Background
Angiogenesis is a multi-step process essential for tumor growth and metastasis, which involves endothelial cell proliferation, migration and capillary formation [
1‐
4]. Among many soluble and matrix-derived angiogenic growth factors and regulators of angiogenesis involved in neovascularization, vascular endothelial growth factor (VEGF) plays a crucial role in the proliferation, migration and survival of vascular endothelial cells [
2,
5‐
7].
The VEGF family consists of six members, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and the placenta growth factor (PLGF) [
4,
7,
8]. Among them, VEGF-A is known as the most important factor for many angiogenic processes. VEGF-A binds to two tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) [
2,
8‐
11]. Signaling through VEGFR-1 is related to embryonic angiogenesis and acts as a regulator of VEGFR-2 [
7,
8,
12,
13]. Although the affinity of VEGFR-2 for VEGF is lower than that of VEGFR-1, VEGFR-2 is more potent than VEGFR-1 in stimulating endothelial cell proliferation and migration [
11,
14]. VEGFR-2 expression is almost restricted to vascular endothelial cells and it has been reported that VEGFR-2 expression was markedly up-regulated during chronic inflammation, wound repair and tumor growth [
5,
15,
16].
Differentiation-inducing factors (DIFs) were identified in
Dictyostelium discoideum as morphogens required for stalk cell differentiation [
17]. In the DIF family, DIF-1 (1-(3, 5-dichloro-2, 6-dihydroxy-4-methoxyphenyl)-1-hexanone) was the first to be identified. The actions of DIFs are not limited to
Dictyostelium and they strongly inhibit the proliferation of human cells [
18,
19]. Previously, we reported that DIFs inhibited the Wnt/β-catenin signaling pathway via glycogen synthase kinase-3β (GSK-3β) activation, leading to cell cycle arrest at G
0/G
1 phase through suppression of cyclin D1 expression in various human tumor cells [
20‐
23]. It is well known that the Wnt/β-catenin signaling pathway plays a number of key roles in embryonic development and maintenance of homeostasis in matured tissues. And also, this signaling pathway has been reported to play important roles in the proliferation and migration of endothelial cells, resulting in the promotion of angiogenesis [
24‐
28].
In this study, we investigated the effect of DIF-1 on angiogenesis in in vitro and in vivo systems. We revealed that DIF-1 decreased the expression of VEGFR-2 in protein and mRNA levels via the suppression of the promoter activity by a Wnt/β-catenin signaling pathway-independent mechanism. Our results suggest that the suppression of VEGFR-2 expression could be one mechanism of the inhibition of angiogenesis induced by DIF-1 and that DIF-1 suppressed not only the Wnt/β-catenin signaling pathway but also neovascularization.
Discussion
In this study, we demonstrated that DIF-1 strongly inhibited angiogenesis
in vitro and
in vivo. As it is known that VEGF-A signal plays a prominent role in angiogenesis, we paid special attention to two types of VEGF receptors, VEGFR-1 and VEGFR-2. Although DIF-1 decreased the levels of protein expression of both receptors on HUVECs, the effects were faster and stronger in VEGFR-2 than VEGFR-1. Activation of VEGFR-2 by VEGF-A depends on the phosphorylation status of several tyrosine residues (such as 951, 1059, 1175, and 1214) in VEGFR-2. Among these tyrosine residues, Tyr
1175 is the binding site of phospholipase-Cγ, a main signal transducer of VEGFR-2 [
32‐
34]. However, as shown in Figure
6B, DIF-1 did not have significant effects on the Tyr
1175 phosphorylation status, suggesting that DIF-1 did not affect VEGFR-2 activation. Since VEGFR-2 is a direct signal transducer for pathological angiogenesis as observed in cancers, the powerful reduction of VEGFR-2 protein levels may be involved in DIF-1 induced anti-angiogenic effects.
