Quercetin modulates Nrf2 and glutathione-related defenses in HepG2. cells. Involvement of p38

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1 Quercetin modultes Nrf2 nd glutthione-relted defenses in HepG2 cells. Involvement of p38 An Belén Grndo-Serrno, Mrí Angeles Mrtín, Lur Brvo, Luis Goy nd Soni Rmos* Deprtment of Metolism nd Nutrition Institute of Food Science, Technology nd Nutrition-ICTAN (former Instituto del Frío) Consejo Superior de Investigciones Científics (CSIC) José Antonio Novis 1 Ciudd Universitri, 284, Mdrid Spin Phone: Fx: * Corresponding uthor: e-mil: s.rmos@if.csic.es 1

2 Astrct 1 Dietry flvonoid quercetin hs een suggested s cncer chemopreventive gent, ut the mechnisms of ction remin uncler. This study investigted the influence of quercetin on p38- MAPK nd the potentil regultion of the nucler trnscription fctor erythroid-2p45-relted fctor (Nrf2) nd the cellulr ntioxidnt/detoxifying defense system relted to glutthione (GSH) y p38 in HepG2 cells. Incution of HepG2 cell with quercetin t rnge of concentrtions (5-5μM) for 4 or 18h induced differentil effect on the modultion of p38 nd Nrf2 in HepG2 cells, showing 5 M quercetin the highest ctivtion of p38 nd vlues similr to those of control cells fter 4 nd 18h tretment, respectively, together with the inhiition of Nrf2 t oth incution times. Quercetin (5 M) induced time-dependent ctivtion of p38, which ws in concert with trnsient stimultion of Nrf2 to provoke its inhiition fterwrd. Quercetin lso incresed GSH content, mrna levels of glutmylcysteine-synthetse (GCS) nd expression nd/or ctivity of glutthioneperoxidse, glutthione-reductse nd GCS fter 4h of incution, nd glutthione-s-trnsferse fter 18h of exposure. Further studies with the p38 specific inhiitor SB2358 showed tht the p38 lockge restored the inhiited Nrf2 trnscription fctor nd the enzymtic expression nd ctivity of ntioxidnt/detoxificnt enzymes fter 4h exposure. In conclusion, p38-mapk is involved in the 1 Arevitions used: AKT/PKB, protein kinse B; AP-1, ctivtor protein-1; ARE, ntioxidnt response element; CHX, cycloheximide; DAPI, 6-dimidino-2-phenylindole; DTT, dithiothreitol; ERK, extrcellulr regulted kinse; FBS, fetl ovine serum; GAPDH, glycerldehyde-3-phosphte dehydrogense; GCS, glutmylcysteine synthetse; GPx, glutthione peroxidse; GR, glutthione reductse; GRB2, growth fctor receptor-ound protein 2; GSH, glutthione; GST, glutthione S-trnsferse; JNK, c-jun mino-terminl kinse; Kep-1, Kelch-like ECH-ssocited protein-1; MAPK, mitogen-ctivted protein kinse; MCB, monochloroimne; NAC, N-cetyl-cysteine; Nrf2, nucler trnscription fctor erythroid 2p45 (NF-E2)-relted fctor; PARP, poly(adpriose)polymerse; PI3K, phosphtidylinositol-3-kinse; PKC, protein kinse C; PMSF, phenylmethylsulfonyl fluoride; ROS, rective oxygen species. 2

3 mechnisms of the cell response to quercetin through the modultion of Nrf2 nd glutthionerelted enzymes in HepG2 cells. Keywords: Glutthione, Glutthione-relted enzymes, Nrf2, p38, Quercetin. 3

4 1. Introduction Quercetin (3, 3, 4, 5, 7-penthydroxylflvone) is flvonoid, one of the most undnt polyphenolic groups in plnts (fruits nd vegetles) [1], which cn e extensively metolized during sorption in the smll intestine nd in the liver [2, 3]. Mny studies hve demonstrted tht quercetin exerts potentil nticrcinogenic ctivities, since it is potent inhiitor of tumor initition in vivo [4], is powerful rdicl scvenger le to prevent or dely conditions which fvor cellulr oxidtive stress [5, 6], possesses nti-prolifertive ctivities ginst tumor cells in vitro [4, 7, 8], nd induces poptosis in different cncer cell lines y modulting numer of key elements in cellulr signl trnsduction pthwys linked to the poptotic cell deth [1, 7-12]. Two possile mechnisms hve een suggested to explin the chemoprotective effects of quercetin. The first includes the removl of preinitited cells from dmged tissues through cell cycle rrest nd poptosis [1-13]. Alterntively, the modultion of metolizing enzymes hs een proposed s nother chemopreventive mechnism tht cn lock crcinogen ctivtion (inhiition of cytochrome P45) or enhnce the detoxifiction of ctivted crcinogens (ctivtion of phse II detoxifying enzymes) [14]. Thus, quercetin is found to interct with the cellulr defense system such s NADPH:quinone oxidoreductse, inos, monooxygense, COX, xnthine oxidse, lipoxygense, hemeoxygense-1, etc. [8, 15-17]. In this regrd, the induction of ntioxidnt/detoxifying enzymes s well s glutthione (GSH) levels provides significnt iologicl mechnisms for protection ginst toxic effects of endogenous rective oxygen species (ROS) nd exogenous crcinogens nd/or their rective intermedites [16, 18-22]. Nucler fctor-erythroid 2p45-relted fctor-2 (Nrf2) plys centrl role in the induction of ntioxidnt nd detoxifying enzymes through its inding to the ntioxidnt response element (ARE) [23, 24]. Nrf2 is sequestered in the cytoplsm s n inctive complex with its cytosolic repressor Kelch-like ECH ssocited protein-1 (Kep-1). The dissocition of Nrf2 from Kep-1 is crucil for 4

