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*
Department of Food Science and Human Nutrition, Michigan State University, E. Lansing, MI 48824;
College of Health and Human Sciences, University of Southern Mississippi, Hattiesburg MS 39406; and
**
Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • MDA-MB-231 • athymic mice • genistein • breast cancer
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Miller 1977
, Wynder and Hirayama 1977
).
Incidence rates are as high as 1 in 8 in the United States and as low
as 1 in 30 in Japan. Furthermore, the incidence of breast cancer in
migrant Japanese women to the U.S. increases in the first and second
generations of offspring to approach that in the U.S. (Buell 1973
, Hirayama 1978
). These data suggest
environmental factors, such as diet, may play a primary role in the
onset and development of breast cancer.
Soy products contain a number of compounds with the potential to
inhibit carcinogenesis, including protease inhibitors (Kennedy and Manzone 1995
), phytates (Shamsuddin 1995
)
and isoflavones (Barnes et al. 1990
). Studies presented
in this report focus on genistein, which is present in soy products at
concentrations as high as 1.5 mg/g (Coward et al. 1993
,
Eldridge and Kwolek 1983
, Murphy and Wang 1993
).
Genistein is chemopreventive in the carcinogen-induced rat breast
cancer model. When, genistein is administered prepubertally to rats, a
reduction in dimethylbenzanthracene-initiated tumor number is
observed. Additionally, this reduction in tumor number has been
observed when genistein was injected (Lamartiniere et al. 1995
, Murrill et al. 1996
) or fed at 250
µg/g in the diet (Fritz et al. 1998
). We
have demonstrated (Hsieh et al. 1998
) that dietary
genistein can stimulate growth of existing estrogen-dependent
tumors in ovariectomized athymic mice. Thus it is possible that
genistein can be preventative when administered early in an animal’s
life (before a chemical carcinogen) but can stimulate growth of an
existing estrogen-dependent tumor.
Genistein inhibits the proliferation of a number of transformed cell
lines in cell culture. Inhibition of cell growth by genistein may be
due to inhibition of topoisomerase II (Markovits et al. 1989
, Okura et al. 1988
) and protein tyrosine
kinases (Akiyama et al. 1987
, Geissler et al. 1990
). Genistein concentrations as low as 12
µmol/L can inhibit topoisomerase II activity, whereas
concentrations as low as 3 µmol/L inhibit tyrosine kinase
activity. Both of these enzymes are involved in cell proliferation.
Concentrations of genistein within this range have been shown to
inhibit proliferation of a number of tumor cell lines, e.g., genistein
inhibited MCF-7 adriamycin resistant, MCF-7/WT and MDA-MB-231 human
breast cancer cells with concentrations of genistein that inhibit 50%
of cell proliferation
(IC50)3
of between 7 and 37 µmol/L (Monti and Sinha 1994
), MCF-7 and MDA-468 human breast cancer cells with
IC50 of 24–44 µmol/L
(Peterson and Barnes 1993
), stomach and colon cancer
cell lines with IC50 of ~25 µmol/L
(Yanagihara et al. 1993
) and AGS human gastric cells
with an IC50 of between 7 and 23
µmol/L (Piontek et al. 1993
).
Genistein (60 µmol/L) has been shown to reversibly arrest
cell cycle progression of human gastric cancer (HGC-27) cells at
G2/M (Matsukawa et al. 1993
) and
of Jurkat T-leukemia cells at G2/M
(18.5–37.0 µmol/L), whereas higher doses (74–110
µmol/L) blocked cell cycle progression through the S phase
(Spinozzi et al. 1994
). Blocking progression of the cell
cycle is likely due to the inhibition of tyrosine kinase activity by
genistein at key regulatory points in the cell cycle, thus preventing
progression through mitosis, resulting in an inhibition of cell
proliferation.
