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* 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, University of Valencia, Avda. Blasco Ibáñez 17 46010 Valencia, Spain. E-mail: jose.vina{at}uv.es
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 µM, 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 (RT-PCR). 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 NFB-signaling 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.
1. Physiological plasma concentrations of genistein lower peroxide levels in MCF-7 cells
Genistein, at concentrations of 0.5 µM, lowered peroxide levels in cultured MCF-7 cells (control, 1.81±0.42; genistein 0.5 µM, 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 µM) 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.
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3. Genistein induces EKR1/2 phosphorylation in MCF-7 cells
Incubation of MCF-7 cells with 0.5 µM genistein for 3 min induces a rapid phosphorylation of ERK1/2 (see Fig. 2
). This effect was maintained for at least 30 min (data not shown). Coincubation of MCF-7 cells with 1 µM U0126 (an inhibitor of MEK1/2, an upstream activator of ERK1/2) prevented acute phosphorylation of ERK1/2 in response to genistein.
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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 µM 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 µM) 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 µM), 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 µM genistein, peroxide levels decreased significantly. However, coincubation of cells with 0.5 µM genistein and 1 µM 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 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 ERßbeta;. 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 isoflavone 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 ERßbeta; 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.
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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 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.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5522fje
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