Death Receptor-mediated Apoptosis Pathway Research Articles

Carbon monoxide induces a late preconditioning-mimetic cardioprotective and antiapoptotic milieu in the myocardium

A growing body of evidence indicates that carbon monoxide (CO), once perceived merely as a poisonous gas, exerts antiapoptotic and cytoprotective effects. Using a water-soluble CO-releasing molecule (CORM) tricarbonylchloro(glycinato)ruthenium(II) (CORM-3), we previously reported that CO induces a delayed protection against myocardial infarction similar to that observed in the late phase of ischemic preconditioning (PC). In the current study, we investigated the molecular mechanisms underlying this cardioprotective effect. The impact on apoptotic signaling pathways was first examined in the setting of ischemia/reperfusion injury. Mice were pretreated with CORM-3 or iCORM-3 (which does not release CO) and subjected to coronary occlusion/reperfusion 24h later. In mice that received CORM-3, there was a significant reduction in markers of apoptosis (cleaved lamin A, cleaved caspase-3, and cleaved PARP-1) after ischemia/reperfusion injury. To elucidate the mechanism of CORM-3-induced cardioprotection we further examined the activation of transcription factors and induction of cardioprotective and apoptosis modulating proteins. Infusion of CORM-3 rapidly activated the stress-responsive transcription factors nuclear factor kappaB (NF-κB), signal transducers and activators of transcription (STAT)1, STAT3, and NF-E2-related factor-2 (Nrf2). This was followed 24h later by upregulation of cardioprotective proteins (heme oxygenase-1 [HO-1], cyclooxygenase-2 [COX-2], and extracellular superoxide dismutase [Ec-SOD]) and antiapoptotic proteins involving both the mitochondria-mediated (Mcl-1) and the death receptor-mediated (c-FLIPS and c-FLIPL) apoptosis pathways. We conclude that CO released by CORM-3 triggers a cardioprotective signaling cascade that recruits the transcription factors NF-κB, STAT1/3, and Nrf2 with a subsequent increase in cardioprotective and antiapoptotic molecules in the myocardium leading to the late PC-mimetic infarct-sparing effects. This article is part of a Special Issue entitled ‘Possible Editorial’.

HsTRAIL/Apo2L Induces Apoptosis in Enteropathy-Associated T-Cell Lymphoma

Abstract 1665EATL is an intestinal tumor of aberrant intraepithelial T-lymphocytes (IELs) and may be preceded by refractory celiac disease type II (RCD II). Current therapies include surgery, chemotherapy and autologous and/or allogeneic stem cell transplantation. Despite these therapies, the overall outcome of EATL is very poor with 1- and 5-year survival rates in the range of 31–39% and 8–20%, respectively. Therefore, new therapeutic options are needed. Human soluble tumor necrosis factor-related apoptosis-inducing ligand, hsTRAIL/Apo2L, a member of the TNF family, has proven to selectively kill tumor cells via an alternative, death-receptor mediated apoptosis pathway. In this present study we evaluated if hsTRAIL/Apo2L induces apoptosis in both isolated lymphoma cells of EATL biopsies and isolated cells of RCD II biopsies.hsTRAIL/Apo2L induced apoptosis in isolated EATL lymphoma cells. RCD II cells were less sensitive to hsTRAIL/Apo2L compared to EATL cells. hsTRAIL/Apo2L induced apoptosis in EATL cells was caspase-9 dependent, but unexpectedly active caspase-8 involvement could not be detected. RT-MLPA analysis on EATL samples confirmed this observation by showing increased levels of c-Flip in EATL cells, which suggests a blockage in the extrinsic apoptosis pathway. Both EATL and RCDII cells showed expression of TRAIL receptors R1 and R2 and almost no expression of R3 and R4.In conclusion, our study showed that hsTRAIL/Apo2L induces apoptosis in EATL cells through the intrinsic apoptosis pathway. Moreover, we showed that TRAIL receptor R1 and R2 are expressed in EATL cells. Based on these results, hsTRAIL/Apo2L may be a new therapeutic option for EATL patients. Disclosures:No relevant conflicts of interest to declare.

