自噬在A549肺腺癌细胞耐厄洛替尼中的作用研究

Research on the role of autophagy in A549′s resistance of erlotinib

目的探讨自噬在A549肺腺癌细胞耐厄洛替尼中的作用。方法不同水平厄洛替尼处理A549细胞及其获得性耐厄洛替尼细胞(A549/ER)后,细胞免疫荧光和Western blot观察自噬标记蛋白微血管相关蛋白1轻链3-Ⅱ(MAP1-LC3-Ⅱ)的变化;应用自噬抑制剂3-甲基腺嘌呤(3-MA)抑制自噬,流式细胞仪(FCM)检测用药后细胞周期及凋亡率。结果低水平(1、5μmol/L)厄洛替尼作用下两种细胞MAP1-LC3-Ⅱ表达水平较对照组(0.1%DMSO)均升高,高水平(25μmol/L)厄洛替尼作用下A549细胞MAP1-LC3-Ⅱ表达水平较低水平时降低,而A549/ER细胞MAP1-LC3-Ⅱ表达水平较低水平时仍升高。厄洛替尼水平提高至100μmol/L可诱导A549/ER细胞凋亡,3-MA抑制自噬后A549/ER细胞的凋亡率从7.76%上升至18.85%。结论自噬增强了A549/ER的耐厄洛替尼作用,抑制自噬可增强厄洛替尼的抗肿瘤作用。

Objective To investigate the effect of autophagy in A549＇s resistance of erlotinib. Methods After A549 cells were treated with different levels of erlotinib,autophagy was determined by cell immunofluorescence and protein lever of LC3-Ⅱ was de-termined by Western blot at certain times. And then afer inhibiting autophagy by the pharmacological inhibitor 3-methyladenine（3-MA） ,the cell cycle and apoptosis rate were determined by flow cytometry（FCM）. Results Compared to control group （0. 1%DM-SO） ,the protein levers of MAP1-LC3-Ⅱ in both A549 and A549/ER were upregulated at low levels（1,5 μmol/L）. The protein le-ver of MAP1-LC3-Ⅱ in A549/ER was still upregulated while the protein lever of LC3-Ⅱ in A549 was downregulated at the high level（25 μmol/L）. Moreover,A549/ER＇s apoptosis induced by erlotinib was enhanced after inhibiting autophagy by 3-MA, the ap-optosis rate rose from 7. 76% to 18.85%. Conclusion Autophagy thus represents a role in A549/ER resistance of erlotinib TKIs and inhibiting autophagy may be an approach to improve the efficacy of erlotinib.