China Center for Type Culture Collection supplied the human OC cell lines SKOV3, A2780 and HEY-T30. SKOV3 cells were grown in McCoy's 5A medium (Cienry Biotechnology, Inc.), whereas A2780 and HEY-T30 cells were grown in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.). Both media were supplemented with 10% (v/v) fetal bovine serum (FBS; Invigentech, Inc.), 100 U/ml penicillin and 100 μg/ml streptomycin (Beyotime Institute of Biotechnology). The cells were grown at 37°C in an atmosphere containing 5% CO₂ with saturated humidity.

PFG (cat. no. B31203; molecular weight: 318.326; purity ≥98%; lot no. D15GB171320; Fig. 1A) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. PFG was mixed with dimethyl sulfoxide and stored at -80°C. To reduce the effects of serum and other chemicals in the culture medium on pharmacological action, PFG was diluted in cell culture medium containing 2% FBS and applied in all experiments. Docetaxel (cat. no. HY-B0011; purity 99.42%) was purchased from MedChemExpress, and was used to treat SKOV3 and A2780 cells for 24 h at 37°C at a dose of 80 nM. The Wnt/β-catenin pathway inhibitor XAV939 (cat. no. S1180; molecular weight: 312.31; purity 99.95%) was purchased from Selleck Chemicals, which was used to treat SKOV3 and A2780 cells for 24 h at 37°C at a dose of 10 μM. The MUC1 (cat. no. 4538S), N-cadherin (cat. no. 13116S), Vimentin (cat. no. 5741S), Snail (cat. no. 3879S), β-catenin (cat. no. 8480S) and cyclin D1 (cat. no. 55506S) antibodies were purchased from Cell Signaling Technology, Inc. Antibodies against MMP2 (cat. no. ab92536), MMP9 (cat. no. ab76003) and CDK2 (cat. no. ab32147) were acquired from Abcam. Antibodies against c-Myc (cat. no. sc-40), cyclin A (cat. no. sc-239) and cyclin E (cat. no. sc-247) were obtained from Santa Cruz Biotechnology, Inc. The β-actin antibody (cat. no. AC026) was obtained from ABclonal, Inc. HRP-conjugated goat anti-mouse IgG H&L (cat. no. BK-M050) and anti-rabbit IgG H&L (cat. no. BK-R050) antibodies were purchased from BIOKER Biotechnology, Inc. For western blotting, antibodies were used at a 1:1,000 dilution.

The Research Collaboratory for Structural Bioinformatics Protein Data Bank database (https://www.rcsb.org/) and the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) were utilized to find the three-dimensional structures of the MUC1 protein and molecular ligand structures, respectively. The Dockthor tool (https://docthor.lncc.br/v2/) was used to process proteins and ligands, including removing water molecules, performing hydrogenation and calculating protein charges. The final docking conformation should have the strongest affinity. According to this principle, the conformation was selected and visualized using Pymol 2.3 software (33).

Cell proliferation and the drug half maximal inhibitory concentration (IC₅₀) were examined by CCK-8 assay (Biosharp Life Sciences). SKOV3 and A2780 cells were inoculated in 96-well plates at a density of 4×10³ cells/well. The cells were incubated overnight with 5%CO₂ at 37°C in 5% CO₂, and were then treated with various doses of PFG (0, 0.5, 1, 2, 5, 10, 20 and 40 μM) for 24, 48 and 72 h at 37°C. As recommended, 10 μl CCK-8 reagent was then added per well and incubated for 1 h at 37°C in 5% CO₂. A New Epoch™ 2 Epoch Microplate Spectrophotometer (Thermo Fisher Scientific, Inc.) was used to measure the absorbance at 450 nm.

