Genistein action in the prepubertal mammary gland in a chemoprevention model
Michelle S. Cotroneo1,
Jun Wang1,
Wayne A. Fritz1,
Isam-Eldin Eltoum2,3 and
Coral A. Lamartiniere1,2,4
1 Department of Pharmacology and Toxicology,
2 Comprehensive Cancer Center, University of Alabama at Birmingham, 1670 University Blvd, Birmingham, AL 35294, USA and
3 Department of Pathology, University of Alabama at Birmingham, 1670 University Blvd, Birmingham, AL 35294, USA
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Abstract
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A diet high in soy is associated with many health benefits, including reduced incidence of breast cancer. The soy phytoestrogen, genistein, is hypothesized to contribute to mammary chemoprevention via interaction with estrogen receptors (ERs) alpha and/or beta. These steroid signaling pathways are believed to exert control over proliferation and differentiation of the mammary gland by a complex bidirectional interaction with the epidermal growth factor (EGF) signaling pathway. The current work was designed to study the role of these two pathways in prepubertal mammary gland growth. Female SpragueDawley CD rats were injected with genistein (500 µg/g body wt) or estradiol benzoate (EB) (500 ng/g body wt) on days 16, 18 and 20. Whole mount analysis of mammary glands from 21-day-old rats showed that both treatments resulted in significantly increased terminal end buds (TEBs), and increased ductal branching, compared with animals given the vehicle, dimethylsulfoxide (DMSO). Both effects were inhibited by blockage of ER function by pre-treating with 2 mg ICI 182,780/kg body wt, a steroidal anti-estrogen. Immunoblotting analyis of mammary gland extracts demonstrated increased epidermal growth factor receptor (EGFR) and progesterone receptor (PR) expression following treatment with EB or genistein. Tyrosine-phosphorylated EGFR, as measured by immunoprecipitation/immunoblotting was also increased, but when normalized to total receptors, there was no net effect. The expression and phosphorylation of downstream targets of the EGFR, mitogen activating kinase kinase (MEK 1 and 2) and extracellular signal regulated kinases 1 and 2 (ERK 1 and 2) were not significantly affected. Anti-estrogen pre-treatment prevented the increase in EGFR, phospho-EGFR and PR. The data indicate an ER-based mechanism of action for genistein in mammary gland proliferation and differentiation, which can lead to protection against mammary cancer.
Abbreviations: DMSO, dimethylsulfoxide; EB, estradiol benzoate; EGF, epidermal growth factor; ERK, extracellular signal regulated kinases; ERs, estrogen receptors; MEK, mitogen activating kinase kinase; PR, progesterone receptor; TEB, terminal end buds.
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Introduction
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Asian women have a lower incidence of breast cancer than Western women (1,2). A major factor, which is thought to contribute to this protection is the Asian diet, which is rich in soy products. Of the components of soy, the phytoestrogen, genistein, is the most studied. Our laboratory has shown that exposure of female rats to genistein either by injection (3) or via the diet (4) prior to puberty resulted in protection from dimethylbenz[a]anthracene-induced mammary carcinogenesis. The protection was derived from an early proliferative phase of mammary gland growth during the prepubertal period, resulting in an increase in TEBs (3), the most immature terminal ductal structures (5,6). The mitogenic effects of genistein treatment led to differentiation of the terminal end buds (TEBs) into lobules later in life (3); the latter are the most resistant terminal ductal structures to chemical carcinogenesis, due to their high degree of differentiation (5,6). The chemopreventative effect of accelerated glandular differentiation by genistein is similar to the protection derived from pregnancy early in life in humans (7), whereby hormonal exposure results in glandular maturity.
Of the multitude of biological effects identified for genistein, an important molecular basis for its action is binding to estrogen receptors (ER) alpha and ER beta (reviewed in ref. 8), a property that is conferred by its structural similarity to estradiol 17-beta. Another important action of genistein, which may contribute to its chemopreventative properties, is inhibition of the epidermal growth factor receptor (EGFR) tyrosine kinase (9). The EGF signaling cascade is important in the development of the rodent mammary gland (reviewed in ref. 10). Previous work using a chemoprevention protocol indicated that the expression of the EGFR and its ligand, TGF-alpha, were initially increased in the terminal ductal structures of the mammary glands of genistein-treated rats (11).
