Available online at www.sciencedirect.com
Toxicology Letters 177 (2008) 48–58
Denbinobin induces apoptosis in human lung adenocarcinoma cells via Akt
inactivation, Bad activation, and mitochondrial dysfunction
Chen-Tzu Kuo
a
, Ming-Jen Hsu
a
,
b
, Bing-Chang Chen
c
, Chien-Chih Chen
d
,
Che-Ming Teng
e
, Shiow-Lin Pan
e
, Chien-Huang Lin
a
,
∗
aGraduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan bDepartment of Pharmacology, College of Medicine, Taipei Medical University, Taipei, Taiwan
cSchool of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan dNational Institute of Chinese Medicine, Taipei, Taiwan
ePharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan Received 9 August 2007; received in revised form 15 December 2007; accepted 15 December 2007
Available online 28 December 2007
Abstract
Increasing evidence demonstrated that denbinobin, isolated from Ephemerantha lonchophylla, exert cytotoxic effects in cancer cells. The purpose
of this study was to investigate whether denbinobin induces apoptosis and the apoptotic mechanism of denbinobin in human lung adenocarcinoma
cells (A549). Denbinobin (1–20
M) caused cell death in a concentration-dependent manner. Flow cytometric analysis and annexin V labeling
demonstrated that denbinobin increased the percentage of apoptotic cells. A549 cells treated with denbinobin showed typical characteristics of
apoptosis including morphological changes and DNA fragmentation. Denbinobin induced caspase 3 activation, and
N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, prevented denbinobin-induced cell death. Denbinobin induced the
loss of the mitochondrial membrane potential and the release of mitochondrial apoptotic proteins including cytochrome c, second mitochondria
derived activator of caspase (Smac), and apoptosis-inducing factor (AIF). In addition, denbinobin-induced Bad activation was accompanied by the
dissociation of Bad with 14-3-3 and the association of Bad with Bcl-xL. Furthermore, denbinobin induced Akt inactivation in a time-dependent
manner. Transfection of A549 cells with both wild-type and constitutively active Akt significantly suppressed denbinobin-induced Bad activation
and cell apoptosis. These results suggest that Akt inactivation, followed by Bad activation, mitochondrial dysfunction, caspase 3 activation, and
AIF release, contributes to denbinobin-induced cell apoptosis.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Denbinobin; Apoptosis; Akt; Bad; Lung adenocarcinoma cell
Abbreviations: AIF, apoptosis-inducing factor; BCIP, 5-bromo-4-chloro-3-indolyl-phosphate; Apaf1, apoptosis-activating factor 1; DiOC6, 3,3-dihexyloxacarbocyanine; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; FCS, fetal calf serum; IAPs, inhibitors of apoptosis pro-teins; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NBT, 4-nitro blue tetrazolium; NP-40, Nonidet P-40; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; PI, propidium iodide; PI3K, phosphoinositide-3-OH-kinase; PMSF, phenylmethylsulphonyl fluoride; SDS, sodium dodecylsulfate; Smac, second mitochondria-derived activator of caspase; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling; zVAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone.
∗Corresponding author. Tel.: +886 2 27361661x7100; fax: +886 2 27399145.
E-mail address:[email protected](C.-H. Lin).
1. Introduction
Lung cancer is a major cause of cancer-related death
world-wide. Adenocarcinoma is the most common type, making up
30–40% of lung cancer. The curative treatments for lung cancer
include surgery, radiation therapy or chemotherapy depending
on the cancer stage. However, the therapeutic outcome,
includ-ing survival and relapse rate, is disappointinclud-ing. Thus, numerous
researchers devoted their efforts to the development of novel
therapeutic agents inducing lung cancer cell death.
Denbinobin
(5-hydroxy-3,7-dimethoxy-1,4-phenanthraqui-none), a compound extracted and purified from several
Dendro-bium or Ephemerantha (Orchidaceae) species, such as D. nobile
(
Lee et al., 1995
), D. moniliforme (
Lin et al., 2001
), and E.
lon-chophylla (
Chen et al., 1999, 2000
), was recently demonstrated
0378-4274/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.to induce cell death in several cancer cell lines including lung
adenocarcinoma cells (A549), ovary adenocarcinoma cells
(SK-OV-3), promyelocytic leukemia cells (HL60), human leukemia
cells (K562), and colon adenocarcinoma cells (COLO 205) (
Lee
et al., 1995; Huang et al., 2005; Yang et al., 2005
).
Denbi-nobin has also been reported to have other biological effects
such as antiplatelet aggregation (
Chen et al., 2000
),
antioxida-tion (
Chen et al., 1999
), and anti-inflammation (
Lin et al., 2001
).
Alterations of tubulin polymerization and Bcr–Abl activity have
been shown to contribute to denbinobin-induced cell death in
human K562 leukemia cells (
Huang et al., 2005
). However, the
precise molecular mechanism of denbinobin-induced cell death
and whether debinobin induces cell apoptosis has not been fully
delineated.
Cell death by apoptosis plays a critical role in both the
nor-mal development and pathology of a wide variety of tissues
(
Jacobson et al., 1997; Nagata, 1997
). Initiation of apoptosis is
controlled by regulation of the balance between the death and
survival signals perceived by a cell (
Musci et al., 1997; Wang
et al., 1999
). The characterization of survival signal
transduc-tion pathways stimulated by various growth factors has revealed
that phosphoinositide-3-OH-kinase (PI3K) is involved in
pro-tecting cells from undergoing apoptotic cell death (
Jung et al.,
2000; Mathieu et al., 2001
). The prominent target of PI3K is the
serine/threonine kinase, Akt, also termed protein kinase B. Akt
mediates many PI3K-regulated biological responses including
glucose uptake, protein synthesis, and inhibition of apoptosis
(
Cong et al., 1997; Cichy et al., 1998; Mathieu et al., 2001;
Dijkers et al., 2002
). Ectopic expression of Akt, especially
constitutively active Akt, induces cell survival and malignant
transformation, whereas inhibition of Akt activity stimulates
apoptosis in a range of mammalian cells (
Goswami et al., 1999
).
