Human OS cells lines (143B and MG-63) were purchased from the American type culture collection (Manassas, VA, USA). Cell lines were cultured in RPMI-1640 containing 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a 5% CO₂ humidified atmosphere, as previously described (14). SSd (purity, >95%; authenticated by Shanghai Academy of Life Sciences, Shanghai, China) was mixed with RPMI-1640 medium and stored at room temperature.

143B and MG-63 cells (1×10⁴ cells/well) were cultured in 6-well plates in RPMI-1640 medium supplemented with 10% FBS for 24 h at room temperature. The medium was replaced with RPMI-1640 supplemented with 10% FBS containing 80 µmol/l SSd and cells were cultured for 48 h at 37°C prior to harvesting. RNA was extracted as reported previously (15). Total RNA was isolated from the cells using TRIzol reagent (Thermo Fisher Scientific, Inc.). Isolated RNA samples were quantified using spectrophotometry and stored at −80°C until further use. RNA concentrations were detected by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) and 2 µl samples were separated using gel electrophoresis on a 5% agarose gel to examine the purity of the RNA as reported previously (15,16).

Cell viability was assessed by MTS assay (Beyotime Institute of Biotechnology, Haimen, China). Cells (143B and MG-63) were cultured in 96-well plates (2,000 cells/well) RMPI-1640 supplemented with 10% FBS for 24 h at room temperature (14,17). Cells were incubated with RMPI-1640 plus 10% FBS and varying concentrations of SSd (20, 40, 60 and 80 µmol/l) for 24, 48 and 72 h at room temperature. Following, 20 µl MTS solution was added to each well and samples were incubated at room temperature for 3 h prior to absorbance measurements at 450 nm using a microplate reader. Each experiment was repeated three times.

EdU assays were performed using the Click-iT EdU Imaging kit (Promega Corporation, Madison, WI, USA) and an apollo fluorescent dye (Guangzhou RiboBio Co., Ltd., Guangzhou, China) in accordance with the manufacturer's instructions. 143B and MG-63 cells (3×10³ cells/well) were incubated in RMPI-1640 medium plus 10% FBS with 80 µmol/l SSd for 24 and 48 h at room temperature. Cells were exposed to 100 µl EdU at 37°C for 3 h and fixed at room temperature for 20 min with 4% paraformaldehyde. The number of EdU-positive and total cells was counted using a fluorescence microscope (magnification in blue light, ×20) with four non-overlapping fields per coverslip. Each experiment was repeated three times.

Cell cycle and apoptosis were determined by flow cytometry as previously described (18). For cell cycle assays, cells were plated in six-well plates at 1×10⁵ cells/well and cultured overnight at room temperature. RMPI-1640 medium plus 10% FBS with 80 µmol/l SSd were added to the plates for 48 h and cells were collected, digested and centrifuged (800 × g at room temperature for 10 min). Following, cells were incubated with ice-cold 70% ethanol at room temperature for 24 h and treated with propidium iodide (PI) for 30 min at 4°C in the dark. Annexin V-fluorescein isothiocyanate (FITC)/PI staining was performed for apoptosis analysis following the manufacturer's guidelines (BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA) for 30 min at 4°C in the dark. Samples were analyzed using a flow cytometer. Data were analyzed using CellQuest 16.0 software (BD Biosciences). Each experiment was repeated three times. Apoptosis assays were performed using the described procedure and staining was based on FITC-Annexin V and PI using the FITC-Annexin V Apoptosis Detection kit (BD Biosciences) according to manufacturer's instructions. Experiments were performed in triplicate.

RNA samples extracted from control and SSd-treated cells were reverse transcribed using a Go-Taq DNA polymerase kit (Promega Corporation) as reported previously (18,19). RT was performed with the following protocol: 37°C for 15 min and 85°C for 5 sec. β-actin functioned as the internal control. RT-qPCR reactions were performed using the HT7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and qPCR was performed using the SYBR⁻Green PCR Master mix (Thermo Fisher Scientific, Inc.). The PCR program began with an initial denaturation at 95°C for 2 min, followed by 40 amplification reaction cycles (95°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec), with a final extension at 72°C for 3 min. Primer sequences are reported in Table I. Each sample was tested in triplicate and relative amounts of mRNA were determined using the 2^−ΔΔCt method (20).

