IUBMB Life, 56(3): 145–151, March 2004
Research Communication
Nutritional Flavonoids Modulate Estrogen Receptor a Signaling
Fabio Virgili2, Filippo Acconcia1, Roberto Ambra2, Alessandra Rinna2, Pierangela Totta1 and Maria Marino1
1
Department of Biology, University ‘Roma Tre’, Viale G. Marconi, 446, I-00146 Rome, Italy
2
National Institute for Food and Nutrition Research (INRAN), Via Ardeatina, 546, I-00178 Rome, Italy
Summary
Estrogen receptor a (ERa) mediates 17b-estradiol (E2) actions
through the transcription of E2-sensitive target genes. In addition,
rapid non-genomic signaling (e.g., MAPK/ERK) occurs. It is now
well accepted that these rapid membrane-initiated responses account
for E2-related cancer. Beside many beneficial effects on human
health, nutritional flavonoids exert protective and anticarcinogenic
effects on E2-related cancer. The mechanism underlying these effects
seems to be related to flavonoids antioxidant properties and/or to
their ability to alter signal transduction protein kinases. In addition,
an antiestrogenic activity has been proposed but not yet defined.
However, the identification and characterization of the responsible
mechanisms for flavonoid antitumoral effects is poorly understood.
Here, we investigated the possibility that the antimitogenic effects of
flavonoids are transduced by modulating ERa-mediated rapid
signaling. The ability of two flavonoids, the flavanone naringenin
and the flavanol quercetin, with respect of E2, to induce ERa
activities has been studied in the human cervix epitheloid carcinoma
cell line (HeLa) devoid of any estrogen receptors and rendered E2-
sensitive by transient transfection with a human ERa expression
vector. Our results indicate that flavonoids act as E2 mimetic on ERa
transcriptional activity, whereas they impair the activation of rapid
signaling pathways committed to E2-induced proliferation. The
resulting decoupling of ERa signal transduction could be proposed as
a new mechanism in the protective effects of flavonoids against E2-
related cancer.
IUBMB Life, 56: 145–151, 2004
Keywords Estrogen receptor a; 17b-estradiol; quercetin; naringenin;
transcriptional activity; ERK; cell proliferation.
Received 3 March 2004; accepted 3 March 2004
Address correspondence to: Dr. Maria Marino, Department of
Biology, University ‘Roma Tre’, Viale G. Marconi, 446, I-00146
Rome, Italy. Tel: + 390655176345; Fax: + 390655176321;
E-mail: m.marino@uniroma3.it
Abbreviations: AP-1, activator protein-1; E2, 17b-estradiol;
EMSA, electromobility shift assay; ERa, estrogen receptor a; ERE,
estrogen responsive element; ERK, extracellular signal regulated
kinase; MAPK, mitogen activated protein kinase; NF-kB, nuclear
factor-kB; PMA, phorbol 12-myristate 13-acetate; SERM, selective
estrogen receptor modulator
ISSN 1521-6543 print/ISSN 1521-6551 online # 2004 IUBMB
DOI: 10.1080/15216540410001685083
INTRODUCTION
Estrogen receptors (ERa, ERb) mediate the pleiotropic
effects of 17b-estradiol (E2) (e.g., growth, development, and
homeostasis of different organs and tissues). ERs transcrip-
tional activity is elicited by inducing the direct receptor
binding to its estrogen responsive sequence (ERE) on the
promoter of target genes (i.e., genomic mechanism) (1).
Furthermore, ERs associate to DNA-bound trascription
factors which activate (e.g., activator protein-1 AP-1, stimu-
lator protein 1 SP1) or inactivate (e.g., nuclear factor-kB NF-
kB) the transcription of E2-dependent-ERE-devoid genes (1).
In addition, a third mechanism, involving rapid membrane-
initiated responses, serves largely for mitogenic E2-induced
effects (i.e., non-genomic mechanism) (1 – 4).
