Investigation of inhibition effect of daidzein on osteosarcoma cells based on experimental validation and systematic pharmacology analysis

Reagents

Daidzein [7-hydroxy-3- (4-hydroxyphenyl) chromen-4-one] was purchased from Aladdin Industrial, Inc. (Shanghai, China), which with a purity over 98%. It was dissolved in DMSO at a final concentration of 100 mM in medium and stored at −20 °C. Dimethyl sulfoxide (DMSO) was obtained from Sinopharm Chemical Reagent Co., Ltd. (China).

Cell culture

Human osteosarcoma cell lines (143B and U2OS) and human osteoblast cell line hFOB 1.19 were obtained from the China Centre for Type Culture Collection (Wuhan, China). 143B cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat - inactivated FBS (Gibco) and 1% antibiotics in a 37 °C incubator with 5% CO₂. U2OS cells were cultured in McCoy’s 5A medium (modified) when hFOB 1.19 was cultured in α-MEM (HyClone) and the other conditions were the same as those used for 143B cells.

Cell proliferation analysis

Cells were plated in 96-well plates at a density of 2000 cells per well and incubated at 37 °C for 12 h. Then, the cells were treated with different concentrations of daidzein diluted in medium containing 10% FBS. After incubation, the supernatants were removed, and MTT solution (Beyotime, Shanghai, China) was added to each well (100 µL per well). Following incubation at 37 °C in a dark environment for an additional 4 h, the supernatants were removed again, and 200 µL DMSO was added to each well to solubilize formazan. After 10 min, the 96-well plates were placed in a microplate reader, and the absorbance was recorded at 570 nm. The data came from three independent experiments, each with six copies. The mean cell viability of the treated cells was used to calculate the survival rate.

Colony formation assay

The cells were evenly seeded in a 6-well plate at a density of 500 cells per well. After 24 h, the cells were treated with different concentrations of daidzein for approximately 7 days until the cells grew into visible colonies. Then, the supernatant was discarded, and the cells were washed with PBS three times. Colonies were then fixed with 4% paraformaldehyde for 30 min and stained with 1% crystal violet for 10 min. The number of colonies (more than 50 cells/colony) was counted and quantified.

Cell apoptosis and cycle detection

Following exposure to daidzein for a specified period, osteosarcoma cells were collected for apoptosis and cycle detection. Hoechst 33342 (Beyotime, Shanghai, China) was used to detect cell apoptosis by observing the cell morphology. After daidzein treatment, the medium was discarded, and the cells were washed twice with PBS and then fixed with sufficient 4% paraformaldehyde at 4 °C for 10 min. Then, sufficient Hoechst 33342 staining solution was added to cover the cells. After incubation in the dark for 10 min, the cells were washed with PBS and then immediately observed and imaged by fluorescence microscopy. In addition, cell apoptosis and cell cycle progression were analyzed by flow cytometry. Cell apoptosis of human osteosarcoma cells was analyzed based on Annexin V-FITC/propidium iodide (PI) dual staining when the cell cycle was assessed by staining cells with 20 µg/mL PI.

Migration ability detection

The migration ability of osteosarcoma cells was detected by Transwell migration assay and scratch wound assay. The Transwell experimental procedure was as follows: Approximately 5 ×10⁴ cells suspended in 100 µL serum-free RPMI 1640 or McCoy’s 5A medium were added to each upper chamber, and 600 µL of medium containing 10% FBS was added to the lower chamber. Cells were cultured at 37 °C for 24 h, fixed with 4% paraformaldehyde for 30 min and stained with 1% crystal violet for 10 min. Cotton swabs were used to remove the unmigrated cells on the upper surface of the membrane, and the cells that migrated on the lower surface of the film were counted. As for scratch wound assays, cells were plated in a 6-well plate at a density of 5 ×10⁵ cells per well and then incubated at 37 °C until almost complete confluence. Three parallel wounds were made with a 10 µL pipette tip per well. Cells were cultured in serum-free medium supplemented with different concentrations of daidzein. At 0 h and 24 h, photos were taken at the same position under an optical microscope, and the wound width of each group was compared.

