Figure 2

PPAR and RXR ligands inhibit human breast cancer cell proliferation in vitro. (A) Expression of SRC-1, CBP, RXRα, and PPAR mRNA in human breast cancer cell lines. (B) Expression of SRC-1, CBP, RXRα, and PPAR proteins in human breast cancer cell lines. (C, D). Triplicate cultures of MCF7 or MDA-MB-468 cells were treated with 100 μM of the PPARα agonist clofibrate (clo) or the PPARγ agonist ciglitazone (cig) alone or in combination with 100 nM of the RXR selective compound AGN194204 or vehicle for 6 days. At 2 day intervals, cells were trypsinized and counted using a hemacytometer. Error bars represent SEM of 3 independent experiments. (E) Human breast cancer cell lines MCF7, T47 D, MDA-MB-231, and MDA-MB-468 were treated with 100 nM AGN194204, 100 μM ciglitazone (cig) or clofibrate (clo), or vehicle for 16 hours. Protein extracts from these treated cells were subjected to western blotting using cell cycle antibodies indicated at left. These experiments were performed 3 times with similar results. (F, G) PPAR and RXR ligands inhibit coactivator occupancy of the proximal EGFR promoter. MCF7 or MDA-MB-468 cells were treated with 100 nM AGN194204, 100 μM ciglitazone (cig) or clofibrate (clo), or vehicle for 16 hours followed by chromatin immunoprecipitation (ChIP) with antibodies to SRC1, CBP, or acetylated histone H3 (acH3). Nuclease digested DNA from treated cells was amplified to detect nucleosomal loading of the EGFR promoter. The proximal promoter region was amplified by quantitative PCR and amplification products normalized to input DNA. (H, I) SRC-1 overexpression blocks PPAR and RXR ligand mediated suppression of EGFR promoter activity. The EGFR-luc promoter/reporter vector was transiently transfected with SRC-1, CBP, or control expression vectors into triplicate cultures of MCF7 or MDA-MB-468 cells. Cultures were treated with AGN194204, clofibrate, or ciglitazone alone or combination. Luciferase activity was determined as described in Materials and Methods. Error bars indicate SEM of 3 independent experiments.