Stable knock-down of efflux transporters leads to reduced glucuronidation in UGT1A1-overexpressing HeLa cells: the evidence for glucuronidation-transport interplay.
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.
†Division of Pharmaceutics, College of Pharmacy, and ‡Guangzhou Jinan Biomedicine Research and Development Center, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China.
Xingwang
X
Zhang
Dong
D
Dong
†Division of Pharmaceutics, College of Pharmacy, and ‡Guangzhou Jinan Biomedicine Research and Development Center, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China.
Huailing
H
Wang
†Division of Pharmaceutics, College of Pharmacy, and ‡Guangzhou Jinan Biomedicine Research and Development Center, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China.
Zhiguo
Z
Ma
Yifei
Y
Wang
†Division of Pharmaceutics, College of Pharmacy, and ‡Guangzhou Jinan Biomedicine Research and Development Center, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China.
Baojian
B
Wu
D047310:Apigenin / Q000737:chemistry; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000737:chemistry; D004642:Emodin / Q000737:chemistry; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D019833:Genistein / Q000737:chemistry; D020719:Glucuronides / Q000737:chemistry*; D014453:Glucuronosyltransferase / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D016086:Lentivirus / Q000235:genetics; D008969:Molecular Sequence Data; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D014162:Transfection; D000099148:UGT1A1 Enzyme
D047310:Apigenin; D017931:DNA Primers; D004642:Emodin; D019833:Genistein; D020719:Glucuronides; D014453:Glucuronosyltransferase; D034741:RNA, Small Interfering; D000099148:UGT1A1 Enzyme
BCRP; HeLa cells; MRPs; UGT; glucuronidation