WA, bovine serum albumin (BSA), ribonuclease A (RNase A), propidium iodide (PI), sulforhodamine B (SRB) and a rabbit polyclonal anti-β-actin antibody (1:1,000; cat. no. A2668) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), RPMI 1640 medium, trypsin-EDTA and antibiotic-antimycotic were purchased from Gibco Life Technologies (Grand Island, NY, USA). The following antibodies: Rabbit polyclonal Cdk2 (1:1,000; cat. no. sc-163), rabbit polyclonal cyclin A (1:1,000; sc-751), mouse monoclonal cyclin B1 (1:1,000; cat. no. sc-245) and mouse monoclonal Cdc2 (1:1,000; cat. no. sc-54) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The following antibodies: Rabbit polyclonal p-Cdc2 (Tyr15; 1:1,000; cat. no. P06493), rabbit polyclonal p-Chk1 (Ser345; 1:1,000; cat. no. 2341S), rabbit polyclonal p-Chk2 (Thr68; 1:1,000; cat. no. 2661), rabbit polyclonal Chk1 (1:1,000; cat. no. 2344S) and rabbit polyclonal Chk2 (1:1,000; cat. no. 2662) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

U2OS and MG-63 human osteosarcoma cell lines were obtained from the China Center for Type Culture Collection (Wuhan, China), following standard ethical procedures (27). Following isolation, the osteosarcoma cells were identified based on histological type and grade. The cell lines were grown in high glucose Dulbecco's modified Eagle's medium supplemented with 10% FBS, 100 U/ml penicillin G and 100 U/ml streptomycin (Sigma-Aldrich). The cells were subsequently incubated in a humidified incubator at 37°C with 5% CO₂. All cells used in the experiments were in the logarithmic phase.

The antiproliferation potential of WA in U2OS and MG-63 cells was examined using an SRB assay (11). Cell lines were seeded at a density of 9×10³ cells/well into 96-well plates and allowed to adhere for 72 h. The cells were subsequently treated with WA at various concentrations (0.1, 1 and 10 µM). Dimethyl sulfoxide (DMSO) was used as the experimental control. The results were presented as percentages relative to the solvent-treated control incubations. Using nonlinear regression analysis, the IC₅₀ values were calculated.

U2OS and MG-63 cell lines were plated into 35-mm culture dishes at a density of 1×10⁶ cells/100 mm and incubated for 24 h. The medium was replaced with fresh medium containing WA (at concentrations of 0.1, 1 and 10 µM) and 0.1% DMSO alone. Following incubation for 24 h at 37°C, the cells were harvested, washed with phosphate-buffered saline and fixed with 70% (v/v) ice-cold ethanol for 1 h at 4°C. Subsequently, 50 µg/ml RNase A and 50 µg/ml PI were added to the fixed cells. In total, ~10,000 cells were analyzed by flow cytometry (BD Accuri™ C6; BD Biosciences, Franklin Lakes, NJ, USA). All experiments were repeated three times and data analysis was recorded using BD CellQuest™ cell cycle analysis software (BD Biosciences).

U2OS and MG-63 cell lines were incybated with varying concentrations of WA (0.1, 1 and 10 µM) for 24 h. The cell samples were lysed in sample buffer [150 mM Tris (pH 6.8), 8 M urea, 50 mM DTT, 2% sodium dodecyl sulfate, 15% sucrose, 2 mM EDTA, 0.01% bromophenol blue and 1% protease and phosphatase inhibitor cocktails], sonicated (Ultrasonic Homogenizer 300MP; BioLogics, Manassas, VA, USA) and the protein concentrations were recorded using the Lowry protein assay method (28). Subsequently, samples of 40–45 µg were refluxed, run through an 8–12% Bis/Tris gel (Invitrogen Life Technologies, Carlsbad, CA, USA) using 5X 2-(N-morpholino)ethanesulfonic buffer (Sigma-Aldrich) and transferred to an Immobilon-nitrocellulose membrane (Sigma-Aldrich). The membrane was blocked with 5% skimmed milk or 3% BSA in Tris-buffered saline Tween-20 [TBST; 150 mM NaCl, 50 mM Tris (pH 7.5) and 0.1% Tween-20] (blocking buffer). Next, the membrane was probed with antibodies targeted against cyclin A, cyclin B1, Cdk2, p-Cdc2 (Tyr15), Cdc2, p-Chk1 (Ser345), Chk1, p-Chk2 (Thr68), Chk2 and β-actin (1:1,000) overnight at 4°C in blocking buffer. Following washing with TBST, the membranes were probed with anti-rabbit (cat. no. 7074; Cell Signalling Technology, Inc.) and goat anti-mouse (cat. no. 11011MP; Life Technologies, Grand Island, NY, USA) immunoglobulin G secondary antibodies (1:5,000) in blocking buffer at room temperature for 90 min, and after washing again, the fluorescence was subsequently detected using a Bio-Rad imaging system (Bio-Rad Laboratories, Hercules CA, USA).

