The MCF-7 human breast cancer cell line (HTB-22™; American Type Culture Collection, Manassas, VA, USA) was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (HyClone Laboratories, Inc., Logan, UT, USA). The MTT reagent was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit polyclonal Bcl-2 (cat. no. ab7973), rabbit polyclonal MMP9 (cat. no. ab38898) and mouse monoclonal TIMP1 (cat. no. ab1827) antibodies were purchased from Abcam (Cambridge, UK) and rabbit monoclonal Bax antibody (cat. no. AJ1079a) was obtained from Abgent (Suzhou, China).

The dried red flower and leaves of the safflower (Tongrentang Co., Ltd., Beijing, China) were used to extract the polysaccharide. The dried red flower was fully impregnated into water, and was boiled four times for 1 h each time. The filtrate was combined and concentrated to a volume of 1/4 prior to adding 95% ethanol (Jinhua Chemical Reagent Co., Ltd., Guangzhou, China) to a volume of four-five times and incubated overnight at 4°C. The SPS was precipitated by alcohol precipitation and collected by centrifugation at 200 × g/min for 5 min. The SPS was dissolved in deionized water and the SPS was precipitated twice using ethanol. The SPS content was determined using a sulfuric acid-phenol method, as described previously (29–32), and the polysaccharide purity was up to 76.05%.

Cell proliferation analysis was performed using an MTT assay. Briefly, the MCF-7 breast cancer cells were seeded into 96-well plates (5×10⁴ cells/well), and following adherence, the cells were treated with SPS at concentrations of 0.04, 0.08, 0.17, 0.34, 0.68 or 1.36 mg/ml for 24, 48 and 72 h at 37°C. A total of 20 μl MTT (5 mg/ml) was then added to the medium in each well for 4 h and 150 μl dimethylsulfoxide was added. The absorbance of the 96-well plates was read using a microplate reader (Ultraviolet Spectrophotometer AquaMate-Plus; Thermo Fisher Scientific, Waltham, MA, USA) at a test wavelength of 490 nm and a reference wavelength of 570 nm.

The breast cancer cells were seeded into 48-well plates (5×10⁵ cells/well) for 8 h, prior to treatment with different concentrations of SPS (0.04, 0.20 and 0.60 mg/ml) for 24, 48 and 72 h. The cell lysates were prepared using sodium dodecyl sulfate (SDS) loading buffer. The lysates were separated using 10% SDS-PAGE gels and the proteins were transferred from the gel onto a nitrocellulose membrane (Biodee Corporation, Beijing, China) at 35 V for 3 h (constant voltage). The membrane was then blocked with TBST supplemented with 5% bovine serum albumin for 30 min prior to incubating the membrane with the specific antibodies in Tris-buffered saline with Tween-20 (TBST) containing 5% bovine serum albumin at 4°C overnight. The membrane was washed three times with TBST and then incubated with the goat anti-mouse IgG (SN133) and goat anti-rabbit (BA1054) secondary antibodies [Sunshine Biotechnology (Nanjing) Co., Ltd., Nanjing, China] for 1 h at room temperature. The bands were detected in a dark room using an Immobilon Western Chemiluminescent kit (EMD Millipore, Billerica, MA, USA).

The apoptotic rates of the MCF-7 breast cancer cells were determined using annexin V-propidium iodide (PI) staining, according to the manufacturer’s instructions (Santa Cruz Biotechnolgy, Inc., Dallas, TX, USA). Briefly, the cells (5×10⁵ cells/well) were seeded into 6-well plates for 6 h prior to treatment with SPS at the concentrations of 0.04 and 0.68 mg/ml for 48 h. The cells were then washed and resuspended in phosphate-buffered saline (HyClone Laboratories, Inc.). Annexin V (0.1 μg/μl) and PI (0.05 μg/μl) were added to the cells and incubated in the dark for 30 min on ice. The apoptotic rate was determined using fluorescent activated cell sorting (FACS) analysis (FACSCalibur Cell Sorting system; BD Biosciences, Franklin Lakes, NJ, USA).

