The transcription factors aryl hydrocarbon receptor and MYC cooperate in the regulation of cellular metabolism.

The transcription factor AHR (aryl hydrocarbon receptor) drives the expression of genes involved in detoxification pathways in cells exposed to pollutants and other small molecules. Moreover, AHR supports transcriptional programs that promote ribosome biogenesis and protein synthesis in cells stimulated to proliferate by the oncoprotein MYC. Thus, AHR is necessary for the proliferation of MYC-overexpressing cells. To define metabolic pathways in which AHR cooperates with MYC in supporting cell growth, here we used LC-MS-based metabolomics to examine the metabolome of MYC-expressing cells upon AHR knockdown. We found that AHR knockdown reduced lactate, S-lactoylglutathione, N-acetyl-l-alanine, 2-hydroxyglutarate, and UMP levels. Using our previously obtained RNA sequencing data, we found that AHR mediates the expression of the UMP-generating enzymes dihydroorotate dehydrogenase (quinone) (DHODH) and uridine monophosphate synthetase (UMPS), as well as lactate dehydrogenase A (LDHA), establishing a mechanism by which AHR regulates lactate and UMP production in MYC-overexpressing cells. AHR knockdown in glioblastoma cells also reduced the expression of LDHA (and lactate), DHODH, and UMPS but did not affect UMP levels, likely because of compensatory mechanisms in these cells. Our results indicate that AHR contributes to the regulation of metabolic pathways necessary for the proliferation of transformed cells.

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

PURPOSE: 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. METHOD: 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. RESULTS: 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). CONCLUSIONS: OPRT and DPD expression can be treated as a potential predictive biomarker for S-1 response in gastric cancer patients. Further investigation is warranted.

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 cross­complementing 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 drug­metabolizing genes.

¹8F-FDG uptake on PET is a predictive marker of thymidylate synthase expression in patients with thoracic neoplasms.

The aim of this study was to investigate the relationship between the expression levels of thymidylate synthase (TS) and 2-[¹8F]-fluoro-2-deoxy-D-glucose (¹8F-FDG) uptake on positron emission tomography (PET) in various thoracic neoplasms. In total, 392 patients [non-small cell lung cancer (NSCLC) (n=140), malignant pleural mesothelioma (MPM) (n=21), pulmonary metastatic tumors (PMT) (n=148), thymic epithelial tumors (n=49) and pulmonary neuroendocrine (NE) tumor (n=34)] who underwent ¹8F-FDG PET before treatment were included in this study. Tumor sections were stained using immunohistochemistry for determination of TS, orotate phosphoribosyltransferase (OPRT), dihydropyrimidine dehydrogenase (DPD), vascular endothelial growth factor (VEGF), microvessel density (MVD), CD34 and p53. The expression of TS in thoracic neoplasms had a positivity of 58% (230/392), and the positive rates of TS expression in NSCLC, PMT, thymic epithelial tumor, NE tumor and MPM samples were 56, 57, 57, 85 and 47%, respectively. The positivity of TS expression was significantly higher in NE tumors compared to that in other thoracic tumors. A statistically significant correlation between TS expression and ¹8F-FDG uptake was observed in thoracic neoplasms, in particular primary lung adenocarcinomas, high-grade NE tumors, thymomas and MPMs. Moreover, TS expression was closely associated with angiogenesis, DPD, OPRT and p53. Our results indicated that SUV(max) by ¹8F-FDG uptake may be an alternative biomarker for predicting TS expression in patients with primary lung adenocarcinoma, high-grade NE tumor, thymoma and MPM.

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 pro­drug 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.

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.

Expression of dihydropyrimidine dehydrogenase, orotate phosphoribosyl transferase and thymidylate synthase in patients with primary colorectal cancer, and associations with site of first metastasis.

AIM: The activity of the widely used anticancer drug 5-fluorouracil (5-FU) is determined by the presence of several enzymes, including dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), and thymidylate synthase (TS). This study compared the expression levels of these enzymes between primary colorectal cancer with and without distant metastases, and examined whether these expression patterns are associated with hematogenous metastasis. MATERIALS AND METHODS: Among 40 patients with colorectal cancer, 20 had no metastasis and 20 had distant metastasis. Strong expression on immunohistochemistry was classified as positive, while weak, moderate or no expression was classified as negative. RESULTS: Positive expressions of DPD, OPRT and TS in primary colorectal cancer tissue were seen in 47.5%, 75% and 20%, respectively. However, no relationships were observed among the expressions of DPD, OPRT and TS. Expressions of OPRT (p=0.029) and TS (p=0.017) in primary tissues were significantly associated with hematogenous metastasis. Patterns of the expression of the three enzymes varied, and were classified in six ways. A tendency was seen for primary colorectal cancer with DPD-high expression to have liver metastasis and for that with DPD-low expression, to have lung metastasis. CONCLUSION: High expression levels of OPRT and TS in colorectal cancer appear to be significantly involved in metastasis after curative surgery. The organs in which metastases arise may be controlled by the expression of DPD.

