Norihiko Suzuki

Mode of action of trifluorothymidine (TFT) against DNA replication and repair enzymes.

Trifluorothymidine (TFT) is well known to be converted to TFT-monophosphate by thymidine kinase and to inhibit thymidylate synthase. In addition, TFT-triphosphate (TFT-TP) is also incorporated into DNA, resulting in cytocidal effects. However, the precise mechanism of TFT-induced DNA damage is still unclear. Therefore, we investigated the modes of action of TFT against DNA replication and repair enzymes, as compared with those of 5FU and FdUrd. When HeLa cells were treated with TFT at a concentration of 1 µM (IC50 value), the concentration of TFT in the DNA was calculated as 62.2±0.9 pmol/1x106 cells for 4 h. On the other hand, following treatment of the cells with FdUrd (0.5 µM) and 5FU (10 µM) at their IC50 doses, the drug concentrations in the DNA were 7.53, and 0.17 pmol/1 x 10⁶ cells for 4 h, respectively. These results show the markedly greater degree of incorporation of TFT into the DNA of the HeLa cells compared with that of 5FU (approximately more than 300-fold for 4 h) or FdUrd (approximately more than 8-fold for 4 h). The primer extension assay demonstrated that TFT-TP was also incorporated into the T-sites of the growing DNA strand, however, it competed only weakly with thymidine triphosphate. The DNA glycosylase assay was performed using commercially available DNA glycosylase and fractionated HeLa cell extracts obtained by gel filtration. There was no detectable excision of the TFT pairing to adenine by uracil DNA glycosylase (UDG), thymine DNA glycosylase (TDG), methyl-CpG binding domain 4 (MBD4) or the fractionated HeLa cell extracts, however, TDG and MBD4 were able to excise the TFT pairing to guanine. Additional data indicate that small-interfering RNA-mediated knockdown of TDG or MBD4 significantly increased the resistance to the cytotoxic effects of FdUrd, but not to that of TFT. These studies show the greater degree of incorporation of TFT into the DNA than that of 5FU or FdUrd, and that such a high degree of incorporation of TFT residues into the DNA might be related to exhibit potent cytotoxic activity to be refractory to cleavage by these DNA glycosylases; thus, the DNA-directed cytotoxic effect of the compound is quite different from that of 5FU.

Simple and Rapid Enzymatic Method for the Synthesis of Single-Strand Oligonucleotides Containing Trifluorothymidine

To investigate the mechanism of trifluorothymidine (TFT)-induced DNA damage, we developed an enzymatic method for the synthesis of single-strand oligonucleotides containing TFT-monophosphate residues. Sixteen-mer oligonucleotides and 14-mer 5′-phosphorylated oligonucleotides were annealed to the template of 25-mer, so as to empty one nucleotide site. TFT-triphosphate was incorporated into the site by DNA polymerase and then ligated to 5′-phosphorylated oligonucleotides by DNA ligase. The synthesized 31-mer oligonucleotides containing TFT residues were isolated from the 25-mer complementary template by denaturing polyacrylamide electrophoresis. Using these single-strand oligonucleotides containing TFT residues, the cleavage of TFT residues from DNA, using mismatch uracil-DNA glycosylase (MUG) of E.coli origin, was compared with that of 5-fluorouracil (5FU) and 5-bromodeoxyuridine (BrdU). The TFT/A pair was not cleaved by MUG, while the other pairs, namely, 5FU/A, 5FU/G, BrdU/A, BrdU/G, and TFT/G, were easily cleaved from each synthesized DNA. Thus, this method is useful for obtaining some site-specifically modified oligonucleotides.