In ER-positive mammary cancer cells, treatment with estradiol induced synthesis of the EGFR mRNA and protein, an effect that was blocked in the presence of an anti-estrogen (12). In mice, treatment with estradiol increased tyrosine-phosphorylated EGFR in the mammary gland (13), suggesting a link between ERs and EGF signaling. Genistein was hypothesized to alter the EGFR tyrosine kinase activity by direct binding in multiple sites of the reaction (9), making it possible for growth regulation through non-ER-dependent mechanisms. Therefore, it is possible that the mechanism of action for genistein and estrogen in the growth of the pre-pubertal mammary gland may differ. The aim of the present research was to compare the mechanism of action of genistein and estradiol benzoate (EB) and to determine the role of ERs in the regulation of EGFR expression and mammary gland growth, which leads to differentiation.
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Materials and methods
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Animal treatment
All animal studies were conducted in accordance with the UAB Guidelines for Animal Use and Care. Animals were housed in a temperature-controlled facility with a 12 h on/off light cycle. Female SpragueDawley rats (Charles River, Raleigh, NC) were fed standard laboratory chow LM485 (Harlan Teklad, Madison, WI) and bred in our facility. Litters were reduced to 11 offspring/dam at parturition; at this time standard lab chow was replaced with AIN-76A pellets (Harlan Teklad), which are devoid of phytoestrogens. Female offspring (ovary-intact) were assigned to the following groups: vehicle (dimethylsulfoxide) (DMSO) (Sigma, St Louis, MO); genistein (98.5% purity; Hoffmann-LaRoche, Basel, Switzerland); EB (Sigma); ICI 182,780 (Zeneca Pharmaceuticals, Cheshire, UK); genistein with ICI 182,780 pre-treatment; and EB with ICI 182,780 pre-treatment. On postnatal days 16, 18 and 20, female offspring were injected s.c. with 500 µg genistein/g body wt, 500 ng EB/g body wt or an equivalent volume of the vehicle. The treatment protocol for genistein is one that resulted in significant chemoprevention of chemically induced mammary tumors (3). An additional study using this protocol was done on prepubertally ovariectomized rats. On postnatal day 15, female rats were anesthetized using ketamine (10 mg/100 g)/xylazine (1.5 mg/100 g) and bilaterally ovariectomized. Injections of genistein, EB and vehicle were given on days 16, 18 and 20; rats were killed on day 21.
For animals with ICI 182,780 pre-treatment, 2.0 mg ICI 182,780/kg body wt was injected 30 min prior to genistein or EB treatments. Animals not receiving ICI 182,780 pre-treatment were injected with the vehicle. An additional dose of ICI 182,780 or vehicle was given on days 17 and 19. Rats were killed on postnatal day 21, 1820 h after the last injection.
Evaluation of apoptosis
To ensure that the selected ICI 182,780 dose was growth-inhibitory without excessively triggering cell death, mammary glands from ovary-intact vehicle-treated and ICI 182,780-treated rats were analyzed for the presence of apoptotic cells, using six animals per group. The fourth abdominal mammary glands from vehicle- and anti-estrogen-treated animals were fixed overnight in 10% neutral-buffered formalin and embedded in paraffin blocks. Sections were placed onto glass slides and placed in xylene to remove paraffin, re-hydrated in a series of alcohols with increasing water content and immersed in Tris-buffered saline. DNA strand breakage was detected by labeling of the 3'-OH ends with biotinylated deoxynucleotides, catalyzed by terminal deoxynucleotidyl transferase (TdT-FragEL, Oncogene Research Products, Boston, MA), according to the manufacturers instructions. Apoptotic nuclei were stained brown with 3,3'-diaminobenzidine and normal cell nuclei stained with methyl green counter stain. A positive control slide was treated with DNase (Promega, Madison, WI); negative control tissue received no TdT enzyme. Apoptotic and normal nuclei were enumerated in all epithelial cells using light microscopy; the number of apoptotic nuclei per section was expressed as a percentage of total cells. The median percentage of apoptotic nuclei for the two groups was compared using MannWhitney Rank Sum test (Sigma Stat., Jandel Scientific, San Rafael, CA).
