细胞自噬对吉非替尼抑制肺癌细胞增殖的影响

Effects of autophagy on growth inhibition of Gefitinib in non-small cell lung cancer cell lines

目的探讨细胞自噬对吉非替尼(Gefitinib)抑制非小细胞肺癌(NSCLC)细胞增殖的影响。方法采用Western blotting和LC3抗体检测自噬标志物LC3Ⅱ的含量,判断Gefitinib是否诱导NSCLC Calu6和H460细胞自噬。采用增强绿色荧光蛋白(EGFP)-LC3b质粒转染NSCLC细胞系Calu6和H460,再用40μmol/L Gefitinib处理细胞48 h,观察绿色荧光蛋白(GFP)的分布情况。分别采用细胞自噬诱导剂依维莫司(Everolimus)、Gefitinib和Gefitinib联合Everolimus处理H460细胞,药物处理10 d后用结晶紫染色,显微镜下统计克隆数,计算不同药物刺激后细胞克隆形成数和药物对H460细胞的抑制率。结果 Westernblotting检测显示,Gefitinib 40μmoL/L处理细胞48 h后,明显增加LC3Ⅱ的积累,绿色荧光呈散点状分布。经Everolimus、Gefitinib和Gefitinib联合Everolimus处理后,H460细胞克隆形成数差异有统计学意义(P<0.05),其中Gefitinib联合Everolimus处理对H460细胞的抑制率最强,两者之间有协同作用。结论 Gefitinib能够诱导NSCLC细胞自噬,并且增加细胞自噬能增强Gefitinib对肺癌细胞增殖的抑制率。

Objective To investigate the effects of autophagy on growth inhibition of Gefitinib in non-small cell lung cancer （NSCLC） cell lines. Methods The content of autophagy marker LC3II was detected by Western blotting and LC3 antibody, and whether Gefitinib could induce autophagy in NSCLC Calu6 and H460 cells were determined. H460 and Calu6 cells were transfeeted with enhanced green fluorescence protein （EGFP）-LC3b plasmid, and were treated with 40 μmol/L Gefitinib for 48 h respectively. The distribution of green fluorescence protein （GFP） was observed. In addition, H460 cells were treated with Everolimus, Gefitinib, and Everolimus combined with Gefitinib respectively. Ten days later, cells were stained with crystal violet, cell clonies were counted under microscope, and growth inhibition rates were calculated. Results Western blotting revealed that LC3 Ⅱ was significantly accumulated after treatment with 40 μmol/L Gefitinib for 48 h, and green fluorescence was dispersedly distributed. There were significant differences in numbers of clony formation among H460 cells treated with Everolimus, Gefitinib, and Everolimus combined with Gefitinib （P 〈 0.05）, and the growth inhibition rate of Everolimus combined with Gefitinib was the highest. Conclusion Gefitinib induces autophagy in NSCLC cells, and autophagy induction enhances growth inhibition of Gefitinib on NSCLC cells.