Induction of apoptosis and inhibition of angiogenesis by COX-2 inhibitors in hepatocellular carcinomas

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In this project, we have investigated anti-tumor effect of NS-398, a selective COX-2 inhibitor, on human hepatoma cells, focusing on the agent's ability to induce apoptosis in those cells and the ability to inhibit VEGF production from those cells. Constitutive expression of COX-2 was found by RT-PCR and immunocytochemistry in the hepatoma cells examined. A significant growth-inhibitory effect was demonstrated in NS-398-treated hepatoma cells, however, the effect was not attributable to an increase in apoptotic cells nor to inactivation of COX-2 by NS-398, suggesting involvement of a COX-2-independent mechanism in growth inhibition of hepatoma cells treated with COX-2 inhibitors. While the study was going on, NS-398 has been reported to be not only a COX-2 inhibitor but also a PPAR γ ligand (J Biol Chem, 1999). This finding encouraged us to perform subsequent studies to elucidate the involvement of PPAR γ in growth inhibition in hepatoma cells. PPAR γ was constitutively expressed in all the cell lines (HLF, HuH-7, HAK- 1A, HAK-1B, and HAK-5) and the HCC tissues used in this study. A cytostatic effect of PPAR γ ligands was found in those cell lines, and this inhibition of cell growth was dosage-dependent. 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.However, HuH-7, lacking p21 protein expression, did not demonstrate clear arrest in the cell cycle analysis. HLF, which was pRb-deficient, responded most profoundly to a PPAR γ ligand, showing an increased expression in not only p21 but also in p27 and p18. These findings suggested that p21, p27, and p18 might be involved in PPAR γ ligand-induced cell cycle arrest.