With stimulation by growth factors or cytokines, Akt is
phos-phorylated at two key regulatory sites, threonine
308(Thr
308) and
serine
473(Ser
473) (
Alessi et al., 1996
). Fully activated Akt, in
turn, functions to promote cell survival by phosphorylating
sev-eral downstream targets including the Bcl-2 family member Bad,
IB kinase, caspase family member caspase-9, and forkhead
family transcription factor FKHRL1 (
Datta et al., 1997; del Peso
et al., 1997; Brunet et al., 1999; Fujita et al., 1999; Ozes et al.,
1999; Pastorino et al., 1999; Dijkers et al., 2002
).
Bad is a member of 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 the mitochondria from
other cellular compartments, leading to cell death by apoptosis
(
Pastorino et al., 1999; Huang and Strasser, 2000
). Bad is capable
of forming heterodimers with the antiapoptotic proteins, Bcl-xL
and Bcl-2, and antagonizes their antiapoptotic activity
result-ing in mitochondrial permeabilization (
Zha et al., 1996, 1997;
Kelekar et al., 1997
). Subsequently, mitochondrial apoptogenic
proteins, including cytochrome c, the second
mitochondria-derived activator of caspase (Smac), and apoptosis-inducing
factor (AIF), are released into the cytosol, leading to caspase
activation and eventual cell death (
Kluck et al., 1997; Lorenzo
et al., 1999; Du et al., 2000
). Cytochrome c triggers caspase
activation through interactions with apoptosis-activating factor
1 (Apaf1) (
Hu et al., 1998
). Smac relieves inhibitors of
apop-tosis proteins (IAPs) from inhibiting caspases (
Du et al., 2000;
Wu et al., 2000
), thus ensuring the caspase-dependent pathway.
However, the mitochondrial release of AIF causes nuclear
chro-matin condensation and fragmentation in a caspase-independent
manner (
Lorenzo et al., 1999
). The preferential release of select
proapoptotic proteins from mitochondria might be subject to
dif-ferential upstream regulation. The Akt cascade, via its control of
Bad activity, has emerged as an important regulatory mechanism
upstream of mitochondria in the maintenance of cell viability
(
Datta et al., 1997
).
In the present study, we explored the roles of Akt and Bad in
denbinobin-induced mitochondrial dysfunction and cell death
in human lung adenocarcinoma cells (A549). We demonstrated
that the Akt inactivation, followed by Bad activation,
mitochon-drial dysfunction, mitochonmitochon-drial molecules such as cytochrome
c, smac, and AIF release, and caspase 3 activation contributes
to denbinobin-induced cell apoptosis.
2. Materials and methods
2.1. Materials
Denbinobin was kindly provided by Dr. Chien-Chih Chen (National Research Institute of Chinese Medicine, Taipei, Taiwan). The methods of extrac-tion and isolaextrac-tion of denbinobin were reported previously (Chen et al., 2000), and the purity is over 98% based on the HPLC analysis. Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12, fetal calf serum (FCS), penicillin/streptomycin, OptiMEM, and lipofectamin plusTMreagent were purchased from Invitrogen (Carlsbad, CA). Antibodies specific for Bcl-2, Bax, and procaspase 3 were pur-chased from Transduction Laboratories (Lexington, KY). Protein A/G beads, antibodies specific for cytochrome c, Smac, AIF, Bcl-xL, poly (ADP-ribose) polymerase (PARP), Bad, phospho-Bad (Ser136), 14-3-3 and Akt, as well as horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An antibody specific for phospho-Akt (Ser473) was purchased from New England Biolabs (Beverly, MA). Anti-mouse IgG-conjugated alkaline phosphatase or flourescein isothiocyanate (FITC) was purchased from Jackson Immuno Research Labo-ratories (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 SDS-PAGE were obtained from Bio-Rad (Hercules, CA). The pUSEamp-Akt1 cDNA (wild-type Akt, wt-Akt), and pUSEamp-myr-Akt1 cDNA (constitutively active Akt, myr-Akt) were purchased from Upstate Biotechnology (Lake Placid, NY). 3,3-Dihexyloxacarbocyanine (DiOC6) was purchased from Molecular Probes (Eugene, OR, USA). Annexin V-FITC apoptosis detection kit and cas-pase 3/CPP32 colormetric assay kit were purchased from BioVision (Mountain View, CA). A TdT Fragel DNA fragmentation detection kit was purchased from Oncogene Research Products (San Diego, CA). All materials for agarose gel electrophoresis and [␥-32P] ATP were obtained from GE Healthcare (Little Chalfont, UK). Propidium iodide (PI), N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), dithiothreitol (DTT), phenylmethylsulphonyl fluoride (PMSF), pepstatin A, leupeptin, sodium dodecylsulfate (SDS), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium (MTT), and other chemicals were obtained from Sigma (St. Louis, MO).
2.2. Cell culture
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 100g/ml streptomycin). Cells were cultured at 37◦C in a humid-ified 5% CO2atmosphere.
2.3. Cell viability assay
Cell viability was measured by a previously described colorimetric MTT assay (Goswami et al., 1999; Chen et al., 2002). Briefly, cells (2× 105cells/well) were cultured in 24-well plates and incubated with vehicle or various concentra-tions (1, 3, 10, 20M) of denbinobin for 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 500l 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.
