Role of quercetin and its in vivo metabolites in protecting H9c2 cells against oxidative stress

Biochimie 89 (2007) 73e82 www.elsevier.com/locate/biochi

Role of quercetin and its in vivo metabolites in protecting H9c2 cells against oxidative stress C. Angeloni a, J.P.E. Spencer b, E. Leoncini a, P.L. Biagi a, S. Hrelia a,* a

Department of Biochemistry ‘‘G. Moruzzi’’, Nutrition Research Centre, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy b Department of Food Biosciences, Molecular Nutrition Group, University of Reading, Whiteknights, Reading RG2 9AR, UK Received 5 April 2006; accepted 5 September 2006 Available online 26 September 2006

Abstract The aim of this study was to investigate the potential of quercetin and two of its ‘‘in vivo‘‘ metabolites, 30 -O-methyl quercetin and 40 -Omethyl quercetin, to protect H9c2 cardiomyoblasts against H2O2-induced oxidative stress. As limited data are available regarding the potential uptake and cellular effects of quercetin and its metabolites in cardiac cells, we have evaluated the cellular association/uptake of the three compounds and their involvement in the modulation of two pro-survival signalling pathways: ERK1/2 signalling cascade and PI3K/Akt pathway. The three flavonols associated with cells to differing extents. Quercetin and its two O-methylated metabolites were able to reduce intracellular ROS production but only quercetin was able to counteract H2O2 cell damage, as measured by MTT reduction assay, caspase-3 activity and DNA fragmentation assays. Furthermore, only quercetin was observed to modulate pro-survival signalling through ERK1/2 and PI3K/Akt pathway. In conclusion we have demonstrated that quercetin, but not its O-methylated metabolites, exerts protective effects against H2O2 cardiotoxicity and that the mechanism of its action involves the modulation of PI3K/Akt and ERK1/2 signalling pathways. Ó 2006 Elsevier Masson SAS. All rights reserved. Keywords: Quercetin; Oxidative stress; H9c2 cells; Uptake

1. Introduction Many investigations support the view that reactive oxygen species (ROS) contribute to the pathophysiological alterations following ischemia/reperfusion in the heart [1]. During reoxygenation, the excessive formation of free radicals may overwhelm endogenous antioxidant defences leading to damage of myocardial cells. Indeed, the activity of antioxidant enzymes in the heart has been reported to be lower than in other tissues such as the liver [2], while the susceptibility to oxidative stress is relatively high compared to other tissues [3]. Abbreviations: 30 Q, 30 -O-methyl quercetin; 40 Q, 40 -O-methyl quercetin; Ac-DEVD-AMC, Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; DCFHDA, dichlorodihydrofluorescein diacetate; FBS, fetal bovine serum; MTT, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; Q, Quercetin. * Corresponding author. Tel.: þ39 051 209 1233; fax: þ39 051 209 1235. E-mail address: [email protected] (S. Hrelia). 0300-9084/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2006.09.006

Flavonoids are a large group of polyphenolic compounds found in a variety of commonly consumed fruits, vegetables and beverages [4]. Flavonoid intake, particularly that of quercetin, has been inversely associated with incidences of heart disease, cerebrovascular disease, and several types of cancer [5]. Other studies have also shown an inverse relationship between coronary heart disease and flavonoid intake, particularly when subjects consumed fruits and vegetables rich in quercetin [6e8]. The flavonol quercetin has been shown to possess a broad range of pharmacological properties, including anti-proliferative effects on cancer cells [9] and protective effects against oxidative stress by virtue of its strong antioxidant capacity [10]. However, quercetin is extensively metabolised to O-methylated, glucuronide and sulphate metabolites during absorption in the small intestine and in the liver [11,12]. Therefore, it is essential to investigate the biological effects not only of quercetin, but also of these physiological flavonol metabolites.

