Augmentation of methotrexate cytotoxicity in human colon cancer cells achieved through inhibition of thymidine salvage by dipyridamole

In HCT 116 cells, a human colon cancer cell line, the levels of thymidine (0.6 μM) and hypoxanthine (9 μM) contributed to the tissue culture medium by the fetal bovine serum significantly reduced the growth inhibition and lethality produced by 0.1 μM methotrexate. Dipyridamole, an inhibitor of nucleoside transport, potentiated the growth inhibitory effects of methotrexate when the cells were grown in medium that was changed daily. However, when the medium was supplemented with dialyzed serum, methotrexate cytotoxicity was not increased by dipyridamole. Similarly, in cloning experiments, dipyridamole increased the cell killing produced by methotrexate. The potentiation of methotrexate toxicity produced by dipyridamole was mediated through inhibition of thymidine uptake. The uptake of 1 μM thymidine was inhibited 50% by 0.12 μM dipyridamole but neither hypoxanthine nor guanine uptake was decreased by dipyridamole (5 μM). As a result, the decrease in dTTP pools produced by methotrexate was augmented by dipyridamole. In contrast, dipyridamole did not influence the effect of methotrexate on ribonucleoside triphosphate pools. HCT 116 cells avidly salvaged low concentrations of thymidine, and methotrexate increased this capacity. Conversion of 0.11 μM thymidine to thymidine triphosphate was increased by 55%, from 16.6 to 25.7 pmoles/10⁶ cells, following exposure to 1.0 μM methotrexate. Dipyridamole blocked this pool expansion. This study suggests that the salvage of physiological levels of thymidine may diminish the cytotoxic effects of methotrexate on human colon cancer cells. Inhibition of thymidine uptake by dipyridamole may be an effective strategy to increase the cytotoxicity of methotrexate.