Involvement of Nrf2 and JNK1 in the activation of antioxidant responsive element (ARE) by chemopreventive agent phenethyl isothiocyanate (PEITC).
OBJECTIVE
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 beta-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.


CONCLUSIONS
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.
Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA.
Young-Sam
YS
Keum
Edward D
ED
Owuor
Bok-Ryang
BR
Kim
Rong
R
Hu
A N Tony
AN
Kong
D016588:Anticarcinogenic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D000975:Antioxidants / Q000378:metabolism*; D018890:Chemoprevention; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004790:Enzyme Induction; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D017879:Isothiocyanates / Q000494:pharmacology* / Q000627:therapeutic use; D008156:Luciferases / Q000378:metabolism; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D051267:NF-E2-Related Factor 2; D020218:Response Elements; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection
D016588:Anticarcinogenic Agents; D000975:Antioxidants; D004268:DNA-Binding Proteins; D017879:Isothiocyanates; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D015534:Trans-Activators; C058305:phenethyl isothiocyanate; D008156:Luciferases; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases