm, Eun-Ryeong; Arlotti, Julie A; Eiseman, Julie; Shiva, Sruti S; Rigatti, Lora H; Singh, Shivendra V

    2017-02-01

    The present study is the first to report inhibition of breast cancer cell growth in vitro and in vivo and suppression of self-renewal of breast cancer stem cells (bCSC) by a pine bark component (leelamine). Except for a few recent publications in melanoma, anticancer pharmacology of this interesting phytochemical is largely elusive. Leelamine (LLM) dose-dependently inhibited viability of MDA-MB-231 (triple-negative), MCF-7 (estrogen receptor-positive), and SUM159 (triple-negative) human breast cancer cells in association with apoptotic cell death induction. To the contrary, a normal mammary epithelial cell line derived from fibrocystic breast disease and spontaneously immortalized (MCF-10A) was fully resistant to LLM-mediated cell growth inhibition and apoptosis induction. LLM also inhibited self-renewal of breast cancer stem cells. Apoptosis induction by LLM in breast cancer cells was accompanied by a modest increase in reactive oxygen species production, which was not due to inhibition of mitochondrial electron transport chain complexes. Nevertheless, ectopic expression of manganese superoxide dismutase conferred partial protection against LLM-induced cell death but only at a lower yet pharmacologically relevant concentration. Exposure of breast cancer cells to LLM resulted in (a) induction and/or activation of multidomain proapoptotic proteins Bax and Bak, (b) caspase-9 activation, and (c) cytosolic release of cytochrome c. Bax and Bak deficiency in immortalized fibroblasts conferred significant protection against cell death by LLM. Intraperitoneal administration of LLM (7.5 mg/kg; 5 times/wk) suppressed the growth of orthotopic SUM159 xenografts in mice without any toxicity. In conclusion, the present study provides critical preclinical data to warrant further investigation of LLM. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Bax and Bak Pores: Are We Closing the Circle?

    PubMed

    Cosentino, Katia; García-Sáez, Ana J

    2017-04-01

    Bax and its homolog Bak are key regulators of the mitochondrial pathway of apoptosis. On cell stress Bax and Bak accumulate at distinct foci on the mitochondrial surface where they undergo a conformational change, oligomerize, and mediate cytochrome c release, leading to cell death. The molecular mechanisms of Bax and Bak assembly and mitochondrial permeabilization have remained a longstanding question in the field. Recent structural and biophysical studies at several length scales have shed light on key aspects of Bax and Bak function that have shifted how we think this process occurs. These discoveries reveal an unexpected molecular mechanism in which Bax (and likely Bak) dimers assemble into oligomers with an even number of molecules that fully or partially delineate pores of different sizes to permeabilize the mitochondrial outer membrane (MOM) during apoptosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Charge Profile Analysis Reveals That Activation of Pro-apoptotic Regulators Bax and Bak Relies on Charge Transfer Mediated Allosteric Regulation

    PubMed Central

    Ionescu, Crina-Maria; Svobodová Vařeková, Radka; Prehn, Jochen H. M.; Huber, Heinrich J.; Koča, Jaroslav

    2012-01-01

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

  3. Rapamycin-enhanced mitomycin C-induced apoptotic death is mediated through the S6K1–Bad–Bak pathway in peritoneal carcinomatosis

    PubMed Central

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

    2014-01-01

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

  4. Rapamycin-enhanced mitomycin C-induced apoptotic death is mediated through the S6K1-Bad-Bak pathway in peritoneal carcinomatosis.

    PubMed

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

    2014-06-05

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

  5. Knockdown of miR-221 promotes the cisplatin-inducing apoptosis by targeting the BIM-Bax/Bak axis in breast cancer.

    PubMed

    Ye, Zhiqiang; Hao, Rutian; Cai, Yefeng; Wang, Xiaobo; Huang, Guanli

    2016-04-01

    Accumulating evidence shows that microRNAs (miRNAs) have a critical role in the initiation and progression of types of human cancer, including breast cancer. Recent research indicated that miRNAs are also related with the chemotherapy on cancers. In this study, the expression of miR-221 in breast cancer (BC) patients' serum and cancer tissues was found to be significantly up-regulated. The results of in vitro MTT assay indicated that although the anti-miR-221 oligonucleotide alone did not influence the viability of BC cell lines markedly, it significantly promoted the cytotoxicity of cisplatin (DDP) to BC cells. Mechanistic studies demonstrated that the gene of BIM (Bcl-2 interacting mediator of cell death), a pro-apoptotic Bcl-2 family protein, was up-regulated by the knockdown of miR-221. We found that the synergetic effect of anti-miR-221 on increasing the sensitivity of MDA-MB-231 was BIM dependant. Furthermore, results of immunoprecipitation showed the up-regulated BIM directly combined with the Bax and Bak, leading to mitochondrial dysfunction. Our results suggest the anti-miR-221 could promote the cisplatin-inducing apoptosis by targeting the Bim-Bax/Bak axis in breast cancer.

  6. Alterations in the characteristic size distributions of subcellular scatterers at the onset of apoptosis: effect of Bcl-xL and Bax/Bak

    NASA Astrophysics Data System (ADS)

    Zheng, Jing-Yi; Boustany, Nada N.

    2010-07-01

    Optical scatter imaging is used to estimate organelle size distributions in immortalized baby mouse kidney cells treated with 0.4 μM staurosporine to induce apoptosis. The study comprises apoptosis competent iBMK cells (W2) expressing the proapoptotic proteins Bax/Bak, apoptosis resistant Bax/Bak null cells (D3), and W2 and D3 cells expressing yellow fluorescent protein (YFP) or YFP fused to the antiapoptotic protein Bcl-xL (YFP-Bcl-xL). YFP expression is diffuse within the transfected cells, while YFP-Bcl-xL is localized to the mitochondria. Our results show a significant increase in the mean subcellular particle size from approximately 1.1 to 1.4 μm in both Bax/Bak expressing and Bax/Bak null cells after 60 min of STS treatment compared to DMSO-treated control cells. This dynamic is blocked by overexpression of YFP-Bcl-xL in Bax/Bak expressing cells, but is less significantly inhibited by YFP-Bcl-xL in Bax/Bak null cells. Our data suggest that the increase in subcellular particle size at the onset of apoptosis is modulated by Bcl-xL in the presence of Bax/Bak, but it occurs upstream of the final commitment to programmed cell death. Mitochondrial localization of YFP-Bcl-xL and the finding that micron-sized particles give rise to the scattering signal further suggest that alterations in mitochondrial morphology may underlie the observed changes in light scattering.

  7. Suture compression induced midpalatal suture chondrocyte apoptosis with increased caspase-3, caspase-9, Bad, Bak, Bax and Bid expression.

    PubMed

    Lan, Tingting; Zhao, Hanchi; Xiang, Bilu; Wang, Jun; Liu, Yang

    2017-07-22

    Previous studies found bone resorption and chondrocytes loss in mouse models of mid-palatal suture when given continuous compressive force, although chondrocytes response remained unknown. Herein, we design this study to determine how continuous compression force induces chondrocytes apoptosis. Thirty C57BL/6 male mice (aged 6 weeks) were randomly assigned into controls (not ligated to a spring), blank controls (ligated with no compression) and the compression group (ligated with 20-g compression). After 4 d, palatal tissues were sampled and stained by TB and safranin-O. Tunel staining measured the percentage of apoptotic chondrocytes, and immunohistochemistry was performed to label apoptosis-associated proteins (e.g., Bcl-2, Bcl-xl, Bax, Bak, Bid, Bad, caspase-3, caspase-8 and caspase-9). Intergroup comparison was made by the rank sum test, and P < 0.05 was defined as statistical significance. After 7d of induction, TB and safranin-O staining revealed that the cartilage area in the compression group was significantly decreased, while the control group remained largely unaltered. Tunel staining showed that apoptotic cell numbers in the mid-palatal suture were significantly higher than the control group. Immunohistochemistry showed that mice in the compression group had significantly increased expression of caspase-3, caspase-9, Bad, Bak, Bax and Bid; However, caspase-8 remained unaltered. No expression of Bcl-2 and Bcl-xl was detected. Continuous compression force induces chondrocytes apoptosis in the mid-palatal suture. This process might be associated with the mitochondrial pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Repression of Mcl-1 and disruption of the Mcl-1/Bak interaction in myeloma cells couple ER stress to mitochondrial apoptosis.

    PubMed

    Gomez-Bougie, Patricia; Halliez, Maxime; Moreau, Philippe; Pellat-Deceunynck, Catherine; Amiot, Martine

    2016-12-28

    As myeloma cells actively produce and secrete immunoglobulins, they are prone to ER stress, which if unresolved leads to apoptosis. We found that myeloma cell death induced by the ER stressor Thapsigargin was highly variable, ranging from 2 to 89%. Induction of ATF4 and CHOP was observed in myeloma cells under Thapsigargin independently of cell death. The decrease in Mcl-1 was associated with protein translation inhibition and identified as a crucial factor in Thapsigargin sensitivity, since it was the only Bcl-2 family protein differentially modified between sensitive and resistant myeloma cells. Bak but not Bax was found to contribute to Thapsigargin-induced apoptosis. Appropriately, a basal Mcl-1/Bak interaction was demonstrated in Thapsigargin-sensitive cells. Of note, the only pro-apoptotic protein freed from Mcl-1 under Thapsigargin was Bak, whereas Mcl-1/Noxa or Mcl-1/Bim complexes were simultaneously increased. Thus, the disruption of the basal Mcl-1/Bak complex in Thapsigargin-sensitive cells seemed to be an essential event in cell death induction, probably favored by the induced Noxa and Bim BH3-only proteins. These findings underscore the implication of the Mcl-1/Bak axis in myeloma cell death triggered by Thapsigargin. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  9. Bax/Bak-dependent, Drp1-independent Targeting of X-linked Inhibitor of Apoptosis Protein (XIAP) into Inner Mitochondrial Compartments Counteracts Smac/DIABLO-dependent Effector Caspase Activation.

    PubMed

    Hamacher-Brady, Anne; Brady, Nathan Ryan

    2015-09-04

    Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the

  10. The combination of thioxodihydroquinazolinones and platinum drugs reverses platinum resistance in tumor cells by inducing mitochondrial apoptosis independent of Bax and Bak

    PubMed Central

    Qian, Wei; Salamoun, Joseph; Wang, Jingnan; Roginskaya, Vera; Van Houten, Bennett; Wipf, Peter

    2015-01-01

    The effective management of tumors resistant to platinum drugs-based anticancer therapies is a critical challenge in current clinical practices. The proapoptotic Bcl-2 family proteins Bax and Bak are essential for cisplatin-induced apoptosis. Unfortunately, Bax and its related upstream endogenous apoptotic signaling pathways are often dysregulated in cancer cells. Strategies that are able to bypass Bax- and Bak-dependent apoptotic pathways will thus provide opportunities to overcome platinum drug resistance. We have identified the thioxodihydroquinazolinone mdivi-1 as a member of a novel class of small molecules that are able to induce Bax- and Bak-independent mitochondrial outer membrane permeabilization when combined with cisplatin, thereby efficiently triggering apoptosis in platinum resistant tumor cells. In the present structure activity relationship (SAR) study of a computationally selected library of mdivi-1 related small molecules, we established a pharmacophore model that can lead to the enhancement of platinum drug efficacy and Bax/Bak-independent mitochondrial apoptosis. Specifically, we found that a thiourea function is necessary but not sufficient for the synergism of this class of thioxodihydroquinazolinones with cisplatin. We were also able to identify more potent mdivi-1 analogs through this SAR study, which will guide future designs with the goal to develop novel combination regimens for the treatment of platinum and multidrug resistant tumors. PMID:25582599

  11. Cotton GhBAK1 mediates Verticillium wilt resistance and cell death.

    PubMed

    Gao, Xiquan; Li, Fangjun; Li, Maoying; Kianinejad, Ali S; Dever, Jane K; Wheeler, Terry A; Li, Zhaohu; He, Ping; Shan, Libo

    2013-07-01

    Virus-induced gene silencing (VIGS) offers a powerful approach for functional analysis of individual genes by knocking down their expression. We have adopted this approach to dissect gene functions in cotton resistant to Verticillium wilt, one of the most devastating diseases worldwide. We showed here that highly efficient VIGS was obtained in a cotton breeding line (CA4002) with partial resistance to Verticillium wilt, and GhMKK2 and GhVe1 are required for its resistance to Verticillium wilt. Arabidopsis AtBAK1/SERK3, a central regulator in plant disease resistance, belongs to a subfamily of somatic embryogenesis receptor kinases (SERKs) with five members, AtSERK1 to AtSERK5. Two BAK1 orthologs and one SERK1 ortholog were identified in the cotton genome. Importantly, GhBAK1 is required for CA4002 resistance to Verticillium wilt. Surprisingly, silencing of GhBAK1 is sufficient to trigger cell death accompanied with production of reactive oxygen species in cotton. This result is distinct from Arabidopsis in which AtBAK1 and AtSERK4 play redundant functions in cell death control. Apparently, cotton has only evolved SERK1 and BAK1 whereas AtSERK4/5 are newly evolved genes in Arabidopsis. Our studies indicate the functional importance of BAK1 in Verticillium wilt resistance and suggest the dynamic evolution of SERK family members in different plant species.

  12. Cotton GhBAK1 Mediates Verticillium Wilt Resistance and Cell DeathF

    PubMed Central

    Gao, Xiquan; Li, Fangjun; Li, Maoying; Kianinejad, Ali S.; Dever, Jane K.; Wheeler, Terry A.; Li, Zhaohu; He, Ping; Shan, Libo

    2015-01-01

    Virus-induced gene silencing (VIGS) offers a powerful approach for functional analysis of individual genes by knocking down their expression. We have adopted this approach to dissect gene functions in cotton resistant to Verticillium wilt, one of the most devastating diseases worldwide. We showed here that highly efficient VIGS was obtained in a cotton breeding line (CA4002) with partial resistance to Verticillium wilt, and GhMKK2 and GhVe1 are required for its resistance to Verticillium wilt. Arabidopsis AtBAK1/SERK3, a central regulator in plant disease resistance, belongs to a subfamily of somatic embryogenesis receptor kinases (SERKs) with five members, AtSERK1 to AtSERK5. Two BAK1 orthologs and one SERK1 ortholog were identified in the cotton genome. Importantly, GhBAK1 is required for CA4002 resistance to Verticillium wilt. Surprisingly, silencing of GhBAK1 is sufficient to trigger cell death accompanied with production of reactive oxygen species in cotton. This result is distinct from Arabidopsis in which AtBAK1 and AtSERK4 play redundant functions in cell death control. Apparently, cotton has only evolved SERK1 and BAK1 whereas AtSERK4/5 are newly evolved genes in Arabidopsis. Our studies indicate the functional importance of BAK1 in Verticillium wilt resistance and suggest the dynamic evolution of SERK family members in different plant species. PMID:23675706

  13. Targeted therapy of the XIAP/proteasome pathway overcomes TRAIL-resistance in carcinoma by switching apoptosis signaling to a Bax/Bak-independent 'type I' mode.

    PubMed

    Gillissen, B; Richter, A; Richter, A; Overkamp, T; Essmann, F; Hemmati, P G; Preissner, R; Belka, C; Daniel, P T

    2013-05-23

    TRAIL is a promising anticancer agent, capable of inducing apoptosis in a wide range of treatment-resistant tumor cells. In 'type II' cells, the death signal triggered by TRAIL requires amplification via the mitochondrial apoptosis pathway. Consequently, deregulation of the intrinsic apoptosis-signaling pathway, for example, by loss of Bax and Bak, confers TRAIL-resistance and limits its application. Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes. However, active caspase-3 was degraded by the proteasome and not detectable unless the XIAP/proteasome pathway was inhibited. Direct or indirect inhibition of XIAP by RNAi, Mithramycin A or by the SMAC mimetic LBW-242 as well as inhibition of the proteasome by Bortezomib overcomes TRAIL-resistance of Bax/Bak double-deficient tumor cells. Moreover, activation and stabilization of caspase-3 becomes independent of mitochondrial death signaling, demonstrating that inhibition of the XIAP/proteasome pathway overcomes resistance by converting 'type II' to 'type I' cells. Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the 'type II' phenotype. Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.

  14. Bax/Bak-independent mitochondrial depolarization and reactive oxygen species induction by sorafenib overcome resistance to apoptosis in renal cell carcinoma.

    PubMed

    Gillissen, Bernhard; Richter, Anja; Richter, Antje; Preissner, Robert; Schulze-Osthoff, Klaus; Essmann, Frank; Daniel, Peter T

    2017-04-21

    Renal cell carcinoma (RCC) is polyresistant to chemo- and radiotherapy and biologicals, including TNF-related apoptosis-inducing ligand (TRAIL). Sorafenib, a multikinase inhibitor approved for the treatment of RCC, has been shown to sensitize cancer cells to TRAIL-induced apoptosis, in particular by down-regulation of the Bak-inhibitory Bcl-2 family protein Mcl-1. Here we demonstrate that sorafenib overcomes TRAIL resistance in RCC by a mechanism that does not rely on Mcl-1 down-regulation. Instead, sorafenib induces rapid dissipation of the mitochondrial membrane potential (ΔΨm) that is accompanied by the accumulation of reactive oxygen species (ROS). Loss of ΔΨm and ROS production induced by sorafenib are independent of caspase activities and do not depend on the presence of the proapoptotic Bcl-2 family proteins Bax or Bak, indicating that both events are functionally upstream of the mitochondrial apoptosis signaling cascade. More intriguingly, we find that it is sorafenib-induced ROS accumulation that enables TRAIL to activate caspase-8 in RCC. This leads to apoptosis that involves activation of an amplification loop via the mitochondrial apoptosis pathway. Thus, our mechanistic data indicate that sorafenib bypasses central resistance mechanisms through a direct induction of ΔΨm breakdown and ROS production. Activation of this pathway might represent a useful strategy to overcome the cell-inherent resistance to cancer therapeutics, including TRAIL, in multiresistant cancers such as RCC. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Proapoptotic Bax and Bak Proteins Form Stable Protein-permeable Pores of Tunable Size

    PubMed Central

    Bleicken, Stephanie; Landeta, Olatz; Landajuela, Ane; Basañez, Gorka; García-Sáez, Ana J.

    2013-01-01

    The Bcl-2 proapoptotic proteins Bax and Bak mediate the permeabilization of the mitochondrial outer membrane during apoptosis. Current models consider that Bax and Bak form pores at the mitochondrial outer membrane that are responsible for the release of cytochrome c and other larger mitochondrial apoptotic factors (i.e. Smac/DIABLO, AIF, and endoglycosidase G). However, the properties and nature of Bax/Bak apoptotic pores remain enigmatic. Here, we performed a detailed analysis of the membrane permeabilizing activity of Bax and Bak at the single vesicle level. We directly visualized that cBid-activated Bax and BakΔC21 can form membrane pores large enough to release not only cytochrome c, but also allophycocyanine, a protein of 104 kDa. Interestingly, the size of Bax and BakΔC21 pores is not constant, as typically observed in purely proteinaceous channels, but evolves with time and depends on protein concentration. We found that Bax and BakΔC21 formed long-lived pores, whose areas changed with the amount of Bax/BakΔC21 but not with cardiolipin concentration. Altogether, our results demonstrate that Bax and BakΔC21 follow similar mechanisms of membrane permeabilization characterized by the formation of protein-permeable pores of dynamic size, in agreement with the proteolipidic nature of these apoptotic pores. PMID:24100034

  16. Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins

    PubMed Central

    Goodall, Katharine J.; Finch-Edmondson, Megan L.; van Vuuren, Joanne; Yeoh, George C.; Gentle, Ian E.; Vince, James E.; Ekert, Paul G.; Vaux, David L.; Callus, Bernard A.

    2016-01-01

    Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax-/-Bak-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins. PMID:27806040

  17. Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins.

    PubMed

    Goodall, Katharine J; Finch-Edmondson, Megan L; van Vuuren, Joanne; Yeoh, George C; Gentle, Ian E; Vince, James E; Ekert, Paul G; Vaux, David L; Callus, Bernard A

    2016-01-01

    Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax-/-Bak-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.

  18. Differential retrotranslocation of mitochondrial Bax and Bak

    PubMed Central

    Todt, Franziska; Cakir, Zeynep; Reichenbach, Frank; Emschermann, Frederic; Lauterwasser, Joachim; Kaiser, Andrea; Ichim, Gabriel; Tait, Stephen WG; Frank, Stephan; Langer, Harald F; Edlich, Frank

    2015-01-01

    The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death. PMID:25378477

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

    PubMed

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

    2014-01-01

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

  20. Induction apoptosis of luteolin in human hepatoma HepG2 cells involving mitochondria translocation of Bax/Bak and activation of JNK

    SciTech Connect

    Lee, H.-J.; Wang, C.-J.; Kuo, H.-C.; Chou, F.-P.; Jean, L.-F.; Tseng, T.-H. . E-mail: tht@csmu.edu.tw

    2005-03-01

    Since hepatocellular carcinoma remains a major challenging clinical problem in many parts of the world including Eastern Asia and Southern Africa, it is imperative to develop more effective chemopreventive and chemotherapy agents. Herein, we present an investigation regarding the anticancer potential of luteolin, a natural flavonoid, and the mechanism of its action in human hepatoma HepG2 cells. Using DNA fragmentation assay and nuclear staining assay, it showed that luteolin induced apoptosis of HepG2 cells. Luteolin induced the cytosolic release of cytochrome c and activated CPP32. We found that Bax and Bak translocated to mitochondria apparently, whereas Fas ligand (FasL) was unchanged after a treatment with luteolin for 3 h. In addition, it showed that c-Jun NH{sub 2}-terminal kinase (JNK) was activated after the treatment of luteolin for 3-12 h. Further investigation showed that a specific JNK inhibitor, SP600125, reduced the activation of CPP 32, the mitochondrial translocation of Bax, as well as the cytosolic release of cytochrome c that induced by luteolin. Finally, the apoptosis induced by luteolin was suppressed by a pretreatment with SP600125 via evaluating annexin V-FITC binding assay. These data suggest that luteolin induced apoptosis via mechanisms involving mitochondria translocation of Bax/Bak and activation of JNK.

  1. Bik reduces hyperplastic cells by increasing Bak and activating DAPk1 to juxtapose ER and mitochondria.

    PubMed

    Mebratu, Yohannes A; Leyva-Baca, Ivan; Wathelet, Marc G; Lacey, Neal; Chand, Hitendra S; Choi, Augustine M K; Tesfaigzi, Yohannes

    2017-10-06

    Bik reduces hyperplastic epithelial cells by releasing calcium from endoplasmic reticulum stores and causing apoptosis, but the detailed mechanisms are not known. Here we report that Bik dissociates the Bak/Bcl-2 complex to enrich for ER-associated Bak and interacts with the kinase domain of DAPk1 to form Bik-DAPk1-ERK1/2-Bak complex. Bik also disrupts the Bcl2-IP3R interaction to cause ER Ca(2+) release. The ER-associated Bak interacts with the kinase and calmodulin domains of DAPk1 to increase the contact sites of ER and mitochondria, and facilitate ER Ca(2+) uptake by mitochondria. Although the Bik BH3 helix was sufficient to enrich for ER-Bak and elicit ER Ca(2+) release, Bik-induced mitochondrial Ca(2+) uptake is blocked with reduced Bak levels. Further, the Bik-derived peptide reduces allergen- and cigarette smoke-induced mucous cell hyperplasia in mice and in differentiated primary human airway epithelial cultures. Therefore, Bik peptides may have therapeutic potential in airway diseases associated with chronic mucous hypersecretion.Bcl-2 interacting killer (Bik) decreases airway epithelial hyperplasia via apoptosis mediated by calcium release from the endoplasmic reticulum (ER), but the mechanism is unclear. Here the authors show that Bik promotes Bak enrichment at the ER to tether mitochondria for efficient calcium transfer.

  2. Bcl-XL specifically activates Bak to induce swelling and restructuring of the endoplasmic reticulum

    PubMed Central

    Klee, Martina; Pimentel-Muiños, Felipe X.

    2005-01-01

    Bcl-2 family members Bak and Bax constitute a mitochondrial gateway for multiple death pathways. Both proteins are also present in the endoplasmic reticulum where they control apoptosis through the regulation of calcium levels. We show here that reticular Bak has the additional capacity of modulating the structure of this organelle. Coexpression of Bak and Bcl-XL provokes extensive swelling and vacuolization of reticular cisternae. A Bak version lacking the BH3 domain suffices to induce this phenotype, and reticular targeting of this mutant retains the activity. Expression of upstream BH3-only activators in similar conditions recapitulates ER swelling and vacuolization if ryanodine receptor calcium channel activity is inhibited. Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant. These results reveal a previously unidentified role of Bak in regulating reticular conformation. Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues. PMID:15728194

  3. Fenugreek induced apoptosis in breast cancer MCF-7 cells mediated independently by fas receptor change.

    PubMed

    Alshatwi, Ali Abdullah; Shafi, Gowhar; Hasan, Tarique Noorul; Syed, Naveed Ahmed; Khoja, Kholoud Khalid

    2013-01-01

    Trigonella foenum in graecum (Fenugreek) is a traditional herbal plant used to treat disorders like diabetes, high cholesterol, wounds, inflammation, gastrointestinal ailments, and it is believed to have anti-tumor properties, although the mechanisms for the activity remain to be elucidated. In this study, we prepared a methanol extract from Fenugreek whole plants and investigated the mechanism involved in its growth-inhibitory effect on MCF- 7 human breast cancer cells. Apoptosis of MCF-7 cells was evidenced by investigating trypan blue exclusion, TUNEL and Caspase 3, 8, 9, p53, FADD, Bax and Bak by real-time PCR assays inducing activities, in the presence of FME at 65 μg/mL for 24 and 48 hours. FME induced apoptosis was mediated by the death receptor pathway as demonstrated by the increased level of Fas receptor expression after FME treatment. However, such change was found to be absent in Caspase 3, 8, 9, p53, FADD, Bax and Bak, which was confirmed by a time-dependent and dose-dependent manner. In summary, these data demonstrate that at least 90% of FME induced apoptosis in breast cell is mediated by Fas receptor-independently of either FADD, Caspase 8 or 3, as well as p53 interdependently.

  4. Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function

    PubMed Central

    Ma, S B; Nguyen, T N; Tan, I; Ninnis, R; Iyer, S; Stroud, D A; Menard, M; Kluck, R M; Ryan, M T; Dewson, G

    2014-01-01

    In non-apoptotic cells, Bak constitutively resides in the mitochondrial outer membrane. In contrast, Bax is in a dynamic equilibrium between the cytosol and mitochondria, and is commonly predominant in the cytosol. In response to an apoptotic stimulus, Bax and Bak change conformation, leading to Bax accumulation at mitochondria and Bak/Bax oligomerization to form a pore in the mitochondrial outer membrane that is responsible for cell death. Using blue native-PAGE to investigate how Bax oligomerizes in the mitochondrial outer membrane, we observed that, like Bak, a proportion of Bax that constitutively resides at mitochondria associates with voltage-dependent anion channel (VDAC)2 prior to an apoptotic stimulus. During apoptosis, Bax dissociates from VDAC2 and homo-oligomerizes to form high molecular weight oligomers. In cells that lack VDAC2, constitutive mitochondrial localization of Bax and Bak was impaired, suggesting that VDAC2 has a role in Bax and Bak import to, or stability at, the mitochondrial outer membrane. However, following an apoptotic stimulus, Bak and Bax retained the ability to accumulate at VDAC2-deficient mitochondria and to mediate cell death. Silencing of Bak in VDAC2-deficient cells indicated that Bax required either VDAC2 or Bak in order to translocate to and oligomerize at the mitochondrial outer membrane to efficiently mediate apoptosis. In contrast, efficient Bak homo-oligomerization at the mitochondrial outer membrane and its pro-apoptotic function required neither VDAC2 nor Bax. Even a C-terminal mutant of Bax (S184L) that localizes to mitochondria did not constitutively target mitochondria deficient in VDAC2, but was recruited to mitochondria following an apoptotic stimulus dependent on Bak or upon over-expression of Bcl-xL. Together, our data suggest that Bax localizes to the mitochondrial outer membrane via alternate mechanisms, either constitutively via an interaction with VDAC2 or after activation via interaction with Bcl-2 family

  5. Cisplatin-induced apoptosis in non-small-cell lung cancer cells is dependent on Bax- and Bak-induction pathway and synergistically activated by BH3-mimetic ABT-263 in p53 wild-type and mutant cells.

    PubMed

    Matsumoto, Masaru; Nakajima, Wataru; Seike, Masahiro; Gemma, Akihiko; Tanaka, Nobuyuki

    2016-04-29

    Cisplatin is a highly effective anticancer drug for treatment of various tumors including non-small-cell lung cancer (NSCLC), and is especially useful in cases nonresponsive to molecular-targeted drugs. Accumulating evidence has shown that cisplatin activates the p53-dependent apoptotic pathway, but it also induces apoptosis in p53-mutated cancer cells. Here we demonstrated that DNA-damage inducible proapoptotic BH3 (Bcl-2 homology region 3)-only Bcl-2 family members, Noxa, Puma, Bim and Bid, are not involved in cisplatin-induced apoptosis in human NSCLC cell lines. In contrast, the expression of proapoptotic multidomain Bcl-2-family members, Bak and Bax, was induced by cisplatin in p53-dependent and -independent manners, respectively. Moreover, in wild-type p53-expressing cells, cisplatin mainly used the Bak-dependent apoptotic pathway, but this apoptotic pathway shifted to the Bax-dependent pathway by loss-of-function of p53. Furthermore, both Bak- and Bax-induced apoptosis was enhanced by the antiapoptotic Bcl-2 family member, Bcl-XL knockdown, but not by Mcl-1 knockdown. From this result, we tested the effect of ABT-263 (Navitoclax), the specific inhibitor of Bcl-2 and Bcl-XL, but not Mcl-1, and found that ABT-263 synergistically enhanced cisplatin-induced apoptosis in NSCLC cells in the presence or absence of p53. These results indicate a novel regulatory system in cisplatin-induced NSCLC cell apoptosis, and a candidate efficient combination chemotherapy method against lung cancers.

  6. Apoptosis in immune-mediated diseases

    PubMed Central

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

    2015-01-01

    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

  7. 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.

  8. Stimulatory heterotrimeric G protein augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 human lung cancer cells.

    PubMed

    Choi, Yoon Jung; Kim, So Young; Oh, Jung Min; Juhnn, Yong Sung

    2009-08-31

    Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the alpha subunit of Gs (GalphasQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GalphasQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked GalphasQL-stimulated Bak reporter luciferase activity. Expression of GalphasQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Galphas activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Galphas augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.

  9. Mitochondrial oligomers boost glycolysis in cancer stem cells to facilitate blebbishield-mediated transformation after apoptosis

    PubMed Central

    Jinesh, GG; Molina, JR; Huang, L; Laing, NM; Mills, GB; Bar-Eli, M; Kamat, AM

    2016-01-01

    Apoptosis culminates in secondary necrosis due to lack of ATP. Cancer stem cells form spheres after apoptosis by evoking the blebbishield emergency program. Hence, determining how blebbishields avoid secondary necrosis is crucial. Here we demonstrate that N-Myc and VEGFR2 control transformation from blebbishields, during which oligomers of K-Ras, p27, BAD, Bax, and Bak boost glycolysis to avoid secondary necrosis. Non-apoptotic cancer cells also utilize oligomers to boost glycolysis, which differentiates the glycolytic function of oligomers from their apoptotic action. Smac mimetic in combination with TNF-α or TRAIL but not in combination with FasL abrogates transformation from blebbishields by inducing secondary necrosis. Thus blebbishield-mediated transformation is dependent on glycolysis, and Smac mimetics represent potential candidates to abrogate the blebbishield emergency program. PMID:27551498

  10. Mitochondrial oligomers boost glycolysis in cancer stem cells to facilitate blebbishield-mediated transformation after apoptosis.

    PubMed

    Jinesh, G G; Molina, J R; Huang, L; Laing, N M; Mills, G B; Bar-Eli, M; Kamat, A M

    2016-01-01

    Apoptosis culminates in secondary necrosis due to lack of ATP. Cancer stem cells form spheres after apoptosis by evoking the blebbishield emergency program. Hence, determining how blebbishields avoid secondary necrosis is crucial. Here we demonstrate that N-Myc and VEGFR2 control transformation from blebbishields, during which oligomers of K-Ras, p27, BAD, Bax, and Bak boost glycolysis to avoid secondary necrosis. Non-apoptotic cancer cells also utilize oligomers to boost glycolysis, which differentiates the glycolytic function of oligomers from their apoptotic action. Smac mimetic in combination with TNF-α or TRAIL but not in combination with FasL abrogates transformation from blebbishields by inducing secondary necrosis. Thus blebbishield-mediated transformation is dependent on glycolysis, and Smac mimetics represent potential candidates to abrogate the blebbishield emergency program.

  11. Semaphorins as mediators of neuronal apoptosis.

    PubMed

    Shirvan, A; Ziv, I; Fleminger, G; Shina, R; He, Z; Brudo, I; Melamed, E; Barzilai, A

    1999-09-01

    Shrinkage and collapse of the neuritic network are often observed during the process of neuronal apoptosis. However, the molecular and biochemical basis for the axonal damage associated with neuronal cell death is still unclear. We present evidence for the involvement of axon guidance molecules with repulsive cues in neuronal cell death. Using the differential display approach, an up-regulation of collapsin response mediator protein was detected in sympathetic neurons undergoing dopamine-induced apoptosis. A synchronized induction of mRNA of the secreted collapsin-1 and the intracellular collapsin response mediator protein that preceded commitment of neurons to apoptosis was detected. Antibodies directed against a conserved collapsin-derived peptide provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. Moreover, neuronal apoptosis was inhibited by antibodies against neuropilin-1, a putative component of the semaphorin III/collapsin-1 receptor. Induction of neuronal apoptosis was also caused by exposure of neurons to semaphorin III-alkaline phosphatase secreted from 293EBNA cells. Anti-collapsin-1 antibodies were effective in blocking the semaphorin III-induced death process. We therefore suggest that, before their death, apoptosis-destined neurons may produce and secrete destructive axon guidance molecules that can affect their neighboring cells and thus transfer a "death signal" across specific and susceptible neuronal populations.

  12. Apoptosis repressor with a CARD domain (ARC) restrains Bax-mediated pathogenesis in dystrophic skeletal muscle.

    PubMed

    Davis, Jennifer; Kwong, Jennifer Q; Kitsis, Richard N; Molkentin, Jeffery D

    2013-01-01

    Myofiber wasting in muscular dystrophy has largely been ascribed to necrotic cell death, despite reports identifying apoptotic markers in dystrophic muscle. Here we set out to identify the contribution of canonical apoptotic pathways to skeletal muscle degeneration in muscular dystrophy by genetically deleting a known inhibitor of apoptosis, apoptosis repressor with a card domain (Arc), in dystrophic mouse models. Nol3 (Arc protein) genetic deletion in the dystrophic Sgcd or Lama2 null backgrounds showed exacerbated skeletal muscle pathology with decreased muscle performance compared with single null dystrophic littermate controls. The enhanced severity of the dystrophic phenotype associated with Nol3 deletion was caspase independent but dependent on the mitochondria permeability transition pore (MPTP), as the inhibitor Debio-025 partially rescued skeletal muscle pathology in Nol3 (-/-) Sgcd (-/-) double targeted mice. Mechanistically, Nol3 (-/-) Sgcd (-/-) mice showed elevated total and mitochondrial Bax protein levels, as well as greater mitochondrial swelling, suggesting that Arc normally restrains the cell death effects of Bax in skeletal muscle. Indeed, knockdown of Arc in mouse embryonic fibroblasts caused an increased sensitivity to cell death that was fully blocked in Bax Bak1 (genes encoding Bax and Bak) double null fibroblasts. Thus Arc deficiency in dystrophic muscle exacerbates disease pathogenesis due to a Bax-mediated sensitization of mitochondria-dependent death mechanisms.

  13. In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins

    PubMed Central

    Senft, D; Weber, A; Saathoff, F; Berking, C; Heppt, M V; Kammerbauer, C; Rothenfusser, S; Kellner, S; Kurgyis, Z; Besch, R; Häcker, G

    2015-01-01

    Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak. PMID:26610208

  14. Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak

    PubMed Central

    Lindqvist, Lisa M.; Heinlein, Melanie; Huang, David C. S.; Vaux, David L.

    2014-01-01

    Antiapoptotic B-cell lymphoma 2 (Bcl-2) family members such as Bcl-2, myeloid cell leukemia 1 (Mcl-1), and B-cell lymphoma-X large (Bcl-xL) are proposed to inhibit autophagy by directly binding to the BH3 domain of Beclin 1/Atg6. However, these Bcl-2 family proteins also block the proapoptotic activity of Bcl-2–associated X (Bax) and Bcl-2 homologous antagonist/killer (Bak), and many inducers of autophagy also cause cell death. Therefore, when the mitochondrial-mediated apoptosis pathway is functional, interpretation of such experiments is complicated. To directly test the impact of the endogenous antiapoptotic Bcl-2 family members on autophagy in the absence of apoptosis, we inhibited their activity in cells lacking the essential cell death mediators Bax and Bak. We also used inducible lentiviral vectors to overexpress Bcl-2, Bcl-xL, or Mcl-1 in cells and subjected them to treatments that promote autophagy. In the absence of Bax and Bak, Bcl-2, Bcl-xL, and Mcl-1 had no detectable effect on autophagy or cell death in myeloid or fibroblast cell lines. On the other hand, when Bax and Bak were present, inhibiting the prosurvival Bcl-2 family members stimulated autophagy, but this correlated with increased cell death. In addition, inhibition of autophagy induced by amino acid starvation, etoposide, or interleukin-3 withdrawal did not affect cell death in the absence of Bax and Bak. These results demonstrate that the antiapoptotic Bcl-2 family members do not directly inhibit components of the autophagic pathway but instead affect autophagy indirectly, owing to their inhibition of Bax and Bak. PMID:24912196

  15. Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak.

    PubMed

    Lindqvist, Lisa M; Heinlein, Melanie; Huang, David C S; Vaux, David L

    2014-06-10

    Antiapoptotic B-cell lymphoma 2 (Bcl-2) family members such as Bcl-2, myeloid cell leukemia 1 (Mcl-1), and B-cell lymphoma-X large (Bcl-xL) are proposed to inhibit autophagy by directly binding to the BH3 domain of Beclin 1/Atg6. However, these Bcl-2 family proteins also block the proapoptotic activity of Bcl-2-associated X (Bax) and Bcl-2 homologous antagonist/killer (Bak), and many inducers of autophagy also cause cell death. Therefore, when the mitochondrial-mediated apoptosis pathway is functional, interpretation of such experiments is complicated. To directly test the impact of the endogenous antiapoptotic Bcl-2 family members on autophagy in the absence of apoptosis, we inhibited their activity in cells lacking the essential cell death mediators Bax and Bak. We also used inducible lentiviral vectors to overexpress Bcl-2, Bcl-xL, or Mcl-1 in cells and subjected them to treatments that promote autophagy. In the absence of Bax and Bak, Bcl-2, Bcl-xL, and Mcl-1 had no detectable effect on autophagy or cell death in myeloid or fibroblast cell lines. On the other hand, when Bax and Bak were present, inhibiting the prosurvival Bcl-2 family members stimulated autophagy, but this correlated with increased cell death. In addition, inhibition of autophagy induced by amino acid starvation, etoposide, or interleukin-3 withdrawal did not affect cell death in the absence of Bax and Bak. These results demonstrate that the antiapoptotic Bcl-2 family members do not directly inhibit components of the autophagic pathway but instead affect autophagy indirectly, owing to their inhibition of Bax and Bak.

  16. Organization of the mitochondrial apoptotic BAK pore: oligomerization of the BAK homodimers.

    PubMed

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

    2014-01-31

    The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX "BH3-in-groove homodimer." Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a "worm hole."

  17. Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane

    PubMed Central

    O'Neill, Katelyn L.; Huang, Kai; Zhang, Jingjing; Chen, Yi; Luo, Xu

    2016-01-01

    The mechanism of Bax/Bak activation remains a central question in mitochondria-dependent apoptotic signaling. While it is established that all proapoptotic Bcl-2 homology 3 (BH3)-only proteins bind and neutralize the anti-apoptotic Bcl-2 family proteins, how this neutralization leads to Bax/Bak activation has been actively debated. Here, genome editing was used to generate cells deficient for all eight proapoptotic BH3-only proteins (OctaKO) and those that lack the entire Bcl-2 family (Bcl-2 allKO). Although the OctaKO cells were resistant to most apoptotic stimuli tested, they underwent Bax/Bak-dependent and p53/Rb-independent apoptosis efficiently when both Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins, were inactivated or eliminated. Strikingly, when expressed in the Bcl-2 allKO cells, both Bax and Bak spontaneously associated with the outer mitochondrial membrane (OMM) through their respective helix 9, and this association triggered their homo-oligomerization/activation. Together, these results strongly suggest that the OMM, not BH3-only proteins or p53/Rb, is the long-sought-after direct activator of Bax/Bak following BH3-only-mediated neutralization of anti-apoptotic Bcl-2 proteins. PMID:27056669

  18. Nigericin-induced impairment of autophagic flux in neuronal cells is inhibited by overexpression of Bak.

    PubMed

    Lim, Junghyun; Lee, Yunsu; Kim, Hyun-Wook; Rhyu, Im Joo; Oh, Myung Sook; Youdim, Moussa B H; Yue, Zhenyu; Oh, Young J

    2012-07-06

    Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K(+)/H(+) ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.

  19. Nigericin-induced Impairment of Autophagic Flux in Neuronal Cells Is Inhibited by Overexpression of Bak*

    PubMed Central

    Lim, Junghyun; Lee, Yunsu; Kim, Hyun-Wook; Rhyu, Im Joo; Oh, Myung Sook; Youdim, Moussa B. H.; Yue, Zhenyu; Oh, Young J.

    2012-01-01

    Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K+/H+ ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis. PMID:22493436

  20. 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

  1. Comparison of E-cadherin with STAT3 and apoptosis regulators: Bak and Bcl-xL in endometrioid adenocarcinomas of different ER-alpha immunoprofile.

    PubMed

    Sulkowska, Urszula; Wincewicz, Andrzej; Kanczuga-Koda, Luiza; Koda, Mariusz; Sulkowski, Stanislaw

    2017-09-22

    E-cadherin is a factor of good prognosis in endometrioid adenocarcinomas, while STAT3 is an oncogenic driver of carcinogenesis. E-cadherin, Bak, Bcl-xL and STAT3 were immunohistochemically detected in 78 human endometrioid adenocarcinomas. E-cadherin correlated with STAT3 (p <. 001, r = 0.537) as well as Bak (p = .005, r = 0.314) and Bcl-xL (p = .002, r = 0.340) in the whole study group. In G2 tumors, E-cadherin associated with Bak (p = .021, r = 0.319), Bcl-xL (p = .026, r = 0.309) and STAT3 (p <.001, r = 0.513) but not in G3 adenocarcinomas. E-cadherin correlated with Bak and Bcl-xL in both G1- and estrogen receptor (ER)-negative tumors with significant relation of E-cadherin and STAT3 in G1- and ER-negative tumors. Antigrowth synergy of expression was preserved for antiapoptotic Bak and proliferation-suppressing E-cadherin in IA adenocarcinomas (p = .031, r = 0.342) with no significance between Bak and E-cadherin or STAT3 and emerging correlation between E-cadherin and Bcl-xL in IB + II tumors instead (p = .003, r = 0.472). E-cadherin correlated with Bak and Bcl-xL in ER-positive adenocarcinomas (p =  .002, r = 0.382 and p <.001, r = 0.439, respectively) but not in ER-negative tumors. In conclusion, expression deregulation of studied proteins is reflected in selective loss of correlation between suppressors of tumor growth (E-cadherin and Bak) presumably due to progressing impairment of growth-inhibitory properties of clone of neoplastic cells within higher staging and poorer differentiation.

  2. The X-ray Structure of a BAK Homodimer Reveals an Inhibitory Zinc Binding Site

    SciTech Connect

    Modoveanu,T.; Liu, Q.; Tocilj, A.; Watson, M.; Shore, G.; Gehring, K.

    2006-01-01

    BAK/BAX-mediated mitochondrial outer-membrane permeabilization (MOMP) drives cell death during development and tissue homeostasis from zebrafish to humans. In most cancers, this pathway is inhibited by BCL-2 family antiapoptotic members, which bind and block the action of proapoptotic BCL proteins. We report the 1.5 {angstrom} crystal structure of calpain-proteolysed BAK, cBAK, to reveal a zinc binding site that regulates its activity via homodimerization. cBAK contains an occluded BH3 peptide binding pocket that binds a BID BH3 peptide only weakly . Nonetheless, cBAK requires activation by truncated BID to induce cytochrome c release in mitochondria isolated from bak/bax double-knockout mouse embryonic fibroblasts. The BAK-mediated MOMP is inhibited by low micromolar zinc levels. This inhibition is alleviated by mutation of the zinc-coordination site in BAK. Our results link directly the antiapoptotic effects of zinc to BAK.

  3. Benzo[a]pyrene-7,8-diol-9,10-epoxide causes caspase-mediated apoptosis in H460 human lung cancer cell line.

    PubMed

    Xiao, Hui; Rawal, Malika; Hahm, Eun-Ryeong; Singh, Shivendra V

    2007-11-15

    We have shown previously that wild-type p53 renders H460 human lung cancer cells more sensitive to apoptosis induction by environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), but the mechanism of cell death is not fully understood. The present study provides insights into the mechanism by which BPDE causes apoptosis in H460 cells. Exposure of H460 cells to BPDE resulted in a concentration-dependent apoptotic cell death characterized by cleavage of poly(ADP-ribose)polymerase, DNA condensation, and apoptotic histone-associated DNA fragments released into the cytosol. The BPDE-mediated release of apoptotic histone-associated DNA fragments into the cytosol was also observed in a normal bronchial epithelial cell line BEAS-2B. The BPDE-induced apoptosis in H460 cells correlated with up-regulation of pro-apoptotic protein Bak, downregulation of anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, release of cytochrome c from mitochondria to the cytosol without a change in mitochondrial membrane potential or mitochondrial morphology (electron microscopy), and cleavage of caspase-8, -9, and -3. Ectopic expression of Bcl-2 failed to confer significant protection against BPDE-induced apoptosis in H460 cells. The SV40 immortalized mouse embryonic fibroblasts (MEFs) derived from Bak and Bax double knockout mice, but not Bid knockout mice, were significantly more resistant to BPDE-induced apoptosis compared with the MEFs derived from wild-type mice. The BPDE-induced apoptosis was partially but statistically significantly attenuated in the presence of specific inhibitors of caspase-9 (z-LEHDfmk) and caspase-8 (z-IETDfmk). In conclusion, the present study reveals that BPDE-induced apoptosis in H460 cells is associated with Bak induction and caspase activation but independent of Bcl-2.

  4. Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner.

    PubMed

    Guo, W; Zhang, Y; Ling, Z; Liu, X; Zhao, X; Yuan, Z; Nie, C; Wei, Y

    2015-10-15

    Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.

  5. Host and Viral Factors in HIV-Mediated Bystander Apoptosis

    PubMed Central

    Garg, Himanshu; Joshi, Anjali

    2017-01-01

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

  6. The BH3 α-Helical Mimic BH3-M6 Disrupts Bcl-XL, Bcl-2, and MCL-1 Protein-Protein Interactions with Bax, Bak, Bad, or Bim and Induces Apoptosis in a Bax- and Bim-dependent Manner*

    PubMed Central

    Kazi, Aslamuzzaman; Sun, Jiazhi; Doi, Kenichiro; Sung, Shen-Shu; Takahashi, Yoshinori; Yin, Hang; Rodriguez, Johanna M.; Becerril, Jorge; Berndt, Norbert; Hamilton, Andrew D.; Wang, Hong-Gang; Sebti, Saïd M.

    2011-01-01

    A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-XL, and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-XL and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-XL, Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-XL/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-XL, Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612. PMID:21148306

  7. Nortriptyline induces mitochondria and death receptor-mediated apoptosis in bladder cancer cells and inhibits bladder tumor growth in vivo.

    PubMed

    Yuan, Sheau-Yun; Cheng, Chen-Li; Ho, Hao-Chung; Wang, Shian-Shiang; Chiu, Kun-Yuan; Su, Chung-Kuang; Ou, Yen-Chuan; Lin, Chi-Chen

    2015-08-15

    Nortriptyline (NTP), an antidepressant, has antitumor effects on some human cancer cells, but its effect on human bladder cancer cells is not known. In this study, we used a cell viability assay to demonstrate that NTP is cytotoxic to human TCCSUP and mouse MBT-2 bladder cancer cells in a concentration and time-dependent manner. We also performed cell cycle analysis, annexin V and mitochondrial membrane potential assays, and Western blot analysis to show that NTP inhibits cell growth in these cells by inducing both mitochondria-mediated and death receptor-mediated apoptosis. Specifically, NTP increases the expression of Fas, FasL, FADD, Bax, Bak, and cleaved forms of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. In addition, NTP decreases the expression of Bcl-2, Bcl-xL, BH3 interacting domain death agonist, X-linked inhibitor of apoptosis protein, and survivin. Furthermore, NTP-induced apoptosis is associated with reactive oxygen species (ROS) production, which can be reduced by antioxidants, such as N-acetyl-L-cysteine. Finally, we showed that NTP suppresses tumor growth in mice inoculated with MBT-2 cells. Collectively, our results suggest that NTP induces both intrinsic and extrinsic apoptosis in human and mouse bladder cancer cells and that it may be a clinically useful chemotherapeutic agent for bladder cancer in humans. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Proapoptotic Activities of Protein Disulfide Isomerase (PDI) and PDIA3 Protein, a Role of the Bcl-2 Protein Bak*

    PubMed Central

    Zhao, Guoping; Lu, Huayi; Li, Chi

    2015-01-01

    Protein disulfide isomerase (PDI) family proteins are classified as enzymatic chaperones for reconstructing misfolded proteins. Previous studies have shown that several PDI members possess potential proapoptotic functions. However, the detailed molecular mechanisms of PDI-mediated apoptosis are not completely known. In this study, we investigated how two members of PDI family, PDI and PDIA3, modulate apoptotic signaling. Inhibiting PDI and PDIA3 activities pharmacologically alleviates apoptosis induced by various apoptotic stimuli. Although a decrease of PDIA3 expression alleviates apoptotic responses, overexpression of PDIA3 exacerbates apoptotic signaling. Importantly, Bak, but not Bax, is essential for PDIA3-induced proapoptotic signaling. Furthermore, both purified PDI and PDIA3 proteins induce Bak-dependent, but not Bax-dependent, mitochondrial outer membrane permeabilization in vitro, probably through triggering Bak oligomerization on mitochondria. Our results suggest that both of PDI and PDIA3 possess Bak-dependent proapoptotic function through inducing mitochondrial outer membrane permeabilization, which provides a new mechanism linking ER chaperone proteins and apoptotic signaling. PMID:25697356

  9. Loss-of-function of Arabidopsis receptor-like kinase BIR1 activates cell death and defense responses mediated by BAK1 and SOBIR1.

    PubMed

    Liu, Yanan; Huang, Xingchuan; Li, Meng; He, Ping; Zhang, Yuelin

    2016-11-01

    The Arabidopsis receptor-like kinase (RLK) BIR1 (BAK1-INTERACTING RECEPTOR-LIKE KINASE 1) functions as a negative regulator of plant immunity. Previous work showed that loss-of-function of BIR1 leads to constitutive activation of cell death and defense responses. These autoimmune phenotypes are partially dependent on another RLK, SOBIR1. In order to identify additional components involved in the BIR1-regulated plant defense signaling pathway, a suppressor screen was carried out in the bir1-1 pad4-1 mutant background. Mutations in the suppressor mutants were identified by genetic mapping and re-sequencing of the mutant genomes. A number of suppressor mutants were found to carry mutations in an additional RLK, BAK1, indicating that BAK1 is required for activation of cell death and defense responses in bir1-1. Co-immunoprecipitation analysis revealed that BAK1 and SOBIR1 associate with each other in planta when the function of BIR1 is compromised. Although BAK1 was previously characterized as a negative regulator of cell death, our study highlights a novel role of BAK1 in promoting cell death and defense responses in conjunction with SOBIR1. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  10. Heterodimerization of BAK and MCL-1 activated by detergent micelles.

    PubMed

    Liu, Qian; Gehring, Kalle

    2010-12-24

    BAK is a key protein mediating mitochondrial outer membrane permeabilization; however, its behavior in the membrane is poorly understood. Here, we characterize the conformational changes in BAK and MCL-1 using detergents to mimic the membrane environment and study their interaction by in vitro pulldown experiments, size exclusion chromatography, titration calorimetry, and NMR spectroscopy. The nonionic detergent IGEPAL has little impact on the structure of MCL-1 but induces a conformational change in BAK, whereby its BH3 region is able to engage the hydrophobic groove of MCL-1. Although the zwitterionic detergent CHAPS induces only minor conformational changes in both proteins, it is still able to initiate heterodimerization. The complex of MCL-1 and BAK can be disrupted by a BID-BH3 peptide, which acts through binding to MCL-1, but a mutant peptide, BAK-BH3-L78A, with low affinity for MCL-1 failed to dissociate the complex. The mutation L78A in BAK prevented binding to MCL-1, thus demonstrating the essential role of the BH3 region of BAK in its regulation by MCL-1. Our results validate the current models for the activation of BAK and highlight the potential value of small molecule inhibitors that target MCL-1 directly.

  11. The peroxisomes strike BAK: Regulation of peroxisome integrity by the Bcl-2 family.

    PubMed

    Chipuk, Jerry Edward; Luna-Vargas, Mark P A

    2017-03-06

    Within the mitochondrial pathway of apoptosis, VDAC2 controls both the localization and proapoptotic activity of BAK. In this issue, Hosoi et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201605002) find that loss of VDAC2 diverts BAK into peroxisome membranes, revealing the ability of BAK to control peroxisome membrane integrity and the release of soluble peroxisomal matrix proteins. © 2017 Chipuk and Luna-Vargas.

  12. Constitutive BAK activation as a determinant of drug sensitivity in malignant lymphohematopoietic cells

    PubMed Central

    Dai, Haiming; Ding, Husheng; Meng, X. Wei; Peterson, Kevin L.; Schneider, Paula A.; Karp, Judith E.; Kaufmann, Scott H.

    2015-01-01

    Mitochondrial outer membrane permeabilization (MOMP), a key step in the intrinsic apoptotic pathway, is incompletely understood. Current models emphasize the role of BH3-only BCL2 family members in BAX and BAK activation. Here we demonstrate concentration-dependent BAK autoactivation under cell-free conditions and provide evidence that this autoactivation plays a key role in regulating the intrinsic apoptotic pathway in intact cells. In particular, we show that up to 80% of BAK (but not BAX) in lymphohematopoietic cell lines is oligomerized and bound to anti-apoptotic BCL2 family members in the absence of exogenous death stimuli. The extent of this constitutive BAK oligomerization is diminished by BAK knockdown and unaffected by BIM or PUMA down-regulation. Further analysis indicates that sensitivity of cells to BH3 mimetics reflects the identity of the anti-apoptotic proteins to which BAK is constitutively bound, with extensive BCLXL•BAK complexes predicting navitoclax sensitivity, and extensive MCL1•BAK complexes predicting A1210477 sensitivity. Moreover, high BAK expression correlates with sensitivity of clinical acute myelogenous leukemia to chemotherapy, whereas low BAK levels correlate with resistance and relapse. Collectively, these results inform current understanding of MOMP and provide new insight into the ability of BH3 mimetics to induce apoptosis without directly activating BAX or BAK. PMID:26494789

  13. DPI induces mitochondrial superoxide-mediated apoptosis.

    PubMed

    Li, Nianyu; Ragheb, Kathy; Lawler, Gretchen; Sturgis, Jennie; Rajwa, Bartek; Melendez, J Andres; Robinson, J Paul

    2003-02-15

    The iodonium compounds diphenyleneiodonium (DPI) and diphenyliodonium (IDP) are well-known phagocyte NAD(P)H oxidase inhibitors. However, it has been shown that at high concentrations they can inhibit the mitochondrial respiratory chain as well. Since inhibition of the mitochondrial respiratory chain has been shown to induce superoxide production and apoptosis, we investigated the effect of iodonium compounds on mitochondria-derived superoxide and apoptosis. Mitochondrial superoxide production was measured on both cultured cells and isolated rat-heart submitochondrial particles. Mitochondria function was examined by monitoring mitochondrial membrane potential. Apoptotic pathways were studied by measuring cytochrome c release and caspase 3 activation. Apoptosis was characterized by detecting DNA fragmentation on agarose gel and measuring propidium iodide- (PI-) stained subdiploid cells using flow cytometry. Our results showed that DPI could induce mitochondrial superoxide production. The same concentration of DPI induced apoptosis by decreasing mitochondrial membrane potential and releasing cytochrome c. Addition of antioxidants or overexpression of MnSOD significantly reduced DPI-induced mitochondrial damage, cytochrome c release, caspase activation, and apoptosis. These observations suggest that DPI can induce apoptosis via induction of mitochondrial superoxide. DPI-induced mitochondrial superoxide production may prove to be a useful model to study the signaling pathways of mitochondrial superoxide.

  14. Active Bax and Bak are functional holins

    PubMed Central

    Pang, Xiaming; Moussa, Samir H.; Targy, Natalie M.; Bose, Jeffrey L.; George, Nicholas M.; Gries, Casey; Lopez, Hernando; Zhang, Liqiang; Bayles, Kenneth W.; Young, Ry; Luo, Xu

    2011-01-01

    The mechanism of Bax/Bak-dependent mitochondrial outer membrane permeabilization (MOMP), a central apoptotic event primarily controlled by the Bcl-2 family proteins, remains not well understood. Here, we express active Bax/Bak in bacteria, the putative origin of mitochondria, and examine their functional similarities to the λ bacteriophage (λ) holin. As critical effectors for bacterial lysis, holin oligomers form membrane lesions, through which endolysin, a muralytic enzyme, escapes the cytoplasm to attack the cell wall at the end of the infection cycle. We found that active Bax/Bak, but not any other Bcl-2 family protein, displays holin behavior, causing bacterial lysis by releasing endolysin in an oligomerization-dependent manner. Strikingly, replacing the holin gene with active alleles of Bax/Bak results in plaque-forming phages. Furthermore, we provide evidence that active Bax produces large membrane holes, the size of which is controlled by structural elements of Bax. Notably, lysis by active Bax is inhibited by Bcl-xL, and the lysis activity of the wild-type Bax is stimulated by a BH3-only protein. Together, these results mechanistically link MOMP to holin-mediated hole formation in the bacterial plasma membrane. PMID:22006182

  15. Type I Interferon Upregulates Bak and Contributes to T Cell Loss during Human Immunodeficiency Virus (HIV) Infection

    PubMed Central

    Fraietta, Joseph A.; Mueller, Yvonne M.; Yang, Guibin; Boesteanu, Alina C.; Gracias, Donald T.; Do, Duc H.; Hope, Jennifer L.; Kathuria, Noshin; McGettigan, Shannon E.; Lewis, Mark G.; Giavedoni, Luis D.; Jacobson, Jeffrey M.; Katsikis, Peter D.

    2013-01-01

    The role of Type I interferon (IFN) during pathogenic HIV and SIV infections remains unclear, with conflicting observations suggesting protective versus immunopathological effects. We therefore examined the effect of IFNα/β on T cell death and viremia in HIV infection. Ex vivo analysis of eight pro- and anti-apoptotic molecules in chronic HIV-1 infection revealed that pro-apoptotic Bak was increased in CD4+ T cells and correlated directly with sensitivity to CD95/Fas-mediated apoptosis and inversely with CD4+ T cell counts. Apoptosis sensitivity and Bak expression were primarily increased in effector memory T cells. Knockdown of Bak by RNA interference inhibited CD95/Fas-induced death of T cells from HIV-1-infected individuals. In HIV-1-infected patients, IFNα-stimulated gene expression correlated positively with ex vivo T cell Bak levels, CD95/Fas-mediated apoptosis and viremia and negatively with CD4+ T cell counts. In vitro IFNα/β stimulation enhanced Bak expression, CD95/Fas expression and CD95/Fas-mediated apoptosis in healthy donor T cells and induced death of HIV-specific CD8+ T cells from HIV-1-infected patients. HIV-1 in vitro sensitized T cells to CD95/Fas-induced apoptosis and this was Toll-like receptor (TLR)7/9- and Type I IFN-dependent. This sensitization by HIV-1 was due to an indirect effect on T cells, as it occurred in peripheral blood mononuclear cell cultures but not purified CD4+ T cells. Finally, peak IFNα levels and viral loads correlated negatively during acute SIV infection suggesting a potential antiviral effect, but positively during chronic SIV infection indicating that either the virus drives IFNα production or IFNα may facilitate loss of viral control. The above findings indicate stage-specific opposing effects of Type I IFNs during HIV-1 infection and suggest a novel mechanism by which these cytokines contribute to T cell depletion, dysregulation of cellular immunity and disease progression. PMID:24130482

  16. Type I interferon upregulates Bak and contributes to T cell loss during human immunodeficiency virus (HIV) infection.

    PubMed

    Fraietta, Joseph A; Mueller, Yvonne M; Yang, Guibin; Boesteanu, Alina C; Gracias, Donald T; Do, Duc H; Hope, Jennifer L; Kathuria, Noshin; McGettigan, Shannon E; Lewis, Mark G; Giavedoni, Luis D; Jacobson, Jeffrey M; Katsikis, Peter D

    2013-01-01

    The role of Type I interferon (IFN) during pathogenic HIV and SIV infections remains unclear, with conflicting observations suggesting protective versus immunopathological effects. We therefore examined the effect of IFNα/β on T cell death and viremia in HIV infection. Ex vivo analysis of eight pro- and anti-apoptotic molecules in chronic HIV-1 infection revealed that pro-apoptotic Bak was increased in CD4+ T cells and correlated directly with sensitivity to CD95/Fas-mediated apoptosis and inversely with CD4+ T cell counts. Apoptosis sensitivity and Bak expression were primarily increased in effector memory T cells. Knockdown of Bak by RNA interference inhibited CD95/Fas-induced death of T cells from HIV-1-infected individuals. In HIV-1-infected patients, IFNα-stimulated gene expression correlated positively with ex vivo T cell Bak levels, CD95/Fas-mediated apoptosis and viremia and negatively with CD4+ T cell counts. In vitro IFNα/β stimulation enhanced Bak expression, CD95/Fas expression and CD95/Fas-mediated apoptosis in healthy donor T cells and induced death of HIV-specific CD8+ T cells from HIV-1-infected patients. HIV-1 in vitro sensitized T cells to CD95/Fas-induced apoptosis and this was Toll-like receptor (TLR)7/9- and Type I IFN-dependent. This sensitization by HIV-1 was due to an indirect effect on T cells, as it occurred in peripheral blood mononuclear cell cultures but not purified CD4+ T cells. Finally, peak IFNα levels and viral loads correlated negatively during acute SIV infection suggesting a potential antiviral effect, but positively during chronic SIV infection indicating that either the virus drives IFNα production or IFNα may facilitate loss of viral control. The above findings indicate stage-specific opposing effects of Type I IFNs during HIV-1 infection and suggest a novel mechanism by which these cytokines contribute to T cell depletion, dysregulation of cellular immunity and disease progression.

