Abstract
Purpose: To investigate the effect of ursolic acid (UA)-incorporated glycol chitosan (GC) nanoparticles on inhibition of human osteosarcoma.Methods: U2OS and Saos-2 osteosarcoma cells were transfected with ursolic acid (UA) incorporated glycol chitosan (GC) nanoparticles. Ultraviolet (UV) spectrophotometry was used to measure drug contents in nanoparticles at 365 nm with empty GC vehicles as blank. Bicinchoninic acid assay (BCA) method was employed to determine protein concentration. Identification of apoptosis and necrosis in osteosarcoma cells was performed by propidium iodide and FITC-annexin V reagents, respectively. FAC Scan flow cytometry was used to analyse apoptotic cells.Results: Among the range of UA concentrations tested, the minimum effective concentration was 10 μM with half inhibitory concentration IC50 of 25 μM. In U2OS cells, treatment with 10 and 25 μM UAinduced apoptosis in 5.89 ± 3.90 and 60.54 ± 5.40 % cells, respectively, compared to 2.05 ± 1.01 % cells for control. In Saos-2 cells, exposure to 10 and 25 μM UA induced apoptosis in 9.86 ± 8.89 and 47.54 ± 14.5 % cells, respectively, compared to 1.79 ± 0.23 % for control cells. Western blot analysis revealed translocation of Bax and Bcl-2 proteins from mitochondria to cell cytosol. Increase in UA concentration from 10 μM to 25 μM led to increase in the proportion of cells in G0/G1 phase and decrease in the number of cells in S and G2/M phases. These results confirm that UA transfection arrests cell cycle in G0/G1 phase in human osteosarcoma cell lines.Conclusion: UA transfection resulted in the inhibition of cell proliferation, Ezh2 expression inhibition, and apoptosis via mitochondrial pathway due to decrease in membrane potential and release of cytochrome C, as well as cell cycle arrest in G0/G1 phase.Keywords: Osteosarcoma, Cell cycle arrest, Palliation, Glycol chitosan, Ursolic acid
Highlights
Osteosarcoma, a common primary bone sarcoma has a 5-year survival rate of ~70 % in children and adolescents [1]
The results showed a significant decrease in Enhancer of Zeste homolog 2 (Ezh2) expression level after 24 h of UAincorporated glycol chitosan (GC) nanoparticles (25 μM) transfection compared to control
The results clearly showed that increasing in concentration of ursolic acid (UA) in UA-incorporated GC nanoparticle from 10 to 25 μM significantly reduced the mitochondrial membrane potential in U2OS cells (Fig 5A)
Summary
Osteosarcoma, a common primary bone sarcoma has a 5-year survival rate of ~70 % in children and adolescents [1]. Mechanistic studies revealed that ursolic acid blocks the cell cycle progression in G1 phase and its treatment results in the triggering of apoptosis as determined by DNA fragmentation assay. In the present study effect of UA-incorporated glycol chitosan nanoparticles on osteosarcoma were investigated. Treatment of cells with various concentrations of UA-incorporated GC nanoparticles for 24 h was followed by washing with PBS.
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