Progressive resistance of BTK-143 osteosarcoma cells to Apo2L/TRAIL-induced apoptosis is mediated by acquisition of DcR2/TRAIL-R4 expression: resensitisation with chemotherapy.
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ABSTRACT:
SUBMITTER: Bouralexis S
PROVIDER: S-EPMC2394221 | biostudies-other | 2003 Jul
REPOSITORIES: biostudies-other
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Project description:TNF-related apoptosis-inducing ligand or Apo2L (Apo2L/TRAIL) is a promising anti-cancer drug owing to its ability to trigger apoptosis by binding to TRAIL-R1 or TRAIL-R2, two membrane-bound receptors that are often expressed by tumor cells. TRAIL can also bind non-functional receptors such as TRAIL-R4, but controversies still exist regarding their potential to inhibit TRAIL-induced apoptosis. We show here that TRAIL-R4, expressed either endogenously or ectopically, inhibits TRAIL-induced apoptosis. Interestingly, the combination of chemotherapeutic drugs with TRAIL restores tumor cell sensitivity to apoptosis in TRAIL-R4-expressing cells. This sensitization, which mainly occurs at the death-inducing signaling complex (DISC) level, through enhanced caspase-8 recruitment and activation, is compromised by c-FLIP expression and is independent of the mitochondria. Importantly, TRAIL-R4 expression prevents TRAIL-induced tumor regression in nude mice, but tumor regression induced by TRAIL can be restored with chemotherapy. Our results clearly support a negative regulatory function for TRAIL-R4 in controlling TRAIL signaling, and unveil the ability of TRAIL-R4 to cooperate with c-FLIP to inhibit TRAIL-induced cell death.
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Project description:Human cholangiocarcinomas evade apoptosis by overexpression of Mcl-1. The drug obatoclax (GX15-070) inhibits antiapoptotic members of the Bcl-2 family including Mcl-1. The purpose of this study is to determine if obatoclax sensitizes human cholangiocarcinoma cells to apoptosis. The human cholangiocarcinoma cell lines, KMCH, KMBC, and TFK, were employed for these studies. Protein expression was assessed by immunoblot and protein-protein interactions detected by coprecipitation of the polypeptide of interest with S-tagged Mcl-1. Activation of Bak and Bax was observed by immunocytochemistry with conformation-specific antisera. Obatoclax induced minimal apoptosis alone; however, it increased apoptosis 3- to 13-fold in all three cancer cell lines when combined with Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Obatoclax did not alter cellular expression of Bid, Bim, Puma, Noxa, Bak, Bax, Mcl-1, or cFLIP. Mcl-1 binding to Bak was readily identified in untreated cells, and this association was disrupted by treating the cells with obatoclax. Additionally, Bim binding to Mcl-1 was markedly decreased by obatoclax treatment. We also identified alterations in Bak and Bax conformation following treatment with obatoclax plus Apo2L/TRAIL but not with either Apo2L/TRAIL or obatoclax alone. In conclusion, obatoclax releases Bak and Bim from Mcl-1 and sensitizes human cholangiocarcinoma cells to Apo2L/TRAIL-induced apoptosis. Obatoclax is a potentially promising adjunctive agent for the treatment of this cancer.
| S-EPMC2562222 | biostudies-literature
Project description:TRAIL/Apo2L is a pro-apoptotic ligand of the TNF family that engages the apoptotic machinery through two pro-apoptotic receptors, TRAIL-R1 and TRAIL-R2. This cell death program is tightly controlled by two antagonistic receptors, TRAIL-R3 and TRAIL-R4, both devoid of a functional death domain, an intracellular region of the receptor, required for the recruitment and the activation of initiator caspases. Upon TRAIL-binding, TRAIL-R4 forms a heteromeric complex with the agonistic receptor TRAIL-R2 leading to reduced caspase-8 activation and apoptosis.We provide evidence that TRAIL-R4 can also exhibit, in a ligand independent manner, signaling properties in the cervical carcinoma cell line HeLa, through Akt. Ectopic expression of TRAIL-R4 in HeLa cells induced morphological changes, with cell rounding, loss of adherence and markedly enhanced cell proliferation in vitro and tumor growth in vivo. Disruption of the PI3K/Akt pathway using the pharmacological inhibitor LY294002, siRNA targeting the p85 regulatory subunit of phosphatidylinositol-3 kinase, or by PTEN over-expression, partially restored TRAIL-mediated apoptosis in these cells. Moreover, the Akt inhibitor, LY294002, restituted normal cell proliferation index in HeLa cells expressing TRAIL-R4.Altogether, these results indicate that, besides its ability to directly inhibit TRAIL-induced cell death at the membrane, TRAIL-R4 can also trigger the activation of signaling pathways leading to cell survival and proliferation in HeLa cells. Our findings raise the possibility that TRAIL-R4 may contribute to cervical carcinogenesis.
