ALT and Cell Counting Kit (CCK)-8 were obtained from MedChem Express. Primary antibodies against PI3K (4249), phosphorylated (p)-AKT (4060), AKT (9272), cyclin D1 (2978), p27 (3686), Bax (5023), Bcl-2 (15071) and β-actin (3700) were purchased from Cell Signaling Technology, Inc., primary antibodies against cleaved caspase-3 and cleaved caspase-8 were purchased from Abcam, and primary antibodies against MMP-2 and MMP-9 were obtained from ProteinTech Group, Inc. The Annexin V-FITC/propidium iodide (PI) apoptosis detection kit and was purchased from Nanjing KeyGen Biotech Co., Ltd. RPMI-1640 medium and FBS were purchased from Hyclone; GE Healthcare Life Sciences.

The human osteosarcoma U2OS and HOS cell lines were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences. U2OS and HOS cells were cultured in RPMI-1640 medium and DMEM (Gibco; Thermo Fisher Scientific, Inc.), respectively, supplemented with 10% FBS and 1% penicillin/streptomycin, and maintained in a humidified atmosphere of 37°C and 5% CO₂.

The U2OS and HOS cell lines were seeded at a density of 8×10³ cells/well in 96-well plates and cells were subsequently exposed to a range of concentrations of ALT (2.5, 5, 10, 20, 40, 80 or 160 µM) for 24 and 48 h at 37°C with 5% CO₂. The CCK-8 assay was used to assess the viability of osteosarcoma cells following drug treatment. A total of 10 µl CCK-8 kit solution was added to each well and the cells were subsequently incubated for a further 4 h at 37°C with 5% CO₂, after which the optical density of the cell lysates was measured at 450 nm. GraphPad Prism version 7.0 software (GraphPad Software, Inc.) was used to calculate the median lethal concentration of ALT (IC₅₀) for osteosarcoma cells.

U2OS and HOS cells were collected and seeded in 6-well plates at a density of 1×10³ cells/well. Following cell adherence, the culture medium was replaced with each cell line's respective media containing a range of ALT concentrations (U2OS, 0, 5, 10 or 20 µM; HOS, 0, 15, 30 or 60 µM) and the cells were further incubated for a further 8 days in a humidified cell incubator at 37°C with 5% CO₂. Following incubation, colonies were first fixed with 4% paraformaldehyde for 30 mins at room temperature and then stained with 0.1% crystal violet for 5 min at room temperature. Colonies (>50 cells) were visualized using an optical microscope (magnification, ×10).

To evaluate the apoptotic rates of ALT-treated U2OS cells, the Hoechst 33258 kit (Beyotime Institute of Biotechnology) was used for nuclear staining. Bright blue nuclear staining indicated nuclear pyknosis, which is a characteristic of apoptotic cells (23). Following treatment with ALT (0, 5, 10 or 20 µM) for 48 h, the U2OS cells were fixed with 4% paraformaldehyde for 30 min at room temperature. The U2OS cells were rinsed with PBS three times both before and after staining with Hoechst 33258 (10 µg/ml; 5 min at room temperature)) in the dark. An Eclipse TS100 fluorescence microscope (Nikon Corporation; magnification, ×20 and ×40) was used to visualize the changes in the nuclear morphology of ALT-treated U2OS cells.

A total of 5×10⁵ U2OS cells/well were seeded in 6-well plates and incubated with a range of ALT concentrations (0, 5, 10 or 20 µM) for 48 h at 37°C with 5% CO₂. Following cellular adherence to the plates, the U2OS cells were harvested and rinsed with pre-chilled PBS (4°C). To further evaluate ALT-induced apoptosis of the U2OS cells, an Annexin V-FITC/ PI kit was used, according to the manufacturer's protocol. Flow cytometry cell sorting equipment (Navios EX flow cytometer; Beckman Coulter, Inc. and FlowJo v. 10.4; FlowJo LLC) was used to analyze the apoptosis of ALT-treated U2OS cells. The cells stained with Annexin V (+) were considered early apoptotic and the cells stained with PI (+) were late apoptotic cells.

