| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics |
Department of Pathology [M. C. d. J., G. L. S., A. B. S., R. J. S.] and Medical Oncology [H. J. B.], University Hospital Vrije Universiteit, 1081 HV Amsterdam; Department of Molecular Recognition, Institute for Animal Science and Health (ID-DLO), 8200 AB Lelystad [J. W. S., W. C. P., R. D., R. H. M.]; and Division of Molecular Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam [M. K.], the Netherlands
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Despite their common involvement in MDR, there are clear differences in function and substrate specificity of Pgp and MRP1. Pgp binds and transports neutral, or positively charged, hydrophobic compounds. In contrast, MRP1 transports conjugated organic anions such as the leukotriene C4 and GSH S-conjugates of prostaglandin A2 and aflatoxin B1. Therefore MRP1 is known as multispecific organic anion transporter (MOAT; Ref. 4 ) or GS-X pump (5) . For transport by MRP1 of cytostatics such as the anthracyclines, GSH is required (6) , but GSH is not necessarily conjugated to the drug. Instead, GSH may be cotransported (7) .
In humans, a well-known ABC protein involved in peptide transport is the transporter associated with antigen presentation. It plays a pivotal role in MHC class I restricted antigen presentation by mediating peptide translocation across the endoplasmic reticulum membrane (8) . Regarding Pgp, among the diverse range of substrates, peptides have also been reported to interact with this protein. Sharma et al. (9) selected CHO cells with the cytotoxic tripeptide ALLN, which resulted in a cell line that showed the classical MDR phenotype with overexpression of Pgp. Also, hydrophobic peptides stimulate Pgp ATPase activity (10) and a radiolabeled tripeptide (Nac-LLY-amide) was shown to be transported by Pgp into both membrane vesicles and reconstituted proteoliposomes (11) . Furthermore, several peptides can reverse the Pgp-mediated resistance to cytostatic drugs. Well-known cyclic chemosensitizing peptides are cyclosporin A and its nonimmunosuppressive derivative, PSC833, but linear peptides are also able to antagonize Pgp-mediated drug transport (10 , 12) .
We decided to investigate the potential role of MRP1 as a transmembrane transporter for peptides. To this goal, we selected cytotoxic peptides of different origins and structures for study as possible substrates of MRP1 (Table 1)
. Both ALLN and valinomycin are known substrates of Pgp (9)
. 4A6 is a synthetic peptide derivative [Ac-Thr(tBu)-His(Bzl)-Thr(Bzl)-Nle-Glu(OtBu)-Gly-Bza; for structure see Fig. 1
] that was discovered by us in a cytotoxicity-based screen for potential MDR antagonists. The present results show that MRP1 is an effective peptide transporter. This may give a lead to the design of peptide-based selective and potent reverting agents.
|
|
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All of the other chemicals and drugs used, including the peptide ALLN, were from Sigma Chemical Co. (St. Louis, MO) except for doxorubicin, which was purchased from Farmitalia Carlo Erba (Brussels, Belgium), trichloroacetic acid, which was from ICN Biomedicals Inc. (Aurora, OH), and MK571 (L-660,711), obtained from Dr. Robert Zamboni (Merck-Frosst, Pointe-Claire, Quebec, Canada).
Peptide Synthesis.
For 4A6 synthesis, DMF (1 ml) was added to 100 mg of Fmoc-Gly-Sasrin-resin (0.69 mmol Fmoc-Gly per g resin) and mixed (1 h). After washing with DMF, the Fmoc-group was removed using 2 ml of 20% piperidine in DMF (10 min). The first amino acid Fmoc-Glu(OtBu)-OH (0.5 mmol in 1 ml DMF) was coupled to the resin using DIEA (1.25 mmol in 0.5 ml DMF) and HBTU/HOBt (0.5 mmol in 1.1 ml DMF). The same procedure was used for coupling of the second to the fifth amino acids, i.e., Fmoc-Nle-OH, Fmoc-Thr(Bzl)-OH, Fmoc-His(Bzl)-OH, and Fmoc-Thr(tBu)-OH. To obtain acetyl-4A6 the peptide-resin was acetylated, after Fmoc cleavage and washing, using acetic anhydride/DMF/DIEA 1/10/0.1 (30 min). For fluorescein-labeled 4A6 the peptide-resin was, after Fmoc cleavage and washing, incubated with a solution of fluorescein (0.14 mmol in 290 µl of DMF), DIEA (0.36 mmol in 143 µl of DMF), and HBTU/HOBt (0.14 mmol in 350 µl of DMF). After washing with DCM, the peptide-resin was treated with 1% trifluoric acid in DCM, which cleaved the protected peptide from the resin. To the free COOH terminus of the protected peptide (0.069 mmol in 690 µl of DMF) benzylamine (0.1 mmol) was coupled using DIEA (0.175 mmol in 70 µl of DMF) and HBTU/HOBt (0.069 mmol in 155 µl of DMF). Subsequently, the protected peptide was precipitated from the previous solution using water. Finally the protected peptides were purified on HPLC (Waters 2690 Alliance Waters PrepLC4000 07234). 4A6 analogous peptides were synthesized as described above, replacing the different 4A6 amino acid building blocks one by one by either Ala or Lys(Z). The NH2-terminal groups pyrrole-2-carboxylic acid and 2-pyrazinecarboxylic acid were coupled to the peptides like true Fmoc amino acids (concentration, 0.1 M during synthesis).