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

    PubMed

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

    2009-02-19

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

  18. Penfluridol suppresses pancreatic tumor growth by autophagy-mediated apoptosis

    PubMed Central

    Ranjan, Alok; Srivastava, Sanjay K.

    2016-01-01

    Pancreatic tumors exhibit enhanced autophagy as compared to any other cancer, making it resistant to chemotherapy. We evaluated the effect of penfluridol against pancreatic cancer. Penfluridol treatment induced apoptosis and inhibited the growth of Panc-1, BxPC-3 and AsPC-1, pancreatic cancer cells with IC50 ranging between 6–7 μM after 24 h of treatment. Significant autophagy was induced by penfluridol treatment in pancreatic cancer cells. Punctate LC3B and autophagosomes staining confirmed autophagy. Inhibiting autophagy by chloroquine, bafilomycin, 3-methyladenine or LC3BsiRNA, significantly blocked penfluridol-induced apoptosis, suggesting that autophagy lead to apoptosis in our model. Penfluridol treatment suppressed the growth of BxPC-3 tumor xenografts by 48% as compared to 17% when treated in combination with chloroquine. Similarly, penfluridol suppressed the growth of AsPC-1 tumors by 40% versus 16% when given in combination with chloroquine. TUNEL staining and caspase-3 cleavage revealed less apoptosis in the tumors from mice treated with penfluridol and chloroquine as compared to penfluridol alone. Penfluridol treatment also suppressed the growth of orthotopically implanted Panc-1 tumors by 80% by inducing autophagy-mediated apoptosis in the tumors. These studies established that penfluridol inhibits pancreatic tumor growth by autophagy-mediated apoptosis. Since penfluridol is already in clinic, positive findings from our study will accelerate its clinical development. PMID:27189859

  19. Functional divergence of BAK1 genes from Brassica rapa in regulating plant architecture.

    PubMed

    Zhang, S; Li, C; Li, Q; Wang, Q N; Huang, S H; Zhang, Y F; Wang, X F

    2015-11-19

    BAK1 is a co-receptor of BRI1 in early signaling pathways mediated by brassinosteroids (BRs) and is thought to play a major role in plant growth and development. As the role of BAK1 has not yet been fully elucidated then further research is required to explore its potential for use in genetic modification to improve crops. In this study, three BAK1 genes from the amphidiploid species Brassica rapa were isolated and their kinase functions were predicted following DNA sequence analysis. A bioinformatic analysis revealed that two genes, BrBAK1-1 and BrBAK1-8, shared a conserved kinase domain and 5 tandem leucine-rich repeats (LRRs) that are characteristic of a BAK1 receptor for BR perception, whereas the third gene, BrBAK1-3, was deficient for a signal peptide, but had 4 leucine zippers and 3 leucine-rich repeats (LRRs) in an extracellular domain. All three BrBAK1 kinases localized on the cellular membrane. Ectopic expression of each BrBAK1 gene in BR-insensitive (bri1-5 mutant) Arabidopsis plants indicated that BrBAK1-1 and BrBAK1-8 were functional homologues of AtBAK1 based on the rescue of growth in the bri1-5 mutant. Overexpression of BrBAK1-3 caused a severe dwarf phenotype resembling the phenotype of null BRI1 alleles. The results here suggest there are significant differences among the three BrBAK1 kinases for their effects on plant architecture. This conclusion has important implications for genetic modification of B. rapa.

  20. Caspase-9 mediates Puma activation in UCN-01-induced apoptosis.

    PubMed

    Nie, C; Luo, Y; Zhao, X; Luo, N; Tong, A; Liu, X; Yuan, Z; Wang, C; Wei, Y

    2014-10-30

    The protein kinase inhibitor 7-hydroxystaurosporine (UCN-01) is one of the most potent and frequently used proapoptotic stimuli. The BH3-only molecule of Bcl-2 family proteins has been reported to contribute to UCN-01-induced apoptosis. Here we have found that UCN-01 triggers Puma-induced mitochondrial apoptosis pathway. Our data confirmed that Akt-FoxO3a pathway mediated Puma activation. Importantly, we elucidate the detailed mechanisms of Puma-induced apoptosis. Our data have also demonstrated that caspase-9 is a decisive molecule of Puma induction after UCN-01 treatment. Caspase-9 mediates apoptosis through two kinds of feedback loops. On the one hand, caspase-9 enhances Puma activation by cleaving Bcl-2 and Bcl-xL independent of caspase-3. On the other hand, caspase-9 directly activated caspase-3 in the presence of caspase-3. Caspase-3 could cleave XIAP in an another positive feedback loop to further sensitize cancer cells to UCN-01-induced apoptosis. Therefore, caspase-9 mediates Puma activation to determine the threshold for overcoming chemoresistance in cancer cells.

  1. A Bak-dependent mitochondrial amplification step contributes to Smac mimetic/glucocorticoid-induced necroptosis.

    PubMed

    Rohde, Katharina; Kleinesudeik, Lara; Roesler, Stefanie; Löwe, Oliver; Heidler, Juliana; Schröder, Katrin; Wittig, Ilka; Dröse, Stefan; Fulda, Simone

    2017-01-01

    Necroptosis is a form of programmed cell death that critically depends on RIP3 and MLKL. However, the contribution of mitochondria to necroptosis is still poorly understood. In the present study, we discovered that mitochondrial perturbations play a critical role in Smac mimetic/Dexamethasone (Dexa)-induced necroptosis independently of death receptor ligands. We demonstrate that the Smac mimetic BV6 and Dexa cooperate to trigger necroptotic cell death in acute lymphoblastic leukemia (ALL) cells that are deficient in caspase activation due to absent caspase-8 expression or pharmacological inhibition by the caspase inhibitor zVAD.fmk, since genetic silencing or pharmacological inhibition of RIP3 or MLKL significantly rescue BV6/Dexa-induced necroptosis. In addition, RIP3 or MLKL knockout mouse embryonic fibroblasts (MEFs) are protected from BV6/Dexa/zVAD.fmk-induced cell death. In contrast, antagonistic antibodies against the death receptor ligands TNFα, TRAIL or CD95 ligand fail to rescue BV6/Dexa-triggered cell death. Kinetic studies revealed that prior to cell death BV6/Dexa treatment causes hyperpolarization of the mitochondrial membrane potential (MMP) followed by loss of MMP, reactive oxygen species (ROS) production, Bak activation and disruption of mitochondrial respiration. Importantly, knockdown of Bak significantly reduces BV6/Dexa-induced loss of MMP and delays cell death, but not ROS production, whereas ROS scavengers attenuate Bak activation, indicating that ROS production occurs upstream of BV6/Dexa-mediated Bak activation. Consistently, BV6/Dexa treatment causes oxidative thiol modifications of Bak protein. Intriguingly, knockdown or knockout of RIP3 or MLKL protect ALL cells or MEFs from BV6/Dexa-induced ROS production, Bak activation, drop of MMP and disruption of mitochondrial respiration, demonstrating that these mitochondrial events depend on RIP3 and MLKL. Thus, mitochondria might serve as an amplification step in BV6/Dexa-induced necroptosis

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

    PubMed Central

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

    2014-01-01

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

  3. Impact of the combined loss of BOK, BAX and BAK on the hematopoietic system is slightly more severe than compound loss of BAX and BAK.

    PubMed

    Ke, F; Grabow, S; Kelly, G L; Lin, A; O'Reilly, L A; Strasser, A

    2015-10-22

    It is well established that BAX and BAK play crucial, overlapping roles in the intrinsic pathway of apoptosis. Gene targeted mice lacking both BAX and BAK have previously been generated, but the majority of these animals died perinatally. BOK is a poorly studied relative of BAX and BAK that shares extensive amino acid sequence homology to both proteins, but its function remains largely unclear to date. To determine whether BOK plays an overlapping role with BAX and BAK, we utilized a hematopoietic reconstitution model where lethally irradiated wild type mice were transplanted with Bok(-/-)Bax(-/-)Bak(-/-) triple knockout (TKO) fetal liver cells, and compared alongside mice reconstituted with a Bax(-/-)Bak(-/-) double knockout (DKO) hematopoietic compartment. We report here that mice with a TKO and DKO hematopoietic system died at a similar rate and much earlier than control animals, mostly due to severe autoimmune pathology. Both TKO and DKO reconstituted mice also had altered frequencies of various leukocyte subsets in the thymus, bone marrow and spleen, displayed leukocyte infiltrates and autoimmune pathology in multiple tissues, as well as elevated levels of anti-nuclear autoantibodies. Interestingly, the additional deletion of BOK (on top of BAX and BAK loss) led to a further increase in peripheral blood lymphocytes, as well as enhanced lymphoid infiltration in some organs. These findings suggest that BOK may have some functions that are redundant with BAX and BAK in the hematopoietic system.

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

    PubMed

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

    2016-02-01

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

  5. Essential role of BAX,BAK in B cell homeostasis and prevention of autoimmune disease

    PubMed Central

    Takeuchi, Osamu; Fisher, Jill; Suh, Heikyung; Harada, Hisashi; Malynn, Barbara A.; Korsmeyer, Stanley J.

    2005-01-01

    B cell homeostasis is maintained by a balance between the continual generation of new cells and their elimination. Here we show proapoptotic BCL-2 family members BAX and BAK are essential for regulating the number of B cells at both immature and mature developmental stages. BAX and BAK are critical mediators of B cell death induced by multiple stimuli. In addition, BAX- and BAK-deficient B cells display defective cell cycle progression to B cell receptor crosslinking and lipopolysaccharide, but not to CpG–DNA. Furthermore, inducible deletion of Bax and Bak in adult mice results in the development of severe autoimmune disease. PMID:16055554

  6. Artemisinin induces A549 cell apoptosis dominantly via a reactive oxygen species-mediated amplification activation loop among caspase-9, -8 and -3.

    PubMed

    Gao, Weijie; Xiao, Fenglian; Wang, Xiaoping; Chen, Tongsheng

    2013-10-01

    This report is designed to explore the roles of caspase-8, -9 and -3 in artemisinin (ARTE)-induced apoptosis in non-small cell lung cancer cells (A549 cells). ARTE induced reactive oxygen species (ROS)-mediated apoptosis in dose- and time-dependent fashion. Although ARTE treatment did not induce Bid cleavage and significant loss of mitochondrial membrane potential, it induced release of Smac and AIF but not cytochrome c from mitochondria, and silencing of Bak but not Bax significantly prevented ARTE-induced cytotoxicity. Moreover, ARTE treatment induced ROS-dependent activation of caspase-9, -8 and -3. Of the utmost importance, silencing or inhibiting any one of caspase-8, -9 and -3 almost completely prevented ARTE-induced activation of all the three caspases and remarkably abrogated the cytotoxicity of ARTE, suggesting that ARTE triggered an amplification activation loop among caspase-9, -8 and -3. Collectively, our data demonstrate that ARTE induces a ROS-mediated amplification activation loop among caspase-9, -8 and -3 to dominantly mediate the apoptosis of A549 cells.

  7. Mechanisms of strain-mediated mesenchymal stem cell apoptosis.

    PubMed

    Kearney, E M; Prendergast, P J; Campbell, V A

    2008-12-01

    Mechanical conditioning of mesenchymal stem cells (MSCs) has been adopted widely as a biophysical signal to aid tissue engineering applications. The replication of in vivo mechanical signaling has been used in in vitro environments to regulate cell differentiation, and extracellular matrix synthesis, so that both the chemical and mechanical properties of the tissue-engineered construct are compatible with the implant site. While research in these areas contributes to tissue engineering, the effects of mechanical strain on MSC apoptosis remain poorly defined. To evaluate the effects of uniaxial cyclic tensile strain on MSC apoptosis and to investigate mechanotransduction associated with strain-mediated cell death, MSCs seeded on a 2D silicone membrane were stimulated by a range of strain magnitudes for 3 days. Mechanotransduction was investigated using the stretch-activated cation channel blocker gadolinium chloride, the L-type voltage-activated calcium channel blocker nicardipine, the c-jun NH(2)-terminal kinase (JNK) blocker D-JNK inhibitor 1, and the calpain inhibitor MDL 28170. Apoptosis was assessed through DNA fragmentation using the terminal deoxynucleotidyl transferase mediated-UTP-end nick labeling method. Results demonstrated that tensile strains of 7.5% or greater induce apoptosis in MSCs. L-type voltage-activated calcium channels coupled mechanical stress to activation of calpain and JNK, which lead to apoptosis through DNA fragmentation. The definition of the in vitro boundary conditions for tensile strain and MSCs along with a proposed mechanism for apoptosis induced by mechanical events positively contributes to the development of MSC biology, bioreactor design for tissue engineering, and development of computational methods for mechanobiology.

  8. Betulin induces reactive oxygen species-dependent apoptosis in human gastric cancer SGC7901 cells.

    PubMed

    Li, Yang; Liu, Xiaokang; Jiang, Dan; Lin, Yingjia; Wang, Yushi; Li, Qing; Liu, Linlin; Jin, Ying-Hua

    2016-09-01

    Betulin, an abundant natural compound, significantly inhibited the cell viability of advanced human gastric cancer SGC7901 cells. Mechanism study demonstrated that betulin induced apoptosis through mitochondrial Bax and Bak accumulation-mediated intrinsic apoptosis pathway. Downregulation of the anti-apoptosis proteins Bcl-2 and XIAP was involved during betulin-induced cell apoptosis. Reactive oxygen species (ROS) was generated in cells after betulin treatment in a time- and dose-dependent manner. Addition of antioxidant N-acetyl-L-cysteine (NAC) significantly attenuated betulin-induced ROS generation as well as Bcl-2 and XIAP downregulation. The mitochondrial accumulation of Bax and Bak, as well as caspase activity, was also remarkably inhibited by NAC treatment, indicating that ROS are important signaling intermediates that lead to betulin-induced apoptosis by modulating multiple apoptosis-regulating proteins in SGC7901 cells.

  9. MicroRNA-125b Confers the Resistance of Breast Cancer Cells to Paclitaxel through Suppression of Pro-apoptotic Bcl-2 Antagonist Killer 1 (Bak1) Expression*

    PubMed Central

    Zhou, Ming; Liu, Zixing; Zhao, Yuhua; Ding, Yan; Liu, Hao; Xi, Yaguang; Xiong, Wei; Li, Guiyuan; Lu, Jianrong; Fodstad, Oystein; Riker, Adam I.; Tan, Ming

    2010-01-01

    Paclitaxel (Taxol) is an effective chemotherapeutic agent for treatment of cancer patients. Despite impressive initial clinical responses, the majority of patients eventually develop some degree of resistance to Taxol-based therapy. The mechanisms underlying cancer cells resistance to Taxol are not fully understood. MicroRNA (miRNA) has emerged to play important roles in tumorigenesis and drug resistance. However, the interaction between the development of Taxol resistance and miRNA has not been previously explored. In this study we utilized a miRNA array to compare the differentially expressed miRNAs in Taxol-resistant and their Taxol-sensitive parental cells. We verified that miR-125b, miR-221, miR-222, and miR-923 were up-regulated in Taxol-resistant cancer cells by real-time PCR. We further investigated the role and mechanisms of miR-125b in Taxol resistance. We found that miR-125b was up-regulated in Taxol-resistant cells, causing a marked inhibition of Taxol-induced cytotoxicity and apoptosis and a subsequent increase in the resistance to Taxol in cancer cells. Moreover, we demonstrated that the pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) is a direct target of miR-125b. Down-regulation of Bak1 suppressed Taxol-induced apoptosis and led to an increased resistance to Taxol. Restoring Bak1 expression by either miR-125b inhibitor or re-expression of Bak1 in miR-125b-overexpressing cells recovered Taxol sensitivity, overcoming miR-125-mediated Taxol resistance. Taken together, our data strongly support a central role for miR-125b in conferring Taxol resistance through the suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming Taxol resistance in a number of different tumor histologies. PMID:20460378

  10. Evaluation of the BH3-only protein Puma as a direct Bak activator.

    PubMed

    Dai, Haiming; Pang, Yuan-Ping; Ramirez-Alvarado, Marina; Kaufmann, Scott H

    2014-01-03

    Interactions among Bcl-2 family proteins play critical roles in cellular life and death decisions. Previous studies have established the BH3-only proteins Bim, tBid, and Noxa as "direct activators" that are able to directly initiate the oligomerization and activation of Bak and/or Bax. Earlier studies of Puma have yielded equivocal results, with some concluding that it also acts as a direct activator and other studies suggesting that it acts solely as a sensitizer BH3-only protein. In the present study we examined the interaction of Puma BH3 domain or full-length protein with Bak by surface plasmon resonance, assessed Bak oligomerization status by cross-linking followed by immunoblotting, evaluated the ability of the Puma BH3 domain to induce Bak-mediated permeabilization of liposomes and mitochondria, and determined the effect of wild type and mutant Puma on cell viability in a variety of cellular contexts. Results of this analysis demonstrate high affinity (KD = 26 ± 5 nM) binding of the Puma BH3 domain to purified Bak ex vivo, leading to Bak homo-oligomerization and membrane permeabilization. Mutations in Puma that inhibit (L141E/M144E/L148E) or enhance (M144I/A145G) Puma BH3 binding to Bak also produce corresponding alterations in Bak oligomerization, Bak-mediated membrane permeabilization and, in a cellular context, Bak-mediated killing. Collectively, these results provide strong evidence that Puma, like Bim, Noxa, and tBid, is able to act as a direct Bak activator.

  11. Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice.

    PubMed

    Vandenberg, C J; Josefsson, E C; Campbell, K J; James, C; Lawlor, K E; Kile, B T; Cory, S

    2014-05-01

    Bax and Bak are critical effectors of apoptosis. Although both are widely expressed and usually functionally redundant, recent studies suggest that Bak has particular importance in certain cell types. Genetic and biochemical studies indicate that Bak activation is prevented primarily by Mcl-1 and Bcl-xL, whereas Bax is held in check by all pro-survival Bcl-2 homologues, including Bcl-2 itself. In this study, we have investigated whether loss of Bak or elevated Mcl-1 modulates haemopoietic abnormalities provoked by overexpression of Bcl-2. The Mcl-1 transgene had little impact, probably because the expression level was insufficient to effectively reduce Bak activation. However, loss of Bak enhanced lymphocytosis in vavP-BCL-2 transgenic mice and increased resistance of their thymocytes to some cytotoxic agents, implying that Bak-specific signals can be triggered in certain lymphoid populations. Nevertheless, lack of Bak had no significant impact on thymic abnormalities in vavP-BCL-2tg mice, which kinetic analysis suggested was due to accumulation of self-reactive thymocytes that resist deletion. Intriguingly, although Bak(-/-) mice have elevated platelet counts, Bak(-/-)vavP-BCL-2 mice, like vavP-BCL-2 littermates, were thrombocytopaenic. To clarify why, the vavP-BCL-2 platelet phenotype was scrutinised more closely. Platelet life span was found to be elevated in vavP-BCL-2 mice, which should have provoked thrombocytosis, as in Bak(-/-) mice. Analysis of bone marrow chimaeric mice suggested the low platelet phenotype was due principally to extrinsic factors. Following splenectomy, blood platelets remained lower in vavP-BCL-2 than wild-type mice. However, in Rag1(-/-) BCL-2tg mice, platelet levels were normal, implying that elevated lymphocytes are primarily responsible for BCL-2tg-induced thrombocytopaenia.

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

    PubMed

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

    2015-01-01

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

  13. Parthenolide suppresses pancreatic cell growth by autophagy-mediated apoptosis

    PubMed Central

    Liu, Weifeng; Wang, Xinshuai; Sun, Junjun; Yang, Yanhui; Li, Wensheng; Song, Junxin

    2017-01-01

    Pancreatic cancer is an aggressive malignancy and is unresponsive to conventional chemotherapies. Parthenolide, a sesquiterpene lactone isolated from feverfew, has exhibited potent anticancer effects against various cancers. The purpose of this report was to investigate the effect and underlying mechanism of parthenolide in human pancreatic cancer Panc-1 and BxPC3 cells. The results demonstrated that parthenolide suppressed the growth and induced apoptosis of Panc-1 and BxPC3 pancreatic cancer cells with the half maximal inhibitory concentration (IC50) ranging between 7 and 9 μM after 24 h of treatment. Significant autophagy was induced by parthenolide treatment in pancreatic cancer cells. Parthenolide treatment concentration-dependently increased the percentage of autophagic cells and significantly increased the expression levels of p62/SQSTM1, Beclin 1, and LC3II in Panc-1 cells. Punctate LC3II staining confirmed autophagy. Furthermore, inhibiting autophagy by chloroquine, 3-methyladenine, or LC3II siRNA significantly blocked parthenolide-induced apoptosis, suggesting that parthenolide induced apoptosis through autophagy in this study. In conclusion, these studies established that parthenolide inhibits pancreatic cell growth by autophagy-mediated apoptosis. Data of the present study suggest that parthenolide can serve as a potential chemotherapeutic agent for pancreatic cancer. PMID:28176967

  14. Parthenolide suppresses pancreatic cell growth by autophagy-mediated apoptosis.

    PubMed

    Liu, Weifeng; Wang, Xinshuai; Sun, Junjun; Yang, Yanhui; Li, Wensheng; Song, Junxin

    2017-01-01

    Pancreatic cancer is an aggressive malignancy and is unresponsive to conventional chemotherapies. Parthenolide, a sesquiterpene lactone isolated from feverfew, has exhibited potent anticancer effects against various cancers. The purpose of this report was to investigate the effect and underlying mechanism of parthenolide in human pancreatic cancer Panc-1 and BxPC3 cells. The results demonstrated that parthenolide suppressed the growth and induced apoptosis of Panc-1 and BxPC3 pancreatic cancer cells with the half maximal inhibitory concentration (IC50) ranging between 7 and 9 μM after 24 h of treatment. Significant autophagy was induced by parthenolide treatment in pancreatic cancer cells. Parthenolide treatment concentration-dependently increased the percentage of autophagic cells and significantly increased the expression levels of p62/SQSTM1, Beclin 1, and LC3II in Panc-1 cells. Punctate LC3II staining confirmed autophagy. Furthermore, inhibiting autophagy by chloroquine, 3-methyladenine, or LC3II siRNA significantly blocked parthenolide-induced apoptosis, suggesting that parthenolide induced apoptosis through autophagy in this study. In conclusion, these studies established that parthenolide inhibits pancreatic cell growth by autophagy-mediated apoptosis. Data of the present study suggest that parthenolide can serve as a potential chemotherapeutic agent for pancreatic cancer.

  15. Ursolic acid mediates photosensitization by initiating mitochondrial-dependent apoptosis

    NASA Astrophysics Data System (ADS)

    Lee, Yuan-Hao; Wang, Exing; Kumar, Neeru; Glickman, Randolph D.

    2013-02-01

    The signaling pathways PI3K/Akt and MAPK play key roles in transcription, translation and carcinogenesis, and may be activated by light exposure. These pathways may be modulated or inhibited by naturally-occurring compounds, such as the triterpenoid, ursolic acid (UA). Previously, the transcription factors p53 and NF-kB, which transactivate mitochondrial apoptosis-related genes, were shown to be differentially modulated by UA. Our current work indicates that UA causes these effects via the mTOR and insulin-mediated pathways. UA-modulated apoptosis, following exposure to UV radiation, is observed to correspond to differential levels of oxidative stress in retinal pigment epithelial (RPE) and skin melanoma (SM) cells. Flow cytometry analysis, DHE (dihydroethidium) staining and membrane permeability assay showed that UA pretreatment potentiated cell cycle arrest and radiation-induced apoptosis selectively on SM cells while DNA photo-oxidative damage (i.e. strand breakage) was reduced, presumably by some antioxidant activity of UA in RPE cells. The UA-mediated NF-κB activation in SM cells was reduced by rapamycin pretreatment, which indicates that these agents exert inter-antagonistic effects in the PI3K/Akt/mTOR pathway. In contrast, the antagonistic effect of UA on the PI3K/Akt pathway was reversed by insulin leading to greater NF-κB and p53 activation in RPE cells. MitoTracker, a mitochondrial functional assay, indicated that mitochondria in RPE cells experienced reduced oxidative stress while those in SM cells exhibited increased oxidative stress upon UA pretreatment. When rapamycin administration was followed by UA, mitochondrial oxidative stress was increased in RPE cells but decreased in SM cells. These results indicate that UA modulates p53 and NF-κB, initiating a mitogenic response to radiation that triggers mitochondria-dependent apoptosis.

  16. High Bak Expression Is Associated with a Favorable Prognosis in Breast Cancer and Sensitizes Breast Cancer Cells to Paclitaxel

    PubMed Central

    Luo, Yanwei; Wang, Xinye; Wang, Heran; Xu, Yang; Wen, Qiuyuan; Fan, Songqing; Zhao, Ran; Jiang, Shihe; Yang, Jing; Liu, Yukun; Li, Xiayu; Xiong, Wei; Ma, Jian; Peng, Shuping; Zeng, Zhaoyang; Li, Xiaoling; Phillips, Joshua B.; Li, Guiyuan; Tan, Ming; Zhou, Ming

    2015-01-01

    Breast cancer has become the leading cause of cancer-related death among women. A large number of patients become resistant to drug chemotherapy. Paclitaxel (Taxol) is an effective chemotherapeutic agent used to treat cancer patients. Taxol has been widely used in human malignancies including breast cancer because it can stabilize microtubules resulting in cell death by causing an arrest during the G2/M phase of the cell cycle. Pro-apoptotic Bcl-2 antagonist killer 1 (Bak) plays an important role in Taxol-induced apoptosis in breast cancer. In our present study, we investigated the expression of the Bak protein and clinicopathological correlations in a large sample of breast cancer tissues by immunohistochemistry. We found that the percentage of high scores of Bak expression in breast cancer was significantly lower than that of the non-cancerous breast control tissue. In addition, lower Bak expression was positively associated with the clinical TNM stage of breast cancer with a significant decrease in overall survival compared with those with higher Bak expression especially in the Luminal and HER2 subtypes. Importantly, higher Bak expression predicted a favorable clinical outcome in the cases treated with Taxol indicated by a higher overall survival than that of patients with lower Bak expression especially in Luminal and HER2 subtypes. Furthermore, these results were confirmed in vitro since overexpression of Bak sensitized breast cancer cells to Taxol by inhibiting proliferation and promoting apoptosis; in contrast, downregulation of Bak through siRNA transfection inhibited Taxol induced-apoptosis. Therefore, our results demonstrate that Bak acts as a sensitive biomarker and favorable prognostic factor for Taxol treatment in breast cancer. The restoration of Bak expression would be therapeutically beneficial for Taxol resistant breast cancer patients. PMID:26406239

  17. High Bak Expression Is Associated with a Favorable Prognosis in Breast Cancer and Sensitizes Breast Cancer Cells to Paclitaxel.

    PubMed

    Luo, Yanwei; Wang, Xinye; Wang, Heran; Xu, Yang; Wen, Qiuyuan; Fan, Songqing; Zhao, Ran; Jiang, Shihe; Yang, Jing; Liu, Yukun; Li, Xiayu; Xiong, Wei; Ma, Jian; Peng, Shuping; Zeng, Zhaoyang; Li, Xiaoling; Phillips, Joshua B; Li, Guiyuan; Tan, Ming; Zhou, Ming

    2015-01-01

    Breast cancer has become the leading cause of cancer-related death among women. A large number of patients become resistant to drug chemotherapy. Paclitaxel (Taxol) is an effective chemotherapeutic agent used to treat cancer patients. Taxol has been widely used in human malignancies including breast cancer because it can stabilize microtubules resulting in cell death by causing an arrest during the G2/M phase of the cell cycle. Pro-apoptotic Bcl-2 antagonist killer 1 (Bak) plays an important role in Taxol-induced apoptosis in breast cancer. In our present study, we investigated the expression of the Bak protein and clinicopathological correlations in a large sample of breast cancer tissues by immunohistochemistry. We found that the percentage of high scores of Bak expression in breast cancer was significantly lower than that of the non-cancerous breast control tissue. In addition, lower Bak expression was positively associated with the clinical TNM stage of breast cancer with a significant decrease in overall survival compared with those with higher Bak expression especially in the Luminal and HER2 subtypes. Importantly, higher Bak expression predicted a favorable clinical outcome in the cases treated with Taxol indicated by a higher overall survival than that of patients with lower Bak expression especially in Luminal and HER2 subtypes. Furthermore, these results were confirmed in vitro since overexpression of Bak sensitized breast cancer cells to Taxol by inhibiting proliferation and promoting apoptosis; in contrast, downregulation of Bak through siRNA transfection inhibited Taxol induced-apoptosis. Therefore, our results demonstrate that Bak acts as a sensitive biomarker and favorable prognostic factor for Taxol treatment in breast cancer. The restoration of Bak expression would be therapeutically beneficial for Taxol resistant breast cancer patients.

  18. bak deletion stimulates gastric epithelial proliferation and enhances Helicobacter felis-induced gastric atrophy and dysplasia in mice.

    PubMed

    Duckworth, C A; Abuderman, A A; Burkitt, M D; Williams, J M; O'Reilly, L A; Pritchard, D M

    2015-09-15

    Helicobacter infection causes a chronic superficial gastritis that in some cases progresses via atrophic gastritis to adenocarcinoma. Proapoptotic bak has been shown to regulate radiation-induced apoptosis in the stomach and colon and also susceptibility to colorectal carcinogenesis in vivo. Therefore we investigated the gastric mucosal pathology following H. felis infection in bak-null mice at 6 or 48 wk postinfection. Primary gastric gland culture from bak-null mice was also used to assess the effects of bak deletion on IFN-γ-, TNF-α-, or IL-1β-induced apoptosis. bak-null gastric corpus glands were longer, had increased epithelial Ki-67 expression, and contained fewer parietal and enteroendocrine cells compared with the wild type (wt). In wt mice, bak was expressed at the luminal surface of gastric corpus glands, and this increased 2 wk post-H. felis infection. Apoptotic cell numbers were decreased in bak-null corpus 6 and 48 wk following infection and in primary gland cultures following cytokine administration. Increased gastric epithelial Ki-67 labeling index was observed in C57BL/6 mice after H. felis infection, whereas no such increase was detected in bak-null mice. More severe gastric atrophy was observed in bak-null compared with C57BL/6 mice 6 and 48 wk postinfection, and 76% of bak-null compared with 25% of C57BL/6 mice showed evidence of gastric dysplasia following long-term infection. Collectively, bak therefore regulates gastric epithelial cell apoptosis, proliferation, differentiation, mucosal thickness, and susceptibility to gastric atrophy and dysplasia following H. felis infection. Copyright © 2015 the American Physiological Society.

  19. In Situ Characterization of Bak Clusters Responsible for Cell Death Using Single Molecule Localization Microscopy

    PubMed Central

    Nasu, Yusuke; Benke, Alexander; Arakawa, Satoko; Yoshida, Go J.; Kawamura, Genki; Manley, Suliana; Shimizu, Shigeomi; Ozawa, Takeaki

    2016-01-01

    Apoptosis plays a pivotal role in development and tissue homeostasis in multicellular organisms. Clustering of Bak proteins on the mitochondrial outer membrane is responsible for the induction of apoptosis by evoking a release of pro-apoptotic proteins from mitochondria into cytosol. However, how the protein cluster permeabilizes the mitochondrial membrane remains unclear because elucidation of the cluster characteristics such as size and protein density has been hampered by the diffraction-limited resolution of light microscopy. Here, we describe an approach to quantitatively characterize Bak clusters in situ based on single molecule localization. We showed that Bak proteins form densely packed clusters at the nanoscale on mitochondria during apoptosis. Quantitative analysis based on the localization of each Bak protein revealed that the density of Bak protein is uniform among clusters although the cluster size is highly heterogeneous. Our approach provides unprecedented information on the size and protein density of Bak clusters possibly critical for the permeabilization and is applicable for the analysis of different cluster formations. PMID:27293178

  20. Acidosis Promotes Bcl-2 Family-mediated Evasion of Apoptosis

    PubMed Central

    Ryder, Christopher; McColl, Karen; Zhong, Fei; Distelhorst, Clark W.

    2012-01-01

    Acidosis arises in solid and lymphoid malignancies secondary to altered nutrient supply and utilization. Tumor acidosis correlates with therapeutic resistance, although the mechanism behind this effect is not fully understood. Here we show that incubation of lymphoma cell lines in acidic conditions (pH 6.5) blocks apoptosis induced by multiple cytotoxic metabolic stresses, including deprivation of glucose or glutamine and treatment with dexamethasone. We sought to examine the role of the Bcl-2 family of apoptosis regulators in this process. Interestingly, we found that acidic culture causes elevation of both Bcl-2 and Bcl-xL, while also attenuating glutamine starvation-induced elevation of p53-up-regulated modulator of apoptosis (PUMA) and Bim. We confirmed with knockdown studies that these shifts direct survival decisions during starvation and acidosis. Importantly, the promotion of a high anti- to pro-apoptotic Bcl-2 family member ratio by acidosis renders cells exquisitely sensitive to the Bcl-2/Bcl-xL antagonist ABT-737, suggesting that acidosis causes Bcl-2 family dependence. This dependence appears to be mediated, in part, by the acid-sensing G protein-coupled receptor, GPR65, via a MEK/ERK pathway. PMID:22685289

  1. Polymeric mechanical amplifiers of immune cytokine-mediated apoptosis

    PubMed Central

    Mitchell, Michael J.; Webster, Jamie; Chung, Amanda; Guimarães, Pedro P. G.; Khan, Omar F.; Langer, Robert

    2017-01-01

    Physical forces affect tumour growth, progression and metastasis. Here, we develop polymeric mechanical amplifiers that exploit in vitro and in vivo physical forces to increase immune cytokine-mediated tumour cell apoptosis. Mechanical amplifiers, consisting of biodegradable polymeric particles tethered to the tumour cell surface via polyethylene glycol linkers, increase the apoptotic effect of an immune cytokine on tumour cells under fluid shear exposure by as much as 50% compared with treatment under static conditions. We show that targeted polymeric particles delivered to tumour cells in vivo amplify the apoptotic effect of a subsequent treatment of immune cytokine, reduce circulating tumour cells in blood and overall tumour cell burden by over 90% and reduce solid tumour growth in combination with the antioxidant resveratrol. The work introduces a potentially new application for a broad range of micro- and nanoparticles to maximize receptor-mediated signalling and function in the presence of physical forces. PMID:28317839

  2. Polymeric mechanical amplifiers of immune cytokine-mediated apoptosis

    NASA Astrophysics Data System (ADS)

    Mitchell, Michael J.; Webster, Jamie; Chung, Amanda; Guimarães, Pedro P. G.; Khan, Omar F.; Langer, Robert

    2017-03-01

    Physical forces affect tumour growth, progression and metastasis. Here, we develop polymeric mechanical amplifiers that exploit in vitro and in vivo physical forces to increase immune cytokine-mediated tumour cell apoptosis. Mechanical amplifiers, consisting of biodegradable polymeric particles tethered to the tumour cell surface via polyethylene glycol linkers, increase the apoptotic effect of an immune cytokine on tumour cells under fluid shear exposure by as much as 50% compared with treatment under static conditions. We show that targeted polymeric particles delivered to tumour cells in vivo amplify the apoptotic effect of a subsequent treatment of immune cytokine, reduce circulating tumour cells in blood and overall tumour cell burden by over 90% and reduce solid tumour growth in combination with the antioxidant resveratrol. The work introduces a potentially new application for a broad range of micro- and nanoparticles to maximize receptor-mediated signalling and function in the presence of physical forces.

  3. Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies

    PubMed Central

    Iyer, Sweta; Anwari, Khatira; Alsop, Amber E.; Yuen, Wai Shan; Huang, David C. S.; Carroll, John; Smith, Nicholas A.; Smith, Brian J.; Dewson, Grant; Kluck, Ruth M.

    2016-01-01

    During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the α2–α5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the α1–α2 loop. A monoclonal antibody (clone 7D10) binds close to α1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release. Anti-FLAG antibodies also activate Bak containing a FLAG epitope close to α1. An antibody (clone 3C10) to the Bax α1–α2 loop activates mitochondrial Bax, but blocks translocation of cytosolic Bax. Tethers within Bak show that 7D10 binding directly extricates α1; a structural model of the 7D10 Fab bound to Bak reveals the formation of a cavity under α1. Our identification of the α1–α2 loop as an activation site in Bak paves the way to develop intrabodies or small molecules that directly and selectively regulate these proteins. PMID:27217060

  4. Multiple mechanisms repress N-Bak mRNA translation in the healthy and apoptotic neurons.

    PubMed

    Jakobson, M; Jakobson, M; Llano, O; Palgi, J; Arumäe, U

    2013-08-22

    N-Bak is a neuron-specific BH3-only splice variant of pro-apoptotic Bcl-2 family member Bak. We have shown that its mRNA is stable in the neurons, whereas the protein cannot be detected by antibodies, suggesting a strong translational arrest of the mRNA. Here we identify two regulatory elements in the N-Bak mRNA that significantly repress translation in the luciferase reporter assay: an upstream open reading frame in the 5'-untranslated region (UTR) and naturally spliced exon-exon junction downstream of the premature translation termination codon in the 3'UTR. We also show that N-Bak mRNA is stored in granular structures in the sympathetic neurons and stays in these granules during intrinsic apoptosis. Finally, we confirm the absence of N-Bak protein by quantitative mass spectrometry analysis in the healthy, apoptotic or stressed sympathetic and cortical neurons. We conclude that N-Bak mRNA is translationally repressed by multiple mechanisms, and the protein does not participate in the classical apoptosis or cellular stress response.

  5. Multiple mechanisms repress N-Bak mRNA translation in the healthy and apoptotic neurons

    PubMed Central

    Jakobson, M; Jakobson, M; Llano, O; Palgi, J; Arumäe, U

    2013-01-01

    N-Bak is a neuron-specific BH3-only splice variant of pro-apoptotic Bcl-2 family member Bak. We have shown that its mRNA is stable in the neurons, whereas the protein cannot be detected by antibodies, suggesting a strong translational arrest of the mRNA. Here we identify two regulatory elements in the N-Bak mRNA that significantly repress translation in the luciferase reporter assay: an upstream open reading frame in the 5′-untranslated region (UTR) and naturally spliced exon–exon junction downstream of the premature translation termination codon in the 3′UTR. We also show that N-Bak mRNA is stored in granular structures in the sympathetic neurons and stays in these granules during intrinsic apoptosis. Finally, we confirm the absence of N-Bak protein by quantitative mass spectrometry analysis in the healthy, apoptotic or stressed sympathetic and cortical neurons. We conclude that N-Bak mRNA is translationally repressed by multiple mechanisms, and the protein does not participate in the classical apoptosis or cellular stress response. PMID:23969856

  6. Assembly of Bak homodimers into higher order homooligomers in the mitochondrial apoptotic pore

    PubMed Central

    Mandal, Tirtha; Shin, Seungjin; Aluvila, Sreevidya; Chen, Hui-Chen; Grieve, Carter; Choe, Jun-Yong; Cheng, Emily H.; Hustedt, Eric J.; Oh, Kyoung Joon

    2016-01-01

    In mitochondrial apoptosis, Bak is activated by death signals to form pores of unknown structure on the mitochondrial outer membrane via homooligomerization. Cytochrome c and other apoptotic factors are released from the intermembrane space through these pores, initiating downstream apoptosis events. Using chemical crosslinking and double electron electron resonance (DEER)-derived distance measurements between specific structural elements in Bak, here we clarify how the Bak pore is assembled. We propose that previously described BH3-in-groove homodimers (BGH) are juxtaposed via the ‘α3/α5’ interface, in which the C-termini of helices α3 and α5 are in close proximity between two neighboring Bak homodimers. This interface is observed concomitantly with the well-known ‘α6:α6’ interface. We also mapped the contacts between Bak homodimers and the lipid bilayer based on EPR spectroscopy topology studies. Our results suggest a model for the lipidic Bak pore, whereby the mitochondrial targeting C-terminal helix does not change topology to accommodate the lining of the pore lumen by BGH. PMID:27488021

  7. Over-expression of p53/BAK in aseptic loosening after total hip replacement.

    PubMed

    Landgraeber, Stefan; Toetsch, Martin; Wedemeyer, Christian; Saxler, Guido; Tsokos, Michael; von Knoch, Fabian; Neuhäuser, Markus; Löer, Franz; von Knoch, Marius

    2006-05-01

    Particle-induced osteolysis is a major cause of aseptic loosening after total joint replacement. The possible induction of apoptosis has not been addressed in great detail. Thus far, it has been shown that ceramic and polyethylene particles can induce apoptosis of macrophages in vitro. The purpose of this study was to test the hypothesis that wears debris generated from total hip arthroplasty could induce cellular damage and apoptosis in vivo. We therefore determined by immunohistochemical methods if increased expression of p53, an important transcription factor, and BAK and Bcl-2, two important regulators of apoptosis, can be found in interface membranes and capsules of hips with aseptically loose implants. Strongly positive immunohistochemical staining for p53 and BAK was found in peri-implant tissues from patients with aseptic hip implant loosening. Differentiation of various cell types showed that macrophages stained positive for p53 in all capsule and interface specimens. p53 was frequently detected in giant cells. Positive staining of BAK in macrophages and giant cells was seen in all specimens. Some positive reactions were observed in fibroblasts, only two of 19 cases stained for p53 and three cases for BAK within synovial cells. Positive macrophages and giant cells were localized around polyethylene particles. While T-lymphocytes showed a regular BAK-staining, the other leukocytes were negative. Statistical analyses showed significant positive correlations (p < 0.001) between the presence of polyethylene and metal debris and the expression of BAK and p53. Polyethylene particles were surrounded by more positive macrophages and giant cells than were metal particles, indicating that polyethylene debris may be a stronger inductor of cell cycle arrest and apoptosis than metal debris. In this study apoptosis of macrophages, giant cells and T-lymphocytes in capsules and interface membranes of patients with aseptic hip implant loosening has been demonstrated in

  8. Janus kinase-2 signaling mediates apoptosis in rat cardiomyocytes.

    PubMed

    Mascareno, Eduardo; Beckles, Daniel L; Siddiqui, M A Q

    2005-11-01

    We tested the hypothesis that activation Jak2, which is prominently involved in the up-regulation of the renin-angiotensin system (RAS), constitutes a focal point in relaying signals triggered by a Angiotensin II (Ang II) and hypoxia/reoxygenation separately to cause an enhanced susceptibility of cardiac myocyte to apoptotic cell death. Ang II-treated adult cardiomyocytes in culture exhibited an increased level of apoptosis that accompanied activation of pro-apoptotic as well as anti-apoptotic signaling pathways. We observed increased phosphorylation of Jak2 kinase, Stat1, JNK, with increased expression of Bax protein, followed by an increase in caspase-1 and caspase-3 activity. Activation of these pro-apoptotic pathways was blocked by the Jak2 pharmacological inhibitor, Tyrphostin AG490. We also observed an increase in phosphorylation of cardioprotective pathway components, namely S6 ribosomal protein, and heat shock protein 27 (HSP27). Likewise, the oxidative stress, via the hypoxia/reoxygenation treatment of rat adult cardiomyocytes, produced apoptosis that was dependent upon activation of Jak2. The apoptotic response was not only reduced by Losartan, an inverse agonist of the AT1, receptor, but by treatment with AG490 as well. Taken together, these observations provide clear evidence in favor of Jak2 signaling as mediator of the apoptotic response in cardiomyocytes. However, there was a concomitant induction of cytoprotective signaling that presumably provides a negative feed-back to the deleterious effects of the agonist.

  9. Overexpression of BAK1 causes salicylic acid accumulation and deregulation of cell death control genes.

    PubMed

    Kim, Sun Young; Shang, Yun; Joo, Se-Hwan; Kim, Seong-Ki; Nam, Kyoung Hee

    2017-03-18

    Since the BRI1-Associated Receptor Kinase 1 (BAK1) was firstly identified as a co-receptor of BRI1 that mediates brassinosteroids (BR) signaling, the functional roles of BAK1, as a versatile co-receptor for various ligand-binding leucine-rich repeat (LRR)-containing receptor-like kinase (RLKs), are being extended to involvement with plant immunity, cell death, stomatal development and ABA signaling in plants. During more than a decade of research on the BAK1, it has been known that transgenic Arabidopsis plants overexpressing BAK1 tagged with various reporters do not fully represent its natural functions. Therefore, in this study, we characterized the transgenic plants in which native BAK1 is overexpressed driven by its own promoter. We found that those transgenic plants were more sensitive to BR signaling but showed reduced growth patterns accompanied with spontaneous cell death features that are different from those seen in BR-related mutants. We demonstrated that more salicylic acid (SA) and hydrogen peroxide were accumulated and that expressions of the genes that are known to regulate cell death, such as BONs, BIRs, and SOBIR, were increased in the BAK1-overexpressing transgenic plants. These results suggest that pleiotropic phenotypic alterations shown in the BAK1- overexpressing transgenic plants result from the constitutive activation of SA-mediated defense responses. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. miR-326 targets antiapoptotic Bcl-xL and mediates apoptosis in human platelets.

    PubMed

    Yu, Shifang; Huang, Huicong; Deng, Gang; Xie, Zuoting; Ye, Yincai; Guo, Ruide; Cai, Xuejiao; Hong, Junying; Qian, Dingliang; Zhou, Xiangjing; Tao, Zhihua; Chen, Bile; Li, Qiang

    2015-01-01

    Platelets play crucial roles in hemostasis, thrombosis, wound healing, inflammation, angiogenesis, and tumor metastases. Because they are anucleated blood cells, platelets lack nuclear DNA, but they do contain mitochondrial DNA, which plays a key role in regulating apoptosis. Recent evidence has suggested that miRNAs are also involved in regulating gene expression and apoptosis in platelets. Our previous study showed that the expression of miR-326 increased visibly when apheresis platelets were stored in vitro. The antiapoptotic Bcl-2 family regulator Bcl-xL has been identified as a putative target of miR-326. In the present study, dual reporter luciferase assays were used to characterize the function of miR-326 in the regulation of the apoptosis of platelet cells. These assays demonstrated that miR-326 bound to the 3'-translated region of Bcl-xL. To directly assess the functional effects of miR-326 expression, levels of Bcl-xL and the apoptotic status of stored apheresis platelets were measured after transfection of miR-326 mimic or inhibitor. Results indicated that miR-326 inhibited Bcl-xL expression and induced apoptosis in stored platelets. Additionally, miR-326 inhibited Bcl-2 protein expression and enhanced Bak expression, possibly through an indirect mechanism, though there was no effect on the expression of Bax. The effect of miR-326 appeared to be limited to apoptosis, with no significant effect on platelet activation. These results provide new insight into the molecular mechanisms affecting differential platelet gene regulation, which may increase understanding of the role of platelet apoptosis in multiple diseases.

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

    PubMed

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

    2014-03-13

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

  12. Patulin induces apoptosis through ROS-mediated endoplasmic reticulum stress pathway.

    PubMed

    Boussabbeh, Manel; Ben Salem, Intidhar; Prola, Alexandre; Guilbert, Arnaud; Bacha, Hassen; Abid-Essefi, Salwa; Lemaire, Christophe

    2015-04-01

    Patulin (PAT) is a toxic metabolite produced by several filamentous fungi of the genera of Penicillium, Aspergillus, and Byssochlamys. PAT is the most common mycotoxin found in apples and apple-based products including juice, compotes, cider, and baby food. Exposure to this mycotoxin has been reported to induce intestinal and kidney injuries. This study investigated the mechanism of PAT-induced toxicity in human colon carcinoma (HCT116) and embryonic kidney cells (HEK293). We demonstrated that PAT activated endoplasmic reticulum (ER) and unfolded protein response as evidenced by up-regulation of GRP78 and GADD34, splicing of XBP1 mRNA, and expression of the proapoptotic factor CHOP. This ER stress response was accompanied by the induction of the mitochondrial apoptotic pathway. Apoptosis occurred with ROS production, drop in mitochondrial membrane potential and caspase activation. Further, we showed that deficiency of the proapoptotic protein Bax or Bak protected cells against PAT-induced apoptosis. The treatment of cells with the ROS scavenger N-acetyl cysteine inhibits the ER stress response and prevents mitochondrial apoptosis. Collectively, our data provide new mechanistic insights in the signaling pathways of the cell death induced by PAT and demonstrate that PAT induces cytotoxicity through a ROS-dependent mechanism involving ER stress and activation of mitochondrial apoptotic pathway in human intestinal and kidney cells. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  13. Proapoptotic BAX and BAK: A Requisite Gateway to Mitochondrial Dysfunction and Death

    PubMed Central

    Wei, Michael C.; Zong, Wei-Xing; Cheng, Emily H. -Y.; Lindsten, Tullia; Panoutsakopoulou, Vily; Ross, Andrea J.; Roth, Kevin A.; MacGregor, Grant R.; Thompson, Craig B.; Korsmeyer, Stanley J.

    2011-01-01

    Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a “BH3-domain–only” BCL-2 family member, triggers the homooligomerization of “multidomain” conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a “multidomain” proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli. PMID:11326099

  14. Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice

    PubMed Central

    Vandenberg, C J; Josefsson, E C; Campbell, K J; James, C; Lawlor, K E; Kile, B T; Cory, S

    2014-01-01

    Bax and Bak are critical effectors of apoptosis. Although both are widely expressed and usually functionally redundant, recent studies suggest that Bak has particular importance in certain cell types. Genetic and biochemical studies indicate that Bak activation is prevented primarily by Mcl-1 and Bcl-xL, whereas Bax is held in check by all pro-survival Bcl-2 homologues, including Bcl-2 itself. In this study, we have investigated whether loss of Bak or elevated Mcl-1 modulates haemopoietic abnormalities provoked by overexpression of Bcl-2. The Mcl-1 transgene had little impact, probably because the expression level was insufficient to effectively reduce Bak activation. However, loss of Bak enhanced lymphocytosis in vavP-BCL-2 transgenic mice and increased resistance of their thymocytes to some cytotoxic agents, implying that Bak-specific signals can be triggered in certain lymphoid populations. Nevertheless, lack of Bak had no significant impact on thymic abnormalities in vavP-BCL-2tg mice, which kinetic analysis suggested was due to accumulation of self-reactive thymocytes that resist deletion. Intriguingly, although Bak−/− mice have elevated platelet counts, Bak−/−vavP-BCL-2 mice, like vavP-BCL-2 littermates, were thrombocytopaenic. To clarify why, the vavP-BCL-2 platelet phenotype was scrutinised more closely. Platelet life span was found to be elevated in vavP-BCL-2 mice, which should have provoked thrombocytosis, as in Bak−/− mice. Analysis of bone marrow chimaeric mice suggested the low platelet phenotype was due principally to extrinsic factors. Following splenectomy, blood platelets remained lower in vavP-BCL-2 than wild-type mice. However, in Rag1−/− BCL-2tg mice, platelet levels were normal, implying that elevated lymphocytes are primarily responsible for BCL-2tg-induced thrombocytopaenia. PMID:24464220

  15. NF-κB p65 recruited SHP regulates PDCD5-mediated apoptosis in cancer cells.

    PubMed

    Murshed, Farhan; Farhana, Lulu; Dawson, Marcia I; Fontana, Joseph A

    2014-03-01

    Transcription factor NF-κB promotes cell proliferation in response to cell injury. Increasing evidence, however, suggests that NF-κB can also play an apoptotic role depending on the stimulus and cell type. We have previously demonstrated that novel retinoid 4-[3-Cl-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC)-mediated apoptosis in breast carcinoma cells requires activation of canonical and non-canonical NF-κB pathways. The mechanism NF-κB uses to induce apoptosis remains largely unknown. NF-κB subunit p65 (RelA) was identified as one potent transcriptional activator in 3-Cl-AHPC-mediated apoptosis in cells. Here we used ChIP-on-chip to identify NF-κB p65 genes activated in 3-Cl-AHPC mediated apoptosis. This paper focuses on one hit: pro-apoptotic protein programmed cell death 5 (PDCD5). 3-Cl-AHPC mediated apoptosis in MDA-MB-468 had three related effects on PDCD5: NF-κB p65 binding to the PDCD5 gene, enhanced PDCD5 promoter activity, and increased PDCD5 protein expression. Furthermore, 3-Cl-AHPC increased orphan nuclear receptor small heterodimer partner (SHP) mRNA expression, increased SHP protein bound to NF-κB p65, and found the SHP/NF-κB p65 complex attached to the PDCD5 gene. PDCD5 triggered apoptosis through increased Bax protein and release of cytochrome C from mitochondria to cytosol. Lastly, knockdown of PDCD5 protein expression blocked 3-Cl-AHPC mediated apoptosis, while over-expression of PDCD5 enhanced apoptosis, suggesting PDCD5 is necessary and sufficient for NF-κB p65 mediated apoptosis. Our results demonstrate a novel pathway for NF-κB p65 in regulating apoptosis through SHP and PDCD5.

  16. The Makes Caterpillars Floppy (MCF)-Like Domain of Vibrio vulnificus Induces Mitochondrion-Mediated Apoptosis

    PubMed Central

    Agarwal, Shivangi; Zhu, Yeuming; Gius, David R.

    2015-01-01

    The multifunctional-autoprocessing repeats-in-toxin (MARTXVv) toxin of Vibrio vulnificus plays a significant role in the pathogenesis of this bacterium through delivery of up to five effector domains to the host cells. Previous studies have established that the MARTXVv toxin is linked to V. vulnificus dependent induction of apoptosis, but the region of the large multifunction protein essential for this activity was not previously identified. Recently, we showed that the Makes Caterpillar Floppy-like MARTX effector domain (MCFVv) is an autoproteolytic cysteine protease that induces rounding of various cell types. In this study, we demonstrate that cell rounding induced by MCFVv is coupled to reduced metabolic rate and inhibition of cellular proliferation. Moreover, delivery of MCFVv into host cells either as a fusion to the N-terminal fragment of anthrax toxin lethal factor or when naturally delivered as a V. vulnificus MARTX toxin led to loss of mitochondrial membrane potential, release of cytochrome c, activation of Bax and Bak, and processing of caspases and poly-(ADP-ribose) polymerase (PARP-γ). These studies specifically link the MCFVv effector domain to induction of the intrinsic apoptosis pathway by V. vulnificus. PMID:26351282

  17. The Makes Caterpillars Floppy (MCF)-Like Domain of Vibrio vulnificus Induces Mitochondrion-Mediated Apoptosis.

    PubMed

    Agarwal, Shivangi; Zhu, Yeuming; Gius, David R; Satchell, Karla J F

    2015-11-01

    The multifunctional-autoprocessing repeats-in-toxin (MARTXVv) toxin of Vibrio vulnificus plays a significant role in the pathogenesis of this bacterium through delivery of up to five effector domains to the host cells. Previous studies have established that the MARTXVv toxin is linked to V. vulnificus dependent induction of apoptosis, but the region of the large multifunction protein essential for this activity was not previously identified. Recently, we showed that the Makes Caterpillar Floppy-like MARTX effector domain (MCFVv) is an autoproteolytic cysteine protease that induces rounding of various cell types. In this study, we demonstrate that cell rounding induced by MCFVv is coupled to reduced metabolic rate and inhibition of cellular proliferation. Moreover, delivery of MCFVv into host cells either as a fusion to the N-terminal fragment of anthrax toxin lethal factor or when naturally delivered as a V. vulnificus MARTX toxin led to loss of mitochondrial membrane potential, release of cytochrome c, activation of Bax and Bak, and processing of caspases and poly-(ADP-ribose) polymerase (PARP-γ). These studies specifically link the MCFVv effector domain to induction of the intrinsic apoptosis pathway by V. vulnificus.

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

    DTIC Science & Technology

    2005-01-01

    AD Award Number: W81XWH-04-1-0052 TITLE: Modulating TRAIL-Mediated Apoptosis in Prostate Cancer Using Synthetic Triterpenoids PRINCIPAL INVESTIGATOR...Modulating TRAIL-Mediated Apoptosis in Prostate Cancer W81XWH-04-1-0052 Using Synthetic Triterpenoids 6. AUTHOR(S) Marc L. Hyer, Ph.D. 7. PERFORMING...dien-28-oic acid (CDDO) and its derivative 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im), which induce apoptosis in breast and

  19. PERK-mediated Autophagy in Osteosarcoma Cells Resists ER Stress-induced Cell Apoptosis

    PubMed Central

    Ji, Guang-rong; Yu, Nai-chun; Xue, Xiang; Li, Zong-guang

    2015-01-01

    Osteosarcoma is a bone cancer that develops commonly in children and adolescents. However, osteosarcoma treatments often fail by the development of chemoresistance to apoptosis, and the molecular mechanisms remain unclear. In this study, we propose that autophagy is responsible for osteosarcomatous resistance to apoptosis. We implicate PERK-mediated autophagy as a significant contributor to apoptosis resistance due to ER stress in osteosarcoma cells. By immunostainings and western blots, we identified that PERK activated osteosarcomatous autophagy via inhibiting mTORC1 pathway, thereby preventing cell apoptosis. While using RNAi, we knocked down PERK and found that autophagy was suppressed, result in osteosarcomatous apoptosis. Our results identify a novel role of PERK-mediated autophagy as a significant mechanism for osteosarcoma cell survival. These results will help to understand the mechanism of chemoresistance in osteosarcoma cells, and indicate a novel target for improving osteosarcoma therapy. PMID:26078722

  20. PERK-mediated Autophagy in Osteosarcoma Cells Resists ER Stress-induced Cell Apoptosis.

    PubMed

    Ji, Guang-rong; Yu, Nai-chun; Xue, Xiang; Li, Zong-guang

    2015-01-01

    Osteosarcoma is a bone cancer that develops commonly in children and adolescents. However, osteosarcoma treatments often fail by the development of chemoresistance to apoptosis, and the molecular mechanisms remain unclear. In this study, we propose that autophagy is responsible for osteosarcomatous resistance to apoptosis. We implicate PERK-mediated autophagy as a significant contributor to apoptosis resistance due to ER stress in osteosarcoma cells. By immunostainings and western blots, we identified that PERK activated osteosarcomatous autophagy via inhibiting mTORC1 pathway, thereby preventing cell apoptosis. While using RNAi, we knocked down PERK and found that autophagy was suppressed, result in osteosarcomatous apoptosis. Our results identify a novel role of PERK-mediated autophagy as a significant mechanism for osteosarcoma cell survival. These results will help to understand the mechanism of chemoresistance in osteosarcoma cells, and indicate a novel target for improving osteosarcoma therapy.

  1. How do viruses control mitochondria-mediated apoptosis?

    PubMed

    Neumann, Simon; El Maadidi, Souhayla; Faletti, Laura; Haun, Florian; Labib, Shirin; Schejtman, Andrea; Maurer, Ulrich; Borner, Christoph

    2015-11-02

    There is no doubt that viruses require cells to successfully reproduce and effectively infect the next host. The question is what is the fate of the infected cells? All eukaryotic cells can "sense" viral infections and exhibit defence strategies to oppose viral replication and spread. This often leads to the elimination of the infected cells by programmed cell death or apoptosis. This "sacrifice" of infected cells represents the most primordial response of multicellular organisms to viruses. Subverting host cell apoptosis, at least for some time, is therefore a crucial strategy of viruses to ensure their replication, the production of essential viral proteins, virus assembly and the spreading to new hosts. For that reason many viruses harbor apoptosis inhibitory genes, which once inside infected cells are expressed to circumvent apoptosis induction during the virus reproduction phase. On the other hand, viruses can take advantage of stimulating apoptosis to (i) facilitate shedding and hence dissemination, (ii) to prevent infected cells from presenting viral antigens to the immune system or (iii) to kill non-infected bystander and immune cells which would limit viral propagation. Hence the decision whether an infected host cell undergoes apoptosis or not depends on virus type and pathogenicity, its capacity to oppose antiviral responses of the infected cells and/or to evade any attack from immune cells. Viral genomes have therefore been adapted throughout evolution to satisfy the need of a particular virus to induce or inhibit apoptosis during its life cycle. Here we review the different strategies used by viruses to interfere with the two major apoptosis as well as with the innate immune signaling pathways in mammalian cells. We will focus on the intrinsic mitochondrial pathway and discuss new ideas about how particular viruses could activately engage mitochondria to induce apoptosis of their host.

  2. Altered mitochondrial morphology and defective protein import reveal novel roles for Bax and/or Bak in skeletal muscle.

    PubMed

    Zhang, Yuan; Iqbal, Sobia; O'Leary, Michael F N; Menzies, Keir J; Saleem, Ayesha; Ding, Shuzhe; Hood, David A

    2013-09-01

    The function Bax and/or Bak in constituting a gateway for mitochondrial apoptosis in response to apoptotic stimuli has been unequivocally demonstrated. However, recent work has suggested that Bax/Bak may have unrecognized nonapoptotic functions related to mitochondrial function in nonstressful environments. Wild-type (WT) and Bax/Bak double knockout (DKO) mice were used to determine alternative roles for Bax and Bak in mitochondrial morphology and protein import in skeletal muscle. The absence of Bax and/or Bak altered mitochondrial dynamics by regulating protein components of the organelle fission and fusion machinery. Moreover, DKO mice exhibited defective mitochondrial protein import, both into the matrix and outer membrane compartments, which was consistent with our observations of impaired membrane potential and attenuated expression of protein import machinery (PIM) components in intermyofibrillar mitochondria. Furthermore, the cytosolic chaperones heat-shock protein 90 (Hsp90) and binding immunoglobulin protein (BiP) were markedly increased with the deletion of Bax/Bak, indicating that the cytosolic environment related to protein folding may be changed in DKO mice. Interestingly, endurance training fully restored the deficiency of protein import in DKO mice, likely via the upregulation of PIM components and through improved cytosolic chaperone protein expression. Thus our results emphasize novel roles for Bax and/or Bak in mitochondrial function and provide evidence, for the first time, of a curative function of exercise training in ameliorating a condition of defective mitochondrial protein import.

