Acceleration of MRP-associated efflux of rhodamine 123 by genistein and related compounds.

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 (Rh123) 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 Rh123 vs daunorubicin transport in the MRP-mediated MDR cell lines: the accumulation of daunorubicin was increased, whereas the accumulation of Rh123 was decreased by the flavonoids. The depolarisation of the membrane potential caused by genistein might be involved in the acceleration of the efflux of Rh123 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.

Treatment of cancer cell lines with one of a group of natural cytotoxic drugs, such as the anthracyclines, vinca alkaloids and epipodophyllotoxins, frequently results in cross-resistance to the other drugs. In many of these multidrugresistant (MDR) cells, resistance is caused by reduced intracellular drug levels owing to the overexpression of plasma membrane drug transporters. Up to now, two different plasma membrane drug transporters have been shown to confer MDR in human tumour cell lines, namely P-glycoprotein (P-gp), encoded by the MDR-J gene (Gottesman and Pastan, 1993), and the multidrug resistance-associated protein, MRP (Cole et al., 1992). In addition to the cytotoxic drugs themselves, a number of fluorescent dyes are being used as probes in the study of transporter activity. One such probe, Rh 123, is very efficiently transported by P-gp, resulting in a larger accumulation deficit than that for doxorubicin and daunorubicin. The use of Rhl23 has, therefore, been suggested to be a useful approach for the determination of P-gp activity in human haemopoietic malignancies (Chaudhary and Roninson, 1993). Recently, we have shown that Rhl23 is a substrate not only for P-gp but also for MRP . Expression of both P-gp and MRP has been reported to occur in malignant haemopoietic cells (Schuurhuis et al., 1995). Therefore, transport of Rhl23 in such cells may be influenced both by MRP and by P-gp.
Recently, it has been shown that, in addition to the hydrophobic agents which are effluxed from both P-gp-and MRP-overexpressing cells, anions, such as leukotriene C4 and glutathione S-conjugates, are transported by MRP (Jedlitschky et al., 1994;Muller et al,. 1994). MRP has, therefore, been suggested to be the glutathione S-conjugate transporter present in a variety of normal cell types. Furthermore, glutathione depletion inhibits MRP-but not P-gp-mediated drug transport (Lutzky et al., 1989;Versantvoort et al., 1995). On the other hand, Pgp-MDR modifiers, such as verapamil, cyclosporin A and PSC833, are less effective in MRP-overexpressing cell lines (Barrand et al., 1993). Thus, methods to circumvent resistance show such an important difference between the two transporters.
We have shown previously that the efflux of daunorubicin  ., 1993). In contrast, the activity of P-gp appears to be up-regulated by several flavonoids (Critchfield et al., 1994). Therefore, we thought that genistein might be a useful agent in facilitating discrimination between P-gp-and MRP-mediated Rh123 transport. In this study, we have examined the modulation of Rhl23 transport by genistein and three other (iso)flavonoids in two MDR lung cancer cell lines that overexpress MRP. The study showed that the transport of Rhl23 and of daunorubicin in MRP-overexpressing MDR cell lines is affected differently by (iso)flavonoids.

Cells
In this study, the following human lung tumour cell lines were used: the large-cell lung cancer cell line COR-L23/P, the adenocarcinoma cell line MOR/P and the small-cell lung cancer cell line H69/P, together with their doxorubicinselected MDR variants COR-L23/R, MOR/R and H69/LX4 (Twentyman et al., 1986;Barrand et al., 1994). The MDR COR-L23/R and MOR-R cell lines overexpress the MRP but not the MDR-1 gene (Barrand et al., 1994). For comparison, the P-gp-overexpressing H69/LX4 cell line was used (Twentyman et al, 1986). Cell lines were cultured in RPMI-1640 medium supplemented with penicillin (100 U ml-1), streptomycin (100 U ml-') and 10% fetal bovine serum (all from Sigma). The resistant sublines were cultured in doxorubicin-containing medium until 2-7 days before experiments. Cellular drug transport Cells (0.1 x 106 per sample) were incubated with 0.5 gM [3H]daunorubicin or 0.1 Mg ml-' Rh123 for various time periods at 37°C as described previously (Versantvoort et al., 1995). The accumulation of drugs was then stopped by two ice-cold washes with PBS, and cellular drug content was determined by liquid scintillation counting (for daunorubicin) or by flow cytometry (for Rh123, excitation at 488 nm and emission above 630 nm). Values were corrected for amount of cell-associated drugs at time zero at 0°C. For determination of Rh123 efflux, cells were resuspended in drug-free medium in the presence or absence of modifier after loading for 60 min with 0.1 Mug ml-' Rh123.
