Modulation of Pro-survival Akt/Protein Kinase B and ERK1/2Signaling Cascades by Quercetin and Its in Vivo MetabolitesUnderlie Their Action on Neuronal Viability*

Received for publication, May 14, 2003Published, JBC Papers in Press, June 24, 2003, DOI 10.1074/jbc.M305063200

Jeremy P. E. Spencer, Catherine Rice-Evans, and Robert J. Williams‡

From the Wolfson Centre for Age-related Diseases, Guy’s, King’s and St. Thomas’ School of Biomedical Sciences,Hodgkin Building, King’s College, Guy’s Campus, London, SE1 9RT, United Kingdom

Much recent interest has focused on the potential offlavonoids to interact with intracellular signaling path-ways such as with the mitogen-activated protein kinasecascade. We have investigated whether the observedstrong neurotoxic potential of quercetin in primary cor-tical neurons may occur via specific and sensitive inter-actions within neuronal mitogen-activated protein ki-nase and Akt/protein kinase B (PKB) signaling cascades,both implicated in neuronal apoptosis. Quercetin in-duced potent inhibition of both Akt/PKB and ERK phos-phorylation, resulting in reduced phosphorylation ofBAD and a strong activation of caspase-3. High querce-tin concentrations (30 �M) led to sustained loss of Aktphosphorylation and subsequent Akt cleavage bycaspase-3, whereas at lower concentrations (<10 �M) theinhibition of Akt phosphorylation was transient andeventually returned to basal levels. Lower levels of quer-cetin also induced strong activation of the pro-survivaltranscription factor cAMP-responsive element-bindingprotein, although this did not prevent neuronal damage.O-Methylated quercetin metabolites inhibited Akt/PKBto lesser extent and did not induce such strong activa-tion of caspase-3, which was reflected in the loweramount of damage they inflicted on neurons. In con-trast, neither quercetin nor its O-methylated metabo-lites had any measurable effect on c-Jun N-terminal ki-nase phosphorylation. The glucuronide of quercetin wasnot toxic and did not evoke any alterations in neuronalsignaling, probably reflecting its inability to enter neu-rons. Together these data suggest that quercetin and toa lesser extent its O-methylated metabolites may induceneuronal death via a mechanism involving an inhibitionof neuronal survival signaling through the inhibition ofboth Akt/PKB and ERK rather than by an activation ofthe c-Jun N-terminal kinase-mediated death pathway.

Recent epidemiological and dietary intervention studies inboth humans and animals have suggested that diet-derivedphenolics, in particular the flavonoids, may play a beneficialrole in the prevention of neurodegeneration, age-related cogni-tive and motor decline (1, 2), and brain ischemia/reperfusioninjury (3). For example, the neuroprotective action of one of thegreen tea flavonoids, epigallocatechin gallate, has been shownin both oxidative stress- (4) and A�-induced (5) neuronal death

models. Protective effects in both systems were linked to amodulation in signaling through protein kinase C and/or mod-ulation of cell survival/cell cycle genes (4, 6). Much evidencealso exists to support the potential beneficial and neuromodu-latory effects of flavonoid-rich ginkgo biloba extracts, such asEGb 761, in the central nervous system (7–9). Clinical trialswith EGb 761, which contains kaempferol and quercetin, haveindicated beneficial effects on brain function, particularly inconnection with age-related dementias and Alzheimer’s disease(9, 10). However, although there is growing evidence in favor ofthe beneficial affects of flavonoids, there is still uncertaintyabout their actions in vivo and concern about their potentialtoxic side effects at higher concentrations. For instance, epigal-locatechin gallate has also been shown to exert pro-apoptotic aswell as anti-apoptotic effects in neuroblastoma cells (11),whereas quercetin has been observed to express no protectionagainst A�-induced neurotoxicity, whereas the structurally re-lated flavonols, apigenin and kaempferol, expressed significantprotection (12).

Although the flavonol quercetin is one of the most frequentlyresearched flavonoids, with evidence for both its beneficial (13,14) and deleterious effects (15, 16) on different cell types, itsmechanism of action remains unclear. Furthermore, recentevidence has shown that quercetin is extensively metabolizedto O-methylated and glucuronide metabolites during absorp-tion in the small intestine and in the liver (17, 18), and suchmetabolites should be taken into consideration to provide invivo relevance for any mechanism. Quercetin itself has beenthoroughly investigated for its abilities to express anti-prolif-erative effects (19, 20) and induce death predominantly by anapoptotic mechanism in cancer cell lines (21–23). For example,it has been observed to induce caspase-3 activation in themalignant cell line HPB-ALL (24), activate caspase-3 andcaspase-9, release cytochrome c in HL-60 cells (25), and inducechromatin and nuclear fragmentation in colonic cancer cells(20). On the other hand, quercetin treatment has been shown tosuppress the c-Jun N-terminal kinase (JNK)1 activity and apo-ptosis induced by hydrogen peroxide (26) and 4-hydroxy-2-nonenal (27). Furthermore, quercetin may evoke anti-apoptoticeffects via the suppression of the peroxide-induced JyNK-c-Jun/AP-1 pathway and the ERK-c-Fos/AP-1 pathway in cul-tured mesangial cells (28). The ability of quercetin to inhibitboth AP-1 activation and the JNK pathway (29) has beenshown to have relevance in both phorbol 12-myristate 13-ace-

* This work was supported by Biotechnology and Biological SciencesResearch Council Grant BBSRC 18/D14751. The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

‡ To whom correspondence should be addressed. E-mail: robert.williams@kcl.ac.uk.

1 The abbreviations used are: JNK, c-Jun N-terminal kinase; ERK,extracellular signal-regulated kinase; MAPK, mitogen-activated pro-tein kinase; PI, phosphatidylinositol; CREB, cAMP-responsive element-binding protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl; pAb,polyclonal antibody; PBS, phosphate-buffered saline; TBS, Tris-buff-ered saline; AM, acetoxymethyl ester; PKB, protein kinase B.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 37, Issue of September 12, pp. 34783–34793, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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tate- and tumor necrosis factor-�-induced intercellular adhe-sion molecule 1 (ICAM-1) expression.

The toxic effects of quercetin, or indeed other flavonoids, arelikely to involve an apoptotic mode of death in which membersof the mitogen-activated protein kinase (MAPK) family, such asJNK, may play a role (30). Alternatively, the modulation ofsignaling through the serine/threonine kinase, Akt/PKB, one ofthe main downstream effectors of phosphatidylinositol (PI)3-kinase and a pivotal kinase in neuronal survival (31–34),may also be important. There is considerable evidence linkingthe activation of the JNK pathway to neuronal apoptosis (35,36), and stress signaling through ERK has also been observedto be detrimental to neurons (37), although the latter has alsobeen shown to be pro-survival partly through the activation ofthe cAMP-responsive element-binding protein (CREB) (38, 39).Generally, an activation of JNK is considered pro-apoptotic,whereas activation of ERK and Akt are viewed as being pro-survival in neurons (4, 31).

