Involvement of P21Waf1/Cip1, P27Kip1, and P18Ink4c in Troglitazone–Induced Cell–Cycle Arrest in Human Hepatoma Cell Lines

Abstract
Peroxisome proliferator-activated receptor γ (PPARγ) regulates cell growth and differentiation. Recent evidence has suggested that PPARγ ligands had anti–tumor effects through inhibiting cell growth and inducing cell differentiation in several types of malignant neoplasm. In the present study, we investigated: 1) the expression of PPARγ in both human hepatoma cell lines and 5 resected human hepatocellular carcinoma (HCC) tissues; 2) the growth–inhibitory effect of troglitazone, a PPARγ ligand, on those hepatoma cells; and 3) the molecular mechanisms of troglitazone–induced cell–cycle arrest. Five hepatoma cell lines, HLF, HuH–7, HAK–1A, HAK–1B, and HAK–5, were used. The mRNA expression levels of PPARγ, p21WAF1/Cip1, and p27Kip1 were determined by real–time quantitative reverse transcription–polymerase chain reaction. The expression of cell cycle-regulating proteins, such as p21, p27, p18INK4c, cyclin E, and pRb, was examined using Western blotting. PPARγ was constitutively expressed in all the cell lines and the HCC tissues used in this study. A cytostatic effect of troglitazone was found in those cell lines, and this inhibition of cell growth was dosage–dependent. G0/G1 arrest was apparently demonstrated in flow cytometric analysis in HLF, HAK–1A, HAK–1B, and HAK–5, all of which showed an increased expression of p21 protein. However, HuH–7, lacking p21 protein expression, did not demonstrate clear arrest in the cell–cycle analysis. HLF, which was deficient in the protein product of the retinoblastoma tumor–suppressor gene (pRb), responded most profoundly to troglitazone, showing an increased expression in not only p21, but also in p27 and in p18. These findings suggested that p21, p27, and p18 might be involved in troglitazone–induced cell–cycle arrest in human hepatoma cells.