Molecular evidence for increased antitumor activity of gemcitabine in combination with a cyclin-dependent kinase inhibitor, P276-00 in pancreatic cancers

Abstract

Background: P276-00 is a novel cyclin-dependent kinase inhibitor currently in Phase II clinical trials. Gemcitabine is a standard of care for the treatment of pancreatic cancer. The present study investigated the effect of the combination of P276-00 and gemcitabine in five pancreatic cancer cell lines.

Methods: Cytotoxic activity was evaluated by Propidium Iodide assay. Cell cycle and apoptosis was analyzed by flow cytometry. Genes and proteins known to inhibit apoptosis and contribute to chemoresistance were analysed using western blot analysis and RT-PCR. In vivo efficacy was studied in PANC-1 xenograft model.

Results: The combination of gemcitabine followed by P276-00 was found to be highly to weakly synergistic in various pancreatic cancer cell lines as assessed by the combination index. Enhancement of apoptosis in PANC-1 cells and decrease in the antiapoptotic protein Bcl-2 and survivin was seen. P276-00 potentiated the gemcitabine-induced cytotoxicity by modulation of proteins involved in chemoresistance to gemcitabine and cell cycle viz. antiapoptotic proteins p8 and cox-2, proapoptotic protein BNIP3 and cell cycle related proteins Cdk4 and cyclin D1. The above results could explain the novel mechanisms of action of the combination therapy. We also show here that gemcitabine in combination with P276-00 is much more effective as an antitumor agent compared with either agent alone in the PANC-1 xenograft tumor model in SCID mice.

Conclusions: 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. Phase IIb clinical trials of P276-00 in combination with gemcitabine in pancreatic cancer patients are ongoing.

Figure 1

The effect of P276-00 and gemcitabine treatment on growth of five pancreatic cancer cell lines. Cells were seeded in 96-well plates and incubated overnight. P276-00 or gemcitabine was added at the indicated concentrations and cells were further incubated for 48 h. Cell proliferation was determined using PI assay. (A &B) The 50% inhibitory concentration of P276-00 in five different pancreatic cancer cell lines. (C &D) The 50% inhibitory concentration of gemcitabine in five different pancreatic cancer cell lines.

Figure 2

Effect of P276-00 and gemcitabine used singly or in combination on survival of BxPC-3, AsPC-1, PANC-1, Capan-1 and MIA PACA-2 pancreatic cancer cell lines. The cells were treated as described under Materials and Method section. There was significantly higher growth inhibition of the cells treated with gemcitabine (24 h) followed by P276-00 at IC₅₀ concentration (1.5, 1.5, 1.6, 0.7 and 0.6 μM for AsPC-1, BxPC-3, Capan-1, MIA PaCa-2 and PANC-1 respectively) for 72 h or 96 h compared to the cells treated with either agent alone. P276-00 treatment alone in BxPC-3, AsPC-1, PANC-1, Capan-1 and MIA PaCa-2 showed 78%, 48%, 38%, 69% and 47% survival respectively.

Figure 3

The effect of P276-00, gemcitabine and the combination of gemcitabine followed by P276-00 on (A). Cells were treated with 300 nM P276-00 for 72 h or 70 nM of gemcitabine for 24 h followed by only medium for additional 72 h or combination treatment of gemcitabine for 24 h followed by P276-00 for 72 h. (B) Cells were treated with 300 nM P276-00 for 96 h or 70 nM of gemcitabine for 24 h followed by only medium for additional 96 h or combination treatment of gemcitabine for 24 h followed by P276-00 for 96 h. Cell cycle analysis using Flow cytometry shows the percentage apoptosis in drug alone and combination treatment in comparison to untreated control. The percent distribution of cells in various phases of the cell cycle is represented as bar plot (C) Western blot analysis in PANC-1 cells of the antiapoptotic proteins Bcl-2 and survivin. Samples were obtained from PANC-1 cells treated with 300 nM of P276-00 for 72 h or gemcitabine for 24 h or the combination of gemcitabine for 24 h followed by P276-00 for 72 h. (D) Densitometric analysis of the Bcl-2 and survivin bands using the software Image J.

Figure 4

Western blot analysis in PANC-1 cells of the cell cycle proteins Cdk4 and cyclin D1. (A) Samples were obtained from PANC-1 cells treated with 300 nM of P276-00 for 72 h or 70 nM gemcitabine for 24 h or the combination of gemcitabine for 24 h followed by P276-00 for 72 h. (B) Densitometric analysis of the Cdk4 and cyclin D1 bands using the software Image J.

Figure 5

RT-PCR and Western blot analysis in PANC-1 cells. Samples were obtained from PANC-1 cells treated with 300 nM of P276-00 and/or 70 nM gemcitabine at various time points as indicated in the figure. (A) Electrophoretic analysis of BNIP3, p8 and COX-2 mRNA. (B) protein expression analysis of antiapoptotic protein COX-2 and proapoptotic protein BNIP-3. (C) Densitometric analysis of the COX-2 and BNIP3 bands using the software Image J.

Figure 6

The combination treatment of gemcitabine with P276-00 showed significant tumor growth inhibition in PANC-1 xenograft model. (A) Differences in the Tumour weight (mg) in the different treatment groups. (B) Percent growth inhibition after treatment. Statistically significant difference (p < 0.005) of the combination treatment of gemcitabine and P276-00 compared with the control was seen.