Proteins from cells cultured in RMPI-1640 medium plus 10% FBS with 80 µmol/l SSd were collected using RIPA buffer containing fresh protease and phosphatase inhibitor cocktails (all EMD Millipore, Billerica, MA, USA). To determine the concentration of proteins, a BCA protein kit (Thermo Fisher Scientific, Inc.) was used. Extracted proteins (50 µg/lane) were separated as previously described (21). Proteins was separated by SDS-PAGE on a 10% gel and transferred to polyvinylidene difluoride membranes (EMD Millipore). Aliquoted proteins were separated with 12% gels using SDS-PAGE and then transferred to polyvinylidene fluoride membranes. After blocking with 5% nonfat milk at room temperature for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. The following antibodies were included: Tumor protein 53 (p53; cat. no. ab131442); p21 (cat. no. ab188224); p27 (cat. no. ab190851); cyclinD1 (cat. no. ab137875); B-cell lymphoma (Bcl)-2-like protein 4 (Bax; cat. no. ab182733); cleaved caspase-3 (cat. no. ab198447; all 1:1,000); and β-actin (cat. no. ab8227; 1:2,000; all Abcam, Cambridge, UK) as a control. Membranes were then incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit antibodies (cat. no. sc-2004; 1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h. The bound antibodies were detected using enhanced chemiluminescence kit (EMD Millipore) and protein blots were visualized with the Las4000 Imaging system (GE Healthcare Life Sciences, Little Chalfont, UK).

SPSS 17.0 (SPSS, Inc. Chicago, IL, USA) was used for statistical analyses. All data are expressed as means ± standard deviation. Differences between two samples were assessed by two-tailed Student's t-test and one-way analysis of variance followed by the Bonferroni post hoc test to compare between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

143B and MG-63 cell viability and proliferation were tested by MTS and EdU assays. MTS assays assessed effects of varying concentration of SSd (20, 40, 60 and 80 µmol/l) on cell viability and results demonstrated that SSd had no significant effects at 20–60 µmol/l when compared with the control group (P>0.05; Fig. 1A). At 80 µmol/l SSd significantly inhibited 143B and MG-63 cell viability following 24 and 48 h treatment compared with the control (P<0.001; Fig. 1A). EdU incorporation assays further suggested that SSd significantly inhibited cell proliferation at 80 µmol/l compared with the control (P<0.001; Fig. 1B). In 143B and MG-63 lower doses of SSd (20, 40 and 60 µmol/l) had no significant influence on proliferation following 24 or 48 h treatment compared with the control (P>0.05; Fig. 1B). Subsequent experiments were performed using 80 µmol/l SSd as cell treatment.

Flow cytometric analyses of the cell cycle were used to evaluate the underlying mechanisms of proliferation suppression by SSd. It was observed that 80 µmol/l SSd significantly increased the percentage of 143B and MG-63 cells in G₀-G₁ phase by 18 and 23%, respectively (P<0.001; Fig. 2).

A significant increase in apoptosis was observed compared with the control group following the treatment of 143B and MG-63 with 80 µmol/l SSd for 48 h (P<0.001; Fig. 3), indicating that SSd may serve a vital role in the apoptosis of OS cells.

While cell cycle arrest and apoptosis are known to be associated with the activation of the p53 signaling pathway, SSd has been demonstrated to lead to p53 accumulation in ovarian cancer cell lines, Hey and SKOV3 (9). A potential association of SSd and p53 was evaluated. p53 expression was assessed in 143B and MG-63 treated with 80 µmol/l SSd. Western blot analysis revealed that SSd promoted p53 accumulation in OS cells. Furthermore, it was demonstrated that SSd treatment was associated with the upregulation of p53 downstream targets, including p21, p27, Bax, cleaved caspase-3 and the downregulation of cyclinD1 (Fig. 4A). RT-qPCR was performed to evaluate the mRNA expression of p53 signaling pathway targets. In 143B and MG-63, SSd treatment (80 µmol/l) significantly decreased mRNA levels of cyclinD1 and significantly increased mRNA levels of p21, p27, Bax and cleaved caspase-3 (P<0.001; Fig. 4B). Thus, SSd increased the expression of p53 in protein and mRNA levels in 143B and MG-63.