Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker

We developed an assay method for measuring dihydroorotate dehydrogenase (DHODH) activity in cultured HeLa cells and fibroblasts, and in stage III stomach cancer and adjacent normal tissues from the same patient. The assay comprised enzymatic reaction of DHODH with a large amount of dihydroorotic aci...

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Autores principales: Yin, Sheng, Kabashima, Tsutomu, Zhu, Qinchang, Shibata, Takayuki, Kai, Masaaki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2017
Materias:
Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5233952/
https://www.ncbi.nlm.nih.gov/pubmed/28084471
http://dx.doi.org/10.1038/srep40670
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author Yin, Sheng
Kabashima, Tsutomu
Zhu, Qinchang
Shibata, Takayuki
Kai, Masaaki
author_facet Yin, Sheng
Kabashima, Tsutomu
Zhu, Qinchang
Shibata, Takayuki
Kai, Masaaki
author_sort Yin, Sheng
collection PubMed
description We developed an assay method for measuring dihydroorotate dehydrogenase (DHODH) activity in cultured HeLa cells and fibroblasts, and in stage III stomach cancer and adjacent normal tissues from the same patient. The assay comprised enzymatic reaction of DHODH with a large amount of dihydroorotic acid substrate, followed by fluorescence (FL) detection specific for orotic acid using the 4-trifluoromethyl-benzamidoxime fluorogenic reagent. The DHODH activities in the biologically complex samples were readily measured by the assay method. Our data indicate significantly higher DHODH activity in HeLa cells (340 ± 25.9 pmol/10(5) cells/h) than in normal fibroblasts (54.1 ± 7.40 pmol/10(5) cells/h), and in malignant tumour tissue (1.10 ± 0.19 nmol/mg total proteins/h) than in adjacent normal tissue (0.24 ± 0.11 nmol/mg total proteins/h). This is the first report that DHODH activity may be a diagnostic biomarker for cancer.
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spelling pubmed-52339522017-01-17 Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker Yin, Sheng Kabashima, Tsutomu Zhu, Qinchang Shibata, Takayuki Kai, Masaaki Sci Rep Article We developed an assay method for measuring dihydroorotate dehydrogenase (DHODH) activity in cultured HeLa cells and fibroblasts, and in stage III stomach cancer and adjacent normal tissues from the same patient. The assay comprised enzymatic reaction of DHODH with a large amount of dihydroorotic acid substrate, followed by fluorescence (FL) detection specific for orotic acid using the 4-trifluoromethyl-benzamidoxime fluorogenic reagent. The DHODH activities in the biologically complex samples were readily measured by the assay method. Our data indicate significantly higher DHODH activity in HeLa cells (340 ± 25.9 pmol/10(5) cells/h) than in normal fibroblasts (54.1 ± 7.40 pmol/10(5) cells/h), and in malignant tumour tissue (1.10 ± 0.19 nmol/mg total proteins/h) than in adjacent normal tissue (0.24 ± 0.11 nmol/mg total proteins/h). This is the first report that DHODH activity may be a diagnostic biomarker for cancer. Nature Publishing Group 2017-01-13 /pmc/articles/PMC5233952/ /pubmed/28084471 http://dx.doi.org/10.1038/srep40670 Text en Copyright © 2017, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Yin, Sheng
Kabashima, Tsutomu
Zhu, Qinchang
Shibata, Takayuki
Kai, Masaaki
Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker
title Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker
title_full Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker
title_fullStr Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker
title_full_unstemmed Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker
title_short Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker
title_sort fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5233952/
https://www.ncbi.nlm.nih.gov/pubmed/28084471
http://dx.doi.org/10.1038/srep40670
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