RPMI-1640 medium (cat. no. C11875500BT), fetal bovine serum (FBS; cat. no. 10099-141), penicillin-streptomycin (cat. no. SV30010), 0.25% trypsin-EDTA (cat. no. 25200-072), Pierce radioimmunoprecipitation assay buffer (cat. no. 89901), Pierce BCA Protein Assay kit (cat. no. 23227) and SuperSignal™ West Pico Chemiluminescent Substrate (cat. no. 34080) were all purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). MTT was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The BD BioCoat Matrigel Invasion Chamber was purchased from BD Biosciences (San Jose, CA, USA). The PrimeScript RT Reagent kit was provided by Takara Biotechnology Co., Ltd. (Dalian, China). TRIzol reagent was obtained from Thermo Fisher Scientific, Inc. Anti-neural (N)-cadherin (cat. no. ab98952) and epithelial (E)-cadherin (cat. no. ab128804) antibodies were purchased from Abcam (Cambridge, UK). Anti-TGF-β (cat. no. 3711), Mothers against decapentaplegic homolog 4 (SMAD4; cat. no. 3716) and β-actin (cat. no. 4967) antibodies were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (cat. no. E030120) was purchased from EarthOx Life Science (Millbrae, CA, USA).

EEHDW was prepared as described previously (25). Stock solutions of EEHDW were prepared by dissolving the EEHDW powder in 100% dimethyl sulfoxide (DMSO) to a final concentration of 500 mg/ml and stored at −20°C. The working concentrations of EEHDW were made by diluting the stock solution in the culture medium. The final concentrations of DMSO in the medium were <0.5%.

The human colorectal 5-FU resistant cell line HCT-8/5-FU and its parental cell line HCT-8 were obtained from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Cells were maintained in RPMI-1640 medium containing 10% (v/v) FBS, 100 U/ml penicillin and 100 g/ml streptomycin, while the HCT/5-FU cells were cultured with an additional 15 g/ml 5-FU, at 37°C in a humidified atmosphere containing 5% CO₂.

Cell viability was assessed by MTT assay. HCT-8, HCT-8/5-FU or HCT-8 cells were seeded into 96-well plates at a density of 1×10⁴ cells/well in 0.1 ml media and were treated with various concentrations of 5-FU (0, 25, 50, 100, 200, 400, 800 and 1600 mM) for 48 h. HCT-8/5-FU cells were seeded into 96-well plates at a density of 8×10³ cells/well in 0.1 ml medium. Cells were treated with various concentrations (0, 0.5, 1 and 2 mg/ml) of EEHDW for different periods of time. A total of 100 ml MTT (0.5 mg/ml in PBS) was added to each well and the samples were incubated for an additional 4 h at 37°C. The purple-blue MTT formazan precipitate was dissolved in 100 µl DMSO. The absorbance was measured at 570 nm using an ELISA reader (ELX800; BioTek Instruments, Inc., Winooski, VT, USA). The resistance index (RI) of the HCT-8/5-FU cells to 5-FU was calculated by dividing the drug concentration required to inhibit growth by 50% (IC₅₀) for HCT-8/5-FU cells by the IC₅₀ value for the parental cells (HCT-8). IC₅₀ values were determined using nonlinear regression analysis.

HCT-8/5-FU cells were seeded into 6-well plates at a density of 5×10⁵ cells/well in 2 ml medium. After 24 h of incubation, cells were scratched vertically in each well using a P200 pipette tip. A phase-contrast inverted microscope at a magnification of ×100 was used to observe three randomly-selected fields of view along the scraped line and images of each well were captured. Cells were then treated with indicated concentrations (0, 0.5, 1 and 2 mg/ml) of EEHDW for 24 h, and another set of images were captured by the same method. A reduction in the width of the scratch indicates a sign of migration.

