Research Communication

Naringenin and 17b-Estradiol Coadministration PreventsHormone-Induced Human Cancer Cell Growth

Pamela Bulzomi1, Alessandro Bolli1,2, Paola Galluzzo1, Stefano Leone1, Filippo Acconcia1

and Maria Marino1,2*1Department of Biology, University Roma Tre, Viale G. Marconi, Roma, Italy2National Institute of Biostructures and Biosystems, Viale Medaglie d’Oro, Roma, Italy

Summary

Flavonoids have been described as health-promoting, disease-preventing dietary components. In vivo and in vitro experimentsalso support a protective effect of flavonoids to reduce the inci-dence of certain hormone-responsive cancers. In particular, ourprevious results indicate that the flavanone naringenin (Nar),decoupling estrogen receptor a (ERa) action mechanisms, drivescancer cells to apoptosis. Because these studies were conducted inthe absence of the endogenous hormone 17b-estradiol (E2), thephysiological relevance of these findings is not clear. We investi-gate whether the antiproliferative Nar effect persists in the pres-ence of physiological E2 concentration (i.e. 10 nM), using bothERa-transfected (HeLa cells) and ERa-containing (HepG2 cells)cancer cell lines. Ligand saturation experiments indicate that Nardecreases the binding of E2 to ERa without impairing the estro-gen response element (ERE)-driven reporter plasmid activity. Incontrast, Nar stimulation prevents E2-induced extracellular regu-lated kinases (ERK1/2) and AKT activation and still induces theactivation of p38, the proapoptotic member of mitogen-activatingprotein kinase (MAPK) family. As a consequence, Nar stimula-tion impedes the E2-induced transcription of cyclin D1 promoterand reverts the E2-induced cell proliferation, driving cancer cellto apoptosis. Thus, these results suggest that coexposure to thislow-affinity, low-potency ligand for ERa specifically antagonizesthe E2-induced ERa-dependent rapid signals by reducing theeffect of the endogenous hormone in promoting cellular prolifera-tion. As a whole, these data indicate that Nar is an excellentcandidate as a chemopreventive agent in E2-dependentcancers. � 2009 IUBMB

IUBMBLife, 62(1): 51–60, 2010

Keywords 17b-estradiol; estrogen receptor a; cell proliferation; nar-ingenin.

INTRODUCTION

Flavonoids, plant secondary metabolites, are defined chemi-

cally as substances composed of a common phenylchromanone

structure (C6-C3-C6) with one or more hydroxyl substituents

(1). Flavonoids are present in fruits, vegetables and beverages

derived from plants (e.g. tea, red wine, orange, and grapefruit

juices), and in many dietary supplements or herbal remedies

(1). Flavonoids have been described as health-promoting, dis-

ease-preventing dietary components; moreover, in vivo and

in vitro experiments support a protective effect of flavonoids to

reduce the incidence of certain hormone-responsive cancers (1–

5). In addition, they are extremely safe and associated with low

toxicity, making them good candidates as chemopreventive

agents.

The cancer-protective effects of flavonoids have been attrib-

uted to a wide variety of mechanisms (6). These include pro-

and/or antioxidant effects, and the modulation of kinase

activities as well as protein functions through competitive or

allosteric interactions. However, flavonoid-dependent kinases

modulation and antioxidant effects are only reported after

administration of high flavonoid concentration ([50 lM) (see

Ref. 6 and literature cited therein). Currently, the relative

importance of these pathways and their putative cross-talk

remain to be established. Furthermore, their clinical significance

at nutritionally relevant concentrations remains unsolved (7). Atconcentrations more physiologically achievable in the plasma

after the consumption of meals rich in flavonoids (i.e. 0.1–10lM), these compounds interact with estrogen receptors (ERaand ERb) and affect their resulting cellular responses (3, 8),thus leading to estrogenic or antiestrogenic effects. Because of

this ability to interfere with E2 action, flavonoids are actually

defined as dietary phytoestrogens (9).

We recently demonstrated that the flavanone naringenin

(Nar, 5,7,40-trihydroxyflavanone) hampers ERa-mediated rapid

activation of signaling kinases [i.e. extracellular regulated ki-

nases (ERK1/2) member of mitogen-regulated protein kinase

Address correspondence to: Maria Marino, Department of Biology,

University ‘‘Roma Tre’’, Viale G. Marconi, 446, I-00146 Roma, Italy.

Tel: 139 06 55176345. Fax: 139 06 55176321.

