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 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.

The 4-TFMBAO fluorogenic reagent selectively reacted with the orotic acid product at 80 °C for 4 min in the presence of K 3 [Fe(CN) 6 ] and K 2 CO 3 (pH [10][11][12], and provided a strong FL compound for orotic acid, but not for the large amount of DHO substrate or other biogenic substances such as nucleobases, nucleosides, nucleotides, amino acids, vitamins, or sugars 22 . Thus, the present assay comprises the enzymatic reaction of DHO with DHODH, followed by the FL chemical reaction of the produced orotic acid with 4-TFMBAO.
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.

Conditions for assay of DHODH activity in HeLa cells.
To optimise the assay conditions, cell lysate was used as the DHODH source. The effects of buffers on DHODH activity were investigated: pH 7.0-9.5, 50-250 mM K 2 CO 3 -HCl, and 50-200 mM Tris-HCl (Fig. 3). For 200 mM K 2 CO 3 -HCl (Fig. 3a), the FL intensity due to the orotic acid product was highest at a pH of between 7.0 and 8.5 (Fig. 3b). As previously reported 22 , the 4-TFMBAO reagent produced the highest FL intensity for orotic acid in the presence of 10-40 mM K 2 CO 3 (pH ca. 11). Therefore, 200 mM K 2 CO 3 -HCl (pH 8.0) was chosen for the DHODH reaction and a final K 2 CO 3 concentration of 40 mM (pH 11) was used for the FL reaction.
The enzymatic reaction was performed at 37 °C for 1.0 h to produce orotic acid from the DHO substrate. Figure 4a shows the effect of substrate concentration on DHODH activity and the Km value for the DHODH enzyme. Based on this result, a sufficient concentration (500 μ M) of DHO could be selected for the present DHODH assay.
DHODH activity per 1.0 × 10 5 cells was expressed as the amount (pmol) of orotic acid produced in 1.0 h. Cell numbers were normalised to 1.0 × 10 5 cells in each DHODH reaction to account for differences in sample size. Usually, 2.0-3.0 × 10 5 cells were used per assay. To measure enzyme activity, we determined the relationship between cell number and amount of orotic acid produced in a 1.0-mL reaction mixture (Fig. 4b). For the calculation of the production, the preexisting level of orotic acid at non-incubation time was subtracted from total amount of orotic acid observed at the end of the hour incubation. DHODH activity was proportional to the cell number (0.5-3.0 × 10 5 cells). Thus, the specific activity of DHODH in the HeLa cells was 323 pmol/10 5 cells/h. DHODH activity in HeLa and fibroblast cells. FL intensity due to orotic acid increased with increasing incubation time (Fig. 5a) when either HeLa or fibroblast cells as the DHODH source were used. The specific activity of DHODH in HeLa and fibroblast cells was measured (Fig. 5b). The specific activity of DHODH was approximately 6 times higher in HeLa cells (340 ± 25.9 pmol/10 5 cells/h) than in fibroblasts (54.1 ± 7.40 pmol/10 5 cells/h).
We also investigated the relationship between the endogenous orotic acid content (Fig. 5c) and DHODH activity in HeLa and fibroblast cells. Endogenous orotic acid concentration was 130 ± 9.72 pmol/10 5 for HeLa cells and 151 ± 18.6 pmol/10 5 for fibroblasts (n = 5 each). Endogenous orotic acid levels were similar, indicating that DHODH activity is significantly different in these cell types.
DHODH activity in malignant tumour and adjacent normal tissues. We measured DHODH activity and endogenous orotic acid concentration in malignant stomach tumour and adjacent normal tissue from a patient with stage III cancer (Fig. 6). Tissue samples were normalised by measuring the total protein concentration to avoid variations in wet tissue weight. The total proteins in each reaction mixture were adjusted to ca. 0.75 mg.
Orotic acid was enzymatically produced in the reaction using both cancer and adjacent normal tissue samples (Fig. 6a). The FL intensity due to orotic acid increased according to the incubation time, with a similar profile to that of cultured cells. Figure 6b shows the specific activity of DHODH in malignant tumour and matched normal tissues (n = 5 each). The specific activity of DHODH in the malignant tumour tissue (1.10 ± 0.19 nmol/mg total proteins/h) was approximately 5 times higher than that in adjacent normal tissue (0.24 ± 0.11 nmol/mg total proteins/h). However, the concentration of endogenous orotic acid was similar in both malignant tumour (1.09 ± 0.16 nmol/mg total proteins) and adjacent normal (1.28 ± 0.19 nmol/mg total proteins) tissues (Fig. 6c). Comparison with other assay method. Enzyme activity of recombinant DHODH has been measured for the evaluation of synthetic inhibitors of DHODH by a conventional colourimetric method [18][19][20] . Figure 7a shows the principle of the coluorimetric assay method. In the method, DHO is oxidized by the enzymatic reaction, and then reduction of DCPIP is occurred. Thus, absorbance of DCPIP in the reaction mixture should be decreased by the DHODH activity. As the results (Fig. 7b) for the assay of endogenous DHODH activity, however, DCPIP in the reaction mixture did not show the decrease of its absorbance at 610 nm depending on the incubation time. It means that DCPIP could not be reduced by the enzymatic reaction. It was thus suggested that the reductive efficiency for DCPIP was inhibited by some oxidants co-existing in the sample lysates 21 . This colourimetric assay method may require purification in advance of DHODH from the complex lysate samples. On the contrary, the present FL assay method can measure the DHODH activity in the lysates without purification of the enzyme.

