Inhibition of the P-glycoprotein- and multidrug resistance protein-mediated efflux of anthracyclines and calceinacetoxymethyl ester by PAK-104P

Multidrug resistance phenotype in mammalian cells is often correlated with overexpression of P-glycoprotein or Multidrug Resistance-Associated protein (MRP(1)). Both proteins are energy-dependent drug efflux pumps that efficiently reduce the intracellular accumulation and hence the cytotoxicity of many natural cytotoxins. Overexpression of these transporters by tumor cells is thought to be a significant factor in both intrinsic and acquired resistance to anticancer drugs. Consequently a great deal of interest is focused on identifying chemical agents that can either antagonise drug transport by these proteins or that can inhibit the proliferation of tumors cells despite the expression of these transporters. P-glycoprotein-mediated multidrug resistance is reversed by a variety of compounds, but surprisingly, few agents reverse the MRP(1)-mediated multidrug resistance. However, it has recently been shown that 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl-5-(trans-4,6-dimethyl-1, 3, 2-dioxaphosphorinan-2-yl)-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P oxide (PAK-104P) was able to inhibit the P-glycoprotein and MRP(1)-mediated efflux of several compounds. Understanding of the interactions between transporters and multidrug resistance reversing agents is important in the design of more effective multidrug resistance modulators. We now examined the effect of PAK-104P on Pgp-and MRP1-mediated efflux of three anthracyclines, daunorubicin, pirarubicin, hydroxydoxorubicin and of calcein acetoxymethyl ester and calcein. Our data show that PAK-104P non-competitively inhibits the P-glycoprotein-mediated efflux of anthracycline derivatives and calcein acetoxymethyl ester with an inhibitory constant K(I)=0. 25+/-0.05 microM. PAK-104P also non-competitively inhibits the MRP(1)-mediated efflux of daunorubicin, pirarubicin, hydroxyrubicin, calcein acetoxymethyl ester and calcein. However, surprisingly, in this case the K(I) values obtained were very different ranging from 0.06 for hydroxyrubicin to 10 microM for calcein. These data strongly suggested the existence of two different mechanisms for the inhibition by PAK-104P of the MRP(1)-mediated efflux of molecules: a first mechanism, involving a low-affinity site for PAK-104P, and which would concern molecules such as calcein, cysteinyl leukotriene LCT(4) etc. whose efflux do not depend on glutathione. A second mechanism involving a high-affinity site for PAK-104P and which would concern molecules such as anthracyclines, calcein acetoxymethyl ester whose efflux depends on the presence of glutathione.     

Kinetic analysis of fluorescein and dihydrofluorescein effluxes in tumour cells expressing the multidrug resistance protein,MRP1

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of two transporters the P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP1) which actively pump out multiple chemically unrelated substrates across the plasma membrane. A clear distinction in the mechanism of translocation of substrates by MRP1 or P-gp is indicated by the finding that, in most of cases, the MRP1-mediated transport of substrates is inhibited by depletion of intracellular glutathione (GSH), which has no effect on their P-gp-mediated transport. The aim of the present study was to quantitatively characterise the transport of anionic compounds dihydrofluorescein and fluorescein (FLU). We took advantage of the intrinsic fluorescence of FLU and performed a flow cytometric analysis of dye accumulation in the wild-type drug sensitive GLC4 that do not express MRP1 and its MDR subline which display high level of MRP1. The measurements were made in real time using intact cells. The kinetics parameters, k(a)=V(M)/K(m), which is a measure of the efficiency of the transporter-mediated efflux of a substrate, was very similar for the two FLU analogues. They were highly comparable with values for k(a) of other negatively charged substrates, such as GSH and calcein. The active efflux of both FLU derivatives was inhibited by GSH depletion.     

