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Endocrinology, doi:10.1210/en.2007-1173
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Endocrinology Vol. 149, No. 5 2567-2575
Copyright © 2008 by The Endocrine Society

The Nutritional Flavanone Naringenin Triggers Antiestrogenic Effects by Regulating Estrogen Receptor {alpha}-Palmitoylation

Paola Galluzzo, Paolo Ascenzi, Pamela Bulzomi and Maria Marino

Department of Biology (P.G., P.A., P.B., M.M.), University "Roma Tre," I-00146 Roma, Italy; and National Institute for Infectious Diseases Istituto di Ricovero e Cura a Carattere Scientifico "Lazzaro Spallanzani" (P.A.), I-00149 Roma, Italy

Address all correspondence and requests for reprints to: Maria Marino, Department of Biology, University "Roma Tre," Viale G. Marconi, 446, I-00146 Roma, Italy. E-mail: m.marino{at}uniroma3.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Naringenin (Nar) is a component of fruits and vegetables associated with healthful benefits, such as in osteoporosis, cancer, and cardiovascular diseases. These protective effects have been linked with Nar antiestrogenic as well as estrogenic activities. Previous studies indicate that Nar impaired estrogen receptor (ER) {alpha} signaling by interfering with ER{alpha}-mediated activation of ERK and phosphoinositide 3-kinase signaling pathways in the absence of effects at the transcriptional level. The present studies evaluated the hypothesis that these Nar antagonistic effects occur at the level of the plasma membrane. Our results indicate that Nar induces ER{alpha} depalmitoylation faster than 17β-estradiol, which results in receptor rapid dissociation from caveolin-1. Furthermore, Nar impedes ER{alpha} to bind adaptor (modulator of nongenomic actions of the ER) and signaling (c-Src) proteins involved in the activation of the mitogenic signaling cascades (i.e. ERK and phosphoinositide 3-kinase). On the other hand, Nar induces the ER-dependent, but palmitoylation-independent, activation of p38 kinase, which in turn is responsible for Nar-mediated antiproliferative effects in cancer cells. Altogether, these data highlight new ER-dependent mechanisms on the root of antiproliferative and antiestrogenic effects of Nar. Moreover, the different modulation of ER{alpha} palmitoylation exerted by different ligands represents a pivotal mechanism that drives cancer cell to proliferation or apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FLAVONOIDS ARE plant-derived chemicals primarily recognized as the pigments responsible for the many shades of yellow, orange, and red in flowers (1). To date, more than 4000 flavonoids, categorized as flavonols, flavones, flavanols, flavanonols, flavanones, and isoflavones, have been identified in edible plants and are consumed regularly with the human diet (2, 3).

Although the affinity of flavonoids for the estrogen receptors (ERs) (ER{alpha} and ERβ) is 1,000–10,000 times lower than that of the natural ligand 17β-estradiol (E2) (4), in the 1940s they were considered as the antiestrogenic principle in red clover that caused infertility in sheep in Western Australia (5). This adverse effect of flavonoids, currently confirmed in vivo in laboratory animals, placed these substances in the class of endocrine-disrupting chemicals (6, 7).

Recent evidence has indicated that an adult human diet rich in flavonoids leads to a decrease of total cholesterol, low-density lipoproteins, and triglycerides in plasma (8), as well as a reduced incidence of cardiovascular diseases (9) and osteoporosis (10). These protective effects of dietary compounds, recognized as estrogen mimetic, are currently being explored to prevent osteoporosis (10), the risk of coronary artery disease (11, 12), and the vasomotor flushing related to E2 deficiency in women during menopause (13). Thus, in mammalian cells, flavonoids behave both as estrogen antagonists by acting as endocrine disruptors and as estrogen agonists by mimicking hormone protective effects on some degenerative diseases. Nonetheless, the mechanism(s) underlying the contrasting effects of flavonoids remains poorly understood.

Epidemiological evidence supports a protective effect of high phytoestrogen diets to reduce the incidence of several E2-responsive cancers, such as breast, colon, and prostate cancer (2, 14, 15, 16). Thus, the study of the anticancer action of nutritional flavonoids, a well-established antiestrogenic effect, could help to clarify the basis of flavonoid opposite effects. Notably, the main studies on flavonoid effects on cancer growth have been conducted on soy derived isoflavones (e.g. genistein, daidzein) (15), whereas information about the other classes of flavonoids found in the principal aliments of Western diet (e.g. flavonols, flavones, and flavanones) is still scarce.

