Inhibition of radiation induced migration of human head and neck squamous cell carcinoma cells by blocking of EGF receptor pathways

The cell lines HN, BHY [26] and CAL-27 [27] were used (DSMZ, Braunschweig, Germany). Cells were grown in Dulbecco's modified Eagle medium (DMEM) or Roswell park memorial institute medium (RPMI 1640) (Invitrogen, Karlsruhe, Germany) containing 10% fetal calf serum, 2 mM glutamine, and 100 μg/ml penicillin/streptomycin and maintained at 37°C in an atmosphere of 5% CO₂ grown to a 70-90% confluence.

Irradiation was performed at the Department of Radiotherapy (Technical University of Munich). Cells were X-irradiated with single doses of 2, 5 or 8 Gy with a Philips RT 100 (Philips, Amsterdam) operated at 300 kV with 1.4 mm copper half-value layer at a dose rate of approximately 1 Gy/min. The dose inhomogeneity was ± 2%. The sham-treated group (0 Gy, control) was subjected to the same protocol as exposed cells.

Investigation of cell migration capability after irradiation treatment was performed by a modified wound healing assay, as described before [28]: Briefly, treated and untreated cells were grown to confluent monolayers. Immediately before irradiation the inhibitors rapamycin (100 nM) (Biomol, Hamburg, Germany), LY294002 (50 μM) (Calbiochem, Darmstadt, Germany), PD98059 (50 μM) (Biomol, Hamburg, Germany), tyrphostin AG1478 (10 μM) (Merck, Darmstadt, Germany) or the epidermal growth factor (EGF) (10 ng/ml) (Upstate, Billerica, USA) were separately added to the medium. After that, the monolayers were wounded by scratching the surface as uniformly as possible with a 200 μl pipette tip (Sarstedt AG & Co., Nümbrecht, Germany). After irradiation cells were cultivated for another 12 hrs. This initial wounding (0 hr) and the movement of the cells in the scratched area were photographically monitored under an inverted light microscope (field of view by a 40 fold magnification - Axiovert 25, Carl Zeiss AG, Göttingen, Germany, equipped with an Olympus SC 35 Camera, Volketswil, Switzerland). Migrating cells were counted 12 hours after irradiation (Figure 2). These time points were chosen because in former experiments all cells were grown to confluence after 36 hours (data not shown).

To confirm the results of the scratch test we analyzed the migration by a modified Boyden chamber. Cells were given to a transwell permeable polycarbonate membrane with a pore size of 0.8 μm (Corning Incorporated, New York, USA). Inhibitors were added in the medium above and under the membrane then irradiation were done. 12 hrs later cells above the membrane were removed by a cotton drill, and fixed with DAPI. Thereafter cells were counted under a microscope. These experiments were performed to show consistent and comparable results of radiation induced migration.

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Roche Diagnostics, Penzberg, Germany) was used to assess cell proliferation, as previously described [29]. Briefly, cells were plated on 96-well plates at a concentration of 1000 cells/well. The above-mentioned inhibitors were added 12 hours prior to irradiation. After incubation, and 12, 24 and 72 hours after irradiation, 10 μl of MTT solution was added to each well for four hours (37°C). Subsequently, 100 μl of dimethylsulfoxide was added to each well, yielding purple solution. The optical density was measured at 590 nm using an ELISA reader (ASYS Hitech, Eugendorf, Germany) and ratios in relation to controls were made. All experiments were performed eight times (n = 8).