  3. Caffeic acid phenethyl ester induces mitochondria-mediated apoptosis in human myeloid leukemia U937 cells.

    PubMed

    Jin, Un-Ho; Song, Kwon-Ho; Motomura, Muneo; Suzuki, Ikukatsu; Gu, Yeun-Hwa; Kang, Yun-Jeong; Moon, Tae-Chul; Kim, Cheorl-Ho

    2008-03-01

    Caffeic acid phenyl ester (CAPE), a biologically active ingredient of propolis, has several interesting biological properties including antioxidant, anti-inflammatory, antiviral, immunostimulatory, anti-angiogenic, anti-invasive, anti-metastatic and carcinostatic activities. Recently, several groups have reported that CAPE is cytotoxic to tumor cells but not to normal cells. In this study, we investigated the mechanism of CAPE-induced apoptosis in human myeloid leukemia U937 cells. Treatment of U937 cells with CAPE decreased cell viability in a dose-dependent and time-dependent manner. DNA fragmentation assay revealed the typical ladder profile of oligonucleosomal fragments in CAPE-treated U937 cells. In addition, as evidenced by the nuclear DAPI staining experiment, we observed that the nuclear condensation, a typical phenotype of apoptosis, was found in U937 cells treated with 5 microg/ml of CAPE. Therefore, it was suggested that CAPE is a potent agent inducing apoptosis in U937 cells. Apoptotic action of the CAPE was accompanied by release of cytochrome C, reduction of Bcl-2 expression, increase of Bax expression, activation/cleavage of caspase-3 and activation/cleavage of PARP in U937 cells, but not by Fas protein, an initial mediator in the death signaling, or by phospho-eIF2 alpha and CHOP, crucial mediators in ER-mediated apoptosis. From the results, it was concluded that CAPE induces the mitochondria-mediated apoptosis but not death receptors- or ER-mediated apoptosis in U937 cells.

  4. Label-Free Classification of Bax/Bak Expressing vs. Double-Knockout Cells.

    PubMed

    Naser, Mohammad; Graham, Michelle T; Pierre, Kamau; Boustany, Nada N

    2016-11-01

    We combine optical scatter imaging with principal component analysis (PCA) to classify apoptosis-competent Bax/Bak-expressing, and apoptosis resistant Bax/Bak-null immortalized baby mouse kidney cells. We apply PCA to 100 stacks each containing 236 dark-field cell images filtered with an optically implemented Gabor filter with period between 0.3 and 2.9 μm. Each stack yields an "eigencell" image corresponding to the first principal component obtained at one of the 100 Gabor filter periods used. At each filter period, each cell image is multiplied by (projected onto) the eigencell image. A Feature Matrix consisting of 236 × 100 scalar values is thus constructed with significantly reduced dimension compared to the initial dataset. Utilizing this Feature Matrix, we implement a supervised linear discriminant analysis and classify successfully the Bax/Bak-expressing and Bax/Bak-null cells with 94.7% accuracy and an area under the curve (AUC) of 0.993. Applying a feature selection algorithm further reveals that the Gabor filter period ranges most significant for the classification correspond to both large (likely nuclear) features as well as small sized features (likely organelles present in the cytoplasm). Our results suggest that cells with a genetic defect in their apoptosis pathway can be differentiated from their normal counterparts by label-free multi-parametric optical scatter data.

  5. Bax/Bak activation in the absence of Bid, Bim, Puma, and p53.

    PubMed

    Zhang, J; Huang, K; O'Neill, K L; Pang, X; Luo, X

    2016-06-16

    How BH3-only proteins activate Bax/Bak, the two gateway proteins of the mitochondria-dependent apoptotic pathway, remains incompletely understood. Although all pro-apoptotic BH3-only proteins are known to bind/neutralize the anti-apoptotic Bcl-2 proteins, the three most potent ones, Bid (tBid), Bim, and Puma, possess an additional activity of directly activating Bax/Bak in vitro. This latter activity has been proposed to be responsible for triggering Bax/Bak activation following apoptotic stimulation. To test this hypothesis, we generated Bid(-/)(-)Bim(-/)(-)Puma(-/)(-) (TKO), TKO/Bax(-/)(-)/Bak(-/)(-) (PentaKO), and PentaKO/Mcl-1(-/-) (HexaKO) HCT116 cells through gene editing. Surprisingly, although the TKO cells were resistant to several apoptotic stimuli, robust apoptosis was induced upon the simultaneous inactivation of Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins known to suppress Bax/Bak activation and activity. Importantly, such apoptotic activity was completely abolished in the PentaKO cells. In addition, ABT-737, a BH3 mimetic that inhibits Bcl-xL/Bcl-w/Bcl-2, induced Bax activation in HexaKO cells reconstituted with endogenous level of GFP-Bax. Further, by generating TKO/p53(-/-) (QKO) cells, we demonstrated that p53, a tumor suppressor postulated to directly activate Bax, is not required for Bid/Bim/Puma-independent Bax/Bak activation. Together, these results strongly suggest that the direct activation activities of Bid (tBid), Bim, Puma, and p53 are not essential for activating Bax/Bak once the anti-apoptotic Bcl-2 proteins are neutralized.

  6. Apoptosis and autophagy induction in mammalian cells by small interfering RNA knockdown of mRNA capping enzymes.

    PubMed

    Chu, Chun; Shatkin, Aaron J

    2008-10-01

    Addition of a 5' cap to RNA polymerase II transcripts, the first step of pre-mRNA processing in eukaryotes from yeasts to mammals, is catalyzed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine-N-7)methyltransferase. The effects of knockdown of these capping enzymes in mammalian cells were investigated using T7 RNA polymerase-synthesized small interfering RNA and also a lentivirus-based inducible, short hairpin RNA system. Decreasing either guanylyltransferase or methyltransferase resulted in caspase-3 activation and elevated terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining characteristic of apoptosis. Induction of apoptosis was independent of p53 tumor suppressor but dependent on BAK or BAX. In addition, levels of the BH3 family member Bim increased, while Mcl-1 and Bik levels remained unchanged during apoptosis. In contrast to capping enzyme knockdown, apoptosis induced by cycloheximide inhibition of protein synthesis required BAK but not BAX. Both Bim and Mcl-1 levels decreased in cycloheximide-induced apoptosis while Bik levels were unchanged, suggesting that apoptosis in siRNA-treated cells is not a direct consequence of loss of mRNA translation. siRNA-treated BAK(-/-) BAX(-/-) double-knockout mouse embryonic fibroblasts failed to activate capase-3 or increase TUNEL staining but instead exhibited autophagy, as demonstrated by proteolytic processing of microtubule-associated protein 1 light chain 3 (LC3) and translocation of transfected green fluorescent protein-LC3 from the nucleus to punctate cytoplasmic structures.

  7. Prenatal exposure to PFOS caused mitochondia-mediated apoptosis in heart of weaned rat.

    PubMed

    Zeng, Huai-Cai; He, Qing-Zhi; Li, Yuan-Yuan; Wu, Cheng-Qiu; Wu, Yi-Mou; Xu, Shun-Qing

    2015-09-01

    Perfluorooctanyl sulfonate (PFOS), a cardiac toxicity compound, has been widely detected in the environment and in organisms. However, the toxic mechanism is not clear. Our previous study indicated that prenatal PFOS exposure led to swollen mitochondrial with vacuolar structure and loss of cristae in offsping's heart. The purpose of this study was to investigate the effect of PFOS on the apoptosis in developing heart and mitochondria-mediated apoptosis pathway. Pregnant Sprague-Dawley (SD) rats were exposed to PFOS at doses of 0.1, 0.6, and 2.0 mg/kg-d and 0.05% Tween 80 as control by gavage from gestation day 2 (GD 2) to GD 21. Apoptosis, as well as expression of apoptosis related genes associated with mitochondrial-mediated apoptosis pathway, including p53, bcl-2, bax, cytochrome c, caspase-9, and caspase-3 were analyzed in heart tissues from weaned (postnatal day 21, PND 21) offspring. The results showed that prenatal PFOS exposure resulted in apoptosis in the offspring's heart. The mRNA and protein expression levels of p53, bax, cytochrome c, caspase-9, and caspase-3 in the offspring's heart were enhanced in various PFOS-treated groups, meanwhile, the bcl-2 expression levels were decreased. Our results indicated that prenatal PFOS exposure induced the apoptosis of weaned offspring rat heart tissue via mitochondria-mediated apoptotic pathway. © 2014 Wiley Periodicals, Inc.

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

    PubMed

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

    2015-12-01

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

  9. Endogenous Bak inhibitors Mcl-1 and Bcl-xL: differential impact on TRAIL resistance in Bax-deficient carcinoma

    PubMed Central

    Gillissen, Bernhard; Wendt, Jana; Richter, Antje; Richter, Anja; Müer, Annika; Overkamp, Tim; Gebhardt, Nina; Preissner, Robert; Belka, Claus; Dörken, Bernd

    2010-01-01

    Tumor necrosis factor (α)–related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that preferentially kills tumor cells with limited cytotoxicity to nonmalignant cells. However, signaling from death receptors requires amplification via the mitochondrial apoptosis pathway (type II) in the majority of tumor cells. Thus, TRAIL-induced cell death entirely depends on the proapoptotic Bcl-2 family member Bax, which is often lost as a result of epigenetic inactivation or mutations. Consequently, Bax deficiency confers resistance against TRAIL-induced apoptosis. Despite expression of Bak, Bax-deficient cells are resistant to TRAIL-induced apoptosis. In this study, we show that the Bax dependency of TRAIL-induced apoptosis is determined by Mcl-1 but not Bcl-xL. Both are antiapoptotic Bcl-2 family proteins that keep Bak in check. Nevertheless, knockdown of Mcl-1 but not Bcl-xL overcame resistance to TRAIL, CD95/FasL and tumor necrosis factor (α) death receptor ligation in Bax-deficient cells, and enabled TRAIL to activate Bak, indicating that Mcl-1 rather than Bcl-xL is a major target for sensitization of Bax-deficient tumors for death receptor–induced apoptosis via the Bak pathway. PMID:20308427

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

    PubMed

    Feng, Jie; Meng, Chengbo; Xing, Da

    2015-02-01

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

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

    PubMed

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

    2016-04-01

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

  12. Nicotine induces Nme2-mediated apoptosis in mouse testes.

    PubMed

    Gu, Yunqi; Xu, Wangjie; Nie, Dongsheng; Zhang, Dong; Dai, Jingbo; Zhao, Xianglong; Zhang, Meixing; Wang, Zhaoxia; Chen, Zhong; Qiao, Zhongdong

    2016-04-15

    In mouse testes, germ cell apoptosis can be caused by cigarette smoke and lead to declining quality of semen, but the exact molecular mechanisms remain unclear. To evaluate the effects of nicotine exposure on apoptosis during spermatogenesis, we first constructed a nicotine-treated mouse model and detected germ cell apoptosis activity in the testes using the TUNEL method. Then we analyzed the variation of telomere length and telomerase activity by real-time PCR and TRAP-real-time PCR, respectively. Further, we investigated a highly expressed gene, Nme2, in mouse testes after nicotine treatment from our previous results, which has close correlation with the apoptosis activity predicted by bioinformatics. We performed NME2 overexpression in Hela cells to confirm whether telomere length and telomerase activity were regulated by the Nme2 gene. Finally, we examined methylation of CpG islands in the Nme2 promoter with the Bisulfite Sequencing (BSP) method. The results showed that apoptosis had increased significantly, and then telomerase activity became weak. Further, telomere length was shortened in the germ cells among the nicotine-treated group. In Hela cells, both overexpression of the Nme2 gene and nicotine exposure can suppress the activity of telomerase activity and shorten telomere length. BSP results revealed that the Nme2 promoter appeared with low methylation in mouse testes after nicotine treatment. We assume that nicotine-induced apoptosis may be caused by telomerase activity decline, which is inhibited by the up expression of Nme2 because of its hypomethylation in mouse germ cells.

  13. The Effect of Selenium on the Cd-Induced Apoptosis via NO-Mediated Mitochondrial Apoptosis Pathway in Chicken Liver.

    PubMed

    Zhang, Runxiang; Yi, Ran; Bi, Yanju; Xing, Lu; Bao, Jun; Li, Jianhong

    2017-01-06

    Cd-induced apoptosis and the protective effects of Se against Cd-induced injury have been reported in previous studies. However, little is known regarding the effects of Cd-induced apoptosis in hepatic cells and the antagonistic effects of Se on Cd in poultry. In the present study, 128 healthy 31-week-old laying hens were randomly divided into four groups, which were fed basic diets, with the addition of Se (Na2SeO3, 2 mg/kg), Cd (CdCl2, 150 mg/kg), or Se + Cd (150 mg/kg of CdCl2 and 2 mg/kg of Na2SeO3) for 90 days. Ultrastructural changes, nitric oxide (NO) concentrations, inducible nitric oxide synthase (iNOS) activities, results of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of apoptosis, and the expression of iNOS and apoptosis-related genes in livers were determined. It was observed that Cd treatment significantly increased the concentrations of NO and iNOS activity in chicken livers. The production of excessive NO initiated the mitochondrial apoptotic pathway. Exposure to Cd increased the mRNA and the protein expression levels of iNOS, caspase-3, Bax, p53, and Cyt-c. Furthermore, the ratio of Bax/Bcl-2 increased, while the expression of Bcl-2 decreased. Treatment with Se significantly alleviated Cd-induced apoptosis in chicken livers, as evidenced by a reduction in the production of NO, iNOS activity, the number of apoptotic cells, and mRNA and protein expression levels of iNOS, caspase-3, Bax, and Cyt-c. It indicated that Cd induced NO-mediated apoptosis through the mitochondrial apoptotic pathway and Se exerted antagonizing effects. The present study provides new insights as to how Se affects Cd-induced toxicity in the chicken liver.

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

    PubMed Central

    Chang, Wei-An; Tsai, Pei-Hsun; Wu, Cheng-Ying; Ho, Ya-Wen; Yen, Meng-Chi; Lin, Yi-Shiuan; Kuo, Po-Lin; Hsu, Ya-Ling

    2017-01-01

    Developing novel chemo-prevention techniques and advancing treatment are key elements to beating lung cancer, the most common cause of cancer mortality worldwide. Our previous cohort study showed that cysteinyl leukotriene receptor antagonists, mainly montelukast, decreased the lung cancer risk in asthma patients. In the current study, we conducted in vivo and in vitro experiments to demonstrate the inhibiting effect of montelukast on lung cancer and to investigate the underlying mechanisms. Using Lewis lung carcinoma-bearing mice, we showed that feeding montelukast significantly delayed the tumor growth in mice (p < 0.0001). Montelukast inhibited cell proliferation and colony formation and induced the cell death of lung cancer cells. Further investigation showed the down-regulation of B-cell lymphoma 2 (Bcl-2), up-regulation of Bcl-2 homologous antagonist/killer (Bak), and nuclear translocation of apoptosis-inducing factor (AIF) in montelukast-treated lung cancer cells. Montelukast also markedly decreased the phosphorylation of several proteins, such as with no lysine 1 (WNK1), protein kinase B (Akt), extracellular signal-regulated kinase 1/2 (Erk1/2), MAPK/Erk kinase (MEK), and proline-rich Akt substrate of 40-kDa (PRAS40), which might contribute to cell death. In conclusion, montelukast induced lung cancer cell death via the nuclear translocation of AIF. This study confirmed the chemo-preventive effect of montelukast shown in our previous cohort study. The utility of montelukast in cancer prevention and treatment thus deserves further studies. PMID:28672809

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

    PubMed

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

    2013-01-01

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

  16. Oxidative Stress Mediates Radiation Lung Injury by Inducing Apoptosis

    SciTech Connect

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

    2012-06-01

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

  17. Influenza Virus Induces Apoptosis via BAD-Mediated Mitochondrial Dysregulation

    PubMed Central

    Tran, Anh T.; Cortens, John P.; Du, Qiujiang; Wilkins, John A.

    2013-01-01

    Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication. PMID:23135712

  18. Infection-induced bystander-apoptosis of monocytes is TNF-alpha-mediated.

    PubMed

    Dreschers, Stephan; Gille, Christian; Haas, Martin; Grosse-Ophoff, Julia; Schneider, Marion; Leiber, Anja; Bühring, Hans-Jörg; Orlikowsky, Thorsten W

    2013-01-01

    Phagocytosis induced cell death (PICD) is crucial for controlling phagocyte effector cells, such as monocytes, at sites of infection, and essentially contributes to termination of inflammation. Here we tested the hypothesis, that during PICD bystander apoptosis of non-phagocyting monocytes occurs, that apoptosis induction is mediated via tumor necrosis factor-alpha (TNF-α and that TNF-α secretion and -signalling is causal. Monocytes were infected with Escherichia coli (E. coli), expressing green fluorescent protein (GFP), or a pH-sensitive Eos-fluorescent protein (EOS-FP). Monocyte phenotype, phagocytic activity, apoptosis, TNF-receptor (TNFR)-1, -2-expression and TNF-α production were analyzed. Apoptosis occured in phagocyting and non-phagocyting, bystander monocytes. Bacterial transport to the phagolysosome was no prerequisite for apoptosis induction, and desensitized monocytes from PICD, as confirmed by EOS-FP expressing E. coli. Co-cultivation with non-infected carboxyfluorescein-succinimidyl-ester- (CFSE-) labelled monocytes resulted in significant apoptotic cell death of non-infected bystander monocytes. This process required protein de-novo synthesis and still occurred in a diminished way in the absence of cell-cell contact. E. coli induced a robust TNF-α production, leading to TNF-mediated apoptosis in monocytes. Neutralization with an anti-TNF-α antibody reduced monocyte bystander apoptosis significantly. In contrast to TNFR2, the pro-apoptotic TNFR1 was down-regulated on the monocyte surface, internalized 30 min. p.i. and led to apoptosis predominantly in monocytes without phagocyting bacteria by themselves. Our results suggest, that apoptosis of bystander monocytes occurs after infection with E. coli via internalization of TNFR1, and indicate a relevant role for TNF-α. Modifying monocyte apoptosis in sepsis may be a future therapeutic option.

  19. Myonuclear apoptosis in dystrophic mdx muscle occurs by perforin-mediated cytotoxicity.

    PubMed Central

    Spencer, M J; Walsh, C M; Dorshkind, K A; Rodriguez, E M; Tidball, J G

    1997-01-01

    Myonuclear apoptosis is an early event in the pathology of dystrophin-deficient muscular dystrophy in the mdx mouse. However, events that initiate apoptosis in muscular dystrophy are unknown, and whether elimination of apoptosis can ameliorate subsequent muscle wasting remains a major question. We have tested the hypothesis that cytotoxic T-lymphocytes initiate myonuclear apoptosis in dystrophic muscle, and examined whether perforin-mediated cytotoxicity plays a role in the pathophysiology of muscular dystrophy. Mdx mice showed muscle invasion by cytotoxic T cells and helper T cells at the onset of histologically detectable muscle fiber pathology. At this time, perforin-expressing cells were also present at elevated concentration. Mdx mice depleted of CD8(+) cells showed a significant reduction of apoptotic myonuclei concentration and a reduction in necrosis, judged by macrophage invasion of muscle fibers. Double-mutant mice, deficient in dystrophin and perforin, showed nearly complete absence of myonuclear apoptosis, and a significant reduction in the concentration of macrophages in the connective tissue surrounding muscle fibers. However, muscle fiber invasion by macrophages was not reduced significantly in double mutant mice. Thus, cytotoxic T-lymphocytes contribute significantly to apoptosis and necrosis in mdx dystrophy, and perforin-mediated killing is primarily responsible for myonuclear apoptosis. PMID:9169505

  20. P53-Mediated Rapid Induction of Apoptosis Conveys Resistance to Viral Infection in Drosophila melanogaster

    PubMed Central

    Liu, Bo; Behura, Susanta K.; Clem, Rollie J.; Schneemann, Anette; Becnel, James; Severson, David W.; Zhou, Lei

    2013-01-01

    Arthropod-borne pathogens account for millions of deaths each year. Understanding the genetic mechanisms controlling vector susceptibility to pathogens has profound implications for developing novel strategies for controlling insect-transmitted infectious diseases. The fact that many viruses carry genes that have anti-apoptotic activity has long led to the hypothesis that induction of apoptosis could be a fundamental innate immune response. However, the cellular mechanisms mediating the induction of apoptosis following viral infection remained enigmatic, which has prevented experimental verification of the functional significance of apoptosis in limiting viral infection in insects. In addition, studies with cultured insect cells have shown that there is sometimes a lack of apoptosis, or the pro-apoptotic response happens relatively late, thus casting doubt on the functional significance of apoptosis as an innate immunity. Using in vivo mosquito models and the native route of infection, we found that there is a rapid induction of reaper-like pro-apoptotic genes within a few hours following exposure to DNA or RNA viruses. Recapitulating a similar response in Drosophila, we found that this rapid induction of apoptosis requires the function of P53 and is mediated by a stress–responsive regulatory region upstream of reaper. More importantly, we showed that the rapid induction of apoptosis is responsible for preventing the expression of viral genes and blocking the infection. Genetic changes influencing this rapid induction of reaper-like pro-apoptotic genes led to significant differences in susceptibility to viral infection. PMID:23408884

  1. Inhibitors of XIAP sensitize CD40-activated chronic lymphocytic leukemia cells to CD95-mediated apoptosis

    PubMed Central

    Kater, Arnon P.; Dicker, Frank; Mangiola, Massimo; Welsh, Kate; Houghten, Richard; Ostresh, John; Nefzi, Adel; Reed, John C.; Pinilla, Clemencia; Kipps, Thomas J.

    2005-01-01

    Patients with chronic lymphocytic leukemia (CLL) treated with adenovirus CD154 (Ad-CD154, CD40 ligand [CD40L]) gene therapy experienced rapid reductions in leukemia cell counts and lymph node size associated with the induced expression of Fas (CD95). However, CLL cells initially resist CD95-mediated apoptosis within the first 3 days after CD40 ligation in vitro. Thereafter, they become sensitive, which is associated with the CD40-induced expression of the proapoptotic protein B-cell leukemia 2 homology 3 (BH3) interacting domain death agonist (Bid). We hypothesized that the initial resistance to CD95-mediated apoptosis may be due to the high-level expression of X-linked inhibitor of apoptosis protein (XIAP) by CLL cells. Consistent with this, CLL cells from patients 1 day after treatment with autologous Ad-CD154-transduced CLL cells became sensitive to CD95-mediated apoptosis following treatment with a novel XIAP inhibitor, 1540-14. Similarly, 1540-14 specifically enhanced CD95-mediated apoptosis of CLL cells following CD40 ligation in vitro. Immunoblot analyses demonstrated that treatment with 1540-14 allowed CD40-stimulated CLL cells to experience high-level activation of caspases-8 and -3 and cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase following CD95 ligation. This study demonstrates that distal apoptosis regulators contribute to the initial resistance of CD40-activated CLL cells to CD95-mediated apoptosis and suggests that XIAP inhibitors might enhance the effectiveness of immune-based treatment strategies that target CD40, such as CD154 gene therapy. (Blood. 2005;106:1742-1748) PMID:15914559

  2. Inhibition of Bak Activation by VDAC2 Is Dependent on the Bak Transmembrane Anchor*

    PubMed Central

    Lazarou, Michael; Stojanovski, Diana; Frazier, Ann E.; Kotevski, Aneta; Dewson, Grant; Craigen, William J.; Kluck, Ruth M.; Vaux, David L.; Ryan, Michael T.

    2010-01-01

    Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death. PMID:20851889

  3. Alginate Oligosaccharide Prevents Acute Doxorubicin Cardiotoxicity by Suppressing Oxidative Stress and Endoplasmic Reticulum-Mediated Apoptosis.

    PubMed

    Guo, Jun-Jie; Ma, Lei-Lei; Shi, Hong-Tao; Zhu, Jian-Bing; Wu, Jian; Ding, Zhi-Wen; An, Yi; Zou, Yun-Zeng; Ge, Jun-Bo

    2016-12-20

    Doxorubicin (DOX) is a highly potent chemotherapeutic agent, but its usage is limited by dose-dependent cardiotoxicity. DOX-induced cardiotoxicity involves increased oxidative stress and activated endoplasmic reticulum-mediated apoptosis. Alginate oligosaccharide (AOS) is a non-immunogenic, non-toxic and biodegradable polymer, with anti-oxidative, anti-inflammatory and anti-endoplasmic reticulum stress properties. The present study examined whether AOS pretreatment could protect against acute DOX cardiotoxicity, and the underlying mechanisms focused on oxidative stress and endoplasmic reticulum-mediated apoptosis. We found that AOS pretreatment markedly increased the survival rate of mice insulted with DOX, improved DOX-induced cardiac dysfunction and attenuated DOX-induced myocardial apoptosis. AOS pretreatment mitigated DOX-induced cardiac oxidative stress, as shown by the decreased expressions of gp91 (phox) and 4-hydroxynonenal (4-HNE). Moreover, AOS pretreatment significantly decreased the expression of Caspase-12, C/EBP homologous protein (CHOP) (markers for endoplasmic reticulum-mediated apoptosis) and Bax (a downstream molecule of CHOP), while up-regulating the expression of anti-apoptotic protein Bcl-2. Taken together, these findings identify AOS as a potent compound that prevents acute DOX cardiotoxicity, at least in part, by suppression of oxidative stress and endoplasmic reticulum-mediated apoptosis.

  4. Alginate Oligosaccharide Prevents Acute Doxorubicin Cardiotoxicity by Suppressing Oxidative Stress and Endoplasmic Reticulum-Mediated Apoptosis

    PubMed Central

    Guo, Jun-Jie; Ma, Lei-Lei; Shi, Hong-Tao; Zhu, Jian-Bing; Wu, Jian; Ding, Zhi-Wen; An, Yi; Zou, Yun-Zeng; Ge, Jun-Bo

    2016-01-01

    Doxorubicin (DOX) is a highly potent chemotherapeutic agent, but its usage is limited by dose-dependent cardiotoxicity. DOX-induced cardiotoxicity involves increased oxidative stress and activated endoplasmic reticulum-mediated apoptosis. Alginate oligosaccharide (AOS) is a non-immunogenic, non-toxic and biodegradable polymer, with anti-oxidative, anti-inflammatory and anti-endoplasmic reticulum stress properties. The present study examined whether AOS pretreatment could protect against acute DOX cardiotoxicity, and the underlying mechanisms focused on oxidative stress and endoplasmic reticulum-mediated apoptosis. We found that AOS pretreatment markedly increased the survival rate of mice insulted with DOX, improved DOX-induced cardiac dysfunction and attenuated DOX-induced myocardial apoptosis. AOS pretreatment mitigated DOX-induced cardiac oxidative stress, as shown by the decreased expressions of gp91 (phox) and 4-hydroxynonenal (4-HNE). Moreover, AOS pretreatment significantly decreased the expression of Caspase-12, C/EBP homologous protein (CHOP) (markers for endoplasmic reticulum-mediated apoptosis) and Bax (a downstream molecule of CHOP), while up-regulating the expression of anti-apoptotic protein Bcl-2. Taken together, these findings identify AOS as a potent compound that prevents acute DOX cardiotoxicity, at least in part, by suppression of oxidative stress and endoplasmic reticulum-mediated apoptosis. PMID:27999379

  5. Thiourea compound AW00178 sensitizes human H1299 lung carcinoma cells to TRAIL-mediated apoptosis.

    PubMed

    Ryu, Byung Jun; Hwang, Mi-Kyung; Park, Mikyung; Lee, Kyunghee; Kim, Seong Hwan

    2012-06-15

    The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a wide variety of cancer cells. Recently, cancer cell resistance to TRAIL-mediated apoptosis has become a challenging issue in the development of TRAIL-based anti-cancer therapies. In this study, we found that 1-(5-chloro-2-methyl-phenyl)-3-[4-(5-trifluoromethyl-pyrazol-1-yl)-phenyl]-thiourea (AW00178) was able to sensitize TRAIL-resistant human lung cancer H1299 cells to TRAIL-mediated apoptosis. Treatment with AW00178, either alone or in combination with TRAIL, induced the expression of CHOP, a protein related to TRAIL sensitivity, and reduced the expression of survivin, an anti-apoptotic protein involved in TRAIL resistance. Additionally, AW00178, alone or in combination with TRAIL, induced the activation of c-Jun and inactivation of Akt. A pharmacologic inhibition study revealed that c-Jun activation and Akt inactivation were strongly related to CHOP induction and survivin down-regulation, respectively. In summary, these results suggested that AW00178 mediated sensitization to TRAIL-mediated apoptosis in H1299 cells by increasing sensitivity and decreasing resistance to TRAIL via the induction of c-Jun-dependent CHOP expression and the reduction of Akt-dependent survivin expression, respectively.

  6. Megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained to survive and produce platelets

    PubMed Central

    Josefsson, Emma C.; James, Chloé; Henley, Katya J.; Debrincat, Marlyse A.; Rogers, Kelly L.; Dowling, Mark R.; White, Michael J.; Kruse, Elizabeth A.; Lane, Rachael M.; Ellis, Sarah; Nurden, Paquita; Mason, Kylie D.; O’Reilly, Lorraine A.; Roberts, Andrew W.; Metcalf, Donald; Huang, David C.S.

    2011-01-01

    It is believed that megakaryocytes undergo a specialized form of apoptosis to shed platelets. Conversely, a range of pathophysiological insults, including chemotherapy, are thought to cause thrombocytopenia by inducing the apoptotic death of megakaryocytes and their progenitors. To resolve this paradox, we generated mice with hematopoietic- or megakaryocyte-specific deletions of the essential mediators of apoptosis, Bak and Bax. We found that platelet production was unperturbed. In stark contrast, deletion of the prosurvival protein Bcl-xL resulted in megakaryocyte apoptosis and a failure of platelet shedding. This could be rescued by deletion of Bak and Bax. We examined the effect on megakaryocytes of three agents that activate the intrinsic apoptosis pathway in other cell types: etoposide, staurosporine, and the BH3 mimetic ABT-737. All three triggered mitochondrial damage, caspase activation, and cell death. Deletion of Bak and Bax rendered megakaryocytes resistant to etoposide and ABT-737. In vivo, mice with a Bak−/− Bax−/− hematopoietic system were protected against thrombocytopenia induced by the chemotherapeutic agent carboplatin. Thus, megakaryocytes do not activate the intrinsic pathway to generate platelets; rather, the opposite is true: they must restrain it to survive and progress safely through proplatelet formation and platelet shedding. PMID:21911424

  7. Cerulenin-mediated apoptosis is involved in adenine metabolic pathway

    SciTech Connect

    Chung, Kyung-Sook; Sun, Nam-Kyu; Lee, Seung-Hee; Lee, Hyun-Jee; Choi, Shin-Jung; Kim, Sun-Kyung; Song, Ju-Hyun; Jang, Young-Joo; Song, Kyung-Bin; Yoo, Hyang-Sook; Simon, Julian . E-mail: jsimon@fhcrc.org; Won, Misun . E-mail: misun@kribb.re.kr

    2006-10-27

    Cerulenin, a fatty acid synthase (FAS) inhibitor, induces apoptosis of variety of tumor cells. To elucidate mode of action by cerulenin, we employed the proteomics approach using Schizosaccharomyces pombe. The differential protein expression profile of S. pombe revealed that cerulenin modulated the expressions of proteins involved in stresses and metabolism, including both ade10 and adk1 proteins. The nutrient supplementation assay demonstrated that cerulenin affected enzymatic steps transferring a phosphoribosyl group. This result suggests that cerulenin accumulates AMP and p-ribosyl-s-amino-imidazole carboxamide (AICAR) and reduces other necessary nucleotides, which induces feedback inhibition of enzymes and the transcriptional regulation of related genes in de novo and salvage adenine metabolic pathway. Furthermore, the deregulation of adenine nucleotide synthesis may interfere ribonucleotide reductase and cause defects in cell cycle progression and chromosome segregation. In conclusion, cerulenin induces apoptosis through deregulation of adenine nucleotide biosynthesis resulting in nuclear division defects in S. pombe.

  8. Impact of Mitochondria-Mediated Apoptosis in U251 Cell Cycle Arrest in G1 Stage and Caspase Activation.

    PubMed

    Zhang, Lei; Liang, Peng; Zhang, Rui

    2015-11-23

    BACKGROUND Most mitochondria-mediated apoptosis has some relevance to the cell cycle, but there is still a lack of investigations about U251 cell cycle in human brain glioma cells. In this study, we aimed to clarify the correlation of mitochondria-mediated apoptosis with the U251 cell cycle and its influence on apoptosis, through observing the impact of mitochondria-mediated apoptosis in U251cell specificity cycle arrest and Caspase activation. MATERIAL AND METHODS AnnexinV/PI and API were used to label the brain glioma cells for flow cytometry analysis of U251 cell apoptosis and cell cycle. RT-PCR and Western blot were performed to detect Caspase-3 and Caspase-9 activation. RESULTS Peripheral blood in stationary phase is not sensitive to apoptosis induction, but U251 cells have obvious apoptosis. Mitochondria-mediated apoptosis mainly occurs in the G1 phase of the cell cycle. Caspase-3 and Caspase-9 mRNAs and proteins expression increased significantly after the cells were treated by mitochondrial apoptosis-related gene Bax induction. CONCLUSIONS Mitochondria-mediated apoptosis is related to the U251 cell cycle with specific G1 stage arrest. Caspase activation occurs in the process of cell apoptosis.

  9. Stem cells are resistant to TRAIL receptor-mediated apoptosis.

    PubMed

    Szegezdi, Eva; O'Reilly, Aoife; Davy, Yeung; Vawda, Reaz; Taylor, Deanna L; Murphy, Mary; Samali, Afshin; Mehmet, Huseyin

    2009-01-01

    New therapeutic approaches aim to eradicate tumours by expression of tumouricidal proteins in the tumour stroma. One such anti-neoplastic protein is tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) because it induces apoptosis in cancerous cells, but not in non-transformed cells. Stem cells can migrate to, survive and proliferate in tumours. We examined the suitability of bone marrow-derived adult mesenchymal stem cells (bmMSC), foetal-MSC and umbilical cord matrix stem cells (Wharton's Jelly MSCs) as TRAIL-delivery vehicles. Although all MSC types expressed DR4 and/or DR5, none of them were sensitive to TRAIL-induced apoptosis. Selective activation of DR4 or DR5 with agonistic antibodies or DR5-selective TRAIL-mutant (D269H/E195R) revealed that the TRAIL receptors are inactive in MSCs. In fMSC DR5 was not fully inactivated, its activity however was minimal in comparison to the colon carcinoma cell, Colo205. The intracellular components of the TRAIL-apoptotic pathway, such as pro-caspase-8 and -9 were also expressed at very low; almost undetectable levels in all three MSC types. In conclusion, the MSC species examined are resistant to TRAIL and thus can be suitable tools for TRAIL delivery to tumours.

  10. Stem cells are resistant to TRAIL receptor-mediated apoptosis

    PubMed Central

    Szegezdi, Eva; O’Reilly, Aoife; Davy, Yeung; Vawda, Reaz; Taylor, Deanna L; Murphy, Mary; Samali, Afshin; Mehmet, Huseyin

    2009-01-01

    New therapeutic approaches aim to eradicate tumours by expression of tumouricidal proteins in the tumour stroma. One such anti-neoplastic protein is tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) because it induces apoptosis in cancerous cells, but not in non-transformed cells. Stem cells can migrate to, survive and proliferate in tumours. We examined the suitability of bone marrow-derived adult mesenchymal stem cells (bmMSC), foetal-MSC and umbilical cord matrix stem cells (Wharton’s Jelly MSCs) as TRAIL-delivery vehicles. Although all MSC types expressed DR4 and/or DR5, none of them were sensitive to TRAIL-induced apoptosis. Selective activation of DR4 or DR5 with agonistic antibodies or DR5-selective TRAIL-mutant (D269H/E195R) revealed that the TRAIL receptors are inactive in MSCs. In fMSC DR5 was not fully inactivated, its activity however was minimal in comparison to the colon carcinoma cell, Colo205. The intracellular components of the TRAIL-apoptotic pathway, such as pro-caspase-8 and -9 were also expressed at very low; almost undetectable levels in all three MSC types. In conclusion, the MSC species examined are resistant to TRAIL and thus can be suitable tools for TRAIL delivery to tumours. PMID:19604313

  11. Suppression of GLI sensitizes medulloblastoma cells to mitochondria-mediated apoptosis.

    PubMed

    Lin, Zhongxiao; Li, Sisi; Sheng, Hansong; Cai, Ming; Ma, Lin Yuan Si; Hu, Liuxun; Xu, Shangyu; Yu, Li Sheng; Zhang, Nu

    2016-12-01

    The sonic hedgehog (SHH) signalling pathway plays the important role in medulloblastoma (MB). Altered GLI expression plays a key role in these processes, and the inhibition of GLI may be a good cancer-targeted therapy. This study aimed to investigate whether GANT61, a GLI inhibitor, may inhibit the SHH signalling pathway promoting cell mitochondria-mediated apoptosis and enhance cisplatin apoptosis antineoplastic therapy. In our study, we determined the effect of GANT61-mediated inhibition of GLI in Daoy MB cells. Cells were treated with different concentrations of GANT61 alone or in combination with cisplatin. Cell proliferation was assessed with CCK-8 assays, and cell invasion and migration were performed using 8-µm transwell inserts. Cell apoptosis was assessed with flow cytometric analysis and rhodamine 123. qPCR was used to complete RNA experiments. Protein expression was assessed with Western blotting. The GANT61 significantly inhibited cell proliferation. GANT61 decreased the cell migration and invasion, impairing these crucial steps in tumour progression. Cell apoptosis was significantly increased in Daoy cells. Rhodamine 123 assay showed that GANT61 could decrease the mitochondrial membrane potential promoting cell mitochondria-mediated apoptosis. GANT61 inhibited the expression of GLI and Bcl-2 at both the mRNA and protein levels and might affect the expression of Bax, caspase-3 and caspase-9 to promote cell intrinsic apoptosis. Furthermore, GANT61 could enhance cisplatin-induced apoptosis to decrease the IC50 value of cisplatin. Finally, data suggest that GANT61 could enhance cisplatin-induced apoptosis through promoting the expression of Bax, caspase-3 and caspase-9 protein levels. Our data suggest that the SHH signalling pathway plays an important role in MB. GLI is an oncogenic transcription factor in the SHH pathway, and targeting GLI with GANT61 results in favourable antitumour activity and targeted therapy.

  12. Nitric oxide-mediated apoptosis of neutrophils through caspase-8 and caspase-3-dependent mechanism

    PubMed Central

    Dubey, Megha; Nagarkoti, Sheela; Awasthi, Deepika; Singh, Abhishek K; Chandra, Tulika; Kumaravelu, J; Barthwal, Manoj K; Dikshit, Madhu

    2016-01-01

    Neutrophils play an indispensable role in killing of invading pathogens by enhancing reactive oxygen species (ROS) and NO generation, and subsequently undergoing apoptosis. Unlike ROS/NOX2, role of NO/NOS still remains undefined in the apoptosis of neutrophils (PMNs) and the present study attempts to decipher the importance of NO/NOS in the neutrophil apoptosis. Prolonged treatment of human PMNs or mice bone marrow derived neutrophils (BMDN) with NO led to enhanced ROS generation, caspase-8/caspase-3 cleavage, reduced mitochondrial membrane potential and finally cellular apoptosis. NO-induced ROS generation led to caspase-8 deglutathionylation and activation, which subsequently activated mitochondrial death pathway via BID (Bcl-2 family protein) cleavage. NO-mediated augmentation of caspase-8 and BID cleavage was significantly prevented in BMDN from neutrophil cytosolic factor-1 (NCF-1) knockout (KO) mice, implying the involvement of NOX2 in NO-induced apoptosis of PMNs. Furthermore, ROS, NO generation and inducible nitric oxide synthase (iNOS) expression were enhanced in a time-dependent manner in human PMNs and mice BMDN undergoing spontaneous apoptosis. Pharmacological and genetic ablation of iNOS in human PMNs and mice BMDN significantly reduced the levels of apoptosis. Impaired apoptosis of BMDN from iNOS KO mice was due to reduced caspase-8 activity which subsequently prevented caspase-3 and -9 activation. Altogether, our results suggest a crucial role of NO/iNOS in neutrophil apoptosis via enhanced ROS generation and caspase-8 mediated activation of mitochondrial death pathway. PMID:27584786

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

    SciTech Connect

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

    2011-07-08

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

  14. Prevention of Trauma and Hemorrhagic Shock-Mediated Liver Apoptosis by Activation of Stat3α

    PubMed Central

    Moran, Ana; Akcan Arikan, Ayse; Mastrangelo, Mary-Ann A.; Wu, Yong; Yu, Bi; Poli, Valeria; Tweardy, David J.

    2008-01-01

    Trauma is a major cause of mortality in the United States. Death among those surviving the initial insult is caused by multiple organ failure (MOF) with the liver among the organs most frequently affected. We previously demonstrated in rodents that trauma complicated by hemorrhagic shock (trauma/HS) results in liver injury that can be prevented by IL-6 administration at the start of resuscitation; however, the contribution of the severity of HS to the extent of liver injury, whether or not resuscitation is required and the mechanism for the IL-6 protective effect have not been reported. In the experiments reported here, we demonstrated that the extent of liver apoptosis induced by trauma/HS depends on the duration of hypotension and requires resuscitation. We established that IL-6 administration at the start of resuscitation is capable of completely reversing liver apoptosis and is associated with increased Stat3 activation. Microarray analysis of the livers showed that the main effect of IL-6 was to normalize the trauma/HS-induced apoptosis transcriptome. Pharmacological inhibition of Stat3 activity within the liver blocked the ability of IL-6 to prevent liver apoptosis and to normalize the trauma/HS- induced liver apoptosis transcriptome. Genetic deletion of a Stat3β, a naturally occurring, dominant-negative isoform of the Stat3, attenuated trauma/HS-induced liver apoptosis, confirming a role for Stat3, especially Stat3α, in preventing trauma/HS-mediated liver apoptosis. Thus, trauma/HS-induced liver apoptosis depends on the duration of hypotension and requires resuscitation. IL-6 administration at the start of resuscitation reverses HS-induced liver apoptosis, through activation of Stat3α, which normalizes the trauma/HS-induced liver apoptosis transcriptome. PMID:18997875

  15. Involvement of Bcl-2 and Bax in photodynamic therapy-mediated apoptosis. Antisense Bcl-2 oligonucleotide sensitizes RIF 1 cells to photodynamic therapy apoptosis.

    PubMed

    Srivastava, M; Ahmad, N; Gupta, S; Mukhtar, H

    2001-05-04

    Photodynamic therapy (PDT), a promising treatment modality, is an oxidative stress that induces apoptosis in many cancer cells in vitro and tumors in vivo. Understanding the mechanism(s) involved in PDT-mediated apoptosis may improve its therapeutic efficacy. Although studies suggest the involvement of multiple pathways, the triggering event(s) responsible for PDT-mediated apoptotic response is(are) not clear. To investigate the role of Bcl-2 in PDT-mediated apoptosis, we employed Bcl-2-antisense and -overexpression approaches in two cell types differing in their responses toward PDT apoptosis. In the first approach, we treated radiation-induced fibrosarcoma (RIF 1) cells, which are resistant to silicon phthalocyanine (Pc 4)-PDT apoptosis, with Bcl-2-antisense oligonucleotide. This treatment resulted in sensitization of RIF 1 cells to PDT-mediated apoptosis as demonstrated by i) cleavage of poly(ADP-ribose) polymerase, ii) DNA ladder formation, iii) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, and iv) DEVDase activity. This treatment also resulted in oligonucleotide concentration-dependent decrease in cell viability and down-regulation of Bcl-2 protein with a concomitant increase in apoptosis. However, the level of Bax, a pro-apoptotic member of Bcl-2 family, remained unaltered. In the second approach, an overexpression of Bcl-2 in PDT apoptosis-sensitive human epidermoid carcinoma (A431) cells resulted in enhanced apoptosis and up-regulation of Bax following PDT. In both the approaches, the increased Bax/Bcl-2 ratio was associated with an increased apoptotic response of PDT. Our data also demonstrated that PDT results in modulation of other Bcl-2 family members in a way that the overall ratio of pro-apoptotic and anti-apoptotic member proteins favors apoptosis.

  16. The Rac activator Tiam1 prevents keratinocyte apoptosis by controlling ROS-mediated ERK phosphorylation.

    PubMed

    Rygiel, Tomasz P; Mertens, Alexander E; Strumane, Kristin; van der Kammen, Rob; Collard, John G

    2008-04-15

    Tiam1 is a ubiquitously expressed activator of the small GTPase Rac. Previously, we found that Tiam1 knockout (KO) mice are resistant to DMBA-induced skin tumorigenicity, which correlated with increased apoptosis in keratinocytes of the skin epidermis. Here, we have studied the mechanisms by which Tiam1 protects against apoptosis. We found that Tiam1-KO keratinocytes show increased apoptosis in response to apoptotic stimuli, including growth factor deprivation and heat-shock treatment. Expression of catalytically active Tiam1, but not inactive Tiam1, rescues the apoptosis susceptibility of Tiam1-KO keratinocytes, indicating that this defect is caused by impaired Tiam1-mediated Rac activation. Apoptosis induced by growth factor starvation correlates with impaired ERK phosphorylation in Tiam1-KO keratinocytes. Moreover, Tiam1-KO keratinocytes contain lower levels of intracellular reactive oxygen species (ROS) when compared with wild-type cells. The ROS content of keratinocytes is dependent on both Tiam1 and the activity of NADPH oxidase (Nox), and is required for ERK-mediated survival signaling. Indeed, Tiam1 deficiency or the inhibition of intracellular ROS production blocks ERK phosphorylation and sensitizes wild-type keratinocytes to apoptotic stimuli. Our results indicate that the Rac activator Tiam1 controls the intracellular redox balance by Nox-mediated ROS production, which regulates ERK phosphorylation and the susceptibility of keratinocytes to apoptotic signaling.

  17. Mitochondria-independent induction of Fas-mediated apoptosis by MSSP.

    PubMed

    Nomura, Jun; Matsumoto, Ken-Ichi; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2005-11-01

    Fas-mediated apoptosis has been proposed to play an important role in homeostasis. Fas triggers apoptosis after stimulation by its ligand FasL or the Fas ligand agonist anti-Fas antibody through a mitochondria-dependent or -independent pathway, and MSSP has been identified as a transcription factor that regulates the c-myc gene and was later found to positively or negatively regulate a variety of genes, including alpha-smooth actin, MHC class I, MHC class 2 and the thyrotropin receptor. We further found that expression of the Fas gene was repressed, resulting in abrogation of the Fas-mediated induction of apoptosis both in Mssp-knockout mice and primary thymocytes. MSSP was then found to stimulate promoter activity of the Fas gene by binding to a specific region. In this study, to identify the MSSP-dependent Fas-induced apoptosis pathway, primary fibroblasts from MSSP (+/+) and MSSP (-/-) cells were treated with the combination of interleukin 1-beta and interferon-gamma and expression of the Fas gene was examined. The results showed that the Fas gene was expressed at the same levels in the two cell types. Furthermore, when these cells were treated with the anti-Fas antibody, it was found that cytochrome C was not released in the cytosol and that activations of caspase 8 and caspase 3 occurred in primary fibroblasts from MSSP (+/+) cells but not from MSSP (-/-) cells. These results indicate that Fas-mediated apoptosis induced by MSSP occurs independently of mitochondria.

  18. NFAT2 mediates high glucose-induced glomerular podocyte apoptosis through increased Bax expression

    SciTech Connect

    Li, Ruizhao; Zhang, Li; Shi, Wei; Zhang, Bin; Liang, Xinling; Liu, Shuangxin; Wang, Wenjian

    2013-04-15

    Background: Hyperglycemia promotes podocyte apoptosis and plays a key role in the pathogenesis of diabetic nephropathy. However, the mechanisms that mediate hyperglycemia-induced podocyte apoptosis is still far from being fully understood. Recent studies reported that high glucose activate nuclear factor of activated T cells (NFAT) in vascular smooth muscle or pancreatic β-cells. Here, we sought to determine if hyperglycemia activates NFAT2 in cultured podocyte and whether this leads to podocyte apoptosis. Meanwhile, we also further explore the mechanisms of NFAT2 activation and NFAT2 mediates high glucose-induced podocyte apoptosis. Methods: Immortalized mouse podocytes were cultured in media containing normal glucose (NG), or high glucose (HG) or HG plus cyclosporine A (a pharmacological inhibitor of calcinerin) or 11R-VIVIT (a special inhibitor of NFAT2). The activation of NFAT2 in podocytes was detected by western blotting and immunofluorescence assay. The role of NFAT2 in hyperglycemia-induced podocyte apoptosis was further evaluated by observing the inhibition of NFAT2 activation by 11R-VIVIT using flow cytometer. Intracellular Ca{sup 2+} was monitored in HG-treated podcocytes using Fluo-3/AM. The mRNA and protein expression of apoptosis gene Bax were measured by real time-qPCR and western blotting. Results: HG stimulation activated NFAT2 in a time- and dose-dependent manner in cultured podocytes. Pretreatment with cyclosporine A (500 nM) or 11R-VIVIT (100 nM) completely blocked NFAT2 nuclear accumulation. Meanwhile, the apoptosis effects induced by HG were also abrogated by concomitant treatment with 11R-VIVIT in cultured podocytes. We further found that HG also increased [Ca{sup 2+}]i, leading to activation of calcineurin, and subsequent increased nuclear accumulation of NFAT2 and Bax expression in cultured podocytes. Conclusion: Our results identify a new finding that HG-induced podocyte apoptosis is mediated by calcineurin/NFAT2/Bax signaling pathway

  19. Overcoming Resistance of Prostate Cancer to TRAIL - Mediated Apoptosis

    DTIC Science & Technology

    2005-12-01

    DR5 receptor in prostate cancer cell lines. We used histidine-tagged TRAIL prepared in E . coli added to lysed cells at 1 ug/ml for 20 min on ice...normal ones. However, we found that in normal human prostate epithelial cells (PrEC) TRAIL is capable of inducing apoptosis as e �ciently as in some tumor...measured by tetrazolium conversion but had little or no e �ect on other TRAIL-induced apoptotic responses. Although cycloheximide did not further accelerate

  20. Nitric oxide-mediated apoptosis in rat macrophages subjected to Shiga toxin 2 from Escherichia coli.

    PubMed

    Baronetti, José Luis; Villegas, Natalia Angel; Paraje, María Gabriela; Albesa, Inés

    2011-04-01

    Shiga toxin-producing Escherichia coli are important food-borne pathogens. The main factor conferring virulence on this bacterium is its capacity to secrete Shiga toxins (Stxs), which have been reported to induce apoptosis in several cell types. However, the mechanisms of this apoptosis have not yet been fully elucidated. In addition, Stxs have been shown to stimulate macrophages to produce nitric oxide (NO), a well-known apoptosis inductor.The aim of this study was to investigate the participation of NO in apoptosis of rat peritoneal macrophages induced by culture supernatants or Stx2 from E. coli. Peritoneal macrophages incubated in the presence of E. coli supernatants showed an increase in the amounts of apoptosis and NO production. Furthermore, inhibition of NO synthesis induced by addition of aminoguanidine (AG) was correlated with a reduction in the percentage of apoptotic cells, indicating participation of this metabolite in the apoptotic process. Similarly, treatment of cells with Stx2 induced an increase in NO production and amount of apoptosis, these changes being reversed by addition of AG. In summary, these data show that treatment with E. coli supernatants or Stx2 induces NO-mediated apoptosis of macrophages. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

  1. Mitochondrion-mediated apoptosis is involved in reproductive damage caused by BPA in male rats.

    PubMed

    Wang, Peng; Luo, Chunhua; Li, Qianyuan; Chen, Sai; Hu, Yong

    2014-11-01

    Bisphenol A (BPA) is a widely used environmental endocrine disruptor. Many studies have reported that BPA exposure shows reproductive toxicity and causes apoptosis in spermatogenic cells. However, few studies have investigated the relationship between the mitochondrial pathway and BPA-induced apoptosis. This study investigated the role of the mitochondrial pathway in apoptosis induced by BPA, which resulted in compromised male rat spermatogenesis and reproductive damage. Rats were exposed to various BPA concentrations (0, 50, 100, or 200mg of BPA/kg body weight per day), and factors in the mitochondrial signal transduction pathway and the apoptosis indices of spermatogenic cells were measured and sperm characteristics were analyzed. Our data revealed that BPA exposure increased the protein and mRNA levels of cytochrome C, apoptosis-inducing factor, caspase-3/9, and Bax; caspase-3 and caspase-9 activities; and the apoptosis indices of spermatogenic cells. In addition, abnormal structure of mitochondria and decreased protein and gene levels of Bcl-2 were observed following BPA exposure. These results suggest that apoptosis in the mitochondrial pathway mediates compromised reproductive system function caused by BPA exposure.

  2. Astaxanthin prevents pulmonary fibrosis by promoting myofibroblast apoptosis dependent on Drp1-mediated mitochondrial fission

    PubMed Central

    Zhang, Jinjin; Xu, Pan; Wang, Youlei; Wang, Meirong; Li, Hongbo; Lin, Shengcui; Mao, Cuiping; Wang, Bingsi; Song, Xiaodong; Lv, Changjun

    2015-01-01

    Promotion of myofibroblast apoptosis is a potential therapeutic strategy for pulmonary fibrosis. This study investigated the antifibrotic effect of astaxanthin on the promotion of myofibroblast apoptosis based on dynamin-related protein-1 (Drp1)-mediated mitochondrial fission in vivo and in vitro. Results showed that astaxanthin can inhibit lung parenchymal distortion and collagen deposition, as well as promote myofibroblast apoptosis. Astaxanthin demonstrated pro-apoptotic function in myofibroblasts by contributing to mitochondrial fission, thereby leading to apoptosis by increasing the Drp1 expression and enhancing Drp1 translocation into the mitochondria. Two specific siRNAs were used to demonstrate that Drp1 is necessary to promote astaxanthin-induced mitochondrial fission and apoptosis in myofibroblasts. Drp1-associated genes, such as Bcl-2-associated X protein, cytochrome c, tumour suppressor gene p53 and p53-up-regulated modulator of apoptosis, were highly up-regulated in the astaxanthin group compared with those in the sham group. This study revealed that astaxanthin can prevent pulmonary fibrosis by promoting myofibroblast apoptosis through a Drp1-dependent molecular pathway. Furthermore, astaxanthin provides a potential therapeutic value in pulmonary fibrosis treatment. PMID:26119034

  3. Fluid shear stress sensitizes cancer cells to receptor-mediated apoptosis via trimeric death receptors

    NASA Astrophysics Data System (ADS)

    Mitchell, Michael J.; King, Michael R.

    2013-01-01

    Cancer metastasis, the process of cancer cell migration from a primary to distal location, typically leads to a poor patient prognosis. Hematogenous metastasis is initiated by intravasation of circulating tumor cells (CTCs) into the bloodstream, which are then believed to adhere to the luminal surface of the endothelium and extravasate into distal locations. Apoptotic agents such as tumor necrosis factor apoptosis-inducing ligand (TRAIL), whether in soluble ligand form or expressed on the surface of natural killer cells, have shown promise in treating CTCs to reduce the probability of metastasis. The role of hemodynamic shear forces in altering the cancer cell response to apoptotic agents has not been previously investigated. Here, we report that human colon cancer COLO 205 and prostate cancer PC-3 cells exposed to a uniform fluid shear stress in a cone-and-plate viscometer become sensitized to TRAIL-induced apoptosis. Shear-induced sensitization directly correlates with the application of fluid shear stress, and TRAIL-induced apoptosis increases in a fluid shear stress force- and time-dependent manner. In contrast, TRAIL-induced necrosis is not affected by the application fluid shear stress. Interestingly, fluid shear stress does not sensitize cancer cells to apoptosis when treated with doxorubicin, which also induces apoptosis in cancer cells. Caspase inhibition experiments reveal that shear stress-induced sensitization to TRAIL occurs via caspase-dependent apoptosis. These results suggest that physiological fluid shear forces can modulate receptor-mediated apoptosis of cancer cells in the presence of apoptotic agents.

  4. Astaxanthin prevents pulmonary fibrosis by promoting myofibroblast apoptosis dependent on Drp1-mediated mitochondrial fission.

    PubMed

    Zhang, Jinjin; Xu, Pan; Wang, Youlei; Wang, Meirong; Li, Hongbo; Lin, Shengcui; Mao, Cuiping; Wang, Bingsi; Song, Xiaodong; Lv, Changjun

    2015-09-01

    Promotion of myofibroblast apoptosis is a potential therapeutic strategy for pulmonary fibrosis. This study investigated the antifibrotic effect of astaxanthin on the promotion of myofibroblast apoptosis based on dynamin-related protein-1 (Drp1)-mediated mitochondrial fission in vivo and in vitro. Results showed that astaxanthin can inhibit lung parenchymal distortion and collagen deposition, as well as promote myofibroblast apoptosis. Astaxanthin demonstrated pro-apoptotic function in myofibroblasts by contributing to mitochondrial fission, thereby leading to apoptosis by increasing the Drp1 expression and enhancing Drp1 translocation into the mitochondria. Two specific siRNAs were used to demonstrate that Drp1 is necessary to promote astaxanthin-induced mitochondrial fission and apoptosis in myofibroblasts. Drp1-associated genes, such as Bcl-2-associated X protein, cytochrome c, tumour suppressor gene p53 and p53-up-regulated modulator of apoptosis, were highly up-regulated in the astaxanthin group compared with those in the sham group. This study revealed that astaxanthin can prevent pulmonary fibrosis by promoting myofibroblast apoptosis through a Drp1-dependent molecular pathway. Furthermore, astaxanthin provides a potential therapeutic value in pulmonary fibrosis treatment.

  5. Bid chimeras indicate that most BH3-only proteins can directly activate Bak and Bax, and show no preference for Bak versus Bax

    PubMed Central

    Hockings, C; Anwari, K; Ninnis, R L; Brouwer, J; O'Hely, M; Evangelista, M; Hinds, M G; Czabotar, P E; Lee, E F; Fairlie, W D; Dewson, G; Kluck, R M

    2015-01-01

    The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-xL according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax. PMID:25906158

  6. The immunity regulator BAK1 contributes to resistance against diverse RNA viruses.

    PubMed

    Kørner, Camilla Julie; Klauser, Dominik; Niehl, Annette; Domínguez-Ferreras, Ana; Chinchilla, Delphine; Boller, Thomas; Heinlein, Manfred; Hann, Dagmar R

    2013-11-01

    The plant's innate immune system detects potential biotic threats through recognition of microbe-associated molecular patterns (MAMPs) or danger-associated molecular patterns (DAMPs) by pattern recognition receptors (PRR). A central regulator of pattern-triggered immunity (PTI) is the BRI1-associated kinase 1 (BAK1), which undergoes complex formation with PRR upon ligand binding. Although viral patterns inducing PTI are well known from animal systems, nothing similar has been reported for plants. Rather, antiviral defense in plants is thought to be mediated by post-transcriptional gene silencing of viral RNA or through effector-triggered immunity, i.e., recognition of virus-specific effectors by resistance proteins. Nevertheless, infection by compatible viruses can also lead to the induction of defense gene expression, indicating that plants may also recognize viruses through PTI. Here, we show that PTI, or at least the presence of the regulator BAK1, is important for antiviral defense of Arabidopsis plants. Arabidopsis bak1 mutants show increased susceptibility to three different RNA viruses during compatible interactions. Furthermore, crude viral extracts but not purified virions induce several PTI marker responses in a BAK1-dependent manner. Overall, we conclude that BAK1-dependent PTI contributes to antiviral resistance in plants.

  7. Vitamin D-mediated apoptosis in cancer and obesity.

    PubMed

    Sergeev, Igor N

    2014-11-01

    Low vitamin D status has been associated with increased risk of several cancers and obesity; concurrently, obesity and cancer have been linked to impaired vitamin D status. In both cancer and obesity, selective elimination of cancer cells and adipocytes can result in decreasing tumor size and a long-term reduction in adipose tissue mass. These effects can be achieved through induction of apoptotic cell death. The vitamin D-derived hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) triggers apoptosis in epithelial cancer cells and mature adipocytes via induction of apoptotic Ca2+ signal - a sustained, prolonged increase in concentration of intracellular Ca2+. This Ca2+ signal functions as an apoptotic initiator that directly recruits apoptotic effectors, Ca2+-dependent proteases, in cancer cells and adipocytes. The 1,25(OH)2D3 - cellular Ca2+ - apoptosis link in cancer and obesity supports the rationale to include vitamin D compounds modulating intracellular Ca2+ and Ca2+-dependent apoptotic proteases as promising targets for discovery of new therapeutic and preventive agents for cancer and obesity. The concept of maintaining an increased vitamin D status for protecting against cancer and decreasing adiposity also warrants further evaluation.

  8. Iris movement mediates vascular apoptosis during rat pupillary membrane regression.

    PubMed

    Morizane, Yuki; Mohri, Satoshi; Kosaka, Jun; Toné, Shigenobu; Kiyooka, Takahiko; Miyasaka, Takehiro; Shimizu, Juichiro; Ogasawara, Yasuo; Shiraga, Fumio; Minatogawa, Yohsuke; Sasaki, Junzo; Ohtsuki, Hiroshi; Kajiya, Fumihiko

    2006-03-01

    In the course of mammalian lens development, a transient capillary meshwork known as the pupillary membrane (PM) forms, which is located at the pupil area; the PM nourishes the anterior surface of the lens and then regresses to make the optical path clear. Although the involvement of apoptotic process has been reported in the PM regression, the initiating factor remains unknown. We initially found that regression of the PM coincided with the development of iris motility, and iris movement caused cessation and resumption of blood flow within the PM. Therefore, we investigated whether the development of the iris's ability to constrict and dilate functions as an essential signal that induces apoptosis in the PM. Continuous inhibition of iris movement with mydriatic agents from postnatal day 7 to day 12 suppressed apoptosis of the PM and migration of macrophage toward the PM, and resulted in the persistence of PM in rats. The distribution of apoptotic cells in the regressing PM was diffuse and showed no apparent localization. These results indicated that iris movement induced regression of the PM by changing the blood flow within it. This study suggests the importance of the physiological interactions between tissues-in this case, the iris and the PM-as a signal to advance vascular regression during organ development, and defines a novel function of the iris during ocular development in addition to the well-known function, that is, optimization of light transmission into the eye.

