Down-regulation of PPARgamma1 suppresses cell growth and induces apoptosis in MCF-7 breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear hormone receptor superfamily and is highly expressed in many human tumors including breast cancer. PPARγ has been identified as a potential target for breast cancer therapy based on the fact that its activation by synthetic ligands affects the differentiation, proliferation, and apoptosis of cancer cells. However, the controversial nature of current studies and disappointing results from clinical trials raise questions about the contribution of PPARγ signaling in breast cancer development in the absence of stimulation by exogenous ligands. Recent reports from both <it>in vitro </it>and <it>in vivo </it>studies are inconsistent and suggest that endogenous activation of PPARγ plays a much more complex role in initiation and progression of cancer than previously thought.</p> <p>Results</p> <p>We have previously demonstrated that an increase in expression of PPARγ1 in MCF-7 breast cancer cells is driven by a tumor-specific promoter. Myc-associated zinc finger protein (MAZ) was identified as a transcriptional mediator of PPARγ1 expression in these cells. In this study, using RNA interference (RNAi) to inhibit PPARγ1 expression directly or via down-regulation of MAZ, we report for the first time that a decrease in PPARγ1 expression results in reduced cellular proliferation in MCF-7 breast cancer cells. Furthermore, we demonstrate that these changes in proliferation are associated with a significant decrease in cell transition from G₁to the S phase. Using a dominant-negative mutant of PPARγ1, Δ462, we confirmed that PPARγ1 acts as a pro-survival factor and showed that this phenomenon is not limited to MCF-7 cells. Finally, we demonstrate that down-regulation of PPARγ1 expression leads to an induction of apoptosis in MCF-7 cells, confirmed by analyzing Bcl-2 expression and PARP-1 cleavage.</p> <p>Conclusion</p> <p>Thus, these findings suggest that an increase in PPARγ1 signaling observed in breast cancer contributes to an imbalance between proliferation and apoptosis, and may be an important hallmark of breast tumorigenesis. The results presented here also warrant further investigation regarding the use of PPARγ ligands in patients who are predisposed or already diagnosed with breast cancer.</p

Specific Thiazolidinediones Inhibit Ovarian Cancer Cell Line Proliferation and Cause Cell Cycle Arrest in a PPARγ Independent Manner

Peroxisome Proliferator Activated Receptor gamma (PPARγ) agonists, such as the thiazolinediones (TZDs), have been studied for their potential use as cancer therapeutic agents. We investigated the effect of four TZDs--Rosiglitazone (Rosi), Ciglitazone (CGZ), Troglitazone (TGZ), and Pioglitazone (Pio)--on ovarian cancer cell proliferation, PPARγ expression and PPAR luciferase reporter activity. We explored whether TZDs act in a PPARγ dependent or independent manner by utilizing molecular approaches to inhibit or overexpress PPARγ activity.Treatment with CGZ or TGZ for 24 hours decreased proliferation in three ovarian cancer cell lines, Ovcar3, CaOv3, and Skov3, whereas Rosi and Pio had no effect. This decrease in Ovcar3 cell proliferation was due to a higher fraction of cells in the G(0)/G(1) stage of the cell cycle. CGZ and TGZ treatment increased apoptosis after 4 hours of treatment but not after 8 or 12 hours. Treatment with TGZ or CGZ increased PPARγ mRNA expression in Ovcar3 cells; however, protein levels were unchanged. Surprisingly, luciferase promoter assays revealed that none of the TZDs increased PPARγ activity. Overexpression of wild type PPARγ increased reporter activity. This was further augmented by TGZ, Rosi, and Pio indicating that these cells have the endogenous capacity to mediate PPARγ transactivation. To determine whether PPARγ mediates the TZD-induced decrease in proliferation, cells were treated with CGZ or TGZ in the absence or presence of a dominant negative (DN) or wild type overexpression PPARγ construct. Neither vector changed the TZD-mediated cell proliferation suggesting this effect of TZDs on ovarian cancer cells may be PPARγ independent.CGZ and TGZ cause a decrease in ovarian cancer cell proliferation that is PPARγ independent. This concept is supported by the finding that a DN or overexpression of the wild type PPARγ did not affect the changes in cell proliferation and cell cycle

Effect of PPARγ antagonists on Ovcar3 proliferation.

<p>Cells were treated with GW9662 (<b>A</b>) or T007 (<b>B</b>) for 24 h and cell proliferation was assessed. Results are the means ± SEM for at least 4 measurements. Bars that do not share a letter designation are significantly different (<i>p</i><0.05).</p

Effect of TZD treatment on Ovcar3 cell proliferation.

<p>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 µM of CGZ (<b>A</b>), TGZ (<b>B</b>), Rosi (<b>C</b>), Pio (<b>D</b>). Cell proliferation was assessed with the BrdU assay. Results are the means ± 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 (<i>p</i><0.05).</p

Cell cycle kinetics using flow cytometry.

<p>Ovcar3 cells were serum starved for 24 hours and treated for an additional 24 hours with vehicle control (DMSO) or 10 µM of CGZ or TGZ. (<b>A</b>) G₀/G₁, (<b>B</b>) G₂, (<b>C</b>) S phase. Results are the means ± SEM for at least 3 measurements from three individual experiments. Bars that do not share a letter or number designation are significantly different (<i>p</i><0.05).</p