Cell Lines.
The SW-1573, HL60, and A2780 cell lines and their multidrug resistant sublines have been described previously (13, 14, 15)
. The cells were grown in RPMI 1640 or DMEM, supplemented with 10% FCS, 2 mM glutamine, penicillin, and streptomycin. Multidrug resistant cell lines SW-1573/2R120 (MRP1), SW-1573/2R160 (Pgp), and 2780AD (Pgp), but not HL60/ADR (MRP1), were cultured in the presence of doxorubicin, until 3–10 days before experiments.
The 2008 ovarian carcinoma cell line (16) was obtained from Dr. Howell from the University of California, San Diego, CA. 2008-M1–4 and 2008-M1–6 cells are 2008-derived MRP1-transfected cells and are described elsewhere4 (17) .
Cytotoxicity Assays.
For cytotoxicity experiments, exponentially growing cells were plated in triplicate in 96-well plates (number of cells per well: 5,000 SW-1573; 6,000 SW-1573/2R120 and SW-1573/2R160; 7,500 HL60; 10,000 HL60/ADR; 3,750 2008 and 2008-M1–4; 5,000 2008-M-6; 10,000 A2780; 10,000 2780AD; and 12,500 A2780/ALLN1) and were cultured in the presence of 6–8 different concentrations of the compounds to be tested. In some experiments, MRP1 modulators (MK571, probenecid, and BSO) were added, but always at nontoxic (less than 20% growth inhibitory) concentrations. Appropriate controls were included to check for cytotoxicity of DMSO that was used to dissolve the peptides and probenecid. Total amount of DMSO in the cell cultures never exceeded 0.5%. After 96 h, cell survival was determined using the sulforhodamine B method (18)
for the adherent cell lines (SW-1573, A2780, and 2008) and the XTT method (19)
for the suspension HL60 cells. The optimal XTT concentrations for the HL60 and HL60/ADR cells were 50 µg of XTT mixed with 8 µM phenazine methosulfate per well for 3–4 h. The RF was calculated as the ratio of the IC50 (concentration of the compound that inhibits growth of the cells for 50%) of the resistant cells:IC50 of the parental cells.
Peptide Accumulation Assay.
HL60 and HL60/ADR (105 cells) were incubated with 20 µM 4A6-Flu in culture medium in the presence or absence of 30 µM MK571 and were left overnight in a 5% CO2 incubator. The cells were then centrifuged and washed two times with ice-cold PBS. Fluorescence was analyzed on a FACS-star flow cytometer (Becton and Dickinson, San Jose, CA). Before fluorescence-activated cell sorting analysis, the cell viability was checked using trypan blue exclusion (viability, >90%).
Selection of ALLN-resistant Cell Lines.
A2780 cells were cultured in 24-well plates with a continuous IC75 dose of ALLN in the presence or absence of 10 µM of the Pgp antagonist verapamil. As described before for CHO cells (9)
, A2780 cells were found to be 2-fold more sensitive to ALLN in the presence of verapamil. Therefore the IC75 concentration of ALLN that was used for the initial selection of resistant cells with verapamil was 2-fold lower than the concentration used for selection without verapamil. When surviving cells had grown to 
75% confluency, they were replated, and some of them were immediately used for further selection by incubating them with a doubled concentration of ALLN. Selected cell lines were characterized using cytotoxicity assays and immunocytochemistry.
