A novel synthetic compound PHID (8-Phenyl-6a, 7, 8, 9, 9a, 10-hexahydro-6H-isoindolo [5, 6-g] quinoxaline-7, 9-dione) protects SH-SY5Y cells against MPP+-induced cytotoxicity through inhibition of reactive oxygen species generation and JNK sign

Article abstract of DOI:10.1016/j.ejphar.2010.09.063

1-Methyl-4-phenylpyridinium ion (MPP⁺), a neurotoxin selective to dopaminergic neurons and an inhibitor of mitochondrial complex I, has been widely used as an etiologic model of Parkinson's disease. In this study, we investigated the protective

Full text of DOI:10.1016/j.ejphar.2010.09.063

European Journal of Pharmacology 650 (2011) 48–57
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Molecular and Cellular Pharmacology
A novel synthetic compound PHID (8-Phenyl-6a, 7, 8, 9, 9a, 10-hexahydro-6H-isoindolo
[5, 6-g] quinoxaline-7, 9-dione) protects SH-SY5Y cells against MPP+-induced
cytotoxicity through inhibition of reactive oxygen species generation and JNK signaling
In Su Kim a, Sushruta Koppula a, Byung Wook Kim a, Min Dong Song a, Ju Yeon Jung a, Gwang Lee b,
Hee Soon Lee c, Dong-Kug Choi a,
⁎
a
Department of Biotechnology, Konkuk University, Chungju, 380-701, South Korea
Department of Molecular Science and Technology, Ajou University, Suwon, South Korea
Department of Medicinal Chemistry, Chungbuk National University, Chungbuk, South Korea
b
c
a r t i c l e i n f o
a b s t r a c t
1-Methyl-4-phenylpyridinium ion (MPP+), a neurotoxin selective to dopaminergic neurons and an inhibitor of
mitochondrial complex I, has been widely used as an etiologic model of Parkinson's disease. In this study, we
investigated the protective effects of a novel synthetic compound, 8-Phenyl-6a,7,8,9,9a,10-hexahydro-6H-isoindolo
[5,6-g]quinoxaline-7,9-dione (PHID), on MPP+-induced cytotoxicity in SH-SY5Y cells. MPP+ induced apoptosis
characterized by generation of reactive oxygen species, caspase-3 activation, poly ADP ribose polymerase proteolysis
and increase in Bax/Bcl-2 ratio were blocked by PHID in a dose-dependent fashion. Furthermore, MPP+-mediated
activation of stress-activated protein kinase/c-Jun N-terminal kinase (JNK) was also inhibited by PHID in a dose-
dependent manner. The results indicate that PHID protects against MPP+-induced apoptosis by blocking reactive
oxygen species stimulation and JNK signaling pathways in SH-SY5Y cells, implicating the novel compound in the
prevention of progressive neurodegenerative diseases such as Parkinson's disease.
Article history:
Received 15 March 2010
Received in revised form 17 September 2010
Accepted 20 September 2010
Available online 12 October 2010
Keywords:
PHID
MPP+
Reactive oxygen species
Apoptosis
Parkinson's disease
Cytotoxicity
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
suppression of dopaminergic neuronal cell death by regulation of
intracellular reactive oxygen species and modification of the apoptotic
Neuronal cell death is an important feature of both acute and chronic
neurodegenerative diseases (Yuan and Yankner, 2000; Mattson, 2000).
The hallmark of Parkinson's disease is a loss of dopaminergic neurons in
the substantia nigra. Although the etiology of Parkinson's disease
remains unclear, accumulating evidence strongly suggests the involve-
ment of oxidative stress and mitochondrial dysfunction (Mattson, 2000;
Pieczenik and Neustadt, 2007; Szeto, 2006). The major mitochondrial
defect in Parkinson's disease appears to be associated with inhibition of
respiratory chain complex I. 1-Methyl-4-phenylpyridinium ion (MPP+),
the active metabolite of 1-methyl-4-phenyl-2, 3, 6-tetrahydropyridine
(MPTP), is a neurotoxin that selectively and potently inhibits complex I
of the mitochondrial electron transport chain (Singer and Ramsay,
1990) and induces a condition closely resembling Parkinson's disease in
cellular and animal models (Eberhardt and Schulz, 2003; Przedborski
et al., 2004; Przedborski and Jackson-Lewis, 1998). It is now believed
that the dramatic loss of dopaminergic neurons involving oxidative
stress and/or mitochondrial impairment results from the activation of
anapoptotic cascade (Mattson, 1990; Fiskum etal., 2003). Therefore, the
cascade could be an effective therapeutic approach in the alleviation of
the progression of neurodegeneration.
For several years, our group has synthesized compounds from
isoindoloquinoxaline, an antitumor agent (Diana et al., 2008; Jung et al.,
2006), and screened for anti-apoptotic activities by neuron cell-based
assays. One of these compounds, 8-Phenyl-6a,7,8,9,9a,10-hexahydro-
6H-isoindolo[5,6-g]quinoxaline-7,9-dione (PHID), was presently inves-
tigated. Here, we report the inhibitory effects of PHID upon cell viability
loss, increase in reactive oxygen species generation, caspase-3 activa-
tion, poly ADP ribose polymerase (PARP) proteolysis and Bax/Bcl-2 ratio
induced by MPP+ in SH-SY5Y cells. Further, we have examined MPP+-
mediated activation of stress-activated protein kinase (SAPK)/c-Jun
N-terminal kinase (JNK) in SH-SY5Y cells.
2. Materials and methods
2.1. Reagents
MPP+, 3-(3,4-dimehylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bro-
mide (MTT), poly-D-lysine and caspase-3 assay kit were obtained from
Sigma-Aldrich (St Louis, MO, USA). Six-well and 96-well tissue culture
plates and 100 mm-diameter culture dishes were purchased from Nunc
⁎
Corresponding author. Tel.: +82 43 840 3610; fax: +82 43 840 3872.
E-mail address: choidk@kku.ac.kr (D.-K. Choi).
0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2010.09.063

I.S. Kim et al. / European Journal of Pharmacology 650 (2011) 48–57
49
(North Aurora Road, IL, USA). Dulbecco's modified Eagle's medium
2.4. Isolation of total RNA and expression analysis
(DMEM) and fetal bovine serum (FBS) were obtained from Gibco-BRL
Technologies (Carlsbad, CA, USA). Lactate dehydrogenase (LDH) assay kits
were purchased from Dojindo (Rockville, MD, USA). 2, 7-Dichlorofluo-
rescein diacetate (DCFH-DA) and propidium iodide (PI) were supplied by
BD Clontech (Mountain View, CA, USA). Antibodies against apoptosis
signal-regulating kinase 1 (ASK1), phospho-ASK1, stress-activated protein
kinase/c-Jun N-terminal kinase (SAPK/JNK), phospho-SAPK/JNK, c-Jun,
phospho-c-Jun, p38 MAPK kinase, phospho-p38 and poly ADP ribose
polymerase (PARP) were obtained from Cell Signaling Technology
(Danvers, MA. USA). Horseradish peroxidase (HRP)-conjugated secondary
antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). All other chemicals used in this study were of analytical grade and
were obtained, unless otherwise noted, from Sigma-Aldrich (St Louis, MO,
USA).
