JOURNAL OF CELLULAR PHYSIOLOGY 199:47–56 (2004)
Ceramide Induces Caspase-Dependent and -Independent
Apoptosis in A-431 Cells
SHENG ZHAO, YA-NAN YANG, AND JIAN-GUO SONG*
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
We investigated the ceramide-induced apoptosis and potential mechanism in A431 cells. Ceramide treatment causes the round up and the death of A-431 cells that
is associated with p38 activation and can be observed in 10 h. Short-time ceramide
treatment-induced cell death is not associated with the typical apoptotic
phenotypes, such as the translocation of phosphatidylserine (PS) from inner layer
to outer layer of the plasma membrane, loss of mitochondrial membrane potential, DNA fragmentation, caspase activation, and PARP or PKC-d degradation.
SB202190, a specific inhibitor of p38 mitogen-activated protein (MAP) kinase, but
not caspase inhibitor, blocks the cell death induced by short-time ceramide treatment (within 12 h). Whereas neither inhibition of p38 MAP kinase nor inhibition of
caspases blocks cell death induced by prolonged ceramide treatment. Moreover,
incubation of cells with ceramide for a long time (over 12 h) results in the reduction
of proportion of S phase accompanied with typical apoptotic cell death phenotypes
that are different from the cell death induced by short-time ceramide treatment. Our
data demonstrated that ceramide-induced apoptotic cell death involves both
caspase-dependent and caspase-independent signaling pathways. The caspaseindependent cell death that occurred in relatively early stage of ceramide treatment
is mediated via p38 MAP kinase, which can progress into a stage that is associated
with changes of cell cycle events and involves both caspase-dependent and
-independent mechanisms. J. Cell. Physiol. 199: 47–56, 2004.
ß 2003 Wiley-Liss, Inc.
Apoptosis is a form of cell death with unique cellular
phenotypes including appearance of phosphatidylserine
(PS) on the external cell membrane, loss of mitochondrial transmembrane potential, the activation of the
caspase, cleavage of the poly (ADP) ribose polymerase
(PARP), and protein kinase C-d (PKC-d), nuclear chromatin condensation, and DNA fragmentation (Blatt and
Glick, 2001). Apoptosis induced by ceramide through
either caspase-dependent or -independent mechanisms
has been reported in recent years. However, the
mechanism of caspase-independent apoptosis was still
poorly understood. In lymphoblast cells, it was shown
that ceramide and radiation exposure can induce
distinct types of apoptosis via different mechanisms
which can be determined from their phenotypes (Shi
et al., 2001). In human glioma cells, Akt protein kinase, a
well-known factor for cell survival, was found to be
involved in the negative regulation of ceramide-induced
caspase-independent apoptosis (Mochizuki et al., 2002).
Ceramide-induced cell death of T lymphocyte and U937
was also shown to be a caspase-independent apoptosis
(Belaud-Rotureau et al., 1999). In human cervix carcinoma cells (Lopez-Marure et al., 2002) and prostate
cancer cell line LNCaP (Engedal and Saatcioglu, 2001),
ceramide induces necrosis-like cell death without the
biochemical and morphological markers characteristic
of apoptosis. Nevertheless, in contrast to caspase-dependent apoptosis where the central mechanism and
ß 2003 WILEY-LISS, INC.
molecules involved in the transduction of death signals
have been extensively studied and largely delineated,
information regarding the players participating in the
regulation of caspase-independent apoptosis is very
limited.
Sphingolipids are not only structural components of
cell membrane but also a ubiquitous and evolutionarily
Abbreviations: ERK, extracellular signal-regulated kinase; MAP
kinase, mitogen-activated protein kinase; PARP, poly (ADP)
ribose polymerase; PKC-d, protein kinase C-d.
Sheng Zhao and Ya-Nan Yang contributed equally to this work.
