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Food Chemistry 141 (2013) 39673976

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Modulation of the antioxidant/pro-oxidant balance, cytotoxicity and antiviral actions of grape seed extracts
Codrut a Ignea a,, Cristina Mihaela Dorobant u b, Christopher Paul Mintoff c, Norica Branza-Nichita b, Michael R. Ladomery d, Panagiotis Kefalas a, Veronica Sanda Chedea a,d,e,
a Department of Food Quality and Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania, Centre International de Hautes Etudes Agronomiques Mditerranennes, Chania, PO Box 85, 73100 Chania, Crete, Greece b Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, Splaiul Independentei 296, Sector 6, Bucuresti 77700, Romania c Peter MacCallum Cancer Centre, St. Andrews Pl, East Melbourne, VIC 3002, Australia d Centre for Research in Bioscience, Faculty of Health and Life Sciences, University of the West of England, Coldharbour Lane, Frenchay, Bristol BS16 1QY, UK e Laboratory of Animal Biology, National Research Development Institute for Animal Biology and Nutrition Balotes ti (IBNA), Calea Bucuresti 1, Balotes ti, Ilfov 077015, Romania

a r t i c l e

i n f o

a b s t r a c t
Grape seed extracts (GSEs) were investigated in yeast cells harbouring defects in their antioxidant system (regarding the cellular growth and growth recovery from H2O2 insult). GSEs antioxidant activity was detected in wild-type and mutant strains Dcta1, Dgsh1 and Doye2glr1, while pro-oxidant activity in Dsod1 cells was seen. Assessment of proliferation of prostate cancer PC3 and HBV-replicating HepG2 2.2.15 cells treated with GSEs has shown higher cytotoxicity of red grape seed extract (RW) than white grape seed extract (WW) subjective to dose and period of administration. No antiviral effect was detected by measuring the secreted virion particles in HepG2 2.2.15 cells treated with GSEs. The GSEs play a dual antioxidant/pro-oxidant role in vivo according with the cellular antioxidant system deciencies and exhibit cytotoxic properties in PC3 and HepG2 2.2.15 cell lines, but no antiviral action against HBV. 2013 Elsevier Ltd. All rights reserved.

Article history: Received 4 March 2013 Received in revised form 10 May 2013 Accepted 20 June 2013 Available online 29 June 2013 Keywords: Antioxidant/prooxidant activity Antiviral action Cytotoxicity Grape seed extracts Yeast

1. Introduction Winemaking by-products are of particular interest because grape is the worlds largest fruit crop, with more than 68 million tons produced per year, of which the European Union produce approximately 24.5 million tons per year in 2010 (FAO STAT Database in http://faostat.fao.org (March. 27th, 2012). Grape seeds are waste products of
Abbreviations: FRAP, ferric reducing antioxidant power; FBS, fetal bovine serum; GAE, gallic acid equivalents; GSE, grape seed extract; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HCC, hepatocellular carcinoma; HBV, human hepatitis B; MAPK, mitogen-activated protein kinase; OYE, old yellow enzymes; GSSG, oxidised glutathione; PI3K, phosphatidylinositide 3-kinase; PCD, programmed cell death; ROS, reactive oxigen species; RW, red grape seed aqueous extract; GSH, reduced glutathione; TP, total polyphenolic content; WW, white grape seed aqueous extract; Dcta1, Dsod1, and Dgsh1, yeast mutants derived from the wild-type (wt) strain BY474, harbouring deletion of the genes CTA1, SOD1, or GSH1 respectively. Corresponding authors. Address: Laboratory of Animal Biology, National Research Development Institute for Animal Biology and Nutrition Balotes ti (IBNA), Calea Bucuresti nr. 1, Balotes ti, Ilfov 077015, Romania. Tel.: +40 21 351 20 81 (V.S. Chedea). Department of Food Quality and Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania, Centre International de Hautes Etudes Agronomiques Mditerranennes, Chania, PO Box 85, 73100 Chania, Crete, Greece. Tel.: +30 28210 35051 (C. Ignea). E-mail addresses: igneacodruta@yahoo.com (C. Ignea), chedea.veronica@ibna.ro (V.S. Chedea). 0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.06.094

