Orotate phosphoribosyltransferase levels measured by a newly established enzyme‐linked immunosorbent assay in gastric carcinoma

Abstract

A number of enzymes have been shown to be involved in the process of activation and/or degradation of 5-fluorouracil (5-FU), and are potential candidates for predicting chemosensitivity to 5-FU. Among these, orotate phosphoribosyltransferase (OPRT EC 2.4.2.10) is a key enzyme related to the first-step activation process of 5-FU and has been shown to be an important enzyme that helps to predict sensitivity to 5-FU and its related derivatives. We developed a new enzyme-linked immunosorbent assay (ELISA) to accurately assess intratumoral activity of OPRT. A new sandwich ELISA was established using anti-OPRT polyclonal antibodies obtained from the rabbit immunized with the recombinant human peptides of the OPRT molecule. OPRT levels were measured in eight human cancer xenografts and in 75 gastric cancer tissues using both a newly established ELISA and a conventional enzyme assay, using radiolabeled 5-FU as a substrate. There was a significant correlation between OPRT levels measured by this ELISA and OPRT enzyme activity the in eight human cancer xenografts (r 2 = 0.782) and gastric carcinoma tissue (r 2 = 0.617). The ELISA system for OPRT requires a minimal amount of carcinoma tissue, making it an easy-to-use assay system to predict sensitivity to 5-FU and its derivatives in gastric carcinoma. There was a significant correlation between tumor growth inhibition rates against the oral administration of oral-uracil/tegafur (UFT) and OPRT enzyme activity in the human cancer xenografts (r 2 = 0.574). These results suggest that this newly developed sandwich ELISA system for the quantification of OPRT levels is technically simple, feasible and a useful tool to predict sensitivity to fluoropyrimidine-based anticancer chemotherapy in patients with gastric carcinoma and other cancers. A lthough 5-fluorouracil (5-FU) and its derivatives have been important anticancer agents and have been widely applied for patients with advanced gastrointestinal cancers, the overall response rate of 5-FU alone is still at best 10 -20% for gastric carcinoma.(1) Mechanisms of the anticancer effect, metabolic pathway and the enzymes involved in the metabolic process of 5-FU have been studied extensively (Fig. 1). A variety of attempts have been made to enhance anticancer effects and to reduce toxic effects by modulating enzymes involved in the metabolic pathway of 5-FU.(2) Some of the enzymes involved in the metabolic process of 5-FU, including thymidylate synthase and dihydropyrimidine dehydrogenase, have been shown to predict sensitivity to 5-FU and/or prognosis. (3,4) Based on the fact that the inhibitor of dihydropyrimidine dehydrogenase (DPD) has been shown to enhance the anticancer effects of 5-FU, (2) DPD-inhibitory fluoropyrimidines (DIF) have been developed to enhance anticancer effects.(2) To date, DIFs have played a major role in neoadjuvant and/or adjuvant chemotherapy for patients with advanced gastric carcinoma. (5) 5-FU administered in vivo is taken up by cancer cells and is phosphorylated to 5-fluorouridine-5′-monophosphate (FUMP...

Levels and expressions of orotate phosphoribosyltransferase in gastric carcinoma and normal gastric mucosa tissues

“…OPRT levels were signifi cantly higher in gastric carcinoma tissue than in normal gastric mucosa. These results, along with our previous observations [11], suggest that OPRT plays an important role in the activation of fl uoropyrimidines in carcinoma tissues and that the OPRT level in gastric carcinoma tissues is an independent variable that can predict sensitivity to dihydropyrimidine dehydrogenase-inhibitory fl uoropyrimidines (DIFs). Evidence for a signifi cant anticancer effect of S-1 was shown by the recent interim analysis of adjuvant chemotherapy for gastric cancer with S-1 (ACTS-GC), randomized phase III multi-institutional clinical trial, as presented at the 2007 Gastrointestinal Cancers Symposium (ASCO-GI).…”

“…We showed previously that a signifi cant correlation existed between OPRT levels and OPRT enzymatic activity [11]; therefore, the OPRT level determined by the ELISA directly refl ects enzymatic activity. The ELISA is described in detail elsewhere [11]. Briefl y, the assay was performed with anti-OPRT-A and anti-OPRT-C antibodies as the catcher and the tracer, respectively.…”

“…A recently established sandwich-type ELISA [11][12][13] was used to measure OPRT levels in gastric carcinoma tissue and normal gastric mucosa. We showed previously that a signifi cant correlation existed between OPRT levels and OPRT enzymatic activity [11]; therefore, the OPRT level determined by the ELISA directly refl ects enzymatic activity.…”

“…We previously showed that sensitivity to a DIF such as UFT (Taiho Pharmaceutical, Tokyo, Japan) was correlated with OPRT levels in human cancer xenografts [11]; therefore, OPRT may be an important enzyme for determining the anticancer effects of DIFs and for predicting the postoperative survival of patients with gastric carcinoma. We recently established an enzyme-linked immunosorbent assay (ELISA) that uses antibodies against the OPRT molecule to measure tissue OPRT levels [11][12][13]. The assay allows us to process large numbers of tissue samples simultaneously, with minimal amounts of tissue [11].…”

“…We recently established an enzyme-linked immunosorbent assay (ELISA) that uses antibodies against the OPRT molecule to measure tissue OPRT levels [11][12][13]. The assay allows us to process large numbers of tissue samples simultaneously, with minimal amounts of tissue [11]. Our preAbstract Background.…”