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Introduction
Alzheimer's disease (AD) is a neurodegenerative disease
characterized by progressive and irreversible memory loss
due to neuronal death in aged people[1]. One of the major
histopathological features of AD is the presence of senile
plaques and concomitant neuronal loss in specific areas of
the brain. β-Amyloid peptide (Aβ) is the main constituent of
senile plaques and plays a critical role in the
pathophysiology of AD[2_5]. Evidence has suggested that the neurotoxic
effect of Aβ is related to the activation of the apoptosis
pathway[6_9]. Neuronal apoptosis is the main cause of
neuronal loss in patients with AD, thus, it has been proposed
that neuroprotection may be a therapeutic strategy for
slowing down the apoptotic process of related brain cells of AD
patients[10].
The natural squamosamide was isolated from the leaves
of Annona squamosa. The compound FLZ is a novel
synthetic cyclic derivative of natural squamosamide. Its
chemical name is N-(2-[4-hydroxy-phenyl]-ethyl)-2-(2,5-dimethoxy-
phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide and its
molecular weight is 449.5 (Figure 1). Previous studies have
demonstrated that FLZ has a potent neuroprotective
property against experimental Parkinsonism and memory and
learning deficits in mice[11,12]. In an
in vitro study, FLZ was shown to protect against damage and apoptosis of primary
cultured rat brain neurons, PC12, and SH-SY5Y cell lines
exposed to hydrogen peroxide, glutamate,
N-methyl-D-aspartate, dopamine, 1-methyl, 4-phenyl-pyridinium ion
(MPP+), and ischemia-reoxygenation, indicating that
FLZ possess a neuroprotective
property[11,13]. Based on these results, the objective of this paper was to study the
protective effect of FLZ on Aβ25_35-induced toxicity in human
neuroblastoma SH-SY5Y cells and its active mechanism.
Materials and methods
Materials The compound FLZ was kindly provided by
Professor Xiao-tian LIANG (Department of Pharmaceutical
Chemistry, Institute of Materia Medica, Chinese Academy
of Medical Sciences, Beijing, China). It is a white powder
with 99% purity. The compound was first dissolved in
absolute ethanol and then diluted with 0.9% saline; the final
ethanol concentration was <0.1% for use.
Aβ25_35(Sigma, St Louis, MO, USA) was dissolved in sterile double-distilled water at
a concentration of 1 mmol/L stock solution, was aged at 37 °C for 4 d, and then stored at -20 °C before use.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H
-tetrazolium bromide (MTT), trypsin, 2',7'-dichlorofluoroescin diacetate
(DCFH_DA), EDTA, propidium iodide (PI), rhodamine 123,
agarose, HEPES, dithiothreitol (DTT), phenylmethylsul-fonyl
fluoride (PMSF), Nonidet P40, and DNase were all purchased
from Sigma (USA). Dulbecco's Modified Eagle's medium
(DMEM), F-12, and fetal bovine serum (FBS) were obtained
from Hyclone (Logan, UT, USA). The primary mouse
monoclonal antibodies Bcl-2, Bax, and cytochrome c were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). All other regents were purchased from Beijing
Chemical Company (Beijing, China).
SH-SY5Y cell cultures Human neuroblastoma
SH-SY5Y cells were purchased from the Cell Center of the
Institute of Basic Medical Science Research (Chinese Academy
of Medical Sciences, China). The SH-SY5Y cells were
cultured in DMEM:F12 (1:1) supplemented with 10% FBS, 100
IU/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified 95% oxygen and 5%
CO2 atmosphere. The medium was changed every other day. The cells were
cultured for 3_4 d until a confluence of 70%_80% was
achieved. The trypan blue assay was used to count the cells
and evaluate their viability as a percentage of viable and
non-viable cells. The cell viability was >97% prior to the
following experimental procedure. The cells were plated
at an appropriate density according to each experimental
protocol.
Treatment of SH-SY5Y cells On the day of the
experi-ment, the medium was removed and the cells were then
cultured in the same medium without FBS and exposed to
the aged peptide Aβ25_35 for 48 h in the presence or absence
of FLZ. Before adding Ab25_35, different concentrations of
FLZ (0.1, 1, and 10 µmol/L) were incubated with the cells for
30 min at 37 °C.
Cell viability assay After the cells were cultured with
Aβ25_35 in the presence or absence of FLZ for 48 h, the cell
viability was assayed with the MTT
method[14]. Briefly, MTT was dissolved in the medium without FBS and added to cells
grown in 96-well plates at a final concentration of 0.5 g/L.
