Narasin was purchased from Sigma-Aldrich (Merck KGaA), dissolved in 100% DMSO to generate a 10 mM solution and stored at −80°C in small aliquots. Recombinant human TGF-β and IL-6 were purchased from PeproTech, Inc. Antibodies against phosphorylated (p)-STAT3 (Tyr705; cat. no. 9145; 1:1,000), STAT3 (cat. no. 9139; 1:1,000), p-AKT (Ser473; cat. no. 4060; 1:1,000), AKT (cat. no. 4691; 1:1,000), p-SMAD2 (Ser465/467)/SMAD3 (Ser423/425; cat. no. 8828; 1:1,000), SMAD3 (cat. no. 9523; 1:1,000), β-catenin (cat. no. ARG52651; 1:800; Arigo Biolaboratories Corp.), ZEB1 (cat. no. ab203829; 1:500; Abcam) and β-actin (cat. no. M1210-2; 1:10,000; Hangzhou HuaAn Biotechnology Co., Ltd.), as well as an EMT Antibody Sampler kit (cat. no. 9782; 1:1,000) were purchased from Cell Signalling Technology, Inc. All other reagents were acquired from Sigma-Aldrich (Merck KGaA).

Human ER⁺ BC cell lines, MCF-7 and T47D, and human triple-negative BC cell line, MDA-MB-231, were purchased from the American Type Culture Collection. All cells were cultured in DMEM or RPMI-1640 medium supplemented with 10% fetal bovine serum, and the medium was replaced every 48 h. Cells were maintained in a humidified 37°C incubator with 5% CO₂.

Human BC cells were incubated in a 96-well plate at a density of 4.5×10⁴ to 5.5×10⁴ cells per well and treated with narasin at a final concentration of 0, 1, 2.5, 5, 10 µM for 72 h at 37°C. Cell viability was determined using an MTS kit (CellTiter 96 Aqueous One Solution Reagent; Promega Corporation), as previously described (23,24). MTS solution (20 µl) was added to each well, the plates were incubated for 2 h, and the absorbance was measured at 490 nm with a microplate reader (Bio-Rad Laboratories, Inc.). All experiments were repeated three times.

The wound healing assay was performed as previously described (23,24). Briefly, human BC cell lines were incubated in 6-well plates and allowed to grow to 80% confluence. Cells were then washed three times with the medium, scratched with a 200 µl pipette tip, and incubated in the growth medium with 10% FBS and different concentrations of narasin. Images of cells were taken at 24 h at ×40 magnification with an inverted microscope (TE2000; Nikon Corporation). The migrated cells were counted in three randomly selected fields. Cells in the growth medium without narasin treatment served as the vehicle control. The inhibition percentage was expressed as a percentage of vehicle control (100%). The assay was repeated three times.

The cell invasion assay was performed using Matrigel-coated Boyden inserts (8 µm; BD Biosciences), as previously described (23,24). Cells in the logarithmic growth phase were digested using trypsin. Subsequently, 150 µl serum-free culture medium containing 1×10⁶ cells was plated into the upper chamber, and 600 µl culture medium containing different doses of narasin was plated into the lower chamber. The assays were performed for 12 h. Invaded cells were fixed with 4% paraformaldehyde at 4°C for 20 min, stained with 0.5% crystal violet for 10 min at room temperature, and counted under a light microscope in 4–7 randomly selected fields of view. Three independent experiments were performed in triplicate.

ER⁺ BC MCF-7 cells (2×10⁶) were seeded in 100-mm tissue culture dishes with or without narasin treatment (0.05 µM) for 24 h. TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to isolate RNA. The mRNA microarray analysis was carried out with the Affymetrix GeneChip^® PrimeView™ Human Gene Expression Array (Affymetrix; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions by the Gene Tech Company Limited (Shanghai, China). Data were analyzed in The Database for Annotation, Visualization and Integrated Discovery (DAVID) pathway enrichment analysis for Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (25–27) (https://david.ncifcrf.gov/), Search Tool for the Retrieval of Interacting Genes (STRING; http://string-db.org/) and the heatmap package in R (28). The differentially expressed genes (DEGs) were determined based on a false discovery rate threshold of 0.05.