Contract grant sponsor: The Chinese Academy of Sciences;
Contract grant number: KSCX2-SW-203; Contract grant sponsor:
The Virtual Research Institute of Aging of Nippon Boehringer
Ingelheim; Contract grant sponsor: The ‘‘973’’ Project of China;
Contract grant number: 2002CB513000.
*Correspondence to: Jian-Guo Song, Laboratory of Molecular Cell
Biology, Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences,
Box 25, 320 Yue-Yang Road, Shanghai 200031, China.
E-mail: jgsong@sibs.ac.cn
Received 6 May 2003; Accepted 3 September 2003
DOI: 10.1002/jcp.10453
48
ZHAO ET AL.
conserved signaling system in which ceramide and other
sphingolipid derivatives can be formed. Ceramide can be
generated through the hydrolysis of sphingomyelin or
de novo synthesis. Inhibition of the degradation of
ceramide by ceramidase also has an impact on the
intracellular level of ceramide (Testi, 1996; Hannun and
Luberto, 2000; Ohanian and Ohanian, 2001; Gomez
et al., 2002). In addition, the cellular compartmentalization restricts the site of ceramide production and
subsequent interaction with target proteins (Kolesnick
and Goni, 2000; Ohanian and Ohanian, 2001; Vielhaber
et al., 2001). Increasing evidence suggests that branching pathways of sphingolipid metabolism mediate either
cell death or mitogenic responses depending on cell type
and the nature of the stimulus. Although the main
biological function of ceramide appears to be linked to its
potency to induce cell death, its actual relevance as a
regulator of cell death has been the subject of controversial discussions. Previous reports have shown
that the mitogen-activated protein (MAP) kinase cascade, a key signal transduction pathway, contributes to
ceramide-induced physiologic effects and usually determines the fate of the cells. The extracellular signalregulated kinase (ERK) pathway, activated by growth
factors and hormones, is involved in mediating cellular proliferation, transformation, and differentiation
(Grewal et al., 1999). In contrast, the c-Jun N-terminal
kinase (JNK) and the p38 cascades are implicated in cell
death triggered by cytokines, growth factor withdrawal,
and environmental stresses (Davis, 2000; MartinBlanco, 2000). Ceramide was shown to inhibit ERK
activity but activate JNK and p38 MAPK in some types
of cell lines (Westwick et al., 1995; Kitatani et al., 2001;
Willaime et al., 2001; Won et al., 2001). While in other
cases, it was reported that ERK can be activated by
ceramide (Raines et al., 1993; Reunanen et al., 1998;
Subbaramaiah et al., 1998; Chen et al., 2001). The
implications of JNK and p38 in ceramide-induced cell
death in various cell types have been shown through
some pharmacological and molecular assays (Brenner
et al., 1997; Hida et al., 1999; Caricchio et al., 2002).
Nevertheless it has also been demonstrated that p38
may be not essential for ceramide-induced cell death in
U937 and MC/9 cells (Jarvis et al., 1997; Scheid et al.,
1999).
It has been suggested that cells have intrinsic
biochemical and molecular machinery functioning primarily to sense the injury and insult that lead the cells to
execute appropriate programs of response. Mammalian
cells can respond to such stimuli by undergoing cell cycle
arrest to allow adequate time for repair of damage or by
undergoing apoptosis if the damage is too severe and
irreparable. Besides cell death, ceramide is also an
important mediator of cell cycle arrest in G1/G0 phase
(Dbaibo et al., 1995; Hannun, 1996; Lee et al., 1998).
Moreover, it was reported that the cell cycle-dependent
caspase activation may be involved in ceramidemediated apoptosis in neuro tumor cells (Di Bartolomeo
et al., 2000). High correlation between the G1 blockade of
the cell cycle and the following apoptotic cleavage in HL60 cells suggests the cell cycle arrest in G1 phase may
render cells to undergo apoptosis (Bartova et al., 1997).
We report in this study, that ceramide induces both
caspase-dependent and -independent apoptosis through
different signaling pathways in A-431 cells: p38 MAPK
plays an important role in the caspase-independent
apoptosis, whereas ceramide-induced caspase-dependent apoptosis may be related to cell cycle events. The
data indicate that a complex interactive signaling event
with multiple control points is involved in the regulation
of ceramide-induced cell death.
