Department of Veterinary Physiology & Pharmacology, Texas A&M University, 4466 TAMU, College Station, Texas 77843
Received June 13, 2000; accepted August 9, 2000
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ABSTRACT |
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Key Words: AhR; antagonist; breast cancer cells; 3',4'-dimethoxyflavone.
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INTRODUCTION |
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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., 1991; Nair et al., 1991
; Gillner et al., 1989
; Jellinck et al., 1993
; Chen et al., 1996
; Gradelet et al., 1997
; Ciolino et al., 1998a
,b
; Casper et al., 1999
; Ciolino and Yeh, 1999
).
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., 1988; Astroff and Safe, 1989
; Bannister et al., 1989
; Harris et al., 1989
; Yao and Safe, 1989
), and DIM (Chen et al., 1996
), are also selective AhR modulators (SAhRMs) that inhibit induced CYP1A1-dependent responses but exhibit both antiestrogenic and antitumorigenic activity in mammary cancer models (Safe, 1992
; Safe et al., 1999
; McDougal et al., 1997
; Chen et al., 1998
). Previous studies have demonstrated the AhR antagonist activities of substituted 3'-methoxyflavones (Lu et al., 1995
; Gasiewicz et al., 1996
; Reiners et al., 1998
; Henry et al., 1999
) 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.
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MATERIALS AND METHODS |
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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 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., 1997). 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, 0.110 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 6070% 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., 1993). 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 [-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 56% 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.
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RESULTS |
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DISCUSSION |
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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., 1995, 1996
; Gasiewicz et al., 1996
; Henry et al., 1999
), and similar results have been observed for 2'-amino-3'-methoxyflavone (Reiners et al., 1998
, 1999
). However, many of these substituted flavones are also protein tyrosine kinase inhibitors and cytotoxic at doses > 10 µM (Reiners et al., 1998
, 1999
; Cunningham et al., 1992
). 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., 1999
) than previously observed in this laboratory in breast cancer cell lines (Lu et al., 1995
), 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., 1996
), 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. 5). 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. 2
), which could be due to partial ER antagonist activities or inhibition of protein kinasedependent pathways (Cunningham et al., 1992
; Reiners et al., 1998
). 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. 4
). Thus, 3',4'-DMF does not resemble MCDF, which acts as an AhR agonist for this response (Safe et al., 1999
), but resembles
-naphthoflavone, which inhibits AhR-mediated CYP1A1 induction (Merchant et al., 1990
; Gasiewicz and Rucci, 1991
) and inhibition of E2-induced transactivation in breast cancer cells (Merchant et al., 1993
). The major advantage of 3',4'-DMF over
-naphthoflavone is that the latter compound is an AhR antagonist at concentrations < 1 µM, whereas at higher concentrations (110 µM),
-naphthoflavone is an AhR agonist (Santostefano et al., 1993
). 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., 1998
, 1999
; Lu et al., 1995
, 1996
; Gasiewicz et al., 1996
; Henry et al., 1999
). 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., 1999
). In contrast, 3'-methoxyflavones that contain 4'-methoxy (this study) and 4'-amino groups (Lu et al., 1995
) 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.
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ACKNOWLEDGMENTS |
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NOTES |
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