SH-SY5Y cells (1×106cells/well) were cultured in 6-well plates, and
the total RNA was isolated by extraction with TRIzol (Invitrogen, Carlsbad,
CA, USA). For the reverse transcription-polymerase chain reaction
(RT-PCR), 2.5 μg of total RNA were reverse transcribed using a First
Strand cDNA synthesis kit (Invitrogen) and the resulting complimentary
DNA was used as the template for PCR. The following primers were used
for PCR: Bcl-2 sense, 5′- ACTTTGCAGAGATGTCCAGT-3′; Bcl-2 anti-sense,
5′-CGGTTCAGGTACTCAGTCAT-3′; Bax sense, 5′-CTGGACAGTAACATG-
GAGC-3′; Bax anti-sense, 5′-TCTTCTTCCAGATGGTGAGT-3′; glyceralde-
hyde 3-phosphate dehydrogenase (GAPDH; which was used as an
internal control to evaluate the relative expressions of Bcl-2 and Bax)
sense, 5′-GCAGTGGCAAAGTGGAGATTG-3′; and GAPDH anti-sense 5′-TG
CAGGATGCATTGCTGACA-3′. RT-PCR products were electrophoresed on a
1% (w/v) agarose gel, stained with ethidium bromide, and bands were
visualized by ultraviolet light.
2.2. Cell culture and treatments
Human neuroblastoma SH-SY5Y and mouse neuroblastoma
Neuro-2a cells were obtained from the American Type Culture
Collection (ATCC) and cultured in DMEM supplemented with 10%
(v/v) inactivated fetal bovine serum, and 100 U/ml penicillin/strepto-
mycin. Cells were maintained at 37 °C in 5% CO2 and 95% humidified air
incubator for the indicated time. Mouse dopaminergic MN9D cells were
kindly provided by Dr. Young J Oh (Yonsei University, Seoul, f Korea).
MN9D cells were plated on 96-well plates or 6-well plates coated with
25 μg/ml poly-D-lysine. Cultures were maintained in DMEM supple-
mented with 10% fetal bovine serum (Invitrogen, San Diego, CA) in an
incubator with an atmosphere of 10% CO2 at 37 °C for 3 days. Cells were
subsequentlyswitched to serum-freeN2medium (Bottenstein and Sato,
1979). All experiments were carried out 24–48 h aftercells were seeded.
SH-SY5Y, Neuro-2a and MN9D cells were pretreated for 4 h with 0.1, 1,
or 10 μM PHID before incubation in medium containing 1 mM, 0.5 mM
and 25 μM MPP+, respectively. For morphological studies, SH-SY5Y cells
maintained in DMEM were kept in a humidified atmosphere at 37 °C
and 5% CO2. Cells were allowed to attach for 1 h before the addition of
10 μM (final concentration) all trans-retinoic acid (RA, made in ethanol
as a 10 mM stock solution; Sigma-Aldrich, St Louis, MO, USA) that was
used to induce neuronal differentiation.
2.5. Immunoblot analysis
To obtain the total cell lysate, 0.1 or 0.05 ml of RIPA buffer (1× PBS,
1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate
(SDS), with freshly added protease inhibitor cocktail; (Calbiochem,
San Diego, CA, USA) was added to SH-SY5Y and Neuro-2a cells cultured
in 6-well plates. The cells were scraped from the well surface,
incubated for 10 min on ice, and centrifuged at 14,000×rpm for
10 min at 4 °C. The protein concentration was determined by the DC
protein assay (Bio-Rad, Hercules, CA, USA), and 15 μg of whole cell
lysate was loaded for 10% SDS-polyacrylamide gel electrophoresis
(SDS-PAGE). Electrophoresis was performed and the proteins were
transferred to polyvinylidene fluoride membranes (Millipore, Billerica,
MA, USA) using an electroblotting apparatus (Bio-Rad, Hercules, CA,
USA). Each membrane was blocked for 1 h in Tris-buffered saline (TBS)
containing 0.1% Tween-20 and 5% dry milk prior to incubation
overnight with a 1:1000 dilution of anti-PARP or anti-phosphor-c-
Jun primary antibody followed by incubation for 1 h with a 1:10000
dilution of HRP-conjugated secondary antibody. The optical densities
of the antibody-specific bands were analyzed by a LAS-3000
luminescent image analyzer (Fuji, Tokyo, Japan).
2.3. Assessment of cell viability
2.6. Flow cytometry detection of apoptotic cells
Cell viability was measured by using a previously described
quantitative colorimetric MTT assay of mitochondrial activity in living
cells (Datki et al., 2003). MTT dissolved in phosphate-buffered saline
(PBS) was added at the end of incubation to a final concentration of
0.5 mg/ml. After incubation for 4 h at 37 °C and 5% CO2, each
supernatant was removed and the formazan crystals that had formed
in the viable cells were measured at 550 nm using a microplate reader
(Molecular Devices, Sunnyvale, CA, USA). The release of the
intracellular enzyme LDH into the medium was also used as a
quantitative measurement of cell viability. Cells were incubated in
96-well plates with the indicated concentration of PHID and 1 mM of
MPP+ for 48 h and each assay included control cells that were not
treated with PHID. Each cell suspension was centrifuged (4000×g,
5 min, 4 °C) and then the supernatant was collected. LDH activity in
the supernatant was performed by using a cytotoxicity assay kit
according to the manufacturer's instructions (Takara Bio, Shiga,
Japan). Absorbance was read at 440 nm and cytotoxicity (%) was
calculated as:
SH-SY5Y cells (1×106cells/well) were collected by centrifugation
following MPP+ exposure for 60 h and washed two times with ice-cold
PBS. The pellets were resuspended in ice-cold 70% ethanol and fixed at
4 °C for 24–48 h. Cells were washed and resuspended in 1 ml of DNA
staining reagent containing 50 μg/ml RNase, 0.1% Triton X-100, 0.1 mM
EDTA (pH 7.4), and 50 μg/ml PI. The staining was stable at 4 °C for
30 min (Telford et al., 1991). Red fluorescence (DNA) was detected
through a 563–607 nm band pass filter by using a FACSCalibur flow
cytometer (Becton Dickinson, San Jose, CA, USA). Ten thousand cells in
each sample were analyzed and the percentage of apoptotic cells
accumulating in the sub-G1 peak was calculated by Cell Quest software
(Becton Dickinson San Jose, CA, USA).
2.7. Caspase-3 activity assay
Caspase-3 activity was detected by use of the colorimetric caspase-
3 assay kit according to the manufacturer's protocol. In brief, the
reaction mixture (total volume 100 μl) contained 5 μl of cell lysate and
10 μl of caspase-3 substrate (Ac-DEVD-pNA; final concentration,
200 μM) in assay buffer, and the assay was carried out in a 96-well
plate. To account for non-specific hydrolysis of substrate, a control
reaction mixture contained 5 μl of cell lysate and 10 μl of the specific
caspase-3 inhibitor (Ac-DEVD-CHO; final concentration, 20 μM) in
È
Â
ðsupernatant value−blank valueÞ ꢀ ðsupernatant value−blank valueÞ
+ ðupper control value−blank valueÞꢁg × 100: Each experiment
was performed in triplicate:

50
I.S. Kim et al. / European Journal of Pharmacology 650 (2011) 48–57
assay buffer. Both mixtures were incubated for 90 min at 37 °C and
the absorbance was read at 405 nm.
3.2. PHID ameliorated MPP+-induced reduction in cell viability
Treatment of SH-SY5Y cells with 1 mM MPP+ alone for up to 48 h
resulted in neuronal cell loss in almost half of the cells as evaluated by
MTTassay. When thecells werepretreated withnon-toxic doses of PHID
(0.1, 1, and 10 μM) for 4 h prior to the addition of 1 mM MPP+, cell
viability was significantly improved in a dose-dependent fashion. PHID
alone did not affect the overall cell viability. When cell viability under
serum-free conditions was defined as 100% survival, viability of cells
treated with 1 mM MPP+ decreased to 50.2 0.4%. The viability of cells
incubated with 0.1, 1 and 10 μM of PHID was 53.3 3.4%, 61.4 1.3%,
and 71 0.7% of the control, respectively (Fig. 2A).
