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.
Highlights
Multidrug resistance relates to resistance of tumor cells to a whole range of chemotherapy drugs with different structures and cellular targets [1]
Flow cytometry data were analyzed by comparison of median fluorescence, by using Kolmogorov-Smirnov statistics (D-value) [26] or by calculating multi drug resistance (MDR) Activity Factor (MAF) values using the following formula: multidrug resistance activity factor (MAF) = 100*((MFIinh2MFI0)/MFIinh), wherein MFIinh and MFI0 are the mean fluorescence intensity values measured in the presence and absence of inhibitor [7]
To confirm drug resistance of the cell lines used as model systems in these studies, HeLa, U-2 OS, CHO K1 and A549 cells were seeded in 96-well plates at a density of 56103 cells/well and 24 h later, treated with increasing doses of different drugs that are commonly used for cancer chemotherapy and are known to be associated with the phenomenon of MDR
Summary
Multidrug resistance relates to resistance of tumor cells to a whole range of chemotherapy drugs with different structures and cellular targets [1]. One of the underlying molecular mechanisms responsible for MDR is the up-regulation of a family of MDR transporter proteins that lead to chemotherapy resistance in cancer by actively extruding a wide variety of therapeutic compounds from the malignant cells. MDR transporters belong to an evolutionarily conserved family of ATP binding cassette (ABC) proteins, expressed in practically all living organisms from prokaryotes to mammals [2]. Additional members of the MRP/ ABCC family have been indicated to be involved in cancer multidrug resistance [for details, see [3]]. The transport properties of BCRP overlap both with that of MDR1 and the MRP type proteins, these three proteins form a special network involved with chemo-defense mechanisms
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