Naringenin (5,7,4'-trihydroxyflavanone) (Nar) (Fig. 1Go), especially abundant in citrus fruits and tomatoes, is reported to have antiproliferative effects in different cancer cell lines (e.g. colon, breast, and uterus cancer cell lines) (2, 17, 18, 19, 20, 21, 22, 23). Among several mechanisms proposed for Nar-induced antiproliferative effects (i.e. antioxidant activities and kinase and glucose uptake inhibition) (21, 24), the ability of Nar to hamper cell proliferation by binding to ERs is particularly intriguing. We previously showed that Nar concentrations, physiologically achieved in the plasma (1–10 µM) after the consumption of meals rich in Nar, enhanced ERβ-mediated signals important for cancer cell apoptosis and impaired ER{alpha}-mediated signaling important for E2-induced cancer cell proliferation (22, 23). In the presence of ER{alpha}, Nar prevented the activation of ERK/MAPK and phosphoinositide 3-kinase (PI3K)/AKT signaling without impairing the transcription of an estrogen responsive element-containing gene construct. Moreover, Nar activated the rapid phosphorylation of p38/MAPK, which in turn induced a pro-apoptotic cascade (e.g. caspase-3 activation) (22, 25). These data raised the possibility that Nar antagonistic effects on E2-related cancers were dependent on the flavonoid ability to modulate ER{alpha} association with the plasma membrane. Because {alpha}-palmitoylation is the major determinant for receptor localization at the plasma membrane (26, 27) and for ER{alpha} ability to rapidly activate signal transduction pathways important for cell proliferation (26, 28), we hypothesize that Nar binding to ER{alpha} could selectively modulate this receptor posttranslational lipid modification. Here, this possibility has been investigated comparing the effect of E2 and Nar on ER{alpha} palmitoylation and on ER{alpha} association with either membrane (i.e. caveolin-1) or adaptors [i.e. modulator of nongenomic actions of the ER (MNAR)], or signaling proteins (i.e. c-Src). Finally, the influence of Nar-induced regulation of ER{alpha} palmitoylation on signaling cascade activation has been evaluated.


Figure 1
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  		FIG. 1. Chemical structures of E2 and Nar.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Nar, E2, gentamicin, penicillin and other antibiotics, the GenElute plasmid maxiprep kit, DMEM, RPMI 1640 medium (without phenol red), charcoal-stripped fetal calf serum, and the palmitoylacil transferase inhibitor 2-bromohexadecanoic acid (2-Br) were purchased from Sigma-Aldrich (St. Louis, MO). The ER inhibitor ICI 182,780 was obtained from Tocris (Ballwin, MO). The p38/MAPK inhibitor, SB 203,580, was obtained from Calbiochem (San Diego, CA). 9,10-[3H]palmitic acid (specific activity 57.00 Ci/mmol) was purchased from DuPont New England Nuclear Life Science Products (Boston, MA). Lipofectamine reagent was obtained from Gibco-BRL Life Technologies (Gaithersburg, MD). Bradford protein assay was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). The anti-phospho-ERK, anti-phospho-p38, anti-AKT, anti-β-actin, anti-ERK, anti-p38, anti-caveolin-1, anti-c-Src-B12, anti-ER{alpha} MC20 (C terminus), and anti-ER{alpha} D12 (N terminus) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-PELP1/MNAR and anti-phospho-c-Src (Tyr416) antibodies were purchased from Bethyl Laboratories Inc. (Montgomery, TX). The polyclonal anti-phospho-AKT antibody was purchased from New England Biolabs (Ipswich, MA). CDP-Star, chemiluminescence reagent for Western blot was obtained from New England Nuclear Life Science Products. All the other products were from Sigma-Aldrich. Analytical or reagent grade products were used without further purification.

Cell culture
HepG2 and HeLa cells were routinely grown in 5% CO2 in modified, phenol red-free RPMI 1640 or DMEM media containing 10% (vol/vol) charcoal-stripped fetal calf serum, L-glutamine (2.0 mM), gentamicin (10.0 µg/ml), and penicillin (100.0 U/ml). Cells were passaged every 2 d. Cells were simultaneously treated with either E2 (final concentration, 10.0 nM in ethanol/PBS, 1:10, vol/vol) or Nar (final concentration, 10 µM in dimethylsulfoxide/PBS 1:10, vol/vol), or vehicle (ethanol/PBS 1:10, vol/vol). In some experiments cells were treated with E2 (10.0 nM) and different concentrations of Nar (0.01–100 µM). When indicated, the PAT inhibitor 2-Br (final concentration 10.0 µM) or p38/MAPK cascade inhibitor SB 203,580 (final concentration 5.0 µM), or the ER inhibitor ICI 182,780 (final concentration 1.0 µM) was added 30 min before E2 administration. Cells, grown to approximately 70% confluence in six-well plates, were stimulated and then harvested with trypsin, centrifuged, and stained with the Trypan blue solution, and counted in a hemocytometer (improved Neubauer chamber) in quadruplicate.