Thymidylate synthase expression is closely associated with outcome in patients with pulmonary adenocarcinoma.

The aim of this study is to elucidate the prognostic significance of thymidylate synthase (TS), orotate phosphoribosyltransferase (OPRT) and dihydropyrimidine dehydrogenase (DPD) in completely resected non-small cell lung cancer (NSCLC). One hundred and sixty patients with NSCLC were included in this study. Tumor sections were stained by immunohistochemistry for TS, OPRT, DPD, glucose transporter 1 (Glut1), hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), microvessel density (MVD) determinated by CD34, epidermal growth factor receptor (EGFR), phosph-Akt, phosph-mammalian target of rapamycin (mTOR) and p53. TS, OPRT and DPD were positively expressed in 46, 71 and 54%, respectively. The expression of TS and OPRT was significantly higher in patients with non-adenocarcinoma (non-AC) (n = 53) than adenocarcinoma (AC) (n = 107), and DPD expression was higher in adenocarcinoma as compared with non-adenocarcinoma. A positive TS expression was an independent prognostic factor for predicting a poor outcome in patients with AC, but not in those with non-AC. In AC patients, TS expression was significantly associated with advanced stage, lymph node metastases, vascular invasion, Glut1, HIF-1α, angiogenesis, EGFR signaling pathway and p53. In patients with non-AC, TS expression was not closely correlated with outcome and these biomarkers. A positive TS expression was a powerful prognostic factor to predict a poor outcome in completely resected AC patients.

Prognostic impact of dihydropyrimidine dehydrogenase expression on pancreatic adenocarcinoma patients treated with S-1-based adjuvant chemotherapy after surgical resection.

BACKGROUND AND OBJECTIVES: The aim of this study is to investigate the prognostic value of intratumoral expression of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT) in patients treated with S-1-based chemotherapy after surgical resection for pancreatic adenocarcinoma. METHODS: Intratumoral TS, DPD, and OPRT expression was investigated in 106 patients with resected pancreatic adenocarcinoma by immunohistochemistry. Associations between clinicopathological factors, including intratumoral TS, DPD, and OPRT expression, and survival were evaluated by univariate and multivariate analyses. RESULTS: Of 106 patients, 72 had received S-1-based adjuvant chemotherapy (S-1(+) group), and 34 had not (S-1(-) group). High TS, DPD, and OPRT expression was observed in 64%, 37%, and 66% of patients, respectively. Among S-1(+) group patients, survival was significantly better for patients with low DPD expression than for patients with high DPD expression (P = 0.022). Intratumoral DPD expression was the only independent prognostic factor for patients treated with S-1-based adjuvant chemotherapy by multivariate analysis (P = 0.037). Intratumoral TS and OPRT expression did not appear to influence survival. CONCLUSIONS: Intratumoral DPD expression may be a relevant predictive marker of survival benefit associated with S-1-based adjuvant chemotherapy for pancreatic adenocarcinoma.

Biomarker analysis in patients with advanced gastric cancer treated with S-1 plus cisplatin chemotherapy: orotate phosphoribosyltransferase expression is associated with treatment outcomes.

This study was performed to analyze the impact of protein expression related to fluoropyrimidine and cisplatin metabolism (thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, orotate phosphoribosyltransferase [OPRT], excision repair cross-complementation 1, Fanconi anemia complementation group D2, glutathione S-transferase P1, and X-ray repair cross-complementing group 1) on treatment outcomes in patients with metastatic or relapsed gastric cancer (MRGC) receiving S-1/cisplatin chemotherapy. Protein expression was measured by immunohistochemistry (IHC). Of the 43 patients who had received S-1 (80 mg/m2/day; days 1-14) and cisplatin (60 mg/m2; day 1) every 3 weeks and had available tissue blocks, IHC was successfully performed in 41 patients. Patients with high OPRT levels in tumor tissues (IHC score≥6) had superior progression-free survival (PFS) (23.3 vs. 14.1 weeks [median]) and overall survival (OS) (72.4 vs. 55.4 weeks [median]) to those with low OPRT levels (IHC score≤5; P-values<.05). Expression levels of other proteins were not predictive of treatment outcomes. In multivariate analysis, both a good performance status and a high OPRT level were independently associated with prolonged PFS and OS. The OPRT expression level may be a good predictive marker in S-1/cisplatin-treated patients with MRGC.