5 its nucler trnsloction, followed y inding to DNA nd ctivtion of cytoprotective genes [23]. To dte, multiple signling kinses relted to cell survivl/prolifertion hve een reported to regulte Nrf2, which include extrcellulr signl-regulted kinse (ERK), c-jun NH2-terminl kinse (JNK), phosphoinositide 3-kinse (PI3K) nd protein kinse C (PKC) [21, 23]. Indeed, the phosphoryltion of Nrf2 y these different kinses t multiple sites seems to e n importnt mechnism in Nrf2-medited ARE ctivtion nd in regulting the stility of this trnscription fctor [25], ut few dt hve een reported out Nrf2 regultion nd p38 MAPK. p38 MAPK consists of four isoforms: p38, p38, p38 nd p38. p38 is the most undnt nd uiquitously expressed fmily protein nd hs well estlished role in stress response nd inflmmtion [26]. Once p38-mapk is phosphorylted nd ctivted, it phosphoryltes nd/or ctivtes downstrem sustrtes (kinses, trnscriptionl fctors, etc.), resulting in vrious cellulr responses such s prolifertion, poptosis, cell cycle rrest, nd inflmmtion [26]. In line with this, up-regultion of the hemeoxygense-1 detoxifiction enzyme vi Nrf2 hs een reported to involve p38-mapk in renl cells [27]. Similrly, Yo et l [17] hve demonstrted tht p38 nd ERK medited quercetin-derived Nrf2 trnsloction into nuclei nd susequent induction of hemeoxygense-1 ctivity. Although different studies hve consistently demonstrted tht quercetin is strong inducer of GSH content nd ntioxidnt/detoxificnt enzymes, the detiled upstrem signling mechnisms re still uncler. In the present study, quercetin modultion of p38 in reltion to Nrf2 function nd the role of p38 on the regultion of cellulr ntioxidnt/detoxifying defense system relted to glutthione hve een nlyzed. 5

6 2. Mterils nd methods 2.1. Mterils nd chemicls Quercetin, SB2358, cycloheximide (CHX), gentmicin, penicillin G nd streptomycin were purchsed from Sigm Chemicl (Mdrid, Spin). Anti-phospho-p38 recognizing phosphorylted Thr18/Tyr182 (9216) nd nti- -ctin (4697) were otined from Cell Signlling Technology (Izs, Mdrid, Spin). Anti-p38 (sc-535), nti-nrf2 (C-2, sc-722), nti-nrf2 (H-3, sc-1332), nti-parp (sc-715) nd nti-grb2 (sc-255) were purchsed from Snt Cruz Biotechnology Inc. (Snt Cruz, CA, USA). Mterils nd chemicls for electrophoresis were from BioRd (BioRd Lortories S.A., Mdrid, Spin). Cell culture dishes nd cell culture medium were from Flcon (Cjl, Mdrid, Spin) nd Biowhittker Europe (Lonz, Mdrid, Spin), respectively Cell culture nd quercetin tretment Humn heptom HepG2 cells were grown in DMEM F-12 medium from Biowhitker (Lonz, Mdrid, Spin), supplemented with 2.5% Biowhitker fetl ovine serum (FBS) nd the following ntiiotics: gentmicin, penicillin nd streptomycin (5 mg/l). Cells were mintined t 37ºC in humidified tmosphere of 5% CO 2. Cells were seeded nd routinely grown in DMEM-F12 medium nd 2.5% FBS, ut they were chnged to serum-free medium 24 h efore the ssy in order to void the influence of the growth fctors contined in the FBS on the results. To study the time-course effects of quercetin, cells were treted with 5 µm quercetin nd then hrvested t different times (, 1, 3, 6, 12, 24 nd 18 min). In the experiments with quercetin nd the selective phrmcologicl inhiitor of p38, cells were preincuted with SB2358 (1 µm) for 2 h prior to 5 µm quercetin tretment for 24 min or 18 min. 6

7 To nlyze the effect of quercetin nd p38 inhiition on Nrf2 stiliztion, HepG2 cells were incuted with SB2358 for 2 h efore incuting with 5 µm quercetin for 18 min nd then treted with 1 µg/ml CHX for 1 h Fluorescence microscopy Fluorescence ssy ws performed s previously descried [28]. Briefly, HepG2 cells were seeded (25. cells/well) on glss coverslips with DMEM F-12 supplemented with FBS for 24 h nd chnged to serum-free medium 24 h efore the ssy. After incution with quercetin for the indicted times, cells were wshed with PBS t room temperture nd then fixed with 3.7% prformldehyde for 1 min t room temperture. Nuclei were visulized y using DAPI stining. The coverslips were mounted in Vectshield nd imges were tken with Zeiss Axiovert 2M fluorescence microscope (Crl Zeiss Microimging GmH, Munich, Germny) t 63x mgnifiction. AxioVisionRel 4.6 softwre ws used for the nlysis of the imges otined Preprtion of totl cell lystes To detect p38 nd phospho-p38, cells were lysed t 4ºC in uffer contining 25 mm HEPES (ph 7.5),.3 M NCl, 1.5 mm MgCl 2,.2 mm EDTA,.5 mm dithiothreitol (DTT),.1% Triton X- 1, 2 mm β-glycerolphosphte,.1 mm N 3 VO 4, 2 µg/ml leupeptin nd 1mM phenylmethylsulfonyl fluoride (PMSF). The superntnts were collected, ssyed for protein concentrtion y using the Bio-Rd (Bio-Rd, Mdrid, Spin) protein ssy kit ccording to the mnufcture s specifictions, liquoted nd stored t -8ºC until used for Western lot nlyses Preprtion of nucler nd cytosolic extrcts To nlyze Nrf2, cells were resuspended t 4ºC in 1 mm HEPES ph 7.9, 1.5 mm MgCl 2, 1 mm KCl,.5 mm DTT,.2 mm PMSF (uffer A), llowed to swell on ice for 1 min, nd then 7

