Zenya Naito

Association between Helicobacter bilis in bile and biliary tract malignancies: H. bilis in bile from Japanese and Thai patients with benign and malignant diseases in the biliary tract

Japan and Thailand have high incidences of bile duct carcinoma and gallstones. The presence of Helicobacter bilis (H. bilis) detected by polymerase chain reaction (PCR) and 16S rRNA analysis in bile samples from Chileans with chronic cholecystitis was reported. The association between H. bilis in bile and biliary tract malignancies has not been investigated, and therefore the aim of this study is to determine whether malignant diseases of the biliary tract are associated with the presence of H. bilis in bile samples obtained from two high‐risk populations. Bile samples from 45 Japanese and 40 Thai patients were subjected to PCR analysis using H. bilis‐specific primers, and six of the H. bilis amplicons were sequenced. Thirteen out of 15 (87%) Japanese and 11 out of 14 (79%) Thai patients with bile duct or gallbladder cancer tested positive for the presence of H. bilis in their bile. Eight out of 16 (50%) Japanese and 10 out of 26 (38%) Thai patients with gallstone and/or cholecystitis tested positive for H. bilis. Only 4 out of 14 (29%) subjects without biliary disease tested positive for H. bilis among the Japanese. Bile duct and gallbladder cancer showed significantly higher positive rates for H. bilis than did the non‐biliary diseases among the Japanese (P<0.01) and the odds ratios for bile duct or gallbladder cancer with H. bilis in comparison with gallstone and/or cholecystitis were 6.50 (95%CI 1.09–38.63) in the Japanese and 5.86 (1.31–26.33) in the Thai patients. In conclusion, H. bilis infection in bile was associated with biliary tract and gallbladder cancers in two high risk populations, Japanese and Thai.

Expression and roles of heat shock proteins in human breast cancer

Heat shock proteins (hsps) are thought to play important roles in the cell cycle and various processes of carcinogenesis. Therefore, we evaluated the expression of hsps, mainly hsp90 and hsp70, in human breast cancer tissues. Hsp90α mRNA was expressed at much higher levels in the cancerous tissue than in the non‐cancerous tissue. In addition, a close correlation between hsp90α mRNA expression and the proliferating‐cell‐nuclear‐antigen labeling index (PCNA LI) was observed for the cancerous tissue. These findings suggest that increased expression of the hsp90α isoform may play a role in cell proliferation. On the other hand, hsp90β mRNA expression was significantly higher in poorly differentiated carcinomas than in well differentiated carcinomas of the breast. The intracellular localization of hsp70 was consistent with that of ubiquitin. In specimens showing hsp70 in the nucleus, the PCNA LI was significantly high, Hsc73 mRNA, a member of the hsp70 family, was also expressed at higher levels in cancerous tissues associated with a high PCNA LI than in non‐cancerous tissues. These results suggest that hsp90α may play a role in cancer cell proliferation and that hsp90β may contribute to cell differentiation and structural constitution. In addition, hsp70, especially hsc73, is related to ubiquitin and seems to be a marker for cancer proliferation.

Nestin in gastrointestinal and other cancers: Effects on cells and tumor angiogenesis

Nestin is a class VI intermediate filament protein that was originally described as a neuronal stem cell marker during central nervous system (CNS) development, and is currently widely used in that capacity. Nestin is also expressed in non-neuronal immature or progenitor cells in normal tissues. Under pathological conditions, nestin is expressed in repair processes in the CNS, muscle, liver, and infarcted myocardium. Furthermore, increased nestin expression has been reported in various tumor cells, including CNS tumors, gastrointestinal stromal tumors, pancreatic cancer, prostate cancer, breast cancer, malignant melanoma, dermatofibrosarcoma protuberances, and thyroid tumors. Nestin is reported to correlate with aggressive growth, metastasis, and poor prognosis in some tumors; however, the roles of nestin in cancer cells have not been well characterized. Furthermore, nestin is more specifically expressed in proliferating small-sized tumor vessels in glioblastoma and gastric, colorectal, and prostate cancers than are other tumor vessel markers. These findings indicate that nestin may be a marker for newly synthesized tumor vessels and a therapeutic target for tumor angiogenesis. It has received a lot of attention recently as a cancer stem cell marker in various cancer cells including brain tumors, malignant rhabdoid tumors, and uterine, cervical, prostate, bladder, head and neck, ovarian, testicular, and pancreatic cancers. The purpose of this review is to clarify the roles of nestin in cancer cells and in tumor angiogenesis, and to examine the association between nestin and cancer stem cells. Nestin has the potential to serve as a molecular target for cancers with nestin-positive cancer cells and nestin-positive tumor vasculature.

