Inhibition of thymidine phosphorylase expression by Hsp90 inhibitor potentiates the cytotoxic effect of salinomycin in human non-small-cell lung cancer cells.

Salinomycin is a polyether ionophore antibiotic having anti-tumorigenic property in various types of cancer. Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is responsible for the stabilization and maturation of many oncogenic proteins. In this study, we report whether Hsp90 inhibitor 17-AAG could enhance salinomycin-induced cytotoxicity in NSCLC cells through modulating TP expression in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1975. We found that salinomycin increased TP expression in a MKK3/6-p38 MAPK activation manner. Knockdown of TP using siRNA or inactivation of p38 MAPK by pharmacological inhibitor SB203580 enhanced the cytotoxic and growth inhibition effects of salinomycin. In contrast, enforced expression of MKK6E (a constitutively active form of MKK6) reduced the cytotoxicity and cell growth inhibition of salinomycin. Moreover, Hsp90 inhibitor 17-AAG enhanced cytotoxicity and cell growth inhibition of salinomycin in NSCLC cells, which were associated with down-regulation of TP expression and inactivation of p38 MAPK. Together, the Hsp90 inhibition induced TP down-regulation involved in enhancing the salinomycin-induced cytotoxicity in A549 and H1975 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.

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.

Association between mRNA expression of chemotherapy-related genes and clinicopathological features in colorectal cancer: A large-scale population analysis.

To establish the individualized treatment of patients with colorectal cancer, factors associated with chemotherapeutic effects should be identified. However, to the best of our knowledge, few studies are available on this topic, although it is known that the prognosis of patients and sensitivity to chemotherapy depend on the location of the tumor and that the tumor location is important for individualized treatment. In this study, primary tumors obtained from 1,129 patients with colorectal cancer were used to measure the mRNA expression levels of the following genes associated with the effects of standard chemotherapy for colorectal cancer: 5-fluorouracil (5-FU)-related thymidylate synthase (TYMS), dihydropyrimidine dehydrogenase (DPYD) and thymidine phosphorylase (TYMP); folate-related dihydrofolate reductase (DHFR), folylpolyglutamate synthase (FPGS) and gamma-glutamyl hydrolase (GGH); irinotecan-related topoisomerase I (TOP1); oxaliplatin-related excision repair cross-complementing 1 (ERCC1); biologic agent-related vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). Large-scale population analysis was performed to determine the association of gene expression with the clinicopathological features, in particular, the location of the colorectal cancer. From the results of our analysis of the mRNA expression of these 10 genes, we noted the strongest correlation between DPYD and TYMP, followed by TYMS and DHFR. The location of the colorectal cancer was classified into 4 regions (the right­ and left-sided colon, rectosigmoid and rectum) and was compared with gene expression. A significant difference in all genes, apart from VEGF, was noted. Of the remaining 9 genes, the highest expression of TYMS and DPYD was observed in the right­sided colon; the highest expression of GGH and EGFR was noted in the left-sided colon; the highest expression of DHFR, FPGS, TOP1 and ERCC1 was noted in the rectosigmoid, whereas TYMP expression was approximately equivalent in the right-sided colon and rectum, and higher than that in other locations. The data generated from this study may prove to be useful for the development of individualized chemotherapeutic treatments for patients with colorectal cancer, and will mean that the tumor location is taken into account.