8 vortexed for 1s. Smples were centrifuged t 1, g for 2 min nd the superntnt contining the cytosolic frction ws stored t -8 C. The pellet ws resuspended in cold uffer B (2 mm HEPES ph 7.9, 25% glycerol, 42 mm NCl, 1.5 mm MgCl 2,.2 mm EDTA,.5 mm DTT,.2 mm PMSF, 2.5 µg/ml leupeptin, 2.5 µg/ml protinin) nd incuted on ice for 2 min for high slt extrction. Cellulr deris ws removed y centrifugtion t 13, g for 1 min t 4 C, nd the superntnt frction contining nucler protein extrct ws stored t -8 C. Proteins were mesured using the Brdford regent (Bio-Rd, Mdrid, Spin) following the recommendtions of the supplier Western lot nlysis Equl mounts of proteins (1 µg) were seprted y SDS-polycrylmide gel electrophoresis nd trnsferred to polyvinylidene difluoride (PVDF) filters (Protein Sequencing Memrne, Bio-Rd, Mdrid, Spin). Memrnes were proed with the corresponding primry ntiody followed y incution with peroxide-conjugted nti-rit immunogloulin (GE Helthcre, Mdrid, Spin) or peroxide-conjugted nti-mouse immunogloulin only for p-p38 (Sigm, Mdrid, Spin). Blots were developed with the ECL system (GE Helthcre, Mdrid, Spin). Antigrowth fctor receptoround protein-2 (nti-grb2) nd nti-poly(adpriose) polymerse (nti-parp) ntiodies were used s mrkers for the cytosolic nd nucler extrcts, respectively. Normliztion of Western lot ws ensured y β-ctin nd nds were quntified using scnner nd ccompnying softwre Mesurement of the Nrf2 DNA inding ctivity The DNA inding of Nrf2 ws mesured with colorimetric non-rdioctive Nrf2 Trnscription Fctor ELISA Assy (Active Motif, Rixensrt, Belgium) ccording to the mnufcturer s instructions. Briefly, nucler extrcts were incuted in the oligonucleotide-coted wells. Then, wells were wshed nd incuted with the ntiody ginst Nrf2. Addition of the secondry 8

9 ntiody conjugted to horserdish peroxidse provided colorimetric redout. The sornce of ech well ws mesured using microplte reder t 45 nm (Bio-Tek, Winooski, VT, USA) Cell viility ssy Cell viility ws determined using the crystl violet ssy [11]. HepG2 cells were seeded t low density (1 4 cells per well) in 96-well pltes, grown for 2 h nd incuted with crystl violet (.2% in ethnol) for 2 min. Pltes were rinsed with wter, llowed to dry, nd 1% sodium dodecylsulfte (SDS) dded. The sornce of ech well ws mesured using microplte reder t 57 nm (Bio- Tek, Winooski, VT, USA) RNA semiquntittion y reverse trnscriptse-polymerse chin rection Totl RNA from cultured HepG2 cells ws extrcted y RNesy kit (Qigen, IZASA, Brcelon, Spin). One microgrm of totl RNA ws used for reverse trnscription polymerse chin rection (RT-PCR) nlysis. PCR ws performed using the following couples of primers: 5 - TCCGCTGCAAATACATCTCC-3 nd 5 -TGTTTCCCGTTGCCATTGAT-3 (T=55ºC) for glutthione S-trnsferse (GST); 5 -GGCACAGGTAAAACCAAATAGTAAC-3 nd 5 - CAAATTGTTTAGCAAATGCAGTCA-3 (T=55ºC) for glutmylcysteine synthetse (GCS); 5 - CAGTGGGACTCACGGAAGAT-3 nd 5 -TTCACTGCAACAGCAAAACC-3 (T=52ºC) for glutthione reductse (GR); 5 -CCTCAAGTACGTCCGACCTG-3 nd 5 - TAGGAGTTGCCAGACTGCTG-3 (T=58ºC) for glutthione peroxidse (GPx). Glycerldehyde- 3-phosphte dehydrogense (GAPDH) ws used s housekeeping gene. Smples were incuted in n utomted het lock (PCR express system, Thermo Hyid, Middlesex, UK). The PCR products were electrophoresed on 1.5% grose gel contining ethidium romide. Densitometric nlysis ws performed using scnner nd ccompnying softwre. Bnd intensity ws normlized to vlues for GAPDH tht ws used s n internl control. 9

10 2.1. Determintion of reduced glutthione (GSH) The content of GSH ws quntified y the fluorometric ssy of Hissin nd Hilf [29]. The method tkes dvntge of the rection of GSH with o-phtlldehyde t ph 8.. After the different tretments, cells (4x1 6 ) were detched nd homogenized y ultrsound with 5% trichlorocetic cid contining 2 mm EDTA. Following centrifugtion of cells for 3 min t 1, g, 5 µl of the cler superntnt ws trnsferred to 96-multiwell plte for the ssy. Fluorescence ws mesured t n excittion wvelength of 34 nm nd n emission wvelength of 46 nm. The results of the smples were referred to those of stndrd curve of GSH. The precise protocol hs een descried elsewhere [5, 6] Determintion of GPx nd GR ctivities Treted cells were collected in PBS nd centrifuged t 3 g for 5 min. Cell pellets were resuspended in 2 mm Tris contining 5 mm EDTA nd.5 mm mercptoethnol, sonicted nd centrifuged t 3, g for 15 min. Enzymtic ctivities were mesured in the superntnts. The determintion of the GPx ctivity is sed on the oxidtion of GSH y GPx, using 3 mm tertutylhydroperoxide s sustrte, coupled to the disppernce of NADPH y GR [3]. A cuvette contining ssy uffer plus.1 M GSH, 5 U/mL GR nd 9.6 mm NADPH ws used to lnk the spectrophotometer prior to ddition of the smple nd 3 mm tert-utyl hydroperoxide. Additionlly, smple with cells treted with 4 μm tert-utylhydroperoxide for 3 hours ws used s n internl positive control for GPx overctivity (dt not shown) [5]. GR ctivity ws determined y following the decrese in sornce due to the oxidtion of NADPH utilized in the reduction of oxidized GSH [31]. Ech smple ws lnked t the spectrophotometer in the presence of ssy uffer plus 15 mm EDTA nd 65 mm oxidted glutthione prior to ddition of 9.6 mm NADPH. As for GPx ctivity, smple with cells treted with 4 μm tert-utylhydroperoxide for 1

