Stable Knock-down of Efflux Transporters Leads to Reduced Glucuronidation in UGT1A1-Overexpressing HeLa Cells: The Evidence for Glucuronidation-Transport Interplay

Abstract

Efflux of glucuronide is facilitated by the membrane transporters including BCRP and MRPs. In this study, we aimed to determine the effects of transporter expression on glucuronide efflux and cellular glucuronidation. Single efflux transporter (i.e., BCRP, MRP1, MRP3, or MRP4) was stably knocked-down in UGT1A1-overexpressing HeLa cells. Knock-down of transporters was performed by stable transfection of short-hairpin RNA (shRNA) using lentiviral vectors. Glucuronidation and glucuronide transport in the cells were characterized using three different aglycones (i.e., genistein, apigenin, and emodin) with distinct metabolic activities. BCRP knock-down resulted in significant reductions in excretion of glucuronides (42.9% for genistein glucuronide (GG), 21.1% for apigenin glucuronide (AG) , and 33.7% for emodin glucuronide (EG); p < 0.01) and in cellular glucuronidation (38.3% for genistein, 38.6% for apigenin, and 34.7% for emodin; p < 0.01). Knock-down of a MRP transporter led to substantial decreases in excretion of GG (32.3% for MRP1, 36.7% for MRP3, and 36.6% for MRP4; p < 0.01) and AG (59.3% for MRP1, 24.7% for MRP3, and 34.1% for MRP4; p < 0.01). Also, cellular glucuronidation of genistein (38.3% for MRP1, 32.3% for MRP3, and 31.1% for MRP4; p < 0.01) and apigenin (40.6% for MRP1, 32.4% for MRP3, and 34.6% for MRP4; p < 0.001) was markedly suppressed. By contrast, silencing of MRPs did not cause any changes in either excretion of EG or cellular glucuronidation of emodin. In conclusion, cellular glucuronidation was significantly altered by decreasing expression of efflux transporters, revealing a strong interplay of glucuronidation with efflux transport.

Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Controls Efflux Transport of Hesperetin Sulfates in Sulfotransferase 1A3–Overexpressing Human Embryonic Kidney 293 Cells

“…MRP4 was knocked down by transient transfection of shRNA. The selected shRNA was shown to significantly decrease the expression of target transporter MRP4 by ;65% in our previous studies (Quan et al, 2015;Zhang et al, 2015). MRP4 knockdown led to substantial reductions (.47.1%; P , 0.01) in the rates of sulfate excretion (Fig.…”

“…The experimental procedures for sulfate excretion were similar to those for glucuronide excretion detailed previously (Quan et al, 2015;Zhang et al, 2015).…”

“…After sampling at the last time point, the cells were collected and processed to measure the intracellular amounts of sulfate conjugates. The excretion rate of intracellular sulfate was calculated exactly as described in our publications (Quan et al, 2015;Zhang et al, 2015). The apparent efflux clearance (CL ef,app ) was derived as the excretion rate divided by C i (C i was the intracellular concentration of sulfate).…”

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

“…1) and f met (fraction of drug metabolized; eq. 2) value were determined exactly as described (Quan et al, 2015;Zhang et al, 2015). The f met value measures the extent of compound sulfonation (or formation of sulfate metabolites) in the cell system.…”

“…A better understanding of transporter-enzyme interplay contributes to improved predictions of in vivo drug disposition and drug-drug interactions (Benet et al, 2003;Lam et al, 2006). In addition to P-glycoprotein/cytochrome p450 3A, the interplay is also applicable to the efflux transporters (i.e., breast cancer resistance protein and MRPs) and phase II enzymes (Jiang et al, 2012;Zhang et al, 2015). The dependence of phase II metabolism on efflux transporters is underpinned by the fact that the hydrophilic phase II metabolites (lacking in passive transport ability) require the efflux transporters for cellular excretion (Wu, 2012).…”