  9. Necrosis and apoptosis in Trichinella spiralis-mediated tumour reduction

    PubMed Central

    Vasilev, Sasa; Ilic, Natasa; Gruden-Movsesijan, Alisa; Vasilijic, Sasa; Bosic, Martina

    2015-01-01

    It is known that infection with different pathogens, including helminths, can alter the progression of malignant or other diseases. We studied the effect of chronic Trichinella spiralis infection or muscle larvae excretory-secretory (ES L1) antigens on the malignant tumour growth in the mouse melanoma model system in vivo and in vitro. Our results confirmed that chronic infection with T. spiralis possesses the capacity to slow down the progression of tumour growth, resulting in an impressive reduction in tumour size. We found that the phenomenon could, at least partially, be related to a lower level of tumour necrosis compared to necrosis present in control animals with progressive malignancy course. An increased apoptotic potential among the low percentage of cells within the total tumour cell number in vivo was also observed. ES L1 antigen, as a parasitic product that is released during the chronic phase of infection, reduced the survival and slightly, but significantly increased the apoptosis level of melanoma cells in vitro. Our results imply that powerful Trichinella anti-malignance capacity does not rely only on necrosis and apoptosis but other mechanisms through which infection or parasite products manipulate the tumor establishment and expansion should be considered. PMID:26155183

  10. Aortic valvular interstitial cells apoptosis and calcification are mediated by TNF-related apoptosis-inducing ligand.

    PubMed

    Galeone, Antonella; Brunetti, Giacomina; Oranger, Angela; Greco, Giovanni; Di Benedetto, Adriana; Mori, Giorgio; Colucci, Silvia; Zallone, Alberta; Paparella, Domenico; Grano, Maria

    2013-11-15

    Calcific aortic valvular disease (CAVD) is an actively regulated process characterized by the activation of specific osteogenic signaling pathways and apoptosis. We evaluated the involvement in CAVD of the TNF-related apoptosis-inducing ligand (TRAIL), an apoptotic molecule which induces apoptosis by interacting with the death receptor (DR)-4 and DR5, and whose activity is modulated by the decoy receptor (DcR)-1 and DcR2. Sections of calcific and normal aortic valves, obtained at surgery time, were subjected to immunohistochemistry and confocal microscopy for TRAIL immunostaining. Valvular interstitial cells (VICs) isolated from calcific (C-VICs) and normal (N-VICs) aortic valves were investigated for the gene and protein expression of TRAIL receptors. Cell viability was assayed by MTT. Von Kossa staining was performed to verify C-VIC ability to produce mineralized nodules. TRAIL serum levels were detected by ELISA. Higher levels of TRAIL were detected in calcific aortic valves and in sera from the same patients respect to controls. C-VICs express significantly higher mRNA and protein levels of DR4, DR5, DcR1, DcR2 and Runx2 compared to N-VICs. C-VICs and N-VICs, cultured in osteogenic medium, express significantly higher mRNA levels of DR4, Runx2 and Osteocalcin compared to baseline. C-VICs and N-VICs were sensitive to TRAIL-apoptotic effect at baseline and after osteogenic differentiation, as demonstrated by MTT assay and caspase-3 activation. TRAIL enhanced mineralized matrix nodule synthesis by C-VICs cultured in osteogenic medium. TRAIL is characteristically present within calcific aortic valves, and mediates the calcification of aortic valve interstitial cells in culture through mechanism involving apoptosis. © 2013.

  11. A Small-Molecule Inhibitor of Bax and Bak Oligomerization Prevents Genotoxic Cell Death and Promotes Neuroprotection.

    PubMed

    Niu, Xin; Brahmbhatt, Hetal; Mergenthaler, Philipp; Zhang, Zhi; Sang, Jing; Daude, Michael; Ehlert, Fabian G R; Diederich, Wibke E; Wong, Eve; Zhu, Weijia; Pogmore, Justin; Nandy, Jyoti P; Satyanarayana, Maragani; Jimmidi, Ravi K; Arya, Prabhat; Leber, Brian; Lin, Jialing; Culmsee, Carsten; Yi, Jing; Andrews, David W

    2017-04-20

    Aberrant apoptosis can lead to acute or chronic degenerative diseases. Mitochondrial outer membrane permeabilization (MOMP) triggered by the oligomerization of the Bcl-2 family proteins Bax/Bak is an irreversible step leading to execution of apoptosis. Here, we describe the discovery of small-molecule inhibitors of Bax/Bak oligomerization that prevent MOMP. We demonstrate that these molecules disrupt multiple, but not all, interactions between Bax dimer interfaces thereby interfering with the formation of higher-order oligomers in the MOM, but not recruitment of Bax to the MOM. Small-molecule inhibition of Bax/Bak oligomerization allowed cells to evade apoptotic stimuli and rescued neurons from death after excitotoxicity, demonstrating that oligomerization of Bax is essential for MOMP. Our discovery of small-molecule Bax/Bak inhibitors provides novel tools for the investigation of the mechanisms leading to MOMP and will ultimately facilitate development of compounds inhibiting Bax/Bak in acute and chronic degenerative diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. The c-Jun N-terminal kinase signaling pathway mediates chrysotile asbestos-induced alveolar epithelial cell apoptosis

    PubMed Central

    LI, PENG; LIU, TIE; KAMP, DAVID W.; LIN, ZIYING; WANG, YAHONG; LI, DONGHONG; YANG, LAWEI; HE, HUIJUAN; LIU, GANG

    2015-01-01

    Exposure to chrysotile asbestos exposure is associated with an increased risk of mortality in combination with pulmonary diseases including lung cancer, mesothelioma and asbestosis. Multiple mechanisms by which chrysotile asbestos fibers induce pulmonary disease have been identified, however the role of apoptosis in human lung alveolar epithelial cells (AEC) has not yet been fully explored. Accumulating evidence implicates AEC apoptosis as a crucial event in the development of both idiopathic pulmonary fibrosis and asbestosis. The aim of the present study was to determine whether chrysotile asbestos induces mitochondria-regulated (intrinsic) AEC apoptosis and, if so, whether this induction occurs via the activation of mitogen-activated protein kinases (MAPK). Human A549 bronchoalveolar carcinoma-derived cells with alveolar epithelial type II-like features were used. The present study showed that chrysotile asbestos induced a dose- and time-dependent decrease in A549 cell viability, which was accompanied by the activation of the MAPK c-Jun N-terminal kinases (JNK), but not the MAPKs extracellular signal-regulated kinase 1/2 and p38. Chrysotile asbestos was also shown to induce intrinsic AEC apoptosis, as evidenced by the upregulation of the pro-apoptotic genes Bax and Bak, alongside the activation of caspase-9, poly (ADP-ribose) polymerase (PARP), and the release of cytochrome c. Furthermore, the specific JNK inhibitor SP600125 blocked chrysotile asbestos-induced JNK activation and subsequent apoptosis, as assessed by both caspase-9 cleavage and PARP activation. The results of the present study demonstrated that chrysotile asbestos induces intrinsic AEC apoptosis by a JNK-dependent mechanism, and suggests a potential novel target for the modulation of chrysotile asbestos-associated lung diseases. PMID:25530474

  13. The c-Jun N-terminal kinase signaling pathway mediates chrysotile asbestos-induced alveolar epithelial cell apoptosis.

    PubMed

    Li, Peng; Liu, Tie; Kamp, David W; Lin, Ziying; Wang, Yahong; Li, Donghong; Yang, Lawei; He, Huijuan; Liu, Gang

    2015-05-01

    Exposure to chrysotile asbestos exposure is associated with an increased risk of mortality in combination with pulmonary diseases including lung cancer, mesothelioma and asbestosis. Multiple mechanisms by which chrysotile asbestos fibers induce pulmonary disease have been identified, however the role of apoptosis in human lung alveolar epithelial cells (AEC) has not yet been fully explored. Accumulating evidence implicates AEC apoptosis as a crucial event in the development of both idiopathic pulmonary fibrosis and asbestosis. The aim of the present study was to determine whether chrysotile asbestos induces mitochondria‑regulated (intrinsic) AEC apoptosis and, if so, whether this induction occurs via the activation of mitogen‑activated protein kinases (MAPK). Human A549 bronchoalveolar carcinoma‑derived cells with alveolar epithelial type II‑like features were used. The present study showed that chrysotile asbestos induced a dose‑ and time‑dependent decrease in A549 cell viability, which was accompanied by the activation of the MAPK c‑Jun N‑terminal kinases (JNK), but not the MAPKs extracellular signal‑regulated kinase 1/2 and p38. Chrysotile asbestos was also shown to induce intrinsic AEC apoptosis, as evidenced by the upregulation of the pro‑apoptotic genes Bax and Bak, alongside the activation of caspase‑9, poly (ADP‑ribose) polymerase (PARP), and the release of cytochrome c. Furthermore, the specific JNK inhibitor SP600125 blocked chrysotile asbestos‑induced JNK activation and subsequent apoptosis, as assessed by both caspase‑9 cleavage and PARP activation. The results of the present study demonstrated that chrysotile asbestos induces intrinsic AEC apoptosis by a JNK‑dependent mechanism, and suggests a potential novel target for the modulation of chrysotile asbestos‑associated lung diseases.

  14. Bcl-xL is overexpressed in hormone-resistant prostate cancer and promotes survival of LNCaP cells via interaction with proapoptotic Bak.

    PubMed

    Castilla, Carolina; Congregado, Belén; Chinchón, David; Torrubia, Francisco J; Japón, Miguel A; Sáez, Carmen

    2006-10-01

    Androgen-sensitive prostate cancer cells turn androgen resistant through complex mechanisms that involve dysregulation of apoptosis. We investigated the role of antiapoptotic Bcl-xL in the progression of prostate cancer as well as the interactions of Bcl-xL with proapoptotic Bax and Bak in androgen-dependent and -independent prostate cancer cells. Immunohistochemical analysis was used to study the expression of Bcl-xL in a series of 139 prostate carcinomas and its association with Gleason grade and time to hormone resistance. Expression of Bcl-xL was more abundant in prostate carcinomas of higher Gleason grades and significantly associated with the onset of hormone-refractory disease. In vivo interactions of Bcl-xL with Bax or Bak in untreated and camptothecin-treated LNCaP and PC3 cells were investigated by means of coimmunoprecipitation. In the absence of any stimuli, Bcl-xL interacts with Bax and Bak in androgen-independent PC3 cells but only with Bak in androgen-dependent LNCaP cells. Interactions of Bcl-xL with Bax and Bak were also evidenced in lysates from high-grade prostate cancer tissues. In LNCaP cells treated with camptothecin, an inhibitor of topoisomerase I, the interaction between Bcl-xL and Bak was absent after 36 h, Bcl-xL decreased gradually and Bak increased coincidentally with the progress of apoptosis. These results support a model in which Bcl-xL would exert an inhibitory effect over Bak via heterodimerization. We propose that these interactions may provide mechanisms for suppressing the activity of proapoptotic Bax and Bak in prostate cancer cells and that Bcl-xL expression contributes to androgen resistance and progression of prostate cancer.

  15. Isoalantolactone Induces Reactive Oxygen Species Mediated Apoptosis in Pancreatic Carcinoma PANC-1 Cells

    PubMed Central

    Khan, Muhammad; Ding, Chuan; Rasul, Azhar; Yi, Fei; Li, Ting; Gao, Hongwen; Gao, Rong; Zhong, Lili; Zhang, Kun; Fang, Xuedong; Ma, Tonghui

    2012-01-01

    Isoalantolactone, a sesquiterpene lactone compound possesses antifungal, antibacteria, antihelminthic and antiproliferative activities. In the present study, we found that isoalantolactone inhibits growth and induces apoptosis in pancreatic cancer cells. Further mechanistic studies revealed that induction of apoptosis is associated with increased generation of reactive oxygen species, cardiolipin oxidation, reduced mitochondrial membrane potential, release of cytochrome c and cell cycle arrest at S phase. N-Acetyl Cysteine (NAC), a specific ROS inhibitor restored cell viability and completely blocked isoalantolactone-mediated apoptosis in PANC-1 cells indicating that ROS are involved in isoalantolactone-mediated apoptosis. Western blot study showed that isoalantolactone increased the expression of phosphorylated p38 MAPK, Bax, and cleaved caspase-3 and decreased the expression of Bcl-2 in a dose-dependent manner. No change in expression of phosphorylated p38 MAPK and Bax was found when cells were treated with isoalantolactone in the presence of NAC, indicating that activation of these proteins is directly dependent on ROS generation. The present study provides evidence for the first time that isoalantolactone induces ROS-dependent apoptosis through intrinsic pathway. Furthermore, our in vivo toxicity study demonstrated that isoalantolactone did not induce any acute or chronic toxicity in liver and kidneys of CD1 mice at dose of 100 mg/kg body weight. Therefore, isoalantolactone may be a safe chemotherapeutic candidate for the treatment of human pancreatic carcinoma. PMID:22532787

  16. Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization.

    PubMed

    Matroule, J Y; Carthy, C M; Granville, D J; Jolois, O; Hunt, D W; Piette, J

    2001-07-05

    Pyropheophorbide-a methylester (PPME) is a second generation of photosensitizers used in photodynamic therapy (PDT). We demonstrated that PPME photosensitization triggered apoptosis of colon cancer cells as measured by using several classical parameters such as DNA laddering, PARP cleavage, caspase activation and mitochondrial release of cytochrome c. Preincubation of cells with N-acetyl cysteine (NAC) or pyrolidine dithiocarbamate (PDTC) protected against apoptosis mediated by PPME photosensitization showing that reactive oxygen species (ROS) are involved as second messengers. On the other hand, photosensitization carried out in the presence of deuterium oxide (D2O) which enhances singlet oxygen (1O2) lifetime only increases necrosis without affecting apoptosis. Since PPME was localized in the endoplasmic reticulum (ER)/Golgi system and lysosomes, other messengers than ROS were tested such as calcium, Bid, Bap31, phosphorylated Bcl-2 and caspase-12 but none was clearly identified as being involved in triggering cytochrome c release from mitochondria. On the other hand, we demonstrated that the transduction pathways leading to NF-kappaB activation and apoptosis were clearly independent although NF-kappaB was shown to counteract apoptosis mediated by PPME photosensitization.

  17. Evasion of TNF-α-mediated apoptosis by hepatitis C virus.

    PubMed

    Kim, Hangeun; Ray, Ranjit

    2014-01-01

    Hepatitis C virus (HCV) often causes chronic infection in humans, although the mechanisms for viral chronicity are not clearly understood. Tumor necrosis factor-α (TNF-α)-mediated apoptosis is a key element in a host organism's defense inhibiting viral spread and persistence. HCV has evolved mechanisms that antagonize host cell death signals so that virus propagation can continue unabated in infected cells. HCV core protein blocks TNF-α-mediated apoptosis signaling and inhibits caspase-8 activation by sustaining the expression of cellular FADD-like interleukin-1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP). HCV core protein also blocks TNF-induced proteolytic cleavage of the death substrate poly (SDP-ribose) polymerase from its native 116-kDa protein to the characteristic 85-kDa polypeptide. A decrease in endogenous c-FLIP by specific small-interfering RNA induces TNF-α-mediated apoptotic cell death and caspase-8 activation. However, HCV core neither affects the association between TNF receptor 1 (TNFR1) and TNFR1-associated death domain protein (TRADD) nor TRADD-Fas-associated death domain protein (FADD) and procaspase-8. Thus, HCV core protein appears to play a role in the inhibition of TNF-α-mediated cell death. This chapter describes methods to identify inhibitory mechanism of HCV for TNF-α-mediated apoptosis.

  18. Kupffer cells of cirrhotic rat livers sensitize colon cancer cells to Fas-mediated apoptosis.

    PubMed

    Song, E; Chen, J; Ouyang, N; Wang, M; Exton, M S; Heemann, U

    2001-05-04

    Metastasis of colorectal carcinomas rarely occurs in cirrhotic livers. Our study investigated the influence of activated Kupffer cells from cirrhotic rat livers on hepatic colonization and FasR-mediated apoptosis of colon cancer cells. A rat colon cancer cell line, RCN-9, was used to inoculate rat livers. Treatment with conditioned media of Kupffer cells isolated from CCl(4)-induced cirrhotic rat livers (cirrhotic KCM) significantly reduced the incidence of hepatic colonization of RCN-9 cells. In vitro cytotoxicity of Kupffer cells and tumour infiltrating lymphocytes (TILs) on RCN-9 cells was evaluated using [(3)H]-release assay. RCN-9 cells were resistant to cytotoxicity mediated by cirrhotic Kupffer cells, but were sensitized to TIL-mediated killing after treatment with cirrhotic KCM. The specific killing induced by TILs was FasR-mediated, as it was inhibited by ZB4, an antagonistic anti-FasR antibody. In agreement, cirrhotic KCM increased recombinant Fas ligand-induced apoptosis of RCN-9 cells, and up-regulated FasR expression on RCN-9 cells as evaluated by RT-PCR and flow cytometry. These findings suggest that Kupffer cells in cirrhotic livers sensitize metastatic colon cancer cells to FasR-mediated apoptosis by up-regulating the receptors, which thus prepare them to be eliminated by infiltrating lymphocytes.

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

    SciTech Connect

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

    2010-08-13

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

  20. Potential role for laminin 5 in hypoxia-mediated apoptosis of human corneal epithelial cells.

    PubMed

    Esco, M A; Wang, Z; McDermott, M L; Kurpakus-Wheater, M

    2001-11-01

    Laminin 5 functions to promote cell-matrix adhesion and therefore is hypothesized to abrogate apoptosis initiated through the loss of epithelial cell contact with extracellular matrix. Laminin 5 levels are decreased in epithelial cells cultured in a hypoxic environment. Exposure of epithelial cells to hypoxia may induce apoptotic pathways transmitted through changes in mitochondrial membrane potential. Using an apoptosis assay based on mitochondrial membrane integrity, the effect of hypoxia (2% oxygen) on human corneal epithelial cell viability was determined. Both a virally transformed corneal epithelial cell line and third passage corneal epithelial cells were resistant to hypoxia-mediated apoptosis for up to 5 days in culture. However, at 7 days in culture, a statistically significant increase in apoptosis was noted in hypoxic corneal epithelial cells compared to normoxic (20% oxygen) controls. Increased apoptosis in hypoxic epithelium at 7 days in culture correlated with decreased deposition of laminin 5 into the extracellular matrix, as determined by western blot analysis and immunofluorescence microscopy. Additionally, the extracellular processing of the alpha3 and gamma2 chains of laminin 5 was negatively impacted by corneal epithelial cell exposure to hypoxia for 7 days. Treatment of human corneal epithelial cells cultured in 20% oxygen with function-inhibiting antibodies to laminin 5 for 2 or 3 days resulted in a statistically significant decrease in proliferation, and concomitant increase in apoptosis, compared with untreated normoxic controls. Based on these results, it appears that mechanisms of hypoxia-mediated apoptosis in human corneal epithelial cells may be initiated by the loss of processed laminin 5 in the extracellular matrix or by the loss of laminin 5-epithelial cell communication and transmitted through mitochondria.

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

    SciTech Connect

    Agarwal, Rakhee; Ali, Nawab

    2010-04-12

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

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

    NASA Astrophysics Data System (ADS)

    Agarwal, Rakhee; Ali, Nawab

    2010-04-01

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

  3. Notch protection against apoptosis in T-ALL cells mediated by GIMAP5.

    PubMed

    Chadwick, Nicholas; Zeef, Leo; Portillo, Virginia; Boros, Joanna; Hoyle, Sarah; van Doesburg, Jaap C L; Buckle, Anne-Marie

    2010-10-15

    Recent studies have highlighted the role of Notch signalling in the development of T cell acute lymphoblasic leukaemia (T-ALL). Over-expression of Notch3 and gain of function mutations in the Notch1 gene have been reported. The aims of this study were to determine the effect of Notch signalling on apoptosis in human T-ALL cell lines and to identify targets of Notch signalling that may mediate this effect. Functional studies showed that inhibition of Notch signalling using gamma secretase inhibitors promoted glucocorticoid-induced apoptosis in cells carrying gain of function mutations in Notch1. Moreover, ectopic expression of constitutively activated Notch provided protection against glucocorticoid-induced apoptosis, indicating that signalling via Notch may also contribute to the development of T-ALL by conferring resistance to apoptosis. Microarray analysis revealed that GIMAP5, a gene coding for an anti-apoptotic intracellular protein, is upregulated by Notch in T-ALL cell lines. Knockdown of GIMAP5 expression using siRNA promoted glucocorticoid-induced apoptosis in T-ALL cells carrying gain of function mutations in Notch1 and in T-ALL cells engineered to express ectopic constitutively activated Notch indicating that Notch signalling protects T-ALL cells from apoptosis by upregulating the expression of GIMAP5.

  4. Secreted tyrosine sulfated-eIF5A mediates oxidative stress-induced apoptosis

    PubMed Central

    Seko, Yoshinori; Fujimura, Tsutomu; Yao, Takako; Taka, Hikari; Mineki, Reiko; Okumura, Ko; Murayama, Kimie

    2015-01-01

    Oxidative stress plays a critical role in ischemia/reperfusion-injury, atherosclerosis, and aging. It causes cell damage that leads to apoptosis via uncertain mechanisms. Because conditioned medium from cardiac myocytes subjected to hypoxia/reoxygenation induces extensive apoptosis of cardiac myocytes under normoxia, we hypothesized that a humoral factor released from the hypoxic/reoxygenated cardiac myocytes mediates apoptosis. We identified an apoptosis-inducing humoral factor in the hypoxia/reoxygenation-conditioned medium. Here, we found that eIF5A undergoes tyrosine sulfation in the trans-Golgi and is rapidly secreted from cardiac myocytes in response to hypoxia/reoxygenation; then, eIF5A induces apoptosis by acting as a pro-apoptotic ligand. The apoptosis of cardiac myocytes induced by hypoxia/reoxygenation or ultraviolet irradiation was suppressed by anti-eIF5A neutralizing monoclonal antibodies (mAbs) in vitro. Myocardial ischemia/reperfusion (but not ischemia alone) markedly increased the plasma levels of eIF5A, and treatment with anti-eIF5A neutralizing mAbs significantly reduced myocardial injury. These results identify an important, novel specific biomarker and a critical therapeutic target for oxidative stress-induced cell injury. PMID:26348594

  5. Induction of mitosis-mediated apoptosis by sodium arsenite in HeLa S3 cells.

    PubMed

    Huang, S; Huang, C F; Lee, T

    2000-09-15

    Arsenic has been used effectively as a chemotherapeutic drug for the treatment of acute promyelocytic leukemia patients. Numerous studies have demonstrated that arsenic induces apoptosis in various cell types. In the present study, we showed that approximately 35% of arsenite-treated HeLa S3 cells arrested in mitosis. After release from arsenite treatment, more than 80% of arsenite-arrested mitotic cells subsequently underwent apoptosis, as indicated by anachronistic nuclear envelope reformation, DNA ladder occurrence, chromatin condensation, and activation of caspases 3 and 9. In exploring how these cells entered apoptosis mechanistically, we found an inverse correlation between mitotic indexes and apoptotic frequencies. As shown by using Percoll density gradient fractionation and flow cytometric analysis, the mitosis-mediated apoptosis induced by arsenite was accompanied by delayed cyclin B degradation and altered mitotic exit. Furthermore, treatment of arsenite-arrested mitotic cells with staurosporine or 2-aminopurine resulted in a rapid degradation of cyclin B, moved these cells forward to interphase without cell division, and abrogated apoptosis. These results suggest that apoptosis occurs in arsenite-arrested mitotic cells that exit mitosis abnormally.

  6. Anticancer activity of hispidin via reactive oxygen species-mediated apoptosis in colon cancer cells.

    PubMed

    Lim, Ji-Hong; Lee, Yoon-Mi; Park, Sa Ra; Kim, Da Hye; Lim, Beong Ou

    2014-08-01

    Few studies have been performed on the anticancer activity of hispidin, a phenolic compound produced from the medicinal mushroom Phellinus linteus. Herein, we studied hispidin-induced apoptosis, which is associated with the generation of reactive oxygen species (ROS) in colon cancer cells. Hispidin was found to reduce cell viability both in mouse and human colon cancer cells. Apoptotic cell morphological changes were observed by microscopy, and apoptosis was assessed in hispidin-treated cells using a biochemical method. The results showed accumulation of the sub-G1 cell population and increase in early apoptosis in a dose-dependent manner. In addition, hispidin induced apoptosis through up-regulation of both intrinsic and extrinsic apoptotic pathways. Although the molecular mechanism underlying hispidin-induced apoptosis is known to involve the generation of ROS, however hispidin did not show any apoptosis in the pre-treatment with a ROS scavenger, N-acetyl-L-cysteine. In conclusion, hispidin induces both intrinsic and extrinsic apoptotic pathways mediated by ROS in colon cancer cells, thereby suggesting that hispidin could be a promising new anticancer agent. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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

    NASA Astrophysics Data System (ADS)

    Bauer, G.

    2011-01-01

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

  8. P53 mediates amosite asbestos-induced alveolar epithelial cell mitochondria-regulated apoptosis.

    PubMed

    Panduri, Vijayalakshmi; Surapureddi, Sailesh; Soberanes, Saul; Weitzman, Sigmund A; Chandel, Navdeep; Kamp, David W

    2006-04-01

    Asbestos causes pulmonary toxicity in part by generating reactive oxygen species that cause DNA damage. We previously showed that the mitochondria-regulated (intrinsic) death pathway mediates alveolar epithelial cell (AEC) DNA damage and apoptosis. Because p53 regulates the DNA damage response in part by inducing intrinsic cell death, we determined whether p53-dependent transcriptional activity mediates asbestos-induced AEC mitochondrial dysfunction and apoptosis. We show that inhibitors of p53-dependent transcriptional activation (pifithrin and type 16-E6 protein) block asbestos-induced AEC mitochondrial membrane potential change (DeltaPsim), caspase 9 activation, and apoptosis. We demonstrate that asbestos activates p53 promoter activity, mRNA levels, protein expression, and Bax and p53 mitochondrial translocation. Further, pifithrin, E6, phytic acid, or rho(0)-A549 cells (cells incapable of mitochondrial reactive oxygen species production) block asbestos-induced p53 activation. Finally, we show that asbestos augments p53 expression in cells at the bronchoalveolar duct junctions of rat lungs and that phytic acid prevents this. These data suggest that p53-dependent transcription pathways mediate asbestos-induced AEC mitochondria-regulated apoptosis. This suggests an important interactive effect between p53 and the mitochondria in the pathogenesis of asbestos-induced pulmonary toxicity that may have broader implications for our understanding of pulmonary fibrosis and lung cancer.

  9. Mouse mammary tumor virus suppresses apoptosis of mammary epithelial cells through ITAM-mediated signaling.

    PubMed

    Kim, Hyoung H; Grande, Shannon M; Monroe, John G; Ross, Susan R

    2012-12-01

    Many receptors in hematopoietic cells use a common signaling pathway that relies on a highly conserved immunoreceptor tyrosine-based activation motif (ITAM), which signals through Src family tyrosine kinases. ITAM-bearing proteins are also found in many oncogenic viruses, including the mouse mammary tumor virus (MMTV) envelope (Env). We previously showed that MMTV Env expression transformed normal mammary epithelial cells and that Src kinases were important mediators in this transformation. To study how ITAM signaling affects mammary cell transformation, we utilized mammary cell lines expressing two different ITAM-containing proteins, one encoding a MMTV provirus and the other a B cell receptor fusion protein. ITAM-expressing cells were resistant to both serum starvation- and chemotherapeutic drug-induced apoptosis, whereas cells transduced with these molecules bearing ITAM mutations were indistinguishable from untransduced cells in their sensitivity to these treatments. We also found that Src kinase was activated in the MMTV-expressing cells and that MMTV-induced apoptosis resistance was completely restored by the Src inhibitor PP2. In vivo, MMTV infection delayed involution-induced apoptosis in the mouse mammary gland. Our results show that MMTV suppresses apoptosis through ITAM-mediated Src tyrosine kinase signaling. These studies could lead to the development of effective treatment of nonhematopoietic cell cancers in which ITAM-mediated signaling plays a role.

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  11. TRAIL mediates apoptosis in cancerous but not normal primary cultured cells of the human reproductive tract.

    PubMed

    Sadarangani, Anil; Kato, Sumie; Espinoza, Natalia; Lange, Soledad; Llados, Carmen; Espinosa, Marisol; Villalón, Manuel; Lipkowitz, Stanley; Cuello, Mauricio; Owen, Gareth I

    2007-01-01

    Cancer of the reproductive tract encompasses malignancies of the uterine corpus, cervix, ovary, Fallopian tube, among others and accounts for 15% of female cancer mortalities. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates apoptosis by binding to death receptors and offers a promising cancer treatment. The goal of this study was to investigate and characterize the effect of TRAIL in endometrial cancer cell lines and normal (non-cancerous) epithelial cells of endometrial origin. We also examined the effect of TRAIL in other primary cultured cancers and normal cells of the human female reproductive tract and evaluated if TRAIL mediated apoptosis correlated with death receptors and decoy receptors 1 and 2.Herein, we demonstrate that TRAIL at concentrations which kill cancerous cells, does not mediate apoptosis or alter cell viability in normal human endometrium, ovary, cervix or Fallopian tube. The partial inhibition by a caspase 9 inhibitor and the total inhibition by a caspase 8 inhibitor demonstrates the dependency on the extrinsic apoptotic pathway. The selective mortality does not correlate with the presence of death or decoy receptors. These results suggest that TRAIL may be an effective treatment for endometrial cancer and other female reproductive cancers, with minimal secondary effects on healthy tissue.

  12. BRCA1 regulates PIG3-mediated apoptosis in a p53-dependent manner.

    PubMed

    Zhang, Wenwen; Luo, Jiayan; Chen, Fengxia; Yang, Fang; Song, Wei; Zhu, Aiyu; Guan, Xiaoxiang

    2015-04-10

    BRCA1 plays a key role in the regulation of p53-dependent target gene transcription activation. Meanwhile, the p53 inducible gene 3 (PIG3) is a downstream target of p53 and is involved in p53-initiated apoptosis. However, little is known about whether BRCA1 can regulate PIG3-mediated apoptosis. Using a tissue microarray containing 149 breast cancer patient samples, we found that BRCA1 and PIG3 expression status were significantly positively correlated (r = 0.678, P < 0.001) and identified a significant positive correlation between high expression of BRCA1 and/or PIG3 and overall survival (OS). Moreover, we reveal that overexpression of BRCA1 significantly increased expression of PIG3 in cells with intact p53, whereas no increase in PIG3 was observed in p53-null MDA-MB-157 cells and p53-depleted HCT116p53-/- cells. Meanwhile, ectopic expression of BRCA1 could not lead to an increase expression level of prohibitin (PHB), which we have previously identified to induce PIG3-mediated apoptosis. Finally, ChIP analysis revealed that PHB can bind to the PIG3 promoter and activate PIG3 transcription independent of p53, although p53 presence did enhance this process. Taken together, our findings suggest that BRCA1 regulates PIG3-mediated apoptosis in a p53-dependent manner, and that PIG3 expression is associated with a better OS in breast cancer patients.

  13. Cyclin C mediates stress-induced mitochondrial fission and apoptosis

    PubMed Central

    Wang, Kun; Yan, Ruilan; Cooper, Katrina F.; Strich, Randy

    2015-01-01

    Mitochondria are dynamic organelles that undergo constant fission and fusion cycles. In response to cellular damage, this balance is shifted dramatically toward fission. Cyclin C–Cdk8 kinase regulates transcription of diverse gene sets. Using knockout mouse embryonic fibroblasts (MEFs), we demonstrate that cyclin C directs the extensive mitochondrial scission induced by the anticancer drug cisplatin or oxidative stress. This activity is independent of transcriptional regulation, as Cdk8 is not required for this activity. Furthermore, adding purified cyclin C to unstressed permeabilized MEF cultures induced complete mitochondrial fragmentation that was dependent on the fission factors Drp1 and Mff. To regulate fission, a portion of cyclin C translocates from the nucleus to the cytoplasm, where it associates with Drp1 and is required for its enhanced mitochondrial activity in oxidatively stressed cells. In addition, although HeLa cells regulate cyclin C in a manner similar to MEF cells, U2OS osteosarcoma cultures display constitutively cytoplasmic cyclin C and semifragmented mitochondria. Finally, cyclin C, but not Cdk8, is required for loss of mitochondrial outer membrane permeability and apoptosis in cells treated with cisplatin. In conclusion, this study suggests that cyclin C connects stress-induced mitochondrial hyperfission and programmed cell death in mammalian cells. PMID:25609094

  14. Grape seed proanthocyanidins (GSPs) inhibit the growth of cervical cancer by inducing apoptosis mediated by the mitochondrial pathway.

    PubMed

    Chen, Qing; Liu, Xiao-Fang; Zheng, Peng-Sheng

    2014-01-01

    Grape seed proanthocyanidins (GSPs), a biologically active component of grape seeds, have been reported to possess a wide array of pharmacological and biochemical properties. Recently, the inhibitory effects of GSPs on various cancers have been reported, but their effects on cervical cancer remain unclear. Here, we explored the effect of GSPs on cervical cancer using in vitro and in vivo models. In vitro, the treatment of HeLa and SiHa cells with GSPs resulted in a significant inhibition of cell viability. Further investigation indicated that GSPs led to the dose-dependent induction of apoptosis in cancer cells. The underlying mechanism was associated with increased expression of the pro-apoptotic protein Bak-1, decreased expression of the anti-apoptotic protein Bcl-2, the loss of mitochondrial membrane potential, and the activation of caspase-3, suggesting that GSPs induced cervical cancer cell apoptosis through the mitochondrial pathway. In addition, the administration of GSPs (0.1%, 0.2%, and 0.4%, w/v) as a supplement in drinking water significantly inhibited the tumor growth of HeLa and SiHa cells in athymic nude mice, and the number of apoptotic cells in those tumors was also increased significantly. Taken together, our studies demonstrated that GSPs could inhibit the growth of cervical cancer by inducing apoptosis through the mitochondrial pathway, which provides evidence indicating that GSPs may be a potential chemopreventive and/or chemotherapeutic agent for cervical cancer.

  15. Cyclic adenosine monophosphate-mediated protection against bile acid-induced apoptosis in cultured rat hepatocytes.

    PubMed

    Webster, C R; Anwer, M S

    1998-05-01

    Cyclic adenosine monophosphate (cAMP) has been shown to modulate apoptosis. To evaluate the role of cAMP in bile acid-induced hepatocyte apoptosis, we studied the effect of agents that increase cAMP on the induction of apoptosis by glycochenodeoxycholate (GCDC) in cultured rat hepatocytes. GCDC induced apoptosis in 26.5%+/-1.1% of hepatocytes within 2 hours. Twenty-minute pretreatment of hepatocytes with 100 micromol/L 8-(4-chlorothiophenyl) cAMP (CP-cAMP) resulted in a reduction in the amount of apoptosis to 35.2%+/-3.8% of that seen in hepatocytes treated with GCDC alone. Other agents that increase intracellular cAMP, including dibutyryl cAMP (100 micromol/L), glucagon (200 nmol/L), and a combination of forskolin (20 micromol/L) and 3-isobutyl-1-methylxanthine (20 micromol/L), also inhibited GCDC-induced apoptosis to a similar extent. Pretreatment with the protein kinase A (PKA) inhibitor, KT5720, prevented the protective effect of CP-cAMP and inhibited CP-cAMP-induced activation of PKA activity. Inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin (50 nmol/L), or Ly 294002 (20 micromol/L) also prevented the cytoprotective effect of cAMP. PI3K assays confirmed that wortmannin (50 nmol/L) inhibited PI3K activity, while CP-cAMP had no effect on the activity of this lipid kinase. GCDC increased mitogen-activated protein kinase (MAPK) activity, but had no effect on stress-activated protein kinase (SAPK) activity in hepatocytes. cAMP decreased basal and GCDC-induced MAPK activity and increased SAPK activity. The MAPK kinase inhibitor, PD 98059, inhibited both GCDC-mediated MAPK activation and GCDC-induced apoptosis. 1) agents that increase intracellular cAMP protect against hepatocyte apoptosis induced by hydrophobic bile acids; 2) activation of MAPK by GCDC may be involved in bile acid-induced apoptosis; and 3) cAMP-mediated cytoprotection against bile acid-induced apoptosis appears to involve PKA, MAPK, and PI3K.

  16. Mechanism of apoptosis induction by inhibition of the anti-apoptotic BCL-2 proteins.

    PubMed

    Chipuk, Jerry E; Fisher, John C; Dillon, Christopher P; Kriwacki, Richard W; Kuwana, Tomomi; Green, Douglas R

    2008-12-23

    Normal cellular lifespan is contingent upon preserving outer mitochondrial membrane (OMM) integrity, as permeabilization promotes apoptosis. BCL-2 family proteins control mitochondrial outer membrane permeabilization (MOMP) by regulating the activation of the pro-apoptotic BCL-2 effector molecules, BAX and BAK. Sustainable cellular stress induces proteins (e.g., BID, BIM, and cytosolic p53) capable of directly activating BAX and/or BAK, but these direct activators are sequestered by the anti-apoptotic BCL-2 proteins (e.g., BCL-2, BCL-xL, and MCL-1). In the event of accumulated or marked cellular stress, a coordinated effort between previously sequestered and nascent BH3-only proteins inhibits the anti-apoptotic BCL-2 repertoire to promote direct activator protein-mediated MOMP. We examined the effect of ABT-737, a BCL-2 antagonist, and PUMA, a BH3-only protein that inhibits the entire anti-apoptotic BCL-2 repertoire, with cells and mitochondria that sequestered direct activator proteins. ABT-737 and PUMA cooperated with sequestered direct activator proteins to promote MOMP and apoptosis, which in the absence of ABT-737 or PUMA did not influence OMM integrity or cellular survival. Our data show that the induction of apoptosis by inhibition of the anti-apoptotic BCL-2 repertoire requires "covert" levels of direct activators of BAX and BAK at the OMM.

  17. Intracellular mechanisms mediating tocotrienol-induced apoptosis in neoplastic mammary epithelial cells.

    PubMed

    Sylvester, Paul W; Shah, Sumit

    2005-01-01

    Tocotrienols and tocopherols represent the two subgroups that make up the vitamin E family of compounds. However, tocotrienols display significantly more potent apoptotic activity in neoplastic mammary epithelial cells than tocopherols. Studies were conducted to determine the intracellular mechanism(s) mediating tocotrienol-induced apoptosis in neoplastic +SA mouse mammary epithelial cells in vitro. An initial step in apoptosis is the activation of 'initiator' caspases (caspase-8 or -9) that subsequently activate 'effector' caspases (caspase-3, -6 and -7) and induce apoptosis. Treatment with cytotoxic doses of alpha-tocotrienol (20 microM) resulted in a time-dependent increase in caspase-8 and caspase-3 activity. Combined treatment with specific caspase-8 or caspase-3 inhibitors completely blocked alpha-tocotrienol-induced apoptosis and caspase-8 or caspase-3 activity, respectively. In contrast, alpha-tocotrienol treatment had no effect on caspase-9 activation, and combined treatment with a specific caspase-9 inhibitor did not block alpha-tocotrienol-induced apoptosis in (+)SA cells. Since caspase-8 activation is associated with the activation of death receptors, such as Fas, tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL) receptors, studies were conducted to determine the exact death receptor(s) and ligand(s) involved in mediating tocotrienol-induced caspase-8 activation and apoptosis. Treatment with Fas-ligand (FasL), Fas-activating antibody, or TRAIL failed to induce cell death in (+)SA neoplastic mammary epithelial cells, suggesting that these cells are resistant to death receptor-induced apoptosis. Moreover, treatment with cytotoxic doses of alpha-tocotrienol did not alter the intracellular levels of Fas, FasL, or Fas-associated death domain (FADD) in these cells. Western blot analysis also showed that alpha-tocotrienol did not induce FasL or FADD translocation from the cytosolic to membrane fraction in these cells. Finally

  18. Intrinsic Pathway-Mediated Apoptosis in Elastase-Induced Aneurysms in Rabbits

    PubMed Central

    Kadirvel, Ramanathan; Ding, Yong Hong; Dai, Daying; Lewis, Debra A; Kallmes, David F

    2009-01-01

    Background and Objectives The pathophysiology of saccular aneurysms is complex and multifactorial. The aim of the present study was to understand the mechanism of apoptosis in elastase-induced model aneurysms in rabbits. Methods Elastase-induced saccular aneurysms were created at the origin of the right common carotid artery in 20 rabbits. Aneurysm samples were harvested at 2 and 12 weeks after creation. Expression of apoptosis-associated proteins, including caspases and Bcl-2 proteins, were assessed by Western blot analysis (n=5 at both time points). TUNEL staining, which indicates the presence of apoptosis, was performed in tissue sections (n=5 at both time points). The unoperated, contralateral common carotid artery was used as a control. Results Expression of active caspase-3, the final executioner of apoptosis, and caspase-9, the mediator of the intrinsic mitochondrial pathway, was observed in aneurysms at 2 weeks, whereas the expression of activated caspase-8, the mediator of the extrinsic death receptor pathway, was absent at both time points. Expression of anti-apoptotic proteins, Bcl-2 and phospho-Bad, was down-regulated in aneurysms as compared to controls at 2 weeks. None of these proteins was differentially expressed at 12 weeks. These results were confirmed by the presence of TUNEL positive cells in some aneurysms at the early time point. Conclusion In this study of elastase induced aneurysms in a rabbit model, activation of apoptosis in elastase-induced model aneurysms is mediated predominantly by the Bcl-2 mediated-intrinsic pathway through the activation of caspase-9. PMID:19749227

  19. An Allelic Series of bak1 Mutations Differentially Alter bir1 Cell Death, Immune Response, Growth, and Root Development Phenotypes in Arabidopsis thaliana

    PubMed Central

    Wierzba, Michael P.; Tax, Frans E.

    2016-01-01

    Receptor-like kinases (RLKs) mediate cell-signaling pathways in Arabidopsis thaliana, including those controlling growth and development, immune response, and cell death. The RLK coreceptor BRI1-ASSOCIATED KINASE-1 (BAK1) partners with multiple ligand-binding RLKs and contributes to their signaling in diverse pathways. An additional RLK, BAK1-INTERACTING RECEPTOR-1 (BIR1), physically interacts with BAK1, and loss-of-function mutations in BIR1 display constitutive activation of cell death and immune response pathways and dwarfism and a reduction in lateral root number. Here we show that bir1 plants display defects in primary root growth, characterize bir1 lateral root defects, and analyze expression of BIR1 and BAK1 promoters within the root. Using an allelic series of bak1 mutations, we show that loss of BAK1 function in immune response pathways can partially suppress bir1 cell death, immune response, and lateral root phenotypes and that null bak1 alleles enhance bir1 primary root phenotypes. Based on our data, we propose a model in which BIR1 functions to regulate BAK1 participation in multiple pathways. PMID:26680657

  20. Brassinosteroids inhibit pathogen-associated molecular pattern–triggered immune signaling independent of the receptor kinase BAK1

    PubMed Central

    Albrecht, Catherine; Boutrot, Freddy; Segonzac, Cécile; Schwessinger, Benjamin; Gimenez-Ibanez, Selena; Chinchilla, Delphine; Rathjen, John P.; de Vries, Sacco C.; Zipfel, Cyril

    2012-01-01

    Plants and animals use innate immunity as a first defense against pathogens, a costly yet necessary tradeoff between growth and immunity. In Arabidopsis, the regulatory leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1 combines with the LRR-RLKs FLS2 and EFR in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and the LRR-RLK BRI1 in brassinosteroid (BR)-mediated growth. Therefore, a potential tradeoff between these pathways mediated by BAK1 is often postulated. Here, we show a unidirectional inhibition of FLS2-mediated immune signaling by BR perception. Unexpectedly, this effect occurred downstream or independently of complex formation with BAK1 and associated downstream phosphorylation. Thus, BAK1 is not rate-limiting in these pathways. BRs also inhibited signaling triggered by the BAK1-independent recognition of the fungal PAMP chitin. Our results suggest a general mechanism operative in plants in which BR-mediated growth directly antagonizes innate immune signaling. PMID:22087006

  1. Effects of Fluoride on DNA Damage and Caspase-Mediated Apoptosis in the Liver of Rats.

    PubMed

    Song, Guo Hua; Huang, Fu Bing; Gao, Ji Ping; Liu, Mao Lin; Pang, Wen Biao; Li, Wei bin; Yan, Xiao Yan; Huo, Mei Jun; Yang, Xia

    2015-08-01

    Fluoride compounds are abundant and widely distributed in the environment at a variety of concentrations. Further, fluoride induces toxic effects in target organs such as the liver. In this study, we investigated liver histopathology, DNA damage, apoptosis, and the mRNA and protein expressions of caspase-3 and -9 in the rat livers by administering varying concentrations of fluoride (0, 50, 100, 200 mg/L ) for 120 days. The results showed fluoride-induced morphological changes and significantly increased apoptosis and DNA damage in rats exposed to fluoride, especially in response to higher doses. The immunohistochemical and qRT-PCR results indicated that caspase-3, caspase-9 protein positive expression and mRNA relative expression enhanced with increasing NaF concentration. In summary, our findings suggest that chronic exposure to fluoride causes damages to liver histopathology and leads to liver apoptosis through caspase-mediated pathways.

  2. Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress.

    PubMed

    Lin, Pingdong; Weng, Xiaping; Liu, Fayuan; Ma, Yuhuan; Chen, Houhuang; Shao, Xiang; Zheng, Wenwei; Liu, Xianxiang; Ye, Hongzhi; Li, Xihai

    2015-12-01

    Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type Ⅱ collagen. The ER stress-mediated apoptosis of tunicamycin (TM)‑stimulated chondrocytes was detected using 4-phenylbutyric acid (4‑PBA). We found that 4‑PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4',6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM‑induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X‑box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP‑homologous protein (Chop), caspase‑9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase

  3. Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress

    PubMed Central

    LIN, PINGDONG; WENG, XIAPING; LIU, FAYUAN; MA, YUHUAN; CHEN, HOUHUANG; SHAO, XIANG; ZHENG, WENWEI; LIU, XIANXIANG; YE, HONGZHI; LI, XIHAI

    2015-01-01

    Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type II collagen. The ER stress-mediated apoptosis of tunicamycin (TM)-stimulated chondrocytes was detected using 4-phenylbutyric acid (4-PBA). We found that 4-PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4′,6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM-induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X-box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), caspase-9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase-9, caspase-3

  4. Mitochondrial-derived free radicals mediate asbestos-induced alveolar epithelial cell apoptosis.

    PubMed

    Panduri, Vijayalakshmi; Weitzman, Sigmund A; Chandel, Navdeep S; Kamp, David W

    2004-06-01

    Asbestos causes pulmonary toxicity by mechanisms that in part involve reactive oxygen species (ROS). However, the precise source of ROS is unclear. We showed that asbestos induces alveolar epithelial cell (AEC) apoptosis by a mitochondrial-regulated death pathway. To determine whether mitochondrial-derived ROS are necessary for causing asbestos-induced AEC apoptosis, we utilized A549-rho(omicron) cells that lack mitochondrial DNA and a functional electron transport. As expected, antimycin, which induces an oxidative stress by blocking mitochondrial electron transport at complex III, increased dichlorofluoroscein (DCF) fluorescence in A549 cells but not in A549-rho(omicron) cells. Compared with A549 cells, rho(omicron) cells have less asbestos-induced ROS production, as assessed by DCF fluorescence, and reductions in total glutathione levels as well as less caspase-9 activation and apoptosis, as assessed by TdT-mediated dUTP nick end labeling staining and DNA fragmentation. A mitochondrial anion channel inhibitor that prevents ROS release from the mitochondria to the cytoplasm also blocked asbestos-induced A549 cell caspase-9 activation and apoptosis. Finally, a role for nonmitochondrial-derived ROS with exposure to high levels of asbestos (50 microg/cm(2)) was suggested by our findings that an iron chelator (phytic acid or deferoxamine) or a free radical scavenger (sodium benzoate) provided additional protection against asbestos-induced caspase-9 activation and DNA fragmentation in rho(omicron) cells. We conclude that asbestos fibers affect mitochondrial DNA and functional electron transport, resulting in mitochondrial-derived ROS production that in turn mediates AEC apoptosis. Nonmitochondrial-associated ROS may also contribute to AEC apoptosis, particularly with high levels of asbestos exposure.

  5. Nickel nanowires induced and reactive oxygen species mediated apoptosis in human pancreatic adenocarcinoma cells

    PubMed Central

    Hossain, Md. Zakir; Kleve, Maurice G

    2011-01-01

    Background The ability to evade apoptosis is one of the key properties of cancer. The apoptogenic effect of nickel nanowires (Ni NWs) on cancer cell lines has never been adequately addressed. Due to the unique physicochemical characteristics of Ni NWs, we envision the development of a novel anticancer therapeutics specifically for pancreatic cancer. Thus, we investigated whether Ni NWs induce ROS-mediated apoptosis in human pancreatic adenocarcinoma (Panc-1) cells. Methods In this study Ni NWs were fabricated using the electrodeposition method. Synthesized Ni NWs were physically characterized by energy dispersive X-ray analysis, UV-Vis spectroscopy of NanoDrop 2000 (UV-Vis), magnetization study, scanning electron microscopy, and transmission electron microscopy. Assessment of morphological apoptotic characteristics by phase contrast microscopy (PCM), Ni-NWs-induced apoptosis staining with ethidium bromide (EB) and acridine orange (AO) followed by fluorescence microscopy (FM) was performed. For molecular biological and biochemical characterization, Panc-1 cell culture and cytotoxic effect of Ni NWs were determined by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Quantitative apoptosis was analyzed by flow cytometry staining with propidium iodide through cell cycle arrest and generation of ROS using 2′, 7′-dichlorofluorescein diacetate fluorescence intensity. In all experiments, Panc-1 cancer cells without any treatment were used as the negative controls. Results The intracellular uptake of Ni NWs through endocytosis by Panc-1 cells was observed by PCM. EB and AO staining of FM and MTT assay qualitatively and quantitatively confirmed the extent of apoptosis. Flow cytometric cell cycle arrest and ROS generation indicated Ni NWs as inducers of apoptotic cell death. Conclusion We investigated the role of Ni NWs as inducers of ROS-mediated apoptosis in Panc-1 cells. These results suggested that Ni NWs could be an effective

  6. The Mitochondrial Fission Protein hFis1 Requires the Endoplasmic Reticulum Gateway to Induce Apoptosis

    PubMed Central

    Alirol, Emilie; James, Dominic; Huber, Denise; Marchetto, Andrea; Vergani, Lodovica

    2006-01-01

    Mitochondrial fission ensures organelle inheritance during cell division and participates in apoptosis. The fission protein hFis1 triggers caspase-dependent cell death, by causing the release of cytochrome c from mitochondria. Here we show that mitochondrial fission induced by hFis1 is genetically distinct from apoptosis. In cells lacking the multidomain proapoptotic Bcl-2 family members Bax and Bak (DKO), hFis1 caused mitochondrial fragmentation but not organelle dysfunction and apoptosis. Similarly, a mutant in the intermembrane region of hFis1-induced fission but not cell death, further dissociating mitochondrial fragmentation from apoptosis induction. Selective correction of the endoplasmic reticulum (ER) defect of DKO cells restored killing by hFis1, indicating that death by hFis1 relies on the ER gateway of apoptosis. Consistently, hFis1 did not directly activate BAX and BAK, but induced Ca2+-dependent mitochondrial dysfunction. Thus, hFis1 is a bifunctional protein that independently regulates mitochondrial fragmentation and ER-mediated apoptosis. PMID:16914522

  7. Platelets induce apoptosis via membrane-bound FasL

    PubMed Central

    Schleicher, Rebecca I.; Reichenbach, Frank; Kraft, Peter; Kumar, Anil; Lescan, Mario; Todt, Franziska; Göbel, Kerstin; Hilgendorf, Ingo; Geisler, Tobias; Bauer, Axel; Olbrich, Marcus; Schaller, Martin; Wesselborg, Sebastian; O’Reilly, Lorraine; Meuth, Sven G.; Schulze-Osthoff, Klaus; Gawaz, Meinrad; Li, Xuri; Kleinschnitz, Christoph; Edlich, Frank

    2015-01-01

    After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL△m/△m) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre+ FasLfl/fl mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis. PMID:26232171

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

    PubMed Central

    Wang, Kewei; Wang, Minhua; Gannon, Maureen

    2016-01-01

    Background and Aims Growth hormone (GH) not only supports hepatic metabolism but also protects against hepatocyte cell death. Hnf6 (or Oc1) belonging to the Onecut family of hepatocyte transcription factors known to regulate differentiated hepatic function, is a GH-responsive gene. We evaluate if GH mediates Hnf6 activity to attenuate hepatic apoptotic injury. Methods We used an animal model of hepatic apoptosis by bile duct ligation (BDL) with Hnf6 -/- (KO) mice in which hepatic Hnf6 was conditionally inactivated. GH was administered to adult wild type WT and KO mice for the 7 days of BDL to enhance Hnf6 expression. In vitro, primary hepatocytes derived from KO and WT liver were treated with LPS and hepatocyte apoptosis was assessed with and without GH treatment. Results In WT mice, GH treatment enhanced Hnf6 expression during BDL, inhibited Caspase -3, -8 and -9 responses and diminished hepatic apoptotic and fibrotic injury. GH-mediated upregulation of Hnf6 expression and parallel suppression of apoptosis and fibrosis in WT BDL liver were abrogated in KO mice. LPS activated apoptosis and suppressed Hnf6 expression in primary hepatocytes. GH/LPS co-treatment enhanced Hnf6 expression with corresponding attenuation of apoptosis in WT-derived hepatocytes, but not in KO hepatocytes. ChiP-on-ChiP and electromobility shift assays of KO and WT liver nuclear extracts identified Ciap1 (or Birc2) as an Hnf6-bound target gene. Ciap1 expression patterns closely follow Hnf6 expression in the liver and in hepatocytes. Conclusion GH broad protective actions on hepatocytes during liver injury are effected through Hnf6, with Hnf6 transcriptional activation of Ciap1 as an underlying molecular mediator. PMID:27936029

  9. Glutamine deprivation sensitizes human breast cancer MDA-MB-231 cells to TRIAL-mediated apoptosis.

    PubMed

    Dilshara, Matharage Gayani; Jeong, Jin-Woo; Prasad Tharanga Jayasooriya, Rajapaksha Gedara; Neelaka Molagoda, Ilandarage Menu; Lee, Seungheon; Park, Sang Rul; Choi, Yung Hyun; Kim, Gi-Young

    2017-04-01

    Tumor cell metabolism is a promising target for various cancer treatments. Apart from aerobic glycolysis, cancer cell growth is dependent on glutamine (Gln) supply, leading to their survival and differentiation. Therefore, we examined whether treatment with TNF-related apoptosis-inducing ligand (TRAIL) sensitizes MDA-MB-231 cells to apoptosis under Gln deprivation condition (TRAIL/Gln deprivation). Gln deprivation decreased cell proliferation as expected, but did not induce remarkable cell death. TRAIL/Gln deprivation, however, significantly increased growth inhibition and morphological shrinkage of MDA-MB-231 cells compared to those induced by treatment with either Gln deprivation or TRAIL alone. Moreover, TRAIL/Gln deprivation upregulated the apoptotic sub-G1 phase accompanied with a remarkable decrease of pro-caspase-3, pro-caspase-9, and anti-apoptotic xIAP, and Bcl-2. Increased cleavage of PARP and pro-apoptotic Bid protein expression suggests that TRAIL/Gln deprivation triggers mitochondrion-mediated apoptosis in MDA-MB-231 cells. Additionally, TRAIL/Gln deprivation upregulated the expression of endoplasmic reticulum (ER) stress markers such as ATF4 and phosphorylated eIF2α, thereby enhancing the C/EBP homologous protein (CHOP) protein level. Transient knockdown of CHOP partically reversed TRAIL/Gln deprivation-mediated apoptosis. Accordingly, TRAIL/Gln deprivation enhanced the expression of death receptor 5 (DR5) and transient knockdown of DR5 completely restored TRAIL/Gln deprivation-mediated apoptosis. Taken together, our results suggest that Gln deprivation conditions can be used for the development of new therapies for TRAIL-resistant cancers.

  10. Involvement of apoptosis in mediating mitomycin C-induced teratogenesis in vitro.

    PubMed

    Singh, Gyanendra; Sinha, Neeraj

    2010-05-01

    Mitomycin C (MMC) is among the most commonly used drugs worldwide and is known to cause congenital malformations and fetal death in animals. In this study, the effect of MMC on major organogenesis period and the role of apoptosis in mediating congenital malformations have been carried out. In the present study, post-implantation rat embryos of day 11 were cultured for 24 h with various concentrations of MMC, i.e. 1, 10, and 100 microg/ml cultures. The growth and developmental of each embryo was evaluated and compared with control ones for the presence of any malformations. The MMC decreased all growth and developmental parameters in a concentration-dependent manner, when compared with control. However, exposure to MMC at 1 microg/ml culture did not show any significant effect on embryonic growth and development. Parallel to this, flow cytometric analysis (cell cycle and annexin V binding) and DNA fragmentation assay were carried out followed by quantitation by 3'-OH labeling of cultured rat embryos to evaluate the role of apoptosis in bringing about MMC-induced teratogenesis. All results were found to be dose-dependent and an increase in apoptosis in embryonic tissues may be related to the increased risk of congenital malformations. The data suggested that apoptosis might be involved in mediating teratogenesis of MMC in vitro.

  11. Role of apoptosis in mediating salicylic acid-induced teratogenesis in vitro.

    PubMed

    Singh, Gyanendra; Sinha, Neeraj; Mahipag G, S N R

    2009-02-01

    Salicylic acid (SAL) is among the most commonly used drugs worldwide and is known to cause congenital malformations and fetal death in animals. In this study, the effect of SAL on major organogenesis period and the role of apoptosis in mediating congenital malformations have been carried out. In the present study, post-implantation rat embryos of day 11 were cultured for 24 h with various concentrations of SAL, i.e. 10, 100, and 1000 microg/ml cultures, respectively. The growth and developmental of each embryo was evaluated and compared with control ones for the presence of any malformations. The SAL decreased all growth and developmental parameters in a concentration-dependent manner, when compared with control. However, exposure to SAL at 10 microg/ml culture did not show any significant effect on embryonic growth and development. Parallel to this, flow cytometric analysis (cell cycle and annexin V binding) and DNA fragmentation assay were carried out followed by quantitation by 3'-OH labeling of cultured rat embryos to evaluate the role of apoptosis in bringing about SAL-induced teratogenesis. All results were found to be dose-dependent and an increase in apoptosis in embryonic tissues may be related to the increased risk of congenital malformations. The data suggested that apoptosis might be involved in mediating teratogenesis of SAL in vitro.

  12. The intrinsic apoptosis pathway mediates the pro-longevity response to mitochondrial ROS in C. elegans.

    PubMed

    Yee, Callista; Yang, Wen; Hekimi, Siegfried

    2014-05-08

    The increased longevity of the C. elegans electron transport chain mutants isp-1 and nuo-6 is mediated by mitochondrial ROS (mtROS) signaling. Here we show that the mtROS signal is relayed by the conserved, mitochondria-associated, intrinsic apoptosis signaling pathway (CED-9/Bcl2, CED-4/Apaf1, and CED-3/Casp9) triggered by CED-13, an alternative BH3-only protein. Activation of the pathway by an elevation of mtROS does not affect apoptosis but protects from the consequences of mitochondrial dysfunction by triggering a unique pattern of gene expression that modulates stress sensitivity and promotes survival. In vertebrates, mtROS induce apoptosis through the intrinsic pathway to protect from severely damaged cells. Our observations in nematodes demonstrate that sensing of mtROS by the apoptotic pathway can, independently of apoptosis, elicit protective mechanisms that keep the organism alive under stressful conditions. This results in extended longevity when mtROS generation is inappropriately elevated. These findings clarify the relationships between mitochondria, ROS, apoptosis, and aging.

  13. Ketoconazole induces apoptosis in rat cardiomyocytes through reactive oxygen species-mediated parkin overexpression.

    PubMed

    Won, Kyung Jong; Lee, Kang Pa; Yu, Suyeol; Lee, Donghyen; Lee, Dong-Youb; Lee, Hwan Myung; Kim, Junghwan; Jung, Seung Hyo; Baek, Suji; Kim, Bokyung

    2015-10-01

    Azole antifungals such as ketoconazole are generally known to induce a variety of heart function side effects, e.g., long-QT syndrome and ventricular arrhythmias. However, a clear mechanism for the action of ketoconazole in heart cells has not been reported. In the present study, we assessed the correlation between ketoconazole-induced apoptosis and the alteration of genes in response to ketoconazole in rat cardiomyocytes. Cardiomyocyte viability was significantly inhibited by treatment with ketoconazole. Ketoconazole also stimulated H2O2 generation and TUNEL-positive apoptosis in a dose-dependent manner. DNA microarray technology revealed that 10,571 genes were differentially expressed by more than threefold in ketoconazole-exposed cardiomyocytes compared with untreated controls. Among these genes, parkin, which encodes a component of the multiprotein E3 ubiquitin ligase complex, was predominantly overexpressed among those classified as apoptosis- and reactive oxygen species (ROS)-related genes. The expression of parkin was also elevated in cardiomyocytes treated with exogenous H2O2. Moreover, cell viability and apoptosis in response to ketoconazole were inhibited in cardiomyocytes treated with ROS inhibitors and transfected with parkin siRNA. From the present findings, we concluded that ketoconazole may increase the expression of parkin via the ROS-mediated pathway, which consequently results in the apoptosis and decreased viability of cardiomyocytes.

  14. PTEN Reduced UVB-Mediated Apoptosis in Retinal Pigment Epithelium Cells

    PubMed Central

    He, Jia; Long, Chongde; Huang, Zixin; Zhou, Xin; Kuang, Xielan; Liu, Lanying; Liu, Huijun; Tang, Yan; Fan, Yuting; Ning, Jie; Ma, Xinqi; Zhang, Qingjiong

    2017-01-01

    Age-related macular degeneration (AMD) is a leading cause of blindness and progressive loss of central vision in the elderly population. The important factor of AMD pathogenesis is the degeneration of retinal pigment epithelial (RPE) cells by oxidative stress. Inactivation of PTEN can disrupt intercellular adhesion in the RPE cells, but the mechanism of oxidative stress is less known. Here we presented evidence that UVB-mediated oxidative stress induced apoptosis in ARPE-19 cells. Downregulation of the expression of PTEN in UVB-irradiative RPE cells triggered DNA damage and increased the level of UVB-induced apoptosis by activating p53-dependent pathway. However, overexpression of PTEN increased cell survival by suppressing p-H2A in response to DNA damage and apoptosis. When using Pifithrin-α (one of p53 inhibitors), the level of p53-dependent apoptosis was significantly lower than untreated, which suggested that p53 was possibly involved in PTEN-dependent apoptosis. Thus, it elucidated the molecular mechanisms of UVB-induced damage in RPE cells and may offer an alternative therapeutic target in dry AMD. PMID:28321407

  15. Immunohistochemical detection of extrinsic and intrinsic mediators of apoptosis in porcine paraffin-embedded tissues.

