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B
* 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
1Correspondence: Department of Physiology, Universidad de Valencia, Avda. Blasco Ibáñez 17 46010 Valencia, Spain. Email: jose.vina{at}uv.es
ABSTRACT
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 µM 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.—Borrás, C., Gambini, J., Gómez-Cabrera, M. C., Sastre, J., Pallardó, F. V., Mann, G. E., Viña, J. Genistein, a soy isoflavone, upregulates expression of antioxidant genes: involvement of estrogen receptors, ERK1/2, and NFB.
Key Words: phytoestrogens • aging • oxidative stress • redox signaling
THE LONGEVITY OF FEMALES is longer than males (1)
, and previous studies from our laboratories have shown that estrogens up-regulate longevity-associated genes (2
3
4
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
7
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
10
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 ß (ER ß), 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
14
15)
.
Here, we investigate whether genistein, an isoflavone of similar structure to 17ß-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 concentrations 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 µg/ml streptomycin and 0.3 µg/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 µg) 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).
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 real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using the Tth DNA polymerase kit (Roche Diagnostics-Boehringer Mannheim, Penzburg and Mannheim, Germany), as described by the manufacturer. 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 = 2^(-
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 
0.05. Second, sets of data in which F was significant were further examined using a modified t test with P 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
14
15)
, genistein (0.5 µM) 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 higher, nonphysiological concentrations of genistein (5–15 µM) 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 µM), 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 17ß-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 µM 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 µM) completely abolished genistein-stimulated 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)
.
|
Mitogen-activated protein kinase activation by genistein induces NFB translocation
In our previous studies with MCF-7 cells, we demonstrated that 17ß-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 µM genistein also increases the level of the p50 subunit of NFB in nuclear lysates (Fig. 4
. Further experiments established that stimulation of NFB involved genistein-mediated activation of ERK1/2, as treatment of cells with U0126 (1 µM) prevented the nuclear accumulation of the p50 subunit.
|
Genistein induces up-regulation of MnSOD gene expression via the ERK1/2
Similar to our previous findings with 17ß-estradiol in MCF-7 cells (3)
, we have now established that genistein (0.5 µM, 48 h) also up-regulates the expression of 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 µM), 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 µM genistein, peroxide levels fell significantly (see Table 1
). However, when cells were coincubated with 0.5 µM genistein in the presence of 1 µM U0126, the peroxide-lowering 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
14
15)
, it is highly unlikely that the antioxidant properties 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
19
20)
, with isoflavones exhibiting a higher affinity for ERß 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
19
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 ß (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 17ß-estradiol
We hypothesize that the beneficial effects of genistein (and other isoflavones) may be mediated via their interaction with estrogen receptor ß, 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 µM, 3–30 min) involve 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 µM) 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 17ß-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 17ß-estradiol, involving interactions with estrogen receptor(s), activation of ERK1/2 and NFB and up-regulation 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 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 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.
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ACKNOWLEDGMENTS
We thank D. Royo for skillful technical assistance and gratefully acknowledge the support of the Comisión Interministerial de Ciencia y Tecnología (SAF2004–03755 to J.V. and SAF2002/00885 to F.V.P) and by Instituto de Salud Carlos III, RCMN (C03/08), Madrid, Spain.
Received for publication January 26, 2006. Accepted for publication May 25, 2006.
REFERENCES
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  J. D. Gardner, G. L. Brower, T. G. Voloshenyuk, and J. S. Janicki Cardioprotection in female rats subjected to chronic volume overload: synergistic interaction of estrogen and phytoestrogens Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H198 - H204. [Abstract] [Full Text] [PDF]
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 J. P. Konhilas and L. A. Leinwand The Effects of Biological Sex and Diet on the Development of Heart Failure Circulation, December 4, 2007; 116(23): 2747 - 2759. [Full Text] [PDF]
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 G. E. Mann, D. J. Rowlands, F. Y.L. Li, P. de Winter, and R. C.M. Siow Activation of endothelial nitric oxide synthase by dietary isoflavones: Role of NO in Nrf2-mediated antioxidant gene expression Cardiovasc Res, July 15, 2007; 75(2): 261 - 274. [Abstract] [Full Text] [PDF]
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