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Soy Diets Containing Varying Amounts of Genistein Stimulate
Growth of Estrogen-dependent (MCF-7) Tumors in a
Dose-dependent Manner
Clinton D. Allred, Kimberly F. Allred, Young H. Ju, et al.
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To request permission to re-use all or part of this article, contact the AACR PublicationsDepartment at [CANCER RESEARCH 61, 5045–5050, July 1, 2001] Soy Diets Containing Varying Amounts of Genistein Stimulate Growth of Estrogen-
dependent (MCF-7) Tumors in a Dose-dependent Manner1
Clinton D. Allred,2 Kimberly F. Allred,2 Young H. Ju, Suzanne M. Virant, and William G. Helferich3
Department of Food Science and Human Nutrition [C. D. A., K. F. A., Y. H. J., S. M. V., W. G. H.], and Division of Nutritional Sciences [C. D. A., W. G. H.], University of Illinois,Urbana, Illinois 61801 ABSTRACT
various forms, including supplement capsules, which contain mixturesof isoflavones derived from soy. Isoflavones are also present in We have demonstrated that the isoflavone, genistein, stimulates growth
bioactive concentrations in food products such as soy protein isolates, of estrogen-dependent human breast cancer (MCF-7) cells in vivo (C. Y.
which can contain varying isoflavone content. It is generally accepted Hsieh et al., Cancer Res., 58: 3833–3838, 1998). The isoflavones are a
group of phytoestrogens that are present in high concentrations in soy.
that consuming the phytochemical components of soy, particularly the Whether consumption of genistein from soy protein will have similar
isoflavones, in pure form, as in supplements, may pose some health effects on estrogen-dependent tumor growth as pure genistein has not
concerns but that consumption of more whole foods containing these been investigated in the athymic mouse tumor implant model. Depending
are natural and, as such, safe. It is important to note that the concen- on processing, soy protein isolates vary widely in concentrations of genis-
tration of these compounds in soy protein isolates is dependent on the tein. We hypothesize that soy isolates containing different concentrations
method of processing, and specific processing methods can be used to of genistein will stimulate the growth of estrogen-dependent cells in vivo in
enrich the isoflavone content in soy protein isolates.
a dose-dependent manner. To test this hypothesis we conducted experi-
Clinical and preclinical laboratory animal and in vitro studies have ments in which these soy protein isolates were fed to athymic mice
demonstrated the hormonal activity of dietary isoflavones. Our labo- implanted s.c. with estrogen-dependent tumors. Genistein content (agly-
cone equivalent) of the soy isolate diets were 15, 150, or 300 ppm. Positive
ratory has examined the estrogenic activity of genistein. In vitro, when (with 17␤-estradiol pellet implant) and negative (no 17␤-estradiol) control
human estrogen-dependent breast cancer (MCF-7) cells were treated groups received casein-based (isoflavone-free) diets. Tumor size was meas-
with increasing concentrations of genistein (1–10 ␮M), the cells ex- ured weekly. At completion of the study animals were killed and tumors
pressed increased mRNA levels of pS2, an estrogen-responsive gene, collected for evaluation of cellular proliferation and estrogen-dependent
in a dose-dependent manner. In vivo, 25-day-old ovariectomized, gene expression. Incorporation of bromodeoxyuridine into cellular DNA
athymic mice consuming dietary genistein for 5 days had increased was used as an indicator of cell proliferation, and pS2 mRNA was used as
number and size of terminal end buds in the mammary gland when an estrogen-responsive gene. Soy protein diets containing varying
compared with control animals (5). Not only do the isoflavones have amounts of genistein increased estrogen-dependent tumor growth in a
estrogenic activity on various tissues when given in pure form but also dose-dependent manner. Cell proliferation was greatest in tumors of
animals given estrogen or dietary genistein (150 and 300 ppm). Expression
when consumed in products such as soy protein isolate. In humans, of pS2 was increased in tumors from animals consuming dietary genistein
Petrakis et al. (6) demonstrated that consumption of soy protein (150 and 300 ppm). Here we present new information that soy protein
isolate had stimulatory effects on the breast tissue of premenopausal isolates containing increasing concentrations of genistein stimulate the
women. They found that duct fluid aspirates contained greater num- growth of estrogen-dependent breast cancer cells in vivo in a dose-depen-
bers of hyperplastic epithelial cells in women consuming soy protein dent manner.
isolate. Nipple aspirate apolipoprotein D and pS2 expression has alsobeen shown to be elevated in women consuming 60 g of soy in the INTRODUCTION
form of ground, textured vegetable protein for as little as 2 weeks (7).
