BH3-only protein mimetic obatoclax sensitizes cholangiocarcinoma cells to Apo2L/TRAIL-induced apoptosis.

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

Sustained IL-6/STAT-3 signaling in cholangiocarcinoma cells due to SOCS-3 epigenetic silencing.

BACKGROUND & AIMS: Interleukin 6 (IL-6)-mediated signal transducers and activators of transcription 3 (STAT-3) phosphorylation (activation) is aberrantly sustained in cholangiocarcinoma cells resulting in enhanced myeloid cell leukemia 1 (Mcl-1) expression and resistance to apoptosis. Because suppressor of cytokine signaling 3 (SOCS) controls the IL-6/STAT-3 signaling pathway by a classic feedback loop, the aims of this study were to examine SOCS-3 regulation in human cholangiocarcinoma. METHODS: SOCS-3 expression was assessed in human cholangiocarcinoma tissue and the Mz-ChA-1 and CCLP1 human cholangiocarcinoma cell lines. RESULTS: An inverse correlation was observed between phospho-STAT-3 and SOCS-3 protein expression in cholangiocarcinoma. In those cancers failing to express SOCS-3, extensive methylation of the SOCS-3 promoter was demonstrated in tumor but not in paired nontumor tissue. Likewise, methylation of the socs-3 promoter was also identified in 2 cholangiocarcinoma cell lines. Treatment with a demethylating agent, 5-aza-2\u27-deoxycytidine (DAC), restored IL-6 induction of SOCS-3, terminated the phospho-STAT-3 response, and reduced cellular levels of Mcl-1. Enforced expression of SOCS-3 also reduced IL-6 induction of phospho-STAT-3 and Mcl-1. Either DAC treatment or enforced SOCS-3 expression sensitized the cells to TRAIL-mediated apoptosis. CONCLUSIONS: SOCS-3 epigenetic silencing is responsible for sustained IL-6/STAT-3 signaling and enhanced Mcl-1 expression in cholangiocarcinoma

miR-25 targets TNF-related apoptosis inducing ligand (TRAIL) death receptor-4 and promotes apoptosis resistance in cholangiocarcinoma.

It has been established that microRNA expression and function contribute to phenotypic features of malignant cells, including resistance to apoptosis. Although targets and functional roles for a number of microRNAs have been described in cholangiocarcinoma, many additional microRNAs dysregulated in this tumor have not been assigned functional roles. In this study, we identify elevated miR-25 expression in malignant cholangiocarcinoma cell lines as well as patient samples. In cultured cells, treatment with the Smoothened inhibitor, cyclopamine, reduced miR-25 expression, suggesting Hedgehog signaling stimulates miR-25 production. Functionally, miR-25 was shown to protect cells against TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Correspondingly, antagonism of miR-25 in culture sensitized cells to apoptotic death. Computational analysis identified the TRAIL Death Receptor-4 (DR4) as a potential novel miR-25 target, and this prediction was confirmed by immunoblot, cell staining, and reporter assays. CONCLUSION: These data implicate elevated miR-25 levels in the control of tumor cell apoptosis in cholangiocarcinoma. The identification of the novel miR-25 target DR4 provides a mechanism by which miR-25 contributes to evasion of TRAIL-induced cholangiocarcinoma apoptosis

A smac mimetic reduces TNF related apoptosis inducing ligand (TRAIL)-induced invasion and metastasis of cholangiocarcinoma cells.

UNLABELLED: Cholangiocarcinoma (CCA) cells paradoxically express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand that, failing to kill CCA cells, instead promotes their tumorigenicity and especially the metastatic behaviors of cell migration and invasion. Second mitochondria-derived activator of caspase (smac) mimetics are promising cancer therapeutic agents that enhance proapoptotic death receptor signaling by causing cellular degradation of inhibitor of apoptosis (IAP) proteins. Our aim was to examine the in vitro and in vivo effects of the smac mimetic JP1584 in CCA. Despite JP1584-mediated loss of cellular inhibitor of apoptosis-1 (cIAP-1) and cIAP-2, TRAIL failed to induce apoptosis in KMCH-1, TFK-1, and BDEneu CCA cells; a finding consistent with a downstream block in death signaling. Because cIAP-1 and cIAP-2 also promote nuclear factor kappa B (NF-kappaB) activation by the canonical pathway, the effect of JP1584 on this signaling pathway was examined. Treatment with JP1584 inhibited TRAIL-induced NF-kappaB activation as well as TRAIL-mediated up-regulation of the NF-kappaB target gene, matrix metalloproteinase 7 (MMP7). JP1584 also reduced TRAIL-mediated CCA cell migration and invasion in vitro. Finally, in a syngeneic rat orthotopic CCA model, JP1584 administration reduced MMP7 messenger RNA levels and extrahepatic metastases. CONCLUSION: : Although the smac mimetic JP1584 does not sensitize cells to apoptosis, it reduces TRAIL-induced CCA cell metastatic behavior. These data support the emerging concept that IAPs are prometastatic and represent targets for antimetastatic therapies

