异橙黄酮通过AKT/GSK-3β/β-catenin信号通路诱导胃癌AGS细胞凋亡和周期阻滞研究

Isosinensetin Promoting the Apoptosis and Cell Cycle Arrest of Gastric Carcinoma Cell AGS by AKT/GSK-3β/β-catenin Pathway

目的:探讨异橙黄酮对胃癌细胞AGS增殖、凋亡和周期的影响及相关作用机制。方法:用2.5,5和10μmol·L^(-1);的异橙黄酮处理胃癌AGS细胞,另设阴性对照组,加入等体积的DMSO。应用CCK-8、EDU和流式细胞数检测各组AGS细胞增殖、凋亡和周期情况;分子对接技术明确异橙黄酮的可能作用靶点;Western blot检测各组AGS细胞中蛋白激酶B(AKT)、磷酸化蛋白激酶B(p-AKT)、糖原合酶激酶3β(GSK-3β)、磷酸化糖原合酶激酶3β(9号丝氨酸位点)[p-GSK-3β(ser9)]、β-连环蛋白(β-catenin)、B细胞淋巴瘤/白血病-2蛋白(Bcl-2)、Bcl-2相关X调控因子(Bax)、半胱天冬酶-8剪切体(cleave-caspase-8)、周期素依赖性激酶4(CDK4)和周期素依赖性激酶6(CDK6)的表达。以10μmol·L^(-1);的异橙黄酮、10μmol·L^(-1);的异橙黄酮联合10μmol·L^(-1);AKT激动剂SC79以及等体积的DMSO(对照)处理胃癌AGS细胞,应用Western blot检测AKT、p-AKT、GSK-3β、p-GSK-3β(ser9)和β-catenin的表达,以及应用CCK-8、EDU和流式细胞数检测各组AGS细胞增殖、凋亡和周期情况。结果:与阴性对照组相比,2.5,5和10μmol·L^(-1);异橙黄酮处理组AGS细胞增殖均明显减弱,凋亡率均明显增加,显著阻滞在G1期(P<0.05或P<0.01),异橙黄酮能与AKT的活性口袋稳定结合,显著减少胃癌AGS细胞中p-AKT、p-GSK-3β(ser9)、β-catenin、Bcl-2、CDK4和CDK6的表达以及增加Bax和cleave-caspase-8的表达(P<0.05或P<0.01)。与10μmol·L^(-1);的异橙黄酮处理组相比,10μmol·L^(-1);的异橙黄酮联合10μmol·L^(-1);的AKT激动剂SC79处理组细胞p-AKT、p-GSK-3β(ser9)和β-catenin的表达显著增加,细胞增殖率明显增加,凋亡率明显减少,且G1期细胞阻滞程度明显减弱(P<0.05或P<0.01)。结论:异橙黄酮可能通过AKT/GSK-3β/β-catenin信号通路抑制AGS细胞的增殖以及诱导其凋亡和周期阻滞。

Objective:To explore the effects and molecular mechanism of isosinensetin on the proliferation,apoptosis and cell cycle of gastric carcinoma cell AGS.Methods:2.5,5 and 10μmol·L^(-1);Isosinensetin were used to treat AGS cells,while the cells treated with DMSO were set as negative control group.CCK-8 method,EDU assay and flow cytometry were used to detect the proliferation,apoptosis and cell cycle of AGS cells in each group.After analyzing the potential targets of isosinensetin via molecular docking,Western blot was used to detect the expression levels of AKT,p-AKT,GSK-3β,p-GSK-3β(ser9),β-catenin,Bcl-2,Bax,cleave-caspase-8,CDK4 and CDK6 in each group.10μmol·L^(-1);sosinensetin,10μmol·L^(-1);isosinensetin combined with 10μmol·L^(-1);AKT activator SC79 and DMSO(control)were respectively used to treat AGS cells.Western blot was used to detect the expression levels of AKT,p-AKT,GSK-3β,p-GSK-3β(ser9)andβ-catenin in each group.CCK-8 method,EDU assay and flow cytometry were used to detect the proliferation,apoptosis and cell cycle of each group.Results:Compared with the negative control group,2.5,5 and 10μmol·L^(-1);isosinensetin all significantly inhibited the proliferation of AGS cells,and promoted the apoptosis and G1 phase arrest(P<0.05 or P<0.01);isosinensetin could bind to the active site of AKT,and decrease the expression levels of p-AKT,p-GSK-3β(ser9),β-catenin,Bcl-2,CDK4 and CDK6 in AGS cells,as well as increase the expression levels of Bax and cleave-caspase-8(P<0.05 or P<0.01).Compared with those in 10μmol·L^(-1);isosinensetin treatment group,the expression levels of p-AKT,p-GSK-3β(ser9)andβ-catenin were increased in 10μmol·L^(-1);AKT activator SC79 combined with 10μmol·L^(-1);treatment group.Compared with those in 10μmol·L^(-1);isosinensetin treatment group,the proliferation in 10μmol·L^(-1);AKT activator SC79 combined with 10μmol·L^(-1);treatment group was significantly increased,while the apoptosis and cell cycle arrest were significantly decreased(P<0.05 or P<0.01).Conclusion:Isosinensetin might inhibit the proliferation and induce the apoptosis and G1 phase arrest of gastric carcinoma AGS cells via AKT/GSK-3β/β-catenin pathway.