Differential sensitivities of MRP1-overexpressing lung tumor cells to cytotoxic metals

The human multidrug-resistance protein (MRP1), known to mediate cellular efflux of a wide range of xenobiotics, including anticancer drugs, has also been shown to transport antimony, thereby conferring resistance to this heavy metal. The aim of the present study was to investigate whether other cytotoxic metals could be handled by MRPI using MRP1-overexpressing lung tumor GLC4/Sb30 cells. Such cells were found to be 3.4-, 12.7- and 16.3-fold more resistant than parental GLC4 cells to mercuric ion, arsenite and arsenate, respectively, whereas they remained sensitive to other cytotoxic metals tested such as copper, chromium, cobalt or aluminium. MK571, a potent inhibitor of MRP1 activity, almost totally reversed resistance of GLC4/Sb30 cells to mercuric ions and arsenic while it did not significantly alter sensitivity of GLC4 cells to metals. Arsenate-treated GLC4/Sb30 cells were found to poorly accumulate arsenic through increased MK571-inhibitable efflux of the metal. Arsenate, however, failed to alter MRP1-mediated transport of known MRP1 substrates such as calcein and vincristine. In conclusion, these findings demonstrated that MRP1 likely handled some, but not all, cytotoxic metals such as arsenic and mercuric ions in addition to antimony, therefore resulting in reduced toxicity of these compounds towards MRP1-overexpressing cells.     

Resistance of human multidrug resistance-associated protein 1-overexpressing lung tumor cells to the anticancer drug arsenic trioxide

The human multidrug-resistance protein (MRP1) confers resistance to some heavy metals such as arsenic and antimony, mainly through mediating an increased cellular efflux of metal. However, it was recently suggested that arsenic, used under its trioxide derivative form as anticancer drug, is not handled by MRP1. The aim of the present study was to test this hypothesis in MRP1-overexpressing human lung tumor GLC4/Sb30 cells. Using the cytotoxicity MTT assay, GLC4/Sb30 cells were found to be 10.8-fold more resistant to arsenic trioxide (As2O3) than parental GLC4 cells. MK571, a potent inhibitor of MRP1 activity, almost totally reversed resistance of GLC4/Sb30 cells, but did not alter the sensitivity of GLC4 cells. Moreover, As2O3-loaded GLC4/Sb30 cells poorly accumulated arsenic through an increased MK571-sensitive efflux of metal. Finally, depletion of cellular glutathione levels in buthionine sulfoximine-treated GLC4/Sb30 cells was found to result in increased accumulation and reduced efflux of arsenic in cells exposed to As2O3, outlining the glutathione-dependence of MRP1-mediated transport of the metal. These results indicate that MRP1 overexpression in human tumor cells can confer resistance to As2O3, which may limit the clinical use of this anticancer drug for treatment of MRP1-positive tumors.     

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.     

Rat intestinal precision-cut slices as an in vitro model to study xenobiotic interaction with transporters

ATP-binding cassette (ABC) proteins play key role in tissue defence by transporting metabolic waste and toxic chemicals out of the cells. Consequently, intact cell systems are required to study xenobiotic interactions with ATP-dependent transporters. The aim of the present study was to set up an intestinal precision-cut slice technique to study the interactions of ABC transporters with xenobiotics. Rat intestinal slices were incubated with verapamil, indomethacin and glibenclamide, and the ability of the above-mentioned drugs to inhibit the multidrug resistance glycoprotein (MDR) and/or multidrug-resistance-associated protein (MRP) was assessed by measuring the intracellular conversion of calcein-AM to fluorescent calcein. The ABC transporters’ inhibitors caused a time-dependent florescence increase which reached the maximum value at 30 min. Verapamil and glibenclamide promoted a concentration-dependent intracellular accumulation of calcein (IC₅₀ 8.1 × 10⁻⁶ M, 1.9 × 10⁻⁴ M, respectively). The effect of glibenclamide was fully reversed by washing the slices, suggesting the reversible nature of calcein accumulation. These data suggest that the precision-cut intestinal slices are a reliable, simple and fast system to evaluate xenobiotic interactions with ABC transporters in rat and, hopefully, in human intestine.     

