Kuguacin J isolated from Momordica charantia leaves inhibits P-glycoprotein (ABCB1)-mediated multidrug resistance

Multidrug resistance (MDR) is a major factor in the failure of chemotherapy in cancer patients. Resistance to chemotherapy has been correlated to the overexpression of ABC drug transporters including P-glycoprotein (P-gp) that actively efflux chemotherapeutic drugs from cancer cells. Our previous study showed that bitter melon (Momordica charantia) leaf extract (BMLE) was able to reverse the MDR phenotype by increasing the intracellular accumulation of chemotherapeutic drugs. In the present study, bioguided fractionation was used to identify the active component(s) of BMLE that is able to modulate the function of P-gp and the MDR phenotype in a human cervical carcinoma cell line (KB-V1). We found that kuguacin J, one of the active components in BMLE, increased sensitivity to vinblastine and paclitaxel in KB-V1 cells. A flow cytometry assay indicated that kuguacin J inhibits the transport function of P-gp and thereby significantly increases the accumulation of rhodamine 123 and calcein AM in the cells. These results were confirmed by [³H]-vinblastine transport assay. Kuguacin J significantly increases intracellular [³H]-vinblastine accumulation and decreased the [³H]-vinblastine efflux in the cells. Kuguacin J also inhibited the incorporation of [¹²⁵I]-iodoarylazidoprazosin into P-gp in a concentration-dependent manner, indicating that kuguacin J directly interacts with the drug-substrate-binding site on P-gp. These results indicate that kuguacin J modulates the function of P-gp by directly interacting at the drug-substrate-binding site, and it appears to be an effective inhibitor of P-gp activity in vitro and thus could be developed as an effective chemosensitizer to treat multidrug-resistant cancers.     

Effect of Stemona curtisii root extract on P-glycoprotein and MRP-1 function in multidrug-resistant cancer cells

Multidrug resistance (MDR) is the result of overexpression of membrane bound proteins that efflux chemotherapeutic drugs from the cells. Two proteins, P-glycoprotein (P-gp) and multidrug-resistance associated protein-1 (MRP-1) efflux chemotherapeutic agents out of the cancer cell that decrease intracellular drug accumulation, thereby decreasing the effectiveness of many chemotherapeutic agents. In the present study, the ethanolic extract of the roots of Stemona curtisii Hook. was tested for the potential ability to modulate the MDR phenotype and function of P-gp and MRP-1. The S. curtisii extract reversed the resistance to putative chemotherapeutic agents, including vinblastine, paclitaxel and colchicine of KB-V1 cells (MDR human cervical carcinoma with high P-gp expression) in a dose-dependent manner, but not in KB-3-1 cells (drug sensitive human cervical carcinoma, which lack P-gp expression). The root extract also increased the intracellular uptake and retention of (3)[H]-vinblastine in KB-V1 cells dose dependently. The extract did not influence MDR phenotype-mediated MRP-1 in MRP1-HEK293 (human embryonic kidney cells stably transfected with pcDNA3.1-MRP1-H10 which show high MRP-1 expression) and pcDNA3.1-HEK293 (wild type). In summary, the S. curtisii root extract modulated P-gp activity but not MRP-1 activity. The result obtained from this study strongly indicated that S. curtisii extract may play an important role as a P-gp modulator as used in vitro and may be effective in the treatment of multidrug-resistant cancers. The purified form of the active components of S. curtisii extract should be investigated in more details in order to explain the molecular mechanisms involved in P-gp modulation. This is the first report of new biological activity in this plant, which could be a potential source of a new chemosensitizer.     

Role of multidrug resistance P-glycoprotein in the secretion of aldosterone by human adrenal NCI-H295 cells

We determinedthe role of the multidrug resistance (MDR1) gene product,P-glycoprotein (PGP), in the secretion of aldosterone by the adrenalcell line NCI-H295. Aldosterone secretion is significantly decreased bythe PGP inhibitors verapamil, cyclosporin A (CSA), PSC-833, andvinblastine. Aldosterone inhibits the efflux of the PGP substraterhodamine 123 from NCI-H295 cells and from human mesangial cells(expressing PGP). CSA, verapamil, and the monoclonal antibody UIC2significantly decreased the efflux of fluorescein-labeled (FL)-aldosterone microinjected into NCI-H295 cells. In MCF-7/VP cells,expressing multidrug resistance-associated protein (MRP) but not PGP,and in the parental cell line MCF7 (expressing no MRP andno PGP), the efflux of microinjected FL-aldosterone was slow. In BC19/3cells (MCF7 cells transfected with MDR1), the efflux of FL-aldosteronewas rapid and it was inhibited by verapamil, indicating thattransfection with MDR1 cDNA confers the ability to transportFL-aldosterone. These results strongly indicate that PGP plays a rolein the secretion of aldosterone by NCI-H295 cells and in other cellsexpressing MDR1, including normal adrenal cells.

