Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker

Qinchang Zhu,Sheng Yin,Tsutomu Kabashima,Masaaki Kai,Takayuki Shibata

doi:10.1038/srep40670

Qinchang Zhu, Sheng Yin + Show 3 more

Open Access

https://doi.org/10.1038/srep40670

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Journal: Scientific Reports	Publication Date: Jan 13, 2017
Citations: 18	License type: open-access

Affiliation: Nagasaki University

Abstract

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/105 cells/h) than in normal fibroblasts (54.1 ± 7.40 pmol/105 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.

Highlights

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
Coenzyme Q 10 is necessary to activate Dihydroorotate dehydrogenase (DHODH) because it works as electron acceptor in the redox reaction[18], and triton X-100 increases the solubility of coenzyme Q 10
This assay format enabled the specific assay of DHODH activity in biologically complex samples such as cultured cells and tissues, and had sufficient selectivity and sensitivity for measuring the DHODH activity

Summary

Introduction

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. We applied this FL reaction to the assay of DHODH activity in cultured cells and in human stomach tissues. We determined the relationship between cell number and amount of orotic acid produced in a 1.0-mL reaction mixture (Fig. 4b).

Results

Conclusion