MATERIALS AND METHODS
Materials
A-431 human epidermoid carcinoma cell line was purchased from American Type Culture Collection (ATCC,
Manassas, VA). Cell culture reagents and fetal bovine
serum (FCS) were purchased from Life Technologies
(Grand Island, NY). [3H-methyl]-thymidine (83.60 Ci/
mmol) was from NEN Life Science Products (Boston,
MA). PD98059, SB202190, C8-ceramide, caspase inhibitor I (Z-VAD-fmk, a specific caspase inhibitor), caspase
inhibitor III (BD-fmk, a broad caspase inhibitor),
caspase substrate set I (Colorimetric), and horseradish
peroxidase (HRP)-conjugated anti-mouse and anti-goat
secondary antibodies were purchased from Calbiochem
(La Jolla, CA). Nitrocellulose membrane was obtained
from Amersham Pharmacia Biotech (Buckinghamshire,
UK). Mouse monoclonal antibody against phosphorylated ERK (p-ERK) and rabbit polyclonal antibodies
against ERK, p38, PARP, PKC-d, and goat polyclonal
antibody against actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal antibody against phosphorylated p38 (p-p38) was obtained
from New England Biolabs, Inc. (Beverly, MA). Super
signal reagents were purchased from Pierce (Rockford,
IL). Annexin V-FITC apoptosis detection kit I was
obtained from Pharmingen (San Diego, CA). 3,30 Dihexyloxacarbocyanine iodide (DiOC6(3), propidium
iodide (PI), hydroxyurea, nocodazole, and daunorubicin
were from Sigma (St. Louis, MO).
Cell culture
A-431 cells cultured in 100-mm master plates in
Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FCS, 100 U/ml of penicillin, and 100 mg/ml of
streptomycin in a humidified atmosphere of 5% CO2
at 378C. Medium were renewed every 2–3 days until
confluence was reached. For experiment, cells were
seeded into 60 or 35-mm plates and cultured under the
same condition. The photographs of cell morphology
were taken directly under the inverted phase-contrast
microscope after indicated treatments.
Detection of the loss of plasma
membrane asymmetry
In the early stage of apoptosis, the membrane PS is
translocated from the inner to the outer leaflet of the
plasma membrane, thereby exposing PS to the external
cellular environment. Annexin V is a 35–36 kDa Ca2þdependent phospholipid-binding protein that has a high
affinity for PS, which can bind to cells with exposed
PS (Raynal and Pollard, 1994). Annexin V-FITC staining precedes the loss of membrane integrity that
accompanies the latest stages of cell death resulting
from either apoptotic or necrotic processes, which can be
determined via PI staining. Thus, we used the annexin
V-FITC in conjunction with PI staining to identify the
49
CERAMIDE-INDUCED CELL APOPTOSIS
early apoptotic cells (Annexin V-FITC positive and PI
negative) as per Vendor’s instruction of the kit.
Detection of the loss of mitochondrial
membrane potential
The loss of mitochondrial membrane potential is
another early event of apoptosis. This phenomena can
be examined by several potent sensitive dyes such as
DiOC6(3) to which normal cells will be stained (positive).
It can also be used in conjunction with PI staining to
determine the early apoptotic cells (DiOC6(3) negative
and PI negative) (Joza et al., 2001). After indicated
treatment, cells were trypsinized and stained by 40 nM
DiOC6(3) and 500 ng/ml PI in phosphate-buffered saline
(PBS) at 378C for 30 min and then analyzed by FACS
(Becton Dickinson FACSCalibur, Franklin Lakes, NJ).
