Dulbecco's modified Eagle's medium (DMEM), DMEM/F12, fetal bovine serum (FBS), penicillin, streptomycin, 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), 50X B27 supplement, Pierce radioimmunoprecipitation assay Buffer, Pierce bicinchoninic acid (BCA) Protein assay kit, SuperSignal™ West Pico Chemiluminescent Substrate and DreamTaq Green PCR Master mix (2X) were all purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were obtained from PeproTech (Rocky Hill, NJ, USA). Hoechst 33342 and verapamil were purchased from Sigma Chemicals (St. Louis, MO, USA). Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5; catalog. no. ab75732) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; catalog. no. ab181602) rabbit polyclonal antibodies were purchased from Abcam (Cambridge, UK). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (catalog. no. E030120-01) was purchased from Earthox (Millbrae, CA, USA). RNAiso Plus reagent and PrimeScript™ Reverse Transcription (RT) Reagent kit were purchased from Takara Bio, Inc. (Dalian, Liaoning, China). Water-soluble tetrazolium salts (WST)-1 assay kit and Blocking buffer were purchased for Beyotime Institute of Biotechnology (Shanghai, China).

EEHDW was prepared as previously described (16). Stock solutions of EEHDW were prepared by dissolving the EEHDW powder in a concentration of 40% dimethyl sulfoxide (DMSO; catalog. no. 67-68-5; Amresco, Solon, OH, USA) to achieve a final concentration of 400 mg/ml. Working concentrations of EEHDW were created by diluting the stock solution in culture medium (DMEM for HT-29 cells or DMEM/F12 for SP cells). The final concentration of DMSO in the culture medium was <0.5%.

Human CRC HT-29 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in DMEM containing 10% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml streptomycin in a 37°C humidified incubator with an atmosphere of 5% CO₂.

The SP assay is based on the efflux of Hoechst dye from cells via the ATP-binding cassette (ABC) family of transporter proteins expressed within the cell membrane. In the two-dimensional flow analysis chart, the cells are located on the side of a main cell population in a comet-like distribution; these cells are termed the ‘side population’. The verapamil control is an ABC transporter inhibitor. The SP from HT-29 cells was isolated and analyzed using MoFloTM XDP cell sorter flow cytometry (Beckman Coulter, Inc., Brea, CA, USA) as described previously (17). Briefly, the HT-29 cells were digested with 0.25% trypsin-EDTA and re-suspended in DMEM (supplemented with 2% FBS) at a concentration of 2.5×106 cells/ml. Fresh Hoechst 33342 (10 µg/ml final concentration) was added for 30 min at 37°C in a rotary shaker. As a control, certain cells were incubated with Hoechst 33342 in the presence of 50 µM verapamil. At the end of incubation, the cells were washed and re-suspended in cold phosphate-buffered saline (PBS), then 1 mg/ml propidium iodide was added, and the cells were kept at 4°C in the dark. Excitation of Hoechst dye was performed with an ultraviolet laser (catalog. no. CY-355-100; JDSU, Milpitas, CA, USA) at 355 nm, and the fluorescence was measured using a 450±25 nm filter (Hoechst blue) and a 620±15-nm filter (Hoechst red). Sorted SP cells were cultured in serum-free stem cell DMEM/F12 culture medium, containing B27 supplement (1X), 20 ng/ml EGF and 20 ng/ml bFGF.

Isolated HT-29 SP cells were seeded at a density of 2×10⁵ cells/well into 6-well plates (NEST Biotechnology Co., Ltd., Wuxi, Jiangsu, China) in 2 ml DMEM culture medium. Following treatment with various concentrations of EEHDW (0, 0.5, 1 and 2 mg/ml) for 24 h at 37°C, cells were digested with 0.25% trypsin-EDTA and seeded at a density of 1.0×10³ cells/well into Costar^® 6-well Ultra-Low attachment plates (Corning, Inc., Corning, NY, USA), and cultured in DMEM/F12 serum-free stem cell culture medium. The medium was replaced every 2 days. Following 15 days of incubation, cells were collected and transferred into a new well in a 96-well plate (catalog. no. 205512; BD Diagnostics, Sparks Glencoe, MD, USA); and images were captured using a BD Pathway™ 855 at magnification, ×100 (BD Biosciences, Franklin Lakes, NJ, USA). A spheroid with >50 cells inside was considered to be a full sphere.

Cell viability was analyzed by WST-1 assay. Sorted SP cells were seeded at a density of 2.0×10⁴ cells/well into 96-well plates, and incubated with serum-free stem cell culture medium for a total of 48 h at 37°C. Subsequently, cells were treated with various concentrations of EEHDW (0, 0.5, 1 and 2 mg/ml) for 24 h at 37°C. At the conclusion of the treatment period, 10 µl WST-1 were added to each well, and the samples were incubated for an additional 2 h at 37°C. The absorbance was measured at 450 nm using a microplate reader (ELx800 Absorbance Reader; BioTek Instruments, Inc., Winooski, VT, USA).

