Thymidine Phosphorylase Protein Research Articles

Insights from the SNP analysis of TYMP gene linking MNGIE.

TYMP gene, which codes for thymidine phosphorylase (TP) is also known as platelet-derived endothelial cell growth factor (PD-ECGF). TP plays crucial roles in nucleotide metabolism and angiogenesis. Mutations in the TYMP gene can lead to Mitochondrial Neurogastrointestinal Encephalopathy (MNGIE) syndrome, a rare genetic disorder. Our main objective was to evaluate the impact of detrimental non-synonymous single nucleotide polymorphisms (nsSNPs) on TP protein structure and predict harmful variants in untranslated regions (UTR). We employed a combination of predictive algorithms to identify nsSNPs with potential deleterious effects, followed by molecular modeling analysis to understand their effects on protein structure and function. Using 13 algorithms, we identified 119 potentially deleterious nsSNPs, with 82 located in highly conserved regions. Of these, 53 nsSNPs were functional and exposed, while 79 nsSNPs reduced TP protein stability. Further analysis of 18 nsSNPs through 3D protein structure analysis revealed alterations in amino acid interactions, indicating their potential impact on protein function. This will help in the development of faster and more efficient genetic tests for detecting TYMP gene mutations.

Thymidine phosphorylase induction by ionizing radiation antagonizes 5-fluorouracil resistance in human ductal pancreatic adenocarcinoma

Chemoresistance in pancreatic ductal adenocarcinoma (PDAC) frequently contributes to failure of systemic therapy. While the radiosensitizing properties of 5-fluorouracil (FU) are well known, it is unknown whether ionizing radiation (IR) sensitizes towards FU cytotoxicity. Here, we hypothesize that upregulation of thymidine phosphorylase (TP) by IR reverses FU chemoresistance in PDAC cells. The FU resistant variant of the human PDAC cell line AsPC-1 (FU-R) was used to determine the sensitizing effects of IR. Proliferation rates of FU sensitive parental (FU-S) and FU-R cells were determined by WST-1 assays after low (0.05 Gy) and intermediate dose (2.0 Gy) IR followed by FU treatment. TP protein expression in PDAC cells before and after IR was assessed by Western blot. To analyze the specificity of the FU sensitizing effect, TP was ablated by siRNA. FU-R cells showed a 2.7-fold increase of the half maximal inhibitory concentration, compared to FU-S parental cells. Further, FU-R cells showed a concomitant IR resistance towards both doses applied. When challenging both cell lines with FU after IR, FU-R cells had lower proliferation rates than FU-S cells, suggesting a reversal of chemoresistance by IR. This FU sensitizing effect was abolished when TP was blocked by anti-TP siRNA before IR. An increase of TP protein expression was seen after both IR doses. Our results suggest a TP dependent reversal of FU-chemoresistance in PDAC cells that is triggered by IR. Thus, induction of TP expression by low dose IR may be a therapeutic approach to potentially overcome FU chemoresistance in PDAC.

Demethoxycurcumin sensitizes the response of non-small cell lung cancer to cisplatin through downregulation of TP and ERCC1-related pathways

Excision repair cross-complementary 1 (ERCC1) overexpression in lung cancer cells is strongly correlated with its resistance to platinum-based chemotherapy. Overexpression of thymidine phosphorylase (TP) reverts platinum-induced cancer cell death. Curcumin has been reported to enhance antitumor properties through the suppression of TP and ERCC1 in non-small cell lung carcinoma cells (NSCLC). Nevertheless, whether two other curcuminoids, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) from Curcuma longa demonstrate antitumor activity like that of curcumin remain unknown. MTT assay was conducted to determine the cell cytotoxicity. Western blotting was used to determine the protein expressions. Docking is the virtual screening of a database of compounds and predicting the strongest binders based on various scoring functions. BIOVIA Discovery Studio 4.5 (D.S. 4.5) were used for docking. Firstly, when compared with curcumin and BDMC, DMC exhibited the most potent cytotoxic effect on NSCLC, most importantly, MRC-5, a lung fetal fibroblast, was insensitive to DMC (under 30µM). Secondly, DMC alone significantly inhibited on-target cisplatin (CDDP) resistance protein, ERCC1, via PI3K-Akt-snail pathways, and TP protein expression in A549 cells. Thirdly, DMC treatment markedly increased post-target CDDP resistance pathway including Bax and cytochrome c. DMC significantly decreased Bcl-2 protein expressions. Finally, MTT assay indicated that DMC significantly increased CDDP-induced cytotoxicity and was confirmed with an increased Bax/Bcl-2 ratio, indicating upregulation of caspase-3. We concluded that enhancement of the cytotoxicity to CDDP by coadminstration with DMC was mediated by down-regulation of the expression of TP and ERCC1, regulated by PI3K-Akt-Snail pathway inactivation.