E-mail: m.marino@ uniroma3.it

Received 26 June 2009; accepted 8 October 2009

ISSN 1521-6543 print/ISSN 1521-6551 online

DOI: 10.1002/iub.279

IUBMB Life, 62(1): 51–60, January 2010

(MAPK) and phosphatidyl inositol 3 kinase (PI3K)/AKT] and

cyclin D1 transcription, important for cell cycle progression,

only when HeLa cells, devoid of any ER isoforms, were

endowed with human ERa (8, 10, 11). On the other hand, in

the presence of ERb, Nar does not impair the ERb-mediated

activities. Rather, Nar acts as an estrogen mimetic (8). These

results increase the possibility that Nar could reduce the effect

of the potent endogenous 17b-estradiol (E2) in promoting cellu-

lar proliferation when administrated in sufficient quantities, with

the net effect of antagonizing the ERa-dependent E2 effects.

Thus, the aim of the study was to evaluate the antagonistic

effect of Nar by investigating the effects of physiological E2

concentration (i.e. 10 nM) in the presence of different concen-

trations of Nar in ERa-expressing cells. In particular, the HeLa

cell line was chosen because it is devoid of endogenous expres-

sion of ERs but it can be rendered E2 sensitive after the tran-

sient transfection with ERa expression vector without any com-

plication because of the presence of ERb. Moreover, we previ-

ously reported that the full complement of coactivators,

corepressors, and signalling kinases necessary for the full ER

activity are present in this line (8, 12). In addition, the hepa-

toma cell line (HepG2), which endogenously express low levels

of ERa (13), was also used.

MATERIALS AND METHODS

Reagents

Naringenin, 17b-estradiol, gentamicin, penicillin and other

antibiotics, GenElute plasmid maxiprep kit, Dulbecco Modified

Eagle Medium (DMEM) and RPMI-1640 media without phenol

red, and charcoal-stripped fetal calf serum were purchased from

Sigma–Aldrich (St. Louis, MO). Lipofectamine reagent was

obtained from GIBCO-BRL Life-technology (Gaithersburg,

MD). The luciferase kit was obtained from Promega (Madison,

WI). Bradford protein assay was obtained from BIO-RAD Lab-

oratories (Hercules, CA). [6,7-3H]E2 (specific activity 5 44.8

Ci/mmol) was purchased from Perkin-Elmer Life Sciences

(Cambridge, UK). The human recombinant ERa was obtained

by PanVera (Madison, WI). The antiphospho-ERK1/2, anti-

AKT, anti-b-tubulin, anti-ERa, anticaspase-3, antipoly(ADP-

ribose)polymerase (PARP), and anti-ERK1/2 antibodies were

obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The

polyclonal antiphospho-AKT, anti-phospho-p38, and anti-p38

antibodies were purchased from New England Biolabs (Beverly,

MA). ECL, chemiluminescence reagent for Western blot was

obtained from Amersham Biosciences, (Little Chalfont, UK).

All the other products were from Sigma–Aldrich. Analytical or

reagent grade products were used without further purification.

Ligand Binding Analysis

The reversible binding of E2 to human recombinant ERawas studied by ligand saturation experiments. Recombinant

ERa (final concentration 1.0 3 10210 M) was incubated for 2 h

at 25 8C in the binding buffer (Tris–HCl 4.0 3 1022 M, EDTA

1.0 3 1023 M, DDT 1.0 3 1023 M, 1% (w/v) yeast extract

and 10% (v/v) glycerol, pH 7.4) with [3H]E2 (final concentra-

tion, ranging between 1.0 3 10210 and 4.0 3 1028 M). In par-

allel, recombinant ERa (final concentration, 1.0 3 10210 M)

was incubated for 2 h at 25 8C in the binding buffer with

[3H]E2 (final concentration, ranging between 1.0 3 10210 and

4.0 3 1028 M) in the presence of 1.0 3 1026 or 1.0 3 1025

M Nar. In all saturation ligand-binding experiments, the free

and ERa-bound radioligand were separated by vacuum filtration

through a 12-sample Millipore filter manifold (Bedford, MA),

holding glass microfibre filters (Whatman Ltd, UK) (14). Radio-

activity retained on each filter was counted in 5 mL of the scin-

tillation cocktail (Perkin Elmer, Cambridge, UK) with a

2100TR Tri-Carb liquid scintillation analyzer (Packard Instru-

ments CO., Meriden, CT).

The value of the apparent dissociation equilibrium constant

for [3H]E2 binding to ERa (Kd0), in the absence and presence of

1.0 3 1026 M and 1.0 3 1025 M Nar, was determined from

the dependence of the radioactivity retained on filters (i.e. R) on

the [3H]E2 concentration, according to Equation (1) (14):

Y ¼ R=Rtot ¼ ð13½L�Þ=ðK0d þ ½L�Þ (1)

where Rtot is the maximum asymptotic value of radioactivity

measured when the complete saturation of the receptor was

achieved and [L] denotes the free radioligand concentration (i.e.