11 3 hours ws used s n internl positive control for GR overctivity (dt not shown) [5]. Protein ws mesured y the Brdford regent (Bio-Rd, Mdrid, Spin) Determintion of GST ctivity GST ctivity ws determined using commercil fluorimetric ctivity ssy kit (Biovision Reserch Products, CA, USA). Treted cells were collected in PBS nd centrifuged t low speed (3 g) for 5 min. Cell pellets were resuspended in smple uffer, sonicted nd centrifuged t 3, g for 15 min nd enzyme ctivity ws mesured in the superntnts. The ssy utilizes monochloroimne (MCB) s n rtificil sustrte nd glutthione to determine totl GST ctivity. GST ctlyses the MCB-glutthione rections nd the fluorescence levels re proportionl to the mount of GST present in the rection medium. A fluorescent microplte reder t n excittion wvelength of 38 nm nd n emission wvelength of 46 nm ws used (Bio-Tek, Winooski, VT, USA). The results of the smples were referred to those of stndrd curve of GST ctivity, nd the GST stndrd ws provided y the commercil kit. Protein ws mesured y the Brdford regent Sttistics Prior to sttisticl nlysis, dt were tested for homogeneity of vrinces y the test of Levene; for multiple comprisons, one-wy ANOVA ws followed y the Bonferroni test when vrinces were homogeneous or y the Tmhne test when vrinces were not homogeneous. P<.5 ws considered significnt. A SPSS version 17. progrm ws used. 11

12 3. Results 3.1. Dose-effect of quercetin on Nrf2 nd p38 To investigte whether quercetin tretment in HepG2 cells ws le to regulte key proteins relted to the cellulr response to chemicl/oxidtive stress [23, 26], p38-mapk nd Nrf2 protein expressions were nlyzed. HepG2 cells were treted with 5-5 μm quercetin for 4 nd 18 h, s short- nd long-term tretments, in order to study the regultory effect of the flvonoid on p38- MAPK nd the ntioxidnt defense system oth in potentil non-toxic nd toxic conditions, respectively. Figures 1A nd 1B illustrte tht 4 h tretment with 1-5 μm quercetin incresed the phosphorylted levels of p38 in HepG2 cells. In ddition, 18 h of incution with 5-25 μm flvonoid induced the phosphoryltion of p38, wheres 5 μm quercetin showed comprle levels of phosphorylted p38 to those of control cells. Likewise, there ws no difference in the totl levels of p38. Similrly, it ws investigted whether quercetin tretment ws le to regulte the trnscription fctor Nrf2 in HepG2 cells. No phosphorylted Nrf2 (1 kd) ws detected in the cytosolic frction of HepG2 cells, s previously shown [28]. A 4h-tretment with 1 nd 25 μm quercetin significntly incresed the nucler trnsloction of Nrf2 (57 kd) nd the nucler content of phosphorylted Nrf2 (1 kd), wheres 5 μm quercetin decresed oth the Nrf2 phosphorylted levels nd the nucler/cytosolic Nrf2 rtio (Figures 1C-1E). On the other hnd, 18 h tretment with 5-1 μm quercetin incresed the nucler trnsloction of Nrf2 (57 kd) nd the nucler content of phosphorylted Nrf2 (1 kd), nd oth prmeters decresed in the presence of 5 μm quercetin. The integrity of the cytosolic nd nucler frctions ws confirmed y the nlysis of the comprtment-specific cytosolic GRB2 nd nucler PARP proteins. These results suggest tht the durtion of the tretment nd the concentrtion of quercetin hve prominent effect on the modultion of p38 nd Nrf2 in HepG2 cells. 12

13 Given tht 5 μm quercetin ws the concentrtion tht provoked the strongest induction of p38 fter 4 h of tretment returning the phosphorylted levels of p38 to control vlues fter 18h of incution, nd showed n inhiitory effect on Nrf2 t oth incution times, this ws the concentrtion selected for studying the contriution of p38 to the modultion of Nrf2 y quercetin in HepG2 cells Sucellulr locliztion of quercetin By using the fluorogenic properties of quercetin, the cellulr locliztion of the flvonoid ws studied upon its internliztion, HepG2 cells were treted for different times with 5 M quercetin. Cell incution resulted in yellow-green fluorescent signl tht ws detected in fluorescence microscope upon excittion t nm nd emission t nm (Figure 2). The fluorescent signl of quercetin ws minly locted in the nucleus of HepG2 cells s erly s fter 1min of incution (Figure 2). The signl ws oserved up to 24 min of tretment nd only fter the longest incution time (18 min) ws not detected (Figure 2). These results indicted tht quercetin exhiited specific fluorescence nd showed minly nucler locliztion in HepG2 cells Quercetin ctivtes p38 in time-dependent mnner The ctivtion of p38-mapks hs trditionlly een ssocited with the stress response nd some poptotic processes, lthough p38-mapks hve lso een implicted s positive regultors in cell survivl [26]. Since vritions in the phosphorylted levels of p38 could occur in time-dependent mnner, it ws importnt to study the effect induced y quercetin on this protein through the time in HepG2 cells. To this end, totl nd phosphorylted (ctive form) protein expressions of p38 were nlyzed. Tretment of HepG2 cells with quercetin resulted in n enhnced phosphoryltion of p38 t 1-24 min, returning to control levels t the longest time tested (18 h) (Figures 3A nd 3B). Totl p38 protein levels did not chnge during quercetin exposure. 13