Following a 4-h incubation to allow its conversion into
formazan crystals, the media was removed and cells were
lysed with DMSO (Me2SO) to allow the crystals to dissolve.
The absorbance was read at 570 nm using a Bio-Rad 450
microplate reader (Hercules, CA, USA). The results were
expressed as a percentage of MTT reduction. The
absorbance of the control cells (no FLZ and no
Aβ25_35) was used as 100%.
Measurement of lactate dehydrogenase The medium was
collected from the cells treated with
Aβ25_35 for 48 h in the presence or absence of FLZ and centrifuged at 14
000×g for 5 min. The 50 µL of the supernatant was transferred to a
tube, and the activity of lactate dehydrogenase
(LDH) was determined using an LDH kit (Beijing Chemical Reagents
Company, Beijing, China).
Apoptotic ratio assay by flow cytometry analysis
Flow cytometry was used to assess the percentage of genomic
DNA fragmentation in nuclei[15]. The cultured cells were
trypsinized and harvested, centrifuged for 5 min at
200×g, washed twice with phosphate-buffered saline (PBS) and
fixed in 70% alcohol overnight at 4 °C. The cells were
centrifuged and washed with PBS, resuspended in 0.5 mL
PBS containing 50 mg/L RNase A, and incubated for 1 h at
37 °C. The samples were stained with 50 mg/L PI for 30 min
at 4 °C in the dark. The flow cytometry analysis was
performed by FACScan (BD Biosciences, San Jose, CA, USA).
Calculations of the percentage of apoptotic cells were based
on the cumulative frequency curves of the appropriate DNA
histograms. Debris was excluded from the collection of 10
000 nuclei by empirically setting the forward-scatter channel
and side-scatter channel threshold levels.
Measurement of intracellular reactive oxygen species
by flow cytometry analysis The production of intracellular
reactive oxygen species (ROS) was determined using the
fluorescent probe DCFH_DA[16]. DCFH_DA is a membrane
permeable, non-fluorescent compound. In the presence of
peroxides in cells, it is converted to the fluorescent
derivative dichlorofluorescein (DCF). Following treatment with
Aβ25_35 (25 µmol/L) for 48 h in the presence or absence of
FLZ (0.1, 1, and 10 µmol/L), the cells were rinsed with PBS
and incubated with DCFH_DA (20 µmol/L, final
concen-tration) in Me2SO for 30 min at 37 °C. Loaded cells were
washed 3 times, and the fluorescence intensity of DCF was
determined by using flow cytometry (excitation=485 nm,
emission=535 nm).
Measurement of intracellular glutathione
The level of glutathione (GSH) was determined using
o-phthalaldehyde (OPT)[17]. On the day of the experiment, the cultured medium
was removed, the cells were washed 3 times with PBS, and
lysed by vigorous shaking in the 500 µL buffer containing
0.2% Triton-X100 and 5 mmol/L EDTA (pH =8.3). After
lysis, 0.3 mL of the buffer was removed and mixed with
0.1 mL 20% trichloric acetic acid, and centrifuged at
200×g for10 min. The supernatant was incubated with 50 mg/L
OPT (dissolved in Me2SO and diluted to the final
concentration with PBS) for 15 min at room temperature. The
fluorescence was measured at an excitation wavelength of
350 nm and emission wavelength of 420 nm. The protein
content was determined by the Lowry
method[18].
Western blot analysis of cytochrome c, Bax, and Bcl-2
in SH-SY5Y cells Following the treatment of
Aβ25_35, the cells were collected and washed with PBS. After
centri-fugation, cell lysis was carried out at 4 °C by vigorous
shaking for 30 min in RIPA buffer (25 mmol/L Tris-HCl, 150
mmol/L NaCl, 5 mmol/L EDTA, 5 mmol/L EGTA, 1 mmol/L PMSF, 1%
TritonX-100, 0.5% Nonidet P40, 10 mg/L aprotinin, and 10
mg/L leupeptin). After centrifugation at 12
000×g for 15 min, the supernatant was separated and stored at -70 °C for the
measurement of Bcl-2 and Bax[19].
The analysis of the cytochrome c release was performed
as previously described[20]. The cells were collected and
washed twice with PBS. After centrifugation, the cell pellets
were suspended in 5 mL extraction buffer (in mmol/L: HEPES
50, KCl 50, EGTA 5, MgCl2 2, DTT 1, and PMSF 0.1, pH=7.4),
and centrifuged after 15 min on ice. The cell pellets were
resuspended in the above buffer, and homogenized in a
Teflon-glass homogenizer (up and down, 50 strokes) after 45
min on ice. The homogenized buffer was transferred into the
Eppendorf tube and centrifuged at 15
000×g for 20 min at 4 °C. The supernatants were removed and stored at -70 °C until
use[20].