Valproic acid potentiates the anticancer activity of capecitabine in vitro and in vivo in breast cancer models via induction of thymidine phosphorylase expression.

The prognosis of patients with metastatic breast cancer remains poor, and thus novel therapeutic approaches are needed. Capecitabine, which is commonly used for metastatic breast cancer in different settings, is an inactive prodrug that takes advantage of elevated levels of thymidine phosphorylase (TP), a key enzyme that is required for its conversion to 5-fluororacil, in tumors. We demonstrated that histone deacetylase inhibitors (HDACi), including low anticonvulsant dosage of VPA, induced the dose- and time-dependent up-regulation of TP transcript and protein expression in breast cancer cells, but not in the non-tumorigenic breast MCF-10A cell line. Through the use of siRNA or isoform-specific HDACi, we demonstrated that HDAC3 is the main isoform whose inhibition is involved in the modulation of TP. The combined treatment with capecitabine and HDACi, including valproic acid (VPA), resulted in synergistic/additive antiproliferative and pro-apoptotic effects in breast cancer cells but not in TP-knockout cells, both in vitro and in vivo, highlighting the crucial role of TP in the synergism observed. Overall, this study suggests that the combination of HDACi (e.g., VPA) and capecitabine is an innovative antitumor strategy that warrants further clinical evaluation for the treatment of metastatic breast cancer.

Abstract 2569: Synergistic antitumor interaction between valproic acid, capecitabine and radiotherapy in colorectal cancer as a rationale for the innovative V-shoRT-R3 trial in locally advanced rectal cancer patients

Abstract We have recently demonstrated that the histone deacetylase-inhibitor (HDACi) vorinostat induces synergistic antitumour effects in combination with capecitabine by up-regulating, in vitro and in vivo, in colorectal cancer cells but not in ex vivo treated peripheral blood lymphocytes, the mRNA and protein expression of thymidine phosphorylase (TP), the key enzyme converting capecitabine to 5-FU (Di Gennaro, Brit J Cancer 2010). We confirmed a time and dose-dependent induction of TP mRNA and protein expression by several other HDACi, including valproic acid (VPA). We investigated potential antitumor interaction between capecitabine metabolite 5′-deoxy-5-fluorouridine (5′-DFUR) and several HDACi showing synergistic/additive antiproliferative and pro-apoptotic effects in all cancer cells tested, with good results with VPA. Interestingly, TP protein induction is achieved also at low doses of VPA (0.5-1 mM), corresponding to a plasma level between 50 and 100 μg/ml, easily reached in patients with normal anticonvulsant doses. Although at these doses VPA did not induce growth inhibition as single agents, a significant synergistic antitumor effect was still demonstrated in combination with 5′-DFUR, suggesting a specific mechanism of interaction. TP knockdown experiments confirmed a crucial role of TP protein modulation in the observed synergism. Radiotherapy further potentiated in colorectal cancer cells the antiproliferative, pro-apoptotic and DNA damage effects induced by 5′-DFUR/VPA combination, as demonstrated by clonogenic assay, Caspase-3 cleavage and γH2AX foci formation, respectively. On these bases we launched a phase I/II clinical study (V-ShoRT-R3 trial) to explore whether the addition of both VPA and capecitabine to short-course radiotherapy (SCRT) before optimal radical surgery, might increase the pathologic complete tumor regression rate in low-moderate risk rectal cancer patients (ClinicalTrials.gov number NCT01898104). Several biomarkers will be evaluated comparing normal mucosa with tumor and on blood samples. Tumor metabolism will be measured by 18FDG-PET at baseline and 11 days after the beginning of SCRT. Currently phase I clinical study is ongoing. We have also optimized a protocol to evaluate histones and proteins acetylation in peripheral blood mononuclear cells of recruited patients by flow cytometry, as pharmacodynamic/predictive specific marker of VPA HDACi activity and preliminary results will be presented. Citation Format: Manuela Terranova Barberio, Biagio Pecori, Serena Imbimbo, Alessandra Leone, Francesca Bruzzese, Maria Carmela Piccirillo, Paolo Delrio, Franco Bianco, Luigi Aloj, Antonio Sorrentino, Fabiana Tatangelo, Antonella Petrillo, Secondo Lastoria, Paolo Muto, Francesco Perrone, Antonio Avallone, Alfredo Budillon, Elena Di Gennaro. Synergistic antitumor interaction between valproic acid, capecitabine and radiotherapy in colorectal cancer as a rationale for the innovative V-shoRT-R3 trial in locally advanced rectal cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2569. doi:10.1158/1538-7445.AM2015-2569