We also attempted to clarify the mechanism by which DIF-1 reduced the amount of VEGFR-2 protein. DIF-1 affected the synthesis rather than proteolysis of VEGFR-2. This was consistent with the result that DIF-1 inhibited the mRNA expression and promoter activity of VEGFR-2. However, degrees of suppression of the mRNA expression (29%) and promoter activity (24%) were relatively small compared to VEGFR-2 protein quantity suppression (93%) after 24 h-treatment with DIF-1. The same sort of phenomenon was also observed by Wnt3a (16% promoter activity suppression vs. 34% protein quantity suppression). Although we could not explain this difference at present, the short half-life of VEGFR-2 protein of about 1 h [
35] could be associated with this phenomenon. In other words, as the proteolysis of VEGFR-2 is quick and rapid synthesis is required to restore VEGFR-2, even weak inhibition of promoter activity may significantly affect the quantity of VEGFR-2 protein.
Since we have shown that DIF-1 inhibits the Wnt/β-catenin signaling pathway in various cells, the effects of DIF-1 on the Wnt/β-catenin signaling pathway in HUVECs were examined. We found that DIF-1 also inhibited this signaling pathway via GSK-3β activation in HUVECs. Although the Wnt/β-catenin signaling pathway has been reported to be important to promote angiogenesis
in vitro [
24‐
28], the role of Wnt/β-catenin signaling pathway in endothelial cells and angiogenesis is controversial. Cheng
et al. reported that Wnt1 signaling inhibits HUVEC proliferation [
36]. On the other hand, it has been reported that Wnt1 and 3a mediated induction of VEGFR-2 (Quek-1) expression during avian somite development [
37]. In this study, we showed that Wnt3a slightly but significantly reduced promoter activity and VEGFR-2 protein expression. Therefore, suppression of VEGFR-2 expression induced by DIF-1 may not be due to suppression of the Wnt/β-catenin signaling pathway. Our results might suggest that activation of the Wnt/β-catenin signaling pathway suppressed the promotion of angiogenesis. However, Samarzija
et al. showed that although Wnt3a stimulated HUVEC proliferation and migration independent of VEGFR signaling [
38]. Therefore, further studies are needed to elucidate the relationship between the Wnt/β-catenin signaling pathway and angiogenesis.
Cyclin D1 plays a key role in the initiation and progression of the G
1 phase [
39]. We previously showed that DIF-1 and DIF-3 reduced cyclin D1 quantity and induced cell cycle arrest in G
0/G
1 phase using various mammalian cells [
19,
21,
22]. In this study, we also demonstrated that DIF-1 inhibited HUVECs proliferation and induced restriction of cell cycle in the G
0/G
1 phase by degrading cyclin D1. This result is consistent with that published in our previous reports, and indicates that cyclin D1 also plays an important role in HUVEC proliferation. Furthermore, it has been reported that antisense to cyclin D1 inhibited tumor-associated neovascularization [
40]. As such, suppression of cyclin D1 expression may be one of the anti-angiogenesis mechanisms induced by DIF-1.
Conclusions
In summary, we found that DIF-1 reduced the expression of cyclin D1 and VEGFR-2 in HUVECs. The reduction of cyclin D1 and VEGFR-2 expression may inhibit proliferation, and reduction of VEGFR-2 may cause inhibition of migration and tube formation. These effects may explain the powerful anti-angiogenic properties of DIF-1.
We previously reported that DIF-1 showed anti-tumor activity by inhibiting cyclin D1 expression and the Wnt/β-catenin signaling pathway. In addition to these effects, this study demonstrated that DIF-1 also exhibited anti-angiogenic effects independent of the Wnt/β-catenin signaling pathway. Elucidation of the target molecule of DIF-1 will facilitate the development of potent novel anti-tumor agents which suppresses not only the Wnt/β-catenin signaling pathway but also angiogenesis.
Methods
Cell Culture
Human umbilical vein endothelial cells (HUVECs) were purchased from DS Pharma Biomedical (Osaka, Japan). The cells were grown in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St Louis, Mo, USA) supplemented with 20% fetal bovine serum, 5 ng/ml (0.29 nM) recombinant human basic fibroblast growth factor (PeproTech, Rocky Hill, NJ, USA), 100 units/ml penicillin G, and 100 μg/ml streptomycin using 0.1% gelatin coated dishes. HeLa cells (human cervical carcinoma cell line) and bovine aortic endothelial cells (BAECs) were grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum, 100 units/ml penicillin G and 100 μg/ml streptomycin.
