Multidrug-Resistant Tumor Cells Remain Sensitive to a Recombinant Interleukin-4-Pseudomonas Exotoxin, Except When Overexpressing the Multidrug ...
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Vol. 9, 5009 –5017, October 15, 2003 Clinical Cancer Research 5009
Multidrug-Resistant Tumor Cells Remain Sensitive to a
Recombinant Interleukin-4-Pseudomonas Exotoxin,
Except When Overexpressing the Multidrug
Resistance Protein MRP1
Mariska C. de Jong, George L. Scheffer, IL-4 toxin with resistance factors of 4.3 to 8.4. MRP1-
Henk J. Broxterman, Jan H. Hooijberg, overexpressing cells were not resistant to PE itself. IL-4
toxin resistance in MRP1-overexpressing cells could be re-
Jerry W. Slootstra, Rob H. Meloen,
versed by the MRP1 inhibitors probenecid or MK571 and
Robert J. Kreitman, Syed R. Husain, were not affected by glutathione depletion by DL-buthionine-
Bharat H. Joshi, Raj K. Puri, and Rik J. Scheper1 S,R-sulfoximine. In a transport assay using plasma mem-
Departments of Pathology [M. C. d. J., G. L. S., R. J. S.], Medical brane vesicles prepared from MRP1-overexpressing cells,
Oncology [H. J. B.], and Pediatric Hematology/Oncology [J. H. H.], IL-4 toxin and IL-4, but not PE, inhibited the translocation
VU University Medical Center, 1081 HV Amsterdam, the
of the known MRP1 substrate 17-estradiol 17-(-D-glucu-
Netherlands; Department of Molecular Recognition, Institute for
Animal Science and Health (ID-DLO), 8200 AB Lelystad, the ronide) (E217G). These data suggest that MRP1-overex-
Netherlands [J. W. S., R. H. M.]; Laboratory of Molecular Biology, pressing cells are resistant to IL-4 toxin because of extrusion
Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, of this agent by MRP1. Still, the results of this study dem-
Maryland 20892 [R. J. K.]; and Laboratory of Molecular Tumor onstrate that IL-4 toxin effectively kills most MDR tumor
Biology, Division of Cellular and Gene Therapies, Center for
Biologics Evaluation and Research, Food and Drug Administration, cells and, therefore, represents a promising anticancer drug.
Bethesda, Maryland 20892 [S. R. H., B. H. J., R. K. P.]
INTRODUCTION
Cytostatic drug resistance in cancer patients may be attrib-
ABSTRACT utable to a phenomenon called MDR.2 MDR is caused by the
Tumor cells may become resistant to conventional an- presence of drug pumps in the plasma membrane of cancer cells
ticancer drugs through the occurrence of transmembrane that transport structurally and functionally unrelated drugs out
transporter proteins such as P-glycoprotein (ABCB1), of the cell. Proteins best known for their cytostatic drug trans-
breast cancer resistance protein (ABCG2), or members of port activity are the MDR1 gene-encoded Pgp (ABCB1; re-
the multidrug resistance-associated protein family (MRP1– viewed in Ref. 1), proteins of the MRP family (MRP1–5;
MRP5; ABCC1–ABCC5). In this report, we studied whether ABCC1-ABCC5; reviewed in Ref. 2), and BCRP [ABCG2 (3)].
tumor cells that are cytostatic drug resistant because of They all are members of the ATP-binding cassette transporter
overexpression of one of the above mentioned proteins are superfamily and have different, but overlapping, drug specific-
sensitive to a new anticancer agent, interleukin-4 toxin (IL-4 ities. Conventional anticancer drugs that are known to be trans-
toxin). IL-4 toxin is a fusion protein composed of circularly ported by these transporters include the anthracyclines (Pgp,
permuted IL-4 and a truncated form of Pseudomonas exo- MRP1–2, BCRP), the Vinca alkaloids (Pgp, MRP1–2), epipo-
toxin (PE) [IL-4(38 –37)-PE38KDEL]. Ninety-six-h cytotox- dophyllotoxins (Pgp, MRP1–3), camptothecins (BCRP), mitox-
icity assays and 10-day clonogenic assays showed that drug- antrone (Pgp, MRP1–2, BCRP), cisplatin (MRP2), methotrexate
selected multidrug resistant (MDR) tumor cells that (MRP1– 4), and the purine analogues 6-mercaptopurine and
overexpress P-glycoprotein or breast cancer resistance pro- thioguanine (MRP4 –5; Refs. 1–3).
teins are still sensitive to IL-4 toxin. Also, tumor cells trans- A different class of anticancer therapeutics are the immu-
fected with cDNA for MRP2–5 showed no resistance, or notoxins. IL-4 toxin is a fusion protein composed of circular
marginal resistance, only to the toxin as compared with the permuted IL-4 and a truncated form of a bacterial toxin called
parent cells. In contrast, MRP1-overexpressing cells, both Pseudomonas exotoxin [IL-4(38-37)-PE38KDEL; Ref. 4]. We
drug selected and MRP1 transfected, are clearly resistant to have shown before that various kinds of tumor cells express the
IL-4R, including renal cell carcinoma, breast carcinoma, ovarian
Received 10/18/02; revised 5/12/03; accepted 5/13/03.
