Apoptosis In Osteosarcoma Cells Induced By TRAIL And TRAIL Combined With Chemotherapeutic Agents

Objective Osteosarcoma is one of the most common primary malignant tumors of bone.TRAIL(TNF-related apoptosis inducing ligand) is a new member of TNF superfamily.One aim of this study is to explore the potential mechanism of apoptosis induced by TRAIL through investigating its effect on osteosarcoma cell line HOS and the expression o f c -myc d uring t he c ourse. A nother aim i s t o i nvestigate w hether t here i s a synergetic effect when TRAIL combined with doxorubicin(ADM) treated.Methods HOS cells were cultured ,then divided into the following groups xontrol group,TRAIL groups, TRAIL+caspase3 inhibitor group,.ADM groups,ADM+TRAIL groups. The morphologic changes and characters of apoptosis was observed by cytochemical staining and transmission electron microscopy (TEM)..The inhibitive rate was analysed by MTT assay and the apoptosis rate was ascertained by FCM.The expression of TRAIL receptors and c-myc was examined by RT-PCR. Synergetic effect was determinded by Webb coefficient. Statistic analysis was processed by SPSS10.0 software. P values less than 0.05 was considered to be significant.Results The result of MTT reductive experiment and FCM showed TRAIL inhibited Hos cells in the dose dependent manner, and this effect could be blocked by caspase-3 inhibitor Z-DEVD-FMK. 0.5μg/ml TRAIL and 1μg/ml ADM show synergistic cytotoxin effect in combined treatment.Under phase contrast microscope, some cells became to be small and round with intact cellular membrane 3 hours after treated with TRAIL (dose>0.5 μg/ml). A large member of cells exfoliated and drifted into culture medium when treated with TRAIL combing ADM. TEM showed shrinking cellular morphology, chromatin condensation and crescent nucleus of HOS cell.Semi-quantitative RT-PCR detected the expression of TRAIL R1~R3 mRNA in HOS cells. The expression of TRAIL R2 mRNA significantly increased in ADM+TRAIL groups,while the expression of c-myc decreased during apoptosis in HOS cells induced byTRAIL.Conclusion Proliferation inhibition and apoptosis induction of HOS cells could be detected when treated with TRAIL in vitro, which were correlated with the down-regulation of c-myc gene, c-myc and caspase-3 signals may have feedback inhibitory regulation during this process, c-myc may be one of the important intermedial links inside the mechanisms of apoptosis of HOS induced by TRAIL. TRAIL in combination with subtoxic ADM could synergisticly kill HOS cells. The up-regulation of TRAIL R2 expression may be the mechanism of this syneristicly effect.