Cell cycle analysis
The cell cycle was quantitatively determined by flow
cytometry analysis (Blatt and Glick, 2001; Shi et al.,
2001). The percentage of cells with sub-G1 DNA content
was taken as a measure of the apoptotic rate of cell
population. After indicated treatment, cells were trypsinized and fixed with 70% ethanol for over 1 h. Cells
were then pelleted and washed with PBS plus 20 mM
EDTA. RNA was removed by adding RNase (1 mg/ml) at
378C for at least 2 h. Cells were finally stained with PI
(final concentration: 30 mg/ml), and the DNA contents of
cells were then analyzed by FACS. To synchronize the
cells to the entry of S phase, we first incubated the cells
in serum-free medium for 24 h, followed by incubating
the cells in 10% FCS containing medium plus 1 mM
hydroxyurea for another 24 h. When the medium was
replaced again with the normal culture medium, the
cells were released from the entry of S phase. Six hours
later, cells that were going to enter the G2 phase were
treated with nocodazole (50 ng/ml) for 4 h. The cells were
then synchronized in the entry of M phase.
DNA fragmentation assay
DNA fragmentation of apoptotic cells was detected as
previously described (Chen et al., 2000) with minor
modifications. The cells were rinsed with PBS twice and
lysed on ice for 30 min in 10 mM Tris-Cl (pH 8.0), 25 mM
EDTA, and 0.25% Triton X-100. After centrifugation at
13,800g for 15 min, the supernatant was incubated with
RNase at 378C for 60 min and then with proteinase K at
568C overnight. The contents were extracted sequentially with phenol, phenol:chloroform (1:1), and chloroform. The DNA in aqueous phase was precipitated and
analyzed by 1.5% agarose gel electrophoresis. Gel was
visualized and photographed under transmitted UV
light.
Caspase activity assay
A-431 cells cultured in 60-mm plates with 10% FCS
were treated with C8-ceramide as indicated, trypsinized, and washed once with PBS. Cells were resuspended in ice-cold lysis buffer (50 mM HEPES,
pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM DTT, 0.1 mM
EDTA) for 5 min and centrifuged at 10,000g for 10 min.
The supernatants (cytosolic extract) were collected as
the sample for determining the caspase activity. Each
assay contains 10 ml samples with 10–30 mg proteins,
10 ml substrate (2 mM), and 80 ml assay buffer (50 mM
HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM
DTT, 0.1 mM EDTA, and 10% glycerol). The reactions
were proceeded at 378C for 120 min, followed by
measuring the optical density (OD) at 405 nm.
Statistical analysis
Results are presented as means standard deviations (SD) for the number of experiments indicated. For
statistical analysis, Student’s t test was performed.
Differences were considered significant at a level of
P < 0.05.
Preparation of cell lysates and immunoblotting
RESULTS
C8-ceramide induces the apoptosis in A-431 cells
A-431 cells cultured in 60-mm plates were treated
with C8-ceramide in the presence of 1% NCS (for
detecting MAP kinase activation) or 10% FCS as
indicated and then lysed in 0.4 ml of ice-cold lysis buffer
(50 mM HEPES pH 7.4, 5 mM EDTA, 50 mM NaCl, 1%
Triton X-100, 50 mM NaF, 1 mM Na3VO4, 10 mM
Na4P2O7 10 H2O, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM PMSF) followed by shearing using 2 ml
needle for three times. The lysates were centrifuged at
14,000g for 10 min. The supernatants were analyzed by
Western blotting after SDS–PAGE. The proteins were
transferred onto nitrocellulose membrane followed by
blocking with 5% BSA in Tris-buffered saline (TBS)
containing 0.1% Tween-20 for 1 h at room temperature
and subsequently incubated with the primary antibody
(1:2,000 dilution) overnight at 48C. After being washed
for another 1 h at room temperature, the membrane was
further incubated with a horseradish peroxidase conjugated secondary antibody for 2 h and washed for 1 h.
The immunoreactive bands were visualized by super
signal reagents (Pierce) and the protein level was detected under the same condition after striping the
membrane.
