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J. Biol. Chem., Vol. 278, Issue 37, 34783-34793, September 12, 2003
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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
Received for publication, May 14, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-induced
(5) neuronal death models.
Protective effects in both systems were linked to a modulation in signaling
through protein kinase C and/or modulation of cell survival/cell cycle genes
(4,
6). Much evidence also exists
to support the potential beneficial and neuromodulatory effects of
flavonoid-rich ginkgo biloba extracts, such as EGb 761, in the central nervous
system
(7–9).
Clinical trials with EGb 761, which contains kaempferol and quercetin, have
indicated beneficial effects on brain function, particularly in connection
with age-related dementias and Alzheimer's disease
(9,
10). However, although there
is growing evidence in favor of the beneficial affects of flavonoids, there is
still uncertainty about their actions in vivo and concern about their
potential toxic side effects at higher concentrations. For instance,
epigallocatechin gallate has also been shown to exert pro-apoptotic as well as
anti-apoptotic effects in neuroblastoma cells
(11), whereas quercetin has
been observed to express no protection against A-induced neurotoxicity,
whereas the structurally related flavonols, apigenin and kaempferol, expressed
significant protection
(12).
Although the flavonol quercetin is one of the most frequently researched
flavonoids, with evidence for both its beneficial
(13,
14) and deleterious effects
(15,
16) on different cell types,
its mechanism of action remains unclear. Furthermore, recent evidence has
shown that quercetin is extensively metabolized to O-methylated and
glucuronide metabolites during absorption in the small intestine and in the
liver (17,
18), and such metabolites
should be taken into consideration to provide in vivo relevance for
any mechanism. Quercetin itself has been thoroughly investigated for its
abilities to express anti-proliferative effects
(19,
20) and induce death
predominantly by an apoptotic mechanism in cancer cell lines
(21–23).
For example, it has been observed to induce caspase-3 activation in the
malignant cell line HPB-ALL
(24), activate caspase-3 and
caspase-9, release cytochrome c in HL-60 cells
(25), and induce chromatin and
nuclear fragmentation in colonic cancer cells
(20). On the other hand,
quercetin treatment has been shown to suppress the c-Jun N-terminal kinase
(JNK)1 activity and
apoptosis induced by hydrogen peroxide
(26) and 4-hydroxy-2-nonenal
(27). Furthermore, quercetin
may evoke anti-apoptotic effects via the suppression of the peroxide-induced
JyNK-c-Jun/AP-1 pathway and the ERK-c-Fos/AP-1 pathway in cultured mesangial
cells (28). The ability of
quercetin to inhibit both AP-1 activation and the JNK pathway
(29) has been shown to have
relevance in both phorbol 12-myristate 13-acetate- and tumor necrosis
factor-
-induced intercellular adhesion molecule 1 (ICAM-1)
expression.
The toxic effects of quercetin, or indeed other flavonoids, are likely to involve an apoptotic mode of death in which members of the mitogen-activated protein kinase (MAPK) family, such as JNK, may play a role (30). Alternatively, the modulation of signaling through the serine/threonine kinase, Akt/PKB, one of the 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 linking the activation of the JNK pathway to neuronal apoptosis (35, 36), and stress signaling through ERK has also been observed to be detrimental to neurons (37), although the latter has also been shown to be pro-survival partly through the activation of the 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 mechanisms underlying
the neurotoxic effects of quercetin and its major in vivo
metabolites, 3'-O-methyl quercetin, 4'-O-methyl
quercetin, and quercetin-7-O--D-glucuronide, on
primary cortical neurons in terms of the balance of influence between
pro-survival pathways, such as the Akt/PKB cascade, and pro-death pathways,
namely apoptotic signaling through JNK and caspase-3.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-D-glucuronide was synthesized as
described previously (40). The
antibodies used were anti-phospho-Akt (Ser473) pAb and total Akt
pAb (Cell Signaling Technology Inc.); anti-phospho-Akt (Thr308)
pAb, anti-phospho-BAD (Ser136) pAb, and anti-BAD monoclonal
antibody (Upstate Biotechnology, Inc., Lake Placid, NY); anti-phospho-CREB
(Ser133) pAb (Calbiochem, La Jolla, CA); anti-ACTIVE MAPK (ERK1/2)
pAb and anti-ACTIVE JNK pAb (Promega, Madison, WI); total ERK2 pAb and total
JNK1 pAb (Santa Cruz Biotechnology, Santa Cruz, CA). Horseradish
peroxidase-conjugated goat anti-rabbit secondary antibody (Sigma) and ECL
reagent and Hyperfilm-ECL were purchased from Amersham Biosciences. Elgastat
UHP double distilled water (18.2 M
grade) was used
throughout the study. All other reagents used were of the analytical grade and
obtained from Sigma. All of the other reagents were obtained from Sigma or
Merck.
