Antitumor mechanisms of 3′-ethynyluridine and 3′-ethynylcytidine as RNA synthesis inhibitors: development and characterization of 3′-ethynyluridine- resistant cells
Introduction
The molecular targets of nucleoside anti-metabolites which can be used as anti-viral and anti-cancer drugs, have been identified by recent progress in molecular biology. To develop a novel nucleoside anti-metabolite that has a new strategy as the mechanism of action for anti-tumor activity will contribute to the treatment of human cancers. We reported two cytosine nucleosides with a 2′-substituent in the sugar moiety, 1-(2-deoxy-2-methylene-β-d-erythro-pentofuranosyl)cytosine (DMDC) 1, 2and 1-(2-C-cyano-2-deoxy-β-d-arabino-pentofuranosyl)cytosine (CNDAC) 3, 4. They have potent anti-tumor activity against solid tumors in murine models and human cancer xenografts in nude mice. This wide spectrum of anti-tumor activity is in contrast to that of 1-β-d-arabinofuranosylcytosine (ara-C), which is inactive against solid tumors. They are phosphorylated by deoxycytidine kinase to 5′-monophosphate and subsequently to the related 5′-diphosphate and -triphosphate for the inhibition of DNA synthesis. These nucleosides slightly but non-negligibly inhibit RNA synthesis. The inhibition of RNA synthesis would contribute to the anti-tumor activity against slow-growing solid tumors, because a large population of tumor cells is in other than S-phase in vivo, in which only RNA synthesis and DNA repair are done. Therefore, as more active compounds against solid tumors, we have designed and synthesized such multifunctional nucleosides with an ethynyl residue at the 3′-β position of the ribose moiety of cytidine or uridine, 1-(3-C-ethynyl-β-d-ribo-pentofuranosyl)cytosine (ECyd) and 1-(3-C-ethynyl-β-d-ribo-pentofuranosyl)uracil (EUrd). Both nucleosides show strong anti-tumor effects on human cancers in vitro and in vivo. The cytotoxic effects of ECyd and EUrd are prevented by both cytidine and uridine, suggesting that phosphorylation by uridine/cytidine kinase may be required for the anti-tumor activity. These nucleosides strongly inhibit RNA synthesis and slightly inhibit DNA synthesis 5, 6, 7.
In this report, we describe the establishment and characterization of the resistant cells from human fibrosarcoma HT-1080 to EUrd to discover the mechanisms of action of ECyd and EUrd.
Section snippets
Cells and drugs
Human fibrosarcoma HT-1080 cells (American Type Culture Collection, Rockville, MD) were propagated in RPMI 1640 (Nissui Pharmaceutical Co., Osaka, Japan) with 10% heat-inactivated fetal bovine serum (Gibco Lab., Grand Island, NY), 100 units/ml penicillin G, and 100 μg/ml streptomycin, at 37°C in a humidified atmosphere containing 5% CO2. EUrd and ECyd were synthesized as described in our previous reports 5, 6, 7. Ara-C was a product of Yamasa Shoyu Co. Ltd. (Choshi, Japan). 5-Fluorouracil
Establishment of EUrd-resistant cell
EUrd was first used on parental HT-1080 cells at the concentration of 0.01 ng/ml, and then the concentration of EUrd was increased by steps. The resistant cells (HT-1080/EUrd) were finally able to grow at a 50-fold higher concentration (2 μg/ml) of the parental IC50. The resistant-sensitive ratio was estimated to be approximately 2000 for EUrd as shown in Table 1. The cells were cross-resistant to ECyd (approximately 700-fold) and FUrd (176-fold), but not to 5FU, FdUrd, ara-C, ADM, MMC, or CDDP.
Discussion
Nucleoside antimetabolites are still one of the most important drugs for cancer treatment and the mechanisms of action of these nucleosides are believed to be due mainly to their inhibition of DNA synthesis in tumor cells. However, almost all solid tumors grow slowly, and the drugs have few chances to encounter the S-phase of the cell cycle, in which DNA synthesis occurs. Thus, it is not enough for nucleoside antimetabolites to inhibit only DNA synthesis in human solid cancers. To overcome the
Acknowledgements
S. T. is deeply indebted to Prof. Y. Watanabe, Kanazawa University School of Medicine, for his continuous interest and support. This work was supported in part by Grants-in-Aid for Cancer Research and 2nd-Term Comprehensive Ten-Year Strategy for Cancer Control from the Ministry of Health and Welfare, and also Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Sciences, Sports, and Culture, Japan.
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Induction of apoptosis in cervical cancer cells by the duplex drug 5-FdU-ECyd, coupling 2′-deoxy-5-fluorouridine and 3′-C-ethinylcytidine
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