| S-EPMC3098831 | biostudies-literature
Project description:Approximately half of tumor cell lines are resistant to the tumor-selective apoptotic effects of tumor necrosis factor-related apoptosis-inducing ligand (Apo22L/TRAIL). Previously, we showed that combining Apo2L/TRAIL with sorafenib, a multikinase inhibitor, results in dramatic efficacy in Apo2L/TRAIL-resistant tumor xenografts via inhibition of Mcl-1. Soluble Apo2L/TRAIL is capable of binding to several surface receptors, including the pro-apoptotic death receptors, DR4 and DR5, and decoy receptors, DcR1 and DcR2. Monoclonal antibodies targeting either of these death receptors are being investigated as antitumor agents in clinical trials. We hypothesized that sorafenib and Apo2L/TRAIL or Apo2L/TRAIL death receptor agonist (TRA) antibodies against DR4 (mapatumumab) and DR5 (lexatumumab) will overcome resistance to Apo2L/TRAIL-mediated apoptosis and as increase antitumor efficacy in Apo2L/TRAIL-sensitive solid tumors.We found that Apo2L/TRAIL or TRA antibodies combined with sorafenib synergistically reduce cell growth and increase cell death across a panel of solid tumor cell lines in vitro. This panel included human breast, prostate, colon, liver and thyroid cancers. The cooperativity of these combinations was also observed in vivo, as measured by tumor volume and TUNEL staining as a measure of apoptosis. We found that sorafenib inhibits Jak/Stat3 signaling and downregulates their target genes, including cyclin D1, cyclin D2 and Mcl-1, in a dose-dependent manner.The combination of sorafenib with Apo2L/TRAIL or Apo2L/TRAIL receptor agonist antibodies sensitizes Apo2L/TRAIL-resistant cells and increases the sensitivity of Apo2L/TRAIL-sensitive cells. Our findings demonstrate the involvement of the Jak2-Stat3-Mcl1 axis in response to sorafenib treatment, which may play a key role in sorafenib-mediated sensitization to Apo2L/TRAIL.
| S-EPMC3784419 | biostudies-other
Project description:BackgroundMacroautophagy is a catabolic process that can mediate cell death or survival. Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment (TR) is known to induce autophagy. Here we investigated whether SQSTM1/p62 (p62) overexpression, as a marker of autophagic flux, was related to aggressiveness of human prostate cancer (PCa) and whether autophagy regulated the treatment response in sensitive but not resistant PCa cell lines.MethodsImmunostaining and immunoblotting analyses of the autophagic markers p62 [in PCa tissue microarrays (TMAs) and PCa cell lines] and LC3 (in PCa cell lines), transmission electron microscopy, and GFP-mCherry-LC3 were used to study autophagy induction and flux. The effect of autophagy inhibition using pharmacologic (3-methyladenine and chloroquine) and genetic [(short hairpin (sh)-mediated knock-down of ATG7 and LAMP2) and small interfering (si)RNA-mediated BECN1 knock-down] approaches on TR-induced cell death was assessed by clonogenic survival, sub-G1 DNA content, and annexinV/PI staining by flow cytometry. Caspase-8 activation was determined by immunoblotting.ResultsWe found that increased cytoplasmic expression of p62 was associated with high-grade PCa, indicating that autophagy signaling might be important for survival in high-grade tumors. TR-resistant cells exhibited high autophagic flux, with more efficient clearance of p62-aggregates in four TR-resistant PCa cell lines: C4-2, LNCaP, DU145, and CWRv22.1. In contrast, autophagic flux was low in TR-sensitive PC3 cells, leading to accumulation of p62-aggregates. Pharmacologic (chloroquine or 3-methyladenine) and genetic (shATG7 or shLAMP2) inhibition of autophagy led to cell death in TR-resistant C4-2 cells. shATG7-expressing PC3 cells, were less sensitive to TR-induced cell death whereas those shLAMP2-expressing were as sensitive as shControl-expressing PC3 cells. Inhibition of autophagic flux using chloroquine prevented clearance of p62 aggregates, leading to caspase-8 activation and cell death in C4-2 cells. In PC3 cells, inhibition of autophagy induction prevented p62 accumulation and hence caspase-8 activation.ConclusionsWe show that p62 overexpression correlates with advanced stage human PCa. Pharmacologic and genetic inhibition of autophagy in PCa cell lines indicate that autophagic flux can determine the cellular response to TR by regulating caspase-8 activation. Thus, combining various autophagic inhibitors may have a differential impact on TR-induced cell death.
