Naringenin and 17β-estradiol coadministration prevents hormone-induced human cancer cell growth

Authors


Abstract

Flavonoids have been described as health-promoting, disease-preventing dietary components. In vivo and in vitro experiments also support a protective effect of flavonoids to reduce the incidence of certain hormone-responsive cancers. In particular, our previous results indicate that the flavanone naringenin (Nar), decoupling estrogen receptor α (ERα) action mechanisms, drives cancer cells to apoptosis. Because these studies were conducted in the absence of the endogenous hormone 17β-estradiol (E2), the physiological relevance of these findings is not clear. We investigate whether the antiproliferative Nar effect persists in the presence of physiological E2 concentration (i.e. 10 nM), using both ERα-transfected (HeLa cells) and ERα-containing (HepG2 cells) cancer cell lines. Ligand saturation experiments indicate that Nar decreases the binding of E2 to ERα without impairing the estrogen response element (ERE)-driven reporter plasmid activity. In contrast, Nar stimulation prevents E2-induced extracellular regulated kinases (ERK1/2) and AKT activation and still induces the activation of p38, the proapoptotic member of mitogen-activating protein kinase (MAPK) family. As a consequence, Nar stimulation impedes the E2-induced transcription of cyclin D1 promoter and reverts the E2-induced cell proliferation, driving cancer cell to apoptosis. Thus, these results suggest that coexposure to this low-affinity, low-potency ligand for ERα specifically antagonizes the E2-induced ERα-dependent rapid signals by reducing the effect of the endogenous hormone in promoting cellular proliferation. As a whole, these data indicate that Nar is an excellent candidate as a chemopreventive agent in E2-dependent cancers. © 2009 IUBMB IUBMB Life, 62(1):51–60, 2010

INTRODUCTION

Flavonoids, plant secondary metabolites, are defined chemically 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, disease-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 attributed 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 μM) (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). At concentrations more physiologically achievable in the plasma after the consumption of meals rich in flavonoids (i.e. 0.1–10 μM), these compounds interact with estrogen receptors (ERα and ERβ) 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, 5,7,4′-trihydroxyflavanone) hampers ERα-mediated rapid activation of signaling kinases [i.e. extracellular regulated kinases (ERK1/2) member of mitogen-regulated protein kinase (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 ERα (8, 10, 11). On the other hand, in the presence of ERβ, Nar does not impair the ERβ-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 17β-estradiol (E2) in promoting cellular proliferation when administrated in sufficient quantities, with the net effect of antagonizing the ERα-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 concentrations of Nar in ERα-expressing cells. In particular, the HeLa cell line was chosen because it is devoid of endogenous expression of ERs but it can be rendered E2 sensitive after the transient transfection with ERα expression vector without any complication because of the presence of ERβ. Moreover, we previously 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 hepatoma cell line (HepG2), which endogenously express low levels of ERα (13), was also used.

MATERIALS AND METHODS

Reagents

Naringenin, 17β-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 Laboratories (Hercules, CA). [6,7-3H]E2 (specific activity = 44.8 Ci/mmol) was purchased from Perkin-Elmer Life Sciences (Cambridge, UK). The human recombinant ERα was obtained by PanVera (Madison, WI). The antiphospho-ERK1/2, anti-AKT, anti-β-tubulin, anti-ERα, 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 ERα was studied by ligand saturation experiments. Recombinant ERα (final concentration 1.0 × 10−10 M) was incubated for 2 h at 25 °C in the binding buffer (Tris–HCl 4.0 × 10−2 M, EDTA 1.0 × 10−3 M, DDT 1.0 × 10−3 M, 1% (w/v) yeast extract and 10% (v/v) glycerol, pH 7.4) with [3H]E2 (final concentration, ranging between 1.0 × 10−10 and 4.0 × 10−8 M). In parallel, recombinant ERα (final concentration, 1.0 × 10−10 M) was incubated for 2 h at 25 °C in the binding buffer with [3H]E2 (final concentration, ranging between 1.0 × 10−10 and 4.0 × 10−8 M) in the presence of 1.0 × 10−6 or 1.0 × 10−5 M Nar. In all saturation ligand-binding experiments, the free and ERα-bound radioligand were separated by vacuum filtration through a 12-sample Millipore filter manifold (Bedford, MA), holding glass microfibre filters (Whatman Ltd, UK) (14). Radioactivity retained on each filter was counted in 5 mL of the scintillation cocktail (Perkin Elmer, Cambridge, UK) with a 2100TR Tri-Carb liquid scintillation analyzer (Packard Instruments CO., Meriden, CT).

