Pharmaceutical Research

, Volume 20, Issue 9, pp 1351–1356 | Cite as

Involvement of Nrf2 and JNK1 in the Activation of Antioxidant Responsive Element (ARE) by Chemopreventive Agent Phenethyl Isothiocyanate (PEITC)

  • Young-Sam Keum
  • Edward D. Owuor
  • Bok-Ryang Kim
  • Rong Hu
  • A.-N. Tony Kong


Purpose. Phenethyl isothiocyanate (PEITC) has been of great interest as a promising cancer chemopreventive agent. To better understand its chemopreventive activity, we examined the effect of PEITC on the antioxidant responsive element (ARE), which is an important gene regulatory element of many phase II drug-metabolizing/detoxification enzymes as well as cellular defensive enzymes.

Methods. HeLa cells were transiently transfected with different cDNA plasmids using calcium phosphate precipitation. Subsequently, the cells were maintained in fresh media, and various concentrations of PEITC were added to the transfected cells. After harvesting and lysing of the cells, ARE-luciferase reporter gene activity was measured and normalized against β-galactosidase activity.

Results. Treatments of HeLa cells with PEITC transiently stimulated ARE-reporter gene expressions in a dose-dependent manner. Overexpression of wild-type NF-E2 related factor-2 (Nrf2) dramatically increased ARE-reporter gene expression in a dose-dependent manner. Similar effects were seen when wild-type c-Jun N-terminal kinase 1 (JNK1) was transfected, although the transactivating potential of JNK1 was much less than that of Nrf2. Cotransfection of Nrf2 and JNK1 showed additional enhancement of ARE reporter gene expression, implying that JNK1 might be an upstream activator of Nrf2. To support this, overexpression of dominant-negative JNK1 suppressed Nrf2-induced ARE reporter gene expression in a dose-dependent manner. When PEITC was added, slight enhancement of ARE reporter gene expression was observed in either Nrf2- or JNK1-transfected cells. Finally, ARE reporter activity induced by PEITC was substantially attenuated by transfection of either dominant-negative mutant of Nrf2 or dominant-negative mutant of JNK1.

Conclusion. Taken together, these data suggest that JNK1 acts as an upstream activator of Nrf2 and that PEITC activates ARE-mediated phase II drug metabolism gene expressions via the JNK1- and Nrf2-dependent pathways.