In this study, the effect of genistein on the cell cycle and proliferation of estrogen receptor–negative human breast cancer (MDA-MB-231) cells was investigated in culture. It is important to point out that the concentration of genistein required to inhibit cancer cell growth is high, usually >10 µmol/L. Whether dietary genistein can produce plasma levels of genistein equivalent to those required to inhibit tumor cell growth in vitro was also examined. Studies are also presented on the effect of dietary genistein on growth of MDA-MB-231 cells in female athymic mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genistein was synthesized from organic precursors as described by
Chang et al. (1994)
. Chemical identity was assessed by
nuclear magnetic resonance and purity assessed at >98%. Analytical
grade reagents were used for HPLC analysis. All other chemicals, unless
otherwise specified, were purchased from Sigma Chemical (St. Louis,
MO).
Animals and diets.
Athymic female mice were purchased from Harlan Sprague Dawley
(Indianapolis, IN). In all experiments, mice were housed three or four
to a cage. Athymic mice were kept under aseptic conditions (enclosed
laminar flow hood, sterilized cages, bedding and water). All mice were
kept in a temperature-controlled (22 ± 2°C), relative
humidity–controlled (40–70%) and light-controlled (12-h
light:dark cycle) animal facility. All mice were fed the AIN-93G
semipurified diet (corn oil replaced soybean oil) containing genistein
at the levels specified below (Reeves et al. 1993
).
Tetracycline (500 mg/L) was added to the drinking water.
Cell proliferation.
MDA-MB-231 human breast cancer cells were obtained from Dr. Clifford Welsch at Michigan State University. Cells were maintained in minimal essential medium (MEM) with the following: Na-bicarbonate, 2.2 g/L; L-glutamine, 0.292 g/L; fetal bovine serum (FBS), 100 mL/L (10%); Na-pyruvate, 8.8 mg/L; bovine insulin, 10 mg/L; penicillin, 10000 U/L; and streptomycin 10 mg/L. Cells were collected from 100 mm x 20 mm tissue culture plates at 80% confluence by washing two times with 1X PBS followed by trypsinization. Cells were counted using a hemocytometer and the cell suspension diluted to ~5 x 109 cells/L.
The cells were plated in 24-well plates at 15,000 cells per well and incubated for 16 h in maintenance medium (described above). After 16 h, the medium was removed and replaced with similar maintenance medium but with only 1 mL/L FBS. Cells were incubated for 48 h after which the medium was replaced with fresh maintenance medium (MEM + 10% FBS) containing genistein (in ethanol) at 0, 10, 20, 40 and 80 µmol/L. Ethanol vehicle for the treatments did not amount to >0.1% total volume of the medium in the well.
Cells were collected at 1, 3, 5 and 7 d for DNA analysis as
described by West et al. (1985)
with minor
modifications. For the DNA assay, the medium was removed, cells washed
two times with 1X PBS and then lysed with 2.7 mL of 10 mmol/L EDTA, pH
12.3. After a 30-min incubation at 37°C, the pH was adjusted to 7.0
with KH2PO4 (~150 µL), and
20 µL (1000 ng dye) of a stock solution of
Hoechst-33258 dye (200 mg/L diluted 1:4) was added to each well.
Aliquots of 200 µL were transferred to a black 96-well
plate for reading on a fluorescent plate reader (Cytofluor II,
Perspective Biosystems, Framingham, MA.). The fluorophore was excited
at 350 nm and emission measured at 455 nm. Fluorescence values were
converted to DNA values on the basis of data from a DNA standard curve
made from salmon testes DNA.
Cell cycle and cell viability analysis.
MDA-MB-231 cells were collected from 100 mm x 20 mm tissue culture plates at 80% confluence by washing two times with 1X PBS followed by trypsinization. Cell number was determined using a hemocytometer and the cell suspension diluted to ~5 x 109 cells/L. Maintenance media (3 mL; described above) was added to each 60 mm x 15 mm tissue culture plate and 1 mL of the cell suspension added (500,000 cells/plate). The cells were incubated for 16 h and the medium removed and replaced with similar maintenance medium but with only 1 mL/L FBS. Cells were incubated for 48 h; the medium was then replaced with fresh maintenance medium (MEM + 10% FBS) containing genistein at 0, 10, 20, 40 and 80 µmol/L. The ethanol vehicle for the treatments did not amount to >0.1% total volume of the medium in the well.
