Promoter hypermethylation silences cyclooxygenase-2 (Cox-2) and regulates growth of human hepatocellular carcinoma cells.

Cyclooxygenase-2 (COX-2) upregulation is recognized to confer advantage in progression in a wide variety of cancers, with colorectal cancer most intensively investigated. Epidemiologically, chemopreventive effects of COX-2 inhibitors have been proven on numerous cancers, but not on hepatocellular carcinoma (HCC). Although the antiapoptotic feature of COX-2 generally supports cancer cell growth, previous reports have shown that COX-2 expression, upregulated in early HCC, is downregulated in advanced HCC. Therefore, COX-2 downregulation may be somehow advantageous and specific for HCC development. However, its mechanism remains unclear. Since promoter hypermethylation often silences the gene expression, we hypothesized that the epigenetic mechanism might regulate COX-2 expression in HCC. We examined the methylation status of the Cox-2 promoter in six human HCC cell lines (Hep3B, HepG2, SK-Hep1, HuH7, PLC, and FLC-7 cells) using methylation-specific PCR. The promoter was remarkably hypermethylated in Hep3B and FLC-7 cells and moderately in HepG2 and SK-Hep1 cells, but not in HuH7 and PLC cells. In Hep3B cells, coincubation with 5-aza-2'-deoxycytidine, a demethylator, demethylated the promoter and upregulated COX-2 expression as well as prostaglandin E2 production dose dependently. On the other hand, no such effects were observed in HuH7 cells. Additionally, the methylator suppressed growth of Hep3B cells dose dependently, accompanied by cyclin D1 downregulation, and the growth suppression was abrogated by potent COX-2 inhibition with a COX-2 selective inhibitor celecoxib, but these responses were not found in HuH7 cells. These results indicated that cell growth was largely retarded by Cox-2 upregulation via promoter demethylation, rather than the potentially reactivated genes concurrently demethylated by 5-aza-2'-deoxycytidine. In conclusion, promoter hypermethylation transcriptionally silences Cox-2 in HCC cells. Epigenetic alteration of Cox-2, at least in part, modulates the growth of HCC cells.