Expression and localization of ER alpha by immunohistochemistry
Mammary glands of ovary-intact animals treated with genistein, EB or vehicle were analyzed for ER alpha expression. Because of the low expression of ER alpha, and its predominant epithelial expression in the rat mammary gland (14), immunohistochemistry (IHC) analyses were performed instead of immunoblotting. Also, this allowed for the analysis of cell-specific differences in expression. Fourth abdominal mammary glands were fixed in 10% neutral-buffered formalin overnight and embedded in paraffin blocks. Paraffin sections were cut with a microtome (5 µ thick) and placed on glass slides. Sections were stained for ER alpha with an antibody to the N-terminus (Zymed, San Francisco, CA) by an automated immunohistochemistry system (Ventanna Medical Systems, Tuscon, AZ), as described previously (15). Uterine and spleen tissue were used as positive and negative controls, respectively. A section of mammary gland from each animal was run in parallel without primary antibody, also serving as a negative control. Antibodies to the N-terminus are advantageous in IHC because of the lack of homology between ER alpha and ER beta in this region (16). Also, N-terminal antibodies are less susceptible to decreased epitope exposure following hormone binding than those directed to the hormone binding domain or the hinge region (17). Using light microscopy, each individual structure within a section was classified as a TEB, duct or lobule and analyzed for the percentage of positively stained cells and the average staining intensity. As the terminal end proximal to the nipple of all ducts are not seen when a mammary gland is cross-sectioned, and therefore could actually be a TEB, we referred to them as ducts, and not terminal ducts. A scale from 1 (least intense) to 4 (most intense) was used to estimate intensity as described previously (11,18). For each animal, an average was calculated for intensity and percent of positive cells for TEB, ducts and lobules. The average values for each treatment group (n = 10/group) were compared using one-way analysis of variance (Sigma Stat, Jandel Scientific).
Immunoblotting analyses
EGFR signaling proteins and progesterone receptor
A more detailed description of the protocols may be found in our publications (11,15,19). Mammary glands were frozen in liquid nitrogen immediately after dissection. The tissue was homogenized in lysis buffer containing 0.1% Triton X-100 with protease inhibitors, and the protein quantified on the extracts using a BCA assay (Pierce, Rockford, IL). The extracts were boiled in sample buffer containing 10% beta-mercaptoethanol and 10% SDS, and equal quantities of protein were separated by electrophoresis using SDSpolyacrylamide gels. Proteins were electroblotted onto nitrocellulose membranes and non-specific antibody binding was prevented by blocking with 5% non-fat dry milk. The following primary antibodies were used: 1:1000 dilution of anti-EGFR (Calbiochem, La Jolla, CA), 1:250 anti-progesterone receptor (PR) (Neomarkers/Labvision, Fremont, CA), 1:1000 anti-mitogen activating kinase kinase (MEK) 1/2 (Cell Signaling, Beverly, MA), 1:500 anti-phospho-MEK 1/2 (Cell Signaling), anti-extracellular signal regulated kinases (ERK) 1/2 (Cell Signaling) and anti-phospho-ERK 1/2. After incubation with HRP-conjugated secondary antibody, bands were detected using chemiluminescence (Pierce) and exposed to X-ray radiography film. Band intensity was quantified using scanning densitometry. Values were compared using one-way ANOVA (Sigma Stat, Jandel Scientific).
Cytokeratins
Mammary glands from ovary-intact rats were crushed under liquid nitrogen and homogenized in buffer containing 2% SDS, 5 mM beta-mercaptoethanol and protease inhibitors as described above. Samples were centrifuged at 12 000 r.p.m. to remove cellular debris and fat. Total protein was determined using a Bradford-based assay (Bio-Rad, Hercules, CA) using BSA as a standard. Equal amounts (20 µg) of total protein were separated by electrophoresis and transferred as described above. Membranes were incubated with a 1:750 dilution of monoclonal anti-pan cytokeratin (Sigma), which recognizes cytokeratins 1, 4, 5, 6, 8, 10, 13, 18 and 19. The antibody does not react with non-epithelial tissue and, therefore, rat skeletal muscle was used as a negative control. Statistical comparison of band intensity was achieved as described above.
