Pparγ Ligands Inhibit Cholangiocarcinoma Cell Growth Through P53–Dependent Gadd45 and P21Waf1/Cip1 Pathway

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Abstract
Ligands of peroxisome proliferator–activated receptor–γ (PPARγ) induce differentiation and growth inhibition in several human cancers. However, the role of PPARγ ligands in the growth control of human cholangiocarcinoma cells remains unknown. This study was designed to investigate the biological functions and molecular mechanisms of PPARγ ligands in the growth regulation of human cholangiocarcinoma cells. Western blot analysis showed that PPARγ is expressed in all of the three human cholangiocarcinoma cell lines used in this study (SG231, CC–LP–1, and HuCCT1). Transient transfection assays using a peroxisome proliferator response element (PPRE) reporter construct showed that the PPARγ expressed in human cholangiocarcinoma cells is functional as a transcription activator. Exposure of SG231, CC–LP–1, and HuCCT1 cells to PPARγ ligands 15–deoxy–Δ12, 14–prostaglandin J2 (15d–PGJ2) and troglitazone for 24 to 96 hours resulted in a dose–dependent inhibition of cell growth. Flow cytometry analysis showed that 15d–PGJ2 and troglitazone–induced cell cycle arrest at the G2/M checkpoint. Consistent with these findings, both 15d–PGJ2 and troglitazone significantly inhibited the G2/M cyclin–dependent kinase (CDK) Cdc2 activity. Furthermore, cells treated with 15d–PGJ2 and troglitazone showed elevated expression of p53 and two p53–controlled downstream genes, GADD45 and p21WAF1/Cip1. Dominant negative inhibition of p53 in SG231 cells significantly blocked the 15d–PGJ2 and troglitazone–induced growth inhibition, G2/M arrest, and GADD45/p21 induction. 15d–PGJ2 and troglitazone failed to directly inhibit Cdc2 activity in a cell–free system in spite of direct association between GADD45 and PPARγ proteins. In conclusion, these results show a novel p53–dependent mechanism in the PPARγ ligand–mediated inhibition of cholangiocarcinoma growth and suggest a potential therapeutic role of PPARγ ligands in the treatment of human cholangiocarcinoma.