These data collected from both laboratory animals and humans dem- The Food and Drug Administration recently approved a health onstrate the estrogenic activity of the isoflavones whether they are claim for soy protein. The claim states “25 g of soy protein a day, as taken as a pure compound or consumed in soy protein isolate con- part of a diet low in saturated fat and cholesterol, may reduce the riskof heart disease” (1). As a result, the consumption of soy protein by Americans has increased. Soy and soy-derived supplements are not The role that isoflavones play in breast cancer is unclear. Some being used solely to lower serum cholesterol and reduce risk of heart reports indicate that exposure to genistein is preventative in the disease. Soy contains a complex mixture of a variety of phytochemi- development of breast cancer (8 –10), whereas others show that ge- cals; our focus is on the estrogenic isoflavones referred to as phy- nistein stimulates the growth of existing estrogen-dependent tumors toestrogens. Isoflavones have been demonstrated to act as estrogen (5). Studies published over the last 5 years have demonstrated that agonists by binding to the estrogen receptor and generating estrogen- exposure to dietary genistein before puberty reduces the number of induced responses (2– 4). As a result, postmenopausal women may chemically induced mammary tumors formed in female Sprague consume soy for the estrogenic effects of these compounds to relieve Dawley rats. The authors suggest protection against the development menopausal symptoms. Soy products are marketed as a “natural” of breast cancer tumors is attributable to the estrogenic effects of alternative to hormone replacement therapy with the perception that genistein that causes increased cellular differentiation in mammary these phytoestrogens are without the risks associated with hormone gland cells of prepubertal animals. A differentiated cell undergoes less replacement therapy. Isoflavone-containing products are produced in proliferation and therefore is less likely to progress through the cancerprocess (8 –10). If prepubertal exposure to genistein results in earlierdifferentiation of the mammary gland, then the assumption can be Received 8/9/00; accepted 4/27/01.
The costs of publication of this article were defrayed in part by the payment of page made that earlier differentiation would be protective against chemi- charges. This article must therefore be hereby marked advertisement in accordance with cally induced mammary tumors. Cohen et al. (11) found that continual 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by NIH Grant CA77355 (to W. G. H.).
feeding of soy protein isolate (containing 1.67 mg total isoflavones 2 C. D. A. and K. F. A. contributed equally to this paper.
per g isolate) postpubertally for 18 weeks after chemical induction of 3 To whom requests for reprints should be addressed, at 580 Bevier Hall, Department mammary tumors in rats had no detectable effect on the development of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801. Phone:(217) 244-5414; Fax: (217) 244-7877; E-mail: [email protected].
of these tumors. Our research has focused on the effect dietary SOY PROTEIN AND E2-DEPENDENT BREAST CANCER TUMOR GROWTH genistein has on the growth of existing estrogen-dependent (MCF-7) diets. Treatment animals were fed either soy isolate-containing diets or AIN- tumors in animals with low circulating E 4 concentrations. We have 93G diet plus genistein. Three soy protein isolates with varying concentrations demonstrated that genistein fed at 750 ppm stimulates the growth of of genistein were used in this study. Each isolate was added at 20% of the diet MCF-7 cells implanted into athymic mice. Mice consuming 750 ppm as the sole source of protein in the AIN-93G diet. Both the casein-based diet dose of genistein have a total plasma genistein concentration of ϳ2 and the soy-based diet provided ϳ18% protein, but 2.2 g of methionine was added to each kilogram of soy isolate diet to meet the sulfur-containing amino M (5). This is a relevant dietary dosage because women who acid requirements and as a result, all of the essential amino acid requirements consume varying amounts of isoflavones from soy milk have plasma of the mouse were met. The three soy protein isolates (Protein Technologies genistein levels of 0.8-2.2 ␮M (12). In vitro genistein has been shown International, St. Louis, MO) were processed to contain varying amounts of to stimulate the growth of MCF-7 cells at concentrations as low as 200 genistein. There were no significant differences in amino acid content or nM (2, 5). Whereas these data demonstrate genistein can stimulate the proximate analysis among the isolates. Additionally, the isolates were very low growth of estrogen-dependent breast cancer tumors, it has not yet been in fiber and as such the lignin content was negligible. The majority of the determined if soy protein isolate, which contains a complex mixture of isoflavones in the protein isolates were present as the aglycone form (ϳ60%).
