Sensitive and Specific Fluorescent Probes for Functional Analysis of the Three Major Types of Mammalian ABC Transporters

Abstract

An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC2(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments.

Cardiomyocyte H9c2 cells present a valuable alternative to fish lethal testing for azoxystrobin

“…Furthermore, AZX revealed the capacity to inhibit cell proliferation in a concentrationdependent manner in HepG2 cells within 24 h, while most of the cell lines showed the same result only after 48 h. A549 and VSa16 cell lines are likely to need more than 48 h for AZX to pass through the cellular membrane as they both showed low r 2 , with A549 not even fitting the regression model used with 24 h data. On the other hand, most carcinoma cells develop mechanisms of multidrug resistance, based on the existence of transporter proteins (e.g., MDR1, MRP1/2, BCRP/MXR), and the human lung adenocarcinoma epithelial cell line A549 is considered a multidrug resistance cell line expressing MRP1 and BCRP proteins (Lebedeva et al, 2011). The comparison of the six IC 50 results revealed that the H9c2 cell line is more sensitive to AZX than the others, since it showed cytotoxicity at smaller concentrations.…”

Exploring the Carbamazepine Interaction with Human Pregnane X Receptor and Effect on ABCC2 Using in Vitro and in Silico Approach

“…Medium used for this experiment was opti-MEM and each solution was prepared in buffer at pH of 7.4, and fluorescence readings were performed using Infinite 200 PRO multimode plate reader (Tecan) with excitation and emission wavelengths of 492 and 518 nm, respectively. Efflux activity was calculated using formula: % Efflux activity = 100*((FI inh − FI 0 )/FI inh ), wherein FI inh and FI 0 are the fluorescence intensity values measured in the presence and absence of inhibitor (29). Fluorescence intensities were normalized with total protein content.…”

Characterization of multidrug transporter-mediated efflux of avermectins in human and mouse neuroblastoma cell lines

“…CMFDA is a non-fluorescent lipophilic compound which is cleaved by intracellular esterases then modified to generate a hydrophilic membrane impermeant fluorescent product, GSMF, within the cells. GSMF is a specific substrate for MRP/Mrp isoforms (Forster et al, 2008;Lebedeva et al, 2011Lebedeva et al, 2011. SH-SY5Y or N2a cells were exposed to a range of concentrations of CMFDA (0-1.5 mM) and intracellular fluorescence was measured in the absence and presence of MK571 (10 mM), a specific inhibitor of MRP/Mrp isoforms (Luders et al, 2009).…”