Presentation Authors: Akinori Sato, Takako Asano, Makoto Isono, Kazuki Okubo*, Tokorozawa, Japan
Introduction: Inhibition of histone deacetylase (HDAC) 6 by the pan-deacetylase inhibitor panobinostat hinders the refolding of unfolded proteins by increasing the acetylation of molecular chaperones and increases the amount of unfolded proteins. The human immunodeficiency virus (HIV) protease inhibitor ritonavir suppresses proteasomes and inhibits degradation of unfolded proteins. We thought that panobinostat combined with ritonavir would kill bladder cancer cells by causing unfolded proteins to accumulate and thereby inducing endoplasmic reticulum (ER) stress.
Methods: A panel of bladder cancer cells (UMUC3, T24, J82, 5637) was treated with panobinostat (50-200 nM) and/or ritonavir (25-50 ÂµM). The cell viability and clonogenicity were assessed by MTS assay and colony formation assay. The induction of ER stress and the expression of acetylated histone, acetylated Î±-tubulin, cyclin D1, and cyclin-dependent kinase (CDK) 4 were assessed using western blot analysis. Flow cytometry was used for annexin V assay, measuring the mitochondrial membrane potential, and cell cycle analysis. Combination indexes were calculated using the Chou-Talalay method.
Results: Panobinostat and ritonavir induced drastic apoptosis (up to 93.7% annexin V-positive cells) and inhibited the growth of bladder cancer cells synergistically (combination index < 1). The combination cooperatively caused depolarization of the mitochondrial membrane potential, confirming that the combination activated the mitochondrial apoptotic pathway. The combination also decreased the expression of cyclin D1 and CDK4, leading to the accumulation of the cells in the sub-G1 fraction. Acetylation of the HDAC6 substrate Î±-tubulin confirmed the suppression of HDAC6 activity by panobinostat, and the combination was shown to induce ER stress synergistically. This ER stress induction was shown to play a pivotal role in the combination&[prime]s cytotoxic action because the ER stress inhibitor cycloheximide markedly attenuated the combination-induced apoptosis. Interestingly, we also found that panobinostat-induced histone acetylation was synergistically enhanced by ritonavir, which is another important mechanism of action.
Conclusions: The combination of panobinostat and ritonavir causes bladder cancer apoptosis by inducing ER stress and increasing histone acetylation synergistically.
Source of Funding: JSPS KAKENHI Grant Number JP18K09183