组蛋白去乙酰化酶抑制剂F321-2治疗肿瘤的机制研究

Study on mechanisms of histone deacetylase inhibitors F321-2 in treatment of tumors

目的探讨组蛋白去乙酰化酶(HDAC)抑制剂F321-2治疗肿瘤的机制。方法 MTT法测定F321-2对多类肿瘤细胞的抗增殖活性;划痕实验检验F321-2对A2780s细胞的迁移影响;流式细胞术测定F321-2处理Hct116细胞24 h后的周期影响; Western blot检测F321-2作用Hct116细胞后Ac-α-tubulin、 Ac-H3蛋白的表达情况。采用Hct116异种移植和4T1移植小鼠模型评估F321-2的体内抗肿瘤活性,将模型小鼠随机分为溶剂组,伏立诺他(SAHA, 100 mg·kg-1)阳性对照组, F321-2低、高剂量(25、50 mg·kg-1)组,每组7只,每周3次灌胃给药,记录小鼠肿瘤的体积和重量。结果 F321-2对21种肿瘤细胞的半数抑制浓度(IC50)范围主要在50~200 nmol·L-1内,具有较好的抗增殖活性; 1 000 nmol·L-1F321-2几乎完全抑制A2780s细胞的迁移; F321-2 (200、 1 000、 5 000 nmol·L-1)处理Hct116细胞24 h后, G2/M期的细胞比例随药物浓度增加而逐渐增多(P <0.05), F321-2能阻滞细胞于G2/M期; F321-2能介导Hct116细胞内Ac-H3蛋白水平的上调,且同剂量下比SAHA效果显著(P <0.05); Hct116异种移植和4T1移植小鼠模型分别给药16 d和19 d, F321-2高剂量组肿瘤体积均明显小于溶剂组和SAHA阳性对照组(P <0.01)。结论 F321-2在体内和体外均具有显著的抗肿瘤作用,抗肿瘤活性可能与HDAC标志蛋白Ac-H3表达水平变化有关。

AIM To explore the mechanism of histone deacetylase (HDAC)inhibitors F321-2 in the treatment of tumors.METHODS Anti-proliferative activity of F321-2 against a variety of tumor cells was tested by MTT method.Scratch assay was used to test the effect of F321-2 on the migration of A2780s ceils.Cyclic experiments were performed by flow cytometry after F321-2 treatment on Hct116 cells for 24h.Western blot was used to detect the expression of Ac-α-tubulin and Ac-H3 protein expression.In v ivo anti-tumor activity of F321- 2was evaluated by the Hct116 xenografted Balb/c mouse model and the 4T1 transplantation of Balb/c mouse model.The model mice were randomly assigned into vehicle group,vorinostat (SAHA,100mg·kg^-1)positive control group,F321-2 low dose and high dose (25and 50mg·kg^-1)groups,and each group with 7 mice by oral administration (3 times a week).The tumor volume and weight should be recorded.RESULTS The halfinhibitory concentration (IC50)of F321-2 in vitro treatment of 21 tumor cells was ranged from 50to 200nmol· L^-1,had a good anti-proliferative activity.F321-21000nmol ·L^-1completely inhibited the migration of A2780s cells.After Hct116 cells were treated with F321-2(200,1000,5000nmol·L^-1)for 24h,the proportion of cells in G2/M phase gradually increased with the increasing drug concentration (P <0.05),indicating that F321-2 can arrest cells in G2/M phase.F321-2 could mediate the up-regulation of Ac-H3 protein in Hct116 cells,and it was more effective than SAHA at the same dose (P <0.05).After Hct116 xenografted Balb/c mouse and 4T1 transplanted Balb/c mouse models were treated 16 days and 19 days respectively,the tumor volume of F321-2 high dose group was significantly lower than that of the vehicle group and the SAHA positive control group (P <0.01).CONCLUSION F321-2 achieves a remarkable anti-tumor effect in vitro and in vivo, which may be related to the change of HDAC marker protein Ac-H3 expression level.