Activity Of Multidrug Resistance Protein Research Articles

MDR1 mediated chemoresistance: BMI1 and TIP60 in action

Chemotherapy-induced emergence of drug resistant cells is frequently observed and is exemplified by the expression of family of drug resistance proteins including, multidrug resistance protein 1 (MDR1). However, a concise mechanism for chemotherapy-induced MDR1 expression is unclear. Mechanistically, mutational selection, epigenetic alteration, activation of the Wnt pathway or impaired p53 function have been implicated. The present study describes that the surviving fraction of cisplatin resistant cells co- upregulate MDR1, BMI1 and acetyl transferase activity of TIP60. Using complementary gain and loss of function approaches, we demonstrate that the expression of MDR1 is positively regulated by BMI1, a stem-cell factor classically known as a transcriptional repressor. Our study establishes a functional interaction between TIP60 and BMI-1 resulting in upregulation of MDR1 expression. Chromatin immunoprecipitation (ChIP) assays further establish that the proximal MDR1 promoter responds to cisplatin in a BMI1 dependent manner. BMI1 interacts with a cluster of E-box elements on the MDR1 promoter and recruits TIP60 resulting in acetylation of histone H2A and H3. Collectively, our data establish a hitherto unknown liaison among MDR1, BMI1 and TIP60 and provide mechanistic insights into cisplatin-induced MDR1 expression resulting in acquired cross-resistance against paclitaxel, doxorubicin and likely other drugs. In conclusion, our results advocate utilizing anti-BMI1 strategies to alleviate acquired resistance to chemotherapy.

Inhibition of ABC transport proteins by oil sands process affected water

The ATP-binding cassette (ABC) superfamily of transporter proteins is important for detoxification of xenobiotics. For example, ABC transporters from the multidrug-resistance protein (MRP) subfamily are important for excretion of polycyclic aromatic hydrocarbons (PAHs) and their metabolites. Effects of chemicals in the water soluble organic fraction of relatively fresh oil sands process affected water (OSPW) from Base Mine Lake (BML-OSPW) and aged OSPW from Pond 9 (P9-OSPW) on the activity of MRP transporters were investigated in vivo by use of Japanese medaka at the fry stage of development. Activities of MRPs were monitored by use of the lipophilic dye calcein, which is transported from cells by ABC proteins, including MRPs. To begin to identify chemicals that might inhibit activity of MRPs, BML-OSPW and P9-OSPW were fractionated into acidic, basic, and neutral fractions by use of mixed-mode sorbents. Chemical compositions of fractions were determined by use of ultrahigh resolution orbitrap mass spectrometry in ESI+ and ESI− mode. Greater amounts of calcein were retained in fry exposed to BML-OSPW at concentration equivalents greater than 1× (i.e., full strength). The neutral and basic fractions of BML-OSPW, but not the acidic fraction, caused greater retention of calcein. Exposure to P9-OSPW did not affect the amount of calcein in fry. Neutral and basic fractions of BML-OSPW contained relatively greater amounts of several oxygen-, sulfur, and nitrogen-containing chemical species that might inhibit MRPs, such as O+, SO+, and NO+ chemical species, although secondary fractionation will be required to conclusively identify the most potent inhibitors. Naphthenic acids (O2−), which were dominant in the acidic fraction, did not appear to be the cause of the inhibition. This is the first study to demonstrate that chemicals in the water soluble organic fraction of OSPW inhibit activity of this important class of proteins. However, aging of OSPW attenuates this effect and inhibition of the activity of MRPs by OSPW from Base Mine Lake does not occur at environmentally relevantconcentrations.

Multidrug resistance protein 1 reduces the aggregation of mutant huntingtin in neuronal cells derived from the Huntington's disease R6/2 model.