The costs of publication of this article were defrayed in part by the
2
payment of page charges. This article must therefore be hereby marked The abbreviations used are: MDR, multidrug resistance/multidrug
advertisement in accordance with 18 U.S.C. Section 1734 solely to resistant; Pgp, P-glycoprotein; BCRP, breast cancer resistance protein;
indicate this fact. MRP, multidrug resistance protein; IL-4, interleukin-4; PE, Pseudomo-
Supported by Grant KWF-VU96-1256 of the Dutch Cancer Society. nas exotoxin; GSH, glutathione; BSO, DL-buthionine-S,R-sulphoximine;
1
To whom requests for reprints should be addressed, at Department of E217G, 17-estradiol 17-(-D-glucuronide); IL-4R, IL-4 receptor;
Pathology, VU University Medical Center, De Boelelaan 1117, 1081 HEK, human embryonic kidney; MFI, mean fluorescence index; XTT,
HV Amsterdam. Phone: 31-20-444-4031; Fax: 31-20-444-2964; E-mail: 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxani-
rj.scheper@VUmc.nl. lide; RF, resistance factor.
Downloaded from clincancerres.aacrjournals.org on January 24, 2021. © 2003 American Association for
Cancer Research.5010 IL-4 Toxin Activity in MDR Tumor Cells
carcinoma, melanoma, Kaposi sarcoma, head and neck cancer, term assays, although to a lower extent than MRP1 cells (RF,
glioma, and glioblastoma cells (5–10). These IL-4R-positive 21.4; Ref. 25). 2008-MRP3 cells are low-level resistant to
tumor cells could effectively and specifically be killed using VP-16 (RF, 3.3 and 1.7 for 2008-M3-8 and 2008-M3-4, respec-
IL-4 toxin, both in vitro (9 –12) and in vivo (8, 13, 14). On the tively), but highly resistant to short-exposure methotrexate (RF,
basis of these results, this IL-4 toxin was recently tested in nine 75 and 33 for 2008-M3-8 and 2008-M3-4, respectively; 21).
patients with recurrent, malignant high-grade gliomas, which HEK293/MRP5 cells were shown to be low-level resistant to the
are tumors with very poor prognosis. It was shown that direct drugs 6-mercaptopurine (RF, 3.1) and thioguanine (RF, 2.1; Ref.
glioma injection was safe, without systemic cytotoxicity and, in 23). The same was found for HEK293/MRP4 cells, which are
seven patients, resulted in necrosis of tumor, but not brain tissue. ⬃3–5-fold more resistant to 6-mercaptopurine and thioguanine
One patient was still a complete responder 18 months after drug than to HEK293 parental cells (24).
infusion (15). The cells were grown in RPMI 1640 or DMEM (both from
Thus, IL-4 toxin appears to be a promising new drug in Bio-Whittaker, Verviers, Belgium) supplemented with 10%
cancer treatment. It would be especially important if it shows FCS (Integro, Zaandam, the Netherlands), 2 mM L-glutamine
itself to be active in tumor cells intrinsically resistant, or with (Life Technologies, Inc., Paisley, Scotland), 50 I.U./ml penicil-
acquired resistance, to conventional chemotherapy. Therefore, lin and 50 g/ml streptomycin. MDR cell lines SW-1573/2R120
we investigated the activity of IL-4 toxin in tumor cells that are (MRP1), SW-1573/2R160 (Pgp), GLC4/ADR (MRP1), MCF7/
drug resistant because of overexpression of any of the above D40 (Pgp), MCF7/MR (BCRP), 8226/DOX40 (Pgp), and 8226/
mentioned drug pumps. MR20 (BCRP) were cultured in the presence of either doxoru-
bicin or mitoxantrone, until 3–10 days before experiments. Cell
lines were routinely tested to ensure the absence of Myco-
MATERIALS AND METHODS plasma.
Chemicals. All chemicals and drugs were obtained from IL-4R Expression. IL-4R expression was determined
Sigma Chemical Co. (St. Louis, MO) except for doxorubicin, using a Fluorokine human IL-4R detection kit (R&D systems,
which was purchased from Farmitalia Carlo Erba (Brussels, Minneapolis, MN) according to the manufacturer’s recommen-
Belgium), trichloroacetic acid and Pseudomonas aeruginosa dations. Adherent cells were washed with PBS and were de-
toxin A, which were from ICN Biomedicals Inc. (Aurora, OH), tached from the culture flask bottom by using 5 mM EDTA. The
recombinant IL-4 (CLB, Amsterdam, the Netherlands), and suspension cells were washed thoroughly with PBS to remove
MK571 (L-660,711), which was obtained from Dr. Robert Zam- any residual growth factors that may have been present in the
boni (Merck-Frosst, Pointe-Claire, Quebec, Canada). culture medium. Then 105 cells/sample were incubated with
Recombinant IL-4 Toxin. The IL-4 toxin IL-4(38-37)- biotinylated IL-4 for 60 min and were stained using FITC-
PE38KDEL, containing the circularly permuted IL-4 mutant in conjugated avidin. For negative controls, cells were incubated
which amino acids 38 –129 were linked to amino acids 1–37 via with an irrelevant protein that was biotinylated to the same
a GGNGG linker and then fused to truncated toxin PE38KDEL, degree as IL-4 (soybean trypsin inhibitor, delivered with the
consisting of amino acids 253–364 and 381– 608 of PE, fol- IL-4R detection kit). Fluorescence was analyzed on a FACS-star
lowed by KDEL, was expressed in Escherichia coli and purified flow cytometer (Becton and Dickinson, San Jose, CA). The MFI
as described previously (4, 11, 13). was calculated as the ratio of the mean fluorescence of biotiny-
Cell Lines. The SW-1573 non-small cell lung carcinoma, lated IL-4 treated samples to the mean fluorescence of the
GLC4 small cell lung carcinoma, MCF7 breast carcinoma, and negative control samples.
