| Osteosarcoma, a common primary bone sarcoma has five-year survival rate of-70% in children and adolescents. Patients with osteosarcoma have a poor prognosis, with overall survival rates of <20%. Osteosarcoma is a well-defined clinical entity with a characteristic radiographic appearance, histologic features, a relatively consistent spectrum of clinical presentations, and established standard treatments. These features have been the subject of many prior book chapters and reviews. However, all the present treatments are less efficient. Therefore, the discovery of molecules with roles in the osteosarcoma inhibition is highly desired to improve the clinical treatment.Polycomb group genes (PcG) which are involved epigenetically in regulating gene transcription programs during development and cellular differentiation harbour a catalytic subunit, Enhancer of Zeste homolog 2 (Ezh2). Knowledge regarding tumor stem cells has demonstrated that Ezh2 similarly controls expansion and differentiation of tumor initiating cells and plays an important role in the development and progression of cancer. Myelodysplastic syndromes involving inhibition mutations in Ezh2 gene proves that Ezh2 functions as a tumor suppressor. Ezh2 inhibits cell differentiation to maintain sternness of tumor cells.Triterpenes and their derivatives have been reported to possess activities such as anti-HIV, inhibition of HIV protease and cytotoxicity to tumor cell lines. Ursolic acid, a pentacyclic triterpene isolated in abundance from the peels of Malus pumila Mill has been reported to possess a wide range of pharmacological properties, including anti-inflammatory, antiallergic, antibacterial, antiviral, antitumor and cytotoxic activities being the most intriguing. Owing to its biological potential, it was ranked as one of the most promising tumorpreventive medications by Japanese researchers. Mechanistic studies revealed that ursolic acid blocks the cell cycle progression in the G1 phase and its treatment results in the triggering of apoptosis as determined by DNA fragmentation assay. Despite its in vitro biological promise, its poor bioavailability under in vivo restricts its clinical applications.One of the techniques to improve the bioavailability of this molecule is the development of polymeric micelles, like glycol chitosan micelle. So we investigated the effect of ursolic acid (UA)-incorporated glycol chitosan (GC) nanoparticles on inhibition of human osteosarcoma.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 a blank. Bicinchoninic acid assays (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. Among the range of UA concentrations tested, the minimum effective concentration was 10 ?M with half inhibitory concentration IC 50 of 25 ?M. In U2OS cells, treatment with 10 and 25 ?M UA-induced 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 ?M 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 ?M to 25 ?M 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. UA-incorporated GC nanoparticles formed by electrostatic interaction between-COOH group of UA and-NH2 group of glycol chitosan were prepared. The presence of reactive-NH2 group makes chitosan a suitable substrate for drug conjugation and ion complex formation with anionic drugs. Thunemann and Beyermann initially developed the concept of nanoparticle formation between acid and positively charged macromolecules. The present study demonstrates the effect of UA-incorporated GC nanoparticles on inhibition of osteosarcoma cell proliferation and cell cycle arrest. The results showed a marked inhibition in cell proliferation on treatment with UA-incorporated GC nanoparticles after 24 h. The proliferation inhibition continued for 72 h and the effect was seen to be maximum on day It has been shown that polycomb group genes are epigenetically associated with the regulation of gene transcription programs during cellular differentiation and development. Ezh2 controls expansion and differentiation of tumor initiating cells and plays an important role in the development and progression of cancer. The results from the present study revealed that UA transfection in U2OS and Saos-2 human osteosarcoma cells caused inhibition of Ezh2 expression. The results from apoptosis and necrosis assay showed increase in the percentage of apoptosis on increasing the concentration of UA. It was observed that osteosarcoma cells undergo apoptosis through mitochondrial pathway. The Bax and Bcl-2 proteins were seen to translocate from mitochondria into cytoplasm where they led to release of cytochrome c. Cytochrome c then activates caspase 9 and caspase 3, which play key roles in the apoptosis pathway. The increase in concentration of UA in UA-incorporated GC nanoparticle from 10 ?M to 25 ?M significantly reduced the mitochondrial membrane potential in U2OS cells. Therefore, these results suggest that the UA inhibition of Ezh2 expression induces apoptosis through the mitochondrial pathway in human osteosarcoma cells. Our results from flow cytometry also suggest that UA induces cell cycle arrest in G0/G1 phase. UA-incorporated GC nanoparticles resulted in the inhibition of human osteosarcoma cell proliferation, induces cell apoptosis and induces cell cycle arrest in G0/G1 phase.These effects are achieved by inhibiting the expression of Ezh2 in osteosarcoma cells.Therefore, UA combined with GC nanoparticles may provide a new strategy for the treatment of osteosarcoma. |
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