Toxicology 262 (2009) 199–206

Contents lists available at ScienceDirect

Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Arachidonic acid-induced apoptosis of human neuroblastoma SK-N-SH cells is

mediated through mitochondrial alteration elicited by ROS and Ca2+ -evoked
activation of p38␣ MAPK and JNK1
Ku-Chung Chen, Long-Sen Chang ∗
Institute of Biomedical Sciences, National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center,
National Sun Yat-Sen University, Kaohsiung 804, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: Arachidonic acid (AA)-induced apoptosis of human neuroblastoma SK-N-SH cells was characteristic of
Received 12 May 2009 elevation of intracellular Ca2+ concentration ([Ca2+ ]i), ROS generation, activation of 38 MAPK and JNK
Received in revised form 10 June 2009 and loss of mitochondrial membrane potential (m). Subsequent modulation of Bcl-2 family members
Accepted 11 June 2009
and cytochrome c release accompanied with activation of caspase-9 and -3 were involved in the death
Available online 21 June 2009
of SK-N-SH cells. BAPTA-AM (Ca2+ chelator) pretreatment rescued viability of AA-treated cells through
abolishing phosphorylation of p38 MAPK and JNK, m loss and ROS generation. N-Acetylcysteine
Keywords:
(ROS scavenger) pretreatment reduced the dissipation of m, but insignificantly affected AA-induced
Arachidonic acid
Apoptosis
p38 MAPK and JNK activation. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) attenu-
p38␣ MAPK ated mitochondrial depolarization, degradation of Bcl-2/Bcl-xL, and mitochondrial translocation of Bax.
JNK1 Transfection of specific siRNA proved that p38␣ MAPK and JNK1 were involved in modulating Bcl-2
Mitochondrial alteration family proteins. Taken together, our data suggest that the cytotoxicity of AA toward SK-N-SH cells is
Reactive oxygen species mediated through mitochondria-dependent death pathway, eliciting by AA-induced ROS generation and
Ca2+ -evoked activation of p38␣ MAPK and JNK1.
© 2009 Published by Elsevier Ireland Ltd.

1. Introduction ber of signal transduction pathways (Rizzo and Carlo-Stella, 1996;

Arachidonic acid (AA), a cis-polyunsaturated fatty acid (20:4,
cis-5, 8, 11, 14) enriched in the central nervous system (CNS), is
liberated from plasma membrane through activation of phospho-
lipase A2 after physiological, pharmacological, and pathological
stimulations (Gijon and Leslie, 1997). Although it is difficult to
determine the actual concentration of AA in physiological tissues,
concentrations of 30–300 ␮M have been suggested to be attainable
under pathological stimulations (Lipton, 1999). Free AA is metab-
olized by cyclooxygenase (COX) to prostaglandins, lipoxygenase
(LOX) to leukotrienes and cytochrome P450 (CYP450) to epoxye-
icosatrienoic products, respectively (Pompeia et al., 2003a,b). AA
and its eicosanoid metabolites are markedly produced from the
neurons and glial cells in a variety of neurodegenerative condi-
tions such as ischemia, epilepsy, and Alzheimer’s disease (Bazan et
al., 2002; Candelario-Jalil et al., 2003; Phillis and O’Regan, 2003).
Although many effects of AA on cell function are mediated by
its conversion into eicosanoids (Rizzo, 2002), several studies have
shown that AA per se is involved in the regulation of a num-
∗ Corresponding author. Fax: +886 7 5250197.
E-mail address: lschang@mail.nsysu.edu.tw (L.-S. Chang).
Rizzo et al., 1999; Rao et al., 1994). Neurotoxic effects of AA, such
as induction of oxidative stress, activation of protein kinase C,
increased intracellular calcium levels, and decreased cell survival,
are observed in hippocampal neurons, spinal cord neurons, and cor-
tical neurons (Okuda et al., 1994; Li et al., 1997; Toborek et al., 1999).
Additionally, AA is also reported to possess stimulatory or inhibitory
effects on a variety of ion channels, including the voltage-gated Na+ ,
Ca2+ , K+ channels, NMDA receptor channel, and Ca2+ -dependent K+
channels (Meves, 1994; Denson et al., 2000; Liu and Rittenhouse,
2003; Kwon et al., 2005).
Neuroblastoma, a childhood malignancy of the sympathetic
nervous system, shows very complex biological and clinical het-
erogeneity (Brodeur, 2003). Given that neuroblastoma has poor
prognosis and often develops resistance to conventional therapy,
alternative treatment options are important for the treatment of
this disease. In terms of a role of AA in signaling cell death of
neurons, it is interesting to explore whether AA-induced death of
neuroblastoma cells. Thus, studies on the cytotoxic effect of AA
toward human neuroblastoma SK-N-SH cells were conducted in the
present study. Elucidation of molecular mechanisms and signal-
ing pathways responsible for cell death of neuroblastoma induced
by exogenous AA may suggest additional targets and/or rational
strategies for neuroblastoma therapy.

0300-483X/$ – see front matter © 2009 Published by Elsevier Ireland Ltd.

