淫羊藿素通过Akt/mTOR调控糖酵解抑制肝内胆管癌细胞增殖的作用机制研究

Effect and mechanism of icaritin on inhibiting proliferation of intrahepatic cholangiocarcinoma cells by Akt/mTOR-mediated glycolysis

目的探究淫羊藿素对人肝内胆管癌HuCCT1细胞增殖的影响及其作用机制。方法CCK-8法检测淫羊藿素对HuCCT1细胞增殖活性的影响;平板克隆法检测淫羊藿素对HuCCT1细胞集落形成能力的影响;流式细胞术检测淫羊藿素对HuCCT1细胞周期的影响;分光光度法检测淫羊藿素对HuCCT1细胞葡萄糖摄取量、乳酸生成量、三磷酸腺苷(adenosine triphosphate,ATP)生成量以及己糖激酶(hexokinase,HK)和丙酮酸激酶(pyruvate kinase,PK)活性的影响;Western blotting法检测淫羊藿素对HuCCT1细胞中增殖相关蛋白和蛋白激酶B(protein kinase B,Akt)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)通路及糖酵解相关蛋白表达的影响;Western blotting检测淫羊藿素对瞬时转染Akt基因质粒的HuCCT1细胞中Akt/mTOR及糖酵解相关蛋白表达的影响。结果淫羊藿素显著抑制HuCCT1细胞的活力,并呈时间和剂量相关性;淫羊藿素呈剂量相关性地抑制HuCCT1细胞的集落形成(P<0.001);淫羊藿素可以将HuCCT1细胞周期阻滞在G 1期,并显著降低增殖相关蛋白的表达水平(P<0.05);淫羊藿素可以显著降低HuCCT1细胞葡萄糖摄取量、乳酸生成量及ATP生成量(P<0.05、0.01、0.001);淫羊藿素显著抑制糖酵解相关酶(HK、PK)的活性以及蛋白表达水平(P<0.05、0.01、0.001);淫羊藿素显著降低磷酸化Akt(phosphorylated Akt,p-Akt)、磷酸化mTOR(phosphorylated mTOR,p-mTOR)、磷酸化核糖体蛋白S6(phosphorylated ribosomal protein S6,p-RPS6)的蛋白表达水平(P<0.05、0.01);淫羊藿素显著降低Akt基因过表达的HuCCT1细胞中p-Akt、p-mTOR、p-RPS6、HK1、HK2、PKM1、PKM2的蛋白表达水平(P<0.05、0.01、0.001)。结论淫羊藿素能够抑制HuCCT1细胞的增殖,其作用机制可能与Akt/mTOR信号通路调控的糖酵解途径有关。

Objective To investigate the effect and mechanism of icaritin on proliferation of human intrahepatic cholangiocarcinoma HuCCT1 cells.Methods CCK-8 method was used to detect the effect of icaritin on proliferation activity of HuCCT1 cells.Plate cloning method was used to detect the effect of icaritin on colony formation ability of HuCCT1 cells.Flow cytometry was used to detect the effect of icaritin on cell cycle of HuCCT1 cells.Spectrophotometric method was used to detect the effect of icaritin on glucose uptake,lactate production and adenosine triphosphate(ATP)production of HuCCT1 cells and activities of hexokinase(HK)and pyruvate kinase(PK)in cells.Western blotting was used to detect the effect of icaritin on expression levels of proliferation-related proteins,protein kinase B(Akt)/mammalian target of rapamycin(mTOR)pathway and glycolysis-related proteins in HuCCT1 cells.Western blotting was used to detect the effect of icaritin on expression levels of Akt/mTOR and glycolysis-related proteins in HuCCT1 cells transiently transfected with Akt plasmids.Results Icaritin significantly inhibited the viability of HuCCT1 cells in a time-and dose-dependent manner.Icaritin significantly inhibited the colony formation of HuCCT1 cells in a dose-dependent manner(P<0.001).Icaritin blocked cell cycle of HuCCT1 in G 1 phase,and significantly reduced the expression levels of proliferation-related proteins(P<0.05).Icaritin significantly reduced glucose uptake,lactate production and ATP production in HuCCT1 cells(P<0.05,0.01,0.001).Icaritin significantly inhibited the activities of glycolysis-related enzymes and protein expression levels(P<0.05,0.01,0.001).Icaritin significantly reduced the expression levels of phosphorylated Akt(p-Akt),phosphorylated mTOR(p-mTOR)and phosphorylated ribosomal protein S6(p-RPS6)(P<0.05,0.01).Icaritin significantly reduced the expression levels of p-Akt p-mTOR,p-RPS6,HK1,HK2,PKM1 and PKM2 in HuCCT1 cells over-expressing Akt gene(P<0.05,0.01,0.001).Conclusion Icaritin can inhibit the proliferation of HuCCT1 cells,and its mechanism may be related to Akt/mTOR-mediated glycolysis pathway.