Reversal of resistance by GF120918 in cell lines expressing the ABC half-transporter, MXR
Introduction
The emergence of multiple members of the ABC transporter gene family has raised anew the question of effective reversal of clinical drug resistance. While P-glycoprotein (Pgp) and the multidrug resistance associated protein (MRP) are the most extensively studied drug resistance transporters, the number of newly reported genes suggests the potential involvement of many others in clinical resistance [1], [2]. The canalicular multispecific organic anion transporter (cMOAT or MRP2), responsible for hepatic transport of bilirubin glucuronide, has been correlated with resistance to cisplatinum and to SN38, the active metabolite of CPT 11 [3]. A number of MRP and cMOAT homologues (MRP3, MRP4, MRP5, MRP6) have been described [4], [5]. Examination of an EST database revealed evidence of 21 previously unknown ABC transporter genes [6]. Finally, the breast cancer resistance gene (BCRP) was identified in an adriamycin-resistant subline of MCF-7 cells [7], and recently in our laboratory, a mitoxantrone-resistance gene (MXR) was identified in the same subline and in a mitoxantrone-resistant subline of human colon carcinoma cells [8]. Both genes are almost identical to ABCP, an ABC transporter gene cloned from human placenta [9]. Homologies with other genes such as the white eye pigment gene suggest that BCRP/MXR/ABCP encodes a protein which is a half-transporter molecule requiring dimerization in order to function [10], [11].
Given the array of ABC transporters that may play a role in clinical drug resistance, a multispecific antagonist able to inhibit more than one transporter or MDR phenotype would be advantageous. The development of antagonists against Pgp-mediated drug efflux led to a large number of clinical trials attempting the reversal of drug resistance [12], [13]. Yet, despite the relative ease of sensitizing cells to chemotherapy in the laboratory, clinical trials have been disappointing, with no clear support emerging for the use of chemosensitizers in clinical practice [14].
The aim of this study was to assess the potential of the second generation Pgp antagonist, GF120918, as a multispecific antagonist in a cell line with overexpression of the newly described transporter, MXR. Cell lines overexpressing MXR have resistance to mitoxantrone, anthracyclines, topotecan, and the active metabolite of irinotecan, SN38 [8]. Cells transfected with the BCRP/MXR/ABCP gene display reduced rhodamine retention and display a resistance pattern similar to cells that overexpress MXR [7]. While this is a broad resistance pattern, several notable exceptions include the vinca alkaloids, taxanes and VP-16. GF120918 is an acridine carboxamide derivative able to inhibit Pgp-mediated efflux at nanomolar concentrations [15], [16], [17]. The assays used to assess the effectiveness of GF120918 as a potential antagonist were cytotoxicity assays, confocal microscopy, and flow cytometry.
GF120918 was found to be highly effective at reversing both Pgp-mediated and MXR-mediated multidrug resistance, although relatively ineffective in blocking MRP. GF120918, therefore, appears to fit the paradigm of a multispecific blocker.
Section snippets
Materials
GF120918 was obtained from Glaxo Pharmaceuticals (Research Triangle Park, NC). Mitoxantrone, topotecan, and rhodamine 123 were obtained from Sigma Chemical Co. (St. Louis, MO). Calcein AM was obtained from Molecular Probes (Eugene, OR).
Cell lines
The cell lines used were the S1 colon cancer cell line, the MCF-7 breast cancer cell line and several derivative sublines which have been selected in chemotherapeutic drugs for acquired drug resistance. The human colon carcinoma cell line, S1, and its
In vitro cytotoxicity assay
The cytotoxicity of GF120918 was initially tested in the resistant sublines. No evidence of cross-resistance in the resistant sublines was observed. The IC50 for GF120918 alone in each of the cell lines was 15.2, 3.6, 2.5, 40 and 7.5 μM for S1 parental, S1-B1-20, S1-M1-80, MCF-7 parental and MCF-7/VP cell lines, respectively.
Next, the IC50 values of mitoxantrone and topotecan were assessed with or without GF120918 in the different cell lines. Fig. 1A–C shows the results of GF120918 reversal of
Discussion
While Pgp and MRP are best characterized, the number of identified transporters that potentially could play a role in clinical drug resistance has steadily expanded. Chemosensitizers that act on more than one transporter may, therefore, play an important role in the attempts to reverse multidrug resistance. In this report we describe a potential multispecific blocker, GF120918, a second generation Pgp antagonist identified for its potency in reversing Pgp-mediated resistance [15], [28].
Acknowledgements
We would like to thank Paul Wissel for his help in obtaining GF 120918, and Lee Greenberger for the original S1-M1-3.2 cell line from which the S1-M1-80 cell line was derived.
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