title: Hypermethylation of the Keap1 gene inactivates its function, promotes Nrf2 nuclear accumulation, and is involved in arsenite-induced human keratinocyte transformation.
abstract: It is well known that long-term exposure to arsenite leads to human skin cancer, but the underlying mechanisms of carcinogenesis remain obscure. The transcription factor Nrf2-mediated antioxidant response represents a critical cellular defense mechanism; however, emerging data suggest that constitutive activation of Nrf2 is associated with cancer development and chemotherapy resistance. The reasons Nrf2 continuously accumulates in cancer cells remain to be fully understood. By establishing transformed human keratinocyte cells via chronic arsenite treatment, we observed a continuous reduction in reactive oxygen species levels and enhanced levels of Nrf2 and its target antioxidant enzymes in the later stage of arsenite-induced cell transformation. We also revealed that hypermethylation of the Keap1 gene promoter region induced by DNA methyltransferase-3 leading to inactivation of its function was responsible for constitutive activation of Nrf2 and its target enzymes. To validate these observations, the expression of Keap1 protein was restored in arsenite-transformed cells by treatment with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-Aza-dC), and protein levels of Nrf2 and colony formation were then determined after these treatments. Results showed that enhancement of Keap1 expression by 5-Aza-dC significantly reduced Nrf2 and its target antioxidant enzyme levels, and that in turn suppressed cell proliferation and colony formation of the transformed cells. Taken together, the present study strongly suggests that loss of Keap1 function by hypermethylation of its promoter region leading to Nrf2 nuclear accumulation appears to play a role in arsenite-induced human keratinocyte transformation.
authors.affiliation: Department of Toxicology, School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Medical College of Soochow University, Suzhou, Jiangsu 215123, People's Republic of China; Department of Toxicology, School of Public Health, Guizhou Medical University, Guiyang, Guizhou 550025, People's Republic of China.
authors.forename: Dapeng
authors.initials: D
authors.lastname: Wang
authors.affiliation: Department of Toxicology, School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Medical College of Soochow University, Suzhou, Jiangsu 215123, People's Republic of China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Department of Toxicology, School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Medical College of Soochow University, Suzhou, Jiangsu 215123, People's Republic of China.
authors.forename: Xu
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Toxicology, School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Medical College of Soochow University, Suzhou, Jiangsu 215123, People's Republic of China.
authors.forename: Xiguo
authors.initials: X
authors.lastname: Xu
authors.affiliation: Department of Toxicology, School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Medical College of Soochow University, Suzhou, Jiangsu 215123, People's Republic of China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Toxicology, School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Medical College of Soochow University, Suzhou, Jiangsu 215123, People's Republic of China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zhu
authors.affiliation: Department of Toxicology, School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Medical College of Soochow University, Suzhou, Jiangsu 215123, People's Republic of China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Toxicology, School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Medical College of Soochow University, Suzhou, Jiangsu 215123, People's Republic of China.
authors.forename: Hanyi
authors.initials: H
authors.lastname: Deng
authors.affiliation: Department of Toxicology, School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Medical College of Soochow University, Suzhou, Jiangsu 215123, People's Republic of China.
authors.forename: Chunchun
authors.initials: C
authors.lastname: Li
authors.affiliation: Department of Toxicology, School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Medical College of Soochow University, Suzhou, Jiangsu 215123, People's Republic of China.
authors.forename: Fenfang
authors.initials: F
authors.lastname: Gao
authors.affiliation: Department of Toxicology, School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Medical College of Soochow University, Suzhou, Jiangsu 215123, People's Republic of China.
authors.forename: Jian
authors.initials: J
authors.lastname: Tong
authors.affiliation: Laboratory of Environmental Toxicology and Carcinogenesis, School of Pharmacy, Nihon University, Chiba, Japan.
authors.forename: Kenzo
authors.initials: K
authors.lastname: Yamanaka
authors.affiliation: Department of Toxicology, School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Medical College of Soochow University, Suzhou, Jiangsu 215123, People's Republic of China. Electronic address: dranyan@126.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: An
mesh_terms: D000975:Antioxidants / Q000494:pharmacology; D018053:Arsenites / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D002478:Cells, Cultured; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D000077209:Decitabine; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D000072019:Kelch-Like ECH-Associated Protein 1; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013723:Teratogens / Q000494:pharmacology
chemical_list: D000975:Antioxidants; D018053:Arsenites; C000717967:DNMT3A protein, human; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013723:Teratogens; D000077209:Decitabine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D001374:Azacitidine; C015001:arsenite
keywords: Arsenite; Carcinogenesis; Free radicals; Keap1; Methylation; Nrf2