ABBS 2008,40(04): 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

Original Paper

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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