RPMI-1640, Dulbecco's modified Eagle's medium (DMEM) with high glucose, Fetal bovine serum (FBS), penicillin-streptomycin (catalog no. SV30010), 0.25% trypsin-EDTA, Pierce RIPA buffer, Pierce BCA Protein Assay kit (catalog no. 23227), and SuperSignal^™ West Pico Chemiluminescent substrate (catalog no. 34080) were all purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). CellTiter 96^® AQueous Non-Radioactive Cell Proliferation Assay kit (MTS assay; catalog no. G5430) was provided by Promega Corporation (Madison, WI, USA). Matrigel was obtained from BD Biosciences (Franklin Lakes, NJ, USA). TumorTACS in situ Apoptosis kit (catalog no. 4815–30-K) was purchased from R&D Systems Inc. (Minneapolis, MN, USA). VECTASTAIN Elite ABC kit was provided by Vector Laboratories, Inc. (Burlingame, CA, USA). Rabbit monoclonal antibodies against Pim-1 (catalog no. ab75776), B-cell lymphoma 2 (Bcl-2; catalog no. ab32124), cyclooxygenase-2 (COX-2; catalog no. ab62331), inducible nitric oxide synthase (iNOS; catalog no. ab178945), endothelial nitric oxide synthase (eNOS; catalog no. ab66127), rabbit polyclonal antibody against Ki-67 (catalog no. ab15580), Bcl-2-like protein 4 (Bax; catalog no. ab69643) and mouse monoclonal antibody against hypoxia-inducible factor 1-α (HIF1-α; catalog no. ab463) were purchased from Abcam (Cambridge, MA, USA). Rabbit monoclonal antibodies against cytochrome C (catalog no. 4280), caspase-3 (catalog no. 9665), poly (ADP-ribose) polymerase 1 (PARP; catalog no. 9532), mouse monoclonal antibody against caspase-9 (catalog no. 9508), and rabbit polyclonal antibody against β-actin (catalog no. 4967) were obtained from Cell Signaling Technology, Inc., (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG; catalog no. E030120) and goat anti-mouse IgG secondary antibodies (catalog no. E030110) was purchased from Earthox, LLC (Millbrae, CA, USA). Mouse interleukin (IL)-1β, IL-6, IL-4, IL-10 and tumor necrosis factor (TNF)-α ELISA kits were obtained from Shanghai Westang Bio-tech Co., Ltd. (Shanghai, China). Bio-Plex Phosphoprotein Detection Reagent kit (catalog no. 171–304005) were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Culture flask and plates were purchased from Wuxi NEST Biotechnology Co., Ltd. (Wuxi, Jiangsu, China). All the other chemicals used, unless otherwise stated, were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Authentic plant material was purchased from Guo Yi Tang Chinese herbal medicine store (Fujian, China). Identification of the Hedyotis diffusa Willd (HDW) herb was confirmed by Dr Wei Xu (Department of Pharmacology, Fujian University of Traditional Chinese Medicine, Fujian, China). Ethanol extract of HDW (EEHDW) was prepared as previously described (20–22). Briefly, 500 g HDW was extracted with 5,000 ml of 85% ethanol using refluxing method and then filtered. The ethanol solvent was then evaporated on a rotary evaporator. The resultant solution was concentrated to a relative density of 1.05, and the dried powder of EEHDW was obtained using spray desiccation method using a spray dryer.

For in vitro experiments, stock solutions of EEHDW were prepared by dissolving the EEHDW powder in 40% dimethyl sulfoxide (DMSO) to achieve a final concentration of 500 mg/ml. Working concentrations of EEHDW were prepared by diluting the stock solution in culture medium (RPMI-1640 for HCT-8 and HCT-116; DMEM for HT-29 and SW620). The final concentration of DMSO in the medium was <0.2%. For in vivo experiments, working concentrations of EEHDW were prepared by dissolving the EEHDW powder in saline to a concentration of 0.1 g/ml.

Human CRC cell lines, HCT-8, HT-29, HCT-116 and SW620, were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HCT-8 and HCT-116 cells were cultured in RPMI-1640. HT-29 and SW620 cells were cultured in DMEM. All cell media were supplemented with 10% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml streptomycin and cultured at 37°C, with 5% CO₂ in a humidified incubator.

