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1
*
Department of Environmental Preservation and Development, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501 and
Biotics Department, Nichimo Company, Tokyo 140-0002, Japan
1To whom correspondence should be addressed. E-mail: weijun_pan{at}nichimo.co.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • isoflavone • genistein • daidzein • glycitein • smooth muscle cells • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
breast and prostate cancers (Barnes et al. 1995
,
Messina et al. 1994
), osteoporosis (Valente et al. 1994
), as well as in ameliorating menopausal symptoms
(Boulet et al. 1995
). Epidemiologic studies have shown
that the consumption of phytoestrogen-rich diets is associated with
lower risks of cardiovascular diseases and breast and prostate cancers
(Adlercreutz 1998
, Barnes 1998
,
Messina 1999
). Phytoestrogens are naturally found in
many plant foods and are structurally or functionally similar to
estrogens. Soybeans contain large amounts of isoflavones, which can
bind to estrogen receptors and exert estrogenic and antiestrogenic
activities (Adlercreutz et al. 1982
, Bannwart et al. 1984
, Setchell et al. 1984
, Tang and Adams 1977 and 1980
). Isoflavones may reduce the biological
activity and level of estrogen by stimulating the production of plasma
sex hormone–binding globulin and inhibiting human aromatases
(Adlercreutz et al. 1992
). These effects may prevent or
reduce the risk of sex hormone–dependent cancers (Messina and Barnes 1991
). Isoflavones have weak estrogenic properties and
exert biological antioxidant effects such as reducing the oxidation of
LDL (Carroll 1991
). Isoflavones may decrease
plasma total and LDL cholesterol and thereby prevent cardiovascular
diseases (Anthony et al. 1998
, Hamilton and Carroll 1976
).
Soybean isoflavones make up 0.1–0.3 g/100 g soybean (Wang and Murphy 1994a
). Genistein (4',5,7-trihydroxyisoflavone),
daidzein (4',7-dihydroxyisoflavone), glycitein (6-methoxydaidzein) and
their glycosides are the major isoflavones in soybeans in which
glycosides comprise 97–98% of the total isoflavones (Murphy 1982
, Wang and Murphy 1994b
) (Fig. 1
). Recent experimental evidence suggests that soybean isoflavones are
responsible for the beneficial effects of soy in the prevention of
atherosclerosis (Anthony et al. 1998
). The underlying
mechanisms responsible for these effects are not yet completely
understood. Because smooth muscle cells
(SMC)2
are found in both fatty streaks and fibrous plaques in atherosclerosis
and their proliferation is induced by platelet-derived growth
factor (PDGF), SMC proliferation is a key event that determines how
extensive fibrous plaques become and whether clinical sequelae are
likely to develop (Ross 1986
and 1993
). Genistein is a
specific inhibitor of the ability of epidermal growth factor (EGF)
receptor tyrosine kinase to reduce cell proliferation in vitro
(Akiyama et al. 1987
). Soybean isoflavones may inhibit
SMC proliferation and the migration from the media into the intima, and
block the stimulation by growth factors such as PDGF.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dulbecco’s modified Eagle medium (DMEM) and PBS were purchased from Nissui Pharmaceutical (Tokyo, Japan). DMEM (phenol red free) was obtained from Life Technologies (Rockville, MD). Fetal bovine serum (FBS) was purchased from Filtron (Brooklyn, Australia). Fetal bovine albumin (FBA) was obtained from Sigma Chemical (St. Louis, MO). Penicillin and streptomycin were purchased from Meiji Seika (Tokyo, Japan). Trypsin-EDTA solution was obtained from Difco Laboratories (Detroit, MI). Recombinant human PDGF-BB was purchased from Genzyme/Techne (Cambridge, MA). Soybean isoflavones, genistein, daidzein and glycitein were obtained from Extrasynthese (Genay, France), Fujicco (Kobe, Japan) and Nichimo (Tokyo, Japan) respectively. The Biotrak cell proliferation ELISA system, version 2, was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). The sterilizing filter unit (0.22 µm) was obtained from Millipore (Bedford, MA). All tissue-culture dishes and flasks were purchased from Becton Dickinson (Franklin Lakes, NJ).
Aortic smooth muscle cells cultured from SHRSP.
Aortic SMC were cultured from SHRSP and WKY by an explant method
(Yamori et al. 1981
). The cells were cultured in DMEM
with penicillin (1 x 105 U/L),
streptomycin (100 mg/L), and NaHCO3 (24 mmol/L) containing
10% FBS, plated in tissue-culture flasks (75 cm2) and
incubated under standard tissue culture conditions (37°C, 5%
CO2). The cells, which grew to confluent monolayers in the
above medium, were dislodged by treatment with 0.25% trypsin-EDTA
solution and underwent further passaging. SMC in the 5th passage were
used for all studies. All experiments in the present study conformed to
the published guidelines (Physiological Society of Japan 1998
).