The migration assay assay was performed using Transwell cell culture chambers, and the invasion assay was performed using Transwell cell culture chambers coated with Matrigel (BD Biosciences). The inserts were placed within a 24-well chamber containing 0.7 ml RPMI-1640 with 10% FBS as a chemoattractant. A total of 2.5×10⁵ cells were seeded into 6-well plates per well and were treated with different concentrations (0, 0.5, 1 and 2 mg/ml) of EEHDW for 24 h. Cells (5×10⁴ cells) were seeded into the inserts suspended in 0.2 ml serum-free RPMI-1640 medium. Cells were incubated at 37°C with 5% CO₂ for 12 or 24 h for the migration and invasion assays, respectively. The upper surface of the filter was scraped to remove non-migratory cells. Migratory and invasive cells were fixed with ice-cold 4% paraformaldehyde for 10 min and stained with crystal violet at room temperature for 15 min. For quantification, the average number of migratory or invasive cells/field was assessed by counting five random fields under a phase-contrast microscope (FMIL/DFC295; Leica Microsystems GmbH, Wetzlar, Germany) at a magnification of ×200.

HCT-8/5-FU cells were seeded into 6-well plates at a density of 2×10⁵ cells/well in 2 ml medium and were treated with different concentrations (0, 0.5, 1 and 2 mg/ml) of EEHDW for 24 h. Cells were digested and suspended in RPMI-1640 medium. Cells were seeded in 6-well plates at a density of 2×10⁴ cells/well and incubated for 2 h. The supernatant was discarded, and the cells were washed two times with PBS. Adhered cells were stained with 0.1% crystal violet at room temperature for 15 min. The adhered cells were counted under a phase-contrast microscope at a magnification of ×200.

HCT-8/5-FU cells were seeded into 6-well plates in 2 ml medium and were treated with indicated concentrations of EEHDW for 24 h. Total RNA was isolated with TRIzol reagent. Oligo (dT)-primed RNA (1 µg) was reverse-transcribed using the PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. The cDNA was used to determine the mRNA levels of TGF-β, SMAD4, E-cadherin and N-cadherin using sqPCR with PCR kit (Master mix; Applied Biosystems, Thermo Fisher Scientific, Inc.). GAPDH was used as an internal control. The RT-sqPCR conditions were performed for 30 cycles as follows: Denaturation at 94°C for 40 sec, annealing at 60°C for 40 sec and extension at 72°C for 45 sec. The following primers were used for the amplification of transcripts: TGF-β forward, 5′-ACCCACAACGAAATCTATGACA-3′ and reverse, 5′-CTAAGGCGAAAGCCCTCAAT-3′; SMAD4 forward, 5′-GATTTGCGTCAGTGTCATCG-3′ and reverse, 5′-AGTCTAAAGGTTGTGGGTCTG-3′; E-cadherin forward, 5′-CTACAATGCCGCATCGCTT-3′ and reverse, 5′-GTATACGTAGGGAAACTCTCTCGGTC-3′; N-cadherin forward, 5′-AAGAACGCCAGGCCAAACAAC-3′ and reverse, 5′-CTGGCTCAAGTCATAGTCCTGGTCT-3′; and GAPDH forward, 5′-GTCATCCATGACAACTTTGG-3′ and reverse, 5′-GAGCTTGACAAAGTGGTCGT-3′. The PCR was repeated in 3 independent times. A Thermal Cycler (Bio-Rad S1000; Hercules, CA, USA) was used to perform the experiment. Samples were analyzed by 1.5% agarose gel electrophoresis and the DNA bands were examined using a gel documentation system (Gel Doc XR+; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

