Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 297-303
doi:10.1111/j.1745-7270.2008.00401.x
Inhibition of fibroblast
growth factor 2-induced apoptosis involves survivin expression, protein kinase
Ca activation and subcellular translocation
of Smac in human small cell lung cancer cells
Desheng Xiao#,
Kuansong Wang#,
Jianhua Zhou*, Huiqiu Cao, Zhenghao Deng, Yongbin Hu, Xiahui Qu, and Jifang Wen
Department of
Pathology, Xiangya School of Medicine, Central South University, Changsha
410013, China
Received: September
17, 2007
Accepted: February
13, 2008
This work was
supported by the Natural Science Foundation of Hunan Province (No. 06JJ2098)
#These authors
contributed equally to this work
*Corresponding
author: Tel, 86-731-2650406; Fax, 86-731-2650406; E-mail, [email protected]
To
investigate the mechanism by which fibroblast growth factor 2 (FGF-2) inhibits
apoptosis in the human small cell lung cancer cell line H446 subjected to serum
starvation, apoptosis was evaluated by flow cytometry, Hoechst 33258 staining,
caspase-3 activity, and DNA fragmentation. Survivin expression induced by FGF-2
and protein kinase Ca (PKCa) translocation was
detected by subcellular fractionation and Western blot analysis. In addition,
FGF-2-induced release of Smac from mitochondria to the cytoplasm was analyzed
by Western blotting and immunofluorescence. FGF-2 reduced apoptosis induced by
serum starvation and up-regulated survivin expression in H446 cells in a
dose-dependent and time-dependent manner, and inhibited caspase-3 activity.
FGF-2 also inhibited the release of Smac from mitochondria to the cytoplasm
induced by serum starvation and increased PKCa translocation from the
cytoplasm to the cell membrane. In addition, PKC inhibitor inhibited the
expression of survivin. FGF-2 up-regulates the expression of survivin protein
in H446 cells and blocks the release of Smac from mitochondria to the
cytoplasm. PKCa regulated FGF-2-induced survivin expression.
Thus, survivin, Smac, and PKCa might play important roles in the
inhibition of apoptosis by FGF-2 in human small cell lung cancer cells.
Keywords FGF-2; survivin; Smac; PKC; apoptosis; lung cancer
Lung cancer remains one of the leading causes of cancer death and, of
all lung cancers, small cell lung cancer (SCLC) is the most malignant
histological type [1]. Currently, the primary therapy used for patients with
SCLC is chemotherapy. Despite rapid progress in improving the efficacy of
chemotherapy, it has a very poor prognosis as it is refractory to most
chemotherapeutic agents. The 5-year survival rate is the lowest among all
primary lung cancers [1,2].A high expression of fibroblast growth factor 2 (FGF-2) was associated
with poor overall survival in SCLC [3]. FGF-2 induces a broad spectrum of drug
resistance by inhibiting apoptosis in various tumor types, but the molecular
mechanism is not clear [4]. Apoptosis is regulated by a complex balance of
signals, including the pro-apoptotic factors, such as p53 and Bax, and the
anti-apoptotic factors, such as bcl-2 family members and members of the
inhibitor of apoptosis protein (IAP) family [5–7]. Survivin, an IAP family
member, is involved in both inhibition of apoptosis and control of cell
division. Its anti-apoptotic function is related to its ability to inhibit
caspases [8–13]. Smac/DIABLO, a recently identified protein released from
mitochondria after apoptotic stimuli, binds IAPs, allowing caspase activation
and cell death [14–17]. In the present study, we investigated the effect of FGF-2 on the
expression of survivin, and the subcellular location of Smac in the human SCLC
cell line H446, to clarify the molecular mechanisms involved in repression of
apoptosis by this agent.
Materials and Methods
Reagents
Antibodies against survivin, b-actin, and protein kinase
C (PKCa) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA),
the neutralizing anti-FGF-2 antibody was from Upstate Biotechnology (Lake Placid,
USA), and the antibody against Smac was from R&D Systems (Minneapolis,
USA). Recombinant human FGF-2 was from PeproTech EC (London, UK). The SABC-Cy3
Immunofluorescence reagent kit was from Boster Biological Technology Company
(Wuhan, China). The BCA Protein Assay reagent kit was from Pierce (Rockford,
USA) and the In situ cell death detection kit was purchased from Roche
Diagnostics (Indianapolis, USA). The caspase-3 activity assay kit was from
R&D Systems, and calphostin C was from Sigma-Aldrich (St. Louis, USA). The
membrane protein extraction kit was from Keygen (Nanjing, China).