14 3.4. Time-course effect on Nrf2 modultion induced y quercetin Nrf2 is involved in the response to chemicl/oxidtive stress [23]. To ssess the impliction of Nrf2 on quercetin effects, time-course studies on the nucler nd cytosolic levels of this trnscription fctor were performed. Quercetin concurrently incresed nucler trnsloction of Nrf2 (57 kd) nd nucler content of phosphorylted Nrf2 (1 kd) t 6 min of tretment, followed y decrese t 24 min of quercetin exposure (Figures 4A-4C). This effect remined up to 18 min of incution s cn e deduced from the decline in the nucler phosphorylted content nd nucler/cytosolic rtio of Nrf2 (Figures 4A-4C). Therefore, quercetin erly nd trnsiently ctivtes Nrf2 trnsloction to lter decrese its moiliztion. To further investigte the time-course effect of quercetin on the Nrf2 regultion, Nrf2 DNA-inding ctivity ws studied. Quercetin induced trnsient increse of Nrf2 inding ctivity (6 min), nd lter showed reduced inding ctivity (24 nd 18 min) (Figure 4D). The profile of the effect on Nrf2 inding ctivity ws similr to tht oserved for the nucler phosphorylted content nd nucler/cytosolic rtio of this trnscription fctor (Figure 4A-4C). Thus, s previously suggested quercetin ctivted Nrf2 nd DNA inding erly nd trnsiently to lter induce the inhiition of this trnscription fctor Role of p38 in quercetin-induced effects on cell viility nd Nrf2 To elucidte the contriution of p38 to the modultion of Nrf2 y quercetin, the effects of SB2358 specific inhiitor of p38 were evluted. HepG2 cells were previously exposed to SB2358 nd then treted with 5 M quercetin during 4 or 18 h. First, the potentil cytotoxicity ws studied in these experimentl conditions. Tretment of HepG2 cells with SB2358 followed y ddition of quercetin did not modify cell viility fter 4 h of incution (Tle 1). The reduced percentge of vile cells ws prtly restored y the selective lockde of p38 with SB2358 inhiitor fter 18 h of quercetin tretment (Tle 1). 14

15 In ddition, in quercetin-treted cells the selective lockde of p38 completely restored to control levels the Nrf2 phosphorylted vlues nd the rtio of nucler versus cytosolic Nrf2 levels fter 4 h of incution (Figures 5A-5C), wheres t 18 h of tretment prtil recovery in oth Nrf2 phosphorylted levels nd nucler/cytosolic Nrf2 rtio ws oserved (Figures 5E-5G). These results suggested tht p38 is involved in the modultion of Nrf2 induced y quercetin, s the inhiition of p38 completely or prtly reverted the flvonoid-induced effects on Nrf2 depending on the length of the exposure to quercetin. In ddition, cell viility ws restored in prt fter 18 h of incution with quercetin y inhiiting p38-mapk. Nrf2 DNA-inding ctivity ws lso studied fter the inhiition of p38 y using SB2358. Q+SB2358 treted cells recovered the Nrf2 inding ctivity to control vlues fter 4 h of incution (Figure 5D), s occurred with the Nrf2 phosphorylted vlues nd nucler/cytosolic rtio (Figures 5A-5C). In ddition, the decresed Nrf2 DNA-inding ctivity in quercetin-treted cells fter 18 h resulted in slight recovery on the Nrf2 DNA-inding ctivity when incuting cells with quercetin nd SB2358 (Figure 5H). Comprle effects were oserved for the Nrf2 phosphorylted vlues nd the nucler/cytosolic rtio fter treting cells with quercetin (5 μm) nd SB2358 for 18 h (Figures 5E-5G). These dt indicted tht quercetin effects on Nrf2 inding ctivity were totlly or modertely restored, depending on the durtion of the tretment when p38 is inhiited p38 inhiition stilize Nrf2 To clrify whether p38 inhiition contriutes to the stimultion of the trnscription fctor y stilizing Nrf2 protein, cells were pretreted with SB2358 for 2 h efore incuting with 5 µm quercetin during 18 min nd then treted with the inhiitor of protein synthesis CHX for 1 h. The quercetin-decresed nucler/cytosol rtio of Nrf2 ws prtly restored y the inhiition of p38, wheres dditionl tretment with CHX evoked comprle levels to those of untreted cells 15

16 (Figure 6). These results reveled tht the inhiition of p38 pthwy seems to contriute to stilize Nrf2 protein levels Quercetin modultes GSH levels nd ntioxidnt/detoxificnt enzymtic ctivities. Role of p38 Studies on the ntioxidnt enzyme induction y oxidtive stress stimuli hve shown tht Nrf2 lso plys min prt nd tht PI3K nd ERK-MAPK pthwys seem to e involved in the trnsduction of signls inititing gene ctivtion [23]. To further ddress whether similr mechnism is responsile for the regultion of the GSH nd the ctivities of ntioxidnt/detoxifying enzymes induced y quercetin, the effect of the p38 inhiitor ws ssyed. GSH concentrtion ws evluted s n index of the intrcellulr non-enzymtic ntioxidnt defenses. After 4 h tretment, 5 M quercetin evoked n increse in the concentrtion of GSH, wheres pretretment with the p38 inhiitor returned the quercetin-enhnced GSH vlues to similr levels to those of untreted cells (Figure 7A). Quercetin lone or in comintion with SB2358 did not induce ny chnge in the GSH levels fter 18 h of incution (Figure 7A). As shown in Figure 6B, pretretment with SB2358 returned the quercetin-induced ctivity of GPx fter 4 or 18 h of incution to control vlues. Similrly, quercetin-induced GR ctivity ws suppressed y SB2358 pretretment fter 4 or 18 h of incution (Figure 7C). After 18 h of the flvonol exposure, GST ctivity ws enhnced, ut the ctivity of this detoxifying enzyme remined unchnged fter 4 h of tretment with quercetin (Figure 7D). Moreover, the p38 inhiitor did not ffect GST ctivity t oth tested times when compred to quercetin-treted cells (Figure 7D). These results illustrted tht the p38 pthwy seems to e involved in the quercetin-induced ctivtion of cellulr ntioxidnt/detoxificnt defenses. 16

17 3.8. Involvement of p38 in quercetin-regulted ntioxidnt/detoxificnt enzymtic gene expressions The effect of 5 μm quercetin on glutthione-relted enzymes gene expression ws further exmined. The incresed GPx mrna expression induced y quercetin tretment (4 h) ws reverted to control levels y the exposure to the p38 inhiitor (Figures 8A nd 8B). On the other hnd, fter 18h of tretment, quercetin did not modify the expression of GPx, nd its mrna levels remined lso unchnged in cells incuted with the p38 inhiitor in comprison to untreted cells (Figures 8C nd 8D). Pretretment with SB2358 suppressed oth the incresed mrna expression of GR nd GCS t 4 h of incution (Figures 8A nd 8B). However, the expression levels of GCS nd GR were unltered in the presence of quercetin nd/or the p38 inhiitor fter 18 h of incution, showing similr vlues to those of control cells (Figures 8C nd 8D). Conversely, SB2358 significntly inhiited the quercetin-induced mrna expression of GST (18 h), wheres cells treted for 4 h either with quercetin lone or in comintion with the p38 inhiitor did not show ny modifiction on GST gene expression when compred to control cells (Figures 8A nd 8B). All these results suggested n involvement of p38 pthwy in the quercetin-induced modultion of GSH-relted ntioxidnt/detoxificnt enzymes t 4 h, lthough the defensive regultory mechnisms seem to e ttenuted through the time. 17

