Abstract 5442: In vitro and in vivo upregulation of thymidine phosphorylase expression in colon cancer cells by HDAC inhibitor vorinostat resulted in synergistic antitumor effect in combination with capecitabine
Authors: Gennaro Elena Di , Piro Geny , Chianese Maria I. , Cintio Alessandra Di , Moccia Tania , Luciano Antonio , Bruzzese Francesca , Arra Claudio , Budillon Alfredo 
Published: February 2011

Abstract
The oral fluoropyrimidine capecitabine is converted to the active compound 5-florouracil (5-FU) in several steps, the last of which is the conversion of 5′-deoxy-5-fluorouridine (5′DFUR) to 5-FU by thymidine phosphorylase (TP). Capecitabine was designed to take advantage of the increased levels of TP observed in tumors as opposed to normal tissues, potentially allowing for selective toxicity in tumors. In this study we found that the antiproliferative effect induced by the histone deacetylase inhibitor vorinostat in colorectal cancer cells in vitro and in vivo xenograft models, was paralleled by downregulation of thymidilate synthase, an essential enzyme for DNA synthesis and the target of 5-FU, and by upregulation of TP. These effects were evident at the mRNA and at the protein level and were not observed in peripheral blood lymphocyte (PBL) from healthy donors treated ex vivo by vorinostat. On the basis of these results, we have evaluated in vitro the effects of the combination between vorinostat and the capecitabine metabolite 5′-DFUR on the growth of human LoVo, LS174T and SW620 colon cancer cell lines, showing that simultaneous exposure of equipotent doses of the two agents, for 96 hours, resulted in synergistic antiproliferative effect in all cell lines as demonstrated by calculating combination indexes. We also demonstrated that vorinostat and 5′-DFUR in combination, compared to single agents treatment, increased apoptotic cell death, demonstrated by flow cytometry analysis of DNA content after propidium iodide staining, by the induction of BAX protein expression and by the cleavage of PARP. The observed apoptotic effect could be, at least in part, related to the increase in the ROS content induced by vorinostat and 5′-DFUR combination compared to single agents treatment. In order to verify in vivo the synergistic effects on tumor cell growth demonstrated in vitro, we evaluated vorinostat in combination with capecitabine in SW620 colon cancer cells xenograft model. A marked inhibition of tumor growth was observed in mice group treated with vorinostat (100 mg/kg p.o. daily) plus capecitabine (359 mg/Kg p.o. daily) as compared to single agents treatment, with a consequent increase by 2.4-fold of tumor growth delay and a substantial increase in survival. Analysis of xenografts tumor sections by immunohistochemistry showed a significant increase in apoptotic cells in vorinostat plus capecitabine treated group compared with control, or single agent treated groups, as well as the down-regulation TS and upregulation TP proteins expression in mice treated with vorinostat alone or in combination with capecitabine. Overall these data suggested that the association of vorinostat plus capecitabine could be clinically explored in colon cancer patients.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5442.