PPARc Ligands Differentially Impact OVCAR Cells Figure 5. Effect of TZDs on PPARc mRNA and protein expression. PPARc mRNA and protein expression were measured in Ovcar3 cells using (A) Real time PCR and (B) western blot analysis after TZD treatment. Cells were serum starved for 24 hours and treated for an additional 24 hours with vehicle control (DMSO) or one of the following: 10 mM of Rosi, CGZ, TGZ, or Pio. Results are the means 6 SEM for at least 3 measurements from two individual experiments. Bars with different superscripts are significantly different (p,0.05). doi:10.1371/journal.pone.0016179.g005 CGZ and TGZ have anti-proliferative actions on three ovarian cancer cell lines, whilst Rosi and Pio have no effect. The fact that these anti-proliferative actions are seen in all three cell lines suggests that this is a common phenomenon and not confined to a single ovarian cancer cell line. The experiments described herein also demonstrate that the four TZDs have distinct actions on ovarian cancer cells possibly through both PPARc dependent as well as independent pathways. Although several reports indicate that different cell lines respond differently to TZDs, [30,31,32,33], to our knowledge, a direct comparison between these four TZDs in ovarian cancer and delineating whether these effects are PPARc dependent using molecular, physiological and pharmacological approaches has not been investigated. In the present study, 10 mM of CGZ and TGZ decreased cell proliferation in all three of the ovarian cancer cell lines studied. These observations are in concordance with other studies utilizing ovarian cancer cells as well as other cell lines where CGZ and/or TGZ decreased MTT activity [20,21,34]. However, our findings are in contrast to cancer cell lines from other tissues such as breast, thyroid, bladder and others that usually require much higher concentrations of TZDs to elicit a biological response. In fact, doses up to 100 mM, which exceed the reported ligand specific concentrations of 10 mM or less [12], may be required before an inhibition of proliferation is seen [35,36,37,38,39]. However, these differences may be due in part to the use of different cells lines or differences in cell culture conditions where, for example, investigators have used 1% FBS [39], 5% FBS [35,36,37,38,39] or 10% FBS [21]. The present findings demonstrate that this decrease in proliferation is due to an increase in cell cycle arrest rather than an increase in apoptosis. Similarly, other reports have shown that TZDs lead to the degradation of cyclin D1 in prostate cancer [40], which causes cell cycle arrest, and a decrease in tumor cell proliferation. Likewise, in A2780 ovarian cancer cells, CGZ decreases cyclin D1 along with other pro-survival factors resulting in cell cycle arrest [21]. Yang and colleagues [20] reported that CGZ and TGZ treatment of ES-2 and PA-1 ovarian cancer cells resulted in cell cycle arrest; however, this change in cell cycle kinetics was associated with increased levels of apoptosis. Although our studies support the previous findings in different ovarian cancer cells that the TZDs decrease cell proliferation by arresting cells in the G0/G1 stage of the cell cycle, the possibility exists that the higher concentrations of TZDs used in these previous studies results in the induction of apoptosis [20]. The present study demonstrates that each of the TZDs has distinct actions on ovarian cancer cell proliferation. CGZ and TGZ cause a dramatic decrease in ovarian cancer cell proliferation whilst Rosi and Pio had no effect. This is not unexpected as each TZD has a different affinity and binding to PPAR [7,41], recruits different coactivators [42] and may elicit distinct cellular responses. These unique actions have led to the concept that the TZDs are selective PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16179