番木瓜种子提取物异硫氰酸苄酯对肝癌细胞凋亡的影响

Benzyl isothiocyanate induces apoptosis of hepatocarcinoma cells

目的:通过异硫氰酸苄酯(benzyl-isothiocyanate,BITC)对体外培养的人肝癌细胞株HLE和Bel7402的凋亡影响,初步探讨BITC调控肝癌细胞凋亡的可能机制.方法:3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)染色法检测不同浓度BITC对肝癌细胞增殖的影响;选择具有抑制肝癌细胞生长的BITC浓度处理肝癌细胞后4,6-二脒基-2-苯基吲哚二盐酸盐(DAPI)染色法进行凋亡的形态学观察;Caspase3酶活性检测试剂盒检测细胞中的Caspase3酶活性;Western blot法检测具代表性的凋亡相关蛋白Caspase8和Caspase3,细胞周期相关蛋白Cyclin D1的表达.结果:MTT结果显示BITC对人肝癌细胞的增殖有明显的抑制作用,用浓度为40μmol/L的BITC处理时,HLE和Bel 7402细胞存活率分别为71.56%和78.09%,和对照组比较,有统计学差异(P<0.05);当用浓度为80μmol/L的BITC处理时,HLE和Bel 7402生长率分别为32.91%和53.06%,和对照组比较,有统计学显著性差异(P<0.01);经BITC处理后的细胞,DAPI染色显示细胞特征性凋亡变化;Western blot和Caspase活性检测结果显示,随着BITC浓度增加,Caspase8和Caspase3蛋白表达水平及活性呈上升趋势,而细胞周期蛋白Cyclin D1的表达则明显下降.结论:BITC对人肝癌细胞增殖有抑制作用,并能诱导肝癌细胞凋亡;BITC通过激活Caspase8和Caspase3信号途径以及抑制细胞周期进展诱导细胞凋亡.

AIM： To investigate the influence of benzyl isothiocyanate（BITC） on apoptosis of hepatoma carcinoma cell lines HLE and Bel-7402 in vitro, and to explore the possible mechanism involved. METHODS： After HLE and Bel-7402 cells were treated with an optimal concentration of BITC for 24 h, cell proliferation was tested by MTT assay, the morphology of apoptotic hepatoma cells was observed after DAPI staining, the enzymatic activity of caspase 3 was assayed using Caspase3 Activity Assay Kit, and expression of caspase 8, caspase 3 and cell cycle related protein cyclin D1 were detected by Western blot. RESULTS： MTT analysis showed that BITC had an obvious inhibitory effect on the proliferation of hepatoma cells. The growth rates of HLE and Bel-7402 cells treated with 40 μmol/L BITC were 71.56% and 78.09%（P ＆lt; 0.05 vs control groups）, respectively, and the growth rates of HLE and Bel-7402 cells treated with 80 μmol/BITC were 32.91% and 53.06%（P ＆lt; 0.01 vs control groups）, respectively. Hepatoma cells treated with BITC tended to showed characteristic morphological changes of apoptosis. BITC treatment elevated the activity of caspase 3 in hepatoma cells, stimulated the expression of caspase 3 and caspase 8, but inhibited cyclin D1 expression. CONCLUSION： BITC induces apoptosis of hepatoma cells possibly by activating the caspase signal pathway and inhibiting cell cycle progression.