Fumitremorgin C: A Chemosensitizing Agent for Mitoxantrone Resistance
July 1, 2000
Fumitremorgin C: A Chemosensitizing Agent for Mitoxantrone Resistance
By Sridhar K. Rabindran, PhD
The efficacy of chemotherapy is limited by the phenomenon known as multiple drug resistance (MDR). The use of chemosensitizing agents (also known as reversal agents or MDR modulators) to render resistant tumor cells responsive to anticancer agents is an appealing strategy to overcome MDR. Modulators for P-glycoprotein, the best understood mediator of MDR, are currently being evaluated in clinical trials. However, the appearance and co-existence of MDR pathways during therapy complicates this approach. One solution to the problem is to develop reversal agents that are specific for each known resistance pathway, and to combine these in the clinic, depending upon the spectrum of resistance mechanisms that are present. Toward this goal, we set out to identify reversal agents that specifically resensitze a tumor cell line selected for resistance to the antitumor agent, mitoxantrone. The properties of fumitremorgin C (FTC) are described: a chemosensitizing agent specific for the breast cancer resistance protein (BCRP), and a transporter protein that mediates resistance to mitoxantrone and doxorubicin.
Introduction
In experimental systems, selection for resistance to a single cancer chemotherapeutic agent often results in cross-resistance to a variety of structurally and functionally diverse molecules.1 Overexpression of members of the ATP binding cassette family of transporters (ABC transporters), which act as drug efflux pumps, is commonly associated with this phenotype. Among the 33 genes predicted to encode human ABC transporters, five mediate MDR when transfected into drug-sensitive cells: P-glycoprotein (P-gp)/MDR1; the multidrug resistance protein (MRP), and its homologues cMOAT/MRP2 and MRP3; and a newly reported transporter, the breast cancer resistance protein (BCRP).1-5 (BCRP is also known as the mitoxantrone-resistance gene product, [MXR] or the placenta-specific ABC transporter [ABCP].)6,7 These proteins mediate resistance to such commonly used chemotherapeutic drugs as doxorubicin, mitoxantrone, etoposide, topotecan, paclitaxel, and vincristine. However, the exact cross-resistance profile mediated by each transporter is distinct, although overlapping. For example, cells expressing P-gp are often cross-resistant to mitoxantrone and doxorubicin, while MRP-expressing cells are resistant to doxorubicin but not mitoxantrone.3,8 Selection of tumor cells in doxorubicin often results in increased expression of P-gp or MRP, while selection of cells in mitoxantrone or topotecan frequently results in sublines with increased BCRP expression.9,10 As a further complication, multiple transporters may exist together within drug-resistant cells, and a switch in the resistance mechanism may occur as a cell line is cultured in increasing concentration of the selective agent.11
ABC transporters are not the sole mediators of MDR. Mutations in the tumor suppressor p53, loss of cell cycle checkpoints, and alteration in apoptotic pathways clearly determine the response of a cell to antitumor agents.12 Increased expression of LRP/major vault protein, changes in the activity/levels or nuclear localization of topoisomerase II, or modifications in glutathione metabolism are also associated with MDR.1,13 In most cases, however, the precise contribution of these alternate pathways toward the total drug resistance phenotype remains to be elucidated.
Identification and Characterization of Fumitremorgin C
Reversing MDR is a major goal of cancer chemotherapy. ABC transporters, which mediate drug resistance, are membrane-embedded proteins, and are thought to act as drug efflux pumps, preventing the cytotoxic agent from reaching lethal levels within the cell. Inhibition of the ABC transporters by pharmacological agents, therefore, is an attractive strategy to resensitize cells to antitumor agents. Furthest along in clinical development are modulators for P-gp mediated MDR, particularly PSC-833.14 Verapamil, genistein, a 1,4-dihydropyridine analogue (NIK 250), and a PKC inhibitor (GF 109203X), have been reported to reverse MRP-mediated resistance in experimental systems.15 Our original goal was to identify a chemosensitizing agent specific for cells selected for resistance to mitoxantrone, which do not express P-gp or MRP. At that time, the mechanism underlying this resistance phenotype was unknown, although it was suspected to be because of the expression of a novel drug efflux pump.16 Since the publication of that report, the gene for the transporter protein has been cloned, and the "mitoxantrone resistant phenotype" has been attributed to BCRP, the newly identified ABC transporter.5
To identify reversal agents for the mitoxantrone resistance pathway, we used a drug-selected cell line, S1-M1-3.2.16 These cells were obtained from S1 cells (a subclone of LS174T, a human colon carcinoma cell line) by stepwise selection in mitoxantrone. S1-M1-3.2 cells show all the phenotypic features typical of cells selected for mitoxantrone resistance: 1) primary resistance to anthracyclines (mitoxantrone and doxorubicin), with significant cross-resistance to the topoisomerase I poison, topotecan, lower levels of resistance to the topoisomerase II poisons (etoposide, amsacrine), and no cross-resistance to the microtubule-active agents (paclitaxel, colchicine, vinblastine); 2) reduced drug accumulation because of enhanced ATP-dependent drug efflux; and 3) overexpression of BCRP, but not P-gp or MRP.16 Extracts from natural product libraries were incubated with S1-M1-3.2 cells in the absence or presence of 3.2 mcmol mitoxantrone (a concentration to which the cells are completely resistant, and which is used for their routine culture). Extracts that caused cell death when combined with mitoxantrone, but had no effect in its absence, were considered to have reversal activity and were evaluated further. A lysate of Aspergillus fumigatus, grown under solid fermentation conditions, was found to have consistent reversal activity in these cells, with minimal toxicity over a wide dose range. The active agent in the extract was purified by high pressure liquid chromatography and shown to be FTC by spectroscopic methods.