doi:10.1016/j.tox.2009.06.009
200 K.-C. Chen, L.-S. Chang / Toxicology 262 (2009) 199–206
2. Materials and methods
Arachidonic acid (AA), 2-aminoethoxydiphenyl borate (2-APB), N-acetylcysteine
(NAC), antimycin A, cyclosporin A, digitonin, MTT, diphenyleneiodonium chlo-
ride (DPI), MG-132, propidium iodide (PI), rotenone, ruthenium red, SB202190,
SP600125 and U0126 were purchased from Sigma-Aldrich Inc. Anti-caspase-3
and anti-caspase-8 antibodies were obtained from Calbiochem, and anti-␤-actin
antibodies were the products of Chemicon International Inc. PARP, anti-caspase-
9, anti-p38 MAPK, anti-p38␣ MAPK, anti-p38␤ MAPK, anti-phospho-p38 MAPK,
anti-JNK, anti-phospho-JNK, anti-ERK, anti-phospho-ERK, anti-Bcl-2, anti-Bak and
anti-Bax antibodies were the products of Cell Signaling. Anti-Bcl-xL, anti-Bid and
anti-cytochrome c antibodies were obtained from BD Pharmingen. BAPTA-AM, Fluo-
4 AM, dichlorodihydrofluorescein diacetate (H2 DCFDA), annexin V-FITC kit and
rhodamine-123 were the products of Molecular Probes. Horseradish peroxidase-
conjugated secondary antibodies were obtained from Pierce. Cell culture supplies
were purchased from GIBCO/Life Technologies Inc. Unless otherwise specified; all
other reagents were of analytical grade.
2.1. Cell viability assay
Human neuroblastoma SK-N-SH cells obtained from ATCC (Rockville, MD, USA)
were routinely cultured with Dulbecco’s modified Eagle’s medium (DMEM) contain-
ing 10% fetal calf serum, 2 mM glutamine and penicillin (100 units/ml)/streptomycin
(100 ␮g/ml) in an incubator humidified with 95% air and 5% CO2 . Cells were
harvested from subconfluent monolayers grown in bottles of 75 cm2 after their
detachment by trypsin (1500 U/ml) containing 5.3 mM EDTA, for 5 min at 37 ◦ C.
Exponentially growing cells (2 × 104 cells/well) were plated in 96-well plates and
treated with AA in serum-free medium after 24 h of growth. For pharmacological
experiments, culture cells were pre-treated with 10 ␮M U0126, 10 ␮M SB202190,
10 ␮M SP600125, 2 mM NAC, 10 ␮M DPI, 50 ␮M 2-APB, 10 ␮M ruthenium red,
10 ␮M BAPTA-AM, 10 ␮M EGTA, 1 ␮M cyclosporin A, 10 ␮M antimycin A or 1 ␮M
rotenone for 1 h before AA was added. At suitable time intervals, MTT solution
was added to each well at a final concentration of 0.5 mg/ml and incubated for
4 h. Formazan crystals resulting from MTT reduction were dissolved by addition
of 100 ␮l DMSO per well. The absorbance was detected at 595 nm using a plate
reader.
2.2. Detection of apoptotic cells
Annexin V/propidium iodide (PI) staining was carried out according to the manu-
facturer’s protocol (Annexin V-FITC kit from Molecular Probes). After AA treatment
at indicated time periods, SK-N-SH cells were washed with cold PBS and resus-
pended with binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 )
before transferring 1 × 105 cells to a 5 ml tube. Then 5 ␮l of annexin V-FITC and 5 ␮l
of PI were added, and the cells were incubated for 15 min in the dark. Binding buffer
(400 microliters ) was then added to each tube, and the cells were analyzed by a
Beckman Coulter Epics XL flow cytometer.
2.3. Sub-G1 analysis
Sub-G1 distribution was determined by staining DNA with PI. Briefly, 1 × 106 cells
were incubated with AA for 24 h. Cells were then washed in phosphate buffered
saline (PBS) and fixed in 70% ethanol. Cells were again washed with PBS and
then incubated with PI (10 ␮g) with simultaneous treatment of RNase at 37 ◦ C for
30 min. The percentages of cells having the sub-G1 DNA content were measured
with a Beckman Coulter Epics XL flow cytometer and analyzed using EXPO32 ADC
software.
2.4. Detection of ROS generation and mitochondrial membrane potential
AA-treated cells were incubated with 10 ␮M H2 DCFDA for 20 min at room
temperature. ROS was measured using Beckman Coulter Epics XL flow cytome-
ter or Beckman Coulter ParadigmTM Detection Platform with excitation at 485 nm
and emission at 530 nm. Alternatively, AA-treated cells were incubated with
20 nM rhodamine-123 for 20 min prior to harvesting, and then washed with PBS.
Rhodamine-123 intensity was determined by flow cytometry. Cells with reduced
fluorescence (less rhodamine-123) were counted as having lost their mitochondrial
membrane potential.
2.5. Intracellular Ca2+ concentration ([Ca2+ ]i) measurement
The level of intracellular Ca2+ was quantified by fluorescence with Fluo-4 AM. The
cells were treated with AA for time indicated, and the treated cells were washed with
ice-cold PBS. The cells resuspended in 1 ml of PBS were incubated with 5 ␮l of 1 mM
Fluo-4 AM for 1 h. The fluorescence intensity of intracellular Ca2+ concentration was
measured by flow cytometer or Beckman Coulter ParadigmTM Detection Platform
with excitation at 485 nm and emission at 530 nm. Results were shown as increase
in fluorescence intensity per microgram of proteins compared with the control
group.
2.6. DNA transfection
pRSV-MKK3(Ala) encoding dominant-negative MKK3 was obtained from Dr.
R.J. Davis (University of Massachusetts Medical School, USA), and pcDNA3-p38␣
and pcDNA3-p38␤ were kindly provided by Dr. J. Han (Xiamen University, China).
The plasmids were transfected into SK-N-SH cells using Pipette-type Electroporator
(MicroPorator-MP100, Digital Bio Tech. Co., Korea).
2.7. RNA interference
pKD-JNK1 shRNA plasmid was purchased from Upstate, and pSuper-p38␣ shRNA
plasmid was obtained from Dr. A. Porras (Ciudad University, Spain). For the trans-
fection procedure, cells were grown to 60% confluence, and pKD-JNK1 shRNA
or pSuper-p38␣ shRNA were transfected using LipofetamineTM 2000 (Invitrogen)
according to the manufacturer’s instructions. At 24 h post-transfection, the cells
were exposed to AA for an additional 24 h. Afterwards, cells were harvested for
Western blot analyses.
2.8. Separation of cytosolic and mitochondrial fractions
Following induction of apoptosis, cytosolic and pellet (mitochondrial) frac-
tions were generated using a digitonin-based subcellular fractionation technique.
Briefly, 1 × 107 cells were harvested by centrifugation at 800 × g, washed in PBS
and re-pelleted. Cells were digitonin-permeablilized for 5 min on ice at a density
of 3 × 107 /ml in cytosolic extraction buffer (75 mM NaCl, 1 mM NaH2 PO4 , 8 mM
Na2 HPO4 , 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 ␮g/ml
leupeptin, 5 ␮g/ml aprotinin and 0.05% digitonin). Following the centrifugation step
at 800 × g at 4 ◦ C for 10 min, the supernatant was separated from the pellet com-
prising mitochondria and cellular debris. The supernatant containing cytoplasmic
protein was further purified by centrifugation at 13,000 × g at 4 ◦ C for 10 min. The
pellets were solubilized in the same volume of mitochondrial lysis buffer (50 mM
Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton X-100, 0.3% NP-40,
1 mM PMSF, 5 ␮g/ml leupeptin, 5 ␮g/ml aprotinin). After centrifugation at 12,000 × g
at 4 ◦ C for 10 min, the supernatants were collected and used as the mitochondrial
fraction. Cytochrome c and proteins of Bcl-2 family were detected by Western blot
analysis.
2.9. Western blot analysis
After specific treatments, cells were incubated in lysis buffer containing 20 mM
Tris–HCl (pH 7.5), 1% Triton X-100, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 1 mM
Na3 VO4 , 50 mM NaF, 1 mM PMSF and protease inhibitor mixtures for 20 min on ice.
After insoluble debris was precipitated by centrifugation at 13,000 × g for 15 min at
4 ◦ C, the supernatants were collected and assayed for protein concentration using
the Bradford method. An equal amount of protein per sample (15 ␮g) was resolved on
SDS-PAGE and transferred onto a PVDF membrane. The transferred membranes were
blocked for 1 h in 5% nonfat milk in PBST (PBS containing 0.05% Tween 20) and incu-
bated with appropriate primary antibodies and horseradish peroxidase-conjugated
secondary antibodies. The immune complexes were detected by SuperSignal West
Pico Chemiluminescent substrate kit (Pierce).
2.10. Statistical analysis
All data are presented as mean ± S.D. Significant differences among the groups
were determined using the unpaired Student’s t-test. A value of P < 0.05 was taken
as an indication of statistical significance. All the figures shown in this article were
obtained from at least three independent experiments with similar results.
3. Results and discussion
Upon exposure to AA, SK-N-SH cells showed a concentration-
and time-dependent decrease in cell viability (Fig. 1A). Because the
dose required for half-maximum inhibition of viability was around
50 ␮M after AA treatment for 24 h, this single treatment was used
for further assessment of cytotoxicity. As shown in Fig. 1B, flow
cytometric analyses showed that AA induced an increased annexin
V staining, while no evidence of PI staining (upper left quad-
rants) was detected in AA-treated cells Moreover, compared with
untreated control cells (0.2%), an increased accumulation of cells
in the sub-G1 phase (23.8%) was noted (Fig. 1C). Immunoblotting
analyses revealed a marked decrease in the level of procaspase-
3 and PARP after AA treatment (Fig. 1D). These supported that AA
induced apoptotic death of SK-N-SH cells. Noticeably, no significant
degradation of procaspase-8 in AA-treated cells was detected.
Previous studies reported that AA induces increase in [Ca2+ ]i,
which arose from either intracellular stores or Ca2+ influx (Fleming
 K.-C. Chen, L.-S. Chang / Toxicology 262 (2009) 199–206 201

