The osteosarcoma MG-63 (code no: TCHu 124) and U2OS (code no: TCHu 88) cell lines were purchased from the Cell Research Center of the Chinese Academy of Sciences (Shanghai, China). RPMI-1640 medium, Dulbecco's modified Eagle's high glucose medium (DMEM) and trypsin were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Embelin was obtained from Abcam (Cambridge, UK). Pan-caspase inhibitor z-VAD-fmk and caspase-8 inhibitor z-IETD-fmk were purchased from Abcam (Cambridge, UK). Rabbit antibodies against caspases 3 and 8, death receptor (DR) 5, XIAP and MMP-9 were purchased from Abcam.

The U2OS cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) and the MG63 cells were cultured in DMEM with 10% FBS. The two cell lines were cultured in an incubator in a humidified 5% CO₂ atmosphere. The cells entering the logarithmic growth phase were selected. In the pre-experiment, U2OS cells (0.8×105/ml) and MG63 cells (1×105/ml) were inoculated in 96-well plates with culture medium (Gibco; Thermo Fisher Scientific, Inc.) containing TRAIL with concentrations of 1, 10 and 100 ng/ml or containing Embelin with concentrations of 5, 10 and 20 µl/l or experimental control without drug. Following culture for 12, 24 and 48 h, 20 µl MTT was added and incubated at 37°C for 4 h, prior to being dissolved in 150 µl dimethyl sulfoxide. The absorbance was subsequently measured at a wavelength of 570 nm. According to the pre-experiment on the concentration of TRAIL and Embelin using an MTT assay, concentrations of 1, 10 and 100 ng/ml were selected for the TRAIL group, concentrations of 5, 10 and 20 µl/l for the Embelin group, and 100 ng/ml TRAIL combined with 20 µl/Embelin for the combined group. A concentration of 100 ng/ml TRAIL or 20 µmol/l Embelin did not reach the half maximal inhibitory concentration at 12, 24 and 48 h, according to the pre-experiment. The study was performed using the following time intervals: 12, 24 and 48 h.

U2OS cells (0.8×10⁵/ml) and MG63 cells (1×10⁵/ml) were inoculated in 96-well plates, and were added to the culture medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing reagents of different concentrations of 100 µl drug or experimental control without drug. Following culture for 12, 24 and 48 h, 20 µl MTT was added and incubated at 37°C for 4 h, prior to being dissolved in 150 µl dimethyl sulfoxide. The absorbance was subsequently measured at a wavelength of 570 nm. The survival rate of tumor cells (%)=experimental group A value/control group A value ×100 (11).

The apoptotic cells were directly observed under an inverted phase contrast microscope at the magnification of ×400. A cover slide was placed in the 6-well plate, and following apoptosis, cells were fixed using 99.5% absolute ethyl-alcohol at 37°C for 10 min and stained with 0.5 ml Hoechst 33258 staining solution at 37°C for 5 min. Images were subsequently captured using a fluorescence microscope at the magnification of ×400 on the object slide covered by the cover slide and a drop of anti-fading solution from Hoechst Staining kit (Beyotime Institute of Biotechnology, Shanghai, China) was added.

Following culture for 12, 24 or 48 h, apoptosis was detected using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Cells in 6-well plates (5×10⁵/well) were detached by trypsinization and washed three times in phosphate-buffered saline, centrifuged at 4°C at 1,000 × g for 5 min and resuspended in 195 µl Annexin V-FITC binding buffer (BD Biosciences). Annexin V-FITC (5 µl) was added and mixed. Following this, the U2OS and MG63 cells were stained by Annexin V-FITC in binding buffer in the dark for 10 min at room temperature. The cells were subsequently centrifuged at 4°C at 1,000 × g for 5 min and were resuspended in 190 µl Annexin V-FITC binding buffer. Finally, 10 µl propidium iodide (PI) staining solution was added and mixed at 4°C for 15 min. The U2OS and MG63 cells were maintained on ice in the dark and immediately subjected to flow cytometric analysis (12). The data were analyzed using the Cell Quest software (version 7.5.3; BD Biosciences, San Jose, CA, USA).