Mounting evidence indicates that ERb act as a dominant
negative regulator of E2 signaling and in many instances (e.g.,
association to transcription factors, cell proliferation) ERb
opposes the actions of ERa (5, 6). Moreover, ERa activities
can be also modulated by selective estrogen receptor
modulators (SERMs) (e.g., 4-hydroxytamoxifen, raloxifene,
ICI 182,870) which show a high tissue specificity (7). Non-
steroidal plant-derived flavonoids present in the human diet
show high degree of structural resemblance to certain SERMs
(8). In fact, the flavonoids compete with E2 in binding to ERa
and induce the classical genomic activity of ER through the
transcription of ERE-containing genes (9). In addition, this
family of dietary components may have protective roles in
several E2-related diseases (i.e., osteoporosis and cardiovas-
cular diseases in post-menopausal women) (10 – 12). These
observations raise the hypothesis that flavonoids may act as an
estrogen ‘mimetic’ in different tissues and organs. However,
flavonoids display divergent effects from E2 in affecting cancer
growth. Epidemiological and in vitro studies indicate a
significant effect of flavonoids against E2-dependent cancer
(12 – 14).
Several putative mechanisms have been proposed for
antiproliferative effects of flavonoids (i.e., antioxidant activ-
ities, kinases inhibition, antiestrogenic activity) (13, 14). The
146 VIRGILI ET AL.
ability of flavonoids to hamper cell proliferation via their
binding to ERa and to interfere with its rapid signaling is still
scarce and limited to the isoflavonoid class of flavonoids (e.g.,
soy-derived genistein, daidzein, and kampherol) (15 – 17).
Here, the effects of two other flavonoid classes commonly
present in the western diet, the flavanone naringenin (e.g.,
derived by tomato, citrus) and the flavanol quercetin (e.g.,
derived by onion, apple) were analyzed (18). In particular, we
investigated whether the antimitogenic effects of naringenin
and quercetin could be evoked by the impairment of the rapid
ERa-mediated pathways (i.e., extracellular activated kinase
ERK, cyclin D1) committed to cell cycle progression.
This study was conducted on human cervix epitheloid
carcinoma cells (HeLa), devoid of any ERs and rendered E2-
sensitive by transient transfection with a human ERa
expression vector. This model allows analyzing the flavonoids
effects towards ERa activities without any interference of ERb
or ER splice variants.
Our results show that naringenin and quercetin can block
cancer cell growth mainly by exerting an antiestrogenic
activity. In addition, these flavonoids act as an estrogen
mimetic inducing the expression of ERE-containing genes, but
they do not activate gene transcription which depends on the
non-genomic activity of ERa. These findings indicate that
naringenin and quercetin can selectively modulate rapid non-
genomic ERa-induced signaling, thus suggesting a new
potential chemopreventive action for flavonoids on cancer
growth.
MATERIALS AND METHODS
Reagents. 17b-estradiol, naringenin, quercetin, phorbol
12-myristate 13-acetate (PMA), gentamicin, penicillin,
DMEM (without phenol red), and charcoal-stripped fetal calf
serum were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). Lipofectamine reagent was obtained from
GIBCO-BRL Life-technology (Galthersburg, MD, USA).
The luciferase kit was obtained from Promega (Madison,
WI, USA). GenElute plasmid maxiprep kit was obtained from
Sigma Chemical Co. (St. Louis, MO, USA). Bradford Protein
Assay was obtained from BIO-RAD Laboratories (Hercules,
CA, USA). The polyclonal anti-ERK-2 and the monoclonal
anti-phospho-ERK-2, and anti-b-actin antibodies were ob-
tained from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). CDP-Star, chemiluminescence reagent for Western blot
was obtained from NEN (Boston, MA, USA).
All other products were from Sigma Chemical Co. (St.
Louis, MO, USA). Analytical or reagent grade products,
without further purification, were used.