The value of the apparent dissociation equilibrium constant for [3H]E2 binding to ERα (Kd′), in the absence and presence of 1.0 × 10−6 M and 1.0 × 10−5 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):

equation image(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, Kd′ corresponds to the intrinsic dissociation equilibrium constant for E2 binding to recombinant ERα (i.e. Kmath image). According to the competitive inhibition mechanism, the intrinsic equilibrium dissociation constant for Nar binding to ERα (i.e. Kmath image) was determined from the dependence of Kd from the Nar concentration, according to Equation (2) (14):

equation image(2)

As expected from Eq. (2), for [Nar] = 0, Kd′ corresponds to Kmath image.

Cell Culture

The ER devoid of human cervix epitheloid carcinoma cell line (HeLa) and the ERα containing human hepatoma cell line (HepG2) were routinely grown in air containing 5% COmath image in 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-β-galactosidase and the wild-type human ERα 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 μg of plasmids was transfected together with 1.0 μg of pCR3.1-β-galactosidase to normalize for transfection efficiency (∼50–60%). Plasmids were purified for transfection using the GenElute plasmid maxiprep kit according to the manufacturer's instructions. HeLa cells were grown to ∼70% confluence and then transfected using Lipofectamine Reagent according to the manufacturer'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 × 10−6 M) or E2 (1.0 × 10−8 M) or with different concentration (1.0 × 10−8 to 1.0 × 10−4 M) of Nar in the presence of 1.0 × 10−8 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 luminometer (Bad Wildbad, Germany).

Cell Viability and Cell Cycle

HeLa cells were grown to ∼70% confluence in six-well plates, transfected with human pSG5-hERα or pCMV5 (empty vector) and, after 24 h, stimulated with different concentration (1.0 × 10−10 to 1.0 × 10−4 M) of Nar or E2 for 24 h or with different concentration (1.0 × 10−10 to 1.0 × 10−4 M) of Nar in the presence of 1.0 × 10−8 M E2. After treatment, cells were harvested with trypsin, centrifuged, stained with trypan blue solution, and counted in a hemocytometer (improved Neubauer chamber) in quadruplicate. For cell cycle analysis, 106 HeLa cells were transfected with either human pSG5-hERα or pCMV5 expression vectors and, 24 h after, stimulated with Nar 1.0 × 10−6 M in the presence or absence of E2 1.0 × 10−8 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 × 10−8 M in ethanol/phosphate-buffered saline, PBS, 1:10, v/v) or Nar (final concentration, 1.0 × 10−6 M in DMSO/PBS 1:10, v/v) or E2 + Nar (final concentration, 1.0 × 10−8 M and 1.0 × 10−6 M, respectively) or vehicle (ethanol/PBS 1:10, v/v). In some experiments, HepG2 cells were treated with E2 (1.0 × 10−8 M) and different concentrations of Nar (1.0 × 10−6 to 1.0 × 10−4 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 μg/mL leupeptin; then the cell lysates were boiled for 2 min. Total proteins were quantified using the Bradford protein assay. Solubilized proteins (20 μg) were resolved by 7 or 10% SDS-PAGE at 100 V for 1 h at 24 °C and then electrophoretically transferred to nitrocellulose for 45 min at 100 V and 4 °C. The nitrocellulose was treated with 3% (w/v) BSA in 138.0 mM NaCl, 25.0 mM Tris, pH 8.0, at 24 °C for 1 h and then probed overnight at 4 °C with either anti-ERα 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 Stripping 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-β-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 Student′ t test with the GraphPad INSTAT3 software system for Windows. In all cases, P values <0.05 were considered significant.

