Oltipraz inhibits 3-methylcholanthrene induction of CYP1A1 by CCAAT/enhancer-binding protein activation.
Oltipraz, a cancer chemopreventive agent, induces CYP1A1 to a certain extent by transactivation of the gene via the Ah receptor (AhR)-xenobiotic response element (XRE) pathway. Previously, we showed that oltipraz promoted CCAAT/enhancer binding proteinbeta (C/EBPbeta) activation, which leads to the induction of glutathione S-transferase. Given that oltipraz activates C/EBPbeta for gene transactivation and that the putative C/EBP binding site is located in the CYP1A1 promoter region, this study investigated the effect of oltipraz on CYP1A1 induction by 3-methylcholanthrene (3-MC). 3-MC induced CYP1A1 in H4IIE cells in a time- and concentration-dependent manner. Gel shift analysis showed that 3-MC increased the band intensity of protein binding to the XRE. Immunocompetition analysis verified the specificity of AhR-XRE binding. Oltipraz (30 microM) induced CYP1A1 and the CYP1A1 promoter-luciferase gene and increased AhR DNA binding activity, which was 10-20% of those in 3-MC (100 nM)-treated cells. However, AhR-XRE binding was not increased after 10 microM oltipraz treatment. Oltipraz (10 microM) significantly inhibited CYP1A1 and CYP1A1-luciferase gene induction by 3-MC with no increase in AhR DNA binding. Oltipraz enhanced protein binding to the C/EBP binding site in the gene promoter and the binding complex comprised of C/EBPbeta and partly C/EBPdelta. Overexpression of dominant-negative mutant C/EBP significantly abolished the ability of oltipraz to suppress 3-MC-inducible CYP1A1 and the CYP1A1 reporter gene expression. Consistently, C/EBPbeta overexpression blocked CYP1A1 reporter gene induction by 3-MC. These results provide evidence that oltipraz suppresses 3-MC induction of CYP1A1 gene expression and that activation of C/EBPbeta by oltipraz contributes to suppression of 3-MC-inducible AhR-mediated CYP1A1 expression.
Cho
Il Je
IJ
National Research Laboratory, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Korea.
Kim
Sang Geon
SG
D000818:Animals; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D001665:Binding Sites; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis* / Q000235:genetics; D004247:DNA / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D022781:Hepatocytes; D008156:Luciferases / Q000235:genetics; D008748:Methylcholanthrene / Q000494:pharmacology*; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011719:Pyrazines / Q000494:pharmacology*; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics; D011993:Recombinant Fusion Proteins; D013871:Thiones; D013876:Thiophenes; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection
D016588:Anticarcinogenic Agents; D022762:CCAAT-Enhancer-Binding Proteins; D011719:Pyrazines; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; D013871:Thiones; D013876:Thiophenes; D008748:Methylcholanthrene; C026209:oltipraz; D004247:DNA; D008156:Luciferases; D019363:Cytochrome P-450 CYP1A1