MCF-7 human breast cancer cells were obtained from the Department of Pathophysiology, Basic Medical College of Wuhan University (Wuhan, China). Cells were maintained in DMEM (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) at 37°C in a humidified incubator supplemented with 5% CO₂. To inhibit expression of pSTAT3 and VASP, MCF-7 cells were treated with 15 µM NSC74859 for 24 h before western blot analysis. A total of 1.5×10⁵ cells were plated in 2 ml growth medium without antibiotics in 6-well plates. A total of 10 µl Lipofectamine™ 2000 and 4 µg pCGN-HAM-STAT3 or pCGN-HAM-vector diluted in 50 µl Opti-MEMI Reduced Serum medium were mixed gently and incubated for 20 min at room temperature. pCGN-HAM-STAT3 was previously constructed in our laboratory with primer, STAT3, forward, 5′-AATCTCGAGGGATGGCCCAATGGAATCAGCTA-3′, and reverse, 5′-AATGTCGACTCACATGGGGGAGGTAGCGC-3′. The resultant mixture was added to 1.5×10⁵ cells and 100 µl Opti-MEMI medium was added to a blank control groups. Cells were incubated for 48 h prior to analysis of transgene expression. MCF-7 cells were also treated with IL-6 (10 ng/ml, 37°C) for 30 min before western blot analysis to explore the effect of IL-6-induced STAT3 phosphorylation on VASP protein expression.

A total of 16 human breast cancer and matched adjacent tissues were collected between July 2016 to May 2017 at Zhongnan Hospital of Wuhan University (Wuhan, China). Prior to the surgery, patients signed informed consent forms for the present study, which included consent for the use of their tissue for research. The inclusion criteria were: i) Adults aged 18–60; ii) primary invasive tumors; iii) no metastasis; iv) no previous therapy prior to surgical resection. The exclusion criteria were: i) 18> age >60; ii) male breast cancer; iii) with metastasis; iv) recurrent lesions. The study was endorsed by the Ethics Committee of the Zhongnan Hospital of Wuhan University and was conducted in accordance with the principles of the Declaration of Helsinki.

Total RNA was extracted from breast cancer and matched normal tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and was used for first-strand cDNA synthesis using the RevertAid™ kit (Fermentas; Thermo Fisher Scientific, Inc.). For reverse transcription, RNA strand was incubated with 1 µl RNAase inhibition 2 µl 10 mM dNTP mix at 37°C for 5 mins. cDNA was synthetized with reverse transcriptase at 42°C for 60 mins and 72°C for 10 mins. PCR amplification of cDNA was conducted with 10 µl of 2X SYBR Master mix (Toyobo Life Science) using the following primers: STAT3, forward 5′-GCACTTGTAATGGCGTCTTCA-3′ and reverse 5′-TCTAGCTGTTCTGCCTCACCT-3′; vasodilator-stimulated phosphoprotein (VASP), forward 5′-AAAGTCAGCAAGCAGGAGGA-3′ and reverse 5′-ATTCATCCTTGGGGGTTTTC-3′; and internal control GAPDH, forward 5′-TGATGACATCAAGAAGGTGGTGAAG-3′ and reverse 5′-TCCTTGGAGGCCATGTGGGCCAT-3′. Cycling conditions were 95°C for 10 sec, 65°C for 20 sec and 72°C for 15 sec (40 cycles).

MTT assays (Amresco, LLC) were performed to determine proliferation. Cells were seeded in 96-well plates at 2×10³ cells/well and incubated. Cells were treated with various SSb2 concentrations (0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20 and 50 µM) for 48 h. Following treatment with 100 µl MTT (5 mg/ml), cells were incubated for further 4 h at 37°C. Medium was discarded and 150 µl DMSO was added (Sigma-Aldrich; Merck KGaA). Absorbance at 490 nm was determined using an ELISA plate reader (Infinite^® 200 PRO; Tecan Group, Ltd.). The viability of SSb2-treated cells was calculated by comparing the absorbance of each group with DMSO-treated cells, which were arbitrarily assigned 100%.

The effect of SSb2 on breast cancer cell migration was measured using wound-healing assays. Cells were plated at 1×10⁵ cells/well in 6-well plates and allowed to grow to 60–70% confluence. Cells were serum-starved for 12 h, washed twice with PBS and supplied with DMEM supplemented with 10% FBS and various concentrations of SSb2 (0.2, 1 and 5 µM). Wounds were created by scratching across the cell monolayer and cells within the same fields of view were captured at 0, 24 and 48 h under a light microscope (Olympus, Japan). The cell migration area was measured between cut regions using Image J software (V1.8.0; National Institutes of Health) and normalized to control cells.

