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Introduction
Cell proliferation, differentiation and apoptosis are
central features of tissue homeostasis, and inhibition of
apoptosis might be involved in the pathogenesis of cancer
by prolonging cell life and sustaining growth of malignant
tissues[1,2]. Protein kinase C
(PKC) is a family of phospholipid-dependent serine/threonine kinases and participates in
many cellular responses, including cell apoptosis, which can
be inhibited by staurosporine[3-5]. The family contains at
least 12 isoforms, and specific roles in cell cycle progression
and in apoptosis have been hypothesized for the different
PKC isoenzymes. Of these PKC isoforms, PKCa is
distributed in almost all tissues and is involved in various signal
transductions. Considerable evidence suggests that
PKCa plays an important role in the apoptosis of some tumor cells
and that the inhibition of PKCa might induce
apoptosis[5,6]. In contrast, one recent study reported that
elevated expression of PKCa might promote the apoptosis of gastric cancer
cells[7]. However, little is known about the protein level and
subcellular distribution of PKC a in the apoptosis of oral
cancer cells. Elucidating the patterns of PKCa action will
lead to a better understanding of the molecular mechanism
of the PKCa signal pathway involved in the apoptosis of
oral cancer cells.
The activation of caspases, particularly caspase-3,
appears to be a central part in most apoptotic pathways and
most types of cells[8,9]. Caspases have been shown to be
cleaved and possibly activated by PKCs suggesting that
they act downstream of PKCs as effectors in the apoptotic
machinery[6]. Survivin, a recently characterized inhibitor of
apoptosis protein (IAP), is abundantly expressed in oral
squamous cell carcinoma (OSCC), but undetectable in normal oral
tissues[10,11], suggesting a potential role in oral carcinogenesis.
However, no direct molecular interaction has been described
so far between PKCa, survivin and caspase-3 in mediating
apoptosis of oral cancer cells.
In the present study, we investigated the functional and
molecular interactions among PKCa, survivin and caspase-3
in apoptosis induced by staurosporine with the aim of
further highlighting their relevance in OSCC biology.
Materials and methods
Reagents Fetal bovine serum (FBS) and Dulbecco¡¯s
modified Eagle¡¯s medium (DMEM) were obtained from Gibco
Laboratories (Gibro/BRL, Grand Island, NY). MTT, acridine
orange (AO), ethidium bromide (EB), propidium iodide (PI) and
staurosporine were obtained from Sigma Biotechnologies
(Sigma-Aldrich, Inc, Saint Louis, Missouri, USA).
Staurosporine was dissolved in ethanol to make a stock
solution of 1 mmol/L and diluted to their final concentrations in
the culture medium. The final concentration of ethanol never
exceeded 0.01%, a concentration at which there is no
discernible effect on tongue squamous cell carcinoma (TSCCa)
cells in comparison with the control. Primary antibodies
including mouse monoclonal antibodies to PKCa (H-7,
sc-8393), b-actin (C-2, sc-8432), caspase-3 (E-8, sc-7272) and
rabbit polyclonal antibody against survivin (FL-142, sc-1081)
were obtained from Santa Cruz Biotechnology (Santa Cruz,
California, USA). An enhanced chemiluminescent detection
system (ECL kit) from Amersham international Plc (Amersham
Biosciences, Buckinghamshire, UK) was used for Western
blot analysis. All other reagents were analytical reagents.
Cell culture TSCCa cell line was established by Dr
Hui-xi JIN from a patient with OSCC of the
tongue[12]. TSCCa cells were cultured in DMEM, supplemented with 10%
heat-inactivated FBS, 100 kU/L penicillin, and 0.1 g/L streptomycin.
The cells were maintained at 37 °C in a humidified
atmosphere of 5% CO2. TSCCa cells were cultured and treated
with either 0.01% ethanol (vehicle) or 1-100 nmol/L
stauro-sporine. The treated cells were examined by phase contrast
microscope (Nikon Instech Co Ltd, Kanagawa, Japan) for
evidence of morphological changes induced by staurosporine
treatment.
MTT assay Cell viability and activity were detected
using MTT dye assay, in which the dye was converted into
formazan granules in the presence of reactive oxygen. TSCCa
cells were plated at a density of
5×103 cells per well on a
96-well plate. At 24 h after seeding, straurosporine (1-100
nmol/L) was added to the culture medium. At 6 h, 12 h, and
24 h after drug treatment, MTT was added to each well at a
concentration of 500 mg/L and incubated for 4 h at 37 °C.
After that, media were aspirated and cells were
solubilized in 400 mL Me2SO. Cells were incubated for 10 min at 37
°C with gentle shaking. Absorbance was measured at 540 nm using
a computerized microplate analyzer.
AO/EB staining To assess apoptosis, cells were stained
with AO/EB. In brief, after TSCCa cells were treated with 100
nmol/L straurosporine for 24 h, nonadherent cells in the
medium and trypsinized adherent cells were centrifuged at
200×g for 10 min at 4 °C and all but 50 µL media was removed.
The pellet was resuspended with 2 µL for each 0.1 g/L AO
and EB. Cells were immediately viewed using a fluorescence
microscope (Leica Microsystems AG, Wetzlar, Germany).
Morphology was defined according to descriptions from Kern
and Kehrer for general apoptotic
characteristics[13].
Flow cytometry For flow cytometric assessment of
apoptosis and cell cycle phases, TSCCa cells were fixed with
ice-cold 75% ethanol following 100 nmol/L staurosporine
treatment for 6 h, 12 h or 24 h, and washed twice with
phosphate-buffered saline (PBS). Cells were incubated at 37 °C
for 30 min in PBS containing 1 g/L RNase, then stained with
100 mg/L PI. Cellular DNA content was measured by Beckman
Coulter Epics Altra II cytometer (Beckman Coulter, California,
USA).
PKCa subcellular fractionation and Western blot
analysis TSCCa cells treated with 100 nmol/L staurosporine for 12
h and 24 h were washed twice with PBS and were then scraped
from the culture vessels and collected. Harvested cells were
suspended in 0.5 mL of hypotonic buffer [10 mmol/L
N-2-hydroxyethylpiperazine-N¡¯-2-ethanesulfonic acid (HEPES, pH
7.4), 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.2 mmol/L
phenyl-methylsulfonyl fluoride (PMSF), 0.5 mmol/L DTT] and
homo-genized with a Dounce homogenizer at 4°C. Unlysed cells,
nuclei and cell debris were pelleted by centrifugation at
1000×g at 4 °C for 5 min . The supernatant was centrifuged at
100 000×g at 4 °C for 1 h . The
100 000×g supernatant was designated as a cytosol fraction. The
100 000×g pellet was suspended in hypotonic buffer containing 1% Triton X-100
and centrifuged at 10 000×g for 10 min, and the resulting
supernatant is referred to as the membrane fraction. Protein
samples were mixed with an equal volume of 2×sodium
dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) sample buffer, boiled for 5 min, and then separated
using 10% SDS-PAGE gels with 20 µg of protein loaded per
lane. After electrophoresis, proteins were transferred to
PVDF membranes by semi-dry electrophoretic transfer. The
membranes were blocked in 5% dry milk (1 h), rinsed and
then incubated with primary antibody of PKCa (1:1000
dilution) overnight at 4 °C. The primary antibody was
removed, membranes were washed 4 times, and 0.1 mg/L
peroxidase-labeled goat secondary antibody was added for
1 h. Bands were then visualized by ECL kit and exposed to
X-ray film. Percentage of membrane or cytosol band was
calculated by a Bio-Rad (Richmond, CA) model GS-670
Imaging Densitometer.
Immunocytochemistry TSCCa cells were seeded on
8-chamberred glass slides and treated with 100 nmol/L
stauro-sporine for 12 h and 24 h as described above. Cells were
fixed for 10 min in 100% methanol at room temperature. Then,
cells were washed with PBS, treated with 3% hydrogen
peroxide to block endogenous peroxidase activity and incubated
with normal serum. After a 2-h incubation with primary
antibodies, cells were washed 3 times with PBS and
incubated with biotin-labeled anti-mouse or anti-rabbit
immuno-globin G for 20 min. After washing 3 times with PBS, cells
were stained using the UltraSensitive
streptavidin-peroxidase detection system (Maixin Biotechnology, Fuzhou,
China). Positive reaction was seen as brown staining. One
hundred cells were counted to determine the intensities of
protein expression of caspase-3 and survivin by using
HPLAS-2000 analysis software (Qianping Biotechnology,
Wuhan, China).
Statistical analysis Data were expressed as mean±SD.
Statistical significance was assessed with one-way
ANOVA followed by Duncan¡¯s multiple-range test.
P<0.05 was considered statistically significant.
Results
Effect of staurosporine on cell viability in TSCCa cells
In the present study, the effect of staurosporine on TSCCa
cell viability was evaluated using a MTT assay.
Stauro-sporine exerted a marked dose- and time-dependent
inhibitory effect on the viability of TSCCa cells. The effect was
observed at 12 h after treatment, and increased with time, so
that after 24 h with 100 nmol/L staurosporine cell viability
was reduced to only 24% of the control (Figure 1).
Morphological changes in TSCCa cells induced by
staurosporine To address the ability of staurosporine to
induce cell death, we first investigated the effect of
stauro-sporine on cell morphological changes using a phase
contrast microscope. Numerous TSCCa cells exhibited a flat
appearance in the absence of staurosporine (Figure 2A). After
incubation with 100 nmol/L staurosporine, TSCCa cells
presented remarkable morphological changes. TSCCa displayed
a long spindle shape 6 h after stimulation (Figure 2B). Then
the number of TSCCa cells decreased, with an increased,
large amount of intracellular vesicles (Figure 2C). After 24 h
of staurosporine stimulation, we observed that treatment
resulted in cells exhibiting evidence of apoptosis, including
cell detachment, loss of cell processes and membrane
shrin-kage, as evidenced by curling up of cells (Figure 2D).
Morphological changes of TSCCa cells were also
investigated by AO/EB staining using fluorescence microscopy.
Live cells stain only with AO, which shows up green inside
the cell. Apoptotic cells induce fragmentation of the yellow
organelle. Necrotic cells stain with EB, and are detected by
their red color. The images in Figure 3 show that with a dose
of 100 nmol/L staurosporine treatment for 24 h, there were
increasing amounts of swollen and distorted cells coupled
with organelle disintegration and a rise in necrotic cells.
DNA content analysis of TSCCa cells treated with
staurosporine To determine the effects of staurosporine on
cell cycle progression and apoptosis, TSCCa cells were
stained with PI and subjected to flow cytometric analysis. A
representative cell cycle profile of control cells is shown in
Figure 4A. Treatment with 100 nmol/L staurosporine for
various periods of time resulted in changed DNA content
profiles. Staurosporine treatment for 6 h and 12 h caused an
accumulation of the cells in G2/M phase of approximately
26.6% and 34.0%, respectively; and that of the control was
approximately 9.9% (Table 1). This indicates that the
appearance of a G2/M arrest of staurosporine in TSCCa cells
is time-dependent. With the time prolonged, the
G2/M arrest caused by staurosporine released and cells returned to
G0/G1 phase and S phase. After 24 h of staurosporine
treat-ment, the percentage of cells in
G2/M decreased to 19.8%; however, this was still higher than that of the control (Figure
4).
The percentage of cells or cell fragments with DNA
content less than 2 N (hypodiploid or apoptotic shift) increased
in a time-dependent manner, from 2.9% in control cultures to
approximately 27.4% at 100 nmol/L staurosporine exposure
for 24 h (Table 1, Figure 4).
Changes in PKC a content and subcellular distribution
in TSCCa cells Because the translocation of PKC
a from cytosol to the membrane is a hallmark of PKC
a activation, Western blot experiments were performed to detect the
protein level of PKC a in cytosol and membrane of TSCCa cells
treated with 100 nmol/L staurosporine for different periods
of time. We found that PKC a is expressed in both cytosol
and membrane in ethanol-treated control TSCCa cells. After
treatment with staurosporine for various periods of time,
PKC a content in cytosol and membrane decreased in a
time-dependent manner (Figure 5). In addition, the percentage of
PKC a content in membrane versus cytosol decreased quickly
in staurosporine treated cells, from 0.45 in ethanol-treated
control cultures to 0.18 at staurosporine exposure for 24 h
(Table 2).
Effect of staurosporine on the activation of caspase-3
and the protein level of survivin To further characterize the
mechanisms that control apoptosis in TSCCa cells, protein
levels of caspase-3 and survivin in TSCCa cells treated with
100 nmol/L staurosporine for 12 h and 24 h was measured by
immunocytochemical staining. In untreated TSCCa cells,
there was a faint brown cytoplasmic staining for caspase-3
(Figure 6Aa). After incubation with staurosporine for 12 h
and 24 h, cytoplasmic caspase-3 staining was substantially
increased in a time-dependent manner (Figure 6B). TSCCa
cells with fusiform shape, numerous intracellular vesicles
and shrinking cells showed deep brown cytoplasmic
granules of caspase-3 staining (Figure 6Ab).
Furthermore, a strong cytoplasmic staining of survivin
was observed in control cells, especially in nuclear division
cells (Figure 6Ac). After incubation with staurosporine for
24 h, survivin expression was diminished (Figure 6Ad, 6B).
