The role of multidrug resistance efflux transporters in antifolate resistance and folate homeostasis.

Members of the ATP-binding cassette (ABC) transporters including P-glycoprotein (Pgp/ABCB1), multidrug resistance proteins (MRPs/ABCC) as well as breast cancer resistance protein (BCRP/ABCG2) function as ATP-dependent drug efflux transporters, which form a unique defense network against multiple chemotherapeutic drugs and cellular toxins. Among antitumor agents is the important group of folic acid antimetabolites known as antifolates. Antifolates such as methotrexate (MTX), pemetrexed and raltitrexed exert their cytotoxic activity via potent inhibition of folate-dependent enzymes essential for purine and pyrimidine nucleotide biosynthesis and thereby block DNA replication. Overexpression of MRPs and BCRP confers resistance upon malignant cells to various hydrophilic and lipophilic antifolates. Apart from their central role in mediating resistance to antifolates and other anticancer drugs, MRPs and BCRP have been recently shown to transport naturally occurring reduced folates. This was inferred from various complementary systems as follows: (a) Cell-free systems including ATP-dependent uptake of radiolabeled folate/MTX into purified inside-out membrane vesicles from stable transfectants and/or cells overexpressing these transporters, (b) Decreased accumulation of radiolabeled folate/MTX in cultured tumor cells overexpressing these transporters, as well as (c) In vivo rodent models such as Eisi hyperbillirubinemic rats (EHBR) that hereditarily lack MRP2 in their canalicular membrane and thereby display a bile that is highly deficient in various reduced folate cofactors and MTX, when compared with wild type Sprague-Dawley (SD) rats. In all cases, these folate/antifolate transporters functioned as high capacity, low affinity ATP-driven exporters. While the mechanism of cellular retention of (anti)folates is mediated via (anti)folylpolyglutamylation, certain efflux transporters including MRP5 (ABCC5) and BCRP were shown to transport both mono-, di- as well as triglutamate derivatives of MTX and folic acid. Furthermore, overexpression of MRPs and BCRP has been shown to result in decreased cellular folate pools, whereas loss of ABC transporter expression brought about a significant expansion in the intracellular reduced folate pool. The latter finding has important implications to antifolate-based chemotherapy as an augmented cellular folate pool results in a significant level of resistance to certain antifolates. Hence, the aims of the present review are: (a) To summarize and discuss the cumulative evidence supporting a functional role for various multidrug resistance efflux transporters of the ABC superfamily which mediate resistance to hydrophilic and lipophilic antifolates, (b) To describe and evaluate the recent data suggesting a role for these efflux transporters in regulation of cellular folate homeostasis under folate replete and deplete conditions. Furthermore, novel developments and future perspectives regarding the identification of novel antifolate target proteins and mechanisms of action, as well as rationally designed emerging drug combinations containing antifolates along with receptor tyrosine kinase inhibitors are being discussed.