Expression of hsp90 and cyclin D1 in human breast cancer

The integral roles of heat shock proteins (hsps) in the cell cycle and in multistep processes leading to tumorigenesis have been implied. We examined the expression of hsp90alpha, hsp90beta and cyclin D1 in human breast cancer. Levels of mRNAs coding for hsp90alpha and cyclin D1 were significantly higher in cancer tissues than in non-cancer tissues. Moreover, there was a close relationship between the extent of the two mRNA levels, suggesting that increased expression of hsp90alpha, an isoform of the hsp90 family, is associated with the proliferation of human breast cancer. Hsp90beta was expressed in cancer cells, but not associated with cell proliferation.

Nestin is a novel target for suppressing pancreatic cancer cell migration, invasion and metastasis.

Nestin, is a class VI intermediate filament (IF) that is expressed in 30% of pancreatic ductal adenocarcinoma (PDAC) cases, and its expression in PDAC positively correlates with peripancreatic invasion. An expression vector carrying a short hairpin RNA (shRNA) targeting nestin was stably transfected into PANC-1 and PK-45H human pancreatic cancer cells, which express high nestin levels. Alterations in morphology and alignment of actin filaments and α-tubulin were examined by phase-contrast and immunocytochemistry. Effects on cell growth, migration in scratch and Boyden chamber assays, invasion, cell adhesion, and in vivo growth were determined. Differences in mRNA levels were examined by arrays. Nestin shRNA-transfected cells exhibited decreased nestin expression, a sheet-like appearance with tight cell-cell adhesion, increased expression of filamentous F-actin and E-cadherin, and attenuated migration and invasion, both of which were enhanced following nestin re-expression. Expression of α-tubulin, and in vitro cell growth and adhesion were not altered by nestin down-regulation, whereas hepatic metastases were decreased. Thus, nestin plays important roles in pancreatic cancer cell migration, invasion and metastasis by selectively modulating the expression of actin and cell adhesion molecules, and may therefore be a novel therapeutic target in PDAC. See commentary: Pancreatic cancer with Nest-in tendencies

Helicobacter pylori infection-negative gastric cancer in Japanese hospital patients: Incidence and pathological characteristics

We used Helicobacter pylori sero‐positivity and mucosal atrophy as detected by the serum pepsinogen method to identify H. pylori infection‐negative gastric cancer patients with or without atrophy. One hundred and six of 748 (14.2%) primary gastric cancer patients were infection‐negative by a serum antibody detection system. Further, 121 (16.2%) of the 748 were negative for gastric mucosal atrophy by the pepsinogen method, of whom 15/748 (2.0%) were H. pylori‐negative by pepsinogen I level (>70 ng/mL) and pepsinogen I/II ratio (>3.0). Twenty‐seven of 782 (3.6%) gastric cancer patients were H. pylori‐negative by antibodies and severe atrophy as determined by pepsinogen I level (<30 ng/mL) and pepsinogen I/II ratio (<2.0). H. pylori‐negative gastric cancer patients with severe atrophy likely had a previous infection. These results indicate that the actual number of H. pylori‐negative patients is 2.0% at minimum and 10.6% (14.2% minus 3.6%) at maximum in the general Japanese population. Five of 15 (33%) cases displaying neither anti‐H. pylori antibodies nor atrophy were intestinal‐type and 10 (67%) were diffuse‐type adenocarcinomas. Thirteen surgical patients with primary gastric cancer displaying neither antibodies nor mucosal atrophy were further analyzed for pathological and phenotypic characteristics. The mucin phenotype was divided into four gastric, five gastric and intestinal, two intestinal and two null types, independent of histological classification. Intestinal phenotype elements were detected by Cdx2 immunohistochemical methods in nine of 13 (70%) cases examined. We conclude that a small fraction of gastric cancer patients displayed multifactorial carcinogenesis without H. pylori infection, indicating that gastric cancer risk still exists in the absence of H. pylori infection, at an incidence of 2.0% at minimum and 10.6% at maximum in the general Japanese population. (Cancer Sci 2007; 98: 790–794)

Nestin expression correlates with nerve and retroperitoneal tissue invasion in pancreatic cancer

