Identification of 3'-methoxy-4'-nitroflavone as a pure aryl hydrocarbon (Ah) receptor antagonist and evidence for more than one form of the nuclear 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, respectively) 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 characterized 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 induction of ethoxyresorufin O-deethylase (EROD) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells; moreover, in vitro studies with TCDD-induced rat liver microsomes showed that flavones 1 to 4 inhibited EROD activity in the presence or absence of NADPH. In MCF-7 cells cotreated with flavones 1 or 2 (0.01 to 10 microM) plus TCDD or [3H]TCDD, there was a concentration-dependent inhibition of TCDD-induced CYP1A1 mRNA levels and formation of radiolabeled nuclear Ah receptor complex. Velocity sedimentation analysis of nuclear extracts from MCF-7 cells treated with [3H]TCDD plus 1 or 10 microM concentrations of flavones 1 and 2 showed that an early eluting specifically bound nuclear Ah receptor complex was present. However, under these same conditions the flavones inhibited TCDD-induced CYP1A1 gene expression. The apparent molecular mass of this nuclear complex was 190 kDa as determined by cross-linking to a 32P-labeled bromodeoxyuridine-substituted consensus dioxin-responsive element. Similar cross-linking results were obtained using the nuclear extract from MCF-7 cells treated with [3H]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 transcriptionally active form binds [3H]TCDD and flavones 1 or 2 inhibit nuclear uptake of this receptor complex. The other form of the nuclear Ah receptor complex appears to be transcriptionally inactive and ligand binding with [3H]TCDD is not competitively inhibited by flavones 1 and 2.
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station 77843-4466.
Y F
YF
Lu
M
M
Santostefano
B D
BD
Cunningham
M D
MD
Threadgill
S
S
Safe
D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism*; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D004273:DNA, Neoplasm / Q000378:metabolism; D004790:Enzyme Induction; D005260:Female; D005419:Flavonoids / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon / Q000819:agonists / Q000037:antagonists & inhibitors*; D012045:Regulatory Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured
C091768:3'-methoxy-4'-nitroflavone; D004273:DNA, Neoplasm; D005419:Flavonoids; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1