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Originally published In Press as doi:10.1074/jbc.M414041200 on May 26, 2005

J. Biol. Chem., Vol. 280, Issue 33, 29828-29836, August 19, 2005
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Mechanisms for the Apoptosis of Small Cell Lung Cancer Cells Induced by Anti-GD2 Monoclonal Antibodies

ROLES OF ANOIKIS*

Wei Aixinjueluo{ddagger}, Keiko Furukawa{ddagger}, Qing Zhang{ddagger}, Kazunori Hamamura{ddagger}§, Noriyo Tokuda{ddagger}, Shoko Yoshida¶, Ryuzo Ueda¶, and Koichi Furukawa{ddagger}||

From the Departments of {ddagger}Biochemistry II and §Oral and Maxillofacial Surgery, Nagoya University School of Medicine 65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan and Department of Internal Medicine II, Nagoya City University School of Medicine, 1 Kawasumi, Mizuho-ku, Nagoya 467-8601, Japan

Received for publication, December 14, 2004 , and in revised form, May 6, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anti-GD2 ganglioside antibodies could be a promising, novel therapeutic approach to the eradication of human small cell lung cancers, as anti-GD2 monoclonal antibodies (mAbs) induced apoptosis of small cell lung cancer cells in culture. In this study, we analyzed the mechanisms for the apoptosis of these cells by anti-GD2 mAbs and elucidated the mechanisms by which apoptosis signals were transduced via reduction in the phosphorylation levels of focal adhesion kinase (FAK) and the activation of a MAPK family member, p38, upon the antibody binding. Knock down of FAK resulted in apoptosis and p38 activation. The inhibition of p38 activity blocked antibody-induced apoptosis, indicating that p38 is involved in this process. Immunoprecipitation-immunoblotting analysis of immune precipitates with anti-FAK or anti-integrin antibodies using an anti-GD2 mAb revealed that GD2 could be precipitated with integrin and/or FAK. These results suggested that GD2, integrin, and FAK form a huge molecular complex across the plasma membrane. Taken together with the fact that GD2+ cells showed marked detachment from the plate during apoptosis, GD2+ small cell lung cancer cells seemed to undergo anoikis through the conformational changes of integrin molecules and subsequent FAK dephosphorylation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gangliosides have been considered to be tumor markers of neuroectoderm-derived human cancers, such as malignant melanomas, neuroblastomas, and gliomas (1, 2). They have been used as target molecules in antibody therapy, i.e. GD3 in malignant melanomas (3) or GD2 in neuroblastomas (4). Some patients showed significant responses to antibody therapy against gangliosides. In the extended analysis of ganglioside expression in other human cancer cells, we have demonstrated the characteristic expression of GD2 in human T lymphotropic virus type I-infected T lymphocytes (5), GD3 in acute T lymphoblastic leukemia cells (6), and GD2 in small cell lung cancer (SCLC) cells (7).

In many of these studies, no clear implication of individual gangliosides have been elucidated, except that GD2 in small cell lung cancer (SCLC)1 was shown to induce increased cell growth and invasion using the transfectant cells with GD3 synthase cDNA (7). Furthermore, it was shown that anti-GD2 monoclonal antibodies (mAbs) could suppress the cell proliferation of GD2+ SCLC cells and also induce apoptosis with caspase activation. These results indicated that antibody therapy might be a promising approach to overcoming the disastrous disease SCLC.

As for the tumor cell apoptosis with anti-ganglioside antibodies, a few studies have been reported. Hanai and co-workers (8) report apoptosis induction of melanoma cells with anti-GM2 mAb in multicellular heterospheroids. On the other hand, in neuronal tissues, anti-ganglioside antibodies often cause tissue damage mainly in the motor neuron system and trigger serious motor neuron paralysis, such as Guillain-Barre syndrome (9). These facts suggest that gangliosides on malignant tumor cells can be good targets of antibody therapy with mAbs or their modified forms, although not many examples of apoptosis induction with anti-ganglioside mAbs have been reported so far.

To clarify the possibility of therapeutic application of anti-ganglioside mAbs for malignant tumors, molecular mechanisms for the apoptosis of SCLC cells with anti-GD2 ganglioside were studied. The most intriguing issue in understanding the apoptosis signals triggered by an antibody binding to gangliosides express on the cell surface membrane is how the events outside of the cells can induce the sequential activation of molecules involved in the apoptotic pathway inside of the cells. Here we have demonstrated a whole feature of the signaling pathway for apoptosis triggered by anti-GD2 mAbs leading to apoptotic cell death.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines—A GD3 synthase gene transfectant cell line, D-18, and a vector control cell line, C-2, were generated from SK-LC-17 in our laboratory (7) and were maintained in RPMI 1640 medium containing 10% fetal calf serum and G418 (250 µg/ml) in a humidified 5% CO2 atmosphere at 37 °C. Other SCLC cell lines, NCI-417, ACC-LC-171, and ACC-LC-96 were as described previously (7).