Ceramide was shown to induce the time-dependent
apoptosis of A-431 cells as detected by several different
methods. By morphological examination, we observed
that, in response to C8-ceramide treatment, cells became
firstly rounded up and looked much brighter than the
untreated cells. Further incubation of cells with ceramide (10 h and longer) resulted in the death of cells
(Fig. 1A). By using Annexin V or DIOC6(3) staining,
two classical early events of apoptotic cell death, the
translocation of PS from inner side of the cytoplasm
membrane to the outer side and the loss of mitochondrial membrane potential were detected. In Figure 1B,
the annexin V positive but PI negative cells (the right
lower square) were detected 14 h after treatment,
and then moved upward into the annexin V and PI
positive square (end stage apoptosis and death) when
C8-ceramide treatment was prolonged. In Figure 1C, the
DiOC6(3) and PI negative cells (the left lower square)
were also observed 14 h after the treatment, which then
moved upward into the DiOC6(3) negative but PI
positive square (end stage apoptosis and death) when
ceramide treatment was continued. In addition, there is
an increase in the sub G1 DNA content and decrease in
50
ZHAO ET AL.
To investigate the potential mechanism of ceramideinduced apoptosis, we first examined the effect of
ceramide on MAPK activation. As shown in Figure 2A,
ceramide stimulates a rapid increase in the level of
phosphorylated p38 which can be observed from 30 min
to 8 h after the treatment. Incubation of cells with C8-
ceramide also resulted in an activation of ERK as shown
by increased forms of phosphorylated ERK1/ERK2
(Fig. 2B). We further investigated the role of these
MAPK in ceramide-induced cell death. As shown in
Figure 2C, inhibition of p38 by specific inhibitor
SB202190 suppressed cell apoptosis induced by ceramide in the early period of treatment (within 12 h),
which can be easily observed through cell morphological
examination. Nevertheless, SB202190 is unable to inhibit the cell death induced by long-term ceramide
treatment (24 h). In contrast, inhibition of ERK1/ERK2
by the MEK1/MEK2 inhibitor PD 98059 has no effect
on ceramide-induced apoptosis. We also observed that
Fig. 1. Ceramide induced cell death. A-431 cells cultured in medium
containing 10% FCS were treated with C8-ceramide (25 mM) for
indicated times. A: The morphological changes of cells were examined by microscopy (400 magnifications). B: After treatment with
ceramide, cells were trypsinized and collected, then stained with
annexin V/PI. The deaths of cells were determined by FACS. C: Cells
were treated as in B and stained with DiOC6(3)/PI, which exhibited
the early apoptotic events induced by ceramide after 14 h. D: After the
ceramide treatment, cells were stained with PI and fixed with ethanol.
The sub G1 and S phase DNA contents of cells were determined by
FACS. The data represent one of three independent experiments with
same results.
the proportion in cells of S phase (Fig. 1D). The increase
in the sub G1 DNA content became highly pronounced
20 h after the C8-cearmide treatment.
Inhibition of p38 MAP kinase blocks
the apoptosis induced by ceramide in
the early stage of the treatment
CERAMIDE-INDUCED CELL APOPTOSIS
Fig. 1.
51
(Continued)
treatment of cells with Z-VAD-fmk does not inhibit
ceramide-induced cell death. The same results were
obtained with BD-fmk (data not shown). In addition,
inhibition of p38 does not prevent the PS translocation,
and the loss of mitochondrial potential (Fig. 3A,B), and
DNA fragmentation induced by long-term ceramide
treatment (Fig. 3C), implying that p38 activation may
not be involved in the classic apoptotic cell death which
occurred only in long-term ceramide treatment, but is
implicated in atypical apoptotic cell death which played
a role in the early period of ceramide treatment.