Fig. 1. AA-induced apoptotic death of SK-N-SH cells. (A) Time-dependent effect of AA on cell viability. SK-N-SH cells were incubated with 50 ␮M AA for 24 h. (Inset): Dose-
dependent effect of AA on cell viability. Cell viability was determined by MTT after treatment with varying concentrations of AA for 24 h. (B) Flow cytometry analyses of
annexin V–propidium double staining AA-treated cells. SK-N-SH cells were incubated with 50 ␮M AA for 24 h. On the flow cytometric scatter graphs, the left lower quadrant
represents remaining live cells. The right lower quadrant represents the population of early apoptotic cells. The right upper quadrant represents the accumulation of late
apoptotic cells. (C) Cell cycle analysis of SK-N-SH cells treated with AA. Flow cytometry analyses showed an increase in the sub-G1 DNA content of SK-N-SH cells after treatment
with 50 ␮M AA for 24 h. (D) Western blot analyses of degradation of procaspase-3, procaspase-8 and PARP in AA-treated cells. SK-N-SH cells were treated with 50 ␮M AA for
indicated time periods.

and Mellow, 1995; Mignen and Shuttleworth, 2000; Mignen et
al., 2003). Thus, [Ca2+ ]i of AA-treated cells was measured. As
shown in inset of Fig. 2A, flow cytometry analyses showed
AA-induced elevation of [Ca2+ ]i. Cytosolic Ca2+ determined by flu-
orescent ELISA reader revealed that maximal increase in [Ca2+ ]i
was attained after AA treatment for 15 min, and then [Ca2+ ]i grad-
ually declined to basal level. In contrast to the inhibitors of Ca2+
release from intracellular depots (2-APB, ruthenium red), BAPTA-
AM (membrane-permeable Ca2+ chelator) and EGTA (Ca2+ chelator)
notably attenuated AA-induced [Ca2+ ]i increase (Fig. 2B) and res-
cued viability of AA-treated cells to approximately 85% of untreated
control cells (Fig. 2C). Moreover, BAPTA-AM abolished degrada-
tion of procaspase-3 and PARP in AA-treated cells (Fig. 2D). These
suggested that AA-induced Ca2+ influx was a detrimental event
responsible for AA-induced cell death.
Since mitogen activated protein kinases (MAPKs) are common
components of the apoptotic program (Proskuryakov et al., 2003),
phosphorylation of MAPKs was examined in AA-treated SK-N-
SH cells. Fig. 3A shows that the levels of phospho-p38 MAPK
and phospho-JNK were notably increased after 1 h of AA treat-
ment, while that of phospho-ERK was insignificantly affected.
Viability of AA-treated cells was restored by pretreatment with
SB202190 (p38 MAPK inhibitor), SP600125 (JNK inhibitor) or a
combination of SB202190 and SP600125 (Fig. 3B), reflecting that
phospho-p38 MAPK and phospho-JNK were associated with AA-
induced cell death. Alternatively, suppression of ERK by U0126
enhanced cytotoxicity of AA (Fig. 3B). Fig. 3C shows that AA-induced
phosphorylation of p38 MAPK and JNK was abrogated by BAPTA-
AM pretreatment. However, AA-induced [Ca2+ ]i increase was not
affected by SB202190, SP600125 or U0126 (Fig. 3D). Obviously, AA-
elicited [Ca2+ ]i increase was an upstream event responsible for
activation of p38 MAPK and JNK in SK-N-SH cells.
Previous studies have reported that AA induces ROS genera-
tion through activation of NADPH oxidase in neutrophils, HL-60
cells, Jurkat cells, thymocytes and macrophages (Cherny et al.,
2001; Pompeia et al., 2003a,b; Bostan et al., 2003). Thus, ROS level
in AA-treated cells was measured. As shown in inset of Fig. 4A,
flow cytometry analyses showed an increase in the production
of ROS in AA-treated cells. Maximal ROS generation determined
by fluorescent ELISA reader was noted after AA treatment for 4 h.
Compared with DPI (NADPH oxidase inhibitor), NAC (ROS scav-
enger) effectively rescued the cell viability of AA-treated cells and
eliminated generated ROS (Fig. 4B and C). These likely reflected
that AA-induced ROS generation was not exclusively derived from
NADPH oxidase. Alternatively, BAPTA-AM treatment also notably
reduced ROS generation (Fig. 4C), indicating that AA-induced
[Ca2+ ]i increase could induce ROS generation. Previous studies sug-
gested that NADPH oxidase, in response to elevation of cytosolic
Ca2+ , generated large amount of superoxide (Banfi et al., 2001).
Fig. 4D shows that AA-induced p38 MAPK and JNK activation
was not significantly affected by pretreatment with NAC. These
suggested that AA-induced ROS generation was not involved in
activation of p38 MAPK and JNK.
Increasing evidence suggests that altered mitochondrial func-
tion is linked to apoptosis and a decreasing mitochondrial
transmembrane potential (m) is associated with mitochon-
drial dysfunction (Green and Reed, 1998). As shown in Fig. 5A,
flow cytometry analysis showed that, in untreated control SK-N-
SH cells, more than 99% of cells were functionally active with
high rhodamine-123 signals. Increasing population of SK-N-SH cells
exhibited the loss of m after AA treatment for 24 h. BAPTA-
AM showed a stronger effect on suppressing the dissipation of
m than NAC did. Pretreatment with SB202190 or SP202190 sup-
pressed dissipation of m in AA-treated cells. Taken together,
these reflected that Ca2+ -evoked p38 MAPK and JNK activation and
AA-induced ROS generation elicited mitochondrial depolarization
of SK-N-SH cells. To further identify the causal relationship between
mitochondrial alteration and ROS generation, SK-N-SH cells were
pre-treated with cyclosporin A, rotenone or antimycin A. As shown
in Fig. 5B, rotenone and antimycin A, inhibitors of mitochondrial
202 K.-C. Chen, L.-S. Chang / Toxicology 262 (2009) 199–206

Fig. 2. AA-induced [Ca2+ ]i increase in SK-N-SH cells. (A) Elevation of [Ca2+ ]i was noted with notexin-treated SK-N-SH cells. Intracellular Ca2+ concentration was quantified
by fluorescence plate reader after loading the cells with a calcium indicator (Fluo-4 AM). Results were shown as fold-increase in fluorescence intensity compared with the
control group. SK-N-SH cells were treated with 50 ␮M AA for indicated time periods. The data represent the mean ± SD (n = 6). (Inset) Flow cytometric analyses of the level of
[Ca2+ ]i in AA-treated cells. SK-N-SH cells were treated with 50 ␮M AA for 15 min. (B) Effect of BPATA-AM (Ca2+ chelator), EGTA and inhibitors of Ca2+ release from intracellular
depots (2-APB and Ruthenium red) on AA-evoked [Ca2+ ]i increase. The cells were pre-treated with 10 ␮M BAPTA-AM, 10 ␮M EGTA, 50 ␮M 2-APB or 10 ␮M ruthenium red for
1 h before incubation with 50 ␮M AA for 15 min. (*, **P < 0.05) (C) Effect of BPATA-AM (Ca2+ chelator), EGTA and inhibitors of Ca2+ release from intracellular depots (2-APB
and Ruthenium red) on viability of AA-treated cells. SK-N-SH cells were pre-treated with 10 ␮M BAPTA-AM, 10 ␮M EGTA, 50 ␮M 2-APB, 10 ␮M ruthenium red for 1 h before
incubation with 50 ␮M AA for 24 h. Cell viability was determined by MTT (n = 6, *, **P < 0.05). (D) Western blot analyses showed that BAPTA-AM (Ca2+ chelator) suppressed
degradation of procaspase-3 and PARP of AA-treated cells. SK-N-SH cells were pre-treated with 10 ␮M BAPTA-AM for 1 h, and then incubated with 50 ␮M AA for 24 h.

Fig. 3. Effect of AA treatment on the levels of phospho-ERK, phospho-p38 MAPK and phospho-JNK in SK-N-SH cells. (A) Western blot analyses of phosphorylated MAPKs.
SK-N-SH cells were treated with 50 ␮M AA for indicated time periods. (B) Effect of SP600125 (JNK inhibitor), SB202190 (p38 MAPK inhibitor) and U0126 (MEK1 and MEK2
inhibitor) on AA-induced death of SK-N-SH cells. The cells were pre-treated with 10 ␮M SP600125, 10 ␮M SB202190 or 10 ␮M U0126 for 1 h and then incubated with 50 ␮M
AA for 24 h. Cell viability was analyzed by MTT assay, and the data are the mean ± SD of six experiments (*, **, ***P < 0.05). (C) Effect of BAPTA-AM on phosphorylation of
MAPKs. The cells were pre-treated with 10 ␮M BAPTA-AM for 1 h prior to treating with 50 ␮M AA for 24 h. (D) Effect of SP600125, SB202190 and U0126 on AA-evoked [Ca2+ ]i
increase. The cells were pre-treated with 10 ␮M SP600125, 10 ␮M SB202190 or 10 ␮M U0126 for 1 h and then incubated with 50 ␮M AA for 15 min. Results were shown as
fold-increase in fluorescence intensity compared with the control group. The data represent the mean ± SD (n = 6).
 K.-C. Chen, L.-S. Chang / Toxicology 262 (2009) 199–206 203

