Vitamin E抑制胞浆APE1/Beclin1信号途径克服肺癌EGFR T790M突变耐药及机制研究

The incidence of EGFR-TKI resistance for advanced lung cancer is 100%, more than 50% of the first generation EGFR-TKI resistance is caused by T790M mutation. It has been confirmed that enhanced Beclin1-autophagy pathway can restore the sensitivity of T790M mutation to TKI. We previously found that Vitamin E can inhibit cytoplasmic double function gene apurinic/apyrimidinic endonuclease (APE1) expression and APE1-Beclin1 interaction, but promote autophagy; then we propose "Vitamin E can restore the sensitivity of T790M mutation to the first generation of TKI through inhibiting APE1/Beclin1 interaction and enhancing autophagy". The project intends to adopt Co-iP, GST pull-down and yeast hybridization technique to study the interaction of APE1/Beclin1 and the effect of the interaction on T790M mutation; using T790M mutant cells and nude mice model to investigate the molecular mechanism of that Vitamin E reverses of T790M mutation in vitro and in vivo, further researchs the impact of Vitamin E on cytoplasmic APE1/Beclin1 signaling, so as to provide experimental basis for that the combination of Vitamin E and TKI prolongs the period of survival of the advanced lung cancer.

EGFR-TKI治疗特定晚期肺癌耐药发生率为100%，50%以上TKI耐药是由于EGFR T790M突变所致；现已证实，增强Beclin1-自噬途径可逆转T790M突变耐药。我们前期发现，Vitamin E可抑制胞浆双功能基因脱嘌呤脱嘧啶核酸内切酶（APE1）表达及APE1/Beclin1相互作用，并促进自噬；进而我们提出“Vitamin E可能通过抑制APE1/Beclin1相互作用增强自噬，恢复T790M突变耐药对第一代TKI的敏感性”。本项目拟采用Co-iP、GST pull-down和酵母杂交技术研究APE1-Beclin1相互作用及对T790M突变耐药的影响；利用T790M突变耐药细胞及裸鼠模型探讨Vitamin E体内、外逆转T790M突变耐药的分子机制，及Vitamin E对胞浆APE1/Beclin1信号的影响，为Vitamin E联合TKI延缓晚期肺癌生存期提供实验依据。

EGFR-TKI治疗特定晚期肺癌耐药发生率为100%，50%以上TKI耐药是由于EGFR T790M突变所致。我们给与0、2.5、5、10 umol/L 埃克替尼联合30 umol/L Vitamin-E处理HCC-827IR细胞，CCK-8和克隆形成实验检测细胞增殖水平；利用Western blotting实验观察A549、H460、HCC-827、PC-9、HCC-827IR、PC-9ER、H1975细胞浆APE1、PARP1、LC-3I和LC-3II表达情况；通过慢病毒转染实验过表达HCC-827IR胞浆APE1表达。利用免疫荧光检测LC-3II在细胞中表达及定位情况。我们发现，联合Vitamin E可通过抑制NSCLC胞浆APE1表达克服埃克替尼耐药；PARP1介导的自噬是NSCLC细胞中TKI抗性的关键途径，参与了对TKI的抗性。而且，下调APE1表达可通过氧化应激依赖的途径抑制PARP1介导的增殖信号途径。总之，Vitamin-E可通过抑制NSCLC细胞APE1介导的PARP1-增殖信号途径克服第一代埃克替尼耐药。