表没食子儿茶素没食子酸酯诱导人肝癌细胞HepG2凋亡与TGF—β1-Smad通路激活有关

Epigallocatechin gallate induces apoptosis in human hepatocellular carcinoma HepG2 cells via TGF/ Smad signaling pathway

目的探讨表没食子儿茶素没食子酸酯（EGCG）对人肝癌细胞HepG2细胞周期和凋亡的影响及其机制。方法四甲基偶氮唑蓝（MTT）法检测EGCG对HepG2细胞增殖的影响，流式细胞术检测EGCG对HepG2细胞周期的影响以及细胞凋亡情况，逆转录聚合酶链反应（RT—PCR）和萤光素酶报告基因系统检测验证HepG2细胞中TGF—β1-Smad通路的完整性，实时聚合酶链反应（realtime PCR）检测EGCG对HepG2细胞中Sfnad2、Smad3、Smad4和Smad7 mRNA表达的影响。结果高浓度EGCG干预时，HepG2细胞的增殖能力随着EGCG浓度和作用时间的增加而下降，呈明显的时效和量效关系，作用72h，HepG2细胞的IC50为111．76μmoL／L。随EGCG剂量增大，HepG2细胞中G0／G1期细胞的比例逐渐增高，80、120和160μmoL/L EGCG处理组分别为44．9％、54．8％和91．3％；相反，S期细胞比例逐渐降低，80、120和160μmoL/L EGCG处理组依次为38．5％、29．2％和3．0％。随着EGCG剂量增大，HepG2细胞的早期凋亡增加，80、120和160μmol／L EGCG处理组的早期凋亡率分别为1．82％、4．22％和6．83％；晚期凋亡也随之增加，80、120和160μmol／L EGCG处理组的晚期凋亡率分别为7．92％、24．19％和27．92％。HepG2细胞中TGF—β1-Smad通路是完整的。EGCG对HepG2细胞中Smad2、Smad3和Smad4 mRNA的表达无明显影响，但下调Smad7 mRNA的表达。结论EGCG能诱导人肝癌细胞HepG2的凋亡，其机制可能涉及TGF—β1-Smad通路的激活。

Objective To investigate the cytotoxic effect of epigallocateehin gallate （EGCG） on human hepatocellular carcinoma cell line HepG2 ceils and corresponding changes of TGF-β1-Smad pathway. Methods The cytotoxic effect of EGCG on HepG2 cells was determined by MTT assay. Cell cycle and apoptosis rate were detected by flow cytometry. RT-PCR and luciferase assay were used to verify whether TGF-β1-Smad signaling pathway is intact in HepG2. The mRNA expression of Smad 2, Smad3, Smad4 and Smad7 was detected by real-time PCR. Results EGCG induced apoptosis in the HepG2 cells in a time- and concentration-dependent manner. The proportion of Gl phase cells was increased gradually as the concentration increased. However, the percentage of cells in S phase was decreased gradually. Annexin V/ PI assay demonstrated that early apoptosis increased as the concentration increased, and late apoptosis also increased, when treated with high-concentration EGCG. The intact TGF-β1-Smad pathway was verified by lneiferase assay and RT-PCR. There was no significant effect of EGCG on mRNA level of Smad 2, Smad 3, and Smad 4 in HepG2 cells, hut downregulated mRNA level of Smad 7. Conclusion EGCG can reduce apoptosis in human hepatoeellular carcinoma cell line HepG2 cells. The activation of TGF-β1-Smad signaling pathway may be involved in its cytotoxieity mechanisms.