川楝素通过线粒体途径诱导人肝癌细胞凋亡

Human Hepatocarcinoma Cell Apoptosis Induced by Toosendanin through Mitochondria-dependent Pathway

目的探讨川楝素对人肝癌细胞SMMC-7721(P53+)、Hep3B(P53-)凋亡及其对相关蛋白Bcl-2、Bax和Fas表达的影响。方法 MTT法检测川楝素(0.1～0.9μmol/L)对人肝癌细胞增殖的影响;倒置显微镜观察人肝癌细胞形态学改变;Annexin V法检测早期凋亡率;比色法检测Caspase-3、8、9相对活性改变;免疫细胞化学法检测凋亡相关蛋白Bcl-2、Bax和Fas的表达。结果川楝素显著抑制人肝癌细胞增殖,呈时间-剂量依赖性;形态学观察可见细胞数量减少,黏附力降低,皱缩变圆;0.5μmol/L川楝素作用SMMC-7721、Hep3B细胞72h的凋亡率分别为21.55%、18.35%,经Caspase抑制剂z-VAD-fmk处理后,细胞凋亡率降低;川楝素组SMMC-7721细胞Caspase-3、8、9活性均增高,Hep3B细胞Caspase-3、9活性增高,Caspase-8活性无明显变化;免疫细胞化学法显示,川楝素组SMMC-7721细胞Bcl-2表达减弱,Bax和Fas表达较CTX阳性对照组增强,Hep3B细胞Bcl-2表达减弱,Bax表达增强,Fas表达无明显变化。结论川楝素对P53+和P53-的人肝癌细胞均有抑制增殖、诱导凋亡的作用,涉及线粒体途径的参与,可能是一种通过非P53依赖途径发挥抗癌作用的天然药物。

Objective To explore the effects of toosendanin in inducing apoptosis of human hepatocarcinoma cell line SMMC-7721 and Hep3B,and its influence on the related genes,Bcl-2,Bax and Fas. MethodsThe inhibitory rate of cell proliferation and cell growth curve were detected by MTT assay; morphological changes of cells were observed by inverted microscope; early stage apoptosis rate were detected by Annexin Ⅴ-FITC/PI assay; relative activities of Caspase-3,-8 and-9 were analyzed by spectrophotometry; and the expressions of Bcl-2,Bax and Fas were detected using immunohistochemistry assay. Results Toosendanin presented significant inhibitory effect on proliferation of hepatocarcinoma cells in a time-and dose-dependent manner. After toosendanin treatment,the amount of cells was significantly reduced,shrunk in size and rounded in shape,with decreased adhesion ability. The apoptosis rates of SMMC-7721 cells and Hep3B cells treated with 0.5 μmol/L toosendanin for 72 h were 21.55% and 18.35% respectively,which were reduced after z-VAD-fmk （inhibitor of Caspase） treatment. The activities of Caspase-3,-8 and -9 all markedly enhanced after treatment in SMMC-7721 cells,while in Hep3B cells,activities of Caspase-3 and -9 enhanced,but that of Caspase-8 unchanged. As compared with the control group,after toosendanin treatment,expression of Bcl-2 decreased,and that of Bax and Fas increased in SMMC-7721 cells; but in Hep3B cells the expression of Bcl-2 decreased,that of Bax increased,and expression of Fas unchanged. Conclusions Toosendanin could inhibit the proliferation and induce the apoptosis of both P53＋ and P53-human hepatocarcinoma cells,which involved the participation of mitochondria-dependent pathway. So it may be a kind of natural anti-cancer drug,playing its effect through P53 independent pathway.