title: Ginger compound [6]-shogaol and its cysteine-conjugated metabolite (M2) activate Nrf2 in colon epithelial cells in vitro and in vivo.
abstract: In this study, we identified Nrf2 as a molecular target of [6]-shogaol (6S), a bioactive compound isolated from ginger, in colon epithelial cells in vitro and in vivo. Following 6S treatment of HCT-116 cells, the intracellular GSH/GSSG ratio was initially diminished but was then elevated above the basal level. Intracellular reactive oxygen species (ROS) correlated inversely with the GSH/GSSG ratio. Further analysis using gene microarray showed that 6S upregulated the expression of Nrf2 target genes (AKR1B10, FTL, GGTLA4, and HMOX1) in HCT-116 cells. Western blotting confirmed upregulation, phosphorylation, and nuclear translocation of Nrf2 protein followed by Keap1 decrease and upregulation of Nrf2 target genes (AKR1B10, FTL, GGTLA4, HMOX1, and MT1) and glutathione synthesis genes (GCLC and GCLM). Pretreatment of cells with a specific inhibitor of p38 (SB202190), PI3K (LY294002), or MEK1 (PD098059) attenuated these effects of 6S. Using ultra-high-performance liquid chromatography-tandem mass spectrometry, we found that 6S modified multiple cysteine residues of Keap1 protein. In vivo 6S treatment induced Nrf2 nuclear translocation and significantly upregulated the expression of MT1, HMOX1, and GCLC in the colon of wild-type mice but not Nrf2(-/-) mice. Similar to 6S, a cysteine-conjugated metabolite of 6S (M2), which was previously found to be a carrier of 6S in vitro and in vivo, also activated Nrf2. Our data demonstrated that 6S and its cysteine-conjugated metabolite M2 activate Nrf2 in colon epithelial cells in vitro and in vivo through Keap1-dependent and -independent mechanisms.
authors.affiliation: Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, North Carolina Research Campus , 500 Laureate Way, Kannapolis, North Carolina 28081, United States.
authors.forename: Huadong
authors.initials: H
authors.lastname: Chen
authors.forename: Junsheng
authors.initials: J
authors.lastname: Fu
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.forename: Yuhui
authors.initials: Y
authors.lastname: Hu
authors.forename: Dominique N
authors.initials: DN
authors.lastname: Soroka
authors.forename: Justin R
authors.initials: JR
authors.lastname: Prigge
authors.forename: Edward E
authors.initials: EE
authors.lastname: Schmidt
authors.forename: Feng
authors.initials: F
authors.lastname: Yan
authors.forename: Michael B
authors.initials: MB
authors.lastname: Major
authors.forename: Xiaoxin
authors.initials: X
authors.lastname: Chen
authors.forename: Shengmin
authors.initials: S
authors.lastname: Sang
mesh_terms: D000478:Alkylation; D000818:Animals; D002396:Catechols / Q000737:chemistry* / Q000494:pharmacology; D003545:Cysteine / Q000032:analysis / Q000737:chemistry*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D020939:Zingiber officinale / Q000737:chemistry* / Q000378:metabolism; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D045325:HCT116 Cells; D051547:Heme Oxygenase-1 / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D000072019:Kelch-Like ECH-Associated Protein 1; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D051267:NF-E2-Related Factor 2 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D002396:Catechols; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D008565:Membrane Proteins; D051267:NF-E2-Related Factor 2; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; C040115:shogaol; D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse; C560749:GCLM protein, mouse; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D003545:Cysteine