The Relationship Between Modulation of MDR and Glutathione in MRP-Overexpressing Human Leukemia Cells

doi:10.1016/S0006-2952(97)00562-5

Biochemical Pharmacology

Volume 55, Issue 8, 15 April 1998, Pages 1283-1289

https://doi.org/10.1016/S0006-2952(97)00562-5 Get rights and content

Abstract

Multidrug resistance-associated protein (MRP) causes multidrug resistance (MDR) involving the anthracyclines and epipodophyllotoxins. Many studies show modulation of anthracycline levels and cytotoxicity in MRP-overexpressing cells, but there is limited data on the modulation of etoposide levels and cytotoxicity in MRP-overexpressing or in P-glycoprotein-expressing cells. Etoposide accumulation was 50% reduced in both the CEM/E1000 MRP-overexpressing subline and the CEM/VLB₁₀₀ P-glycoprotein-expressing subline compared to the parental CEM cells, correlating with similar resistance to etoposide (200-fold) of the two sublines. For the CEM/VLB₁₀₀ subline, the P-glycoprotein inhibitor SDZ PSC 833, but not verapamil, was able to increase etoposide accumulation and cytotoxicity. For the CEM/E1000 subline, neither SDZ PSC 833 nor verapamil had any effect on etoposide accumulation. However, verapamil caused a 4-fold sensitization to etoposide in this subline, along with an 80% decrease in cellular glutathione (P < 0.05). Buthionine sulfoximine (BSO), which depletes glutathione, also caused a 2.5-fold sensitization to etoposide with no effect on accumulation in the CEM/E1000 subline. In contrast, SDZ PSC 833 was able to increase daunorubicin accumulation in the CEM/E1000 subline (P < 0.05), but had no effect on daunorubicin cytotoxicity, or cellular glutathione. These results show that modulation of etoposide cytotoxicity in MRP-overexpressing cells may be through changes in glutathione metabolism rather than changes in accumulation and confirm that changes in drug accumulation are not related to drug resistance in MRP-overexpressing cells.

Section snippets

Drugs

Daunorubicin and etoposide were from David Bull Laboratories. Etoposide (powder) was obtained from Sigma Chemical Co. and was prepared as a stock solution of 20 mg/mL in DMSO.

Cell Lines

The CCRF-CEM (CEM) human leukemia cell line [10] and its epirubicin selected CEM/E1000 [4] and vinblastine selected CEM/VLB₁₀₀[9] multidrug-resistant sublines were grown in RPMI 1640 medium (Trace) supplemented with 10% fetal calf serum (Trace), 20 mM of HEPES (Trace), and 10 mM of NaHCO₃ at 37° in a humidified atmosphere

Characterization of Etoposide Resistance

Etoposide is a lipophilic drug, the pharmacological preparation being administered in a solvent containing polyethylene glycol 300 and polysorbate 80. Because similar solvents have been reported to sensitize P-glycoprotein-expressing cells to the cytotoxic effects of drugs [13], the cytotoxicity of this preparation was compared to that of etoposide dissolved in DMSO in both the MRP-overexpressing and the P-glycoprotein-expressing MDR sublines. Fig. 1 shows that the solvent had no effect on

Discussion

Both the MRP-overexpressing CEM/E1000 and the P-glycoprotein-expressing CEM/VLB₁₀₀ were similarly resistant to etoposide (Fig. 1). The effect of the solvent for the preparation of etoposide on cytotoxicity suggests that the variability reported on etoposide resistance in MDR cells may be related to the solvent used in the preparation of the drug. The lack of effect of the solvent on the parental CEM cells indicates this effect was specific for both MRP-overexpressing and

