title: Disruption of p73-MDM2 binding synergizes with gemcitabine to induce apoptosis in HuCCT1 cholangiocarcinoma cell line with p53 mutation.
abstract: Chemotherapy to date has not been effective in the treatment of cholangiocarcinoma. However, gemcitabine, a novel nucleoside analog, has shown remarkable biological activity against cholangiocarcinoma in some clinical studies. Combinations of gemcitabine with other agents have also shown promising results, with a tolerable toxicity profile. Nutlin-3 is a small-molecule inhibitor that acts to inhibit murine double minute-2 (MDM2) binding to p53 or p73 and subsequently activates p53- or p73-dependent apoptosis signaling pathways. To investigate their effects in combination, a p53-mutant cholangiocarcinoma line HuCCT1 was treated with Nutlin-3 and/or gemcitabine in the current study. Cell proliferation assay, apoptosis assay, Western blot, coimmunoprecipitation, and small interfering RNA (siRNA) experiments were analyzed in HuCCT1 cells. Antitumoral effects of Nutlin-3 and/or gemcitabine targeting the p73/MDM2 pathways were evaluated. Nutlin-3 and gemcitabine can both inhibit the growth of HuCCT1 cells. Nutlin-3 induces apoptosis and potentiates the cytotoxic effect of gemcitabine through disrupting the binding of p73 with MDM2. Nutlin-3 leads to activation of caspases, increase levels of puma and bax, and decrease the expression of bcl-2. Blocking p73 function with a siRNA attenuates the apoptosis induced by gemcitabine, Nutlin-3, and gemcitabine/Nutlin-3 combination. Our data provide evidence that p73 might compensate for p53 function in gemcitabine-induced apoptosis of HuCCT1 cells. Nutlin-3 acts through the inhibition of p73-MDM2 with subsequent activation of the apoptotic pathway signaling, which leads to the increase in chemosensitivity to gemcitabine. In summary, our findings suggest that Nutlin-3 will be active in the treatment of p53-mutant cholangiocarcinoma, especially when in combination with gemcitabine.
authors.affiliation: Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, No. 23 Youzheng Street, Heilongjiang Province, Harbin, 150001, China.
authors.forename: Tongsen
authors.initials: T
authors.lastname: Zheng
authors.forename: Jiabei
authors.initials: J
authors.lastname: Wang
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Xianzhi
authors.initials: X
authors.lastname: Meng
authors.forename: Xuan
authors.initials: X
authors.lastname: Song
authors.forename: Zhaoyang
authors.initials: Z
authors.lastname: Lu
authors.forename: Hongchi
authors.initials: H
authors.lastname: Jiang
authors.forename: Lianxin
authors.initials: L
authors.lastname: Liu
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001650:Bile Duct Neoplasms / Q000235:genetics / Q000473:pathology; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D018281:Cholangiocarcinoma / Q000235:genetics / Q000473:pathology*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D047468:Immunoprecipitation; D009154:Mutation; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors*; D010879:Piperazines / Q000494:pharmacology*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors*; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D004268:DNA-Binding Proteins; D007093:Imidazoles; D009687:Nuclear Proteins; D010879:Piperazines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C495901:TP53 protein, human; C000606460:TP73 protein, human; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D003841:Deoxycytidine; C482205:nutlin 3; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D000093542:Gemcitabine