Cell lines and cell culture
The human SCLC cell line H446 was a generous gift from Prof. Zhiming
He of the Tumor Research Institute of Central South University (Changsha,
China). Cell cultures were maintained in RPMI 1640 (Gibco Biocult, Paisley, UK)
supplemented with 10% (V/V) fetal calf serum, 50 U/ml penicillin,
and 50 mg/ml streptomycin in an atmosphere of 5% CO2/95% humidified air at 37 ?C. The incubation medium was changed
every 1 or 2 d, and subculture was carried out every 2 or 3 d according to the
cell confluence. For experimental purposes, the cells were grown in medium
contain 1% serum for 36 h then treated with FGF-2 for the indicated time.
Flow cytometry
Approximately 5?106 cells were immediately fixed in 70% ethanol and stored at 4 ?C in
phosphate-buffered saline (PBS) for fluorescence-activated cell sorting. Flow
cytometry analysis was carried out on a FACStar flow cytometer (Becton
Dickinson, Franklin Lakes, USA). Histograms of the number of cells at each
fluorescence intensity increment (on a logarithmic scale) were recorded for
10,000 cells per sample.
Hoechst 33258 staining
Hoechst 33258 staining was carried out as previously described
[18,19]. Briefly, cells were collected by centrifugation and the pellets were
washed twice with PBS. Pellets were then resuspended in PBS and stained with 10
mg/ml
Hoechst 33258 (Sigma) for 10 min at room temperature. Morphological evaluation
of nuclear condensation and fragmentation was carried out immediately with a
Nikon Microphot-FXA fluorescence microscope (Tokyo, Japan). The percentage of
apoptotic nuclei was determined by counting the number of apoptotic nuclei for
every 300 cells in a particular microscopic field, dividing that number by 300,
and multiplying it by 100.
Terminal deoxynucleotidyl
transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay
A modified TUNEL assay was carried out using the In situ cell
death detection kit according to the manufacturer’s protocol. The morphological
changes were observed with the Nikon Microphot-FXA fluorescence microscope.
TUNEL-positive cells revealed nuclei with light green fluorescence. Positive cells
per field were counted.
Caspase-3 activity assay
SCLC cells were harvested and washed with cold PBS followed by
centrifugation. The cell pellet was incubated in buffer A [20 mM HEPES
potassium salt (pH 7. 5), 10 mM KCl, 1.5 mM MgCl2, 1.0 mM EDTA disodium salt, and 250 mM sucrose] for 10 min at 4 ?C.
The cell lysates were centrifuged at 12,000 g for 15 min and the protein
concentration was determined by the Bradford method. Quantitative detection of
caspase-3 activity in cellular lysates was carried out using a caspase-3
activity assay kit according to the manufacturer’s instructions.
Western blot analysis
Total cellular extracts were obtained by lysing the cells in lysis
buffer and membrane protein was isolated as described by Li and colleagues
[20]. Western blot analysis was carried out as previously described [21].
Briefly, 60 mg cell extract from each sample was separated by electrophoresis
on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to an
Immobilon P polyvinylidene difluoride membrane (Millipore, Billerica, USA) with
a semi-dry electroblotter (PatentStorm, Washington, USA). After blocking, the
membrane was incubated overnight with primary antibodies to PKCa, survivin, and
Smac in 5% milk, followed by incubation with the corresponding horseradish
peroxidase-conjugated anti-goat or anti-rabbit IgG. The bands were detected by
diaminobenzidine coloration.
Subcellular fraction
H446 cells were centrifuged in 1.5 ml Eppendorf tubes, resuspended
in 100 ml ice-cold RSB hypotonic buffer [10 mM Tris-HCl (pH 7.4) containing 2.5
mM MgCl2, 10 mM NaCl, 1 ml/ml phenylmethylsulfonyl
fluoride, and 1 ml/ml dithiothreitol], and incubated on ice for 10 min. The cell
suspension was homogenized in a precooled Dounce homogenizer, then resuspended
in 2.5 MS buffer [5 mM Tris-HCl (pH 7.4) containing 210 mM mannitol, 70 mM
sucrose, 1 mM EDTA, 1 ml/ml phenylmethylsulfonyl fluoride, and 1 ml/ml dithiothreitol]. Cells
were then centrifuged at 3750 g at 4 ?C for 5 min. The supernatant was
removed and resuspended, then centrifuged at 15,000 g at 4 ?C for 20
min. The supernatant, containing the cytoplasmic fraction, was separated from
the pellet, containing the mitochondrial fraction.
