A.-N. Tony Kong

p38 Mitogen-activated Protein Kinase Negatively Regulates the Induction of Phase II Drug-metabolizing Enzymes That Detoxify Carcinogens

Phase II drug-metabolizing enzymes, such as glutathione S-transferase and quinone reductase, play an important role in the detoxification of chemical carcinogens. The induction of these detoxifying enzymes by a variety of agents occurs at the transcriptional level and is regulated by a cis-acting element, called the antioxidant response element (ARE) or electrophile-response element. In this study, we identified a signaling kinase pathway that negatively regulates ARE-mediated gene expression. Treatment of human hepatoma HepG2 and murine hepatoma Hepa1c1c7 cells with tert-butylhydroquinone (tBHQ) stimulated the activity of p38, a member of mitogen-activated protein kinase family. Inhibition of p38 activation by its inhibitor, SB203580, enhanced the induction of quinone reductase activity and the activation of ARE reporter gene by tBHQ. In contrast, SB202474, a negative analog of SB203580, had little effect. Consistent with this result, interfering with the p38 kinase pathway by overexpression of a dominant-negative mutant of p38 or MKK3, an immediate upstream regulator of p38, potentiated the activation of the ARE reporter gene by tBHQ, whereas the wild types of p38 and MKK3 diminished such activation. In addition, inhibition of p38 activity augmented the induction of ARE reporter gene activity bytert-butylhydroxyanisole, sulforaphane, and β-naphthoflavone. Thus, p38 kinase pathway functions as a negative regulator in the ARE-mediated induction of phase II detoxifying enzymes.

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

AbstractPurpose. 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.

Phenylethyl Isothiocyanate Induces Apoptotic Signaling via Suppressing Phosphatase Activity against c-Jun N-terminal Kinase

Dietary isothiocyanates induce apoptosis in various cancer cell lines through a c-Jun N-terminal kinase (JNK)-dependent mechanism. We found that phenylethyl isothiocyanate (PEITC) was capable of inducing JNK activation and apoptosis in prostate cancer cell lines with distinct p53 statuses. PEITC induced JNK-mediated apoptotic signaling via a different pathway than that used by DNA-damaging agents, because genotoxicresistant LNCaP prostate cancer cells were equally sensitive to PEITC as parental LNCaP cells. PEITC did not induce significant MKK4 or MKK7 activation and did not activate JNK directly, suggesting that JNK and JNK upstream kinases are not primary targets of PEITC. The JNK dephosphorylation and inactivation rates were decreased in cells exposed to PEITC. Expression levels of M3/6, a JNK-specific phosphatase, were down-regulated by PEITC via a proteasome-dependent mechanism. Taken together, our data suggest that PEITC activates JNK through suppression of JNK dephosphorylation and that PEITC may be an alternative therapeutic agent for cancers that are resistant to genotoxic agents. This study also reveals that JNK phosphatases are potential targets for the development of novel cancer therapeutic agents.