Avaliação de consumo alimentar, medidas antropométricas e tempo de menopausa de mulheres na pós-menopausa / Food consumption in postmenopausal women and its relation with anthropometric measurements and time since menopause

OBJETIVO: Avaliar o hábito alimentar e nutricional de mulheres na pós-menopausa e compará-los com o perfil antropométrico, faixa etária e tempo de menopausa. MÉTODOS: No período de junho a agosto de 2011, 148 mulheres na pós-menopausa residentes no Estado de São Paulo (região Sudeste do Brasil) foram avaliadas com um questionário estruturado contendo dados socioeconômicos, clínicos, antropométricos e alimentares. Avaliou-se nível de atividade física, variáveis bioquímicas, Índice de Massa Corporal (IMC), circunferência abdominal (CA) e consumo alimentar (energia, proteínas, carboidratos e gorduras, fibra, colesterol, vitaminas A e C, minerais, cálcio e ferro) de acordo com a faixa etária e o tempo de pós-menopausa (TPM). RESULTADOS: A média de IMC foi 29,0±5,6 kg/m2 e da CA, 95,7±12,9 cm. O consumo médio calórico diário atingiu 1.406,3±476,5 kcal. A ingestão e a adequação calórica foram significantemente mais apropriadas entre as mulheres eutróficas e com CA<88 cm. O mesmo ocorreu quanto ao consumo de proteínas (p<0,001 e p=0,006, respectivamente). Na análise por faixa etária ou TPM não houve diferenças significantes, exceto a média do consumo proteico, maior no grupo com 5 anos ou menos de menopausa (p=0,048). CONCLUSÃO: O perfil antropométrico de mulheres na pós-menopausa mostrou predominância de sobrepeso ou obesidade. O consumo alimentar apresentou-se adequado quanto às calorias e percentuais de macronutrientes, entre as eutróficas e com CA<88 cm. .

PURPOSE: To evaluate eating in postmenopausal women and its relation to anthropometry, age and time since menopause in São Bernardo do Campo residents. METHODS: During the period from June to August of 2011, 148 postmenopausal women residents in state of São Paulo (Southeast region of Brazil) were evaluated using a structured questionnaire containing socioeconomic, clinical, anthropometric and food data. The level of physical activity, biochemical variables, Body Mass Index (BMI), abdominal circumference (AC) and dietary intake (energy, protein, carbohydrates and fats, fiber, cholesterol, vitamins A and C, minerals, calcium and iron) were analyzed according to age and time after menopause. RESULTS: Mean BMI was 29.0≤5.6 kg/m2 and abdominal circumference was 95.7±12.9 cm. The average daily caloric consumption was 1,406.3±476.5 kcal. The calorie intake was significantly more appropriate in normal-weight women and women with AC<88 cm. The same was observed for protein intake (p<0.001 and p=0.006, respectively). No association was observed with age or duration of the postmenopausal period, except for average protein consumption that was higher in the group with five years or less of menopause (p=0.048). CONCLUSION: The anthropometry of postmenopausal women showed a predominance of overweight and obesity. Dietary intake was adequate in relation to the percentage of calories and macronutrients and calories among most normal-weight women and women with AC<88 cm. .

Radiation recall gastritis secondary to erlotinib in a patient with pancreatic cancer.

BACKGROUND: Radiation recall refers to chemotherapy-triggered inflammation in healthy areas previously exposed to irradiation. Chemotherapeutics known to be associated with radiation recall phenomenon include anthracyclines, taxanes and antimetabolites, such as gemcitabine and capecitabine. Case reports detailing radiation recall dermatitis and pneumonitis associated with erlotinib have been previously described in the literature, however, there are no reported cases describing radiation gastritis associated with erlotinib. We report a patient with pancreatic cancer who developed gastrointestinal bleeding secondary to radiation recall gastritis related to erlotinib exposure. CASE REPORT: A 57-year-old Hispanic male with pancreatic cancer initially received 7 cycles of FOLFIRINOX followed by capecitabine with radiation therapy for 28 fractions for a total of 5,040 cGy. Re-staging with computed tomography demonstrated stable disease. The patient was then treated with erlotinib and capecitabine for approximately two months before restaging demonstrated progressive disease. Shortly after discontinuing erlotinib and capecitabine, the patient reported maroon colored stools. Laboratory studies demonstrated a precipitous drop in hemoglobin and hematocrit from pre-treatment baseline, ultimately requiring transfusion with packed red blood cells. Subsequent esophagogastroduodenoscopy demonstrated findings consistent with radiation gastritis, with oozing in the gastric body and antrum, which was treated therapeutically with argon plasma coagulation. The patient's gastrointestinal bleed was difficult to control. Over the course of a two-month period - the patient required multiple admissions, repeat therapeutic esophagogastroduodenoscopies and transfusions. DISCUSSION: Radiation recall from erlotinib is rare but can potentially arise in any site that has been previously irradiated. There may be an association between the pathogenesis of radiation recall and erlotinib's up-regulation of the angiogenic growth factor thymidine phosphorylase. Treating physicians are reminded of the potential toxicity from erlotinib either given concomitantly or followed by radiation. We suggest discontinuing erlotinib if radiation gastritis is observed. We encourage physicians with similar experiences with erlotinib to report their findings. Further studies are warranted to investigate the pathogenesis of this unique phenomenon and its association with erlotinib.