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Antiretroviral protease inhibitors potentiate chloroquine antimalarial activity in malaria parasites by regulating intracellular glutathione metabolism
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Antiretroviral protease inhibitors significantly potentiated the sensitivity of chloroquine-resistant malaria parasites to the antimalarial drug in vitro and in vivo. Ritonavir was found to be potent in potentiating CQ antimalarial activities in both -resistant and -sensitive lines. The mechanism by which the APIs modulate the CQ resistance in malaria parasites was further investigated. CQ-resistant parasites showed increased intracellular glutathione levels in comparison with the CQ-sensitive parasites. Treatment with APIs significantly reduced the levels of GSH and glutathione S-transferase activities in CQ-resistant parasites. Ritonavir also decreased glutathione reductase activities and glutathione peroxidase activities in CQ-resistant parasite line. Taken together, these results demonstrate that parasite GSH and GST may play an important role in CQ resistance and APIs are able to enhance the sensitivity of CQ-resistant malaria parasite to the drug by influencing the levels of GSH and the activities of the related enzymes.
Increased expression of ABC transport proteins is associated with ivermectin resistance in the model nematode Caenorhabditis elegans
2009, International Journal for Parasitology
Widespread resistance to chemotherapeutic agents is one of the biggest challenges facing human health and the agricultural industry, with resistance to all current anthelmintics now recorded and few new agents or vaccines available. Understanding the development of drug resistance in parasitic nematodes is critical to prolonging the efficacy of current anthelmintics, developing markers for monitoring drug resistance and is beneficial in the design of new chemotherapeutic agents or targets. This study describes the development of ivermectin-resistant strains of the model nematode Caenorhabditis elegans through step-wise exposure to increasing doses of ivermectin commencing with a non-toxic dose of 1 ng/ml. Resistant strains were developed that displayed a multidrug resistance phenotype with cross-resistance to the related drug moxidectin and to other anthelmintics, levamisole and pyrantel, but not albendazole. Resistance was associated with increased expression of the multidrug resistance proteins (MRPs) and P-glycoproteins. Resistance to ivermectin was reversible by the co-administration of MRP, P-glycoprotein and glutathione biosynthesis inhibitors, confirming the involvement of these proteins in resistance. In our model, resistance to low levels of ivermectin (⩽6 ng/ml) was associated with increased expression of mrp-1 and pgp-1 and decreased glutathione, while higher level resistance (10 ng/ml) was primarily associated with the increased expression of P-glycoproteins. Importantly, resistance was stable after 3 months without ivermectin treatment. This clearly demonstrates the involvement of transport proteins in ivermectin resistance and provides a model to understand drug resistance and its reversal.
Harnessing drug resistance: Using ABC transporter proteins to target cancer cells
2007, Biochemical Pharmacology
The ATP-binding cassette (ABC) class of proteins is one of the most functionally diverse transporter families found in biological systems. Although the abundance of ABC proteins varies between species, they are highly conserved in sequence and often demonstrate similar functions across prokaryotic and eukaryotic organisms. Beginning with a brief summary of the events leading to our present day knowledge of ABC transporters, the purpose of this review is to discuss the potential for utilizing ABC transporters as a means for cellular glutathione (GSH) modulation. GSH is one of the most abundant thiol antioxidants in cells. It is involved in cellular division, protein and DNA synthesis, maintenance of cellular redox status and xenobiotic metabolism. Cellular GSH levels are often altered in many disease states including cancer. Over the past two decades there has been considerable emphasis on methods to sensitize cancer cells to chemotherapeutics and radiation therapy by GSH depletion. We contend that ABC transporters, particularly multi-drug resistant proteins (MRPs), may be used as therapeutic targets for applications aimed at modulation of GSH levels. This review will emphasize MRP-mediated modulation of intracellular GSH levels as a potential alternative and adjunctive approach for cancer therapy.
(R)- and (S)-verapamil differentially modulate the multidrug-resistant protein MRP1
2007, Journal of Biological Chemistry
Citation Excerpt :
Indeed, although Cole et al. (9) did not observe any reversion of chemoresistance, Davey et al. (11) and Cullen et al. (10) described a small chemosensitization to daunomycin. However, verapamil has been clearly demonstrated to stimulate GSH transport (10, 12, 26) and to strongly inhibit LTC4 transport by MRP1 in the presence of GSH (12, 13). This duality reflects the complex mechanism of verapamil interaction as suggested previously (27).
