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Stable Knock-down of Efflux Transporters Leads to Reduced Glucuronidation in UGT1A1-Overexpressing HeLa Cells: The Evidence for Glucuronidation-Transport Interplay
journal contribution
posted on 2015-04-06, 00:00 authored by Xingwang Zhang, Dong Dong, Huailing Wang, Zhiguo Ma, Yifei Wang, Baojian WuEfflux 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.
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Keywords
efflux transportUGT 1A HeLa Cellsmembrane transportersGGUGT 1A HeLa cellsEfflux TransportersSingle efflux transporterlentiviral vectorsBCRPEGRNAglucuronide transportemodin glucuronideapigenin glucuronideefflux transportersReduced Glucuronidationtransporter expressiongenistein glucuronideAGMRP transporterglucuronide efflux
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