Influence of Apical Enlargement in Cleaning of Curved Canals Using Negative Pressure System

This study aimed to evaluate, by scanning electron microscopy (SEM), the cleaning of canal walls with moderate curvature subjected to biomechanical preparation with different final diameters using apical negative pressure irrigation. Thirty-two mesiobuccal roots of molars were divided into 4 groups (n=8) according to the instrument's final diameter: GI: 30.02, GII: 35.02, GIII: 40.02 and GIV: 45.02. Irrigating procedure was performed at each change of instrument with 1% NaOCl using the Endovac system. Final irrigation was conducted with 17% EDTA for 5 min. The SEM photomicrographs were evaluated under 35× and 1000× magnification, by three calibrated examiners, in a double-blind design. Data were submitted to Kruskal-Wallis and Dunn's post hoc tests (α=0.05). Canals instrumented with 30.02 and 35.02 final diameters showed more debris, statistically different from the other groups (p<0.05). Comparing each root canal third, for the cervical and apical portions no statistically significant difference (p>0.05) was found among the four groups. Regarding the presence of smear layer, canals with 30.02 final diameter showed the highest scores, statistically different from the 45.02 group (p<0.05) and similar to the 35.02 and the 40.02 groups (p>0.05). Although none of the studied diameters completely removed debris and smear layer, it may be concluded that instrumentation with higher final diameters was more effective in cleaning the root canals with moderate curvature.

Este estudo buscou avaliar, por meio de microscopia eletrônica de varredura (MEV), a limpeza das paredes de canais com curvatura moderada, submetidos ao preparo biomecânico com diferentes diâmetros finais utilizando-se irrigação por pressão apical negativa. Trinta e duas raízes mésio-vestibulares de molares foram divididas em 4 grupos (n=8) de acordo com o diâmetro final dos instrumentos: GI: 30.02, GII: 35.02, GIII: 40.02 e GIV: 45.02. O procedimento de irrigação foi realizado a cada troca de instrumento com NAOCl 1% utilizando o sistema EndoVac. A irrigação final foi conduzida com EDTA 17% por 5 min. As microfotografias de MEV foram avaliadas sob aumentos de 35× e 1000×, por três examinadores calibrados, em estudo duplo-cego. Os dados foram submetidos ao teste de Kruskal-Wallis e pós-teste de Dunn (=0,05). Os canais instrumentados com diâmetros finais de 30.02 e 35.02 demonstraram mais debris, estatisticamente diferente dos demais grupos (p<0,05). Comparando-se cada terço do canal radicular, para as porções cervical e apical não foi encontrada diferença estatisticamente significante (p>0,05) entre os quatro grupos. Com relação à presença de smear layer, canais com diâmetro final de 30.02 demonstraram os maiores scores, estatisticamente diferente do grupo 45.02 (p<0,05) e similar aos grupos 35.02 e 40.02 (p>0,05). Apesar de nenhum dos diâmetros estudados ter removido completamente os debris e a smear layer, pode ser concluído que a instrumentação com diâmetros finais maiores foi mais efetiva na limpeza dos canais radiculares com curvatura moderada.