8226 myeloma cell lines and their MDR sublines have been Cytotoxicity Assays. For cytotoxicity experiments, at
described previously (16 –20). The HT-29 colon carcinoma cell day 0, exponentially growing cells were briefly trypsinized
line was obtained from the American Type Culture Collection. (⬍10 min) and plated in triplicate in 96-well plates (number of
The 2008 ovarian carcinoma cell line was obtained from Dr. cells/well: 5,000 SW-1573; 6,000 SW-1573/2R120 and SW-
Howell from the University of California, San Diego, CA. 1573/2R160; 5,000 GLC4; 6,250 GLC4/ADR; 7,500 MCF7;
MRP1-transfected 2008-M1-4 and 2008-M1-6 cells, MRP2/ 10,000 MCF7/D40 and MCF7/MR; 10,000 8226 and 8226/
cMOAT-transfected 2008-cM-1 and 2008-cM-23 cells and DOX40 and 8226/MR20; 5,000 2008; 6,250 2008-M1– 4 and
MRP3-transfected 2008-M3-4 and 2008-M3-8 cells (21, 22) 2008-M1– 6 and 2008-cM-1 and 2008-cM-23 and 2008-M3– 4
were kindly provided by M. Kool (Netherlands Cancer Institute, and 2008-M3– 8) and cultured for 24 h. Trypsin treatment fa-
Amsterdam, the Netherlands). 293 HEK- and MRP5-transfected cilitated the most accurate plating of the cells, whereas such
HEK293/MRP5 cells (23) were kindly provided by J. Wijnholds short exposure never resulted in a detectable loss of IL-4R.3
(Netherlands Cancer Institute, Amsterdam, the Netherlands). Twenty-four h after plating the cells, toxins (IL-4 toxin, PE)
MRP4-transfected HEK293/MRP4 were kindly provided by G. were added in six to eight different concentrations, and, after
Reid (Netherlands Cancer Institute, Amsterdam, the Nether- another 72 h, cell survival was determined using the sulforho-
lands) and are described elsewhere (24). All of the transfected damine B method for the adherent cell lines (SW-1573, MCF7,
cell lines have been shown previously to overexpress functional and 2008 and sublines) and the XTT method for the semiadher-
drug pumps as reflected in their resistance to cytostatic drugs. In ent GLC4 and GLC4/ADR and suspension 8226, 8226/DOX40,
cytotoxicity assays, 2008-MRP1 cells were reported to be
clearly resistant to VP-16 (RF, 20) and methotrexate in short-
term exposure experiments (RF, 93; Ref. 21). Also, the 2008-
3
MRP2 cells were found to be resistant to methotrexate in short- Puri, R. K., Kreitman, R. J., and Husain, S. R., unpublished data.
Downloaded from clincancerres.aacrjournals.org on January 24, 2021. © 2003 American Association for
Cancer Research.Clinical Cancer Research 5011
and 8226/MR20 cells. GLC4 and GLC4/ADR cells are more RESULTS
efficient metabolizing XTT than the 8226, 8226/DOX40, and Drug-Selected MDR Tumor Cells Express IL-4R. The
8226/MR20 cells. Therefore, the optimal XTT concentrations expression of IL-4R on MDR tumor cells was determined using
were 25 g of XTT mixed with 1.9 g of phenazine methosul- biotinylated IL-4 in a flow cytometry assay. IL-4R expression
fate for the GLC4 and GLC4/ADR cells and 50 g of XTT and was calculated as the ratio of mean fluorescence of cell bound,
1.9 g of phenazine methosulfate for the 8226, 8226/DOX40, stained, IL-4 compared with the mean fluorescence of an irrel-
and 8226/MR20 cells/well for 3– 4 h. The RF was calculated as evant control protein, soybean trypsin inhibitor (MFI). For a
the ratio of the IC50 (concentration of the compound that inhibits colon carcinoma cell line, HT-29, previously reported to express
growth of the cells by 50%) of the resistant cells to the IC50 of ⬃6000 IL-4Rs/cell (28), which is similar to IL-4R numbers of
the parental cells. In some experiments, also MRP1 modulators other, IL-4 toxin sensitive, tumor cell lines (5, 7), we found
(MK571 or probenecid) were added to the cultures. Appropriate positive staining with a MFI of 14.9 ⫾ 7.6.