     

A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2)

The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) in order to develop functional assays for the half-transporter, MXR/BCRP/ABCP1. A measure of FTC-inhibitable efflux was generated for each compound in a series of MXR-overexpressing drug-selected cell lines and in ten unselected cell lines which were used to determine if the four fluorescent compounds were sensitive enough to detect the low MXR levels found in drug-sensitive cell lines. FTC-inhibitable efflux of mitoxantrone and prazosin was found in four of the ten cell lines, SF295, KM12, NCI-H460, and A549, and low but detectable levels of MXR mRNA were also observed by Northern analysis in these cells. FTC-inhibitable mitoxantrone and prazosin efflux in both selected and unselected cell lines was found to correlate well with MXR levels as determined by Northern blotting, r(2)=0.89 and r(2)=0.70 respectively. In contrast, rhodamine and LysoTracker were not able to reliably detect MXR. Cytotoxicity assays performed on two of the four unselected cell lines confirmed increased sensitivity to mitoxantrone in the presence of FTC. FTC was found to be a specific inhibitor of MXR, with half-maximal inhibition of MXR-associated ATPase activity at 1 microM FTC. Short term selections of the SF295, KM12, NCI-H460 and A549 cell lines in mitoxantrone resulted in a small but measurable increase in MXR by both Northern blot and functional assay. These studies show that flow cytometric measurement of FTC-inhibitable mitoxantrone or prazosin efflux is a sensitive and specific method for measuring the function of the MXR half-transporter in both selected and unselected cell lines.     

Lobeline,a piperidine alkaloid from Lobelia can reverse P-gp dependent multidrug resistance in tumor cells

Multidrug resistance (MDR) can limit efficacy of chemotherapy. The best studied mechanism involves P-gp (P-glycoprotein) mediated drug efflux. This study focuses on MDR reversal agents from medicinal plants, which can interfere with P-gp. Rhodamine 123 accumulation assay and flow cytometry analysis were employed to screen for P-gp dependant efflux inhibitors. Lobeline, a piperidine alkaloid from Lobelia inflata and several other Lobelia species, inhibited P-gp activity. MDR reversal potential of lobeline could be demonstrated in cells treated with doxorubicin in that lobeline can sensitize resistant tumor cells at non-toxic concentrations. However, lobeline cannot block BCRP (Breast Cancer Resistance Protein) dependent mitoxantrone efflux. Lobeline could be a good candidate for the development of new MDR reversal agents.     

Discovery of alkoxyl biphenyl derivatives bearing dibenzo[c,e]azepine scaffold as potential dual inhibitors of P-glycoprotein and breast cancer resistance protein

We recently reported alkoxyl biphenyl derivatives bearing dibenzo[c,e]azepine scaffold as novel P-glycoprotein (P-gp, ABCB1) inhibitors. In this study, their ability to reverse breast cancer resistance protein (BCRP, ABCG2)-mediated multidrug resistance was tested in HEK293/BCRP cells which was BCRP-transfected stable HEK293 cells. It was observed that compounds 4d, 4h, 4i increased mitoxantrone accumulation in HEK293/BCRP cells via inhibiting BCRP efflux function. Notably, the inhibitory activity of 4i was comparable to that of the classical BCRP inhibitor Ko143 at an equimolar concentration. Interestingly, 4i had little inhibitory effect on multidrug resistance-associated protein 1 (MRP1, ABCC1), another drug efflux transporter. These results, together with the previous findings, suggest that 4i may be a dual inhibitor of P-gp and BCRP to warrant further investigation.     