Antibody Purification—The anti-GD2 monoclonal antibody (mAb) 220-51 (mouse IgG3), which was generated in our laboratory (10), was purified using a protein G affinity column, and the concentration of the protein was determined by Lowry's method (11).

Flow Cytometry—Cell surface expression of ganglioside GD2 was analyzed by flow cytometry (FACScan, BD Biosciences) using an anti-GD2 mAb, 220-51. Cells were incubated with mAbs for 60 min on ice and then stained with FITC-conjugated anti-mouse IgG (ICN/Cappel, Durham, NC). Control cells for flow cytometry were prepared using the second antibody alone.

MTT Assay—For cell proliferation assay, transfectant cells and control cells (1 x 104 cells/well) were prepared in 48-well plates (CellStar, Carrollton, TX) in serum-containing medium and cultured for 4 days. For cell growth inhibition assay, transfectant cells and control cells (5 x 104 cells/well) were seeded in 24-well plates (CellStar) in serum-containing medium and treated with anti-GD2 mAb 220-51 diluted to the indicated concentrations for 24 h. Freshly prepared medium containing antibodies was used for medium exchange everyday. To quantify the cell proliferation, MTT (Sigma) was added to each well (0.5 mg/ml). After incubation for 4 h at 37 °C, the supernatants were aspirated, and 100 µl of n-propyl alcohol containing 0.1% Nonidet P-40 and 4 mM HCl were added. The color reaction was quantitated using an automatic plate reader, Immuno-Mini NJ-2300 (Nihon InterMed, Tokyo, Japan) at 590 nm with a reference filter of 620 nm, as reported previously (12). MTT assays were carried out in triplicate. To analyze the growth suppression effects of mAb 220-51 in the transfectants, cells (1 x 105 cells/well) were prepared in 24-well plates in serum-containing medium and treated with 0-60 µg/ml of mAb 220-51 for 24 h and then counted with trypan blue.

Analysis of Apoptosis—Cells (1 x 106 cells) were plated in 60-mm tissue culture plates (CellStar) in serum-containing medium and then treated with 60 µg/ml mAb 220-51 for 2 h at 37 °C. After the treatment, the cells were harvested with trypsin/EDTA, resuspended in the binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2) and then incubated with FITC-conjugated Annexin V and propidium iodide (1 µg/ml) (Annexin V-FITC kit; Roche Applied Science), according to the manufacturer's protocol. The numbers of apoptotic cells were monitored with flow cytometry.

DNA Fragmentation Assay—Cells (1 x 106 cells) were treated with 60 µg/ml mAb 220-51. After 42 h, the cells were harvested and lysed in 100 µl of a lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, and 0.5% Triton X-100) for 10 min at 4 °C. After centrifugation, the supernatants were collected, and 6 µl of RNase (10 mg/ml) and 6 µl of proteinase K (10 mg/ml) were added. After incubation for 1 h at 37 °C, the fragmented DNA was precipitated in 2-propanol (360 µl) and electrophoresed at 50 V for 1.6 h on a 2% agarose gel containing 0.2 µg/ml ethidium bromide in TAE buffer (40 mM Tris acetate, 1 mM EDTA). The gel was observed under UV light.

Western Immunoblotting—For the antibody stimulation experiments, cells (8 x 105 cells) were plated in 60-mm tissue culture plates in serum-containing medium and then treated with 60 µg/ml mAb 220-51 for the appropriate times in the individual experiments. After treatment, the cells were harvested with 0.02% EDTA, washed twice with ice-cold phosphate-buffered saline, and solubilized in a lysis buffer (20 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 10% glycerol, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, and 20 units of aprotinin). Soluble proteins (50 µg/lane) were subjected to 12% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with 5% bovine serum albumin in phosphate-buffered saline overnight at 4 °C, washed with phosphate-buffered saline containing 0.05% Tween 20, and then incubated with antibodies reactive with p38, phospho-p38 (Tyr-180/Tyr-182, rabbit anti-human p38 mitogen-activated protein polyclonal antibody; Cell Signaling Technology, Inc., Beverly, MA), and {beta}-actin (mouse monoclonal anti-mouse {beta}-actin; Sigma). Bands were detected with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (HRP-conjugated goat anti-rabbit IgG; Cell Signaling Technology, Inc.) or anti-mouse IgG (anti-mouse IgG, HRP-linked whole antibody from sheep; Amersham Biosciences) combined with an ECL kit (Amersham Biosciences). For immunoprecipitation, the lysates were incubated with 5 µl of anti-FAK (1 µg IgG) (rabbit polyclonal anti-human FAK; Santa Cruz Biotechnology, Inc.) overnight, and then 20 µl of protein G-Sepharose (1:1) (Amersham Biosciences) was added and incubated for 3 h at 4°C. After being washed with immunoprecipitation washing buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.5% Nonidet P-40, 1 mM Na3VO4), the precipitates were solubilized in 50 µl of lysis buffer, and 20 µl was subjected to immunoblotting, as described above. Anti-phosphotyrosine antibody (PY20, mouse monoclonal IgG2b; BD Biosciences) or anti-FAK antibody was used for the immunoblotting, as described above.