Prolonged treatment with ceramide induces
the classic caspase-dependent cell apoptosis
We examined the ceramide-induced cellular caspase
activities with several different caspase substrates for a
better understanding of the mechanism of ceramide-
induced apoptosis. Ceramide-induced caspase activities
were also determined by measuring the degradation of
PARP and PKC-d. As shown in Figure 4A, C8-ceramide
was shown to induce various types of caspase activities,
which can be inhibited efficiently by Z-VAD-fmk and
BD-fmk. Treatment of cells with C8-ceramide also induces a time-dependent PARP (Fig. 4B) and PKC-d
(Fig. 4C) degradation, which became evident after 16 h,
further indicating the ceramide-induced activation of
caspases. Daunorubicin, which was previously shown to
induce the caspase-dependent apoptosis and the degradation of PARP and PKC-d as shown previously (Chen
et al., 2000) was used here as a positive control.
To investigate the involvement of caspases in longterm ceramide treatment-induced apoptosis, cells were
treated in the presence or absence of BD-fmk, and then
treated with C8-ceramide for 24 h. As shown in Figure 5,
52
ZHAO ET AL.
which all the cells are in the same phase of cell cycle. As
shown in Figure 6, the prolonged treatment of cells with
C8-ceramide (24 h) resulted in an obvious decline of the S
phase proportion, but an increase of the sub G1 DNA
content, which is consistent with Figure 1D. Moreover,
it seems that ceramide does not cause the cell cycle
arrest but just delays the progress of the cell cycle,
suggesting that the ceramide-induced programmed cell
death are related with the alterations of signaling
events controlling the cell cycle.
DISCUSSION
Fig. 2. p38 MAP kinase activation is associated with the short-time
ceramide treatment-induced cell apoptosis. A-431 cells were incubated
in 1% NCS-containing medium for 24 h and then treated with C8ceramide (25 mM) for indicated time in the presence or absence of
SB202190, PD98059, or caspase inhibitor, and the MAP kinase
activation or cell apoptosis was then determined. The cells were lysed
and immunoblotted as described in ‘‘Materials and Methods.’’ A:
Ceramide induced time-dependent p38 activation in A-431 cells. B:
Ceramide induced time-dependent activation of ERK. The data
represent one of three independent experiments with same results.
C: Effect of MAP kinase inhibitors on the morphological changes
during the caspase-independent apoptosis induced by C8-ceramide
treatment (150 magnifications). PD98059 (10 mM), SB202190
(10 mM), and the Z-VAD-fmk (10 mM) were pre-added 10 min before
the treatment with C8-ceramide.
BD-fmk, but not the p38 MAP kinase inhibitor
SB202190, is able to block the DNA fragmentation
(Fig. 5A) and PKC-d or PARP degradation (Fig. 5B) of
cells induced by prolonged ceramide treatment. In the
case of PARP or PKC-d, degradation was only partially
inhibited by BD-fmk, which is consistent with our recent
observation that PARP can also be hydrolyzed by
caspase-independent pathway (Yang et al., 2003). Unexpectedly, BD-fmk either used alone or together with
SB202190, failed to block the cell death induced by
prolonged ceramide treatment (Fig. 5C), suggesting that
caspase- and p38-independent mechanisms were also
implicated in ceramide-induced apoptosis which occurred in the later period of the treatment.
C8-ceramide-induced caspase-dependent
programmed cell death is associated with the
decrease in the S phase proportion
By synchronization of cell cycle, we were able to
further observe the effect of ceramide in a condition in
Apoptosis is mediated by various cellular events including protein synthesis and degradation, the alteration in protein phosphorylation states, the activation
of lipid second messenger systems, and disruption of
normal mitochondrial function. It has been shown that
ceramide is able to activate a genetically active and
regulatable suicide program (Belaud-Rotureau et al.,
1999; Engedal and Saatcioglu, 2001; Shi et al., 2001;
Lopez-Marure et al., 2002; Mochizuki et al., 2002),
which is distinct from the classic caspase-dependent
apoptotic cell death. In this study, we demonstrated that
caspase-independent apoptosis was induced by shorttime ceramide treatment, which was followed by both
caspase-dependent and -independent apoptosis. The
caspase-independent apoptosis that occurred in the
early stage of the ceramide-treatment cannot be determined by classic phenotypes of apoptosis. It thus
appears to be an atypical apoptotic cell death. The
caspase-dependent apoptosis was determined by unique
cellular phenotypes including the translocation of PS
from inner membrane to the outer membrane, loss of
mitochondrial transmembrane potential, activation of
caspase, and DNA fragmentation. Since ceramideinduced cell death varies with cell types and the cellular
environment, it is likely that the cellular context and the
environment may contribute to the types of the cell
death induced by ceramide.
