Abstract
ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [125I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC50 value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.
Highlights
ABCB1, known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family
To confirm that calcein AM efflux in KBV1 cells is due to the overexpression of ABCB1, cell lysates from KB-3-1 and KB-V1 cells were subjected to immunoblotting with an anti-ABCB1 antibody
The flow cytometry assay indicated that the ABCB1 specific inhibitor, XR9576, blocked calcein AM efflux in KB-V1 cells, but neither ABCG2 specific inhibitor fumitremorgin C (FTC) nor ABCC1 specific inhibitor MK-571 interfered with ABCB1-mediated calcein AM efflux in KB-V1 cells (Figure 1B), suggesting that ABCC1 and ABCG2 are not involved in calcein AM efflux in KB-V1 cells
Summary
ABCB1, known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. ABCB1 is largely recognized for its role in enabling cancer cells to evade response to treatment via the efflux of chemotherapeutic agents. This multidrug resistance impedes the clinical cure of cancer by chemotherapy [1]. ABCB1 is expressed in many normal cells and tissues, including the kidneys, liver, brain, intestine, and placenta, serving a key role in drug-drug interactions (DDI) [2] and the absorption, distribution, and excretion of a vast array of xenobiotics [3,4]. The presence of an inhibitor for ABCB1 alters the bioavailability of a drug in the intestine and has an impact on the clinical safety of the selected drug [5]. To enhance current knowledge on the functional roles of ABCB1, to discover new compounds for cancer treatment, and to evaluate the interaction between ABCB1 and newly developed therapeutic agents, it is imperative to develop reliable assays that can efficiently and effectively characterize drug candidates
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