Role Of Multidrug Resistance Proteins Research Articles

Role of Multidrug Resistance Proteins in Nonresponders to Immunomodulatory Therapy for Noninfectious Uveitis.

PurposeNearly a third to half of patients with noninfectious uveitis (NIU) fail to achieve control with immunomodulatory therapy (IMT). Multidrug resistance (MDR) proteins are transmembrane proteins that allow efflux of intracellular drugs, leading to drug resistance. The aim of our study was to compare MDR protein function in blood CD4+ cells between responders and nonresponders to IMT.MethodsWe included NIU patients on IMT for ≥6 months and corticosteroid dose ≤10 mg/d. Nonresponders to treatment were those with worsening (two or more steps) of inflammation in the past 3 months on full-dose immunosuppressive therapy. MDR function was assessed by Rhodamine-123 dye retention in blood CD4+ cells. Three nonresponders were treated with adjunctive oral cyclosporine A (CSA, MDR inhibitor) therapy for 2 months and reevaluated.ResultsFourteen NIU patients were recruited. Most (n = 8) had Vogt-Koyanagi-Harada disease. These included nine nonresponders and five responders to IMT. Nonresponders produced significantly higher MDR function and proinflammatory cytokines (interferon γ, tumor necrosis factor α, interleukin 17, and Granulocyte Macrophage Colony Stimulating Factor (GM-CSF)) than responders. In vitro CSA treatment of CD4+ cells inhibited MDR expression and proinflammatory cytokine production while increasing Foxp3. Finally, adjunctive oral CSA therapy led to improvement in clinical inflammatory scores with a concurrent decrease in MDR function and proinflammatory cytokine secretion.ConclusionsMDR function is significantly higher in CD4+ T cells of nonresponders to IMT. Adjunctive CSA therapy may decrease MDR function and allow improvement in treatment response to IMT.Translational RelevanceOur study highlights the need for MDR inhibition strategies in NIU patients not responding to IMT for improving the efficacy of anti-inflammatory therapy.

Investigation of the role of multidrug resistance proteins (MRPs) in vascular homeostasis

Cellular levels of cyclic GMP (cGMP) are tightly controlled by synthetic and degradative mechanisms. Pharmacological manipulation of these processes (e.g. soluble guanylate cyclase stimulators and phosphodiesterase 5 inhibitors) augments cGMP-dependent signalling and is beneficial in treating cardiovascular disease (eg. pulmonary hypertension). An additional mechanism potentially important in the inactivation of cGMP is cellular extrusion, driven by a family of multidrug resistance proteins (MRPs). Herein, we investigated if MRPs modulate vascular reactivity, smooth muscle cell proliferation, and systemic hemodynamics. The functional reactivity of murine aortic rings and proliferation of human pulmonary artery smooth muscle cells (PASMC) were determined in the absence and presence of the MRP inhibitor MK571. Hemodynamic changes in vivo in response to MK571 were analysed acutely by bolus dosing and chronically by radiotelemetry. MK571 (1nM-50µM) caused a concentration-dependent relaxation of mouse aortic r...

Investigation of the Role of Multidrug Resistance Proteins (MRPs) in Vascular Homeostasis

