The Neuroprotective Effects of Phytoestrogens on Amyloid
Protein-induced Toxicity Are Mediated by Abrogating the Activation of
Caspase Cascade in Rat Cortical Neurons*
Chuen-Neu
Wang
,
Chih-Wen
Chi§,
Yun-Lian
Lin
,
Chieh-Fu
Chen
, and
Young-Ji
Shiao
¶
From the
National Research Institute of Chinese
Medicine, Republic of China and the § Institute of
Pharmacology, National Yang-Ming University, Taipei 112, Taiwan,
Republic of China
Received for publication, July 19, 2000, and in revised form, November 2, 2000
 |
ABSTRACT |
Amyloid
protein (A
) elicits a toxic effect
on neurons in vitro and in vivo. In present
study we attempt to elucidate the mechanism by which A
confers its
neurotoxicity. The neuroprotective effects of phytoestrogens on
A
-mediated toxicity were also investigated. Cortical neurons treated
with 5 µM A
-(25-35) for 40 h decreased the cell viability by 45.5 ± 4.6% concomitant with the
appearance of apoptotic morphology. 50 µM kaempferol and
apigenin decreased the A
-induced cell death by 81.5 ± 9.4%
and 49.2 ± 9.9%, respectively. A
increased the activity of
caspase 3 by 10.6-fold and to a lesser extent for caspase 2, 8, and 9. The A
-induced activation of caspase 3 and release of cytochrome
c showed a biphasic pattern. Apigenin abrogated
A
-induced cytochrome c release, and the activation of
caspase cascade. Kaempferol showed a similar effect but to a less
extent. Kaempferol was also capable of eliminating A
-induced accumulation of reactive oxygen species. These two events accounted for
the remarkable effect of kaempferol on neuroprotection. Quercetin and
probucol did not affect the A
-mediated neurotoxicity. However, they
potentiated the protective effect of apigenin. Therefore, these results
demonstrate that A
elicited activation of caspase cascades and
reactive oxygen species accumulation, thereby causing neuronal death.
The blockade of caspase activation conferred the major neuroprotective
effect of phytoestrogens. The antioxidative activity of
phytoestrogens also modulated their neuroprotective effects on
A
-mediated toxicity.
 |
INTRODUCTION |
Senile plaque and neurofilament tangles are the hallmarks of
Alzheimer's disease (AD),1
which is one of the major neurodegenerative diseases. Amyloid
protein (A
), the major protein component of senile plaque, has been
suggested to play an important role in pathogenesis of AD (1, 2). A
,
a 39- to 42-amino acid peptide, is derived proteolytically from amyloid
precursor protein, which is expressed widely throughout the brain (3,
4). Multiple lines of evidence have demonstrated that fibril A
participates in the induction of neuronal death and neuritic changes
(5-8). The A
-related fragments (A
-(1-40), A
-(1-42), and
A
-(25-35)) exhibit toxicity to neurons either in vitro
or in vivo (9-11). The mechanisms by which A
elicits
deleterious effect on neurons remain to be established.
Extensive studies have shown that A
-induced neurotoxicity in
multiple cell types may be mediated by several different mechanisms. The neurotoxic effect may be attributable to the disturbance in calcium
homeostasis (12-14) and consequently inducing the accumulation of
reactive oxygen species (ROS) (13, 15-18). ROS provokes membrane damage compromising membrane integrity and increasing the permeability of ions, including calcium. The increase of calcium influx leads to
generating more ROS, thereby initiating the positive feedback loop.
Cultured neurons treated with A
or transgenic mice expressing A
renders neurons vulnerable to apoptosis, indicating that caspase activation plays a role in A
-induced neurotoxicity. Several caspases involved in apoptosis have been described to be activated by A
(6,
19-22). However, the mechanisms by which A
activates caspase cascade remain unclear. Furthermore, the activation of cyclin-dependent kinase has also been indicated to be a mediator of neurotoxicity induced by A
(23).
Several agents have been shown to be neuroprotective in in
vitro system by targeting to specific pathway responsible for
A
-induced toxicity. These agents include antioxidants or free
radical scavengers (12, 15, 17, 18, 24-26), calcium ion channel
blockers (13, 26); growth factors (27), inhibitor of
cyclin-dependent kinase (23), and caspase inhibitors (15,
19, 21). Recent evidence shows that estrogen deficiency in
postmenopausal women is one of the most significant risk factors for
onset of AD (28, 29). Thus, estrogens have become a research focus. It
has been shown that estrogen protects neurons against a number of toxic
insults, including A
(30-34). The neuroprotective effects of
estrogen are suggested to be independent of their classic nuclear
estrogen receptors (32, 34).
