To proof this hypothesis, we evaluated the influence of the orally available mTOR inhibitor RAD001, applied alone or com bined with the dual EGF and VEGF receptor tyrosine kinase inhibitor AEE788, on RCC cell adhesion and proliferation in vitro. Our results indicate that both AEE788 and RAD001 exert potent exactly anti tumor activity. However, combined use of both compounds seems to be more effective than the single drug application and thus may provide a therapeutic advantage over either agent as monotherapy for RCC treatment. Methods Cell cultures Kidney carcinoma Caki 1 and KTC 26 cells were pur chased from LGC Promochem. A498 cells were derived from CLS. Tumor cells were grown and subcultured in RPMI 1640 medium supplemented with 10% FCS, 100 IU ml penicillin and 100 g ml streptomy cin at 37 C in a humidified, 5% CO2 incubator.
Endothe lial cells were isolated from human Inhibitors,Modulators,Libraries umbilical veins and harvested by enzymatic treatment with chymo trypsin. HUVEC were grown in Medium 199, 10% fetal calf serum, 10% pooled human serum, 20 g ml endothelial cell growth factor, 0. 1% heparin, 100 ng ml gentamycin and 20 mM HEPES buffer. Cell cultures were serially passaged. Subcultures from passages 2 4 were selected for experimental use. Drugs AEE788 and RAD001 were dissolved in DMSO as 10 mM stocks and stored as aliquots at 20 C. RCC cells were treated either with AEE788 or with RAD001 at concentra tions indicated in the results section. Combination treat ment with both compounds was based on 1 M AEE788 and 1 nM RAD001. Controls remained untreated.
In addi tional experiments, AEE788 was compared to tyrosine Inhibitors,Modulators,Libraries kinase inhibitors which are currently in clinical use gefit inib, erlotinib or sunitinib. To exclude toxic effects of the com pounds, cell viability was determined by trypan blue. For apoptosis detection the expres sion of Annexin V propidium iodide Inhibitors,Modulators,Libraries was evaluated using the Annexin V FITC Apoptosis Detection kit. Tumor Inhibitors,Modulators,Libraries cells were washed twice with PBS, and were then incubated with 5 l of Annexin V FITC and 5 l of PI in the dark for 15 min at RT. Cells were analyzed on a FACScalibur. The percentage of apop totic cells in each quadrant was calculated using CellQuest software. Tumor cell adhesion To analyze tumor cell adhesion, HUVEC were transferred to 6 well multiplates in complete HUVEC medium. When confluency was reached, Caki 1, KTC 26 or A498 cells were detached from the culture flasks by accutase treatment and 0.
5 106 cells were then added to the HUVEC monolayer for 60 min. Subsequently, non adherent tumor cells were washed off using warmed Medium 199. Inhibitors,Modulators,Libraries selleckbio The remaining cells were fixed with 1% glutaraldehyde. Adherent tumor cells were counted in five different fields of a defined size using a phase contrast microscope and the mean cellular adhe sion rate was calculated. Attachment to extracellular matrix components 6 well plates were coated with collagen G, lam inin, or fibronectin overnight.