SKOV3 and A2780 cells were exposed to different concentrations of PFG (0.5, 1 and 2 μM) for 24 h at 37°C in 5% CO₂, then grown in McCoy's 5A or RPMI 1640 media supplemented with 10% FBS for 2 weeks, after being plated at a density of 1,000 cells/well in 6-well plates. Subsequently, 4% paraformaldehyde was employed to fix the colonies for 20 min and they were stained with 0.1% crystal violet for 30 min at room temperature. An optical microscope was used to count colonies containing >50 cells.

Using an EdU assay kit (Beyotime Institute of Biotechnology), the cell proliferation capacity was detected. At a density of 4×10⁵ cells/well, cells were plated into 6-well plates and exposed to different concentrations of PFG (0.5, 1 and 2 μM) for 24 h at 37°C in 5% CO₂. After SKOV3 and A2780 cells underwent a 2 h incubation in 50 μM EdU buffer at 37°C in 5% CO₂, they were fixed for 30 min in 4% paraformaldehyde, and then permeabilized for 20 min in 0.1% Triton X-100. After the culture was infused with click reaction solution, prepared according to the manufacturer's instructions, and then incubated in the dark for 30 min at room temperature, the nuclei were stained with 1X Hoechst 33342 at room temperature in the dark for 10 min. A fluorescence microscope was used to visualize the cells.

To analyze cell apoptosis, the Annexin-V FITC/PI Apoptosis Analysis Kit (Beyotime Institute of Biotechnology) was used. Several concentrations of PFG (0.5, 1 and 2 μM) were used to pretreat SKOV3 and A2780 cells for 24 h at 37°C in 5% CO₂. According to the manufacturer's instructions, prior to staining with 5 μl Annexin-V/FITC and 10 μl PI at room temperature in the dark for 15 min, centrifuged cells (1,000 × g for 5 min at 25°C) were rinsed in precooled PBS. In addition, as a positive control, cells were treated with 80 nM docetaxel for 24 h at 37°C in 5% CO₂. To assess cell cycle progression, the Cell Cycle Analysis Kit (Shanghai Yuanye Bio-Technology Co., Ltd.) was performed. The same cells were assessed as for the apoptosis experiment. According to the manufacturer's instructions, centrifuged cells (1,000 × g for 5 min at 25°C) were rinsed gently in precooled PBS before being stained with 500 μl 50 μg/ml PI staining reagent (containing 200 μg/ml RNase) at room temperature in the dark for 15 min. The test was repeated three times for each sample. Finally, FlowJo-V10 software (FlowJo, LLC) was used to investigate the apoptosis and cell cycle data, which were collected using Beckman CytoFlex (Beckman Coulter, Inc.). The apoptotic rate was calculated using the formula: Apoptotic rate=Q1-LR + Q1-UR. LR refers to early apoptotic rate and UR to late apoptotic rate.

DNA fragmentation, which is a hallmark of apoptosis, was studied using the One-Step TUNEL Apoptosis Detection Kit (FITC) (MedChemExpress). SKOV3 and A2780 cells (2×10⁴/well) were seeded in 24-well plates and treated with PFG (0.5, 1 and 2 μM) or docetaxel (80 nM) at 37°C in 5% CO₂ for 24 h. Cells were fixed with 10% neutral buffered formalin at room temperature for 20 min, and stained according to the manufacturer's protocol. Slides were then cover-slipped with antifade mounting medium with DAPI (Vector Laboratories, Inc.). Images were acquired using a fluorescence microscope, and the fluorescence intensity was statistically analyzed by ImageJ v1.48 software (National Institutes of Health). The experiment was repeated three times.

OC cells (3×10⁶ cells/well) were plated on 6-well plates and allowed to adhere to the plates for 24 h. To maintain a healthy cell growth state, when the fusion degree of the cell monolayer reached >80%, a straight line was scratched into the monolayer using a 200-μl pipette tip. After three washes with PBS, PFG (0.5, 1 and 2 μM) with 2% FBS was added to each plate at 37°C in 5% CO₂ for 24 h. The distance that cells had migrated was photographed under a light microscope at 0, 12 and 24 h for the subsequent calculation. Taking 24 h as an example, migration was assessed as follows: Migration rate (%)=(At=0 h)-(At=24 h)/(At=0 h). (At=0 h) refers to the area of the wound measured immediately after scratching; (At=24 h) refers to the area of the wound measured 24 h after the scratch was performed.

