The human umbilical vein endothelial cell line (HUVEC) and mouse mammary tumor cell line, 4T1, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and were routinely cultured in DMEM medium (Gibco-BRL, Rockville, IN, USA) containing 10% FBS (Haoyang Biological Manufacturer Co., Ltd., Tianjin, China), 100 U/ml penicillin and 100 μg/ml streptomycin in 5% CO₂ at 37°C. Anti-p21, anti-extracellular signal-regulated kinase (ERK), anti-phosphorylated (p)-ERK, anti-c-Jun N-terminal kinase (JNK), anti-p-JNK, anti-p65, anti-p-p65, anti-signal transducer and activator of transcription 3 (STAT3) and anti-p-STAT3 (ser727) antibodies were obtained from Cell Signaling Technology, (Beverly, MA, USA). Anti-VEGF antibody was provided by Abcam, (Cambridge, MA, USA). Anti-β-actin (1:5000) antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-mouse and rabbit IgG horseradish peroxidase (HRP) antibodies (1:5000) was from ZhongShan Goldenbridge Biotechnology Co., Ltd. (Beijing, China). The pro-lighting HRP agent for western blot analysis was supplied by Tiangen Biotech Co., Ltd., (Beijing, China).

Electuary ointment of Huaier extract was kindly provided by Gaitianli Medicine Co., Ltd. (Jiangsu, China). The electuary ointment (2 g) was soaked in 20 ml of DMEM. The solid residue of the above dissolved herbs was filtered and discarded through a 0.22-μm filter. The final 100 mg/ml stock solution was kept at −20°C for long storage.

HUVECs were seeded in a 24-well plate. After 12 h, the HUVECs were exposed to various concentrations of Huaier extract for an additional 24 h. Finally, the morphological changes caused by Huaier extract were observed under an Olympus light microscope and photomicrographs were taken with an Olympus digital camera (Olympus, Tokyo, Japan).

MTT assay was performed to measure the viability of the HUVECs and 4T1 cells after treatment with Huaier extract. In brief, the HUVECs (10³ cells/well) and 4T1 cells (700 cells/well) were seeded in cultured medium in 96-well plates and incubated in 5% CO₂ at 37°C. After 12 h, the medium in each well was replaced with the vehicle or different concentrations (2, 4 and 8 mg/ml) of Huaier extract and incubated for another 48 or 72 h. Subsequently, 20 μl of MTT (5 mg/ml in PBS) were added into each well. After 4 h of incubation at 37°C, the supernatants were aspirated carefully and 100 μl of dimethyl sulfoxide (DMSO) were added to each well. Absorbance values at 490 nm were determined by the Microplate Reader (Bio-Rad, Hercules, CA, USA).

After 24 h of starvation in serum-free medium at 37°C, the HUVECs were treated with various concentrations of Huaier extract or complete medium as the negative control. After 24 h, the treated cells were harvested, washed with 1X cold PBS and fixed with 70% ice-cold ethanol overnight. After the ethanol was removed by centrifugation at 1,200 × g for 1 min, the fixed cells were washed with PBS twice. The pellets were then resuspended with 1 ml of DNA staining solution (MultiSciences Biotech Co., Ltd.). After incubation for 30 min at room temperature in the dark, cells were analyzed in the presence of the dye by FACScan flow cytometry (Becton-Dickinson, Franklin Lakes, NJ, USA) and the data were analyzed by ModFitLT V2.0 software (Becton-Dickinson).

The BD Pharmingen™ PE Annexin V Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect the proportion of apoptotic cells, according to the instructions of the manufacturer. Briefly, after treatment with various concentrations of Huaier extract for the indicated times, the HUVECs were harvested and washed with PBS twice. The cells (1×10⁵) were then resuspended in 100 μl of binding buffer, followed by adding 5 μl Annexin V-FITC and 5 μl PI. After incubation for 15 min in the dark, another 400 μl of binding buffer were added before the cells were analyzed by FACScan flow cytometry.

Scratch assay was applied to determine cell mortality caused by Huaier extract. This assay was performed using a standard method (26) with some modifications. Briefly, 2.5×10⁴ HUVECs were seeded on a 12-well plate in complete medium overnight to obtain a full confluent monolayer. After 24 h of starvation, a 20-μl pipette tip was used to create a straight cell-free wound. Each well was washed twice with PBS to remove debris. The cells were then cultured in serum-free medium in the absence or presence of various concentrations of Huaier extract. The distances between the 2 edges of the scratch were analyzed quantitatively.

In vitro cell migration assay was performed using the Transwell system (24-wells, 8-μm pore size with polycarbonate membrane; Corning Costar, Lowell, MA, USA). Cells were starved in serum-free medium for 24 h at 37°C. The HUVECs were then harvested and resuspended in various concentrations of Huaier extract diluted in serum-free medium. The conditional medium from the NIH3T3 fibroblasts and the complete medium were then mixed (v/v 1:1). Subsequently, 1 ml of the mixture was added to the lower well of each chamber, and 100 μl of cell solutions containing 1×10⁴ HUVECs were added to the upper wells. After treatment for 24 h, the cells attached to the lower surface were fixed with methanol and stained with 0.2% Giemsa. The successfully migrated cells were counted on 5 random fields using an Olympus light microscope.

The ability of the HUVECs to form network structures was tested on Matrigel basement membrane matrix (BD Biosciences, San Jose, CA, USA). Firstly, 50 μl of Matrigel were plated per well on 96-well plates and allowed to polymerize at 37°C for 30 min. Subsequently, 100 μl of HUVECs suspended in complete medium at a density of 1×10⁵/ml were added to each well in the absence or presence of Huaier extract. After 9 h, tube-like structures were photographed with an Olympus digital camera.

CAM assay was performed as described previously (27) with small modifications. Briefly, 40 fertilized chicken eggs were incubated at 37°C at constant humidity and randomly divided into 4 groups. On the 9th day of incubation, a square window (1×1 cm²) was opened in the shell. The following day, filter discs loaded with 20 μl complete medium or various concentrations of Huaier extract were placed on the top of the growing CAMs under sterile conditions. Afterwards, the window was sealed with sterilized surgical tape and the eggs were returned to the incubator. After 24 h of incubation, the CAMs were photographed using an Olympus Live View Digital SLR camera.

Angiogenesis ex vivo was also studied by rat aortic ring assay (28). Briefly, a 48-well plate was first covered with Matrigel and incubated for 30 min at 37°C. Subsequently, 2-month old BALB/c mice were sacrificed by cervical dislocation, and the thoracic aortas were dissected and cut into 1–2-mm long sections. Afterwards, aortic rings were placed into wells pre-coated with Matrigel, and then covered with another layer of Matrigel. After 30 min of polymerization, DMEM supplemented with 20% FBS was added into each well. The following day, the supernatants were replaced with medium in the absence or presence of various concentrations of Huaier extract. On day 6, the fields covered by the sprouting from the aortic rings were measured by an Olympus digital camera.

In brief, the HUVECs were allowed to grow to 60–70% confluence in 25 cm² cell culture flasks, and then incubated with gradient concentrations of Huaier extract at 37°C under 5% CO₂. After 2 h of treatment, the cells were harvested, and the proteins were lysed in lysis buffer (1X PBS, 1% NP40, 0.1% sodium dodecyl sulfate, 5 mM EDTA, 0.5% sodium deoxycholate and 1 mM sodium orthovanadate) with protease inhibitors. Subsequently, 50 μg of total cellular protein from each sample were separated by 10% SDS-PAGE and electrotransferred onto a polyvinylidene fluoride (PVDF) membranes by using a semi-dry blotting apparatus (Bio-Rad). After blocking with 5% non-fat milk, the PVDF membranes were covered with specific primary antibodies, followed by incubation with secondary antibodies. The protein bands were then visualized by using Pro-lighting HRP agent and their densities were analyzed using ImageJ software. β-actin was used as the loading control.

Twenty BALB/c female mice, 4–5 weeks old, were purchased from the Center for New Drugs Evaluation of Shandong University, and housed under pathogen-free conditions. All the experiments were approved by the institutional guidelines of the Animal Care and Use Committee at Shandong University. 4T1 cells (1×10⁶) were subcutaneously injected into the left flank of each mouse. After 2 days, each mouse was given 100 μl solution containing 50 mg Huaier extract by gavage daily. After 21 days, the mice were sacrificed, and the xenografts were removed for immunohistochemical staining.