Immunocytochemistry.
Cytocentrifuge preparations were air-dried overnight and fixed in 100% acetone for 10 min. The slides were then incubated with monoclonal antibodies MRPr1 (5 µg/ml; Ref. 20
) or JSB-1 (5 µg/ml; 21
) diluted in PBS containing 1% (w/v) BSA. Antibody binding was detected using a biotinylated rabbit antirat (1:100; Dako A/S, Denmark) or biotinylated rabbit antimouse (1:150, Dako A/S, Denmark), for MRPr1 and JSB-1 respectively, and streptavidin conjugated to horseradish peroxidase (1:500; Zymed, San Francisco, CA). Bound peroxidase was developed with 0.02% (w/v) 3-amino-9-ethylcarbazole and 0.02% (v/v) H2O2 in 0.1 M sodium acetate (pH 5.0). Then, the slides were counterstained with hematoxylin and mounted.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Both Pgp-overexpressing cell lines SW-1573/2R160 and 2780AD were resistant to ALLN and the K+ ionophore valinomycin, confirming that these peptides are substrates of Pgp, which was reported previously (9)
. Also the newly synthesized toxic peptide 4A6 appeared to be a Pgp substrate (Table 2)
. Subsequent experiments with MRP1-overexpressing SW-1573/2R120 and HL60/ADR cell lines revealed that these cells also show distinct resistance to ALLN (RFs, 4.4 and 5.1, respectively) and 4A6 (RFs, 8.3 and 10.8, respectively; Table 2
). The ability of MRP1 to transport these peptides was confirmed with two independent clones of MRP1-transfected 2008 cells. These cell lines also showed a resistant phenotype with resistance factors varying from 3.4 and 2.5 for ALLN and 3.8 and 2.9 for 4A6 (Table 2)
. In MRP1-overexpressing cells, however, no resistance was found to valinomycin (Table 2)
.
|
. The small, but consistent chemosensitizing effects in the HL60 cells are most likely related to a low level of MRP1 in the latter cells.
|
. The addition of MK571 restored the accumulation defect of 4A6-Flu in these cells to the levels observed in HL60 cells, in line with the finding that MK571 reversed the 4A6 resistance in HL60/ADR cells (Fig. 3)
. Although a contribution of the fluorescein moiety of Flu-4A6 to these results cannot be excluded, these findings support the conclusion reached above that MRP1 can mediate peptide resistance.
|
-glutamylcysteine synthetase, the enzyme that catalyzes the first step in GSH synthesis (24)
. Depletion of cellular GSH by BSO potentiates the toxicity of a broad variety of drugs, including vincristine, VP-16, and doxorubicin, to MRP1-, but not Pgp-, overexpressing cells (6)
, which indicated that GSH is needed for the export of these drugs by MRP1. On the other hand, efflux of some MRP1 substrates, for instance calcein (25)
, is not affected by GSH depletion. Here, we found that BSO restores the cytotoxicity of ALLN in HL60/ADR cells almost to the parental level but has only a small effect on the cytotoxicity of 4A6 (Fig. 4)
. These results indicate that, depending on the peptide, GSH can contribute to peptide transport by MRP1.
|
. Thus, with partially reduced resistance for only one of the peptide analogues (Table 3
, last peptide of second group), NH2-terminal group or amino acid substitutions did not seem to affect resistance in MRP1-overexpressing cells. However, they did affect the cytotoxicity of the peptide. The residue Thr(Bzl) on position 3 seems to be particularly critical for toxicity of the peptide (Table 3)
.
|
10 ALLN-resistant cell lines. Interestingly, none of the resistant cell lines that were obtained showed overexpression of Pgp, independent from coculturing with or without verapamil. Instead, most of the cell lines showed low, but distinct, up-regulation of MRP1. When cells were selected for higher resistance levels to ALLN, MRP1 was not further up-regulated, possibly because other resistance mechanisms emerged. Immunocytochemical staining for other MDR-associated proteins [Pgp, MRP2/cMOAT (multispecific organic anion transporter), MRP3, MRP5, BCRP (breast cancer resistance protein); Ref. 26
] and the lung resistance protein (LRP)/major vault protein (MVP; reviewed in Ref. 27
) remained, however, negative (results not shown). One of the ALLN/verapamil coselected cell lines (A2780/ALLN1) was chosen for further analysis. These cells were obtained in a single-step selection by culturing A2780 cells with 10 µM ALLN and 10 µM verapamil continuously. The cell line was found to be 6.4-fold more resistant to ALLN than the parental A2780 cells (Table 4)
. Cytospin stainings of A2780/ALLN1 cells showed up-regulated MRP1 but not Pgp (Fig. 5)
. In cytotoxicity experiments, A2780/ALLN1 cells were found to be cross-resistant to the 4A6 peptide (RF, 7.3), and the cytotoxic drugs VP-16 (RF, 4.7), vincristine (RF, 3.4), and doxorubicin (RF, 2.7; Table 4
). No resistance could be detected to the Pgp substrate valinomycin (RF, 1.2). This cross-resistance pattern is consistent with a MDR phenotype caused by overexpression of MRP1.
|
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Until now, only few, indirect, data have become available about possible interactions of MRP1 with peptides. Of course, GSH, which has been described as a required cosubstrate for transport of substrates by MRP1 (7 , 28) and may even be transported on its own (29) , is a tripeptide itself, consisting of glutamate, cysteine, and glycine residues. In this study, we set out to further investigate putative peptide transport by MRP1, and the results obtained confirm that two neutral peptides, ALLN and 4A6, are efficiently transported both by Pgp and MRP1. This suggests that, at least for peptides, a negative charge is not a required factor in determining MRP1 substrate properties. This view is supported by the experiments with BSO-pretreated cells showing that MRP1-mediated resistance to 4A6, a neutral toxic peptide, was largely independent of intracellular levels of the anionic tripeptide GSH. Because, in contrast, MRP1-mediated resistance to the other neutral, toxic peptide, ALLN, was found to be strongly GSH dependent, cotransport of GSH may facilitate transport of certain neutral substrates but is not an absolute requirement for MRP1 function. Alternatively, certain substrates such as 4A6 may need relatively low concentrations of GSH compared with other substrates (ALLN).
Pgp can interact with circular peptides, because both Pgp overexpressing cell lines showed resistance to the peptide K+ ionophore valinomycin. This is in line with the well-known potencies of the circular peptides cyclosporin and its congener PSC833 as Pgp chemosensitizers (30) . We did not, however, find resistance to valinomycin in MRP1-overexpressing cells. Earlier, it was found that cyclosporin also does not affect MRP1-mediated drug transport (31) .
ALLN and 4A6 are a tri- and hexapeptide, respectively. It will be interesting to determine the maximum size allowed for peptides to function as a MRP1 substrate. Recent findings suggest that MRP1 might be involved in the transport of proteins as large as basic fibroblast growth factor, with an apparent Mr of 16,000 (32)
. Also, Pgp was implicated in the transport of proteins like the cytokines IL-2, IL-4, and IFN- (33)
. However, the latter data were recently disputed by results obtained in T cells derived from Pgp-knockout mice, showing that these cells, when polyclonally activated, displayed equal secretion of cytokines like IL-2, IL-4, IL-5, IL-10, and IFN-, as T cells derived from wild-type mice (34)
. Apparently, additional studies will be required to determine whether, indeed, distinct cytokines can be transported by members of the transmembrane transporter family.
A relative lack of substrate specificity of MRP1 is illustrated in the experiment with peptides that are analogous to 4A6. Although these peptides retained linearity, neutrality, and approximately the same size, toxicity was lost by replacing just one of the 4A6 residues [Thr(Bzl)] by Ala or Lys(Z). In contrast, as could be determined for the peptides that were still cytotoxic, none of the residues Thr(tBu), His(Bzl), Nle or Glu(OtBu) or the NH2-terminal acetyl group appeared to be critical for interaction of MRP1 with the peptide.
Following from our finding that small linear peptides may interact with MRP1, it seems worthwhile to search for linear peptides that are not only transported by MRP1 but also can modulate MRP1 drug transport activity. Using 96-h cytotoxicity assays, we tested ALLN, 4A6, and the 4A6 analogous peptides for their potential of reversing doxorubicin, vincristine, or VP-16 resistance in HL60/ADR or SW-1573/2R120 cells, but they were not efficient resistance modulators (data not shown). One reason for this may be that the affinities of these peptides for MRP1 are not high enough. On the other hand, Sharom et al. (12) suggested that for peptide-mediated reversal of Pgp, not only high affinity but also a high rate of transmembrane equilibration of the peptide is required for efficient modulation. 4A6 may not have this property because, in the present peptide accumulation experiments, several hours were required to achieve sufficient intracellular levels of fluorescein-labeled 4A6 for detection. Therefore, a further search using peptide library technology, may identify potent peptide-based MRP1 modulators.
Taken together, in this study, evidence is presented that peptides are substrates of the MRP1 transmembrane transporter. This adds MRP1 to Pgp, another ABC transmembrane transporter protein that was first identified for its role in mediating cytostatic drug resistance and later was shown to interact with peptides. Additional studies are, therefore, warranted to identify peptides with clinically relevant activity in reversing MRP1-mediated MDR. Also, a putative role of MRP1 in normal physiology in the secretion of peptides and proteins, including cytokines, should be further investigated.
|
FOOTNOTES |
|---|
1 Supported by Dutch Cancer Society Grant KWF-VU96-1256. 
2 To whom requests for reprints should be addressed, at Department of Pathology, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands. Phone: 31-20-444-4031; Fax: 31-20-444-2964; E-mail: rj.scheper{at}azvu.nl
3 The abbreviations used are: MDR, multidrug resistance; MRP, MDR protein; Pgp, P-glycoprotein; ALLN, N-acetyl-Leu-Leu-norleucinal; GSH, glutathione; OtBu, (O-)tert-butyl; Bzl, benzyl; Bza, benzylamine; Z, benzyloxycarbonyl; DMF, N,N-dimethylformamide; DIEA, N,N-diïsopropyl-ethylamine; DCM, dichloromethane; HOBt, 1-hydroxybenzotriazole; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tretramethyluronium hexafluorophosphate; BSO, DL-buthionine-S,R-sulphoximine; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide); RF, resistance factor; VP-16, etoposide; CHO, Chinese hamster ovary; IL, interleukin.
4 M. Kool, M. de Haas, M. van der Linden, F. Baas, and P. Borst. MRP2: Localization, drug resistance and GSH transport in comparison to MRP1 and MRP3, manuscript in preparation.
Received 5/11/00. Accepted 1/16/01.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
in normal human T lymphocytes. Blood, 88: 1747-1754, 1996.This article has been cited by other articles:
|
|
 |
|
  R. Oerlemans, N. E. Franke, Y. G. Assaraf, J. Cloos, I. van Zantwijk, C. R. Berkers, G. L. Scheffer, K. Debipersad, K. Vojtekova, C. Lemos, et al. Molecular basis of bortezomib resistance: proteasome subunit {beta}5 (PSMB5) gene mutation and overexpression of PSMB5 protein Blood, September 15, 2008; 112(6): 2489 - 2499. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. D. Klaassen and H. Lu Xenobiotic Transporters: Ascribing Function from Gene Knockout and Mutation Studies Toxicol. Sci., February 1, 2008; 101(2): 186 - 196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. G. Deeley, C. Westlake, and S. P. C. Cole Transmembrane Transport of Endo- and Xenobiotics by Mammalian ATP-Binding Cassette Multidrug Resistance Proteins. Physiol Rev, July 1, 2006; 86(3): 849 - 899. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Haimeur, G. Conseil, R. G. Deeley, and S. P.C. Cole Mutations of Charged Amino Acids in or near the Transmembrane Helices of the Second Membrane Spanning Domain Differentially Affect the Substrate Specificity and Transport Activity of the Multidrug Resistance Protein MRP1 (ABCC1) Mol. Pharmacol., June 1, 2004; 65(6): 1375 - 1385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. de Jong, G. L. Scheffer, H. J. Broxterman, J. H. Hooijberg, J. W. Slootstra, R. H. Meloen, R. J. Kreitman, S. R. Husain, B. H. Joshi, R. K. Puri, et al. Multidrug-Resistant Tumor Cells Remain Sensitive to a Recombinant Interleukin-4-Pseudomonas Exotoxin, Except When Overexpressing the Multidrug Resistance Protein MRP1 Clin. Cancer Res., October 15, 2003; 9(13): 5009 - 5017. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Savolainen, J. E. Edwards, M. E. Morgan, P. J. McNamara, and B. D. Anderson Effects of a P-Glycoprotein Inhibitor on Brain and Plasma Concentrations of Anti-Human Immunodeficiency Virus Drugs Administered in Combination in Rats Drug Metab. Dispos., May 1, 2002; 30(5): 479 - 482. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