The invasive ability of U2OS and MG63 cells was calculated by the number of cells that passed through a polycarbonate membrane (8 µmol/l pore). The chamber was washed with serum-free medium (Gibco; Thermo Fisher Scientific, Inc.), and then 20 µl Matrigel (dilution, 1:8; BD Biosciences) was added to evenly cover the surface of the polycarbonate membrane. Pre-processed DMEM (200 µl) containing 2×10⁵ cells with 10% FBS was placed into the upper Transwell chamber while 600 µl DMEM with 10% FBS was placed into the lower chamber. The Transwell invasion system was added to the cell incubator for 24 h. The upper chamber was removed and the cells were stained with 1% crystal violet for 15 min at room temperature (13). Invading cells that attached to the surface of the membrane were observed under an inverted phase contrast microscope at a magnification of ×400.

Since MG-63 cells exhibit certain characteristics of osteoblasts and U2OS cells are more malignant and stable than MG-63 cells, U2-OS cells were selected for western blot analysis (14). A total Protein Extraction kit (Beyotime Institute of Biotechnology, Haimen, China) was used to extract the total protein from U2OS cells, according to the manufacturer's protocol. Protein concentration was then determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Protein (40 µg/lane) was separated by 10% SDS-PAGE using an SDS-PAGE Gel kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol, prior to being transferred onto 0.2-µm pore polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology). The membrane was blocked with 5% skimmed milk in TBST at room temperature for 2 h, prior to being incubated with antibodies against the following: rabbit anti-caspase-3 (dilution, 1:5,000; cat. no. ab32351), rabbit anti-caspase-8 (dilution, 1:5,000; cat. no. ab25901), rabbit anti-DR-5 (dilution, 1:1,000; cat. no. ab199357), rabbit anti-XIAP (dilution, 1:1,000; cat. no. ab21278; all Abcam) and rabbit anti-MMP-9 (dilution, 1:1,000; cat. no. 10375–2-AP; ProteinTech Group, Inc., Chicago, IL, USA) in TBST containing 1% bovine serum albumin overnight at 4°C. A rabbit anti-β-actin monoclonal antibody (dilution, 1:5,000; cat. no. ab8227; Abcam) was used as a control for caspase-3 and caspase-8 while a rabbit anti-GAPDH monoclonal antibody (dilution, 1:5,000; cat. no. ab181602; Abcam) was used as a control for DR-5, XIAP and MMP-9. The membranes were subsequently washed three times for 30 min in Tris-buffered saline containing Tween 20 (1×TBST) and were incubated with the horseradish peroxidase-conjugated AffiniPure goat anti-rabbit IgG secondary antibody (dilution, 1:50,000; cat. no. ab182016; Abcam) diluted with TBST for 2 h at room temperature, prior to being washed again in TBST three times for 30 min. A BeyoECL Plus kit (Beyotime Institute of Biotechnology) was used to detect protein bands, according to the manufacturer's protocol. MF-Chemisis 2.0 (DNR Bio-Imaging Systems, Ltd., Neve Yamin, Israel) and GelCapture software (version 2.24, DNR Bio-Imaging Systems, Ltd.) were used to obverse and analyze the protein bands (15,16).

Statistical analysis was performed using Windows SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation of three independent experiments. Data were compared using one-way analysis of variance, followed by the Student-Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference.

While concentrations of 1, 10 or 100 ng/ml TRAIL were used in U2OS or MG63 cells, no significant difference in the dose-inhibiting effect was observed between different groups (P>0.05; Fig. 1A and B). This observation was consistent with the pre-experimental results. In the pre-experiment, it was revealed that the use of TRAIL alone with concentrations ranging between 0.1 and 1,000 ng/ml has little effect on U2OS or MG63 cells. As the purpose of the present study was to examine and verify the effect of combined application of Embelin and TRAIL to osteosarcoma cells, appropriate concentrations were selected. When concentrations of 5 µmol/l Embelin were applied to U2OS or MG63 cells at 12, 24 or 48 h, no significant difference in the dose-inhibiting effect was observed compared with at 0 h (P>0.05; Fig. 1C and D). However, when concentrations of 10 or 20 µmol/l Embelin were applied to U2OS or MG63 cells, cell viability was inhibited (P<0.05; Fig. 1C and D). When a combination of 100 ng/ml TRAIL and 20 µmol/l Embelin was applied to U2OS or MG63 cells, the viability rate was significantly lower (P<0.01), compared with the individual treatment groups (Fig. 1E and F). The results of the present study demonstrated that the combined application of TRAIL and Embelin had a strong inhibitory effect on the viability rates of U2OS and MG63 cells.

