Substrate Of Multidrug Resistance-associated Protein Research Articles

Expression of multidrug resistance-associated protein (MRP) in human retinal pigment epithelial cells and its interaction with BAPSG, a novel aldose reductase inhibitor.

The objective of this study was to determine the expression and activity of multidrug resistance-associated protein (MRP) in the retinal pigment epithelial (RPE) cells and to further assess whether BAPSG, a novel anionic aldose reductase inhibitor, interacts with MRP. Functional and biochemical evidence for MRP was obtained in a human retinal pigment epithelial (ARPE-19) cell line and primary cultures of human retinal pigment epithelial (HRPE) cells. Fluorescein accumulation and efflux in the presence and absence of MRP inhibitors was used to obtain functional evidence for MRP. Western blots and RT-PCR were used to obtain biochemical evidence for MRP1. The influence of MRP inhibitors on BAPSG accumulation and efflux in ARPE-19 cells was determined to understand its interaction with MRP. MRP inhibitors increased fluorescein accumulation and reduced efflux in RPE cells. Both cell types exhibited a 190-kDa western blot band corresponding to MRP1 protein and a 287 bp RT-PCR band corresponding to MRP1 mRNA. MRP inhibitors reduced BAPSG efflux and increased its accumulation in ARPE-19 cells. MRP is functionally and biochemically active in human RPE cells. Anionic BAPSG is a likely substrate for MRP.

Monitoring interactions at ATP-dependent drug efflux pumps.

Chemotherapeutic treatment of cancer patients is often unsuccessful, due to the involvement of various mechanisms, leading to multidrug resistance (MDR). In this review, I describe the mechanisms involved in MDR. Furthermore, results obtained by imaging of P-glycoprotein (P-gp) and the multidrug resistance associated protein (MRP) are reviewed. Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are unique techniques to study P-gp- and MRP-mediated transport. The radiopharmaceutical (99m)Tc-sestamibi is a substrate for both P-gp and MRP. This tracer has been used for tumor imaging in clinical studies, and to visualize blockade of P-gp mediated transport after modulation of the P-gp pump. Other (99m)Tc-radiopharmaceuticals such as (99m)Tc- tetrofosmin and several (99m)Tc-Q-complexes are also substrates for P-gp. Until now, for these compounds only results from in vitro and animal studies are available. For quantification of P-gp mediated transport with PET in vivo, several agents, such as [(11)C]colchicine, [(11)C]verapamil and [(11)C]daunorubicin have been evaluated. In vivo results suggest that these radiopharmaceuticals can be used to image P-gp function in tumors. (124)I and (76)Br radiolabeled doxorubicin analogues are also useful to examine P-gp mediated transport. Leukotrienes are specific substrates for MRP. Therefore, N-[(11)C]acetyl-leukotriene E4 provides the opportunity to study MRP function non-invasively. Results obtained with this radiopharmaceutical in MRP(2) mutated GY/TR- rats indicate visualization of MRP-mediated transport. This tracer enables to study MRP transport function abnormalities in vivo such as in Dubin-Johnson patients, who are MRP(2) gene deficient. In conclusion, it is feasible to study the functionality of MDR transporters in vivo, both with SPECT and with PET. Such imaging techniques may become an important factor in the development of novel chemotherapeutic drugs.

Screening method for substrates of multidrug resistance-associated protein

Multidrug resistance-associated protein (MRP) confers multidrug resistance (MDR); it transports many chemically unrelated cytotoxic reagents to the extracellular side, thereby becoming an obstacle in the chemotherapy of cancer. In this work, a simple kinetic method of analysis is presented to assay the transport selectivity of MRP for its substrates. This method is in contrast to the conventional method in which the absolute amount of transported substrates into MRP-bearing vesicles was measured. MRP is a non-selective ATP-dependent transporter, the only thing common to all processes of transport of various kinds of substrates by MRP is consumption of ATP. Thus, kinetics of ATP consumption by MRP was chosen as a measure for estimating selectivity of MRP for its substrates. NADH oxidation was monitored at 340 nm as an index for ATP consumption. The K m values obtained for LTC4, LTD4, LTE4 (LT, leukotriene), oxidized glutathione (GSSG), daunorubicin (Dau) and vincristine (Vin) were 0.15±0.04, 0.71±0.02, 1.18±0.04, 90.9±5.2, 1.47±0.35 and 2.19±0.16, respectively. This method is thus proposed for screening the substrates of MRP, which is expected to contribute to the chemotherapy of cancer.

