Anti-Angiogenic Effect of Asperchalasine A Via Attenuation of VEGF Signaling

Cytochalasans are a group of structurally diverse fungal polyketide-amino acid hybrid metabolites that exhibit diverse biological functions. Asperchalasine A was identified and isolated from an extract of the marine-derived fungus, Aspergillus. Asperchalasine A is a cytochalasan dimer which consists of two cytochalasan molecules connected by an epicoccine. This study investigated the potential antiangiogenic effects of Aspergillus extract and asperchalasine A, which significantly inhibited cell adhesion and tube formation in human umbilical vein endothelial cells (HUVECs). Aspergillus extract and asperchalasine A decreased the vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR)-2 mRNA expression in a concentration-dependent manner. In addition, Aspergillus extract and asperchalasine A inhibited angiogenesis via downregulation of VEGF, p-p38, p-extracellular signal-regulated protein kinase (ERK), p-VEGFR-2, and p-Akt signaling pathways. Moreover, Aspergillus extract and asperchalasine A significantly inhibited the amount of blood vessel formation in fertilized chicken eggs using a chorioallantoic membrane assay. Our results provide experimental evidence of this novel biological activity of the potential antiangiogenic substances, Aspergillus extract, and asperchalasine A. This study also suggests that Aspergillus extract and its active component asperchalasine A are excellent candidates as adjuvant therapeutic substances for cancer prevention and treatment.


Introduction
All the cells of the human body need blood to supply oxygen and nutrients and to remove metabolic waste. Thus, the microvascular network extends throughout the human body to maintain the homeostatic functions and metabolic activities of the cells [1]. Angiogenesis refers to a series of processes wherein new blood vessels are created from the existing blood vessels. It is required for fetal development, menstruation, and wound healing processes under normal conditions. On the other hand, angiogenesis is also known to be necessary in various diseases, such as cancer growth and metastasis, rheumatoid arthritis, and diabetic blindness [2,3]. Angiogenesis is a very tightly regulated phenomenon that is rarely observed under normal conditions. Usually, the biological factors inducing

Cell Adhesion Assay
A 96-well plate was pre-coated with Matrigel (100 µg/mL) at 4 • C for the cell adhesion assay [4]. Excess amounts of Matrigel solution was removed after 30 min of the coating period, and the plate was air-dried. The cells (2 × 10 2 cells/µL/well) were then plated onto the Matrigel-coated plate, and media containing the samples were added. The plate was then incubated further for 30 min at 37 • C in 5% CO 2 . The cells were fixed and stained with 4% paraformaldehyde and hematoxylin after incubation. The cell adhesion was viewed using a microscope and quantified by counting the number of cells.

Cell Migration Assay
The migration of endothelial cells was determined using Falcon cell culture PET (polyethylene terephthalate) inserts with a pore size of 8 µm in a 24-well dish (No 353097; Falcon, Franklin Lakes, NJ, USA) [17]. The lower surface of the filters was coated with Matrigel (5 µg/mL). Cell suspension (2 × 10 3 cells/µL/well) was added to the upper compartment of the chamber, and 600 µL of culture medium with or without test samples were added to the lower compartment, and the chambers were incubated at 37 • C. Cells which failed to migrate were removed with cotton swabs. The migrated cells on the filter were fixed with methanol and stained with Mayer's hematoxylin and 1% Eosin Y solution (MUTO PURE CHEMICALS CO., LTD., Tokyo, Japan). Cells were photographed, counted, and the percentage of migrated cells was calculated.

Tube Formation Assay
Plates (96-well) were coated with 60 µL of Matrigel (10 mg/mL), which was allowed to polymerize at 37 • C. Cells were seeded at a density of 3 × 10 2 cells/µL/well onto the Matrigel-coated plate. Clonetics EGM-2 MV BulletKit, with or without asperchalasine A, was added. The plates were then incubated at 37 • C for 24 h. After incubation, the cells were fixed with 4% paraformaldehyde, followed by staining with Mayer's hematoxylin. Changes in cellular morphology and tubular structure formation were observed using a light microscope. The degree of tube formation was quantified by measuring the lengths of the tubes in the captured images using ImageJ [18].

