Login to Access Video or Poster Abstract: MP73-10
Sources of Funding: none

Introduction

Histone deacetylase (HDAC) inhibitors not only induce histone acetylation but also inhibit molecular chaperones, causing unfolded proteins to accumulate and thereby inducing endoplasmic reticulum (ER) stress. However, the efficacy of HDAC inhibitors as anticancer agents is limited, especially in solid tumors. The HDAC inhibitor entinostat is partly metabolized by CYP3A4, and the human immunodeficiency virus protease inhibitor ritonavir also inhibits CYP3A4. We therefore thought that combining ritonavir with entinostat would kill cancer cells effectively by enhancing entinostat's activity.

Methods

Renal cancer cells (769-P, 786-O, Caki-2) were treated with ritonavir (20-40 µM) and entinostat (10-20 µM). Cell viability and clonogenicity were assessed by MTS assay and colony formation assay. A murine subcutaneous tumor model was used to evaluate in vivo efficacy. Flow cytometry was used for cell cycle analysis and annexin-V assay. Western blotting was used to evaluate the induction of histone acetylation and ER stress and the expression of cleaved poly(ADP-ribose) polymerase and the autophagy marker light chain (LC) 3-II. Combination indexes were calculated by the Chou-Talalay method.

Results

The combination of entinostat and ritonavir inhibited cancer cell growth synergistically (combination indexes <1) and suppressed colony formation significantly (p <0.05). The combination perturbed the cell cycle, increasing the number of the cells in the sub-G1 fraction (up to 98.2%). Drastic increases in the number of annexin-V positive cells (up to 98.6%) confirmed that the combination induced apoptosis. In murine subcutaneous tumor models using Caki-2 cells, a 10-day treatment with a combination of entinostat (2 mg/kg) and ritonavir (50 mg/kg) was well tolerated and inhibited tumor growth significantly (p <0.05). As expected, entinostat induced histone acetylation in a dose-dependent fashion, and ritonavir enhanced this acetylation synergistically. The combination also synergistically induced ER stress evidenced by increased expression of glucose-regulated protein 78 and endoplasmic reticulum resident protein 44. Interestingly, we also found that entinostat-ritonavir combination increased the expression of LC3-II, confirming that the unfolded protein accumulation due to the combination induced autophagy.

Conclusions

Ritonavir enhanced entinostat's activity, and the combination inhibited renal cancer growth synergistically.

Funding

none