Suppression of multidrug resistance by treatment with TRAIL in human ovarian and breast cancer cells with high level of c-Myc

In this study, we investigated the role of c-Myc in overcoming multidrug resistance (MDR) in human ovarian and breast cancer cells by TRAIL. We showed that P-gp expressing MDR variants (Hey A8-MDR and MCF7-MDR cells) with high level of c-Myc were highly susceptible to TRAIL treatment when compared to their drug-sensitive parental human ovarian cancer Hey A8 and breast MCF-7 cells, respectively. Up-regulation of DR5 TRAIL receptor and down-regulation of c-FLIP and the promotion of caspase-dependent cell death, which contribute to TRAIL sensitization of MDR cells, were regulated by the over-expressed c-Myc in the MDR cells. After targeted inhibition of c-Myc with specific siRNA, these responses to TRAIL disappeared and TRAIL-induced apoptosis was also suppressed in MCF7-MDR cells. Treatment with TRAIL significantly reduced P-glycoprotein (P-gp)-mediated efflux of rhodamine123 in both Hey A8-MDR and MCF7-MDR cells. Furthermore, TRAIL significantly potentiated the cytotoxicity of vinblastine, vincristine, doxorubicin and VP-16 that are P-gp substrate anticancer drugs in both MDR cells, which resulted in the reversal effect of TRAIL on the MDR phenotype. The present study shows for the first time that elevated c-Myc expression in the MDR cells plays a critical role in overcoming MDR by TRAIL that can act as a specific sensitizer for P-gp substrate anticancer drug.     

99mTc(N)-DBODC(5), a potential radiolabeled probe for SPECT of multidrug resistance: in vitro study

[^99mTc(N)(DBODC)(PNP5)]⁺ [DBODC is bis(N-ethoxyethyl)dithiocarbamato; PNP5 is bis(dimethoxypropylphosphinoethyl)ethoxyethylamine], abbreviated as ^99mTc(N)-DBODC(5), is a lipophilic cationic mixed compound investigated as a myocardial imaging agent. The findings that this tracer accumulates in mitochondrial structures through a mechanism mediated by the negative mitochondrial membrane potential and that the rapid efflux of ^99mTc(N)-DBODC(5) from nontarget tissues seems to be associated with the multidrug resistance (MDR) P-glycoprotein (P-gp) transport function open up the possibility to extend its clinical applications to tumor imaging and noninvasive MDR studies. The rate of uptake at 4 and 37 °C of ^99mTc(N)-DBODC(5) was evaluated in vitro in selected human cancer cell lines and in the corresponding sublines before and after P-gp and/or MDR-associated protein (MRP) modulator/inhibitor treatment using ^99mTc-sestamibi as a reference. The results indicated that (1) the uptake of both ^99mTc(N)-DBODC(5) and ^99mTc-sestamibi is correlated to metabolic activity of the cells and (2) the cellular accumulation is connected to the level of P-gp/MRP expression; in fact, an enhancement of uptake in resistant cells was observed after treatment with opportune MDR inhibitor/modulator, indicating that the selective blockade of P-gp/MRP prevented efflux of the tracers. This study provides a preliminary indication of the applicability of ^99mTc(N)-DBODC(5) in tumor imaging and in detecting P-gp/MRP-mediated drug resistance in human cancer. In addition, the possibility to control the hydrophobicity and pharmacological activity of this heterocomplex through the variation of the substituents on the ligands backbone without affecting the P₂S₂ coordinating sphere makes ^99mTc(N)-DBODC(5) a suitable scaffold for the preparation of a molecular probe for single photon emission computed tomography of MDR.     

Breast Cancer-Derived Microparticles Display Tissue Selectivity in the Transfer of Resistance Proteins to Cells

Microparticles (MPs) play a vital role in cell communication by facilitating the horizontal transfer of cargo between cells. Recently, we described a novel “non-genetic” mechanism for the acquisition of multidrug resistance (MDR) in cancer cells by intercellular transfer of functional P-gp, via MPs. MDR is caused by the overexpression of the efflux transporters P-glycoprotein (P-gp) and Multidrug Resistance-Associated Protein 1 (MRP1). These transporters efflux anticancer drugs from resistant cancer cells and maintain sublethal intracellular drug concentrations. By conducting MP transfer experiments, we show that MPs derived from DX breast cancer cells selectively transfer P-gp to malignant MCF-7 breast cells only, in contrast to VLB₁₀₀ leukaemic cell-derived MPs that transfer P-gp and MRP1 to both malignant and non-malignant cells. The observed transfer selectivity is not the result of membrane restrictions for intercellular exchange, limitations in MP binding to recipient cells or the differential expression of the cytoskeletal protein, Ezrin. CD44 (isoform 10) was found to be selectively present on the breast cancer-derived MPs and not on leukaemic MPs and may contribute to the observed selective transfer of P-gp to malignant breast cells observed. Using the MCF-7 murine tumour xenograft model we demonstrated the stable transfer of P-gp by MPs in vivo, which was found to localize to the tumour core as early as 24 hours post MP exposure and to remain stable for at least 2 weeks. These findings demonstrate a remarkable capacity by MPs to disseminate a stable resistant trait in the absence of any selective pressure.     