Western Blotting Analysis
Western blotting was conducted as reported previously [19]. In brief, whole cell lysates were prepared according to the manufacturer's instructions using radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling technology, Danvers, MA, USA) containing phenylmethylsulfonyl fluoride and a protease inhibitor cocktail. Proteins (30 µg/lane) were separated by electrophoresis and blotted onto polyvinylidene fluoride transfer membranes. Bound antibodies were visualized using enhanced chemiluminescence (ECL) reagents (GE Healthcare, Hatfield, UK) and a LAS 4000 imaging system (Fujifilm, Kanagawa, Japan) [20].

Semi-Quantitative Eeverse-Transcriptase PCR
The HUVECs (3 × 10 5 cells/well) were treated with asperchalasine A overnight. After 24 h, the total RNAs were prepared using an RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol [21]. Total RNA was then reverse-transcribed to cDNA using the AccuPower CycleScript RT premix (dT18) (Bioneer, Daejeon, Korea). Specific primers (Table 1) were used to amplify the cDNA encoding of the vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes. To perform PCR, we used the Premix Taq polymerase (Takara Bio Inc., Tokyo, Japan) and PCR conditions as follows: denaturation (95 • C for 1 min), primer-annealing (56 • C for 30 s), and elongation (72 • C for 45 s) for 40 cycles, and one cycle of extension at 72 • C for 10 min. The PCR reactions were carried out using a Biometra T gradient Thermocycler (Göttingen, Germany). Table 1. Primer sequences used for semi-quantitative reverse-transcription polymerase chain reaction (PCR).

Gene Name
Forward Primer Reverse Primer

Chorioallantoic Membrane Assay
Fertilized chicken eggs were incubated in a humidified atmosphere at 37 • C for three days. To separate the developing chorioallantoic membrane (CAM) from the eggshell, albumin was taken out with a syringe. After incubation for 48 h, the eggshell was peeled away to expose CAM, and then the coverslips containing vehicle alone, asperchalasine A or Aspergillus extract, were put on the CAM. After treatment for 48 h, the formed blood vessels were photographed under a microscope.

Statistical Analyses
Statistical significance was determined using analysis of variance (ANOVA), followed by multiple comparison tests with a Bonferroni adjustment. A p-value less than 0.05 was considered statistically significant.

