Regulatory effect of nerve growth factor in alpha9beta1 integrin-dependent progression of glioblastoma

Abstract

In the present study we described the role of alpha9beta1 integrin in glioblastoma progression following its interaction with nerve growth factor (NGF). The level of expression of alpha9beta1 on astrocytomas is correlated with increased grade of this brain tumor and is highest on glioblastoma, whereas normal astrocytes do not express this integrin. Two glioblastoma cell lines, LN229 and LN18, that are alpha9beta1 integrin positive and negative, respectively, were used for alpha9beta1 integrin-dependent NGF-induced tumor progression. NGF was a significant promoter of promigratory and pro-proliferative activities of glioblastoma cells through direct interaction with alpha9beta1 integrin and activation of MAPK Erk1/2 pathway. The level of NGF increases approximately threefold in the most malignant glioma tissue when compared with normal brain. This increase is related to secretion of NGF by tumor cells. Specific inhibitors of alpha9beta1 integrin or gene silencing inhibited NGF-induced proliferation of LN229 cell line to the level shown by LN18 cells. VLO5 promoted alpha9beta1-dependent programmed cell death by induction of intrinsic apoptosis pathway in cancer cells. LN229 cells were rescued from proapoptotic effect of VLO5 by the presence of NGF. This disintegrin significantly inhibited tumor growth induced by implantation of LN229 cells to the chorioallantoic membrane (CAM) of quail embryonic model, and this inhibitory effect was significantly abolished by the presence of NGF. alpha9beta1 integrin appears to be an interesting target for blocking the progression of malignant gliomas, especially in light of the stimulatory effect of NGF on the development of these tumors and its ability to transfer proapoptotic signals in cancer cells.

Fig. 1.

(A) Representative images of immunohistochemical analysis of normal brain and different grades of gliomas stained with anti-α9 polyclonal antibody. The tissue array plates were blocked with 5% normal horse serum and incubated with a primary antibody overnight at room temperature in a humidified chamber. Biotinylated antirabbit immunoglobulin G was added and incubated for 1 h. Color was developed by incubation with avidin-biotin complex and diaminobenzidine. Images were analyzed using an Olympus AX70 light microscope with 100× and 400× magnification. (B) Comparison of expression of α9β1 integrin in normal brain and different grades of glioma tissues by Western blot analysis. Frozen samples of human tissues were obtained from patients’ surgical resection. Tissues were homogenized in a lysis buffer–containing cocktail of protease inhibitors, and soluble fractions were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis with a total amount of 20 μg of protein per sample. After electrophoresis, proteins were transferred onto the polyvinylidene fluoride membrane and incubated first with primary anti-α9 polyclonal antibody, and then horseradish peroxidase–conjugated goat anti-rabbit secondary antibody. The bands were visualized using a chemiluminescent Western detection kit. The numbers above the bands represent value of average pixels, reflecting the intensity of the bands, which were digitalized using Un-Scan-It gel software (Silk Scientific, Inc., Orem, UT, USA). Error values represent SD for normal brain (n = 5), grade II glioma (n = 3), and grade IV glioma (n = 7).

Fig. 2.

Identification of integrins on human glioblastoma cell lines. (A) Monoclonal antibodies against integrins were immobilized overnight at 4°C on a 96-well plate in phosphate-buffered saline. LN18 (light bars) and LN229 (dark bars) cell lines were labeled with 5-chloromethylfluorescein diacetate and added (1 × 10⁵/100 μl) to the wells, previously blocked by bovine serum albumin. Incubation was performed at 37°C for 30 min in Hank’s balanced salt solution buffer containing calcium and magnesium. After washing the unbound cells, Triton X-100 was added to the wells, and the plate was read using a fluorescence microplate reader at an excitation wavelength of 485 nm using a 530-nm emission filter. The number of adhered cells was calculated from the standard curve prepared in parallel on the same plate from a known number of cells. The data represent the mean from three independent, duplicated experiments. (B) Western blot analysis of cell line lysates with polyclonal anti-α9 antibody. The molecular weight markers are indicated by arrows. (C) Immunocytostaining of glioblastoma cell lines by polyclonal antibody against α9 and monoclonal anti-α2 antibody (clone P1E6). Images of cells were analyzed in the presence of 4’,6-diamidino-2-phenylindole using a fluorescent convolution microscope (Nikon TE-300) with 400× magnification. (D) Expression of α9β1 and α2β1 integrins analyzed by flow cytometry in LN229 and LN18 glioblastoma cell lines. Cells were stained with Y9A2 or P1E6 monoclonal antibodies against α9 and α2 integrin subunits, respectively, and analyzed using the Guava EasyCyte flow cytometry system (Guava Technologies, Hayward, CA, USA).

