Silencing of Glutathione Peroxidase 3 through DNA Hypermethylation Is Associated with Lymph Node Metastasis in Gastric Carcinomas

Dun-Fa Peng,Tian-Ling Hu,Zheng Chen,Ze-Kuan Xu,Barbara G Schneider,Wael El-Rifai,Regine Schneider-Stock

doi:10.1371/journal.pone.0046214

Abstract

Gastric cancer remains the second leading cause of cancer-related death in the world. H. pylori infection, a major risk factor for gastric cancer, generates high levels of reactive oxygen species (ROS). Glutathione peroxidase 3 (GPX3), a plasma GPX member and a major scavenger of ROS, catalyzes the reduction of hydrogen peroxide and lipid peroxides by reduced glutathione. To study the expression and gene regulation of GPX3, we examined GPX3 gene expression in 9 gastric cancer cell lines, 108 primary gastric cancer samples and 45 normal gastric mucosa adjacent to cancers using quantitative real-time RT-PCR. Downregulation or silencing of GPX3 was detected in 8 of 9 cancer cell lines, 83% (90/108) gastric cancers samples, as compared to non-tumor adjacent normal gastric samples (P<0.0001). Examination of GPX3 promoter demonstrated DNA hypermethylation (≥10% methylation level determined by Bisulfite Pyrosequencing) in 6 of 9 cancer cell lines and 60% of gastric cancer samples (P = 0.007). We also detected a significant loss of DNA copy number of GPX3 in gastric cancers (P<0.001). Treatment of SNU1 and MKN28 cells with 5-Aza-2′ Deoxycytidine restored the GPX3 gene expression with a significant demethylation of GPX3 promoter. The downregulation of GPX3 expression and GPX3 promoter hypermethylation were significantly associated with gastric cancer lymph node metastasis (P = 0.018 and P = 0.029, respectively). We also observed downregulation, DNA copy number losses, and promoter hypermethylation of GPX3 in approximately one-third of tumor-adjacent normal gastric tissue samples, suggesting the presence of a field defect in areas near tumor samples. Reconstitution of GPX3 in AGS cells reduced the capacity of cell migration, as measured by scratch wound healing assay. Taken together, the dysfunction of GPX3 in gastric cancer is mediated by genetic and epigenetic alterations, suggesting impairment of mechanisms that regulate ROS and its possible involvement in gastric tumorigenesis and metastasis.

Highlights

Gastric cancer (GC) is the fourth most common cancer in the world [1,2] with about 900,000 new cases diagnosed in the world each year [2]
The glutathione peroxidase family (GPXs) is a major antioxidative enzyme family that catalyzes the reduction of hydrogen peroxide, organic hydroperoxide, and lipid peroxides by reduced glutathione [15,16,17,18,19]
These results suggest that the hypermethylation of the Glutathione peroxidase 3 (GPX3) promoter region is one of the factors involved in the suppression of its mRNA expression in gastric cancers

Summary

Introduction

Gastric cancer (GC) is the fourth most common cancer in the world [1,2] with about 900,000 new cases diagnosed in the world each year [2]. It is generally agreed that gastric cancer is a multifactor disease in which Helicobacter pylori (H. pylori) infection plays a crucial role, especially for distal gastric cancer [3,4,5]. Accumulating data indicate that H. pylori infection generates high levels of reactive oxygen species (ROS) from multiple sources [6]. For example, are the main source of ROS production in the H. pylori-infected stomach; H. pylori itself can produce ROS [7]. Extensive recent studies have revealed that H. pylori-induced ROS production in gastric epithelial cells might affect gastric epithelial cell signal transduction, resulting in gastric carcinogenesis [8,9,10]. Excessive ROS production in the stomach promotes DNA damage in gastric epithelial cells, suggesting its involvement in gastric carcinogenesis [7,11,12]

Methods

Results

Conclusion