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Introduction
Ceramide is a membrane sphingolipid that has recently
emerged as a second messenger involved in the induction of
apoptosis[1_4]. It can be generated either by
sphingomye-linases or de novo synthesis. It plays an important role in
activating cell death signals that are initiated by stress stimuli
such as chemotherapeutic agents, cytokines, and ionizing
radiation. Recent research indicates that exogenous ceramide
causes apoptotic cell death in human lung adenocarcinoma
A549 cell cultures[5_7].
The target molecules and terminal effectors of the
ceramide-mediated apoptotic pathway have not been
completely defined, and in lung cells they are unknown.
Mitogen-activated protein kinases (MAPK) are a diverse
superfamily of serine/threonine kinases that are conserved
phylogenetically. There are 3 "classic" MAPK families:
extracellular signal-regulated kinases (ERK1 and 2), the p38
MAPK (p38 α, β, δ, and γ), and c-Jun N-terminal kinases (JNK
1, 2, and 3)[8]. Individual MAPK is phosphorylated and
activated by MEK (mitogen-activated protein kinase kinase,
protein kinase that is usually specific for particular MAPK) and
controls distinct cellular processes. Ceramide regulation of
ERK1/2 activity is complex. There are certain cellular models
where ceramide treatment may cause the activation of
ERK1/2, whereas it appears to have an inhibitory effect in other
models[9,10]. Under certain conditions, ceramide activates p38
and JNK MAPK in postmitotic cells, a step that may be
critical for ceramide-induced apoptosis. Recent studies indicate
that specific protease activation by ceramide may play a
crucial role in the apoptotic process. These proteases, termed
caspases for cysteinyl aspartate-specific protease, are
members of a cysteine protease family. Studies with ceramide
analogs or chemotherapeutic agents suggest that ceramide
accumulation induces the activation of
caspase-3[11,12].
Targeting the ceramide metabolic pathway is an
attractive strategy to induce lung cancer cells to become apoptotic.
However, a better understanding of the mechanisms leading
to ceramide-mediated apoptosis is required. The role of the
p38 MAPK in ceramide-induced apoptosis has been
contro-versial, and a positive association has been identified, but
mainly in neuronal cells[13,14]. On the other hand, although
some reports have demonstrated that exogenous ceramide
causes apoptotic cell death in human lung adenocarcinoma
A549 cell cultures[5_7], the exact roles for MAPK in
ceramide-induced cell death in A549 cells are unknown. Therefore, in
order to provide experimental data for further research on
the signal transduction of apoptosis in lung adenocarcinoma
cells, we examined the effects of C2-ceramide (a non-natural,
but cell-permeable analog of the endogenous long-chain
ceramides) administration on several members of the MAPK
superfamily and caspase-3 in A549 cells.
Materials and methods
Materials Cell culture growth media, buffers, and fetal
bovine serum (FBS) were obtained from Life Technologies
(Grand Island, NY, USA). C2-ceramide was from Sigma
Chemical (St Louis, MO, USA). SB-203580 and U0126 were from
Promega (Madison, WI, USA). Cell counting kit-8 (CCK-8)
was from Wako Fine Chemicals (Osaka, Japan). Propidium
iodide (PI) staining reagents for flow cytometry were
purchased from Sigma Chemical (St
Louis, USA). Bradford
protein assay and Western analysis reagents and equipment
were from Bio-Rad (Hercules, CA, USA). The following
antibodies used for Western blotting were from Cell Signaling
Technology (Beverly, MA, USA): phospho-MEK1/2
(Ser217/221) antibody, phospho-p44/42 MAPK (Thr202/Tyr204)
antibody, phospho- SAPK (stress-activated protein
kinase)/JNK (Thr183/Tyr185) antibody, phospho-p38 MAPK
(Thr180/Tyr182) antibody, caspase-3 antibody, and cleaved
caspase-3 antibody. p38 siRNA used siGenome
SMARTselec-tion™ designed siRNA duplexes against p38 (MAPK14,
Dharmacon, RNA technologies, Lafayette, CO, USA).
