Lopinavir-Ritonavir Combination Induces Endoplasmic Reticulum Stress and Kills Urological Cancer Cells

Abstract

Background/Aim: Induction of endoplasmic reticulum (ER) stress is a novel approach to cancer treatment. This study investigated the ability of the clinically feasible combination of the human immunodeficiency virus protease inhibitors lopinavir and ritonavir to induce ER stress killing urological cancer cells. Materials and Methods: Renal cancer cells (769-P, 786-O) and bladder cancer cells (UMUC-3, T-24) were used to investigate the ability of the combination to induce ER stress and its mechanism of action. Results: The combination inhibited the growth of both renal and bladder cancer cells synergistically by inducing ER stress. The combination-induced ER stress increased the expression of AMP-activated protein kinase and suppressed the mammalian target of rapamycin pathway. It also increased the expression of a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor and thereby sensitized the cancer cells to TRAIL. Conclusion: The combination of lopinavir and ritonavir acts against urological cancer cells by inducing ER stress synergistically. Materials and Methods Cell culture. Human renal cancer cells (769-P, 786-O) and human bladder cancer cells (UMUC-3, T-24) were purchased from the American Type Culture Collection (Rockville, MD, USA). The cells were cultured in either Roswell Park Memorial Institute Medium 1640 (769-P and 786-O cells), Minimum Essential Medium (UMUC-3 cells), or McCoy's 5A medium (T-24 cells) containing 10% fetal bovine serum and 1.0% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37˚C under 5% CO 2 in a humidified incubator. Reagents and antibodies. Ritonavir purchased from Toronto Research Chemicals (North York, ON, Canada) and lopinavir purchased from Selleck (Houston, TX, USA) were dissolved in dimethyl sulfoxide. Cycloheximide purchased from Enzo Life Sciences (Farmingdale, NY, USA) was dissolved in distilled water. Human recombinant TRAIL purchased from R&D Systems (Minneapolis, MN, USA) was dissolved in sterile phosphatebuffered saline (PBS) containing 0.1% bovine serum albumin. These solutions were stored at −80˚C or −20˚C until use. Primary antibodies for western blotting were used against the following: survivin and death receptor 5 (DR5) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); cleaved poly(ADP-ribose) polymerase (PARP), S6 ribosomal protein, phosphorylated S6, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), and 5891 This article is freely accessible online.

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“…Bissinger et al ( 30 ) showed that LPV induced apoptosis in erythrocytes, accompanied by cell shrinkage and phospholipid scrambling ( 30 ). Okubo et al ( 40 ) also showed the anti-proliferative properties of lopinavir/ritonavir (LPV/r) in combination against urological cancer cells. This study used 40/10 μM ratio of LPV/r over 48 h, and indicated that LPV/r treatment induced endoplasmic reticulum (ER) stress and kills urological cancer cells ( 40 ).…”

“…Okubo et al ( 40 ) also showed the anti-proliferative properties of lopinavir/ritonavir (LPV/r) in combination against urological cancer cells. This study used 40/10 μM ratio of LPV/r over 48 h, and indicated that LPV/r treatment induced endoplasmic reticulum (ER) stress and kills urological cancer cells ( 40 ). Lopinavir was also shown to inhibit melanoma cell proliferation, induce morphological changes, apoptosis, and reactive oxygen species production, ( 41 ).…”