Cell Culture and Quercetin Exposure—Primary cultures of
mouse cortical neurons were prepared as described previously
(41,
42). The neurons were plated
onto 6- and 24-well Nunc multiwell plates that had been precoated overnight
with poly-L-ornithine and then with 10% heat-inactivated fetal
bovine serum (Invitrogen) for 2 h. Following removal of the final coating
solution, the cells were plated (106/ml) in a serum-free medium
composed of a mixture of Dulbecco's modified Eagle'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, pH
7.4, 100 µg/ml streptomycin, and 60 µg/ml penicillin (all from
Invitrogen). A mixture of hormones and salts composed of 25 µg/ml insulin,
100 µg/ml transferrin, 60 µg/ml putrescine, 20 nM
progesterone, and 30 nM sodium selenate (all from Sigma) was also
added to the culture medium. The cells were 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 acidic protein
immunocytochemistry, respectively (not shown).
Primary cortical neurons (106 cells/ml; 24-well plates) were
exposed to quercetin, 3'-O-methyl- and
4'-O-methyl-quercetin and
quercetin-7-O--D-glucuronide (0.3–10
µM) for 6 h. The time dependence was also investigated by the
addition of quercetin, 3'-O-methyl- and
4'-O-methyl-quercetin and
quercetin-7-O--D-glucuronide (3–30
µM) for 0.5, 2, or 6 h. Following all exposures, the medium was
removed and replaced with 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
for neuronal damage were also performed immediately following the exposures
(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 were exposed to quercetin, 3'-O-methyl-quercetin,
4'-O-methyl-quercetin, and
quercetin-7-O--D-glucuronide (0.3–30
µM) for 0.5, 2, or 6 h. Directly following exposure, the neurons
were washed twice with ice-cold PBS, pH 7.4, containing EGTA/EDTA (200
µM) and lysed on ice before preparation of samples for
immunoblotting.
Assessment of Neuronal Damage—Cellular damage elicited by treatments was evaluated by measuring MTT reduction (41, 43), by utilizing the Dead or Alive assay (Molecular Probes, Eugene, OR), and by morphological examination (38, 41). Following exposure of neuronal cultures to quercetin and its metabolites, the cultures were washed twice with 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 CaCl2,33mM glucose, 5 mM NaHCO3, 1.1 mM MgCl2, 1.2 mM Na2HPO4). Following incubation (60 min; 37 °C), MTT solutions were removed, and the formazan product was solubilized in Me2SO, and the absorbance read at 505 nm. The results were expressed as the percentages of protection against the decline in MTT reduction.
Cell survival was further assessed using calcein AM and ethidium homodimer (Molecular Probes). Calcein AM is taken up into living cells and is metabolized in the cytoplasm by esterases to the green fluorescent product, calcein, so that viable cells show a uniform green fluorescence (emission, 530 nm) under appropriate excitation (495 nm). Ethidium homodimer is excluded from living cells but can cross the compromised plasma membrane of dying cells and interact with nucleic acids to give a strong red fluorescence (emission, 530 nm) under appropriate excitation (495 nm). At the end of the experiment exposures, the neurons were washed, calcein AM (4 µM) and ethidium homodimer 2 µM) were added, and the neurons were incubated for 30 min at 37 °C. The fluorescence was measured using a SPECTRAmax® Gemini microplate spectrofluorometer (Molecular Devices). The temperature was maintained at 25 °C, and the emission was recorded at 530 and 645 nm for calcein and ethidium, respectively, after exciting at 495 nm. Each well was scanned in the well scan mode accumulating data from 21 independent points/well, which were then transformed in an average signal expressed in relative light units. All data were calculated and normalized with respect to the increase of fluorescence of a control. The percentages of living and dead cells were determined and analyzed statistically by Student's t test.
The morphological assessment of neuronal damage was made by analysis of phenotypic markers under light microscopy using a Nikon eclipse T5100 at 40x magnification. The images were captured using a Nikon E995 digital camera fitted with a Coolpix MDC lens adaptor.
