Characterization of multidrug resistance P-glycoprotein transport function with an organotechnetium cation.
Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an approximately 170 kDa integral membrane efflux transporter, the MDR1 P-glycoprotein. Hexakis (2-methoxyisobutyl isonitrile)technetium(I) (Tc-SESTAMIBI), a gamma-emitting lipophilic cationic metallopharmaceutical, has recently been shown to be a P-glycoprotein transport substrate. Exploiting the negligible lipid membrane adsorption properties of this organometallic substrate, we studied the transport kinetics, pharmacology, drug binding, and modulation of P-glycoprotein in cell preparations derived from a variety of species and selection strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells. Rapid cell accumulation (t1/2 approximately 6 min) of the agent to a steady state was observed which was inversely proportional to immunodetectable levels of P-glycoprotein. Many MDR cytotoxic agents inhibited P-glycoprotein-mediated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation accumulation. Median effective concentrations (EC50; microM) were as follows: vinblastine, 13; daunomycin, 55; idarubicin, 65; actinomycin D, 235; colchicine, minimal inhibition; adriamycin, no effect. P-glycoprotein modulators generally demonstrated significantly greater potency (EC50; microM): SDZ PSC 833, 0.08; cyclosporin A, 1.3; verapamil, 4.1; quinidine, 6.4; prazosin, > 300. Modulator-induced enhancement up to 100-fold was observed with Hill coefficients approximately 1, consistent with simple Michaelis-Menten kinetics. Vanadate was an efficacious transport inhibitor, while agents usually not included in the MDR phenotype were without effect. Scatchard analysis showed quinidine to be a noncompetitive inhibitor of P-glycoprotein-mediated Tc-SESTAMIBI transport, indicating allosteric effector sites on P-glycoprotein. The lipid bilayer adsorbing agents tetraphenyl borate and phloretin induced large increases in final Tc-SESTAMIBI accumulation, showing maximal accumulations 2-fold greater than classic MDR modulators and Hill coefficients >> 2. In V79 and 77A cells, modulators of PKC activity altered Tc-SESTAMIBI accumulation, while there was no indication of modulation of P-glycoprotein-mediated Tc-SESTAMIBI transport by hypotonic buffer, extracellular ATP, Cl-, or K+ (membrane potential). While recognized and avidly transported by the P-glycoprotein at buffer concentrations as low as 7 pM, Tc-SESTAMIBI at up to 100 microM only minimally modulated the cytotoxic action of colchicine, doxorubicin, or vinblastine in MDR cells. In conclusion, transport analysis with Tc-SESTAMIBI is a sensitive assay for detecting functional expression of low levels of P-glycoprotein and for the quantitative characterization of transporter modulation and regulation. The biochemical data favor a high Km, high capacity allosterically modulated translocation mechanism for P-glycoprotein-mediated transport of this organometallic cation.
Piwnica-Worms
D
D
Laboratory of Molecular Radiopharmacology, Mallinckrodt Institute of Radiology, Washington University Medical School, St. Louis, Missouri 63110, USA.
Rao
V V
VV
Kronauge
J F
JF
Croop
J M
JM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D001386:Azides / Q000494:pharmacology; D001692:Biological Transport; D002412:Cations / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D010693:Phloretin / Q000494:pharmacology; D011224:Prazosin / Q000031:analogs & derivatives / Q000494:pharmacology; D011802:Quinidine / Q000494:pharmacology; D017256:Technetium Tc 99m Sestamibi / Q000378:metabolism*; D013775:Tetraphenylborate / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D002412:Cations; D008051:Lipid Bilayers; D004364:Pharmaceutical Preparations; D013775:Tetraphenylborate; D004317:Doxorubicin; D000255:Adenosine Triphosphate; C041552:azidoprazosin; D017256:Technetium Tc 99m Sestamibi; D011802:Quinidine; D010693:Phloretin; D003078:Colchicine; D011224:Prazosin