In the study described here, we have investigated the mech-anisms underlying the neurotoxic effects of quercetin and itsmajor in vivo metabolites, 3�-O-methyl quercetin, 4�-O-methylquercetin, and quercetin-7-O-�-D-glucuronide, on primary cor-tical neurons in terms of the balance of influence betweenpro-survival pathways, such as the Akt/PKB cascade, and pro-death pathways, namely apoptotic signaling through JNKand caspase-3.

EXPERIMENTAL PROCEDURES

Materials—Specialized chemicals used were obtained from Sigma:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT), mammalian proteaseinhibitor mixture, caspase-3 peptide substrate, acetyl-Asp-Glu-Val-Aspp-nitroanilide, caspase-3 inhibitor, acetyl-Asp-Glu-Val-Asp-al, and re-combinant caspase-3 positive control. Quercetin, 3�-O-methyl quercetin(isorhamnetin) and 4�-O-methyl-quercetin (tamarixetin) were pur-chased from Extrasynthese (Genay Cedex, France). Quercetin-O-7-�-D-glucuronide was synthesized as described previously (40). The antibod-ies used were anti-phospho-Akt (Ser473) pAb and total Akt pAb (CellSignaling Technology Inc.); anti-phospho-Akt (Thr308) pAb, anti-phos-pho-BAD (Ser136) pAb, and anti-BAD monoclonal antibody (UpstateBiotechnology, Inc., Lake Placid, NY); anti-phospho-CREB (Ser133) pAb(Calbiochem, La Jolla, CA); anti-ACTIVE MAPK (ERK1/2) pAb andanti-ACTIVE JNK pAb (Promega, Madison, WI); total ERK2 pAb andtotal JNK1 pAb (Santa Cruz Biotechnology, Santa Cruz, CA). Horse-radish peroxidase-conjugated goat anti-rabbit secondary antibody (Sig-ma) and ECL reagent and Hyperfilm-ECL were purchased from Amer-sham Biosciences. Elgastat UHP double distilled water (18.2 M� grade)was used throughout the study. All other reagents used were of theanalytical grade and obtained from Sigma. All of the other reagentswere obtained from Sigma or Merck.

Cell Culture and Quercetin Exposure—Primary cultures of mousecortical neurons were prepared as described previously (41, 42). Theneurons were plated onto 6- and 24-well Nunc multiwell plates that hadbeen precoated overnight with poly-L-ornithine and then with 10%heat-inactivated fetal bovine serum (Invitrogen) for 2 h. Followingremoval of the final coating solution, the cells were plated (106/ml) in aserum-free medium composed of a mixture of Dulbecco’s modified Ea-gle’s medium and F-12 nutrient (1:1 v/v) supplemented with 33 mM

glucose, 2 mM glutamine, 6.5 mM sodium bicarbonate, 5 mM HEPES, pH7.4, 100 �g/ml streptomycin, and 60 �g/ml penicillin (all from Invitrogen).A mixture of hormones and salts composed of 25 �g/ml insulin, 100 �g/mltransferrin, 60 �g/ml putrescine, 20 nM progesterone, and 30 nM sodiumselenate (all from Sigma) was also added to the culture medium. The cellswere cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2,and after 5–7 days the vast majority of cells were neuronal (�98%) with�2% astrocytes as determined by �-tubulin and glial fibrillary acidicprotein immunocytochemistry, respectively (not shown).

Primary cortical neurons (106 cells/ml; 24-well plates) were exposedto quercetin, 3�-O-methyl- and 4�-O-methyl-quercetin and quercetin-7-O-�-D-glucuronide (0.3–10 �M) for 6 h. The time dependence was alsoinvestigated by the addition of quercetin, 3�-O-methyl- and 4�-O-meth-yl-quercetin and quercetin-7-O-�-D-glucuronide (3–30 �M) for 0.5, 2, or6 h. Following all exposures, the medium was removed and replacedwith fresh conditioned medium, and the neurons were incubated for up

to a total of 24 h (exposure and reincubation for 6 and 18 h, respectively)before assessment of neuronal viability. In addition, the assays forneuronal damage were also performed immediately following the expo-sures (i.e. 0.5, 2, or 6 h). For analysis of signaling proteins, the neurons(106 cells/ml) were grown on 6-well Nunc plates. The neurons wereexposed to quercetin, 3�-O-methyl-quercetin, 4�-O-methyl-quercetin,and quercetin-7-O-�-D-glucuronide (0.3–30 �M) for 0.5, 2, or 6 h. Di-rectly following exposure, the neurons were washed twice with ice-coldPBS, pH 7.4, containing EGTA/EDTA (200 �M) and lysed on ice beforepreparation of samples for immunoblotting.

Assessment of Neuronal Damage—Cellular damage elicited by treat-ments was evaluated by measuring MTT reduction (41, 43), by utilizingthe Dead or Alive assay (Molecular Probes, Eugene, OR), and by mor-phological examination (38, 41). Following exposure of neuronal cul-tures to quercetin and its metabolites, the cultures were washed twicewith sterile PBS before the addition of MTT (0.5 mg/ml) in HEPES-buffered medium (5 mM HEPES, 154 mM NaCl, 4.6 mM KCl, 2.3 mM

CaCl2, 33 mM glucose, 5 mM NaHCO3, 1.1 mM MgCl2, 1.2 mM Na2HPO4).Following incubation (60 min; 37 °C), MTT solutions were removed, andthe formazan product was solubilized in Me2SO, and the absorbanceread at 505 nm. The results were expressed as the percentages ofprotection against the decline in MTT reduction.

Cell survival was further assessed using calcein AM and ethidiumhomodimer (Molecular Probes). Calcein AM is taken up into living cellsand is metabolized in the cytoplasm by esterases to the green fluores-cent product, calcein, so that viable cells show a uniform green fluores-cence (emission, 530 nm) under appropriate excitation (495 nm).Ethidium homodimer is excluded from living cells but can cross thecompromised plasma membrane of dying cells and interact with nucleicacids to give a strong red fluorescence (emission, 530 nm) under appro-priate excitation (495 nm). At the end of the experiment exposures, theneurons were washed, calcein AM (4 �M) and ethidium homodimer 2�M) were added, and the neurons were incubated for 30 min at 37 °C.The fluorescence was measured using a SPECTRAmax® Gemini micro-plate spectrofluorometer (Molecular Devices). The temperature wasmaintained at 25 °C, and the emission was recorded at 530 and 645 nmfor calcein and ethidium, respectively, after exciting at 495 nm. Eachwell was scanned in the well scan mode accumulating data from 21independent points/well, which were then transformed in an averagesignal expressed in relative light units. All data were calculated andnormalized with respect to the increase of fluorescence of a control. Thepercentages of living and dead cells were determined and analyzedstatistically by Student’s t test.