The level of cell death was also estimated by LDH assay which
detected the release of LDH in culture medium. Exposure of SH-SY5Y
cells to MPP+ resulted in 1.6 0.1-fold increase in the release of LDH
when compared to the control cultures (Pb0.001 vs. control group).
Pretreatment of PHID at 0.1, 1 and 10 μM for 4 h significantly reduced
MPP+-induced LDH release dose responsibly. The viability of cells
treated with MPP+, incubated with indicated concentrations of PHID
was 1.52 0.03 at 0.1 μM PHID (Pb0.05), 1.45 0.03 at 1 μM PHID
(Pb0.005) and 1.38 0.01 at 10 μM PHID (Pb0.001) respectively,
compared with 1 mM MPP+ only group (Fig. 2B).
2.8. Measurement of intracellular reactive oxygen species
Intracellular reactive oxygen species production based on the
formation of hydrogen peroxide generated by an oxidative metabolic
burst was measured using a non-fluorescent compound (DCFH-DA) as
previously described (Bass et al., 1983). Viable cells can deacetylate
DCFH-DA to 2′,7′-dichlorofluorescin (DCFH), which is not fluorescent.
This compound reacts quantitatively with oxygen species within the
cell to produce a fluorescent dye 2′, 7′-dichlorofluorescein (DCF),
which remains trapped within the cell and can be measured to
provide an index of reactive oxygen species level. Cells (1×106 cells/
well) were collected and loaded with 20 μM DCFH-DA dissolved in
dimethyl sulfoxide for 30 min at 37 °C. After washing out the excess
probe, the cells were measured by a FACS Caliber flow cytometer
(Becton Dickinson, San Jose, CA, USA).
2.9. Statistical analysis
Further, to prove that PHID has similar effect in dopaminergic cells
and independent of the cell line used we performed MPP+-induced
reduction in cell viability loss using MTT assay on mouse dopaminer-
gic MN9D and mouse neuroblastoma Neuro-2a cells. Our data
revealed that exposure of MN9D cells to 25 μM MPP+ induced a
reduction in cell viability. When pretreated with PHID at 0.1, 1, and
10 μM for 4 h prior to the addition of 25 μM MPP+, cell viability was
significantly improved which occurred in a concentration dependent
manner (Fig. 2C). Similar effects were shown with Neuro-2a cells
treated with 0.5 mM MPP+ with or without various concentrations of
PHID (Fig. 2D). PHID treatment alone did not affect the overall cell
viability tested in both these cell lines. These results strongly indicate
that MPP+-induced loss of cell viability can be fully attenuated by
PHID in a dose-dependent manner in all the cell types used.
To investigate the morphology, we observed the effect of PHID in
MPP+-induced cellular morphology changes in differentiated SH-SY5Y
cells. As shown in Fig. 2E (lower panel), concentration dependent
protection to neurite outgrowthwasobservedindifferentiated SH-SY5Y
cells on pretreatment with PHID upon MPP+ insult. However, PHID
alone does not show any morphological changes.
The results were expressed as means S.E.M of at least three
independent experiments run in duplicate or triplicate. Statistical
analysis was performed by one-way analysis of variance (ANOVA)
followed by post hoc multiple comparisons using the Bonferroni
method with the Sigmaplot 11.1 software (Systat Software, San Jose,
CA, USA). Pb0.05 was considered to be statistically significant.
3. Results
3.1. Synthesis of PHID
A solution of quinoxalinosultine (100 mg, 0.45 mM) and N-phenyl-
maleimide (3 equiv) in toluene (4 ml) was sealed in a 40 ml Pyrex tube
and heated at 200 °C for 24 h. The solution was then cooled to room
temperature. The solvent was evaporated under vacuum, and the
residue was subjected to flash silica gel column chromatography using
dichloromethane/ethyl acetate (from 9:1 to 1:4) as the eluent to yield
PHID (purityN98%; Fig. 1). The product was obtained as a white solid in
93% yield; mp 250–251 °C; IR (KBr) 1706, 1502, 1376 cm−1
;
1H-NMR
To verify the inhibitory effect of PHID on the increase in apoptotic cells,
SH-SY5Y cells were labeled with PI and histogram analysis-related nuclear
DNA contents were ascertained by flow cytometry. DNA content
(CDCl3, 300 MHz) δ 8.82 (s, 2H, hetAr-H), 7.96 (s, 2H, Ar-H), 6.82–7.06
(m, 5H, Ar-H), 3.51–3.58 (m, 4H, CH2, CH), and 3.25–3.28 (m, 2H, CH2).
Fig. 1. The structure and synthesis of PHID.

I.S. Kim et al. / European Journal of Pharmacology 650 (2011) 48–57
51
Fig. 2. Effect of PHID on MPP+-induced cell death in SH-SY5Y, MN9D and Neuro-2a cells. Cells were exposed to MPP+ for 48 h and cell viability was assessed by MTT assay or LDH
assay. Cells were treated with indicated doses of MPP+ in the absence or presence of PHID (0.1, 1, and 10 μM). (A) Cell viability assessed by MTT assay in SH-SY5Y cells. (B) Cell
viability assessed by LDH assay in SH-SY5Y cells. (C) Cell viability assessed by MTT assay in MN9D cells. (D) Cell viability assessed by MTT assay in Neuro-2a cells. (E) Microscopic
examination of morphological changes treated with MPP+ with or without PHID in SH-SY5Y cells differentiated with retinoic acid. Data are expressed as the percentage of values in
untreated control cultures. Each value represents mean S.E.M. (n=3). #Pb0.001, compared with control group; and *Pb0.05 and ** Pb0.001 compared with MPP+-treated group
by one-way analysis of variance (ANOVA) and post hoc Bonferroni multiple comparison.

52
I.S. Kim et al. / European Journal of Pharmacology 650 (2011) 48–57
histograms were obtained after cells were exposed to 1 mM MPP+ and
with various concentrations of PHID. Cells with DNA content below G1
phase (defined as hypodiploid sub-G1 peak) were regarded as apoptotic
cells (Liu et al., 2000, 2001; Zhang and Zhao, 2003; Ormerod, 2002). When
cells were incubated in medium alone, a typical single peak of nuclei with
diploid DNA content was observed (Fig. 3A) and there was only about
2%–3% cell death. In the presence of 1 mM MPP+, apoptotic cells
(apparent as a characteristic hypodiploid DNA content peak which
shows sub-G0-G1 apoptotic populations) were distinguishable in the
population of 34.7 0.96% (Fig. 3B). PHID alone did not show any effect on
the overall population of cells (Fig. 3C). Following treatment with PHID
(0.1, 1 and 10 μM), the apoptotic population was reduced to 34.5 0.48%
(Fig. 3D), 26.8 0.56% (Fig. 3E), and 18.25 0.38% (Fig. 3F), respectively.
3.4. PHID suppressed MPP+-induced increase of reactive oxygen species
in SH-SY5Y cells
Mounting evidence suggests that oxidative damage and overpro-
duction of reactive oxygen species can cause severe impairment of
cellular functions, and may contribute to the apoptotic process found in
Parkinson's disease (Zhou et al., 2008; Choi and Suk, 2007; Kehrer and
Smith, 1994). MPP+ stimulates the production of the reactive oxygen
species and induces cell death in SH-SY5Y cells (Adams et al., 1993;
Cassarino et al., 1999; Domingues et al., 2008). Therefore, we
investigated whether PHID was capable of modulating intracellular
reactive oxygen species generation by MPP+. The fluorescent probe
DCFH-DA was used to assess the generation of reactive oxygen species.