Establishment of a nude mouse osteosarcoma xenograft model

Twelve four-week-old male BALB/c nude mice obtained from Hubei Experimental Animal Research Center (Hubei, China) were used for animal experiments. 143B cells were digested and suspended in cold PBS at a final concentration of 1 ×10⁷ cells per mL. In this model, a total of 100 µL of the prepared cell suspension was injected in the right subaxillary area subcutaneously. 7 days after tumor inoculation, when the volume of the tumor was approximately 100 mm³, daidzein (20 mg/kg) was administered by tail vein injection every 2 days in the experimental group (n = 6), while the control group (n = 6) was injected with an equal volume of DMSO. The injection treatment lasted for 9 days, and the tumor volume and mouse body weight were measured every 3 days. Tumor volume (mm³) was calculated using the following equation: length × width² × 0.5. On day 16, mice were sacrificed by dislocation of cervical vertebra after anesthesia with 1% sodium pentobarbital (100 mg/kg body weight, intraperitoneal injection), and the xenografts were dissected and fixed with 4% polyformaldehyde for H&E staining analysis. When the tumor diameter exceeded 20 mm or rupture and infection occurred, as well as when treatment was ineffective after systemic infection occurred, mice were euthanized using the method described above.

This in vivo study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Institutional Animal Care and Use Committee, Wuhan University Center for Animal Experiment (protocol code WP2020-08106. On August 10th, 2020).

Target identification and network construction

The osteosarcoma-related targets were collected and combined from the following databases: DigSeE (Kim et al., 2013), DisGeNET (http://www.disgenet.org/), GeneCards (https://www.genecards.org/), KEGG disease (https://www.kegg.jp/kegg/disease/), MalaCards (http://www.malacards.org/), and Phenopedia (https://phgkb.cdc.gov/PHGKB/startPagePhenoPedia.action). The validated targets of daidzein were extracted from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and PharmMapper (http://www.lilab-ecust.cn/pharmmapper/). Common targets of osteosarcoma and daidzein were discovered and retained by VENNY 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/) for further analysis. A protein-protein interaction (PPI) network was constructed by STRING (https://string-db.org/) and visualized by Cytoscape software. The Database for Annotation, Visualization and Integrated Discovery (DAVID; http://david.abcc.ncifcrf.gov/) was used to perform GO and pathway enrichment analyses, and Cytoscape software (Version 3.8.1) was used to construct the target-function network.

Molecular docking studies

To identify the potential interaction between Src and daidzein, molecular docking simulations were conducted with AutoDock Vina (version 1.1.2) to predict their preferred binding conformation. PubChem was referenced for the SDF file of daidzein, and the SDF file was transformed into a PDB file by Open Babel 2.3.2. The crystal structure of human Src was derived from the Protein Data Bank (PDB) database (https://www.rcsb.org/structure/2H8H). Before molecular docking analysis, PYMOL 2.3.4 software was used to remove the water molecules as well as ligands from the receptor protein, and AutodockTools software was used to balance the charge and affix hydrogen atoms to the receptor protein. Grid Option was used to adjust the number of grid points in each direction, the center of the binding pocket and the spacing between the grid points. The centroid of the daidzein molecule in the crystal structures of the complex was defined as the binding site. AutoDock Vina software was used for molecular docking of the Src protein and daidzein molecule, and the results were output in the form of affinity. The affinity is obtained by calculating the space effect, repulsion, hydrogen bond, hydrophobic interaction and molecular flexibility of the complex, which is an important indicator to measure whether the ligand can effectively bind to the receptor molecule. The lower the value, the better the binding effect.

Analysis of proteins in osteosarcoma cells treated with daidzein

Cold PBS was used to wash the adherent osteosarcoma cells cultured in 6-well plates three times. RIPA buffer containing a proteinase inhibitor (Beyotime, Shanghai, China) was used to extract total protein from osteosarcoma cells. Osteosarcoma cells were scraped with a cell scraper, dissolved in buffer, and transferred to a 1.5 mL EP tube with a pipette. The above samples were treated with an ultrasonic cell breaker, with ultrasonic treatment for 3s and an interval time of 10s, which was repeated three times. Then, the lysates were centrifuged at 4 °C and 13000 g for 15 min. In this way, the protein composition was separated and diluted for denaturation. Protein samples were subjected to SDS-PAGE and then transferred to PVDF membranes. The blocking solution was prepared by dissolving 5% skim milk powder in TBST, and the membrane was put into the solution and blocked for 1 h. The membranes were then incubated with the following rabbit anti-human polyclonal primary antibodies overnight: anti-Src (ab109381), anti-p-Src (ab40660, phospho Y418), anti-JNK (ab179461), anti-p-JNK (ab76572, phospho Y185+Y185+Y223), anti-ERK (ab184699), anti-p-ERK (ab201015, phospho T202+T185), anti-p38 (ab178867, phospho T180), anti-p-p38 (ab195049, phospho T180+Y182) and anti-GAPDH (ab9485) (Abcam, Shanghai, China). GAPDH expression was used as a loading control. After washing three times, the membranes were incubated with the corresponding goat anti-rabbit secondary antibodies (Abcam, Shanghai, China). A Western blot visualization (ECL kit, Pierce, USA) and analysis system (Bio-Rad) was used to detect chemiluminescence.

