Pien Tze Huang (PZH) was obtained from and authenticated by the sole manufacturer Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd., China (Chinese FDA approval No: Z35020242). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, Trypsin-EDTA, TRIzol reagent, were purchased from Invitrogen (Grand Island, NY, USA). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Bio-Plex phosphoprotein assay kits were purchased from Bio-Rad Laboratories (Hercules, CA, USA). All the other chemicals, unless otherwise stated, were obtained from Sigma Chemicals (St. Louis, MO, USA).

Human colon carcinoma HT-29 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in DMEM containing 10% (v/v) FBS, 100 Units/ml penicillin and 100 μg/ml streptomycin in a 37°C humidified incubator with 5% CO₂. The cells were subcultured at 80–90% confluency.

Athymic male nude mice were obtained from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in specific pathogen-free rooms in an environment 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 strictly in accordance with the international ethics 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 the Fujian University of Traditional Chinese Medicine.

Cells (1.5×10⁶) mixed with Matrigel (1:1) were subcutaneously injected in the right flank area of athymic nude mice to initiate tumor growth. After 3 days of xenograft implantation, mice were randomized into two groups (n=10) and given intragastric administration of 234 mg/kg/day dose of PZH or saline daily, 5 days a week for 16 days. Tumor growth were measured every two days. Tumor growth 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 and the tumor tissue was removed and weighed.

Tumor samples were fixed with 10% formaldehyde for 12 h and subsequently processed conventionally for paraffin-embedded tumor slides. The slides were subjected to antigen retrieval and the endogenous peroxidase activity was blocked with 3% hydrogen peroxide in water. For immunohistochemical staining, slides were incubated with rabbit polyclonal antibodies against CD31, VEGF-A, VEGFR2, bFGF, bFGFR, iNOS or eNOS (all in 1:200 dilution; Santa Cruz Biotechnology). After washing with PBS, slides were incubated with biotinylated secondary antibody followed by conjugated horseradish peroxidase (HRP)-labelled streptavidin (Dako) and then washed with PBS. The slides were then incubated with diamino-benzidine (DAB, Sigma) as the chromogen, followed by counterstaining with diluted Harris hematoxylin (Sigma). 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, Silver Spring, MD, USA). To rule out any non-specific staining, PBS was used to replace the primary antibody as a negative control.

Tumors were homogenized and then lysed using a commercially available lysis kit (Bio-Rad Laboratories), followed by centrifugation 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, p-Erk1/2, p-JNK and p-p38 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 Laboratories).

Data are presented as mean ± SD for the indicated number of independently performed experiments. Statistical analysis was carried out with the Student’s t-test. Differences with P<0.05 were considered to be statistically significant.

The therapeutic efficacy of PZH against tumor growth was evaluated through comparison of tumor weight and volume in treated and control CRC xenograft mice. As shown in Fig. 1, tumor growth was significantly suppressed by PZH treatment throughout the experiment. The final tumor volume or tumor weight per mouse in control group was 0.71±0.16 cm³ or 0.47±0.13 g; while that in PZH-treated group was 0.36±0.15 cm³ or 0.23±0.10 g (P<0.05), demonstrating that PZH is effective in suppressing colorectal tumor growth.

Angiogenesis plays an essential role in cancer development; we therefore investigated the effect of PZH on intratumoral microvessel density (MVD) that is an indicator of new blood vessel growth. Tumors from CRC xenograft mice were evaluated by immunohistochemical staining (IHS) for the expression of an endothelial cell-specific marker CD31; and data in Fig. 2 show that the percentage of CD31-positive cells in control or PZH-treated mice was 35.22±3.26 or 23.60±6.73%, respectively (P<0.05), suggesting that PZH-caused inhibition of tumor growth is associated with its anti-angiogenic activity.

As most potent angiogenic stimulators, VEGF-A and bFGF are commonly overexpressed in many kinds of human cancer, which is correlated with tumor progression, invasion and metastasis, and poorer survival and prognosis in patients (33–36). VEGF-A and bFGF exert their biological function primarily through interaction with their specific receptors located on the surface of vascular endothelial cells, such as VEGFR-2 and bFGFR. Binding of VEGF-A and bFGF and their receptors leads to receptor dimerization, which in turn activates downstream signaling cascades such as the Akt and Erk pathways, leading to the proliferation, migration, survival, sprouting and eventually tube formation of endothelial cells (35–37).

By performing IHS analysis we found that PZH treatment profoundly inhibited the expression of VEGF-A and bFGF as well as their receptors in tumor tissues. The percentage of VEGF-A, VEGFR2, bFGF or bFGFR-positive cells in control group was, respectively, 38.77±7.76, 31.38±3.36, 23.80±6.73 and 33.50±5.67%, whereas that in PZH-treated mice was 26.0±5.67, 20.60±4.32, 15.25±3.17 and 22.20±4.07% (Fig. 3; P<0.05).

Nitric oxide synthases (NOSs), particularly the inducible (iNOS) and endothelial (eNOS) isoforms, have been strongly implicated in tumor angiogenesis (21,22,37–41); we therefore determined the effect of PZH on NOS expression. As shown in Fig. 4, the percentage of iNOS- or eNOS-positive cells in control group was, respectively, 45.33±6.93 and 30.33±5.87%, while that in PZH-treated mice was 23.80±4.52 and 22.75±4.34% (P<0.05), suggesting that iNOS and eNOS could be potential molecular targets for the antitumor and anti-angiogenic effects of PZH.

Cancer development is tightly regulated by multiple intracellular signaling pathways, including STAT3, Akt, ERK, JNK and p38. Aberrant activation of these pathways alters the expression of various critical target genes, such as above-mentioned angiogenic factors and NOSs, leading to the promotion of tumor angiogenesis. To further elucidate the underlying mechanisms of antitumor activity of PZH, we determined its effect on the activation of STAT3, Akt, ERK, JNK and p38 pathways. The activation (phosphorylation) of STAT3, Akt, Erk1/2, JNK and p38 in xenograft tumor tissues was determined by Bio-Plex phosphoprotein assay. As shown in Fig. 5, after PZH treatment the phosphorylation levels of STAT3, ERK, Akt, JNK and p38 in tumors were decreased as compared to controls (P<0.05), suggesting that PZH exerts its antitumor activity probably through affecting multiple intracellular targets.

In conclusion, in the present study, we demonstrate for the first time that PZH inhibits colorectal cancer growth via suppression of multiple intracellular signaling pathways leading to the inhibition of tumor angiogenesis, which may in part explain its anticancer activity.