奥沙利铂联合AG1478调控活性氧抑制PI3K/AKT通路对肺癌的影响  

Effect of oxaliplatin combined with AG1478 on lung cancer by regulating reactive oxygen species and inhibiting PI3K/AKT pathway

作　　者：韦东雪 江绍锋 崔珊 李洋[1] 黄金清 WEI Dong-xue;JIANG Shao-feng;CUI Shan;LI Yang;HUANG Jin-qing(Guangxi Key Laboratory of Tumor Immunology and Microenvironmental Regulation,College of Basic Medical Sciences,Guilin Medical University,Guilin 541199,Guangxi Zhuang Autonomous Region,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：《中国临床药理学杂志》2024年第12期1754-1758,共5页The Chinese Journal of Clinical Pharmacology

基　　金：广西自然科学基金资助项目(2021GXNSFBA196002);广西高校中青年教师科研基础能力提升基金资助项目(2021KY0522);广西肿瘤免疫与微环境调控重点实验室基金资助项目(203030302213)。

摘　　要：目的探究表皮生长因子酪氨酸激酶抑制药AG1478联合奥沙利铂(OXA)对非小细胞肺癌细胞H1299的影响。方法将体外培养的H1299细胞分为对照组(常规培养,不加药)、OXA-25组、OXA-50组、OXA-100组(分别用25、50和100μmol·L^(-1)OXA处理)、AG-20组(20μmol·L^(-1)AG1478处理)和OXA+AG组(25μmol·L^(-1)OXA+20μmol·L^(-1)AG1478处理)。用MTT法检测细胞存活率计算半数抑制浓度(IC50);用活性氧(ROS)探针H2DCFDA检测细胞ROS水平;用丹酰尸胺(MDC)法检测细胞自噬情况;用蛋白质印迹法检测磷酸化磷脂酰肌醇3激酶(p-PI3K)、磷酸化蛋白激酶B(p-AKT)、自噬蛋白(Beclin-1)、微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)的表达水平。结果OXA作用于H1299细胞的IC50为91.09μmol·L^(-1);AG1478作用于H1299细胞的IC50为31.83μmol·L^(-1)。对照组、OXA-25组、OXA-50组、OXA-100组、AG-20组和OXA+AG组的ROS水平分别为1.00±0.03、1.15±0.02、1.76±0.04、2.89±0.02、1.05±0.01和3.20±0.03;MDC水平分别为1.00±0.04、1.10±0.02、1.16±0.02、1.46±0.04、1.04±0.01和1.31±0.02;p-PI3K蛋白表达水平分别为1.12±0.05、0.88±0.06、0.72±0.07、0.60±0.05、0.91±0.07和0.64±0.09;p-AKT蛋白表达水平分别为1.09±0.04、0.87±0.08、0.77±0.07、0.63±0.05、0.76±0.05和0.46±0.03;Beclin-1蛋白表达水平分别为0.82±0.03、0.91±0.04、1.06±0.28、1.11±0.03、0.87±0.04和1.27±0.10;LC3-Ⅱ蛋白表达水平分别为0.65±0.08、0.82±0.11、1.08±0.12、1.38±0.09、0.72±0.11和1.38±0.15。OXA+AG组、OXA各剂量组的上述指标与对照组比较,差异均有统计学意义(P<0.05,P<0.01);OXA+AG组的上述指标与OXA各剂量组比较,在统计学上差异均有统计学意义(P<0.05,P<0.01)。结论AG1478联合OXA作用于非小细胞肺癌细胞H1299,可升高ROS水平抑制PI3K/AKT信号通路激活,进而诱导细胞发生自噬。Objective To investigate the effect of epidermal growth factor tyrosine kinase inhibitor AG1478 combined with oxaliplatin(OX A) on non-small cell lung cancer cells H1299.Methods H1299cells were divided into control group(conventional culture,without drug),OXA-25,-50,-100 groups(25,50 and 100 μmol·L^(-1)OXA),AG-20 group(20 μmol·L^(-1)AG1478) and OXA+AG group(25 μmol·L^(-1)OXA and 20 μmol·L^(-1)AG1478).Detection of cell survival rate by MTT assay and calculation of half inhibitory concentration(IC_(50)).Reactive oxygen species(ROS) probe H2DCFDA was used to detect ROS levels.Autophagy of H1299 cells was detected by MDC method.Western blot was used to detect the expression levels of phosphorylated-phosphoinositide 3-kinase(p-PI3 K),phosphorylated-protein kinase B(p-AKT),autophagy protein(Beclin-1) and microtubule associated protein light chain 3-Ⅱ(LC3-Ⅱ).Results The IC_(50) of OXA on H1299 cells was 91.09 μmol·L^(-1).The IC_(50) of AG1478 on H1299 cells was 31.83 μmol·L^(-1).The relative ROS level were 1.00±0.03,1.15±0.02,1.76±0.04,2.89±0.02,1.05±0.01 and 3.20±0.03,respectively;the relative MDC levels were 1.00±0.04,1.10±0.02,1.16±0.02,1.46±0.04,1.04±0.01 and 1.31±0.02,respectively;the relative expression levels of p-PI3K protein were 1.12±0.05,0.88±0.06,0.72±0.07,0.60±0.05,0.91±0.07 and 0.64±0.09,respectively;the relative expression levels of p-AKT protein were1.09±0.04,0.87±0.08,0.77±0.07,0.63±0.05,0.76±0.05 and 0.46±0.03,respectively;the relative expression levels of Beclin-1 protein were 0.82±0.03,0.91±0.04,1.06±0.28,1.11±0.03,0.87±0.04 and1.27±0.10,respectively;the relative expression levels of LC3-Ⅱ protein were 0.65±0.08,0.82±0.11,1.08±0.12,1.38±0.09,0.72±0.11 and 1.38±0.15,respectively.Compared the OXA+AG group and the OXA single group with the control group,compared the OXA+AG group with the OXA single group,there were statistically significant differences in the above indicators(P <0.05,P <0.01).Conclusion AG1478 combined with OXA can increase ROS level

关 键 词：AG1478 奥沙利铂 非小细胞肺癌 活性氧 磷脂酰肌醇3激酶信号通路 自噬 

分 类 号：R979.1[医药卫生—药品]