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Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801 and
National Center for Toxicological Research, Jefferson, AR 72079
*
2To whom correspondence should be addressed. E-mail: helferic{at}uiuc.edu
ABSTRACT
Previously our laboratory has shown that the soy isoflavone, genistein,
stimulates growth of human breast cancer (MCF-7) cells in vivo and in
vitro. In this study, the dose-response analysis of genistein at
the physiologically achievable concentration range between 125 and
1,000 µg/g in the diet was conducted in ovariectomized
athymic nude mice implanted with MCF-7 cells. We hypothesized that
genistein at this concentration range can stimulate
dose-dependently the breast tumor growth, cell proliferation and an
estrogen-responsive pS2 gene induction. Tumor size and body weight
were monitored weekly. At completion of the study, we analyzed cellular
proliferation of tumors using incorporation of BrdU, pS2 expression of
tumors using a Northern blot analysis and total genistein level in
plasma using liquid chromatography–isotope dilution mass spectrometry
(LC-ES/MS). Dietary genistein (
250 µg/g) increased tumor
size in a dose-dependent manner [8.4x the negative control (NC)
group in the 250 µg/g group, 12.0x in the 500
µg/g group, 20.2x in the 1,000 µg/g group
and 23.2x in the positive control (PC) group]. The percentage of
proliferating cells was significantly increased by genistein at and
above 250 µg/g (5.3x the NC group in the 250
µg/g, 5.6x in the 500 µg/g, 5.0x in the
1,000 µg/g and 4.8x in the PC group). Expression
of pS2 mRNA was also significantly increased with increasing dietary
genistein levels (11.25x the NC group in the 500 µg/g
group and 15.84x in the 1,000 µg/g group). Total plasma
genistein concentrations were between 0.39 and 3.36 µmol/L
in mice fed between 125 and 1,000 µg/g genistein. In
conclusion, dietary treatment with genistein at physiological
concentrations produces blood levels of genistein sufficient to
stimulate estrogenic effects, such as breast tumor growth, cellular
proliferation and pS2 expression in athymic mice in a
dose-responsive manner similar to that seen in vitro.
KEY WORDS: • genistein • MCF-7 • athymic nude mouse • pS2 • BrdU
The soy isoflavone, genistein, has been extensively evaluated using in
vitro, preclinical and clinical studies to determine its role in the
prevention and treatment of chronic diseases. Genistein has numerous
biological activities including inhibition of tyrosine kinase
(1
) and DNA topoisomerases I and II (2
, 3
),
antioxidant activity (4
) and estrogenic activity. The
estrogenic activity of genistein has been the reason for its use as a
dietary supplement for postmenopausal women. Genistein binds to the
estrogen receptor
(ER)3
with an affinity 20–100x lower than estradiol (5
–10
).
Genistein is also estrogenic in estrogen-dependent human breast
cancer (MCF-7) cells and can stimulate MCF-7 cell proliferation and
induce an estrogen-responsive gene pS2 at 0.1–1.0
µmol/L (9
, 11
–13
). In vivo genistein can
enhance uterine wet weight in rodents (10
, 14
, 15
).
Additionally, genistein and other isoflavones are known to cause
reproductive failure in sheep consuming subterranean clover
(16
, 17
). Genistein has also been shown to inhibit
7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary cancer by
increasing mammary gland differentiation in Sprague-Dawley rats
(18
). Lamartiniere et al. (19
) demonstrated
that early exposure to genistein promotes cell differentiation
resulting in a less active epidermal growth factor signaling pathway in
adulthood, which suppresses the development of mammary cancer. Whereas
Cohen et al. (20
) showed that there was no difference in
N-nitroso-N-methylurea (NMU)-induced mammary
tumorigenisis (on 50 d) between soy protein and
isoflavone-depleted soy protein–fed animals (from 1 wk before NMU
injection until 18 wk after the injection). These data suggest that
there may be a difference on mammary tumorigenesis dependent on the
timing of genistein administration. Hilakivi-Clarke et al.
(21
) gave direct evidence of a critical role of the timing
of exposure.
