The cell lines MDA-MB-231, MCF-7, SKBR3, MCF-10A and RAW264.7 were purchased from the Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. MDA-MB-231, MCF-7 and SKBR3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (HyClone; Cytiva) and 100 U/ml penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.). MCF-10A were cultured in DMEM/F12 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 5% horse serum (Invitrogen; Thermo Fisher Scientific, Inc.), 10 µg/ml insulin (Invitrogen; Thermo Fisher Scientific, Inc.), 20 ng/ml epidermal growth factors (Invitrogen; Thermo Fisher Scientific, Inc.), 100 ng/ml cholera toxin (Sigma-Aldrich; Merck KGaA) and 0.5 µg/ml hydrocortisone (Invitrogen; Thermo Fisher Scientific, Inc.). RAW264.7 were cultured in α-minimum essential medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 100 U/ml penicillin-streptomycin. All cells were placed at 37°C in a humidified incubator containing 5% CO₂.

A total of 35 BA derivatives were obtained and the compound 20 was named SH-479. To obtain SH-479, a solution of BA (2.28 g, 5 mmol) in tetrahydrofuran (10 ml) was added dropwise to a stirring solution of 2-iodoxybenzoic acid (2.1 g, 7.5 mmol) in DMSO (30 ml) at 23°C. The reaction mixture was stirred for 2 h at 23°C and then diluted with ethyl acetate (AcOEt; 30 ml) and washed with brine (50 ml). The organic layer was dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash chromatography (petroleum ether/AcOEt; 8/1 v/v) to give compound 1 (2.05 g, 90%) as a white solid (melting point, 251–253°C) (23). The concentrations used for BA are 0–25 µM. The concentrations used for SH-479 are 0–10 µM. The durations of the treatments was 72 h. The effect of BA and SH-479 on cell viability was determined using the MTS method following the instructions from the Cell Titer 96 Aqueous One Solution Cell Proliferation assay (Promega Corporation). Briefly, the cell density was adjusted to 10⁵/ml. The cell suspension was inoculated into 96-well plates at 100 µl/well and cultured overnight. Cells were treated with BA (0–25 µM) and SH479 (0–10 µM) and cultured for 72 h. MTS solution (20 µl) was added to each well and incubated for 4 h. The absorbance was read at 490 nm on a VERSA max microplate reader (Molecular Devices, LLC).

The cytotoxicity of SH-479 on MDA-MB-231 cells was assessed using Live and Dead assay kit (Invitrogen; Thermo Fisher Scientific, Inc.) as previously described (29). Briefly, 2,500 cells were seeded into 96-well plates. After cell attachment, the cells were treated with SH479 (0–10 µM) for 30 h. Cells were stained with Live and Dead reagent (5 µM ethidium homodimer and 5 µM calcein-AM) and were incubated at 37°C for 45 min. Live and dead cells were observed under a fluorescence microscope (magnification, ×20).

MDA-MB-231 were seeded into 96-well plates (5,000 cells/well) and treated with SH-479 (0–10 µM). After 3 days, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and subsequently incubated for 5 min at room temperature with PBS containing 0.1% Triton-X 100 and with rhodamine conjugated phalloidin (cat. no. R415; 1:200 in PBS; Thermo Fisher Scientific, Inc.) for 30 min in the dark at room temperature. F-actin staining was observed under an inverted fluorescence microscope (Olympus Corporation; magnification, ×40).

MDA-MB-231 cells were lysed on ice using RIPA buffer (cat. no. R0278; Sigma-Aldrich; Merck KGaA). Protein concentration was evaluated using the BCA method. Proteins (30 µg) were separated by 10% SDS PAGE and transferred onto PVDF membranes. After blocking for 1 h in 5% skimmed milk dissolved in PBS, membranes were incubated with primary antibodies against cyclin E (cat. no. SC248 1:500 Santa Cruz Biotechnology, Inc.), PARP (cat. no. AY 0276; 1:1,000; Abways Technology, Inc.), cleaved PARP (cat. no. CY5035; 1:500; Abways Technology, Inc.) and β-actin (cat. no. A5441; 1:5,000; Sigma-Aldrich; Merck KGaA) at 4°C overnight. The membranes were washed three times with PBST (0.1% Tween) and incubated with infrared dye-labeled secondary antibody (LI-COR Biosciences) for 1 h at room temperature. The signal on the membrane was visualized using LI-COR Odyssey System.

