Generation of a stable antioxidant response element-driven reporter gene cell line and its use to show redox-dependent activation of nrf2 by cancer chemotherapeutic agents.

The NF-E2 p45-related factor 2 (Nrf2) regulates cytoprotective genes that contain an antioxidant response element (ARE) in their promoters. To investigate whether anticancer drugs can induce ARE-driven gene expression, we have developed a stable human mammary MCF7-derived reporter cell line called AREc32, which contains a luciferase gene construct controlled by eight copies of the cis-element. In these cells, luciferase activity was increased up to 50-fold following treatment with 50 mumol/L tert-butylhydroquinone (t-BHQ). Basal and inducible luciferase activities in AREc32 cells were increased by forced overexpression of Nrf2 and reduced by knockdown of endogenous Nrf2 expression with RNA interference. Depletion of cellular reduced glutathione (GSH) by treatment of AREc32 cells with l-buthionine-S,R-sulfoximine (BSO) did not influence basal levels of luciferase activity, but pretreatment with BSO augmented induction of luciferase activity by t-BHQ. Induction of reporter activity by t-BHQ in AREc32 cells was suppressed markedly by the antioxidants N-acetylcysteine and GSH but only modestly by vitamins C or E, suggesting that ARE-luciferase expression is induced primarily by thiol-active electrophiles rather than free radicals. The anticancer drugs cisplatin, etoposide, mitoxantrone, chlorambucil, melphalan, and carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)] weakly induced luciferase activity in AREc32 cells. Moreover, treatment of AREc32 cells with BSO immediately before exposure to anticancer drugs enhanced induction of ARE-driven luciferase activity by cisplatin, BCNU, chlorambucil, and melphalan and also induced endogenous AKR1C (AKR1C refers to AKR1C1 and AKR1C2), a target gene of Nrf2. Our findings show that Nrf2 can be activated by certain anticancer agents, and this will influence the effectiveness of chemotherapy.

[1]  X. Wang,et al.  Relationship between hepatic phenotype and changes in gene expression in cytochrome P450 reductase (POR) null mice. , 2005, The Biochemical journal.

[2]  Mark R. Green,et al.  Co‐ordinated over‐expression of the MRP and γ‐glutamylcysteine synthetase genes, but not MRD1, correlates with doxorubicin resistance in human malignant mesothelioma cell lines , 1998, International journal of cancer.

[3]  D. Moore,et al.  Induction of c-fos and c-jun gene expression by phenolic antioxidants. , 1993, Molecular endocrinology.

[4]  E. Grzesiuk.  The role of mutation frequency decline and SOS repair systems in methyl methanesulfonate mutagenesis. , 1998, Acta biochimica Polonica.

[5]  P. Nioi,et al.  Contribution of NAD(P)H:quinone oxidoreductase 1 to protection against carcinogenesis, and regulation of its gene by the Nrf2 basic-region leucine zipper and the arylhydrocarbon receptor basic helix-loop-helix transcription factors. , 2004, Mutation research.

[6]  T. Rushmore,et al.  The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity. , 1991, The Journal of biological chemistry.

[7]  C. Wolf,et al.  The Nrf2 transcription factor contributes both to the basal expression of glutathione S-transferases in mouse liver and to their induction by the chemopreventive synthetic antioxidants, butylated hydroxyanisole and ethoxyquin. , 2000, Biochemical Society transactions.

[8]  Y. Kan,et al.  Identification of the NF-E2-related Factor-2-dependent Genes Conferring Protection against Oxidative Stress in Primary Cortical Astrocytes Using Oligonucleotide Microarray Analysis* , 2003, The Journal of Biological Chemistry.

[9]  D. Townsend,et al.  The importance of glutathione in human disease. , 2003, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie.

[10]  C. Wolf,et al.  Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice. , 2002, The Biochemical journal.

[11]  T. Kensler,et al.  The role of Keap1 in cellular protective responses. , 2005, Chemical research in toxicology.

[12]  J. Moehlenkamp,et al.  Nrf2-dependent activation of the antioxidant responsive element by tert-butylhydroquinone is independent of oxidative stress in IMR-32 human neuroblastoma cells. , 2001, Biochemical and biophysical research communications.

[13]  Makoto Kobayashi,et al.  Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation. , 2005, Antioxidants & redox signaling.

[14]  J. Hayes,et al.  Dietary indoles and isothiocyanates that are generated from cruciferous vegetables can both stimulate apoptosis and confer protection against DNA damage in human colon cell lines. , 2001, Cancer research.

[15]  D. Harrison,et al.  Cellular response to cancer chemopreventive agents: contribution of the antioxidant responsive element to the adaptive response to oxidative and chemical stress. , 1999, Biochemical Society symposium.

[16]  K. Ozawa,et al.  Ascorbic acid restores sensitivity to imatinib via suppression of Nrf2-dependent gene expression in the imatinib-resistant cell line. , 2004, Experimental hematology.

[17]  K. Itoh,et al.  Identification of a novel Nrf2-regulated antioxidant response element (ARE) in the mouse NAD(P)H:quinone oxidoreductase 1 gene: reassessment of the ARE consensus sequence. , 2003, The Biochemical journal.