SMC from SHRSP and WKY were plated at a density of 1 x 104
cells/well in 24-well tissue-culture dishes. SMC were allowed to
grow to subconfluence in DMEM containing 10% dextran-coated
charcoal (DCC)-FBS under standard tissue culture conditions for 5 d. SMC were dislodged and counted using a Coulter counter (Sysmex
CAD-500, Kobe, Japan) (Fig. 2
).
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For cell number experiments, SMC were plated at a density of 1 x 104 cells/well in 24-well tissue-culture dishes and allowed to grow to subconfluence in DMEM containing 10% FBS under standard tissue culture conditions. After three cell washes with PBS, the growth of SMC was arrested by adding DMEM (phenol red free) containing 0.4% FBA for 48 h. First, SMC were treated with a concentration of 0 (control) or 0.1–30 µmol/L genistein, daidzein and glycitein in fresh DMEM (phenol red free) containing 10% DCC-FBS and allowed to grow for 4 d. Second, in PDGF-BB (20 µg/L), proliferation of SMC was induced; cells were treated with a concentration of 0 (control) or 0.1–30 µmol/L genistein, daidzein or glycitein in fresh DMEM (phenol red free) containing 10% DCC-FBS and allowed to grow for 4 d. On d 5, the cells of both groups were dislodged and counted using a Coulter counter.
DNA synthesis studies.
The cell proliferation ELISA system was based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation to investigate the effects of genistein, daidzein and glycitein on mitogen-induced DNA synthesis. The aortic SMC from SHRSP were plated at a density of 2000 cells/well in 96-well tissue-culture dishes and allowed to grow for 72 h in DMEM containing 10% FBS under standard tissue culture conditions. After three cell washes with PBS, the growth of SMC was arrested by adding DMEM (phenol red free) containing 0.4% FBA for 48 h. First, SMC were treated with a concentration of 0 (control) or 1–30 µmol/L genistein, daidzein and glycitein in fresh DMEM (phenol red free) containing 10% DCC-FBS and allowed to grow for 20 h of incubation (37°C, 5% CO2). Second, in PDGF-BB (20 µg/L), proliferation of SMC was induced; cells were treated with a concentration of 0 (control) or 1–30 µmol/L genistein, daidzein or glycitein in fresh DMEM (phenol red free) containing 10% DCC-FBS and allowed to grow for 20 h of incubation (37°C, 5% CO2).
The BrdU labeling solution was added to cultured cells with a final concentration of 10 µmol/L and reincubated for 2 h at 37°C. After removal of the culture medium, the cells were fixed; DNA was denatured by addition of a fixative and incubation for 30 min at room temperature. The fixative solution was removed by tapping, adding blocking buffer to cells and incubating for 30 min at room temperature, then removing it by tapping. The peroxidase-labeled anti-BrdU working solution was added and incubated for 90 min at room temperature. The cells were washed three times with washing buffer and immediately dispensed at 100 µL/well of substrate reaction for 10 min at room temperature; the reaction was stopped by pipetting 20 µL of 1 mol/L sulfuric acid into each well. The absorbances were determined from the optical density in a microtitre plate reader at 450 nm within 5 min, which correlated directly with the amount of DNA synthesis and thereby to the number of proliferating cells in the culture.
Statistical analysis.
Values are presented as means ± SD (n = 3) for cells treated with genistein, daidzein or glycitein. Data were analyzed by ANOVA and the Tukey-Kramer honestly significant difference test. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SMC proliferation induced by 10% DCC-FBS and cultured from the
aorta of SHRSP was 
100% greater than that from the aorta of WKY on
d 5 (P < 0.001, Fig. 2
). Genistein, daidzein and
glycitein inhibited 10% of the DCC-FBS–induced proliferation of SMC
from SHRSP in a concentration-dependent manner (Fig. 3
). The lowest concentrations with significant inhibition after 4 d
treatment were 3 µmol/L for genistein (P
< 0.01), and 10 µmol/L for both daidzein and
glycitein (P < 0.01). In PDGF-BB–induced
proliferation of SMC from SHRSP, the effective concentration for
significant inhibition was as low as 0.1 µmol/L for each
isoflavone (P < 0.05), and the inhibitions were
concentration dependent (Fig. 4
).
