Psoralidin inhibits osteosarcoma growth and metastasis by downregulating ITGB1 expression via the FAK and PI3K/Akt signaling pathways

Cell culture and drug preparations

Human OS cell lines 143B and MG63 were purchased from the American Type Culture Center (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin were purchased from Gibco (Carlsbad, CA, USA). Psoralidin (20 mg, 99% purity), Cell Counting Kit-8 (CCK8), and dimethyl sulfoxide (DMSO) were purchased from China MCE. Crystal violet staining solution was purchased from Beijing Solarbio Science & Technology, and 5-Ethynyl-2′-deoxyuridine (EdU) 488 and BCA protein concentration kits from Beyotime Biotechnology. Transwell assay kits were purchased from Guangzhou Jet Bio-Filtration, and Matrigel from BD Biosciences, USA. To create a PSO stock solution, 20 mg of PSO was dissolved in 595 µL of DMSO to make a final concentration of 100 mM. The stock solution was stored at –20 °C and diluted in fresh medium before use.

CCK8 assay

MG63 and 143B cells were collected at the logarithmic growth phase, digested, and counted. Next, they were mixed with 10% FBS, seeded in 96-well culture plates at a density of 5 × 103 cells/well, and incubated for 24 h. After cell adhesion, different concentrations of PSO were added, and the plates were incubated for 24 h or 48 h. The cells were then incubated in 10% CCK8 liquid diluted in normal medium at 37 °C for 2 h. The spectroscopic absorbance at 450 nm in each well was measured using an automated plate reader. To investigate the anti-proliferative effect of PSO on OS cells, 143B, and MG63 cells were treated with 0–50 µM PSO, and cell viability was measured at 24 h and 48 h. Control cells were treated with DMSO. After 24 h of treatment, the half-maximal inhibitory concentration (IC50) of PSO in the OS cells was calculated (IC50, MG63 = 31.44 µM, 143B = 29.52 µM). Based on these data, we selected 10, 20, 30, and 40 µM for 143B cells and 20, 30, 40, and 50 µM for MG63 cells as working PSO concentrations for subsequent experiments.

Crystal violet assay

MG63 and 143B cells were seeded in 96-well plates at a density of 5 × 103 cells/well, incubated overnight, and treated with different concentrations of PSO for 24 h or 48 h. Subsequently, the cells were stained with crystal violet. The crystal violet in the plates was completely dissolved with a 20% acetic acid solution. The OD at 590 nm, which approximates the wavelength of maximum absorbance for the enzyme marker, was measured in each well to quantify cell proliferation activity.

EdU cell staining assay

MG63 and 143B cells were cultured in 6-well plates (2 × 105 cells/well) at 37 °C in the presence of 5% CO2 for 24 h. Then, the EdU solution was added to the wells and the plates were incubated at 37 °C for another 4 h. The cells were fixed with 4% paraformaldehyde for 20 min and then permeated with 0.3% Triton-100 for 15 min. They were then incubated with 500 µL of click reaction solution for 30 min. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and the EdU/DAPI-positive rate was calculated. Images were acquired and observed using a fluorescence microscope (Nikon Clipstie, Japan).

Wound healing assay

MG63 and 143B cells were seeded into 6-well plates and grown to confluence. The monolayer in each well was scratched with a 200 μL pipette tip and then washed three times with PBS to remove cell debris and suspended cells. Next, fresh medium with different concentrations of PSO was added to each well. The scratched area was photographed at various time points (6, 12, and 24 h), and the recovery area was calculated using the ImageJ software.

Cell migration and invasion assays

To measure cell invasion capacity, Matrigel was thawed at 4 °C and diluted in serum-free DMEM at a ratio of 1:8. The mixed solution was added to Transwell chambers containing 80 μL of medium/well and the plates were incubated at 37 °C in the presence of 5% CO2 for 4 h. Subsequently, 2.5 × 104 cells of each OS cell line were respectively added to the upper Transwell chambers, and different concentrations of PSO were added to the lower chambers. After incubation for 24 h, the cells that had penetrated the Matrigel coating and migrated to the lower chamber were stained with crystal violet (0.1%) and photographed. To measure cell migration, the experiment was conducted under the same conditions, except without Matrigel in the upper chamber.

Flow-cytometric analysis

Flow cytometry was used to determine the effects of PSO on OS apoptosis and the cell cycle. After treatment with PSO, cells were harvested and washed three times with PBS. To determine the apoptosis rate, cells were resuspended in 500 μL of PBS, stained using an Annexin V-FITC/PI double labeling staining kit according to the manufacturer’s instructions, and analyzed by flow cytometry. For cell-cycle assessment, cells were fixed with 70% ethanol at 4 °C overnight and analyzed using a flow cytometer (CytoFLEX, Beckman Coulter, Fullerton, CA, USA).