Cell culture Human lung adenocarcinoma A549 cells
were obtained from the American Type Culture
Collection (Rockville, MD, USA) and maintained in DMEM (Dulbecco's
Modified Eagle Medium) supplemented with 1%
nonessential amino acids, 1% penicillin-streptomycin, and 10%
FBS. Cells were seeded at a density of
1×106_5×106 cells/mL and
incubated at 37 °C in a humidified atmosphere containing
5% CO2. After C2-ceramide (dissolved in DMSO) treatments,
the cells were removed from the culture plates by incubation
with 0.05% trypsin-EDTA (ethylenediaminetetraacetic
acid) and washed twice with ice-cold phosphate buffered saline
(PBS), and the cell pellets were used for the
different assays.
Analysis of cell viability and apoptosis
Trypan blue exclusion assay The effects of
C2-cera-mide on A549 cell growth were determined by trypan blue
exclusion assay. In short, cells seeded as
3×105 cells/dish were treated in the absence or presence of various
concentrations of C2-ceramide for 24 h. Then the cells were
trypsinized and diluted in PBS. The floating dead cells in the
medium and the cells that remained attached to the plates
were then counted using a hematocytometer in the presence
of trypan blue solution at a 1:1 ratio (v/v) (Sigma, USA) as
described by the manufacturer.
Cell growth assay The measurement of cell
proliferation was performed using 96-well cell culture plates (BD
Falcon, Franklin Lakes, NJ, USA). A549 cells were seeded
onto the plates at an initial density of approximately
0.1×105_0.5×105 cells in DMEM containing 10% FBS per well, and the
total volume was 200 µL after adding different
concentrations of C2-ceramide into each well. Where appropriate, cells
were pretreated for 1 h with SB-203580 (2.5_20 µmol/L, a p38
MAPK inhibitor) or U0126 (1_6 µmol/L, a MEK inhibitor).
Then the cells were incubated for 24 h to allow adhesion.
Cells cultured without test material served as controls. The
number of cells was determined using CCK-8. Ten μL of the
CCK-8 solution was added to each well of the plate. The
plate was incubated for 1 h in the humidified incubator (at
37 °C, 5% CO2). After incubation, cell growth was
determined colorimetrically at 450 nm (Titertec Multiskan Plus,
Flow Laboratory, McLean, VA, USA). The cell number was
calculated from the absorbance value relative to a standard
curve.
Cell apoptosis detected by fluorescence activated
cell sorter (FACS) analysis After treatment with
C2-ceramide(100 µmol/L ) for 24 h, the cells were harvested by
trypsinization and centrifuged at 400
×g for 15 min, stained with PI (50 µg/mL), then immediately analyzed by flow
cytometry (FACSCaliber, Becton Dickson, Franklin Lake, NJ,
USA). Experiments were performed in triplicate and a total of
10 000 cells were analyzed in each individual experiment.
Western blotting Western blotting analysis was done
as described previously[15]. In brief, cells were lysed in lysis
buffer {1% Triton X, 5 mmol/L EDTA, 5 mmol/L EGTA [Ethyleneglycol
bis(2-aminoethylether)-N,N,
N',N'-tetraacetic acid], and protease inhibitor cocktail (Sigma, USA) in PBS},
and the samples were centrifuged at 10 000
×g for 10 min. Protein concentrations in the samples were determined with
the Bradford protein assay kit (Bio-Rad, USA) and bovine
serum albumin as the standard. Samples containing equal
amounts of protein (40 µg )were resolved by SDS-PAGE and
transferred to nitrocellulose membranes (Bio-Rad, USA),
blocked, and incubated overnight with specific primary
antibodies in 0.1% Tween-20, 5% nonfat dried
milk in 10 mmol/L Tris-HCl and 150 mmol/L NaCl (pH 7.5). Immunoreactive
bands were visualized using the enhanced
chemiluminescence Western blotting protocol (Amersham Pharmacia,
Piscataway, New Jersey, USA).
p38 siRNA transfection Equally seeded wells of A549
cells were transiently transfected with 120 nmol/L siRNA
using Lipofectamine Plus TM reagent (Invitrogen, Carlsbad,
CA, USA) according to the manufacturer's instructions. As
the control, cells were treated with Lipofectamine Plus TM
reagent alone.
