RT Journal Article SR Electronic T1 Functional Loss of the Reduced Folate Carrier Enhances the Antitumor Activities of Novel Antifolates with Selective Uptake by the Proton-coupled Folate Transporter JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP mol.112.079004 DO 10.1124/mol.112.079004 A1 Sita Kugel Desmoulin A1 Lei Wang A1 Lisa Polin A1 Kathryn White A1 Juiwanna Kushner A1 Mark Stout A1 Zhanjun Hou A1 Christina Cherian A1 Aleem Gangjee A1 Larry H Matherly YR 2012 UL http://molpharm.aspetjournals.org/content/early/2012/06/26/mol.112.079004.abstract AB Uptake of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with 4 or 3 bridge carbons [compound 1 (C1) and compound 2 (C2), respectively] into solid tumors by the proton-coupled folate transporter (PCFT) represents a novel therapeutic strategy which harnesses the acidic tumor microenvironment. Although these compounds are not substrates for the reduced folate carrier (RFC), the major facilitative folate transporter, RFC expression may alter drug efficacies by impacting cellular tetrahydrofolate (THF) cofactor pools that can compete for polyglutamylation and/or binding to intracellular enzyme targets. Human tumor cells including wild-type (WT) and R5 (RFC-null) HeLa cells express high levels of PCFT protein. C1 and C2 inhibited proliferation of R5 cells 3 to 4 times more potently than WT cells or R5 cells transfected with RFC. Transport of C1 and C2 was virtually identical between WT and R5 cells, establishing that differences in drug sensitivities between sublines were independent of PCFT transport. Steady-state intracellular [3H]THF cofactors derived from [3H]5-formyl-THF were depleted in R5 cells compared to WT cells, an effect exacerbated by C1 and C2. While C1 and C2 polyglutamates accumulated to similar levels in WT and R5 cells, there were differences in polyglutamyl distributions in favor of the longest chain-length forms. In severe combined immunodeficient mice, anti-tumor efficacies of C1 and C2 were greater toward subcutaneous R5 tumors compared to WT tumors, confirming the collateral drug sensitivities observed in vitro. Thus, solid tumor-targeted antifolates with PCFT-selective cellular uptake should have enhanced activities toward tumors lacking RFC function, reflecting contraction of THF cofactor pools.