Oxidative stress causes epigenetic alteration of CDX1 expression in colorectal cancer cells

The intestine-specific transcription factor, caudal type homeobox-1 (CDX1), is a candidate tumor suppressor gene that plays key roles in regulating intestinal epithelial differentiation and proliferation. It is aberrantly down-regulated in colorectal cancers and colon cancer-derived cell lines by promoter hypermethylation. Since the effects of oxidative stress on the transcription of tumor suppressor genes are largely unknown, this study explored the epigenetic alterations that occur during reactive oxygen species (ROS)-induced silencing of CDX1 in colorectal cancer cells. Oxidative stress by hydrogen peroxide (H2O2) down-regulated CDX1 mRNA levels and protein expression in the human colorectal cancer cell line, T-84. This down-regulation was abolished by pretreatment with the ROS scavenger, N-acetylcysteine. In addition, the DNA methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza-dC) markedly attenuated the decrease in mRNA and protein expression levels induced by H2O2. Moreover, methylation-specific PCR data revealed that H2O2 treatment increased CDX1 promoter methylation, and treatment with 5-Aza-dC reversed this effect, suggesting that an epigenetic regulatory mechanism triggered by ROS-induced methylation may be involved in CDX1 expression. Furthermore, H2O2 treatment resulted in up-regulation of DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) expression and activity, and enhanced the association between DNMT1 and HDAC1. Taken together, these results suggest that ROS-induced oxidative stress silences the tumor suppressor CDX1 through epigenetic regulation, and may therefore be associated with the progression of colorectal cancer.