Antitumor mechanisms and metabolism of the novel antitumor nucleoside analogues, 1-(3- C -ethynyl-β- D - ribo -pentofuranosyl)cytosine and 1-(3- C -ethynyl-β- D - ribo -pentofuranosyl)uracil

Abstract

The antitumor ribonucleoside analogues 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd) and 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)uracil (EUrd), first synthesized in 1995, have strong antitumor activity against human cancer xenografts without severe side effects. Here, we studied the antitumor mechanisms of ECyd and EUrd using mouse mammary tumor FM3A cells in vitro and the mechanism of selective cytotoxicity of ECyd using human tumor xenografts in nude rats in vivo. In FM3A cells, ECyd and EUrd were rapidly phosphorylated to ECyd 5'-triphosphate (ECTP) and EUrd 5'-triphosphate (EUTP), which strongly inhibiting RNA synthesis. Cells treated with EUrd were later found to contain both EUTP and ECTP, and ECTP accumulated as the final product. Probably the uracil moieties of EUrd derivatives were efficiently converted to cytosine moieties in the cells. EUrd and its derivatives were minor metabolites in the cells treated with ECyd, so cytidine forms probably were not converted to uridine forms at the nucleoside or nucleotide stage. The ultimate metabolite of ECyd and EUrd, ECTP, is stable in cultured cells with a half-life of at least 3 days, so ECyd and EUrd are on a "closed" metabolic pathway to ECTP. These characteristics of ECyd and EUrd may be important for their antitumor activity. ECyd had strong and selective antitumor activity against the human tumor xenografts. ECyd-phosphorylating activity (uridine/cytidine kinase) in the xenografts was higher than that in the organs of the rats. This finding may account for the strong activity with mild side effects. ECyd and EUrd may be a new kind of antitumor nucleoside analogue for clinical use.

X Irradiation Induces the Proapoptotic State Independent of the Loss of Clonogenic Ability in Chinese Hamster V79 Cells

“…This operation was repeated at least five times until the pH of the solution became 5. The extract was analyzed by HPLC with an anionexchange column (TSK-gel DEAE-2SW, Tosoh Corporation, Tokyo, Japan) according to the method described by Takatori et al (7). The extract was eluted with 100% buffer A [10 mM NH 4 H 2 PO 4 , 10 mM sodium hexanesulfonate and 20% CH 3 CN (pH 3.0)] at a flow rate of 1.0 ml/min for 10 min and subsequently with a linear gradient to 60% buffer B [80 mM Na 2 HPO 4 , 10 mM hexanesulfonate and 20% CH 3 CN (pH 6.0)] for 35 min and finally with 100% buffer B for 80 min.…”

Treatment Combining X-Irradiation and a Ribonucleoside Anticancer Drug, TAS106, Effectively Suppresses the Growth of Tumor Cells Transplanted in Mice

“…This compound is effectively phosphorylated by uridine/cytidine kinase 2 (UCK2), which has higher activity in tumor cells than in normal cells (14,15). The resultant metabolite, ECTP, a 5'-triphosphate form of TAS106, inhibits RNA synthesis by competing with cytidine 5'-triphosphate and results in a compound cytotoxic to tumor cells (3,(13)(14)(15)(16)(17)(18). Since survivin has been reported to attenuate the activities of caspases 3, 7, and/or 9 (19)(20)(21) and to regulate proliferation of tumor cells in association with the mitotic apparatus (19), its down-regulation by TAS106 is expected to induce significant enhancement of radiation-induced cell death not only in vitro but also in vivo.…”

Cellular and Biochemical Mechanisms of the Resistance of Human Cancer Cells to a New Anticancer Ribo‐nucleoside, TAS‐106

“…5) Moreover, in preliminary experiments, RNA polymerase was inhibited competitively by ECTP with a K i value of 21 nM (apparent K m value of CTP, 8.0 µM) in isolated nuclei of FM3A mouse mammary tumor cells. 6,7) ECTP was found to accumulate as the major intracellular metabolite following the exposure of the cells to TAS-106 for 4 h and was then very slowly eliminated from the cells. 5,6) Therefore, intracellular accumulation and retention of the active metabolite ECTP may contribute to the potent cytotoxicity of TAS-106.…”

“…6,7) ECTP was found to accumulate as the major intracellular metabolite following the exposure of the cells to TAS-106 for 4 h and was then very slowly eliminated from the cells. 5,6) Therefore, intracellular accumulation and retention of the active metabolite ECTP may contribute to the potent cytotoxicity of TAS-106. This profile, considered to reflect a unique mechanism of antitumor activity and cellular metabolism, makes TAS-106 different from other antitumor nucleosides and TAS-106 is a promising candidate as a therapeutic agent for cancer patients.…”

“…[1][2][3] The results of several studies show that TAS-106 acts by interfering with RNA metabolism. 2,[4][5][6][7] Further in vitro studies on RNA polymerase II, an enzyme expressed in the nuclear extracts from HeLa cells, revealed that the triphosphate of TAS-106, ECTP, inhibited in a concentration-dependent manner the activity of that enzyme. 4) The action of TAS-106 on RNA polymerase-specific transcription was demonstrated to be without specificity for RNA polymerase I, II or III in K562 human leukemia cells.…”