The sorted SP cells were seeded into 96-well plates at a density of 2.0×10⁴ cells/well in 0.1 ml serum-free stem cell culture medium. The cells were treated with various concentrations of EEHDW (0, 0.5, 1 and 2 mg/ml) for 24 h at 37°C. Cell morphology was observed using a phase-contrast microscope (DMIL/DFC295; Leica Microsystems GmbH, Wetzlar, Germany). Images were captured at ×200 magnification.

HT-29 cells were seeded at a density of 2.5×10⁵ cells/well into 6-well plates in 2 ml DMEM medium, and were treated with various concentrations of EEHDW (0, 0.5, 1 and 2 mg/ml) for a total of 24 h at 37°C. The treated cells were washed with PBS and scraped off into a tube, then lysed using lysis buffer containing protease and phosphatase inhibitor cocktails on ice for 15 min. Following high-speed centrifugation (12,000 × g) for 20 min at 4°C, supernatant containing the sample proteins was collected. The concentration of proteins was determined using the BCA Protein Assay Reagent kit. A total of 50 µg of protein was resolved using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The membranes were blocked with blocking buffer and probed with primary antibodies against Lgr5 or GAPDH (1:1,000) overnight at 4°C, and subsequently with the appropriate HRP-conjugated goat anti-rabbit secondary antibody (1:5,000) for 1 h at room temperature. The chemiluminescence signals were visualized using the SuperSignal™ West Pico Chemiluminescent Substrate. Image Lab™ Software, version 3.0, was used for densitometric analysis/quantification of the western blotting (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Total RNA from the HT-29 cells was isolated with RNAiso Plus reagent. Oligo (dT)-primed RNA (1 µg) was reverse transcribed using the PrimeScript RT Reagent kit according to the manufacturer's protocol. Briefly, the gDNA Eraser (1 µl) contained in the kit was used to remove the genomic DNA following incubation with the total RNA for 2 min at 42°C, then the PrimeScript RT Enzyme mix and RT Primer mix were added to perform RT using incubation for 15 min at 37°C. The obtained complementary DNA was used to determine the messenger (m)RNA quantity of c-Myc, β-catenin, PCNA, survivin and ABCB1 by PCR, using the DreamTaq Green PCR Master mix (2X). PCR was performed by the 3-step method, with a denaturation stage at 95°C for 30 sec, an annealing stage at an appropriate temperature (55°C for c-Myc, β-catenin and survivin, and 58°C for PCNA, ABCB1 and GAPDH) for 30 sec and an extension stage at 60°C for 30 sec for 30 cycles. GAPDH was used as an internal control. The primer were synthesized by Invitrogen, Thermo Fisher Scientific Inc., (Waltham, MA, USA) and the sequences used in the RT-PCR are listed in Table I. A negative control with no DNA and an RT control with no reverse transcription were used as the experimental controls. The PCR was repeated in three independent experiements. A Bio-Rad S1000 Thermal Cycler was used to perform the experiment, and Image Lab™ Software, version 3.0, was used for quantification/densitometric analysis (both Bio-Rad Laboratories Inc.).

Data were analyzed using SPSS for Windows (version 17.0; SPSS, Inc. Chicago, IL, USA). Statistical analysis of the data was performed with Student's t-test and analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

The effect of EEHDW on cancer stem cells was determined by examining the SP proportion in HT-29 cells. As demonstrated in Fig. 1, similar to verapamil, a multi-drug transporter inhibitor that is typically used as a positive control in SP analysis, EEHDW significantly reduced the percentage of SP in HT-29 cells in a dose-dependent manner compared with the control (P<0.05). In order to confirm these results, the expression of Lgr5, which is considered to be a bio-marker of CSCs, was detected. As shown in Fig. 2, EEHDW significantly and dose-dependently downregulated Lgr5 protein expression compared with the control (P<0.05).

In order to investigate EEHDW's effect on CSC growth, the present study evaluated the sphere formation of isolated HT-29 SP cells. As shown in Fig. 3A and B, EEHDW dose-dependently suppressed SP sphere formation compared with the control (P<0.05). In order to verify the growth-suppressive activity of EEHDW in CSCs, the viability of isolated HT-29 SP cells was determined. As demonstrated in Fig. 3C, EEHDW treatment significantly reduced SP viability in a dose-dependent manner compared with the control (P<0.05). The growth inhibition ability of EEHDW was confirmed by its effect on SP cell morphology via phase-contrast microscopy, as cell morphology in culture is indicative of the healthy status of the cells. As shown in Fig. 4, following incubation with various concentrations of EEHDW (0, 0.5, 1 and 2 mg/ml) for 24 h the majority of the cells became shrunken and less confluent. Taken together, these data demonstrate that EEHDW inhibits the growth of isolated HT-29 SP cells.

In order to elucidate the underlying mechanism of the anti-CSC activity of EEHDW, the present study examined the expression of ABCB1, β-catenin, c-Myc, PCNA and survivin in isolated HT-29 SP cells. As demonstrated in Fig. 5, EEHDW treatment markedly reduced the mRNA levels of the aforementioned genes in the SP cells compared with the control (P<0.05).