BackgroundTreatment options for metastatic renal cell carcinoma (RCC) are limited due to resistance to chemo- and radiotherapy. The development of small-molecule multikinase inhibitors has now opened novel treatment options. We evaluated the influence of the receptor tyrosine kinase inhibitor AEE788, applied alone or combined with the mammalian target of rapamycin (mTOR) inhibitor RAD001, on RCC cell adhesion and proliferation in vitro.MethodsRCC cell lines Caki-1, KTC-26 or A498 were treated with various concentrations of RAD001 or AEE788 and tumor cell proliferation, tumor cell adhesion to vascular endothelial cells or to immobilized extracellular matrix proteins (laminin, collagen, fibronectin) evaluated. The anti-tumoral potential of RAD001 combined with AEE788 was also investigated. Both, asynchronous and synchronized cell cultures were used to subsequently analyze drug induced cell cycle manipulation. Analysis of cell cycle regulating proteins was done by western blotting.ResultsRAD001 or AEE788 reduced adhesion of RCC cell lines to vascular endothelium and diminished RCC cell binding to immobilized laminin or collagen. Both drugs blocked RCC cell growth, impaired cell cycle progression and altered the expression level of the cell cycle regulating proteins cdk2, cdk4, cyclin D1, cyclin E and p27. The combination of AEE788 and RAD001 resulted in more pronounced RCC growth inhibition, greater rates of G0/G1 cells and lower rates of S-phase cells than either agent alone. Cell cycle proteins were much more strongly altered when both drugs were used in combination than with single drug application. The synergistic effects were observed in an asynchronous cell culture model, but were more pronounced in synchronous RCC cell cultures.ConclusionPotent anti-tumoral activitites of the multikinase inhibitors AEE788 or RAD001 have been demonstrated. Most importantly, the simultaneous use of both AEE788 and RAD001 offered a distinct combinatorial benefit and thus may provide a therapeutic advantage over either agent employed as a monotherapy for RCC treatment.

integrin α and β subtypes were evaluated by flow cytometry (surface expression) and Western blotting (intracellular protein expression). RESULTSRCC cell treatment with AEE788 and VPA in combination resulted in a stronger inhibition of tumour cell proliferation than that caused by either drug alone. There were also additive effects of the combined treatment on tumour cell adhesion to endothelial cells and to immobilized laminin (but not to immobilized fibronectin and collagen). AEE788 alone or combined with VPA reduced Akt expression and histone H3 acetylation. Both compounds altered integrin α and β subtype expression, in particular α 1, α 3 and β 4, and blocked integrin-dependent integrin-linked kinase and focal-adhesion kinase (total and phosphorylated) signalling. CONCLUSIONSBoth AEE788 and VPA profoundly block the interaction of RCC cells with endothelium and extracellular matrix and reduce tumour growth in vitro . Therefore, this combined regimen warrants further preclinical and possible clinical study for treating advanced RCC.