小分子酪氨酸激酶抑制剂A2B通过调控PI3K/Akt通路诱导胃癌细胞凋亡  

The small-molecule tyrosine kinase inhibitor A2B induces apoptosis of gastric cancer cells by regulating the PI3K/Akt pathway

作　　者：敖嘉怡 叶连宝 马宇昕[1] 褚夫江[3] 周畅[1] AO Jiayi;YE Lianbao;MA Yuxin;CHU Fujiang;ZHOU Chang(Department of Human Anatomy and Histology and Embryology,School of Basic Medical Sciences,Guangdong Pharma-ceutical University,Guangzhou 510006,China;Department of Medicinal chemistry,School of Pharmacy,Guangdong Pharmaceutical University,Guangzhou 510006,China;School of Basic Medical Sciences,Guangdong Pharmaceutical University/Guangdong Provincial Key Laboratory of Pharmaceutical Bioactive Substances,Guangzhou 510006,China)

机构地区：[1]广东药科大学基础医学院人体解剖与组织胚胎学系,广东广州510006 [2]广东药科大学药学院药物化学系,广东广州510006 [3]广东药科大学基础医学院/广东省生物活性药物研究重点实验室,广东广州510006

出　　处：《中国病理生理杂志》2023年第10期1806-1813,共8页Chinese Journal of Pathophysiology

基　　金：广东省创新强校工程-广东省普通高校重点科研平台项目(No.2022ZDZX2030);广东省医学科学技术研究基金项目(No.B2022076);广东省中医药局科研项目(No.20231201)。

摘　　要：目的:探讨小分子酪氨酸激酶抑制剂A2B诱导人胃癌SGC-7901细胞凋亡的分子机制。方法:体外培养人胃癌SGC-7901细胞并加入2.5~160μmol/L梯度浓度的A2B分别干预24、48和72 h,利用CCK-8法检测细胞活力的半数抑制浓度(IC50),筛选出药物最适浓度。后续实验将SGC-7901细胞随机分为对照组(正常培养组)、溶剂组(含0.08%DMSO培养液处理)、A2B 10μmol/L实验组和A2B 20μmol/L实验组,每组每项实验均重复3次。利用EdU细胞染色实验和平板集落形成实验检测各组细胞增殖能力;Annexin V-FITC/PI双染法检测各组细胞凋亡率;线粒体膜电位检测试剂盒(JC-1染色法)检测各组细胞线粒体膜电位变化;Western blot检测各组细胞中表皮生长因子受体(EGFR)、人表皮生长因子受体2(HER2)、细胞间充质-表皮转化因子(c-Met)、B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、caspase-3、cleaved caspase-3、蛋白激酶B(PKB/Akt)和磷酸化Akt(p-Akt)蛋白的表达情况。结果:(1)A2B作用于人胃癌SGC-7901细胞24、48和72 h的IC50值分别为26.85、19.58和12.24μmol/L;(2)20μmol/L A2B能够同时下调EGFR、HER2和c-Met蛋白水平(P<0.01);(3)10和20μmol/L A2B作用于SGC-7901细胞后,EdU+细胞数和集落数均显著减少(P<0.01);(4)经A2B处理后SGC-7901细胞凋亡率增加(P<0.05),JC-1绿色荧光比例增加(P<0.01),同时Bax/Bcl-2比值和cleaved caspase-3蛋白表达水平上调(P<0.05),caspase-3蛋白表达水平下调(P<0.05);(5)A2B还下调了SGC-7901细胞中p-Akt/Akt比值(P<0.05)。结论:A2B能够靶向作用于EGFR、HER2和c-Met,并通过抑制PI3K/Akt信号通路激活,诱导人胃癌SGC-7901细胞发生凋亡。AIM:To investigate the molecular mechanism of which the small molecule tyrosine kinase inhibi‐tor A2B affects apoptosis in the human gastric cancer cell line SGC-7901.METHODS:Human gastric cancer SGC-7901 cells were cultured in vitro and treated with 2.5~160µmol/L A2B for 24,48 and 72 h,and CCK-8 assay was used to de‐termine the half maximal inhibitory concentration(IC50)in order to select the optimal drug concentration.In the following experiments,the SGC-7901 cells were randomly divided into the control group(normal culture group),vehicle group(containing 0.08%DMSO treatment),A2B 10µmol/L group and A2B 20µmol/L group,and each experiment was re‐peated three times.EdU staining and plate colony formation assay were used to assess the proliferation of cells.Apoptosis was detected via Annexin V-FITC/PI double staining.Changes in mitochondrial membrane potential were tested with a mi‐tochondrial membrane potential detection kit(JC-1 staining).The protein expression levels of epidermal growth factor re‐ceptor(EGFR),human epidermal growth factor receptor 2(HER2),cellular mesenchymal-epithelial transition factor(c-Met),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),caspase-3,cleaved caspase-3,protein kinase B(PKB/Akt)and phosphorylated Akt(p-Akt)were measured by Western blot.RESULTS:(1)The IC50 values of A2B to SGC-7901 cells 24,48 and 72 h were 26.85,19.58 and 12.24µmol/L,respectively.(2)A2B(20µmol/L)significant‐ly downregulated the protein expression of EGFR,HER2 and c-Met(P<0.01).(3)The positivity rates of EdU+cells and the numbers of colonies of SGC-7901 cells were decreased significantly after treatment with 10 and 20µmol/L A2B(P<0.01).(4)After treatment with A2B,the apoptosis rate of SGC-7901 cells was increased(P<0.05),and the proportion of JC-1 green fluorescence was increased(P<0.01).Furthermore,the protein expression of cleaved caspase-3 and ratio of Bax/Bcl-2 were upregulated(P<0.05),while the protein expression of caspase-3 was downregulated(P<0.05).(5)A2B inhibited the ratio of p

关 键 词：酪氨酸激酶抑制剂 胃癌 表皮生长因子受体 人表皮生长因子受体2 细胞间充质-表皮转化因子 

分 类 号：R735.2[医药卫生—肿瘤] R363.2[医药卫生—临床医学]