Antagonizing IAPs by SMAC Mimetic TL32711 Induces Apoptosis in AML Cells Including AML Stem/Progenitor Cells Alone and in Combination with Chemotherapy

Abstract 66The antiapoptotic function of the inhibitors of apoptosis family of proteins (IAPs), including cIAP1, cIAP2, and XIAP, is antagonized by SMAC (second mitochondrial-derived activator of caspases). XIAP directly binds and inhibits caspase-9 and caspase-3 and suppresses both mitochondrion-mediated intrinsic and death receptor-mediated extrinsic apoptosis pathways, while the cIAPs are components of the cytoplasmic signaling complex containing members of TNF receptor associated factors and suppress death receptor/caspase-8 mediated extrinsic pathway activation. SMAC mimetics are a new class of anti-cancer agents that induce rapid degradation of cIAP1, relieve XIAP-mediated caspase repression, and promote TRAIL or TNFa-dependent apoptosis in various malignant cell types.To assess the therapeutic potential of SMAC mimetics in AML, we determined the protein levels of cIAP1 and XIAP, which are targets of SMAC mimetics, and caspase-8, the initiator caspase of the death receptor pathway by reverse phase protein array in blasts obtained from 511 newly diagnosed AML patients and in CD34+38− stem/progenitor cells isolated from blasts of these patients. We found that all three proteins were expressed in AML blasts. Importantly, we observed that the protein levels of cIAP1 and caspase-8 in CD34+38− AML stem/progenitor cells were significantly higher than those in bulk AML cells (P < 0.001).TL32711 (TL) is a highly potent and well-tolerated SMAC mimetic in clinical development that promoted rapid degradation of cIAP1 at low nM concentrations and induced pronounced apoptosis in AML cell lines in the presence of TNFα. Cell death was enhanced in the presence of TRAIL, confirming activation of the death receptor pathway as a significant mechanism of apoptosis induction. Caspase-8 mutant Jurkat cells (JurkatI9.2) were completely resistant to TL, further supporting the critical role of caspase-8 in TL-mediated cell death. TL synergistically enhanced apoptosis when combined with various nucleoside analogues clinically used in AML therapy such as Ara-C, clofarabine, and demethylating agents decitabine and 5-azacytidine (5-AC). Mechanistic studies showed that decitabine and 5-AC increased and activated caspase-8, decreased cFLIP, and induced XAF-1, a XIAP antagonist known to be hypermethylated in various malignant cell types. In addition, TL had single agent activity against blasts from primary AML samples with no toxicity in CD34+ cells from normal bone marrows at doses effective against AML cells. Importantly, TL not only induced apoptosis in bulk AML blast but also in CD34+38− AML stem/progenitor cells.Collectively, we showed that cIAP1 and caspase-8 are overexpressed in AML stem/progenitor cells and that inhibition of IAPs by the novel SMAC mimetic TL32711 synergistically enhances drug induced-death of AML cells and also has the potential to eliminate AML stem/progenitor cells. Disclosures:Weng:Tetralogic Pharmaceuticals: Employment. McKinlay:Tetralogic Pharmaceuticals: Employment.

Interplay between Ret and Fap-1 regulates CD95-mediated apoptosis in medullary thyroid cancer cells

Emerging evidence suggests that Ret oncoproteins expressed in medullary thyroid cancer (MTC) might evade the pro-apoptotic function of the dependence receptor proto-Ret by directly impacting the apoptosis machinery. Identification of the molecular determinants of the interplay between Ret signaling and apoptosis might provide a relevant contribution to the optimization of Ret-targeted therapies. Here, we describe the cross-talk between Ret-M918T oncogenic mutant responsible for type 2B multiple endocrine syndrome (MEN2B), and components of death receptor-mediated extrinsic apoptosis pathway. In the human MEN2B-type MTC cell line MZ-CRC-1 expressing Ret-M918T, Ret was found associated with Fap-1, known as inhibitor of the CD95 death receptor trafficking to the cell membrane, and with procaspase-8, the initiator pro-form caspase in the extrinsic apoptosis pathway. Cell treatment with the anti-tumor Ret kinase inhibitor RPI-1 inhibited tyrosine phosphorylation of procaspase-8, likely inducing its local activation, followed by downregulation of both Ret and Fap-1, and translocation of CD95 into lipid rafts. According to the resulting increase of CD95 cell surface expression, the CD95 agonist antibody CH11 enhanced RPI-1-induced cell growth inhibition and apoptosis. RET RNA interference downregulated Fap-1 protein in MZ-CRC-1 cells, whereas exogenous RET-M918T upregulated Fap-1 in HEK293 cells. Overall, these data indicate that the Ret oncoprotein exerts opposing controls on Fap-1 and CD95, increasing Fap-1 expression and decreasing CD95 cell surface expression. The functional interplay of the Ret mutant with the extrinsic apoptosis pathway provides a mechanism possibly contributing to MTC malignant phenotype and a rational basis for novel therapeutic strategies combining Ret inhibitors and CD95 agonists.