Multidrug resistance protein functionality: no effect of intracellular or extracellular pH changes

A major problem in the treatment of cancer is cellular resistance to cytotoxic drugs. In tumor cells in vitro, the development of multidrug resistance is usually accompanied by increased expression of drug transporters, either P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP(1)). Both proteins belong to the superfamily of ATP-binding cassette (ABC) transporter proteins and mediate the transport of a broad range of drugs. Altenberg et al. (Proc Natl Acad Sci USA90: 9735-9738, 1993) have shown that changes in intra- or extracellular pH do not mediate P-gp-dependent multidrug resistance. Therefore, we similarly studied whether changes in intra- or extracellular pH could mediate MRP(1)-dependent multidrug resistance. In particular, we measured the MRP(1)-mediated efflux of hydroxyrubicin from GLC4/ADR cells. Since hydroxyrubicin is a fully neutral anthracycline derivative that has no deprotonable function at pH lower than 10 and so cannot accumulate in non-nuclear compartments under the influence of pH or transmembrane gradients, we hypothesized that any modifications of its kinetics of efflux as a function of pH can be assigned to a modification of the transporter efficiency. However, as our data show, modifications of extra- and/or intracellular pH yielded no modification of the MRP(1)-mediated efflux of hydroxyrubicin.     

Expression of Adenosine Triphosphate-Binding Cassette (ABC) Drug Transporters in Peripheral Blood Cells

Adenosine triphosphate-binding cassette (ABC)-type transport proteins were initially described for their ability to reduce intracellular concentrations of anticancer compounds, thereby conferring drug resistance. In recent years, expression of this type of proteins has also been reported in numerous cell types under physiological conditions; here, these transporters are often reported to alter systemic and local drug disposition (e.g. in the brain or the gastrointestinal tract). In this context, peripheral blood cells have also been found to express several ABC-type transporters. While erythrocytes mainly express multidrug resistance protein (MRP) 1, MRP4 and MRP5, which are discussed with regard to their involvement in glutathione homeostasis (MRP1) and in the efflux of cyclic nucleotides (MRP4 and MRP5), leukocytes also express P-glycoprotein and breast cancer resistance protein. In the latter cell types, the main function of efflux transporters may be protection against toxins, as these cells demonstrate a very high turnover rate. In platelets, only two ABC transporters have been described so far. Besides MRP1, platelets express relatively high amounts of MRP4 not only in the plasma membrane but also in the membrane of dense granules, suggesting relevance for mediator storage.In addition to its physiological function, ABC transporter expression in these structures can be of pharmacological relevance since all systemic drugs reach their targets via circulation, thereby enabling interaction of the therapeutic agent with peripheral blood cells. Moreover, both intended effects and unwanted side effects occur in peripheral blood cells, and intracellular micropharmacokinetics can be affected by these transport proteins. The present review summarises the data available on expression of ABC transport proteins in peripheral blood cells.     

Transport of new non-cross-resistant antitumor compounds of the benzoperimidine family in multidrug resistant cells

Multidrug resistance (MDR) phenotype in mammalian cells is often correlated with overexpression of P-glycoprotein or multidrug resistance-associated protein (MRP1). Both proteins are energy-dependent drug efflux pumps that efficiently reduce the intracellular accumulation and hence the cytotoxicity of many natural cytotoxins. The influx and efflux of drugs across the cell membrane are in large part responsible for their intracellular concentrations, and in the search for new compounds able to overcome MDR, it is of prime importance to determine the molecular parameters whose modification would lead to an increase in the kinetics of uptake and/or to a decrease in the pump-mediated efflux. Here, we studied three members of a new family of benzoperimidine antitumor compounds which exhibit comparable cytotoxicity towards resistant cells expressing P-glycoprotein, or MRP1, and sensitive cells. We used spectrofluorometric methods to determine the kinetics of the uptake and release of these three drugs in different cell lines: the erythroleukemia cell line K562 and the resistant K562/Adr expressing P-glycoprotein, the small-cell lung cancer cell line GLC4 and resistant GLC4/Adr expressing MRP1. We also studied, using confocal microscopy, the intracellular distribution of these drugs in NIH/3T3 cells. Our data show that (i) the kinetics for the uptake of these drugs is very rapid, higher than 2 x 10(-17) mole cell(-1) s(-1), (ii) the drugs are strongly accumulated in the nucleus and lysosomes, (iii) the three drugs are recognized and pumped out by both transporters, as shown by the inhibition of P-glycoprotein- and MRP1-mediated efflux of pirarubicin by benzoperimidine, with inhibitory constants of 1.5 and 2.1 microM for P-glycoprotein and MRP1, respectively, suggesting that benzoperimidine is transported by the two transporters with K(m) approximately 2 microM. In conclusion, the fast uptake kinetics of the benzoperimidines counterbalance their efflux by P-glycoprotein and MRP1.     