phytochemicals, can exhibit similar effects on growth of estrogen- The genistein contents were 0.075, 0.75, and 1.5 mg genistein (aglycone equivalents)/g of product. This resulted in the final concentrations of genistein The study presented here was designed to evaluate the effects of in the three soy isolate diets to be 15, 150, and 300 mg genistein/kg diet dietary soy protein isolates containing varying concentrations of ge- respectively. To compare the genistein content from soy to pure genistein, itwas added to the AIN-93G diet at equal concentrations to that found in the soy nistein: (a) on the growth of MCF-7 tumors in athymic mice; (b) on isolate diets and provided 15, 150, and 300 ppm genistein in a casein-based cellular proliferation of estrogen-dependent tumors in ovariectomized diet. The eight final treatment groups were: positive controls (2 mg E pellet); athymic mice; and (c) on expression of the estrogen-responsive gene, negative controls; low soy isolate [LSI (15 ppm genistein)]; medium soy pS2, in response to dietary exposure to soy protein isolates with isolate [MSI (150 ppm genistein)]; high soy isolate [HSI (300 ppm genistein)]; varying concentrations of genistein.
low genistein [LG (AIN-93G ϩ 15 ppm genistein)]; medium genistein [MG(AIN-93G ϩ 150 ppm genistein)]; and high genistein [HG (AIN- 93G ϩ 300ppm genistein)].
MATERIALS AND METHODS
E Pellet Preparation. MCF-7 cells will not produce tumors in ovariec-
tomized mice unless they are supplemented with estrogen. Therefore, 1 week Effects of Consumption of Soy Protein Isolate and Casein-based Diets
after delivery, animals were implanted with E pellets. E pellets were made Containing Genistein on the Growth of Estrogen-dependent Tumors
containing 2 mg of E mixed with 18 mg of cholesterol as a carrier. A 20-mg mixture containing E and cholesterol was placed into a pellet mold and Animals. Female athymic nude mice were purchased from Harlan Sprague
pressed into a compact pellet ϳ4.5 mm in diameter and ϳ2.5 mm in depth.
Dawley (Indianapolis, IN) and delivered at 28 days of age. Mice were ovari-ectomized at 21 days of age and allowed a week to recover before delivery. A Pellets were then placed s.c. in the interscapular region of mice (14).
2-mg E pellet was placed under the skin of each mouse before MCF-7 cells Tumor Implantation. MCF-7 cells were maintained in 100 mm ϫ 20 mm
were transplanted into the animal. Cells were then injected into the four flanks plastic tissue culture plates in IMEM media (Biofluids) containing 10% fetal on the back of each animal. Within the first 4 weeks, tumors that subsequently bovine serum, penicillin (100 units/ml) and streptomycin (100 units/ml). E2 formed were allowed to develop to an average cross-sectional area of ϳ40 (1.0 nM) was added in the media to keep the cell line estrogen-dependent. Cells mm2. At this point, animals were placed into eight treatment groups (seven were incubated at 37°C in a humidified atmosphere of 5% CO . Cells were animals/group). The treatment groups were: (a) positive control; (b) negative grown to confluence, collected using trypsin-EDTA, and counted. Cells were control; (c) dietary soy protein isolate providing 15 ppm genistein; (d) dietary suspended in matrigel matrix (Becton Dickinson) before being injected (15) soy protein isolate providing 150 ppm genistein; (e) dietary soy protein isolate and then 40 ␮l of the cell suspension 1.5 ϫ 105 cells were injected per site into providing 300 ppm genistein; (f) dietary genistein in casein-based diet at 15 each of the four flanks of the athymic mice.
ppm; (g) 150 ppm; and (h) 300 ppm. The E pellet was removed from the Tumors were measured weekly and cross-sectional area was determined negative control animals and from all of the mice on the various dietary using the formula [length/2 ϫ width/2 ϫ ␲] (5, 16). When tumors reached an treatments. The E pellet was also removed from the positive control group, average cross-sectional area of 40 mm2, animals were divided into treatment but these animals were again implanted with an identical 2-mg E pellet.
groups with each group normalized for tumor number, tumor size, and animal Negative and positive controls were given AIN 93G as a control diet. This diet number. E pellets were removed from all of the animals, and the mice were provided protein from casein. The remaining animals were put on one of the then placed on the treatment diets. Positive control mice were reimplanted with six treatment diets. Tumor area and body weight were measured weekly. At the a fresh pellet containing 2 mg of E. We then resumed measuring tumor areas end of the study, tumors and plasma samples were collected for tissue analysis.
