Xu Zhihong, Hang Junbiao, Hu Jiaan, Gao Beili
Department of Geriatrics, Ruijin Hospital, Shanghai Jiaotong University, Shanghai, China.
J BUON. 2014 Apr-Jun;19(2):466-73.
To investigate the effects of autophagy on growth inhibition by gefitinib in non-small cell lung cancer (NSCLC) cell lines and its probable mechanism.
The mRNA and protein levels of Beclin 1, authophagy related 5 (Atg5) and Atg7 were assessed. H460 and Calu6 NSCLC cell lines were transfected with plasmids expressing green fluorescent protein (GFP)-LC3 and the formation of autophagosome was monitored under fluorescent microscope. In addition, H460 cells were treated with agonists of autophagy (everolimus and 3-methyladenine/ 3MA), AMP-activated protein kinase (AMPK) inhibitor (Compound C) and gefitinib, respectively. Cells were stained and studied under microscope. Cell colonies were counted and growth inhibition was calculated. Phosphorylated acetyl-Coenzyme A carboxylase (ACC) and AMPK were detected. Moreover, H460 cells were transfected with small interfering RNA (siRNA) against AMPK2 subunit and AMPK 2 was knocked down.
LCII was accumulated to a higher level after treatment with gefitinib than that without addition of gefitinib, and gefitinib increased GFP punctuated cells. Besides, everolimus enhanced the autophagic process induced by gefitinib. Consistent with this, everolimus enhanced the growth inhibition of gefitinib on H460 cells. Also, incubation with gefitinib could significantly increase AMPK phosphorylation and phosphorylated ACC. Compound C AMPK inhibitor could reverse the activation of gefitinib on autophagy, as determined by Beclin 1, Atg5 and Atg7 mRNA levels. Knockdown of AMPK2 also significantly inhibited the activation of autophagy by gefitinib.
Inhibition of AMPK by its antagonist (Compound C) or siRNA predominantly blocked the induction of autophagy by gefitinib.
研究自噬对吉非替尼抑制非小细胞肺癌（NSCLC）细胞系生长的影响及其可能机制。
评估Beclin 1、自噬相关蛋白5（Atg5）和Atg7的mRNA及蛋白水平。用表达绿色荧光蛋白（GFP）-LC3的质粒转染H460和Calu6 NSCLC细胞系，在荧光显微镜下监测自噬体的形成。此外，分别用自噬激动剂（依维莫司和3-甲基腺嘌呤/3MA）、AMP激活的蛋白激酶（AMPK）抑制剂（化合物C）和吉非替尼处理H460细胞。对细胞进行染色并在显微镜下观察。计数细胞集落并计算生长抑制率。检测磷酸化乙酰辅酶A羧化酶（ACC）和AMPK。此外，用针对AMPK2亚基的小干扰RNA（siRNA）转染H460细胞以敲低AMPK 2。
与未添加吉非替尼相比，用吉非替尼处理后LCII积累到更高水平，且吉非替尼增加了GFP点状细胞。此外，依维莫司增强了吉非替尼诱导的自噬过程。与此一致，依维莫司增强了吉非替尼对H460细胞的生长抑制作用。而且，用吉非替尼孵育可显著增加AMPK磷酸化和磷酸化ACC。根据Beclin 1、Atg5和Atg7 mRNA水平测定，化合物C AMPK抑制剂可逆转吉非替尼对自噬的激活作用。敲低AMPK2也显著抑制了吉非替尼对自噬的激活作用。
其拮抗剂（化合物C）或siRNA抑制AMPK主要阻断了吉非替尼诱导的自噬。