Mutant huntingtin (mHtt) aggregation in the nucleus is the most readily apparent phenotype and cause of neuronal death in Huntington’s disease (HD). Inhibiting mHtt aggregation reduces cell death in the brain and is thus a promising therapeutic approach. The results of the present study demonstrated that mHtt aggregation in the nucleus was altered by the activity of multidrug resistance protein 1 (MDR1), which was experimentally modulated by verapamil, siRNA and an expression vector. MDR1 detoxifies drugs and metabolites through its excretory functions in the membrane compartment, thereby protecting cells against death or senescence. When they were treated with verapamil, R6/2 mice showed a progressive decline in rotarod performance and increased mHtt aggregation in the brain. Using neuronal stem cells from R6/2 mice, we developed an in vitro HD model to test mHtt accumulation in the nuclei of neurons. When MDR1 activity in cells was decreased by verapamil or siRNA, mHtt aggregation in the nuclei increased, whereas the induction of MDR1 resulted in a decrease in mHtt aggregation. Thus, our data provide evidence that MDR1 plays an important role in the clearance of mHtt aggregation and may thus be a potential target for improving the survival of neurons in Huntington’s disease.

Decreased functional activity of multidrug resistance protein in primary colorectal cancer.

BackgroundThe ATP-Binding Cassette (ABC)-transporter MultiDrug Resistance Protein 1 (MDR1) and Multidrug Resistance Related Protein 1 (MRP1) are expressed on the surface of enterocytes, which has led to the belief that these high capacity transporters are responsible for modulating chemosensitvity of colorectal cancer. Several immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) studies have provided controversial results in regards to the expression levels of these two ABC-transporters in colorectal cancer. Our study was designed to determine the yet uninvestigated functional activity of MDR1 and MRP1 transporters in normal human enterocytes compared to colorectal cancer cells from surgical biopsies.Methods100 colorectal cancer and 28 adjacent healthy mucosa samples were obtained by intraoperative surgical sampling. Activity of MDR1 and MRP1 of viable epithelial and cancer cells were determined separately with the modified calcein-assay for multidrug resistance activity and sufficient data of 73 cancer and 11 healthy mucosa was analyzed statistically.ResultsSignificantly decreased mean MDR1 activity was found in primary colorectal cancer samples compared to normal mucosa, while mean MRP1 activity showed no significant change. Functional activity was not affected by gender, age, stage or grade and localization of the tumor.ConclusionWe found lower MDR activity in cancer cells versus adjacent, apparently, healthy control tissue, thus, contrary to general belief, MDR activity seems not to play a major role in primary drug resistance, but might rather explain preferential/selective activity of Irinotecan and/or Oxaliplatin. Still, this picture might be more complex since chemotherapy by itself might alter MDR activity, and furthermore, today limited data is available about MDR activity of cancer stem cells in colorectal cancers.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1675739129145824

Mutational studies to understand the structure–function relationship in multidrug efflux transporters: Applications for distinguishing mutants with high specificity

Multidrug transporters play key roles for drug resistance, mediating the transport of organic compounds and substrate recognition. The specificity of substrates and functions of multidrug transporters are affected with amino acid substitutions. Hence, it is important to understand the effect of mutations in multidrug resistance proteins on transport function and substrate specificity. In this work, we have analyzed the relationship between amino acid properties and activity of multidrug resistance proteins upon mutations and substrates. We found that the properties for drug activity and kinetic factors depend on amino acid substitutions and specific to substrates. The inclusion of information from neighboring residues from the mutants enhanced our understanding to the activity of multidrug resistant proteins. Further, we have combined amino acid properties using multiple regression technique, which showed a correlation of up to 0.99 between amino acid properties and activity. In addition, we have utilized Naïve Bayes classifier for distinguishing between decrease and increase in IC50 upon mutations using wild type, mutant and neighboring residues, which showed a 10-fold cross-validation accuracy of 86%. Further, we have developed multiple regression models for predicting IC50 upon mutations with a maximum correlation of 0.92. The present method could be used for identifying the mutants in multidrug resistant proteins with enhanced specificity.

Differential involvement of glutathione S-transferase mu 1 and multidrug resistance protein 1 in melanoma acquired resistance to vinca alkaloids.