Fig. 3.

Identification of TrkA and p75^NTR on glioblastoma cell lines. (A) Western blot analysis of cell line lysates using polyclonal serum against TrkA (left panel) and polyclonal antibody against p75^NTR (right panel). The numbers above the bands represent the values of average pixels, reflecting the intensity of the bands, which were digitalized using Un-Scan-It gel software (Silk Scientific, Inc., Orem, UT, USA). (B) Immunocytostaining of LN229 and LN18 glioblastoma cell lines with polyclonal anti-TrkA and anti-p75^NTR. Images of cells were analyzed using a fluorescent convolution microscope (Nikon TE-300) with 400× magnification. Left images show staining with anti-TrkA, whereas right images show staining with anti-p75^NTR polyclonal primary antibodies.

Fig. 4.

Interaction of normal human astrocytes and glioblastoma cell lines with nerve growth factor (NGF) in adhesion assay and effect of integrin antibodies and disintegrins on adhesion of LN229 cell line to immobilized NGF. (A) Adhesion of normal primary human astrocytes (triangles) and LN18 (open circles) and LN229 (filled circles) glioblastoma cell lines to immobilized mouse NGF (mNGF). Different concentrations of mNGF were immobilized on a 96-well, and adhesion of 5-chloromethylfluorescein diacetate (CMFDA)–labeled cell lines was performed as described in the Fig. 2 caption. Error bars represent SD from three independent duplicated experiments. (B) Effect of monoclonal antibodies against various integrins and snake venom disintegrins on the adhesion of LN229 cell line to immobilized mNGF. mNGF (10 μg/ml) was immobilized on a 96-well plate, and CMFDA-labeled cells were added to the wells in the absence or presence of blocking integrin antibodies (10 μg/ml) or disintegrins (1 μM). Error bars represent SD from three independent duplicated experiments.

Fig. 5.

Chemotaxis of astrocytoma cell lines induced by different chemoattractants. Experiment was performed using a Boyden chamber with fluoroblock membranes (3 μm). Cells were labeled with calcein in the culture by incubation for 1 h. After detaching and washing, cells were applied to the upper chamber in Dulbecco’s modified Eagle’s medium, whereas chemoattractants were applied to the lower chamber: 2% fetal bovine serum, VLO5 (1 μM), Y9A2 (10 μg/ml), NGF (1 μg/ml). Incubation was performed on a 24-well plate at 37°C for 1 h. Plate was read by a fluorescence plate reader with bottom reading option at an excitation wavelength of 485 nm using a 530 nm-emission filter. The asterisks indicate p < 0.05 in comparison with random migration, as statistically significant differences.

Fig. 6.