Immunoblotting—Immunoblotting analysis was performed essentially as previously described (44) with minor modifications. Following exposures, the neurons were washed with ice-cold PBS with 200 µM 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 inhibitor mixture (1:100 dilution), 1 mM sodium pyrophosphate, 10 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, and 50 mM sodium fluoride. The lysed cells were scraped and left on ice to solubilize for 45 min. The lysates were centrifuged at 1,000 x g for 5 min at 4 °C to remove unbroken cell debris and nuclei. The protein concentration in the supernatants was determined by the Bio-Rad Bradford protein assay®. 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-mercaptoethanol, 10% glycerol, and 0.0025% bromphenol blue). The boiled samples (20–30 µg/lane) were run on 9–12% SDS-polyacrylamide gels, and the proteins were transferred to nitrocellulose membranes (Hybond-ECL®; Amersham Biosciences) by semi-dry electroblotting (1.5 mA/cm2). The nitrocellulose membrane was then incubated in a blocking buffer (20 mM Tris, pH 7.5, 150 mM NaCl; TBS) containing 4% (w/v) skimmed milk powder for 30 min at room temperature followed by two 5-min washes in TBS supplemented with 0.05% (v/v) Tween 20 (TTBS). The blots were then 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, clone BYC001 monoclonal antibody (1:1000), anti-ERK1/ERK2 pAb (1:1000), or anti-JNK1 pAb (1:1000) in TTBS containing 1% (w/v) skimmed milk powder (antibody buffer) overnight at room temperature on a three-dimensional rocking table. The blots were washed twice for 10 min in TTBS and then incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:1000 dilution), rabbit anti-sheep IgG conjugated to horseradish peroxidase (1:2000 dilution), or goat anti-mouse IgG conjugated to horseradish peroxidase (1:3000 dilution; Upstate Biotechnology, Inc.), in antibody buffer for 60 min. Finally the blots were washed twice for 10 min in TTBS rinsed in TBS and exposed to ECL® 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 cassette and developed. The bands were analyzed using BioImage® Intelligent Quantifier software (Ann Arbor, MI). The molecular weights of the bands were calculated from comparison with prestained molecular weight markers (molecular weight, 27,000–180,000 and 6,500–45,000; Sigma) that were run in parallel with the samples. The equal loading and efficient transfer of proteins was confirmed by staining the nitrocellulose with Ponceau Red (Sigma).
Caspase-3 Activity—The neurons (106/ml) were
pretreated with quercetin, 3'-O-methyl-quercetin,
4'-O-methyl-quercetin, and
quercetin-7--D-glucuronide (0.3–30 µM)
for 6 h. In a separate series of experiments, the time course of activation
was investigated using a fixed 30 µM exposure for 0.5, 2, and 6
h. Following exposures, the medium was removed, and the neurons were incubated
with fresh conditioned medium. After a total of 12 h (exposure and
reincubation for 6 and 6 h, respectively), the cells were washed twice with
ice-cold PBS (+ EGTA 200 µM) and lysed on ice as described
above. The lysates were scraped from the plates and incubated on ice for 45
min before centrifugation (microcentrifuge, 1000 x g), and the
supernatant was collected. Caspase-3-like protease activity in neuronal
lysates was assessed based on the hydrolysis of the peptide substrate
acetyl-Asp-Glu-Val-Asp p- nitroanilide by caspase-3, resulting in the
release of the p-nitroaniline moiety, which absorbs at 405 nm.
Quantification of the absorbance at 405 nm was carried out using a Versa Max
UV microplate reader (Molecular Devices). Suitable control experiments were
performed using the caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-al. All of the
data are 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 Student's t test with a confidence level of 95%. The significance level was set at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-D-glucuronide did not induce toxicity
at any concentration. The neurotoxicity of quercetin was also apparent at
shorter exposure times. For example, quercetin (10 and 30 µM)
expressed significant toxicity following exposure for 0.5, 2, and 6 h, whereas
quercetin (3 µM) only induced significant neuronal injury
following 2- and 6-h exposures (Fig.
1B). Again this toxicity was only apparent 24 h
post-stress, and no damage was apparent immediately following quercetin
exposure. 3'-O-methyl quercetin (30 µM) and
4'-O-methyl quercetin (30 µM) also induced
significant neuronal injury at 2 h (p < 0.01), although this was
small in comparison with quercetin (Fig.
1C).
