Koichiro Nomata

Habitual Intake of Lactic Acid Bacteria and Risk Reduction of Bladder Cancer

Introduction: A kind of lactic acid bacteria, Lactobacillus casei strain Shirota, shows antitumor activity in experimental animals. One clinical trial using L. casei showed a significant decrease in the recurrence of superficial bladder cancer. So, to assess the preventive effect of the intake of L. casei, widely taken as fermented milk products in Japan, against bladder cancer, we conducted a case-control study. Methods: A total of 180 cases (mean age: 67 years, SD 10) were selected from 7 hospitals, and 445 population-based controls matched by gender and age were also selected. Interviewers asked them 81 items. The conditional logistic regression was used to estimate adjusted odds ratios (OR). Results: The OR of smoking was 1.61 (95% confidence interval: 1.10–2.36). Those of previous (10–15 years ago) intake of fermented milk products were 0.46 (0.27–0.79) for 1–2 times/week and 0.61 (0.38–0.99) for 3–4 or more times/week, respectively. Conclusion: It was strongly suggested that the habitual intake of lactic acid bacteria reduces the risk of bladder cancer.

Lymphangiogenesis and Angiogenesis in Bladder Cancer: Prognostic Implications and Regulation by Vascular Endothelial Growth Factors-A, -C, and -D

Purpose: Lymph vessel density (LVD) and microvessel density (MVD) correlate with the malignant potential of tumors and patient survival. Vascular endothelial growth factors (VEGF)-A, VEGF-C, and VEGF-D could modulate LVD and MVD. We investigated the clinical and prognostic significance of LVD and MVD on lymphangiogenic and angiogenic function of VEGF-A, VEGF-C, and VEGF-D in human bladder cancer. Experimental Design: We reviewed tissue samples from patients with nonmetastatic bladder cancer who had undergone transurethral resections (n = 126). The densities of D2-40-positive vessels (LVD) and CD34-positive vessels (MVD) were measured by a computer-aided image analysis system. Expression of VEGF-A, VEGF-C, and VEGF-D was examined by immunohistochemistry; survival analyses and their independent roles were investigated using multivariate analysis models. Results: LVD was associated with tumor grade but not with pT stage. LVD was associated with metastasis-free survival (log rank P = 0.039), but was not an independent prognostic factor. Although MVD affected survival, the combination of high LVD and high MVD in tumors was an independent predictor of metastasis-free survival. Although VEGF-C expression was positively associated with both LVD and MVD, VEGF-D was associated only with LVD. VEGF-A expression was associated with MVD in univariate analysis, however, it was not an independent factor. Conclusions: Lymphangiogenesis and angiogenesis influence metastasis-free survival, and are regulated by VEGF-C and/or VEGF-D. Our results suggest that LVD and MVD are useful tools for the selection of postoperative management and treatment strategies in patients with bladder cancer.

Gene expression and immunohistochemical localization of basic fibroblast growth factor in renal cell carcinoma

Renal cell carcinoma is known as a neoplastic condition of renal tubular cells and usually shows a hypervascular tumor in angiographic examination. We examined the presence of basic fibroblast growth factor (bFGF) in human renal cell carcinoma. To determine if alterations in bFGF gene expression are present in human renal cell carcinoma, paired samples of normal and neoplastic renal tissue from 6 patients were analyzed for bFGF mRNA content by Northern blot hybridization. In 4 out of 6 patients, tumor tissue expressed bFGF mRNA 2 to 4 times greater than corresponding normal tissue. Two patients showed minimal elevation of tumor bFGF mRNA. The localization of bFGF in the renal cell carcinoma tissue was also examined using immunohistochemical staining, and it was found that bFGF was positively stained at the nuclei of tumor cells and the cell surface. These results suggest that increased expression of bFGF may be associated with neoplastic growth in renal tubular epithelial cells and neovascularization.

