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Resistance to 5-fluorouracil and 5-fluoro-2'-deoxyuridine mechanisms and clinical implications
A Sobrero1, C Aschele, A Guglielmi
1Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.
Abstract:
Preliminary to studies on biopsy specimens from colorectal carcinomas, we have investigated the mechanisms of resistance to 5-fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) on a human colon carcinoma cell line, HCT-8 in vitro. After obtaining a high level of resistance, extracts were made from sensitive and resistant HCT-8 cells and the enzymes responsible for the activation or catabolism of fluoropyrimidines were assayed using a thin-layer chromatographic method. The activity of thymidine-kinase, thymidine phosphorylase, uridine-kinase, uridine phosphorylase, orotate phosphoribosyl transferase as well as the activity of the target enzyme thymidylate synthase were not significantly different in sensitive, FUra resistant or FdUrd resistant cells. The activities of these enzymes in sensitive HCT-8 cells were respectively 15.6, 3.4, 40.8, 2.1, 6.9 and 30.2 nmol/mg protein/h. Direct evidence that impaired transport was the mechanism of resistance to FdUrd was obtained using a short term "oil stop" technique, whereas indirect evidence suggests that reduced inhibition of thymidylate synthase is likely to be the mechanism of resistance to FUra. Cells seven hundred-fold resistant to FdUrd were in fact still sensitive to FUra (ED50 value after continuous exposure 2.1 microM) while FUra resistant cells were fully cross-resistant to FdUrd (ED50 value after continuous exposure 0.003 microM). The clinical relevance of these data is discussed in the light of the extensive literature about the potentiation of fluoropyrimidines by leucovorin.
Insights
Mechanisms of resistance to 5-fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) in colon cancer cells were studied. Impaired transport caused FdUrd resistance, while reduced thymidylate synthase inhibition likely caused FUra resistance.
Area of Science:
- Oncology
- Pharmacology
- Biochemistry
Background:
- Colorectal carcinomas are a significant health concern.
- 5-fluorouracil (FUra) and 5-fluoro-2 -deoxyuridine (FdUrd) are key chemotherapeutic agents.
- Understanding drug resistance mechanisms is crucial for effective cancer treatment.
Purpose of the Study:
- To investigate the in vitro mechanisms of resistance to FUra and FdUrd in a human colon carcinoma cell line (HCT-8).
- To compare enzyme activities involved in fluoropyrimidine metabolism between sensitive and resistant cells.
Main Methods:
- Established FUra- and FdUrd-resistant HCT-8 cell lines.
- Assayed key enzymes (thymidine-kinase, thymidine phosphorylase, uridine-kinase, uridine phosphorylase, orotate phosphoribosyl transferase, thymidylate synthase) using thin-layer chromatography.
- Utilized an "oil stop" technique to assess drug transport.
Main Results:
- No significant differences in the activity of key metabolic enzymes were found between sensitive and resistant cells.
- Impaired drug transport was identified as the mechanism for FdUrd resistance.
- Reduced inhibition of thymidylate synthase was suggested as the mechanism for FUra resistance.
- FUra-resistant cells showed cross-resistance to FdUrd, but FdUrd-resistant cells remained sensitive to FUra.
Conclusions:
- Drug transport and target enzyme inhibition, rather than altered enzyme activity, are key mechanisms of FUra and FdUrd resistance in colon cancer cells.
- These findings have implications for combination therapies and understanding clinical resistance to fluoropyrimidines.