3',4'-Dimethoxyflavone as an Aryl Hydrocarbon Receptor Antagonist in Human Breast Cancer Cells

Jeong-Eun Lee and Stephen Safe1

Department of Veterinary Physiology & Pharmacology, Texas A&M University, 4466 TAMU, College Station, Texas 77843

Received June 13, 2000; accepted August 9, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Treatment of MCF-7 and T47D human breast cancer cells with 3',4'-dimethoxyflavone (3',4'-DMF) alone did not induce CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity or reporter gene activity in cells transfected with an aryl hydrocarbon (Ah)-responsive construct (pRNH11c). In contrast, 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced up to a 50- to 80-fold increase in EROD and reporter gene activity in MCF-7 and T47D cells. In cells cotreated with 1 nM TCDD plus 0.1–10 µM 3',4'-DMF, there was a concentration-dependent decrease in the TCDD-induced responses, with 100% inhibition observed at the 10 µM concentration. Gel mobility shift assays using rat liver cytosol and breast cancer cell nuclear extracts showed that 3',4'-DMF alone did not transform the AhR to its nuclear binding form, but inhibited TCDD-induced AhR transformation in rat liver cytosol and blocked TCDD-induced formation of the nuclear AhR complex in MCF-7 and T47D cells. TCDD also inhibited estrogen-induced transactivation in MCF-7 cells, and this response was also blocked by 3',4'-DMF, confirming the AhR antagonist activity of this compound in breast cancer cells.

Key Words: AhR; antagonist; breast cancer cells; 3',4'-dimethoxyflavone.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aryl hydrocarbon receptor (AhR) is a member of the basic helix-loop-helix (bHLH) family of nuclear transcription factors and is the only bHLH protein that is ligand activated (reviewed in Swanson and Bradfield, 1993; Wilson and Safe, 1998). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a high-affinity ligand for the AhR, and this compound has been used as a prototype for determining AhR-mediated biochemical and toxic responses and the molecular mechanisms of AhR action (Poland and Knutson, 1982Go; Goldstein and Safe, 1989Go; Whitlock, 1993Go). The mechanism of CYP1A1 induction by TCDD has been extensively investigated, and the results are consistent with the following pathway: (1) treatment with TCDD results in the rapid accumulation of the heterodimeric nuclear AhR complex containing the AhR and AhR nuclear translocator (Arnt) proteins; (2) the AhR complex interacts with 5'-dioxin response elements (DREs) in target gene (CYP1A1) promoters; and (3) subsequently recruits other nuclear proteins and interacts with basal transcription factors to activate gene expression (Whitlock, 1993Go, 1996Go; Schmidt and Bradfield, 1996Go). Pathways for ligand activation of the AhR are similar to those described for the steroid hormone receptors and other members of the nuclear receptor superfamily (Evans, 1988Go; Beato et al., 1995Go; Mangelsdorf et al., 1995Go; Perlmann and Evans, 1997Go).

Although TCDD and related toxic environmental contaminants have been extensively characterized as AhR ligands, there is an increasing number of structurally diverse synthetic and naturally occurring compounds that interact with the AhR. For example, several phytochemicals bind and/or modulate AhR action, and these include indole-3-carbinol (I3C) and diindolylmethane (DIM), various flavonoids, coumarins, alkaloids, carotenoids, and resveratrol (a polyhydroxylated antioxidant) (Bjeldanes et al., 1991Go; Nair et al., 1991Go; Gillner et al., 1989Go; Jellinck et al., 1993Go; Chen et al., 1996Go; Gradelet et al., 1997Go; Ciolino et al., 1998aGo,bGo; Casper et al., 1999Go; Ciolino and Yeh, 1999Go).

