236 PREVENTION OF RESISTANCE DEVELOPMENT IN RENAL CELL CARCINOMA BY COMBINED INHIBITION OF HISTONE DEACETYLASE AND MAMMALIAN TARGET OF RAPAMYCIN

Abstract

tone acetylation, and the expression of histone deacetylase (HDAC) 1, 2, 3, 6 were evaluated by western blot analysis. Combination indices (CI) were calculated using the Chou-Talalay method. RESULTS: Ritonavir in combination with bortezomib inhibited the growth of renal cancer cells synergistically (CI 1, n 6) and colony formation significantly at clinically feasible concentrations. In subcutaneous tumor models using Caki-1 cells, 10-day combination therapy with ritonavir (50 mg/kg) and bortezomib (60 g/kg) was well tolerated and inhibited tumor growth significantly (P 0.009). Ritonavir itself induced ER stress and in combination with bortezomib it further enhanced ER stress and induced protein ubiquitination synergistically. Interestingly, we also found that the combination of ritonavir and bortezomib enhanced histone acetylation synergistically by inhibiting the expression of HDACs. The combination therapy induced apoptosis and, because the pancaspase inhibitor Z-VAD-FMK reduced the number of annexin-V-positive cells, the apoptosis induced by ritonavir and bortezomib was thought to be caspase-dependent. CONCLUSIONS: The combination of ritonavir and bortezomib induces caspase-dependent apoptosis and inhibits the proliferation of renal cancer cells synergistically in vitro and in vivo. The effectiveness of the combination is due to enhanced ER stress, protein ubiquitination, and histone acetylation. Our results provide a rationale for investigating ritonavir in combination with bortezomib in patients with renal cancer.