Regulation of drug transporter expression by oncostatin M in human hepatocytes

The cytokine oncostatin M (OSM) is a member of the interleukin (IL)-6 family, known to down-regulate expression of drug metabolizing cytochromes P-450 in human hepatocytes. The present study was designed to determine whether OSM may also impair expression of sinusoidal and canalicular drug transporters, which constitute important determinants of drug hepatic clearance. Exposure of primary human hepatocytes to OSM down-regulated mRNA levels of major sinusoidal solute carrier (SLC) influx transporters, including sodium-taurocholate co-transporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, organic cation transporter 1 and organic anion transporter 2. OSM also repressed mRNA expressions of ATP binding cassette (ABC) efflux transporters such as multidrug resistance protein (MRP) 2/ABCC2 and breast cancer resistance protein/ABCG2, without however impairing those of multidrug resistance gene 1/P-glycoprotein/ABCB1, MRP3/ABCC3, MRP4/ABCC4 and bile salt export pump/ABCB11. The cytokine concomitantly reduced NTCP, OATP1B1, OATP2B1 and ABCG2 protein expression and NTCP and OATP transport activities. OSM effects towards transporters were found to be dose-dependent and highly correlated with those of IL-6, but not with those of other inflammatory cytokines such as tumor necrosis factor-α or interferon-γ. In addition, OSM-mediated repression of some transporters such as NTCP, OATP1B1 and OATP2B1, was counteracted by knocking-down expression of the type II OSM receptor subunits through siRNA transfection. This OSM-mediated down-regulation of drug SLC transporters and ABCG2 in human hepatocytes may contribute to alterations of pharmacokinetics in patients suffering from diseases associated with increased production of OSM.     

Effluxing ABC transporters in human corneal epithelium

ATP-binding cassette (ABC) transporters are able to efflux their substrate drugs from the cells. We compared expression of efflux proteins in normal human corneal epithelial tissue, primary human corneal epithelial cells (HCEpiC), and corneal epithelial cell culture model (HCE model) based on human immortal cell line. Expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1–6 (MRP1–6) and breast cancer resistance protein (BCRP) was studied using quantitative RT-PCR, Western blot, and immunohistochemistry. Only MRP1, MRP5, and BCRP were expressed in the freshly excised human corneal epithelial tissue. Expression of MRP1 and MRP5 was localized predominantly in the basal cells of the central cornea and limbus. Functional efflux activity was shown in the cell models, but they showed over-expression of most efflux transporters compared to that of normal corneal epithelium. In conclusion, MRP1, MRP5, and BCRP are expressed in the corneal epithelium, but MDR1, MRP2, MRP3, MRP4, and MRP6 are not significantly expressed. HCE cell model and commercially available primary cells deviate from this expression profile. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1087–1098, 2010     

ATP-binding cassette multidrug transporters in Indian-rock oyster Saccostrea forskali and their role in the export of an environmental organic pollutant tributyltin

ATP-binding cassette (ABC) multidrug transporters confer resistance in human cancer cells as well as in pathogenic microorganisms by mediating the extrusion of various chemotherapeutic drugs out of the cell. In aquatic invertebrates, the presence of ABC transporters which are involved in the multi-xenobiotic resistance has been demonstrated. However, most studies have been confined to the MDR1 subfamily. In the present study, we characterized the expression and localization of the ABC multidrug transporters including MDR1, MRP1 and BCRP subfamily in the Indian-rock oyster Saccostrea forskali. To our knowledge, these data represent one of the first reports on the orthologues of MRP1 and BCRP in marine invertebrates. Furthermore, the observations of (i) the expression of the ABC multidrug proteins in detoxifying tissues; (ii) the induction of these transporters upon exposure to an environmental organic pollutant tributyltin (TBT); and (iii) the concentration-dependent inhibition of rhodamine efflux by TBT imply a possible role of these proteins in the export of TBT. Our findings along with previous studies suggest that the ABC multidrug transporters act as a detoxifying mechanism of various toxic agents including TBT in aquatic organisms.     

Involvement of transporters in the hepatic uptake and biliary excretion of valsartan, a selective antagonist of the angiotensin II AT1-receptor, in humans.

Valsartan is a highly selective angiotensin II AT1-receptor antagonist for the treatment of hypertension. Valsartan is mainly excreted into the bile in unchanged form. Because valsartan has an anionic carboxyl group, we hypothesized that a series of organic anion transporters could be involved in its hepatic clearance. In this study, to identify transporters that mediate the hepatic uptake and biliary excretion of valsartan and estimate the contribution of each transporter to the overall hepatic uptake and efflux, we characterized its transport using transporter-expressing systems, human cryopreserved hepatocytes, and Mrp2-deficient Eisai hyperbilirubinemic rats (EHBRs). Valsartan was significantly taken up into organic anion-transporting polypeptide (OATP) 1B1 (OATP2/OATP-C)- and OATP1B3 (OATP8)-expressing HEK293 cells. We also observed saturable uptake into human hepatocytes. Based on our estimation, the relative contribution of OATP1B1 to the uptake of valsartan in human hepatocytes depends on the batch, ranging from 20 to 70%. Regarding efflux transporters, the ratio of basal-to-apical transcellular transport of valsartan to that in the opposite direction in OATP1B1/MRP2 (multidrug resistance-associated protein 2) double transfected cells was the highest among the three kinds of double transfectants, OATP1B1/MRP2, OATP1B1/multi-drug resistance 1, and OATP1B1/breast cancer resistance protein-expressing MDCKII cells. We observed saturable ATP-dependent transport into membrane vesicles expressing human MRP2. We also found that the elimination of intravenously administered valsartan from plasma was markedly delayed, and the biliary excretion was severely impaired in EHBR compared with normal Sprague-Dawley rats. These results suggest that OATP1B1 and OATP1B3 as the uptake transporters and MRP2 as the efflux transporter are responsible for the efficient hepatobiliary transport of valsartan.     

Modulation of multidrug resistance efflux pump activity to overcome chemoresistance in cancer

Early publications using cultured cancer cells immediately recognized the phenomenon of resistance to anticancer agents. However, it was not until 1973 that it was first demonstrated that a major factor in the resistance of cancer cells was that of reduced drug accumulation. This year marks the 30th anniversary of the discovery by Juliano and Ling that P-glycoprotein mediates this active efflux of chemotherapeutic drugs from cancer cells. Since this seminal finding, the investigation of P-glycoprotein (MDR1, ATP binding cassette [ABC]B1) has proceeded with great vigour. However, it soon became apparent that P-glycoprotein was not expressed in all drug-resistant cells that displayed an accumulation deficiency, which led to the discovery of other ABC transporters involved in drug efflux. In 1992, the multidrug resistance-associated protein (MRP1, ABCC1) was identified in small cell lung cancer followed by breast cancer resistance protein (mitoxantrone resistance protein, ABCG2) in 1999. After three decades of research, can we confidently define the contribution of multidrug resistance transporters to chemoresistance and do we have clinically useful drugs to sensitise cancers?     

Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense

In tumor cell lines, multidrug resistance is often associated with an ATP-dependent decrease in cellular drug accumulation which is attributed to the overexpression of certain ATP-binding cassette (ABC) transporter proteins. ABC proteins that confer drug resistance include (but are not limited to) P-glycoprotein (gene symbol ABCB1), the multidrug resistance protein 1 (MRP1, gene symbol ABCC1), MRP2 (gene symbol ABCC2), and the breast cancer resistance protein (BCRP, gene symbol ABCG2). In addition to their role in drug resistance, there is substantial evidence that these efflux pumps have overlapping functions in tissue defense. Collectively, these proteins are capable of transporting a vast and chemically diverse array of toxicants including bulky lipophilic cationic, anionic, and neutrally charged drugs and toxins as well as conjugated organic anions that encompass dietary and environmental carcinogens, pesticides, metals, metalloids, and lipid peroxidation products. P-glycoprotein, MRP1, MRP2, and BCRP/ABCG2 are expressed in tissues important for absorption (e.g., lung and gut) and metabolism and elimination (liver and kidney). In addition, these transporters have an important role in maintaining the barrier function of sanctuary site tissues (e.g., blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier or placenta). Thus, these ABC transporters are increasingly recognized for their ability to modulate the absorption, distribution, metabolism, excretion, and toxicity of xenobiotics. In this review, the role of these four ABC transporter proteins in protecting tissues from a variety of toxicants is discussed. Species variations in substrate specificity and tissue distribution of these transporters are also addressed since these properties have implications for in vivo models of toxicity used for drug discovery and development.     