On account of its extreme intrinsic resistance to apoptosis and of its strong ability to become chemoresistant after a primary response to drugs, malignant melanoma (MM) is still a therapeutic challenge. We previously showed that glutathione S-transferase mu 1 (GSTM1) acts in synergy with multidrug resistance protein 1 (MRP1) to protect GSTM1-transfected human CAL1 melanoma cells from toxic effects of vincristine (VCR). Herein, we investigated the role of these proteins in the acquired resistance of CAL1 cells to vinca alkaloids (VAs). Resistant lines were established by continuous exposure (>1year) of parental CAL1-wt cells to VCR, vindesine (VDS), or vinorelbine (VRB): CAL1R-VCR, CAL1R-VDS, CAL1R-VRB, respectively. All resistant lines displayed more than 10-fold increase in resistance to their selection VA, and specifically expressed GSTM1. Suggesting a direct interaction between this protein and VAs, each VA specifically decreased the GSTM1-mediated glutathione conjugation activity in cell lysates. Curcumin (GSTM1 inhibitor), BSO (glutathione synthesis inhibitor), and MK571 (MRP1 inhibitor) considerably reversed the acquired resistance to VCR and VDS, but not to VRB. Microarray data analysis revealed similar gene expression patterns of CAL1R-VCR and CAL1R-VDS, and a distinct one for CAL1R-VRB. These data suggest a differential involvement of GSTM1 and MRP1 in acquired resistance to VAs. A coordinated expression and activity of GSTM1 and MRP1 is required to protect CAL1 cells from VCR and VDS, while the simple expression of GSTM1 is sufficient, possibly by a direct drug/protein interaction, to confer resistance against VRB.

Folates provoke cellular efflux and drug resistance of substrates of the multidrug resistance protein 1 (MRP1)

Cellular folate concentration was earlier reported to be a critical factor in the activity and expression of the multidrug resistance protein MRP1 (ABCC1). Since MRP1 mediates resistance to a variety of therapeutic drugs, we investigated whether the cellular folate concentration influences the MRP1-mediated cellular resistance against drugs. As a model system, we used the human ovarian carcinoma cell line 2008wt, and its stably MRP1/ABCC1-transfected subline 2008/MRP1. These cell types have a moderate and high expression of MRP1, respectively. In folate-deprived 2008/MRP1 cells, the MRP1-mediated efflux of its model substrate calcein decreased to ~55 % of the initial efflux rate under folate-rich conditions. In 2008wt cells, only a small decrease in efflux was observed. Folate depletion for 5-10 days markedly increased (~500 %) cellular steady-state accumulation of calcein in 2008/MRP1 cells and moderately in 2008wt cells. A subsequent short (24 h) exposure to 2.3 μM L-leucovorin decreased calcein levels again in MRP1-overexpressing cells. Folate deprivation markedly increased growth inhibitory effects of the established MRP1 substrates daunorubicin (~twofold), doxorubicin (~fivefold), and methotrexate (~83-fold) in MRP1-overexpressing cells, proportional to MRP1 expression. In conclusion, this study demonstrates that increased cellular folate concentrations induce MRP1/ABCC1-related drug efflux and drug resistance. These results have important implications in the understanding of the role of MRP1 and its homologs in clinical drug resistance.

MDR1 expression predicts outcome of Ph+ chronic phase CML patients on second-line nilotinib therapy after imatinib failure

In the face of competing tyrosine kinase inhibitors (TKIs), identification of chronic myeloid leukemia (CML) patients expecting favorable response to second-line treatment is warranted. At the time of imatinib resistance, the investigation of multidrug-resistance protein 1 (MDR1) and BCR-ABL yielded the following results: (i) Patients with high MDR1 transcript levels showed superior response at 48 months as compared with low-level MDR1 patients: major molecular response (MMR) in 41% vs 16% (P=0.014), complete cytogenetic response (CCyR) in 58% vs 39% (P=0.044), and progression-free survival (PFS) in 67% vs 46% (P=0.032). (ii) Patients with BCR-ABL(IS) <28% achieved higher MMR rates (48% vs 21%, P=0.009). (iii) PFS at 48 months was associated with in vitro resistance of BCR-ABL kinase domain mutations: 63% (no mutation) vs 61% (sensitive, intermediately sensitive or unknown IC50 (median inhibitory concentration)) vs 23% (resistant, P=0.01). (iv) Single-nucleotide polymorphisms (SNPs) at positions 1236 and 2677 were associated with higher MDR1 expression in comparison to wild type. (v) Nilotinib was able to impede proliferation of MDR1-overexpressing imatinib-resistant cells. High MDR1 gene expression might identify patients whose mode of imatinib resistance is essentially determined by increased efflux activity of MDR1 and therefore can be overcome by second-line nilotinib treatment.