| S-EPMC3998041 | biostudies-literature
Project description:Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent. Recombinant human Apo2L/TRAIL has been under clinical trials, whereas various kinds of malignant tumors have resistance to Apo2L/TRAIL. We and others have shown that several anticancer agents and flavonoids overcome resistance to Apo2L/TRAIL by upregulating death receptor 5 (DR5) in malignant tumor cells. However, the mechanisms by which these compounds induce DR5 expression remain unknown. Here we show that the dietary flavonoid apigenin binds and inhibits adenine nucleotide translocase-2 (ANT2), resulting in enhancement of Apo2L/TRAIL-induced apoptosis by upregulation of DR5. Apigenin and genistein, which are major flavonoids, enhanced Apo2L/TRAIL-induced apoptosis in cancer cells. Apigenin induced DR5 expression, but genistein did not. Using our method identifying the direct targets of flavonoids, we compared the binding proteins of apigenin with those of genistein. We discovered that ANT2 was a target of apigenin, but not genistein. Similarly to apigenin, knockdown of ANT2 enhanced Apo2L/TRAIL-induced apoptosis by upregulating DR5 expression at the post-transcriptional level. Moreover, silencing of ANT2 attenuated the enhancement of Apo2L/TRAIL-induced apoptosis by apigenin. These results suggest that apigenin upregulates DR5 and enhances Apo2L/TRAIL-induced apoptosis by binding and inhibiting ANT2. We propose that ANT2 inhibitors may contribute to Apo2L/TRAIL therapy.
| S-EPMC3576345 | biostudies-literature
Project description:Osteosarcoma (OS) is the most common bone malignancy in the paediatric population, principally affecting adolescents and young adults. Minimal advancements in patient prognosis have been made over the past two decades because of the poor understanding of disease biology. Runx2, a critical transcription factor in bone development, is frequently amplified and overexpressed in OS. However, the molecular and biological consequences of Runx2 overexpression remain unclear.si/shRNA and overexpression technology to alter Runx2 levels in OS cells. In vitro assessment of doxorubicin (doxo)-induced apoptosis and in vivo chemosensitivity studies. Small-molecule inhibitor of c-Myc transcriptional activity was used to assess its role.Loss of Runx2 sensitises cells to doxo-induced apoptosis both in vitro and in vivo. Furthermore, in conjunction with chemotherapy, decreasing Runx2 protein levels activates both the intrinsic and extrinsic apoptotic pathways. Transplanted tumour studies demonstrated that loss of endogenous Runx2 protein expression enhances caspase-3 cleavage and tumour necrosis in response to chemotherapy. Finally, upon doxo-treated Runx2 knockdown OS cells there was evidence of enhanced c-Myc expression and transcriptional activity. Inhibition of c-Myc under these conditions resulted in decreased activation of apoptosis, therefore insinuating a role for c-Myc in dox-induced activation of apoptotic pathways.Therefore, we have established a novel molecular mechanism by which Runx2 provides a chemoprotective role in OS, indicating that in conjunction to standard chemotherapy, targeting Runx2 may be a new therapeutic strategy for patients with OS.