The data were analyzed using SPSS 11.5 statistical software (SPSS, Inc., Chicago, IL, USA) and the data are expressed as the mean ± standard error of the mean. One way analysis of variance and Student’s t-test were used to analyze the results. P<0.01 was considered to indicate a statistically significant difference.

In order to investigate the antitumor activity of SPS on breast cancer cells, the MCF-7 human breast cancer cell line was used and the antitumor effects of SPS were detected using an MTT assay. As shown in Fig. 1, the MCF-7 cells were treated by SPS at concentrations of 0.04, 0.08, 0.17, 0.34, 0.68 or 1.36 mg/ml. The results demonstrated that the inhibitory rates of SPS on the MCF-7 cells increased significantly as the concentration of SPS increased.

The MCF-7 cells were also treated with various concentrations of SPS for 24, 48 and 72 h, which revealed that the antitumor effects of SPS occurred in a time-dependent manner. As shown in Fig. 1, the inhibitory rate on the breast cancer cells treated with SPS for 72 h was significantly higher compared with those treated for 24 h (P<0.01) and 48 h (P<0.05). SPS had an effective antitumor effect at the concentration of 0.68 mg/ml at various time points. The inhibitory rate at the concentration of 1.36 mg/ml was lower compared with that at 0.68 mg/ml. These data revealed that SPS has an antitumor effect and that these inhibitory effects increased in a time- and dose-dependent manner.

The duration of 72 h was selected as an appropriate time-point to treat the MCF-7 cells with SPS. This was repeated more than three times and all the samples were detected in duplicates. Untreated breast cancer cells were used as a negative control. As shown in Fig. 2, the IC₅₀ value for 72 h treatment was calculated as 0.12 mg/ml.

The MCF-7 cell apoptotic rates were determined by FACS analysis. The MCF-7 breast cancer cell line was treated with various concentrations of SPS for 48 h. The lowest concentration of SPS was 0.04 mg/ml and the highest concentration of SPS was 0.68 mg/ml. As shown in Fig. 3, the apoptotic rates of the MCF-7 cells treated with SPS at the concentrations of 0.68 and 0.04 mg/ml were 54.5 and 28.9%, respectively. As expected, the apoptotic rates of the MCF-7 breast cancer cells treated with SPS were significantly higher compared with the untreated cells. The apoptotic rates also increased in a dose-dependent manner.

Cell apoptosis is induced via the extrinsic pathway and the intrinsic pathway, which are mediated by death receptors and mitochondria, respectively. In the present study, FACS analysis demonstrated that SPS induced the apoptosis of the MCF-7 cells. Subsequent investigation aimed to determine whether the SPS-induced cell apoptosis was induced by the death receptor pathway or by the mitochondria-mediated signaling pathway. The MCF-7 breast cancer cells were treated with SPS at a concentration of 0.68 mg/ml for various time-periods. The cell lysates were prepared and the protein expression levels of Bcl-2 and Bax were detected by western blot analysis. As shown in Fig. 4, the expression of the apoptosis inhibitor, Bcl-2, reduced in a time-dependent manner, however, the expression of the apoptosis promoter, Bax, increased in the SPS-treated MCF-7 cells, suggesting that the SPS-induced cell apoptosis was dependent on the mitochondria-mediated signaling pathway.

In order to investigate the role of SPS on tumor metastasis in breast cancer, the expression levels of MMP-9 and TIMP-1 were detected by western blot analysis. The MCF-7 breast cancer cells were treated with various concentrations of SPS for 72 h and the results demonstrated that the expression of MMP-9 was downregulated and the expression of TIMP-1 was upregulated in the SPS-treated cells at the concentration of 0.6 mg/ml (Fig. 5). All these results suggested that SPS inhibited breast cancer cell metastases.