The morphological assessment of neuronal damage was made byanalysis of phenotypic markers under light microscopy using a Nikoneclipse T5100 at 40� magnification. The images were captured using aNikon E995 digital camera fitted with a Coolpix MDC lens adaptor.

Immunoblotting—Immunoblotting analysis was performed essen-tially as previously described (44) with minor modifications. Followingexposures, the neurons were washed with ice-cold PBS with 200 �M

EGTA and lysed on ice using 50 mM Tris, 0.1% Triton X-100, 150 mM

NaCl, and 2 mM EGTA/EDTA containing mammalian protease inhibi-tor mixture (1:100 dilution), 1 mM sodium pyrophosphate, 10 �g/mlphenylmethylsulfonyl fluoride, 1 mM sodium vanadate, and 50 mM

sodium fluoride. The lysed cells were scraped and left on ice to solubilizefor 45 min. The lysates were centrifuged at 1,000 � g for 5 min at 4 °Cto remove unbroken cell debris and nuclei. The protein concentration inthe supernatants was determined by the Bio-Rad Bradford proteinassay®. The samples were incubated for 5 min at 95 °C in boiling buffer(final concentration, 62.5 mM Tris, pH 6.8, 2% SDS, 5% 2-mercaptoeth-anol, 10% glycerol, and 0.0025% bromphenol blue). The boiled samples(20–30 �g/lane) were run on 9–12% SDS-polyacrylamide gels, and theproteins were transferred to nitrocellulose membranes (Hybond-ECL®;Amersham Biosciences) by semi-dry electroblotting (1.5 mA/cm2). Thenitrocellulose membrane was then incubated in a blocking buffer (20mM Tris, pH 7.5, 150 mM NaCl; TBS) containing 4% (w/v) skimmed milkpowder for 30 min at room temperature followed by two 5-min washesin TBS supplemented with 0.05% (v/v) Tween 20 (TTBS). The blots werethen incubated with either anti-ACTIVE MAPK pAb (1:5000 dilution),anti-ACTIVE JNK pAb (1:5000), anti-phospho-Akt (Ser473) pAb(1:1000), anti-phospho-Akt (Thr308) pAb (1:1000), anti-phospho-CREB(Ser133) (1:1000), anti-phospho-BAD pAb (1:1000), anti-Bad, cloneBYC001 monoclonal antibody (1:1000), anti-ERK1/ERK2 pAb (1:1000),or anti-JNK1 pAb (1:1000) in TTBS containing 1% (w/v) skimmed milkpowder (antibody buffer) overnight at room temperature on a three-dimensional rocking table. The blots were washed twice for 10 min inTTBS and then incubated with goat anti-rabbit IgG conjugated tohorseradish peroxidase (1:1000 dilution), rabbit anti-sheep IgG conju-

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gated to horseradish peroxidase (1:2000 dilution), or goat anti-mouseIgG conjugated to horseradish peroxidase (1:3000 dilution; UpstateBiotechnology, Inc.), in antibody buffer for 60 min. Finally the blotswere washed twice for 10 min in TTBS rinsed in TBS and exposed toECL® reagent for 1–2 min as described in the manufacturer’s protocol(Amersham Biosciences). The blots were exposed to Hyperfilm-ECL®(Amersham Biosciences) for 2–5 min in an autoradiographic cassetteand developed. The bands were analyzed using BioImage® IntelligentQuantifier software (Ann Arbor, MI). The molecular weights of thebands were calculated from comparison with prestained molecularweight markers (molecular weight, 27,000–180,000 and 6,500–45,000;Sigma) that were run in parallel with the samples. The equal loadingand efficient transfer of proteins was confirmed by staining the nitro-cellulose with Ponceau Red (Sigma).

Caspase-3 Activity—The neurons (106/ml) were pretreated with quer-cetin, 3�-O-methyl-quercetin, 4�-O-methyl-quercetin, and quercetin-7-�-D-glucuronide (0.3–30 �M) for 6 h. In a separate series of experiments,the time course of activation was investigated using a fixed 30 �M

exposure for 0.5, 2, and 6 h. Following exposures, the medium wasremoved, and the neurons were incubated with fresh conditioned me-dium. After a total of 12 h (exposure and reincubation for 6 and 6 h,respectively), the cells were washed twice with ice-cold PBS (� EGTA200 �M) and lysed on ice as described above. The lysates were scrapedfrom the plates and incubated on ice for 45 min before centrifugation(microcentrifuge, 1000 � g), and the supernatant was collected.Caspase-3-like protease activity in neuronal lysates was assessed basedon the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide by caspase-3, resulting in the release of the p-nitroanilinemoiety, which absorbs at 405 nm. Quantification of the absorbance at405 nm was carried out using a Versa Max UV microplate reader(Molecular Devices). Suitable control experiments were performed us-ing the caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-al. All of the dataare presented as absorbance readings at 405 nm.

Statistical Analysis—The data are expressed as the means � S.D.Statistical comparisons were made using an unpaired, two-tailed Stu-dent’s t test with a confidence level of 95%. The significance level wasset at p � 0.05.

RESULTS

Neurotoxicity of Quercetin and Its in Vivo Metabolites—Ex-posure of neurons to quercetin (0.3–30 �M) for 6 h resulted in aconcentration-dependent increase in neuronal damage as evi-denced by a decreased ability of neurons to reduce MTT whenmeasured 24 h following the exposure (6-h exposure and 18-hreincubation) (Fig. 1A). Significant neuronal injury was ob-served at quercetin exposures as low as 1 and 3 �M (p � 0.01),although this was increased substantially following treatmentwith 10 and 30 �M concentrations. The two O-methylated me-tabolites of quercetin also induced an impairment of neuronalfunction that was concentration-dependent (Fig. 1A). However,the amount of damage induced was much lower than that seenwith quercetin and only reached significance after exposure to10 and 30 �M (p � 0.01). Notably, the neuronal injury observedfollowing exposure to the flavonols for 6 h was observed only18 h after the exposure and not immediately following the 6-hexposure: quercetin, 95.7% � 3.4%; 3OmeQ, 96.3% � 2.6%;4OmeQ, 97.9% � 1.6%; Q glucuronide, 98.3% � 1.2%; mean �S.D., n � 4). Quercetin-7-O-�-D-glucuronide did not inducetoxicity at any concentration. The neurotoxicity of quercetinwas also apparent at shorter exposure times. For example,quercetin (10 and 30 �M) expressed significant toxicity follow-ing exposure for 0.5, 2, and 6 h, whereas quercetin (3 �M) onlyinduced significant neuronal injury following 2- and 6-h expo-sures (Fig. 1B). Again this toxicity was only apparent 24 hpost-stress, and no damage was apparent immediately follow-ing quercetin exposure. 3�-O-methyl quercetin (30 �M) and4�-O-methyl quercetin (30 �M) also induced significant neuro-nal injury at 2 h (p � 0.01), although this was small in com-parison with quercetin (Fig. 1C).

