Báo cáo khoa học: The importance of polymerization and galloylation for the antiproliferative properties of procyanidin-rich natural extracts doc

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The importance of polymerization and galloylation forthe antiproliferative properties of procyanidin-richnatural extractsD. Lizarraga1, C. Lozano2, J. J. Briede´3, J. H. van Delft3, S. Tourin˜o2, J. J. Centelles1,J. L. Torres2and M. Cascante1,21 Biochemistry and Molecular Biology Department, Biology Faculty, University of Barcelona, Biomedicine Institute from University of Barcelona(IBUB) and Centre for Research in Theoretical Chemistry, Scientific Park of Barcelona (CeRQT-PCB), Associated Unit to CSIC, Spain2 Institute for Chemical and Environmental Research (IIQAB-CSIC), Barcelona, Spain3 Department of Health Risk Analysis and Toxicology, Maastricht University, the NetherlandsColorectal cancer is the third most commonly diagnosedcancer in the world and is one of the major causes ofcancer-associated mortality in the USA [1,2]. Epidemio-logical studies indicate that colon cancer incidence isinversely related to the consumption of fruit, vegetablesand green tea [3,4]. Specifically, the imbalance betweenhigh-level oxidant exposure and antioxidant capacity inthe colon has been linked to increased cancer risk andis strongly influenced by dietary antioxidants [5–7].Several studies have demonstrated that polyphenoliccompounds are capable of providing protection againstcancer initiation and its subsequent development [8–11].A variety of health-promoting products obtainedfrom grape seeds and skins, tea leaves, pine and otherplant byproducts are currently available and a greatdeal of research is being devoted to testing the putativebeneficial effect of these products in relation to theirpolyphenolic content [12–16]. Catechins and their poly-meric forms (proanthocyanidins) are being studied inparticular depth. The composition of monomeric cate-chins and their oligomers and polymers (proantho-cyanidins), as well as the percentage of galloylatedspecies in these natural extracts, differs between tea,grape and pine bark.The antiproliferative activity of catechins and pro-anthocyanidins is associated with their ability to inhi-bit cell proliferation and to induce cell cycle arrest andapoptosis [17,18]. Most of the polyphenols in tea aremonomers of gallocatechins and their gallates [19],whereas grape contains monomers and oligomers ofKeywordsantiproliferative; apoptosis; cell cycle; coloncancer; scavenger capacityCorrespondenceM. Cascante Serratosa, Department ofBiochemistry and Molecular Biology,University of Barcelona, Biology Faculty,Av. Diagonal 645, 08028 Barcelona, SpainFax: +34 934021219Tel: +34 934021593E-mail: martacascante@ub.edu(Received 2 May 2007, revised 3 July 2007,accepted 18 July 2007)doi:10.1111/j.1742-4658.2007.06010.xGrape (Vitis vinifera) and pine (Pinus pinaster) bark extracts are widelyused as nutritional supplements. Procyanidin-rich fractions from grape andpine bark extract showing different mean degrees of polymerization, per-centage of galloylation (percentage of gallate esters) and reactive oxygenspecies-scavenging capacity were tested on HT29 human colon cancer cells.We observed that the most efficient fractions in inhibiting cell proliferation,arresting the cell cycle in G2phase and inducing apoptosis were the grapefractions with the highest percentage of galloylation and mean degree ofpolymerization. Additionally, the antiproliferative effects of grape fractionswere consistent with their oxygen radical-scavenging capacity and theirability to trigger DNA condensation–fragmentation.AbbreviationsDMPO, 5,5-dimethyl-1-pyrolline-N-oxide; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; MTT,3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide; PI, propidium iodide.4802 FEBS Journal 274 (2007) 4802–4811 ª 2007 The Authors Journal compilation ª 2007 FEBScatechins with some galloylation and mainly poly-merized