For the migration and invasion assays, PFG was applied to SKOV3 and A2780 at 0.5, 1 and 2 μM at 37°C in 5% CO₂ for 24 h. Subsequently, the upper chamber of a Transwell system (pore size, 8 μm; Corning, Inc.) was seeded with 2×10⁴ cells resuspended in 100 μl serum-free medium, and the bottom of the chamber contained the cell culture medium with 10% FBS. After the cells had migrated for 24 h at 37°C in 5% CO₂, the chamber was removed, the unmigrated cells were scraped off, and the migrated cells were fixed with 4% paraformaldehyde for 20 min at room temperature and stained with 0.01% crystal violet (Beijing Solarbio Science & Technology Co., Ltd.) for 15 min at room temperature. Using a light microscope (×200 magnification; Olympus Corporation) cell images were captured and cell counts from five random fields were measured. For the invasion assay, the upper chamber of the insert was coated with 100 μl growth factor-reduced Matrigel (Corning, Inc.) overnight at 37°C.

To infect the SKOV3 and A2780 cell lines, Shanghai GeneChem Co., Ltd. provided both the lentivirus-based short hairpin (sh) RNA vector GV493 (hU6 -MCS-CBh-gcGFP-IRES-puromycin) and the lentivirus-based overexpression RNA vector GV721 (CMV enhancer-MCS-3FLAG-EF1a-firefly_ Luciferase-SV40-puromycin). According to the manufacturer's instructions, the recombined GV493/GV721 lentiviral vector plasmid or the negative control lentiviral vector plasmid, and pHelper 1.0 and pHelper 2.0 plasmids (Shanghai Genechem Co., Ltd.) were co-transfected into 293T cells (American Type Culture Collection) using HitransG enhanced infection solution (Shanghai Genechem Co., Ltd.) at 37°C in 5% CO₂ for 6 h. Subsequently, the culture supernatant was removed and replaced with fresh high-glucose DMEM supplemented with 10% FBS and the culture supernatants were collected at 48 h post-transfection. Following centrifugation at 4,000 × g for 10 min at 4°C to remove cell debris, the supernatant was filtered through 0.45-μm filters. The concentrated viral supernatant was aliquoted and maintained at -80°C prior to use. The lentivirus was then diluted with serum-free McCoy's 5A or 1640 medium and used to infect SKOV3 and A2780 cells at a multiplicity of infection of 10. After 8 h at 37°C in 5% CO₂, the medium was refreshed. Subsequently, at 72 h post-infection, puromycin (2 μg/ml) was employed to screen out the stable knockdown and overexpression cell lines at 37°C in 5% CO₂ for 48 h, and then 1 μg/ml puromycin was used for maintenance. Finally, the SKOV3 and A2780 cells were harvested for further analysis. The following shRNA sequences were used: sh-MUC1, 5'-CCG GGA TAC CTA CCA TCC TAT P-3'; sh-Control (non-targeting negative control viral vector), 5'-TTCTCCGAACGTGTCACGT-3'.

According to the manufacturer's protocol, the pGL3 Basic human MUC1 promoter (HIBio Biotechnology Co., Ltd.) was transfected into human OC SKOV3 cells using Lipofectamine^® 3000 Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc.). As an internal control, a pRL-TK plasmid carrying the Renilla luciferase gene (HIBio Biotechnology Co., Ltd.) was co-transfected with pGL3 Basic human MUC1 promoter in SKOV3 cells. Briefly, 24-well plates were used for transfection; the plasmid was transfected into cells at 37°C in 5% CO₂ for 8 h, then the medium was replaced with fresh culture medium for 24 h. Subsequently, PFG (1 and 2 μM) was added for 24 h at 37°C in 5% CO₂. Utilizing the Dual-luciferase Reporter Assay Kit (Promega Corporation), according to the guidelines provided by the manufacturer, cells transfected with vector were added to perform luciferase assays in triplicate in 96-well plates after puromycin resistance screening. GraphPad Prism 8 (Dotmatics) was used to evaluate and plot the exported data.