controls were done to check for the cytotoxicity of DMSO that In general, the tested tumor cell lines, which were from
was used to dissolve the probenecid. The total amount of DMSO different histogenetic origins, including lung carcinoma, breast
in the cell cultures never exceeded 0.5%. For MRP1 modulation carcinoma, and multiple myeloma, and their drug-selected MDR
through GSH depletion, cells were preincubated with BSO 24 h sublines, bound biotinylated IL-4 to a level similar to that of the
before the addition of the cytotoxic compound to be tested. HT-29 colon tumor cell line (Table 1). In some cases, however
Clonogenic Assay. At day 0, SW-1573, SW-1573/ (for instance, the SW-1573/2R120 lung carcinoma cells), drug
2R120, and SW-1573/2R160 were plated in duplicate in 6-well selection had resulted in more binding of IL-4 to the cells
plates to obtain ⬃200 colonies/well in the control (no drug) compared with the parental cells, suggesting up-regulation of
wells. Twenty-four h later, cells were exposed to different the IL-4R (Table 1). On the basis of these data, and because IL-4
concentrations of IL-4 –PE and were left to grow for another 9 toxin sensitivity of cells is dependent on their IL-4R expression,
days. Then cells were washed, fixed, and stained with crystal MDR tumor cells would be expected to be as sensitive, or even
violet (0.1% crystal violet in 20% ethanol). Colonies of ⬎30 more sensitive, to IL-4 toxin than the non-MDR, parental cells
cells were counted. from which they were derived.
Inside-Out Plasma Membrane Vesicle [3H]E217G Up- Drug-Selected MDR Tumor Cells Are Sensitive to IL-4
take Assay. Inside-out vesicles were prepared from plasma Toxin Except When Overexpressing MRP1. In 72-h cyto-
membranes of GLC4/ADR cells as described previously (26) toxicity assays, tumor cells that are resistant to the established
with slight modifications. Cells were collected by centrifugation anticancer drugs doxorubicin or mitoxantrone because of over-
(275 ⫻ g, 5 min) and washed twice in ice-cold PBS (pH 7.4). expression of Pgp (SW-1573/2R160, MCF7/D40) or BCRP-
The cell suspension (107 cells/ml) was incubated in a buffer (MCF7/MR and 8226/MR20) were found to be almost as sen-
containing 100 mM KCl, 5 mM MgCl2, 1 mM phenylmethylsul- sitive to IL-4 toxin as the non-MDR tumor cells with RFs ⬍2
fonyl fluoride, and 50 mM HEPES/KOH (pH 7.4) for 60 min at (Table 1; Fig. 1). Notably, the doxorubicin-selected and highly
0°C. Hereafter, cells were disrupted by sonication at 20% of the Pgp-overexpressing 8226/D40 myeloma cells were found to be
maximum power of an M.SE sonicator (Soniprep 150) for 3 five times more sensitive to the toxin than were the parental
bursts of 15 s each. The suspension was centrifuged at 1500 ⫻ 8226 cells (P ⬍ 0.01; Table 1; Fig. 1). In contrast, the doxoru-
g for 10 min. The supernatant was layered on top of a 46% bicin-selected MRP1-overexpressing cells SW-1573/2R120
sucrose cushion (KCl/HEPES buffer) and was centrifuged at were significantly resistant to IL-4 toxin with a RF of 8.7 (P ⬍
100,000 ⫻ g for 60 min. The interface was removed and washed 0.01). The MRP1-overexpressing GLC4/ADR cells also were
in the buffer described above. The final membrane preparations resistant with RF 4.7, but this difference did not reach statistical
were stored at ⫺80°C at a protein concentration of 4 mg/ml. significance (P ⫽ 0.09; Table 1). In a 10-day clonogenic assay
A rapid filtration method (27) was used to measure the using SW-1573 cells and the MRP1- and Pgp-overexpressing
transport of [3H]E217G into isolated inside-out plasma mem- sublines, the colony formation of the Pgp-overexpressing SW-
brane vesicles. Vesicles were incubated in a buffer containing 1573/2R160 cells was inhibited as well as the colony formation
100 mM KCl/50 mM HEPES/KOH (pH 7.4) at 37°C (0.15 mg/ml of the parental cells (Fig. 1). The MRP1 overexpressing SW-
protein), in the presence of 10 mM MgCl2, 2 mM ATP, and 10 nM 1573/2R120, again, showed resistance because tumor cell col-
[3H]E217G (specific activity, 40.5 Ci/mmol; Perkin-Elmer onies were formed at higher doses of IL-4 toxin.