Research in this laboratory has identified several structural classes of compounds that exhibit AhR antagonist activities and some of these, including alternate substituted polychlorinated dibenzofurans (PCDFs) (Astroff et al., 1988Go; Astroff and Safe, 1989Go; Bannister et al., 1989Go; Harris et al., 1989Go; Yao and Safe, 1989Go), and DIM (Chen et al., 1996Go), are also selective AhR modulators (SAhRMs) that inhibit induced CYP1A1-dependent responses but exhibit both antiestrogenic and antitumorigenic activity in mammary cancer models (Safe, 1992Go; Safe et al., 1999Go; McDougal et al., 1997Go; Chen et al., 1998Go). Previous studies have demonstrated the AhR antagonist activities of substituted 3'-methoxyflavones (Lu et al., 1995Go; Gasiewicz et al., 1996Go; Reiners et al., 1998Go; Henry et al., 1999Go) in multiple cell lines including MCF-7 cells; however, these responses in the latter cell line only determined inhibition of CYP1A1-dependent activities. This study reports that 3',4'-dimethoxyflavone (3',4'-DMF) appears to be a pure AhR antagonist in both T47D and MCF-7 breast cancer cells and inhibits AhR-dependent CYP1A1 induction and AhR-mediated inhibition of estrogen-induced gene expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells, chemicals, biochemicals and plasmids.
TCDD was prepared by Safe in this laboratory (> 98% pure by chromatographic analysis). 3',4'-DMF was purchased from Lancaster Synthesis Inc. (Windham, NH) (97%) carefully stored in the dark to avoid photodecomposition. MCF-7 and T-47D human breast cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Ethoxyresorufin, 17ß-estradiol (E2), Dulbecco's modified Eagle's medium nutrient mixture F-12 Ham (DME F-12) without phenol red, {alpha}-minimum essential media ({alpha}MEM), phosphate-buffered saline (PBS), acetyl-CoA, and 100X antibiotic/antimycotic solution were purchased from Sigma Chemical Co. (St. Louis, MO). Minimum Essential Medium (MEM) was purchased from Life Technologies (Grand Island, NY). [{alpha}-32P]ATP (3000 Ci/mmol) and [14C]chloramphenicol (53 mCi/mmol) were purchased from NEN Research Products (Boston, MA). Poly[d(I-C)] and T4-polynucleotide kinase were purchased from Boehringer Mannheim (Indianapolis, IN). The wild-type double-stranded DRE oligonucleotide 5'-GATCTC CGGTCCTTCTCACGCAACGCCTGGGG-3' and mutant DRE 5'-GATCTCCGGTCC TTCTACATCAACGCCTGGGG-3' were synthesized by Gene Technologies Laboratory (College Station, TX). All other chemicals and biochemicals used in these studies were the highest quality available from commercial sources.

Cell growth.
MCF-7 cells were grown as monolayer cultures in MEM supplemented with 10% fetal bovine serum plus sodium bicarbonate (2.2 g/l), gentamycin (2.5 mg/l), penicillin/streptomycin (10,000 units/l and 10 mg/l), amphotericin B (1.25 mg/l), and 10 µg insulin. T47D cells were grown in {alpha}MEM supplemented with 2.2g/l sodium bicarbonate, 5% fetal bovine serum (FBS), and 10 ml antibiotic-antimycotic solution (Sigma). Cells were maintained in 150 cm2 culture flasks in an air:carbon dioxide (95:5) atmosphere at 37°C. The plasmid pRNH11c contains the regulatory human CYP1A1 region from the Taq I site at –1142 to the BclI site at +2434 fused to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene (kindly provided by Dr. R. Hines, University of Wisconsin at Milwaukee, Milwaukee, WI). The creatine kinase B construct (pCKB) contains the –2900 to +5 promoter insert linked to a CAT reporter gene and was provided by Dr. P. Benfield (Dupont-Merck Pharmaceutical Co., Wilmington, DE). The human ER (hER) expression plasmid was kindly provided by Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston, TX).

Ethoxyresorufin O-deethylase (EROD) activity.
EROD activity was determined essentially as described (Willett et al., 1997Go). Trypsinized cells were plated into 48-well tissue culture plates (2 x 105 cells/ml), allowed to attain 60% confluency, and treated with 1 nM TCDD, 0.1–10 µM 3',4'-DMF or their combination for 24 h. After 24 h, cells were washed by PBS; 185 µl PBS was added to each well, and cells were incubated in a 37°C water bath for 2 min. The reaction was started by adding 50 µl ethoxyresorufin (1 mg ethoxyresorufin/40 ml methanol) in a 37°C water bath for 13 min. After incubation for 13 min, the reaction was stopped by adding 100 µl fluorescamine. The 48-well tissue culture plate was scanned for fluorescence measurements using the cytofluorTM 2350 fluorescence measurement system and results for each treatment group were determined as means ± SE for at least three separate experiments.

