Fig 1.
Potential targets and PPI networks of PA against osteosarcoma.
(A) Venn diagram of PA and potential targets of osteosarcoma. (B) PA- Common target-osteosarcoma interaction network diagram. Red represents osteosarcoma. Purple represents PA and blue line represents 63 common targets. (C) PPI network of common targets of PA and osteosarcoma. (D) PPI network topology analysis showed the top 30 ranked core targets.
Fig 2.
GO and KEGG analysis results of PA against osteosarcoma.
(A) GO enrichment analysis of common targets of PA against osteosarcoma. (B) KEGG enrichment analysis of common targets. Bubble plots show the top 20 enriched pathways. The intensity of red color increases with the level of significance of P value.
Fig 3.
Molecular Docking Analysis of PA with its core target.
(A) Chemical structure and molecular formula of PA. (B) PA binds three amino acid residues (CYS-773, ARG-817, and ASP-831) in the EGFR protein via hydrogen bonds. (C) PA binds to CASP3 protein. (D) PA binds to one amino acid residues (MET-343) in the ESR1 protein via hydrogen bonds. (E) Asiatic acid binds one amino acid residues (GLY-135) in the HSP90AA1 protein via hydrogen bonds. (F) PA binds three amino acid residues (LEU-275, ASP350 and GLY-346) in the SRC protein via hydrogen bonds. Yellow dashed lines represent hydrogen bonds.
Fig 4.
PA inhibits the proliferation of osteosarcoma cells.
(A) Following treatment of osteosarcoma 143B and HOS cells with PA (00, 25, 50, and 100 μM) for 24−48 h, cell viability was determined by using CCK-8 assay. (B) PA inhibited colony formation ability of 143B and HOS cells. (C) Cycle arrest of 143B and HOS cells detected by flow cytometry. *p < 0.05 vs. control group.
Fig 5.
PA-induced apoptosis in osteosarcoma cells.
(A)The ratio of apoptosis in osteosarcoma cells was detected by Annexin V-FITC double staining combined with flow cytometry analysis. (B) Intracellular MPP turnover was detected by staining with probe JC-1 combined with flow cytometry analysis. (C) The expression levels of apoptosis-related proteins Bcl-2 and Bax in osteosarcoma cells (HOS and 143B) were measured by Western blot analysis. *p < 0.05 vs. control group.
Fig 6.
PA triggers autophagy in osteosarcoma cells.
(A) PA-triggered autophagy-related protein LC3I/II and p62 protein expression was detected by Western blot analysis. (B) PA promoted an increased quantities of autophagosomes within 143B and HOS cells as observed by transmission electron microscopy. Red triangles point to autophagosome position. Low magnification, 8000X; High magnification, 30,000X. *p < 0.05 vs. control group.
Fig 7.
PA inhibits PI3K/AKT pathways in osteosarcoma cells.
Following treatment of 143B and HOS cells with PA (00, 25, 50, and 100 μM) for 24h, protein levels of p-PI3K, PI3K, p-Akt, and Akt as measured by western blot analysis. *p < 0.05 vs. control group.
Fig 8.
Potential mechanism of anti-osteosarcoma activity of PA.