The protein concentration was determined by the Lowry
method[18]. After the addition of the sample loading buffer,
the protein samples (equal quantity) were denaturalized by
heating at 100 °C and were separated on the 12% SDS_PAGE.
The proteins were transferred to a polyvinylidene difluoride
(PVDF) membrane. The membrane were blocked for 1 h at
room temperature in fresh blocking buffer (TBST, 0.1%
Tween-20 in Tris-buffered saline, pH=7.4, containing 5%
nonfat dried milk) and incubated with the primary antibody
(dilution: Bcl-2 and Bax 1:200, cytochrome
c 1:1000) for 3 h at room temperature. Following 3 washes with TBST,
the membrane was incubated with alkaline
phosphatase-conjugated secondary antibodies in TBST for 2 h at room
temperature. The membrane was washed again 3 times in
TBST buffer. The protein blot was visualized by
5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt/nitro-blue
tetrazolium chloride. The blot was scanned and analyzed
the density using the software.
Statistical analysis All experiments were repeated at least
3 times using independent culture preparations.
Quantitative data were expressed as mean±SD. The statistical
analysis between various experimental results was performed
using one-way ANOVA followed by the least significant
difference test; P<0.05 was considered statistically significant.
Results
Effect of FLZ on cytotoxicity induced by
Aβ25_35 in SH-SY5Y cells The exposure of SH-SY5Y cells to
Aβ25_35 (10~50 µmol/L) for 48 h resulted in a significant decrease of cell
viability and increase of LDH release into the medium in a
dose-dependent manner (Table 1). The viability of the
SH-SY5Y cells treated with Aβ25_35 (25 µmol/L) for 48 h was
reduced to ~70% of that of the control (P<0.01), and the
activity of LDH in the medium increased 1.5-fold
(P<0.01). Most of the SH-SY5Y cellular morphology became round in shape
and aggregated together (Figure 2). The 25 µmol/L
Aβ25_35 was therefore used in the following study of the protective
action of FLZ against Aβ25_35 neurotoxicity in SH-SY5Y cells.
To select a non-cytotoxic concentration of FLZ in the
study of the effect of FLZ on Aβ25_35 neurotoxicity, we first
evaluated the effect of various concentrations (0.1, 1, and 10
µmol/L) of FLZ itself on the cell viability of SH-SY5Y cells.
FLZ at concentrations of 0.1, 1, and 10 µmol/L showed no
apparent cytotoxicity to SH-SY5Y cells (Table 2).
Pretreatment of SH-SY5Y cells with 1 and 10
µmol/L FLZ for 30 min significantly protected the cells from
Aβ25_35-induced cytotoxicity as demonstrated by increasing cell viability
(P<0.01), and a concomitant reduction of LDH release
(P<0.05) into the cultured medium (Table 3). The morphological injury of the
SH-SY5Y cells were also reduced by FLZ treatment (Figure 2).
Effect of FLZ on apoptosis in SH-SY5Y cells induced by
Aβ25_35 The hypodiploid
sub-G1 peak is regarded as apoptotic cells. The results of the flow cytometry assay showed that
the apoptotic ratio of the Aβ25_35-treated SH-SY5Y cells
markedly increased as compared with that of untreated cells. FLZ
(1 and 10 µmol/L) treatment significantly decreased the
apoptotic cell accumulation in the
sub-G1 peak in comparison with the
Aβ25_35-treated SH-SY5Y cells,
indicating that FLZ attenuated Ab-induced apoptosis (Figure 3).
Effect of FLZ on ROS production and the GSH level
induced by Aβ25_35in SH-SY5Y cells
Hydrogen peroxide was reported to mediate Ab neurotoxicity. In our experiments,
the exposure of SH-SY5Y cells to
Aβ25_35 (25 µmol/L) induced a 2-fold increase in DCF fluorescence intensity, indicating
that Aβ25_35 stimulated the production of ROS. The count of
the high fluorescence intensity of the
Aβ25_35-treated cells was also more than that of the untreated cells. The
pretreatment of FLZ (10 µmol/L) almost completely inhibited the
increase in DCF fluorescence in the SH-SY5Y cells (Figure 4).