the winery and grape juice industry, containing lipid, protein, carbohydrates, and 58% polyphenols, depending on the variety. Grape seed polyphenols contain, aside from phenolic acid precursors (gallic acid), the monomeric avan-3-ols (catechin, epicatechin, gallocatechin, epigallocatechin and epicatechin 3-O-gallate) and procyanidin dimers, trimers, and highly polymerised procyanidins (Chedea et al., 2011). The pharmacological and health benets of grape seed extracts (GSEs) include antioxidant (Chedea, Braicu, & Socaciu, 2010), cardioprotective (Schewe, Sadik, Klotz, Yoshimoto, Khn, & Sies, 2001), hepatoprotective, neuroprotective, anti-inammatory, antidiabetic, anti-carcinogenic, and anti-ageing effects (Nassiri-Asl & Hosseinzadeh, 2009; Xia, Deng, Guo, & Li, 2010). Oxidative stress, as a gap between production of reactive oxygen species (ROS) and cellular antioxidant defence, is a key phenomenon in chronic disorders: diabetes mellilitus, cardiovascular diseases, and cancer (Schewe et al., 2001). The harmful effects of oxidative processes in living organisms can be diminished by the dietary intake of polyphenols like avan-3-ols and procyanidins, present in GSEs (Chedea et al., 2010). Redox cell signalling and the antioxidant potential of various natural compounds have been widely evaluated in vivo using yeast, as an eukaryotic model system (Dani, Bonatto, Salvador, Pereira, Henriques, &, Eleutherio, 2008; Zyracka, Zadrag, Koziol, Krzepilko, Bartosz, &, Bilinski, 2005). This takes advantage of the existing elaborate enzymatic

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redox machinery, consisting of catalases, superoxide dismutases, reductases, peroxiredoxins, glutaredoxins, and glutathione transferases (Herrero, Ros, Belli, & Cabiscol, 2007). Saccharomyces cerevisiae strains lacking the antioxidant machinery mimic the altered intracellular redox circumstances that are frequently encountered in human pathological conditions, and can be used to assess the ability of antioxidant compounds to protect against oxidative damage (Zyracka et al., 2005). The consumption of polyphenol-rich foods exerts valuable outcomes in oxidative stress-related chronic diseases, including some forms of cancer (Surh, 2003). The anticancer effects of dietary polyphenols occur via a variety of mechanisms, such as the inhibition of cell growth proliferation, the modulation of cancer cell signalling and antioxidant enzymatic machinery, the induction of apoptosis and cell cycle arrest or the elimination of carcinogenic agents (Ramos, 2008). Paradoxically, dietary polyphenols may also perform anti-cancer effects as a result of their pro-oxidant activity (Halliwell, 2008; Surh, 2008). Polyphenols are among the 400 compounds that have been listed as potential chemopreventive agents, 40 of these being currently under clinical evaluation (Cimino et al., 2012). To address the antioxidant/pro-oxidant capacity of aqueous GSEs to promote/prevent cell survival from oxidative damage we focused on yeast model systems, to simulate common cellular dysfunctions that occur in oxidative stress using strains compromised in important components of their antioxidant machinery. The cytotoxic effect on tumour cells, the antiviral potential of GSEs and the possible link between these two effects was also investigated. This study is ultimately aimed at increasing the use of wine industry waste through inexpensive processing with a view to exploit the material in nutrition to prevent disease, and even to develop novel pharmacological uses.

2.4. Cell lines The PC3 cell line derived from prostate metastatic androgen independent tumour was purchased from the Health Protection Agency Culture Collections (http://www.hpacultures.org.uk/collections/ecacc.jsp). PC3 cells were seeded at 25% conuence (i.e. 3 105 in a 6-well plate), fed with RPMI medium supplemented with L-glutamine, containing 10% foetal bovine serum (FBS), 50 units/ml penicillin, and 50 lg/ml streptomycin. The hepatocarcinomaderived HepG2 2.2.15 cells (kind gift from Dr. David Durantel, INSERM U871, Lyon, France), stably transfected with two head-to-tail dimers of the Human Hepatitis B (HBV) genome, were grown in RPMI 1640 medium (Euroclone) containing 10% foetal bovine serum (FBS), 50 units/ml penicillin, 50 lg/ml streptomycin and 2 mM Glutamax (Invitrogen), supplemented with 200 lg/ml of G418 (Gibco).

2.5. Growth recovery curves Yeast strains were routinely cultivated at 30 C in YEPD medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose. Single yeast colonies from fresh YEPD plates were used to inoculate starter cultures in 50 ml YEPD broth at A600 = 0.1, which were incubated at 30 C with shaking (160 rpm) for 1418 h. Aliquots were taken at regular intervals and the absorbance was measured. When A600 > 1, aliquots were serially diluted and new measurements were taken. The assay was conducted as previously described (Amari, Fettouche, Samra, Kefalas, Kampranis, &, Makris, 2008).

