Activation of PPARγ leads to inhibition of anchorage-independent growth of human colorectal cancer cells  Jeffrey A. Brockman, Rajnish A. Gupta, Raymond N. DuBois  Gastroenterology  Volume 115, Issue 5, Pages 1049-1055 (November 1998) DOI: 10.1016/S0016-5085(98)70072-1 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 PPARγ is expressed and functionally active in colon cancer cells. (A) PPARγ is expressed at the mRNA level in colon cancer cells. Total RNA (10 μg) from either HCA-7, HCT-15, HCT-116, or Caco-2 cells was subjected to an RT reaction either with (+) or without (−) reverse transcriptase. A 2-μL aliquot from each reaction was subjected to 40 cycles of PCR. Amplification products were resolved in a 2% agarose gel. The identity of the amplicon was confirmed by restriction mapping and DNA sequence analysis. (B) PPARγ protein is present in colon cancer cells. Whole-cell extracts (500 μg) were immunoprecipitated with a PPARγ peptide–specific affinity-purified antibody (1 μg) for 4 hours. Immunoprecipitates were eluted with 50 μL PPARγ peptide (200 μg/mL) overnight. Eluted proteins were resolved on a 10% SDS-PAGE gel, transferred to a polyvinylidene difluoride membrane, and immunoblotted with the same antibody. (C) Cells were transiently transfected with 1 μg PPRE3-tk-luciferase and 0.01 μg pRL-SV40 by lipid transfection. Transfections were treated with 10 μmol/L BRL 49653 (■) or DMSO (control; □) for 12 hours. Cells were harvested and a dual-luciferase assay was performed as described in the Materials and Methods. The means of normalized relative light units from 3 independent transfections are shown. Error bars = SEM. Gastroenterology 1998 115, 1049-1055DOI: (10.1016/S0016-5085(98)70072-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 The PPARγ selective agonist BRL 49653 inhibits anchorage-independent growth of cells that overexpress functional PPARγ. (A) Inhibition of anchorage-independent growth by BRL 49653. HCT-15 or HCT-15-G25 cells (3 × 104) were plated into media containing 0.4% (wt/vol) agar, supplemented with either DMSO (control; □) or 10 μmol/L BRL 49653 (■). After 7 days of growth, the number of colonies was determined. The values shown are the means of 3 independent experiments performed in triplicate. Error bars = SEM. (B) BRL 49653 is a selective PPARγ agonist in HCT-15 cells. HCT-15 cells were transiently transfected with expression vectors encoding either PPARα, β, or γ (1.5 μg), PPRE3-tk-luciferase (1 μg), and pRL-SV40 (0.01 μg) by lipid transfection. Transfected cells were treated with 10 μmol/L BRL 49653 (■) or DMSO (control; □) for 12 hours and harvested, and a dual-luciferase assay was performed as described in Materials and Methods. Gastroenterology 1998 115, 1049-1055DOI: (10.1016/S0016-5085(98)70072-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 The PPARγ selective agonist BRL 49653 inhibits anchorage-independent growth of cells that overexpress functional PPARγ. (A) Inhibition of anchorage-independent growth by BRL 49653. HCT-15 or HCT-15-G25 cells (3 × 104) were plated into media containing 0.4% (wt/vol) agar, supplemented with either DMSO (control; □) or 10 μmol/L BRL 49653 (■). After 7 days of growth, the number of colonies was determined. The values shown are the means of 3 independent experiments performed in triplicate. Error bars = SEM. (B) BRL 49653 is a selective PPARγ agonist in HCT-15 cells. HCT-15 cells were transiently transfected with expression vectors encoding either PPARα, β, or γ (1.5 μg), PPRE3-tk-luciferase (1 μg), and pRL-SV40 (0.01 μg) by lipid transfection. Transfected cells were treated with 10 μmol/L BRL 49653 (■) or DMSO (control; □) for 12 hours and harvested, and a dual-luciferase assay was performed as described in Materials and Methods. Gastroenterology 1998 115, 1049-1055DOI: (10.1016/S0016-5085(98)70072-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 BRL 49653 inhibits cell growth of colon cancer cells. (A) Cells (1 × 105/well) were grown in the continuous presence of either 0, 1, 10, or 25 μmol/L BRL 49653 with equal amounts of DMSO. After 5 days of growth, the number of cells per well was determined. The values shown represent the means of 3 independent wells normalized to the DMSO control, set at 100%. Error bars = SEM. The experiment was performed twice with similar results. (B) Cells were transiently transfected with 1 μg PPRE3-tk-luciferase, and 0.01 μg pRL-SV40 by lipid transfection. Transfected cells were treated with either 0, 1, 10, or 25 μmol/L BRL 49653 for 12 hours and harvested, and a dual-luciferase assay was performed as described in Materials and Methods. ■ HCA-7, HCT-116, ● HCT-15, ▴ HCT-15-G25. Gastroenterology 1998 115, 1049-1055DOI: (10.1016/S0016-5085(98)70072-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 BRL 49653 inhibits cell growth of colon cancer cells. (A) Cells (1 × 105/well) were grown in the continuous presence of either 0, 1, 10, or 25 μmol/L BRL 49653 with equal amounts of DMSO. After 5 days of growth, the number of cells per well was determined. The values shown represent the means of 3 independent wells normalized to the DMSO control, set at 100%. Error bars = SEM. The experiment was performed twice with similar results. (B) Cells were transiently transfected with 1 μg PPRE3-tk-luciferase, and 0.01 μg pRL-SV40 by lipid transfection. Transfected cells were treated with either 0, 1, 10, or 25 μmol/L BRL 49653 for 12 hours and harvested, and a dual-luciferase assay was performed as described in Materials and Methods. ■ HCA-7, HCT-116, ● HCT-15, ▴ HCT-15-G25. Gastroenterology 1998 115, 1049-1055DOI: (10.1016/S0016-5085(98)70072-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 PPARγ activation induces G1 cell cycle arrest. Either HCT-15 or HCT-15-G25 cells (0.25 × 106) were plated into 6-well plates and allowed to grow to 30% confluence. Cells were then treated with either DMSO or BRL 49653 (10 μmol/L) for 5 days in serum-free media. Cells were harvested, stained with propidium iodide, and analyzed for DNA content by fluorescence-activated cell sorter analysis. The values represent the number of cells in the G1 phase of the cell cycle as a percentage of total cells. Gastroenterology 1998 115, 1049-1055DOI: (10.1016/S0016-5085(98)70072-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions