Caudatin was purchased from Sigma-Aldrich (St. Louis, MO, USA), and dissolved with dimethyl sulfoxide (DMSO). Cell culture reagents and fetal bovine serum (FBS) were procured from Invitrogen (Carlsbad, CA, USA). The second antibody IgG (cat. no. 3452) and all of the primary antibodies used in this study were both purchased from Cell Signaling Technology (Beverly, MA, USA), including p-FAK (cat. no. 3281), VEGF (cat. no. 2463), VEGFR2 (cat. no. 9698), p-VEGFR2 (cat. no. 2478S), AKT (cat. no. 9272), p-AKT (cat. no. 4060), Ki67 (cat. no. 9027) and CD34 (cat. no. 3569). LY294002 was obtained from Calbiochem (San Diego, CA, USA). PF-562271 was obtained from Selleck Chemicals (Houston, TX, USA). All solvents used were of high-performance liquid chromatography (HPLC) grade. Water used in this study was provided by a Milli-Q water purification system from Merck Millipore (Billerica, MA, USA).

HUVECs were purchased from the KeyGen Biotech (Nanjing, China) and cultured in DMEM-F12 medium supplemented with 10% FBS, 100 U/ml penicillin and 50 U/ml streptomycin. Cells were maintained in a humidified incubator of 5% CO₂ at 37°C.

Cell viability was measured with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) assay. Briefly, HUVECs were cultured in a 96-well plate (6×10³ cells/well) for 24 h and then were treated with indicated concentrations of caudatin for 48 h. After that, 20 µl MTT (5 mg/ml) solution/well was added and maintained for 4 h. Then, the supernatant was removed, and 200 µl DMSO/well was added, followed by 20 min shake to dissolve the formazan crystals. Subsequently, the absorbance at 570 nm was measured with a microplate reader (Molecular Device, USA). Cell viability was expressed as percentage of control (as 100%). Cell morphology was observed by inverted microscope (magnification, ×200).

Wound healing assay was used to evaluate cell migration. HUVECs seeded in 6-well culture plate were incubated to full monolayer. Monolayer HUVECs were wounded by scratching with a pipette tip and washed twice with phosphate buffer solution (PBS). Fresh medium containing 1% FBS was then added together with caudatin or other reagents as designed. Cells were photographed under a Nikon Ti-S inverted microscope at the beginning and after incubation for 48 h (magnification, ×100; Nikon Corporation, Tokyo, Japan). Cell migration distance was measured by image plus software, and the migrated rate was expressed as percentage of control. Three independent experiments were performed.

Transwell assay was used to determine the effect of caudatin on HUVECs invasion. Transwell chambers (BD Biosciences, Bedford, MA, USA) containing a 6.5-mm-diameter polycarbonate filter with a pore size of 8 µm were used. Briefly, each filter was coated with 60 µl Matrigel (1 mg/ml; BD Biosciences) diluted with DMEM-F12 and incubated at 37°C for 45 min. HUVECs (4×10⁴ cells) suspended in 100 µl DMEM-F12 (FBS-free) were seeded in the filter, and treated with or without 100 µM caudatin for 24 h. Complete DMEM-F12 (600 µl, 10% FBS) was added into the chamber below. VEGF (200 ng/ml) and cisplatin (5 µM) were employed as the positive and negative control, respectively. After incubation, the cells on the filter were wiped away, and the invaded cells below the filter were washed with PBS, fixed with 90% ethanol and stained with 0.1% crystal violet. The invaded cells were calculated by manual counting with a Nikon Ti-S inverted microscope (magnification, ×200). The invaded rate was expressed as percentage of the control. Three independent experiments were performed.

Capillary-like tube formation assay was used to further detect the antiangiogenesis effect of caudatin. Matrigel was pipetted into 48-well plate (60 µl/well) and polymerized at 37°C for 45 min. HUVECs (100 µl, 1×10⁴ cells/well) mixed with 100 µM caudatin were seeded into Matrigel pre-coated wells and incubated at 37°C with 5% CO₂ for 24 h. Cells were photographed and the tube number was calculated by manual counting using a Nikon Ti-S inverted microscope (magnification, ×100). Tube formation was scored as follows: A three branch point event was defined as one tubular structure. Eight random fields per well were quantified by manual counting. Three independent experiments were performed.

Western blot analysis was performed as described before (8). HUVECs were cultured in dishes (10 cm) and treated with indicated concentrations of caudatin or other reagents for 48 h. Then, cells were washed twice by PBS and lysed in RIPA lysis buffer (1xPBS, 1% NP40, 0.1% SDS, 5 mM EDTA, 0.5% sodium deoxycholate and 1 mM sodium orthovanadate) for 20 min in the ice environment. Lysates were centrifuged and supernatants were kept as total cell protein. The concentration of total cell protein was quantified by bicinchoninic acid assay (BCA; Beyontime, Beijing, China) according to the manufacturer's instructions. Total protein was added with sample loading buffer, boiled for 10 min to be denatured, and stored at −80°C environment for subsequent SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Briefly, total protein (40 µg/lane) was separated by SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore). After that, the membranes were blocked with 5% bovine serum albumin (BSA) at room temperature for 2 h. Then, the membranes were incubated with specific primary antibodies overnight at 4°C and secondary antibodies for 2 h at room temperature sequentially. At last, target protein bands were visualized with ECL under an Imaging System (ChemiDoc MP, Bio-Rad). β-actin plays as a positive control.