2.4. Flow cytometric analysis
A549 cells were cultured in 10-cm Petri dishes. After reaching confluence, cells were treated with vehicle or 20M denbinobin for 12, 24, 36, or 48 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 25g/ml PI for 30 min in the dark before the flow cytometric analysis. Samples were ana-lyzed by FACScan and the Cellquest program (Becton Dickinson, San Jose, CA).
2.5. Annexin V-propidium iodide staining
Apoptosis was assessed using annexin V, a protein that binds to phos-phatidylserine (PS) residues which are exposed on the cell surface of apoptotic cells, as previously described (Dijkers et al., 2002). Cells were treated with vehicle or 20M denbinobin for 12, 24, 36, or 48 h. After treatment, A549 cells were washed twice with PBS (pH 7.4), and resuspended in staining buffer containing 1g/ml PI and 0.025 g/ml annexin V-FITC. Double-labeling was performed at room temperature for 10 min in the dark before the flow cytomet-ric analysis. Cells were immediately analyzed using FACScan and the Cellquest program (Becton Dickinson). Viable cells were determined from non-apoptotic and non-necrotic cell populations.
2.6. DNA fragmentation
Genomic DNA was isolated as described previously (Chen et al., 2002). Briefly, cells were treated with vehicle, denbinobin (1, 3, or 20M) or tumor necrosis factor-␣ (TNF-␣, 100 ng/ml) for 24 h or 48 h. After treatment, cells were washed with PBS and lysed in cell lysis buffer containing 50 mM Tris–HCl (pH 7.4), 10 mM EDTA, 0.2% Triton X-100, 1 mM PMSF, 0.1 mM aprotinin, 1 mM leupeptin, and 0.5 mg/ml proteinase K. The supernatant was incubated with RNase A (0.2 mg/ml) at 37◦C for 2 h. An equal volume of phenol/chloroform was then added with intermittent gentle agitation for 10 min. Following cen-trifugation at 12,000× g for 20 min, DNA in the upper phase was collected. Then the genomic DNA was precipitated by adding sodium acetate (0.3 M, pH 5.4) and 99.9% ethanol. The DNA pellet was washed with 70% (v/v) ethanol and dissolved in Tris–EDTA buffer. The DNA concentration was determined at 260 nm by spectrophotometry. DNA sample, of∼0.2 mg each, were elec-trophoresed on a 2% (w/v) agarose gel containing 0.5g/ml ethidium bromide. DNA fragmentation bands were photographed under UV light.
2.7. The terminal deoxynucleotidyl transferase (TdT)-mediated
dUTP nick end labeling (TUNEL) assay
A549 cells were plated on glass cover slides. Cells were then treated with vehicle, 20M denbinobin or 100 ng/ml TNF-␣ for 24 h. After treat-ment, cells were fixed with freshly prepared paraformaldehyde (4% in PBS, pH 7.4). The slides were rehydrated in PBS and incubated in 0.3% (v/v) H2O2 in methanol for 30 min to block endogenous peroxidase. DNA nicks were determined using an in situ TdT-FragEL DNA fragmentation detection
kit (Oncogene Research, Cambridge, MA). Briefly, the specimens were covered for 1.5 h at 37◦C with the TUNEL reaction mixture containing TdT enzyme solution and label solution (modified nucleotide mixture in reaction buffer). The reaction was terminated by washing with PBS, and the slides covered with Converter-POD (streptavidin conjugated with horseradish peroxidase, POD) for 30 min and visualized using 3,3-diaminobenzidine for 10 min. Counterstaining was performed with 5% (w/v) methyl green in a 0.1 M sodium acetate solu-tion (pH 4.0) for 2 min, after which the stained cells were analyzed by light microscopy.
2.8. Determination of the mitochondrial membrane potential
The mitochondrial membrane potential was assessed using a fluorometric probe, DiOC6 (Molecular Probes), with a positive charge of a mitochondrial-specific fluorophore, as previously described (Susin et al., 1997; Ye et al., 1999). Briefly, A549 cells were plated in 6-well culture dishes. After reaching conflu-ence, cells were treated with vehicle or 20M denbinobin for 12 h or 24 h. After incubation, cells were stained with DiOC6 (40 nM) for 15 min at 37◦C. Cells were collected, washed twice in PBS, and analyzed by FACScan flow cytometry. The probes were excited with a laser at 488 nm, and emission was monitored through a 530-nm bandpass filter. At least 10,000 cells were analyzed per sample.
2.9. Immunoblot analysis
To determine the levels of procaspase 3, PARP, Bcl-xL, Bad, phospho-Bad (Ser136) Bax,␣-tubulin, phospho-Akt (Ser473), and Akt in A549 cells, the proteins were extracted as previously described (Chang et al., 2002), with modifications. Briefly, A549 cells were cultured in 6-cm dishes. After reach-ing confluence, cells were treated with vehicle or 20M denbinobin for 12, 24, 36, or 48 h. 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 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 amount of protein (60g) in each sample were boiled in SDS sample loading buffer, and then fractionated on SDS-PAGE before blotting 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 anti-bodies. After washing with PBS, blots were developed using NBT/BCIP or an enhanced chemiluminescence kit according to the vendor’s instruction before exposure to photographic film.
