XIAP Antagonist Embelin Inhibited Proliferation of Cholangiocarcinoma Cells

Abstract

Cholangiocarcinoma cells are dependent on antiapoptotic signaling for survival and resistance to death stimuli. Recent mechanistic studies have revealed that increased cellular expression of the E3 ubiquitin-protein ligase X-linked inhibitor of apoptosis (XIAP) impairs TRAIL- and chemotherapy-induced cytotoxicity, promoting survival of cholangiocarcinoma cells. This study was undertaken to determine if pharmacologic antagonism of XIAP protein was sufficient to sensitize cholangiocarcinoma cells to cell death. We employed malignant cholangiocarcinoma cell lines and used embelin to antagonize XIAP protein. Embelin treatment resulted in decreased XIAP protein levels by 8 hours of treatment with maximal effect at 16 hours in KMCH and Mz-ChA-1 cells. Assessment of nuclear morphology demonstrated a concentration-dependent increase in nuclear staining. Interestingly, embelin induced nuclear morphology changes as a single agent, independent of the addition of TNF-related apoptosis inducing ligand (TRAIL). However, caspase activity assays revealed that increasing embelin concentrations resulted in slight inhibition of caspase activity, not activation. In addition, the use of a pan-caspase inhibitor did not prevent nuclear morphology changes. Finally, embelin treatment of cholangiocarcinoma cells did not induce DNA fragmentation or PARP cleavage. Apoptosis does not appear to contribute to the effects of embelin on cholangiocarcinoma cells. Instead, embelin caused inhibition of cell proliferation and cell cycle analysis indicated that embelin increased the number of cells in S and G2/M phase. Our results demonstrate that embelin decreased proliferation in cholangiocarcinoma cell lines. Embelin treatment resulted in decreased XIAP protein expression, but did not induce or enhance apoptosis. Thus, in cholangiocarcinoma cells the mechanism of action of embelin may not be dependent on apoptosis.

Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA)

“…Among anti-apoptotic factors, several members of the Bcl-2 protein family are upregulated in CCA cell lines 310 , causing chemoresistance to cisplatin and 5-fluorouracil 311 . The E3 ubiquitin protein ligase (XIAP, IAP3 or BIRC4), which inhibits caspases 3 and 9 and TRAIL pathway, shows increased activity in CCA-enhancing chemoresistance in vitro 312 . Likewise, the anti-apoptotic protein survivin is also highly expressed in CCA 279 .…”

Key necroptotic proteins are required for Smac mimetic-mediated sensitization of cholangiocarcinoma cells to TNF-α and chemotherapeutic gemcitabine-induced necroptosis

“…Preclinical studies have also demonstrated that Smac mimetics can trigger cancer cell death as single agents or as sensitizers with other anticancer drugs, including chemotherapeutic agents [30]. In addition, the combination of a Smac mimetic and TNF-related apoptosisinducing ligand (TRAIL) has been reported to decrease cell proliferation, invasion and metastasis but did not sensitize CCA cells to cell death [36,37].…”

miR-106b-responsive gene landscape identifies regulation of Kruppel-like factor family

“…Treated cells were assayed for apoptosis by nuclear morphology, and for cell proliferation by 3 (4,5 dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay, as described [28]. All experiments were repeated at least three times.…”