Thymidine Phosphorylase Expression Levels Research Articles

Vesicular IFN-γ as a cooperative attacker to enhance anti-cancer effect of 5-fluorouracil via thymidine phosphorylase upregulation and tumor microenvironment normalization

On the basis of immuno-modulating effect and upregulating the activity of thymidine phosphorylase (TP), interferon-γ (IFN-γ) as a cooperative attacker was explored to enhance the anticancer activity of 5-fluorouracil (5-FU). We designed and prepared a self-assembled nano-vesicular system IFN-γ-EDP formulated by amphiphilic poly((polyethylene glycol)(dodecylphosphoethanolamine)phosphazene) (EDP) to entrap IFN-γ in the hydrophilic cavity. The IFN-γ-EDP vesicles allowed IFN-γ to accumulate at the tumor site and be taken up by tumor cells, resulting in significantly upregulated expression level of TP, distinct inhibition of cell growth, more cellular apoptosis and more serious cell cycle arrest when administrated combined with 5-FU. Moreover, IFN-γ-EDP could normalize the tumor microenvironment by enhancing the CD4+ and CD8+ T cell populations, promoting the IL-12 secretion and suppressing the IL-10 secretion in tumor. As a consequence, the combination therapy of IFN-γ-EDP with 5-FU achieved remarkably enhanced tumor inhibition rate of 56.9% against CT26 colorectal cancer.

Thymidine phosphorylase affects clinical outcome following surgery and mRNA expression levels of four key enzymes for 5‑fluorouracil metabolism in patients with stage�I and II non‑small cell lung cancer

The expression levels of thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), thymidylate synthase (TS) and orotate phosphoribosyltransferase (OPRT) may predict the clinical efficacy of 5-fluorouracil-based chemotherapy in patients with cancer. We herein investigated the differences in the mRNA levels of these enzymes in non-small-cell lung cancer (NSCLC) and evaluated their prognostic value for NSCLC treated by surgical resection. The intratumoral mRNA levels of TP, DPD, TS, and OPRT were quantified in 66 patients with pathological stage I and II NSCLC (adenocarcinoma or squamous cell carcinoma) following complete resection according to the Danenberg Tumor Profile method. The TP level was the only significant prognostic factor for disease-specific survival (DSS) following complete resection; the mean TP mRNA level differed significantly between the high and low mRNA expression groups. The DSS at 5 years was significantly higher in the low TP mRNA compared with that in the high TP mRNA expression group (83.4 vs. 58.6%, respectively; P=0.005). A Cox proportional hazards model revealed that pathological stage, sex, and TP expression were independent prognostic factors for DSS in patients with stage I and II NSCLC following complete resection. Thus, TP level may be used to monitor treatment efficacy and predict the outcome of NSCLC patients.

Suppression of TWIST1 enhances the sensitivity of colon cancer cells to 5-fluorouracil

The 5-fluorouracil (5FU)-based adjuvant chemotherapy improves the survival of patients with colorectal cancer, however the main obstacle affecting its effectiveness is a drug resistance. Our study aimed to investigate the impact of TWIST1 silencing on the sensitivity of cancer cells to 5FU. The suppression of TWIST1 expression in human colon cancer HT29 and HCT116 cell lines was achieved by transduction with lentiviral vector carrying the TWIST1 silencing sequence (pLL3.7-shTWIST1). The suppression of TWIST1 expression induced changes in the expression pattern of epithelial to mesenchymal transition markers, reduced the cells proliferation rate, increased their sensitivity to serum withdrawn, and increased the cytotoxic effect of 5FU. However, significantly higher 5FU cytotoxicity was observed in HT29 cell cultures. Cells with silenced TWIST1 displayed altered expression of enzymes metabolizing 5FU. The expression level of dihydropyrimidine dehydrogenase, and thymidylate synthase decreased significantly in HT29 shTWIST1 cells, but not in HCT116 shTWIST1 cells. On the other hand, significant increases in the expression levels of thymidine phosphorylase, and uridine phosphorylase 1 were seen in both cell lines with suppressed expression of TWIST1. The changes in enzymes expression were mirrored by enzymatic activities. In conclusion, our observations point to TWIST1 as a target protein to enhance the sensitivity of colorectal cancer cells to 5FU.

