It is has been recognized since shortly after antifolates were introduced in the clinics that the membrane transport of these agents is an important determinant of their pharmacological activity and that impaired transport is an important mechanism of resistance. These observations were based both in studies of tumor cell growth in vitro
and tumor cells derived from patients treated with MTX (Matherly and Goldman, 2003
). The focus of this work has been directed virtually exclusively on RFC which has been shown to undergo profound changes in expression and amino acid residue integrity under antifolate selective pressure (Zhao and Goldman, 2003
;Matherly and Goldman, 2003
). With the introduction of pemetrexed, the role of membrane transport became more complicated when studies demonstrated that the activity of this agent could be fully preserved in the complete absence of RFC due, in part, to the presence of another genetically distinct transport mechanism (Zhao and Goldman, 2003
). That transporter was recently identified as PCFT, a carrier with very high affinity for folate that has optimal activity at low pH (Qiu et al., 2006
). In this paper, pemetrexed transport properties were assessed as a function of pH, along with the impact of PCFT relative to RFC under physiological pH conditions in which PCFT function is far from its optimum.
H]pemetrexed, these studies confirmed the high affinity of this agent for PCFT suggested from earlier studies. Hence, the influx Km
for pemetrexed at pH 5.5 of 0.8 µM was one fourth that for MTX, 3.4 µM, in PCFT-transfected HepG2 cells while the influx Km
was 0.26 µM and 2 µM µM, respectively, for these substrates in Xenopus oocytes injected with PCFT cDNA. The major difference between PCFT-mediated pemetrexed transport and transport of the other antifolates studied is the small changes in pemetrexed influx Km
as the pH was increased. Hence, at pH 7.4 the pemetrexed influx Km
was 13 µM as compared to a MTX Km
of 130 µM, values consistent with influx Ki
’s estimated for the low-pH folate transport activity in an RFC-null HeLa cell line (Zhao et al., 2004b
). The similarity between the substrate specificity for PCFT and the low-pH folate transport activities studied in human cell lines provides additional evidence that the low-pH transport activity, ubiquitously present in tissues and cells, is encoded by PCFT.
This retention of PCFT-mediated pemetrexed transport at physiological pH translated into enhanced pharmacologic activity. Hence, using a cell line with a complete lack of constitutive carrier expression, PCFT and RFC were shown to produce equally potent pemetrexed growth inhibition when cells were grown with 25 nM 5-formylTHF as the folate growth source. This was not the case for ZD1694, PT523, and MTX; in this case, RFC restored the levels of activity to that of wild-type HeLa cells, PCFT did not. These results are consistent with what was observed when RFC was inactivated in HeLa or HCT-15 cells; preservation of pemetrexed activity but high-level resistance to MTX, ZD1694, PT523 or PT632 (an analog of PT523) (Zhao et al., 2004b
;Chattopadhyay et al., 2006
These findings relate to the marked difference in affinities of the antifolates for PCFT and RFC at physiological pH and the high degree of preference of PCFT for pemetrexed (Westerhof et al., 1995
;Zhao et al., 2004b
). The lesser decrease in the pemetrexed IC50
produced by PCFT versus RFC in cells grown with folic acid is attributed to the greater increase in cell folates produced by the former and the inhibitory effect this has on the formation of pemetrexed polyglutamate derivates (Andreassi and Moran, 2002
;Zhao et al., 2004b
). Based upon these finding: (i) It is highly unlikely that tumors will become resistant to pemetrexed due to a loss of transport since this would require two separate events affecting two genetically unrelated carriers that have comparable ability to transport this agent into cells. (ii) Tumors that become resistant to MTX in vivo
due to loss of RFC activity should remain transport-competent for pemetrexed, although activity would be determined on the basis of different parameters since these agents have quite different mechanisms of action. Likewise, sensitivities to the other antifolates studied will be influenced by cellular properties such as activating enzymes, in some cases, and specific intracellular targets.
High-level PCFT expression also increased pemetrexed activity, but not the activities of other antifolates, even in HepG2 that constitutively express these transporters. However, the level of enhancement of pemetrexed activity was less than observed in cells that lack endogenous carriers. It remains unclear, however, as to the extent to which PCFT might influence the transport of a broader spectrum of antifolates under the acid pH conditions of solid tumors (Wike-Hooley et al., 1984
;Helmlinger et al., 1997
) where the affinity of these drugs for this transporter would be higher than at physiological pH.
It was recognized early-on, from work in other laboratories, that there must a folic acid transporter independent of RFC (Yang et al., 1983
;Henderson and Strauss, 1990
). It now appears that PCFT is that elusive folic acid transporter that functions most efficiently at low pH but delivers sufficient levels of this folate to sustain folate pools even at physiological pH. Indeed, PCFT is much more efficient than RFC under these conditions (). Hence, the marked reduction of the low-pH transport activity and PCFT transcript in HeLa R1 cells (that lack RFC and PCFT) is accompanied by a reduction in pemetrexed transport and folic acid accumulation at neutral pH (Zhao et al., 2005
). PCFT transfection in this study restored not only the low-pH folate transport activity but almost fully restored folic acid accumulation at neutral pH. The impact of RFC on folic acid accumulation was much smaller in comparison.
In all HeLa cell transfectants used in this study folate receptor was expressed at comparable levels so that it does not appear that this uptake route played a role as a determinant of folate pool differences, sensitivities to antifolates, or folate growth requirements. This does not preclude their importance under other conditions since folate receptor expression may have been too low to be of consequence, or the receptors that were expressed were not functional, a phenomenon that has been reported previously in some cell lines (Kamen and Smith, 2004
). It is possible, however, that the low level of folate receptor expression in R1-11-Mock cells was sufficient to provide the very low levels of folate necessary to permit cell replication in the complete absence of PCFT and RFC.