procyanidins [20]. In contrast, procyanidinfractions from pine bark extracts do not contain gallo-catechins or gallates.The influence of polyphenolic structure on antioxi-dant activity, protective capacity and, particularly, onthe mechanism of action remains open to debate andfurther study is required. Research with different celllines has shown that the most widely studied naturalpolyphenol, epigallocatechin-3-gallate from green tea, isa potent antioxidant and chemopreventive agent [21,22].These and other results suggest that the galloylation ofcatechins and the presence of gallocatechin moieties innatural extracts could be important chemical character-istics. They may be useful indicators in evaluating thepotential of natural plant extracts for colon cancer pre-vention or treatment and the degree of polymerizationrelated to the bioavailability in the colon.Procyanidins and monomeric catechins (Fig. 1) arethe main active polyphenols in grape and pine bark.The difference between grape and pine catechins andprocyanidins is found in the presence of gallate estersin position 3 (galloylation). Whereas grape flavanolsare galloylated to some extent [23,24], pine barkappears to be devoid of gallate esters [25,26]. It hasbeen reported that oligomeric procyanidins are not sig-nificantly absorbed in the intestinal tract, and reachthe colon mainly intact [27]. They are therefore bio-available to the epithelial cells in the intestinal wall,where procyanidins and other phenolics are extensivelydegraded, metabolized and absorbed. In a first stage,the oligomers are depolymerized and the constitutivecatechin units are partially absorbed as glucuronates,sulfates and methyl esthers, as described for the smallintestine [28]. They are also, in part, extensively metab-olized to phenolic acids such as 3-hydroxyphenylvalericacid and 3-hydroxyphenylpropionic acid, which arethen absorbed as glucuronates and sulfates [27,29].The gallate esters are more stable than the simple cate-chins upon being metabolized [30] and may be morebioavailable in the colon. Gallates have been reportedto inhibit cell growth, trigger cell cycle arrest in tumorcell lines and induce apoptosis [31,32]. Furthermore,studies have shown that they also offer protection byscavenging reactive oxygen species such as superoxideanion, hydrogen peroxide and hydroxyl radicals, whichcause destruction of biochemical components that areimportant in physiological metabolism [33,34]. Thiscapacity to prevent the imbalance between high-leveloxidant exposure and antioxidant capacity, whichleads to several pathological processes, may contributeto the chemopreventive effect of the gallic acid deriva-tives. Because grape is a rich source of procyanidinsand contains some galloylation, procyanidin fractionsfrom grape could be potential antiproliferative com-pounds of interest in the prevention of colon cancer.In the present study, we investigated the relationshipof different structural factors of procyanidins, such asthe mean degree of polymerization and percentage ofgalloylation, with their antiproliferative potential andtheir scavenging capacity for hydroxyl and superoxideanion radicals.Results and DiscussionGrowth inhibition capacityTable 1 shows that pine bark extracts containingoligomers (XIP, VIIIP, IVP, VIP and OWP) reducedproliferation of the carcinoma cell line HT29 dose-dependently with IC50values between 100 and 200 lmand IC80values between 200 and 300 lm, whereas theIC50and IC80values of fraction VP containing mono-mers were almost one order of magnitude higher (1551and 2335 lm, respectively). If we consider that the pineFig. 