Cells were collected at 12, 24, 48 and 72 h for cell cycle and viability analysis. Cells were collected by removing the medium from the tissue culture plates into flow cytometry tubes (13 mm x 75 mm), trypsinizing the cells, collecting them into the same tubes and centrifuging at 350 x g for 5 min. The supernatant fraction was aspirated and the pellets washed with MEM containing 10% FBS. For analysis of cell viability, two 90-µL aliquots were collected into 0.5-mL microfuge tubes, 10 µL of 40% trypan blue added, the samples incubated at room temperature for 5 min and cell viability determined by examining the ability of the cells to exclude trypan blue.
For flow cytometry, the remaining samples were centrifuged at 350 x g for 5 min and the supernatant removed. The cellular pellet was fixed in 1.5 mL of 70% ethanol and then placed at 4°C for 96 h. The cells were removed from 4°C, centrifuged at 350 x g, the ethanol removed and the cellular pellet washed with 1X PBS. The following flow cytometry staining reagents were then added: 945 µL reagent A (stock solution of reagent A containing 100 µL of 100 mmol/L EDTA, pH 7.4, 100 µL of Triton X-100 in 99.8 mL 1X PBS), 50 µL reagent B (stock solution of reagent B containing propidium iodide, 1 g/L in H2O) and 10 µL of RNase that had been boiled for 6 min. The tubes were gently vortexed and placed in the dark at 4°C until reading on the flow-activated cell sorter. Fluorescence was assessed by excitation with an Argon laser at 488 nm and the emission measured at 630 ± 10 nm. The percentage of cells in each phase of the cell cycle was calculated with MPLUS software (Phoenix, AZ).
Genistein fed to mice inoculated with MDA-MB-231 cells.
To evaluate the effect of dietary genistein on the growth of solid
tumor masses, the following study was conducted. Female athymic mice
(n = 30) were received at 3–4 wk of age and
allowed unrestricted access to water and the AIN-93G diet for 7 d
before subcutaneous injection of MDA-MB-231 cells. These cells had been
collected from 80% confluent 100 mm x 20 mm plates by washing
two times with 1X PBS, trypsinizing, and then collecting and pooling
the cells in MEM supplemented with 10% FBS. Cell counts were
determined using a hemocytometer and the cells diluted to 5 x 109 cells/L. The mice (n = 30) were
anesthetized and injected subcutaneously with 200 µL
of the cell suspension (106 cells/site), using a 1-mL
syringe with a 25-gauge needle (1.6 cm), in each of their four flanks.
After 5 wk, to equalize tumor number and cross-sectional area, the
mice were grouped into a 750 µg genistein/g AIN-93G
diet group (tumors = 27) and an AIN-93G diet control group (tumors
= 33) and treatment began. Only mice with tumors >3 mm x 3
mm (measured by caliper) at the time of grouping were used in the
study. Tumor length (l) and width (w)
were measured weekly for 5 wk and cross-sectional area determined
by the following formula (l/2) · (w/2) · 
. Food
intake was measured during the last 3 d of the study. At the end
of the study, mice were anesthetized, weighed, bled via cardiac
puncture and killed by overexposure to anesthesia.
To evaluate the effect of dietary genistein on initial tumor formation and growth, the following study was conducted. Female athymic mice were received at 3–4 wk of age and allowed unrestricted access to water and the AIN-93G diet for 7 d. One group of mice (n = 9) were then fed 750 µg genistein/g diet 3 d before injection of the MDA-MB-231 cells; the control group (n = 9) was fed the AIN-93G diet. The genistein-treated mice developed 25 tumors and the control group 32 tumors. Injection of tumor cells and tumor measurements were performed as described above.
Plasma genistein concentration in response to dietary genistein.
To assess plasma genistein concentration in mice fed different amounts of genistein, the following study was conducted. Female Balb/C mice (n = 36) were received at 4 wk of age and allowed unrestricted access to water and the AIN-93G diet. After 2 wk, mice were weighed, sorted three mice to a cage, two cages per group, and provided 6 g AIN-93G diet/(mouse · d). Cages and food cups were collected each day for 4 d, the bedding sifted for food and baseline control food intake determined. Genistein was then mixed into the AIN-93G diet in the following amounts: 0, 375, 750, 1500, 3000 and 6000 µg/g and fed to each group for 4 d. Food intake was determined each day as described above. After 5 d of genistein consumption, mice were anesthetized, weighed and plasma collected by cardiac puncture for genistein analysis.