Plasmids. The gene reporter plasmids pC3-luciferase
(pC3), pXP2-D1-2966-luciferase (pD1), and the expression
vectors for pCR3.1-b-galactosidase (3), and human pSG5-
hERa have been used. Furthermore an empty plasmid,
pCMV5, was used as control (3). Plasmids were purified for
transfection using a plasmid preparation kit according to
manufacturer’s instructions. A luciferase dose response curve
showed that the maximum effect was present when 1 mg of
plasmid was transfected together with 1 mg of pCR3.1-b-
galactosidase to normalize for transfection efficiency (* 55 –
65%).
Cell culture. HeLa cells were routinely grown in air
containing 5% CO2 in modified, phenol red-free, DMEM
medium, containing 10% (v/v) charcoal-stripped fetal calf
serum, L-glutamine (2 mM), gentamicin (10 mg/ml) and
penicillin (100 U/ml). Cells were diluted and media changed
every 2 days.
Transfection and luciferase assay. Cells were grown to
*70% confluence, then transfected using Lipofectamine
Reagent according to the manufacturer’s instructions. Six
hours after transfection the medium was changed and 24 h
thereafter cells were stimulated with different concentration of
E2 or naringenin or quercetin for 6 h. The cell lysis procedure
as well as the subsequent measurement of luciferase gene
expression was performed using the luciferase kit according to
the manufacturer’s instructions with a EC & G Berthold
luminometer (Bad Wildbad, Germany).
Cell viability. Cells were grown to *70% confluence in 6
wells plates, then transfected and, 24 h after, stimulated with
10 nM E2 or 10 mM naringenin or quercetin for 6, 24, 30, and
54 h. After treatment cells were harvested with trypsin and
centrifuged. After that, cells were stained with trypan blue
solution and counted in a hemocytometer (improved Neu-
bauer chamber) in quadruplicate.
Electrophoresis and immunoblotting. After treatment with
hormone or flavonoids (5 and 15 min) transfected cells were
lysed as described (19) and solubilized in 0.125 M Tris HCl
(pH 6.8) containing 10% SDS (w/v), 1 mM phenylmethylsul-
fonyl fluoride and 5 mg/ml leupeptin and boiled for 2 min.
Proteins were quantified using the Bradford Protein Assay.
Twenty mg solubilized proteins were resolved using 10% SDS-
PAGE at 100 V for 1 h. The proteins were then electrophor-
etically transferred to nitrocellulose for 45 min at 150 V at
48C. The nitrocellulose was treated with 3% bovine serum
albumin in 138 mM NaCl, 26.8 mM KCl, 25 mM Tris HCl
(pH 8.0), 0.05% Tween-20, 0.1% BSA, and then probed at
48C overnight with anti-phospho-ERK-2 antibody (1 mg/ml).
This antibody recognizes both ERK-1 and ERK-2. The
nitrocellulose was stripped by Restore Western blot stripping
buffer (Pierce Chemical Company, Rockford, IL, USA) for
10 min at room temperature and then probed with anti-ERK-
2 (1 mg/ml). Anti-b-actin antibody (1 mg/ml) was used to
normalize the sample loading. Antibody reaction was visua-
lized with chemiluminescence reagent for Western blot.
EMSA. After transfection, cells were stimulated for 3 h
with hormone, flavonoids, or PMA (1 mM) as positive control.
Nuclear extracts were prepared according to Suzuki and
Packer (20). Briefly, cells were lysed for 20 min at 28C in a
hypotonic buffer [10 mM HEPES, 1 mM MgCl2, 10 mM KCl,
 NUTRITIONAL FLAVONOIDS AND ERa RAPID SIGNALS
0.1 mM EDTA, 0.5 mM EGTA, and 5% (v/v) glycerol,
pH 7.8], containing a cocktail of protease inhibitors. Nuclei
were treated with 0.65% Nonidet 40 for 5 min and pelleted by
centrifugation at 20,000 6 g for 30 s. Nuclear proteins were
obtained by incubating with a hypertonic buffer [50 mM
HEPES, 400 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 1 mM
EGTA, and 10% (v/v) glycerol, pH 7.8] also containing a
cocktail of protease inhibitors. After treatment, nuclei were
centrifuged at 20,000 6 g for 5 min, and the supernatant
retained for use in the DNA binding assay. Two double-
stranded deoxy-oligonucleotides were end-labeled with [g-32P]-
ATP (3000 Ci/mmol; NEN, Boston, MA, USA) using a T4
kinase (Amersham Pharmacia Biotech, Uppsala, Sweden).
