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Figure 1 | BMC Medicine

Figure 1

From: The cytoprotective drug amifostine modifies both expression and activity of the pro-angiogenic factor VEGF-A

Figure 1

Amifostine enhances vascular endothelial growth factor A (VEGF-A) mRNA and protein expression in cancer cells. MCF7 and HCT116 carcinoma cells, as well as U87 glioma cells, were grown up to 70% confluence in 10-cm culture dishes. Cells were then incubated in freshly added complete medium under classical conditions of oxygen (NT, 20% 02) or low oxygen conditions (LO, low O2, 3% 02), or in the presence of the indicated concentrations of WR-1065 under 20% of oxygen. (A) Amifostine increases VEGF-A mRNA levels in U87, MCF7 and HCT116 cells. Following a 16-h (MCF7 cells, grey bars) or a 24-h (HCT116 cells, white bars; U87 cells, black bars) incubation time, total mRNA was isolated. Expression of VEGF-A mRNA in cells was measured by quantitative polymerase chain reaction using primers amplifying all the VEGF-A isoforms. Histogram values represent the level of expression of all VEGF-A splice variants, normalized to α-tubulin. Results are the mean values ± standard error of mean of three independent experiments. (B) Amifostine increases specific VEGF-A mRNA isoforms. Shown is a representative gel electrophoresis pattern of the different VEGF-A splice variants in U87, MCF7 and HCT116 cells, β-actin being used as standard. (C) Amifostine increases VEGF-A protein secretion by MCF7 cells. Cells were treated up to 2 days in complete medium, and conditioned media were collected after 24 h, 36 h and 48 h of treatment. VEGF-A protein secretion was measured by ELISA using supernatants of cells grown under low levels of oxygen (3% O2,) or 20% of oxygen, and of cells treated with 1 mM WR-1065 in the presence of aminoguanidine (AG) or with AG alone. Results shown are representative of three independent experiments done in triplicates.

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