莫特塞尼与EZH2抑制剂GSK126联合应用对肝癌Huh7细胞增殖和凋亡的影响及其机制  

Effect of motesanib combined with EZH2 inhibitor GSK126 on proliferation and apoptosis of liver cancer Huh7 cells and its mechanism

作　　者：梁媛媛 赵颂 胡静 安妮 魏验芦 苏荣健 LIANG Yuanyuan;ZHAO Song;HU Jing;AN Ni;WEI Yanu;SU Rongjian(Department of Cell Biology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121001,China;Department of Rheumatoid Immunity,First Affiliated Hospital,Jinzhou Medical University,Jinzhou 121000,China)

机构地区：[1]锦州医科大学基础医学院细胞生物学教研室,辽宁锦州121000 [2]锦州医科大学附属第一医院风湿免疫科,辽宁锦州121000

出　　处：《吉林大学学报（医学版）》2023年第4期896-904,共9页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金项目(81903109);辽宁省教育厅自然科学基金项目(2020-MS-299)。

摘　　要：目的:探讨多激酶抑制剂莫特塞尼联合果蝇zeste基因增强子的人类同源物2 (EZH2)抑制剂GSK126对肝癌Huh7细胞体外增殖和凋亡的影响,初步阐明其相关机制。方法:不同浓度(0、1、5、10、20、40和60μmol·L^(-1))莫特塞尼处理Huh7细胞,采用CCK-8法检测各组Huh7细胞增殖率,采用Western blotting法检测不同浓度(0、2.5、5.0、10.0和20.0μmol·L^(-1))莫特塞尼组Huh7细胞中EZH2和磷酸化EZH2 (p-EZH2)蛋白表达水平。莫特塞尼和GSK126联合作用于Huh7细胞,采用CCK-8法和克隆形成实验确定后续实验合适的药物浓度。Huh7细胞分为对照组、莫特塞尼(10μmol·L^(-1))组、GSK126 (5μmol·L^(-1))组和联合组(莫特塞尼+GSK126),采用5-乙炔基-2’脱氧尿嘧啶核苷(EdU)荧光染色法检测各组Huh7细胞增殖率,流式细胞术检测各组Huh7细胞凋亡率,Western blotting法检测各组Huh7细胞中细胞外信号调节激酶(ERK)、磷酸化ERK (p-ERK)、蛋白激酶B (AKT)和磷酸化AKT (p-AKT)蛋白表达水平。结果:CCK-8法检测,不同浓度莫特塞尼组Huh7细胞存活率随着药物浓度升高逐渐降低,与0μmol·L^(-1)莫特塞尼组比较,20、 40和60μmol·L^(-1)莫特塞尼组Huh7细胞增殖率明显降低(P<0.05或P<0.01);Western blotting法检测,与0μmol·L^(-1)莫特塞尼组比较,5.0、10.0和20.0μmol·L^(-1)莫特塞尼组Huh7细胞中EZH2和p-EZH2蛋白表达水平明显升高(P<0.01)。通过CCK-8法和克隆形成实验确定10μmol·L^(-1)莫特塞尼和5μmol·L^(-1) GSK126用于后续实验。与对照组比较,莫特塞尼组、GSK126组和联合组Huh7细胞增殖率明显降低(P<0.01),细胞凋亡率明显升高(P<0.05或P<0.01);与莫特塞尼组和GSK126组比较,联合组Huh7细胞增殖率明显降低(P<0.01),细胞凋亡率明显升高(P<0.01)。与对照组、莫特塞尼组和GSK126组比较,联合组Huh7细胞中p-AKT和p-ERK蛋白表达水平明显降低(P<0.05或P<0.01)。结论:单独应用莫特塞尼抑制肝癌Huh7细胞增殖和促进凋�Objective:To discuss the effect of multi-kinase inhibitor motesanib combined with enhancer of zeste homolog 2(EZH2)inhibitor GSK126 on the proliferation and apoptosis of the liver cancer Huh7 cells in vitro,and to clarify its related mechanism.Methods:The Huh7 cells were treated with different concentrations(0,1,5,10,20,40,and 60μmol·L^(-1) )of motesanib.The proliferation rates of Huh7 cells in various groups were detected by CCK-8 assay;Western blotting method was used to detect the expression levels of EZH2 and phosphorylated EZH2(p-EZH2)proteins in the Huh7 cells in different concentrations(0,2.5,5.0,10.0,20.0μmol·L^(-1) )of motesanib groups.The Huh7 cells were treated with motesanib and GSK126 in various groups;the appropriate drug concentrations were selected by CCK-8 assay and colony formation assay.The Huh7 cells were divided into control group,motesanib(10μmol·L^(-1) )group,GSK126(5μmol·L^(-1) )group,and combination(motesanib+GSK126)group.The 5-ethynyl-2'-deoxyu ridine(EdU)fluorescence staining assay was used to detect the proliferation rates of Huh7 cells in various groups;the apoptotic rates of Huh7 cells in various groups were detected by flow cytometry;Western blotting method was used to detect the expression levels of extracellular regulated protein kinases(ERK),phosphorylated ERK(p-ERK),protein kinase B(AKT),and phosphorylated AKT(p-AKT)proteins in the Huh7 cells in various groups.Results:The CCK-8 assay results showed that proliferation rate of Huh7 cells in different concentrations of motesenib groups were decreased gradually with the increasing of drug concentrations;compared with motesenib(0μmol·L^(-1) )group,the proliferation rates of the Huh7 cells in 20,40,and 60μmol·L^(-1) motesenib groups were decreased significantly(P<0.05 or P<0.01).The Western blotting results showed that compared with 0μmol·L^(-1) motesanib group,the expression levels of EZH2 and p-EZH proteins in the Huh7 cells in 5,10,and 20μmol·L^(-1) motesanib groups were increased(P<0.05 or P<0.01).The CCK-8 results

关 键 词：莫特塞尼 肝肿瘤 细胞增殖 细胞凋亡 果蝇zeste基因增强子的人类同源物2 GSK126 

分 类 号：R735.7[医药卫生—肿瘤]