Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells.

Abstract

In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants (N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

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BioMed Central
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Journal of Biomedical Science
Open Access
Research
Apoptosis signal-regulating kinase 1 mediates denbinobin-induced
apoptosis in human lung adenocarcinoma cells
Chen-Tzu Kuo1, Bing-Chang Chen2, Chung-Chi Yu1, Chih-Ming Weng1,
Ming-Jen Hsu1,3, Chien-Chih Chen4, Mei-Chieh Chen5, Che-Ming Teng6,
Shiow-Lin Pan6, Mauo-Ying Bien2, Chung-Hung Shih2 and Chien-
Huang Lin*1,7,8
Address: 1Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan, ROC, 2School of Respiratory
Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan, ROC, 3Department of Pharmacology, College of Medicine, Taipei Medical
University, Taipei, Taiwan, ROC, 4National Institute of Chinese Medicine, Taipei, Taiwan, ROC, 5Department of Microbiology and Immunology,
College of Medicine, Taipei Medical University, Taipei, Taiwan, ROC, 6Pharmacological Institute, College of Medicine, National Taiwan
University, Taipei, Taiwan, ROC, 7Taipei Medical University-Municipal Wang-Fang Hospital, Taipei, Taiwan, ROC and 8Taipei Medical University-
Shuang-Ho Hospital, Taipei County, Taiwan, ROC
Email: Chen-Tzu Kuo - bigkuo7@hotmaol.com; Bing-Chang Chen - bcchen@tmu.edu.tw; Chung-Chi Yu - b8604032@tmu.edu.tw; Chih-
Ming Weng - m109090013@tmu.edu.tw; Ming-Jen Hsu - aspirin@tmu.edu.tw; Chien-Chih Chen - ccchen@nricm.edu.tw; Mei-
Chieh Chen - mcchen@tmu.edu.tw; Che-Ming Teng - cmteng@ntu.edu.tw; Shiow-Lin Pan - psl0826@ms13.hinet.net; Mauo-
Ying Bien - mybien@tmu.edu.tw; Chung-Hung Shih - CHSHIH43@tmu.edu.tw; Chien-Huang Lin* - chlin@tmu.edu.tw
* Corresponding author
Abstract
In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in
denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced
cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants
(N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor
(SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with
denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and
these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused
JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin
induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim
expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-
induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim
expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These
results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1
activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.
Background
Denbinobin
thraquinone) is purified from several Dendrobium or
(5-hydroxy-3,7-dimethoxy-1,4-phenan-
Ephemerantha (Orchidaceae) species, such as D. nobile [1],
D. moniliforme [2], and E. lonchophylla [3,4]. It has been
shown that denbinobin possesses many biological effects
Published: 1 May 2009
Journal of Biomedical Science 2009, 16:43doi:10.1186/1423-0127-16-43
Received: 26 August 2008
Accepted: 1 May 2009
This article is available from: http://www.jbiomedsci.com/content/16/1/43
© 2009 Kuo et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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such as antioxidation, antiplatelet aggregation, and anti-
inflammation. It was also demonstrated to induce cell
death in colon adenocarcinoma (COLO 205) cells, ovary
adenocarcinoma (SK-OV-3) cells, human leukemia
(K562) cells, promyelocytic leukemia (HL60) cells, and
human lung adenocarcinoma (A549) cells [1,5-7]. Previ-
ous studies demonstrated that alterations in tubulin
polymerization and Bcr-Abl activity are involved in den-
binobin-induced human K562 leukemia cell death [5].
Recently, we demonstrated that Akt inactivation, followed
by Bad activation, mitochondrial dysfunction, the release
of mitochondrial molecules such as cytochrome c, second
mitochondria-derived activator of caspase (Smac), and
apoptosis-inducing factor (AIF), and caspase 3 activation
contributes to denbinobin-induced A549 cell apoptosis
[6]. However, the precise molecular mechanism of den-
binobin-induced A549 cell apoptosis has not been fully
delineated.