It has been reported by several groups that p38
MAPK was involved in the mediation of ceramideinduced cell death (Brenner et al., 1997; Hida et al.,
1999; Shimizu et al., 1999; Willaime et al., 2001). In
ultraviolet B irradiation-induced apoptosis of human
keratinocyte HaCaT cells (Shimizu et al., 1999), p38
MAP kinase was considered as a signaling component
upstream of the caspase. In contrast, reported evidence
that does not support the role of p38 MAPK in the
mediation of ceramide-induced apoptosis has also
emerged (Jarvis et al., 1997; Scheid et al., 1999).
Although ERK MAP kinase can be down-regulated by
ceramide in some cell lines, our data demonstrated that
C8-ceramide can induce the activation of both ERK and
p38 MAP kinases in A-431 cells, which is consistent with
the reports that sphingomyelinase and ceramide activate ERK and p38 in HL-60 cells (Raines et al., 1993),
dermal fibroblasts (Reunanen et al., 1998), 184B5/HER
cells (Subbaramaiah et al., 1998), and NCI-H292 cells
(Chen et al., 2001). Since the inhibition of ERK
activation by MEK inhibitor PD98059 has no effect on
ceramide-induced cell death, whereas inhibition of p38
MAP kinase by specific inhibitor SB202190 suppressed
the ceramide-induced death in A431 cells, it thus
appears that the activation of p38 but not ERK pathway
CERAMIDE-INDUCED CELL APOPTOSIS
53
plays an important role in cell apoptosis induced by
short-term ceramide treatment. Short-time ceramide
treatment induces the cell death without the appearance of the typical apoptotic phenotypes including the
DNA fragmentation, caspase activation, PKC-d and
PARP degradation, and cannot be inhibited by caspase
inhibitor, suggesting that this type of cell death is a
caspase-independent atypical apoptosis. Since the inhibition of p38 MAP kinase strongly suppresses the shorttime ceramide treatment-induced cell death, the result
indicates that p38 MAP kinase plays an important role
in the caspase-independent cell death induced by shorttime ceramide treatment. Longer time treatment of cells
with ceramide (over 12 h) will induce the classic
apoptotic phenotypes, such as PS translocation from
inner side to the outer side of the plasma membrane, loss
of mitochondrial potential, caspase activation, and DNA
fragmentation. Because caspase inhibitor effectively
inhibits the long-term ceramide treatment-induced
DNA fragmentation and partially inhibits PKC-d and
PARP degradation, but has no effect on the ceramideinduced cell death, it suggests that both caspasedependent and -independent mechanisms were involved
in the apoptosis induced by long-term ceramide treatment. The fact that ceramide-induced caspase-independent cell death can be blocked by inhibiting p38 MAP
kinase also implies that such cell death is not a passive
process (necrosis) but an active and regulated apoptotic
cell death.
It has been reported that ceramide acted as a cell cycle
suppressor which can lead to G1/G0 arrest (Dbaibo et al.,
1995; Hannun, 1996; Lee et al., 1998). In this study, we
observed a reduction in S phase proportion of cells when
ceramide treatment was conducted after cell cycle
synchronization. Moreover, cells that decreased in
S phase proportion in response to ceramide treatment
was associated with an increase in sub-G1 DNA
contents, indicating that DNA cleavage and therefore
the caspase-dependent apoptosis was induced. The dying of cells in response to ceramide may contribute to the
drop of S phase proportion because the cell cycle was just
delayed but not arrested. The results imply that the G1
cells cannot pass through the G1/S check point normally,
which may render the cells more sensitive to ceramide
stimuli and the subsequent cell apoptosis. The data
presented by two groups on the doxorubicin-induced
apoptosis in SH-SY5Y cells (Di Bartolomeo et al., 2000)
and the C2-ceramide-induced apoptosis in HL-60 cells
(Bartova et al., 1997) also suggested these possibilities.
