Received for publication October 4, 2007.
Revised January 10, 2008.
Accepted for publication January 10, 2008.

Molecular mechanisms underlying the synergistic interaction of erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, with the multitargeted antifolate pemetrexed in non-small-cell lung cancer cells

Abstract

Since the EGFR tyrosine-kinase inhibitor erlotinib and the multitargeted antifolate pemetrexed are registered in the treatment of second-line non-small-cell lung cancer (NSCLC), empirical combinations of these drugs are being tested. This study investigated molecular mechanisms underlying their combination in six NSCLC cell lines. Cells were characterized by heterogeneous expression of pemetrexed determinants, including thymidylate synthase (TS) and dihydrofolate reductase (DHFR), and mutations potentially affecting chemosensitivity. Pharmacologic interaction was studied using the combination-index (CI) method, while cell cycle, apoptosis induction and EGFR, ERK1/2 and Akt phosphorylation were studied by flow cytometry, fluorescence microscopy, and ELISAs. RT-PCR, western blot and activity assays were performed to assess whether erlotinib influenced TS. MTT assays demonstrated that EGFR and k-Ras mutations were related to erlotinib sensitivity, while TS and DHFR expression were related to pemetrexed sensitivity. Synergistic cytotoxicity was found in all cells, most pronounced with pemetrexed+erlotinib(24h)erlotinib(48h) sequence (CI, 0.09-0.40), which was associated with a significant induction of apoptosis. Pemetrexed increased EGFR phosphorylation and reduced Akt phosphorylation, which was additionally reduced by drug combination (-70.6% in H1650). Erlotinib significantly reduced TS expression and activity, possibly via E2F-1 reduction, as detected by RT-PCR and western blot, and the combination decreased TS in situ activity in all cells. Erlotinib and pemetrexed showed a strong synergism in NSCLC cells, regardless of their genetic characteristics. Induction of apoptosis, modulation of EGFR and Akt phosphorylation and changes in the expression of critical genes involved in pemetrexed activity contribute to this synergistic interaction, and support the clinical investigation of these markers.

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

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