| S-EPMC4815801 | biostudies-literature
Project description:Previously, we have reported that overexpression of IHPK2 (inositol hexakisphosphate kinase 2) sensitized NIH-OVCAR-3 ovarian carcinoma cell lines to the growth-suppressive and apoptotic effects of IFN-beta (interferon-beta) treatment and gamma-irradiation. In the present study, we demonstrate that Apo2L/TRAIL (Apo2L/tumour-necrosis-factor-related apoptosis-inducing ligand) is a critical mediator of IFN-induced apoptosis in these cells. Compared with IFN-alpha2, IFN-beta is a more potent inducer of Apo2L/TRAIL and IHPK2 activity. Overexpression of IHPK2 converts IFN-alpha2-resistant cells into cells that readily undergo apoptosis in response to IFN-alpha2. In untreated cells transfected with IHPK2-eGFP (where eGFP stands for enhanced green fluorescent protein), the fusion protein is localized to the cytoplasm and perinuclear region. After treatment with IFN-beta, IHPK2-eGFP translocated to the nucleus. In cells transfected with mutant IHPK2-NLS-eGFP (where NLS stands for nuclear localization sequence), containing point mutations in the NLS, the fusion protein remained trapped in the cytoplasm, even after IFN-beta treatment. Cells expressing mutant NLS mutation were more resistant to IFN-beta. The IC50 value of IHPK2-expressing cells was 2-3-fold lower than vector control. The IC50 value of NLS-mutant-expressing cells was 3-fold higher than vector control. Blocking antibodies to Apo2L/TRAIL or transfection with a dominant negative Apo2L/TRAIL receptor (DR5Delta) inhibited the antiproliferative effects of IFN-beta. Thus overexpression of IHPK2 enhanced apoptotic effects of IFN-beta, and expression of the NLS mutant conferred resistance to IFN-beta. Apo2L/TRAIL expression and nuclear localization of IHPK2 are both required for the induction of apoptosis by IFN-beta in ovarian carcinoma.
| S-EPMC1134734 | biostudies-other
Project description:The synthetic cannabinoid WIN 55,212-2 is a potent cannabinoid receptor agonist with anticancer potential. Experiments were performed to determine the effects of WIN on proliferation, cell cycle distribution, and programmed cell death in human osteosarcoma MG63 and Saos-2 cells. Results show that WIN induced G2/M cell cycle arrest, which was associated with the induction of the main markers of ER stress (GRP78, CHOP and TRB3). In treated cells we also observed the conversion of the cytosolic form of the autophagosome marker LC3-I into LC3-II (the lipidated form located on the autophagosome membrane) and the enhanced incorporation of monodansylcadaverine and acridine orange, two markers of the autophagic compartments such as autolysosomes. WIN also induced morphological effects in MG63 cells consisting in an increase in cell size and a marked cytoplasmic vacuolization. However, WIN effects were not associated with a canonical apoptotic pathway, as demonstrated by the absence of specific features, and only the addition of TRAIL to WIN-treated cells led to apoptotic death probably mediated by up-regulation of the tumor suppressor factor PAR-4, whose levels increased after WIN treatment, and by the translocation of GRP78 on cell surface.
| S-EPMC4007360 | biostudies-literature
Project description:Apo2L/TRAIL stimulates cancer-cell death through the proapoptotic receptors DR4 and DR5, but the determinants of tumor susceptibility to this ligand are not fully defined. mRNA expression of the peptidyl O-glycosyl transferase GALNT14 correlated with Apo2L/TRAIL sensitivity in pancreatic carcinoma, non-small cell lung carcinoma and melanoma cell lines (P < 0.00009; n=83), and up to 30% of samples from various human malignancies displayed GALNT14 overexpression. RNA interference of GALNT14 reduced cellular Apo2L/TRAIL sensitivity, whereas overexpression increased responsiveness. Biochemical analysis of DR5 identified several ectodomain O-GalNAc-Gal-Sialic acid structures. Sequence comparison predicted conserved extracellular DR4 and DR5 O-glycosylation sites; progressive mutation of the DR5 sites attenuated apoptosis signaling. O-glycosylation promoted ligand-stimulated clustering of DR4 and DR5, which mediated recruitment and activation of the apoptosis-initiating protease caspase-8. These results uncover a novel link between death receptor O-glycosylation and apoptosis signaling, providing potential predictive biomarkers for Apo2L/TRAIL-based cancer therapy. Keywords: cell type comparison
2007-08-31 | GSE8332 | GEO