Chemical inhibition and stable knock-down of efflux transporters leads to reduced glucuronidation of wushanicaritin in UGT1A1-overexpressing HeLa cells: the role of breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs) in the excretion of glucuronides

Abstract

Active efflux transport of glucuronides out of cells is a critical process in elimination of drugs and food-derived compounds. Wushanicaritin, a natural polyphenol from Epimedium species, has shown many biological activities. However, the transporters responsible for excretion of wushanicaritin glucuronides still remain undefined. Herein, chemical inhibitors (Ko143, MK571, dipyridamole and leukotriene C4) and single stable knocked-down efflux transporters (BCRP, MRP1, MRP3 and MRP4) were used to determine the contributions of efflux transporters to glucuronide efflux and cellular glucuronidation in UGT1A1-overexpressing HeLa cells (HeLa1A1). Knock-down of transporters was performed by stable transfection of short hairpin RNA (shRNA) using lentiviral vectors. The HeLa1A1 cell lysate catalyzed wushanicaritin glucuronidation, generating wushanicaritin-3-O-glucuronide and wushanicaritin-7-O-glucuronide. Ko143 (a dual inhibitor of BCRP, 5-20 μM) caused a marked decrease in excretion rate (maximal 53.4%) and increase of intracellular glucuronides (maximal 86.0%), while MK-571 (an inhibitor of MRPs, 5-20 μM) resulted in a significant reduction in excretion rate (maximal 64.6%) and rise of intracellular glucuronides (maximal 98.0%). By contrast, dipyridamole and leukotriene C4 showed no inhibitory effects on glucuronide excretion. Furthermore, shRNA-mediated silencing of a target transporter led to a marked reduction in the excretion rate of wushanicaritin glucuronides (maximal 33.8% for BCRP; 25.9% for MRP1; 26.7% for MRP3; 39.3% for MRP4). Transporter silencing also led to substantial decreases in efflux clearance (maximal 61.5% for BCRP; 48.7% for MRP1; 35.1% for MRP3; 63.1% for MRP4). In conclusion, chemical inhibition and gene silencing results suggested that BCRP, MRP1, MRP3 and MRP4 were significant contributors to excretion of wushanicaritin glucuronides.

Metabolism and disposition of corylifol A from Psoralea corylifolia: metabolite mapping, isozyme contribution, species differences and identification of efflux transporters for corylifol A-O-glucuronide in HeLa1A1 cells

“…(2) compared with other kinetic parameters such as K m and V max values, CL int value is more relevant in an attempt to predict hepatic clearance in vivo (Wu et al, 2013). And this calculation protocol for CL int values has been widely applied in previous studies (Zhu et al, 2012;Qin et al, 2018a;Qi et al, 2019).…”

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

“…Human UGT1A1 cDNA was synthesized and subcloned into the BamHI and MluI sites of the pLVXmCMV‐ZsGreen‐PGK‐Puro vector. The recombinant vector was stably transfected into HeLa cells as previously described . The expressions of UGT1A1 at mRNA and protein levels in HeLa1A1 cells were determined using the methods (qPCR and Western blotting) described in our previous studies .…”

“…The recombinant vector was stably transfected into HeLa cells as previously described . The expressions of UGT1A1 at mRNA and protein levels in HeLa1A1 cells were determined using the methods (qPCR and Western blotting) described in our previous studies . The multiplicity of infection (MOI) value was determined by pilot experiments, and the optimal value was obtained when the cells were best transfected as described previously.…”

“…Glucuronide excretion assays were performed as described previously . Briefly, HeLa1A1 cells were incubated with 2 mL Hank's buffered salt solution (HBSS) containing 5 μM BDMC.…”

“…In this study, we aimed to determine the efflux transporters for the excretion of BDMC‐ O ‐glucuronide. Toward this goal, we adopted an previously established UGT1A1‐overexpressing HeLa cells, which proved rather active in generation of the glucuronides , to evaluate the roles of BCRP and MRPs in the excretion of BDMC‐ O ‐glucuronide. In addition, chemical inhibitors and short hairpin RNA‐mediated silencing of BCRP and MRPs transporters assays were both employed to identify the active glucuronide transporters.…”