title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.
authors.forename: Cody J
authors.initials: CJ
authors.lastname: Wehrkamp
authors.affiliation: Department of Biochemistry and Molecular Biology, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.
authors.forename: Ashley R
authors.initials: AR
authors.lastname: Gutwein
authors.affiliation: Department of Biochemistry and Molecular Biology, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.
authors.forename: Sathish Kumar
authors.initials: SK
authors.lastname: Natarajan
authors.affiliation: Department of Biochemistry and Molecular Biology, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.
authors.forename: Mary Anne
authors.initials: MA
authors.lastname: Phillippi
authors.affiliation: Department of Biochemistry and Molecular Biology, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.
authors.forename: Justin L
authors.initials: JL
authors.lastname: Mott
mesh_terms: D016227:Benzoquinones / Q000494:pharmacology*; D001650:Bile Duct Neoplasms / Q000378:metabolism / Q000473:pathology; D001653:Bile Ducts, Intrahepatic; D002453:Cell Cycle / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D018281:Cholangiocarcinoma / Q000378:metabolism / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D059748:Proteolysis / Q000187:drug effects; D016196:S Phase / Q000187:drug effects; D013997:Time Factors; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D016227:Benzoquinones; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C010945:embelin