Using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), RNA was extracted from the cells. The Nanodrop 2000 system (Thermo Fisher Scientific, Inc.) was employed to measure the quality and quantity of RNA. Subsequently, 1 μg total RNA was used to generate cDNA using the PrimeScript RT reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol. qPCR was conducted on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) using the SYBR Premix Ex Taq™ kit (Takara Bio, Inc.). The thermocycling conditions were as follows: An initial denaturation step at 95°C for 10 sec, followed by 40 cycles, (denaturation at 95°C for 10 sec, annealing at 64°C for 25 sec), and a final extension step at 55°C for 1 min and 95°C for 10 sec. GAPDH was used as an internal control. To obtain the relative gene expression levels, the 2^-ΔΔCq method (34) was used. The following primer sequences were used: MUC1, forward 5'-ATA CCT ACC ATC CTA TGA G CG A-3', reverse 5'-CTG CTG GGT TTG TGT AAG AGA-3'; and GAPDH, forward 5'-GGT GGT CTC CTC TGA CTT CAA CA-3' and reverse 5'-GTT GCT GTA GCC AAA TTC GTT GT-3'.

Cells were lysed in RIPA buffer with a proteinase inhibitor cocktail (Beyotime Institute of Biotechnology), and the BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.) was used to quantify the amount of protein in the extracts. Proteins (20 μg) were separated by SDS-PAGE on 8% gels and were then transferred to polyvinylidene fluoride membranes (MilliporeSigma). To prevent nonspecific binding, the membranes were blocked for 2 h with 5% skim milk (Bio-Rad Laboratories, Inc.) diluted in Tris-buffered saline. Prior to being incubated with HRP-conjugated secondary antibodies at room temperature for 2 h, membranes were pre-incubated with a specific primary antibody for an overnight period at 4°C. An ECL Western Blotting Detection kit (Biosharp Life Sciences) was utilized for detecting protein signals. After standardization to β-actin, ImageJ v1.48 software (National Institutes of Health) was used to measure the optical density of the blot.

GraphPad Prism version 8 software was used to investigate statistical significance via a Student's unpaired t-test, one-way ANOVA or two-way ANOVA with a Bonferroni multiple comparisons test (if the variance is homogeneous) or Games-Howell multiple comparison test (if the variance is uneven). All studies were conducted at least in triplicate, and all data are presented as the mean ± SEM. P<0.05 was considered to indicate a statistically significant difference.

The binding mode of PFG to MUC1 was predicted by molecular docking, and it was revealed that PFG formed three hydrogen bonds with HIS-1104, LYS-43 and TRP-1105 amino acid residues (Fig. 1B). These results indicated that MUC1 is a potential target protein of PFG. Next, the effects of PFG on the promoter activity of MUC1 were assessed using a dual-luciferase assay. Compared with the human MUC1 promoter-pGL3 Basic group, the PFG (1 and 2 μM) groups showed a significant decrease in relative fluorescence activity, indicating that PFG treatment affected the binding of transcription factors to the MUC1 promoter in SKOV3 cells (Fig. 1C). Subsequently, quantification of MUC1 expression in SKOV3 and A2780 cells was performed using RT-qPCR and western blotting. The results suggested that PFG inhibited MUC1 expression and exhibited a concentration-dependent effect on the downregulation of MUC1 expression at concentrations of 0.5, 1 and 2 μM (Fig. 1D and E). According to these findings, PFG may inhibit MUC1 expression by decreasing MUC1 promoter activity in OC cells.