Reagents and antibodies
DIF-1 (1-(3,5-dichloro-2, 6-dihydroxy-4-methoxyphenyl)-1-hexanone) was synthesized as described previously [
41]. MG132 was obtained from the Peptide Institute (Osaka, Japan). Cycloheximide was obtained from Sigma-Aldrich. Polyclonal anti-cyclin D1 antibody, polyclonal anti-PECAM-1 (CD31) antibody and the polyclonal anti-VEGFR-1/Flt-1 antibody were purchased from Santa Cruz Biotechnology (CA, USA). Monoclonal anti-VEGFR-2 antibody and the monoclonal anti-phospho-VEGFR-2 (Tyr
1175) antibody were from Cell Signaling Technology (Danvers, MA, USA). The monoclonal GAPDH antibody was obtained from Abcam (Cambridge, MA, USA). Growth factor reduced Matrigel was obtained from BD Biosciences (San Jose, CA, USA). TOPflash (TCF reporter plasmid) and FOPflash (negative control of TOPflash) were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Human Wnt3a was from R&D Systems (Minneapolis, MN, USA).
Cell proliferation assay
The cells were plated on 24-well plates (1.0×104 cells/well) and treated with or without various amounts of DIF-1 for defined periods. Cells were harvested by trypsin/EDTA treatment and enumerated using Coulter Counter (Beckman Coulter, Brea, CA, USA).
Flow Cytometry
Cells harvested by trypsin/EDTA treatment were suspended in hypotonic fluorochrome solution containing 50 μg/ml of propidium iodide, 0.1% sodium citrate, and 0.1% Triton X-100. Cells (5×103) for each sample were analyzed for fluorescence by a Becton-Dickinson FACScalibur (Franklin Lakes, NJ, USA).
Western blotting
Samples were separated by 10 or 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a semidry transfer system (1 h, 12 V). After blocking with 5% skim milk, the membrane was probed with a first antibody. Incubation was carried out overnight at 4°C. The membrane was then washed three times and incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Bio-Rad, Hercules, CA, USA) for 1 h. Immunoreactive proteins on the membrane were visualized by treatment with a detection reagent (LumiGLO, Cell Signaling Technology). Optical densitometric scan was performed using NIH Image J software.
Tube formation assay was performed as previously described [
42] with slight modification. Briefly, Matrigel was thawed at 4°C and 250 μl of the solution were added to each well in a 24-well plate and formed a gel at 37°C for 30 min. HUVECs were suspended at 3×10
4 cells in 500 μl of 3% FBS with or without 30 μM DIF-1, and then added to each well. After 8 h-incubation, the degree of tube formation was determined by counting the number of areas surrounded by tubes contained in 10 random fields, and expressed as mean ± SE.
Cell migration assay
The effect of DIF-1 treatment on
in vitro migration of HUVECs was determined using a Boyden Chamber [
43]. The PET membrane (8 μm pore size, Greiner Bio-One, Frickenhausen, Germany) was pre-coated with 10 μg of Matrigel. HUVECs were suspended at 5×10
4 cells in 100 μl of serum free DMEM with or without 30 μM DIF-1 and seeded into the upper part of each chamber, whereas the lower compartments were filled with 600 μl of DMEM supplemented with 0.1% bovine serum albumin and 20 ng/ml (0.52 nM) VEGF. After incubation for 10 h at 37°C, non-migrated cells were scraped off with a cotton swab. Migrated cells on the lower surface of the membrane were fixed with 1% glutaraldehyde for 10 min and stained with 4% crystal violet for 30 min. HUVEC migration was quantified by counting the number of cells in ten random fields per membrane. Data are expressed as mean ± SE of cells/fields.