Collateral sensitivity of resistant MRP1-overexpressing cells to flavonoids and derivatives through GSH efflux

The multidrug resistance protein 1 (MRP1) is involved in multidrug resistance of cancer cells by mediating drug efflux out of cells, often in co-transport with glutathione (GSH). GSH efflux mediated by MRP1 can be stimulated by verapamil. In cells overexpressing MRP1, we have previously shown that verapamil induced a huge intracellular GSH depletion which triggered apoptosis of the cells. That phenomenon takes place in the more global anticancer strategy called “collateral sensitivity” and could be exploited to eradicate some chemoresistant cancer cells. Seeking alternative compounds to verapamil, we screened a library of natural flavonoids and synthetic derivatives. A large number of these compounds stimulate MRP1-mediated GSH efflux and the most active ones have been evaluated for their cytotoxic effect on MRP1-overexpressing cells versus parental cells. Interestingly, some are highly and selectively cytotoxic for MRP1-cells, leading them to apoptosis. However, some others do not exhibit any cytotoxicity while promoting a strong GSH efflux, indicating that GSH efflux is necessary but not sufficient for MRP1-cells apoptosis. In support to this hypothesis, structure activity relationships show that the absence of a hydroxyl group at position 3 of the flavonoid C ring is an absolute requirement for induction of MRP1-cells death, but is not for GSH efflux stimulation. Chrysin (compound 8) and its derivatives, compounds 11 and 22, exhibit a high selectivity toward MRP1-cells with a IC₅₀ value of 4.1 μM for compound 11 and 4.9 μM for chrysin and compound 22, making them among the best described selective killer compounds of multidrug ABC transporter-overexpressing cells.     

MRP1 mutated in the L0 region transports SN-38 but not leukotriene C4 or estradiol-17 (beta-D-glucuronate)

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that confers multidrug resistance on tumor cells. Much convincing evidence has accumulated that MRP1 transports most substances in a GSH-dependent manner. On the other hand, several reports have revealed that MRP1 can transport some substrates independently of GSH; however, the importance of GSH-independent transport activity is not well established and the mechanistic differences between GSH-dependent and -independent transport by MRP1 are unclear. We previously demonstrated that the amino acids W261 and K267 in the L0 region of MRP1 were important for leukotriene C4 (LTC4) transport activity of MRP1 and for GSH-dependent photolabeling of MRP1 with azidophenyl agosterol-A (azidoAG-A). In this paper, we further tested the effect of W222L, W223L and R230A mutations in MRP1, designated dmL0MRP1, on MRP1 transport activity. SN-38 is an active metabolic form of CPT-11 that is one of the most promising anti-cancer drugs. Membrane vesicles prepared from cells expressing dmL0MRP1 could transport SN-38, but not LTC4 or estradiol-17 (beta-D-glucuronate), and could not be photolabeled with azidoAG-A. These data suggested that SN-38 was transported by a different mechanism than that of GSH-dependent transport. Understanding the GSH-independent transport mechanism of MRP1, and identification of drugs that are transported by this mechanism, will be critical for combating MRP1-mediated drug resistance. We performed a pairwise comparison of compounds that are transported by MRP1 in a GSH-dependent or -independent manner. These data indicated that it may be possible to predict compounds that are transported by MRP1 in a GSH-independent manner.     

Modulation of GSH levels in ABCC1 expressing tumor cells triggers apoptosis through oxidative stress

The over-expression of ABCC1 transmembrane protein has been shown to cause multidrug resistance in tumor cell lines. ABCC1 is a member of the ABC transmembrane proteins that function as efflux pumps with diverse substrate specificity. Several endogenous cell metabolites, including the leukotriene C4 (LTC(4)) and glutathione (GSH) are substrates for ABCC1 protein. ABCC1 expression in certain tumor cells was demonstrated to confer hypersensitivity to glutathione modulating agents. In this report we have investigated the mechanism of collateral sensitivity seen in tumor cells over-expressing ABCC1 protein. The results of this study show that ABCC1 expression in tumor cells correlates with their hypersensitivity to various glutathione modulating agents, as demonstrated in H69AR-drug selected and HeLa/ABCC1-transfectant cells. This effect was triggered either through inhibition of GSH synthesis with BSO or by increasing ABCC1-mediated GSH transport with verapamil or apigenin. In addition, our results show that the hypersensitivity of ABCC1-expressing cells to BSO, verapamil or apigenin was preceded by an increase in reactive oxygen species (or ROS). A decrease in GSH level is also observed prior the increase in ROS. In addition, we show that hypersensitivity to the BSO, verapamil or apigenin leads to tumor cell death by apoptosis. Together, the results of this study demonstrate that ABCC1 potentiates oxidative stress in tumor cells through reductions in cellular GSH levels.     