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.

Transfection of thymidine phosphorylase cDNA with lentiviral vector enhances the anticancer effect of 5'-deoxy-5-fluorouridine on colorectal cancer cell lines HT29 and LS174T

To explore the changes of anticancer efficiency of 5'-deoxy-5-fluorouridine (5'-DFUR) and 5-fluorouracil (5-Fu) in colorectal cancer cell line HT29 and LS174T cells after transfection of thymidine phosphorylase (TP) cDNA with a lentiviral vector. TP cDNA was transfected into human colorectal cancer cell lines HT29 and LS174T with the lentiviral vector pLenti6.3-MCS-IRES2-EGFP, and the transfection efficiency of the two cell lines passed 5 generations was analyzed by flow cytometry. The expression of TP protein and the relative quantitative expression of TP mRNA in these 2 cell lines were detected by Western blot and RT-PCR, respectively. The 50% inhibitory concentration (IC50) of 5'-DFUR and 5-Fu in both HT29 and LS174T parent cells and TP-transfected cells were assessed by MTS assay. Finally, the concentration of converted 5-Fu was detected by high performance liquid chromatography (HPLC) either in the medium containing a series of concentrations of 5'-DFUR, in which HT29/HT29-TP or LS174T/LS174T-TP cells were cultured, or in the cell culture lysates. The HT29 and LS174T cells transfected with human TP cDNA were monitored for 5 generations, and their transfection efficiency was about 95.0%. Immunohistochemical staining showed that both the parent cells and TP-transfected HT29 and LS174T cells were TP-positive, while vector-transfected cells were TP-negative. Western blotting showed that the TP protein expression in HT29-TP and LS174T-TP cells were significantly increased compared with that in their parents cells. The relative quantity (RQ) values of TP mRNA in HT29-TP and LS174T-TP cells were 8.45 ± 0.15 and 2 615.02 ± 253.97, respectively, which were significantly higher than that in their parents cells (P < 0.01). The IC50 values of 5'-DFUR on HT29-TP cells and its parents cell were (14.33 ± 0.74) µmol/L and (707.66 ± 5.66) µmol/L, respectively (P < 0.05), while (0.59 ± 0.11) µmol/L in LS174T-TP cells and (239.20 ± 21.83) µmol/L in its parent cells, respectively (P < 0.05). The IC50 values of 5-Fu of HT29-TP cells and its parents cells were (5.42 ± 0.75) µmol/L and (14.19 ± 0.97) µmol/L, respectively (P < 0.05), while (4.41 ± 0.96)µmol/L in LS174T-TP cells and (16.42 ± 2.12)µmol/L in its parents cells, respectively (P < 0.05). The HPLC results showed that the 5-Fu concentration detected from media contained a series of concentrations of 5'-DFUR for culturing HT29-TP and LS174T-TP cells were 12.2 to 28.7-folds and 13.1 to 23.6-folds, respectively, higher than that in their parents cells, (P < 0.01 for all). Otherwise, just a little of 5-Fu was detected in the two TP-transfected cells lysate, about 0.9% to 4.2% of 5-Fu detected in the media of the same cultured cells, whereas no 5-Fu was detected in the two parent cell lysates. Transfection of TP cDNA into colorectal cancer cell lines HT29 and LS174T with lentiviral vector can improve the expression of both TP mRNA and TP protein levels in passaged cells, enhance the conversion of 5-Fu from 5'-DFUR in the medium, and result in an enhanced anticancer effect on these two cell lines.