2.6. Viability colony assay 2. Materials and methods 2.1. Plant material Varieties of red and white grapes cultivated in Greece were used for all seed samples studied (Chedea, Moussouni, Socaciu, &, Kefalas, 2012), red grape seed aqueous extract (RW) and white grape seed aqueous extract (WW). The grapes were harvested at optimum technological maturity. Grape berries were manually deseeded and seeds were mixed, frozen in liquid nitrogen, and stored in the freezer (20 C) until analysed. BY4741 and the mutant Dsod1 strains were freshly grown overnight in rich YEPD medium. Viability colony assays were performed as previously described (Amari et al., 2008). All experiments were performed independently in triplicate. The results were statistically analysed by the GraphPad statistical software.

2.7. Cytotoxicity (MTS) assay PC3 and HepG2.2.2.15 cells were seeded at conuence in 96 wells plates (Greiner) in a nal volume of 100 ll/well culture medium. Wells loaded with 0.1 ll milliQ water were used as negative control. GSEs to the nal concentration of 1 lM GAE, 5 lM GAE, 10 lM GAE, 25 lM GAE, 50 lM GAE and 100 lM GAE were added to each well. Supplemented cells were left to incubate at 37 C for 24 and 48 h. The cell proliferation was assayed using WST-1 reagent (Roche Diagnostics Gmbh, Manheim, Germany). Cells were incubated with 10 ll of WST-1 for 30 min. The measurements were done in triplicate for each sample. Colour change was assayed by measuring the absorbance at 420 nm with a microplate reader (optima BMG labtech). The assay quanties enzymatic activities in metabolically active cells; therefore, dead cells do not contribute to the colour change.

2.2. Polyphenol extraction 5 g of seeds were ground with liquid nitrogen, and extracted for 1 h by adding 40 ml of boiled water to the seed powder. After sedimentation of the large solid particles, the supernatant was centrifuged for 10 min at 1500g, ltered, and then aliquoted and stored at 20 C until further analysis. The total polyphenolic (TP) content was measured by the FolinCiocalteu method and expressed as mg of gallic acid equivalents (GAE)/g seeds (Chedea et al., 2012).

2.3. Yeast strains The wild-type (wt) strain BY4741 and the derived mutants Dcta1, Dsod1, and Dgsh1, harbouring deletion of the genes CTA1, SOD1, or GSH1, respectively, were obtained from Research Genetics. The double-knockout strain Doye2glr1 was as previously described (Odat et al., 2007). The yeast strains employed show defects in free radical-scavenging enzymatic system and glutathione metabolism.

2.8. Quantication of HBV subviral particles secretion by ELISA Aliquots of supernatants from GSE-treated HepG2 2.2.15 cells were analysed for the secreted HBsAg, using the Monolisa HBsAg Ultra Kit (Bio-Rad). The results were registered as ratios of signal to cutoff and were converted to percentages of HBsAg secretion form control, untreated samples.

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2.9. Quantication of HBV viral DNA secretion by real-time PCR Secretion of HBV virions by GSE-treated HepG2 2.2.15 cells was monitored by purication of encapsidated viral DNAs from supernatants using phenolchloroform extraction, as previously described (Lazar et al., 2007). The DNA was quantied using the Corbett Rotor Gene 6000 real-time PCR system and the Maxima SYBR Green qPCR Master Mix (Fermentas). Primers were designed to amplify a 279-bp HBV-specic fragment: HBV3575-3854_for, 50 TCCAGGATCCTCAACAACCAGCACG-30 and HBV3575-3854_rev, 50 TGGCCCCCAATACCACATCATCC-30 . 2.10. Statistical analysis Statistical analysis was performed by using GraphPad Prism six Software. Experimental data was analysed by RM one-way ANOVA. Equal variability of differences was assumed with no correction. Fishers LSD test was used for multiple comparisons of treated samples with untreated-control samples. 3. Results The chemical composition of the GSEs assessed in the present work for their in vivo antioxidant/pro-oxidant activity, cytotoxicity and antiviral actions was characterised in a previous study (Chedea et al., 2012). The polyphenol composition of RW and WW extracts was determined by LCDADMS in ESI+ mode (Chedea et al., 2012). Gallic acid, catechin, epicatechin, procyanidine dimers, trimers, tetramers, pentamers and hexamers were identied. Procyanidin oligomers with a low degree of galloylation were extracted using hot water (Chedea et al., 2012). TP for RW was 40.11 0.90 and for WW 38.05 0.86 mg GAE/g seeds (Chedea et al., 2012). The antioxidant activity of the two extracts was assessed by the ferric reducing antioxidant power (FRAP), as well as by the DPPH radical and luminol chemiluminescence methods. The behaviour in bulk oil medium was evaluated by the Rancimat test (Chedea et al., 2012). 3.1. Effect of GSEs on yeast cell growth and H2O2-induced oxidative stress Antioxidant and pro-oxidant activities of natural (dietary) agents are important health-promoting properties, ensuring protective mechanisms or inducing important detoxifying enzymes (Azam, Hadi, Khan, & Hadi, 2004). Our previous study showed that the oxidation products of polyphenols, either as pure molecules or in GSEs, are formed within the cellular matrix and the extracellular medium; the tested GSEs, considered as an antioxidant nutritive supplement, might have pro-oxidant activity as well, depending on dose, duration of administration and other dietary components (Chedea et al., 2010). To evaluate the capacity of the two GSEs to assist yeast cells growth and protect cells compromised in their enzymatic antioxidant machinery, we used four deletion strains and their isogenic wild-type counterpart in cell growth and growth recovery assays. Cells deleted in superoxide dismutase (Dsod1), catalase A (Dcta1), glutathione synthase (Dgsh1) and double knockout in old yellow enzyme 2 and glutathione reductase 1 (Doye2glr1) were subjected to 1.25 mM H2O2 pro-oxidant insult, which induces programmed cell death (PCD) in a percentage of the population. The cell growth and the growth recovery of the surviving cells in complete minimal media supplemented with low (5 lM GAE) or high (50 lM GAE) antioxidants was monitored for a period of 1218 h. All cell growth and recovery assays were performed independently in triplicate. Statistically signicant differences in cell growth could be seen

