The taccalonolides are a class of structurally and mechanistically distinct microtubule-stabilizing agents isolated from Tacca chantrieri. A crucial feature of the taxane family of microtubule stabilizers is their susceptibility to cellular resistance mechanisms including overexpression of P-glycoprotein (Pgp), multidrug resistance protein 7 (MRP7), and the BIII isotype of tubulin. The ability of four taccalonolides, A, E, B, and N, to circumvent these multidrug resistance mechanisms was studied. Taccalonolides A, E, B, and N were effective in vitro against cell lines that overexpress Pgp and MRP7. In addition, taccalonolides A and E were highly active in vivo against a doxorubicin-and paclitaxel-resistant Pgp-expressing tumor, Mam17/ADR. An isogenic HeLa-derived cell line that expresses the BIII isotype of tubulin was generated to evaluate the effect of BIII-tubulin on drug sensitivity. When compared with parental HeLa cells, the BIII-tubulin-overexpressing cell line was less sensitive to paclitaxel, docetaxel, epothilone B, and vinblastine. In striking contrast, the BIII-tubulinoverexpressing cell line showed greater sensitivity to all four taccalonolides. These data cumulatively suggest that the taccalonolides have advantages over the taxanes in their ability to circumvent multiple drug resistance mechanisms.

MRP8 (ABCC11) is a recently identified cDNA that has been assigned to the multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporters, but its functional characteristics have not been determined. Here we examine the functional properties of the protein using transfected LLC-PK1 cells. It is shown that ectopic expression of MRP8 reduces basal intracellular levels of cAMP and cGMP and enhances cellular extrusion of cyclic nucleotides in the presence or absence of stimulation with forskolin or SIN-1A. Analysis of the sensitivity of MRP8-overexpressing cells revealed that they are resistant to a range of clinically relevant nucleotide analogs, including the anticancer fluoropyrimidines 5-fluorouracil (ϳ3-fold), 5-fluoro-2-deoxyuridine (ϳ5-fold), and 5-fluoro-5-deoxyuridine (ϳ3-fold), the anti-human immunodeficiency virus agent 2,3-dideoxycytidine (ϳ6-fold) and the anti-hepatitis B agent 9-(2-phosphonylmethoxynyl)adenine (PMEA) (ϳ5-fold). By contrast, increased resistance was not observed for several natural product chemotherapeutic agents. In accord with the notion that MRP8 functions as a drug efflux pump for nucleotide analogs, MRP8-transfected cells exhibited reduced accumulation and increased efflux of radiolabeled PMEA. In addition, it is shown by the use of in vitro transport assays that MRP8 is able to confer resistance to fluoropyrimidines by mediating the MgATP-dependent transport of 5-fluoro-2-deoxyuridine monophosphate, the cytotoxic intracellular metabolite of this class of agents, but not of 5-fluorouracil or 5-fluoro-2-deoxyuridine. We conclude that MRP8 is an amphipathic anion transporter that is able to efflux cAMP and cGMP and to function as a resistance factor for commonly employed purine and pyrimidine nucleotide analogs.Cellular extrusion of cyclic nucleotides has been described in prokaryotic and eukaryotic cells (1-4). This process provides extracellular cAMP involved in intercellular signaling, as determined for Dictyostelium discoideum, in which cAMP effluxed by solitary amoebae under low nutrient conditions mediates cellular aggregation and differentiation, and has also been proposed as a potential mechanism that may contribute to the attenuation of intracellular signaling mediated by these second messengers (5). Investigations employing cultured cells and membrane vesicle preparations have established that cyclic nucleotide efflux is energy-dependent, and the susceptibility of this process to inhibition by antagonists of organic anion pumps indicates that it is mediated by amphipathic anion transporters (2, 3, 6 -16). Recently, insights into the identities of the cellular components that mediate cyclic nucleotide efflux have come from studies of the MRP 1 family of ABC transporters. MRP4 and MRP5, two members of this extended family of amphipathic anion transporters (17), have been determined to be competent in the transport of cyclic nucleotides (18 -20). By contrast, other characterized MRP family members are able to transport a variety of lipophilic anions, ...

