AEE788 was additionally compared to kinase inhibitors currently in clinical use. The EGF receptor tyro sine kinase inhibitors erlotinib and gefitinib, each applied at 1 M, significantly reduced RCC cell proliferation. selleck chemical Brefeldin A However, both agents were not as potent as was 1 M AEE788. Furthermore, erlotinib Inhibitors,Modulators,Libraries RAD001 and gefitinib RAD001 combination reduced cell growth to a lesser extent than the AEE788 RAD001 com bination. The same was true when the VEGF receptor inhibitor sunitinib was applied. Even, cell growth of A498 was not diminished at all by sunitinib. In all experiments, the Annexin V FITC assay did not reveal any signs of apoptosis. Therefore, cell growth reduction due to apoptotic events could be excluded. Ongoing studies con centrated on the influence of AEE788 and RAD001 on cell cycle progression and cell cycle regulating proteins.

AEE788 and RAD001 impair cell cycle progression Cell cycle analysis was carried out on A498 and Caki 1 cells. Based on asynchronous A498 cell populations, AEE788 and RAD001 significantly decreased the amount of S phase and enriched the amount of G0 G1 cells. Inhibitors,Modulators,Libraries Both compounds evoked similar effects on A498 cells, inde pendent Inhibitors,Modulators,Libraries on the concentration used. Cell cycle progression of asynchronous Caki 1 cells were also affected by AEE788 and RAD001, how ever AEE788 was more potent than RAD001 in this matter. The maximum cell cycle blockade was achieved when 1 M AEE788 and 1 nM RAD001 were added to RCC cells in combination. Subsequently, A498 or Caki 1 cells were released from an aphidicolin block to enrich the mitotic population.

In doing so, 1 M AEE788 or 1 nM RAD001 distinctly delayed cell cycle entry, AEE788 being Inhibitors,Modulators,Libraries more effective than RAD001. Combined application of both agents in the 1 M 1 nM formulation induced much stronger alterations on the cell cycle than the 1 M AEE788 or 1 nM RAD001 single drug application. Effects induced by the 1 M 1 nM drug combination were then similar to those seen under 5 M AEE788 and even more intense than seen under 5 nM RAD001 single drug appli cation. AEE788 and RAD001 alter expression level of cell cycle proteins Alteration of cell cycle regulating proteins strongly depended on the drug exposure time, the drug dosage and the RCC cell line Inhibitors,Modulators,Libraries used. With respect to asynchronous A498 cells, cdk2 was lowered after 6 h by 1 or 5 M AEE788 or by 5 nM RAD001 but enhanced by 1 nM RAD001, compared to the controls.

24 h analysis revealed cdk2 reduction by both AEE788 and RAD001. Cdk4 was found to be up regulated, notably by 1 M AEE788 or 1 nM RAD001 after a 6 h exposure. selleck chemicals llc Cyclin D1 was mainly diminished by AEE788 after 6 and 24 h, whereas cyclin E was enhanced after the same time period mainly by RAD001. p27 was drastically elevated after 6 and 24 h by both compounds, compared to the non treated controls. AEE788 and RAD001 also manipulated protein expres sion in asynchronous Caki 1 and KTC 26 cell cultures.