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Recent studies have speculated that the classical hydrogendonating antioxidant activity of many flavonoids [13,14] is unlikely to be the sole explanation for their cellular effects [15e17]. Accumulating evidence suggests that the cellular effects of flavonoids may be mediated by their interaction with intracellular signalling cascades [18] such as mitogenactivated protein kinases (MAPK) cascade [19,20] and the Akt/Protein Kinase B (PKB) signalling cascade [21]. Interestingly, recent studies have implicated modulations in intracellular signalling in the damage associated with ischemia/ reperfusion injury. For example, the inhibition of the extracellular signal-regulated kinase (ERK) during sustained ischemia/ reperfusion significantly increased the size of myocardial infarction in pig myocardium [22], exaggerated reperfusion injury in isolated rat hearts and enhanced ischemia/reperfusion-induced apoptosis in neonatal cardiomyocytes [23]. Furthermore, Matsui et al. showed that adenoviral expression of constitutively active mutants of either phosphoinositide-3kinase (PI3K) or Akt reduces hypoxia-induced apoptosis in cardiomyocytes in vitro [24]. Studies have also indicated that hydrogen peroxide induces apoptosis of H9c2 cardiomyoblasts via activation of c-jun N-terminal kinase (JNK) [25] and that quercetin may reduce peroxide-induced injury via modulation of mitochondrial function and inhibition of caspase-3 activity [26]. However, there is little information regarding the cellular effects of quercetin metabolites. In this study, we investigated the potential protective effects of quercetin and two of its in vivo metabolites, 30 -O-methyl and 40 -O-methyl quercetin, against H2O2-induced H9c2 cardiomyoblast injury. We highlight the association of quercetin, 30 -O-methyl and 40 -O-methyl quercetin with these cells and evaluate their cytoprotective effects in terms of their ability to modulate two pro-survival signalling pathways, namely the PI3K/Akt pathway and the MAPK signalling cascade through ERK.

from Cell Signalling Technology (Beverly, MA, USA). All the other chemicals were of the highest analytical grade.

2.2. Cell cultures H9c2 myoblast cell line was obtained from the European Collection of Cell Cultures (Salisbury, UK). The cells were routinely grown in DMEM with 10% (v/v) heat-inactivated FBS, penicillin (100 U/mL), streptomycin (100 mg/mL), and  L-glutamine (2 mM) and grown at 37 C in a atmosphere of 5% CO2 and 95% air, at a relative humidity of 95% and split 1 to 4 at sub-confluence (80%). Before each experiments, cells were seeded at the density of 5  104 cells/cm2.

2.3. Assessment of cardiomyoblast uptake/association To evaluate the cell-associated levels of quercetin, 30 -Omethyl quercetin and 40 -O-methyl quercetin, the three flavonols were dissolved in ethanol and supplemented to the cells at a final concentration of 30 mM (the ethanol content should never exceed 0.1%) for different periods (1, 6, 24 h). Following exposure, cells were washed four times with ice-cold PBS and rapidly lysed on ice using aqueous methanol (50% v/v) containing HCl (0.1%). Lysed cells were scraped off and left on ice to solubilize for 45 min, then centrifuged at 2000  g for 5 min at 4  C to remove unbroken cell debris and nuclei. The supernatants were recovered and analysed by HPLC with photodiode array detection as previously reported [16]. The protein concentration in the supernatants was determined by the Bio-Rad Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA).

2. Materials and methods

2.4. Assessment of cell damage

2.1. Materials

Cells were exposed to 150 mM H2O2 for 1 h in PBS. To evaluate the potential protective effects of quercetin and its O-methylated metabolites, H9c2 cells were supplemented with 1e30 mM quercetin, 30 -O-methyl quercetin and 40 -Omethyl quercetin for 1e24 h. After, cells were washed twice with PBS (to prevent direct extracellular interactions between the compounds and H2O2) prior to the addition of H2O2. Vehicle controls containing equivalent volumes of ethanol were carried out. Cellular damage elicited by H2O2 treatment was evaluated by measuring MTT reduction. MTT was added to the medium (final concentration 0.5 mg/mL) and incubated for 1 h at 37  C. After incubation, MTT solutions were removed, DMSO was added and the absorbance was measured using a microplate spectrophotometer (VICTOR3 VÔ Multilabel Counter, Perkin Elmer e Wellesley, MA, USA) at a wavelength of 595 nm. Additional experiments to assess the direct toxic effects of quercetin and its metabolites (30 mM; 24 h) were also carried out.

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), quercetin, H2O2, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), LY294002, PD98059, mammalian protease inhibitor mixture, sodium pyrophosphate, phenylmethylsulfonyl fluoride, sodium vanadate, sodium fluoride, 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA), Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC), were purchased from Sigma Chemical (St. Louis, MO, USA). 30 -O-methyl quercetin and 40 -O-methyl quercetin were from Extrasynthese (Genay Cedex, France). Cell Death Detection ELISAPLUS was obtained from Roche Diagnostic (Mannheim, Germany). Penicillin, streptomycin, and L-glutamine from Euroclone (Pero, MI, Italy). Anti-Akt, anti-phospho-Akt (Ser473), anti-p42/44 MAPK, anti-phospho-p42/44 MAPK (Thr202/Tyr204), goat anti-rabbit IgG, Prestained Protein Marker, were purchased

