MP35-10 RITONAVIR SYNERGIZES WITH CARFILZOMIB TO INDUCE ENDOPLASMIC RETICULUM STRESS AND AUTOPHAGY IN RENAL CANCER CELLS

Abstract

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2014MP35-10 RITONAVIR SYNERGIZES WITH CARFILZOMIB TO INDUCE ENDOPLASMIC RETICULUM STRESS AND AUTOPHAGY IN RENAL CANCER CELLS Akinori Sato, Takako Asano, Makoto Isono, Keiichi Ito, and Tomohiko Asano Akinori SatoAkinori Sato More articles by this author , Takako AsanoTakako Asano More articles by this author , Makoto IsonoMakoto Isono More articles by this author , Keiichi ItoKeiichi Ito More articles by this author , and Tomohiko AsanoTomohiko Asano More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1053AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Inducing endoplasmic reticulum (ER) stress is a novel strategy used to treat malignancies. The HIV protease inhibitor ritonavir has recently been shown to increase unfolded proteins by suppressing the function of heat shock protein 90. We postulated that combining the novel proteasome inhibitor carfilzomib with ritonavir would kill cancer cells effectively by inhibiting the degradation of the ritonavir-increased unfolded proteins and thereby inducing ER stress. METHODS Renal cancer cells (ACHN, 769-P, 786-O) were treated with carfilzomib (20-100 nM) and/or ritonavir (25-50 µM). Cell viability and clonogenicity were evaluated by MTS assay and colony formation assay. Combination indexes were calculated using the Chou-Talalay method. Apoptosis was assayed using flow cytometry and by detecting the expression of cleaved poly(ADP-ribose) polymerase. ER stress and changes in the expression of ubiquitinated proteins and the autophagy marker light chain (LC) 3 were evaluated by western blotting. RESULTS Ritonavir in combination with carfilzomib inhibited renal cancer growth synergistically (combination indexes <1) and suppressed colony formation significantly (P <0.05). The combination induced drastic apoptosis (annexin-V fluorescence intensity increased 9.6-fold), and this apoptosis was thought to be mainly caspase-dependent because the number of annexin-V-positive cells was reduced by co-treatment with the pancaspase inhibitor Z-VAD-FMK. As expected, the combination synergistically induced ER stress evidenced by increased expression of glucose-regulated protein (GRP) 78: neither 20 nor 50 nM carfilzomib alone increased GRP78 expression, but either concentration combined with 50 µM ritonavir increased GRP78 expression markedly. We also found that ritonavir-carfilzomib combinations decreased the expression of LC3-I and increased the expression of LC3-II, confirming that they induced autophagy. This autophagy was thought to be due to aggresome formation caused by extensive protein ubiquitination. CONCLUSIONS Ritonavir combined with carfilzomib inhibits the growth of renal cancer cells by synergistically inducing ER stress and autophagy. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e374 Peer Review Report Advertisement Copyright & Permissions© 2014MetricsAuthor Information Akinori Sato More articles by this author Takako Asano More articles by this author Makoto Isono More articles by this author Keiichi Ito More articles by this author Tomohiko Asano More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...