Transcriptional silencing of Cyclooxygenase-2 by hyper-methylation of the 5 ' CpG island in human gastric carcinoma cells

It has been well established that overexpression of Cyclooxygenase-2 (Cox-a ) in epithetial cells inhibits apoptosis and increases the invasiveness of malignant cells, favoring tumorigenesis and metastasis, However, the molecu lar mechanism that regulates Cox-2 expression has not been well defined in gastric carcinoma. In this study, we examined whether the Cox-2 expression could be regulated by hyper-methylation of the Cox-2 CPG bland (spanning fr om -590 to +186 with respect to the transcription initiation site) in human gastric carcinoma cell lines. By Southern analysis, we found that three ga stric cells (SNU-601, -620, and -719) without Cox-2 expression demonstrated hyper-methylation at the Cox-2 CpG island, A detailed methylation pattern using bisulfite sequencing analysis revealed that all of the CpG sites were completely methylated in SNU-601. Treatment with demethylating agents effe ctively reactivated the expression of Cox-2 and restored IL-1 beta sensitiv ity in the previously resistant SNU-601. By transient transfection experime nts, we demonstrate that constitutively active Cox-2 promoter activities we re exhibited even without an exogenous stimulation in SNU-601, Furthermore, when the motif of the nuclear factor for interleukin-6 expression site, th e cyclic AMP response element, or both was subjected to point mutation, the constitutive luciferase activity was markedly reduced. In addition, Cox-2 promoter activity was completely blocked by irt vitro methylation of all of the CpG sites in the Cox-2 promoter region with SssI (CpG) methylase in SN U-601, Taken together, these results indicate that transcriptional repressi on of Cox-2 is caused by hyper-methylation of the Cox-2 CpG island in gastr ic carcinoma cell lines.