In vitro and in vivo effects of the PPAR-alpha agonists fenofibrate and retinoic acid in endometrial cancer

Abstract

Fenofibrate, an agonist of PPAR-alpha, in doses above 25 microM, inhibits proliferation and induces apoptosis in Ishikawa endometrial cancer cells. We show that these effects are potentiated by retinoic acid, an agonist of the retinoid-X-receptor. DNA content analysis shows that G1/S phase progression through the cell cycle is inhibited. Independent Component Analysis of gene microarray experiments demonstrated downregulation of Cyclin D1 (CCND1) and associated changes in cell cycle gene expression. Expression of PPAR-alpha mRNA was reduced by >75% using RNA-interference but this resulted in only minor changes in biological effects. A nude mouse model of endometrial carcinoma was used to investigate the effect of fenofibrate in vivo but failed to show consistent inhibition of tumour growth.

Conclusion: The combination of fenofibrate and retinoic acid is a potent inhibitor of Ishikawa endometrial cancer cell growth in vitro.

Figure 1

Effect of fenofibrate and retinoic acid (48 hours exposure) on endometrial cancer cell growth, using cell counting (Top) and MTS assay (Bottom).

Figure 2

Top – DNA content analysis. The bars represent the ratio of cells in G1:G2M phase of the cell cycle after 12 hours of treatment with drug (RA+ = ATRA 10 μM. RA++ = ATRA 30 μM, FN+ = Fenofibrate 30 μM, FN++ = Fenofibrate 100 μM). Bottom – Apoptosis by FACS analysis using Annexin-FITC staining. Cells were treated for 3 hours. FN = fenofibrate, RG = rosiglitazone, St = staurosporine, a potent apoptogen.

Figure 3

Top – mRNA expression as quantified using Taqman RT-PCR following RNAi for PPARα using RNAi concentration shown. Middle – Cell growth following RNAi and treatment with fenofibrate (FN) and ATRA (RA) for 48 hours. Cell numbers were corrected to adjust for the reduced cell growth following PPARα RNAi compared to control RNAi. Bottom – Apoptosis measured by FACS analysis using Annexin-FITC staining following RNAi and treatment with fenofibrate and retinoic acid for 3 hours.

Figure 4

Genes identified as differentially expressed after treatment with high-dose (100 μM) fenofibrate. The plots show upregulation (red) and downregulation (green) of expression compared to DMSO control-treated cells. Cells were treated with 10 μM ("low dose") or 100 μM ("high dose") fenofibrate for 48 hours. Left – Normalised uncorrected data. Right – Data corrected using ICA-based filtering and removal of artefactual components. The data is improved but reveals an aberrant sample in the high-dose group (the first).

Figure 5

Comparison between array data and gene expression (as measured using Taqman RT-PCR). The "532 control" column represents the green-channel array data from the pooled cDNA (from DMSO-treated control samples) and is used as an internal control. Three genes were chosen for verification of the array: top – Cyclin D1, middle – Methionine Adenosyltransferase 2-alpha, bottom – Phosphoenolpyruvate carboxykinase 2.

Figure 6

Tumour weights in nude mice following endometrial cancer cell xenografting and treatment with "high dose" (0.25%) or "low dose" (0.1%) fenofibrate. Top – n = 6 mice per group treated for 21 days. Middle – n = 8 mice per group treated for 14 days and Bottom – n = 8 mice per group treated for a further 10 days with fenofibrate alone. Tumour weights were lower in the high-dose fenofibrate-treated mice in the first experiment, but this was not repeated in the second experiment.