Use of the anionic dye carboxy-2',7'-dichlorofluorescein for sensitive flow cytometric detection of multidrug resistance-associated protein activity.

Multidrug resistance-associated protein (MRP) and P-glycoprotein are drug efflux pumps conferring multidrug resistance to tumor cells and sharing numerous substrates. In order to determine a flow cytometric assay allowing to analyse MRP activity in cancerous cells in a sensitive and specific manner, cellular accumulation and efflux of the anionic fluorescent dye carboxy-2',7'-dichlorofluorescein (CDF) were studied by flow cytometry using mainly MRP-overexpressing lung GLC4/Sb30 cells and parental GLC4 cells. GLC4/Sb30 cells were found to display reduced accumulation and enhanced efflux of the dye when compared to their parental counterparts. Probenecid, a well known blocker of MRP, strongly enhanced CDF accumulation in GLC4/Sb30 cells through inhibiting efflux of the dye; it also increased CDF levels in GLC4 cells, although to a lesser extent, which may likely be linked to the low, but detectable, expression of MRP in these cells. Comparison of CDF retention with that of calcein demonstrated that the former dye was the most efficiently effluxed by GLC4/Sb30 cells. In contrast to MRP overexpression, that of P-glycoprotein was not found to alter cellular CDF labelling whereas it strongly impaired calcein staining. These results indicate that CDF is a substrate for MRP, but not for P-gp, which may likely be useful for sensitive and specific flow cytometric determination of MRP activity in clinical samples.

Interactions of HIV protease inhibitors with ATP-dependent drug export proteins.

We used renal proximal tubules from a teleost fish (killifish; Fundulus heteroclitus), fluorescent substrates and confocal microscopy to study the interactions between human immunodeficiency virus protease inhibitors and drug-transporting ATPases. Both saquinavir and ritonavir inhibited luminal accumulation of a fluorescent cyclosporin A derivative (a substrate for P-glycoprotein) and of fluorescein methotrexate [a substrate for multidrug resistance-associated protein 2 (Mrp2)]. Of the two protease inhibitors, ritonavir was the more potent inhibitor of transport by a factor of at least 20. Ritonavir was at least as good an inhibitor of P-glycoprotein- and Mrp2-mediated transport as cyclosporin A and leukotriene C4, respectively. Inhibition of P-glycoprotein- and Mrp2-mediated transport was not due to toxicity or impaired metabolism, because neither saquinavir nor ritonavir inhibited transport of fluorescein on the renal organic anion system. Experiments with a fluorescent saquinavir derivative showed strong secretion into the tubular lumen that was inhibited by verapamil, leukotriene C4, saquinavir, and ritonavir. Together, the data demonstrate that saquinavir, and especially ritonavir, are potent inhibitors of P-glycoprotein- and Mrp2-mediated transport. The experiments with the fluorescent saquinavir derivative suggest that these protease inhibitors may also be substrates for both P-glycoprotein and Mrp2.

The multidrug resistance-associated protein (MRP) is over-expressed and functional in rat hepatoma cells.

Expression of multidrug-resistance-associated protein (MRP), a drug efflux pump transporting a wide range of xenobiotics, including anti-cancer drugs and chemical carcinogens, and present at low levels in normal hepatocytes, was investigated in rat hepatoma cells. Northern-blot analysis allowed detection of high levels of MRP mRNA in rat diethylnitrosamine-induced hepatocarcinomas when compared with normal liver. Similarly, elevated expression of MRP transcripts were evidenced in 6 rat hepatoma cell lines of different origins, especially in HTC cells, that, in contrast, failed to express mRNA of the canalicular multispecific organic anion transporter (cMOAT), an efflux pump sharing numerous substrates with MRP. HTC cells were also found by Western blotting to display much higher amounts of MRP than those observed in normal hepatocytes. In contrast, the MRP gene copy number was similar both in hepatoma HTC cells and in hepatocytes, as assessed by Southern blotting. Analysis of MRP-related transport using 3 types of MRP substrates, namely, the fluorescent glutathione-bimane, the anionic dye calcein and the cationic anti-cancer drug vincristine, demonstrated that HTC cells displayed cellular efflux of these 3 compounds, an efflux strongly inhibited by MRP modulators such as indomethacin. These results indicate that MRP is over-expressed and functional in rat hepatoma cells and may therefore be included in the de-toxifying pathways that are altered during hepatocarcinogenesis and are thus thought to contribute to the known multidrug resistance of liver tumors.

Reversal of MRP-Mediated Multidrug Resistance in Human Lung Cancer Cells by the Antiprogestatin Drug RU486

Multidrug resistance-associated protein (MRP) and P-glycoprotein (P-gp) are drug efflux pumps conferring multidrug resistance to tumor cells. RU486, an antiprogestatin drug known to inhibit P-gp function, was examined for its effect on MRP activity in MRP-overexpressing lung tumor GLC4/Sb30 cells. In such cells, the antihormone compound was found to increase intracellular accumulation of calcein, a fluorescent compound transported by MRP, in a dose-dependent manner, through inhibition of cellular export of the dye ; in contrast, it did not alter calcein levels in parental GLC4 cells. RU486, when used at 10 μM, a concentration close to plasma concentrations achievable in humans, strongly enhanced the sensitivity of GLC4/Sb30 cells towards two known cytotoxic substrates of MRP, the anticancer drug vincristine and the heavy metal salt potassium antimonyl tartrate. Vincristine accumulation levels were moreover up-regulated in RU486-treated GLC4/Sb30 cells. In addition, such cells were demonstrated to display reduced cellular levels of glutathione which is required for MRP-mediated transport of some anticancer drugs. These findings therefore demonstrate that RU486 can down-modulate MRP-mediated drug resistance, in addition to that linked to P-gp, through inhibition of MRP function.

Inhibition of multidrug resistance-associated protein (MRP) activity by rifampicin in human multidrug-resistant lung tumor cells

The multidrug resistance-associated protein (MRP) is a drug efflux membrane pump conferring multidrug resistance on tumor cells. In order to look for compounds that can lead to reversal of such a resistance, the antituberculosis compound rifampicin, belonging to the chemical class of rifamycins, was examined for its effect on MRP activity in human multidrug resistant lung cancer GLC4/ADR cells. Rifampicin was shown to increase accumulation of the MRP substrate calcein in GLC4/ADR cells in a dose-dependent manner by inhibiting its MRP-mediated efflux from the cells; it also enhanced intracellular retention of another substrate of MRP such as the anticancer drug vincristine in the resistant cells. By contrast, the antituberculosis drug did not alter cellular levels of accumulation of either calcein or vincristine in parental drug-sensitive GLC4 cells. Other rifamycins such as rifamycin B and rifamycin SV were also demonstrated to increase intracellular accumulation of calcein in GLC4/ADR cells. These results therefore indicate that rifamycins, including rifampicin, probably constitute a new chemical class of modulators down-regulating MRP-mediated drug transport.

Altered expression of the MYCN oncogene modulates MRP gene expression and response to cytotoxic drugs in neuroblastoma cells.

We have recently shown a close correlation between expression of the Multidrug Resistance-associated Protein (MRP) gene and the MYCN oncogene and provided evidence that high MRP expression is a powerful independent predictor of poor outcome in neuroblastoma (Norris et al., New Engl. J. Med., 334, 231-238, 1996). The effect of MYCN down-regulation on MRP expression and response to cytotoxic drugs was investigated in NBL-S neuroblastoma cells transfected with MYCN antisense RNA constructs. Concomitant with MYCN down-regulation, the level of MRP expression was decreased in the NBAS-4 and NBAS-5 antisense transfectants. These cells demonstrated significantly increased sensitivity to the high affinity MRP substrates vincristine, doxorubicin, sodium arsenate and potassium antimony tartrate, but not to the poor MRP substrates, taxol or cisplatin. Similarly, transfection of full-length MYCN cDNA into SH-EP neuroblastoma cells resulted in increased MRP expression and significantly increased resistance specifically to MRP substrates. The results provide evidence for the MYCN oncogene influencing cytotoxic drug response via regulation of MRP gene expression. Our data also provide a link between the malignant and chemoresistant phenotypes of this childhood malignancy.