Apoptosis signal-regulating kinase 1 (ASK1) is a multi-
functional serine/threonine protein kinase that partici-
pates in diverse physiological processes, including cell
differentiation and apoptosis [8,9]. ASK1 has been
reported to be activated by many stress signals, including
H2O2, tumor necrosis factor (TNF)-, and endoplasmic
reticular stress [10-12]. However, the role of ASK1 in den-
binobin-induced A549 cell apoptosis is still unknown.
Bim is a member of the "BH3-only proteins", a subgroup
of Bcl-2 apoptotic regulators which contain only one of
the bcl-2 homologous regions (BH3). In response to
apoptotic stimuli, BH3-only proteins are translocated to
mitochondrial membranes from other cellular compart-
ments where they interfere with the function of antiapop-
totic Bcl-2 family members, leading to apoptotic cell
death [13,14].
In the present study, we explored the roles of ASK1 in den-
binobin-induced cell death in human lung adenocarci-
noma (A549) cells. Our data demonstrate for the first
time that denbinobin might activate ASK1 through reac-
tive oxygen species (ROS) production to cause JNK/AP-1
activation, which in turn induces Bim expression, and
ultimately results in A549 cell apoptosis.
Materials and methods
Materials
Denbinobin was kindly provided by Dr. Chien-Chih
Chen (National Research Institute of Chinese Medicine,
Taipei, Taiwan). The methods of extraction and isolation
of denbinobin were previously reported [3], and the
purity exceeded 98% based on a high-performance liquid
chromatographic (HPLC) analysis. Dulbecco's modified
Eagle's medium (DMEM)/Ham's F-12, fetal calf serum
(FCS), penicillin/streptomycin, OptiMEM, Lipofectamine
plus™ reagent, and TRIzol reagent were purchased from
Invitrogen (Carlsbad, CA). Antibodies specific for phos-
pho-JNK1/2 (Thr183/Tyr185), JNK1/2, and phospho-Akt
(Ser473) were purchased from New England Biolabs (Bev-
erly, MA). The -tubulin antibody was purchased from
Novus Biologicals (Littleton, CO). Protein A/G beads,
antibodies specific for JNK1, phospho-c-Jun, c-Jun, c-Fos,
Akt1/2, and GAPDH, as well as horseradish peroxidase-
conjugated anti-mouse and anti-rabbit antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Anti-mouse immunoglobulin G (IgG)-conjugated
alkaline phosphatase was purchased from Jackson
Immuno Research Laboratories (West Grove, PA). The
enhanced chemiluminescence detection agent was pur-
chased from PerkinElmer Life Sciences (Boston, MA). 4-
Nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-
indolyl-phosphate (BCIP) were purchased from Boe-
hringer Mannheim (Mannheim, Germany). All materials
for sodium dodecylsulfate polyacrylamide gel electro-
phoresis (SDS-PAGE) were obtained from Bio-Rad (Her-
cules, CA). SP600125 was purchased from Calbiochem-
Novabiochem (San Diego, CA). 2',7'-Dichlorodihydroflu-
orescein diacetate (H2DCFDA) was purchased from
Molecular Probes (Eugene, OR). ASK1DN, JNK1DN, and
JNK2DN were kindly provided by Dr. Mei-Chieh Chen
(Taipei Medical University, Taipei, Taiwan). All materials
for agarose gel electrophoresis and [-32P] ATP were
obtained from GE Healthcare (Little Chalfont, UK). The
RNAi-Ready pSIREN-RetroQ-ZsGreen vector was pur-
chased from BD Biosciences-Clontech (Palo Alto, CA). All
materials for sqRT-PCR were obtained from Protech Tech-
nology (Taipei, Taiwan). N-Acetyl-L-cysteine (NAC), glu-
tathione (GSH), propidium iodide (PI), dithiothreitol
(DTT), phenylmethylsulphonyl fluoride (PMSF), pepsta-
tin A, leupeptin, SDS, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-
diphenyltetrazolium (MTT), and other chemicals were
obtained from Sigma (St. Louis, MO).
