EGCG对A431细胞NF-κB表达的表观遗传修饰效应及对光动力的影响

Cancer preventive/therapeutic effects via NF-κB activation epigenetic modulation and coincident ALA-PDT co-effects in human A431 squamous cancer cells induced by EGCG

目的:研究表没食子儿茶素没食子酸酯(EGCG)对人表皮细胞癌A431细胞内核转录因子(NF-κB)表达的表观遗传修饰效应,探讨联合5-氨基酮戊酸介导光动力疗法(ALA-PDT)的增敏作用。方法:低氧诱导培养A431细胞,用MTT比色法检测不同浓度EGCG对A431细胞增殖的影响,MS(methylation specific)-PCR检测p16INK4ɑ基因甲基化变化,免疫印迹检测NF-κBp65,DNMT1,HDAC1,Cyclin D1,HIF-1α,VEGF,p16INK4ɑ表达,并用免疫细胞化学染色观察A431细胞E-钙黏蛋白(E-cadherin);用流式细胞仪检测EGCG联合ALA-PDT诱导的A431细胞凋亡。结果:EGCG具有抑制A431细胞增殖并诱导凋亡的作用,抑制效应呈浓度依赖性; p16INK4ɑ基因甲基化水平减低显著;免疫印迹显示NF-κBp65,DNMT1,HDAC1,Cyclin D1,HIF-1α,VEGF表达下调,p16INK4ɑ表达上调; E-cadherin表达上调显著,呈浓度依赖性。EGCG联合ALAPDT治疗组促凋亡作用显著。结论:EGCG对A431细胞具有抑制增殖、血管生成、侵袭的作用,通过影响NF-κB表观遗传修饰诱导细胞凋亡。EGCG联合ALA-PDT的促凋亡作用,增强了A431细胞对ALA-PDT的敏感性。

Objective：The preventive/therapeutic effect via NF-κB activation epigenetic modulation induced by EGCG and co-effect on ALA-PDT by combined with EGCG in human A431 squamous cancer cells were investigated.Methods：In A431 cancer cells induced by EGCG,the p16INK4ɑ gene was detected by MS （methylation specific）-PCR.The modulated epigenetic effects in A431 cancer cells induced by EGCG,including antiproliferation effect detected by MTT assay.Expressions of NF-κBp65,DNMT1,HDAC1,CyclinD1,HIF-1α,VEGF,p16INK4ɑ were detected by immunoblotting,and anti-metastatic effect displayed by cadherin-immunocytochemistry and the apoptosis induced by EGCG detected by Annexin V-FITC.The therapeutic co-effect of ALA-PDT combined with EGCG on A431 cancer cells apoptosis was detected by Annexin V-FITC.Results：The epigenetic pattern of A431 cancer cells could be modulated via demethylation of p16INK4ɑ gene by EGCG.The down-regulated NF-κBp65,DNMT1, HDAC1,CyclinD1,HIF-1α,VEGF and upregulated p16INK4ɑ induced by EGCG were showed in immunoblotting.The anti-metastatic effect of enhanced cadherin-immunocytochemistry induced by EGCG also showed dose/time dependant.The apoptosis of A431 cancer cells could be induced by EGCG or ALA-PDT,further enhanced apoptosis of the cancer cells was induced by ALA-PDT combined with EGCG.Conclusion：EGCG with anti-oxidative activity can not only inhibit proliferative,vasculogenesis and metastatic effects via epigenetic-NF-κB signaling pathway to induce apoptosis of A431 cancer cells,but also can further promote the apoptosis induced by ALA-PDT combined with EGCG via pro-oxidant activity as cancer preventive/therapeutic agent.