The OS cell lines (143B, CRL-8303; MG63, CRL-1427; Saos2, HTB-85l; and U2OS, HTB-96) and normal cells (HEB, CRL-8621; and HS5, CRL-11882) used were provided by Professor Tongchuan He (University of Chicago, USA), and were originally obtained from the American Type Culture Collection. The cells were cultured in DMEM (HyClone; Cytiva), supplemented with 10% FBS (Shanghai ExCell Biology, Inc.) and 1% penicillin-streptomycin (100 IU/ml; Hyclone; Cytiva). The OS cells were cultured in 90- or 60-mm cell culture dishes, in a humidified incubator at 37°C with 5% CO₂. The cells to be passaged were washed with PBS (Gibco; Thermo Fisher Scientific, Inc.) and treated with 0.25% trypsin-EDTA (Thermo Fisher Scientific, Inc.) for subsequent subculturing.

Sch B (purity ≥98%) was purchased from Chengdu Herburify Co., Ltd. (http://www.herbpurify.com/). A total of 20 mg Sch B was dissolved in 1.5 ml DMSO (SigmaAldrich; Merck KGaA). Sch B was stored at −80°C and used for subsequent use in in vitro cell experiments by diluting immediately prior to application. For the in vivo experiments, 200 mg Sch B were dissolved in 40 ml carboxymethyl cellulose (SigmaAldrich; Merck KGaA) and stored at −80°C following ultrasonication overnight.

The OS cells (143B, MG63, Saos2 and U2OS) were seeded into 24 well-plates at a concentration of 2.5×10⁴ cells/well. The OS cells were first treated with various concentrations of Sch B (0, 20, 40, 60, 80 or 100 µM), in order to determine the effective concentration range (data not shown). The cells (at 50% confluency) were then treated with various concentrations of Sch B (0, 20, 40, 60 or 80 µM) or DMSO (vehicle control). Following treatment for 24, 48 and 72 h, the cells were washed with 4°C PBS three times and fixed with 4% paraformaldehyde for 20 min at 25°C. After removing the 4% paraformaldehyde, crystal violet dye (Beyotime Institute of Biotechnology) was added to the 24-well plate for 3 min in the dark at 25°C. Subsequently, the excess crystal violet dye was washed with double distilled water. The 24 well-plates were dried and imaged using an Epson Perfection V200 Photo (Epson). For quantification analysis, 200 µl 20% acetic acid solution were added to each well to dissolve the crystal violet for 10 min with constant shaking in the dark at 25°C. The absorbance was measured at 595 nm in a multifunctional enzyme labeled detector (BioTek_Synergy4; Gene Company, Ltd.).

MTT powder was purchased from Sigma-Aldrich; Merck KGaA. The OS cells were seeded in 96-well plates (3×10³ cells/well). OS cells were first treated with various concentrations of Sch B (0, 20, 40, 60, 80 or 100 µM) to determine the effective concentration range (data not shown). The OS cells (at 50% confluency) were treated with various concentrations of Sch B (0, 20, 40, 60 or 80 µM) or DMSO. Normal cells were treated in the same manner, but with a larger range of concentrations (0–200 µM). Following 24, 48 and 72 h treatment, 10 µl of 5 mg/ml MTT solution (Sigma-Aldrich; Merck KGaA) were added to each well of the 96-well plate and incubated for 4 h at 37°C. The solution was then removed using a 1-ml syringe and 100 µl DMSO were added to each well. Following a 15-min agitation at 25°C, the absorbance in the wells was detected at 492 nm using a multifunctional enzyme labeled detector (BioTek_Synergy4 Gene Company, Ltd.). The following formula was used to determine the IC₅₀ values: lgIC=Xm-I[P-(3-Pm-Pn)/4], where Xm is the maximum drug concentration, I is the maximum drug concentration/adjacent drug concentration, P is the sum of positive reaction rates, Pm is the maximum positive reaction rate and Pn is the minimum positive reaction rate.