    PubMed

    Barranco, Inmaculada; Gómez-Laguna, Jaime; Rodríguez-Gómez, Irene M; Salguero, Francisco J; Pallarés, Francisco J; Bernabé, Antonio; Carrasco, Librado

    2011-02-15

    Apoptosis is a strictly regulated mechanism of cell death that involves a complex network of biochemical pathways. Whether a cell undergoes apoptosis or not depends on a delicate balance of anti- and pro-apoptotic stimuli. This phenomenon can be induced by two different pathways: intrinsic and extrinsic pathways. The main aim of this study was to determine the ideal fixative and antigen retrieval method in porcine paraffin embedded tissues for the immunohistochemical detection of apoptosis mediators, from both extrinsic and intrinsic pathways. Tonsil, retropharyngeal lymph node and lung tissue samples were fixed in 10% neutral buffered formalin, Bouin solution and zinc salts fixative (ZSF) and different unmasking methods were carried out. Both 10% neutral buffered formalin and ZSF resulted as the fixatives of election to study apoptosis phenomena. Tween 20 (0.01% in PBS), citrate buffer (microwave, pH 6.0) and/or protease type XIV were the antigen retrieval methods which displayed better labelling. Our results allow to deep in the knowledge of apoptosis and its role in the pathogenesis of porcine diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

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

    SciTech Connect

    Chen Chen; Shimizu, Shigeomi; Tsujimoto, Yoshihide; Motoyama, Noboru . E-mail: motoyama@nils.go.jp

    2005-07-29

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

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

    PubMed

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

    2017-10-01

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

  18. Apigenin induces c-Myc-mediated apoptosis in FRO anaplastic thyroid carcinoma cells.

    PubMed

    Kim, Si Hyoung; Kang, Jun Goo; Kim, Chul Sik; Ihm, Sung-Hee; Choi, Moon Gi; Yoo, Hyung Joon; Lee, Seong Jin

    2013-04-30

    Apigenin promotes apoptosis in cancer cells. We studied the effect of apigenin on cell survival and c-Myc expression in FRO anaplastic thyroid carcinoma (ATC) cells. Apigenin caused apoptosis via the elevation of c-Myc levels in conjunction with the phosphorylation of p38 and p53. In the c-Myc siRNA-transfected and apigenin-treated cells, compared with the apigenin-treated control cells, apoptosis and phosphorylation of p38 and p53 were ameliorated. In the presence of apigenin, diminution of p38 and p53 did not affect cell survival although apigenin activated the phosphorylation of p38 and p53 via increased c-Myc levels. In conclusion, our results indicate that apigenin induces apoptosis mediated via c-Myc with concomitant phosphorylation of p53 and p38 in FRO ATC cells. These findings suggest that augmented c-Myc acts as a core regulator and is necessary for apigenin-induced apoptosis in FRO ATC cells. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

    NASA Astrophysics Data System (ADS)

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

    2010-10-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

  1. l-Cystathionine Inhibits the Mitochondria-Mediated Macrophage Apoptosis Induced by Oxidized Low Density Lipoprotein

    PubMed Central

    Zhu, Mingzhu; Du, Junbao; Chen, Siyao; Liu, Angie Dong; Holmberg, Lukas; Chen, Yonghong; Zhang, Chunyu; Tang, Chaoshu; Jin, Hongfang

    2014-01-01

    This study was designed to investigate the regulatory role of l-cystathionine in human macrophage apoptosis induced by oxidized low density lipoprotein (ox-LDL) and its possible mechanisms. THP-1 cells were induced with phorbol 12-myristate 13-acetate (PMA) and differentiated into macrophages. Macrophages were incubated with ox-LDL after pretreatment with l-cystathionine. Superoxide anion, apoptosis, mitochondrial membrane potential, and mitochondrial permeability transition pore (MPTP) opening were examined. Caspase-9 activities and expression of cleaved caspase-3 were measured. The results showed that compared with control group, ox-LDL treatment significantly promoted superoxide anion generation, release of cytochrome c (cytc) from mitochondrion into cytoplasm, caspase-9 activities, cleavage of caspase-3, and cell apoptosis, in addition to reduced mitochondrial membrane potential as well as increased MPTP opening. However, 0.3 and 1.0 mmol/L l-cystathionine significantly reduced superoxide anion generation, increased mitochondrial membrane potential, and markedly decreased MPTP opening in ox-LDL + l-cystathionine macrophages. Moreover, compared to ox-LDL treated-cells, release of cytc from mitochondrion into cytoplasm, caspase-9 activities, cleavage of caspase-3, and apoptosis levels in l-cystathionine pretreated cells were profoundly attenuated. Taken together, our results suggested that l-cystathionine could antagonize mitochondria-mediated human macrophage apoptosis induced by ox-LDL via inhibition of cytc release and caspase activation. PMID:25514411

  2. BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy.

    PubMed

    Pedro, Jose Manuel Bravo-San; Wei, Yongjie; Sica, Valentina; Maiuri, Maria Chiara; Zou, Zhongju; Kroemer, Guido; Levine, Beth

    2015-01-01

    Disruption of the complex of BECN1 with BCL2 or BCL2L1/BCL-XL is an essential switch that turns on cellular autophagy in response to environmental stress or treatment with BH3 peptidomimetics. Recently, it has been proposed that BCL2 and BCL2L1/BCL-XL may inhibit autophagy indirectly through a mechanism dependent on the proapoptotic BCL2 family members, BAX and BAK1. Here we report that the BH3 mimetic, ABT-737, induces autophagy in parallel with disruption of BCL2-BECN1 binding in 2 different apoptosis-deficient cell types lacking BAX and BAK1, namely in mouse embryonic fibroblasts cells and in human colon cancer HCT116 cells. We conclude that the BH3 mimetic ABT-737 induces autophagy through a BAX and BAK1-independent mechanism that likely involves disruption of BECN1 binding to antiapoptotic BCL2 family members.

  3. Cell cycle specificity of Fas-mediated apoptosis in WIL-2 cells.

    PubMed

    Beletskaya, I V; Nikonova, L V; Beletsky, I P

    1997-07-21

    Antibodies to Fas/APO1 receptor induce effective apoptosis in WIL-2 cells of the human B-lymphoid line. Quantitative assessment of the extent of the death in cells synchronized by thymidine block revealed a significant increase in their sensitivity to the cytocidal effect mediated by Fas/APO1 during the G1 phase of the cell cycle. Western analysis of the content of the p53 antigen in the cytoplasm and nuclei of the cells showed that the Fas/APO1-induced death is accompanied by massive translocation of the p53 from the cytoplasm to the nucleus. These findings suggest that cell vulnerability to the Fas/APO1-mediated apoptosis is subjected to regulation by cell cycle-dependent mechanisms, one of which is probably the function of the p53 antigen.

  4. CTLA4 mediates antigen-specific apoptosis of human T cells.

    PubMed Central

    Gribben, J G; Freeman, G J; Boussiotis, V A; Rennert, P; Jellis, C L; Greenfield, E; Barber, M; Restivo, V A; Ke, X; Gray, G S

    1995-01-01

    The regulation of T cell-mediated immune responses requires a balance between amplification and generation of effector function and subsequent selective termination by clonal deletion. Although apoptosis of previously activated T cells can be induced by signaling of the tumor necrosis factor receptor family, these molecules do not appear to regulate T-cell clonal deletion in an antigen-specific fashion. We demonstrate that cross-linking of the inducible T-cell surface molecule CTLA4 can mediate apoptosis of previously activated human T lymphocytes. This function appears to be antigen-restricted, since a concomitant signal T-cell receptor signal is required. Regulation of this pathway may provide a novel therapeutic strategy to delete antigen-specific activated T cells. Images Fig. 3 PMID:7846057

  5. P62 Regulates resveratrol-mediated Fas/Cav-1 complex formation and transition from autophagy to apoptosis

    PubMed Central

    Zhang, Jun; Ma, Ke; Qi, Tingting; Wei, Xiaoning; Zhang, Qing; Li, Guanwu; Chiu, Jen-Fu

    2015-01-01

    Resveratrol is a potential polyphenol drug used in cancer treatment. We examined the relationship between autophagy and apoptosis in RSV-treated non-small lung adenocarcinoma A549 cells. Resveratrol treatment increased autophagy and autophagy-mediated degradation of P62. Immunocytochemistry revealed P62 co-localized with Fas/Cav-1 complexes, known to induce apoptosis. However, siRNA-mediated P62 downregulation enhanced formation of Fas/Cav-1 complexes, suggesting that P62 inhibited Fas/Cav-1 complex formation. Fas/Cav-1 complexes triggered caspase-8 activation and cleavage of Beclin-1, releasing a C-terminal Beclin-1 peptide that translocated to the mitochondria and initiate apoptosis. Inhibition of autophagy by siRNA-mediated repression of Beclin-1 also blocked RSV-induced apoptosis, showing a dependence of apoptosis on autophagy. P62 knockdown by siRNA accelerated the activation of caspase-8 and initiate apoptosis, while Cav-1 knockdown inhibited apoptosis, but increased autophagy. Inhibition of autophagy by 3-MA prevented both P62 degradation and induction of apoptosis, whereas inhibition of apoptosis by z-IETD-FMK or z-DEVD-FMK enhanced both P62 induction and autophagic cell death. In conclusion, P62 links resveratrol-induced autophagy to apoptosis. P62 blocks apoptosis by inhibiting Fas/Cav-1 complex formation, but RSV-induced autophagic degradation of P62 enables formation of Fas/Cav-1 complexes which then activate caspase-8-mediated Beclin-1 cleavage, resulting in translocation of the Beclin-1 C-terminal fragment to the mitochondria to initiate apoptosis. PMID:25596736

  6. Dexamethasone-induced apoptosis of osteocytic and osteoblastic cells is mediated by TAK1 activation.

    PubMed

    Ding, Heyuan; Wang, Tao; Xu, Dongli; Cha, Bingbing; Liu, Jun; Li, Yiming

    2015-05-01

    Increased apoptosis of osteoblasts and osteocytes is the main mechanism of glucocorticoid (GC)-induced osteonecrosis. In the current study, we investigated whether dexamethasone (Dex)-induced osteoblastic and osteocytic cell apoptosis is mediated through activation of transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), and whether TAK1 inhibition could promote survival opposing the deleterious effects of Dex. We found that TAK1 was activated by Dex in both osteocytic MLO-Y4 and osteoblastic OB-6 cells, which was prevented by two known anti-oxidants N-acetylcysteine (NAC) and ebselen. TAK1 inhibitors, including LYTAK1 and 5Z-7-oxozeaenol (57-OZ), inhibited Dex-induced apoptosis of MLO-Y4 and OB-6 cells. Meanwhile shRNA-mediated knockdown of TAK1 also suppressed Dex-induced damages to MLO-Y4 and OB-6 cells. On the other hand, exogenously over-expressing TAK1 enhanced Dex-induced MLO-Y4 and OB-6 cell apoptosis. At the molecular level, we found that TAK1 mediated Dex-induced pro-apoptotic Pyk2-JNK activation. Inhibition or silencing of TAK1 almost abolished Pyk2-JNK phosphorylations by Dex in MLO-Y4 and OB-6 cells. TAK1 over-expression, on the other hand, increased Dex's activity on Pyk2-JNK phosphorylations in above cells. We conclude that part of the pro-apoptotic actions of Dex on osteoblastic and osteocytic cells are mediated through TAK1 activation, and that inhibition of TAK1 might protect from GC-induced damages to osteoblasts and osteocytes.

  7. Wip1 inhibitor GSK2830371 inhibits neuroblastoma growth by inducing Chk2/p53-mediated apoptosis

    PubMed Central

    Chen, Zhenghu; Wang, Long; Yao, Dayong; Yang, Tianshu; Cao, Wen-Ming; Dou, Jun; Pang, Jonathan C.; Guan, Shan; Zhang, Huiyuan; Yu, Yang; Zhao, Yanling; Wang, Yongfeng; Xu, Xin; Shi, Yan; Patel, Roma; Zhang, Hong; Vasudevan, Sanjeev A.; Liu, Shangfeng; Yang, Jianhua; Nuchtern, Jed G.

    2016-01-01

    Neuroblastoma (NB) is the most common extracranial tumor in children. Unlike in most adult tumors, tumor suppressor protein 53 (p53) mutations occur with a relatively low frequency in NB and the downstream function of p53 is intact in NB cell lines. Wip1 is a negative regulator of p53 and hindrance of Wip1 activity by novel inhibitor GSK2830371 is a potential strategy to activate p53’s tumor suppressing function in NB. Yet, the in vivo efficacy and the possible mechanisms of GSK2830371 in NB have not yet been elucidated. Here we report that novel Wip1 inhibitor GSK2830371 induced Chk2/p53-mediated apoptosis in NB cells in a p53-dependent manner. In addition, GSK2830371 suppressed the colony-formation potential of p53 wild-type NB cell lines. Furthermore, GSK2830371 enhanced doxorubicin- (Dox) and etoposide- (VP-16) induced cytotoxicity in a subset of NB cell lines, including the chemoresistant LA-N-6 cell line. More importantly, GSK2830371 significantly inhibited tumor growth in an orthotopic xenograft NB mouse model by inducing Chk2/p53-mediated apoptosis in vivo. Taken together, this study suggests that GSK2830371 induces Chk2/p53-mediated apoptosis both in vitro and in vivo in a p53 dependent manner. PMID:27991505

  8. Mitochondria-cytochrome C-caspase-9 cascade mediates isorhamnetin-induced apoptosis.

    PubMed

    Lee, Hyo-Jung; Lee, Hyo-Jeong; Lee, Eun-Ok; Ko, Seong-Gyu; Bae, Hyun-Soo; Kim, Cheol-Ho; Ahn, Kyoo-Seok; Lu, Junxuan; Kim, Sung-Hoon

    2008-11-08

    Isorhamnetin is a flavanoid present in plants of the Polygonaceae family and is also an immediate metabolite of quercetin in mammals. Since the plasma level of isorhamnetin is maintained longer than quercetin, isorhamnetin may be a key metabolite to mediate the anti-tumor effect of quercetin. In the present study, we investigated the apoptotic mechanism of isorhamnetin in Lewis lung cancer (LLC) cells in vitro and established its in vivo anti-cancer efficacy. In cell culture, isorhamnetin significantly increased DNA fragmentation, and TUNEL positive apoptotic bodies and sub-G(1) apoptotic population in time- and dose-dependent manners. Western blot analyses revealed increased cleavage of caspase-3, and caspase-9 and PARP and increased cytosolic cytochrome C in isorhamnetin-treated cells. These events were accompanied by a reduced mitochondrial potential. Apoptosis was blocked by a general caspase inhibitor or the specific inhibitor of caspase-3 or -9. These in vitro results support mitochondria-dependent caspase activation to mediate isorhamnetin-induced apoptosis. Furthermore, an animal study revealed for the first time that isorhamnetin given by i.p. injection at a dose that is at least one order of magnitude lower than quercetin significantly suppressed the weights of tumors excised from LLC bearing mice. The in vivo anti-tumor efficacy was accompanied by increased TUNEL-positive and cleaved-caspase-3-positive tumor cells. Our data therefore support isorhamnetin as an active anti-cancer metabolite of quercetin in part through caspase-mediated apoptosis.

  9. Tnfaip8 is an essential gene for the regulation of glucocorticoid-mediated apoptosis of thymocytes.

    PubMed

    Woodward, M J; de Boer, J; Heidorn, S; Hubank, M; Kioussis, D; Williams, O; Brady, H J M

    2010-02-01

    Glucocorticoids have significant immunoregulatory actions on thymocytes and T cells and act by binding and activating cytosolic glucocorticoid receptors, which translocate to the nucleus and control gene expression through binding to specific response elements in target genes. Glucocorticoids promote cell death by activating an apoptotic program that requires transcriptional regulation. We set out to identify genes that are crucial to the process of glucocorticoid-mediated thymocyte apoptosis. Freshly isolated murine primary thymocytes were treated with dexamethasone, mRNA isolated and used to screen DNA microarrays. A set of candidate genes with upregulated expression was identified and selected members assayed in reconstituted fetal thymic organ culture (FTOC). Fetal liver-derived hematopoietic progenitor cells (HPCs) were infected with retroviruses expressing individual genes then used to repopulate depleted fetal thymic lobes. Reconstituted FTOCs expressing the gene Tnfaip8 were treated with dexamethasone and shown to be greatly sensitized to dexamethasone. Retrovirus-mediated RNA interference was applied to knock down Tnfaip8 expression in HPCs and these were used to reconstitute FTOCs. We observed that downregulating the expression of Tnfaip8 alone was sufficient to effectively protect thymocytes against glucocorticoid-induced apoptosis. We propose that Tnfaip8 is crucial in regulating glucocorticoid-mediated apoptosis of thymocytes.

  10. Arsenate-induced Apoptosis in Murine Embryonic Maxillary Mesenchymal Cells via Mitochondrial Mediated Oxidative Injury

    PubMed Central

    Singh, Saurabh; Greene, Robert M.; Pisano, M. Michele

    2009-01-01

    Background Arsenic is a ubiquitous element that is a potential carcinogen and teratogen and can cause adverse developmental outcomes. Arsenic exerts its toxic effects through the generation of reactive oxygen species (ROS) that include hydrogen peroxide (H2O2), superoxide-derived hydroxyl ion, and peroxyl radicals. However, the molecular mechanisms by which arsenic induces cytotoxicity in murine embryonic maxillary mesenchymal (MEMM) cells are undefined. Methods MEMM cells in culture were treated with different concentrations of pentavalent sodium arsenate [As (V)] for 24 or 48 hours and various end points measured. Results We show that treatment of MEMM cells with the pentavalent form of inorganic arsenic resulted in caspase-mediated apoptosis, accompanied by generation of ROS and disruption of mitochondrial membrane potential. Treatment with caspase inhibitors markedly blocked apoptosis. In addition, the free radical scavenger N-acetylcysteine dramatically attenuated arsenic-mediated ROS production and apoptosis, and exposure to arsenate increased Bax and decreased Bcl protein levels in MEMM cells. Conclusions Taken together, these findings suggest that in MEMM cells, arsenate-mediated oxidative injury acts as an early and upstream initiator of the cell death cascade, triggering cytotoxicity, mitochondrial dysfunction, altered Bcl/Bax protein ratios, and activation of caspase-9. PMID:19739150

  11. CrmA, a poxvirus-encoded serpin, inhibits cytotoxic T-lymphocyte-mediated apoptosis.

    PubMed

    Tewari, M; Telford, W G; Miller, R A; Dixit, V M

    1995-09-29

    Cytotoxic T-lymphocytes (CTLs), by virtue of their ability to recognize and induce apoptotic death of virus-infected cells, comprise a major antiviral defense mechanism. The induction of apoptosis by CTLs can be completely accounted for by two mechanisms: (i) a Ca(2+)-dependent component that involves the exocytotic release of serine proteases known as granzymes from CTL granules and their subsequent insertion into the target cell to induce apoptosis and (ii) a Ca(2+)-independent component that involves the activation of Fas, a receptor on the target cell membrane that triggers apoptosis. Although viruses have evolved several indirect mechanisms for evading the CTL response, direct inhibition of the apoptotic cascade has never been described. We now show for the first time that the cowpox virus protein CrmA, a protease inhibitor of the serpin family, is capable of inhibiting CTL-mediated cytolysis. The inhibitory effect is largely the result of blockade of the Ca(2+)-independent (i.e. Fas-mediated) component of CTL killing. CrmA thus represents the first example of a viral gene product capable of directly blocking CTL-mediated cell death.

  12. Cigarette Smoke–Induced CXCR3 Receptor Up-Regulation Mediates Endothelial Apoptosis

    PubMed Central

    Green, Linden A.; Petrusca, Daniela; Rajashekhar, Gangaraju; Gianaris, Tom; Schweitzer, Kelly S.; Wang, Liang; Justice, Matthew J.; Petrache, Irina

    2012-01-01

    Endothelial monocyte–activating polypeptide II (EMAP II) and interferon-inducible protein (IP)–10 are proinflammatory mediators, which in addition to their chemokine activities, selectively induce apoptosis in endothelial cells and are up-regulated in the lungs of cigarette smoke–exposed humans. Previously, we showed that EMAP II is an essential mediator of cigarette smoke–induced lung emphysema in mice linking endothelial cell apoptosis with inflammation. Here we addressed the role of the CXCR3 receptor in EMAP II–induced and IP-10–induced apoptosis in endothelial cells and its regulation by cigarette smoke. We found that both neutralizing antibodies and small inhibitory RNA to CXCR3 abrogated EMAP II–induced and IP-10–induced endothelial caspase-3 activation and DNA fragmentation. CXCR3 receptor surface expression in human lung microvascular endothelial cells and in lung tissue endothelium was up-regulated by exposure to cigarette smoke. In tissue culture conditions, EMAP II–induced and IP-10–induced apoptosis was enhanced by preincubation with cigarette smoke extract. Interestingly, serum starvation also induced CXCR3 up-regulation and enhanced EMAP II–induced endothelial apoptosis. Signal transduction via p38 mitogen-activated protein kinase activation was essential for CXCR3-induced cell death, but not for CXCR3 receptor up-regulation by cigarette smoke. In turn, protein nitration was required for CXCR3 receptor up-regulation by cigarette smoke and consequently for subsequent CXCR3-induced cell death. In conclusion, the concerted up-regulation of proinflammatory EMAP II, IP-10, and CXCR3 by cigarette smoke could sustain a cascade of cell death that may promote the alveolar tissue loss noted in human emphysema. PMID:22936405

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

    PubMed

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

    2013-01-01

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

  14. ASC Induces Apoptosis via Activation of Caspase-9 by Enhancing Gap Junction-Mediated Intercellular Communication.

    PubMed

    Kitazawa, Masato; Hida, Shigeaki; Fujii, Chifumi; Taniguchi, Shun'ichiro; Ito, Kensuke; Matsumura, Tomio; Okada, Nagisa; Sakaizawa, Takashi; Kobayashi, Akira; Takeoka, Michiko; Miyagawa, Shin-Ichi

    2017-01-01

    ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy.

  15. Role of the Chemokine MCP-1 in Sensitization of PKC-Mediated Apoptosis in Prostate Cancer Cells

    DTIC Science & Technology

    2009-02-01

    cells of hairy cell leukemia . Am. J. Pathol. 170: 745-754 (2007). 14. Kampfer S, Windegger M, Hochholdinger F, Schwaiger W, Pestell RG, Baier G...0119 TITLE: Role of the chemokine MCP-1 in sensitization of PKC-mediated apoptosis in prostate cancer cells ...of 5a. CONTRACT NUMBER PKC-mediated apoptosis in prostate cancer cells b. GRANT NUMBER W81XWH-07-1-0119 5c. PROGRAM ELEMENT

  16. Autophagy and apoptosis: where do they meet?

    PubMed

    Mukhopadhyay, Subhadip; Panda, Prashanta Kumar; Sinha, Niharika; Das, Durgesh Nandini; Bhutia, Sujit Kumar

    2014-04-01

    Autophagy and apoptosis are two important cellular processes with complex and intersecting protein networks; as such, they have been the subjects of intense investigation. Recent advances have elucidated the key players and their molecular circuitry. For instance, the discovery of Beclin-1's interacting partners has resulted in the identification of Bcl-2 as a central regulator of autophagy and apoptosis, which functions by interacting with both Beclin-1 and Bax/Bak respectively. When localized to the endoplasmic reticulum and mitochondria, Bcl-2 inhibits autophagy. Cellular stress causes the displacement of Bcl-2 from Beclin-1 and Bax, thereby triggering autophagy and apoptosis, respectively. The induction of autophagy or apoptosis results in disruption of complexes by BH3-only proteins and through post-translational modification. The mechanisms linking autophagy and apoptosis are not fully defined; however, recent discoveries have revealed that several apoptotic proteins (e.g., PUMA, Noxa, Nix, Bax, XIAP, and Bim) modulate autophagy. Moreover, autophagic proteins that control nucleation and elongation regulate intrinsic apoptosis through calpain- and caspase-mediated cleavage of autophagy-related proteins, which switches the cellular program from autophagy to apoptosis. Similarly, several autophagic proteins are implicated in extrinsic apoptosis. This highlights a dual cellular role for autophagy. On one hand, autophagy degrades damaged mitochondria and caspases, and on the other hand, it provides a membrane-based intracellular platform for caspase processing in the regulation of apoptosis. In this review, we highlight the crucial factors governing the crosstalk between autophagy and apoptosis and describe the mechanisms controlling cell survival and cell death.

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

    SciTech Connect

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

    2012-09-01

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

  18. Inhibition of autophagy increased AGE/ROS-mediated apoptosis in mesangial cells

    PubMed Central

    Xu, Li; Fan, Qiuling; Wang, Xu; Zhao, Xue; Wang, Lining

    2016-01-01

    The aim of our study was to investigate the role of autophagy, a homeostatic process involved in the lysosomal degradation of damaged cell organelles and proteins, in regulating the survival of mesangial cells treated with advanced glycation end products (AGEs). In the present study, AGEs induced mitochondrial depolarization and led to mitochondrial-dependent apoptosis in mesangial cells, as shown by the loss of the mitochondrial membrane potential; increased Bax processing; increased Caspase-9, Caspase-3 and PARP cleavage; and decreased Bcl-2 expression. Meanwhile, AGEs also triggered autophagy flux in mesangial cells, as confirmed by the presence of autophagic vesicles, the conversion of LC3II/LC3I and the increase/decrease in Beclin-1/p62 expression. Interestingly, this study reported apparent apoptosis and autophagy that were dependent on reactive oxygen species (ROS) production. Scavenging ROS with N-acetyl-l-cysteine could prevent the appearance of the autophagic features and reverse AGE-induced apoptosis. Moreover, AGE-triggered mitophagy, which was confirmed by the colocalization of autophagosomes and mitochondria and Parkin translocation to mitochondria, played a potential role in reducing ROS production in mesangial cells. Additionally, inhibition of autophagy significantly enhanced AGE-induced cell apoptosis. Taken together, our data suggest that ROS were the mediators of AGE-induced mesangial cell apoptosis and that autophagy was likely to be the mechanism that was triggered to repair the ROS-induced damage in the AGE-treated cells and thereby promote cell survival. This study provides new insights into the molecular mechanism of autophagy involved in AGE-induced apoptosis in mesangial cells. PMID:27809300

  19. Proteasome Dysfunction Mediates High Glucose-Induced Apoptosis in Rodent Beta Cells and Human Islets

    PubMed Central

    Broca, Christophe; Varin, Elodie; Armanet, Mathieu; Tourrel-Cuzin, Cécile; Bosco, Domenico; Dalle, Stéphane; Wojtusciszyn, Anne

    2014-01-01

    The ubiquitin/proteasome system (UPS), a major cellular protein degradation machinery, plays key roles in the regulation of many cell functions. Glucotoxicity mediated by chronic hyperglycaemia is detrimental to the function and survival of pancreatic beta cells. The aim of our study was to determine whether proteasome dysfunction could be involved in beta cell apoptosis in glucotoxic conditions, and to evaluate whether such a dysfunction might be pharmacologically corrected. Therefore, UPS activity was measured in GK rats islets, INS-1E beta cells or human islets after high glucose and/or UPS inhibitor exposure. Immunoblotting was used to quantify polyubiquitinated proteins, endoplasmic reticulum (ER) stress through CHOP expression, and apoptosis through the cleavage of PARP and caspase-3, whereas total cell death was detected through histone-associated DNA fragments measurement. In vitro, we found that chronic exposure of INS-1E cells to high glucose concentrations significantly decreases the three proteasome activities by 20% and leads to caspase-3-dependent apoptosis. We showed that pharmacological blockade of UPS activity by 20% leads to apoptosis in a same way. Indeed, ER stress was involved in both conditions. These results were confirmed in human islets, and proteasome activities were also decreased in hyperglycemic GK rats islets. Moreover, we observed that a high glucose treatment hypersensitized beta cells to the apoptotic effect of proteasome inhibitors. Noteworthily, the decreased proteasome activity can be corrected with Exendin-4, which also protected against glucotoxicity-induced apoptosis. Taken together, our findings reveal an important role of proteasome activity in high glucose-induced beta cell apoptosis, potentially linking ER stress and glucotoxicity. These proteasome dysfunctions can be reversed by a GLP-1 analog. Thus, UPS may be a potent target to treat deleterious metabolic conditions leading to type 2 diabetes. PMID:24642635

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

    SciTech Connect

    Boosen, Meike; Vetterkind, Susanne; Koplin, Ansgar; Illenberger, Susanne; Preuss, Ute . E-mail: u.preuss@uni-bonn.de

    2005-12-10

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

  1. HAX1 deletion impairs BCR internalization and leads to delayed BCR-mediated apoptosis

    PubMed Central

    Wolkerstorfer, Susanne; Schwaiger, Elisabeth; Rinnerthaler, Mark; Karina Gratz, Iris; Zoegg, Thomas; Brandstetter, Hans; Achatz-Straussberger, Gertrude

    2016-01-01

    Deletion of HAX1 in mice causes a severe reduction in the numbers of lymphocytes in the bone marrow and in the spleen. Additionally, B220+ B progenitor cells in the bone marrow are reduced, suggesting an important function of HAX1 in B cell development. HAX1 is thought to play a protective role in apoptotic processes; therefore, we investigated the role of HAX1 in bone marrow B progenitor cells and splenic B cells. We did not observe an effect on the survival of Hax1−/− bone marrow cells but detected enhanced survival of splenic Hax1−/− B cells upon in vitro starvation/growth-factor withdrawal. To explain this apparent inconsistency with previous reports of HAX1 function, we also studied the B cell receptor (BCR)-induced apoptosis of IgM-stimulated splenic naïve B cells and found that apoptosis decreased in these cells. We further found impaired internalization of the BCR from Hax1−/− splenic B cells after IgM crosslinking; this impaired internalization may result in decreased BCR signaling and, consequently, decreased BCR-mediated apoptosis. We measured HAX1 binding to the cytoplasmic domains of different Ig subtypes and identified KVKWI(V)F as the putative binding motif for HAX1 within the cytoplasmic domains. Because this motif can be found in almost all Ig subtypes, it is likely that HAX1 plays a general role in BCR-mediated internalization events and BCR-mediated apoptosis. PMID:25864916

  2. Advanced oxidation protein products induce chondrocyte apoptosis via receptor for advanced glycation end products-mediated, redox-dependent intrinsic apoptosis pathway.

    PubMed

    Wu, Qian; Zhong, Zhao-Ming; Zhu, Si-Yuan; Liao, Cong-Rui; Pan, Ying; Zeng, Ji-Huan; Zheng, Shuai; Ding, Ruo-Ting; Lin, Qing-Song; Ye, Qing; Ye, Wen-Bin; Li, Wei; Chen, Jian-Ting

    2016-01-01

    Pro-inflammatory cytokine-induced chondrocyte apoptosis is a primary cause of cartilage destruction in the progression of rheumatoid arthritis (RA). Advanced oxidation protein products (AOPPs), a novel pro-inflammatory mediator, have been confirmed to accumulate in patients with RA. However, the effect of AOPPs accumulation on chondrocyte apoptosis and the associated cellular mechanisms remains unclear. The present study demonstrated that the plasma formation of AOPPs was enhanced in RA rats compared with normal. Then, chondrocyte were treated with AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. Exposure of chondrocyte to AOPPs activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and increased expression of NADPH oxidase subunits, which was mediated by receptor for advanced glycation end products (RAGE), but not scavenger receptor CD36. Moreover, AOPPs challenge triggered NADPH oxidase-dependent ROS generation which induced mitochondrial dysfunction and endoplasmic reticulum stress resulted in activation of caspase family that eventually lead to apoptosis. Lastly, blockade of RAGE, instead of CD36, largely attenuated these signals. Our study demonstrated first time that AOPPs induce chondrocyte apoptosis via RAGE-mediated and redox-dependent intrinsic apoptosis pathway in vitro. These data implicates that AOPPs may represent a novel pathogenic factor that contributes to RA progression. Targeting AOPPs-triggered cellular mechanisms might emerge as a promising therapeutic option for patients with RA.

  3. Fas/Fas Ligand–mediated Apoptosis in Different Cell Lineages and Functional Compartments of Human Lymph Nodes

    PubMed Central

    Kokkonen, Tuomo S.; Karttunen, Tuomo J.

    2010-01-01

    We have optimized an immunohistochemical double-staining method combining immunohistochemical lymphocyte lineage marker detection and apoptosis detection with terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling. The method was used to trace Fas-mediated apoptosis in human reactive lymph nodes according to cell lineage and anatomical location. In addition to Fas, we also studied the expression of Fas ligand (FasL), CD3, CD20, CD19, CD23, and CD68 of apoptotic cells. The presence of simultaneous Fas and FasL positivity indicated involvement of activation-induced death in the induction of paracortical apoptosis. FasL expression in the high endothelial venules might be an inductor of apoptosis of Fas-positive lymphoid cells. In addition to B-lymphocyte apoptosis in the germinal centers, there was often a high apoptosis rate of CD23-expressing follicular dendritic cells. In summary, our double-staining method provides valuable new information about the occurrence and mechanisms of apoptosis of different immune cell types in the lymph node compartments. Among other things, we present support for the importance of Fas/FasL–mediated apoptosis in lymph node homeostasis. (J Histochem Cytochem 58:131–140, 2010) PMID:19826071

  4. Fas/Fas ligand-mediated apoptosis in different cell lineages and functional compartments of human lymph nodes.

    PubMed

    Kokkonen, Tuomo S; Karttunen, Tuomo J

    2010-02-01

    We have optimized an immunohistochemical double-staining method combining immunohistochemical lymphocyte lineage marker detection and apoptosis detection with terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling. The method was used to trace Fas-mediated apoptosis in human reactive lymph nodes according to cell lineage and anatomical location. In addition to Fas, we also studied the expression of Fas ligand (FasL), CD3, CD20, CD19, CD23, and CD68 of apoptotic cells. The presence of simultaneous Fas and FasL positivity indicated involvement of activation-induced death in the induction of paracortical apoptosis. FasL expression in the high endothelial venules might be an inductor of apoptosis of Fas-positive lymphoid cells. In addition to B-lymphocyte apoptosis in the germinal centers, there was often a high apoptosis rate of CD23-expressing follicular dendritic cells. In summary, our double-staining method provides valuable new information about the occurrence and mechanisms of apoptosis of different immune cell types in the lymph node compartments. Among other things, we present support for the importance of Fas/FasL-mediated apoptosis in lymph node homeostasis.

  5. Antigen receptor-induced B lymphocyte apoptosis mediated via a protease of the caspase family.

    PubMed

    Andjelic, S; Liou, H C

    1998-02-01

    An extensive body of data, in a variety of systems, denoted the caspase family of proteases as a key player in the execution of programmed cell death. This family consists of cysteine proteases that cleave after asparagine-containing motifs. It is well established that the caspases are essential for the apoptosis mediated by Fas (CD95) and TNF receptor p55, molecules that contain the "death domain" in the cytoplasmic tail. However, little is known about the mechanisms underlying the antigen receptor-mediated cell death in B lymphocytes, a process instrumental in negative selection of potentially autoreactive B cells. Here, we investigated the involvement of caspases in cell death triggered via the antigen receptor in B lymphocytes (BCR) by using specific inhibitors. Initially, we used a well-established cell line, CH31, which is a model of B cell tolerance, to demonstrate that these proteases indeed participate in the BCR-induced apoptotic pathway. Next, we confirmed the physiological relevance of the caspase-mediated cell death pathway in splenic B cell populations isolated ex vivo that were induced to undergo apoptosis by extensive cross-linking of their BCR. Most interestingly, our data demonstrated that caspases regulate not only the nuclear DNA fragmentation, but also the surface membrane phosphatidylserine translocation as well as the degradation of a specific nuclear substrate. Taken together, this report supports the hypothesis that regulation of the caspase family is crucial in controlling the life/death decision in B lymphocytes mediated by the antigen receptor signal transduction.

  6. The role of DNA damage and repair in decitabine-mediated apoptosis in multiple myeloma

    PubMed Central

    Maes, Ken; Smedt, Eva De; Lemaire, Miguel; Raeve, Hendrik De; Menu, Eline; Van Valckenborgh, Els; McClue, Steve

    2014-01-01

    DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) are under investigation for the treatment of cancer, including the plasma cell malignancy multiple myeloma (MM). Evidence exists that DNA damage and repair contribute to the cytotoxicity mediated by the DNMTi decitabine. Here, we investigated the DNA damage response (DDR) induced by decitabine in MM using 4 human MM cell lines and the murine 5T33MM model. In addition, we explored how the HDACi JNJ-26481585 affects this DDR. Decitabine induced DNA damage (gamma-H2AX foci formation), followed by a G0/G1- or G2/M-phase arrest and caspase-mediated apoptosis. JNJ-26481585 enhanced the anti-MM effect of decitabine both in vitro and in vivo. As JNJ-26481585 did not enhance decitabine-mediated gamma-H2AX foci formation, we investigated the DNA repair response towards decitabine and/or JNJ-26481585. Decitabine augmented RAD51 foci formation (marker for homologous recombination (HR)) and/or 53BP1 foci formation (marker for non-homologous end joining (NHEJ)). Interestingly, JNJ-26481585 negatively affected basal or decitabine-induced RAD51 foci formation. Finally, B02 (RAD51 inhibitor) enhanced decitabine-mediated apoptosis. Together, we report that decitabine-induced DNA damage stimulates HR and/or NHEJ. JNJ-26481585 negatively affects RAD51 foci formation, thereby providing an additional explanation for the combinatory effect between decitabine and JNJ-26481585. PMID:24833108

  7. Stearoyl-CoA desaturase-1 mediated cell apoptosis in colorectal cancer by promoting ceramide synthesis

    PubMed Central

    Chen, Ling; Ren, Jie; Yang, Longhe; Li, Yanting; Fu, Jin; Li, Yuhang; Tian, Yifeng; Qiu, Funan; Liu, Zuguo; Qiu, Yan

    2016-01-01

    Inhibition of stearoyl-CoA desaturase 1 (SCD1) has been found to effectively suppress tumor cell proliferation and induce apoptosis in numerous neoplastic lesions. However, mechanism underlying SCD1-mediated anti-tumor effect has maintained unclear. Herein, we reported endo-lipid messenger ceramides played a critical role in tumor fate modulated by SCD1 inhibition. In vitro study in colorectal cancer cells demonstrated inhibition of SCD1 activity promoted apoptosis attributed to mitochondria dysfunctions, upregulation of reaction oxygen species (ROS), alteration of mitochondrial transmembrane potential and translocation of mitochondrial protein cytochrome C. While these effects were mediated by intracellular ceramide signals through induction of ceramide biosynthesis, rather than exclusive SFA accumulation. In vivo study in xenograft colorectal cancer mice showed pharmacologic administration of SCD1 inhibitor A939 significantly delayed tumor growth, which was reversed by L-cycloserine, an inhibitor of ceramide biosynthesis. These results depicted the cross-talk of SCD1-mediated lipid pathway and endo-ceramide biosynthesis pathway, indicating roles of ceramide signals in SCD1-mediated anti-tumor property. PMID:26813308

  8. Induction of mitochondrial-mediated apoptosis by Morinda citrifolia (Noni) in human cervical cancer cells.

    PubMed

    Gupta, Rakesh Kumar; Banerjee, Ayan; Pathak, Suajta; Sharma, Chandresh; Singh, Neeta

    2013-01-01

    Cervical cancer is the second most common cause of cancer in women and has a high mortality rate. Cisplatin, an antitumor agent, is generally used for its treatment. However, the administration of cisplatin is associated with side effects and intrinsic resistance. Morinda citrifolia (Noni), a natural plant product, has been shown to have anti-cancer properties. In this study, we used Noni, cisplatin, and the two in combination to study their cytotoxic and apoptosis-inducing effects in cervical cancer HeLa and SiHa cell lines. We demonstrate here, that Noni/Cisplatin by themselves and their combination were able to induce apoptosis in both these cell lines. Cisplatin showed slightly higher cell killing as compared to Noni and their combination showed additive effects. The observed apoptosis appeared to be mediated particularly through the up-regulation of p53 and pro-apoptotic Bax proteins, as well as down- regulation of the anti-apoptotic Bcl-2, Bcl-XL proteins and survivin. Augmentation in the activity of caspase-9 and -3 was also observed, suggesting the involvement of the intrinsic mitochondrial pathway of apoptosis for both Noni and Cisplatin in HeLa and SiHa cell lines.

  9. NRAGE mediates p38 activation and neural progenitor apoptosis via the bone morphogenetic protein signaling cascade.

    PubMed

    Kendall, Stephen E; Battelli, Chiara; Irwin, Sarah; Mitchell, Jane G; Glackin, Carlotta A; Verdi, Joseph M

    2005-09-01

    Understanding the molecular events that govern neural progenitor lineage commitment, mitotic arrest, and differentiation into functional progeny are germane to our understanding of neocortical development. Members of the family of bone morphogenetic proteins (BMPs) play pivotal roles in regulating neural differentiation and apoptosis during neurogenesis through combined actions involving Smad and TAK1 activation. We demonstrate that BMP signaling is required for the induction of apoptosis of neural progenitors and that NRAGE is a mandatory component of the signaling cascade. NRAGE possesses the ability to bind and function with the TAK1-TAB1-XIAP complex facilitating the activation of p38. Disruption of NRAGE or any other member of the noncanonical signaling cascaded is sufficient to block p38 activation and thus the proapoptotic signals generated through BMP exposure. The function of NRAGE is independent of Smad signaling, but the introduction of a dominant-negative Smad5 also rescues neural progenitor apoptosis, suggesting that both canonical and noncanonical pathways can converge and regulate BMP-mediated apoptosis. Collectively, these results establish NRAGE as an integral component in BMP signaling and clarify its role during neural progenitor development.

  10. NRAGE Mediates p38 Activation and Neural Progenitor Apoptosis via the Bone Morphogenetic Protein Signaling Cascade

    PubMed Central

    Kendall, Stephen E.; Battelli, Chiara; Irwin, Sarah; Mitchell, Jane G.; Glackin, Carlotta A.; Verdi, Joseph M.

    2005-01-01

    Understanding the molecular events that govern neural progenitor lineage commitment, mitotic arrest, and differentiation into functional progeny are germane to our understanding of neocortical development. Members of the family of bone morphogenetic proteins (BMPs) play pivotal roles in regulating neural differentiation and apoptosis during neurogenesis through combined actions involving Smad and TAK1 activation. We demonstrate that BMP signaling is required for the induction of apoptosis of neural progenitors and that NRAGE is a mandatory component of the signaling cascade. NRAGE possesses the ability to bind and function with the TAK1-TAB1-XIAP complex facilitating the activation of p38. Disruption of NRAGE or any other member of the noncanonical signaling cascaded is sufficient to block p38 activation and thus the proapoptotic signals generated through BMP exposure. The function of NRAGE is independent of Smad signaling, but the introduction of a dominant-negative Smad5 also rescues neural progenitor apoptosis, suggesting that both canonical and noncanonical pathways can converge and regulate BMP-mediated apoptosis. Collectively, these results establish NRAGE as an integral component in BMP signaling and clarify its role during neural progenitor development. PMID:16107717

  11. p75NTR Mediates Neurotrophin-Induced Apoptosis of Vascular Smooth Muscle Cells

    PubMed Central

    Wang, Shiyang; Bray, Paula; McCaffrey, Timothy; March, Keith; Hempstead, Barbara L.; Kraemer, Rosemary

    2000-01-01

    The development of atherosclerotic lesions results from aberrant cell migration, proliferation, and extracellular matrix production. In advanced lesions, however, cellular apoptosis, leading to lesion remodeling, predominates. During lesion formation, the neurotrophins and the neurotrophin receptor tyrosine kinases, trks B and C, are induced and mediate smooth muscle cell migration. Here we demonstrate that a second neurotrophin receptor, p75NTR, is expressed by established human atherosclerotic lesions and late lesions that develop after balloon injury of the rat thoracic aorta. The p75NTR, a member of the tumor necrosis factor/FAS receptor family, can modulate trk receptor function as well as initiate cell death when expressed in cells of the nervous system that lack kinase-active trk receptors. p75NTR expression colocalizes to neointimal cells, which express smooth muscle cell α-actin and are expressed by cultured human endarterectomy-derived cells (HEDC). Areas of the plaque expressing p75NTR demonstrate increased TUNEL positivity, and HEDC undergo apoptosis in response to the neurotrophins. Finally, neurotrophins also induced apoptosis of a smooth muscle cell line genetically manipulated to express p75NTR, but lacking trk receptor expression. These studies identify the regulated expression of neurotrophins and p75NTR as an inducer of smooth muscle cell apoptosis in atherosclerotic lesions. PMID:11021829

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

    PubMed

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

    2017-05-11

    Nano-sized copper particles are widely used in various chemical, physical, and biological fields. However, earlier studies have shown that nano copper particles (40-100 μg/mL) can induce cell toxicity and apoptosis. Therefore, this study was conducted to investigate the role of nano copper in mitochondrion-mediated apoptosis in PK-15 cells. The cells were treated with different doses of nano copper (20, 40, 60, and 80 μg/mL) to determine the effects of apoptosis using acridine orange/ethidium bromide (AO/EB) fluorescence staining and a flow cytometry assay. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in the PK-15 cells were examined using commercially available kits. Moreover, the mRNA levels of the Bax, Bid, Caspase-3, and CYCS genes were assessed by real-time PCR. The results revealed that nano copper exposure induced apoptosis and changed the mitochondrial membrane potential. In addition, nano copper significantly altered the levels of the Bax, Bid, Caspase-3, and CYCS genes at a concentration of 40 μg/mL. To summarize, nano copper significantly (P < 0.05) decreased the level of SOD and increased the level of MDA in PK-15 cells. Altogether, these results suggest that nano copper can play an important role in inducing the apoptotic pathway in PK-15 cells, which may be the mechanism by which nano copper induces nephrotoxicity.

  13. NLRP3 inflammasome mediates contrast media-induced acute kidney injury by regulating cell apoptosis

    PubMed Central

    Shen, Jianxiao; Wang, Ling; Jiang, Na; Mou, Shan; Zhang, Minfang; Gu, Leyi; Shao, Xinghua; Wang, Qin; Qi, Chaojun; Li, Shu; Wang, Wanpeng; Che, Xiajing; Ni, Zhaohui

    2016-01-01

    Iodinated contrast media serves as a direct causative factor of acute kidney injury (AKI) and is involved in the progression of cellular dysfunction and apoptosis. Emerging evidence indicates that NLRP3 inflammasome triggers inflammation, apoptosis and tissue injury during AKI. Nevertheless, the underlying renoprotection mechanism of NLRP3 inflammasome against contrast-induced AKI (CI-AKI) was still uncertain. This study investigated the role of NLRP3 inflammasome in CI-AKI both in vitro and in vivo. In HK-2 cells and unilateral nephrectomy model, NLRP3 and NLRP3 inflammasome member ASC were significantly augmented with the treatment of contrast media. Moreover, genetic disruption of NLRP3 notably reversed contrast-induced expression of apoptosis related proteins and secretion of proinflammatory factors, similarly to the effects of ASC deletion. Consistent with above results, absence of NLRP3 in mice undergoing unilateral nephrectomy also protected against contrast media-induced renal cells phenotypic alteration and cell apoptosis via modulating expression level of apoptotic proteins. Collectively, we demonstrated that NLRP3 inflammasome mediated CI-AKI through modulating the apoptotic pathway, which provided a potential therapeutic target for the treatment of contrast media induced acute kidney injury. PMID:27721494

  14. Cyclovirobuxine D Inhibits Cell Proliferation and Induces Mitochondria-Mediated Apoptosis in Human Gastric Cancer Cells.

    PubMed

    Wu, Jie; Tan, Zhujun; Chen, Jian; Dong, Cheng

    2015-11-19

    Gastric cancer is one of the most common malignant cancers, with high death rates, poor prognosis and limited treatment methods. Cyclovirobuxine D (CVB-D) is the main active component of the traditional Chinese medicine Buxus microphylla. In the present study, we test the effects of CVB-D on gastric cancer cells and the underlying mechanisms of action. CVB-D reduced cell viability and colony formation ability of MGC-803 and MKN28 cells in a time- and concentration-dependent manner. Flow cytometry showed that cell cycle of CVB-D treated cells was arrested at the S-phase. CVB-D also induced apoptosis in MGC-803 and MKN28 cells, especially early stage apoptosis. Furthermore, mitochondria membrane potential (Δψm) was reduced and apoptosis-related proteins, cleaved Caspase-3 and Bax/Bcl-2, were up-regulated in CVB-D-treated MGC-803 and MKN28 cells. Taken together, our studies found that CVB-D plays important roles in inhibition of gastric tumorigenesis via arresting cell cycle and inducing mitochondria-mediated apoptosis, suggesting the potential application of CVB-D in gastric cancer therapy.

  15. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL.

    PubMed Central

    Walczak, H; Degli-Esposti, M A; Johnson, R S; Smolak, P J; Waugh, J Y; Boiani, N; Timour, M S; Gerhart, M J; Schooley, K A; Smith, C A; Goodwin, R G; Rauch, C T

    1997-01-01

    TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL-R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL-R2, by ligand-based affinity purification and subsequent molecular cloning. TRAIL-R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL-R2 contains two extracellular cysteine-rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell-surface-expressed TRAIL-R2, and TRAIL-induced apoptosis is inhibited by a TRAIL-R2-Fc fusion protein. TRAIL-R2 mRNA is widely expressed and the gene encoding TRAIL-R2 is located on human chromosome 8p22-21. Like TRAIL-R1, TRAIL-R2 engages a caspase-dependent apoptotic pathway but, in contrast to TRAIL-R1, TRAIL-R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family. PMID:9311998

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

    PubMed

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

    2016-02-15

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

  17. HIF-1 antagonizes p53-mediated apoptosis through a secreted neuronal tyrosinase.

    PubMed

    Sendoel, Ataman; Kohler, Ines; Fellmann, Christof; Lowe, Scott W; Hengartner, Michael O

    2010-06-03

    Hypoxia-inducible factor (HIF) is a transcription factor that regulates fundamental cellular processes in response to changes in oxygen concentration. HIFalpha protein levels are increased in most solid tumours and correlate with patient prognosis. The link between HIF and apoptosis, a major determinant of cancer progression and treatment outcome, is poorly understood. Here we show that Caenorhabditis elegans HIF-1 protects against DNA-damage-induced germ cell apoptosis by antagonizing the function of CEP-1, the homologue of the tumour suppressor p53. The antiapoptotic property of HIF-1 is mediated by means of transcriptional upregulation of the tyrosinase family member TYR-2 in the ASJ sensory neurons. TYR-2 is secreted by ASJ sensory neurons to antagonize CEP-1-dependent germline apoptosis. Knock down of the TYR-2 homologue TRP2 (also called DCT) in human melanoma cells similarly increases apoptosis, indicating an evolutionarily conserved function. Our findings identify a novel link between hypoxia and programmed cell death, and provide a paradigm for HIF-1 dictating apoptotic cell fate at a distance.

  18. HIF-1 antagonizes p53-mediated apoptosis through a secreted neuronal tyrosinase

    PubMed Central

    Sendoel, Ataman; Kohler, Ines; Fellmann, Christof; Lowe, Scott W.; Hengartner, Michael O.

    2012-01-01

    Hypoxia-inducible factor (HIF) is a transcription factor that regulates fundamental cellular processes in response to changes in oxygen concentration. HIFα protein levels are increased in most solid tumours and correlate with patient prognosis. The link between HIF and apoptosis, a major determinant of cancer progression and treatment outcome, is poorly understood. Here we show that Caenorhabditis elegans HIF-1 protects against DNA-damage-induced germ cell apoptosis by antagonizing the function of CEP-1, the homologue of the tumour suppressor p53. The antiapoptotic property of HIF-1 is mediated by means of transcriptional upregulation of the tyrosinase family member TYR-2 in the ASJ sensory neurons. TYR-2 is secreted by ASJ sensory neurons to antagonize CEP-1-dependent germline apoptosis. Knock down of the TYR-2 homologue TRP2 (also called DCT) in human melanoma cells similarly increases apoptosis, indicating an evolutionarily conserved function. Our findings identify a novel link between hypoxia and programmed cell death, and provide a paradigm for HIF-1 dictating apoptotic cell fate at a distance. PMID:20520707

  19. Agonist-mediated activation of STING induces apoptosis in malignant B cells

    PubMed Central

    Tang, Chih-Hang Anthony; Zundell, Joseph A.; Ranatunga, Sujeewa; Lin, Cindy; Nefedova, Yulia; Del Valle, Juan R.; Hu, Chih-Chi Andrew

    2016-01-01

    Endoplasmic reticulum (ER) stress responses through the IRE-1/XBP-1 pathway are required for the function of STING (TMEM173), an ER-resident transmembrane protein critical for cytoplasmic DNA sensing, interferon production and cancer control. Here we show that the IRE-1/XBP-1 pathway functions downstream of STING and that STING agonists selectively trigger mitochondria-mediated apoptosis in normal and malignant B cells. Upon stimulation, STING was degraded less efficiently in B cells, implying that prolonged activation of STING can lead to apoptosis. Transient activation of the IRE-1/XBP-1 pathway partially protected agonist-stimulated malignant B cells from undergoing apoptosis. In Eμ-TCL1 mice with chronic lymphocytic leukemia, injection of the STING agonist 3′3′-cGAMP induced apoptosis and tumor regression. Similarly efficacious effects were elicited by 3′3′-cGAMP injection in syngeneic or immunodeficient mice grafted with multiple myeloma. Thus, in addition to their established ability to boost anti-tumoral immune responses, STING agonists can also directly eradicate malignant B cells. PMID:26951929

  20. Mesenchymal Stem Cell-Induced Immunoregulation Involves Fas Ligand/Fas-Mediated T Cell Apoptosis

    PubMed Central

    Akiyama, Kentaro; Chen, Chider; Wang, DanDan; Xu, Xingtian; Qu, Cunye; Yamaza, Takayoshi; Cai, Tao; Chen, WanJun; Sun, Lingyun; Shi, Songtao

    2012-01-01

    SUMMARY Systemic infusion of bone marrow mesenchymal stem cells (BMMSCs) shows therapeutic benefit for a variety of autoimmune diseases, but the underlying mechanisms are poorly understood. Here we show that in mice systemic infusion of BMMSCs induced transient T-cell apoptosis via the Fas ligand (FasL)-dependent Fas pathway and could ameliorate disease phenotypes in fibrillin-1 mutated systemic sclerosis (SS) and dextran sulfate sodium-induced experimental colitis. FasL−/− BMMSCs did not induce T-cell apoptosis in recipients, and could not ameliorate SS and colitis. Mechanistic analysis revealed that Fas-regulated monocyte chemotactic protein 1 (MCP-1) secretion by BMMSCs recruited T-cells for FasL-mediated apoptosis. The apoptotic T-cells subsequently triggered macrophages to produce high levels of TGFβ which in turn led to the upregulation of Tregs and, ultimately, to immune tolerance. These data therefore demonstrate a previously unrecognized mechanism underlying BMMSC-based immunotherapy involving coupling via Fas/FasL to induce T-cell apoptosis. PMID:22542159

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

    PubMed Central

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

    2016-01-01

    Caspase-2 plays an important role in apoptosis induced by several stimuli, including oxidative stress. However, the subcellular localization of caspase-2, particularly its presence in the mitochondria, is unclear. It is also not known if cytosolic caspase-2 translocates to the mitochondria to trigger the intrinsic pathway of apoptosis or if caspase-2 is constitutively present in the mitochondria that then selectively mediates this apoptotic effect. Here, we demonstrate the presence of caspase-2 in purified mitochondrial fractions from in vitro-cultured cells and in liver hepatocytes using immunoblots and confocal microscopy. We show that mitochondrial caspase-2 is functionally active by performing fluorescence resonance energy transfer analyses using a mitochondrially targeted substrate flanked by donor and acceptor fluorophores. Cell-free apoptotic assays involving recombination of nuclear, cytosolic and mitochondrial fractions from the livers of wild type and Casp2−/− mice clearly point to a direct functional role for mitochondrial caspase-2 in apoptosis. Furthermore, cytochrome c release from Casp2−/− cells is decreased as compared with controls upon treatment with agents inducing mitochondrial dysfunction. Finally, we show that Casp2−/− primary skin fibroblasts are protected from oxidants that target the mitochondrial electron transport chain. Taken together, our results demonstrate that caspase-2 exists in the mitochondria and that it is essential for mitochondrial oxidative stress-induced apoptosis. PMID:27019748

  2. Thiamine deficiency caused by thiamine antagonists triggers upregulation of apoptosis inducing factor gene expression and leads to caspase 3-mediated apoptosis in neuronally differentiated rat PC-12 cells.

    PubMed

    Chornyy, Sergiy; Parkhomenko, Julia; Chorna, Nataliya

    2007-01-01

    Recent evidence suggests that alterations in oxidative metabolism induced by thiamine deficiency lead to neuronal cell death. However, the molecular mechanisms underlying this process are still under extensive investigation. Here, we report that rat pheochromocytoma PC-12 cells differentiated in the presence of NGF into neurons undergo apoptosis due to thiamine deficiency caused by antagonists of thiamine - amprolium, pyrithiamine and oxythiamine. Confocal laser scanning fluorescence microscopy revealed that annexin V binds to PC-12 cells in presence of thiamine antagonists after 72 h incubation. Results also show that thiamine antagonists trigger upregulation of gene expression of mitochondrial-derived apoptosis inducing factor, DNA fragmentation, cleavage of caspase 3 and translocation of active product to the nucleus. We therefore propose that apoptosis induced by amprolium, pyrithiamine or oxythiamine occurs via the mitochondria-dependent caspase 3-mediated signaling pathway. In addition, our data indicate that pyrithiamine and oxythiamine are more potent inducers of apoptosis than amprolium.

  3. TRESK contributes to pain threshold changes by mediating apoptosis via MAPK pathway in the spinal cord.

    PubMed

    Zhou, Jun; Lin, Wenjing; Chen, Hongtao; Fan, Youling; Yang, Chengxiang

    2016-12-17

    The mechanism underlying neuropathic pain (NP) is complex and has not been fully elucidated. The TWIK-related spinal cord K(+) (TRESK) is the major background potassium current in dorsal root ganglia (DRG), we found that mitogen-activated protein kinase (MAPK) signal pathway were activated in spinal cord accompanied by TRESK down regulation in response to NP. Therefore, we investigated whether TRESK mediates inflammation and apoptosis by MAPK pathway in the spinal cord of NP rats. SNI rats exhibited reduced TRESK expression in DRG and spinal cord and higher sensitivity to mechanical stimuli but no effect on thermal stimuli. Intrathecal injections of TRESK overexpressing adenovirus alleviated mechanical allodynia, inhibited phosphorylation of extracellular signal-regulated kinase (ERK) and p38, and decreased inflammatory reactions and apoptosis in the spinal cords of SNI rats. Down regulation of TRESK in DRG and spinal cord was detected in normal rats after intrathecal TRESK shRNA lentivirus injection, which induced mechanical allodynia but had no effect on pain thresholds for heat stimulation. Phosphorylated ERK and p38 were increased in the spinal cord. Intrathecal injection of an ERK antagonist (PD98059) and p38 antagonist (SB203580) prevented ERK and p38 activation in the spinal cord and mechanical allodynia induced by TRESK shRNA lentivirus. In conclusion, our study clearly demonstrated an important role for TRESK in NP and that TRESK regulation contributes to pain sensitivity mediates inflammation and apoptosis by ERK and p38 MAPK signaling in the spinal cord.

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

    PubMed

    Ray, Tanusree; Pal, Amit

    2016-05-01

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

  5. FLIP switches Fas-mediated glucose signaling in human pancreatic cells from apoptosis to cell replication

    NASA Astrophysics Data System (ADS)

    Maedler, Kathrin; Fontana, Adriano; Ris, Frédéric; Sergeev, Pavel; Toso, Christian; Oberholzer, José; Lehmann, Roger; Bachmann, Felix; Tasinato, Andrea; Spinas, Giatgen A.; Halban, Philippe A.; Donath, Marc Y.

    2002-06-01

    Type 2 diabetes mellitus results from an inadequate adaptation of the functional pancreatic cell mass in the face of insulin resistance. Changes in the concentration of glucose play an essential role in the regulation of cell turnover. In human islets, elevated glucose concentrations impair cell proliferation and induce cell apoptosis via up-regulation of the Fas receptor. Recently, it has been shown that the caspase-8 inhibitor FLIP may divert Fas-mediated death signals into those for cell proliferation in lymphatic cells. We observed expression of FLIP in human pancreatic cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro exposure of islets from nondiabetic organ donors to high glucose levels decreased FLIP expression and increased the percentage of apoptotic terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)-positive cells; FLIP was no longer detectable in such TUNEL-positive cells. Up-regulation of FLIP, by incubation with transforming growth factor or by transfection with an expression vector coding for FLIP, protected cells from glucose-induced apoptosis, restored cell proliferation, and improved cell function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-Fas antibody, indicating their dependence on Fas receptor activation. The present data provide evidence for expression of FLIP in the human cell and suggest a novel approach to prevent and treat diabetes by switching Fas signaling from apoptosis to proliferation.

  6. Hypoxia-mediated autophagic flux inhibits silver nanoparticle-triggered apoptosis in human lung cancer cells

    PubMed Central

    Jeong, Jae-Kyo; Gurunathan, Sangiliyandi; Kang, Min-Hee; Han, Jae Woong; Das, Joydeep; Choi, Yun-Jung; Kwon, Deug-Nam; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Song, Hyuk; Kim, Jin-Hoi

    2016-01-01

    Solid tumors are frequently associated with resistance to chemotherapy because the fraction of hypoxic tumor cells is substantial. To understand the underlying mechanism of hypoxia on silver nanoparticle (AgNPs)-induced apoptosis, the expression of hypoxia-inducible factor (HIF)-1α, a hallmark of hypoxia, was measured in the presence and absence of AgNPs. The results showed that HIF-1α expression was upregulated after AgNPs treatment under both hypoxic and normoxic conditions. Cell viability assays showed that AgNPs promoted cell death in cancer cells but not in non-cancer cells, as cancer cells are slightly more acidic than normal cells. However, reactive oxygen species generation induced by AgNPs in lung cancer cells caused high susceptibility to oxidative stress, whereas pre-exposure to hypoxia blocked AgNPs-induced oxidative stress. Notably, HIF-1α inhibited AgNPs-induced mitochondria-mediated apoptosis by regulating autophagic flux through the regulation of ATG5, LC3-II, and p62. Further, cell viability after treatment of cancer cells with AgNPs under hypoxic conditions was lower in HIF-1α siRNA-transfected cells than in control siRNA-transfected cells, indicating that HIF-1α knockdown enhances hypoxia induced decrease in cell viability. Our results suggest that hypoxia-mediated autophagy may be a mechanism for the resistance of AgNPs-induced apoptosis and that strategies targeting HIF-1α may be used for cancer therapy. PMID:26867977

  7. Role of apoptosis and necrosis in cell death induced by nanoparticle-mediated photothermal therapy

    NASA Astrophysics Data System (ADS)

    Pattani, Varun P.; Shah, Jay; Atalis, Alexandra; Sharma, Anirudh; Tunnell, James W.