The Thymidine Phosphorylase Imaging Agent 123I-IIMU Predicts the Efficacy of Capecitabine.

Recently, companion diagnostics with nuclear medicine techniques have been anticipated as more suitable means than biopsy for predicting treatment efficacy. The anticancer effect of capecitabine, an orally administered chemotherapeutic agent activated by thymidine phosphorylase (TP), is positively associated with tumor TP expression levels. This study aimed to assess whether TP imaging using a radiolabeled uracil derivative, (123)I-5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil ((123)I-IIMU), could predict the efficacy of capecitabine treatment. Sensitivity to doxifluridine, a metabolite of capecitabine and direct substrate for TP, was assessed by water-soluble tetrazolium salt assays in vitro for 3 human colon cancer cell lines with different TP expression profiles. The intracellular uptake and retention of (123)I-IIMU were evaluated. Mice inoculated with each cell line were treated with capecitabine for 2 wk, and tumor growth was compared. In vivo distribution studies and SPECT/CT imaging of (123)I-IIMU were performed in inoculated mice. In vitro experiments showed a positive relation between TP expression levels and doxifluridine sensitivity. In vitro studies revealed that intracellular uptake and retention of (123)I-IIMU were dependent on TP expression levels. In vivo experiments in inoculated mice showed that (123)I-IIMU accumulation in tumor tissue was in line with TP expression levels and susceptibility to capecitabine treatment. Moreover, SPECT/CT imaging of (123)I-IIMU in tumor-inoculated mice showed that (123)I-IIMU reflects TP expression levels in tumor tissues. (123)I-IIMU could be used as an in vivo companion diagnostic for predicting the efficacy of capecitabine treatment.

Clinicopathological significance of vascular endothelial growth factor, thymidine phosphorylase and microvessel density in colorectal cancer.

Colorectal cancer is a common malignant disease, the incidence of which is increasing worldwide, therefore, identifying novel prognostic factors to improve adjuvant therapeutic strategies or postoperative monitoring is required. Angiogenesis, which is assessed by microvessel density (MVD), is significant in tumor growth and metastasis. However, the association between angiogenesis and clinical outcome remains controversial. In the present study, 84 surgically resected cases of colorectal cancer were examined to clarify the clinicopathological significance of vascular endothelial growth factor (VEGF), thymidine phosphorylase (TP) and cluster of differentiation (CD)34 expression levels. VEGF expression was identified to be significantly correlated with TP expression (r=0.45; P<0.0001) and MVD in the high VEGF expression group was observed to be significantly greater than that in the low VEGF expression group (P=0.0194). In the Dukes' stage D group, the MVD in the high TP expression group was significantly greater than that in the low TP expression group (P=0.0149). High VEGF expression was subsequently correlated with a short overall survival rate for patients exhibiting lymph node metastasis (P=0.0128); however, there was no significant difference in overall survival rate regarding the expression levels of TP and CD34. The results of the present study indicate that VEGF expression may serve as a prognostic factor for colorectal cancer patients exhibiting lymph node metastasis. Furthermore, angiogenesis, as assessed by MVD, is an important prognostic factor for tumor growth at the primary site.

Relationship between biodistribution of a novel thymidine phosphorylase (TP) imaging probe and TP expression levels in normal mice.

