THE JOURNAL OF BIOLOGICAL CHEMISTRY

2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 280, No. 25, Issue of June 24, pp. 23758 23765, 2005
Printed in U.S.A.

Bcl-2 Rescues Ceramide- and Etoposide-induced Mitochondrial

S
Apoptosis through Blockage of Caspase-2 Activation*
Received for publication, October 29, 2004, and in revised form, February 17, 2005
Published, JBC Papers in Press, April 6, 2005, DOI 10.1074/jbc.M412292200

Chiou-Feng Lin, Chia-Ling Chen, Wen-Tsan Chang, Ming-Shiou Jan**, Li-Jin Hsu,
Ren-Huang Wu, Yi-Ting Fang, Ming-Jer Tang, Wen-Chang Chang, and Yee-Shin Lin
From the Departments of Microbiology and Immunology, Biochemistry, Physiology, and Pharmacology and the
Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan 701, Taiwan and the
**Department of Microbiology and Immunology, Chung-Shan Medical University, Taichung 402, Taiwan

During the process of apoptosis, there is, in general, a reduction of mitochondrial transmembrane potential (m) followed
by the release of cytochrome c, which binds to Apaf-1 and

* This work was supported by Grant 91-B-FA09-1-4 from the Ministry of Education (MOE) Program for Promoting Academic Excellence of
University (Taiwan). The costs of publication of this article were defrayed in part by the payment of page charges. This article must
therefore be hereby marked advertisement in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.

S The on-line version of this article (available at http://www.jbc.org)

contains supplemental Figs. S1S3.
A postdoctoral fellow supported by the National Health Research
Institutes, Taiwan, ROC (PD9403).
To whom correspondence should be addressed. Tel.: 886-6-235-3535
(ext. 5646); Fax: 886-6-208-2705; E-mail: yslin1@mail.ncku.edu.tw.

promotes caspase-9 and -3 activation (13). Bcl-2 family proteins serve as critical regulators of mitochondrial apoptosis,
functioning as either inhibitors or promoters of cell death (4).
Bcl-2 inhibits apoptosis by blocking cytochrome c release from
mitochondria (5) through prevention of channel formation,
which is mediated by proapoptotic Bax and Bid (6 8). A recent
study (9) indicated that, in healthy cells, Bcl-2 adopts a typical
tail-anchored topology. Induction of apoptosis by ceramide or
etoposide triggered a change of Bcl-2 to the multispanning
transmembrane topology. In addition to membrane topology,
Bcl-2 phosphorylation is required for its full anti-apoptotic
function (10, 11).
Caspase-2 acts upstream of mitochondria to promote cytochrome c release and apoptosis (1218), although caspase-2
may also act downstream of mitochondria (19, 20). One study in
the mechanisms of caspase-2 activation showed that caspase-2
complex formation occurs independently of an Apaf-1/apoptosome pathway and that the recruitment of caspase-2 into this
complex is sufficient to mediate its activation upstream of
mitochondria (21). A recent report (22) demonstrated that activation of caspase-2 occurs in the complex that contains the
p53-induced death-domain-containing protein and the adapter
protein RAIDD (RIP (ribosome-inactivating protein)-associated
ICH-1/CED-homologous protein with death domain). Increased
p53-induced death-domain-containing protein may result in
caspase-2 activation to regulate apoptosis induced by genotoxic
stress. Interestingly, Bcl-2 suppresses p53-dependent apoptosis that requires Bax and caspase-2 as essential apoptotic
mediators (23).
We previously demonstrated sequential caspase-2 and -8
activation upstream of mitochondria during ceramide- and etoposide-induced apoptosis (24). In the present study, the relationship between Bcl-2 and caspase-2 in mitochondrial apoptosis induced by ceramide and etoposide was investigated. Bcl-2
overexpression rescued ceramide- and etoposide-induced apoptosis (2532). Furthermore, ceramide caused Bcl-2 dysfunction through its dephosphorylation at serine 70 mediated by
protein phosphatase 2A (33). Chemotherapeutic etoposide
caused Bcl-2 cleavage, which led to cell apoptosis (34). In addition, protein phosphatase facilitated cells undergoing etoposide-induced apoptosis (35, 36). Using Bcl-2 short interfering
RNA or Bcl-2 inhibitor ethyl 2-amino-6-bromo-4-(1-cyano-2ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1),1 we

1
The abbreviations used are: HA14-1, ethyl 2-amino-6-bromo-4(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; MDCK,
Madin-Darby canine kidney; z, benzyloxycarbonyl; fmk, fluoromethyl
ketone; PI, propidium iodide; GFP, green fluorescent protein; EGFP,
enhanced GFP; siRNA, short interfering RNA; OA, okadaic acid; DAPI,
4,6-diamidino-2-phenylindole; OD, optical density.