1. Structure of the major polyphenols found in white grapepomace.D. Lizarraga et al. Antiproliferative properties of natural extractsFEBS Journal 274 (2007) 4802–4811 ª 2007 The Authors Journal compilation ª 2007 FEBS 4803fractions are not galloylated, it can clearly be con-cluded that oligomers are much more efficient thanmonomers at inhibiting colon carcinoma cell prolifera-tion.Under the same experimental conditions, the grapepolyphenolic fractions with an equivalent degree ofpolymerization but also with a percentage of galloyla-tion ‡ 15% (VIIIG, IVG, VIG and OWG) producedIC50and IC80values that were approximately halfthose of the homologous pine fractions. Moreover, aswas observed for pine fractions, the grape oligomerswere much more efficient than the monomers.These results clearly show that both polymerizationand galloylation enhance the antiproliferative capacityof polyphenolic fractions, which suggests that naturalpolyphenolic extracts with a high degree of galloyla-tion and containing oligomers are more suitable aspotential antiproliferative agents than those containingmonomers.Cell cycle analysisTo examine the effects of grape and pine fractions onthe cell cycle pattern at concentrations equal to theirIC50and IC80values (Table 1), HT29 cells were treatedwith each fraction for 72 h and then analyzed with afluorescence-activated cell sorter (FACS) (Fig. 2). Thecell cycle distribution pattern induced after grape poly-phenolic treatments showed that, at IC50, the fractionswith the highest mean degree of polymerization andpercentage of galloylation (VIIIG and IVG) induced aG2-phase cell cycle arrest, whereas the rest of the frac-tions did not have a significant effect on the cell cycledistribution. At IC80, the G2-phase arrest induced byfractions VIIIG and IVG was enhanced, and fractionVIG displayed a significant effect (Fig. 2A). FractionVIG is chemically classified in Table 1 as having thethird highest mean degree of polymerization andgalloylation, situated below fractions VIIIG and IVG,respectively.To determine whether galloylation was required toinduce the G2-phase arrest, we also examined the non-galloylated pine fractions with high mean degrees ofpolymerization (VIIIP and IVP) and observed thatthey also induced a G2-phase arrest at their respectiveIC50values (Fig. 2B). These results showed that pro-cyanidin polymerization plays a more important rolethan galloylation in cell cycle arrest.Apoptosis inductionHT29 cell incubations with polyphenolic fractionswere performed at the concentrations described inExperimental procedures. As show in Fig. 3A, atIC50, the grape polyphenolic fractions VIIIG andIVG induced significant percentages of apoptosis inHT29 cells (approximately 25% and 17%, respec-tively) as measured by FACS analysis. Fraction VI-IIG also induced a significant percentage of necrosis(approximately 5%), which could be due to a pro-oxidant effect at high concentration [35,36]. More-over, this percentage is negligible in comparison tothe apoptotic effect induced by fraction VIIIG onHT29 cells. At a concentration equal to IC80, frac-tions VIIIG and IVG induced significant percentagesof apoptosis in HT29 cells (approximately 24% and18%, respectively) and fraction VIG also displayeda significant effect (approximately 22%) (Fig. 3A).Fraction VIG is chemically classified in Table 1 ashaving the third highest mean degree of polymeriza-tion and galloylation, situated below fractions VIIIGand IVG, respectively.The pine fractions VIIIP and IVP were analyzedto determine whether galloylation enhanced the apop-totic induction observed; a significant percentage ofapoptosis was induced, but the percentages wereTable 1. Comparative chemical characteristics and HT29 cell growth inhibition of grape and pine fractions. Percentage of galloylation (%G),mean degree of polymerization (mDP) and mean relative molecular mass (mMr) from Torres et al. [50] and Tourin˜o et al. [26].Fraction %G mDP mMrIC50(lM)IC80(lM)Grape VIIIG 34 3.4 1160 55 ± 3 76 ± 3IVG 25 2.7 880 67 ± 3 100 ± 3VIG 16 2.4 751 56 ± 7 113 ± 7OWG 15 1.7 552 99 ± 18 134 ± 18VG 0 1 290 410 ± 10 483 ± 10Pine XIP 0 3.4 999 108 ± 4 308 ± 4VIIIP 0 3 876 123 ± 6 199 ± 6IVP 0 2.9 833 127 ± 6 204 ± 6VIP 0 2.7 777 143 ± 7 230 ± 7OWP 0 2.1 601 190 ± 5 305 ± 5VP 0 1 290 1551 ± 14 2335 ± 14Antiproliferative properties of natural extracts D. Lizarraga