Thymidine phosphorylase (TP) is a key enzyme in the pyrimidine nucleoside salvage pathway and its expression is upregulated in a wide variety of solid tumors. In mice, we previously observed high and specific accumulation levels of our TP imaging probe, radioiodinated 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil (IIMU) not only in high-TP-expressing tumors, but also in the liver and small intestine. To clarify the reason for the high accumulation levels of radioiodinated IIMU in the liver and small intestine, we investigated the expression levels of TP in mice in comparison with the biodistribution of radioiodinated IIMU ((123)I-IIMU). BALB/cCrSlc mice were injected with (123)I-IIMU, and the radioactivity levels [%ID/g (normalized to a mouse of 25 g body weight)] in the tissues of interest were determined 0.5, 1, 3 and 24 h after the injection (n = 5, each time point). To determine the expression levels of TP, BALB/cCrSlc and ddy mice (n = 3/each strain) were euthanized, and the heart, liver, lung, spleen, kidney, stomach, small intestine, large intestine and brain were collected. The mRNA and protein expression levels of TP in these organs were examined by quantitative reverse transcription-polymerase chain reaction and western blot analyses, respectively. In BALB/cCrSlc mice administered (123)I-IIMU, markedly high radioactivity levels were observed in the liver [1.568 ± 0.237 (%ID/g)] and small intestine [0.506 ± 0.082 (%ID/g)], whereas those in the other tissues were fairly low [<0.010 ± 0.003 (%ID/g)] 30 min after the injection. The highest expression levels of TP mRNA were also observed in the liver and small intestine among the tissues tested. Immunoblotting showed intense immunoreactive bands of the TP protein for the liver and small intestine, whereas no notable bands were detected for other tissues. Similar expression profiles of TP mRNA and protein were observed in ddy mice. We confirmed TP expression in various tissues of mice at the mRNA and protein levels: high TP expression levels were observed in the liver and small intestine. These high TP expression levels are consistent with the high accumulation levels of (123)I-IIMU in these tissues. Our results may provide important information about the physiological accumulation of (123)I-IIMU, which may be useful for the clinical diagnostic imaging of TP.

Relative mRNA expression of prostate-derived E-twenty-six factor and E-twenty-six variant 4 transcription factors, and of uridine phosphorylase-1 and thymidine phosphorylase enzymes, in benign and malignant prostatic tissue.

Prostate cancer is the most frequent urological tumor, and the second most common cancer diagnosed in men. Incidence and mortality are variable and appear to depend on behavioral factors and genetic predisposition. The prostate-derived E-twenty-six factor (PDEF) and E-twenty-six variant 4 (ETV4) transcription factors, and the thymidine phosphorylase (TP) and uridine phosphorylase-1 (UP-1) enzymes, are reported to be components of the pathways leading to tumorigenesis and/or metastasis in a number of tumors. The present study aimed to analyze the mRNA expression levels of these proteins in prostatic cancerous and benign tissue, and their association with clinical and pathological variables. Using quantitative reverse transcription polymerase chain reaction, the mRNA expression levels of PDEF, ETV4, TP and UP-1 were studied in 52 tissue samples (31 of benign prostatic hyperplasia and 21 of prostate adenocarcinomas) obtained from patients treated by transurethral resection of the prostate or by radical prostatectomy. Relative expression was assessed using the ∆-CT method. Data was analyzed using Spearman's tests for correlation. P<0.05 was considered to indicate a statistically significant difference. The results revealed that PDEF, ETV4, UP-1 and TP were expressed in 85.7, 90.5, 95.2 and 100% of the prostate cancer samples, and in 90.3, 96.8, 90.3 and 96.8% of the benign samples, respectively. PDEF and ETV4 exhibited a significantly higher relative expression level in the tumor samples compared with their benign counterparts. The relative expression of TP and UP-1 did not differ significantly between benign and cancerous prostate tissues. The relative expression of TP was moderately and significantly correlated with the expression of ETV4 in the benign tissues. The relative expression of UP-1 was significantly lower in T3 compared with T1 and T2 cancers. These findings indicate that PDEF, ETV4, TP and UP-1 are typically expressed in benign and malignant prostatic tissues. Further studies are necessary to define the role of these proteins as therapeutic targets in prostate cancer.

Thymidine phosphorylase expression is associated with time to progression in patients with metastatic colorectal cancer.

