S_nary Previous studies have shown that multidrug resistance (MDR) in the doxorubicin-selected lung tumour cel lines COR-L23/R, GLC4 'ADR and MOR/R is associated with overexpression of the MRP gene.In this study we report that resistance to daunorubicin, vincristine and rhodamine 123 can be partially reversed in these cell lines by exposing the cells to buthionine sulphoximine (BSO), an inhibitor of glutathione (GSH) synthesis. This effect of BSO on drug resistance was associated with an increased intracellular accumulation of daunorubicin and rhodamine 123, owing to inhibition of the enhanced drug efflux. In contrast, the accumulation of daunorubicin was not increased by BSO treatment in a P-glycoprotein (P-gp)-mediated MDR cell line. BSO treatment (25 FM, 20 h) of the cell lines resulted in 60-80% depletion of cellular GSH levels. The effects of BSO on daunorubicin accumulation in the COR-L23/R and GLC4/ADR cells were associated with cellular GSH depletion. In addition, increase of cellular GSH levels in BSO-treated COR-L23/R and GLC4/ADR cells as a result of incubation with 5 mM GSH ethyl ester restored the accumulation deficit of daunorubicin. However, the transport of daunorubicin did not increase the GSH release in any of the cell lines. These results demonstrate that drug transport in MRP-but not in P-gp-overexpressing MDR tumour cell lines can be regulated by intracellular GSH levels.

In tumour cells the pharmacological basis for multidrug resistance (MDR) often appears to be a reduced cellular cytostatic drug accumulation caused by the drug efflux protein, P-glycoprotein (Pgp MDR), or by other drug transporters (non-Pgp MDR). Here we report the reversal of the decreased daunorubicin (DNR) accumulation in five non-Pgp MDR cell lines (GLC4/ADR, SW-1573/2R120, HT1080/DR4, MCF7/Mitox and HL60/ADR) by genistein. Genistein inhibited the enhanced DNR efflux in the GLC4/ADR cells. In these cells the decreased VP-16 accumulation was also reversed by genistein. Three other (iso)flavonoids biochanin A, apigenin and quercetin also increased the DNR accumulation in the GLC4/ADR cells. In contrast to the effects on non-Pgp MDR cells, 200 microM genistein did not increase the reduced DNR accumulation in three Pgp MDR cell lines (SW-1573/2R160, MCF7/DOX40 and KB8-5) or in the parental cell lines. In conclusion the use of genistein provides a means to probe non-Pgp related drug accumulation defects. Images Figure 1 Figure 6

Previous studies have shown that the in vitro-selected adriamycin-resistant human small-cell lung-carcinoma cell line GLC4-ADR150 displays multidrug resistance as the result of 3-fold decreased DNA-topoisomerase II (topo II) activity and a 6-fold reduction in adriamycin accumulation. Not the MDR1 gene, but the MRP gene, was over-expressed in this cell line. The aim of our study was to establish which of these drug-resistance-associated factors are already involved in drug resistance occurring at early steps of selection with adriamycin. To address this question, changes in expression of topo II alpha/topo II beta, MRP and drug accumulation were measured along with adriamycin resistance (from 2- to 10- to 150-fold) and in a partial revertant cell line (10-fold resistant). Topo II alpha and II beta mRNA and protein levels were decreased in the resistant sub-lines, except in the 10-fold-resistant cell line. Cellular daunorubicin accumulation was decreased 1.2- to 5-fold with increasing resistance. MRP mRNA was over-expressed in all resistant sub-lines, with a marked increase in the 10-fold-resistant cells (level of expression as high as in the GLC4-ADR150 cells). Expression of an ATP-binding 190-kDa membrane protein and Western-blot analysis with anti-MRP anti-serum ASPKE, was in accordance with the expression of MRP mRNA in all cell lines. Expression of MRP mRNA and protein, however, was not proportional with the decrease in drug accumulation in all resistant sub-lines. This study also shows that drug accumulation, topo II and MRP expression were all changed at the earliest stage of resistance development of GLC4 cells upon adriamycin selection.

Cells exposed to calcein acetoxymethyl ester (calcein AM) in the growth medium become fluorescent following cleavage of calcein AM by cellular esterases to produce the fluorescent derivative calcein. It has previously been shown by others that multidrug resistant cells which overexpress P-glycoprotein accumulate much less fluorescent calcein than the corresponding parental cells. We have now examined the transport of calcein in multidrug resistant cells which overexpress an alternative transporter, the multidrug resistance-associated protein (MRP). Accumulation of calcein fluorescence was greatly reduced in the MRP-overexpressing human lung cancer cell lines COR-L23/R and MOR/R compared with their parental lines. Energy depletion resulted in a considerably increased accumulation in the resistant lines. Treatment of resistant cells with buthionine sulfoximine (BSO), which depletes cellular glutathione (GSH), did not affect calcein accumulation, in marked contrast to our previous results for daunorubicin or the fluorescent probe rhodamine 123. Genistein, verapamil, cyclosporin A and ouabain were also each able to modify, to some extent, accumulation of daunorubicin, whilst having essentially no effect on calcein accumulation. However, the organic anion transport inhibitor probenecid was able to increase accumulation of both calcein and daunorubicin in the resistant cells. Genistein and verapamil treatment preferentially reduced the GSH content of resistant cells, whilst probenecid did not. However, probenecid caused a clear decrease in release of GSH from resistant cells into the medium.

Summary Multidrug resistance (MDR), caused by overexpression of either P-glycoprotein or the multidrug resistance protein (MRP), is characterised by a decreased cellular drug accumulation due to an enhanced drug efflux. In this study, we examined the effects of genistein and structurally related (iso)flavonoids on the transport of rhodamine 123 (Rhl23) and daunorubicin in the MRP-overexpressing MDR lung cancer cell lines COR-L23/R and MOR/R. Genistein, genistin, daidzein and quercetin showed major differences in effects on Rhl23 vs daunorubicin transport in the MRP-mediated MDR cell lines: the accumulation of daunorubicin was increased, whereas the accumulation of Rhl23 was decreased by the flavonoids. The depolarisation of the membrane potential caused by genistein might be involved in the acceleration of the efflux of Rhl23 measured in the MRP-overexpressing cell lines. These observations should be taken into account when using fluorescent dyes as probes for determination of transporter activity as a measure of MDR.

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Multidrug resistance (MDR) in tumor cells is frequently associated with reduced cellular cytostatic drug accumulation, caused by the drug efflux protein, P-glycoprotein (Pgp). The action of Pgp in tumor cells can be detected by measuring the increase of daunorubicin accumulation upon blocking Pgp with drugs such as verapamil. A number of MDR cell lines have been described, characterized by decreased drug accumulation without Pgp being present. For such non-Pgp MDR cells no gene probes or functional assays are available to study this phenotype in clinical tumor specimens. We have worked out a method which enables the detection of drug-transport-related decreases in cellular daunorubicin accumulations without the need for the use of specific Pgp blockers. The cells used were SW-1573-, GLC4- and HT1080-sensitive cell lines, which accumulated (corrected for DNA content) 272%, 1,288% and 203% more daunorubicin than the non-Pgp MDR sublines SW-1573/2R120, GLC4/ADR and HT1080/DR4. When the plasma membranes of these MDR lines were permeabilized with 20 microM digitonin an increase to 282%, 1,260% and 239% of 14C-daunorubicin control accumulation was measured (at pH = 7.35). The intracellular pH measured with BCECF was the same in parent and corresponding MDR cells, excluding the role of pH differences in the measured effects. This method provides a tool allowing the detection of cellular mechanisms (including Pgp) which are related to active outward transport of daunorubicin.