The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line.
The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line.
复制标题
DOI：
10.1016/j.taap.2013.11.019
复制
发表时间：
2014-01
影响因子：
3.8
通讯作者：
Yanyan Zhang;Yahui Hu;Yi-dong Feng;Nandani Darshika Kodithuwakku;W. Fang;Yunman Li;Wen-long Huang
中科院分区：
文献类型：
--
作者：
Yanyan Zhang;Yahui Hu;Yi-dong Feng;Nandani Darshika Kodithuwakku;W. Fang;Yunman Li;Wen-long Huang
近年来，研究和开发逆转多药耐药现象的药物已成为一个有吸引力的目标，也是提高癌症患者临床生存率的关键方法。尽管已经鉴定出三代P-糖蛋白调节剂，但较差的清除率和代谢使得这些药物毒性太大而无法用于临床应用。 HZ08经过多年的研究，显示出显着的逆转作用和低细胞毒性。我们首次旨在描述 HZ08 与 Caco-2 细胞系中 P-糖蛋白之间的相互作用，在该细胞系中 P-糖蛋白自然过度表达。本研究采用细胞毒性和多药耐药性逆转试验，结合流式细胞术、荧光显微镜和siRNA干扰以及Caco-2单层转运模型来评估HZ08和P-糖蛋白之间的相互作用。这项研究表明，由于细胞内阿霉素积累增强，HZ08能够逆转P-糖蛋白介导的阿霉素耐药性，其效果优于维拉帕米。此外，我们证实HZ08在Caco-2单层模型中抑制罗丹明123的转运，但对P-糖蛋白表达影响不大。 HZ08 的转运被 P-糖蛋白抑制剂（维拉帕米和 LY335979）减弱，并且通过靶向 Caco-2 细胞中 MDR1 的 siRNA 增加了 HZ08 的积累。此外，考虑到 P-糖蛋白的结合位点，维拉帕米可作为 HZ08 的竞争性抑制剂。总之，作为 P-糖蛋白底物，HZ08 抑制 P-糖蛋白活性，并且可能与维拉帕米与 P-糖蛋白具有相同的结合位点。
Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity and multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein.