Salidroside (purity, >99%) was purchased from the National Institute of Pharmaceutical and Biological Products (Beijing, China). Salidroside was dissolved in water and filtered through a 0.22-μm filter before use. Propidium iodide (PI) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of PI and MTT were prepared by dissolving 1 mg of each compound in 1 ml of phosphate-buffered saline (PBS). The solution was protected from light, stored at 4°C, and used within 1 month.

For western blot analysis, the following antibodies were used: mouse monoclonal anti-human β-actin (Sigma-Aldrich), mouse monoclonal anti-human Bcl2, mouse monoclonal anti-human Bax, mouse monoclonal anti-human p21, mouse monoclonal anti-human cyclin D1, mouse monoclonal anti-human cyclin D3, mouse monoclonal anti-human matrix metalloproteinase (MMP)-2, mouse monoclonal anti-human MMP-9, mouse monoclonal anti-human p38MAPK, mouse monoclonal anti-human phosphorylated (p)-p38MAPK, mouse monoclonal anti-human c-Jun N-terminal kinase (JNK), mouse monoclonal anti-human p-JNK, mouse monoclonal anti-human ERK1/2 and mouse monoclonal anti-human p-ERK1/2 (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibody HRP-conjugated goat anti-mouse IgG was purchased from Amersham Biosciences (Uppsala, Sweden).

Human breast cancer cell line, MCF-7, was purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences, (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco-BRL, Gaithersburg, MD, USA), 100 U/ml of penicillin and 0.1 mg/ml of streptomycin at 37°C in a humidified atmosphere of 5% CO₂.

Cell proliferation was determined by MTT assay as previously described (24). Briefly, 1×10⁴ cells/well were cultivated in 100 μl of culture medium in 96-well flat-bottomed plates (Corning Inc., Corning, NY, USA) and incubated with different concentrations of salidroside (0.01–50 μM) for 72 h, followed by the addition of 10 μl of MTT solution (5 mg/ml, dissolved in PBS; Sigma-Aldrich). After a 4-h incubation, 100 μl of SDS (10%, w/v, dissolved in 0.01 M HCl; Sigma-Aldrich) was added and mixed thoroughly to dissolve the formazan crystals at 37°C. After shaking the plates for 10 min, the absorbance was read at 570 nm in an ELISA plate reader (Molecular Devices Corp., Sunnyvale, CA, USA).

Cells were seeded in 6-well culture plates at a density of 1×10⁴ cells/well. After being cultured for 24 h, the cells were treated with different concentrations of salidroside (0, 5, 20 and 40 μM). After 14 days, the cells were washed, fixed by paraformaldehyde, and stained with Giemsa for 10 min. Then extra Giemsa was washed 3 times by ddH₂O, and the colonies were photographed using a digital camera. The visible colonies in each group were counted.

Apoptotic cell death induced by salidroside was quantified by flow cytometry using the Annexin V-fluorescein isothiocyanate (FITC) kit following the manufacturer’s instructions. Briefly, the cells were plated at a density of 3×10⁵ cells/well in a 6-well plate and incubated with different concentrations of salidroside (0, 5, 20 and 40 μM) for 48 h. Floating cells as well as residual attached cells were collected and washed twice with PBS. The cell pellets were resuspended in 500 μl of 1X binding buffer at a concentration of 1×10⁶ cells/ml. Five microliters of Annexin V-FITC and PI was added to the cell suspension for 10 min at room temperature, stained samples were examined using a Coulter Epics XL flow cytometer (Beckman Coulter, Miami, FL, USA), and the data were analyzed using CellQuest software (BD Biosciences, San Jose, CA, USA). Experiments were performed in triplicate. In addition, we also detected Bax and Bcl-2 expression by western blotting as an additional indicator of apoptosis.

Caspase-3, -8 and -9 activity was measured using the Caspase Colorimetric Assay kit (Millipore Corporation, Billerica, MA, USA) according to the manufacturer’s instructions. Briefly, the cells after treatment for 48 h were harvested and lysed in lysis buffer on ice for 10 min and then centrifuged at 10,000 × g for 1 min. After centrifugation, the supernatants were incubated with caspase-3, -8 and -9 substrates in reaction buffer. Samples were seeded into a 96-well flat-bottom microplate at 37°C for 1 h. Samples were analyzed at 405 nm in a microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). The relative caspase activity of the untreated group (0 μM salidroside treatment) was taken as 100. Each assay was conducted in triplicate.

To determine the cell cycle distribution, the cells were plated in 60-mm dishes and treated with different concentrations of salidroside (0, 5, 20 and 40 μM) for 48 h. After treatment, the cells were collected by trypsinization, fixed in 70% ethanol, and kept at −20°C overnight for fixation. Cells were washed twice with PBS, and then resuspended in 1 ml of PBS containing RNase (100 μg/ml) and PI (40 μg/ml) in the dark for 30 min at room temperature. The distribution of cells in the cell cycle phases was analyzed from the DNA histogram with a FACS Caliber flow cytometer (Becton-Dickinson, San Jose, CA, USA). The data were analyzed using CellQuest software (BD Biosciences). Furthermore, we also detected p21, cyclin D1 and cyclin D3 expression by western blotting as an additional indicator of cell cycle arrest.

