New insights into the P‐glycoprotein‐mediated effluxes of rhodamines

Abstract

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...

Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes

“…Uptake of TMRE was more rapid than uptake of R123, because TMRE is more highly cell membrane-permeant (21,32). Accordingly, [R e ] for TMRE reached steady state more rapidly than [R e ] for R123, thereby allowing the time course for TMRE studies to be shorter.…”

“…Because the fractional loss of dye from the extracellular medium in Fig. 1 is dose-independent, Pgpmediated dye efflux from V c to V e is hypothesized to follow linear kinetics (32). Moreover, the model allows for slowly equilibrating nonspecific dye interactions with the cuvette (B e ) within V e and rapidly equilibrating nonspecific dye interactions with binding sites B c and B m within V c and V m , respectively.…”

“…A kinetic model was developed to interpret the data and to obtain parameter values for ⌬ m , ⌬ p , and Pgp for both dyes. R123 and TMRE were selected because, whereas TMRE is more cell membrane-permeable than R123, both have relatively strong fluorescence quantum yields, have sensitivity to changes in the cellular environment, and undergo membrane potential-dependent changes in distribution across the mitochondrial and plasma membranes (21,22,32,58,64). Thus one concept was that the utility of the proposed approach for quantifying ⌬ m and ⌬ p in pulmonary arterial endothelial cells would be revealed by the consistency of the parameter values obtained from the studies with two dyes that have both distinct and common properties.…”

Teaching the basics of cancer metabolism: Developing antitumor strategies by exploiting the differences between normal and cancer cell metabolism

“…Tumor cells have increased levels of a multidrug resistant, plasma-membrane-bound p-glycoprotein that acts as an efflux pump for positively charged compounds or drugs [71], [72]. This is a clinically significant, p-glycoprotein-mediated multidrug resistance (MDR).…”

Accumulation of Eu3+ chelates in cells expressing or not P-glycoprotein: Implications for blood–brain barrier crossing

“…The passive permeability rate constant, k, can then be determined. This rate constant takes into account the permeability constant of the molecule and the membrane exchange area per cell with the relation V + = k Á C e , where C e is the extracellular concentration of [Eu-DOTAM] 3+ [42,44,45]. The k value for [Eu-DOTAM] 3+ is $2 Â 10 À15 L/s/cell, whereas the value is $2 Â 10 À12 L/s/cell for the rhodamine derivative, tetramethylrosamine (TMR) and $8 Â 10 À15 L/s/cell for hexakis(-methoxy-isobutylisonitrile)technetium(I) (TcMIBI).…”