Membrane potential The fluorescent probe, DiOC5, was used to measure the membrane potential. Cells were loaded with 0.1 gM DiOC5 for 15 min (steady state) in the presence or absence of 200 Mm genistein or 100 gM DIDS. Cells were then washed with PBS and the accumulation of DiOC5 was determined by flow cytometry with excitation at 488 nm and fluorescence emission measured above 530 nm.

Results
Effect offiavonoids on daunorubicin accumulation in MDR cells Since genistein was shown to inhibit the efflux of daunorubicin in several MRP-but not in P-gp-overexpressing MDR cells (Versantvoort et al., 1993), we first determined the effects of genistein and three other (iso)flavonoids on the daunorubicin accumulation in two MRP-overexpressing MDR cell lines, COR-L23/R and MOR/R [which do not overexpress P-gp (Barrand et al., 1994)], and in the P-gp-overexpressing MDR cell line, H69/LX4. Structures of the (iso)flavonoids are depicted in Figure 1. Genistein increased the daunorubicin accumulation in a concentration-dependent manner in the MRP-MDR COR-L23/R cell line with a maximal effect at 200-400 LM genistein (data not shown). For further experiments, 200 gM flavonoid was used, since this concentration could be obtained with < 0.5% DMSO. Figure 2 shows the effect of the flavonoids on the daunorubicin accumulation in MRP-and P-gp-MDR cell lines. All four (iso)flavonoids increased the daunorubicin accumulation in the MRP-MDR cell lines, with genistein being the most effective modulator. Only small effects of the flavonoids were seen in the parental cell lines. Genistein, quercetin and daidzein did not increase the daunorubicin accumulation in the P-gp-MDR H69/LX4 cell line, which is in accordance with our previous data for genistein in P-gp-MDR cell lines (Versantvoort et al., 1993). In contrast, genistin almost completely reversed the daunorubicin accumulation deficit in the H69/LX4 cells.

Effect offlavonoids on Rh 123 transport
We then examined the effects of genistein on the accumulation and efflux of Rhl23 in the COR-L23 cells. It can be seen from Figure 3a that genistein decreased the accumulation of Rh123 in the MRP-MDR COR-L23/R cell line. This is in contrast to the effects of genistein on the daunorubicin accumulation ( Figure 2). Since genistein had no effect on the accumulation of Rh123 in the parental COR-L23/P cells during this time period, it is unlikely that the decrease in Rh123 accumulation in the resistant cells is a result of a change in the passive transport of Rh123 by genistein.
Since the accumulation deficit of Rhl23 in the COR-L23/ R cells is caused by an enhanced Rh 123 efflux from the resistant cells , we measured the effect of genistein on the efflux of Rhl23. Figure 3b shows that genistein immediately accelerated the efflux of Rh123 from the resistant COR-L23/R cells. A similar efflux experiment was performed in MOR cells and genistein also accelerated the Rh123 efflux in the resistant cells of this line ( Figure 4). The effects on Rh123 efflux were apparent within 5 min of administration of genistein in the resistant cell lines, whereas genistein reduced the retention of Rh123 in the parental cell lines significantly at time points beyond 90 min. Semi-logarithmic plotting of the retention data revealed that the efflux of Rh123 followed first-order kinetics in the resistant cells. Genistein enhanced the efflux of Rhi23 3-to 5-fold in the resistant COR-L23/R and MOR/R cell lines, as well to some extent (<2-fold) in the parental MOR/P cells (Table I).