Plasma genistein analysis.
Mice were anesthetized and bled by cardiac puncture. Blood (~800 µL/mouse) was placed in microfuge tubes containing 10 µL of 15% EDTA, centrifuged at 12,000 x g and the plasma removed and stored at -20°C. To determine conjugated and unconjugated genistein, 50 µL of plasma was divided into duplicate aliquots; one set received 5 µL (515 U) ß-glucoronidase Type H-1 (Sigma Chemical). All aliquots were incubated in 0.5-mL microfuge tubes at 37°C for 24 h. After the incubation, 50 µL of absolute methanol was added to each tube, the tubes vortexed and then centrifuged at 15,000 x g for 10 min. Approximately 75 µL was removed and placed at -20°C until analysis. For analysis of genistein, the microfuge tubes were centrifuged at 15,000 x g for 10 min and 20 µL of the content injected onto a C-18 column Rainin Instrument, Woburn, MA) with a flow rate of 1.0 mL/min of 50:50 methanol/water with 1 mL/L of acetic acid. Sample absorbance was monitored at 260 nm. Recovery was determined by adding a known amount of genistein (28 µL of 21.1 mg/L in ethanol) to 600 µL plasma from control mice and diluting this with plasma to a final concentration of 1.82 µmol/L genistein. Mean recoveries were determined to be 96 ± 5.87%. Genistein was not detected in the plasma of control mice fed the AIN-93G diet. Plasma genistein concentrations are reported only for samples in which genistein was detected. The level of detection for plasma genistein was ~130 nmol/L.
Statistical analyses.
All statistical tests were performed using a PC-based version of SPSS (Version SPSS/PC 9.0, Chicago, IL). Significant differences in tumor area, food intake per cage and mouse weight were assessed by Student’s t test at the end of each experiment. Significant differences in cell culture studies were assessed by ANOVA. When a significant (P < 0.05) treatment effect was detected, treatment means were compared using Tukey’s post-hoc comparisons. Significant differences in the genistein feeding study were assessed by ANOVA. Variances in plasma genistein concentration were nonhomogeneous with respect to treatment; thus, these data (excluding the 0 µg genistein/g diet group) were log transformed before ANOVA. When a significant (P < 0.05) treatment effect was detected, treatment means were compared using Tukey’s post-hoc comparisons. The 0 µg genistein/g diet group was compared with the 375 µg genistein/g diet by Student’s t test. Values in the text and figures are means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genistein inhibited cell proliferation by ~50% at a concentration of
~20 µmol/L (Fig. 1
). Genistein at 10 µmol/L had no apparent effect on cell
proliferation, whereas 20, 40 and 80 µmol/L resulted in a
dose-dependent decline in cell proliferation.
|
). Cell viability, as assessed by the cellular exclusion of trypan blue
dye, was > 86% at all concentrations of genistein.
|
Dietary genistein (750 µg genistein/g diet), whether fed
before or after the injection of tumor cells, did not significantly
(P > 0.05) alter the growth of tumors (Figs. 2
,
3
). Plasma genistein (free + conjugated) was 0.93 ± 0.11
µmol/L and the free form was 0.31 ± 0.14
µmol/L.
|
|
Effect of dietary genistein on plasma genistein concentration.
Plasma genistein increased in mice fed 0 to 6000 µg/g diet
in a dose-dependent manner with total plasma genistein
concentrations reaching 7 µmol/L (conjugated + free) at
the highest dosage. Free genistein in female mice fed 6000
µg/g diet was ~2 µmol/L (Fig. 4
).
|
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and that
the probable mechanism is inhibition of the cell cycle at
G2/M (Table 1)
. We have also shown that 40 and 80
µmol/L genistein blocked cultured cells in the
G2/M phase of the cell cycle. These data are
consistent with the published work of others using different
transformed cell lines (Hunakova et al. 1994
,
Matsukawa et al. 1993
, Monti and Sinha 1994
, Peterson and Barnes 1991
, Piontek et al. 1993
, Spinozzi et al. 1994
,
Traganos et al. 1992
, Yanagihara et al. 1993
). In the studies presented here, we did not see any
evidence of a G1 block in cells treated with
genistein at concentrations up to 80 µmol/L. It is likely
that genistein is inhibiting one or more tyrosine kinases required for
transition through the different phases of the cell cycle and that the
specific perturbation in the cell cycle produced by genistein depends
on the cell line utilized.
