摘要
目的:观察新型哺乳动物雷帕霉素靶蛋白(mammalia target of rapamycin,mTOR)抑制剂含磷西罗莫司衍生物FIM-A对人骨肉瘤MG-63细胞增殖及凋亡的影响。方法:不同浓度(1×10-9～1×10-5mol/L)FIM-A处理MG-63细胞后,采用CCK-8法检测MG-63细胞的增殖,流式细胞术检测MG-63细胞周期和凋亡情况,ELISA法检测血管内皮细胞生长因子(vascu-lar endothelial cell growth factor,VEGF)和低氧诱导因子(hypoxia inducible factor-1α,HIF-1α)的分泌量,RT-PCR和Western blot-ting分别检测FIM-A对MG-63细胞中mTOR、p70核糖体S6激酶(p70S6 kinase protein,p70s6k)及4E结合蛋白1(4E-bindingprotein 1,4E-BP1)mRNA和蛋白表达的影响。结果:与人成骨hF-OB1.19细胞相比,人骨肉瘤MG-63细胞中mTOR、p70s6k及4E-BP1 mRNA的表达水平明显升高(P<0.05)。FIM-A可有效抑制MG-63细胞的增殖(P<0.05),且呈剂量依赖性(r=0.940,P<0.01)。1×10-6mol/L FIM-A处理24 h后与对照组相比,G0/G1期MG-63细胞比例明显增加[(56.4±3.2)%vs(43.4±6.9)%,P<0.05],而MG-63细胞的凋亡率没有明显改变。不同浓度FIM-A作用24 h后,MG-63细胞中HIF-1α和VEGF表达均明显低于对照组(P<0.05),且具有剂量依赖性(HIF-1α,r=-0.988,P<0.01;VEGF,r=-0.998,P<0.01)。同时,FIM-A对MG-63细胞中mTOR(r=-0.919,P<0.01)、p70s6k(r=-0.843,P<0.01)及4EBP1(r=-0.818,P<0.01)蛋白的磷酸化也具有浓度依赖性抑制作用。结论:FIM-A能抑制人骨肉瘤MG-63细胞的增殖,并阻滞细胞周期于G0/G1期,其机制可能与影响mTOR信号通路蛋白磷酸化有关。
Objective： To investigate the effect of phosphorus sirolimus derivatives FIM-A, a new mammalian mammalia target of rapamycin （mTOR） inhibitor, on the proliferation and apoptosis of human MG-63 osteosarcoma cell line. Meth- ods ： Human MG-63 osteosarcoma ceils and hF-OB1.19 osteoblasts were cultured in vitro and incubated with different con- centrations of FIM-A （ 1 x 10 ^-9 - 1 x 10 ^-5 mol/L） for 24 hours. CCK-8 assay was used to evaluate the cell proliferation. The cell cycle and apoptosis were analyzed using flow cytometry. ELISA was used to detect the secretions of vascular endo-thelial cell growth factor （VEGF） and hypoxia inducible factor-lot （HIF-lot）. The expressions of mTOR, p70S6 kinase protein （pTOs6k） and 4E-binding protein 1 （4E-BP1） mRNA and protein were detected by RT-PCR and Western blot- ting, respectively. Results： The expressions of roTOR, p70s6k and 4E-BP1 mRNA in MG-63 osteosarcoma cells were sig- nificantly higher than that in the hF-OB1.19 osteoblasts （ P 〈 0.05 ）. The proliferation of the MG-63 osteosarcoma cells were significantly inhibited after FIM-A treatment. The proliferation inhibition rate of MG-63 cells was significantly higher than that of the negative control group after the treatment of 1 x 10 -7 mol/L FIM-A （ [ 37.64 ± 2.07 ] % vs 0, P 〈 0.05 ）, and the cell proliferation inhibition rate increased along with FIM-A concentrations in a dose-dependent manner （r = 0. 940, P 〈 0.01）. After the treatment of 1 ×10^-6 molZL FIM-A for 24 hours, the proportion of MG-63 ceils in G0/G1 phase was significantly increased compared with the control group （ [ 56.4 ± 3.21% vs [ 43.4 ±6.9 ] %, P 〈 0.05 ）. No obvious changes were found in the apoptotic rate of MG-63 cells compared with the control group. The expression levels of HIF-1 ot and VEGF in MG-63 cells were significantly lower than those of the control group after the treatment of different concentrations of FIM-A for 24 hours （ P 〈 0.05 ） , and as concentrations increased,
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2013年第4期419-424,共6页
Chinese Journal of Cancer Biotherapy
基金
福建省科技重大专项资助项目(No.2011YZ0002-1)~~