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Recent studies indicate that caspase-2 is involved in

the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been
shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we
report a regulatory role of Bcl-2 on caspase-2 upstream
of mitochondria. Stress stimuli, including ceramide and
etoposide, caused caspase-2 activation, mitochondrial
damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell
line A549. When A549 cells were pretreated with the
caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected
with caspase-2 short interfering RNA, both ceramideand etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented
ceramide- and etoposide-induced caspase-2 activation
and mitochondrial apoptosis. Furthermore, caspase-2
was activated when A549 cells were introduced with
Bcl-2 short interfering RNA or were treated with Bcl-2
inhibitor, which provided direct evidence of a negative
regulatory effect of Bcl-2 on caspase-2. Cell survival was
observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability
transition pore and caspase-9 demonstrated that Bcl-2modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and
etoposide treatment. A protein phosphatase inhibitor,
okadaic acid, rescued Bcl-2 dephosphorylation and
blocked caspase-2 activation, mitochondrial damage,
and cell death. Taken together, ceramide and etoposide
induced mitochondria-mediated apoptosis by initiating
caspase-2 activation, which was, at least in part, regulated by Bcl-2.

Bcl-2 Modulates Caspase-2 Activation

23759

showed directly that Bcl-2 negatively regulated caspase-2.

Upon ceramide and etoposide stimulation, protein phosphatase-mediated Bcl-2 dephosphorylation led to activation of
caspase-2, mitochondrial damage, and apoptosis.
EXPERIMENTAL PROCEDURES

Cell Cultures and ReagentsThe human lung epithelial cell line

A549, Madin-Darby canine kidney (MDCK) cells, and their bcl-2 transfectants (A549-B2 and MDCK-B6) and vector controls (A549-P2 and
MDCK-C1) were provided by Dr. M. T. Lin, Department of Biochemistry, and Dr. M. J. Tang, Department of Physiology, National Cheng
Kung University, Taiwan. A549 and MDCK cells were cultured in
Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 0.05 mg/ml streptomycin. They
were maintained at 37 C in 5% CO2. Human prostate cancer DU145
cells were cultured in minimum essential medium supplemented with
10% fetal bovine serum. A549-B2, A549-P2, MDCK-B6, and MDCK-C1
cells were constructed using lipofection with a transfection reagent
(Lipofectamine 2000, Invitrogen) and then cultured in Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum and
500 g/ml G418 (Calbiochem). Bcl-2 cDNA constructed in expression
vector, pUSEamp, was purchased from Upstate Biotechnology. Ceramide analogue C2-ceramide (BioMol) and etoposide (BioVision) were
dissolved in Me2SO. The broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(-OMe)-fluoromethyl ketone (z-VAD-fmk) and
caspase-9, -3, and -2 inhibitors benzyloxycarbonyl-Leu-Glu(-OMe)-HisAsp(-OMe)-fluoromethyl ketone (z-LEHD-fmk), benzyloxycarbonylAsp(-OMe)-Glu(-OMe)-Val-Asp(-OMe)-fluoromethyl ketone (z-DEVDfmk),
and
benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)fluoromethyl ketone (z-VDVAD-fmk), respectively, were purchased
from Sigma and dissolved in Me2SO. Bcl-2 inhibitor HA14-1 (Tocris),
bongkrekic acid (Calbiochem), and okadaic acid (OA, Sigma) were dissolved in Me2SO.
Analysis of Cell ApoptosisFor apoptosis detection characterized by
DNA fragmentation, cells were fixed with 70% ethanol in phosphatebuffered saline for propidium iodide (PI, Sigma) staining and then were
analyzed using flow cytometry (FACSCalibur; BD Biosciences). DAPI
(Sigma) was also used for apoptotic cell staining in 5 g/ml for 30 min
at room temperature and was followed by microscopy detection. Apo-

