Figure 2.

EC0746 is a folate-receptor-specific dihydrofolate reductase inhibitor with potent cytostatic effect on RAW264.7 macrophages. (a) RAW264.7 cells were given a 2-hour pulse of 100 nM EC0746 ± 10 μM folic acid (FA) followed by a 22-hour chase. Aminopterin (AMT) and methotrexate (MTX) were allowed to incubate for 24 hours. The dihydrofolate reductase activities in whole cell lysates (in duplicate) were normalized to untreated control cells (mean ± standard error of the mean). *P < 0.05. (b), (c) RAW264.7 cells were subjected to a 2-hour pulse followed by a 70-hour chase of a 10-fold serial dilution of EC0746 ± 100-fold molar excess of FA. Free AMT was allowed to incubate for 72 hours continuously. Four hours prior to the end of incubation, lipopolysaccharide (100 ng/ml) was added to stimulate TNFα production. The (b) cell viability and (c) TNFα in culture media were determined by XTT and ELISA assays, respectively. Results expressed as the percentage of control in absorbance (mean ± standard error of the mean in triplicates). (d) RAW264.7 cells were treated with indicated concentrations of EC0746 ± excess FA (2-hour pulse plus a 70-hour chase). The surviving cells were redistributed in equal numbers in fresh medium and allowed to incubate further for 72 hours. The cell proliferation was again determined by the XTT assay.