P-glycoprotein (P-gp/MDR1)-mediated efflux of sex-steroid hormones and modulation of P-gp expression in vitro.
OBJECTIVE
To determine whether female sex-steroid hormones and their metabolites can modulate P-glycoprotein (P-gp) expression and catalytic activity and to investigate P-gp mediated transport of these sex-steroids across MDR1-transfected Madine-Darby canine kidney (MDCK) cells.


METHODS
Changes in P-gp protein and MDR1 mRNA expression levels were examined in the presence of various estrogens and progestins after a 72-h induction period in the LS180 human colon carcinoma cell line via Western blotting and semiquantitative Reverse-transcription-polymerase chain reaction (RT-PCR), respectively. Concentration-dependent stimulation of vanadate-sensitive P-gp ATPase activity was measured in membranes of Sf9 insect cells infected with a recombinant baculovirus containing the human MDR1 cDNA used with appropriate control membranes. MDCK and MDR1-transfected MDCK cell lines were then used to measure bidirectional P-gp transport of various steroids in the presence and absence of the P-gp inhibitor, GG918. Samples obtained were quantified using LC/MS.


RESULTS
Our findings show that P-gp protein levels are inducible by estrone (4-fold over control), estriol (2-fold), and ethynyl estradiol (3-fold). MDR1 mRNA expression levels were also inducible in a concentration-dependent manner from 25 nM to 10 microM. Bidirectional transport studies indicate that ethynyl estradiol, estrone, and estriol are all substrates for P-gp with respective efflux ratios of 10.3, 6.9, and 2.8. Norethindrone was not found to be a substrate for P-gp. Ethynyl estradiol and progesterone were able to significantly stimulate P-gp ATPase activity in a concentration-dependent manner.


CONCLUSIONS
Our studies indicate that several sex-steroid hormones are substrates for P-gp-mediated transport and are also able to induce P-gp expression at both the protein and mRNA level in vitro. Stimulation of P-gp ATPase catalytic activity by steroid hormones was also observed, suggesting physical interactions and identifying a need for further investigations to understand the in vivo effects of endogenous and synthetic steroid hormones on the expression and function of P-gp.
Kim
Winnie Y
WY
Department of Biopharmaceutical Sciences, University of California, San Francisco, California 94143, USA.
Benet
Leslie Z
LZ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D000166:Acridines / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002460:Cell Line; D002853:Chromatography, Liquid; D004305:Dose-Response Relationship, Drug; D045166:Estradiol Congeners / Q000008:administration & dosage / Q000493:pharmacokinetics*; D005260:Female; D006801:Humans; D013058:Mass Spectrometry; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044005:Tetrahydroisoquinolines / Q000494:pharmacology; D013997:Time Factors
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000166:Acridines; D045166:Estradiol Congeners; D012333:RNA, Messenger; D044005:Tetrahydroisoquinolines; C083501:Elacridar