Construction of Src-overexpressing osteosarcoma cell lines by lentivirus transfection

For Src overexpression, 143B and U2OS cells were transfected with 4 µg of pMSCV-Flag or pMSCV-Flag-Src using Lipofectamine 3000 Transfection Reagent (Thermo Scientific, USA) according to the manufacturer’s instructions. Empty virus vectors were used as negative controls. The primers were 5′-tgttcggaggcttcaactcc-3′(F) and 5′-gacatccaccttcctcgtgt-3′(R). After 72 h of transfection, the expression of the green fluorescent protein EGFP in cells was observed under a fluorescence microscope, and stably transfected cell lines were established by the puromycin (1.5 ng/µL) screening method. The rescue experiment was performed based on stably transfected cells.

Statistical analysis

The results are expressed as the mean ± standard deviation (SD) from at least three independent experiments. Student’s t-test was used to compared the difference between two groups while, one-way analysis of variance (ANOVA) was used to analyze statistical differences between more than two groups. GraphPad Prism software (version 7.04) was used for statistical analysis. A statistically significant difference was defined as p < 0.05.

Daidzein inhibits the proliferation of 143B and U2OS osteosarcoma cells

Firstly, we explored the effect of daidzein on proliferation of 143B and U2OS osteosarcoma cells. 143B and U2OS cells were treated with gradient concentrations of daidzein (0, 10, 20, 50, 100, 200 or 500 µmol/L). After 48 or 72 h treatment, MTT assay was performed to detect the cell proliferation capacity. As shown in Figs. 2A–2B, daidzein significantly inhibited the proliferation capacity of osteosarcoma cells in a dose- and time-dependent manner. According to the absorbance curve, the 48 h 50% inhibiting concentration (IC₅₀) of daidzein on 143B and U2OS cells are 63.59 µmol/L and 125 µmol/L. Meanwhile, we treated normal human hFOB 1.19 osteoblasts with the same daidzein dose in 48 and 72 h (Fig. 2C). According to the analysis results of IC₅₀ (Fig. 2D), daidzein had a much weaker inhibitory effect on the proliferation of hFOB cells compared with osteosarcoma cells (Student’s t-test, p < 0.05). Thus, daidzein should be considered relatively safe when applied clinically. Then, we set three concentration treatment groups (143B: 20 µmol/L for the low concentration group, 48 h IC₅₀ group, and 200 µmol/L for the high concentration group; U2OS: 50 µmol/L for the low concentration group, 48 h IC₅₀ group, and 200 µmol/L for the high concentration group) for the following experiments. And colony formation assay results also showed that daidzein significantly inhibited the colony formation ability of 143B and U2OS osteosarcoma cells (Figs. 2E–2F). Thus, daidzein may inhibits the proliferation of 143B and U2OS osteosarcoma cells in a dose- and time-dependent manner.

Daidzein induces apoptosis and cell cycle arrest in 143B and U2OS osteosarcoma cells

Hoechst 33342 staining and Annexin V-FITC/PI dual staining detection kits were used to determine the effect of daidzein on apoptosis 143B and U2OS cells. The results indicated that 24 h of treatment with daidzein (at low, IC₅₀, and high concentrations) considerably increased apoptosis in a dose-dependent manner compared to that of the control group. The results of the flow cytometric analysis showed that the early and late apoptosis rate of daidzein treatment group (at low, IC₅₀, and high concentrations) were significantly lower than that of the control group (Figs. 3A–3B). Next, we determined whether daidzein was involved in the cell cycle of osteosarcoma cells. According to the cell cycle distribution analyzed by flow cytometry, we found that daidzein increased the percentage of S phase cells but decreased the percentage of G₀/G₁ phase cells in 143B and U2OS cells (Figs. 3C–3D). These results suggest that daidzein may induce apoptosis and cell cycle arrest in 143B and U2OS osteosarcoma cells.

Daidzein suppresses migration ability of and 143B and U2OS osteosarcoma cells

We also explored the influence of daidzein on migration ability of osteosarcoma. 143B and U2OS cells were pretreated with daidzein for 24 h before the Transwell assays. Daidzein significantly suppressed cell migration in 143B and U2OS cells, as shown in Fig. 3E. Moreover, scratch wound assay revealed that the cell mobility rates in daidzein treatment group were significantly decreased when compared with the control group (Fig. 3G). Therefore, daidzein can suppress migration ability of and 143B and U2OS osteosarcoma cells.