Co-precipitation Experiments—To analyze the co-precipitation of FAK and integrin, cells (2 x 106) were solubilized in 50 µl of the lysis buffer, and the lysates were immunoprecipitated separately with 5 µl (1 µg of IgG) of anti-FAK or anti-integrin {beta}1 (rabbit polyclonal anti-human integrin {beta}1; Santa Cruz Biotechnology, Inc.) or normal rabbit IgG (Cell Signaling) following the methods described under "Western Immunoblotting." The immunoprecipitates were subjected to SDS-PAGE and then blotted onto polyvinylidene difluoride membrane, as described above. An antibody against integrin {beta}1 was used in the immunoblotting combined with HRP-conjugated anti-mouse IgG (Cell Signaling) as a second antibody. To examine the co-precipitation of GD2 with FAK and integrin, 2 x 107 cells were solubilized in 100 µl of the lysis buffer and were immunoprecipitated separately with 5 µl (1 µg of IgG) of anti-FAK, anti-integrin {beta}1, or normal rabbit IgG. The immunoprecipitates were subjected to SDS-PAGE and then blotted onto polyvinylidene difluoride membrane, as described above. mAb 220-51 was used at a dilution of 1:100 in the immunoblotting combined with peroxidase-conjugated anti-rabbit IgG as a second antibody.

Knock Down of FAK—To suppress FAK expression, we prepared five kinds of small interfering RNAs (siRNAs). siRNA duplexes were directed toward the following mRNA targets (nucleotide 1 is A of ATG): nucleotides 510-531, 5'-AAG UUG GGU UGU CUA GAA AUA-3'; nucleotides 920-941, 5'-AAG UGA AGA CAA GGA CAG AAA-3'; nucleotides 1011-1032, 5'-AAG GAG AAU AUG GCU GAC CUA-3'; nucleotides 2030-2051, 5'-AAG CAC AAU CCU GGA GGA AGA-3'; nucleotides 2307-2328, 5'-AAC CAA ACA GAU UCA UGG AAU-3'. Fluorescein-labeled luciferase GL2 Duplex (DHARMACOM RNA Technologies, Lafayette, CO) was used as a control. The double-stranded RNAs were introduced into the cells with Lipofectamine 2000TM (Invitrogen) according to the manufacturer's directions. The cells were used after 48 h for immunoblotting of total FAK protein and for apoptosis induction experiments.

p38 Inhibition—p38 activity was inhibited via exposure to SB203580 (Promega Corporation, Madison, WI) and diluted to the indicated concentrations from 0 to 100 µM for 24 h at 37 °C. An equal volume of the Me2SO vehicle (1%) was used as a control. Inhibition was confirmed by the suppression of p38 phosphorylation after UV light exposure (100 J/m2) with immunoblotting of phospho-p38 as well as p38 and {beta}-actin. Cells with ~70% inhibition of p38 phosphorylation were used in the experiment for apoptosis induction.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GD2 Expression in the Transfectant Cells and SCLC Cell Lines—It was known that the GD3 synthase gene was highly expressed in the SCLC cell lines (7). The GM2/GD2 synthase gene was broadly expressed in the majority of lung cancer cell lines examined, whereas SCLC lines showed slightly higher expression levels. The transfection of GD3 synthase cDNA can, therefore, induce GD2 expression, as shown in Fig. 1A. D-18 was generated by the transfection of GD3 synthase cDNA into SK-LC-17. GD2 expression was also detected in three SCLC cell lines at slightly lower levels than D-18 (Fig. 1B).

