Basic Fibroblast Growth Factor-Mediated Overexpression of Vascular Endothelial Growth Factor in 1F6 Human Melanoma Cells is Regulated by Activation of PI-3K and p38 MAPK

Dennis Fontijn,Monique C A Duyndam,Epie Boven,Maria P A Van Berkel,Linda J W Bosch,Maarten L Janmaat

doi:10.1155/2009/387242

Abstract

Background: 1F6 human melanoma xenografts overexpressing either the 18 kD (18kD) form or all (ALL) forms of human basic fibroblast growth factor (bFGF) demonstrate an abundant number of microvessels and accelerated growth. We now examined whether bFGF mediates vascular endothelial growth factor (VEGF) expression.Methods: Quantitative RT-PCR was used to determine bFGF and VEGF mRNA, VEGF protein secretion was measured by ELISA and VEGF promoter activation was assessed by a dual luciferase activity assay. Western blot was carried out to detect phosphorylation of bFGF-regulated target proteins.Results: In 1F6-18kD and 1F6-ALL clones VEGF mRNA was increased 4- to 5-fold and VEGF protein secretion was highly stimulated due to activation of the VEGF promotor. PI-3K, p38 MAPK and ERK1/2 MAPK pathways were activated, while inhibition of PI-3K or p38 resulted in, respectively, 55% and up to 70% reduction of VEGF mRNA overexpression. A concurrent 60% decrease in VEGF protein secretion was mostly apparent upon inhibition of PI-3K. Inhibition of ERK1/2 hardly affected VEGF mRNA or protein secretion. Two unselected human melanoma cell lines with high metastatic potential contained high bFGF and VEGF, while three non- or sporadically metastatic cell lines displayed low bFGF and VEGF.Conclusion: These data indicate that stimulation of VEGF protein secretion in response to bFGF overexpression may contribute to increased vascularization and enhanced aggressiveness in melanoma.

Highlights

Melanoma cells express a wide variety of growth factors, such as basic fibroblast growth factor and vascular endothelial growth factor (VEGF). bFGF is considered to be a key protein involved in melanoma growth
We demonstrate that activation of the PI-3K and p38 MAPK signal transduction pathways are involved in the increased VEGF production in bFGF-overexpressing melanoma clones
High endogenous bFGF rewas not detectable in conditioned media of 1F6 and 1F6-pcDNA3 cells collected for a period of 24 h, VEGF protein secretion of 4000 and 2000 pg/ml/106 cells was measured in clones 1F6-18 kD (18kD) and 1F6-ALL, respectively

Summary

Introduction

Melanoma cells express a wide variety of growth factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). bFGF is considered to be a key protein involved in melanoma growth. Melanoma cells express a wide variety of growth factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Since melanoma cells express bFGF receptors, bFGF can act as a paracrine and autocrine growth stimulator in each sequential step of melanoma tumor progression [15,18]. VEGF is another important growth factor in melanoma development, since it is upregulated during the course of melanoma progression and will stimulate angiogenesis as well [22,23,30,40]. The VEGF receptors Kinase Domain Region (KDR; VEGFR-2) and fmsrelated tyrosine kinase 1 (Flt-1; VEGFR-1) are not widely distributed on melanoma cells [12,19] Graeven et al [13] have suggested that, in contrast to bFGF, VEGF expression has a beneficial, but non-essential role in melanoma development and progression. We examined whether bFGF mediates vascular endothelial growth factor (VEGF) expression

Methods

Results

Conclusion