Background5-Fluorouracil (5-FU) is the cornerstone of chemotherapeutic treatment for patients with colorectal cancer. The enzyme thymidine phosphorylase (TP) catalyzes the conversion of 5-FU to its active metabolite, 5-fluoro-2’-deoxyuridine. TP is expressed in tumour epithelial cells and stromal cells, particularly in tumour-associated macrophages. These macrophages may affect sensitivity to chemotherapy. Previously, we identified TP as a predictive factor in microdissected tumour samples of patients with advanced colorectal cancer. In the present study, we analysed TP expression in tissues and associated stromal cells from patients with advanced colorectal cancer and associated TP levels to tumour response and time-to-event variables during first-line chemotherapy treatment. We also investigated the association between serum TP levels at the time of surgery and gene expression in primary tumour tissues.MethodsThis study included 125 patients with metastatic colorectal cancer treated with first-line 5-FU-based chemotherapy. To quantify TP gene expression levels in tumour tissues, real-time polymerase chain reaction was performed using the 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). TP protein concentration in matched serum samples was determined using an enzyme-linked immunosorbent assay system (USCN Life Science Inc.).ResultsThe tumour response rate was 31%, and 30% of patients exhibited stable disease. No associations between TP expression level and age or gender were observed. Levels of TP mRNA in mucosa and tumours were positively correlated (r = 0.41, p < 0.01). No correlation between TP expression and tumour response rate was observed. Time to progression was significantly longer in patients with high TP expression (p < 0.01). Serum TP protein levels were not associated with tumour response or time-to-event variables and did not correlate with gene expression in tumour tissues.ConclusionsHigh TP gene expression in non-microdissected tumour tissues of patients with advanced colorectal cancer correlates with longer time to progression, which could be related to treatment. These results are in contrast to previous studies where microdissected tumour cells were analysed and may be due to the presence of adjacent stromal cells. Serum TP protein expression does not correlate to TP gene expression in tissues of patients with advanced colorectal cancer.

Comparison of site-specific gene expression levels in primary tumors and synchronous lymph node metastases in advanced gastric cancer

Many malignant tumors consist of heterogeneous subpopulations of cells. This heterogeneity is associated with genetic characteristics. However, it remains unclear whether gene expression levels differ among specific sites of tumors in gastric cancer. We studied differences in gene expression levels among specific sites of primary tumors and synchronous lymph node metastases, using formalin-fixed, paraffin-embedded specimens resected surgically from 48 patients with previously untreated advanced gastric cancer. Specimens were obtained by laser-captured microdissection from five regions: (1) nonneoplastic mucosa, (2) surface layer (mucosa) of the primary tumor (surface sections), (3) middle layer (submucosa) of the primary tumor (middle sections), (4) the deepest layer of the primary tumor (muscularis propria or deeper) at the site of deepest invasion (deep sections), and (5) level 1 synchronous lymph node metastasis (lymph node metastases). Expression levels of the following target genes were determined by quantitative real-time polymerase chain reaction: thymidylate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor-1α (HIF1α). TP, DPD, EGFR, and HIF1α gene expression levels were significantly higher in deep sections than in surface sections. TP, EGFR, VEGF, and HIF1α gene expression levels were significantly higher in lymph node metastases than in surface sections. TP, DPD, EGFR, VEGF, and HIF1α gene expression levels were positively correlated with the specific samples harvested from the tumors. Our results show that the expression levels of some genes in tumor cells can change in specific sites of tumors and can become higher in association with tumor progression.

Clinical significance of the thymidylate synthase, dihydropyrimidine dehydrogenase, and thymidine phosphorylase mRNA expressions in hepatocellular carcinoma patients receiving 5-fluorouracil-based transarterial chemoembolization treatment

PurposeTo determine whether 5-fluorouracil (5-FU) sensitivity is associated with the mRNA expressions of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and thymidine phosphorylase (TP) in patients with hepatocellular carcinoma (HCC) treated with 5-FU-based transarterial chemoembolization (TACE).MethodsFormalin-fixed, paraffin-embedded tumor specimens from 40 patients treated with 5-FU-based TACE were selected for the examination of TS, DPD, and TP expression level by a quantitative real-time reverse transcription- polymerase chain reaction (PCR) technique. Patients were categorized into high and low expression groups according to the median expression level of each enzyme. Associations between the mRNA expression levels of TS, DPD, and TP and clinical parameters including treatment efficacies, clinicopathological factors, and prognosis were assessed.ResultsHigh DPD expression was associated with worse treatment outcome, including intrahepatic disease progression rate (hazard ratio [HR] for high DPD versus low DPD, 2.212; 95% confidence interval [CI], 1.030–4.753; P = 0.042), extrahepatic disease progression rate (HR for high versus low DPD, 3.171; 95% CI, 1.003–10.023; P = 0.049), and progression-free survival (HR for high versus low DPD, 2.308; 95% CI, 1.102–4.836; P = 0.027). No correlation was found between the mRNA expression of TS/TP and treatment outcome.ConclusionDPD mRNA expression level was negatively correlated with the clinical outcomes of HCC patients treated with 5-FU-based TACE. These results provide indirect evidence that high DPD mRNA expression is a predictive marker of treatment resistance for 5-FU.