Cell viability was assessed using MTS assay. The cells (10,000/well) were incubated in 96-well plates with culture medium at 37°C for 12 h and then treated with various concentrations of EEHDW (0, 0.5, 1 and 2 mg/ml) for 24 and 48 h; the control cells were treated with DMSO without EEHDW at 37°C for 24 and 48 h. Next, 10 µl MTS was added to each well, and the samples were incubated for 1 h at 37°C. The resulting absorbance was measured at 490 nm using an ELISA reader (BioTek Instruments, Inc., Winooski, VT, USA).

A total of 20-week-old athymic BALB/c nu/nu male mice (initial body weight, 20±2 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and housed under pathogen-free conditions (22°C; 12-h light/dark cycle). Food and water were given ad libitum throughout the experiment. All animal experiments were approved by the Institutional Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine (Fujian, China).

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, the mice were randomly divided into two groups (n=10) and given daily intra-gastric administration with 1 g/kg EEHDW or saline for 6 days per week for a total of 16 days. The diameters of the tumors were measured every second day 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 mice were sacrificed using 100 mg/kg pelltobarbitalum natricum (Sigma-Aldrich; Merck KGaA), and the tumor tissues were removed and fixed in 4% paraformaldehyde (China National Medicines Corporation Ltd., Beijing, China) or stored at −80°C. Blood was collected aseptically from the orbital sinus. Blood collection tubes were allowed to stand at room temperature for 5 h prior to serum collection by centrifugation at 2,000 × g for 20 min at room temperature, and then stored at −80°C.

Tumor tissues were analyzed by IHC as previously described (24). Briefly, following fixation at room temperature with 4% paraformaldehyde for 24 h, the tumor samples were paraffin-embedded using 100% liquid paraffin at 56°C until the paraffin had fully hardened, and sliced into 4 µm-thick sections. The slides were subjected to antigen retrieval and endogenous peroxidase activity was quenched using hydrogen peroxide. IHC staining was performed using the VECTASTAIN Elite ABC kit according to the manufacturer's instructions. Briefly, following blocking of non-specific proteins with normal serum in PBS (0.1% Tween 20), the slides were incubated with Ki-67, Pim-1, Bcl-2, Bax, COX-2, iNOS, eNOS, HIF1-α (all 1:200 diluted in PBS) primary antibodies or PBS (negative control) overnight at 4°C. After washing with PBS, the slides were incubated for 30 min at room temperature with biotinylated secondary antibody (goat anti-rabbit immunoglobulin G; cat. no. E030120) followed by conjugated HRP-labeled streptavidin (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) and then washed with PBS. The slides were then incubated with 3,3′-diaminobenzidine (DAB, Sigma-Aldrich, Merck KGaA) as the chromogen, followed by counterstaining with diluted hematoxylin (Sigma-Aldrich, Merck KGaA) at room temperature for 30 sec. After staining, five high-power fields (magnification, ×400) were randomly selected in each slide and the mean proportion of positive cells in each field were counted using the true color multi-functional cell image analysis management system (version 6.0; Image-Pro Plus; Media Cybernetics, Inc., Rockville, MD, USA).

Apoptosis in tumor tissues were analyzed by TUNEL staining using TumorTACS in situ Apoptosis kit as described previously (22). Briefly, apoptotic cells were examined by light microscopy (DM4000, Leica Microsystems GmbH, Wetzlar, Germany) and counted as DAB-positive cells (brown staining) at five arbitrarily selected microscopic fields (magnification, ×400). TUNEL-positive cells were counted as a percentage of the total cells.

