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Genistein, a soy isoflavone, up-regulates expression of antioxidant genes: involvement of estrogen receptors, ERK1/2, and NFB Consuelo Borra´s,† Juan Gambini,* M. Carmen Go´mez-Cabrera,† Juan Sastre,* Federico V. Pallardo´,† Giovanni E. Mann,‡ and Jose Vin˜a*,1 *Department of Physiology, School of Medicine, University of Valencia, Valencia, Spain; † Catholic University of Valencia, Valencia, Spain; and ‡Cardiovascular Division, King’s College London, London, UK We have previously reported that estrogens up-regulate longevity-associated genes. As recent evidence has shown that estrogen replacement therapy is associated with an increased risk of cardiovascular disease, we have studied the effects of genistein, a soy isoflavone with a similar structure to estradiol, on the expression of antioxidant, longevity-related genes. MCF-7 cells (human mammary gland tumor cell line) were incubated for 48 h with 0.5 M genistein, a concentration found in the plasma of populations consuming diets rich in soy protein. Peroxide levels were determined by fluorimetry, activation of extracellular-signal regulated kinase (ERK1/2), and nuclear factor B (NFB)-signaling pathways by Western blot analysis and ELISA, respectively, and mRNA expression of antioxidant genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Inhibition of basal peroxide levels in MCF-7 cells by genistein was prevented by pretreatment of cells with the estrogen receptor antagonist tamoxifen. Phosphorylation of extracellular regulated kinase (ERK)1/2 led to an activation of NFB, as indicated by increased p50 subunit expression in nuclear extracts, and increased mRNA levels of the antioxidant enzyme manganese-superoxide dismutase (MnSOD). Inhibition of ERK1/2 abrogated genistein-mediated NFB activation and elevated expression of MnSOD. Our molecular studies may provide a basis to determine the effects of genistein and other soy protein-derived products on longevity in both animals and the human population.—Borra´s, C., Gambini, J., Go´mez-Cabrera, M. C., Sastre, J., Pallardo´, F. V., Mann, G. E., Vin˜a, J. Genistein, a soy isoflavone, upregulates expression of antioxidant genes: involvement of estrogen receptors, ERK1/2, and NFB. FASEB J. 20, E1476 –E1481 (2006)
ABSTRACT
Key Words: phytoestrogens 䡠 aging 䡠 oxidative stress 䡠 redox signaling
(2–5). Epidemiological evidence suggests that estrogens are cardioprotective, with premenopausal women having a lower incidence of coronary heart disease (CHD) compared to age-matched men (6 – 8). The incidence of CHD increases significantly after menopause, with loss of cardiovascular protection attributed to estrogen deficiency. Observational studies in postmenopausal women have concluded that estrogen therapy reduces cardiovascular risk (8), although recent clinical trials have highlighted an increased incidence of stroke and cancer in women receiving prolonged hormone replacement therapy (9 –11). In a search for alternatives to conventional hormone replacement therapy, recent studies have focused on the potential benefits of selective estrogen receptor modulators (SERMs), including the synthetic compound raloxifene, antiestrogen tamoxifen, and the natural isoflavone phytoestrogens genistein and daidzein. Genistein, one of the major isoflavones in soy protein, binds to estrogen receptor  (ER ), with higher affinity than to ER␣ (12). Plasma concentrations of genistein range between 50 and 800 ng/ml in adults consuming soy-rich foods and can achieve levels found in Japanese consuming their traditional soy-rich diet (13–15). Here, we investigate whether genistein, an isoflavone of similar structure to 17-estradiol, can mimic the actions of estrogen in regulating the expression of antioxidant, longevity-related genes and peroxide levels in cultured MCF-7 cells via ERK1/2 and nuclear factor B (NFB) (3). We report that micromolar concentrations of genistein reduce oxidative stress by up-regulating the expression of manganese-superoxide dismutase (MnSOD) via activation of ERK1–2 and NFB signaling pathways. The importance of our findings is that genistein induces an up-regulation of antioxidant gene expression at physiologically relevant plasma concen1
The longevity of females is longer than males (1), and previous studies from our laboratories have shown that estrogens up-regulate longevity-associated genes E1476
Correspondence: Department of Physiology, Universidad de Valencia, Avda. Blasco Iba´n ˜ ez 17 46010 Valencia, Spain. Email:
[email protected] doi: 10.1096/fj.05-5522fje 0892-6638/06/0020-1476 © FASEB
trations and most likely independent of undesirable side effects associated with long-term estrogen therapy.
