Competition between innate multidrug resistance and intracellular binding of rhodamine dyes

Abstract

The present study aimed to elucidate the contribution of the intracellular binding of drugs to multidrug resistance. For this purpose, uptake of rhodamines was studied in cells whose mitochondria had been uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Surprisingly, in a variety of drug-untreated cells, presumed to be sensitive to multidrug resistance-type drugs, rhodamines were excluded from entering the cells. Thus, the amount of rhodamine 123 taken up into parental untreated K562 cells was less than the amount bound to the cell exterior. Rhodamine uptake was prevented by an active efflux pump. The efflux was inhibited by 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) and MK571 and, to a lesser extent, by ATP depletion, indomethacin, probenecid and vanadate. All the inhibitors, apart from NBD-Cl, are known to modulate multidrug resistance-associated protein (MRP) 1. Because MRP1 was expressed in all the cell lines tested and the efflux of rhodamines in MRP1 over-expressing cells was abolished by NBD-Cl, it appears that rhodamines are excluded from these cells by MRP1. On the other hand, the uptake of rhodamines into cells respiring with their coupled mitochondria demonstrated diminished sensitivity to NBD-Cl and MK571. Thus, active pumping into the mitochondria allowed enhanced uptake into the cells, overcoming the innate resistance. The innate resistance provided by MRP1 to cells prevents rhodamine dyes, and possibly drugs such as doxorubicin, from achieving equilibration of their concentration in the cytoplasm with their concentration in the external medium. The protection provided to multidrug resistance cells by ABC transporters has to overcome competition by passive uptake of the drugs and binding/uptake of the drugs into intracellular targets.