Selective Preservation of Pemetrexed Pharmacological Activity in HeLa Cells Lacking the Reduced Folate Carrier

Abstract

A methotrexate (MTX)-resistant HeLa subline (R5), developed in this laboratory, with impaired transport due to a genomic deletion of the reduced folate carrier (RFC) was only 2-fold resistant to pemetrexed (PMX), but 200-and 400-fold resistant to raltitrexed (ZD1694) and N ␣ -(-4-amino-4-deoxypteroyl)-N ␦ -hemiphthaloyl-1-ornithine (PT523), respectively, compared with parental HeLa cells when grown with 2 M folic acid. When folic acid was replaced with the more physiological 25 nM 5-formyltetrahydrofolate, R5 cells were 2-fold collaterally sensitive to PMX but still 40-and 200-fold resistant to ZD1694 and PT523, respectively. Sensitivity to PT523 and PMX could be completely restored, and sensitivity to ZD1694 nearly restored, by transfection of RFC cDNA into R5 cells, indicating that the defect in drug transport was the only, or major, factor in resistance. The preserved PMX activity in R5 cells could not be related to the very low expression of folate receptors. Rather, retained PMX activity in R5 cells was associated with residual transport by another process that exhibits good affinity for PMX (K t ‫؍‬ 12 M) with much lower affinities for ZD1694, MTX, and PT523 (K i s of ϳ 90, 100, and 250 M, respectively). PMX transported by this route was rapidly converted to higher polyglutamates and, when grown with 25 nM 5-formyltetrahydrofolate, the rate of formation of these derivatives and their net accumulation in R5 cells was comparable to that of wild-type cells. These data suggest that selective preservation of PMX pharmacological activity in RFC-null R5 cells is due, in part, to partial preservation of transport by secondary process with a higher affinity for PMX than the other antifolates evaluated.

Characterization of a Folate Transporter in HeLa Cells with a Low pH Optimum and High Affinity for Pemetrexed Distinct from the Reduced Folate Carrier

“…This RFC-independent transport activity at pH 7.4 also had a relatively higher affinity for PMX (K i ϳ12 mol/L) than for MTX, ZD1694, and folic acid (K i ϳ90 -100 mol/L) and a very low affinity (K i ϳ250 mol/L) for PT523 (37). R5 cells were, in fact, collaterally sensitive to PMX when grown in medium in which 5-CHO-THF was the folate growth source.…”

“…R5 cells were, in fact, collaterally sensitive to PMX when grown in medium in which 5-CHO-THF was the folate growth source. This was attributed to the partial preservation of transport along with the marked contraction of folate cofactor pools under these conditions (9,37,38). The mechanism underlying this transport activity at neutral pH was not clarified, but it is possible that this may represent residual activity of the low pH route within the physiologic range with pH-dependent alterations in binding constants for folates and antifolates.…”

Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function

“…Cells were maintained in RPMI 1640 medium containing 2.3 M folic acid supplemented with 10% fetal calf serum (Hyclone), 2 mM glutamine, 20 M 2-mercaptoethanol, penicillin (100 U/ml), and streptomycin (100 g/ml). Wild-type HeLa and RFC-null R5 cells were maintained in cell culture as described previously (38).…”

“…Folate receptor expression has not been detected in murine or human intestinal cells, although it has been reported to be present in human cancer cell lines of intestinal origin, such as Caco-2 cells (5, 24). Folate receptor expression was assessed in IEC-6, IEC-6/A4, and IEC-6/PT1 cells with the [ 3 H]folic acid binding assay and found to be very low in all three cell lines (0.028, 0.011, 0.025 nmol/g protein, respectively), far below the level of expression in HeLa cells (0.22 nmol/g protein) (38). Hence, folate transport activity observed at pH 5.5 was not related at all to a folate receptormediated process.…”

Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition

“…More recently, the emphasis in folatemediated drug therapy has shifted to obtain a better understanding of transport mechanisms of antifolates because dose-limiting toxicities arise from their transport via RFC, and possibly PCFT, into normal cells (6,11,(15)(16)(17). To date, antifolates approved for clinical use are transported primarily via RFC, although one of the most recently developed antifolates, pemetrexed (PMX), may also transit through PCFT (6,11,(18)(19)(20)(21). Given the toxicities associated with transport via facilitative transporters, the development of therapeutic molecules specifically transported via the FRs has been explored over the last two decades (22)(23)(24).…”