用于筛选组蛋白去乙酰化酶抑制剂细胞模型的验证

目的验证已建立的组蛋白去乙酰化酶(HDAC)抑制剂筛选细胞模型,并采用该模型进行药物筛选。方法采用脂质体转染法将含有TA1和TA2启动子元件的荧光素酶报道基因真核表达载体pTA1-Luc和pTA2-Luc导入COS-7细胞,G418筛选获得稳定转染上述报告基因系统的细胞克隆。以特异性HDAC抑制剂缩酯环肽FK228,N-辛二酰苯胺异羟肟酸(SAHA)和曲古抑菌素A(TSA)为阳性对照,通过测定荧光素酶活性评估TA1和TA2启动子对HDAC抑制剂的反应;并采用此细胞模型对其他抗肿瘤药物和植物提取物进行初步筛选。结果稳定转染的COS-pTA1及COS-pTA2细胞对HDAC抑制剂具有良好的浓度依赖性(FK228:相关系数为0.7236;SAHA:相关系数为0.7997;TSA为相关系数为0.9815;P<0.01),其中COS-pTA1的特点是灵敏度高,可以有效避免因样品活性低而出现漏检。COS-pTA2细胞的特点是检测背景低,特异性强,可以有效排除假阳性的标本。两种细胞模型的联合筛选,可以鉴定具有HDAC抑制活性的化合物。结论该细胞模型可用于筛选具有HDAC抑制活性的先导化合物。

Verification of cell models for screening histone deacetylase inhibitors

OBJECTIVE To verify cell models which can be used for screening candidates for histone deacetylase (HDAC) inhibitors. METHODS Two eukaryotic luciferase report vectors containing TA1 and TA2 promoters which can be activated specifically by HDAC inhibitors were constructed and named pTA1-Luc and pTA2-Luc. These plasmids were transfected into COS-7 cells, and the stable transfectants were selected by G418 and named COS-pTA1 and COS-pTA2 respectively. The responsiveness of COS-pTA1 and COS-pTA2 cells to either HDAC inhibitors 〔depsipeptide FK228, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA)〕 or other anticancer reagents were evaluated via luciferase assay and other natural anticancer drugs were preliminarily screened. RESULTS The COS-pTA1 and COS-pTA2 cells showed time- and concentration-dependent response to different HDAC inhibitors by luciferase assay. The coeffienent of correlation of FK228, SAHA and TSA was 0.7236, 0.7997 and 0.9815 respectively(P<0.01). Various HDAC inhibitors induced higher luciferase activities in COS-pTA1 cells than in COS-pTA2 cells. The data indicated that COS-pTA1 cells were more sensitive, while COS-pTA2 cells had better specificity to identify possible HDAC inhibitors. The combination of these two cell models could offer optimal potential to screen drugs with HDAC inhibitory activity. CONCLUSION COS-pTA1 and COS-pTA2 cell models may be useful tools for discovering lead compounds of HDAC inhibitors through library screening.