PPARc Ligands Differentially Impact OVCAR Cells Figure 8. PPRE Luciferase promoter activity assay. Ovcar3 cells were double transfected with a PPRE construct and one of the following: Empty vector, DN, or OE PPARc. After the double transfections, cells were treated for 24 hours with vehicle control (DMSO) or 10 mM of (A) CGZ, (B) TGZ, (C) Rosi, (D) or Pio. 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 (p,0.05). doi:10.1371/journal.pone.0016179.g008 modulators of PPARc and therefore target distinct genes resulting in tissue selectivity [12,42,43,44]. In addition, TZDs have different specificities for PPARs. For example, Rosi and Pio are considered the most potent PPARc agonists out of the four TZDs used in this study [11]. The findings that these potent PPARc agonists do not inhibit cell proliferation further support our hypothesis that the actions of the TZDs may be independent of PPAR. Furthermore, Rosi, CGZ and TGZ are specific to PPARc while Pio has been shown to also activate PPARa [45,46]. Hence, our data may reflect the differences in the selective modulation of PPARc, differences in the specificity for PPARc, or the activation of distinct PPARc independent pathways. To begin to understand the selective modulation of PPARc by TZDs in ovarian cancer cells, we examined the mRNA and protein expression profiles of PPARc and the activation of PPARc’s promoter following TZD treatment. There is a dramatic increase in PPARc mRNA expression when Ovcar3 cells were treated with TGZ and to a lesser extent with CGZ and Rosi. In contrast, the highest expression of PPARc protein was observed when cells were treated with Rosi. This discordance between the expression of PPARc mRNA and protein after TZD treatment has not been observed nor examined previously, however, the levels of PPARc protein after TZD treatment have been shown to be highly variable between ES-2 and PA-1 ovarian cancer cells [21]. We postulated that examination of a static 24 hour time point might not accurately capture the induction of PPARc mRNA and protein. We therefore assessed the level of PPARc mRNA as well as protein patterns at different times after TZD treatment and observed a lack of correlation between PPARc mRNA and protein expression at all time points. An alternative theory to explain this discordance is that the increase in expression of PPARc mRNA when cells are treated with TGZ may increase the turnover and degradation of PPARc protein thereby reducing protein expres- sion. This has been previously demonstrated in other systems where it was shown that with increasing doses of TZDs, there was an increase in PPARc protein degradation which resulted in a PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e16179