Drug Resistance Mechanisms in Non-Small Cell Lung Carcinoma

Lung cancer is the most commonly diagnosed cancer in the world. “Driver” and “passenger” mutations identified in lung cancer indicate that genetics play a major role in the development of the disease, progression, metastasis and response to therapy. Survival rates for lung cancer treatment have remained stagnant at ~15% over the past 40 years in patients with disseminated disease despite advances in surgical techniques, radiotherapy and chemotherapy. Resistance to therapy; either intrinsic or acquired has been a major hindrance to treatment leading to great interest in studies seeking to understand and overcome resistance. Genetic information gained from molecular analyses has been critical in identifying druggable targets and tumor profiles that may be predictors of therapeutic response and mediators of resistance. Mutated or overexpressed epidermal growth factor receptor (EGFR) and translocations in the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) genes (EML4-ALK) are examples of genetic aberrations resulting in targeted therapies for both localized and metastatic disease. Positive clinical responses have been noted in patients harboring these genetic mutations when treated with targeted therapies compared to patients lacking these mutations. Resistance is nonetheless a major factor contributing to the failure of targeted agents and standard cytotoxic agents. In this review, we examine molecular mechanisms that are potential drivers of resistance in non-small cell lung carcinoma, the most frequently diagnosed form of lung cancer. The mechanisms addressed include resistance to molecular targeted therapies as well as conventional chemotherapeutics through the activity of multidrug resistance proteins.

Targeting Multidrug Resistance Protein 1 (MRP1, ABCC1): Past, Present, and Future

The human ATP-binding cassette transporter multidrug resistance protein 1 (MRP1), encoded by ABCC1, was initially identified because of its ability to confer multidrug resistance in lung cancer cells. It is now established that MRP1 plays a role in protecting certain tissues from xenobiotic insults and that it mediates the cellular efflux of the proinflammatory cysteinyl leukotriene C4 as well as a vast array of other endo- and xenobiotic organic anions. Many of these are glutathione (GSH) or glucuronide conjugates, the products of Phase II drug metabolism. MRP1 also plays a role in the cellular efflux of the reduced and oxidized forms of GSH and thus contributes to the many physiological and pathophysiological processes influenced by these small peptides, including oxidative stress. In this review, the pharmacological and physiological aspects of MRP1 are considered in the context of the current status and future prospects of pharmacological and genetic modulation of MRP1 activity.

Inhibition of Human MDR1 and BCRP Transporter ATPase Activity by Organochlorine and Pyrethroid Insecticides

Despite the growing evidence suggesting that pesticides contribute to chronic diseases, there is a limited understanding of how these chemicals are removed from cells and whether pesticides can alter the disposition of drugs. The present study examined the effects of two classes of insecticides (organochlorine and pyrethroid) on the ATPase activity of the human multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) efflux transporters. Using plasma membranes from cells overexpressing MDR1 and BCRP, it was demonstrated that the organochlorine pesticide dichlorodiphenyltrichloroethane (DDT) (o,p'-DDT and p,p'-DDT isomers) as well as its metabolite (p,p'-dichlorodiphenyldichloroethane), inhibit both MDR1 and BCRP ATPase activity. In addition, p,p'-dichlorodiphenyldichloroethylene, and two pyrethroid pesticides inhibited BCRP ATPase activity between 4 and 7 μM. Additional research is necessary to further characterize the functional inhibition of MDR1 and BCRP activity and determine whether pesticides alter the transporter-mediated disposition of other chemicals.