Abstract 5503: Targeting thymidine metabolic pathways in cancer cells with a novel thymidine conjugate.

Abstract Cellular thymidine metabolism involves two pathways: the catabolic and salvage pathways. The catabolic pathway breaks down thymidine to carbon dioxide for energy while the salvage pathway converts thymidine to its 5′-triphosphate as one of building blocks for DNA synthesis. Cytosolic level of thymidine is regulated by both thymidine phosphorylase (TYMP) of the catabolic pathway and thymidine kinase (TK) of the salvage pathway. In tumor tissues and cancer cells, abnormally high levels of these two proteins have been consistently reported. Therefore, thymidine metabolic pathways in cancer cells are a potential therapeutic target for novel drug design and development. We recently developed a novel thymidine analog, 3′-deoxythymidine phenylquinoxaline conjugate (dT-QX) that selectively killed a series of cancer cell lines including human liver carcinoma HepG2 and Hep3B, breast adenocarcinoma MCF-7, rat brain glioma C6 and mouse liver cancer H22 cells and showed low cytotoxicity on the normal liver hepatocyte HL-7702 cells. dT-QX is a conjugate of 3′-deoxythymidine and a DNA intercalating moiety with a triazole linkage achieved by click chemistry. The selective killing of cancer cells was attributed to the selective inhibition of DNA synthesis in the cancer cells but not in normal liver HL-7702 cells, resulting in extensive production of mitochondrial superoxide stress. The conjugation of 3′-deoxythymidine was found to be critical for dT-QX to achieve the selective killing of cancer cells. The killing by dT-QX was revealed to be directly due to the expression levels of TYMP and TKs in cancer cells, confirming that dT-QX successfully targeted the thymidine metabolic pathways as designed. Finally, a preliminary mouse study with subcutaneous liver tumors confirmed that dT-QX effectively inhibited the growth of tumors in vivo. Citation Format: Qibing Zhou, Qiong Wei, Dejun Zhang. Targeting thymidine metabolic pathways in cancer cells with a novel thymidine conjugate. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5503. doi:10.1158/1538-7445.AM2013-5503

The role of TP, TS, and DPD as potential predictors of outcome following capecitabine plus oxaliplatin (XELOX) versus bolus 5-fluorouracil/leucovorin (5-FU/LV) as adjuvant therapy for stage III colon cancer: Biomarker findings from study NO16968 (XELOXA).

3578 Background: In NO16968, XELOX was superior in terms of disease-free survival (DFS) and overall survival (OS) to bolus 5-FU/LV as adjuvant therapy for stage III colon cancer (Schmoll et al. ASCO GI 2012). Three key enzymes appear to have the potential to predict efficacy and/or safety of fluoropyrimidine-based treatment: thymidine phosphorylase (TP), thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD). We evaluated the association between baseline TP, TS and DPD and outcome (DFS and OS). Methods: Pts with stage III colon cancer received either XELOX (8 cycles, 24w) or bolus 5-FU/LV (Mayo Clinic, 6 cycles, 24w; Roswell Park, 4 cycles, 32w). The primary study endpoint was DFS; secondary endpoints included OS. TP, TS and DPD expression levels were determined in formalin-fixed, paraffin-embedded tissues by RT-PCR, and the median used as a cut-off point: high (above median) vs. low (below median). Results: The biomarker population included 498 (26%) of 1886 pts entered (XELOX, n=242; 5-FU/LV, n=256). Baseline demographics, tumor characteristics, cancer history and efficacy (DFS and OS) were similar to those in the main study population. Cox regression analysis for DFS (Table). In the XELOX group pts with low DPD and TP levels and a high TP/DPD ratio appeared to have significantly better DFS; this effect was not observed with 5-FU/LV. Subgroup analysis shows that the difference between XELOX and 5-FU/LV was also higher in pts with low DPD levels. Conclusions: These exploratory findings suggest that tumor DPD and TP RNA levels could be used to predict outcomes of adjuvant treatment with fluoropyrimidine/oxaliplatin combinations, and should be validated prospectively. Analysis of the current dataset is ongoing and further details on potential biomarkers will be available. [Table: see text]

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.

