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: Tongsen Zheng, Jiabei Wang, Xi Chen, Xianzhi Meng, Xuan Song, Zhaoyang Lu, Hongchi Jiang, Lianxin Liu
• Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine (Tumour Biol) Vol. 31 Issue 4 Pg. 287-95 (Aug 2010) ISSN: 1423-0380 [Electronic] Netherlands
• PMID: 20422343 (Publication Type: Journal Article)

Chemical References

• Antimetabolites, Antineoplastic
• DNA-Binding Proteins
• Imidazoles
• Nuclear Proteins
• Piperazines
• RNA, Messenger
• RNA, Small Interfering
• TP53 protein, human
• TP73 protein, human
• Tumor Protein p73
• Tumor Suppressor Protein p53
• Tumor Suppressor Proteins
• Deoxycytidine
• nutlin 3
• MDM2 protein, human
• Proto-Oncogene Proteins c-mdm2
• Gemcitabine

Topics

• Antimetabolites, Antineoplastic (pharmacology)
• Apoptosis (drug effects)
• Bile Duct Neoplasms (genetics, pathology)
• Blotting, Western
• Cell Proliferation (drug effects)
• Cholangiocarcinoma (genetics, pathology)
• DNA-Binding Proteins (antagonists & inhibitors)
• Deoxycytidine (analogs & derivatives, pharmacology)
• Drug Synergism
• Drug Therapy, Combination
• Humans
• Imidazoles (pharmacology)
• Immunoprecipitation
• Mutation
• Nuclear Proteins (antagonists & inhibitors)
• Piperazines (pharmacology)
• Proto-Oncogene Proteins c-mdm2 (antagonists & inhibitors)
• RNA, Messenger (genetics)
• RNA, Small Interfering (pharmacology)
• Reverse Transcriptase Polymerase Chain Reaction
• Tumor Cells, Cultured
• Tumor Protein p73
• Tumor Suppressor Protein p53 (genetics)
• Tumor Suppressor Proteins (antagonists & inhibitors)
• Gemcitabine