Valproic acid synergistically enhances the cytotoxicity of gossypol in DU145 prostate cancer cells: An iTRTAQ-based quantitative proteomic analysis Dong-yun Ouyang a, 1 , Yu-hua Ji a, b, 1 , Mark Saltis c , Li-hui Xu a , Yan-ting Zhang a , Qing-bing Zha a , Ji-ye Cai d , Xian-hui He a, ⁎ a Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China b Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, China c MSI, Essington PA 19029, USA d Department of Chemistry, Jinan University, Guangzhou 510632, China ARTICLE INFO ABSTRACT Article history: Received 2 April 2011 Accepted 15 June 2011 Available online 25 June 2011 Gossypol (GOS), a BH3 mimetic, has been investigated as a sensitizing co-therapy to radiation and chemotherapy in treatment of metastatic prostate cancer. In this study, we found that valproic acid (VPA), a histone deacetylase inhibitor (HDACI), counteracted the suppressive effect of GOS on histone H3 acetylation and enhanced the cytotoxicity of GOS to DU145 prostate cancer cells. Significant synergistic effects were observed in combined GOS and VPA treatment, culminating in more DNA damage and cell death. The iTRAQ-based quantitative proteomic analysis revealed differential proteomic profiles in cells treated with VPA, GOS or their combination. In GOS-treated cells, oxidative phosphorylation-related proteins were depressed and endoplasmic reticulum stress markers were upregulated. In the presence of VPA, the GOS-induced mitochondrial stress was further enhanced since glycolysis- and hypoxia-associated proteins were upregulated, suggesting a disruption of energy metabolism in these cells. Furthermore, the DNA damage repair ability of cells co-treated with GOS and VPA was also decreased, as evidenced by the downregulation of DNA damage repair proteins and the enhancement of DNA fragmentation and cell death. These findings suggest that GOS in combination with an HDACI has the potential to increase its clinical efficacy in the treatment of prostate cancer. © 2011 Elsevier B.V. All rights reserved. Keywords: Valproic acid Gossypol Synergistic activity Prostate cancer DNA damage 1. Introduction Prostate cancer remains a significant health threat. According to the most recent statistics available, the disease has affected nearly 204, 000 men in the United States and resulted in nearly 29, 000 deaths in 2006 (http://www.cdc.gov/Features/ ProstateCancer/. Accessed 10 March, 2011). Progress in treat- ment of prostate cancer after metastasis has been limited by resistance to chemotherapies, and development of androgen- independent recurrent tumors through the upregulation of numerous signaling and survival pathways [1]. These somatic adaptations necessitate investigation into new drug combi- nations to combat the epigenetic changes that occur in prostate cancer after recurrence of drug-resistant tumors. Gossypol (GOS), a natural product from the seeds of gossypium species (cotton), has been used extensively to lower JOURNAL OF PROTEOMICS 74 (2011) 2180 – 2193 ⁎ Corresponding author. Tel.: +86 20 85220679; fax: +86 20 85220679. E-mail address: thexh@jnu.edu.cn (X. He). 1 These two authors contributed equally to this work. 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.06.016 available at www.sciencedirect.com www.elsevier.com/locate/jprot