Orotate Phosphoribosyltransferase Expression Research Articles

Mechanism of acquired 5FU resistance and strategy for overcoming 5FU resistance focusing on 5FU metabolism in colon cancer cell lines.

Fluorouracil (5FU) is converted to its active metabolite fluoro-deoxyuridine monophosphate (FdUMP) through the orotate phosphoribosyl transferase (OPRT)-ribonucleotide reductase (RR) pathway and thymidine phosphatase (TP)-thymidine kinase (TK) pathway and inhibits thymidylate synthase (TS), leading to inhibition of thymidine monophosphate (dTMP) synthesis through a de novo pathway. We investigated the mechanism of 5FU resistance and strategies to overcome it by focusing on 5FU metabolism. Colon cancer cell lines SW48 and LS174T and 5FU-resistant cell lines SW48/5FUR and LS174T/5FUR were used. FdUMP amount was measured by western blotting. The FdUMP synthetic pathway was investigated by combining TP inhibitor (tipiracil hydrochloride; TPI) or RR inhibitor (hydroxyurea; HU) with 5FU. Drug cytotoxicity was observed by crystal violet staining assay. FdUMP was synthesized through the OPRT-RR pathway in SW48 cells but was scarcely synthesized through either the OPRT-RR or TP-TK pathway in SW48/5FUR cells. FdUMP amount in SW48/5FUR cells was reduced by 87% vs. SW48 cells. Expression levels of OPRT and TP were lower in SW48/5FUR when compared with these levels in the SW48 cells, indicating decreased synthesis of FdUMP-led 5FU resistance. These results indicated that fluoro-deoxyuridine (FdU) rather than 5FU promotes FdUMP synthesis and overcomes 5FU resistance. Contrastingly, FdUMP was synthesized through the OPRT-RR and TP-TK pathways in LS174T cells but mainly through the TP-TK pathway in LS174T/5FUR cells. FdUMP amount was similar in LS174T/5FUR vs. the LS174T cells. OPRT and RR expression was lower and TK expression was higher in LS174T/5FUR vs. the LS174T cells, indicating that dTMP synthesis increased through the salvage pathway, thus leading to 5FU resistance. LS174T/5FUR cells also showed cross-resistance to FdU and TS inhibitor, suggesting that nucleoside analogs such as trifluoro-thymidine should be used to overcome 5FU resistance in these cells. 5FU metabolism and mechanisms of 5FU resistance are different in each cell line. Both synthesized FdUMP amount and FdUMP sensitivity should be considered in 5FU-resistant cells.

5-Fluorouracil-based adjuvant chemotherapy improves the clinical outcomes of patients with lymphovascular invasion of upper urinary tract cancer and low expression of dihydropyrimidine dehydrogenase.

Lymphovascular invasion (LVI) by urothelial carcinoma of the upper urinary tract (UC-UUT) is associated with an unfavorable prognosis. However, a high proportion of patients with UC-UUT are unable to receive the recommended doses of cisplatin-based adjuvant chemotherapy due to advanced age or renal dysfunction resulting from nephroureterectomy. Tegafur-uracil is an oral form of 5-fluorouracil whose efficacy is influenced by the activities of enzymes associated with its metabolism, such as dihydropyrimidine dehydrogenase (DPD), orotatephosphoribosyltransferase (OPRT) and thymidylate synthase (TS). The aim of the present study was to investigate the efficacy of adjuvant 5-fluorouracil chemotherapy for UC-UUT with LVI, and to assess the expression of enzymes associated with 5-fluorouracil metabolism as promising biomarkers of therapy efficacy. The present study retrospectively investigated 52 cases of UC-UUT. Following nephroureterectomy, tegafur-uracil was administered to 15 out of 30 patients with LVI who were not eligible for cisplatin-based adjuvant chemotherapy. Levels of DPD, OPRT and TS expression in tumor specimens were determined by reverse transcription-quantitative polymerase chain reaction, and their associations with the efficacy of adjuvant 5-fluorouracil chemotherapy were analyzed. The levels of DPD, OPRT and TS expression were not associated with pathological factors or outcome, although a higher expression of TS was associated with a poorer outcome. Adjuvant 5-fluorouracil chemotherapy significantly improved the outcome of patients with lower DPD expression. However, the levels of OPRT and TS expression did not influence therapeutic efficacy. Adjuvant 5-fluorouracil chemotherapy appears to be effective for lymphovascular-invasive UC-UUT in patients with lower DPD expression.

