表没食子儿茶素没食子酸酯通过下调表皮生长因子受体发挥抑制喉癌细胞的作用机制研究

Study on the Inhibitory Effect of Gallocatechin-3-gallate on Laryngeal Cancer Cells by Downregulating Epidermal Growth Factor Receptors

关键词:  表皮生长因子受体  表没食子儿茶素没食子酸酯  喉癌细胞  凋亡  自噬  细胞周期阻滞

Key words:  epidermal growth factor receptor  epigallocatechin-3-gallate  laryngeal carcinoma cell  apoptosis  autophagy  cell cycle arrest

摘要:

目的 探究表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate，EGCG)抑制喉癌细胞的作用机制。方法 利用Western blotting检测喉癌细胞株AMC-HN-8、TU686和TU212中表皮生长因子受体(epidermal growth factor receptor，EGFR)的表达，并采用CCK-8法检测西妥昔单抗和EGCG对3种喉癌细胞的抑制作用；构建含有EGFR启动子以及Luc报告基因的慢病毒载体并感染TU686细胞，获得EGFR启动子调控表达荧光素酶的TU686-EGFR-Luc报告基因细胞系，检测EGCG处理后的荧光素酶活性；利用流式细胞术检测EGCG作用后的喉癌细胞的细胞周期和凋亡情况，并用Western blotting检测分析EGFR及下游信号通路分子ERK的表达与活化、细胞周期相关分子P53及P27、凋亡相关分子BCL2及PARP、自噬相关指标LC3A/B的水平情况。结果 喉癌细胞株对西妥昔单抗不敏感，但EGCG能有效抑制喉癌细胞的生长；EGCG能有效抑制EGFR启动子的转录活性；在亚IC₅₀剂量的EGCG作用下，TU686细胞出现明显的S期细胞周期阻滞和部分凋亡；EGFR和下游信号通路的表达和激活受到明显抑制。结论 EGCG可有效下调喉癌细胞的EGFR表达并抑制喉癌细胞生长，其进一步的体内作用及机制有待研究。

Abstract:

OBJECTIVE To explore the mechanism of action of epigallocatechin-3-gallate(EGCG) in inhibiting laryngeal cancer cells. METHODS The expression of epidermal growth factor receptor(EGFR) in laryngeal cancer cell lines AMC-HN-8, TU686 and TU212 was detected by Western blotting, and the inhibitory effects of cetuximab and EGCG on three laryngeal cancer cells were detected by CCK-8 assay. A lentiviral vector containing EGFR promoter and Luc reporter gene was constructed to generate a TU686-EGFR-Luc cell line that could steadily express Luc activity. Luciferase assay was performed to evaluate the effect of EGCG on the transcription activity of EGFR promoter. Cell cycle and apoptosis of EGCG-treated laryngeal carcinoma cells were analyzed by flow cytometry, and changes of the levels of EGFR and downstream ERK1/2, cell cycle-associated proteins P53 and P27, apoptosis-associated proteins BCL2 and PART, and autophagy marker LC3A/B were further examined. RESULTS The laryngeal carcinoma cell lines were insensitive to cetuximab but could be effectively suppressed by EGCG. EGCG effectively inhibited the transcription activity of EGFR promoter. Treatment of TU686 cells at sub-IC₅₀ dose EGCG resulted in significant cell cycle arrest at S phase with partial apoptosis. Significant inhibition of expression and activation of EGFR and downstream signaling pathway were observed. CONCLUSION EGCG can effectively downregulate EGFR and suppress laryngeal carcinoma cells, further investigation on in vivo effect and mechanisms are anticipated.