Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, Trizol reagent were purchased from Invitrogen (Carlsbad, NM, USA). SuperScript II reverse transcriptase was provided by Promega (Madison, WI, USA). PCNA, Bcl-2, Bax, P21, cyclinD1, CDK4 antibodies, horseradish peroxidase (HRP)-conjugated secondary anti-bodies were obtained from Cell Signaling (Beverly, MA, USA). pcna assay, TUNEL assay kit was purchased from R&D Systems (Minneapolis, MN, USA), VECTASTAIN Elite ABC kit was provided by VECTOR Laboratories (Burlingame, CA, USA). All the other chemicals used, 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 (named EEHDW) was prepared as previously described [21]. Briefly, 500 g of HDW was extracted with 5000 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.

Chromatographic fingerprint was employed as quality control for different batches [23]. Samples were analyzed on an Agilent 1200 HPLC system (Agilent, Santa Clara, CA, USA) using a Sepax Sapphire-C₁₈ column (250 mm × 4.60 mm, 5 μm). The absorbance was measured at 355 nm. The mobile phase consisted of solvent A (methanol) and solvent B (water) at a flow rate of 1 mL/min with an injection volume of 10 μL. The gradient procedure was used as follows: 20–30% A at 0–15 min and 40–60% A at 15–40 min. Based on the fingerprint as shown in Figure 1, we established an optimum and easily controlled procedure for preparing the EEHDW extract as mentioned above. If the peak areas of 1, 2, and 3 are in an optimum ratio of 1:6.4:9.3, all results from the study would readily be reproducible.

Human colon carcinoma HT-29 cells were obtained from 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.

Athymic BALB/c nu/nu male 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 a 12 h light/dark cycle. Food and water were given ad libitum throughout the experiment. 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 were grown in culture and then detached by trypsinization, washed, and resuspended in serum-free DMEM. 1.5 × 10⁶ of cells 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 = 12) and given intra-gastric administration of 6 g/kg of EEHDW or saline daily, 5 days a week for 16 days. Body weight and 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 experiment, the animals were anaesthetized with pelltobarbitalum natricum, and the tumor tissue was removed and weighed.

Tumor samples were fixed in 10% buffered formalin for 12 h, and subsequently processed conventionally for paraffin-embedded tumor sections (4 μm thick). The tumor sections were then deparaffinized and rehydrated via treatment with a series of xylenes and graded alcohols. Endogenous peroxidase activity of the sections was quenched by incubating in water containing 0.3% H₂O₂ for 30 min. Immunohistochemical staining was performed using the VECTASTAIN elite ABC kit according to the manufacturer’s instructions. Briefly, after blocking non-specific proteins with normal serum in PBS (0.1% Tween 20), the sections were treated with PCNA antibody (at a dilution of 1:200) for 1 h. The sections were incubated with a biotinylated anti-rabbit IgG antibody for 30 min and then treated with the ABC reagent for 30 min. They were finally treated with DAB for 10 min. After the sections were washed and air-dried, cover slips were applied to the sections using permount slide mounting medium. PCNA was quantified by counting respective positive cells and total number of cells in five high-power fields (×400) 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).

The 4-μm-thick sections of tumor samples were analyzed by TUNEL staining using TumorTACS in situ Apoptosis kit (R&D Systems, Minneapolis, MN, usa). Apoptotic cells were counted as DAB-positive cells (brown stained) at five arbitrarily selected microscopic fields at a magnification of 400×. TUNEL-positive cells were counted as a percentage of the total cells.