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.

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.

Liver as a source for thymidine phosphorylase replacement in mitochondrial neurogastrointestinal encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive mitochondrial disease associated with mutations in the nuclear TYMP gene. As a result, the thymidine phosphorylase (TP) enzyme activity is markedly reduced leading to toxic accumulation of thymidine and therefore altered mitochondrial DNA. MNGIE is characterized by severe gastrointestinal dysmotility, neurological impairment, reduced life expectancy and poor quality of life. There are limited therapeutic options for MNGIE. In the attempt to restore TP activity, allogenic hematopoietic stem cell transplantation has been used as cellular source of TP. The results of this approach on ∼20 MNGIE patients showed gastrointestinal and neurological improvement, although the 5-year mortality rate is about 70%. In this study we tested whether the liver may serve as an alternative source of TP. We investigated 11 patients (7M; 35–55 years) who underwent hepatic resection for focal disorders. Margins of normal liver tissue were processed to identify, quantify and localize the TP protein by Western Blot, ELISA, and immunohistochemistry, and to evaluate TYMP mRNA expression by qPCR. Western Blot identified TP in liver with a TP/GAPDH ratio of 0.9±0.5. ELISA estimated TP content as 0.5±0.07 ng/μg of total protein. TP was identified in both nuclei and cytoplasm of hepatocytes and sinusoidal lining cells. Finally, TYMP mRNA was expressed in the liver. Overall, our study demonstrates that the liver is an important source of TP. Orthotopic liver transplantation may be considered as a therapeutic alternative for MNGIE patients.

Tamoxifen enhances erlotinib-induced cytotoxicity through down-regulating AKT-mediated thymidine phosphorylase expression in human non-small-cell lung cancer cells

Tamoxifen is a triphenylethylene nonsteroidal estrogen receptor (ER) antagonist used worldwide as an adjuvant hormone therapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Thymidine phosphorylase (TP) is an enzyme of the pyrimidine salvage pathway which is upregulated in cancers. In this study, tamoxifen treatment inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with tamoxifen decreased TP mRNA and protein levels through AKT inactivation. Furthermore, expression of constitutively active AKT (AKT-CA) vectors significantly rescued the decreased TP protein and mRNA levels in tamoxifen-treated NSCLC cells. In contrast, combination treatment with PI3K inhibitors (LY294002 or wortmannin) and tamoxifen further decreased the TP expression and cell viability of NSCLC cells. Knocking down TP expression by transfection with small interfering RNA of TP enhanced the cytotoxicity and cell growth inhibition of tamoxifen. Erlotinib (Tarceva, OSI-774), an orally available small molecular inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for clinical treatment of NSCLC. Compared to a single agent alone, tamoxifen combined with erlotinib resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-AKT and phospho-ERK1/2, and reduced TP protein levels. These findings may have implications for the rational design of future drug regimens incorporating tamoxifen and erlotinib for the treatment of NSCLC.

Abstract 1301: Metformin induces cytotoxicity by TP and ERCC1 decrease.