2.10. Isolation of the cytosolic fraction
The procedure for isolation of the cytosolic fraction was previously described (Pastorino et al., 1999). Briefly, cells were lysed with 40 strokes of a Wheaten Dounce glass homogenizer (type B pestle) in ice-cold lysis buffer (250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1.5 mM MgCl2, 10 mM KCl, 20 mM HEPES (pH 7.4), 0.4M aprotinin, and 10 M leupeptin). The lysate was centrifuged at 600× g for 10 min at 4◦C to remove nuclei and unbroken cells. The supernatant was removed and centrifuged at 14,000× g for 15 min at 4◦C to eliminate mitochondria. The supernatant was then re-centrifuged at 100,000× g for 1 h at 4◦C. The protein concentration of the resulting super-natant, which represented the cytosolic fraction, was assayed by the Bradford protein assay (Bio-Rad). Equal amount of total protein (20g) in each sample was boiled in SDS sample loading buffer and then fractionated on SDS-PAGE. The protein levels of cytochrome c, Smac, AIF and␣-tubulin in the cytosolic fraction were then analyzed by immunoblotting.
2.11. Caspase 3 activity assay
Caspase 3 activity was determined using caspase 3/CPP32 colormetric assay kit (BioVision). Briefly, A549 cells were cultured in 6-cm dishes. After reaching confluence, cells were treated with vehicle or 20M denbinobin for 24 h or 48 h.
Fig. 1. Denbinobin-induced A549 cell death. (A) Cells were treated with vehicle or denbinobin (1–20M) for 24 h, and cell viability was determined by the MTT assay. Each column represents the mean± S.E.M. of at least three independent experiments performed in duplicate.*p < 0.05, compared with the control group. The
percentage of apoptotic cells was also analyzed by flow cytometric analysis of annexin V/PI double staining (B) and PI staining (C) as described in Section2. Data are representative of three independent experiments with similar results. Compiled results are shown in the graph. Apo, apoptotic region.
Cells were harvested, and then cell lysates (100g) were incubated with DEVD-pNA (50M) for 2 h at 37◦C. The absorbance at 405 nm was measured on a microplate reader.
2.12. Immunofluorescence microscopy
A549 cells were grown on glass coverslips. After incubation in the absence or presence of 20M denbinobin for 24 h, cells were washed twice with PBS. Subsequently, cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were then permeabilized for 2 min in 0.02 % (v/v) Triton X-100, and incubated with 5% (v/v) bovine serum albumin (BSA) in PBS for 1 h before staining. To observe the distribution of AIF, cells were stained with an anti-AIF antibody for 2 h at room temperature. After washing with PBS containing 0.02% Tween-20, cells were incubated with FITC-labeled secondary antiserum for an additional 1 h at room temperature. Cells were then washed with PBS containing 0.02% Tween-20 and incu-bated with 20 nM PI for another 15 min at room temperature. After staining, cells were observed under inverted laser scanning confocal microscopy (Olym-pus).
2.13. Co-immunoprecipitation (Co-IP)
A549 cells were grown in 6-cm dishes. After reaching confluence, cells were treated with 20M denbinobin for 12, 24, or 36 h. Cells were harvested, lysed in 1 ml of PD buffer (40 mM Tris–HCl (pH 8.0), 500 mM NaCl, 0.1% NP-40,
6 mM EGTA, 10 mM-glycerophosphate, 10 mM NaF, 300 M Na3VO4, 2 mM PMSF, 10g/ml aprotinin, 1 g/ml leupeptin, and 1 mM DTT), and centrifuged. The supernatant was immunoprecipitated overnight with specific antibodies against Bad in the presence of protein A/G-agarose beads at 4◦C. The immuno-precipitated complex was washed three times with PD buffer. Samples were fractionated on 15% SDS-PAGE, transferred to a PVDF membrane and sub-jected to immunoblotting with antibodies specific for phospho-Bad (Ser136), 14-3-3 or Bcl-xL.
2.14. Measurement of Akt kinase activity
A549 cells were grown in 6-cm dishes. After reaching confluence, cells were treated with 20M denbinobin for 6, 12, or 24 h. After incubation, cells were washed twice with ice-cold phosphate-buffered saline, 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, 10g/ml leupeptin, 10 g/ml aprotinin, 25 mM -glycerophosphate, 50 mM NaF, and 100 M Na3VO4, and centrifuged at 4◦C and 12,000× g for 30 min. The supernatant was then immunoprecipitated with a polyclonal antibody against Akt 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 immunoprecip-itated beads with 20l of kinase buffer supplemented with 50 g/ml of histone 2B (H2B), 20M ATP, and 3 Ci of [␥-32P] ATP at 30◦C for 30 min. The reaction mixtures were analyzed by 15% SDS-PAGE followed by autoradiog-raphy.
2.15. Plasmid DNA transfection
A549 cells were seeded at a density of 2× 105cells/ml into 12-well plates. Cells were transfected on the following day with the Lipofectamine plusTM reagent containing 1g/well of pUSEamp (mock), pUSEamp-Akt1 (wt-Akt), or pUSEamp-myr-Akt1 (myr-Akt) cDNA 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 20M denbinobin for another 24 h before harvesting.
2.16. Statistical analysis
Results are presented as the mean± S.E.M. from at least three independent experiments. One-way analysis of variance, 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.
3. Results
3.1. Induction of cell apoptosis by denbinobin
Treatment of A549 cells for 24 h with denbinobin (1–20
M)
decreased cell viability in a concentration-dependent manner.