The multidrug-resistant protein MRP1 (involved in the cancer cell multidrug resistance phenotype) has been found to be modulated by racemic verapamil (through stimulation of glutathione transport), inducing apoptosis of human MRP1 cDNA-transfected baby hamster kidney 21 (BHK-21) cells and not of control BHK-21 cells. In this study, we show that the two enantiomers of verapamil have different effects on MRP1 activity. Only the S-isomer (not the R-isomer) potently induced the death of MRP1-transfected BHK-21 cells. The decrease in cellular glutathione content induced by the S-isomer, which was not observed with the R-isomer, was stronger than that induced by the racemic mixture, indicating that the R-isomer antagonized the S-isomer effect. Both enantiomers altered leukotriene C₄ and calcein transport by MRP1. Thus, the R-isomer behaved as an inhibitor, which was confirmed by its ability to revert the multidrug resistance phenotype toward vincristine. Molecular studies on purified MRP1 using fluorescence spectroscopy showed that both enantiomers bound to MRP1 with high affinity, with the binding being prevented by glutathione. Furthermore, conformational changes induced by the two enantiomers (monitored by sodium iodide accessibility of MRP1 tryptophan residues) were quite different, correlating with their distinct effects. (S)-Verapamil induces the death of potentially resistant tumor cells, whereas (R)-verapamil sensitizes MRP1-overexpressing cells to chemotherapeutics. These results might be of great potential interest in the design of new compounds able to modulate MRP1 in chemotherapy.
Fractionated irradiation of H69 small-cell lung cancer cells causes stable radiation and drug resistance with increased MRP1, MRP2, and topoisomerase IIalpha expression
2002, International journal of radiation oncology, biology, physics
$Purpose$ : After standard treatment with chemotherapy and radiotherapy, small-cell lung cancer (SCLC) often develops resistance to both treatments. Our aims were to establish if fractionated radiation treatment alone would induce radiation and drug resistance in the H69 SCLC cell line, and to determine the mechanisms of resistance.
$Methods and Materials$ : H69 SCLC cells were treated with fractionated X-rays to an accumulated dose of 37.5 Gy over 8 months to produce the H69/R38 subline. Drug and radiation resistance was determined using the MTT (3,-4,5 dimethylthiazol-2,5 diphenyltetrazolium bromide) cell viability assay. Protein expression was analyzed by Western blot.
$Results$ : The H69/R38 subline was resistant to radiation (2.0 ± 0.2-fold, p < 0.0001), cisplatin (14 ± 7-fold, p < 0.001), daunorubicin (6 ± 3-fold, p < 0.05), and navelbine (1.7 ± 0.15-fold, p < 0.02). This was associated with increased expression of the multidrug resistance-associated proteins, MRP1 and MRP2, and topoisomerase IIα and decreased expression of glutathione-S-transferase π (GSTπ) and bcl-2 and decreased cisplatin accumulation. Treatment with 4 Gy of X-rays produced a 66% decrease in MRP2 in the H69 cells with no change in the H69/R38 cells. This treatment also caused a 5-fold increase in topoisomerase IIα in the H69/R38 cells compared with a 1.5-fold increase in the H69 cells.
$Conclusions$ : Fractionated radiation alone can lead to the development of stable radiation and drug resistance and an altered response to radiation in SCLC cells.
Plasmodium berghei: Analysis of the γ-glutamylcysteine synthetase gene in drug-resistant lines
2002, Experimental Parasitology
The rapid emergence of multidrug-resistant Plasmodium falciparum is a worldwide concern. Despite the magnitude of the problem, the mechanisms involved in this phenomenon are not well understood. One current proposal suggests that toxic heme molecules are degraded by glutathione (GSH), and that anti-malarial drugs, such as chloroquine (CQ), inhibit this degradation, thus implicating GSH in drug resistance. Furthermore, in some strains of Plasmodium berghei and P. falciparum, chloroquine resistance is accompanied by an increase in glutathione levels and increased activity in GSH-related enzymes. We are investigating the relationship between the γ-glutamylcysteine synthetase (ggcs) gene, the rate-limiting enzyme in de novo synthesis of GSH, and drug resistance in P. berghei at the molecular level. In this report, we have demonstrated an increase in pbggcs mRNA levels associated with CQ and mefloquine (MFQ) resistance. In addition, the pbggcs gene locus structure was shown to be similar and localized to chromosome 8 in four parasite lines of P. berghei with different drug resistance profiles. This work suggests a link between increased GSH levels and drug resistance in Plasmodium.
Index Descriptors and Abbreviations: Apicomplexa; Malaria; Plasmodium berghei; Drug resistance; γ-Glutamylcysteine synthetase; Glutathione; Chloroquine; MDR, multidrug resistance phenotype; AMO, amodiaquine; MFQ, mefloquine; CQ, chloroquine; GSH, glutathione; ggcs, γ-glutamylcysteine synthetase; GB, GenBank; RBC, red blood cell; BSO, buthionine sulfoximine; pfggcs, Plasmodium falciparum ggcs gene; pbggcs, Plasmodium berghei ggcs gene; RPA, ribonuclease protection assay.

