Broad-spectrum modulation of ATP-binding cassette transport proteins by the taxane derivatives ortataxel (IDN-5109, BAY 59-8862) and tRA96023
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- Minderman, H., Brooks, T.A., O’Loughlin, K.L. et al. Cancer Chemother Pharmacol (2004) 53: 363. doi:10.1007/s00280-003-0745-2
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The taxanes paclitaxel and docetaxel are substrates for P-glycoprotein (Pgp), an ATP-binding cassette (ABC) transport protein associated with multidrug resistance (MDR). In contrast, the synthetic taxane ortataxel (BAY 59-8862, IDN-5109) is effective against Pgp-expressing cells by virtue of modulation of Pgp-mediated transport. The synthetic taxane tRA96023 also modulates Pgp and is noncytotoxic due to removal of the tubulin-binding side chain at the C-13 position of the taxane backbone. We studied the effects of ortataxel and tRA96023 on the other MDR-associated ABC transport proteins, multidrug resistance protein (MRP-1) and breast cancer resistance protein (BCRP, MXR, ABCG2).
Modulation of mitoxantrone, daunorubicin and doxorubicin retention and cytotoxicity by ortataxel and tRA96023 was studied in established cell lines overexpressing Pgp, MRP-1 and wild type (BCRPR482) and mutant (BCRPR482T) BCRP, and was compared with modulation by the established Pgp-, MRP-1- and BCRP-specific modulators PSC-833, probenecid and fumitremorgin C, respectively.
Ortataxel effectively modulated drug retention and cytotoxicity in cell lines overexpressing MRP-1 and BCRPR482, in addition to Pgp. tRA96023 modulated drug retention and cytotoxicity in cell lines overexpressing BCRPR482 and Pgp, but not those overexpressing MRP-1. Neither ortataxel nor tRA96023 modulated BCRPR482T.
The synthetic taxane derivatives ortataxel and tRA96023 are broad-spectrum ABC protein modulators. Further studies will seek to identify a noncytotoxic synthetic taxane that modulates Pgp, MRP-1 and BCRP.
KeywordsTaxanesP-glycoproteinMultidrug resistance proteinBreast cancer resistance proteinModulation
Tumor cells frequently exhibit multidrug resistance (MDR) mediated by the ATP-binding cassette (ABC) membrane proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP-1) and breast cancer resistance protein (BCRP) . Noncytotoxic molecules, termed MDR modulators, inhibit drug efflux mediated by these proteins, restoring cellular drug sensitivity. PSC-833 , probenecid  and fumitremorgin C  modulate Pgp, MRP-1 and BCRP, respectively; cyclosporin A (CsA) has activity against MRP-1 and BCRP, as well as Pgp . Structural properties responsible for broad-spectrum, versus ABC protein-specific, modulation are unknown.
The taxanes paclitaxel and docetaxel, semisynthetic chemotherapy drugs that inhibit microtubule depolymerization [34, 38], are substrates for Pgp [3, 16, 39]. Limited availability of natural resources led to the development of synthetic taxanes , and active efflux of natural product taxanes by Pgp prompted attempts to synthesize analogs that are not susceptible to Pgp-mediated drug resistance . Ortataxel (formerly BAY 59-8862 and IDN 5109)  is a cytotoxic taxane  that modulates Pgp, blocking its own efflux from Pgp-overexpressing cells [17, 28, 29, 30, 31, 32, 45]. tRA96023 also modulates Pgp , but is noncytotoxic due to removal of the side chain at the C-13 position of the taxane backbone, which is required for tubulin binding . Ortataxel is in clinical trials , and tRA96023 is in preclinical development.
To determine whether ortataxel and tRA96023 might be broad-spectrum MDR modulators, we studied their effects on drug retention and cytotoxicity in resistant cell lines overexpressing MRP-1 and BCRP, as well as Pgp. The drug retention assay measures short-term effects on drug transport, while the cytotoxicity assay measures long-term effects on cell survival.
Materials and methods
Established resistant cell lines overexpressing Pgp (8226/Dox6, MCF7/R), MRP-1 (HL60/Adr) or wild type (8226/MR20) or mutant (MCF7/AdVp3000) BCRP were studied [8, 14, 24, 25]. Wild type (BCRPR482) and mutant (BCRPR482T) BCRP, with arginine and threonine in the amino acid 482 position, respectively, both efflux mitoxantrone, but only BCRPR482T effluxes anthracyclines . Wild type HL60 cells served as negative controls for all three proteins  since the parental 8226 and MCF7 cell lines express low levels of BCRPR482 [25, 27].
ABC protein expression
Drug retention studies
Drug retention was studied as previously described . Briefly, drug uptake was achieved by incubating cells with drug (3 μM) for 30 min. Drug efflux was then studied by comparing drug retention following a 90-min incubation in drug-free medium in the presence and absence of modulator. All experiments were performed in triplicate. Cellular drug content was measured by flow cytometry and analyzed using the WinList program (Verity Software House, Topsham, Me.). In experiments with ortataxel, cellular fluorescence was compensated to account for that associated with the modulator.