The effect of ALT on the migratory ability of U2OS cells was assessed using a scratch wound healing assay. A total of 5×10⁵ U2OS cells/well were seeded into 6-well plates and cultured to 90% confluence prior to incubation at 37°C with 5% CO₂ with 5% serum and a range of ALT concentrations (0, 5, 10 or 20 µM) for 48 h. The wound scratch was performed by 10 µl pipette tip. The wound area was visualized at the 0, 12, 24 and 48 h time points using an optical microscope (magnification, ×10) and the width of the wound was analyzed using ImageJ software (v. d 1.47; National Institutes of Health). The Recovered wound area (%) of each time point (12, 24 and 48 h) was calculated as The wound area of 0 h-the wound area of each time point)/The wound area of 0 h.

Matrigel-coated (BD Biosciences) Transwell chambers were used to detect the invasive ability of U2OS cells exposed to 0, 5, 10 or 20 µM ALT. A total of 2×10⁴ U2OS cells/well were plated in the upper chambers of Transwell plates in 100 µl FBS-free RPMI-1640 medium containing the previously indicated concentrations of AL, 200 µl RPMI-1640 medium containing 20% FBS was placed in the lower chambers. Following incubation for 48 h at 37°C with 5% CO₂, the non-invasive cells remaining in the upper chambers were removed. The invasive cells in the lower chambers were fixed with 4% paraformaldehyde for 30 min and subsequently stained with 0.1% crystal violet for 20 min at room temperature. Stained cells were counted using an optical microscope (magnification, ×20).

A total of 5×10⁵ U2OS cells/well were seeded into 6-well plates and incubated with a range of ALT concentrations (0, 5, 10 or 20 µM) for 48 h at 37°C with 5% CO₂. Total protein was extracted from U2OS cells using RIPA lysis buffer containing 1% protease and phosphorylase inhibitor (all Beyotime Institute of Biotechnology). Protein samples were maintained on ice for 30 min and subsequently centrifuged (15,000 × g for 10 min at 4°C). Total protein was quantified using a bicinchoninic acid kit (Beyotime Institute of Biotechnology) and the extracted proteins were mixed with loading buffer and boiled at 95°C for 5 min for denaturation. Proteins (40 µg) were separated via SDS-PAGE on a 12% gel. The separated proteins were subsequently transferred onto a 0.22-µm PVDF membrane and blocked for 2 h with 5% non-fat dry milk at room temperature. The membranes were incubated with the following primary antibodies overnight at 4°C: Anti-PI3K, anti-AKT, anti-p-AKT, anti-cyclin D1, anti-p27, anti-Bax, anti-Bcl-2, anti-cleaved caspase-3, anti-cleaved caspase-8, anti-MMP-2, anti-MMP-9 and anti-β-actin, all the primary antibodies were diluted in the primary antibody diluent (Beyotime Institute of Biotechnology) at a concentration of 1%. Membranes were washed three times with TBS-0.1% Tween 20. Following the primary antibody incubation, membranes were incubated with anti-rabbit antibodies (14708, Cell Signaling Technology, Inc.) which were diluted in the secondary antibody diluent (Beyotime Institute of Biotechnology) at a concentration of 0.02%, for 1 h at room temperature. Protein bands were visualized using the BioSpectrum Imaging system and ImageJ software (v. d 1.47; National Institutes of Health) was used for densitometry.

All data are expressed as the mean ± SD from ≥3 independent experimental repeats. Statistical analyses were performed using GraphPad Prism software (version 7.0; GraphPad Software, Inc.). Significant differences between groups were determined by using one-way ANOVA, followed by a Tukey's post hoc test, P<0.05 was considered to indicate a statistically significant difference.

The chemical structure of ALT is presented in Fig. 1A. Cellular viability of U2OS and HOS cells was evaluated in the presence of a range of ALT concentrations for 24 and 48 h using the CCK-8 assay. Concentrations of 20 or 60 µM ALT significantly reduced the viability of U2OS cells and HOS cells respectively, compared with the untreated cells (0 µM; Fig. 1B). The IC₅₀ of ALT in U2OS and HOS cell lines was determined as 29.63 and 67.74 µM at 24 h, and 19.57 and 56.86 µM at 48 h, respectively, for each cell line. Concentrations of 20 or 60 µM ALT presented the half maximal inhibitory concentration, so these concentrations were chosen as the high concentrations in the subsequent experiment.

