Mechanistic studies of a protein kinase C inhibitor in combination with an antifolate in non-small cell lung cancer cells

Abstract

Introduction: Non-small cell lung cancer (NSCLC) is one of the most commonly occurring malignancies worldwide. Platinum based regimens are standard first line treatment against this disease. However, poor response rates, and the limiting toxicity profile of the platinum compounds, makes novel strategies and new combination regimens against NSCLC warranted. The present study investigated the cellular aspects of the interaction between enzastaurin (a selective PKC inhibitor) and pemetrexed (an antifolate) against SW1573 and A549 cells.

Methods: The cells were treated with the indicated drugs, and their pharmacological interaction was assessed using the Combination Index (Calcusyn ® software). The role of the drugs in the modulation of the cell cycle and their effects on cell death was analyzed by the use of flow cytometry (FACS), whereas the effect on the protein expression and phosphorylation of different targets was investigated by western blot technique. The thymidylate synthase (TS) catalytic activity upon drug treatment was measured by the use of a radioactive TS activity assay.

Results: Synergistic interaction with respect to growth inhibition was demonstrated for all the treatment regimens tested. Cell cycle analysis showed that pemetrexed was the dominating drug in the combinations, but still enzastaurin enhanced the amount of cell death with respect to the amount caused by pemetrexed alone. Enzastaurin treatment reduced the phosphorylation of targets in cell signaling pathways; Akt and GSK3. The latter also provided evidence that inhibition of GSK3 phosphorylation can serve as a pharmacodynamic marker for enzastaurin activity in NSCLC cells. Furthermore, enzastaurin was able to abrogate the G2/M checkpoint due to inhibition of Cdc25C phosphorylation, thus being able to promote pemetrexed-damaged cells to undergo apoptosis. Finally, enzastaurin was able to downregulate TS expression, potentially facilitating pemetrexed activity.

Conclusion: These data provide evidence that enzastaurin was able to enhance the effects of pemetrexed in NSCLC cells in vitro and suggests that this combination might be a new approach in the treatment against NSCLC.