Fig. 4. AA-induced ROS generation in SK-N-SH cells. (A) Time-dependent increase in ROS generation. SK-N-SH cells were incubated with 50 ␮M AA for indicated time periods.
Results were shown as fold-increase in fluorescence intensity compared with the control group. The data represent the mean ± SD (n = 6). ROS was quantified by fluorescence
plate reader. (Inset) Flow cytometric analyses of ROS level in AA-treated cells. SK-N-SH cells were incubated with 50 ␮M AA for 4 h. (B) Effect of NAC and DPI on viability of
AA-treated cells. SK-N-SH cells were pre-treated with 2 mM NAC or 10 ␮M DPI for 1 h, and then incubated with 50 ␮M AA for 24 h. Cell viability was analyzed by MTT assay,
and the data are the mean ± SD of six experiments (*, **P < 0.05). (C) Effect of BAPTA-AM, NAC and DPI on AA-induced ROS generation. SK-N-SH cells were pre-treated with
10 ␮M BAPTA-AM, 2 mM NAC or 10 ␮M DPI for 1 h, and then incubated with 50 ␮M AA for 4 h. ROS was quantified by fluorescence plate reader, and the data represent the
mean ± SD (n = 6, *, **, ***P < 0.05). (D) Effect of NAC on phosphorylation of MAPKs. The cells were pre-treated with 2 mM NAC, and then treated with 50 ␮M AA for 24 h.

electron transport chain complexes I and III, significantly atten-
uated ROS generation in AA-treated cells. Likewise, cyclosporin
A, an inhibitor of mitochondria permeability transition pore, also
reduced the production of ROS. These results suggested that, in
addition to NADPH oxidase, mitochondrial alteration was also con-
tributed to ROS generation in AA-treated cells.
During mitochondrion-mediated apoptosis, cytochrome c is
released from the mitochondria into the cytosol, where it pro-
motes caspase activation (Wang, 2001). As seen in Fig. 5C, a
time-dependent release of cytochrome c into cytosol was detected
relative to gradual decrease in mitochondrial cytochrome c. More-
over, activation of caspase-9 was noted after AA treatment. Fig. 5C
shows that downregulation of Bcl-2/Bcl-xL and marked translo-
cation of Bax to mitochondria were observed after AA treatment
for 1 h. Alternatively, expression of apoptotic proteins Bax/Bak was
not appreciably altered, and the cleaved product of Bid was not
observed. Pretreatment with SB202190, SP600125 or BAPTA-AM
markedly abolished downregulation of Bcl-2 and Bcl-xL in response
to AA treatment (Fig. 5D), reflecting that Ca2+ -elicited p38 MAPK
and JNK activation was involved in this event. Several lines of
evidence show that phosphorylation of Bcl-2 and Bcl-xL by p38
MAPK enhances their degradation by proteasome-dependent path-
way (Breitschopf et al., 2000; Grethe et al., 2004; De Chiara et al.,
2006). Fig. 5D reveals that pretreatment with MG-132 (proteasome
inhibitor) notably attenuated Bcl-2/Bcl-xL downregulation in AA-
treated cells. In addition, phosphorylation of Bax by p38 MAPK and
JNK has been reported to initiate its activation and mitochondrial
translocation (Kim et al., 2006). Consistent with this, mitochon-
dria translocation of Bax was inhibited by SB202190, SP600125
and BAPTA-AM (Fig. 5D). Noticeably, mitochondrial translocation
of Bax was also abolished by MG-132 pretreatment. Previous stud-
ies showed that MG-132-induced Bax translocation and inhibition
of protein synthesis by cycloheximide suppressed MG-132-induced
Bax activation (Ding et al., 2007). However, several lines of evidence
showed that proteasome inhibition altered AA metabolism (Levine,
1998, 2004). Given that many effects of AA on cell function are
mediated by its metabolites (Rizzo, 2002), alterations in the pro-
duction of AA metabolites may affect mitochondrial translocation
of Bax in SK-N-SH cells treated with a combination of MG-132 and
AA. Alternatively, NAC pretreatment significantly affected expres-
sion of Bcl-2 family proteins (Fig. 5E), suggesting that ROS-elicited
m loss was partly mediated through modulation of Bcl-2 family
proteins. This was in line with previous studies showing the involve-
ment of ROS in Bax/Bak-dependent release of cytochrome c (Li et
al., 2004; Hail, 2005).
p38 MAPK has four isomers including p38␣, p38␤, p38␥ and
p38␦ (Krishna and Narang, 2008). Previous studies reveal that
SB202190 mainly inhibits p38␣ MAPK and p38␤ MAPK, but it has
no effect on p38␥ and ␦ (Kumar et al., 1997). Given that SB202190
repressed AA-induced downregulation of Bcl-2/Bcl-xL and mito-
chondrial translocation of Bax (Fig. 5D), p38␣ MAPK and/or p38␤
MAPK were suggested to be involved in these effects. Enslen et
al. (1998, 2000) demonstrated that MKK3 activates only p38␣,
p38␥ and p38␦. Thus, expression of dominant negative mutant
of MKK3 should inactivate p38␣ but did not affect p38␤. Com-
pared with that of control cells, expression of p38␣ and p38␤ in
pRSV-MKK3(Ala)-transfected cells was not significantly changed
(Fig. 6A). Fig. 6A shows that AA treatment was unable to evoke
p38 MAPK, degradation of Bcl-2/Bcl-xL and mitochondrial translo-
cation of Bax in pRSV-MKK3(Ala)-transfected cells. This suggested
that p38␣ MAPK was crucial for modulation of Bcl-2 family
proteins in AA-treated cells. In sharp contrast to pcDNA3-p38␤-
transfected cells, pcDNA3-p38␣-transfected cells showed marked
downregulation of Bcl-2/Bcl-xL, mitochondrial translocation of Bax,
cytochrome c release and activation of caspase-9 and -3 after AA
treatment (Fig. 6B). Fig. 7A shows that transfection of pSuper-p38␣
shRNA plasmid attenuated AA-induced Bcl-2/Bcl-xL degradation
and mitochondrial translocation of Bax, reinforcing the role of p38␣
204 K.-C. Chen, L.-S. Chang / Toxicology 262 (2009) 199–206