Quantification of phosphorylated EGFR
Mammary gland extracts from ovary-intact and ovariectomized rats were prepared as described above. Immunoprecipitation of tyrosine-phosphorylated proteins was achieved by incubating equal amounts of protein with anti-phosphotyrosine antibody (Signal Transduction Laboratories, Carpinteria, CA) for 30 min at 4°C while rotating. Protein Aagarose beads were added (Calbiochem) and the samples rotated for an additional 3 h. The immunocomplexes were pelleted by centrifugation and washed four times with lysis buffer (described above), resuspended in loading buffer containing 5% SDS, 0.5 mM Tris and 5% beta-mercaptoethanol, boiled for 5 min and subjected to SDSPAGE western blotting as described above. Membranes were blotted with a 1:1000 dilution of anti-EGFR (Calbiochem).
Mammary gland whole mounts
Whole mounts from ovary-intact animals were prepared as described previously (20,21). Briefly, mammary glands were fixed in 10% neutral-buffered formalin, defatted in acetone, re-hydrated, stained in alum carmine, dehydrated in a series of graded alcohols, cleared in xylene and coverslipped with mounting media. Light microscopy (200x magnification) was used to classify and quantify the terminal ductal structures within an area 1.5 mm from the edge of the gland, distal to the nipple as described previously (3,11). Criteria used for the classification of the terminal ductal structures as TEBs, terminal ducts (TDs) and lobules I are based on those described elsewhere (5,6). For quantitative branching analysis, mammary glands were photographed using a digital camera (Polaroid DMCIe) connected to a dissecting microscope (Leica MZ6). Measurements of ductal length and total gland area were taken using Scion Images Software (Scion, Frederick, MD, through the NIH). For assessment of the degree of side branching, each gland was divided into thirds, and one branch per area was selected. Starting at the portion of the gland most distal to the nipple, each branch was traced to the primary duct. The number of branches, including secondary branches, lateral buds and lobules were counted. The length of each branch was recorded, and the ratio of the total number of branch points per unit length (arbitrary units) was calculated. The average of the three values was taken for each gland (n = 4/group), and the mean values for each group were compared with controls using a one-way ANOVA. Pair-wise multiple comparisons were made using Tukey test (Jandel Scientific).
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Results
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Immunolocalization of ER alpha by IHC
Expression of ER alpha was localized mainly in the nuclei of the mammary epithelium; few positive stromal cells were present. The average percentage of positively stained cells in the TEBs, ducts and lobules of genistein- or EB-treated animals was not significantly different from controls (Table I
). Average staining intensity was significantly decreased in the TEBs and ducts of genistein- and EB-treated rats, while staining intensity was slightly decreased in the lobules.
Analysis of EGFR and downstream kinases by western blotting
Evaluation of mammary gland extracts for proteins involved in EGF signaling revealed that genistein and EB treatments significantly increased expression of total EGFR and phosphorylated EGFR compared with controls (Figure 1
). Normalization of the band intensity of phospho-EGFR to total EGFR for each sample resulted in no significant difference compared with controls. Expression of the downstream kinases MEK 1/2 and ERK 1 and 2 were not significantly altered by genistein or EB; analysis using phospho-specific antibodies revealed no significant difference, compared with controls (data not shown).

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Fig. 1. Measurement of total and tyrosine-phosphorylated EGFR from mammary gland extracts from ovary-intact 21-day-old rats. Total EGFR was measured by western blot analysis using anti-EGFR. Tyrosine-phosphory-lated EGFR was immunoprecipitated with anti-phosphotyrosine and immunoblotted with anti-EGFR. The percentage of phosphorylated EGFR was calculated by dividing phosphorylated into total EGFR. Densitometric values were reported as a percentage of the controls ± SEM. aP < 0.001, compared with controls.
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As circulating levels of estradiol are increased by injections of genistein in this model (15), we evaluated EGFR expression in the mammary gland of ovariectomized prepubertal rats to rule out indirect action of genistein via estradiol. Expression of EGFR and tyrosine-phosphorylated EGFR was increased in the mammary gland of EB- and genistein-treated ovariectomized rats (Figure 2
). As seen in ovary-intact rats, there was no net effect on the ratio of phosphorylated to total EGFR. As similar results were obtained in ovariectomized and ovary-intact rats, analysis of downstream kinases was restricted to ovary-intact rats only.