Total RNA was reverse transcribed to cDNA using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). The following temperature protocol was used for reverse transcription: 42°C for 60 min, 70°C for 5 min and 4°C maintenance. PCR was performed using the SuperScript III Platinum One-Step Quantitative RT-PCR System (Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for qPCR: 94°C for 3 min; followed by 35 cycles of 94°C for 30 sec, annealing temperature for 30 sec, 72°C for 35 sec; 72°C for 10 min; and maintained at 4°C. The DNA products were run on a 2% agarose gel containing ethidium bromide. DNA bands were visualized using an ultraviolet imager and a Gel Imaging Analysis System (Bio-Rad Laboratories, Inc.). GAPDH was used as the loading control. The sequences of the primers used for qPCR were as follows: E-cadherin forward, 5′-ACAGGATGGCTGAAGGTGAC-3′ and reverse, 3′-TCAGGATCTTGGCTGAGGAT-5′; N-cadherin forward, 5′-GGACCGAGAATCACCAAATG-3′ and reverse, 3′-CCATTAAGCCGAGTGATGGT-5′; vimentin forward, 5′-TCGCCAACTACATCGACAAG-3′ and reverse, 3′-GACGCATTGTCAACATCCTG-5′; and GAPDH forward, 5′-AGGTCGGTGTGAACGGATTTG-3′ and reverse, 3′-TGTAGACCATGTAGTTGAGGTCA-5′. mRNA expression levels were quantified using GeneSpan (version 7.04.02; SynGene).

Molecular docking analysis was performed to analyze the binding ability of narasin to the TGF-β receptor using Autodock 4.2 (The Scripps Research Institute), as previously described (23,24). TGF-β receptor (PDB ID, 3TZM) was obtained from RCSB Protein Data Bank (http://www.rcsb.org/) and processed with UCSF Chimera software (version 1.13.1; National Institutes of Health), which included removal of water molecules, ions and ligands. The molecular structure of hydrophilic narasin was obtained from NCBI (https://www.ncbi.nlm.nih.gov/) without the molecular optimization process.

Western blotting was performed as previously described (23,24). Briefly, cells were harvested and lysed with RIPA buffer (4.25 g NaCl, 3.03 g Tris, 5 g SDS, 5 ml NP-20, 2.5 g 10% sodium deoxycholate, pH 8.0). Protein concentration was determined using a BCA assay kit (Sigma-Aldrich; Merck KGaA). Samples containing 30–50 µg total proteins were separated via SDS-PAGE on 8–12% gels, and subsequently transferred to PVDF membranes by electroblotting (Bio-Rad Laboratories, Inc.). Membranes were then blocked with 5% BSA (Sangon Biotech Co., Ltd.) for 2 h, and then incubated overnight at 4°C with the following primary antibodies: p-STAT3, STAT3, p-AKT, AKT, p-SMAD2/SMAD3 and an EMT antibody kit. The next day, the membranes were incubated with the appropriate anti-rabbit (1:2,500; cat. no. A21010; Abbkine Scientific Co., Ltd.), anti-mouse (1:2,500; cat. no. A21020; Abbkine Scientific Co., Ltd.) secondary antibodies for 1 h at room temperature. After several washes, specifically bound antibodies were visualized using an enhanced chemiluminescence system (Bio-Rad Laboratories, Inc.). Protein expression levels were semi-quantified using Image Lab software (version 5.2.1; Bio-Rad Laboratories, Inc.).

