Oxidative Stress Induces Hypomethylation of LINE-1 and Hypermethylation of the RUNX3 Promoter in a Bladder Cancer Cell Line

Wikrom Wongpaiboonwattana,Piyaratana Tosukhowong,Chanchai Boonla,Thasinas Dissayabutra,Apiwat Mutirangura

doi:10.7314/apjcp.2013.14.6.3773

Wikrom Wongpaiboonwattana, Piyaratana Tosukhowong + Show 3 more

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https://doi.org/10.7314/apjcp.2013.14.6.3773

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Abstract

Increased oxidative stress and changes in DNA methylation are frequently detected in bladder cancer patients. We previously demonstrated a relationship between increased oxidative stress and hypomethylation of the transposable long-interspersed nuclear element-1 (LINE-1). Promoter hypermethylation of a tumor suppressor gene, runt-related transcription factor 3 (RUNX3), may also be associated with bladder cancer genesis. In this study, we investigated changes of DNA methylation in LINE-1 and RUNX3 promoter in a bladder cancer cell (UM-UC-3) under oxidative stress conditions, stimulated by challenge with H2O2 for 72 h. Cells were pretreated with an antioxidant, tocopheryl acetate for 1 h to attenuate oxidative stress. Methylation levels of LINE-1 and RUNX3 promoter were measured by combined bisulfite restriction analysis PCR and methylation-specific PCR, respectively. Levels of LINE-1 methylation were significantly decreased in H2O2-treated cells, and reestablished after pretreated with tocopheryl acetate. Methylation of RUNX3 promoter was significantly increased in cells exposed to H2O2. In tocopheryl acetate pretreated cells, it was markedly decreased. In conclusion, hypomethylation of LINE-1 and hypermethylation of RUNX3 promoter in bladder cancer cell line was experimentally induced by reactive oxygen species (ROS). The present findings support the hypothesis that oxidative stress promotes urothelial cell carcinogenesis through modulation of DNA methylation. Our data also imply that mechanistic pathways of ROS-induced alteration of DNA methylation in a repetitive DNA element and a gene promoter might differ.

Highlights

Stepwise accumulations of genetic mutations and epigenetic alterations are vital events in the carcinogenic process
DNA methyltransferase (DNMT) requires S-adenosylmethionine (SAM) as a methyl donor for Increase in reactive oxygen species (ROS) production and alterations of DNA methylation reaction, and SAM is synthesized from methylation are well recognized to vitally involve in the methionine
Of H2O2, a representative of ROS, on methylation changes synthesis under oxidative stress condition, leads of long-interspersed nuclear element-1 (LINE-1) and RUNX3 promoter in bladder cancer UM- to depletion of SAM to be used in the DNA methylation

Summary

Introduction

Stepwise accumulations of genetic mutations and epigenetic alterations are vital events in the carcinogenic process. Beside genetic mutations and epigenetic alterations, oxidative stress is critically involved in the bladder cancer carcinogenesis. Increase in oxidative stress in patients with bladder cancer has been reported (Akcay et al, 2003, Opanuraks et al, 2010). Reactive oxygen species (ROS) directly damage the cellular DNA and promote tumor development through genetic mutations, and through epigenetic alterations (Wachsman, 1997). Global hypomethylation and regional (site-specific CpG island promoter) hypermethylation of the tumor suppressor genes are demonstrated in bladder cancer (Sanchez-Carbayo, 2012). We reported that patients with bladder cancer had increased oxidative stress and LINE-1 hypomethylation relative to the healthy controls (Patchsung et al, 2012). Whether LINE-1 hypomethylation is a cause or effect of oxidative stress

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