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Fig. 2. Measurement of total and tyrosine-phosphorylated EGFR from mammary gland extracts from ovariectomized 21-day-old rats. Total EGFR were measured by western blot analysis using anti-EGFR. Tyrosine-phosphorylated EGFR were immunoprecipitated with anti-phosphotyrosine and immunoblotted with anti-EGFR. The percentage of phosphorylated EGFR was calculated by dividing phosphorylated into total EGFR. Densitometric values were reported as a percentage of the controls ± SEM. aP < 0.05, compared with controls.
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Cytokeratin expression
As western blotting analyses are reflective of the amount of target protein per total protein, an increase in the number of epithelial structures in the mammary gland after treatment with genistein or EB could result in a greater percentage of epithelial protein loaded onto the gel, compared with control animals. This would result in falsely increased EGFR and PR, which are expressed highly in the epithelium. Therefore, epithelial-specific markers were used to rule out a potential artifact. There was no significant difference in the expression of cytokeratins following genistein or EB treatment, compared with controls (data not shown). There were no cytokeratins in the skeletal muscle.
Evaluation of ICI 182,780 dose
Uterine wet weight was used as an indicator of anti-estrogenic activity by ICI 182,780. Genistein and EB significantly increased uterine wet weight in ovary-intact rats (Figure 3
). The increase was blocked when 2.0 mg ICI 182,780/kg body wt was given prior to either compound; ICI 182,780 alone induced a slight decrease in uterine weights, when compared with vehicle-treated rats. However, the change was not statistically significant. There were no significant alterations in body weight in ICI 182,780-treated rats, compared with controls (data not shown).

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Fig. 3. Uterine to body weight ratio in ovary-intact rats treated ± genistein, EB and/or ICI 182,780 (ICI). Uterine wet weights (mg) were divided by body weights (g) and the result multiplied by a factor of 100. aP < 0.001, when compared with vehicle-treated rats.
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Evaluation of apoptosis
Apoptotic nuclei were enumerated and compared in mammary glands from ovary-intact vehicle- and ICI 182,780-treated animals to determine if the anti-estrogenic action of ICI 182,780 resulted in cell death. DNase-treated positive control tissue exhibited brown staining in the nuclei. The negative control, which lacked the enzyme required to label fragmented DNA, showed no positive staining. A small percentage of apoptotic nuclei were observed in the ductal epithelium and the TEBs. The mean percentage of apoptotic cells in mammary glands from the vehicle- and ICI 182,780-treated animals were 0.35 ± 0.10 and 0.25 ± 0.06%, respectively. Statistical comparison of the median values (0.34% for controls and 0.22% for ICI 182,780-treated animals) did not indicate a significant difference MannWhitney Rank Sum test (Sigma Stat, Jandel Scientific).
Effect of anti-estrogen pre-treatment on EGFR and PR expression
To provide biochemical evidence of inhibition of the ER function by ICI 182,780, measurement of an estrogen-responsive protein, the progesterone receptor, was performed on mammary extracts by immunoblotting analysis. Genistein or EB injections resulted in significantly increased PR (A and B isoforms) protein expression in ovary-intact rats (Figure 4
) compared with the vehicle. In ovariectomized rats, the effect on PR expression was similar to ovary intact rats. PR isoform A expression was 150 and 165% for genistein- and EB-treated rats, respectively, when normalized to control mean (data not shown). PR B expression for genistein- and EB-treated mammary glands was 170 and 161% of controls (data not shown). The increase in PR isoforms in the mammary glands of ovary-intact rats was inhibited by pre-treatment with ICI 182,780. ICI 182,780 alone had no significant effect on PR expression.

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Fig. 4. Immunoblotting analysis for PR (A and B isoforms) from mammary gland extracts from ovary-intact rats treated ± genistein, EB and/or ICI 182,780 (ICI) (n = 8/group). Densitometric values were reported as a percentage of the controls ± SEM. aP < 0.05, compared with controls.
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The increase in EGFR expression by genistein and EB in ovary-intact females was inhibited by pre-treatment with ICI 182,780 (Figure 5
). The anti-estrogen alone had no significant effect on EGFR expression when compared with vehicle-treated animals. There was no significant effect on the amount of tyrosine-phosphorylated EGFR following treatment with ICI 182,780, ICI 182,780 + genistein or ICI 182,780 + EB, when compared with vehicle-treated rats (data not shown).