MATERIALS AND METHODS Cell culture Human mammary gland tumor cells (MCF-7) were cultured in Iscove’s modified Dulbecco⬘s medium (IMDM) without phenol red, supplemented with 10% fetal calf serum, antibiotics (25 U/ml penicillin and 25 g/ml streptomycin and 0.3 g/ml amphotericin B) in 5% CO2 in air at 37°C in 25 or 75 cm2 flasks. All the experiments were performed with confluent cultures. Determination of peroxide levels in MCF-7 cells Intracellular levels of hydrogen peroxide were determined using a modification of methods described by Barja (16). Briefly, cells were washed twice with PBS and then incubated at 37°C with a PBS solution containing 0.1 mM homovanilic acid and 6 U/ml horseradish peroxidase. The incubation was stopped at 5 min with 1 ml of cold 2 M glycine buffer containing 50 mM EDTA and 2.2 M NaOH. The fluorescence of supernatants was measured using 312 nm as an excitation wavelength and 420 nm as an emission wavelength. The levels of peroxides were calculated using a standard curve for H2O2 and were expressed per milligram of protein content. ERK 1/2 phosphorylation Immediately after harvesting, aliquots of whole cell lysates (40 g) were boiled for 10 min to inactivate proteases and phosphatases, electrophoresed on SDS–12.5% polyacrylamide gels, and electroblotted (Bio-Rad) onto an Immobilon-P nylon membrane (Gibco). Membranes were incubated with primary antibodies against phosphoERK1 and phosphoERK2 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Blots were then washed three times with buffer (PBS– 0.2% Tween 20) for 5 min at room temperature and then incubated for 1 h with a secondary horseradish peroxidase (HRP)-linked anti-rabbit IgG antibody (Ab) (Cell Signaling Technologies, Danvers, MA). Blots were washed three times, as above, and developed using LumiGLO® reagent as specified by the manufacturer (Cell Signaling Technologies). Autoradiographic bands were assessed using a Fujifilm scanning densitometer (Fujifilm LAS-1000 plus).
Real-time quantitation of glutathione peroxidase (GPx) and manganese-superoxide dismutase (MnSOD) mRNA relative to glyceraldehyde-3P-dehydrogenase (GAPDH) mRNA levels was performed using the SYBR Green I assay and evaluated in an iCycler detection system (Bio-Rad, Hercules, CA). Target cDNAs were amplified in separated tubes using the following procedure: 10 min at 95°C and then 40 cycles of: denaturation 95°C for 30 s and annealing and extension at 64.2°C for 1 min per cycle. The increase in fluorescence was measured in real-time during extension step. The threshold cycle (Ct) was determined, and relative gene expression was then expressed as fold change ⫽ 2ˆ(-⌬⌬Ct). The specific primers used for MnSOD were CGT GCT CCC ACA chloroamphenicol acetyltransferase (CAT) CAA TC and TGA ACG TCA CCG AGG AGA AG and for the housekeeping gene, GAPDH, CCT GGA GAA acetyl-coenzyme A carboxylase (ACC) TGC CAA GTA TG, and GGT CCT CAG TGT automatic gain control (AGC) CCA AGA TG. Statistics Data are expressed as means ⫾ sd An ANOVA was performed, and the null hypothesis was accepted for all numbers in sets in which F was nonsignificant at the level of P ⱕ 0.05. Second, sets of data in which F was significant were further examined using a modified t test with P ⱕ 0.05 as the critical limit. For real-time RT-PCR, differences between means were analyzed using a one-way ANOVA. The Tukey multiple-comparisons test for all pairs of columns was applied as a posttest and P ⬍ 0.05 taken as an indication of significance. A commercial software package (Kaleida Graph 3.6 Software) was used to perform the statistical analyses.