    2015-01-01

    Current cancer therapies can cause significant collateral damage due to a lack of specificity and sensitivity. Therefore, we explored the cell death pathway response to gold nanorod (GNR)-mediated photothermal therapy as a highly specific cancer therapeutic to understand the role of apoptosis and necrosis during intense localized heating. By developing this, we can optimize photothermal therapy to induce a maximum of `clean' cell death pathways, namely apoptosis, thereby reducing external damage. GNRs were targeted to several subcellular localizations within colorectal tumor cells in vitro, and the cell death pathways were quantitatively analyzed after photothermal therapy using flow cytometry. In this study, we found that the cell death response to photothermal therapy was dependent on the GNR localization. Furthermore, we demonstrated that nanorods targeted to the perinuclear region irradiated at 37.5 W/cm2 laser fluence rate led to maximum cell destruction with the `cleaner' method of apoptosis, at similar percentages as other anti-cancer targeted therapies. We believe that this indicates the therapeutic potential for GNR-mediated photothermal therapy to treat cancer effectively without causing damage to surrounding tissue.

  8. p53 causes butein-mediated apoptosis of chronic myeloid leukemia cells

    PubMed Central

    WOO, SANG-MI; CHOI, YOUN KYNUG; KIM, AH JEONG; CHO, SUNG-GOOK; KO, SEONG-GYU

    2016-01-01

    Progression of chronic myeloid leukemia, marked by the oncogenic Bcr-Abl mutation, is tightly associated with an alteration of the p53 pathway. It is known that butein extracted from various plants represses cancer growth. Although the anticancer effects of butein are widely accepted, the mechanisms by which butein induces apoptosis of chronic myeloid leukemia cells remains to be elucidated. The present study demonstrated that butein-induced apoptosis was mediated by p53. KBM5 chronic myeloid leukemia (CML) cells expressing wild-type p53 were more sensitive to butein compared with p53-null K562 CML cells in terms of apoptotic cell death. In addition, butein arrested KBM5 cells at S-phase and altered the expression levels of certain cyclins and the p53-downstream targets, MDM2 and p21. In addition, while butein reduced the protein expression of MDM2 in the KBM5 and K562 cells, it resulted in proteasome-independent MDM2 degradation in p53-expressing KBM5 cells, however, not in p53-null K562 cells. Therefore, the present study suggested that p53 causes the butein-mediated apoptosis of leukemic cells. PMID:26676515

  9. Increased expression of FLIP, an inhibitor of Fas-mediated apoptosis, in stomach cancer.

    PubMed

    Lee, Sug Hyung; Kim, Hong Sug; Kim, Su Young; Lee, Yun-Sil; Park, Won Sang; Kim, Sang Ho; Lee, Jung Young; Yoo, Nam Jin

    2003-02-01

    Despite the cell surface expression of Fas (Apo-1/CD95), many types of tumor cells, including stomach cancer cells, are resistant to Fas-mediated apoptosis, indicating the presence of inactivating mechanisms of Fas signaling. Expression of FLICE-like inhibitory protein (FLIP), one of the inhibitory proteins of Fas-mediated apoptosis, has been reported in several cancer types, but not in stomach cancer. In the present study, we analyzed the expression of Fas and FLIP in 60 advanced gastric adenocarcinomas by immunohistochemistry using a tissue microarray approach. Immunopositivity (defined as >/=30% of the neoplastic cells) was observed for Fas in 58 (97%) and FLIP in 54 (90%) of the 60 cancers. All of the tumors with FLIP immunostaining also showed Fas immunostaining. Loss of cell surface Fas immunostaining, another mechanism of Fas resistance, was observed in 45 tumors (75%). By contrast, normal gastric mucosal cells showed no or weak expression of both Fas and FLIP. Taken together, these results indicate that increased expression of FLIP is a frequent event in stomach carcinomas, and suggest that for evading apoptosis stomach carcinoma cells in vivo may need FLIP expression, which might contribute to tumor development.

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

    PubMed

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

    2015-09-01

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

  11. c-Abl mediates angiotensin II-induced apoptosis in podocytes

    PubMed Central

    Chen, Xinghua; Ren, Zhilong; Liang, Wei; Zha, Dongqing; Liu, Yipeng; Chen, Cheng; Singhal, Pravin C.; Ding, Guohua

    2013-01-01

    findings indicate that c-Abl may mediates Ang II-induced podocyte apoptosis, and inhibition of c-Abl expression can protect podocytes from Ang II-induced injury. PMID:23515840

  12. Platelet production proceeds independently of the intrinsic and extrinsic apoptosis pathways.

    PubMed

    Josefsson, Emma C; Burnett, Deborah L; Lebois, Marion; Debrincat, Marlyse A; White, Michael J; Henley, Katya J; Lane, Rachael M; Moujalled, Diane; Preston, Simon P; O'Reilly, Lorraine A; Pellegrini, Marc; Metcalf, Donald; Strasser, Andreas; Kile, Benjamin T

    2014-03-17

    BH3 mimetic drugs that target BCL-2 family pro-survival proteins to induce tumour cell apoptosis represent a new era in cancer therapy. Clinical trials of navitoclax (ABT-263, which targets BCL-2, BCL-XL and BCL-W) have shown great promise, but encountered dose-limiting thrombocytopenia. Recent work has demonstrated that this is due to the inhibition of BCL-XL, which is essential for platelet survival. These findings raise new questions about the established model of platelet shedding by megakaryocytes, which is thought to be an apoptotic process. Here we generate mice with megakaryocyte-specific deletions of the essential mediators of extrinsic (Caspase-8) and intrinsic (BAK/BAX) apoptosis. We show that megakaryocytes possess a Fas ligand-inducible extrinsic apoptosis pathway. However, Fas activation does not stimulate platelet production, rather, it triggers Caspase-8-mediated killing. Combined loss of Caspase-8/BAK/BAX does not impair thrombopoiesis, but can protect megakaryocytes from death in mice infected with lymphocytic choriomeningitis virus. Thus, apoptosis is dispensable for platelet biogenesis.

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

    PubMed

    Tsai, Hwei-Fang; Hsu, Ping-Ning

    2017-02-01

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

  14. Novel X-linked inhibitor of apoptosis inhibiting compound as sensitizer for TRAIL-mediated apoptosis in chronic lymphocytic leukaemia with poor prognosis.

    PubMed

    Frenzel, Lukas P; Patz, Michaela; Pallasch, Christian P; Brinker, Reinhild; Claasen, Julia; Schulz, Alexandra; Hallek, Michael; Kashkar, Hamid; Wendtner, Clemens-Martin

    2011-01-01

    Given that aggressive DNA damaging chemotherapy shows suboptimal efficacy in chronic lymphocytic leukaemia (CLL), alternative therapeutic approaches are needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce tumour-specific apoptosis. However, apoptosis might be inhibited by elevated levels of X-linked inhibitor of apoptosis (XIAP). Use of XIAP-inhibiting compounds might sensitize primary CLL cells towards TRAIL-mediated apoptosis. A novel small molecule, compound A (CA), an inhibitor of XIAP, was used in combination with TRAIL to induce apoptosis in primary CLL cells (n = 48). XIAP was significantly more highly expressed in primary CLL cells (n = 28) compared to healthy B cells (n = 16) (P = 0·02). Our data obtained by specific knock-down of XIAP by siRNA identified XIAP as the key factor conferring resistance to TRAIL in CLL. Combined treatment with CA/TRAIL significantly increased apoptosis compared to untreated (P = 8·5 × 10⁻¹⁰), solely CA (P = 4·1 × 10⁻¹²) or TRAIL treated (P = 4·8 × 10⁻¹⁰) CLL cells. CA rendered 40 of 48 (83·3%) primary CLL samples susceptible to TRAIL-mediated apoptosis. In particular, cells derived from patients with poor prognosis CLL (ZAP-70(+) , IGHV unmutated, 17p-) were highly responsive to this drug combination. Our highly-effective XIAP inhibitor CA, in concert with TRAIL, shows potential for the treatment of CLL cases with poor prognosis and therefore warrants further clinical investigation.

  15. Baicalin induces apoptosis in human osteosarcoma cell through ROS-mediated mitochondrial pathway.

    PubMed

    Wan, Daqian; Ouyang, Huoniu

    2017-09-05

    Baicalin is extracted from a traditional Chinese herb, Scutellaria baicalensis. In this study, the anticancer activity and underlying mechanisms of baicalin towards human osteosarcoma cell (HOS) were investigated. Baicalin could inhibit HOS cell proliferation in a concentration-dependent manner. Mitochondrial membrane potential decreased obviously after treated with different concentration of baicalin by flow cytometry assay and revealed that baicalin triggered a significant generation of reactive oxygen species (ROS). Western blotting assay further revealed that baicalin-induced cell apoptosis by suppressing Bcl-2 level, then activating caspase-9 and caspase-3. In vivo experiment, baicalin significantly suppressed tumour growth in female BALB/C nude mice bearing HOS tumours. In addition, baicalin did show toxicity to treated animal by comparing the body weight increase and mortality. In general, the present results demonstrated that baicalin-induced apoptosis in human osteosarcoma cell via a ROS-mediated mitochondrial pathway. The paper indicated that baicalin is a promising candidate for the treatment of HOS.

  16. Probiotic-derived ferrichrome inhibits colon cancer progression via JNK-mediated apoptosis

    PubMed Central

    Konishi, Hiroaki; Fujiya, Mikihiro; Tanaka, Hiroki; Ueno, Nobuhiro; Moriichi, Kentaro; Sasajima, Junpei; Ikuta, Katsuya; Akutsu, Hiroaki; Tanabe, Hiroki; Kohgo, Yutaka

    2016-01-01

    Previous reports have suggested that some probiotics inhibit tumorigenesis and cancer progression. However, the molecules involved have not yet been identified. Here, we show that the culture supernatant of Lactobacillus casei ATCC334 has a strong tumour-suppressive effect on colon cancer cells. Using mass spectrometry, we identify ferrichrome as a tumour-suppressive molecule produced by L. casei ATCC334. The tumour-suppressive effect of ferrichrome is greater than that of cisplatin and 5-fluorouracil, and ferrichrome has less of an effect on non-cancerous intestinal cells than either of those agents. A transcriptome analysis reveals that ferrichrome treatment induces apoptosis, which is mediated by the activation of c-jun N-terminal kinase (JNK). Western blotting indicates that the induction of apoptosis by ferrichrome is reduced by the inhibition of the JNK signalling pathway. This we demonstrate that probiotic-derived ferrichrome exerts a tumour-suppressive effect via the JNK signalling pathway. PMID:27507542

  17. CSB ablation induced apoptosis is mediated by increased endoplasmic reticulum stress response

    PubMed Central

    Caputo, Manuela; Balzerano, Alessio; Arisi, Ivan; D’Onofrio, Mara; Brandi, Rossella; Bongiorni, Silvia; Brancorsini, Stefano; Frontini, Mattia; Proietti-De-Santis, Luca

    2017-01-01

    The DNA repair protein Cockayne syndrome group B (CSB) has been recently identified as a promising anticancer target. Suppression, by antisense technology, of this protein causes devastating effects on tumor cells viability, through a massive induction of apoptosis, while being non-toxic to non-transformed cells. To gain insights into the mechanisms underlying the pro-apoptotic effects observed after CSB ablation, global gene expression patterns were determined, to identify genes that were significantly differentially regulated as a function of CSB expression. Our findings revealed that response to endoplasmic reticulum stress and response to unfolded proteins were ranked top amongst the cellular processes affected by CSB suppression. The major components of the endoplasmic reticulum stress-mediated apoptosis pathway, including pro-apoptotic factors downstream of the ATF3-CHOP cascade, were dramatically up-regulated. Altogether our findings add new pieces to the understanding of CSB mechanisms of action and to the molecular basis of CS syndrome. PMID:28253359

  18. Bacopa monnieri-Induced Protective Autophagy Inhibits Benzo[a]pyrene-Mediated Apoptosis.

    PubMed

    Das, Durgesh Nandini; Naik, Prajna Paramita; Nayak, Aditi; Panda, Prashanta Kumar; Mukhopadhyay, Subhadip; Sinha, Niharika; Bhutia, Sujit K

    2016-11-01

    Benzo[a]pyrene (B[a]P) is capable of inducing oxidative stress and cellular injuries leading to cell death and associates with a significant risk of cancer development. Prevention of B[a]P-induced cellular toxicity with herbal compound through regulation of mitochondrial oxidative stress might protect cell death and have therapeutic benefit to human health. In this study, we demonstrated the cytoprotective role of Bacopa monnieri (BM) against B[a]P-induced apoptosis through autophagy induction. Pretreatment with BM rescued the reduction in cell viability in B[a]P-treated human keratinocytes (HaCaT) cells indicating the cytoprotective potential of BM against B[a]P. Moreover, BM was found to inhibit B[a]P-mediated reactive oxygen species (ROS)-induced apoptosis activation in HaCaT cells. Furthermore, BM was found to preserve mitochondrial membrane potential and inhibited release of cytochrome c in B[a]P-treated HaCaT cells. Bacopa monnieri induced protective autophagy; we knocked down Beclin-1, and data showed that BM was unable to protect from B[a]P-induced mitochondrial ROS-mediated apoptosis in Beclin-1-deficient HaCaT cells. Moreover, we established that B[a]P-induced damaged mitochondria were found to colocalize and degraded within autolysosomes in order to protect HaCaT cells from mitochondrial injury. In conclusion, B[a]P-induced apoptosis was rescued by BM treatment and provided cytoprotection through Beclin-1-dependent autophagy activation. Copyright © 2016 John Wiley & Sons, Ltd.

  19. Inhibition of histone deacetylases by chlamydocin induces apoptosis and proteasome-mediated degradation of survivin.

    PubMed

    De Schepper, Stefanie; Bruwiere, Hélène; Verhulst, Tinne; Steller, Ulf; Andries, Luc; Wouters, Walter; Janicot, Michel; Arts, Janine; Van Heusden, Jim

    2003-02-01

    The naturally occurring cyclic tetrapeptide chlamydocin is a very potent inhibitor of cell proliferation. Here we show that chlamydocin is a highly potent histone deacetylase (HDAC) inhibitor, inhibiting HDAC activity in vitro with an IC(50) of 1.3 nM. Like other HDAC inhibitors, chlamydocin induces the accumulation of hyperacetylated histones H3 and H4 in A2780 ovarian cancer cells, increases the expression of p21(cip1/waf1), and causes an accumulation of cells in G(2)/M phase of the cell cycle. In addition, chlamydocin induces apoptosis by activating caspase-3, which in turn leads to the cleavage of p21(cip1/waf1) into a 15-kDa breakdown product and drives cells from growth arrest into apoptosis. Concomitant with the activation of caspase-3 and cleavage of p21(cip1/waf1), chlamydocin decreases the protein level of survivin, a member of the inhibitor of apoptosis protein family that is selectively expressed in tumors. Although our data indicate a potential link between degradation of survivin and activation of the apoptotic pathway induced by HDAC inhibitors, stable overexpression of survivin does not suppress the activation of caspase-3 or cleavage of p21(cip1/waf1) induced by chlamydocin treatment. The decrease of survivin protein level is mediated by degradation via proteasomes since it can be inhibited by specific proteasome inhibitors. Taken together, our results show that induction of apoptosis by chlamydocin involves caspase-dependent cleavage of p21(cip1/waf1), which is strikingly associated with proteasome-mediated degradation of survivin.

  20. Studying significance of apoptosis in mediating tolbutamide-induced teratogenesis in vitro.

    PubMed

    Singh, Gyanendra; Kumar, Akhilesh; Sinha, Neeraj

    2012-08-01

    The incidence of type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus) is growing worldwide and poses a serious public health problem in a current paradigm of changing life style and food habits. Tolbutamide (sulfonylurea) is among the commonly used anti-diabetic drugs worldwide for treating type 2 diabetes and is known to cause congenital malformations in animals. In this study, the effect of tolbutamide on major organogenesis period and the possible involvement of apoptosis in mediating congenital malformations have been carried out. In the present study design, post-implantation rat embryos of day 11 were cultured for 24 h with various concentrations of tolbutamide, i.e., 10, 100, and 1000 μg/mL cultures, respectively. The growth and developmental of each embryo was evaluated and compared with control ones for the presence of any malformations. The tolbutamide decreased all growth and developmental parameters in a concentration-dependent manner, when compared with control. However, exposure to tolbutamide at 10 μg/mL culture did not show any significant effect on embryonic growth and development in vitro. In parallel to this, flow cytometric analysis (cell cycle and annexin V binding) and DNA fragmentation assay were carried out followed by quantitation by 3'-OH labeling of cultured rat embryos to examine the role of apoptosis in bringing about tolbutamide-induced teratogenesis. All results were found to be dose dependent and an increase in apoptosis in embryonic tissues may be related to the increased risk of congenital malformations. The outcome of the research suggested that apoptosis might be involved in mediating teratogenesis of tolbutamide in vitro. Further research is warranted to fully understand this mechanism.

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

    PubMed

    Chen, Gang; Gong, Ming; Yan, Min; Zhang, Xiaoming

    2013-01-01

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

  2. Autophagy as a Survival Mechanism for Squamous Cell Carcinoma Cells in Endonuclease G-Mediated Apoptosis

    PubMed Central

    Masui, Atsushi; Hamada, Masakazu; Kameyama, Hiroyasu; Wakabayashi, Ken; Takasu, Ayako; Imai, Tomoaki; Iwai, Soichi; Yura, Yoshiaki

    2016-01-01

    Safingol, L- threo-dihydrosphingosine, induces cell death in human oral squamous cell carcinoma (SCC) cells through an endonuclease G (endoG) -mediated pathway. We herein determined whether safingol induced apoptosis and autophagy in oral SCC cells. Safingol induced apoptotic cell death in oral SCC cells in a dose-dependent manner. In safingol-treated cells, microtubule-associated protein 1 light chain 3 (LC3)-I was changed to LC3-II and the cytoplasmic expression of LC3, amount of acidic vesicular organelles (AVOs) stained by acridine orange and autophagic vacuoles were increased, indicating the occurrence of autophagy. An inhibitor of autophagy, 3-methyladenine (3-MA), enhanced the suppressive effects of safingol on cell viability, and this was accompanied by an increase in the number of apoptotic cells and extent of nuclear fragmentation. The nuclear translocation of endoG was minimal at a low concentration of safingol, but markedly increased when combined with 3-MA. The suppressive effects of safingol and 3-MA on cell viability were reduced in endoG siRNA- transfected cells. The scavenging of reactive oxygen species (ROS) prevented cell death induced by the combinational treatment, whereas a pretreatment with a pan-caspase inhibitor z-VAD-fmk did not. These results indicated that safingol induced apoptosis and autophagy in SCC cells and that the suppression of autophagy by 3-MA enhanced apoptosis. Autophagy supports cell survival, but not cell death in the SCC cell system in which apoptosis occurs in an endoG-mediated manner. PMID:27658240

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

    PubMed

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

    2014-01-01

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

  4. The Interferon Stimulated Gene 54 Promotes Apoptosis*

    PubMed Central

    Stawowczyk, Marcin; Van Scoy, Sarah; Kumar, K. Prasanna; Reich, Nancy C.

    2011-01-01

    The ability of interferons (IFNs) to inhibit viral replication and cellular proliferation is well established, but the specific contribution of each IFN-stimulated gene (ISG) to these biological responses remains to be completely understood. In this report we demonstrate that ISG54, also known as IFN-induced protein with tetratricopeptide repeats 2 (IFIT2), is a mediator of apoptosis. Expression of ISG54, independent of IFN stimulation, elicits apoptotic cell death. Cell death and apoptosis were quantified by propidium iodide uptake and annexin-V staining, respectively. The activation of caspase-3, a key mediator of the execution phase of apoptosis, was clearly apparent in cells expressing ISG54. The anti-apoptotic B cell lymphoma-xl (Bcl-xl) protein inhibited the apoptotic effects of ISG54 as did the anti-apoptotic adenoviral E1B-19K protein. In addition, ISG54 was not able to promote cell death in the absence of pro-apoptotic Bcl family members, Bax and Bak. Analyses of binding partners of ISG54 revealed association with two homologous proteins, ISG56/IFIT1 and ISG60/IFIT3. In addition, ISG60 binding negatively regulates the apoptotic effects of ISG54. The results reveal a previously unidentified role of ISG54 in the induction of apoptosis via a mitochondrial pathway and shed new light on the mechanism by which IFN elicits anti-viral and anti-cancer effects. PMID:21190939

  5. Sodium butyrate induces DRP1-mediated mitochondrial fusion and apoptosis in human colorectal cancer cells.

    PubMed

    Tailor, Dhanir; Hahm, Eun-Ryeong; Kale, Raosaheb K; Singh, Shivendra V; Singh, Rana P

    2014-05-01

    Sodium butyrate (NaBt) is the byproduct of anaerobic microbial fermentation inside the gastro-intestinal tract that could reach up to 20mM, and has been shown to inhibit the growth of various cancers. Herein, we evaluated its effect on mitochondrial fusion and associated induction of apoptosis in colorectal cancer cells (CRC). NaBt treatment at physiological (1-5mM) concentrations for 12 and 24h decreased the cell viability and induced G2-M phase cell cycle arrest in HCT116 (12h) and SW480 human CRC cells. This cell cycle arrest was associated with mitochondria-mediated apoptosis accompanied by a decrease in survivin and Bcl-2 expression, and generation of reactive oxygen species (ROS). Furthermore, NaBt treatment resulted in a significant decrease in the mitochondrial mass which is an indicator of mitochondrial fusion. Level of dynamin-related protein 1 (DRP1), a key regulator of mitochondrial fission and fusion where its up-regulation correlates with fission, was found to be decreased in CRC cells. Further, at early treatment time, DRP1 down-regulation was noticed in mitochondria which later became drastically reduced in both mitochondria as well as cytosol. DRP1 is activated by cyclin B1-CDK1 complex by its ser616 phosphorylation in which both cyclin B1-CDK1 complex and phospho-DRP1 (ser616) were strongly reduced by NaBt treatment. DRP1 was observed to be regulated by apoptosis as pan-caspase inhibitor showing rescue from NaBt-induced apoptosis also caused the reversal of DRP1 to the normal level as in control proliferating cells. Together, these findings suggest that NaBt can modulate mitochondrial fission and fusion by regulating the level of DRP1 and induce cell cycle arrest and apoptosis in human CRC cells.

  6. Hepatoprotective properties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathway.

    PubMed

    Ma, Jie-Qiong; Ding, Jie; Zhang, Li; Liu, Chan-Min

    2014-02-01

    Sesamin (Ses), one of the major lignan derived from sesame seeds, has been reported to have many benefits and medicinal properties. However, its protective effects against carbon tetrachloride (CCl4) induced injury in liver have not been clarified. The aim of the present study was to investigate the hepatoprotective effects of sesamin on oxidative stress and apoptosis in mice exposed to CCl4. Our data showed that sesamin significantly prevented CCl4-induced hepatotoxicity in a dose-dependent manner, indicated by both diagnostic indicators of liver damage (serum aminotransferase activities) and histopathological analysis. Moreover, CCl4-induced profound elevation of reactive oxygen species (ROS) production and oxidative stress, as evidenced by increasing of lipid peroxidation level and depleting of the total antioxidant capacity (TAC) in liver, were suppressed by treatment with sesamin. Furthermore, TUNEL assay showed that CCl4-induced apoptosis in mouse liver was significantly inhibited by sesamin. In exploring the underlying mechanisms of sesamin action, we found that activities of caspase-3 were markedly inhibited by the treatment of sesamin in the liver of CCl4 treated mice. Sesamin increased expression levels of phosphorylated Jun N-terminal kinases (JNK) in liver, which in turn inactivated pro-apoptotic signaling events restoring the balance between mitochondrial pro- and anti-apoptotic Bcl-2 proteins and decreasing the release of mitochondrial cytochrome c in liver of CCl4 treated mice. JNK was also involved in the mitochondrial extrinsic apoptotic pathways of sesamin effects against CCl4 induced liver injury by regulating the expression levels of phosphorylated c-Jun proteins, necrosis factor-alpha (TNF-α) and Bak. In conclusion, these results suggested that the inhibition of CCl4-induced apoptosis by sesamin is due at least in part to its anti-oxidant activity and its ability to modulate the JNK signaling pathway.

  7. JNJ-26481585 primes rhabdomyosarcoma cells for chemotherapeutics by engaging the mitochondrial pathway of apoptosis

    PubMed Central

    Heinicke, Ulrike; Kupka, Johanna; Fulda, Simone

    2015-01-01

    Rhabdomyosarcoma (RMS) is a common soft-tissue sarcoma in childhood with a poor prognosis, highlighting the need for new treatment strategies. Here we identify a synergistic interaction of the second-generation histone deacetylase inhibitor (HDACI) JNJ-26481585 and common chemotherapeutic drugs (i.e. Doxorubicin, Etoposide, Vincristine, Cyclophosphamide and Actinomycin D) to trigger apoptosis in RMS cells. Importantly, JNJ-26481585/Doxorubicin cotreatment also significantly suppresses long-term clonogenic survival of RMS cells and tumor growth in vivo in a preclinical RMS model. Mechanistically, JNJ-26481585/Doxorubicin cotreatment causes upregulation of the BH3-only proteins Bim and Noxa as well as downregulation of the antiapoptotic proteins Mcl-1 and Bcl-xL. These changes in the ratio of pro- and antiapoptotic Bcl-2 proteins contribute to JNJ-26481585/Doxorubicin-mediated apoptosis, since knockdown of Bim or Noxa significantly inhibits cell death. Also, JNJ-26481585 and Doxorubicin cooperate to stimulate activation of Bax and Bak, which is required for JNJ-26481585/Doxorubicin-induced apoptosis, since silencing of Bax or Bak protects against apoptosis. Consistently, overexpression of Bcl-2 significantly reduces JNJ-26481585/Doxorubicin-mediated apoptosis. JNJ-26481585/Doxorubicin cotreatment leads to caspase activation and caspase-dependent apoptosis, since the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) rescues cells from apoptosis. In conclusion, the second-generation HDACI JNJ-26481585 cooperates with chemotherapeutics to engage mitochondrial apoptosis in RMS cells, demonstrating that JNJ-26481585 represents a promising strategy for chemosensitization of RMS. PMID:26473375

  8. Hantavirus-infection Confers Resistance to Cytotoxic Lymphocyte-Mediated Apoptosis

    PubMed Central

    Gupta, Shawon; Braun, Monika; Tischler, Nicole D.; Stoltz, Malin; Sundström, Karin B.; Björkström, Niklas K.; Ljunggren, Hans-Gustaf; Klingström, Jonas

    2013-01-01

    Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardio-pulmonary syndrome (HCPS; also called hantavirus pulmonary syndrome (HPS)), both human diseases with high case-fatality rates. Endothelial cells are the main targets for hantaviruses. An intriguing observation in patients with HFRS and HCPS is that on one hand the virus infection leads to strong activation of CD8 T cells and NK cells, on the other hand no obvious destruction of infected endothelial cells is observed. Here, we provide an explanation for this dichotomy by showing that hantavirus-infected endothelial cells are protected from cytotoxic lymphocyte-mediated induction of apoptosis. When dissecting potential mechanisms behind this phenomenon, we discovered that the hantavirus nucleocapsid protein inhibits the enzymatic activity of both granzyme B and caspase 3. This provides a tentative explanation for the hantavirus-mediated block of cytotoxic granule-mediated apoptosis-induction, and hence the protection of infected cells from cytotoxic lymphocytes. These findings may explain why infected endothelial cells in hantavirus-infected patients are not destroyed by the strong cytotoxic lymphocyte response. PMID:23555267

  9. p53 Mediates Colistin-Induced Autophagy and Apoptosis in PC-12 Cells.

    PubMed

    Zhang, Ling; Xie, Daoyuan; Chen, Xueping; Hughes, Maria L R; Jiang, Guozheng; Lu, Ziyin; Xia, Chunli; Li, Li; Wang, Jinli; Xu, Wei; Sun, Yuan; Li, Rui; Wang, Rui; Qian, Feng; Li, Jian; Li, Jichang

    2016-09-01

    -induced autophagy and apoptosis are associated with the p53-mediated pathway. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  10. p53 Mediates Colistin-Induced Autophagy and Apoptosis in PC-12 Cells

    PubMed Central

    Zhang, Ling; Xie, Daoyuan; Chen, Xueping; Hughes, Maria L. R.; Jiang, Guozheng; Lu, Ziyin; Xia, Chunli; Li, Li; Wang, Jinli; Xu, Wei; Sun, Yuan; Li, Rui; Wang, Rui; Qian, Feng

    2016-01-01

    -induced autophagy and apoptosis are associated with the p53-mediated pathway. PMID:27324771

  11. Activating transcription factor 4 is involved in endoplasmic reticulum stress-mediated apoptosis contributing to vascular calcification.

    PubMed

    Duan, Xiao-Hui; Chang, Jin-Rui; Zhang, Jing; Zhang, Bao-Hong; Li, Yu-Lin; Teng, Xu; Zhu, Yi; Du, Jie; Tang, Chao-Shu; Qi, Yong-Fen

    2013-09-01

    Our previous work reported that endoplasmic reticulum stress (ERS)-mediated apoptosis was activated during vascular calcification (VC). Activating transcription factor 4 (ATF4) is a critical transcription factor in osteoblastogenesis and ERS-induced apoptosis. However, whether ATF4 is involved in ERS-mediated apoptosis contributing to VC remains unclear. In the present study, in vivo VC was induced in rats by administering vitamin D3 plus nicotine. Vascular smooth muscle cell (VSMC) calcification in vitro was induced by incubation in calcifying media containing β-glycerophosphate and CaCl2. ERS inhibitors taurine or 4-phenylbutyric acid attenuated ERS and VSMC apoptosis in calcified rat arteries, reduced calcification and retarded the VSMC contractile phenotype transforming into an osteoblast-like phenotype in vivo. Inhibition of ERS retarded the VSMC phenotypic transition into an osteoblast-like cell phenotype and reduced VSMC calcification and apoptosis in vitro. Interestingly, ATF4 was activated in calcified aortas and calcified VSMCs in vitro. ATF4 knockdown attenuated ERS-induced apoptosis in calcified VSMCs. ATF4 deficiency blocked VSMC calcification and negatively regulated the osteoblast phenotypic transition of VSMCs in vitro. Our results demonstrate that ATF4 was involved at least in part in the process of ERS-mediated apoptosis contributing to VC.

  12. Diallyl disulfide induces apoptosis in human leukemia HL-60 cells through activation of JNK mediated by reactive oxygen.

    PubMed

    Yi, Lan; Ji, Xiao-Xia; Lin, Min; Tan, Hui; Tang, Yi; Wen, Ling; Ma, Yan-Hua; Su, Qi

    2010-09-01

    Diallyl disulfide (DADS) is a chemopreventive agent that can induce apoptosis in many tumor cells. Reactive oxygen species (ROS) are important mediators in apoptosis induced by various stimuli, including chemopreventive agents. The phosphotransferase c-JUN N-terminal kinase (JNK) has been shown to regulate apoptosis. In this study, we found that DADS-induced apoptosis in human leukemia HL-60 cells is mediated by ROS-activated JNK. The DADS-treated HL-60 cells showed a dose- and time-dependent decrease in cell viability and proliferation. Agarose gel electrophoresis of cells treated with 10.0 or 20.0 mg/L DADS for 24 h showed a characteristic ladder pattern in their DNA. Levels of DADS-induced ROS, as measured by 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescence, also showed dose- and time-dependent increases in HL-60 cells. Activity of JNK was induced by DADS in a dose-dependent manner; HL-60 cells exposed to 10.0 mg/L DADS for 8 h showed maximum levels of phosphorylated JNK, which decreased when exposed for additional 4h. In contrast, Sp600125, a specific inhibitor of JNK, blocked apoptosis of HL-60 cells exposed to DADS. N-Acetylcysteine (NAC), a known antioxidant, also decreased ROS generation, effectively blocked apoptosis, and decreased DADS-induced phosphorylated JNK levels. These results suggest that JNK is involved in DADS-induced ROS-mediated apoptosis in HL-60 cells.

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

    PubMed

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

    2015-03-01

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

  14. Caspase-mediated apoptosis in neuronal excitotoxicity triggered by nitric oxide.

    PubMed Central

    Leist, M.; Volbracht, C.; Kühnle, S.; Fava, E.; Ferrando-May, E.; Nicotera, P.

    1997-01-01

    BACKGROUND: Excitotoxicity and excess generation of nitric oxide (NO) are believed to be fundamental mechanisms in many acute and chronic neurodegenerative disorders. Disturbance of Ca2+ homeostasis and protein nitration/nitrosylation are key features in such conditions. Recently, a family of proteases collectively known as caspases has been implicated as common executor of a variety of death signals. In addition, overactivation of poly-(ADP-ribose) polymerase (PARP) has been observed in neuronal excitotoxicity. We therefore designed this study to investigate whether triggering of caspase activity and/or activation of PARP played a role in cerebellar granule cell (CGC) apoptosis elicited by peroxynitrite (ONOO-) or NO donors. MATERIALS AND METHODS: CGC from wild-type or PARP -/- mice were exposed to various nitric oxide donors. Caspase activation and its implications for membrane alterations, Ca2+ homeostasis, intracellular proteolysis, chromatin degradation, and cell death were investigated. RESULTS: CGC exposed to NO donors undergo apoptosis, which is mediated by excess synaptic release of excitotoxic mediators. This excitotoxic mechanism differs from direct NO toxicity in some other neuronal populations and does not involve PARP activation. Inhibition of caspases with different peptide substrates prevented cell death and the related features, including intracellular proteolysis, chromatin breakdown, and translocation of phosphatidylserine to the outer surface of the cell membrane. Increased Ca2+ influx following N-methyl-D-aspartate (NMDA) receptor (NMDA-R) activation was not inhibited by caspase inhibitors. CONCLUSIONS: In CGC, NO donors elicit apoptosis by a mechanism involving excitotoxic mediators, Ca2+ overload, and subsequent activation of caspases. Images Fig. 4 FIG. 5 FIG. 6 FIG. 7 PMID:9407551

  15. Pin1-FADD interactions regulate Fas-mediated apoptosis in activated eosinophils#

    PubMed Central

    Oh, Jiyoung; Malter, James S.

    2013-01-01

    Abnormally long-lived eosinophils (Eos) are the major inflammatory component of allergic responses in the lungs of active asthmatics. Eos recruited to the airways after allergen exposure produce and respond to IL-5 and GM-CSF, enhancing their survival. Pro-survival signaling activates Pin1, a cis-trans peptidyl isomerase (PPIase) that binds to Bax and prevents it activation. How long-lived Eos, despite the continued presence of GM-CSF or IL-5, eventually undergo apoptosis to end allergic inflammation remains unclear. Here we show that Pin1 location, activity and protein interactions are jointly influenced by Fas and pro-survival cytokine IL-5. Fas signaling strongly induced the phosphorylation of FADD at Ser194 and Pin1 at Ser16 as well as their nuclear accumulation. Phospho-mimic Ser194Glu FADD mutants accelerated Eos apoptosis compared to WT or Ser194Ala mutants. Downstream of FADD phosphorylation, Caspase 8, 9 and 3 cleavage as well as Eos apoptosis induced by Fas were reduced by constitutively active Pin1 and enhanced by Pin1 inhibition. Pin1 was activated by IL-5 while simultaneous IL-5 and anti-Fas treatment modestly reduced PPIase activity but induced Pin1 to associate with FADD after its phosphorylation at Ser194. Mechanistically, Pin1 mediated isomerization facilitated the subsequent dephosphorylation of Ser194 FADD and maintenance of cytoplasmic location. In vivo activated bronchoalvelolar (BAL) Eos obtained after allergen challenge showed elevated survival and Pin1 activity that could be reversed by anti-Fas. Therefore, our data suggest that Pin1 is a critical link between FADD mediated cell death and IL-5 mediated pro-survival signaling. PMID:23606538

  16. RNase L Cleavage Products Promote Switch from Autophagy to Apoptosis by Caspase-Mediated Cleavage of Beclin-1

    PubMed Central

    Siddiqui, Mohammad Adnan; Mukherjee, Sushovita; Manivannan, Praveen; Malathi, Krishnamurthy

    2015-01-01

    Autophagy and apoptosis share regulatory molecules enabling crosstalk in pathways that affect cellular homeostasis including response to viral infections and survival of tumor cells. Ribonuclease L (RNase L) is an antiviral endonuclease that is activated in virus-infected cells and cleaves viral and cellular single-stranded RNAs to produce small double-stranded RNAs with roles in amplifying host responses. Activation of RNase L induces autophagy and apoptosis in many cell types. However, the mechanism by which RNase L mediates crosstalk between these two pathways remains unclear. Here we show that small dsRNAs produced by RNase L promote a switch from autophagy to apoptosis by caspase-mediated cleavage of Beclin-1, terminating autophagy. The caspase 3-cleaved C-terminal fragment of Beclin-1 enhances apoptosis by translocating to the mitochondria along with proapoptotic protein, Bax, and inducing release of cytochrome C to the cytosol. Cleavage of Beclin-1 determines switch to apoptosis since expression of caspase-resistant Beclin-1 inhibits apoptosis and sustains autophagy. Moreover, inhibiting RNase L-induced autophagy promotes cell death and inhibiting apoptosis prolongs autophagy in a cross-inhibitory mechanism. Our results demonstrate a novel role of RNase L generated small RNAs in cross-talk between autophagy and apoptosis that impacts the fate of cells during viral infections and cancer. PMID:26263979

  17. Inhibition of Cathepsin S Induces Mitochondrial ROS That Sensitizes TRAIL-Mediated Apoptosis Through p53-Mediated Downregulation of Bcl-2 and c-FLIP.

    PubMed

    Seo, Bo Ram; Min, Kyoung-Jin; Woo, Seon Min; Choe, Misun; Choi, Kyeong Sook; Lee, Young-Kyung; Yoon, Gyesoon; Kwon, Taeg Kyu

    2017-08-01

    Cathepsin S is highly expressed in various cancer cells, and it has protumoral effects, including promotion of migration, invasion, and neovascularization. In this study, we show that inhibition of cathepsin S could sensitize cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. An inhibitor of cathepsin S (Z-FL-COCHO; ZFL) markedly induced apoptosis in human renal cancer cells treated with TRAIL. In contrast, combined treatment with ZFL and TRAIL had no effect on normal cells. ZFL downregulated Bcl-2 expression at the transcriptional level in a p53-dependent manner, and overexpression of Bcl-2 also markedly blocked apoptosis induced by combined treatment with ZFL and TRAIL. In addition, ZFL induced downregulation of c-FLIP, and overexpression of c-FLIP blocked the apoptosis induced by ZFL plus TRAIL. Moreover, ZFL increased the expression of Cbl, an E3 ligase of c-FLIP, in a p53-dependent manner, and knockdown of Cbl markedly prevented c-FLIP downregulation and the apoptosis induced by ZFL plus TRAIL. Interestingly, ZFL induced p53 expression via production of mitochondrial reactive oxygen species (ROS). We also demonstrated that downregulation of cathepsin S by small interfering RNA sensitized TRAIL-mediated apoptosis in Caki cells. These results reveal the importance of cathepsin S on resistance against TRAIL, and inhibition of cathepsin S activity plays a crucial role in TRAIL-mediated cell death of cancer cells. Our results indicated that inhibition of cathepsin S stimulates TRAIL-induced apoptosis through downregulation of Bcl-2 and Cbl-mediated c-FLIP by ROS-mediated p53 expression. Antioxid. Redox Signal. 27, 215-233.

  18. Evodiamine Induces Apoptosis and Enhances TRAIL-Induced Apoptosis in Human Bladder Cancer Cells through mTOR/S6K1-Mediated Downregulation of Mcl-1

    PubMed Central

    Zhang, Tao; Qu, Shanna; Shi, Qi; He, Dalin; Jin, Xunbo

    2014-01-01

    The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), either alone or in combination with other anti-cancer agents, has been considered as a new strategy for anti-cancer therapy. In this study, we demonstrated that evodiamine, a quinolone alkaloid isolated from the fruit of Evodia fructus, induced apoptosis and enhanced TRAIL-induced apoptosis in human bladder cancer cells. To elucidate the underlying mechanism, we found that evodiamine significantly reduced the protein levels of Mcl-1 in 253J and T24 bladder cancer cells, and overexpression of this molecule attenuated the apoptosis induced by evodiamine alone, or in combination with TRAIL. Further experiments revealed that evodiamine did not affect the mRNA level, proteasomal degradation and protein stability of Mcl-1. On the other hand, evodiamine inhibited the mTOR/S6K1 pathway, which usually regulates protein translation; moreover, knockdown of S6K1 with small interfering RNA (siRNA) effectively reduced Mcl-1 levels, indicating evodiamine downregulates c-FLIP through inhibition of mTOR/S6K1 pathway. Taken together, our results indicate that evodiamine induces apoptosis and enhances TRAIL-induced apoptosis possibly through mTOR/S6K1-mediated downregulation of Mcl-1; furthermore, these findings provide a rationale for the combined application of evodiamine with TRAIL in the treatment of bladder cancer. PMID:24566141

  19. Feline immunodeficiency virus envelope glycoprotein mediates apoptosis in activated PBMC by a mechanism dependent on gp41 function

    SciTech Connect

    Garg, Himanshu; Joshi, Anjali; Tompkins, Wayne A. . E-mail: Wayne_Tompkins@ncsu.edu

    2004-12-20

    Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which parallels HIV-1-induced immunodeficiency in humans. It has been established that HIV envelope (Env) glycoprotein mediates T cell loss via a mechanism that requires CXCR4 binding. The Env glycoprotein of FIV, similar to HIV, requires CXCR4 binding for viral entry, as well as inducing membrane fusion leading to syncytia formation. However, the role of FIV Env in T cell loss and the molecular mechanisms governing this process have not been elucidated. We studied the role of Env glycoprotein in FIV-mediated T cell apoptosis in an in vitro model. Our studies demonstrate that membrane-expressed FIV Env induces apoptosis in activated feline peripheral blood mononuclear cells (PBMC) by a mechanism that requires CXCR4 binding, as the process was inhibited by CXCR4 antagonist AMD3100 in a dose-dependent manner. Interestingly, studies regarding the role of CD134, the recently identified primary receptor of FIV, suggest that binding to CD134 may not be important for induction of apoptosis in PBMC. However, inhibiting Env-mediated fusion post CXCR4 binding by FIV gp41-specific fusion inhibitor also inhibited apoptosis. Under similar conditions, a fusion-defective gp41 mutant was unable to induce apoptosis in activated PBMC. Our findings are the first report suggesting the potential of FIV Env to mediate apoptosis in bystander cells by a process that is dependent on gp41 function.

  20. Targeting acute myeloid leukemia by dual inhibition of PI3K signaling and Cdk9-mediated Mcl-1 transcription.

    PubMed

    Thomas, Daniel; Powell, Jason A; Vergez, Francois; Segal, David H; Nguyen, Nhu-Y N; Baker, Adele; Teh, Tse-Chieh; Barry, Emma F; Sarry, Jean-Emmanuel; Lee, Erwin M; Nero, Tracy L; Jabbour, Anissa M; Pomilio, Giovanna; Green, Benjamin D; Manenti, Stéphane; Glaser, Stefan P; Parker, Michael W; Lopez, Angel F; Ekert, Paul G; Lock, Richard B; Huang, David C S; Nilsson, Susie K; Récher, Christian; Wei, Andrew H; Guthridge, Mark A

    2013-08-01

    Resistance to cell death is a hallmark of cancer and renders transformed cells resistant to multiple apoptotic triggers. The Bcl-2 family member, Mcl-1, is a key driver of cell survival in diverse cancers, including acute myeloid leukemia (AML). A screen for compounds that downregulate Mcl-1 identified the kinase inhibitor, PIK-75, which demonstrates marked proapoptotic activity against a panel of cytogenetically diverse primary human AML patient samples. We show that PIK-75 transiently blocks Cdk7/9, leading to transcriptional suppression of MCL-1, rapid loss of Mcl-1 protein, and alleviation of its inhibition of proapoptotic Bak. PIK-75 also targets the p110α isoform of PI3K, which leads to a loss of association between Bcl-xL and Bak. The simultaneous loss of Mcl-1 and Bcl-xL association with Bak leads to rapid apoptosis of AML cells. Concordantly, low Bak expression in AML confers resistance to PIK-75-mediated killing. On the other hand, the induction of apoptosis by PIK-75 did not require the expression of the BH3 proteins Bim, Bid, Bad, Noxa, or Puma. PIK-75 significantly reduced leukemia burden and increased the survival of mice engrafted with human AML without inducing overt toxicity. Future efforts to cotarget PI3K and Cdk9 with drugs such as PIK-75 in AML are warranted.

  1. IRF-1 transcriptionally upregulates PUMA, which mediates the mitochondrial apoptotic pathway in IRF-1-induced apoptosis in cancer cells.

    PubMed

    Gao, J; Senthil, M; Ren, B; Yan, J; Xing, Q; Yu, J; Zhang, L; Yim, J H

    2010-04-01

    Interferon regulatory factor-1 (IRF-1) is a transcription factor that acts as a tumor suppressor and causes apoptosis in cancer cells. We evaluated IRF-1-induced apoptosis in gastric cancer cell lines. We established stable clones in AGS cells that have a tetracycline-inducible IRF-1 expression system. We used these clones and recombinant adenovirus expressing IRF-1 to explore the mechanism of IRF-1-induced apoptosis in gastric cancer. Expression of IRF-1 causes apoptosis in gastric cancer cell lines as shown by phosphatidylserine exposure and cleavage of caspase-8, caspase-3, and Bid with the mitochondrial release of cytochrome c. However, inhibition of caspase-8 and Bid did not inhibit apoptosis and did not decrease cleaved caspase-9 or mitochondrial release of cytochrome c. We then show that IRF-1 upregulates PUMA (p53 upregulated modulator of apoptosis), which is known to activate apoptosis by the intrinsic pathway; this can be p53-independent. IRF-1 binds to distinct sites in the promoter of PUMA and activates PUMA transcription. Moreover, molecular markers of mitochondrial apoptosis are eliminated in PUMA knockout and knockdown cells and phosphatidylserine exposure is decreased dramatically. Finally, we show that IFN-gamma induces IRF-1-mediated upregulation of PUMA in cancer cells. We conclude that IRF-1 can induce apoptosis by the intrinsic pathway independent of the extrinsic pathway by upregulation of PUMA.

  2. A mitochondria-dependent pathway mediates the apoptosis of GSE-induced yeast.

    PubMed

    Cao, Sishuo; Xu, Wentao; Zhang, Nan; Wang, Yan; Luo, YunBo; He, Xiaoyun; Huang, Kunlun

    2012-01-01

    Grapefruit seed extract (GSE), which has powerful anti-fungal activity, can induce apoptosis in S. cerevisiae. The yeast cells underwent apoptosis as determined by testing for apoptotic markers of DNA cleavage and typical chromatin condensation by Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) and 4,6'-diaminidino-2-phenylindole (DAPI) staining and electron microscopy. The changes of ΔΨmt (mitochondrial transmembrane potential) and ROS (reactive oxygen species) indicated that the mitochondria took part in the apoptotic process. Changes in this process detected by metabonomics and proteomics revealed that the yeast cells tenaciously resisted adversity. Proteins related to redox, cellular structure, membrane, energy and DNA repair were significantly increased. In this study, the relative changes in the levels of proteins and metabolites showed the tenacious resistance of yeast cells. However, GSE induced apoptosis in the yeast cells by destruction of the mitochondrial 60 S ribosomal protein, L14-A, and prevented the conversion of pantothenic acid to coenzyme A (CoA). The relationship between the proteins and metabolites was analyzed by orthogonal projections to latent structures (OPLS). We found that the changes of the metabolites and the protein changes had relevant consistency.

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

    PubMed

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

    2015-03-01

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

  4. A Mitochondria-Dependent Pathway Mediates the Apoptosis of GSE-Induced Yeast

    PubMed Central

    Cao, Sishuo; Xu, Wentao; Zhang, Nan; Wang, Yan; Luo, YunBo; He, Xiaoyun; Huang, Kunlun

    2012-01-01

    Grapefruit seed extract (GSE), which has powerful anti-fungal activity, can induce apoptosis in S. cerevisiae. The yeast cells underwent apoptosis as determined by testing for apoptotic markers of DNA cleavage and typical chromatin condensation by Terminal Deoxynucleotidyl Transferase–mediated dUTP Nick End Labeling (TUNEL) and 4,6′-diaminidino-2-phenylindole (DAPI) staining and electron microscopy. The changes of ΔΨmt (mitochondrial transmembrane potential) and ROS (reactive oxygen species) indicated that the mitochondria took part in the apoptotic process. Changes in this process detected by metabonomics and proteomics revealed that the yeast cells tenaciously resisted adversity. Proteins related to redox, cellular structure, membrane, energy and DNA repair were significantly increased. In this study, the relative changes in the levels of proteins and metabolites showed the tenacious resistance of yeast cells. However, GSE induced apoptosis in the yeast cells by destruction of the mitochondrial 60 S ribosomal protein, L14-A, and prevented the conversion of pantothenic acid to coenzyme A (CoA). The relationship between the proteins and metabolites was analyzed by orthogonal projections to latent structures (OPLS). We found that the changes of the metabolites and the protein changes had relevant consistency. PMID:22403727

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

    PubMed

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

    2011-01-01

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

  6. The pesticide rotenone induces caspase-3-mediated apoptosis in ventral mesencephalic dopaminergic neurons.

    PubMed

    Ahmadi, Ferogh A; Linseman, Daniel A; Grammatopoulos, Tom N; Jones, Susan M; Bouchard, Ron J; Freed, Curt R; Heidenreich, Kim A; Zawada, W Michael

    2003-11-01

    In vivo, the pesticide rotenone induces degeneration of dopamine neurons and parkinsonian-like pathology in adult rats. In the current study, we utilized primary ventral mesencephalic (VM) cultures from E15 rats as an in vitro model to examine the mechanism underlying rotenone-induced death of dopamine neurons. After 11 h of exposure to 30 nm rotenone, the number of dopamine neurons identified by tyrosine hydroxylase (TH) immunostaining declined rapidly with only 23% of the neurons surviving. By contrast, 73% of total cells survived rotenone treatment, indicating that TH+ neurons are more sensitive to rotenone. Examination of the role of apoptosis in TH+ neuron death, revealed that 10 and 30 nm rotenone significantly increased the number of apoptotic TH+ neurons from 7% under control conditions to 38 and 55%, respectively. The increase in apoptotic TH+ neurons correlated with an increase in immunoreactivity for active caspase-3 in TH+ neurons. The caspase-3 inhibitor, DEVD, rescued a significant number of TH+ neurons from rotenone-induced death. Furthermore, this protective effect lasted for at least 32 h post-rotenone and DEVD exposure, indicating lasting neuroprotection achieved with an intervention prior to the death commitment point. Our results show for the first time in primary dopamine neurons that, at low nanomolar concentrations, rotenone induces caspase-3-mediated apoptosis. Understanding the mechanism of rotenone-induced apoptosis in dopamine neurons may contribute to the development of new neuroprotective strategies against Parkinson's disease.

  7. HCV-Mediated Apoptosis of Hepatocytes in Culture and Viral Pathogenesis

    PubMed Central

    Silberstein, Erica; Ulitzky, Laura; Lima, Livia Alves; Cehan, Nicoleta; Teixeira-Carvalho, Andréa; Roingeard, Philippe; Taylor, Deborah R.

    2016-01-01

    Chronic Hepatitis C Virus (HCV) infection is associated with progressive liver injury and subsequent development of fibrosis and cirrhosis. The death of hepatocytes results in the release of cytokines that induce inflammatory and fibrotic responses. The mechanism of liver damage is still under investigation but both apoptosis and immune-mediated processes may play roles. By observing the changes in gene expression patterns in HCV-infected cells, both markers and the causes of HCV-associated liver injury may be elucidated. HCV genotype 1b virus from persistently infected VeroE6 cells induced a strong cytopathic effect when used to infect Huh7.5 hepatoma cells. To determine if this cytopathic effect was a result of apoptosis, ultrastructural changes were observed by electron microscopy and markers of programmed cell death were surveyed. Screening of a human PCR array demonstrated a gene expression profile that contained upregulated markers of apoptosis, including tumor necrosis factor, caspases and caspase activators, Fas, Bcl2-interacting killer (BIK) and tumor suppressor protein, p53, as a result of HCV genotype 1b infection. The genes identified in this study should provide new insights into understanding viral pathogenesis in liver cells and may possibly help to identify novel antiviral and antifibrotic targets. PMID:27280444

  8. Calcium and oxidative stress mediate perillaldehyde-induced apoptosis in Candida albicans.

    PubMed

    Tian, Hui; Qu, Su; Wang, Yanzhen; Lu, Zhaoqun; Zhang, Man; Gan, Yeyun; Zhang, Peng; Tian, Jun

    2017-02-21

    New anti-Candida albicans drugs are needed due to the emergence of resistant cases in recent years. Perillaldehyde (PAE) is a natural monoterpenoid compound derived from Perilla frutescens. The minimum inhibitory concentration of PAE against C. albicans was 0.4 μL/mL. We aimed to elucidate the antifungal mode of action of PAE against C. albicans. The antifungal activity of PAE against C. albicans was found to correlate with an elevation in intracellular Ca(2+) and accumulation of ROS. Several downstream apoptosis events such as the disruption of mitochondrial membrane potential, phosphatidylserine externalization, cytochrome c release, and metacaspase activation were observed in PAE-treated cells. DNA damage and nuclear fragmentation assays also revealed apoptosis of C. albicans cells. In summary, by means of fluorescent microscopy, flow cytometer analysis, and Western blot, our data uncovered that PAE exerts its antifungal activity through Ca(2+) and oxidative stress-mediated apoptosis mechanisms. This study deciphered the mode of action of PAE, which will be useful in the design of improved antifungal therapies.

  9. Adiponectin-induced antiangiogenesis and antitumor activity involve caspase-mediated endothelial cell apoptosis

    PubMed Central

    Bråkenhielm, Ebba; Veitonmäki, Niina; Cao, Renhai; Kihara, Shinji; Matsuzawa, Yuji; Zhivotovsky, Boris; Funahashi, Tohru; Cao, Yihai

    2004-01-01

    Obesity is a risk factor for the development of many severe human diseases such as cardiovascular disorders, diabetes, and cancer, which are tightly linked to angiogenesis. The adipose tissue produces several growth factors/hormones including leptin, tumor necrosis factor α, and adiponectin. It has been found that adiponectin levels are reduced in obesity. Here, we report a unique function of adiponectin as a negative regulator of angiogenesis. In vitro, adiponectin potently inhibits endothelial cell proliferation and migration. In the chick chorioallantoic membrane and the mouse corneal angiogenesis assays, adiponectin remarkably prevents new blood vessel growth. Further, we demonstrate that the antiendothelial mechanisms involve activation of caspase-mediated endothelial cell apoptosis. Adiponectin induces a cascade activation of caspases-8, -9, and -3, which leads to cell death. In a mouse tumor model, adiponectin significantly inhibits primary tumor growth. Impaired tumor growth is associated with decreased neovascularization, leading to significantly increased tumor cell apoptosis. These data demonstrate induction of endothelial apoptosis as an unique mechanism of adiponectin-induced antiangiogenesis. Adiponectin, as a direct endogenous angiogenesis inhibitor, may have therapeutic implications in the treatment of angiogenesis-dependent diseases. PMID:14983034

  10. Transferrin mediated solid lipid nanoparticles containing curcumin: enhanced in vitro anticancer activity by induction of apoptosis.

    PubMed

    Mulik, Rohit S; Mönkkönen, Jukka; Juvonen, Risto O; Mahadik, Kakasaheb R; Paradkar, Anant R

    2010-10-15

    Photodegradation and low bioavailability are major hurdles for the therapeutic use of curcumin. Aim of the present study was to formulate transferrin-mediated solid lipid nanoparticles (Tf-C-SLN) to increase photostability, and enhance its anticancer activity against MCF-7 breast cancer cells. Tf-C-SLN were prepared by homogenization method and characterized by size, zeta potential, entrapment efficiency and stability, transmission electron microscopy (TEM), X-ray diffraction (XRD) and in vitro release study. Microplate analysis and flow cytometry techniques were used for cytotoxicity and apoptosis study. The physical characterization showed the suitability of method of preparation. TEM and XRD study revealed the spherical nature and entrapment of curcumin in amorphous form, respectively. The cytotoxicity, ROS and cell uptake was found to be increased considerably with Tf-C-SLN compared to curcumin solubilized surfactant solution (CSSS) and curcumin-loaded SLN (C-SLN) suggesting the targeting effect. AnnexinV-FITC/PI double staining, DNA analysis and reduced mitochondrial potential confirmed the apoptosis. The flow cytometric studies revealed that the anticancer activity of curcumin is enhanced with Tf-C-SLN compared to CSSS and C-SLN, and apoptosis is the mechanism underlying the cytotoxicity. The present study indicated the potential of Tf-C-SLN in enhancing the anticancer effect of curcumin in breast cancer cells in vitro.

  11. Telmisartan ameliorates cisplatin-induced nephrotoxicity by inhibiting MAPK mediated inflammation and apoptosis.

    PubMed

    Malik, Salma; Suchal, Kapil; Gamad, Nanda; Dinda, Amit Kumar; Arya, Dharamvir Singh; Bhatia, Jagriti

    2015-02-05

    Nephrotoxicity is a major adverse effect of the widely used anticancer drug cisplatin. Oxidative stress, inflammation and apoptosis are implicated in the pathophysiology of cisplatin-induced acute renal injury. Moreover, cisplatin activates many signal transduction pathways involved in cell injury and death, particularly mitogen activated protein kinase (MAPK) pathway. With this background, we aimed to investigate the protective effect of telmisartan, a widely used antihypertensive drug, in cisplatin-induced nephrotoxicity model in rats. To accomplish this, male albino wistar rats (150-200 g) were divided into 6 groups: Normal, cisplatin-control, telmisartan (2.5, 5 and 10 mg/kg) and telmisartan per se treatment groups. Normal saline or telmisartan was administered orally to rats for 10 days and cisplatin was given on 7th day (8 mg/kg; i.p.) to induce nephrotoxicity. On 10th day, rats were killed and both the kidneys were harvested for biochemical, histopathological and molecular studies. Cisplatin injected rats showed depressed renal function, altered proxidant-antioxidant balance and acute tubular necrosis which was significantly normalized by telmisartan co-treatment. Furthermore, cisplatin administration activated MAPK pathway that caused tubular inflammation and apoptosis in rats. Telmisartan treatment significantly prevented MAPK mediated inflammation and apoptosis. Among the three doses studied telmisartan at 10 mg/kg dose showed maximum nephroprotective effect which could be due to maintenance of cellular redox status and inhibition of MAPK activation. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Sinularin induces oxidative stress-mediated G2/M arrest and apoptosis in oral cancer cells.

    PubMed

    Chang, Yung-Ting; Wu, Chang-Yi; Tang, Jen-Yang; Huang, Chiung-Yao; Liaw, Chih-Chuang; Wu, Shih-Hsiung; Sheu, Jyh-Horng; Chang, Hsueh-Wei

    2017-09-01

    Soft corals-derived natural product, sinularin, was antiproliferative against some cancers but its effect and detailed mechanism on oral cancer cells remain unclear. The subject of this study is to examine the antioral cancer effects and underlying detailed mechanisms in terms of cell viability, oxidative stress, cell cycle analysis, and apoptosis analyses. In MTS assay, sinularin dose-responsively decreased cell viability of three oral cancer cells (Ca9-22, HSC-3, and CAL 27) but only little damage to oral normal cells (HGF-1). This cell killing effect was rescued by the antioxidant N-acetylcysteine (NAC) pretreatment. Abnormal cell morphology and induction of reactive oxygen species (ROS) were found in sinularin-treated oral cancer Ca9-22 cells, however, NAC pretreatment also recovered these changes. Sinularin arrested the Ca9-22 cells at G2/M phase and dysregulated the G2/M regulatory proteins such as cdc2 and cyclin B1. Sinularin dose-responsively induced apoptosis on Ca9-22 cells in terms of flow cytometry (annexin V and pancaspase analyses) and western blotting (caspases 3, 8, 9) and poly (ADP-ribose) polymerase (PARP). These apoptotic changes of sinularin-treated Ca9-22 cells were rescued by NAC pretreatment. Taken together, sinularin induces oxidative stress-mediated antiproliferation, G2/M arrest, and apoptosis against oral cancer cells and may be a potential marine drug for antioral cancer therapy. © 2017 Wiley Periodicals, Inc.

  13. ASC Induces Apoptosis via Activation of Caspase-9 by Enhancing Gap Junction-Mediated Intercellular Communication

    PubMed Central

    Hida, Shigeaki; Fujii, Chifumi; Taniguchi, Shun’ichiro; Ito, Kensuke; Matsumura, Tomio; Okada, Nagisa; Sakaizawa, Takashi; Kobayashi, Akira; Takeoka, Michiko; Miyagawa, Shin-ichi

    2017-01-01

    ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy. PMID:28056049

  14. Involvement of nitric oxide during phthalocyanine (Pc4) photodynamic therapy-mediated apoptosis.

    PubMed

    Gupta, S; Ahmad, N; Mukhtar, H

    1998-05-01

    Photodynamic therapy (PDT), a new treatment modality, uses a combination of photosensitizing agent and visible light for the therapy of many solid malignancies. The hallmark of PDT is intracellular oxidative stress mediated by reactive oxygen species, which, through a cascade of events, results in a cell kill that induces apoptosis in some cells. To better understand the mechanism of apoptosis, we hypothesized the role of nitric oxide (NO), which is considered to be involved in a variety of physiological and pathological processes, during PDT. The model photosensitizer we have been working with is a silicon-phthalocyanine compound termed Pc4. Here, we investigated the involvement of NO during Pc4 PDT in PDT of apoptosis-resistant radiation-induced fibrosarcoma (RIF-1) cells and in PDT of apoptosis-sensitive human epidermoid carcinoma (A431) cells. Pc4 PDT resulted in a rapid increase in nitrite production in A431 cells, starting as early as 15 s post-PDT, and showed a progressive increase up to 15 min post-PDT. This increase in nitrite production was observed in cell lysates as well as in the cell culture medium. RIF-1 cells did not show an increase in nitrite production in either the cell lysates or the culture medium. At this time, a majority of the cells were viable. The Western blot analysis also showed a rapid increase in the expression of the constitutive form of NO synthase as early as 15 s post-PDT when compared to that of the controls. This response showed a dose dependency up to 5 min after Pc4 PDT. This observation was confirmed by a [3H]L-citrulline assay, which also showed a similar pattern for constitutive NO-synthase activity. RIF-1 cells did not show any change in protein expression or enzyme activity after the same treatment. These data, for the first time, demonstrate the generation of NO during PDT and suggest that it may be involved in PDT-mediated apoptosis. This may have relevance in improving the therapeutic efficacy of PDT using

  15. Apoptosis Inhibitor 5 Increases Metastasis via Erk-mediated MMP expression.

    PubMed

    Song, Kwon-Ho; Kim, Seok-Ho; Noh, Kyung Hee; Bae, Hyun Cheol; Kim, Jin Hee; Lee, Hyo-Jung; Song, Jinhoi; Kang, Tae Heung; Kim, Dong-Wan; Oh, Se-Jin; Jeon, Ju-Hong; Kim, Tae Woo

    2015-06-01

    Apoptosis inhibitor 5 (API5) has recently been identified as a tumor metastasis-regulating gene in cervical cancer cells. However, the precise mechanism of action for API5 is poorly understood. Here, we show that API5 increases the metastatic capacity of cervical cancer cells in vitro and in vivo via up-regulation of MMP-9. Interestingly, API5-mediated metastasis was strongly dependent on the Erk signaling pathway. Conversely, knock-down of API5 via siRNA technology decreased the level of phospho-Erk, the activity of the MMPs, in vitro invasion, and in vivo pulmonary metastasis. Moreover, the Erk-mediated metastatic action was abolished by the mutation of leucine into arginine within the heptad leucine repeat region, which affects protein-protein interactions. Thus, API5 increases the metastatic capacity of tumor cells by up-regulating MMP levels via activation of the Erk signaling pathway.

