Received for publication December 18, 2007.
Revised June 3, 2008.
Accepted for publication June 3, 2008.

Erlotinib Induces Mitochondrial-Mediated Apoptosis in Human H3255 Non-Small Cell Lung Cancer Cells with EGFR^L858R Mutation through Mitochondrial Oxidative Phosphorylation-Dependent Activation of Bax and Bak

Abstract

EGFR TKI erlotinib shows potent anti-tumor activity in some NSCLC cell lines and is approved by FDA as second/third line treatment for NSCLC. However, the molecular mechanisms by which erlotinib induces apoptosis remain to be elucidated. Here we investigated the effect of erlotinib on apoptotic signal pathways in H3255 cells with EGFR^L858R mutation. Erlotinib induces apoptosis associated with the activation of caspases in a dose- and time-dependent manner. Erlotinib did not alter the expression of apoptotic receptors FAS and TRAIL, although it induced caspase-8 activation and BID cleavage. Additionally, cell death caused by erlotinib was not prevented by co-incubation with FAS and TRAIL antagonists, ZB-4 mAb and TRAIL/Fc recombinant, suggesting that erlotinib-induced apoptosis is not associated with receptor-mediated pathways. Erlotinib induces loss of mitochondrial membrane potential (m), and release of cytochrome c and Smac/DIABLO from mitochondria. Furthermore, erlotinib causes BAX translocation to mitochondria, BAX and BAK conformational changes and oligomerization. Erlotinib did not induce ROS generation, and co-treatment with antioxidants did not alter erlotinib-induced activation of BAX and BAK and apoptosis. However, co-treatment with inhibitors of mitochondrial oxidative phosphorylation significantly blocked erlotinib-induced activation of BAX and BAK and cell death. Z-VAD-fmk had no effect on erlotinib-induced BAX and BAK activation, but effectively prevented apoptosis. Over-expression of BCL-2 caused a significant attenuation of erlotinib-induced cell death, but not effect on BAX and BAK activation. Down-regulation of BAX and BAK gene expression with siRNA led to an effective reduction of erlotinib-induced apoptosis. Our data indicate that activation of BAX and BAK plays a critical role in the initiation of erlotinib-induced apoptotic cascades.

Key words: NGF/EGF, Mechanisms of cell killing/apoptosis