Multicenter phase II trial of S-1 in patients with cytokine-refractory metastatic renal cell carcinoma.

PURPOSE: This phase II multicenter trial was conducted to evaluate the activity and safety of S-1 in Japanese patients with metastatic renal cell carcinoma (mRCC). We also examined the relation between response and mRNA expression levels of enzymes involved in the metabolism of fluorouracil (FU). METHODS: Patients with mRCC who had received nephrectomy in whom cytokine-based immunotherapy was ineffective or contraindicated were studied. S-1 was administered orally at 80-, 100-, or 120-mg daily, assigned according to body surface area, on days 1 to 28 of a 42-day cycle. The primary end point was the objective response rate. The mRNA expression levels of FU-related enzymes were measured by reverse-transcriptase polymerase chain reaction in formalin-fixed, paraffin-embedded specimens of tumors obtained at nephrectomy. RESULTS: A total of 45 eligible patients were enrolled. Eleven (24.4%) of 45 patients had partial responses to S-1, and 28 (62.2%) had stable disease. Median progression-free survival was 9.2 months. The severity of most adverse events was mild to moderate. The most common grade 3/4 drug-related adverse events were neutropenia (8.9%) and anorexia (8.9%). The expression level of thymidylate synthase (TS) mRNA was significantly lower in patients who responded to treatment (t-test, P = .048), and progression-free survival was significantly longer in patients whose TS mRNA expression levels were below the median value, as compared with those with higher levels (log-rank test, P = .006). CONCLUSION: S-1 is active against cytokine-refractory mRCC. Quantification of TS mRNA levels in tumors before treatment may facilitate prediction of the response of mRCC to S-1.

Microdissection is essential for gene expression analysis of irradiated rectal cancer tissues.

Microdissection is a reliable technique and is extensively used in many cancer studies. We sought to verify the importance of the microdissection technique in molecular analysis of irradiated rectal cancer specimens. Forty patients with rectal cancer underwent 5-fluorouracil based chemoradiotherapy followed by curative surgery. We compared gene expressions that had previously been shown to be involved in chemotherapy or radiation effects; one obtained using RNA extracted from cancer cells by microdissection, and the other from bulky cancer tissues in all patients. More than 50% regression of the primary tumor was seen in 16 patients (40.0%). There was no significant difference in candidate gene expression profiles between tumor and stromal cells except for thymidine phosphorylase (TP). Without microdissection, there was no significant association between distant recurrence and gene expression in specimens. With microdissected sample analysis, however, patients who developed distant recurrence were found to have significantly higher intratumoral thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT) compared with patients without recurrence. It is possible that microdissection is essential for gene expression analysis of clinically irradiated rectal specimens because preoperative chemoradiotherapy for rectal cancer affects the tumor-stroma balance in irradiated rectal cancer specimen.

Overexpression of orotate phosphoribosyl transferase in hormone-refractory prostate cancer.

Orotate phosphoribosyl transferase (OPRT) is the initial enzyme of 5-fluorouracil (5-FU) activation, in which 5-FU is converted to 5-fluorouridinemonophosphate. Dihydropyrimidine dehydrogenase (DPD) is a degrading enzyme that catabolizes 5-FU. In this study, we investigated the expression of these enzymes in normal prostate gland (NP), hormone-sensitive prostate cancer (HSPC) and hormone-refractory prostate cancer (HRPC). Forty-two prostatic tissue specimens were obtained from patients who had undergone prostate needle biopsies without any treatments or with PSA failure after initial androgen deprivation. The tissue samples derived from formalin-fixed, paraffin-embedded sections were made by laser-captured microdissection and from those RNA was extracted. The levels of OPRT and DPD mRNA expression were examined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The level of OPRT mRNA expression in the HSPC or the HRPC specimens was significantly higher than that in the NP specimens. Immunohistochemical staining for OPRT revealed strong expression of OPRT in prostate cancer cells. There was a significant correlation between OPRT mRNA expression levels and the tumor pathological grade. Furthermore, the OPRT/DPD expression ratio, a powerful predictive factor to evaluate 5-FU sensitivity, in the HRPC group was significantly higher than that in the low grade HSPC group. Thus, 5-FU may be an effective option for some HRPC patients.