The 143B cells were seeded into 6-well plates (200 cells/well). Considering the high cell density of the colony formation assay and the purpose of the experiment, the OS cells were first treated with lower concentrations of Sch B (0, 3, 5, 7, 9, 11, 13, 15, 17 or 19 µM) to determine the effective concentration range (data not shown). The cells were then treated with various concentrations of Sch B (0, 3, 5, 7 or 9 µM) or DMSO when clusters of 2–3 cells could be observed under a light microscope (ECLIPSE Ti, Nikon Corporation, White Light, ×100 magnification). Following 2 weeks of culture, all cells were stained with crystal violet (Beyotime Institute of Biotechnology) as aforementioned, and dried and imaged using an Epson Perfection V200 Photo (Epson). The number of colonies were counted, and a cluster of cells was considered a colony if it consisted of >50 cells.

The 143B and MG63 were seeded into 6-well plates to evaluate the changes in the cell migratory capability in Sch B-treated cells. When the cells had grown to 90% confluency, a sterile pipette tip was used to create a wound in the cell monolayer. The floating cells were washed using PBS. Serum-starved medium (HyClone; Cytiva) was used during the wound healing assay. Considering the high cell density required for the wound healing assay, the OS cells were first treated with large concentrations of Sch B (0, 30, 50, 70, 90, 110, 130, 150, 170 or 190 µM) to determine the effective concentration range before formal experiments (data not shown). All cells were treated with various concentrations of Sch B (0, 30, 50, 70 or 90 µM) or DMSO, and incubated for 24 h at 37°C. The wound width was recorded using an inverted microscope (ECLIPSE Ti; Nikon Corporation, White Light, ×100 magnification) after 0, 12 and 24 h. The following formula was used to determine the healing ability of the treated cells as a percentage: [(Start scratch width-end scratch width)/(start scratch width)] ×100.

Transwell chamber inserts (8-µm pores; MilliporeSigma) were placed in 24-well plates. For the migration assay, 2.5×10⁴ cells were resuspended in 400 µl FBS-free medium (HyClone; Cytiva) and added to the upper chamber. The OS cells were first treated with various concentrations of Sch B (0, 20, 40, 60, 80 or 100 µM) to determine the effective concentration range (data not shown). Subsequently, media (HyClone; Cytiva) supplemented with 10% FBS as a chemotactic agent with various Sch B concentrations (0, 20, 40, 60 or 80 µM) were placed in the lower compartment. For the invasion assay, 30 µl Matrigel (BD Biosciences) diluted with 270 µl DMEM was placed in each filter (50 µl per) to create the matrix adhesive film. Subsequently, all remaining experimental steps were performed in the same manner as those described above for the migration assay. Following 24 h of incubation at 37°C, the cells that had not invaded or migrated were removed using a cotton swab. Filters were stained with crystal violet as aforementioned and subsequently wiped with a cotton swab for the removal of the excess dye. Finally, the number of cells which had migrated or invaded were imaged (ECLIPSE Ti; Nikon Corporation) and counted in a random field of view (magnification, White Light, ×100 magnification).

The OS cells were first treated with various concentrations of Sch B (0, 20, 40, 60, 80 or 100 µM) to determine the effective concentration range (data not shown). The OS cells were seeded into 6-well plates and treated with various concentrations of Sch B (0, 20, 40, 60 or 80 µM) or DMSO when the cell density reached 50%. Following 24 h of treatment, the cells were fixed with 4% paraformaldehyde for 20 min, washed with cold PBS, and stained with 10 ng/ml Hoechst 33258 (Beijing Solarbio Science & Technology Co., Ltd.) for 10 min in the dark at 25°C. Finally, the apoptotic cells were imaged (ECLIPSE Ti; Nikon Corporation) and counted in a random field of view (magnification, fluorescence, ×100 magnification).