18 4. Discussion There hs een recent surge in interest in phytochemicls s medicinl nticncer gents due to their potentil fvorle efficcy [1]. Quercetin is consumed in the diet nd lrge ody of evidence indictes tht it hs potent chemopreventive effects ginst vriety of mlignnt cell lines nd in murine crcinom models [4, 7, 1]. Quercetin dily intke rnges etween 5-4 mg/dy, ut it hs een estimted tht its dily ingestion cn rech 2-5 mg/dy in people with high intke of fruit nd vegetles [32]. Pulished dt on quercetin phrmcokinetics in humns suggest tht dietry supplement of 1-2 g of quercetin my result in plsm concentrtions rnging etween 1-5 μm [33]. In ddition, it hs een reported tht fter dministrtion of quercetin in high doses, its metolism minly tkes plce in the liver insted of the smll intestine [3]. In plsm, the min quercetin metolites found re sulphted nd glucuronidted derivtives, s well s O-methylquercetin nd 3'-O-methylquercetin-3-Oglucuronide [32]. In ddition, quercetin degrdtion y the microflor produces different phenolic cids, which re minly 3,4-dihydroxyphenylcetic, 3-methoxy-4-hydroxyphenylcetic (homovnillic cid), nd 3-hydroxyphenylcetic cid [32]. However, t present the potentil contriution of quercetin metolites to the iologicl ctivity is uncler nd it should not e underestimted; its evlution will require further studies. In the present study, it is demonstrted tht quercetin differently modultes p38 nd Nrf2 depending on the concentrtion used nd the length of the tretment. Thus, 5 μm quercetin regultes GSHrelted ntioxidnt/detoxifying enzymes nd Nrf2 y trgeting p38-mapk. Quercetin rpidly ctivtes Nrf2 y up-regulting the phosphoryltion nd trnsloction of Nrf2 to lter inhiit oth effects nd in prllel lso trnsiently induces p38-mapk ctivtion. These ctions cused y quercetin seem to ply n importnt role oth in the regultion of importnt glutthione-relted enzymes nd in the finl induction of cell deth. 18

19 The intrcellulr iotrnsformtion of quercetin y HepG2 cells ws evidenced y the grdul disppernce of its intrinsic fluorescence. However, it should e considered tht quercetin nd/or its metolites could contriute to the oserved fluorescence. This signl of fluorescence ws minly locted in the nucleus fter short incution time, in greement with Nifli et l. [34], suggesting tht quercetin internliztion is coupled to its trnsient inding to specific trget proteins. Activtion of p38-mapk hs een positively relted to cell survivl nd more trditionlly to the stress response nd poptosis, nd its role in the progrmmed cell deth depends on the cell type nd the stimuli [26]. Consequently, p38-mapk my ct s meditor of poptosis in firolsts [35], crdiomyocytes [35] nd in heptic cells [36]. Thus, quercetin-induced ctivtion of p38 could e ssocited to the up-regultion of propoptotic proteins nd down-regultion of survivl pthwys, which would led to cell deth, in greement with the present nd previous studies [1-12, 35]. Nrf2 seems to hve n importnt role in the protection ginst induced liver injury [23, 37]. Thus, Nrf2 regultes the response to cellulr stress nd cell survivl/prolifertion [8, 37-39]. In line with this, it hs een demonstrted tht polyphenols lone, s well s few glycosylted such s quercitrin, could ctivte Nrf2 [8, 16, 17, 24, 28], which is in greement with the present study. Nrf2 phosphoryltion hs een descried s criticl event for the nucler trnsloction of this trnscription fctor nd its trnscriptionl ctivity [23, 25, 28]. Moreover, Nrf2 DNA-inding ctivity could e linked to the nucler phosphorylted Nrf2 levels (1 kd) descried in the present study. Nrf2 my lso define the initil threshold for toxicity y controlling, t lest in prt, constitutive spects of cell defense [24, 37, 38]. In this regrd, it hs een descried tht n gent could stimulte the nucler ccumultion of Nrf2 t non-cytotoxic concentrtions or fter short time of incution, lthough it could induce significnt cytotoxicity t longer times of exposure [37]. 19

20 Therefore, the Nrf2-ARE pthwy could ct s sensor nd respond to chemicl stress efore the onset of the cytotoxicity. In line with this, it could e suggested tht Nrf2 is ctivted in response to mild nd cute quercetin tretment s n dptive response to gurd ginst oxidtive nd inflmmtory cell dmge, ut Nrf2 might not ply protective role ginst long nd cytotoxic flvonoid exposure. Post-trnsltionl modifiction of Nrf2 y vrious protein kinse signling pthwys cn ffect its nucler trnsloction. ERK, JNK, PI3K nd PKC re some of the kinses identified s responsile for Nrf2 phosphoryltion [23]. However, the effect of p38 on Nrf2 seems to e controversil, s it hs een reported tht Nrf2 ctivtion could e ttenuted y the overexpression of p38-mapks [39, 4]. On the contrry, it hs lso een descried tht p38 ctivtion leds to ARE induction [41, 42]. A possile explntion for these different findings is tht while certin ARE inducers my ctivte p38-mapk, others my inhiit p38-mapk [43]. In line with this, quercetin hs een reported to upregulte Nrf2 through the ctivtion of oth its trnscription nd post-trnscription modifictions [16]. In the present study, it seems tht Nrf2 is regulted y posttrnsltionl ltertions since the use of p38-mapk pthwy inhiitor increses the stility of Nrf2, s previously shown [43]. The cpcity of cells to mintin homeostsis during oxidtive stress resides in the induction of protective enzymes, s well s non-enzymtic defenses such s GSH [5, 7, 8, 18-2, 22, 44], plying Nrf2 n importnt role in the regultion of these processes [8, 16, 21, 24]. Consequently, the enhncement of GSH concentrtion nd glutthione-relted enzymes seem to prepre cells to prevent potentil oxidtive insult nd suppress oxidtive-stress induced dmge [5, 6, 19, 21, 22, 45]. Consistent with this, our results reveled tht 4 nd 18 h tretment of HepG2 cells with quercetin incresed the ctivity of GPx nd GR, s well s GSH content fter 4 h of incution. In this regrd, n increse in GSH concentrtion induced y quercetin hs een previously found in HepG2 cells nd in the liver [6, 17, 46]. Furthermore, the enhncement in GPx 2