Flavonoids, the so-called phytoestrogens, occur ubiquitously in food
plants and herbal medicines. They not only bind to estrogen receptor
but also exert antioxidative and antiproliferative activity (35-37).
Furthermore, evidence has demonstrated that phytoestrogens interfere
with a number of intracellular processes, including enzyme activation
and cAMP accumulation (38, 39). Flavonoids are typically classified
into four groups. Those are flavone, flavonol, flavanone, and
isoflavone. Thus, apigenin, luteolin, kaempferol, quercetin,
naringenin, 2,3-dihydroluteolin, genistein, and prunetin were
chosen for the study of neuroprotection (Fig. 1).
In the present report, we demonstrate that apigenin blocked the release
of cytochrome c and activation of caspase cascade induced by
A
. Kaempferol only inhibited the activation of caspase cascade, and
the effect was less potent than that of apigenin. However, kaempferol
was more effective than apigenin in counteract the deleterious effect
of A
on cortical neurons. Kaempferol exhibited antioxidative
activity and decreased the ROS accumulation induced by A
, whereas
apigenin lacked antioxidative activity and showed a marginal effect on
ROS level. Furthermore, quercetin or probucol facilitated the
neuroprotective effect of apigenin on A
-mediated toxicity.
Therefore, these results indicated that blockade of activation of
caspase cascade conferred the neuroprotective effects of phytoestrogens
on A
-mediated neurotoxicity. The inhibition of caspase cascade in
combination with antioxidative activity will further eliminate
A
-mediated neurotoxicity.
 |
EXPERIMENTAL PROCEDURES |
Materials
Media and supplements for cell culture were from Life
Technologies (Gaithersburg, MD). Amyloid
peptide-(25-35)
(A
-(25-35)), poly-D-lysine,
cytosine-
-D-arabinofuranoside, probucol,
genistein, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT),
trypan blue, dimethyl sulfoxide (Me2SO), and fluorescein diacetate were purchased from Sigma Chemical Co. (St. Louis, MO). Enhanced chemiluminescence detection reagents and anti-mouse IgG antibody conjugated with horseradish peroxidase were obtained from
Amersham Pharmacia Biotech (Buckinghamshire, UK).
2,2'-Azinobis(3-ethylbenzthiazoline sulfonic acid) was purchased
from Roche Molecular Biochemicals (Mannheim, Germany). Fluorescein
diacetate (FDA) and
5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate
(CM-H2DCFDA) were purchased from Molecular Probes (Leiden, The Netherlands). Ac-DEVD-CHO, Ac-LEHD-CHO, AC-IETD-CHO, and a casepase-3 cellular activity assay kit were obtained from Calbiochem (Darmstadt, Germany). Colorimetric caspase substrates were from BIOSOURCE (Nivelles, Belgium). Monoclonal
antibodies for cytochrome c were obtained from PharMingen
(San Diago, CA). All other reagents were purchased from Sigma or Merck
(Darmstadt, Germany).
Methods
Cell Culture--
Primary cultures of neonatal cortical neurons
were prepared from the cerebral cortex of Harlan Sprague-Dawley rat
pups at postnatal day 1 (40, 41). Briefly, each pup was decapitated and
the cortex was digested in 0.5 mg/ml papain at 37 °C for 15 min. The
tissue was dissociated in Hypernate A medium (containing B27
supplement) by aspirating trituration. Cells were plated (5 × 104 cells/cm2) onto
poly-D-lysine-coated dishes and maintained in Neurobasal medium containing B27 supplement (40), 10 units/ml penicillin, 10 µg/ml streptomycin, and 0.5 µg/ml glutamine (5%
CO2/9% O2) for 3 days. Cells were then exposed
to cytosine-
-D-arabinofuranoside (5 µM)
for 1 day to inhibit proliferation of non-neuronal cells. The cells
were used for the experiment on the fifth day.
Measurement of Cell Viability--
The reduction of MTT,
cleavage of FDA, and trypan blue exclusion were used to evaluate the
cell viability. Cells were incubated with minimum essential medium
containing 0.5 mg/ml MTT for 1 h. The medium was aspirated, and
the formazan particle was dissolved with lysis buffer (10% sodium
dodecyl sulfate, 3.3 mM HCl, 50% dimethylformamide).
A600 nm was measured by using
enzyme-linked immunosorbent assay reader (42). Cells were loaded with
15 µM FDA for 5 min at 25 °C, and then 1 ml of 1%
deoxycholate was added to lyse the cells. The fluorescent intensity of
the lysate was determined by using a spectrofluorometer with excitation
and emission wavelength of 490 nm and 514 nm, respectively (26, 43).
Cell viability was also assessed by using trypan blue exclusion as described previously (42).