Next, we measured the concentration-dependent effect of genistein on Rh 123 retention. Figure 5 shows a gradual decrease in Rh 123 retention with increasing genistein concentrations in COR-L23/R cells with a maximal effect at 100-200 gM genistein. Only the highest genistein concentration had a significant effect in the COR-L23/P cells.
We then examined the effect of the other flavonoids on the retention of Rh 123 and compared the effects with those of the resistance modifiers, verapamil, cyclosporin A and buthionine sulphoximine (BSO), as well as the cytotoxic agent vinblastine. Results for the COR-L23/R cells are shown in Figure 6. Treatment with BSO was given for 20 h before Rh123 retention was determined; the other modulators were 9$ 1951 added only during the efflux period. It can be seen that all modifiers, as well as the cytotoxic agent vinblastine, inhibited the efflux of Rhl23 from the COR-L23/R cells. All the (iso)flavonoids tested decreased the retention of Rhl23 in COR-L23/R cells, although genistin was only slightly active. Quercetin decreased the retention in the parental COR-L23/P cells to some extent, although less than in the resistant cells.
Effect of membrane potential on Rh123 transport Since the enhancement of the Rhl23 efflux by the (iso)flavonoids is in contrast with the inhibition of the efflux of the cytotoxic agents, daunorubicin, doxorubicin and VP-16     Versantvoort et al., 1993), we considered the possibility that alterations in the accumulation of Rhl23 rather than stimulation of the activity of the drug transporter causes the accelerated efflux of Rhl23 by genistein. Because Rhl23 depends for its accumulation on the mitochondrial membrane potential, we compared the effects of sodium azide, which is known to disrupt the mitochondrial membrane potential, with the effects of genistein. Sodium azide concentrations were chosen such that cellular ATP levels were not depleted to such a degree as to influence the transport of drugs (Versantvoort et al., 1994). The effects of sodium azide on Rhl23 efflux are shown in Figure 7. It can be seen that 25 mm sodium azide accelerated the efflux of Rhl23 to a degree similar to the effect of genistein. This effect of sodium azide might suggest that the acceleration of the Rhl23 efflux by genistein is caused by alterations in the membrane potential. Therefore, we measured the membrane potential with the fluorescent probe DiOC5. The accumulation of DiOC5 was rapid (steady state was reached in 15 min) and similar in the parental and the resistant COR-L23 cell lines (not shown). We then examined the effects of genistein on the accumulation of DiOC5. As a comparison 100 jgM DIDS, which is known to depolarise the membrane potential, was included in the experiments. DIDS, as well as genistein, decreased the accumulation of DiOC5 to 60-72% of control in the COR-L23 cell lines. Depolarisation of the membrane potential by genistein was similar in parental and resistant cells, 66% and 60% respectively.

Discussion
The plasma membrane protein P-gp is well known for its prominent role as a drug efflux pump in the MDR phenotype. Overexpression of MRP in tumour cell lines involves cross-resistance to similar cytotoxic drugs, such as daunorubicin, doxorubicin, vincristine, colchicine and etoposide, owing to an enhanced efflux of the drugs out of the cells Grant et al., 1994). Absolute discrimination between P-gpand MRP-mediated resistance appears currently not to be achievable based on functional drug transport assays. However, the effects of various resistance modifiers vary considerably between the two types of MDR. Recently, we have shown that the isoflavonoid genistein and cellular glutathione depletion are potent inhibitors of MRP-but not P-gp-mediated daunorubicin transport (Versantvoort et al., 1993(Versantvoort et al., , 1995. Moreover, Phang et al. (1993) showed that P-gp-mediated efflux was accelerated by flavonoids. Since glutathione depletion by buthionine sulphoximine takes several hours (Versantvoort et al., 1995), genistein is potentially more useful in a functional assay to discriminate between P-gpand MRPmediated resistance.