  16. Aeromonas hydrophila induced head kidney macrophage apoptosis in Clarias batrachus involves the activation of calpain and is caspase-3 mediated.

    PubMed

    Banerjee, Chaitali; Goswami, Ramansu; Verma, Gaurav; Datta, Malabika; Mazumder, Shibnath

    2012-07-01

    The mechanism of macrophage cytotoxicity induced by Aeromonas hydrophila is yet unresolved. We observed A. hydrophila induces Head Kidney Macrophage (HKM) apoptosis in Clarias batrachus, as evident from Hoechst 33342 and AnnexinV-Propidium Iodide staining and presence of oligonucleosomal DNA ladder. Initiation of apoptosis required the bacteria to be alive, be actively phagocytosed into HKM and was dependent on host proteins. Elevated cytosolic calcium and consequent calpain activity that declined following pre-incubation with EGTA, verapamil and nifedipine implicates the role of calcium influx through voltage gated calcium channels and calpain in A. hydrophila-induced HKM apoptosis. Though, calpain-1 and -2 were involved, calpain-2 appeared to be more important in the process. EGTA, verapamil, nifedipine and calpain-2 inhibitor reduced caspase-3 activity and apoptosis. We conclude that A. hydrophila alters cytosolic calcium homeostasis initiating the activation of calpains, more specifically calpain-2, which leads to caspase-3 mediated HKM apoptosis in C. batrachus.

  17. DNA-PK Mediates AKT Activation and Apoptosis Inhibition in Clinically Acquired Platinum Resistance12

    PubMed Central

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-01-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  18. Compound 21 and Telmisartan combination mitigates type 2 diabetic nephropathy through amelioration of caspase mediated apoptosis.

    PubMed

    Pandey, Anuradha; Gaikwad, Anil Bhanudas

    2017-06-10

    The current study aimed to understand the role of novel, highly selective, orally active, non-peptide Angiotensin II type 2 receptor (AT2R) agonist, Compound 21 and its potential additive effect with Telmisartan on apoptosis and underlying posttranslational modifications in a non-genetic murine model for type 2 diabetic nephropathy (T2DN). An experimental model for T2DN was developed by administering low dose Streptozotocin in high fat diet fed male Wistar rats, followed by their treatment with Telmisartan, C21 or their combination. Our results demonstrated that C21 and Telmisartan combination attenuated metabolic and renal dysfunction, renal morphological and micro-architectural aberrations and hemodynamic disturbances in type 2 diabetic rats. The anti-apoptotic and anti-inflammatory effects of Telmisartan were significantly accentuated by C21 indicated by expression of apoptotic markers (Parp1, Caspase 8, Caspase 7, cleaved PARP and cleaved Caspase 3) and NF-κB mediated inflammatory molecules like interleukin 6, tumour necrosis factor alpha; monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1. C21 was found to improve Telmisartan mediated reversal of histone H3 acetylation at lysine 14 and 27 and expression of histone acetyl transferase, p300/CBP-associated factor also known to regulate NF-κB activity and DNA damage response. C21 in combination with Telmisartan markedly mitigates caspase mediated apoptosis and NF-κB signalling in T2D kidney, which could be partially attributed to its influence on PCAF mediated histone H3 acetylation. Hence further research should be done to develop this combination to treat T2DN. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Induction of Mitochondria Mediated Apoptosis in Human Breast Cancer Cells (T-47D) by Annona reticulata L. Leaves Methanolic Extracts.

    PubMed

    Roham, Pratiksha H; Kharat, Kiran R; Mungde, Priyanka; Jadhav, Mahadev A; Makhija, Surinder J

    2016-01-01

    Annona reticulata Linn. (Common name: Bullock's-heart) (Annonaceae family) is a semi-evergreen and small deciduous tree. The extracts of various parts of Annona reticulata L. have been reported as cytotoxic to many cancer cells. Annona reticulata L. leaves' methanolic extract (ARME) was prepared and used against the breast cancer cells. The breast cancer cells (T-47D) viability and IC50 were evaluated by Vybrant® MTT Cell Proliferation Assay Kit. Detection of phosphatidylserine on membranes of apoptotic cells was done by Attune flow cytometer. RNA transcripts were quantified in ARME treated and untreated cells. Finally, the Vybrant® FAM Poly Caspases assay kit was used for analysis of polycaspases activity in T-47D cells. The IC50 (5 ± 0.5 µg/mL) of the ARME was found against breast cancer cells (T-47D). The Paclitaxel was used as a control standard drug for the study. The downregulation of Bcl-2 and upregulation of Bax and Bak, and caspases activation suggested induction of apoptosis in T-47D cells by ARME through mitochondrial pathway. The cell cycle halted at G2/M phase in the ARME treated cells. The ARME was found to be effective against Breast cancer cells (T-47D).

  20. AMPK/mTOR-mediated inhibition of survivin partly contributes to metformin-induced apoptosis in human gastric cancer cell.

    PubMed

    Han, Gang; Gong, Hangjun; Wang, Yidong; Guo, Shaowen; Liu, Kun

    2015-01-01

    Recent studies demonstrated that metformin exerts anti-neoplastic effect in a spectrum of malignancies. However, the mechanism whereby metformin affects various cancers, including gastric cancer, is poorly elucidated. Considering apoptosis plays critical role in tumorigenesis, we, in the present study, investigated the in vitro apoptotic effect of metformin on human gastric cancer cell and the underlying mechanism. Three differently-differentiated gastric cancer cell lines, MKN-28, SGC-7901 and BGC-823, along with one noncancerous gastric cell line GES-1 were used. We found that metformin treatment selectively induces apoptosis in the 3 cancer cell lines, but not the noncancerous one, as confirmed by flow cytometry, Caspase-Glo assay and western blotting against PARP and cleaved caspase 3. Moreover, the apoptotic effect of metformin seems to correlate negatively with the differentiation degree of gastric cancer. Metformin-induced apoptosis may be partially mediated through inhibition of anti-apoptotic survivin. Additionally, AMPK and mTOR, 2 important regulatory molecules responsible for metformin action, were investigated for their possible involvements in metformin-induced apoptosis of gastric cancer cell. AMPK knockdown by siRNA restores metformin-inhibited survivin expression and partially abolishes metformin-induced apoptosis. Similarly, forced overexpression of mTOR downstream effector p70S6K1 relieves metformin-induced inhibition of survivin and partly attenuates metformin-induced apoptosis. More importantly, survivin overexpression alleviates metformin-induced apoptosis. Xenograft nude mouse experiment also confirmed that AMPK/mTOR-mediated decrease of suvivin is in vivo implicated in metformin-induced apoptosis. Taken together, these evidences suggest that AMPK/mTOR-mediated inhibition of survivin may partly contribute to metformin-induced apoptosis of gastric cancer cell.

  1. AMPK/mTOR-mediated inhibition of survivin partly contributes to metformin-induced apoptosis in human gastric cancer cell

    PubMed Central

    Han, Gang; Gong, Hangjun; Wang, Yidong; Guo, Shaowen; Liu, Kun

    2015-01-01

    Recent studies demonstrated that metformin exerts anti-neoplastic effect in a spectrum of malignancies. However, the mechanism whereby metformin affects various cancers, including gastric cancer, is poorly elucidated. Considering apoptosis plays critical role in tumorigenesis, we, in the present study, investigated the in vitro apoptotic effect of metformin on human gastric cancer cell and the underlying mechanism. Three differently-differentiated gastric cancer cell lines, MKN-28, SGC-7901 and BGC-823, along with one noncancerous gastric cell line GES-1 were used. We found that metformin treatment selectively induces apoptosis in the 3 cancer cell lines, but not the noncancerous one, as confirmed by flow cytometry, Caspase-Glo assay and western blotting against PARP and cleaved caspase 3. Moreover, the apoptotic effect of metformin seems to correlate negatively with the differentiation degree of gastric cancer. Metformin-induced apoptosis may be partially mediated through inhibition of anti-apoptotic survivin. Additionally, AMPK and mTOR, 2 important regulatory molecules responsible for metformin action, were investigated for their possible involvements in metformin-induced apoptosis of gastric cancer cell. AMPK knockdown by siRNA restores metformin-inhibited survivin expression and partially abolishes metformin-induced apoptosis. Similarly, forced overexpression of mTOR downstream effector p70S6K1 relieves metformin-induced inhibition of survivin and partly attenuates metformin-induced apoptosis. More importantly, survivin overexpression alleviates metformin-induced apoptosis. Xenograft nude mouse experiment also confirmed that AMPK/mTOR-mediated decrease of suvivin is in vivo implicated in metformin-induced apoptosis. Taken together, these evidences suggest that AMPK/mTOR-mediated inhibition of survivin may partly contribute to metformin-induced apoptosis of gastric cancer cell. PMID:25456211

  2. Role of apoptosis in mediating diclofenac-induced teratogenesis: An in vitro approach.

    PubMed

    Singh, Gyanendra; Maurya, Ranjeeta; Kumar, Akhilesh; Sinha, Neeraj

    2015-07-01

    Diclofenac (DCF) is among the most commonly used nonsteroidal anti-inflammatory drugs worldwide for the treatment of various conditions in postpubertal women. However, very limited information is available regarding its safety during pregnancy and teratogenecity. The present study was designed to elucidate the effects of DCF on the developing rat embryos during the major organogenesis period and investigate the critical role of apoptosis in bringing about these congenital anomalies. Embryos were exposed in vitro to various concentrations of DCF, that is, 0, 3.75, 7.5 and 15 µg/ml for 24 h, respectively, and examined for the growth and differentiation at the end of the culture period for the presence of any specific malformations. Growth and developmental parameters such as weight of embryos, crown-rump length and number of somites were found to be lower in the embryos exposed to high concentrations of DCF (7.5 and 15.0 μg/ml) when compared with the untreated control. However, no significant difference in growth parameters was found between embryos exposed to 3.75 µg/ml and the control group. In parallel to this, flow cytometric analysis and DNA quantitation of cultured rat embryos were performed to verify the involvement of apoptosis in mediating DCF-induced teratogenesis. A concentration-dependent increase in apoptosis in embryos suggests a possible engagement of apoptosis in the role of DCF as a teratogenic agent. A detailed analysis of the actual effect of DCF on cellular apoptotic machinery necessitates further evaluation.

  3. IRF8 Regulates Acid Ceramidase Expression to Mediate Apoptosis and Suppresses Myelogeneous Leukemia

    PubMed Central

    Hu, Xiaolin; Yang, Dafeng; Zimmerman, Mary; Liu, Feiyan; Yang, Jine; Kannan, Swati; Burchert, Andreas; Szulc, Zdzislaw; Bielawska, Alicja; Ozato, Keiko; Bhalla, Kapil; Liu, Kebin

    2011-01-01

    IFN regulatory factor 8 (IRF8) is a key transcription factor for myeloid cell differentiation and its expression is frequently lost in hematopoietic cells of human myeloid leukemia patients. IRF8-deficient mice exhibit uncontrolled clonal expansion of undifferentiated myeloid cells that can progress to a fatal blast crisis, thereby resembling human chronic myelogeneous leukemia (CML). Therefore, IRF8 is a myeloid leukemia suppressor. While the understanding of IRF8 function in CML has recently improved, the molecular mechanisms underlying IRF8 function in CML is still largely unknown. In this study, we identified acid ceramidase (A-CDase) as a general transcription target of IRF8. We demonstrated that IRF8 expression is regulated by IRF8 promoter DNA methylation in myeloid leukemia cells. Restoration of IRF8 expression repressed A-CDase expression, resulting in C16 ceramide accumulation and increased sensitivity of CML cells to FasL-induced apoptosis. In myeloid cells derived from IRF8-deficient mice, A-CDase protein level was dramatically increased. Furthermore, we demonstrated that IRF8 directly bind to the A-CDase promoter. At the functional level, inhibition of A-CDase activity, silencing A-CDase expression or application of exogenous C16 ceramide sensitized CML cells to FasL-induced apoptosis, whereas, overexpression of A-CDase decreased CML cells sensitivity to FasL-induced apoptosis. Consequently, restoration of IRF8 expression suppressed CML development in vivo at least partially through a Fas-dependent mechanism. In summary, our findings determine the mechanism of IRF8 downregulation in CML cells and they determine a primary pathway of resistance to Fas-mediated apoptosis and disease progression. PMID:21487040

  4. Proteomic evaluation of pathways associated with dexamethasone-mediated apoptosis and resistance in multiple myeloma.

    PubMed

    Rees-Unwin, Karen S; Craven, Rachel A; Davenport, Emma; Hanrahan, Sarah; Totty, Nicholas F; Dring, Ann M; Banks, Rosamonde E; J Morgan, Gareth; Davies, Faith E

    2007-11-01

    We have used global protein expression analysis to characterize the pathways of dexamethasone-mediated apoptosis and resistance in myeloma. Analysis of MM.1S cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) identified a series of proteins that were up- and downregulated following dexamethasone treatment. Downregulated proteins included proteins involved in cell survival and proliferation, whereas upregulated proteins were involved in post-translational modification, protein folding and trafficking. A comparison with published gene expression studies identified FK binding protein 5 (FKBP5) (also known as FKBP51), a key regulatory component of the Hsp90-steroid-receptor complex to be increased at the mRNA and protein level postdexamethasone exposure. Quantitative real time polymerase chain reaction and 2D-PAGE analysis of the dexamethasone resistant cell line MM.1R demonstrated no increase in FKBP5, consistent with its association with dexamethasone-mediated apoptosis. Western blot analysis of FKBP5 and other members of the Hsp90-receptor complex showed an increase in FKBP5 whilst FKBP4 (also known as FKBP52) and Hsp90 expression remained constant. No changes were observed in MM.1R. In conclusion, we demonstrated that following steroid receptor signalling, the cell carries out a number of adaptive responses prior to cell death. Interfering with these adaptive responses may enhance the myeloma killing effect of dexamethasone.

  5. Deacetylation of Ku70 by SIRT6 attenuates Bax-mediated apoptosis in hepatocellular carcinoma.

    PubMed

    Tao, Na-Na; Ren, Ji-Hua; Tang, Hua; Ran, Long-Kuan; Zhou, Hong-Zhong; Liu, Bo; Huang, Ai-Long; Chen, Juan

    2017-04-15

    SIRT6 is a class III histone deacetylase that has been implicated in HCC development. We previously reported that SIRT6 potentiated apoptosis evasion in hepatocellular carcinoma by inhibiting both Bax expression and mitochondrial translocalization. However, the mechanism underlying SIRT6-mediated inhibition of Bax mitochondrial localization remains elusive. In this study, we found that although SIRT6 had no effect on the expression level of Ku70, SIRT6 could interact with Ku70 and deacetylate it. The increased acetylation of Ku70 in SIRT6-depleted cells disrupt its interaction with Bax, which finally resulted in Bax mitochondrial translocalization. Furthermore, lysine K542 on Ku70 was the target for deacetylation by SIRT6. Ku70(K542Q) mutation abolished suppression of association between Ku70 and Bax and caused redistribution of Bax to the cytosol in SIRT6-depleted cells. Finally, Ku70(K542Q) mutation could reversed the inhibition of growth and apoptosis promotion mediated by SIRT6 silencing. Together, our findings revealed SIRT6 could block the mitochondrial translocation of Bax and decrease the apoptotic ratio of HCC cells by deacetylation of Ku70. SIRT6 may serve as a promising target for developing targeted therapies for HCC in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Protein kinase C-δ isoform mediates lysosome labilization in DNA damage-induced apoptosis

    PubMed Central

    PARENT, NICOLAS; SCHERER, MAX; LIEBISCH, GERHARD; SCHMITZ, GERD; BERTRAND, RICHARD

    2013-01-01

    A lysosomal pathway, characterized by the partial rupture or labilization of lysosomal membranes (LLM) and cathepsin release into the cytosol, is evoked during the early events of 20-S-camptothecin lactone (CPT)-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. Recently, in a comparative proteomics analysis performed on highly-enriched lysosomal extracts, we identified proteins whose translocation to lysosomes correlated with LLM induction after CPT treatment, including protein kinase C-δ (PKC-δ). In this study, we show that the PKC-δ translocation to lysosomes is required for LLM, as silencing its expression with RNA interference or suppressing its activity with the inhibitor, rottlerin, prevents CPT-induced LLM. PKC-δ translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase in ceramide (CER) content in lysosomes. The accumulation of endogenous CER in lysosomes is a critical event for CPT-induced LLM as suppressing PKC-δ or ASM activity reduces both the CPT-mediated CER generation in lysosomes and CPT-induced LLM. These findings reveal a novel mechanism by which PKC-δ mediates ASM phosphorylation/activation and CER accumulation in lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment. PMID:21174057

  7. Endoplasmic reticulum stress-mediated hippocampal neuron apoptosis involved in diabetic cognitive impairment.

    PubMed

    Zhang, Xiaoming; Xu, Linhao; He, Daqiang; Ling, Shucai

    2013-01-01

    Poor management of DM causes cognitive impairment while the mechanism is still unconfirmed. The aim of the present study was to investigate the activation of C/EBP Homology Protein (CHOP), the prominent mediator of the endoplasmic reticulum (ER) stress-induced apoptosis under hyperglycemia. We employed streptozotocin- (STZ-) induced diabetic rats to explore the ability of learning and memory by the Morris water maze test. The ultrastructure of hippocampus in diabetic rats and cultured neurons in high glucose medium were observed by transmission electron microscopy and scanning electron microscopy. TUNEL staining was also performed to assess apoptotic cells while the expression of CHOP was assayed by immunohistochemistry and Western blot assay in these hippocampal neurons. Six weeks after diabetes induction, the escape latency increased and the average frequency in finding the platform decreased in diabetic rats (P < 0.05). The morphology of neuron and synaptic structure was impaired; the number of TUNEL-positive cells and the expression of CHOP in hippocampus of diabetic rats and high glucose medium cultured neurons were markedly altered (P < 0.05). The present results suggested that the CHOP-dependent endoplasmic reticulum (ER) stress-mediated apoptosis may be involved in hyperglycemia-induced hippocampal synapses and neurons impairment and promote the diabetic cognitive impairment.

  8. Anticancer activity of sesquiterpenoids extracted from Solanum lyratum via the induction of mitochondria-mediated apoptosis

    PubMed Central

    Chen, Min; Wu, Jian; Zhang, Xing-Xing; Wang, Qiong; Yan, Shi-Hai; Wang, Hai-Dan; Liu, Sheng-Lin; Zou, Xi

    2017-01-01

    Sesquiterpenoids are a major type of compound found in Solanum lyratum (S. lyratum). The present study aimed to investigate whether sesquiterpenoids from S. lyratum demonstrated cytotoxicity against the MCF-7, HCT-8, A-549, SGC-7901 and BEL-7402 cell lines, and the mechanism of solajiangxin H and lyratol D, which exhibited high cytotoxicity against SGC-7901 cells (half maximal inhibitory concentration, IC50=4.8 and 5.9 µg/ml), was associated with mitochondria-mediated apoptosis. The results of the Cell Counting Kit-8 assay indicated that 15 sesquiterpenoids had cytotoxicity against the aforementioned cultured cells. The results of DAPI staining and western blot analysis, used to study the anticancer mechanisms of solajiangxin H and lyratol D in SGC-7901 cells, suggested that solajiangxin H and lyratol D induced the apoptosis of SGC-7901 cells significantly (P<0.01), downregulated the expression of the antiapoptotic proteins B-cell lymphoma (Bcl)-2 and survivin, and upregulated the expression of the proapoptotic proteins Bcl-2-like protein 4, second mitochondria-derived activator of caspase, cleaved (c)-caspase-3 and c-caspase-9. The present study therefore demonstrated that 15 sesquiterpenoids from S. lyratum exhibited anticancer activity in MCF-7, HCT-8, A-549, SGC-7901 and BEL-7402 cells, and that the anticancer mechanisms of solajiangxin H and lyratol D may be associated with mitochondria-mediated apoptosis. Additionally, the present study provides evidence in support of the hypothesis that S. lyratum may be a promising candidate for the development of novel cancer therapies. PMID:28123569

  9. Eosinophil resistance to glucocorticoid-induced apoptosis is mediated by the transcription factor NFIL3.

    PubMed

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

    2016-04-01

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

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

    PubMed Central

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

    2016-01-01

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

  11. MTOR inhibition attenuates DNA damage and apoptosis through autophagy-mediated suppression of CREB1.

    PubMed

    Wang, Ying; Hu, Zhongdong; Liu, Zhibo; Chen, Rongrong; Peng, Haiyong; Guo, Jing; Chen, Xinxin; Zhang, Hongbing

    2013-12-01

    Hyperactivation of mechanistic target of rapamycin (MTOR) is a common feature of human cancers, and MTOR inhibitors, such as rapamycin, are thus becoming therapeutics in targeting certain cancers. However, rapamycin has also been found to compromise the efficacy of chemotherapeutics to cells with hyperactive MTOR. Here, we show that loss of TSC2 or PTEN enhanced etoposide-induced DNA damage and apoptosis, which was blunted by suppression of MTOR with either rapamycin or RNA interference. cAMP response element-binding protein 1 (CREB1), a nuclear transcription factor that regulates genes involved in survival and death, was positively regulated by MTOR in mouse embryonic fibroblasts (MEFs) and cancer cell lines. Silencing Creb1 expression with siRNA protected MTOR-hyperactive cells from DNA damage-induced apoptosis. Furthermore, loss of TSC2 or PTEN impaired either etoposide or nutrient starvation-induced autophagy, which in turn, leads to CREB1 hyperactivation. We further elucidated an inverse correlation between autophagy activity and CREB1 activity in the kidney tumor tissue obtained from a TSC patient and the mouse livers with hepatocyte-specific knockout of PTEN. CREB1 induced DNA damage and subsequent apoptosis in response to etoposide in autophagy-defective cells. Reactivation of CREB1 or inhibition of autophagy not only improved the efficacy of rapamycin but also alleviated MTOR inhibition-mediated chemoresistance. Therefore, autophagy suppression of CREB1 may underlie the MTOR inhibition-mediated chemoresistance. We suggest that inhibition of MTOR in combination with CREB1 activation may be used in the treatment of cancer caused by an abnormal PI3K-PTEN-AKT-TSC1/2-MTOR signaling pathway. CREB1 activators should potentiate the efficacy of chemotherapeutics in treatment of these cancers.

  12. Hibiscus anthocyanins-rich extract inhibited LDL oxidation and oxLDL-mediated macrophages apoptosis.

    PubMed

    Chang, Yun-Ching; Huang, Kai-Xun; Huang, An-Chung; Ho, Yung-Chyuan; Wang, Chau-Jong

    2006-07-01

    The oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathogenesis of atherosclerosis. Anti-oxidative reagents, which can effectively inhibit LDL oxidation, may prevent atherosclerosis via reducing early atherogenesis, and slowing down the progression to advance stages. As shown in previous studies Hibiscus sabdariffa L. is a natural plant containing a lot of pigments that was found to possess anti-oxidative of activity. Therefore, in this study, we evaluated the anti-oxidative activity of Hibiscus anthocyanins (HAs) by measuring their effects on LDL oxidation (in cell-free system) and anti-apoptotic abilities (in RAW264.7 cells). HAs have been tested in vitro examining their relative electrophoretic mobility (REM), Apo B fragmentation, thiobarbituric acid relative substances (TBARS) and radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity assay. The anti-oxidative activity of HAs was defined by relative electrophoretic mobility of oxLDL (decrease of 50% at 2 mg/ml), fragmentation of Apo B (inhibition of 61% at 1mg/ml), and TBARS assay (IC(50): 0.46 mg/ml) in the Cu(2+)-mediated oxidize LDL. Furthermore, the addition of >0.1 mg/ml of HAs could scavenge over 95% of free DPPH radicals, HAs showed strong potential in inhibiting LDL oxidation induced by copper. In addition, to determine whether oxLDL-induced apoptosis in macrophages is inhibited by HAs, we studied the viability, morphology and caspase-3 expression of RAW 264.7 cells. MTT assay, Leukostate staining analysis and Western blotting reveals that HAs could inhibit oxLDL-induced apoptosis. According to these findings, we suggest that HAs may be used to inhibit LDL oxidation and oxLDL-mediated macrophage apoptosis, serving as a chemopreventive agent. However, further investigations into the specificity and mechanism(s) of HAs are needed.

  13. Mitochondrial mediated thimerosal-induced apoptosis in a human neuroblastoma cell line (SK-N-SH).

    PubMed

    Humphrey, Michelle L; Cole, Marsha P; Pendergrass, James C; Kiningham, Kinsley K

    2005-06-01

    Environmental exposure to mercurials continues to be a public health issue due to their deleterious effects on immune, renal and neurological function. Recently the safety of thimerosal, an ethyl mercury-containing preservative used in vaccines, has been questioned due to exposure of infants during immunization. Mercurials have been reported to cause apoptosis in cultured neurons; however, the signaling pathways resulting in cell death have not been well characterized. Therefore, the objective of this study was to identify the mode of cell death in an in vitro model of thimerosal-induced neurotoxicity, and more specifically, to elucidate signaling pathways which might serve as pharmacological targets. Within 2 h of thimerosal exposure (5 microM) to the human neuroblastoma cell line, SK-N-SH, morphological changes, including membrane alterations and cell shrinkage, were observed. Cell viability, assessed by measurement of lactate dehydrogenase (LDH) activity in the medium, as well as the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, showed a time- and concentration-dependent decrease in cell survival upon thimerosal exposure. In cells treated for 24 h with thimerosal, fluorescence microscopy indicated cells undergoing both apoptosis and oncosis/necrosis. To identify the apoptotic pathway associated with thimerosal-mediated cell death, we first evaluated the mitochondrial cascade, as both inorganic and organic mercurials have been reported to accumulate in the organelle. Cytochrome c was shown to leak from the mitochondria, followed by caspase 9 cleavage within 8 h of treatment. In addition, poly(ADP-ribose) polymerase (PARP) was cleaved to form a 85 kDa fragment following maximal caspase 3 activation at 24 h. Taken together these findings suggest deleterious effects on the cytoarchitecture by thimerosal and initiation of mitochondrial-mediated apoptosis.

  14. G Protein Inactivator RGS6 Mediates Myocardial Cell Apoptosis and Cardiomyopathy Caused by Doxorubicin

    PubMed Central

    Yang, Jianqi; Maity, Biswanath; Huang, Jie; Gao, Zhan; Stewart, Adele; Weiss, Robert M.; Anderson, Mark E.; Fisher, Rory A.

    2013-01-01

    Clinical use of the widely used chemotherapeutic agent doxorubicin is limited by life-threatening cardiotoxicity. The mechanisms underlying Dox-induced cardiomyopathy and heart failure remain unclear, but are thought to involve p53-mediated myocardial cell apoptosis. The tripartite G protein inactivating protein RGS6 has been implicated in reactive oxygen species (ROS) generation, ATM/p53 activation and apoptosis in Dox-treated cells. Thus, we hypothesized that RGS6, the expression of which is enriched in cardiac tissue, might also be responsible for the pathological effects of Dox treatment in heart. In this study, we show that RGS6 expression is induced strongly by Dox in the ventricles of mice and isolated ventricular myocytes (VCM) via a post-transcriptional mechanism. While Dox-treated wild type (WT) mice manifested severe left ventricular dysfunction, loss of heart and body mass, along with decreased survival five days after Dox administration, mice lacking RGS6 were completely protected against these pathogenic responses. Activation of ATM/p53-apoptosis signaling by Dox in ventricles of WT mice was also absent in their RGS6−/− counterparts. Dox-induced ROS generation was dramatically impaired in both the ventricles and VCM isolated from RGS6−/− mice, and the apoptotic response to Dox in VCM required RGS6-dependent ROS production. These results identify RGS6 as an essential mediator of the pathogenic responses to Dox in heart, and they argue that RGS6 inhibition offers a rational means to circumvent Dox cardiotoxicity in human cancer patients. PMID:23338613

  15. Oncogenic mutations in intestinal adenomas regulate Bim-mediated apoptosis induced by TGF-β

    PubMed Central

    Wiener, Zoltán; Band, Arja M.; Kallio, Pauliina; Högström, Jenny; Hyvönen, Ville; Kaijalainen, Seppo; Ritvos, Olli; Haglund, Caj; Kruuna, Olli; Robine, Sylvie; Louvard, Daniel; Ben-Neriah, Yinon; Alitalo, Kari

    2014-01-01

    In the majority of microsatellite-stable colorectal cancers (CRCs), an initiating mutation occurs in the adenomatous polyposis coli (APC) or β-catenin gene, activating the β-catenin/TCF pathway. The progression of resulting adenomas is associated with oncogenic activation of KRas and inactivation of the p53 and TGF-β/Smad functions. Most established CRC cell lines contain mutations in the TGF-β/Smad pathway, but little is known about the function of TGF-β in the early phases of intestinal tumorigenesis. We used mouse and human ex vivo 3D intestinal organoid cultures and in vivo mouse models to study the effect of TGF-β on the Lgr5+ intestinal stem cells and their progeny in intestinal adenomas. We found that the TGF-β–induced apoptosis in Apc-mutant organoids, including the Lgr5+ stem cells, was mediated by up-regulation of the BH3-only proapoptotic protein Bcl-2–like protein 11 (Bim). BH3-mimetic compounds recapitulated the effect of Bim not only in the adenomas but also in human CRC organoids that had lost responsiveness to TGF-β–induced apoptosis. However, wild-type intestinal crypts were markedly less sensitive to TGF-β than Apc-mutant adenomas, whereas the KRas oncogene increased resistance to TGF-β via the activation of the Erk1/2 kinase pathway, leading to Bim down-regulation. Our studies identify Bim as a critical mediator of TGF-β–induced apoptosis in intestinal adenomas and show that the common progression mutations modify Bim levels and sensitivity to TGF-β during intestinal adenoma development. PMID:24825889

  16. Plasminogen activator inhibitor-1 impairs plasminogen activation-mediated vascular smooth muscle cell apoptosis

    PubMed Central

    Rossignol, Patrick; Angles-Cano, Eduardo; Lijnen, Henri Roger

    2006-01-01

    SUMMARY The role of plasminogen activator inhibitor-1 (PAI-1) in vascular smooth muscle cell (VSMC) apoptosis mediated by plasminogen activation was studied with the use of aortic VSMC derived from mice with deficiency of PAI-1 (PAI-1−/−), tissue-type (t-PA−/−) or urokinase-type (u-PA−/−) plasminogen activator or from wild-type (WT) mice with corresponding genetic background. Plasminogen incubated with confluent VSMC was activated in a concentration-dependent and saturable manner for all 4 cell types, with maximal activation rates that were comparable for WT, u-PA−/− and t-PA−/− cells, but about 2- fold higher for PAI-1−/− cells. Plasminogen activation was impaired by addition of the lysine analogue 6-aminohexanoic acid, and by addition of t-PA and u-PA neutralizing antibodies, suggesting that it depends on binding to cell surface COOH-terminal lysine residues, and on plasminogen activator activity. Morphological alterations consistent with apoptosis were observed much earlier in PAI-1−/− than in WT VSMC. Without addition of plasminogen, the apoptotic index was similar for all 4 cell types, whereas after incubation with physiological plasminogen concentrations, it was greater in PAI-1−/− VSMC, as compared to WT, t-PA−/− or u-PA−/− VSMC. Furthermore, the apoptotic rate paralleled the release of plasmin. Thus, plasmin-mediated apoptosis of VSMC occurs via plasminogen activation by either t-PA or u-PA and is impaired by PAI-1. PMID:17080225

  17. The BIR Motifs Mediate Dominant Interference and Oligomerization of Inhibitor of Apoptosis Op-IAP

    PubMed Central

    Hozak, Rebecca R.; Manji, Gulam A.; Friesen, Paul D.

    2000-01-01

    The defining structural motif of the inhibitor of apoptosis (iap) protein family is the BIR (baculovirus iap repeat), a highly conserved zinc coordination domain of ∼70 residues. Although the BIR is required for inhibitor-of-apoptosis (IAP) function, including caspase inhibition, its molecular role in antiapoptotic activity in vivo is unknown. To define the function of the BIRs, we investigated the activity of these structural motifs within Op-IAP, an efficient, virus-derived IAP. We report here that Op-IAP1–216, a loss-of-function truncation which contains two BIRs but lacks the C-terminal RING motif, potently interfered with Op-IAP's capacity to block apoptosis induced by diverse stimuli. In contrast, Op-IAP1–216 had no effect on apoptotic suppression by caspase inhibitor P35. Consistent with a mechanism of dominant inhibition that involves direct interaction between Op-IAP1–216 and full-length Op-IAP, both proteins formed an immunoprecipitable complex in vivo. Op-IAP also self-associated. In contrast, the RING motif-containing truncation Op-IAP183–268 failed to interact with or interfere with Op-IAP function. Substitution of conserved residues within BIR 2 caused loss of dominant inhibition by Op-IAP1–216 and coincided with loss of interaction with Op-IAP. Thus, residues encompassing the BIRs mediate dominant inhibition and oligomerization of Op-IAP. Consistent with dominant interference by interaction with an endogenous cellular IAP, Op-IAP1–216 also lowered the survival threshold of cultured insect cells. Taken together, these data suggest a new model wherein the antiapoptotic function of IAP requires homo-oligomerization, which in turn mediates specific interactions with cellular apoptotic effectors. PMID:10669762

  18. Mitochondrial dysfunction as a mediator of hippocampal apoptosis in a model of hepatic encephalopathy.

    PubMed

    Bustamante, J; Lores-Arnaiz, S; Tallis, S; Roselló, D M; Lago, N; Lemberg, A; Boveris, A; Perazzo, J C

    2011-08-01

    In this study, we describe the presence of apoptosis, associated with a mitochondrial dysfunction in the hippocampus of animals in an experimental model defined as minimal hepatic encephalopathy (MHE). This experimental model was studied after 10 days of induced portal vein calibrated stricture, leading to portal hypertension and to a moderate hyperammonemia, without the presence of other evident central nervous system changes. The molecular mechanisms here proposed indicate the presence of apoptotic intrinsic pathways that point to hippocampal mitochondria as an important mediator of apoptosis in this experimental model. In this model of MHE, the presence of DNA fragmentation is documented by 2.3-times increased number of TUNEL-positive cells. These findings together with a higher ratio of the Bcl-2 family members Bax/Bcl-xL in the outer mitochondrial membrane of the MHE animals together with 11% of cytochrome c release indicate the presence of apoptosis in this experimental model. A detailed analysis of the hippocampal mitochondrial physiology was performed after mitochondrial isolation. The determination of the respiratory rate in the presence of malate plus glutamate and ADP showed a 45% decrease in respiratory control in MHE animals as compared with the sham group. A marked decrease of cytochrome oxidase (complex IV of the electron transport chain) was also observed, showing 46% less activity in hippocampal mitochondria from MHE animals. In addition, mitochondria from these animals showed less ability to maintain membrane potential (ΔΨ (m)) which was 13% lower than the sham group. Light scattering experiments showed that mitochondria from MHE animals were more sensitive to swell in the presence of increased calcium concentrations as compared with the sham group. In addition, in vitro studies performed in mitochondria from sham animals showed that mitochondrial permeability transition (MPT) could be a mitochondrial mediator of the apoptotic signaling in the

  19. RIPK1 promotes death receptor-independent caspase-8-mediated apoptosis under unresolved ER stress conditions

    PubMed Central

    Estornes, Y; Aguileta, M A; Dubuisson, C; De Keyser, J; Goossens, V; Kersse, K; Samali, A; Vandenabeele, P; Bertrand, M J M

    2014-01-01

    Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and results in the activation of the unfolded protein response (UPR), which aims at restoring ER homeostasis. However, when the stress is too severe the UPR switches from being a pro-survival response to a pro-death one, and the molecular mechanisms underlying ER stress-mediated death have remained incompletely understood. In this study, we identified receptor interacting protein kinase 1 (RIPK1)—a kinase at the crossroad between life and death downstream of various receptors—as a new regulator of ER stress-induced death. We found that Ripk1-deficient MEFs are protected from apoptosis induced by ER stressors, which is reflected by reduced caspase activation and PARP processing. Interestingly, the pro-apoptotic role of Ripk1 is independent of its kinase activity, is not regulated by its cIAP1/2-mediated ubiquitylation, and does not rely on the direct regulation of JNK or CHOP, two reportedly main players in ER stress-induced death. Instead, we found that ER stress-induced apoptosis in these cells relies on death receptor-independent activation of caspase-8, and identified Ripk1 upstream of caspase-8. However, in contrast to RIPK1-dependent apoptosis downstream of TNFR1, we did not find Ripk1 associated with caspase-8 in a death-inducing complex upon unresolved ER stress. Our data rather suggest that RIPK1 indirectly regulates caspase-8 activation, in part via interaction with the ER stress sensor inositol-requiring protein 1 (IRE1). PMID:25476903

  20. SGLT-1-mediated glucose uptake protects human intestinal epithelial cells against Giardia duodenalis-induced apoptosis.

    PubMed

    Yu, Linda C H; Huang, Ching-Ying; Kuo, Wei-Ting; Sayer, Heather; Turner, Jerrold R; Buret, Andre G

    2008-07-01

    Infection with Giardia duodenalis is one of the most common causes of waterborne diarrheal disease worldwide. Mechanisms of pathogenesis and host response in giardiasis remain incompletely understood. Previous studies have shown that exposure to G. duodenalis products induce apoptosis in enterocytes. We recently discovered that sodium-dependent glucose cotransporter (SGLT)-1-mediated glucose uptake modulates enterocytic cell death induced by bacterial lipopolysaccharide. The aim of this study was to examine whether enhanced epithelial SGLT-1 activity may constitute a novel mechanism of host defense against G. duodenalis-induced apoptosis. SGLT-1-transfected Caco-2 cells were exposed to G. duodenalis products in low (5mM) or high (25mM) glucose media. In low glucose environments, G. duodenalis-induced caspase-3 activation and DNA fragmentation in these cells. These apoptotic phenomena were abolished in the presence of high glucose. A soluble proteolytic fraction of G. duodenalis was found to upregulate SGLT-1-mediated glucose uptake in a dose- and time-dependent manner, in association with increased apical SGLT-1 expression on epithelial cells. Kinetic analysis showed that this phenomenon resulted from an increase in the maximal rate of sugar transport (V(max)) by SGLT-1, with no change in the affinity constant (K(m)). The addition of phloridzin (a competitive inhibitor for glucose binding to SGLT-1) abolished the anti-apoptotic effects exerted by high glucose. Together, the findings indicate that SGLT-1-dependent glucose uptake may represent a novel epithelial cell rescue mechanism against G. duodenalis-induced apoptosis.

  1. Differential Expression of Bcl-2 Family Proteins Determines the Sensitivity of Human Follicular Lymphoma Cells to Dexamethasone-mediated and Anti-BCR-mediated Apoptosis.

    PubMed

    Adem, Jemal; Ropponen, Antti; Eeva, Jonna; Eray, Mine; Nuutinen, Ulla; Pelkonen, Jukka

    2016-01-01

    Bcl-2 family comprises proapoptotic and antiapoptotic proteins. The balance between these proteins is critical for the survival of the cells. Overexpression of the antiapoptotic protein, Bcl-2, is the hallmark of follicular lymphoma (FL). High expression of Bcl-2 provides survival advantage and may facilitate chemotherapeutic resistance in FL. In the present study, we examined expression profile of Bcl-2 family proteins such as Bcl-2, Bcl-xL, and Bim in human FL cell lines, HF1A3 and HF28. We assessed the correlation between the expression levels of these proteins and cells' sensitivity to dexamethasone (Dex)-mediated and B-cell receptor (BCR)-mediated apoptosis. Here, we show that Dex and anti-BCR-induced synergistic apoptosis which correlated with significant downregulation of Bcl-xL, inhibition of ERK1/2 phosphorylation and accumulation of nonphosphorylated Bim. However, HF28 cells were less sensitive than HF1A3 cells to Dex-induced and anti-BCR-induced apoptosis due to high Bcl-2 protein level. It is interesting to note that, a Bcl-2-specific inhibitor, ABT-199, sensitized HF28 cells to Dex-induced or anti-BCR-induced apoptosis. In addition, overexpression of Bcl-xL prevented Dex-mediated, anti-BCR-mediated, and ABT-199-mediated apoptosis, indicating that mitochondria were involved. In conclusion, these data show that the expression levels of Bcl-2 family proteins may serve to predict tumor response to BH3 mimetics and the sensitivity of FL cells to Dex-induced and anti-BCR-induced apoptosis. Moreover, our results show that BCR-targeted apoptosis might have therapeutic benefit against FL and B-cell lymphomas.

  2. The interaction between HIV-1 Nef and adaptor protein-2 reduces Nef-mediated CD4(+) T cell apoptosis.

    PubMed

    Jacob, Rajesh Abraham; Johnson, Aaron L; Pawlak, Emily N; Dirk, Brennan S; Van Nynatten, Logan R; Haeryfar, S M Mansour; Dikeakos, Jimmy D

    2017-09-01

    Acquired Immune Deficiency Syndrome is characterized by a decline in CD4(+) T cells. Here, we elucidated the mechanism underlying apoptosis in Human Immunodeficiency Virus-1 (HIV-1) infection by examining host apoptotic pathways hijacked by the HIV-1 Nef protein in the CD4(+) T-cell line Sup-T1. Using a panel of Nef mutants unable to bind specific host proteins we uncovered that Nef generates pro- and anti-apoptotic signals. Apoptosis increased upon mutating the motifs involved in the interaction of Nef:AP-1 (NefM20A or NefEEEE62-65AAAA) or Nef:AP-2 (NefLL164/165AA), implying these interactions limit Nef-mediated apoptosis. In contrast, disrupting the Nef:PAK2 interaction motifs (NefH89A or NefF191A) reduced apoptosis. To validate further, apoptosis was measured after short-hairpin RNA knock-down of AP-1, AP-2 and PAK2. AP-2α depletion enhanced apoptosis, demonstrating that disrupting the Nef:AP-2α interaction limits Nef-mediated apoptosis. Collectively, we describe a mechanism by which HIV-1 regulates cell survival and demonstrate the consequence of interfering with Nef:host protein interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Daxx Upregulation within the Cytoplasm of Reovirus-Infected Cells Is Mediated by Interferon and Contributes to Apoptosis

    PubMed Central

    Dionne, Kalen R.; Zhuang, Yonghua; Leser, J. Smith; Tyler, Kenneth L.

    2013-01-01

    Reovirus infection is a well-characterized experimental system for the study of viral pathogenesis and antiviral immunity within the central nervous system (CNS). We have previously shown that c-Jun N-terminal kinase (JNK) and the Fas death receptor each play a role in neuronal apoptosis occurring in reovirus-infected brains. Death-associated protein 6 (Daxx) is a cellular protein that mechanistically links Fas signaling to JNK signaling in several models of apoptosis. In the present study, we demonstrate that Daxx is upregulated in reovirus-infected brain tissue through a type I interferon-mediated mechanism. Daxx upregulation is limited to brain regions that undergo reovirus-induced apoptosis and occurs in the cytoplasm and nucleus of neurons. Cytoplasmic Daxx is present in Fas-expressing cells during reovirus encephalitis, suggesting a role for Daxx in Fas-mediated apoptosis following reovirus infection. Further, in vitro expression of a dominant negative form of Daxx (DN-Daxx), which binds to Fas but which does not transmit downstream signaling, inhibits apoptosis of reovirus-infected cells. In contrast, in vitro depletion of Daxx results in increased expression of caspase 3 and apoptosis, suggesting that Daxx plays an antiapoptotic role in the nucleus. Overall, these data imply a regulatory role for Daxx in reovirus-induced apoptosis, depending on its location in the nucleus or cytoplasm. PMID:23302889

  4. 2,4-dichlorophenol induces ER stress-mediated apoptosis via eIF2α dephosphorylation in vitro.

    PubMed

    Zhang, Xiaoning; Zhang, Xiaona; Qi, Yongmei; Huang, Dejun; Zhang, Yingmei

    2016-02-01

    2,4-Dichlorophenol (2,4-DCP) has been widely used to produce herbicides and pharmaceutical intermediates, which exhibits various toxic effects including apoptosis. However, the mechanisms underlying 2,4-DCP-induced apoptosis, especially mediated by endoplasmic reticulum (ER) stress, are still unknown. In the present study, the mouse embryonic fibroblasts (MEFs) were used as an in vitro model system to figure out whether 2,4-DCP could induce ER stress, and further to elucidate the role of ER stress in 2,4-DCP-induced apoptosis. The results showed that 2,4-DCP dramatically caused the decrease of cell viability, the increase of apoptotic cells, the collapse of mitochondrial membrane potential (MMP) and the activation of caspase-3, suggesting that 2,4-DCP did induce apoptosis. Meanwhile, 2,4-DCP acted similarly as ER stress agonist tunicamycin (Tu) to activate all three branches (IRE1α, ATF6 and eIF2α) of ER stress. Furthermore, repression of ER stress or inhibition of eIF2α dephosphorylation significantly alleviated 2,4-DCP-induced apoptosis. Taking these results together, the present study firstly showed that 2,4-DCP induced ER stress-mediated apoptosis via eIF2α dephosphorylation in mammalian cells. These findings will provide new insights into the mechanisms underlying apoptosis after chlorophenols exposure.

  5. Dihydroartemisinin and its derivative induce apoptosis in acute myeloid leukemia through Noxa-mediated pathway requiring iron and endoperoxide moiety

    PubMed Central

    Zhao, Xuan; Zhong, Hang; Wang, Rui; Liu, Dan; Waxman, Samuel; Zhao, Linxiang; Jing, Yongkui

    2015-01-01

    Anti-apoptotic protein Mcl-1 plays an important role in protecting cell from death in acute myeloid leukemia (AML). The apoptosis blocking activity of Mcl-1 is inhibited by BH3-only protein Noxa. We found that dihydroartemisinin (DHA) and its derivative X-11 are potent apoptosis inducers in AML cells and act through a Noxa-mediate pathway; X-11 is four-fold more active than DHA. DHA and X-11-induced apoptosis is associated with induction of Noxa; apoptosis is blocked by silencing Noxa. DHA and X-11 induce Noxa expression by upregulating the transcription factor FOXO3a in a reactive oxygen species-mediated pathway. Interfering with the integrity of the endoperoxide moiety of DHA and X-11, as well as chelating intracellular iron with deferoxamine, diminish apoptosis and Noxa induction. AML cells expressing Bcl-xL, or with overexpression of Bcl-2, have decreased sensitivity to DHA and X-11-induced apoptosis which could be overcome by addition of Bcl-2/Bcl-xL inhibitor ABT-737. DHA and X-11 represent a new group of AML cells-apoptosis inducing compounds which work through Noxa up-regulation utilizing the specific endoperoxide moiety and intracellular iron. PMID:25714024

  6. Heat stress induces apoptosis through transcription-independent p53-mediated mitochondrial pathways in human umbilical vein endothelial cell.

    PubMed

    Gu, Z T; Wang, H; Li, L; Liu, Y S; Deng, X B; Huo, S F; Yuan, F F; Liu, Z F; Tong, H S; Su, L

    2014-03-26

    Cells apoptosis induced by intense heat stress is the prominent feature of heat-related illness. However, little is known about the biological effects of heat stress on cells apoptosis. Herein, we presented evidence that intense heat stress could induce early apoptosis of HUVEC cells through activating mitochondrial pathway with changes in mitochondrial membrane potential(ΔΨm), release of cytochrome c, and activation of caspase-9 and -3. We further revealed that p53 played a crucial role in heat stress-induced early apoptosis, with p53 protein rapidly translocated into mitochondria. Using pifithrin-α(PFT), a p53's mitochondrial translocation inhibitor, we found that pretreated with PFT, heat stress induced mitochondrial p53 translocation was significantly suppressed, accompanied by a significant alleviation in the loss of ΔΨm, cytochrome c release and caspase-9 activation. Furthermore, we also found that generation of reactive oxygen species (ROS) was a critical mediator in heat stress-induced apoptosis. In addition, the antioxidant MnTMPyP significantly decreased the heat stress-induced p53's mitochondrial translocation, followed by the loss of ΔΨm, cytochrome c release, caspase-9 activation and heat stress-mediated apoptosis. Conclusively, these findings indicate the contribution of the transcription-independent mitochondrial p53 pathway to early apoptosis in HUVEC cells induced by oxidative stress in response to intense heat stress.

  7. Activation of PI3K/Akt pathway limits JNK-mediated apoptosis during EV71 infection.

    PubMed

    Zhang, Hua; Li, Fengqi; Pan, Ziye; Wu, Zhijun; Wang, Yanhong; Cui, Yudong

    2014-11-04

    Apoptosis is frequently induced to inhibit virus replication during infection of Enterovirus 71 (EV71). On the contrary, anti-apoptotic pathway, such as PI3K/Akt pathway, is simultaneously exploited by EV71 to accomplish the viral life cycle. The relationship by which EV71-induced apoptosis and PI3K/Akt signaling pathway remains to be elucidated. In this study, we demonstrated that EV71 infection altered Bax conformation and triggered its redistribution from the cytosol to mitochondria in RD cells. Subsequently, cytochrome c was released from mitochondria to cytosol. We also found that c-Jun NH2-terminal kinase (JNK) was activated during EV71 infection. The JNK specific inhibitor significantly inhibited Bax activation and cytochrome c release, suggesting that EV71-induced apoptosis was involved into a JNK-dependent manner. Meanwhile, EV71-induced Akt phosphorylation involved a PI3K-dependent mechanism. Inhibition of the PI3K/Akt pathway enhanced JNK phosphorylation and the JNK-mediated apoptosis upon EV71 infection. Moreover, PI3K/Akt pathway phosphorylated apoptosis signal-regulating kinase 1 (ASK1) and negatively regulated the ASK1 activity. Knockdown of ASK1 significantly decreased JNK phosphorylation, which implied that ASK1 phosphorylation by Akt inhibited ASK1-mediated JNK activation. Collectively, these data reveal that activation of the PI3K/Akt pathway limits JNK-mediated apoptosis by phosphorylating and inactivating ASK1 during EV71 infection.

  8. Cx43 Mediates Resistance against MPP+-Induced Apoptosis in SH-SY5Y Neuroblastoma Cells via Modulating the Mitochondrial Apoptosis Pathway

    PubMed Central

    Kim, In-Su; Ganesan, Palanivel; Choi, Dong-Kug

    2016-01-01

    Neuronal apoptosis in the substantia nigra par compacta (SNpc) appears to play an essential role in the pathogenesis of Parkinson’s disease. However, the mechanisms responsible for the death of dopaminergic neurons are not fully understood yet. To explore the apoptotic mechanisms, we used a well-known parkinsonian toxin, 1-methyl-4-phenylpyridine (MPP+), to induce neuronal apoptosis in the human dopaminergic SH-SY5Y cell line. The most common method of interaction between cells is gap junctional intercellular communication (GJIC) mediated by gap junctions (GJs) formed by transmembrane proteins called connexins (Cx). Modulation of GJIC affects cell viability or growth, implying that GJIC may have an important role in maintaining homeostasis in various organs. Here, we hypothesized that increasing the level of the gap junction protein Cx43 in SH-SY5Y neuroblastoma cells could provide neuroprotection. First, our experiments demonstrated that knocking down Cx43 protein by using Cx43-specific shRNA in SH-SY5Y neuroblastoma cells potentiated MPP+-induced neuronal apoptosis evident from decreased cell viability. In another experiment, we demonstrated that over-expression of Cx43 in the SH-SY5Y cell system decreased MPP+-induced apoptosis based on the MTT assay and reduced the Bax/Bcl-2 ratio and the release of cytochrome C based on Western blot analysis. Taken together, our results suggest that Cx43 could mediate resistance against MPP+-induced apoptosis in SH-SY5Y neuroblastoma cells via modulating the mitochondrial apoptosis pathway. PMID:27809287

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

    PubMed

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

    2014-01-01

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

  10. Interdependence of Bad and Puma during Ionizing-Radiation-Induced Apoptosis

    PubMed Central

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

    2014-01-01

    Ionizing radiation (IR)-induced DNA double-strand breaks trigger an extensive cellular signaling response that involves the coordination of hundreds of proteins to regulate DNA repair, cell cycle arrest and apoptotic pathways. The cellular outcome often depends on the level of DNA damage as well as the particular cell type. Proliferating zebrafish embryonic neurons are highly sensitive to IR-induced apoptosis, and both p53 and its transcriptional target puma are essential mediators of the response. The BH3-only protein Puma has previously been reported to activate mitochondrial apoptosis through direct interaction with the pro-apoptotic Bcl-2 family proteins Bax and Bak, thus constituting the role of an “activator” BH3-only protein. This distinguishes it from BH3-only proteins like Bad that are thought to indirectly promote apoptosis through binding to anti-apoptotic Bcl-2 family members, thereby preventing the sequestration of activator BH3-only proteins and allowing them to directly interact with and activate Bax and Bak. We have shown previously that overexpression of the BH3-only protein Bad in zebrafish embryos supports normal embryonic development but greatly sensitizes developing neurons to IR-induced apoptosis. While Bad has previously been shown to play only a minor role in promoting IR-induced apoptosis of T cells in mice, we demonstrate that Bad is essential for robust IR-induced apoptosis in zebrafish embryonic neural tissue. Moreover, we found that both p53 and Puma are required for Bad-mediated radiosensitization in vivo. Our findings show the existence of a hierarchical interdependence between Bad and Puma whereby Bad functions as an essential sensitizer and Puma as an essential activator of IR-induced mitochondrial apoptosis specifically in embryonic neural tissue. PMID:24516599

  11. Mast cell chymase induces smooth muscle cell apoptosis by disrupting NF-{kappa}B-mediated survival signaling

    SciTech Connect

    Leskinen, Markus J.; Heikkilae, Hanna M.; Speer, Mei Y.; Hakala, Jukka K.; Laine, Mika; Kovanen, Petri T.; Lindstedt, Ken A. . E-mail: ken.lindstedt@wri.fi

    2006-05-01

    Chymase released from activated mast cells induces apoptosis of vascular smooth muscle cells (SMCs) in vitro by degrading the pericellular matrix component fibronectin, so causing disruption of focal adhesion complexes and Akt dephosphorylation, which are necessary for cell adhesion and survival. However, the molecular mechanisms of chymase-mediated apoptosis downstream of Akt have remained elusive. Here, we show by means of RT-PCR, Western blotting, EMSA, immunocytochemistry and confocal microscopy, that chymase induces SMC apoptosis by disrupting NF-{kappa}B-mediated survival signaling. Following chymase treatment, the translocation of active NF-{kappa}B/p65 to the nucleus was partly abolished and the amount of nuclear p65 was reduced. Pretreatment of SMCs with chymase also inhibited LPS- and IL-1{beta}-induced nuclear translocation of p65. The chymase-induced degradation of p65 was mediated by active caspases. Loss of NF-{kappa}B-mediated transactivation resulted in downregulation of bcl-2 mRNA and protein expression, leading to mitochondrial swelling and release of cytochrome c. The apoptotic process involved activation of both caspase 9 and caspase 8. The results reveal that, by disrupting the NF-{kappa}B-mediated survival-signaling pathway, activated chymase-secreting mast cells can mediate apoptosis of cultured arterial SMCs. Since activated mast cells colocalize with apoptotic SMCs in vulnerable areas of human atherosclerotic plaques, they may participate in the weakening and rupture of atherosclerotic plaques.

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

    PubMed

    Urresti, Jorge; Ruiz-Meana, Marisol; Coccia, Elena; Arévalo, Juan Carlos; Castellano, José; Fernández-Sanz, Celia; Galenkamp, Koen M O; Planells-Ferrer, Laura; Moubarak, Rana S; Llecha-Cano, Núria; Reix, Stéphanie; García-Dorado, David; Barneda-Zahonero, Bruna; Comella, Joan X

    2016-01-15

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

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

    PubMed Central

    Urresti, Jorge; Ruiz-Meana, Marisol; Coccia, Elena; Arévalo, Juan Carlos; Castellano, José; Fernández-Sanz, Celia; Galenkamp, Koen M. O.; Planells-Ferrer, Laura; Moubarak, Rana S.; Llecha-Cano, Núria; Reix, Stéphanie; García-Dorado, David; Barneda-Zahonero, Bruna; Comella, Joan X.

    2016-01-01

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

  14. MK-STYX, a catalytically inactive phosphatase regulating mitochondrially dependent apoptosis.

    PubMed

    Niemi, Natalie M; Lanning, Nathan J; Klomp, Jeff A; Tait, Stephen W; Xu, Yong; Dykema, Karl J; Murphy, Leon O; Gaither, L Alex; Xu, H Eric; Furge, Kyle A; Green, Douglas R; MacKeigan, Jeffrey P

    2011-04-01

    Evasion of apoptosis is a significant problem affecting an array of cancers. In order to identify novel regulators of apoptosis, we performed an RNA interference (RNAi) screen against all kinases and phosphatases in the human genome. We identified MK-STYX (STYXL1), a catalytically inactive phosphatase with homology to the mitogen-activated protein kinase (MAPK) phosphatases. Despite this homology, MK-STYX knockdown does not significantly regulate MAPK signaling in response to growth factors or apoptotic stimuli. Rather, RNAi-mediated knockdown of MK-STYX inhibits cells from undergoing apoptosis induced by cellular stressors activating mitochondrion-dependent apoptosis. This MK-STYX phenotype mimics the loss of Bax and Bak, two potent guardians of mitochondrial apoptotic potential. Similar to loss of both Bax and Bak, cells without MK-STYX expression are unable to release cytochrome c. Proapoptotic members of the BCL-2 family (Bax, Bid, and Bim) are unable to trigger cytochrome c release in MK-STYX-depleted cells, placing the apoptotic deficiency at the level of mitochondrial outer membrane permeabilization (MOMP). MK-STYX was found to localize to the mitochondria but is neither released from the mitochondria upon apoptotic stress nor proximal to the machinery currently known to control MOMP, indicating that MK-STYX regulates MOMP using a distinct mechanism.