Characterization and Resistance Mechanisms of A 5-fluorouracil-resistant Hepatocellular Carcinoma Cell Line

The chemoresistance of human hepatocellular carcinoma (HCC) to cytotoxic drugs, especially intrinsic or acquired multidrug resistance (MDR), still remains a major challenge in the management of HCC. In the present study, possible mechanisms involved in MDR of HCC were identified using a 5-fluorouracil (5-FU) -resistant human HCC cell line. BEL-7402/5-FU cells were established through continuous culturing parental BEL-7402 cells, imitating the pattern of chemotherapy clinically. Growth curves and chemosensitivity to cytotoxic drugs were determined by MTT assay. Doubling times, colony formation and adherence rates were calculated after cell counting. Morphological alteration, karyotype morphology, and untrastructure were assessed under optical and electron microscopes. The distribution in the cell cycle and drug efflux pump activity were measured by flow cytometry. Furthermore, expression of potential genes involved in MDR of BEL-7402/5-FU cells were detected by immunocytochemistry. Compared to its parental cells, BEL-7402/5-FU cells had a prolonged doubling time, a lower mitotic index, colony efficiency and adhesive ability, and a decreased drug efflux pump activity. The resistant cells tended to grow in clusters and apparent changes of ultrastructures occurred. BEL-7402/5-FU cells presented with an increased proportion in S and G2/M phases with a concomitant decrease in G0/G1 phase. The MDR phenotype of BEL-7402/5-FU might be partly attributed to increased drug efflux pump activity via multidrug resistance protein 1 (MRP1), overexpression of thymidylate synthase (TS), resistance to apoptosis by augmentation of the Bcl-xl/Bax ratio, and intracellular adhesion medicated by E-cadherin (E-cad). P-glycoprotein (P-gp) might play a limited role in the MDR of BEL-7402/5-FU. Increased activity or expression of MRP1, Bcl-xl, TS, and E-cad appear to be involved in the MDR mechanism of BEL-7402/5-FU.

Gender-related differences in drug effects: The role of multidrug resistance proteins

Mechanisms of the involvement of multidrug resistance proteins (MRPs) in the formation of gender-related differences in drug effectiveness and adverse effects have been analyzed. The structure, tissue and cellular localization, substrate specificity, and functions of MRPs are discussed. The regulation of MRP expression and activity by endogenous metabolites, signaling compounds, including sex hormones, and pharmaceutical agents is described. Nuclear receptors have been shown to play a role in the regulation of MRP expression. Data on gender-related differences in the expression of MRPs and nuclear receptors that are involved in the induction of MRPs in the liver and kidney are presented. It is concluded that gender-related differences in the expression and functional activity of MRPs in excretory organs (the liver and kidney) may significantly contribute to gender dependence of drug pharmacokinetics and effectiveness.

Expression and Function of Multidrug Resistance Protein 1 and Multidrug Resistance-Associated Protein 1 in Lung Dendritic Cells From Aging Mice

Multidrug resistance protein 1 and multidrug resistance-associated protein 1 are transporters that efflux diverse xenobiotics from cells. We investigated changes in the expression and activity of multidrug resistance protein 1 and multidrug resistance-associated protein 1 in highly purified lung dendritic cells (LDCs) during aging using magnetic and flow cytometric cell sorting. Multidrug resistance protein 1 blockade by the specific inhibitor reduced the percentage of rhodamine 123(low) cells in LDCs from aged mice (54.8% ± 2.6% to 13.2% ± 2.5%, p < .01). The difference in the proportions of rhodamine 123(low) cells in aged LDCs was more apparent than that in LDCs from young mice (p < .05). The multidrug resistance-associated protein 1-specific inhibitor reduced the percentage of Fluo-3(low) cells in aged LDCs (60.8% ± 5.3% to 25.8% ± 7.5%, p < .01). The difference in the proportions of Fluo-3(low) cells in aged LDCs was smaller than that in young LDCs (p < .05). These data showed that LDCs from aged mice exhibited multidrug resistance protein 1- and multidrug resistance-associated protein 1-mediated efflux and that the age-associated changes differed according to transporters.

Detection and characterisation of multi-drug resistance protein 1 (MRP-1) in human mitochondria.

