Identification of the subcellular localization of daunorubicin in multidrug-resistant K562 cell line

We examined the subcellular distribution of daunorubicin (DNR) in resistant K562 cell line which overexpress the P-glycoprotein by confocal laser scanning microscopy. Three fluorescent probes - Rhodamine123, neutral red, NBD-ceramide, which stain the mitochondria, lysosomes, Golgi apparatus respectively, were used to identify the nature of the subcellular compartment sequestering daunorubicin. In sensitive k562 cell line, nuclear and cytoplasmic DNR fluorescence was intense and diffuse. In contrast, resistant K562 cell line showed a different DNR distribution. A bright fluorescence signal was located in the perinuclear region and peripheral plasma, the nucleus and other cytoplasmic region appear as empty, as suggested by the distribution of fluorescent probe Rhodamine123 specifically for mitochondria. Verapamil, an effective resistance modulator in P-glycoprotein MDR cells, restored the DNR distribution closer to that in the parent cells. Golgic inhibitor brefeldin A and lysosomotropic agent chloroquine had little effect on drug sequestration. Our studies demonstrate that daunorubicin may be sequestered in mitochondrial compartment in the resistant cells and P-glycoprotein plays an important role on mediating DNR transport.