Cell culture
A549 cells, a human pulmonary type II epithelial adeno-
carcinoma cell line, were obtained from the American
Type Culture Collection and cultured in DMEM/Ham's F-
12 nutrient mixture with 10% FCS and antibiotics (100
U/ml penicillin and 100 g/ml streptomycin). Cells were
cultured at 37°C in a humidified 5% CO2 atmosphere.
Flow cytometric analysis
A549 cells were cultured in 10-cm Petri dishes. After
reaching confluence, cells were transfected with different
plasmid DNAs for 6 h or pretreated with specific inhibi-
tors for 30 min as indicated followed by treatment with 20
M denbinobin for additional 24 h. After treatment, cells
were harvested and washed twice with phosphate-buff-
ered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4, and 1.5 mM KH2PO4; pH 7.4), and re-sus-
pended in ice-cold 70% ethanol at -20°C overnight. Cells

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were washed for 5 min with 0.4 ml phosphate-citric acid
buffer (pH 7.8) containing 50 mM Na2HPO4, 25 mM cit-
ric acid, and 0.1% Triton X-100 and subsequently stained
with 1.5 ml PI staining buffer containing 0.5% Triton X-
100, 10 mM PIPES, 100 mM NaCl, 2 mM MgCl2, 0.1 U/
ml RNase A, and 25 g/ml PI for 30 min in the dark before
the flow cytometric analysis. Samples were analyzed by
FACScan and the Cellquest program (Becton Dickinson,
San Jose, CA).
Cell viability assay
Cell viability was measured by a previously described
colorimetric MTT assay [15]. Briefly, cells (2 × 105 cells/
well) were cultured in 24-well plates and transfected with
different plasmid DNAs for 6 h or pretreated with specific
inhibitors for 30 min as indicated followed by incubation
with 20 M denbinobin for another 24 h. After various
treatments, 1 mg/ml MTT was added to the culture plates
and incubated at 37°C for an additional 4 h. Then cells
were lysed in 500 l dimethyl sulfoxide. The absorbance
at 550 nm was measured on a microplate reader. Each
experiment was performed in triplicate and repeated at
least three times.
Plasmid DNA transfection
A549 cells were seeded at a density of 2 × 105 cells/ml into
12-well plates. Cells were transfected on the following day
with the Lipofectamine plus™ reagent containing 1 g/
well of pcDNA (mock), ASK1DN, JNK1DN, JNK2DN, pSi-
REN-NC, or pSiREN-bim cDNA in Opti-MEM for 6 h. At
the end of transfection, the medium was aspirated and
replaced with fresh culture medium for 24 h. Cells were
treated with 20 M denbinobin for various time periods
dependent on different experiments before harvesting. To
determine the transfection efficiency, cells were trans-
fected with 1 g of pEGFP, a green fluorescence protein
(GFP) expression vector for 24 h. After treatment, the
medium was aspirated and replaced with fresh DMEM/
Ham's F12 containing 10% FBS for another 24 h. Cells
were observed under inverted laser scanning confocal
microscopy (Olympus). The transfection efficacy was
defined as the percentage of cells expressing GFP. The
transfection rate of GFP was about 33% (data not shown).
Measurements of ASK1 and JNK kinase activity
A549 cells were seeded in 6-cm dishes. After reaching con-
fluence, cells were treated with 20 M denbinobin for 10,
30, 60, or 120 min. After treatment, cells were lysed with
lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM
MgCl2, 125 mM NaCl, 1% Triton X-100, 1 mM PMSF, 10
g/ml leupeptin, 10 g/ml aprotinin, 25 mM -glycero-
phosphate, 50 mM NaF, and 100 M Na3VO4, and centri-
fuged at 4°C and 12,000 × g for 30 min. The supernatant
was then immunoprecipitated with a specific antibody
against ASK1 or JNK1/2 in the presence of A/G-agarose
beads overnight. The beads were washed three times with
lysis buffer and two times with kinase buffer containing
20 mM HEPES (pH 7.4), 20 mM MgCl2, and 2 mM DTT.