Statistical analysis All statistical analyses were
performed by SPSS software. We used t-test. In all cases, the
minimal requirement for establishing significance was 0.05.
Results
Detection of the effects of C2-ceramide on A549 cell
survival and apoptosis The effects of C2-ceramide on A549
cells survival were examined by trypan blue exclusion assay,
CCK-8 assay and FACS analysis. Treatment of A549 cells
with increasing concentrations of C2-ceramide was done.
At 24 h, the cells were harvested, and the survival rate of the
A549 cells was measured and compared to time-matched
controls. As shown in Figure 1, it was found that at the
concentration of 10 µmol/L C2-ceramide, a 17.93% decrease
in the cell survival rate was detected. Furthermore, in the
presence of 50 µmol/L and 100 µmol/L C2-ceramide,
substantial drop in the survival rate was observed, since 72.31%
and 78.68% of cells, respectively, were killed. On the other
hand, incubation with C2-ceramide (100 µmol/L) for 3, 6, 12,
and 24 h resulted in the survival rate decreasing to 68.36%,
36.11%, 34.72%, and 21.32%, respectively (Figure 1). As
demonstrated in Figure 2, the absorbance of
C2-ceramide-treated cells decreased with the increasing concentration of
C2-ceramide. Compared with the control (DMEM with A549
cells), at the concentration of 50 µmol/L C2-ceramide, the
absorbance of A549 cells reduced to 85.26%
(P<0.01). With concentrations of 100 and 200 µmol/L, there was a
significant decrease in the absorbance of the A549 cells, which
was 63.82% and 61.37%, respectively, versus the control
(P<0.01). Through the dose-response experiments, we
established that 100 µmol/L is one of the lowest concentrations of
C2-ceramide capable of inducing a significant degree of
apoptosis in A549 cells after 24 h of treatment. Therefore,
this C2-ceramide concentration was used throughout our
studies. As shown in Figure 3, C2-ceramide (100 µmol/L)
induced apoptosis in A549 cells in a time-dependent fashion.
Changes of MAPK during C2-ceramide induced A549
cell death or apoptosis By Western blotting analysis, we
examined the changes of MAPK during C2-ceramide induced
A549 cell death or apoptosis. As demonstrated in Figure 4,
C2-ceramide treatment of A549 cells caused major changes
in the phosphorylation state of MAPK. Compared to the
control samples (DMEM with A549 cells), in the
C2-ceramide-treated A549 cells, the phosphorylation of MEK1/2 (Figure
4A) and p38 (Figure 4D) increased significantly in a
time-dependent manner. These changes were evident as early as
3 h after C2-ceramide administration and were maximal
between 6 and 12 h. But JNK (Figure 4B) and ERK1/2 (Figure
4C) phosphorylation showed no significant alteration
during the same time frame.
Changes of caspase-3 during C2-ceramide induced A549
cell death or apoptosis The role of caspases in the execution
phase of apoptosis is well established. The report that
exposure of primary human lung cancer A549 airway epithelial
cells to H2O2 induce a rapid accumulation of cellular ceramide
and increase the apoptotic populations of these cells led us
to examine the effects of C2-ceramide on caspase activation,
focusing on caspase-3, the last caspase to be activated in
the execution phase of
apoptosis[2,16,17]. Compared with the control samples, in the C2-ceramide-treated A549 cells,
procaspase-3 (an inactive form of caspase-3) increased
remarkably as early as 6 h after C2-ceramide administration
and was maximal at 12 h. The active form of caspase-3,
cleaved caspase-3, was detected at 6 h and enhanced
notably at 12 h after C2-ceramide treatment (Figure 5).