For the IF assay, cultured cells (2×10⁴) were treated with the indicated concentrations of narasin for 24 h, followed by stimulation with 100 ng/ml IL-6 or 5 ng/ml TGF-β for 2 h at 37°C. Cells were fixed with 4% paraformaldehyde at 0°C for 20 min, permeabilized with 0.1% Triton X-100, and blocked with 1% BSA in PBS at room temperature for 1 h. The cells were washed 3 times with PBS after each step, followed by incubation with primary antibodies (1:1,000) overnight at 4°C. After thorough washing, the cells were stained with FITC-conjugated goat anti-mouse (1:1,000; cat. no. A21020; Abbkine Scientific Co., Ltd.) or anti-rabbit IgG (1:1,000; cat. no. A22120; Abbkine Scientific Co., Ltd.). Cells were then washed and incubated with DAPI (Beijing Solarbio Science & Technology Co., Ltd.) for 5–10 min at room temperature. After the final wash, the samples were mounted with Antifade solution (Beijing Solarbio Science & Technology Co., Ltd.) and visualized under a fluorescence microscope (DM2000; Leica Microsystems GmbH; magnification, ×20).

A total of 46 four-week-old female BALB/c athymic nude mice (weight, 20 g) were obtained from the Experimental Animal Center of Ningxia Medical University. All mouse studies were performed according to animal protocols approved by the Institutional Animal Care and Use Committee of Ningxia Medical University (Yinchuan, China). To measure metastasis, MCF-7-Luc cells (1×10⁵; Shanghai Key Regulatory Institute of Biomedical Sciences) were injected into the left ventricle of female BALB/c athymic nude mice (5-7 per group). Mice were treated with narasin [0.5 mg/kg (n=6) or 1.5 mg/kg (n=5); i.p.] or DMSO (control group; n=7) every 2 days for 50 consecutive days. The IVIS detection system (Caliper Life Sciences) was used to detect tumor growth and metastasis.

For the xenograft assays, MCF-7 cells (1×10⁷ cells per mouse; resuspended in compete medium) were subcutaneously implanted into the axilla of the mice. When tumors reached ~100 mm³, the mice were randomly divided into three groups (n=9) and injected with narasin (0.5 or 1.5 mg/kg) or DMSO (control group) every 2 days for 2 consecutive weeks (14 days). After 2 weeks of treatment, the maximal tumor volume was 1,400 mm³. Then, the mice were euthanized, and tumor tissues were harvested, weighed and photographed. Mice were first anesthetized with 2% pentobarbital 0.2 ml (20 mg/kg/per mouse) by intraperitoneal injection, and then euthanized by cervical dislocation. Cessation of breathing and heartbeat was used to confirm death. There were no accidental deaths during this experiment.

All mice were kept in specific-pathogen-free animal laboratories at Ningxia Medical University, air purification degree was at least 10,000 grade according to the standard requirements, ammonia concentration ≤14 mg/m³, temperature was 18–29°C, daily temperature difference was ≤3°C, relative humidity was 40–70%, air flow speed ≤0.18 m/sec, and room air pressure gradient 20–50 Pa. There was a total of 6 mice in each cage, they were fed and given water regularly every day, and the cage was cleaned every 3 days.

Tumor tissues were removed, fixed with 10% formaldehyde at room temperature overnight, and embedded in paraffin (0.4–0.5 mm-thick sections) for immunohistochemical staining analysis to detect the expression of CD31. Sections were blocked with 30% sheep serum (JinJiang ZhongYi JinQiao Machinery Co., Ltd.) at room temperature for 1 h. Subsequently, sections were incubated with an anti-CD31 primary antibody (1:500; cat. no. ab134168; Abcam) at 4°C overnight. Following primary incubation, sections were incubated with a HRP-conjugated secondary antibody (1:400; cat. no. A21020; Abbkine Scientific Co., Ltd.) for 1 h at room temperature. Images were taken using a Leica DM4000 B photo microscope (magnification, ×400; Leica Microsystems GmbH).

Statistical analyses were performed using GraphPad Prism software (version 6.0; GraphPad Software, Inc.). Comparisons among groups were analyzed using one-way ANOVA followed by a Dunnett's test. Data are presented as the mean ± SD. All experiments were repeated three times. P<0.05 was considered to indicate a statistically significant difference.