Measurement of ROS--
Intracellular reactive oxygen species
were measured by CM-H2DCFDA assay (26). In brief, cells
were loaded with 50 µM CM-H2DCFDA for 30 min,
and then 1 ml of 1% deoxycholate was added to lyse the cells. The
fluorescent intensity of the lysate was determined by using a
spectrofluorometer with excitation and emission wavelength of 488 nm
and 525 nm, respectively.
Measurement of Cytochrome c Release--
Treated cells were
collected in harvest buffer (50 mM Hepes, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 10 µg/ml
leupeptin) and suspended by passage through a 200-µl pipette tip. The
cell suspensions were centrifuged at 10,000 × g for 10 min, and supernatants were diluted with the same volume of
immunoprecipitation buffer (harvest buffer containing 2% Nonidet
P-40). Immunoprecipitation of cytochrome c was performed by
using anti-cytochrome c antibody. The immunoprecipitates
were subjected to SDS-polyacrylamide gel electrophoresis.
Immunoreactive proteins were detected by enhanced chemiluminescence
detection reagents.
Measurement of Cellular Activity of Caspases--
Treated cells
were harvested in cell lysis buffer (50 mM Hepes, pH 7.4, 1 mM dithiothreitol, 0.1 mM EDTA, 0.1% Chaps,
and 0.1% Triton X-100). The detailed experiments were performed
according to the manufacturer's protocol.
Other Methods--
The antioxidant activity of phytoestrogens
were determined by trolox equilibrium antioxidant capacity (TEAC)
method as described by Pellegrini et al. (44). The activity
of lactate dehydrogenase (LDH) was determined by a method described
previously (45). Results are expressed as means ± S.D. and were
analyzed by ANOVA with post hoc multiple comparison with a Bonferroni test.
 |
RESULTS |
Phytoestrogens Protected Neurons from A
-induced
Neurotoxicity--
Treatment of rat cortical neurons with the toxic
fragment of fibril A
(f-A
-(25-35)) for 40 h decreased cell
viability in a concentration-dependent manner as determined
by MTT reduction or trypan blue exclusion (Fig.
2A). 10 µM A
decreased the MTT reduction and trypan blue exclusion by 58.2 ± 3.9% and 63.3 ± 11.5%, respectively. However, treatment with
A
elicited a marginal effect on the ability to cleave fluorescein
diacetate and had no effect on the release of LDH. The effect of A
on cell viability of cortical neurons was time-dependent as
measured by MTT reduction (Fig. 2B). The appearance of
irregularly shaped cell bodies and discontinuous neurite was
concomitant with the decrease of cell viability (Fig. 2C).
The percentage of cells with injured morphology was elevated as the
concentration of A
was increased.

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Fig. 2.
Effects of
A -(25-35) on the cell viability of cortical
neurons. Cortical neurons were incubated with various
concentrations of A -(25-35) for 40 h (A), and
neurotoxicity was measured by the FDA assay (closed
circles), MTT assay (closed squares), trypan blue
exclusion (opened squares), and LDH release (closed
triangles). Neurons were exposed to 5 µM of
A -(25-35) for 0-40 h, after the incubation periods, and cell
viability was determined by the MTT assay (B). Results are
means ± S.D. (where large enough to be shown) from four
independent experiments and expressed relative to the cells treated
with vehicle alone. C, the representative photographs of
neurons treated with 0 (a), 5 (b), 10 (c), and 20 µM (d) of A -(25-35)
for 24 h.
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The effects of a series of phytoestrogens on A
-induced neurotoxicity
were investigated. Cells were incubated with various concentrations of
pytoestrogens for 2 h then exposed to 5 µM A
for
40 h. Cell viability was verified by MTT reduction analysis. Results showed that kaempferol and apigenin reduced the A
-induced neurotoxicity in a concentration-dependent manner (Fig.
3, A and B).
Kaempferol at 50 µM decreased the percentage of cell
death from 45.5 ± 4.6% to 7.9 ± 3.6%. Apigenin was less
potent to attenuate A
-induced neurotoxicity (Fig. 3B).
For the morphology, 50 µM kaempferol diminished the
extent of cells with injured morphology induced by A
(Fig.
3C). The protective effect of kaempferol on A
-mediated
neurotoxicity was further confirmed by the measurement of trypan blue
exclusion (data not shown). Luteolin, quercetin, flavanones,
isoflavones, and
-estradiol did not show any significant effects on
A
-induced neurotoxicity (Table I).

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Fig. 3.