Suppression of Cell Growth by Anti-GD2 Antibodies—The effects of anti-GD2 mAbs on cell growth were then examined by adding antibodies to the culture medium. The increased cell growth after GD3 synthase gene transfection was strongly suppressed in the presence of an anti-GD2 mAb 220-51. The suppression effects were dependent on the concentration of the added antibody and were significant even at 10 µg/ml (Fig. 2A).

Apoptosis Induction by Anti-GD2 mAbs—To examine the induction of apoptosis during the growth suppression of SCLC cell lines with anti-GD2 mAb, double staining of cells with Annexin V (FITC) and propidium iodide was performed. A transfectant line treated with anti-GD2 mAb was 38.7% positive for Annexin V binding at 2 h after the addition of the mAb (Fig. 2B), indicating the induction of apoptotic cell death. The vector control line C-2 showed no staining for both reagents. Three SCLC cell lines expressing GD2 treated with anti-GD2 mAb were also positive for Annexin V and/or propidium iodide (Fig. 2B), indicating that the native GD2-expressant cells are also sensitive to apoptosis induction. To confirm the DNA degradation in the anti-GD2 mAb-treated cells, cytoplasmic DNA was prepared from 1 x 106 cells and used for agarose gel electrophoresis. Only D-18 cells treated with the mAb showed definite DNA ladder formation (Fig. 2C), suggesting that apoptosis was really induced by the anti-GD2 mAb. The transfectant cells treated with anti-GD2 mAb showed marked shrinkage of cytoplasm and formation of spaces between cells, whereas vector control cells showed no change in morphology (Fig. 2D). As for the native GD2-expressant cell lines, similar morphological changes were observed (Fig. 2D), although the changes were less obvious due to their original morphology.



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  		FIG. 1.
Expression of GD2 in the transfectant cell and SCLC cell lines. GD2 expression was analyzed with flow cytometry, as described under "Materials and Methods." Results of a transfectant line (D-18) and a control (C-2) were shown. Results of three SCLC cell lines are also shown as labeled in the figure.

 
Phosphorylation of MAPKs, ERK1/2, and p38 after Addition of the Antibody—For the antibody stimulation experiments, cells (8 x 105) were plated in 60-mm tissue culture plates in serum-containing medium and then treated with 60 µg/ml mAb 220-51 for the appropriate times in the individual experiments. Phosphorylation of ERK1/2 was reduced from 240 min after the addition of the antibody in GD2-expressing D-18 (but not in the vector control cells) (Fig. 3A). Phosphorylation of p38 was induced from 120 min after the addition of the antibody in GD2-expressing D-18 (but not in the vector control cells) (Fig. 3B). Phosphorylation of p38 was also examined in a native GD2-expressant line, ACC-LC-171, and a very similar activation pattern of p38 was observed (Fig. 3C).

Phosphorylation of FAK after Addition of the Antibody—Cells (8 x 105) were plated in 60-mm tissue culture plates in serum-containing medium and then treated with 60 µg/ml of mAb 220-51 for the appropriate times in the individual experiments. The lysates were incubated with anti-FAK (1 µg IgG) and immunoprecipitated and then used for immunoblotting with anti-phosphotyrosine antibody (PY20) or anti-FAK antibody. Phosphorylation of FAK was reduced along with the time of the antibody treatment from 10 min in GD2-expressing D-18, whereas no reduction in the FAK phosphorylation level was observed in vector control cells (Fig. 4A). In the case of a native GD2-expressant line, ACC-LC-171, a similar or even faster reduction of FAK phosphorylation was observed after the antibody treatment (Fig. 4B).

GD2, FAK, and Integrin Formed a Complex in the Transfectant Cells—To analyze the association of GD2 with integrin or FAK, immunoprecipitates with anti-integrin or those with anti-FAK were used for immunoblotting with anti-GD2 mAb. First of all, integrin was co-precipitated with FAK, as shown in Fig. 5A. Thereafter, it was shown that GD2 was co-precipitated with FAK and/or integrin (Fig. 5B). This was also the case in the native GD2-expressant lines (Fig. 5C). Taken together, GD2 seemed to form a complex with integrin and FAK molecules across the plasma membrane.

Suppression of FAK Expression Resulted in Apoptosis—Five kinds of siRNAs were prepared based on the cDNA sequence and used to suppress FAK expression. The effects were examined with immunoblotting of anti-phospho-FAK as well as anti-FAK antibodies (Fig. 6A). More than 60% of suppression in FAK expression was achieved in siRNA1-treated cells. Bands of tyrosine-phosphorylated FAK also reduced in intensity in parallel with total FAK bands. Therefore, this was used for the apoptosis induction experiment. After siRNA transfection, cells were used for an apoptosis assay by FACS. After the transfection of FAK siRNA, expression of FAK was definitely reduced, and apoptosis was found in both D-18 and C-2 cells (Fig. 6B, top panels). The apoptosis induction was also detected in two native GD2-expressant lines after the transfection of FAK siRNA (Fig. 6B, bottom panels).