The OS cells were cultured in 60-mm dishes. The cells were treated with various concentrations of Sch B (0, 20, 40, 60 or 80 µM) or DMSO when the cell density reached 50%, for 24 h. The cells in each dish were digested with trypsin and placed in 500 µl PBS for 1 min at 25°C, stained with annexin V-FITC/PI (Beyotime Institute of Biotechnology) and analyzed using a flow cytometer (BD Biosciences) to detect the apoptotic rate at each stage and analyzed using FlowJo software (version 10.4.0; BD Biosciences). For determining the cell cycle distribution, the cells were digested with trypsin and placed in 500 µl 70% ethanol solution overnight at 4°C, with the subsequent application of all previously described experimental procedures. A CytoFLEX flow cytometer (Beckman Coulter, Inc.) was used to detect the cell cycle distribution.

Firstly, the 3D chemical structure of Sch B was obtained using PubChem (https://pubchem.ncbi.nlm.nih.gov/), and the drug target of Sch B was then obtained using the PharmMapper (http://www.lilab-ecust.cn/pharmmapper/), CTD (http://ctdbase.org/) and STITCH databases (http://stitch.embl.de/). The OS target genes were then collected from the GeneCards database (https://genecards.weizmann.ac.il/v3/), and the drug disease intersection genes were finally obtained. The protein interaction network was constructed using the STRING database (https://string-db.org/) (14), and the Sch B OS target protein regulatory network was visualized using Cytoscape (15). The node degree value was calculated and the key genes were screened. The target gene Gene Ontology (GO) functional annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis were performed using R and R studio (version 3.4.0) (16–20).

The 143B cells cultured in 90-mm dishes were treated with Sch B (0, 40, 60 or 80 µM) or DMSO for 24 h. For protein extraction, the cells were washed with cold PBS three times and 1 ml pre-cooled lysate was added (Wuhan Boster Biological Technology, Ltd.) to lyse the cells. After boiling with 1X SDS loading buffer for 5 min, the protein concentration was measured using BCA protein assays (Beyotime Institute of Biotechnology). For electrophoresis, 35 µg protein samples were loaded on 10% SDS gels and resolved using SDS-PAGE. When the target protein was ~1 cm away from the lower edge of the gel, electrophoresis was terminated, and the resolved proteins were transferred to PVDF membranes (MilliporeSigma). The membranes were blocked in 5% BSA (Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at 37°C, and the membranes were incubated with specific primary antibodies [β-actin (cat. no. 3700), proliferating cell nuclear antigen (PCNA; cat. no. 13110) β-catenin (cat. no. 8480), c-Myc (cat. no. 18583), cyclin D1 (cat. no. 55506), GSK-3β (cat. no. 12456), Bcl-2 (cat. no. 15071), Bad (cat. no. 9268), Bax (cat. no. 14796), caspase-3 (cat. no. 9662), cleaved caspase-3 (cat. no. 9661), PARP (cat. no. 9532), cleaved-PARP (cat. no. 5625), MMP-2 (cat. no. 40994), MMP-7 (cat. no. 3801), MMP-9 (cat. no. 13667), vimentin (cat. no. 5741), E-cadherin (cat. no. 14472), N-cadherin (cat. no. 13116), cyclin E (cat. no. 4129), Akt (cat. no. 4685), p-Akt (Ser473; cat. no. 4060), phosphorylated (p-)Akt (Thr308; cat. no. 4070); dilution 1:1,000 for all the aforementioned antibodies; all from Cell Signaling Technology, Inc.] at 4°C overnight in 5% BSA (Cell Signaling Technology, Inc.). The secondary antibodies used (anti-mouse IgG cat. no. 7076, anti-rabbit IgG, cat. no. 7074; dilution 1:1,000; both from Cell Signaling Technology, Inc.) was selected based on the source of the primary antibody, and the PVDF membranes were incubated with the secondary antibodies at room temperature for 1 h. The membranes were then washed with TBS-Tween-20 three times, 10 min each time, and an enhanced chemiluminescent kit (MilliporeSigma) was used to visualize the proteins with a ChemiDoc MP Imaging system and Image Lab Software (version 5.2.1; Bio-Rad Laboratories, Inc.). All bands presented together were probed on the same membrane in order for the proteins to share the same loading control.