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2.5. 20 ,70 -Dichlorodihydrofluorescein diacetate assay for oxidative stress The formation of intracellular ROS was evaluated using a fluorescent probe, DCFH-DA, as described by Wang et al. [27]. Briefly, H9c2 cells were incubated for 24 h with quercetin, 30 -O-methyl quercetin and 40 -O-methyl quercetin (1e30 mM). The cells were washed with PBS and then incubated with 5 mM DCFH-DA in PBS for 30 min. After removal of the DCFH-DA and further washing, the cells were incubated with H2O2 150 mM for 1 h. The fluorescence of the cells from each well was measured using 485 nm excitation and 535 nm emission with a microplate spectrofluorometer (VICTOR3 VÔ Multilabel Counter, Perkin Elmer e Wellesley, MA, USA). Intracellular antioxidant activity was expressed as the percentage of inhibition of intracellular ROS following H2O2 exposure. 2.6. Western immunoblotting H9c2 cells were supplemented for 24 h with 30 mM quercetin, 30 -O-methyl quercetin, 40 -O-methyl quercetin. The cells were then washed with PBS and incubated with 150 mM H2O2 for 1 h. Cells were washed with ice-cold PBS and lysed on ice using 50 mM Tris, 0.1% Triton X-100, 150 mM NaCl, and 2 mM EGTA/EDTA containing mammalian protease inhibitor mixture (1:100 dilution), 1 mM sodium pyrophosphate, 10 mg/mL phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, and 50 mM sodium fluoride. The cells were scraped off and left on ice to solubilize for 45 min. The lysates were centrifuged at 5000  g for 5 min at 4  C to remove unbroken cell debris and nuclei. The samples were boiled at 98  C for 3 min in boiling buffer (62.5 mM Tris, pH 6.8 containing 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, and 0.0025% bromophenol blue). The boiled samples were run on 8% SDS-polyacrylamide gels (20 mg/lane), and the proteins were transferred to nitrocellulose membranes (Hybond-ECL; Amersham Biosciences, Buckinghamshire, UK) by semi-dry electroblotting (1.5 mA/cm2). The nitrocellulose membrane was then incubated in a blocking buffer (TBS supplemented with 0.05% (v/v) Tween 20 (TTBS)) containing 5% (w/v) skimmed milk powder for 60 min at room temperature followed by three 5 min washes in TTBS. The blots were then incubated with either anti-Akt (1:1000 dilution), antiphospho-Akt (Ser473) (1:1000 dilution), anti-p42/44 MAPK (1:1000 dilution), anti-phospho-p42/44 MAPK (Thr202/ Tyr204) (1:1000 dilution), in TTBS containing 5% (w/v) skimmed milk powder (antibody buffer) overnight at room temperature on a three-dimensional rocking table. The blots were washed twice for 10 min in TTBS and then incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:2000 dilution) in antibody buffer for 60 min. Finally, the blots were washed twice for 10 min in TTBS and exposed to ECLÒ reagent for 1e2 min as described in the manufacturer’s protocol (Amersham Biosciences, Buckinghamshire, UK). The blots were exposed to Hyperfilm-ECL (Amersham Biosciences, Buckinghamshire, UK) for 2e5 min in an