Results and Discussion
In general, natural substances containing toxins can affect cell proliferation, growth, and cell death [22]. Therefore, assessment of the effects of natural substances on cell viability is very important. We investigated the proliferation of HUVECs, which are commonly used as in vitro angiogenesis models, to determine the effects of the Aspergillus extract and asperchalasine A ( Figure 1) on endothelial cell growth. As shown in Figure 1, Aspergillus extract and asperchalasine A exhibited no significant effect on cell growth in HUVECs. As a result, we assert that Aspergillus extract and asperchalasine A do not cause any cytotoxicity in HUVECs.
Angiogenesis is a complex process involving endothelial cell migration, proliferation, adhesion, tube formation, and survival [23]. Adhesion of endothelial cells to the extracellular matrix has been known to be important for endothelial cell growth, migration, differentiation, and survival [24]. The effect of the Aspergillus extract and asperchalasine A on cell adhesion in HUVECs was investigated using a cell adhesion assay. As shown in Figure 2, cell adhesion was significantly reduced after treatment of HUVECs. Compared with the control, 56.26 and 67.39% inhibition of cell adhesion was observed with 50 and 100 µg/mL Aspergillus extract, and 62.81 and 75.14% inhibition was seen with 50 and 100 µM asperchalasine A, respectively.
Directional migration of endothelial cells is an essential element of angiogenesis [25], so we investigated the effect of treatment with Aspergillus extract and aspochalasine A on HUVEC migration. As shown in Figure 3, the treatments with Aspergillus extract and aspochalasine A showed a tendency to decrease cell migration in HUVECs. Compared with the control, 10.88 and 17.06% inhibition of cell migration was observed upon treatments with 50 and 100 µg/mL Aspergillus extract, and 16.28 and 20.82% inhibition was noted with 50 and 100 µM aspochalasine A treatment, respectively. Biomolecules 2019, 9, x FOR PEER REVIEW 5 of 16 Angiogenesis is a complex process involving endothelial cell migration, proliferation, adhesion, tube formation, and survival [23]. Adhesion of endothelial cells to the extracellular matrix has been known to be important for endothelial cell growth, migration, differentiation, and survival [24]. The effect of the Aspergillus extract and asperchalasine A on cell adhesion in HUVECs was investigated using a cell adhesion assay. As shown in Figure 2, cell adhesion was significantly reduced after treatment of HUVECs. Compared with the control, 56.26 and 67.39% inhibition of cell adhesion was observed with 50 and 100 μg/mL Aspergillus extract, and 62.81 and 75.14% inhibition was seen with 50 and 100 μM asperchalasine A, respectively. Directional migration of endothelial cells is an essential element of angiogenesis [25], so we investigated the effect of treatment with Aspergillus extract and aspochalasine A on HUVEC migration. As shown in Figure 3, the treatments with Aspergillus extract and aspochalasine A showed a tendency to decrease cell migration in HUVECs. Compared with the control, 10.88 and 17.06% inhibition of cell migration was observed upon treatments with 50 and 100 μg/mL Aspergillus extract, and 16.28 and 20.82% inhibition was noted with 50 and 100 μM aspochalasine A treatment, respectively.  Angiogenesis entails the creation of new blood vessels from existing blood vessels, and is a complex process of basement membrane dissolution, endothelial cell proliferation, and migration [26,27]. As shown in Figure 4, treatment with Aspergillus extract and aspochalasine A decreased tube formation in HUVECs, as was quantified by the number of branching points. Compared with the control, a 51.71 and 69.34% decrease in tube formation was achieved with 50 and 100 μg/mL Aspergillus extract, and a 66.98 and 72.24% decrease in tube formation was observed with 50 and 100 μM aspochalasine A treatment, respectively. These results suggest that the adhesion and migration of cells are reduced by treatment with Aspergillus extract and aspochalasine A, respectively, thereby inhibiting tube formation. Angiogenesis entails the creation of new blood vessels from existing blood vessels, and is a complex process of basement membrane dissolution, endothelial cell proliferation, and migration [26,27]. As shown in Figure 4, treatment with Aspergillus extract and aspochalasine A decreased tube formation in HUVECs, as was quantified by the number of branching points. Compared with the control, a 51.71 and 69.34% decrease in tube formation was achieved with 50 and 100 µg/mL Aspergillus extract, and a 66.98 and 72.24% decrease in tube formation was observed with 50 and 100 µM aspochalasine A treatment, respectively. These results suggest that the adhesion and migration of cells are reduced by treatment with Aspergillus extract and aspochalasine A, respectively, thereby inhibiting tube formation.  