Применение метода LSA-алгоритма для осуществления интеллектуального поиска комплекса упражнений развития компетенций

BACKGROUND & AIMS: Interleukin 6 (IL-6)-mediated signal transducers and activators of transcription 3 (STAT-3) phosphorylation (activation) is aberrantly sustained in cholangiocarcinoma cells resulting in enhanced myeloid cell leukemia 1 (Mcl-1) expression and resistance to apoptosis. Because suppressor of cytokine signaling 3 (SOCS) controls the IL-6/STAT-3 signaling pathway by a classic feedback loop, the aims of this study were to examine SOCS-3 regulation in human cholangiocarcinoma. METHODS: SOCS-3 expression was assessed in human cholangiocarcinoma tissue and the Mz-ChA-1 and CCLP1 human cholangiocarcinoma cell lines. RESULTS: An inverse correlation was observed between phospho-STAT-3 and SOCS-3 protein expression in cholangiocarcinoma. In those cancers failing to express SOCS-3, extensive methylation of the SOCS-3 promoter was demonstrated in tumor but not in paired nontumor tissue. Likewise, methylation of the socs-3 promoter was also identified in 2 cholangiocarcinoma cell lines. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine (DAC), restored IL-6 induction of SOCS-3, terminated the phospho-STAT-3 response, and reduced cellular levels of Mcl-1. Enforced expression of SOCS-3 also reduced IL-6 induction of phospho-STAT-3 and Mcl-1. Either DAC treatment or enforced SOCS-3 expression sensitized the cells to TRAIL-mediated apoptosis. CONCLUSIONS: SOCS-3 epigenetic silencing is responsible for sustained IL-6/STAT-3 signaling and enhanced Mcl-1 expression in cholangiocarcinoma

Hedgehog Inhibition Promotes a Switch from Type II to Type I Cell Death Receptor Signaling in Cancer Cells

TRAIL is a promising therapeutic agent for human malignancies. TRAIL often requires mitochondrial dysfunction, referred to as the Type II death receptor pathway, to promote cytotoxicity. However, numerous malignant cells are TRAIL resistant due to inhibition of this mitochondrial pathway. Using cholangiocarcinoma cells as a model of TRAIL resistance, we found that Hedgehog signaling blockade sensitized these cancer cells to TRAIL cytotoxicity independent of mitochondrial dysfunction, referred to as Type I death receptor signaling. This switch in TRAIL requirement from Type II to Type I death receptor signaling was demonstrated by the lack of functional dependence on Bid/Bim and Bax/Bak, proapoptotic components of the mitochondrial pathway. Hedgehog signaling modulated expression of X-linked inhibitor of apoptosis (XIAP), which serves to repress the Type I death receptor pathway. siRNA targeted knockdown of XIAP mimics sensitization to mitochondria-independent TRAIL killing achieved by Hedgehog inhibition. Regulation of XIAP expression by Hedgehog signaling is mediated by the glioma-associated oncogene 2 (GLI2), a downstream transcription factor of Hedgehog. In conclusion, these data provide additional mechanisms modulating cell death by TRAIL and suggest Hedgehog inhibition as a therapeutic approach for TRAIL-resistant neoplasms

Sustained IL-6/STAT-3 signaling in cholangiocarcinoma cells due to SOCS-3 epigenetic silencing.

