Original Paper
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file on Synergy |
Acta Biochim Biophys
Sin 2008, 40: 125-132
doi:10.1111/j.1745-7270.2008.00386.x
Protective effects of a new
metalloporphyrin on paraquat-induced oxidative stress and apoptosis in N27
cells
Ping Chen1, Ang Li1,
Mengjie Zhang1, Meilan He1, Zhen Chen2,
Xiaokang Wu2, Chunjun Zhao1, Shilong Wang1,
and Liping Liang1*
1 School of Life
Science and Technology and 2 Department of Neurosurgery, Shanghai
Tenth People’s Hospital, Tongji University, Shanghai 200092, China
Received: September
10, 2007
Accepted: October
15, 2007
*Corresponding
author: Tel, 86-21-65986852; E-mail, [email protected]
Paraquat (PQ,
1,1‘-dimethyl-4,4‘-bipyridinium), a widely-used herbicide, has
been suggested as a potential etiologic factor for the development of
Parkinson’s disease. In recent years, many studies have focused on the
mechanism(s) of PQ neurotoxicity. In this study, we examined the
neuroprotective effect of manganese (III) meso-tetrakis(N,N‘-diethylimidazolium)porphyrin
(MnTDM), a superoxide dismutase/catalase mimetic, on PQ-induced oxidative
stress and apoptosis in 1RB3AN27 (N27) cells, a dopaminergic neuronal cell line. The results
indicated that MnTDM significantly attenuated PQ-induced loss of cell
viability, glutathione depletion, and reactive oxygen species production. MnTDM
also ameliorated PQ-induced morphological nuclear changes of apoptosis and
increased rates of apoptosis. In addition, our data provide direct evidence
that MnTDM suppressed PQ-induced caspase-3 cleavage, possibly a key event of PQ
neurotoxicity. These observations suggested that oxidative stress and apoptosis
are implicated in PQ-induced neurotoxicity and this toxicity could be
prevented by MnTDM. These findings also proposed a novel therapeutic approach
for Parkinson’s disease and other disorders associated with oxidative stress.
Keywords Parkinson’s
disease; oxidative stress; apoptosis; paraquat; metalloporphyrin; antioxidant;
caspase-3; glutathione
Although the etiology of Parkinson’s disease (PD) is unknown, there
is a general consensus that environmental factors are important causative
agents. Paraquat, (PQ; 1,1‘-dimethyl-4,4‘-bipyridinium) has been
used as a herbicide worldwide for more than 50 years. Epidemiological studies
indicated a strong correlation between the incidence of PD and the level of PQ
exposure [1–3]. Systemic treatment of rodents with PQ induces selective
dopaminergic neuronal loss and intracellular a-synuclein deposits in the substantial
nigra [4–8], compatible with PD. Some studies revealed that the cytotoxicity
of PQ is correlated with reactive oxygen species (ROS) production and
apoptosis. However, the precise mechanism(s) of PQ toxicity is still unclear. Superoxide dismutase (SOD) and catalase are important antioxidant
enzymes that scavenge superoxide anion (O2–) and H2O2 to protect cells from
oxidative damage. If abnormal formation of O2– and H2O2 is over the capability
of SOD/catalase defenses or the activities of SOD and catalase decrease
abnormally, the production of ROS will induce cell death. This process has been
implicated in the pathogenesis of some diseases including inflammation, cancer,
and neurodegenerative diseases. The therapeutic strategy for these diseases
using SOD/catalase has not been successful. The main problems of these native
enzymes are their large size, which limits their accessibility into the cell,
short circulation half-life, and antigenicity. To overcome this issue, a class of
catalytic SOD/catalase mimetics, metalloporphyrins, were developed that can
scavenge a wide range of ROS, not only O2– and H2O2, but also peroxynitrite
(ONOO–) and lipid peroxyl radicals. It
has been shown that metalloporphyrins have much higher SOD/catalase activity
and potencies as inhibitors of lipid peroxidation than native Cu-Zn SOD and
catalase [9]. Because they are small molecule antioxidant analogs, the
bioavailability of metalloporphyrins is high and they can get into cell and