The biological activity of genistein may be different once an
estrogen-dependent cancer exists. We have demonstrated that
genistein at 750 µg/g can stimulate growth of MCF-7 tumors in
ovariectomized athymic mice (11
). The dosage utilized in
that study produced a plasma genistein level of 
2.1
µmol/L, similar to what is observed in humans consuming a
diet containing soy products (22
). In fact, when a large
amount of an isoflavone containing product is consumed, the levels can
reach 6 µmol/L (23
, 24
).
In vitro, genistein (10 µmol/L) inhibits the growth of
both ER-dependent and ER-independent breast cancer cells
(10
, 11
, 13
). Animal studies have shown that genistein
inhibits mammary tumor promotion (25
, 26
).
Cassidy et al. (27
) showed that a daily intake of soy
products (containing 50 mg of isoflavone) prolonged the menstrual
cycle, which may be related to lower breast cancer risk in
premenopausal women. Lu et al. (28
) showed that
consumption of isoflavone-containing soy diet (113–202 mg/d of
total isoflavones) for one menstrual cycle reduced levels of ovarian
steroids. Intake of soy protein beverage (containing 42 mg genistein
and 27 mg daidzein) for 6 mo increased nipple aspirate fluid volume in
premenopausal women but not in postmenopausal women (29
).
McMichael-Phillips et al. (30
) reported that
short-term dietary soy stimulates breast cancer cell proliferation.
Another study in humans showed an increase in pS2 expression after soy
consumption (31
). Together these studies suggest that
genistein is estrogenic in vitro, in animals and humans. It is possible
that postmenopausal women with estrogen-dependent breast cancer
consume diets high in genistein or soy products, believing that the
phytochemical may be beneficial for prevention of breast cancer when in
fact circulating genistein may stimulate growth of an existing
estrogen-responsive breast tumor.
We have demonstrated that genistein (
5 µmol/L) induced
cell proliferation of MCF-7 cells and pS2 gene expression. Our previous
study showed that 750 µg/g dietary genistein
(11
) enhanced tumor growth and epithelial proliferation in
the mammary gland in ovariectomized athymic nude mice transplanted with
MCF-7 cells. In this study, we tested the hypothesis that dietary
treatment with a broader concentration range of genistein would
stimulate growth of human estrogen-dependent breast cancer cells
transplanted into athymic mice in a dose-responsive manner similar
to that seen in vitro.
MATERIALS AND METHODS
Materials.
Improved Minimal Essential Medium (IMEM) (without gentamicin and with
glutamine) and phenol red–free IMEM were purchased from Biofluids
(Rockville, MD). Bovine calf serum (BCS) was purchased from Hyclone
(Logan, UT). Penicillin and streptomycin, trypsin/EDTA and Random
Primer DNA Labeling System were purchased from Gibco-BRL (Grand
Island, NY). [
-32P]dCTP was purchased from Amersham
(Arlington Heights, IL). Reagents for Northern blot assay and BrdU
incorporation were purchased from Amersham, Fisher Scientific (Fair
Lawn, NJ.), Gibco-BRL and Sigma Chemical (St. Louis, MO).
Athymic nude mice.
Female athymic BALB/c (nude) mice were purchased from Charles River Laboratories (Wilmington, MA) and acclimated for 1 wk. Mice were ovariectomized at 21 d of age by the vendor and were allowed to recover for 7 d.
Estrogen pellet preparation.
An estradiol pellet contained 2 mg of 17ß-estradiol and 18 mg of
cholesterol. A 17ß-estradiol pellet was placed under the skin of each
mouse before MCF-7 cells were transplanted into the animal
(32
).
Maintenance of human breast cancer cells.
MCF-7 cells were maintained in IMEM supplemented with 5% BCS, 1% pen
(1,000 U/L)/strep (1 mg/L) and 1 nmol/L 17ß-estradiol. MCF-7 cells
were maintained at 37°C in a humidified atmosphere of 5%
CO2 in air as a monolayer culture in plastic culture plates
(100 mm diameter). One week before the injection of MCF-7 cells, the
media was switched to phenol red–free IMEM containing 5%
charcoal-dextran–stripped BCS (CD-BCS) (33
) and 1%
penicillin/streptomyocin.
Diet formulation.