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To establish further the neurotoxic properties of quercetin, the percentage of dead and alive cells were measured following exposure. The percent of "alive" neurons was established based on the cytoplasmic esterase conversion of calcein AM to the green fluorescent product, calcein, by living cells. The percentage of "dead" neurons was estimated on the basis that ethidium homodimer is excluded from living cells and will only cross compromised plasma membranes of dying cells and interact with nucleic acids to give a strong red fluorescence. Quercetin exposure (0.3–30 µM) for 6 h resulted in a concentration-dependent reduction in the percentage of viable neurons and a corresponding concentration-dependent increase in the percentage of dead cells at 24 h (Fig. 2A). The increase in dead cells indicates the loss of membrane integrity after exposure to quercetin. However, as with the MTT measurements, damage was only observed 18 h after the completion of the flavonoid treatment with no increase apparent immediately following the quercetin 6-h exposure (Fig. 2A), indicating that at this time there has been no alteration in neuronal membrane integrity. Morphological analyses using light microscopy also highlighted this delayed neuronal damage in that the morphological appearance of cortical neurons was dramatically affected 24 h post-quercetin exposure (30 µM; 6- and 18-h reincubation) but not immediately following the addition (Fig. 2B). Control cultures of cortical neurons exhibit a large intact cell body as well as a finely developed dendritic network (38, 42) (Fig. 2B, panel I). Following exposure to quercetin (10 or 30 µM) for 6 h and reincubation for 18 h, the neuronal cell bodies appeared shrunken, and there was a significant loss of dendrites (Fig. 2B, panels III and IV). In contrast, no significant loss of the dendritic network was observable immediately after the 6-h quercetin exposure, and similarly there was no alteration in cell body morphology (Fig. 2B, panel II).
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Effects of Quercetin on Akt/PKB Signaling—To begin
exploring the mechanism of quercetin-induced neurotoxicity, we examined the
effect of quercetin on Akt/PKB, an essential kinase in neuronal survival and a
downstream effecter of PI 3-kinase
(31,
32). Activation of the PI
3-kinase/Akt signaling pathway in neurons has been strongly implicated in the
regulation of neuronal survival and/or protection
(4,
45). We investigated whether
quercetin could influence Akt phosphorylation in cortical neurons by
immunoblotting neuronal homogenates with an anti-phospho-Akt polyclonal
antibody that detects Akt when it is phosphorylated at Ser473, an
event known to be essential for full activation of the kinase. Exposure of
cortical neurons to quercetin (10 and 30 µM) for 0.5, 2, or 6 h
resulted in a marked decrease in Akt phosphorylation relative to control
neurons, as demonstrated by a robust decrease in the relative intensity of the
immunodetectable band relating to phosphoryated Akt/PKB (
60 kDa)
(Fig. 3, A and
D). The changes in Akt phosphorylation were both time-
and concentration-dependent. Levels of phosphoryated Akt in neurons exposed to
quercetin (10 µM) were greatly reduced at early exposure times
(0.5 and 2 h) but showed some recovery at the 6-h time point with levels
significantly higher (p < 0.01) at this time than at the earlier
exposure times. In contrast, exposure to quercetin (30 µM)
resulted in a concentration-dependent reduction in phospho-Akt levels with
pAkt undetectable following a 6-h exposure. A similar decrease in the relative
intensity of the immunodetectable band relating to phosphoryated threonine 308
(within the catalytic domain of Akt) was also observed following a 2-h
quercetin exposure (Fig.
3C), which matched the decrease in pAkt
(Ser473). Parallel immunoblots with an antibody that detects total
Akt protein levels (nonphosphorylated and phosphorylated Akt) were performed.
There was no change in total Akt at 0.5 h of exposure. Interestingly, there
were changes in total levels of Akt in response to high quercetin
concentrations and at longer exposure times
(Fig. 3B), most
dramatically in neurons exposed to quercetin (30 µM) for 6
h.
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Effect of Quercetin on Phosphorylation of ERK1/2 and JNK1/2—There was a clear concentration-dependent inhibition of pAkt (Ser473) following exposure of cortical neurons to quercetin for 0.5, 2, and 6 h (Fig. 4, A and B). However, at the 6-h exposure time there was significant recovery (p < 0.01) in Akt/PKB phosphorylation toward control levels in neurons exposed to 3 and 10 µM of quercetin (Fig. 4B) compared with the earlier exposure times. ERK has also been implicated in neuronal death/survival signaling (37, 39). Therefore it was of relevance to investigate the effect of quercetin and its metabolites on this kinase. The phosphorylation state of ERK1/2 was probed using a phospho-specific antibody, which recognizes the dually phosphorylated motif pTEpY within activated ERK1/2. Incubation of cortical neurons with quercetin (0.3–30 µM; 0.5 h) resulted in a strong dose-dependent decrease in the phosphorylation below basal levels in the two bands corresponding to ERK1 (44 kDa) and ERK2 (42 kDa) (Fig. 4C).
