Involvement of p21(WAF1/Cip1), p27(Kip1), and p18(INK4c) in troglitazone-induced cell-cycle arrest in human hepatoma cell lines

Peroxisome proliferator-activated receptor gamma (PPAR gamma) regulates cel l growth and differentiation. Recent evidence has suggested that PPAR gamma ligands had anti-tumor effects through inhibiting cell growth and inducing cell differentiation in several types of malignant neoplasm. In the presen t study, we investigated: 1) the expression of PPAR gamma in both human hep atoma cell lines and S resected human hepatocellular carcinoma (HCC) tissue s; 2) the growth-inhibitory effect of troglitazone, a PPAR gamma ligand, on those hepatoma cells; and 3) the molecular mechanisms of troglitazone-indu ced cell-cycle arrest. Five hepatoma cell lines, HLF, HuH-7, HAK-1A, HAK-1B , and HAK-5, were used. The mRNA expression levels of PPAR gamma, p21(WAF1/ Cip1), and p27(Kip1) were determined by real-time quantitative reverse tran scription-polymerase chain reaction. The expression of cell cycle-regulatin g proteins, such as p21, p27, p18(INK4c), cyclin E, and pRb, was examined u sing Western blotting. PPAR gamma was constitutively expressed in all the c ell lines and the HCC tissues used in this study. A cytostatic effect of tr oglitazone was found in those cell lines, and this inhibition of cell growt h was dosage-dependent. G0/G1 arrest was apparently demonstrated in flow cy tometric 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 e xpression, did not demonstrate clear arrest in the cell-cycle analysis. HLF , which was deficient in the protein product of the retinoblastoma tumor-su ppressor gene (pRb), responded most profoundly to troglitazone, showing an increased expression in not only p21, but also in p27 and in p18. These fin dings suggested that p21, p27, and p18 might be involved in troglitazone-in duced cell-cycle arrest in human hepatoma cells.