et al.4804 FEBS Journal 274 (2007) 4802–4811 ª 2007 The Authors Journal compilation ª 2007 FEBSlower than those induced by the grape fractions(Fig. 3B).These results show that galloylation plays a moreimportant role than polymerization in apoptosis induc-tion. Next, apoptosis induction by the two most highlygalloylated and polymerized fractions (VIIIG andIVG) was analyzed by Hoescht staining, whichrevealed early membrane alterations at the beginningof the apoptotic process. Chromatin condensation wasalso seen, and confirmed the induction of apoptosis byfractions VIIIG and IVG (Fig. 4A). Finally, DNAfragmentation was detected as a late marker of apop-tosis by observing the pattern of DNA laddering atIC50and IC80(Fig. 4B).Oxygen radical scavenging activity as detectedby ESR spectroscopyThe next series of experiments used ESR spectroscopyto test the radical-scavenging capacity of the fractions.The results show that the oligomeric fractions (VIIIG,IVG, VIIIP and IVP), which were the most effective inthe previous assays using HT29 cells, were also themost efficient as hydroxyl radical and superoxide scav-engers at 50 lm (Fig. 5A). Fraction VIIIG was themost potent radical scavenger, followed by fractionIVG and the pine fractions VIIIP and IVP. The samelevels of efficiency were also observed in the inductionof cell cycle arrest and apoptosis. When fractions weretested at their respective IC50values, fractions VIIIG,IVG, VIIIP and IVP were again the most effective(Fig. 5B). There is a clear relationship between highscavenger capacity ⁄ lower IC50and a high level ofapoptosis induction. Grape fractions proved to bemore potent scavengers than pine fractions in bothradical generation systems. The apparent high effi-ciencies detected for the monomers (VG and VP) canbe largely attributed to the high concentrations used(410 lm and 1551 lm, respectively).Interestingly, the efficiencies observed for grape oligo-meric fractions, which proved to be better apoptoticinducers and better ROS scavengers than pine oligo-meric fractions, are apparently related to the degree ofgalloylation and are enhanced by the polymerizationof the fractions. Hydroxyl radical (OH) is the mostreactive product of reactive oxygen species formed bysuccessive one-electron reductions of molecular oxygen(O2) in cell metabolism, is primarily responsible for theCell cycle at IC50 (Grape fraction)ctctctctctctVIIIGVIIIGVIIIGVIIIGVIIIGVIIIGG1SG2 G1SG2IEC-6 IEC-18Cell cycle at IC50 (Grape fractions)ABC0 10203040506070ctVIIIGIVGVIGOWGVGctVIIIGIVGVIGOWGVGctVIIIGIVGVIGOWGVGG1 S G2G1 S G2Cell cycle stagesCell cycle stagesCell cycle stagesCell cycle stages% Cell distribution (HT29) 0 10203040506070% Cell distribution (HT29) 0 10203040506070% Cell distribution (HT29) 0 10203040506070% Cell distribution * ******Cell cycle at IC50 (Pine fractions)ctctIVPctVIIIPVIIIPVIIIPIVPIVPG1SG2****Cell cycle at IC80 (Grape fractions)ctVIIIGIVGVIGOWGVGctVIIIGIVGVIGOWGVGctVIIIGIVGVIGOWGVG********Fig. 2. Cell cycle analysis of HT29, IEC-6 and IEC-18 cells treated with grape and pine polyphenolic fractions. (A) HT29 cells at their respec-tive grape IC50and IC80values. (B) HT29 cells at pine IC50. (C) IEC-6 and IEC-18 cells treated with grape fraction VIIIG at HT29 IC50. Percent-ages of cells in different cell stages are shown. Cell phases analyzed: G1, S and G2(% cells ± SEM, *P < 0.05, **P < 0.001). Experimentswere performed in triplicate.D. Lizarraga et al. Antiproliferative properties of natural extractsFEBS Journal 274 (2007) 4802–4811 ª 2007 The Authors Journal compilation ª 2007 FEBS 4805cytotoxic effects observed in aerobic organisms frombacteria to plants and animals, and has been identifiedas playing a role in the development of many humancancers [37,38].Cancer chemoprevention conducted by administeringchemical and dietary components to interrupt the initi-ation, promotion and progression of tumors is consid-ered to be a new and promising approach in cancerprevention [39–41]. However, the development