Background:Overexpression of plasma membrane multi-drug resistance protein 1 (MRP-1) can lead to multidrug resistance. In this study, we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues.Methods:MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased using retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-propidium iodide labelling of cells.Results:MRP-1 was detected in the mitochondria of cancer and normal cells and tissues. The efflux activity of mitochondrial MRP-1 was more efficient (55–64%) than that of plasma membrane MRP-1 (11–22% P<0.001). Induced MRP-1 expression resulted in a preferential increase in mitochondrial MRP-1, suggesting selective targeting to this organelle. Treatment with a non-lethal concentration of doxorubicin (0.85 nM, 8 h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. For the first time, we have identified MRP-1 with efflux activity in human mitochondria.Conclusion:Mitochondrial MRP-1 may be an exciting new therapeutic target where historically MRP-1 inhibitor strategies have limited clinical success.

Quercetin increased bioavailability and decreased methylation of green tea polyphenols in vitro and in vivo

The extensive methylation of green tea polyphenols (GTPs) in vivo may limit their chemopreventive potential. We investigated whether quercetin, a natural inhibitor of catechol-O-methyltransferase (COMT) and multidrug resistance proteins (MRPs), will differentially increase the intracellular concentration and decrease the methylation of GTPs in different cancer cell lines. Intrinsic COMT activity was lowest in lung cancer A549 cells, intermediate in kidney 786-O cells and highest in liver HepG2 cells. Quercetin increased the cellular absorption of epigallocatechin gallate (EGCG) four-fold in A549 cells with a decreased methylation rate from 63 to 19%, 2-fold in 786-O cells with a decreased methylation from 97% to 56%, while no significant effect was observed in HepG2 cells. The combination significantly decreased the activity and protein expression of COMT and decreased the protein expression of MRP1 compared to individual treatments. The combination exhibited the strongest increase in antiproliferation in A549 cells, an intermediate effect in 786-O cells and lowest effect in HepG2 cells. The effect of quercetin on bioavailability and metabolism of GTPs was confirmed in vivo. Severe combined immunodeficiency (SCID) mice were administered brewed green tea (GT) and a diet supplemented with 0.4% quercetin alone or in combination for 2 weeks. We observed a 2- to 3-fold increase of total and non-methylated EGCG in lung and kidney and an increasing trend in liver. In summary, combining quercetin with GT provides a promising approach to enhance the chemoprevention of GT. Responses of different cancers to the combination may vary by tissue depending on the intrinsic COMT and MRP activity.

Abstract B79: Multidrug resistance protein 1 (MRP-1) is expressed in the mitochondria of normal and cancer cells and may play a role in intrinsic drug resistance of ESFT cells.

Abstract Overexpression of plasma membrane MRP-1 can lead to multidrug resistance. In this study we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues and investigate the functional significance of mitochondrial MRP-1 with a view to identifying a new therapeutic starategy. MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Protein extracts were de-glycosylated using the Protein Deglycosylation Mix (NEB). Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased by retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-PI labelling of cells. Mitochondrial membranes were solubilised by incubation with increasing concentrations of the detergent digitonin and MRP-1 localisation within the mitochondria evaluated by Western blot; protein markers of the mitochondrial outer and inner membranes, the matrix and intermembrane space were used to confirm localisation within the mitochondria. For the first time we have detected MRP-1 in the mitochondria of untreated cancer and normal cells and tissues by Western blot and confirmed these findings by immunofluorescence and confocal microscopy. The size of the MRP-1 protein in the mitochondria of cancer and normal cells was between 150–250 kDa, reflecting differences in glycosylation. The efflux activity of mitochondrial MRP-1 was more efficient (55–64%) than plasma membrane MRP-1 (11–22%; p&amp;lt;0.001). Specific overexpression of MRP-1 in TC-32 cells by retroviral infection induced a preferential increase in mitochondrial MRP-1 (150%), resulting in an 85% increase in MRP-1 efflux activity (p&amp;lt;0.0001), suggesting selective targeting to this organelle. This was associated with an increase in the IC50 of the MRP-1 substrates vincristine (4.5nM to 14.7nM; p&amp;lt;0.001) and etoposide (101.7nM to 121.6nM (p&amp;lt;0.001) but not the non MRP-1 substrate actinomycin D (0.5nM). Treatment with a non-lethal concentration of doxorubicin (0.85nM, 8h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. The glycosylation of MRP-1 and the efflux of calcein-F from whole mitochondria are consistent with localisation of MRP-1 to the outer mitochondrial membrane. This was confirmed by the increased expression of MRP-1 protein in the mitochondrial supernatant after treatment with digitonin and centrifugation. The expression pattern of MRP-1 was similar to that of porin, an outer mitochondrial membrane marker. Analysis of the MRP-1 sequence (using Predator and MitoProtII) failed to reveal published mitochondrial targeting sequences, and so we are currently investigating the mechanism of MRP-1 transport to the mitochondria to identify a druggable therapeutic target. MRP-1 expressed in the outer mitochondrial membrane has increased efflux activity compared to plasma membrane MRP-1, and so represents an exciting new therapeutic target where historically conventional MRP-1 inhibitor strategies targeting plasma membrane MRP-1 have had limited clinical success. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B79.