Denbinobin at 10
M and 20 M significantly decreased the
cell viability by 48.4
± 13.8% and 56.1 ± 11.0%, respectively
(n = 4) (
Fig. 1
A). We next investigated whether denbinobin
induces cell death through an apoptotic mechanism. Annexin
V-PI double-labeling was used for the detection of PS
external-ization, a hallmark of early phase of apoptosis. As compared
to vehicle-treated cells, a high proportion of annexin V
+label-ing was detected in cells treated with 20
M denbinobin for
12, 24, 36, or 48 h (
Fig. 1
B), and two levels of labeling were
observed: annexin V
+cells which remained PI
−(lower right
quadrant), corresponding to early apoptotic cells, and annexin
V
+/PI
+cells, corresponding to advanced apoptotic cells. In
addi-tion, cells were unaffected by the treatment with vehicle for 12,
24, 36, or 48 h (data not shown). Flowcytometry analysis of
PI-stained cells was also used to further confirm that denbinobin
induces cell death by apoptosis. Similar to
Fig. 1
B, the
percent-age of PI-stained cells in the apoptotic region (Apo, sub-G0/G1
peak) time-dependently increased following 20
M denbinobin
treatment. The ratio of apoptotic cells was significantly increased
by 16.3
± 2.4%, 32.1 ± 2.0%, and 38.5 ± 2.2% after denbinobin
exposure for 24, 36, and 48 h, respectively (
Fig. 1
C). It appears
that cells treated with vehicle for 12, 24, 36 or 48 h show no
difference (data not shown).
Degradation of DNA into a specific fragmentation pattern
(consisting of DNA ladders) is a characteristic feature of
apopto-sis. After 24 h or 48 h of exposure to denbinobin (1, 3, or 20
M),
the genomic DNA from A549 cells was subjected to agarose gel
electrophoresis. As shown in
Fig. 2
A, clear DNA
fragmenta-tion ladders were detected in samples from cells treated with
denbinobin (
Fig. 2
A, upper panel, lanes 5, and 7–9) as
com-pared to vehicle-treated cells (
Fig. 2
A, upper panel, Lanes 2
and 6). DNA fragmentation ladders were also shown in cells
treated with TNF-␣ (100 ng/ml) for 24 h (
Fig. 2
A, bottom panel,
Lane 3) as compared to vehicle-treated cells (
Fig. 2
A, bottom
panel, Lane 2). DNA laddering marker (M) was also shown in
Fig. 2
A. The TUNEL assay was also used to stain nuclei that
con-Fig. 2. Denbinobin-induced apoptosis in A549 cells. (A) Cells were treated with vehicle or denbinobin (1–20M) for 24 h or 48 h. DNA fragmentation was then evaluated as described in Section2. Lane ‘M’ indicates the DNA laddering marker. Data are representative of three independent experiments with similar results. (B) Cells were treated with vehicle (a), 20M denbinobin (b), or 100 ng/ml TNF-␣ (c) for 24 h; TUNEL-positive cells were visualized using a peroxidase–substrate system as having condensed nuclei indicated by arrow. Data are representative of three independent experiments with similar results.
tained nick-ended DNA, a characteristic exhibited by apoptotic
cells. Vehicle-treated cells were almost completely negative for
TUNEL staining (
Fig. 2
B, a). In contrast, following exposure to
20
M denbinobin for 24 h, a large number of TUNEL-positive
cells were observed (
Fig. 2
B, b). TUNEL-positive cells were
also seen following 100 ng/ml TNF-␣ treatment, considered to
be a positive control group (
Fig. 2
B, c). Taken together, these
results suggested that denbinobin-induced A549 cells death by
apoptotic mechanism.
3.2. Denbinobin induced caspase activation and PARP
cleavage
We next attempted to determine whether caspase
activa-tion contributes to denbinobin-induced A549 cell apoptosis.
As shown in
Fig. 3
A, zVAD-fmk (100
M), a broad-spectrum
Fig. 3. Involvement of caspase activation in denbinobin-induced cell death in A549 cells. (A) Cells were pretreated with vehicle or zVAD-fmk (100M) for 4 h before the addition with denbinobin (20M) for an additional 24 h. Cell viability was then determined by the MTT assay. Each column represents the mean± S.E.M. of at least three independent experiments performed in tripli-cate.*p < 0.05, compared with the group treated with denbinobin alone. (B) Cells were treated with vehicle or denbinobin (20M) for the indicated time intervals. Procaspase-3 and PARP levels were then determined by immunoblot-ting as described in Section2. Typical traces representative of three independent experiments with similar results are shown. (C) Cells were treated with vehicle or 20M denbinobin for 24 h and 48 h. After treatment, the caspase 3 activity was assessed as described in Section2. Each column represents the mean± S.E.M. of at least three independent experiments.*p < 0.05, compared with the control group.
caspase inhibitor, significantly inhibited the decrease of cell
via-bility after exposure to 20
M denbinobin for 24 h. Caspase 3
has been shown to lay downstream of the apoptotic signaling
pathway regardless of whether intrinsic- or extrinsic-mediated
apoptotic signaling occurs (
Cohen, 1997; Susin et al., 1997
).
We then examined whether denbinobin is able to induce
procas-pase 3 degradation leading to casprocas-pase 3 activation. Denbinobin
(20
M) time-dependently induced procaspase 3 degradation
(
Fig. 3
B, upper panel). A specific caspase 3 substrate, PARP, was
then used to confirm that denbinobin activates caspase 3
result-ing in PARP cleavage (
Cohen, 1997; Nagata, 1997; Boulares
et al., 1999
). As shown in
Fig. 3
B (bottom panel), denbinobin
induced the cleavage of PARP from 115 to 85-kDa fragment. In
addition, caspase 3 activity is markedly increased in cells after
exposure to denbinobin (20
M) for 24 h or 48 h (
Fig. 3
C). These
results suggested that caspase 3 is involved, at least in part, in
denbinobin-induced A549 cell death.
