EGCG影响TNF-α诱导的NF-κB/p65入核及促凋亡效应  

Effects of EGCG on regulating NF-κB/p65 induced by TNF-α and on inducing apoptosis

作　　者：王强[1] 陈晓艳[1] 罗招阳[1] 赵慧[1] 

机构地区：[1]南华大学肿瘤研究所,湖南衡阳421001

出　　处：《中国癌症杂志》2008年第1期6-10,共5页China Oncology

摘　　要：背景与目的:研究表明,表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)抗肿瘤作用机制尚不十分清楚,本研究旨在观察EGCG调节TNF-α诱导的胃癌SGC-7901细胞核转录因子κB/p65(NF-κB/p65)的入核并发挥诱导凋亡的作用。方法:Hoechst染色法检测EGCG处理SGC-7901细胞前后凋亡的形态学改变;Westernblot方法观察EGCG作用前后NF-κB/p65蛋白在胞质内及核内的变化;流式细胞术检测EGCG调节NF-κB/p65入核前后细胞凋亡的变化;DNAladder方法观察EGCG诱导细胞DNA断裂情况。结果:Hoechst染色法显示EGCG处理后细胞形态改变:核致密浓染或呈碎块状、苍白色。Westernblot方法检测发现,10ng/ml的TNF-α作为NF-κB/p65入核的诱导剂分别作用细胞30、60、90和120min后,核内NF-κB/p65明显增多并在60min达高峰;60μg/ml的EGCG预处理细胞不同时间(0、12、24、36和48h)后,再以TNF-α处理60min,检测发现核内NF-κB/p65明显下降并在24h达低谷;不同浓度EGCG(40、60、80和100μg/ml)预处理细胞24h后,再加TNF-α处理60min,核内NF-κB/p65呈时间依赖性下降。流式细胞术显示,10ng/ml的TNF-α处理细胞60min后,细胞凋亡率为3.7%,60μg/mlEGCG处理细胞24h后再以TNF-α处理细胞,凋亡率为16.6%;NF-κB通路阻断剂吡咯啉烷二甲基硫脲(PDTC)100μmol/L预处理30min后再以TNF-α处理细胞60min,细胞凋亡率为21.4%。EGCG60μg/ml处理细胞24h、PDTC处理细胞30min后再以TNF-α处理细胞60min,DNA凝胶电泳出现明显梯形条带。结论:EGCG能够抑制TNF-α诱导的NF-κB/p65入核,并可能通过此机制发挥诱导细胞凋亡的作用。Background and purpose： Anticancer mechanism of epigallocatechin-3-gallate （EGCG） remains unclear. This study was to investigate the effects of EGCG on induction of apoptosis by TNF-α through translocation of NF- KB/p65 into SGC-7901 cellular nucleus. Methods： Hoechst staining method was used to display the apoptosis form of SGC- 7901 induced by EGCG. The disposition of NF-KB/p65 was detected by Western blot. The effect of EGCG on induction of apoptosis was analyzed by flow cytometry （FCM） and DNA agarose gel electrophoresis. Results： Hoechst staining method showed that EGCG could significantly induce apoptosis of SGC-7901. After being exposed to EGCG, the cell nucleus changed to shiver and pallor. After being treated by 10 ng/ml TNF-α at various times （30, 60, 90 and 120 min）, an inductor of regulating NF-KB/p65, the expression of NF-KB/p65 in the cell nucleus increased obviously. It displayed a platform after being treated for 60 minutes. Incubation with 60 μg/ml EGCG at various times （0, 12, 24, 36, 48 h） before TNF-α（ 10 ng/ml, 60 min） treatment, the expression of NF-KB/p65 in the cell nucleus decreased in a time-depended manner that reached plateau after treated for 24 h. Being treated with EGCG for 24 h at various concentrations （40, 60, 80, 100 μg/ml） before the TNF-α（ 10 ng/ml, 60 min）, the expression of NF-KB/p65 in the cell nucleus decreased in a dose-depended manner. Analysis by flow cytometry showed that the percentages of apoptosis was 3.7% in cells exposed to 10 ng/ml TNF- α,60 min, apoptotic percentage was 16.6% after ceils exposed to 60 μg,/ml EGCG （24 h） before TNF-α（ 10ng,/ml, 60 min）. It could reach to 21.4% after cells were exposed to PDTC （ 100 μmol/L , 30 min, before TNF-α）, a suppressor of NF-KB. In DNA agarose gel electrophoresis , “Ladder”bands occurred after the treatment with EGCG（60 μg/ml, 24 h） before TNF-α（ 10 ng/ml, 60 min）. The “Ladder”bands also occurred after incubation with PDTC（ 100 μmol/L, 30 min）

关 键 词：EGCG 胃肿瘤 NF—KB/p65 细胞凋亡 TNF NF—KB通路阻断剂 

分 类 号：R73-354[医药卫生—肿瘤]