The Role of α-Folate Receptor-Mediated Transport in the Antitumor Activity of Antifolate Drugs

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Abstract
Purpose: Raltitrexed, pemetrexed, lometrexol, and ZD9331 are antifolate drugs transported into cells via the ubiquitously expressed reduced-folate carrier. They display also high affinity for the α-folate receptor (α-FR), a low capacity folate transporter that is highly overexpressed in some epithelial tumors. The role of α-FR in the activity of the antifolates has been evaluated in two α-FR-overexpressing cell lines grown in a physiological concentration of folate (20 nm R,S-Leucovorin). Experimental Design and Results: A431-FBP cells (transfected with the α-FR) were 3–5-fold more sensitive to the antifolates than A431 cells. KB cells (constitutive α-FR overexpression) were less sensitive to the drugs when coexposed to 1 μm folic acid to competitively inhibit binding to the α-FR. Raltitrexed, pemetrexed, and lometrexol are polyglutamated in cells leading to drug retention, e.g., the raltitrexed 4- and 24-h IC50s in A431 cells were ∼0.6 and 0.008 μm, respectively, compared with 0.003 μm for 72-h continuous exposure. A431-FBP cells were ∼3-fold more sensitive to raltitrexed and pemetrexed at all exposure times. ZD9331 is not polyglutamated, and the 4- and 24-h IC50s in A431 cells were >100 and ∼100 μm, respectively, reducing to 2 and 0.1 μm, respectively, in A431-FBP cells. The ZD9331 4- and 24-h IC50s in KB cells were 20 and 1 μm, respectively, and reversible by coaddition of 1 μm folic acid. An in situ thymidylate synthase assay demonstrated continued thymidylate synthase inhibition after ZD9331-treated A431-FBP and KB, but not A431, cells were placed in drug-free medium for 16 h. A model is proposed in which the antifolates accumulate in the α-FR/endosomal apparatus, leading to slow release into the cytoplasm. In particular, this leads to cellular retention of the nonpolyglutamatable ZD9331. Conclusions: Antifolate drugs, particularly ZD9331, have the potential for increased efficacy in tumors that highly overexpress the α-FR.