U251 human glioma cells xenograft model was constructed as described previously (13). Briefly, 1×10⁷ U251 cells in 100 µl serum-free medium were subcutaneously injected into the right oxter of male nude mice. When tumors grew to 60 mm³ on average after 8 days, mice were randomly divided into control group and the caudatin-treated groups (25 and 100 mg/kg). Drugs were given through caudal vein injection every other day for 16 days. At the termination of the experiment, tumors were collected and used for western blotting and immunohistochemical (IHC) assay (magnification, ×200). All animal experiments were approved by the Animal Experimentation Ethics Committee.

Firstly, MTT assay was employed to examine the anti-proliferation activity of caudatin against HUVECs. As shown in Fig. 1, treatment of HUVECs with caudatin (below 12 µM) showed no cytotoxicity. Treatment of cells with 6 µM caudatin slightly promoted U251 cells growth. However, caudatin (25–200 µM) significantly inhibited U251 cells growth in a dose-dependent manner. For instance, cells exposed to 50, 100 and 200 µM of caudatin for 48 h markedly inhibited the HUVECs viability to 71.5, 53.6 and 38.1%, respectively. This result suggests that caudatin may act as a potential cytostatic agent in hunting HUVECs growth.

Wound-healing assay, Transwell assay and capillary-like tube formation were used to evaluate the inhibition effect of caudatin on cell migration, invasion and tube formation, respectively. VEGF and cisplatin were employed as positive and negative control respectively in these assays to evaluate caudatin's potential. Cell migration and invasion is critical for endothelial cells to form blood vessels during angiogenesis. As shown in Fig. 2, VEGF obviously promoted cell migration (Fig. 2A and B), invasion (Fig. 2C and D) and capillary-like tube formation (Fig. 2E and F). However, caudatin (100 µM) treatment effectively blocked HUVECs migration, invasion and capillary-like tube formation (Fig. 2). The statistical data of migrated rate, invaded rate and the tube formation further confirmed caudatin's inhibitory effects, which is similar with that of cisplatin. These results showed that caudatin has the potential to block the capillary-like tube formation in vitro by inhibiting the HUVECs migration and invasion.

FAK is an upstream kinase that has a key role in the regulation of cell migration and invasion. Therefore, it was interested to investigate whether FAK was involved in the caudatin-induced inhibition of HUVEC migration and invasion. A specific antibody against the phosphorylated (activated) form of FAK was used (Fig. 3). The results showed that caudatin treatment caused significant dephosphorylation of FAK in a dose- and time-dependent manner (Fig. 3A and B). To further confirm the role of FAK, wound healing assay was operated using the specific FAK inhibitor (PF562271). As shown in Fig. 3D and E, pretreatment of HUVECs with PF562271 (10 nM) for 1 h markedly enhanced caudatin-induced inhibition against cell migration, which confirmed the effect of inactivation FAK on cell migration inhibition. Results in protein levels further confirmed this conclusion. Treatment of HUVECs with caudatin caused significant inactivation of FAK, and this effect was enhanced at the presence of PF562271 (Fig. 3C). Taken together, the results above suggest that caudatin can inhibit cell migration of HUVECs through FAK dephosphorylation.

The VEGF-VEGFR2- AKT signal axis has key roles in regulation of cell proliferation, cell growth, survival and angiogenesis. Therefore, it was of interest to investigate whether the VEGF-VEGFR2-AKT signal axis was involved in the caudatin-induced inhibition against HUVECs proliferation and angiogenesis. Firstly, western blotting assay was employed and different concentrations of caudatin on the VEGF-VEGFR2-AKT signal axis expression was assayed, and the results showed that caudatin treatment significantly suppressed the expression of VEGF and p-VEGFR2 in a dose-dependent manner (Fig. 4A), but caused no significant changes in the expression of total-VEGFR2 and total-AKT. To further confirm the role of the VEGF-VEGFR2- AKT signal axis, cell viability was detected by MTT assay using a specific AKT inhibitor (LY294002). As shown in Fig. 4B, pre-treatment of HUVECs with LY294002 (an AKT-upstream inhibitor) for 1 h markedly enhanced caudatin-induced cell growth inhibition against HUVECs, indicating that caudatin inhibited HUVEC proliferation with AKT-dependent manner. These results indicated that the disturbance of VEGF-VEGFR2-AKT signal axis contributed to caudatin-induced inhibition against proliferation and angiogenesis.

To investigate the antiangiogenesis effect of caudatin in vivo, immune-deficient nude mice bearing U251 xenograft tumors were employed (Fig. 5). The results demonstrated that caudatin treatment in vivo significantly inhibited U251 cell xenograft tumor growth, as convinced by the decrease of tumour volume (Fig. 5A) and tumor weight (Fig. 5B) and ki-67 expression (Fig. 5D). Moreover, CD-34, one of the endothelial cell markers, was detected in xenograft tumors by IHC method, and the results clearly demonstrated that caudatin treatment obviously abolished the tumor angiogenesis in vivo. VEGF expression as one of the most important pro-angiogenic factors also showed dramatically decrease (Fig. 5D). Furthermore, caudatin treatment caused the dephosphorylation of AKT and FAK detected by western blotting, which further affirmed the inhibitory effect of caudatin on glioma angiogenesis. Taken together, these results above suggest that caudatin can inhibit glioma cells growth by suppression of the in vivo angiogenesis involving the FAK and AKT dephosphorylation and inhibition of VEGF expression.