From the Department of Endocrinology and Metabolic
Diseases, Leiden University Medical Center, Albinusdreef 2, 2300 RC, Leiden, The Netherlands and the § Division de
Pharmacologie Moleculaire et Cellulaire, Institut de Recherches
Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, France
Received for publication, September 16, 2002, and in revised form, October 28, 2002
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ABSTRACT |
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The principal soy phytoestrogen genistein has an
array of biological actions. It binds to estrogen receptor (ER) In recent years, soy phytoestrogens have attracted wide attention
due to their potential beneficial effects on some common medical
disorders (1-3). Genistein, the principal soy phytoestrogen, has an
array of biological actions and is widely available in herbal tablets
(3-5). It binds to estrogen receptors
(ERs),1 ER Peroxisome proliferator-activated receptor- PPAR In the present study, we examined the effects of genistein on
osteogenesis and adipogenesis and explored its molecular mechanisms of
action. Our results show that genistein, in addition to its estrogenic
activity, activates PPAR Cell Cultures and Assays--
The methods for cell culture have
been described before (23). In brief, KS483 cells and mouse bone marrow
cells were cultured in phenol red-free Membrane-bound PPAR Transient Gene Expression Assays in KS483 Cells--
The
estrogen-responsive reporter gene construct (2XERE-TATA-luc), which
contains two copies of a consensus estrogen response element, and the
empty control TATA-luc plasmids were kindly provided by Dr. E. Kalkhoven and Dr. M. G. Parker. The peroxisome
proliferator-responsive element (3XPPRE-tk-luc) containing three copies
of a consensus peroxisome proliferator-responsive element and the human
PPAR Statistics--
Data are presented as means ± S.E.
Differences between groups were accepted at p < 0.05, which were assessed by one-way analysis of variance or related test
using software Instat.
Osteogenesis--
As shown in Fig.
1, genistein added to cultures of KS483
cells had a clear biphasic effect on osteogenesis, similar to that of
E2 (23). At concentrations from 0.1 to 10 µM,
genistein stimulated ALP activity, nodule formation, and calcium
deposition, with a maximal effect at 1 µM. In contrast,
at concentrations of 25 µM or higher, genistein inhibited
ALP activity, nodule formation and Ca2+ deposition. These
changes were paralleled by mRNA expression of the osteoblastic
markers, Cbfa1, osteocalcin, and PTH/PTHrP receptor that, relative to
control, were increased by 1 µM genistein and decreased
by 25 µM (Fig. 1). Similar stimulatory and inhibitory effects of genistein on bone formation were also observed in mouse bone
marrow cell cultures (Fig. 2). In those
cultures, genistein stimulated ALP activity and Ca2+
deposition at concentrations between 0.1 and 10 µM,
whereas it inhibited osteogenesis at concentrations of 25 µM or higher. These data demonstrate that genistein
affects osteogenesis of progenitor cells in a biphasic way; namely, it
increases osteogenesis at low concentrations and inhibits osteogenesis
at high concentrations.
Adipogenesis--
Genistein had also a biphasic effect on
adipogenesis, which was, however, different to that of E2
(23). At low concentrations between 0.1 and 1 µM, it
decreased adipocyte numbers, while at higher concentrations (>10
µM) it stimulated adipogenesis (Fig. 3A). The effects of genistein
on adipogenesis were paralleled by changes in mRNA expression of
the adipocyte markers, PPAR ER-dependent and ER-independent Effects of
Genistein--
Both ER-dependent and ER-independent
effects were observed in KS483 cells treated with different
concentrations of genistein (Fig. 4). At
a concentration of 1 µM, the effects were mediated by ERs
because stimulation of ALP activity and inhibition of adipogenesis were
both blocked by 1 µM ICI 164,382, a specific
antiestrogen. In contrast, at higher concentrations of genistein the
effects observed were ER-independent because ICI 164.382 at
concentrations from 0.01 to 100 µM did not affect the
action of genistein on osteogenesis or adipogenesis. In addition,
E2 (10 Activation of PPAR
To determine whether genistein activates PPAR Balance between Activated ERs and PPAR
The question that arises is whether activation of ERs by genistein can
also alter the transcriptional regulation of PPAR We show here that PPAR and
and has ER-mediated estrogenic effects. In addition, it has
antiestrogenic effects as well as non-ER-mediated effects such as
inhibition of tyrosine kinase. Because of its complex biological
actions, the molecular mechanisms of action of genistein are poorly
understood. Here we show that genistein dose-dependently
increases estrogenic transcriptional activity in mesenchymal progenitor
cells, but its biological effects on osteogenesis and adipogenesis are
different. At low concentrations (
1 µM),
genistein acts as estrogen, stimulating osteogenesis and inhibiting
adipogenesis. At high concentrations (>1
µM), however, genistein acts as a ligand of PPAR
,
leading to up-regulation of adipogenesis and down-regulation of
osteogenesis. Transfection experiments show that activation of PPAR
by genistein at the micromolar concentrations down-regulates its
estrogenic transcriptional activity, while activation of ER
or ER
by genistein down-regulates PPAR
transcriptional activity. Genistein
concurrently activates two different transcriptional factors, ERs and
PPAR
, which have opposite effects on osteogenesis or adipogenesis.
