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

Arch Biochem Biophys. 1995 Jan 10;316(1):470-7. doi: 10.1006/abbi.1995.1062.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Breast Neoplasms / enzymology
  • Breast Neoplasms / metabolism*
  • Cytochrome P-450 CYP1A1
  • Cytochrome P-450 Enzyme System / biosynthesis*
  • Cytochrome P-450 Enzyme System / genetics
  • Cytosol / enzymology
  • Cytosol / metabolism
  • DNA, Neoplasm / metabolism
  • Enzyme Induction
  • Female
  • Flavonoids / chemistry
  • Flavonoids / pharmacology*
  • Humans
  • Liver / enzymology
  • Liver / metabolism*
  • Male
  • Molecular Sequence Data
  • Oxidoreductases / biosynthesis*
  • Oxidoreductases / genetics
  • RNA, Messenger / analysis
  • Rats
  • Receptors, Aryl Hydrocarbon / agonists
  • Receptors, Aryl Hydrocarbon / antagonists & inhibitors*
  • Regulatory Sequences, Nucleic Acid
  • Tumor Cells, Cultured


  • 3'-methoxy-4'-nitroflavone
  • DNA, Neoplasm
  • Flavonoids
  • RNA, Messenger
  • Receptors, Aryl Hydrocarbon
  • Cytochrome P-450 Enzyme System
  • Oxidoreductases
  • Cytochrome P-450 CYP1A1