starting at 4 h for wild-type and Doye2glr1 cells, at 6 h for the slower growing Dsod1 and Dcta1 cells and 8 h for the very slow growing Dgsh1. The H2O2 stress applied to the yeast cultures appears to last for up to 10 h, as judged by the lag in cell division time compared to untreated cells. Unlike the mutant strains, treatment of the wt strain with GSEs exhibited only a minor inuence on cellular growth (Figs. 1A and 2A). An improved growth rate of wt cells, statistically signicant when compared with untreated cells, was acquired in the presence of 50 lM GAE WW, exhibiting a 12% enhancement over the untreated cells (Fig. 1A). Addition of WW extract determined an increased recovery from the oxidative insult in a dose-dependent fashion, ranging from 6 to 15.5% at 5 and 50 lM GAE, respectively (Fig. 3A). Under H2O2 toxic stress, the wt strain cells attained the same tolerance to the induced injury after administration of 50 lM GAE RW. By contrast, a pro-oxidant effect was observed at low dose (5 lM GAE) RW, statistically signicant, which increased stress by 17% compared to control cells (Fig. 4A). The rst candidate strain carries a deletion in the catalase A gene (Dcta1). This enzyme is responsible for neutralizing H2O2 and is primarily found in the peroxisomal matrix and mitochondria (Petrova, Drescher, Kujumdzieva, & Schmitt, 2004). The Dcta1 mutant strain showed evidence of healthier growth in the presence of both extracts. The overall benet gained from GSE supplementation was found to be dose-independent, around 15% (Figs. 1B and 2B), but only 50 lM GAE WW treatment appeared statistically signicant when compared with untreated cells. Both extracts were highly protective in the compromised deletion strain Dcta1 after H2O2 treatment. A low dose of provided GSEs improved recovery by around 35%, while addition of 50 lM GAE WW and RW rescued Dcta1 cells from oxidative injury by 25% and 38%, respectively as compared to control cells (Figs. 3B and 4B). The antioxidant activity of GSEs in Dcta1 cell was found to be statistically signicant in all treatments. The free radical-scavenging enzyme, superoxide dismutase Sod1 is one of the rst line of cell defence against oxidative injury, catalysing the dismutation of superoxide O2 to H2O2. Deletion of sod1 sensitises cells to pro-oxidant insult, increases endogenous ROS, and causes methionine auxotrophy (Amari et al., 2008). Supplementation of Dsod1 cells with WW did not contributed to cell growth (Fig. 1C), with a statistically signicant inhibition of 10% at 5 lM GAE WW being observed. RW rendered cells more sensitive to endogenous ROS and a dose-independent growth inhibition between 13% and 16% was measured (Fig. 2C). A signicant prooxidant effect, reducing cell recovery in a range of 2226%, was shown in H2O2-treated Dsod1 cells incubated with WW (Fig. 3C). The same effect was monitored for RW, which inhibited cell growth recovery in a dose-depended manner by 11% at 5 lM GAE RW and 28% at 50 lM GAE RW (Fig. 4C). Overall the observed growth inhibition and pro-oxidant effect generated by GSEs in Dsod1 cells were statistically signicant by comparison with untreated Dsod1 cells. According to these ndings, the protection provided by GSEs against H2O2 insult requires the cytosolic superoxide dismutase, since the Dsod1 strain was not able to acquire tolerance when treated with GSEs. Glutathione reductase is an enzyme responsible for recycling oxidised glutathione back to the reduced form. Old yellow enzymes (OYE) have been implicated in modulating oxidative stress responses by altering actin polymerisation (Odat et al., 2007). Deletion of glr1 and oye2 causes cellular sensitivity to oxidative stress and H2O2-induced programmed cell death (PCD), respectively (Odat et al., 2007). The double-knockout strain Doye2glr1, exhibits a radically altered ratio of reduced glutathione to oxidised glutathione (GSH/GSSG) in favour of the latter, higher than single mutant Dglr1 cells (Odat et al., 2007). Growth enhancement, varying from 15% to 18%, was monitored for the Doye2glr1 cells in the presence