Kaempferol and apigenin prevented cell death
of cortical neurons induced by
A -(25-35). Cortical neurons were
pretreated with various concentrations of kaempferol (A) or
apigenin (B) for 2 h and then exposed to 5 µM of A -(25-35) for 40 h. Cell death was
measured by MTT assay. Results are means ± S.D. (where large
enough to be shown) from four independent experiments and expressed
relative to cells treated with vehicle alone. Significant differences
between cells treated with A -(25-35) and A -(25-35) plus
phytoestrogen are indicated by **, p < 0.01 and ***,
p < 0.001. C, neurons were pretreated with
vehicle (0.1% Me2SO) (a, b) or 50 µM kaempferol (c, d) for 2 h
and then exposed to 5 µM of A -(25-35) (b,
d) for 40 h. The morphology was visualized by
phase-contrast microscope. The results are representative photographs,
and similar results were repeated three times.
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Table I
Protection of cortical neurons against A -induced cell death by
phytoestrogens and estrogen
Cortical neurons were incubated with vehicle (0.1% Me2SO),
various phytoestrogens, or -estradiol at highest nontoxic
concentration for 2 h then exposed to 5 µM A for
40 h. The cell viability was assessed by MTT reduction analysis.
The data are means ± S.D. and expressed relative to cells treated
with vehicle alone. Significant differences between cells treated with
A and A plus phytoestrogen or estrogen are indicated in
footnotes.
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A
Induced the Activation of Caspase Cascade--
Inhibitors of
caspases were employed to investigate whether apoptosis was involved in
A
-mediated toxicity. Ac-DEVD-CHO and Ac-LEHD-CHO are the
cell-permeable inhibitors for caspases 3 and 9, respectively. These
inhibitors block the activity of caspases but do not interfere with its
activation. Both inhibitors reduced A
-induced cell death in a
concentration-dependent manner (Fig. 4, A and B). In
contrary, Ac-IETD-CHO, the cell-permeable inhibitor of caspase 8, did
not decrease A
-induced cell death (data not shown). Morphological
study also showed that Ac-DEVD-CHO and Ac-LEHD-CHO eliminated the cells
with injured morphology induced by A
(Fig. 4C).

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Fig. 4.
Ac-DEVD-CHO and Ac-LEHD-CHO prevented cell
death of cortical neurons induced by
A -(25-35). Cortical neurons were
incubated with various concentrations of Ac-DEVD-CHO (caspase 3 inhibitor) (A) and Ac-LEHD-CHO (caspase 9 inhibitor)
(B) for 2 h then exposed to 5 µM of
A -(25-35) for 40 h. Cell death were measured by MTT assay.
Results are means ± S.D. from four independent experiments and
expressed relative to cells treated with vehicle alone. Significant
differences between cells treated with A -(25-35) and A -(25-35)
plus phytoestrogen are indicated by ***, p < 0.001. C, neurons were pretreated with vehicle (0.1%
Me2SO) (a, b), 20 µM of
Ac-DEVD-CHO (c, d), or 20 µM of
Ac-LEHD-CHO (e, f) for 2 h and then exposed
to 5 µM of A -(25-35) (b, d,
f) for 40 h. The morphology was visualized by
phase-contrast microscope. The results are representative photographs,
and similar results were repeated three times.
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Analysis of the activity of caspases were performed to further
determine whether the activation of caspase cascade was involved in the
A
-mediated neurotoxicity. Treatment of neurons with A
induced
activation of caspase 2, 3, 8, and 9 in a time-dependent manner as measured by substrate cleavage (Fig.
5). Exposure of neurons to A
for
24 h, the specific activity of caspase 2, 3, 8, and 9 were
increased by 6.4-, 11.6-, 6.0-, and 4.7-fold of control, respectively.
The activity of caspase 3 was enhanced to a larger extent by A
in
comparison with caspase 2, 8, and 9. A
exhibited a biphasic effect
on the activation of caspase 3. There was a transit activation of
caspase 3 at 2 h and followed by a sustained activation from 8 to
24 h. The similar result for caspase 9 was also obtained. However,
the first wave of activation of caspase 9 at 2 h was not
statistically significant. Caspases 2 and 8 did not show early phase of
activation. The significant activation of caspases 2 and 8 occurred
from 12 to 24 h.

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Fig. 5.
