title: Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype.
abstract: A duplex 21 nucleotide small interfering RNA (siRNA) against human Keap1 is described that represents a unique class of cancer chemopreventive agent. This siRNA can knockdown Keap1 mRNA and thereby relieve negative regulation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-mediated gene expression. The siRNA lowered endogenous Keap1 mRNA to <30% of control levels between 24 and 72 h after transfection in human HaCaT keratinocyte cells and was capable of blocking ectopic expression of FLAG-tagged human Keap1 protein but not that of ectopic V5-tagged mouse Keap1 protein. Transfection of human HaCaT cells with Keap1 siRNA markedly enhanced endogenous levels of nuclear factor erythroid 2 p45-related factor 2 (Nrf2) protein and increased transcription of an antioxidant response element-driven reporter gene by 2.3-fold. Furthermore, 48 h after transfection of these cells with Keap1 siRNA, expression of aldo-keto reductase 1C1/2 and the glutamate cysteine ligase catalytic and modifier subunits was elevated between 5- and 14-fold. A modest increase of 3-fold in NAD(P)H:quinone oxidoreductase 1 was also observed. The Keap1 siRNA produced a 1.75-fold increase in intracellular glutathione 48 h after transfection. Thus, antagonism of Keap1 by siRNA can be used to preadapt human cells to oxidative stress without the need to expose them to redox stressors.
authors.affiliation: Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.
authors.forename: Tim W P
authors.initials: TW
authors.lastname: Devling
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Lindsay
authors.forename: Lesley I
authors.initials: LI
authors.lastname: McLellan
authors.forename: Michael
authors.initials: M
authors.lastname: McMahon
authors.forename: John D
authors.initials: JD
authors.lastname: Hayes
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D015153:Blotting, Western; D002478:Cells, Cultured; D018890:Chemoprevention; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter / Q000235:genetics; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D000072019:Kelch-Like ECH-Associated Protein 1; D008156:Luciferases; D051267:NF-E2-Related Factor 2; D009369:Neoplasms / Q000517:prevention & control*; D010957:Plasmids / Q000235:genetics; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011808:Quinone Reductases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D011506:Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D015534:Trans-Activators; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; C000614209:AKR7A5 protein, mouse; D008156:Luciferases; D011808:Quinone Reductases; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione