GSK3β对骨肉瘤细胞增殖的影响

目的 研究糖原合成激酶3β(GSK3β)在骨肉瘤细胞增殖中的作用及其分子机制。方法 通过Western blot法和NIRKA法,检测正常成骨细胞hFO及骨肉瘤细胞中GSK3β的表达和活性;使用两种GSK3β小分子抑制剂处理骨肉瘤细胞,检测GSK3β活性受抑制下骨肉瘤细胞增殖率和凋亡率;通过GSK3β siRNA降低内源性GSK3β表达,Western blot法检测p27及其下游cyclinD1-CDK-Rb通路因子的蛋白表达及磷酸化;建立骨肉瘤细胞荷瘤小鼠模型,分别给予DMSO作为对照,以及GSK3抑制剂SB216763、AR-A014418,每周3次腹腔给药,观察肿瘤生长和裸鼠的体重变化情况。结果 与正常人成骨细胞相比,骨肉瘤细胞中存在GSK3超表达及活性调节异常;内源性GSK3活性受抑制后抑制骨肉瘤细胞增殖,诱导细胞的凋亡;降低内源性GSK3活性,肿瘤体积与对照组比较,SB216763,AR-A014418组肿瘤增长受到抑制,有统计学意义(P＜0.05);降低骨肉瘤MG-63细胞内源性GSK3蛋白表达,使p27蛋白表达水平明显上调,Rb及其在S780、795部位的磷酸化水平下降,CDK2、4、6蛋白水平降低,cyclinD1的表达上调但其磷酸化水平没有影响。结论 GSK3β通过对p27及cyclinD1-CDK-Rb径路的调节促进骨肉瘤细胞的增殖,可能为骨肉瘤的临床治疗开辟一个潜在的靶点。

Effects of glycogen synthase Kinase-3 on proliferation of human osteosarcoma cells

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.