Orotate phosphoribosyltransferase as a predictor of benefit from S‐1 adjuvant chemotherapy for cholangiocarcinoma patients

To improve the prognosis of cholangiocarcinoma, we investigated potential biomarkers that may enable the selection of patients for whom postoperative adjuvant chemotherapy is likely effective. The cohort of this retrospective study included 170 surgically resected cholangiocarcinoma patients, 26 with gemcitabine adjuvant chemotherapy (GEM group), 36 with S-1 adjuvant chemotherapy (S-1 group), and 103 receiving no adjuvant chemotherapy (NC group). Propensity score matching was performed to adjust patient backgrounds; 36 patients from the NC group then were selected. Immunohistochemistry of orotate phosphoribosyltransferase (OPRT) and human equilibrative nucleoside transporter 1 (hENT1) was performed to determine the correlation between their expression and disease-free survival (DFS). After matching, the backgrounds of these three groups were unbiased. No significant improvement of DFS by adjuvant chemotherapy was observed in the whole cohort. However, among the high-OPRT-expression patients, DFS of GEM, S-1, and NC groups at 5years was 28.8%, 53.8%, and 25.5%, respectively. The DFS of the S-1 group was significantly longer than that of the NC group (P=0.034). On the other hand, no significant differences in DFS were observed among the low OPRT expression patients. hENT1 expression was shown to have no predictive value. Multivariate analysis of the high-OPRT-expression patients demonstrated that S-1 adjuvant chemotherapy can reduce tumor recurrence (HR, 0.303; P=0.013). Cholangiocarcinoma patients with high OPRT expression would benefit from postoperative adjuvant S-1 therapy, which increases the DFS. Assessment of OPRT expression may contribute to the optimization of adjuvant chemotherapy for cholangiocarcinoma.

Expression of thymidylate synthase predicts clinical outcomes of S-1-based chemotherapy in squamous cell lung cancer.

Non-small cell lung cancer (NSCLC) patients with squamous cell carcinoma (SCC) histology have limited chemotherapeutic options. Treatment with S-1 combined with carboplatin (CBDCA) has been shown to provide a significant survival benefit in SCC patients compared with treatment with combined CBDCA and paclitaxel. The aim of the present study was to investigate the association between the expression of molecular markers related to the pharmacological action of S-1, including thymidylate synthase (TS), orotate phosphoribosyltransferase (OPRT) and dihydropyrimidine dehydrogenase (DPD), and the clinical efficacy of S-1-based chemotherapy in SCC patients. The immunohistochemical expression of TS, OPRT and DPD were retrospectively analyzed in tumor biopsy and resection specimens from patients with advanced SCC (n=32). Immunohistochemical H-scores were calculated and their association with S-1/CBDCA response was evaluated. Median progression-free survival time was significantly longer in patients with low TS H-scores than in those with high TS H-scores (162.5 vs. 97 days; P=0.004); by contrast, overall survival time was not observed to differ significantly between these groups (P=0.185). In the multivariate analysis, low TS expression was a significant positive factor for progression-free survival rate (hazard ratio, 0.40; P=0.021). A low TS H-score was also associated with an increased response to S-1-based chemotherapy compared with a high TS H-score (P=0.002). This indicates that SCC patients with low TS expression can benefit significantly from S-1-based chemotherapy, and that H-score measurement of intratumoral TS expression may represent a useful predictive biomarker for response to S-1-based chemotherapy by patients with SCC-type NSCLC.