ptotic cell membrane disruption characterized by the presence of phosphatidylserine was performed using the annexin V-phycoerythrin detection kit (BioVision).
Detection of Caspase ActivationCellular caspase activation was
determined using the ApoAlert caspase colorimetric assay kit (Clontech) for caspase-3 and an ApoAlert caspase fluorescent assay kit for
caspase-9 according to the manufacturers instructions. Caspase-2 activity was detected using a caspase-2 assay kit (Calbiochem). Optical
density (OD) measurements were performed using a microplate reader,
and the substrate activities shown as p-nitroanilide (nmol) were calculated for caspase-3 and -9. For caspase-2, the relative substrate activity
was shown by the OD values. Caspase-3 activation monitored in cells
was performed using PhiPhiLux-G2D2 staining (OncoImmunol) and
detected with fluorescent microscopy. The activation of caspases was
also detected using Western blot analysis as described below.
Western Blot AnalysisTo detect cytochrome c release, cytosolic extract without the mitochondrial fraction was separated using an ApoAlert cell fractionation kit (Clontech) according to the manufacturers
instructions. To detect other proteins, total cell lysate was used. Western blotting was then performed (BD Biosciences). Briefly, cell extract
was separated by SDS-PAGE and then transferred to a polyvinylidene
difluoride membrane (Millipore). After blocking, blots were developed
with a series of antibodies as indicated. Rabbit antibodies specific for
human caspase-9 and -3 (Cell Signaling Technology), cytochrome c
(Santa Cruz Biotechnology), green fluorescent protein (GFP) (Santa
Cruz Biotechnology), Bcl-2, and phospho-Bcl-2 serine 70 (R&D) were
used. Monoclonal antibodies against human caspase-2 (R&D) and -actin (Sigma) were used. Finally, blots were hybridized with horseradish
peroxidase-conjugated goat anti-rabbit IgG or anti-mouse IgG (Calbiochem) and developed using an AEC substrate kit (Zymed Laboratories
Inc.).
Mitochondrial Functional AssayThe loss of mitochondrial transmembrane potential (m) value was determined using rhodamine 123
(Sigma). Cells were incubated with rhodamine 123 (50 M) for 30 min in
cultured medium. After being washed with phosphate-buffered saline,
cells were resuspended in cold phosphate-buffered saline and immediately underwent flow cytometric analysis. Mitochondrial dehydrogenase activity was determined using a WST-8 assay kit (Dojindo Laboratories, Kumamoto, Japan).

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FIG. 1. Ceramide and etoposide induce mitochondria-mediated apoptosis in A549 cells. A, human A549 epithelial cells were treated
with or without 50 M C2-ceramide or 25 M etoposide for 48 h. The changes in cell morphology (characterized by cell rounding up) and in nuclear
morphology (characterized by nuclear fragmentation) are shown by phase-contrast microscopic observation and DAPI staining, respectively. B,
time- and dose-dependent (as indicated) ceramide- or etoposide-induced A549 cell apoptosis was detected using PI staining followed by flow
cytometric analysis. The percentages of apoptotic cells are shown (means S.D. of triplicate cultures). C, using rhodamine 123 followed by flow
cytometric analysis, the percentages of ceramide- or etoposide-treated cells with m (MTP) reduction at different times are shown (means S.D.
of triplicate cultures). D, the activation of caspase-9 and -3 induced by ceramide or etoposide time dependently as determined by caspase activity
assay kits are shown (means S.D. of triplicate cultures). pNA, p-nitroanilide. E, with or without caspase inhibitors as indicated, ceramide- or
etoposide-induced cell apoptosis at 48 h was detected using PI staining. The percentages of apoptotic cells are shown (means S.D. of triplicate
cultures).

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Bcl-2 Modulates Caspase-2 Activation

ImmunostainingFor intracellular immunostaining, cells were

fixed with 1% formaldehyde in phosphate-buffered saline and permeabilized with 0.01% saponin in phosphate-buffered saline. A series of
antibodies was used as indicated, followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Calbiochem) staining. Rabbit
anti-Bcl-2 and anti-phospho-Bcl-2 serine 70 (R&D) antibodies were
used for flow cytometric analysis. For confocal microscopy, rabbit
anti-truncated Bid (tBid) (Calbiochem) antibodies were used. Mito
Tracker Red CMXRos (Molecular Probes) was used for mitochondrial
staining.
Short Interfering RNA (siRNA) PreparationPlasmids expressing
short hairpin RNA were constructed using standard techniques. The
pSUPER/enhanced GFP (EGFP) contained the GFP gene from
pEGFP-N1 (Clontech) inserted into pSUPER vector (37) (kindly provided by Dr. R. Agami, The Netherlands Cancer Institute, Amsterdam,
The Netherlands). To generate pSUPER-Casp2/EGFP and pSUPERBcl2/EGFP, pSUPER/EGFP was digested with BglII and HindIII, and
the annealed targeting oligonucleotides ACAGCTGTTGTTGAGCGAA
for caspase-2 (15) and GCTGCACCTGACGCCCTTC for Bcl-2 (23) were

