Figure 3.

Colorectal carcinoma (CRC) cell-secreted VEGF induces human endothelial cell (HUVEC) proliferation.

(A) Presence of VEGF in the supernatant of CRC cell cultures. VEGF was detected with specific ELISA according to manufacturer’s instructions (R&D Systems GmbH, Germany). For this, 1 million CRC cells and HUVEC were cultured in 6-well plates with serum reduced (1%) RPMI 1640 medium for 24 hours. Levels of detected VEGF ranged from 1000 pmol/l for T84 to over 7000 pmol/l for HT29. In supernatant of HUVEC, detected VEGF levels ranged below 100 pmol/l.

(B) E7080 suppresses HUVEC proliferation in the presence of CRC cell-secreted VEGF. Representative results shown for CaCo2 cells with CRC cell supernatant and/or E7080 (1 μmol/l). See also Supplementary Figure 2. HUVEC (1.5 × 10⁴ cells) were seeded in 96-well plates coated with attachment factor (#S-006-100, Gibco Life Technologies) for 24 hours. The cells were washed with PBS and incubated with 100 μl CRC cell line supernatant, 100 μl fresh serum-reduced (1%) RPMI 1640 medium and E7080 (0.1 μmol/l) for 72 hours. Cell viability was determined with WST-8 assay. ***P < 0.001 to HUVEC control proliferation (without CRC cell supernatant and E7080).

(B) E7080 suppresses HUVEC proliferation in the presence of CRC cell-secreted VEGF. Representative results shown for CaCo2 cells with CRC cell supernatant and/or E7080 (1 μmol/l). See also Supplementary Figure 2. HUVEC (1.5 × 10⁴ cells) were seeded in 96-well plates coated with attachment factor (#S-006-100, Gibco Life Technologies) for 24 hours. The cells were washed with PBS and incubated with 100 μl CRC cell line supernatant, 100 μl fresh serum-reduced (1%) RPMI 1640 medium and E7080 (0.1 μmol/l) for 72 hours. Cell viability was determined with WST-8 assay. ***P < 0.001 to HUVEC control proliferation (without CRC cell supernatant and E7080).