of effec-tive and safe agents for the prevention and treatmentof cancer remains inefficient and costly, and falls shortof the requirements for primary prevention among thehigh-risk population and for prevention in cancer sur-vivors [42].In recent years, many popular, polyphenol-enricheddietary supplements have been commercialized, such astea catechins, grape seed proanthocyanidins and othernatural antioxidant extracts, each of which has beenclaimed to exert chemopreventive activity in cellularmodels of cancer [43,44]. Recent publications have sta-ted that the antiproliferative activity of flavonoids isdependent on particular structure motifs, such as gal-late groups and degree of polymerization [45,46].Our results suggest that polymerization plays agreater role than galloylation in cell cycle arrest inHT29 cells. Interestingly, galloylation appears to bemore influential than polymerization in the biologicalapoptosis activities tested and in the hydroxyl andsuperoxide anion radical-scavenging capacity of thefractions when compared at the same concentration of50 lm (Fig. 5A). The galloylated and polymerizedgrape procyanidins were the most effective hydroxylradical scavengers and also triggered cell cycle arrestand apoptosis, and although this does not necessarilyindicate that both effects are mechanistically related,such as relationship cannot be ruled out. The presentresults are in general agreement with previouslyreported data for pure compounds [47]. Essentially, theinduction of apoptosis seems to be related to the elec-tron transfer capacity of the phenolic extracts. Otherantioxidants with anti-inflammatory and anticanceractivities have been reported, such as edaravone [48]and the flavonoid silydianin [49], both of which induceapoptosis and act as radical scavengers.It was also observed that the most efficient procyani-din fraction, VIIIG, which induced approximatelyApoptosis at IC50 (Pine fractions)ctVIIIPIVPctVIIIPIVPctVIIIPIVPEarly Late NecroticCell stage * *Apoptosis at IC50 (Grape fractions)ABC05101520ctVIIIGIVGVIGOWGVGctVIIIGIVGVIGOWGVGctVIIIGIVGVIGOWGVGEarly Late NecroticCell stage% Cell distribution (HT29) 05101520% Cell distribution (HT29) 05101520% Cell distribution (HT29) 05101520% Cell distribution ** * * * *Apoptosis at IC80 (Grape fractions)ctVIIIGIVGVIGOWGVGctVIIIGIVGVIGOWGVGctVIIIGIVGVIGOWGVGEarly Late NecroticCell stage* ** * *Apoptosis at IC50 (Grape fraction)ctVIIIGctVIIIGctVIIIGctVIIIGctVIIIGctVIIIGEarly Late Necrotic Early Late NecroticIEC-6 IEC-18Cell stage*Fig. 3. Apoptosis was induced in HT29 tumor cells and did not affect normal epithelial cells. (A) HT29 cells after treatment with grape poly-phenolic fractions at their respective IC50and IC80values. (B) HT29 cells after treatment with pine polyphenolic fractions at their respectiveIC50values. (C) IEC-6 and IEC-18 cells treated with grape fraction VIIIG at HT29 IC50. Percentages of cells in different cell stages are shown(cell stages shown on the x-axis). (% cells ± SEM, *P < 0.05, **P < 0.001). Experiments were performed in triplicate.Antiproliferative properties of natural extracts D. Lizarraga et al.4806 FEBS Journal 274 (2007) 4802–4811 ª 2007 The Authors Journal compilation ª 2007 FEBS30% apoptosis in HT29 cells, did not induce apoptosisor affect the cell cycle of the intestinal nontumoral celllines IEC-18 and IEC-6, and even induced 10% necro-sis in the IEC-6 cell line (Figs 2C and 3C). The resultsobtained provide information about the activities ofprocyanidin mixtures with different origins and struc-tures on colon epithelial cells. These results should beuseful in defining the putative benefits of plant poly-phenols in nutritional supplements. Additionally, thisstudy provides useful insights into the polyphenolicstructure, which should help in the rational design offormulations for potent chemopreventive or antiprolif-erative natural vegetable products on the basis ofapoptosis-inducing activity.Experimental proceduresMaterialsDMEM and Dulbecco’s phosphate-buffered saline (NaCl ⁄Pi) were obtained from Sigma Chemical Co. (St Louis,MO, USA), antibiotics (10 000 UÆmL)1penicillin, 10 000lgÆmL)1streptomycin) were obtained from Gibco-BRL(Eggenstein, Germany), and fetal bovine serum wasobtained from Invitrogen (Carlsbad, CA, USA). Tryp-sin ⁄ EDTA solution C (0.05% trypsin ⁄ 0.02% EDTA) waspurchased from Biological Industries (Kibbutz Beit Ha-emet, Israel). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl-tetra-zolium bromide (MTT), dimethylsulfoxide, propidiumiodide (PI) and Igepal CA-630 were obtained from SigmaChemical Co. NADH disodium salt (grade I) was suppliedby Boehringer (Mannheim, Germany). RNase and agaroseMP were obtained from Roche Diagnostics (Mannheim,Germany). Iron(II) sulfate heptahydrate was obtained fromMerck (Darmstadt, Germany) a-a-a-Tris(hydroxymeth-yl)aminomethane was obtained from Aldrich-Chemie(Steinheim, Germany) and moviol from Calbiochem (LaJolla, CA, USA). The annexin V ⁄ fluorescein isothiocyanate(FITC) kit was obtained from Bender System (Vienna, Aus-tria), the Realpure DNA extraction kit, including protein-ase K, was obtained from Durviz S.L. (Paterna, Spain),and Blue ⁄ Orange Loading dye and the 1 kb DNA ladderwere purchased from Promega (Madison, WI, USA).5,5-Dimethyl-1-pyrolline-N-oxide (DMPO), hydrogen per-oxide, phenazine methosulfate and Hoescht were obtainedfrom Sigma (St Louis, MO). DMPO was further purifiedby charcoal treatment.FractionsThe polyphenolic mixtures were obtained previously in ourlaboratories [26,50] and contain mainly procyanidins.OWG and OWP are composed of species that are solublein both ethyl acetate and water, and the rest of the frac-tions (G for grape, P for pine) were generated by a combi-nation of preparative RP-HPLC and semipreparativechromatography on a Toyopearl TSK HW-40F column(TosoHass, Tokyo, Japan), which separated the compo-nents by size and hydrophobicity. The phenolics wereeluted from the latter column with MeOH (fractions VG,VP) and water ⁄ acetone 1 : 1 (fractions IVG, VIG, VIIIG,IVP, VIP, VIIIP and XIP), evaporated almost to dryness,redissolved in Milli-Q water, and freeze-dried. The secondand third columns of Table 1 show the average chemicalcomposition of the fractions.Cell cultureHuman colorectal adenocarcinoma HT29 cells(ATCC HTB-38) and two nontumoral intestinal rat celllines, IEC-6 (ECCAC no. 88071401) and IEC-18 (EC-CAC no. 88011801), were used in all of the experiments.HT29, IEC-6 and IEC-18 cells were maintained in mono-layer culture in an incubator with 95% humidity and 5%CO2at 37 °C. HT29, IEC-6 and IEC-18 cells were passagedat preconfluent densities using trypsin ⁄ EDTA solution C.M Ct1Ct2IVGAIVGBVIIIGAVIIIGBControl48H (VIIIG)72H (VIIIG)48H (IVG)72H (IVG)ControlABA= IC50B= IC80Fig. 4. Induction of apoptosis by grape fractions VIIIG and IVG inHT29 cells. (A) Nuclear condensation of HT29 cells. Arrows indicatethe apoptotic cells with condensed and fragmented nuclei. (B) DNAladdering induced in both treatments.D. Lizarraga et al. Antiproliferative properties of natural extractsFEBS Journal 274 (2007) 4802–4811 ª 2007 The Authors Journal compilation ª 2007 FEBS 4807Cells were cultured and passaged in DMEM supplementedwith 10% heat-inactivated fetal bovine serum and 0.1%streptomycin ⁄ penicillin.Cell growth inhibitionHT29, IEC-6 and IEC-18 cells were seeded densitiesof 3 · 103cells per well, 5 · 103cells per well and1 · 103cells per well, respectively, in 96-well flat-bottomedplates. After 24 h of incubation at 37 °C, the polyphenolicmixtures were added to the cells at different concentra-tions from 5 lm to 2300 lm in fresh medium. The culturewas incubated for 72 h, after which the medium wasremoved and 50 lL of MTT (5 mgÆmL)1in NaCl ⁄ Pi) with50 lL of fresh medium was added to each well and incu-bated for 1 h. The blue MTT formazan precipitated wasdissolved in 100 lL of dimethylsulfoxide, and the absor-bance values at 550 nm were measured on an ELISA platereader (Tecan Sunrise