Equilibrative nucleoside transporters (ENTs) 1 and 2 reportedly accept fluorouracil as a substrate. Here, we evaluated ENT1/2 expression at the messenger RNA (mRNA), protein, and functional levels in a panel of four triple-negative breast cancer (TNBC) cell lines, BT-549, Hs578T, MDA-MB-231, and MDA-MB-435, and we examined the relationship of the observed profiles to fluorouracil sensitivity. Nitrobenzylthioinosine (NBMPR) at 0.1 μM inhibits only ENT1, while dipyridamole at 10 μM or NBMPR at 100 μM inhibits both ENT1 and ENT2. We found that the uptake of [3H]uridine, a typical substrate of ENT1 and ENT2, was decreased to approximately 40% by 0.1 μM NBMPR. At 100 μM, NBMPR almost completely blocked the saturable uptake of [3H]uridine, but this does not imply a functional role of ENT2, because 10 μM dipyridamole showed similar inhibition to 0.1 μM NBMPR. Expression of ENT1 mRNA was almost 1 order of magnitude higher than that of ENT2 in all TNBC cell lines. Liquid chromatography-tandem mass spectrometry(LC-MS/MS) LC-MS/MS-based targeted protein quantification showed that ENT1 protein levels were in the range of 9.3–30 fmol/μg protein in plasma membrane fraction of TNBC cell lines, whereas ENT2 protein was below the detection limit. [3H]Fluorouracil uptake was insensitive to 0.1 μM NBMPR and 10 μM dipyridamole, suggesting a negligible contribution of ENT1 and ENT2 to fluorouracil uptake. The levels of ENT1 mRNA, ENT1 protein, ENT2 mRNA, and ENT1-mediated [3H]uridine uptake in the four TNBC cell lines showed no correlation with fluorouracil sensitivity. These results indicate that neither ENT1 nor ENT2 contributes significantly to the fluorouracil sensitivity of TNBC cell lines.
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