The molecular structure of asiatic acid was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and entered into the PharmMapper (http://www.lilab-ecust.cn/pharmmapper/), Swiss Target Prediction (http://swisstargetprediction.ch/) and Uniprot (https://www.uniprot.org/) databases to identify drug targets with scores >0 after eliminating repeated targets. In addition, the Online Mendelian Inheritance in Man (OMIM; http://omim.org/), Therapeutic Target Database (TTD; http://db.idrblab.net/ttd/) and Genecards (https://www.genecards.org/) databases were searched to identify disease targets based on the keyword ‘osteosarcoma’. Duplicated disease targets were removed and Venn diagrams of common drug-disease targets were drawn using the Venny software mapping tool (https://bioinfogp.cnb.csic.es/tools/venny/). Finally, Cytoscape software (3.8.2 version) was used to draw a network diagram of ‘disease-target-drug’ interactions.

The aforementioned common targets were imported into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://string-db.org/) and Cytoscape software (https://cytoscape.org/; version 3.8.2) was used to draw the resulting PPI network maps. Network analyzer was used to perform a topological analysis of the PPI networks. The core targets selected according to a degree value were mapped using R software (http://www.R-project.org).

GO and KEGG functional enrichment analyses of critical target genes were performed using the Bioconductor bioinformatics package (https://www.bioconductor.org/; version 3.17) of R software (http://www.R-project.org). GO and KEGG enrichment analysis results were visualized in bar and bubble plots. P-value <0.05 was employed to identify statistically significant GO terms and KEGG pathways. The significance of GO and KEGG enrichment was represented by employing the fold change threshold=-log₁₀ (P-value) for a more comprehensive depiction of the figure.

The asiatic acid structure obtained from the PubChem database was imported into Chem3D software and the optimized structures were further imported into Schrodinger software (https://www.schrodinger.com/; version 2019.1) for hydrogenation and energy minimization. The protein structures of EGFR (1M17), SRC proto-oncogene, non-receptor tyrosine kinase (SRC; 1YOL), Caspase-3 (2CNK), heat shock protein 90α family class A member 1 (HSP90AA1; 4BQG), Estrogen Receptor 1 (ESR1; 7UJF) and Interleukin 6 (IL-6; 4O9H) were obtained from the RCSB database (https://www.rcsb.org/), and entered into Schrodinger Maestro's Protein Preparation Wizard for hydrogenation, peptide repair, energy minimization and structure optimization. The molecular docking was then optimized in the Glide platform of the software by determining sites on the basis of protein structure and ligands in 15 Å × 15 Å × 15 Å boxes. Finally, molecular docking and screening were conducted using the Standard Precision Glide Docking method.

MG63 human osteosarcoma cells (cat. no. TCHu124), HOS human osteosarcoma cells (cat. no. TCHu167) and MC3T3-E1 mouse-derived normal osteoblast cells (cat. no. GNM15) were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. 143B human osteosarcoma cells (cat. no. CRL-8303) were obtained from American Type Culture Collection. Human osteosarcoma cells were cultured in Minimum Essential Medium (MEM; Gibco; Thermo Fisher Scientific, Inc.) containing 1% penicillin-streptomycin (Beijing Solarbio Science & Technology Co., Ltd.) and 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in a sterile incubator containing 5% CO₂ at 37°C. MC3T3-E1 cells were cultured in α-MEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS and 1% penicillin-streptomycin under the same controlled culture conditions. The osteosarcoma and MC3T3-E1 cells were passaged when reaching 80–90% confluency in culture flasks.

Asiatic acid (high-performance liquid chromatography ≥98%; Shanghai Yuanye Biotechnology Co., Ltd.) was dissolved in DMSO. An appropriate concentration of DMSO was used for the experimental controls. Antibodies against Bcl-2 (cat. no. 4223), Bax (cat. no. 2772), phosphorylated (p-)ERK1/2 (cat. no. 4370), ERK1/2 (cat. no. 4695), p38 (cat. no. 8690), p-p38 (cat. no. 4511), JNK (cat. no. 9252), p-JNK (cat. no. 4668), p-PI3K (cat. no. 17366), CDK2 (cat. no. 18048) and cyclin A2 (cat. no. 67955) and the goat anti-rabbit IgG (cat. no. 7074) and anti-mouse IgG (cat. no. 7076) secondary antibodies were purchased from Cell Signaling Technology, Inc. The voltage dependent anion channel 1 (VDAC1; cat. no. ab14734), p-AKT (cat. no. ab192623), AKT (cat. no. ab179463), PI3K (cat. no. ab191606), LC3 (cat. no. ab48394) and p62 (cat. no. ab56416) antibodies were obtained from Abcam. The GAPDH antibody (cat. no. 21612) was purchased from Signalway Antibody LLC.