A -(25-35)-induced
activation of caspases in cortical neurons. Cortical neurons were
treated with 5 µM of A -(25-35) for indicated time periods. Activity of caspase 9, 3, 2, and 8 was
determined by the cleavage ability of Ac-LEHD-pNA (a),
Ac-DEVD-pNA (b), Ac-VDVAD-pNA (c), and
Ac-IETD-pNA (d) in cell extracts, respectively. Results are
means ± S.D. (where large enough to be shown) from four
independent experiments. Significant differences between control and
A -treated cells are indicated by *, p < 0.05 and
***, p < 0.001.
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The Effects of Caspase Inhibitors and Phytoestrogens on Caspase
Cascade--
The inhibitor of caspases 3, 9, and 8 decreased the
activity of caspase 3 and 9 at 2 h (Table
II). Both inhibitors of caspase 3 and 9 inhibited the activity of caspase 2, 3, and 9 at 24 h (Table
III). The inhibitor of caspase 3 diminished the activity of caspase 2, 3, and 9 by 72.8 ± 8.7, 90.8 ± 4.4, and 60.9 ± 15.0%, respectively, and the
inhibitor of caspase 9 decreased the activity of caspases to a lesser
extent. However, both inhibitors did not show significant effect on the
activity of caspase 8. The inhibitor of caspase 8 decreased the
activity of caspase 8 and 9 by 17.0 and ± 4.0% and 43.4 ± 4.0%, respectively.
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Table II
The inhibition of caspase activation at 2 h by phytoestrogens and
caspase inhibitors
Cortical neurons were incubated with phytoestrogens (50 µM) or caspase inhibitor (10 µM) for 2 h and exposed to 5 µM A for 2 h. Cell viability
was assessed by MTT reduction analysis. The data are means ± S.D.
of four independent experiments and expressed relative to cell treated
with A alone. Significant differences between cells treated with
A and A plus phytoestrogen or caspase inhibitor are indicated in
footnotes.
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Table III
The inhibition of caspase activation at 24 h by either
phytoestrogen or caspase inhibitors
Cortical neurons were incubated with phytoestrogens (50 µM) or caspase inhibitor (10 µM) for 2 h and exposed to 5 µM A for 24 h. Cell viability
was assessed by MTT reduction analysis. The data are means ± S.D.
of four independent experiments and expressed relative to cell treated
with A alone. Significant differences between cells treated with
A and A plus phytoestrogen or caspase inhibitor are indicated in
footnotes.
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Apigenin and kaempferol did not show significant effects on the
activation of caspase cascade at 2 h (Table II). However, apigenin
inhibited the activity of caspase 2, 3, 8, and 9 at 24 h by about
55 to 60% (Table III). Kaempferol also diminished the activity of
these caspases but to a lesser extent. Quercetin only showed inhibitory
effect on the activity of caspase 9 at 24 h (Table III).
Apigenin Inhibited A
-induced Release of Cytochrome
c--
Kaempferol and apigenin attenuated A
-induced activation of
caspases 9 and 3. The release of cytochrome c from
mitochondria, the upstream signaling component of these two caspases,
was therefore investigated (Fig. 6). The
release of cytochrome c induced by A
exerted a biphasic
pattern similar to the activation of caspase 3. The first phase
occurred at 2 h (Fig. 6A) and followed by the second
phase from 12 to 24 h (data not shown). Apigenin significantly inhibited A
-induced cytochrome c release at 2 and 12 h by 34.1 and 55.7%, respectively. However, kaempferol did not affect
A
-induced cytochrome c release either at 2 or 12 h.

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Fig. 6.
Apigenin blocked
A -(25-35)-induced release of cytochrome
c from mitochondria in cortical neurons.
A, cortical neurons were treated with 5 µM of
A -(25-35) for different time period. B, cells were
incubated with 50 µM kaempferol or 50 µM
apigenin for 2 h then exposed to 5 µM A -(25-35)
for 2 h (opened column) or 12 h (closed
column). After treatment, cytochrome c release was
analyzed by immunoblotting. The top parts of A
and B are the representative immunoblots of cytochrome
c. The bottom parts are the level of cytochrome
c relative to the cells treated with vehicle alone. Results
are means ± S.D. from three independent experiments. Significant
differences between control and A -treated cells (A), or
cells treated with A -(25-35) and A -(25-35) plus phytoestrogen
(B) are indicated by *, p < 0.05 and ***,
p < 0.001.
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Antioxidative Activity Potentiated the Neuroprotection of
Apigenin--
The effect of A
on intracellular level of ROS was
examined by using
5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate
(CM-H2DCFDA). A
induced ROS accumulation significantly from 8 to 24 h (Fig. 7A).
Treatment with 5 µM A
for 8 and 16 h elevated the
level of ROS to 129 ± 5% and 158 ± 10% of control, respectively. A
increased the level of ROS in a
concentration-dependent manner (Fig. 7B).
Kaempferol, luteolin, and quercetin reduced the level of ROS by
30-50% in control cells and by 50-60% in A
-treated cells (Fig. 7C). Apigenin decreased the ROS level by 25% in
A
-treated cells but did not affect that in control cells. The
antioxidant capacity of phytoestrogens was also determined by TEAC
method (Table IV). The results showed
that apigenin had lower antioxidant capacity than quercetin and
luteolin. Kaempferol also showed higher antioxidant capacity than
apigenin. Thus, the sequence of antioxidant capacity for these
phytoestrogens did not correlate well to their neuroprotective ability.