Immediately after excision, the tumor tissues were stored in 10% neutral-buffered formalin. After 24 h, the samples were paraffin-embedded and then sliced into 4-μm section for hematoxylin and eosin (H&E) staining according to the standard techniques. The relative areas of necrosis in tumors were analyzed.

To quantify the microvessel density (MVD), the SP-9000 Histostain™-Plus Kits (ZhongShan Goldenbridge Biotechnology Co.) were used to detect CD34 expression using the standard steps. Briefly, the sections were deparaffinized and rehydrated, followed by antigen retrieval with pH 6.0 citrate buffer. Endogenous peroxidase activity was inhibited with 3% H₂O₂ for 15 min and the sections were incubated with 10% normal goat serum to block non-specific binding. After incubation with anti-CD34 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, the sections were washed, treated with biotinylated anti-immunoglobulin antibody for 20 min and reacted with horseradish peroxidase-conjugated streptavidin. Then the liquid DAB substrate/chromogen system (Maixin Bio, Fuzhou, China) was used, followed by counterstaining with hematoxylin. The representative images of tumor tissues were taken by an Olympus light microscope.

TUNEL assay was performed to identify the apoptotic cells in the paraffin-embedded sections using the One Step TUNEL Apoptosis Assay kit (Beoytime, Beijing, China) according to the manufacturer’s instructions. TUNEL-positive cells were visualized with red fluorescent staining observed by a fluorescence microscope (Olympus).

The results are presented as means ± standard deviation (SD) and differences between groups were compared by one-way ANOVA and considered significant at P<0.05. The statistical analysis was carried out by using SSPS edition 16.0.

To investigate the effect of Huaier extract on angiogenesis, we first observed the cell morphology of the HUVECs after exposure to Huaier extract. Following 24 h of incubation with various concentrations of Huaier extract, the morphological changes in the HUVECs were observed (Fig. 1A). Obvious morphological changes were observed in the HUVECs. Compared with the untreated cells, the majority of the cells in the Huaier-treated groups became enlongated and star-shaped with sharp outlines. These results suggest that Huaier extract causes cell skeleton rearrangement in HUVECs.

We then examined the cell viability by using MTT assay. As shown in Fig. 1B, Huaier extract suppressed the proliferation of the HUVECs in a time- and dose-dependent manner. The inhibitory rates of Huaier extract varied from 2.3±3.7% to a maximum of 87.7±0.5% after 48-h incubation, with an IC₅₀ of 8.1±0.9 mg/ml. After treatment with increasing concentrations of Huaier extract for 72 h, the viabilities of the HUVECs were suppressed by 6.9±2.2, 29.4±0.9, 83.7±0.5, 87.9±4.5 and 88.0±0.2%, respectively. A significant reduction was firstly observed at 4 mg/ml (P<0.05).

To further explore the underlying mechanism of the antiproliferative effect of Huaier extract, we applied flow cytometry to analyze the apoptotic rate and cell cycle distribution after treatment with Huaier extract. The data demonstrated that the ratios of apoptotic HUVECs were 4.4±0.6, 6.4±1.4 and 10.9±2.6% in the presence of increasing concentrations of Huaier extract for 48 h, respectively (Fig. 2A and C). In addition, as shown in Fig. 2B and D, after 24 h of treatment, the proportion of cells at the G0/G1 phase was dose-dependently increased (from 36.79±2.25% in control group to 62.41±9.77% in 8 mg/ml Huaier group). Furthermore, treatment with Huaier extract resulted in the accumulation of p21 with a maximum at 4 mg/ml (Fig. 2E and F), and this partly contributed to the cell cycle arrest mentioned above.

We then examined the influence of Huaier extract on cell motility by using modified scratch assay (26) and cell migration assay (29). As shown in Fig. 3A and B, after treatment with Huaier extract, the migration of the HUVECs was dose- and time-dependently inhibited. This result was consistent with the results presented in Fig. 3C and D. At 8 mg/ml, the number of cells which had successfully migrated to the lower side of the filter was reduced by 66.2±2.8% (P<0.01) after 24 h of treatment with Huaier extract.