RESULTS Micromolar genistein concentrations inhibit peroxide levels in cultured MCF-7 cells At concentrations equivalent to those found in the plasma of Eastern populations consuming a soy protein-rich diet (13–15), genistein (0.5 M) lowered peroxide levels in MCF-7 cells (see Fig. 1). The reduction in intracellular peroxide levels was only detected in cells pretreated with genistein for at least 48 h. Treatment of MCF-7 cells for shorter time periods required
NFB activation-p50 levels in nuclear lysates Nuclear MCF-7 cell lysates were immunoblotted for p50, an index of NFB activation (3). p50 activation was detected in nuclear lysates by ELISA, according to manufacturer instructions (TransAM NFB p50 Chemi. Active Motif North America). Quantitative real-time reverse transcriptase polymerase chain reaction RNA was isolated from MCF-7 cells using the RNeasy® Mini Kit (Qiagen Distributors, Valencia, CA). Quantitative realtime reverse transcriptase-polymerase chain reaction (RTPCR) was performed using the Tth DNA polymerase kit (Roche Diagnostics-Boehringer Mannheim, Penzburg and Mannheim, Germany), as described by the manufacturer. GENISTEIN UPREGULATES ANTIOXIDANT GENES
Figure 1. Genistein diminishes peroxide levels in MCF-7 cells at concentrations detected in the plasma of populations consuming soy protein diets rich in isoflavones. Peroxide levels were determined by a fluorimetric method using homovanilic acid (see Materials and Methods). Data are expressed as means ⫾ sd for 8 –10 different experiments, *P ⬍ 0.05; **P ⬍ 0.01 vs. control. E1477
higher, nonphysiological concentrations of genistein (5–15 M) to detect antioxidant effects (data not shown). This indicates that, at low micromolar concentrations, genistein does not act as an antioxidant per se, because of its phenolic nature, but involves other signaling pathways that require prolonged exposure of cells to this soy isoflavone. Antioxidant actions of genistein are mediated by estrogen receptors As shown in Fig. 2, estrogen receptors appear to mediate the antioxidant actions of genistein. When MCF-7 cells were pretreated with tamoxifen (15 M), an estrogen receptor modulator known to act as an estrogen receptor antagonist in this tumor cell type, genistein-mediated decreases in basal peroxide levels were largely prevented. These findings suggest that the antioxidant actions of genistein involve the classical activation of genomic responses by estrogen receptors. Genistein induces ERK1/2 phosphorylation in MCF-7 cells We have previously shown that treatment of MCF-7 cells with 17-estradiol for 48 h is associated with activation of the extracellular signal-regulated kinases ERK1/2 (3). Fig. 3 shows that incubation of MCF-7 cells with 0.5 M genistein for 3 min leads to a rapid phosphorylation of ERK1/2. Activation of ERK1/2 was sustained for up to 30 min (data not shown). In subsequent experiments, coincubation of MCF-7 cells with the MEK inhibitor U0126 (1 M) completely abolished genisteinstimulated activation of ERK1/2. These findings are reminiscent of similar studies in human umbilical vein endothelial cells, in which low nanomolar concentrations of genistein and other isoflavones were found to rapidly (⬍2 min) activate phosphorylation of ERK1/2 and eNOS (17).