Fig. 5. AA induced m loss, cytochrome c release, caspase-9 activation, downregulation of Bcl-2/Bcl-xL and mitochondrial translocation of Bax on SK-N-SH cells. (A) AA-
induced dissipation of m. Cells were treated with 50 ␮M AA for 24 h and then incubated with 20 nM rhodamine-123 for 20 min at 37 ◦ C. The loss of m was analyzed by
flow cytometry. SK-N-SH cells were pre-treated with 10 ␮M BAPTA-AM, 10 ␮M SB202190, 10 ␮M SP600125 or 2 mM NAC for 1 h prior to incubation with 50 ␮M AA for 24 h.
(B) Effect of cyclosporin A, rotenone and antimycin A on ROS generation in AA-treated cells. SK-N-SH cells were incubated with 50 ␮M AA for 4 h. Alternatively, SK-N-SH cells
were pre-treated with 1 ␮M cyclosporin A (CSA), 1 ␮M rotenone (Rote) or 10 ␮M antimycin A (Ant A) for 1 h, and then incubated with 50 ␮M AA for 4 h. The data represent
the mean ± SD (n = 6, *, **, ***P < 0.05 vs. AA-treated cells). (C) Western blot analyses of degradation of procaspase-9, cytochrome c release and expression of Bcl-2 family
proteins in AA-treated cells. SK-N-SH cells were treated with 50 ␮M AA for indicated time periods. (D) Effect of BAPTA-AM, SB202190, SP600125 and MG-132 on AA-induced
Bcl-2/Bcl-xL downregulation and mitochondrial translocation of Bax. SK-N-SH cells were pre-treated with 10 ␮M BAPTA-AM, 10 ␮M SB202190, 10 ␮M SP600125 or 10 ␮M
MG-132 for 1 h, and then incubated with 50 ␮M AA for 24 h. (E) Effect of NAC on protein expression of Bcl-2, Bcl-xL and Bax in AA-treated cells. SK-N-SH cells were pre-treated
with 2 mM NAC for 1 h, and then incubated with 50 ␮M AA for 24 h.

Fig. 6. Involvement of p38␣ MAPK in AA-induced Bcl-2/Bcl-xL downregulation and mitochondrial translocation of Bax. (A) The inability of AA to induce p38 MAPK activation,
Bcl-2/Bcl-xL downregulation and mitochondrial translocation of Bax in SK-N-SH cells that expressed dominant negative mutant of MKK3. SK-N-SH cells were transfected with
an empty expression vector and pRSV-MKK3(Ala), respectively. After 24 h post-transfection, cells were treated with 50 ␮M AA for 24 h. (B) The ability of AA to induce p38␣
MAPK activation, p38␤ MAPK activation, Bcl-2/Bcl-xL downregulation, mitochondrial translocation of Bax, cytochrome c release and activation of caspase-9 and -3 in SK-N-SH
cells that transfected with pcDNA3-p38␣ or pcDNA3-p38␤. SK-N-SH cells were transfected with an empty expression vector, pcDNA3-p38␣ or pcDNA3-p38␤, respectively.
After 24 h post-transfection, cells were treated with 50 ␮M AA for 24 h. (Left panel) Compared with that in control cells, transfection of pcDNA3-p38␣ or pcDNA3-p38␤
increased the expression of p38␣ MAPK or p38␤ MAPK, respectively. (Right panel) Western blot analyses of p38 MAPK activation, Bcl-2/Bcl-xL downregulation, mitochondrial
translocation of Bax, cytochrome c release and activation of caspase-9 and -3 in pcDNA3-p38␣- or pcDNA3-p38␤-transfected SK-N-SH cells after treatment with 50 ␮M AA
for 24 h.
 K.-C. Chen, L.-S. Chang / Toxicology 262 (2009) 199–206 205

Fig. 7. Effect of downregulation of p38␣ MAPK and JNK1 on AA-induced degradation of Bcl-2/Bcl-xL and mitochondrial translocation of Bax. (A) Downregulation of p38␣
MAPK attenuated AA-induced Bcl-2/Bcl-xL downregulation and mitochondrial translocation of Bax. SK-N-SH cells were transfected with control vector or pSuper-p38␣ shRNA
plasmid, respectively. After 24 h post-transfection, cells were treated with 50 ␮M AA for 24 h. (B) Downregulation of JNK1 attenuated AA-induced Bcl-2/Bcl-xL downregulation
and mitochondrial translocation of Bax. SK-N-SH cells were transfected with control vector or pKD-JNK1 shRNA plasmid, respectively. After 24 h post-transfection, cells were
treated with 50 ␮M AA for 24 h.