We felt that weekly tumor area was the most critical outcome evaluated.
Tissue/Tumor Collection. At the completion of the study, mice were
Measurements were made over a total of 29 weeks after removal of E pellets.
killed by cervical dislocation, and tumors were harvested. Tumors from each However, at the conclusion of the study, tumors from the negative control, low mouse were fixed in 10% formalin and embedded for immunohistochemical soy isolate, and low genistein groups were small, making it impossible to staining. Additional tumors from each mouse were immediately frozen in collect representative samples for mRNA and cell proliferation data. There- liquid nitrogen for subsequent RNA isolation and analysis.
fore, to obtain these measurements for the negative control group, a second setof negative control animals were used. These animals were killed 11 weeks Analysis of Estrogen-responsive pS2 mRNA
after removal of the E pellet when their tumors were at an average cross- sectional area of 25 mm2. We feel this was the most accurate method to obtain RNA Preparation. The mRNA was isolated using procedures routinely
tissues from the negative control group that were regressing but still large used in our laboratory (17). Briefly, frozen tumors (Յ200 mg) from liquid enough to obtain data for mRNA and immunohistochemical analysis.
nitrogen were smashed and the coarse tumor powder was transferred into Diet Formulation. AIN-93G semipurified diet was selected as a base diet
TRIZOL (Life Technologies, Inc., Grand Island, NY) in a 15-ml tube and was for control animals as it has been established as meeting all of the nutritional homogenized using a Polytron-Aggregate (Luzern, Switzerland). Chloroform requirements of mice (13). Soy oil was removed from all of the diets and corn was added into a homogenized tumor sample, shaken vigorously, and then oil added to eliminate any additional components of soy being added to the incubated for 10 min at 24°C. The reaction tube was centrifuged at 12,000 ϫ gfor 15 min at 4°C. The upper portion was removed and transferred into a freshtube. An equal volume of isopropyl alcohol was added, shaken, and incubated 4 The abbreviations used are: E , 17␤-estradiol; BrdUrd, 5-bromo-2Ј-deoxyuridine; for 10 min at 24°C. The mixture was centrifuged at 12,000 ϫ g for 10 min at LSI, low soy isolate; MSI, medium soy isolate; HSI, high soy isolate; LG, low genistein; 4°C. The RNA pellet was washed with ice-cold 75% ethanol and centrifuged MG, medium genistein; HG, high genistein; AIN 93G, American Institute of Nutrition 93growth diet.
at 7,500 ϫ g for 5 min at 4°C. The RNA pellet was air-dried then dissolved SOY PROTEIN AND E2-DEPENDENT BREAST CANCER TUMOR GROWTH with RNase-free dH O. RNA was stored at Ϫ80°C. RNA concentration was ϭ 40 ␮g of single-stranded RNA/ml).
Northern Blot Analysis. Expression of pS2 was used as a biomarker of
estrogenic activity (18). For the detection of pS2 expression, 10 ␮g of RNAwere separated on 1.2% formaldehyde denaturing agarose gels and transferredto a Magnacharge, Nylon, Transfer membrane (Osmonics, Westboro, MA).
The RNA was UV cross-linked onto the membrane. The membrane wasprehybridized in a formamide prehybridization solution containing denaturedsalmon sperm DNA for 3 h at 42°C. After 3-h prehybridization, the DNAprobe was labeled using Random Primers DNA Labeling System (Life Tech-nologies, Inc.). For the estrogen-responsive pS2 gene, a 25 ng of pS2 cDNA,or for the control, a 25 ng of glyceraldehyde-3-phosphate dehydrogenasecDNA probe was labeled with 50 ␮Ci ␣-[ 32P]dCTP. The membrane wasincubated with 32P-labeled probe overnight at 42°C. The membrane waswashed twice with 5 ml of 0.2% SSC/0.1% SDS at 24°C for 5 min each, threetimes with 5 ml of 0.1% SSC/0.1% SDS at 68°C for 15 min each, and rinsedwith 2% SSC. The membrane was exposed to X-ray film for 6 h, andhybridizing RNA molecules were detected by performing autoradiography.
Film was analyzed using Collage (version 4.0) software (Image DynamicsCorp.) with Foto Analyst (Futodyne).