Abstract Metformin is an anti-diabetic drug recently shown to inhibit cancer cell proliferation and growth, although the involved molecular mechanisms have not been elucidated. In many cancer cells, highly expression of thymidine phosphorylase (TP) and ERCC1 are associated with poor prognosis. We used A549 and H1975 huamn non-small cell lung cancer (NSCLC) cell lines to investigate the role of TP and ERCC1 expression in metformin-induced cytotoxicity. Metformin treatment decreased cellular TP and ERCC1 protein and mRNA levels by down-regulating phosphorylated MEK1/2-ERK1/2 protein levels in a dose- and time-dependent manner. The enforced expression of the constitutively active MEK1 (MEK1-CA) vectors significantly restored cellular TP and ERCC1 protein levels and cells viability. Specific inhibition of TP and ERCC1 expression by siRNA enhanced the metformin-induced cytotoxicity and growth inhibition. In conclusion, metformin induces cytotoxicity by down-regulating TP and ERCC1 expression in NSCLC cells. Citation Format: Yun-Wei Lin, Yu-Ching Huang, Sheng-Chieh Tseng, Hsien-Chun Chiu, Huang-Jen Chen, Yi-Jhen Huang, Ting-Yu Wo, Shao-Hsing Weng, Jen-Chung Ko. Metformin induces cytotoxicity by TP and ERCC1 decrease. [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 1301. doi:10.1158/1538-7445.AM2013-1301

Effect of thymidine phosphorylase cDNA transfection on the inhibition of human colon carcinoma cell line by 5'-deoxy-5-fluorouridine

To investigate the inhibiting impact of 5'-deoxy-5-fluorouridine (5'-DFUR) on human colon carcinoma cell line LOVO after transfection of thymidine phosphorylase (TP) cDNA. TP cDNA was transfected into human colon carcinoma cell line LOVO with lentiviral vector pLenti6.3_MCS_IRES2-EGFP, and the transfection efficiency was analyzed by flow cytometry. TP mRNA and protein expressions were detected by RT-PCR and Western blotting respectively. The IC50 of 5'-DFUR on TP-transfected LOVO and parental cell were evaluated by MTT assay. The volumes of 5-FU converted from 5'-DFUR in media, where TP-transfected and parental LOVO were cultured, were detected by HPLC. The stable transfectants passed 5 generations were obtained and the transfection rate was 95%. Compared with parental cell, the RQ values of mRNA expression in TP-transfected LOVO was (282.5±86.8) folds higher significantly (P<0.01), also the TP protein expression of TP-transfected LOVO was obviously up-regulated as compared to parental cells. The IC50 value of 5'-DFUR of TP-transfectants was (1087.7±89.1) μmol/L, less than (1607.3±56.8) μmol/L of parental cells significantly (P<0.01), while there was no significant difference between parental cells and vector-transfectants [(1699.5±38.7) μmol/L, P>0.05]. HPLC revealed that when medium was added with 0, 500, 1000, and 2000 μmol/L of 5'-DFUR respectively, 0, 2.10, 3.13, and 7.19 μmol/L of 5-FU was found in the parental cells culture, while 0, 22.16, 30.94 and 40.02 μmol/L of 5-FU was found in TP-transfectants culture, but no 5-FU was found in the vector-transfectants culture. TP cDNA transfection into LOVO can up-regulate the TP mRNA and protein expressions, increase the 5-FU converted from 5'-DFUR, and enhance the cytotoxic effect of 5'-DFUR on the LOVO cells.

Metformin Induces Cytotoxicity by Down‐Regulating Thymidine Phosphorylase and Excision Repair Cross‐Complementation 1 Expression in Non‐Small Cell Lung Cancer Cells

Metformin is an antidiabetic drug recently shown to inhibit cancer cell proliferation and growth, although the involved molecular mechanisms have not been elucidated. In many cancer cells, high expression of thymidine phosphorylase (TP) and Excision repair cross-complementation 1 (ERCC1) is associated with poor prognosis. We used A549 and H1975 human non-small cell lung cancer (NSCLC) cell lines to investigate the role of TP and ERCC1 expression in metformin-induced cytotoxicity. Metformin treatment decreased cellular TP and ERCC1 protein and mRNA levels by down-regulating phosphorylated MEK1/2-ERK1/2 protein levels in a dose- and time-dependent manner. The enforced expression of the constitutively active MEK1 (MEK1-CA) vectors significantly restored cellular TP and ERCC1 protein levels and cell viability. Specific inhibition of TP and ERCC1 expression by siRNA enhanced the metformin-induced cytotoxicity and growth inhibition. Arachidin-1, an antioxidant stilbenoid, further decreased TP and ERCC1 expression and augmented metformin's cytotoxic effect, which was abrogated in lung cancer cells transfected with MEK1/2-CA expression vector. In conclusion, metformin induces cytotoxicity by down-regulating TP and ERCC1 expression in NSCLC cells.

