Azaspirene, a fungal product, inhibits angiogenesis by blocking Raf-1 activation

Abstract

Angiogenesis is an inevitable event in tumor progression and metastasis, and thus has been a compelling target for cancer therapy in recent years. Effective inhibition of tumor progression and metastasis could become a promising way to treat tumor-induced angiogenesis. We discovered that a fungus, Neosartorya sp., isolated from a soil sample, produced a new angiogenesis inhibitor, which we designated azaspirene. Azaspirene was previously shown to inhibit human umbilical vein endothelial cell (HUVEC) migration induced by vascular endothelial growth factor (VEGF) at an effective dose, 100% of 27 micromol/L without significant cell toxicity. In the present study, we investigated the antiangiogenic activity of azaspirene in vivo. Azaspirene treatment reduced the number of tumor-induced blood vessels. Administration of azaspirene at 30 microg/egg resulted in inhibition of angiogenesis (23.6-45.3% maximum inhibition relative to the controls) in a chicken chorioallantoic membrane assay. Next, we elucidated the molecular mechanism of antiangiogenesis of azaspirene. We investigated the effects of azaspirene on VEGF-induced activation of the mitogen-activated protein kinase signaling pathway in HUVEC. In vitro experiments indicated that azaspirene suppressed Raf-1 activation induced by VEGF without affecting the activation of kinase insert domain-containing receptor/fetal liver kinase 1 (VEGF receptor 2). Additionally, azaspirene preferentially inhibited the growth of HUVEC but not that of the non-vascular endothelial cells NIH3T3, HeLa, MSS31, and MCF-7. Taken together, these results demonstrate that azaspirene is a novel inhibitor of angiogenesis and Raf-1 activation that contains a unique carbon skeleton in its molecular structure.

Figure 1

Effect of azaspirene on tumor‐induced angiogenesis in vivo. (a) The chemical structure of azaspirene. (b) Photographs of tumor‐induced vessel formation after treatment with vehicle, azaspirene, and paclitaxel. (c) Quantification of newly formed blood vessels. The statistical significance of differences between the control and experimental groups was determined using one‐way ANOVA, Tukey method analysis, repeated measures. *P < 0.05; **P < 0.01 was taken as the level of statistical significance.

Figure 2

Suppression of blood vessel formation within chorioallantoic membrane (CAM) by azasprirene. (a) The 4.5‐day‐old CAM were treated with increasing concentrations of azaspirene for 48 h, and then patterns of angiogenesis were photographed. (b) The total area of blood vessels was analyzed with angiogenesis‐measuring software and is shown under each panel. Solid column, vehicle (10% dimethyl sulfoxide); hatched column, 30 µg/egg azaspirene. The statistical significance of differences between control and experimental groups was determined using two‐group two‐tailed Student's t‐test. *P < 0.05 was taken as the level of statistical significance.

Figure 3

Effect of azaspirene on the mitogen‐activated protein (MAP) kinase signaling pathways in human umbilical vein endothelial cells (HUVEC). Azaspirene inhibited the phosphorylation of MEK1 and 2 and ERK1 and 2 induced by (a) vascular endothelial growth factor (VEGF), (b) epidermal growth factor (EGF), (c) basic fibroblast growth factor (bFGF), and (d) phorbol 12, 13‐dibutyrate (PDBu). HUVEC were pretreated for 60 min with various concentrations (8, 27, or 81 µmol/L) of azaspirene and PD98059 (25 µmol/L) before exposure to VEGF (12.5 ng/mL) for 5 min, EGF (10 ng/mL) for 5 min, bFGF (25 ng/mL) for 15 min, or PDBu (10 ng/mL) for 5 min. After stimulation the cells were harvested and western blotting was carried out. IB, antibodies used for western blotting. The results shown are representative of three experiments.

