Figure 8
Vascular endothelial growth factor secretion in U138 and A172 glioma cell lines. (A) Effects of WAY and E4031 on VEGF secretion in U138 cells. The amount of VEGF secreted into the medium was determined with an ELISA performed in triplicate (see Materials and Methods) after cells were incubated for 24 h in serum-free medium. The kit used allows detection of both VEGF121 and VEGF165. WAY and E4031 were added at time zero, at the final concentrations indicated below. Values are expressed as pg of secreted VEGF/103 cells during 24 h incubation. The number of cells per well was the following: Control: 5.98±0.59 × 105 (n=11); WAY 1 μ M: 6.31±0.49 × 105 (n=3); WAY 5 μ M: 5.16±1.12 × 105 (n=5); WAY 20 μ M: 5.09±1.08 × 105 (n=4); E4031 5 μ M: 7.58±0.5 × 105 (n=3). Data reported are means±s.e.m. of three to five separate experiments, each carried out on triplicate samples. *Significantly different from the control (Student's t-test for paired samples): WAY 5 μ M: P=0.03; WAY 20 μ M: P=0.01; WAY 40 μ M: P=0.003. Inset: bFGF secretion in U138 cells in the absence or presence of WAY. The same supernatants as those used for VEGF measurement in panel A were used for the detection of the secreted bFGF, using an ELISA (see Materials and Methods). Note the different amount of secreted bFGF as compared to VEGF. (B) Effect of WAY and E4031 on VEGF secretion in A172 cells. Vascular endothelial growth factor was quantified as reported in (A). WAY (5 and 20 μ M, final concentrations) and E4031 (20 μ M, final concentration) were added at time zero. The number of cells per well was the following: Control: 2.21±0.03 × 105 (n=3); WAY 5 μ M: 2.18±0.15 × 105 (n=2); WAY 20 μ M: 2.18±0.04 × 105 (n=2); E4031 5 μ M: 2.06 × 105 (n=1). Data reported are means±s.e.m. of two to three separate experiments, each carried out on triplicate samples. It is worth pointing out that the difference in the amount of basal VEGF secretion between U138 and A172 cells can be attributed to the different cell size. (C) Semiquantitative analysis of vegf mRNA expression after WAY addition. Vegf expression was determined after 24 h of cell incubation in the absence or presence of increasing concentrations of WAY (5, 20 and 40 μ M). PCR conditions were adjusted in order to determine the number of cycles corresponding to the exponential phase of amplification, when semiquantitative analysis is possible. Densitometry was performed with Scion Image software by comparing the intensity of vegf (25 cycles) and gapdh (20 cycles) PCR products. Vegf expression was inhibited by approximately 30% by treatment with 40 μ M WAY.