Biophysical characterization of MDR breast cancer cell lines reveals the cytoplasm is critical in determining drug sensitivity

Dielectrophoresis (DEP) was used to examine a panel of MCF-7 cell lines comprising parental MCF-7 cells and MDR derivatives: MCF-7TaxR (paclitaxel-resistant, P-glycoprotein (P-gp) positive), MCF-7DoxR (doxorubicin-resistant MRP2 positive) plus MCF-7MDR1 (MDR1 transfected, P-gp positive). MCF-7DoxR and MCF-7MDR1 were broadly cross-resistant to natural product anticancer agents, whereas MCF-7TaxR cells were not, contrary to P-gp expression. Whilst DEP revealed modest membrane changes in MDR sub-lines, we saw significant changes in their cytoplasmic conductivity: MCF-7TaxR相似文献   

A novel flavonoid from Fissistigma cupreonitens, 5‑hydroxy‑7,8‑dimethoxyflavanone,competitively inhibited the efflux function of human P-glycoprotein and reversed cancer multi-drug resistance

BackgroundP-glycoprotein (P-gp) over-expression plays a vital role in not only systemic drug bioavailability but also cancer multi-drug resistance (MDR). Develop functional inhibitors of P-gp can conquer both problems.Purpose and study designThe aim of the present study was to research the P-gp modulating effects and MDR reversing ability of a novel flavonoid from Fissistigma cupreonitens, the underlying inhibitory mechanisms were further elucidated as well.MethodsCalcein-AM, rhodamine 123, and doxorubicin were fluorescent substrates for the evaluation of P-gp inhibitory function and detailed drug binding modes. Docking simulation was performed to reveal the in silico molecular bonding. ATPase assay and MDR1 shift assay were adopted to reveal the ATP consumption and conformational change of P-gp. The MDR reversing effects were demonstrated through cytotoxicity, cell cycle, and apoptosis analyses.Results5‑hydroxy‑7,8‑dimethoxyflavanone inhibited the efflux of rhodamine 123 and doxorubicin in a competitive manner, and increased the intracellular fluorescence of calcein at a concentration as low as 2.5 μg/ml. 5‑hydroxy‑7,8‑dimethoxyflavanone slightly changed P-gp's conformation and only stimulated ATPase at very high concentration (100 μg/ml). The docking results showed that 5‑hydroxy‑7,8‑dimethoxyflavanone and verapamil exhibited similar binding affinity to P-gp. The MDR reversing effects were prominent in the vincristine group, the reversal folds were 23.01 and 13.03 when combined with 10 μg/ml 5‑hydroxy‑7,8‑dimethoxyflavanone in the P-gp over-expressing cell line (ABCB1/Flp-In™-293) and MDR cancer cell line (KB/VIN), respectively.ConclusionThe present study demonstrated that 5‑hydroxy‑7,8‑dimethoxyflavanone was a novel effective flavonoid in the P-gp efflux inhibition and in vitro cancer MDR reversion.     

Enhanced glutathione depletion, protein adduct formation, and cytotoxicity following exposure to 4-hydroxy-2-nonenal (HNE) in cells expressing human multidrug resistance protein-1 (MRP1) together with human glutathione S-transferase-M1 (GSTM1)

