Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells
© Kuo et al; licensee BioMed Central Ltd. 2009
Received: 26 August 2008
Accepted: 01 May 2009
Published: 01 May 2009
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.
Denbinobin (5-hydroxy-3,7-dimethoxy-1,4-phenanthraquinone) is purified from several Dendrobium or Ephemerantha (Orchidaceae) species, such as D. nobile , D. moniliforme , and E. lonchophylla [3, 4]. It has been shown that denbinobin possesses many biological effects 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]. Previous studies demonstrated that alterations in tubulin polymerization and Bcr-Abl activity are involved in denbinobin-induced human K562 leukemia cell death . 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 . However, the precise molecular mechanism of denbinobin-induced A549 cell apoptosis has not been fully delineated.
Apoptosis signal-regulating kinase 1 (ASK1) is a multifunctional serine/threonine protein kinase that participates 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)-α, and endoplasmic reticular stress [10–12]. However, the role of ASK1 in denbinobin-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 compartments where they interfere with the function of antiapoptotic Bcl-2 family members, leading to apoptotic cell death [13, 14].
In the present study, we explored the roles of ASK1 in denbinobin-induced cell death in human lung adenocarcinoma (A549) cells. Our data demonstrate for the first time that denbinobin might activate ASK1 through reactive 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
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 , 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 phospho-JNK1/2 (Thr183/Tyr185), JNK1/2, and phospho-Akt (Ser473) were purchased from New England Biolabs (Beverly, MA). The α-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 purchased from PerkinElmer Life Sciences (Boston, MA). 4-Nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) were purchased from Boehringer Mannheim (Mannheim, Germany). All materials for sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) were obtained from Bio-Rad (Hercules, CA). SP600125 was purchased from Calbiochem-Novabiochem (San Diego, CA). 2',7'-Dichlorodihydrofluorescein 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 [γ-32P] ATP were obtained from GE Healthcare (Little Chalfont, UK). The RNAi-Ready pSIREN-RetroQ-ZsGreen vector was purchased from BD Biosciences-Clontech (Palo Alto, CA). All materials for sqRT-PCR were obtained from Protech Technology (Taipei, Taiwan). N-Acetyl-L-cysteine (NAC), glutathione (GSH), propidium iodide (PI), dithiothreitol (DTT), phenylmethylsulphonyl fluoride (PMSF), pepstatin A, leupeptin, SDS, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium (MTT), and other chemicals were obtained from Sigma (St. Louis, MO).
A549 cells, a human pulmonary type II epithelial adenocarcinoma 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 μg/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 inhibitors for 30 min as indicated followed by treatment with 20 μM denbinobin for additional 24 h. After treatment, cells were harvested and washed twice with phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.5 mM KH2PO4; pH 7.4), and re-suspended in ice-cold 70% ethanol at -20°C overnight. Cells were washed for 5 min with 0.4 ml phosphate-citric acid buffer (pH 7.8) containing 50 mM Na2HPO4, 25 mM citric 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 μg/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 . 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 μM 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 μl 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 μg/well of pcDNA (mock), ASK1DN, JNK1DN, JNK2DN, pSiREN-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 μM denbinobin for various time periods dependent on different experiments before harvesting. To determine the transfection efficiency, cells were transfected with 1 μg 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 confluence, cells were treated with 20 μM 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 μg/ml leupeptin, 10 μg/ml aprotinin, 25 mM β-glycerophosphate, 50 mM NaF, and 100 μM Na3VO4, and centrifuged 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 μl of kinase buffer supplemented with substrate (50 μg/ml myelin basic protein (MBP) for ASK1 activity or 50 μg/ml c-Jun-GST fusion protein for JNK1/2 activity), 20 μM ATP, and 3 μCi of [γ-32P] ATP at 30°C for 30 min. The reaction mixtures were analyzed by 15% SDS-PAGE followed by autoradiography.