[[3H]E2]). In the absence of Nar, Kd0 corresponds to the intrinsic

dissociation equilibrium constant for E2 binding to recombinant

ERa (i.e. KE2d ). According to the competitive inhibition mecha-

nism, the intrinsic equilibrium dissociation constant for Nar

binding to ERa (i.e. KNard ) was determined from the dependence

of K0d from the Nar concentration, according to Equation (2)

(14):

K0d ¼ ðKE2

d

�KNard Þ 3 ½Nar� þ KE2

d (2)

As expected from Eq. (2), for [Nar] 5 0, Kd0 corresponds to

KE2d .

Cell Culture

The ER devoid of human cervix epitheloid carcinoma cell

line (HeLa) and the ERa containing human hepatoma cell line

(HepG2) were routinely grown in air containing 5% CO2in

modified, phenol red-free, DMEM (HeLa cells) or RPMI-1640

medium (HepG2 cells) containing 10% (v/v) charcoal-stripped

fetal calf serum, L-glutamine (2.0 mM), gentamicin (10 mg/

mL), and penicillin (100 U/mL). Cells were passaged every

2 days (HeLa cells) or every 3 days (HepG2 cells).

Plasmids, Cell Transfection, and Luciferase Assay

The gene reporter plasmids complement 3-luciferase (pC3),

Cyclin D1-luciferase (pXP2-D1-2966-luciferase, pD1), and the

plasmids containing the vector expression for pCR3.1-b-galacto-

52 BULZOMI ET AL.

sidase and the wild-type human ERa pSG5-HE0 have been

described elsewhere (8). Furthermore, an empty vector,

pCMV5, was used as control. A luciferase dose response curve

showed that the maximum effect was obtained when 1.0 lg of

plasmids was transfected together with 1.0 lg of pCR3.1-b-ga-lactosidase to normalize for transfection efficiency (�50–60%).

Plasmids were purified for transfection using the GenElute plas-

mid maxiprep kit according to the manufacturer’s instructions.

HeLa cells were grown to �70% confluence and then trans-

fected using Lipofectamine Reagent according to the manufac-

turer’s instructions. Six hours after transfection, the medium

was changed and 24 h after the cells were stimulated for 24 h

with either Nar (1.0 3 1026 M) or E2 (1.0 3 1028 M) or with

different concentration (1.0 3 1028 to 1.0 3 1024 M) of Nar

in the presence of 1.0 3 1028 M E2. 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 lumi-

nometer (Bad Wildbad, Germany).

Cell Viability and Cell Cycle

HeLa cells were grown to �70% confluence in six-well

plates, transfected with human pSG5-hERa or pCMV5 (empty

vector) and, after 24 h, stimulated with different concentration

(1.0 3 10210 to 1.0 3 1024 M) of Nar or E2 for 24 h or with

different concentration (1.0 3 10210 to 1.0 3 1024 M) of Nar

in the presence of 1.0 3 1028 M E2. After treatment, cells

were harvested with trypsin, centrifuged, stained with trypan

blue solution, and counted in a hemocytometer (improved Neu-

bauer chamber) in quadruplicate. For cell cycle analysis, 106

HeLa cells were transfected with either human pSG5-hERa or

pCMV5 expression vectors and, 24 h after, stimulated with Nar

1.0 3 1026 M in the presence or absence of E2 1.0 3 1028 M

for 24 h. After stimulation, cells were fixed with 1 mL ice-cold

70% ethanol and subsequently stained with 2 mg/mL DAPI/

PBS solution. The fluorescence of DNA was measured with

DAKO Galaxy flow-cytometer equipped with HBO mercury

lamp, and the percentage of cells present in sub-G1, G1, S and

G2/M phases was calculated using a FloMax� Software.

Western Blot

Cells were stimulated with either E2 (final concentration, 1.0

3 1028 M in ethanol/phosphate-buffered saline, PBS, 1:10, v/v)

or Nar (final concentration, 1.0 3 1026 M in DMSO/PBS 1:10,

v/v) or E2 1 Nar (final concentration, 1.0 3 1028 M and 1.0

3 1026 M, respectively) or vehicle (ethanol/PBS 1:10, v/v). In

some experiments, HepG2 cells were treated with E2 (1.0 31028 M) and different concentrations of Nar (1.0 3 1026 to 1.0

3 1024 M). After stimulation, cells were lysed and solubilized

in 0.125 M Tris, pH 6.8, containing 10% (w/v) SDS, 1.0 mM

phenylmethylsulfonyl fluoride, and 5.0 lg/mL leupeptin; then

the cell lysates were boiled for 2 min. Total proteins were quan-

tified using the Bradford protein assay. Solubilized proteins

(20 lg) were resolved by 7 or 10% SDS-PAGE at 100 V for 1

h at 24 8C and then electrophoretically transferred to nitrocellu-

lose for 45 min at 100 V and 4 8C. The nitrocellulose was

treated with 3% (w/v) BSA in 138.0 mM NaCl, 25.0 mM Tris,

pH 8.0, at 24 8C for 1 h and then probed overnight at 4 8C with

either anti-ERa or anticaspase-3 or anti-PARP or antiphospho-

ERK1/2 or antiphospho-AKT or antiphospho-p38 antibodies.