mitochondria and cross the blood-brain barrier. Manganese (III) meso-tetrakis
(N,N‘-diethylimidazolium) porphyrin (MnTDM) is a new generation of
manganese porphyrin that is designed to optimize its antioxidant properties
and minimize its potential toxicity [10]. It has been indicated that MnTDM
does not have any significant side-effects and is tolerated by patients in a
phase I clinical trial for therapy of amyotrophic lateral sclerosis [11].The 1RB3AN27 (N27) cell line,
derived from rat fetal mesencephalon dopaminergic neurons, is a homogenous
population of tyrosine hydroxylase-positive neuronal cells that express
dopamine transporter and most in the enzymes for dopamine synthesis and
metabolism. It has been verified that the cellular organelles, including
mitochondria, in N27 cells are better developed than those in tumorous cell
lines [12]. N27 cells have been fully applied in neurodegenerative disease
research, especially in PD study, for their characteristic features of dopamine
neurons [13–15]. The goals of this study were to determine whether PQ neurotoxicity
is involved in oxidative stress and apoptosis, and whether MnTDM protects
PQ-induced neurotoxicity. Evaluation of the protective effects of MnTDM on
PQ-induced oxidative stress and apoptosis can help further understand the
mechanism(s) of PQ neurotoxicity and pathogenesis of PD and provide a new
therapeutic strategy for this disease.
Materials and Methods
Cell culture and treatment
N27 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine
serum, 100 U/ml penicillin and 100 U/ml streptomycin in a water-saturated
atmosphere of 5% CO2 at 37 ?C. The cells (3?105 cells/ml) were incubated with PQ alone or in
the presence of different concentrations of MnTDM (a kind gift from Dr. Jun
CHEN, Cornell University, New York, USA) for the indicated times. MnTDM was
added 1 h prior to PQ treatment
Determination of cell
viability
The cell viability was assessed by the method described previously
[16] based on the reduction of the yellow dye MTT into blue formazan product,
mainly reduced by the mitochondrial dehydrogenases. Briefly, MTT (0.5 mg/ml)
was added to each well in 96-well plates and incubated at 37 ?C for an
additional 3 h. At the end of the incubated period, the medium with MTT was
removed and 200 ml dimethyl sulfoxide was added to each well. The plate was shaken on
a microplate shaker to dissolve the blue MTT formazan. Absorbance was read at
570 nm on a microplate reader. Cell viability was expressed as a percentage of
the control culture.
Measurement of glutathione
(GSH)
N27 cells were homogenized and deproteinated by 0.1 N perchloric
acid. The supernatant after centrifugation at 16,000 g for 10 min was
collected to determine total GSH levels according to the manufacturer’s
protocol (Glutathione assay kit; Cayman Chemical, Ann Arbor, USA). The total
GSH levels were expressed as nmol/mg protein.
Measurement of ROS production
Intracellular ROS formation was measured with oxidation-sensitive
fluorescent probe 2‘,7‘-dihydrodichlorofluorescin-diacetate
(H2DCF-DA; Molecular Probes, Eugene, USA) following the protocol described
previously [17]. Briefly, approximately 5105 cells
per well on a 48-well plate were cultured for at least 24 h and H2DCF-DA was
added to the cells at a final concentration of 20 mM. After incubation for 30
min, PQ alone or with various concentrations of MnTDM was added and incubated
for another 60 min. The fluorescence was analyzed in a fluorescent plate
reader (PerkinElmer Life Sciences, Wellesley, USA) at an excitation wavelength
of 485 nm and emission at 535 nm. The resulting fluorescence was expressed as
relative ROS production.
Assessment of morphological
nuclear change
Assessment of morphological
nuclear change
The nuclear morphological change was evaluated using Hoechst 33258
from Sigma (St. Louis, USA) following the method described before [18]. Cells
were incubated with 10 mg/ml Hoechst 33258 for 3 min at room temperature in the dark. The
stained cells were observed with a fluorescence microscope (Olympus, Tokyo,
Japan).