ligated into the vector. To generate the double knockdown construct

pSUPER-Bcl2/Casp2/EGFP, the Casp2 short hairpin RNA expression
cassette from pSUPER-Casp2/EGFP was inserted into pSUPER-Bcl2/
EGFP. The pSUPER-Casp2/EGFP/Neo, pSUPER-Bcl2/EGFP/Neo, and
pSUPER-Bcl2/Casp2/EGFP/Neo were generated by inserting the Neor
gene from the pIRESneo2 (Clontech) into the pSUPER-Casp2/EGFP,
pSUPER-Bcl2/EGFP, and pSUPER-Bcl2/Casp2/EGFP vectors, respectively (supplemental Fig. S1).
A549 and DU145 cells were cultured in 6-well plastic plates in
Dulbeccos modified Eagles medium and minimum essential medium,
respectively, supplemented with 10% fetal bovine serum (5 105/
well). Before short hairpin RNA-expression vector transfection, cells
were washed with serum-free medium and cultured with 2 l of
Lipofectamine 2000 and 1 g of DNA. After 6 h of incubation, cells
were maintained in cultured medium containing 10% fetal bovine
serum for an additional 24 h before experiments. A FACSAria cell
sorter (BD Biosciences) was used to sort EGFP-positive cells for
vector control and siRNA-expressing cells in some experiments as
indicated.

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FIG. 2. Requirement of caspase-2 activation during ceramide- or etoposide-induced mitochondrial apoptosis. A, A549 cells were
treated with 50 M C2-ceramide or 25 M etoposide for various time periods followed by a caspase-2 activity assay; the substrate relative activities
(OD) are shown (top, means S.D. of triplicate cultures). Also, ceramide (C)- or etoposide (E)-induced caspase-2 processing was determined using
Western blot analysis (bottom). Protein expression of -actin was used as an internal control. B, ceramide or etoposide induced caspase-2-mediated
mitochondrial disruption. With or without caspase-2 inhibitor z-VDVAD-fmk or broad spectrum caspase inhibitor z-VAD-fmk, m (MTP)
reduction and cell apoptosis induced by 50 M C2-ceramide or 25 M etoposide for 48 h were determined using rhodamine 123 and PI staining,
respectively, followed by flow cytometric analysis (means S.D. of triplicate cultures). The activities of caspase-9 and -3 (means S.D. of triplicate
cultures) were determined using caspase activity assay kits (top). Ceramide- or etoposide-induced cytosolic cytochrome c expression with or without
caspase inhibitors was determined using Western blot analysis (bottom). Protein expression of -actin was used as an internal control. pNA,
p-nitroanilide. C, blockage of ceramide- or etoposide-induced cell apoptosis by caspase-2 siRNA. A549 cells were transfected with siRNA to
caspase-2 as described under Experimental Procedures. The expression of procaspase-2 (45 kDa) in vector-transfected cells (Vector) and caspase-2
siRNA-transfected cells (Casp-2 siRNA) was detected using Western blot analysis (top). Protein expression of EGFP was used as an internal
control. The changes in morphology of transfected cells with 50 M C2-ceramide or 25 M etoposide treatment for 48 h were determined using
fluorescent microscopy (EGFP) plus phase-contrast microscopy (Merge). The apoptotic cells are shown in transfected cells (filled arrowheads) or
untransfected cells (open arrowheads). D, ceramide- or etoposide-treated transfected cells were stained with annexin V and caspase-3-specific
fluorescence substrate PhiPhiLux-G2D2 as described under Experimental Procedures. The percentages of annexin V-positive and active-caspase3-positive cells are shown (means S.D. of triplicate cultures).