Stromal Anti-Apoptotic Androgen Receptor Target Gene c-FLIP in Prostate Cancer

The tumor microenvironment significantly influences prostate cancer progression. Androgen receptor exerts its effect through downstream target genes to regulate prostate cancer cell proliferation. The c-FLIP gene was recently shown to be an androgen receptor target gene. c-FLIP is an inactive homologue of caspase-8 and, thus, it inhibits the death receptor mediated apoptosis pathway. c-FLIP over expression was shown to accelerate the progression of prostate cancer cells to androgen independence. We evaluated the role of c-FLIP expression in stromal cells in prostate cancer development. We examined c-FLIP expression in 53 androgen dependent and 21 androgen independent prostate cancer stromal cells by immunohistochemical analysis. The effects of c-FLIP over expression in stromal cells on the growth and invasion of LNCaP and PC3 prostate cancer cells were determined in indirect coculture systems. At the androgen dependent stage the stromal c-FLIP level was increased in prostate cancer tissue. The expression level of stromal c-FLIP was associated with tumor differentiation. However, stromal c-FLIP expression was not increased in androgen independent human prostate cancer. c-FLIP over expression in stromal cells stimulated the growth and invasion of prostate cancer, including LNCaP and PC3 cells in vitro. These results indicate the over expression of stromal c-FLIP and its function for promoting prostate cancer growth and invasion.

Selenocystine induces apoptosis of A375 human melanoma cells by activating ROS-mediated mitochondrial pathway and p53 phosphorylation.

Selenocystine (SeC), a naturally occurring selenoamino acid, has been shown to be a novel compound with broad-spectrum anticancer activity. In this study, we showed that SeC triggered time- and dose-dependent apoptosis in A375 human melanoma cells by activating the mitochondria-mediated and death receptor-mediated apoptosis pathways. Pretreatment of cells with a general caspase inhibitor z-VAD-fmk significantly prevented SeC-induced apoptosis. A375 cells exposed to SeC showed an increase in levels of total p53 and phosphorylated p53 (serine-15). Silencing of p53 expression with RNA interference significantly suppressed SeC-induced p53 phosphorylation, caspase activation and apoptotic cell death. Moreover, generation of reactive oxygen species and subsequent induction of DNA strand breaks were found to be upstream mediators of p53 activation induced by SeC. In a nude mice xenograft experiment, SeC significantly inhibited the tumor growth of A375 cells via induction of apoptosis. Taken together, these results suggest the potential applications of SeC in cancer chemoprevention.

Silencing of caspase-8 and caspase-3 by RNA interference prevents vascular endothelial cell injury in mice with endotoxic shock

Septic shock and sequential multiple organ failure remain the cause of death in septic patients. Vascular endothelial cell apoptosis may play a role in the pathogenesis of the septic syndrome. Caspase-8 is presumed to be the apex of the death receptor-mediated apoptosis pathway, whereas caspase-3 belongs to the "effector" protease in the apoptosis cascade. Synthetic small interfering RNAs (siRNAs) specifically suppress gene expression by RNA interference. Therefore, we evaluated the therapeutic efficacy of caspase-8/caspase-3 siRNAs in a murine model of polymicrobial endotoxic shock. Polymicrobial endotoxic shock was induced by cecal ligation and puncture (CLP) in BALB/c mice. In vivo delivery of siRNAs was performed by using a transfection reagent (Lipofectamine 2000) at 10 h after CLP. As a negative control, animals received non-sense (scrambled) siRNA. Marked increases in caspase-8 and caspase-3 protein expression in CLP aortic tissues were strongly suppressed by treatment with caspase-8/caspase-3 siRNAs. This siRNA treatment prevented DNA ladder formation and less phosphorylation of the pro-apoptotic protein Bad seen in CLP aortic tissues. Transferase-mediated dUTP nick end labeling (TUNEL) revealed that the appearance of apoptosis in aortic endothelium after CLP was eliminated by this siRNA treatment. Although all of the control animals subjected to CLP died within 2 days, administration of caspase-8/caspase-3 siRNAs indefinitely (>7 days) improved the survival of CLP mice. Gene silencing of caspase-8 and caspase-3 with siRNAs provided profound protection against polymicrobial endotoxic shock. The prevention of vascular endothelial cell apoptosis appears to be, at least in part, responsible for their beneficial effects in endotoxic shock.