The results also implied that high antioxidative activity of flavonoid
per se was not able to protect neuron against A
-induced neurotoxicity.

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Fig. 7.
Effects of A -(25-35) on the level of ROS
in cortical neurons. Cells were incubated with 5 µM
of A -(25-35) for the time periods indicated (A) or
treated with various concentrations of A -(25-35) for 16 h
(B). Cortical neurons were incubated with or without 50 µM of various phytoestrogens for 18 h (opened
column). Cells were incubated with or without 50 µM
of various phytoestrogens for 2 h and then exposed to 5 µM of A -(25-35) (closed column) for
16 h (C). Thereafter, the level of ROS was measured by
the CM-DCFDA. Results are means ± S.D. from four independent
experiments and expressed as percentage relative to the cells treated
with vehicle alone. Significant differences between vehicle- and
phytoestrogen-treated cells (opened column) or cells treated
with A and A plus phytoestrogen (closed column) are
indicated by ***, p < 0.001.
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Table IV
Antioxidant capacity of phytoestrogens and estrogen
The unit of antioxidant capacity (TEAC) is defined as the concentration
of Trolox having the equivalent antioxidant activity to 1 mM of the phytoestrogens or estrogen. The data are
means ± S.D. of five independent experiments.
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Apigenin was much more able than kaempferol to block caspase activation
(Table III). Apigenin, however, exhibited the neuroprotective effect to
a lesser extent (Fig. 3). We speculated, therefore, that the
antioxidative activity and ability for ROS reducing may modulate the
neuroprotective effect of kaempferol. To address the hypothesis that
antioxidative activity may enhance the neuroprotective effect of
apigenin, the effect of antioxidants on neuroprotection of apigenin was
evaluated (Fig. 8). Quercetin did not
reduce A
-mediated cell death from 1 to 50 µM.
Cotreatment with quercetin and apigenin enhanced the neuroprotective
effect of apigenin (Fig. 8A). Probucol, an antioxidant, was
more potent at potentiating the neuroprotection effect of apigenin
(Fig. 8B). The results demonstrated that, although antioxidant activity of quercetin and probucol per se did
not show neuroprotective effect, they did modulate the neuroprotective effect of apigenin.

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Fig. 8.
Quercetin and probucol facilitated the
neuroprotective effect of apigenin. Cortical neurons were
pretreated with various concentrations of quercetin (A) or
probucol (B) in the presence (closed symbol) or
absence (opened symbol) of 50 µM of apigenin
for 2 h, and then exposed to 5 µM of A -(25-35)
for 40 h. Cell death was measured by the MTT assay. Results are
means ± S.D. (where large enough to be shown) from four
independent experiments and expressed relative to cells treated with
vehicle alone. Significant differences between cells treated with
A -(25-35) plus apigenin and A -(25-35) plus apigenin and
antioxidant are indicated by ***, p < 0.001.
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 |
DISCUSSION |
In the present report, we demonstrate that kaempferol and apigenin
prevented death of cultured neurons exposed to fibril A
-(25-35). The action modes of these phytoestrogens were neither due to an activation of the nuclear estrogen receptor nor solely based on an
antioxidative mechanism. The anti-apoptotic signaling activity conferred neuroprotective effect of kaempferol and apigenin.
Antioxidative activity of flavonoids or other antioxidants did not
exhibit direct effects on neuroprotection. However, antioxidative
activity facilitated the neuroprotective effect of apigenin.
Kaempferol and apigenin protected neurons from A
-mediated toxicity,
whereas quercetin and luteolin failed to protect neurons. This result
suggests that the substitution of hydroxyl group at C-3' position
severely impairs the neuroprotective ability of kaempferol. The
deficiency of neuroprotective effects of narigenin and
2,3-dihydroluteolin also implicates that the double bond between the
C-2 and C-3 positions is required for apigenin and kaempferol to exert
their neuroprotective effects. 4-Hyroxytamoxifen, an estrogen receptor
antagonist, did not abrogate the protective effects, suggesting that
the mode of action is independent of nuclear estrogen receptor (data
not shown). That 17
-Estradiol did not protect cortical neurons
against A
-induced toxicity further confirms the hypothesis that the
effect of phytoestrogen segregates from the action of estrogen
receptor. However, some studies have shown that estrogens counteract
the toxic effect of A
on PC12 phenochromocytoma cells and human
neuroblastoma cells (32, 33, 45, 46). A possible explanation of these
discrepancies could be A
exerting toxic effect on cell line and
primary culture of cortical neurons via distinct mechanisms.