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JNK has been implicated in pro-apoptotic signaling in neurons (35), and consequently we investigated the ability of quercetin and its metabolites to modulate the phosphorylation of this kinase. The phosphorylation state of JNK was investigated by immunoblotting of neuronal homogenates with anti-active JNK1/2 antibody. This antibody detects JNK when it is dually phosphoryated within the Thr138-Pro-Tyr185 motif (pTPpY) in the catalytic core of active JNK. Treatment of cortical neurons with quercetin (0.3–30 µM) for 2 h resulted in no measurable alteration in the relative intensities of the two immunodetectable 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 Phosphorylation of Akt and
JNK1/2—When considering the effects of flavonoids in
culture models, it is vital to investigate the effects of its actual in
vivo metabolite forms. In contrast to the dramatic inhibition of
phospho-Akt (Ser473) induced by quercetin (30 µM; 2
h), the two O-methylated metabolites of quercetin induced a much
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 µM) for 2 h resulted in
67.2 and 62.4% reductions in Akt phosphorylation, respectively, relative to
control cells, as demonstrated by the decrease in the relative intensity of
the immunodetectable band relating to phosphoryated Akt/PKB
(Ser473) (Fig. 5, A and
D). In contrast,
quercetin-7--D-glucuronide had no significant effect on the
basal state of Akt (Ser473) phosphorylation in cortical neurons. In
addition, the metabolites did not significantly alter the levels of total Akt
in neurons following exposure (Fig.
5B), and neither quercetin nor any of its metabolites
induced a change in basal levels of phospho-JNK1/2
(Fig. 5C).
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Effect on the Phosphorylation of BAD and CREB—Akt maintains neuronal viability by regulating the actions of downstream effectors such as BAD and CREB (46). The pro-apoptotic Bcl-2 family member, BAD, was the first cell death component to be identified as a regulatory target of survival signaling (47). It has been shown to be under direct regulatory control by Akt/PKB, which specifically phosphorylates BAD at the Ser136 (46, 48) an event essential for the inhibition of apoptosis. We were interested to investigate the effect of quercetin on this important anti-apoptotic molecule, especially because a marked inhibition of Akt/PKB had been observed by quercetin. BAD phosphorylation in cortical neurons was undertaken by immunoblotting neuronal homogenates with anti-phospho-BAD polyclonal antibody that detects BAD when phosphorylated at Ser136. Exposure of cortical neurons for 6 h to quercetin (10 or 30 µM) led to a significant reduction in BAD phosphorylation (Fig. 6A). Interestingly, the inhibition of BAD phosphorylation by quercetin paralleled the changes seen in the phosphorylation state of Akt/PKB at 6 h (Fig. 3A). Parallel blots were run and probed with an antibody that detects total levels of BAD. The exposure to quercetin did not alter the total levels of BAD (Fig. 6A), which also confirmed equal protein loading. In contrast to experiments with quercetin, there were no observable changes in the BAD phosphorylation state when cortical neurons were exposed to either 3'-O-methyl quercetin, 4'-O-methyl quercetin or the glucuronidated quercetin (30 µM; 6h) (data not shown). This lack of alteration in the BAD phosphorylation state in the presence of Akt/PKB inhibition by the O-methylated metabolites may reflect a requirement for a threshold of Akt deactivation to occur prior to BAD inhibition.
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Another possible downstream target of Akt/PKB, and indeed ERK, is CREB, a pro-survival transcription factor of importance in neurons (39, 49). We were interested in whether quercetin-induced inhibition of both Akt/PKB and ERK could transduce downstream to affect CREB phosphorylation/activation. However, exposure of cortical neurons to quercetin (3, 10, and 30 µM) for 0.5 h resulted in an increase in CREB phosphorylation relative to control neurons, as demonstrated by a significant increase in the relative intensity of the immunodetectable band relating to phosphoryated CREB (Fig. 6, B and C). The activation of CREB was most significant in neurons exposed to quercetin (3 or 10 µM) for 2 h. The increase in CREB activation was unexpected and did not reflect upstream quercetin-induced Akt/PKB and ERK1/2 inhibition, although neuronal exposure to quercetin (30 µM) 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 JNK activation in response to quercetin or its metabolites, it
is possible that caspase-activation may occur following the reduction of Akt
and BAD phosphorylation, which lead to the release of cytochrome c,
the formation of the apoptosome and subsequent activation of effecter caspases
such as caspase-3 and caspase-7 (reviewed in Refs.