Interactions of mefloquine with ABC proteins, MRP1 (ABCC1) and MRP4 (ABCC4) that are present in human red cell membranes

Human erythrocyte membranes express the multidrug resistance-associated proteins, MRP1, MRP4 and 5, that collectively can efflux oxidised glutathione, glutathione conjugates and cyclic nucleotides. It is already known that the quinoline derivative, MK-571, is a potent inhibitor of MRP-mediated transport. We here examine whether the quinoline-based antimalarial drugs, amodiaquine, chloroquine, mefloquine, primaquine, quinidine and quinine, also interact with erythrocyte MRPs with consequences for their access to the intracellular parasites or for efflux of oxidised glutathione from infected cells. Using inside-out vesicles prepared from human erythrocytes we have shown that mefloquine and MK-571 inhibit transport of 3 microM [(3)H]DNP-SG known to be mediated by MRP1 (IC(50) 127 and 1.1 microM, respectively) and of 3.3 microM [(3)H]cGMP thought but not proven to be mediated primarily by MRP4 (IC(50) 21 and 0.41 microM). They also inhibited transport in membrane vesicles prepared from tumour cells expressing MRP1 or MRP4 and blocked calcein efflux from MRP1-overexpressing cells and BCECF efflux from MRP4-overexpressing cells. Both stimulated ATPase activity in membranes prepared from MRP1 and MRP4-overexpressing cells and inhibited activity stimulated by quercetin or PGE(1), respectively. Neither inhibited [alpha-(32)P]8-azidoATP binding confirming that the interactions are not at the ATP binding site. These results demonstrate that mefloquine and MK-571 both inhibit transport of other substrates and stimulate ATPase activity and thus may themselves be substrates for transport. But at concentrations achieved clinically mefloquine is unlikely to affect the MRP1-mediated transport of GSSG across the erythrocyte membrane.     

The accumulation and efflux of lead partly depend on ATP-dependent efflux pump–multidrug resistance protein 1 and glutathione in testis Sertoli cells

Since lead accumulation is toxic to cells, its excretion is crucial for organisms to survive the toxicity. In this study, mouse testis sertoli (TM4) and Mrp1 lower-expression TM4-sh cells were used to explore the lead accumulation characteristics, and the role of ATP-dependent efflux pump–multidrug resistance protein 1 (Mrp1) in lead excretion. TM4 cells possess Mrp-like transport activity. The expression levels of mrp1 mRNA and Mrp1 increased after lead treatments at first and then decreased. The maximum difference of relative mRNA expression reached 10 times. In the presence of lead acetate, the amount of cumulative lead in TM4-sh was much higher than that in TM4. After the treatment with lead acetate at 10–40 μM for 12 h or 24 h, the differences were about 2–8 times. After with the switch to lead-free medium, the cellular lead content in TM4-sh remains higher than that in TM4 cells at 1,3, 6, and 9 h time points (P < 0.01). Energy inhibitor sodium azide, Mrp inhibitors MK571 and glutathione (GSH) biosynthesis inhibitor BSO could block lead efflux from TM4 cells significantly. These results indicate that lead excretion may be mediated by Mrp1 and GSH in TM4 cells. Mrp1 could be one of the important intervention points for lead detoxification.     