Previous studies have shown that 750 µg genistein/g
AIN-93G diet exerts biological effects in estrogen-responsive
tissues in rats (Santell et al. 1997
). In addition, 750
µg genistein/g AIN-93G diet produces a plasma genistein
concentration of ~1 µmol/L in mice, which is similar to
that seen in humans consuming soymilk containing a total of 36 mg of
genistein (Xu et al. 1994
). Tumor growth in mice fed
this amount of genistein (750 µg genistein/g AIN-93G) did
not differ significantly from tumor growth in control mice (Fig. 2)
.
In the first study, 750 µg genistein/g AIN-93G diet was
administered to the mice after the formation of solid tumor masses.
Solid tumors > 2–3 mm in diameter require a vasculature for
nourishment. This vasculature is formed (angiogenesis) through an
intricate balance of protease activity, resulting in degradation of the
basement membrane in a controlled fashion, thus permitting the
generation and infiltration of blood vessels. Genistein has been shown
to inhibit this process, although the concentration required in vitro
was ~150 µmol/L (Fotsis et al. 1993
).
This concentration is much greater than that observed in the plasma of
mice fed genistein; however, the effect of lower concentrations of
genistein on the initial stages of solid tumor development in vivo has
not been investigated. In addition, in vitro endothelial cell
proliferation was inhibited with IC50 of 5
(Fotsis et al. 1993
) and 12 µmol/L
genistein (Koroma and de Juan 1994
). In the studies
presented here, when genistein was fed at an amount that would produce
a plasma concentration of 1 µmol/L (750 µg
genistein/g AIN-93G diet) either before or after inoculating tumor
cells, the growth of tumors was not altered.
Shao et al. (1998)
administered genistein by injection
(500 µg/kg body weight) to athymic mice implanted with
MDA-231 tumors and demonstrated a significant reduction in tumor growth
with genistein. Although the authors did not determine plasma genistein
concentration, it is possible that the injection of genistein produced
plasma concentrations of genistein high enough to inhibit tumor growth.
In mice fed different doses of genistein (0–6000 µg/g
AIN-93G), we observed a dose-dependent increase in plasma
concentration from 0 to 7 µmol/L (free + conjugated forms)
with ~2 µmol/L free genistein at the highest dose. Even
if we assume that all forms of genistein are biologically active, it is
unlikely, based on the IC50 from our in vitro
data (Fig. 1)
, that we will see any reduction in cancer cell growth in
vivo. We have fed dietary genistein to athymic mice with MDA-231 tumors
at a concentration of 3000 µg/g AIN-93G diet (data not
shown). It is important to note that at this high level of dietary
genistein, we observed reduced food intake along with a reduction in
tumor growth rate. Mice fed 3000 µg genistein/g AIN-93G
consumed ~11% less food. Thus it was not possible to determine
whether the reduction in tumor growth rate was due to the dietary
genistein or energy restriction (Welsch et al. 1994
).
The long-term physiologic effects of dietary genistein are unknown.
Genistein is a known phytoestrogen and at the doses used in these
studies, does produce estrogenic responses in various organs, including
the mammary gland, in ovariectomized rodents (Santell et al. 1997
). Furthermore, genistein has been shown to induce estrogen
receptor positive human breast cancer cell (MCF-7) proliferation in
vitro and (MCF-7) tumor growth in vivo (Hsieh et al. 1998
). The effect of administering genistein to humans,
particularly those susceptible to estrogen-dependent cancers, is
unknown.
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FOOTNOTES |
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3 FBS, fetal bovine serum; IC50, concentration that inhibits 50% of cell proliferation; MEM, minimal essential medium.
Manuscript received September 10, 1999. Initial review completed October 22, 1999. Revision accepted February 24, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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