Overexpression of the Orotate Phosphoribosyl-Transferase Gene Enhances the Effect of 5-Fluorouracil on Gastric Cancer Cell Lines

Abstract

Objective: Orotate phosphoribosyl-transferase (OPRT) is the initial enzyme of the 5-fluorouracil (5-FU) metabolic pathway, converting 5-FU into 5-fluorouridinemonophosphate, which is the most important mechanism of 5-FU activation. We therefore investigated whether overexpression of the OPRT gene enhances sensitivity to 5-FU. Methods: An expression vector of the OPRT gene (pTARGET-OPRT) was transfected into two gastric cancer cell lines, TMK-1 and MKN-45, with low baseline expression levels of OPRT. The sensitivity to and anti-tumor activity of 5-FU were then investigated in vitro and in vivo in these two transfected clones (TMK-OPRT and MKN-OPRT). Results: Although cell growth was unaltered compared to parent cells, overexpression of the OPRT gene was confirmed by Western blotting in both the TMK-OPRT and MKN-OPRT cells. OPRT enzyme activity increased 38-fold in TMK-OPRT cells and 8.0 fold in MKN-OPRT cells compared to their parent cells. Interestingly, although the sensitivity to Adriamycin, cis-platinum, mitomycin C and paclitaxel was unaltered in the transfected clones, the sensitivity to 5-FU was increased 14.2- and 6.0-fold in TMK-OPRT and MKN-OPRT cells, respectively, compared to their parent cells. Moreover, enhanced sensitivity was also confirmed in the in vivo study. Conclusion: The results indicate that overexpression of the OPRT gene plays an important role in the antiproliferative effect of 5-FU and might therefore be a predictive factor of response to 5-FU in gastric cancer patients.

Overexpression of the Orotate Phosphoribosyl-Transferase Gene Enhances the Effect of 5-Fluorouracil in Head and Neck Squamous Cell CarcinomaIn Vitro

“…Inaba et al reported a significantly low activity of OPRT in the 5-FU-resistant cell line by measuring the enzyme activities in the human cancer cell lines, [11]. Taomoto et al also indicated that OPRT overexpression plays an important role in the increased sensitivity of gastric carcinomas to 5-FU chemotherapy [4]. The present result is compatible with those of the earlier studies.…”

In vivo evidence for a significant role of folylpolyglutamate synthase in combined chemotherapy with oral fluoropyrimidine, UFT or S-1, and leucovorin

“…tumors were placed in 3-time volumes of ice-cold 10 mM tris-Hcl buffer (pH 7.4), containing 1 mM eDtA and 0.5 mM Dtt and homogenized. oprt activity was determined using a previously described 5-FU phosphorylation assay (14). the supernatant of tumor tissue homogenates was incubated with 20 µM [6-14 c] 5-FU; 50 mM tris-Hcl, pH 8.0; 5 mM Mgcl 2 ; 10 mM naF; 0.5 mM DTT and 4 mM phosphoribosylpyrophosphate at 37˚C for 10 min.…”

5-Fluorouracil derivatives: a patent review (2012 – 2014)

“…Low intratumor 5-FU levels and its high degradation depend on dihydropyrimidine dehydrogenase (DPYD) activity, an enzyme responsible of drug catabolism [36]. On the other hand, it is important to highlight the key role of an anabolic enzyme, the orotate phosphoribosyltransferase (OPRT), which metabolizes 5-FU to 5-fluorouridine monophosphate in the presence of 5-phosphoribosyl-1-pyrophosphate [37]. In the patent WO2011/052554 [38], inventors claim the discovery of a series of compounds with a DPYD and OPRT inhibitory activity, achieving a decrease in 5-FU gastrointestinal damage and powerful antitumor effects ( Figure 1B).…”