Human soluble TRAIL/Apo2L induces apoptosis in a subpopulation of chemotherapy refractory nodal diffuse large B‐cell lymphomas, determined by a highly sensitive in vitro apoptosis assay

Resistance to chemotherapy in therapy-refractory diffuse large B-cell lymphomas (DLBCL) is related to inhibition of the intrinsic apoptosis pathway. Human soluble tumour necrosis factor (TNF)-related apoptosis-inducing ligand (hsTRAIL/Apo2L) induces apoptosis via the alternative, death-receptor mediated apoptosis pathway and might be an effective alternative form of therapy for these lymphomas. This study investigated whether hsTRAIL/Apo2L could actually induce apoptosis in isolated lymphoma cells of DLBCL biopsies of patients with chemotherapy-refractory DLBCL. Twelve out of a total of 22 DLBCL samples were sensitive to hsTRAIL/Apo2L. These sensitive lymphomas included seven clinically chemotherapy-refractory lymphomas. Furthermore, hsTRAIL/Apo2L induced apoptosis in DLBCL cells and in B-cell lines that showed high expression levels of inhibitors of the intrinsic apoptosis pathway: Bcl-2 and/or X-linked inhibitor of apoptosis (XIAP). hsTRAIL/Apo2L-sensitive lymphoma cells showed expression of the TRAIL receptors R1 and/or R2 and absence of R3 and R4. We conclude that hsTRAIL/Apo2L induced apoptosis in a subpopulation of chemotherapy-refractory nodal DLBCL and that disruption of the intrinsic apoptosis-mediated pathway and expression of Bcl-2 and XIAP did not confer resistance to hsTRAIL/Apo2L-induced apoptosis in DLBCL. Thus, based on our results, further exploration of hsTRAIL/Apo2L as an alternative treatment for patients with chemotherapy-refractory DLBCL should be considered.

Analysis of the in vitro synergistic effect of 5-fluorouracil and cisplatin on cervical carcinoma cells

5-Fluorouracil (5-FU) is currently being used as an anticancer drug to reduce tumor bulk in order to increase the operability rate and postoperative survival in patients with cervical cancer, which has been combined with cisplatin (CP) because of its superior activities observed in human carcinoma cells. However, the combined anticancer effect of 5-FU and CP in cervical carcinoma cells is poorly understood. Therefore, we conducted a study to investigate whether anticancer drugs 5-FU and CP may exhibit the combined antiproliferative effect in cervical carcinoma cells. Using proteomics analysis, including two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), we investigated the antiproliferative effect-related proteins after treatment with 5-FU and/or CP. Our experiments showed that the combination of 5-FU and CP engaged both the apoptotic pathways: the membrane death receptor-mediated apoptosis pathway and the mitochondrial apoptotic pathway. Moreover, the combination of 5-FU and CP resulted in remarkable increasing susceptibility to apoptosis. We suggest that the combination of 5-FU and CP suppresses the growth of cervical carcinoma cells by synergistic effect with the induction of apoptosis. In vitro synergistic effect of 5-FU and CP supports the basis of the clinical application of the combination chemotherapy to the patients with cervical cancer.

Analysis of differential protein expression by cisplatin treatment in cervical carcinoma cells

Cisplatin (cis-diaminedichloroplatinum), a DNA-damaging agent, which readily induces apoptosis in vitro, is one of the widely used anticancer drug in the treatment of human malignancies. Cisplatin has played an important role in cervical cancer management for effective chemotherapeutic regimen, but the underlying mechanisms inducing cell death at protein level are unknown. Using proteome analysis, an investigation aimed at a better understanding of the antiproliferative mechanisms by cisplatin was carried out in HeLa cervical carcinoma cells. In total, 21 protein spots were found to be differentially expressed following cisplatin treatment, of which 12 were upregulated (eg, regulator of G-protein signaling, TRAF:TNF (tumor necrosis factor) receptor-associated factor-interacting protein [I-TRAF], and cyclin-dependent kinase inhibitor p27 [p27(kip1)]) and 9 were downregulated (eg, myc proto-oncoprotein [c-myc] and proliferating cell nuclear antigen). Interestingly, we found the upregulation of proliferating cell nuclear antigen, which used molecular marker in cervical cancer screening. On the basis of proteomic data, we showed that cisplatin induced TRAF2-mediated NF-kappaB downregulation. In addition, our study demonstrated that cisplatin induced membrane death receptor-mediated and mitochondria-mediated apoptosis pathway. Our findings may offer new insights into the antiproliferative mechanism by cisplatin and its mode of action in cervical carcinoma cells.

