Characterization of Chrysin Glucuronidation in UGT1A1-Overexpressing HeLa Cells: Elucidating the Transporters Responsible for Efflux of Glucuronide

Abstract

Active transport of glucuronide out of cells is a critical process in elimination of drugs via the glucuronidation pathway. Here, HeLa cells were stably transfected with UGT1A1 and the contributions of BCRP and MRP family transporters to the cellular efflux of chrysin glucuronide (CG) were determined. The cDNA of UGT1A1 was introduced into HeLa cells using the lentiviral transfection method. The modified cells were functional in generation of the glucuronide from chrysin. Ko143 at 10-20 mM (a dual inhibitor of BCRP and UGT1A1) caused a marked decrease (51.3%-59.7%, P < 0.01) in the excretion rate and efflux clearance of CG. Likewise, MK-571 at 5-20 mM (an inhibitor of MRPs but an activator of UGT1A1) resulted in a significant reduction in the excretion rate (18.2%-64.0%, P < 0.01) and efflux clearance (37.0%-90.2%, P < 0.001). By contrast, dipyridamole and leukotriene C4 showed no inhibitory effects on CG excretion. The chemical inhibition indicated that excretion of CG was contributed by the MRP family transporters, whereas the role of BCRP was unclear. Furthermore, short hairpin RNA-mediated silencing of a target transporter led to a marked reduction in the excretion rate of CG (38.6% for BCRP, 39.3% for MRP1, 36.4% for MRP3, and 28.7% for MRP4; P < 0.01). Transporter silencing also led to substantial decreases in the efflux clearance (44.7% for BCRP, 60.4% for MRP1, 36.7% for MRP3, and 28.7% for MRP4; P < 0.01). The gene silencing results suggested that BCRP, MRP1, MRP3, and MRP4 were significant contributors to excretion of CG.

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

“…Cell lysate were prepared using the sonication method as described previously (Quan et al, 2015). In brief, the cells were collected and suspended in potassium phosphate buffer (50 mM, pH 7.4).…”

“…HEK293 cells were stably transfected with the cDNA of human ARSC using the lentiviral transduction approach (Quan et al, 2015). The ARSC cDNA (1752 bp) was polymerase chain reaction (PCR) amplified from the pCMV6-XL5-ARSC plasmid (OriGene Technologies).…”

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

“…MRP4 was knocked down by short-hairpin RNA-mediated gene silencing. The selected short-hairpin RNA was able to decrease the expression of MRP4 by 65% in HeLa cells (Quan et al, 2015). Knockdown of MRP4 caused a significant reduction (P , 0.05) in excretion of resveratrol glucuronides (Supplemental Fig.…”

“…Glucuronide excretion experiments were performed following the published procedures (Quan et al, 2015). Resveratrol and its glucuronides (R3G and R49G) were quantified using the Waters ACQUITY ultra-performance liquid chromatography system (Milford, MA).…”

“…Establishment of UGT1A1-and GUSB-overexpressing HeLa Cells. UGT1A1-or GUSB-overexpressing HeLa cells were generated using the lentiviral transfection method as previously described (Quan et al, 2015;Sun et al, 2015). Western blotting against GUSB was performed as previously described (Quan et al, 2015).…”