Regulation of cellular glutathione modulates nuclear accumulation of daunorubicin in human MCF7 cells overexpressing multidrug resistance associated protein.

Multidrug resistance (MDR) is frequently associated with the overexpression of P-glycoprotein (Pgp) and/or multidrug resistance associated protein (MRP1), both members of the ABC superfamily of transporters. Pgp and MRP1 function as ATP-dependent efflux pumps that extrude cytotoxic drugs from tumour cells. Glutathione (GSH) has been considered to play an important role in the MRP1-mediated MDR. In our study, we examined the effects of buthionine sulphoximine (BSO), an inhibitor of GSH biosynthesis, on the nuclear accumulation of daunorubicin (DNR), in etoposide (VP16) and doxorubicin (ADR) resistant MCF7 cell lines, overexpressing respectively MRP1 (MCF7/VP) and Pgp (MCF7/ADR). The study of DNR transport was carried out using scanning confocal microspectrofluorometry. This technique allows the determination of the nuclear accumulation of anthracyclines in single living tumour cells. Treatment of MCF7/VP cells with BSO increased the sensitivity of these cells to DNR whilst the cytotoxicity of the drug in MCF7/ADR cells remained unchanged. In MCF7 resistant cells treated with BSO, their GSH level decreased as observed by confocal microscopy. DNR nuclear accumulation in MCF7/VP cells was increased by BSO whereas in MCF7/ADR cells BSO was unable to significantly increase the DNR nuclear accumulation. These data suggest a requirement for GSH in MRP1-mediated resistance whilst the nuclear efflux of GSH conjugates is probably not the primary mechanism of Pgp-mediated MDR. Finally, BSO might be a useful agent in clinical assays for facilitating detection of MRP1 expression.

Evidence for functional discrimination between leukemic cells overexpressing multidrug-resistance associated protein and P-glycoprotein.

Multidrug-resistance (MDR), caused by overexpression of either P-glycoprotein (Pgp) or the multidrug-resistance associated protein (MRP), is characterised by a decreased cellular drug accumulation. One form of MDR is the sequestration of the drug inside cytoplasmic vesicles followed by an a exocytotic and/or efflux process. In some studies, increased intracellular glutathione (GSH) has been associated with MDR. In this study, we examined the effects of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole or NBD (a H(+)-ATPase pump inhibitor) and buthionine sulphoximine or BSO (an inhibitor of GSH biosynthesis) on the subcellular distribution of daunorubicin or DNR in two leukemic homoharringtonine-resistant K562 cell lines, overexpressing MRP (K-H30) and Pgp (K-H300). DNR nuclear accumulation was carried out using microspectrofluorometry. Our results show that DNR nuclear accumulation and sensitivity of K-H30 cells were increased by NBD and BSO whereas in K-H300 cells, NBD and BSO were unable to increase the DNR nuclear accumulation and sensitivity of these cells. This study demonstrates 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 addition, GSH plays an important part in the pathway of drug transport in cells overexpressing MRP. Data entrain also the notion of functional discrimination between the MDR and MRP phenotype.

Characterization of H+-ATPase-dependent activity of multidrug resistance-associated protein in homoharringtonine-resistant human leukemic K562 cells.

Multidrug resistance (MDR), caused by overexpression of either P-glycoprotein or the multidrug resistance-associated protein (MRP), is characterized by a decreased cellular drug accumulation due to an enhanced drug efflux. Many studies on cells overexpressing MRP and/or Pgp, have shown a concentration of the drug inside cytoplasmic acidic vesicles followed by an exocytotic process. In this study, we examined the effects of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole or NBD (a H+-ATPase pump inhibitor), buthionine sulphoximine or BSO (an inhibitor of glutathione (GSH) biosynthesis) and verapamil or VPL (a calcium channel blocker) on the subcellular distribution of daunorubicin or DNR in K562 cells overexpressing MRP (K-H30) and Pgp (K-H300) and A549 cells overexpressing spontaneously MRP. Nucleo-cytoplasmic distribution of DNR was carried out using scanning confocal microspectrofluorometry. This technique allows determination of nuclear accumulation of anthracyclines. Our results show that nuclear accumulation of DNR in K-H30 and A549 cells was increased by NBD, BSO and VPL while in K-H300 cells, only VPL was able to increase nuclear accumulation of DNR. Similarly, NBD, BSO and VPL could reverse DNR resistance in K-H30 cells whereas, in K-H300 cells, only VPL increased the sensitivity of these cells. These data suggest a requirement for GSH in MRP-mediated resistance and suggest that even if vesicular sequestration can happen in cells overexpressing MRP and Pgp proteins, probably only the MRP protein is able to extrude the drug through intracellular vesicles and efflux. Finally, NBD and BSO might be a useful agents in facilitating discrimination between Pgp and MRP phenotypes and prognosis in patients.

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.