Visualization of multidrug resistance in vivo.

Various mechanisms are involved in multidrug resistance (MDR) for chemotherapeutic drugs, such as the drug efflux pumps, P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP). In this review the mechanisms involved in MDR are described and results are reviewed with particular attention to the in vivo imaging of Pgp and MRP. Various detection assays provide information about the presence of drug efflux pumps at the mRNA and protein levels. However, these methods do not yield information about the dynamic function of Pgp and MRP in vivo. For the study of Pgp- and MRP-mediated transport, single-photon emission tomography (SPET) and positron emission tomography (PET) are available. Technetium-99m sestamibi is a substrate for Pgp and MRP, and has been used in clinical studies for tumour imaging, and to visualize blockade of Pgp-mediated transport after modulation of the Pgp pump. Other 99mTc radiopharmaceuticals, such as 99mTc-tetrofosmin and several 99Tc-Q complexes, are also substrates for Pgp, but to date only results from in vitro and animal studies are available for these compounds. Several agents, including [11C]colchicine, [11C]verapamil and [11C]daunorubicin, have been evaluated for the quantification of Pgp-mediated transport with PET in vivo. The results suggest that radiolabelled colchicine, verapamil and daunorubicin are feasible substrates with which to image Pgp function in tumours. Uptake of [11C]colchicine and [11C]verapamil is relatively high in the chest area, reducing the value of both tracers for monitoring Pgp-mediated drug transport in tumours located in this region. In addition, it has to be borne in mind that only comparison of Pgp-mediated transport of radioalabelled substrates in the absence and in the presence of Pgp blockade gives quantitative information on Pgp-mediated pharmacokinetics. Leukotrienes are specific substrates for MRP. Therefore, N-[11C]acetyl-leukotriene E4 provides an opportunity to study MRP function non-invasively. Results obtained in MRP2 mutated GY/TR rats have demonstrated visualization of MRP-mediated transport. This tracer permits the study of MRP transport function abnormalities in vivo, e.g. in Dubin-Johnson patients, who are MRP2 gene deficient. Results obtained show the feasibility of using SPET and PET to study the functionality of MDR transporters in vivo.

Substrates of multidrug resistance-associated proteins block the cystic fibrosis transmembrane conductance regulator chloride channel.

1. The effects of physiological substrates of multidrug resistance-associated proteins (MRPs) on cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel currents were examined using patch clamp recording from CFTR-transfected mammalian cell lines. 2. Two MRP substrates, taurolithocholate-3-sulphate (TLCS) and beta-estradiol 17-(beta-D-glucuronide) (E217betaG) caused a voltage-dependent block of macroscopic CFTR Cl- currents when applied to the intracellular face of excised membrane patches, with mean apparent dissociation constants (KDs) of 96+/-10 and 563+/-103 microM (at 0 mV) respectively. The unconjugated bile salts taurocholate and cholate were also effective CFTR channel blockers under these conditions, with KDs of 453+/-44 and 3760+/-710 microM (at 0 mV) respectively. 3. Reducing the extracellular Cl- concentration from 154 to 20 mM decreased the KD for block intracellular TLCS to 54+/-1 microM, and also significantly reduced the voltage dependence of block, by suggesting that TLCS blocks Cl- permeation through CFTR by binding within the channel pore. 4. Intracellular TLCS reduced the apparent amplitude of CFTR single channel currents, suggesting that the duration of block is very fast compared to the gating of the channel. 5. The apparent affinity of block by TLCs is comparable to that of other well-known CFTR channel blockers, suggesting that MRP substrates may comprise a novel class of probes of the CFTR channel pore. 6. These results also suggest that the related proteins CFTR and MRP may share a structurally similar anion binding site at the cytoplasmic face of the membrane.

Expression of the multidrug resistance-associated protein (MRP) and chemoresistance of human non-small-cell lung cancer cells.

