How ever, major differences were seen in KTC 26 cells, since cdk4 and cyclin D1 became all elevated by AEE788 or RAD001, whereas cyclin E was reduced by AEE788 after a Dasatinib CAS 6 and 24 h drug exposure. The AEE788 RAD001 combination experiments yielded ambiguous results. Additive effects became obvious in A498 cells with respect to cdk2 expression, in Caki 1 cells with respect to p27 expression. This was not true in the KTC 26 cell model. However, cyclin E was diminished to a greater extent in these cells by the AEE788 RAD001 combination compared to the single drug application. When drug treatment and protein analysis was carried out in the synchronous cell culture model, a clearer picture was obtained. As a general rule, cdk2, cdk4, cyclin D1 and cyclin E were all found to be down regu lated by AEE788 or RAD001.

Still, few exceptions remained demonstrating no changes or even elevated pro tein expression, compared to the controls. Alterations of the p27 expression level took place 6 and 24 h after the experimental start, becoming enhanced in A498 and Caki 1 cells by AEE788. The same effect was evoked by RAD001 in Caki 1. Interestingly, AEE788 reduced p27 expression in KTC Inhibitors,Modulators,Libraries 26 cells, whereas RAD001 enhanced it. AEE788 RAD001 combination treatment strongly aug Inhibitors,Modulators,Libraries mented the effects of the Inhibitors,Modulators,Libraries single drug treatment in all cell lines investigated. In particular, cdk2, cdk4, cyclin D1 and cyclin E were drastically reduced or even lost at specific time points in A498 and KTC 26 cells when both agents were used together.

Analysis of mTOR and EGF receptor signaling Finally, we evaluated if AEE788 and or RAD001 effects are linked to the inhibition of their primary targets. Total EGF receptor, ERK1 2, Akt and p70S6K were not changed by both agents. However, amount of activated EGF receptor was diminished by AEE788 in Caki 1 and A498 cells. Activated EGF receptor was also found to be reduced in Inhibitors,Modulators,Libraries presence of the AEE788 RAD001 drug combination. Phosphorylated ERK1 2 became lost by AEE788 or the AEE788 RAD001 drug combination in A498 cells. This phenomenon Inhibitors,Modulators,Libraries was not seen in Caki 1 cells. Interestingly, activation of Akt was only slightly down regulated by RAD001 in A498 cells, and the response of Caki 1 cells to RAD001 was only marginal in this matter. However, RAD001 strongly inhibited p70S6K activation in both Caki 1 and A498 cells.

Very strong deactivation of p70S6K was achieved by the AEE788 RAD001 drug combination in A498 cells. Discussion AEE788 is a 7H pyrrolo pyrimidine class receptor tyrosine kinase inhibitor that potently inhibits the EGFR associated kinase activity with additional inhibition of VEGFR 1 and VEGFR 2 at higher con centrations. Anti proliferative effects of this com pound have already been full report demonstrated on prostate, colon, pancreatic, lung, ovarian, and gliob lastoma cell lines.