Effects of Combined Valproic Acid and the Epidermal Growth Factor/vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor AEE788 on Renal Cell Carcinoma Cell Lines in Vitro

Objective: To evaluate adhesion and growth inhibiting effects of the multiple receptor tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) on renal cell carcinoma (RCC) cells.

Materials And Methods: Caki-1 cells were treated with AEE788 and VPA, either alone or in combination, to investigate RCC cell adhesion to vascular endothelial cells or to immobilized extracellular matrix proteins. Tumour cell proliferation was examined by MTT dye reduction assay. Effects of drug treatment on cell signalling pathways were determined by Western blotting. The expression levels of integrin alpha and beta subtypes were evaluated by flow cytometry (surface expression) and Western blotting (intracellular protein expression).

Results: RCC 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 alpha and beta subtype expression, in particular alpha1, alpha3 and beta4, and blocked integrin-dependent integrin-linked kinase and focal-adhesion kinase (total and phosphorylated) signalling.

Conclusions: Both 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.