P3.03-026 Orotate Phosphoribosyltransferase is Overexpressed in Malignant Pleural Mesothelioma: Dramatically Responds One Case in High OPRT Expression: Topic: Mesothelioma Transitional

Malignant pleural mesothelioma (MPM) is a rare and aggressive, treatment-resistant cancer. Pemetrexed, an inhibitor of thymidylate synthase (TS), is used worldwide for MPM as a first-line chemotherapy regimen. However, there is little consensus for a second-line chemotherapy. S-1, a highly effective dihydropyrimidine dehydrogenase (DPD)-inhibitory fluoropyrimidine, mainly acts via a TS inhibitory mechanism similar to pemetrexed. Orotate phosphoribosyltransferase (OPRT) is a key enzyme related to the first step activation of 5-fluorouracil (5-FU) for inhibiting RNA synthesis. We investigated 5-FU related-metabolism proteins, especially focusing on OPRT expression, in MPM. Fifteen MPM patients who were diagnosed between July 2004 and December 2013 were enrolled. We examined the protein levels of 5-FU metabolism-related enzymes (TS, DPD, OPRT, and thymidine phosphorylase [TP]) in 14 cases. High TS, DPD, OPRT, and TP expressions were seen in 28.6%, 71.4%, 85.7%, and 35.7% of patients, respectively. We found that OPRT expression was extremely high in MPM tissue. We experienced one remarkable case of highly effective S-1 combined therapy for pemetrexed refractory MPM. This case also showed high OPRT protein expression. The present study suggests that OPRT expression is high in MPM tumors. Although pemetrexed is mainly used for MPM chemotherapy as a TS inhibitor, S-1 has potential as an anticancer drug not only as a TS inhibitor but also inhibiting RNA synthesis through the OPRT pathway. This is the first report investigating OPRT protein expressions in MPM.

High/positive expression of 5-fluorouracil metabolic enzymes predicts better response to S-1 in patients with gastric cancer: a meta-analysis.

To provide an assessment by meta-analysis of the relationship between the expression variations of 5-fluorouracil metabolic enzymes and clinical outcomes in patients with gastric cancer treated with S-1. Databases were searched electronically from inception to April 19th, 2015. Studies in gastric cancer patients treated with S-1 investigating the expression variations of 5-fluorouracil metabolic enzymes were included after having been identified systematically. Pooled odds ratios (OR) for the objective response rate (ORR) and median survival ratio were calculated using the Review Manager 5.3 and Stata 12.0 software separately. A total of 555 patients in 10 studies met our inclusion criteria. There was a significant difference in ORR between patients with high/+ and low/- expression of orotate phosphoribosyl transferase (OPRT) (OR = 8.06; 95% CI, 4.06-16.02; p<0.001) and dihydropyrimidine dehydrogenase (DPD) (OR = 1.95; 95% CI, 1.21-3.13; p = 0.006). There was no significant difference in ORR between different expression levels of thymidylate synthase (TS) and thymidine phosphorylase (TP). Although patients with low/- TS expression, low/- TP expression and high/+ DPD expression showed a trend towards longer survival, no statistical significance was found. The median OS was significantly longer in patients with high/+ expression of OPRT (p = 0.076). OPRT and DPD expression can be treated as a potential predictive biomarker for S-1 response in gastric cancer patients. Further investigation is warranted.

Orotate phosphoribosyltransferase is overexpressed in malignant pleural mesothelioma: Dramatically responds one case in high OPRT expression.

ABSTRACTObjective: Malignant pleural mesothelioma (MPM) is a rare and aggressive, treatment-resistant cancer. Pemetrexed, an inhibitor of thymidylate synthase (TS), is used worldwide for MPM as a first-line chemotherapy regimen. However, there is little consensus for a second-line chemotherapy. S-1, a highly effective dihydropyrimidine dehydrogenase (DPD)-inhibitory fluoropyrimidine, mainly acts via a TS inhibitory mechanism similar to pemetrexed. Orotate phosphoribosyltransferase (OPRT) is a key enzyme related to the first step activation of 5-fluorouracil (5-FU) for inhibiting RNA synthesis. We investigated 5-FU related-metabolism proteins, especially focusing on OPRT expression, in MPM Methods and Patients: Fifteen MPM patients who were diagnosed between July 2004 and December 2013 were enrolled. We examined the protein levels of 5-FU metabolism-related enzymes (TS, DPD, OPRT, and thymidine phosphorylase [TP]) in 14 cases Results: High TS, DPD, OPRT, and TP expressions were seen in 28.6%, 71.4%, 85.7%, and 35.7% of patients, respectively. We found that OPRT expression was extremely high in MPM tissue. We experienced one remarkable case of highly effective S-1 combined therapy for pemetrexed refractory MPM. This case also showed high OPRT protein expression Conclusion: The present study suggests that OPRT expression is high in MPM tumors. Although pemetrexed is mainly used for MPM chemotherapy as a TS inhibitor, S-1 has potential as an anticancer drug not only as a TS inhibitor but also inhibiting RNA synthesis through the OPRT pathway. This is the first report investigating OPRT protein expressions in MPM.