The 143B cells were treated with the Wnt signaling pathway agonist (BML284; cat. no. S8178) and Akt phosphorylation activator (SC79; cat. no. S7863) purchased from Selleck Chemicals in combination with Sch B. Subsequently, the effects of BML284 and SC79 were observed using crystal violet, wound healing and Transwell assays, as well as flow cytometric analysis.

In the present study, all experimental procedures involving animals were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (approval no. 2020-503), and experiments were performed in accordance with the guidelines of the Declaration of Helsinki (21). A total of 15 BALB/c nude mice (female, 28 days old, weighing 16.5-18.5 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. The mice were reared under specific pathogen-free conditions and allowed to acclimatize for 1 week without any treatment. The environment was maintained at a constant temperature of 24°C and at 55% humidity, with a 12 h light/dark cycle. All the mice had free access to food and water. 143B cells (2.5×10⁶) in 60 µl PBS were injected into the tibial plateau of the mice through the proximal tibia. At 3 days after the injection of the tumor cells, the mouse vital signs were observed. Afterwards, 15 mice were randomly divided into five groups, with 3 mice per group. One group was selected as the control group, and the remaining four groups were administered various doses of Sch B (25, 50 or 75 mg/kg) or 0.5% sodium carboxymethyl cellulose by intra-gastric administration every other day. The mouse weight and tumor size (length and width) were recorded prior to each administration. Mouse survival was judged by observing the respiratory movements and heartbeat of the mice. Mouse death was verified when no breathing and heartbeat were observed for >3 min. The animal survival rate was evaluated for up to 40 days. The experimental animals were euthanized when obvious cachexia, infection and excessive tumor volume were observed. However, there were no animals that required euthanasia during the experiment due to the reasons stated above. In addition, no animals died during the experiment. Following 15 treatments, all mice were euthanized by CO₂ with a flow rate of 30% volume displacement/min. Tumor tissue and lungs were removed and fixed in 4% paraformaldehyde. The maximum observed length and width of the tumors were 18 and 16 mm, respectively, and the tumor volume did not exceed 2,500 mm³.

Tumor and lung tissues were fixed with 4% paraformaldehyde at 4°C for 72 h, and embedded in paraffin. Paraffinembedded tumor samples were cut into 5-µm-thick sections. The wax on the section surface was removed by heating and soaking in various concentrations of alcohol (100, 90, 80 and 70%). The OS tumor sections and lung tissues were then stained with H&E (Beyotime Institute of Biotechnology) at 37°C for 1 min, to visualize tumor tissue lung metastasis. The same embedded sections and dewaxing methods used for the tumor and lung tissues in H&E staining were used also to perform immunohistochemistry. For immunohistochemistry, the OS tumor tissue sections were incubated with specific histochemical antibodies [PCNA (cat. no. 13110), Bcl-2 (cat. no. 15071), Vimentin (cat. no. 5741), p-Akt (Ser473; cat. no. 4060) and β-catenin (cat. no. 8480); dilution for all antibodies, 1:100; Cell Signaling Technology, Inc.] at 4°C overnight, and probed with HRPconjugated secondary antibody (anti-rabbit IgG cat. no. ab6721; dilution 1:350; Abcam) at 37°C for 30 min, and then imaged under a light microscope (ECLIPSE Ti, Nikon Corporation, magnification, ×200 and ×400).