Design, synthesis and structure-activity relationships of novel taxane-based multidrug resistance reversal agents

A series of novel taxane-based multidrug resistance (MDR) reversal agents (TRAs) has been designed and synthesized. Structure-activity relationship (SAR) study clearly indicates that modification of the C-7 position with hydrophobic arenecarbonylcinnamoyl groups brings about high potency against drug efflux mediated by P-glycoprotein (P-gp). Six TRAs exhibit ability to modulate a wide range of ATP-binding cassette (ABC) transporters, such as P-gp, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP), which may serve as novel broad-spectrum modulators of ABC transporters.     

Characterization and inhibition by a wide range of xenobiotics of organic anion excretion by primary human hepatocytes

Organic anion secretion by human hepatocytes was characterized using primary liver parenchymal cell cultures and the anionic fluorescent dye carboxy-2′,7′-dichlorofluorescein (CF). Probenecid, a well-known common blocker of the membrane transport process for anions, was shown to increase CF accumulation in primary human hepatocytes by inhibiting cellular CF efflux in a dose-dependent manner, thereby establishing the presence of an efflux system for organic anions in cultured hepatocytes. Outwardly directed transport of CF from hepatocytes was found to be temperature-dependent; it was not altered by changes in the ionic composition of the incubation medium used in efflux experiments. In addition to probenecid, various structurally and functionally unrelated xenobiotics such as glibenclamide, rifampicin, vinblastine, MK-571, indomethacin, and cyclosporin A were shown to inhibit secretion of CF by primary human hepatocytes, thus suggesting that organic anion excretion by human liver may be impaired by various drugs. Northern blot and Western blot analyses of the expression of multidrug resistance proteins (MRP), such as MRP1 and MRP2, which are known to mediate cellular outwardly directed transport of organic anions indicated that MRP2 was present at substantial levels in cultured human hepatocytes as well as in their in vivo counterparts, whereas MRP1 expression was only barely detectable. These results therefore suggest that MRP2, unlike MRP1, may contribute to the organic anion efflux system displayed by primary human hepatocytes and inhibited by a wide range of xenobiotics.     

Unaltered expression of multidrug resistance transporters in polycyclic aromatic hydrocarbon-resistant rat liver cells

Rat liver epithelial cells resistant to the chemical carcinogen 3MC, termed F258/3MC cells and generated by long-term exposure of parental F258 cells to the PAH, were characterized, especially with respect to expression of multidrug resistance transporters such as P-glycoprotein, MRP1 and MRP2. F258/3MC cells were found to be cross-resistant to other PAHs such as BP and dimethylbenz(a)anthracene but remained sensitive to known substrates of multidrug resistance efflux pumps such as doxorubicin and vincristine. They did not display either decreased cellular PAH accumulation or increased PAH efflux. In addition, P-glycoprotein and MRP2 mRNA levels were not, or only barely detected, in F258/3MC cells and in their parental counterparts whereas these PAH-resistant and sensitive cells showed closed levels of MRP1 mRNAs and activity. Moreover, P-gp- and MRP1-overexpressing cells were shown to display similar accumulation and efflux of BP than those found in P-gp- and MRP1-negative control cells. These data therefore suggest that multidrug resistance transporters do not contribute to PAH resistance in PAH-selected liver cells.     