In contrast to the increase of ROS, the intracellular GSH
level decreased dramatically in the SH-SY5Y cells after
exposure to Aβ25_35 (25 µmol/L) for 48 h. The pretreatment of FLZ
significantly attenuated the decrease of the GSH level
induced by 25 µmol/L Aβ25_35 (Table 4).
Effect of FLZ on cytochrome c release, Bax, and Bcl-2
protein expressions in SH-SY5Y cells treated with
Aβ25_35The Western blot analysis showed that the treatment of
SH-SY5Y cells with Aβ25_35 resulted in an increase of the
pro-apoptotic Bax protein expression, while the expression of
the anti-apoptosis Bcl-2 protein decreased. When the
immunoblots were quantified by densitometry analysis, the
ratio of Bax to Bcl-2 in the Aβ25_35-treated cells significantly
increased as compared with the untreated cells. The
pretreatment of SH-SY5Y cells with 1 and 10 µmol/L FLZ
reversed the alternations of Bax and Bcl-2 expressions induced
by Aβ25_35, and substantially reduced the ratio of Bax/Bcl-2
(Figure 5). The effect of FLZ at 0.1 µmol/L concentration
based on the above criteria was weak.
Aβ25_35 caused an increase of cytochrome
c release from the mitochondria of SH-SY5Y cells. FLZ at concentrations of
1 and 10 µmol/L effectively blocked cytochrome
c release from mitochondria of SH-SY5Y cells induced by
Aβ25_35 (Figure 5).
Discussion
The accumulation of plaques containing Ab in the brain
is an invariant feature of AD pathology, and abundant
evidence suggests that Ab contributes to the etiology of
AD[21]. Neuronal apoptosis was observed in human AD
brains[22,23]. Several investigators reported that
Aβ induced apoptosis in multiple cell types in
vitro[19,24_26].
Aβ25_35 is considered to be the shorter toxic fragment exerting neurotoxic
effects similar with Aβ1_40/42,
such as learning and memory impairment, neuronal apoptosis, cholinergic dysfunction,
and oxidative stress[27_29], so
Aβ25_35 is usually used to establish the
in vitro model of AD for the study of the neurotoxic
properties of Aβ and for drug screening. The human
dopaminergic neuroblastoma cell line SH-SY5Y is widely applied in
different neurochemical research. Aβ was uptaken
rapidly into the SH-SY5Y cells and reserved for several
days[30]. Some evidence has been accumulated that suggests that
Aβ25_35induces neurotoxic effects in SH-SY5Y cells similar to the
pathological changes of neurons in the mouse
brain[24_26]. The results of the present paper also indicated that the
exposure of SH-SY5Y cells to Aβ25_35 (25
µmol/L) for 48 h displayed remarkable injuries. The cell viability decreased and
LDH release from the cells increased in a dose-dependent
manner. Most of the cells became round in shape and
aggregated together. The cell apoptotic ratio also increased
significantly. When the SH-SY5Y cells were precultured with
FLZ (1 and 10 µmol/L) for 30 min, all of the above
Aβ25_35-induced injuries were significantly reduced. The cell viability,
LDH release, and apoptotic ratio were all improved. The
morphology of the FLZ-treated cells were close to that of the
control cells. These results suggest that FLZ has protective
action against Aβ25_35-induced neurocytotoxicity.
It is very important to study the mechanism by which
FLZ exerts its protective action against
Aβ25-35-induced neurocytotoxicity. Although which signaling pathway
mediated Aβ-induced neurotoxicity is not fully defined,
oxidative stress has been proposed to play a key
role[31_33]. Aβ stimulates the production of ROS by a direct or indirect
pathway[31_34]. Several investigators have demonstrated that ROS
is involved in the apoptotic mechanism of Aβ-mediated
neurotoxicity and may contribute to the increase in the apoptotic
processes found in AD[25,29,31_33]. The production of ROS
can occur very early and cause damage to cardinal cellular
components, such as lipid, protein, and nuclei acids,
resulting in cell death by modes of apoptosis or necrosis. The
high metabolic rate, a low concentration of GSH and the
antioxidant enzyme catalase, and the large proportion of
polyunsaturated fatty acids in the brain make brain tissue
particularly vulnerable to oxidative
damage[7]. Some studies have reported that free-radical scavengers or antioxidants, such as
melatonin, EGb-761, vitamin E, and estrogen could attenuate
the Aβ-induced apoptosis and
neurocytotoxicity[25,29,31,35]. Some antioxidants were reported to be effective in the
treatment of mild-to moderate dementia of AD
patients[36,37]. It seems reasonable that antioxidants will play an important
role in the search of drugs as pharmacotherapy of AD. Data
from this present study showed that 25 µmol/L
Aβ25_35 resulted in a significant increase of the ROS level in SH-SY5Y
cells. This result is consistent with previous descriptions of
the Ab-mediated generation of ROS. In addition, the authors
found that GSH, the most abundant antioxidant in cells, was
depleted by the addition of Aβ25_35. The results suggested
that oxidative stress was involved in Aβ-induced toxicity in
SH-SY5Y cells.
