Abstract A67: Molecular evidence of increased antitumor activity of gemcitabine in combination with a Cdk inhibitor, P276 in pancreatic cancer

Abstract Purpose: Gemcitabine is an inhibitor of ribonucleotide reductase and DNA synthesis and is a standard of care for the treatment of pancreatic cancer. The present study investigates whether the cdk inhibitor P276 would be synergistic with gemcitabine in pancreatic cancers. Experimental design: Various pancreatic cancer cell lines were treated with gemcitabine and P276 and the cytotoxicity was assessed using the combination index. The effect of gemcitabine and P276 on cell cycle and apoptosis was analyzed by flow cytometry. Several genes that are known to inhibit apoptosis and contribute to chemoresistance were analyzed using western blot analysis and RT-PCR. Finally, in vivo efficacy of the combination was studied in Panc-1 xenograft model in SCID mice. Results: The combination of gemcitabine followed by P276 was found to be synergistic in various pancreatic cancer cell lines Panc-1, Capan-1, BxPc-3, AsPc-1 and MiaPaca-2 as assessed by the combination index using the Compusyn software. Mechanistic studies indicated enhancement of apoptosis in Panc-1 cells as seen by flow cytometry and increase in the antiapoptotic Bcl-2 protein by western blot analysis. Several genes that are known to inhibit apoptosis and contribute to chemoresistance such as p8, cox-2 and survivin were assessed to investigate the basis for the observed chemosensitizing effects of P276. P276 potentiated the gemcitabine induced cytotoxicity by downregulation of p8, cox-2 and survivin. In contrast, cox-2 was found to be significantly up regulated when the cells were exposed to gemcitabine alone, while there was no effect on p8 or survivin as compared to control, suggesting the potential mechanism of acquired chemoresistance to gemcitabine. BNIP3 the tumor suppressor is also downregulated in most pancreatic cancer cell lines and is associated with chemoresistance to gemcitabine. Interestingly BNIP3 was significantly up regulated in the combination and P276 alone but not by gemcitabine alone. In addition to in vitro results, we show here that gemcitabine in combination with P276 is much more effective as an antitumor agent compared with either agent alone in our Panc-1 xenograft tumor model in SCID mice. Conclusion: These data demonstrate that the gemcitabine and a Cdk inhibitor, P276 combination displays synergistic cytotoxic activity, induces apoptosis and sensitizes the pancreatic cells to gemcitabine by modulating the pro- and anti apoptotic proteins. In vivo evidence of the synergistic combination is also demonstrated in Panc-1 xenograft model. The chemosensitzation of pancreatic tumors to gemcitabine would likely be an important and novel strategy for treatment of pancreatic cancer and enable the use of lower and safer concentrations, to pave the way for a more effective treatment in this devastating disease. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A67.