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). CD31, SHH, PTCH-1, SMO, Gli-1, VEGF-A and VEGFR2 antibodies, secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals, unless otherwise stated, were obtained from Sigma Chemicals (St. Louis, MO, USA).

Authentic plant material was purchased from Guo Yi Tang Chinese Herbal medicine store, Fujian, China. The original herb was identified as Hedyotis diffusa Willd (HDW) by Dr Wei Xu at Department of Pharmacology, Fujian University of Traditional Chinese Medicine, China. Ethanol extract of HDW (EEHDW) was prepared as previously described (3). Briefly, 500 g of HDW was extracted with 5,000 ml of 85% ethanol using a refluxing method and filtered. The ethanol solvent was then evaporated on a rotary evaporator (Yarong, Model RE-2000, Shanghai, China). The resultant solution was concentrated to a relative density of 1.05 and the dried powder of EEHDW was obtained by a spraying desiccation method using a spray dryer (Buchi, Model B-290, Flawil, Switzerland). The working concentrations of EEHDW were made by dissolving the extract in saline to a concentration of 0.6 g/ml.

Human colon carcinoma cell line HT-29 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in DMEM medium supplemented with 10% serum (Gibco-BRL), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified environment with 5% CO₂ at 37°C. Male BALB/C athymic nude mice (with an initial body weight of 20–22 g) were purchased from Shanghai Si-Lai-Ke Experimental Animal Ltd (Shanghai, China). The mice were housed five per cage in specific pathogen-free room in an environment with controlled temperature (22°C), humidity and a 12 h light/dark cycle with free access to water and standard laboratory food. All animal treatments were strictly in accordance with international ethical guidelines and the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals, and the experiments were approved by the Institutional Animal Care and Use committee of Fujian University of Traditional Chinese Medicine.

HT-29 cells (5×10⁶) mixed with Matrigel (1:1) were subcutaneously injected in the right flank area of athymic nude mice to initiate tumor growth. After 5 days of xenograft implantation, mice were randomly divided into two groups (n=10) and given intra-gastric administration with 3 g/kg of EEHDW or saline daily, 6 days a week for 16 days. Tumor diameters were measured at regular intervals with digital calipers and the tumor volume (V) was calculated using the formula: V = (width)² × length × π/6. At the end of experiment, the animals were anaesthetized and the tumor issue was removed. Blood was obtained aseptically from orbit. Blood-containing tubes were allowed to stand at room temperature for 5 h and serums were obtained by centrifugation at 3, 000 × g for 20 min.

Total RNA was isolated from fresh tumor tissues with TRIzol Reagent. Oligo(dT)-primed RNA (1 μg) was reverse-transcribed with SuperScript II reverse transcriptase (Promega) according to the manufacturer’s instructions. The obtained cDNA was used to determine the mRNA amount of SHH, PTCH, SMO, Gli-1, VEGF-A and VEGFR2 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 (Bio-Rad, Model Gel Doc 2000, Hercules, CA, USA).

After fixing with 10% formaldehyde for 12 h, tumor samples were processed conventionally for 5-μm-thick paraffin-embedded tumor slides. The slides were subjected to antigen retrieval and the endogenous peroxidase activity was quenched with hydrogen peroxide. After blocking non-specific proteins with normal serum in PBS, slides were incubated with rabbit polyclonal antibodies against CD31, SHH, PTCH-1, SMO, Gli-1, VEGF-A and VEGFR2 (all in 1:100 dilution, Santa Cruz Biotechnology). After washing with PBS, slides were incubated with biotinylated secondary antibody followed by conjugated HRP-labelled streptavidin (Dako) and then washed with PBS. The slides were then incubated with diaminobenzidine (DAB, Sigma Chemicals) as the chromogen, followed by counterstaining with diluted Harris hematoxylin (Sigma Chemicals). After staining, five high-power fields (×400) were randomly selected in each slide, and the average proportion of positive cells in each field were counted using the true color multi-functional cell image analysis management system (Image-Pro Plus, Media Cybernetics, Bethesda, MD, USA). To rule out any non-specific staining, PBS was used to replace the primary antibody as a negative control.

All data are the means of three determinations and data were analyzed using the SPSS package for Windows (Version 11.5). Statistical analysis of the data was performed with Student’s t-test and ANOVA. Differences with P<0.05 were considered statistically significant.

The inhibitory effect of EEHDW on cancer growth was evaluated by measuring tumor volume. We found that administration with EEHDW significantly inhibited tumor growth throughout the study, as compared with the control group (Fig. 1). At the end of experiments, tumor volume per mouse in EEHDW treatment group was 0.37±0.11 cm³, whereas that in control group was 0.82±0.23 cm³ (Fig. 1, P<0.01), demonstrating the therapeutic efficacy of EEHDW against CRC in vivo.

We performed immunohistochemical staining (IHS) for endothelial cell-specific marker CD31, to determine the effect of EEHDW on intratumoral microvessel density (MVD) that is considered as an indicator of tumor angiogenesis and is associated with cancer growth and progression (17). As shown in Fig. 2, the percentage of CD31-positive cells in tumors from EEHDW-treated or control mice was 24.67±5.43 or 35.33±2.88%, respectively (P<0.01), suggesting that inhibition of tumor angiogenesis by EEHDW could have contributed to the inhibition of colorectal tumor growth.

To determine the effect of EEHDW on SHH pathway, we evaluated mRNA and protein expression levels of the key mediators of this pathway in CRC xenograft tumors using RT-PCR and IHS assay. Data from RT-PCR showed that EEHDW profoundly reduced the mRNA expression of SHH, Ptch-1, Smo and Gli-1 in CRC tumor tissues (Fig. 3A, P<0.01). In a consistent manner, the protein expression of these factors was significantly downregulated by EEHDW treatment. The percentage of cells in the CRC xenograft tumors expressing SHH, Ptch-1, Smo or Gli-1 in the EEHDW-treated mice was 25.67±7.81, 18.00±3.96, 26.00±4.98 or 25.17±5.12%, whereas that in control group was 34.83±5.31, 35.33±5.24, 37.33±5.99 or 35.83±7.30%, respectively (Fig. 3B, P<0.01). Collectively, these data suggest that EEHDW inhibits the activation of SHH signaling in colorectal cancer.

To further investigate the mechanism of anti-angiogenic activity of EEHDW, we examined the expression of VEGF-A, a critical target gene of SHH pathway, as well as the expression of its specific receptor VEGFR2. As shown in Fig. 4, EEHDW treatment obviously decreased the mRNA and protein expression levels of VEGF-A and VEGFR2 in CRC tumor tissues (P<0.01).