基于EGFR/PI3K/AKT/mTOR通路探讨表没食子儿茶素-3-没食子酸酯调控非小细胞肺癌细胞周期和凋亡的机制

Mechanism of Epigallocatechin-3-gallate Regulating Cell Cycle and Apoptosis of Non-small Cell Lung Cancer(NSCLC)Based on EGFR/PI3K/AKT/mTOR Pathway

目的:基于表皮生长因子受体(epidermal growth factor receptor,EGFR)/磷脂酰肌醇激酶(phosphoinositide 3-kinase,PI3K)/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)通路探讨表没食子儿茶素-3-没食子酸酯(epigallocatechin-3-gallate,EGCG)调控非小细胞肺癌(non-small cell lung cancer,NSCLC)A549细胞周期和凋亡的作用机制。方法:将A549细胞随机分为空白对照组、模型组、EGCG组、吉非替尼组、EGCG+吉非替尼组。采用MTT法检测A549细胞的增殖情况,流式细胞术检测细胞周期和凋亡情况。通过分子对接和细胞热力学迁移实验(cellular thermal shift assay,CETSA)检测EGCG与EGFR的靶向结合情况。采用Western blot检测细胞周期蛋白依赖性激酶4(cyclin-dependent kinase 4,CDK4)、B-细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)、p-EGFR/EGFR、p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR表达水平。结果:与模型组比较,EGCG和吉非替尼均对细胞表现出显著的抑制作用。与空白对照组比较,模型组在G1/G0期细胞百分比降低,在G2/M期细胞百分比升高(P<0.01);与模型组比较,EGCG组、吉非替尼组和EGCG+吉非替尼组在G1/G0期细胞百分比升高(P<0.01),吉非替尼组和EGCG+吉非替尼组在G2/M期细胞百分比降低(P<0.01)。与空白对照组比较,模型组细胞凋亡率降低(P<0.01);与模型组比较,EGCG组、吉非替尼组和EGCG+吉非替尼组的细胞凋亡率升高(P<0.05)。与空白对照组比较,模型组细胞CDK4、Bcl-2、Bcl-2/Bax蛋白表达水平升高(P<0.01),Bax蛋白表达水平降低(P<0.01);与模型组比较,EGCG组、吉非替尼组和EGCG+吉非替尼组细胞CDK4、Bcl-2、Bcl-2/Bax蛋白表达水平降低(P<0.01),Bax蛋白表达水平增加(P<0.05)。分子对接结果显示,EGCG和吉非替尼与EGFR的结合能均小于-5.0 kcal·mol^(-1)。CETSA结果显示,与RPMI 1640组比较,EGCG组细胞EGFR蛋白的CETSA曲线向右漂移。与空白对照组比较,模型组细胞p-EGFR/EGFR、p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR蛋白表达水平升高(P<0.05);与模型组比较,EGCG组、吉非替尼组、EGCG+吉非替尼组细胞的p-EGFR/EGFR、p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR蛋白表达水平降低(P<0.01)。结论:EGCG可能通过靶向调控EGFR/PI3K/AKT/mTOR信号通路,诱导A549细胞周期阻滞和凋亡,从而发挥抗癌作用。

Objective:Based on epidermal growth factor receptor(EGFR)/phosphoinositide 3-kinase(PI3K)/protein kinase B,Discussion on the AKT/mammalian target of rapamycin(mTOR)Pathway of epigallocatechin-3-gallate,EGCG regulates cell cycle and apoptosis in non-small cell lung cancer(NSCLC)A549.Methods:A549 cells were randomly divided into blank control group,model group,EGCG group,gefitinib group and EGCG+gefitinib group.The proliferation of A549 cells was detected by MTT assay,and the cell cycle and apoptosis were detected by flow cytometry.Molecular docking and cellular thermal shift assay(CETSA)were used to detect the targeted binding of EGCG to EGFR.cyclin-dependent kinase 4(CDK4),B-cell lymphoma-2(Bcl-2)were detected by Western blot.Expression levels of Bcl-2,Bcl2-associated X protein(Bax),p-EGFR/EGFR,p-PI3K/PI3K,p-AKT/AKT,and p-mTOR/mTOR.Results:Compared with the model group,EGCG and gefitinib both showed significant inbibitory effects on A594 cells.Compared with the blank control group,the cell percentage in the model group was decreased in G1/G0 phase and increased in G2/M phase(P<0.01).Compared with model group,the cell percentage of EGCG group,gefitinib group and EGCG+gefitinib group was increased in G1/G0 stage(P<0.01),while the cell percentage of gefitinib group and EGCG+gefitinib group was decreased in G2/M stage(P<0.01).Compared with blank control group,apoptosis rate of model group was decreased(P<0.01).Compared with model group,the apoptosis rate of EGCG,gefitinib and EGCG+gefitinib groups was increased(P<0.05).Compared with blank control group,CDK4,Bcl-2 and Bcl-2/Bax protein expression levels in model group were increased(P<0.01),while Bax protein expression levels were decreased(P<0.01).Compared with model group,the expression levels of CDK4,Bcl-2 and Bcl-2/Bax in EGCG group,gefitinib group and EGCG+gefitinib group were decreased(P<0.01),while the expression levels of Bax were increased(P<0.05).Molecular docking results showed that the binding energy of EGCG and gefitinib with EGFR was less than-5.0 kcal·mol^(-1).The CETSA results showed that compared with RPMI 1640 group,the CETSA curve of EGFR protein in EGCG group shifted to the right.Compared with blank control group,the protein expression levels of p-EGFR/EGFR,p-PI3K/PI3K,p-Akt/AKT and p-mTOR/mTOR in model group were increased(P<0.05).Compared with model group,the protein expression levels of p-EGFR/EGFR,p-PI3K/PI3K,p-Akt/AKT and p-mtor/mTOR in EGCG group,gefitinib group and EGCG+gefitinib group were decreased(P<0.01).Conclusion:EGCG may regulate A549 cell cycle and apoptosis through targeted regulation of EGFR/PI3K/AKT/mTOR signaling pathway,thus playing an anticancer role.