4-Hydroxy-2-nonenal (HNE) is one of the most reactive products of lipid peroxidation and has both cytotoxic and genotoxic effects in cells. Several enzymatic pathways have been reported to detoxify HNE, including conjugation by glutathione-S-transferases (GSTs). Removal of the resulting HNE-glutathione conjugate (HNE-SG) by an efflux transporter may be required for complete detoxification. We investigated the effect of expression of GSTM1 and/or the ABC efflux transporter protein, multidrug-resistance protein-1 (MRP1), on HNE-induced cellular toxicity. Stably transfected MCF7 cell lines were used to examine the effect of GSTM1 and/or MRP1 expression on HNE-induced cytotoxicity, GSH depletion, and HNE-protein adduct formation. Co-expression in the MCF7 cell line of GSTM1 with MRP1 resulted in a 2.3-fold sensitization to HNE cytotoxicity (0.44-fold IC(50) value relative to control) rather than the expected protection. Expression of either GSTM1 or MRP1 alone also resulted in slight sensitization to HNE cytotoxicity (0.79-fold and 0.71-fold decreases in IC(50) values, respectively). Co-expression of GSTM1 and MRP1 strongly enhanced the formation of HNE-protein adducts relative to the non-expressing control cell line, whereas expression of either MRP1 alone or GSTM1 alone yielded similarly low levels of HNE-protein adducts to that of the control cell line. Glutathione (GSH) levels were reduced by 10-20% in either the control cell line or the MCF7/GSTM1 cell line with the same HNE exposure for 60min. However, HNE induced >80% depletion of GSH in cells expressing MRP1 alone. Co-expression of both MRP1 and GSTM1 caused slightly greater GSH depletion, consistent with the greater protein adduct formation and cytotoxicity in this cell line. Since expression of GSTM1 or MRP1 alone did not strongly sensitize cells to HNE, or result in greater HNE-protein adducts than in the control cell line, these results indicate that MRP1 and GSTM1 collaborate to enhance HNE-protein adduct formation and HNE cytotoxicity, facilitated by GSH depletion mediated by both MRP1 and GSTM1.     

Effects of plant sterols on human multidrug transporters ABCB1 and ABCC1

The effects of dietary plant sterols on human drug efflux transporters P-glycoprotein (P-gp, ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) were investigated using P-gp-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural phytosterols found in foods, herbs, and dietary supplements such as β-sitosterol, campesterol, stigmasterol, fucosterol, and z-guggulsterone were investigated. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-gp, increased in the presence of guggulsterone in KB-C2 cells. The efflux of rhodamine 123 from KB-C2 cells was inhibited by guggulsterone. Guggulsterone also increased the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. The ATPase activities of P-gp and MRP1 were stimulated by guggulsterone. These results suggest that guggulsterone, a natural dietary hypolipidemic agent have dual inhibitory effects on P-gp and MRP1 and the potencies to cause food-drug interactions.     

Loss of multidrug resistance protein 1 expression and folate efflux activity results in a highly concentrative folate transport in human leukemia cells

We studied the molecular basis of the up to 46-fold increased accumulation of folates and methotrexate (MTX) in human leukemia CEM-7A cells established by gradual deprivation of leucovorin (LCV). CEM-7A cells consequently exhibited 10- and 68-fold decreased LCV and folic acid growth requirements and 23-25-fold hypersensitivity to MTX and edatrexate. Although CEM-7A cells displayed a 74-86-fold increase in the reduced folate carrier (RFC)-mediated influx of LCV and MTX, RFC overexpression per se cannot induce a prominently increased folate/MTX accumulation because RFC functions as a nonconcentrative anion exchanger. We therefore explored the possibility that folate efflux activity mediated by members of the multidrug resistance protein (MRP) family was impaired in CEM-7A cells. Parental CEM cells expressed substantial levels of MRP1, MRP4, poor MRP5 levels, whereas MRP2, MRP3 and breast cancer resistance protein were undetectable. In contrast, CEM-7A cells lost 95% of MRP1 levels while retaining parental expression of MRP4 and MRP5. Consequently, CEM-7A cells displayed a 5-fold decrease in the [(3)H]folic acid efflux rate constant, which was identical to that obtained with parental CEM cells, when their folic acid efflux was blocked (78%) with probenecid. Furthermore, when compared with parental CEM, CEM-7A cells accumulated 2-fold more calcein fluorescence. Treatment of parental cells with the MRP1 efflux inhibitors MK571 and probenecid resulted in a 60-100% increase in calcein fluorescence. In contrast, these inhibitors failed to alter the calcein fluorescence in CEM-7A cells, which markedly lost MRP1 expression. Replenishment of LCV in the growth medium of CEM-7A cells resulted in resumption of normal MRP1 expression. These results establish for the first time that MRP1 is the primary folate efflux route in CEM leukemia cells and that the loss of folate efflux activity is an efficient means of markedly augmenting cellular folate pools. These findings suggest a functional role for MRP1 in the maintenance of cellular folate homeostasis.     