Phytoestrogens reducing the loss of MTT reduction by A
has also been
described in PC12 phenochromocytoma cells. The report (45),
however, concludes that phytoestrogens affect the plasma membrane
sensitivity to formazan crystal rather than protect cells against
A
-induced cell death (45). Furthermore, quercetin shows protective
effect to PC12 cells but not to primary culture of cortical neurons. These results suggest that cell lines and the primary culture of
cortical neurons are protected by pytoestrogens through distinct mechanism.
Both caspase 3 inhibitor and caspase 9 inhibitor prevented neuronal
death induced by A
, suggesting that A
-exerted neurotoxicity is
mediated by an apoptotic pathway. Evidence supporting this speculation includes the facts that A
elevated the activity of caspase 3, 9, 8, and 2 and promoted the release of cytochrome c from mitochondria. The results also showed that caspase 3 displayed a biphasic activation by A
treatment. This activation
consisted of a transient activation at 2 h and a sustained
activation after 8 h. For caspase 9, the first wave activation was
not supported by the statistics. Nevertheless, the elevation of caspase
9 activity at 2 h may be biologically relevant. The release of
cytochrome c also showed a similar pattern as the activation
of caspase 3 and 9, although A
activated caspase 2 and 8 after
16 h and to a lesser extent. These data suggest that the apoptosis
signaling induced by A
is mediated primarily by activation of
caspase 3 and 9 and cytochrome c release. The activation of
caspase 2 and 8 and the production of ROS may be the secondary
responses. Caspase 8 inhibitor was found unable to prevent neuronal
death induced by A
, which further confirms that caspase 8 may not
play the major role in A
-induced neuronal death.
Although the A
-mediated neurotoxicity becomes the focus
of intense interest. The underlining mechanisms are still
controversial. The study of Giovanni et al. shows that
cortical neurons from caspase 3 knockout mice are resistant to
A
-mediated cell death, suggesting that caspase 3 is the major
component mediating A
-induced apoptosis (22). However, the studies
of Troy et al. (21) show down-regulation of caspase 3 does
not block A
-(1-42)-induced cell death. They also show that
sympathetic neurons from caspase 2 null mice are resistant to
A
-(1-42)-mediated cell death, implicating an important role of
caspase 2 in A
-(1-42)-induced apoptosis (21). Moreover, cell death
is blocked by the down-regulation of caspase 2 in hippocampal neurons,
sympathetic neurons, or neuronal PC12 cells with antisense
oligonucleotides. Beside caspase 3 and 2, other caspases are also
thought to be involved in A
-induced apoptosis. Nakagawa et
al. (20) shows that caspase-12-deficient cortical neurons are not
susceptible to the apoptosis induced by A
-(1-40) (20). Another
study, however, shows that the apoptotic pathway activated by A
requires both caspase 8 and Fas-associated death domain (FADD)
(47). Our results demonstrate that A
elicited the activation of
caspase 2, 3, 8, and 9 in cortical neurons. The release of cytochrome
c and activation of caspase 9 and caspase 3 may be the major
pathway mediating the A
-induced apoptosis of neurons. Kaempferol and
apigenin abrogated the activation of all four caspases (Table III).
Apigenin was more potent than kaempferol in blocking the release of
cytochrome c and activation of caspase cascade induced by
A
. However, kaempferol was more effective than apigenin to protect
neurons from A
-induced cell death. On the basis of these data, we
speculate that there may be other factors involved in the protective
effect of kaempferol. ROS scavenging activity of kaempferol may be the
most possible candidate to promote its neuroprotective effect.
Many reports have demonstrated or proposed that ROS is
responsible for A
-induced neurotoxicity (13, 15-18). Behl et
al. (17) shows that A
increases the intracellular level of
H2O2 and lipid peroxide. This result suggests
that free radical damage is one factor accounting for A
cytotoxicity. The studies of Kruman et al. (48) further
provide evidence that 4-hydroxynonenal, an aldehydic product of
membrane lipid peroxidation, is a key mediator of neuronal apoptosis
induced by A
. Therefore, it is possible that scavenging of ROS may
also contribute to the neuroprotective activity of kaempferol and
apigenin. The level of ROS in A
-treated neurons was elevated after
8-h incubation, implying that ROS production may be another mediator
for A
-induced cell death. The level of ROS in A
-treated neurons
were reduced by both neuroprotective and nonprotective flavonoids.
Furthermore, nonprotective flavonoids, quercetin and luteolin, were
more potent in reducing the level of ROS and had higher antioxidative
activity. These results indicate that the antioxidative activity of
flavonoids per se do not confer the neuroprotective effect
on A
-mediated toxicity. Nevertheless, quercetin and probucol, which
possess antioxidative activity, did show a significant facilitating
effect on the neuroprotection of apigenin. These data provide the
convincing explanation for the inconsistent results between inhibition
of caspase cascade and the neuroprotective effect of kaempferol and apigenin.
