The human MCF-7 and MDA-MB-231 breast cancer cells were cultured in DMEM/F12 and L15 medium supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (TransGen Biotech, China) at 37°C in humidified air containing 5% CO₂, respectively.

Fentanyl citrate was obtained from Northeast Pharmaceutical Group (China). The sources of reagents were as follows: Sox2, Oct4, N-cadherin, Vimentin, E-cadherin, β-catenin, GSK-3β, CD44, Cyclin D1, and GAPDH antibody (Proteintech, China); Nanog antibody (Abcam, USA); FUT8, c-Jun and VEGF antibody (Santa Cruz, USA); Lens culinaris agglutinin (LCA) Lectin, binding of α1, 6-fucosylation epitope. (Vector Laboratories, USA); p-GSK-3β (Ser⁹) antibody (Elabscience, China); LGK-974 (inhibitor of Wnt ligands) (Selleck, USA).

Cells were seeded onto ultra low attachment 6-well plate (Corning, USA) at a density of 500 cells per well. The medium was supplemented with 2% B27, 20 ng/ml bFGF, and 20 ng/ml EGF. After culture for 12 days at 37°C in 5% CO₂, the number of tumor-spheres with a diameter of ≥50 μm was counted under a microscope (Olympus BX51, Japan).

500 cells were seeded onto a 35 mm dish. After culture for 10 days, surviving colonies were counted with crystal violet staining. Triplicate independent experiments were carried out.

The proteins from cells and tumor tissues were extracted. Protein concentration was determined with Coomassie Protein Assay Reagent using bovine serum albumin as a standard. Total protein was separated by a 12% SDS-PAGE gel followed by transferring to a nitrocellulose membrane (NC). The membrane was blocked with 5% non-fat dry milk in TBST for 2 h and incubated overnight with primary antibody or LCA Lectin, and then membrane was incubated with HRP-secondary antibodies or HRP-Streptavidin for 1 h. The HRP conjugates were exposed with the ECL (Amersham, USA) detection system, and data was analyzed by Quantity One software (Bio-Rad, USA).

Migrated breast cancer cells were determined by transwell migration assay. Briefly, 1 × 10⁴ cells/well were cultured in triplicate in the top chambers of 24-well transwell plates (8.0 mm-pore, Corning, USA) and 10% FBS medium were added into the bottom chambers. After culture for 20 h, the migrated cells on the bottom surface of the top chamber membranes were fixed with methanol, stained with crystal violet, and imaged under a microscope.

Quantitative PCR (qPCR) was used to measure mRNA level. Total RNAs were extracted using Trizol (Takara, Japan) according to the manufacturer's protocol. RNA was reversely transcribed into cDNA using PrimeScriptTMRT reagent kit (TransGen Biotech, China), and then qPCR was performed using TransStart TipTop Green qPCR SuperMix (TransGen Biotech, China) according to the manufacturer's instructions. The primer sequences were as follows: Nanog: 5′-CCT GTG ATT TGT GGG CCT GA-3′ (F), 5′-CTC TGC AGA AGT GGG TTG TTT G-3′ (R); Sox2: 5′-GTG AGC GCC CTG CAG TAC AA-3′ (F), 5′-GCG AGT AGG ACA TGC TGT AGG TG-3′ (R); Oct4: 5′-GCA GAT CAG CCA CAT CGC CC-3′ (F), 5′-GCC CAG AGT GGT GAC GGA GA-3′ (R); N-cadherin: 5′-AAA GAA CGC CAG GCC AAA C-3′(F), 5′-GGC ATC AGG CTC CAC AGT GT-3′(R); E-cadherin: 5′-CAA CGA CCC AAC CCA AGA A-3′(F), 5′-CCG AAG AAA CAG CAA GAG CA-3′(R); Vimentin: 5′-CGT CTC TGG CAC GTC TTG AC-3′(F), 5′-GCT TGG AAA CAT CCA CAT CGA-3′(R). Quantified data was normalized to GAPDH, and the relative quantity was calculated using the 2-ΔΔCT method. Triplicate independent experiments were carried out.

Immunofluorescent staining was performed after fixing the cells with 4% paraformaldehyde for 20 min. After blocking with 1% goat serum (Beyotime, China) for 2 h, the cells were incubated with primary antibodies overnight at 4°C. After washing with PBS, the cells were incubated with FITC-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG for 1 h at room temperature. Then slides were incubated with DAPI for 10 min at room temperature. Images were obtained under a fluorescence microscope (Olympus BX83, Japan).