The nitrocellulose was stripped by Restore Western Blot Strip-

ping Buffer (Pierce Chemical Company, Rockford, IL) for 10

min at room temperature and then probed with either anti-

ERK1/2 or anti-AKT or anti-p38 and anti-b-tubulin antibodies.

Antibody reaction was visualized with chemiluminescence

Western blot detection reagent (Amersham Biosciences, Little

Chalfont, UK). Densitometric analyses were performed by

ImageJ software for Windows.

Statistical Analysis

A statistical analysis was performed by using Student0 t testwith the GraphPad INSTAT3 software system for Windows. In

all cases, P values\0.05 were considered significant.

Figure 1. Naringenin (Nar) effect on 17b-estradiol (E2) bindingto human recombinant ERa. Panel a: Dependence of the intrin-

sic molar fraction (Y) of [3H]E2-bound to ERa from [3H]E2

concentration in the absence (squares) and in the presence of 2

representative Nar concentrations (1.0 3 1026 M [diamonds]

and 1.0 3 1025 M [circles]). Data are the means 6 SD of five

different experiments. Panel b: Dependence of the apparent dis-

sociation equilibrium constant for [3H]E2 binding to ERa (Kd0)

from Nar concentrations (ranging from 1.0 3 1026 M to 1.0 31025 M). Data are means 6 SD of five different experiments.

For details see the text.

53E2 AND NARINGENIN ANTAGONIST EFFECTS ON PROLIFERATION

RESULTS

E2 and Nar Binding to ERa

To assess the Nar ability to compete with E2 for binding to

human recombinant ERa, E2 saturation experiments have been

performed in the absence and presence of 1.0 3 1026 and

1.0 3 1025 M Nar. Both in the absence and in the presence of

Nar, E2 binding to ERa follows a simple equilibrium as postu-

lated by Equation (1), the Hill coefficient being 1.0 6 0.1. In

the absence of Nar, E2 binding to ERa is characterized by an

intrinsic equilibrium dissociation constant (KE2d ) of (2.0 6 0.5)

3 10210 M. In the presence of unlabeled Nar, the apparent

equilibrium constant for E2 binding to ERa increased to Kd0 5

(5.2 6 0.6) 3 1029 M and (3.1 6 0.4) 3 1028 M in the pres-

ence of 1.0 3 1026 and 1.0 3 1025 M of Nar, respectively

(Fig. 1). The linear dependence of Kd0 on the Nar concentration

(Fig. 1b) indicates that a simple competition mechanism is oper-

ative (15, 16). Data reported in Figure 1b, analyzed according

to Eq. (2), allowed the determination of the intrinsic dissocia-

tion constant for Nar binding to ERa (KNard 5 1.4 6 0.3 3

1027 M). This confirms that Nar binds to ERa with an affinity

lower by about three orders of magnitude than that of E2. These

data indicate that Nar and E2 bind competitively to ERa; more-

over, in the presence of nutritionally relevant Nar concentra-

tions, the molar fraction of E2 bound to ERa decreases.

ERa Transcriptional Activities

The result of Nar binding to ERa prompted us to evaluate

the effect of co-stimulation of E2 and Nar on the ERa activities.

We first assessed the ERa-mediated direct gene transcription

(i.e. estrogen responsive element (ERE)-dependent) (13). HeLa

cells, transiently transfected with ERa or empty vector, and the

ERE-containing reporter plasmid (pC3) were incubated with

either E2 alone (1.0 3 1028 M) or Nar alone (1.0 3 1026 M)

or in the presence of E2 (1.0 3 1028 M) and different Nar

Figure 2. Naringenin (Nar) effect on 17b-estradiol (E2)-induced promoter activities. Panel a: Luciferase assay detection on HeLa

cells cotranfected with ERa (left panel) or empty vector (right panel) and pC3-luciferase construct and then treated 24 h with either

vehicle or E2 (1.0 3 1028 M) or Nar (1.0 3 1026 M) or with different Nar concentrations (1.0 3 1028 M to 1.0 3 1024 M) in

the presence of 1028 M E2. Data are the means 6 S.D. of four independent experiments. *P \ 0.001, compared with vehicle val-

ues, was determined using Student’s t test. Panel b: Luciferase assay detection on HeLa cells co-tranfected with ERa (left panel) or

empty vector (right panel) and cyclin D1 (pD1)-luciferase construct and then treated 24 h with either vehicle or E2 (1.0 3 1028

M) or Nar (1.0 3 1026 M) or with different Nar concentrations (1.0 3 1028 M to 1.0 3 1024 M) in the presence of 1028 M E2.