Tumor Lymphangiogenesis in Transitional Cell Carcinoma of the Upper Urinary Tract: Association With Clinicopathological Features and Prognosis

PURPOSEnLymph node metastasis is an important prognostic factor in many types of cancer. Recently several specific markers for lymphatic endothelium were developed that facilitate the quantification of lymphangiogenesis in human cancer tissues. We investigated the clinical and prognostic significance of lymphangiogenesis in patients with transitional cell carcinoma of the upper urinary tract.nnnMATERIALS AND METHODSnWe measured lymph vessel density and relative lymphatic vascular area in 125 specimens by quantitative immunohistochemical staining for D2-40 antibody (DakoCytomation, Glostrup, Denmark). These parameters were examined in the intratumor and peritumor areas, and measured using image analysis software.nnnRESULTSnPeritumor lymph vessel density and peritumor lymphatic vascular area correlated with lymph node metastasis and tumor grade. In the intratumor area lymphatic vessels were detected in only 16.0% of specimens. However, the presence of intratumor lymphatic vessels was associated with lymph node metastasis (p = 0.002). Multivariate analysis identified high peritumor lymphatic vascular area and the presence of intratumor lymphatic vessels as significant and independent factors of metastasis-free survival after surgery (OR = 5.11, p = 0.020 and OR = 2.92, p = 0.025, respectively). Multivariate analysis also identified the presence of intratumor lymphatic vessels as the only independent predictive factor of cause specific survival (OR = 3.89, p = 0.049).nnnCONCLUSIONSnLymphangiogenesis may have important roles in tumor metastasis and survival in patients with transitional cell carcinoma of the upper urinary tract. Quantification of lymphatic vessels, especially peritumor lymphatic vascular area and intratumor lymphatic vessels, was useful for predicting metastasis-free survival. In addition, the presence of intratumor lymphatic vessels was an independent predictor of cause specific survival.

Growth factor regulation of the renal cortical tubular cells by epidermal growth factor, insulin-like growth factor-I, acidic and basic fibroblast growth factor, and transforming growth factor-β in serum free culture

Abstract of cultured rabbit renal cortical cells containing more than 50% of proximal tubular cells have been investigated. It was consequently found that epidermal growth factor exhibited the most potent growth promoting activity. In addition, insulin-like growth factor-I and acidic and basic fibroblast growth factor also stimulated the growth of cultured tubular cells, though no effect was observed in platelet derived growth factor. In contrast, transforming growth factor-β was found to be the only growth inhibitor of the tubular cells in the presence of insulin or fibroblast growth factor.

Green tea polyphenol suppresses tumor invasion and angiogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-induced bladder cancer

BACKGROUNDnGreen tea polyphenol (GTP) suppresses malignancy in bladder cancer cell lines. However, the detail of its anti-carcinogenic effect in vivo is not fully understood. This study investigated the effect of GTP on bladder tumor size and angiogenesis in mice given N-butyl-(-4-hydroxybutyl) nitrosamine (BBN), with and without GTP.nnnMETHODSnEight-week-old female C3H/He mice were treated with and without 0.05% BBN solution for 14 or 24 weeks. In addition, they were also treated with and without 0.5% GTP solution for the same periods. Histopathological diagnosis was established using hematoxylin and eosin staining, and microvessel density (MVD) was estimated by counting CD34- and von Willebrand factor-positive vessels in the tumor area.nnnRESULTSnAt 14 weeks, cancer cells were detected in BBN and BBN+GTP mice [5/14 (35.7%) and 3/14 (21.4%), respectively, p=0.678]. At 24 weeks, the incidence of cancer cells was also similar between the groups (BBN+GTP: 61.9% vs. BBN: 82.6%; p=0.179). However, the frequency of invasive tumors in BBN+GTP mice was significantly lower (23.8%; p=0.030) than in those given BBN alone (65.2%). Tumor volume and MVD of intratumoral and stromal region in the BBN+GTP group were also significantly lower than in BBN mice.nnnCONCLUSIONnThe results showed that GTP had no anti-carcinogenic effect, but inhibited tumor growth and invasion in mice with established bladder cancer, at least in part through the regulation of angiogenesis. Our data suggest that GTP seems to suppress tumor development in bladder cancer.