Life Science, Boston, MA). The final reaction volume was 50 MRP1-Transfected Cells Are Resistant to IL-4 Toxin.
l. The transport was stopped after 2 min by the addition of 2 Because these results with drug-selected cell lines suggested a
ml of ice-cold KCl/HEPES buffer. Thereafter, the mixture was possible role for MRP family members in IL-4 toxin resistance,
rapidly filtrated through OE67 membrane filters (Schleicher & we selected a panel of MRP transfectants and performed cyto-
Schuell, Dassel, Germany). The filters were washed twice with toxicity assays. Two independent clones of MRP1-transfected
2 ml of KCl/HEPES buffer. The radioactivity associated with cells, 2008-M1– 4 and 2008-M1– 6, showed clear resistance to
the filters was measured by liquid scintillation counting. IL-4 toxin with RFs of 5.9 and 4.3 (P ⬍ 0.01; Table 2). In
Statistical Analysis. Statistical analyses of the data were contrast, the cells that were transfected with the full-length
performed using the unpaired two-tailed Student’s t test. cDNA of other members of the MRP protein family, MRP2–5,
Differences were considered statistically significant when were not (MRP3–5), or only slightly (MRP2), resistant to IL-4
P ⬍ 0.05. toxin (Table 2). Similarly as seen for the drug-selected MRP1-
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Cancer Research.5012 IL-4 Toxin Activity in MDR Tumor Cells
Table 1 IL-4R expression and IL-4 toxin sensitivity of drug-sensitive and drug-selected MDR tumor cell lines
Results are shown as means ⫾ SD (in ng/ml and nM) of two to six experiments. Respective RFs and number of measurements (n) are shown
within parentheses.
IC50 IL-4 toxin
Phenotype MFI IL-4R ng/ml nM
Non-small cell lung carcinoma
SW-1573 Parental 13.4 ⫾ 9.8 (n ⫽ 2) 82.4 ⫾ 48.7 1.6 ⫾ 1.0 (n ⫽ 3)
SW-1573/2R120 doxa selected, MRP1 21.9 ⫾ 18.0 (n ⫽ 2) 718.3 ⫾ 83.0 14.0 ⫾ 1.6 (8.7) (n ⫽ 3)b
SW-1573/2R160 dox selected, Pgp 11.9 ⫾ 5.1 (n ⫽ 2) 120.0 ⫾ 21.2 2.3 ⫾ 0.4 (1.5) (n ⫽ 2)
Small cell lung carcinoma
GLC4 Parental 10.0 ⫾ 7.7 (n ⫽ 3) 273.3 ⫾ 99.4 5.3 ⫾ 1.9 (n ⫽ 3)
GLC4/ADR dox selected, MRP1 18.2 ⫾ 8.1 (n ⫽ 3) 1033.0 ⫾ 628.2 20.0 ⫾ 12.2 (4.7) (n ⫽ 3)
Breast carcinoma
MCF7 Parental 7.4 ⫾ 0.3 (n ⫽ 2) 1.4 ⫾ 1.0 0.027 ⫾ 0.019 (n ⫽ 6)
MCF7/D40 dox selected, Pgp 9.4 ⫾ 1.6 (n ⫽ 2) 2.0 ⫾ 1.2 0.039 ⫾ 0.023 (1.4) (n ⫽ 4)
MCF7/MR Mitox selected, BCRP 7.1 ⫾ 0.9 (n ⫽ 2) 2.6 ⫾ 2.3 0.050 ⫾ 0.044 (1.9) (n ⫽ 4)
Multiple myeloma
8226 Parental 16.5 ⫾ 10.0 (n ⫽ 3) 603.0 ⫾ 188.8 11.7 ⫾ 3.7 (n ⫽ 5)
8226/D40 dox selected, Pgp 23.9 ⫾ 15.6 (n ⫽ 3) 99.4 ⫾ 56.8 1.9 ⫾ 1.1 (0.2) (n ⫽ 3)b
8226/MR20 Mitox selected, BCRP 17.8 ⫾ 13.1 (n ⫽ 2) 603.2 ⫾ 137.9 11.7 ⫾ 2.7 (1.0) (n ⫽ 3)
a
dox, doxorubicin; mitox, mitoxantrone.
b
Statistically significantly (P ⬍ 0.01) different from parent cell line in unpaired two-tailed Student’s t test.
