title: Pregnane X receptor mediates the induction of P-glycoprotein by spironolactone in HepG2 cells.
abstract: We evaluated the effect of spironolactone (SL), a well-known inducer of biotransformation and elimination pathways, on the expression and activity of P-glycoprotein (P-gp/ABCB1/MDR1), a major xenobiotic transporter, in HepG2 cells, as well as the potential mediation of pregnane X nuclear receptor (PXR). Cells were exposed to SL (1, 5, 10, 20 or 50 μM) for 48 h. Expression of P-gp and its mRNA levels were estimated by Western blotting and real time PCR, respectively. P-gp activity was inversely correlated with the ability of the cells to accumulate the model substrate rhodamine 123 (Rh123, 5 μM), in the presence or absence of verapamil (50 μM), a P-gp inhibitor. At the highest dose of SL tested, P-gp and MDR1 mRNA levels were significantly increased (73 and 108%) with respect to control cells. Rh123 accumulation was concomitantly reduced and verapamil was able to abolish this effect, confirming P-gp participation. Additionally, we tested the cytotoxicity of doxorubicin, a model substrate of P-gp, under inducing conditions. HepG2 cells treated with SL exhibited higher viability, i.e. less doxorubicin toxicity, than control cells, consistent with P-gp up-regulation. When HepG2 cells were treated with SL in the presence of ketoconazole (KTZ), a non-specific nuclear receptor inhibitor, the up-regulation of P-gp was suppressed. To further identify the nuclear receptor involved, cells were transfected with a siRNA directed against human PXR, leading to a 74% decrease in PXR protein levels, which totally abolished SL induction of P-gp. We conclude that SL up-regulates P-gp expression, likely at transcriptional level, and its efflux activity in HepG2 cells. This effect is mediated by PXR. Thus, ligands of PXR such as SL may alter the disposition and toxicity of other xenobiotics, including drugs of therapeutic use, that are P-gp substrates.
authors: Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario, Argentina.
authors: Juan Pablo
authors: JP
authors: Rigalli
authors: María Laura
authors: ML
authors: Ruiz
authors: Virginia Gabriela
authors: VG
authors: Perdomo
authors: Silvina Stella Maris
authors: SS
authors: Villanueva
authors: Aldo Domingo
authors: AD
authors: Mottino
authors: Viviana Alicia
authors: VA
authors: Catania
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000633:toxicity; D004347:Drug Interactions; D056945:Hep G2 Cells; D006801:Humans; D000451:Mineralocorticoid Receptor Antagonists / Q000008:administration & dosage / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D000077297:Pregnane X Receptor; D012333:RNA, Messenger / Q000378:metabolism; D011987:Receptors, Steroid / Q000378:metabolism*; D020112:Rhodamine 123 / Q000493:pharmacokinetics; D013148:Spironolactone / Q000008:administration & dosage / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects*; D014700:Verapamil / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000451:Mineralocorticoid Receptor Antagonists; D000077297:Pregnane X Receptor; D012333:RNA, Messenger; D011987:Receptors, Steroid; D020112:Rhodamine 123; D013148:Spironolactone; D004317:Doxorubicin; D014700:Verapamil