Effects of inhibitors of the p38 MAPK and MEK1/2
We used CCK-8 assay to determine the intervening effects of
SB-203580 (pyridine imidazole compounds that specifically
inhibit p38 MAPK α and β, but not p38 δ and γ) and U0126
(a specific inhibitor of the ERK1/2 activator kinases, MEK1/2).
In contrast to the C2-ceramide-treated cells, SB-203580
pretreatment (at a concentration of 20 µmol/L) improved cell
viability significantly after C2-ceramide treatment, whereas
U0126 pretreatment had no significant effect on C2-ceramide
induced cell death even at the concentration of 6 µmol/L
(Figures 6, 7).
To investigate the vital role of the p38 MAPK in
C2-ceramide induced apoptosis, we employed p38 siRNA. We
confirmed our findings by transfecting the cells with p38
siRNA oligonucleotides and determining the rates of
apoptosis following treatment with 100 µmol/L C2-ceramide.
To determine the efficiency of p38 siRNA on inhibiting p38
expression, Western blotting was performed in the presence
and absence of C2-ceramide. We found a marked decrease
in p38 expression in the A549 cells following transfection
with the siRNA at baseline and after treatment with 100
µmol/L C2-ceramide (Figure 8).We found that p38 siRNA had no
effect on basal rates of apoptosis, but significantly decreased
apoptosis induced by 100 µmol/L C2-ceramide compared to
nontransfected cells (Figure 9). These data suggest that p38
activation mediates apoptosis induced by 100 µmol/L
C2-ceramide in A549 cells.
Discussion
Recent experimental evidence supports a role for
C2-ceramide (an exogenous analog of ceramide) in the
induction and progression of human lung adenocarcinoma A549
cell death[5,6]. In the present report, we examine cell survival
in A549 cells exposed to exogenous C2-ceramide and changes
in MAPK phosphorylation and caspase-3 activation.
C2-ceramide induced evident alterations in the
phosphorylation of MEK1/2, and the p38 MAPK, but had no effect on
ERK1/2 and JNK. The p38 inhibitor, but not the MEK1/2
inhibitor, partially attenuated ceramide-induced cell death.
Our data also show C2-ceramide-induced apoptosis through
the activation of caspase-3.
Cell cycle checkpoints are important mechanisms that
ensure the proper execution of cell cycle events. MAPK are
the major molecular players in cell cycle progression.
Recently, many studies have shown that the inhibition of
MAPK activity, which leads to cell cycle arrest, has turned
out to be the productive strategy for the discovery and
design of novel anticancer of cancer cells by causing cell cycle
arrest and apoptosis. Therefore, we used specific inhibitors
for the MEK1/2 and p38 MAPK pathways. The p38 MAPK
inhibitor (SB-203580) partially attenuated ceramide-induced
apoptosis, whereas the MEK1/2 inhibitor (U0126) had no
significant effect on C2-ceramide-induced cell death. These
results indicated that SB-203580, but not U0126, partially
rescued cell death induced by C2-ceramide. Thus, the p38
MAPK was partially involved in C2-ceramide-induced A549
cell death or apoptosis. The high specificity demonstrated
by these inhibitors for their enzyme targets, as well as the
fact that we used moderate concentrations and included
inactive controls, is consistent with our conclusion
regarding the modulatory roles of MAPK in ceramide-induced
apoptosis.
Although often associated with promoting cell survival,
ERK1/2 activation has also been linked to cell
death[9,10]. It appears that the ERK1/2 pathway can influence the balance
between cell survival and cell death in both ways,
depending of the specific cellular system and its condition. To date,
the mechanisms underlying the deleterious effects of
ERK1/2 remain unclear. In our experiments, although the
phosphorylation of MEK1/2 was increased, C2-ceramide did not
affect ERK1/2 in A549 cells. Moreover, U0126 did not
attenu-ate A549 cell death significantly. This is partly because the
MEK1/2 and ERK1/2 pathways were not crucial in the
signaling cascades of C2-ceramide-induced A549 apoptosis.
Our results support and extend, but also differ in several
significant regards, from those reported by another group.
Although we confirmed a role for the p38 MAPK in
ceramide-induced A549 cell death, Newton et
al[7] found that ceramide was an efficient inducer of the ERK, but not the p38 MAPK.
We believe that these dissimilarities reflect differences in
ceramide treatment conditions and A549 culture systems. In
our research, A549 cells were administrated C2-ceramide (100
µmol/L) for 3, 6, and 12 h, respectively. Newton
et al only treated cells with C2-ceramide (100 µmol/L) for 15, 30, or 45
min, respectively. So the unobvious change of the p38
MAPK was probably due to the short duration of the ceramide
treatment. Furthermore, the difference of the A549 culture
system is also possibly responsible for the diversity of the
results. We employed DMEM supplemented with 10% FBS
in the cell culture system, whereas they used serum-free
media in order to concentrate on observing the effects of
ceramide on cyclooxygenase-2 prostaglandin E2 and nuclear
factor-kappaB (a kind of intracellular signaling protein).
In our experiments, C2-ceramide did not influence the
phosphorylation of JNK. This indicates that JNK did not
participate in ceramide-induced apoptosis. Kurinna
et al[5] reported that ceramide promoted apoptosis in A549 cells by
a mechanism involving JNK. The JNK inhibitor SP 600125
proved effective at protecting cells from the lethal effects of
ceramide. To understand which JNK-mediated pathway may
be involved, a number of JNK target proteins were examined.
At last they found that ceramide promoted the
phosphorylation of Bim (a pro-apoptotic protein in Bcl-2 family members)
and induced translocation of active JNK from the nucleus to
the cytoplasm and mitochondrial fraction.
Ceramide-mediated changes in the localization of JNK were consistent with
the observed changes in the phosphorylation status of
c-Jun and Bim. Furthermore, ceramide promoted Bim
translocation to the mitochondria. The mitochondrial localization
of Bim has been shown recently to promote apoptosis. These
results suggest that JNK may participate in ceramide-induced
apoptosis in A549 cells by a mechanism involving Bim. In
conjunction with their results, JNK may play a role in
ceramide-induced A549 cells apoptosis by other manners,
but does not influence the activation of JNK.
Our results also show that C2-ceramide promoted the
expressions of procaspase-3 and caspase-3 that is known to
involve cell cycle arrest and apoptosis. Similarly, Ravid
et al[5] elucidated the link between caspase-3 activation at the
execution phase, and ceramide accumulation, at the
commitment phase of apoptosis in A549 human lung
adenocarcinoma cells. They concluded that ceramide accumulation
precedes caspase-3 activation during apoptosis of A549
human lung adenocarcinoma cells. Caspases play critical
roles in the initiation of apoptosis. It is well known that 2
apoptotic pathways called caspases dependent and
independent are being reported in mammalian cells. Based on
the substrate specificities and target proteins of caspases,
caspases can be grouped as "apoptotic initiators", such as
caspase-8, and "apoptotic effector", such as caspase-3. In
our study, cleaved caspase-3 induced by C2-ceramide was
observed at 6 h, and then up to 12 h (Figure 6). We also
found that C2-ceramide decreased the expression of the
pro-apoptotic proteins in Bcl-2 family members such as Bax and
Bad in A549 cells. We also used flow cytometry assay for
caspase-3 activity where it was indicated that C2-ceramide
promoted the activity of caspase-3 (data not shown).
In summary, our results indicate that exogenous
C2-ceramide induces apoptosis in human lung adenocarcinoma
A549 cells. Several numbers of MAPK and caspase-3 were
involved in the mechanisms of ceramide-induced apoptosis
in A549 cells. Our research provides experimental data for
further research on the signal transduction of apoptosis in
lung adenocarcinoma cells. The further research on
ceramide-induced apoptosis is a promising and encouraging career
and will contribute to the exploration and development of
new anticancer therapeutic drugs[18_23].
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