Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker

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.

Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells

“…DHODH Activity Assay DHODH activity measurements were performed using a modified protocol according to Yin et al (2017). In brief, cells were collected, washed with PBS, re-suspended in potassium phosphate buffer (0.1 M, pH 7.0) and lysed using three freeze-thaw cycles; the lysates were incubated in the solution of 160 mM K 2 CO 3 -HCl (pH 8.0), 400 mM DHO, 80 mM decylubiquinone at 37 C for 60 min.…”

Replication and ribosomal stress induced by targeting pyrimidine synthesis and cellular checkpoints suppress p53-deficient tumors

“…DHODH activity was evaluated using a modified protocol according to Yin et al 56 . In brief, cells were collected, washed with PBS, re-suspended in potassium phosphate buffer (0.1 M, pH 7.0) and lysed using three freeze-thaw cycles; the lysates were incubated in the solution of 160 mM K 2 CO 3 /HCl (pH 8.0), 400 μM dihydroorotate, 80 μM decylubiquinone at 37°C for 60 min.…”

Elevated DHODH expression promotes cell proliferation via stabilizing β-catenin in esophageal squamous cell carcinoma

“…DHODH polymorphism was reported to be linked with rheumatoid arthritis and lung cancer as well 12 – 14 . Additionally, enhanced activity of DHODH has been implicated as a biomarker of malignant tumor including gastric cancer and skin cancer 15 , 16 . Ultraviolet radiation could transcriptionally induce DHODH expression by activating STAT3 17 .…”