Study On The Effect And Mechanism Of Celastrol In Proliferation And Apopptosis Of Human Osteosarcoma Cells

Objective:To investigate the effect and mechanism of celastrol in proliferation and apopptosis of human osteosarcoma cells.Methods:The MTT colorimetric assay was performed to evaluate the effect of celastrol with different concentrations at different time on the viability of human osteosarcoma cells;Hoechst 33258 staining was used to observe the morphological change in human osteosarcoma cells following treatment with celastrol at different concentrations in fluorescence microscopy;The effect of celastrol on the apoptotic rate was determined by flow cytometry analysis of human osteosarcoma cells using Annexin V-PE/7-AAD staining assay at different concentrations;The expression levels of apoptosis related proteins in human osteosarcoma cells,such as Bcl-2 family proteins,caspase-3,caspase-8,procaspase-9,cytochrome c and PARP were measured by western blot assay.Results:The results of MTT assay revealed that celastrol inhibited proliferation of human osteosarcoma cells(MG-63,U-2OS and HOS)in a time-and dose-dependent manner.Through analyzing the growth curve,we found that human osteosarcoma U-2OS cells were the most sensitive to celastrol.Celastrol-treated human osteosarcoma U-2OS cells stained by Hoechst 33258 showed condensed and fragmented nuclei,which were typical morphological characteristics of apoptotic cells.The results of flow cytometry analysis revealed that there was a dose-dependent increase in the apoptosis rate of human osteosarcoma U-2OS cells following treatment with celastrol.The results of the western blot analysis showed that celastrol treatment caused a profoundly marked change in the mitochondrial apoptosis related proteins in a dose-dependent manner: the ratio of Bax to Bcl-2 increased;expression levels of procaspase-9 were downregulated;the release of cytochrome c was also observed.However,there was no obvious change of expression levels of death receptor apoptosis related proteins,such as caspase-8.Caspase-3,a vital protein in the execution of apoptosis,was obviously cleaved and activated.Besides,death substrate(PARP)was cleaved and degradated.Conclusion:1.Celastrol induces apoptosis and inhibits proliferation of human osteosarcoma cells;2.Celastrol induces apoptosis of human osteosarcoma cells via the mitochondrial apoptotic pathway.