Tumor Cell Proliferation Immunohistochemistry
BrdUrd Analysis. BrdUrd incorporation into cellular DNA was used as an
indicator of cells that were actively proliferating (19). Four h before killing theanimals each mouse was injected i.p. with 50 mg BrdUrd/kg body weight.
Tumors were excised, skin and fat removed, and processed for tissue staining.
Prepared sections were then stained for the presence of BrdUrd using amodified immunohistochemistry protocol (20). Briefly, slides were deparaf-finized and hydrated by immersing in xylene twice for 12 min and immersingin a series of alcohol/water solutions for 5 min each. To block endogenousperoxidase, slides were immersed in 0.3% H O for 20 min then washed with distilled water. Slides were then microwaved in a Pyrex dish in 18 mM citratebuffer at pH 6.0 for 20 min and cooled. Then slides were washed in PBS (pH7.1–7.4) for 5 min, and tissue sections were rimmed with wax. 50-␮l anti-BrdUrd primary antibody (Amersham) was added to slides and incubated for1 h at room temperature in a humidity chamber. Slides were washed in PBSand 50 ␮l of diluted secondary antibody (Sigma Chemical Co.) was added toslides and incubated for 30 min at room temperature. Slides were then washed Fig. 1. A and B, effects of soy protein isolates and genistein on MCF-7 tumor growth in athymic mice. Female ovariectomized athymic mice were implanted with a 2-mg E in PBS. One drop of 3,3Ј-diaminobenzidine ϩ Ni enhancer solution, freshly pellet. The animals were then injected with 1.5 ϫ 105 MCF-7 cells/site in four locations.
prepared, was added to each slide with a Pasteur pipette. Slides were then Subsequently, tumors developed and were allowed to grow to an average cross-sectional washed in water twice, in PBS, and counterstained with 20% hematoxylin for area of 40 mm2. At this time, E pellets were removed from all of the mice, and they were 1 min. The slides were then dehydrated by placing them in 80% alcohol for 5 assigned to one of eight treatment groups: positive controls that were reimplanted with anew 2-mg E pellet (8 mice; n ϭ 32 tumors), negative controls that were fed AIN 93G min, 95% ethanol for 5 min, and 100% ethanol for 5 min followed by xylene rodent diet alone (6 mice; n ϭ 24 tumors), LSI containing 15 ppm genistein (7 mice; four times for 5 min each. Slides were then coverslipped and analyzed by light n ϭ 27 tumors), MSI containing 150 ppm genistein (8 mice; n ϭ 31 tumors), HSI microscope. Both positive and background stained cells were counted in a containing 300 ppm genistein (6 mice; n ϭ 23 tumors), LG AIN 93G ϩ 15 ppm genistein given area of tissue. The data were then presented as percentage of cells (7 mice; n ϭ 27 tumors), MG AIN 93G ϩ 150 ppm genistein (6 mice; n ϭ 23 tumors),and HG AIN 93G ϩ 300 ppm genistein (7 mice; n ϭ 27 tumors). A, the effects of soy protein isolate on the growth of MCF-7 tumors. B, the effect of consumption genistein in Statistical Analysis. Tumor area data were analyzed using one-way or
AIN 93G on the growth of MCF-7 tumors. In soy-containing diets, the soy protein isolates repeated-measures ANOVA according to the characteristics of the data set were the sole source of protein whereas casein was the protein source in AIN93G diets using the SAS program. pS2 gene expression data were analyzed using supplemented with genistein. Both soy- and casein-based diets were formulated to meetall of the nutritional requirements of the mice. The day animals were started on experi- one-way treatment ANOVA. If the overall treatment F-ratio was significant mental diets and were designated as measurement 0. Tumors were then measured weekly.