To establish further the neurotoxic properties of quercetin,the percentage of dead and alive cells were measured followingexposure. The percent of “alive” neurons was established based

on the cytoplasmic esterase conversion of calcein AM to thegreen fluorescent product, calcein, by living cells. The percent-age of “dead” neurons was estimated on the basis that ethidium

FIG. 1. Effects of quercetin and its major in vivo metabolites onneuronal damage as assessed by the MTT assay. A, neurons wereexposed to quercetin 3�-O-methyl quercetin, 4�-O-methyl quercetin, andquercetin-7-O-�-D-glucuronide (0.3–30 �M) for 6 h, and neuronal dam-age was assessed by the MTT assay 24 h post-initial stress. The datapoints are the means of three separate experiments, each performed intriplicate and presented � S.D. B, time course of neuronal damageinduced by quercetin as measured by the MTT assay. The neurons wereexposed to quercetin (3 �M, white bars; 10 �M, black bars; and 30 �M,gray bars) for 0.5, 2, or 6 h, and the damage was assessed 24 hpost-initial stress. C, time course of neuronal damage induced by quer-cetin metabolites as measured by the MTT assay. Quercetin, 3�-O-methyl quercetin, 4�-O-methyl quercetin, and quercetin-7-O-�-D-glucu-ronide (30 �M) were exposed to neurons for 0.5, 2, or 6h and damage wasassessed 24 h post-initial stress. f, quercetin; ●, 3 �-O-methyl quercetin;Œ, 4�-O-methyl quercetin; �, quercetin-7-�-D-glucuronides.

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homodimer is excluded from living cells and will only crosscompromised plasma membranes of dying cells and interactwith nucleic acids to give a strong red fluorescence. Quercetinexposure (0.3–30 �M) for 6 h resulted in a concentration-de-pendent reduction in the percentage of viable neurons and acorresponding concentration-dependent increase in the per-centage of dead cells at 24 h (Fig. 2A). The increase in dead cellsindicates the loss of membrane integrity after exposure toquercetin. However, as with the MTT measurements, damagewas only observed 18 h after the completion of the flavonoidtreatment with no increase apparent immediately following thequercetin 6-h exposure (Fig. 2A), indicating that at this timethere has been no alteration in neuronal membrane integrity.Morphological analyses using light microscopy also highlightedthis delayed neuronal damage in that the morphological ap-

pearance of cortical neurons was dramatically affected 24 hpost-quercetin exposure (30 �M; 6- and 18-h reincubation) butnot immediately following the addition (Fig. 2B). Control cul-tures of cortical neurons exhibit a large intact cell body as wellas a finely developed dendritic network (38, 42) (Fig. 2B, panelI). Following exposure to quercetin (10 or 30 �M) for 6 h andreincubation for 18 h, the neuronal cell bodies appearedshrunken, and there was a significant loss of dendrites (Fig.2B, panels III and IV). In contrast, no significant loss of thedendritic network was observable immediately after the 6-hquercetin exposure, and similarly there was no alteration incell body morphology (Fig. 2B, panel II).

Effects of Quercetin on Akt/PKB Signaling—To begin explor-ing the mechanism of quercetin-induced neurotoxicity, we ex-amined the effect of quercetin on Akt/PKB, an essential kinasein neuronal survival and a downstream effecter of PI 3-kinase(31, 32). Activation of the PI 3-kinase/Akt signaling pathway inneurons has been strongly implicated in the regulation of neu-ronal survival and/or protection (4, 45). We investigatedwhether quercetin could influence Akt phosphorylation in cor-tical neurons by immunoblotting neuronal homogenates withan anti-phospho-Akt polyclonal antibody that detects Akt whenit is phosphorylated at Ser473, an event known to be essentialfor full activation of the kinase. Exposure of cortical neurons toquercetin (10 and 30 �M) for 0.5, 2, or 6 h resulted in a markeddecrease in Akt phosphorylation relative to control neurons, asdemonstrated by a robust decrease in the relative intensity ofthe immunodetectable band relating to phosphoryated Akt/PKB (60 kDa) (Fig. 3, A and D). The changes in Akt phos-phorylation were both time- and concentration-dependent. Lev-els of phosphoryated Akt in neurons exposed to quercetin (10�M) were greatly reduced at early exposure times (0.5 and 2 h)but showed some recovery at the 6-h time point with levelssignificantly higher (p � 0.01) at this time than at the earlierexposure times. In contrast, exposure to quercetin (30 �M)resulted in a concentration-dependent reduction in phospho-Akt levels with pAkt undetectable following a 6-h exposure. Asimilar decrease in the relative intensity of the immunodetect-able band relating to phosphoryated threonine 308 (within thecatalytic domain of Akt) was also observed following a 2-hquercetin exposure (Fig. 3C), which matched the decrease inpAkt (Ser473). Parallel immunoblots with an antibody thatdetects total Akt protein levels (nonphosphorylated and phos-phorylated Akt) were performed. There was no change in totalAkt at 0.5 h of exposure. Interestingly, there were changes intotal levels of Akt in response to high quercetin concentrationsand at longer exposure times (Fig. 3B), most dramatically inneurons exposed to quercetin (30 �M) for 6 h.

Effect of Quercetin on Phosphorylation of ERK1/2 andJNK1/2—There was a clear concentration-dependent inhibi-tion of pAkt (Ser473) following exposure of cortical neurons toquercetin for 0.5, 2, and 6 h (Fig. 4, A and B). However, at the6-h exposure time there was significant recovery (p � 0.01) inAkt/PKB phosphorylation toward control levels in neurons ex-posed to 3 and 10 �M of quercetin (Fig. 4B) compared with theearlier exposure times. ERK has also been implicated in neu-ronal death/survival signaling (37, 39). Therefore it was ofrelevance to investigate the effect of quercetin and its metab-olites on this kinase. The phosphorylation state of ERK1/2 wasprobed using a phospho-specific antibody, which recognizes thedually phosphorylated motif pTEpY within activated ERK1/2.Incubation of cortical neurons with quercetin (0.3–30 �M; 0.5 h)resulted in a strong dose-dependent decrease in the phospho-rylation below basal levels in the two bands corresponding toERK1 (44 kDa) and ERK2 (42 kDa) (Fig. 4C).