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Genistein, daidzein and glycitein inhibited 10% DCC-FBS–induced DNA
synthesis of SMC from SHRSP in a concentration-dependent manner
(Fig. 5
). The effective concentrations for significant inhibition were 10
µmol/L for genistein (P < 0.05), 30
µmol/L for daidzein (P < 0.05) and 3
µmol/L for glycitein (P < 0.05). In
PDGF-BB–induced DNA synthesis of SMC from SHRSP, significant
inhibition was noted at 30 µmol/L for both genistein
(P < 0.05) and glycitein (P < 0.01;
Fig. 6
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A meta-analysis has shown that coronary heart
disease risk is nearly 50% lower in women who take postmenopausal
estrogen compared with those who do not (Stampfer and Colditz 1991
). Epidemiologic studies have shown that the prevalence of
coronary heart disease is much higher in women living in Western
countries with a diet rich in animal fat than those living in Southeast
Asian countries with a diet high in soy protein (Barnes 1998
). Consumption of soy protein rather than animal protein
significantly decreases serum concentrations of total cholesterol, LDL
cholesterol and triglycerides (Anderson et al. 1995
). A
soy protein diet decreased both plasma cholesterol concentrations and
intestinal cholesterol absorption in ovariectomized cynomolgous monkeys
(Greaves et al. 2000
).
Recent evidence indicates that soybean isoflavones possess estrogenic
(Karas et al. 1994
), antioxidant (Carroll 1991
), hypocholesterolemic (Anonymous 1980
) and
cell proliferation inhibitory (Akiyama et al. 1987
)
activities. They may have different modes of action, but similarly
block multiple steps in cell proliferation to protect against
atherosclerosis.
In this study, we observed the effect of isoflavones on the
proliferation of SMC from SHRSP, which genetically proliferate faster
than those from WKY (Fig. 2)
. Soybean isoflavones inhibited in a
concentration-dependent manner 10% DCC/FBS–induced cell
proliferation and DNA synthesis as well as PDGF-BB–induced
proliferation and DNA synthesis in SMC from SHRSP. This is the first
report that glycitein inhibits proliferation of SMC from SHRSP. The
inhibitory effects of soybean isoflavones were observed even at low
concentrations, especially the inhibitory concentration of
PDGF-BB–induced SMC proliferation (0.1 µmol/L of all
isoflavones) (P < 0.05). In humans administered a
single dose of 30 mg of isoflavone aglycones (genistein and daidzein),
the plasma concentrations of isoflavones (genistein and daidzein)
reached their highest level (2 µmol/L) 2 h after
intake. Isoflavone aglycones were absorbed faster and in greater
quantity than their glucosides in humans (Izumi et al. 2000
). A supplement of isoflavone aglycones may be useful to
prevent atherosclerotic cardiovascular diseases.
Soybeans contain large amounts of glycitein and its glucosides. Soy
flour and germ contain 10 and 40% of all soybeans isoflavones,
respectively, as glycitein and its glucosides (Wang and Murphy 1994a
). This compound also has effects on growth and DNA
synthesis of SMC.
Genistein is a protein tyrosine kinase inhibitor that inhibits the
activation of receptor tyrosine phosphorylation of EGF, thus
inducing DNA synthesis for cell proliferation in vitro and arresting
the cell cycle progression. Genistein arrests the cell cycle
progression at G2-M of human gastric cancer
(HGC-27) cells with a 50% inhibitory concentration
(IC50) of 20 µmol/L on d 4 of
culture. Quercetin and the structurally similar daidzein arrest the
cell cycle at G1 of HGC-27 cells with an
IC50 of 32–55 µmol/L on d 2 of
culture (Akiyama et al. 1987
, Matsukawa et al. 1993
, Yoshida et al. 1990
). In SMC,
soybean isoflavones may arrest the cell cycle progression at the
G0 to G1 transitions by the
inhibition of mitogen-activated protein (MAP) kinases (Dubey et al. 1999
, Langan et al. 1994
). MAP kinases
function as cytosolic serine/threonine kinases, and may be the point of
convergence for diverse growth factors utilizing these signaling
pathways. Soybean isoflavones can inhibit MAP kinase activities and
thereby inhibit SMC proliferation (Dubey et al. 1999
).
17ß-Estradiol inhibits PDGF-
mRNA expression in human SMC in
vitro; the expression of PDGF- mRNA was reduced 80% by 10 nmol/L of
17ß-estradiol. 17ß-Estradiol also inhibits c-myc mRNA
expression in human SMC in vitro. Estrogenic activity may also inhibit
cell cycle progressions at the G0 to
G1 transitions (Urabe 1997
). These
inhibitory effects suggest that soybean isoflavones play a role similar
to that of estrogen in inhibiting DNA synthesis in SMC.
The soybean isoflavones, genistein, daidzein and glycitein, may have potential benefits in human health maintenance due to their biological effects. This study suggests that soybean isoflavones may inhibit proliferation of vascular SMC and thus contribute to the prevention of atherosclerotic cardiovascular diseases.
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FOOTNOTES |
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Manuscript received April 27, 2000. Initial review completed July 10, 2000. Revision accepted December 29, 2000.
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