Calreticulin Represses E-cadherin Gene Expression in Madin-Darby Canine Kidney Cells via Slug

Calreticulin (CRT) is a multifunctional Ca2+-binding molecular chaperone in the endoplasmic reticulum. In mammals, the expression level of CRT differs markedly in a variety of organs and tissues, suggesting that CRT plays a specific role in each cell type. In the present study, we focused on CRT functions in the kidney, where overall expression of CRT is quite low, and established CRT-overexpressing kidney epithelial cell-derived Madin-Darby canine kidney cells by gene transfection. We demonstrated that, in CRT-overexpressing cells, the morphology was apparently changed, and the original polarized epithelial cell phenotype was destroyed. Furthermore, CRT-overexpressing cells showed enhanced migration through Matrigel®-coated Boyden chamber wells, compared with controls. E-cadherin expression was significantly suppressed at the protein and transcriptional levels in CRT-overexpressing cells compared with controls. On the other hand, the expression of mesenchymal protein markers, such as N-cadherin and fibronectin, was up-regulated. We also found that the expression of Slug, a repressor of the E-cadherin promoter, was up-regulated by overexpression of CRT through altered Ca2+ homeostasis, and this led to enhanced binding of Slug to the E-box element in the E-cadherin promoter. Thus, we conclude that CRT regulates the epithelial-mesenchymal transition-like change of cellular phenotype by modulating the Slug/E-cadherin pathway through altered Ca2+ homeostasis in cells, suggesting a novel function of CRT in cell-cell interaction of epithelial cells.

Fas‐Fas ligand system as a possible mediator of spermatogenic cell apoptosis in human maturation‐arrested testes

To elucidate the mechanism of maturation arrest, known as one of the male infertility, we addressed whether germ cell apoptosis occurs during maturation arrest, and if so, whether Fas and Fas ligand expressions are involved in the apoptosis. By electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), typical apoptotic features were frequently found around the spermatocytic stage in maturation arrest, compared to that in normal testes. When paraffin-embedded sections reacted with anti-Fas antiserum, staining for Fas was found in the plasma membranes of spermatocytes in the maturation-arrested testes, while no positive spermatogenic cells were seen in the normal testes. On the other hand, positive immunostaining for Fas ligand was restricted to Sertoli cells in the maturation-arrested testes as well as in the normal testes, although the intensity of staining for Fas ligand in normal testicular Sertoli cells was much weaker than that of maturation-arrested ones. Thus, these findings demonstrate that “maturation arrest” is characterized by frequent apoptosis of spermatocytes, and that Fas and Fas ligand staining are associated with a high frequency of apoptosis.

All-trans retinoic acid enhances gap junctional intercellular communication among renal epithelial cells in vitro treated with renal carcinogens.

Epidemiological and clinical studies imply that retinoids have a chemopreventative action against cancer and can suppress the growth of cancer cells. The regulation of connexin (Cx) expression by retinoids varies among tissues and organs. In this study, we investigated whether all-trans retinoic acid (ATRA) upregulates gap junctional intercellular communication (GJIC) in renal epithelial cells exposed to renal carcinogens. Madin Darby canine kidney (MDCK) cells were incubated with ATRA for 3 days, then briefly exposed to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or renal carcinogens potassium bromate (KBrO3) and dimethylnitrosamine (DMN). ATRA increased the expression of connexin 43 mRNA and protein without affecting Cx 43 phosphorylation and prevented inadequate Cx 43 localisation caused by TPA/KBrO3 or DMN. Consequently, ATRA prevented the disruption of GJIC in MDCK cells. These data suggest that ATRA enhanced GJIC by upregulating Cx 43 expression and that ATRA might be useful for prevention of renal cell carcinoma.

Anti-epidermal growth factor antibody inhibits compensatory renal hyperplasia but not hypertrophy after unilateral nephrectomy in mice

The effect of anti-epidermal growth factor (EGF) antibody on the compensatory renal growth including hyperplasia and hypertrophy was investigated. The antibody used in this study blocked the stimulatory effect of EGF on the DNA synthesis of cultured rat hepatocytes. When this antibody was injected into mice intravenously after unilateral nephrectomy, the labeling index of the renal cortical tubular cells decreased significantly at the second day after uninephrectomy, but the antibody caused no decrease in remaining kidney weight. Immunohistochemical study revealed that injected anti-mouse EGF rabbit IgG was positively stained at the renal cortical tubular cells. EGF would thus appear importantly essential to compensatory renal hyperplasia.