1, 25-二羟基维生素D_(3)通过下调葡萄糖转运蛋白-1表达抑制膀胱癌细胞增殖

1,25-(OH)_(2)-VitD_(3) inhibits the proliferation of bladder cancer cells by down-regulating the expression of glucose transporter-1

目的:研究1,25-二羟基维生素D_(3)(1,25-(OH)_(2)-VitD_(3))对膀胱癌细胞增殖的影响,并探讨其作用机制。方法:体外培养膀胱癌细胞T24,MTT法检测不同浓度1,25-(OH)_(2)-VitD_(3)对细胞活力的影响,并筛选1,25-(OH)_(2)-VitD_(3)干预细胞的最佳时间及半数抑制浓度。将细胞分为4组:对照组、葡萄糖转运蛋白-1(glucose transporter-1,GLUT1)抑制组、1,25-(OH)_(2)-VitD_(3)组、1,25-(OH)_(2)-VitD_(3)+GLUT1抑制组。采用Hoechst 33258染色观察各组细胞形态变化;试剂盒检测细胞内葡萄糖摄取、三磷酸腺苷(adenosine triphosphate,ATP)产生及乳酸形成水平;Real-time PCR及Western blot检测GLUT1、己糖激酶2(hexokinase 2,HK2)、6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3,PFKFP3)、丙酮酸激酶同工酶2(pyruvate kinase isozyme type M2,PKM2)、乳酸脱氢酶(lactate dehydrogenase A,LDHA)和低氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)mRNA和蛋白表达水平。结果:1,25-(OH)_(2)-VitD_(3)可降低T24细胞增殖能力,且呈剂量依赖性。1,25-(OH)_(2)-VitD_(3)组、GLUT1抑制组及1,25-(OH)_(2)-VitD_(3)+GLUT1抑制组细胞凋亡水平高于对照组,而GLUT1、HK2、PFKFP3、PKM2、LDHA和HIF-1αmRNA及蛋白表达水平明显低于对照组。与GLUT1抑制组比较,1,25-(OH)_(2)-VitD_(3)+GLUT1抑制组GLUT1、HK2、PFKFP3、PKM2、LDHA和HIF-1αmRNA(均P=0.000)和蛋白(均P=0.000)表达水平明显降低。结论:1,25-(OH)_(2)-VitD_(3)可抑制膀胱癌细胞增殖,诱导细胞凋亡,其作用机制可能与下调GLUT1表达抑制细胞糖酵解过程有关。

Objective:To study the effect of 1,25-(OH)_(2)-VitD_(3) on the proliferation of bladder cancer cells and explore its mechanism.Methods:Bladder cancer cells T24 were cultured in vitro.The effects of 1,25-(OH)_(2)-VitD_(3) at different concentrations on cell viability were detected by MTT assay.The optimal time and half inhibitory concentration of 1,25-(OH)_(2)-VitD_(3) on bladder cancer cells were screened.Cells were divided into four groups:control group,glucose transporter-1(GLUT1)inhibitor group,1,25-(OH)_(2)-VitD_(3) group and 1,25-(OH)_(2)-VitD_(3)+GLUT1 inhibitor group.Hoechst 33258 staining was used to observe the morphological changes of cells in each group.The levels of glucose uptake,adenosine triphosphate(ATP)production and lactate formation were detected by kits.The mRNA and protein expression levels of GLUT1,hexokinase 2(HK2),6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFP3),pyruvate kinase isozyme type M2(PKM2),lactate dehydrogenase A(LDHA)and hypoxia-inducible factor-1α(HIF-1α)were detected by real-time PCR and Western blot.Results:1,25-(OH)_(2)-VitD_(3) could reduce the proliferation of T24 cells in a dose-dependent manner.The number of apoptotic cells in 1,25-(OH)_(2)-VitD_(3) group,GLUT1 inhibitor group and 1,25-(OH)_(2)-VitD_(3)+GLUT1 inhibitor group were higher than that in control group,while the mRNA and protein expression levels of GLUT1,HK2,PFKFP3,PKM2,LDHA and HIF-1αwere significantly lower than those in control group.Compared with GLUT1 inhibitor group,the mRNA(all P=0.000)and protein(all P=0.000)expression levels of GLUT1,HK2,PFKFP3,PKM2,LDHA and HIF-1αwere significantly lower in 1,25-(OH)_(2)-VitD_(3)+GLUT1 inhibitor group.Conclusion:1,5-(OH)_(2)-VitD_(3) can inhibit the proliferation and induce apoptosis of bladder cancer cells.Its mechanism may be related to down-regulation of GLUT1 expression to inhibit cell glucolusis.