The HCT116 and SW480 human colon cancer cell lines were purchased from the American Type Culture Collection and maintained in high glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 100 U/ml streptomycin/penicillin (all purchased from Gibco; Thermo Fisher Scientific, Inc.), at 37°C in 5% CO₂.

Fn (25586) was purchased from the American Type Culture Collection and cultured at Wuhan Research Institute of First Light Industry (Wuhan, China). The methods of bacterial pellets and conditioned medium were performed, as previously described (13). The following groups were classified: Control group, Fn group (supernatant of Fn was added to the cells) and 0.5% fucose+Fn (Fnf) group. Fucose (Sigma-Aldrich; Merck KGaA) was added when Fn was cultured and the supernatant was subsequently added to the cells. In total, 100 nM S3I-201 (Selleck Chemicals), an inhibitor of stat3, was incubated for 1 h prior to bacteria treatment to access the role of stat3 pathway.

The CCK-8 assay was performed to assess cell proliferation. HCT116 and SW480 cells were seeded into a 96-well plate at a density of 1×10⁴ with 100 µl conditioned medium and allowed to adhere overnight. Following treatment with Fn or Fnf for 12 h at 37°C, the culture medium was removed and 10 µl CCK-8 solution (Beyotime Institute of Biotechnology) along with 90 µl medium was added to each well. Following incubation for 1 h at 37°C, cell proliferation was analyzed at a wavelength of 450 nm, using a microplate reader (Biotek Instruments, Inc.).

The colon cancer cells were stimulated with Fn or Fnf for 12 h at 37°C and subsequently seeded into 6-well plates at a density of 500 cells/well. Cells were cultured for 14 days at 37°C in 5% CO₂. Following incubation, cells were washed with PBS for three times, fixed with 4% paraformaldehyde for 30 min and stained with 0.5% crystal violet for 15 min both at room temperature. Cell colonies were counted using ImageJ1 software (National Institutes of Health).

Cells from each group were respectively seeded into 6-well plates at a density of 5×10⁵ with serum-free DMEM medium and incubated overnight at 37°C. Cell monolayers were scratched using a sterile pipette tip. Cells were washed with PBS three times to remove the cellular debris, and cell migration was subsequently analyzed after 24 h, using ImageJ1 software (National Institutes of Health).

For the cell migration assay, cells from each group were resuspended with serum-free DMEM medium and plated in the upper chambers of 24-well Transwell plates at a density of 1×10⁵. DMEM medium supplemented with 10% FBS was plated in the lower chambers. Following incubation for 24 h at 37°C, the migratory cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.5% crystal violet for 15 min both at room temperature. Cells were observed under a light microscope (magnification ×200; Olympus Corporation). For the cell invasion assay, Transwell membranes were precoated with Matrigel at 37°C for 4 h.

Total protein was extracted from cells or tissues using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with phenylmethyl sulfonyl fluoride protease inhibitor (Beyotime Institute of Biotechnology) and phosphatase inhibitor (Beyotime Institute of Biotechnology). Total protein was quantified using the BCA assay (Thermo Fisher Scientific, Inc.) and equal amounts of protein (40 µg/lane) were separated by 10% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes (EMD Millipore) and blocked with 5% BSA at room temperature for 1 h. The membranes were incubated with primary antibodies (all 1:1,000 dilution) against: GAPDH (ABclonal Biotech Co., Ltd.; cat. no. AC001), ACTB (ABcloanl Biotech Co., Ltd.; cat. no. AC006), phospho-stat3 (Tyr705; Cell Signaling Technology, Inc.; cat. no. 9145S), stat3 (ABclonal Biotech Co., Ltd.; cat. no. A11216), phosphor-jak2 (Tyr1007/1008; ABclonal Biotech Co., Ltd.; cat. no. AP0531), jak2 (ABclonal Biotech Co., Ltd.; cat. no. A7694), β-catenin (Cell Signaling Technology, Inc.; cat. no. 8480S), E-cadherin (Cell Signaling Technology, Inc.; cat. no. 3195S), N-cadherin (GeneTex, Inc.; cat. no. GTX127345) and Vimentin (Cell Signaling Technology, Inc.; cat. no. 5741S) overnight at 4°C. Following the primary incubation, membranes were incubated with HRP-labelled secondary antibodies (AntGene; cat. no. ANT020; 1:2,000) at room temperature for 1 h. Protein bands were visualized using enhanced chemiluminescent reagents (Beyotime Institute of Biotechnology).

Statistical analysis was performed using SPSS 25.0 software (IBM Corp.) and GraphPad Prism 7.0 software (GraphPad Software, Inc.). All experiments were performed in triplicate and data are presented as the mean ± standard error of the mean. One-way analysis of variance and Tukey's post hoc test were used to compare differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether there were differences between the impacts that Fn and Fnf exerted on colon cancer cells, the CCK-8 assay was performed on HCT116 and SW480 cells following treatment for 12 h. The results demonstrated that cells treated with Fn proliferated faster compared with the control group (HCT116 control, 0.42±0.01; Fn, 0.60±0.02, P<0.0001; SW480 control, 0.36±0.02; Fn, 0.42±0.01, P=0.005; Fig. 1A). However, following treatment with fucose, Fn exerted a weaker pro-proliferative ability (HCT116 Fn, 0.60±0.02; Fnf, 0.54±0.01, P=0.014; SW480 Fn, 0.42±0.01; Fnf, 0.38±0.01, P=0.009; Fig. 1A). The colony formation assay was subsequently performed to assess the long-term effect on cell proliferation. The results demonstrated that the number of HCT116 colonies treated with Fn increased (control, 52.0±7.0; Fn, 125.3±11.3; P=0.005), while less colonies were observed in the Fnf group (Fn, 125.3±11.3; Fnf, 81.0±9.9, P=0.042; Fig. 1B and C). Similar results were observed in SW480 cells (control, 96±10; Fn, 170±5; Fnf, 131.5±6.5; P=0.022 and P=0.043; Fig. 1B and C). Collectively, these results suggest that L-fucose may ameliorate the pre-proliferative ability of Fn on colon cancer cells.