Plasmids and cell transfection
The expression vector pCR3.1-β-galactosidase, wild-type human ER{alpha} pSG5-HE0, and pSG5-Cys447Ala (human ER{alpha} Cys447Ala mutant) have been described elsewhere (26). A luciferase dose-response curve showed that the maximum effect was obtained when 1.0 µg plasmids was transfected together with 1.0 µg 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 approximately 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 as described elsewhere.

Cell labeling with [3H]palmitate and immunoprecipitation
Twenty-four hours after transfection with plasmid containing wild-type ER{alpha}, HeLa cells and untransfected HepG2 cells were incubated with 0.5 mCi/ml [3H]palmitate at 37 C for different times (0–240 min). Where indicated, HepG2 and HeLa cells were stimulated with E2 or Nar at different times (10, 60, and 240 min) in the presence of [3H]palmitate. Cells were then washed in ice-cold PBS, harvested by scraping, and lysed in 50 µl lysis buffer [10.0 mM Tris (pH 7.5), 1.0 mM EDTA, 0.5 mM EGTA, 10.0 mM NaCl, 1% (vol/vol) Triton X-100, and 1% (vol/vol) sodium cholate] containing protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1.0 µg/ml leupeptin, and 5.0 µg/ml aprotinin). The cell lysates were then clarified by centrifugation and immunoprecipitated as described previously (26). Briefly, equal amounts of soluble cell extracts were incubated with 1.0 µg anti-ER{alpha} D12 (N terminus) together with 1.0 µg anti-ER{alpha} MC20 (C terminus) antibodies. The lysates and antibodies were incubated for 90 min at 4 C, and then 20 µl protein-A agarose was added for 30 min at 4 C. After centrifugation (50,000 x g for 15 min), the supernatant and immunoprecipitated proteins were separated in 7 or 10% SDS-PAGE. Proteins were electrophoretically transferred to nitrocellulose and then probed overnight at 4 C with anti-ER{alpha} MC20 antibody. In some experiments the radioactivity present in immunoprecipitated proteins and in the supernatant was monitored by counting with a Camberra Packard Liquid β-counter (Milan, Italy).

True-blot immunoprecipitation
The cell lysates, prepared as described previously, were clarified by centrifugation and immunoprecipitated with TrueBlot (eBioscience, San Diego, CA), which preferentially detects the native disulfide form of mouse or rabbit IgG, reducing interference by the approximate 55 kDa heavy and approximate 23 kDa light chains of the immunoprecipitating antibody (29). Briefly, after stimulation equal amounts of soluble cell extracts were incubated with either 2.0 µg anti-caveolin-1 or anti-c-Src or anti-MNAR or anti-MC20 ER{alpha} antibodies. The lysates and antibodies were incubated at 4 C for 1 h, then 20 µl antimouse IgG beads (eBioscience) were added, and samples incubated for 1 h on a rocking platform at 4 C. Samples were centrifuged at 10,000 x g for 10 min, the supernatant was removed completely, and beads (pelleted) were washed three times with 100 µl lysis buffer. Sodium dodecyl sulfate-reducing sample buffer (20 µl, containing 50 mM dithiothreitol) was added, and samples were boiled at 100 C for 5 min. Proteins were resolved using 7 or 10% SDS-PAGE at 100 V for 1 h and then electrophoretically transferred to nitrocellulose for 45 min at 100 V at 4 C. The nitrocellulose was treated with 5% (wt/vol) nonfat dry milk (Bio-Rad Laboratories) in 150 mM NaCl, 50.0 mM Tris HCl (pH 8.0), 0.1% (wt/vol) Tween 20, and then probed at 4 C overnight with either 2.0 µg anti-caveolin-1 or anti-c-Src or anti-MNAR and anti-MC20 ER{alpha} antibodies. The antibody reaction was visualized with the chemiluminescence reagent for Western blot (Amersham Biosciences, Little Chalfont, UK).

Electrophoresis and immunoblotting
After stimulation, cells were lysed and solubilized in 0.125 M Tris (pH 6.8), containing 10% (wt/vol) sodium dodecyl sulfate, 1.0 mM phenylmethylsulfonyl fluoride, and 5.0 µg/ml leupeptin; the cell lysates were then 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% (wt/vol) 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-ER{alpha} or anti-phospho-ERK or anti-phospho-AKT or anti-phospho-p38 or anti-phospho-c-Src antibodies. The nitrocellulose was stripped by Restore Western Blot Stripping Buffer (Pierce, Rockford, IL) for 10 min at room temperature, and then probed with either anti-ERK or anti-AKT or anti-p38 or anti-c-Src and anti-β-actin antibodies. Antibody reaction was visualized with chemiluminescence Western blotting detection reagent (Amersham Biosciences).