The kinase reactions were performed by incubating
immunoprecipitated beads with 20 l of kinase buffer
supplemented with substrate (50 g/ml myelin basic pro-
tein (MBP) for ASK1 activity or 50 g/ml c-Jun-GST fusion
protein for JNK1/2 activity), 20 M ATP, and 3 Ci of [-
32P] ATP at 30°C for 30 min. The reaction mixtures were
analyzed by 15% SDS-PAGE followed by autoradiogra-
phy.
ROS generation assay
Intracellular ROS formation was measured using the
fluorogenic probe, H2DCFDA (Molecular Probes,
Eugene, OR). Briefly, A549 cells were loaded with 10 M
H2DCFDA for 30 min at 37°C in the dark. After incuba-
tion, cells were treated with 20 M denbinobin for the
indicated time intervals, or pretreated with specific inhib-
itors as indicated followed by denbinobin. Cells then har-
vested, and ROS generation was measured by FACScan
flow cytometry to detect the log of the mean fluorescence
intensity (MFI) with an acquisition of fluorescence chan-
nel 1 (FL1). A minimum number of 10,000 events was
collected and analyzed by the Cellquest program.
Immunoblot analysis
An immunoblot analysis was performed as previously
described [16]. A549 cells were cultured in 6-cm dishes.
After reaching confluence, cells were treated with 20 M
denbinobin for the indicated time intervals, or pretreated
with specific inhibitors, or transiently transfected with
pcDNA, ASK1DN, JNK1DN, or JNK2DN for 6 h followed
by denbinobin. After incubation, cells were washed twice
with ice-cold PBS and solubilized in extraction buffer con-
taining 10 mM Tris (pH 7.0), 140 mM NaCl, 3 mM MgCl2,
2 mM PMSF, 5 mM DTT, 0.5% NP-40, 0.01 mg/ml apro-
tinin, 0.01 mg/ml leupeptin, 1 mM benzamidine, and 1
mM Na3VO4. Protein concentrations of cell lysates were
determined by the Bradford protein assay (Bio-Rad).
Equal amounts of protein (60 g) in each sample were
boiled in SDS sample loading buffer, and then fraction-
ated on SDS-PAGE before being blotted onto a polyvinyli-
dene difluoride (PVDF) membrane. Blots were then
incubated in 150 mM NaCl, 20 mM Tris, and 0.02%
Tween (pH 7.4) containing 5% non-fat milk. Proteins
were visualized by specific primary antibodies and then
incubated with alkaline phosphatase- or horseradish per-
oxidase-conjugated second antibodies. After washing with
PBS, blots were developed using NBT/BCIP or an
enhanced chemiluminescence kit according to the ven-
dor's instructions before exposure to photographic film.

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Preparation of nuclear extracts and the electrophoretic
mobility shift assay (EMSA)
Cells were grown in 6-cm dishes. After reaching conflu-
ence, cells were treated with 20 M denbinobin for the
indicated time intervals. The nuclear protein fractions
were then prepared as described previously [17]. Briefly,
cells were washed with ice-cold PBS, and then centrifuged.
The cell pellet was resuspended in hypotonic buffer (10
mM HEPES, 10 mM KCl, 0.5 mM DTT, 10 mM aprotinin,
10 mM leupeptin, and 20 mM PMSF) for 15 min on ice,
and vortexed for 10 sec. The nuclei were collected by cen-
trifugation at 15,000 × g for 1 min. The pellet containing
nuclei was resuspended in hypertonic buffer (20 mM
HEPES (pH 7.6), 25% glycerol, 1.5 mM MgCl2, 4 mM
EDTA, 0.05 mM DTT, 20 mM PMSF, 10 mM aprotinin,
and 10 mM leupeptin) for 30 min on ice. The superna-
tants containing the nuclear proteins were collected by
centrifugation at 15,000 × g for 30 min and then stored at
-80°C.