Daidzein effectively suppresses osteosarcoma growth in xenograft mice model

To investigate the effect of daidzein on osteosarcoma in vivo, an osteosarcoma xenograft mice model was established by subcutaneous implantation of 143B cells in BALB/c nude mice. As shown in Fig. 4, the volume and weight of the tumors and tumor growth rate in the daidzein-treated group significantly decreased when compared with those in the control group. H&E staining of the tumor revealed that the number of necrotic cells in the daidzein-treated group was significantly higher than that in the control group (Fig. 4E). Furthermore, the body weight of the nude mice was not significantly affected (Fig. 4B), indicating that there was little systemic toxicity of daidzein in vivo.

Target prediction of daidzein on osteosarcoma through systemic pharmacology analysis

To reveal the mechanism of daidzein regulating biological process of osteosarcoma cells, systemic pharmacology analysis was performed on PharmMapper platform. A total of 243 human genes associated with daidzein were predicted by the PharmMapper platform (Table S1). As shown in Fig. 5A, a “daidzein-daidzein targets” network was constructed. Osteosarcoma-related genes were collected and combined from DigSeE, DisGeNET, GeneCards, KEGG disease, MalaCards, and Phenopedia (Table S2). After intersecting the two gene sets (Fig. 5B), there were 133 common targets of osteosarcoma and daidzein through which daidzein might play a role in regulating the progression of osteosarcoma. After that, 133 target genes associated with daidzein and osteosarcoma were imported into the STRING database for PPI network construction. As shown in Fig. 5C and Table S3, there were 133 nodes and 295 interaction edges (interaction score>0.9). To analyze the PPI diagram, the analysis tool in Cytoscape was applied, and the top 30 hub genes were identified and are shown in Fig. 5D. Among the top 30 hub genes, Src was the node that interacted with the most numerous edges, suggesting that Src may play a vital role of daidzein in regulating biological process of osteosarcoma.

Src protein may be the target of daidzein

In order to explore whether daidzein can effectively combine with Src and play a regulatory role, we assessed the potential interaction between daidzein and Src through molecular modeling and docking simulation. The 3D structure of daidzein was obtained from the PubChem database (Fig. 5E). After adjustment and analysis, the grid size was set to 40 × 40 × 40, and we obtained an affinity of −9.1 kcal/mol. As shown in Fig. 5F, hydrophobic interactions were formed between daidzein and the following amino acid residues: Ile336, Thr338, Met314, Val323, Phe405, Ala403, Leu393, Asp404, Val281 and Ala293. In brief, these results suggest a strong potential of direct binding between daidzein and Src. Thereafter, we employed GO annotation for the functions of the key target genes. The top 20 highly enriched functional terms are shown in Fig. 6A. The key target genes could regulate multiple biological processes, including steroid hormone receptor activity, nuclear receptor activity, ligand-activated transcription factor activity, etc. The KEGG results indicated that these key target genes were involved in multiple signaling pathways, and the top 20 pathways are shown in Fig. 6B. Among them, the MAPK signaling pathway was the highest-ranked pathway, with the highest gene ratio as well as the third minimum adjusted P-value (less than 1e⁻⁰⁷). According to previous studies, the MAPK (JNK/ERK/p38) signaling pathway is a downstream molecule of Src kinase (Cui et al., 2017; Gao et al., 2019). Therefore, we further predicted that the Src and MAPK pathways may be involved in the regulation of daidzein on biological process of osteosarcoma.

Daidzein inhibits phosphorylation of the Src and ERK signaling pathways osteosarcoma cells

According to the results of previous systemic pharmacology analysis and molecular docking studies, we detected the protein expression and phosphorylation levels of Src, JNK, ERK and p38 to elucidate the molecular mechanisms by which daidzein affects osteosarcoma cells. As shown in Figs. 6C–6D, daidzein treatment decreased the expression of p-Src and p-ERK, but the expression levels of Src, JNK, p-JNK, ERK, p38 and p-p38 were not significantly changed. Therefore, we determined that Src and ERK phosphorylation was suppressed in daidzein-treated cells, whereas JNK and p38 phosphorylation was not altered.

Overexpression of Src rescues inhibitory effect of daidzein on osteosarcoma cells

As shown in Figs. 7B–7C, overexpression of Src partially restituted the phosphorylation of Src and ERK in osteosarcoma cells treated with daidzein. Moreover, according to MTT assay (Fig. 7D) and apoptosis analysis by flow cytometry (Figs. 7E–7F), decreased viability and in osteosarcoma cells treated with daidzein was partially reversed by overexpression of Src. Therefore, it suggests that daidzein may exert anti-osteosarcoma efficiency through the inhibition of the Src kinase-mediated ERK pathway.