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Open Access 01-12-2012 | Research article

Methylation of the KEAP1 gene promoter region in human colorectal cancer

Authors: Naoyuki Hanada, Takenori Takahata, Qiliang Zhou, Xulu Ye, Ruowen Sun, Jugoh Itoh, Atsushi Ishiguro, Hiroshi Kijima, Junsei Mimura, Ken Itoh, Shinsaku Fukuda, Yasuo Saijo

Published in: BMC Cancer | Issue 1/2012

Abstract

Background

The Keap1-Nrf2 pathway has been reported to be impaired in several cancers. However, the status of Keap1-Nrf2 system in human colorectal cancer (CRC) has not been elucidated.

Methods

We used colorectal cancer (CRC) cell lines and surgical specimens to investigate the methylation status of the KEAP1 promoter region as well as expression of Nrf2 and its downstream antioxidative stress genes, NQO-1 and AKR1C1.

Results

DNA sequencing analysis indicated that all mutations detected were synonymous, with no amino acid substitutions. We showed by bisulfite genomic sequencing and methylation-specific PCR that eight of 10 CRC cell lines had hypermethylated CpG islands in the KEAP1 promoter region. HT29 cells with a hypermethylated KEAP1 promoter resulted in decreased mRNA and protein expression but unmethylated Colo320DM cells showed higher expression levels. In addition, treatment with the DNA methyltransferase inhibitor 5-Aza-dC combined with the histone deacetylase inhibitor trichostatin A (TSA) increased KEAP1 mRNA expression. These result suggested that methylation of the KEAP1 promoter regulates its mRNA level. Time course analysis with the Nrf2-antioxidant response element (ARE) pathway activator t-BHQ treatment showed a rapid response within 24 h. HT29 cells had higher basal expression levels of NQO-1 and AKR1C1 mRNA than Colo320DM cells. Aberrant promoter methylation of KEAP1 was detected in 53% of tumor tissues and 25% of normal mucosae from 40 surgical CRC specimens, indicating that cancerous tissue showed increased methylation of the KEAP1 promoter region, conferring a protective effect against cytotoxic anticancer drugs.

Conclusion

Hypermethylation of the KEAP1 promoter region suppressed its mRNA expression and increased nuclear Nrf2 and downstream ARE gene expression in CRC cells and tissues.

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The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/66/prepub

Title: Methylation of the KEAP1 gene promoter region in human colorectal cancer
Authors: Naoyuki Hanada
Takenori Takahata
Qiliang Zhou
Xulu Ye
Ruowen Sun
Jugoh Itoh
Atsushi Ishiguro
Hiroshi Kijima
Junsei Mimura
Ken Itoh
Shinsaku Fukuda
Yasuo Saijo
Publication date: 01-12-2012
Publisher: BioMed Central
Published in: BMC Cancer / Issue 1/2012
Electronic ISSN: 1471-2407
DOI: https://doi.org/10.1186/1471-2407-12-66

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