JNK has been implicated in pro-apoptotic signaling in neu-

FIG. 2. Quercetin-induced neuronal injury assessed by meas-urement of dead or alive cells and morphological changes. Theneurons were exposed to quercetin (0.3–30 �M) for 6 h, and living anddead cells were assessed either immediately (�, dead cells; Œ, alivecells) or following a reincubation for 18 h (f, dead cells; ●, alive cells).Living cells were characterized by calcein AM uptake and metabolismto calcein (emission, 530 nm; excitation, 495 nm), and dead cells werecharacterized by the uptake and interaction of ethidium homodimerwith nucleic acids (emission, 530 nm; excitation, 495 nm). B, morpho-logical assessment of neuronal damage induced by quercetin was madeby analysis of phenotypic markers under light microscopy. Panel I,methanol vehicle (6 and 18 h of reincubation); panel II, quercetin (30�M; 6 h); panel III, quercetin (10 �M; 6 and 18 h of reincubation; panelIV, quercetin (30 �M; 6 and 18 h of reincubation). Scale bar, 40 �m.

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rons (35), and consequently we investigated the ability of quer-cetin and its metabolites to modulate the phosphorylation ofthis kinase. The phosphorylation state of JNK was investigatedby immunoblotting of neuronal homogenates with anti-activeJNK1/2 antibody. This antibody detects JNK when it is duallyphosphoryated within the Thr138-Pro-Tyr185 motif (pTPpY) inthe catalytic core of active JNK. Treatment of cortical neuronswith quercetin (0.3–30 �M) for 2 h resulted in no measurablealteration in the relative intensities of the two immunodetect-able bands, corresponding to the activated JNK isoforms, JNK(54 kDa) and JNK (46 kDa), as compared with basal levels(Fig. 4D).

The Effects of Quercetin Metabolites on the Phosphorylationof Akt and JNK1/2—When considering the effects of flavonoidsin culture models, it is vital to investigate the effects of itsactual in vivo metabolite forms. In contrast to the dramaticinhibition of phospho-Akt (Ser473) induced by quercetin (30 �M;2 h), the two O-methylated metabolites of quercetin induced amuch smaller reduction in the basal phosphorylation of Akt(Ser473) (Fig. 5A). Exposure of cortical neurons to either 3�-O-methyl quercetin or 4�-O-methyl quercetin (30 �M) for 2 hresulted in 67.2 and 62.4% reductions in Akt phosphorylation,respectively, relative to control cells, as demonstrated by thedecrease in the relative intensity of the immunodetectableband relating to phosphoryated Akt/PKB (Ser473) (Fig. 5, A andD). In contrast, quercetin-7-�-D-glucuronide had no significanteffect on the basal state of Akt (Ser473) phosphorylation incortical neurons. In addition, the metabolites did not signifi-cantly alter the levels of total Akt in neurons following expo-sure (Fig. 5B), and neither quercetin nor any of its metabolitesinduced a change in basal levels of phospho-JNK1/2 (Fig. 5C).

Effect on the Phosphorylation of BAD and CREB—Akt main-tains neuronal viability by regulating the actions of down-stream effectors such as BAD and CREB (46). The pro-apo-

ptotic Bcl-2 family member, BAD, was the first cell deathcomponent to be identified as a regulatory target of survivalsignaling (47). It has been shown to be under direct regulatorycontrol by Akt/PKB, which specifically phosphorylates BAD atthe Ser136 (46, 48) an event essential for the inhibition ofapoptosis. We were interested to investigate the effect of quer-cetin on this important anti-apoptotic molecule, especially be-cause a marked inhibition of Akt/PKB had been observed byquercetin. BAD phosphorylation in cortical neurons was under-taken by immunoblotting neuronal homogenates with anti-phospho-BAD polyclonal antibody that detects BAD whenphosphorylated at Ser136. Exposure of cortical neurons for 6 hto quercetin (10 or 30 �M) led to a significant reduction in BADphosphorylation (Fig. 6A). Interestingly, the inhibition of BADphosphorylation by quercetin paralleled the changes seen inthe phosphorylation state of Akt/PKB at 6 h (Fig. 3A). Parallelblots were run and probed with an antibody that detects totallevels of BAD. The exposure to quercetin did not alter the totallevels of BAD (Fig. 6A), which also confirmed equal proteinloading. In contrast to experiments with quercetin, there wereno observable changes in the BAD phosphorylation state whencortical neurons were exposed to either 3�-O-methyl quercetin,4�-O-methyl quercetin or the glucuronidated quercetin (30 �M;6h) (data not shown). This lack of alteration in the BAD phos-phorylation state in the presence of Akt/PKB inhibition by theO-methylated metabolites may reflect a requirement for athreshold of Akt deactivation to occur prior to BAD inhibition.

Another possible downstream target of Akt/PKB, and indeedERK, is CREB, a pro-survival transcription factor of impor-tance in neurons (39, 49). We were interested in whether quer-cetin-induced inhibition of both Akt/PKB and ERK could trans-duce downstream to affect CREB phosphorylation/activation.However, exposure of cortical neurons to quercetin (3, 10, and30 �M) for 0.5 h resulted in an increase in CREB phosphorylation

FIG. 3. Inhibition of Akt/PKB phos-phorylation by quercetin in primarycortical neurons exposed to querce-tin. A, crude lysates (20 �g) preparedfrom cultured cortical neurons exposed tovehicle (MeOH) or quercetin (10 or 30 �M)for 0.5, 2, or 6 h were immunoblotted withan antibody that specifically recognizesphosphorylated Akt (Ser473). B, the samecrude lysates (20 �g) immunoblotted withan antibody that recognizes total levels ofAkt (Total Akt). C, crude lysates (20 �g)prepared from cultured cortical neuronsexposed to vehicle (MeOH) or quercetin(10 or 30 �M) for 2 h were immunoblottedwith an antibody that specifically recog-nizes phosphorylated Akt (Thr308). BlotsB and C are representative blots of threeindependent experiments on different cul-tures that yielded similar results. D, dataobtained from immunoblot experimentsrepresented in A were analyzed usingBioimage Intelligent Quantifier software.Each column represents the mean � S.D.of four independent experiments. *, p �0.01.

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relative to control neurons, as demonstrated by a significantincrease in the relative intensity of the immunodetectable bandrelating to phosphoryated CREB (Fig. 6, B and C). The activationof CREB was most significant in neurons exposed to quercetin (3or 10 �M) for 2 h. The increase in CREB activation was unex-pected and did not reflect upstream quercetin-induced Akt/PKBand ERK1/2 inhibition, although neuronal exposure to quercetin(30 �M) for 2 h did result in a strong inhibition of CREB.