AIN-93G semipurified diet (Dyets, Bethlehem, PA) was selected as a base
diet for control mice as it has been established to meet all of the
nutritional requirements of mice (34
). Soy bean oil was
removed from all diets and corn oil was added to eliminate any
additional components of soy being added to the diets. Treatment mice
were fed AIN-93G diet plus different dosages of genistein (125, 250,
500 or 1,000 µg/g).
Analysis of tumor growth.
One week after the estrogen pellets were inserted into mice, MCF-7
cells were harvested using 500 µL trypsin-EDTA
(0.5% Trypsin and 5.3 mmol/L EDTA · 4Na) (Gibco-BRL) per 100 mm
culture plate. Cells were adjusted to 1 x 105 cells
per 40 µL of Matrigel (Collaborative Biomedical
Products, Bedford, MA) and injected at 40 µL per site
into each of the four flanks of the athymic mice. Tumors were grown
until their average cross-sectional area reached 38
mm2. One week before grouping, animal diets were switched
to the casein based AIN-93G diet. Mice were divided into six treatment
groups (10–11 mice/group): negative control (NC), positive control
(PC) (estradiol control), and 125, 250, 500 and 1000
µg/g of genistein treatment groups. Estradiol pellets
were removed from all mice except the PC group, and dietary genistein
treatment was started. Tumor growth and body weight were measured
weekly for 22 wk, and cross-sectional area was determined using the
formula [length/2 x width/2 x 
]
(11
, 32
, 35
). Food intake was measured once throughout the
study. At the end of the study, BrdU incorporation was used as an index
of cell proliferation. Four hours before termination, 50 mg BrdU in
phosphate-buffered saline (PBS)/kg body wt were injected
intraperitoneally (36
). Mice were anesthetized and killed
by cervical dislocation. Tissues and blood samples were collected for
analysis. Some tumors were removed and fixed in 10% formalin for cell
proliferation assay; others were stored in liquid nitrogen for RNA
analysis. The protocols used in these experiments were approved by the
Institutional Animal Care and Use Committee at University of Illinois
at Urbana-Champaign.
Cell proliferation.
Cell proliferation in tumors was determined using immunohistochemical
analysis. BrdU incorporation into cellular DNA was used as an indicator
of cells that were actively proliferating (37
). We
utilized a method that we have determined to accurately identify
proliferating cells in tumors in the transplant model that is an
adaptation of a method previously described for use in other models
(38
). Tumors in 10% formalin were embedded in paraffin
blocks, cut into 5-µm sections and placed on
microscope slides. Slides were deparaffinized by immersing in xylene
twice for 12 min each and hydrated by immersing in a series of 100%
ethanol, 95% ethanol, and three times in dH2O for 5 min
each. To block endogenous peroxidase, slides were immersed in 0.3%
H2O2 for 20 min then washed with
dH2O. Slides were placed in citrate buffer (pH 6) and
microwaved for 20 min and cooled at 24°C. Then slides were washed in
PBS (pH 7.4) for 5 min. Anti-BrdU primary antibody (Amersham,
Arlington Heights, IL) was added to slides and incubated for 1 h
at 24°C in a humidity chamber. Slides were washed in PBS and 50
µl of goat antimouse secondary antibody (Sigma
Chemical) was added to slides and incubated for 30 min at 24°C.
Slides were then washed in PBS. One drop of 3,3'-diaminobenzidine
tetrahydrachloride (DAB) and Ni enhancer mix prepared right before use
was added to each slide. Slides were then washed twice in
dH2O and PBS and were counterstained with 20% hematoxlin
for 1 min. The slides were then dehydrated by immerging them in 80%
ethanol for 5 min, 95% ethanol for 5 min and 100% ethanol for 5 min
followed by immerging them in xylene four times for 5 min each. Slides
were mounted and analyzed by light microscope. Both positive and
background stained cells were counted in a given area of tissue. The
data were then presented as a percentage of cells proliferating in a
given area of tumor.
RNA preparation.
RNA from frozen tumor was isolated using Trizol reagent and a protocol.
Frozen tumor (200 mg) from liquid nitrogen was smashed using a
BioPulverizer (Thomas Scientific, Swedesboro, NJ), and a coarse tumor
powder was transferred into 2 mL Trizol (Gibco-BRL). The coarse tumor
powder was homogenized using a Polytron-Aggregate (Littau, Luzern,
Switzerland). Chloroform (400 µL) was added
into a homogenized tumor sample and was incubated for 10 min at 24°C.