Discussion
We have developed an alternative assay method for DHODH activity in biological samples, including cultured HeLa cells and fibroblasts, and normal and malignant human stomach tissues. The assay utilised a previously reported FL reaction 22 , with 4-TFMBAO as a fluorogenic reagent. This reagent is non-FL and provided a selective, strong FL intensity for both endogenous orotic acid and enzymatically produced orotic acid under the chemical reaction conditions at 80 °C for 4.0 min in aqueous alkaline solution.
The present assay method enabled the specific activity of DHODH to be determined in the biologically complex samples. DHODH activity was apparently higher in cultured HeLa cells than in fibroblasts (Fig. 5), and also significantly higher in malignant tumour tissue than in adjacent normal tissue (Fig. 6). On the other hand, a protein, survivin has been studied for its role as a prognostic biomarker in cancer therapy [23][24][25] . Significantly higher expression of survivin was reported in gastric tumor tissues than in its adjacent normal tissues in both of its mRNA (ca. 2 folds) and protein (ca. 4 folds) levels 25 . In our research, the specific activity of DHODH in the gastric  tumor (stage III) tissue was approximately 5 folds higher than that in the adjacent normal tissue. Therefore, DHODH may become a potential biomarker of cancer.
Several DHODH inhibitors have been suggested to have anti-cancer effects 16,17 . Our data suggest that the anti-cancer effect of DHODH inhibitors might be associated with the higher level of DHODH activity in cancer cells. Large amounts of pyrimidines are required for the rapid proliferation of cancer cells and/or malignant tissues. Thus, the pyrimidine biosynthetic pathway is activated, resulting in high DHODH expression in these cells; however, endogenous orotic acid might be rapidly metabolised to UMP 1,26 .
Further diagnostic studies are necessary to evaluate DHODH activity as a general cancer biomarker in other biological samples such as bloods and tissues from patients with different cancer types and different stages. The newly developed, convenient DHODH assay will technically contribute to these research approaches. Human tissues. Tumour and adjacent normal tissues from a patient with stage III stomach cancer were purchased from Tissue Solutions Ltd (Glasgow, UK). The supplier declared that the tissues have been obtained according to the legal and ethical requirements with the approval of an ethics committee and with anonymous consent from the donor or nearest relative. All experiments in this study were carried out in accordance with our university's ethical guidelines.
Preparation of cell and tissue lysates. Cells were counted and lysed in water (10 6 cells/mL) at 4 °C by sonication for 10 min. Lysates were clarified at 16000 g for 20 min. Wet tissue was cut into tiny pieces and homogenised with a glass homogeniser (ca. 60 mg wet weight in 1.0 mL of H 2 O) for 10 min, followed by sonication for 10 min. The homogenate was clarified at 16000 g for 20 min. Lysates were used in the DHODH reaction and for determining endogenous orotic acid levels.

Enzyme reaction of DHODH and FL detection of orotic acid for the assay of DHODH activity.
Lysate (300 μ L) was incubated in an aqueous solution (total volume, 1.    Data analysis. Statistical analysis was performed using two-tailed Student's t test. P < 0.05 was considered to be significant.