Discussion
The molecular mechanisms associated with apoptosis
have been widely explored, but are not yet precisely
understood[14]. Staurosporine has been shown to induce apoptosis
in a wide variety of cell types, such as rat cardiomyocytes,
human dermal papilla fibroblasts and luteinized granulose
cells. Therefore, staurosporine-induced apoptosis has been
recognized as a useful model for investigating the
mechanism of apoptosis in mammalian
cells[15]. To assess whether staurosporine induces apoptosis in oral cancer cells, 3
specific methods were used in the present study to evaluate
apoptosis. The presented data demonstrate that apoptotic
changes induced by staurosporine were confirmed by
morphological changes observed under phase contrast
micro-scopy, AO/EB staining and flow cytometric analyses of
cellular DNA content. A significant proportion of cells at
G2/M phase were also observed after 6 h treatment with
stauro-sporine, and appeared at peak after 12 h of treatment. With
the time prolonged, the G2/M arrest caused by staurosporine
released and cells returned to
G0/G1 phase and S phase after
24 h of treatment, whereas a significant proportion of cells
appeared at sub-G0/G1 apoptotic peak. Another report showed
that staurosporine induced apoptosis in Chang liver cells by
a mitochondria-caspase-dependent pathway, which can be
suppressed by z-VAD-fmk, a general inhibitor of caspases;
whereas the arrest of cells in G2/M phase of cell cycle was
not modified by z-VAD-fmk, suggesting that apoptosis and
G2/M arrest caused by staurosporine might be controlled in
different independent pathways[16]. Although cells with
typical apoptotic morphology were observed after the treatment,
we cannot exclude the possibility that some necrosis
occurr-ed with the high staurosporine dose. However, our findings
were consistent with other reports showing that
stauro-sporine at 100 nmol/L concentration can trigger apoptosis
regardless of the cell cycle
status[15].
To explore more precisely the mechanisms of
stauro-sporine-induced TSCCa cells apoptosis, we investigated
whether the PKCa signal pathway was involved, as it has
been shown that perturbation of PKCa activity can induce
or repress apoptosis[5-7]. We determined, using Western blot
analysis, that cultured TSCCa cells expressed PKC
a both in cytosol and membrane under 10% serum concentration.
When TSCCa cells were treated with staurosporine, content
of PKC a in cytosol and membrane decreased dramatically,
especially in membrane. One important factor in determining
specific functions of PKC isoforms is their intracellular
localization. PKC isoforms show different patterns of
subcellular localization, which can vary for the various isoforms
according to tissue and cell type. PKC a typically reside in
the cytosol in an inactive state. After cell stimulation, they
are often translocated to other compartments. Our results
suggest that inhibition of PKC a translocated from cytosol
to membrane might mediate staurosporine-induced TSCCa
cells apoptosis. It is not clear, however, how the complex
isoform-specific subcellular distribution and
stimulus-induced redistribution can be achieved. As staurosporine was
a wide inhibitor of PKC, we could not exclude the possibility
that other PKC isoenzymes could also mediate
staurosporine-induced TSCCa cells¡¯ apoptosis.
Caspase-3 has been reported to play a key role in
apopto-sis[8,9]. Caspase-3 normally exists in the cytosolic fraction of
cells as an inactive precursor, activating proteolytically when
cells are signaled to undergo apoptosis. Multiple apoptotic
signals, including serum withdrawal and treatment with a
variety of pharmacological agents, activate caspase-3. The
role of caspase-3 in apoptosis of oral cancer cells has not yet
been reported. Lewis et al report that PKC inhibition
induces DNA fragmentation in the colon cancer cell line, COLO
205 cells, which is blocked by cysteine protease inhibition
but not mediated through caspase-3[17]. However, we have
examined the caspase-3 content changes in TSCCa cells
after staurosporine exposure for various periods of time, and
have shown that staurosporine activates caspase-3 in a
time-dependent manner. These results suggest that caspase-3
was activated by staurosporine in TSCCa cells, and might
mediate staurosporine-induced TSCCa cells apoptosis.
Survivin is associated with the microtubules of the
mitotic spindle. Disruption of the survivin-microtubule
interaction leads to loss of survivin function and increased
proapoptotic caspase-3 activity[18]. In the present study,
the majority of ethanol-treated control TSCCa cells
over-expressed survivin, but this was not enough to protect
TSCCa cells from staurosporine-induced apoptosis.
Further-more, a dose-related decrease of survivin content in TSCCa
cells was associated with the induction of apoptosis by
staurosporine; these results suggest that survivin in TSCCa
cells could not protect TSCCa cells from the lethal effects of
staurosporine.
In conclusion, the present study demonstrates that
staurosporine induces apoptosis in TSCCa cells. Our
results document, for the first time, the potential roles of the
inhibition of PKC a in TSCCa cells¡¯ apoptosis. This
property of PKC a appears to be linked to the cleavage of survivin
and activation of caspase-3. Previous studies have reported
that PKC activation could cause stimulation of the
transcription factor NF-kB, which in turn induced expression of the
IAP[19-22]. As discussed in the present study, the IAPs are
known to block caspase-3 activity and apoptosis. Therefore,
it is possible that PKC a inhibition-mediated staurosporine
causes reduction of survivin and results in the activation of
the caspase-3 signaling pathway. Further study is needed
to understand the role of other caspases in
staurosporine-induced TSCCa cells apoptosis, which might be a potential
target in the treatment of oral cancer diseases.
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