As an essential step for angiogenesis, the formation of tube-like structures involves matrix degradation, rearrangement and apoptosis of endothelial cells. As shown in Fig. 4A, untreated HUVECs formed organized capillary tubes within 9 h. While in the presence of Huaier extract, the HUVECs rounded up and rendered incomplete network structures.

To verify the anti-angiogenic effect of Huaier extract ex vivo, CAM assay and aortic ring assay were also applied. On day 9 of embryo development, fertilized chick eggs were treated with various concentrations of Huaier extract. After 24 h of incubation, normal vascular pattern with numerous branchings was observed in the control group. However, Huaier extract significantly distorted the vasculature architecture on the chorioallantoic membrane in a dose-dependent manner (Fig. 4B).

The results demonstrated that Huaier extract caused a dramatic decrease in sprout length and density from the aortic ring in a dose-dependent manner (Fig. 4C). In conclusion, Huaier exhibited anti-angiogenic activity both in vitro and ex vivo.

To identify whether Huaier extract can regulate multiple molecules involved in angiogenesis, we used western blot analysis to investigate the changes between the vehicle- and Huaier-treated groups. The results showed that Huaier extract regulated the ERK pathway by down-regulating the phosphorylation of ERK without affecting overall ERK expression levels (Fig. 5). In addition, the expression of VEGF was significantly reduced by 46.4±2.1% after incubation with 8 mg/ml Huaier extract for 24 h (P<0.01). Similarly, Huaier extract reduced the phosphorylation of JNK, STAT3 and p65. However, no effect was observed on Akt signaling and Huaier extract was unable to suppress the level of HIF (data not shown).

The antiproliferative effect of Huaier extract on 4T1 cells was examined by MTT assay. The results demonstrated that Huaier extract significantly inhibited the proliferation of 4T1 cells in a time- and dose-dependent manner (Fig. 6A). With 2 mg/ml of Huaier extract, a significant suppression on 4T1 cell proliferation was observed with a 7.7±0.9% (at 48 h, P<0.05) and 15.2±1.8% (at 72 h, P<0.01) reduction. The IC₅₀ for 4T1 cells was 7.9±0.9 mg/ml (at 48 h) or 4.4±0.6 mg/ml (at 72 h). These data suggest that 4T1 cells are more sensitive to Huaier extract than HUVECs.

Taking into account the anti-angiogenic and antitumor effects of Huaier extract in vitro and ex vivo, we then examined the antitumor effect of Huaier extract in BALB/c mice. As shown in Fig. 6B and C, the administration of Huaier extract by gavage delayed the tumor volume at concentration of 2.5 g/kg per day. Compared with the control group (667.0±52.6 mm³), tumor volumes in the Huaier-treated group were significantly smaller (488.9±86.5 mm³, P<0.05) at day 21. The antitumor activity of Huaier extract in vivo was confirmed by measuring the tumor weights after the mice were sacrificed. The weights of tumors isolated from the Huaier-treated groups were significantly decreased by 23.6±5.1% (0.81±0.13 g in the control group, and 0.62±0.05 g in the treated group, P<0.05). However, we observed no significant difference between the 2 groups in body weight, which indicated no obvious toxicity to mice at the curative dose (Fig. 6D). Our data prove the antitumor effect of Huaier extract on 4T1 mouse mammary cancer without causing marked toxicity in vivo.

To elucidate the mechanisms behind the effect of Huaier extract in vivo, the tumor tissues from the animal models were stained with H&E, CD34 and TUNEL (Fig. 7). The H&E-stained sections revealed large areas of necrosis occurring in both groups. The necrotic part in the untreated tumors was hemorrhagic. However, an ischemic type of necrosis with little or no blood was observed in the Huaier-treated tumors. We then performed TUNEL staining to explore the apoptotic effect induced by Huaier extract in vivo. The increased ratio of TUNEL-positive cells clearly demonstrated that the induction of apoptosis was involved in the antitumor activity of Huaier extract. Furthermore, CD34 staining was performed to evaluate the MVD in the tumor. Under microscopic analysis, a marked decrease in the expression of CD34 was observed in the treated group compared with the control group.