Cepharanthine is a potent reversal agent for MRP7(ABCC10)-mediated multidrug resistance

Multidrug resistance protein 7 (MRP7; ABCC10) is an ABC transporter that confers resistance to anticancer agents such as the taxanes. We previously reported that several inhibitors of P-gp and MRP1 were able to inhibit the in vitro transport of E₂17βG by MRP7 in membrane vesicles transport assays. However, compounds that are able to reverse MRP7-mediated cellular resistance have not been identified. In this study, we examined the effects of cepharanthine (6′,12′-dimethoxy-2,2′-dimethyl-6,7-[methylenebis(oxy)]oxyacanthan), an herbal extract isolated from Stephania cepharantha Hayata, to reverse paclitaxel resistance in MRP7-transfected HEK293 cells. Cepharanthine, at 2 μM, completely reversed paclitaxel resistance in MRP7-transfected cells. In contrast, the effect of cepharanthine on the parental transfected cells was significantly less than that on the MRP7-transfected cells. In addition, cepharanthine significantly increased the accumulation of paclitaxel in MRP7-transfected cells almost to the level of control cells in the absence of cepharanthine. The efflux of paclitaxel from MRP7-transfected cells was also significantly inhibited by cepharanthine. The ability of cepharanthine to inhibit MRP7 was analyzed in membrane vesicle assays using E₂17βG, an established substrate of MRP7, as a probe. E₂17βG transport was competitively inhibited by cepharanthine with a K_i value of 4.86 μM. These findings indicate that cepharanthine reverses MRP7-mediated resistance to paclitaxel in a competitive manner.     

ATP-Dependent efflux of CPT-11 and SN-38 by the multidrug resistance protein (MRP) and its inhibition by PAK-104P

Non-P-glycoprotein-mediated multidrug-resistant C-A120 cells that overexpressed multidrug resistance protein (MRP) were 10.8- and 29. 6-fold more resistant to 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) and SN-38, respectively, than parental KB-3-1 cells. To see whether MRP is involved in CPT-11 and SN-38 resistance, MRP cDNA was transfected into KB-3-1 cells. The transfectant, KB/MRP, which overexpressed MRP, was resistant to both CPT-11 and SN-38. 2-[4-Diphenylmethyl)-1-piperazinyl]ethyl-5-(trans-4,6-dimethyl-1,3 , 2-dioxaphosphorinan-2-yl)-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P) and MK571, which reversed drug resistance in MRP overexpressing multidrug-resistant cells, significantly increased the sensitivity of C-A120 and KB/MRP cells, but not of KB-3-1 cells, to CPT-11 and SN-38. The accumulation of both CPT-11 and SN-38 in C-A120 and KB/MRP cells was lower than that in KB-3-1 cells. The treatment with 10 microM PAK-104P increased the accumulation of CPT-11 and SN-38 in C-A120 and KB/MRP cells to a level similar to that found in KB-3-1 cells. The ATP-dependent efflux of CPT-11 and SN-38 from C-A120 and KB/MRP cells was inhibited by PAK-104P. DNA topoisomerase I expression, activity, and sensitivity to SN-38 were similar in the three cell lines. Furthermore, the conversion of CPT-11 to SN-38 in KB-3-1 and C-A120 cell lines was similar. These findings suggest that MRP transports CPT-11 and SN-38 and is involved in resistance to CPT-11 and SN-38 and that PAK-104P reverses the resistance to CPT-11 and SN-38 in tumors that overexpress MRP.     

Mechanisms of glutathione disulfide efflux from erythrocytes

Glutathione (GSH) plays numerous critical protective roles in the erythrocyte and GSH turnover is likely an important factor in regulating susceptibility to oxidative stress and toxins. Efflux of glutathione disulfide (GSSG) from erythrocytes is an important component in the regulation of GSH levels; however, little is known of the mechanisms involved. We hypothesize that multidrug resistance associated protein 1 (MRP1) is responsible, in part, for GSSG transport from erythrocytes. To test this, we determined the levels of MRP1 protein in erythrocyte membranes from healthy adults and compared them with intracellular levels of GSH. MRP1 levels varied substantially from person to person and were inversely correlated with levels of GSH (r = -0.39, P < 0.05). In contrast, activity levels of glutamyl cysteine ligase, the rate limiting GSH biosynthetic enzyme, were unrelated to GSH levels. To directly determine the role of MRP1 in GSSG transport, in vitro studies were conducted examining the effects of MRP1 inhibitors MK571 and verapamil on GSSG efflux. Both compounds resulted in significant but not complete inhibition (20-53%) of GSSG efflux from cells. Overall, these findings support a role for MPR1 in the regulation of erythrocyte GSH levels through the transport and elimination of GSSG from cells.     