Angiogenesis is regulated by positive or negative effectors, such as members of the fibroblast growth factor (FGF) family, VEGF, angiogenin, and TNF-α [28]. Among these, VEGF has been reported to be the most potent regulator of angiogenesis [29]. In endothelial cells, VEGF activates downstream effectors, such as phosphoinositide-3-kinase, extracellular signal-regulated protein kinase (ERK), and AKT [30]. The phosphorylation of p38 and MAP kinase signaling pathways is known to be involved in the cell proliferation and migration of endothelial cells [31]. As shown in Figure 5, the western blot analysis indicated that the expression levels of VEGF (0.52 ± 0.02 and 0.50 ± 0.01-fold at 50 and 100 μg/mL, respectively), p-ERK (0.63 ± 0.01 and 0.66 ± 0.02-fold at 50 and 100 μg/mL, respectively), and p38 (0.68 ± 0.02 and 0.69 ± 0.01-fold at 50 and 100 μg/mL, respectively) decreased in the HUVECs treated with Aspergillus extract, as compared to the control. Angiogenesis is regulated by positive or negative effectors, such as members of the fibroblast growth factor (FGF) family, VEGF, angiogenin, and TNF-α [28]. Among these, VEGF has been reported to be the most potent regulator of angiogenesis [29]. In endothelial cells, VEGF activates downstream effectors, such as phosphoinositide-3-kinase, extracellular signal-regulated protein kinase (ERK), and AKT [30]. The phosphorylation of p38 and MAP kinase signaling pathways is known to be involved in the cell proliferation and migration of endothelial cells [31]. As shown in Figure 5, the western blot analysis indicated that the expression levels of VEGF (0.52 ± 0.02 and 0.50 ± 0.01-fold at 50 and 100 µg/mL, respectively), p-ERK (0.63 ± 0.01 and 0.66 ± 0.02-fold at 50 and 100 µg/mL, respectively), As shown in Figure 6, western blot analysis indicated that the expression levels of VEGF (0.56 ± 0.02 and 0.52 ± 0.01-fold at 50 and 100 μM, respectively), pERK (0.80 ± 0.01 and 0.81 ± 0.02-fold at 50 and 100 μM, respectively), and p38 (0.78 ± 0.02 and 0.64 ± 0.01-fold at 50 and 100 μM, respectively) were lower in HUVECs treated with aspochalasine A than in the control. Thus, this study suggests that Aspergillus extract and aspochalasine A may inhibit angiogenesis by the downregulation of VEGF, phosphorylated p38, and pERK in HUVECs, and may be useful in the prevention of diseases, such as cancer. As shown in Figure 6, western blot analysis indicated that the expression levels of VEGF (0.56 ± 0.02 and 0.52 ± 0.01-fold at 50 and 100 µM, respectively), pERK (0.80 ± 0.01 and 0.81 ± 0.02-fold at 50 and 100 µM, respectively), and p38 (0.78 ± 0.02 and 0.64 ± 0.01-fold at 50 and 100 µM, respectively) were lower in HUVECs treated with aspochalasine A than in the control. Thus, this study suggests that Aspergillus extract and aspochalasine A may inhibit angiogenesis by the downregulation of VEGF, phosphorylated p38, and pERK in HUVECs, and may be useful in the prevention of diseases, such as cancer. VEGF and VEGFR-2 play an important role in angiogenesis, and are well-characterized signaling pathways [32]. In the process of angiogenesis, VEGF binds to the cognate receptor VEGFR-2 and results in the activation of downstream signaling molecules, including MAPK, Akt, PlC-γ, PI3K, and small GTPases [33,34]. The PI3K/Akt pathway is important for signal transduction in normal cells as well as cancer cells. This pathway plays a diverse role in angiogenesis, including cell proliferation, attachment, migration, invasion, metabolism, and survival [35,36]. Thus, VEGF/VEGFR-2 signaling activates cell survival, migration, and endothelial proliferation [37]. We investigated the effects of the Aspergillus extract and aspochalasine A on the phosphorylation of VEGFR-2 and PI3K/Akt. As shown in Figure 7, treatment of Aspergillus extract and aspochalasine A in HUVECs decreased the VEGF and VEGFR-2 play an important role in angiogenesis, and are well-characterized signaling pathways [32]. In the process of angiogenesis, VEGF binds to the cognate receptor VEGFR-2 and results in the activation of downstream signaling molecules, including MAPK, Akt, PlC-γ, PI3K, and small GTPases [33,34]. The PI3K/Akt pathway is important for signal transduction in normal cells as well as cancer cells. This pathway plays a diverse role in angiogenesis, including cell proliferation, attachment, migration, invasion, metabolism, and survival [35,36]. Thus, VEGF/VEGFR-2 signaling activates cell survival, migration, and endothelial proliferation [37]. We investigated the effects of the Aspergillus extract and aspochalasine A on the phosphorylation of VEGFR-2 and PI3K/Akt. As shown in Figure 7, treatment of Aspergillus extract and aspochalasine A in HUVECs decreased the phosphorylation of VEGFR-2 and Akt, whereas PI3K phosphorylation was not significantly decreased by the Aspergillus extract and aspochalasine A in HUVECs. It is possible that decreased phosphorylation of VEGFR-2, VEGF, phosphorylation of ERK, p38, and Akt may play a sufficient role in angiogenesis.