RESULTS

E2 and Nar Binding to ERα

To assess the Nar ability to compete with E2 for binding to human recombinant ERα, E2 saturation experiments have been performed in the absence and presence of 1.0 × 10−6 and 1.0 × 10−5 M Nar. Both in the absence and in the presence of Nar, E2 binding to ERα follows a simple equilibrium as postulated by Equation (1), the Hill coefficient being 1.0 ± 0.1. In the absence of Nar, E2 binding to ERα is characterized by an intrinsic equilibrium dissociation constant (Kmath image) of (2.0 ± 0.5) × 10−10 M. In the presence of unlabeled Nar, the apparent equilibrium constant for E2 binding to ERα increased to Kd′ = (5.2 ± 0.6) × 10−9 M and (3.1 ± 0.4) × 10−8 M in the presence of 1.0 × 10−6 and 1.0 × 10−5 M of Nar, respectively (Fig. 1). The linear dependence of Kd′ on the Nar concentration (Fig. 1b) indicates that a simple competition mechanism is operative (15, 16). Data reported in Figure 1b, analyzed according to Eq. (2), allowed the determination of the intrinsic dissociation constant for Nar binding to ERα (Kmath image = 1.4 ± 0.3 × 10−7 M). This confirms that Nar binds to ERα with an affinity lower by about three orders of magnitude than that of E2. These data indicate that Nar and E2 bind competitively to ERα; moreover, in the presence of nutritionally relevant Nar concentrations, the molar fraction of E2 bound to ERα decreases.

Figure 1.

Naringenin (Nar) effect on 17β-estradiol (E2) binding to human recombinant ERα. Panel a: Dependence of the intrinsic molar fraction (Y) of [3H]E2-bound to ERα from [3H]E2 concentration in the absence (squares) and in the presence of 2 representative Nar concentrations (1.0 × 10−6 M [diamonds] and 1.0 × 10−5 M [circles]). Data are the means ± SD of five different experiments. Panel b: Dependence of the apparent dissociation equilibrium constant for [3H]E2 binding to ERα (Kd′) from Nar concentrations (ranging from 1.0 × 10−6 M to 1.0 × 10−5 M). Data are means ± SD of five different experiments. For details see the text.

ERα Transcriptional Activities

The result of Nar binding to ERα prompted us to evaluate the effect of co-stimulation of E2 and Nar on the ERα activities. We first assessed the ERα-mediated direct gene transcription (i.e. estrogen responsive element (ERE)-dependent) (13). HeLa cells, transiently transfected with ERα or empty vector, and the ERE-containing reporter plasmid (pC3) were incubated with either E2 alone (1.0 × 10−8 M) or Nar alone (1.0 × 10−6 M) or in the presence of E2 (1.0 × 10−8 M) and different Nar concentrations. Nar, alone or with E2, induced the ERE-containing 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 ERα ligands (Fig. 2a), thus demonstrating the ERα dependence of this effect. The indirect transcriptional activity of ERα [i.e. through interaction with activator 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 × 10−7 M Nar reduced the E2 effect, and higher Nar concentrations (i.e. 1.0 × 10−6 to 1.0 × 10−4 M) completely prevented E2-induced pD1 promoter activity (Fig. 2b). To determine the ER involvement in the ligand-induced cyclin D1 promoter activity, experiments were performed also in HeLa cells transfected 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).

Figure 2.

Naringenin (Nar) effect on 17β-estradiol (E2)-induced promoter activities. Panel a: Luciferase assay detection on HeLa cells cotranfected with ERα (left panel) or empty vector (right panel) and pC3-luciferase construct and then treated 24 h with either vehicle or E2 (1.0 × 10−8 M) or Nar (1.0 × 10−6 M) or with different Nar concentrations (1.0 × 10−8 M to 1.0 × 10−4 M) in the presence of 10−8 M E2. Data are the means ± S.D. of four independent experiments. *P < 0.001, compared with vehicle values, was determined using Student's t test. Panel b: Luciferase assay detection on HeLa cells co-tranfected with ERα (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 × 10−8 M) or Nar (1.0 × 10−6 M) or with different Nar concentrations (1.0 × 10−8 M to 1.0 × 10−4 M) in the presence of 10−8 M E2. Data are the means ± S.D. of four independent experiments. P < 0.001, determined by using Student's t test, was compared with vehicle (*) or E2 (°) or Nar (+) values.

ERα-Dependent Rapid Signals

The E2-induced cyclin D1 promoter activity requires rapid signal transduction pathways. In particular, the rapid (15 min) E2-induced activation of ERK1/2 and PI3K/AKT cascades are fundamental 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 × 10−6 M Nar was evaluated in HeLa cells transfected with the empty vector or with ERα expression vector. No kinase activation was detected in HeLa cells devoid of ERα 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 ERα-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).

Figure 3.