Immunoblot analysis was performed to determine EGFR expression including its downstream proteins ERK and Akt. 12 hours after irradiation, cells were harvested in lysis buffer (Cell Lysis Buffer, New England Biolabs, Ipswich, USA) at 4°C. Lysates were centrifuged (10000 rpm) for 15 minutes at 4°C to remove insoluble components. Protein content was quantified by the Bio-Rad Dc protein assay (Bio Rad, Hercules, USA). Equal amounts of protein were separated on SDS-PAGE 10% or 12.5% gels. Proteins were transferred to Immobilon-P PVDF membrane (Millipore, Billerica, USA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0,1% Tween 20 (TBST) and afterwards incubated with primary antibody in 5% nonfat dry milk in TBST, followed by secondary antibody linked to rabbitradish peroxidase diluted in 5% nonfat dry milk in TBST. ECL Detection System for Western blot Analysis (Amersham, Freiburg, Germany) was used according to the manufacturer's instructions. The Imager SRX-101° (Konica Minolta, Langenhagen, Germany) was used to detect bands of appropriate sizes. The following antibodies were used: phospho-EGFR (Tyr1068), phospho-Akt (Ser473), PKB/Akt, phospho-p44/42 ERK (Thr202/Tyr204), p44/42 ERK, phospho-Raf (Ser259), phospho-MEK1/2, and MEK1/2. All antibodies were obtained from Cell Signaling Technology, (Boston, USA) and used at a dilution of 1:1000.

For the investigation of cell migration, a two-factorial design was considered with the factors "treatment" (control, EGF, LY294002, PD98059, rapamycin, AG1478) and radiation dose (ranging from 0 Gy to 8 Gy). The whole analysis was repeated n = 9 times. Cell proliferation was investigated for all six treatments, for doses 0 Gy and 8 Gy only. For each dose and each group, the sample size was n = 8. In the whole study, the cells were randomly assigned to the treatment groups and radiation doses.

Radiation induced migration was assessed in a linear regression model were migration was set as dependent variable and the radiation dose as the metric predictor. The potential dose dependent inhibition or enhancement of migration through stimulation was investigated based on a generalized least squares model fitted with the R function 'gls' with the migration as dependent variable. The predictors were: the radiation dose (metric predictor, coefficient β_rad), the treatment (categorical predictor with coefficients β_EGF, β_AG, etc and controls as reference category), and their interactions (coefficients β_rad.EGF, β_rad.AG, etc). A uniform correlation structure was assumed within each of the n = 9 experiments, corresponding to a linear mixed model with a random forest for each experiment. Residual analysis showed that this model can reasonably be applied to the data at hand. Additionally, linear hypotheses tests were performed in the above GLS model to test the effect of the radiation dose in the presence of treatment (tested hypotheses: β_rad+β_rad.EGF = 0, β_rad+β_rad.AG = 0, etc). Confidence intervals for the estimated coefficients were calculated, and all hypotheses were tested based on the Wald test. Separate analyses were conducted for the three cell lines BHY, CAL-27 and HN for the time point 12 hours. The t-test was used to compare proliferation in two conditions. Confidence intervals for the difference of means were calculated. All statistical analyses were performed using the R statistical software http://​www.​r-project.​org, version 2.6.1.

The cultured cell lines BHY, CAL-27 and HN were irradiated with 2, 5 and 8 Gy and monitored during 12 hours. The means and standard errors of nine tests per time point and dose were calculated for each cell line. Radiation induced a significant dose dependent migration of tumor cells after irradiation compared to the control group (BHY: β_rad = 16, CI:[6;26], p = 0.003, CAL-27: β_rad = 20, CI:[10;30], p < 0.001, HN: β_rad = 37, CI:[22;51], p < 0.001) (Figure 3).

The findings of the wound healing assay were consistent with the results of the modified Boyden chamber where a radiation induced migration was also observed.

The stimulation of not irradiated (0 Gy) cells with EGF lead to a significant increase of migration in all cell lines (tested hypothesis β_EGF = 0, BHY: β_EGF = 137 CI:[66;209], p < 0.001, CAL-27: β_EGF = 79, CI:[1;156], p = 0.048, HN: β_EGF = 211, CI:[113;308], p < 0.001). In contrast, the EGFR inhibitor AG1478 significantly decreased migration (Figure 4).