Folate concentration dependent transport activity of the Multidrug Resistance Protein 1 (ABCC1)

The Multidrug Resistance Protein MRP1 (ABCC1) can confer resistance to a variety of therapeutic drugs. In addition, MRP1/ABCC1 mediates cellular export of natural folates, such as folic acid and l-leucovorin. In this study we determined whether cellular folate status affected the functional activity of MRP1/ABCC1 mediated efflux of an established substrate, the anthracycline daunorubicin (DNR). As a model system we used the human ovarian carcinoma cell line 2008wt, and its MRP1/ABCC1 transfected subline 2008/MRP1. Both types of these moderate- and high-MRP1/ABCC1 expressing cells displayed efflux of DNR when maintained in standard culture media (2.3microM folic acid). The initial total cellular DNR efflux rate in 2008/MRP1 cells was approximately 2-fold higher compared to 2008wt cells. This efflux consisted of MRP1/ABCC1 mediated transport, possibly non-MRP1 mediated transport, as well as passive diffusion. Benzbromarone, a specific MRP1 inhibitor, decreased the initial efflux rate in 2008/MRP1 cells (4-fold) and in 2008wt cells (2-fold). When 2008/MRP1 cells were challenged for 2 days in folate-free medium, total cellular DNR efflux was decreased to 43% of the initial efflux rate under folate-rich conditions. In 2008wt cells DNR efflux was decreased to 84% of the folate-rich conditions. Benzbromarone did not inhibit DNR efflux after the folate-free period in both cell lines. Repletion of folate by a 2-24hr exposure to 2.5microM l-leucovorin or folic acid resulted in a complete restoration of DNR efflux. In contrast, expression of MRP1/ABCC1 protein was not changed significantly during the folate-free period or the repletion-period, nor were cellular ATP or ADP pools. In conclusion, this study demonstrates that the cellular folate status can influence the transport activity of MRP1/ABCC1. These results have potentially important implications in the understanding of the (patho-)physiological roles of MRP1/ABCC1, and possibly other ABC transporter proteins in cellular folate homeostasis and drug resistance.     

Multidrug resistance protein (MRP) 4 attenuates benzo[a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells

Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (−)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2′-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (−)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (−)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen.     

Evaluation of the binding of the tricyclic isoxazole photoaffinity label LY475776 to multidrug resistance associated protein 1 (MRP1) orthologs and several ATP- binding cassette (ABC) drug transporters

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB(100) cells to both vincristine and doxorubicin. LY475776 photolabeled 170kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C(4), vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.     

Non-destructive micromethod for MRP1 functional assay in human lung tumor cells

Defense against toxic endo- and xenobiotics is a major concern of all living species and ABC transporters play a vital role in this defense system. Multidrug resistance associated proteins 1 (MRP1) is a cellular detoxifying factor supposed to transport a wide range of compounds across cell membranes either as GSH conjugates or as co-transport accompanying glutathione transposition. The cellular localization of MRP1 is a determining factor whether the transport function can take place. In this study we have undertaken experiments on the transport activity of MRP1 in cultured human lung tumor cells in order to check whether MRP1 is expressed as a functionally active protein. For this purpose we have adapted a quantitative fluorescence imaging assay to conditions where a small number of attached cells should be repeatedly measured by a non-destructive method. In cultured A549, H358 and H322 cells MRP1 is located in the cell membrane as observed by immunocytochemistry. Efflux of 5,6-carboxy-2′-7′-dichloro-fluorescein (CDF) from lung cells was sensitive toward the MRP1 inhibitor MK571 while verapamil had no effect. On the other hand, efflux of Rhodamin 123, a Pgp-glycoprotein substrate, from lung cells reacted to inhibition by verapamil, while MK571 had no effect. Modulation of glutathion content of lung cells by N-acetyl cystein and buthionine sulfoximine shifted CDF efflux toward higher or lower rates, respectively. These experiments confirm that MRP1 function can be followed in the attached cells in vitro under non-toxic concentrations of the substrates without the need to harvest and destroy the cells. Abdelrahman Torky and Ekkehard Stehfest have contributed equally to this study     

Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

We hypothesized that flavonoid-induced glutathione (GSH) efflux through multi-drug resistance proteins (MRPs) and subsequent intracellular GSH depletion is a viable mechanism to sensitize cancer cells to chemotherapies. This concept was demonstrated using chrysin (5-25 μM) induced GSH efflux in human non-small cell lung cancer lines exposed to the chemotherapeutic agent, doxorubicin (DOX). Treatment with chrysin resulted in significant and sustained intracellular GSH depletion and the GSH enzyme network in the four cancer cell types was predictive of the severity of chrysin induced intracellular GSH depletion. Gene expression data indicated a positive correlation between basal MRP1, MRP3 and MRP5 expression and total GSH efflux before and after chrysin exposure. Co-treating the cells for 72 h with chrysin (5-30 μM) and DOX (0.025-3.0 μM) significantly enhanced the sensitivity of the cells to DOX as compared to 72-hour DOX alone treatment in all four cell lines. The maximum decrease in the IC₅₀ values of cells treated with DOX alone compared to co-treatment with chrysin and DOX was 43% in A549 cells, 47% in H157 and H1975 cells and 78% in H460 cells. Chrysin worked synergistically with DOX to induce cancer cell death. This approach could allow for use of lower concentrations and/or sensitize cancer cells to drugs that are typically resistant to therapy.     

Lapatinib and erlotinib are potent reversal agents for MRP7 (ABCC10)-mediated multidrug resistance

In recent years, a number of TKIs (tyrosine kinase inhibitors) targeting epidermal growth factor receptor (EGFR) family have been synthesized and some have been approved for clinical treatment of cancer by the FDA. We recently reported a new pharmacological action of the 4-anilinoquinazoline derived EGFR TKIs, such as lapatinib (Tykerb^®) and erlotinib (Tarceva^®), which significantly affect the drug resistance patterns in cells expressing the multidrug resistance (MDR) phenotype. Previously, we showed that lapatinib and erlotinib could inhibit the drug efflux function of P-glycoprotein (P-gp, ABCB1) and ABCG2 transporters. In this study, we determined if these TKIs have the potential to reverse MDR due to the presence of the multidrug resistance protein 7 (MRP7, ABCC10). Our results showed that lapatinib and erlotinib dose-dependently enhanced the sensitivity of MRP7-transfected HEK293 cells to several established MRP7 substrates, specifically docetaxel, paclitaxel, vinblastine and vinorelbine, whereas there was no or a less effect on the control vector transfected HEK293 cells. [³H]-paclitaxel accumulation and efflux studies demonstrated that lapatinib and erlotinib increased the intracellular accumulation of [³H]-paclitaxel and inhibited the efflux of [³H]-paclitaxel from MRP7-transfected cells but not in the control cell line. Lapatinib is a more potent inhibitor of MRP7 than erlotinib. In addition, the Western blot analysis revealed that both lapatinib and erlotinib did not significantly affect MRP7 expression. We conclude that the EGFR TKIs, lapatinib and erlotinib reverse MRP7-mediated MDR through inhibition of the drug efflux function, suggesting that an EGFR TKI based combinational therapy may be applicable for chemotherapeutic practice clinically.     