Phosphorylation of p38 Was Induced by the Suppression of FAK—The phosphorylation level of p38 increased by the suppression of FAK via FAK siRNA (Fig. 6C). Further increase in p38 phosphorylation was found by the addition of anti-GD2 mAb in immunoblotting with anti-phospho-p38.

Apoptosis Induced by Anti-GD2 mAb Was Protected with Inhibition of p38 —p38 activity after UV light exposure (100 J/m2) was inhibited via pre-exposure to SB203580 at 100 µM (but not at concentrations of 50 µM or less, as determined by Western immunoblotting of phospho-p38) (Fig. 7A). After treatment with SB203580 (100 µM) or Me2SO (vehicle), cells were treated with mAb 220-51 (60 µg/ml) for 1 h at 37 °C and used for apoptosis assay by FACS. Apoptosis in D-18 was almost completely protected by SB203580 treatment (Fig. 7B, right panels). In the vector control cells, no apoptosis was induced by anti-GD2 mAb regardless of SB203580 treatment (Fig. 7B, left panels). As for the native GD2-expressant lines, both of the two cell lines examined showed marked suppression of apoptosis by SB203580 treatment (Fig. 7C).

SB203580 Showed No Effect on the Phosphorylation Levels of FAK—The effects of p38 inhibition with SB203580 on the phosphorylation levels of FAK was examined by immunoblotting. It was shown that SB203580 had almost no effect on the phosphorylation levels of FAK, suggesting that p38 was positioned at the downstream of FAK in the apoptosis signaling pathway (Fig. 8).

Apoptosis Signaling Pathway Triggered by Anti-GD2 mAbs—An apoptosis signaling pathway triggered by anti-GD2 mAbs was proposed based on the results demonstrated in this study in Fig. 9.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is unusual that only an antibody binding to a surface molecule can induce apoptotic cell death, except for anti-Fas antibodies, which bind the Fas antigen associated with sequential death signaling molecules, such as FADD (13, 14). On the other hand, gangliosides are embedded in the outer layer of the plasma membrane, and there appears to be difficulty in transmitting signals from outside the cell into the cell nucleus. Therefore, we need to consider a quite novel mechanism for apoptosis induced via gangliosides. Although Cheresh et al. (15) already report the cell rounding and detachment of melanoma cells from fibronectin substrate with anti-GD2 mAbs, we demonstrated in this study that the apoptosis of SCLC cells induced with anti-GD2 mAbs was caused by dephosphorylation of FAK with various results, i.e. reduction of phosphorylated FAK and morphological changes of GD2+ cells after the treatment with anti-GD2 mAbs and apoptosis induction with knock down of FAK. The formation of spaces between cells under antibody treatment seemed to be caused by the detachment of apoptotic cells. However, the precise cause of the cell shrinkage that simultaneously occurred (probably due to detachment) remains to be clarified. Apoptosis induced with disruption of cell-matrix interaction has been called "anoikis" (16). The anti-apoptotic or anti-anoikis activity of FAK has been reported by a number of studies using overexpression of wild type FAK (17), introduction of the constitutively activated form of FAK (18), or enforced activation of {beta}1 integrin (19). The mechanisms for these anti-apoptotic activities of FAK include its binding to a death domain of receptor-interacting protein, augmentation of phosphatidylinositol 3-kinase (p85), phosphorylation, and Akt activity. Interestingly, FAK phosphorylation is also critical in the protection of apoptosis induced with ultraviolet irradiation (20). Moreover, FAK induces resistance to apoptosis induced with hydrogen peroxide in glioma cells (21) and even in a leukemia cell line (HL-60) (22). In turn, apoptosis induction with disruption of FAK has also been reported using a peptide of the FAK binding site of the integrin {beta}1 tail, microinjection of anti-FAK antibody (23), mutated FAK at a tyrosine-phosphorylation site (24), and the N-terminal domain of FAK (25). All of these results indicate that the activation levels of FAK are considered to be a crucial and universal factor in determining the fates of those cells treated with preapoptotic stresses in both monolayer cells and suspension cells.