The relative fluorescent intensity graph and the typical flow cytometric
histogram of DCF were shown (Fig. 5). Cells exposed to MPP+ showed
the obvious increase in DCF fluorescent intensity at 24 h when
compared to the control cultures (Fig. 5B). PHID treatment at 10 μM
alone did not show any significant changes in the reactive oxygen
species production (Fig. 5C). Whereas the reactive oxygen species
production in cells exposed to 1 mM MPP+ with 0.1, 1, and 10 μM of
PHID were suppressed to 89.45 5.06%, 79.93 2.06%, and 64.70
3.25% respectively in a dose-dependent fashion as compared to that in
MPP+-treated group (Fig. 5D–F). Flow cytometric analysis also indicated
that PHID effectively blocked ROS production mediated by MPP+, as
evident by a shift to the left of the fluorescence intensity thus reflecting a
reduction in reactive oxygen species generation, and suggesting that
PHID possesses reactive oxygen species scavenging properties.
3.3. PHID suppresses MPP+-induced caspase-3 activation and PARP
proteolysis
Caspases are the molecular machinery that drives apoptosis
(Grutter, 2000). Presently caspase-3, a crucial biomarker of the neuronal
apoptosis that also act as an apoptotic executor (Hartmann et al., 2000)
was investigated in both SH-SY5Y and MN9D cells. Treatment with
1 mMMPP+ markedly increased caspase-3 activity and addition of PHID
attenuated MPP+-induced caspase-3 expression to 349.33 15.14%,
266 18.68%, and 191.33 20.25%, respectively, in SH-SY5Y cells in a
dose-dependent manner (Fig. 4A).
Caspase-3 also plays a major role in PARP cleavage early during
apoptosis in many different cell lines (Lazebnik et al., 1994; Le et al.,
2002). We further examined the cleavage of PARP. A previous study
reported that MPP+ induces obvious increase in PARP proteolysis at
48 h when compared to the control cultures (Kitamura et al., 1998).
Cleavage of PARP was detected using polyclonal antibody against
cleaved PARP fragments (85 kDa). Following the treatment with 1 mM
MPP+, PARP proteolysis markedly increased to 2.34 0.43 fold, while
PHID at concentrations of 0.1, 1, and 10 μM, attenuated MPP+-induced
PARP proteolysis to 2.36 0.47, 1.89 0.48, and 1.67 0.39 fold,
respectively in SH-SY5Y cells dose-dependently. The western blot data
and the band density ratio to β-actin were represented in Fig. 4B. Similar
effects were observed in attenuation of MPP+-induced caspase-3
activation and PARP proteolysis in MN9D cells and the data was
represented in Fig. 4C and D, respectively.
3.5. PHID suppresses MPP+-induced increase in JNK and p38 activation,
but not extracellular signal-regulated kinase
Stress-activated protein kinases are considered as one of the important
upstream signals which lead to neuronal degeneration. Neuronal
apoptosis in neurodegenerative diseases was observed in response to
various stimuli via the c-Jun N-terminal kinases (JNK) and p38 kinases
(Mielke and Herdegen, 2000). Phosphorylated forms of JNK and p38 are
recognized to be highly expressed in postmortem brains of PD (Ferrer
et al., 2001). Activation of SAPK/JNK and its transcription factor targets are
prominent mediators of dopaminergic toxicity in a MPP+-induced model
of Parkinson's disease (Luo et al., 1998; Oo et al., 1999; Choi et al., 1999;
Saporito et al., 1999, 2000; Gearan et al., 2001; Chun et al., 2001; Xia et al.,
Fig. 3. Effect of PHID against MPP+-induced neurotoxicity in cultured SH-SY5Y cells as detected by flow cytometric DNA analysis. (A) Control cells; (B) cells exposed to 1 mM MPP+
alone; (C) cells exposed to 10 μM PHID alone; and cells pretreated with PHID at 0.1 (D), 1 (E), and 10 μM (F), in the presence of 1 mM MPP+. Bar ( ) represents a sub-G0/G1 peak or
├┤
hypodiploid DNA fraction. The results are representative of one of three independent experiments.

I.S. Kim et al. / European Journal of Pharmacology 650 (2011) 48–57
53
Fig. 4. PHID inhibits MPP+-induced activation of caspase-3 and PARP proteolysis SH-SY5Y.and MN9D cells. Cells were treated with MPP+ in the absence or presence of PHID.
Caspase-3 activity in SH-SY5Y cells was measured by a colorimetric assay kit (A) and PARP proteolysis was measured by immunoblot analysis in SH-SY5Y cells. The band intensity
was quantified and is presented as relative to the level of β-actin (B). Caspase-3 activity in MN9D cells was measured by a colorimetric assay kit (C) and PARP proteolysis was
measured by immunoblot analysis. The band intensity was quantified and is presented as relative to the level of β-actin (D). Cell lysate samples (20 μg protein/lane) were subjected
to immunoblot with specific antibodies against the cleaved site of human PARP. Data represent mean S.E.M. (n=3). #Pb0.001 compared with control group; and *Pb0.05,
**Pb0.005 and *** Pb0.001 compared with MPP+-treated group by one-way analysis of variance (ANOVA) and post hoc Bonferroni multiple comparison.
2001). Also, overexpression of ASK1 activates JNK and induces apoptosis
in nerve growth factor-differentiated PC12 cells and primary rat
sympathetic neurons. While expression of dominant-negative form of
ASK1 inhibits such induced apoptosis in these cells (Kanamoto et al.,
2000). To investigate a possible effect of PHID on MPP+-stimulated JNK
signaling cascade, we measured the changes in phosphorylation levels of
ASK1, SAPK/JNK, c-Jun and p38 in SH-SY5Y cells (Fig. 6). Western blot
analysis of the total protein levels and the expression of phospho-ASK1
(Thr845), phospho-SAPK/JNK (Thr183/Tyr185), phospho-c-Jun (Ser73)
and p38 were performed to provide an estimate of the relative level of
expression of the proteins. After 24 h of incubation, 1 mM MPP+
significantly increased phosphorylation of ASK1 to 1.95 0.25 fold
(Fig. 6A), p54-SAPK/JNK to 3.23 0.25 fold (Fig. 6B), c-Jun to 3.15
0.33-fold (Fig. 6C) and p38 to 4.24 0.24 (Fig. 6D) as compared to
controls. Following PHID treatment (0.1, 1, and 10 μM), p-ASK1 was
reduced to 1.91 0.09, 1.51 0.12 and 1.20 0.05 fold, phosphor-p54-
SAPK/JNK to 3.08 0.15, 2.89 0.09, 1.92 0.14 fold, phospho-c-Jun to
2.81 0.22, 2.53 0.15, 1.88 0.38 fold and phosphor-p38 to 3.97 0.33,
3.30 0.15, 2.61 0.26 fold (Fig. 6A–D) respectively, in a concentration
dependent fashion. PHID pretreatment alone did not affect the total
expression of these proteins but only inhibited the activated forms
indicating that PHID suppresses the MPP+-induced increase in JNK
activation.
using phospho-ERK1/2 antibody. PHID treatment alone and in the
presence of 1 mM MPP+ did not significantly affect phosphorylation of
ERK 1/2, indicating that the protective effect of PHID was not regulated via
the mitogen-activated protein kinase (MAPK)/ERK signaling pathway
(Fig. 6E). Our result correlated with earlier study that treatment with
1 mM MPP+ for 24 h did not induce any activation in the expression of
phosphorylated ERK in SH-SY5Y cells (Gómez-Santos et al., 2002) andwas
only activated when high doses of MPP+ (N2.5 mM) was administered
(Zhu et al., 2007; Halvorsen et al., 2002).