To understand the anticancer effect of PFG on OC cells, SKOV3 and A2780 cells were treated with PFG (0.5-40 μM) for 24, 48 and 72 h, and cell viability was detected by CCK-8 assay. According to the results, compared with in the control group, PFG suppressed the viability of SKOV3 and A2780 cells, and the inhibition was dose-dependent (Fig. 2A). The IC₅₀ value at 24 h for SKOV3 cells was 10.79 μM, and the IC₅₀ value at 24 h for A2780 cells was 3.845 μM. To rule out the potential bias in subsequent experiments caused by the cytotoxicity of high doses of PFG, 0.5, 1 and 2 μM concentrations were selected for subsequent experiments.

The EdU assay also indicated that PFG treatment inhibited the proliferation of both cell lines (Fig. 2B). Moreover, the colony formation assay revealed that PFG significantly inhibited the clonogenic ability of both cell lines (Fig. 2C).

To further assess the mechanism underlying the anti-proliferative effects of PFG, flow cytometry was performed to detect its effects on the apoptosis and cell cycle progression of the two cell lines. The results showed that in SKOV3 and A2780 cells, the apoptotic rate was significantly different in the PFG (2 μM) and docetaxel (80 nM) treatment groups compared with that in the control group (Fig. 3A), TUNEL detection indicated the same trend change in SKOV3 and A2780 cells (Fig. S1). However, the maximum apoptotic rate detected by the two experimental methods was <10%, which indicates that the effect of PFG on the apoptosis of SKOV3 and A2780 cells may have no biological significance. Cell cycle experiments demonstrated that PFG treatment could block the cell cycle in S phase, resulting in shortening of the G₀/G₁ phase and prolonging the S phase (Fig. 3B), with no significant change in the proportion of cells in G₂ phase. To explore the molecular mechanism underlying PFG-induced S-phase blockade, the expression levels of specific proteins associated with S-phase regulation were examined by western blotting. Notably, PFG treatment had a marked dose-dependent inhibitory effect on the protein expression levels of cyclin A, cyclin E and CDK2 (Fig. 3C). These results suggested that PFG may induce S-phase cell cycle arrest in OC cell lines through controlling the levels of S-phase cell cycle regulatory proteins.

OC frequently exhibits distant metastasis, and patients with metastatic OC exhibit a low survival rate when compared with primary OC (35). In order to determine if PFG has anti-metastatic effects, the migration of SKOV3 and A2780 cells was assessed using a wound-healing assay. Compared with in the control group, PFG significantly inhibited the migration of both cell lines in a dose-dependent manner (Fig. 4A). Furthermore, the effects of PFG on migration and invasion were assessed using the Transwell assay, and it was revealed that PFG markedly inhibited the migration and invasion of both cell lines in a dose-dependent manner (Fig. 4B).

Due to the importance of the EMT in the distant metastases of OC, identifying a new therapy that can target EMT is critical. To the best of our knowledge, the specific mechanism underlying the effects of PFG on the EMT of OC cells has not been reported. Therefore, after 24 h of PFG treatment, the expression levels of EMT-related proteins in the two OC cell lines were examined via western blot analysis. It was revealed that PFG could significantly inhibit the expression levels of MMP2 and MMP9, and downregulate the expression levels of mesenchymal markers, such as N-cadherin, Vimentin and Snail (Fig. 4C). These results indicated that PFG could block the activation of EMT in OC cells. Overall, it may be concluded that PFG suppresses OC cell migration and invasion via blocking EMT.