Biomolecules 2019, 9, x FOR PEER REVIEW 11 of 16 phosphorylation of VEGFR-2 and Akt, whereas PI3K phosphorylation was not significantly decreased by the Aspergillus extract and aspochalasine A in HUVECs. It is possible that decreased phosphorylation of VEGFR-2, VEGF, phosphorylation of ERK, p38, and Akt may play a sufficient role in angiogenesis.  We also investigated the changes in VEGF and VEGFR-2 mRNA expression when treated with Aspergillus extract and aspochalasine A in HUVEC. As shown in Figure 8, treatment with Aspergillus extract and aspochalasine A decreased VEGF and VEGFR-2 mRNA expression in a concentrationdependent manner. We also investigated the changes in VEGF and VEGFR-2 mRNA expression when treated with Aspergillus extract and aspochalasine A in HUVEC. As shown in Figure 8, treatment with Aspergillus extract and aspochalasine A decreased VEGF and VEGFR-2 mRNA expression in a concentration-dependent manner. To verify the anti-angiogenic effects of asperchalasine A and Aspergillus extract in vivo, a CAM assay was carried out (Figure 9). Coverslips containing asperchalasine A (10 and 20 μM) and Aspergillus extract (10 and 20 μg/mL) were placed onto a CAM for 48 h, and inhibitory effects of neovascularization were observed under a microscope. Blood vessel formation in control conditions was inhibited by 12.5% at 0.5% DMSO vehicle, n = 8); however, treated asperchalasine A and Aspergillus extract significantly inhibited the number of blood vessel formation (57.1% at 10 μM asperchalasine A, n = 7; 75.0% at 20 μM asperchalasine A, n = 8; 42.9% at 10 μg/mL Aspergillus extract, n = 7; 50.0% at 20 μg/mL Aspergillus extract, n = 6). They showed no toxicity against pre-existing vessels. To verify the anti-angiogenic effects of asperchalasine A and Aspergillus extract in vivo, a CAM assay was carried out (Figure 9). Coverslips containing asperchalasine A (10 and 20 µM) and Aspergillus extract (10 and 20 µg/mL) were placed onto a CAM for 48 h, and inhibitory effects of neovascularization were observed under a microscope. Blood vessel formation in control conditions was inhibited by 12.5% at 0.5% DMSO vehicle, n = 8); however, treated asperchalasine A and Aspergillus extract significantly inhibited the number of blood vessel formation (57.1% at 10 µM asperchalasine A, n = 7; 75.0% at 20 µM asperchalasine A, n = 8; 42.9% at 10 µg/mL Aspergillus extract, n = 7; 50.0% at 20 µg/mL Aspergillus extract, n = 6). They showed no toxicity against pre-existing vessels.
In order to overcome the disadvantages of anticancer drug therapy, such as side effects and drug resistance, many studies have been carried out using natural substances. Previous studies have shown that 7α, 15-dihydroxydehydroabietic acid isolated from Pinus koraiensis pinecone extract has anti-angiogenic effects [23]. Additionally, Calvatia nipponica extract inhibited angiogenesis through molecular mechanisms involving inhibition of VEGF/VEGFR-2, p38, MEK/ERK, and Akt signaling pathways in HUVECs [29]. However, there is a long way to go for the use of these natural products as therapeutics due to safety, bioavailability, mass synthesis, structural optimization, and clinical trials. Further studies, including animal experiments, are required for the validation of the use of these compounds. Biomolecules 2019, 9, x FOR PEER REVIEW 14 of 16 In order to overcome the disadvantages of anticancer drug therapy, such as side effects and drug resistance, many studies have been carried out using natural substances. Previous studies have shown that 7α, 15-dihydroxydehydroabietic acid isolated from Pinus koraiensis pinecone extract has anti-angiogenic effects [23]. Additionally, Calvatia nipponica extract inhibited angiogenesis through molecular mechanisms involving inhibition of VEGF/VEGFR-2, p38, MEK/ERK, and Akt signaling pathways in HUVECs [29]. However, there is a long way to go for the use of these natural products as therapeutics due to safety, bioavailability, mass synthesis, structural optimization, and clinical trials. Further studies, including animal experiments, are required for the validation of the use of these compounds.

Conclusions
In conclusion, we have confirmed the toxic effects of Aspergillus extract and aspochalasine A in HUVECs and the inhibitory effect on cell adhesion. Both Aspergillus extract and aspochalasine A inhibited tube formation through the downregulation of VEGF, p-ERK, p-p38, p-VEGFR-2, and p-Akt in HUVECs. This study suggests the possibility of the use of the Aspergillus extract and aspochalasine A as therapeutic agents for the inhibition of cancer metastasis.

Conclusions
In conclusion, we have confirmed the toxic effects of Aspergillus extract and aspochalasine A in HUVECs and the inhibitory effect on cell adhesion. Both Aspergillus extract and aspochalasine A inhibited tube formation through the downregulation of VEGF, p-ERK, p-p38, p-VEGFR-2, and p-Akt in HUVECs. This study suggests the possibility of the use of the Aspergillus extract and aspochalasine A as therapeutic agents for the inhibition of cancer metastasis.