(P Ͻ 0.05), the differences between treatment means were tested with Fisher’s Data are expressed as average cross-sectional tumor area for all of the tumors in each and LG groups were similar to the negative control group resulting in Effect of Soy Isolates and Genistein on MCF-7 Tumor Growth
average tumor areas of 16 mm2 and 14 mm2, respectively. The in Athymic Mice. By week 3 after retreatment with a new 2-mg E
average tumor area in the MSI and MG groups were significantly pellet, the average cross sectional area of the tumors in the positive higher than the negative control group with final cross sectional areas control group was 126 mm2 (Fig. 1, A and B). At this point, these mice of 60 mm2 and 54 mm2 (P Ͻ 0.01) respectively. The MSI and MG groups were not significantly different from one another. The tumors negative control tumors regressed to an average area of 12 mm2. They from both the HSI and HG groups were significantly larger than the then maintained this size until termination of the study. Soy protein negative control and other dietary treatment groups (P Ͻ 0.01). The isolates stimulated growth of MCF-7 tumors in a dose-dependent HSI average area was 112 mm2 whereas the HG group had a final fashion (Fig. 1A). Also, casein-based diets containing equivalent average area of 97 mm2. These (HSI and HG) were not significantly levels of genistein stimulated tumor growth in a dose-dependent different from one another and were similar to that obtained from the manner (Fig. 1B). By week 29 after E pellet removal, both the LSI positive control group 3 weeks after retreating them with E pellets SOY PROTEIN AND E2-DEPENDENT BREAST CANCER TUMOR GROWTH (Fig. 2). Body weight was monitored weekly, and no significantdifference was observed among the treated and control groups (datanot shown). These data indicate that dietary soy isolates containingincreasing concentrations of genistein and casein-based diets contain-ing equalized concentrations of genistein act in a dose-dependentmanner to stimulate growth of human estrogen-dependent breastcancer cells transplanted into athymic mice. Also, it is important tonote that there were no significant differences in growth of tumors inthe negative control, low soy isolate, and low genistein groups. Thesedata suggest that there is a threshold level of dietary genistein belowwhich no increase in estrogen-dependent tumor growth is observed.
Effect of Soy Isolates and Genistein on Estrogen-responsive pS2
mRNA Expression in MCF-7 Tumors. To evaluate the ability of
soy isolates and genistein in the diet to enhance the expression of an
Fig. 3. Effect of soy and genistein treatments on pS2 gene expression in MCF-7 estrogen-responsive gene, pS2, we conducted Northern blot analysis tumors. At the end of the study, tumors were collected for analysis. For the detection of using mRNA isolated from tumors excised from animals in each pS2 expression, mRNA was isolated from each tumor. Northern blot analysis was used toquantify the amount of mRNA produced as a result of the treatments. Three tumors from treatment group. At the conclusion of the study, tumors from the each treatment group were used for pS2 analysis. pS2 expression is presented as the negative control, low soy isolate, and low genistein groups were too relative pS2 mRNA level. Glyceraldehyde-3-phosphate dehydrogenase was used as astandard. Bars, SE.
small to obtain sufficient RNA for Northern blot analysis. Negativecontrol tumors were collected from a separate set of tumor-bearingathymic mice in which the tumors were regressed for 11 weeks after ing and nonproliferating cells in a given field of view were counted, E pellets were removed. This allowed us tumors that were regressing and final values were expressed as percentage of proliferating cells.
(attributable to low E ) but were large enough for RNA isolation.
The percentage of proliferating cells value for the negative control Expression of pS2 was not detectable in the RNA isolated from group was 1.4%. Both the MSI and MG groups had a significantly negative control animals. Expression of pS2 was very low in the higher percentage of cellular proliferation when compared with the isoflavone-treated animals, ϳone-tenth that of the E -treated mice.
negative control group with values of 9.4% and 8.2% (P Ͻ 0.001).
However, pS2 expression was significantly (P Ͻ 0.05) higher in The animals consuming the high soy isolate with the highest concen- animals consuming the medium and high soy protein isolates when tration of genistein and the animals consuming the highest concen- compared with the negative control group. Levels of pS2 expression tration of genistein in control diet had proliferation of 11.9% and in tumors from animals that were consuming medium and high 11.8% respectively. These values were significantly higher than all of concentrations of genistein in the AIN93G diet were also significantly the other groups (P Ͻ 0.05; Fig. 4).