Flow cytometry detection of
apoptotic cells
Apoptotic and necrotic cells were quantitated by annexin V binding
and propidium iodide (PI) uptake following the manufacturer? instructions. An annexin
V-fluorescein-isothiocyanate (FITC) apoptosis detection kit was purchased from
KeyGen Biotech (Nanjing, China). Cells were collected by centrifugation (350 g),
washed with phosphate-buffered saline, adjusted to approximately 5106 cells/ml, and labeled with 5 ml annexin V (50 mg/ml) and 10 ml PI (100 mg/ml). After
labeling, cells were analyzed by flow cytometry. At each experiment, 20,000
cells were examined and CellQuest software (BD CellQuest, Franklin Lakes, USA)
was used for the acquisition and analysis of the data. Fluorescence microscopy
analysis of annexin V-biotin/streptavidin-FITC staining was also carried out to
confirm the effects of these compounds on the induction of early apoptosis.
Western blot analysis for
cleaved caspase-3 protein
The cleaved caspase-3 protein was determined by Western blot
analysis. The cells were collected, washed with phosphate-buffered saline at
pH 7.4, and lysed by lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM
EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, and 1 mM glycerophosphate]
with protease inhibitors. Protein concentration was determined by Bradford
assay. For Western blot analysis, 50 mg protein was separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis on 12% gel (Bio-Rad Laboratories,
Hercules, USA). Then the protein in gel was transferred onto a nitrocellulose
membrane. The membrane was blocked with 5% non-fat milk, and immunoblotted with
anti-caspase-3 antibody (1:200; Cell Signaling Technology, Beverly, USA)
followed by incubation with horseradish peroxidase-conjugated goat anti-rat
immunoglobulin G secondary antibody (1:10,000). The membrane was developed
using an ECL Plus Western blot detection kit (Amersham Pharmacia Biotech,
Little Chalfont, UK). The densitometry analysis of protein bands was carried
out using Image J (National Institutes of Health, Bethesda, USA), an open
source image manipulation tool widely used for biomedical image processing
analysis systems. The density of each band was normalized by b-actin.
Statistical analysis
The data are shown as the mean±SEM. For comparison of three or more
treatment groups, one-way anova with Tukey’s post-hoc test was used. P<0.05 was considered significant.
Results
MnTDM ameliorated PQ-induced
loss of neuronal cell viability
To determine whether MnTDM itself has any toxic effect on N27 cells,
the cells were exposed to various concentrations of MnTDM from 0.1 to 10 mM for 24 h. MTT
assay showed that MnTDM resulted in no loss of viability in N27 cells,
suggesting that 24 h exposure to MnTDM (10 mM) had no toxic effect on
N27 cells (data not shown). Incubation with various concentrations of PQ (100–400 mM) for 24 h
reduced the cell viability in a concentration-dependent manner. With 400 mM of PQ
treatment, the cell viability was 63.4%1.6% of the control [Fig. 1(A)].
When cells were pre-incubated with 0.5, 1.0, or 2.0 mM MnTDM for 1 h before PQ
(400 mM) treatment, the cell viability was significantly restored to
81.3%7.0%, 84.4%5.0%, and 92.5%1.0% of the control, respectively [Fig. 1(B)].
The lethal concentration of PQ determined by cell viability after 24 h
incubation in N27 cells was 504 mM (95% confidence interval: 435–572 mM).
MnTDM protected N27 cells
against PQ-induced GSH depletion and ROS production
GSH is the most abundant thiol-containing antioxidant and an
indicator of cellular redox status in tissues including the brain [19]. It also
plays an important role in preventing ROS damage. To determine whether the
cytotoxicity of PQ involved in the production of ROS and to further link the
neuroprotective effects of MnTDM with antioxidant functions, the levels of GSH
and ROS production were measured in N27 cells with PQ (400 mM) alone or in
the presence of various concentrations of MnTDM. The level of GSH was depleted
to approximately 45% of the control with PQ treatment. MnTDM treatment
significantly restored the levels of GSH in N27 cells in a
concentration-dependent manner [Fig. 2(A)]. The treatment of cells with
MnTDM (10 mM) alone had no effect on the level of GSH (data not shown). The
relative ROS production measured by H2DCF-DA was increased by approximately
100% by PQ treatment compared with the control. MnTDM treatment significantly
reduced PQ-induced ROS production in N27 cells in a concentration-dependent
manner as well, and the increased ROS production by PQ treatment was diminished
when treated with 2 mM MnTDM [Fig. 2(B)].