A total of three tumors were randomly selected from the control and the EEHDW group, washed with PBS 3 times and homogenized in Pierce RIPA buffer containing protease inhibitor and phosphatase inhibitor cocktails. The samples were then centrifuged at 17,000 × g for 20 min at 4°C, and the resulting protein concentrations were determined using BCA Protein Assay reagent kit. A total of 50 µg protein for each sample was loaded onto 10% SDS-PAGE and resolved at 20 V for 10 min, at 80 V for 30 min and at 120 V for 1 h. The proteins were then transferred onto nitrocellulose membranes. Following blocking with 5% non-fat milk powder at room temperature for 2 h with the 5% non-fat milk powder dissolved using TBS with Tween-20 (TBST, pH8.0) containing 0.1% Tween, the membranes were incubated with cytochrome C, caspase-3, caspase-9, PARP and β-actin (all 1:1,000) primary antibodies overnight at 4°C, and then incubated with the aforementioned HRP-conjugated anti-rabbit secondary antibodies to bind the antibodies of cytochrome C, caspase-3, PARP and β-actin, or anti-mouse secondary antibody to bind the caspase-9 antibody (all 1:5,000) for 1 h at room temperature. The membranes were then subjected to enhanced chemiluminescence (ECL) detection using SuperSignal^™ West Pico Chemiluminescent substrate. Image Lab^™ software (version 3.0; Bio-Rad Laboratories, Inc.) was used for densitometric analysis.

Blood were collected and stored at −80°C until further analysis. The level of IL-1β, IL-6, IL-4, IL-10 and TNF-α in the serum was measured using IL-1β (cat. no. F10770), IL-6 (cat. no. F10830), IL-4 (cat. no. F10810), IL-10 (cat. no. F10870) and TNF-α (cat. no. F11630) ELISA kits (Xitang Biological Technology Co., Ltd., Shanghai, China), respectively, according to the manufacturer's protocol. Briefly, 100 µl diluted standard and test samples were added in each well. Plates were covered and incubated for 2 h at 37°C, then washed 5 times prior to incubation with 50 µl biotinylated antibody solution for 1 h at 37°C. The Plates were washed three times, and 100 µl streptavidin-HRP solution was distributed to all wells prior to incubation for 1 h at 37°C. After washing, the substrate was added and incubated for 15 min at room temperature in a darkened room. Finally, the reactions were stopped and the absorbance was measured at 450 nm. Wells with no biotinylated antibody solutions or streptavidin-HRP solution were used as the negative controls. The concentrations of the aforementioned cytokines were determined by comparing to serial dilutions of the purified standards.

A total of 8 tumors were randomly selected from the EEHDW and control groups, and homogenized. For analysis of phosphorylation of protein kinase B (AKT), mitogen-activated protein kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK), p38, p53 and p70S6K in vitro, HT-29 cells (2.5×10⁵) were seeded into 25 cm² flasks with 5 ml RPMI-1640 medium and treated with 2 mg/ml EEHDW for 24 h; the control cells were treated with RPMI-1604 with 0.16% DMSO without EEHDW at 37°C for 24 h. To detect signal transducer and activator of transcription 3 (STAT3) phosphorylation in vitro, HT-29 cells were first cultured at 37°C in complete DMEM (10% FBS) until ~70% confluence, and subsequently cultured at 37°C in FBS-free medium overnight. The medium was replaced with DMEM with 10% FBS, and the cells were pre-treated with EEHDW (2 mg/ml) for 1 h at 37°C, followed by stimulation with 10 ng/ml IL-6 for 15 min at 37°C. Tumor tissues and treated cells were lysed using a commercially available lysis kit (Bio-Rad Laboratories) and centrifuged at 17,000 × g for 20 min at 4°C, and the resulting protein concentrations were determined by BCA protein assay. The presence of phosphorylated (p-)AKT, p-Erk1/2, p-JNK, p-p38, p-p53, p-p70S6K and p-STAT3 were detected using a bead-based multiplex assay for phosphoproteins (Bio-Plex Phosphoprotein assay kit; Bio-Rad Laboratories), according to the manufacturer's instructions. Data were collected and analyzed using the Bio-Plex 200 suspension array system (Bio-Rad Laboratories).

All data were expressed as the mean ± standard deviation. Statistical analysis was performed using the SPSS software (version 17.0) for Windows (SPSS, Inc. Chicago, IL, USA) using one-way analysis of variance. Fisher's least significant difference (for equal variances) or Dunnett's correction (for unequal variances) were used in post-hoc tests. P<0.05 was considered to be statistically significant.