3.3. Denbinobin caused mitochondrial dysfunction and
release of cytochrome c, Smac, and AIF
To explore whether denbinobin-induced cell apoptosis is
mediated through mitochondrial dysfunction, we determined
the mitochondrial membrane potential with the
mitochondria-sensitive dye, DiOC6, using flow cytometry. As shown in
Fig. 4
A, treatment of A549 cells with 20
M denbinobin for
12 h and 24 h induced the loss of the mitochondrial
mem-brane potential in a time-dependent manner. Several molecules
Fig. 4. Denbinobin induced the loss of mitochondrial membrane potential and the release of cytochrome c, Smac, and AIF in A549 cells. (A) Cells were treated with vehicle or 20M denbinobin for 12 h and 24 h. After treatment, the mitochondrial membrane potential was assessed as described in Section2. Data are representative of three independent experiments with similar results. (B) Cells were treated with vehicle or denbinobin (20M) for the indicated time intervals. After treatment, the cytosolic fraction was collected and subjected to immunoblotting. Data are representative of three independent experiments with similar results. (C) Cells were treated with vehicle or 20M denbinobin for 24 h. After treatment, AIF (green) translocation was determined using Olympus confocal microscopy as described in Section2. The red nuclei counter-stained with PI indicate nuclear localization. Data are representative of three independent experiments with similar results. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
including cytochrome c, Smac, and AIF are known to be
liberated from mitochondria and to induce apoptosis via
caspase-dependent and caspase-incaspase-dependent pathways (
Saleh et al.,
1999; Daugas et al., 2000; Du et al., 2000; Murahashi et al.,
2003
). To establish whether denbinobin induces cytochrome
c, Smac, or AIF protein release from mitochondria to the
cytosol, the levels of these proteins in the cytosolic fraction
were determined using Western blotting analysis. As shown in
Fig. 4
B, denbinobin caused a marked increase in the
cytoso-lic levels of cytochrome c, Smac, and AIF in A549 cells.
Cytochrome c and Smac have been reported to induce
apop-tosis via caspase-dependent pathways (
Du et al., 2000
). In
contrast, the mitochondrial death-executing molecule, AIF, can
translocate to the nucleus and induce caspase-independent cell
death upon apoptotic stimuli (
Lorenzo et al., 1999
). We thus
investigated whether AIF was translocated to the nucleus after
denbinobin exposure in A549 cells. Results from a
confo-cal microscopic analysis indicated that AIF immunoreactivity
(shown in green fluorescence) and nuclear staining (PI staining,
shown in red fluorescence) were co-localized in
denbinobin-treated A549 cells, whereas those of undenbinobin-treated A549 cells were
mutually exclusively localized (
Fig. 4
C). These findings suggest
that AIF is also involved in denbinobin-induced cell apoptosis
in A549 cells.
3.4. Denbinobin caused Bad activation and the association
of Bad and Bcl-xL
Bcl-2 family proteins regulate mitochondria-dependent
apoptosis with the balance of the anti- and pro-apoptotic
mem-bers arbitrating life-or-death decisions (
Tsujimoto and Shimizu,
2000; Tsujimoto, 2002
). Thus, we examined whether the
pro-tein levels of Bcl-2 family propro-teins are altered after denbinobin
exposure. Treatment of A549 cells with 20
M denbinobin for
12, 24, 36, or 48 h did not alter the protein levels of Bad, Bax,
or Bcl-xL (data not shown). Next, we examined the effects
of denbinobin on the phosphorylation status of Bad, which
is crucial for activation of caspase 3 through a
mitochondria-dependent pathway (
Kennedy et al., 1999
). Bad was highly
phosphorylated at Ser
136in untreated A549 cells, but it was
dephosphorylated in a time-dependent manner after exposure
to 20
M denbinobin (
Fig. 5
, upper panel). The complied data
are shown in the bottom of
Fig. 5
. Under normal
physiolog-ical conditions, Bad, in both its phosphorylated and inactive
form, is known to exist almost exclusively in the cytoplasm
(
Cross et al., 1995
). The interaction of Bad phosphorylated at
Ser
136and 14-3-3 avoided the proapoptotic function of Bad
by translocation from the cytosol to the mitochondrial
outer-membrane (
Zha et al., 1996, 1997
). Activated Bad, recognized
as an essential initiator of the apoptotic cascade, is capable of
forming heterodimers with the anti-apoptotic mitochondrial
pro-tein, Bcl-xL
,and antagonizes its antiapoptotic activity (
Kelekar
et al., 1997; Kelekar and Thompson, 1998
). Therefore, a
co-immunoprecipitation analysis was employed to confirm the
hypothesis that denbinobin-induced Bad Ser
136dephosphory-lation was accompanied by dissociation of the Bad-14-3-3
complex. As shown in
Fig. 6
, denbinobin (20
M) induced
Fig. 5. Denbinobin induced Bad dephosphorylation and the association with Bcl-xL in A549 cells. (A) Cells were treated with 20M denbinobin for the indi-cated time intervals, then immunoprecipitated with the anti-Bad antibody. The immunoprecipitated complex was subjected to immunoblotting with antibodies against phospho-Bad (Ser136), 14-3-3, or Bcl-xL. Each column represents the mean± S.E.M. of at least three independent experiments.*p < 0.05, compared with the control group. IP, immunoprecipitation; IB, immunoblotting.
the dissociation of Bad from 14-3-3. In addition, Bad-14-3-3
complex dissociation was accompanied by the association of
Bad and Bcl-xL (
Fig. 5
). The complied data are shown in the
bottom of
Fig. 5
. The maximal response was seen after 24 h
exposure to denbinobin. These results suggest that Bad
dephos-phorylation followed by the dissociation of Bad from 14-3-3
and the subsequent association of Bad and Bcl-xL is involved in
denbinobin-induced mitochondrial dysfunction.
