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.
Highlights
Cholangiocarcinoma is a liver tumor with cellular features of bile duct epithelial cells and is the second most common primary liver cancer
Cellular Xlinked inhibitor of apoptosis protein (XIAP) protein levels decreased with time in Mz-ChA-1 and KMCH cells, while XIAP was essentially unchanged in HuCCT cells treated with embelin for up to 32 hours (Fig. 1B)
The cellular thermal shift assay measures heat-induced protein denaturation in the absence and presence of the small molecule ligand. In this case, lysed Mz-ChA-1 cells were incubated with vehicle or embelin and XIAP denaturation was measured by loss of solubility upon heat treatment
Summary
Cholangiocarcinoma is a liver tumor with cellular features of bile duct epithelial cells and is the second most common primary liver cancer. Chemotherapy has been shown to prolong survival, but only modestly [1], and five-year survival remains less than 10%. This may be due to decreased tumor cell death in response to chemotherapy. XIAP protects cholangiocarcinoma cells from apoptosis induced by chemotherapeutic drugs [5] and by the death receptor ligand TNF-related apoptosis-inducing ligand (TRAIL) [6]. Treatment of cholangiocarcinoma cells with the small molecule triptolide resulted in decreased XIAP protein levels and increased sensitivity to TRAIL [7]. Together, these data suggest that targeting XIAP in cholangiocarcinoma cells increases sensitivity to apoptosis. XIAP’s antiapoptotic effects are overcome upon mitochondrial membrane permeabilization and release of SMAC/DIABLO [8], a protein that binds the BIR3 domain of XIAP [9,10]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