As a result, the balance between activated ERs and PPAR
determines the biological effects of genistein on osteogenesis and adipogenesis. Our findings may explain distinct effects of genistein in different tissues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and ER
, and
has ER-mediated effects (6, 7). In addition, it has antiestrogenic
effects, but the underlying mechanism is still unknown (1, 2, 4).
Non-ER mediated genistein actions such as an inhibition of protein
tyrosine kinase, DNA topoisomerases I and II and ribosomal S6
kinase have also been reported (8-10). These actions are most likely
mediated through transcriptional processes rather than via direct
effects on enzyme activity (11, 12).
(PPAR
), one of the
subtypes of PPARs, is a ligand-dependent transcription
factor of the nuclear hormone receptor superfamily (13). PPAR
is
most highly expressed in adipose tissue and is involved in critical physiological functions such as adipogenesis and glucose and
cholesterol metabolism (14). It is a target for therapeutic
intervention in cardiovascular diseases, various cancers, and diabetes
(15).
is the essential transcriptional factor for adipogenesis
(16-18). Adipocytes and the bone-forming cells, the osteoblasts, arise
from the same bone marrow mesenchymal precursor cells (19, 20). The
osteoprogenitor KS483 cells, which are cloned from mouse calvaria (21,
22), have been shown to differentiate into both osteoblasts and
adipocytes. Using this cell line, we recently showed that
17
-estradiol (E2) stimulates osteogenesis and
concurrently inhibits adipogenesis in these precursor cells (23).
Whether the phytoestrogen genistein has similar effects is unknown.
, resulting in a down-regulation of
osteogenesis and an up-regulation of adipogenesis. This action is
concentration-dependent. Our data show that the balance
between activated ERs and PPAR
determines the biological effects of genistein.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimum essential medium
(
-MEM) supplemented with 10% fetal bovine serum (Invitrogen)
or 15% fetal bovine serum (for mouse bone marrow), 50 µg/ml ascorbic
acid, 10 mM
-glycerophosphate, and 10
8
M dexamethasone (only for mouse bone marrow). Cells were
continuously exposed to genistein 1 day after plating until the end of
the experiment at day 21. Assays for ALP activity and DNA content, mRNA expression by RT-PCR, and Oil-Red-O staining for adipocytes were performed as described previously (23).
Binding Assay--
Binding assays, using
a human full-length PPAR
construct expressed in bacteria, were
performed in 96-well plates (24). Binding buffer consisted of 10 mM Tris/HCl, pH 8.2, containing 50 mM KCl and 1 mM dithiothreitol. Membrane preparations (5 µg/ml) were
incubated for 180 min at 4 °C in the presence of
[3H]rosiglitazone (BRL49653, Amersham Biosciences)
(10 nM) and the tested compounds. Nonspecific binding was
defined using an excess of unlabelled rosiglitazone (10 µM). Incubation was terminated by the addition of
ice-cold 50 mM Tris/HCl buffer, pH 7.4, followed by rapid
filtration under reduced pressure through Whatman GF/C filter plates
presoaked with ice-cold buffer, followed by three successive washes
with the same buffer. Radioactivity was measured in a TopCount
apparatus (Packard). The receptor preparation used during these
experiments presented a Bmax of 49 pmol/mg proteins and a Kd of 5.58 nM for [3H]rosiglitazone. Genistein was
solubilized in Me2SO and diluted to the appropriate working
concentrations (100 µM-0.1 nM).