Enzymatic synthesis of 2'-deoxyuridine by whole cell catalyst co-expressing uridine phosphorylase and thymidine phosphorylase through auto-induction system.

Genes encoding uridine phosphorylase (UP) and thymidine phosphorylase (TP) from Escherichia coli K12 were cloned and recombined respectively into plasmids pET-21a(+) and pET-28a(+). The recombinant plasmids BL21/pET21a-UP and BL21/pET28a-TP were co-transformed into E. coli BL21(DE3) to construct highly effective BTU strain (BL21/pET28a-TP, pET21a-UP) overexpressing both of UP and TP. BTU was cultivated in ZYM-Fe-5052 medium for 10 h and used as catalyst to synthesize 2'-deoxyuridine (dUR). It was found to increase the productivity of dUR by 8-9 fold when compared to wild E. coli K12 and E. coli BL21(DE3) strains. A series of experiments were carried out to find out the optimal conditions for synthesis of dUR. At 50°C, with 0.25‰ dry wt./v to catalyze the reaction of 2:1 ß-thymidine: uracil (60 mM ß-thymidine, 30 mM uracil), the conversion rate of dUR would reach 61.6% at 1 h, which was much higher than the rates obtained by BTU strain cultured in LB medium and induced by IPTG. This result proved co-expression and auto-induction were efficient methods in enhancing the expression quantity and activity of nucleoside phosphorylases, and they also had significant implications for large-scale industrial production of dUR and synthesis of other nucleoside derivatives.

Gene therapy using a liver-targeted AAV vector restores nucleoside and nucleotide homeostasis in a murine model of MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in TYMP, enconding thymidine phosphorylase (TP). TP deficiency results in systemic accumulation of thymidine and deoxyuridine, which interferes with mitochondrial DNA (mtDNA) replication and leads to mitochondrial dysfunction. To date, the only treatment available for MNGIE patients is allogeneic hematopoietic stem cell transplantation, which is associated with high morbidity and mortality. Here, we report that AAV2/8-mediated transfer of the human TYMP coding sequence (hcTYMP) under the control of a liver-specific promoter prevents the biochemical imbalances in a murine model of MNGIE. hcTYMP expression was restricted to liver, and a dose as low as 2 × 10(11) genome copies/kg led to a permanent reduction in systemic nucleoside levels to normal values in about 50% of treated mice. Higher doses resulted in reductions to normal or slightly below normal levels in virtually all mice treated. The nucleoside reduction achieved by this treatment prevented deoxycytidine triphosphate (dCTP) depletion, which is the limiting factor affecting mtDNA replication in this disease. These results demonstrate that the use of AAV to direct TYMP expression in liver is feasible as a potentially safe gene therapy strategy for MNGIE.

Lidamycin up-regulates the expression of thymidine phosphorylase and enhances the effects of capecitabine on the growth and pulmonary metastases of murine breast carcinoma.