The radiation-induced increase of migration was significantly less pronounced after stimulation with EGF (tested hypothesis β_rad.EGF = 0, BHY: β_rad.EGF = -26, CI:[-41;-11], p < 0.001, CAL-27: β_rad.EGF = -26, CI:[-42,-10], p = 0.002, HN: β_rad.EGF = -21, CI:[-41,-1;], p = 0.042) as well as after inhibition with AG1478 (tested hypothesis β_rad.AG = 0, BHY: β_rad.AG = -17, CI:[-32;-2], p = 0.028, CAL-27: β_rad.AG = -19, CI:[-35;-3], p = 0.021, HN: β_rad.AG = -31, CI:[-52,-11], p = 0.003) than in control cells (Figure 3). More precisely, migration did not increase significantly with radiation dose in the cells stimulated with EGF or inhibited with AG1478 (tested hypotheses: β_rad+β_rad.EGF = 0, β_rad+β_rad.AG = 0, p > 0.05), in contrast to what happens in control cells.

Additionally to the above mentioned inhibition of the EGF receptor, we blocked the downstream pathways of EGFR: PI3K by LY294002 (30 minutes before radiation), mTOR by rapamycin (1 hour before radiation) and MEK1 by PD98059 (30 minutes before radiation). A significant negative interaction between irradiation and the inhibitors was seen in the HN cell line after 12 hours. These findings indicate that the radiation-induced migration of tumor cells was decreased significantly by downstream inhibitors of the EGFR (Table 1). Migration was most effectively decreased by blocking of PI3K (LY294002) (Figure 5, Table 1).

After inhibition migration did not significantly increase with radiation dose (tested hypotheses: β_rad+β_rad.LY = 0, β_rad+β_rad.Rapa = 0, β_rad+β_rad.PD = 0, p > 0.05) in all 3 cell lines, in contrast to what happens in controls. Thus, inhibition seems to attenuate the influence of radiation on migration.

The strongest migration ability was observed at the dose of 8 Gy. Therefore we focused on studying the effects elicited at this radiation dose. We found a significant decrease of proliferation after radiation with 8 Gy after 72 hours (BHY: CI:[-0.19,-0.12], p < 0.001; CAL-27: CI:[-0.13,-0.05], p < 0.001; HN: CI:[-0.07,-0.01], p = 0.014). Stimulation with EGF showed no significant effect on proliferation (BHY: CI:[-0.11,0.06], p = 0.5; CAL-27: CI:[-0.09,-0.01], p = 0.02; HN: CI:[0.02,0.09], p = 0.005) without radiation, and no significant effect by simultaneously radiation with 8 Gy (BHY: CI:[-0.06,0.10], p = 0.57; CAL-27: CI:[-0.05,0.01], p = 0.19; HN: CI:[-0.05,0.005], p = 0.10). After EGF receptor blockade with AG1478 a significant decrease in proliferation was observed, compared to the control group (BHY: CI:[-0.27,-0.15],, CAL-27: CI:[-0.20,-0.14],, HN: CI:[-0.13,-0.07], p < 0.001).

Inhibition of MEK1 with PD98059 or inhibition of mTOR with rapamycin reduced significantly proliferation (BHY: CI:[-0.25,-0.12], CAL-27: CI:[-0.17,-0.10], HN: CI:[-0.15,-0.07], p < 0.001). Blocking of PI3K with LY294002 also reduced proliferation (BHY: CI:[-0.36,-0.31], CAL-27: CI:[-0.26,-0.20], HN: CI:[-0.24,-0.18], p < 0.001) (Figure 6).

Protein was isolated at 0 hours and 24 hours after radiation with 8 Gy. In all 3 cell lines we found a constitutive activation of Akt and ERK. The stimulation with EGF preceded an up-regulation of EGFR phosphorylation and a phosphorylation of the downstream pathways. Blockade of the EGFR by AG1478 provoked a down regulation of the receptor, the PI3K/Akt and the Raf/MEK/ERK pathways. This effect continued during 24 hours. After radiation an up regulation of the EGFR phosphorylation was observed. The western blot results are presented in Figure 7.