Inhibition of thymidine phosphorylase expression by using an HSP90 inhibitor potentiates the cytotoxic effect of cisplatin in non-small-cell lung cancer cells

Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. In this study, we examined the role of TP expression in relation to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced cytotoxicity in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1650. Treatment with 17-AAG (0.1–1μM) resulted in a decrease in cellular TP protein and mRNA levels, which was accompanied by a downregulation of phosphorylated MKK1/2-ERK1/2 and AKT protein levels. The 17-AAG treatment disrupted the interaction between HSP90 and TP and triggered TP protein degradation through the ubiquitin-26S proteasome pathway. Specific inhibition of TP expression by siRNA further enhanced the cell death and growth inhibition that had been induced by 17-AAG. An enhancement of ERK1/2 or AKT activation by transfecting the cancer cells with constitutively active MKK1/2 or AKT expression vectors significantly restored the 17-AAG-reduced TP protein levels as well as cell viability. In contrast, a combination of U0126 (MKK1/2 inhibitors) or LY294002 (PI3K inhibitor) further decreased the TP expression and cell viability induced by 17-AAG. Moreover, 17-AAG enhanced the cisplatin-induced cytotoxic effect through downregulation of the cisplatin-induced TP expression and ERK1/2 and AKT activation. Taken together, our results suggest that the down-modulation of TP protein induced by 17-AAG represents a key factor in enhancing the cytotoxic effects of cisplatin in NSCLC cells.

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.

Enhancement of Mitomycin C-Induced Cytotoxicity by Curcumin Results from Down-Regulation of MKK1/2-ERK1/2-Mediated Thymidine Phosphorylase Expression

Curcumin (diferuloylmethane), a phenolic compound obtained from the rhizome of Curcuma longa, has been found to inhibit cell proliferation in various human cancer cell lines, including non-small cell lung cancer (NSCLC). Thymidine phosphorylase (TP) is considered an attractive therapeutic target, because increased TP expression can suppress cancer cell death induced by DNA-damaging agents. Mitomycin C (MMC), a chemotherapeutic agent used to treat NSCLC, inhibits tumour growth through DNA cross-linking and breaking. Whether MMC can affect TP expression in NSCLC is unknown. Therefore, in this study, we suggested that curcumin enhances the effects of MMC-mediated cytotoxicity by decreasing TP expression and ERK1/2 activation. Exposure of human NSCLC cell lines H1975 and H1650 to curcumin decreased MMC-elicited phosphorylated MKK1/2-ERK1/2 protein levels. Moreover, curcumin significantly decreased MMC-induced TP protein levels by increasing TP mRNA and protein instability. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased TP protein levels and cell viability in curcumin- and MMC-co-treated cells. In contrast, U0126, a MKK1/2 inhibitor, augmented the cytotoxic effect and the down-regulation of TP by curcumin and MMC. Specific inhibition of TP by siRNA significantly enhanced MMC-induced cell death and cell growth inhibition. Our results suggest that suppression of TP expression or administration of curcumin along with MMC may be a novel lung cancer therapeutic modality in the future.

Histone deacetylase inhibitors induce thymidine phosphorylase expression in cultured breast cancer cell lines

Thymidine phosphorylase (TP) is an enzyme involved in thymidine synthesis and degradation. The expression of this enzyme has been proposed as a predictive factor for the therapeutic benefit of capecitabine, which is a precursor of the drug 5'-fluorouracil. In fact, TP is the rate-limiting enzyme in the activation of capecitabine. Therefore, higher levels of TP are expected to sensitize cancer cells to capecitabine treatment. In the present study, using breast cancer cell lines, we found a correlation between TP mRNA and protein levels, suggesting that compounds able to increase TP gene expression also increase protein levels. Accordingly, we demonstrated that treatment of breast cancer MCF7 and MDA231 cell lines with histone deacetylase inhibitors, tricostatin A and suberoylanilide hydroxamic acid, increased TP both at the mRNA and protein level. The effects of histone deacetylase inhibitors were not found to occur via the cytokine TNFα, a known inducer of TP expression. Our findings suggest a strategy to sensitize breast cancer cells to capecitabine treatment.

