We read with interest the article by Kremer (1) reviewing the mechanisms of action of methotrexate (MTX) and potential mechanisms of MTX resistance. One facet that Kremer highlighted is the role of drug efflux transporters in mediating resistance to MTX. Although he highlights the role of multidrug resistance-associated proteins (MRPs) in mediating MTX transport, he also emphasizes a study examining permeable glycoprotein (P-gp) expression and response to MTX (2,3). Although it is now established that MRP-1 transports MTX (4), whether P-gp transports MTX is less clear.
In order to clarify this issue, we undertook a drug accumulation study using radiolabeled MTX in an in vitro T lymphoblastoid cell line (CEM cells). Parent CEM cells do not express any efflux transporters, but CEM cells pretreated with vinblastine (VBL) constitutively express the efflux transporter P-gp. CEM cells pretreated with epirubicin (E1000) constitutively express the efflux transporter MRP-1. This in vitro system is well characterized and has been used to determine the impact of the multidrug resistant transporters on the cellular accumulation of other compounds (5). We incubated 1 Ο« 10 6 cells/ml with radiolabeled MTX for 18 hours at 37Β°C across a range of drug concentrations. The excess supernatant was discarded and the cells were extracted in methanol. The intracellular drug concentrations were calculated for the 3 cell lines and results compared using the Kruskal-Wallis test for multiple comparisons. The extent of accumulation of MTX in the MRP-1-expressing cells was significantly reduced (P Ο½ 0.001), compared with the CEM and VBL cells (Figure 1).
These data support the balance of published evidence suggesting that MTX is a substrate for MRP-1 (4) but is not a substrate for P-gp (6). This is also consistent with the observation that P-gp preferentially transports neutral hydrophobic compounds, whereas MRP-1 preferentially transports anionic compounds such as MTX (6). Instead of P-gp it is likely that the transporters MRPs 1-4 (4,6,7), together with breast cancer resistance protein (8), are more important in mediating MTX resistance. Studies suggest that breast cancer resistance protein is also capable of transporting di-and triglutamate forms of MTX (9). This is important, as Kremer (1) highlights, since the polyglutamated forms of MTX are critical to its intracellular activity and efficacy. Further studies are required to clarify the relative importance of the different cellular efflux proteins in mediating an individual's response to MTX in rheumatoid arthritis.