31 and
32). We therefore investigated
the potential activation of caspase-3 by the quercetin compounds. Exposure of
neurons to quercetin (0.3–30 µM) for 6 h led to an
enhanced activity of caspase-3 in neurons, which was apparent as an increase
in absorbance at 405 nm reflecting the increased cleavage of the caspase-3
specific substrate Ac-DEVD-p-nitroanalide
(Fig. 7A). Levels of
caspase-3 activity were increased markedly in neurons exposed to quercetin, in
particular those exposed to concentrations of >3 µM. In
contrast, exposure of neurons to 3'-O-methyl quercetin and
4'-O-methyl quercetin led only to increases in caspase-3
activity at concentrations of 3 µM and above, whereas
quercetin-7-O--D-glucuronide failed to stimulate
significant caspase-3 activation at any concentration. Measurable increases in
caspase-3 activity were detected after a 30-min exposure to quercetin (30
µM), and this level increased markedly after 2 h but did not
significantly 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 of quercetin
was ineffective at inducing the activity of caspase-3 at any time point.
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Effects of Caspase-3 Activation on Loss of Total
Akt—Apoptotic proteases have been observed to cleave and inactivate
survival signaling molecules such as Akt/PKB
(50,
51), phospholipase C
1
(52), and Bcl-2. To
investigate a possible link between the increases in caspase-3 activity in
neurons seen in our experiments and the observed loss of total Akt in neurons
exposed to quercetin (30 µM), a specific caspase-3 inhibitor was
employed. Pretreatment of neurons with the caspase-3 inhibitor for 15 min
prior to the addition of quercetin (30 µM) significantly reduced
the depletion in total Akt after 2 or 6 h of exposure
(Fig. 8A). This was
particularly striking at 6 h, where Akt loss was most apparent. This
prevention of total Akt loss was paralleled by a decrease in the activity of
caspase-3 in neuronal lysates treated with the inhibitor
(Fig. 8B).
Furthermore, neurons pretreated with the inhibitor prior to quercetin exposure
(3, 10, and 30 µM; 6 h) suffered significantly less neuronal
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.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The toxicity of quercetin at higher concentrations (50 µM to
100 mM) toward cells in culture, in particular cancer cells, is
well established and has been linked to an apoptotic mode of death
(15,
16,
21–23).
However, little is known regarding the precise mechanism of quercetin-induced
toxicity in other cell systems, and nothing is known of its effects on
neurons. In addition, it is difficult to relate this to what occurs in
vivo because during absorption from the gastrointestinal tract to the
circulation quercetin and its glycosides are metabolized to
3'-O-methyl quercetin, 4'-O-methyl quercetin,
and quercetin-7--D-glucuronide (as well as sulfates) by the
action of phase I and II enzymes present in the small intestine and liver
(17). These metabolic
processes lead to relatively low amounts of quercetin and higher levels of the
metabolites in the circulation. Furthermore, the cells in culture readily
accumulate relatively high amounts of quercetin and its O-methylated
metabolites, which result in further intracellular oxidative metabolism,
glutathionylation, and conversion of O-methylated metabolites to free
quercetin (40). Potential
toxicity of flavonoids such as quercetin toward neuronal cells in
vivo may be limited by the action of the blood-brain barrier preventing
the entry of such compounds to the brain. However, recent evidence has
demonstrated that flavonoids and some of their metabolites are able to
traverse the blood-brain barrier and that the potential for permeation is
consistent with compound lipophilicity
(55). Indeed quercetin, and
especially its O-methylated metabolites, have relatively high
lipophilicity, indicating the potential brain access of these compounds.
Whereas earlier investigations into flavonoid bioactivity in vitro and in vivo concentrated mainly on their ability to act as either as free radical scavengers and antioxidants or as prooxidants, much recent interest has concentrated on their potential to interact with intracellular signaling pathways such as the MAPK cascade (29, 41, 56). For example, epicatechin and its 3'-O-methylated metabolite protect primary striatal neurons against oxidized low density lipoprotein-induced death by potently inhibiting oxidized low density lipoprotein-induced activation of JNK1/2, c-Jun, and caspase-3 (41). These data suggest that flavonoids might exert effects on neurons via direct interactions with signaling proteins in reactions independent of their antioxidant ability. The speculation that their actions may occur via interactions with neuronal signaling cascades implicated in apoptosis led us to investigate whether the observed strong neurotoxic actions of quercetin and its metabolites toward primary cortical neurons reflect their sensitive interactions within pro-survival MAPK and Akt/PKB signaling cascades.