As major sources of ROS, mitochondrial structures are
exposed to high concentrations of ROS and might therefore
be particularly susceptible to oxidative injury. It was
reported that mitochondrial damage plays a pivotal role in cell
apoptosis[7]. The present results show that
Aβ25_35induced mitochondrial dysfunction in the MTT analysis, because the
MTT underwent conversion of the yellow MTT to purple
formazan crystals by mitochondrial succinate
dehydrogenase in viable cells, which primarily reflects the
mitochondrial metabolic capacity of viable cells and the intracellular
redox state[14]. Overproduction of ROS induced the opening
of the mitochondrial permeability pore and caused the
mitochondrial intermembrane space soluble protein (cytochrome
c, apoptosis-inducing factor) release into the
cytoplasm[38]. In most pathways of apoptosis, the release of mitochondrial
cytochrome c and apoptosis-inducing factor are also key
events in initiating the cascade of reactions leading to
apoptotic cell death[39]. The release of cytochrome
c is clearly regulated by the pro- and anti-apoptotic proteins of the
Bcl-2 family (Bax, bak, bad, bim, and bid as pro-, and bcl-2 and
bcl-xL as anti-apoptotic proteins). Bax promotes the release
of cytochrome c from the mitochondria, and Bcl-2 inhibits
the release of cytochrome c. The relative ratio of
pro-apoptotic and anti-apoptotic proteins is important to
determine cell survival or death[40_42]. The overexpression of
Bcl-2 or Bcl-xL can inhibit free-radical generation and protects
cells from apoptosis induced by various
stimuli[43,44]. In the present study,
Aβ25_35 treatment decreased the expression of
Bcl-2, increased the expression of Bax, and promoted the
cytochrome c release from mitochondria. The cell apoptotic
ratio also significantly increased. In the previous study at
our laboratory, Aβ25_35-induced cell apoptosis was also
identified by other methods, such as the DNA ladder (data not
shown).
In the present study, the pretreatment of FLZ
significantly inhibited the increase of ROS generation and the
decrease of GSH content in SH-SY5Y cells. In our previous
study, FLZ was shown to inhibit microsomal lipid peroxidation induced by
Fe2+-cysteine, and also to scavenge oxygen free radicals, indicating that FLZ has an
antioxidant property[45]. Moreover,
H2O2 is a donor of hydroxyl
radical that has been reported to mediate Aβ protein
toxicity[46]. The pretreatment of FLZ inhibited the
H2O2-induced apoptosis of
cells[11]. The pretreatment of FLZ also
protected PC12/SH-SY5Y cells against
dopamine/MPP+-induced apoptosis through inhibiting cytochrome
c release and caspase 3
activation[11,13]. The present study further
confirmed that FLZ reduced the Aβ-induced relative ratio of Bax
and the Bcl-2 protein by increasing Bcl-2 and decreasing the
Bax expressions; and also decreased the cell apoptotic ratio
of SH-SY5Y cells. Taken together, it appears that the
protective action of FLZ against Aβ25_35neuron toxicity may be in part due to its antioxidant property.
There is debate as to whether treatment with
antioxidants might theoretically act to prevent propagation of
tissue damage and improve both survival and neurological
outcomes[48]. The great pharmacological disadvantage of
most antioxidants is their very limited passage through the
blood_brain barrier. Therefore, antioxidants with much
better blood_brain barrier permeability are required for
their potential application in treating neurodegenerative
diseases, such as AD[47]. Fortunately, the results of a
pharmacokinetic study of FLZ in rats indicated that the oral
administration of FLZ penetrates through the blood_brain
barrier very well (data to be published).
Because apoptosis is the main cause in
neurodegenera-tion, such as AD and Parkinson's disease (PD), and
oxidative stress is an early event in the apoptosis process, it
appears that FLZ is a novel neuroprotectant to protect against
oxidative injury and apoptosis, and a good candidate for
neurodegeneration therapy. Further pharmacological and
toxicological studies on FLZ are in progress.
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