Identification of transporters of 5-fluorouracil

Abstract

5-fluorouracil (5-FU) is a nucleobase analogue used to treat used to treat cancers of the head and neck, breast, skin, pancreas and gastrointestinal tract and skin. The amount of 5- FU that reaches its intracellular sites of action is mitigated by anabolic and catabolic enzymes, and by a cell’s drug transport capacity. While the pathways of biotransformation of 5-FU by metabolic enzymes are largely known, the mechanisms through which it is transported through the cell membrane remain elusive and are an avenue where further study is warranted. In this project, candidate transporters were investigated for their capacity to transport 5-FU in the gastrointestinal cancer cell line, Cancer coli-2, or Caco-2, which possesses the capacity to spontaneously differentiate into a phenotype highly resemblant of a polarised enterocyte upon the cells reaching confluency. Undifferentiated and differentiated monolayers of Caco-2 cells were incubated in [14C] 5-FU-spiked uptake buffer for transport studies. The composition of ions, substrates, and inhibitors in the uptake buffer was manipulated to assess the role of candidate transporters in 5-FU transport. Two inhibitors of drug transporter activity with broad specificity reduced 5-FU uptake: phloretin (900 µM) and indomethacin (2000 µM). These effects were only statistically significant in undifferentiated Caco-2 cells. Cell viability assessments pointed to these effects not being a result of a deleterious effect of the high concentrations of inhibitor used on cell viability. These results suggest a role of the transporters inhibited by phloretin and indomethacin in the transport of 5-FU. Neither the addition of L-Ascorbic acid or BCH,or the omission of glucose and/or sodium ion to incubations resulted in a significant change in 5- FU uptake, and no single candidate transporter could be identified from these results. However, further experimentation would be prudent to rule out these findings as artefactual of this work. To investigate candidate transporters for the uptake of 5-FU, a systematic review of the literature was carried out. A search string was constructed to catch articles with associations of transporters with 5-FU cytotoxicity in pre-clinical studies, as well as with patient outcomes and toxicities. The search returned 6437 articles, 167 of which met the inclusion/exclusion criteria for the review. The transporter that turned up most frequently was the equillibrative nucleoside transporter encoded by the gene, SLC29A1, ENT1, although there was no strong evidence for the transport of 5-FU by this transporter. The only direct evidence found in this review, for the transport of 5-FU by a transporter, was for the organic anion transporter 2, OAT2, encoded by the gene, SLC22A7. All other transporter families had weak evidence at best for a role in drug transport. In conclusion, it is indicated that further investigation of transporters inhibited by phloretin and indomethacin may be a valuable avenue for investigation of the identity of transporters of 5-FU. Collectively the strongest case to be made from the findings of the systematic review and experimental work of this project is for the transport of 5-FU by the OAT2 transporter in humans. This is a sodium-independent transporter that can be inhibited by indomethacin. In this project, omission of sodium ion from uptake media did not significantly alter the uptake of 5-FU, whereas the addition of 900 µM indomethacin significantly inhibited uptake. The strongest literature evidence for a transporter having a role in the uptake of 5-FU in humans found in the systematic review in this project, was for the uptake transport of 5-FU by OAT2.