overexpressing cells, the resistance in the MRP1-transfected cell MRP1-Expressing Cells Are Not Resistant to PE. To
lines was not related to a decreased IL-4R expression (Table 2). define the structural requirements for resistance to IL-4 toxin in
IL-4 Toxin Resistance Can Be Reversed by MRP1 An- MRP1-overexpressing cells, cytotoxicity assays were performed
tagonists MK571 and Probenecid, but not by BSO. To test with IL-4 and PE. IL-4 did not inhibit the growth of 2008
whether the mechanism leading to resistance to the IL-4 toxin ovarian, and SW-1573 and GLC4 lung carcinoma cells up until
fusion protein behaves similarly to the more classical MRP1 a 100-ng/ml dose (results not shown), even though IL-4 at high
substrates, we tested two established strategies for MRP1-resist- doses (10 –100 ng/ml) may be cytostatic to some tumor cell lines
ance reversal, by pump modulators and by GSH depletion. (5, 32). PE was highly toxic and, in the case of SW-1573 cells,
Probenecid and MK571 are known antagonists of MRP1. They was found to be even more toxic than the IL-4 toxin (IC50 of
were previously shown to reverse the resistance to cytostatic 50.3 ng/ml versus 82.4 ng/ml; Tables 1 and 3). This is probably
drugs in MRP1-, but not in Pgp-, overexpressing cells in a because of high expression of the ␣2 macroglobulin receptor,
concentration-dependent way (29, 30). BSO inhibits the enzyme which binds PE and internalizes PE into cells. This receptor is
␥-glutamylcysteine synthetase, which catalyzes the first step in widely expressed in different normal human tissues, and re-
GSH synthesis. GSH depletion by BSO results in the reversal of placement of the ␣2 macroglobulin receptor-binding fragment of
MRP1-mediated resistance to drugs like doxorubicin and vin- PE by other ligands, e.g., IL-4, facilitates more specific targeting
cristine, indicating that GSH is needed for the efflux of these of the toxin to tumor cells only. Cells with overexpression of
drugs from the cell (31). MRP1, like SW-1573/2R120 and 2008-M1– 4 and 2008-M1– 6,
In our experiments, the addition of probenecid or MK571, were slightly resistant [SW-1573, RF 1.9 (P ⬍ 0.01) and
in concentrations routinely used to antagonize MRP1 (1 mM and 2008-M1– 4, RF 1.4; Table 3] or even more sensitive to PE
30 M, respectively; Refs. 29, 30), reversed IL-4 toxin resist- [2008-M1– 6, RF 0.6 (P ⬍ 0.05); Table 3]. This indicates that
ance in MRP1-transfected 2008-M1– 4 cells (Fig. 2). This is in the main difference between PE and IL-4 toxin, the above
line with the involvement of MRP1 in IL-4 toxin resistance. The mentioned replacement of the ␣2 macroglobulin receptor bind-
smaller, but consistent, effect of probenecid and MK571, seen in ing domain of PE (amino acids 1 to 252, of a total of 613 amino
2008 parental cells, has been attributed to the basal expression acids) by IL-4, is a structural requirement for resistance to the
level of MRP1 in 2008 cells (22). cytotoxic activity of IL-4 toxin mediated by MRP1.
Because 2008, 2008-M1– 4, and GLC4 cells showed high Inhibition by IL-4 Toxin and IL-4, but not by PE, of
intrinsic sensitivity to BSO treatment (results not shown) GLC4/ MRP1-Mediated [3H]E217G Transport. To further delin-
ADR cells were used to test the effect of GSH depletion on eate the mechanism of MRP1-mediated IL-4 toxin resistance,
MRP1-mediated IL-4 toxin resistance. GLC4/ADR cells were we used an inside-out vesicle system, developed earlier for
incubated with 25 M BSO for 24 h before the addition of IL-4 studying transmembrane transporter functions (26, 27, 33). We
toxin. Previously, we reported that this procedure results in measured the inhibition of transport of the established MRP1
reduced GSH levels to 18% of control cultures of this cell line substrate E217G (34, 35) by IL-4 toxin. Vesicles prepared
(31). This GSH depletion by BSO, however, did not affect the from MRP1-overexpressing GLC4/ADR cells were used for
IL-4 toxin resistance of GLC4/ADR cells, whereas it did affect these experiments. At a concentration of 10 nM, the uptake of
the doxorubicin resistance as expected (Fig. 3). This indicates [3H]E217G was 2.1 ⫾ 0.2 pmol/mg protein/min. In the pres-
that a physiological level of GSH is not required for MRP1- ence of different concentrations of IL-4 toxin the uptake of
mediated IL-4 toxin resistance. [3H]E217G was inhibited (Fig. 4). Importantly, also IL-4 in-
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Cancer Research.Clinical Cancer Research 5013
DISCUSSION
The success rate of chemotherapy in cancer treatment is
limited. Tumors can be intrinsically resistant or acquire resist-
ance during treatment with cytostatic drugs. In these cases, other
therapies like immunotherapy may be useful. Most immunotox-
ins, composed of either a monoclonal antibody or another tumor
cell-binding ligand, and a toxin, like PE, are proteins of rather
large size and do not resemble the classical chemical structures
of substrates of drug pumps like Pgp and the MRP proteins.
Therefore, they may be good alternatives or additives to con-
ventional chemotherapy.
A number of studies reported on the effects of immuno-
toxins on drug-resistant, primarily Pgp-overexpressing, cells
with some conflicting results. Some reported a beneficial effect
of an immunotoxin alone or in combination with conventional
drugs in cell kill of MDR tumor cells (36 –38). Others argue for
using immunotoxins in combination with primary conventional
chemotherapy before drug resistance develops, because an ad-
ditive effect of a saporin toxin-conjugated anti-CD138 immu-
notoxin on chemotherapy could be measured only in drug-
sensitive, non-Pgp-overexpressing, cells (39). Notably, two
independent studies reported on immunotoxins that specifically
target Pgp-overexpressing cells (40, 41). Both compounds con-
sist of an antibody (fragment) recognizing Pgp and (truncated)
PE. In both reports, it was concluded that, independent of the
question as to whether treating patients with such an immuno-
toxin will be feasible because of potential toxicity to normal
tissues expressing Pgp, the sensitivity of MDR cells to the toxin
was sufficient.