Orotate phosphoribosyltransferase localizes to the Golgi complex and its expression levels affect the sensitivity to anti-cancer drug 5-fluorouracil.

Orotate phosphoribosyltransferase (OPRT) is engaged in de novo pyrimidine synthesis. It catalyzes oronitine to uridine monophosphate (UMP), which is used for RNA synthesis. De novo pyrimidine synthesis has long been known to play an important role in providing DNA/RNA precursors for rapid proliferative activity of cancer cells. Furthermore, chemotherapeutic drug 5-fluorouracil (5-FU) is taken up into cancer cells and is converted to 5-fluoro-UMP (FUMP) by OPRT or to 5-fluoro-dUMP (FdUMP) through intermediary molecules by thymidine phosphorylase. These 5-FU metabolites are misincorporated into DNA/RNA, thereby producing dysfunction of these information processing. However, it remains unclear how the subcellular localization of OPRT and how its variable expression levels affect the response to 5-FU at the cellular level. In this study, immunocytochemical analysis reveals that OPRT localizes to the Golgi complex. Results also show that not only overexpression but also downregulation of OPRT render cells susceptible to 5-FU exposure, but it has no effect on DNA damaging agent doxorubicin. This study provides clues to elucidate the cellular response to 5-FU chemotherapy in relation to the OPRT expression level.

Prognostic significance of thymidylate synthase in postoperative non-small cell lung cancer patients.

The aim of the present study was to investigate the clinicopathologic/prognostic significance of thymidylate synthase (TS), orotate phosphoribosyltransferase (OPRT), and thymidine phosphorylase (TP) proteins in postoperative non-small cell lung cancer (NSCLC) patients. Microarray slides from a set of 178 NSCLC patients were used for the detection of TS, OPRT, and TP expression by immunohistochemistry. The correlation between clinicopathologic factors and protein expression of three proteins was analyzed. Ninety seven carcinomas (57.4%) were TS-positive, 90 carcinomas (53.9%) were OPRT-positive, and 102 carcinomas (69.4%) were TP-positive. Compared with the TS-positive patients, the overall survival (OS) was significantly lower in the TS-negative patients (hazard ratio [HR] =1.766, 95% confidence interval [CI] =1.212–2.573, P=0.003). Significant differences between TS-positive and TS-negative patients was also observed in the following stratified analyses: 1) adenocarcinoma subgroup (HR =2.079, 95% CI =1.235–3.500, P=0.006); 2) less than 60-year-old subgroup (HR =1.890, 95% CI =1.061–3.366, P=0.031); 3) stage II/III subgroup (HR =1.594, 95% CI =1.036–2.453, P=0.034); and 4) surgery plus adjuvant therapy subgroup (HR =1.976, 95% CI =1.226–3.185, P=0.005). However, the OS was not significantly correlated with OPRT or TP protein expression. This study demonstrates that the TS level in tumor tissues may be a useful marker to predict the postoperative OS in NSCLC patients.

A COX-2 inhibitor enhances the antitumor effects of chemotherapy and radiotherapy for esophageal squamous cell carcinoma

Cyclooxygenase-2 (COX-2) is a key enzyme of prostaglandin (PG) synthesis that has been demonstrated to be overexpressed in several types of cancers. The function of COX-2 in tumor progression has been recently elucidated. In tumors in which COX-2 is overexpressed, the antitumor effects are suppressed. We examined the effects of celecoxib, a COX-2 inhibitor, in enhancing the antitumor effects of chemotherapy and radiotherapy for esophageal squamous cell carcinoma (ESCC) by reducing the COX-2 activity. We used the human esophageal squamous cell lines TE2 and T.Tn treated with celecoxib and 5-FU/radiation, after which cell viability assays were performed. Changes in the expressions of dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT) mRNA and PGE2 were also measured. In addition, apoptotic changes, and the invasion and migration activity in both the celecoxib and 5-FU treated cells were evaluated. The experiments showed that T.Tn and TE2 proliferation was strongly inhibited by the combination of 5-FU/radiation and the COX-2 inhibitor. Inhibiting the COX-2 activity induced a reduction in PGE2 levels in TE2/T.Tn cells. Following treatment with the COX-2 inhibitor and 5-FU, the OPRT expression was upregulated and the DPD expression was downregulated in the resistant cells. In addition, the combination treatment with the COX-2 inhibitor and 5-FU markedly inhibited both the cell invasion and migration activity. Therefore, COX-2 inhibitors can be useful enhancers of antitumor drugs and radiotherapy for ESCC.

