Abstract 4040: Short-term romidepsin treatment combined with clotrimazole or bifonazole leads to decreased mitochondrial hexokinase 2 and apoptosis in cancer cells

Abstract

Abstract In light of disappointing clinical trials in solid tumors, we have sought novel combinations to increase the efficacy of the histone deacetylase inhibitor (HDI) romidepsin. Hexokinase 2 (HK2) expression is increased in cancer cells and is postulated to prevent Bax-mediated cytochrome c release from mitochondria. Since romidepsin treatment induces apoptosis via the mitochondrial pathway, combining romidepsin with agents that detach mitochondrial HK2 might result in synergistic apoptotic effects. HCT-116 colon carcinoma cells were treated with 25 ng/ml romidepsin alone for 6h, or with romidepsin in the presence of 25 µM clotrimazole or bifonazole, compounds known to detach mitochondrial HK2. The medium was removed and cells were treated for an additional 42 h with or without clotrimazole or bifonazole. Cells were also treated with clotrimazole or bifonazole alone for 48 h. Apoptosis was then quantitated by annexin staining. While short-term romidepsin treatment alone induced modest annexin staining and 25 µM clotrimazole or bifonazole had little effect, the combination resulted in significant apoptosis. Similar effects were observed for A549 lung cancer cells, MDA-MB-231 breast cancer cells and 786-0 renal carcinoma cells. We also treated HCT-116 and A549 cells lacking Bak, Bax or both with the clotrimazole/romidepsin combination. Apoptosis was slightly increased in Bak-/- cells, reduced in Bax-/- cells and nearly completely abrogated in cells lacking both proteins compared to wild-type cells suggesting apoptosis occurred through the intrinsic pathway. The clotrimazole analog TRAM-34 was similarly active to clotrimazole. Protein expression was determined in cytoplasmic and mitochondrial fractions of cells treated with romidepsin, clotrimazole or both and we found a >60% decrease in mitochondrial HK2 in cells treated with the combination versus untreated cells. Our results suggest that combining romidepsin with a compound that leads to decreased mitochondrial hexokinase, such as clotrimazole or bifonazole, results in increased apoptosis. We postulate that cell death following combined romidepsin and clotrimazole treatment requires an intact intrinsic apoptotic pathway and that HK2 serves an anti-apoptotic function at the mitochondria. Citation Format: Robert W. Robey, Andrew J. McDonald, Hanna Kozlowski, Michael M. Gottesman, Susan E. Bates. Short-term romidepsin treatment combined with clotrimazole or bifonazole leads to decreased mitochondrial hexokinase 2 and apoptosis in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4040. doi:10.1158/1538-7445.AM2017-4040