IDENTIFICATION OF 3'-METHOXY-4'-NITROFLAVONE AS A PURE ARYL-HYDROCARBON (AH) RECEPTOR ANTAGONIST AND EVIDENCE FOR MORE THAN ONE FORM OF THENUCLEAR AH RECEPTOR IN MCF-7 HUMAN BREAST-CANCER CELLS

The competitive binding of 3'-methoxy-4'-nitro, 4'amino-3'-methoxy, 4' -methoxy-3'-nitro, and 3'-amino-4'-methoxyflavone (compounds 1 to 4, r espectively) to the rat cytosolic aryl hydrocarbon (Ah) receptor gave IC50 values of 2.27, 86.1, 872, and 19.4 nM. Flavones 3 and 4 were cha racterized as Ah receptor agonists in MCF-7 human breast cancer cells and induced CYP1A1 gene expression, whereas the 3-methoxy-substituted flavones (1 and 2) were inactive. All four compounds inhibited inducti on of ethoxyresorufin O-deethylase (EROD) activity by 2,3,7,8-tetrachl orodibenzo-p-dioxin (TCDD) in MCF-7 cells; moreover, in vitro studies with TCDD-induced rat liver microsomes showed that flavones 1 to 4 inh ibited EROD activity in the presence or absence of NADPH. In MCF-7 cel ls cotreated with flavones 1 or 2 (0.01 to 10 mu M) plus TCDD or [H-3] TCDD, there was a concentration-dependent inhibition of TCDD-induced C YP1A1 mRNA levels and formation of radiolabeled nuclear Ah receptor co mplex. Velocity sedimentation analysis of nuclear extracts from MCF-7 cells treated with [H-3]TCDD plus 1 or 10 mu M concentrations of flavo nes 1 and 2 showed that an early eluting specifically bound nuclear Ah receptor complex was present. However, under these same conditions th e flavones inhibited TCDD-induced CYP1A1 gene expression. The apparent molecular mass of this nuclear complex was 190 kDa as determined by c ross-linking P-32-labeled bromodeoxyuridine-substituted consensus diox in-responsive element. Similar cross-linking results were obtained usi ng the nuclear extract from MCF-7 cells treated with [H-3]TCDD alone. The results of this study suggest that there are at least two forms of the nuclear Ah receptor complex in MCF-7 cells; the major transcripti onally active form binds [H-3]TCDD and flavones 1 or 2 inhibit nuclear uptake of this receptor complex. The other form of the nuclear Ah rec eptor complex appears to be transcriptionally inactive and ligand bind ing with [H-3]TCDD is not competitively inhibited by flavones 1 and 2. (C) 1995 Academic Press, Inc.