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

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

[Solidago sp.]

原著和名: [記載なし]科名: キク科 = Compositae採集地: 徳島県 那賀郡 鷲敷町 那賀川畔 (阿波 那賀郡 鷲敷町 那賀川畔)採集日: 1972/11/30採集者: 萩庭丈壽整理番号: JH023172国立科学博物館整理番号: TNS-VS-97317

The 18 kDa isoform of basic fibroblast growth factor is sufficient to stimulate human melanoma growth and angiogenesis

Basic fibroblast growth factor is the best-characterized autocrine growth factor in melanoma development and progression. We hypothesized that basic fibroblast growth factor might induce a more aggressive phenotype dependent on the amount of protein expressed in melanoma. Two human melanoma cell lines, M14 and 1F6, known to have low endogenous basic fibroblast growth factor expression and slow growth as subcutaneous xenografts, were stably transfected with vectors encoding either the 18 kDa or all (ALL) isoform proteins of human basic fibroblast growth factor. Different clones overexpressing the 18 kDa or ALL basic fibroblast growth factor proteins were easily obtained. Increased levels of basic fibroblast growth factor were secreted in conditioned medium and stored on the extracellular membrane. Biological activity of the overexpressed basic fibroblast growth factor was confirmed in a human umbilical vein endothelial cell proliferation assay. In 1F6 cells, overexpression of either 18 kDa or ALL basic fibroblast growth factor proteins resulted in up to two-fold shorter in-vitro doubling times (P<0.05). In addition, in vivo, both 18 kDa and ALL basic fibroblast growth factor-overexpressing 1F6 subcutaneous xenografts displayed significantly higher growth rates (P<0.05). In contrast, no major differences in in-vitro and in-vivo doubling times were observed when 18 kDa or ALL isoforms of basic fibroblast growth factor were overexpressed in M14 cells. Interestingly, basic fibroblast growth factor overexpression only affected the microvasculature in 1F6 xenografts. Although blood vessels in 1F6 parent tumors were large, 1F6 tumors overexpressing basic fibroblast growth factor contained numerous small, compressed vessels. Taken together, overexpression of the 18 kDa basic fibroblast growth factor protein only can promote autocrine melanoma cell growth and paracrine-driven angiogenesis

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

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

Prolonged hypoxia inhibits sprouting of endothelial cells into 3D fibrin matrices.

<p>hMVECs were precultured at 20% (black and dark grey bars) or 1% oxygen (light grey and white bars) for 14 days before seeded on top of 3D fibrin matrices. Subsequently, the hMVECs were stimulated with the combination of VEGF-A/TNFα (4 donors in 7 experiments) or bFGF/TNFα (5 donors in 10 experiments) either at 20% oxygen (black and white bars) or at 1% oxygen (dark grey and light grey bars) (each in quadruple). <b>(A)</b> Representative photos are shown of hMVECs 7 days after seeding and stimulation with VEGF-A/TNFα. Scale bars represent 1 mm. Photos are focused on the sprouts. <b>(B)</b> Tube length was quantified by using Optimas software and expressed as percentage of 20% O₂ with SEM. For statistical analysis repeated measures ANOVA with Bonferroni post-hoc test was used (** p<0.01 *** p<0.001).</p

Effect of hypoxia on human microvascular endothelial cell proliferation.

<p>hMVECs were cultured in normoxia (20% O₂, grey circles) or hypoxia (1% O₂, black squares) for 17 days. <b>(A)</b> Photos of confluent monolayers of hMVECs on day 17 are shown. The white scale bar represents 1 mm and the black scale bar represents 0.1 mm. <b>(B)</b> Photos were taken every day and analyzed with ImageJ software, the arrows indicate days that cells were passaged 1:3. Representative experiment out of 3 independent hMVEC donors, showing number of cells/cm² as average of triplicate wells with SD.</p

Addition of recombinant uPA increases endothelial sprouting.

<p>hMVECs were seeded on top of 3D fibrin matrices and stimulated with the combination of VEGF-A/TNFα and the indicated concentration of human recombinant uPA either in normoxia (grey circles) or hypoxia (black squares) (each in quadruple). <b>(A)</b> Representative photos are shown of hMVECs 7 days after seeding and stimulation with VEGF-A/TNFα. Scale bars represent 1 mm. Photos are focused on the sprouts. <b>(B)</b> Tube length of a representative experiment was quantified by using Optimas software and expressed as mm/cm² with SEM. After addition of 20 ng/ml u-PA lysis of fibrin was observed, which interfered with the stability of the tubular structures.</p

Effect of hypoxia on relative mRNA expression (hypoxia/normoxia) of DLL4-NOTCH signaling genes in different human microvascular endothelial cells.

<p>Effect of hypoxia on relative mRNA expression (hypoxia/normoxia) of DLL4-NOTCH signaling genes in different human microvascular endothelial cells.</p

Effect of prolonged hypoxia on the expression of genes involved in pericellular proteolysis.

<p><b>(A)</b> hMVECs were cultured in normoxia (Un (20%), white bar) or prolonged hypoxia (Un (1%), black bar) for 14 days and transfected with si-HIF-2α (si-H2 (1%), light grey bar) or scrambled (scr (1%), dark grey bar) and stimulated for 24 hours with VEGF/TNFα. mRNA was isolated for analysis by qRT-PCR and the relative mRNA levels were expressed as mean fold change with SEM (4 independent donors). Data was normalized to 1% O₂. <b>(B)</b> uPA and PAI-1 antigens were measured in hMVECs cultured in 20% O₂ (white bar) or 1% O₂ (black bar). Levels were expressed as ng/72h/10⁵ cells with SEM (4 independent donors). <b>(C)</b> uPA antigen was measured in hMVECs cultured in 1% O₂ and either untransfected (Un, black bar), or transfected with scrambled si-RNA (scr, dark grey bar), si-HIF-1α (si-H1, hatched bar), or si-HIF-2α (si-H2, light grey bar). Levels were expressed as ng/72h/10⁵ cells with SEM (3 independent donors). For statistical analysis TWO-way ANOVA with Bonferroni post-hoc test (A), paired student t test (B), or repeated measures ANOVA with Bonferroni post-hoc test was used (C) (** p<0.005, *** p<0.001).</p

Effect of hypoxia on the regulation of HIF mRNA and protein.

<p>mRNA was isolated from hMVECs precultured for 14 days at 20% O₂ that were incubated for 24 hours in 20% O₂ (black bar) or in 1% O₂ (white bar) or precultured for 14 days at 1% O₂ and incubated for 24 hours in 1% O₂ (grey bar). Cells were either <b>(A)</b> not stimulated (4 independent donors) or <b>(B)</b> stimulated with VEGF-A/TNFα for 24 hours (4 independent donors) and expressed as mean fold change with SEM. Data was normalized to 20% O₂. <b>(C)</b> Western blot analysis of whole cells lysates collected from cells precultured at 20% O₂ for 14 days and incubated in hypoxia with or without VEGF-A/TNFα for indicated hours (2 independent donors) or <b>(D)</b> western blot analysis of whole cell lysates collected from hMVECs precultured at 20% O₂ for 14 days and incubated in hypoxia with VEGF-A/TNFα for indicated hours or hMVECs precultured in hypoxia for 14 days and stimulated with VEGF-A/TNFα for 24 hours (3 independent donors). For statistical analysis TWO-way ANOVA with Bonferroni post-hoc test was used (A and B) (* p<0.05 **p<0.01 *** p<0.001).</p