Decreased orotate phosphoribosyltransferase activity produces 5-fluorouracil resistance in a human gastric cancer cell line.

To elucidate the mechanism of resistance to 5-fluorouracil (5-FU) in human gastric cancer cells, we established a cell line MKN45/F2R, which acquired 5-FU resistance as a result of continuous exposure to increasing dosages of 5-FU over a year. The cell line showed 157-fold elevated 5-FU resistance compared to the MKN45 human gastric cancer parental cell line. Furthermore, the cells acquired crossresistance to paclitaxel and docetaxel. To identify the mechanism of 5-FU resistance, the expressions of 5-FU metabolic enzymes were examined. Although protein expression and activity of thymidylate synthase and dihydropyrimidine dehydrogenase did not change, orotate phosphoribosyl-transferase (OPRT) protein expression and activity significantly decreased in the 5-FU resistant MKN45/F2R cells. Interestingly, expression of proteins related to taxane resistance including P-glycoprotein, class III beta-tubulin and Bcl-2 increased in MKN45/F2R cells. OPRT-knockout MKN45 parent cells using small interfering RNA demonstrated 15.8-fold increased resistance to 5-FU compared to the control cells. However, resistance to paclitaxel and docetaxel was not observed. These results strongly indicate that decreased activity of OPRT plays an important role in the acquired resistance of gastric cancer cells towards 5-FU; however, it does not play a direct role in paclitaxel and docetaxel resistance. Further studies are now underway to identify genes related to crossresistance to these chemotherapeutic agents.

Expression of orotate phosphoribosyltransferase in colorectal carcinoma: an immunohistochemical analysis in several components of neoplastic lesions.

The purpose of this study was to evaluate the pattern of the expression of orotate phosphoribosyltransferase (OPRT) in several components of colorectal carcinoma (CRC). Fifty-six surgically-resected samples of CRC were subjected to immunohistochemistry with a polyclonal anti-OPRT antibody. Grading was performed independently for several components of CRC, including mucosal carcinoma lesions (n=56), infiltrative lesions (n=53), lymphovascularly invasive lesions (n=34) and metastatic lymph nodes (n=17). The expression of OPRT in mucosal carcinoma and infiltrative lesions correlated significantly only with the presence of lymphovascular invasion (p=0.0007 and <0.0001, respectively). The frequency of OPRT expression in mucosal carcinoma, infiltrative and lymphovascularly invasive lesions as well as metastatic lymph nodes was 32.1, 69.8, 88.2 and 88.8%, respectively. In addition, nuclear staining of OPRT was observed in metastatic lymph nodes and lymphovascularly invasive lesions. Our results suggest that OPRT is involved in the invasion and metastasis of CRC.

Efficacy of laser capture microdissection plus RT-PCR technique in analyzing gene expression levels in human gastric cancer and colon cancer.

BACKGROUND: Thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase gene expressions are reported to be valid predictive markers for 5-fluorouracil sensitivity to gastrointestinal cancer. For more reliable predictability, their expressions in cancer cells and stromal cells in the cancerous tissue (cancerous stroma) have been separately investigated using laser capture microdissection. METHODS: Thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase mRNA in cancer cells and cancerous stroma from samples of 47 gastric and 43 colon cancers were separately quantified by reverse transcription polymerase chain reaction after laser capture microdissection. RESULTS: In both gastric and colon cancers, thymidylate synthase and orotate phosphoribosyltransferase mRNA expressions were higher (p < 0.0001, p <0.0001 respectively in gastric cancer and P = 0.0002, p < 0.0001 respectively in colon cancer) and dihydropyrimidine dehydrogenase mRNA expressions were lower in cancer cells than in cancerous stroma (P = 0.0136 in gastric cancer and p < 0.0001 in colon cancer). In contrast, thymidine phosphorylase mRNA was higher in cancer cells than in cancerous stroma in gastric cancer (p < 0.0001) and lower in cancer cells than in cancerous stroma in colon cancer (P = 0.0055). CONCLUSION: By using this method, we could estimate gene expressions separately in cancer cells and stromal cells from colon and gastric cancers, in spite of the amount of stromal tissue. Our method is thought to be useful for accurately evaluating intratumoral gene expressions.

