Yosuke Tsurudome

Lymphatic Microvessel Density is an Independent Prognostic Factor in Colorectal Cancer

PurposeAlthough lymph node metastasis via lymphatic vessels often is related with an adverse outcome, it is not well known whether lymphatic spread to lymph node needs the development of the new lymphatic formation. In addition, the correlation between lymphangiogenesis and prognosis has not been well documented. This study was designed to assess the prognostic value of lymphangiogenesis and lymphatic vessel invasion in colorectal cancer.MethodsWe examined 106 colorectal cancer specimens by immunostaining for podoplanin, lymphatic endothelial specific marker. We evaluated lymphangiogenesis, as measured by lymphatic microvessel density, and lymphatic vessel invasion. We next investigated the association of these two parameters with the clinicopathologic findings and prognosis.ResultsA significant correlation was observed between high lymphatic microvessel density and positive lymphatic vessel invasion (P = 0.0003). Positive lymphatic vessel invasion was significantly associated with the presence of lymph node metastasis (P = 0.0071). The survival curves demonstrated that both high lymphatic microvessel density and positive lymphatic vessel invasion were correlated with an adverse outcome (P = 0.0004 and P = 0.009, respectively). In a univariate analysis, high lymphatic microvessel density and positive lymphatic vessel invasion were negatively associated with the overall survival (P = 0.0011 and P = 0.0118, respectively). Furthermore, high lymphatic microvessel density, but not lymphatic vessel invasion, correlated with a poor outcome in a multivariate analysis (P = 0.0114).ConclusionsOur data suggested that lymphatic vessel invasion was related with lymph node metastasis and that both lymphatic microvessel density and lymphatic vessel invasion were related with an adverse outcome in colorectal cancer. Furthermore, lymphatic microvessel density may be a useful prognostic factor in colorectal cancer.

Increased 8-Hydroxyguanine in DNA and Its Repair Activity in Hamster and Rat Lung after Intratracheal Instillation of Crocidolite Asbestos

Asbestos and man‐made‐mineral fibers are known to increase one type of oxidative DNA damage, 8‐hydroxyguanine (8‐OH‐Gua), in vitro. In this study, we analyzed the 8‐OH‐Gua level in DNA and its repair activity after a single intratracheal instillation of fibers (crocidolite or glass) or saline to Syrian hamsters or Wistar rats. The 8‐OH‐Gua level was measured with a high‐performance liquid chromatography‐electrochemical detector (HPLC‐ECD) system. The 8‐OH‐Gua repair enzyme activity was determined with an endonuclease nicking assay using a 32P‐labeled or fluorescently labeled 22mer DNA that contains 8‐OH‐Gua at a specific position. A significant increase in the 8‐OH‐Gua level in the lung DNA was observed 1 day after the exposure to crocidolite, as compared to the saline control. The repair activity was increased significantly at 7 days. On the other hand, after exposure to glass fibers, little or no increase of these carcinogenicity indicators was detected. These assays of 8‐OH‐Gua and its repair activity in short‐term animal experiments will be useful for evaluating the carcinogenicity of fibers. This is the first report of the increase of 8‐OH‐Gua and its repair activity in the animal lung after the instillation of asbestos fibers.

Analyses of Oxidative DNA Damage and Its Repair Activity in the Livers of 3′-Methyl-4-dimethylaminoazobenzene-treated Rodents

We measured the levels of 8‐hydroxyguanine (8‐OH‐Gua) and its repair activity in the livers of the Donryu rat, the carcinogen‐resistant DRH rat, and the ddy mouse, which were fed a 0.06% 3′‐ methyl‐4‐dimethylaminoazobenzene (3′‐MeDAB)‐containing diet. In a short‐term rat experiment (maximum 2 months), 3′‐MeDAB did not increase the 8‐OH‐Gua levels in the livers of the two rat strains, although it significantly increased the repair activity in only the Donryu rat liver at 1 and 2 months. After long‐term 3′‐MeDAB administration to the ddy mouse (8 months), the levels of 8‐OH‐Gua and its repair activity were increased in the liver by 3.6‐fold and 1.6‐fold, respectively. These experiments suggest that 3′‐MeDAB increases 8‐OH‐Gua generation in rodent liver DNA and the 8‐OH‐Gua repair assay is a reliable marker of cellular oxidative stress induced by carcinogens.