Cellular levels of cyclic GMP (cGMP) are tightly controlled by synthetic and degradative mechanisms. Pharmacological manipulation of these processes (e.g. soluble guanylate cyclase stimulators and phosphodiesterase 5 inhibitors) augments cGMP-dependent signalling and is beneficial in treating cardiovascular disease (eg. pulmonary hypertension). An additional mechanism potentially important in the inactivation of cGMP is cellular extrusion, driven by a family of multidrug resistance proteins (MRPs). Herein, we investigated if MRPs modulate vascular reactivity, smooth muscle cell proliferation, and systemic hemodynamics. The functional reactivity of murine aortic rings and proliferation of human pulmonary artery smooth muscle cells (PASMC) were determined in the absence and presence of the MRP inhibitor MK571. Hemodynamic changes in vivo in response to MK571 were analysed acutely by bolus dosing and chronically by radiotelemetry. MK571 (1nM-50µM) caused a concentration-dependent relaxation of mouse aortic rings. In the presence of a threshold concentration of MK571 (3µM) vasorelaxant responses to NO and atrial natriuretic peptide were significantly augmented. MK571 (1nM-3µM) also caused a significant inhibition of PASMC growth. In vivo, MK571 (0.001-10mg/kg; iv.) elicited an acute, dose-dependent hypotensive activity and when delivered via the drinking water, caused a more sustained drop in mean arterial pressure (~5mmHg). These data suggest that extrusion by MRPs contributes to the dynamic equilibrium regulating intracellular levels of cGMP, and may represent a target amenable to drug intervention for the treatment of cardiovascular disease. Funded by the MRC

Oncogenic BRAF inhibitor UAI-201 induces cell cycle arrest and autophagy in BRAF mutant glioma cells

AimsAn activating mutation of BRAF (BRAF-V600E) has been reported in a subset of malignant brain tumors. Thus, the aim of the present study was to identify the antiproliferative effect of the new oncogenic B-Raf targeting drug UAI-201 on 6 types of glioma cell lines with differing B-Raf mutational status. Main methodsThe IC50 values of UAI-201 were determined using crystal violet assays in six glioma cell lines. Real-time RT-PCR was performed to assess the functional role of multidrug resistance proteins in response to UAI-201. The effects of UAI-201 on six glioma cells were further examined by immunoblotting analysis, cell cycle analysis, flow cytometric apoptotic assay and autophagy assay. To identify the role of autophagy in UAI-201-induced growth inhibition, Atg5 and Beclin 1 were knocked down by RNA interference. Key findingsReal-time RT-PCR analysis showed a poor correlation between UAI-201 activity and the expression level of multidrug resistance proteins. The growth inhibitory effects of UAI-201 correlated with the BRAF-V600E genotype of the glioma cell lines. BRAF blockade with UAI-201 resulted in dose-dependent inhibition of MEK/ERK phosphorylations and increased G0/G1 arrest in glioma cells with BRAF-V600E. Interestingly, UAI-201 preferentially induced autophagy in BRAF-V600E cells, but not in BRAF-WT cells. More notably, autophagy inhibition through siRNA-mediated Beclin 1 knockdown partially attenuated the growth inhibition induced by UAI-201 in BRAF-V600E cells. SignificanceThe pro-death autophagic processes could be one of the underlying mechanisms for the sensitization of BRAF-V600E glioma cells toward UAI-201.

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.

Abstract 3241: The selective inhibition of GST by CAPE enhances the cytotoxicity of doxorubicin and camptotechin in SK-MEL-28 melanoma cells

