Abstract:

Objective Overexpression of cyclooxygenase-2(COX-2) is known to be associated with the carcinogenesis of esophageal squamous cell carcinoma(ESCC), but in some ESCC cell lines the expression level of COX-2 may be low. In this study, methylation state of cyclooxygenase-2 gene promoter in ESCC was examined in xenografts to investigate the relationship between promoter methylation and COX-2 expression. We also studied the effect of COX-2 selective inhibitor nimesulide in conjunction with 5-aza-2 deoxycytidine on the growth of xenografts. Methods KYSE150 cell line with low COX-2 expression was used to establish ESCC xenograft in nude mice. The mice were divided into four groups, nimesulide(NIM)+5-Aza-DC(5-Aza) (group 1), NIM group(group 2), 5-Aza group(group 3), and sodium chloride control group(group 4). The growth of those xenografts in nude mice was observed. Methylation state of cyclooxygenase-2 gene promoter in xenografts was monitored using bisulfate sequencing method. RT-PCR and immunohistochemistry were employed to determine the expression level of COX-2 mRNA and protein in xenograft samples, respectively. PGE₂ concentration in xenografts was measured by ELISA. Results During the trial, mice grew well and no infection or death was observed. No difference of weight was found between groups(P>0.05). Xenograft volumes were different between groups with that of the sodium chloride group smaller than other groups, and the difference was significant(P<0.01). It was showed that methylation degree of COX-2 gene promoter was higher in NIM group and sodium choloride group than that in NIM combining 5-Aza group and 5-Aza group. For 10 CpG sites, methylation degrees were 30% and 58.3% in group 3 and group 4, respectively. The expressions of COX-2 mRNA and protein were coincidently low in group 1 and high in group 3. PGE₂ values were 0.37, 0.91 and 1.22 in group 1, 2 and 3 compared with group 4, respectively. Conclusion Promoter methylation of COX-2 gene is one of the important mechanisms to regulate COX-2 expression in low COX-2 expression ESCC xenografts. Combination of selective COX-2 inhibitor and 5-Aza has synergistic effect on ESCC xenografts.

Key words: esophageal squamous cell carcinoma, cyclooxygenase-2, DNA methylation, xenografts