Abstract:
5-Fluorouracil (5-FU) is an effective chemotherapeutic agent widely used in the treatment of gastrointestinal cancers. However, it is associated with severe (grade 3-4) side effects including inflammation of the gastrointestinal mucosa (mucositis) and severe diarrhoea, which are highly debilitating and dose-limiting. Inherited deficiencies in dihydropyrimidine dehydrogenase, key for the hepatic elimination of 5-FU, can predict only one-third of the individuals who are at risk of severe toxicity. It is hypothesised that inter-individual variation in the uptake of 5-FU into gastrointestinal mucosal cells may be an additional mechanism for these severe side effects in some patients. Little is known about how 5-FU enters cells, or which transporters are involved. The related endogenous nucleobases uracil and thymine have recently been suggested to be substrates for SLC23A1-2 and SLC29A1-4 transporters. The aim of this thesis was to investigate transport of 5-FU into cells, and to characterise the role of these candidate transporters in the uptake of this drug. Cells derived from the gastrointestinal mucosa (HCT116 and CaCo-2) were grown as monolayers and uptake of 14C 5-FU across the apical membrane assessed. Expression of the candidate transporters in these cell lines was confirmed by RT-PCR. The Michaelis-Menten kinetics of uptake were determined for in HCT-116 (Vmax; 405.8 ± 56.9 pmol/min/105 cells, KM; 1.42 mM) and CaCo-2 (Vmax; 425.8 ± 170 pmol/min/105 cells, KM; 6.12 mM) cells. This saturable process contrasted with the simple linear uptake observed when cells were assessed under ice-cold conditions. The sodium and pH dependency of 5-FU uptake was assessed, as these factors are known to affect the activity of the SLC23A symporter family and SLC29A4 respectively. The effect of known substrates and selective inhibitors of SLC23A1-2 and SLC29A1, 2 & 4 on the apical uptake of 14C 5-FU into these cells was determined. Based on these experiments it was found that SLC23A1-2 and SLC29A1-3 were not likely to be involved in this process. However in HCT 116 cells 5-FU uptake was pH sensitive and inhibited by D-22, an inhibitor of SLC29A4. Lopinavir a more selective inhibitor of SLC29A4 did not have an effect. Thus, a role for the candidate transporters in the apical uptake of 5-FU in these cells has been discounted. The identity of the apical uptake transporter(s) as well as the characterisation of basolateral uptake of 5-FU in human cells requires further study.