ABCB1介导HDAC非选择性抑制剂panobinostat耐药的机制研究  

Mechanism study on ABCB1-mediated resistance of pan-HDAC inhibitor panobinostat

作　　者：窦永海 高晓丽 Dou Yonghai;Gao Xiaoli(Tianjin Yongjiu Hospital,Tianjin300450;Tianjin Fifth Central Hospital,Tianjin 300450)

机构地区：[1]天津市永久医院,天津300450 [2]天津市第五中心医院,天津300450

出　　处：《天津药学》2022年第4期12-19,共8页Tianjin Pharmacy

摘　　要：目的:探究ABCB1介导非选择性HDAC抑制剂panobinostat(PBST)在肿瘤细胞中耐药的机制。方法:以KB-3-1、SW620、HEK293/pcDNA3.1以及其对应的耐药细胞株KB-C2、SW620/Ad300、HEK293/ABCB1细胞为研究对象,利用MTT法评价panobinostat对上述细胞系中的增殖抑制活性,并利用基于MTT的逆转实验评价已报道的ABCB1逆转药物对PBST的耐药逆转效果。将PBST作用于耐药细胞,利用Western blot方法检测ABCB1蛋白表达情况。并通过免疫荧光法探究PBST对耐药细胞中ABCB1质膜定位的影响。以氚标记的紫杉醇为探针评价PBST在上述细胞中蓄积水平的差异。通过钒敏感的ATP酶活性实验探究PBST对ABCB1的ATP酶活性影响,并采用计算机辅助分子对接模拟PBST和ABCB1活性口袋的结合情况。结果:PBST的抗肿瘤活性在肿瘤多药耐药细胞中显著下调,已报道的ABCB1逆转药物,如verapamil、ibrutinib、nilotinib、lapatinib以及erlotinib,均可不同程度地逆转PBST耐药。在PBST处理后,肿瘤多药耐药细胞中ABCB1的蛋白表达水平上调,但ABCB1的质膜定位未发生显著改变。PBST作用于KB-C2细胞可显著增加氚标紫杉醇的蓄积水平,但不改变亲本细胞KB-3-1中的蓄积。此外,PBST上调ABCB1的ATP酶活性,并可与ABCB1活性口袋稳定结合。结论:PBST可通过上调ABCB1在肿瘤细胞中的表达水平,下调其药物蓄积,介导其耐药,联合ABCB1抑制剂是潜在的逆转耐药策略。Objective:To determine the mechanisms of ABCB1-mediated resistance of pan-HDAC inhibitor panobinostat(PBST)in cancer cells.Methods:MTT assay was conducted to evaluate PBST on the proliferation of KB-3-1,KB-C2,SW620,SW620/Ad300,HEK293/pcDNA3.1,and HEK293/ABCB1 cell lines.MTT-based reversal assay was conducted to determine the reversal activity of documented reversal agents in combination with PBST.Western blot was used to detect the expression of ABCB1 in resistant cells after panobinostat treatment.Immunofluorescence was conducted to determine the subcellular localization of ABCB1 after panobinostat treatment.[3H]-paclitaxel was used as a probe to evaluate the accumulation of panobinostat in KB-C2 cells.The ATPase activity of ABCB1 was determined by vanadium-ATPase assay,and docking analysis was conducted in silico to evaluate the simulated docking of panobinostat with drug-binding pocket of ABCB1.Results:In ABCB1-overexpression cell lines,the sensitivity of panobinostat significantly decreased,which could be reversed by a series of documented ABCB1 reversal agents,such as verapamil,ibrutinib,nilotinib,lapatinib,as well as erlotinib.In addition,panobinostat significantly increased the expression of ABCB1 in resistant cells without alteration of its subcellular localization.Panobinostat could upregulate the accumulation of[3H]-paclitaxel in KB-C2 but not in KB-3-1 cells.Moreover,panobinostat upregulated ATPase activity of ABCB1 and docked with residues in drug binding pocket of ABCB1.Conclusion:Overexpression is a potential mechanism of action on PBST resistance.Combining panobinostat with ABCB1reversal agents is a potential strategy to overcome panobinostat resistance.

关 键 词：PANOBINOSTAT 肿瘤耐药 ABC转运蛋白B1亚家族 药物蓄积 

分 类 号：R96[医药卫生—药理学]