Data are the means 6 S.D. of four independent experiments. P \ 0.001, determined by using Student’s t test, was compared with

vehicle (*) or E2 (8) or Nar (1) values.

54 BULZOMI ET AL.

concentrations. Nar, alone or with E2, induced the ERE-contain-

ing promoter activity to a level comparable with that of E2

alone (Fig. 2a). No pC3 promoter activity was present when

HeLa cells, transiently transfected with the empty plasmid, were

stimulated with different ERa ligands (Fig. 2a), thus demon-

strating the ERa dependence of this effect. The indirect tran-

scriptional activity of ERa [i.e. through interaction with activa-

tor protein-1 (AP-1) or stimulating protein 1 (Sp1) transcription

factors] (13) was assessed by transfection with cyclin D1 (pD1)

promoter. In fact, cyclin D1 is a well-known E2-responsive

gene, even if ERE-like sequence in its promoter has not been

detected (17). As expected, cell treatment with E2 resulted in a

significant increase in cyclin D1 promoter activity (Fig. 2b)

comparable with those previously reported (13). Notably, 1.0 31027 M Nar reduced the E2 effect, and higher Nar concentra-

tions (i.e. 1.0 3 1026 to 1.0 3 1024 M) completely prevented

E2-induced pD1 promoter activity (Fig. 2b). To determine the

ER involvement in the ligand-induced cyclin D1 promoter ac-

tivity, experiments were performed also in HeLa cells trans-

fected with the empty plasmids (Fig. 2b). Results indicate that

no pD1 promoter activity was present when these cells were

stimulated with E2 or Nar (Fig. 2b).

ERa-Dependent Rapid Signals

The E2-induced cyclin D1 promoter activity requires rapid sig-

nal transduction pathways. In particular, the rapid (15 min) E2-

induced activation of ERK1/2 and PI3K/AKT cascades are funda-

mental for E2-induced pD1 promoter activity (13, 18). On the

other hand, Nar stimulation induces the rapid and persistent

(15 min to 24 h) activation of p38, another component of MAPK

family (8, 11). Thus, the ability of E2 to still induce rapid signal

kinase cascades even in the presence of 1.0 3 1026 M Nar was

evaluated in HeLa cells transfected with the empty vector or with

ERa expression vector. No kinase activation was detected in

HeLa cells devoid of ERa stimulated with E2 or Nar (data not

shown), whereas E2 ability to induce the rapid (15 min) ERK1/2

and AKT activation without any effect on the persistent (24 h)

p38 activation has been confirmed in ERa-containing HeLa cells

(Fig. 3). Remarkably, Nar stimulation prevents E2-induced

ERK1/2 and AKT activation and still induces the persistent p38

phosphorylation even in the presence of E2 (Fig. 3).

ERa-Dependent E2-Induced Cell Proliferation

Cyclin D1 represents the upstream sensor of E2-induced pro-

liferative signals, which, in turn, depends on the rapid activation

of upstream E2-induced kinase (12, 13). However, in the pres-

ence of ERa, Nar prevents cell proliferation inducing a proa-

poptotic cascade (8, 11). Figure 4 confirms that 1.0 3 1026, 1.0

3 1025, and 1.0 3 1024 M Nar reduced cell number only in

ERa-containing HeLa cells, whereas physiological E2 concen-

trations (i.e. 1.0 3 1029, and 1.0 3 1028 M) doubled the cell

numbers in 24 h (Fig. 4a). Note that high Nar or E2 concentra-

tion (1.0 3 1024 M) reduced cell numbers also in empty vec-

tor-transfected HeLa cells, suggesting an ERa-independent cyto-toxic effects for both substances (Fig. 4a). Intriguingly, Nar

stimulation reverted the E2-induced effect on cell proliferation

significantly reducing the number of cells in a dose-dependent

manner (Fig. 4b). Furthermore, 1.0 3 1026 M Nar changed the

E2-induced distribution of cell population in the cell cycle

phases (Fig. 4c), decreasing the cells present in G1 phase and

increasing the number of cell present in sub-G1 phase of the

cell cycle as follows 15.0 6 1.3 % (Vehicle), 20.2 6 0.5%

(E2), 42.0 6 0.7 % (Nar), and 43.4 6 1.0 % (E21Nar) (Fig.

