The human osteosarcoma cells (HOS, MG63 and U2OS) and human osteoblast line (hFOB1.19) used in this study were obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All of the cell lines were authenticated by Short Tandem Repeat (STR) profiling. The simian virus 40-transfected hFOB1.19 cells were applied to detect whether CTAB is toxic to normal human osteoblasts. The cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM; HyClone) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), and 1% penicillin/streptomycin (Sigma-Aldrich; Merck KGaA), and incubated at 37°C with 5% CO₂. BALB/c nude mice (male, 4-5 weeks, 20-22 g) were obtained from the Shanghai SLAC Laboratory Animal Co and housed in a standard animal environment (22-26 °C, 40-70% humidity) with free access to water and food. All animal experiments were approved by the Animal Research Committee of the First Affiliated Hospital of Chinese Medical University.

CTAB was purchased from Sigma-Aldrich; Merck KGaA. PI3K agonist 740 Y-P were purchased from MedChemExpress (MCE). Antibodies against caspase-3 (1:1,000 dilution; cat. no. 9662), cleaved caspase-3 (1:1,000 dilution; cat. no. 9661), caspase-8 (1:1,000 dilution; cat. no. 9746), cleaved caspase-8 (1:1,000 dilution; cat. no. 9496), caspase-9 (1:1,000 dilution; cat. no. 9502), cleaved caspase-9 (1:1,000 dilution; cat. no. 20750), PARP (1:1,000 dilution; cat. no. 9532), cleaved PARP (1:1,000 dilution; cat. no. 32563), AKT (1:1,000 dilution; cat. no. 9272) and p-AKT (1:1,000 dilution; cat. no. 9271) were purchased from Cell Signaling Technology. Antibodies against PI3K (1:1,000 dilution; cat. no. 180967), p-PI3K (1:1,000 dilution; cat. no. 278545), Bcl-2 (1:1,000 dilution; cat. no. 32124), Bax (1:1,000 dilution; cat. no. 32503), cytochrome c (1:5,000 dilution; cat. no. 133504), and β-actin (1:1,000 dilution; cat. no. 8226) and secondary antibodies (1:5,000 dilution; cat. no. 96899 and 96879) were obtained from Abcam. Phosphate-buffered saline (PBS) was obtained from Gibco; Thermo Fisher Scientific, Inc. Radioimmunoprecipitation assay (RIPA) lysis buffer was purchased from Santa Cruz Biotechnology.

OS cells and osteoblasts were cultured (1×10⁴ cells/well) in 96-well plates (Thermo Fisher Scientific, Inc.) and treated with CTAB at different concentrations (0, 1, 2, 3, 4, 5, 6, 7, and 8 µM) for 24, 48 and 72 h in vitro. After the specified incubation time, 10 µl of CCK-8 (Dojindo Molecular Technologies, Inc.) was added to the plate, the cells were incubated at 37°C for 1-4 h, and the absorbance was measured at 450 nm using an ELISA microplate reader (Bio-Rad). Cells without CTAB treatment were used as negative controls.

After incubation with a concentration gradient of CTAB at 37°C for 48 h, the cells were collected, washed twice with PBS, and then fixed overnight with 70% cold ethanol at 4°C. After the cells had been washed with PBS again, they were resuspended in 0.5 ml stain buffer containing 100 µg/ml RNase A and 50 µg/ml PI (Beyotime Biotech) in the dark at room temperature for 30 min and analyzed by a flow cytometer (Becton-Dickinson). Moreover, CytExpert (version 2.3) was used for subsequent data analysis.

OS cell migration and invasion were detected by the wound-healing and Transwell assays, respectively. The cells were seeded in 6-well plates at a density of 5×10⁵ cells/well. After the cell monolayer was formed, a micropipette tip was used to create a wound. Then, the cells were washed with PBS, and the medium was replaced with serum-free high-glucose DMEM containing different concentrations (0, 1, 2 and 4 µM) of CTAB. Cells migrating to the wound area were photographed with an inverted microscope at 0, 6, 18 and 24 h (the average wound size represented the relative migration of cells). A 24-well Transwell chamber (Corning Costar) coated with matrix gel (Sigma-Aldrich; Merck KGaA) was used for cell invasion analysis. HOS and MG63 cells (5×10⁴ cells/well) were inoculated with medium containing different concentrations (0 and 4 uM) of CTAB (without FBS) in the upper part of the Transwell chamber, and the lower chamber was filled with complete medium containing 10% FBS. After 24 h of treatment, the cells in the upper chamber were removed, and the remaining cells that had invaded through the Matrigel matrix were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted under an inverted fluorescence microscope (Nikon Eclipse Ti-S).

