Background: The accumulation of reactive oxygen species and subsequent oxidative DNA damage underlie the development of Barrett's esophagus (BE) and its progression to Barrett's dysplasia (BD) and adenocarcinoma (BAC).
Methods: We systematically analyzed the promoter regions of 23 genes of the Glutathione S-transferase (GST) and Glutathione peroxidase (GPX) families. Quantitative bisulfite pyrosequencing, real-time RT-PCR (qRT-PCR), Western blot, and immunohistochemical (IHC) analysis methods were utilized in this study.
Results: We identified 14 genes that have CpG islands around their transcription start sites; GSTs (M2-M5, A4, P1, Z1, T2, O1-O2) and GPXs (GPX1, GPX3, GPX4, GPX7). Analysis of an initial set of 20 primary samples demonstrated promoter DNA hypermethylation and mRNA down-regulation of GPX3, GPX7, GSTM2, GSTM3, and GSTM5 in more than half of the BACs samples. Further analysis of 159 primary human samples (37 normal, 11 BE, 11 BD, and 100 BACs) indicated frequent hypermethylation (≥10% methylation) of GPX3 (62%), GPX7 (67%), GSTM2 (69.1%), and GSTM3 (15%) in BACs. A significant inverse correlation between DNA methylation and mRNA expression level was shown for GPX3 (P<.0001), GPX7 (P=.002), GSTM2 (P<.0001), and GSTM5 (P=.01). Treatment of esophageal cancer cell lines with 5-Aza-2'-deoxycytidine and Trichostatin-A led to reversal of the methylation pattern and re-expression of these genes at the mRNA and protein levels. The IHC analysis of GPX3, GPX7, and GSTM2 on a tissue microarray that contained 75 BACs with normal squamous esophageal samples demonstrated an absent-to-weak staining in tumors (52% for GPX3, 57% for GPX7, and 45% for GSTM2) and a moderate-to-strong immunostaining in normal samples.
Conclusion: Epigenetic inactivation of members of the glutathione pathway can be an important mechanism in Barrett's tumorigenesis.