In the RT-qPCR analysis, the two cell lines were treated with 10 µM WA for 12 h. Extraction of the total cellular RNA was performed using TRIzol® reagent (Invitrogen Life Technologies), according to the manufacturer's instructions. For reverse transcription of the RNA into cDNA, a SuperScript III First-Strand Synthesis kit (Invitrogen Life Technologies) was used. Subsequently, the qPCR was conducted using an ABI Prism 7900HT Real-Time PCR system (PerkinElmer, Inc., Waltham, MA, USA) with SYBR Green PCR Master Mix (Applied Biosystems Life Technologies, Foster City, CA, USA). The primers used in the RT-qPCR analysis are presented in Table I. The mRNA expression levels were recorded as the fold change relative to the control. Following completion of the RT-qPCR, cycle numbers (Ct values) in which the signal intensity was equal to the threshold value were obtained from the software (SDS Plate Utility v2.3; Applied Biosystems Life Technologies). The relative expression values were calculated using the 2^−ΔΔCt method (29).

All experiments were repeated a minimum of three times. Data are expressed as the mean ± standard deviation. The treated groups were compared using one-way analysis of variance with SPSS 12.0 software (SPSS, Inc., Chicago, IL, USA), where P<0.05 was considered to indicate a statistically significant difference. Relative percentages were calculated using GraphPad Prism software, version 4.0 for Windows (GraphPad Software, Inc., San Diego, CA, USA).

Two human osteosarcoma cells lines, MG-63 (Fig. 2A) and U2OS (Fig. 2B), were treated with varying concentrations of WA for 72 h to assess the antiproliferative effect of WA. The cell proliferation of each of the tested samples was recorded using an SRB assay. Higher concentrations of WA were shown to exhibit potent antiproliferative effects. Thus, the results revealed that WA produced a potent antiproliferative effect by significantly inhibiting the growth of the tested cell lines.

MG-63 and U2OS cells were treated with varying concentrations of WA for 24 h and the underlying antiproliferative mechanisms were investigated via flow cytometric analysis with PI staining. Cell cycle distribution analysis revealed that WA administration resulted in the significant arrest of MG-63 and U2OS human osteosarcoma cells at the G2/M phase in a dose-dependent manner (Fig. 3). At a concentration of 10 µM WA, more than half of the cells were arrested at the G2/M phase.

Western blot analysis was used to examine the expression levels of G2/M cell cycle regulatory proteins in MG-63 (Fig. 4A) and U2OS (Fig. 4B) human osteosarcoma cell lines treated with WA, in order to determine whether the cell cycle arrest was associated with the regulation of cell cycle checkpoint proteins. There was a significant decrease in the levels of cyclin A, cyclin B1, Cdk2 and p-Cdc2 (Tyr15) in the MG-63 and U2OS cells; however, the protein expression levels of p-Chk1 (Ser345) and p-Chk2 (Thr68) increased. No statistically significant changes were observed in the levels of Cdc2 and Chk2 in the two cell lines following WA treatment.

In order to determine the effect of WA on the mRNA expression levels of cell cycle checkpoint proteins, the expression levels of G2/M cell cycle regulatory proteins, including cyclin A, cyclin B1, Cdk2, Cdc2, Chk1 and Chk2, were elucidated by RT-qPCR analysis for the MG-63 (Fig. 5A) and U2OS (Fig. 5B) cell lines. There was a significant decrease in the mRNA expression levels of cyclin A, cyclin B1 and Cdk2, while no marked changes were observed in the mRNA expression levels of Cdc2, Chk1 and Chk2 in the two cell lines.