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Fig. 5. Immunoblotting analysis for EGFR (phosphorylated and dephosphorylated forms) from mammary gland extracts from ovary-intact rats treated ± genistein, EB and/or ICI 182,780 (ICI) (n = 8/group). Densitometric values were reported as a percentage of the controls ± SEM. aP < 0.05, compared with controls.
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Whole mount analysis
Analysis of mammary terminal ductal structures indicated approximately twice the number of TEBs in genistein- or EB-treated rats, compared with the vehicle (Table II
). Pre-treatment with ICI 182,780 inhibited this effect, resulting in approximately two-thirds fewer TEBs than vehicle-treated animals. The number of TEBs in mammary glands from animals treated with ICI 182,780 + genistein or ICI 182,780 + EB were significantly fewer than those of animals treated with genistein or EB alone, respectively. Animals given ICI 182,780 alone had significantly fewer TEBs when compared with vehicle-treated rats. Only EB treatment altered the number of TDs, resulting in a significant decrease (40% fewer). When ICI 182,780 was given alone or prior to genistein or EB, the number of TDs was not significantly different from controls. There were no significant effects on the number of lobules I in any treatment group.
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Table II. Quantification of mammary terminal ductal structures of rats treated ± genistein, EB and/or ICI 182,780 (ICI)
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Figure 6
depicts mammary gland whole mounts representative of each treatment. The mammary glands from animals treated with the anti-estrogen were minimally branched, with very few tertiary branches and small lateral buds. The branching density of mammary glands of vehicle-treated rats was greater than those of anti-estrogen-treated rats, with many secondary/tertiary branches and alveolar units. Glands from animals treated with genistein or EB were more extensively branched than controls, with numerous alveolar units present. The interior branching of glands of animals treated with ICI 182,780 + EB and ICI 182,780 + genistein appeared less dense than controls, but not as sparse as animals given ICI 182,780 alone. Glands from animals treated with ICI 182,780 + genistein and ICI 182,780 + EB were composed mainly of short branches and lateral buds, while control glands were composed primarily of longer branches and alveolar units.

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Fig. 6. Representative mammary gland whole mounts taken from ovary-intact rats treated ± genistein, EB and/or ICI 182,780 (ICI). Photographs were taken at 2x magnification. TEB = terminal end bud, LN = lymph node, PD = primary duct, which leads to the nipple.
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In order to provide quantitative data to support our visual observations, the numbers of branch points along three random ducts per gland were counted. The degree of branching in the EB- and genistein-treated rats was significantly greater than that of controls and animals pre-treated with ICI 182,780 (Figure 7
). When ICI 182,780 was given prior to genistein or EB, the degree of branching was similar to controls. ICI 182,780 alone significantly decreased the degree of side branching by 30%, compared with controls.

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Fig. 7. Analysis of branching units per length (arbitrary units) from digital photographs of mammary gland whole mounts taken from ovary-intact rats treated ± genistein, EB and/or ICI 182,780 (ICI). Computerized measurements were taken of the length of three branches per gland (n = 4 animals/group), from the main lactiferous duct to the distal terminal structure. All side structures, including secondary/tertiary ductal branches, lateral buds, and alveolar units were counted. The average number of side structures was divided by the length of the duct. aP < 0.05, bP < 0.001, compared with vehicle-treated rats (one-way ANOVA); cP < 0.001, compared with genistein treated rats (Tukey test); dP < 0.001, compared with EB-treated rats (Tukey test).
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Discussion
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Decreased expression of ER alpha in response to genistein has been observed in ER-positive tissues, including the uterus (15) and prostate (22). Similarly, others have shown that estrogen treatment reduced the expression of ER alpha in breast cancer cells (2325). In the mammary gland, semi-quantitative immunohistochemical analysis showed decreased ER alpha staining intensity in the TEBs and ducts following treatment with genistein and EB. The decrease in staining intensity may indicate receptor down-regulation, which is hypothesized to occur as part of the cyclic nature of ER expression. While the significance of the down-regulation and cycling of ERs is not clear, it may represent a mechanism of regulatory control of estrogen responsiveness of the tissue. The induction of the estrogen-inducible protein, PR, by genistein and EB suggests that decreased immunoreactivity of ER correlated with activation of the receptor. Likewise, the down-regulation of ER may be explained on the basis of PR to repress ER transcriptional activity (26). Decreased expression of ER alpha may also reflect proliferation, as proliferating epithelial cells of the mammary glands of young virgin rats do not express the receptor (14,27). This is consistent with our previous results, which indicated that prepubertal genistein treatment resulted in a proliferative mammary gland at day 21, which led to a more differentiated structure evident at day 50 (3). These actions resulted in subsequent down-regulated EGFR expression (11) and reduced cell proliferation (3) in the TEBs of adult rats. The similar decrease in immunoreactivity between genistein and EB suggests a common molecular action.