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autoradiographic cassette and developed. The molecular weights of the bands were calculated from comparison with prestained molecular weight markers (molecular weight range 6500e175,000) that were run in parallel with the samples. Semiquantitative analysis of specific immunolabeled bands was performed using a Fluor S image analyzer (BioRad Laboratories, Hercules, CA, USA). 2.7. Caspase-3 activity assay The activity of caspase-3 was measured by the cleavage of the fluorogenic peptide substrate Ac-DEVD-AMC [28]. At the end of each experiment, H9c2 cells were collected, washed in PBS, suspended in 150 mL of lysis buffer (50 mM Tris, 0.1% Triton X-100, 150 mM NaCl, 2 mM EGTA/EDTA, 1 mM sodium pyrophosphate, 10 mg/mL phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 50 mM sodium fluoride, 1 mg/mL aprotinin), vortexed and left 45 min on ice. The lysates were centrifuged 5 min at 5000  g and the supernatant was used as enzyme source. 10 mL of this extract (containing about 1 mg of protein) were combined with 20 mL of assay buffer containing 100 mM HEPES pH 7.0, 5 mM dithiothreitol, 0.1% CHAPS, 10% sucrose and 0.15 mM DEVD-AMC and incubated 15 min at 37  C. The reaction was stopped by adding 0.1 mL of 2% sodium acetate in 0.2 M acetic acid. The samples were diluted with 2.5 mL of water and the specific cleavage of the fluorogenic peptide DEVD-AMC was monitored following AMC cleavage at 370 nm excitation and 455 nm emission wavelengths. One unit is defined as the amount of enzyme activity cleaving 1.0 nmol of substrate per min under the conditions described. 2.8. Quantification of DNA fragmentation Quantification of DNA fragmentation was determined by Cell Death Detection ELISAPLUS following the manufacturer’s instructions. Briefly, cells were seeded in 96 multiwells plates at the density of 1  103 cells/well. After each experiment, cells were homogenized in 100 mL of lysis buffer and incubated for 30 min at room temperature. After centrifugation to remove nuclei and cellular debris, the supernatants were diluted 1:2 (v/v) with lysis buffer. Then 20 mL of supernatant from each sample was transferred to a 96-well plate pre-coated with anti-histone antibody to which 80 mL of immunoreagent mix was added. After incubation and washes, the wells were treated with the chromogen 2,20 -azinobis(3-ethylbinzthiazoline) sulfonic acid as a substrate. The absorbance was measured using a microplate spectrophotometer (VICTOR3 VÔ Multilabel Counter, Perkin Elmer e Wellesley, MA, USA) at a wavelength of 405 nm. 2.9. Data presentation and statistics Each experiment was performed at least three times, and all values are represented as means  SD. Student’s t-test was used to analyse a statistical significance of the results (Prism

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4, GraphPad Software Inc., San Diego, CA, U.S.A.). Values of p < 0.05 were considered as statistically significant. 3. Results 3.1. Cellular association of quercetin and its metabolites In order to provide information regarding the potential protective effects of quercetin and its O-methylated metabolites we initially assessed their cellular association following 1, 6 and 24 h exposure (Fig. 1). Quercetin levels were observed to peak at 1 h of treatment after which cellular association decreased significantly with undetectable levels at 24 h. Similarly, 30 -O-methyl quercetin levels peaked at 1 h treatment and decreased following 6 and 24 h, although 30 -O-methyl quercetin levels remained higher than the corresponding quercetin levels. In contrast, 40 -O-methyl quercetin peaked at 6 h and its level at 24 h was reduced only slightly relative to the maximum level and was always higher than the corresponding uptake of both quercetin and 30 -O-methyl quercetin. 3.2. Effects of quercetin and its metabolites on cell viability H9c2 cells were treated with increasing concentrations (1e30 mM) of quercetin and its two O-methylated metabolites for 24 h to investigate their direct effect on cell integrity/damage. There was no observed decrease in the ability of H9c2 cells to reduce MTT (evaluated as % cell viability in comparison to controls) following exposure to quercetin, 30 -O-methyl quercetin or 40 -O-methyl quercetin up to 30 mM, indicating no toxicity of the flavonols (Fig. 2). To investigate whether quercetin, 30 -O-methyl quercetin and 40 -O-methyl quercetin were able to protect H9c2 cardiomyoblasts against oxidative stress-induced injury, we pretreated cells (1e24 h) with the flavonols (1e30 mM) prior to

Fig. 2. Protection against peroxide-induced cell damage by quercetin. H9c2 cells were supplemented with 1e30 mM Q (A), 30 Q (B) and 40 Q (C) for 1e24 h before the addition of H2O2 (150 mM, 1 h) and cellular damage was assessed by the MTT assay and reported as % cell viability in comparison to controls. Each column represents the mean  S.D. of four independent experiments. Statistical analysis was performed by the Student’s t test: * at least p < 0.05 in comparison to H2O2 treated cells.

Fig. 1. Uptake/association of quercetin, 30 -O-methyl quercetin, and 40 -O-methyl quercetin with H9c2 cells. Cells were supplemented with 30 mM Q, 30 Q and 40 Q for 1, 6, 24 h, then lysed, and the extracts were analysed by HPLC with photodiode array detection as reported in Section 2. Each point represents the mean  S.D. of three independent experiments.

the addition of H2O2 (150 mM) for 1 h (Fig. 2). The concentration of 150 mM of hydrogen peroxide was used as it represents the IC50 value for peroxide toxicity, i.e. the concentration that evoked an approximate 50% loss in the ability of cells to reduce MTT (53.5  3.3%). Fig. 2A shows the time and concentration dependent protective effect of quercetin against peroxide-induced cellular damage. Quercetin supplementation counteracted cell damage following a short pre-treatment (1 h) at 10 and 30 mM concentrations, and also significantly protected cells after 18 h of treatment at 1 and 3 mM

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concentrations. Exposure of cells to 30 mM quercetin for 24 h evoked complete protection against peroxide-induced injury. In particular, at the 24 h exposure time, a significant correlation was found between quercetin concentration and the ability of the cells to reduce MTT (r2 ¼ 0.884, p < 0.001). In contrast, pre-treatment of cells with 30 -O-methyl quercetin (Fig. 2B) and 40 -O-methyl quercetin (Fig. 2C) did not protect cells against oxidative injury at any concentrations or at any time exposures.