A double-stranded oligonucleotide probe containing the
recombinant AP-1 consensus sequence (5'-TTC CGG CTG
ACT CAT CAA GCG-3'; Promega) was end-labeled with [-
32P] ATP using the T4 polynucleotide kinase. The nuclear
extract (2.5~5 g) was incubated with 1 ng of a 32P-
labeled AP-1 probe (50,000~75,000 cpm) in 10 l of
binding buffer containing 1 g poly(dI-dc), 15 mM
HEPES (pH 7.6), 80 mM NaCl, 1 mM EDTA, 1 mM DTT,
and 10% glycerol at 30°C for 25 min. DNA/nuclear pro-
tein complexes were separated from the DNA probe by
electrophoresis on 6% polyacrylamide gels. The gels were
vacuum-dried and subjected to autoradiography with an
intensifying screen at -80°C. For competition experi-
ments, 1 ng of the labeled oligonucleotide was mixed with
50 ng of AP-1 or NF-B unlabeled competitor oligonucle-
otides prior to protein addition. For the supershift experi-
ments, 4 g of the anti-c-Jun or anti-c-Fos antibody was
mixed with the nuclear extract proteins.
Interference of bim expression
To generate siRNAs targeting bim mRNA for degradation,
we produced the pSiREN-bim plasmid to suppress bim
expression. To create pSiREN-bim, artificial complemen-
tary oligonucleotides were annealed and cloned into the
pSIREN-RetroQ-ZsGreen vector (BD Biosciences Clon-
tech, San Diego., CA): the sense oligonucleotide sequence
for bim was 5'-GATCCGTTCTGAGTGTGACCGAGAttcaa-
gagaTCTCGGTCACACTCAGAACTTTTTTG-3'; and the
antisense oligonucleotide sequence for bim was 3'-
GCAAGACTCACACTGGCTCTaagttctctAGAGCCAGTGT-
GAGTCTTGAAAAAACTTAA-5'. A negative control RNAi
(pSiREN-NC) was also constructed by cloning custom-
synthesized oligonucleotides into the pSIREN-RetroQ-
ZsGreen vector. The accuracies of the pSiREN-bim and pSi-
REN-NC plasmids were confirmed based on the sequenc-
ing analysis.
RNA isolation and semiquantitative (sq)RT-PCR
A549 cells were seeded in 6-cm dishes. After reaching con-
fluence, cells were treated with 20 M denbinobin or 5
mM H2O2 for the indicated time intervals, or pretreated
with specific inhibitors (1 mM NAC, 100 M GSH, or 10
M SP600125) for 30 min, or transiently transfected with
pcDNA, ASK1DN, JNK1DN, or JNK2DN for 6 h followed
by denbinobin. Total RNA was purified using the TRIzol
reagent (Invitrogen) following the manufacturer's proto-
col and quantified by spectrophotometry at a 260 nm
wavelength. RNA was reverse-transcribed, and the cDNA
product was amplified by PCR using 500 ng of specific
forward and reverse primers. -Actin was normalized in
parallel in the same samples. PCR conditions included
pre-incubation at 95°C for 5 min followed by 40 amplifi-
cation cycles (denaturation at 95°C for 1 min, annealing
at 53°C for 1 min, elongation at 72°C for 1 min, and a
final extension for 10 min at 72°C). Forward and reverse
primers for bim were designed for all isoforms except for
bim-: sense, 5'-AAG CAA CCT TCT GAT GTA-3'; anti-
sense, 5'-CAA TGC ATT CTC CAC ACC AG-3'. The sense
primer of -actin was 5'-GAC TAC CTC ATG AAG ATC CT-
3'; and the reverse primer of -actin was 5'-CCA CAT CTG
CTG GAA GGT GG-3'. PCR products were visualized by
UV-illuminated agarose gel electrophoresis.
Statistical analysis
Results are presented as the mean ± SEM from at least
three independent experiments. One-way analysis of vari-
ance (ANOVA), followed by Bonferroni's multiple-range
tests when appropriate, was used to determine the statisti-
cal significance of the difference between the means. A p
value of < 0.05 was considered statistically significant.
