Epigenetic Changes Induced by Reactive Oxygen Species in Hepatocellular Carcinoma: Methylation of the E-cadherin Promoter

Abstract

In addition to genetic alterations, epigenetic changes underlie tumor progression and metastasis. Promoter methylation can silence tumor suppressor genes, and reactive oxygen species (ROS) promote DNA damage, although the relationship between ROS and epigenetic changes in cancer cells is not clear. We sought to determine whether ROS promote hypermethylation of the promoter region of E-cadherin, a regulator of the epithelial-to-mesenchymal transition, in hepatocellular carcinoma (HCC) cells. HCC cells were exposed to H(2)O(2) or stably transfected to express Snail, a transcription factor that down-regulates E-cadherin expression. E-cadherin and Snail expression levels were examined by real-time reverse-transcriptase polymerase chain reaction and immunoblot analyses. The methylation status of E-cadherin was examined by methyl-specific polymerase chain reaction, bisulfite sequencing, and chromatin immunoprecipitation. The interactions between Snail, histone deacetylase 1, and DNA methyltransferase 1 were assessed by immunoprecipitation/immunoblot and immunofluorescence analyses. ROS-induced stress, E-cadherin expression, Snail expression, and E-cadherin promoter methylation were confirmed in HCC tissues by immunoblot, immunohistochemistry, and methyl-specific polymerase chain reaction analyses. We demonstrated that ROS induce hypermethylation of the E-cadherin promoter by increasing Snail expression. Snail induced DNA methylation of the E-cadherin promoter by recruiting histone deacetylase 1 and DNA methyltransferase 1. In human HCC tissues, we observed a correlation among ROS induction, E-cadherin down-regulation, Snail up-regulation, and E-cadherin promoter methylation. These findings provide novel mechanistic insights into epigenetic modulations induced by ROS in the process of carcinogenesis. They are potentially relevant to understanding the activity of ROS in silencing various tumor suppressor genes and in subsequent tumor progression and metastasis.