2 constructs were kind gifts from Dr. J. Auwerx. The luciferase reporter construct (5XPPRE-TATA-luc) contained five copies of a
consensus PPRE and a TATA box and were provided by Dr. M. Karperien. The pT-109 FARE PPRE construct was a kind gift from Dr. K. van der Lee
and Dr. M. van Bilsen. The ACO-luc PPRE construct was kindly supplied
by Dr. K. W. Kinzler and Dr. B. Vogelstein. The human ER
construct was kindly provided by Dr. G. Kuiper. KS483 cells were seeded
into 24-well plates. After 24 h, they were transfected using a
lipid-based FuGENE 6 transfection reagent according to the manufacturer
(Roche Molecular Biochemicals). For each triplicate of sample, 100 ng
of luciferase reporter and 500 ng of
-galactosidase expression
vector were applied. The transfection medium was changed after 16 h into the different medium as indicated. After 48 h, cells were
washed twice with PBS, lysed in PBS containing 1% Triton X-100 and
sonicated. Luciferase activity was measured and expressed as fold
induction ± S.E., which was corrected for transfection efficiency
using
-galactosidase activity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effects of genistein on osteogenesis in KS483
cells. KS483 cells were cultured in 12-well plates in the medium
containing charcoal-stripped serum and continuously exposed to
different concentrations of genistein for 18 days. Cellular ALP
activity (A), the number of nodules (B), calcium
deposition (C), and mRNA expression of osteogenic
markers Cbfa1, osteocalcin, and PTHrP-R (D) were quantified.
Each value is the mean ± S.E. of the results from three different
wells and is representative of results from at least five different
experiments. Significant differences (* < 0.05) were indicated.
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Fig. 2.
Effects of genistein on osteogenesis in mouse
bone marrow cells. Mouse bone marrow cells were cultured in
12-well plates in the medium containing charcoal-stripped serum and
continuously exposed to different concentrations of genistein for 21 days. Cellular ALP activity (A) and calcium deposition
(B) were quantified. Each value is the mean ± S.E. of
the results from three different wells and is representative of results
from at least five different experiments. Significant differences (* < 0.05) were indicated.
2, aP2, and lipoprotein lipase
(Fig. 3B). Adipogenic responses of mouse bone marrow cells
to different doses of genistein are shown in Fig. 3C. Mouse
bone marrow cultures treated with genistein concentrations of 25 µM or higher did not reach confluence, and there were no
adipocytes during the cultures. However, compared with control an
increase in adipocyte numbers was observed at the concentration of 10 µM, whereas a decrease in adipocyte numbers was found at
the concentrations of 0.1 and 1 µM. These data show that
genistein affects adipogenesis of progenitor cells in a biphasic way,
i.e. an inhibition of adipogenesis at low concentrations and
a stimulation of adipogenesis at high concentrations.
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Fig. 3.
Effects of genistein on adipogenesis in KS483
cells and in mouse bone marrow cells. KS483 cells or mouse bone
marrow cells were cultured in 12-well plates in the medium containing
charcoal-stripped serum and continuously exposed to different
concentrations of genistein for 18 days or 21 days, respectively. The
number of adipocytes in KS483 cell cultures (A), mRNA
expression of adipogenic markers PPAR 2, aP2, and LPL (B),
and the number of adipocytes in mouse bone marrow cell cultures
(C) were quantified. Each value is the mean ± S.E. of
the results from three different wells and is representative of results
from at least five different experiments. Significant differences (* < 0.05) were indicated.
10 M to 10
5
M) did not reverse the effects of genistein at 25 µM on osteogenesis or adipogenesis. These data suggest
that the action of genistein at low concentrations is likely
ER-mediated, whereas its effects at high concentrations are not
ER-mediated.
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Fig. 4.
Effects of ICI 164,382 on genistein-induced
osteogenesis and adipogenesis in KS483 cells. KS483 cells were
cultured in 12-well plates in the medium containing charcoal-stripped
serum and continuously exposed to genistein, ICI 164.382 (1 µM) or in combination of both substances for 18 days.
