ABSTRACT

No abstract

Background and aims-IL-6 mediated STAT-3 phosphorylation (activation) is aberrantly sustained in cholangiocarcinoma cells resulting in enhanced Mcl-1 expression and resistance to apoptosis. Because SOCS-3 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.

The present studies were performed to determine whether lysosomal permeabilization contributes to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity and to reconcile a role for lysosomes with prior observations that Bcl-2 family members regulate TRAIL-induced apoptosis. In KMCH cholangiocarcinoma cells stably expressing Mcl-1 small interference RNA (siRNA), treatment with TRAIL induced a redistribution of the cathepsin B from lysosomes to the cytosol. Pharmacological and small hairpin RNA-targeted inhibition of cathepsin B attenuated TRAIL-mediated apoptosis as assessed by morphological, biochemical, and clonogenic assays. Neither Bid siRNA nor Bak siRNA prevented cathepsin B release. In contrast, treatment of the cells with Bim siRNA or the JNK inhibitor SP600125 attenuated lysosomal permeabilization and cell death. Moreover, Bim and active Bax co-localized to lysosomes in TRAIL-treated cells in a JNK-dependent manner, and Bax siRNA reduced TRAIL-induced lysosomal permeabilization and cell death. Finally, BH3 domain peptides permeabilized isolated lysosomes in the presence of Bax. Collectively, these data suggest that TRAIL can trigger an apoptotic pathway that involves JNK-dependent activation of Bim, which in turn induces Bax-mediated permeabilization of lysosomes. Tumor necrosis factor (TNF)2 -related apoptosis-inducing ligand (TRAIL), a death ligand that preferentially induces apoptosis in transformed and virally infected cells, is under evaluation as an anti-cancer agent in humans (1-3). TRAIL induces apoptosis by binding to death receptors 4 and 5 (DR4/ TRAIL-R1 and DR5/TRAIL-R2), type I transmembrane proteins that contain death domains in their cytosolic tails. TRAIL binding results in receptor oligomerization (4) followed by recruitment of Fas-associated protein with death domain. This polypeptide in turn recruits procaspases-8 and -10 to the receptor complex through homotypic interactions involving death effecter domains in Fas-associated protein with death domain and these two apical caspases (4). The resulting activation of the bound caspases is essential for TRAIL-induced apoptosis (5).The cytotoxicity of TRAIL is also regulated by members of the Bcl-2 family of polypeptides (6). In particular, the multidomain proapoptotic Bcl-2 family member Bax is essential for TRAIL-induced killing of a colon cancer cell line that has low level expression of Bak (7). Other Bcl-2 family members also affect TRAIL action. We and others have demonstrated that the multidomain antiapoptotic protein Mcl-1 blocks TRAILmediated apoptosis in various cell lines (8 -10). The related proteins Bcl-2 and Bcl-X L have also been reported to inhibit TRAIL-induced killing (11,12). Conversely, certain proapoptotic BH3-only family members, which function as sensors of various cellular stresses (13), participate in TRAIL-induced killing. Specifically, Bid and Bim have both been implicated in TRAIL cytotoxicity (10,14). How these Bcl-2 family members integrate into a composite signaling pathway involving apica...

It has been established that microRNA expression and function contribute to phenotypic features of malignant cells, including resistance to apoptosis. While 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 TRIAL-induced cholangiocarcinoma apoptosis.