TAS-102 is an oral anticancer drug composed of
trifluorothymidine (TFT) and TPI (an inhibitor of
thymidine phosphorylase that strongly inhibits the biodegradation of TFT). Similar to
5-fluorouracil (
5FU) and
5-fluoro-2'-deoxyuridine (FdUrd), TFT also inhibits
thymidylate synthase (TS), a rate-limiting
enzyme of
DNA biosynthesis, and is incorporated into
DNA. TFT exhibits an anticancer effect on
colorectal cancer cells that have acquired
5FU and/or FdUrd resistance as a result of the overexpression of TS. Therefore, we examined the mode of action of TFT-induced DNA damage after its incorporation into
DNA. When HeLa cells were treated with TFT, the number of ring-open
aldehyde forms at apurinic/apyrimidinic sites increased in a dose-dependent manner, although we previously reported that no detectable excisions of TFT paired to
adenine were observed using
uracil DNA glycosylases,
thymine DNA glycosylase or methyl-CpG binding domain 4 and HeLa whole
cell extracts. To investigate the functional mechanism of TFT-induced DNA damage, we measured the phosphorylation of ATR, ATM, BRCA2, chk1 and chk2 in nuclear extracts of HeLa cells after 0, 24, 48 or 72 h of exposure to an IC(50) concentration of TFT, FdUrd or
5FU using Western blot analysis or an
enzyme-linked
immunosorbent assay (ELISA). Unlike FdUrd and
5FU, TFT resulted in an earlier phosphorylation of ATR and chk1
proteins after only 24 h of exposure, while phosphorylated ATM, BRCA2 and chk2
proteins were detected after more than 48 h of exposure to TFT. These results suggest that TFT causes single-strand breaks followed by double-strand breaks in the
DNA of TFT-treated cells. TFT (as TAS-102) showed a more potent antitumor activity than oral
5FU on CO-3
colon cancer xenografts in mice, and such antitumor potency was supported by the increased number of double-strand breaks occurring after single-strand breaks in the
DNA of the TFT-treated
tumors. These results suggest that TFT causes single-strand breaks after its incorporation into
DNA followed by double-strand breaks, resulting in DNA damage. This effect of TFT on
DNA may explain its potent anticancer activity in
cancer therapy.