Abstract Glutathione S-transferase (GST) and multidrug resistance proteins (MRPs) play major roles in the detoxification of anti-melanoma drugs; hence diminishing their toxicity towards melanoma cells. In this study, we investigated i) the selective inhibition of GST by Caffeic Acid Phenethyl Ester (CAPE) in presence of tyrosinase, ii) the role of MRP in CAPE induced toxicity, and iii) the effect of CAPE in combination with probenecid, doxorubicin and camptotechin. The inhibition of GST was investigated using CDNB method. LC/MS/MS was used to identify a CAPE glutathione conjugate formation by tyrosinase. MTT assay, Annexin V apoptosis assay and TMRM florescence method were used in the investigation of biochemical mechanism of CAPE efficacy in human SK-MEL-28 melanoma cells. At 30 min incubation, CAPE was bioactivated 65% by tyrosinase as measured by glutathione depletion. LC/MS/MS analyses identified CAPE-glutathione conjugate m/z [MH]+ 590 as the major metabolite formed as the result of CAPE bioactivation by tyrosinase. In the presence of tyrosinase, CAPE 10-25 µM showed 47-90% GST inhibition whereas in the absence of tyrosinase, CAPE 10-25 µM led to minimal GST inhibition. Both CAPE-SG conjugate and CAPE-quinone (25 µM) demonstrated ≥90% GST inhibition via reversible and irreversible competitive mechanisms with Ki of 5 µM with respect to GSH and with Ki of 6 µM with respect to CDNB, respectively. CAPE, a non-substrate for GST, only at concentrations >50 µM acted as a weak reversible non-competitive and competitive GST inhibitor with Ki of 277 µM with respect to GSH and with Ki of 234 µM with respect to CDNB, respectively. Moreover, computational auto docking analyses suggest that CAPE binds to the GST catalytic active site. CAPE (15-100 µM) showed 61-86% GST inhibition in human SK-MEL-28 cell homogenate. Furthermore, probenecid, a MRP inhibitor, increased CAPE (15, 30 µM) cell toxicity by 27-29%, apoptotic cell death by 10-14%, and decreased mitochondrial membrane potential by 27-40% in SK-MEL-28 melanoma cells, suggesting that the inhibition of MRP increases CAPE induced toxicity. The combination of CAPE (5 µM) with doxorubicin (0.1 µM) demonstrated synergistic effects as the combination index was 0.53 (<1). Similarly, CAPE (7.5 µM) showed synergistic effects with camptotechin (0.05 µM) with a combination index of 0.75. In summary, our results suggest that CAPE was bioactivated by tyrosinase to form a CAPE-quinone and CAPE glutathione conjugate, which played a major role in the inhibition of GST. Our study also elucidates the role of MRP in the biochemical mechanism of CAPE induced toxicity in melanoma cells. For the first time, we showed that CAPE in the presence of tyrosinase acts as a selective inhibitor of GST. CAPE therapy in combination with probencid, doxorubicin and camptotechin has synergistic effects in human SK-MEL-28 melanoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3241. doi:10.1158/1538-7445.AM2011-3241

The membrane transport of flavonoids from Crossostephium chinense across the Caco‐2 monolayer

The permeation and efflux of six polysubstituted flavonoids isolated from Crossostephium chinense, a Chinese traditional and herbal drug for the treatment of diabetes, were investigated using the Caco-2 cell monolayer. The six flavonoids (selagin, apometzgerin, tricetin-3',4',5'-trimethylether, quercetagetin-3,6,7-trimethylether, hispidulin and quercetagetin) sharing a similar parent skeleton structure with varied substituents in the heterocyclic ring B were selected for the study. Quercetagetin exhibited a low bi-directional permeability comparable to that of atenolol, suggesting a paracellular diffusion mechanism. The remaining compounds exhibited time- and concentration- dependent permeation with apparent permeability coefficient (P(app)) values in the range 10⁻⁶-10⁻⁵ cm/s, suggesting transcellular diffusion pathways. Selagin exhibited significantly larger basolateral to apical P(app) than that of the reverse direction, suggesting the existence of efflux mechanisms. ATP-depletion and probenecid pretreatment led to a significant inhibition of the efflux of selagin, whereas verapamil had no effect on the basolateral to apical transport of selagin, suggesting that multidrug resistance proteins (MRPs) and not P-glycoprotein play a role in the intestinal efflux of selagin. Based on present and previous results, the structure-permeation relationship and the role of MRPs in mediating the efflux of flavonoids are discussed. Experimental results in this study provide useful information for pharmacological applications of the flavonoids from C. chinense.

