GSPs, consisting of at least 95% proanthocyanidins, 1.8% proanthocyanidin B2 and 60% oligomers were purchased from Jianfeng Co. (Tianjin, China). Leibovitz’s L-15 medium, MCDB131 medium, epithelial growth factor (EGF), hydrocortisone, sulforhodamine B (SRB) and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Lanzhou National HyClone Bio-Engineering Co., Ltd. (Lanzhou, China). Millicell cell culture inserts were purchased from Millipore Corp. (Bedford, MA, USA). VEGF and Ang1 ELISA kits were ordered from R&D Systems (Minneapolis, MN, USA). Rabbit polyclonal anti-VEGF and rabbit polyclonal anti-Ang1 were purchased from Boster Bio-engineering Limited Co., Ltd. (Wuhan, China). MaxVision TM HRP-Polymer anti-Mouse/Rabbit IHC kit and DAB kit were purchased from Maixin Biological Technology, Ltd. (Fuzhou, China).

Human colon cancer SW620 cells were obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and were cultured in Leibovitz’s L-15 medium supplemented with 10% FBS. Human microvascular endothelial cells (HMEC-1) were cultured in MCDB131 medium supplemented with 1.18 mg/ml NaHCO₃, 20% inactivated FBS, 10 ng/ml EGF and 1 μg/ml hydrocortisone. All cells were cultured in a highly humidified atmosphere of 5% CO₂ at 37°C. Fertilized White Leghorn chicken eggs were obtained from the Lanzhou Institute of Biological Products (Lanzhou, China) and were incubated at 37°C in a humidified egg incubator.

The viability of SW620 cells was determined in 96-well plates by the SRB method with some modifications (27). Briefly, exponentially growing cells were seeded in 96-well plates with the final volume 100 μl/well. After 24 h of incubation, cells were treated with various concentrations of GSPs for the indicated times. The cultures were then fixed at 4°C for 1h with ice-cold 50% trichloroacetic acid to give a final concentration of 10%. Fixed cells were rinsed 5 times with deionized water and stained for 10 min with 0.4% SRB dissolved in 0.1% acetic acid. The wells were then washed 5 times with 0.1% acetic acid and left to dry overnight. The absorbed SRB was dissolved in 150 μl unbuffered 1% Tris base (pH 10.5). The absorbance of extracted SRB at 515 nm was measured on a microplate reader.

Fertilized White Leghorn chicken eggs were incubated under conditions of constant humidity at a temperature of 37°C. On Day 10 of incubation, a small hole was punched over the air sac in order to detach the CAM from the eggshell by gently exhausting, and then a square window was opened on the broad side of the egg, exposing the CAM. After the CAM was exposed, 40 μl serum-free culture medium containing 1×10⁶ SW620 cells were deposited on the CAM. The window was sealed with tape and the eggs were returned to the incubator. When the solid tumor began to vascularize after implantation for two days, GSP or vehicle was deposited locally each day. Five days later, CAM was photographed in ovo under a stereomicroscope and tumors were resected and weighed.

For the measurement of VEGF and Ang1 secretion, confluent SW620 cells (90–100%) were cultured in serum-free media for 24 h in the absence or presence of GSP. Cell-free culture supernatants were harvested and used for the determination of VEGF and Ang1 levels using a human VEGF or Ang1 ELISA kit according to the manufacturer’s instructions. The concentration of the VEGF and Ang1 in the samples was then determined by comparing the optical density of the samples to the standard curve.

Tumor conditioned medium was prepared from the SW620 cell culture as follows: SW620 cells were grown to subconfluency (approximately 90%). After being washed twice with D-Hanks, cells were incubated in MCDB131 medium supplemented with or without 100 μg/ml GSP for 24 h. The supernatant was then harvested, centrifuged at 2000 × g at 4°C for 10 min, filter-sterilized through 0.22-μm pore size filters and stored at −20°C prior to use.

Cell migration was performed in millicell cell culture inserts using a polycarbonate filter with a pore size of 8 μm. HMEC-1 cells (2×10⁵) suspended in 0.4 ml serum-free MCDB131 culture medium were added to the upper compartment of cell culture inserts. The lower compartment contained 0.6 ml conditioned medium. Following incubation for 24 h at 37°C, the nonmigrated cells on the upper surface of the membrane were removed with a cotton swab. The migrated cells on the lower surface of the membrane were fixed with methanol and then stained with 0.1% crystal violet. Images from randomly selected microscopic fields were obtained under light microscopy. Each sample was repeated three times.

Intracellular ROS levels in both GSP-treated and control cells were measured by DCFH-DA assay as previously described (31). Briefly, sub-confluent SW620 cells were treated with different concentrations of GSP for 24 h. Following incubation, cells were washed once with D-Hanks and stained with DCFH-DA (10 μM) for 30 min. Subsequently, cells were washed twice with D-Hanks to remove the excess dye, and then 100 μl of D-Hanks was added. The images were visualized and photographed under a fluorescent microscope. During the entire procedure with DCFH-DA, the plate was kept out of light to avoid fading of the fluoroprobe.

Following pretreatment with or without 100 μg/ml GSP for 2 h and further incubation with or without 100 μM H₂O₂ for 24 h, SW620 cells seeded on glass coverslips were fixed with 4% paraformaldehyde. The expression of both VEGF and Ang1 was detected with the immunohistochemical staining kit according to the manufacturer’s instructions.

Results are expressed as the means ± SD, and were analyzed using the Student’s t-test. Values of P<0.05 were considered to indicate statistically significant differences.

GSP treatment for 72 h significantly inhibited cell viability in a dose-dependent manner with an IC₅₀ value of 116.44 μg/ml. However, treatment with GSP (25–200 μg/ml) for 24 h did not change cell viability (Fig. 1). Therefore, all subsequent experiments were carried out with the treatment time not exceeding 24 h.

SW620 cells inoculated on chick CAM formed solid, avascular tumor within the first day. Two days after inoculation, the tumor became vascularized and grew rapidly. Five days later, numerous vessels developed radially around the tumor. Vessels at the tumor surface were clearly visible in the control group (Fig. 2A). Local treatment of the tumor from Day 3 to Day 7 (the day when SW620 cells were inoculated was designated as Day 1) with GSPs markedly inhibited tumor-induced angiogenesis. The tumor appeared white (Fig. 2B and C). The growth of the tumor was also inhibited by GSP treatment (Fig. 2D). During the experiment, some chick embryos from both the control and the GSP-treated groups died before the end of the experiment and were not included in the results shown. However, there was no significant difference in the incidence of embryonic death between GSP-treated and control groups, indicating that the inhibitory effect of GSP on tumor-induced angiogenesis was not related to toxic effects.

Solid tumors secrete various proangiogenic factors, such as VEGF and Ang1, to activate the nearest endothelial cells in the host tissue for neoangiogenesis (32). In the present study, we investigated whether GSPs could inhibit proangiogenic attributes of colon cancer SW620 cells. GSP treatment for 24 h inhibited both VEGF and Ang1 expression in a dose-dependent manner (Fig. 3). The inhibitory effect on VEGF expression was stronger than on Ang1 expression. These results suggest that the inhibitory effect of GSPs on tumor-induced angiogenesis is partly through suppressing the expression of angiogenic factors that initiate tumor angiogenesis in SW620 cells.

Both VEGF and Ang1 are chemotactic factors specific for endothelial cells. To further verify that GSPs inhibit tumor-induced angiogenesis by suppressing the expression of both VEGF and Ang1 in tumor cells, we conducted endothelial cell migration assay to examine the effect of conditioned medium from either GSP-treated or untreated SW620 cells on endothelial cell migration. Conditioned medium collected from SW620 cells induced endothelial cell migration (Fig. 4). However, endothelial cell migration was suppressed by conditioned medium collected from 100 μg/ml GSP-treated SW620 cells. These results further verified that the inhibitory effect of GSP on tumor-induced angiogenesis is mediated by reducing the expression of proangiogenic factors in SW620 cells.

Excessive ROS production has been confirmed to have a vital role in the induction of VEGF or Ang1 expression in many tumor cell lines. To investigate whether the antioxidant activity is part of the mechanisms by which GSPs suppress the expression of angiogenic factors in SW620 cells, we first used fluorescent probes DCFH-DA to detect the effect of GSPs on ROS production in SW620 cells. As shown in Fig. 5, colon cancer SW620 cells had a high level of ROS. Pretreatment with GSPs significantly inhibited ROS production in a concentration-dependent manner. Thus, our data suggest that GSPs can significantly reduce the high levels of intracellular produced ROS. To further verify that the antioxidant activity is part of the mechanisms by which GSPs suppress the expression of angiogenic factors, the effects of GSPs on H₂O₂-induced VEGF and Ang1 expression were examined with the immunohistochemical assay. As shown in Fig. 6, 100 μM of H₂O₂ markedly induced both VEGF and Ang1 expression in SW620 cells. However, the expression levels of VEGF and Ang1 induced by H₂O₂ were suppressed by pretreatment with 100 μg/ml GSP.