The effect of CTAB on the apoptosis of OS cells was analyzed by using a membrane protein V-FITC apoptosis detection kit (BD Biosciences). HOS and MG63 cells were inoculated in 6-well plates for 24 h and then treated with CTAB at a concentration of 0, 2, 4 or 6 µM for 48 h. The cells were washed twice with precooled PBS, resuspended in 100 µl 1X buffer and then incubated with FITC-labeled Annexin V as well as PI for 15 min at room temperature in the dark. Finally, 400 µl 1X binding buffer was added to the reaction system, and cell apoptosis was assessed by flow cytometry (Becton-Dickinson) and CytExpert (version 2.3).

After treatment of HOS and MG63 cells with different doses of CTAB (0, 2, 4 and 6 µM) for 48 h, the cells were washed twice with PBS and lysed with RIPA buffer comprising protease/phosphatase inhibitors to extract total protein. Mitochondrial and cytosolic proteins were isolated according to the protocol provided by the mitochondrial extract kit (Thermo Fisher Scientific Inc.). A BCA protein assay kit (Beyotime) was used to determine the protein concentration. Then, 10 and 12% SDS-PAGE gels were used to separate the proteins (30 µg per lane), which were then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% bovine serum albumin solution at room temperature for 2 h and incubated with all the primary antibodies overnight at 4°C. Then, the membranes were incubated with secondary antibody for 2 h at room temperature. Subsequently, the results were visualized with an enhanced chemiluminescence (ECL) system (UVP Inc.) and a Protein Blotting Detection and Imaging Scanner (Bio-Rad). ImageJ (NIH) software was used for quantitative analysis.

The change in the mitochondrial membrane potential (MMP) of osteosarcoma cells after intervention with CTAB was assessed by the JC-1 Analysis Kit (Beyotime). HOS and MG63 cells were seeded in 6-well plates (5×10⁵/well) overnight and then treated with different doses of CTAB for 24 h. The next day, the supernatant was removed, and the cells were washed with PBS and 1X JC-1 buffer. Then, the cells were resuspended in 500 µl JC-1 staining solution in an incubator for 20 min and analyzed by flow cytometry (Beckham).

HOS cells (2×10⁶) suspended in 100 µl PBS were subcutaneously inoculated into the dorsal area of 4-week-old male nude mice. Three days after inoculation, the mice were randomly divided into 3 groups (n=4). Then, CTAB (10 or 20 mg/kg/d) was injected intraperitoneally every 3 days into each mouse for a total of 20 days. The control group was treated with the same volume of physiological saline. During the administration period, the body weights and tumor sizes of the mice were monitored every 3 days. The mice were sacrificed by cervical dislocation after 20 days of CTAB treatment, and the tumors and major organs, including the liver, lung, kidney, spleen and heart, were collected from each group of mice and immersed in 4% formalin for immunohistochemical staining and hematoxylin and eosin (H&E) staining. The following formula was used to calculate the tumor volume: Volume=1/2 (length x width²). The tissue samples were fixed with 10% formalin (for 24 h at room temperature), embedded in paraffin, and cut into 4 µm sections. The sections were deparaffinized with xylene and then dehydrated with gradient ethanol solutions. Then, the slides were stained with hematoxylin for 10 min and eosin for 5 min. Tissue damage was observed under a microscope (Nikon) after the slides were sealed with neutral resin.

Data are expressed as the mean ± standard deviation (SD). GraphPad Prism 7.0 software was used for statistical analysis, and Student's t-test or one-way analysis of variance (ANOVA) followed with Tukey post-hoc test was used to analyze differences between groups. P<0.05 was considered statistically significant. All the cell experiments were performed in triplicate.

To investigate the effect of CTAB on the proliferation of OS cells, we treated HOS, MG63 and U2OS cells with different concentrations of CTAB in vitro for 24, 48 or 72 h. The results showed that CTAB significantly inhibited the activity of osteosarcoma cells in a time- and concentration-dependent manner (Fig. 1A), and the IC50 values of HOS, MG63, and U2OS cells were 4.949, 3.500, and 4.212 µM, respectively (Fig. 1A). Notably, CTAB treatment did not affect the viability of hFOB1.19 cells (Fig. 1A), indicating that CTAB at a concentration of 1-8 µM had no significant toxicity compared to normal human osteoblasts. Based on the above results, 2, 4 and 6 µM were selected as the effective drug concentrations for subsequent analysis. To determine whether CTAB inhibits cell proliferation by inducing cell cycle arrest, we evaluated the cell cycle distribution of osteosarcoma cells using flow cytometry. The proportion of G1 phase cells increased from 30.29 to 53.34%, 40.23 to 61.55% and 39.95 to 60.58% in HOS, MG63 and U2OS cells treated with CTAB (2, 4 or 6 µM), respectively (Fig. 1B). These data suggest that CTAB has an inhibitory effect on OS cells and induces G1 phase arrest.

