Induction of apoptosis in MG-63 cell line by human soluble TRAIL / 中华骨科杂志

ABSTRACT

Objective TNF-related apoptosis-inducing ligand(TRAIL)was a new member of TNF family.It could quickly inactivate tumor cell originated from different tissues but no effects on normal tissue in vitro.Osteosarcoma was the most common primary malignant bone tumor with the survival rate of 5years no more than20%after simple surgical management.The induced apoptosis and selective cytotoxicity of human soluble TRAIL for MG -63cell line were investigated in order to explore the feasibility of sTRAIL in clinical treatment of osteosarcoma.Methods sTRAIL-mediated cytotoxicity by MTT assay in MG-63osteosarcoma cell line,MRC-5human diploid lung cell line and L-02fetus hepar cell line were assessed,apoptotic cellular morphological transformation by phase contrast microscope and electron micro-scope was observed.The expression of TRAIL receptors mRNA in the cell lines was examined by RT -PCR.The apoptotic rates of MG-63cell line were shown by flow cytometry.Moreover,the apoptosis of MG-63cell line induced by sTRAIL was confirmed by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL).Results MG-63,MRC-5and L-02cell lines were treated for 24hours with dif ferent concentrations of sTRAIL and the inhibitive rates of sTRAIL were evaluated with MTT assay,the in hibitive rates of 500ng /ml,1?g /ml ,2?g /ml and5?g /ml TRAIL inducing MG-63osteosarcoma cell line were10.1%,24.3%,50.6%and more than95%respectively.By flow cytometry,an obvious apoptosis peak ahead the diploid peak was obtained when MG-63cell line incubating with2?g/ml TRAIL for 6hours,howev er,the consequences of MRC-5and L-02cell line were non-differentiated.Therefore,MG-63cell line was significantly sensitive to sTRAIL-mediated apoptosis,but MRC-5and L-02cell line were resistant to sTRAIL-induced cell death.Under phase contrast microscope,when MG-63cell line were treated by sTRAIL,some cells began to became small and round in3to8hours.In24hours a large number of cells exfoliated and drifted in culture medium.Moreover,karyopyknosis and crescent aggregation of chro-matin were observed by electron microscope.By RT-PCR,the expression of TRAIL-R1,R2and R3but not R4mRNA on MG -63and MRC -5cell line were observed,and there were the expression of TRAIL-R1,R2,R3and R4mRNA on L-02cell line.In TUNEL assay,the nucleus of the apoptotic cells became brown or yellow.Conclusion TRAIL is able quickly to kill MG-63osteosarcoma cell line in vit-ro.Fur thermore,TRAIL is non significant cy-totoxic for normal tissue.TRAIL or TRAIL com bined with chemotherapy and radiotherapy is a potential and valuable proposal for clinical treatment of osteosarcoma.