Effect Of Celecoxib On Inducing Apoptosis Of Human Osteosarcoma Cell MG-63 Line Through PKB/AKT Signaling Transduction Pathway

[Objective] this study was to investigate effect of selective cyclooxygenase-2 inhibitor Celecoxib on inducing apoptosis of human osteosarcoma cells MG-63 line through PKB/AKT/ signaling transduction pathway and to explore the mechanism of Celecoxib inducing cells apoptosis and anoikis in vitro and inhibiting angiogeneisis in vivo.[Methods] Manners of concentration and time-dependent were detected by MTT assay and flow cytometry (FCM). The characteristic changes of apoptosis in morphology were observed by Hoechst 33258 staining and fluorescence microscopy and TDT-mediated dUTP-biotin nick end-labelling (TUNEL) assay, the ultrastructure observed by transmission electron microscopy (TEM). Anoikis resistance of MG-63 cells was determined by DNA ladder assay, flow cytometry analysis, and soft agar colony formation growth assay upon suspending culture. The effect of Celecoxib on cells cycle of anoikis resistance MG-63 cell line was analyzed by AnnexinV-FITC/PI labeling. In order to probe the approach of Celecoxib inducing tumor cells apoptosis and anoikis, expression of mRNA and protein of some molecule effectors relative to signaling transduction pathway PKB/AKT/Bad and Mtor/4EBP1 was analyzed by Reverse transcription polymerase reaction(RT-PCR) and Western Blot. Then nude mice models by subcutaneously implanting MG-63 cells were successfully copied andtreatment with Celecoxib, and then the micro-vascular density, COX-2 and VEGF mRNA protein expression were deteced by immunohistochemistry technology, RT-PCR and Western Blot.[Results] After treatment with different concentration Celecoxib, the apoptosis rates were obviously different, respectively (P<0.05, P<0.01) . Celecoxib could induce MG-63 apoptosis upon concentration and time-dependent manners. Through TEM and TUNEL assay, the special morphological changes were observed, such as nuclear chromatin condensation, fragmentation, cell shrinkage, and the formation of apoptotic bodies. Representative DNA ladders appeared in VEC cells but not in MG-63 and MCF-7 cells. Then the property of anoikis resistance of MG-63 cell line was revealed by some features including low apoptosis rate, cell cycle arrested in G\. We analyzed effect of Celecoxib on signaling transduction pathway PKB/AKT/Bad and mTOR/4EBP1, and found that Celecoxib could regulate molecule effectors relative to apoptosis events by inhibiting AKT, Bad phosphorylation and mTOR protein expression, increasing mRNA and protein expression of caspase9, cytochrome C and 4EBP1. Then in vivo, Celecoxib could decrease MVD in nude mice model of osteosarcoma, and inhibit mRNA and protein expression of COX-2 and VEGF.[Conclusion] MG-63 cell line has anoikis resistance property. But Celecoxib could induce human osteosarcoma cell MG-63 line apoptosis and anoikis upon concentration and time-dependent manners. Celecoxib could regulate PKB/AKT/Bad and mTOR/4EBP1 signaling transduction pathway by regulating some molecule effectors relative to apoptosis events such as AKT, Bad phosphorylation and caspase9, cytochrome C, mTOR and 4EBP1 et al. In vivo, through modulating expression of COX-2 and VEGF mRNA and protein, Celecoxib could reduce MVD and inhibit angiogenesis in osteosarcoma model of subcutaneously implanting MG-63 cells into nude mice.