Transient transfection assay.
Cells were seeded at ~60–70% confluency in 100-mm tissue culture dishes in DME-F12 without phenol red medium supplemented with 5% FBS treated with dextran-coated charcoal (DCC), 1.2 g/l sodium bicarbonate, and 10 ml/l antibiotic solution. After 24 h, cells were transiently cotransfected with 10 µg pCKB and 5 µg hER or 10 µg pRNH11c using the calcium phosphate method. After 6 h, cells were shocked with 25% DMSO in PBS. Cells were treated with chemicals dissolved in DMSO (0.1%) for 48 h, and DMSO served as a control. After 48 h, cells were washed twice with PBS and scraped from the plates. Cell lysates were prepared in 0.1 ml 0.25 M Tris-HCl, pH 7.8 by three freeze-thaw-sonication cycles. Protein concentrations were determined using bovine serum albumin (BSA) as a standard, and aliquots of cell lysate were incubated with 1 µl [14C]chloramphenicol (52 mCi/mol) and 42 µl of 4 mM acetyl-CoA at 37°C. Ethyl acetate was then added; the extract was dried and redissolved in 20 µl ethyl acetate, and metabolites were separated by thin-layer chromatography (TLC) in 95:5 chloroform:methanol. Following TLC, acetylated products were visualized and quantitated using Packard Instant Imager (Meridian, CT). CAT activity was calculated as the percentage of that observed in cells treated with DMSO alone (arbitrarily set at 100%).

Cell proliferation assay.
Cells were seeded at 7.5 x 104 cells/well in 6-well plates in media containing 2 ml DME/F12 without phenol red, supplemented with 5% FBS treated with dextran-coated charcoal (FBS-DCC). After 24 h, the media were changed (5% FBS-DCC) and cells were treated with E2, TCDD, 3',4'-DMF or their combinations for 11 days. The media were changed and cells were treated with the same chemicals every 48 h. Cells were then trypsinized, harvested, and counted using Coulter Z1 cell counter (Beckman Coulter, Brea, CA).

Preparation of cytosolic and nuclear extracts.
Cytosol from male Sprague-Dawley rat liver was essentially prepared as described (Liu et al., 1993Go). Cells grown in 100-mm Petri dishes and treated with DMSO and the test compounds (3',4'-DMF or TCDD) dissolved in DMSO were harvested and washed twice in 5 ml HE buffer (25 mM HEPES, 1.5 mM EDTA; pH 7.6). Then 0.5 ml HEGD (25 mM HEPES, 1.5 mM EDTA 10% glycerol, 1.0 mM dithiothreitol; pH 7.6) buffer was added to each plate and cells were scraped and incubated on ice, then processed with a Dounce homogenizer. Homogenates were centrifuged at 12,000 x g for 5 min. Supernatants were discarded, and the pelleted fractions were resuspended in 0.1 ml of HEGD buffer containing 0.5 M potassium chloride (pH 7.6), allowed to stand for 30 min to 1 h at 4°C, and nuclear extracts were prepared by centrifuging at 12,000 x g for 10 min at 4°C. The supernatants representing nuclear extracts were collected and stored in –80°C until used. The protein concentrations were determined using BSA as a standard. Nuclei prepared by this method were found to be intact and were greater than 90% free of extranuclear cellular contamination, as determined by microscopic examination and trypan-blue staining.

Gel electrophoretic mobility shift assay.
Nine picomoles of synthetic human DRE oligonucleotide was labeled at the 5' end using T4-polynucleotide kinase and [{alpha}-32P] ATP. Nuclear extracts from MCF-7 (5 µg) or T47D (2 µg) cells treated with DMSO (control), 5 nM TCDD, 5 µM 3',4'-DMF alone or in combination were incubated in HEGD buffer with poly[d(I-C)] for 15 min at 20°C. The mixture was incubated for an additional 15 min (20°C) after the addition of [32P]-labeled DNA. Reaction mixtures were loaded into a 5% polyacrylamide gel (acrylamide:bisacrylamide, 30:0.8) and electrophoresed at 110 V in 0.9 M Tris borate and 2 mM EDTA, pH 8.0, and analyzed as described below for the transformed cytosolic AhR-DRE complex. Rat liver cytosol was incubated with different concentrations of 3',4'-DMF alone or in combination with TCDD at 20°C for 2 h. Cytosol (80 µg) in HEGDK buffer [HEDG + 0.4 M potassium chloride) with 1 µg of poly[d(I-C)] was further incubated for 15 min at 20°C. A 100-fold excess of unlabeled wild-type and mutant DRE oligonucleotides was added for the competition experiments and incubated at 20°C for 5 min. Following addition of [32P]-labeled DNA, the mixture was incubated for an additional 15 min at 20°C. Protein-DNA complexes were resolved on a 5–6% polyacrylamide gel (acrylamide:bisacrylamide ration, 30:0.8) and run in 1X TBE buffer (0.9 M Tris, 0.09 boric acid, 2 mM EDTA, pH 3.8) at 110 V. Bound complexes were visualized by autoradiography and quantitated by densitometry using the Molecular Dynamics Zero-D software package (Sunnyvale, CA) and Sharp JX-330 scanner (Sharp, Mahwah, NJ), and subjected to autoradiography using a Kodak X-Omat film (Eastman Kodak, Rochester, NY) for appropriate times at –80°C.