Specific interactions of chloroacetanilide herbicides with human ABC transporter proteins

Chloroacetanilide herbicides are among the most commonly used herbicides in agriculture. Several studies have demonstrated a number of them to be carcinogenic. ATP binding cassette (ABC) transporters are efflux pumps expressed in cell membranes, which form an important wall of defense against xenobiotics from different sources. We tested the interaction of the herbicides acetochlor, alachlor, dimetachlor, metazachlor, metolachlor, propachlor and prynachlor with human multidrug resistance transporters MDR1, MRP1, MRP2 and BCRP. A number of metabolites were studied for interaction with MRP1, MRP2 and MRP3. Transporter interactions were studied by measuring ATPase activity, inhibition of fluorescent dye efflux and vesicular transport. Also inhibition of MDR1 was monitored by measuring digoxin transport on Caco-2 monolayers and paclitaxel toxicity on K562-MDR cells. Acetochlor, alachlor, metolachlor and metazachlor showed specific interactions with MDR1. Digoxin permeability and paclitaxel cytotoxicity studies revealed that these herbicides are potent inhibitors of MDR1 that can modulate drug absorption and cause chemosensitization of cells. MRP1 was demonstrated to transport an important intermediate of the acetochlor detoxification pathway. Several specific interactions were shown when studying the interaction of chloroacetanilides with human transporter proteins. This study suggests an important role for transporter proteins in hazard prediction of agrochemicals and demonstrates how transporter interactions can be easily detected using in vitro screening methods.     

Inhibitors of cancer cell multidrug resistance mediated by breast cancer resistance protein (BCRP/ABCG2)

Breast cancer resistance protein (BCRP/ABCG2) belongs to the ATP-binding cassette (ABC) transporter superfamily. It is able to efflux a broad range of anti-cancer drugs through the cellular membrane, thus limiting their anti-proliferative effects. Due to its relatively recent discovery in 1998, and in contrast to the other ABC transporters P-glycoprotein (MDR1/ABCB1) and multidrug resistance-associated protein (MRP1/ABCC1), only a few BCRP inhibitors have been reported. This review summarizes the known classes of inhibitors that are either specific for BCRP or also inhibit the other multidrug resistance ABC transporters. Information is presented on structure-activity relationship aspects and how modulators may interact with BCRP.     

Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development

Human contains 49 ATP-binding cassette (ABC) transporter genes and the multidrug resistance associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP4/ABCC4, MRP5/ABCC5, MRP6/ABCC6, MRP7/ABCC10, MRP8/ABCC11 and MRP9/ABCC12) belong to the ABCC family which contains 13 members. ABCC7 is cystic fibrosis transmembrane conductance regulator; ABCC8 and ABCC9 are the sulfonylurea receptors which constitute the ATP-sensing subunits of a complex potassium channel. MRP10/ABCC13 is clearly a pseudo-gene which encodes a truncated protein that is highly expressed in fetal human liver with the highest similarity to MRP2/ABCC2 but without transporting activity. These transporters are localized to the apical and/or basolateral membrane of the hepatocytes, enterocytes, renal proximal tubule cells and endothelial cells of the blood-brain barrier. MRP/ABCC members transport a structurally diverse array of important endogenous substances and xenobiotics and their metabolites (in particular conjugates) with different substrate specificity and transport kinetics. The human MRP/ABCC transporters except MRP9/ABCC12 are all able to transport organic anions, such as drugs conjugated to glutathione, sulphate or glucuronate. In addition, selected MRP/ABCC members may transport a variety of endogenous compounds, such as leukotriene C(4) (LTC(4) by MRP1/ABCC1), bilirubin glucuronides (MRP2/ABCC2, and MRP3/ABCC3), prostaglandins E1 and E2 (MRP4/ABCC4), cGMP (MRP4/ABCC4, MRP5/ABCC5, and MRP8/ABCC11), and several glucuronosyl-, or sulfatidyl steroids. In vitro, the MRP/ABCC transporters can collectively confer resistance to natural product anticancer drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and in concert with alterations in phase II conjugating or biosynthetic enzymes, classical alkylating agents, alkylating agents. Several MRP/ABCC members (MRPs 1-3) are associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. Drug targeting of these transporters to overcome MRP/ABCC-mediated multidrug resistance may play a role in cancer chemotherapy. Most MRP/ABCC transporters are subject to inhibition by a variety of compounds. Based on currently available preclinical and limited clinical data, it can be expected that modulation of MRP members may represent a useful approach in the management of anticancer and antimicrobial drug resistance and possibly of inflammatory diseases and other diseases. A better understanding of their substrates and inhibitors has important implications in development of drugs for treatment of cancer and inflammation.