Nuclear extracts (5 mg) were pre-incubated for 15 min at 28C
with 2 mg poly (dI-dC) (Amersham Pharmacia Biotech,
Uppsala, Sweden) and incubated with [g-32P]-labeled either
NF-kB or AP-1 DNA probe in binding buffer [25 mM
HEPES, 5 mM MgCl2, 50 mM NaCl, 1 mM DTT, and 10%
(v/v) glycerol, pH 7.8] for 20 min at room temperature. DNA
binding activity was separated from the free probe using 5%
polyacrylamide gel in 0.5 6 TBE buffer run at 200 V.
Following electrophoresis, the gel was dried and autoradio-
graphed.
RESULTS
Our first target was to assess the effects of naringenin and
quercetin, in comparison with E2, on two well known E2 : ERa
complex modulated cell functions: ERE-containing gene
transcription and promotion of cell growth.
Figure 1 (panel a) shows the dose-dependent effect of
naringenin and quercetin on pC3 promoter activity. Although
at higher concentration both flavonoids induce ERE-contain-
ing promoter to a level comparable to that of E2 in HeLa cells
transfected with ERa expression vector. No transcriptional
activity was present when HeLa cells, transiently transfected
with the empty plasmid, were stimulated with the different
ERa ligands (data not shown). These results confirm that both
flavonoids can trigger the ERa-mediated genomic mechanism
mimicking E2 effects. However, in a different way from E2, the
treatment of ERa-transfected HeLa cells with naringenin and
quercetin significantly decreased cell number (Fig. 1, panel b,
c, and d). These effects of naringenin and quercetin, in line
with results reported for other flavonoids in several cell lines
(9), were time and dose-dependent within the range utilized
(0.01 mM – 100 mM) (data not shown). However, none of
flavonoid concentration utilized, as well as E2, influenced the
growth of Hela cells transfected with the empty plasmid (Fig.
1, panel b, c, and d).
The E2-induced proliferation in several cell lines requires
ERa, the rapid phosphorylation of ERK, the activation of AP-
1 transcription factor, and the increase of cyclin D1 expression
(1, 3, 21). Here, we have evaluated the ability of naringenin
and quercetin in modulating these ERa-mediated cell func-
147
tions. In ERa-transfected HeLa cells both naringenin and
quercetin failed to induce ERK-2 phosphorylation whereas the
treatment of cells with E2 promptly increased the level of
ERK-2 phosphorylation (Fig. 2, panel a). Notably, neither
flavonoids decreased the basal, constitutive, phosphorylation
status of ERK-2 nor the expression level of total ERK-1/2
(i.e., phosphorylated and non phosphorylated) (Fig. 2, panel
a). Moreover, naringenin and quercetin treatment of ERa-
transfected HeLa cells did not significantly change AP-1 DNA
binding activity (Fig. 2, panel b). On the contrary, stimulation
of ERa-containing cells with E2 led to an evident increase in
AP-1 DNA binding (Fig. 2, panel b). It has been reported that
E2:ERa complex associates to c-rel subunit of NF-kB
transcription factor to inactivate NF-kB-dependent gene
transcription (1). Thus, we evaluated the possibility that
flavonoids could modulate this ERa-indirect action. Remark-
ably, in the presence of ERa, quercetin but not naringenin
increased NF-kB binding to DNA; on the contrary, E2
stimulation induced a decrease of NF-kB binding activity
(Fig. 2, panel c). Moreover, neither flavonoids nor E2
modified the AP-1 and NF-kB DNA binding activity, whereas
PMA, used as positive control, induced an increase of both
transcription factors binding to DNA in empty plasmid
transfected-HeLa cells (Fig. 2, panel b and c).