Folate deprivation results in the loss of breast cancer resistance protein (BCRP/ABCG2) expression. A role for BCRP in cellular folate homeostasis

Breast cancer resistance protein (BCRP/ABCG2) is currently the only ABC transporter that exports mono- and polyglutamates of folates and methotrexate (MTX). Here we explored the relationship between cellular folate status and BCRP expression. Toward this end, MCF-7 breast cancer cells, with low BCRP and moderate multidrug resistance protein 1 (MRP1/ABCC1) levels, and their mitoxantrone (MR)-resistant MCF-7/MR subline, with BCRP overexpression and low MRP1 levels, were gradually deprived of folic acid from 2.3 microm to 3 nm resulting in the sublines MCF-7/LF and MCF-7/MR-LF. These cell lines expressed only residual BCRP mRNA and protein levels and retained a poor MRP2 (ABCC2) through MRP5 (ABCC5) expression. Furthermore, MCF-7/MR-LF cells also displayed 5-fold decreased MRP1 levels relative to MCF-7/MR cells. In contrast, BCRP overexpression was largely retained in MCF-7/MR cells grown in MR-free medium containing 2.3 microm folic acid. Loss of BCRP expression in MCF-7/LF and MCF-7/MR-LF cells resulted in the following: (a) a prominent decrease in the efflux of Hoechst 33342, a BCRP substrate; (b) an approximately 2-fold increase in MR accumulation as revealed by flow cytometry; this was accompanied by a 2.5- and approximately 84-fold increased MR sensitivity in these cell lines, respectively. Consistently, Ko143, a specific BCRP inhibitor, rendered MCF-7 and MCF-7/MR cells 2.1- and approximately 16.4-fold more sensitive to MR, respectively. Loss of BCRP expression also resulted in the following: (c) an identical MTX sensitivity in these cell lines thereby losing the approximately 28-fold MTX resistance of the MCF-7/MR cells; (d) an approximately 2-fold increase in the 4- and 24-h accumulation of [(3)H]folic acid. Furthermore, MCF-7/MR-LF cells displayed a significant increase in folylpoly-gamma-glutamate synthetase activity. Hence, consistent with the mono- and polyglutamate folate exporter function of BCRP, down-regulation of BCRP and increased folylpoly-gamma-glutamate synthetase activity appear to be crucial components of cellular adaptation to folate deficiency conditions. This is the first evidence for the possible role of BCRP in the maintenance of cellular folate homeostasis.     

The role of multidrug resistance protein 1 (MRP1) in transport of fluorescent anions across the human erythrocyte membrane

We employed human red blood cells as a model system to check the affinity of MRP1 (Multidrug Resistance-associated Protein 1) towards fluorescein and a set of its carboxyl derivatives: 5/6-carboxyfluorescein (CF), 2,7-bis-(2-carboxyethyl)-5/6-carboxyfluorescein (BCECF) and calcein (CAL). We found significant differences in the characteristics of transport of the dyes tested across the erythrocyte membrane. Fluorescein is transported mainly in a passive way, while active efflux systems at least partially contribute to the transport of the other compounds. Inside-out vesicle studies revealed that active transport of calcein is masked by another, ATP-independent, transport activity. Inhibitor profiles of CF and BCECF transport are typical for substrates of organic anion transporters. BCECF is transported mainly via MRP1, as proven by the use of QCRL3, a monoclonal antibody known to specifically inhibit MRP1-mediated transport. Lack of effect of QCRL3 on CF uptake excludes the possibility of MRP1 being a transporter of this dye. No inhibition of CF accumulation by cGMP, thioguanine and 6-mercaptopurine suggests also that this fluorescent marker is not a substrate for MRP5, another ABC transporter identified in the human erythrocyte membrane.     

Kinetic analysis of rhodamines efflux mediated by the multidrug resistance protein (MRP1)

Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells. The formation of a substrate concentration gradient was observed. MRP1-mediated transport of rhodamine was glutathione-dependent. The kinetics parameter, k(a) = V(M)/k(m), was very similar for the four rhodamine analogs but approximately 10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes.     

Novel structure–activity relationships and selectivity profiling of cage dimeric 1,4-dihydropyridines as multidrug resistance (MDR) modulators

Synthesized series of cage dimeric 1,4-dihydropyridines have been systematically evaluated as MDR modulators in in vitro assays to investigate structure-dependent selectivity properties of inhibiting most cancer-relevant efflux pump proteins. Structure–activity relationships of each P-glycoprotein (P-gp) and multidrug resistance associated protein (MRP) 1 and MRP2 inhibition are discussed and prove to be mainly determined by certain aromatic substitution patterns. The characterization of breast cancer resistance protein (BCRP) inhibition results in the discovery of benzyloxy substituted derivatives as selective P-gp inhibitors.