All experiments were repeated three times to eliminate errors. All data are presented as the mean ± standard deviation and were analyzed using GraphPad Prism version 5.0 (GraphPad Software, Inc.). A one-way ANOVA followed by a Tukey's post hoc test was used for conducting comparisons between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Firstly, the inhibitory effects of Sch B on OS cell (143B, MG63, Saos2 and U2OS) proliferation were observed using crystal violet staining, and quantitative analysis demonstrated that the inhibitory effects of Sch B on OS cell proliferation were concentration-dependent (Fig. 1A and B). An MTT assay was used to further verify the effects of Sch B on OS cell viability and evaluate its toxic effects on normal cells from the perspective of safety. Following 48 h of treatment, the IC₅₀ values in the OS cells were 70.32 µM (143B cells), 58.68 µM (MG63 cells), 65.33 µM (Saos2 cells) and 72.38 µM (U2OS cells) (Fig. 1C), whereas for the normal cells (HEB and HS5 cells), these values were 286.98 and 366.95 µM respectively, which further illustrated the safety of the use of Sch B (Fig. 1D). In addition, it was found that Sch B inhibited the colony formation ability of the 143B cells at very low concentrations (3, 6 and 9 µM), which indicated that the OS cells were highly sensitive to Sch B, suggesting that it has the ability to inhibit the initiation of OS (Fig. 1E and F). Moreover, Sch B decreased the protein expression levels of PCNA (Fig. 1G and H), which is a recognized marker of cell proliferation. Together, these results indicated that Sch B exerted satisfactory OS cell proliferation inhibitory effects, whilst it did not exert notable toxic effects on normal cells at therapeutic concentrations (0–100 µM).

OS metastasis has been associated with a poor prognosis (3,4). Therefore, in the present study, the effects of Sch B on OS cell migration were assessed using wound healing and Transwell assays. In the wound healing assay, the healing rate of the different groups at different time periods was calculated to analyze the inhibitory effects of Sch B on OS cell migration. Compared with the control and DMSO groups, Sch B significantly inhibited OS cell migration in a concentration-dependent manner (Fig. 2A-F). The prominent invasive ability of OS often leads to the destruction of surrounding tissues, which gives rise to the hematogenous metastasis of OS. Herein, Matrigel Transwell invasion assays revealed that Sch B suppressed OS cell invasive potential as well (Fig. 2G and H). However, considering that Sch B can effectively inhibit cell proliferation, when the cells exhibit different proliferative abilities under the different experimental conditions, this may affect the study of cell migration and invasion. Therefore, the expression levels of well-known markers of tumor invasion and metastasis were also assessed. Epithelial-mesenchymal transition (EMT) is a biological process in which epithelial cells transform into cells with mesenchymal phenotype through specific procedures (22). EMT is important for tumor invasion and metastasis. The expression of the EMT-related proteins, N-cadherin, Snail and Vimentin, was observed using western blot analysis, and was found to be decreased by Sch B, whereas the opposite effect was observed as regards E-cadherin expression (Fig. 2I and J). MMPs can degrade almost all types of protein components in the extracellular matrix; thus, increasing attention has been given due to their role in destroying the histological barrier (23). Sch B also significantly decreased the expression of MMP-2, MMP-7 and MMP-9 (Fig. 2I and J). Therefore, Sch B may effectively suppress OS cell migration and invasion.

Through flow cytometry analysis, it was revealed that Sch B hindered OS cell mitosis progression in the G₁ phase (Fig. 3A-D). Therefore, the expression levels of ccyclin D1 and cyclin E were detected using western blot analysis, and it was demonstrated that the cyclin levels, which promote cell cycle progression from the G₁ to the S phase, decreased in a concentration-dependent manner (Fig. 3E and F). Following Hoechst 33258 staining, it was observed under a fluorescence microscope that following treatment with Sch B, nuclear pyknosis, nuclear fragmentation and a large number of apoptotic bodies were present in OS cells (Fig. 4A). In addition, flow cytometric analysis for apoptosis, it was demonstrated that Sch B significantly increased the OS cell apoptotic rate, particularly that of late apoptosis (Fig. 4B-E). In order to explore the molecular mechanisms underlying the promoting effects of Sch on the apoptosis of OS cells, the expression of a large number of apoptosis-related proteins was assessed. It was observed that Sch B increased the expression of certain apoptosis-promoting proteins, including the Bcl-2 protein family members Bad and Bax. However, Sch B decreased Bcl-2 expression. Moreover, the expression of the apoptotic biomarkers, cleaved caspase-3, PARP and cleaved PARP was increased following treatment with Sch B, and the expression of caspase-3 decreased (Fig. 4F and G).