To investigate the effects of MUC1 on OC cells, MUC1 protein expression levels were compared in three OC cell lines, and SKOV3 cells with the highest relative expression levels and A2780 cells with the lowest relative expression levels were selected for the construction of cells with stable MUC1 knockdown and overexpression for further mechanistic studies (Fig. 5A). Accordingly, RT-qPCR and western blot experiments verified the knockdown and overexpression of MUC1 (Fig. 5B and C). Compared with in the sh-Control cells, the results demonstrated that MUC1 knockdown had a significant effect on inhibiting the proliferation of OC cells (Fig. 6A and B), inducing cell cycle arrest at G₀/G₁ phase (Fig. 6C), and reducing cell migration and invasion (Fig. 6D). By contrast, compared with in the overexpression control (OE-control) cells, MUC1 overexpression promoted OC cell proliferation, accelerated cell cycle progression, and increased cell migration and invasion. These results implied that MUC1 is essential for OC cell proliferation and metastasis.

To clarify whether PFG-induced antitumor effects were mediated by MUC1, the changes in migration and invasion were detected in cells with MUC1 knockdown and overexpression after treatment with various concentrations of PFG. The migration and invasion rate of sh-MUC1 cells was more affected by PFG than that of sh-control cells (Fig. 7A and B). Compared with the sh-control cells, in sh-MUC1 cells, the expression levels of MUC1 were significantly decreased and the inhibitory effect of PFG on EMT-related proteins (including N-cadherin, Vimentin and Snail) was enhanced (Fig. 7C).

By contrast, MUC1 overexpression restored and enhanced the migratory and invasive ability of SKOV3 and A2780 cells, while also activating EMT. Compared with the OE-control cells, the inhibitory effect of different concentrations of PFG supplementation on cell migration and invasion was reduced in MUC1-overexpressing cells (Fig. 8A and B). Compared with the OE-control cells, the expression levels of MUC1 were significantly increased in OE-MUC1 cells, and MUC1 overexpression partially reversed the PFG-induced decrease in N-cadherin, Vimentin and Snail protein expression levels. (Fig. 8C). These results indicated that MUC1 may be the dominant target protein through which PFG inhibits SKOV3 and A2780 cells migration, invasion and EMT.

The Wnt/β-catenin pathway is closely related to the migration and invasion of tumor cells (36). To evaluate the role of the Wnt/β-catenin pathway in the anti-OC effect of PFG, the Wnt/β-catenin pathway inhibitor XAV939 was used. Western blot analysis revealed that PFG and AXV939 exhibited similar inhibitory effects on SKOV3 and A2780 cells. The combination of PFG and XAV939 further suppressed the expression levels of β-catenin in SKOV3 cells, as well as the downstream target protein MMP9 in SKOV3 and A2780 cells (Fig. S2A). The results of the wound-healing assay showed that combining PFG and XAV939 markedly improved the inhibitory effect of PFG on OC cell migration (Fig. S2B). These results suggested that the Wnt/β-catenin pathway may have an important role in the inhibition of OC by PFG.

To elucidate whether PFG regulates the Wnt/β-catenin pathway and cancer motility via inhibiting the expression of MUC1, the protein expression levels of Wnt/β-catenin-related proteins and their downstream target proteins, which are linked to tumor cell metastasis and invasion were assessed. PFG (2 μM) treatment significantly reduced the protein expression levels of β-catenin, c-Myc, cyclin D1 and MMP9 in the sh-control cells and OE-control cells, when compared with the control cells (0 μM PFG), indicating that PFG inhibited Wnt/β-catenin pathway signaling in both OC cell lines. In addition, compared with sh-control cells, the silencing of MUC1 accelerated the decrease in β-catenin, c-Myc, MMP9 and cyclin D1 protein expression levels induced by PFG in SKOV3 and A2780 cells (Fig. 9A).

By contrast, compared with in the OE-control group, overexpression of MUC1 partially reversed the decrease in β-catenin protein expression levels in SKOV3 and A2780 cells induced by PFG, as well as c-Myc and cyclin D1 protein expression levels in SKOV3 cells (Fig. 9B). In summary, it may be suggested that PFG can prevent the EMT process by blocking the Wnt/β-catenin signaling pathway through inhibiting MUC1 in OC cells (Fig. 10).