Kazutaka Miyadera

Correlation between thymidine phosphorylase expression and prognosis in human renal cell carcinoma.

PURPOSEThymidine phosphorylase (TP) is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and has angiogenic activity. We examined whether TP expression in renal cell carcinoma (RCC) is associated with microvessel density as a marker of angiogenesis, clinicopathologic characteristics, and outcome.PATIENTS AND METHODSThe enzymatic activity and expression of TP were examined in 18 RCCs and 19 kidney tissues not grossly involved with tumor from 24 patients with 13 paired samples and 11 unpaired samples by spectrophotometry and immunoblotting. The relationship between TP expression and microvessel density was assessed by immunohistochemistry in 133 RCCs.RESULTSThe median enzymatic activity of TP in RCCs was nine fold higher than that in nonneoplastic kidney tissues (P < .001). Similar results were obtained by immunoblot analysis. According to the TP staining profile, tumors were classified as no or low, intermediate, or high TP-expressing tumors. TP positivity was significantly correlated ...

Expression of Thymidine Phosphorylase in Human Gastric Carcinoma

The activity of thymidine phosphorylase (dThdPase) has heen reported to increase in several types of malignant tumors. Experimental evidence has shown that dThdPase is identical to platelet‐derived endothelial cell growth factor, and that dThdPase has angiogenic activity. We examined the expression of dThdPase to investigate whether the expression of dThdPase correlates with angiogenesis, clinicopathologic features and the prognosis of patients with human gastric carcinomas. Microvessels were assessed by immnnostaining endothelial cells for factor VIII. We counted microvessels in the tumors of 158 patients whose tumors were completely removed surgically. Microvessels were counted in a × 400 field in the most active areas of neovascularization. We purified a monoclonal antibody (TMA‐1) against dThdPase and studied the expression of dThdPase using TMA‐1 in the same serial sections as those used for the detection of factor VIII. The correlation between angiogenesis and dThdPase, and the clinicopathological significance of dThdPase, in patients with gastric carcinoma were examined. The positive expression of dThdPase was more frequent (P<0.001) in gastric carcinomas (67/158, 43.4%) than that in normal tissues (12/158, 7.6%). The average microvessel count in dThdPase‐positive gastric carcinomas was higher (P<0.001) than that in dThdPase‐negative carcinomas. The percentage of gastric carcinoma cells expressing dThdPase was significantly correlated with the microvessel count (P<0.001). Further, the average size of dThdPase‐positive carcinomas was significantly larger (P<0.001) than that of negative carcinomas and the mean microvessel count in dThdPase‐positive gastric carcinomas was also significantly higher (P<0.001) than that in dThdPase‐negative carcinomas. There was a significant correlation between the positive expression of dThdPase and microvessel count (P<0.001) or lymph node metastasis (P=0.013) by multivariate logistic analysis. Further, patients with dThdPase‐positive carcinoma showed a significantly worse prognosis than those with dThdPase‐negative carcinoma overall and in stage III. These findings indicate that the expression of dThdPase in gastric carcinomas is related to progression and metastasis, and this enzyme affects the prognosis of some patients with the disease.

γ-Hydroxybutyric acid and 5-fluorouracil, metabolites of UFT, inhibit the angiogenesis induced by vascular endothelial growth factor

UFT, a drug composed of uracil and tegafur at the molar ratio of 4:1, is an orally active agent for the treatment of a wide variety of malignant tumours. Using a murine dorsal air sac (DAS) assay, we have previously shown that UFT and its metabolites, γ-hydroxybutyric acid (GHB) and 5-fluorouracil (5-FU), inhibited the angiogenesis induced by murine renal cell carcinoma. Here we report that UFT was more effective than other fluorinated pyrimidines such as 5-FU and doxifluridine (5′-DFUR) in blocking the angiogenic responses elicited by five human cancer cell lines which produced high levels of vascular endothelial growth factor (VEGF), but no detectable fibroblast growth factor-2 (FGF-2) in vitro. In contrast, UFT was unable to block the angiogenic response to one human gastric cancer cell line which produced both VEGF and FGF-2 in vitro. However, the production or secretion of VEGF by these cells was unaffected by GHB and 5-FU treatment. Interestingly, GHB suppressed the chemotactic migration and tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by VEGF, without inhibiting their DNA synthesis. Since GHB did not affect the FGF-2-driven activities in HUVECs, its action appears to be VEGF-selective. On the other hand, 5-FU inhibited DNA synthesis and migration of HUVECs stimulated by both VEGF and FGF-2, and tube formation driven by VEGF, suggesting that 5-FU is cytotoxic to endothelial cells. The inhibitory effects of 5-FU, and especially those GHB, were reproduced under in vivo condition using the DAS assay. The VEGF-mediated angiogenesis was significantly inhibited by UFT, 5-FU, and especially by GHB. We propose that the selective inhibitory effects of GHB on VEGF-mediated responses of endothelial cells are involved in the anti-angiogenic activity of UFT.

