Huaier aqueous extract was obtained from Gaitianli Pharmacy Co. (Qidong, China). Dulbecco’s modified Eagle’s medium, nutrient mixture F-12 (DMEM/F12), was purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was supplied by Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China). The anti-β-catenin (1:3,000), anti-cyclin D1 (1:3,000) and anti-β-actin (1:1,000) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The collagenase and hyaluronidase were obtained from Sigma Chemical (Balcatta, WA, Australia).

Primary CRC cells (T1 and T2 cells) were established from patients’ cancer tissues following surgery as described previously (26). In brief, resected CRC tissues were obtained in accordance with the Research Ethics Board on Human Experimentation at the Second Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China) from two patients with informed consent. The histological diagnosis was based on microscopic features of the carcinoma cells. The cancer tissues were intensively washed four times in PBS solution containing antibiotics. Enzymatic digestion was performed using collagenase (1.5 mg/ml) and hyaluronidase (20 mg/ml) in PBS for 1 h. The cancer cells were then used for culturing in DMEM/F12 supplemented with 10% FBS and 1X antibiotic-antimycotic. The cells were finally incubated at 37°C in a 5% CO₂ humidified incubator. Cultures contaminated with fibroblasts were removed and cancer cells were identified in NOD/SCID mice.

The proliferation rates and sensitivity to Huaier extract were assessed by MTS assays using the CellTiter 96 Aqueous MTS kit (Promega, Fitchburg, WI, USA). The colorectal primary cancer cells were seeded in 100 μl medium at a density of 2-5×10⁴ cells per well in 96-well plates (Corning, New York, NY, USA). Following exposure to the Huaier extract for 48 h, the MTS assay was performed according to the manufacturer’s instructions.

CRC cells were plated in 24-chamber culture plates at 10,000 cells per well, allowed to adhere and incubated with the Huaier aqueous extract at 8 mg/ml for 48 h. The morphology of the cells was visualized and photomicrographs were obtained with an Olympus light microscope (×10).

Cells were cultured in DMEM/F12 basal serum-free medium supplemented with 20 ng/ml EGF (Invitrogen), 10 ng/ml bFGF, B27, 100 U/ml penicillin and 100 μg/ml streptomycin. Single cells were prepared by enzymatic dissociation and seeded at low densities (100–200 cells/well) in 96-well low-adhesion plates (Corning). Various concentrations of the Huaier extract were added. After 14 days of culturing, the number and size of spheroids were assessed.

Colorectal CSCs are highly enriched with cells having a high ALDH enzyme activity (27). The Aldefluor assay was performed using an Aldefluor kit (Stemcell Technologies, Durham, NC, USA). Single cells obtained from cell cultures were incubated in an Aldefluor assay buffer containing an ALDH substrate for 40 min at 37°C according to the manufacturer’s instructions. A fraction of the cells was incubated with the ALDH inhibitor diethylaminobenzaldehyde under the same conditions as a negative control. The cells were analyzed with a FACSCalibur instrument (Becton Dickinson, Franklin Lakes, NJ, USA).

The cells were treated with Huaier aqueous extract (2 or 4 mg/ml) for 48 h. At the end of the incubation period, the cells were harvested and lysed using the radioimmunoprecipitation assay buffer [20 mmol/l Tris-HCl, 150 mmol/l NaCl, 1% NP-40, 5 mmol/l EDTA and 1 mmol/l Na₃VO₄ (pH 7.5)] supplemented with a protease inhibitor cocktail. Proteins were detected by western blot analysis using anti-β-catenin, anti-cyclin D1 and anti-β-actin. Goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) was used as the secondary antibody. Immunoreactive bands were detected using a chemiluminescent substrate.

To evaluate the TCF/LEF transcriptional activity induced by activated β-catenin, TOPflash and the negative control FOPflash, a pair of luciferase reporter constructs, were used. The reporter system was a gift from Dr Yongliang Zhu (Zhejiang University School of Medicine). In brief, the cells were transiently infected with either TOPflash reporter plasmids (10 μg/100 μl) or FOPflash plasmids (10 μg/100 μl) together with Renilla-tk plasmids (1 μg/100 μl; encoding Renilla luciferase). After 24 h, various concentrations of the Huaier extract were added and the cells were continually incubated in the same medium for 24 h. Cells were washed with PBS and the luciferase activity was measured with the dual-luciferase reporter assay system (Promega).

The statistical analysis was performed with GraphPad Prism 5.0 software and statistical differences were determined by one-way ANOVA. The data were expressed as the mean ± standard deviation and all experiments were performed in triplicate. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the biological activity of Huaier aqueous extract on cancer cells, cell viability was measured using the MTS assay following treatment with various concentrations of the extract. Cell survival decreased with increasing concentrations of the Huaier extract and the IC₅₀ was determined to be ∼8 mg/ml for T1 and T2 cells (colorectal primary cells; Fig. 1A). The results showed the Huaier extract to be an antiproliferative agent against CRC cells.

In addition to the antiproliferative effects, the morphological effects of the Huaier extract on CRC cells were also investigated. The control cells were epithelial cells with radial spread and large cell size, indicating typical epithelial morphology (Fig. 1B). Following the addition of the Huaier extract, the cell morphology was markedly altered. In the majority of the Huaier-treated colorectal cells, the membranes shrank and the cells became irregularly shaped or round with no visible radial spread. These alterations were accompanied by a decrease in cell number.

To evaluate its direct effects on the formation of spheroids, spheroidal growing cells were treated with Huaier aqueous extract. CRC cells were exposed to varying concentrations of the Huaier extract and then cultured for 14 days. The Huaier extract inhibited spheroid formation by the cancer cells (Fig. 2). Not only did the number of spheroids decline significantly, but also the size of the spheroids was reduced (P<0.05). It was noteworthy that the concentrations of Huaier capable of suppressing spheroid formation were ∼15-fold lower than those exhibiting antiproliferative effects in the MTS assay (IC₅₀, ∼8 mg/ml for T1 and T2 cells).

ALDH1 activity, a marker associated with CSCs, was further tested by the Aldefluor assay. Cell populations with high ALDH activity have been demonstrated to enrich CSCs. Long-term treatment with Huaier extract for seven days led to a selective decrease in the ALDH-positive populations of the CRCs (Fig. 3; P<0.05). This finding demonstrated that the Huaier extract was able to eliminate CSCs in vitro. A notable observation was that the Huaier extract was able to kill CSCs at concentrations (0.25–0.5 mg/ml) that hardly affected the bulk cancer cells, suggesting that it may preferentially target CSCs compared with the bulk cancer cells.

The Wnt/β-catenin pathway is a key pathway in CSC self-renewal that is activated via β-catenin and results in the phosphorylation of numerous downstream molecules. Whether the Huaier extract altered the Wnt/β-catenin pathway in human CRC cells was studied. The β-catenin protein was constitutively expressed in cancer cells. Treating the CRC cells with Huaier led to dose-dependent downregulation of the levels of total β-catenin protein and also decreased the expression of cyclin D1 (Fig. 4A), one of the Wnt/β-catenin target genes. To further investigate whether the downregulation of β-catenin reduced the activity of downstream molecules, a TOP/FOP flash reporter system was used. Activation of TCF/LEF in the nucleus mediated by β-catenin was detected and quantified with a luciferase assay system. A decrease in the TOP levels was observed in the CRC cells treated with Huaier extract (Fig. 4B; P<0.05). These data indicate that the downregulation of the Wnt/β-catenin self-renewal pathway may be a potential target of the Huaier extract.