Abstract: Objective To investigate the molecular mechanisms of effect of glycogen synthase kinase-3beta(GSK3β)on proliferation of human osteosarcoma cells.Methods Normal osteoblast hFO and osteosarcoma cell lines were examined for GSK3β expression and activity by Western blot and in vitro kinase assay(NIRKA).The effects of small molecule GSK3β inhibitors on cell proliferation and apoptosis were examined.Depletion of endogenous GSK3β by GSK3β siRNA detected the expression and phosphorylation of p27 and its downstream cyclinD1-CDK-Rb pathway factor by Western blot.Human osteosarcoma cell xenografts,in athymic mice model,were treated with DMSO as control or with GSK3β inhibitor SB-216763 or AR-A014418 by intraperitoneal injection,3 times a week.The tumor growth and body weight were observed in nude mice.Results Osteosarcoma cell lines showed increased GSK3β expression and decreased serine 9 phosphorylation compared with normal osteoblast cells.Inhibition of GSK3β resulted in attenuated cell proliferation and increased apoptosis in most osteosarcoma cell lines in vitro and in vivo in MG63 xenografts in rodents but not in hFOB cells.We decreased endogenous GSK3b activity,tumor growth was inhibited in SB216763,AR-A014418 group compared with control group.There was statistical significance(P＜0.05).GSK3β inhibition in osteosarcoma cells was associated with decreased p27 expression,Rb expression and phosphorylation level of decline,CDK2,4,6 protein level decreased,the upregulation of cyclin D1 expression but the phosphorylation level of no effect.Conclusion In this research,we demonstrate that deregulated GSK3β sustains osteosarcoma cells survival through modulation of p27and cyclinD1-CDK-Rb pathway.The result will open up a potential target for clinical treatment of osteosarcoma.

Key words: Osteosarcoma, GSK3β, Cell proliferation, p27, Rb signaling pathway