Figure 3. Genistein activates extracellular signal-regulated kinase pathway. A representative Western blot is shown of phospho-ERK 1/2 in MCF-7 cells after 3-min incubation with genistein (0.5 M) alone or following coincubation with 1 M U0126. Histograms represent densitometric measurement of specific bands of phospho-ERK 1/2 content using total ERK levels as housekeeping controls. Data are expressed as means ⫾ sd for 5 independent experiments, *P ⬍ 0.05; **P ⬍ 0.01 vs. control.
Mitogen-activated protein kinase activation by genistein induces NFB translocation In our previous studies with MCF-7 cells, we demonstrated that 17-estradiol induces the translocation of the NFB to the nucleus (3). In the present study, we have shown that treatment of MCF-7 cells with 0.5 M genistein also increases the level of the p50 subunit of NFB in nuclear lysates (Fig. 4. Further experiments established that stimulation of NFB involved genisteinmediated activation of ERK1/2, as treatment of cells with U0126 (1 M) prevented the nuclear accumulation of the p50 subunit.
Figure 2. Antioxidant effects of genistein involve estrogen receptor(s). MCF-7 cells were treated with genistein (0.5 M) and/or tamoxifen (15 M) for 48 h. Data are expressed as means ⫾ sd for 6 – 8 different experiments, *P ⬍ 0.05 vs. control and ˆˆP ⬍ 0.01 vs. genistein. E1478
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Genistein induces up-regulation of MnSOD gene expression via the ERK1/2 Similar to our previous findings with 17-estradiol in MCF-7 cells (3), we have now established that genistein (0.5 M, 48 h) also up-regulates the expression of
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TABLE 1. Inhibition of MAP kinase activation using UO126 abolishes the antioxidant effect of genistein in MCF-7 cells H2O2 nmol/ mg prot
Figure 4. Genistein activates the NFB signaling pathway in MCF-7 cells. Levels of the active p50 subunit of NFB were measured in nuclear lysates from cells treated for 48 h with 0.5 M genistein alone or after coincubation with 1 M U0126. Data are expressed as means ⫾ sd for 5 different experiments, *P ⬍ 0.05; **P ⬍ 0.01 vs. control.
MnSOD but not glutathione peroxidase (see Fig. 5). Up-regulation of MnSOD expression was prevented when cells were coincubated with the MEK inhibitor U0126 (1 M), thereby implicating activation of ERK1/2 in the upstream signaling cascade(s), leading to genistein-mediated antioxidant gene expression. Inhibition of ERK1/2 prevents genistein-mediated decreases in basal peroxide levels When MCF-7 cells were treated for 48 h with 0.5 M genistein, peroxide levels fell significantly (see Table 1). However, when cells were coincubated with 0.5 M genistein in the presence of 1 M U0126, the peroxidelowering effect of genistein was abolished.
DISCUSSION We have demonstrated that the antioxidant effects of low micromolar concentrations of genistein are mediated via the up-regulation of antioxidant gene expression, involving activation of ERK1/2 and NFB. Although the antioxidant properties of genistein might be the result of direct chemical actions due mainly to its phenolic structure, it is worth highlighting that the isoflavone genistein has a structure similar to that of estradiol. Moreover, as plasma concentrations of genistein found in plasma range from 50 to 800 ng/ml (13–15), it is highly unlikely that the antioxidant prop-
Control 0.5 M Genistein 0.5 M Genistein ⫹ 1 M UO126 1 M UO126
1.81 ⫾ 0.42 1.49 ⫾ 0.40* 2.25 ⫾ 0.22# 1.99 ⫾ 0.66
Data are expressed as means ⫾ sd for 6 – 8 different experiments, *P ⬍ 0.05 vs. control; #P ⬍ 0.05 vs. genistein.