4-[3,5-Bis(trimethylsilyl)benzamido] benzoic acid inhibits angiogenesis in colon cancer through reduced expression of vascular endothelial growth factor.

4-[3,5-bis(trimethylsilyl)benzamido] Benzoic acid (TAC-101) has potent antiproliferative, antiangiogenic, and antitumor effects in vitro and in vivo. These effects might be due to TAC-101 binding to retinoic acid receptor alpha (RAR-alpha) and interfering with the binding of activator protein-1 (AP-1) to DNA. However, little is known about the detailed mechanism of TAC-101 function. We investigated the mechanism of the antiangiogenic effect of TAC-101 using a rat hepatic metastatic model in vivo and DLD-1 human colon cancer cells in vitro. Liver metastases were induced by portal injection of RCN-9 rat colonic cancer cells into F344 rats. TAC-101 (8 mg/kg) was orally administered 5 days per week for 4 weeks and then hepatic tumors were immunohistochemically evaluated for microvessel density (MVD) and vascular endothelial growth factor (VEGF). TAC-101 significantly reduced both MVD and VEGF expression. Northern blot analysis and ELISA indicated that TAC-101 efficiently inhibited production of VEGF mRNA and protein in DLD-1 cells in a time- and dose-dependent manner. These findings suggest that TAC-101 may inhibit progression and metastasis in colon cancer by interfering with tumor production of VEGF.

Increase in 8-hydroxyguanine and its repair activity in the esophagi of rats given long-term ethanol and nutrition-deficient diet

Epidemiological studies have shown that an increased risk of esophageal cancer is associated with the chronic consumption of alcoholic beverages, although alcohol itself is not a carcinogen in animal models. Reactive oxygen species produced by the metabolism of ethanol or by chronic inflammation may play an important role in the carcinogenic process. In this study, we analyzed one type of oxidative DNA damage, 8‐hydroxyguanine (8‐OH‐Gua), and its repair activity in the esophagus as indicators of cellular oxidative stress in rats given long‐term ethanol and an autoclaved diet (nutrition‐deficient diet). Three‐week‐old male Sprague‐Dawley rats were fed an ethanol beverage whose concentration was increased from 12 to 70% over 20 weeks. When the concentration reached 50%, the diet of one group was changed from the regular diet to an autoclaved diet. At the feeding periods of 20, 25, 30, and 35 weeks, the rats were sacrificed and the 8‐OH‐Gua levels and repair activities within the esophagi were measured. After 30 weeks of ethanol‐ and autoclaved diet‐feeding, significant increases of 8‐OH‐Gua and its repair activity were observed in the esophagi, but not in those of the ethanol‐ and normal diet‐fed rats. This result indicates that the combined effects of long‐term ethanol consumption and nutritional deficiency may be involved in inducing oxidative stress in the rat esophagus.

Detection of a smaller, 32-kDa 8-oxoguanine DNA glycosylase 1 in 3'-methyl-4-dimethylamino- azobenzene-treated mouse liver

