TY - JOUR
T1 - Fluorouracil uptake in triple-negative breast cancer cells
T2 - Negligible contribution of equilibrative nucleoside transporters 1 and 2
AU - Noguchi, Saki
AU - Takagi, Akinori
AU - Tanaka, Takahiro
AU - Takahashi, Yu
AU - Pan, Xiaole
AU - Kibayashi, Yuka
AU - Mizokami, Ryo
AU - Nishimura, Tomohiro
AU - Tomi, Masatoshi
N1 - Funding Information:
The authors would like to thank Dr. Yoshikazu Sugimoto (Keio University, Tokyo, Japan) for his help in handling TNBC cell lines BT‐549, Hs578T, MDA‐MB‐231, and MDA‐MB‐435 obtained from the DCTD Tumor Repository, National Cancer Institute, United States. This work was supported in part by JSPS KAKENHI grant numbers 18H03183. It was also funded in part by Keio Gijuku Academic Development Fund and the Hoansha Foundation.
Publisher Copyright:
© 2021 John Wiley & Sons Ltd.
PY - 2021/3
Y1 - 2021/3
N2 - 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.
AB - 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.
KW - breast cancer
KW - fluorouracil
KW - nitrobenzylthioinosine
KW - nucleoside transporter
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U2 - 10.1002/bdd.2261
DO - 10.1002/bdd.2261
M3 - Article
C2 - 33426680
AN - SCOPUS:85100457706
SN - 0142-2782
VL - 42
SP - 85
EP - 93
JO - Biopharmaceutics and Drug Disposition
JF - Biopharmaceutics and Drug Disposition
IS - 2-3
ER -