Radiolabeled uracil derivative as a novel SPECT probe for thymidine phosphorylase

We have developed a radiolabeled uracil derivative, 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil (IIMU) as a novel single photon emission computed tomography probe for thymidine phosphorylase (TP). This radioiodinated IIMU has a high affinity for TP and highly accumulates in the TP-expressing tumor cell line A431 (human epidermoid carcinoma). To evaluate the specificity of the cellular uptake of IIMU to TP expression, we examined the effects of TP knockdown on the uptake of ¹²⁵I-labeled IIMU (¹²⁵I-IIMU) in the tumor cells. TP-specific small interfering RNA (siRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific siRNA (positive control), and negative control siRNA were transfected into A431 cells, respectively. Target-mRNA and protein expression levels of TP and GAPDH were examined 48 and 72 h after transfection, respectively. The cellular uptake level of ¹²⁵I-IIMU was also evaluated 72 h after transfection. The results were compared after normalization with the corresponding negative controls. After TP-specific and GAPDH-specific siRNA transfection, the expression levels of TP and GAPDH mRNA decreased significantly to 41 and 29%, respectively, compared with the negative control (P<0.001 for both). The expression levels of TP and GAPDH protein also significantly decreased to 34 and 30%, respectively (P<0.001 for both). After TP-specific siRNA transfection, the cellular uptake level of ¹²⁵I-IIMU decreased significantly to 66% (P<0.001). In contrast, GAPDH siRNA transfection did not significantly affect the cellular uptake level of ¹²⁵I-IIMU. siRNA-mediated TP knockdown significantly decreased the cellular uptake level of ¹²⁵I-IIMU. This finding indicates that the uptake of IIMU in tumor cells is TP specific and directly corresponds to TP expression levels.

Thymidylate Synthase, Dihydropyrimidine Dehydrogenase, Thymidine Phosphorylase, Orotate Phosphoribosyltransferase mRNA Expression in Lung Cancer Metastatic Lymph Node Samples Obtained by Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration – A Pilot Study

Introduction/BackgroundPyrimidine antimetabolites' target molecules thymidylate synthase (TS) and flopropyrimidine-metabolising enzymes such as dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyltransferase (OPRT) are known as biomarkers of 5-fluorouracil (5-FU) treatment. The purpose of this study was to evaluate the feasibility of mRNA expression analysis for TS, DPD, TP, and OPRT using metastatic lymph node samples obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in patients with non–small-cell lung cancer (NSCLC). Materials and MethodsMetastatic lymph nodes from 20 patients with NSCLC diagnosed by EBUS-TBNA were analyzed. After pathological diagnosis, additional sections were made and confirmed the existence of tumor cells by hematoxylin and eosin staining. Tumor cells were extracted using laser-captured microdissection and the mRNA levels were measured by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) for each marker. ResultsThere were 17 mediastinal and 3 hilar lymph nodes. The histology included 13 cases of adenocarcinoma and 7 cases of squamous cell carcinoma. DPD and TP could be successfully analyzed in all samples and TS and OPRT could be measured in 18 samples. The median values of the relative TS, DPD, TP, and OPRT mRNA expression levels were 10.68, 6.23, 18.32, and 1.77, respectively. ConclusionTS, DPD, TP, and OPRT mRNA expression levels could be analyzed using metastatic lymph node samples obtained by EBUS-TBNA. The result of this pilot study may be applicable for further translational research for the treatment of locally advanced NSCLC.

