Combinatorial Effect of Non-Steroidal Anti-inflammatory Drugs and NF-kB Inhibitors in Ovarian Cancer Therapy Luiz F. Zerbini1,4*, Rodrigo E. Tamura1, Ricardo G. Correa2, Akos Czibere3, Jason Cordeiro4, Manoj Bhasin4, Fernando M. Simabuco4, Yihong Wang4, Xuesong Gu4, Linglin Li5, Devanand Sarkar6, Jin-Rong Zhou5, Paul B. Fisher6, Towia A. Libermann4 1 Medical Biochemistry Division, Faculty of Health Sciences, International Center for Genetic Engineering and Biotechnology (ICGEB), University of Cape Town, Cape Town, South Africa, 2 Sanford-Burnham Institute for Medical Research, La Jolla, California, United States of America, 3 Department of Haematology, Oncology and Clinical Immunology, Heinrich Heine-University, D u¨sseldorf, Germany, 4 BIDMC Genomics and Proteomics Center, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States of America, 5 Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States of America, 6 Department of Human and Molecular Genetics, School of Medicine, VCU Institute of Molecular Medicine, VCU Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia, United States of America Abstract Several epidemiological studies have correlated the use of non-steroidal anti-inflammatory drugs (NSAID) with reduced risk of ovarian cancer, the most lethal gynecological cancer, diagnosed usually in late stages of the disease. We have previously established that the pro-apoptotic cytokine melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24) is a crucial mediator of NSAID-induced apoptosis in prostate, breast, renal and stomach cancer cells. In this report we evaluated various structurally different NSAIDs for their efficacies to induce apoptosis and mda-7/IL-24 expression in ovarian cancer cells. While several NSAIDs induced apoptosis, Sulindac Sulfide and Diclofenac most potently induced apoptosis and reduced tumor growth. A combination of these agents results in a synergistic effect. Furthermore, mda-7/IL-24 induction by NSAIDs is essential for programmed cell death, since inhibition of mda-7/IL-24 by small interfering RNA abrogates apoptosis. mda-7/IL-24 activation leads to upregulation of growth arrest and DNA damage inducible (GADD) 45 a and c and JNK activation. The NF-kB family of transcription factors has been implicated in ovarian cancer development. We previously established NF-kB/IkB signaling as an essential step for cell survival in cancer cells and hypothesized that targeting NF-kB could potentiate NSAID-mediated apoptosis induction in ovarian cancer cells. Indeed, combining NSAID treatment with NF- kB inhibitors led to enhanced apoptosis induction. Our results indicate that inhibition of NF-kB in combination with activation of mda-7/IL-24 expression may lead to a new combinatorial therapy for ovarian cancer. Citation: Zerbini LF, Tamura RE, Correa RG, Czibere A, Cordeiro J, et al. (2011) Combinatorial Effect of Non-Steroidal Anti-inflammatory Drugs and NF-kB Inhibitors in Ovarian Cancer Therapy. PLoS ONE 6(9): e24285. doi:10.1371/journal.pone.0024285 Editor: Irina V. Lebedeva, Enzo Life Sciences, Inc., United States of America Received April 12, 2011; Accepted August 5, 2011; Published September 12, 2011 Copyright: 2011 Zerbini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: RET is a recipient of the ICGEB post-doctoral fellowship. This research was supported by National Institutes of Health grants 1R01 CA85467, P50 CA090381, P50 CA105009 and the Hershey Foundation (TAL), NIH grants 1R01 CA097318, 1R01 CA127641, P01 CA104177, the Samuel Waxman Cancer Research Foundation (PBF) and Department of Defense grants PC051217 and OC0060439 (LFZ). DS is a Harrison Scholar in Cancer Research and a Blick Scholar in the VCU Massey Cancer Center and the VCU School of Medicine. PBF holds the Thelma Newmeyer Corman Chair in Cancer Research in the VCU Massey Cancer Center and is a SWCRF Investigator. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: luiz.zerbini@uct.ac.za Introduction Ovarian cancer represents the most lethal gynecological cancer and the 5th leading cause of women death related to cancer in the United States [1]. Late diagnosis is one of the main hurdles to treat ovarian cancer, as nearly 70% of women present with an advanced stage of the disease at diagnosis [2]. Several epidemi- ological studies have suggested that the use of NSAIDs at clinically relevant concentrations reduces colorectal [3], breast [4] and ovarian cancer risks [5,6,7], although controversy still remains [8,9]. One major target of NSAID action is inhibition of cyclooxygenase (COX), which are responsible for the conversion of arachidonic acids into prostaglandins and employ a variety of different mechanisms. The two COX genes, COX-1 and COX-2, are almost identical; however, one relevant difference is that COX-1 expression is constitutive, whereas COX-2 expression is induced by growth factors and pro-inflammatory stimuli [10]. NSAIDs are typically classified as specific COX-2 inhibitors or non-specific COX inhibitors. In ovarian cancer COX-1, but not COX-2 has been found to be overexpressed [11,12], nevertheless other studies reported that COX-2 is also upregulated [13,14]. High COX-1 expression in ovarian cancer strongly correlates with high levels of Vascular Endothelial Growth Factor (VEGF) [15,16,17] and NSAIDs inhibit VEGF production in ovarian cancer cell lines [12,18] indicating that COX-1 may regulate VEGF expression. Angiogenesis and VEGF expression are implicated in ascites formation [19] and metastasis of ovarian cancer [20], while its inhibition prevents ascites formation and inhibits disseminated cancer growth [21]. Our previous study demonstrates that NSAIDs also induce apoptosis of cancer cells via induction of mda-7/IL-24 expression [22], leading to enhanced expression of two members of the Growth Arrest and DNA-Damage 45 (GADD45) family [23]. The GADD45 gene family encodes three structurally highly related PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24285