Statistical analysis
A statistical analysis was performed using the Student’s t test with the InStat.3 software system (GraphPad Software Inc., San Diego, CA). P values less than 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nar decreases ER{alpha} palmitoylation
ER{alpha} is a palmitoylated protein (26, 28). In fact, [3H]palmitate incorporated into ER{alpha}-transfected HeLa cells for up to 240 min reaches a steady-state regimen after 10 min; the half-time of ER{alpha} palmitoylation is about 1.5 min (Fig. 2AGo). This is consistent with a rapid turnover of fatty acid on ER{alpha}, supporting the idea that ER{alpha}-palmitoylation is a dynamic event involving cycles of acylation and deacylation (30). As previously reported (26), 10 nM E2 stimulation induces the decrease of [3H]palmitate incorporation with a half-time of about 30 min. Two hundred forty minutes after E2 stimulation, 25 ± 0.5% of ER{alpha} was still palmitoylated (Fig. 2BGo). A similar kinetics behavior was obtained pretreating cells with the PAT inhibitor 2-Br 30 min before E2 stimulation. The Nar concentration achievable in human plasma after a meal rich in flavonoids (i.e. 1–10 µM) rapidly decreased the amount of [3H]palmitate incorporated in HeLa cells transfected with wild-type ER{alpha}; the same result was obtained at higher Nar concentrations (i.e. 100 µM), whereas a lower Nar concentration (i.e. 0.01 and 0.1 µM) was ineffective (data not shown). The half-time of Nar-induced ER{alpha}-depalmitoylation (Nar concentration = 10 µM) in cells containing transfected ER{alpha} (HeLa) (Fig. 2BGo) was about 8 min. In cells containing endogenous ER{alpha} (HepG2), the half-time of Nar-induced ER{alpha}-depalmitoylation was about 1 min (Fig. 2CGo). Two hundred forty minutes after Nar stimulation, 6.0 ± 0.5% and 8.1 ± 1.2% of ER{alpha} were still palmitoylated in HeLa and HepG2 cells, respectively (Fig. 2Go, B and C). No change in the ER{alpha} protein level accompanied the E2- and Nar-induced decrease of [3H]palmitate incorporation into ER{alpha} in both cell lines (Fig. 2DGo).


Figure 2
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  		FIG. 2. Effect of Nar on ER{alpha} palmitoylation. A, Time course of [3H]palmitate incorporation in ER{alpha}-transfected HeLa cells. Data are the mean of six independent experiments ± SD. B, Effect of ligands on [3H]palmitated ER{alpha} in transfected HeLa cells. After [3H]palmitate incorporation (120 min), cells were stimulated with either 10 nM E2 or 10 µM Nar or vehicle (control), or 10 µM PAT inhibitor 2-Br (added 30 min before E2 administration). At different times, ER{alpha} was immunoprecipitated and radioactivity determined. Data are the mean of five independent experiments ± SD. C, Effect of Nar on [3H]palmitated ER{alpha} in HepG2 cells. After [3H]palmitate incorporation (120 min), cells were stimulated with 10 µM Nar. At different times, ER{alpha} was immunoprecipitated and radioactivity determined. Data are the mean of five independent experiments ± SD. D, Western blot; and D', densitometric analysis of immunoprecipitated ER{alpha} in transfected HeLa cells and in HepG2 cells. Data, normalized by comparison with β-actin expression (data not shown), are the mean of five independent experiments ± SD. For details, see Materials and Methods.

 
Nar rapidly impairs ER{alpha}-caveolin-1 association
At steady-state ER{alpha} palmitoylation is necessary for receptor association with the scaffold protein caveolin-1 (26). Thus, we would assess if ligand-induced ER{alpha} depalmitoylation could affect the association between receptor and this membrane protein. As expected (31), ER{alpha} was constitutively associated with caveolin-1; this association was still present 30 min after E2 stimulation. ER{alpha}-caveolin-1 association decreased, by half, 60 min after hormone treatment, and it was barely detectable 240 min after stimulation (Fig. 3AGo). On the other hand, 10 min after Nar treatment, the residual ER{alpha}-caveolin-1 complex was only approximately 10% (Fig. 3Go, B and C). This tendency corresponds to the time of E2-induced and Nar-induced ER{alpha} depalmitoylation, suggesting that receptor depalmitoylation and ER{alpha}-caveolin-1 dissociation are parallel events driven by ligand binding to ER{alpha} (compare Fig. 2BGo with Fig. 3Go, A and B).