Cells treated with varying concentrations of SSb2 (0.2, 2, 10, 20 and 50 µM) for 48 h were plated at 200 cells/dish and incubated at 37°C in a 5% CO₂ incubator. Following treatment with different concentrations of SSb2, the cells were incubated for two additional weeks and the clones stained with crystal violet (0.5%, m/v) for 30 mins at room temperature. Cells were washed with PBS (4×5 min), clones were counted and images were captured. Clone formation rate (%)=(number of clones/number of inoculated cells) ×100.

Following washes with PBS, cells were incubated in ice-cold RIPA assay buffer (20 mM Tris-HCl pH 7.5, 1 mM phenyl-methylsulfonylfluoride, 150 mM NaCl, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.25% deoxycholate, 1.5 mM MgCl₂, 1 mM egtazic acid, 10 mM NaF, 1% Triton X-100 and 10 mM pervanadate). After 15 min on the ice, mixtures were centrifuged (15,000 × g, 15 min) to extract proteins. A Coomassie Brilliant Blue assay was used to determine the protein concentration in the supernatant. Lysates were mixed with 5X sample buffer containing 2-mercaptoethanol and boiled for 5 min prior to loading onto 12% gels and separation by SDS-PAGE. Proteins were then transferred to nitrocellulose membranes over 2 h. Membranes were incubated at 4°C overnight with antibodies, following a blocking with 5% (w/v) BSA in Tris-buffer for 1.5 h at room temperature. Anti-VASP (1:1,000; cat. no. sc-376226), anti-STAT3 (1:2,000; cat. no. sc-293151), anti-phospho (p)STAT3 (1:2,000; cat. no. sc-56747), anti-c-myc (1:1,000; cat. no. sc-53854), anti-cyclin D1 (1:1,000; cat. no. sc-450), anti-MMP2 (1:1,000; cat. no. sc-13594), anti-MMP9 (1:1,000; cat. no. sc-21733) and anti-GAPDH (1:2,000; cat. no. sc-66163) primary antibodies (Santa Cruz Biotechnology, Inc.) were diluted in 1X TBS containing 0.1% Tween 20 with 5% BSA. Following three washes with TBS containing 0.1% Tween 20, blots were incubated with horseradish peroxidase-conjugated secondary antibody (1:2,000; cat: 516102; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. An ECL reagent kit (Axygen; Corning Inc., Corning, NY, USA) was used to detect protein bands. Band Scan (version 5.0, Glyko lnc.) was used to quantitatively analyze each band for statistical comparison.

H&E staining was used to assess the hepatotoxicity and nephrotoxicity in Kunming mice treated with SSb2. Kunming mice (n=12, 8-weeks old, 18–24 g) were maintained under sterile conditions and fed with sterile feed and water at 18°C and a 12-h light/dark cycle. The mice were obtained from Animal Biosafety Level 3 Laboratory of Wuhan University and randomly divided into 2 groups with 6 females in each. Mice in the experimental group were administered SSb2 (30 mg/kg) daily by intraperitoneal injection for 1 month and the control group was administered same volume of saline. The experimental mice were kept at a constant 20°C with 50% humidity and with regular clean food and water. Fresh liver and kidney tissues were obtained from the mice and tissues were cut into small pieces, fixed with 4% paraformaldehyde for 24 h at room temperature, embedded in paraffin and stained with H&E. The slices were stained with hematoxylin for 10 min and eosin for 30 sec at 60°C. Paraffin sections were observed under a light microscope (magnification, ×400). All experiments followed the Guidelines for Animal Experimentation of the Wuhan University (Wuhan, China) and protocols were approved by the Ethics Committee for Animal Experimentation.

Data are presented as the mean ± SEM representing multiple experiments. Student's t-tests were performed to compare data from two groups, and differences between three or more groups were analysed using one-way ANOVA analysis. Tukey's honestly significant difference procedure was used for post hoc analysis. P<0.05 was considered to indicate a statistically significant difference.

To detect STAT3 and VASP mRNA expression in breast cancer tissues, RT-qPCR analysis was performed. As presented in Fig. 1, STAT3 and VASP mRNA expression in cancer tissues were significantly increased compared with the adjacent normal tissues (P<0.05).