Tumor metastasis is the most common factor affecting the survival of patients. Therefore, inhibiting tumor migration and invasion may be an effective strategy to prevent tumor metastasis. In this study, the wound-healing assay showed that CTAB treatment significantly reduced the wound closure rate of HOS and MG63 cells in a dose-dependent manner (Fig. 2A). The invasion of tumor cells was analyzed by the Transwell assay, in which Matrigel matrix effectively simulates the invasion microenvironment of tumor cells. The digestion and penetration of Matrigel matrix by tumor cells is a good reflection of invasion ability. Compared with the control, CTAB significantly reduced the number of invasive HOS and MG63 cells (Fig. 2B).

The inhibitory effect of CTAB on the growth of osteosarcoma cells resulting from apoptosis was verified by flow cytometry. The percentage of apoptotic OS cells increased significantly after treatment with different concentrations of CTAB compared with control treatment for 48 h (Fig. 3A). The expression level of downstream apoptotic proteins was further measured by western blot analysis, and as shown in Fig. 3B and C, CTAB-treated OS cells exhibited downregulated expression of the antiapoptotic protein PARP and Bcl-2 but upregulated expression of cytosolic cytochrome c (cyto-c) and the proapoptotic protein Bax. In addition, CTAB significantly promoted the cleavage of PARP, caspase-3, caspase-8 and caspase-9. These results suggest that CTAB triggers apoptosis in OS cell lines. Disturbance of the mitochondrial membrane potential (MMP, ΔΨm) is an early sign of apoptosis that affects the permeability of mitochondrial membranes (14). An increased mitochondrial membrane permeability leads to the release of mitochondrial apoptotic factors, such as cyto-c, which is transferred from the mitochondria to the cytoplasm and activates a series of apoptotic enzymes (15). The results of flow cytometry (Fig. 3D) showed that after 24 h of treatment with 4 µM CTAB, the percentage of HOS and MG63 cells with a normal MMP was significantly reduced. These results indicate that CTAB may promote OS apoptosis by interfering with the MMP and disrupting mitochondrial membrane permeability.

The PI3K/AKT signaling pathway is the main intracellular signaling pathway that regulates the proliferation, apoptosis and migration of tumor cells (16). Previous findings have reported that excessive activation of the PI3K/AKT pathway is closely related to the negative regulation of tumor cell apoptosis (17). Therefore, we further analyzed whether CTAB-induced OS cell apoptosis depends on the PI3K/AKT signaling pathway. As shown in Fig. 4A, the expression levels of p-PI3k and p-AKT in HOS and MG63 cells treated with different concentrations of CTAB decreased significantly. We further investigated whether the antitumor effect of CTAB is mediated through inhibition of the PI3K/AKT signaling pathway, and we found that 740Y-P (a PI3K agonist) partially reversed the inhibitory effect of CTAB on OS cells (Fig. 4B). Specifically, 740Y-P reversed CTAB-induced alterations in the abundance of p-PI3K, p-Akt, Bax and cleaved caspase-9 in HOS and MG63 cells (Fig. 4B). In addition, the flow cytometry showed that 740Y-P decreased the apoptosis rate of OS cells (Fig. 4C). After treatment with CTAB (4 µM), the apoptosis rates of HOS and MG63 cells were 41.9 and 50.2%, respectively, but the combination of CTAB and 740Y-P reduced the apoptosis rate to 18.7 and 21.5%, respectively. These results confirmed that CTAB induces apoptosis by inhibiting the PI3K/AKT signaling pathway.

Since CTAB can inhibit OS cell proliferation and metastasis and induce apoptosis in vitro, we examined whether CTAB can inhibit the growth of OS (HOS) in xenograft tumors. As shown in Fig. 5A-D, CTAB treatment resulted in a significant reduction in tumor volume and weight in a dose-dependent manner. The average tumor volume of the control group was 1718±97 mm³, while that of the 10 mg/kg/d CTAB-treated group was 987±107 mm³ and that of the 20 mg/kg/d CTAB-treated group was 545±105 mm³. In addition, the mean tumor weight of the control group was 1380±87 mg, while that of the 10 mg/kg/d CTAB-treated group was 566±101 mg and that of the 20 mg/kg/d-treated group was 270±75 mg. In addition, there was no significant organ toxicity in the CTAB group, indicating that it safely exerts antitumor effects in vivo (Fig. 5E).