The present analysis of MRP7 activity provides surprising detail on the resistance profile of the pump. In a previous study, we focused our analysis on natural product anti-cancer agents because resistance towards at least some agents of this family is characteristic of MRP family members that possess three membrane spanning domains. However, the relatively low degree of similarity between MRP7 and other MRP family members raised the possibility that its activity might not be restricted to natural product agents. By investigating this conjecture, we determined that the MRP7 resistance profile includes nucleoside-based agents, a class of compounds which are not known to be components of the resistance profiles of other large members of the MRP family, but instead are agents towards which the small members, such as MRP4, MRP5 and MRP8 have activity (35
). The nucleoside-based agents towards which MRP7 is able to confer resistance included anticancer agents such as Ara-C, a mainstay in the treatment of acute myelogenous leukemia, and gemcitabine, an agent with utility in pancreatic and lung cancers. This is the first example of an MRP family member that is able to confer resistance to either of these widely employed agents. In addition, MRP7 is also able to confer resistance to antiviral agents such as ddC, similar to MRP8 and PMEA, as observed with MRP4, MRP5 and MRP8. It is likely that MRP7, which we have established as being competent in the transport of amphipathic anions (18
), effluxes the negatively charged monophosphate metabolites of Ara-C, gemcitabine and ddC, as opposed to the uncharged parent nucleosides. This mechanism of resistance would be in accord with the ability, for example, of MRP8 to transport the monophosphate of 5-FU but not the uncharged parent nucleoside analog (38
). By contrast with the former nucleoside analogs, PMEA is a charged nucleotide analog which is likely to be a direct substrate of MRP7.
The present study also revealed that the range of MRP7 towards natural product agents is much broader than had been previously inferred from our prior analysis of MRP7-transfected HEK293 cells (17
). Confirming our previous reported findings, we show that expression of MRP7 in the context of Pgp/Mrp1 null fibroblasts confers resistance to taxanes and vinca alkaloids. However in this genetically deficient context, MRP7 also protected cells from anthracyclines (daunorubicin), camptothecins (SN-38) and epipodophyllotoxins (etoposide). The ability to detect increased activity of MRP7 towards the former agents, and emergence of activity towards the latter agents when the pump is expressed in Pgp/Mrp1 deficient fibroblasts compared to HEK293 cells (), is consistent with the phenotype of MEF3.8 cells. This cell line is reported to be 11–51, 2–12, 4–6, 5–9 and 19–41-fold more sensitive to paclitaxel, docetaxel, SN-38, daunorubucin, etoposide and vincristine, respectively, compared to wild-type fibroblasts (21
). The lack of significant enhancement of MRP7 activity for nucleoside analogs such as Ara-C and the alkylating agent cisplatin in Pgp/Mrp1 deficient cells compared to HEK293 cells is also consistent with the absence of sensitization of MEF3.8 cells towards these two agents (0.8–1.5 and 0.6–1.3-fold, respectively). That MRP7 activity (e.g. docetaxel) is enhanced in MEF3.8 cells towards agents for which this cell line is sensitized, whereas its activity (e.g. Ara-C) is not increased towards agents for which MEF3.8 cells are not sensitized, tends to support the validity of the MRP7 phenotype we describe here.
Our previous study disclosed that MRP7 is able to confer resistance to two classes of agents that target microtubules (vinca alkaloids and taxanes). It was therefore of interest to determine if MRP7 might also confer resistance to newer anti-microtubule agents. A notable feature of MRP7 that emerged from this line of investigation is that it is able to confer resistance to epothilone B. Of the non-taxane microtubule-stabilizing agents that are in clinical development, epothilones are the most advanced (39
). Epothilones, which have broad antitumor activity, are considered to be particularly attractive agents because they are not susceptible in vitro or in vivo to transport by P-glycoprotein or any previously tested drug efflux pumps (39
). In accord with this situation, the absence of enhanced levels of MRP7-conferred resistance towards epothilone B in the MEF3.8 system as compared to HEK293 cells is expected, in that MEFs that are deficient in P-glycoprotein and MRP1 should not be rendered sensitive to this agent. The finding that MRP7-transfected HEK293 cells are resistant to epothilone B indicates that there is at least one pump capable of effluxing this agent. By contrast with epothilone B, we did not detect resistance towards epothilone A, which differs from the former compound by the absence of a methyl group at C12. In addition, MRP7-transfected HEK293 cells exhibited either relatively modest or no resistance to the taxane analogs MAC-321 and MST-997, and the microtubule destabilizing agents phomoposin A and HTI-286.
Another feature of MRP7 that was investigated in this study is the involvement of glutathione in MRP7-mediated resistance. The finding that BSO had no effect on resistance to docetaxel or Ara-C suggests that transport of anticancer agents by MRP7 does not require glutathione. This finding, in combination with the observations that glutathione levels are not significantly decreased in MRP7-transfected HEK293 cells (19
) or MRP7-transfected MEF3.8 cells (present study), suggests that glutathione is not a substrate of MRP7. In this regard, MRP7 is distinct from MRP1 and MRP2, both of which require glutathione for transport of natural product agents and are competent in the transport this tripeptide (3
). Instead, MRP7 is similar to MRP3 which appears to transport etoposide in a glutathione-independent fashion (42
). We also show in the present study that in apparent contrast to MRP7, all three of the latter pumps are unable to confer resistance to docetaxel. However, with respect to MRP3, in contrast to studies using MRP3-transfected HEK293 cells, ovarian cancer 2008 cells, and P-glycoprotein and Mrp1-deficient MEF cells (42
), a recent report found that when expressed in a breast cancer cell line MRP3 is able to confer resistance to paclitaxel (45
). This suggests that the activity of MRP3 may depend on cell context. Therefore, with regard to the activity of MRP3 towards docetaxel, further studies are warranted using additional cell lines to the HEK293 cells used here.
In order to better understand the potential impact of MRP7 on the sensitivity of normal tissues and tumors detailed information will be needed on the protein expression pattern of the pump. Our laboratory and others have detected MRP7 transcript in a variety of tissues including pancreas, liver, placenta, kidney, brain, ovary, spleen, heart, skeletal muscle, testis, intestine, prostate, and white blood cells, as well as in various fetal tissues (17
) (Supplemental Figure 1
). Transcript has also been detected in several adenocarcinomas, including tumors such as breast, ovary and lung (46
). This is of potential interest in that the latter tumors are treated with taxanes. In addition, MRP7 may be induced by anti-cancer agents towards which the pump confers resistance. MRP7 transcript and protein were reported to be induced by vincristine exposure in two salivary gland adenocarcinoma cell lines that are cross resistant to docetaxel (48
), and transcript was reported to be increased in MCF7 cells by exposure to doxorubicin (46
). In addition, it was recently reported that MRP7 is induced by paclitaxel in a non-small cell lung cancer cell line (49
). MRP7 antibodies and an Mrp7 gene-disrupted mouse we have generated (E.H-B. and G.D.K, unpublished) should be valuable tools for understanding the potential impact of MRP7 on drug sensitivity.