MAPK in modulating expression of Bcl-2 family proteins. In contrast
to JNK2, several lines of evidence revealed that JNK1 is involved
in Bcl-2 phosphorylation (Wei et al., 2008; Pattingre et al., 2009).
Thus, the contribution of JNK1 to modulate expression of Bcl-2,
Bcl-xL and Bax was evaluated by transfection of pKD-JNK1 shRNA
plasmid. Fig. 7B reveals that degradation of Bcl-2/Bcl-xL and mito-
chondrial translocation of Bax were attenuated by downregulation
of JNK1. Several studies on how increased mitochondrial permeabil-
ity and m loss may be involved in the release of cytochrome c,
and the crucial role of Bax/Bcl-2 in mitochondrial alteration is well
established (Gogvadze et al., 2006; Reed, 2006). Thus, our data sug-
gested that p38␣ MAPK and JNK1 were involved in mitochondrial
alteration through modulation of Bcl-2 family proteins.
In conclusion, our data show that AA-induced death of SK-N-SK
cells is mediated through mitochondria-dependent death pathway,
eliciting by generated ROS and elevation of cytosolic Ca2+ (Fig. 8).
The effect of [Ca2+ ]i on mitochondrial alteration is evoked by activa-
tion of p38␣ MAPK and JNK1, which modulate Bcl-2 family proteins
and facilitate mitochondrial permeability. Meanwhile, AA-induced
ROS generation further aggravates the collapse of m. Finally,
cytochrome c release activates caspase-9 and -3, thus resulting in
apoptotic death of human neuroblastoma SK-N-SH cells. Although
AA and its eicosanoid metabolites are suggested to be involved
in neurodegenerative conditions (Bazan et al., 2002; Candelario-
Jalil et al., 2003; Phillis and O’Regan, 2003), the detailed signaling
pathway remains elusive. Thus, the results of present study should
provide clues to interpret the cytotoxic mechanism of the released
AA during neurodegenerative conditions. Moreover, understand-
ing of the signal pathways responsible for AA-induced apoptotic
cell death may afford the benefit of searching effective strategies
in improving neuroblastoma treatment and overcoming therapy
resistance.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by grant NSC95-2320-B110-007-MY3

from the National Science Council, ROC (to L.S. Chang), and grant
of National Sun Yat-Sen University-Kaohsiung Medical University
Joint Research Center.