3.6. PHID affected the expression of Bcl-2 and Bax in MPP+-treated cells
One of the main mechanisms involved in the induction of the
mitochondrial apoptotic pathway is a decrease in the levels of Bcl-2 or,
alternatively, an increase in the levels of Bax (Cory and Adams, 2002). In
this study, we investigated whether PHID has any effect on the
expression of Bcl-2 and Bax in MPP+-treated cells using expression
analysis. As shown in Fig. 7A, Bax expression was increased significantly
in the MPP+-treated group compared with control cells, a finding
consistent with previous studies (Gao et al., 2008; Cheng et al., 2009).
However, PHID treatment suppressed Bax mRNA expression in a dose-
dependent manner. In contrast, the level of Bcl-2 in the MPP+-treated
group was significantly decreased compared with that of control cells,
while expression of Bcl-2 was recovered following PHID treatments. The
Bax/Bcl-2 ratio in cells exposed to 1 mM MPP+ was increased 3.2
0.4 fold than the control group, while in cells pretreated with 0.1, 1, and
10 μM PHID, the ratio decreased in a dose-dependent manner,
suggesting that PHID treatment shifted the balance between pro- and
anti-apoptotic members towards cell survival (Fig. 7B). PHID treatment
alone did not significantly alter the Bax/Bcl-2 ratio (Fig. 7B).
Experiments in cell culture and animal model systems showed that
ERK1/2 play a key role in a variety of cellular signaling including
dopaminergic cell death (Hunot et al., 2004; Peng et al., 2004). Activation
of ERK1/2 by the neurotoxin MPP+ has been well documented (Gómez-
Santos et al., 2002; Zhang et al., 2007). Given that ERK1/2 confers survival/
neuroprotective effects (Kyriakis and Avruch, 2001), we investigated
activation of ERK1/2 in MPP+-induced cell death by Western blot analysis

54
I.S. Kim et al. / European Journal of Pharmacology 650 (2011) 48–57
Fig. 5. Effects of PHID on reactive oxygen species generation in SH-SY5Y cells exposed to MPP+. Cells were exposed to 1 mM MPP+ in the absence or presence (0.1, 1, or 10 μM) of PHID.
Reactive oxygen species generation was detected by fluorometric analysis of the fluorescent intensity of DCF following conversion of DCFH-DA. Upper panel: The relative fluorescent
intensity graph. Lower panels: (A) control cells; (B) cells exposed to 1 mM MPP+ only; (C) cells exposed to 10 μM PHID only; and cells pretreated with 0.1 (D), 1 (E), and 10 μM (F) PHID in
the presence of 1 mM MPP+. Data represent mean S.E.M. (n=3). #Pb0.001 compared with control group; and *Pb0.05 and **Pb0.005 compared with MPP+-treated group. FL1-H:
relative DCF fluorescence intensity, and counts: cell number.
4. Discussion
inhibited the MPP+-induced morphological changes and cell loss in
SH-SY5Y cells differentiated with retinoic acid. Further, PHID signifi-
cantly suppressed MPP+-induced reactive oxygen species production,
decreased the Bax/Bcl-2 ratio, and inhibited SAPK/JNK and c-Jun
phosphorylation in SH-SY5Y cells. When compared with a known
drug apomorphine at 5 μM concentration in SH-SY5Y cells, the
inhibition of MPP+-induced neuronal cell death, Bax/Bcl-2 ratio,
caspase-3 activation and PARP proteolysis were similar with that of
PHID treated at 10 μM, proving the compound as a promising candidate
and can be further evaluated for development as a potent therapeutic
agent in treating neurodegenerative diseases (supplementary data).
It is well-known that many types of chemical and physiological
neurotoxins including MPP+ induce oxidative stress are able to cause an
initial reactiveoxygen species increase and initiate oxidative-dependent
cascades in apoptotic dopaminergic cell death (Wong et al., 1999).
Previous reports indicated that oxidative damage occurs in the
Parkinsonian brain (Mattson, 2000) and overproduction of reactive
oxygen species can cause severe impairment of cellular functions and
may contribute to the apoptotic process found in Parkinson's disease
(Kehrer and Smith, 1994). Presently, treatment with MPP+ resulted in
significantcellviabilityloss and reactiveoxygen species overproduction.
Pretreatment with PHID decreased the MPP+-induced cytotoxicity
and accumulation of reactive oxygen species in a dose-dependent
SH-SY5Y cells treated with MPP+ have been widely used as an in
vitro cellular model for studying neurodegenerative events that occur in
Parkinson's disease (Sheehan et al., 1997). It is interesting to note that
several compounds like flavopiridol, 17-(Allylamino)-17-demethoxy-
geldanamycin, and protocatechuic acid having profound antitumor
activities also possess marked neuroprotective function (Pallas et al.,
2005; Waza et al., 2006; Tseng et al., 1996). In search for developing
novel therapeutic agents for treating neurodegenerative diseases
including Parkinson's disease, we systematically screened isoindolo-
quinoxaline. Isoindoloquinoxaline and its derivatives have been earlier
reported for their potent antitumor activities (Diana et al., 2008), but
their use in neuroprotection was untouched. Our search yielded a
synthetic compound PHID, derived from isoindoloquinoxaline showing
significant neuroprotective effects in MPP+-induced cytotoxicity in
SH-SY5Y cells.
Here, we present evidence that non-toxic concentrations of PHID
protect SH-SY5Y cells against MPP+-induced cytotoxicity in several
aspects. At sub-toxic doses, PHID attenuated MPP+-induced cell loss in
human neuroblastoma SH-SY5Y, mouse neuroblastoma neuro-2a and
mouse dopaminergic MN9D cells. Further, PHID prevented caspase-3
activation and PARP proteolysis in SH-SY5Y and MN9D cells. PHID also
Fig. 6. Effects of PHID on JNK signaling cascade in MPP+-induced apoptosis in SH-SY5Y cells. Cells were treated with 1 mM MPP+ in the absence or presence of PHID. The immunoblot
analysis using specific antibodies and the densitometric analysis of total and phosphorylated forms of each protein (20 μg protein/lane) in cell lysate samples was represented. (A) Total
ASK1 and phosphorylated form of ASK1. (B) Total SAPK/JNK and phospho-SAPK/JNK. (C) Total c-Jun and phospho-c-Jun. (D) Total p38 and phospho-p38 levels and (E) total ERK and
phospho-ERK levels. Data represent mean S.E.M. (n=3). #Pb0.001 compared with control group; *Pb0.05 and **Pb0.005 compared with MPP+-treated group; and $no significant
difference compared with MPP+ treated group and control group by one-way analysis of variance (ANOVA) and post hoc Bonfferoni multiple comparison.

I.S. Kim et al. / European Journal of Pharmacology 650 (2011) 48–57
55

56
I.S. Kim et al. / European Journal of Pharmacology 650 (2011) 48–57
signaling pathway regulates cell proliferation, differentiation, and
survival, while the SAPK/JNK and p-38 MAPK pathways are activated
in response to cytokines, growth factors, and a variety of environ-
mental stresses (Ryan et al., 2007). Reactive oxygen species can
activate both SAPK/JNK and apoptosis in a variety of cell systems
(Chen et al., 2003; Verheij et al., 1996). Recent studies have also linked
activation of the JNK signaling pathway converging on the phosphor-
ylation of c-Jun with the induction of apoptosis in 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine-treated mice and blocking of the JNK
signaling pathway may be effective in the control of the disorder
(Saporito et al., 2000; Xia et al., 2001).
