MP18-15 RITONAVIR AND OPROZOMIB INHIBIT RENAL CANCER GROWTH BY INDUCING ENDOPLASMIC RETICULUM STRESS

Abstract

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II (MP18)1 Apr 2020MP18-15 RITONAVIR AND OPROZOMIB INHIBIT RENAL CANCER GROWTH BY INDUCING ENDOPLASMIC RETICULUM STRESS Takako Asano*, Kazuki Okubo, and Akinori Sato Takako Asano*Takako Asano* More articles by this author , Kazuki OkuboKazuki Okubo More articles by this author , and Akinori SatoAkinori Sato More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000843.015AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Inducing endoplasmic reticulum (ER) stress is a novel strategy used to treat malignancies. The human immunodeficiency virus protease inhibitor ritonavir inhibits molecular chaperones and increases the amount of unfolded proteins in cells. We postulated that combining ritonavir with the novel proteasome inhibitor oprozomib would kill renal cancer cells effectively by inhibiting the degradation of these unfolded proteins, thereby inducing ER stress triggering apoptosis. METHODS: The viability and clonogenicity of renal cancer cells (769-P, Caki-1, Caki-2) treated with ritonavir (20–40 μM) and/or oprozomib (50–200 nM) were assessed by MTS assay and colony formation assay. Flow cytometry was used to detect and quantify apoptosis. The induction of ER stress and the expression of acetylated histone, ubiquitinated proteins, AMP-activated protein kinase (AMPK), S6 ribosomal protein (S6), and histone deacetylases (HDACs) were assessed using western blot analysis. Combination indexes were calculated using the Chou-Talalay method. RESULTS: The combination of ritonavir and oprozomib inhibited renal cancer growth synergistically (combination indexes < 1) and suppressed colony formation significantly (p < 0.05). The combination caused renal cancer cell apoptosis synergistically: either 40 μM ritonavir or 50 nM oprozomib alone induced only moderate apoptosis, but in combination they induced drastic apoptosis (up to 97.6% annexin V-positive cells). Mechanistically, the combination caused ubiquitinated unfolded proteins to accumulate and thereby induced ER stress synergistically. This ER stress induction was essential to the combination’s cytotoxic action because inhibition of unfolded protein accumulation by the protein synthesis inhibitor cycloheximide markedly attenuated the combination-induced apoptosis. Furthermore, the combination-induced ER stress caused dephosphorylation of S6 by increasing the expression of mammalian target of rapamycin (mTOR) inhibitor AMPK, showing that the combination also inhibited the mTOR pathway. Interestingly, we also found that the combination increased histone acetylation synergistically by decreasing the expression of HDACs 1, 3, and 6. CONCLUSIONS: The combination of ritonavir and oprozomib inhibits renal cancer growth by synergistically inducing ER stress. Histone acetylation is also an important mechanism of its cytotoxicity. Source of Funding: None © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e241-e241 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takako Asano* More articles by this author Kazuki Okubo More articles by this author Akinori Sato More articles by this author Expand All Advertisement PDF downloadLoading ...