The effect of SH-479 on cell cycle was determined by flow cytometry. Briefly, cells were serum-starved for 24 h and treated with 5 µM SH-479 for 12 h. Subsequently, cells were fixed in 70% ethanol at 4°C overnight. Cells were stained with propidium iodide (cat. no. R37108; Thermo Fisher Scientific, Inc.) for 30 min at room temperature. Cell cycle analysis was conducted using a flow cytometry (Becton, Dickinson and Company). The software used for the analysis was FlowJo VX10.0 (FlowJo LLC).

The migration assay was performed in Boyden chambers (Corning Inc.). MDA-MB-231 cells (80,000) were suspended in 100 µl DMEM medium and treated with SH-479 (0–5 µM). The mixture was added to the upper well of Boyden chambers. DMEM medium (600 µl) supplemented with 2% FBS was added in the lower chamber. After 8 h incubation, cells that have migrated to the lower chambers were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet 5 min at room temperature. Pictures of the cells were taken under an inverted microscope (Olympus Corporation; magnification, ×20) and quantification was performed using Image-Pro 6.0 software (Media Cybernetics, Inc.).

Since osteoclasts originate from monocytes/macrophages in the bone marrow, monocytes were studied in the present study. A total of 2×10³ MDA-MB-231 cells and 5×10³ RAW264.7 cells were co-cultured in 24-well plate (30). Cells were cultured in α-MEM medium containing 10% FBS and SH-479 (0–5 µM). After 7 days, cells were fixed with 4% paraformaldehyde 5 min at room temperature and incubated for 5 min with PBS containing 0.1% Triton-X 100 at room temperature. Cells were subsequently stained with tartrate-resistant acid phosphatase (TRAP) using Leukocyte acid phosphatase kit (Sigma-Aldrich; Merck KGaA) for 5 min at 37°C. TRAP positive cells were imaged under an inverted microscope (Olympus Corporation; magnification, ×40) and counted using Image-Pro software 6.0 (Media Cybernetics, Inc.).

National Institutes of Health guide for the care and use of Laboratory animals has been followed for these experiments. Animals experiments were approved by the Ethics Committee of East China Normal University (ECNU; approval no. m20151002). The C57bl/6 wild type male mice (8 weeks; n=3) were purchased from the ECNU, had free access to food pellets and tap water. They were maintained under the standard 12-h dark/light cycle, with controlled temperature (24±2°C) and relative humidity (55–60%). Mice were euthanized by intraperitoneal injection of 3% pentobarbital sodium (200 mg/kg) to collect bone marrow. Femurs and tibias were collected from mice and the femoral head and ankle joint were preserved in order to ensure the integrity of the bone marrow cavity. The bone marrow cavities of the femurs and tibias were opened, and the bone marrow was flushed with PBS solution containing 1% FBS (31,32). The rinsing solution was collected and centrifuged at 125 × g for 5 min at 4°C. After centrifugation, cells collected were resuspended in PBS solution containing 1% FBS and counted. Cells were then transferred into Eppendorf tube at the concentration of 10⁶/100 µl for later use and seeded in a 6 cm plate and cultured in a cell incubator. Cells were treated with 10 µM SH-479 for 48 h at 37°C in the 6 cm plate. After 48 h, one million cells were incubated with 0.5 µl antibody (CD3-FITC, cat. no. 100204; CD4-PerCP-Cy5.5, cat. no. 10043; CD8-APC, cat. no. 344722; CD11b-APC, cat. no. 101212; Ly6C-PerCP-Cy5.5, cat. no. 128012; Ly6G-PE, cat. no. 127608; BioLegend, Inc.) at 4°C for 30 min. Cells were then washed with 2% PBS, and T cells and MDSCs (CD11b+) were detected by flow cytometry (Becton, Dickinson and Company). Results were analyzed using FlowJo VX 10.0 software (FlowJo LLC).

