Localization of anthracyclines in drug resistant human MCF-7 breast cancer cells

Abstract

Multidrug resistance (MDR) commonly occurs during the treatment of cancer. Current research has focused mostly on the role of drug transporters, as the main mechanism of MDR; however, few have demonstrated a definite link between the expression or function of drug transporters and MDR in cancer patients. Anthracyclines such as doxorubicin and epirubicin, autofluoresce and can be monitored by confocal microscopy. Two of the four resistant cell fines generated in our lab: the MCF-7EPI cells and to some extent MCF-7 DOX cells, exhibit a localization defect, whereby epirubicin is localized primarily in the cytoplasm rather than the nucleus. This drug localization defect temporally correlated with the onset of drug-resistance during selection for drug resistance in these cell lines. Consistent with the possible sequestration of drugs into acidic vesicles, acridine orange staining has revealed the presence of aggregates of acidified vesicles in the perinuclear region of MCF-7EPI cells. However, co-localization experiments using a number of intracellular organelle markers determined that epirubicin was localized to lysosomes and not consistently to acidic vesicles. An inhibitor of vacuolar H+ ATPase, was unable to restore the localization of epirubicin to the nucleus. Immunofluorescence using an ABCB1 antibody revealed the localization of ABCB1 predominantly in the plasma membrane and to some extent in the perinuclear region of MCF-7EPI cells. Nevertheless, inhibitors of this transporter failed to restore localization of epirubicin to the nucleus. Taken together, these findings strongly suggest that the acquisition of epirubicin resistance in breast tumour cells may involve the P-glycoprotein independent sequestration of drug into lysosomes. These lysosomes need not be acidic, nor does the removal of acid vesicles by inhibition of vacuolar H+ ATPases block the sequestration of drug into lysosomes.