Analysis of the in vitro synergistic effect of 5-fluorouracil and cisplatin on cervical carcinoma cells

5-Fluorouracil (5-FU) is currently being used as an anticancer drug to reduce tumor bulk in order to increase the operability rate and postoperative survival in patients with cervical cancer, which has been combined with cisplatin (CP) because of its superior activities observed in human carcinoma cells. However, the combined anticancer effect of 5-FU and CP in cervical carcinoma cells is poorly understood. Therefore, we conducted a study to investigate whether anticancer drugs 5-FU and CP may exhibit the combined antiproliferative effect in cervical carcinoma cells. Using proteomics analysis, including two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), we investigated the antiproliferative effect–related proteins after treatment with 5-FU and/or CP. Our experiments showed that the combination of 5-FU and CP engaged both the apoptotic pathways: the membrane death receptor–mediated apoptosis pathway and the mitochondrial apoptotic pathway. Moreover, the combination of 5-FU and CP resulted in remarkable increasing susceptibility to apoptosis. We suggest that the combination of 5-FU and CP suppresses the growth of cervical carcinoma cells by synergistic effect with the induction of apoptosis. In vitro synergistic effect of 5-FU and CP supports the basis of the clinical application of the combination chemotherapy to the patients with cervical cancer.

Indomethacin-induced activation of the death receptor-mediated apoptosis pathway circumvents acquired doxorubicin resistance in SCLC cells

Small-cell lung cancers (SCLCs) initially respond to chemotherapy but are often resistant at recurrence. A potentially new method to overcome resistance is to combine classical chemotherapeutic drugs with apoptosis induction via tumour necrosis factor (TNF) death receptor family members such as Fas. The doxorubicin-resistant human SCLC cell line GLC4-Adr and its parental doxorubicin-sensitive line GLC4 were used to analyse the potential of the Fas-mediated apoptotic pathway and the mitochondrial apoptotic pathway to modulate doxorubicin resistance in SCLC. Western blotting showed that all proteins necessary for death-inducing signalling complex formation and several inhibitors of apoptosis were expressed in both lines. The proapototic proteins Bid and caspase-8, however, were higher expressed in GLC4-Adr. In addition, GLC4-Adr expressed more Fas (3.1x) at the cell membrane. Both lines were resistant to anti-Fas antibody, but plus the protein synthesis inhibitor cycloheximide anti-Fas antibody induced 40% apoptosis in GLC4-Adr. Indomethacin, which targets the mitochondrial apoptotic pathway, induced apoptosis in GLC4-Adr but not in GLC4 cells. Surprisingly, in GLC4-Adr indomethacin induced caspase-8 and caspase-9 activation as well as Bid cleavage, while both caspase-8 and caspase-9 specific inhibitors blocked indomethacin-induced apoptosis. In GLC4-Adr, doxorubicin plus indomethacin resulted in elevated caspase activity and a 2.7-fold enhanced sensitivity to doxorubicin. In contrast, no effect of indomethacin on doxorubicin sensitivity was observed in GLC4. Our findings show that indomethacin increases the cytotoxic activity of doxorubicin in a doxorubicin-resistant SCLC cell line partly via the death receptor apoptosis pathway, independent of Fas.

Taxol Induces Caspase-10-dependent Apoptosis

Taxol (paclitaxel) is known to inhibit cell growth and trigger significant apoptosis in various cancer cells. Although taxol induces apoptosis of cancer cells, its exact mechanism of action is not yet known. In this study we investigated death receptors, FAS-associated death domain protein (FADD), the activation of caspases-10 and -8 as well as the downstream caspases, and reactive oxygen species (ROS) in taxol-induced apoptosis in the CCRF-HSB-2 human lymphoblastic leukemia cell line. Pretreating the cells with neutralizing antibodies to Fas, tumor necrosis factor (TNF)-alpha receptor 1, or TNF-related apoptosis-inducing ligand receptors (DR4 and DR5) did not affect taxol-induced apoptosis, but transfection of the cells with a dominant negative FADD plasmid resulted in inhibition of taxol-induced apoptosis, revealing that taxol induces apoptosis independently of these death receptors but dependently on FADD. Furthermore, the drug induced activation of caspases-10, -8, -6, and -3, cleaved Bcl-2, Bid, poly(ADP-ribose) polymerase, and lamin B, and down-regulated cellular levels of FLICE-like inhibitory protein (FLIP) and X-chromosome-linked inhibitor of apoptosis protein (XIAP). However, despite the release of cytochrome c from the mitochondria in taxol-treated cells, caspase-9 was not activated. Inhibitors of caspases-8, -6, or -3 partially inhibited taxol-induced apoptosis, whereas the caspase-10 inhibitor totally abrogated this process. Taxol-induced apoptosis was also associated with decreased mitochondrial membrane potential (Deltapsim) and a significant increase in ROS generation. However, increased ROS production was not directly involved in taxol-triggered apoptosis. Therefore, these results demonstrate for the first time that taxol induces FADD-dependent apoptosis primarily through activation of caspase-10 but independently of death receptors.