ROS generation assay
Intracellular ROS formation was measured using the fluorogenic probe, H2DCFDA (Molecular Probes, Eugene, OR). Briefly, A549 cells were loaded with 10 μM H2DCFDA for 30 min at 37°C in the dark. After incubation, cells were treated with 20 μM denbinobin for the indicated time intervals, or pretreated with specific inhibitors as indicated followed by denbinobin. Cells then harvested, and ROS generation was measured by FACScan flow cytometry to detect the log of the mean fluorescence intensity (MFI) with an acquisition of fluorescence channel 1 (FL1). A minimum number of 10,000 events was collected and analyzed by the Cellquest program.
An immunoblot analysis was performed as previously described . A549 cells were cultured in 6-cm dishes. After reaching confluence, cells were treated with 20 μM 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 containing 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 aprotinin, 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 μg) in each sample were boiled in SDS sample loading buffer, and then fractionated on SDS-PAGE before being blotted onto a polyvinylidene 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 peroxidase-conjugated second antibodies. After washing with PBS, blots were developed using NBT/BCIP or an enhanced chemiluminescence kit according to the vendor's instructions before exposure to photographic film.
Preparation of nuclear extracts and the electrophoretic mobility shift assay (EMSA)
Cells were grown in 6-cm dishes. After reaching confluence, cells were treated with 20 μM denbinobin for the indicated time intervals. The nuclear protein fractions were then prepared as described previously . 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 centrifugation 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 supernatants 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 [γ-32P] ATP using the T4 polynucleotide kinase. The nuclear extract (2.5~5 μg) was incubated with 1 ng of a 32P-labeled AP-1 probe (50,000~75,000 cpm) in 10 μl of binding buffer containing 1 μg 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 protein 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 experiments, 1 ng of the labeled oligonucleotide was mixed with 50 ng of AP-1 or NF-κB unlabeled competitor oligonucleotides prior to protein addition. For the supershift experiments, 4 μg 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 complementary oligonucleotides were annealed and cloned into the pSIREN-RetroQ-ZsGreen vector (BD Biosciences Clontech, San Diego., CA): the sense oligonucleotide sequence for bim was 5'-GATCCGTTCTGAGTGTGACCGAGAttcaagagaTCTCGGTCACACTCAGAACTTTTTTG-3'; and the antisense oligonucleotide sequence for bim was 3'-GCAAGACTCACACTGGCTCTaagttctctAGAGCCAGTGTGAGTCTTGAAAAAACTTAA-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 pSiREN-NC plasmids were confirmed based on the sequencing analysis.
RNA isolation and semiquantitative (sq)RT-PCR
A549 cells were seeded in 6-cm dishes. After reaching confluence, cells were treated with 20 μM denbinobin or 5 mM H2O2 for the indicated time intervals, or pretreated with specific inhibitors (1 mM NAC, 100 μM GSH, or 10 μM 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 protocol 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. β-Actin was normalized in parallel in the same samples. PCR conditions included pre-incubation at 95°C for 5 min followed by 40 amplification 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-γ: sense, 5'-AAG CAA CCT TCT GAT GTA-3'; antisense, 5'-CAA TGC ATT CTC CAC ACC AG-3'. The sense primer of β-actin was 5'-GAC TAC CTC ATG AAG ATC CT-3'; and the reverse primer of β-actin was 5'-CCA CAT CTG CTG GAA GGT GG-3'. PCR products were visualized by UV-illuminated agarose gel electrophoresis.
Results are presented as the mean ± SEM from at least three independent experiments. One-way analysis of variance (ANOVA), followed by Bonferroni's multiple-range tests when appropriate, was used to determine the statistical significance of the difference between the means. A p value of < 0.05 was considered statistically significant.
ASK1 mediates denbinobin-induced A549 cell apoptosis
Involvement of ROS generation in denbinobin-induced A549 cell apoptosis
JNK is involved in denbinobin-induced A549 cell apoptosis
AP-1 is involved in denbinobin-induced A549 cell apoptosis
Bim upregulation is involved in denbinobin-induced apoptosis
Our previous study showed that Akt inactivation, followed by Bad activation, mitochondrial dysfunction, caspase 3 activation, and AIF release, contributes to denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. However, the precise molecular mechanism responsible for the apoptotic action of denbinobin remains to be fully characterized. The findings of this study demonstrated that activation of the ROS-ASK1-JNK signaling cascade followed by AP-1 activation and Bim expression contributes to denbinobin-induced apoptosis in A549 cells.