Finally, we investigated the effects of flavonoids toward the
transactivation of the ERE-devoid cyclin D1 gene. Figure 2
(panel d) shows that neither naringenin nor quercetin were
able to affect the cyclin D1 promoter activity both in the
presence and in the absence of ERa. Note that flavonoids did
not induce any promoter activity in cells transfected with the
empty plasmid, therefore non expressing any ERs.
DISCUSSION
Flavonoids have a basic 2-phenyl-benzo-g-pyrone structure
bearing one or more hydroxyl groups. Owing to this basic
chemical structure, the most obvious feature of flavonoids is in
their ability to quench free radicals by forming resonance-
stabilized phenoxyl radicals (22). Not necessarily related to its
antioxidant capacity, and due to their high affinity for
proteins, flavonoids have been reported to inhibit several
kinases involved in signal transduction (23). However, the
final identification and characterization of these mechanisms,
as the agent responsible for the multiple beneficial effects of
flavonoids in human health and, in particular, for the
antitumoral effect, is not yet clearly understood. Our findings
indicate that the reduction of the cell growth exerted by the
two flavonoids, naringenin and quercetin, in HeLa cells occurs
only in the presence of ERa. Furthermore, in HeLa cells
transfected with the empty plasmid, flavonoid stimulation
does not decrease the phosphorylation levels of ERK-2. Thus
it seems to be very improbable that antioxidant effects and the
inhibition of protein kinases phosphorylation, which would be
present also in the absence of ERa at high concentration of
148 VIRGILI ET AL.

Figure 1. Effect of 17b-estradiol and flavonoids on pC3 promoter activity and cell growth. Panel a, dose-response curves
(0.01 mM to 100 mM) of luciferase assay detection on HeLa cells co-tranfected with ERa and pC3-luciferase construct and then
treated 6 h with vehicle (C), 17b-estradiol (E2), naringenin (N), and quercetin (Q). Data are the means + SD of four
independent experiments. *P 5 0.001, compared with respective control values, was determined using Student’s t-test. Time
course analysis of cell growth in empty plasmid or ERa transfected-HeLa cells in the absence (C) or in the presence of naringenin
(10 mM) (Panel b), or quercetin (10 mM) (Panel c), or E2 (10 nM) (Panel d). The data are the mean values + SD of three
independent experiments carried out in duplicate. *P 5 0.001, compared with un-stimulated cell values (C), was determined
using Student’s t-test. For details see the text.

flavonoids ( 4 50 mM) (14, 24), may underlie the antiproli-
ferative effects of flavonoids reported in Fig. 1.
Our data indicate that the two flavonoids display a
significant estrogenic activity related to the modulation of
ERa signaling committed to cell cycle progression. The
flavonoid concentration used in this study (10 mM) could be
physiologically achieved in the human plasma after the
consumption of meals rich in flavonoids (1 – 18 mM) (24, 25).
The treatment of ERa-transfected HeLa cells with 10 mM
of naringenin or quercetin impairs both ERK-2 phosphor-
ylation, or AP-1 binding to DNA, and cyclin D1 promoter
activity. In particular, although quercetin treatment is
associated with an increase of NF-kB binding to DNA,
which is involved in cyclin D1 transcription (26), quercetin
itself does not induce cyclin D1 promoter activity, sustaining
that TPA-responsive element (TRE motif) activated by
ERa-AP-1 complex is dominant in driving cyclin D1
transcription (3, 4). However, both flavonoids still induce
the ERE-containing promoter activity. This suggests their
ability to decouple ERa action mechanisms impairing the
activation of rapid signaling pathways but evoking ERa
genomic activities.