FTC belongs to a class of diketopiperazines, which are potent mycotoxins.17 In vitro, FTC shows little toxicity by itself up to 80 mcmol, but can potently resensitize cells S1-M1-3.2 cells to mitoxantrone, doxorubicin, and topotecan at concentrations between 1 and 10 mcmol. No chemosensitization is observed in drug sensitive parental cells. Reversal of resistance is associated with an increase in the amount of antitumor drug accumulated by the resistant cells, suggesting that FTC resensitizes drug-resistant cells by inhibiting BCRP-mediated drug transport.16 Since the drug selected S1-M1-3.2 cells also show reduced topoisomerase II activity, which may contribute to the MDR phenotype, it was important to test the activity of FTC in BCRP-transfected cells, where drug resistance is solely due to the expression of the transporter protein.16 FTC almost completely resensitizes BCRP-transfected MCF-7 cells to mitoxantrone, doxorubicin, and topotecan.18 As in the drug-selected cells, chemosensitization is associated with an increase in the accumulation of chemotherapeutic drugs.18 This demonstrates that the reversal activity of FTC is exclusively due to modulation of BCRP function.
FTC Efficacy
FTC is a selective reagent for BCRP-mediated MDR, since it does not reverse resistance in P-gp or MRP-positive cells.16 FTC is broadly effective against several other drug-selected cell lines overexpressing BCRP: two breast cancer cell lines (MCF-7/mtrR and MCF-7/AdrVp); and a multiple myeloma line (8226/MR4).16,18 Four cell lines that have not been previously selected for resistance in chemotherapeutic agents, (HeLa, MCF-7, and two non-small cell lung carcinoma cell lines [H460 and A549]), can be sensitized to mitoxantrone by FTC16 (and unpublished observations). This suggests that inherent resistance to mitoxantrone may occur in some cells. Here, FTC is a useful tool to identify the presence of low levels of BCRP, using a functional assay.
In vivo, FTC causes tremors in cockerels at 25 mcg/kg (oral).17 Two compounds highly related to FTC, fumitremorgin A and fumitremorgin B, are tremorgenic in rabbits at 100-200 mg/kg (intravenous), probably because of their effect on the spinal cord or brain stem.19 In nude mice bearing S1-M1-3.2 xenografts, no tremors or other toxic effects were observed with FTC at doses up to 50 mg/kg, administered intraperitonially on days 1, 5, 9, and 13 after tumor cell implantation. However, no significant inhibition of xenograft growth was observed when FTC was co-administered with doxorubicin (5 mg/kg) using the same dose schedule (personal communication and SKR unpublished observations). This lack of chemosensitization may be because of the high levels of resistance of the S1-M1-3.2, the relatively weak response of the parental unselected cells (S1) to doxorubicin in vivo, or the poor pharmacokinetics of FTC in the animals. The use of cells with lower levels of resistance (obtained during the step-wise selection of the parental cells in mitoxantrone) may alleviate some of these problems. Alternatively, since MCF-7 cells respond well to doxorubicin in mouse xenograft models, BCRP-transfected MCF-7 cells, which are cross-resistant to doxorubicin, may be useful.5 A number of analogs of FTC have been synthesized. Although none of these compounds had superior reversal activity compared with FTC in vitro, some of them may have improved pharmacokinetic properties, and warrant being tested in animal models.20
The mechanism by which FTC reverses drug resistance is unknown. Since FTC increases the accumulation of antitumor drugs in resistant cells, it is likely that it blocks the action of BCRP, and allows the cytotoxic agent to reach levels high enough to kill cells. Mitoxantrone and doxorubicin, like FTC, are multi-ring, planar molecules, and therefore, FTC may compete with these molecules for the binding site on the transporter. This mechanism would be similar to P-gp, where it has been shown that inhibitors and substrates that are transported by P-gp directly interact with the transporter protein.1 Competition studies between FTC and chemotherapeutic drugs in BCRP-containing membrane vesicles or whole cells may resolve this issue.
Concluding Remarks
FTC is a potent and selective pharmacological agent for BCRP-mediated MDR. This compound, along with antibodies and nucleic acid probes for BCRP, will be invaluable to evaluate the role of BCRP in clinical MDR. FTC or its analogs will also be useful in animal tumor xenograft models to address the feasibility of reversing clinical drug resistance mediated by this transporter. Determination of the structure of FTC bound to BCRP will provide insights into the molecular interactions between other chemotherapeutic drugs and BCRP. Unlike P-gp and MRP, which contain two sets of transmembrane domains and two ATP binding sequences, BCRP structurally resembles a half-transporter because it contains only a single putative transmembrane region and ATP binding motif.5-7 In this case, the relative simplicity of BCRP compared to the other larger ABC transporters should make it an ideal model system to study drug transport mediated by this protein family. (Dr. Rabindran is Senior Research Scientist, Oncology and Immunoinflammatory Research, Wyeth-Ayerst Research, Pearl River, NY.)
References
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The breast cancer resistance protein (BCRP) is a transporter protein that mediates resistance to:
a. mitoxantrone.
b. doxorubicin.
c. taxol.
d. Both A and B
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