Lectin fluorescent staining was performed after fixing the cells with 4% paraformaldehyde for 20 min. After blocking with 1% goat serum (Beyotime, China) for 2 h, the cells were incubated with rhodamine-labeled LCA Lectin for 2 h at room temperature. After washing with PBS, the slides were incubated with DAPI for 10 min at room temperature. Images were obtained under a fluorescence microscope (Olympus BX83, Japan).

MCF-7 cells (1 × 10⁵) were trypsinized, and seeded onto 6-well plate. When cells reached 70–80% confluence, short interfering RNA of FUT8 (siFUT8): 5′-GUG GAG UGA UCC UGG AUA UTT-3′ (F), 5′-AUA UCC AGG AUC ACU CCA CTT-3′ (R) (Genepharma, China) was transiently transfected into MCF-7 cells using Lipofectamine™ 2000 Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The cells were collected after 48 h transfection for further experiments.

Visible tumors were removed from the mice, and immunohistochemistry was performed on paraffin-embedded sections. Serial sections (4 μm each) were prepared, deparaffinized in xylene and rehydrated in a graded alcohol. After microwaving for 20 min in citrate buffer to expose the antigen, the slides were washed with PBS and incubated in 3% H₂O₂ for 10 min at room temperature to block endogenous peroxidase activity. Non-specific binding was blocked with goat serum at room temperature for 30 min before incubation overnight at 4°C with primary antibody. After extensive washing with PBS, slides were incubated with biotinylated secondary antibody for 15 min at room temperature. The slides were then incubated with a streptavidin-peroxidase complex. The signal was visualized with DAB (3, 3′-diaminobenzidine) kit, and the slides were briefly counterstained with hematoxylin. Images were captured with a microscope.

Female nude mice (Balb/c-nu/nu) were obtained from Animal Center (Dalian Medical University). The animals (4–6 weeks) were maintained under sterile conditions during the entire experimental period. MCF-7 cells (5 × 10⁶) suspended in 0.2 ml PBS were injected subcutaneously into the right flank. After 7 days, mice were randomly divided into 2 different groups (n = 5/group). Fentanyl (0.02 mg/kg of body weight) was subcutaneously administered for 3 weeks with time interval of 24 h. The mice treated with citrate buffer only served as controls. The tumor dimensions were measured every 3 days using a digital caliper. The tumor volume was calculated according to the formula (Volume = 1/2 Length × Width²). At the end of the experiment (the 30th day), the tumor mass was weighed. All animal procedures were approved by the Institutional Review Board of Dalian Medical University.

Data were expressed as mean ± SEM of three independent experiments using GraphPad Prism v5.01 (GraphPad Software, La Jolla, CA, USA). The Student's t-test was used to make a statistical comparison between groups. Statistical significance was defined as P < 0.05.

To analyze the role of fentanyl on stem-like characteristics in breast cancer cells, we performed sphere formation assay in MCF-7 and MDA-MB-231 cells under serum-free and non-adherent conditions with treatment of different concentrations of fentanyl (0, 0.01 μM, 0.1 μM). The results showed that fentanyl induced a significant increase in the size (P < 0.01) and number (P < 0.05) of tumor-spheres in a dose-dependent manner (Figures 1A,B). Representative images of colony forming assay were shown (Figures 1C,D). There was an obvious stimulating effect of fentanyl on colony formation ability as compared with the untreated cells (P < 0.01). We then examined the expression of stemness-related markers. As shown in Figures 1E,F, fentanyl upregulated the expression of Nanog, Sox2 and Oct4 genes by qPCR. In addition, the effect of fentanyl on the protein level was confirmed by Western blot (Figures 1G,H). Significant difference in both mRNA and protein levels normalized to the GAPDH was observed in the control compared to treatment groups (P < 0.05). Collectively, these results suggest that fentanyl promotes the stemness of breast cancer cells.

To determine whether fentanyl plays a role in EMT, MCF-7, and MDA-MB-231 cells were treated with different concentrations of fentanyl (0, 0.01 μM, 0.1 μM) for 48 h. The expression of EMT markers N-cadherin, E-cadherin and Vimentin was assessed by qPCR and Western blot. As shown in Figures 2A–D, fentanyl significantly enhanced the expression of N-cadherin (P < 0.05) and Vimentin (P < 0.01), whereas decreased the expression of E-cadherin (P < 0.01). Alterations of N-cadherin and E-cadherin in MCF-7 cells was determined by immunofluorescent staining (Figure 2E). Furthermore, migrated cells were increased in fentanyl treated group compared with the control, confirming the stimulating effect of fentanyl on EMT (Figures 2F,G). These results indicate that fentanyl induces EMT in breast cancer cells.

