Usolic acid (UA) was purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). Matrigel was provided by Becton-Dickinson (San Jose, CA, USA). Roswell Park Memorial Institute medium-1640 (RPMI-1640), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA and TRIzol reagent were purchased from Invitrogen (Carlsbad, CA, USA). SuperScript II reverse transcriptase was obtained from Promega (Madison, WI, USA). The In Vitro Angiogenesis Assay kit was purchased from Millipore (Billerica, MA, USA). Human VEGF-A and bFGF ELISA kits were obtained from Shanghai Xitang Biological Technology Ltd. (Shanghai, China). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). BCA Protein assay kit was purchased from Tiangen Biotech (Beijing) Co., Ltd. Bio-Plex phosphoprotein assay kits were purchased from Bio-Rad (Hercules, CA, USA). All other chemicals, unless otherwise stated, were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Human colon carcinoma HT-29 cells were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Human umbilical vein endothelial cells (HUVECs) were purchased from Xiangya Cell Center, University of Zhongnan (Hunan, China). HT-29 cells and HUVECs were grown in DMEM and RPMI-1640, respectively. Both DMEM and RPMI-1640 were supplemented with 10% (v/v) FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. All cell lines were cultured at 37°C, 5% CO₂ and in a humidified environment.

Male BALB/c athymic (nude) mice (with an initial body weight of 20–22 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and housed under pathogen-free conditions with controlled temperature (22°C), humidity, and a 12-h light/dark cycle. Food and water were given ad libitum throughout the experiment. All animal treatments were performed strictly in accordance with international ethical guidelines and the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals. The experiments were approved by the Institutional Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine.

CRC xenograft mice were produced with HT-29 cells. The cells were grown in culture and then detached by trypsinization, washed, and resuspended in serum-free DMEM. Resuspended cells (1.5×10⁶) mixed with Matrigel (1:1) were subcutaneously injected into the right flank of mice to initiate tumor growth. At 5 days following xenograft implantation (tumor size ∼3 mm in diameter), mice were randomized into two groups (n=10) and treated with UA (dissolved in PBS) 12.5 mg/kg or saline by daily intraperitoneal injection, 6 days a week for 16 days. Body weight and tumor size were measured. Tumor size was determined by measuring the major (L) and minor (W) diameter with a caliper. The tumor volume was calculated according to the following formula: tumor volume = π/6 × L × W². At the end of the experiment, the animals were anaesthetized with pelltobarbitalum natricum, and tumors were excised. A portion of each tumor was fixed in 10% buffered formalin and the remaining tissue snap-frozen in liquid nitrogen and stored at −80°C.

Six tumors were randomly selected from UA-treatment or control groups. Tumor tissues were fixed in 10% formaldehyde for 12 h, paraffin-embedded, sectioned and placed on slides. The slides were subjected to antigen retrieval and endogenous peroxidase activity was quenched with hydrogen peroxide. Non-specific binding was blocked with normal serum in PBS (0.1% Tween-20). Rabbit polyclonal antibodies against CD31, SHH, Gli-1, VEGF-A and bFGF (all in 1:200 dilution, Santa Cruz Biotechnology) were used to detect the relevant proteins. The binding of the primary antibody was demonstrated with a biotinylated secondary antibody, horseradish peroxidase (HRP)-conjugated streptavidin (Dako) and diaminobenzidine (DAB) as the chromogen. The tissues were counterstained with diluted Harris hematoxylin. After staining, five high-power fields (at magnification of ×400) were randomly selected in each slide. The proportion of positive cells in each field was determined using the true color multi-functional cell image analysis management system (Image-Pro Plus, Media Cybernetics, USA). To control for non-specific staining, PBS was used to replace the primary antibody as a negative control.

A CAM assay was performed to determine the in vivo anti-angiogenic activity of UA. Briefly, 10 μl of UA (25 μg/μl) was loaded onto a 0.5-cm diameter Whatman filter paper. The filter was then applied to the CAM of a 7-day embryo. After incubation for 72 h at 37°C, the region surrounding the filter was photographed with a digital camera. The number of blood vessels was quantified manually in a circular perimeter surrounding the implants, at a distance of 0.25 cm from the edge of the filter. Assays were performed twice with a final total of 10 eggs for each data point.

UA was dissolved in DMSO and diluted to working concentrations with culture medium. The final concentration of DMSO in the medium for all cell-based experiments was 0.1% HUVECs were seeded into 96-well plates at a density of 1.0×10⁴ cells/well in 0.1 ml medium and incubated at 37°C under a 5% CO₂ for 24 h. The cells were treated with various concentrations of UA for 24 h or with 40 μM of UA for different periods of time. Treatment with 0.1% DMSO was included as the vehicle control. At the end of the treatment, 10 μl MTT [5 mg/ml in phosphate buffered saline (PBS)] were added to each well, and the samples were incubated for an additional 4 h at 37°C. The purple-blue MTT formazan precipitate was dissolved in 100 μl DMSO. Absorbance was measured at 570 nm using an ELISA reader (BioTek, Model EXL800, USA).

Migration of HUVECs was performed by the wound healing method. HUVECs were seeded into 12-well plates at a density of 2×10⁵ cells/well in 1 ml medium. After 24 h of incubation, cells were scraped away vertically in each well using a P100 pipette tip. Three randomly selected views along the scraped line were photographed in each well using phase-contrast inverted microscopy at a magnification of ×100. Cells were treated with various concentrations of UA for 24 h, and a second set of images were taken using the same method. A reduction in the size of the scraped region is indicative of cell migration.

HUVEC tube formation was examined using the ECMatrix assay kit (Millipore) following the manufacturer’s instructions. Briefly, confluent HUVECs were harvested and diluted (1×10⁴ cells) in 50 μl of medium containing various concentrations of UA. The harvested cells were seeded with ECMatrix gel (1:1 v/v) into 96-well plates and incubated for 9 h at 37°C. The cells were photographed using phase-contrast inverted microscopy at a magnification of ×100. The level of HUVEC tube formation was quantified by calculating the length of the tubes in three randomly chosen fields from each well.

Total RNA was isolated from tumor tissues (three tumors were randomly selected from UA-treatment or control groups) or HT-29 cells with TRIzol reagent. Oligo(dT)-primed RNA (1 μg, isolated from tumor tissues or cells) was reverse-transcribed with SuperScript II reverse transcriptase (Promega) according to the manufacturer’s instructions. The obtained cDNA was used to determine the level of VEGF-A, bFGF, Shh and Gli-1 mRNA by PCR with Taq DNA polymerase (Fermentas). GAPDH was used as an internal control. Samples were analyzed by gel electrophoresis (1.5% agarose). The DNA bands were examined using a gel documentation system (Model Gel Doc 2000, Bio-Rad).

HT-29 cells (2.5×10⁵) in 5 ml medium were seeded into 25 cm² flasks and treated with the indicated concentrations of UA for 24 h. Treated cells were lysed in mammalian cell lysis buffer (M-PER, Thermo Scientific, Rockford, IL, USA) containing protease (EMD Biosciences) and phosphatase inhibitor (Sigma-Aldrich) cocktails and centrifuged at 14,000 × g for 15 min. Protein concentrations in cell lysate supernatants were determined by BCA protein assay. Equal amounts of protein from each tumor or cell lysate were resolved on 12% Tris-glycine gels and transferred onto PVDF membranes. The membranes were blocked for 2 h with 5% non-fat dry milk and incubated with the desired primary antibody directed against Shh, Gli-1, or β-actin (all in 1:1,000 dilutions) overnight at 4°C. Appropriate HRP-conjugated secondary antibodies with chemiluminescence detection were used to image the antibody-detected proteins.

Eight tumors were randomly selected from UA-treatment or control groups and homogenized. For analysis of phosphorylation of Akt and p70S6K in vitro, HT-29 cells (2.5×10⁵) were seeded into 25-cm² flasks in 5 ml medium and treated with 40 μM of UA for 24 h. To detect STAT3 phosphorylation in vitro, HT-29 cells were first grown in complete DMEM (10% FBS) until ∼70% confluency and subsequently cultured in FBS-free medium overnight. The medium was replaced with DMEM with 10% FBS and cells were pre-treated with UA (40 μM) for 1 h followed by stimulation with 10 ng/ml of IL-6 for 15 min. Tumor tissues and treated cells were lysed using a commercially available lysis kit (Bio-Rad Laboratories) and centrifuged at 14,000 × g for 15 min. Protein concentrations of the clarified supernatants were determined by BCA protein assay. The presence of p-STAT3, p-Akt and p-p70S6K was detected using a bead-based multiplex assay for phosphoproteins (Bio-Plex Phosphoprotein assay, Bio-Rad Laboratories) according to the manufacturer’s protocol. Data were collected and analyzed using the Bio-Plex 200 suspension array system (Bio-Rad).

