Zeng Xuejun, Shi Jian, Zhao Min, Chen Qingwei, Wang Liping, Jiang Huangyu, Luo Feifei, Zhu Lijun, Lu Linlin, Wang Xinchun, Liu Zhongqiu
Department of Pharmacy, First Hospital Affiliated to Shihezi University, Shihezi, Xinjiang, 832002, China.
International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, China.
PLoS One. 2016 Nov 10;11(11):e0166239. doi: 10.1371/journal.pone.0166239. eCollection 2016.
This study aimed to determine the reaction kinetics of the regioselective glucuronidation of diosmetin and chrysoeriol, two important methylated metabolites of luteolin, by human liver microsomes (HLMs) and uridine-5'-diphosphate glucuronosyltransferase (UGTs) enzymes. This study also investigated the effects of breast cancer resistance protein (BCRP) on the efflux of diosmetin and chrysoeriol glucuronides in HeLa cells overexpressing UGT1A9 (HeLa-UGT1A9). After incubation with HLMs in the presence of UDP-glucuronic acid, diosmetin and chrysoeriol gained two glucuronides each, and the OH-in each B ring of diosmetin and chrysoeriol was the preferable site for glucuronidation. Screening assays with 12 human expressed UGT enzymes and chemical-inhibition assays demonstrated that glucuronide formation was almost exclusively catalyzed by UGT1A1, UGT1A6, and UGT1A9. Importantly, in HeLa-UGT1A9, Ko143 significantly inhibited the efflux of diosmetin and chrysoeriol glucuronides and increased their intracellular levels in a dose-dependent manner. This observation suggested that BCRP-mediated excretion was the predominant pathway for diosmetin and chrysoeriol disposition. In conclusion, UGT1A1, UGT1A6, and UGT1A9 were the chief contributors to the regioselective glucuronidation of diosmetin and chrysoeriol in the liver. Moreover, cellular glucuronidation was significantly altered by inhibiting BCRP, revealing a notable interplay between glucuronidation and efflux transport. Diosmetin and chrysoeriol possibly have different effects on anti-cancer due to the difference of UGT isoforms in different cancer cells.
本研究旨在确定木犀草素的两种重要甲基化代谢产物香叶木素和 Chrysoeriol 通过人肝微粒体（HLMs）和尿苷 - 5'-二磷酸葡萄糖醛酸转移酶（UGTs）进行区域选择性葡萄糖醛酸化的反应动力学。本研究还调查了乳腺癌耐药蛋白（BCRP）对过表达 UGT1A9 的 HeLa 细胞（HeLa-UGT1A9）中香叶木素和 Chrysoeriol 葡萄糖醛酸苷外排的影响。在 UDP - 葡萄糖醛酸存在下与 HLMs 孵育后，香叶木素和 Chrysoeriol 各自获得两种葡萄糖醛酸苷，并且香叶木素和 Chrysoeriol 的每个 B 环中的 OH 是葡萄糖醛酸化的优选位点。用 12 种人表达的 UGT 酶进行的筛选试验和化学抑制试验表明，葡萄糖醛酸苷的形成几乎完全由 UGT1A1、UGT1A6 和 UGT1A9 催化。重要的是，在 HeLa-UGT1A9 中，Ko143 显著抑制香叶木素和 Chrysoeriol 葡萄糖醛酸苷的外排，并以剂量依赖性方式增加它们的细胞内水平。这一观察结果表明，BCRP 介导的排泄是香叶木素和 Chrysoeriol 处置的主要途径。总之，UGT1A1、UGT1A6 和 UGT1A9 是肝脏中香叶木素和 Chrysoeriol 区域选择性葡萄糖醛酸化的主要贡献者。此外，抑制 BCRP 可显著改变细胞葡萄糖醛酸化，揭示了葡萄糖醛酸化和外排转运之间的显著相互作用。由于不同癌细胞中 UGT 同工型的差异，香叶木素和 Chrysoeriol 可能对抗癌有不同的作用。