The reaction tube was centrifuged at 12,000g for 15 min
at 4°C. The upper part was taken out and transferred into a fresh
tube. Equal volume of isopropyl alcohol was added and incubated for 10
min at 24°C. The mixture was centrifuged at 12,000g
for 10 min at 4°C. The RNA pellet was washed with 1 mL ice-cold
75% ethanol and centrifuged at 7,500g for 5 min at
4°C. The RNA pellet was air-dried and dissolved with
RNase-free dH2O. RNA was stored at -80°C. RNA
concentration was measured at 260 and 280 nm (1 O.D.260
= 40 µg of single-stranded RNA/mL).
Estrogen-responsive pS2 mRNA expression.
For the analysis of induction endogenous estrogen-regulated gene
expression, we utilized a Northern blot assay, as described in Ju et
al. (33
). pS2, a reliable marker for estrogenicity, was
used as a marker gene, and the GADPH cDNA probe was used as a standard.
Plasma analysis.
Blood samples were collected by cardiac puncture and placed into
EDTA-containing tubes and centrifuged at 500g for 5
min. Aliquots (100 µL each) of plasma were stored at
-20°C until analyzed. Plasma samples were analyzed by liquid
chromatography–and isotope dilution electrospray mass spectrometry
(LC-ES/MS) using a modification of the method previously described by
Chang et al. (39
). Offline solid-phase extraction
(SPE) was substituted for online SPE, and the performance
specifications were comparable (i.e., intraday and interday
precision/accuracy in the range of 5–10%).
Statistics.
Data from tumor area, cell proliferation, Northern blot analysis and plasma analysis were analyzed accordingly using one-way or repeated-measures analysis of variance according to the characteristics of the data set using the SAS program. If the overall treatment F ratio was significant (P < 0.05), the differences between treatment means were tested with Fisher’s LSD test.
RESULTS
Tumor growth.
When tumors reached an average cross-sectional area of 38
mm2, mice were divided into six treatment groups
(NC, PC, and 125, 250, 500 and 1,000 µg/g genistein).
Tumors in the PC group (which still had estradiol implants) reached
141.5 mm2 4 wk after the dietary treatment was
initiated in the other groups, and mice were terminated due to tumor
burden. Tumors in the rest of the treatment groups were monitored for
22 wk (Fig. 1
). Body weight was also monitored weekly, and no significant difference
was observed among the treated and control groups (data not shown).
Food intake was measured during the study, and no significant
difference was observed among any of the treatment groups (data not
shown). Tumors in the NC and 125 µg/g groups regressed
after the estradiol pellets were taken out. The average tumor size of
the 125 µg/g treatment group did not differ from the NC
group (3.0-fold that of the NC group) (Fig. 2
). Tumor area size of the other treatment groups were different from the
NC group (by 8.4x the NC group in the 250 µg/g group,
12.0x in the 500 µg/g or 20.2x in the 1,000
µg/g and 23.2x in the PC group, P < 0.05).
|
|
To evaluate the cellular proliferation effects induced by dietary
genistein, BrdU incorporation was used (Fig. 3
). Genistein at or above 250 µg/g stimulated cellular
proliferation of MCF-7 cells implanted in athymic mice at a level
similar to that of the PC group. There were significant differences in
cell proliferation induced by genistein (250 µg/g) and
the PC group when compared with the NC group (5.3x the NC group in the
250 µg/g, 5.6x in the 500 µg/g, 5.0x in the
1,000 µg/g and 4.8x in the PC group).
|
pS2 is a well-established estrogen-responsive gene and an excellent
marker for estrogen-dependent growth. To evaluate the ability of
genistein to stimulate the expression of an estrogen-responsive pS2
gene, mRNA from tumors were analyzed using a Northern Blot assay
(Fig. 4
top). pS2 expression is presented as the relative pS2 mRNA level.
