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Chemical inhibition and stable knock-down of efflux transporters leads to reduced glucuronidation of wushanicaritin in UGT1A1-overexpressing HeLa cells: the role of breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs) in the excretion of glucuronides†
Food & Function ( IF 6.1 ) Pub Date : 2017-12-13 00:00:00 , DOI: 10.1039/c7fo01298e
Zifei Qin 1, 2, 3, 4, 5 , Shishi Li 1, 2, 3, 4 , Zhihong Yao 1, 2, 3, 4, 5 , Xiaodan Hong 1, 2, 3, 4, 6 , Baojian Wu 1, 2, 3, 4, 5 , Kristopher W. Krausz 7, 8, 9, 10, 11 , Frank J. Gonzalez 7, 8, 9, 10, 11 , Hao Gao 1, 2, 3, 4, 5 , Xinsheng Yao 1, 2, 3, 4, 5
Affiliation  

Active efflux transport of glucuronides out of cells is a critical process in elimination of drugs and food-derived compounds. Wushanicaritin, a natural polyphenol from Epimedium species, has shown many biological activities. However, the transporters responsible for excretion of wushanicaritin glucuronides still remain undefined. Herein, chemical inhibitors (Ko143, MK571, dipyridamole and leukotriene C4) and single stable knocked-down efflux transporters (BCRP, MRP1, MRP3 and MRP4) were used to determine the contributions of efflux transporters to glucuronide efflux and cellular glucuronidation in UGT1A1-overexpressing HeLa cells (HeLa1A1). Knock-down of transporters was performed by stable transfection of short hairpin RNA (shRNA) using lentiviral vectors. The HeLa1A1 cell lysate catalyzed wushanicaritin glucuronidation, generating wushanicaritin-3-O-glucuronide and wushanicaritin-7-O-glucuronide. Ko143 (a dual inhibitor of BCRP, 5–20 μM) caused a marked decrease in excretion rate (maximal 53.4%) and increase of intracellular glucuronides (maximal 86.0%), while MK-571 (an inhibitor of MRPs, 5–20 μM) resulted in a significant reduction in excretion rate (maximal 64.6%) and rise of intracellular glucuronides (maximal 98.0%). By contrast, dipyridamole and leukotriene C4 showed no inhibitory effects on glucuronide excretion. Furthermore, shRNA-mediated silencing of a target transporter led to a marked reduction in the excretion rate of wushanicaritin glucuronides (maximal 33.8% for BCRP; 25.9% for MRP1; 26.7% for MRP3; 39.3% for MRP4). Transporter silencing also led to substantial decreases in efflux clearance (maximal 61.5% for BCRP; 48.7% for MRP1; 35.1% for MRP3; 63.1% for MRP4). In conclusion, chemical inhibition and gene silencing results suggested that BCRP, MRP1, MRP3 and MRP4 were significant contributors to excretion of wushanicaritin glucuronides.

中文翻译:

化学抑制和外排转运蛋白的稳定敲低导致过表达UGT1A1的HeLa细胞中Wushanicaritin的葡萄糖醛酸化作用降低:乳腺癌抗性蛋白(BCRP)和多药抗性相关蛋白(MRPs)在葡萄糖苷类排泄中的作用

葡萄糖醛酸苷主动外排运出细胞是消除药物和食物衍生化合物的关键过程。Wushanicaritin,从天然多酚淫羊藿物种,已显示出许多生物活性。然而,负责乌山菌素葡萄糖醛酸苷排泄的转运蛋白仍然不确定。本文中,使用化学抑制剂(Ko143,MK571,双嘧达莫和白三烯C4)和单个稳定的敲除外排转运蛋白(BCRP,MRP1,MRP3和MRP4)来确定外转运转运蛋白对过表达UGT1A1的葡萄糖醛酸外排和细胞葡萄糖醛酸化的贡献。 HeLa细胞(HeLa1A1)。通过使用慢病毒载体稳定转染短发夹RNA(shRNA)进行转运蛋白的敲低。HeLa1A1细胞裂解物催化乌山菌素葡萄糖醛酸化,生成乌山菌素-3- O-葡萄糖醛酸和乌山菌素-7 - O。-葡糖苷酸。Ko143(BCRP的双重抑制剂,5-20μM)引起排泄率显着下降(最大53.4%)和细胞内葡糖醛酸苷的增加(最大86.0%),而MK-571(MRP的抑制剂,5-20μM) )导致排泄率显着降低(最大64.6%)和细胞内葡糖醛酸苷的升高(最大98.0%)。相比之下,双嘧达莫和白三烯C4对葡糖醛酸排泄没有抑制作用。此外,shRNA介导的靶转运蛋白沉默导致乌山菌素葡萄糖醛酸苷的排泄率显着降低(BCRP最高33.8%; MRP1最高25.9%; MRP3最高26.7%; MRP4最高39.3%)。转运蛋白沉默也导致外排清除率显着降低(BCRP最大值为61.5%; MRP1最大值为48.7%; MRP3最大值为35.1%; MRP4最大值为63.1%)。综上所述,
更新日期:2017-12-13
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