Thymidine phosphorylase in human esophageal squamous cell carcinoma

Experimental evidence has shown that thymidine phosphorylase (dThdPase) is identical to platelet‐derived endothelial cell growth factor (PD‐ECGF) and has angiogenic activity. The enzymatic activity of dThdPase was needed for the angiogenesis by the enzyme. These observations were catalysts for the current study.

Targeted Deletion of Both Thymidine Phosphorylase and Uridine Phosphorylase and Consequent Disorders in Mice

ABSTRACT Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP−/− UP−/−) mice. TP activities were inhibited in TP−/− UP−/− mice, and the level of thymidine in the plasma of TP−/− UP−/− mice was higher than for TP−/− mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP−/− UP−/− mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T2 maps in the brain and axonal edema by electron microscopic study of the brain in TP−/− UP−/− mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.

Expression of thymidine phosphorylase and vascular endothelial cell growth factor in human head and neck squamous cell carcinoma and their different characteristics.

Thymidine phosphorylase (dThdPase) is identical to platelet‐derived endothelial cell growth factor (PD‐ECGF). dThdPase is known to promote the development of new blood vessels, which are fundamental to tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is a 34–42 kilodalton (kD) protein that induces both angiogenesis and vascular permeability. Proliferating cell nuclear antigen (PCNA) is a 36 kD nuclear protein, and its expression is associated with DNA synthesis and cell proliferation.

The correlation of thymidine phosphorylase activity with the expression of interleukin 1α, interferon α and interferon γ in human colorectal carcinoma

Abstract Thymidine phosphorylase (dThdPase) is an angiogenic enzyme and seems to be related to an angiogenesis in human colorectal carcinoma. The incidence of dThdPase-positive cells was significantly correlated with microvessel count in 21 human colorectal carcinomas. Interleukin 1α (IL-1α), tumor necrosis factor α (TNF-α), interferon α (IFN-α) interferon γ (IFN-γ) induce dThdPase activity in human cancer cell lines. To study whether this phenomenon occurs in the human colorectal carcinomas, we examined the correlation between dThdPase activity and the expression levels of IL-1α, TNF-α, IFN-α and IFN-γ in colorectal carcinoma tissues. dThdPase activity was assayed by the methods of Friedkin and Robert, and the expression level of IL-1α, TNF-α, IFN-α and IFN-γ was determined by ELISA. dThdPase activity was significantly correlated with the amount of IL-1α (n = 19, r = 0.347, P = 0.0001), INF-α (n = 18, r = 0.717, P = 0.0008), and IFN-γ (n = 4, r = 0.9777, P = 0.0234) in human colorectal carcinomas. However, the dThdPase activity was not correlated with the amount of TNF-α (n = 21, r = 0.235, P = 0.2682). These results suggested that the expression levels of IL-1α, IFN-α and IFN-γ are correlated with dThdPase activity in human colorectal carcinomas and that these cytokines may cause angiogenesis by inducing the expression of dThdPase.