References

Fig. 8. Signaling pathway responsible for AA-induced apoptotic death of SK-N-SH
cells. AA treatment induced increase in [Ca2+ ]i, leading to activation of p38 MAPK and
JNK. Activated p38 MAPK and JNK modulated degradation of Bcl-2,/Bcl-xL and mito-
chondrial translocation of Bax, resulting in mitochondrial depolarization. Moreover,
generated ROS induced by AA further aggravated mitochondrial alteration. Finally,
released cytochrome c triggered activation of caspase-9 and -3, and apoptotic death
of SK-N-SH cells.
Banfi, B., Molnar, G., Maturana, A., Steger, K., Hegedus, B., Demaurex, N., Krause, K.H.,
2001. A Ca2+ -activated NADPH oxidase in testis, spleen and lymph nodes. J. Biol.
Chem. 276, 37594–37601.
Bazan, N.G., Colangelo, V., Lukiw, W.J., 2002. Prostaglandins and other lipid mediators
in Alzheimer’s disease. Prostaglandins Other Lipid Mediat. 68–69, 197–210.
Bostan, M., Galatiuc, C., Hirt, M., Constantin, M.C., Brasoveanu, L.I., Iordachescu, D.,
2003. Phospholipase A2 modulates respiratory burst developed by neutrophils
in patients with rheumatoid arthritis. J. Cell. Mol. Med. 7, 57–66.
Breitschopf, K., Haendeler, J., Malchow, P., Zeiher, A.M., Dimmeler, S., 2000. Posttrans-
lational modification of Bcl-2 facilitates its proteasome-dependent degradation:
molecular characterization of the involved signaling pathway. Mol. Cell. Biol. 20,
1886–1896.
206 K.-C. Chen, L.-S. Chang / Toxicology 262 (2009) 199–206
Brodeur, G.M., 2003. Neuroblastoma: biological insights into a clinical enigma. Nat.
Rev. Cancer 3, 203–216.
Candelario-Jalil, E., Gonzalez-Falcon, A., Garcia-Cabrera, M., Alvarez, D., Al-Dalain, S.,
Martinez, G., Leon, O.S., Springer, J.E., 2003. Assessment of the relative contri-
bution of COX-1 and COX-2 isoforms to ischemia induced oxidative damage and
neurodegeneration following transient global cerebral ischemia. J. Neurochem.
86, 545–555.
Cherny, V.V., Henderson, L.M., Xu, W., Thomas, L.L., DeCoursey, T.E., 2001. Activation
of NADPH oxidase-related proton and electron currents in human eosinophils
by arachidonic acid. J. Physiol. 535, 783–794.
De Chiara, G., Marcocci, M.E., Torcia, M., Lucibello, M., Rosini, P., Bonini, P., Higadshi-
moto, Y., Damonte, G., Armirotti, A., Amodei, S., Palamara, A.T., Russo, T., Garsci, E.,
Cozzolino, F., 2006. Bcl-2 phosphorylation by p38 MAPK, Identification of target
sites and biologic consequences. J. Biol. Chem. 281, 21353–21361.
Denson, D.D., Wang, X., Worrell, R.T., Eaton, D.C., 2000. Effects of fatty acids on BK
channels in GH3 cells. Am. J. Physiol. 279, 1211–1219.
Ding, W.X., Ni, H.M., Chen, X., Yu, J., Zhang, L., Yin, X.M., 2007. A coordinated action
of Bax, PUMA, and p53 promotes MG132-induced mitochondria activation and
apoptosis in colon cancer cells. Mol. Cancer Ther. 6, 1062–1069.
Enslen, H., Raingeaud, J., Davis, R.J., 1998. Selective activation of p38 mitogen-
activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and
MKK6. J. Biol. Chem. 273, 1741–1748.
Enslen, H., Brancho, D.M., Davis, R.J., 2000. Molecular determinants that mediate
selective activation of p38 MAP kinase isoforms. EMBO J. 19, 1301–1311.
Fleming, N., Mellow, L., 1995. AA stimulates intracellular calcium mobilization and
regulates protein synthesis, ATP levels, and mucin secretion in submandibular
gland cells. J. Dent. Res. 74, 1295–1302.
Gogvadze, V., Orrenius, S., Zhivotovsky, B., 2006. Multiple pathways of cytochrome c
release from mitochondria in apoptosis. Biochim. Biophys. Acta 1757, 639–647.
Grethe, S., Ares, M.P.S., Anderson, T., Pore-Ares, M.I., 2004. p38 MAPK mediates TNF-
induce apoptosis in endothelia cells via phosphorylation and downregulation of
Bcl-xL. Exp. Cell Res. 298, 632–642.
Green, D.R., Reed, J.C., 1998. Mitochondria and apoptosis. Science 281, 1309–1312.
Gijon, M.A., Leslie, C., 1997. Phospholipase A2 . Cell Dev. Biol. 8, 297–303.
Hail Jr., N., 2005. Mitochondria: a novel target for the chemoprevention of cancer.
Apoptosis 10, 687–705.
Kim, B.J., Ryu, S.W., Song, B.J., 2006. JNK and p38 kinase-mediated phosphorylation
of Bax leads to its activation and mitochondrial translocation and to apoptosis
of human hepatoma HepG2 cells. J. Biol. Chem. 281, 21256–21265.
Krishna, M., Narang, H., 2008. The complexity of mitogen activated protein kinases