BACKGROUND & AIMS: Interleukin 6 (IL-6)-mediated signal transducers and activators of transcription 3 (STAT-3) phosphorylation (activation) is aberrantly sustained in cholangiocarcinoma cells resulting in enhanced myeloid cell leukemia 1 (Mcl-1) expression and resistance to apoptosis. Because suppressor of cytokine signaling 3 (SOCS) controls the IL-6/STAT-3 signaling pathway by a classic feedback loop, the aims of this study were to examine SOCS-3 regulation in human cholangiocarcinoma. METHODS: SOCS-3 expression was assessed in human cholangiocarcinoma tissue and the Mz-ChA-1 and CCLP1 human cholangiocarcinoma cell lines. RESULTS: An inverse correlation was observed between phospho-STAT-3 and SOCS-3 protein expression in cholangiocarcinoma. In those cancers failing to express SOCS-3, extensive methylation of the SOCS-3 promoter was demonstrated in tumor but not in paired nontumor tissue. Likewise, methylation of the socs-3 promoter was also identified in 2 cholangiocarcinoma cell lines. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine (DAC), restored IL-6 induction of SOCS-3, terminated the phospho-STAT-3 response, and reduced cellular levels of Mcl-1. Enforced expression of SOCS-3 also reduced IL-6 induction of phospho-STAT-3 and Mcl-1. Either DAC treatment or enforced SOCS-3 expression sensitized the cells to TRAIL-mediated apoptosis. CONCLUSIONS: SOCS-3 epigenetic silencing is responsible for sustained IL-6/STAT-3 signaling and enhanced Mcl-1 expression in cholangiocarcinoma

A hedgehog survival pathway in 'undead' lipotoxic hepatocytes

BACKGROUND & AIMS: Ballooned hepatocytes in nonalcoholic steatohepatitis (NASH) generate sonic hedgehog (SHH). This observation is consistent with a cellular phenotype in which the cell death program has been initiated but cannot be executed. Our aim was to determine if ballooned hepatocytes have potentially disabled the cell death execution machinery, and if so, can their functional biology be modeled in vitro. METHODS: Immunohistochemistry was performed on human NASH specimens. In vitro studies were performed using Huh-7 cells with shRNA targeted knockdown of caspase 9 (shC9 cells) or primary hepatocytes from caspase 3(−/−) mice. RESULTS: Ballooned hepatocytes in NASH display diminished expression of the caspase 9. This phenotype was modeled using shC9 cells; these cells were resistant to lipoapoptosis by palmitate (PA) or lysophosphatidylcholine (LPC) despite lipid droplet formation. During lipid loading by either PA or LPC, shC9 cells activate JNK which via AP-1 induces SHH expression. An autocrine hedgehog survival signaling pathway was further delineated in both shC9 and caspase 3(−/−) cells during lipotoxic stress. CONCLUSION: Ballooned hepatocytes in NASH downregulate caspase 9, a pivotal caspase executing the mitochondrial pathway of apoptosis. Hepatocytes engineered to reduce caspase 9 expression are resistant to lipoapoptosis, in part, due to a hedgehog autocrine survival signaling pathway

XIAP promoter predicted GLI-binding sites exhibit sequence-specific binding to nuclear proteins.

<p><i>A,</i> Putative GLI binding sites in the XIAP promoter region. Nucleotide positions were counted from the transcription start site (TSS). Potential binding sites were observed as indicated by arrows on the schematic (I and II). <i>B,</i> Nuclear protein extracts were isolated from KMCH cells. EMSA was performed using Cy5.5-labeled double-stranded oligonucleotides containing the putative GLI binding sequences within the human XIAP promoter, and competition experiments with 200-fold molar excess cold oligonucleotides as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018330#s2" target="_blank">MATERIALS AND METHODS</a>”. <i>C</i>, Total cellular protein was isolated 48 hours after transient transfection with the indicated siRNA against GLI family members. XIAP protein expression was determined by immunoblotting and the signal was quantified by densitometry as a ratio to actin expression. <i>D,</i> Chromatin immunoprecipitation using antiserum to GLI1, GLI2, GLI3, or a control IgG was performed followed by PCR using primers flanking site I (341 bp) within the <i>XIAP</i> promoter region, as indicated. As a positive control, ChIP was performed using primers flanking the <i>Bcl-2</i> promoter GLI-binding site (GLI1 and GLI2), or primers flanking the GLI3 binding site within the <i>GLI1</i> promoter.</p