Hsp90抑制剂WH-4对肝癌细胞株SK-HEP-1增殖、凋亡及耐药基因表达的影响观察  

Effects of Hsp90 inhibitor WH-4 on proliferation, apoptosis, and drug-resistant gene expression of hepatocarcinoma SK-HEP-1 cells

作　　者：徐单单 王颖 陈素红[1,2] XU Dandan;WANG Ying;CHEN Suhong(Guangdong Food and Drug Vocational College,Guangzhou 510520,China)

机构地区：[1]广东食品药品职业学院生物技术学院,广州510520 [2]暨南大学生命科学技术学院 [3]仲恺农业工程学院食品学院

出　　处：《山东医药》2019年第33期10-14,共5页Shandong Medical Journal

基　　金：广东医学科研基金项目(A2018238);广东食品药品职业学院校级课题(2016YZ001);广州市科技计划项目(201904010050)

摘　　要：目的探讨Hsp90抑制剂WH-4对肝癌细胞SK-HEP-1增殖、凋亡及耐药基因表达的影响。方法将肝癌细胞SK-HEP-1随机分为A、B、C组,A组分别加入0.625、1.25、2.5、5、10μmol/L WH-4,B组分别加入0.625、1.25、2.5、5、10μmol/L 17-AAG,C组加入等量生理盐水,培养48 h后,采用MTT法测算各组肝癌细胞SK-HEP-1增殖抑制率,并计算IC 50。将肝癌细胞SK-HEP-1随机分为A1、B1、C1组,分别加入2.3μmol/L WH-4、2.6μmol/L 17-AAG及生理盐水,培养48 h后,采用BrdU法观察肝癌细胞SK-HEP-1增殖活性。将肝癌细胞SK-HEP-1随机分为A2、B2、C2组,分别加入2.25μmol/L WH-4、2.80μmol/L 17-AAG、生理盐水,培养48 h后,采用软琼脂平板实验检测肝癌细胞SK-HEP-1克隆形成率,流式细胞术检测肝癌细胞SK-HEP-1凋亡率,采用Western blotting法检测肝癌细胞SK-HEP-1凋亡蛋白Bcl2、Bax、Bcl-xL。将肝癌细胞SK-HEP-1随机分为A3、B3、C3组,分别加入2.30μmol/L WH-4、2.65μmol/L 17-AAG及生理盐水。培养48 h后,采用qPCR法检测耐药基因ABCB1、ABCG2。结果WH-4对肝癌细胞SK-HEP-1有抑制作用,且呈浓度依赖性(R 2=0.647(48h),P均<0.05),IC 50为(2.35±0.25)μmol/L。与C1组相比,A1、B1组SK-HEP-1绿色荧光减弱。A2、B2组克隆形成率相比,P<0.05。与B2组比较,A2组细胞凋亡率升高(t=0.342,P<0.05),Bax蛋白相对表达量升高,B淋巴细胞瘤-2基因及Bcl-xL蛋白相对表达量降低。A3组ABCB1、ABCG2基因相对表达量低于B3组(t分别为-8.88、-13.00,P均<0.05)。结论Hsp90抑制剂WH-4对肝癌细胞SK-HEP-1具有增殖抑制、诱导凋亡作用,同时可降低耐药基因的表达。Objective To investigate the effects of heat shock protein 90(Hsp90)inhibitor WH-4 on the proliferation,apoptosis,and drug-resistant gene expression of the hepatocarcinoma SK-HEP-1 cells.Methods The liver cancer cells SK-HEP-1 were randomly divided into groups A,B,and C.The cells in the group A were treated with different concentration of WH-4(0.625,1.25,2.5,5,and 10μmol/L),group B were treated with different concentration of 17-AAG(0.625,1.25,2.5,5,and 10μmol/L),and group C with equal volume of normal saline for 48 h.MTT assay was employed to evaluate the proliferation inhibition rates of WH-4,and IC 50 values were calculated.SK-HEP-1 cells were randomly divided into groups A1,B1,and C1.Cells in the groups A1,B1,and C1 were treated with 2.3μmol/L WH-4,2.6μmol/L 17-AAG,and equal volume of normal saline for 48 h,respectively.BrdU assay was used to detect the proliferation activity of SK-HEP-1 cells.SK-HEP-1 cells were randomly divided into groups A2,B2,and C2,which were treated with 2.25μmol/L WH-4,2.80μmol/L 17-AAG,and equal volume of normal saline for 48 h,respectively.The cloning efficiency was evaluated by soft agar plate experiment assay.Flow cytometry was used to detect the apoptosis of SK-HEP-1 cells.The expression levels of B lymphocytoma-2(Bcl2),Bax,and Bcl-xL in SK-HEP-1 cells were analyzed by Western blotting.SK-HEP-1 cells were randomly divided into groups A3,B3,and C3,which were treated with 2.30μmol/L WH-4,2.65μmol/L 17-AAG,and equal volume of normal saline for 48 h,respectively.The resistance genes ABCB1 and ABCG2 were evaluated by qPCR.Results WH-4 inhibited the viability of the SK-HEP-1 cells in a dose-dependent manner and the IC 50 value was 2.35±0.25μmol/L[R 2=0.647(48 h)and 0.524(24 h)].Compared with the group C1,the green fluorescence of SK-HEP-1 in the groups A1 and B1 was weakened.Significant difference was found in the clone formation rates between the groups A2 and B2(P<0.05).Compared with group B2,the apoptosis rate of group A2 increased(t=0.342,P<0.05),the relative expression o

关 键 词：热休克蛋白90抑制剂 WH-4 肝癌 B淋巴细胞瘤-2基因 B淋巴细胞瘤-2基因相关蛋白 多药耐药基因 ABCB1 乳腺癌耐药蛋白 

分 类 号：R446.6[医药卫生—诊断学]