Trifluridine/tipiracil overcomes the resistance of human gastric 5-fluorouracil-refractory cells with high thymidylate synthase expression

Abstract

Trifluridine/tipiracil (FTD/TPI or TFTD, also known as TAS-102) is a combination of the antineoplastic thymidine analog, FTD, and thymidine phosphorylase inhibitor, TPI (molar ratio 1:0.5). FTD/TPI was approved in Japan, the United States, and the European Union for the treatment of unresectable advanced or recurrent colorectal cancer. We evaluated the in vitro and in vivo efficacy and mechanisms of action of FTD and FTD/TPI against 5-fluorouracil (5-FU)-resistant MKN45/5FU, MKN74/5FU, and KATOIII/5FU human gastric cancer cells overexpressing thymidylate synthase (TS) and their respective parent cell lines. MKN45/5FU and KATOIII/5FU cells were not cross-resistant to FTD, whereas MKN45/5FU cells were 3.7-fold more resistant than the parental cells in vitro. FTD was also incorporated into genomic DNA in a concentration-dependent manner in 5-FU-resistant and parental cells. Additionally, deoxyuridine monophosphate levels in MKN45/5FU cells after 24-h FTD treatment were 3.0-fold higher than those in parental cells, and FTD treatment for 72 h induced G2/M arrest in MKN45/5FU cells, unlike the S phase arrest in MKN45 cells. Thus, TS-overexpressing MKN45/5FU cells, but not MKN74/5FU and KATOIII/5FU cells, showed partial cross-resistance to FTD. However, FTD/TPI (administered orally twice a day) exhibited antitumor activity to the same extent in MKN45 and MKN45/5FU xenograft mouse models, overcoming in vitro cross-resistance to FTD. DNA incorporation rather than TS inhibition seems to be the main action of FTD under these in vivo conditions. Thus, FTD/TPI is a promising chemotherapeutic agent against gastric cancers recurring following 5-FU therapy.

Keywords: 5-FU resistant cells; TAS-102; gastric cancer; trifluridine/tipiracil.

Figure 1. Inhibitory activity of FTD and 5-FU against cancer cells sensitive and resistant to 5-FU

Cell lines were cultured with various concentrations of FTD and 5-FU for 72 h. Data are represented as the mean + SD of three independent experiments, normalized to the control. (A) and (B) MKN45 and MKN45/5FU, (C) and (D) MKN74 and MKN74/5FU, (E) and (F) KATOIII and KATOIII/5FU.

Figure 2. Relative tumor volume (RTV) of xenografted tumors after daily oral administration of FTD/TPI and S-1

Xenografted mice were randomized on day 0. FTD/TPI (150 mg/kg) and S-1 (10 mg/kg) were administered orally twice and once daily, respectively, from days 1 to 14. Data are represented as the mean + SD (n = 8). The horizontal dotted line indicates a relative tumor volume of 5. (A) MKN45, (B) MKN45/5FU, (C) MKN74, and (D) MKN74/5FU.

Figure 3. Incorporation of FTD into DNA

DNA incorporation of FTD is shown as time-dependent (A–C) and dose-dependent (D–F) responses. (A) MKN45 and MKN45/5FU, (B) MKN74 and MKN74/5FU, and (C) KATOIII and KATOIII/5FU cells were treated with FTD for 24-72 h. Double-stranded DNA was extracted and FTD incorporation was determined by LC/MS/MS. Data are represented as the mean + SD (n = 3). FTD concentration was positively correlated with incorporation (Pearson correlation coefficients: (D) R² = 0.85 (MKN45), 0.77 (MKN45/5FU). (E) R² = 0.97 (MKN74), 0.97 (MKN74/5FU). (F) R² = 0.92 (KATOIII), 0.93 (KATOIII/5FU)).

Figure 4. FTD-induced accumulation of dUMP

The cells were exposed for 4, 8, and 24 h to indicated concentration of FTD. The cell dUMP levels were measured using LC/MS/MS. Data are represented as the mean + SD (n = 3). (A) MKN45 and MKN45/5FU, (B) MKN74 and MKN74/5FU, (C) KATOIII and KATOIII/5FU.

Figure 5. Effect of FTD on cell cycle distribution in parental and 5-FU resistant cells

The cells were exposed for 72 h to indicated concentration of FTD and assayed using flow cytometry. Data are represented as the mean + SD of three independent experiments. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. (A) and (B) MKN45 and MKN45/5FU, (C) and (D) MKN74 and MKN74/5FU, (E) and (F) KATOIII and KATOIII/5FU.

Figure 6. Hypothetical model of action of FTD with continuous exposure or intermittent exposure

(A) When tumor cells are continuously exposed to FTD, such as during in vitro growth inhibition assay, TS inhibition significantly affects cytotoxicity. Therefore, it is presumed that MKN45/5FU cells with high TS expression show partial cross-resistance to FTD. (B) When FTD/TPI is administered orally twice a day (i.e., intermittent exposure), since plasma concentrations of FTD decrease between doses, TS inhibition recovers between doses. It is presumed that DNA incorporation of FTD predominantly induces antitumor effects, since FTD is incorporated into DNA in a concentration-dependent manner under this oral administration condition. Therefore, the cross-resistance to FTD was overcome even in the MKN45/5FU cells with high TS expression. F indicates FTD.