erties genistein can be attributed to direct chemical effects. Classical actions of estrogens and isoflavone phytoestrogens are mediated via transcriptional activation of estrogen-responsive genes, involving intracellular estrogen receptors (18 –20), with isoflavones exhibiting a higher affinity for ER than for ER␣ (12). The receptor-hormone complex binds to a specific estrogen response element (ERE) in the promoter region of target genes, leading to transcriptional activation (12, 18 –20). However, activation of target genes by estrogens may also be mediated by other transcription factors, including activating protein (AP)-1 and NFB (19), independent of the ERE. Recent evidence also implicates cell surface receptors in the rapid responses to estrogen and phytoestrogens in a number of different cell types (20). Previous work from our laboratory has shown that the antioxidant properties of estrogens are not due to their chemical structure but rather to the modulation of the expression of antioxidant genes via the interaction of estradiol with estrogen receptors (3). Thus, we hypothesized that genistein may act similarly. Genistein binds preferentially to estrogen receptor  (13), and interestingly, we found that genistein decreases the basal peroxide levels in MCF-7 cells. In this context, our results are consistent with the findings that diets rich in soy isoflavones do not necessarily increase the antioxidant capacity of plasma (21). Although genistein and other isoflavones are slowly absorbed across the gastrointestinal tract, our experiments have established that the effects of isoflavones are catalytic, i.e., they increase transcriptional activation of defense genes. As in the case of other hormones, we have found that in MCF-7 cells genistein acts via specific estrogen receptors. Beneficial effects of genistein are mediated via binding to estrogen receptors mimicking actions of 17-estradiol
Figure 5. Genistein up-regulates the expression of MnSOD in MCF-7 cells. Genistein (0.5 M) induced increases in mRNA levels of Mn-superoxide dismutase (P⬍0.01 vs. control) are abrogated after coincubation of cells with 1 M U0126. Data are expressed as means ⫾ sd for three experiments. GENISTEIN UPREGULATES ANTIOXIDANT GENES
We hypothesize that the beneficial effects of genistein (and other isoflavones) may be mediated via their interaction with estrogen receptor , leading to the subsequent activation of intracellular signaling pathways involved in the up-regulation of MnSOD expression. As shown in Figs. 3 and 4, the signaling cascades activated by genistein (0.5 M, 3–30 min) involve E1479
activation of ERK1/2 and NFB. Increased translocation of the p50 subunit of the NFB complex to the nucleus in response to genistein treatment confirms the activation of NFB. As activation of NFB was abolished after coincubation of MCF-7 cells with genistein and an inhibitor of ERK1/2 activation, this suggests that activation of ERK1/2 lies upstream of NFB-mediated signaling. In the present study, we have shown that genistein increases the expression of MnSOD (Fig. 5). Inhibition of ERK1/2 with U0126 abolished the up-regulation of MnSOD mRNA levels in response to genistein. Our findings in MCF-7 cells thus support the hypothesis that low concentrations of genistein (0.5 M) increase antioxidant capacity in cells via 1) an interaction with estrogen receptors, 2) activation of ERK1/2, 3) nuclear translocation of NFkB leading to an overexpression of MnSOD and lower intracellular peroxide levels. The beneficial effects of genistein occur at nutritionally relevant concentrations The present study extends our previous work on the actions of 17-estradiol in rats in vivo (2) and in vitro with MCF-7 cells (3). Furthermore, our findings are consistent with our earlier study of the beneficial actions of a soy isoflavone-rich diet on vascular function and antioxidant gene expression in rats in vivo (22). In this latter study, we demonstrated that increased MnSOD and eNOS gene expression in male rats fed a soy protein diet rich in genistein/daidzein for up to 16 mo is associated with decreased reactive oxygen species (ROS) production, improved endothelial function, and lower blood pressure in vivo. Because the beneficial effects of soy isoflavones on vascular reactivity and blood pressure were also observed in soy-deficient rats fed a soy protein diet for 6 mo, this suggests that soy isoflavones reduce cellular oxidative stress. We thus hypothesize that a diet rich in soy isoflavones will increase the expression of antioxidant, longevity-related genes, leading to reduced oxidative stress. The main conclusion that can be drawn from our present study is that genistein up-regulates the expression of longevity-related genes in a manner similar to 17-estradiol, involving interactions with estrogen receptor(s), activation of ERK1/2 and NFB and upregulation of longevity-related gene expression. The nutritional relevance of our study is that physiologically relevant concentrations of genistein, found in the plasma of Eastern populations consuming soy protein rich diets, decrease cellular oxidative stress. In contrast, plasma concentrations of genistein in Western populations whose diet does not include large amounts of soy protein may be less effective in up-regulating antioxidant gene expression. In summary, we have shown that physiologically relevant plasma concentrations of genistein (low micromolar) modulate the expression of longevity-related genes. As illustrated in Fig. 6, genistein interacts with E1480
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Figure 6. Genistein exerts its antioxidant effect by binding to estrogen receptor(s), leading to the rapid activation of ERK1/2 and NFB signaling pathways and a delayed upregulation of MnSOD gene expression in MCF-7 cells.
