Induction of neuropilin-1 and vascular endothelial growth factor by epidermal growth factor in human gastric cancer cells

Abstract

The epidermal growth factor receptor (EGF-R) pathway plays a pivotal role in the progression of human gastric cancer. The angiogenic factor vascular endothelial growth factor (VEGF) has been shown to be induced by EGF in various cancer cell lines. Neuropilin-1 (NRP-1) acts as a coreceptor for VEGF-165 and increases its affinity for VEGF receptor 2 (VEGFR-2) in endothelial cells. Furthermore, NRP-1 has been found to be expressed by tumour cells and has been shown to enhance tumour angiogenesis and growth in preclinical models. We examined the expression of NRP-1 mRNA and EGF-R protein in seven human gastric cancer cell lines. NRP-1 expression was expressed in five of seven cell lines, and EGF-R expression closely mirrored NRP-1 expression. Moreover, in EGF-R-positive NCI-N87 and ST-2 cells, EGF induced both NRP-1 and VEGF mRNA expression. C225, a monoclonal antibody to EGF-R, blocked EGF-induced NRP-1 and VEGF expression in NCI-N87 cells in a dose-dependent manner. The treatment of NCI-N87 cells with EGF resulted in increases in phosphorylation of Erk1/2, Akt, and P38. Blockade of the Erk, phosphatidylinositol-3 kinase/Akt, or P38 pathways in this cell line prevented EGF induction of NRP-1 and VEGF. These results suggest that regulation of NRP-1 expression in human gastric cancer is intimately associated with the EGF/EGF-R system. Activation of EGF-R might contribute to gastric cancer angiogenesis by a mechanism that involves upregulation of VEGF and NRP-1 expression via multiple signalling pathways.

Figure 1

Expression of NRP-1 and EGF-R by seven human gastric cancer cell lines. Cells were grown to 80% confluence in 10% serum-containing medium. RT–PCR with NRP-1-specific primers was performed to detect NRP-1 RNA expression. Western blots were performed to detect EGF-R protein expression. NRP-1 expression closely mirrored EGF-R expression.

Figure 2

Induction of NRP-1 (A) and VEGF (B) in NCI-N87 and ST-2 cells after treatment with EGF. The cells were incubated in 5% serum-containing medium overnight and then were incubated with or without 50 ng ml⁻¹ EGF for 4 or 24 h in 1% serum-containing medium. Total RNA was extracted for Northern blot analysis. EGF induced NRP-1 and VEGF mRNA expression at 4 and 24 h.

Figure 3

Inhibitory effect of C225 on NRP-1 (A) and VEGF (B) mRNA induction by EGF in NCI-N87 cells. The cells were incubated in 5% serum-containing medium overnight and then were pretreated with or without C225 (10 or 50 μg ml⁻¹) in 1% FBS-containing medium for 24 h. EGF (50 ng ml⁻¹) then was or was not added, and cells were harvested after 4 or 24 h. Relative expression levels of VEGF and NRP-1 mRNA were determined by Northern blot analysis. Pretreatment of the cells with C225 inhibited EGF's effect in a dose-dependent manner.

Figure 4

Effect of EGF on Erk1/2, Akt, and P38 phosphorylation in NCI-N87 cells. The cells were incubated in 5% serum-containing medium overnight and then were incubated with 50 ng ml⁻¹ EGF for the indicated duration in 1% serum-containing medium. Phosphorylated and total protein levels were determined by Western blot analyses. EGF led to induction of phosphorylated Erk1/2, Akt, and P38.

Figure 5

Effect of Erk1/2, Akt, and P38 MAPK inhibition on NRP-1 and VEGF induction by EGF in NCI-N87 cells. The cells were incubated in 5% serum-containing medium overnight and then were pretreated with 50 μM PD98059, 10 μM U0126, 200 nM wortmannin, or 25 μM SB203580 for 1 h in 1% FBS-containing medium. EGF (50 ng ml⁻¹) was then added for 24 h. Control cells were not treated with EGF (lane 1) and cells treated with EGF without addition of signalling inhibitors served as another internal control (lane 2). Total RNA was extracted, and Northern blot analysis was performed. Blockade of the Erk1/2, Akt or P38 pathways all led variable decreases in NRP-1 and VEGF mRNA expression.

Figure 6

Immunohistochemical staining of intestinal-type human gastric cancer for EGF-R and NRP-1. EGF-R and NRP-1 were colocalised in a glandular pattern in a moderately differentiated gastric cancer specimen. Original magnification × 100.