Trifluorothymidine exhibits potent antitumor activity via the induction of DNA double-strand breaks

Abstract

Abstract. 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 singlestrand 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.

Trifluridine Induces p53-Dependent Sustained G2 Phase Arrest with Its Massive Misincorporation into DNA and Few DNA Strand Breaks

“…13) or cotreatment with FTD and TPI (i.e., TAS-102; refs. 13, 34) could result in DNA fragmentation detectable by alkaline gel electrophoresis, in situ nick translation (13), or pulse field gel electrophoresis under the neutral condition (34).…”

Development of an immobilized biocatalyst based on Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine

“…5-Trifluorothymidine, commercially known as trifluridine, is an antiviral agent employed in ophthalmic solutions for the treatment of herpes virus simple (HVS) [22]. This compound can also induce double-strand DNA breaks and inhibit the thymidylate synthase when it is transformed to its phosphorylated form, displaying antitumoral activity [23]. The combination of this compound with an inhibitor of the thymidine phosphorylase, to inhibit degradation of trifluridine, is currently undergoing a Phase III clinical trial with patients with refractory metastatic colorectal cancer (NCT01607957) [24].…”

TAS-102: A Novel Antimetabolite for the 21st Century

“…There are also now conflicting data regarding TFT-induced DNA fragmentation. While Suzuki et al demonstrated that TFT incorporation into DNA resulted in single-and double-strand breaks and subsequent activation of DNA repair cascades, DNA fragmentation was not observed with a more recent study by Matsuoka et al [30,32]. Both studies, however, demonstrated that TAS-102 treatment resulted in massive TFT incorporation into DNA and in activation of similar DNA damage response pathways, which involve phosphorylation of Chk1 and cycle arrest during the G2/M-phase.…”

“…Inhibition of TS does not correlate with the antitumor activity of FTD but there is a positive correlation between the antitumor activity of TAS-102 and the amount of TFT incorporated into human cancer xenograft DNA [26,28]. It has been shown previously that DNA incorporation of TFT results in increased DNA fragmentation due to single-and double-stranded DNA breaks [29,30]. However, the mechanisms underlying this process have not been fully elucidated.…”

“…TFT also does not appear to be a substrate for DNA glycosylases when it is paired with adenine and incorporated into the T sites of DNA helix as it was not subject to excision by uracil DNA glycosylases (UDG), thymine DNA glycosylase (TDG) and methylCpG-binding domain 4 (MBD4) [27]. TDG and MBD4 glycosylases were able to excise the TFT paired to guanine in DNA but the pairing with guanine occurs much less frequently than with adenine [30]. Cells deficient in the TDG and MBD4 glycosylases have been reported to be resistant to 5FU.…”