ER-dependent and ER-independent effects of genistein
occurred at 1 and 25 µM, respectively, as shown by
changes in ALP activity (A) and the number of adipocyte
(B). Each value is the mean ± S.E. of the results from
three different wells and is representative of results from at least
five different experiments. Significant differences (* < 0.05) were
indicated. C, control; G, genistein;
ICI, specific antiestrogen compound ICI164.384.
--
We transiently transfected
KS483 cells with a luciferase reporter construct containing five copies
of a consensus PPRE inserted in front of a TATA box together with
expression plasmids encoding human PPAR
2. PPRE-luc
reporter activity was measured after incubation of transfected cell
cultures with different doses of genistein. As shown in Fig
5A, genistein in the
micromolar range increased PPRE-luc reporter activity
dose-dependently. Furthermore, in the same concentration
range, genistein increased PPRE-luc reporter activity in ER-positive
and ER-negative breast cancer cell lines, T47D and MDA-MD-231,
respectively (not shown). These results were confirmed with three other
reporter constructs including the PPAR
response element ACO-luc
(25). Thus, genistein transcriptionally activates PPRE-luc reporter
activity independent of the cell lines and constructs used.
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Fig. 5.
Genistein is a PPAR
ligand. KS483 cells were seeded into 24-well plates. After
24 h, they were transiently transfected with a luciferase reporter
construct containing five copies of a consensus PPRE inserted in front
of TATA box together with expression plasmids encoding human PPAR
2.
PPRE-luc reporter activity was measured after incubation of transfected
cell cultures with different doses of genistein for 48 h. Gene
reporter assay for PPRE-luc shows that genistein stimulates PPAR
transcriptional activity (A). Binding assays, using a human
full-length PPAR
construct expressed in bacteria, were performed in
96-well plates. The binding assay shows that genistein at the
micromolar concentrations binds to PPAR
(B).
through direct
interaction with this receptor, we performed a membrane-bound PPAR
binding assay. Genistein had a measurable Ki of 5.7 µM (Fig. 5B), which is comparable to that of
the known PPAR
ligands (24). We have checked whether genistein bound
competitively with [3H]rosiglitazone to the same PPAR
site. Indeed the dissociation constant (Kd)
of [3H]rosiglitazone in saturation experiments in the
presence of a high dose of genistein was significantly reduced as
compared with that in the absence of genistein. The maximal number of
sites labeled was not altered. These data demonstrate that both
genistein and [3H]rosiglitazone bind to the same PPAR
site (data not shown). Therefore, genistein can interact directly with
the PPAR
ligand-binding domain and thus act as a PPAR
ligand.
--
As both the
antiestrogenic effects and the activation of PPAR
were increased by
micromolar concentrations of genistein, we tested whether activation of
PPAR
is involved in the antiestrogenic action. When KS483 cells were
treated for 18 days either with a specific PPAR
agonist ciglitazone,
genistein, E2, or a combination of genistein and
E2, a decrease in ALP activity was observed with all
treatments, except for E2 alone that increased ALP activity (Fig. 6A). When, however, we
transiently transfected KS483 cells with a luciferase reporter
construct containing two copies of a consensus ERE inserted in front of
a TATA box and exposed these cells to different concentrations of
genistein, a dose-related increase of ERE-luc reporter activity was
observed at a concentration between 0.1 and 50 µM (Fig.
6B). Furthermore, the estrogenic potency of genistein at the
micromolar range was greater than that of E2
(10
8 M), and anti-estrogenic effects of
genistein were not observed (Fig. 6C). The lack of an
antiestrogenic effect in the gene reporter assays could be due to low
amount of endogenous PPAR
2 in KS483 cells during the first 5 days
(23), the period in which the gene reporter assays were performed. To
investigate this further, we transiently transfected vectors expressing
human PPAR
2 or empty vector along with a ERE-luc construct and
exposed KS483 cells to genistein. The transient co-transfection of
PPAR
2 resulted in a decrease of ERE-luc reporter activity at high
genistein concentrations. Taken together, our data show that the
antiestrogenic effects of genistein are due to an activation of
PPAR
2, leading to down-regulation of ER-mediated transcriptional
activity and osteogenesis.
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Fig. 6.