Improved sensitivity of gastric carcinoma cells to fluorouracil-related drugs by transfection of thymidine phosphorylase gene

To investigate the relationship between the expression of thymidine phosphorylase (TP) and the sensitivity of gastric carcinoma to 5-fluorouracil (5-FU) and its prodrugs. Gastric carcinoma cell line AGS was transfected with recombinant plasmid pEGFP-N1-TP or control plasmid pEGFP-N1 by lipofectamin 2000. The expression of green fluorescence labeled protein was observed under fluorescence microscope. Positive clones AGS-p and AGS-pTP were selected by G418 treatment. Expression of TP protein and mRNA was detected by immunocytochemistry and RT-PCR, respectively. Drug sensitivity to 5-FU and its prodrugs was assessed by MTT assay. Cell clones with the expression of green fluorescent protein (AGS-p) and a clone with TP and green fluorescent fusion protein (AGS-pTP) were established. Immunostaining of TP protein was strongly positive in AGS-pTP and negative in AGS-p and AGS. The expression of TP mRNA was significantly higher in AGS-pTP (0.8090 ± 0.0450) than that in AGS (0.0490 ± 0.0046) and AGS-p (0.0610 ± 0.0069; P < 0.01). The sensitivity to doxifluridine and capecitabine in AGS-pTP was significantly increased, as compared with that in AGS-p. IC50 values of AGS-pTP to doxifluridine and capecitabine were estimated 1.7 folds and 2.2 folds as much as that of AGS-p, respectively. The sensitivity to 5-FU was not different between AGS-pTP and AGS-p. Enhancement of TP expression improves the sensitivity of gastric carcinoma cells to doxifluridine and capecitabine. But it does not affect the sensitivity to 5-FU.

Abstract 3519: Histone deacetylase inhibitors upregulates thymidine phosphorylase gene and protein expression and synergize with capecitabine in breast cancer cells

Abstract Capecitabine is a pro-drug which was designed to take advantage of the increased levels of thymidine phosphorylase (TP), a key enzyme for its conversion to 5-florouracil (5-FU), observed in tumours as opposed to normal tissues, potentially allowing for selective toxicity. Capecitabine is a valuable substitute for bolus or infusion 5-FU either as monotherapy or in combination with other cytotoxic drugs in the treatment of several tumor types including breast cancer. We have recently demonstrated for the first time, that the histone deacetylase-inhibitor (HDAC-I) vorinostat induces synergistic antitumour effects in combination with capecitabine by up-regulating, in vitro and in vivo, in colorectal cancer cells but not in ex vivo treated peripheral blood lymphocytes, the mRNA and protein expression of TP. We have also demonstrated that TP knockdown by a specific siRNA significantly impairs the synergistic apoptotic cell death induced by vorinostat/capecitabine combination. In this study we demonstrated a time and dose-dependent induction of TP mRNA and protein expression by several HDAC-Is in different breast cancer cell lines such MCF-7, SKBR3, MDA-MB-231and MDA-MB-468 cells. Interestingly, this effect was shared by pan-HDAC-Is such as panobinostat (LBH589), trichostatin A (TSA) or vorinostat and by class I HDAC-Is such as valproic acid (VPA) and entinostat (MS275), but not by the specific inhibitor of HDAC-6 tubacin, indicating that targeting class I HDACs is crucial for TP induction. Moreover, the mechanism of TP induction is dependent on a transcriptional effect since preliminary data showed that HDAC-Is did not interfere with TP protein stability. On these bases we investigated potential antitumor interaction between capecitabine and either vorinostat or panobinostat or VPA, demonstrating synergistic antiproliferative effects in all breast cancer cells independently of p53, ER or Her2 status. Overall, this study suggests that HDAC-Is, by upregulating TP expression, may improve the therapeutic index of capecitabine representing an innovative antitumour strategy for the treatment of breast cancer that warrants further clinical evaluation. 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 3519. doi:10.1158/1538-7445.AM2011-3519