Thus, we examined whether the protective effects of PHID could be
involved via the SAPK/JNK, p38 and ERK signaling pathways. Although
MPP+ treatment to SH-SY5Y cells induced the activation of these
signaling cascades, treatment with PHID suppressed the phosphoryla-
tion of SAPK/JNK, c-Jun and p38, but did not affect the phosphorylation
of ERK 1/2. These observations support with the earlier published
reports that treatment at 1 mM concentration of MPP+ did not alter the
expression of p-ERK but increased activation was observed only at high
doses (Zhu et al., 2007; Halvorsen et al., 2002). Another study also
reported that CEP-1347 (KT7515), a neuroprotective agent inhibits JNK
but not ERK 1/2 activation in motor neurons (Maroney et al., 1998).
The discovery of new drug molecules that act by interrupting the
signaling pathways that mediate cell death or activate cell survival
pathways are an effective way of protecting dopaminergic cells from
apoptotic insult in Parkinson's disease. Our present observations that
pretreatment with PHID inhibited phosphorylation of ASK1, SAPK/
JNK, c-Jun and p38 indicates that PHID may act at an early stage of
apoptosis. PHID pretreatment also enhanced the expression of Bcl-2
and inhibited caspase-3 activation in a dose-dependent manner.
In conclusion, our results show that PHID protects SH-SY5Y cells
against MPP+-induced cytotoxicity. The data obtained was highly
convincing with our supporting experiments conducted in MN9D,
Neuro-2a and differentiated SH-SY5Y cells. The compound's antiox-
idative and anti-apoptotic properties render this molecule potentially
protective against MPP+-induced cytotoxicity. Furthermore, PHID
exerts its potent anti-apoptotic effects by blocking intracellular
reactive oxygen species production up-regulated by the neurotoxin
MPP+, and inhibited phosphorylation of JNK signaling in SH-SY5Y
cells. Further studies of the neuroprotective mechanism of PHID in
MPTP-intoxicated animal models, its dosage regimen, and better
understanding of the delivery to the brain through the blood brain
barrier will be required. The present data should spur research on
PHID as a potential therapeutic strategy for the prevention of
progressive neurodegenerative diseases such as Parkinson's disease.
Supplementary data to this article can be found online at
doi:10.1016/j.ejphar.2010.09.063.
Fig. 7. Effects of PHID on the expressions of Bcl-2 and Bax in SH-SY5Y. Cells were treated
with 1 mM MPP+ in the absence or presence of PHID, and total RNA was collected for
semi-quantitative RT-PCR. The levels of Bax and Bcl-2 were quantified by densitometric
analysis (A) and the Bax/Bcl-2 ratio was determined (B). Data represent mean S.E.M.
(n=3). #Pb0.001 compared with control group; and *Pb0.05, **Pb0.005 and Pb0.001
compared with MPP+-treated group by one-way analysis of variance (ANOVA) and
post hoc Bonferroni multiple comparison.
manner. Treatment with up to 10 μM PHID did not produce any
cytotoxic effects on overall cell viability as evaluated by MTT and
LDH assays.
Caspase-3, an important biomarker which plays an important role in
apoptotic processes is well documented. Caspase-3 activation can act
directly on its substrate, PARP, causing hydrolysis (Hartmann et al.,
2000; Nicholson et al., 1995). PARP is an abundant nuclear enzyme that
normally functions in DNA repair, but extensive PARP activation can
promote cell death (Li et al., 2008). In this study, the observation that
treatment with MPP+ lead to an increase in caspase-3 activity is in
agreement with previous findings (Blum et al., 2001). However,
co-treatment with PHID effectively suppressed MPP+-induced activa-
tion of capase-3, and prevented activation of PARP proteolysis in both
the cell types.
Acknowledgements
Cell survival in the apoptotic cascade depends on the balance
between the pro- and anti-apoptotic members of the Bcl-2 family. Bax
and Bcl-2, the two main members of this family, influence the
permeability of the mitochondrial membrane, and play a crucial role
in the mitochondrial apoptotic pathway (Cory and Adams, 2002).
Thus, the Bax/Bcl-2 ratio may better predict the decision between cell
survival and death, and any shift in the balance of pro- and
anti-apoptotic members may affect cell death (Cory and Adams,
2002). Bcl-2 family members are intimately involved in cell death
processes caused by MPP+ (Blum et al., 2001). Presently, the ratio of
the pro-apoptotic Bax to the anti-apoptotic Bcl-2 increased signifi-
This work was supported by the Regional Innovation Center
Program of the Ministry of Knowledge Economy through the Bio-Food
& Drug Research Center at Konkuk University, Korea.
References
Adams Jr., J.D., Klaidman, L.K., Leung, A.C., 1993. MPP+ and MPP+ induced oxygen
radical formation with mitochondrial enzymes. Free Radic. Biol. Med. 15, 181–186.
Bass, D.A., Parce, J.W., Dechatelet, L.R., Szejda, P., Seeds, M.C., Thomas, M., 1983. Flow
cytometric studies of oxidative product formation by neutrophils:
a graded
response to membrane stimulation. J. Immunol. 130, 1910–1917.
cantly in a dose-dependent manner, thereby ameliorating the MPP+
-
Blum, D., Torch, S., Lambeng, N., Nissou, M., Benabid, A.L., Sadoul, R., Verna, J.M., 2001.
Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP:
contribution to the apoptotic theory in Parkinson's disease. Prog. Neurobiol. 65,
135–172.
Bottenstein, J.E., Sato, G.H., 1979. Growth of a rat neuroblastoma cell line in serum-free
supplemented medium. Proc. Natl. Acad. Sci. U.S.A. 76, 514–517.
induced Bax/Bcl-2 ratio elevation in SH-SY5Y cells. Therefore, the
effect of PHID on MPP+-induced apoptosis may be, at least in part,
mediated by regulation of Bax and Bcl-2 expressions.
Among the many signaling pathways that respond to stress, MAPK
family members are crucial for the maintenance of cells. The ERK
Cassarino, D.S., Parks, J.K., Parker Jr., W.D., Bennett Jr., J.P., 1999. The parkinsonian
neurotoxin MPP+ opens the mitochondrial permeability transition pore and

I.S. Kim et al. / European Journal of Pharmacology 650 (2011) 48–57
57
releases cytochrome
Biochim. Biophys. Acta 1453, 49–62.
c
in isolated mitochondria via an oxidative mechanism.
Liu, A.-X., Cerniglia, G.J., Bernhard, E.J., Prendergast, G.C., 2001. Proc. Natl. Acad. Sci. U.S.
A. 98, 6192–6197.
Chen, F., Castranova, V., Li, Z., Karin, M., Shi, X., 2003. Inhibitor of nuclear factor kappaB
kinase deficiency enhances oxidative stress and prolongs c-Jun NH2-terminal
kinase activation induced by arsenic. Cancer Res. 63, 7689–7693.
Luo, Y., Umegaki, H., Wang, X., Abe, R., Roth, G.S., 1998. Dopamine induces apoptosis
through an oxidation-involved SAPK/JNK activation pathway. J. Biol. Chem. 273,
3756–3764.
Cheng, Y.F., Zhu, G.Q., Wang, M., Cheng,H., Zhou, A., Wang, N., Fang, N., Wang,X.C., Xiao, X.Q.,
Chen, Z.W., Li, Q.L., 2009. Involvement of ubiquitin proteasome system in protective
mechanisms of Puerarin to MPP +-elicited apoptosis. Neurosci. Res. 63, 52–58.
