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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 investigate the effects of toosendanin on the apoptosis and the expression of related proteins Bcl-2, Bax and Fas in human hepatocellular carcinoma cell line SMMC-7721 (P53 +) and Hep3B (P53-). Methods MTT assay was used to detect the effect of toosendanin (0.1 ~ 0.9μmol / L) on the proliferation of human hepatocellular carcinoma cells. The morphological changes of human hepatocellular carcinoma cells were observed with inverted microscope. The rate of early apoptosis was detected by Annexin V assay. 8,9, respectively. The expressions of Bcl-2, Bax and Fas were detected by immunocytochemistry. Results Toosendanin significantly inhibited the proliferation of human hepatocellular carcinoma cells in a time-and-dose-dependent manner. Morphological observation showed that the number of cells decreased, the adhesion decreased, and the wrinkles became round. The effect of TSN on SMMC-7721 and Hep3B cells 72h The apoptotic rates of TSMCs were decreased by 21.55% and 18.35%, respectively. The apoptosis rates of the cells were decreased after treatment with z-VAD-fmk, a caspase inhibitor. The activities of Caspase-3, The activity of Caspase-3 and 9 in Caspase-3 cells increased while the activity of Caspase-8 did not change significantly. Immunocytochemistry showed that the expression of Bcl-2 and Bax and Fas in SMMC-7721 cells were decreased, while the expression of Bax and Fas in Hep3B cells increased Bcl-2 expression decreased, Bax expression increased, Fas expression did not change significantly. CONCLUSION Toosendanin can inhibit proliferation and induce apoptosis in P53 + and P53- -induced human hepatocarcinoma cells and involve in mitochondrial pathway, which may be a natural drug that exerts anticancer effects through non-P53-dependent pathway.