However, in vitro studies suggest that the molecular action of phytoestrogens may differ from estradiol. The binding affinity of genistein for ER beta is greater than that for ER alpha, while estradiol-17-beta binds to both receptors with similar affinity (28,29). Recent work has shown that genistein is more capable of regulating transcriptional activity of ER beta than ER alpha, while estradiol appears to be equally effective on both receptors (30). Therefore, genistein may exert control of ER-dependent gene expression primarily through ER beta. Despite the preference of genistein for ER beta, ER alpha may play a larger role in mammary gland development, as the structure and function of the mammary gland in the ER beta knockout mouse appears to be normal (31), while mammary gland development is severely impaired in ER alpha knockout mice (32,33). Interestingly, the expression of ER beta in the mammary glands of prepubertal rats is greater than that of ER alpha (27), but the exact function of ER beta in mammary development remains to be elucidated.
Immunoblotting analyses for EGFR indicated an increase in the expression of receptors after injection with genistein and EB, again indicating a similarity in the mechanism of action between the two compounds. These results are in agreement with data reported previously indicating increased EGFR in the mammary glands of genistein-treated adult rats (11). As EGFR and PR up-regulation also occurred in the mammary glands of ovariectomized, pre-pubertal rats treated with either genistein or EB, an indirect action via increased circulating estradiol following genistein treatment (15) can be ruled out. Alternatively, the possibility of stimulation of local estrogen production in the mammary gland by genistein cannot be excluded. There was no net effect on tyrosine-phosphorylation of the receptors, making a direct action on the EGFR by genistein unlikely. A similar observation was reported where dietary genistein decreased prostatic EGFR and tyrosine-phosphorylated EGFR (19), indicating no net effect on phosphorylation. Therefore, it is not likely that growth enhancement through direct interaction with the EGFR by genistein is occurring. Rather, genistein is acting in a manner similar to EB, and the increase in EGFR is a consequence of ER activation. This conclusion is further supported by the ablation of this response in the presence of the ICI anti-estrogen. Similar results were shown by Ankrapp et al. (34), who reported the inhibition of EGF-induced stimulation of TEB growth in anti-estrogen-treated prepubertal mice. Collectively, the data support a requirement for ERs in EGF signaling in the rat mammary gland.
Stimulation of the EGFR signaling cascade following ligand binding, receptor dimerization and autophosphorylation involves the phosphorylation of downstream kinases, including Raf, MEK and ERK (reviewed in refs 35,36). Therefore, increased autophosphorylation should correlate with increased receptor tyrosine kinase activity, resulting in increased phosphorylation of downstream targets. The lack of an observable effect on the expression and/or phosphorylation of MEKs1/2 and ERKs 1/2 may simply be explained by the absence of any net effect on autophosphorylation. Alternatively, a single time point following multiple injections was chosen to measure expression and may not reflect overall fluctuations in phosphorylation and dephosphorylation. Differential cell-specific effects may not be detected by immunoblotting analysis of proteins extracted from the entire gland, which may be affected by a larger percentage of epithelial-derived protein in the treated animals, due to epithelial proliferation. This was not an issue for the EGFR, which is highly expressed in the epithelium (11), as indicated by the lack of a treatment-related increase in cytokeratins.
