STI571 (Glivec) suppresses the expression of vascular endothelial growth factor in the gastrointestinal stromal tumor cell line, GIST-T1

Figure 1 4×10⁶ of GIST-T1 cells were treated with STI571 (1 μg/mL) for 1 h. Treated cells were lysed in RIPA buffer and immunoprecipitation with anti-c-KIT antibody as described in “Materials and methods”. Western blotting was performed using anti-phosphotyrosine antibody (upper panel) or anti-c-KIT antibody (lower panel). Lane 1, non-treated; lane 2, STI571 1 μg/mL. WB, Western blot; IP, immunoprecipitation.

Figure 2 RT-PCR, 1×10⁶ GIST-T1 cells were treated with STI571 (0. 01, 0.1, and 1 μg/mL) for 6 h (A) or 1 μg/mL of STI571 for 60, 120, and 360 min (B). A: Lane 1, non-treated; lane 2, 0.01 μg/mL; lane 3, 0.1 μg/mL; lane 4, 1 μg/mL; B: lane 1, 0 min; lane 2, 60 min; lane 3, 120 min; lane 4, 360 min. GIST-T1 cells were treated with SCF (10 ng/mL) for 1, 2, 6 h (C). C: lane 1, 0 h; lane 2, 1 h; lane 3, 2 h; lane 4, 6 h. GIST-T1 cells were treated with STI571 (1 μg/mL), LY294002 (50 μmol/L), PD98059 (50 μmol/L), or Wortmannin (100 nmol/L) for 6 h (D). D: Lane 1, non-treated; lane 2, STI571; lane 3, LY294002; lane 4, PD98059; lane 5, Wortmannin. 1×10⁶ DLD-1 cells were treated with SCF in different concentrations for 6 h (E). E: Lane 1, non-treated; lane 2, SCF 5 ng/mL; lane 3, SCF 10 ng/mL; lane 4, SCF 15 ng/mL; lane 5, SCF 20 ng/mL; lane 6, SCF 100 ng/mL. GIST-T1 cells were treated with STI571, SCF, or combination of both for 6 h to determine the transcriptional level of HIF-1 alpha (F). F: Lane 1, non-treated; lane 2, STI571 1 μg/mL; lane 3, SCF 10 ng/mL; lane 4, SCF 10 ng/mL+STI571 1 μg/mL; lane 5, SCF 10 ng/mL+STI571 10 μg/mL.

Figure 3 Expression of HIF-1 alpha was determined by flow cytometry. 2 × 10⁶ GIST-T1 cells were treated with/without STI571 as described in “Materials and the methods. A: Control (normal rabbit IgG); B: non-treated (HIF-1 alpha antibody); C: STI571 (1 μg/mL). MFI, mean fluorescent intensity.

Figure 4 Expression of VEGF determined by flow cytometry. 2×10⁶ GIST-T1 cells were treated with/without STI571 or SCF as described in “Materials and methods”. A: Control (normal rabbit IgG); B: non-treated (VEGF antibody); C: STI571 (1 μg/mL); D: SCF (10 ng/mL); E: SCF (10 ng/mL)+STI571 (1 μg/mL). MFI, mean fluorescent intensity.

Figure 5 Assessment of expression of VEGF by Western blotting. 3×10⁶ cells were treated with STI571 (0.01, 0.1, and 1 μg/mL) for 12 h, and then cells were rinsed with ice-cold PBS and lysed in RIPA buffer as described in “Materials and methods”. Fifty micrograms of protein was applied in each lane. Lane 1: non-treated; lane 2: STI571 0.01 μg/mL; lane 3: STI571 0.1 μg/mL; lane 4: STI571 1 μg/mL.