Caspase-3 Activation—Both Akt/PKB (31) and MAPK (35)signaling have been implicated in the activation of CED-3/ICE-like proteases. Although we observed no increases in JNKactivation in response to quercetin or its metabolites, it ispossible that caspase-activation may occur following the reduc-tion of Akt and BAD phosphorylation, which lead to the releaseof cytochrome c, the formation of the apoptosome and subse-quent activation of effecter caspases such as caspase-3 andcaspase-7 (reviewed in Refs. 31 and 32). We therefore investi-gated the potential activation of caspase-3 by the quercetincompounds. Exposure of neurons to quercetin (0.3–30 �M) for

6 h led to an enhanced activity of caspase-3 in neurons, whichwas apparent as an increase in absorbance at 405 nm reflectingthe increased cleavage of the caspase-3 specific substrate Ac-DEVD-p-nitroanalide (Fig. 7A). Levels of caspase-3 activitywere increased markedly in neurons exposed to quercetin, inparticular those exposed to concentrations of �3 �M. In con-trast, exposure of neurons to 3�-O-methyl quercetin and 4�-O-methyl quercetin led only to increases in caspase-3 activity atconcentrations of 3 �M and above, whereas quercetin-7-O-�-D-glucuronide failed to stimulate significant caspase-3 activationat any concentration. Measurable increases in caspase-3 activ-ity were detected after a 30-min exposure to quercetin (30 �M),and this level increased markedly after 2 h but did not signif-icantly differ from that measured at 6h (Fig. 7B). In contrast,the two O-methylated metabolites of quercetin induced a time-dependent increase in caspase-3 activity. The glucuronide ofquercetin was ineffective at inducing the activity of caspase-3at any time point.

Effects of Caspase-3 Activation on Loss of Total Akt—Apo-

FIG. 4. Phosphorylation of Akt,ERK1/2, and JNK1/2 in cortical neu-rons exposed to quercetin (0.3–30�M). Crude lysates (20 �g) prepared fromcultured cortical neurons exposed to vehi-cle (MeOH) or quercetin (0.3, 1, 3, 10, or30 �M) for 0.5, 2, or 6 h were immuno-blotted with an antibody that specificallyrecognizes phosphorylated Akt (Ser473).B, data obtained from immunoblot exper-iments represented in A were analyzedusing Bioimage Intelligent Quantifiersoftware. Each column represents themean � S.D. of four independent experi-ments. *, p � 0.01. C and D, the samelysates (20 �g) (quercetin 30 �M; 2 h) im-munoblotted with an antibody that specif-ically recognizes the dually phosphoryl-ated region of the active form of ERK1and ERK2 (pERK1/2) (C) and the duallyphosphorylated region of the active formof JNK1 and JNK2 (pJNK) (D).

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ptotic proteases have been observed to cleave and inactivatesurvival signaling molecules such as Akt/PKB (50, 51), phos-pholipase C�1 (52), and Bcl-2. To investigate a possible linkbetween the increases in caspase-3 activity in neurons seen inour experiments and the observed loss of total Akt in neuronsexposed to quercetin (30 �M), a specific caspase-3 inhibitor wasemployed. Pretreatment of neurons with the caspase-3 inhibi-tor for 15 min prior to the addition of quercetin (30 �M) signif-icantly reduced the depletion in total Akt after 2 or 6 h ofexposure (Fig. 8A). This was particularly striking at 6 h, whereAkt loss was most apparent. This prevention of total Akt losswas paralleled by a decrease in the activity of caspase-3 inneuronal lysates treated with the inhibitor (Fig. 8B). Further-more, neurons pretreated with the inhibitor prior to quercetinexposure (3, 10, and 30 �M; 6 h) suffered significantly lessneuronal damage, as measured by their ability to reduce MTT,compared with neurons treated with quercetin alone (Fig. 8C),which was most significant at the lower quercetin exposures.

DISCUSSION

Much effort to characterize the potential activities of fla-vonoids in vivo has centered on the flavonol quercetin, foundwidely in the human diet and generally regarded be theflavonoid with the greatest antioxidant potential (53). How-ever, although quercetin is a highly effective radical scav-enger, its low redox potential has also been used to ascribe toits occasional pro-oxidative (54) and cytotoxic (40) behavior.We have shown that quercetin and its O-methylated metab-

olites are toxic to cortical neurons via inhibition of pro-sur-vival protein kinase cascades. Low concentrations (�10 �M)were observed to induce a reversible inhibition of Akt phos-phorylation and an enhancement of a potential pro-survivalresponse in the form of increased CREB phosphorylation. Incontrast, higher concentrations (30 �M) induced a sustaineddeactivation of Akt/PKB, extensive caspase-3 activation, andsubsequent caspase-dependent cleavage of anti-apoptoticAkt/PKB. This concentration-dependent effect on neuronalsignaling pathways is important because the levels accumu-lated in vivo are unclear.

The toxicity of quercetin at higher concentrations (50 �M to100 mM) toward cells in culture, in particular cancer cells, iswell established and has been linked to an apoptotic mode ofdeath (15, 16, 21–23). However, little is known regarding theprecise mechanism of quercetin-induced toxicity in other cellsystems, and nothing is known of its effects on neurons. Inaddition, it is difficult to relate this to what occurs in vivobecause during absorption from the gastrointestinal tract tothe circulation quercetin and its glycosides are metabolized to3�-O-methyl quercetin, 4�-O-methyl quercetin, and quercetin-7-�-D-glucuronide (as well as sulfates) by the action of phase Iand II enzymes present in the small intestine and liver (17).These metabolic processes lead to relatively low amounts ofquercetin and higher levels of the metabolites in the circula-tion. Furthermore, the cells in culture readily accumulate rel-atively high amounts of quercetin and its O-methylatedmetabolites, which result in further intracellular oxidative

FIG. 5. Phosphorylation of Akt andJNK1/2 in cortical neurons exposedto quercetin and its metabolites,3�-O-methyl quercetin (3OMeQ), 4�-O-methyl quercetin (4OMeQ), and quer-cetin-7-O-�-D-glucuronide (Q glucu-ron). A, crude lysates (20 �g) preparedfrom cultured cortical neurons exposed tovehicle (MeOH), quercetin (30 �M),3OMeQ (30 �M), 4OMeQ (30 �M), or Qglucuron (30 �M) for 6 h were immuno-blotted with an antibody that specificallyrecognizes phosphorylated Akt (Ser473)(A), total Akt (B), or the dually phospho-rylated region of the active form of JNK1and JNK2 (pJNK1/2) (C). D, data ob-tained from immunoblot experiments rep-resented in A were analyzed using Bioim-age Intelligent Quantifier software. Eachcolumn represents the mean � S.D. offour independent experiments. *, p �0.01.