4b). In line with these results, Nar increased the level of the

active caspase-3 (i.e. 17 kDa band, Fig. 5a) as demonstrated by

the increased level of poly(ADP-ribose)polymerase (PARP)

cleavage, a caspase-3 substrate, even in the presence of 1.0 31028 M E2 (Fig. 5b), thus demonstrating the strong antagonistic

effects of this flavanone on E2-induced proliferation.

Figure 3. Naringenin (Nar) effects on 17b-estradiol (E2)-

induced rapid ERa activities. ERa-transfected HeLa cells were

treated with either vehicle or E2 (1.0 3 1028 M) or Nar (1.0 31026 M) or with a mixture of Nar (1.0 3 1026 M) 1 E2 (1.0

3 1028 M). After 15 min (left panel, ERK1/2 and AKT) or af-

ter 24 h (right panel, p38), the phosphorylation of the kinases

was evaluated. The amounts of protein were normalized by

comparison with un-phosphorylated ERK1/2 or AKT or p38

and tubulin antibodies. Upper panels show representative West-

ern blots, lower panel shows the densitometric analysis. Data

are the means 6 S.D. of four independent experiments. P \0.001, determined by using Student’s t test, was compared with

vehicle (*) or E2 (8) values.

55E2 AND NARINGENIN ANTAGONIST EFFECTS ON PROLIFERATION

To avoid any problem because of the receptor overexpres-

sion in HeLa cells, the Nar effect on p38 phosphorylation and

on the activation of a proapoptotic cascade was performed in

parallel in cancer cells that express endogenous ERa (HepG2).

These cells, derived from liver, could be one of main targets of

flavonoid action after oral administration. Moreover, HepG2

cells represent an E2-dependent proliferative model (13). The

level of endogenous ERa was assessed in HepG2 by Western

blot analysis, which confirmed the presence of a unique band at

67 kDa corresponding to ERa (data not shown). In HepG2 cells,

Nar stimulation, both alone or in the presence of E2, increased

p38 phosphorylation, caspase-3 activation, and PARP cleavage

(Fig. 6), confirming that, also in the presence of endogenous re-

ceptor, Nar reverts the E2-dependent proliferative effects as

obtained in Hela cells.

DISCUSSION

E2 influences many physiological processes in mammals,

including reproduction, cardiovascular health, bone integrity,

cognition, and behaviour, to name a few. Given this widespread

role in human physiology, it is not surprising that E2 is also

implicated in the development or progression of numerous dis-

eases which include various types of cancers such as breast,

ovarian, colorectal, prostate, and endometrial cancer (13, 19).

It is now accepted that the mechanisms at the root of E2-

related cancer mainly depend on the ERa-mediated membrane-

starting rapid effects (13, 18). ERb seems to act as a negative

regulator of E2-induced proliferation (12, 13, 20–23). As an

example, ERa-positive MCF-7 breast cancer cells respond to E2

with increased proliferation, but when ERb is introduced into

these cells, E2-induced proliferation is inhibited (21). These

findings are relevant in view of the possible action of ERb as a

tumor suppressor. In search for agents that would be useful in

preventing and treating E2-dependent cancer, the interest in fla-

vonoids has increased markedly (5, 24–30). In fact, contrary to

E2, flavonoids bind to ERb with up to five times higher affin-

ities compared with ERa (31, 32). As ERb ligands, flavonoids

may be able to trigger beneficial responses through their prefer-

ential interaction with this ER isoform. However, this protective

Figure 4. Naringenin (Nar) effects on 17b-estradiol (E2)-induced HeLa cells proliferation. a: Number of HeLa cells tranfected with

ERa or empty vector and treated 24 h with either vehicle (0) or different concentrations of E2 or Nar (1.0 3 10210 M to 1.0 31024 M). b: ERa-tranfected HeLa cells were stimulated for 24 h with either vehicle or E2 (1.0 3 1028 M) or Nar (1.0 3 1026 M)

or with different Nar concentrations (1.0 3 1029 M to 1.0 3 1024 M) in the presence of 1.0 3 1028 M E2. Data are the means 6S.D. of five independent experiments. P \ 0.001, determined by using Student’s t test, was compared with vehicle (0, *) or E2 (8)values. c: Flow cytometric analysis of ERa tranfected HeLa cells after 24 h of treatment with vehicle or E2 (1.0 3 1028 M) or

Nar (1.0 3 1026 M) or E2 1 Nar (1.0 3 1028 M and 1.0 3 1026 M, respectively). The plots indicate a typical cell cycle distribu-

tion present in sub-G1, G1, S, and G2/M phases, respectively (left panels) and the percentage of cell present in sub-G1 phases

(right panels).

