Modulation of gene expression as mechanism of synergism between gemcitabine and pemetrexed in pancreatic cancer cell lines.

Abstract

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Gemcitabine is a pyrimidine analogue that inhibits ribonucleotide reductase (RR) as well as DNA synthesis and is now an established effective agent in the treatment of pancreatic cancer. The present study investigates whether pemetrexed, a new multitargeted antifolate that blocks folate metabolism and DNA synthesis, would be synergistic with gemcitabine against the MIA PaCa-2, PANC-1 and Capan-1 pancreatic cancer cell lines. Cells were treated with gemcitabine (1 h), and pemetrexed (24 h), alone or in sequence, and the analysis by the combination index demonstrated synergism mainly with the sequence pemetrexed-gemcitabine. To asses the role of drug metabolism on gemcitabine cytotoxicity, further studies were performed with inhibitors of the activating enzyme deoxycytidine kinase (dCK), and the inactivating enzymes 5′-nucleotidase (5′-NT), and cytidine deaminase (CDA). The crucial role of dCK in gemcitabine citotoxicity was confirmed by a ten-fold increase in IC₅₀ by adding the dCK inhibitor 2′-deoxycytidine in all cell lines, while there was a modest increase in cytotoxicity by inhibition of 5′-NT and CDA, with respectively diethylpyrocarbonate and tetrahydrouridine. Cell cycle analysis by flow cytometry demonstrated that pemetrexed increased cell population in S phase (from 15.3 to 46.6% in MIA PaCa-2, from 10.6 to 80.1% in PANC-1 and from 46.4 to 63.2% in Capan-1 cells), which is the most sensitive phase of cell cycle to gemcitabine. Furthermore, all the pemetrexed-gemcitabine combinations significantly enhanced the occurence of apoptosis, as detected by fluorescence microscopy. Finally, quantitative RT-PCR analysis demonstrated that there was a correlation between the expression ratio dCK/RR and gemcitabine sensitivity; pemetrexed, at the IC₅₀ level, significantly enhanced the expression of dCK(+227.9%, +86.0% and +135.5% in MIA PaCa-2, PANC-1 and Capan-1 cells, respectively), potentially facilitating gemcitabine activation. These data provide evidence that the combination of gemcitabine and pemetrexed displays schedule-dependent synergistic cytotoxic activity in vitro against pancreatic cancer cells, associated with favorable modulation of cell cycle, induction of apoptosis and enhanced expression of dCK.