Transport of Methotrexate into PC-3 Human Prostate Cancer Cells

Transport of methotrexate (MTX) into human prostatic PC-3 cells was studied. Uptake of MTX vs concentration was saturable at pH 7.4 in cells grown in normal medium and in cells incubated for 24 h in folate-free medium (Km = 3.24 and 4.84 μM, respectively (P > 0.05, n = 3) and Vmax = 0.64 and 0.92 nmol min−1 · 10−9 cells, respectively (P < 0.05, n = 3)). In contrast, uptake at pH 4.5 showed both a saturable component (Km = 1.03 μM, Vmax = 0.42 nmol min−1 · 10−9 cells) and a nonsaturable, linear component. Uptake was inhibited by the structural analogs 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and folic acid (Ki = 6.8, 10.9, and 89.6 μM, respectively). Uptake was inhibited by increasing concentrations of chloride ion, suggesting that MTX transport in PC-3 cells may be via an anion-exchange mechanism. Uptake was significantly decreased by high concentrations of sodium cyanide and sodium arsenate but not by sodium azide. Uptake was inhibited by the sulfhydryl inhibitor p-chloromercuriphenylsulfonate and by the anions probenecid and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. Uptake of MTX was independent of sodium ions in the medium. It is concluded that PC-3 human prostate cancer cells have a carrier-mediated system for the uptake of MTX and other folates.