21 nd GR enzyme ctivities fter 4 h of tretment ws correlted with prllel ugment in their mrna levels, s well s GCS expression, suggesting tht quercetin cn modulte GSH-relted enzymes gene expression s previously descried for quercetin nd other phenolic compounds [18, 46]. Interestingly, the enhnced ctivities of GPx nd GR were not ccompnied y incresed mrna levels fter 18 h of quercetin exposure, nd similr vlues to untreted cells were oserved in these conditions. This feture could e relted to the fct tht the Nrf2 ctivtion occurs s n erly event; in ddition, the elevted GPx ctivity hs een oserved fter sumitting HepG2 cells to oxidtive chllenges [19, 44], nd n ugmented GR ctivity hs een relted to n enhncement in the metolism of the xenoiotic nd flvonoids [14, 44]. Similrly, fter n cute stress GSH levels could e temporlly decresed nd lter recovered due to n increse in GCS ctivity nd mrna [46]; this hs een considered s n indiction of cellulr self-protection ginst sulethl toxic insult [14]. All these results suggest tht the finl cytotoxicity ssocited to quercetin tretment nd the unchnged levels of mrna expression for GPx, GR nd GCS (18 h) might imply impirment in the mchinery involved in gene trnscription nd mrna synthesis of the ntioxidnt enzymes. In fct, cell deth cused y quercetin my indicte tht the toxic insult hs overwhelmed the defense mechnisms, such s Nrf2 response, s previously shown for other gents [38]. In line with this, it hs een reported the ctivtion of Nrf2 nd relted genes to this trnscription fctor in cells undergoing poptosis, suggesting tht the tretment with dietry polyphenolic might not e enough to protect ginst cell deth [24]. The detoxifiction enzyme GST ctlyses the rection of endogenous GSH with numerous electrophiles to yield less toxic conjugtes nd fcilitte their elimintion [18]. GST ctivity nd mrna expression incresed fter 18 h of tretment with quercetin. Different nturl compounds, nd lso quercetin nd its glycosylted form, induce GST ctivity s mechnism of protection ginst oxidtive stress [8, 18, 19, 45, 47]. On the other hnd, numer of studies hve shown correltion etween overexpression of GST nd the development of resistnce towrds vrious 21

22 nticncer drugs [48]. Therey, induction of GST my not e fvorle for cncer chemotherpy nd significnt inverse correltion hs een demonstrted etween GST enzyme ctivity nd tumor incidence [45, 49]. In this line, it hs een recently reported tht epiglloctechin-3-gllte nd sulforphne ugment Nrf2 nd detoxifying enzyme levels, which hs een ssocited with chemoresistnce to cytotoxic drugs [5]. However, other uthors suggest tht the effect of polyphenols on GST ctivity my depend on the mount of phenolic compound incorported into the cells [47]. In ny cse, it should e highlighted tht mny reserches hve shown tht quercetin possesses nticncer effects on different models through different mechnisms [1, 1, 14]. Tken ll these results together, it could e proposed tht the incresed endogenous defense cpcity evoked y quercetin could e criticl determinnt in cell protection/resistnce ginst the induced injury. The pprent correltion etween p38 nd Nrf2-medited ntioxidnt/detoxifying enzyme suggests tht Nrf2 could e downstrem trget of this MAPK fter quercetin tretment. In this line, pretretment with the p38-mapk inhiitor prior to exposure to quercetin ctivted Nrf2 [4], which suggests tht p38-mapk signling pthwy my e implicted in the nucler locliztion nd stility of Nrf2 protein during quercetin-induced gene expression. Thus, this study demonstrtes tht p38 pthwy is involved in the cell defense through the modultion of Nrf2 nd ntioxidnt/detoxificnt enzymes fter quercetin exposure. In summry, the present study demonstrtes tht possile mechnism of ction of quercetin my e y regulting the GSH-relted ntioxidnt/detoxifying enzymes. Thus, p38 is implicted in the modultion of Nrf2 nd GSH-relted enzymes in quercetin-treted cells t shorter time thn tht cusing cell dmge. However, this response is impired t longer times of incution, when the toxic insult hs overwhelmed the cellulr defense mechnisms. These results might hve implictions for humns in terms of use of flvonoids in n ttempt to support cncer chemotherpy. Consequently, further efforts re needed to elucidte the enefits nd risks of flvonoids since they re used incresingly in dietry supplements. 22

23 Conflict of interests The uthors declre tht there re no conflicts of interest. Acknowledgements This work ws supported y the grnts 287I198 (CSIC), nd AGL24-32, AGL nd CSD27-63 from the Spnish Ministry of Science nd Innovtion (CICYT). A.B. Grndo-Serrno ws predoctorl fellow of the Spnish Ministry of Science nd Eduction. 23

