Primary cultures of HUVECs were purchased from Lonza Walkersville Inc. (Walkersville, MD, USA) and used between passages 4 and 6 for all experiments. Cells were cultured in EGM-2^® BulletKit media, according to the manufacturer’s instructions (Lonza).

The following pharmacological agents and antibodies were purchased from commercial sources: VEGF-A and LY294002 [an inhibitor of the phosphatidylinositol3-kinase (PI3-K)/Akt pathway; Merck Millipore, Billerica, MA, USA]; rapamycin [an inhibitor of the mammalian target of rapamycin (mTOR)/p70^S6K pathway; Sigma-Aldrich, St. Louis, MO, USA]; anti-phospho-extracellular signal-regulated kinase (ERK) (T202/Y204), anti-phospho-Akt (S473), anti-phospho-p70^S6K (T421/S424) and anti-phospho-pRb (S780) (Cell Signaling Technology Inc., Beverly, MA, USA); anti-p27^Kip1 (BD Biosciences, Bedford, MA, USA); anti-ERK, anti-Akt, anti-p70^S6K, anti-Cdk4, anti-Cdk2, anti-cyclin D, anti-cyclin E, anti-p27^WAF1/Cip1, anti-actin antibodies, and mouse and rabbit IgG-horseradish peroxidase conjugates (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

SG was purchased from Dae Kwang Herb Medicine Co. (Chuncheon, Gangwon-do, Korea) and deposited at the herbarium of the Radiant Research Institute (Radiant Inc., Chuncheon, Gangwon-do, Korea). One hundred grams of SG was extracted with 1 liter of ethanol and stirred for 90 min. The extract of SG was obtained as previously reported (11,12).

Subconfluent cells, plated on 6-well plates (1×10⁵ cells/well; BD Biosciences), were serum-starved for 14 h to synchronize cells in the G₁/G₀ phase of the cell cycle, pretreated with SG (0.01–1 μg/ml) for 30 min in the presence or absence of LY294002 (10 μM) or rapamycin (50 nM) as indicated and further incubated with VEGF-A (10 ng/ml) for 24 h. Following culture for 24 h, cell viability was determined by a Muse™ cell analyzer using a cell count and viability assay kit (Merck Millipore), and the cell proliferation was quantified as previously described (14). The results from triplicate determinations (mean ± standard deviation) are presented as the fold-increase of the untreated controls or the percentage of viable cells of the total cell count.

Serum-starved cells were pretreated with SG (1 μg/ml) for 30 min, followed by VEGF-A (10 ng/ml) for 24 h. Cells were harvested with trypsin-EDTA, rinsed with phosphate-buffered saline (PBS, pH 7.4) and then fixed with ice-cold 70% ethanol for 3 h. After washing with PBS, cells were stained with Muse™ cell cycle reagent. The profile of cells in the G₁/G₀, S and G₂/M phases of the cell cycle was analyzed with a Muse™ cell analyzer (Merck Millipore) (15).

Subconfluent cells in 100-mm dishes (1×10⁶ cells/dish; BD Biosciences) were serum-starved for 14 h, pretreated with SG for 30 min followed by VEGF-A (10 ng/ml) for 15 min or 24 h, as indicated. Cells were rinsed twice with ice-cold PBS and lysed by incubation in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 100 μg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml pepstatin A, 0.5 μg/ml leupeptin, 80 mM β-glycerophosphate, 25 mM sodium fluoride and 1 mM sodium orthovanadate for 30 min at 4°C. Cell lysates were clarified at 12,500 × g for 20 min at 4°C, and the supernatants were subjected to western blot analysis as described previously (16,17). All western blot analyses were performed at least in triplicate experiments and representative gels are shown. Bands of interest were integrated and quantified by the use of National Institutes of Health (NIH) ImageJ Version 1.34s software.

Subconfluent cells were detached with trypsin-EDTA and allowed to recover in EGM-2^® BulletKit media for 1 h at 37°C with gentle rocking. After recovery, the cells were collected by low-speed centrifugation and resuspended in serum-free EBM-2 media (Lonza). The cell suspension was pretreated with or without SG (1 μg/ml) for 30 min followed by VEGF-A (10 ng/ml) treatment. The cells were plated on 96-well plates (1.5×10⁴ cells/well) and further incubated for 2 h at 37°C. Following the incubation, the unattached cells were removed by washing the wells 3 times with ice-cold PBS. Attached cells were fixed with methanol and then stained with 0.04% Giemsa staining solution (Sigma-Aldrich). The cells were photographed and counted. The results (mean ± standard deviation) are presented as the numbers of adherent cells (18).

Cell migration was quantified in the in vitro wound-healing assay as described previously (14,19). After cells were plated on 48-well plates and grown to confluence, a single wound was created in the center of the cell monolayer by the gentle removal of the attached cells with a sterile plastic pipette tip. Following serum starvation with EBM-2 for 2 h, cells were pretreated with SG (0.01–1 μg/ml) for 30 min in the presence or absence of LY294002 (10 μM) or rapamycin (50 nM) as indicated, followed by VEGF-A (10 ng/ml) stimulation for 15 h. Cells were fixed with methanol and then stained with 0.04% Giemsa solution. The migration of the cells into the wound was observed with still images captured at the indicated time-point.