estrogen receptor(s), leading to a rapid (3–30 min) phosphorylation of ERK1/2 and IB and translocation of the p50 subunit of NFB to the nucleus and transactivation of MnSOD expression. The increased MnSOD mRNA expression in response to genistein treatment accounts for the reduced level of peroxides measured in MCF-7 cells. Thus, our molecular studies of the signal transduction pathways involved in genistein mediation antioxidant gene expression provide a basis for evaluating the effects of soy proteinderived products on longevity in both animal and the human populations. Moreover, our findings strongly suggest that changes in nutritional habits and/or supplementation of Western-type diets with isoflavones may be beneficial in decreasing oxidative stress as a consequence of increased expression of antioxidant defense genes. We thank D. Royo for skillful technical assistance and gratefully acknowledge the support of the Comisio´n Interministerial de Ciencia y Tecnologı´a (SAF2004 – 03755 to J.V. and SAF2002/00885 to F.V.P) and by Instituto de Salud Carlos III, RCMN (C03/08), Madrid, Spain.
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Genistein, a soy isoflavone, upregulates expression of antioxidant genes: involvement of estrogen receptors, ERK1/2, and NFB Consuelo Borra´s,† Juan Gambini,* M. Carmen Go´mez-Cabrera,† Juan Sastre,* Federico V. Pallardo´,* Giovanni E. Mann,‡ and Jose Vin˜a*,1 *Department of Physiology, School of Medicine, University of Valencia, Valencia, Spain; † Catholic University of Valencia, Valencia, Spain; and ‡Cardiovascular Division, King’s College London, London, UK To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5522fje SPECIFIC AIMS Females live longer than males, and recent studies from our laboratories have shown that estrogens up-regulate longevity-associated genes. As estrogen replacement therapy is associated with an increased risk of cardiovascular disease, current research is focusing on the potential benefits of estrogen receptor modulators and natural soy-derived isoflavones such as genistein. Here, we investigate the actions of genistein, a soy isoflavone of similar structure to estradiol, on the expression of antioxidant, longevity-related genes in the human MCF-7 cell line. Cells were incubated with a physiologically relevant concentration of genistein (0.5 M, 48 h), and peroxide levels were determined by fluorimetry, activation of extracellular-signal regulated kinase (ERK1/2) and nuclear factor B (NFB) signaling pathways by Western blot analysis and ELISA, respectively, and antioxidant mRNA expression by real time reverse transcriptase-polymerase chain reaction (RTPCR). We report here that genistein, at nutritionally relevant concentrations, exerts its antioxidant effect by up-regulating the expression of antioxidant defense genes and have established that ERK1/2 and NFBsignaling pathways mediate the actions of genistein.
