Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma drug transporter P-glycoprotein (P-gp). This protein is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Despite the advances of recent years, we still have an unclear view of the molecular mechanism by which P-gp transports such a wide diversity of compounds across the membrane. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. The aim of the present study was to further characterize the transport of several rhodamine analogues, either positively charged or zwitterionic. We took advantage of the intrinsic fluorescence of rhodamines and performed a flow-cytometric analysis of dye accumulation in the wild-type drug sensitive K562 that do not express P-gp and its MDR subline that display high levels of MDR. The measurements were made in real time using intact cells. The kinetic parameter, k a ¼ V M /k m , which is a measure of the efficiency of the P-gp-mediated efflux of a substrate was similar for almost all the rhodamine analogues tested. In addition these values were compared with those determined previously for the P-gp-mediated efflux of anthracycline. Our conclusion is that the compounds of these two classes of molecules, anthracyclines and rhodamines, are substrates of P-gp and that their pumping rates at limiting low substrate concentration are similar. The findings presented here are the first to show quantitative information about the kinetic parameters for P-gp-mediated efflux of rhodamine analogues in intact cells.Keywords: P-glycoprotein; multidrug resistance; membrane transport; rhodamine; efflux. Since 1940, a broad variety of antibiotics active against many infectious organisms were discovered or developed. The widespread, and sometimes uncontrolled, use of these drugs has led to the emergence of defence mechanisms that, at present, are the major drawback of the drug-based treatment of infectious diseases and cancers. Such resistance is not restricted to the drugs (or analogues) used in the treatment but also involves several structurally and functionally unrelated compounds. This phenomenon, which has been termed multidrug resistance (MDR), can be caused by various mechanisms. However, over expression of the P-glycoprotein multidrug transporter (P-gp) in the plasma membrane is believed to be a major cause of resistance to multiple chemotherapeutic drugs [1][2][3][4]. P-gp is an unusual ABC protein in that it appears to be highly promiscuous: hundreds of compounds have been identified as substrates. The spectrum of MDR compounds includes a large number of anticancer drugs (e.g. anthracyclines, vinca alkaloids, taxanes) as well as steroids, fluorescent dyes, rhodamines, and the c-emitting radio pharmaceutical 99m Tc-MIBI. P-gp can transport neutral and positively charged molecules but not negatively charged ones. Despite the advances of recent years, we still h...

Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells. The formation of a substrate concentration gradient was observed. MRP1-mediated transport of rhodamine was glutathione-dependent. The kinetics parameter, k(a) = V(M)/k(m), was very similar for the four rhodamine analogs but approximately 10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes.

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