Flow cytometry data analysis
Labeling with specific antibody and with isotype control was compared by the Kolmogorov-Smirnov (KS) statistic, expressed as a D-value ranging from 0 to 1 . Antibody positivity was defined by D-values ≥0.1 and ≥0.2 for staining of unfixed (MRK-16 ) and fixed (other antibodies ) cells, respectively. Drug retention in the presence and absence of modulator was also compared by the KS statistic; D-values ≥0.2 were considered to be indicative of modulation .
Cells were cultured for 96 h in 96-well plates at densities of 10,000 (suspension) or 1,500 (adherent) cells per well in RPMI 1640 medium with 10% FBS, 2 mM l-glutamine, 20 U/ml penicillin, 20 μg/ml streptomycin and drugs at a range of doses in the absence and presence of modulators, in triplicate. Cell growth was assessed by the WST-1 assay (Roche Diagnostics, Mannheim, Germany) for suspension cells [36, 42] and the sulforhodamine-B (SRB) assay for adherent cells . IC50 values, or drug concentrations inhibiting cell growth by 50% compared to untreated cells, were determined using curve-fitting software, as previously described . Relative resistance (RR) was calculated as the ratio (IC50 resistant cells/IC50 wild type cells). The resistance modifying factor (RMF) was calculated as the ratio (IC50 drug/IC50 drug+modulator).
Effects of ortataxel and tRA96023 on drug efflux mediated by Pgp, MRP-1 and BCRP
Effects of ortataxel and tRA96023 on resistance mediated by Pgp, MRP-1 and BCRP
Modulation of cytotoxicity of mitoxantrone, daunorubicin and doxorubicin by tRA96023 (10 μM) in cell lines overexpressing Pgp, MRP-1, BCRPR482 or BCRPR482T and the corresponding parental cell lines. Resistance modifying factors (RMF) were calculated as the ratios of the IC50 values in the absence and presence of tRA. Each IC50 and RMF was calculated as the mean±SD of triplicate experiments. IC50 values are in micromoles
We demonstrated that synthetic taxanes modulate resistance mediated by the MDR-associated ABC transport proteins MRP-1 and BCRPR482, in addition to their known modulation of Pgp. Ortataxel, which is cytotoxic, increased mitoxantrone and anthracycline retention and was more cytotoxic than paclitaxel and docetaxel in cell lines overexpressing Pgp, MRP-1 and BCRPR482. tRA96023, which is noncytotoxic, enhanced mitoxantrone and anthracycline retention and cytotoxicity in cell lines overexpressing Pgp and BCRPR482, but not those overexpressing MRP-1. Thus synthetic taxane modulators may sensitize cancers with MDR mediated by multiple ABC proteins. The finding that neither taxane modulated drug transport mediated by BCRPR482T, in contrast to their effective modulation of BCRPR482, indicates that specific mutations in this transport protein may affect not only substrate specificity , but also modulator specificity.
The relevance of MDR proteins has been most fully studied in acute myeloid leukemia (AML), because of the ease of obtaining tumor cells for study and the ability to correlate MDR protein expression and function with heterogeneous treatment response. Pgp [7, 13, 18, 19, 20, 21, 23] and MRP-1 [18, 19, 21] have clinical relevance in AML, and the relevance of BCRP has also been suggested [37, 41, 43, 44]. BCRP has been wild type (BCRPR482) in all cases of AML studied to date ; whether mutant BCRP is present in other tumor types, or only in cell lines, is yet unknown. Broad-spectrum modulators have an obvious theoretical advantage in this and other malignancies with expression of multiple ABC transport proteins.
Most MDR modulation clinical trials to date have yielded disappointing results. Trials have only targeted Pgp, despite the fact that additional ABC proteins contribute to clinical MDR. The synthetic taxanes may be promising agents for clinical application because of their broad spectrum of modulation. Nevertheless, the safety and efficacy of clinical application of broad-spectrum modulators remains to be demonstrated.
The data presented here serve to demonstrate that taxane derivatives have the potential to modulate all three MDR pumps. The efficacy of ortataxel as a cytotoxic chemotherapeutic agent is currently being evaluated . The present data suggest that this drug should be effective against tumors expressing MRP-1 and BCRP, as well as those expressing Pgp . Nevertheless, due to the cytotoxic properties of ortataxel, it is not strictly an MDR modulator. tRA96023 is noncytotoxic, but its lack of modulation of MRP-1 makes it less than optimal for development as a broad-spectrum clinical modulator. However, tRA96023 is one of a large library of noncytotoxic taxane derivatives lacking the C13 tubulin-binding side chain [30, 32]. This library is currently being screened to identify noncytotoxic modulators of all three MDR-associated ABC proteins.