MG-132诱导Tca-8113细胞凋亡的机制探讨

Apoptosis induced by MG-132 in Tca-8113 cells

目的:探讨内质网应激在蛋白酶抑制剂MG-132诱导人舌鳞癌细胞株Tca-8113细胞凋亡中的作用。方法:使用含10%胎牛血清的RPMI-1640培养基常规培养Tca-8113细胞,实验分为5组,3个实验组分别加入MG-132,终浓度为10.0、20.0、30.0μmol/L;阳性对照组加入毒胡萝卜素,终浓度为5.0μmol/L;阴性对照组加入1640培养液。培养24h后,Hoechst 33258染色法观察凋亡细胞形态学变化;Annexin V-FITC/PI双染法检测细胞凋亡率,RT-PCR检测GRP78 mRNA,Western印迹检测caspase-12蛋白的表达,ELISA法检测人泛素蛋白连接酶E3浓度。应用SPSS16.0软件包对结果进行统计学分析。结果:MG-132作用Tca-8113细胞24 h后,细胞出现典型的凋亡形态;MG-132实验组细胞凋亡率随MG-132浓度升高而逐渐升高,存在浓度依赖性;MG-132组GRP78的mRNA及caspase-12蛋白表达增强;MG-132组人泛素连接酶E3的浓度分别为28.75±2.28、18.16±0.65、8.85±0.72。结论:MG-132可通过内质网应激途径诱导Tca-8113细胞的凋亡,MG-132可抑制人泛素连接酶E3的表达。

PURPOSE： To explore the apoptotic effect of ubiquitin-proteasome inhibitor（PI） MG-132 on human tongue squamous cell carcinoma cell line（Tca-8113 cell） through endoplasmic reticulum stress.METHODS： Tca-8113 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum,the exponential cells were divided into 5 groups.The cell culture medium was added to 1640（negative control group）,thapsigargin 5μmol / L（positive control group）,and 10,20,30 μmol / L（MG-132 group）.After 24h,the following experiments were carried out： morphological change of apoptotic cells was observed by Hoechst 33258 fluorescent staining under fluorescent microscope,apoptotic rate of cells was determined with annexinⅤ-FITC/PI double staining by flow cytometry,the GRP78 mRNA level was determined by RT-PCR,the expression of caspase-12 protein was evaluated by Western blot,the human ubiquitin-protein ligase E3 concentration was detected by ELISA.The data was analyzed using SPSS16.0 software package.RESULTS： Typical morphological change of apoptosis in Tca-8113 cells was observed 24 hours after treating with 10.0,20.0,30.0μmol/L MG-132 and positive control group;The apoptotic rate of MG-132 groups gradually increased with MG-132 concentration;The GRP78 mRNA level was up-regulated;The expression of caspase-12 increased was demonstrated by Western blot;The expression of the human ubiquitin-protein ligase E3 in MG-132 groups was 28.75±2.28,18.16±0.65 and 8.85±0.72.CONCLUSIONS： MG-132 can induce apoptosis of Tca-8113 cells through endoplasmic reticulum stress;MG-132 can inhibit the expression of human ubiquitin ligase E3.