UDP-Glucuronosyltransferase (UGT) 1A9-Overexpressing HeLa Cells Is an Appropriate Tool to Delineate the Kinetic Interplay between Breast Cancer Resistance Protein (BRCP) and UGT and to Rapidly Identify the Glucuronide Substrates of BCRP
UDP-Glucuronosyltransferase (UGT) 1A9-Overexpressing HeLa Cells Is an Appropriate Tool to Delineate the Kinetic Interplay between Breast Cancer Resistance Protein (BRCP) and UGT and to Rapidly Identify the Glucuronide Substrates of BCRP
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DOI：
10.1124/dmd.111.041467
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发表时间：
2012-02-01
影响因子：
3.9
通讯作者：
Hu, Ming
中科院分区：
文献类型：
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作者：
Jiang, Wen;Xu, Beibei;Hu, Ming
II相酶和外排转运蛋白之间的相互作用导致了类黄酮的广泛代谢和低生物利用度。为了研究UDP-葡萄糖醛酸基转移酶亚型和外排转运体在类黄酮类代谢中的最简单相互作用，建立了稳定高表达UGT1A9的HeLa工程细胞，并对其进行了鉴定，并进一步应用于研究两种模型类黄酮类化合物(染料木素和芹菜素)的代谢和其葡萄糖醛酸苷的排泄。结果表明，高表达UGT1A9的HeLa工程细胞能迅速分泌染料木素和芹菜素的葡萄糖醛酸苷。染料木素或芹菜素葡萄糖醛酸化反应的动力学特征与在HeLa细胞中过表达的UGT1A9或商业上可获得的UGT1A9相似。小干扰(SiRNA)介导的UGT1A9沉默导致葡萄糖醛酸苷排泄显著减少(>；75%，p<；0.01)。此外，乳腺癌耐药蛋白的有效抑制剂3-(6-isobutyl-9-methoxy-1，4-dioxo-1，2，3，4，6，7，12，12a-octahydropyrazino[1‘，2’：1，6]吡啶并[3，4-b]吲哚-3-基)-丙酸叔丁酯(Ko143)，以剂量依赖的方式引起实质和显著的清除减少(74-94%，p<；0.01)，并显著增加细胞内葡萄糖醛酸苷水平(4-8倍，p<；0.01)，导致葡萄糖醛酸排泄量适度下降(19-59%，p<；0.01)。此外，观察到在Ko143存在的情况下，染料木素代谢的部分(f(MET))显著减少，尽管幅度不大。相反，白三烯C(4)和针对多药耐药蛋白(MRP)2和MRP3的siRNA不影响黄酮类葡萄糖醛酸苷的排泄。总之，高表达UGT1A9的HeLa工程细胞是研究UGT1A9和BCRP在黄酮类化合物II相处置中的动力学相互作用的合适模型。这个简单的细胞模型对于快速确定II相代谢产物是否为BCRP的底物也非常有用。
The interplay between phase II enzymes and efflux transporters leads to extensive metabolism and low bioavailability for flavonoids. To investigate the simplest interplay between one UDP-glucuronosyltransferase isoform and one efflux transporter in flavonoid disposition, engineered HeLa cells stably overexpressing UGT1A9 were developed, characterized, and further applied to investigate the metabolism of two model flavonoids (genistein and apigenin) and excretion of their glucuronides. The results indicated that the engineered HeLa cells overexpressing UGT1A9 rapidly excreted the glucuronides of genistein and apigenin. The kinetic characteristics of genistein or apigenin glucuronidation were similar with the use of UGT1A9 overexpressed in HeLa cells or the commercially available UGT1A9. Small interfering (siRNA)-mediated UGT1A9 silencing resulted in a substantial decrease in glucuronide excretion (> 75%, p < 0.01). Furthermore, a potent inhibitor of breast cancer resistance protein (BCRP), 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2' : 1,6] pyrido[3,4-b] indol-3-yl)-propionic acid tert-butyl ester (Ko143), caused, in a dose-dependent manner, a substantial and marked reduction of the clearance (74-94%, p < 0.01), and a substantial increase in the intracellular glucuronide levels (4-8-fold, p < 0.01), resulting in a moderate decrease in glucuronide excretion (19-59%, p < 0.01). In addition, a significant, albeit moderate, reduction in the fraction of genistein metabolized (f(met)) in the presence of Ko143 was observed. In contrast, leukotriene C(4) and siRNA against multidrug resistance protein (MRP) 2 and MRP3 did not affect excretion of flavonoid glucuronides. In conclusion, the engineered HeLa cells overexpressing UGT1A9 is an appropriate model to study the kinetic interplay between UGT1A9 and BCRP in the phase II disposition of flavonoids. This simple cell model should also be very useful to rapidly identify whether a phase II metabolite is the substrate of BCRP.