Fluorouracil uptake in triple‐negative breast cancer cells: Negligible contribution of equilibrative nucleoside transporters 1 and 2

Fluorouracil uptake in triple‐negative breast cancer cells: Negligible contribution of equilibrative nucleoside transporters 1 and 2

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...

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Published in:Biopharmaceutics & drug disposition Vol. 42; no. 2-3; pp. 85 - 93
Main Authors: Noguchi, Saki, Takagi, Akinori, Tanaka, Takahiro, Takahashi, Yu, Pan, Xiaole, Kibayashi, Yuka, Mizokami, Ryo, Nishimura, Tomohiro, Tomi, Masatoshi
Format: Journal Article
Language:English
Published: England Wiley Subscription Services, Inc 01-03-2021
Subjects:
5-Fluorouracil
Breast cancer
Dipyridamole
Ent1 protein
Ent2 protein
fluorouracil
Gene expression
Kinases
Liquid chromatography
Mass spectroscopy
Membrane proteins
nitrobenzylthioinosine
nucleoside transporter
Nucleosides
Proteins
Tumor cell lines
Uridine
breast cancer
nitrobenzylthioinosine
fluorouracil
nucleoside transporter
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Summary: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. Equilibrative nucleoside transporter (ENT) 1, but not ENT2, was functionally expressed (uridine uptake activity) in triple‐negative breast cancer (TNBC) cell lines. However, ENT1 lacked the ability to transport fluorouracil. These results suggest neither ENT1 nor ENT2 affects the chemosensitivity of TNBC cells.
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ISSN:0142-2782
1099-081X
DOI:10.1002/bdd.2261