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metabolism, glutathionylation, and conversion of O-methyl-ated metabolites to free quercetin (40). Potential toxicity offlavonoids such as quercetin toward neuronal cells in vivo maybe limited by the action of the blood-brain barrier preventingthe entry of such compounds to the brain. However, recentevidence has demonstrated that flavonoids and some of theirmetabolites are able to traverse the blood-brain barrier andthat the potential for permeation is consistent with compoundlipophilicity (55). Indeed quercetin, and especially its O-meth-ylated metabolites, have relatively high lipophilicity, indicat-ing the potential brain access of these compounds.

Whereas earlier investigations into flavonoid bioactivity invitro and in vivo concentrated mainly on their ability to act aseither as free radical scavengers and antioxidants or as pro-oxidants, much recent interest has concentrated on their po-tential to interact with intracellular signaling pathways suchas the MAPK cascade (29, 41, 56). For example, epicatechinand its 3�-O-methylated metabolite protect primary striatalneurons against oxidized low density lipoprotein-induced deathby potently inhibiting oxidized low density lipoprotein-inducedactivation of JNK1/2, c-Jun, and caspase-3 (41). These datasuggest that flavonoids might exert effects on neurons viadirect interactions with signaling proteins in reactions inde-pendent of their antioxidant ability. The speculation that theiractions may occur via interactions with neuronal signalingcascades implicated in apoptosis led us to investigate whetherthe observed strong neurotoxic actions of quercetin and itsmetabolites toward primary cortical neurons reflect their sen-

sitive interactions within pro-survival MAPK and Akt/PKBsignaling cascades.

These investigations provide evidence that low micromolarconcentrations of quercetin, and to a lesser extent its O-meth-ylated metabolites, are potently neurotoxic toward cortical neu-rons, whereas quercetin-7-O-�-D-glucuronide has no effect. Thetoxicity caused by quercetin and its O-methylated metaboliteswas characterized both by reductions in neuronal viability andloss of membrane integrity, although both markers of neuronaldamage were only observed 24 h post-exposure and not directlyfollowing the treatments. Prior to measurable losses of neuro-nal viability and membrane integrity, quercetin stimulated astrong inhibition of basal Akt phosphorylation in cortical neu-rons that was both time- and concentration-dependent. Thisinhibition of Akt phosphorylation was apparent at both theregulatory serine 473 and catalytic threonine 308 sites, render-ing it inactive. High quercetin concentrations (30 �M) led torapid inhibition of Akt phosphorylation, which was sustainedup to 6 h and was accompanied by reductions in total Aktprotein levels. In contrast, lower concentrations of quercetinresulted in a transient inhibition of pAkt, with levels of pAkt(Ser473) and total Akt not significantly different from controllevels at 6 h, despite a strong initial inhibition of pAkt. Thistransient effect could be due to reversible inhibition and/ormetabolism of quercetin intracellularly, as we have shown pre-viously (40). Although the two O-methylated metabolites ofquercetin also induced reductions in basal phospho-Akt(Ser473), the level of inhibition was less than that caused by

FIG. 6. Effect of quercetin exposureon the downstream partners of Aktand ERK. A, effect of quercetin on BADphosphorylation. Phosphorylation of BADin cortical neurons exposed to quercetin(10 or 30 �M) for 6 h. Crude homogenates(30 �g) prepared from cultured corticalneurons exposed to vehicle (MeOH), quer-cetin (10 �M), or quercetin (30 �M) for 6 hwere immunoblotted with an antibodythat specifically recognizes phosphoryl-ated BAD (Ser136). B, CREB phosphoryl-ation in cortical neurons exposed to quer-cetin (3, 10, or 30 �M) for 0.5 or 2 h. Crudehomogenates (20 �g) prepared from cul-tured cortical neurons exposed to vehicle(MeOH) or quercetin (3, 10, or 30 �M) for0.5, 2, or 6 h were immunoblotted with anantibody that specifically recognizesphosphorylated CREB (Ser133). C, dataobtained from immunoblot experimentsrepresented in B were analyzed usingBioimage Intelligent Quantifier software.Each column represents the mean � S.D.of four independent experiments. *, p �0.01.

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quercetin and was not accompanied by changes in total Akt.The inhibition of Akt phosphorylation by quercetin and itsO-methylated metabolites paralleled the neuronal toxicitydata, with the O-methylated metabolites being less neurotoxicthan quercetin. This was further highlighted by the fact thatthe glucuronide of quercetin, too polar to enter cells, expressedno significant toxicity and no ability to inhibit the phosphoryl-ation of Akt/PKB. The inhibition of Akt/PKB phosphorylationby quercetin seen in our neuronal system may reflect potentialinhibition of its upstream partner PI 3-kinase, as has previ-ously been described (57). This inhibition appears to be directedtoward the ATP-binding site of the kinase, and analogues ofquercetin such as LY294002 have been developed as potent PI3-kinase inhibitors (58). However, the inhibition of PI 3-kinaseby quercetin in neurons may be influenced by potential intra-cellular metabolism known to occur in other cell systems (40)and also observed in cortical neurons.2 Oxidative metabolismand glutathionylation of quercetin and its O-methylated me-tabolites may act to hinder or enhance the potency of PI 3-ki-nase inhibition, and consequently our data cannot directly becompared with those studies conducted in a cell-free environ-

ment (57, 58). The potential inhibition of neuronal PI 3-kinaseby quercetin and its O-methylated metabolites and by the novelintracellular metabolite 2�-glutathionyl quercetin (40) is thesubject of further investigation.

Akt/PKB has been strongly implicated in cell survival path-ways (31, 32, 39, 59) and along with ERK has been proposed tobe central to neuronal survival responses and neuroprotection(4, 34). Activation of Akt in some neuronal types has beenshown to lead to an inhibition of proteins central to the celldeath machinery, such as the pro-apoptotic Bcl-2 family mem-ber, BAD (47), and members of the caspase-family (31, 39) thatspecifically cleave poly(ADP-ribose) polymerase (32, 60), thuspromoting cell survival. BAD is regulated by phosphorylationof two serine residues, Ser112 and Ser136 (47), and severalstudies have revealed that the Ser136 site can be specificallyphosphorylated by Akt/PKB (46, 48). In our studies, quercetin-induced inhibition of Akt phosphorylation was coupled with asignificant loss of BAD (Ser136) phosphorylation at 6 h. Thisinhibition of BAD phosphorylation through Akt may underliequercetin-induced cortical neuron damage because active orun-phosphorylated BAD is known to induce apoptosis by inhib-iting anti-apoptotic Bcl-2 family members such as Bcl-XL, al-lowing pro-apoptotic proteins such as BAX and BAK to aggre-gate and initiate cytochrome c release and subsequent caspase-activation (61, 62).