The different ERa activities induced by the presence of
different ligands reinforce the view that ERa is an allosteric
protein (27). In fact, ERa activities can be modulated by
several non steroidal compounds, now called selective estrogen
 NUTRITIONAL FLAVONOIDS AND ERa RAPID SIGNALS 149

Figure 2. Role of flavonoids in ERa-mediated non-genomic activities. Panel a, time course of ERK-2 phosphorylation of
empty plasmid and ERa-transfected HeLa cells were performed on un-stimulated controls (0), 17b-estradiol (E2) (10 nM),
naringenin (N) (10 mM) and quercetin (Q) (10 mM) treated cells at 5 and 15 min. The amounts of protein were normalized by
comparison with ERK-2 and b-actin antibodies. DNA binding activity of AP-1 (Panel b) and NF-kB (Panel c) in empty plasmid
and ERa-transfected HeLa cells treated 3 h with vehicle (C), 17b-estradiol (E2) (10 nM), naringenin (N) (10 mM) or quercetin
(Q) (10 mM). PMA (1 mM) was used as positive control. Panels b and c show one representative experiment. Panel d, luciferase
assay detection on HeLa cells co-transfected with empty plasmid or ERa and cyclin D1-luciferase construct and then treated 6 h
with vehicle (C), 17b-estradiol (E2) (10 nM), naringenin (N) (10 mM) and quercetin (Q) (10 mM). Data are the means + SD of
four independent experiments. *P 5 0.001, compared with respective control values, was determined using Student’s t-test. For
details see the text.
150 VIRGILI ET AL.
receptor modulators (SERMs) (e.g., 4-hydroxytamoxifen,
raloxifene) (7, 28, 29). The SERMs, through 3D-changes in
ERa ligand binding pocket (30 – 32) function as ERa agonist
in some tissues and antagonist in other by recruiting specific
ERa transcription coactivators or corepressors (5, 33, 34).
Although flavonoids show a high degree of structural
resemblance to certain SERMs they are not considered as
SERMs (8). Present data indicate that flavonoids, unlike
SERMs, may function by modulating the specific ERa
mechanism of action. Thus, the preventive effects elicited by
flavonoids on E2-dependent cancer may be related to the
impairment of ERa-evoked proliferative pathways. The
different NF-kB activation by naringenin and quercetin
further sustains that small structural differences are associated
with significantly different biological activities and action
mechanisms. Therefore, it is most likely that flavonoids may
induce other specific ERa-mediated signaling that could drive
cells out of proliferation. This hypothesis is currently under
active investigation in our laboratory.
In conclusion, naringenin and quercetin can act as E2
mimetic inducing ERa genomic mechanism while impairing
ERa non-genomic activity. Thus, flavonoids can specifically
and negatively modulate ERa membrane starting rapid
signaling (i.e., ERK), responsible for E2-induced cell prolif-
eration. This observation implies a new action mechanism for
the antiproliferative effects of nutritional flavonoids unrelated
to flavonoid antioxidant properties and to their ability to alter
signal transduction protein kinase activation.
ACKNOWLEDGEMENTS
The editorial assistance of Mr. Peter De Muro is acknowl-
edged. R.A. is supported by the EC grant ISOHEART LRT-
2001-00221. This work was supported by grants from FIRB
2001, and 2003 Università ‘Roma Tre’ to M.M.
REFERENCES
1. Nilsson, S., Makela, S., Treuter, E., Tujague, M., Thomsen, J.,
Andersson, G., Enmark, E., Pettersson, K., Warner, M., and
Gustafsson, J.-Å. (2001) Mechanisms of estrogen action. Physiol.
Rev. 81, 1535 – 1565.
2. Foster, J. S., Henley, D. C., Ahamed, S., and Wimalasena, J. (2001)
Estrogens and cell-cycle regulation in breast cancer. Trends Endocri-
nol. Metab. 12, 320 – 327.
3. Marino, M., Acconcia, F., Bresciani, F., Weisz, A., and Trentalance,
A. (2002) Distinct nongenomic signal transduction pathways con-
trolled by 17b-estradiol regulate DNA synthesis and cyclin D1 gene
transcription in HepG2 cells. Mol. Biol. Cell 13, 3720 – 3729.