Bcl-2 Modulates Caspase-2 Activation

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RESULTS AND DISCUSSION

Ceramide and Etoposide Induce Mitochondria-mediated Apoptosis in A549 CellsApoptotic cell death induced by ceramide
and etoposide has been widely reported (28 32, 38, 39). To
investigate the involvement of mitochondrial dysfunction and
caspase activation, C2-ceramide and etoposide were used to
induce apoptosis in A549 human lung epithelial cells. First, cell
growth inhibition after ceramide and etoposide treatment was
demonstrated using a WST-8 assay to detect mitochondrial
dehydrogenase activity (data not shown). A549 cells that had
been exposed to ceramide and etoposide for 48 h were fixed and
stained with DAPI. In ceramide- and etoposide-treated cultures, but not in untreated cultures, cells exhibited apoptotic
morphology (Fig. 1A). The dose and time kinetics of ceramideand etoposide-induced cell apoptosis were shown using PI
staining and then flow cytometric analysis (Fig. 1B). To further
investigate the involvement of mitochondrial damage, the loss
of mitochondrial transmembrane potential (m) was determined. Using lipophilic cationic fluorochrome rhodamine 123
staining, we found that ceramide and etoposide time dependently induced m reduction in A549 cells (Fig. 1C). Using a
caspase activity assay and Western blot analysis, the activity of
caspase-9 and -3 (Fig. 1D) and the processing of caspase-3 from
proform (35 kDa) to active form (17 kDa) (data not shown) were
observed time dependently after exposure to ceramide and
etoposide. An increase in cytosolic cytochrome c expression was
also observed (data not shown). Furthermore, pretreatment
with the irreversible caspase-9 and -3 inhibitors z-LEHD-fmk
and z-DEVD-fmk, respectively, blocked cell death detected using PI staining (Fig. 1E). We therefore ascertained the involvement of mitochondrial damage, cytochrome c release, and

caspase-9 and -3 activation in ceramide- and etoposide-induced

A549 cell apoptosis.
Requirement of Caspase-2 Activation before Mitochondrial
DamageOur previous study (24) demonstrated that activation of caspase-2 was required for ceramide- and etoposideinduced T cell apoptosis before mitochondrial damage. We thus
confirmed the involvement of caspase-2 in response to ceramide
and etoposide stimulation in A549 cells. Time-dependent activation of caspase-2 was shown by the activity assay (Fig. 2A,
top). The caspase-2 cleavage was also detected using Western
blot analysis (Fig. 2A, bottom). To examine whether caspase-2
was required for the mitochondrial intrinsic pathway of apoptosis, we inactivated caspase-2 in A549 cells by pretreatment
with the inhibitor z-VDVAD-fmk. The results showed the inhibition of cell apoptosis, m reduction, and caspase-9 and -3
activation after z-VDVAD-fmk pretreatment in response to
ceramide and etoposide stimulation (Fig. 2B, top). Accordingly,
cytosolic cytochrome c (Fig. 2B, bottom) and cleavage fragments
of active caspase-9 and -3 (data not shown) were shown in the
ceramide- and etoposide-treated cultures, but not in the cultures pretreated with z-VDVAD-fmk. The broad-spectrum
caspase inhibitor z-VAD-fmk caused an effect similar to that of
z-VDVAD-fmk (Fig. 2B). We further introduced the short hairpin RNA specific for caspase-2 into A549 cells for interference
with caspase-2 expression. The mean transfection efficiency of
caspase-2 siRNA was 52.3% and of vector control was 60.8%
according to a flow cytometric analysis of EGFP-positive cells
in a representative experiment. An 50% inhibition of
caspase-2 expression was observed in the caspase-2 siRNAtransfected cells compared with the vector control (Fig. 2C,
top). After ceramide or etoposide treatment, apoptotic cells

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FIG. 3. Overexpression of Bcl-2 inactivates caspase-2, which rescues ceramide- or etoposide-induced apoptosis. A, A549 cells with
Bcl-2 overexpression were established as described under Experimental Procedures. The protein expression of Bcl-2 in wild-type (WT),
vector-transfected (P2), and Bcl-2-transfected (B2) was detected using Western blot analysis (top) and immunostaining followed by flow cytometric
analysis (bottom). The relative mean fluorescence intensity (MFI) is shown. For Western blotting, protein expression of -actin was used as an
internal control. B, cell apoptosis induced by 50 M C2-ceramide or 25 M etoposide at 48 h was detected using DAPI staining (left) or PI staining
(middle) followed by fluorescent microscopy or flow cytometric analysis, respectively. The percentages of apoptotic cells are shown. For m
reduction detection, ceramide- or etoposide-treated cells were detected using rhodamine 123 followed by flow cytometric analysis (right). The
percentages of cells with m reduction are shown. C, caspase activation in A549-P2 and A549-B2 cells induced by 50 M C2-ceramide (top) or 25
M etoposide (bottom) for various time periods was determined using activity assay kits. The substrate activities for caspase-9 and -3 (pNA,
p-nitroanilide) (left, means S.D. of triplicate cultures) and the relative activity for caspase-2 using OD (right) are shown. D, the processing of
caspase-2 in these cells after ceramide (top) or etoposide (bottom) treatment was detected using Western blot analysis.