Marked activation of caspase-3 was also observed in corticalneurons exposed to quercetin. As well as contributing directlyto the apoptotic mechanism, it was postulated that this distinctactivation of caspase-3 by quercetin might result in the proc-essing of Akt/PKB seen during high quercetin exposures (30�M). Apoptotic proteases have been observed to cleave andinactivate survival-signaling molecules such as Akt/PKB (50,51), phospholipase C�1 (52), and Bcl-2. In our experiments,cortical neurons exposed to quercetin in the presence of aspecific caspase-3 inhibitor did not suffer the same reduction intotal Akt/PKB protein levels at 6 h. Concurrently, there was areduction in the overall level of caspase-3 activity in neuronsexposed to quercetin and the inhibitor compared with neuronsexposed to quercetin alone. Furthermore, the toxicity inducedby quercetin was reduced in the presence of the caspase-3inhibitor.

The activation of Akt/PKB and ERK1/2 in neurons has alsobeen linked at the transcriptional level to the phosphorylationof CREB (Ser133) (39, 49) a transcription factor linked to pro-survival through the up-regulation of genes such as BDNF andBcl-2. An inhibition of Akt activation by quercetin might there-fore be expected to lead to a reduction in the phosphorylation ofCREB at Ser133, a site critical for its activation. Indeed at thehigher concentration of quercetin (30 �M) at 2 h, we observedinhibition of CREB phosphorylation. However, lower levelquercetin exposures (3 and 10 �M) resulted in an increasedphosphorylation of CREB, indicating that CREB activation byquercetin may occur by a separate mechanism or that highconcentrations trigger dephosphorylation. ERK has also beenshown to be pro-survival partly through the activation of CREB(38, 39). The fact that we observe an activation of CREB atconcentrations of quercetin where we see potent inhibition ofERK1/2 strengthens the concept that quercetin-induced acti-vation of CREB is likely to be through a pathway independentof PI3K/Akt or ERK cascades, for example through calcium/calmodulin kinase or protein kinase A. However, inhibition ofERK phosphorylation by quercetin may contribute to the ob-served neuronal damage and may also reflect upstream inhi-bition of PI 3-kinase, which is known to signal to ERK inneurons (38, 63).

In contrast to the marked effects of quercetin on survival2 J. P. E. Spencer, C. Rice-Evans, and R. J. Williams, unpublished

observations.

FIG. 7. Increased activity of caspase-3-like proteases inducedby quercetin. A, level of caspase-3 reaction product p-nitroaniline (405nm) measured in neuronal lysates exposed to quercetin (0.3–30 �M),3�-O-methyl quercetin, 4�-O-methyl quercetin, and quercetin glucuro-nide (3, 10, or 30 �M). Treatment with compounds was for 6 h, andcaspase-like protease activity was assessed 12 h after exposure. Thecells were lysed, and the activity of caspase-3-like proteases was meas-ured spectrophotometrically method by monitoring the cleavage of thecaspase-3 substrate acetyl-Asp-Glu-Val-Asp-p-nitroanilide to p-nitro-aniline (405 nm). B, time course of caspase-3 activation by quercetin(black bars), 3�-O-methyl quercetin (white bars), and 4�-O-methyl quer-cetin (gray bars) (all 30 �M).

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signaling through Akt/PKB and ERK, neither quercetin norany of its metabolites affected the pro-apoptotic JNK pathwayat any concentration. This is interesting because there is con-siderable evidence linking the activation of the JNK pathwayto neuronal apoptosis (35, 36), and consequently the lack ofactivation of JNK by quercetin suggests that this pathway isnot a potential route to the observed cortical neuron damageobserved in our system. This strengthens the prospect of amechanism that acts through inhibition of survival signalingrather than stimulation of death signaling. Quercetin has beenobserved to suppress JNK activity induced by hydrogen perox-ide (26, 28) and 4-hydroxy-2-nonenal (27), suggesting thatquercetin is able to prevent stress-induced activation of JNK

but not basal activation. This agrees with previous investiga-tions where the flavonoids epicatechin, 3�-O-methyl epicat-echin, and kaempferol inhibited JNK activation by oxidized lowdensity lipoprotein but had no effect on basal JNK phosphoryl-ation (41).

Together these data suggest that quercetin, and to a lesserextent its O-methylated metabolites, may induce neuronaldeath via a mechanism involving direct inhibition of survivalsignaling through Akt/PKB and ERK rather than by an induc-tion of the JNK-mediated death pathway. The observation ofCREB activation in neurons, where we also observe potentinhibition of Akt and ERK and inactivation of BAD, indicatesthat both pro-apoptotic and potentially anti-apoptotic path-

FIG. 8. Effect of the specificcaspase-3 inhibitor on levels of totalAkt, caspase-3 activation and neuro-nal damage in neurons exposed toquercetin. A, crude homogenates (20 �g)prepared from cultured cortical neuronsexposed to vehicle (MeOH), quercetin (30�M), and quercetin (30 �M) and inhibitorfor 2 or 6 h were immunoblotted with anantibody that recognizes total levels ofAkt (Total Akt). B, increased activity ofcaspase-3-like proteases at 2 and 6 h in-duced by quercetin (30 �M) and inhibitionof caspase-3 activation in the presence ofthe specific caspase-3 inhibitor. Treat-ment with compounds was for 6 h, andcaspase-like protease activity was as-sessed 12 h after exposure. Capsase-3 wasassayed as described in the legend forFig. 6C.

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ways are activated in neurons in response to quercetin stimu-lus. However, because overall neuronal death results, it ap-pears that quercetin-induced inhibition of the Akt/BADsurvival pathway is dominant here in determining the fate ofthe neurons. We propose that in our cells, high concentrationsof quercetin produce a sustained deactivation of Akt/PKB,which leads to extensive caspase-3 activation and subsequentcaspase-dependent cleavage of anti-apoptotic Akt/PKB, anevent that effectively turns off the major survival signal andresults in the acceleration of apoptotic neuronal death. How-ever, at lower concentrations reversible inhibition of Akt phos-phorylation is observed, and there is evidence of an attemptedsurvival response reflected in the increase in CREB phospho-rylation. Thus, not all flavonoids should be simply regarded asbeing potentially beneficial in the treatment of neurologicaldisease, and due consideration must be given to the in vivoconcentrations of flavonoids and to the bioactivity of relevant invivo metabolites when studying the effects of dietary flavonoidsin cell culture models.

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Quercetin Effects on Akt and MAPK 34793

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Jeremy P. E. Spencer, Catherine Rice-Evans and Robert J. WilliamsViability

Metabolites Underlie Their Action on Neuronalin Vivoby Quercetin and Its Modulation of Pro-survival Akt/Protein Kinase B and ERK1/2 Signaling Cascades

doi: 10.1074/jbc.M305063200 originally published online June 24, 20032003, 278:34783-34793.J. Biol. Chem. 

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