Immunofluorescence
After treatment, SCLC cells were centrifuged in 1.5 ml Eppendorf
tubes. The cell pellet was resuspended in Ca2+-free and Mg2+-free PBS
containing 4% formaldehyde for 15 min at room temperature. Cells were washed
twice in Ca2+-free and Mg2+-free PBS and were resuspended in blocking buffer [PBS containing 3%
bovine serum albumin (BSA)] for 30 min. After three washes in PBS, the
corresponding primary antibody was added (in PBS containing 1% BSA) and
incubated for 1 h at 37 ?C, then overnight at 4 ?C. After three washes in PBS,
the biotinylated secondary antibody was added (in PBS containing 1% BSA) and
incubated for 30 min at room temperature. After three washes in PBS, the
Cy3-labeled streptavidin was added (in PBS containing 1% BSA) and incubated for
30 min at room temperature in the dark. Samples were then washed four times in
PBS, tiled onto glass slides, and observed with the Nikon Microphot-FXA
fluorescence microscope.
Statistical analysis
The results were statistically evaluated by the least significant
difference, Student-Newman-Keuls, and 2-tests using SPSS version
12.0 software (SPSS, Chicago, USA). A P value of less than 0.05
was regarded as significant.
Results
Influence of fibroblast growth
factor 2 (FGF-2) on apoptosis, survivin expression and caspase-3 activity in
human small cell lung cancer cell line H446
To confirm the effect of FGF-2 on apoptosis, the cells were examined
by flow cytometry. As shown in Fig. 1(A), after serum starvation, the
percentage of apoptotic cells increased from 7.29% to 22.6%. FGF-2 exposure
reduced apoptosis to 12.5%. The effect of FGF-2 exposure on the appearance of
apoptotic morphology was further investigated by nuclear staining and DNA
fragmentation assay. After the cells were deprived of serum for 36 h, the
typical morphological changes of apoptotic cells (condensed chromatin and
fragmented nucleus) were apparent, an effect that was attenuated by FGF-2
exposure [Fig. 1(B)]. Control cells had minimal apoptotic nuclei (2.42%±0.85%),
whereas the starved cells showed a significant increase (14.51%±2.68%). FGF-2
exposure decreased the percentage of apoptotic nuclei (6.13%1.89%) in
serum-starved cells, but not to the levels seen in the control cells. The TUNEL
assay, which assessed DNA fragmentation, showed that the number of apoptotic
cells/high power was 2.52±1.15 in the control cultures, 7.65±2.21 in the
serum-deprived cultures, and 5.32±1.25 in the serum-deprived cells pretreated
with FGF-2. The difference between the serum-starved group and the FGF-2
stimulation group was significant (P<0.05). Therefore, FGF-2 could prevent apoptosis of H446 cells. After the cells were serum-starved for 36 h, caspase-3 activity was increased and survivin expression was decreased, but FGF-2 pretreatment inhibited the caspase-3 activity and upregulated the survivin expression [Fig. 1(C,D)].
FGF-2 up-regulated expression
of survivin protein but inhibited caspase-3 activity
Although H446 cells express the basal level of survivin, the levels
in FGF-2 treated groups were significantly higher. After treatment with FGF-2
for 4 h, survivin expression was induced in a dose-dependent manner [from 12.5
to 75 ng/ml; Fig. 2(A)]. When the cells were exposed to 12.5 ng/ml
FGF-2, the expression of survivin protein peaked at 4 h [Fig. 2(B)]. The
FGF-2-induced survivin expression was attenuated after treatment with FGF-2 neutralizing
antibody [Fig. 2(C)].
FGF-2 inhibited Smac release
from mitochondria
Next, we investigated the effect of FGF-2 on Smac from mitochondria
in H446 cells. In this study, we identified that serum starvation for 36 h could
induce Smac release from mitochondria in H446 cells, compared with the control
in which cells were incubated in medium contain 1% serum. Furthermore, when the
H446 cells were preincubated with recombinant human FGF-2 (25100 ng/ml) for 4
h, the release of Smac protein from the mitochondria to the cytoplasm was
inhibited from approximately 53% to approximately 62% [Fig. 3(A)]. The
expression level of Smac protein in the cytoplasm of the FGF-2 pretreated cells
was lower than in the serum-starved cells (P<0.05). Immunofluorescent detection of
Smac in untreated cells showed a punctate staining consistent with
mitochondrial localization. This staining became diffuse on serum starvation.
However, Smac localization was not altered in cells pretreated with 75 ng/ml
FGF-2 [Fig. 3(B)], confirming the Western blot
results.