Unveiling the Role of Multidrug Resistance Proteins in Hypertension

Hypertension is a chronic condition associated with an increased risk of mortality and morbidity. It is estimated to cause 4.5% of current global disease burden, and its prevalence is similar in both developing and developed countries.1 According to a recent review, ≈54% of stroke, 47% of ischemic heart disease, 75% of hypertensive disease, and 25% of other cardiovascular disease worldwide can be attributable to high blood pressure.2 Altered levels of angiotensin and aldosterone are common findings in hypertension. Aldosterone has been shown to play an important role in the pathophysiology of numerous cardiovascular disorders, including heart failure and hypertension,3 and the renin-angiotensin-aldosterone system is currently an important target for 5 antihypertensive drug classes: β-blockers, renin inhibitors, angiotensin-conversion enzyme inhibitors, angiotensin receptor blockers, and aldosterone antagonists.4 In the last few years, increasing evidence has pointed to an involvement of multidrug resistance (MDR)-related proteins with hypertension. Both ABCB1 (P-glycoprotein) and ABCC1 (MDR-associated protein 1), the 2 main proteins first described in multidrug-resistant tumors, are known to physiologically transport several endobiotics, including hormones.5,6 Moreover, ABCB1 and ABCG2 (the third protein related to MDR), seem to also be related to the secretion of several drugs to urine, including some antihypertensives.7,8 This review summarizes the recent findings regarding the relationship between MDR-related proteins and hypertension. MDR is still the main cause of failure in cancer chemotherapy. Although it is a multifactorial phenomenon, virtually all MDR cells present an ATP-dependent reduction in intracellular drug accumulation because of the overexpression of ≥1 of 3 proteins belonging to the ATP-binding cassette (ABC) family of transporters.9,10 The ABC superfamily is one of the largest protein families, and its members have been found in virtually all organisms examined up to now. It is consisted of 7 subfamilies, ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, …

Multidrug resistance proteins affect drug transmission across the placenta

We studied the role of multidrug resistance proteins in regulating transplacental transmission of corticosteroids and protease inhibitors. We performed quantitative polymerase chain reaction and FACS analyses to study MDR1 (encodes P-glycoprotein) and MRP-1 expression in extravillous (HTR-8/SVneo) and villous (BeWo) trophoblast cells treated with saquinavir, a multidrug resistance protein substrate. We measured H3-dexamethasone and H3-ritonavir transfer across confluent, syncytialized BeWo cells before and after treatment with agents that inhibit multidrug resistance proteins. Compared with baseline expression, messenger RNA and protein levels were increased significantly in trophoblast cells after treatment with saquinavir. H3-dexamethasone and H3-ritonavir levels increased in BeWo cells after treatment with anti-P-glycoprotein antibodies or cyclosporine A. Transfer of H3-labeled drugs from the apical (eg, maternal) to basolateral (eg, fetal) side of the syncytialized BeWo cell monolayer was increased significantly when cells were pretreated with anti-P-glycoprotein antibodies. Multidrug resistance proteins regulate drug levels in trophoblast cells and may mediate transmission of therapeutic agents across the placenta.

Heterologous expression of bacterial and human multidrug resistance proteins protect Escherichia coli against mercury and zinc contamination

In order to determine the role of multidrug resistance proteins in mercury and zinc resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA genes were expressed in an Escherichia coli tolC mutant which is hypersensitive to metals. The three transporters conferred an increased mercury and zinc resistance to E. coli as compared to the control bacteria. This improved resistance correlated with a decreased zinc and mercury bioaccumulation. Indeed, quantification of intracellular metal concentrations by atomic absorption spectrometry (AAS) showed a 2.1-, 3-, and 5.1-fold decrease in zinc in cells expressing hMDR1, omrA, and lmrA, respectively, and a 2.7-, 7.5-, and 7.7-fold decrease in mercury in cells expressing omrA, lmrA, and hMDR1, respectively, as compared to the control bacteria. This means that hMDR1, LmrA, and OmrA proteins which are specialised in xenobiotic scavenging, their main known function, are nevertheless able to confer some resistance against metals. Our results show that the tolC mutated strain is well adapted to the study of MDR transporter activity and could be used to screen substrates and competitive hMDR1 inhibitors.

Molecular mechanisms of azole resistance in fungi.