Growing osteosarcoma and MC3T3-E1 cells were trypsinized, centrifuged (180 × g; 25°C; 3 min) and resuspended in complete MEM or α-MEM to prepare cell suspensions. Cells (~4.0×10³) were seeded into 96-well plates and incubated at 37°C for 24 h. Subsequently, the osteosarcoma cells were treated with asiatic acid (0, 10, 20 and 40 µM) at 37°C for 24 and 48 h. The culture medium was replaced with 10 µl CCK8 (Beyotime Institute of Biotechnology) in MEM and cells were incubated at 37°C for 2–4 h. OD values were determined using a microplate reader (MK3; Thermo Fisher Scientific, Inc.) at 450 nm.

The osteosarcoma cells were treated with asiatic acid (0, 20 and 40 µM) at 37°C for 24 h, then collected by centrifugation (180 × g; 4°C; min) and fixed with 70% chilled ethanol at 4°C for 12 h. The fixed cells were stained with PI dye (Beyotime Institute of Biotechnology) and incubated at 37°C for 30 min. A flow cytometer (FACSCelesta; BD Biosciences) and Modfit LT software (5.0 version; Verity Software House, Inc.) were used to analyze the cell cycle phases.

The osteosarcoma cells (~15.0×10⁴) were seeded into six-well plates and incubated with asiatic acid (0, 10, 20 and 40 µM) at 37°C for 24 h. The adherent cells from each well were collected, and suspended and placed in a flow tube. To stain the cells, the cells were resuspended in 5 µl annexin V-FITC and 10 µl PI (Beyotime Institute of Biotechnology) and incubated at room temperature for 30 min. Finally, the stained cells were analyzed by flow cytometry (FACSCelesta; BD Biosciences) and BD FACSDiva Software (version 6.1.3; BD Biosciences). The apoptotic rate was calculated by the percentage of early + late apoptotic cells.

The osteosarcoma cells were treated with asiatic acid for 24 h and then resuspended in 0.5 ml cell culture medium. JC-1 (0.5 ml; Beyotime Institute of Biotechnology) staining working solution was added to the culture medium and the cultures were placed in a cell incubator at 37°C for 20 min. Subsequently, the cells were washed twice with staining buffer and analyzed using a flow cytometer (FACSCelesta; BD Biosciences) and BD FACSDiva Software (version 6.1.3; BD Biosciences).

The 143B and HOS human osteosarcoma cell lines were treated with asiatic acid (0 and 40 µM) at 37°C for 24 h. Dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology) was then diluted with serum-free MEM to a final working solution concentration of 10 µM, and the cells were resuspended in the diluted DCFH-DA and incubated at 37°C for 20 min. After washing the cells three times with serum-free MEM, intracellular ROS levels were measured using a flow cytometer (FACSCelesta; BD Biosciences) and BD FACSDiva Software (version 6.1.3; BD Biosciences).

The preparation and observation of electron microscope specimens was completed in the following seven steps. In the first step, the collected cells were fixed with 3% glutaraldehyde (SPI-CHEM Inc.) for 4 h at 4°C, and then fixed with 0.1 M sodium dimethylarsenate buffer at 4°C for three times, with a 2 h interval between changes. In the second step, the samples were subsequently immersed with 1% osmium acid (SPI-CHEM Inc.) for 2 h at 4°C, then rinsed with 0.1 M sodium dimethylarsenate buffer twice, each time at 4°C for 15 min. Step three was staining with saturated uranyl acetate dye (SPI-CHEM Inc.) for 2 h at room temperature. In the fourth step, the stained samples were soaked in 50% alcohol at 4°C for 10 min, 70% alcohol at 4°C for 10 min, 80% alcohol at room temperature for 10 min, 90% alcohol at room temperature for 10 min, anhydrous ethanol soaks twice at room temperature for 10 min, times acetone permeations twice at room temperature for 15 min, a complete encapsulation solution (Eponate12 epoxy resin; Ted Pella, Inc.) and acetone in a 1:1 ratio at room temperature for 3 h, then changing the above ratio to a 1:2 penetration for 3 h, and then overnight penetration of the complete embedding solution at room temperature. In the fifth step, the samples soaked with the complete embedding solution were placed in a 40°C incubator for 12 h, and then transferred to an embedding plate still soaked with the complete embedding solution and placed in a 60°C incubator for 24 h. In the sixth step, the samples were sectioned by using an ultrathin sectioning machine (UC7; Leica Microsystems GmbH), with a section thickness of 90 nm. Finally, the samples were observed by a JEM-1400 transmission electron microscope (JEOL Ltd.) with an operating voltage of 80 kV, and image acquisition was performed with DigitalMicrograph Software version 832 (Gatan, Inc.; Thermo Fisher Scientific, Inc.).