Cinnamaldehyde/chemotherapeutic agents interaction and drug-metabolizing genes in colorectal cancer

Cinnamaldehyde is an active monomer isolated from the stem bark of Cinnamomum cassia, a traditional oriental medicinal herb, which is known to possess marked antitumor effects in vitro and in vivo. The aim of the present study was to examine the potential advantages of using cinnamaldehyde in combination with chemotherapeutic agents commonly used in colorectal carcinoma (CRC) therapy, as well as to investigate the effect of cinnamaldehyde on chemotherapeutic-associated gene expression. The synergistic interaction of cinnamaldehyde and chemotherapeutic agents on human CRC HT-29 and LoVo cells was evaluated using the combination index (CI) method. The double staining with Annexin V conjugated to fluorescein-isothiocyanate and phosphatidylserine was employed for apoptosis detection. The expression of drug-metabolizing genes, including excision repair cross‑complementing 1 (ERCC1), orotate phosphoribosyltransferase (OPRT), thymidylate synthase (TS), breast cancer susceptibility gene 1 (BRCA1) and topoisomerase 1 (TOPO1), all in HT-29 and LoVo cells, with or without the addition of cinnamaldehyde, was examined by quantitative polymerase chain reaction (PCR). Cinnamaldehyde had a synergistic effect on the chemotherapeutic agents cytotoxicity in HT-29 and LoVo cells. In addition, cinnamaldehyde suppressed BRCA1, TOPO1, ERCC1 and TS mRNA expression, except for OPRT expression, which was markedly upregulated. Our findings indicate that cinnamaldehyde appears to be a promising candidate as an adjuvant in combination therapy with 5-fluorouracil (5-FU) and oxaliplatin (OXA), two chemotherapeutic agents used in CRC treatment. The possible mechanisms of its action may involve the regulation of drug‑metabolizing genes.

Substitution of anti-androgens and tegafur-uracil combination therapy for castration-resistant prostate cancer: Results of a multi-center randomized phase II study

We conducted this study to determine whether substitution with anti-androgen (SOA) and tegafur-uracil (a pro‑drug of 5-FU) combination therapy is more effective than SOA alone after relapse from initial hormonal therapy. Patients who were histologically confirmed and relapsed after initial hormonal therapy were included. All patients were randomly allocated into two groups: SOA alone (group A) or SOA combined with tegafur-uracil (group B). The mRNA expression of four enzymes, including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phospho-ribosyltransferase (OPRT) and thymidine phosphorylase (TP), in prostate cancer cells was analyzed by quantitative reverse-transcription polymerase chain reaction. Fifty-two patients were enrolled in this study. The median age was 77 (range: 47-92) years. The PSA response rate in group B (61.5%) tended to be higher compared to that in group A (34.6%) (p=0.095). Group B (median: 15.9 months) had a significantly longer time to PSA progression (TTP) compared to group A (6.4 months) (p=0.014). In patients with a lower TS expression or a higher OPRT expression, group B demonstrated a higher PSA response rate compared to group A (p=0.019 and p=0.041, respectively). In addition, in the patients with a lower TS expression, group B demonstrated a significantly longer TTP compared to group A (p=0.018). There were no severe adverse events in either treatment group. After relapse from initial hormonal therapy, SOA combined with tegafur-uracil is effective and well tolerated. The TS mRNA expression level may be a predictive factor for this combination therapy.