This paper reviews the current status of our understanding of azole antifungal resistance mechanisms at the molecular level and explores their implications. Extensive biochemical studies have highlighted a significant diversity in mechanisms conferring resistance to azoles, which include alterations in sterol biosynthesis, target site, uptake and efflux. In stark contrast, few examples document the molecular basis of azole resistance. Those that do refer almost exclusively to mechanisms in laboratory mutants, with the exception of the role of multi-drug resistance proteins in clinical isolates of Candida albicans. It is clear that the technologies required to examine and define azole resistance mechanisms at the molecular level exist, but research appears distinctly lacking in this most important area.

The Role of Multidrug Resistance Proteins and Drug Sensitivity in Relation to Poor Prognosis Identified by ZAP-70 Expression and IgVH Mutation Status in B-CLL.

A proportion of haematological malignancies over express the multidrug resistance (MDR) efflux proteins - P-glycoprotein (P-gp), multidrug resistance protein (MRP1) and lung resistance proteins (LRP) that confer resistance to a number of structurally unrelated cytotoxic drugs. The clinical significance of the observed over expression of P-gp in B-CLL regardless of stage or treatment of the disease is unclear, although a protective effect on cell survival has been reported. Recent evidence suggest that a sub-group of B-CLL exist with an aggressive clinical course that is often resistant to treatment. B-CLL cells from this subgroup of patients have unmutated immunoglobulin heavy chain variable region (IgVH) and express zeta-associated protein (ZAP-70). We analysed the association between the expression of MDR proteins and the prognosis indicator ZAP-70 in cells from 46 B-CLL patients (30M/16F, 33 stage A, 7 B, 6 C) for (i) ZAP-70 expression using an antibody (clone 2F3.2)-Alexa Fluor 488 conjugate by flow cytometry and expressed as a ratio of B to T-cell mean cell fluorescence, (ii) the expression of P-gp, MRP1 and LRP protein by flow cytometry using specific antibodies and expressed as MCF ratio to isotype controls, (iii) flow cytometric analysis of functional activity of P-gp expressed as MCF ratio in the presence and absence of verapamil, a known inhibitor of P-gp, (iv) IC50 (μg/ml) for cytotoxic drugs 2-chlorodeoxyadenosine (CdA), chlorambucil (Chl) and fludarabine (FdR) using the cytotoxicity MTT assay and (v) IgVH mutational status by sequence analysis of FR1/JH polymerase chain reaction products in 29/46 (63%) of patients. 13/46 (28%) patients were ZAP-70+ve and the remaining 33/46 (72%) were ZAP-70−ve. P-gp expression (median 1.59 in both groups p=0.85) and P-gp functional activity (median 2.01 vs 1.84 p=0.77) were not statistically different between ZAP-70+ve and ZAP-70−ve groups. There was no significant difference in the expressions of MRP1 (median 2.77 vs 3.48 p=0.31) or LRP (median 4.87 vs 5.18 p=0.56) between ZAP-70+ve and ZAP-70−ve patients. The IC50 levels for CdA, Chl and FdR between ZAP+ve (median 0.04, 6.4 & 0.54mg/ml respectively) and ZAP−ve groups (median 0.065, 12.15 & 0.72 respectively) were not statistically significant (p=0.67, 0.45, 0.36 respectively). 9 ZAP-70+ve and 15 ZAP-70−ve patients were sequentially investigated over a median period of 3 years for expression of P-gp, MRP1, LRP and P-gp function. There were no significant changes in P-gp expression and P-gp function between presentation and follow samples over this period. However, a significant increase in expression of MRP1 (median at presentation 1.65 and follow up 5.23 p= 0.0001) and LRP (median at presentation 3.92 and follow up 5.84 p= 0.0002 respectively) were observed for the ZAP-70−ve group. IgVH mutation analysis was concordant with ZAP-70 expression in 6 patients with poor prognosis (ZAP-70+ve/IgVH unmutated) and in 16 with good prognosis (ZAP-70−ve/IgVH mutated). Statistical analysis of MDR protein expression and IC50 in this smaller cohort of patients provided data similar to that in the main group. MDR proteins, their activity and in vitro drug sensitivity appear not to significantly contribute to the poor prognosis subset of B-CLL identified by their ZAP-70+ve/IgVH unmutated status. The significance of increased MRP1 and LRP expression in the good prognosis ZAP−ve group is unclear.