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Figure 1. Effect of white grape seeds extract (WW) on yeast cell growth. BY4741 wild type-cells (A), Dcta1 cells (B), Dsod1 cells (C), Doye2glr1 cells (D), and Dgsh1 cells (E) were assessed untreated (N). Cells were supplemented with WW at 5 lM GAE (h) and 50 lM GAE (j). Growth was monitored by measuring OD600 every 2 h. Statistically signicant changes in growth assays conducted in triplicate are observed for multiple comparisons of treated samples with untreated-control samples: wt 5 lM P = 0.0673 (ns), 50 lM P = 0.0005 (); Dcta1 5 lM P = 0.0269 (), 50 lM P = 0.1230 (ns); Dsod1 5 lM P = 0.0116 (), 50 lM P = 0.4929 (ns); Doye2glr1 5 lM P = 0.0945 (ns), 50 lM P = 0.0348 (); Dgsh1 5 lM P = 0.1071 (ns), 50 lM P = 0.1513 (ns).

of WW (Fig. 1D) and a favourable, dose-independent, effect of 26% was seen after RW supplementation (Fig. 2D), being statistically signicant for both RW treatments and 50 lM GAE WW treatment. The generated damage of Doye2glr1 cell exposure to H2O2 oxidative stress was more efciently counteracted by addition of 5 lM GAE WW and 50 lM GAE RW, by 14% and 20% respectively (Figs. 3D and 4D), only the last being statistically signicant when compared with untreated cells. Other tested doses did not affect cell recovery. The gene GSH1 encondes a c-glutamyl cisteine synthetase catalysing the rst step in glutathione biosynthesis. Yeast cells harbouring a gsh1 deletion are unable to grow in CM media unless extensively supplemented with GSH. Medium supplementation with 100 nM GSH enables Dgsh1 cells to grow at a lower rate (Amari et al., 2008). The capacity of GSEs to improve the growth properties and recover Dgsh1 cells from oxidative injury was subsequently assayed. Supplementation with WW brought a minor improvement in growth properties of Dgsh1 cells ranging from 7 to 9% (Fig. 1E), while RW addition shown a constant benet of 8% in cell growth (Fig. 2E) of which only the treatment with

5 lM GAE RW was statistically signicant when compared with untreated cells. The antioxidant ability of WW was accomplished by increasing the tolerance of Dgsh1 cells to H2O2 insult by 11% at 5 lM GAE and 29% at 50 lM GAE (Fig. 3E). Peroxide stress was more aggressive in Dgsh1 cells supplemented with a low concentration of RW, a minor cell recovery of 5% being monitored, but a signicant protective effect of 22% was evident at 50 lM GAE RW (Fig. 4E). Antioxidant activity in Dgsh1 was found statistically signicant only after treatment with high concentration of GSEs. To evaluate the comparability of the growth recovery assay data with the frequently employed viability colony assay, wild-type and Dsod1 strains were selected as reference and highly responsive, respectively. Cells were grown to midlog phase at OD600 = 0.5 in glucose CM media. Fresh media containing 50 lM GSEs was inoculated with aliquots of cells. After addition of 1.5 mM H2O2, cells were incubated at 30 C for 2 h. The numbers of viable cells were counted prior and subsequent to H2O2 treatment. As seen in Fig. 5A and B the percentage of surviving colonies after H2O2 treatment was reduced by more than half to an average of 41% for wildtype cells and 38% for Dsod1 cells. Supplementation with GSEs was