MR20-301; TECAN, Salzburg, Aus-tria). Absorbance was proportional to the number of liv-ing cells. The growth inhibition concentrations that caused50% (IC50) and 80% (IC80) cell growth inhibition werecalculated using grafit 3.0 software. The assay was per-formed using a variation of the MTT assay described byMosmann [51].Cell cycle analysisThe assay was carried out using flow cytometry with aFACS. HT29, IEC-6 and IEC-18 cells were plated in six-well flat-bottomed plates at densities of 87.3 · 103cells perwell, 146 · 103cells per well and 29.1 · 103cells per well,respectively. The number of cells was determined as cellsper area of well, as used in the cell growth inhibition assay.The culture was incubated for 72 h in the absence or pres-ence of the polyphenolic mixture at its respective IC50values. The cells were then trypsinized, pelleted by centri-fugation [371 g for 3 min at room temperature (RT) usinga 5415D centrifuge (Eppendorf, Hamburg, Germany) and a24-place fixed angle rotor] and stained in Tris-bufferedsaline (NaCl ⁄ Tris) containing 50 lgÆmL)1PI, 10 lgÆmL)1RNase free of DNase and 0.1% Igepal CA-630 in the darkfor 1 h at 4 °C. Cell cycle analysis was performed with aFACS (Epics XL flow cytometer; Coulter Corporation,Hialeah, FL, USA) at 488 nm. All experiments wereperformed in triplicate, as described previously [47].Apoptosis analysis by FACSAnnexin V ⁄ FITC and PI staining were measured by FACS.Cells were seeded, treated and collected as described inSuperoxide anion radical scavenger capacity ** ** ****** ** **Hydroxyl radical scavenger capacityAB020406080100120 CtVIIIG IVG OWGVGVIIIPIVP OWPVPPolyphenolic fractions at 50 µM 020406080100120 CtVIIIG IVG OWGVGVIIIPIVP OWPVPPolyphenolic fractions at 50 µM Percentage hydroxyl radical system020406080100120 CtVIIIG IVG OWGVGVIIIPIVP OWPVPPercentage hydroxyl radical system** ****** ** ** ** *Superoxide anion radical scavenger capacity Percentage superoxyde anionradical system020406080100120140160Percentage superoxyde anionradical system** **** ** ** **Hydroxyl radical scavenger capacityPolyphenolic fractions at IC50 CtVIIIG IVG OWGVGVIIIPIVP OWPVPPolyphenolic fractions at IC50 ** **** ** ** ** ** **Fig. 5. Scavenging activity of OH and OÁÀ2analyzed by ESR. Grape and pine fractions were evaluated at: (A) 50 lM and (B) IC50in HT29 cellsin hydroxyl radical- and superoxide anion radical-generating systems, as described in Experimental procedures. Experiments were performedin duplicate (*P < 0.05, **P < 0.001).Antiproliferative properties of natural extracts D. Lizarraga et al.4808 FEBS Journal 274 (2007) 4802–4811 ª 2007 The Authors Journal compilation ª 2007 FEBSthe previous section. Following centrifugation [371 g for3 min at RT using a 5415D centrifuge (Eppendorf) with24-place fixed angle rotor], cells were washed in bindingbuffer (10 mm Hepes, pH 7.4, 140 mm sodium chloride,2.5 mm calcium chloride) and resuspended in the samebuffer. Annexin V ⁄ FITC was added using the annex-in V ⁄ FITC kit. Following 30 min of incubation at roomtemperature and in the dark, PI was added 1 min beforethe FACS analysis at 20 lgÆmL)1. Experiments were per-formed in triplicate.Apoptosis detection by DNA ladderingDNA isolation and purification were performed after 72 hin the presence and absence of grape fractions VIIIG andIVG. The fractions were assayed at their respective IC50and IC80values. After treatment, cells were scraped offslides and collected by centrifugation at 14 000 g for 10 sat RT using a 5415D centrifuge (Eppendorf) and 24-placefixed angle rotor. Cells were then lysed by adding 600 lLof Realpure kit lysis buffer and 10 lL of proteinase K,and incubated for 1 h at 55 °C. RNA digestion was per-formed with 1.5 lL of RNase for 1 h at 37 °C, and thiswas followed by protein precipitation with 360 lLofRealpure kit buffer and centrifugation at 14 000 g for10 min at RT using a 5415D centrifuge (Eppendorf) and24-place fixed angle rotor. The DNA sample wasextracted with isopropanol ⁄ ethanol, dried, and