1. Erlotinib induces mitochondrial-mediated apoptosis in human H3255 non-small-cell lung cancer cells with epidermal growth factor receptorL858R mutation through mitochondrial oxidative phosphorylation-dependent activation of BAX and BAK.
- Author
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Ling YH, Lin R, and Perez-Soler R
- Subjects
- Amino Acid Substitution, Arginine, Carcinoma, Non-Small-Cell Lung enzymology, Carcinoma, Non-Small-Cell Lung pathology, Caspase Inhibitors, Cell Line, Tumor, Down-Regulation, Drug Screening Assays, Antitumor, Enzyme Inhibitors pharmacology, Erlotinib Hydrochloride, Humans, Leucine, Lung Neoplasms enzymology, Lung Neoplasms pathology, Mitochondria enzymology, Mitochondria pathology, Mutation genetics, Protein Structure, Quaternary, Protein Transport, RNA, Small Interfering metabolism, Reactive Oxygen Species, bcl-2 Homologous Antagonist-Killer Protein chemistry, bcl-2 Homologous Antagonist-Killer Protein genetics, bcl-2-Associated X Protein chemistry, bcl-2-Associated X Protein genetics, Apoptosis drug effects, ErbB Receptors genetics, Mitochondria metabolism, Oxidative Phosphorylation drug effects, Quinazolines pharmacology, bcl-2 Homologous Antagonist-Killer Protein metabolism, bcl-2-Associated X Protein metabolism
- Abstract
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib shows potent antitumor activity in some non-small-cell lung cancer (NSCLC) cell lines and is approved by the Food and Drug Administration as second and 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 the 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 tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although it induced caspase-8 activation and BID cleavage. In addition, cell death caused by erlotinib was not prevented by coincubation with FAS and TRAIL antagonists, ZB-4 monoclonal antibody and TRAIL/Fc recombinant, suggesting that erlotinib-induced apoptosis is not associated with receptor-mediated pathways. Erlotinib induces loss of mitochondrial membrane potential and release of cytochrome c and second mitochondria-derived activator of caspases/direct IAP binding protein with low pI from mitochondria. Furthermore, erlotinib causes BAX translocation to mitochondria, BAX and BAK conformational changes, and oligomerization. Erlotinib did not induce reactive oxygen species generation, and cotreatment with antioxidants did not alter erlotinib-induced activation of BAX and BAK and apoptosis. However, cotreatment with inhibitors of mitochondrial oxidative phosphorylation significantly blocked erlotinib-induced activation of BAX and BAK and cell death. Benzyloxycarbiny-VAD-fluoromethyl ketone had no effect on erlotinib-induced BAX and BAK activation but effectively prevented apoptosis. Overexpression of BCL-2 caused a significant attenuation of erlotinib-induced cell death, but no effect on BAX and BAK activation. Down-regulation of BAX and BAK gene expression with small interfering RNA 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.
- Published
- 2008
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