Predictive value of orotate phosphoribosyltransferase in colorectal cancer patients receiving 5-FU-based chemotherapy

Pretreatment knowledge of chemosensitivity and side-effects of chemotherapy for colorectal cancer (CRC) patients are likely to ensure the best chemotherapeutic outcome. The aim of this study was to identify additional predictive factors of chemosensitivity to the key CRC treatment drug 5-fluorouracil (5-FU). Surgically obtained specimens from 106 patients treated for CRC were immunohistochemically assessed to investigate the correlation between the protein expression of the 5-FU metabolic enzymes orotate phosphoribosyltransferase (OPRT), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD), and clinicopathological characteristics as well as the correlation between the protein expression and outcomes of 5-FU-based chemotherapy. A correlation was detected between the high expression of the 5-FU metabolic enzyme OPRT and negative lymph node metastasis (P=0.0496), as well as between DPD and advanced Tumor-Node-Metastasis (TNM) grade cases (IIIA-IVB) and positive lymph node metastases (P=0.0414, respectively). In all 106 patients and in 79 patients undergoing 5-FU-based chemotherapy, survival was improved in those patients with a positive OPRT expression (P=0.0144 and 0.0167, respectively). OPRT expression was higher in the 79 patients with no recurrence (P=0.0179) as well as in patients treated with R0 surgery and 5-FU-based chemotherapy without side-effects (P=0.0126). Disease-free survival (DFS) rate was higher in patients without side-effects, and in patients with a positive OPRT expression without side-effects (P=0.0021 and 0.0031, respectively). Findings of this study demonstrated that OPRT expression positively correlated with fewer side-effects of 5-FU-based chemotherapy and longer patient survival.

Better efficacy of S-1 treatment for thymic carcinoma: case report and review of the literature

Primary thymic carcinoma is a rare tumor of the thymus gland. The role of chemotherapy in treating advanced thymic carcinoma is unclear. It has recently been reported that thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT) expression may be important factors when predicting the effectiveness of 5-fluorouracil-based chemotherapy (Kaira et al., 74:419–425, 2011). In this report, we describe three cases in which S-1 or S-1 combination treatment was given as second-line therapy for thymic carcinoma, and examine the relationships between the immunohistochemical expression of TS, DPD, and OPRT and treatment effectiveness. There were no deaths due to toxicity. Two patients achieved partial remission (PR), and one patient achieved stable disease (SD). These results suggest that S-1 is a safe and promising regimen for thymic carcinoma. Immunohistochemical staining for OPRT expression tended to be stronger in the two PR cases than in the SD case. Based on the present results, it appears that S-1 may be useful for treating unresectable thymic carcinoma, and that OPRT expression may predict the response to S-1 treatment. Further clinical studies are needed to confirm these findings.

Expression of thymidylate synthase and orotate phosphoribosyltransferase in thymic carcinoma

Thymic carcinoma is a rare thymic epithelial tumor in which chemotherapy for advanced disease has not yet been established. Thymidylate synthase (TS) and orotate phosphoribosyltransferase (OPRT) protein expression levels in thymic carcinoma were evaluated as possible indicators of the anticancer activity of 5-fluorouracil (5-FU) drugs using immunohistochemistry (IHC). A total of 24 samples of thymic carcinoma were used in the present study. The tumor sections were immunohistochemically stained for TS and OPRT. As a comparison with thymic carcinoma, we also assessed the TS and OPRT protein expression levels in 55 lung cancer samples. The TS expression was positive in 12 of 24 thymic carcinoma samples (50%) and OPRT expression was positive in 10 (42%). The association between TS and OPRT expression and Masaoka stages of thymic carcinoma was analyzed. The TS and OPRT expressions in stage IV were significantly higher compared to that in stages I, II or III. We also compared the TS and OPRT expression levels between thymic carcinoma and lung cancer (33 adenocarcinomas and 22 squamous cell carcinomas). TS expression in thymic carcinoma was significantly lower compared with lung squamous cell carcinoma. OPRT expression in thymic carcinoma was significantly higher compared to lung adenocarcinoma. The combination of a relatively low expression of TS and high expression of OPRT suggests an improved antitumor effect of 5-FU drugs in thymic carcinoma compared to in lung carcinoma.

