Previously, we reported that the 5-fluorourldlne derivative, 2′,3′,5′-tris-O-[N-(2-n-propyl-n-pentanoylglycyl]-5-fluorouridine (UK-21), is a newly synthesized lowly immunosuppressive and potent antitumor drug in comparison with other fluorouridine derivatives such as 5-fluorouracil (5-FU), 5-fluorouridine (5-FUR) and 5-fluorodeoxyuridine (5-FUDR). In order to elucidate the molecular mechanism of antitumor activity of UK-21, we compared the effect of the four drugs on cell proliferation, cell cycle progression and macromolecular syntheses. When KB cells were subjected to a colony-forming inhibition assay designed to expose the cells to the drugs for 4–96 h and wash out, UK-21 and 5-FUR inhibited the colony formation at concentrations ranging from 0.01 to 0.1 μM, whereas 1 100 μM was needed for the cytotoxicity of 5-FU and 5-FUDR. By exposure for 24–48 h, all these drugs inhibited cell growth and caused accumulation of the cells in S or G2 phase at almost the same concentrations of 0.32–8μM. These results suggest that the cytotoxic effects of UK-21 and 5-FUR are irreversible, while those of 5-FU and 5-FUDR are reversible. To confirm this, KB cells were treated with UK-21 and/or 5-FU for 1 h, and continued to be cultured for 1–7 days, resulting in the inhibition of the cell growth by UK-21 in a dose-dependent manner at concentrations of 10–100 μM, but not by 5-FU even at 100 μM. UK-21, 5-FUR and 5-FU showed a linear relationship between exposure time and IC50 in the colony formation assay with a slope of almost −1, but 5-FUDR did not, suggesting that UK-21, 5-FUR and 5-FU are cell cycle non-specific inhibitors, while 5-FUDR is a cell cycle-specific inhibitor. UK-21 and 5-FUR, but not 5-FU and 5-FUDR Inhibited the incorporation of [3H]uridine into the acid insoluble fraction, while UK-21 and 5-FUDR, but not 5-FUR and 5-FU inhibited the incorporation of [3H]thymidine. These results suggest that irreversible cytotoxic effects of UK-21 like 5-FUR are exerted through inhibition of RNA synthesis.
