Hypermethylation of the Keap1 gene inactivates its function, promotes Nrf2 nuclear accumulation, and is involved in arsenite-induced human keratinocyte transformation

• Published in: Free radical biology & medicine 2015-12, Vol.89, p.209-219
• Main Authors: Wang, Dapeng, Ma, Yuan, Yang, Xu, Xu, Xiguo, Zhao, Yingying, Zhu, Zhen, Wang, Xiaojuan, Deng, Hanyi, Li, Chunchun, Gao, Fenfang, Tong, Jian, Yamanaka, Kenzo, An, Yan
• Format: Article
• Language: English
• Subjects: Antioxidants - pharmacology; Arsenite; Arsenites - pharmacology; Azacitidine - analogs & derivatives; Azacitidine - pharmacology; Blotting, Western; Carcinogenesis; Cell Nucleus - metabolism; Cell Proliferation - drug effects; Cell Transformation, Neoplastic - drug effects; Cell Transformation, Neoplastic - metabolism; Cell Transformation, Neoplastic - pathology; Cells, Cultured; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases - genetics; DNA (Cytosine-5-)-Methyltransferases - metabolism; DNA Methylation; Free radicals; Humans; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Keap1; Kelch-Like ECH-Associated Protein 1; Keratinocytes - cytology; Keratinocytes - drug effects; Keratinocytes - metabolism; Methylation; NF-E2-Related Factor 2 - genetics; NF-E2-Related Factor 2 - metabolism; Nrf2; Promoter Regions, Genetic - genetics; Reactive Oxygen Species - metabolism; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; Teratogens - pharmacology
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2015.07.153

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. •Long term arsenite exposure reduces ROS and lipid peroxidation levels.•Long term arsenite exposure enhances Nrf2-mediated antioxidant levels.•Long term arsenite exposure induces hypermethylation of Keap1 gene promoter region.•Long term arsenite exposure down-regulates DNMT-1 but up-regulates DNMT-3 expression.•Demethylation of Keap1 gene reduces malignant degree of arsenite-transformed cells.