These investigations provide evidence that low micromolar concentrations of
quercetin, and to a lesser extent its O-methylated metabolites, are
potently neurotoxic toward cortical neurons, whereas
quercetin-7-O--D-glucuronide has no effect. The
toxicity caused by quercetin and its O-methylated metabolites was
characterized both by reductions in neuronal viability and loss of membrane
integrity, although both markers of neuronal damage were only observed 24 h
post-exposure and not directly following the treatments. Prior to measurable
losses of neuronal viability and membrane integrity, quercetin stimulated a
strong inhibition of basal Akt phosphorylation in cortical neurons that was
both time- and concentration-dependent. This inhibition of Akt phosphorylation
was apparent at both the regulatory serine 473 and catalytic threonine 308
sites, rendering it inactive. High quercetin concentrations (30
µM) led to rapid inhibition of Akt phosphorylation, which was
sustained up to 6 h and was accompanied by reductions in total Akt protein
levels. In contrast, lower concentrations of quercetin resulted in a transient
inhibition of pAkt, with levels of pAkt (Ser473) and total Akt not
significantly different from control levels at 6 h, despite a strong initial
inhibition of pAkt. This transient effect could be due to reversible
inhibition and/or metabolism of quercetin intracellularly, as we have shown
previously (40). Although the
two O-methylated metabolites of quercetin also induced reductions in
basal phospho-Akt (Ser473), the level of inhibition was less than
that caused by quercetin and was not accompanied by changes in total Akt. The
inhibition of Akt phosphorylation by quercetin and its O-methylated
metabolites paralleled the neuronal toxicity data, with the
O-methylated metabolites being less neurotoxic than quercetin. This
was further highlighted by the fact that the glucuronide of quercetin, too
polar to enter cells, expressed no significant toxicity and no ability to
inhibit the phosphorylation of Akt/PKB. The inhibition of Akt/PKB
phosphorylation by quercetin seen in our neuronal system may reflect potential
inhibition of its upstream partner PI 3-kinase, as has previously been
described (57). This
inhibition appears to be directed toward the ATP-binding site of the kinase,
and analogues of quercetin such as LY294002 have been developed as potent PI
3-kinase inhibitors (58).
However, the inhibition of PI 3-kinase by quercetin in neurons may be
influenced by potential intracellular metabolism known to occur in other cell
systems (40) and also observed
in cortical neurons.2
Oxidative metabolism and glutathionylation of quercetin and its
O-methylated metabolites may act to hinder or enhance the potency of
PI 3-kinase inhibition, and consequently our data cannot directly be compared
with those studies conducted in a cell-free environment
(57,
58). The potential inhibition
of neuronal PI 3-kinase by quercetin and its O-methylated metabolites
and by the novel intracellular metabolite 2'-glutathionyl quercetin
(40) is the subject of further
investigation.
Akt/PKB has been strongly implicated in cell survival pathways (31, 32, 39, 59) and along with ERK has been proposed to be central to neuronal survival responses and neuroprotection (4, 34). Activation of Akt in some neuronal types has been shown to lead to an inhibition of proteins central to the cell death machinery, such as the pro-apoptotic Bcl-2 family member, BAD (47), and members of the caspase-family (31, 39) that specifically cleave poly(ADP-ribose) polymerase (32, 60), thus promoting cell survival. BAD is regulated by phosphorylation of two serine residues, Ser112 and Ser136 (47), and several studies have revealed that the Ser136 site can be specifically phosphorylated by Akt/PKB (46, 48). In our studies, quercetininduced inhibition of Akt phosphorylation was coupled with a significant loss of BAD (Ser136) phosphorylation at 6 h. This inhibition of BAD phosphorylation through Akt may underlie quercetin-induced cortical neuron damage because active or un-phosphorylated BAD is known to induce apoptosis by inhibiting anti-apoptotic Bcl-2 family members such as Bcl-XL, allowing pro-apoptotic proteins such as BAX and BAK to aggregate and initiate cytochrome c release and subsequent caspase-activation (61, 62).
Marked activation of caspase-3 was also observed in cortical neurons
exposed to quercetin. As well as contributing directly to the apoptotic
mechanism, it was postulated that this distinct activation of caspase-3 by
quercetin might result in the processing of Akt/PKB seen during high quercetin
exposures (30 µM). Apoptotic proteases have been observed to
cleave and inactivate survival-signaling molecules such as Akt/PKB
(50,
51), phospholipase C1
(52), and Bcl-2. In our
experiments, cortical neurons exposed to quercetin in the presence of a
specific caspase-3 inhibitor did not suffer the same reduction in total
Akt/PKB protein levels at 6 h. Concurrently, there was a reduction in the
overall level of caspase-3 activity in neurons exposed to quercetin and the
inhibitor compared with neurons exposed to quercetin alone. Furthermore, the
toxicity induced by quercetin was reduced in the presence of the caspase-3
inhibitor.