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Bcl-2 Modulates Caspase-2 Activation

characterized by destructive morphology (Fig. 2C, all arrowheads) were seen in both EGFP-positive vector control (filled
arrowheads) and EGFP-negative cells (open arrowheads). In
the caspase-2 siRNA-transfected group, however, only EGFPnegative cells underwent apoptosis (open arrowheads),
whereas EGFP-positive caspase-2 siRNA-expressing cells
showed resistance to ceramide and etoposide stimulation with
intact morphology. Similarly, ceramide- and etoposide-induced
apoptosis was blocked in caspase-2 siRNA-expressing cells,
detected using annexin V staining and caspase-3 activity in
EGFP-positive cells (Fig. 2D). These results showed that interference with the expression of caspase-2 blocked the mitochondria-dependent pathway of apoptosis induced by ceramide and
etoposide.
Bcl-2 Blocks Caspase-2-mediated Ceramide- and Etoposideinduced ApoptosisBcl-2 acts as an anti-apoptotic factor to
block death signals, including those from ceramide and etoposide (2532). To investigate the relation between Bcl-2 and
caspase-2 activity during ceramide- and etoposide-induced apoptosis, we used Bcl-2-overexpressing cells. First, an elevated
expression of Bcl-2 in A549 cells transfected with bcl-2 (A549B2), compared with the wild-type (WT) and vector control
(A549-P2), was confirmed using Western blot analysis (Fig. 3A,
top), intracellular immunostaining, and then flow cytometric
analysis (Fig. 3A, bottom). Cell apoptosis, characterized by
DNA fragmentation, was detected using DAPI staining (Fig.
3B, left) or PI staining followed by flow cytometric analysis
(Fig. 3B, middle). Compared with A549-P2 cells, which had
45.1 and 35.7% apoptotic cells after ceramide and etoposide
stimulation, respectively, apoptotic cells were reduced to 15.1
and 8.3% in A549-B2 cells. Using rhodamine 123 staining, the

m reductions in A549-P2 and A549-B2 cells were 39.2 and

7.1%, respectively, after ceramide treatment and 45.6 and 4.8%
after etoposide treatment (Fig. 3B, right). Ceramide-induced
tBid expression was detected using tBid-specific antibody in
combination with Mito Tracker Red dye. Results showed Bid
cleavage and translocation to mitochondria in ceramidetreated A549-P2 but not in A549-B2 cells (data not shown). A
caspase substrate activity assay revealed a time-dependent
increase in caspase-9, -3, and -2 activities in A549-P2 but not in
A549-B2 cells (Fig. 3C). Also, cleavage of procaspase-2 was seen
in A549-P2 but not in A549-B2 cells after ceramide and etoposide treatment (Fig. 3D). MDCK cells with or without Bcl-2
overexpression after ceramide and etoposide stimulation
showed similar results (supplemental Fig. S2).
Role of Bcl-2 in Regulating Caspase-2 ActivationBcl-2 has
blocked caspase-3 and -2 sequential activation after mitochondrial damage (19). In that case, caspase-2 acted downstream of
mitochondria and caspase-3, and Bcl-2 acted at a point downstream from the release of mitochondrial cytochrome c. Bcl-2
had also acted upstream or downstream of caspase-8 in Fas- or
staurosporine-induced apoptosis (4, 40, 41). Our previous study
demonstrated caspase-2 and -8 sequential activation in ceramide- and etoposide-induced mitochondrial damage in concomitant with the production of tBid and the release of cytochrome
c (24). To directly investigate the regulation of Bcl-2 on
caspase-2, we used the gene-silencing technique and Bcl-2 inhibitor. siRNA transfection in A549 cells induced Bcl-2 or
caspase-2 knockdown, or both. After cell sorting, flow cytometric analysis showed, and Western blot analysis confirmed, that
the EGFP-positive cells with Bcl-2 siRNA, caspase-2 siRNA,
Bcl-2 plus caspase-2 siRNA, and vector control were 82.2, 84,

