Efflux Transport Characterization of Resveratrol Glucuronides in UDP-Glucuronosyltransferase 1A1 Transfected HeLa Cells: Application of a Cellular Pharmacokinetic Model to Decipher the Contribution of Multidrug Resistance-Associated Protein 4

Abstract

Resveratrol undergoes extensive metabolism to form biologically active glucuronides in humans. However, the transport mechanisms for resveratrol glucuronides are not fully established. Here, we aimed to characterize the efflux transport of resveratrol glucuronides using UGT1A1-overexpressing HeLa cells (HeLa1A1 cells), and to determine the contribution of multidrug resistanceassociated protein (MRP) 4 to cellular excretion of the glucuronides. Two glucuronide isomers [i.e., resveratrol 3-O-glucuronide (R3G) and resveratrol 49-O-glucuronide (R49G)] were excreted into the extracellular compartment after incubation of resveratrol (1-100 mM) with HeLa1A1 cells. The excretion rate was linearly related to the level of intracellular glucuronide, indicating that glucuronide efflux was a nonsaturable process. MK-571 (a dual inhibitor of UGT1A1 and MRPs) significantly decreased the excretion rates of R3G and R49G while increasing their intracellular levels. Likewise, short-hairpin RNA (shRNA)-mediated silencing of MRP4 caused a significant reduction in glucuronide excretion but an elevation in glucuronide accumulation. Furthermore, b-glucuronidase expressed in the cells catalyzed the hydrolysis of the glucuronides back to the parent compound. A cellular pharmacokinetic model integrating resveratrol transport/metabolism with glucuronide hydrolysis/excretion was well fitted to the experimental data, allowing derivation of the efflux rate constant values in the absence or presence of shRNA targeting MRP4. It was found that a large percentage of glucuronide excretion (43%-46%) was attributed to MRP4. In conclusion, MRP4 participated in cellular excretion of R3G and R49G. Integration of mechanistic pharmacokinetic modeling with transporter knockdown was a useful method to derive the contribution percentage of an exporter to overall glucuronide excretion.

Efflux excretion of bisdemethoxycurcumin‐O‐glucuronide in UGT1A1‐overexpressing HeLa cells: Identification of breast cancer resistance protein (BCRP) and multidrug resistance‐associated proteins 1 (MRP1) as the glucuronide transporters

“…Previous studies have proved that numerous UGT1As and 2Bs enzymes participated in the glucuronidation of curcumin analogs . Traditionally, the glucuronides were formed by UGT enzymes in cell, and the glucuronides were further transferred from intracellular to extracellular by efflux transporters . Except the UGT enzymes, efflux transporters (e.g.…”

Arylsulfatase B Mediates the Sulfonation-Transport Interplay in Human Embryonic Kidney 293 Cells Overexpressing Sulfotransferase 1A3

“…For instance, MK-571 was a mixed modifier (i.e., activation at low concentrations and inhibition at high concentrations) of UGT1A1-mediated glucuronidation of chrysin (Quan et al, 2015). MK-571 significantly inhibited glucuronidation of resveratrol by UGT1A1 through its binding to the reaction site of the enzyme (Wang et al, 2016). However, we have confirmed that MK-571 did not affect the sulfonation activity of SULT1A3 at all; thus, the reduced excretion of hesperetin sulfates was solely ascribed to the suppression of MRP transporter (Sun et al, 2015a).…”

Differential Proteomics Analysis of Colonic Tissues in Patients of Slow Transit Constipation

“…MRP4 belongs to transmembrane protein family; its main function is to transport intracellular cyclic nucleotides that include cyclic adenosine monophosphate and cyclic guanosine monophosphate. Some studies have confirmed that MRP4 participated in cellular excretion of resveratrol 3-O-glucuronide and resveratrol 4′-O-glucuronide [ 18 ], the inhibition of urinary excretion of methotrexate [ 19 ], cellular excretion of the raloxifene sulfates in breast cancer patients [ 20 ], the process of human obstructive cholestasis [ 21 ], and so on. Recently studies have revealed that MRP4 can be involved in maintaining the homeostasis of smooth muscle cell (SMC) by the regulation of intracellular cyclic nucleotide levels on the intracellular cyclic nucleotide signaling pathways (PKC, PKA, etc.).…”