The migratory ability of colon cancer cells is a functional characteristic to assess aggressiveness (14). The Transwell migration assay was performed to assess the effect of Fn and Fnf on the migratory ability of HCT116 and SW480 cells. The results demonstrated that treatment with Fn significantly enhanced the migratory ability of HCT116 and SW480 cells (HCT116 control, 119.30±3.84; Fn, 148.30±4.41, P=0.008; SW480 control, 123.00±4.93; Fn, 216.50±3.30; P<0.0001; Fig. 2A), while the migratory ability decreased in cells treated with Fnf (HCT116 Fn, 148.30±4.41; Fnf, 128.80±2.29, P=0.008; SW480 Fn, 1216.50±3.30; Fnf, 157.70±2.96, P=0.002; Fig. 2A).

The results of the wound healing assay demonstrated that treatment with Fn increased the migratory rate in HCT116 cells compared with both the control cells and Fnf treated cells (control, 0.152±0.006; Fn, 0.313±0.020; Fnf, 0.221±0.011; P=0.002 and P=0.018; Fig. 2B). Similar trends were observed in SW480 cells (control, 0.190±0.011; Fn, 0.410±0.016; Fnf, 0.259±0.029; P<0.0001 and P=0.004; Fig. 2C). Taken together, these results suggest that L-fucose may inhibit the pro-migratory ability of Fn on colon cancer cells.

The Transwell invasive assay was performed to assess the effect of Fn and Fnf on the invasive ability of HCT116 and SW480 cells. The number of HCT116 and SW480 cells that passed through the membrane was significantly higher in the Fn group compared with the control group (HCT116 control, 111.50±15.16; Fn, 234.00±14.70, P=0.001; Fig. 3A and B; SW480 control, 500.00±44.77; Fn, 857.80±50.50, P=0.002; Fig. 3C and D). As expected, the number of cells in the Fnf group significantly decreased compared with the Fn group (HCT116 Fn, 234.00±14.70; Fnf, 137.30±27.53, P=0.021; Fig. 3A and B; SW480 Fn, 857.80±50.50; Fnf, 676.00±51.14, P=0.042; Fig. 3C and D). Collectively, these results suggest that L-fucose may ameliorate the pro-invasive ability of Fn on colon cancer cells.

The effect of fucose on Fn potentiation on activation of the pathway that promotes CRC progression was assessed in the present study. Stat3 plays a crucial role in tumorigenesis, and progression and invasion of cancer cells (15). Thus, the expression and phosphorylation of stat3 were detected via western blot analysis. As presented in Fig. 4, the protein levels of p-stat3 and p-jak2 significantly increased following treatment with Fn (for p-stat3/stat3, HCT116 control, 1.000±0.020; Fn, 1.851±0.063; P=0.002; SW480 control, 1.000±0.006; Fn, 1.759±0.075; P=0.004. For p-jak2/jak2, HCT116 control, 1.000±0.127; Fn, 2.272±0.098; P=0.016; SW480 control, 1.000±0.052; Fn, 1.400±0.057; P=0.002). However, the protein levels significantly decreased following treatment with fucose (for p-stat3/stat3, HCT116 Fn, 1.851±0.063; Fnf, 1.490±0.095; P=0.044; SW480 Fn, 1.759±0.075; Fnf, 1.318±0.031; P=0.005. For p-jak2/jak2, HCT116 Fn, 2.272±0.098; Fnf, 1.327±0.157; P=0.036; SW480 Fn, 1.400±0.057; Fnf, 1.082±0.108; P=0.040).

Stat3 activation is upstream of epithelial-to-mesenchymal transition (EMT) in CRC, which is associated with tumor progression (16). The present study assessed the specific protein markers of EMT, which exist in different types of tumors, such as prostate and colon cancer, and contribute to tumor metastasis (17). As presented in Fig. 5A and B, the expression levels of N-cadherin, β-catenin and vimentin were higher in HCT116 cells treated with Fn compared with the control group (N-cadherin control, 1.00±0.01; Fn, 1.34±0.03, P=0.007; β-catenin control, 1.00±0.02; Fn, 1.44±0.03, P=0.006; vimentin control, 1.00±0.01; Fn, 2.19±0.09; P=0.006). As expected, the expression levels of N-cadherin, β-catenin and vimentin significantly decreased following treatment with fucose (N-cadherin Fn, 1.34±0.03; Fnf, 1.20±0.01, P=0.048; β-catenin Fn, 1.44±0.03; Fnf, 1.26±0.03, P=0.042; vimentin Fn, 2.19±0.09; Fnf, 1.67±0.07, P=0.045). E-cadherin was expressed at low levels in HCT116 cells treated with Fn compared with the other two groups, which demonstrated the change of the EMT pathway (E-cadherin control, 1.00±0.04; Fn, 0.37±0.03; Fnf, 0.72±0.07; P=0.005 and P=0.040). Similar findings were observed in SW480 cells (Fig. 5C and D).

The stat3 inhibitor, S3I-201, was used to assess the association between stat3 activation and EMT following treatment with Fn. Western blot analysis demonstrated that inhibition of stat3 activation significantly suppressed the activation of EMT in both HCT116 and SW480 cells (Fig. 5E-H). Taken together, these results suggest that fucose inhibits the carcinogenic properties of Fn to activate the stat3 pathway and EMT.