eluted in100 lL of Realpure kit DNA hydration solution. Equalamounts of DNA (20 lg), estimated by measuring absorp-tion at 260 ⁄ 280 nm, were electrophoretically separated on1% TAE agarose gel and viewed under a UV transillumi-nator (Vilber Lourmat, Marne-la-Valle´e, France).Apoptosis detection by Hoescht stainingApoptotic induction was also studied using Hoescht stain-ing. Samples were incubated with grape fractions VIIIGand IVG at 0, 48 and 72 h. After incubation, cells weretrypsinized and fixed with cold methanol for 1 h at ) 20 °C.After being rinsed with NaCl ⁄ Pithree times, cells werestained in the dark with Hoescht (50 ngÆmL)1in NaCl ⁄ Pi)for 50 min. Finally, cells were rinsed, suspended in NaCl ⁄ Piand diluted 1 : 2 with moviol. The samples were mountedon a slide and observed with a fluorescent microscope at anexcitation wavelength of 334 nm and an emission wave-length of 365 nm.ESR spectroscopyESR measurements were performed at concentrations thatcaused 50% cell growth inhibition (IC50) and 50 lm grapeand pine fractions (VIIIG, IVG, OWG, VG, VIIIP, IVP,OWP and VP). Molar concentrations were calculated fromthe mean molecular mass of the fractions estimated by thiol-ysis with cysteamine, as described in [52]. OH and O2–forma-tion were detected by ESR spectroscopy using DMPO(100 mm) as a spin trap. ESR spectra were recorded at roomtemperature in glass capillaries (100 lL; Brand AG,Wertheim, Germany) on a Bruker EMX 1273 spectrometer(Bruker, Karlsruhe, Germany) equipped with an ER 4119HShigh-sensitivity cavity and a 12 kW power supply operatingat X-band frequencies. The modulation frequency of thespectrometer was 100 kHz. Instrumental conditions for therecorded spectra were: magnetic field, 3490 G; scan range,60 G; modulation amplitude, 1 G; receiver gain, 1 · 105;microwave frequency, 9.85 GHz; power, 50 mW; timeconstant, 40.96 ms; scan time, 20.97 s; number of scans, 25.Spectra were quantified by peak surface measurements usingthe WIN-EPR spectrum manipulation program (Bruker).All incubations were done at room temperature; thehydroxyl radical generation system used 500 lm FeSO4and550 lm H2O2,and hydroxyl radicals generated in this systemwere trapped by DMPO, forming a spin adduct detected bythe ESR spectrometer. The typical 1 : 2 : 2 : 1 ESR signalof DMPO-OH was observed. The superoxide radical genera-tion system used performed using 50 lm of the reduced formof b-NADH and 3.3 lm phenazine methosulfate, and thesuperoxide radicals generated in this system were trapped byDMPO, forming a spin adduct detected by the ESR spec-trometer. The typical ESR signal of DMPO-OOH ⁄ DMPO-OH was observed. The OH and O2-scavenging activity wascalculated on the basis of decreases in the DMPO-OH orDMPO-OOH ⁄ DMPO-OH signals, respectively, in which thecoupling constant for DMPO-OH was 14.9 G.Data presentation and statistical analysisAssays were analyzed using the Student’s t-test andwere considered statistically significant at P<0.05 andP<0.001. The data shown are representative of threeindependent experiments, with the exception of ESR experi-ments, which were performed in duplicate. ESR experi-ments were analyzed separately by radicals, Two-wayanova was applied (day was a block factor; due to thenonsignificant effect of the day factor, we reanalyzed with aone-way anova), and finally, a multicomparison betweencompounds with respect to the control was performed.anova with Bonferroni and Scheffe post hoc test was per-formed in ESR experiments.AcknowledgementsThis work was supported by grants PPQ 2003-06602-C04-01, PPQ 2003-06602-C04-04, AGL2004-07579-C04-02 and AGL2004-07579-C04-03 from the SpanishMinistry of Education and Science, and ISCIII-RTICC(RD06 ⁄ 0020 ⁄ 0046) from the Spanish government andD. Lizarraga et al. Antiproliferative properties of natural extractsFEBS Journal 274 (2007) 4802–4811 ª 2007 The Authors Journal compilation ª 2007 FEBS 4809the European Union FEDER funds. 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