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10 mM and 30 mM quercetin and 30 -O-methyl quercetin capable of reducing levels of intracellular oxidants to a value comparable with control cells. 3.4. Effects of quercetin and its metabolites on Akt and ERK1/2 pathways

One possible mechanism of the protective action of flavanols is their ability to reduce the level of intracellular oxidative stress generated during peroxide exposure. To investigate the ability of quercetin ant its O-methylated metabolites to reduce intracellular oxidative stress, cells were pre-treated with the three flavonols (1e30 mM; 24 h) prior to the addition of H2O2 and the level of intracellular ROS was determined using the peroxide-sensitive fluorescent probe DCFH-DA. As expected, an increase in intracellular ROS was observed following exposure to peroxide (Fig. 3). Pre-treatment of cardiomyocytes with flavonols decreased intracellular ROS production induced by H2O2, although there were no differences between that seen with quercetin, or either of its O-methylated metabolites. Decreases were statistically significant at lower concentrations of the flavonols (1e3 mM), with

To begin investigating the mechanism of quercetin-induced protection of H9c2 cells against oxidative stress, we evaluated the effect of quercetin on Akt/PKB, an essential kinase which regulates myocyte growth and promotes cytoprotection [29] and on MAPK ERK1/2, another important kinase subfamily involved in cardiomyocyte protection against oxidative stress. To investigate whether these kinases are important in protection against oxidative injury, we exposed cells to an inhibitor of Akt-phosphorylation (20 mM LY294002) [30] or to an inhibitor of ERK1/2-phosphorylation (20 mM PD98059) [31] for 30 min, prior to quercetin supplementation and subsequent H2O2 addition. The pre-treatment with the two inhibitors significantly decreased the protective effects of quercetin against oxidative stress (Fig. 4), suggesting that quercetin’s protective effects in this cell line may be mediated by interactions within both ERK and Akt pathways. The influence of quercetin and its O-methylated metabolites on Akt phosphorylation in H9c2 cells was determined by immunoblotting analysis with an anti-phospho-Akt (Ser473) polyclonal antibody (Fig. 5). Supplementation of H9c2 cells with 30 mM quercetin for 24 h resulted in a marked

Fig. 3. Effect of quercetin, 30 -O-methyl quercetin, and 40 -O-methyl quercetin on intracellular ROS production. H9c2 cells were supplemented with 1e30 mM Q, 30 Q and 40 Q for 24 h before the addition of H2O2 (150 mM, 1 h) and then the level of intracellular ROS was determined using the peroxide-sensitive fluorescent probe DCFH-DA as reported in Section 2. Data are expressed as % inhibition of ROS produced compared to H2O2 treatment. Each column represents the mean  S.D. of four independent experiments. Statistical analysis was performed by the Student’s t test:  at least p < 0.05 in comparison to H2O2 untreated cells and * at least p < 0.05 in comparison to H2O2 treated cells.

Fig. 4. Effect of PD98059 and LY294002 on quercetin-protection against peroxide-induced cell damage. H9c2 cells were supplemented with 20 mM PD98059 or 20 mM LY294002 for 30 min before the addition of 30 mM Q. After 24 h the cells were exposed to 150 mM H2O2 for 1 h and cellular damage was assessed by the MTT assay as reported in Section 2. Each column represents the mean  S.D. of four independent experiments. Statistical analysis was performed by the Student’s t test:  at least p < 0.05 in comparison to control cells and * at least p < 0.05 in comparison to H2O2 treated cells.

3.3. Inhibition of ROS production by quercetin and its metabolites

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Fig. 5. Phosphorylation of Akt in H9c2 cells supplemented with quercetin, 30 -O-methyl quercetin, and 40 -O-methyl quercetin and exposed to H2O2. H9c2 cells were supplemented with 30 mM Q, 30 Q and 40 Q for 24 h and then exposed to 150 mM H2O2 for 1 h prior to immunoblotting. The cell lysates were immunoblotted with antibodies specific for phospho-Akt (A), total-Akt (B) as described under Section 2. Results of scanning densitometric analysis performed on three independent autoradiographs for phospho-Akt are presented (C). Relative amounts (means  S.D.) are in arbitrary units. Statistical analysis was performed by the Student’s t test:  at least p < 0.05 in comparison to control cells and * at least p < 0.05 in comparison to H2O2 treated cells.

