新型组蛋白去乙酰化酶抑制剂DWP0016诱导神经胶质瘤细胞U251周期阻滞及凋亡作用研究

目的：观察新型组蛋白去乙酰化酶（Histone Deacetylases,HDACs）抑制剂DWP0016对神经胶质瘤U251细胞株的作用,探讨诱导U251细胞周期阻滞及凋亡作用的机制。方法：采用噻唑蓝（MTT）法检测DWP0016对U251细胞株的增殖抑制作用;采用流式细胞术观察DWP0016对U251细胞周期的影响及凋亡诱导作用;采用实时定量PCR（real-time PCR）检测抑癌因子P21,P53的mRNA水平变化;蛋白免疫印迹法（Western blotting）测定Ac-H3,P21,P53,Pi3K,p-Pi3K,Akt,p-Akt的蛋白表达并进行光密度定量。结果：DWP0016抑制U251细胞增殖的半数抑制浓度（IC50=0.531μmol.L-1）明显低于阳性对照奥沙利铂（IC50=4.792μmol.L-1）,组蛋白H3乙酰化水平显著上升;DWP0016作用后,U251细胞中细胞周期阻滞于G1期并产生凋亡,P21,P53的mRNA水平和蛋白水平明显上升,Pi3K/Akt通路中的Pi3K,Akt的磷酸化水平下降。结论：DWP0016能明显抑制U251细胞增殖,诱导细胞周期阻滞和凋亡产生,其机制与促进抑癌因子P21,P53的转录及蛋白表达,抑制细胞中Pi3K/Akt生长信号通路有关,具有良好的抗神经胶质瘤潜力和开发应用前景。

Research on the Block of Cell Cycle and Apoptosis Induction on Glioma Cell Line U251 of a Novel HDAC Inhibitor Named DWP0016

Objective： Examine the effects of a novel HDAC inhibitor named DWP0016 on glioma cell line U251 and explore the molecular mechanisms underlying the cell cycle arrest and apoptosis induction.Methods： The anti-proliferative effects of DWP0016 were detected by MTT assays and the effects on the cell cycle arrest and apoptosis induction were examined by flow cytometry.The regulations on mRNA level of P21 and P53 were measured by Real-time PCR assay;the protein expressions of Ac-H3,P21,P53,Pi3K,p-Pi3K,Akt and p-Akt were detected by Western blotting and measured by densitometry.Results： DWP0016 was found to inhibit the proliferation of U251 cell line at a much lower concentration（IC50=0.531 μmol·L-1） than Oxaliplatin（IC50=4.792 μmol·L-1）.DWP0016 induced G1 phase cell cycle arrest and apoptosis（P0.05） while the acetylation of histone H3 was significantly up-regulated.The mRNA level and protein expression of tumor suppressor P21,P53 were up regulated whereas the protein expression of phosphorylation of Pi3K and Akt were down regulated.Conclusions： DWP0016 could effectively inhibit the proliferation of glioma cell line U251 and induce cell cycle arrest and apoptosis in U251 cells.The molecular mechanisms might relate to activate tumor suppressor P21,P53 and inhibit Pi3K/Akt cell signal pathway.All the results suggested that DWP0016 was a potent compound to be developed as an anti-glioma agent for clinic application in the future.