to impact cell proliferation. Irrespective, this approach provided evidence that there was a lack of an effect of endogenous PPARc on cell proliferation since the expression of a DN or OE form of PPARc did not affect cell proliferation. In summary, our findings demonstrate that CGZ and TGZ decrease cell proliferation mainly through cell cycle arrest. The current study establishes that these effects appear to be TZD specific as these changes are not seen in cells treated with Rosi or Pio, unlike previous reports [20,21], yet all four TZDs drive the same levels of PPRE activity. This could be due to several hypothetical explanations. Rosi and Pio may be recruiting different coactivators than those of CGZ and TGZ. Alternatively, the effects of TZDs on proliferation may be via changes in the Akt/PTEN/mTOR pathway [50]. Rosi has been previously shown to suppress the AKT pathway via increasing the expression of PTEN in cells such as MCF7 [51] and NSCLC [27]. Thus, it is possible that CGZ and TGZ cause a more pronounced effect on the AKT/mTOR pathway than Rosi and Pio. Another plausible explanation is that variations in the specificity of these ligands for PPARc, thus, causing changes in promoter activity such as a prolonged DNA binding activity rather than an increase in magnitude. Alternatively, the changes in cell proliferation with CGZ and TGZ may be independent of PPARc. For example, adding DN in combination to CGZ or TGZ did not rescue Ovcar3 cells from the decrease in proliferation, indicating that these compounds are at least partially acting in a PPARc independent manner. Our findings also demonstrate that PPARc agonists stimulate PPARc expression, but to varying degrees in different ovarian cancer cells. This stimulation of PPARc mRNA is not mirrored by PPARc protein levels and interestingly, the levels of PPARc protein do not accurately predict PPRE reporter activity. These data suggest that TZD actions may be beneficial in hindering the proliferative capacity of tumor cells albeit potentially in a PPARc independent manner. This observation is the first report to our knowledge that utilized both molecular and pharmacological approaches to demonstrate that TZDs convey their actions on ovarian cancer cells independent of PPARc. Since TZDs have often been suggested as plausible therapeutic agents due to their effects on proliferation and antitumor potential in ovarian cancer [52], it will be crucial to further investigate potential targets of TZDs in order to understand their mechanism of action and uncover novel therapeutic targets. Materials and Methods Ethics Statement This study does not involve human participants or animal work. As such, an ethics statement is not required. Cells, Media and Reagents All cell lines (Ovcar3, CaOv3, Skov3 and MCF7) were obtained from the American Type Culture Collection ATCC (Rockville, MD). Ovcar3, CaOv3 and Skov3 cell culture media were obtained from the American Type Culture Collection ATCC (Rockville, MD). Ovcar3 cells were grown in Rossman-Park-Memorial- Institute (RPMI) 1640 media supplemented with 20% fetal bovine serum (FBS) containing penicillin/streptomycin/amphotericin B (Gibco-Invitrogen, Carlsbad, CA). CaOV3 and Skov3 cells were grown in Dulbecco’s Modified Medium (DMEM) and McCoy’s 5A medium respectively. MCF7 media (DMEM-31053) was purchased from Invitrogen. DMEM, McCoy’s and MCF7 media were supplemented with 10% FBS and antibiotics as above. Ciglitazone (CGZ), Rosiglitazone (Rosi), Troglitazone (TGZ), Pioglitazone (Pio) GW9662 and T007 (T0070907 (N-(49-amino- pyridyl-2-chloro-5-nitrobenzamide)) were purchased from Cay- PPARc Ligands Differentially Impact OVCAR Cells man Chemicals (Ann Arbor, MI), while 9-cis-retinoic acid (9-Cis- RA) was a purchased from MP Biomedicals (Solon, OH). Cell Line propagation Cells were grown at 37uC in a 5% CO2 environment until cells reached the desired confluence described below. Cells were then serum starved for 24 hours and treated for an additional 24 hours with vehicle control (DMSO), or 0.1–10 mM of one of the following: Rosi, CGZ, TGZ, or Pio. The maximum concentration of TZDs used was 10 mM as PPARc specific actions are reported with TZDs at concentrations #10 mM [20]. All experiments were performed as outlined above unless otherwise noted. All experiments were done at least 3 times, with 3 replicates each. RNA Isolation In order to examine cellular PPARc expression, cells were grown to 60–90% confluence and treated as described above for 24 hours. Total RNA was isolated using the RNeasy RNA isolation kit from Qiagen (Valencia, CA) according to the manufacturer’s protocol. RNA samples were stored at 280uC until used for real time PCR. Real time PCR The reverse transcription reaction was accomplished using the TaqManH one step rtPCR Master Mix kit from Applied Biosystems (Foster City, CA). Quantitative PCR was performed using a Stratagene Mx3000P real-time thermal cycler and the TaqMan methodology. Pre-optimized primers for PPARc and 18S mRNA probes with a 59 fluorescent reporter (FAM) were purchased from Applied Biosystems. Each RT reaction was performed using 200 ng of RNA at 48uC for 30 minutes. The RT reaction product was then subjected to real time PCR by incubating the reaction at 95uC for 10 min followed by 40 cycles of denaturing (95uC for 15 s) and re- annealing (60uC for 1 min). The amount of mRNA present was recorded as a cycle number (Ct) where the message reaches a fixed threshold. Ct was normalized to ribosomal RNA 18S which was then compared to control by the equation 22DDCt. Western blot analysis Western blot analysis was performed in order to examine the expression of PPARc protein in the different cell lines as well as to evaluate the effect of experimental treatment on protein expression. To accomplish this, cells were grown to 60–90% confluence, treated as previously described and lysed using a lysis buffer containing a protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations were quantified using the Bradford assay (BioRad, Hercules, CA). A total of 50 mg of protein was run on a 10% SDS gel, which was then transferred to a nitrocellulose membrane (BioRad). Membranes were incubated with antibodies against PPARc NR1C3 (1:250, R&D systems, Minneapolis, MN) (Figure 1B) or PPARc E-8 (1:150, Santa Cruz Biotechnology Santa Cruz, CA) (Figure 6B), overnight at 4uC in 5% milk (TBST) or actin a (1:1000, Sigma-Aldrich, A 5060) for 1 hour. Membranes were washed and incubated with secondary anti-mouse or anti rabbit horseradish peroxidase (HRP)-conjugat- ed antibody respectively (Santa Cruz). PPARc and actin a were visualized using an enhanced chemiluminescence detection system (Pierce, Rockford, IL) and exposure to x-ray film. Proliferation assays: MTS and BrdU assays The effect of TZDs on cell proliferation was assessed with the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium) assay (Promega, Madison, WI), PLoS ONE | www.plosone.org 12 January 2011 | Volume 6 | Issue 1 | e16179