56 BULZOMI ET AL.

effect could be abrogated in cells that selectively express ERa,such as mammary gland, ovary, and liver (13, 19, 20), which

only contain minimal residual amounts of ERb.Our previous studies indicate that Nar, a flavanone compo-

nent of citrus fruits and tomatoes, impaired E2-induced prolifer-

ative signals interfering with ERa-mediated activation of ERK1/

2 and PI3K pathways without affecting the transcription of an

ERE-containing reporter gene (8, 10). Moreover, at the plasma

membrane level, Nar induced ERa de-palmitoylation faster than

E2, which resulted in receptor rapid dissociation from mem-

brane caveolin-1 and in impaired receptor binding to the mito-

genic signaling proteins (i.e. ERK1/2 and PI3K/AKT) (11). On

the other hand, Nar induced the ERa-dependent, but palmitoyl-

ation-independent, activation of p38 kinase, which, in turn, was

responsible for Nar-mediated anti-proliferative effects in cancer

cells (8, 11). These results imply that, besides its effects in the

presence of ERb, Nar works as a selective inhibitor of ERa-mediated proliferation (5 and literature cited therein). In addi-

tion, Nar could also acts as an antagonist of E2, preventing E2-

induced cell proliferation when ERa is present. Although it is

essential to evaluate the Nar antiproliferative potential, this

topic has been focused in a very limited number of publications

(33). The aim of this work was to verify this hypothesis and to

extend our knowledge about the mechanisms involved in

Figure 5. Effect of Naringenin (Nar) on the induction of pro-

apoptotic proteins in HeLa cells. Analysis of caspase-3 activa-

tion (panel a) and PARP cleavage (panel b) were performed in

HeLa cells transfected with human ERa and treated 24 h with

vehicle or 17b-estradiol (E2, 1.0 3 1028 M) or Nar (1.0 31026 M) or E2 1 Nar (1.0 3 1028 M and 1.0 3 1026 M,

respectively). Typical Western blots of three independent

experiments are presented in panels a and b. In panel c, the rel-

ative densitometric analysis is reported. The data are the mean

values 6 S.D. P \ 0.001, determined by using Student’s t test,

was compared with vehicle (*) or E2 (8) values.

Figure 6. Effect of Naringenin (Nar) on the induction of proa-

poptotic proteins in HepG2 cells. Analysis of p38 phosphoryla-

tion, caspase-3 activation, and PARP cleavage was performed

in HepG2 cells treated 24 h with vehicle or 17b-estradiol (E2,1.0 3 1028 M) or Nar (1.0 3 1026 M) or with different Nar

concentrations (1.0 3 1026 M to 1.0 3 1024 M) in the pres-

ence of 1.0 3 1028 M E2. Typical Western blots of three inde-

pendent experiments are presented in panel a. In panel a0 are

presented the relative densitometric analyses. The data are the

mean values 6 SD. P\ 0.001, determined by using Student’s t

test, was compared with vehicle (*) or E2 (8) values.

57E2 AND NARINGENIN ANTAGONIST EFFECTS ON PROLIFERATION

Nar-mediated antiproliferative activity by investigating the

effect of this flavonoid on cancer cells growth in the presence

of an E2 background.

Current findings confirm that the Nar concentration required

to half-saturate ERa is about 1000-fold higher than that reported

for E2; however, in the presence of Nar, the Kd of E2 for its re-

ceptor linearly increased, suggesting a decreased affinity

between E2 and ERa. Remarkably, this decreased affinity

between E2:ERa did not impair the E2 ability, in the presence

of Nar, to trigger gene transcription through the direct binding

of ERa to ERE-containing reporter gene (i.e. pC3). This result

implies that the coactivator recruitment on the ligand-bound

ERa is not prevented by Nar as well as the arrangement of a

macromolecular complex, which provides the platform on which

the components of transcriptional machinery are assembled.

However, ligand bound to ERa could mediate gene transcription

even in a manner that does not require the ERa direct binding

to DNA. This is referred to as ‘‘indirect genomic mechanism’’

which requires the ERa interaction with specific transcription

factors such as Sp1 and AP-1. The ERa-Sp1 and ERa-AP-1complexes interact with response elements (GC-rich and TRE,

respectively) within target promoters. Genes activated by E2

through this genomic pathway include cathepsin D, c-fos, reti-

noic acid receptor a1, adenosine deaminase, IGF-binding pro-

tein 4, Bcl2, E2f1, thymidylate synthase, vascular endothelial

growth factor (Vegf), and cyclin D1 (34).

