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Gallbladder carcinoma (GBC) is the most common and aggressive cancer of the biliary tract.Liensinine has been proved to have hypotensive effect.However,the effect of liensinine on GBC is still unknown.The aim of this study is to investigate the effect and mechanism of liensinine in human GBC cells.Cell viability assay and colony formation assay were performed to assess cell growth and proliferation.Flow cytometry analysis was used to investigate cell cycle apoptosis in vitro.Hoechst 33342 staining was also used to evaluate cell apoptosis.Weste blot analysis was used to determine the expression of proteins corresponding to the related cell cycle and apoptosis.The effect of liensinine treatment in vivo was experimented with xenografted tumors.We found that liensinine significantly inhibited the growth of GBC cells both in vivo and in vitro.In vitro,cell growth and proliferation were significantly suppressed by liensinine in a dose-and time-dependent manner.In vivo,liensinine inhibited tumor growth.Liensinine could induce GBC cells G2/M phase arrest by up-regulating the levels of Cyclin B1 and CDK1 proteins.Liensinine also affected GBC cell cycle progression and induced apoptosis by down-regulating phosphorylated protein kinase B (AKT),phosphorylated protein kinase B (p-AKT),phosphatidylinositol 3-kinase (PI3K),and Zinc finger X-chromosomal protein (ZFX)proteins.Liensinine induced G2/M arrest and apoptosis in gallbladder cancer,suggesting that liensinine might represent a novel and effective agent against gallbladder cancer.