Activated ASK1 plays a critical role in regulating diverse physiological processes, including cell differentiation and apoptosis, by stimulating downstream signaling events including activation of MKK4/7 and JNK in sequence [24, 25]. JNK is thought to play a role in AP-1-mediated transcriptional activation of the target genes [26, 27]. However, whether the ASK1-JNK-AP-1 signaling pathway participates in denbinobin-induced A549 cell apoptosis has not been demonstrated. In this study, we found that treatment of A549 cells with denbinobin caused sequential activations of ASK1, JNK, c-Jun, and AP-1, and that the dominant-negative mutants of ASK1, JNK1, and JNK2, and the inhibitors of JNK and AP-1 attenuated denbinobin-induced cell apoptosis in A549 cells. The dominant-negative mutant of ASK1 suppressed denbinobin-induced JNK1/2 activation. Moreover, denbinobin-induced c-Jun phosphorylation was inhibited by the dominant-negative mutants of ASK1, JNK1, and JNK2, and the JNK inhibitor. These results suggest that denbinobin might activate ASK1, causing JNK and c-Jun activation, which in turn induces AP-1 activation, and ultimately leads to A549 cell apoptosis.
ASK1 activity is regulated by interactions with various proteins. When free of oxidative stress, the inactive form of ASK1 is phosphorylated at the serine at residue 967 and is suppressed by interactions with its repressors, such as thioredoxin (Trx), glutaredoxin (Grx), and the 14-3-3 protein. Previous studies showed that ROS might activate ASK1 through oxidizing thioredoxin (Trx) and glutaredoxin [20–22]. Recent reports also indicated that ASK1 is response to oxidative stress such as H2O2 through decreasing phosphorylation at serine 967 parallel to increasing the dissociation with inhibitory proteins leading to activation of the apoptotic function [20, 28]. In this study, we found that denbinobin induced ROS formation, and that two antioxidants (NAC and GSH) inhibited denbinobin-induced ASK1 activation, JNK activation, c-Jun phosphorylation, and cell apoptosis. In addition, treatment of A549 cells with H2O2 induced ASK1 activation. These findings suggest that ROS may play a pivotal role in denbinobin-induced ASK1 activation and subsequent signaling events.
Bcl-2 family proteins regulate mitochondria-dependent apoptosis with the balance of the anti- and proapoptotic members arbitrating life-or-death decisions. Bim, a proapoptotic member of the Bcl-2 family, causes apoptosis by disrupting mitochondrial integrity. bim gene expression is induced by AP-1 in response to selected stress signals [29, 30]. In the present study, we found that denbinobin induced Bim expression and that Bim siRNA attenuated denbinobin-induced A549 cell apoptosis, suggesting that Bim expression is causally related to denbinobin-induced A549 cell apoptosis. Furthermore, denbinobin-induced Bim expression was inhibited by antioxidants, a JNK inhibitor, and the dominant-negative mutants of ASK1, JNK1, and JNK2. Similarly, treatment of cells with H2O2 induced ASK1 activation. Thus, it is plausible that denbinobin activates the ROS-ASK1-JNK cascade causing Bim expression and subsequent cell apoptosis. Recent reports have indicated that in addition to Bim, BH3-only members of the Bcl-2 family, such as Bad, also participate in denbinobin-induced A549 cell apoptosis . These observations explain, at least in part, why blocking the ASK1 signaling cascade did not completely abolish denbinobin-induced A549 cell apoptosis.
apoptosis signal-regulating kinase 1
bovine serum albumin
Dulbecco's modified Eagle's medium
electrophoretic mobility shift assay
fetal calf serum
c-Jun N-terminal kinase
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
4-nitro blue tetrazolium
reactive oxygen species
short interfering RNA
This work was supported by a research grant (NSC95-2314-B-038-014-MY2) from the National Science Council of Taiwan.
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