Total RNA was isolated from fresh tumor with Trizol reagent. Oligo(dT)-primed RNA (1 μg) was reverse transcribed with SuperScript II reverse transcriptase according to the manufacturer’s instructions. The obtained cDNA was used to determine the mRNA amount of Bcl-2, Bax, p21, Cyclin D1 and CDK4 by PCR with Taq DNA polymerase (Fermentas). GAPDH was used as an internal control. The primers used for amplification of Bcl-2, Bax, p21, Cyclin D1, CDK4 and GAPDH transcripts are as follows: Bcl-2 forward 5′-CAG CTG CAC CTG ACG CCC TT-3 and reverse 5′-GCC TCC GTT ATC CTG GAT CC-3′; Bax forward 5′-TGC TTC AGG GTT TCA TCC AGG-3′ and reverse 5′-TGG CAA AGT AGA AAA GGG CGA-3′; p21 forward 5′-GCG ACT GTG ATG CGC TAA TGG-3′ and reverse 5′-TAG AAA TCT GTC ATG CTG GTC TGC-3′; Cyclin D1 forward 5′-TGG ATG CTG GAG GTC TGC GAG GAA-3′ and reverse 5′-GGC TTC GAT CTG CTC CTG GCA GGC-3′; CDK4 forward 5′-CAT GTA GAC CAG GAC CTA AGC-3′ and reverse 5′-AAC TGG CGC ATC AGA TCC TAG-3′; GAPDH forward 5′-GT CAT CCA TGA CAA CTT TGG-3′ and reverse 5′-GA GCT TGA CAA AGT GGT CGT-3′.

Three tumors were selected randomly from control or EEHDW group, homogenized in nondenaturing lysis buffer using homogeniser and centrifuged at 15,000× g for 15 min followed by determination of protein concentration in supernatants. Equal protein per lysate was resolved on Tris-glycine gel, transferred onto PVDF membrane, and blocked for 2 h with 5% nonfat dry milk. Membranes were incubated with desired primary antibody Bcl-2, Bax, p21, Cyclin D1, CDK4, and β-actin (at a dilution of 1:1000) overnight at 4 °C and then with appropriate HRP-conjugated secondary antibody followed by enhanced chemiluminescence detection.

Data were presented as mean ± SD for the indicated number of independently performed experiments. The statistical analysis between groups was carried out using the Student’s t test and p < 0.05 was considered to be statistically significant.

The anti-tumor effect of EEHDW in vivo was evaluated by measuring tumor volume and weight in CRC xenograft mice, while its adverse effect was determined by measuring the body weight gain. As shown in Figures 2A,B, administration of EEHDW decreased tumor volume and weight by 52% and 45%, respectively, compared with control. However, EEHDW treatment had no effect on the body weight gain in experimental animals (Figure 2C). These data suggest that EEHDW can inhibit colorectal tumor growth in vivo, without apparent signs of toxicity.

The in vivo effect of EEHDW on cell apoptosis and proliferation was determined by immunohistochemical (IHC) staining for TUNEL and PCNA. Data in Figure 3 showed 22.0 ± 2.59% and 34.2 ± 3.19% TUNEL-positive cells in control and EEHDW-treated mouse groups, and the percentage of PCNA-positive cells in control and EEHDW-treated mice was 37.8 ± 3.08% and 21.5 ± 4.22%, respectively. Taken together, it is demonstrated that EEHDW inhibits the proliferation of colorectal cancer cells and promotes cell apoptosis in vivo.

The activation of STAT3 is mediated by phosphorylation at tyrosine residue 705. We therefore examined the effect of EEHDW on STAT3 phosphoralytion in tumor tissue by Western blot analysis using antibody that recognizes STAT3 phosphorylation at Tyr⁷⁰⁵. As shown in Figure 4, EEHDW inhibited the phosphorylation of STAT3 in CRC mice, whereas the level of non-phosphorylated STAT3 remained unchanged after EEHDW treatment, suggesting that EEHDW significantly suppresses the activation of STAT3 in vivo.

To further explore the mechanism of EEHDW’s pro-apoptotic and anti-proliferative activities, we performed RT-PCR and Western Blot analyses to examine the mRNA and protein expression, respectively, of the pro-proliferative Cyclin D1, CDK4, anti-proliferative p21 and the pro-apoptotic Bax, anti-apoptotic Bcl-2. The results of the RT-PCR assay showed that EEHDW treatment profoundly reduced the mRNA expression of Bcl-2, p21, Cyclin D1, CDK4 but increased that of Bax and p21 in CRC mice (Figures 5A and C). Data from Western blot analysis showed that the pattern of protein expression was similar to the respective mRNA levels (Figures 5 B,D).