Figure 3
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  		FIG. 3. Effect of E2 and Nar on ER{alpha} association with caveolin-1 (cav). ER{alpha}-transfected HeLa cells were stimulated with 10 nM E2 (A and A') or 10 µM Nar (B, B', C, and C'). Cells were then lysated and subjected to caveolin-1 immunoprecipitation (A, A', B, and B') or ER{alpha} immunoprecipitation (C and C'), followed by Western blot (Wb) with anti-caveolin-1 or anti-ER{alpha} antibodies. Typical Western blots are shown in panels A–C. Densitometric analyses of four different experiments are shown in panels A'–C'; data are the mean ± SD. *, P < 0.001, calculated with the Student’s t test, compared with respective unstimulated values (0 min). For details, see Materials and Methods.

 
Nar prevents the association of ER{alpha} with signaling protein involved in proliferation
ER{alpha}, like other steroid hormone receptors, does not possess kinase activity, thus its ability to mediate E2-induced proliferation depends on protein-protein complex formation, which in turn triggers the activation of proliferative signals (32, 33, 34). We and others hypothesized that the E2-dependent ER{alpha} depalmitoylation and ER{alpha}-caveolin-1 dissociation could be a prerequisite for ER{alpha} docking to the partner proteins (27, 35). To verify this hypothesis, the Nar ability to drive ER{alpha} association with the nonreceptor tyrosine kinase c-Src and with the adaptor protein MNAR has been evaluated and compared with the effect of E2. In the presence of E2, there is a rapid (10 min; data not shown) increase in ER{alpha} association with c-Src that was still detectable 60 min after hormone stimulation (Fig. 4AGo). Nar stimulation of ER{alpha} transfected HeLa cells prevents such an increase. Moreover, prolonged stimulation with Nar induced the dissociation of the ER{alpha}-c-Src complex. A similar result was obtained by immunoprecipitation experiments with the adaptor protein MNAR (Fig. 4BGo). The E2-induced ER{alpha}-c-Src association was paralleled by an increase of c-Src activation (Fig. 4CGo). On the other hand, Nar stimulation was unable to induce c-Src activation. These data suggest that the very fast Nar-induced ER{alpha} depalmitoylation and ER{alpha}-caveolin-1 dissociation impede ER{alpha} relocalization by preventing ER{alpha} binding to other molecular partners. To verify this hypothesis, HeLa cells were transiently transfected with the unpalmitoylable Cys447Ala ER{alpha} point mutant (28) and stimulated with E2. E2 did not increase ER{alpha}-c-Src (Fig. 5AGo) or ER{alpha}-MNAR (Fig. 5BGo) association in the presence of the unpalmitoylable ER{alpha} mutant, thus strongly confirming that the slow E2-induced ER{alpha} depalmitoylation is required for molecular complex formation (i.e. ER{alpha}-c-Src and ER{alpha}-MNAR). Accordingly, Nar stimulation does not allow the activation of downstream kinases involved in E2-induced cell proliferation (36, 37). In fact, no activation of ERK and AKT phosphorylation was observed after Nar stimulation (Fig. 6Go), suggesting an antagonistic role of Nar on this ER{alpha} activity.


Figure 4
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  		FIG. 4. Effect of E2 and Nar on ER{alpha} association with c-Src and MNAR. ER{alpha}-transfected HeLa cells were stimulated with either vehicle [60 min; control (C)] or 10 nM E2 (60 min), or with 10 µM Nar (1, 10, and 60 min). ER{alpha} was immunoprecipitated with anti-ER{alpha} antibody, followed by Western blot (Wb) with anti-c-Src (A) or with anti-MNAR (B) and anti-ER{alpha} antibodies (A and B). In panels C and C', the cells were lysed after stimulation, and Western blot was performed. Typical Western blots are shown in panels A–C. Densitometric analyses of four different experiments are shown in panels A'–C'; data are the mean ± SD. *, P < 0.001, calculated with the Student’s t test, compared with control values. For details, see Materials and Methods.

 

Figure 5
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  		FIG. 5. Effect of E2 on the association of the Cys447Ala ER{alpha} mutant with c-Src and MNAR. HeLa cells were transfected with the unpalmitoylable Cys447Ala ER{alpha} mutant and were stimulated with either vehicle [60 min; control (C)] or 10 nM E2 (1, 10, and 60 min). ER{alpha} was immunoprecipitated with anti-ER{alpha} antibody, followed by Western blot (Wb) with anti-c-Src (A) or with anti-MNAR (B) and anti-ER{alpha} antibodies (A and B). Typical Western blots are shown in panels A and B. Densitometric analyses of four different experiments are shown in panels A' and B'; data are the mean ± SD. For details, see Materials and Methods.