HT-29 cells were seeded into 6-well plates at a density of 2×10⁵ cells/well in 2 ml medium and treated with the indicated concentrations of UA for 24 h. The medium and cells from each well were collected and stored at −80°C until analyzed. The level of VEGF-A or bFGF in the media was measured using an ELISA kit (Xitang Biological Technology Ltd.) according to the manufacturer’s instructions. The concentrations of VEGF-A and bFGF were determined by comparison to serial dilutions of VEGF-A and bFGF purified standards.

Data are presented as the mean ± SD for the indicated number of independently performed experiments. The data were analyzed using the SPSS package for Windows (Version 17.0). Statistical analysis was carried out with Student’s t-test and ANOVA. Differences with P<0.05 were considered to be statistically significant.

The in vivo efficacy of UA against tumor growth was evaluated by measuring tumor volume in CRC xenograft mice. As shown in Fig. 1, administration of UA significantly inhibited tumor growth throughout the study, as compared with the control group (P<0.05), whereas the body weight gain in experimental animals was not affected by UA treatment, suggesting that UA is potent in suppressing colon tumor growth in vivo, without apparent adverse effects.

To determine the effect of UA on tumor angiogenesis, we examined the intratumoral microvessel density (MVD) in CRC mice by evaluating expression of the endothelial cell-specific marker CD31. Data from immunohistochemical staining (IHC) assay showed that the percentage of CD31-positive cells in control or UA-treated mice was 36.0±4.8 or 25.0±2.8%, respectively (Fig. 2A, P<0.01), demonstrating UA’s inhibitory activity on tumor angiogenesis. The in vivo anti-angiogenic activity of UA was further confirmed using a chick chorioallantoic membrane (CAM) model. As shown in Fig. 2B, UA treatment significantly reduced the total number of blood vessels in chicken embryos as compared to untreated controls (P<0.01).

The processes of angiogenesis include endothelial cell (EC) proliferation, migration, and alignment into tubular structures. To evaluate the anti-angiogenic effect of UA we modeled each of these processes with HUVECs in vitro. As shown in Fig. 3A, UA treatment dose- and time-dependently decreased the proliferation (viability) of HUVECs compared to untreated control cells (P<0.01). In addition, UA treatment inhibited HUVEC migration after monolayer wounding (Fig. 3B). Moreover, we examined the effect of UA on capillary tube formation of HUVECs using an extracellular matrix, in which cultured ECs rapidly align and form hollow tube-like structures. As shown in Fig. 3C, untreated HUVECs formed elongated tube-like structures, whereas UA treatment resulted in a significant decrease in capillary tube formation (P<0.01). Taken together, these data suggested that UA-caused inhibition of colon tumor growth is accompanied by its anti-angiogenic activity.

To explore the underlying mechanisms of anti-angiogenic activities of UA, we determined its effect on the activation of several CRC-related signal transduction cascades. Activation of STAT3, Akt and p70S6K is mediated by its phosphorylation, we therefore investigated the effect of UA on STAT3, Akt and p70S6K activation in CRC xenograft tumor tissues and HT-29 cells by Bio-Plex Phosphoprotein assay. We found after UA treatment the phosphorylation level of STAT3, Akt and p70S6K in both tumors (Fig. 4A) and HT-29 cells (Fig. 4B) was decreased as compared to controls (P<0.01). The activation of SHH pathway was evaluated by examining the expression of the key mediators of SHH pathway in CRC xenograft tumors and HT-29 cells. As shown in Fig. 5A, the percentage of cells in the CRC xenograft tumors expressing SHH and Gli-1 in the control group was 32.8±5.3 and 36.0±8.4%, respectively, whereas the levels in UA-treated mice were 22.8±4.8 and 21.3±8.9%, respectively (P<0.01). Similarly, UA treatment significantly reduced the protein expression of SHH and Gli-1 in HT-29 cells (Fig. 5B). Data from RT-PCR showed that the pattern of mRNA expression was similar to their respective protein levels (Fig. 5C and D). Collectively, these data suggest that UA significantly suppresses the activation of multiple signaling pathways mediating tumor angiogenesis.

VEGF-A and bFGF are critical target gene of the above-mentioned pathways (30,31,43,44). As most potent angiogenic stimulators, VEGF-A and bFGF are commonly overexpressed in many kinds of human cancer correlating with tumor progression and poorer prognosis (45–48). To further investigate the mechanism whereby UA inhibited angiogenesis, we determined its effect on VEGF-A and bFGF expression. Data from RT-PCR, IHC and ELISA analyses indicated that UA treatment profoundly decreased mRNA and protein levels of VEGF-A and bFGF in both CRC xenograft tumor tissues and HT-29 cells (Fig. 6).