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Figure 2. Effect of red grape seeds extract (RW) on yeast cell growth. BY4741 wild type-cells (A), Dcta1 cells (B), Dsod1 cells (C), Doye2glr1 cells (D), and Dgsh1 cells (E) were assessed untreated (N). Cells were supplemented with RW at 5 lM GAE (h) and 50 lM GAE (j). Growth was monitored by measuring OD600 every 2 h. Statistically signicant changes in growth assays conducted in triplicate are observed for multiple comparisons of treated samples with untreated-control samples: wt 5 lM P = 0.9728 (ns), 50 lM P = 0.1316 (ns); Dcta1 5 lM P = 0.0018 (), 50 lM P = 0.0049 (); Dsod1 5 lM P = 0.0013 (), 50 lM P = 0.0004 (); Doye2glr1 5 lM P = 0.0002 (), 50 lM P = 0.0004 (); Dgsh1 5 lM P = 0.0307 (), 50 lM P = 0.1802 (ns).

benecial to cell survival for the wild type strain which increased to 67% for WW and 58% for RW. Treatment with both extracts resulted in a signicant reduction in viability of H2O2 treated Dsod1 cells, between 15% and 20%. The effects of supplementation were statistically signicant in all cases and the results of colony viability assays are highly comparable to the corresponding growth recovery assays (Fig 5). 3.2. Cytotoxicity assessment of PC3 and HepG2.2.2.15 cells treated with GSEs There is a growing body of evidence suggesting a correlation of oxidative stress with pathological conditions including various forms of cancer, such as those from the prostate, liver, gut, skin, and so forth (Kumar, Koul, Khandrika, Meacham, & Koul, 2008; Stehbens, 2004). Cell proliferation plays a central role in the development of severe diseases, and it has been shown that the use of specic natural (dietary) or synthetic agents may prevent, delay, or slow down this process. Prostate cancer is a model target disease

for the evaluation of chemopreventive properties of natural products, due to high incidence, long latency and identiable preneoplastic lesions (Cimino et al., 2012). Chronic infection with hepatitis HBV causes hepatocellular carcinoma (HCC), one of the most common cancers worldwide. Phenolic antioxidants have been identied as HCC chemopreventive agents, which induce cytoprotective genes (Nair et al., 2006). In this context, the present study aimed to also determine the cytotoxic activity of GSEs against two different cell lines, PC3 which is an in vitro model for prostate cancer and HepG2.2.2.15, which is a liver tumour-derived cell line, replicating high amounts of HBV (Sells, Chen, & Acs 1987). To this end, we employed the MTS assay as a measure of cell viability, this assay being routinely used to assess the chemosensitivity of tumour cells by measuring the inhibition of proliferation. The human prostate carcinoma PC3 cell line is widely investigated in drug resistance studies (Kumar et al., 2008). Untreated cells were considered to exhibit 100% viability. Fig 6A and B presents the percentage of inhibition or stimulation of cell

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Figure 3. Effect of white grape seed extract (WW) on growth in H2O2-treated cells. BY4741 wild type-cells (A), Dcta1 cells (B), Dsod1 cells (C), Doye2glr1 cells (D), and Dgsh1 cells (E) were treated with 1.25 mM H2O2 (N). Cells were supplemented with WW at 5 lM GAE (h) and 50 lM GAE (j). Growth was monitored by measuring OD600 every 2 h. Statistically signicant changes in growth assays conducted in triplicate are observed for multiple comparisons of treated samples with untreated-control samples: wt 5 lM P = 0.2088 (ns), 50 lM P = 0.0156 (); Dcta1 5 lM P = 0.0054 (), 50 lM P = 0.0405 (); Dsod1 5 lM P = 0.0260 (), 50 lM P = 0.0037 (); Doye2glr1 5 lM P = 0.2708 (ns), 50 lM P = 0.9674 (ns); Dgsh1 5 lM P = 0.3186 (ns), 50 lM P = 0.0154 ().

proliferation, measured by the MTS reduction to formazan, for cells co-incubated with GSEs. The proliferation rate of PC3 cells treated with 1 lM GAE WW was about 70% after 24 h incubation and 80% after 48 h incubation, compared to control. The cytotoxic effect increased after treatment with 5 lM GAE WW, to only 60% cell proliferation after 24 h incubation and 50% after 48 h. No response to treatment was observed for PC3 cells incubated with 10 lM GAE WW for 24 h, but signicant cytotoxic effect was monitored after 48 h (more than 60% inhibition of cell proliferation). Addition of increased concentrations of 25 and 50 lM GAE WW resulted in 80% cell proliferation after 24 h incubation, while at 100 lM GAE WW, 60% cell proliferation was monitored. The cytotoxic effect of WW at higher doses was constantly stronger after 48 h incubation, resulting in 50% cell proliferation. Together, the toxicity prole of WW exhibited the highest activity on PC3 tumour cells at doses between 10 and 25 lM GAE, after a longer incubation time (48 h) (Fig 6A). In the case of RW treatment of PC3 tumour cells the duration of incubation inuences the cytotoxic effect at concentrations of 1 lM GAE, 25 lM GAE and slightly at 100 lM GAE. Generally, the cell proliferation occurs at half rate compared with untreated