HCT-8/5-FU cells were seeded into 25 cm² flasks at a density of 2.5×10⁵ cells/ml in 5 ml medium. Cells were treated with the indicated concentrations of EEHDW for 24 h. The treated cells were lyzed with radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitor cocktails. Total protein concentrations were determined by BCA assay. Equal amounts of total protein (50 µg) were resolved via SDS-PAGE on a 10% gel and electroblotted onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk at room temperature for 2 h, and probed with primary antibodies TGF-β (1:1,000 dilution), SMAD4 (1:1,000 dilution), E-cadherin (1:1,000 dilution), N-cadherin (1:1,000 dilution), and β-actin (1:1,000 dilution) overnight at 4°C. Membranes were subsequently incubated with the HRP-conjugated secondary antibody (1:2,000 dilution) at room temperature for 1 h and followed by enhanced chemiluminescence detection using SuperSignal West Pico Chemiluminescent Substrate. Image Lab™ software (version 3.0; Bio-Rad Laboratories, Inc.) was used for densitometric analysis and quantification of western blots.

All data are presented as the mean of three repeats and were analyzed using the SPSS package for Windows (version 22.0; IBM Corp., Armonk, NY, USA). Statistical analysis of the data was performed using the Student's t-test and one-way analysis of variance, followed by Dunnett's and the Least Significant Difference post hoc tests, as appropriate. Differences with P<0.05 was considered to indicate a statistically significant difference.

To verify the 5-FU resistance profiles of the CRC cell lines, MTT assays were used to detect the cell viability and the resistance index (RI) was used to evaluate the degree of resistance. HCT-8 and HCT-8/5-FU cells were exposed to different concentrations of 5-FU for 48 h. As shown in Fig. 1, the results demonstrated that the viability of the HCT-8 cells was significantly decreased following treatment with ≥25 µM 5-FU compared with the untreated cells, whereas the viability of the HCT-8/5-FU cells was significantly decreased following treatment with ≥800 µM 5-FU. The half-maximal inhibitory concentration of 5-FU was 119.48 mM in HCT-8 cells and 2.803 mM in HCT-8/5-FU cells, and the RI for 5-FU was 23.45 (>1.5) (data not shown). These results indicated that the HCT-8/5-FU cells used in the present study can be used as a 5-FU resistance model.

The effect of EEHDW on the viability of HCT-8/5-FU cells was determined by MTT assay. As demonstrated in Fig. 2, the cell viability was decreased in response to different concentrations (0.5, 1.0 and 2.0 mg/ml) of EEHDW for 12, 24 and 48 h. The results demonstrated that treatment with EEHDW resulted in a time- and dose-dependent inhibitory effect in HCT-8/5-FU cells.

The effect of EEHDW on the migration of HCT-8/5-FU cells was determined using a wound healing assay. As demonstrated in Fig. 3, 24 h following the introduction of a wound, the untreated HCT-8/5-FU cells migrated into the clear area, whereas treatment with EEHDW inhibited the migration of HCT-8/5-FU cells in a dose-dependent manner. In order to investigate further, Transwell assays were performed to determine the effects of EEHDW on the migration and invasion of HCT-8/5-FU cells. As demonstrated in Fig. 4, following treatment with different concentrations of EEHDW, the number of migratory and invasive cells decreased in a dose-dependent manner. These results suggested that EEHDW can inhibit metastasis in HCT-8/5-FU cells.

The effect of EEHDW on adhesion in HCT-8/5-FU cells was determined using the adhesion assay. As demonstrated in Fig. 5, following treatment with different concentration of EEHDW, compared with the control, the adhesive ability of the HCT-8/5-FU cells was attenuated.

To further study the mechanism of EEHDW's antimetastatic effect, the mRNA and protein expression of TGF-β pathway-associated factors in HCT-8/5-FU cells was determined using RT-sqPCR and western blotting, respectively. As demonstrated in Fig. 6, treatment with EEHDW downregulated the expression of mRNA and protein levels of TGF-β, SMAD4 and N-cadherin, and upregulated the mRNA and protein levels of E-cadherin, in a dose-dependent manner, suggesting that EEHDW may inhibit the metastasis of HCT-8/5-FU cells through the suppression of the TGF-β signaling pathway.