Modulation of N rf2/ K eap1 system by W asabi 6‐methylthiohexyl isothiocyanate in ARE ‐mediated NQO 1 expression

Scope 6‐Methylthiohexyl isothiocyanate (6‐ MTITC ), one of the major bioactive ingredients in J apanese W asabi, has revealed cytoprotective and cancer chemopreventive effects. This study aims to clarify the molecular mechanisms how 6‐ MTITC modulates nuclear factor E2‐related factor 2 ( N rf2)/ K elchlike ECH ‐associating protein 1 ( K eap1) system in antioxidant‐responsive element ( ARE )‐mediated nicotinamide adenine dinucleotide phosphate ( NADP ): quinone oxidoreductase 1 ( NQO 1 ) expression. Methods and results H ep G 2 cells were treated with 6‐ MTITC with varying time and dose. NQO 1, N rf2, and K eap1 proteins were detected by W estern blotting. ARE transactivation was detected by electrophilic mobility shift assay and reporter gene assay. Nuclear localization of N rf2 was determined by immunocytochemistry assay. Ubiquitination of N rf2 and K eap1 was detected using immunoprecipitation after treatment with MG 132. Small interfering RNA was used to knockdown N rf2 or K eap1. The results revealed that 6‐ MTITC modulated N rf2/ ARE pathway by stimulating K eap1 modification, and inhibiting N rf2 ubiquitination and protein turnover. These actions finally increased nuclear N rf2 accumulation and ARE ‐binding activity. Moreover, silencing N rf2 markedly reduced ARE ‐driven activity induced by 6‐ MTITC . Conclusion 6‐ MTITC modulated ARE ‐driven NQO 1 expression by stabilizing N rf2 with enhanced K eap1 modification and decreased N rf2 degradation.