Inhibition of the MRP1-mediated transport of the menadione-glutathione conjugate (thiodione) in HeLa cells as studied by SECM.
Oxidative stress induced in live HeLa cells by menadione (2-methyl-1,4-napthaquinone) was studied in real time by scanning electrochemical microscopy (SECM). The hydrophobic molecule menadione diffuses through a living cell membrane where it is toxic to the cell. However, in the cell it is conjugated with glutathione to form thiodione. Thiodione is then recognized and transported across the cell membrane via the ATP-driven MRP1 pump. In the extracellular environment, thiodione was detected by the SECM tip at levels of 140, 70, and 35 µM upon exposure of the cells to menadione concentrations of 500, 250, and 125 µM, respectively. With the aid of finite element modeling, the kinetics of thiodione transport was determined to be 1.6 10(-7) m/s, about 10 times faster than menadione uptake. Selective inhibition of these MRP1 pumps inside live HeLa cells by MK571 produced a lower thiodione concentration of 50 µM in presence of 500 µM menadione and 50 µM MK571. A similar reduced (50% drop) thiodione efflux was observed in the presence of monoclonal antibody QCRL-4, a selective blocking agent of the MRP1 pumps. The reduced thiodione flux confirmed that thiodione was transported by MRP1, and that glutathione is an essential substrate for MRP1-mediated transport. This finding demonstrates the usefulness of SECM in quantitative studies of MRP1 inhibitors and suggests that monoclonal antibodies can be a useful tool in inhibiting the transport of these MDR pumps, and thereby aiding in overcoming multidrug resistance.
Center for Electrochemistry, Department of Chemistry and Biochemistry, University of Texas at Austin, 1 University Station A5300, Austin, TX 78712-0165, USA.
Dipankar
D
Koley
Allen J
AJ
Bard
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D055664:Electrochemical Techniques; D020342:Finite Element Analysis; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D020527:Microscopy, Scanning Probe / Q000379:methods*; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism / Q000494:pharmacology*; D018384:Oxidative Stress / Q000502:physiology*; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D024483:Vitamin K 3 / Q000031:analogs & derivatives* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D005978:Glutathione; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011422:Propionates; D011804:Quinolines; D024483:Vitamin K 3; C495859:menadione-S-glutathione conjugate; C059141:verlukast; C421109:multidrug resistance-associated protein 1