 

Figure 6
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  		FIG. 6. Effect of E2 and Nar on ERK and AKT activation. ER{alpha}-transfected HeLa cells were stimulated with either vehicle [60 min; control (C)] or with 10 nM E2 (15 min) or with 10 µM Nar (1, 10, and 60 min). Cells were lysed, and Western blot (Wb) was performed. The amount of protein levels was normalized by comparison with β-actin expression. Typical Western blots are shown in panels A and B. Densitometric analyses of four different experiments are shown in panels A' and B'; data are the mean ± SD. P < 0.001, calculated with the Student’s t test, was compared with control (*) or E2-stimulated (°) values. For details, see Materials and Methods. P-AKT, Phosphorylated AKT; P-ERK, phosphorylated ERK.

 
Nar rapidly stimulates p38/MAPK
The results reported here indicate, for the first time, the mechanism(s) underlying the different effects of the ER{alpha}-Nar and ER{alpha}-E2 complexes on cell proliferation, but they do not explain the Nar-mediated antiproliferative effect(s). Nar rapidly induces the ER{alpha}-dependent activation of the p38 member of the MAPK family, which in turn activates a pro-apoptotic cascade (22). Here, we verified the impact of Nar-induced rapid ER{alpha} depalmitoylation on this signal pathway. Figure 7Go, A, A’, and A "confirms that both Nar and E2 stimulation induced a rapid (0.25 h) p38 phosphorylation. In addition, Nar, but not E2, induced the persistent (24 h) activation of this kinase. The Nar-induced p38 activation was ER{alpha} dependent, being prevented by cell pretreatment with the pure antiestrogen ICI (Fig. 7BGo). On the contrary, cell pretreatment with the PAT inhibitor 2-Br did not affect Nar-induced p38 activation. A similar effect was observed by stimulating HeLa cells transiently transfected with the unpalmitoylable Cys447Ala ER{alpha} point mutant with both E2 or Nar (Fig. 7BGo). This palmitoylation independent mechanism allows the Nar-induced block of cell cancer growth (Fig. 8AGo). Moreover, Nar hampered the ER{alpha} transfected HeLa cell proliferation even in the presence of E2, confirming the antagonistic role played by this flavonoid on E2-induced proliferation (Fig. 8BGo).


Figure 7
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  		FIG. 7. Effect of E2 and Nar on p38/MAPK activation. A, A', and A'', ER{alpha}-transfected HeLa cells were unstimulated (zero) or stimulated with either 10 nM E2 (0.25–24 h) or with 10 µM Nar (0.25–24 h) or for 1 h with different Nar concentrations (0.01–100 µM). Cells were lysed, and Western blot was performed. The amount of protein levels was normalized by comparison with β-actin expression. Typical Western blots are shown. B and B', HeLa cells were transfected with ER{alpha} wild-type (HE0) or with the unpalmitoylable Cys447Ala ER{alpha} mutant (Cys447Ala). Cells were stimulated with either vehicle (15 min; control) or 10 nM E2 (15 min) or 10 µM Nar (15 min) or 10.0 µM PAT inhibitor 2-Br (added 30 min before Nar administration), or 1 µM ER inhibitor ICI 182,780 (added 30 min before Nar administration). Typical Western blots are shown in panel B. Densitometric analysis of four different experiments is shown in panel B'; data are the mean ± SD. P < 0.001, calculated with the Student’s t test, compared with respective control values (*) or the Nar-stimulated values (°).

 

Figure 8
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  		FIG. 8. Effect of E2 and Nar on cell proliferation. A, ER{alpha}-transfected HeLa cells were grown for 30 h in the presence of either vehicle (control) or E2 (10 nM) or 10 µM Nar and/or 5 µM p38 inhibitor, SB 203,580 (SB) (added 30 min before E2 or Nar administration) and then counted; data are the mean ± SD of five independent experiments performed in duplicate. P < 0.001, calculated with the Student’s t test, compared with the control values (*) or with Nar-stimulated values (°). B, ER{alpha}-transfected HeLa cells were grown for 30 h in the presence of either vehicle (control) or E2 (10.0 nM) and different concentrations of Nar (0.01–100 µM), and then counted; data are the mean ± SD of four independent experiments performed in duplicate. P < 0.001, calculated with the Student’s t test, compared with the control values (*) or E2-stimulated values (°). For details, see Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Flavonoids are amphipathic molecules known for their ability to partition into lipid bilayers. Their permeability across the cell membranes and their membrane affinity depend on the number of hydroxyl groups present in the structure. For Nar, with three hydroxyl groups (Fig. 1Go), the membrane association is strong and the transport across membrane slow. Quercetin (five OH groups) has a stronger interaction with the membrane. However, flavonoids are removed from the membrane and accumulate in the cells by interaction with cell proteins (38, 39).