Transport of dietary phenethyl isothiocyanate is mediated by multidrug resistance protein 2 but not P-glycoprotein

We demonstrated recently that phenethyl isothiocyanate (PEITC), a potent anticarcinogen present in cruciferous vegetables, inhibited P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) and that MRP1 can transport PEITC and/or its metabolites. In this study, we have examined whether PEITC is transported by P-gp and MRP2, two transporters with high expression in human intestine, liver and kidney. Using (14)C-PEITC, no significant difference was observed for the intracellular accumulation of PEITC in human breast cancer MCF-7/sensitive (control) and MCF-7/ADR (P-gp overexpressing) cells at PEITC concentrations of 1, 10 and 50 microM. Moreover, the presence of verapamil or PSC833, two P-gp inhibitors, had no significant effect on the intracellular accumulation of PEITC in P-gp overexpressing MCF-7/ADR and MDA435/LCC6MDR1 cells, indicating that PEITC may not be a substrate for P-gp. In contrast, (14)C-PEITC intracellular accumulation in the kidney epithelial MDCK II/MRP2 cells (transfected with human MRP2) was significantly lower than in the wild-type MDCK II/wt cells at PEITC concentrations of 1, 5, 10 and 50 microM. The presence of MK571, an MRP inhibitor, significantly enhanced (14)C-PEITC accumulation in MDCK II/MRP2 but not MDCK II/wt cells. Furthermore, depletion of intracellular glutathione (GSH) following treatment with buthionine sulphoximine, an inhibitor of GSH biosynthesis, significantly increased (14)C-PEITC intracellular accumulation in a concentration-dependent manner. Transcellular transport studies also demonstrated that depletion of intracellular GSH reduced the mean ratio of basal-to-apical transport to apical-to-basal transport of PEITC in MDCK II/MRP2, but not MDCK II/wt cell monolayers. These results indicate that GSH plays an important role in the MRP2-mediated transport of PEITC. The findings provide new information concerning the interactions between PEITC and membrane transporters and suggest the possibility of PEITC interactions with xenobiotics that are MRP2 substrates.     

Structural requirements for the flavonoid-mediated modulation of glutathione S-transferase P1-1 and GS-X pump activity in MCF7 breast cancer cells

The objective of this study was to investigate the structural requirements necessary for inhibition of glutathione S-transferase P1-1 (GSTP1-1) and GS-X pump (MRP1 and MRP2) activity by structurally related flavonoids, in GSTP1-1 transfected MCF7 cells (pMTG5). The results reveal that GSTP1-1 activity in MCF7 pMTG5 cells can be inhibited by some flavonoids. Especially galangin was able to inhibit almost all cellular GSTP1-1 activity upon exposure of the cells to a concentration of 25microM. Other flavonoids like kaempferol, eriodictyol and quercetin showed a moderate GSTP1-1 inhibitory potential. For GSTP1-1 inhibition, no specific structural requirements necessary for potent inhibition could be defined. Most flavonoids appeared to be potent GS-X transport inhibitors with IC(50) values ranging between 0.8 and 8microM. Luteolin and quercetin were the strongest inhibitors with IC(50) values of 0.8 and 1.3microM, respectively. Flavonoids without a C2-C3 double bond like eriodictyol, taxifolin and catechin did not inhibit GS-X pump activity. The results of this study demonstrate that the structural features necessary for high potency GS-X pump inhibition by flavonoids are (1) the presence of hydroxyl groups, especially two of them generating the 3',4'-catechol moiety; and (2) a planar molecule due to the presence of a C2-C3 double bond. Other factors, like lipophilicity and the total number of hydroxyl groups do not seem to be dominating the flavonoid-mediated GS-X pump inhibition. To identify the GS-X pump responsible for the DNP-SG efflux in MCF7 cells, the effects of three characteristic flavonoids quercetin, flavone and taxifolin on MRP1 and MRP2 activity were studied using transfected MDCKII cells. All three flavonoids as well as the typical MRP inhibitor (MK571) affected MRP1-mediated transport activity in a similar way as observed in the MCF7 cells. In addition, the most potent GS-X pump inhibitor in the MCF7 cells, quercetin, did not affect MRP2-mediated transport activity. These observations clearly indicate that the GS-X pump activity in the MCF7 cells is likely to be the result of flavonoid-mediated inhibition of MRP1 and not MRP2. Altogether, the present study reveals that a major site for flavonoid interaction with GSH-dependent toxicokinetics is the GS-X pump MRP1 rather than the conjugating GSTP1-1 activity itself. Of the flavonoids shown to be most active especially quercetin is frequently marketed in functional food supplements. Given the physiological levels expected to be reached upon supplement intake, the IC(50) values of the present study point at possible flavonoid-drug and/or flavonoid-xenobiotic interactions especially regarding transport processes involved in toxicokinetics.     

Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins

The initial step in reduced glutathione (GSH) turnover in all mammalian cells is its transport across the plasma membrane into the extracellular space; however, the mechanisms of GSH transport are not clearly defined. GSH export is required for the delivery of its constituent amino acids to other tissues, detoxification of drugs, metals, and other reactive compounds of both endogenous and exogenous origin, protection against oxidant stress, and secretion of hepatic bile. Recent studies indicate that some members of the multidrug resistance-associated protein (MRP/CFTR or ABCC) family of ATP-binding cassette (ABC) proteins, as well as some members of the organic anion transporting polypeptide (OATP or SLC21A) family of transporters contribute to this process. In particular, five of the 12 members of the MRP/CFTR family appear to mediate GSH export from cells namely, MRP1, MRP2, MRP4, MRP5, and CFTR. Additionally, two members of the OATP family, rat Oatp1 and Oatp2, have been identified as GSH transporters. For the Oatp1 transporter, efflux of GSH may provide the driving force for the uptake of extracellular substrates. In humans, OATP-B and OATP8 do not appear to transport GSH; however, other members of this family have yet to be characterized in regards to GSH transport. In yeast, the ABC proteins Ycf1p and Bpt1p transport GSH from the cytosol into the vacuole, whereas Hgt1p mediates GSH uptake across the plasma membrane. Because transport is a key step in GSH homeostasis and is intimately linked to its biological functions, GSH export proteins are likely to modulate essential cellular functions.     

Mechanistic differences between GSH transport by multidrug resistance protein 1 (MRP1/ABCC1) and GSH modulation of MRP1-mediated transport

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent polytopic membrane protein that transports many anticancer drugs and organic anions. Its transport mechanism is multifaceted, especially with respect to the participation of GSH. For example, vincristine is cotransported with GSH, estrone sulfate transport is stimulated by GSH, or MRP1 can transport GSH alone, and this can be stimulated by compounds such as verapamil or apigenin. Thus, the interactions between GSH and MRP1 are mechanistically complex. To examine the similarities and differences among the various GSH-associated mechanisms of MRP1 transport, we have measured first the effect of GSH and several GSH-associated substrates/modulators on the binding and hydrolysis of ATP by MRP1 using 8-azidoadenosine-5'-[(32)P]-triphosphate ([(32)P]azidoATP) analogs, and second the initial binding of GSH and GSH-associated substrates/modulators to MRP1. We observed that GSH or its nonreducing derivative S-methylGSH (S-mGSH), but none of the GSH-associated substrate/modulators, caused a significant increase in [gamma-(32)P]azidoATP labeling of MRP1. Moreover, GSH and S-mGSH decreased levels of orthovanadate-induced trapping of [alpha-(32)P]azidoADP. [alpha-(32)P]azidoADP.Vi trapping was also decreased by estone sulfate, whereas vincristine, verapamil, and apigenin had no apparent effects on nucleotide interactions with MRP1. Furthermore, estrone sulfate and S-mGSH enhanced the effect of each other 15- and 10-fold, respectively. Second, although GSH binding increased the apparent affinity of MRP1 for all GSH-associated substrates/modulators tested, only estrone sulfate had a reciprocal effect on the apparent affinity of MRP1 for GSH. Overall, these results indicate significant mechanistic differences between MRP1-mediated transport of GSH and the ability of GSH to modulate MRP1 transport.