Matrigel^® basement membrane matrix (10.4 mg/ml, BD Biosciences) was thawed overnight at 4°C, and each well of pre-chilled 24-well plates was coated with 200 μl Matrigel and then incubated at 37°C for 30 min. Following serum starvation with EBM-2 for 2 h, cells (4×10⁴ cells/ml) were added to the Matrigel-coated plates and pretreated with SG (1 μg/ml) for 30 min, followed by VEGF-A (10 ng/ml) for 6 h. Tube formation was observed with an Olympus CKX41 inverted microscope (CAchN 10/0.25php objective) and ToupTek Toupview software (version x86, 3.5.563, Hangzhou ToupTek Photonics Co., Zhejiang, China).

Statistical analysis was performed using the Student’s t-test and was based on at least 3 different experiments. The results were considered to be statistically significant at P<0.05.

We first examined the effect of SG on cell proliferation of HUVECs. SG treatment suppressed VEGF-A-stimulated endothelial cell proliferation in a dose-dependent manner (Fig. 1A). In addition, treatment of non-stimulated HUVECs with SG at the highest concentration used in this study did not alter the viability or the proliferative response of HUVECs (Fig. 1B), indicating that SG inhibition of cell proliferation was not mediated by induction of apoptosis or cytotoxicity. This finding is similar to the patterns of SG in other cell types as previously reported (11,12). We next investigated the effect of SG on the cell cycle by DNA content analysis (Fig. 2A). VEGF-A stimulation for 24 h increased the percentage of cells in the S phase, compared with the untreated controls (8.8 vs. 14.0%) and resulted in the concomitant decrease of cells in the G₁ phase (73.5 vs. 68.3%). SG treatment prevented the increase in the percentage of cells in the S phase (14.0 vs. 8.6%) and the decrease in the percentage of cells in the G₁ phase (68.3 vs. 72.5%) associated with VEGF-A stimulation, similar to those of the untreated controls. These findings indicate that SG inhibits the transition from G₁ to S phase, leading to G₁ arrest, which is well correlated with inhibition of cell proliferation (Fig. 1A). SG has previously been reported to inhibit proliferation by downregulation of cyclin-dependent kinase 4 (Cdk4), cyclin D and cyclin E and upregulation of p27^Kip1 in SKOV-3 ovarian cancer cells (11). In addition, SG-mediated inhibition of proliferation in A549 and H1299 non-small cell lung cancer cells was mediated by suppression of Cdk4 and Cdk2 (12). Based on these observations, we analyzed the changes in cell cycle-related proteins such as Cdks, cyclins and Cdk inhibitors in the SG-treated HUVECs. As shown in Fig. 2B, SG treatment markedly suppressed the expression of cyclin D and induced the levels of Cdk inhibitors such as p21^WAF1/Cip1 and p27^Kip1, resulting in inhibition of pRb phosphorylation in response to VEGF-A stimulation. Although the molecular mechanism of SG in regulating cell cycle progression appears slightly different in various cell types, these findings clearly demonstrate the antiproliferative activity of SG against various types of cells.

Interaction of endothelial cells with extracellular matrix molecules plays important roles in cell migration, adhesion and capillary-like structure formation which are associated with cancer growth and progression (1,2). SG treatment inhibited VEGF-A-stimulated cell migration in a dose-dependent manner (Fig. 3A). In contrast to cell migration, SG did not alter the VEGF-A-induced endothelial cell adhesion (Fig. 3B), indicating that stable adhesiveness may contribute, at least in part, to reduced cell migration as previously reported (20,21). However, in SKOV-3 ovarian cancer cells SG was found to markedly simultaneously block mitogen-induced cell adhesion as well as migration (11). Collectively, these findings indicate that SG may differentially regulate cell adhesion and migration, depending on the type of cells or growth factor. We next investigated the ability of SG to regulate the formation of capillary-like structures in HUVECs. As shown in Fig. 4, SG completely inhibited VEGF-A-stimulated tube formation to the levels observed in the untreated controls, similar to inhibition of cell migration (Fig. 3A). These observations suggest that the ethanolic extract of SG possesses a variety of biologically active components which act on multiple targets and mechanisms involved in the regulation of cell proliferation, migration and tube formation in HUVECs.

To investigate the molecular mechanisms by which SG regulates VEGF-A-induced endothelial cell responses, we examined the changes in the activation of VEGF-A/VEGFR-2 downstream signaling pathways including ERK, PI3-K/Akt and mTOR/p70^S6K, which play important roles in cellular fate (22). Compared with the unstimulated controls, VEGF-A treatment markedly increased the phosphorylation/activation of ERK, Akt and p70^S6K in HUVECs (Fig. 5). However, SG treatment significantly inhibited VEGF-A-stimulated phosphorylation of Akt and p70^S6K, but not that of ERK. To directly examine the contribution of inactivation of Akt and p70^S6K activity to the anti-angiogenic activity of SG, we studied the changes in cell proliferation and migration in the presence of LY294002 and rapamycin (Fig. 6). Pretreatment of cells with LY294002 or rapamycin mimicked the suppressive effect of SG on VEGF-A-stimulated cell proliferation and migration (Fig. 6A and C). As shown in Fig. 6B, both LY294002 and rapamycin also mimicked the SG-mediated suppression of cyclin D expression, with a high correlation with previous cell proliferation and cell cycle-related protein expression experiments (Fig. 1A and 2B). The addition of LY294002 significantly enhanced the ability of SG to inhibit cell proliferation and cyclin D expression as well as migration (Fig. 6). Rapamycin treatment enhanced the inhibitory effect of SG on cell migration, but not cell proliferation (Fig. 6A and C). Collectively, these observations suggest that SG may contain pharmacologically effective components similar to these inhibitors and share the roles and mechanisms of action in regulating angiogenic responses in vitro.