In vivo mouse Matrigel-plug assay
In vivo angiogenesis was assayed as growth of blood vessels from mouse subcutaneous tissue into the exogenous Matrigel plug induced by VEGF or tumor cells [
43]. For the analysis of VEGF-induced angiogenesis, Matrigel was prepared with 100 ng/ml (2.62 nM) VEGF, 20 units/ml heparin in the presence or absence of 30 μM DIF-1 at 4°C. The liquid Matrigel was injected (final volume; 500 μl) into the flanks of C57BL/6 mice (5~7 weeks, n = 7 for each group) using a cold syringe and allowed to polymerize into a solid gel by body temperature. Seven days later, Matrigel plugs were extracted and samples were prepared for immunohistochemical analysis. To analysis for tumor-induced angiogenesis, 1×10
6 HeLa cells were mixed with liquid Matrigel in the presence or absence of 30 μM DIF-1 (final volume; 500 μl). The mixture was injected subcutaneously in the flanks of 6 week-old nude mice (Kyudo, Saga, Japan). Two weeks later, the tumors were removed and samples were prepared for Western blot and immunohistochemical analyses. The handling and sacrificing of all animals were carried out in accordance with nationally prescribed guidelines, and ethical approval for studies was granted by the Animal Care and Use Committee of Kyushu University.
Immunohistochemical analysis
The removed Matrigel plugs and tumors were fixed in 10% buffered formalin followed by embedding in paraffin. Sections were then stained with hematoxylin-eosin staining and immunofluorescence staining. For immunofluorescence staining, primary PECAM-1/CD31 antibody (1:50 dilution) was applied to the sections and the slides were incubated overnight at 4°C. The secondary antibody (Histofine, Nichirei, Tokyo, Japan) was applied to the sections and incubated for 1 h. The slides were subsequently incubated with streptavidin-FITC (Invitrogen, Carlsbad, CA, USA) and the fluorescence strength was analyzed with Biozero fluorescence microscopy (Keyence, Osaka, Japan).
Real-time quantitative reverse transcriptase-polymerase chain reaction
Total RNAs were extracted from HUVECs using TRIzol (Invitrogen) and SV total RNA isolation system (Promega, Madison, WI, USA). First-strand cDNAs were synthesized from 2 μg of total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA). The 100 ng cDNA products were used for quantitative real-time PCR performed using TaqMan Universal PCR Master Mix (Applied Biosystems) and TaqMan MGB primers [VEGFR-2 (Hs00911700_m1) and GAPDH (Hs99999905_m1)] with an ABI Prism 7500 (Applied Biosystems). The following PCR conditions were used: 50°C for 2 minutes, then 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Cycle threshold (CT) values for each gene were obtained for each sample. Differences in CT values between VEGFR-2 gene and endogenous control (GAPDH) were calculated and used for statistical analyses.
Construction of reporter plasmid
The 5'-flanking region of human VEGFR-2 [
44] was amplified and cloned into PCR 2.1 (Invitrogen) for DNA sequencing. After confirming the sequence, DNA fragments (-1003/-48 bp relative to the transcription start site) were excised with
Sac I and
Bgl II and cloned into pGL3-Basic vectors (Promega).
Luciferase reporter gene assay
Cells were transiently transfected with plasmid DNA (TOPflash, FOPflash orVEGFR-2/pGL-3) and pRL-SV40, a Renilla luciferase expression plasmid (Promega) to control transfection efficacy, using Superfect reagent (Qiagen, Hilden, Germany). To measure luciferase activities, Dual-luciferase Reporter Assay (Promega) and a luminometer (Lumat LB 9507; Berthold Technologies, Bad Wildbad, Germany) were used. Firefly luciferase activities were normalized to that of Renilla luciferase.
Statistics
The results are expressed as mean ± SE. Statistical analysis of the differences between values were conducted using the Student's t-test or the one-way ANOVA with Bonferroni post-hoc tests (GraphPad Prism 5.0, GraphPad Software, La Jolla, CA, USA). A P value < 0.05 was considered statistically significant.
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology. We would like to express our gratitude for the technical support from Ms. Matsumoto (Department of Anatomic Pathology) and the Research Support Center, Graduate School of Medical Sciences, Kyushu University.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TY contributed to the major part of experimental work, analyzed and interpreted data, performed the statistics and drafted the manuscript. FT conceived the study, participated in its design and data analysis, and contributed with scientific discussion and manuscript preparation. FS contributed the production of reporter plasmid. YW provided DIF-1. SM, MH and SH interpreted data and contributed with scientific discussion. TS supervised the project and helped draft the manuscript. All authors read and approved the final manuscript.