4. Marino, M., Acconcia, F., and Trentalance, A. (2003) Biphasic
estradiol-induced AKT-phosphorylation is modulated by PTEN via
MAP kinase in HepG2 cells. Mol. Biol. Cell 14, 2583 – 2591.
5. Matthews, J., and Gustafsson, J.-Å. (2003) Estrogen signaling: a
subtle balance between ERa and ERb. Mol. Inter. 3, 281 – 292.
6. Weihua, Z., Andersson, S., Cheng, G., Simpson, E. R., Warner, M.,
and Gustafsson, J.-Å. (2003) Update on estrogen signaling. FEBS
Lett. 546, 17 – 24.
7. Jordan, C. V. (2003) Antiestrogens and selective estrogen receptor
modulators as multifunctional medicines. 1. Receptor interactions. J.
Med. Chem. 46, 883 – 908.
8. Brzezinski, A., and Debi, A. (1999) Phytoestrogens: the ‘‘natural’’
selective estrogen receptor modulators? Eur. J. Obstet. Gynecol.
Reprod. Biol. 85, 47 – 51.
9. Kuiper, G. G., Lemmen, J. G., Carlsson, B., Corton, J. C., Safe, S. H.,
van der Saag, P. T., van der Burg, B., and Gustafsson, J.-Å. (1998)
Interaction of estrogenic chemicals and phytoestrogens with estrogen
receptor b. Endocrinology 139, 4252 – 4263.
10. Lissin, L. W., and Cooke, J. P. (2000) Phytoestrogens and
cardiovascular health. J. Am. Coll. Cardiol. 35, 1403 – 1410.
11. Ewies, A. A. (2002) Phytoestrogens in the management of the
menopause: up-to-date. Obstet. Gynecol. Surv. 57, 306 – 313.
12. Wuttke, W., Jarry, H., Becker, T., Schultens, A., Christoffel, V.,
Gorkow, C., and Seidlová-Wuttke, D. (2003) Phytoestrogens: endo-
crine disrupter or replacement for hormone replacement therapy?
Maturitas 44, S9 – S20.
13. Middleton, E., Kandaswami, C., and Theoharides, T. C. (2000) The
effects of plant flavonoids on mammalian cells: implications for
inflammation, heart disease, and cancer. Pharmacol. Rev. 52, 673 – 751.
14. Kris-Etherton, P. M., Hecker, K. D., Bonanome, A., Coval, S. M.,
Binkoski, A. E., Hilpert, K. F., Griel, A. E., and Etherton, T. D.
(2002) Bioactive compounds in foods: their role in the prevention of
cardiovascular disease and cancer. Am. J. Med. 113, 71S – 88S.
15. Birt, D. F., Hendrich, S., and Wang, W. (2001) Dietary agents in
cancer prevention: flavonoids and isoflavonoids. Pharmacol. Ther. 90,
157 – 177.
16. Chen, W. F., Huang, M. H., Tzang, C. H., Yang, M., and Wong, M.
S. (2003) Inhibitory actions of genistein in human breast cancer
(MCF-7) cells. Biochim. Biophys. Acta 1638, 187 – 196.
17. Hung, H. (2004) Inhibition of estrogen receptor a expression and
function in MCF-7 cells by kaempferol. J. Cell Physiol. 198, 197 – 208.
18. Hollman, P. C., and Katan, M. B. (1999) Dietary flavonoids: intake,
health effects and bioavailability. Food Chem. Toxicol. 37, 937 – 942.
19. Marino, M., Distefano, E., Caporali, S., Ceracchi, G., Pallottini, V.,
and Trentalance, A. (2001) 17b-estradiol stimulation of DNA
synthesis requires different PKC isoforms in HepG2 and MCF7 cells.
J. Cell Physiol. 188, 170 – 177.
20. Suzuki, Y. J., and Packer, L. (1993) Inhibition of NF-kB activation by
vitamin E derivatives. Biochem. Biophys. Res. Comm. 193, 277 – 283.
