Crucial roles of thymidine kinase 1 and deoxyUTPase in incorporating the antineoplastic nucleosides trifluridine and 2′-deoxy-5-fluorouridine into DNA

Abstract

Trifluridine (FTD) and 2′-deoxy-5-fluorouridine (FdUrd), a derivative of 5-fluorouracil (5-FU), are antitumor agents that inhibit thymidylate synthase activity and their nucleotides are incorporated into DNA. However, it is evident that several differences occur in the underlying antitumor mechanisms associated with these nucleoside analogues. Recently, TAS-102 (composed of FTD and tipiracil hydrochloride, TPI) was shown to prolong the survival of patients with colorectal cancer who received a median of 2 prior therapies, including 5-FU. TAS-102 was recently approved for clinical use in Japan. These data suggest that the antitumor activities of TAS-102 and 5-FU proceed via different mechanisms. Thus, we analyzed their properties in terms of thymidine salvage pathway utilization, involving membrane transporters, a nucleoside kinase, a nucleotide-dephosphorylating enzyme, and DNA polymerase α. FTD incorporated into DNA with higher efficiency than FdUrd did. Both FTD and FdUrd were transported into cells by ENT1 and ENT2 and were phosphorylated by thymidine kinase 1, which showed a higher catalytic activity for FTD than for FdUrd. deoxyUTPase (DUT) did not recognize dTTP and FTD-triphosphate (F3dTTP), whereas deoxyuridine-triphosphate (dUTP) and FdUrd-triphosphate (FdUTP) were efficiently degraded by DUT. DNA polymerase α incorporated both F3dTTP and FdUTP into DNA at sites aligned with adenine on the opposite strand. FTD-treated cells showed differing nuclear morphologies compared to FdUrd-treated cells. These findings indicate that FTD and FdUrd are incorporated into DNA with different efficiencies due to differences in the substrate specificities of TK1 and DUT, causing abundant FTD incorporation into DNA.

Thymidine Kinase 1 Loss Confers Trifluridine Resistance without Affecting 5-Fluorouracil Metabolism and Cytotoxicity

“…Why does loss of TK1 confer FTD resistance? Similar to thymidine, once FTD is transported into the cytoplasm of tumor cells by nucleoside transporters, hENT1, hENT2 (5,6), or hCNT1 (21), it is activated by sequential phosphorylation by TK, TYMK, and NDK. FTD cytotoxicity is possibly mediated by the inhibition of TS activity by FTD-MP (10) and the incorporation of FTD-TP into DNA (7)(8)(9).…”

“…Trifluridine (FTD) is a fluorinated nucleoside analogue and a cytotoxic component of TFTD, and tipiracil hydrochloride is a thymidine phosphorylase (TP) inhibitor that suppresses the degradation of FTD in vivo and maintains the FTD concentration in the bloodstream (3). Once FTD is transported into the cytoplasm of tumor cells by nucleoside transporters, such as equilibrative nucleoside transporter 1 (hENT1) or hENT2 (4)(5)(6), it is phosphorylated to monophosphate (FTD-MP), diphosphate, and triphosphate (FTD-TP) forms by thymidine kinase (TK), thymidylate kinase (TYMK), and nucleoside diphosphate kinase (NDK), respectively, exerting cytotoxic effects via incorporation into DNA (7)(8)(9). In addition, FTD-MP inhibits thymidylate synthase (TS; refs.…”

Bevacizumab in Combination with TAS-102 Improves Clinical Outcomes in Patients with Refractory Metastatic Colorectal Cancer: A Retrospective Study

“…TAS‐102 is an oral antitumor drug that contains the thymidine‐based nucleic acid analog trifluridine (FTD) and the thymidine phosphorylase inhibitor tipiracil hydrochloride (TPI) at a molar ratio of 1:0.5. FTD becomes incorporated into DNA and causes DNA dysfunction and damage . Although FTD is rapidly degraded to its inactive form in the intestines and liver, TPI assists in maintaining the blood concentration of FTD by inhibiting thymidine phosphorylase .…”

Contribution of equilibrative nucleoside transporter(s) to intestinal basolateral and apical transports of anticancer trifluridine

“…An immunohistochemical study indicated that ENT1 and ENT2 are predominantly expressed in the lateral membrane of human enterocytes (Govindarajan et al, ). In addition, the uptake of FTD by a colorectal cancer cell line was inhibited by the ENT inhibitors, S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR) and dipyridamole (Kitao et al, ; Sakamoto et al, ). Moreover, the systemic exposure of FTD was shown to increase linearly in patients with advanced solid tumor (Doi et al, ).…”