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目的观察半边旗提取物Ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid(5F)对人肝癌细胞HepG2增殖的影响,并探讨p53、血管内皮生长因子(VEGF)及Caspase-3在5F诱导的HepG2细胞凋亡中的作用。方法采用MTT分析检测5F对HepG2细胞增殖的影响,并通过细胞凋亡检测ELISA试剂盒分析经5F处理的HepG2细胞胞浆核小体片段,以确定5F能否诱导细胞凋亡,采用Hoechst/PI分析鉴定凋亡细胞核形态。通过免疫印迹(Western blotting)分析测定p53及VEGF蛋白表达水平,并通过Caspases-3分光光度法检测试剂盒检测Caspase-3活性。结果通过细胞活性分析证实,5F对HepG2的细胞毒作用随着5F质量浓度的升高而增强。5F诱导HepG2产生胞浆核小体片段,并且该诱导作用具有剂量依赖性。5F处理后,凋亡变化,如染色质浓缩,被Hoechst/PI染色所确定。5F处理后HepG2细胞核内p53表达水平显著提高,而胞浆VEGF表达水平却下降,同时,Caspase-3活性通过浓度依赖方式增强。结论5F所诱导的HepG2细胞凋亡与p53及Caspase-3活化、VEGF负调控有关。5F可能具有抗癌尤其是抗肝细胞癌价值。
OBJECTIVE: To observe the effect of Ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) on the proliferation of human hepatocellular carcinoma HepG2 cells, and to explore p53 and vascular endothelial growth factor (VEGF). The role of Caspase-3 in the 5F-induced apoptosis of HepG2 cells. Methods The effect of 5F on the proliferation of HepG2 cells was detected by MTT assay. The cytoplasmic nucleosome fragments of HepG2 cells treated with 5F were analyzed by the cell apoptosis detection ELISA kit to determine whether 5F could induce apoptosis. Hoechst/PI was used. Analyze and identify the apoptotic nuclear morphology. Western blotting analysis was used to determine the expression of p53 and VEGF protein, and Caspase-3 activity was measured by Caspases-3 spectrophotometric assay kit. Results It was confirmed by cell activity analysis that the cytotoxic effect of 5F on HepG2 increased with increasing 5F mass concentration. 5F induced HepG2 production of cytoplasmic nucleosome fragments, and this induction was dose-dependent. After 5F treatment, changes in apoptosis, such as chromatin condensation, were determined by Hoechst/PI staining. After 5F treatment, the expression of p53 in HepG2 cells was significantly increased, while the expression of cytoplasmic VEGF was decreased. At the same time, the activity of Caspase-3 was enhanced in a concentration-dependent manner. Conclusion The apoptosis of HepG2 cells induced by 5F is related to the activation of p53 and Caspase-3 and the negative regulation of VEGF. 5F may have anti-cancer, especially anti-hepatocellular carcinoma value.