To assess the effect of salidroside on cell migration and invasion, the migration and invasion assays were performed using Transwell insert chambers (Corning Inc.). For the migration assay, the cells were incubated with different concentrations of salidroside (0, 5, 20 and 40 μM) for 48 h. After treatment, a total of 1×10⁵ cells were plated into the upper chamber in serum-free DMEM. Medium containing 20% FBS in the lower chamber served as chemoattractant. After being cultured for 24 h, the media were removed from the upper chamber by wiping with a cotton swab and cells that migrated to the lower surface of the filter were fixed in 70% ethanol for 30 min followed by staining with 0.2% crystal violet for 10 min. Cell migration was counted by counting five random fields per filter under a light microscope (Olympus, Tokyo, Japan).

For the invasion assay, after treatment, 3×10⁵ cells were incubated in the upper chambers pre-coated with Matrigel (BD Biosciences) in serum-free DMEM, and the subsequent steps were consistent with the migration assay. The number of cells invading the Matrigel was counted in five randomly selected fields using a light microscope (Olympus). All experiments were performed in triplicate.

Intracellular changes in ROS were determined using the MGT Live Cell Fluorescent ROS Detection kit (MGT, Inc., USA) as described previously (24). Briefly, cells were plated in a 96-well plate (25×10³ cells/well) and were treated with different concentrations of salidroside (0, 5, 20 and 40 μM) for 48 h. After treatment, the cells were further incubated with 20 μM 2′,7′-dichlorofluorescein diacetate in Hank’s balanced salt solution (HBSS) at 37°C for 30 min in the dark. Subsequently, the cells were harvested and washed with HBSS and analyzed for DCF fluorescence using a Synergy HT Multi-Mode microplate reader (BioTek Instruments, San Jose, CA, USA).

The cells were collected by trypsinization and lysed in radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich) with the addition of protease inhibitors (Roche) and phosphatase inhibitors (Sigma-Aldrich) for 30 min on ice. Then the homogenates were centrifuged at 14,000 rpm at 4°C for 30 min to remove insoluble material, and the supernatants were collected for protein concentration determination using the BCA protein assay kit (Sigma-Aldrich). The cell extracts (20 μg of protein) were separated on a sodium dodecyl sulfate-polyacrylamide electrophoretic gel (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked with 5% dry milk in PBS and incubated with the primary antibodies. After incubation with primary antibodies, the membranes were washed in PBS and incubated with secondary horseradish peroxidase-coupled goat anti-mouse antibodies. The proteins were detected and protein bands were visualized with enhanced chemiluminescence reagent (ECL; Sigma-Aldrich). The integrated density value (IDV) was analyzed with a computerized image analysis system (Fluor Chen 2.0) and normalized with that of β-actin.

Female BALB/c nude mice at 6–7 weeks of age were obtained from the Experimental Animal Center of Jilin University (Changchun, China) and were housed under standard conditions. MCF-7 cells were trypsinized and washed with PBS and suspended in DMEM-free serum. A total of 2×10⁶ cells were injected into the flanks of nude mice. Tumor growth was measured every 7 days, and tumor volume was estimated as length × width × height × 0.5236. When tumors grew to an average volume of 100 mm³, the mice were divided randomly into 2 groups (10 mice/group). The control group received 1% PBS in deionized water. The treatment group was treated with salidroside (50 mg/kg body weight) intraperitoneally on alternate days for 3 weeks. The doses were selected based on a previous experiment (22). The tumor size was measured using a caliper on days 7, 14 and 21 of treatment. On day 21, the animals were sacrificed using chloroform, and tumor tissues were isolated and weighed. In addition, spleen tissues were collected and cultured for a splenocyte surveillance study to assay splenocyte proliferation as previously described (25). All procedures were in agreement with Jilin University Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee, Jilin University (Changchun, China).

All experiments were performed at least three independent times, and the results are expressed as the mean ± standard deviation (SD). For statistical comparison of quantitative data between groups, analysis of variance (ANOVA) or Student’s t-test was performed. All statistical analyses were performed using the GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA, USA) and the SPSS software (version 16.0; SPSS Inc., Chicago, IL, USA) for Windows^®. P-values <0.05 were considered to be statistically significant.

To determine the cytotoxic effects of salidroside on MCF-7 cells, MTT assay was carried out. It was found that salidroside significantly inhibited the viability of the MCF-7 cells in a dose-dependent manner (Fig. 1B). In the MCF-7 cells, as shown in Fig. 1B, a significant inhibitory effect was observed at 1 μM, which reached a maximum level at 50 μM. The IC₅₀ value (the effective dose that inhibits 50% of growth) for the treatment of MCF-7 cells by salidroside was 19.48 (P<0.05). Based on the results, we chose concentrations of 5, 20 (IC₅₀) and 40 μM (2x IC₅₀) salidroside for further treatments throughout the study.

