In the last two decades, life expectancy at birth has increased by 5–10 years [
One of the antioxidant effects attributed to resveratrol is the ability to protect cells against H2O2-induced oxidative stress [
We have previously reported that oestradiol and genistein are able to decrease hydrogen peroxide levels in MCF-7 cells [
Figure
Resveratrol diminishes hydrogen peroxide levels in MCF-7 cells. Peroxide levels were determined by fluorimetry using homovanillic acid (see Section
In order to find out the mechanism by which resveratrol acts as an antioxidant, we tested if PTEN signaling pathway could be involved in its antioxidant effect. We found that resveratrol treatment for 48 h increased PTEN protein levels in MCF-7 (Figure
Resveratrol activates the PTEN signaling pathway. Levels of PTEN were measured in cells treated for 48 h with resveratrol (1 nM). Data are expressed as means + SD for 4 different experiments;
Figure
Resveratrol inactivates the Akt signaling pathway through PTEN activation in MCF-7 cells. Phospho-Akt levels were measured by Western blotting, after 48 h incubation with resveratrol (1 nM) alone, potassium bisperoxo (bipyridine) oxovanadate (V) as a PTEN inhibitor alone (20 nM), or both together. Histograms represent densitometric measurement of specific bands of phospho-Akt content using tubulin levels as housekeeping control. Data are expressed as means + SD for 4 independent experiments;
Nutritional concentrations of resveratrol upregulate the expression of catalase (Figure
Resveratrol upregulates the expression of catalase (a) and MnSOD (b) in MCF-7 cells. Resveratrol (1 nM) increased mRNA levels of catalase and Mn-superoxide dismutase (
As stated before, 1 nM resveratrol pretreatment led to a reduction in intracellular hydrogen peroxide levels. However, Figure
Resveratrol diminishes peroxide levels in MCF-7 cells, and this effect is mediated by the PTEN/Akt signaling pathway. MCF-7 cells were treated with resveratrol (1 nM) alone, potassium bisperoxo (bipyridine) oxovanadate (V) as a PTEN inhibitor alone (20 nM), or both together. Data are expressed as means + SD for 4–8 different experiments;
Human mammary gland tumor cells (MCF-7) were cultured in Iscove’s modified Dulbecco’s medium (IMDM) without phenol red, supplemented with 10% (v/v) heat-inactivated fetal bovine serum. Cells were plated in 25 or 75 cm² culture flasks and maintained at 37°C with 5% CO2 in air. All the experiments were performed once cells reached confluence.
Based on previous experiments of our laboratory [
Intracellular levels of hydrogen peroxide were determined by fluorimetry using a modification of the method described by Barja [
Briefly, cells were washed twice with PBS and then incubated at 37°C with a PBS solution containing 0.1 mM homovanillic 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 H2O2 standard curve and results were expressed per milligram of protein.
After 48 h of pretreatment with resveratrol, cells were washed twice with cold PBS and lysed in cold lysis buffer (62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% v/v glycerol), which was supplemented with a protease inhibition cocktail (10
Catalase (Cat) and manganese superoxide dismutase (MnSOD) mRNA expression was determined by real-time PCR with glyceraldehyde-3P-dehydrogenase (GAPDH) as the endogenous control, according to previously published results [
For this purpose, total RNA was isolated from cultures by extraction with TRIzol Reagent (Invitrogen), according to the manufacturer’s instructions. RNA was quantified by measuring the absorbance at 260 nm. The purity of the RNA preparations was assessed by the 260/280 ratio.
cDNA was synthesized from 1
The quantitative PCR was performed using the detection system 7900HT Fast Real-Time PCR System (Applied Biosystems) with Maxima SYBR Green/ROX qPCR Master Mix (2X) (Fermentas). Target and control were run in separate wells.
Specific primers employed, sense and antisense for each gene, respectively, were MnSOD, 5′-CGT GCT CCC ACA CAT CAA TC-3′ and 5′-TGA ACG TCA CCG AGG AGA AG-3′; Catalase, 5′-ACG TTG GAT GGA GAA GTG CGG AGA TTC AAC-3′ and 5′-ACG TTG GAT GTT CAC ATA GAA TGC CCG CAC-3′; and GAPDH, 5′-CCT GGA GAA ACC TGC CAA GTA TG-3′ and 5′-GGT CCT CAG TGT AGC CCA AGA TG-3′. Target cDNAs were amplified in separated tubes using the following procedure: 10 min at 95°C and then 40 cycles of denaturation at 95°C for 15 s and annealing and extension at 62°C for 1 min per cycle.
The standard curve method was used to evaluate the relative expression levels of catalase and MnSOD in resveratrol pretreated MCF-7 cells. Briefly, the threshold cycle (Ct) was determined and converted to a relative amount through the use of a standard curve prepared from dilutions of cDNA mix of all samples. The logarithmic formula used to transform Ct values was
Quantitative variables are expressed as means and standard deviation of different experiments. Once the normality of the variables was tested by Kolmogorov-Smirnov test, the statistical analysis was performed using the one-way analysis of variance (ANOVA) test to check any possible statistically significant difference between groups and the adequate post hoc tests. The level of significance was chosen at
Antioxidant supplementation is a common medical practice among the elderly [
Numerous studies have reported the beneficial antioxidant properties of resveratrol. For example, in human blood platelets treated with peroxynitrite, resveratrol inhibited protein carbonylation and nitration, as well as lipid peroxidation [
In our cellular model, resveratrol also acts as an antioxidant by increasing MnSOD and Cat mRNA levels, which in turn decreases H2O2 levels. This H2O2 decrease is in agreement with studies reporting the ability of resveratrol to protect PC12 cells against H2O2-induced cytotoxicity [
In any case, the mechanism by which resveratrol can exert its antioxidant effect has not been fully elucidated. Resveratrol is able to act as a phytoestrogen and mimic estrogen biological effects [
Here we show that PTEN, an antagonist to the PI3K/Akt pathway, is involved in resveratrol antioxidant effects. Resveratrol increases PTEN and decreases phospho-Akt levels. In this regard, Waite et al. observed that preincubation with resveratrol or other phytoestrogens for 48 h had been able to increase PTEN levels and decrease phospho-AKT levels in MCF-7, at concentrations ranging from 0.1 nM to 1
As stated before, resveratrol bioavailability is very low and its absorption is highly variable, depending on the way it is consumed and the kind of food ingested [
The major conclusion of the current study is that nutritionally relevant concentrations of resveratrol can decrease oxidative stress within the cell by upregulating antioxidant genes. As illustrated in Figure
Proposed mechanism for resveratrol to upregulate antioxidant gene expression.
All authors have indicated that they have no financial/commercial conflict of interests.
This work was supported by grants SAF2010-19498 from the Spanish Ministry of Education and Science (MEC); ISCIII2006-RED13-027 and ISCIII2012-RED-43-029 from the Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF); PROMETEO2010/074 from “Conselleria d’Educació, Cultura i Esport de la Generalitat Valenciana”; 35NEURO Gent x Gent from “Fundacio Gent Per Gent de la Comunitat Valenciana”; and RS2012-609 Intramural Grant from INCLIVA and EU Funded CM1001 and FRAILOMIC-HEALTH.2012.2.1.1-2. This study has been cofinanced by FEDER funds from the European Union.