Low concentrations (5 µM) of ALT slightly decreased the number of cells and induced the shrinkage of HOS and U2OS cells compared with untreated cells, whereas higher concentrations (10 and 20 µM) not only decreased the number of osteosarcoma cells, but also made the shrinkage more serious (Fig. 1C). In addition, the colony formation assay demonstrated that ALT markedly reduced the colony-forming ability of U2OS and HOS cells compared with untreated cells (Fig. 1D).

Therapeutic candidates with antitumor activity often exert cellular cytotoxicity through promoting apoptosis of cancer cells (24,25). As U2OS and HOS cell lines were both collected from young Caucasian female patients with osteosarcoma, this peculiarity also lead to some similarities on tumorigenicity and drug resistance, therefore, subsequent experiments were only performed on U2OS cells. Hoechst 33258 staining, which detects condensed chromatin, demonstrated that the number of apoptotic U2OS cells increased with increasing concentrations of ALT compared with untreated cells (Fig. 2A and B). The rate of apoptosis of U2OS cells following ALT administration was also evaluated using Annexin V-FITC/PI double staining and flow cytometry (Fig. 2C and D). The percentage of apoptotic cells was 6.7±0.12 (5 µM), 13.9±0.36 (10 µM) and 37.4±0.45 (20 µM) compared to 0 µM (5.33±0.20)-for ALT concentrations of 0, 5, 10 and 20 µM respectively (Fig. 2C and D). These data demonstrated that ALT markedly enhanced the apoptosis of osteosarcoma cells in a dose-dependent manner.

A wound-healing assay was performed to evaluate the effect of ALT on the migratory ability of U2OS cells. ALT significantly suppressed the migration of the U2OS cells in a dose-dependent manner compared with untreated cells (Fig. 3A and B). Subsequently, a Matrigel assay was performed to assess the effect of ALT on the invasive potential of U2OS cells. The number of invading cells significantly decreased following exposure to ALT for 48 h compared with the untreated cells (Fig. 3C and D). Overall, these results demonstrated that ALT significantly suppressed the invasion and migration of U2OS cells.

To evaluate the mechanisms underlying ALT-mediated inhibition of osteosarcoma, western blotting was performed. High concentration of ALT-treated cells demonstrated significantly reduced expression levels of cyclin D1 and increased expression of p27 compared with the untreated cells (Fig. 4A and B). Cyclin D1 and p27 modulate the progression through the G1 and S phases of the cell cycle (26,27), and thus these changes may suggest the reason for inhibition of cell proliferation.

It has previously been reported that ALT promotes apoptosis in osteosarcoma (28–30); therefore, the effect of ALT on the expression levels on Bcl-2, Bax, cleaved caspase-3 and cleaved caspase-8, which all serve important roles in the process of apoptosis was explored. The expression levels of cleaved caspase-3 and cleaved caspase-8 were significantly upregulated in the ALT-treated U2OS cells compared with the untreated cells (Fig. 4C and D). In addition, ALT treatment reduced the expression levels of Bcl-2 and increased the expression levels of Bax compared with untreated cells (Fig. 4C and D).

MMPs are strongly associated with cellular invasion in osteosarcoma (31–33); therefore, the effect of ALT on MMP-2 and MMP-9 was evaluated. The data revealed that ALT decreased the expression of MMP-2 and MMP-9 in a dose-dependent manner compared with untreated cells (Fig. 4E and G).

Previous studies have reported that the PI3K/AKT signaling pathway serves an important role in osteosarcoma (11,34,35); PI3K/AKT modulates the proliferation, invasion, migration and apoptosis of osteosarcoma cell lines (36–39). Thus, the effect of ALT on the PI3K/AKT signaling pathway was investigated to confirm whether it is the mechanism by which ALT affects osteosarcoma cells. ALT demonstrated a significant ability to reduce the activation of p-AKT, which was observed through significantly decreased protein expression levels in U2OS cells compared with untreated cells (Fig. 4F and H) although no effect on the expression of total PI3K was observed. These findings suggested that the cytotoxic effect of ALT is closely associated with regulation of the PI3K/AKT signaling pathway.