半边旗提取物5F诱导HepG2细胞凋亡与p53活化及血管内皮生长因子抑制有关

Apoptosis in HepG2 cells induced by Pteris semipinnata extract 5F involves p53 activation and VEGF inhibition

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 Pteris semipinnata extract, ent-lla-hydroxy-15-oxo-kaur-16-en-19-oic-acid （SF）, on human hepatoma HepG2 cells about the proliferation and to investigate the role of p53, VEGF, and Caspase-3 in 5F-induced HepG2 cell apoptosis. Methods MTT Assay was used tO determine the effect of 5F on proliferation of HepG2 cells, and cytoplasmic mono- and oligonucleosomes of HepG2 cells treated with 5F was assessed with Cell Death Detection ELISA Kit for determining whether 5F can induce apoptosis. Apoptotic nuclear morphology was assessed using Hoechst/PI assay. The effect on the expression levels of p53 and VEGF protein in HepG2 cells was detected by Western blotting analysis, and Caspase-3 activity was measured by a Caspase-3 Colorimetric Assay Kit. Results The cytotoxicity of 5F on HepG2 cells was elevated with 5F concentration increasing. Cytoplasmic mono- and oligonucleosomes were induced by 5F in HepG2 cells, and the induction was in a concentration-dependent manner. After treatment with 5F, the apoptotic change such as chromatin condensation was confirmed by Hoechst/ PI staining. The level of p53 in nucleus was significantly elevated in HepG2 cells after 5F treatment, but the level of VEGF in cytosol was decreased. Moreover, the activity of Caspase-3 increased in a concentration-dependent manner. Conclusion 5F mediated apoptosis involves p53 and Caspase-3 activation, VEGF down regulation in HepG2 cells. 5F might have a therapeutic value against human cancer cell lines and especially on hepatocellular carcinoma （HCC） cells.