Western blotting

OS cells were treated with different concentrations of PSO for 24 h and then lysed with RIPA buffer. The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (0.45 μm). After blocking with 5% skim milk for 1 h and washing three times with Tris-buffered saline (TBS), the membranes were probed with primary antibodies at 4 °C overnight. After washing three times with TBS-Tween, the membranes were incubated with a secondary antibody (goat anti-rabbit/mouse IgG, 1:5000) at room temperature for 2 h. Protein bands were imaged and analyzed using a ChemiDoc MP Imaging System and Image Lab Software (Bio-Rad, Hercules, CA, USA). The following antibodies were used: anti-PCNA (ab92552, Abcam), anti-matrix metalloproteinase (MMP) 2 (ab92536, Abcam), anti-MMP9 (ab76003, Abcam), snail rabbit mAb (#3879, CST), bcl-2 rabbit mAb (#4223, CST), bax rabbit mAb (#41,162, CST), cleaved caspase-3 rabbit mAb (#9654, CST), β-actin rabbit mAb (#4970, CST), anti-FAK (ab40794, Abcam), anti-p-FAK (phospho Y397) (ab81298, Abcam), anti-PI3 kinase p85 alpha (ab191606, Abcam), anti-p-PI3 kinase p85 alpha (phospho Y607) (ab182651, Abcam), anti-Akt (ab179463, Abcam), and anti-p-Akt (phospho S472 + S473 + S474) (ab192623, Abcam).

RNA-sequencing

143B and MG63 cells were treated with 30 μM PSO for 24 h. Total RNA was extracted using an RNA Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA). The RNA samples were sent to Beijing Qingke Biotechnology (Beijing, China) for RNA-sequencing, which was performed on a DNBSEQ-T7 sequencer to determine changes in mRNA expression.

Kaplan–meier analysis

The GSE21257 OS microarray dataset was downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). This dataset included 53 OS biopsy samples collected before chemotherapy, with metastatic status and time information (34 metastatic and 19 non-metastatic samples), and transition status and time information. Kaplan–Meier curves were constructed in the R package survminer (version 0.4.6) to analyze survival.

Molecular docking

The core protein structures of the protein–protein interaction network were downloaded from the PDB database (http://www.rcsb.org/) and ligands and water molecules were removed with PyMOL. The core ingredients were downloaded as an SDF file from the PubChem database (https://pubchem.ncbi.nim.nih.gov) and converted into mol2 format using the OpenBabel software. Finally, the docking of the obtained protein structures (receptors) and active components (ligands) with PSO was predicted using AutoDock software, and the docking results were visualized using PyMOL software.

Real-time PCR

Total RNA was isolated using TRIzol Reagent according to the manufacturer’s instructions, cDNA was synthesized, and qPCR was performed. The primer sequences are listed in Additional file 1: Table S1.

Small interfering RNA transfection

Small interfering RNA (siRNA) transfection was performed according to the manufacturer's instructions described previously. The siRNA sequences used to knock down ITGB1 expression are mentioned in Additional file 1: Table S2.

Orthotopic OS model establishment

BALB/c nude female mice (3–4 weeks old, 15–20 g) were purchased from Hunan SJA Laboratory Animal. After adaption for 1 week, 50 µL of 143B suspension (2 × 107cells/mL) was injected into the proximal tibia of the mice. The animals were subsequently administered different doses of PSO (5, 10, or 20 mg/kg) or DMSO via oral gavage at two-day intervals. After three weeks of treatment, the animals were euthanized. Tumor volumes were calculated using the following formula: 0.5 × L × W2, where L and W represent the length and width of the tumor, respectively. For subcutaneous tumor measurement, the length (L), the longer dimension, and width (W), which is the shorter dimension perpendicular to the plane of the length and parallel to the plane of the animal's body, were measured using a caliper.All animal experiments were approved by the Animal Care and Use Organization Committee IACUC of Chongqing Medical University.

Hematoxylin and eosin staining and immunohistochemistry

Tumor tissues were collected, fixed in 4% paraformaldehyde for 24 h, and washed with PBS. The fixed tissue samples were embedded in paraffin, dewaxed, and stained with hematoxylin and eosin (H&E). Immunohistochemical staining of tumor sections was performed using antibodies against proliferating cell nuclear antigen (PCNA; 1:100), bax (1:100), and MMP2 (1:100). All sections were imaged using a light microscope at 40 × and 100 × magnifications.

Statistical analysis

The SPSS 22.0 software package (IBM Corp., Armonk, NY, USA) was used for statistical analysis. The in vitro experiments were repeated three times, and the in vivo experiment was repeated five times. To test for differences among multiple groups, one-way analyses of variance were performed, and Tukey’s test was used for between-group comparisons. The experimental data are expressed as mean ± SD, and P < 0.05 was considered statistically significant.

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