Figure 4

Effect of azaspirene on the autophosphorylation of kinase insert domain‐containing receptor/fetal liver kinase 1 (KDR/Flk‐1), phosphorylation of Raf‐1, and disruption of Raf‐1 complexes. (a) Azaspirene did not inhibit the autophosphorylation of KDR/Flk‐1 induced by vascular endothelial growth factor (VEGF). Human umbilical vein endothelial cells (HUVEC) were pretreated for 60 min with various concentrations (8, 27, or 81 µmol/L) of azaspirene and SU5614 (10 µmol/L) before exposure to VEGF (12.5 ng/mL) for 5 min. (b) Azaspirene inhibited the phosphorylation of Raf‐1 induced by VEGF (12.5 ng/mL, 5 min), but did not disrupt the Raf‐1, Hsp90, MEK1, or MEK2 complexes. HUVEC were pretreated for 60 min with various concentrations (8, 27, or 81 µmol/L) of azaspirene and geldanamycin (10 µmol/L) before exposure to VEGF (12.5 ng/mL) for 5 min. IB, western blotting analysis; IP, immunoprecipitation experiments. The results shown are representative of three experiments.

Figure 5

Effect of azaspirene on the mitogen‐activated protein (MAP) kinase signaling pathways in NIH3T3, HeLa, MSS31, and MCF‐7 cells. Azaspirene did not inhibit the phosphorylation of ERK1 and 2 induced by platelet‐derived growth factor (PDGF), epidermal growth factor (EGF), or phorbol 12, 13‐dibutyrate (PDBu) in other cell lines. (a) NIH3T3 cells were pretreated for 60 min with various concentrations (27, 81, or 270 µmol/L) of azaspirene and PD98059 (25 µmol/L) before exposure to basic fibroblast growth factor (bFGF) (10 ng/mL) for 5 min, PDGF (30 ng/mL) for 5 min, or PDBu (10 ng/mL) for 5 min. (b) Azaspirene did not inhibit the phosphorylation of ERK1 and 2 induced by EGF (10 ng/mL, 5 min) or PDBu (10 ng/mL, 5 min) in HeLa cells. (c) MSS31 and MCF‐7 cell lines were pretreated for 60 min with various concentrations (27, 81, or 270 µmol/L) of azaspirene and PD98059 (25 µmol/L) before exposure to PDBu (10 ng/mL) for 5 min. After stimulation, the cells were harvested and western blotting was carried out. IB, western blotting analysis. The results shown are representative of three experiments.

Figure 6

Effects of azaspirene on the growth of human umbilical vein endothelial cells (HUVEC) and on mitogen‐activated protein kinase (MAPK) activation in the HEK293T cell system. (a) Effects of azaspirene on the growth of NIH3T3, HeLa, MSS31, and MCF‐7 cells and HUVEC in a proliferation assay. The azaspirene‐induced growth inhibitions for the different cell lines were as follows: , NIH3T3 (IC₅₀ = 216 µmol/L); , HeLa (IC₅₀ = 189 µmol/L); , MSS31 (IC₅₀ = 173 µmol/L); , MCF‐7 (IC₅₀ = 75.6 µmol/L); and , HUVEC (IC₅₀ = 62.1 µmol/L). Each value is expressed relative to the 1% dimethyl sulfoxide (DMSO) control group; bars, SD. The statistical significance of differences between the growth inhibition (%) of HUVEC with azaspirene at 81 µmol/L was determined using one‐way ANOVA, Tukey method analysis, repeated measures. *P < 0.05; **P < 0.01 was taken as the level of statistical significance. (b) Effects of azaspirene on vascular endothelial growth factor (VEGF)‐induced ERK1 and 2 phosphorylation in HEK293T cells expressing kinase insert domain‐containing receptor/fetal liver kinase 1 (KDR/Flk‐1). HEK293T cells transfected with KDR/Flk‐1 were incubated for 1 h in the absence (DMSO) or presence of various concentrations of azaspirene (27, 81, or 270 µmol/L). These cells were then treated with 50 ng/mL VEGF for 5 min. After stimulation, the cells were harvested, and western blotting was carried out. IB, western blotting analysis. The results shown are representative of three experiments.