Irradiation differentially affects substratum-dependent survival, adhesion, and invasion of glioblastoma cell lines

Abstract

Effects of ionising radiation on extracellular matrix (ECM)-modulated cell survival and on adhesion and invasion are not well understood. In particular, the aggressiveness of glioblastoma multiforme has been associated with tumour cell invasion into adjacent normal brain tissue. To examine these effects in more depth, four human glioblastoma cell lines (A-172, U-138, LN-229 and LN-18) were irradiated on fibronectin (FN), Matrigel, BSA or polystyrene. Major findings of this study include a significantly increased survival of irradiated A-172 but not of irradiated U-138, LN-229, and LN-18 cells on FN or Matrigel compared to cells irradiated on polystyrene or BSA. Irradiation induced a dose-dependent increase in functional beta 1- and beta 3-integrins in all four glioma cell lines. This integrin induction caused improved cell adhesion to FN or Matrigel. In contrast to U-138, LN-229 and LN-18 cells, irradiation strongly impaired A-172 cell invasion. Invasion of all cell lines was inhibited by anti-integrin antibodies, the disintegrin echistatin and the MMP-2/-9 inhibitor III. Additionally, beta 1- and beta 3-integrins modulated basal and radiation-altered gelatinolytic activity of MMP-2. Tested glioblastoma cell lines showed a differential cellular susceptibility to FN or Matrigel which affected the cellular radiosensitivity. Three out of four glioma cell lines demonstrated a combination of a substratum-independent survival after irradiation and an invasive potential which was not affected by irradiation. beta 1- and beta 3-integrins were identified to play a substantial, regulatory role in survival, adhesion, invasion and MMP-2 activity. Detailed insights into radioresistance and invasion processes might offer new therapeutic strategies to enhance cell killing of lethal high-grade astrocytoma.

Figure 1

(A) Plating human A-172, U-138, LN-229 or LN-18 glioblastoma cells on FN, Matrigel, polystyrene or BSA differentially altered the cellular radiosensitivity. Survival of A-172 cells grown on FN or Matrigel was significantly (P<0.01) improved at doses ⩾4 Gy. In contrast, irradiated U-138, LN-229 or LN-18 cells showed no substratum-dependent alteration of cell survival. Each data point represents the mean ±s.d. of three independent experiments (n=18). (B) The surviving fractions after 2 (SF2) or 6 Gy (SF6) of A-172 and U-138 cells were significantly (P<0.01) reduced after single or combined β1- and β3-integrin depletion using siRNA transfection (see Western blot) as compared to irradiated cells transfected with unspecific Duplex II RNA (DII). Single Duplex II RNA (Unirr. DII) did not significantly reduce the plating efficiency of A-127 and U-138 cells. ^* P<0.01.

Figure 2

48-h flow cytometric analysis of β1- and β3-integrin cell surface expression in nonirradiated and irradiated A-172 and U-138 cells showed a dose-dependent induction of these two integrin receptors. Each data point represents the fold induction (mean ±s.d. of three independent experiments) of irradiated samples in comparison with unirradiated controls. P, polystyrene; FN, fibronectin; M, Matrigel.

Figure 3

Radiation (6 Gy) improved the capability of A-172 and U-138 cells to attach to fibronectin (FN) and Matrigel after 48 h in comparison with BSA or polystyrene (P). To determine functional β1- and β3-integrin engagement in radiation-altered adhesion in these two cell lines, the cells were incubated with specific adhesion-blocking antibodies against β1- (mAb13) or β3-integrins (RUU-PL7F12) or with the disintegrin echistatin. Control experiments were performed using equivalent concentrations of unspecific anti-mouse IgG2a or IgG1 antibodies. Columns represent the mean ±s.d. of the optical densities (OD) at 630 nm representing cell adhesion to the different substrates of three independent experiments. Statistical analysis compared adhesion to FN or Matrigel of nonirradiated cells with adhesion to FN or Matrigel of irradiated cells. ^* P<0.01.

Figure 4

The in-vitro invasion assay indicated that irradiation (6 Gy) is able to inhibit strongly A-172 but not U-138, LN-229 and LN-18 cell invasion into Matrigel. To analyse the dependence of the invasion process on MMP-2 or β1- or β3-integrins, nonirradiated as well as irradiated cells were either incubated with the MMP-2/-9 inhibitor III (MI), anti-β1- (mAb13) and anti-β3-integrin (RUU-PL7F12) antibodies or the β1-/β3-integrin-blocking disintegrin echistatin alone or in combination. Additionally, control experiments were performed using equivalent concentrations of unspecific anti-mouse IgG2a or IgG1 antibodies. The data were collected by counting the number of cells per field (four high-powered fields). Columns represent the calculated mean ±s.d. of three independent experiments. Statistical significance was calculated by comparing the rate of invasion after anti-MMP and/or anti-integrin treatment to the rate of invasion of untreated cells. Furthermore, statistics were calculated by comparing the rate of invasion of treated cells after irradiation to the rate of invasion after irradiation alone. nt, no treatment; ^*P<0.01.

Figure 5

Western blot analysis of MMP-2, MT1-MMP and TIMP-2 in nonirradiated (co) and irradiated cells showed that irradiation induced MMP-2 and MMT1-MMP expression within 48 h. Treatment of cells with MMP-2/-9 inhibitor III (MI), anti-β1- (mAb13) or anti-β3-integrins (RUU-PL7F12, RUU) or echistatin (Echi) prevented induction of MMP-2 and MT1-MMP and promoted induction of TIMP-2 after irradiation. In contrast, TIMP-2 expression was downregulated after irradiation and induced by anti-integrin treatment. nt, no treatment.

Figure 6

Double immunofluorescence staining of β1-integrin plus MMP-2 in A-172 and U-138 cells. β1-integrin and MMP-2 were visualised by confocal scanning microscopy after fixing and staining of cells with the appropriate antibodies. Arrows indicate β1-integrin plus MMP-2 colocalisation within focal adhesion sites in the cell membrane. Bar, 10 μm.

Figure 7

Conditioned media of 48-h nonirradiated (co) and irradiated A-172 (A) or U-138 (B) cells were analysed on 10% PAGE-gelatine. Effects of MMP-2/-9 inhibitor III (MI), anti-β1-integrin (mAb13) and anti-β3-integrin (RUU-PL7F12, RUU) antibodies and the β1-/β3-blocking disintegrin echistatin on the gelatinolytic activity of MMP-2 and its conversion from the pro (M_r 66 000) to the active form (M_r 62 000) are shown in nonirradiated and irradiated cells. Additionally, irradiation control experiments were performed using equivalent concentrations of unspecific anti-mouse IgG2a or IgG1 antibodies. Following β1- and/or β3-integrin siRNA transfection of A-172 and U-138 cells, the gelatinolytic activity of MMP-2 was strongly decreased in both cell lines. The combined depletion of both β1- and β3-integrin had the greatest effect. nt, no treatment; DII, unspecific Duplex II control.