The multidrug resistance protein (MRP) family consists of nine members that can be categorized according to whether or not a third (NH 2 -terminal) membrane-spanning domain is present. Three (MRP1, MRP2, and MRP3) of the four members that have this structural feature are able to confer resistance to natural product anticancer agents. We previously established that MRP7, the remaining family member that has three membrane-spanning domains, possesses the cardinal biochemical activity of MRPs in that it is able to transport amphipathic anions such as 17␤-estradiol 17-(␤-D-glucuronide). However, the drug resistance profile of the pump has not been determined. In this study, the drug resistance capabilities of MRP7 are evaluated by analyzing the resistance profiles of two clones of HEK293 cells in which the pump was ectopically expressed. MRP7-transfected HEK293 cells exhibited the highest levels of resistance toward docetaxel (9 -13-fold). In addition, lower levels of resistance were observed for paclitaxel (3-fold), vincristine (3-fold), and vinblastine (3-4-fold). Consistent with the operation of an ATP-dependent efflux pump, MRP7-transfected cells exhibited reduced accumulation of radiolabeled paclitaxel compared with HEK293 cells transfected with parental plasmid. These results indicate that MRP7, unlike other MRPs, is a resistance factor for taxanes.

Multidrug resistance protein 7 (MRP7; ABCC10) is an ATPbinding cassette transporter which is able to transport amphipathic anions and confer resistance to docetaxel and, to a lesser extent, vincristine and paclitaxel. Whereas some detail on the resistance profile of MRP7 is known, the activities of the pump have not been completely determined. Here, it is shown by the analysis of MRP7-transfected HEK293 cells that, in addition to natural product agents, MRP7 is also able to confer resistance to nucleoside-based agents, such as the anticancer agents cytarabine (Ara-C) and gemcitabine, and the antiviral agents 2 ¶,3 ¶-dideoxycytidine and PMEA. Consistent with the operation of an efflux pump, expression of MRP7 reduced the accumulation of Ara-C and PMEA. In addition, MRP7 is also able to confer resistance to the microtubule-stabilizing agent epothilone B. Ectopic expression of MRP7 in mouse embryo fibroblasts deficient in P-glycoprotein and Mrp1 revealed that MRP7 has a broad resistance profile for natural product agents. In this drug-sensitive cellular background, MRP7 conferred high levels of resistance to docetaxel (46-fold), paclitaxel (116-fold), SN-38 (65-fold), daunorubicin (7.5-fold), etoposide (11-fold), and vincristine (56-fold). Buthionine sulfoximine did not attenuate MRP7-conferred resistance to docetaxel or Ara-C. These experiments indicate that the resistance capabilities of MRP7 include nucleoside-based agents and a range of natural product anticancer agents that includes nontaxane antimicrotubule agents that are not susceptible to P-glycoprotein-mediated transport and that, unlike MRP1 and MRP2, MRP7-mediated drug transport does not involve glutathione. [Cancer Res 2009;69(1):178-84]

Multidrug resistance protein (MRP)7, MRP8, and MRP9 (gene symbols ABCC10, ABCC11, and ABCC12) are recently identified members of the MRP family that are at relatively early stages of investigation. Of these proteins, a physiological function has only been established for MRP8, for which a single nucleotide polymorphism determines wet vs dry earwax type. MRP7 and MRP8 are lipophilic anion pumps that are able to confer resistance to chemotherapeutic agents. MRP7 is competent in the transport of the glucuronide E 2 17βG, and its resistance profile, which includes several natural product anticancer agents, is distinguished by the taxane docetaxel. MRP8 is able to transport a diverse range of lipophilic anions, including cyclic nucleotides, E 2 17βG, steroid sulfates such as dehydroepiandrosterone (DHEAS) and E 1 S, glutathione conjugates such as leukotriene C4 and dinitrophenyl-S-glutathione, and monoanionic bile acids. However, the constituent of earwax that is susceptible to transport by MRP8 has not been identified. MRP8 has complex interactions with its substrates, as indicated by the nonreciprocal ability of DHEAS to stimulate E 2 17βG transport. Similar to the case for other MRPs that possess only two membrane spanning domains (MRP4 and MRP5), MRP8 is a cyclic nucleotide efflux pump that is able to confer resistance to nucleoside-based agents, such as PMEA and 5FU. The functional characteristics of MRP9 are currently unknown.