MnTDM protected N27 cells
against PQ-induced changes in nuclear morphology
The nuclear morphological change was assessed by Hoechst 33258 stain.
Nuclei of normal N27 cells have a regular and oval shape [Fig. 3(A)].
With PQ treatment at a concentration of 400 mM for 24 h, the appearance
of collections of multiple chromatin condensation and fragmented apoptotic
nuclei were significantly increased [Fig. 3(B)]. Pretreatment with
increasing concentration (0.5, 1.0, or 2.0 mM) of MnTDM reversed the
PQ-induced changes in nuclear morphology [Fig. 3(C–E)].
MnTDM attenuated PQ-induced
increase of apoptotic rate
To investigate whether PQ induces the increase in the percentage of
apoptotic cells, treated cells were stained with PI and FITC-labeled annexin V
and analyzed by flow cytometry. Fig. 4 illustrates PI versus annexin
V-FITC fluorescence. The lower left quadrants of the cytograms show the viable (intact)
cells that exclude PI and are negative for annexin V-FITC binding. The lower
right quadrants represent the apoptotic cells that are annexin V-FITC positive
and PI negative. The upper right quadrants contain necrotic cells that are
positive for both annexin V-FITC and PI. In the control, 92.4% of cells
excluded PI and were negative for annexin V-FITC binding, indicating intact
cells [Fig. 4(A)]. After exposure to 400 mM PQ for 24 h, 17.8% of
cells showed annexin V-positive, including 15.1% PI-negative and 2.7%
PI-positive, indicating apoptosis and necrosis, respectively [Fig. 4(B)].
Pre-incubation with 0.5, 1, or 2 mM MnTDM for 1 h resulted in a significant
decrease in the percentage of apoptotic cells to 12.2%, 11.0%, and 10.3%,
respectively [Fig. 4(C–E)]. Statistical results are shown in Fig.
4(F).
MnTDM suppressed PQ-induced
caspase-3 cleaved product protein
To examine whether caspase-3 activation is involved in PQ-induced
cell death, Western blot analysis of the caspase-3 proteolytic cleavage protein
from procaspase-3 was carried out 18 h after PQ treatment. Treatment with PQ
causes 5-fold activation of cleaved caspase-3 when compared with control cells.
When pre-treated with 2 mM MnTDM, the activation of cleaved caspase-3 was significantly
abolished (Fig. 5).
Discussion
It has been proposed that PQ cytotoxicity is mediated through ROS
production. PQ continuously produces superoxide anion that could lead to the
formation of more toxic ROS by redox cycling between PQ and PQ radicals coupled
with cellular reductases and molecular oxygen [20]. Therefore, PQ has great
potential to yield large amounts of ROS from relatively low concentrations. In
this study, our results showed that the level of GSH was depleted in PQ-treated
N27 cells. The depletion of GSH levels has been observed in the substantial
nigra of PD patients [21,22]. Although GSH is not the only antioxidant reported
to be decreased in PD, it has been established that GSH loss is an early event
in PD, which precedes decreases in both mitochondrial complex I activity and
dopamine levels [23], and occurs in pre-symptomatic patients [22], suggesting
that GSH depletion might be a crucial factor in the progression of PD. In this
study, our data also provide direct evidence that ROS production was
significantly increased in PQ-treated N27 cells measured by H2DCF-DA.