Next, the effect of salidroside on the cell colony formation of MCF-7 cells was also analyzed. As shown in Fig. 1C, salidroside significantly inhibited the colony formation of MCF-7 cells in a dose-dependent manner (P<0.05).

To determine whether the growth inhibitory effect of salidroside is associated with the induction of apoptotic cell death, flow cytometry was performed. MCF-7 cells were exposed to various concentrations (0, 5, 20 and 40 μM) of salidroside for 48 h and analyzed by flow cytometry. As shown in Fig. 2A, we observed a dose-dependent increase in apoptotic cells in the presence of salidroside in MCF-7 cells.

To examine the contribution of caspases in the salidroside-induced apoptosis, the role of caspase-3, -8 and -9 was investigated. The results demonstrated that treatment of MCF-7 cells with salidroside resulted in a significant increase in the activities of caspase-3, -8 and -9 in a dose-dependent manner (Fig. 2B–D).

To determine the potential mechanism involved in the effect on cell apoptosis by salidroside, expression levels of apoptosis-related proteins, Bax and Bcl-2, were examined by western blot analysis. Western blot analysis displayed a significant upregulation of Bax expression, and downregulation of Bcl-2 expression in the MCF-7 cells following treatment with salidroside in a dose-dependent manner (P<0.05, Fig. 2E and F).

To determine whether the growth inhibitory effect of salidroside is associated with cell cycle arrest, flow cytometry was performed to determine the cell cycle distribution. The MCF7 cells were treated with various concentrations of salidroside (0, 5, 20 and 40 μM) for 48 h. It was found that salidroside significantly increased the percentage of cells in the G0/G1 phase in a dose-dependent manner (P<0.05), while the percentage of cells in the S phase significantly decreased following salidroside treatment (P<0.05, Fig. 3A and B).

Next, we analyzed the effects of salidroside on the expression of cell cycle-associated proteins, such as cyclin D1, cyclin D3 and p21 by western blotting. As shown in Fig. 3C and D, salidroside treatment markedly increased p21 expression, while cyclin D1 and cyclin D3 expression was significantly decreased in the MCF-7 cells in dose-dependent manner (P<0.05).

We were particularly interested in whether salidroside affects cell vitality, demonstrated by migration or invasion activity. Thus, cell migration and invasion assays were performed by Transwell assay. It was found that salidroside significantly decreased the migration of MCF-7 cells in a dose-dependent manner (P<0.05, Fig. 4A). The ability of salidroside to reduce the invasiveness of MCF-7 cells was further investigated. Transwell matrix penetration (coated with Matrigel) assay showed that salidroside markedly reduced the invasiveness of MCF-7 cells in dose-dependent manner (P<0.05, Fig. 4B).

To determine the potential mechanism involved in the effect on cell migration and invasion by salidroside, expression of MMP-2 and MMP-9 protein was determined by western blotting. Western blot analysis showed that salidroside significantly reduced MMP-2 and MMP-9 expression in the MCF-7 cells in a dose-dependent manner (P<0.05, Fig. 4C and D).

ROS are produced particularly when cells undergo chemical or environmental stress and could be one of the factors leading to cell apoptosis. Therefore, the ROS level after a 48-h treatment with salidroside was examined in the breast cancer cells by the fluorescent probe DCFH-DA. It was found that salidroside treatment significantly reduced the intracellular ROS level in the MCF-7 cells in a dose-dependent manner (Fig. 5A).

It is well known that the MAPK signaling pathway plays a crucial role in cell proliferation and survival in various cancers. In addition, ROS production has been shown to be coupled with the sustained activation of the MAPK signaling pathway for a variety of cellular effects (26). Therefore, in the present study, we next evaluated the effect of salidroside on several key downstream molecules involved in the MAPK signaling pathway. Measurements of the phosphorylation/activation pattern of p38MAPK, ERK1/2 and JNK were performed by western blotting 12 h after treatment with salidroside. It was found that salidroside treatment resulted in a marked reduction in phosphorylated p38MAPK, ERK1/2 and JNK in the MCF-7 cells in a dose-dependent manner, without altering the total protein levels of p38MAPK, ERK1/2 and JNK at different concentrations of salidroside (Fig. 5B).

We aimed to ascertain whether salidroside treatment inhibits tumor growth in a xenograft tumor model. Tumor growth was monitored for 3 weeks. On day 21, mice were sacrificed, and the tumor weight was measured. It was found that tumor weight were significantly lower in the salidroside treatment group relative to the control group (untreated group) (P<0.05, Fig. 6A and B). In addition, tumor volume was also determined at different times. The tumor volume in the salidroside treatment group was significantly diminished when compared with that in the control group (Fig. 6A and C). In addition, we employed MTT assays in modulating splenocyte proliferation to demonstrate the antitumor activities of salidroside in vivo. It was found that splenocyte cell proliferation in the salidroside treatment group significantly decreased relative to the control group (P<0.05, Fig. 6D). These data suggest that salidroside treatment suppresses the tumor growth of breast cancer in vivo.