Taken together, the results presented here provide a
plausible mechanism by which A
provokes death of cortical neurons.
In this model (see Fig. 9), A
-mediated
apoptosis consists of the first and second waves of caspases
activation. A
primarily induces the release of cytochrome
c from mitochondria and subsequent activation of caspase 9 and 3 at 2 h (Figs. 5 and 6). The inhibitor of caspase 8 blocked
the activation of caspase 9 and 3 at 2 h (Table II), indicating
caspase 8 may be involved in the first wave of caspase activation.
Thereafter, caspase 3 evokes a second wave of cytochrome c
release and activation of caspase cascade from 12 to 24 h. In the
second wave of caspase activation, there is a positive feedback between
caspase 3 and cytochrome c release, thereby establishing a
signaling cascade and amplifying the signal (49). The inhibitor of
caspase 3 abrogated the activation of caspase 2 at 24 h (Table III), indicating that caspase 2 may be the downstream factor of caspase
3 and complicated in the second wave of caspase activation. The
sustained activation of caspase 8 occurred after 12 h, indicating that caspase 8 may also participate in the second wave of caspase activation. Furthermore, The damaged mitochondria in the second wave of
caspase activation may also cause the accumulation of ROS, or
alternatively, the generation of ROS after 8 h may also be
involved in the release of cytochrome c and the second wave activation of caspase cascade. Both activation of caspase cascade and
elevation of ROS may account for the A
-mediated toxicity. Both
apigenin and kaempferol, but not quercetin, abrogated the second wave
of cytochrome c release or activation of caspase 2, 3, 8, and 9 (Table III, Fig. 6). Despite apigenin abrogating the initial wave
of cytochrome c release, both flavonoids did not affect the
initial wave activation of caspase 3 and 9. These results suggest that
apigenin and kaempferol protect neurons against A
-induced toxicity
by diminishing the second wave activation of the caspase cascade. Our
results clearly demonstrate that kaempferol and apigenin exhibited
differentially neuroprotective effects on A
-induced toxicity by
abrogating the release of cytochrome c and activation of the
caspase cascade. Antioxidative activity of flavonoids or other
antioxidants facilitated the caspase-dependent
neuroprotective effect of phytoestrogen, thereby conferring a
significant neuroprotective ability against A
-mediated toxicity.

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|
Fig. 9.
Proposed model for role of caspase cascade,
ROS accumulation, and the protective effect of flavonoids on cell death
of cortical neurons evoked by A -(25-35).
Schematic illustration describes the pathways contributing to cell
death by A -(25-35). Opened arrowheads indicate the
putative target sites of apigenin and kaempferol. The closed
arrowhead indicates the target side for antioxidants. For a
summary see "Discussion."
|
|
Our results demonstrate that inhibition of caspase and
scavenging of ROS act cooperatively to save neurons from A
-mediated toxicity. Therefore, inhibition of caspase cascade and decrease in the
level of ROS are proposed as neuroprotective strategies in AD. Base on
these results, the development of neuroprotective agents such as a
compound that combines potent antioxidant and caspase inhibitory
properties may prevent the incidence of AD or retard the progression of
AD. Thus, our findings point toward new approach to drug discovery for
clinical therapies of AD.
 |
FOOTNOTES |
*
This study was supported by the National Science Council
(Grants NSC88-2314-B-077-014 and NSC89-2314-B-077-007), Taiwan,
Republic of China.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: National Research
Institute of Chinese Medicine, No. 155-1 Sec. 2, LiNung St., Peitou,
Taipei 112, Taiwan, Republic of China. Tel.: 886-2-2820-1999 (ext. 4212); Fax: 886-2-2826-4266; E-mail:
yshiao@cma23.nricm.edu.tw.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M006406200
 |
ABBREVIATIONS |
The abbreviations used are:
AD, Alzheimer's
disease;
A
, amyloid
protein;
Ac-DEVD-CHO, acetyl-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-aldehyde;
Ac-LEDH-CHO, acetyl-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Glu-Asp-His-aldehyde;
Ac-IETD-CHO, acetyl-Ala- Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Ile- Glu-Thr-Asp-aldehyde;
CM-H2DCFDA, 5-(and-6)-chloromethyl-2',7'- dichlorodihydrofluorescein diacetate;
LDH, lactate dehydrogenase;
FDA, fluorescein diacetate;
MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;
ROS, reactive oxygen species;
TEAC, trolox equilibrium antioxidant
capacity.
 |
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