Elsevier

European Journal of Cancer

Volume 37, Issue 13, September 2001, Pages 1681-1687
European Journal of Cancer

Thymidylate synthase (TS) and ribonucleotide reductase (RNR) may be involved in acquired resistance to 5-fluorouracil (5-FU) in human cancer xenografts in vivo

https://doi.org/10.1016/S0959-8049(01)00174-5Get rights and content

Abstract

A human tumour sub-line resistant to 5-fluorouracil (5-FU) was established by once a day and every 5, with at least 50 administrations of 5-FU to KM12C human colorectal xenografts in nude mice. KM12C tumours treated with 5-FU showed less sensitivity to 5-FU with an inhibition rate (IR) of 7.9%, while non-treated tumours were highly sensitive to 5-FU with an IR of 81.8%. To clarify the mechanism of 5-FU-resistance, the activities of various enzymes and gene expressions involved in the metabolism of 5-FU in both parental and 5-FU-treated KM12C tumours were measured. A 2- to 3-fold increase in thymidylate synthase (TS) activity and 4- to 5-fold decrease in ribonucleotide reductase (RNR) activity were observed in 5-FU-resistant KM12C tumours, while the activities of orotate phosphoribosyltransferase (OPRT) thymidine and uridine phosphorylases (TP,UP) and thymidine kinase (TK) were not markedly changed as a consequence of repeated treatment of KM12C tumours with 5-FU. The expression of TS mRNA was also amplified in accordance with the increased TS activity in a 5-FU-treated tumour sub-line (KM12C/5-FU) compared with that in parental tumours, but changed expressions of both RNR-R1 and RNA-R2 mRNA could not be detected in the 5-FU-resistant tumour sub-line compared with the parental tumours, suggesting possible post-transcriptional regulation of RNR. Moreover, RNR, in addition to TS and OPRT, seemed to be related to the inherent insensitivity to 5-FU in human cancer xenografts. From these results, it may be concluded that RNR activity is one of the acquired or inherent resistant factors, including TS, to 5-FU in human cancer xenografts in vivo.

Introduction

5-Fluorouracil (5-FU) is used to treat patients with gastrointestinal, breast and head and neck cancers. Further attempts to potentiate 5-FU cytotoxicity, by improving the dosing schedule and biochemical modulation of 5-FU, are ongoing. However, the most negative factor in clinical use of 5-FU, whether alone or combined with other anticancer drugs is the development of 5-FU resistance by tumours and the existence of tumours innately resistant to 5-FU. To date, a number of in vitro studies have demonstrated the major mechanism of acquired-resistance to 5-FU to be an increase in thymidylate synthase (TS) activity and/or its gene expression 1, 2, 3, 4, 5.

TS has been recognised as the rate-limiting enzyme in de novo pyrimidine biosynthesis, and as being inhibited by 5-fluoro-2′-deoxyuridylate (FdUMP) formed from 5-FU, which thereby leads to inhibition of DNA synthesis 6, 7, 8, 9. Accordingly, high expression of TS induced by continuous treatment of cancer patients with 5-FU results in a decrease in the cytotoxic effect of 5-FU on the tumour cells. Spears and colleagues [10] reported that innate resistance to 5-FU in cancer patients receiving intravenous (i.v.). 5-FU may be attributable to the low formation of FdUMP and high TS expression. A number of clinical evaluations subsequently referred to the relationships of TS expression in tumours to clinical response and survival of cancer patients receiving 5-FU-based chemotherapy 11, 12, 13, 14, 15. On the other hand, in several in vitro studies, the major mechanism of 5-FU-resistance was suggested to be a marked decrease in 5-FU-metabolising enzymes such as orotate phosphoribosyltransferase (OPRT; EC 2.4.2.10) and uridine kinase (UK; EC 2.7.1.48) in human tumour cells treated with relatively high doses of 5-FU 16, 17.

There are, however, no reports concerning in vivo establishment of 5-FU-resistant human tumours and the characterisation of the resistance mechanism, despite there being several descriptions of in vitro 5-FU-resistance mechanisms.

We have endeavoured herein to establish 5-FU-resistant human tumour xenografts by consecutive and repeated administrations of 5-FU to human cancer-bearing nude mice. Herein, we report the enzymatic and genetic changes involved in 5-FU metabolism in human tumour xenografts showing acquired resistance to 5-FU and possible mechanisms of 5-FU-resistance in vivo.

Section snippets

Chemicals

[6-3H]-5-FU (525 GBq/mmol), [6-3H]-thymidine (dThd; 2.41 TBq/mmol), [6-3H]-uridine (Urd; 0.88 TBq/mmol), [6-3H]-FdUMP (625 GBq/mmol), and [(U)-14C]-cytidine-5′-diphosphate (CDP; 2.04 GBq/mmol) were obtained from Moravek Biochemicals, Inc. (CA, USA). 5-FU was obtained from Sigma Co. (MO, USA). S-1, 5-FU preparations composed by tegafur (as 5-FU analogue), 2,4-dihydroxypyridine (as a DPD inhibitor) and potassium oxonate (as an OPRT inhibitor) was prepared in our laboratory. All other chemicals

Effects of 5-FU on parental and 5-FU-treated KM12C tumours in mice

When 5-FU, 20 mg/kg, was administered i.v. to parental and 5-FU-treated KM12C (F-58) tumour-bearing mice, parental KM12C tumours showed a high sensitivity to 5-FU with an IR value of 82.1%, while KM12C/5-FU (F-58) tumours were almost resistant to 5-FU, as shown in Table 1. Thus, we were able to establish a human colorectal tumour sub-line highly resistant to 5-FU. Furthermore, the rate of tumour growth was nearly the same in the KM12C and KM12C/5-FU (F-58) tumour xenografts.

Activities of pyrimidine-metabolising enzymes in KM12C and KM12C/5-FU tumours

To elucidate the

Discussion

The appearance of acquired resistance or intrinsic resistance to anticancer drugs is a challenging problem in the long-term treatment of cancer patients. In the case of 5-FU, overexpression of TS has been suggested to be the main factor in acquired-resistance to 5-FU in human cancer cells 1, 2, 3. Since Spears and colleagues [10] reported the mechanism of innate resistance to 5-FU in cancer patients receiving this drug, a number of reports also have discussed the relationship of intratumoral TS

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