Human non-small-cell lung cancer (NSCLC) is considered to be a chemotherapy-refractory malignancy. The underlying mechanisms remain rather obscure. The multidrug resistance-associated protein (MRP), mediating a multidrug resistance (MDR) phenotype, has been reported to be overexpressed in several drug-selected lung cancer cell lines. A few previous studies have described intrinsic MRP expression in both NSCLC and normal lung tissues. However, the drug-transporting activity as well as the correlation with chemoresistance is unclear. Using 15 unselected cell lines, we show that MRP (mRNA and protein as detected by reverse transcriptase polymerase chain reaction and immunoblot) is frequently expressed intrinsically, with markedly varying intensity, in NSCLC. Two cell lines expressed high MRP levels, one comparable to the drug-selected controls (GLC4/ADR, HL-60/AR) without, however, amplification of the MRP gene (Southern hybridization). Using 3H-daunomycin (3H-DM) and calcein as MRP substrates and probenecid (PRO), genistein (GEN), benzbromarone (BB), N-ethylmaleimide (NEM) and verapamil (VP) as MRP modulators, drug accumulation studies revealed a transporting activity of MRP that correlated significantly with the gene expression data. Moreover, a significant correlation between MRP expression and chemoresistance against daunomycin (DM), doxorubicin (DOX), etoposide (VP-16) and vinblastine (VBL), but not cisplatin (CDDP) and bleomycin (Bleo) (MTT-based survival assay), was detected. Correlations mainly rested on the pronounced chemoresistance of 2 highly MRP-expressing cell lines and did not reach significance when these cell lines were excluded.

810 - Expression of the multidrug resistance-associated protein (MRP) and chemoresistance of human non-small-cell lung cancer cells

Human non-small-cell lung cancer (NSCLC) is considered to be a chemotherapy-refractory malignancy. The underlying mechanisms remain rather obscure. The multidrug resistance-associated protein (MRP), mediating a multidrug resistance (MDR) phenotype, has been reported to be overexpressed in several drug-selected lung cancer cell lines. A few previous studies have described intrinsic MRP expression in both NSCLC and normal lung tissues. However, the drug-transporting activity as well as the correlation with chemoresistance is unclear. Using 15 unselected cell lines, we show that MRP (mRNA and protein as detected by reverse transcriptase polymerase chain reaction and immunoblot) is frequently expressed intrinsically, with markedly varying intensity, in NSCLC. Two cell lines expressed high MRP levels, one comparable to the drug-selected controls (GLC4/ADR, HL-60/AR) without, however, amplification of the MRP gene (Southern hybridization). Using 3H-daunomycin (3H-DM) and calcein as MRP substrates and probenecid (PRO), genistein (GEN), benzbromarone (BB), N-ethylmaleimide (NEM) and verapamil (VP) as MRP modulators, drug accumulation studies revealed a transporting activity of MRP that correlated significantly with the gene expression data. Moreover, a significant correlation between MRP expression and chemoresistance against daunomycin (DM), doxorubicin (DOX), etoposide (VP-16) and vinblastine (VBL), but not cisplatin (CDDP) and bleomycin (Bleo) (MTT-based survival assay), was detected. Correlations mainly rested on the pronounced chemoresistance of 2 highly MRP-expressing cell lines and did not reach significance when these cell lines were excluded.

Possible role of the multidrug resistance-associated protein (MRP) in chemoresistance of human melanoma cells.

