PPARc Ligands Differentially Impact OVCAR Cells Figure 2. Effect of 9-cis-Retinoic Acid (9-Cis-RA) treatment on Ovcar3, CaOv3 and Skov3 PPRE luciferase activity. Cells were double transfected with a PPRE construct and one of the following: Empty vector (PGL3), DN form of PPARc (DN) or wildtype form of PPARc (OE). After the double transfections, cells were treated for 24 hours with vehicle control (DMSO) or 1 mM of 9-Cis-RA in the dark. PPRE luciferase activity is illustrated for (A) Ovcar3, (B) CaOv3, and (C) Skov3. Controls represent cells that were transfected with the empty vector and treated with vehicle control, shown as the first bar in each panel. Results for the PPRE luciferase assay are the means 6 SEM for at least 9 measurements. Bars that do not share a letter designation are significantly different (p,0.05). Insert Panel A: Western blot of PPARc protein in Ovcar3 cells double transfected with a PPRE construct and either empty vector, DN or OE PPARc constructs and treated with vehicle control for 24 hours. doi:10.1371/journal.pone.0016179.g002 10 mM. CaOv3 cells show an 80% and 30% decrease in cell proliferation when treated with CGZ and TGZ, respectively (Figure 3A–B). Skov3 cells show a 45% and 35% decrease when treated with 10 mM CGZ and TGZ, respectively (Figure 3A–B). Similar to Ovcar3 cells, CaOv3 and Skov3 cells treated with Rosi or Pio did not exhibit a change in proliferation (Figure 3C–D). CGZ and TGZ decrease BrdU DNA incorporation The MTS assay measures mitochondrial activity in cells, which is indicative of their viability [22] and considered an indirect assessment of cell proliferation. However, there is a report that the TZDs can interfere with the MTS assay [23]. Therefore, we also measured the rate of DNA replication in Ovcar3 cells using BrdU incorporation as another index of cell proliferation. Ovcar3 cells were treated with 0, 0.1, 1 or 10 mM of Rosi, CGZ, TGZ or Pio for 24 hours. BrdU assays show similar results as those of the MTS assay. There was approximately a 70% decrease in proliferation in cells treated with 10 mM of CGZ or TGZ, and no effect of Rosi or Pio on cell proliferation (Figure 4A–D). PPARc mRNA expression is enhanced by TZD treatment of ovarian cancer cells To evaluate the association of these ligands with regulation of PPARc, we examined the effect of TZDs on expression of PPARc in Ovcar3 cells. Cells were treated with or without 10 mM of TZDs and PPARc mRNA and protein levels were analyzed 24 hours later. PPAR(expression was increased in Ovcar3 cells treated with CGZ (6.9 fold) and TGZ (18.1 fold) (Figure 5A). Surprisingly, there was an increase in PPAR(protein following Rosi but not CGZ or TGZ treatment (Figure 5B). In an effort to explain this discrepancy between mRNA and protein expression, we investigated a time course of PPAR(mRNA and protein expression. Cells were treated with the different TZDs for 4, 8, 12 or 24 hours and both real time PCR and Western blot analysis were performed. We did not observe a correlation between the expression patterns for PPAR(mRNA and protein across time which indicates that there are potentially other mechanisms affecting the levels of PPAR(protein in these cells. One possibility is that the TZDs increase the turnover and degradation of PPAR(protein as seen in other systems [24,25]. Since CGZ and TGZ caused a decrease in cell proliferation in CaOv3 and Skov3 cells, we measured the mRNA expression of PPARc in these cells. In CaOv3 cells, the mRNA expression of PPARc after CGZ and TGZ treatment was 3.6 and 4.4 fold above the vehicle control respectively although these changes did not reach significance. Skov3 cells showed no changes in PPARc expression after treatment with the TZDs (data not shown). Collectively, our data indicates that PPAR(mRNA is present and is regulated by TZDs in ovarian cancer cells albeit to different degrees depending upon the cell type and the ligand used to activate PPARg. CGZ and TGZ do not induce apoptosis In this and subsequent experiments we examined only Ovcar3 cells as these cells showed the most notable effects when treated with TZDs. Furthermore, these cells are commonly used in the study of ovarian cancer, facilitating comparison with previous studies. In an attempt to better understand the mechanism mediating the decrease in cell proliferation, we examined whether apoptosis contributes to this decrease. We observed a decrease in cell proliferation after 24 hours of treatment (Figures 3 and 4). If this decrease is due to apoptosis, then this cell death should have occurred at an earlier time point. Hence, we examined the effect of TZDs on Ovcar3 cells and determined whether cells were either viable or undergoing apoptosis and were dead at 4, 8 or 12 hours after treatment using FACS analysis. There was a slight increase in apoptotic and dead cells after 4 hours of treatment with CGZ and TGZ (Figure 6A). However, this increase was not detected in samples treated for 8 or 12 hours (Figure 6B and 6C). Trypan blue experiments to confirm live versus dead cells showed similar results (data not shown). CGZ and TGZ cause cell cycle arrest In an effort to clarify the reason underlying the decrease in proliferation, the effects of TZDs on cell cycle progression were also assessed. A significant increase in cells in the G0/G1 phase was seen following treatment with TGZ and CGZ (Figure 7A). CGZ administration resulted in a significant decrease in G2/M phase of the cell cycle (Figure 7B), while no changes in the S phase were seen in cells treated with CGZ or TGZ (Figure 7C). Cells treated with Rosi or Pio did not show any change in cell cycle distribution compared to control (data not shown). Effects of TZDs on PPRE luciferase activity To clarify whether the antiproliferative and cell cycle arrest effects seen with select TZDs are a direct effect of PPARc transactivation, we transiently transfected cells with a 3XPPRE- mTK-pGL3-reporter plasmid. Two additional constructs were cotransfected, namely, a DN form of PPARc and an overexpres- sion (OE) of a wildtype PPARc construct. Cells were then treated for 24 hours with 10 mM of Rosi, CGZ, TGZ, or Pio. None of the TZD treatments alone increased PPRE activity (Figure 8). However, cells transfected with DN showed an approximate 40% decrease in PPRE mediated activity in both untreated and TZD treated cells. Furthermore, increasing the expression of the wild type PPARc showed an increase in luciferase activity when cells were treated with any of the four different TZDs compared to cells that were only treated with TZDs (Figure 8A–8D). Analysis of TZD mediated PPARc dependent and independent actions In order to determine whether the effects of TZDs are PPARc dependent, cells were treated with 10 mM TZDs in the absence or presence of PPARc antagonists (GW9662, T007). The MTS assay was performed in order to determine whether the presence of antagonists could reverse the effects of TZDs on ovarian cancer cell proliferation. Results showed that there was a partial ‘rescue’ when cells were treated with the GW9662 (Figure 9A) or the T007 (Figure 9B) compounds in combination with CGZ or TGZ. However, the pattern of action was inconsistent between the two antagonists and even with the same antagonist in the presence of PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16179