Fig. 6. Phosphorylation of ERK1/2 in H9c2 cells supplemented with quercetin, 30 -O-methyl quercetin, and 40 -O-methyl quercetin. H9c2 cells were supplemented with 30 mM Q, 30 Q and 40 Q for 24 h and then exposed to 150 mM H2O2 for 1 h prior to immunoblotting. The cell lysates were immunoblotted with antibodies specific for phospho-ERK1/2 (A), total-ERK1/2 (B) as described under Section 2. Results of scanning densitometric analysis performed on three independent autoradiographs for phospho-ERK are presented (C). Relative amounts (means  S.D.) are in arbitrary units. Statistical analysis was performed by the Student’s t test:  at least p < 0.05 in comparison to control cells.

increase in Akt phosphorylation relative to control cells, as demonstrated by an increase in the relative intensity of the immunodetectable band relating to phosphorylated Akt/PKB (60 kDa) (Fig. 5A,C). In contrast, exposure to 30 mM 30 -Omethyl quercetin and 40 -O-methyl quercetin for 24 h did not cause any significant difference in Akt phosphorylation in respect to control cells. H2O2 (150 mM) treatment resulted in a marked decrease in Akt phosphorylation as demonstrated by a highly significant decrease in the relative intensity band. Pre-treatment with quercetin, but not 30 -O-methyl quercetin or 40 -O-methyl quercetin prior to oxidative stress was able to maintain Akt phosphorylation to a level similar to that of control cells. Parallel immunoblots with a polyclonal antibody against total Akt protein levels were performed (Fig. 5B) and indicated that there was no changes in total levels of Akt. We also investigated whether quercetin and its O-methylated metabolites could influence ERK1/2 activation (phosphorylation) in H9c2 cells by immunoblotting with an anti-phosphoERK1/2 polyclonal antibody (Fig. 6). After exposure of cells to quercetin and its O-methylated metabolites, only quercetin significantly increased ERK1/2 phosphorylation in respect to control cells (Fig. 6A,C). Oxidative stress did not cause any significant modification in ERK1/2 phosphorylation in respect to control cells and quercetin supplementation prior to H2O2 exposure resulted in phosphorylation of ERK1/2 to levels

significantly higher than that observed in control cells. Parallel immunoblots with an antibody against total ERK1/2 protein levels indicated no differences in the total levels of ERK1/2 (Fig. 6B).

3.5. Effects of quercetin and its metabolites on apoptosis Since many studies have established the involvement of hydrogen peroxide in apoptotic H9c2 cell death [27,32,33], we investigated whether quercetin and its in vivo metabolites were able to reduce peroxide-induced caspase-3 activation in H9c2 cells. Fig. 7 highlights the caspase-3 activity expressed as U/mg of protein. In the absence of oxidative stress no significant differences were observed between the cells supplemented with the three compounds and control cells (data not shown). Cardiomyoblasts were exposed to quercetin, 30 -O-methyl quercetin or 40 -O-methyl quercetin (30 mM) for 24 h prior to the addition of 150 mM H2O2. Staurosporine (1 mM, 4 h) was used as positive control [34,35]. Peroxide was able to induce a significant increase in caspase-3 activity in respect to control cells, and only quercetin was able to reduce caspase-3 activity to a value comparable to control cells. To reinforce caspase-3 activation data, the extent of DNA fragmentation was determined by the Cell Death Detection ELISAPLUS assay according to the manufacturer’s

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Fig. 7. Caspase-3 activity in H9c2 cells in the presence of quercetin, 30 -O-methyl quercetin, and 40 -O-methyl quercetin. H9c2 cells were supplemented with 20 mM PD98059 or 20 mM LY294002 for 30 min before the addition of 30 mM Q, 30 Q and 40 Q for 24 h and then exposed to 150 mM H2O2 for 1 h. Staurosporine (1 mM, 4 h) was used as positive control. Caspase-3 activity was measured spectrofluorimetrically in cell lysates as reported in Section 2. Each column represents the mean  S.D. of four independent experiments. Statistical analysis was performed by the Student’s t test:  at least p < 0.05 in comparison to control cells and * at least p < 0.05 in comparison to H2O2 treated cells.

protocol (Fig. 8). Data obtained are in agreement with the caspase-3 activity values.