Osteosarcoma cells were trypsinized and collected by centrifugation (180 × g; 4°C; 3 min) before mixing with appropriate amounts of protein lysate (PMSF; RIPA, 1:100; Beyotime Institute of Biotechnology). The cells were lysed on ice, sonicated and centrifuged at high speed (13,800 × g; 4°C; 15 min) to collect the supernatant protein. Next, the protein concentration of each sample was measured using a BCA protein assay kit (Beyotime Institute of Biotechnology) and each protein sample was adjusted to the same concentration. Subsequently, 5X SDS-PAGE protein loading buffer was added to the protein solutions and these were boiled for 10 min. Proteins (20 µg/lane) were separated by SDS-PAGE on 10 and 12% separation gels and then transferred (250 mA; 2.5 h) to PVDF membranes (pore size, 0.2 µm; Merck KGaA). Fresh 5% skimmed milk was used to block the membranes for 60 min. Subsequently, the membranes were cut and incubated with primary antibodies (dilution, 1:1,000) at 4°C for 12 h and with secondary antibodies (dilution, 1:3,000) at room temperature for 2 h. The ECL reagents (Zeta-Life Inc.) and Fluorescent Imaging System (Tanon 5200; Tanon Science and Technology Co., Ltd.) were used to visualize the protein bands. In addition, grey value intensities were calculated using ImageJ software (v1.8.0; National Institutes of Health).

All data are presented as the mean ± SD of at least three independent experiments, and all data were analyzed and graphs were plotted using the Figdraw Platform (https://www.figdraw.com/), SPSS Statistics 25.0 (IBM Corp.) and GraphPad Prism 8.0 (GraphPad Software; Dotmatics) software. Unpaired Student's t-tests were used for comparisons between 2 groups. Statistical differences among ≥3 groups were determined using one-way ANOVA followed by Tukey's post hoc tests. P<0.05 was considered to indicate a statistically significant difference.

The asiatic acid structure was obtained from the PubChem database, 341 drug targets after de-duplication were obtained from the PharmMapper, Swiss Target Prediction and Uniprot databases (Table SI), and 911 disease targets were obtained from the OMIM, TTD and Genecards databases (Table SII). A Venn diagram (Fig. 1A) identified 78 common targets, which were entered into Cytoscape to draw a network diagram of ‘osteosarcoma-target-asiatic acid’ interactions (Fig. 1B). The common targets of asiatic acid were entered into the STRING database and a PPI network graph with 78 nodes, 969 edges and 4 concentric circles was obtained (Fig. 1C). Node size, color and depth represent the degree values. The PPI network data were then subjected to topological analysis and 30 core targets were identified based on mean degree values. Among the core target genes, EGFR (64 edges), Caspase-3 (55 edges), ESR1 (54 edges), HSP90AA1 (54 edges), IL-6 (54 edges) and SRC (54 edges) had a high node degree, indicating their close association with the effect of asiatic acid against osteosarcoma (Fig. 1D).

Common targets were analyzed via GO enrichment analysis after running the R programming language to select 1,581 biological process (BP) pathways, 27 items associated with cellular component (CC) expression processes and 110 items associated with molecular function (MF) processes. Fig. 2A shows the top 10 BP, CC and MF items. KEGG analysis of the common targets revealed 134 related pathways, enriched in cancer-related signaling pathways such as ‘Proteoglycans in cancer’ (hsa05205), ‘PI3K-AKT signaling pathway’ (hsa04151), ‘Rap1 signaling pathway’ (hsa04015) ‘MAPK signaling pathway’ (hsa04010) and ‘Ras signaling pathway’ (hsa04014). Fig. 2B shows the top 20 results from a KEGG enrichment bubble map. The present results suggested that the PI3K-AKT and MAPK signaling pathways may be associated with the effects of asiatic acid against osteosarcoma.

