PURPOSE: To investigate the effects of autophagy on growth inhibition by gefitinib in non-small cell lung cancer (NSCLC) cell lines and its probable mechanism. METHODS: 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. RESULTS: 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. CONCLUSION: Inhibition of AMPK by its antagonist (Compound C) or siRNA predominantly blocked the induction of autophagy by gefitinib.
PURPOSE: To investigate the effects of autophagy on growth inhibition by gefitinib in non-small cell lung cancer (NSCLC) cell lines and its probable mechanism. METHODS: 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. RESULTS: 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. CONCLUSION: Inhibition of AMPK by its antagonist (Compound C) or siRNA predominantly blocked the induction of autophagy by gefitinib.