Intriguingly, in the presence of Nar, E2 lacks its ability to

activate cyclin D1 promoter, suggesting that E2-induced ERainteraction to Sp1 and AP-1 is impaired. Our previous data

indicate that E2 stimulation of ERa-containing HeLa cells

induced an increase in AP-1 binding to DNA, whereas Nar or

other flavonoids (i.e. quercetin) were unable to do this (10).

Although evidence indicate the ability of flavonoids to bind

both ER isoforms maintaining the ERs gene transcriptional

ability (31, 32, 35), current data indicate that Nar only allows

the E2-induced direct transcriptional activity of ERa, highlight-ing a role for Nar as an antagonist of E2-induced indirect

gene expression.

The physiological role played by rapid membrane-starting

pathways has been clarified at least for some E2 targets (19,

36–38). Among other cellular functions, the mechanisms by

which E2 exerts proliferative effects is assumed to be exclu-

sively mediated by rapid membrane-starting actions (13, 18). In

HepG2 cells, multiple and parallel membrane-starting pathways

are rapidly activated by the E2:ERa complex (13), and the

blockade of ERK1/2 and PI3K/AKT pathways completely pre-

vents the E2-induced DNA synthesis (13). ERK1/2/MAPK and

PI3K/AKT pathways, rapidly activated by E2:ERa complex,

also have a critical role in E2 action as a survival agent. In

fact, these pathways enhance the expression of the antiapoptotic

protein Bcl-2, block the activation of the p38/MAPK, reduce

the proapoptotic caspase-3 activation, and promote G1-to-S

phase transition through the enhancement of the cyclin D1

expression. Thus, in both ERa-transfected HeLa cells and in

HepG2 cells, the E2 inability to activate rapid signal transduc-

tion pathways, in the presence of Nar, was paralleled by the

block of E2-induced proliferation and by the induction of the

apoptotic cascade (i.e. caspase-3 activation and PARP cleav-

age).

As a whole, the assays with Nar against a background

level of E2 allowed us to assess the estrogenic versus anties-

trogenic activity of this flavanone. The results of this study

demonstrate that Nar treatment do not impair E2-induced

ERE-dependent ERa transcriptional activity, whereas Nar

reverts the proliferative effects of E2 impairing ERa-mediated

rapid signals and inducing different proapoptotic signal trans-

duction pathways. Moreover, the preventive effects elicited by

Nar on E2-dependent cancers may be enhanced, in some tis-

sues, through the induction of specific ERb-dependent proa-

poptotic signalling (8). In addition, these results increase the

list of Nar effects on human health adding up a possible ther-

apeutic benefit of regular consumption of these flavonoids,

which may counteract the E2 proliferative action. Collectively,

our data suggest that the regular consumption of Nar may

slow the rate at which E2-dependent cancer cell proliferate. In

addition, this study indicates that the studies, which only focus

on the transactivation capacity of various naturally derived es-

trogenic ligands, could be misleading in that they are actually

assaying just one of the diverse action mechanisms elicited by

the ERs.

Finally, a number of pleiotropic molecular effects of Nar

have been reported in cancer cells, which include the modula-

tion of cell signalling pathways, the regulation of the cell

cycle, the inhibition of glucose uptake, and antioxidant activ-

ities (1, 6). Some of these mechanisms may even occur inde-

pendently of ER binding (39, 40), but requires high plasma

Nar concentrations (i.e. 0.8 to 25 3 1025 M), which are diffi-

cult to obtain by the oral ingestion of food rich in this biofla-

vonoid. In the best case scenario, only 15% of ingested Nar

will get absorbed in the human gastrointestinal tract. A full

glass of orange juice would supply about enough Nar to

achieve a concentration of about 0.5 3 1026 M. In a study

conducted by Erlund et al. (41) in which five subjects drank

grapefruit juice containing approximately 200 mg Nar (similar

to the Nar content in one medium-sized grapefruit), the peak of

plasma naringenin concentrations ranged from 0.7 to 14.8

3 1026 M, demonstrating that Nar plasma concentration

depends on the ability of each individual to adsorb and metabo-

lize this compound (42). These concentrations could be

increased by the use of flavonoids as dietary supplements. A

huge number of plant extracts or mixtures containing varying

amounts of isolated flavonoids are commercially available on

the market as dietary supplements and healthy products. The

commercial success of these supplements is evident, even

though the health consequences of flavonoids exposure may be

not universally beneficial and, in certain physiological phases

of human life, could even increase the risk of diseases (43,

44). Thus, further investigations on the complex role of

58 BULZOMI ET AL.

nutritional molecules in human beings are warranted before

including flavonoids in specific nutritional recommendations.

ACKNOWLEDGEMENTS

Dr. Alessandro Bolli is supported by a grant from National

Institute of Biostructures and Biosystems (INBB). This work

was supported by grants from Ateneo Roma Tre and Italian

Health Ministry to M.M.

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