Statistical analysis.
Statistical differences between different treatment groups were determined using Student's t test or ANOVA (Scheffe's), and the levels of significance were noted (p < 0.05). The results were expressed as means ± SE for at least three replicate determinations for each experiment.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
3'4'-DMF (0.1–1.0 µM) alone did not significantly induce EROD activity in MCF-7 or T47D breast cancer cells, whereas the AhR agonist TCDD was a potent inducer in both cell lines (Fig. 1Go). In cells cotreated with 1 nM TCDD by 0.1–10 µM 3'4'-DMF, there was significant inhibition of induced EROD activity by 3',4'-DMF at the 1.0-µM and 10-µM doses, and almost complete inhibition was observed at the highest concentration of 3',4'-DMF. To ensure that this inhibitory response was not due just to direct interactions of 3',4'-DMF with CYP1A1 protein, the effects of 3',4'-DMF on TCDD-induced CAT activity in MCF-7 or T47D cells transfected with Ah-responsive pRNH11c were also investigated. The results (Fig. 2Go) show that 3',4'-DMF alone did not exhibit AhR agonist activity, but 3',4'-DMF significantly inhibited TCDD-induced CAT activity in MCF-7 and T47D cells cotreated with 3',4'-DMF + TCDD.



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FIG. 1. Induction of EROD activity by TCDD and inhibition by 3',4'-DMF. MCF-7 (A) or T47D (B) cells were treated with 1 nM TCDD, 0.1–10 µM 3',4'-DMF alone, or 1 nM TCDD plus 3',4'-DMF (0.1–10 µM), and EROD activity was determined. TCDD alone significantly (p < 0.05) induced EROD, and 1.0 and 10 µM 3',4'-DMF significantly (*p < 0.05) inhibited this induced response in both cell lines. The results are means ± SE for three replicate experiments for each treatment group.

 


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FIG. 2. Induction of CAT activity by TCDD and inhibition by 3',4'-DMF. MCF-7 (A) or T47D (B) cells were transiently transfected with pRNH11c and treated with TCDD, 3',4'-DMF or their combination; CAT activity was determined. TCDD alone significantly (p < 0.05) induced CAT activity, and cotreatment with 10 µM 3',4'-DMF significantly (*p < 0.05) inhibited induction of CAT activity by TCDD in both cell lines. The results are means ± SE for three replicate experiments for each treatment group.

 
Previous studies with AhR agonists that inhibit induction of CYP1A1 by TCDD have shown that some of these compounds such as alkyl-substituted PCDFs and I3C/DIM exhibit AhR agonist activity in breast cancer cell lines and inhibit E2-induced growth and transactivation (Safe et al., 1999Go; Chen et al., 1996Go, 1998Go). Therefore, we initially investigated the growth inhibitory effects of 3',4'-DMF alone or in combination with E2 in MCF-7 and T47D breast cancer cells. 3',4'-DMF alone significantly induced T47D cell proliferation (0.1 and 1.0 µM) and appeared to exhibit weak estrogen or growth-stimulatory activity in both cell lines; however, this response was not observed at the highest concentration (10 µM) (Fig. 3Go). In cells treated with 3',4'-DMF plus E2, proliferation was not enhanced by 3',4'-DMF, and 10 µM 3',4'-DMF inhibited E2-induced proliferation of both MCF-7 and T47D cells. Previous studies have also reported growth inhibitory effects of 3'-methoxy–substituted flavones (Reiners et al., 1998Go), and this may be related to their inhibition of constitutive and hormone-induced kinase activities (Cunningham et al., 1992Go). 3',4'-DMF did not reverse the inhibition of E2-induced proliferation by TCDD (data not shown) and this is probably related to the dose-dependent stimulatory and inhibitory effects of this compound. Therefore, we also investigated the AhR antagonist activity of 3',4'-DMF in MCF-7 cells transfected with the E2-responsive pCKB construct and treated with E2, TCDD, or their combination. The results (Fig. 4Go) show that E2 induced CAT activity in MCF-7 cells transfected with pCKB, whereas 10 µM 3',4'-DMF or 10 nM TCDD alone were inactive. TCDD, but not 3',4'-DMF, significantly inhibited E2-induced activity, and 3',4'-DMF reversed the inhibition of E2-induced activity by TCDD, which is consistent with an AhR antagonist effect by 3',4'-DMF. E2-induced activation of pCKB is not inhibited by AhR agonists in T47D cells (Ramamoorthy et al., 1999Go), and therefore, the AhR antagonist activity of 3',4'-DMF was not determined for this response in T47D cells.