chemoprevention isothiocyanate (ITC) phenethyl isothiocyanates (PEITC) antioxidant response element (ARE) Nrf2 JNK1 signal transduction 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    D. T. Verhoeven, H. Verhagen, R. A. Goldbohm, P. A. van den Brandt, and G. van Poppel. A review of mechanisms underlying anticarcinogenicity by brassica vegetables. Chem. Biol. Interact. 103:79–129 (1997).Google Scholar
  2. 2.
    P. Talalay and J. W. Fahey. Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism. J. Nutr. 131:3027S–3033S (2001).Google Scholar
  3. 3.
    S. S. Hecht. Chemoprevention of cancer by isothiocyanates, modifiers of carcinogen metabolism. J. Nutr. 129:768S–774S (1999).Google Scholar
  4. 4.
    E. W. Boberg, E. C. Miller, J. A. Miller, A. Poland, and A. Liem. Strong evidence from studies with brachymorphic mice pentachlorophenol that 1′–sulfooxysafrole is the major ultimate electrophilic carcinogenic metabolite of 1′–hydroxysafrole in mouse liver. Cancer Res. 43:5163–5173 (1983).Google Scholar
  5. 5.
    K. Itoh, T. Chiba, S. Takahashi, T. Ishii, K. Igarashi, Y. Katoh, T. Oyake, N. Hayashi, K. Satoh, I. Hatayama, M. Yamamoto, and Y. Nabeshima. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem. Biophys. Res. Commun. 236:313–322 (1997).Google Scholar
  6. 6.
    T. Xie, M. Belinsky, Y. Xu, and A. K. Jaiswal. ARE–TRE–mediated regulation of gene expression. Response to xenobiotics antioxidants. J. Biol. Chem. 270:6894–6900 (1995).Google Scholar
  7. 7.
    T. H. Rushmore, M. R. Morton, and C. B. Pickett. The antioxidant responsive element. Activation by oxidative stress identification of the DNA consensus sequence required for functional activity. J. Biol. Chem. 266:11632–11639 (1991).Google Scholar
  8. 8.
    J. D. Hayes and M. McMahon. Molecular basis for the contribution of the antioxidant responsive element to cancer chemoprevention. Cancer Lett. 174:103–113 (2001).Google Scholar
  9. 9.
    R. Yu, W. Lei, S. Mandlekar, M. J. Weber, C. J. Der, J. Wu, and A. T. Kong. Role of a mitogen–activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals. J. Biol. Chem. 274:27545–27552 (1999).Google Scholar
  10. 10.
    M. Ramos–Gomez, M. K. Kwak, P. M. Dolan, K. Itoh, M. Yamamoto, P. Talalay, and T. W. Kensler. Sensitivity to carcinogenesis is increased chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor–deficient mice. Proc. Natl. Acad. Sci. USA 98:3410–3415 (2001).Google Scholar
  11. 11.
    A. N. Kong, E. Owuor, R. Yu, V. Hebbar, C. Chen, R. Hu, and S. Mandlekar. Induction of xenobiotic enzymes by the MAP kinase pathway the antioxidant or electrophile response element (ARE/EpRE). Drug Metab. Rev. 33:255–271 (2001).Google Scholar
  12. 12.
    K. Itoh, N. Wakabayashi, Y. Katoh, T. Ishii, K. Igarashi, J. D. Engel, and M. Yamamoto. Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino–terminal Neh2 domain. Genes Dev. 13:76–86 (1999).Google Scholar
  13. 13.
    R. Yu, C. Chen, Y. Y. Mo, V. Hebbar, E. D. Owuor, T. H. Tan, and A. N. Kong. Activation of mitogen–activated protein kinase pathways induces antioxidant response element–mediated gene expression via a Nrf2–dependent mechanism. J. Biol. Chem. 275:39907–39913 (2000).Google Scholar
  14. 14.
    H. C. Huang, T. Nguyen, and C. B. Pickett. Regulation of the antioxidant response element by protein kinase C–mediated phosphorylation of NF–E2–related factor 2. Proc. Natl. Acad. Sci. USA 97:12475–12480 (2000).Google Scholar
  15. 15.
    J. M. Lee, J. M. Hanson, W. A. Chu, and J. A. Johnson. Phosphatidylinositol 3–kinase, not extracellular signal–regulated kinase, regulates activation of the antioxidant–responsive element in IMR–32 human neuroblastoma cells. J. Biol. Chem. 276:20011–20016 (2001).Google Scholar
  16. 16.
    J. Jeyapaul and A. K. Jaiswal. Nrf2 c–Jun regulation of antioxidant response element (ARE)–mediated expression induction of gamma–glutamylcysteine synthetase heavy subunit gene. Biochem. Pharmacol. 59:1433–1439 (2000).Google Scholar
  17. 17.
    H. J. Prochaska and P. Talalay. Regulatory mechanisms of monofunctional bifunctional anticarcinogenic enzyme inducers in murine liver. Cancer Res. 48:4776–4782 (1988).Google Scholar
  18. 18.
    J. D. Brooks, V. G. Paton, and G. Vidanes. Potent induction of phase 2 enzymes in human prostate cells by sulforaphane. Cancer Epidemiol. Biomarkers Prev. 10:949–954 (2001).Google Scholar
  19. 19.
    Y. Nakamura, H. Ohigashi, S. Masuda, A. Murakami, Y. Morimitsu, Y. Kawamoto, T. Osawa, M. Imagawa, and K. Uchida. Redox regulation of glutathione S–transferase induction by benzyl isothiocyanate: correlation of enzyme induction with the formation of reactive oxygen intermediates. Cancer Res. 60:219–225 (2000).Google Scholar
  20. 20.
    P. Rose, K. Faulkner, G. Williamson, and R. Mithen. 7–Methylsulfinylheptyl 8–methylsulfinyloctyl isothiocyanates from watercress are potent inducers of phase II enzymes. Carcinogenesis 21:1983–1988 (2000).Google Scholar
  21. 21.
    C. C. Conaway, D. Jiao, and F. L. Chung. Inhibition of rat liver cytochrome P450 isozymes by isothiocyanates their conjugates: a structure–activity relationship study. Carcinogenesis 17:2423–2427 (1996).Google Scholar
  22. 22.
    R. Yu, S. Mandlekar, K. J. Harvey, D. S. Ucker, and A. N. Kong. Chemopreventive isothiocyanates induce apoptosis caspase–3–like protease activity. Cancer Res. 58:402–408 (1998).Google Scholar
  23. 23.
    R. Yu, S. Mandlekar, W. Lei, W. E. Fahl, T. H. Tan, and A. T. Kong. p38 mitogen–activated protein kinase negatively regulates the induction of phase II drug–metabolizing enzymes that detoxify carcinogens. J. Biol. Chem. 275:2322–2327 (2000).Google Scholar
  24. 24.
    L. M. Zipper and R. T. Mulcahy. Inhibition of ERK p38 MAP kinases inhibits binding of Nrf2 induction of GCS genes. Biochem. Biophys. Res. Commun. 278:484–492 (2000).Google Scholar
  25. 25.
    A. T. Dinkova–Kostova, M. A. Massiah, R. E. Bozak, R. J. Hicks, and P. Talalay. Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups. Proc. Natl. Acad. Sci. USA 98:3404–3409 (2001).Google Scholar
  26. 26.
    M. K. Kwak, K. Itoh, M. Yamamoto, and T. W. Kensler. Enhanced expression of the transcription factor Nrf2 by cancer chemopreventive agents: role of antioxidant response element–like sequences in the nrf2 promoter. Mol. Cell. Biol. 22:2883–2892 (2002).Google Scholar
  27. 27.
    Y. Zhang and P. Talalay. Mechanism of differential potencies of isothiocyanates as inducers of anticarcinogenic Phase 2 enzymes. Cancer Res. 58:4632–4639 (1998).Google Scholar
  28. 28.
    P. M. Chaudhary, M. T. Eby, A. Jasmin, and L. Hood. Activation of the c–Jun N–terminal kinase/stress–activated protein kinase pathway by overexpression of caspase–8 its homologs. J. Biol. Chem. 274:19211–19219 (1999).Google Scholar

Copyright information

© Plenum Publishing Corporation 2003

Authors and Affiliations

  • Young-Sam Keum
    • 1
  • Edward D. Owuor
    • 1
  • Bok-Ryang Kim
    • 1
  • Rong Hu
    • 1
  • A.-N. Tony Kong
    • 1
  1. 1.Department of Pharmaceutics, Ernest Mario School of Pharmacy, RutgersThe State University of New JerseyPiscataway

Personalised recommendations