Because the
epidermal growth factor receptor (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. Pharmacological interaction was studied using the combination index (CI) method, whereas cell cycle, apoptosis induction, and EGFR,
extracellular signal-regulated kinases 1 and 2, and Akt phosphorylation were studied by flow cytometry, fluorescence microscopy, and
enzyme-linked
immunosorbent assays.
Reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, and activity assays were performed to assess whether
erlotinib influenced TS. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays demonstrated that EGFR and k-Ras mutations were related to
erlotinib sensitivity, whereas TS and DHFR expression were related to
pemetrexed sensitivity. Synergistic cytotoxicity was found in all cells, most pronounced with
pemetrexed +
erlotinib (24 h) -->
erlotinib (48 h) 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.