(P Ͻ 0.05) higher than the negative control group (Fig. 3). However,there was no significant difference between the MSI and HSI groups DISCUSSION
Cellular Proliferation in MCF-7 Tumors Excised from Animals
The purpose of this study was to determine the influence of dietary Consuming Soy Protein Isolates and Genistein. Cellular incorpo-
soy protein isolates containing increasing concentrations of genistein ration of BrdUrd was used as an indicator of cellular proliferation in on the growth of estrogen-dependent human breast cancer cells trans- MCF-7 tumors. Cells that stained positive after immunohistochemical planted into athymic mice. The results presented here demonstrate that analysis were considered actively proliferating cells. Both proliferat- soy protein isolates containing varying concentrations of genistein canstimulate growth of estrogen-dependent tumors similar to that seenwith pure dietary genistein. Soy protein isolates stimulated the growthof MCF-7 tumors in a dose-dependent manner as the concentration ofgenistein increased in the isolates. Tumor growth was significantlyincreased in animals consuming genistein at concentrations of 150 and300 ppm in both the casein- and soy-based diets. Dietary concentra-tions in this study are lower than previous studies in which MCF-7tumor growth was observed in athymic mice consuming dietarygenistein at 750 ppm (5). In the study presented here, tumors tooklonger to reach a maximum size when compared with the previousstudy, which was likely attributable to the lower concentrations ofgenistein in the diet. Cellular proliferation was increased in a dose-dependent manner as genistein concentrations increased in both thesoy protein and casein-based diets consistent with the tumor growthdata. The increase in MCF-7 cell proliferation was likely attributableto an estrogenic effect as indicated by the modest increase in pS2 Fig. 2. Average tumor area for week 29. Female ovariectomized athymic mice were expression. These findings suggest that genistein, when fed to athymic implanted with a 2-mg E pellet. The animals were then injected with 1.5 ϫ 105 MCF-7 mice in pure form or in a food source rich in the isoflavone such as cells/site in four locations. Subsequently, tumors developed and were allowed to grow toan average cross-sectional area of 40 mm2. At this time, E pellets were removed from all soy protein isolate, can stimulate estrogen-dependent tumor growth in of the mice and they were assigned to one of eight treatment groups: positive controls that were reimplanted with a new 2-mg E pellet, negative controls that were fed AIN 93G rodent diet alone, LSI containing 15 ppm genistein, MSI containing 150 ppm genistein, The estrogenic activity of isoflavones has been well documented in HSI containing 300 ppm genistein, LG AIN 93G ϩ 15 ppm genistein, MG AIN in vitro, in vivo, and clinical studies. Genistein can bind to the 93G ϩ 150 ppm genistein, and HG AIN 93G ϩ 300 ppm genistein. Tumors from each estrogen receptor ␣ with an affinity 100-1000 times lower than E2 treatment group were evaluated by measuring tumor area. Measurements from all of thetumors in each treatment group were combined and averaged. Bars, SE.
(21). Additionally, in ovariectomized Sprague Dawley rats, dietary SOY PROTEIN AND E2-DEPENDENT BREAST CANCER TUMOR GROWTH growth of MCF-7 cells in vitro (2, 5) and in vivo at 1 ␮M (5). InMCF-7 cells, at concentrations from 20 –90 ␮M genistein inhibitedDNA synthesis, but stimulated DNA synthesis at concentrations of0.1–10 ␮M (31). In this study we demonstrated that dietary genisteinconsumed at 15 ppm, whether from soy protein or as a pure com-pound, does not stimulate growth that is significantly different fromthe negative control group lacking any form of estrogen exposure inregard to stimulation of MCF-7 tumor growth. These results suggestthat there is a level of dietary genistein that will not lead to an increasein tumor growth. Additional investigation will be necessary to deter-mine the concentration of this threshold dose.
Numerous studies have focused on the chemopreventative effects of genistein and demonstrate that if exposed to isoflavones beforepuberty a rat has a reduced risk of developing breast cancer when Fig. 4. Effect of soy and genistein on the cellular proliferation within MCF-7 tumors.
Tumors were removed from the mice for immunohistochemical analysis. Incorporation of exposed to a chemical carcinogen (8 –10). It is reasonable then to BrdUrd into cellular DNA was used as a marker of cellular proliferation. Immunohisto- assume a similar protective effect might be seen in women if they are chemistry was used to stain for cells containing BrdUrd. Positively staining as well as exposed to isoflavones before puberty. Early exposure of women in background cells were counted to give a final count on both proliferating and total cellsin a given area of tissue. Cell counts from each treatment group were then combined and Asia to isoflavones may explain in part why Asian women have a averaged (n ϭ 25 fields/five tumors/treatment group). The data are presented as the lower rate of breast cancer incidence when compared with American percentage of cells actively proliferating in a given area of tissue. Bars, SE.
women. It is hypothesized that a high soy diet throughout life protectsagainst development of breast cancer. This idea is supported by the genistein (750 ppm) enhanced lobular-alveolar mammary gland de- fact that upon moving to the United States, Asian women have a velopment, increased uterine weight, and at 750 ppm, increased pitu- similar breast cancer rate as that of American women after two itary prolactin secretion and serum prolactin levels. In the uterus, generations, indicating early exposure is critical (32–34). In evaluat- dietary genistein increased c-fos mRNA expression (21). In vitro ing the correlation between urinary phytoestrogen levels and breast studies showing an increase in the levels of the estrogen-responsive cancer incidence in postmenopausal women, a recent report showed genes pS2 and c-fos when cells are treated with genistein, provide high urinary genistein excretion was weakly and nonsignificantly additional evidence of the estrogenic property of genistein (5, 22).