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  		FIG. 2.
Suppression of cell growth and apoptosis induction by anti-GD2 mAb. A, suppression of cell growth. D-18 and C-2 were treated with mAb 220-51 diluted at the concentrations indicated for 24 h and then counted with the trypan blue exclusion method. B, apoptosis induction in the GD3 synthase gene transfectant cells (left) and three native GD2-expressant cell lines (right) by anti-GD2 mAb. Shown is double staining of Annexin V and propidium iodide (PI) in the cells after anti-GD2 mAb treatment. D-18 and C-2 were treated with 60 µg/ml mAb 220-51 for 2 h and stained with FITC-conjugated Annexin V (x-axis) and PI (y-axis) and then analyzed by flow cytometry, as described under "Materials and Methods." Three cell lines were also similarly treated with mAb 220-51 and analyzed by flow cytometry. C, cytoplasmic DNA prepared from D-18 after treatment with or without 60 µg/ml of mAb 220-51 for 42 h was analyzed by agarose gel electrophoresis. Similar results were obtained in at least three experiments. D, morphological changes of a transfectant and a control treated with or without 60 µg/ml of mAb 220-51 for 5 h were observed under a phase-contrast microscope at x400 (left). Those of the native cell lines were also examined after treatment with/without mAb 220-51, and typical pictures are shown (right).

 



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  		FIG. 3.
Time course of phosphorylation levels of MAPKs (ERK1/2), and p38 after the addition of anti-GD2 mAb. A, D-18 (right) and C-2 (left) were treated with 60 µg/ml of mAb 220-51 for the times indicated and then used for immunoblotting with an antiphosphorylated ERK1/2 antibody (top). The intensity of bands was measured and corrected with those of total ERK1/2 (middle), and plotted (bottom). B, cells treated with anti-GD2 mAb as in A were used for immunoblotting with an anti-p38 antibody (top), and band intensities were plotted after correction (bottom) with those of total p38 (middle). C, two SCLC cell lines with native GD2 expression were also analyzed for the activation of p38 under the same condition as in B, and the results were presented in the same way. Similar results were obtained in at least three experiments.

 
In addition to the dephosphorylation of FAK, increased phosphorylation of p38 was one more conspicuous change during the apoptosis induction. The signaling pathway between FAK dephosphorylation and p38 activation is not known at this moment. In the previous report, we have found that JNK activation was observed at 6 h after anti-GD2 antibody treatment (26). In this study, activation of another MAPK, p38, could be detected at a much earlier time, suggesting p38 is primarily involved in the apoptosis induction. It has been reported that inhibition of FAK resulted in the activation of p38 and anoikis in a colon cancer cell line (27) and in myotube (28). However, it is not clear whether dephosphorylation of FAK causes p38 activation in a single direction. In our results, knock down of FAK induced p38 activation, and p38 inhibition failed to affect the phosphorylation levels of FAK, suggesting that p38 is located at the downstream of FAK, although the linking pathway between them remains to be investigated. Involvement of various adaptor molecules associating with FAK, such as p130Cas (29, 30) and paxillin (31), should be examined as intervening molecules leading to the activation of p38.



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  		FIG. 4.
Dephosphorylation of FAK in GD2+ cells after treatment with an anti-GD2 mAb. A, D-18 (right) and C-2 (left) were treated with 60 µg/ml mAb 220-51 for the times indicated, as described in the legend to Fig. 3, and the lysates were incubated with anti-FAK (1 µg of IgG) and immunoprecipitated, as described under "Materials and Methods" and then used for immunoblotting with the anti-phosphotyrosine antibody (top) or anti-FAK antibody (middle). The band intensities were normalized with those of total FAK and plotted (bottom). Similar results were obtained in at least three experiments. B, two SCLC cell lines with native GD2 expression were also treated with mAb 220-51 and used for immunoprecipitation/immunoblotting for FAK dephosphorylation, as described in A. Results were obtained and presented as in A.

 



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  		FIG. 5.
GD2, integrin, and FAK form a complex in the transfectant cells and cell lines. A, co-precipitation of FAK and integrin. Cells were solubilized in the lysis buffer, and the lysates were immunoprecipitated (IP) separately with 5 µl (1 µg of IgG) of anti-FAK, anti-integrin {beta}1, or normal rabbit IgG, as described under "Materials and Methods." After SDS-PAGE of the immunoprecipitates, immunoblotting (IB) was performed with an antibody against integrin {beta}1 combined with HRP-conjugated anti-mouse IgG as a second antibody. B, co-precipitation of GD2 with FAK and integrin. Cells were solubilized in 100 µl of the lysis buffer and were immunoprecipitated separately with 5 µl (1 µg of IgG) of anti-FAK, anti-integrin {beta}1, or normal rabbit IgG. Thereafter, mAb 220-51 was used in the immunoblotting combined with peroxidase-conjugated anti-rabbit IgG as a second antibody to detect co-precipitated GD2. C, three native GD2-expressant cell lines were also used for the immunoprecipitation and subsequent immunoblotting to detect the co-precipitated GD2, as described in B.