Abstract 5249: Analysis of gene expression levels associating with metabolism of 5-fluorouracil and folate in primary colorectal cancer and corresponding synchronous or metachronous liver metastasis

Abstract Background Gene expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT) have been shown to be associated with response to 5-fluorouracil-based therapies. Folylpolyglutamate synthase (FPGS), gamma-glutamyl hydrolase (GGH) and dihydrofolate reductase (DHFR) are associated with folate metabolism which regulates intracellular folate level. Analyzing these gene expression levels in liver metastases is important to obtain the prediction of therapy, however, only surgical specimens of the primary tumor are readily available for analysis in many cases. Our aim is to determine how TS, DPD, TP, OPRT, FPGS, GGH and DHFR gene expression levels in primary colorectal cancer (CRC) are related to those in liver metastases. Methods Formalin-fixed, paraffin-embedded tumor specimens from 50 pairs of primary CRC and corresponding liver metastases, including 30 synchronous and 20 metachronous metastases, were dissected by using laser-captured microdissection. Total RNA was extracted and cDNA was prepared by reverse-transcription. Quantitation of target genes and internal reference gene, beta-2-microglobulin, was performed using real-time RT-PCR. Results Significant difference was seen between mRNA expression levels of TS, DPD, FPGS and DHFR in primary tumor and those in corresponding synchronous liver metastases (median value: TS 1.18 vs. 0.05; p&amp;lt;0.0001, DPD 0.18 vs.0.05; p&amp;lt;0.0001, FPGS 0.07 vs. 0.02; p=0.0005, DHFR 0.31 vs. 0.03; p&amp;lt;0.0001), TS, DPD, TP, OPRT, FPGS and DHFR in primary tumor and those in corresponding metachronous liver metastases (TS 0.23 vs. 0.06; p&amp;lt;0.0001, DPD .0.31 vs. 0.03; p&amp;lt;0.0001, TP 0.21 vs. 0.14, p=0.021, OPRT 0.35 vs. 0.17; p=0.016, FPGS 0.11 vs. 0.04; p=0.0003, DHFR 0.41 vs. 0.03, p&amp;lt;0.0001). When matched tissue sets were compared on an individual basis, there was a significant correlation for TP, OPRT and FPGS mRNA expression between primary tumor and corresponding synchronous liver metastases (TP rs=0.64; p=0.018, OPRT rs=0.62; p=0.006, FPGS rs=0.51; p=0.004), DPD and GGH in primary tumor and corresponding metachronous liver metastases (DPD rs=0.54; p=0.014, GGH rs=0.59; p=0.007). Conclusion A good prediction of TP, OPRT and FPGS mRNA levels in synchronous liver metastases as well as DPD and GGH mRNA levels in metachronous liver metastasis can be obtained by measuring those of primary CRC, although no correlation was seen for other genes. We are currently investigating more number of cases and genes related with sensitivity to antitumor drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5249. doi:10.1158/1538-7445.AM2011-5249

Synergistic Effect of Curcumin and Cisplatin via Down-Regulation of Thymidine Phosphorylase and Excision Repair Cross-Complementary 1 (ERCC1)

Curcumin (diferuloylmethane), a phenolic compound obtained from the rhizome of Curcuma longa, is known to have antiproliferative and antitumor properties. Thymidine phosphorylase (TP), an enzyme of the pyrimidine salvage pathway, is considered an attractive therapeutic target, and its expression could suppress cancer cell death induced by DNA damage agents. Excision repair cross-complementary 1 (ERCC1) is a protein involved the process of nucleotide excision repair. The ERCC1 gene is expressed at high levels in cancers and has been associated with resistance to platinum-based chemotherapy. In this study, the effects of curcumin on TP and ERCC1 expression induced by cisplatin in non-small-cell lung cancer (NSCLC) cell lines was investigated. Exposure of the NSCLC cells to various concentrations of curcumin (5-40 μM) down-regulates the mRNA and protein levels of TP and ERCC1 through destabilization of the mRNA and proteins via a mechanism involving inactivation of MKK1/2-extracellular signal-regulated kinase (ERK1/2). Depletion of endogenous TP or ERCC1 expression by transfection with specific small interfering RNAs significantly decreases cell viability in curcumin-exposed NSCLC cells. Curcumin enhances the sensitivity of cisplatin treatment for NSCLC through inactivation of ERK1/2 and by decreasing the TP and ERCC1 protein levels. Enhancement of ERK1/2 signaling by constitutively active MKK1/2 causes an increase in TP and ERCC1 protein levels and promotes cell viability after cotreatment with curcumin and cisplatin. Enhancement of the cytotoxicity to cisplatin by administration of curcumin is mediated by down-regulation of the expression levels of TP and ERCC1 and by inactivation of ERK1/2.