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FIG. 3. Effects of 3',4'-DMF on growth of breast cancer cells. MCF-7 (A) or T47D (B) cells were treated with different concentrations of 3',4'-DMF alone or in combination with 1 nM E2. Significant induction of T47D cell growth (p < 0.05)a was observed only at concentrations of 0.1 and 1.0 µM 3',4'-DMF and significant inhibition (p < 0.05)b of E2-induced growth of both cell lines was observed only at the highest concentration (10 µM) of 3',4'-DMF. The results are means ± SE for three replicate experiments for each treatment group.

 


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FIG. 4. Interactive effects of 3',4'-DMF and TCDD on the induction of pCKB by E2. MCF-7 cells were transfected with pCKB, treated with E2, TCDD, 3',4'-DMF, or their combinations, and CAT activity was determined. E2 significantly induced CAT activitya; this response was inhibited by TCDDb and the inhibitory response was reversedc after cotreatment with 3',4'-DMF. The results are means ± SE for three replicate experiments for each treatment group.

 
Thus, 3',4'-DMF blocks TCDD-induced CYP1A1 and inhibitory AhR-ER interactions in breast cancer cells, and the results in Figure 5Go illustrate the effects of TCDD, 3',4'-DMF, and their combination on the formation of a nuclear AhR complex in MCF-7 and T47D cells. Nuclear extracts from MCF-7 or T47D cells treated with DMSO gave weak to nondetectable binding to [32P]DRE in a gel mobility shift assay. In contrast, an intense band was observed in extracts from cells treated with TCDD; this band was competitively decreased after competition with a 100-fold excess unlabeled wild-type DRE but was unaffected by competition with a mutant DRE oligonucleotide. In contrast, incubation of [32P]DRE with nuclear extracts from MCF-7 or T47D cells treated with 5 nM 3',4'-DMF alone or in combination with TCDD gave minimal to nondetectable retarded bands, demonstrating that 3',4'-DMF blocked TCDD-induced formation of the nuclear AhR complex in breast cancer cell lines. Previous studies show that TCDD induced transformation and DRE binding of rat hepatic cytosolic AhR, as illustrated in Figure 6Go. In contrast, 0.5–50 µM 3',4'-DMF did not transform the rat cytosolic AhR; however, in cytosols cotreated with TCDD plus 3',4'-DMF, there was significant inhibition of TCDD-induced transformation. These results suggest the 3',4'-DMF competitively binds the cytosolic AhR complex and blocks formation of the transformed nuclear AhR complex, and these results are consistent with previous reports on other 3'-methoxy-substituted flavones (Lu et al., 1995Go; Gasiewicz et al., 1996Go; Reiners et al., 1998Go; Henry et al., 1999Go).



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FIG. 5. Effects of 3',4'-DMF on formation of the nuclear AhR complex. MCF-7 (A) or T47D (B) breast cancer cells were treated with DMSO, TCDD, 3',4'-DMF or TCDD plus 3',4'-DMF, and nuclear extracts were isolated and analyzed by gel mobility shift assays. TCDD induced a specifically bound complex (see arrow) in both cell lines, whereas nuclear extracts from cells treated with 3',4'-DMF alone or in combination with TCDD gave a minimal to nondetectable bound complex. The mobility of the specifically bound band was comparable to that observed using in vitro translated AhR/Arnt incubated with [32P]DRE (data not shown).

 


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FIG. 6. Transformation of rat hepatic cytosolic AhR. Rat hepatic cytosol was treated with 5 nM TCDD, 3',4'-DMF (0.05–50 µM) or their combination and analyzed by gel mobility shift assays. TCDD but not 3',4'-DMF transformed the AhR complex, and coincubation of TCDD with 3',4'-DMF resulted in inhibition of TCDD-induced transformation.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The AhR is a ligand-activated transcription factor that binds structurally diverse chemicals that include highly toxic halogenated aromatics (Denison et al., 1998Go; Seidel et al., 2000Go) and chemoprotective phytochemicals such as I3C, DIM, and bioflavonoids (Ciolino et al., 1998bGo; Bjeldanes et al., 1991Go; Chen et al., 1996Go). Research in this laboratory identified a series of alternate-substituted (1,3,6,8- or 2,4,6,8-) alkyl PCDFs typified by the AhR antagonist 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) that inhibited TCDD-induced CYP1A1, porphyria, immunotoxicity, and cleft palate formation in mice (Bannister et al., 1989Go; Harris et al., 1989Go; Yao and Safe, 1989Go). Subsequent studies showed that MCDF was an AhR agonist and inhibited E2-induced responses in the rodent uterus and breast cancer cells and mammary tumor growth in carcinogen-induced female Sprague-Dawley rats (Safe, 1992Go; Zacharewski et al., 1992Go; Dickerson et al., 1995Go; McDougal et al., 1997Go; Sun and Safe, 1997Go). It has also been reported that synthetic and natural flavonoids exhibit AhR antagonist activity and some of the most effective AhR antagonists are substituted 3'-methoxyflavones (Lu et al., 1995Go, 1996Go; Gasiewicz et al., 1996Go; Henry et al., 1999Go; Reiners et al., 1998Go).