Naringenin (Nar) effects on 17β-estradiol (E2)-induced rapid ERα activities. ERα-transfected HeLa cells were treated with either vehicle or E2 (1.0 × 10−8 M) or Nar (1.0 × 10−6 M) or with a mixture of Nar (1.0 × 10−6 M) + E2 (1.0 × 10−8 M). After 15 min (left panel, ERK1/2 and AKT) or after 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 Western blots, lower panel shows the densitometric analysis. Data are the means ± S.D. of four independent experiments. P < 0.001, determined by using Student's t test, was compared with vehicle (*) or E2 (°) values.

ERα-Dependent E2-Induced Cell Proliferation

Cyclin D1 represents the upstream sensor of E2-induced proliferative signals, which, in turn, depends on the rapid activation of upstream E2-induced kinase (12, 13). However, in the presence of ERα, Nar prevents cell proliferation inducing a proapoptotic cascade (8, 11). Figure 4 confirms that 1.0 × 10−6, 1.0 × 10−5, and 1.0 × 10−4 M Nar reduced cell number only in ERα-containing HeLa cells, whereas physiological E2 concentrations (i.e. 1.0 × 10−9, and 1.0 × 10−8 M) doubled the cell numbers in 24 h (Fig. 4a). Note that high Nar or E2 concentration (1.0 × 10−4 M) reduced cell numbers also in empty vector-transfected HeLa cells, suggesting an ERα-independent cytotoxic 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 × 10−6 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 ± 1.3 % (Vehicle), 20.2 ± 0.5% (E2), 42.0 ± 0.7 % (Nar), and 43.4 ± 1.0 % (E2+Nar) (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 × 10−8 M E2 (Fig. 5b), thus demonstrating the strong antagonistic effects of this flavanone on E2-induced proliferation.

Figure 4.

Naringenin (Nar) effects on 17β-estradiol (E2)-induced HeLa cells proliferation. a: Number of HeLa cells tranfected with ERα or empty vector and treated 24 h with either vehicle (0) or different concentrations of E2 or Nar (1.0 × 10−10 M to 1.0 × 10−4 M). b: ERα-tranfected HeLa cells were stimulated for 24 h with either vehicle or E2 (1.0 × 10−8 M) or Nar (1.0 × 10−6 M) or with different Nar concentrations (1.0 × 10−9 M to 1.0 × 10−4 M) in the presence of 1.0 × 10−8 M E2. Data are the means ± S.D. of five independent experiments. P < 0.001, determined by using Student's t test, was compared with vehicle (0, *) or E2 (°) values. c: Flow cytometric analysis of ERα tranfected HeLa cells after 24 h of treatment with vehicle or E2 (1.0 × 10−8 M) or Nar (1.0 × 10−6 M) or E2 + Nar (1.0 × 10−8 M and 1.0 × 10−6 M, respectively). The plots indicate a typical cell cycle distribution 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).

Figure 5.

Effect of Naringenin (Nar) on the induction of pro-apoptotic proteins in HeLa cells. Analysis of caspase-3 activation (panel a) and PARP cleavage (panel b) were performed in HeLa cells transfected with human ERα and treated 24 h with vehicle or 17β-estradiol (E2, 1.0 × 10−8 M) or Nar (1.0 × 10−6 M) or E2 + Nar (1.0 × 10−8 M and 1.0 × 10−6 M, respectively). Typical Western blots of three independent experiments are presented in panels a and b. In panel c, the relative densitometric analysis is reported. The data are the mean values ± S.D. P < 0.001, determined by using Student's t test, was compared with vehicle (*) or E2 (°) values.

To avoid any problem because of the receptor overexpression 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 ERα (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 ERα was assessed in HepG2 by Western blot analysis, which confirmed the presence of a unique band at 67 kDa corresponding to ERα (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 receptor, Nar reverts the E2-dependent proliferative effects as obtained in Hela cells.

Figure 6.

Effect of Naringenin (Nar) on the induction of proapoptotic proteins in HepG2 cells. Analysis of p38 phosphorylation, caspase-3 activation, and PARP cleavage was performed in HepG2 cells treated 24 h with vehicle or 17β-estradiol (E2, 1.0 × 10−8 M) or Nar (1.0 × 10−6 M) or with different Nar concentrations (1.0 × 10−6 M to 1.0 × 10−4 M) in the presence of 1.0 × 10−8 M E2. Typical Western blots of three independent experiments are presented in panel a. In panel a′ are presented the relative densitometric analyses. The data are the mean values ± SD. P < 0.001, determined by using Student's t test, was compared with vehicle (*) or E2 (°) values.

