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PMID: 15883370 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 102 ·No. 20 ·2005-05-17 ·Pages 7280-7285A

Devling TW, Lindsay CD, McLellan LI, McMahon M, Hayes JD

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.

MeSH Terms
Alcohol Oxidoreductases/metabolism Aldehyde Reductase Aldo-Keto Reductases Blotting, Western Cells, Cultured Chemoprevention DNA Primers DNA-Binding Proteins/metabolism Gene Expression Regulation, Neoplastic Genes, Reporter/genetics Glutamate-Cysteine Ligase/metabolism Glutathione/metabolism Humans Intracellular Signaling Peptides and Proteins Kelch-Like ECH-Associated Protein 1 Luciferases NF-E2-Related Factor 2 Neoplasms/prevention & control Plasmids/genetics Proteins/antagonists & inhibitors,metabolism Quinone Reductases/metabolism RNA, Messenger/metabolism RNA, Small Interfering/genetics,metabolism,pharmacology Trans-Activators/metabolism Transfection
Chemicals
DNA Primers DNA-Binding Proteins Intracellular Signaling Peptides and Proteins KEAP1 protein, human Kelch-Like ECH-Associated Protein 1 NF-E2-Related Factor 2 NFE2L2 protein, human Proteins RNA, Messenger RNA, Small Interfering Trans-Activators Alcohol Oxidoreductases Aldo-Keto Reductases Aldehyde Reductase AKR7A5 protein, mouse Luciferases Quinone Reductases Glutamate-Cysteine Ligase Glutathione
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Devling Tim W P
Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.
Lindsay Christopher D
McLellan Lesley I
McMahon Michael
Hayes John D
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.0501475102
Published
2005-05-17
Epub
2005-00-09
Pages
7280-7285A
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC1091750
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.0501475102 PMC Full Text View journal details
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