cells, but very strong cytotoxicity was observed in cells treated with 100 lM GAE RW especially after 24 h of incubation, when only 10% of cell proliferation occurred (Fig. 6B). The cytotoxicity of GSEs was also assessed on the HepG2 2.2.15 cell line supporting replication, but also assembly and secretion of both subviral and infectious HBV particles. The results, shown in Fig. 6C and D, indicate a similar cytotoxicity pattern following treatment of HepG2 2.2.15 cells with both extracts, wth cell proliferation being slightly stimulated at 10 lM GAE and decreasing proportionally with increased concentrations. The highest cytotoxicity was observed at 100 lM GAE GSEs, independent of the incubation period, the treatment resulting in reduced proliferation rate, to around 40% to 60% from that of control. 3.3. Anti HBV activity of GSEs The HepG2 2.2.15 cell line is a useful system to assess the antiviral activity of GSEs due to the capacity to produced and secrete HBV DNA, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in supernatants. The HBsAg secretion was

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Figure 4. Effect of red grape seed extract (RW) on growth in H2O2-treated cells. BY4741 wild type-cells (A), Dcta1 cells (B), Dsod1 cells (C), Doye2glr1 cells (D), and Dgsh1 cells (E) were treated with 1.25 mM H2O2 (N). Cells were supplemented with RW at 5 lM GAE (h) and 50 lM GAE (j). Growth was monitored by measuring OD600 every 2 h. Statistically signicant changes in growth assays conducted in triplicate are observed for multiple comparisons of treated samples with untreated-control samples: wt 5 lM P = 0.0457 (), 50 lM P = 0.8239 (ns); Dcta1 5 lM P = 0.0045 (), 50 lM P = 0.0007 (); Dsod1 5 lM P = 0.6696 (ns), 50 lM P = 0.0008 (); Doye2glr1 5 lM P = 0.5871 (ns), 50 lM P = 0.0189 (); Dgsh1 5 lM P = 0.7882 (ns), 50 lM P = 0.0414 ().

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Figure 5. Viability colony assay in H2O2-treated cells suplemented with GSEs. Wild type (A) and Dsod1 (B) cells grown at midlog phase were used to inoculate fresh media supplemented with 50 lM GAE GSEs. Cells were immediately challenged with 1.5 mM H2O2 and incubated for 2 h. Viable cells were counted prior and subsequent to treatment by plating dilutions on YPD plates. Statistically signicant changes in viability colony assays conducted in triplicate are observed WW: wt P = 0.001 (), Dsod1 P = 0.0007 (); RW: wt P = 0.0005 (), Dsod1 P = 0.0004 ().

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Figure 6. Assessment of cell proliferation by the MTS assay on tumour PC3 and HepG2 2.2.15 cells and the antiviral activity of GSEs against HBV. PC3 cells were treated with WW (A) and RW (B) and on HepG2 2.2.15 infected cells treated with WW (C) and RW (D) incubated for 24 h (j) and 48 h (N). Statistically signicant changes in MTS assays conducted in triplicate are observed WW: PC3 P = 0.006 (), HepG2 2.2.15 P = 0.007 (); RW: PC3 P = 0.0001 (), HepG2 2.2.15 P = 0.001 (). ELISA assay of HBsAg secretion (E) and HBV viral DNA quantication by RT-PCR (F) in GSE-adapted HepG2 2.2.15 cells after 72 h of incubation. All experiments were performed independently in triplicate.

determined by ELISA (Fig. 6E) and quantication of viral DNA secretion was performed by real-time PCR (Fig. 6F). As shown in Fig. 6E, HBsAg secretion was not affected in cells treated with WW, and only a minor inhibition was detected in cells treated with 15 lM GAE RW. The amount of viral DNA measured by real-time PCR in the cell supernatant did not change between the control and supplemented samples, indicating that neither replication, nor assembly or trafcking of the virions was affected by the treatment (Fig. 6F). 4. Discussion The antioxidant potential of GSEs has been investigated through in vitro analysis (Chedea. et al., 2012), although previous

studies have shown that polyphenols are extensively metabolized in vivo, resulting in signicant alteration of their redox potential (Amari et al., 2008). Therefore, it is essential to screen the efcacy of these compounds in vivo. The physiological role of dietary antioxidants depends on their absorption and biotransformation mechanisms. In this study, the action of GSEs was assessed both in vivo, using yeast as a model organism and in vitro in two different biological systems: prostate cancer cells and HBVreplicating hepatoma cells. The active role of GSEs in oxidative stress or cell proliferation is thus supported by in vivo and in vitro studies. Despite the limitations inherent to the use of such a simple single cell experimental model, S. cerevisiae is very useful as a rst screening tool to investigate oxidative stress at the cellular

