Histone deacetylase (HDAC) inhibitors represent a promising class of antineoplastic agents which affect tumour growth, differentiation and invasion. The effects of the HDAC inhibitor valproic acid (VPA) were tested in vitro and in vivo on pre‐clinical renal cell carcinoma (RCC) models. Caki‐1, KTC‐26 or A498 cells were treated with various concentrations of VPA during in vitro cell proliferation 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assays and to evaluate cell cycle manipulation. In vivo tumour growth was conducted in subcutaneous xenograft mouse models. The anti‐tumoural potential of VPA combined with low‐dosed interferon‐α (IFN‐α) was also investigated. VPA significantly and dose‐dependently up‐regulated histones H3 and H4 acetylation and caused growth arrest in RCC cells. VPA altered cell cycle regulating proteins, in particular CDK2, cyclin B, cyclin D3, p21 and Rb. In vivo, VPA significantly inhibited the growth of Caki‐1 in subcutaneous xenografts, accompanied by a strong accumulation of p21 and bax in tissue specimens of VPA‐treated animals. VPA–IFN‐α combination markedly enhanced the effects of VPA monotherapy on RCC proliferation in vitro, but did not further enhance the anti‐tumoural potential of VPA in vivo. VPA was found to have profound effects on RCC cell growth, lending support to the initiation of clinical testing of VPA for treating advanced RCC.

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.

Introduction Renal cell carcinoma (RCC) accounts for 2-3% of adult cancers worldwide, with the highest rates observed in the United was combined with low-dosed interferon-␣ (IFN-␣) and the effects of the combination regimen compared to the single drug application. The experimental strategy was based on earlier reports demonstrating that IFN-␣ may enhance VPA's potency both in vivo and in vitro [9][10][11]. (IgG, clone Y28, dilution 1:500), anti-histone H4 (polyclonal IgG, dilution 1:250), anti-acetylated H4 (Lys8, polyclonal IgG, dilution 1:500) and anti-HDAC3 (polyclonal IgG, dilution 1:2000) were all from Biomol GmbH (Hamburg, Germany). (Taufenkirchen, Germany). VPA was shown to potently block RCC tumour cell adhesion in vitro and prevent RCC tumour growth in vivo. VPA's activity was associated with reduction of HDAC and elevated acetylation of histones H3 and H4. VPA altered integrin-␣ and -␤ subtype expression and blocked integrin-dependent signalling. It is of particular interest that VPA-IFN-␣ combination induced Anti-␤-actin monoclonal antibody was obtained from Sigma Cell culturesKidney carcinoma Caki-1 and KTC-26 cells were purchased from LGC Promochem (Wesel, Germany). A498 were derived from CLS (Heidelberg, Germany). Tumour cells were grown and subcultured in RPMI 1640 medium (Seromed, Berlin, Germany)