3'-Methoxy-4'-nitro- and 4'-amino-3'-methoxyflavone have been extensively characterized as AhR antagonists that act by inhibiting formation of the nuclear AhR complex (Lu et al., 1995Go, 1996Go; Gasiewicz et al., 1996Go; Henry et al., 1999Go), and similar results have been observed for 2'-amino-3'-methoxyflavone (Reiners et al., 1998Go, 1999Go). However, many of these substituted flavones are also protein tyrosine kinase inhibitors and cytotoxic at doses > 10 µM (Reiners et al., 1998Go, 1999Go; Cunningham et al., 1992Go). Henry and coworkers (1999) recently investigated a series of 3'-methoxy-4'-substituted flavones and showed that the most active AhR antagonists contained 4' substituents with high electron density (nitro, azido, and thiocyanate). It was hypothesized that this structural feature facilitated critical hydrogen bonding with the AhR. In contrast, 3'-methoxy-4'-aminoflavone was less active as an AhR antagonist (Henry et al., 1999Go) than previously observed in this laboratory in breast cancer cell lines (Lu et al., 1995Go), and this may be due, in part, to cell context. We also observed that although 4'-methoxyflavone did not inhibit induction of CYP1A1 by TCDD, this compound blocked TCDD-induced transformation of the rat cytosolic AhR (Lu et al., 1996Go), and we therefore hypothesized that 3',4'-DMF, which contains two vicinal methoxy groups, may be an effective AhR antagonist in breast cancer cell lines despite the lack of a 4'-substituent with high electron density.

Most previous studies have characterized the activity of AhR antagonists by determining their effects on CYP1A1-dependent (gene/promoter) activities and their interactions with the cytosolic and nuclear AhR complex. Results obtained for 3',4'-DMF in breast cancer cells are similar to those previously reported for other 3'-methoxyflavones in different cell lines, indicating that inhibition of TCDD-induced CYP1A1 or related activities is due to competitive interaction of the ligand with the cytosolic AhR that does not undergo transformation or nuclear translocation (Fig. 5Go). 3',4'-DMF did not block inhibition of E2-induced proliferation by TCDD (data not shown) and this may be related to the growth inhibitory effects of 3',4'-DMF alone (Fig. 2Go), which could be due to partial ER antagonist activities or inhibition of protein kinase–dependent pathways (Cunningham et al., 1992Go; Reiners et al., 1998Go). However, in transcriptional activation assays using the E2-responsive pCKB construct, 3',4'-DMF exhibited AhR antagonist activity and reversed the antiestrogenic effects of TCDD in this assay (Fig. 4Go). Thus, 3',4'-DMF does not resemble MCDF, which acts as an AhR agonist for this response (Safe et al., 1999Go), but resembles {alpha}-naphthoflavone, which inhibits AhR-mediated CYP1A1 induction (Merchant et al., 1990Go; Gasiewicz and Rucci, 1991Go) and inhibition of E2-induced transactivation in breast cancer cells (Merchant et al., 1993Go). The major advantage of 3',4'-DMF over {alpha}-naphthoflavone is that the latter compound is an AhR antagonist at concentrations < 1 µM, whereas at higher concentrations (1–10 µM), {alpha}-naphthoflavone is an AhR agonist (Santostefano et al., 1993Go). Results obtained in this study demonstrate that 3',4'-DMF inhibits both AhR-mediated CYP1A1 induction and antiestrogenic activity in breast cancer cell lines by blocking transformation of the cytosolic AhR complex and formation of the nuclear AhR complex, and this is consistent with results of previous studies with other 3'-methoxy-substituted flavones (Reiners et al., 1998Go, 1999Go; Lu et al., 1995Go, 1996Go; Gasiewicz et al., 1996Go; Henry et al., 1999Go). Previous studies demonstrated that 3'-methoxyflavone exhibited minimal activity as an inhibitor of TCDD-induced transformation of rat hepatic cytosolic receptor, whereas introduction of high electron density 4' substituents gave compounds that inhibited TCDD-induced transformation and transcription in mouse liver cells (Henry et al., 1999Go). In contrast, 3'-methoxyflavones that contain 4'-methoxy (this study) and 4'-amino groups (Lu et al., 1995Go) that do not have high electron densities are also relatively potent AhR antagonists in breast cancer cells, suggesting that the AhR antagonist activity of these substituted flavones may be influenced by cell context. An important advantage in using 3',4'-DMF is the commercial availability of this compound.


