reductase; EGFR, epidermal growth factor receptor; FA, serum; glycinamide ribonucleotide MRPs, 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. The present work was aimed at evaluating molecular mechanisms underlying the synergistic between erlotinib and the antimetabolite pemetrexed through the in vitro assessment of pathway may modulate the effects of the erlotinib-pemetrexed combination, we studied the pharmacological interaction of LY294002 with erlotinib, pemetrexed and their combination. The cell growth inhibitory effect was detected in cells treated with erlotinib (0.001-50 µ M), pemetrexed (0.001-100 µ M), LY294002 (30 µ M) and their combinations for 72 hours, while drug interaction with LY294002 was assessed, at a non-constant concentration ratio, using the CI method.