To determine the effect of narasin on BC cells, two ER⁺ BC cell lines, MCF-7 and T47D, and one triple-negative BC cell line, MDA-MB-231, were treated with narasin at various concentrations for 72 h. Cell viability was assessed using the MTS cell viability assay kit. As shown in Fig. 1B, narasin significantly inhibited cell viability after treatment with different concentrations of narasin (1–10 µM) for 72 h, with a half-maximal inhibitory concentration (IC₅₀) of 11.76 µM in MDA-MB-231 cells. However, lower concentrations of narasin significantly reduced the viability of MCF-7 and T47D cells, with IC₅₀ values of 2.219 and 3.562 µM, respectively. Therefore, the data suggested that narasin may have increased inhibitory effects on MCF-7 and T47D ER⁺ BC cell lines compared with MDA-MB-231 triple-negative BC cell line.

Cell migration and invasion are essential steps in metastasis. Thus, the effect of narasin on the migration of BC cells was investigated using a wound healing assay. Of note, it was found that narasin concentrations ranging from 2.5 to 5 µM could inhibit the migration of MDA-MB-231 cells. Whereas, narasin at concentrations between 0.005 and 0.05 µM significantly inhibited the migration of MCF-7 and T47D cells in a dose-dependent manner (Fig. 2A). The Transwell invasion assay further revealed that narasin dose-dependently suppressed the invasive ability of BC cells in a similar manner (Fig. 2B). The concentrations of narasin used in the migration and invasion experiments did not affect cell viability (Fig. 2A and B). Thus, this indicated that narasin can significantly inhibit the migration and invasion of ER⁺ BC cells at lower concentrations due to inhibition of cell migration rather than inhibition of cell viability. Collectively, these findings suggested that narasin may be a candidate drug for ER⁺ BC treatment.

EMT is considered to be the initial and critical step towards the metastasis of cancer cells (29). To explore the potential regulatory mechanisms of narasin in migration and invasion of ER⁺ BC cells, the expression of commonly known EMT-associated biomarkers, including E-cadherin, N-cadherin and vimentin, were examined by RT-semi-quantitative PCR and western blotting. At concentrations between 0.005 and 0.05 µM, narasin downregulated the expression of mesenchymal markers (N-cadherin and vimentin), and upregulated the expression of epithelial cell marker E-cadherin in MCF-7 cells (Fig. 2C). Moreover, the EMT transcription factors β-catenin and zinc finger E-box-binding homeobox 1 (ZEB1) were downregulated by narasin compared with the control (Fig. 2C). Consistent with this result, an IF assay confirmed that exposure to narasin reversed EMT, as indicated by the decreased membrane-localized N-cadherin and increased E-cadherin (Figs. 2D and S1A and B). These results confirmed that narasin had inhibitory effects on the migration and invasion of ER⁺ BC cells by impairing the process of EMT.

A number of reports have demonstrated that human BC cell line MCF-7 is a more suitable model for studying pre-clinical ER⁺ BC in vitro and in vivo (30–32). Therefore, to identify the potential mechanisms of narasin-mediated inhibition of ER⁺ BC metastasis and proliferation, the ER⁺ breast cancer line, MCF-7, was chosen to investigate the gene expression profiles involved in the inhibitory effects of narasin by conducting a microarray analysis. Pathway enrichment analysis, an analysis that maps genes to the KEGG pathways (25–27), revealed the involvement of several key signaling pathways, including ‘TGF-β signaling pathway’, ‘mTOR signaling pathway’, ‘JAK-STAT signaling pathway’, ‘p53 signaling pathway’ and ‘HIF-1 signaling pathway’, which play essential roles in tumor progression and metastasis (Fig. 3A and B). Carcinogenic signaling networks are usually represented as crosstalk between multiple signaling pathways, rendering single protein measurements ineffective in predicting complex cellular responses to drugs. Further enrichment analysis was performed to predict the underlying interaction network based on the DEG profiles (33). The data showed that there were some interactions between these signaling pathways (Fig. 3B and C). Then, the protein-protein interaction networks were examined using data from the STRING database. The results showed that the DEGs of signaling pathways formed tightly connected networks (Fig. 3C), suggesting that these signaling pathways work as a functional module at the protein level. Essential genes that are involved in the function of TGF-β and JAK/STAT3 signaling, including ‘SMAD3’ and ‘STAT3’, are highlighted in Fig. 3C, and were significantly downregulated by narasin treatment in the presence of 0.05 µM in the heatmap (Fig. 3D). Further pathway analysis of these DEGs revealed several pathways that were affected by narasin inhibition. Among these pathways we chose to focus on the top-ranked TGF-β/SMAD3 and IL-6/STAT3 signaling pathways.

