Univariate survival analysis By univariate survival analysis, patients outcome was correlated with both tumor TNM and WHO stage. A borderline significance was observed for histological grade. AZD9291 The Kaplan Meier analysis of grouped Sirt1 expression was highly prognostic of poor overall survival for those patients with high Sirt1 expression with a mean postsurgical survival of 13. 0 vs. 54. 1 months. Multivariate survival analysis In multivariate Cox regression analysis, high Sirt1 expression was significantly related to shorter over all survival, in dependently of the degree of histological differentiation and WHO stage. Cellular effects of Sirt1 overexpression To test whether high Sirt1 expression also has a cellular ef fect in vitro, we performed overexpression experiments in both cell lines, MiaPaCa 2 and PANC 1, respectively, using cells upon transfection with flag tagged Sirt1 as determined by MTT assay and Xcelligence proliferation assays.

Nicotinamide and gefitinib treatment in cells with endogenous or overexpressed Sirt1 Inhibition of Sirt1 by increasing concentrations of nico tinamide led to a stepwise decrease of viable cells as depicted in Figure 5. Gefitinib treatment with concentra tions of 50 uM showed similar effects as observed for the application of 25 mM nicotinamide. Interestingly, combinatory treatment with 50 uM gefitinib and 25 mM or 40 mM nicotinamide showed a synergistic effect on cell viability, which was observed in both cell lines. Next, we asked whether inhibition of Sirt 1 by nicotina mide may counterbalance the beneficial effect on cell sur vival triggered by Sirt1 overexpression.

We found that application of 10 mM and lower concentrations of nicotina mide, which in untransfected cells already showed a strong flag tagged Sirt1. Overexpression of GFP served as control. Figure 3A shows immunoblots for endogenous and overexpressed Sirt1 in both cell lines. Cells overexpressing Sirt1 showed a markedly stronger immunosignal compared to their untransfected counterparts, which can also be depicted quantitatively as displayed in Figure 3B. Compared to GFP transfected cells, both cell lines showed statistically significantly increased amounts of viable, proliferating decrease of viable cell fractions compared to controls did not influence cell viability in cells overexpressing Sirt1, while higher concentrations of nicotinamide abrogated increased cell viability mediated by overexpressed Sirt1.

Cellular Cilengitide effects of cambinol, gemcitabine and gefitinib treatment Proliferation assay Real time proliferation assays revealed an inhibition of cell growth of Mia PaCa 2 cells and PANC 1 cells over a time period of 72 hrs upon treatment with cambinol. While for Mia PaCa 2 comparably lower concentrations of cambinol were necessary to achieve this effect, for PANC 1 cells concentrations up to 100 uM had to be applied.