The activation of Akt/PKB and ERK1/2 in neurons has also been linked at the transcriptional level to the phosphorylation of CREB (Ser133) (39, 49) a transcription factor linked to pro-survival through the up-regulation of genes such as BDNF and Bcl-2. An inhibition of Akt activation by quercetin might therefore be expected to lead to a reduction in the phosphorylation of CREB at Ser133, a site critical for its activation. Indeed at the higher concentration of quercetin (30 µM) at 2 h, we observed inhibition of CREB phosphorylation. However, lower level quercetin exposures (3 and 10 µM) resulted in an increased phosphorylation of CREB, indicating that CREB activation by quercetin may occur by a separate mechanism or that high concentrations trigger dephosphorylation. ERK has also been shown to be pro-survival partly through the activation of CREB (38, 39). The fact that we observe an activation of CREB at concentrations of quercetin where we see potent inhibition of ERK1/2 strengthens the concept that quercetin-induced activation of CREB is likely to be through a pathway independent of PI3K/Akt or ERK cascades, for example through calcium/calmodulin kinase or protein kinase A. However, inhibition of ERK phosphorylation by quercetin may contribute to the observed neuronal damage and may also reflect upstream inhibition of PI 3-kinase, which is known to signal to ERK in neurons (38, 63).
In contrast to the marked effects of quercetin on survival signaling through Akt/PKB and ERK, neither quercetin nor any of its metabolites affected the pro-apoptotic JNK pathway at any concentration. This is interesting because there is considerable evidence linking the activation of the JNK pathway to neuronal apoptosis (35, 36), and consequently the lack of activation of JNK by quercetin suggests that this pathway is not a potential route to the observed cortical neuron damage observed in our system. This strengthens the prospect of a mechanism that acts through inhibition of survival signaling rather than stimulation of death signaling. Quercetin has been observed to suppress JNK activity induced by hydrogen peroxide (26, 28) and 4-hydroxy-2-nonenal (27), suggesting that quercetin is able to prevent stress-induced activation of JNK but not basal activation. This agrees with previous investigations where the flavonoids epicatechin, 3'-O-methyl epicatechin, and kaempferol inhibited JNK activation by oxidized low density lipoprotein but had no effect on basal JNK phosphorylation (41).
Together these data suggest that quercetin, and to a lesser extent its O-methylated metabolites, may induce neuronal death via a mechanism involving direct inhibition of survival signaling through Akt/PKB and ERK rather than by an induction of the JNK-mediated death pathway. The observation of CREB activation in neurons, where we also observe potent inhibition of Akt and ERK and inactivation of BAD, indicates that both pro-apoptotic and potentially anti-apoptotic pathways are activated in neurons in response to quercetin stimulus. However, because overall neuronal death results, it appears that quercetin-induced inhibition of the Akt/BAD survival pathway is dominant here in determining the fate of the neurons. We propose that in our cells, high concentrations of quercetin produce a sustained deactivation of Akt/PKB, which leads to extensive caspase-3 activation and subsequent caspase-dependent cleavage of anti-apoptotic Akt/PKB, an event that effectively turns off the major survival signal and results in the acceleration of apoptotic neuronal death. However, at lower concentrations reversible inhibition of Akt phosphorylation is observed, and there is evidence of an attempted survival response reflected in the increase in CREB phosphorylation. Thus, not all flavonoids should be simply regarded as being potentially beneficial in the treatment of neurological disease, and due consideration must be given to the in vivo concentrations of flavonoids and to the bioactivity of relevant in vivo metabolites when studying the effects of dietary flavonoids in cell culture models.
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FOOTNOTES |
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To whom correspondence should be addressed. E-mail:
robert.williams{at}kcl.ac.uk.
1 The abbreviations used are: JNK, c-Jun N-terminal kinase; ERK,
extracellular signal-regulated kinase; MAPK, mitogen-activated protein 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-buffered saline; AM, acetoxymethyl ester;
PKB, protein kinase B.
2 J. P. E. Spencer, C. Rice-Evans, and R. J. Williams, unpublished
observations.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, quercetin;
•,3'-O-methyl quercetin; 
,
4'-O-methyl quercetin; 
,
quercetin-7-