The data were expressed as the means ± standard deviation. Differences between two groups were determined using paired Student's t-test. Comparisons between multiple groups were determined by one-way ANOVA followed by Dunnett's post hoc test. All statistical analyses are performed using GraphPad Prism 5.0. (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

The effect of BA (Fig. 1A) and the 35 BA derivatives on TNBC cell viability was assessed. Because only SH-479 showed a significant effect, the results from other BA derivatives were not presented. The results demonstrated that SH-479 (Fig. 1B) significantly inhibited the viability of MDA-MB-231 cells in a concentration-dependent manner after 72 h compared with the control group (Fig. 1D). However, the viability of MDA-MB-231 cells was significantly inhibited by more than 50% when higher concentration of BA was added (Fig. 1C).

When using drugs to treat tumors, the common side effects of drugs are toxicity to normal cells. The results showed that only 30% of MCF-10A viability was inhibited after SH-479 treatment (0.1–10 µM; Fig. 2A). Furthermore, SH-479 did not significantly inhibit the viability of the luminal type cell line MCF-7 and Her2 type cell line SKBR3 at low concentration (Fig. 2A). In addition, SH-479 had no effect on the morphology of the normal breast tissue cells MCF-10 (Fig. 2B). However, when MDA-MB-231 cells were treated with SH-749, cells seemed to shrink and their morphology changed in a dose-dependent manner (Fig. 2B).

The morphological changes of cell shrinkage might be caused by cell death or by structural changes within the cells. The results demonstrated that after 30 h treatment with SH-479 (0.1–10 µM), MDA-MB-231 cells presented morphological shrinkage, although no cell death was observed. Green fluorescence represents living cells and red fluorescence represents dead cells (Fig. 3A). After 72 h treatment with SH-479 (0.1–10 µM), the microfilaments of MDA-MB-231 cells were altered according to F-actin staining. The red fluorescence indicates the cytoskeleton and the blue fluorescence indicates the nucleus (Fig. 3B). Because SH-479 can inhibit TNBC cell viability, cell cycle and apoptosis were determined. The expression of cyclin E starts in the middle phase of G1 phase and gradually decreases after reaching the peak in G1/S phase. Cyclin E is therefore the key protein of G1 to S phase. The results demonstrated that SH-479 had no effect on cell cycle transition from G1 phase to S phase. There was no change in the expression of proteins involved in cell cycle and apoptosis in MDA-MB-231 cells treated with SH-479 (Fig. 3C). In order to further verify the effect of SH-479 on MDA-MB-231 cell cycle, cell cycle was determined by flow cytometry. The results demonstrated that after 12 h treatment with 5 µM SH-479, the cell cycle of MDA-MB-231 cells was not modified (Fig. 3D).

The incidence of bone metastases in breast cancer is high, and TNBC usually leads to osteolytic bone metastasis. In the present study, MDA-MB-231 cell treatment with SH-479 induced a significant decrease in cell migratory ability in a dose-dependent manner (Fig. 4A). Furthermore, SH-479 treatment led to a decrease in the capacity of MDA-MB-231 cells to induce osteoclast differentiation in a dose-dependent manner (Fig. 4B).

When bone metastasis occurs in patients with breast cancer, immune cells in the bone marrow microenvironment play an important role in the colonization and development of cancer cells. The results from the present study demonstrated that the number of CD3+CD4+T lymphocytes in the bone marrow microenvironment was significantly increased following treatment with SH-479 for 48 h (Fig. 5A). Furthermore, the number of MDSCs in the bone marrow microenvironment also serves a role in breast cancer metastasis to bones, and it can indirectly promote the development of breast cancer cells in the bone microenvironment by inhibiting the function of immune cells. The results from the present study demonstrated that SH-479 treatment decreased the number of MDSCs in bone microenvironment (Fig. 5B).