PPARc Ligands Differentially Impact OVCAR Cells Figure 4. Effect of TZD treatment on Ovcar3 cell proliferation. Ovcar3 cells were serum starved for 24 hours and treated for an additional 24 hours with vehicle control (DMSO) or 0.1, 1.0 or 10 mM of CGZ (A), TGZ (B), Rosi (C), Pio (D). Cell proliferation was assessed with the BrdU assay. Results are the means 6 SEM for at least 3 measurements from three individual experiments. Bars that do not share a letter designation are significantly different within a treatment group (p,0.05). doi:10.1371/journal.pone.0016179.g004 different TZDs. For example, T007 was able to completely block the effects of CGZ on cell proliferation but had no effect on the TGZ mediated decrease in proliferation. In addition, the use of the GW9662 or the T007 compounds alone did not affect prolifer- ation on their own as was expected. This may be due to the fact that these antagonists may be mixed agonists or not PPARc specific [9,26]. These findings indicate that the use of a pharmacological approach employing reported PPARc antago- nists alone does not answer the question as to whether the effect of the TZDs act through PPARc. This led us to use a molecular approach using DN or OE PPARc constructs to investigate whether the effects seen on cell proliferation and cell cycle were PPARc dependent or independent. Transfecting cells with DN or OE forms of PPARc without the presence of ligands did not change cell proliferation as indicated by BrdU assays (Figure 10). More importantly, the effects of CGZ and TGZ on proliferation were not overcome by the presence of the DN form of PPARc (Figure 10). Luciferase assays were run on samples in the same plates in order to ensure that cells were being transfected and expression of the DN caused the expected decrease while overexpression of wild type PPARc increased PPRE activity, respectively (data not shown). In concordance with the BrdU assays, cell cycle data also indicate that the effect of CGZ and TGZ are not PPARc dependent (data not shown). Discussion TZDs have been shown to have a wide range of effects on cancer cells. The presumed target of TZDs, PPARc, is overexpressed in a variety of cancers including breast, lung, colon, prostate and ovary [2,27,28,29]. Although, the exact role TZDs and PPARc play in ovarian cancer remains unknown, this study demonstrates that PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e16179