In this study, the accumulation of daunorubicin was increased by genistein in the MRP-overexpressing MDR cell lines only (Figure 2), which is in accordance with our previous results (Versantvoort et al., 1993). Of note was the reversal of the accumulation deficit of daunorubicin in the Pgp-overexpressing H69/LX4 cell line by genistin (Figure 2), since none of the (iso)flavonoids tested by Critchfield et al. (1994) was able to increase the accumulation of doxorubicin in the P-gp-expressing HCT-15 colon cells efficiently. The fact that genistin and genistein differ only by a glucose unit might have important implications, since many of the flavonoids found in fruits and vegetables are present as conjugates/ glycosides (Hermann, 1976).
Furthermore, we were surprised by our finding that the efflux of Rhl23 in the MRP-MDR cells was accelerated by genistein and the other (iso)flavonoids. This is in marked contrast to our previous results for daunorubicin, doxorubicin and VP-16 (Versantvoort et al., 1993), indicating that the interaction between genistein and Rh123 is clearly different from that involving the cytotoxic drugs. We have shown in the GLC4/ADR MRP-MDR cells that genistein is a competitive inhibitor of the daunorubicin efflux, indicating an interaction of genistein at the drug-binding site (Versantvoort et al., 1994). The different effects of genistein might suggest that the drug-binding site at the transporter is different for daunorubicin and Rh123. Since other modifiers affect Rh123 transport in a similar way to the effects previously found for daunorubicin and vincristine transport (Barrand et al., 1993), other mechanisms might evoke the acceleration of Rh123 transport in MRP-MDR cells.
An alternative mode of interaction between genistein and Rh123 was suggested by the observation that sodium azide, which lowers the mitochondrial membrane potential, was able to stimulate the efflux of Rh123 (Figure 7). The depolarisation of the membrane potential caused by genistein is then likely to affect the transport of Rh123. The different effects of genistein on the transport of Rh123 and daunorubicin in MRP-overexpressing MDR cells can be explained by the fact that Rh123, but not daunorubicin, is depending for its accumulation on the membrane potential. However, if depolarisation of the membrane potential rather than stimulation of MRP activity causes the alterations in Rh123 accumulation, it is then necessary to account for the different effects of genistein in parental and resistant COR-L23 cells, as depolarisation of the membrane potential by genistein was similar in the parental and resistant cells. The answer might be found in the different kinetics of Rhl23 in the parental and resistant cells. Transport of Rhl23 over the plasma membrane is determined by passive diffusion in the COR-L23/P cells and by a passive and active component in the COR-L23/R cells. Depolarisation of the membrane potential will affect passive as well as active transport of RhI23. Since the efflux of Rh123 is 6to 10-fold faster from the resistant cells (active and passive transport) than from the parental cells (passive transport), the effects caused by depolarisation of the membrane potential will be apparent much faster in the resistant cells. As shown in Figures 3 and   4, the effects of genistein on Rh123 efflux are apparent in the resistant cells within 5 min of administration, whereas the effects on the parental cells were only significant at 120 min efflux or longer (data not shown).
It may be concluded from our results that the mechanism(s) by which flavonoids interact with drug transport is rather complex; not only was the daunorubicin transport mediated by P-gp and MRP affected differently by the flavonoids, but genistein and genistin had opposite effects on the daunorubicin transport mediated by P-gp, and the flavonoids had opposite effects on the transport of daunorubicin and Rh123 in MRP-overexpressing cells. These results show that the use of a more sensitive and/or cheaper probe (in this case Rh123) instead of the cytotoxic agent itself for selection of the most efficient resistance modifier must be regarded with caution, since the effects of modifiers on the probe may not predict for effects on cytotoxic agents.