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

    SciTech Connect

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

    2013-11-08

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

  16. Enrichment of Nur77 mediated by RARβ leads to apoptosis of human hepatocellular carcinoma cells induced by fenretinide and HDACi

    PubMed Central

    Yang, Hui; Zhan, Qi; Wan, Yu-Jui Yvonne

    2010-01-01

    Synthetic retinoid fenretinide is one of the most promising clinically tested retinoids. Previously, we have shown that fenretinide induces apoptosis of Huh7 cells, but HepG2 cells are relatively resistant to fenretinide-induced apoptosis. The current study examines the interactive role of fenretinide and histone deacetylase inhibitors (HDACi) in inducing apoptosis of human hepatocellular carcinoma (HCC) cells and the underlying mechanism. Trichostatin A (TSA) and scriptaid can either enhance fenretinide-induced apoptosis in the fenretinide sensitive HCC cells (Huh7 and Hep3B) or sensitize the fenretinide resistant cells (HepG2) to become sensitive to the apoptotic effect of fenretinide in a cancer-cell specific manner. The sensitivity of cells to fenretinide-induced apoptosis was neither associated with ROS production nor anti-oxidant gene expression. However, the level of RARβ and Nur77 was important for inducing apoptosis. Upon fenretinide and HDACi treatment, the expression of RARβ and Nur77 were induced and co-localized in the cytosol. The induction of Nur77 protein level, but not the mRNA level, was RARβ-dependent. In addition, RARβ interacted with Nur77. Nur77 was essential for fenretinide- and HDACi-induced apoptosis of Huh7 cells. Induction of the expression, the interaction, and the nuclear export of RARβ and Nur77 mediate fenretinide and HDACi induced apoptosis. Our findings suggest that targeting Nur77 and RARβ simultaneously provides an effective way to induce HCC cell death. PMID:21319187

  17. BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20-hydroxyecdysone or starvation

    PubMed Central

    Xie, Kun; Tian, Ling; Guo, Xinyu; Li, Kang; Li, Jianping; Deng, Xiaojuan; Li, Qingrong; Xia, Qingyou; Zhong, Yangjin; Huang, Zhijun; Liu, Jiping; Li, Sheng; Yang, Wanying; Cao, Yang

    2016-01-01

    ABSTRACT Autophagy and apoptosis, which could be induced by common stimuli, play crucial roles in development and disease. The functional relationship between autophagy and apoptosis is complex, due to the dual effects of autophagy. In the Bombyx Bm-12 cells, 20-hydroxyecdysone (20E) treatment or starvation-induced cell death, with autophagy preceding apoptosis. In response to 20E or starvation, BmATG8 was rapidly cleaved and conjugated with PE to form BmATG8–PE; subsequently, BmATG5 and BmATG6 were cleaved into BmATG5-tN and BmATG6-C, respectively. Reduction of expression of BmAtg5 or BmAtg6 by RNAi decreased the proportion of cells undergoing both autophagy and apoptosis after 20E treatment or starvation. Overexpression of BmAtg5 or BmAtg6 induced autophagy but not apoptosis in the absence of the stimuli, but promoted both autophagy and apoptosis induced by 20E or starvation. Notably, overexpression of cleavage site-deleted BmAtg5 or BmAtg6 increased autophagy but not apoptosis induced by 20E or starvation, whereas overexpression of BmAtg5-tN and BmAtg6-C was able to directly trigger apoptosis or promote the induced apoptosis. In conclusion, being cleaved into BmATG5-tN and BmATG6-C, BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20E or starvation in Bombyx Bm-12 cells, reflecting that autophagy precedes apoptosis in the midgut during Bombyx metamorphosis. PMID:26727186

  18. RAS signaling promotes resistance to JAK inhibitors by suppressing BAD-mediated apoptosis

    PubMed Central

    Winter, Peter S.; Sarosiek, Kristopher A.; Lin, Kevin H.; Meggendorfer, Manja; Schnittger, Susanne; Letai, Anthony

    2015-01-01

    Myeloproliferative neoplasms (MPNs) frequently have an activating mutation in the gene encoding Janus kinase 2 (JAK2). Thus, targeting the pathway mediated by JAK and its downstream substrate, signal transducer and activator of transcription (STAT), may yield clinical benefit for patients with MPNs containing the JAK2V617F mutation. Although JAK inhibitor therapy reduces splenomegaly and improves systemic symptoms in patients, this treatment does not appreciably reduce the number of neoplastic cells. To identify potential mechanisms underlying this inherent resistance phenomenon, we performed pathway-centric, gain-of-function screens in JAK2V617F hematopoietic cells and found that the activation of the guanosine triphosphatase (GTPase) RAS or its effector pathways (mediated by the kinases AKT and ERK) renders cells insensitive to JAK inhibition. Resistant MPN cells became sensitized to JAK inhibitors when also exposed to inhibitors of the AKT or ERK pathways. Mechanistically, in JAK2V617F cells a JAK2-mediated inactivating phosphorylation of the pro-apoptotic protein BAD [B-cell lymphoma 2 (BCL-2)-associated death promoter] promoted cell survival. In sensitive cells, exposure to a JAK inhibitor resulted in dephosphorylation of BAD, enabling BAD to bind and sequester the pro-survival protein BCL-XL (also known as BCL2-like 1), thereby triggering apoptosis. In resistant cells, RAS effector pathways maintained BAD phosphorylation in the presence of JAK inhibitors, yielding a specific dependence on BCL-XL for survival. BCL-XL inhibitors potently induced apoptosis in JAK inhibitor-resistant cells. In patients with MPNs, activating mutations in RAS co-occur with the JAK2V617F mutation in the malignant cells, suggesting that RAS effector pathways likely play an important role in clinically observed resistance. PMID:25538080

  19. ERECTA and BAK1 Receptor Like Kinases Interact to Regulate Immune Responses in Arabidopsis.

    PubMed

    Jordá, Lucía; Sopeña-Torres, Sara; Escudero, Viviana; Nuñez-Corcuera, Beatriz; Delgado-Cerezo, Magdalena; Torii, Keiko U; Molina, Antonio

    2016-01-01

    ERECTA (ER) receptor-like kinase (RLK) regulates Arabidopsis thaliana organ growth, and inflorescence and stomatal development by interacting with the ERECTA-family genes (ERf) paralogs, ER-like 1 (ERL1) and ERL2, and the receptor-like protein (RLP) TOO MANY MOUTHS (TMM). ER also controls immune responses and resistance to pathogens such as the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto) and the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM). We found that er null-mutant plants overexpressing an ER dominant-negative version lacking the cytoplasmic kinase domain (ERΔK) showed an enhanced susceptibility to PcBMM, suggesting that ERΔK associates and forms inactive complexes with additional RLKs/RLPs required for PcBMM resistance. Genetic analyses demonstrated that ER acts in a combinatorial specific manner with ERL1, ERL2, and TMM to control PcBMM resistance. Moreover, BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) RLK, which together with ERf/TMM regulates stomatal patterning and resistance to Pto, was also found to have an unequal contribution with ER in regulating immune responses and resistance to PcBMM. Co-immunoprecipitation experiments in Nicotiana benthamiana further demonstrated BAK1-ER protein interaction. The secreted epidermal pattern factor peptides (EPF1 and EPF2), which are perceived by ERf members to specify stomatal patterning, do not seem to regulate ER-mediated immunity to PcBMM, since their inducible overexpression in A. thaliana did not impact on PcBMM resistance. Our results indicate that the multiproteic receptorsome formed by ERf, TMM and BAK1 modulates A. thaliana resistance to PcBMM, and suggest that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering.

  20. ERECTA and BAK1 Receptor Like Kinases Interact to Regulate Immune Responses in Arabidopsis

    PubMed Central

    Jordá, Lucía; Sopeña-Torres, Sara; Escudero, Viviana; Nuñez-Corcuera, Beatriz; Delgado-Cerezo, Magdalena; Torii, Keiko U.; Molina, Antonio

    2016-01-01

    ERECTA (ER) receptor-like kinase (RLK) regulates Arabidopsis thaliana organ growth, and inflorescence and stomatal development by interacting with the ERECTA-family genes (ERf) paralogs, ER-like 1 (ERL1) and ERL2, and the receptor-like protein (RLP) TOO MANY MOUTHS (TMM). ER also controls immune responses and resistance to pathogens such as the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto) and the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM). We found that er null-mutant plants overexpressing an ER dominant-negative version lacking the cytoplasmic kinase domain (ERΔK) showed an enhanced susceptibility to PcBMM, suggesting that ERΔK associates and forms inactive complexes with additional RLKs/RLPs required for PcBMM resistance. Genetic analyses demonstrated that ER acts in a combinatorial specific manner with ERL1, ERL2, and TMM to control PcBMM resistance. Moreover, BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) RLK, which together with ERf/TMM regulates stomatal patterning and resistance to Pto, was also found to have an unequal contribution with ER in regulating immune responses and resistance to PcBMM. Co-immunoprecipitation experiments in Nicotiana benthamiana further demonstrated BAK1-ER protein interaction. The secreted epidermal pattern factor peptides (EPF1 and EPF2), which are perceived by ERf members to specify stomatal patterning, do not seem to regulate ER-mediated immunity to PcBMM, since their inducible overexpression in A. thaliana did not impact on PcBMM resistance. Our results indicate that the multiproteic receptorsome formed by ERf, TMM and BAK1 modulates A. thaliana resistance to PcBMM, and suggest that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering. PMID:27446127

  1. Inhibitor of Apoptosis Protein-1 Regulates Tumor Necrosis Factor-Mediated Destruction of Intestinal Epithelial Cells.

    PubMed

    Grabinger, Thomas; Bode, Konstantin J; Demgenski, Janine; Seitz, Carina; Delgado, M Eugenia; Kostadinova, Feodora; Reinhold, Cindy; Etemadi, Nima; Wilhelm, Sabine; Schweinlin, Matthias; Hänggi, Kay; Knop, Janin; Hauck, Christof; Walles, Heike; Silke, John; Wajant, Harald; Nachbur, Ueli; W Wei-Lynn, Wong; Brunner, Thomas

    2017-03-01

    Tumor necrosis factor (TNF) is a cytokine that promotes inflammation and contributes to pathogenesis of inflammatory bowel diseases. Unlike other cells and tissues, intestinal epithelial cells undergo rapid cell death upon exposure to TNF, by unclear mechanisms. We investigated the roles of inhibitor of apoptosis proteins (IAPs) in the regulation of TNF-induced cell death in the intestinal epithelium of mice and intestinal organoids. RNA from cell lines and tissues was analyzed by quantitative polymerase chain reaction, protein levels were analyzed by immunoblot assays. BIRC2 (also called cIAP1) was expressed upon induction from lentiviral vectors in young adult mouse colon (YAMC) cells. YAMC cells, the mouse colon carcinoma cell line MC38, the mouse macrophage cell line RAW 264.7, or mouse and human organoids were incubated with second mitochondrial activator of caspases (Smac)-mimetic compound LCL161 or recombinant TNF-like weak inducer of apoptosis (TNFSF12) along with TNF, and cell death was quantified. C57BL/6 mice with disruption of Xiap, Birc2 (encodes cIAP1), Birc3 (encodes cIAP2), Tnfrsf1a, or Tnfrsf1b (Tnfrsf1a and b encode TNF receptors) were injected with TNF or saline (control); liver and intestinal tissues were collected and analyzed for apoptosis induction by cleaved caspase 3 immunohistochemistry. We also measured levels of TNF and alanine aminotransferase in serum from mice. YAMC cells, and mouse and human intestinal organoids, died rapidly in response to TNF. YAMC and intestinal crypts expressed lower levels of XIAP, cIAP1, cIAP2, and cFLIP than liver tissue. Smac-mimetics reduced levels of cIAP1 and XIAP in MC38 and YAMC cells, and Smac-mimetics and TNF-related weak inducer of apoptosis increased TNF-induced cell death in YAMC cells and organoids-most likely by sequestering and degrading cIAP1. Injection of TNF greatly increased levels of cell death in intestinal tissue of cIAP1-null mice, compared with wild-type C57BL/6 mice, cIAP2-null mice, or

  2. Paclitaxel sensitivity of breast cancer cells requires efficient mitotic arrest and disruption of Bcl-xL/Bak interaction.

    PubMed

    Flores, M Luz; Castilla, Carolina; Ávila, Rainiero; Ruiz-Borrego, Manuel; Sáez, Carmen; Japón, Miguel A

    2012-06-01

    Taxanes are being used for the treatment of breast cancer. However, cancer cells frequently develop resistance to these drugs with the subsequent recurrence of the tumor. MDA-MB-231 and T-47D breast cancer cell lines were used to assess the effect of paclitaxel treatment on apoptosis and cell cycle, the possible mechanisms of paclitaxel resistance as well as the enhancement of paclitaxel-induced apoptosis based on its combination with phenylethyl isothiocyanate (PEITC). T-47D cells undergo apoptosis in response to paclitaxel treatment. The induction of apoptosis was associated with a robust mitotic arrest and the disruption of Bcl-xL/Bak interaction. By contrary, MDA-MB-231 cells were insensitive to paclitaxel-induced apoptosis and this was associated with a high percentage of cells that slip out of paclitaxel-imposed mitotic arrest and also with the maintenance of Bcl-xL/Bak interaction. The sequential treatment of MDA-MB-231 cells with PEITC followed by paclitaxel inhibited the slippage induced by paclitaxel and increased the apoptosis induction achieved with any of the drugs alone. In breast cancer tissues, high Bcl-xL expression was correlated with a shorter time of disease-free survival in patients treated with a chemotherapeutic regimen that contains paclitaxel, in a statistically significant way. Thus, resistance to paclitaxel in MDA-MB-231 cells is related to the inability to disrupt the Bcl-xL/Bak interaction and increased slippage. In this context, the combination of a drug that induces a strong mitotic arrest, such as paclitaxel, with another that inhibits slippage, such as PEITC, translates into increased apoptotic induction.

  3. Apoptotic Caspases Suppress mtDNA-Induced STING-Mediated Type I IFN Production

    PubMed Central

    McArthur, Kate; Metcalf, Donald; Lane, Rachael M.; Cambier, John C.; Herold, Marco J.; van Delft, Mark F.; Bedoui, Sammy; Lessene, Guillaume; Ritchie, Matthew E.; Huang, David C.S.

    2015-01-01

    SUMMARY Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-β. In vivo, this precipitates an elevation in IFN-β levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent. PMID:25525874

  4. Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production.

    PubMed

    White, Michael J; McArthur, Kate; Metcalf, Donald; Lane, Rachael M; Cambier, John C; Herold, Marco J; van Delft, Mark F; Bedoui, Sammy; Lessene, Guillaume; Ritchie, Matthew E; Huang, David C S; Kile, Benjamin T

    2014-12-18

    Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-β. In vivo, this precipitates an elevation in IFN-β levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.

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

    PubMed

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

    2012-06-01

    The present study investigated effects of apoptosis observed during seasonal testicular regression in Japanese Jungle Crows. The study was conducted during January to June 2008, 2009. Testes from adults captured during non-breeding (January), prebreeding (February to mid-March), main-breeding (late March to early May), transition (mid-May to late May), and post-breeding (June) seasons were analyzed. Apoptosis was assessed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Paired-testis volume increased 95-fold from the non-breeding to the main-breeding season (P < 0.05), and subsequently decreased 26-fold from the main breeding to the post-breeding season (P < 0.05). Testicular activity was evaluated from the total germ cell count and sperm index, which increased 42- and 5-fold, respectively, in the main-breeding season, and subsequently decreased 33- and 5-fold in the post-breeding season. In testes, TUNEL-positive germ cells were at low levels in the non-breeding season, absent in the prebreeding and the main-breeding seasons, and highest in mid-May (P < 0.05). In contrast, TUNEL-positive Sertoli cells occurred only in late-April. In addition, TUNEL-positive fibroblast-like cells were observed in the outer zone of the tunica albuginea in the post-breeding season. Collectively, these data suggested that the seasonal rise in the testicular competence occurred slowly in Japanese Jungle Crows; however, testis function was terminated rapidly after the breeding season. Furthermore, we concluded, similar to other avian species, Sertoli cell apoptosis followed by massive germ cell death was responsible for rapid testicular regression in Jungle Crows. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Photolon™ --photosensitization induces apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes.

    PubMed

    Ali-Seyed, Mohamed; Bhuvaneswari, Ramaswamy; Soo, Khee Chee; Olivo, Malini

    2011-10-01

    The localization of photosensitizers in the subcellular compartments during photodynamic therapy (PDT) plays a major role in the cell destruction; therefore, the aim of this study was to investigate the intracellular localization of Chlorin e6-PVP (Photolon™) in malignant and normal cells. Our study involves the characterization of the structural determinants of subcellular localization of Photolon, and how subcellular localization affects the selective toxicity of Photolon towards tumor cells. Using confocal laser scanning microscopy (CLSM) and fluorescent organelle probes; we examined the subcellular localization of Photolon™ in the murine colon carcinoma CT-26 and normal fibroblast (NHLC) cells. Our results demonstrated that after 30 min of incubation, the distribution of Photolon was localized mainly in the cytoplasmic organelles including the mitochondria, lysosomes, Golgi apparatus, around the nuclear envelope and also in the nucleus but not in the endo-plasmic reticulum whereas in NHLC cells, Photolon was found to be localized minimally only in the nucleus not in other organelles studied. The relationship between subcellular localization of Photolon and PDT-induced apoptosis was investigated. Apoptotic cell death was judged by the formation of known apoptotic hallmarks including, the phosphatidylserine externalization (PS), PARP cleavage, a substrate for caspase-3 and the formation of apoptotic nuclei. At the irradiation dose of 1 J/cm2, the percentage of apoptotic cells was 80%, respectively. This study provided substantial evidence that Photolon preferentially localized in the subcellular organelles in the following order: nucleus, mitochondria, lysosomes and the Golgi apparatus and subsequent photodamage of the mitochondria and lyso-somes played an important role in PDT-mediated apoptosis CT-26 cells. Our results based on the cytoplasmic organelles and the intranuclear localization extensively enhance the efficacy of PDT with appropriate

  7. Oxidative stress and cyclooxygenase-2 induction mediate cyanide-induced apoptosis of cortical cells.

    PubMed

    Li, L; Prabhakaran, K; Shou, Y; Borowitz, J L; Isom, G E

    2002-11-15

    Cyanide (KCN)-induced generation of reactive oxygen species (ROS) involves cyclooxygenase-2 (COX-2)-mediated reactions in some neurons. The present study examines the extent to which COX isoforms are involved in KCN-induced apoptotic cell death processes of cultured cortical cells. After treatment with KCN (10-300 microM), COX-2 was expressed in a time- and concentration-dependent manner increasing markedly over a 4-h period. However, no significant changes were observed in COX-1 levels at any cyanide concentration. Correlated with COX-2 up-regulation, KCN induced a time-dependent apoptotic death. TUNEL staining showed that the COX-2 inhibitor NS-398 (30 microM) blocked KCN-induced apoptosis, whereas the selective COX-1 inhibitor valeryl salicylate did not affect the level of apoptotic cell death. Exposure of cells to KCN (300 microM) for 24 h resulted in DNA fragmentation, which was also reduced by NS-398. Prostaglandin E(2) (PGE(2)) accumulation in cell culture supernatants was increased by KCN and NS-398 blocked PGE(2) generation. PCR studies further confirmed that COX-2 expression was increased by KCN. Antioxidants phenyl-N-test-butylnitrone, superoxide dismutase, and catalase significantly inhibited KCN-induced COX-2 up-regulation and subsequent apoptosis. N(G)-nitro-L-arginine methylester an inhibitor of nitric oxide synthase, blocked KCN-induced PGE(2) production and apoptosis, but not COX-2 expression. Increased nitric oxide levels caused by cyanide may directly activate the COX-2 enzyme. These data show that cyanide treatment of cortical cells involves increased COX-2 expression, PGE(2) accumulation, and ROS generation, resulting in apoptotic cell death.

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

    PubMed

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

    2012-04-01

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

  9. Neem (Azadirachta indica L.) leaf extract deteriorates oocyte quality by inducing ROS-mediated apoptosis in mammals.

    PubMed

    Chaube, Shail K; Shrivastav, Tulsidas G; Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ajai K

    2014-01-01

    Neem (Azadirachta indica L.) leaf has been widely used in ayurvedic system of medicine for fertility regulation for a long time. The molecular mechanism by which neem leaf regulates female fertility remains poorly understood. Animal studies suggest that aqueous neem leaf extract (NLE) induces reactive oxygen species (ROS) - mediated granulosa cell apoptosis. Granulosa cell apoptosis deprives oocytes from nutrients, survival factors and cell cycle proteins required for the achievement of meiotic competency of follicular oocytes prior to ovulation. Under this situation, follicular oocyte becomes more susceptible towards apoptosis after ovulation. The increased level of hydrogen peroxide (H2O2) inside the follicular fluid results in the transfer of H2O2 from follicular fluid to the oocyte. The increased level of H2O2 induces p53 activation and over expression of Bax protein that modulates mitochondrial membrane potential and trigger cytochrome c release. The increased cytosolic cytochrome c level induces caspase-9 and caspase-3 activities that trigger destruction of structural and specific proteins leading to DNA fragmentation and thereby oocyte apoptosis. Based on these animal studies, we propose that NLE induces generation of ROS and mitochondria-mediated apoptosis both in granulosa cells as well as in follicular oocyte. The induction of apoptosis deteriorates oocyte quality and thereby limits reproductive outcome in mammals.

  10. Histones-mediated lymphocyte apoptosis during sepsis is dependent on p38 phosphorylation and mitochondrial permeability transition.

    PubMed

    Liu, Zhan-Guo; Ni, Shu-Yuan; Chen, Gui-Ming; Cai, Jing; Guo, Zhen-Hui; Chang, Ping; Li, Yu-Sheng

    2013-01-01

    Lymphocyte apoptosis is one reason for immunoparalysis seen in sepsis, although the triggers are unknown. We hypothesized that molecules in plasma, which are up-regulated during sepsis, may be responsible for this. In this study, peripheral lymphocyte apoptosis caused by extracellular histones was confirmed both in mouse and human primary lymphocytes, in which histones induced lymphocyte apoptosis dose-dependently and time-dependently. To identify which intracellular signal pathways were activated, phosphorylation of various mitogen-activated protein kinases (MAPKs) were evaluated during this process, and p38 inhibitor (SB203580) was used to confirm the role of p38 in lymphocyte apoptosis induced by histones. To investigate the mitochondrial injury during these processes, we analyzed Bcl2 degradation and Rhodamine 123 to assess mitochondrial-membrane stability, via cyclosporin A as an inhibitor for mitochondrial permeability transition (MPT). Then, caspase 3 activation was also checked by western-blotting. We found that p38 phosphorylation, mitochondrial injury and caspase 3 activation occurred dose-dependently in histones-mediated lymphocyte apoptosis. We also observed that p38 inhibitor SB203580 decreased lymphocyte apoptotic ratio by 49% (P<0.05), and inhibition of MPT protected lymphocytes from apoptosis. Furthermore, to investigate whether histones are responsible for lymphocyte apoptosis, various concentrations of histone H4 neutralization antibodies were co-cultured with human primary lymphocytes and plasma from cecal ligation and puncture (CLP) mice or sham mice. The results showed that H4 neutralization antibody dose-dependently blocked lymphocyte apoptosis caused by septic plasma in vitro. These data demonstrate for the first time that extracellular histones, especially H4, play a vital role in lymphocyte apoptosis during sepsis which is dependent on p38 phosphorylation and mitochondrial permeability transition. Neutralizing H4 can inhibit lymphocyte

  11. An ethanolic extract of leaves of Piper betle (Paan) Linn mediates its antileishmanial activity via apoptosis.

    PubMed

    Sarkar, Avijit; Sen, Rupashree; Saha, Piu; Ganguly, Sudipto; Mandal, Goutam; Chatterjee, Mitali

    2008-05-01

    An unprecedented increase in the incidence of unresponsiveness to antimonial compounds has highlighted the urgent need to develop new antileishmanial agents. The leaves of Piper betle (locally known as Paan) have long been in use in the Indian indigenous system of medicine for its antimicrobial properties but its antileishmanial potential has not been studied. Accordingly, an ethanolic extract of leaves of Piper betle (PB) was tested for its antileishmanial activity that was evidenced in both promastigotes and amastigotes, with IC50 values of 9.8 and 5.45 microg/ml, respectively; importantly, it was accompanied by a safety index of >12-fold. This leishmanicidal activity of PB was mediated via apoptosis as evidenced by morphological changes, loss of mitochondrial membrane potential, in situ labeling of DNA fragments by terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling, and cell-cycle arrest at the sub-G0/G1 phase. Taken together, the data indicate that PB has promising antileishmanial activity that is mediated via programmed cell death and, accordingly, merits consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.

  12. Butyrate induces ROS-mediated apoptosis by modulating miR-22/SIRT-1 pathway in hepatic cancer cells.

    PubMed

    Pant, Kishor; Yadav, Ajay K; Gupta, Parul; Islam, Rakibul; Saraya, Anoop; Venugopal, Senthil K

    2017-03-07

    Butyrate is one of the short chain fatty acids, produced by the gut microbiota during anaerobic fermentation of dietary fibres. It has been shown that it can inhibit tumor progression via suppressing histone deacetylase and can induce apoptosis in cancer cells. However, the comprehensive pathway by which butyrate mediates apoptosis and growth arrest in cancer cells still remains unclear. In this study, the role of miR-22 in butyrate-mediated ROS release and induction of apoptosis was determined in hepatic cells. Intracellular expression of miR-22 was increased when the Huh 7 cells were incubated with sodium butyrate. Over-expression of miR-22 or addition of sodium butyrate inhibited SIRT-1 expression and enhanced the ROS production. Incubation of cells with anti-miR-22 reversed the effects of butyrate. Butyrate induced apoptosis via ROS production, cytochrome c release and activation of caspase-3, whereas addition of N-acetyl cysteine or anti-miR-22 reversed these butyrate-induced effects. Furthermore, sodium butyrate inhibited cell growth and proliferation, whereas anti-miR-22 inhibited these butyrate-mediated changes. The expression of PTEN and gsk-3 was found to be increased while p-akt and β-catenin expression was decreased significantly by butyrate. These data showed that butyrate modulated both apoptosis and proliferation via miR-22 expression in hepatic cells.

  13. Novel MDM2 inhibitor SAR405838 (MI-773) induces p53-mediated apoptosis in neuroblastoma

    PubMed Central

    Lu, Jiaxiong; Guan, Shan; Zhao, Yanling; Yu, Yang; Wang, Yongfeng; Shi, Yonghua; Mao, Xinfang; Yang, Kristine L.; Sun, Wenjing; Xu, Xin; Yi, Joanna S.; Yang, Tianshu; Yang, Jianhua; Nuchtern, Jed G.

    2016-01-01

    Neuroblastoma (NB), which accounts for about 15% of cancer-related mortality in children, is the most common childhood extracranial malignant tumor. In NB, somatic mutations of the tumor suppressor, p53, are exceedingly rare. Unlike in adult tumors, the majority of p53 downstream functions are still intact in NB cells with wild-type p53. Thus, restoring p53 function by blocking its interaction with p53 suppressors such as MDM2 is a viable therapeutic strategy for NB treatment. Herein, we show that MDM2 inhibitor SAR405838 is a potent therapeutic drug for NB. SAR405838 caused significantly decreased cell viability of p53 wild-type NB cells and induced p53-mediated apoptosis, as well as augmenting the cytotoxic effects of doxorubicin (Dox). In an in vivo orthotopic NB mouse model, SAR405838 induced apoptosis in NB tumor cells. In summary, our data strongly suggest that MDM2-specific inhibitors like SAR405838 may serve not only as a stand-alone therapy, but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact MDM2-p53 axis. PMID:27764791

  14. The anthracenedione compound bostrycin induces mitochondria-mediated apoptosis in the yeast Saccharomyces cerevisiae.

    PubMed

    Xu, Chunling; Wang, Jiafeng; Gao, Ye; Lin, Huangyu; Du, Lin; Yang, Shanshan; Long, Simei; She, Zhigang; Cai, Xiaoling; Zhou, Shining; Lu, Yongjun

    2010-05-01

    Bostrycin is an anthracenedione with phytotoxic and antibacterial activity that belongs to the large family of quinones. We have isolated bostrycin from the secondary metabolites of a mangrove endophytic fungus, no. 1403, collected from the South China Sea. Using the yeast Saccharomyces cerevisiae as a model, we show that bostrycin inhibits cell proliferation by blocking the cell cycle at G1 phase and ultimately leads to cell death in a time- and dose-dependent manner. Bostrycin-induced lethal cytotoxicity is accompanied with increased levels of intracellular reactive oxygen species and hallmarks of apoptosis such as chromatin condensation, DNA fragmentation and externalization of phosphatidylserine. We further show that bostrycin decreases mitochondrial membrane electric potential and causes mitochondrial destruction during the progression of cell death. Bostrycin-induced cell death was promoted in YCA1 null yeast strain but was partially rescued in AIF1 null mutant both in fermentative and respiratory media, strongly indicating that bostrycin induces apoptosis in yeast cells through a mitochondria-mediated but caspase-independent pathway.

  15. p38 MAP kinase mediates nitric oxide-induced apoptosis of neural progenitor cells.

    PubMed

    Cheng, A; Chan, S L; Milhavet, O; Wang, S; Mattson, M P

    2001-11-16

    Neural progenitor cells (NPC) can proliferate, differentiate into neurons or glial cells, or undergo a form of programmed cell death called apoptosis. Although death of NPC occurs during development of the nervous system and in the adult, the underlying mechanisms are unknown. Here we show that nitric oxide (NO) can induce death of C17.2 NPC by a mechanism requiring activation of p38 MAP kinase, poly(ADP-ribose) polymerase, and caspase-3. Nitric oxide causes release of cytochrome c from mitochondria, and Bcl-2 protects the neural progenitor cells against nitric oxide-induced death, consistent with a pivotal role for mitochondrial changes in controlling the cell death process. Inhibition of p38 MAP kinase by SB203580 abolished NO-induced cell death, cytochrome c release, and activation of caspase-3, indicating that p38 activation serves as an upstream mediator in the cell death process. The anti-apoptotic protein Bcl-2 protected NPC against nitric oxide-induced apoptosis and suppressed activation of p38 MAP kinase. The ability of nitric oxide to trigger death of NPC by a mechanism involving p38 MAP kinase suggests that this diffusible gas may regulate NPC fate in physiological and pathological settings in which NO is produced.

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

    PubMed

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

    2017-01-01

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

  17. Bre1p-mediated histone H2B ubiquitylation regulates apoptosis in Saccharomyces cerevisiae.

    PubMed

    Walter, David; Matter, Anja; Fahrenkrog, Birthe

    2010-06-01

    BRE1 encodes an E3 ubiquitin protein ligase that is required for the ubiquitylation of histone H2B at lysine 123 (K123). Ubiquitylation of this histone residue is involved in a variety of cellular processes including gene activation and gene silencing. Abolishing histone H2B ubiquitylation also confers X-ray sensitivity and abrogates checkpoint activation after DNA damage. Here we show that Saccharomyces cerevisiae Bre1p exhibits anti-apoptotic activity in yeast and that this is linked to histone H2B ubiquitylation. We found that enhanced levels of Bre1p protect from hydrogen-peroxide-induced cell death, whereas deletion of BRE1 enhances cell death. Moreover, cells lacking Bre1p show reduced lifespan during chronological ageing, a physiological apoptotic condition in yeast. Importantly, the resistance against apoptosis is conferred by histone H2B ubiquitylation mediated by the E3 ligase activity of Bre1p. Furthermore, we found that the death of Deltabre1 cells depends on the yeast caspase Yca1p, because Deltabre1 cells exhibit increased caspase activity when compared with wild-type cells, and deletion of YCA1 leads to reduced apoptosis sensitivity of cells lacking Bre1p.

  18. ER stress regulates myeloid-derived suppressor cell fate through TRAIL-R-mediated apoptosis.

    PubMed

    Condamine, Thomas; Kumar, Vinit; Ramachandran, Indu R; Youn, Je-In; Celis, Esteban; Finnberg, Niklas; El-Deiry, Wafik S; Winograd, Rafael; Vonderheide, Robert H; English, Nickolas R; Knight, Stella C; Yagita, Hideo; McCaffrey, Judith C; Antonia, Scott; Hockstein, Neil; Witt, Robert; Masters, Gregory; Bauer, Thomas; Gabrilovich, Dmitry I

    2014-06-01

    Myeloid-derived suppressor cells (MDSCs) dampen the immune response thorough inhibition of T cell activation and proliferation and often are expanded in pathological conditions. Here, we studied the fate of MDSCs in cancer. Unexpectedly, MDSCs had lower viability and a shorter half-life in tumor-bearing mice compared with neutrophils and monocytes. The reduction of MDSC viability was due to increased apoptosis, which was mediated by increased expression of TNF-related apoptosis-induced ligand receptors (TRAIL-Rs) in these cells. Targeting TRAIL-Rs in naive mice did not affect myeloid cell populations, but it dramatically reduced the presence of MDSCs and improved immune responses in tumor-bearing mice. Treatment of myeloid cells with proinflammatory cytokines did not affect TRAIL-R expression; however, induction of ER stress in myeloid cells recapitulated changes in TRAIL-R expression observed in tumor-bearing hosts. The ER stress response was detected in MDSCs isolated from cancer patients and tumor-bearing mice, but not in control neutrophils or monocytes, and blockade of ER stress abrogated tumor-associated changes in TRAIL-Rs. Together, these data indicate that MDSC pathophysiology is linked to ER stress, which shortens the lifespan of these cells in the periphery and promotes expansion in BM. Furthermore, TRAIL-Rs can be considered as potential targets for selectively inhibiting MDSCs.

  19. Phenothiazines induce PP2A-mediated apoptosis in T cell acute lymphoblastic leukemia

    PubMed Central

    Gutierrez, Alejandro; Pan, Li; Groen, Richard W.J.; Baleydier, Frederic; Kentsis, Alex; Marineau, Jason; Grebliunaite, Ruta; Kozakewich, Elena; Reed, Casie; Pflumio, Francoise; Poglio, Sandrine; Uzan, Benjamin; Clemons, Paul; VerPlank, Lynn; An, Frank; Burbank, Jason; Norton, Stephanie; Tolliday, Nicola; Steen, Hanno; Weng, Andrew P.; Yuan, Huipin; Bradner, James E.; Mitsiades, Constantine; Look, A. Thomas; Aster, Jon C.

    2014-01-01

    T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that is frequently associated with activating mutations in NOTCH1 and dysregulation of MYC. Here, we performed 2 complementary screens to identify FDA-approved drugs and drug-like small molecules with activity against T-ALL. We developed a zebrafish system to screen small molecules for toxic activity toward MYC-overexpressing thymocytes and used a human T-ALL cell line to screen for small molecules that synergize with Notch inhibitors. We identified the antipsychotic drug perphenazine in both screens due to its ability to induce apoptosis in fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled with mass spectrometry, we identified protein phosphatase 2A (PP2A) as a perphenazine target. T-ALL cell lines treated with perphenazine exhibited rapid dephosphorylation of multiple PP2A substrates and subsequent apoptosis. Moreover, shRNA knockdown of specific PP2A subunits attenuated perphenazine activity, indicating that PP2A mediates the drug’s antileukemic activity. Finally, human T-ALLs treated with perphenazine exhibited suppressed cell growth and dephosphorylation of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurring identification of phenothiazines as a class of drugs with anticancer effects. Furthermore, these data suggest that pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates has therapeutic potential. PMID:24401270

  20. Carvacrol induces mitochondria-mediated apoptosis in HL-60 promyelocytic and Jurkat T lymphoma cells.

    PubMed

    Bhakkiyalakshmi, Elango; Suganya, Natarajan; Sireesh, Dornadula; Krishnamurthi, Kannan; Saravana Devi, Sivanesan; Rajaguru, Palanisamy; Ramkumar, Kunka Mohanram

    2016-02-05

    The aim of the present study was to investigate the effect of carvacrol, a phenolic monoterpenoid on the induction of apoptosis in HL-60 (Human acute promyelocytic leukemia cells) and Jurkat (human T lymphocyte cells) cells. Carvacrol showed a potent cytotoxic effect on both cells with dose-dependent increase in the level of free radical formation as measured by an oxidation sensitive fluorescent dye, 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) levels. The reduction in the level of antioxidants such as catalase (CAT) and superoxide dismutase (SOD) (P<0.05) was observed in carvacrol-treated cells. The major cytotoxic effect appears to be intervened by the induction of apoptotic cell death as assessed by annexin-V labeling assay using flow cytometry. Western blot analysis showed that Bax expression was increased, whereas Bcl-2 expression was significantly decreased in carvacrol exposed HL-60 cells and Jurkat cells. Further studies revealed that the dissipation of mitochondrial membrane potential of intact cells was accompanied by the activation of caspase-3. Our results found that the potential mechanism of cellular apoptosis induced by carvacrol is mediated by caspase-3 and is associated with the collapse of mitochondrial membrane potential, generation of free radicals, and depletion of the intracellular antioxidant pool.

  1. The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.

    PubMed

    Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

    2014-04-01

    Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival.

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

    PubMed Central

    Cesnekova, Jana; Spacilova, Jana; Hansikova, Hana; Houstek, Josef; Zeman, Jiri; Stiburek, Lukas

    2016-01-01

    p53 is a major cellular tumor suppressor that in addition to its nuclear, transcription-dependent activity is also known to function extranuclearly. Cellular stressors such as reactive oxygen species can promote translocation of p53 into mitochondria where it acts to protect mitochondrial genome or trigger cell death via transcription-independent manner. Here we report that the mammalian homologue of yeast mitochondrial Afg1 ATPase (LACE1) promotes translocation of p53 into mitochondria. We further show that LACE1 exhibits significant pro-apoptotic activity, which is dependent on p53, and that the protein is required for normal mitochondrial respiratory function. LACE1 physically interacts with p53 and is necessary for mitomycin c-induced translocation of p53 into mitochondria. Conversely, increased expression of LACE1 partitions p53 to mitochondria, causes reduction in nuclear p53 content and induces apoptosis. Thus, LACE1 mediates mitochondrial translocation of p53 and its transcription-independent apoptosis. PMID:27323408

  3. Natural pyrethrins induces apoptosis in human hepatocyte cells via Bax- and Bcl-2-mediated mitochondrial pathway.

    PubMed

    Yang, Yun; Zong, Mimi; Xu, Wenping; Zhang, Yang; Wang, Bo; Yang, Mingjun; Tao, Liming

    2017-01-25

    Natural pyrethrins have been widely used for pest control in organic farming and for residential indoor pest managements. Although the specific mechanisms underlying their activity are incompletely understood, natural pesticides are considered the safest based on their target specificity and rapid degradation in the environment. Here, we used in vitro bioassays to characterize the cytotoxic effects of natural pyrethrins and attempted to delineate the cellular and molecular mechanisms of their cytotoxicity against human hepatocytes. The results demonstrate that natural pyrethrins reduce cell viability and enhance apoptosis in HepG2 cells. In addition, the current data indicate that natural pyrethrins cause a reduction in the mitochondrial membrane potential (Δψm), increase reactive oxygen species production, and up-regulate the Bax/Bcl-2 expression, leading to the release of cytochrome-c into the cytosol, activation of caspase-9 and caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). Taken together, the results indicate that natural pyrethrins has potentially exert adverse effects on human health by inducing caspase-dependent apoptosis in hepatocytes through Bax- and Bcl-2-mediated mitochondrial pathway.

  4. Radiotherapy induced Lewis lung cancer cell apoptosis via inactivating β-catenin mediated by upregulated HOTAIR.

    PubMed

    Chen, Jianxiang; Shen, Zhuping; Zheng, Yuanda; Wang, Shengye; Mao, Weimin

    2015-01-01

    HOTAIR, a long intervening non-coding Hox transcript antisense intergenic RNA, negatively regulates transcription on another chromosome and is reported to reprogram chromatin organization and promote tumor progression. Nevertheless, little is known about its roles in the development of radiation therapy of lung cancer. In this study, we established a xenografed model of Lewis lung carcinoma in C57BL/6 mice and investigated the possible involvement of HOTAIR in this radiotherapy. C57BL/6 mice were subcutaneously transplanted with Lewis lung carcinoma cells and locally irradiated followed by measurement in tumor volume. Levels of HOTAIR and WIF-1 mRNA expression were determined by using Quantitative Real-Time PCR. Levels of WIF-1 and β-catenin were determined by using western blot assay. Cell viability was evaluated by MTT assay. Cell apoptosis was examined by using TUNEL assay. In mice bearing Lewis lung carcinoma tumor, local radiotherapy suppressed tumor growth and it also reduced level of HOTAIR but increased WIF-1 expression. When HOTAIR was overexpressed, radio-sensitivity was reduced. In vitro experiments, irradiation inhibited HOTAIR transportation to the nucleus. However, it was reversed by over-expressed HOTAIR. Cells transfected with pcDNA-HOTAIR or siRNA-HOTAIR resulted in decline or increase in radiosensitivity, which was abrogated by co-tansfected with siRNA-β-catenin. Radiotherapy induced Lewis lung cancer cell apoptosis via inactivating β-catenin mediated by upregulated HOTAIR.

  5. Adenovirus mediated BIMS transfer induces growth supression and apoptosis in Raji lymphoma cells.

    PubMed

    Zhao, Ya Ning; Li, Qiang

    2014-09-01

    To transfer pro-apoptotic BIM directly into tumor cells bypass the complicated biological processes of BIM activation so as to reverse the chemoresistance of cancer cells. BIMS was specifically amplified from HL-60 cells by RT-PCR, confirmed to be correct by sequencing and cloned into shuttle vector pAdTrack-CMV carrying a green fluorescence protein gene to generate a recombinant plasmid pAdTrack-CMV-BIMS. This plasmid and adenovirus backbone plasmid pAdEasy-1 were linearized and electroporated into E.coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restrict endonuclease digestion. The recombinant pAdEasy-CMV-BIMS was transferred into HEK293 cells for packaging and amplification. The successful construction of recombinant human BIMS adenovirus (Ad-BIMS) was demonstrated by Western blot. To test whether Ad-BIMS has the capability of inducing apoptosis of tumor cells, Ad-BIMS was used to infect GC resistant Burkitt lymphoma Raji cells. After infected for 2-5 days, BIMS expression in Raji cells was detected by RT-PCR and Western blot. The significant growth retardation and apoptosis of Raji cells were also observed by MTT and flow cytometry. These results indicated that BIMS might be a potential candidate of gene therapy for chemoresistant tumor cells. Copyright © 2014 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  6. EF24 induces ROS-mediated apoptosis via targeting thioredoxin reductase 1 in gastric cancer cells

    PubMed Central

    Chen, Weiqian; Chen, Xi; Ying, Shilong; Feng, Zhiguo; Chen, Tongke; Ye, Qingqing; Wang, Zhe; Qiu, Chenyu; Yang, Shulin; Liang, Guang

    2016-01-01

    Gastric cancer (GC) is one of the leading causes of cancer mortality in the world, and finding novel agents for the treatment of advanced gastric cancer is of urgent need. Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, exhibits potent anti-tumor activities by arresting cell cycle and inducing apoptosis. Although EF24 demonstrates potent anticancer efficacy in numerous types of human cancer cells, the cellular targets of EF24 have not been fully defined. We report here that EF24 may interact with the thioredoxin reductase 1 (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme, to induce reactive oxygen species (ROS)-mediated apoptosis in human gastric cancer cells. By inhibiting TrxR1 activity and increasing intracellular ROS levels, EF24 induces a lethal endoplasmic reticulum stress in human gastric cancer cells. Importantly, knockdown of TrxR1 sensitizes cells to EF24 treatment. In vivo, EF24 treatment markedly reduces the TrxR1 activity and tumor cell burden, and displays synergistic lethality with 5-FU against gastric cancer cells. Targeting TrxR1 with EF24 thus discloses a previously unrecognized mechanism underlying the biological activity of EF24, and reveals that TrxR1 is a good target for gastric cancer therapy. PMID:26919110

  7. Role for Caspase-Mediated Cleavage of Rad51 in Induction of Apoptosis by DNA Damage

    PubMed Central

    Huang, YinYin; Nakada, Shuji; Ishiko, Takatoshi; Utsugisawa, Taiju; Datta, Rakesh; Kharbanda, Surender; Yoshida, Kiyotsugu; Talanian, Robert V.; Weichselbaum, Ralph; Kufe, Donald; Yuan, Zhi-Min

    1999-01-01

    We report here that the Rad51 recombinase is cleaved in mammalian cells during the induction of apoptosis by ionizing radiation (IR) exposure. The results demonstrate that IR induces Rad51 cleavage by a caspase-dependent mechanism. Further support for involvement of caspases is provided by the finding that IR-induced proteolysis of Rad51 is inhibited by Ac-DEVD-CHO. In vitro studies show that Rad51 is cleaved by caspase 3 at a DVLD/N site. Stable expression of a Rad51 mutant in which the aspartic acid residues were mutated to alanines (AVLA/N) confirmed that the DVLD/N site is responsible for the cleavage of Rad51 in IR-induced apoptosis. The functional significance of Rad51 proteolysis is supported by the finding that, unlike intact Rad51, the N- and C-terminal cleavage products fail to exhibit recombinase activity. In cells, overexpression of the Rad51(D-A) mutant had no effect on activation of caspase 3 but did abrogate in part the apoptotic response to IR exposure. We conclude that proteolytic inactivation of Rad51 by a caspase-mediated mechanism contributes to the cell death response induced by DNA damage. PMID:10082566

  8. Non-viable Borrelia burgdorferi induce inflammatory mediators and apoptosis in human oligodendrocytes.

    PubMed

    Parthasarathy, Geetha; Fevrier, Helene B; Philipp, Mario T

    2013-11-27

    In previous studies, exposure to live Borrelia burgdorferi was shown to induce inflammation and apoptosis of human oligodendrocytes. In this study we assessed the ability of non-viable bacteria (heat killed or sonicated) to induce inflammatory mediators and cell death. Both heat-killed and sonicated bacteria induced release of CCL2, IL-6, and CXCL8 from oligodendrocytes in a dose dependent manner. In addition, non-viable B. burgdorferi also induced cell death as evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and another cell viability assay. These results suggest that spirochetal residues left after bacterial demise, due to treatment or otherwise, may continue to be pathogenic to the central nervous system.

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

    PubMed

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

    2014-10-30

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

  10. Quercetin sensitizes pancreatic cancer cells to TRAIL-induced apoptosis through JNK-mediated cFLIP turnover.

    PubMed

    Kim, Ji Hye; Kim, Min Joo; Choi, Kyung-Chul; Son, Jaekyoung

    2016-09-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that can selectively kill cancer cells. Nonetheless, many cancers are resistant to TRAIL, and the molecular mechanisms of TRAIL resistance in cancer, particularly pancreatic cancer, are still unclear. In this study, we tested the hypothesis that quercetin, a flavonoid, induces apoptosis in TRAIL-resistant pancreatic cancer cells. Although quercetin alone had no significant cytotoxic effect, when combined with TRAIL, it promoted TRAIL-induced apoptosis that required mitochondrial outer membrane permeabilization. A BH3-only protein BID knockdown dramatically attenuated TRAIL/quercetin-induced apoptosis. The expression levels of cellular FLICE-like inhibitory protein (cFLIP) decreased in a dose-dependent manner in the presence of quercetin, and overexpression of cFLIP was able to robustly rescue pancreatic cancer cells from TRAIL/quercetin-induced apoptosis. Additionally, quercetin activated c-Jun N-terminal kinase (JNK) in a dose-dependent manner, which in turn induced the proteasomal degradation of cFLIP, and JNK activation also sensitized pancreatic cancer cells to TRAIL-induced apoptosis. Thus, our results suggest that quercetin induces TRAIL-induced apoptosis via JNK activation-mediated cFLIP turnover. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. A whole-genome RNAi screen identifies an 8q22 gene cluster that inhibits death receptor-mediated apoptosis.

    PubMed

    Dompe, Nicholas; Rivers, Celina Sanchez; Li, Li; Cordes, Shaun; Schwickart, Martin; Punnoose, Elizabeth A; Amler, Lukas; Seshagiri, Somasekar; Tang, Jerry; Modrusan, Zora; Davis, David P

    2011-10-25

    Deregulation of apoptosis is a common occurrence in cancer, for which emerging oncology therapeutic agents designed to engage this pathway are undergoing clinical trials. With the aim of uncovering strategies to activate apoptosis in cancer cells, we used a pooled shRNA screen to interrogate death receptor signaling. This screening approach identified 16 genes that modulate the sensitivity to ligand induced apoptosis, with several genes exhibiting frequent overexpression and/or copy number gain in cancer. Interestingly, two of the top hits, EDD1 and GRHL2, are found 50 kb apart on chromosome 8q22, a region that is frequently amplified in many cancers. By using a series of silencing and overexpression studies, we show that EDD1 and GRHL2 suppress death-receptor expression, and that EDD1 expression is elevated in breast, pancreas, and lung cancer cell lines resistant to death receptor-mediated apoptosis. Supporting the relevance of EDD1 and GRHL2 as therapeutic candidates to engage apoptosis in cancer cells, silencing the expression of either gene sensitizes 8q22-amplified breast cancer cell lines to death receptor induced apoptosis. Our findings highlight a mechanism by which cancer cells may evade apoptosis, and therefore provide insight in the search for new targets and functional biomarkers for this pathway.

  12. RIP1 is required for IAP inhibitor-mediated sensitization of childhood acute leukemia cells to chemotherapy-induced apoptosis.

    PubMed

    Löder, S; Fakler, M; Schoeneberger, H; Cristofanon, S; Leibacher, J; Vanlangenakker, N; Bertrand, M J M; Vandenabeele, P; Jeremias, I; Debatin, K-M; Fulda, S

    2012-05-01

    Evasion of apoptosis may contribute to poor treatment response in pediatric acute lymphoblastic leukemia (ALL), calling for novel treatment strategies. Here, we report that inhibitors of apoptosis (IAPs) at subtoxic concentrations cooperate with various anticancer drugs (that is, AraC, Gemcitabine, Cyclophosphamide, Doxorubicin, Etoposide, Vincristine and Taxol) to induce apoptosis in ALL cells in a synergistic manner as calculated by combination index and to reduce long-term clonogenic survival. Importantly, we identify RIP1 as a critical regulator of this synergism of IAP inhibitors and AraC that mediates the formation of a RIP1/FADD/caspase-8 complex via an autocrine/paracrine loop of tumor necrosis factor-α (TNFα). Knockdown of RIP1 abolishes formation of this complex and subsequent activation of caspase-8 and -3, mitochondrial perturbations and apoptosis. Similarly, inhibition of RIP1 kinase activity by Necrostatin-1 or blockage of TNFα by Enbrel inhibits IAP inhibitor- and AraC-triggered interaction of RIP1, FADD and caspase-8 and apoptosis. In contrast to malignant cells, IAP inhibitors and AraC at equimolar concentrations are non-toxic to normal peripheral blood lymphocytes or mesenchymal stromal cells. Thus, our findings provide first evidence that IAP inhibitors present a promising strategy to prime childhood ALL cells for chemotherapy-induced apoptosis in a RIP1-dependent manner. These data have important implications for developing apoptosis-targeted therapies in childhood leukemia.

  13. TNF-α Mediates Diabetes-Enhanced Chondrocyte Apoptosis During Fracture Healing and Stimulates Chondrocyte Apoptosis Through FOXO1

    PubMed Central

    Kayal, Rayyan A; Siqueira, Michelle; Alblowi, Jazia; McLean, Jody; Krothapalli, Nanarao; Faibish, Dan; Einhorn, Thomas A; Gerstenfeld, Louis C; Graves, Dana T

    2010-01-01

    To gain insight into the effect of diabetes on fracture healing, experiments were carried out focusing on chondrocyte apoptosis during the transition from cartilage to bone. Type 1 diabetes was induced in mice by multiple low-dose streptozotocin injections, and simple transverse fractures of the tibia or femur was carried out. Large-scale transcriptional profiling and gene set enrichment analysis were performed to examine apoptotic pathways on total RNA isolated from fracture calluses on days 12, 16, and 22, a period of endochondral bone formation when cartilage is resorbed and chondrocyte numbers decrease. Tumor necrosis factor α (TNF-α) protein levels were assessed by ELISA and caspase-3 by bioactivity assay. The role of TNF was examined by treating mice with the TNF-specific inhibitor pegsunercept. In vitro studies investigated the proapoptotic transcription factor FOXO1 in regulating TNF-induced apoptosis of chondrogenic ATDC5 and C3H10T1/2 cells as representative of differentiated chondrocytes, which are important during endochondral ossification. mRNA profiling revealed an upregulation of gene sets related to apoptosis in the diabetic group on day 16 when cartilage resorption is active but not day 12 or day 22. This coincided with elevated TNF-α protein levels, chondrocyte apoptosis, enhanced caspase-3 activity, and increased FOXO1 nuclear translocation (p < .05). Inhibition of TNF significantly reduced these parameters in the diabetic mice but not in normoglycemic control mice (p < .05). Silencing FOXO1 using siRNA in vitro significantly reduced TNF-induced apoptosis and caspase activity in differentiated chondrocytes. The mRNA levels of the proapoptotic genes caspase-3, caspase-8, caspase-9, and TRAIL were significantly reduced with silencing of FOXO1 in chondrocytic cells. Inhibiting caspase-8 and caspase-9 significantly reduced TNF-induced apoptosis in chondrogenic cells. These results suggest that diabetes causes an upregulation of proapoptotic genes

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

    PubMed

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

    2014-07-17

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

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

    PubMed Central

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

    2014-01-01

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

  16. Combination of Cinobufacini and Doxorubicin Increases Apoptosis of Hepatocellular Carcinoma Cells through the Fas- and Mitochondria-Mediated Pathways.

    PubMed

    Xia, Jufeng; Inagaki, Yoshinori; Gao, Jianjun; Qi, Fanghua; Song, Peipei; Han, Guohua; Sawakami, Tatsuo; Gao, Bo; Luo, Chuan; Kokudo, Norihiro; Hasegawa, Kiyoshi; Sakamoto, Yoshihiro; Tang, Wei

    2017-09-25

    Cinobufacini, a traditional Chinese medicine, has been used widely for cancer treatment, such as hepatocellular carcinoma (HCC), sarcoma, and leukemia. Previous studies done by our lab indicated that cinobufacini could suppress HCC cells through mitochondria-mediated and Fas-mediated apoptotic pathways. Here, we use a combination of cinobufacini and doxorubicin to inhibit the growth of HCC cells. The combination group induced more significant apoptosis by affecting proteins and RNA of apoptosis-related elements, such as Bcl-2, Bax, Bid, and cytochrome c. Furthermore, cinobufacini, as a mixture of a number of components, had stronger apoptosis-inducing activity than particular individual components or a simple mixture of a few components. Overall, these results suggested that the combination of cinobufacini and doxorubicin may provide a new strategy for inhibiting the proliferation of HCC cells.

  17. Apoptosis triggered by pyropheophorbide-α methyl ester-mediated photodynamic therapy in a giant cell tumor in bone

    NASA Astrophysics Data System (ADS)

    Li, K.-T.; Zhang, J.; Duan, Q.-Q.; Bi, Y.; Bai, D.-Q.; Ou, Y.-S.

    2014-06-01

    A giant cell tumor in bone is the common primary bone tumor with aggressive features, occurring mainly in young adults. Photodynamic therapy is a new therapeutic technique for tumors. In this study, we investigated the effects of Pyropheophorbide-α methyl ester (MPPa)-mediated photodynamic therapy on the proliferation of giant cell tumor cells and its mechanism of action. Cell proliferation was evaluated using an MTT assay. Cellular apoptosis was detected by Hoechst nuclear staining, and flow cytometric assay. Mitochondrial membrane potential changes and cytochrome c, caspase-9, caspase-3, and Bcl-2 expression was assessed. Finally, we found that MPPa-mediated photodynamic therapy could effectively suppress the proliferation of human giant cell tumor cells and induce apoptosis. The mitochondrial pathway was involved in the MPPa-photodynamic therapy-induced apoptosis.

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

    SciTech Connect

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

    2014-09-26

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

  19. Bax/Bak promote sumoylation of DRP1 and its stable association with mitochondria during apoptotic cell death.

    PubMed

    Wasiak, Sylwia; Zunino, Rodolfo; McBride, Heidi M

    2007-05-07

    Dynamin-related protein 1 (DRP1) plays an important role in mitochondrial fission at steady state and during apoptosis. Using fluorescence recovery after photobleaching, we demonstrate that in healthy cells, yellow fluorescent protein (YFP)-DRP1 recycles between the cytoplasm and mitochondria with a half-time of 50 s. Strikingly, during apoptotic cell death, YFP-DRP1 undergoes a transition from rapid recycling to stable membrane association. The rapid cycling phase that characterizes the early stages of apoptosis is independent of Bax/Bak. However, after Bax recruitment to the mitochondrial membranes but before the loss of mitochondrial membrane potential, YFP-DRP1 becomes locked on the membrane, resulting in undetectable fluorescence recovery. This second phase in DRP1 cycling is dependent on the presence of Bax/Bak but independent of hFis1 and mitochondrial fragmentation. Coincident with Bax activation, we detect a Bax/Bak-dependent stimulation of small ubiquitin-like modifier-1 conjugation to DRP1, a modification that correlates with the stable association of DRP1 with mitochondrial membranes. Altogether, these data demonstrate that the apoptotic machinery regulates the biochemical properties of DRP1 during cell death.

  20. High glucose induces apoptosis via upregulation of Bim expression in proximal tubule epithelial cells.

    PubMed

    Zhang, Xiao-Qian; Dong, Jian-Jun; Cai, Tian; Shen, Xue; Zhou, Xiao-Jun; Liao, Lin

    2017-04-11

    Diabetic nephropathy is the primary cause of end-stage renal disease. Apoptosis of tubule epithelial cells is a major feature of diabetic nephropathy. The mechanisms of high glucose (HG) induced apoptosis are not fully understood. Here we demonstrated that, HG induced apoptosis via upregulating the expression of proapoptotic Bcl-2 homology domain 3 (BH3)-only protein Bim protein, but not bring a significant change in the baseline level of autophagy in HK2 cells. The increase of Bim expression was caused by the ugregulation of transcription factors, FOXO1 and FOXO3a. Bim expression initiates BAX/BAK-mediated mitochondria-dependent apoptosis. Silence of Bim by siRNA in HK2 cells prevented HG-induced apoptosis and also sensitized HK2 cells to autophagy during HG treatment. The autophagy inhibitor 3-MA increased the injury in Bim knockdown HK2 cells by retriggering apoptosis. The above results suggest a Bim-independent apoptosis pathway in HK2 cells, which normally could be inhibited by autophagy. Overall, our results indicate that HG induces apoptosis via up-regulation of Bim expression in proximal tubule epithelial cells.

  1. High glucose induces apoptosis via upregulation of Bim expression in proximal tubule epithelial cells

    PubMed Central

    Zhang, Xiao-Qian; Dong, Jian-Jun; Cai, Tian; Shen, Xue; Zhou, Xiao-Jun; Liao, Lin

    2017-01-01

    Diabetic nephropathy is the primary cause of end-stage renal disease. Apoptosis of tubule epithelial cells is a major feature of diabetic nephropathy. The mechanisms of high glucose (HG) induced apoptosis are not fully understood. Here we demonstrated that, HG induced apoptosis via upregulating the expression of proapoptotic Bcl-2 homology domain 3 (BH3)-only protein Bim protein, but not bring a significant change in the baseline level of autophagy in HK2 cells. The increase of Bim expression was caused by the ugregulation of transcription factors, FOXO1 and FOXO3a. Bim expression initiates BAX/BAK-mediated mitochondria-dependent apoptosis. Silence of Bim by siRNA in HK2 cells prevented HG-induced apoptosis and also sensitized HK2 cells to autophagy during HG treatment. The autophagy inhibitor 3-MA increased the injury in Bim knockdown HK2 cells by retriggering apoptosis. The above results suggest a Bim-independent apoptosis pathway in HK2 cells, which normally could be inhibited by autophagy. Overall, our results indicate that HG induces apoptosis via up-regulation of Bim expression in proximal tubule epithelial cells. PMID:28445931

  2. The dilution of benzalkonium chloride (BAK) in the tear film.

    PubMed

    Friedlaender, Mitchell H; Breshears, Daphne; Amoozgar, Bahram; Sheardown, Heather; Senchyna, Michelle

    2006-01-01

    The claim that benzalkonium chloride (BAK) synergistically enhances the antibiotic efficacy of gatifloxacin ophthalmic solution 0.3% (preserved with 0.005% BAK [50 microg/mL), Zymar; Allergan Inc., Irvine, Calif, USA) has been the subject of several studies. The purpose of this prospective clinical study was to test the hypothesis that BAK would be significantly diluted shortly after topical ocular administration and would thereafter have little or no effect on the enhancement of the antibiotic efficacy of commercial gatifloxacin on the ocular surface. This hypothesis was tested by investigators who measured the concentration of tear film BAK at successive time points after topical administration of commercial gatifloxacin. After subjects (N=10) received 5 separate instillations of a single 35-microL drop of gatifloxacin 0.3% ophthalmic solution in each eye, tear samples were collected at 30 sec, 1 min, 3 min, 5 min, and 20 min, with the use of graduated 5-microL glass microcapillaries. A validated high-performance liquid chromatography method was used to measure the concentration of BAK in each tear sample. The results showed rapid BAK dilution to 6.4 microg/mL, 3.2 microg/mL, 1.4 microg/mL, below the detection limit, and below the detection limit at 30 sec, 1 min, 3 min, 5 min, and 20 min after instillation of a single 35-microL drop of gatifloxacin. Because such rapid dilution reduces the concentration of BAK to near zero in minutes and does not allow the time (1 h) required for effective bacterial kill power, BAK is not expected to have a clinically significant effect on enhancement of the antimicrobial efficacy of gatifloxacin on the human ocular surface.

  3. Apoptosis inhibitor 5 increases metastasis via Erk-mediated MMP expression

    PubMed Central

    Song, Kwon-Ho; Kim, Seok-Ho; Noh, Kyung Hee; Bae, Hyun Cheol; Kim, Jin Hee; Lee, Hyo-Jung; Song, Jinhoi; Kang, Tae Heung; Kim, Dong-Wan; Oh, Se-Jin; Jeon, Ju-Hong; Kim, Tae Woo

    2015-01-01

    Apoptosis inhibitor 5 (API5) has recently been identified as a tumor metastasis-regulating gene in cervical cancer cells. However, the precise mechanism of action for API5 is poorly understood. Here, we show that API5 increases the metastatic capacity of cervical cancer cells in vitro and in vivo via up-regulation of MMP-9. Interestingly, API5-mediated metastasis was strongly dependent on the Erk signaling pathway. Conversely, knock-down of API5 via siRNA technology decreased the level of phospho-Erk, the activity of the MMPs, in vitro invasion, and in vivo pulmonary metastasis. Moreover, the Erk-mediated metastatic action was abolished by the mutation of leucine into arginine within the heptad leucine repeat region, which affects protein-protein interactions. Thus, API5 increases the metastatic capacity of tumor cells by up-regulating MMP levels via activation of the Erk signaling pathway. [BMB Reports 2015; 48(6): 330-335] PMID:25248562

  4. Sophoridinol derivative 05D induces tumor cells apoptosis by topoisomerase1-mediated DNA breakage

    PubMed Central

    Zhao, Wuli; Zhang, Caixia; Bi, Chongwen; Ye, Cheng; Song, Danqing; Liu, Xiujun; Shao, Rongguang

    2016-01-01

    Sophoridine is a quinolizidine natural product of Sophora alopecuroides and has been applied for treatment of malignant trophoblastic tumors. Although characterized by low toxicity, the limited-spectrum antitumor activity hinders its further applications. 05D, a derivative of sophoridine, exhibits a better anticancer activity on diverse cancer cells, including solid tumors, and hematologic malignancy. It could inhibit topoisomerase 1 (top1) activity by stabilizing DNA–top1 complex and induce mitochondria-mediated apoptosis by promoting DNA single- and double-strand breakage mediated by top1. Also, 05D induced HCT116 cells arrest at G1 phase by inactivating CDK2/CDK4–Rb–E2F and cyclinD1–CDK4–p21 checkpoint signal pathways. 05D suppressed the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) activation and decreased 53BP level, which contributed to DNA damage repair, suggesting that the novel compound 05D might be helpful to improve the antitumor activity of DNA damaging agent by repressing ATM and ATR activation and 53BP level. In addition, the priorities in molecular traits and druggability, such as a simple structure and formulation for oral administration, further prove 05D to be a promising targeting topoisomerase agent. PMID:27274276

  5. Telekin induces apoptosis associated with the mitochondria-mediated pathway in human hepatocellular carcinoma cells.

    PubMed

    Zheng, Beibei; Wu, Lehao; Ma, Lisha; Liu, Shanshan; Li, Lin; Xie, Weidong; Li, Xia

    2013-01-01

    Telekin, a eudesmane-type sesquiterpene lactone compound isolated from Chinese folk medicine Carpesium divaricatum, has been reported to strongly inhibit the proliferation of cancer cells. In this study, the involvement of a mitochondria-mediated pathway in the pro-apoptotic action of telekin was investigated in human hepatocellular carcinoma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that telekin exhibited excellent anti-proliferation activity in hepatocellular carcinoma cells and low cytotoxicity to normal hepatocyte cells. Telekin-induced apoptosis was characterized by chromatin condensation, formation of apoptotic bodies, and exposure of phosphatidylserine on the extracellular surface, as revealed by 4,6-diamidino-2-phenylindole (DAPI) nuclear staining and flow cytometry. Flow cytometry analysis showed that telekin induced the loss of mitochondrial membrane potential (MMP), as well as increased the levels of intracellular free calcium and reactive oxygen species (ROS). Additionally, Western blot resul