Flavonoids have been studied for more than 50 yr, and now it is definite that they exert a wide range of biochemical and pharmacological effects. Among others, the most investigated effects refer to their cancer preventive activities, which have been predominantly associated with their antioxidant proprieties (40), and their inhibitory effects on many enzyme activities such as those of drug metabolism (24), aromatase (41), signal kinases (21), and cyclooxygenase (42).

However, much of these studies have been conducted in vitro using pharmacological doses of flavonoids (e.g. 100 µM) with little regard to the bioavailability and metabolism of the compounds studied. In addition, the cellular mechanisms involved in the flavonoid anticancer activities are still largely unknown.

Although flavonoids have estrogenic activity, bind weakly to ERs, and initiate E2-dependent transcription (4), the impact of these mechanisms on cancer protective effects is largely confused. Note that the weak estrogenicity of flavonoids has been used to advantage by herbalist medicine to promote flavonoids, mainly isoflavones, as a natural alternative to conventional hormone replacement therapy (HRT) (see Ref. 43). Such usage could increase in light of recent evidence that long-term HRT may be associated with an increased risk of E2-related cancer (44). In accordance with Rice and Whitehead (43), we can ask "Are phytoestrogens safe as a natural alternative to HRT and could they be promoters or protectors of cancer?" As weak estrogen, flavonoids should promote proliferation, whereas if they are protectors, they should act as antagonists of ER or elicit other, ER-independent, actions.

The mechanisms through which phytoestrogens may stimulate or inhibit growth of ER-positive cancer cells are controversial, probably due to the different growth-stimulatory properties that E2 mediates in the presence of ER{alpha} or ERβ. Whereas E2 activation of ER{alpha} is known to promote cancer growth, the specific functions of ERβ are less well understood, although ERβ inhibits proliferation of several cell lines (29). By using the flavanone Nar as a flavonoid model, we recently reported that Nar concentration (10 µM), achievable in human plasma after a meal rich in flavonoids (i.e. Mediterranean diet), does not impair the transcriptional activity of an estrogen responsive element-containing gene construct but exerts for all time ER-dependent antiproliferative effects. The mechanisms at the root of this effect differ depending on the ER isoform present (22). In the presence of ER{alpha}, Nar acts as an antiestrogen by specifically altering ER{alpha}-mediated rapid signal transduction pathways important for E2-induced cell proliferation (22, 23). In contrast, in the presence of ERβ, Nar is an agonist that mimics the antiproliferative effects of E2 (22). The present study was undertaken to determine the potential mechanism(s) underlying the Nar antiproliferative effect in human cancer cells containing ER{alpha}. The data reported here strongly indicate that Nar binding to ER{alpha} induces fast receptor depalmitoylation, the major determinant of ER{alpha} localization at the plasma membrane. In turn, the ER{alpha} association with membrane (i.e. caveolin-1) and signaling (i.e. c-Src) proteins is prevented.

Protein modification with fatty acids is a universal feature of eukaryotic cells. Historically, fatty acylation of eukaryotic proteins was divided into two classes: cotranslational addition of myristate (C14:0) to the N-terminal glycine residue through amide linkage (myristoylation), and posttranslational addition of palmitate (C16:0) through thioester linkage to cysteine residues (palmitoylation) (30). Both ER{alpha} and ERβ, which do not possess any consensus sequence for myristoylation, have been recently added to the palmitoylproteoma (26, 29, 35, 45).

There are several fates for thioester-linked palmitate during the lifetime of a protein. Viral membrane glycoproteins are palmitoylated at a site near the cytoplasmic face of the membrane shortly after they are synthesized, and then they remain palmitoylated (46). However, palmitoylation function is more than a simple membrane association of otherwise soluble proteins. Palmitoylation could be an essential step in the protein degradative processes (47). In addition, for many proteins, including ERs, constitutive cycles of palmitoylation and depalmitoylation occur (30) (present data). In fact, as for other signaling proteins (48, 49), the turnover of ER palmitoylation could also be regulated. In accordance, upon E2 binding, ERs undergo conformational changes that promote their depalmitoylation (26, 29, 35, 45) with a slow comparable kinetics (29). On the other hand, Nar binding endorsed a rapid and exhaustive ER{alpha} depalmitoylation, probably due to a different conformational change.