Choi, D.K., Suk, K., 2007. Glial reaction in Parkinson's diseases: inflammatory activation
signaling of glia as a potential therapeutictarget. Curr. Signal Transduct. Ther. 2, 91–104.
Choi, W.S., Yoon, S.Y., Oh, T.H., Choi, E.J., O'Malley, K.L., Oh, Y.J., 1999. Two distinct
mechanisms are involved in 6-hydroxydopamine- and MPP+-induced dopaminergic
neuronal cell death: role of caspases, ROS and JNK. J. Neurosci. Res. 57, 86–94.
Chun, H.S., Gibson, G.E., DeGiorgio, L.A., Zhang, H., Kidd, V.J., Son, J.H., 2001.
Dopaminergic cell death induced by MPP+, oxidant and specific neurotoxicants
shares the common molecular mechanism. J. Neurochem. 76, 1010–1021.
Cory, S., Adams, J.M., 2002. The Bcl-2 family: regulators of cellular life-or-death switch.
Nat. Rev. Cancer 2, 647–656.
Datki, Z., Juhász, A., Gálfi, M., Soós, K., Papp, R., Zádori, D., Penke, B., 2003. Method for
measuring neurotoxicity of aggregating polypeptides with the MTT assay on
differentiated neuroblastoma cells. Brain Res. Bull. 62, 223–229.
Diana, P., Martorana, A., Barraja, P., Montalbano, A., Dattolo, G., Cirrincione, G.,
Dall'acqua, F., Salvador, A., Vedaldi, D., Basso, G., Viola, G., 2008. Isoindolo[2, 1-a]
quinoxaline derivatives, novel potent antitumor agents with dual inhibition of
tubulin polymerization and topoisomerase I. J. Med. Chem. 51, 2387–2399.
Domingues, A.F., Arduíno, D.M., Esteves, A.R., Swerdlow, R.H., Oliveira, C.R., Cardoso, S.M.,
2008. Mitochondria and ubiquitin-proteasomal system interplay: relevance to
Parkinson's disease. Free Radic. Biol. Med. 45, 820–825.
Maroney, A.C., Glicksman, M.A., Basma, A.N., Walton, K.M., Knight, E.J., Murphy, C.A.,
Bartlett, B.A., Finn, J.P., Angeles, T., Matsuda, Y., Neff, N.T., Dionne, C.A., 1998.
Motoneuron apoptosis is blocked by CEP-1347 (KT 7515), a novel inhibitor of the
JNK signaling pathway. J. Neurosci. 18, 104–111.
Mattson, M.P., 1990. Antigenic changes similar to those seen in neurofibrillary tangles
arc elicited by glutamate and Ca2+ influx in cultured hippocampal neurons.
Neuron. 2, 105–117.
Mattson, M.P., 2000. Apoptosis in neurodegenerative disorders. Nat. Rev. Mol. Cell Biol.
1, 120–129.
Mielke, K., Herdegen, T., 2000. JNK and p38 stress kinases — degenerative effectors of
signal transduction cascades in the nervous system. Prog. Neurobiol. 61, 45–60.
Nicholson, D.W., Ali, A., Thornbery, N.A., Vaillancourt, J.P., Ding, C.K., Gallant, M., Gareau,
Y., Griffin, P.R., Labelle, M., Lazebnic, Y.A., Munday, N.A., Raju, S.M., Smulson, M.E.,
Yamin, T.T., Yu, V.L., Miller, D.K., 1995. Identification and inhibition of the ICE/CED-3
protease necessary for mammalian apoptosis. Nature 376, 37–43.
Oo, T.F., Henchcliffe, C., James, D., Burke, R.E., 1999. Expression of c-fos, c-jun, and c-jun
N-terminal kinase (JNK) in a developmental model of induced apoptotic death in
neurons of the substantia nigra. J. Neurochem. 72, 557–564.
Ormerod, M.G., 2002. Investigating the relationship between the cell cycle and
apoptosis using flow cytometry. J. Immunol. Meth. 265, 73–80.
Pallas, M., Verdaguer, E., Jorda, E.G., Jimenez, A., Canudas, A.M., Camins, A., 2005.
Flavopiridol: an antitumor drug with potential application in the treatment of
neurodegenerative diseases. Med. Hypotheses 64, 120–123.
Eberhardt, O., Schulz, J.B., 2003. Apoptotic mechanisms and antiapoptotic therapy in the
MPTP model of Parkinson's disease. Toxicol. Lett. 139, 135–151.
Peng, J., Mao, X.O., Stevenson, F.F., Hsu, M., Andersen, J.K., 2004. The herbicide paraquat
induces dopaminergic nigral apoptosis through sustained activation of the JNK
pathway. J. Biol. Chem. 279, 32626–32632.
Pieczenik, S.R., Neustadt, J., 2007. Mitochondrial dysfunction and molecular pathways
of disease. Exp. Mol. Pathol. 83, 84–92.
Ferrer, I., Blanco, R., Carmona, M., Puig, B., Barrachina, M., Gomez, C., Ambrosio, S., 2001.
Active, phosphorylation-dependent mitogen-activated protein kinase (MAPK/ERK),
stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38 kinase
expression in Parkinson's disease and dementia with Lewy bodies. J. Neural Transm.
108, 1383–1396.
Przedborski, S., Jackson-Lewis, V., 1998. Mechanisms of MPTP toxicity. Mov. Disord. 13,
35–38.
Fiskum, G., Starkov, A., Polster, B.M., Chinopoulos, C., 2003. Mitochondrial mechanisms
of neural cell death and neuroprotective interventions in Parkinson's disease. Ann.
NY Acad. Sci. 991, 111–119.
Gao, M., Zhang, W.C., Liu, Q.S., Hu, J.J., Liu, G.T., Du, G.H., 2008. Pinocembrin prevents
glutamate-induced apoptosis in SH-SY5Y neuronal cells via decrease of bax/bcl-2
ratio. Eur. J. Pharmacol. 591, 73–79.
Przedborski, S., Tieu, K., Perier, C., Vila, M., 2004. MPTP as a mitochondrial neurotoxic
model of Parkinson's disease. J. Bioenerg. Biomembr. 36, 375–379.
Ryan, S., McNicholas, W.T., Taylor, C.T., 2007. A critical role for p38 map kinase in
NF-kappaB signaling during intermittent hypoxia/reoxygenation. Biochem. Biophys.
Res. Commun. 355, 728–733.
Saporito, M.S., Brown, E.M., Miller, M.S., Carswell, S., 1999. CEP-1347/KT-7515, an
inhibitor of c-jun N-terminal kinase activation, attenuates the 1-methyl-4-phenyl
tetrahydropyridine-mediated loss of nigrostriatal dopaminergic neurons in vivo.
J. Pharmacol. Exp. Ther. 288, 421–427.
Gearan, T., Castillo, O.A., Schwarzschild, M.A., 2001. The parkinsonian neurotoxin,
MPP+ induces phosphorylated c-Jun in dopaminergic neurons of mesencephalic
cultures. Parkinsonism Relat. Disord. 8, 19–22.
Gómez-Santos, C., Ferrer, I., Reiriz, J., Viñals, F., Barrachina, M., Ambrosio, S., 2002. MPP+
increases alpha-synuclein expression and ERK/MAP-kinase phosphorylation in
human neuroblastoma SH-SY5Y cells. Brain Res. 935, 32–39.
Saporito, M.S., Thomas, B.A., Scott, R.W., 2000. MPTP activates c-Jun NH(2)-terminal
kinase (JNK) and its upstream regulatory kinase MKK4 in nigrostriatal neurons in
vivo. J. Neurochem. 75, 1200–1208.