Expression of pS2 mRNA by the 125 or 250 µg/g dietary
genistein groups was not statistically different from the NC group
(1.02x the NC group in the 125 µg/g and 4.65x in the 250
µg/g). Genistein treatment (at or above 500
µg/g) increased pS2 expression (11.25x the NC group in
the 500 µg/g, 15.84x in the 1,000 µg/g and
20.15x in the PC group). There were no differences in GAPDH mRNA
levels among the treatment groups (data not shown).
|
Total genistein (aglycone + conjugated forms) concentration in plasma
was measured using LC-ES/MS. While no genistein was detected in plasma
from NC mice (detection limit 0.02 µmol/L), the
concentration of total genistein in plasma from animals treated ranged
from 0.39 to 3.36 µmol/L (Fig. 5
).
|
DISCUSSION
The purpose of this study was to determine the effects of
long-term exposure to the dietary soy isoflavone, genistein, at a
broad concentration range on the growth of estrogen-dependent human
breast cancer cells implanted in athymic nude mice. The animal model
used in this study, ovariectomized athymic mice implanted with MCF-7
cells, is an appropriate model of postmenopausal women with
estrogen-dependent breast cancer. MCF-7 cells are
estrogen-dependent tumor cells isolated from postmenopausal women
(40
). Additionally, blood estradiol levels in
ovariectomized athymic mice are 10 pmol/L, which is similar to that
observed in postmenopausal women (41
, 42
).
Genistein binds to ER and ERß with affinities of 4 and 87%,
respectively, that of estradiol (5
–8
), and its induction
of ER-dependent transcriptional expression characterizes
genistein as an agonist for ER and ERß (43
, 44
). The
concentration of genistein in individuals consuming soy products is
nearly 1,000-fold higher than levels of endogenous estradiol in
premenopausal women (5
, 45
, 46
), suggesting that these ER
agonists have significant estrogenic potential due to the high
concentration present in isoflavone-containing foods. There is
considerable controversy regarding whether genistein can alter the
growth of an estrogen-dependent breast tumor in women. Breast
cancer rates increase with age and the majority of women with breast
cancer are postmenopausal. The estrogenic isoflavones and, in
particular, genistein are consumed by postmenopausal women for relief
of menopausal symptoms. This additional estrogen agonist may pose a
risk to postmenopausal women if they are at a high risk for breast
cancer. There are very few preclinical studies in the scientific
literature that address the effect of genistein on
estrogen-dependent breast cancer. We conducted a dietary
dose-response study to evaluate various dietary dosages of
genistein for its ability to stimulate growth of estrogen-dependent
breast cancer cells implanted in ovariectomized athymic mice.
We have demonstrated that dietary concentrations of 125–1,000
µg genistein/g of diet stimulate MCF-7 tumor growth in
athymic mice in a dose-dependent manner. It is very important to
note that dietary genistein is estrogenic; however, it is much less
potent than the implanted estradiol pellet. Because genistein can be
consumed in high amounts from soy diets or isoflavone supplementation,
it is possible to consume sufficient quantity of genistein to stimulate
estrogen-dependent tumor growth. In this study, dietary genistein
was fed at 125, 250, 500 and 1,000 µg/g. Tumors began to
grow in the treatment groups 10 wk after the removal of the estradiol
pellet (Fig. 1)
. As the level of genistein was increased in the diet,
growth of MCF-7 tumors increased in a dose-dependent manner and
tumor growth was significantly increased in mice consuming genistein at
and above 250 µg/g (Fig. 2)
.
Dietary genistein also increased tumor cell proliferation as indicated
by enhanced BrdU incorporation. However, cellular proliferation in the
breast tumors was not increased in a dose-dependent manner, and the
induction of cell proliferation by genistein at and above 250
µg/g was significantly different from the NC group (Fig. 3)
. Tumor growth is a function of numerous cellular mechanisms,
including proliferation and apoptosis. We only measured cell
proliferation. Other mechanisms, such as apoptosis, may contribute to
this difference. We and others (9
, 11
) have demonstrated
using cultured MCF-7 cells that genistein can stimulate cell
proliferation in a dose-dependent manner in vitro. The
concentrations of genistein in these studies were between 0.2 and 1
µmol/L, concentrations similar to those of genistein in
the plasma of mice consuming dietary genistein at 125–1,000
µg/g, as used in this study. Our data obtained from this
athymic mice model are consistent with estrogenic effects of genistein
(1 µmol/L) in vitro.