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level. This microorganism conserves not only most enzymatic equipment and cellular structures, but also the redox balance and the response to oxidative stress specic to higher eukaryotes. The defects in the antioxidant yeast system studied here are linked to several human diseases, such as familial amyotrophic lateral sclerosis associated with mutations of Sod1 (Rosen et al., 1993), ageing and Parkinsons disease, connected with glutathione homeostases (Patsoukis et al., 2005), or glutathionylation of actin mediated by Oye2 protein (Odat et al., 2007). The yeast cell growth and growth recovery were the assays of choice, as populations of yeast cells can be maintained in chemically controlled media supplemented with various doses of GSEs during the whole assay period, which closely resembles a real life setting. This enables testing concentrations of GSEs at physiologically relevant levels that the organisms encounter (5 lM GAE low dose and 50 lM GAE high dose), especially when the yeast efux system is taken into consideration. Our results clearly emphasise the role of the GSEs supplementation in yeast cell growth and growth recovery assays, which can vary from antioxidant to prooxidant depending on the cellular antioxidant machinery deciency. Incubation period and GSE concentration have a limited contribution to their antioxidant/pro-oxidant activity. Growth augmentation upon GSE supplementation in cells exhibiting deciency of catalase activity or glutathione level could indicate a potential role in controlling intracellular peroxide concentration and avoiding oxidative damage to membranes (Haliwell, 2006) or a possible involvement in the GSH metabolic pathway. GSEs operated as prooxidant agents in cells lacking superoxide dismutase activity which could comply to the common mechanism for anti-cancer and chemoprotective properties of plant polyphenols. The mutant decient strain Dcta1 treated with GSEs exhibited the highest capacity of recovery from H2O2 damage, indicating that GSEs can either succesfully replace Cta1 activity or trigger the super expression of other antioxidant systems as a form of compensation (Zyracka et al., 2005). In the absence of cytoplasmatic superoxide dismutase, the level of ROS produced was elevated, toxicity rising up by 2.5-fold in a dose-dependent fashion. Sod1 contributes essentially to the elimination of ROS achieved by GSEs under H2O2 stress. Chemoprevention is a promising approach to reducing the incidence of cancer. The two GSEs investigated in the present study, exhibited statistically signicant cytotoxic activity against two human tumour-derived cell lines, prostate cancer PC3 and hepatomaderived HepG2 2.2.15 cells. Generally, higher concentration of extracts increased cytotoxicity, with the most convincing effect appearing in PC3 cells treated with RW. The period of administration clearly inuences the inhibition of cell proliferation in HepG2 2.2.15 infected cells, the cytotoxicity increasing after longer incubation. No antiviral effect was observed by measuring the secreted virion particles in HepG2 2.2.15 cells treated with GSEs. Nevertheless, it would be very interesting to further investigate whether the HBeAg secretion in HepG2 2.2.15 cells is affected by GSEs treatment and to which extent the secreted HBV virions are still infectious. While there has been a major focus on the antioxidant properties of natural products, there is an emerging view that these compounds, and their metabolites, may affect signalling pathways that modulate several cell responses (Cimino et al., 2012), like mitogenactivated protein kinase (MAPK) and phosphatidylinositide 3-kinase (PI3K) pathways involved in cancer cell proliferation, or caspase pathway that leads to apoptosis. Dietary agents, being extractive and not synthetic products, may elicit a great variability of therapeutic consequences.

5. Conclusions The action of GSEs was explored in the present study. Taken together, the antioxidant yeast system deciencies affect the capacity of GSEs in vivo to sustain cellular growth and to protect cell viability from oxidative damage, while insights of the cytotoxic properties of GSEs in PC3 and HepG2 2.2.15 cell lines, but no antiviral action against HBV, were revealed. However, future work is necessary to fully understand these effects and the mechanisms by which they protect the organisms against various injuries. Acknowledgements We thank Dr. Antonios Makris for providing Doye2glr1 yeast strain. Dr Veronica S. Chedea was supported by a Royal Society International Travel Grant (reference TG092176). References
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