Treatment schedule is of importance when gefitinib is combined with irradiation of glioma and endothelial cells in vitro

Figures & data

Figure 1.  (A) Western blot analysis showing expression of EGFR, ErbB2, and actin, in human glioma 251MG, SF-767 cells, rat glioma BT4C cells, rat brain endothelial RBE4 cells, and epidermoid carcinoma A431 cells (used as positive control for EGFR). (B) Dose-response for gefitinib induced cytotoxicity to 251MG, SF-767, BT4C, and RBE4 cells after exposure to gefitinib (0–10 mg/l). (C) Western blot for phosphorylated EGFR and Akt expression of human glioma 251MG, and SF-767 cells, and of epidermoid carcinoma A431 cells (used as positive control for phophorylated EGFR), after stimulation with EGF (50 ng/ml) in the presence or the absence of gefitinib (6 mg/l).

Figure 2.  Cytotoxicity to 251MG, SF-767, BT4C, and RBE4 cells after exposure for gefitinib for 30 min prior to irradiation with single doses at 2, 4, and 6 Gy, respectively, followed by continuous incubation with the drug for 6 days after irradiation of the cells. Data denote mean values±SD (standard deviation) for 16 separate observations and each figure is representative for three individual experiments. Significant difference (*p < 0.05) between irradiation alone and irradiation plus gefitinib are indicated.

Figure 3.  Cytotoxicity to SF-767, BT4C, 251MG, and RBE4 cells after exposure for gefitinib for 24 h prior to irradiation with 2, 4, and 6 Gy, respectively, without following incubation with gefitinib. Data denote mean values±SD (standard deviation) for 16 separate observations and each figure is representative for three individual experiments. Significant difference (* = p < 0.05) between irradiation alone and irradiation plus gefitinib are indicated.

Figure 4.  Cytotoxicity to SF-767, BT4C, RBE4, and 251MG cells after post-irradiation exposure for gefitinib at varying length of time (2–24 h) after irradiation with 2, 4, and 6 Gy, respectively. ♦ Irradiation (RT), ▭ RT + gefitinib (administrated after 2 h), Δ RT + gefitinib (administrated after 4 h),×RT + gefitinib (administrated after 8 h),

RT + gefitinib (administrated after 12 h), ○ RT + gefitinib (administrated after 24 h). The used doses were for SF-767 (3 mg/l), BT4C (6 mg/l), RBE4 (5 mg/l), and 251MG (5 mg/l). Data denotes mean values±SD (standard deviation) for eight separate observations and each figure is representative for three individual experiments. The only cell line where the differences between irradiation alone and irradiation plus gefitinib was not statistically significant (p < 0.05) was in the human glioma cell line 251MG at the single dose of 6 Gy, is indicated by ns = not significant.

Figure 5.  Histogram (A) and representative images (B-E) showing increased nuclear DNA fragmentation in rat glioma BT4C cells, 6 days after irradiation treatment (RT) with 2, 4, and 6 Gy, respectively. In the combined treatment group gefitinib was administrated 24 h after irradiation. As a positive control the cells were incubated with cisplatin (1 mg/l), which is known to induce apoptosis in this cell line. Data denotes mean values±SD (standard deviation) for three individual experiments. Significant difference (*p < 0.05) between irradiation alone and irradiation plus gefitinib are indicated. (B) control (untreated) vs. gefitinib (6 mg/l), (C) RT (2 Gy) alone vs. RT (2 Gy) + gefitinib (6 mg/l), (D) RT (4 Gy) alone vs. RT (4 Gy) + gefitinib (6 mg/l), and (E) RT (6 Gy) alone vs. RT (6 Gy) + gefitinib (6 mg/l).