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FIG. 4. Down-regulation of Bcl-2 results in caspase-2 activation. A, A549 cells were transfected with Bcl-2 siRNA, caspase-2 siRNA, or
Bcl-2 plus caspase-2 siRNA as described under Experimental Procedures. After cell sorting, the expression of Bcl-2 (28 kDa) and caspase-2 (45
kDa) in vector-transfected cells (Vector) and siRNA-transfected cells was detected using Western blotting (top). Protein expression of EGFP was
used as an internal control. In the bottom panel, EGFP-positive cells are indicated as successfully transfected cells (all arrowheads). With or
without z-VDVAD-fmk, the morphology changes of 48-h posttransfected cells were detected using phase-contrast plus fluorescent microscopy. The
apoptotic cells (open arrowheads) are shown. B, for apoptosis and caspase-activation analysis, 24 and 48 h posttransfection, cells were stained with
annexin V or caspase-3-specific fluorescence substrate using PhiPhiLux-G2D2 staining, respectively. The percentages of positive cells in vectortransfected and Bcl-2 siRNA-transfected groups are shown (means of duplicate cultures). After cell sorting, the caspase-2 activity was detected
using a caspase-2 activity assay kit. The substrate relative activity is shown using OD (means of duplicate cultures). C, A549 cells were treated
with 100 M Bcl-2 inhibitor HA14-1 for 18 and 36 h, and cell apoptosis was detected by PI staining followed by flow cytometric analysis. Also,
activation of caspase-3 and -2 was determined using caspase activity assay kits. The substrate activity for caspase-3 (pNA, p-nitroanilide) and the
relative activity for caspase-2 using OD are shown (means of duplicate cultures).

Bcl-2 Modulates Caspase-2 Activation

90, and 89.8%, respectively, in one representative experiment

(Fig. 4A, top). We next showed that the hallmarks of apoptosis
were present in EGFP-positive cells with Bcl-2 siRNA but not
in those with vector control. Cells with Bcl-2 siRNA transfection showed apoptotic morphology compared with untransfected cells or vector control cells (Fig. 4A, open arrowheads).
Bcl-2-silencing cells co-transfected with caspase-2 siRNA or
pretreated with z-VDVAD-fmk rescued apoptosis caused by
Bcl-2 inhibition. Bcl-2 siRNA-transfected cells showed a timedependent increase of apoptotic cell death with annexin V
binding and caspase-3 activation compared with vector control
cells (Fig. 4B). To further investigate caspase-2 activation after
Bcl-2 knockdown, we sorted EGFP-positive cells and determined the activation of caspase-2. A substrate activity detection assay suggested that caspase-2 was activated in Bcl-2
siRNA-transfected cells but not in vector control cells (Fig. 4B).
HA14-1, a small molecule inhibitor of Bcl-2, induced cell apoptosis via the mitochondrial intrinsic pathway through Bcl-2
structural dysfunction (42, 43). HA14-1 caused Bax translocation to mitochondria and cytochrome c release (43). Our results
showed that treatment with HA14-1 induced A549 cells to
undergo apoptosis. The m reduction (data not shown) and
caspase-3 and -2 activation (Fig. 4C) were also observed. HA141-induced cell apoptosis and caspase activation were detected
in Bcl-2-overexpressing cells (data not shown). To verify the
specificity of HA14-1 on Bcl-2 dysfunction, we tested a Bcl-2independent apoptotic pathway in DU145 cells, human pros-

FIG. 6. Phosphatase causes Bcl-2 dysfunction, which facilitates ceramide- and etoposide-induced mitochondrial apoptosis. A, Western blot analysis of phospho-Bcl-2 serine 70 (pBcl-2) and
Bcl-2 protein expression (28 kDa) in total A549 cell extracts at various
time points after treatment with 50 M C2-ceramide (top) or 25 M
etoposide (bottom). Protein expression of -actin was used as an internal control. B, using immunofluorescence staining followed by flow
cytometric analysis, the expression of pBcl-2 in A549 cells after ceramide treatment with or without 100 nM protein phosphatase inhibitor
OA was detected at 24 h. The histogram of protein expression is shown
as a representative of two individual experiments. The mean fluorescence intensity (MFI) of each protein expression is shown. C, A549 cells
were treated with ceramide (solid line) or etoposide (dotted line) with or
without 100 nM OA for 24 and 48 h. Cell apoptosis and m (MTP)
reduction were detected using PI and rhodamine 123 staining, respectively, followed by flow cytometric analysis. The percentages of apoptotic cells and positive cells with m reduction are shown (means of
duplicate cultures). Also, activation of caspase-3 and -2 were determined using caspase activity assay kits. The activity as relative OD is
shown (means of duplicate cultures).