Considering that the level of GSH is relatively lower in substantial nigra
than in any other brain area [24], inducing GSH depletion and ROS production is
most likely one of the most important factors in the mechanism(s) of PQ
neurotoxicity for selective damage of dopaminergic neurons.Accumulating evidence suggests that oxidative stress-mediated
apoptosis is strongly implicated in neurodegenerative diseases [25]. In our
study, the results showed that PQ treatment decreases the number of viable
cells and induces apoptotic cells, as indicated by changes in morphological
characteristics such as cell shrinkage, chromatin condensation, and increased
apoptotic cell percentage rates. It is suggested that PQ toxicity is mediated,
at least in part, through the apoptosis pathway. The results are in agreement
with studies previously reported in the PC12 cell line [26,27], in the SH-5Y5Y
neuroblastoma cell line [28,29], and in cultured striatal [30], cortical [31],
and cerebellar granule cells [32]. Because PQ selectively injures dopaminergic
neuron in the substantial nigra, our observation in the N27 dopaminergic cell
line should be more significant than those in non-dopaminergic cell lines or
cultures. To further investigate the mechanism of PQ-induced apoptosis, we
studied cleaved caspase-3 protein production. Caspase-3 plays an important role
in the apoptotic process in two ways [33], the death receptor pathway, and the
mitochondrial apoptotic pathway. No matter which pathway is involved,
activation of caspase-3 acts as an apoptotic executor. Caspase-3 activates DNA
fragmentation factor, which in turn activates endonucleases to cleave nuclear
DNA, and ultimately leads to cell death. In the mitochondrial pathway, a
variety of stimuli trigger the mitochondrial permeability transition and the
release of cytochrome c, then activate caspase-3. In this study, the
result of PQ-induced increases in cleaved caspase-3 protein indicates that the
triggering of apoptosis cascades leading to cell death is likely one of the
most important mechanisms of PQ cytotoxicity. In contrast,
N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and rotenone, which have been
shown to cause death of dopaminergic neurons and to reproduce most
features of PD in animal and cell models, as PQ does, did not activate
caspase-3 [34]. However, it has been suggested that ROS formation and
consequent oxidative damage are induced, at least in part, from the ability of
these two compounds to inhibit complex I [35]. The differences in the
mechanism(s) between N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, rotenone,
and PQ is under further investigation.MnTDM has been shown to have effective protection against oxidative
damage in animal models of neurological diseases including stroke [36] and
amyotrophic lateral sclerosis [10]. In the present study, we evaluated the
protective effect of MnTDM against PQ-induced oxidative stress and apoptosis.
Our results showed that MnTDM prevents PQ-mediated GSH depletion and ROS
production. The ability of MnTDM to attenuate PQ-induced oxidative stress is
concentration-dependent, consistent with the SOD/catalase activities. As a
catalytic antioxidant, only a relatively small amount of MnTDM is needed to act
as a potent antioxidant. Some studies investigated the effect of other
antioxidants, such as vitamin E and melatonin, on PQ toxicity and found no
significant protection [28]. In a multicenter clinical trial, the Deprenyl and
Tocopherol Antioxidative Therapy of Parkinsonism study also showed that there
was no therapeutic benefit of a-tocopherol (vitamin E) alone, or any synergistic interaction
between a-tocopherol and deprenyl in PD patients taking part in a
double-blinded, placebo-controlled study [37]. The reason is that the
efficiency of these non-catalytic antioxidants is not potent enough and much
higher concentrations might be required. In the current experiment, our results
also showed that MnTDM extensively protects PQ-induced apoptosis in N27 cells
from several different apoptosis evaluation processes. Some studies have
established that PQ-induced apoptosis is mediated by oxidative stress [28,38].
The ability of MnTDM to diminish apoptosis is probably linked to its
antioxidant property. The findings of the current study further suggest that
PQ-induced apoptosis is related to ROS production. Furthermore, our data also
confirmed that MnTDM effectively suppressed PQ-induced activation of capase-3.
Some previous studies indicated that PQ induces phosphorylation and c-Jun
N-terminal kinase (JNK)-mediated caspase-3-dependent apoptosis [15,28]. It has
been suggested that JNK signal transduction pathway activation is induced by
oxidative stress in numerous cell types, including in dopaminergic neurons [39,40].
A synthetic SOD/catalase mimetic Eukarion, whose structure is totally different
from MnTDM, protects against PQ-induced neuronal cell death by inhibition of
JNK-mediated caspase-3 activation [5]. The precise mechanism of MnTDM
inhibition of PQ-induced activation of caspase-3 needs further investigation.
In conclusion, our findings suggest that oxidative stress and
apoptosis play a crucial role in PQ-induced neurotoxicity and provide a novel
therapeutic strategy using catalytic antioxidants for neurodegenerative
disorders associated with oxidative stress such as PD.
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