Effect of nerve growth factor (NGF) on proliferation and signal transduction in glioblastoma cell lines. (A) Effect of different concentrations of mouse NGF (mNGF) on proliferation of LN229 and LN18 cell lines. The asterisks indicate p < 0.05 in comparison with the untreated mNGF control cells, as statistically significant differences. (B) Effect of α9β1 integrin inhibitors on proliferation of LN229 cell line stimulated or not with mNGF. (C) Effect of α9 gene silencing on proliferation of LN229 cell line. Cells were grown on a 96-well plate up to 70% confluence and then treated for 48 h with appropriate concentrations of mNGF in the absence or presence of Y9A2 or VLO5 in the medium containing 2% fetal bovine serum. α9 gene silencing was performed in LN229 cells using two duplexes of pre-design siRNA (Ambion, Inc., Austin, TX, USA). 5-bromodeoxyuridine color development assay was performed according to manufacturer’s instructions (Roche). Error bars represent SD from triplicated experiments. (D) NGF-induced Erk1/2 phosphorylation in LN229 and LN18 cell lines. Cells were cultured to the confluence of about 90% on a 6-well plate and starved for 48 h. Cells were stimulated with NGF at concentrations of 100 ng/ml (line b), 200 ng/ml (line c), and 500 ng/ml (line d), or not (line a) and lysed for 30 min. Lysates were applied on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membrane, and incubated with primary anti-pErk or anti-Erk polyclonal antibodies. The bands were visualized using a chemiluminescent Western detection kit. The numbers above the bands represent values of average pixels, reflecting the intensity of the bands, digitalized using Un-Scan-It gel software (Silk Scientific, Inc., Orem, UT, USA).

Fig. 7.

Activity of VLO5 and nerve growth factor (NGF) in regulation of LN229 cell apoptosis and survival. (A) Effect of treatment of LN229 cells in the culture by NGF and VLO5. Cells were grown on a 6-well plate up to 90% confluence in complete Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Treatment was performed in DMEM containing 2% FBS. After 24 h cells were analyzed under a contrast phase microscope (Nikon TE-300) using 400× magnification. (B) Time-dependent analysis of apoptosis in LN229 cells following treatment with VLO5 and NGF. Cells were grown on a 96-well plate up to 70% confluence, and after washing with serum-free DMEM were incubated with 1 μM VLO5 in the presence or absence of 100-ng/ml NGF in the media containing 2% FBS. Cells were fixed by 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate on ice for 2 min. Terminal deoxy-nucleotidyl transferase mediated dUTP nick end labeling (TUNEL) reaction mixture was added and incubated in the dark for 1 h at 37°C. Finally, the mounting buffer containing 4’,6-diamidino-2-phenylindole was added and slides were analyzed under fluorescence microscopy (Nikon TE-300). Error bars represent cell counts from three different observation fields under microscope, performed by three investigators blinded for experimental protocol. (C) VLO5-dependent activation of caspases in LN229 cells. The LN229 cells were grown in 6-well plates up to 70% confluence. The cells were treated in culture with VLO5 (1 μM), NGF (100 ng/ml), or both reagents together. Vincristine (50 μg/ml) was used as a control. Cells were detached and caspases 3/7, 8, and 9 were detected using CaspaTag In Situ Assay Kit (Chemicon). Error bars represent SD from three experiments. Asterisks show statistically significant differences (p < 0.01) according to control.

Fig. 8.

Effect of mouse nerve growth factor (mNGF) and VLO5 on development of glioma tumor induced by implantation of LN229 cell line on quail chorioallantoic membrane (CAM). (A) Images of embryos with fully developed tumor on day 12 (left panels), and pictures of dissected, fixed CAMs with fully developed tumor on day 12 (right panels). (B) Comparison of weight of tumors treated and untreated with mNGF and VLO5. Embryos were grown on 6-well plates until day 7, and LN229 cells (2 × 10⁷ per embryo per 50 μl) were placed on the top of the CAM. Tumors were allowed to grow for 24 h and then were treated with mNGF and/or VLO5 every day until day 12 by direct topping. Control embryos received vehicle treatment (50 μl phosphate-buffered saline [PBS]). On day 12 embryos were fixed and CAMs containing tumor were dissected. Tumors were dissected from the CAMs and weighed. The areas of the tumors’ growth on the CAMs are framed on the embryos’ pictures. The asterisks indicate p < 0.01 in comparison with control PBS-treated embryos, as statistically significant differences.