 
The most intriguing issue in understanding the apoptotic process with anti-GD2 antibodies is how the antibody binding to gangliosides can trigger the initial signal for cell death. The fact that GD2 was physically associated with integrins (Fig. 5) strongly suggested that the antibody binding to GD2 causes conformational changes in integrin molecules, leading to the dephosphorylation of FAK, because integrins and FAK had been known to be tightly associated in both physical and functional senses (32). The formation of a tertiary complex consisting of GD2, integrin, and FAK seemed to be a critical substantial basis to generate and/or maintain the malignant properties of SCLC cells (7). Insults destroying the intact form of the molecular complex might, therefore, result in the reduction of FAK-mediated anti-anoikis signals with its dephosphorylation. Functional modulation of integrins by the expression of glycosphingolipids was previously suggested by Cheresh et al. (33) and our group (34) using human melanoma cells and murine lung cancer cells, respectively.



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  		FIG. 6.
Suppression of FAK expression induced apoptosis. A, inhibition of FAK expression by FAK siRNAs. Five kinds of siRNAs as well as a control siRNA were prepared based on the cDNA sequence and used to suppress FAK expression. The double-stranded siRNAs were introduced into the cells with Lipofectamine 2000TM according to the manufacturer's directions. The cells were used after 48 h for immunoblotting of total FAK protein, phosphorylated FAK, and for apoptosis induction experiments, as described under "Materials and Methods." B, apoptosis induced by FAK siRNA in transfectant cells (top panels). After FAK siRNA 1 transfection, cells were used for an apoptosis assay by FACS. Expression of FAK was reduced, and apoptosis was found in both D-18 and C-2 cells. Apoptosis induced by FAK siRNA 1 in two native GD2-expressant lines was also examined (bottom panels). C, phosphorylation of p38 was induced by the suppression of FAK. The left two lanes were of cells treated with FAK siRNAs, with or without treatment of anti-GD2 mAb (60 µg/ml) for 2 h, and the middle two lanes of cells introduced with luciferase siRNA control, also with or without treatment of anti-GD2 mAb. The right two lanes were of cells with or without exposure to UV light (100 J/m2) as a control for p38 activation. The phosphorylation level of p38 increased by the suppression of FAK via FAK siRNA. Further increase in p38 phosphorylation was found by the addition of anti-GD2 mAb in immunoblotting with anti-phospho-p38. PI, propidium iodide.

 
Although gangliosides have been considered to be tumor-associated molecules and have been used as tumor markers, no clear and specific biological function in the malignant properties of human cancer cells have been elucidated. Colocalization of GD2/GD3 gangliosides at the adhesion plaque of melanoma cells was reported by Cheresh et al. (35). At that time, they demonstrated that these disialo-gangliosides were involved in the adhesion of melanoma cells to the extracellular matrix (15, 36). The critical effects of a peptide containing the ECM binding motif of integrins in the ganglioside-associated adhesion was also shown (33), indicating that disialo-gangliosides are functionally involved in the cell adhesion mediated by the interaction between integrins and the extracellular matrix. Burns et al. (37) also reported synergism between gangliosides and Arg-Gly-Asp-directed glycoprotein receptors in attachment to the matrix protein by melanoma cells, whereas Kojima and Hakomori (38) interpreted the synergism as the result of a combination of two cell recognition systems, i.e. interaction of glycosphingolipid-glycosphingolipid and that of adhesive matrix protein-integrin.