3.5. Akt inactivation is involved in denbinobin-induced Bad
activation and subsequent cell apoptosis
We thus attempted to elucidate the signaling cascade
lead-ing to Bad dephosphorylation. It has been reported that Bad
phosphorylation at Ser
136is induced by the PI3K/Akt
path-way (
Pastorino et al., 1999
). Since serine phosphorylation of
residue 473 in Akt causes enzymatic activation (
Alessi et al.,
1996
), the antibody specific against phosphorylated Akt (Ser
473)
was used to examine Akt phosphorylation, an index of kinase
activation. Denbinobin (20
M) caused significant Akt Ser
473dephosphorylation after 12 h of treatment (
Fig. 6
A, upper panel).
The protein level of Akt was not affected by the presence of
denbinobin. The complied data are shown in the bottom of
Fig. 6
A. In parallel, using histone H2B as an Akt substrate,
a time-dependent decrease in Akt kinase activity was observed
Fig. 6. Denbinobin-induced Akt inactivation in A549 cells. Cells were treated with 20M denbinobin for the indicated time intervals. Akt dephosphorylation (A) and kinase activity (B) were then assessed as described in Section2. Each column represents the mean± S.E.M. of at least three independent experiments. *p < 0.05, compared with the control group.
in 20
M denbinobin-treated A549 cells (
Fig. 6
B). The
com-plied data are shown in the bottom of
Fig. 6
B. To further
confirm whether Akt inactivation contributes to
denbinobin-induced cell apoptosis, A549 cells were transiently transfected
with empty (mock), wild-type Akt (wt-Akt) or constitutively
active Akt (myr-Akt) prior to denbinobin (20
M) treatment
for 24 h. As compared to the control group (mock-transfected
cells), cells transfected with wt-Akt or myr-Akt significantly
inhibited denbinobin-decreased cell viability by 33.3
± 3.1% or
18.5
± 4.0%, respectively (
Fig. 7
A). In addition, flowcytometric
analysis demonstrated that transfection with wt-Akt or
myr-Akt attenuated cell apoptosis after exposure to denbinobin for
24 h with the extent of cell apoptosis reduced from 48.9
± 4.1%
(denbinobin plus mock) to 28.4
± 8.1% (denbinobin plus
wt-Akt) and 30.4
± 5.0% (denbinobin plus myr-Akt), respectively
(
Fig. 7
B). To ascertain the linkage between Akt inactivation and
Bad activation caused by denbinobin, we examined the effects
of wt-Akt and myr-Akt on denbinobin (20
M)-induced Bad
Ser
136dephosphorylation. After transfection, Bad was
immuno-precipitated (IP) and the status of Bad phosphorylation at Ser
136was determined by immunoblotting using specific antibodies
against Bad Ser
136. Imunoprecipitation using normal IgG is
served as negative control. As shown in
Fig. 7
C, transfection
with either wt-Akt or myr-Akt remarkably reversed the Bad
Ser
136dephosphorylation after exposure to denbinobin for 12 h.
Results of these experiments further imply a causal role of Akt in
regulating denbinobin-induced Bad inactivation and A549 cell
apoptosis.
Fig. 7. Involvement of Akt inactivation in denbinobin-induced Bad dephospho-rylation and cell apoptosis. Cells were transfected with the control vector (mock transfection), wild-type Akt (wt-Akt), or constitutively active Akt (Myr-Akt). Following transfection, cells were treated with the vehicle or 20M denbinobin for another 24 h, and cell viability (A) and apoptosis (B) were determined by the MTT assay and flowcytometry as described in Section2. Each column rep-resents the mean± S.E.M. of at least three independent experiments*p < 0.05, compared with the mock transfection group.#p < 0.05, compared with the mock transfection group in the presence of denbinobin. (C) Following the treatment as described above, cells were harvested and immunoprecipitated with an anti-Bad antibody. The immunoprecipitated complex was then used to determine the level of Bad phosphorylation by immunoblotting using the anti-phospho-Bad (Ser136) antibody. Typical bands representative of three separate experiments with similar results are shown. Equal loading in each lane is reflected by similar intensities of Bad in the lower panel.
4. Discussion
Apoptosis, a form of programmed cell death involved in tissue
morphogenesis and homeostasis, is characterized by
cytoplas-mic shrinkage, nuclear condensation and DNA fragmentation
(
Jacobson et al., 1997; Nagata, 1997
). Specific therapeutic policy
designed to improve or decrease the susceptibility of individual
cell types for undergoing apoptosis could form the basis for
treat-ments of a variety of human diseases, such as cancer (
Thompson,
1995
).
Denbinobin is a newly bioactive extract purified from
Chi-nese herbal plant, Shi-Hu, which was used in oriental medicine
to cure lung disease. Thus, denbinobin was suggested to affect
lung adenocarcinoma cell viability. In the present study, we
demonstrated that denbinobin induced apoptosis in lung
adeno-carcinoma A549 cell. The apoptotic mechanism of denbinobin
shown in this study involves the inactivation of Akt and Bad
dephosphorylation, resulting in mitochondrial dysfunction.
Mitochondrial dysfunction has been implicated as being a key
mechanism in apoptosis in various cell death paradigms (
Susin
et al., 1997
). Two major events have been noted in apoptosis
involving mitochondrial dysfunction. One event is the change in
the membrane permeability and subsequent loss of membrane
potential (
Zamzami et al., 1996, 1998
). The other is the release of
apoptotic proteins including AIF, Smac, and cytochrome c from
the intermembrane space of mitochondria into the cytosol (
Liu
et al., 1996; Green and Reed, 1998; Zamzami et al., 1998; Susin
et al., 1999; Daugas et al., 2000; Du et al., 2000
). In agreement
of these observations, we noted that the denbinobin-induced loss
of mitochondrial membrane potential was accompanied by the
release of AIF, Smac, and cytochrome c. Thus, it is plausible
that mitochondrial dysfunction may be involved in
denbinobin-induced A549 cell apoptosis.
