EGFR抑制剂AG1478联合奥沙利铂抑制PI3K/AKT通路促进H1975细胞自噬的作用机制  

Mechanism of EGFR inhibitor AG1478 combined with oxaliplatin in inhibiting PI3K/AKT pathway and promoting autophagy in H1975 cells

作　　者：黄金清 李洋[1] 韦东雪 江山 江绍锋 HUANG Jin-qing;LI Yang;WEI Dong-xue;JIANG Shan;JIANG Shao-feng(College of Basic Medical Sciences,Guilin Medical University,Guangxi Key Laboratory of Tumor Immunology and Microenvironmental Regulation,Guilin Guangxi 541199,China)

机构地区：[1]桂林医学院基础医学院,广西肿瘤免疫与微环境调控重点实验室,广西桂林541199

出　　处：《中国药理学通报》2024年第2期272-278,共7页Chinese Pharmacological Bulletin

基　　金：广西自然科学基金资助项目(No 2021GXNSFBA196002);广西肿瘤免疫与微环境调控重点实验室资助项目No(203030302213);桂林医学院博士科研基金资助项目(No 31304018011)。

摘　　要：目的探究奥沙利铂(oxaliplatin,OXA)联合表皮生长因子受体酪氨酸激酶抑制剂AG1478对非小细胞肺癌(non-small cell lung cancer,NSCLC)H1975细胞自噬的作用。方法采用梯度浓度的AG1478(0、5、10、15、20、25、30、35、40μmol·L^(-1))和OXA(0、25、50、75、88、100、112、125、150μmol·L^(-1))体外培养H1975细胞,MTT法检测AG1478和OXA对H1975细胞活力的影响,MDC法检测H1975细胞自噬水平,Western blot法检测PI3K/AKT信号通路(PI3K、p-PI3K、AKT、p-AKT)以及自噬相关蛋白(Beclin^(-1)、LC3Ⅱ)的表达水平;ROS探针H2DCFDA检测H1975细胞的ROS水平。结果MTT结果显示,OXA作用于H1975细胞的半数抑制浓度IC 50为70.86μmol·L^(-1);AG1478作用于H1975细胞的IC 50为24.24μmol·L^(-1);MDC实验结果显示,与对照组比较,(OXA-25、OXA-50、OXA^(-1)00)OXA单药组、AG1478单药组及联合用药组自噬水平明显升高;与OXA单药组(OXA-25)比较,联合用药组自噬水平升高;Western blot检测结果显示,与对照组比较,(OXA-25、OXA-50、OXA^(-1)00)OXA单药组、AG1478单药组及联合用药组PI3K、p-PI3K、AKT、p-AKT蛋白表达均下调,应用N-乙酰半胱胺酸(N-acetylcysteine,NAC)预处理后,联合用药组PI3K、p-PI3K、AKT、p-AKT下调水平被抑制;与OXA单药组(OXA-25)比较,联合用药组PI3K、p-PI3K、AKT、p-AKT的蛋白表达水平均下降;Western blot检测结果显示,与对照组比较,(OXA-25、OXA-50、OXA^(-1)00)OXA单药组、AG1478单药组及联合用药组Beclin^(-1)、LC3Ⅱ的蛋白表达上调,应用NAC预处理后,联合用药组Beclin^(-1)、LC3Ⅱ的蛋白表达被抑制,应用PI3K抑制剂(LY294002)预处理后,联合用药组Beclin^(-1)、LC3Ⅱ的蛋白表达明显上调;与OXA单药组(OXA-25)比较,联合用药组Beclin^(-1)、LC3Ⅱ的蛋白表达水平升高;H2DCFDA检测发现,与对照组比较,(OXA-25、OXA-50、OXA^(-1)00)OXA单药组、AG1478单药组及联合用药组ROS水平升高;与对照组比较,联合用药组ROS水平明显升高,NAC处理组ROAim To explore the effect of oxaliplatin combined with epidermal growth factor receptor tyrosine kinase inhibitor AG1478 on autophagy in non-small cell lung cancer H1975 cells.Methods H1975 cells were cultured in vitro using gradient concentrations of AG1478(0,5,10,15,20,25,30,35,40μmol·L^(-1))and OXA(0,25,50,75,88,100,112,125,150μmol·L^(-1)).MTT assay was used to detect the effects of AG1478 and OXA on the viability of H1975 cells.MDC assay was used to detect autophagy levels in H1975 cells.Western blot was used to detect the expression levels of PI3K/AKT pathways(PI3K,p-PI3K,AKT,p-AKT)and autophagy related proteins(Beclin^(-1),LC3Ⅱ).ROS probe H2DCFDA was used to detect ROS levels in H1975 cells.Results The MTT results showed that the half inhibitory concentration IC 50 of OXA was 70.86μmol·L^(-1)on H1975 cells.AG1478 had an IC 50 of 24.24μmol·L^(-1)on H1975 cells.The MDC experiment results showed that compared with the control group,the autophagy levels in the(OXA-25,OXA-50,OXA^(-1)00)OXA single drug group,AG1478 single drug group and combination drug group significantly increased.Compared with the OXA monotherapy group(OXA-25),the autophagy level in the combination therapy group significantly increased.Western blotting showed that compared with the control group,the protein expression of PI3K,p-PI3K,AKT and p-AKT in the(OXA-25,OXA-50,OXA^(-1)00),AG1478,and combination groups were all down-regulated.After pretreatment with N-acetylcysteine(NAC),the down-regulation levels of PI3K,p-PI3K,AKT and p-AKT in the combination group were inhibited.Compared with the OXA monotherapy group(OXA-25),the protein expression levels of PI3K,p-PI3K,AKT and p-AKT in the combination therapy group decreased;Western blot showed that compared with the control group,the protein expression of Beclin^(-1)and LC3Ⅱwas up-regulated in the(OXA-25,OXA-50,OXA^(-1)00)single drug group,AG1478 single drug group,and combination drug group.After pretreatment with N-acetylcysteine(NAC),the up-regulation levels of Beclin^(-1)and LC3Ⅱin

关 键 词：非小细胞肺癌 AG1478 奥沙利铂 ROS 自噬 PI3K/AKT信号通路 

分 类 号：R345.57[医药卫生—基础医学] R392R973.2R979.1