tate cancer cells. A previous study (44) showed that Bcl-2

down-regulation by antisense RNA and siRNA strategies was
unable to facilitate cell apoptosis in DU145 cells. Our results
confirmed that DU145 cells resisted a serial dose of Bcl-2
siRNA (supplemental Fig. S3A). DU145 cells treated with 100
M HA14-1 did not undergo apoptosis, m reduction, or
caspase-3 and -2 activation (supplemental Fig. S3B). However,
HA14-1 may have caused DU145 cell death at higher concentrations (data not shown). These results corresponded with a
previous report (23) demonstrating the involvement of
caspase-2 in Bcl-2-silencing cell apoptosis.
Based on our previous findings (24), ceramide- and etoposide-induced caspase-2 activation acted upstream of caspase-8
activation, Bid cleavage, and m reduction. To further confirm that Bcl-2-modulated caspase-2 activation acted upstream
of mitochondrial damage, we used the mitochondrial permeability transition pore inhibitor bongkrekic acid and caspase-9
inhibitor z-LEHD-fmk. After pretreatment with bongkrekic
acid or z-LEHD-fmk, apoptosis (Fig. 5, top) and caspase-3 activation (middle) caused by Bcl-2 knockdown or HA14-1 was
completely blocked. Most importantly, caspase-2 activation induced by Bcl-2 knockdown or HA14-1 was not inhibited in the
presence of bongkrekic acid or z-LEHD-fmk (bottom). We concluded that Bcl-2-modulated caspase-2 activation functioned
upstream of mitochondria.
Bcl-2 Dysfunction Caused by Phosphatase during Ceramideand Etoposide-induced ApoptosisIt had been reported that
ceramide caused Bcl-2 dephosphorylation at serine 70 and resulted in Bcl-2 functional destruction through protein phospha-

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FIG. 5. Caspase-2 activation acts upstream of mitochondrial

damage after Bcl-2 dysfunction. With or without bongkrekic acid
(BA) or caspase-9 inhibitor z-LEHD-fmk, A549 cells were treated with
50 M C2-ceramide, 25 M etoposide, or 100 M HA14-1, or transfected
with Bcl-2 siRNA. Cell apoptosis and caspase activation were determined at 48 h. Cell apoptosis was detected using PI (filled bar) or
annexin V (open bar) staining followed by flow cytometric analysis or
fluorescent microscopy. The percentages of apoptotic cells and annexin
V-positive cells are shown (means of duplicate cultures). The activation
of caspase-3 and -2 was detected by substrate cleavage using PhiPhiLux-G2D2 staining or an activity assay kit. The quantified concentrations for caspase-3 (filled bar) and the percentages of active caspase-3positive cells (open bar) are shown. Bcl-2 siRNA-transfected cells were
sorted and used for caspase-2 activity detection. The substrate-relative
activity is shown using OD (means of duplicate cultures).

23763

23764

Bcl-2 Modulates Caspase-2 Activation

findings shed light on the role of Bcl-2 in the inhibition of
caspase-2 before mitochondrial damage.
AcknowledgmentsWe thank Wan-Hua Tsai from the Institute of
Basic Medical Sciences (NCKU, Taiwan) for conducting the Bcl-2 overexpression system and Ming-Chen Yang and Wen-Wei Chang from the
Department of Microbiology and Immunology (NCKU, Taiwan) for assistance with cell sorting. We also thank Dr. C. W. Chiang for comments
on the manuscript and Bill Franke for editorial assistance.
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related to caspase-2 activation. Indeed, Bcl-2 was dephosphorylated at serine 70 by 12 and 24 h after ceramide and etoposide treatment, respectively, and the expression level of Bcl-2
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caspase-2 need to be further explored. Taken together, these

Bcl-2 Modulates Caspase-2 Activation

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Mechanisms of Signal Transduction:

Bcl-2 Rescues Ceramide- and
Etoposide-induced Mitochondrial
Apoptosis through Blockage of Caspase-2
Activation
Chiou-Feng Lin, Chia-Ling Chen, Wen-Tsan
Chang, Ming-Shiou Jan, Li-Jin Hsu,
Ren-Huang Wu, Yi-Ting Fang, Ming-Jer
Tang, Wen-Chang Chang and Yee-Shin Lin

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This article cites 54 references, 29 of which can be accessed free at
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J. Biol. Chem. 2005, 280:23758-23765.

doi: 10.1074/jbc.M412292200 originally published online April 6, 2005