Human malignant melanoma is characterised by unresponsiveness to conventional chemotherapy. Melanoma-derived cell lines are often markedly chemoresistant, suggesting that cellular mechanisms mediate the multidrug resistance (MDR) phenotype. The multidrug resistance-associated protein (MRP) is a drug transporter protein associated with resistance to a broad spectrum of lipophilic drugs. To investigate whether MRP is involved in intrinsic drug resistance of human melanoma, we analysed expression and functional activity of MRP as well as its impact on chemoresistance in 40 melanoma cell lines (35 established by us from primary and metastatic lesions and 5 obtained from international sources), as well as in one dysplastic naevus-derived cell line and in normal melanocytes. By reverse transcriptase-polymerase chain reaction various levels of MRP mRNA were detected in all melanoma cell lines, and by immunoblot the corresponding protein in a high percentage of them. Functional activity of MRP was assayed by analysing cellular accumulation of 3H-daunomycin (3H-DM) and calcein in response to MRP-modulators by beta-spectrometric and fluorescence-activated cell sorter analysis, respectively. Probenecid (PRO), N-ethylmaleimide (NEM) and benzbromarone (BB) moderately (< or = 1.43-fold) but significantly enhanced intracellular accumulation of MRP substrate probes corresponding to MRP expression. Moreover, the sensitivity of melanoma cell lines to daunomycin (DM) and doxorubicin (DOX), but not to vinblastine (VBL), etoposide (VP-16) and cisplatin (CDDP), analysed by an MTT-based survival assay, were inversely correlated with MRP-gene expression. Our results imply that MRP may be a component of the intrinsic chemoresistance phenotype characteristic of human malignant melanoma.

Granulocyte-Macrophage colony-Stimulating factor receptor-Targeted therapy of chemotherapy- and radiation-Resistant human myeloid leukemias

Contemporary therapies for acute myeloid leukemia (AML) commonly fail to cure patients because of the emergence of drug resistance. Drug resistance in AML is multifactorial but can be associated with the overexpression of transmembrane transporter molecules, including P-glycoprotein (Pgp) or the multidrug resistance-associated protein (MRP), or associated with inactivation of the p53 tumor suppressor gene, as well as overexpression of the anti-apoptotic protein bcl-2. We are investigating if novel recombinant biotherapeutics can circumvent these resistance mechanisms to effectively treat refractory AML. To target the lethal action of diphtheria toxin (DT) to high affinity granulocyte-macrophage colony-stimulating factor (GMCSF) receptors on AML blasts, we have produced a recombinant chimeric fusion toxin, DTctGMCSF. Since DTctGMCSF enters and kills its target cells by unique mechanisms (GMCSF-receptor binding and protein synthesis inhibition) and is not similar in structure to Pgp or MRP substrates, we postulated that it would be an active agent against therapy-resistant AML. DTctGMCSF was selectively cytotoxic (IC50 1–10ng/ml) to GMCSF-receptor positive AML cells expressing the Pgp- or MRP-associated multi-drug resistant phenotypes, despite high level resistance to conventional chemotherapeutic agents. DTctGMCSF also efficiently killed AML cells deficient in p53 expression, as well as radiation-resistant AML cells and mixed lineage leukemia cells expressing high levels of bcl-2. In addition, DTctGMCSF killed>99% of primary leukemic progenitor cells from therapy-refractory AML patients under conditions that we have previously found to not adversely affect the proliferative capacity or differentiation of pluripotent normal hematopoietic progenitor cells. DTctGMCSF may prove useful in treating myeloid leukemias that are otherwise resistant to a wide range of conventional therapies.

6 O - Intrinsic multidrug resistance-associated protein in human malignant melanoma cell lines

Disseminated malignant melanoma is characterized by unresponsiveness to chemotherapy without overexpressing corresponding P-glycoprotein. Another ABC-transporter protein functioning as transmembrane drug-efflux pump and involved in multidrug resistance (MDR), is the multidrug resistance-associated protein (MRP). We investigated wether MRP plays a role in malignant melanoma chemoresistance. Using the RT-PCR (two different oligonucleotid primer sets) we determined mRNA-expression in 33 cell lines established from primary tumors (n=11), metastatic lesions (n=17) and malignant effusions (n=5). Protein expression was determined by Western blot analysis (MRPm6, QCRL-1) and confirmed by immunocytochemistry. All cell lines scored positiv for MRP mRNA at different levels (5high, 13 intermediate, 15low). Levels of MRP mRNA were significantly higher in primary tumors as compared to metastases and malignant effusions. The respective protein was detectable in Western blot and in immunocytochemistry. Accumulation studies using ³H-daunomycin as MRP substrate and genistein as specific MRP modulator were perfornled and revealed - in contrast to the control cell models GLC4/ADR and HL601/AR - no drug efflux activily of MRP. In conclusion melanoma cell lines express MRP mRNA and protein at different levels. The biological role has to be investigated in further studies.