PRINCIPAL FINDINGS We found that nutritionally relevant concentrations of genistein, detected in Japanese populations consuming a soy protein-rich diet, decreases oxidative stress by up-regulating the expression of mitochondrial manganese-superoxide dismutase (MnSOD) via activation of estrogen receptor(s) and subsequent activation of ERK1/2 and NFB signaling pathways. The importance of our present findings is that genistein may exert the beneficial antioxidant effects of estradiol without its undesirable side effects. 2136
1. Physiological plasma concentrations of genistein lower peroxide levels in MCF-7 cells Genistein, at concentrations of 0.5 M, lowered peroxide levels in cultured MCF-7 cells (control, 1.81⫾0.42; genistein 0.5 M, 1.49⫾0.40 nmol H2O2/mg prot). This occurred only when cells were treated with genistein for at least 48 h. Exposure of cells for shorter periods of time required much higher, nonphysiological concentrations of genistein (5–15 M) to detect antioxidant effects. Thus, at nutritionally relevant concentrations, genistein does not act as an antioxidant per se (as a result of its phenolic nature), but rather by other mechanisms, which require time to become activated. 2. The antioxidant effect of genistein is estrogen receptor mediated Figure 1 shows that classical estrogen receptors are most likely involved in the genomic antioxidant actions of the isoflavone genistein. Tamoxifen, an estrogen receptor modulator (SERM), which in mammary gland cells acts as an antagonist of estrogen receptors, prevented the decrease of peroxide levels detected in MCF-7 cells treated with low concentrations of genistein. 3. Genistein induces EKR1/2 phosphorylation in MCF-7 cells Incubation of MCF-7 cells with 0.5 M genistein for 3 min induces a rapid phosphorylation of ERK1/2 (see Fig. 2). This effect was maintained for at least 30 min 1
Correspondence: Department of Physiology, University of Valencia, Avda. Blasco Iba´n ˜ ez 17 46010 Valencia, Spain. E-mail:
[email protected] doi: 10.1096/fj.05-5522fje 0892-6638/06/0020-2136 © FASEB
Figure 1. Antioxidant effects of genistein involve estrogen receptor(s). MCF-7 cells were treated with genistein (0.5 M) and/or tamoxifen (15 M) for 48 h. Data are expressed as means ⫾ sd for 6 – 8 different experiments, *P ⬍ 0.05 vs. control and ˆˆP ⬍ 0.01 vs. genistein.
(data not shown). Coincubation of MCF-7 cells with 1 M U0126 (an inhibitor of MEK1/2, an upstream activator of ERK1/2) prevented acute phosphorylation of ERK1/2 in response to genistein.
may be due to a direct chemical effect caused mainly by its phenolic structure. However, the concentrations of genistein usually found in plasma, range from 50 to 800 ng/ml, making it highly unlikely that its antioxidant properties are the result of direct chemical effects due to its phenolic ring structure. Previous work from our laboratory has shown that the antioxidant properties of estrogens are not due to their chemical structure but rather to the modulation of the expression of antioxidant genes via the interaction of estradiol with estrogen receptors. Thus, we reasoned that the case for genistein might be the same. Genistein binds preferentially to ER. We here report here that genistein decreases the basal level of peroxide production in MCF-7 cells. In this context, our results are consistent with the view that diets rich in soy protein do not necessarily increase the antioxidant capacity of plasma. Indeed genistein (and very likely other isoflavones) are poorly absorbed across the gastrointestinal tract, with only low levels detected in plasma. However, our experiments establish that the effect of the isofla-
