The Efflux Mechanism of Fraxetin-O-Glucuronides in UGT1A9-Transfected HeLa Cells: Identification of Multidrug Resistance-Associated Proteins 3 and 4 (MRP3/4) as the Important Contributors.

Zifei Qin,Xinsheng Yao,Jing Yang,Frank J Gonzalez,Beibei Zhang,Xiaojian Zhang,Zhihong Yao,Shishi Li,Jinjin Xu

doi:10.3389/fphar.2019.00496

Abstract

Fraxetin, a natural compound present in many dietary supplements and herbs, is useful in the treatment of acute bacillary dysentery and type 2 diabetes. Previously, several metabolic studies have revealed extensive first-pass metabolism causing formation of fraxetin-O-glucuronides (G1 and G2), resulting in poor bioavailability of fraxetin. Active transport processes play an important role in the excretion of fraxetin-O-glucuronides. Nevertheless, the transporters involved are yet to be elucidated. In this study, we aimed to determine the active efflux transporters, including breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs), involved in the excretion of fraxetin-O-glucuronides. A chemical inhibitor, MK571 (5 and 20 μM), a pan-MRP inhibitor, led to a significant decrease in excreted G1 (maximal 59.1%) and G2 levels (maximal 42.4%), whereas Ko143 (5 and 20 μM), a selective BCRP inhibitor, caused moderate downregulation of excreted G1 (maximal 29.4%) and G2 (maximal 28.5%). Furthermore, MRP3 silencing resulted in a marked decrease of excretion rates (by 29.1% for G1 and by 21.1% for G2) and of fraction metabolized (fmet; by 24.1% for G1 and by 18.6% for G2). Similar results, i.e., a significant reduction in excretion rates (by 34.8% for G1 and by 32.3% for G2) and in fmet (by 22.7% for G1 and by 23.1% for G2) were obtained when MRP4 was partially silenced. No obvious modifications in the excretion rates, intracellular levels, and fmet values of glucuronides were observed after short hairpin RNA (shRNA)-mediated silencing of transporters BCRP and MRP1. Taken together, our results indicate that MRP3 and MRP4 contribute more to the excretion of fraxetin-O-glucuronides than the other transporters do.

Highlights

Fraxetin is a natural coumarin analog that is widely distributed in many functional foods and dietary supplements (Jyotshna et al, 2017)
Western blotting results showed that the UGT1A9 protein was abundant in HeLa1A9 cells, whereas UGT1A9 was not expressed in wild-type HeLa cells (Figure 1E)
It was noted that the wildtype and engineered HeLa1A9 cells had an identical pattern of transporter expression (Figure 1E)

Summary

Introduction

Fraxetin is a natural coumarin analog that is widely distributed in many functional foods and dietary supplements (Jyotshna et al, 2017). It has been demonstrated to be effective as a therapeutic agent with multiple activities, such as antioxidant, antibacterial, hypoglycemic, antiosteoporosis, and antiplatelet activities (Kuo et al, 2006; Murali et al, 2013; Wang et al, 2014; Feng et al, 2016; Zaragoza et al, 2016) It was identified as a cystathionine β-synthase inhibitor (IC50 = 134 μM) that plays a critical role in human sulfur metabolism (Thorson et al, 2013). It was reported that fraxetin may serve as a promising candidate drug against type 2 diabetes through antioxidative mechanisms (Yao et al, 2018). These significant biological properties led to increased interest in the in vivo metabolic fate and pharmacokinetic characteristics of fraxetin

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