IL-4 toxin is an immunotoxin that takes advantage of the
finding that, for an as-yet-unknown reason, many tumor cell
types overexpress IL-4R (5–10). IL-4R is also expressed in
several types of normal cells (e.g., immune cells and fibroblast
and endothelial cells) but in lower amounts, and it, therefore, is
Fig. 1 Resistance to IL-4 toxin in doxorubicin-resistant MRP1-over- an attractive feature to use for targeting an anticancer drug to
expressing, but not Pgp-overexpressing, MDR cells, both in 96-well tumor cells. After several years of preclinical testing, IL-4 toxin
plate cytotoxicity and clonogenic assays. Parental drug-sensitive SW-
1573, MRP1-overexpressing SW-1573/2R120, and Pgp-overexpressing has reached clinical testing with promising first results in the
SW-1573/2R160 cells were cultured with doxorubicin for 96 h (A) or treatment of high-grade glioma, a particularly deadly form of
with IL-4 toxin for 72 h (B), and cell survival was determined by using cancer (15).
the sulforhodamine B assay. Results shown are means ⫾ SDs of In this report, we show the results of an in vitro study on
triplicate measurements in a typical experiment. In a clonogenic exper-
iment (C), cells were plated in duplicate in 6-well plates with different the potential use of the IL-4 toxin in drug-resistant tumors. We
concentrations of IL-4 toxin and were allowed to form colonies for 9 show that the majority of MDR cell lines, that is, the Pgp-,
days. Colonies were stained using crystal violet, and colonies larger than MRP2–5-, and BCRP-overexpressing tumor cell lines, remain
30 cells were counted. Percentages shown are numbers of colonies/ sensitive or almost just as sensitive to the toxin, as do the
colonies in control without IL-4 toxin ⫻ 100%. Results of a typical
parental cells. Slight resistance cannot be completely ruled out
experiment are shown.
in MRP2–5-overexpressing cells because the overexpression
levels of functional protein have not been directly compared in
these cells, but only MRP1-overexpressing tumor cells, both
drug-selected and MRP1-transfected, show robust resistance to
hibited this uptake. At the highest concentration used (500 nM), IL-4 toxin. Results of additional experiments with the MRP1
IL-4 toxin and IL-4 inhibited MRP1-mediated E217G transport antagonists probenecid and MK-571 confirmed a role of MRP1-
up to 75 and 55%, respectively, of the control. For comparison, mediated transport in IL-4 toxin resistance.
in our hands, the known MRP1 substrate daunorubicin (500 nM) An intriguing question is how MRP1 can confer IL-4 toxin
inhibited this transport to 51% of the control (result not shown). resistance. The cytotoxicity experiments showing that MRP1-
In the presence of the unmodified PE toxin, no inhibition of overexpressing cells are not resistant to unmodified PE indicate
MRP1-mediated transport could be observed, in line with the that MRP1 does not confer general resistance to PE-based
results showing that MRP1-overexpressing cells are resistant to toxins. For PE to become toxic, several processes are required,
IL-4 toxin, but not to PE. including internalization of the toxin into the endosome com-
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Cancer Research.5014 IL-4 Toxin Activity in MDR Tumor Cells
Table 2 IL-4R expression and IL-4 toxin sensitivity of MRP-transfected cell lines
Results are shown as means ⫾ SD (in ng/ml and nM) of two to four experiments. Respective RFs and number of measurements (n) are shown
within parentheses.
IC50 IL-4 toxin
Phenotype MFI IL-4R ng/ml nM
Ovarian carcinoma
2008 Parental 5.7 ⫾ 2.3 (n ⫽ 3) 2.9 ⫾ 0.6 0.056 ⫾ 0.012 (n ⫽ 4)
2008-M1–4 MRP1 9.0 ⫾ 6.0 (n ⫽ 2) 17.0 ⫾ 2.1 0.329 ⫾ 0.041 (5.9) (n ⫽ 3)ⴱⴱa
2008-M1–6 MRP1 7.0 ⫾ 6.3 (n ⫽ 2) 12.6 ⫾ 4.4 0.244 ⫾ 0.085 (4.3) (n ⫽ 2)ⴱⴱ
2008-cM-1 MRP2/cMOAT 4.3 ⫾ 1.0 (n ⫽ 2) 3.5 ⫾ 0.3 0.068 ⫾ 0.006 (1.2) (n ⫽ 2)
2008-cM-23 MRP2/cMOAT 6.2 ⫾ 4.4 (n ⫽ 2) 6.0 ⫾ 2.2 0.116 ⫾ 0.043 (2.1) (n ⫽ 2)ⴱ
2008-M3–4 MRP3 4.3 ⫾ 1.0 (n ⫽ 2) 2.5 ⫾ 0.1 0.049 ⫾ 0.002 (0.9) (n ⫽ 2)
2008-M3–8 MRP3 4.2 ⫾ 0.6 (n ⫽ 2) 1.8 ⫾ 0.4 0.034 ⫾ 0.008 (0.6) (n ⫽ 2)
HEK
HEK293 Parental NDb 7.6 ⫾ 6.8 0.147 ⫾ 0.132 (n ⫽ 2)
HEK293/MRP4 MRP4 ND 7.4 ⫾ 6.9 0.144 ⫾ 0.134 (1.0) (n ⫽ 2)
HEK293/MRP5 MRP5 ND 6.1 ⫾ 7.9 0.118 ⫾ 0.153 (0.8) (n ⫽ 2)
a
ⴱ/ⴱⴱ Statistically significantly (ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01) different from parent cell line in unpaired two-tailed Student’s t test.
b
ND, not determined.