Antiestrogenic effects of genistein in KS483
cells. KS483 cells were cultured in 12-well plates in the medium
containing charcoal-stripped serum and continuously exposed to
genistein of 25 µM, E2 (10 8
M), and ciglitazone of 25 µM as well as
combination of genistein and E2 for 18 days. ALP activity
(A) was measured, which shows that genistein exerts
antiestrogenic effects on osteogenesis. KS483 cells containing an
integrated ERE-luc reporter gene were exposed to various concentrations
of genistein for 48 h. Genistein dose-dependently
increased ERE-luc activity (B). When these cells were
exposed to E2 or genistein of 25 µM or in
combination of both for 48 h, antiestrogenic effects of genistein
were not observed in this gene reporter assay (C). When
KS483 cells were cotransfected with ERE-luc reporter gene together with
constructs expressing PPAR
2 and exposed to different concentrations
of genistein, down-regulation of ERE-luc activity was observed
(D). C, control; E, E2;
G, genistein; Ci, ciglitazone.
. To investigate
this, we transiently transfected vectors expressing human ER
or
ER
or empty vector along with a PPAR
2 construct and a PPRE-luc
construct. Co-transfection of ER
(Fig. 7A) or ER
(Fig.
7B) decreased PPRE-luc reporter activity in KS483 cells treated with different concentrations of genistein.
Interestingly, genistein at 1 µM suppressed
PPRE-luc reporter activity to a level lower than that of controls,
while at 10 µM it suppressed it to the control level in
the presence of sufficient levels of ERs. In contrast, PPRE-luc
reporter activity was higher than the control levels at 50 µM genistein of and was not influenced by the levels of
ER
or ER
(Fig. 7).
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Fig. 7.
Effects of ER and
ER
on PPAR
transcriptional activity. KS483 cells were seeded into
24-well plates and transiently transfected with a luciferase reporter
construct containing five copies of a consensus PPRE inserted in front
of TATA box together with expression plasmids encoding human PPAR
2
and in combination with ER
or ER
. PPRE-luc reporter activity was
measured after incubation of transfected cell cultures with different
doses of genistein for 48 h. Cotransfection of ER
(A) or ER
(B) down-regulates PPAR
transcriptional activity. Each value is the mean ± S.E. of the
results from three different wells and is representative of results
from at least three different experiments. Significant differences (* < 0.05) were indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is a molecular target for genistein. At
the micromolar range, genistein binds to and transactivates PPAR
,
leading to a decrease of osteogenesis and an increase in adipogenesis.
In addition, genistein dose-dependently transactivates ERs,
resulting in an up-regulation of osteogenesis and a down-regulation of
adipogenesis. Moreover, activation of ERs by genistein could down-regulate PPAR
transcriptional activity and vice
versa. The balance between the activation of ERs and PPAR
is
concentration-related. As a result, the biological effects,
i.e. osteogenesis and adipogenesis, vary according to the
concentrations of genistein (Fig. 8). Our findings can explain the previously reported diverse actions of genistein in different tissues.
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Fig. 8.
Molecular mechanisms of action of
genistein. Genistein concurrently activates two different types of
transcriptional factors, ERs and PPAR , which have opposite effects
on osteogenesis or adipogenesis. These transcriptional factors
influence each other and the balance between activated ERs and PPAR
determines the biological effects of genistein on osteogenesis and
adipogenesis.
At low concentrations (1 µM), genistein has
ER-dependent effects on osteogenesis and adipogenesis; the
effects are similar to those of E2 (23). At high
concentrations (>1 µM), however, genistein has
antiestrogenic actions, namely, it down-regulates osteogenesis, which
is opposite to E2-induced effects. Antiestrogenic effects
of genistein have been reported in many cell types and animal models,
but the mechanism responsible for this is still not known (1, 2, 4,
26). We show here that the antiestrogenic effects are not due to a
decrease of estrogenic activity of genistein. Instead, genistein at
micromolar concentrations dose-dependently increased
estrogenic transcriptional activity, and the levels were even higher
than those induced by E2. These results are in line with
reports using different cell lines or assays (6, 27, 28). Moreover,
antiestrogenic effects of genistein could not be restored or blocked by
E2 or by the antiestrogen compound ICI164,382. Together,
our results implicate that antiestrogenic effects of genistein are
elicited via pathways other than the ER pathway.