We previously reported that 3′‐methyl‐4‐dimethylaminoazobenzene (3′‐MeDAB) increased the 8‐hydroxyguanine (8‐OH‐Gua) content in nuclear DNA and the base excision repair activity in mouse liver. However, to understand the mechanism of 3′‐MeDAB carcinogenesis, a further investigation of the 8‐OH‐Gua repair systems was necessary. In this report, we examined the expression of the repair enzyme, 8‐oxoguanine DNA glycosylase 1 (OGG1), in 3′‐MeDAB‐treated mouse liver. We prepared four kinds of anti‐peptide polyclonal antibodies raised against mouse OGG1 (mOGG1). The sequences used as epitopes were designed from positions located close to the N‐terminus, the nuclear localization signal (NLS), and the regions containing Lys249 and Asp267, which are involved in the catalytic mechanisms of mOGG1 (glycosylase and lyase, respectively). Immunoblotting, using all four antibodies, revealed a 32‐kDa protein (mOGG1‐32) in addition to the 38‐kDa mOGG1 in the 3′‐MeDAB‐treated mouse liver. Moreover, immunostaining with mOGG1 antibody yielded strong, positive signals in the 3′‐MeDAB‐treated mouse liver nuclei. However, we could not detect any difference in the Ogg1 mRNA expression pattern. Although the function of mOGG1‐32 remains unclear, these findings suggest that 3′‐MeDAB may alter the function of the DNA repair protein, and this action may be related to 3′‐MeDAB carcinogenesis.

2-Hydroxyadenine, a mutagenic form of oxidative DNA damage, is not repaired by a glycosylase type mechanism in rat organs

Oxygen radicals are known to play a role in causing cellular DNA damage, which is involved in carcinogenesis. 8-Hydroxyguanine (8-OH-Gua) is a major form of oxidative DNA damage and is known as a useful marker of DNA oxidation. Recently, we found another type of oxidative DNA damage, 2-hydroxyadenine (2-OH-Ade), which has a mutation frequency comparable to that of 8-OH-Gua. We compared the repair activities for two types of oxidative DNA damage, 8-OH-Gua and 2-OH-Ade, in 7-week-old male Sprague-Dawley (SD) rat organs. The repair activities were measured by an endonuclease nicking assay using 22 mer [32P]-end-labeled double-stranded DNA substrates, which contained either 8-OH-Gua (opposite C) or 2-OH-Ade (opposite T or C). In all of the SD rat organs we studied, the nicking activity for 2-OH-Ade was not detected, while that for 8-OH-Gua was clearly detected with the same conditions. Moreover, the 2-OH-Ade nicking activity was not induced in Wistar rat kidney extracts prepared after ferric nitrilotriacetate (Fe-NTA) treatment, which is known to increase 8-OH-Gua repair activity. These results suggest that 2-OH-Ade might not be repaired by the glycosylase type mechanism in mammalian cells.

Analysis of 8-Hydroxy-2′-Deoxyguanosine as a Marker of Oxidatively Damaged DNA in Relation to Carcinogenesis and Aging

Reactive oxygen species (ROS) are well known hazards for living organisms and are believed to be associated with the induction of cancer. ROS induce many forms of oxidative damage to proteins, nucleic acids, and lipids. Therefore, to diagnose or prevent cancer, analyses of oxidative products in patient samples, including tissue, blood, and urine, are very informative. Amongst the products of DNA oxidation, 8-hydroxy-2′-deoxyguanosine (8-OH-dG) is an important form of damage that leads to point mutations in genomic DNA. Since 8-OH-dG is the most abundant form of oxidative DNA damage and is easy to detect in laboratories, using a high performance liquid chromatography (HPLC) system equipped with an electrochemical detector (ECD), many researchers studying cellular oxidative stress have primarily analyzed 8-OH-dG. In this chapter, we will describe our findings regarding 8-OH-dG generation and its repair, as well as our recent urinary 8-OH-dG data.

[A case of low grade ductal carcinoma in situ of the breast with difficulties in making the preoperative diagnosis after detection by FDG-PET].

A 56-year-old woman underwent FDG-PET screening, which demonstrated delayed-phase uptake in the lower part of the left breast. The findings of mammography, ultrasonography, MRI and cytological examination were compatible with ductal carcinoma in situ (DCIS), but core needle biopsy showed no evidence of malignancy. Therefore, partial resection of the left breast with sentinel lymph node biopsy was performed to make a definite diagnosis. Histological examination showed that this tumor was low grade DCIS. FDG-PET is a very useful examination to detect malignant diseases, but it is quite difficult to distinguish them from benign ones. It is suggested that delayed-phase uptake of FDG-PET is useful for diagnosis of DCIS.