Downstream events after cytochrome c release involve a
cytosolic protein known as Apaf1. Apaf1, which functions as
a scaffold protein, may form a complex with cytochrome c and
pro-caspase 9 in the presence of dATP. This complex
forma-tion may result in caspase 9 activaforma-tion, which in turn cleaves
and activates caspase 3 (
Liu et al., 1996; Hu et al., 1998; Saleh
et al., 1999; Zou et al., 1999
). Caspase 3 plays a pivotal role
in numerous apoptotic cascades and is responsible for the
pro-teolytic cleavage of many key proteins involved in apoptosis
(
Cohen, 1997
). In addition, the released Smac, which binds
to a class of antiapoptotic proteins known as IAPs, thereby
neutralizes IAP activity to promote caspase activation and
apop-totic cell death (
Du et al., 2000
). In this study, we found that
treatment of A549 cells with denbinobin caused the release of
cytochrome c and Smac, and the activation of caspase 3, and
that zVAD-fmk, a broad-spectrum caspase inhibitor, partially
prevented denbinobin-induced cell death. These results suggest
that denbinobin might induce, at least in part, the release of
cytochrome c and Smac, which in turn mediates caspase 3
acti-vation, and ultimately leads to A549 cell apoptosis. In addition
to cytochrome c and Smac, AIF is also reported to induce the
degradation of nuclear DNA and subsequently cause cell death
(
Susin et al., 1999
). AIF-induced apoptosis was shown to occur
in a caspase-independent manner. Several reports further
indi-cated that translocation of AIF from mitochondria to the nucleus
occurs during apoptosis (
Daugas et al., 2000; Murahashi et al.,
2003
). In the present study, we noted that denbinobin-induced
A549 cell death could not completely be abolished by the
zVAD-fmk. Moreover, denbinobin induced mitochondrial AIF release
and translocation from the cytosol to nuclei of A549 cells. These
findings suggest that denbinobin-induced A549 cell apoptosis
may occur through both caspase-dependent and -independent
pathways downstream of mitochondrial dysfunction. However,
the link between AIF nuclear translocation and cell apoptotic
pathways remains to be established. Additional work is needed
to characterize the interrelationships between AIF
transloca-tion and apoptotic pathways in denbinobin-induced A549 cell
death.
Bcl-2 family proteins regulate mitochondria-dependent
apoptosis with the balance of anti- and pro-apoptotic members
arbitrating life-and-death decisions (
Tsujimoto and Shimizu,
2000; Adams and Cory, 2001
). However, denbinobin did not
affect the protein levels of Bad, Bax, or Bcl-xL. There is growing
evidence that a number of apoptotic stimuli cause Akt
inactiva-tion (
Luo et al., 2003
) Akt phosphorylates many proteins which
are known to regulate apoptosis (
Cross et al., 1995; Datta et
al., 1997; Brunet et al., 1999; Fujita et al., 1999; Ozes et al.,
1999
). Among these proteins, Bad, the BH3-only member of
Bcl-2 family proteins, is prominent (
Datta et al., 1997
;
del Peso
et al., 1997; Pastorino et al., 1999
). In the present study, we found
that denbinobin induced Akt inactivation and Bad
dephosphory-lation and that both the wild-type Akt and constitutively active
Akt diminished denbinobin-induced Bad dephosphorylation and
cell apoptosis. Furthermore, we also noted that the
denbinobin-induced dissociation of Bad from 14-3-3 was accompanied by
an association of Bad with Bcl-xL. The results suggest that
Akt inactivation, followed by Bad activation, the dissociation
of Bad from 14-3-3, and the subsequent association of Bad
and Bcl-xL, is involved in denbinobin-induced mitochondrial
dysfunction and cell apoptosis. Moreover, transfection of
wild-type Akt and constitutively active Akt only partially inhibited
denbinobin-induced cell apoptosis. Recent reports have
indi-cated that in addition to Bad, other BH3-only proteins, such as
Bid, also participate in denbinobin-induced cell death (
Yang et
al., 2005
). Therefore, the Akt-Bad signaling cascade may
par-tially contribute to the signaling cascade of denbinobin-induced
A549 cell death. In fact, we recently found that apoptosis
signal-regulating kinase 1 (ASK1), a pivotal mechanism in a broad
range of cell death paradigms (
Hatai et al., 2000; Matsuzawa
and Ichijo, 2001
), is also involved in the signaling pathway of
denbinobin-induced cell death (unpublished observations).
Together, these results establish a denbinobin-mediated death
signaling cascade in A549 cells involving Akt inactivation,
resulting in a series of cellular events. The downstream
path-way involves Bad dephosphorylation, dissociation of 14-3-3 and
Bad, and binding to Bcl-xL, resulting in disruption of the
mito-chondrial membrane and release of cytochrome c, Smac, and
AIF. Furthermore, the mechanism of denbinobin-induced A549
cell apoptosis may occur through both caspase-dependent and
-independent (AIF) pathways downstream of mitochondrial
dys-function (
Fig. 8
). Thus, denbinobin may be useful as a potential
Fig. 8. Schematic summary of apoptotic pathway involved in denbinobin-induced A549 cell apoptosis. Denbinobin inactivation of the Akt, leads to Bad dephosphorylation, mitochondrial dysfunction and subsequent in cell apoptosis. Approaches applied in the present studies are shown to support the causal role of each step in the cascade.
template for the development of better chemopreventive and/or
chemotherapeutic agents against lung cancer.
Acknowledgements
This work was supported by research grants
(NSC93-2314-B-038-014 and NSC94-2320-B-038-033) from the National
Science Council of Taiwan.
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