    ACKNOWLEDGMENTS
 
The financial assistance of the National Institutes of Health (ES09106 and ES04176) and the Texas Agricultural Experiment Station is gratefully acknowledged. S. Safe is a Sid Kyle Professor of Toxicology.


    NOTES
 
1 To whom correspondence should be sent. Fax: (409) 862-4929. E-mail: ssafe{at}cvm.tamu.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Astroff, B. and Safe, S. (1989). 6-Substituted-1,3,8-trichlorodibenzofurans as 2,3,7,8- tetrachlorodibenzo-p-dioxin antagonists in the rat: structure-activity relationships. Toxicology 59, 285–296.[ISI][Medline]

Astroff, B., Zacharewski, T., Safe, S., Arlotto, M. P., Parkinson, A., Thomas, P., and Levin, W. (1988). 6-Methyl-1,3,8-trichlorodibenzofuran as a 2,3,7,8- tetrachlorodibenzo-p-dioxin antagonist: inhibition of the induction of rat cytochrome P-450 isozymes and related monooxygenase activities. Mol. Pharmacol. 33, 231–236.[Abstract]

Bannister, R., Biegel, L., Davis, D., Astroff, B., and Safe, S. (1989). 6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8- tetrachlorodibenzo-p-dioxin antagonist in C57BL/6 mice. Toxicology 54, 139–150.[ISI][Medline]

Beato, M., Herrlich, P., and Schutz, G. (1995). Steroid hormone receptors: many actors in search of a plot. Cell 83, 851–857.[ISI][Medline]

Bjeldanes, L. F., Kim, J. Y., Grose, K. R., Bartholomew, J. C., and Bradfield, C. A. (1991). Aromatic hydrocarbon responsiveness-receptor agonists generated from indole-3-carbinol in vitro and in vivo – comparisons with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Proc. Natl. Acad. Sci. U. S. A. 88, 9543–9547.[Abstract]

Casper, R. F., Quesne, M., Rogers, I. M., Shirota, T., Jolivet, A., Milgrom, E., and Savouret, J. F. (1999). Resveratrol has antagonist activity on the aryl hydrocarbon receptor: implications for prevention of dioxin toxicity. Mol. Pharmacol. 56, 784–790.[Abstract/Free Full Text]

Chen, I., McDougal, A., Wang, F., and Safe, S. (1998). Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane. Carcinogenesis 19, 1631–1639.[Abstract]

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Dickerson, R., Keller, L. H., and Safe, S. (1995). Alkyl polychlorinated dibenzofurans and related compounds as antiestrogens in the female rat uterus: structure-activity studies. Toxicol. Appl. Pharmacol. 135, 287–298.[ISI][Medline]

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Goldstein, J. A., and Safe, S. (1989). Mechanism of action and structure-activity relationships for the chlorinated dibenzo-p-dioxins and related compounds. In Halogenated Biphenyls, Naphthalenes, Dibenzodioxins and Related Compounds, (R. D. Kimbrough and A. A. Jensen, Eds.). pp. 239–293. Elsevier-North Holland, Amsterdam.

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Harris, M., Zacharewski, T., Astroff, B., and Safe, S. (1989). Partial antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin- mediated induction of aryl hydrocarbon hydroxylase by 6-methyl-1,3,8-trichlorodibenzofuran: mechanistic studies. Mol. Pharmacol. 35, 729–735.[Abstract]

Henry, E. C., Kende, A. S., Rucci, G., Totleben, M. J., Willey, J. J., Dertinger, S. D., Pollenz, R. S., Jones, J. P., and Gasiewicz, T. A. (1999). Flavone antagonists bind competitively with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) to the aryl hydrocarbon receptor but inhibit nuclear uptake and transformation. Mol. Pharmacol. 55, 716–725.[Abstract/Free Full Text]

Jellinck, P. H., Forkert, P. G., Riddick, D. S., Okey, A. B., Michnovicz, J. J., and Bradlow, H. L. (1993). Ah receptor binding properties of indole carbinols and induction of hepatic estradiol hydroxylation. Biochem. Pharmacol. 43, 1129–1136.

