新型组蛋白去乙酰化酶抑制剂致U251细胞周期阻滞、凋亡及其机制的研究

目的探讨新型组蛋白去乙酰化酶抑制剂(HDACi)2,2,3,3-四甲基环丙酰硫脲(TCCT)诱导人脑胶质瘤U251细胞周期阻滞、凋亡及其作用机制。方法以不同药物浓度与U251细胞共同培养48 h后,采用MTT法检测药物作用48 h后肿瘤细胞的增殖。药物作用24 h后,采用RT-PCR检测肿瘤细胞的p21WAF1/CIP1与Cyclin D1 mRNA的表达,Western blot检测HDAC3、HDAC4、Cyclin D1与p21WAF1/CIP1蛋白的表达,PI单染法分析细胞周期,Annexin V-PI双染法检测肿瘤细胞凋亡。结果 TCCT对U251细胞增殖具有明显抑制作用,药物干预48 h时的IC50为(0.461±0.108)mmol·L-1,并呈现剂量依赖性。TCCT药物干预24 h后U251细胞p21WAF1/CIP1mRNA表达上调,Cyclin D1 mRNA下调;组蛋白去乙酰化酶3(HDAC3)和组蛋白去乙酰化酶4(HDAC4)表达下调,Cyc-lin D1蛋白表达弱下调,p21WAF1/CIP1蛋白表达上调;S期细胞比例明显提高(P<0.05);细胞凋亡率明显增高(P<0.01)。结论 TCCT对U251细胞增殖有明显的抑制作用,引起肿瘤细胞S期阻滞和凋亡。其作用机制可能与其下调HDAC3、HDAC4表达,促进组蛋白乙酰化,而影响p21WAF1/CIP1和Cyclin D1的基因、蛋白的表达有关。

Mechanism of a novel histone deacetylase inhibitor arresting cell cycle and inducing apoptosis in U251 cells

Aim To study the effects of a novel histone deacetylase inhibitor(HDACi) 2,2,3,3-tetramethylcyclopropyl acylthiourea(TCCT) on cell arresting,apoptosis and its mechanism in U251 human glioma cells.Methods U251 cells were respectively cultured with different concentrations of TCCT for 24 or 48 h.MTT assay was used to detect the proliferation of U251 after TCCT treament for 48 h.The mRNA expression of p21 WAF1/CIP1 and Cyclin D1 in U251 after TCCT treament for 24 h were measured with RT-PCR.The protein expression of HDAC3,HDAC4 and p21 WAF1/CIP1 changes were detected by Western blot.The cell cycle was determined by PI staining assay.Apoptosis induced by TCCT treament for 24 h in U251 was detected with flow cytometry using Annexin V-PI double staining assay.Results The proliferation of U251 cells was significantly inhibited by TCCT.The IC 50 was(0.461 ± 0.108) mmol·L-1 after TCCT treatment for 48h,and a dose-dependent manner was shown.In U251 cells,p21 WAF1/CIP1 mRNA was upregulated,while Cyclin D1 mRNA was downregulated after TCCT treatment for 24 h.Histone deacetylase 3(HDAC3),histone deacetylase 4(HDAC4) and cyclin D1 protein expressions were downregulated,but cyclin D1 protein expressions were weak,and p21 WAF1/CIP1 protein expression in U 2 5 1 cells was upregulated,after TCCT treatment for 24 h.PI staining assay suggested that S-phase cells were significantly increased(P < 0.05) after TCCT treatment in U251 cells.Annexin Ⅴ-PI demonstrated that TCCT induced apoptosis in U251 cells.Apoptosis rate was significantly increased(P < 0.01).Conclusions TCCT can inhibit proliferation of U251 cells,and induce U251 cells S-phase arrest and apoptosis.The mechanism may be related to the changes of p21 WAF1/CIP1 and Cyclin D1 expressions which result from the decrease of HDAC3 /4 and the increase of histone acetylation.