Grutter, M.G., 2000. Caspases: key players in programmed cell death. Curr. Opin. Struct.
Biol. 10, 649–655.
Sheehan, J.P., Palmer, P.E., Helm, G.A., Tuttle, J.B., 1997. MPP+ induced apoptotic cell death
in SH-SY5Y neuroblastoma cells: an electron microscope study. J. Neurosci. Res. 48,
226–237.
Halvorsen, E.M., Dennis, J., Keeney, P., Sturgill, T.W., Tuttle, J.B., Bennett Jr., J.B., 2002.
Methylpyridinium (MPP +) - and nerve growth factor-induced changes in pro- and anti-
apoptotic signaling pathways in SH-SY5Y neuroblastoma cells. Brain 952, 98–110.
Hartmann, A., Hunot, S., Michel, P.P., Muriel, M.P., Vyas, S., Faucheux, B.A., Mouatt-Prigent,
A., Turmel, H., Srinivasan, A., Ruberg, M., Evan, G.I., Agid, Y., Hirsch, E.C., 2000. Caspase-
3, a vulnerability factor and final effector in apoptotic death of dopaminergic neurons
in Parkinson's disease. Proc. Natl. Acad. Sci. U.S.A. 97, 2875–2880.
Hunot, S., Vila, M., Teismann, P., Davis, R.J., Hirsch, E.C., Przedborski, S., Rakic, P., Flavell, R.A.,
2004. JNK-mediated induction of cyclooxygenase 2 is required for neurodegeneration
in a mouse model of Parkinson's disease. Proc. Natl. Acad. Sci. U.S.A. 101, 665–670.
Jung, J.K., Jung, E.K., Nam-Goong, K., Cho, J.S., Kim, H.M., Park, S.G., Yoo, Y.A., Kwon, J.H.,
Lee, H.S., 2006. Synthesis and cytotoxic activities of 8-alkyl or 8-aryl-8,9-dihydro-
7H-isoindolo[5,6-g]quinoxaline-7,9-diones. Arch. Pharm. Res. 29, 276–281.
Kanamoto, T., Mota, M., Takeda, K., Rubin, L.L., Miyazono, K., Ichijo, H., Bazenet, C.E., 2000.
Role of apoptosis signal-regulating kinase in regulation of the c-Jun N-terminal kinase
pathway and apoptosis in sympathetic neurons. Mol. Cell. Biol. 20, 196–204.
Kehrer, J.P., Smith, C.V., 1994. Free radicals in biology: sources, reactivities, and roles in
the etiology of human diseases. In: Frei, B. (Ed.), Natural Antioxidants in Human
Health and Disease. Academic Press, New York, pp. 25–62.
Kitamura, Y., Kosaka, T., Kakimura, J.I., Matsuoka, Y., Kohno, Y., Nomura, Y., Taniguchi, T.,
1998. Protective effects of the antiparkinsonian drugs talipexole and pramipexole
against 1-methyl-4-phenylpyridinium-induced apoptotic death in human neuroblas-
toma SH-SY5Y cells. Mol. Pharmacol. 54, 1046–1054.
Kyriakis, J.M., Avruch, J., 2001. Mammalian mitogen-activated protein kinase signal
transduction pathways activated by stress and inflammation. Physiol. Rev. 81, 807–869.
Lazebnik, Y.A., Kaufmann, S.H., Desnoyers, S., Poirier, G.G., Earnshaw, W.C., 1994.
Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE.
Nature 371, 346–347.
Singer, T.P., Ramsay, R.R., 1990. Mechanism of the neurotoxicity of MPTP, an update.
FEBS Lett. 274, 1–8.
Szeto, H.H., 2006. Mitochondria-targeted peptide antioxidants: novel neuroprotective
agents. AAPS J. 8, 521–531.
Telford, W.G., King, L.E., Fraker, P.J., 1991. Evaluation of glucocorticoid-induced DNA
fragmentation in mouse thymocytes by flow cytometry. Cell Prolif. 24, 447–459.
Tseng, T.H., Wang, C.J., Kao, E.S., Chu, H.Y., 1996. Hibiscus protocatechuic acid protects
against oxidative damage induced by tert-butylhydroperoxide in rat primary
hepatocytes. Chem. Biol. Interact. 101, 137–148.
Verheij, M., Bose, R., Lin, X.H., Yao, B., Jarvis, W.D., Grant, S., Birrer, M.J., Szabo, E., Zon, L.I.,
Kyriakis, J.M., Haimovitz-Friedman, A., Fuks, Z., Kolesnick, R.N., 1996. Requirement for
ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 380, 75–79.
Waza, M., Adachi, H., Katsuno, M., Minamiyama, M., Tanaka, F., Sobue, G., 2006. Alleviating
neurodegeneration by an anticancer agent: an Hsp90 inhibitor (17-AAG). Ann. NY
Acad. Sci. 1086, 21–34.
Wong, S.S., Li, R.H., Stadlin, A., 1999. Oxidative stress induced by MPTP and MPP(+):
selective vulnerability of cultured mouse astrocytes. Brain Res. 836, 237–244.
Xia, X.G., Harding, T., Weller, M., Bieneman, A., Uney, J.B., Schulz, J.B., 2001. Gene
transfer of the JNK interacting protein-1 protects dopaminergic neurons in the
MPTP model of Parkinson's disease. Proc. Natl. Acad. Sci. U.S.A. 98, 10433–10438.
Yuan, J., Yankner, B.A., 2000. Apoptosis in the nervous system. Nature 407, 802–809.
Zhang, S., Zhao, B., 2003. Green tea polyphenols enhance sodium nitroprusside-induced
neurotoxicity in human neuroblastoma SH-SY5Y cells. J. Neurochem. 86,
1189–1200.
Zhang, S., Zhou, F., Ding, J.H., Zhou, X.Q., Sun, X.L., Hu, G., 2007. ATP-sensitive potassium
channel opener iptakalim protects against MPP-induced astrocytic apoptosis via
mitochondria and mitogen-activated protein kinase signal pathways. J. Neurochem.
103, 569–579.
Zhou, C., Huang, Y., Przedborski, S., 2008. Oxidative stress in Parkinson's disease: a
mechanism of pathogenic and therapeutic significance. Ann. NY Acad. Sci. 1147,
93–104.
Zhu, J., Horbinski, C., Guo, F., Watkins, S., Uchiyama, Y., Chu, C.T., 2007. Regulation of
autophagy by extracellular signal- regulated protein kinases during 1-methyl-4-
phenylpyridinium-induced cell death. Am. J. Pathol. 170, 75–86.
Le, D.A., Wu, Y., Huang, Z., Matsushita, K., Plesnila, N., Augustinack, J.C., Hyman, B.T.,
Yuan, J., Kuida, K., Flavell, R.A., Moskowitz, M.A., 2002. Caspase activation and
neuroprotection in caspase-3- deficient mice after in vivo cerebral ischemia and in
vitro oxygen glucose deprivation. Proc. Natl. Acad. Sci. U.S.A. 99, 15188–15193.
Li, Y.Y., Lu, J.H., Li, Q., Zhao, Y.Y., Pu, X.P., 2008. Pedicularioside A from Buddleia lindleyana
inhibits cell death induced by 1-methyl-4-phenylpyridinium ions (MPP+) in primary
cultures of rat mesencephalic neurons. Eur. J. Pharmacol. 579, 134–140.
Liu, A.-X., Du, W., Liu, J.-P., Jessell, T.M., Prendergast, G.C., 2000. Mol. Cell. Biol. 20,
6105–6113.