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  • 刘志勇,李林林,刘善荣.AKT抑制剂GSK2141795诱导人肝癌细胞株Huh7凋亡的剂量和时间效应[J].第二军医大学学报,2018,39(11):1196-1201    [点击复制]
  • LIU Zhi-yong,LI Lin-lin,LIU Shan-rong.Time and dose effects of AKT inhibitor GSK2141795 on apoptosis of human hepatocellular carcinoma cell line Huh7[J].Acad J Sec Mil Med Univ,2018,39(11):1196-1201   [点击复制]
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AKT抑制剂GSK2141795诱导人肝癌细胞株Huh7凋亡的剂量和时间效应
刘志勇1,李林林2,刘善荣1*
0
(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
*通信作者)
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、30 μmol/L的GSK2141795处理Huh7细胞24 h,同时选择终浓度为10 μmol/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0~10 μmol/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;48 h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。
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
DOI:10.16781/j.0258-879x.2018.11.1196
投稿时间:2018-06-18修订日期:2018-09-07
基金项目:国家杰出青年科学基金(81425019).
Time and dose effects of AKT inhibitor GSK2141795 on apoptosis of human hepatocellular carcinoma cell line Huh7
LIU Zhi-yong1,LI Lin-lin2,LIU Shan-rong1*
(1. Department of Laboratory Medicine, Changhai Hospital, Navy Medical University(Second Military Medical University), Shanghai 200433, China;
2. Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, Jiangsu, China
*Corresponding author)
Abstract:
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 30 μmol/L for 24 h. A concentration of 10 μmol/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 0-10 μmol/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.
Key words:  hepatocellular carcinoma  protein-serine-threonine kinase AKT  protein kinase inhibitors  apoptosis