PURPOSE: Capecitabine (CAP), a prodrug, needs to be converted to 5-fluorouracil by several key enzymes, including thymidine phosphorylase (TP). To improve the therapeutic index, potentiation of antitumor activity of CAP is required. In this study, we explored whether lidamycin (LDM), an enediyne anticancer antibiotic, can induce synergistic antitumor effects in combination with CAP in murine breast cancer in vitro and in vivo. METHODS: Using MTT, cell migration and invasion, siRNA knockdown, and Western blot assays, the in vitro synergistic effects of LDM plus CAP on 4T1(LUC) cells were evaluated, and the mechanism of this synergy was explored. For in vivo model of orthotopic implantation model of 4T1(LUC) cells, optical molecular imaging system was utilized to evaluate the growth of primary tumor and metastasis. To further understand the mechanism of action of the LDM/CAP combination, immunohistochemistry analysis was carried out to detect thymidine phosphorylase induction and ERK signaling. RESULTS: As determined by MTT and transwell assay, LDM enhanced the inhibitory effects of CAP on cancer cell proliferation, migration, and invasion. Western blot showed that this synergistic effect was attributed to the up-regulated expression of TP induced by LDM. Knocking down TP impaired the synergistic anti-proliferative effect of LDM and CAP. Furthermore, our data suggested that LDM-induced up-regulation of TP both in vitro and in vivo is associated with ERK activation, because the inhibition of ERK activity by ERK inhibitor U0126 abrogated LDM-induced TP up-regulation. In animal models, LDM plus CAP potently inhibited primary tumor growth as well as lung metastasis compared with control or single-agent-treated group. CONCLUSIONS: LDM can potentiate the antitumor effects of CAP on breast cancer line. The synergistic effects suggest that the combination of LDM and CAP is an innovative antitumor strategy for breast cancer therapy.

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.

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.

Expression of thymidine phosphorylase and cyclooxygenase-2 in melanoma.

Several studies have reported an increase in vascular structures in malignant melanoma. Neovascularization can be enhanced by several factors. Among them, thymidine phosphorylase (TP) and cyclooxygenase-2 (COX-2) have been reported to play a role. The expressions of TP and COX-2 were evaluated trough immunohistochemistry in a series of 78 primary cutaneous melanomas diagnosed between 2000 and 2004. The expressions of TP and COX-2 through mRNA and western blot analysis were also evaluated in several melanoma cell lines. TP expression and COX-2 expression were considered positive in 25 cases (32%) and 22 cases (28.2%), respectively. TP-positive melanomas showed a lower mitotic rate (P=0.008), smaller thickness (P=0.01), and absence of lymphovascular invasion (P=0.04). COX-2-positive melanomas showed a higher mitotic rate (P=0.01) and higher thickness (P=0.03). COX-2 expression was associated with reduced disease-free survival (P=0.01). COX-2-positive cases showed a trend toward reduced survival, whereas TP was not correlated with overall survival. COX-2 expression was detected in four of 11 melanoma cell lines both by mRNA and by western blot analysis. Our data show that TP expression is associated with more favorable prognostic factors (such as thin melanoma, low mitotic count, and absence of lymphovascular invasion), whereas COX-2 expression is associated with poor prognostic factors (thicker melanoma and high mitotic count).

Making capecitabine targeted therapy for breast cancer: which is the role of thymidine phosphorylase?

Thymidine phosphorylase (TP) expression has been found to be elevated in various solid tumors where it is likely involved in mechanisms that regulate cell proliferation, apoptosis, and angiogenesis. Based on these properties, it is tempting to hypothesize a potential prognostic role of TP, suggesting that a high TP expression could predict a poor outcome. On the other hand, TP expression has been studied for its role in predicting benefit from treatment with fluoropyrimidine-containing chemotherapy. Several studies have been conducted on breast cancer. The current evidence on the value of TP is not mature enough to allow its translation into clinical practice. However, several findings support the potentially predictive role of TP. In this light, TP appears to be a promising marker that can give helpful information to predict the benefit from capecitabine-based chemotherapy in patients with breast cancer.

DPYD, TYMS, TYMP, TK1, and TK2 genetic expressions as response markers in locally advanced rectal cancer patients treated with fluoropyrimidine-based chemoradiotherapy.

