Role of the vacuolar H+-ATPase in daunorubicin distribution in etoposide-resistant MCF7 cells overexpressing the multidrug-resistance associated protein.

Some multidrug-resistant cell lines efflux anticancer drugs but do not overexpress the well-known P-glycoprotein pump or Pgp. A 190 kDa or multidrug-resistant associated protein (MRP) has been identified and described as an MDR mediator. Many studies on cells overexpressing MRP and Pgp, show a concentration of the drug inside cytoplasmic vesicles followed by an exocytotic process. We studied daunorubicin (DNR) subcellular distribution in the presence of an H+-ATPase pump inhibitor 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) and verapamil (VPL) in two human breast adenocarcinoma MCF7 etoposide-resistant and adriamycin-resistant cell lines, overexpressing respectively MRP (MCF7/VP) and Pgp (MCF7/ADR). Nucleo-cytoplasmic distribution of daunorubicin was carried out using scanning confocal microspectrofluorometry. This technique allows the determination of nuclear accumulation of anthracyclines. Our results show that NBD was able to increase the nuclear accumulation of DNR in MCF7/VP but not in MCF7/ADR cells. Similarly, NBD could reverse DNR resistance in MCF7/VP cells but had no effect on DNR cytotoxicity in MCF7/ADR cells. VPL caused a significant increase in nuclear accumulation of DNR in MCF7/VP and MCF7/ADR cells. Incubation of MCF7/VP and MCF7/ADR cells with VPL, increased the sensitivity of these cells. These data demonstrate clearly that even if vesicular sequestration can happen in cells overexpressing MRP and Pgp proteins, only the MRP protein is able to extrude the drug through intracellular vesicles and efflux. In cells overexpressing Pgp, drug efflux probably takes place directly at the membrane level.