The cell membrane P-glycoprotein (P-gp; MDR1, ABCB1) is an energy-dependent efflux
pump that belongs to the ATP-binding cassette (ABC) family of transporters, and has
been associated with drug resistance in eukaryotic cells. Multidrug resistance (MDR)
is related to an increased expression and function of the ABCB1 (P-gp) efflux pump
that often causes chemotherapeutic failure in cancer. Modulators of this efflux pump,
such as the calcium channel blocker verapamil (VP) and cyclosporine A (CypA), can
reverse the MDR phenotype but in vivo studies have revealed disappointing results
due to adverse side effects. Currently available methods are unable to visualize and
assess in a real-time basis the effectiveness of ABCB1 inhibitors on the uptake and
efflux of ABCB1 substrates. However, predicting and testing ABCB1 modulation activity
using living cells during drug development are crucial. The use of ABCB1-transfected
mouse T-lymphoma cell line to study the uptake/efflux of fluorescent probes like ethidium
bromide (EB), rhodamine 123 (Rh-123), and carbocyanine dye DiOC2, in the presence
and absence of potential inhibitors, is currently used in our laboratories to evaluate
the ability of a drug to inhibit ABCB1-mediated drug accumulation and efflux. Here
we describe and compare three in vitro methods, which evaluate the permeability, transport
kinetics of fluorescent substrates, and inhibition of the ABCB1 efflux pump by drugs
of chemical synthesis or extracted from natural sources, using model cancer cell lines
overexpressing this transporter, namely (1) real-time fluorimetry that assesses the
accumulation of ethidium bromide, (2) flow cytometry, and (3) fluorescent microscopy
using rhodamine 123 and DiOC2.