This study is to investigate multiple chemotherapeutic agent- and radiation-related genetic biomarkers in locally advanced rectal cancer (LARC) patients following fluoropyrimidine-based concurrent chemoradiotherapy (CCRT) for response prediction. We initially selected 6 fluoropyrimidine metabolism-related genes (DPYD, ORPT, TYMS, TYMP, TK1, and TK2) and 3 radiotherapy response-related genes (GLUT1, HIF-1α, and HIF-2α) as targets for gene expression identification in 60 LARC cancer specimens. Subsequently, a high-sensitivity weighted enzymatic chip array was designed and constructed to predict responses following CCRT. After CCRT, 39 of 60 (65%) LARC patients were classified as responders (pathological tumor regression grade 2 ~ 4). Using a panel of multiple genetic biomarkers (chip), including DPYD, TYMS, TYMP, TK1, and TK2, at a cutoff value for 3 positive genes, a sensitivity of 89.7% and a specificity of 81% were obtained (AUC: 0.915; 95% CI: 0.840-0.991). Negative chip results were significantly correlated to poor CCRT responses (TRG 0-1) (P = 0.014, hazard ratio: 22.704, 95% CI: 3.055-235.448 in multivariate analysis). Disease-free survival analysis showed significantly better survival rate in patients with positive chip results (P = 0.0001). We suggest that a chip including DPYD, TYMS, TYMP, TK1, and TK2 genes is a potential tool to predict response in LARC following fluoropyrimidine-based CCRT.

Thymidine phosphorylase expression in B-cell lymphomas and its significance: a new prognostic marker?

OBJECTIVE: To explore thymidine phosphorylase (TP) expression in B-cell lymphomas (BCLs). TP is expressed by tumor and stromal cells in a variety of cancers. STUDY DESIGN: Paraffin-embedded tissues from follicular lymphomas, diffuse large BCLs (DLBCLs), and benign lymph nodes were studied using immunohistochemical staining with antibodies for TP and CD68. Prognostic markers were used to stain DLBCLs. We correlated TP expression in DLBCL indirectly with prognostic immunomarkers and directly with survival data. RESULTS: TP expression in BCLs was noted in a subset of malignant B cells. TP expression in higher-grade lymphoma was identified in 66% of cases and 11% of lower-grade lymphomas. Macrophages/stromal cells demonstrated an intense cytoplasmic and/or nuclear staining pattern in both lymphoma and benign lymph nodes, confirmed by CD68 coexpression. Increased macrophage/ stromal cells in higher-grade lymphomas are associated with enhanced TP expression in neoplastic B cells (observation only). Sixty-eight percent of TP-positive DLBCLs were of nongerminal center origin, indicating poorer prognosis. CONCLUSION: TP is more likely expressed by malignant B cells in higher-grade lymphomas, and expression of TP possibly results from changes intrinsic to the tumor cells or interactions between microenvironment and tumor. TP positivity in DLBCL correlates with nongerminal center origin and worse outcome.

Thymidine phosphorylase inhibits vascular smooth muscle cell proliferation via upregulation of STAT3.

Dysregulated growth and motility of vascular smooth muscle cells (VSMC) play important role in obstructive vascular diseases. We previously reported that gene transfer of thymidine phosphorylase (TP) into rat VSMC inhibits cell proliferation and attenuates balloon injury induced neointimal hyperplasia; however, the mechanism remains unclear. The current study identified a signaling pathway that mediates effect of TP inhibited VSMC proliferation with a TP activity-dependent manner. Rat VSMC overexpressing human TP gene (C2) or control empty vector (PC) were used. Serum stimulation induced constitutive STAT3 phosphorylation at tyrosine705 in C2 cell but not in PC, which was independent of JAK2 signaling pathway. Inhibition of Src family kinases activity inhibited STAT3 phosphorylation in C2 cells. Lyn activity was higher in C2 cell than in PC. SiRNA based gene knockdown of Lyn significantly decreased serum induced STAT3 phosphorylation in C2 and dramatically increased proliferation of this cell, suggesting that Lyn plays a pivotal role in TP inhibited VSMC proliferation. Unphosphorylated STAT3 (U-STAT3) expression was significantly increased in C2 cells, which may be due to the increased STAT3 transcription. Gene transfection of mouse wild-type or Y705F mutant STAT3 into PC cell or mouse primary cultured VSMC significantly reduced proliferation of these cells, suggesting that overexpression of U-STAT3 inhibits VSMC proliferation. We conclude that Lyn mediates TP induced STAT3 activation, which subsequently contributes to upregulate expression of U-STAT3. The U-STAT3 plays a critical role in inhibiting VSMC proliferation.