The effect of EEHDW on the viability of various CRC cell lines was determined using MTS assay. EEHDW treatment at 0.5, 1 and 2 mg/ml for 24 or 48 h was able to significantly reduce the viability of HT-29, SW620, HCT-8 and HCT-116 cells) in a dose- and time-dependent manner compared with the control (Fig. 1A). The in vivo anti-tumor effect of EEHDW was subsequently determined by comparing the tumor weight and volume between EEHDW-treated and control mice. EEHDW-treated mice exhibited a 34.94% decrease in tumor volume (control, 2,516±245 mm³; EEHDW-treated, 1,637±173 mm³; P<0.0037) and a 44.14% decrease in tumor weight (control, 2.25±0.19 g; EEHDW-treated, m1.25±0.16 g; P<0.0012) compared with the control mice (Fig. 1B). These results suggest that EEHDW was able to suppress colorectal tumor growth in vivo and in vitro.

The in vivo effect of EEHDW on proliferation and apoptosis in mice was determined using Ki-67 and TUNEL staining, as well as western blotting for proteins in the mitochondrial cytochrome c release-mediated caspase cascade. There was a significant decrease in the number of Ki-67-positive cells in the EEHDW-treated group compared with the control group (22.20±2.63% vs. 33.50±4.22%; P<0.05), while the percentage of TUNEL-positive cells were increased in the EEHDW-treated group compared with the control group (35.60±5.34% vs. 21.67±3.47%; Fig. 2A, P<0.05). Additionally, the expression of cytochrome c, caspase-3, caspase-9 and PARP were upregulated in the EEHDW-treated group compared with the control group (P<0.05; Fig. 2B). Taken together, these results demonstrated that EEHDW was able to inhibit proliferation and promote apoptosis in vivo.

To further investigate how EEHDW is able to inhibit tumor growth, IHC staining was performed to detect the expression of Pim-1, Bcl-2 and Bax in mice. The percentage of Pim-1, Bcl-2 or Bax-positive cells in the control group was, 37.71±7.31, 27.33±3.67 and 18.50±3.83% respectively, whereas in EEHDW-treated mice the percentage was 21.75±4.57, 16.00±4.31 and 35.00±4.32%, respectively (P<0.05; Fig. 3). These data suggested that that EEHDW treatment was able to significantly (P<0.02) downregulate the expression of Pim-1, a potential oncogene, while decreasing Bcl-2 expression and increasing Bax expression, demonstrating that EEHDW inhibits cell proliferation and induces apoptosis in xenograft tumors. In addition, the percentage of COX-2, iNOS, eNOS or HIF-1α-positive cells in the control group was 33.00±5.67, 45.33±6.93, 30.33±5.87, and 28.50±4.77%, respectively, whereas in EEHDW-treated mice the percentage was 23.75±4.12, 27.25±4.24, 20.25±3.78 and 17.00±2.86%. These results suggested that EEHDW treatment was also able to downregulate the expression of COX-2, iNOS, eNOS and HIF-1α (P<0.05; Fig. 3), which are novel tumor markers and are hypothesized to have important roles during tumor angiogenesis (26).

Dense infiltration of cytokine-producing immune cells is frequently observed in cancer tissues: Each immune cell subset and cytokine involved in the activation of intracellular pathways sustains the growth of cancer cells (27). The effect of EEHDW treatment on secreting cytokines was detected using ELISA, whereby the levels of IL-1β, IL-6 and TNF-α were significantly (P<0.026) decreased following EEHDW treatment, By contrast, the levels of IL-4 and IL-10 were upregulated following EEHDW treatment compared with the control group (P<0.05; Fig. 4).

To investigate the underlying mechanisms of EEHDW in inhibiting tumor growth, the effect of EEHDW on the activation of various CRC-associated signal transduction cascades was determined. The effect of EEHDW treatment on the activation and phosphorylation of AKT, Erk1/2, JNK, p38, p70S6K p53 and STAT3 in CRC xenograft tumor tissues and HT-29 cells was determined using Bio-Plex Phosphoprotein assay.

Treatment with EEHDW was able to significantly decrease the levels of phosphorylated AKT, Erk1/2, JNK, p38, p70S6K and STAT3 in tumor tissues (P<0.05; Fig. 5A) and HT-29 cells compared with the control (P<0.05; Fig. 5B). By contrast, p-p53 expression was significantly increased following EEHDW treatment (P<0.05; Fig. 5A and B).