The mechanisms and implication of the interaction of gangliosides with integrin molecules on the cell surface is not well understood. Wang et al. (39) report that GT1b induced apoptosis of SCC12 cells by binding to integrin {alpha}5{beta}1 and the resultant inhibition of the integrin-linked kinase/protein kinase B/Akt pathway. They also report that GM3 blocks epidermal growth factor receptor activation induced by disrupting the association of integrin {beta}1 with the epidermal growth factor receptor (40). They raised the possibility of the modification of caveolin-1 with GM3 as a mechanism, but no evidences were demonstrated. Kawakami et al. (41) also report the promotion of interaction between tetraspanin CD9 and {alpha}3 integrin in microdomain with GM3, leading to inhibition of laminin-5-dependent cell motility. This regulation seems to be based on the modified organizational status of the glycolipid-enriched microdomain with glycosylation status. Actually, alteration of glycosylation in glycolipids affects intracellular localization of integrin, Src, and caveolin into or out of the glycolipid-enriched microdomain (42). Many of these studies indicate that gangliosides suppress integrin functions in cell adhesion, and mainly monosialyl gangliosides have been analyzed in those studies. However, many so-called "cancer-associated glycolipids" are polysialo compounds, as described in the Introduction. Therefore, modes of interaction with integrin and resulting effects should be different depending whether the glycolipids are monosialyl or polysialyl structures. In previous studies, direct interaction between gangliosides and integrin has never been clearly demonstrated, although the possibility that GD2 associates with integrins was suggested by Cheresh et al. (33) on the basis of their co-purification with an affinity column containing either an Arg-Gly-Asp-containing peptide, concanavalin A, or lentil lectin. However, actual molecular interaction has never been demonstrated. Results in this study elucidated, at least partly, the molecular association of GD2 with a membrane-penetrating molecule and an intracellular kinase molecule, i.e. GD2/integrin/FAK, although it is not yet clear whether the interaction between GD2 and integrins is direct or indirect. Precise modes of interaction between polysialo gangliosides and integrins should be clarified with more effective approaches, such as cross-linking, to further understand the mechanisms for apoptosis with anti-ganglioside mAbs.



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  		FIG. 7.
Apoptosis induced by anti-GD2 mAb was protected with inhibition of p38. A, inhibition of p38 activation with p38 inhibitor SB203580. Cells were treated with SB203580 at various concentrations of 0-100 µM and then exposed to UV light (100 J/m2). The inhibitory effects were examined with immunoblotting of anti-phospho-p38 (top). The band intensities were measured and corrected with those of total p38 (bottom) and then plotted (middle). B and C, cells with ~70% inhibition of p38 activity were used for apoptosis induction experiments. After treatment with SB203580 (100 µM) or Me2SO (vehicle), the cells were treated with mAb 220-51 (60 µg/ml) for 1.5 h and used for apoptosis assay by FACS. The top panels are results of FACS analysis with Annexin V and propidium iodide, and bottom panels are those with side scatter (SSC) and forward scatter (FSC) to show the changes in the ratio of living cells. Apoptosis in D-18 was almost completely protected by the addition of SB203580 (B, right panels). In the vector control cells, no apoptosis was induced by anti-GD2 mAb regardless of SB203580 treatment (B, left panels). Apoptosis in two native GD2-expressant lines was also protected by pretreatment with SB203580 (C). The protection of apoptosis was also shown by the increase of living cell populations in the bottom panels in B and C.

 
Whatever the mechanisms are, the fact that not only GD2-overexpressing cells but native GD2-expressant SCLC cell lines underwent equivalent degrees of apoptosis with anti-GD2 mAb strongly encourages us to apply the antibody therapy for the treatment of SCLC patients. The ganglioside nomenclature followed was that of Svennerholm (43).



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  		FIG. 8.
Inhibition of p38 showed no effect on the dephosphorylation of FAK. FAK phosphorylation levels were analyzed after treatment with anti-GD2 mAb using SB203580-pretreated cells, as described in the legend to Fig. 6. Intensities of phospho-FAK bands (top) were normalized with those of total FAK (middle) and plotted (bottom). SB203580 showed almost no effect on the phosphorylation and dephosphorylation of FAK before and after anti-GD2 mAb treatment, respectively. Similar results were obtained in at least three experiments.

 



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  		FIG. 9.
Proposed apoptosis pathway of small cell lung cancer cells. The binding of anti-GD2 mAbs might trigger the conformational changes of integrin molecules resulting in the dephosphorylation of FAK and subsequent activation of p38. Circled P means phosphorylated tyrosine.

 

   FOOTNOTES
 
* This study was supported by the grants-in-aid for scientific research on priority areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) (14082102, 16390075, 16590243). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. of Biochemistry II, Nagoya University School of Medicine 65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan. Tel.: 81-52-744-2070; Fax: 81-52-744-2069; E-mail: koichi{at}med.nagoya-u.ac.jp.

1 The abbreviations used are: SCLC, small cell lung cancer; mAb, monoclonal antibody; MTT, 3-(4,5-dimethyl-2-tetrazolyl)-2,5-diphenyl-2H tetrazolium bromide; HRP, horseradish peroxidase; FAK, focal adhesion kinase; siRNA(s), small interfering RNA(s); MAPK, mitogen-activated protein kinase; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorting. Back


   ACKNOWLEDGMENTS
 
We thank M. Urano for technical assistance.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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