Vascular endothelial growth factor and thymidine phosphorylase expression in salivary gland tumors with distinct metastatic behavior

Metastasis of salivary gland tumors has a negative impact on survival. Angiogenesis and its factors are potential markers for predicting metastasis in different malignant tumors, but this is not the case for salivary gland tumors. Salivary gland tumors of distinct biologic behavior were analyzed according to the semiquantitative immunoexpression of vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP). Vascular endothelial growth factor expression was predominantly weak in benign tumors. Weak TP expression was observed in 100% cases of benign tumors and in 74.3% of primary malignant tumors. High VEGF and TP expression levels were significantly associated with primary malignant tumors but not with primary non-metastasizing and primary metastasizing malignant tumors or with subtypes of malignant tumors. Vascular endothelial growth factor and TP expression levels discriminate benign and malignant tumors but cannot predict metastasis from non-metastasizing tumors.

A retrospective study on TS mRNA expression and prediction of the effects of adjuvant oral 5-fluorouracil in breast cancer

Nucleic acid-metabolizing enzymes, such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT), have attracted attention as candidates for response determinants of 5-fluorouracil (5-FU). Whether the expression levels of these enzymes can be adopted as valuable parameters for 5-FU sensitivity in breast cancer has yet to be elucidated. In the present study, intratumoral mRNA expression of TS, DPD, TP and OPRT were determined in formalin-fixed paraffin-embedded surgical specimens collected from 217 breast cancer patients, using the Danenberg Tumor Profile method, which combines microdissection and real-time-polymerase chain reaction. The significance of these enzymes as prognostic and 5-FU efficacy-predicting factors was evaluated. Our data showed that a low DPD expression is related to a high nuclear grade and other factors including hormone receptor-negativity. Low expression levels of TP were found in hormone receptor-negative tumors. TS and OPRT expression were not related to various clinicopathological factors, but patients with a high TS mRNA expression showed a significantly poorer prognosis in cases where 5-FU was not administered. The efficacy of 5-FU was more significant when administered for more than 6 months in the group with a high TS mRNA expression. These data suggest that TS mRNA expression in breast cancer tissue is an ideal predictor of outcomes for patients with no administration of 5-FU, and of the efficacy of 5-FU.

Relationship between the expression of thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase and survival in epithelial ovarian cancer

The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-PCR (Taqman) was used to detect the mRNA expression of TS, TP and DPD in formalin-fixed and paraffin-embedded 106 samples of epithelial ovarian cancer and 29 normal ovaries. A TATA box-binding protein (TBP) was used as an endogenous reference gene. A relationship between TS, TP, DPD expression and clinicopathologic features was investigated. The protein location and expression of TS, TP and DPD was examined in the same patients by an avidin-biotin-peroxidase immunohistochemistry. TS and TP mRNA expression levels were significantly higher in tumor group than in normal controls, with the average value of TS and TP mRNA being 6.14+/-0.62 and 0.59+/-0.06 in tumor tissue, and 0.71+/-0.14 and 0.16+/-0.04 in normal tissue, respectively. DPD mRNA expression levels were significantly lower in tumor group (0.11+/-0.02) than in normal controls (0.38+/-0.05). There was statistically significant difference in TS and TP mRNA expression levels among different pathological grades and clinical stages (P<0.05), but histological subtype was not significantly associated with TS and TP mRNA expression. DPD gene expression was not significantly associated with any clinicopathological parameters. Immunohistochemistry revealed that TP protein was mainly distributed in nucleus, and TS and DPD mainly in cytoplasm. The protein expression intensity of TS, TP and DPD was coincided with the mRNA expression levels. It was concluded that TS, TP mRNA and protein expression levels were significantly higher in epithelial ovarian cancer, and DPD mRNA and protein expression levels were significantly lower. The expression levels of TS and DPD were related to the patients' prognosis and survival. Combined gene expression levels of TS, TP and DPD represent a new variable to predict the clinical outcome in ovarian cancer. The association of TS, TP and DPD expression levels with survival suggests an importance of these genes for tumor occurrence and progression.