4. Activation of ERK1/2 by genistein induces NFB translocation Treatment of MCF-7 cells with genistein induced translocation of the NFB to the nucleus. At a concentration of 0.5 M genistein increased the level of the p50 subunit in nuclear extracts of MCF-7 cells compare to controls. Activation of NFB was mediated via the ERK1/2 pathway, since coincubation of cells with genistein and the MEK1/2 inhibitor U0126 (1 M) prevented NFB translocation. 5. Genistein up-regulates MnSOD gene expression via the MAPK pathway Treatment of MCF-7 cells with genistein for 48h led to an up-regulation of the expression of MnSOD. This was prevented when cells were coincubated with U0126 (1 M), confirming that ERK1/2 activation is required for the stimulatory effect of genistein on antioxidant gene expression. 6. Inhibiting MAP kinase phosphorylation prevents the antioxidant effect of genistein When MCF-7 cells were treated (48 h) with 0.5 M genistein, peroxide levels decreased significantly. However, coincubation of cells with 0.5 M genistein and 1 M U0126 abrogated the peroxide lowering effect of genistein. CONCLUSIONS AND SIGNIFICANCE Antioxidant effects of genistein are due to its action in up-regulating the expression of antioxidant genes Genistein is an isoflavone whose structure is similar to that of estradiol. Therefore, its antioxidant properties GENISTEIN UPREGULATES ANTIOXIDANT GENES
Figure 2. Genistein activates extracellular-signal regulated kinase pathway. A representative Western blot is shown of phospho-extracellular-signal regulated kinase (ERK) 1/2 in MCF-7 cells after 3 min incubation with genistein (0.5 M) alone or following coincubation with 1 M U0126. Histograms represent densitometric measurement of specific bands of phospho-ERK 1/2 content using total ERK levels as housekeeping controls. Data are expressed as means ⫾ sd for 5 independent experiments, *P ⬍ 0.05; **P ⬍ 0.01 vs. control. 2137
vone genistein is catalytic, that is, it increases the expression of genes which in turn increase the translation of antioxidant enzymes. As in the case of other hormones, genistein appears to act through an interaction with classical estrogen receptors. Beneficial actions of genistein are estrogen receptor mediated and involve ERK1/2 and NFB signaling pathways The beneficial actions of isoflavone phytoestrogens have been attributed to their preferential interaction with ER and subsequent activation of critical cell signaling pathways, leading to an up-regulation of antioxidant gene expression. We have shown that genistein activates phosphorylation of ERK1/2 and nuclear translocation of the p50 subunit of the NFB complex (Fig. 3). Both of these actions of genistein were prevented when cells were coincubated with genistein and an inhibitor of the MAP kinase pathway. Genistein increased the expression of MnSOD (see Fig. 3), and this effect was abolished when cells were incubated with genistein and an inhibitor of the MAP kinase pathway. Thus, our results indicate that the pathway through which genistein acts to increase antioxidant capacity in cells is via interaction with estrogen receptor(s), activation of MAP kinase, activation and nuclear translocation of NFB, overexpression of MnSOD, and lowering of the intracellular levels of oxidants. Beneficial actions of genistein occur at nutritionally relevant concentrations In summary, we have shown that physiologically relevant plasma concentrations of genistein (low micromolar) modulate the expression of longevity-related genes. As illustrated in Fig. 3, genistein interacts with estrogen receptor(s), leading to a rapid (3–30 min) phosphorylation of ERK1/2 and translocation of the p50 subunit of NFB to the nucleus and transactivation of MnSOD expression. The increased MnSOD mRNA expression in response to genistein treatment accounts for the reduced level of peroxides measured in MCF-7 cells. Thus, our molecular studies of the signal transduction pathways involved in genistein mediation antioxidant
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Figure 3. Genistein exerts its antioxidant effect by binding to estrogen receptor(s), leading to the rapid activation of ERK1/2 and nuclear factor B (NFB) signaling pathways and a delayed up-regulation of manganese-superoxide dismutase (MnSOD) gene expression in MCF-7 cells.
gene expression, provide a basis for evaluating the effects of soy protein-derived products on longevity in both animal and the human populations. Moreover, our findings strongly suggest that changes in nutritional habits and/or supplementation of Western-type diets with isoflavones may be beneficial in decreasing oxidative stress as a consequence of increased expression of antioxidant defense genes.
The FASEB Journal
BORRA´S ET AL.