Antitumor efficacy of sequential treatment with docetaxel and 5-fluorouracil against human oral cancer cells

Docetaxel (DOC) and 5-fluorouracil (5-FU) are important anticancer agents widely used in the treatment of a variety of cancers including oral squamous cell carcinoma (OSCC). The purpose of this study was to determine the antitumor efficacy of the sequential administration of DOC and 5-FU against OSCC cells (B88 and CAL27 cells) in vitro and in vivo. In in vitro growth inhibition assays, sequential treatment with DOC followed by 5-FU was more effective in inhibiting cancer cell growth than 5-FU followed by DOC, single treatment with DOC or 5-FU, or combined treatment with DOC and 5-FU. Furthermore, DOC followed by 5-FU significantly inhibited tumor growth in vivo compared to 5-FU followed by DOC. To understand the mechanisms underlying the enhanced growth inhibitory effect of the administration sequence, DOC followed by 5-FU, we examined the expression of 5-FU metabolic enzymes such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT), which were known to regulate the antitumor effect of 5-FU, by real-time RT-PCR and western blot analysis. Downregulation of TS and DPD expression and upregulation of OPRT expression were induced by DOC treatment, suggesting that DOC enhanced the efficacy of 5-FU by altering the expression of its metabolic enzymes. These results indicate that sequential treatment with DOC followed by 5-FU could be a promising therapeutic strategy for oral cancer.

Impact of the gene expressions of thymidylate synthase and dihydropyrimidine dehydrogenase on survival in patients enrolled in the ACTS-GC study.

52 Background: The efficacy of 5-FU-based therapy is basically related to the expression of enzymes involved in pyrimidine metabolism, such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyltransferase (OPRT). Biomarker analysis was conducted to evaluate the influence of these expressions on the outcomes of patients enrolled in the ACTS-GC study, a randomized phase III trial, which demonstrated the efficacy of adjuvant treatment with S-1 after D2 dissection for stage II and III gastric cancer (Sakuramoto et al., NEJM 2007) Methods: Formalin-fixed paraffin-embedded specimens were available for 829 of a total of 1,059 (78.3%) patients. Expressions of TS, DPD, TP and OPRT were measured by RT-PCR in macrodissected tumor specimens and normalized to β-actin expression as the reference gene. The expression levels of each gene were categorized as low or high at the 33.3rd or 66.7th percentile. If the p-value of the interaction was less than 0.1, statistical significance was assumed. Results: Expressions of the 4 genes were quantifiable in 97% of the 829 specimens and the distributions were balanced across the arms. The hazard ratio (HR) for overall survival (OS) of S-1 to surgery alone was smaller in the high-TS (highest one-third) (HR, 0.370; 95%CI, 0.221-0.619) than in the low-TS group (lower two-thirds) (HR, 0.757; 95%CI, 0.563-1.018). This interaction between TS expression and OS was statistically significant (p=0.015). Similarly, the HR for OS of S-1 to surgery alone was smaller in the high-DPD (higher two-thirds) (HR, 0.520; 95%CI, 0.376-0.720) than in the low-DPD group (lowest one-third) (HR, 0.848; 95%CI, 0.563-1.276). This interaction (p=0.065) was also statistically significant. There was no interaction between the TP or OPRT expression and OS. Conclusions: This large biomarker study showed that intratumoral TS and DPD gene expressions may be predictive markers of the efficacy of S-1 in gastric cancer patients receiving postoperative adjuvant chemotherapy with S-1.

Comment on “Influence of Orotate Phosphoribosyl Transferase Levels on Tumor Prognosis and Response to Chemotherapy”

