AKT抑制剂GSK2141795诱导人肝癌细胞株Huh7凋亡的剂量和时间效应  

Time and dose effects of AKT inhibitor GSK2141795 on apoptosis of human hepatocellular carcinoma cell line Huh7

作　　者：刘志勇[1] 李林林 刘善荣[1] LIU Zhi-yong;LI Lin-lin;LIU Shan-rong(Department of Laboratory Medicine,Changhai Hospital,Navy Medical University(Second Military Medical University),Shanghai 200433,China;Institute of Life Sciences,Jiangsu University,Zhenjiang 212013,Jiangsu,China)

机构地区：[1]海军军医大学(第二军医大学)长海医院实验诊断科,上海200433 [2]江苏大学生命科学研究院,镇江212013

出　　处：《第二军医大学学报》2018年第11期1196-1201,共6页Academic Journal of Second Military Medical University

基　　金：国家杰出青年科学基金(81425019)~~

摘　　要：目的观察不同药物浓度和作用时间下蛋白质丝氨酸/苏氨酸激酶AKT抑制剂GSK2141795对人肝癌细胞株Huh7凋亡的影响及其剂量和时间效应。方法分别使用终浓度为0、0.3、1、3、10、30μmol/L的GSK2141795处理Huh7细胞24 h,同时选择终浓度为10μmol/L的GSK2141795分别处理Huh7细胞0、2、6、12、24和48 h。利用蛋白质印迹法检测Huh7细胞中AKT、磷酸化AKTS473(p-AKTS473)的蛋白表达水平,流式细胞术检测细胞凋亡情况,qPCR和蛋白质印迹法分别检测细胞中凋亡相关分子Bad、Bcl-2、Caspase-9的表达水平。结果蛋白质印迹分析结果显示,在0~10μmol/L范围内,随着GSK2141795终浓度的增加,Huh7细胞中AKT蛋白表达水平逐渐降低,p-AKTS473蛋白表达水平逐渐升高;在0~24 h范围内,随着GSK2141795作用时间的延长,Huh7细胞中AKT蛋白的表达水平呈降低趋势、p-AKTS473蛋白的表达水平呈升高趋势;48h时AKT蛋白和p-AKTS473蛋白的表达水平较0 h均升高。流式细胞术检测结果显示,随着GSK2141795浓度的增加和作用时间的延长,Huh7细胞的凋亡比例逐渐增加。qPCR及蛋白质印迹分析结果提示Huh7细胞中凋亡效应分子Bad、Caspase-9表达水平逐渐增加,凋亡拮抗分子Bcl-2表达水平逐渐降低。结论 AKT抑制剂GSK2141795能有效抑制AKT蛋白表达,并能通过下游Bad-Bcl-2通路诱导Huh7细胞凋亡,其药物效应呈一定的浓度和时间依赖性。此外,长时间使用GSK2141795刺激Huh7细胞,AKT蛋白表达可再次升高,提示存在负反馈信号环路。Objective To explore the time and dose effects of AKT(a kind of protein serine/threonine kinase)inhibitor GSK2141795 on the apoptosis of human hepatocellular cell line Huh7.Methods Huh7 cells were treated with GSK2141795 at the concentrations of 0,0.3,1,3,10 and 30μmol/L for 24 h.A concentration of 10μmol/L GSK2141795 was selected to treat Huh7 cells for 0,2,6,12,24 and 48 h.The protein expression levels of AKT and phosphorylated AKTS473(p-AKTS473)were determined by Western blotting and cell apoptosis was detected by flow cytometry.The expression levels of apoptosis-related proteins(Bad,Bcl-2 and Caspase-9)were measured by qPCR and Western blotting.Results With the increase of GSK2141795 concentration,AKT protein level in Huh7 cells was gradually decreased and the p-AKTS473 protein level was gradually increased within the range of 0-10μmol/L.With the prolongation of GSK2141795 treatment time,the AKT protein level was gradually decreased and the p-AKTS473 protein level was gradually increased within the range of 0-24 h.At 48 h of treatment,the AKT protein and p-AKTS473 protein expression levels were increased compared with 0 h.With the increase of GSK2141795 concentration and treatment time,the proportion of apoptotic cells was gradually increased,the expression levels of apoptotic molecules Bad and Caspase-9 were gradually increased,and the expression level of apoptotic antagonist Bcl-2 was gradually decreased.Conclusion AKT inhibitor GSK2141795 can effectively inhibit AKT protein expression,and induce apoptosis of Huh7 cells through Bad-Bcl-2 pathway in a dose-and time-dependent manner.In addition,the expression level of AKT protein in Huh7 cells can increase again after long-term stimulation by GSK2141795,suggesting the existence of a negative feedback signal loop.

关 键 词：肝细胞癌 蛋白质丝氨酸苏氨酸激酶AKT 蛋白激酶抑制剂 细胞凋亡 

分 类 号：R735.7[医药卫生—肿瘤]