There was a threshold in expression of the estrogen-responsive pS2
gene, which was similar to the results observed with cellular
proliferation. Expression of pS2 was significantly (P
< 0.05) higher in mice consuming 500 and 1,000 µg/g
compared with the NC group, suggesting that the increase in MCF-7 cell
proliferation was likely due to an estrogenic effect. Expression of pS2
was very low in the 125 µg/g genistein–fed mice (0.12%
of PC). Although pS2 expression induced by 250 µg/g
genistein (23.1% of PC) was higher than the NC group, the difference
was not significant due to experimental and animal-to-animal variation
(Fig. 4)
.
The concentration of genistein circulating in plasma of women consuming
soy products is 0.74–6.0 µmol/L
(5
, 13
, 24
, 25
, 45
, 47
, 48
). In this study, similar levels of
total genistein (0.39–3.36 µmol/L) in plasma were
detected in mice fed 125–1,000 µg/g genistein (Fig. 5)
. It is
important to note that only a small percentage of genistein (5% of
total genistein) circulating in plasma is present as the aglycone in
mice (49
), rats (50
) and humans
(51
)and only the aglycone is estrogenically active. It has
been observed that the aglycone bioconcentrated in rat tissues to
levels known to activate the ER (39
). This same mechanism
may be important in determining the effective dose required to produce
tumor levels sufficient to stimulate tumor growth.
Genistein has multiple biochemical effects and can act as an estrogen
at low concentrations (5 µmol/L) and as an
antiproliferative agent at high concentrations (10
µmol/L). Therefore, it is very important to determine how
various intakes of dietary genistein and the plasma concentrations
produced are related to biological activity in humans. Blood levels in
mice increased in a dose-dependent manner with increasing
concentrations of genistein in the diet. These data are consistent with
the treatment-dependent changes observed for tumor growth, cell
proliferation and pS2 expression, suggesting that genistein stimulated
estrogen-dependent tumor growth in a dose-dependent manner. The
dietary dosages in this study produced blood levels that are consistent
with those measured in humans. Plasma levels in humans consuming a diet
high in soy protein can be as high as 6 µmol/L, depending
on the type and amount of soy product consumed (24
). Cell
culture studies have indicated that genistein in the media at
concentrations of 0.2–1.0 µmol/L can increase
estrogen-dependent MCF-7 cell proliferation in a dose-dependent
manner, reaching proliferation rates similar to that observed with 1
nmol/L estradiol (9
, 11
). The enhanced tumor growth is
likely due to increased cellular proliferation as evaluated by
increasing BrdU incorporation. In culture, genistein increased cellular
proliferation as indicated by an increase of cells in S phase of the
cell cycle (12
). This increase in cell proliferation is
likely due to the estrogenic activity of genistein. Hsieh et al.
(11
) demonstrated that genistein at concentrations as low
as 0.1 µmol/L stimulate the estrogen-responsive gene
pS2. Here we have observed that dietary genistein also stimulates pS2
expression in MCF-7 cell tumors in vivo.
In summary, dietary genistein at and above 250 µg/g produces sufficient circulating blood levels of total genistein that are sufficient to stimulate growth of estrogen-dependent human breast tumor (MCF-7), enhance cellular proliferation in vivo and induce estrogen-responsive pS2 expression in vivo. The results of this study indicate that genistein can stimulate growth of estrogen-dependent tumors in athymic mice over a wide range of dietary dosages that have relevance to human exposure by postmenopausal women with estrogen-dependent breast cancer.
FOOTNOTES
1 Supprted by Grant CA77355 (to W.G.H.) from the
National Institutes of Health. 
3 Abbreviations used: BCS, bovine calf serum;
BrdU, bromodeoxyuridine; CD-BCS, charcoal-dextran–stripped BCS;
DAB, 3,3'-diaminobenzidine tetrahydrachloride; DMBA,
7,12-dimethylbenz(a)anthracene; ER, estrogen receptor; IMEM, Improved
Minimal Essential Medium; LC-ES/MS, liquid chromatography–isotope
dilution electrospray mass spectrometry; NC, negative control; NMU,
N-nitroso-N-methylurea; PBS,
phosphate-buffered saline; PC, positive control; pS2, presenelin-2,
SPE, solid-phase extraction.
Manuscript received 6 June 2001. Initial review completed 6 July 2001. Revision accepted 15 August 2001.
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