To explore whether α1, 6-fucosylation catalyzed by FUT8 is involved in EMT and stemness induced by fentanyl, breast cancer cells were treated with different concentrations of fentanyl (0, 0.01 μM, 0.1 μM) for 48 h. The results indicated that fentanyl enhanced FUT8 gene and protein expression compared with the control by qPCR, Western blot and immunofluorescent staining (Figures 3A–C). Furthermore, results showed that fentanyl increased the α1, 6-fucosylation level in a dose-dependent manner by Lectin blot in the treatment groups compared with the control (Figure 3D). The effect of fentanyl on α1, 6-fucosylation level was further confirmed by Lectin fluorescent staining (Figure 3E). In brief, fentanyl promotes FUT8 expression and FUT8-catalyzed α1, 6-fucosylation level in breast cancer cells.

To assess the effects of FUT8 in breast cancer phenotypes, we first knockdown the expression of FUT8 in MCF-7 cells, and the knockdown efficiency was verified by Western blot and immunofluorescent staining (Figures 4A,C). The effect of siFUT8 on the biosynthesis of α1, 6-fucosylation was detected by LCA Lectin blot and Lectin fluorescent staining. The results showed that siFUT8 successfully decreased the level of α1, 6-fucosylation (Figures 4B,D). Furthermore, the results of Western blot showed that decreased or blocked α1, 6-fucosylation by siFUT8 or LCA Lectin treatment, respectively, suppressed the expression of stemness- and EMT-related markers (Figures 4E,F). We then examined whether FUT8 expression and α1, 6-fucosylation level could influence the stemness and EMT of breast cancer cell by sphere formation assay and transwell assay, respectively. As shown in Figures 4G,H, there was a significantly inhibition in size and number of tumor-spheres in siFUT8 transfection (P < 0.05) and LCA Lectin blocking groups (P < 0.05) compared with the control. Similarly, the migrated cells were also inhibited by siFUT8 transfection and LCA Lectin blocking (Figures 4I,J). Meantime, fentanyl reversed the inhibitory effects of stemness and EMT mentioned above (Figures 4A–J). These findings indicate that FUT8-catalyzed α 1, 6-fucosylation regulates stemness and EMT induced by fentanyl.

We investigated whether Wnt/β-catenin signaling pathway could be activated by fentanyl, followed by detecting the influence of Wnt signaling inhibition on FUT8, α1, 6-fucosylation, stemness and EMT. We found that fentanyl increased the expression of key molecules (p-GSK-3β, β-catenin) and target genes (Cyclin D1, CD44, VEGF, and c-Jun) in Wnt/β-catenin signaling pathway (Figure 5A). In addition, we detected β-catenin expression in the cytoplasm and nucleus separately. Results showed that fentanyl facilitated the translocation of β-catenin from cytoplasm to nucleus indicating the accumulated and activiated form of β-catenin in nuleus (Figure 5B). However, LGK-974 (1 μM), an inhibitor of Wnt ligands, decreased fentanyl-activated β-catenin, p-GSK-3β (Ser⁹) and FUT8 expression (Figure 5C). The inhibitory effect of LGK-974 on FUT8 expression and α1, 6-fucosylation level was further confirmed by immunofluorescent staining and Lectin fluorescent staining (Figures 5D,E). The downregulated expression of Sox2, Oct4, N-cadherin, whereas upregulated expression of E-cadherin induced by LGK-974 was also detected by Western blot (Figure 5F). Furthermore, LGK-974 induced a decrease in the size and number of tumor-spheres, as well as migrated cells (Figures 5G,H). These results suggest that Wnt/β-catenin signaling pathway mediate upregulation of FUT8 and α1, 6-fucosylation upon fentanyl exposure and subsequently promote stemness and EMT in breast cancer cells.

To confirm the in vitro results, MCF-7 cells were used to establish mouse xenograft tumor models. The tumor volume and weight were analyzed in the control and fentanyl treatment groups. There were no obvious changes in the body weight of the xenograft mice (Figure 6A). However, as shown in Figures 6B,C, fentanyl treatment significantly increased tumor volume and weight in xenografts (P < 0.01). The analysis of Western blot showed that fentanyl promoted the expression of FUT8, β-catenin, p-GSK-3β (Ser⁹), Sox2 and Oct4 and N-cadherin, as well as inhibited the expression of E-cadherin (Figure 6D). Lectin blot showed that fentanyl enhanced α1, 6-fucosylation level (Figure 6E). We also detected the expression of FUT8, β-catenin, N-cadherin, and Oct4 in mouse xenograft tumor tissues by immunohistochemistry. As shown in Figure 6F, FUT8, β-catenin, N-cadherin, and Oct4 were highly expressed in fentanyl treatment group compared with the control. These results reveal that fentanyl promotes FUT8 expression, α1, 6-fucosylation level, stemness and EMT of breast cancer cells in tumor xenograft.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.