Substrate Phage as a Tool to Identify Novel Substrate Sequences of Proteases

Combinatorial phage peptide libraries have been used to identify the ligands for specific target molecules. These libraries are also useful for identification of the specific substrates of various proteases. A substrate phage library has a random peptide sequence at the N-terminus of the phage coat protein and an additional tag sequence that enables attachment of the phage to an immobile phase. When these libraries are incubated with a specific enzyme, such as a protease, the uncleaved phage is excluded from the solution with tag-binding macromolecules. This provides a novel approach to define substrate specificity. The aim of this review is to summarize recent progress on the application of the substrate phage technique to identify specific substrates of proteolytic enzymes. As an example, some of our own experimental data on the selection and characterization of substrate sequences for thrombin, a serine protease, and membrane type-1 matrix metalloproteinase (MT1-MMP) will be presented. Using this approach, the canonical consensus substrate sequence for thrombin was deduced from the selected clones. As expected from the collagenolytic activity of MT1-MMP, a collagen-like sequence was identified in the case of MT1-MMP. A more selective substrate sequence for MT1-MMP was identified during a substrate phage screen. The delineation of the substrate specificity of proteases will help to elucidate the enzymatic properties and the physiological roles of these enzymes. Comprehensive screening of very large numbers of potential substrate sequences is possible with substrate phage libraries. Thus, this approach allows novel substrate sequences and previously unknown target molecules to be defined.

The expression of thymidine phosphorylase/platelet‐derived endothelial cell growth factor is correlated to angiogenesis in breast cancer

It has been shown that human thymidine phosphorylase (TP) Is Identical to platelet‐derlved endothelial cell growth factor and has angiogenlc actlvhy. In the present study, the expression of TP was examined In 139 mammary carclnomas and 35 benign mammary disorders using biochemical and lmmunohlstochemlcal methods. Moreover, In order to evaluate the significance of TP expression in mammary carcinomas, the relationship between vascular density and various cllnicopathological factors, including age and menopausal status of patients with a mammary carcinoma, were compared wtth the size, nodal status, expression of estrogen receptor (ER), progesterone receptor (PgR), c‐erbB‐2, p53 and TP of a mammary carcinoma. Thymidine phosphorylase expression Increased in both the nuclei and cytoplasm of mammary carclnoma cells in comparison to mammary benign disorder cells. The number of mlcroves‐sels In mammary carcinomas was generally correlated to the number of tumor cells with TP expression in cytoplasm. The number of cells with TP expression in cytoplasm was significantly large In tumors that measured 34 cm In diameter, compared wtth tumors measuring 1–2 and 5–6 cm in diameter. In mammary tumors of 1–4 cm diameter, TP expression and vessel denslty were slgnlficantly high in tumors negative for ER or positive for cerbB2 and In tumors positive for TP or cerbB2, respectively; whereas tumors of 5–6 cm In diameter were not modified by any cllnlcopathological factors. The results lndlcated that TP plays an Important anglogenetic role In mammary carcinomas, especially tumors with a certain progression.

The novel VEGF receptor/MET-targeted kinase inhibitor TAS-115 has marked in vivo antitumor properties and a favorable tolerability profile.

VEGF receptor (VEGFR) signaling plays a key role in tumor angiogenesis. Although some VEGFR signal-targeted drugs have been approved for clinical use, their utility is limited by associated toxicities or resistance to such therapy. To overcome these limitations, we developed TAS-115, a novel VEGFR and hepatocyte growth factor receptor (MET)-targeted kinase inhibitor with an improved safety profile. TAS-115 inhibited the kinase activity of both VEGFR2 and MET and their signal-dependent cell growth as strongly as other known VEGFR or MET inhibitors. On the other hand, kinase selectivity of TAS-115 was more specific than that of sunitinib and TAS-115 produced relatively weak inhibition of growth (GI50 > 10 μmol/L) in VEGFR signal- or MET signal-independent cells. Furthermore, TAS-115 induced less damage in various normal cells than did other VEGFR inhibitors. These data suggest that TAS-115 is extremely selective and specific, at least in vitro. In in vivo studies, TAS-115 completely suppressed the progression of MET-inactivated tumor by blocking angiogenesis without toxicity when given every day for 6 weeks, even at a serum-saturating dose of TAS-115. The marked selectivity of TAS-115 for kinases and targeted cells was associated with improved tolerability and contributed to the ability to sustain treatment without dose reduction or a washout period. Furthermore, TAS-115 induced marked tumor shrinkage and prolonged survival in MET-amplified human cancer–bearing mice. These data suggest that TAS-115 is a unique VEGFR/MET-targeted inhibitor with improved antitumor efficacy and decreased toxicity. Mol Cancer Ther; 12(12); 2685–96. ©2013 AACR.