4. Discussion There is much interest in the potential beneficial effects of flavonoids against cardiovascular disease. However, when attempting to determine how such compounds act in vivo one must consider the extensive metabolism that occurs to these polyphenols, as they are absorbed in the small intestine, and metabolized in the liver, or by the colonic microflora in the large intestine. All flavonoids, and especially the flavonols, are conjugated to form O-glucuronides, sulphate esters and Omethylated ethers [36] during absorption into the circulation. But tissue levels of quercetin and its O-methylated derivatives could be higher than what can be measured in plasma due to the potential cleavage of glucuronide metabolites by tissue beta-glucuronidase as suggested by Shimoi et al. [37] for the flavonol luteolin. Furthermore, when considering the possible cellular mechanism of action of flavonoids and their in vivo metabolites, it is important to consider their uptake and possible further metabolism by the cells. Limited data are available on the cellular association of one of the major dietary flavonols, quercetin and its O-methylated forms. In order to clarify these aspects, we have evaluated the association of quercetin and its two methylated metabolites with H9c2 cardiomyoblasts. Although

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Fig. 8. DNA fragmentation in H9c2 cells in the presence of quercetin, 30 -O-methyl quercetin, and 40 -O-methyl quercetin. H9c2 cells were supplemented with 20 mM PD98059 or 20 mM LY294002 for 30 min before the addition of 30 mM Q, 30 Q and 40 Q for 24 h and then exposed to 150 mM H2O2 for 1 h. DNA fragmentation was measured spectrophotometrically by Cell Death Detection ELISAPLUS as reported in Section 2. Each column represents the mean  S.D. of four independent experiments. Statistical analysis was performed by the Student’s t test:  at least p < 0.05 in comparison to control cells and * at least p < 0.05 in comparison to H2O2 treated cells.

this permanent cell line shows morphological characteristics similar to those of immature embryonic cardiomyocytes, they have been shown to preserve several elements of the electrical and hormonal signal pathway found in adult cardiomyocytes and are therefore a useful model for the investigation of cardiac cell metabolism [38]. Moreover the use of a cell line by-pass the ethical problem linked to animal sacrifice, as requested in the model of neonatal rat primary cardiomyocytes. All the three compounds were observed to associate with cells, but to a different extent in respect to the different chemical structures and exposure times. Initially the uptake/association of quercetin, 30 -O-methyl quercetin and 40 -O-methyl quercetin were similar. However, quercetin association was drastically decreased at later incubation times, compared with its O-methylated metabolites, indicating that quercetin may be subjected to a greater degree of oxidative metabolism in cells. This is in agreement with previous data reported by Spencer et al. [21], who demonstrated a marked reduction of quercetin uptake in dermal fibroblasts after 18 h exposure in respect to its O-methylated metabolites. In this study, the formation of two quercetin-glutathione adducts was also demonstrated indicating that oxidative metabolism may be followed by conjugation to cellular thiols [21]. On the basis of previously reported data [21] we could speculate that cellular metabolites of Q are thiol conjugates and oxidative metabolites. In H4IIE hepatoma cells, the intracellular concentration of quercetin was also observed to decrease exponentially with