FGF-2-induced survivin
expression involved in altered localization of PKCa
Furthermore, the role of PKCa in FGF-2-induced survivin expression was tested. After exposure to 75
ng/ml FGF-2, the PKCa levels in the cell membrane fraction were increased in a
time-dependent manner, but did not significantly change in the cell as a whole,
indicating that PKCa translocated from the cytoplasm to the cell membrane [Fig. 4(A)].
Calphostin C could also inhibit FGF-2-induced survivin expression [Fig. 4(B)].
Discussion
FGF-2 belongs to a large family of 19 structurally related members.
This growth factor was initially purified from bovine pituitary extracts [22],
and later found in a variety of tissues, including tumors. An increasing body
of evidence shows that FGF-2 produced by autosecretion or parasecretion
promotes cell proliferation and inhibits apoptosis [4,15]. In agreement with
prior evidence showing that FGF-2 inhibits apoptosis, treatment with FGF-2
reduced apoptosis induced by serum starvation in human SCLC cells in the
present study. But the molecular mechanism of this effect remains to be
determined [23–25].Apoptosis is regulated by a family of proteases known as caspases,
and IAPs are the most important regulators of caspases. Among IAPs, survivin is
considered the most important. Survivin inhibits apoptosis by directly
inhibiting the effector caspases, caspase-3 and caspase-7 [26–27].
Furthermore, bFGF regulation of survivin expression was found to be
extracellular regulated kinase 1/2 dependent [28]. In the present study,
increased expression of survivin was found in FGF-2 treated human SCLC cells.
Neutralizing FGF-2 with anti-FGF2 antibody attenuated the expression of survivin
significantly. In addition, FGF-2 pretreatment inhibited caspase-3 activity,
indicating that FGF-2-mediated survivin expression inhibits apoptosis through
direct interaction with caspase-3 in H446 cells.FGF-2 functions through three signal transduction pathways, the
mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol
3-kinase pathway, and the PKC pathway [29,30]. Sequence analysis of survivin
suggests that the survivin gene contains three PKC active sites (Thr21, Ser88,
and Thr127). Therefore, we hypothesized that FGF-2 might up-regulate survivin
protein expression through the PKC signal transduction pathway. FGF-2
induces survivin expression in a mammary cancer cell line by activating the
MAPK signaling pathway and recruitment of c-myc [21,22,31]. Other studies have
shown activation of the MAPK signaling pathway in FGF-2-induced proliferation
of coronary artery smooth muscle cells [29], an effect modulated by PKCd. In oral
carcinoma TSCCa cells, survivin expression was down-regulated and caspase-3
expression was up-regulated after inhibition of the PKCa signaling pathway with
staurosporine, a potent kinase inhibitor [26]. Thus, PKC is capable of affecting
cell death in cancer cells, and could therefore be involved in FGF-2-related
carcinogenesis and development.Our results indicate that FGF-2 induces translocation of PKCa from the
cytoplasm to the cell membrane of H446 cells. Treatment with calphostin C, a
specific PKC inhibitor, prevented this translocation. In addition,
FGF-2-induced survivin expression was significantly inhibited. Our findings
suggest that FGF-2 might regulate survivin expression in SCLC cells by altering
the localization of PKCa. Pardo et al. reported that FGF-2 increased expression of
the anti-apoptotic proteins XIAP and Bcl-XL through the PKCe signaling
pathway to inhibit apoptosis of lung cancer cells [32]. However, we are the
first to report that FGF-2 up-regulated survivin to inhibit apoptosis in SCLC
cells.There are a large number of known factors that can enhance apoptosis
by eliminating the inhibitory effects of IAPs such as survivin on caspases,
such as Smac [19,33]. Smac, a direct IAP binding protein with a low isoelectric
point (DIABLO), normally localizes within mitochondria and is released from
mitochondria into the cytosol during apoptosis. In the present study, treatment
of serum-starved H446 cells with FGF-2 inhibited Smac release from
mitochondria, indicating that Smac is involved in FGF-2-prevented apoptosis. In conclusion, to our
knowledge, these data show for the first time that FGF-2 inhibits apoptosis in
human SCLC cells. The underlying mechanism for the inhibition of apoptosis is
closely related to the up-regulation of the survivin protein and the reduced
release of Smac from mitochondria to the cytoplasm. We conclude that survivin,
Smac, and PKCa might play important roles in
inhibiting apoptosis by FGF-2 treatment in human SCLC cells. These results indicate new targets for adjuvant therapy to improve
the effectiveness of conventional therapies for SCLC.
Acknowledgements
We thank Prof. Weijun Cai (Central South
University, Changsha, China) for his comments and suggestions during the
preparation of this manuscript.
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