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 diseases 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 ERα-mediated membrane-starting rapid effects (13, 18). ERβ seems to act as a negative regulator of E2-induced proliferation (12, 13, 20–23). As an example, ERα-positive MCF-7 breast cancer cells respond to E2 with increased proliferation, but when ERβ is introduced into these cells, E2-induced proliferation is inhibited (21). These findings are relevant in view of the possible action of ERβ as a tumor suppressor. In search for agents that would be useful in preventing and treating E2-dependent cancer, the interest in flavonoids has increased markedly (5, 24–30). In fact, contrary to E2, flavonoids bind to ERβ with up to five times higher affinities compared with ERα (31, 32). As ERβ ligands, flavonoids may be able to trigger beneficial responses through their preferential interaction with this ER isoform. However, this protective effect could be abrogated in cells that selectively express ERα, such as mammary gland, ovary, and liver (13, 19, 20), which only contain minimal residual amounts of ERβ.

Our previous studies indicate that Nar, a flavanone component of citrus fruits and tomatoes, impaired E2-induced proliferative signals interfering with ERα-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 ERα de-palmitoylation faster than E2, which resulted in receptor rapid dissociation from membrane caveolin-1 and in impaired receptor binding to the mitogenic signaling proteins (i.e. ERK1/2 and PI3K/AKT) (11). On the other hand, Nar induced the ERα-dependent, but palmitoylation-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 ERβ, Nar works as a selective inhibitor of ERα-mediated proliferation (5 and literature cited therein). In addition, Nar could also acts as an antagonist of E2, preventing E2-induced cell proliferation when ERα 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 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 ERα is about 1000-fold higher than that reported for E2; however, in the presence of Nar, the Kd of E2 for its receptor linearly increased, suggesting a decreased affinity between E2 and ERα. Remarkably, this decreased affinity between E2:ERα did not impair the E2 ability, in the presence of Nar, to trigger gene transcription through the direct binding of ERα to ERE-containing reporter gene (i.e. pC3). This result implies that the coactivator recruitment on the ligand-bound ERα 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 ERα could mediate gene transcription even in a manner that does not require the ERα direct binding to DNA. This is referred to as “indirect genomic mechanism” which requires the ERα interaction with specific transcription factors such as Sp1 and AP-1. The ERα-Sp1 and ERα-AP-1 complexes 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, retinoic acid receptor α1, adenosine deaminase, IGF-binding protein 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 ERα interaction to Sp1 and AP-1 is impaired. Our previous data indicate that E2 stimulation of ERα-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 ERα, highlighting 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 exclusively mediated by rapid membrane-starting actions (13, 18). In HepG2 cells, multiple and parallel membrane-starting pathways are rapidly activated by the E2:ERα complex (13), and the blockade of ERK1/2 and PI3K/AKT pathways completely prevents the E2-induced DNA synthesis (13). ERK1/2/MAPK and PI3K/AKT pathways, rapidly activated by E2:ERα 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 ERα-transfected HeLa cells and in HepG2 cells, the E2 inability to activate rapid signal transduction 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 cleavage).

As a whole, the assays with Nar against a background level of E2 allowed us to assess the estrogenic versus antiestrogenic activity of this flavanone. The results of this study demonstrate that Nar treatment do not impair E2-induced ERE-dependent ERα transcriptional activity, whereas Nar reverts the proliferative effects of E2 impairing ERα-mediated rapid signals and inducing different proapoptotic signal transduction pathways. Moreover, the preventive effects elicited by Nar on E2-dependent cancers may be enhanced, in some tissues, through the induction of specific ERβ-dependent proapoptotic signalling (8). In addition, these results increase the list of Nar effects on human health adding up a possible therapeutic 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 estrogenic 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 modulation of cell signalling pathways, the regulation of the cell cycle, the inhibition of glucose uptake, and antioxidant activities (1, 6). Some of these mechanisms may even occur independently of ER binding (39, 40), but requires high plasma Nar concentrations (i.e. 0.8 to 25 × 10−5 M), which are difficult to obtain by the oral ingestion of food rich in this bioflavonoid. 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 × 10−6 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 × 10−6 M, demonstrating that Nar plasma concentration depends on the ability of each individual to adsorb and metabolize 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 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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