Multidrug resistance proteins and topoisomerase IIα expression in colon cancer: association with metastatic potential

Aims To investigate the role of multidrug resistance proteins and topoisomerase IIα in colon cancer. Methods Tissue sections from 89 Dukes’ stage B–D colon cancer patients were selected. The expression of multidrug resistance proteins and topoisomerase IIα in primary tumour cells was assessed by standard immunohistochemistry. The extent of their expression was measured by image analysis and was correlated with clinicopathological features of the patients. Results P-glycoprotein was associated with the presence of lymph node metastasis (P = 0.005), vessel invasion (P = 0.0001) and perineural invasion (P = 0.020). Conclusions P-glycoprotein is probably involved in the processes of local invasion and metastatic dissemination. To investigate the role of multidrug resistance proteins and topoisomerase IIα in colon cancer. Tissue sections from 89 Dukes’ stage B–D colon cancer patients were selected. The expression of multidrug resistance proteins and topoisomerase IIα in primary tumour cells was assessed by standard immunohistochemistry. The extent of their expression was measured by image analysis and was correlated with clinicopathological features of the patients. P-glycoprotein was associated with the presence of lymph node metastasis (P = 0.005), vessel invasion (P = 0.0001) and perineural invasion (P = 0.020). P-glycoprotein is probably involved in the processes of local invasion and metastatic dissemination.

Multidrug resistance proteins and topoisomerase IIα expression in colon cancer: association with metastatic potential

To investigate the role of multidrug resistance proteins and topoisomerase IIalpha in colon cancer. Tissue sections from 89 Dukes' stage B-D colon cancer patients were selected. The expression of multidrug resistance proteins and topoisomerase IIalpha in primary tumour cells was assessed by standard immunohistochemistry. The extent of their expression was measured by image analysis and was correlated with clinicopathological features of the patients. P-glycoprotein was associated with the presence of lymph node metastasis (P=0.005), vessel invasion (P=0.0001) and perineural invasion (P=0.020). P-glycoprotein is probably involved in the processes of local invasion and metastatic dissemination.

Molecular determinants of glucocorticoid sensitivity and resistance in acute lymphoblastic leukemia.

Glucocorticoids (GC) are probably the most important drugs in the treatment of ALL. Despite the extensive use of GC for many years, little is known about the molecular mechanisms of sensitivity and resistance. This review summarizes the knowledge on GC cytotoxicity in leukemia. The relevance of polymorphisms, splice variants and the number and regulation of the GC receptor are discussed. The role of multidrug resistance proteins, glutathione and glutathione S-transferase is evaluated, as well as the influence of the different heat-shock chaperone (hsp 90 and 70) and co-chaperone proteins (BAG-1 and others) which form a complex together with the GC receptor. Finally, the transactivation and transrepression (via NF-kappa B and AP-1 binding) of a wide range of genes (like c-myc) which initiates the final apoptosis pathway are discussed and suggestions for future directions of research in ALL patients are given.

Relevance of multidrug resistance proteins for intestinal drug absorption in vitro and in vivo.

Multidrug resistance proteins (p-glycoprotein and mrps) are becoming increasingly important to explain the pharmacokinetics and action of drugs. Located in epithelial and endothelial cells of the gastrointestinal tract, liver, kidney, blood brain barrier, choroid plexus and other organs, they are critical determinants for the movement of a large number of commonly prescribed drugs across cellular barriers. Here we provide a brief overview of the role of multidrug resistance proteins in drug absorption from the gastrointestinal tract. We address the different types of multidrug resistance proteins involved, describe experimental models to study the influence of these proteins on transcellular transport and discuss the impact of multidrug resistance proteins on overall drug bioavailability in vivo.