(MAPKs) made simple. Cell. Mol. Life Sci., 10.1007/s00018-008-08170-7.
Kumar, S., McDonnell, P.C., Gum, R.J., Hand, A.T., Lee, J.C., Young, P.R., 1997. Novel
homologues of CSBP/p38 MAP kinase activation, substrate specificity and sen-
sitivity to inhibition by pyridinyl imidazoles. Biochem. Biophys. Res. Commun.
235, 533–538.
Kwon, K.J., Jung, Y.S., Lee, S.H., Moon, C.H., Baik, E.J., 2005. Arachidonic acid
induces neuronal death through lipoxygenase and cytochrome P450 rather than
cyclooxygenase. J. Neurosci. Res. 81, 73–84.
Levine, L., 1998. Proteolysis negatively regulates agonist-stimulated arachidonic acid
metabolism. Cell. Signal. 10, 653–659.
Levine, L., 2004. Proteasome inhibitors: their effects on arachidonic acid release
from cells in culture and arachidonic acid metabolism in rat liver cells. BMC
Pharmacol. 4, 15.
Li, D., Ueta, E., Kimura, T., Yamamoto, T., Osaki, T., 2004. Reactive oxygen species
(ROS) control the expression of Bcl-2 family proteins by regulating their phos-
phorylation and ubiquitination. Cancer Sci. 95, 644–650.
Li, Y., Maher, P., Schubert, D., 1997. A role for 12-lipoxygenase in nerve cell death
caused by glutathione depletion. Neuron 19, 453–463.
Liu, L., Rittenhouse, A.R., 2003. Arachidonic acid mediates muscarinic inhibition and
enhancement of N-type Ca2+ current in sympathetic neurons. Proc. Natl. Acad.
Sci. 100, 295–300.
Lipton, P., 1999. Ischemic cell death in brain neurons. Physiol. Rev. 79,
1431–1568.
Meves, H., 1994. Modulation of ion channels by arachidonic acid. Prog. Neurobiol.
43, 175–186.
Mignen, O., Shuttleworth, T.J., 2000. IARC, a novel arachidonate-regulated, nonca-
pacitative Ca2+ entry channel. J. Biol. Chem. 275, 9114–9119.
Mignen, O., Thompson, J.L., Shuttleworth, T.J., 2003. Ca2+ selectivity and fatty acid
specificity of the noncapacitative, arachidonate-regulated Ca2+ (ARC) channels.
J. Biol. Chem. 278, 10174–10181.
Okuda, S., Saito, H., Katsuki, H., 1994. Arachidonic acid: toxic and trophic effects on
cultured hippocampal neurons. Neuroscience 63, 691–699.
Pattingre, S., Bauvy, C., Carpentier, S., Levade, T., Levine, B., Codogno, P., 2009. Role of
JNK1-dependent Bcl-2 phosphorylation in ceramide-induced macroautophage.
J. Biol. Chem. 284, 2719–2728.
Phillis, J.W., O’Regan, M.H., 2003. The role of phospholipases, cyclooxygenases, and
lipoxygenases in cerebral ischemic/traumatic injuries. Crit. Rev. Neurobiol. 15,
61–90.
Pompeia, C., Lima, T., Curi, R., 2003a. Arachidonic acid cytotoxicity: can arachi-
donic acid be a physiological mediator of cell death. Cell Biochem. Func. 21,
97–104.
Pompeia, C., Cury-Boaventura, M.F., Curi, R., 2003b. Arachidonic acid triggers an
oxidative burst in leukocytes. Braz. J. Med. Biol. Res. 36, 1549–1560.
Proskuryakov, S.Y., Konoplyannikov, A.G., Gabai, V.L., 2003. Necrosis: a specific form
of programmed cell death? Exp. Cell. Res. 283, 1–16.
Rao, N.G., Baas, A., Glasgow, W.C., Eling, T.E., Runge, M.S., Alexander, R.W., 1994.
Activation of mitogen-activated protein kinases by arachidonic acid and its
metabolites in vascular smooth muscle cells. J. Biol. Chem. 269, 32586–32591.
Reed, J.C., 2006. Proapoptotic multidomain Bcl-2/Bax-family protein: mecha-
nisms, physiological roles, and therapeutic opportunities. Cell. Death Differ. 13,
1378–1386.
Rizzo, M.T., 2002. The role of arachidonic acid in normal and malignant
hematopoiesis. Prostaglandins Leukot. Essent. Fatty Acids 66, 57–69.
Rizzo, M.T., Regazzi, E., Garau, D., Akard, L., Dugan, M., Boswell, H.S., Rizzoli, V., Cario-
Stella, C., 1999. Induction of apoptosis by arachidonic acid in chronic myeloid
leukemia cells. Cancer Res. 59, 5047–5053.
Rizzo, M.T., Carlo-Stella, C., 1996. Arachidonic acid mediates interleukin-1 and tumor
necrosis factor-␣ induced activation of the c-jun amino terminal kinase in stro-
mal cells. Blood 88, 3792–3800.
Toborek, M., Malecki, A., Garrido, R., Mattson, M.P., Hennig, B., Young, B., 1999.
Arachidonic acid-induced oxidative injury to cultured spinal cord neurons. J.
Neurochem. 73, 684–692.
Wang, X., 2001. The expanding role of mitochondria in apoptosis. Genes Dev. 15,
2922–2933.
Wei, Y., Sinha, S., Levine, B., 2008. Dual role of JNK1-mediated phosphorylation of
Bcl-2 in autophagy and apoptosis regulation. Autophagy 4, 949–951.