Liu, H., Santostefano, M., Lu, Y., and Safe, S. (1993). 6-Substituted 3,4-benzocoumarins: a new structural class of inducers and inhibitors of CYP1A1-dependent activity. Arch. Biochem. Biophys. 306, 223– 231.[ISI][Medline]

Lu, Y. F., Santostefano, M., Cunningham, B. D. M., Threadgill, M. D., and Safe, S. (1996). Substituted flavones: aryl hydrocarbon (Ah) receptor agonists and antagonists. Biochem. Pharmacol. 51, 1077–1087.[ISI][Medline]

Lu, Y. F., Santostefano, M., Cunningham, B. D., Threadgill, M. D., and Safe, S. (1995). 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. 316, 470–477.[ISI][Medline]

Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., and Evans, R. M. (1995). The nuclear receptor superfamily: the second decade. Cell 83, 835–839.[ISI][Medline]

McDougal, A., Wilson, C., and Safe, S. (1997). Inhibition of 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor growth by aryl hydrocarbon receptor agonists. Cancer Lett. 120, 53–63.[ISI][Medline]

Merchant, M., Arellano, L., and Safe, S. (1990). The mechanism of action of {alpha}-naphthoflavone as an inhibitor of 2, 3,7,8-tetrachlorodibenzo-p-dioxin induced CYP1A1 gene expression. Arch. Biochem. Biophys. 281, 84–89.[ISI][Medline]

Merchant, M., Krishnan, V., and Safe, S. (1993). Mechanism of action of {alpha}-naphthoflavone as an Ah receptor antagonist in MCF-7 human breast cancer cells. Toxicol. Appl. Pharmacol. 120, 179–185.[ISI][Medline]

Nair, R. V., Fisher, E. P., Safe, S., Cortez, C., Harvey, R. G., and DiGiovanni, J. (1991). Novel coumarins as potential anticarcinogenic agents. Carcinogenesis 12, 65–69.[Abstract]

Perlmann, T., and Evans, R. M. (1997). Nuclear receptors in Sicily: all in the famiglia. Cancer Res. Cell 90, 391–397.

Poland, A., and Knutson, J. C. (1982). 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons. Examinations of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. 22, 517–554.[ISI][Medline]

Ramamoorthy, K., Gupta, M. S., Sun, G., McDougal, A., and Safe, S. H. (1999). 3,3',4,,4'-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary and in human breast cancer cells. Carcinogenesis 20, 115–123.[Abstract/Free Full Text]

Reiners, J. J., Jr., Clift, R., and Mathieu, P. (1999). Suppression of cell cycle progression by flavonoids: dependence on the aryl hydrocarbon receptor. Carcinogenesis 20, 1561–1566.[Abstract/Free Full Text]

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Safe, S., Qin, C., and McDougal, A. (1999). Development of selective aryl hydrocarbon receptor modulators (SARMs) for treatment of breast cancer. Expert Opin. Invest. Drugs 8, 1385–1396.

Safe, S. (1992). MCDF, 6-methyl-1,3,8-trichlorodibenzofuran. Drugs Future 17, 564–565.

Santostefano, M., Merchant, M., Arellano, L., Morrison, V., Denison, M. S., and Safe, S. (1993). {alpha}-Naphthoflavone-induced CYP1A1 gene expression and cytosolic Ah receptor transformation. Mol. Pharmacol. 43, 200–206.[Abstract]

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Willett, K. L., Gardinali, P. R., Serican, J. L., Wade, T. L., and Safe, S. (1997). Characterization of the rat H4IIE bioassay for evaluation of environmental samples containing polynuclear aromatic hydrocarbons (PAHs). Arch. Environ. Contam. Toxicol. 32, 442–448.[ISI][Medline]

Wilson, C. L., and Safe, S. (1998). Mechanisms of ligand-induced aryl hydrocarbon receptor-mediated biochemical and toxic responses. Toxicol. Pathol. 26, 657–671.[ISI][Medline]

Yao, C., and Safe, S. (1989). 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced porphyria in genetically inbred mice: partial antagonism and mechanistic studies. Toxicol. Appl. Pharmacol. 100, 208–216.[ISI][Medline]

Zacharewski, T., Harris, M., Biegel, L., Morrison, V., Merchant, M., and Safe, S. (1992). 6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) as an antiestrogen in human and rodent cancer cell lines: evidence for the role of the Ah receptor. Toxicol. Appl. Pharmacol. 13, 311–318.