associated with a reduced breast cancer risk (35). Another study Clinical studies have also confirmed the preclinical studies evaluating shows no effect of soy consumption on the progression of chemically the estrogenicity of genistein. Premenopausal women consuming tex- induced mammary tumors in the rat (11). However, it is important to tured vegetable protein containing 45 mg of isoflavones had pro- note that in this study animals were intact with normal circulating E .
longed menstrual cycles. Additionally, the follicular phase was ex- Paradoxically, we have shown that, in ovariectomized mice trans- tended by suppression of the normal surge of follicle-stimulating planted with human estrogen-dependent breast cancer cells, genistein hormone and luteinizing hormone (23, 24). These data in conjunction and now soy protein isolates containing genistein stimulated tumor with the observations that dietary soy increases cell proliferation in growth (5). Ovariectomized athymic mice implanted with MCF-7 human breast tissue (6) and increases pS2 expression (7) demonstrates cells are an appropriate model of postmenopausal women with estro- biological (estrogenic) activity in humans.
gen-dependent breast cancer. Plasma levels of E in ovariectomized Isoflavones are being marketed to postmenopausal women in both athymic mice are 27–38 pg/ml compared with postmenopausal supplement form and in soy food products for the relief of the women 10 – 40 pg/ml (36). We believe the low endogenous estrogen symptoms of menopause. It is, therefore, critical to understand what environment created in this model by ovariectomizing the mice activities genistein may have in these women. Consideration must be affords genistein the opportunity to be a significant source of estro- given to how genistein may influence the endogenous levels of serum genicity in these animals and, hence, allows for the compound to E in women consuming the compound. There is evidence suggesting stimulate the growth of these tumors. Therefore, if a postmenopausal diets high in soy have the ability to lower serum E concentrations woman with low endogenous E levels has an existing estrogen- (25, 26). A study supporting this finding showed that normally cycling dependent breast tumor it is also possible that consumption of dietary women consuming a soy diet containing ϳ154 mg total isoflavones/ genistein from various food sources including soy protein isolate may day had a 25% reduction in circulating E (27). Contrarily, other produce sufficient plasma levels of genistein to result in enhanced studies in which premenopausal women have consumed soy have estrogen-dependent tumor growth as observed in athymic mice. These found no change in serum E concentrations (6, 28), and one study results collectively suggest that the time of exposure to genistein is showed increased serum E in women consuming soy (23). It is critical to the overall effect genistein will have on estrogen-dependent unclear how isoflavones in soy effect normal circulating E concen- trations in premenopausal women. Postmenopausal women consum- From the discussion presented here it is clear that the isoflavones ing a soy diet have been shown to have no change in serum circulating possess diverse biological activities and potency. These activities are E levels (28). This finding in conjunction with reports that genistein often dependent upon the concentration and timing of administration acts estrogenically, and postmenopausal women naturally have low of the isoflavones. As a result, it is important to focus on what plasma concentrations of E , suggest that in a postmenopausal woman, activities occur at plasma concentrations that are relevant to what is these weak estrogens may have significant estrogenic activity.
observed in humans consuming isoflavone containing diets. In regard At concentrations above 10 ␮M genistein has been demonstrated to to breast cancer, the isoflavones, specifically genistein, have paradox- inhibit in vitro cell proliferation in a variety of cell types including ical effects that can be resolved when you consider dosage and timing estrogen-dependent (MCF-7) and estrogen-independent (MDA-468) of administration. For example, prepubertal exposure to genistein human breast cancer cells (29). Genistein has also been shown to appears to be protective against the development of breast cancer, but inhibit activities of protein tyrosine kinase at concentrations Ͼ20 ␮M consumption of the phytoestrogen in either pure form or in soy protein (30). However, at levels as low as 200 nM, genistein stimulates the isolate, after development of an estrogen-dependent breast cancer may SOY PROTEIN AND E2-DEPENDENT BREAST CANCER TUMOR GROWTH enhance the growth of that tumor as determined by this study. Addi- and provide an in vivo model to assess the responsiveness of cells to estrogen.
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