To study the effect of SSb2 on MCF-7 proliferation, cells were treated with SSb2 (0.1, 0.2, 0.5, 1, 2, 5, 10, 20 and 50 µM) for 48 h. Following treatment, MTT assays were performed to measure cell proliferation. As presented in Fig. 2A, the cell proliferation rate was significant inhibited compared with the control group in a dose-dependent manner (P<0.05). Western blot analysis also was performed following cell treatment with 5 µM SSb2 for 48 h. As presented in Fig. 2B, treatment with 5 µM SSb2 significantly reduced the expression of c-myc and cyclin D1, which are related to cell proliferation in the STAT3 signalling pathway, compared with the control (P<0.05).

To observe the effect of SSb2 on the migration of breast cancer cells, cells were treated with SSb2 (0.2, 1 and 5 µM) for 24 and 48 h. Following treatment, wound-healing assays were performed to evaluate MCF-7 migration. As presented in Fig. 2C, following treatment with SSb2, cell migration rates decreased along with the increased concentration of SSb2 at 24 or 48 h in a dose-dependent manner.

To determine whether SSb2 affects the clonal formation ability of MCF-7 breast cancer cells, colony formation assays were performed. Varying concentrations of SSb2 (0.2, 2, 10, 20 and 50 µM) were used to treat MCF-7 cells for 48 h. As presented in Fig. 2D, the colony formation ability was significantly suppressed using 2, 10, 20 or 50 µM SSb2 compared with the control (P<0.05) in a dose-dependent manner.

To study effects of SSb2 on pSTAT3, STAT3 and VASP protein levels in MCF-7 cells, western blot analysis was performed following cell treatment with SSb2 (0.2, 0.5 and 1 µM) for 48 h. As presented in Fig. 3, treatment with 0.2, 0.5 and 1 µM SSb2 significantly reduced pSTAT3 in MCF-7 cells compared with the control (P<0.05). SSb2 at 1 µM significantly reduced STAT3 expression compared with the control (P<0.05), while 0.2 and 0.5 µM SSb2 had no significant effect on STAT3 expression. Analysis of the pSTAT3/STAT3 ratio demonstrated that STAT3 phosphorylation was reduced by 0.5 and 1 µM SSb2. In addition, 0.5 and 1 µM of SSb2 significantly reduced VASP expression in MCF-7 cells compared with the untreated control (P<0.05).

To investigate whether SSb2 affected VASP protein expression by inhibiting STAT3 phosphorylation, cells were treated with NSC74859 (15 µM) a specific inhibitor of STAT3 phosphorylation and pSTAT3, STAT3 and VASP levels were determined by western blot. As presented in Fig. 4, pSTAT3 and VASP levels in the NSC74859-treated cells were significantly decreased compared with the control (P<0.05), while STAT3 levels exhibited no significant changes.

To observe whether STAT3 phosphorylation affects VASP expression, STAT3, pSTAT3 and VASP levels were determined in STAT3 overexpressing cells or following IL-6 treatment of MCF-7 cells. As presented in Fig. 5, STAT3 and pSTAT3 levels in STAT3 overexpressing MCF-7 cells were significantly increased compared to the control (P<0.05). Stimulation with IL-6 significantly increased STAT3, pSTAT3 and VASP levels compared with the control (P<0.05). Treatment of STAT3 overexpressing cells with IL-6 significantly increased STAT3, pSTAT3 and VASP levels compared with the control (P<0.05). In conclusion, VASP level could be only elevated when STAT3 was phosphorylated by IL-6. Thereby, STAT3 phosphorylation induced by IL-6 could affect VASP levels.

To detect the effect of SSb2 on MMP2 and MMP9 expression, MCF-7 cells were treated with 5 µM SSb2 for 48 h and expression was detected by western blot analysis. As presented in Fig. 6, MMP2 and MMP9 expression was significantly decreased in SSb2-treated cells compared with the control group.

To observe whether SSb2 treatment induces liver or kidney damage, an experimental group of Kunming mice was injected intraperitoneally with 30 mg/kg/day SSb2 for 30 days. Liver and kidney tissues from mice of the experimental and the untreated control groups were H&E stained. As presented in Fig. 7, no hepatocellular degeneration or necrosis was observed in the experimental group compared with the control group and no inflammatory cells were detected to have infiltrated the hepatic lobules or the portal area (Fig. 7A and B). There was no significant increase in the number of cells in the glomerular capillary loop and no neutrophils and lymphocyte invasion in the experimental compared with the control mice (Fig. 7C and D).