The PPI network analysis of asiatic acid and osteosarcoma revealed target genes with a significant degree value of association with anti-osteosarcoma effects. Therefore, the top six highest-ranking target genes were selected for molecular docking. Binding energy and hydrogen bond interactions were assessed to evaluate the affinity and binding capacity between asiatic acid and target proteins. Molecular docking visualization showed hydrogen bonds between asiatic acid and amino acids in the active sites of the target proteins. asiatic acid binds the following proteins: EGFR (binding energy, −7.01 kcal/mol) forming hydrogen bonds with LYS-721, ASP-776, CYS-773 and ASP-831 amino acids in its active site (Fig. 3A); Caspase-3 (binding energy, −6.51 kcal/mol) forming hydrogen bonds with TRP-206, GLY-122 and THR-62 amino acids in its active site (Fig. 3B); ESR1 (binding energy, −6.85 kcal/mol) forming hydrogen bonds with VAL-533, THR-347 and ASP-351 amino acids in its active site (Fig. 3C); HSP90AA1 (binding energy, −5.83 kcal/mol) forming hydrogen bonds with LYS-581, ASN-51, GLY-135 and LEU-107 amino acids in its active site (Fig. 3D); IL-6 (binding energy, −6.69 kcal/mol) forming hydrogen bonds with LYS-86, TYR-97 and ASN-63 amino acids in its active site (Fig. 3E); and SRC (binding energy, −7.02 kcal/mol) forming hydrogen bonds with GLY-346, SER-347 and ASN-393 amino acids in its active site (Fig. 3F). In summary, molecular docking revealed that asiatic acid exhibited strong affinity and binding potential with the target proteins EGFR, Caspase-3, ESR1, HSP90AA1, IL-6 and SRC.

Asiatic acid inhibited the proliferation of 143B, MG63 and HOS human osteosarcoma cell lines in a dose- and time-dependent manner. No cytotoxicity was observed in MC3T3-E1 normal osteoblast cells treated with asiatic acid under the same conditions (Fig. 4A). Cell cycle analysis revealed that the proportion of cells in the G₀/G₁ phase was decreased and that in the G₂/M phase was increased (Fig. 4B). Western blot analysis further demonstrated that asiatic acid suppressed the expression of cyclin A2 and CDK2 in osteosarcoma cells (Fig. 4C).

To investigate whether asiatic acid induced apoptosis in 143B, MG63 and HOS osteosarcoma cells, annexin V-FITC/PI double staining reagent was used to analyze the proportion of apoptotic cells. The present results demonstrated that the proportion of apoptotic cells increased with increasing asiatic acid concentrations (Fig. 5A). MMP level reductions are an essential feature of early apoptosis stages (41). Therefore, the MMP was examined and an increase in green fluorescence was observed in JC-1-labeled mitochondrial membranes, indicating early apoptosis with a reduced MMP (Fig. 5B). In addition, changes in apoptosis-related proteins validated these findings. The present results indicated that asiatic acid increased the expression levels of Bax and VDAC1, and decreased the expression levels of Bcl-2 (Fig. 5C). Thus, asiatic acid promoted mitochondrial dysfunction and induced apoptosis in osteosarcoma cells.

Autophagy is closely related to apoptosis and both processes are involved in tumor cell death mechanisms (42,43). Therefore, the present study next investigated whether asiatic acid triggers autophagy in 143B, MG63 and HOS osteosarcoma cells. Transmission electron microscopy is the gold standard for examining autophagy by confirming the presence of autophagosomes at the subcellular level. Osteosarcoma cells were treated with 40 µM asiatic acid. The transmission electron microscopy images revealed marked autophagosome increases in all the treated cells (Fig. 6A). Furthermore, the present study assessed the effects of asiatic acid on the levels of autophagy-related proteins LC3 and p62, which are marker proteins, and the western blot analysis results demonstrated that asiatic acid treatment markedly increased the LC3-II/I ratio and decreased the p62 levels, indicating that asiatic acid could trigger autophagy in osteosarcoma cells (Fig. 6B).

The KEGG enrichment analysis results suggested that the PI3K/AKT and the MAPK signaling pathways were associated with the anti-osteosarcoma effects of asiatic acid. Thus, the present study examined the expression levels of PI3K/AKT signaling pathway-related proteins. Fig. 7A shows that asiatic acid treatment markedly decreased the levels of p-PI3K/PI3K and p-AKT/AKT. ROS analysis using DCFH-DA staining (Fig. 7B) revealed that asiatic acid treatment markedly increased intracellular ROS content. Western blot analysis results further demonstrated that asiatic acid upregulated the protein levels of p-ERK1/2/ERK, p-p38/p38 and p-JNK/JNK (Fig. 7C). Consequently, asiatic acid may inhibit the PI3K/AKT signaling pathway and activate the ROS/MAPK signaling pathway to regulate osteosarcoma cell death mechanisms.