To further explore whether narasin regulated the TGF-β/SMAD3 signaling pathway in ER⁺ BC cells (34), the effect of narasin on SMAD2/3 phosphorylation, which is a crucial mediator of TGF-β/SMAD3 signaling (35–39), was examined. Narasin at tested concentrations (0.005 to 0.05 µmol/l) inhibited SMAD2/3 phosphorylation without TGF-β in a dose-dependent manner in ER⁺ BC cell lines MCF-7 (Fig. 4A) and with or without TGF-β in a dose-dependent manner in ER⁺ BC cell lines MCF-7 and T47D (Fig. 4B-C). The results showed that TGF-β triggered SMAD2/3 phosphorylation in MCF-7 and T47D cells (Fig. 4B and C). Treatment with narasin (0.005 to 0.05 µmol/l) notably inhibited TGFβ-induced SMAD2/3 phosphorylation in a dose-dependent manner in MCF-7 and T47D cells without affecting total SMAD2/3 expression (Fig. 4B and C).

To explore the interaction between narasin and the TGF-β receptor, a molecular docking experiment was performed using Autodock. The optimized binding model of narasin with the TGF-β receptor is shown in Fig. 4D. The narasin molecule was bound to the ATP binding pocket of the TGF-β receptor and was stabilized by hydrogen bonds and hydrophobic interactions. Hydrogen bonds between the amino of Lys232 and the hydroxyl of narasin, and guanine of Arg294 with oxygen of narasin, were observed between the receptor and ligand, which can significantly increase the binding ability. Moreover, the hydrophobic interaction, formed by ALKYL 1 groups or aromatic ring of Asp351, His285, Asp290, Tyr295, stabilized the binding complex in aqueous solution. Hydrophilic interactions between the protein and ligand, which included Lys337, Arg338, Glu284 and Glu209, impacted the molecular chemical conformation of the ligand. These three types of molecular interactions promoted the biological function of narasin to inhibit tumor cell proliferation and metastasis.

The SMAD2/3 complex translocates into the nucleus to regulate the expression of target genes with other DNA-binding transcription factors (40,41). Therefore, IF staining was used to verify the effect of narasin on SMAD3 nuclear translocation in ER⁺ BC cells. The results showed that narasin suppressed TGF-β-induced SMAD3 nuclear translocation in a concentration-dependent manner, with the effective inhibition concentration at 0.005 to 0.05 µmol/l (Figs. 4E and S1C). To examine whether TGF-β-induced EMT was suppressed by narasin, the TGF-β-induced expression of E-cadherin and N-cadherin was examined by IF staining in MCF-7 cells.

Of note, MCF-7 cells treated with 5 ng/ml TGF-β led to a significant reduction in epithelial phenotype marker E-cadherin and an increase in the mesenchymal phenotype marker N-cadherin (Figs. 4F and S1D and E). However, cells exposed to narasin (0.005 to 0.05 µmol/l) showed a dose-dependent increase in the expression of E-cadherin and a decrease in the expression of N-cadherin compared with the TGF-β only group (Fig. 4F). Collectively these results indicated that narasin could inhibit the phosphorylation of SMAD3, block the binding of the TGF-β receptor with its ligand, and thus inactivate the downstream SMAD2/3-mediated EMT signaling pathway.

