【作者】 盛庆丰； 史玉荣； 郝继辉； 李强；

【Author】 Sheng Qing-Feng Shi Yu-Rong Hao Ji-Hui Li Qiang Department of hepatobiliary,Tianjin Medical University Cancer Institute and Hospital,Tianjin 300060

【机构】 天津医科大学附属肿瘤医院肝胆外科；

【摘要】 目的:探讨蛋白酶体抑制剂MG132对TRAIL诱导肝癌细胞系SMMC-7721凋亡的增敏作用。方法:对肝癌细胞系SMMC-7721分别单用TRAIL(250、500、750ng／ml), MG132(1、2、5 μmol／l),联合应用TRAIL(500 ng／ml)和 MG 132(2 μmol／l)处理,用MTT法检测不同药物不同浓度不同时间各组细胞生长抑制作用,用Annexin V—FITC流式细胞仪检测细胞凋亡作用。结果:(1)单独使用TRAIL,分别检测24、48、72和196小时各组生长抑制率。处理组(250、500、 750ng／ml)与对照组比较,细胞生长抑制明显。500 ng／ml处理组与其他两处理组比较,24和196小时无明显区别,48和72 小时抑制率有差异(8．7％、19．7％和7．9％,15．7%、26．9％和18． 9％)。(2)单独使用MG132,分别检测12、24、48和72小时各组生长抑制率。处理组(1、2、5μmol／l)与对照组比较,细胞生长抑制明显。2 μmol／l处理组与其他两处理组比较,12、 24和72小时无明显区别,48小时抑制率有差异(11．1％、23． 0％和17．6％)。(3)联合应用TRAIL(500 ng／ml)和MG132(1、 2、5 μ mol／I),分别检测12、24、48和72小时各组生长抑制率。MG132(1 μmol／l)联合组与单用组无明显区别。MG132 (2．5 μmol／l)联合组于24、48、72小时抑制率均明显高于单用TRAIL,MG 132组。(4)用Annexin V—FITC流式细胞仪检测细胞凋亡。TRAIL(500 ng／ml)、MG132(2 μ mol／l)、联合应用TRAIL(500 ng／ml)和MG132(2μmol／l)处理,分别检测48和72小时凋亡指数,为7．84、10．25、24．04,和8． 04、11．56、29．8。结论:TRAIL对肿瘤细胞具有诱导凋亡作用,可以作为一种潜在抗肿瘤药物,因其对正常细胞无抑制作用,可以高效特异杀伤体内肿瘤细胞。单独应用TRAIL, 随时间延长,其所诱导的凋亡过程不显著。但合并使用蛋白酶体抑制剂MG132,可以有效增加肿瘤细胞对TRAIL的敏感性。因此,蛋白酶体抑制剂MG132和TRAIL诱导肝癌细胞凋亡具有协同作用,具有很高的临床应用价值。更多还原

【Abstract】 Objective: To investigate the combined effects of proteasome inhibitor MG132 and TRAIL on inducing apoptosis in hepatoma cell lines. Methods: TRAIL(250、 500、 750ng/ml),MG132(1, 2, 5 μmol/l) were used separately in culured hepatocellular carcinoma cell lines. The same cell lines were cotreated with TRAIL(500 ng/ml)and MG 132(1,2, 5 μmol/1), after 24, 48,72 and 96 hours, cell survival and inhibiting rate were analyzed by MTT assay. Apoptosis was detected by flow cytometry with Annexin V -FITC. Results: (1) Hepatocellular carcinoma cell lines were treated with different concentrations of TRAIL(250,500,750ng/ml)and MG132 (1,2,5 μmol/1), the inhibiting rates in both treated groups were higher than the controlled groups.The treatment group (500 ng/ml TRAIL)exhibited a pronounced difference with the other two treatment groups(250, 750ng/ml TRAIL)at 48 and 72 hours. The treatment group (2 μmol/ 1 MG132)was significantly different in inhibiting rates compared with the other two groups(1, 5 μ mol/1 MG132)at 48 hours.(2) To test whether the proteasome inhibitor MG132 influence TRAIL sensitivity,we incubated SMMC-7721 cell lines after pretreatment with different concentrations of MG 132(1,2,5 μmol/l)with TRAIL(500 ng/ml). The findings clearly indicated sensitization for combined effects on inducing apoptosis except low concentration of MG 132(1 μ mol/l)group.(3) Finally,we tested apoptosis in different groups by flow cytometry with Annexin V-FITC. The apoptotic index measured in treated groups(500 ng/ ml TRAIL, 2 μ mol/l MG132,500 ng/ml TRAIL+ 2 μ mol/ I MG132) after 48 and 72 hours were 7.84,10.25, 24.04, and 8.04,11.56, 29.8 respectively. Conclusion: TRAIL exhibits potent anti-tumor activity indicated by our experiments.Because of its proven in vivo efficacy, TRAIL may serve as a novel anti-neoplastic drug.However,ap-proximately half of the tumor cell lines tested so far are TRAIL resistant,and potential toxic side effects of certain recombinant forms of TRAIL on human hepatocytes have been described.Pretreatment with the proteasome inhibitor MG132 rendered TRAIL-resistant hepatocellular carcinoma cell lines sensitive for TRAIL-induced apoptosis.So we may expect the possible therapeutic use of TRAIL in combination with proteasome inhibitors for the treatment of HCC.更多还原