In order to examine the mechanisms underlying the inhibitory effects of Sch B on OS cells, the drug-disease gene interactions of Sch B and OS were determined using network pharmacological analysis (Fig. 5A), and the target protein network was visualized (Fig. 5B), so as to screen the top 30 key genes (Fig. 5C). Then the relevant signal pathways were determined by GO function annotation and KEGG pathway enrichment analysis (Fig. 5D and E). It was found that the core targets may be located in the Wnt/β-catenin and PI3K/Akt signaling pathways.

Subsequently, the changes in key targets in related signaling pathways were assessed using western blot analysis. It was observed that Sch B primarily altered the key targets of the Wnt/β-catenin and PI3K/Akt signaling pathways in OS cells. The Wnt/β-catenin signaling pathway plays a crucial role in early development, organ formation, tissue regeneration and other physiological processes during animal embryonic development. If the key proteins in this signaling pathway mutate, this may culminate in an abnormal signal activation, possibly inducing cancer occurrence (24). In the present study, the components and downstream targets of the Wnt/β-catenin signaling pathway were examined using western blot analysis. The results revealed that compared with the control group, the expression of β-catenin, the core member of the Wnt/β-catenin signaling pathway, was decreased, whereas that of GSK-3β, a negative regulator of the Wnt/β-catenin signaling pathway, was significantly increased in the OS cells treated with Sch B. In addition, the expression of c-Myc, the target gene of Wnt, was also significantly decreased (Fig. 6A and B). As an important signal transduction pathway, the PI3K/Akt signaling pathway plays a key role in inhibiting the apoptosis and promoting the proliferation of cells by affecting the activation of downstream effector molecules (25,26). It is closely related to the occurrence and development of various human tumors (27). Herein, western blot analysis revealed that the expression levels of the PI3K/Akt signaling pathway-related proteins, Akt, p-Akt (Ser473) and p-Akt (Thr308), were all decreased in the OS cells following treatment with Sch B (Fig. 6A and B). In order to further confirm that the changes in the protein expression levels of the targets in the related signaling pathways were involved in the effects of Sch B on OS cells, the cells were treated with the Wnt signaling pathway agonist, BML284, and the Akt phosphorylation activator, SC79, in combination with Sch B. Both BML284 and SC79 partially reversed the anticancer effects of Sch B on OS cells (Fig. 6C-K). Taken together, the anticancer effects of Sch B on OS cells were demonstrated to be related to Wnt/β-catenin and PI3K/Akt signaling pathway inhibition.

An in vivo OS model was established to further examine the effects of Sch B on the growth and metastasis of OS. The results revealed that Sch B inhibited OS growth in a dose-dependent manner, and the body weight of the mice in each group did not change significantly during the administration period (Fig. 7A-C). Using H&E staining, it was demonstrated that compared with the control group, the tumor nuclear heterogeneity in the treatment group was reduced; conversely, nuclear fragmentation and nucleolysis were increased. In addition, the tumor tissue in the treatment group became significantly more detached (Fig. 7D). Immunohistochemical results demonstrated that the expression levels of PCNA, Bcl-2, Vimentin, β-catenin and p-Akt (ser308) were significantly decreased when treated with a high Sch B concentration, in accordance with the in vitro results (Fig. 7E). As regards OS metastasis inhibition in vivo, dense and heterogeneous cell masses were observed in the mouse lung tissue in the control group, suggesting the lung metastasis of OS; however, similar cell masses could not be observed in the high Sch B concentration treatment group. Sch B effectively reduced the tumor mass in the lungs according to lung H&E staining (Fig. 7F). Thus, these results suggested that Sch B inhibited OS growth and lung metastasis in vivo.