Aside from the TGF-β/SMAD signaling-mediated EMT process, STAT3 may also directly mediate EMT in cancer metastasis (9,42). IL-6 is a direct upstream mediator of p-STAT3. In the present study, the effect of narasin on IL-6-induced STAT3 phosphorylation was investigated. The results showed that 100 ng/ml IL-6 notably upregulated STAT3 phosphorylation in ER⁺ BC cells, and that 0.05 µmol/l narasin effectively inhibited STAT3 phosphorylation in MCF-7 and T47D cells without affecting total STAT3 expression (Fig. 5A). As shown in Fig. 5A, with or without IL-6, narasin (0.005 to 0.05 µmol/l) also inhibited AKT phosphorylation and did not affect total AKT expression. Constitutive or inducible phosphorylation at Tyr705 of STAT3 is vital for promoting the biological functions of STAT3, including dimerization and cytoplasmic-to-nuclear translocation of STAT3. It was found that narasin inhibited IL-6-induced STAT3 nuclear accumulation (Figs. 5B and S1F), with the maximum inhibition observed at 0.005 to 0.05 µmol/l.

To further determine whether narasin suppressed IL-6-induced EMT, the IL-6-induced expression and distribution of E-cadherin and N-cadherin were examined by IF staining in MCF-7 cells. Treatment with IL-6 decreased the expression of E-cadherin and increased the expression of N-cadherin (Figs. 5C and S1G and H). In contrast, cells exposed to narasin at concentrations of 0.005 to 0.05 µmol/l showed a dose-dependent increase in E-cadherin expression and a decrease in N-cadherin expression. These data indicated that narasin could inhibit EMT via TGF-β/SMAD3 and IL-6/STAT3 signaling, which suppresses the migratory and invasive abilities of ER⁺ BC cells.

As commonly known, the lungs, brain and bones are the primary sites for BC metastasis (43), although nearly any tissue in the body can be metastasized by BC. In the present study, the widely used mouse left ventricle injection tumor metastasis model with MCF-7-Luc cells was used to investigate the potential inhibitory effects of narasin against BC metastasis. Following cancer cell injection, mice were treated with narasin for the duration of the experiment, and then a bioluminescence imaging assay was used to examine the effects of this treatment. As shown in Fig. 6A and B, intraperitoneal injection of narasin at doses of 0.5 and 1.5 mg/kg reduced brain and lung metastases, achieving ~76.2 and 87.5% inhibition compared with the untreated control group on day 50. Besides, no significant differences in body weight were detected among the three groups during treatment periods (Fig. 6C), which may indicate that the toxicity of narasin was low at the effective dose used to inhibit tumor growth. Besides, Fig. 6D shows that narasin effectively suppressed the phosphorylation of both p-SMAD2/3 and p-STAT3, without altering the total protein expression in the treatment groups as compared with the controls.

As the inhibition of primary BC tumor metastasis was observed following narasin treatment, the in vivo antitumor activities of narasin were investigated using the nude mouse subcutaneous xenograft model with MCF-7 cells. Intraperitoneal administration of narasin (0.5 and 1.5 mg/kg, 14 days) significantly decreased tumor volume (Fig. 7A) and tumor weight (Fig. 7B) without noticeable weight changes (Fig. 7C). The percentage of tumor growth inhibition of narasin was 14.9 and 40.1% at 0.5 and 1.5 mg/kg, respectively. When pulling the skin of each mouse back to expose an entire tumor, it was found that narasin-mediated suppression of tumor growth was associated with angiogenesis inhibition, as shown in the representative image from each group (Fig. 7D and E). The number of CD31⁺ endothelial cells was notably decreased in the high dose treatment group (Fig. S2). Additionally, the skin color and organs of the nude mouse were mostly normal, as shown in Fig. 7E, which may suggest that the mice could tolerate the effective doses of narasin.