MP92-16 RITONAVIR AND DELANZOMIB INHIBIT RENAL CANCER GROWTH IN VITRO AND IN VIVO BY INDUCING ENDOPLASMIC RETICULUM STRESS SYNERGISTICALLY

Abstract

INTRODUCTION AND OBJECTIVES: Induction of endoplasmic reticulum (ER) stress is a novel strategy for treating malignancies. The human immunodeficiency virus (HIV) protease inhibitor ritonavir increases the amount of unfolded proteins by suppressing the heat shock protein 90. We thought that combining ritonavir with the proteasome inhibitor delanzomib would kill renal cancer cells effectively by inhibiting the degradation of these unfolded proteins and thereby inducing ER stress. METHODS: Renal cancer cells (769-P, 786-O, Caki-2, Renca) were treated with ritonavir (25-50 mM) and/or delanzomib (25-50 nM). Cell viability and clonogenicity were assessed by MTS assay and colony formation assay. In vivo efficacy was evaluated using murine subcutaneous tumor models. Flow cytometry was used for cell cycle analysis and annexin-V assay. Western blotting was used to evaluate the induction of ER stress (expression of glucose-regulated protein 78, endoplasmic reticulum resident protein 44, and endoplasmic oxidoreductin-1-like protein) and histone acetylation, as well as the expression of Akt, cyclin D1, cyclin-dependent kinase (CDK) 4, sestrin 2, AMPactivated protein kinase (AMPK), and mammalian target of rapamycin (mTOR). RESULTS: The combination of ritonavir and delanzomib inhibited renal cancer growth synergistically (combination indexes <1) and suppressed colony formation significantly (P <0.05). It decreased the expression of cyclin D1 and CDK4, leading to the accumulation of the cells in the sub-G1 fraction (up to 97.0%). The combination induced robust apoptosis (annexin-V positive cells increased up to 91.8%), and this apoptosis seemed to be caspase-dependent because the pancaspase inhibitor Z-VAD-FMK markedly decreased the number of the annexin-V positive cells. In murine tumor models, a 13-day treatment with 15 mg/kg ritonavir and 30 mg/kg delanzomib was well tolerated and inhibited tumor growth significantly (P 1⁄4 0.009). As expected, the combination increased the expression of the ER stress markers synergistically. It also increased the expression of the mTOR inhibitors sestrin 2 and AMPK and decreased the expression of mTOR and Akt, confirming the mTOR pathway inhibition following the ER stress induction. Interestingly, we also found that the combination-induced ER stress enhanced histone acetylation synergistically. CONCLUSIONS: Ritonavir and delanzomib together inhibit renal cancer growth by inducing ER stress synergistically.