We speculated that the E2-dependent ER{alpha} depalmitoylation, decreasing receptor-caveolin-1 association, could allow ER{alpha} redistribution and its association with adaptors and/or signaling proteins (e.g. MNAR, c-Src, tyrosine kinase receptors), which in turn contribute to rapid signaling cascades (e.g. ERK/MAPK and PI3K/AKT) (27). Present data corroborate the validity of the hypothesis. Actually, E2 stimulation of HeLa cells transfected with the unpalmitoylable ER{alpha} mutant does not increase ER{alpha} association with MNAR or c-Src (compare Figs. 4Go and 5Go), impairing ER{alpha} ability to activate ERK and AKT phosphorylation (26). Correspondingly, Nar rapidly modifies ER{alpha} palmitoylation status, and modulates ER{alpha} nongenomic activities impairing ER{alpha} association with adaptors and/or signaling proteins (i.e. MNAR and c-Src). This prevents the ER{alpha}-dependent activation of mitogenic signaling cascades (e.g. ERK/MAPK and PI3K/AKT). Notably, no decrease of constitutive ERK and AKT phosphorylation was observed, suggesting that the Nar concentration used here was unable to inhibit enzyme activities.

The data reported here specify the molecular bases of Nar-ER{alpha} antagonism and explain the mechanism of Nar-induced apoptosis in cancer cells (22). In fact, both E2 and Nar rapidly stimulate (15 min), via ER{alpha}, the activation of the pro-apoptotic kinase p38/MAPK (22) (present data). The ligand-induced p38 activation results independent from ER{alpha} palmitoylation being activated even in HeLa cells transfected with the ER{alpha} unpalmitoylable mutant, but only Nar induces the persistent activation of such a kinase. Apoptosis signal-regulating kinase 1 (ASK1) is one of the upstream activators of p38. In quiescent cells, ASK1 is a cytosolic kinase in which activity is tightly regulated. Raf-1, ERK activator, and AKT suppress ASK1 death-promoting activity (50, 51, 52, 53). Moreover, E2 induces ASK1 phosphorylation at Ser83 via ER{alpha}-AKT cascade (53). Thus, the ability of the ER{alpha}-E2 complex to activate rapidly ERK and AKT avoiding the persistent p38 activation guarantees both cell proliferation and cell survival. On the other hand, by inducing the rapid ER{alpha} depalmitoylation, Nar does not activate ERK and AKT. Therefore, the persistent activation of p38, pivotal for pro-apoptotic cascade initiation, occurs.

Nar in part impairs the ER{alpha} genomic mechanisms. In fact, Nar stimulates the activity of estrogen responsive element-luciferase reporter gene construct in the presence of ER{alpha} (4, 22, 23), even if to a lesser extent than E2. Conversely, this flavanone prevents the indirect ER{alpha}-mediated transcriptional activity of cyclin D1 promoter (23), which occurs through receptor association with other transcription factors (e.g. activator protein-1) and requires ER{alpha}-mediated nongenomic mechanisms (36, 37). Moreover, Nar influences cancer cell proliferation (Fig. 8Go) by acting as a selective antagonist of ER{alpha}-mediated nongenomic activities. This implies Nar to work as an ER{alpha} antagonist on specific pathways. The Nar antagonistic effects are under active evaluation in our laboratory. In addition, Nar and other nutritional flavonoids (i.e. quercetin and daidzein) act as E2 mimetic, thus mediating the antiproliferative and protective effects mediated by ERβ against cancer (22, 29).

In conclusion, the (anti)estrogenic effects of flavonoids originate from the activation of specific signaling pathways that modulate the membrane and nuclear actions of ERs. Understanding the cross talk within different pathways and the elaborate feedback mechanisms will provide an opportunity to use these chemicals as novel therapeutics to direct specific responses in target cells or to modulate selectively ER activities in specific target tissues and organs.


   Acknowledgments
 
We thank Dr. Filippo Acconcia (IFOM-FIRC Institute of Molecular Oncology-Fondazione Italiana per la Ricerca sul Cancro, Milano, Italy) for the helpful and critical discussions. We also thank Mr. Peter De Muro for his editorial assistance.


   Footnotes
 
This work was supported by grants from Ministero dell’istruzione, dell’università e della ricerca (COFIN-PRIN 2006) (to M.M.).

Disclosure Statement: The authors have nothing to declare.

First Published Online January 31, 2008

Abbreviations: ASK1, Apoptosis signal-regulating kinase 1; 2-Br, 2-bromohexadecanoic acid; E2, 17β-estradiol; ER, estrogen receptor; HRT, hormone replacement therapy; MNAR, modulator of nongenomic actions of the estrogen receptor; Nar, naringenin; PI3K, phosphoinositide 3-kinase.

Received August 23, 2007.

Accepted for publication January 24, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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