Fig. 2 Reversal of IL-4 toxin resistance by MRP1 antagonists probe-
necid and MK-571. 2008- and MRP1-transfected 2008-M1-4 cells were Fig. 3 BSO does not reverse resistance to IL-4 toxin in MRP1-over-
cultured in the presence or absence of probenecid (1 mM) or MK-571 expressing cells. GLC4/ADR cells were cultured in the presence or
(30 M), and survival was determined after 72 h by using the sulforho- absence of BSO (25 M). IL-4 toxin or doxorubicin was added after
damine B assay. Results shown are means ⫾ SDs of triplicate meas- 24 h. Survival was determined after an additional 72 h by using the XTT
urements in a typical experiment. Where not visible, SD error bars are assay. Results shown are means ⫾ SDs of triplicate measurements in a
hidden within the symbol. typical experiment.
partment, followed by cleavage of the toxin into two fragments of E217G uptake into inside-out plasma membrane vesicles
and transport of the enzymatically active part to the endoplasmic prepared from MRP1-overexpressing cells by IL-4 toxin sup-
reticulum, translocation of this part from the endoplasmic retic- ports this view. Previously, we reported that MRP1 can trans-
ulum into the cytoplasm, and the inhibition of protein synthesis port short (5-mer) peptides (43). Little is known, however, about
through ADP-ribosylating elongation factor 2 (42). These pro- the transport of such large-size proteins as this toxin, which has
cesses are likely to be the same for the natural PE and IL-4 484 amino acids and a Mr of 52,000. Two reports have sug-
toxin, and, therefore, MRP1 probably does not affect these gested that MRP1 may play a role in the secretion of basic
processes. fibroblast growth factor (at Mr 16,000), which lacks a signal
Instead, it would be more likely that the resistance to IL-4 sequence (44, 45). Independent and definitive proof for that
toxin is related to the transport function of MRP1. The inhibition finding, however, is lacking.
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Cancer Research.Clinical Cancer Research 5015
Table 3 Pseudomonas (P.) exotoxin A cytotoxicity to MRP1-overexpressing cells
Results are shown as means ⫾ SD (in ng/ml and nM) of two or three experiments. Respective RFs and number of measurements (n) are shown
within parentheses.
IC50 P. exotoxin A
Phenotype ng/ml nM
SW-1573 Parental 50.3 ⫾ 2.1 0.77 ⫾ 0.03 (n ⫽ 3)
SW-1573/2R120 MRP1, doxa selected 95.3 ⫾ 4.7 1.45 ⫾ 0.07 (1.9) (n ⫽ 3)ⴱⴱb
2008 Parental 219.0 ⫾ 9.9 3.33 ⫾ 0.15 (n ⫽ 2)
2008-M1–4 MRP1 transfected 313.0 ⫾ 103.2 4.76 ⫾ 1.57 (1.4) (n ⫽ 2)
2008-M1–6 MRP1 transfected 142.5 ⫾ 7.8 2.17 ⫾ 0.12 (0.7) (n ⫽ 2)ⴱ
a
dox, doxorubicin.
b
ⴱ/ⴱⴱ Statistically significantly (ⴱ, P ⬍ 0.05, ⴱⴱ, P ⬍ 0.01) different from parent cell line in unpaired two-tailed Student’s t test.
natively, IL-4 toxin may interact with MRP1 in a noncatalytic
fashion, i.e., through binding rather than actual transport, still
resulting in interference with substrate transport and IL-4 toxin
resistance. In any case, this transport apparently does not require
physiological levels of GSH as seen for some, but not all, other
substrates (29, 43).
From a clinical point of view, our findings show that
increased expression of MDR proteins, except for MRP1, in
tumor cells does not, or only marginally, reduce the sensitivity
to IL-4 toxin. Still, because MRP1 resistance may be reversed
using MRP1 antagonists, IL-4 toxin may be an effective drug for
cancer patients presenting with MDR tumors.
Fig. 4 Uptake of E217G is inhibited by IL-4 toxin and IL-4, but not
by PE. Plasma membrane vesicles of MRP1-overexpressing GLC4/
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Cancer Research.Multidrug-Resistant Tumor Cells Remain Sensitive to a
Recombinant Interleukin-4- Pseudomonas Exotoxin, Except
When Overexpressing the Multidrug Resistance Protein
MRP1
Mariska C. de Jong, George L. Scheffer, Henk J. Broxterman, et al.
Clin Cancer Res 2003;9:5009-5017.
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