[18]  W. Schaffner,et al.  Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. , 1989, Nucleic acids research.

[19]  J. Farber,et al.  Roles for oxidative stress and poly(ADP-ribosyl)ation in the killing of cultured hepatocytes by methyl methanesulfonate. , 1993, Biochemical pharmacology.

[20]  D. Harrison,et al.  Major differences exist in the function and tissue-specific expression of human aflatoxin B1 aldehyde reductase and the principal human aldo-keto reductase AKR1 family members. , 1999, The Biochemical journal.

[21]  J. Hayes,et al.  The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. , 1995, Critical reviews in biochemistry and molecular biology.

[22]  C. Wolf,et al.  Molecular mechanisms of drug resistance. , 1990, The Biochemical journal.

[23]  R. Mulcahy,et al.  Pyrrolidine dithiocarbamate up-regulates the expression of the genes encoding the catalytic and regulatory subunits of gamma-glutamylcysteine synthetase and increases intracellular glutathione levels. , 1999, The Biochemical journal.

[24]  M. McMahon,et al.  Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[25]  S. Reddy,et al.  Nrf2 defends the lung from oxidative stress. , 2006, Antioxidants & redox signaling.

[26]  H. Ohigashi,et al.  Pivotal role of electrophilicity in glutathione S-transferase induction by tert-butylhydroquinone. , 2003, Biochemistry.

[27]  W. Wasserman,et al.  Functional antioxidant responsive elements. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[28]  C. Wolf,et al.  The Cap'n'Collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes. , 2001, Cancer research.

[29]  H. Huang,et al.  Increased Protein Stability as a Mechanism That Enhances Nrf2-mediated Transcriptional Activation of the Antioxidant Response Element , 2003, The Journal of Biological Chemistry.

[30]  H. Akaza,et al.  Nrf2 Is Essential for the Chemopreventive Efficacy of Oltipraz against Urinary Bladder Carcinogenesis , 2004, Cancer Research.

[31]  D. Townsend,et al.  The role of glutathione-S-transferase in anti-cancer drug resistance , 2003, Oncogene.

[32]  W E Fahl,et al.  Development of a green fluorescent protein microplate assay for the screening of chemopreventive agents. , 2000, Analytical biochemistry.

[33]  M. Massiah,et al.  Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. Liebler,et al.  Identification of sensor cysteines in human Keap1 modified by the cancer chemopreventive agent sulforaphane. , 2005, Chemical research in toxicology.

[35]  R. Ozols,et al.  Evidence for altered regulation of gamma-glutamylcysteine synthetase gene expression among cisplatin-sensitive and cisplatin-resistant human ovarian cancer cell lines. , 1995, Cancer research.

[36]  P. Talalay,et al.  Chemical and molecular regulation of enzymes that detoxify carcinogens. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[37]  T. Rushmore,et al.  Transcriptional regulation of a rat liver glutathione S-transferase Ya subunit gene. Analysis of the antioxidant response element and its activation by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. , 1994, The Journal of biological chemistry.

[38]  Y. Tsuji.  JunD activates transcription of the human ferritin H gene through an antioxidant response element during oxidative stress , 2005, Oncogene.

[39]  C. Wolf,et al.  The role of glutathione-dependent enzymes in drug resistance. , 1991, Pharmacology & therapeutics.

[40]  K. Itoh,et al.  Keap1-dependent Proteasomal Degradation of Transcription Factor Nrf2 Contributes to the Negative Regulation of Antioxidant Response Element-driven Gene Expression* , 2003, Journal of Biological Chemistry.

[41]  Masayuki Yamamoto,et al.  Oxidative Stress Sensor Keap1 Functions as an Adaptor for Cul3-Based E3 Ligase To Regulate Proteasomal Degradation of Nrf2 , 2004, Molecular and Cellular Biology.

[42]  Elias S. J. Arnér,et al.  Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. , 2001, Free radical biology & medicine.

[43]  P. van Bladeren,et al.  Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity. , 2006, Biochemical pharmacology.

[44]  R. Pinkus,et al.  Role of Oxidants and Antioxidants in the Induction of AP-1, NF-κB, and Glutathione S-Transferase Gene Expression* , 1996, The Journal of Biological Chemistry.

[45]  K. Tew,et al.  Cellular thiols and reactive oxygen species in drug-induced apoptosis. , 2001, The Journal of pharmacology and experimental therapeutics.

[46]  J. D. Engel,et al.  Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation , 2003, Nature Genetics.

[47]  C. Wolf,et al.  Glutathione and glutathione-dependent enzymes in cancer drug resistance. , 1999, Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy.

[48]  P. Talalay,et al.  Regulatory mechanisms of monofunctional and bifunctional anticarcinogenic enzyme inducers in murine liver. , 1988, Cancer research.

[49]  Daniel C Liebler,et al.  Specific Patterns of Electrophile Adduction Trigger Keap1 Ubiquitination and Nrf2 Activation* , 2005, Journal of Biological Chemistry.

[50]  S. Bergelson,et al.  Two adjacent AP-1-like binding sites form the electrophile-responsive element of the murine glutathione S-transferase Ya subunit gene. , 1992, Proceedings of the National Academy of Sciences of the United States of America.