EGCG抑制IL-6诱导肿瘤血管生成的作用及机制

Epigallocatechin-3-gallate inhibits angiogenesis of gastric cancer induced by IL-6 and its mechanism

目的探讨表没食子儿茶素没食子酸酯(EGCG)抑制IL-6诱导胃癌血管生成的作用及机制。方法以50 ng/mL IL-6或不同浓度EGCG培养AGS胃癌细胞株,Western blot免疫印迹法检测胃癌细胞血管内皮生长因子(VEGF)的表达;Elisa法检测肿瘤细胞培养液中VEGF的蛋白水平;RT-PCR检测胃癌细胞VEGF mRNA表达;制备胃癌细胞条件培养液培养血管内皮细胞,采用MTT法检测血管内皮细胞的体外增殖;小管形成实验检测血管内皮细胞体外小管形成;基质胶(matrigel)塞实验检测体内血管形成。结果 IL-6诱导胃癌细胞VEGF蛋白表达增加2.4倍,VEGF蛋白分泌增加2.8倍,而VEGF mRNA表达增加3.1倍;EGCG呈剂量依赖性地抑制IL-6诱导的胃癌细胞VEGF蛋白的表达、分泌和VEGF mRNA表达。VEGF中和抗体及EGCG在体外可以显著抑制IL-6诱导的血管内皮细胞增殖(t=3.02,P<0.01;t=2.56,P<0.05)和小管形成(t=3.38,P<0.01;t=3.61,P<0.01)以及在体内的血管生成(t=2.66,P<0.05;t=3.77,P<0.01)。结论 EGCG通过下调胃癌细胞VEGF的表达,而抑制IL-6诱导的肿瘤血管生成。

Objective To investigate the inhibitory effect of（-）-Epigallocatechin-3-gallate（EGCG） on angiogenesis of gastric cancer induced by IL-6 and its molecular mechanism.Methods AGS gastric cancer cells were cultured with IL-6（50 ng/mL） or with a series of concentrations of EGCC.Protein levels of vascular endothelial growth factor（VEGF） in tumor cells were examined by Western blot,the VEGF release in tumor culture medium was determined by ELISA,and VEGF mRNA expression in tumor cells by RT-PCR.The conditioned media from gastric cancer cells were prepared for culturing human umbilical vein endothelial cells（HUVEC）.Vascular endothelial cell proliferation was measured with MTT assay,in vitro angiogenesis was determined with endothelial cell tube formation assay in Matrigel,and angiogenesis in vivo was measured with Matrigel plug assay.Results The VEGF protein level,release and mRNA expression were increased in the IL-6 induced AGS cells by 2.4,2.8 and 3.1 folds,respectively.EGCG treatment significantly inhibited the IL-6 induced increase in level and release of VEGF protein and in mRNA expression in a dose-dependent manner.The proliferation and tube formation of vascular endothelial cells induced by IL-6 in vitro were markedly inhibited by VEGF neutralizing antibody and EGCG.The angiogenesis in vivo induced by IL-6 was also markedly inhibited by VEGF neutralizing antibody and EGCG.Conclusions EGCG reduces expression of VEGF induced by IL-6 in gastric cancer cells,and thereby inhibits tumor angiogenesis.