The analysis of mammary gland morphology in genistein and EB-treated prepubertal rats indicated a growth-stimulatory response, manifested as an increase in TEBs and ductal branching. Increased TEBs in the area of the gland closest to the lymph node following prepubertal genistein treatment has been reported previously by our laboratory (3,11). These proliferative effects were inhibited by pre-treatment with ICI 182,780, suggesting that the main effect of the anti-estrogen was to block ERs in the TEBs, which are located in the most proliferative area of the gland, most distal to the nipple (for review, see ref. 37). TEBs also express a greater percentage of ER alpha (5,14) and ER beta (27) compared with the more differentiated structures. Thus, TEBs may be more sensitive than more differentiated structures to the anti-estrogenic action of ICI 182,780, which binds to both receptors without transcriptional activation (38). The atrophied appearance of the mammary glands of animals treated with ICI 182,780 + vehicle suggests that the actions of endogenous estrogens, detectable in vehicle-treated 21-day-old rats (15), were blocked. However, we cannot rule out the possibility that alterations to other hormone pathways (androgen, progesterone, prolactin) may have contributed to glandular atrophy.
Glandular proliferation was not completely inhibited when ICI 182,780 was given prior to genistein or EB. The branching of the mammary glands of these animals was more dense than that of animals treated with ICI 182,780 alone, but not as dense as that of control animals. This effect was attributed to the presence of lobuloalveolar structures and longer branches in the glands of the controls, while the majority of branch points in animals given ICI 182,780 + genistein or ICI 182,780 + EB were shorter branches or lateral buds. As the anti-proliferative action of ICI 182,780 in the mammary gland is reversible if the treatment is discontinued (39), genistein or EB may stimulate the growth in areas of the gland other than that closest to the lymph node after the effects of ICI 182,780 have begun to subside. Therefore, ICI 182,780 treatments were given on days 1620. When ICI 182,780 treatments were given only on days 16, 18 and 20, no inhibition of genistein- or EB-induced mammary gland growth, PR or EGFR expression was observed. Alternatively, other hormones such as prolactin or progesterone may be responsible for ductal growth and branching occurring within the interior of the gland.
Growth inhibition by ICI 182,780 is not likely to occur through interaction with stromal ER alpha, as Russo et al. (14) have reported ER alpha to be absent in the mammary stroma of rats, and our data show low stromal expression of ER alpha. The minimal expression of stromal ER alpha in rats is in contrast to tissue recombination studies in mice, which demonstrated that stromal, not epithelial ER alpha, was responsible for estrogen-induced epithelial growth through a paracrine mechanism (40). ER beta is expressed in some stromal cells of the rat mammary gland (27), but its function is unknown.
A low percentage of apoptotic nuclei were observed in the mammary glands of both control- and anti-estrogen-treated animals, suggesting that ICI 182,780 achieved growth inhibition by blocking estrogen action, not by inducing cell death. This is in agreement with Wakeling et al. (41), who reported that ICI 182,780, when given at concentrations that resulted in anti-estrogenicity in the absence of toxicity, induced a cytostatic response in MCF-7 cells. Others have shown that implantation of ICI 182,780 pellets directly into the mammary glands of mice resulted in local inhibition of growth, while the contralateral glands were unaffected (39). The data suggested a direct and critical role for local estrogen on ERs in mammary development. The inability of genistein to stimulate mammary gland growth in the presence of the anti-estrogen again supports an estrogen-like, ER-based mechanism, and not direct action on the EGFR.
The pharmacologic model used in the current study involved a short duration of exposure to genistein, a treatment that results in chemoprevention (3). Previous studies have shown that short exposure to estrogenic compounds during development also resulted in chemoprevention of spontaneously originating (42) and chemically induced mammary tumors (43,44). The data are in contrast to the estrogen-dependency of mammary cancer with respect to the processes of initiation, promotion and progression (reviewed in ref. 45). Thus, both timing and duration of exposure play a role in chemoprevention.
In conclusion, genistein and EB act via the ERs to regulate PR and EGFR in the rat mammary gland. Genistein up-regulates EGFR expression, but not phosphorylation of the receptor. Genistein action in the prepubertal mammary gland accounts for initial proliferative action that results in gland differentiation and protection against mammary cancer (3,4).
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Notes
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4 To whom correspondence should be addressed Email: coral.lamartiniere{at}ccc.uab.edu 
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Acknowledgments
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The authors would like to thank Hoffmann-LaRoche, Basel, Switzerland for generously providing us with genistein. This research was supported by NIH-R01 CA61742 and DOD DAMD 1700-1-0118. Ms Cotroneo was supported by graduate fellowships NIH 5 R25 CA4788-10-12 and DOD DAMD 17-00-1-0119.
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Received January 8, 2002;
revised May 9, 2002;
accepted May 15, 2002.