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30 Legends to figures Figure 1. Dose-dependent effects of quercetin on p38 nd Nrf2 fter 4 nd 18 h of tretment. Effects on the sl levels of phosphorylted p38 (Thr18/Tyr182) nd totl p38 fter incuting HepG2 cells for 4 (white rs) or 18 h (lck rs). (A) Representtive lots. (B) Percentge dt of the p-p38/p38 rtio reltive to controls (mens SD, n =5). Normliztion of Western lots ws ensured y -ctin. Effects of quercetin on cytosolic nd nucler Nrf2 levels (1 nd 57 kd) fter for 4 (white rs) or 18 h (lck rs) tretment. (C) Bnds of representtive experiments. Anti- GRB2 nd nti-parp ntiodies were used s mrkers for the cytosolic nd nucler extrcts, respectively. (D) Percentge vlues of nucler levels of phosphorylted Nrf2 (1 kd) reltive to the control condition (mens ± SD, n=6) (E) Nucler/cytosolic Nrf2 (57 kd) rtio of nds densitometric quntifiction (mens SD, n=6). Normliztion of Western lots ws ensured y - ctin nd it ws not included in the plot due to the complexity of the figure. Mens without common letter differ, P<.5. Figure 2. Fluorescence imges of the sucellulr locliztion of quercetin. To visulize nuclei, 6- dimidino-2-phenylindole (DAPI) stining ws used. These results re representtive of two independent experiments. Figure 3. Time-dependent effects of quercetin on the sl levels of phosphorylted p38 (Thr18/Tyr182) nd totl p38. (A) Representtive lots. (B) Percentge dt of the p-p38/p38 rtio reltive to controls (mens SD, n =7). Normliztion of Western lots ws ensured y -ctin. Mens without common letter differ, P<.5. 3

31 Figure 4. Time-course effects of quercetin on cytosolic nd nucler Nrf2 levels (1 nd 57 kd), nd on DNA inding ctivity. (A) Bnds of representtive experiments. Anti-GRB2 nd nti-parp ntiodies were used s mrkers for the cytosolic nd nucler extrcts, respectively. (B) Percentge vlues of nucler levels of phosphorylted Nrf2 (1 kd) reltive to the control condition (mens ± SD, n=7) (C) Nucler/cytosolic Nrf2 (57 kd) rtio of nds densitometric quntifiction (mens SD, n=8). Normliztion of Western lots ws ensured y -ctin nd it ws not included in the plot due to the complexity of the figure. (D) DNA-inding ctivity of Nrf2 (mens SD, n=8). Asornce ws mesured t 45 nm. Mens without common letter differ, P<.5. Figure 5. Effects of quercetin nd SB2358 (SB) plus quercetin tretments on cytosolic nd nucler Nrf2 levels (1 nd 57 kd), nd on DNA inding ctivity t 4 nd 18 h. HepG2 cells were incuted with 5 μm quercetin for 4 h or 18 h in the presence or sence of 1 μm SB. (A) Bnds of representtive experiments fter 4 h tretment. (B) Percentge of 1 kd nucler Nrf2 reltive to control condition, (C) 57 kd nucler/cytosolic Nrf2 rtio (mens SD, n=5). (D) DNAinding ctivity of Nrf2 fter 4 h of exposure (mens SD, n=8). (E) Representtive lot fter 18 h of exposure. (F) Vlues of 1 kd nucler Nrf2 re shown s percent reltive to the control condition nd (G) 57 kd nucler/cytosolic Nrf2 rtio (mens SD, n=6). Anti-GRB2 nd nti- PARP ntiodies were used s mrkers for the cytosolic nd nucler extrcts, respectively. Normliztion of Western lots ws ensured y -ctin nd it ws not included in the plot due to the complexity of the figure. (H) DNA-inding ctivity of Nrf2 fter 18 h of incution (mens SD, n=8). Asornce ws mesured t 45 nm. Different letters on rs denote sttisticlly significnt differences, P<.5. Figure 6. Effects of quercetin, SB2358 (SB) nd cycloheximide (CHX) on cytosolic nd nucler Nrf2 levels (57 kd) t 18 h. HepG2 cells were incuted with 5 μm quercetin for 18 h in the 31

32 presence or sence of 1 μm SB nd/or 1 g/ml CHX. (A) Representtive lots. Anti-GRB2 nd nti-parp ntiodies, used s mrkers for the cytosolic nd nucler extrcts, respectively. (B) Nrf2 nucler/cytosolic rtio (57 kd, mens SD, n=6). Normliztion of Western lots ws ensured y -ctin, yet it ws not included in the plot due to the complexity of the figure. Mens without common letter differ, P<.5. Figure 7. Effects of quercetin nd SB2358 (SB) plus quercetin tretments on (A) GSH levels, (B) GPx, (C) GR nd (D) GST ctivities. HepG2 cells were incuted with 5 μm quercetin for 4 (white rs) or 18 h (lck rs) in the presence or sence of 1 μm SB. Vlues re mens of eight to twelve different smples per condition. Different letters on rs denote sttisticlly significnt differences, P<.5 Figure 8. Effects of quercetin nd SB2358 (SB) plus quercetin tretments on GPx, GR, GCS nd GST mrna levels fter (A) 4 h or (C) 18 h quercetin (5 μm) exposure. Representtive RT-PCR of three different experiments. Percentges of mrna levels of GPx, GR, GCS nd GST fter (B) 4 h or (D) 18 h (mens SD). Mens without common letter differ, P<.5. 32

33 Tle 1. Effect of quercetin on cell viility. HepG2 cells were treted with 5 μm quercetin for 4 nd 18 hours, respectively nd cell viility ws determined s reltive percent of crystl violet stined control cells. Dt represent mens SD of 8-1 seprte experiments. Mens in column without common letter differ, P<.5. Quercetin Time (hours) (5 μm) 4 h (% of vile cells) 18 h (% of vile cells) C Q Q+SB 1. ± ± ± ± ± ± 8.4 c 33

34 Nrf2 (nuc/cyt, 57 kd Opticl density p-p38/ p38 Opticl density (% Nrf2 (nuc, 1 kd Opticl density (% Figure 1. C Nrf2 (cyt, 57 kd) GRB2 A p-p38 p38 β-ctin Nrf2 (nuc, 1 kd) Nrf2 (nuc, 57 kd) PARP μm μm min 18 min B 24 min 18 min 3 c 2 c μm 24 min 18 min D E c c d μm c 24 min 18 min 1 c μm 34

35 Figure 2. (min) Q DAPI Q DAPI (min)