U2OS and MG63 cells were added to the surface of a dish in an angular position (Fig. 2A and B). Following the application of 100 ng/ml TRAIL or 20 µmol/l Embelin, few of the osteosarcoma cells were small and round (Fig. 2A and B). However, with the combined application of 100 ng/ml TRAIL and 20 µmol/l Embelin, chromosomes were condensed, and cells became non-adherent and suspended in the DMEM (Fig. 2A and B). Cell death was blocked by joint application of TRAIL and Embelin following the use of 150 µM pan-caspase inhibitor z-VAD-fmk or 150 µM caspase-8 inhibitor z-IETD-fmk (Fig. 3A and B), which indicated that the process of cell death requires caspases activation. In a field of fluorescence microscope, the majority of U2OS and MG63 cells in the control group were lightly-stained and the morphology of the cells were condensed and exhibited fluorescence (Fig. 2C and D), while in the cells treated with the individual application of 100 ng/ml TRAIL or 20 µmol/l Embelin, only a small number of cells were lightly-stained and condensed (Fig. 2C and D). With the combined application of 100 ng/ml TRAIL and 20 µmol/l Embelin, a large number of cells were condensed and exhibited fluorescence, indicating the apoptosis of numerous cells (P<0.05) (Fig. 2C and D).

The Transwell experiments indicated that the number of U2OS and MG63 cells in the combined treatment group passing through the polycarbonate membrane was significantly less than that in the individual treatment groups and the control group (Fig. 4A and B). Cells that were able to invade to the lower side of the membrane in the Transwell assays after 24-h incubation were quantified (Fig. 4C and D). These data indicated that the combined application of TRAIL and Embelin may diminish the invasion of U2OS and MG63 cell lines more significantly than the individual application of either treatment.

Annexin V and PI staining experiment results demonstrated that, with the combined application of 100 ng/ml TRAIL and 20 µmol/l Embelin, the number of apoptotic cells was more than that in the control group and the individual treatment groups (P<0.01; Fig. 5). As Annexin V and PI staining results demonstrated in Fig. 5A, the early apoptotic rates of the control group, the TRAIL-only group, the Embelin-only group and the combined treatment group in U2OS cells were 0.73, 3.54, 12.33 and 20.71%, respectively. The late apoptotic rates in U2OS cells were 1.87, 6.12, 6.82 and 10.84%, respectively, and the total apoptotic rates in U2OS cells were 2.60, 9.66, 19.15 and 31.55%, respectively. A similar trend was also observed in the MG63 cells (Fig. 5B). The early apoptotic rates of the control group, the TRAIL-only group, the Embelin-only group and the combined treatment group in MG63 cells were 0.94, 3.51, 5.76 and 10.17%, respectively, the late apoptotic rates in MG63 cells were 2.43, 4.18, 10.64 and 15.14%, respectively, and the total apoptotic rates in MG63 cells were 3.37, 7.33, 16.40 and 25.31%, respectively. These data indicated that the combined treatment may increase the apoptosis rate of U2OS and MG63 cells, compared with individual treatment with either drug (Fig. 5C and D).

Following exposure to 100 ng/ml TRAIL, 20 µmol/l Embelin or the combined application of the two for 48 h, expression levels of caspase-3, caspase-8, cleaved caspase 3, DR5, XIAP and MMP-9 in U2OS cells were measured using western blot analysis in the present study. The levels of caspase-3, caspase-8, cleaved caspase 3 and DR5 in the combined treatment group were significantly higher than that of the individual treatment groups and the control group (P<0.05). By contrast, levels of XIAP and MMP-9 in the combined treatment group were significantly lower than in the individual treatments groups and the control group (P<0.05) (Fig. 6).