21. Lobenhofer, E. K., Huper, G., Iglehart, J. D., and Marks, J. R. (2000)
Inhibition of mitogen-activated protein kinase and phosphatidylino-
sitol 3-kinase activity in MCF-7 cells prevents estrogen-induced
mitogenesis. Cell Growth Differ. 11, 99 – 110.
22. Bors, W., Michel, C., and Stettmaier, K. (1997) Antioxidant effects of
flavonoids. Biofactors 6, 399 – 402.
23. Hagiwara, M., Inoue, S., Tanaka, T., Nunoki, K., Ito, M., and
Hidaka, H. (1988) Differential effects of flavonoids as inhibitors of
tyrosine protein kinases and serine/threonine protein kinases. Bio-
chem. Pharmacol. 37, 2987 – 2992.
24. Maggiolini, M., Bonofiglio, D., Marsico, S., Panno, M. L., Cenni, B.,
Picard, D., and Andò, S. (2001) Estrogen receptor a mediates the
proliferative but not the cytotoxic dose-dependent effects of two major
phytoestrogens on human breast cancer cells. Mol. Pharmacol. 60,
595 – 602.
25. Erlund, I., Meririnne, E., Alfthan, A., and Aro, A. (2001) Plasma
kinetics and urinary excretion of the flavanones naringenin and
hespertin in humans after ingestion of orange juice and grapefruit
juice. J. Nutr. 131, 235 – 241.
26. Guttridge, D. C., Albanese, C., Reuther, J. Y., Pestell, R. G., and
Baldwin, A. S. (1999) NF-kB controls cell growth and differentiation
through transcriptional regulation of cyclin D1. Mol. Cell Biol. 19,
5785 – 5799.
 NUTRITIONAL FLAVONOIDS AND ERa RAPID SIGNALS
27. Hall, J. M., McDonnell, D. P., and Korach, K. S. (2002) Allosteric
regulation of estrogen receptor structure, function, and coactivator
recruitment by different estrogen response elements. Mol. Endocrinol.
16, 469 – 486.
28. McDonnell, D. P. (1999) The molecular pharmacology of SERMs.
Trends Endocrinol. Metab. 10, 301 – 311.
29. Saarto, T., Blomqvist, C., Ehnholm, C., Taskinen, M. R., and
Elomaa, I. (1996) Antiatherogenic effects of adjuvant antiestrogens: a
randomized trial comparing the effects of tamoxifen and toremifene
on plasma lipid levels in postmenopausal women with node-positive
breast cancer. J. Clin. Oncol. 14, 429 – 433.
30. Tanenbaum, D. M., Wang, Y., Williams, S. P., and Sigler, P. B. (1998)
Crystallographic comparison of the estrogen and progesterone
receptor’s ligand binding domains. Proc. Natl. Acad. Sci. USA 95,
5998 – 6003.
151
31. Pike, A. C., Brzozowski, A. M., Walton, J., Hubbard, R. E., Thorsell,
A. G., Li, Y. L., Gustafsson, J.-Å., and Carlquist, M. (2001)
Structural insights into the mode of action of a pure antiestrogen.
Structure 9, 145 – 153.
32. Paige, L. A., Christensen, D. J., Gron, H., Norris, J. D., Gottlin, E.
B., Padilla, K. M., Chang, C. Y., Ballas, L. M., Hamilton, P. T.,
McDonnell, D. P., et al. (1999) Estrogen receptor (ER) modulators
each induce distinct conformational changes in ER a and ER b. Proc.
Natl. Acad. Sci. USA 96, 3999 – 4004.
33. McKenna, N. J., and O’Malley, B. W. (2000) An issue of tissues:
divining the split personalities of selective estrogen receptor mod-
ulators. Nature Med. 6, 960 – 962.
34. Rachez, C., and Freedman, L. P. (2001) Mediator complexes and
transcription. Curr. Opin. Cell. Biol. 13, 274 – 280.