Specific Thiazolidinediones Inhibit Ovarian Cancer Cell Line Proliferation and Cause Cell Cycle Arrest in a PPARc Independent Manner Linah Al-Alem1,2, R. Chase Southard1, Michael W. Kilgore1, Thomas E. Curry2* 1 Department of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America, 2 Department of Obstetrics and Gynecology, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America Abstract Background: Peroxisome Proliferator Activated Receptor gamma (PPARc) agonists, such as the thiazolinediones (TZDs), have been studied for their potential use as cancer therapeutic agents. We investigated the effect of four TZDs— Rosiglitazone (Rosi), Ciglitazone (CGZ), Troglitazone (TGZ), and Pioglitazone (Pio)—on ovarian cancer cell proliferation, PPARc expression and PPAR luciferase reporter activity. We explored whether TZDs act in a PPARc dependent or independent manner by utilizing molecular approaches to inhibit or overexpress PPARc activity. Principal Findings: Treatment with CGZ or TGZ for 24 hours decreased proliferation in three ovarian cancer cell lines, Ovcar3, CaOv3, and Skov3, whereas Rosi and Pio had no effect. This decrease in Ovcar3 cell proliferation was due to a higher fraction of cells in the G0/G1 stage of the cell cycle. CGZ and TGZ treatment increased apoptosis after 4 hours of treatment but not after 8 or 12 hours. Treatment with TGZ or CGZ increased PPARc mRNA expression in Ovcar3 cells; however, protein levels were unchanged. Surprisingly, luciferase promoter assays revealed that none of the TZDs increased PPARc activity. Overexpression of wild type PPARc increased reporter activity. This was further augmented by TGZ, Rosi, and Pio indicating that these cells have the endogenous capacity to mediate PPARc transactivation. To determine whether PPARc mediates the TZD-induced decrease in proliferation, cells were treated with CGZ or TGZ in the absence or presence of a dominant negative (DN) or wild type overexpression PPARc construct. Neither vector changed the TZD-mediated cell proliferation suggesting this effect of TZDs on ovarian cancer cells may be PPARc independent. Conclusions: CGZ and TGZ cause a decrease in ovarian cancer cell proliferation that is PPARc independent. This concept is supported by the finding that a DN or overexpression of the wild type PPARc did not affect the changes in cell proliferation and cell cycle. Citation: Al-Alem L, Southard RC, Kilgore MW, Curry TE (2011) Specific Thiazolidinediones Inhibit Ovarian Cancer Cell Line Proliferation and Cause Cell Cycle Arrest in a PPARc Independent Manner. PLoS ONE 6(1): e16179. doi:10.1371/journal.pone.0016179 Editor: Irina Agoulnik, Florida International University, United States Received August 17, 2010; Accepted December 14, 2010; Published January 21, 2011 Copyright: 2011 Al-Alem 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: This work was supported by the National Institutes of Health (grant numbers CA95609, HD057446 and RR15592). 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: tecurry@uky.edu Introduction Ovarian cancer is the fifth leading cause of cancer death in women. Of the three main types of ovarian cancer (epithelial, germ cell, and sex cord stromal cancers), epithelial ovarian cancer accounts for about 90% of all cases, and is the first cause of death from gynecological malignancies [1,2]. Despite intense research on ovarian cancer with new targets being constantly investigated, treatment targets remain sparse. One of the challenges in ovarian cancer research is the absence of an experimental animal model that recapitulates the human disease that can be experimentally manipulated [1,3]. Thus, ovarian cancer cell lines have been employed to understand the fundamental processes involved in cancer cell growth, differentiation, and proliferation. The present study utilized three ovarian cancer cells, Ovcar3, CaOv3 and Skvo3, which are derived from human epithelial ovarian cancer [4,5] to further explore therapeutic modalities in cancer cell growth and proliferation. One of the therapeutic targets under investigation for ovarian cancer is nuclear receptors. Drugs that activate or inhibit nuclear receptors have been used to treat many diseases. Indeed, about 13% of the drugs currently on the market target nuclear receptors [6]. PPARc is a highly conserved nuclear receptor [7] expressed throughout the body [8] and is over expressed in many cancers, including ovarian and breast cancer, making it a potentially important player in the development of cancer. Endogenous PPARc ligands are still unknown, but well charac- terized candidates include polyunsaturated fatty acids, Prostaglan- din J2 (PGJ2) and arachidonic acid [9]. Synthetic PPARc ligands include the thiazolidinediones (TZDs), which consist of Rosiglita- zone (AvandiaH), Troglitazone (RezulinH), Pioglitazone (Glustin H/ ActosH), and Ciglitazone, all of which have been developed and/or used to treat type II diabetes [10,11,12]. The use of TZDs as a therapeutic approach in cancer has been investigated but results have been controversial [13,14,15]. In this study we utilized molecular, physiological and pharmacological approaches to PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16179