Results
ASK1 mediates denbinobin-induced A549 cell apoptosis
ASK1 activation is a pivotal mechanism in a broad range
of cell death paradigms [12]. To explore whether ASK1
activation contributes to denbinobin-induced A549 cell
apoptosis, cells were transiently transfected with the
ASK1DN prior to denbinobin treatment. As shown in Fig.
1A, the percentage of PI-stained cells in the apoptotic
region (Apo, sub-G0/G1 peak, subdiploid peak) was sig-
nificantly increased following 20 M denbinobin treat-
ment, compared to the
Denbinobin-induced A549 cell apoptosis was attenuated
by 48.1 ± 5.2% by transfection with ASK1DN (Fig. 1A). To
further elucidate whether ASK1 activation is involved in
the signaling cascade of denbinobin-induced A549 cell
apoptosis, ASK1 kinase activity was measured after den-
binobin exposure. Treatment of A549 cells with 20 M
denbinobin increased ASK1 kinase activity. This response
vehicle-treated group.

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peaked at 10 min and had returned to the basal level
within 2 h (Fig. 1B). Similarly, 5 mM H2O2, a potent ASK1
stimulator [18], caused an increase in ASK1 kinase activity
in a time-dependent manner (Fig. 1C). These results sug-
gest that ASK1 activation participates in denbinobin-
induced apoptosis in A549 cells.
Involvement of ROS generation in denbinobin-induced
A549 cell apoptosis
Previous report indicated that ROS play an important role
in apoptotic signaling pathway [19]. To explore the role of
ROS in denbinobin-induced A549 cell apoptosis, the anti-
oxidants, NAC and GSH, were used. As shown in Fig. 2A,
pretreatment of A549 cells with 1 mM NAC and 100 M
GSH markedly attenuated denbinobin-induced A549 cell
apoptosis by 95.4 ± 2.8% and 77.0 ± 4.1%, respectively.
Moreover, treatment of cells with 20 M denbinobin
caused a time-dependent increase in ROS production with
a maximum effect at 5~10 min after denbinobin treat-
ment. However, after 60 min of treatment with den-
binobin, the response had decreased (Fig. 2B).
Furthermore, denbinobin-induced ROS formation was
almost completely inhibited by treatment with 1 mM
NAC or 100 M GSH (Fig. 2C). These results suggest that
denbinobin caused intracellular ROS generation, and the
oxidative stress further contributed to A549 cell apoptosis.
Previous studies showed that ROS might activate ASK1
through oxidizing thioredoxin (Trx) and glutaredoxin
[20-22]. We next speculated whether ROS generation
results in ASK1 activation in denbinobin-mediated apop-
tosis. As illustrated in Fig. 2D, the denbinobin-induced
increase in ASK1 activity was markedly inhibited by 1 mM
Figure 1
ASK1 activation is involved in denbinobin-induced apoptosis in A549 cells
Figure 1
ASK1 activation is involved in denbinobin-induced
apoptosis in A549 cells. (A) A549 cells were transiently
transfected with pcDNA (mock) or ASK1DN for 6 h. Fol-
lowing transfection, cells were replaced with fresh culture
medium for 24 h and treated with vehicle or 20 M den-
binobin for another 24 h. The percentage of apoptotic cells
was then determined using flow cytometric analysis of PI-
stained cells as described in "Materials and methods". Each
column represents the mean ± S.E.M. of at least three inde-
pendent experiments. *p < 0.05, compared to the mock
transfection group in the presence of denbinobin. Cells were
treated with 20 M denbinobin (B) or 5 mM H2O2 (C) for
the indicated time intervals. Cell lysates were then immuno-
precipitated with the anti-ASK1 antibody, and the kinase
activity was measured as described in "Materials and meth-
ods". Equal loading in each lane is reflected by similar intensi-
ties of -tubulin. Compiled results are shown in the lower
panel. Each column represents the mean ± S.E.M. of at least
three independent experiments. * p < 0.05, compared to the
control group without denbinobin or H2O2 treatment.