It has been reported recently that p38 and ERK MAP
kinases are activated in ceramide-, taxol-, etoposide-, and
Fig. 3. Effect of MAP kinase inhibitors on ceramide-induced cell
apoptosis. A and B: The effect of SB202190 on the C8-ceramideinduced cell apoptosis as determined by FACS assays. Cells were
treated with C8-ceramide (25 mM) for indicated time in the presence
or absence of SB202190, PD98059. Cell apoptosis were determined
by annexin V/PI (A) or DiOC6(3)/PI (B) staining assay. C: The effect
of p38 inhibitor on ceramide-induced apoptosis as measured by
DNA fragmentation. Cells were treated with C8-ceramide (25 mM) in
the presence or absence of selective p38 MAP kinase inhibitor
SB202190 (mM) for indicated time. DNA was extracted and DNA
fragmentation was examined. The data are the represented results
from two or three independent experiments.
54
ZHAO ET AL.
Fig. 4. Ceramide induced caspase activation and PARP and PKC-d
cleavage. A-431 cells cultured in DMEM containing 10% FCS were
treated with C8-ceramide (25 mM) for 24 h in the presence or absence of
caspase inhibitors pre-added 10 min before the treatment. A: The
effects of different caspase inhibitors on ceramide-induced caspase
activity. Data are the means SD of three independent experiments.
Statistic analysis was performed by comparing the last two bars with
the second bar for each groups. *, **, significant at P < 0.05 and
0.01 respectively. B: Time-dependent PARP degradation induced by
C8-ceramide. C: Time-dependent PKC-d degradation induced by C8ceramide. The data represents one of two or three independent
experiments with the same results. Daunorubicin (DNR, 2.5 mM for
6 h) was used as a positive control.
serum starvation-induced apoptosis in A-431 cells,
though the data indicated that neither p38 nor ERK
MAP kinase play essential roles in those stimuliinduced apoptosis (Boldt et al., 2002). In line with these
reports, our data showed that the inhibition of p38 MAP
kinase and/or caspases failed to inhibit the cell death
induced by prolonged ceramide treatment, further indicating that the A-431 cell apoptosis induced by longterm ceramide treatment is different from that induced
by short-term ceramide treatment, and an unidentified
signaling pathway may be implicated in the former. In
summary, the presented data indicate that ceramideinduced apoptosis of A-431 cells is a progressive biol-
ogical process. In the relative early stage of the ceramide
treatment, only caspase-independent apoptosis was
involved, which can be blocked by inhibiting the p38
MAP kinase but not caspases. As the ceramide treatment continues and the progress of apoptotic events,
both caspase-independent and -dependent cell suicide
program were adopted. The data demonstrate that
differential signaling pathways might be involved in
the regulation of ceramide-induced apoptosis: though
the mechanism remains to be identified, long-term
ceramide treatment-induced apoptosis involves both
caspase-dependent and -independent events and was
associated with cell cycle-related signaling events,
55
CERAMIDE-INDUCED CELL APOPTOSIS
Fig. 6. The decline in the S phase proportion of cell cycle in ceramideinduced caspase-dependent programmed cell death. A-431 cells were
synchronized and then treated with 25 mM C8-ceramide in normal
culture medium containing 10% FCS for indicated time. The cell cycle
was examined as described in ‘‘Materials and Methods.’’ The data are
the represented results from three independent experiments.
whereas short-term ceramide treatment-induced caspase-independent apoptosis was mediated via mechanism in which p38 MAPK plays an important role.
ACKNOWLEDGMENTS
We thank Dr. Hehua Chen and Yiran Zhou for their
helpful discussions.
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