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Biochemical Pharmacology

Abstract

Section snippets

Drugs

Cell Lines

Characterization of Etoposide Resistance

Discussion

References (18)

Multidrug resistance of cancer cells

Adv Drug Res

Drug resistance mechanisms and MRP expression in response to epirubicin treatment in a human leukaemia cell line

Leuk Res

Coordinated induction of mrp/gs-x pump and gamma-glutamylcysteine synthetase by heavy metals in human leukemia cells

J Biol Chem

Multidrug resistance protein (MRP)-mediated transport of leukotriene C₄ and chemotherapeutic agents in membrane vesicles—demonstration of glutathione-dependent vincristine transport

J Biol Chem

The MTT cell viability assay for cytotoxicity testing in multidrug resistant human leukaemic cells

Leuk Res

Comparison of drug accumulation in P-glycoprotein-expressing and MRP-expressing human leukaemia cells

Leuk Res

Potential of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA), idarubicin and paclitaxel to circumvent three multidrug resistance phenotypes in vitro

Cancer Chemother Pharmacol

The role of multidrug resistance-associated protein (MRP) expression in multidrug resistance

Anticancer Drugs

ATP-dependent transport of glutathione S-conjugates by the multidrug resistance-associated protein

Cancer Res

Cited by (30)

Antiretroviral protease inhibitors potentiate chloroquine antimalarial activity in malaria parasites by regulating intracellular glutathione metabolism

Increased expression of ABC transport proteins is associated with ivermectin resistance in the model nematode Caenorhabditis elegans

Harnessing drug resistance: Using ABC transporter proteins to target cancer cells

(R)- and (S)-verapamil differentially modulate the multidrug-resistant protein MRP1

Fractionated irradiation of H69 small-cell lung cancer cells causes stable radiation and drug resistance with increased MRP1, MRP2, and topoisomerase IIalpha expression

Plasmodium berghei: Analysis of the γ-glutamylcysteine synthetase gene in drug-resistant lines

Research PapersThe Relationship Between Modulation of MDR and Glutathione in MRP-Overexpressing Human Leukemia Cells

Abstract

Section snippets

Drugs

Cell Lines

Characterization of Etoposide Resistance

Discussion

Adv Drug Res

Leuk Res

J Biol Chem

J Biol Chem

Leuk Res

Leuk Res

Potential of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA), idarubicin and paclitaxel to circumvent three multidrug resistance phenotypes in vitro

Cancer Chemother Pharmacol

The role of multidrug resistance-associated protein (MRP) expression in multidrug resistance

Anticancer Drugs

ATP-dependent transport of glutathione S-conjugates by the multidrug resistance-associated protein

Cancer Res

Research Papers
The Relationship Between Modulation of MDR and Glutathione in MRP-Overexpressing Human Leukemia Cells