研究探索EGCG是否调节PPARα以及PPARα活化对EGCG的肿瘤抑制作用影响及其机制。首先利用CCK-8试剂盒检测细胞存活率,之后分别采用western blotting和real-time PCR检测蛋白质和mRNA表达水平;采用PPARα的特异性激动剂和特异性拮抗剂来改变PPARα的表达;应用荧光素酶报告基因系统及染色质免疫共沉淀研究PPARα对HO-1的调控。结果表明,随着EGCG浓度的增加,PANC1和A2780细胞的存活率逐渐下降。当EGCG处理肿瘤细胞后,PPARα蛋白水平会随着EGCG剂量的提高而增加。PPARα的特异性激动剂—氯贝特(clofibrate)能增加胰腺癌PANC1和卵巢癌A2780细胞对EGCG的敏感性,其机制是氯贝特能够抑制细胞保护性血红素加氧酶-1(HO-1)的诱导。荧光素酶报告基因实验和染色质免疫共沉淀实验(ChIP)表明,活化的PPARα能结合到HO-1启动子上的PPAR结合元件(PPRE)上并抑制HO-1的表达。研究表明,PPARα的活化能在转录水平对HO-1进行负调控,同时增加肿瘤细胞对EGCG的敏感性。

Whether epigallocatechin-3-gallate (EGCG) regulates the expression of PPARα and the effect of PPARα on EGCG sensitivity. Firstly, CCK-8 kit was used to detect cell viability. Western blotting and real-time PCR was used to measure the protein and mRNA level, respectively. PPARα agonist clofibrate and inhibitor GW6471 were used to alter PPARα expression. Luciferase reporter system and chromatin immunoprecipitation (ChIP) were used to investigate the effect of PPARα on HO-1 expression. EGCG inhibits the viability of cancer cell in a dose-dependent manner. When cancer cells were exposed to EGCG, the expression of PPARα was increased at the protein level in a dose-dependent manner. The PPARα agonist clofibrate attenuated heme oxygenase (HO-1) induction and sensitized cancer cells to EGCG-induced cell death. However, the PPARα inhibitor GW6471 increased HO-1 expression. In vivo chromatin immunoprecipitation (ChIP) confirmed that PPARα interacts with the peroxisome proliferator-responsive sequence of the HO-1 promoter. These results indicate that PPARα is a direct negative regulator of HO-1 activation by EGCG and confers cell susceptibility to EGCG.