I read with great interest the recent article by Yasumatsu et al. [1]. Interestingly, recent data suggests that orotate phosphoribosyl transferase (OPRT) may significantly influence tumor prognosis and response to 5-fluorouracil therapy in a number of systemic malignancies besides head and neck carcinomas. For instance, a close association exists between OPRT levels and prognosis in esophageal malignancies. Early stages such as stage I and stage II express higher levels of orotate phosphoribosyl transferase (OPRT) in contrast to late stages [2]. Hence, OPRT may significantly influence prognosis in esophageal carcinomas. Similarly, increased chances of metastasis following surgery for colorectal carcinomas is seen in primary tumors that show accentuated OPRT expression. An increased risk of hematogeneous metastasis is seen in colorectal tumors with elevated OPRT levels [3]. Similarly, OPRT expression enhances the antineoplastic effects of 5-fluorouracil in gastric carcinomas and markedly improves prognosis in these patients [4]. Similar, relationships are seen in pancreatic malignancies. For instance, OPRT negative tumors are associated with a lower postsurgical resection survival rates in comparison to OPRT-positive pancreatic malignancies [5]. Patients with OPRT-positive tumors who are administered chemotherapy with chemotherapeutic agents such as gemcitabine have higher survival rates in contrast to those who do not receive similar adjuvant therapy. Similarly, there is upregulation of OPRT expression in prostate malignancies. In fact, a close association is seen between grading and tumor OPRT mRNA expression. In addition, hormone-refractory prostate cancers demonstrate a higher OPRT/dihydropyrimidine dehydrogenase (DPD) ratio in contrast to hormone-sensitive prostate cancer [6, 7]. This higher OPRT/DPD ratio explains the higher sensitivity of these tumors to 5-fluorouracil therapy. Similarly, bladder carcinomas demonstrate increased OPRT activity in contrast to normal bladder tissue. In fact, low-grade bladder carcinomas express lower levels of OPRT in contrast to high-grade bladder carcinomas. OPRT expression significantly influences the sensitivity of the tumors to 5-fluorouracil and this is an important factor in assessing prognosis in bladder carcinomas [7]. The above examples illustrate the close association between OPRT and treatment outcome and response to 5-fluorouracil in systemic malignancies and the need for further studies in this regard.

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

5-Fluorouracil (5-FU) is a widely used drug in head and neck squamous cell carcinoma (HNSCC). In the anabolic pathway of 5-FU, the first step in activation of the drug is phosphorylation of 5-FU by orotate phosphoribosyltransferase (OPRT), which directly metabolizes 5-FU to 5-fluorouridine monophosphate (FUMP) in the presence of 5-phosphoribosyl-1-pyrophosphate. To date, OPRT expression in the tumors has been related to the clinical response or survival of cancer patients receiving 5-FU-based chemotherapy. In this study, we examined whether OPRT expression correlates with the chemosensitivity to 5-FU and cell proliferation in HNSCC. We constitutively expressed an OPRT cDNA in an HNSCC cell line. The effects of OPRT expression on in vitro cell growth and 5-FU cytotoxicity were examined. OPRT transfection increases the cytotoxicity of 5-FU without affecting cell proliferation of HNSCC cells in vitro. These results indicate that OPRT expression plays an important role in the sensitivity of HNSCC to 5-FU chemotherapy.

Orotate phosphoribosyl transferase mRNA expression and the response of cholangiocarcinoma to 5-fluorouracil

To determine whether expression of certain enzymes related to 5-fluorouracil (5-FU) metabolism predicts 5-FU chemosensitivity in cholangiocarcinoma (CCA). The histoculture drug response assay (HDRA) was performed using surgically resected CCA tissues. Tumor cell viability was determined morphologically with hematoxylin and eosin- and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-stained tissues. The mRNA expression of thymidine phosphorylase (TP), orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) was determined with real-time reverse transcriptase-polymerase chain reaction. The levels of gene expression and the sensitivity to 5-FU were evaluated. Twenty-three CCA tissues were obtained from patients who had been diagnosed with intrahepatic CCA and who underwent surgical resection at Srinagarind Hospital, Khon Kaen University from 2007 to 2009. HDRA was used to determine the response of these CCA tissues to 5-FU. Based on the dose-response curve, 200 μg/mL 5-FU was selected as the test concentration. The percentage of inhibition index at the median point was selected as the cut-off point to differentiate the responding and non-responding tumors to 5-FU. When the relationship between TP, OPRT, TS and DPD mRNA expression levels and the sensitivity of CCA tissues to 5-FU was examined, only OPRT mRNA expression was significantly correlated with the response to 5-FU. The mean expression level of OPRT was significantly higher in the responder group compared to the non-responder group (0.41 ± 0.25 vs 0.22 ± 0.12, P < 0.05). OPRT mRNA expression may be a useful predictor of 5-FU chemosensitivity of CCA. Whether OPRT mRNA could be used to predict the success of 5-FU chemotherapy in CCA patients requires confirmation in patients.