5-Fluorouracil-related gene expression in primary sites and hepatic metastases of colorectal carcinomas.

UNLABELLED: The aim of this study was to investigate the correlation of the mRNA expressions of 5-fluorouracil (5FU)-related genes in the primary sites and liver metastases of colorectal carcinomas. PATIENTS AND METHODS: Patients with liver metastases from colorectal carcinomas were included (n = 43). The expression ratios to beta-actin of mRNA of thymidine synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and oroteta phosphoribosyl transferase (OPRT) were measured in primary and liver metastases of colorectal carcinomas by laser-captured microdissection and real time PCR. RESULTS: The ratios for the expression of TS, DPD, TP and OPRT mRNAs were significantly correlated between paired primary sites and liver metastases. The mRNA expression ratios of DPD and TP showed a significant correlation both in primary sites and in liver metastases. CONCLUSION: Enzymes of the primary colorectal carcinomas can be used in predicting the therapeutic efficacy of 5FU against liver metastases.

5-Fluorouracil-related gene expression in hepatic artery infusion-treated patients with hepatic metastases from colorectal carcinomas.

AIM: To predict the therapeutic efficacy of hepatic arterial infusion (HAI) with 5-fluorouracil (5FU) for patients with liver metastases from colorectal carcinomas, 5FU-related gene expressions were examined in primary colorectal carcinomas. PATIENTS AND METHODS: Thirty-eight patients with liver metastases from colorectal carcinoma received HAI of 5FU. The expressions of the mRNAs for thymidine synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), and oroteta phophoribosyl transferase (OPRT) in primary colorectal carcinomas were measured by RT-PCR. RESULTS: The response rate was 52.6% (20/38). The overall median survival time was 29.1 months. DPD and TP expression was significantly higher in the progressive disease (PD) group than in the complete response (CR) or partial response (PR) group (p = 0.032, p = 0.014), respectively. The levels of DPD and TP mRNAs showed a significant correlation (r = 0.76, p = 0.0001). CONCLUSION: The expression of DPD and TP mRNAs in primary colorectal carcinomas was significantly predictive of the therapeutic response to 5FU HAI.

Effects of tumor necrosis factor-related apoptosis-inducing ligand alone and in combination with fluoropyrimidine anticancer agent, S-1, on tumor growth of human oral squamous cell carcinoma xenografts in nude mice.

BACKGROUND: Chemotherapy has shown little antitumor activity against advanced oral squamous cell carcinoma (OSCC) patients. Therefore, there is an urgent need to develop more effective therapeutic methods for patients with advanced OSCC. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor ligand family that selectively induces apoptosis of cancer cells. S-1 is a new oral antineoplastic agent that can induce apoptosis in various types of cancer cells, including OSCC. Hence, combined treatment of cancer cells with TRAIL and S-1 might exert dramatic antitumor effects on OSCC cells. MATERIALS AND METHODS: In this study, the response of human OSCC cells to TRAIL alone and in combination with S-1 was examined using nude mouse xenograft models. S-1 (10 mg/kg/day, 5 times/week) was administered orally and TRAIL (1 mg/kg, 5 times/week) was injected into peritumoral tissue for three weeks. Apoptotic cells were detected by a TUNEL method. The protein expression of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyl transferase (OPRT) were assessed using immunohistochemistry; their gene expression was determined using microdissection and RT-PCR, and their protein levels using ELISA. RESULTS: Combined therapy of TRAIL and S-1 exerted antitumor effects on human OSCC xenografts markedly and significantly induced apoptotic cells in tumors treated with TRAIL plus S-1. Immunohistochemistry showed that the expressions of TS and DPD were down-regulated, and that OPRT expression was up-regulated in tumors treated with TRAIL plus S-1. In the same way, microdissection and RT-PCR revealed that the expression of TS and DPD mRNA was down-regulated and that expression of OPRT mRNA was up-regulated in tumors administered the combined treatment. Moreover, ELISA indicated that the protein levels of TS and DPD were down-regulated, and that OPRT was up-regulated in tumors treated with the combined therapy. During the experimental period, no loss of body weight was observed in mice treated with the combined therapy. CONCLUSION: These findings demonstrate that the combination of TRAIL and S-1 is effective against OSCC and has the potential of being a new therapeutic tool for future treatment of these tumors.