Trifluridine/tipiracil increases survival rates in peritoneal dissemination mouse models of human colorectal and gastric cancer

A number of patients exhibit peritoneal dissemination of gastric or colorectal cancer, which is a predominant cause of cancer-associated mortality. Currently, there is no markedly effective treatment available. The present study was designed to determine the efficacy of trifluridine/tipiracil (TFTD), formerly known as TAS-102, which is used for the treatment of patients with unresectable advanced or recurrent colorectal cancer refractory to standard therapies. Four colorectal cancer cell lines and one gastric cancer cell line were intraperitoneally inoculated into nude mice, as models of peritoneal dissemination. TFTD (200 mg/kg/day) was orally administered for 5 consecutive days followed by 2 drug-free days for 6 weeks. The increase in the lifespan (ILS) of the TFTD-treated mice compared with that of the drug-free control mice was 66.7, 43.3, 106.3, 98.3 and 133.3% for DLD-1, DLD-1/5-fluorouracil [5-fluorouracil (5FU)-resistant subline of DLD-1], HT-29 and HCT116 colorectal cancer cell lines, and MKN45 gastric cancer cell line, respectively. This ILS was similar to that of the irinotecan-treated mice (ILS, 70–84%), but was significantly (P<0.05) increased compared with that of the 5FU-, tegafur, gimeracil and potassium oxonate- and cisplatin-treated mice (ILS, 1–53%, 0.8–60% and 85%, respectively). No significant increase in body weight loss was observed during the dosing periods with any of the drugs used. The increase in CEA levels with progressive peritoneal dissemination was inhibited by TFTD treatment. TFTD also exhibited marked anticancer effects against Kirsten rat sarcoma viral oncogene homolog-mutated tumors and 5FU-resistant tumors. The results of the present study indicate that TFTD may be a potential drug against peritoneal dissemination of colorectal and/or gastric cancer in humans and may be utilized for chemo-naïve tumors and recurrent tumors following 5FU treatment.

Abstract 1071: Efficacy of trifluridine/tipiracil + anti-mouse PD-1 antibody combination on mouse colorectal cancer model and related tumor immunomodulatory effects

Background: Trifluridine/tipiracil (FTD/TPI) is an oral nucleoside antitumor agent that is composed of trifluridine and tipiracil hydrochloride at a molecular ratio of 1:0.5. Checkpoint-blockade immunotherapies are particularly effective in patients with tumor T cell infiltrations. In this study, the antitumor effects of FTD/TPI + anti-mouse PD-1 antibody combination were studied in a syngeneic mouse model and the tumor-infiltrating lymphocyte (TIL) subsets were evaluated. Method: The mouse colorectal cancer cell line CMT-93 was subcutaneously implanted into C57BL/6 mice. Vehicle (0.5% Hydroxypropyl methylcellulose, 10 mL/kg, p.o.), FTD/TPI (75, 100, and 150 mg/kg/day, twice daily, days 1-14, p.o.), anti-mouse PD-1 antibody (clone RMP1-14; 0.1 mg/body; once daily, days 1, 5, and 9; i.p.), and FTD/TPI + anti-mouse PD-1 antibody combination were administered, and inhibitory activity was evaluated according to tumor-volume changes. Single-cell suspensions were prepared from collected tumors. Based on cell marker expression, CD4+ T cells, CD8+T cells, and regulatory T cells (Tregs) were identified using antibodies against CD4, CD8, CD25, and Foxp3, and subsets of TILs were evaluated by flow cytometry. Results: Both anti-mouse PD-1 antibody and FTD/TPI monotherapies were effective in vivo. Tumor-growth inhibition by anti-mouse PD-1 antibody was 81.7% at 0.1 mg/kg/day and that by FTD/TPI was 33.4%, 46.1%, and 59.5% at 75, 100, and 150 mg/kg/day, respectively. FTD/TPI + anti-mouse PD-1 antibody combination using FTD/TPI (75, 100, and 150 mg/kg/day) with anti-mouse PD-1 antibody (0.1 mg/kg/day) inhibited tumor growth at significantly higher rates (P Citation Format: Norihiko Suzuki, Hiroshi Tsukihara, Fumio Nakagawa, Takashi Kobunai, Teiji Takechi. Efficacy of trifluridine/tipiracil + anti-mouse PD-1 antibody combination on mouse colorectal cancer model and related tumor immunomodulatory effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1071. doi:10.1158/1538-7445.AM2017-1071