Simvastatin-Romidepsin Combination Kills Bladder Cancer Cells Synergistically
Authors: Okubo Kazuki , Miyai Kosuke , Kato Kimi , Asano Takako , Sato Akinori 
Published: July 2020

Abstract
Background
The HMG-CoA reductase inhibitor simvastatin activates AMP-activated protein kinase (AMPK) and thereby induces histone acetylation. We postulated that combining simvastatin with the histone deacetylase (HDAC) inhibitor romidepsin would kill bladder cancer cells by inducing histone acetylation cooperatively.
Methods
Bladder cancer cells (UMUC-3, T-24, J-82, MBT-2) were treated with simvastatin and romidepsin. Cell viability and clonogenicity were assessed by CCK-8 assay and colony formation assay. In-vivo efficacy was evaluated using murine subcutaneous tumor models. Flow cytometry was used to detect annexin-V positive cells and to evaluate cell cycle distribution and cellular reactive oxygen species (ROS) production. Induction of histone acetylation and the expression of AMPK, HDACs, peroxisome proliferator-activated receptor (PPAR) γ, cell-cycle regulators, and the endoplasmic reticulum (ER) stress markers were evaluated by western blotting.
Results
The combination of romidepsin and simvastatin induced robust apoptosis and killed bladder cancer cells synergistically (combination indexes < 1). It also suppressed colony formation significantly. In murine subcutaneous tumor models using MBT-2 cells, a 15-day treatment with 0.5 mg/kg romidepsin and 15 mg/kg simvastatin was well tolerated and inhibited tumor growth significantly. Mechanistically, the combination induced histone acetylation by activating AMPK. The combination also decreased the expression of HDACs, thus further promoting histone acetylation. This AMPK activation was essential for the combination’s action because compound C, an AMPK inhibitor, suppressed the combination-induced histone acetylation and the combination’s ability to induce apoptosis. We also found that the combination increased the expression of PPARγ, leading to ROS production. Furthermore, the combination induced ER stress and this ER stress was shown to be associated with increased AMPK expression and histone acetylation, thus playing an important role in the combination’s action. Our study also suggests there is a positive feedback cycle between ER stress induction and PPARγ expression.
Conclusions
The combination of simvastatin and romidepsin kills bladder cancer cells synergistically. Its mechanism of action includes ER stress induction, AMPK activation, histone acetylation, and increased PPARγ expression.
Background
There is currently no curative treatment for metastatic bladder cancer and development of novel treatment strategy is urgently needed. AMP-activated protein kinase (AMPK) is a cellular energy sensor, which is activated by impaired energy status such as glucose deprivation, ischemia, hypoxia, and oxidative stress, leading to inhibition of cellular growth to restore energy homeostasis [1]. Therefore, drugs activating AMPK have attracted much attention as novel anticancer agents [2–5]. HMG-CoA reductase inhibitors, which are widely used for treating dyslipidemia [6–9], are known to activate AMPK [10–12]. Simvastatin inhibits HMG-CoA reductase and has been shown to kill cancer cells in vitro [12–14], although its anticancer efficacy has not been proven yet in clinical trials [15].