Different from E2, genistein binds to and transactivates
PPAR, leading to adipogenesis. Moreover, activation of PPAR
may also be due to an inhibition of the MAPK pathway. It is well known that
the A/B domain of PPAR
contains a consensus MAPK site (29-31). Inhibition of PPAR
phosphorylation by the specific MAPK inhibitor PD98059 stimulates adipogenesis (32). Genistein inhibits p42/44 MAPKs
in KS483 cells.2 It is
therefore possible that an inhibition of p42/44 MAPKs contributes to an
activation of PPAR
. By using a pure PPAR
ligand, ciglitazone, we
showed that activation of PPAR
down-regulates osteogenesis in KS483
cells. These results are consistent with observations in MC3T3-E1 cells
and in U33 cells (33, 34). It has been shown that PPAR
2 plays a
dominant role in the determination of the fate of mesenchymal
progenitor cells (35). An increase in adipogenesis and a decrease of
osteogenesis by genistein at concentrations of 25 µM or
higher indicate that PPAR
actions dominate at higher genistein concentrations.
Genistein concurrently activates two different transcriptional factors,
ERs and PPAR. These two transcriptional factors have opposite
effects on osteogenesis or adipogenesis. We showed that activation of
PPAR
by genistein at the micromolar concentrations down-regulates
its estrogenic transcriptional activity, while activation of ER
or
ER
down-regulates PPAR
transcriptional activity. It is plausible
that genistein at certain concentrations activates ERs and PPAR
to a
different extent. The balance between activated ERs and PPAR
determines the biological effects of genistein, i.e.
osteogenesis and adipogenesis, which are fully
concentration-dependent.
Our findings provide the molecular basis of the mechanism of action of
genistein and may have wide implications. Diverse effects of genistein
in different tissues have been explained by the high binding affinity
for ER because ER
can act as a dominant negative regulator of
estrogenic activity. These dominant negative effects were only observed
below the micromolar concentrations of genistein (36). However, the
distinct genistein effects in different tissues are often observed at
the micromolar concentrations (1, 2, 4, 37). We show that the balance
between activated ERs and PPAR
determines the biological effects of
genistein, which might explain its diverse biological effects in
different organs. Therefore, the biological effects of genistein in
certain tissues strongly depend on the concentration of genistein
present and the levels of ERs and PPAR
within that particular
tissue. There is accumulating evidence that health benefits occur only
when phytoestrogens are consumed in sufficient quantities (1, 2, 4). It
has been reported that plasma concentration of genistein is relatively low and generally less than 40 nM in humans consuming diets
without soy, whereas it can reach 4 µM in the plasma of
Japanese who consume high amount of soy products (1, 2, 4). Our
findings might explain why genistein functions only at a certain level.
For example, genistein at the micromolar concentration range inhibits
growth of ER-positive breast cancer cells like MCF7 and T47 D as well as ER-negative breast cancer cells like MDA-MD-231 cells (38). Since it
is now well established that ligand activation of PPAR
inhibits cell
growth and induces apoptosis in these cancer cells (39-41), it is
plausible that only when PPAR
is activated, genistein at certain
levels could inhibit the growth of cancer cells.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. E. Kalkhoven, M. G. Parker, J. Auwerx, G. Kuiper, K. van der Lee, M. van Bilsen, K. W. Kinzler and B. Vogelstein for supplying constructs. We thank colleagues from the Endocrinology department for the technical support and Numico Research B. V. for financial support.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Endocrinology and Metabolic Diseases (C4-R), Leiden University Medical Center, Albinusdreef 2, 2300 RC, Leiden, The Netherlands. Tel.: 0031-71-5263075; Fax: 0031-71-5248136; E-mail: c.w.g.m.lowik@lumc.nl.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M209483200
2 Z.-C. Dang, V. Audinot, S. E. Papapoulos, J. A. Boutin, and C. W. G. M. Löwik, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
ER, estrogen
receptor;
PPAR, peroxisome proliferator-activated receptor-
;
E2, 17
-estradiol, MEM, minimum essential medium;
ALP, alkaline phosphate;
MAPK, mitogen-activated protein
kinase.
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REFERENCES |
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