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time, and the presence of new metabolites of quercetin was demonstrated [39]. Cell association/uptake of 40 -O-methyl quercetin was higher at all exposure times compared to its 30 -O-methyl counterpart. As previously demonstrated [29] this could be due to the inability of 40 -O-methyl quercetin to undergo oxidative metabolism compared to that of quercetin and its 30 -O-methyl form. Quercetin, but not the O-methylated metabolites, evoked significant protective effects against oxidative stress induced-cellular injury, as evaluated by measuring MTT reduction as an index of cell viability. Consequently, the protective actions of quercetin in cardiac cells can not be ascribed to a higher uptake/association level, in fact this compound showed the lowest association/uptake of the three flavonols. Moreover the highest protective effect of quercetin was evidenced after 24 h supplementation when quercetin is no longer detectable in cell lysates. On the other hand, both quercetin and its O-methylated metabolites showed a significant ability to reduce intracellular ROS production, as measured by the DCFH-DA test. Therefore, the cytoprotective effect of quercetin appears dependent on its direct antioxidant potential, as previously demonstrated by Chow et al. [40] and Chen et al. [41], but also other mechanisms could be involved. Similar conclusions were drawn for different antioxidants in different cell systems [42e44]. The ability of Q to protect cardiac cells could be also ascribed to an indirect antioxidant activity. In fact, Q has been demonstrated to be a potent phase II enzyme inducer by elevating the total GSH content and the protein level of gamma glutamylcysteine ligase [45], glutathione S-transferase and NAD(P)H:quinone oxidoreductase [46] in different cell systems. So, although many flavonols can decrease oxidative stress through a radical scavenging mechanism by directly interacting with reactive oxygen species, only a few also reveal an indirect antioxidant activity and many data point to differences in induction mechanism between the various antioxidant molecules, that could be responsible for different protective ability against cell death. Furthermore, flavonoids have been reported to exert modulatory effects in cells independent of their classical antioxidant capacity through selective actions at different components of protein kinase and lipid kinase signalling cascades [17,19,47]. It has been demonstrated that the MAPK and the PI3K super-families play a crucial role in cell growth, differentiation, or programmed cell death in response to diverse extracellular stimuli in cardiovascular cells [29]. The inactivation of Akt is highly correlated with susceptibility to apoptosis. Supporting evidence for an anti-apoptotic role for PI3K is provided by the observation that over-expression of PI3K [24,48] or its downstream effector Akt [49,50] is cardioprotective. Several downstream targets of Akt have been recognized as apoptosis regulatory molecules, including the bcl-2-family member BAD [51], procaspase-9 [52], and cAMP-responsive element-binding protein (CREB) [53]. Some of the protective effects are probably mediated at the level of gene expression, as activated Akt localizes to the nucleus to phosphorylate and inhibit Forkhead

transcription factor activity, and reduce the expression of pro-apoptotic genes [54]. The activation of MAPKs in the heart has been demonstrated in vitro as well in vivo [55,56]. Of the MAPK subfamilies that have been studied in the heart, the best characterized is the ERK1/2 cascade. The role of the ERK1/ 2 signalling cascade pathway in the heart has been extensively studied [29,57,58] demonstrating that ERK plays a role in protecting cardiomyocytes against oxidative stress, shifting the balance between cell death and cell survival. Several studies have shown that the ERK pathway may be protective against cell damage induced by oxidative stress [59], in particular, in a model of ischemia/reperfusion in the intact heart, ERK1/2 activation was shown to attenuate the amount of apoptosis subsequent to reperfusion injury [58]. To dissect the role of quercetin in protecting H9c2 cells against oxidative stress injury, PD98059, an ERK pathway inhibitor, and LY294002, an Akt phosphorylation inhibitor, were used in this study. Interestingly, inhibition of both ERK and Akt resulted in a significant reduction of cell viability following quercetin supplementation and oxidative stress exposure. These data are in agreement with the data obtained with the immunoblotting analysis that revealed that quercetin, but not its methylated metabolites, was able to activate both Akt and ERK1/2. In our studies, exposure of H9c2 cells to H2O2 resulted in a marked decrease in Akt phosphorylation, similar to that demonstrated in L6 rat myoblasts [60] while ERK1/2 phosphorylation was unaffected as demonstrated by Turner et al. [25] who showed that peroxide exposure of H9c2 influenced ERK1/2 activation only at short exposure times. Exposure of cells to quercetin, but not 30 -O-methyl quercetin and 40 -O-methyl quercetin, was able to significantly increase Akt and ERK1/2 phosphorylation in comparison to controls in both the absence and presence of oxidative stress. Some Authors reported that Q inhibits Akt and ERK1/2 pathways [21,61], but others presented data indicating both Akt and ERK1/2 phosphorylation [40,62,63]. So, it seems that Q can cause activation of ERK and Akt in some cells, whereas it inhibits them in others. Therefore, activation/inhibition seems to be strictly dependent on the cell type and also on the flavonol concentration and time of treatment. The caspase-3 activity and the DNA fragmentation assays demonstrated that only quercetin was able to prevent H2O2 induced apoptosis. Since the activation of both Akt and ERK1/2 have been demonstrated to prevent stress induced apoptosis, we can hypothesize that the observed quercetin protection against oxidative stress is mainly mediated through its ability to activate Akt and ERK1/2. In this study we have demonstrated that quercetin exerts its protective effects against H2O2 cardiotoxicity via its well know antioxidant activity, and also through the modulation of intracellular signalling pathways. Quercetin but not its two O-methylated derivatives was able to modulate phospho-Akt, phospho-ERK1/2, and caspase-3 activity demonstrating its potential role in counteracting cardiovascular diseases.

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Acknowledgments Supported by MIUR ex 60%, and Fondazione del Monte di Bologna e Ravenna (Italy).

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