Inactivation of Multidrug Resistance Proteins Disrupts Both Cellular Extrusion and Intracellular Degradation of cAMP

In addition to xenobiotics and several other endogenous metabolites, multidrug-resistance proteins (MRPs) extrude the second-messenger cAMP from various cells. Pharmacological and/or genetic inactivation of MRPs has been shown to augment intracellular cAMP signaling, an effect assumed to be a direct consequence of the blockade of cAMP extrusion. Here we provide evidence that the augmented intracellular cAMP levels are not due exclusively to the prevention of cAMP efflux because MRP inactivation is also associated with reduced cAMP degradation by phosphodiesterases (PDEs). Several prototypical MRP inhibitors block PDE activity at concentrations widely used to inhibit MRPs. Their dose-dependent effects in several paradigms of cAMP signaling are more consistent with their potency in inhibiting PDEs than MRPs. Moreover, genetic manipulation of MRP expression results in concomitant changes in PDE activity and protein levels, thus affecting cAMP degradation in parallel with cAMP efflux. These findings suggest that the effects of MRP inactivation on intracellular cAMP levels reported previously may be due in part to reduced degradation by PDEs and identify MRP-dependent transport mechanisms as novel regulators of cellular PDE expression levels. Mathematical simulations of cAMP signaling predict that selective ablation of MRP-dependent cAMP efflux per se does not affect bulk cytosolic cAMP levels, but may control cAMP levels in restricted submembrane compartments that are defined by small volume, high MRP activity, limited PDE activity, and limited exchange of cAMP with the bulk-cytosolic cAMP pool. Whether this regulation occurs in cells remains to be confirmed experimentally under conditions that do not affect PDE activity.

Expression and Function of Human MRP1 (ABCC1) Is Dependent on Amino Acids in Cytoplasmic Loop 5 and Its Interface with Nucleotide Binding Domain 2

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.

Inhibition of P-glycoprotein and multidrug resistance protein 1 by dietary phytochemicals

For the development of a safe and effective dual inhibitor of anticancer drug efflux transporters P-glycoprotein and multidrug resistance protein 1 (MRP1) to conquer multidrug resistance, we investigated the effects of dietary phytochemicals on the functions of P-glycoprotein and MRP1. The effects of dietary phytochemicals on the functions of P-glycoprotein and MRP1 were investigated using P-glycoprotein-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural compounds found in dietary supplements, herbs, and foods such as sesame, ginkgo, soybean, and licorice were evaluated. The accumulation of daunorubicin, a fluorescent substrate of P-glycoprotein, increased in the presence of sesamin, ginkgolic acid, matairesinol, glycyrrhetinic acid, glabridin, and phyllodulcin in KB-C2 cells. Glycyrrhetinic acid and matairesinol also increased the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. KB-C2 and KB/MRP cells were sensitized to anticancer drugs by glycyrrhetinic acid, showing that glycyrrhetinic acid reverses multidrug resistance. The verapamil-stimulated P-glycoprotein ATPase activity was inhibited by glycyrrhetinic acid. Glycyrrhetinic acid stimulated the ATPase activity of MRP1. These results suggest that dietary phytochemicals, such as glycyrrhetinic acid found in licorice, have dual inhibitory effects on P-glycoprotein and MRP1 and might become useful to enhance the efficacy of cancer chemotherapy.