Evaluations of biomarkers associated with sensitivity to 5-fluorouracil and taxanes for recurrent/advanced breast cancer patients treated with capecitabine-based first-line chemotherapy.

The aim of the present study was to investigate the gene expression of biomarkers associated with the sensitivity to fluoropyrimidine and taxanes in recurrent/advanced breast cancer patients treated with first-line capecitabine chemotherapy. We evaluated the clinicopathological/prognostic significance of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), class III ß-tubulin (ßIII-tubulin), and stathmin-1 or oncoprotein-18 (STMN1). Formalin-fixed, paraffin-embedded tumor specimens from 42 patients were used for analysis of TS, DPD, TP, ßIII-tubulin, and STMN1 expression with a real-time reverse transcription-PCR technique. Patients were classified into the high-expression and low-expression groups according to the median value of the expression level of each biomarker. There was a significantly longer time to progression (TTP) in the high-TP group (P=0.018). The multivariate analysis revealed that the TP expression (hazard ratio for the low-TP group vs. the high-TP group, 2.873; 95% confidence interval, 1.143-7.223; P=0.025) is independent of prognostic factors for TTP. In the subgroup of patients treated with capecitabine plus taxanes as first-line chemotherapy, TTP was significantly longer in the low-ßIII-tubulin group (P=0.047). The gene expression of TS, DPD, and STMN1 failed to have any significant impact on the outcome. These results provide further evidence that the TP expression may be a prognostic factor in breast cancer patients treated with capecitabine-based first-line chemotherapy, and ßIII-tubulin can be used to predict the outcome of capecitabine in combination with taxanes as first-line chemotherapy. Therefore, these potential biomarkers should be further evaluated.

The Sp1 transcription factor is essential for the expression of gliostatin/thymidine phosphorylase in rheumatoid fibroblast-like synoviocytes.

INTRODUCTION: Gliostatin/thymidine phosphorylase (GLS/TP) has angiogenic and arthritogenic activities, and aberrant GLS production has been observed in the active synovial membranes of rheumatoid arthritis (RA) patients. The human GLS gene promoter contains at least seven consensus binding sites for the DNA binding protein Sp1. Here we examined whether Sp1 is necessary for GLS production in RA. We also studied the effects of the Sp1 inhibitor mithramycin on GLS production in RA fibroblast-like synoviocytes (FLSs). METHODS: FLSs from RA patients were treated with specific inhibitors. The gene and protein expression of GLS were studied using the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and an enzyme immunoassay. Intracellular signalling pathway activation was determined by western blotting analysis, a luciferase assay, a chromatin immunoprecipitation (ChIP) assay and a small interfering RNA (siRNA) transfection. RESULTS: The luciferase and ChIP assays showed that Sp1 binding sites in the GLS promoter were essential for GLS messenger RNA (mRNA) expression. GLS production was suppressed in FLSs by siRNA against Sp1 transfection. Mithramycin decreased GLS promoter activity, mRNA and protein expression in FLSs. Tumour necrosis factor-α (TNF-α) significantly increased GLS expression in RA FLSs; this effect was reduced by pre-treatment with cycloheximide and mithramycin. CONCLUSIONS: Pretreatment of mithramycin and Sp1 silencing resulted in a significant suppression of GLS production in TNF-α-stimulated FLSs compared to controls. GLS gene expression enhanced by TNF-α was partly mediated through Sp1. As physiological concentrations of mithramycin can regulate GLS production in RA, mithramycin is a promising candidate for anti-rheumatic therapy.

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 (0.1-1 µM) 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.