Nestin was first described as an intermediate filament protein expressed in neuroepithelial stem cells. Nestin expression has also been reported in brain tumors, schwannomas, gastrointestinal stromal tumors, and melanomas. In the pancreas, Nestin expression has been detected in exocrine and mesenchymal cells, including stellate cells, pericytes, and endothelial cells. In the present study, we examined Nestin expression in human pancreatic ductal adenocarcinoma and sought to determine its role in this malignancy. Reverse transcription-polymerase chain reaction analysis demonstrated the presence of Nestin mRNA in all 10 tested pancreatic cancer cell lines, and quantitative reverse transcription-polymerase chain reaction revealed that Nestin mRNA levels were highest in PANC-1 cells and lowest in PK-8 cells. Immunofluorescent analysis revealed that Nestin localized in the outer cytoplasm of PANC-1 cells. Nestin immunoreactivity was present in the cancer cells in 20 (33.3%) of 60 cancer cases, and its expression was confirmed by in situ hybridization. Nestin expression was also increased in peripheral nerve fibers adjacent to cancer cells and in peripheral nerve fibers invaded by cancer cells. Clinicopathologically, there was a statistically significant association between Nestin expression in pancreatic cancer cells and nerve invasion (P = .010) and the presence of cancer cells in the tumor resection margins (P = .003). Nestin-positive cases exhibited similar survival after resection by comparison with Nestin-negative cases, irrespective of whether they were given adjuvant therapy. These findings indicate that Nestin expression in pancreatic cancer cells may contribute to nerve and stromal invasion in this malignancy.

Expression of lumican in human colorectal cancer cells

Lumican is a member of a small leucine‐rich proteoglycan family and its overexpression in human breast cancer tissues is reported to influence the growth of cancer cells. In the present study, we aimed to clarify the expression of lumican mRNA and its protein in human colorectal cancer cell lines and their localization in normal and cancerous colorectal tissues. Reverse transcription–polymerase chain reaction and western blot analysis revealed lumican mRNA and its protein expression in COLO 205, DLD‐1, HCT‐15, SW 480 and WiDr colorectal cancer cell lines. The lumican in colorectal cancer cells had non‐sulfated or poorly sulfated polylactosamine side chains. Based on its immunoreactivity, the lumican protein was found to be localized in fibroblasts and stromal tissues of normal colorectal tissues, but not in colorectal epithelial cells. In colorectal cancer tissues, the lumican was strongly localized in cancer cells in eight of 12 cancer cases. The lumican protein was also localized in epithelial cells with mild reactive dysplasia and fibroblasts adjacent to cancer cells. Lumican mRNA was expressed in cancer cells and adjacent fibroblasts, and epithelial cells. These findings may indicate that the lumican protein synthesized by cancer cells, fibroblasts and epithelial cells with mild reactive dysplasia found adjacent to cancer cells may affect the growth of human colorectal cancer cells.

Expression of Cathepsin B and Cystatin C in Human Breast Cancer

Abstract Cathepsin B, which was originally found to be a lysosomal cysteine protease, is also an important matrix protease. In this study, we investigated the expression of cathepsin B and cystatin C, the strongest inhibitor of cathepsin B, and measured the relative amounts of each in human breast cancer tissues. Cystatin C expression relative to cathepsin B expression was found to be decreased. This finding could be associated with the looseness of cancerous interstitial tissue, which might play a role in cancer invasion and metastasis. This report documents the first simultaneous investigation of cathepsin B and cystatin C in breast cancer tissues.

Neuroepithelial stem cell marker nestin regulates the migration, invasion and growth of human gliomas

Nestin, a class VI intermediate filament protein, was originally described as a neuronal stem cell marker during central nervous system development. Nestin is expressed in gliomas, and its expression levels are higher in gliomas with high WHO histopathological classification grades than in those with low grades. In the present study, we examined whether nestin regulates the biological activities of human glioma cells. Immunohistochemically, the nestin expression patterns in 10 human glioblastoma patients were examined. The expression levels of nestin in A172, a human high-grade glioma cell line, and KG-1-C, a human low-grade glioma cell line, were examined using real-time PCR, Western blot and immunofluorescence analyses. An expression vector carrying a short hairpin RNA targeting nestin was stably transfected into A172 (Sh) cells. The effects of decreased expression levels of nestin in Sh cells on cell growth, migration, invasion, adhesion to extracellular matrices and fibrillar actin expression on three-dimensional culture plates were examined. The nestin expression vector was transiently transfected into KG-1-C (Nes) cells, and the effects of the nestin overexpression on cell growth and migration were examined. Nestin was expressed in the cytoplasm of the glioblastoma cells in all cases examined. Sh cells showed marked decreases in the expression levels of nestin mRNA and protein, and the growth rate of Sh cells was lower than that of sham (Sc) cells. In contrast, the adhesion activity of Sh cells to types I and IV collagens, fibronectin and laminin was higher than that of Sc cells. Fibrillar actin was clearly detected at the periphery of colonies of Sh cells at the attachment sites on three-dimensional culture plates. The migration and invasion of Sh cells were markedly inhibited compared with those of Sc cells. In contrast, the levels of nestin expression markedly increased in the Nes cells, which were transiently transfected with the nestin expression vector. The growth rate and motility of Nes cells were higher than those of the mock cells. In conclusion, nestin plays important roles in cell growth, migration, invasion and adhesion to extra-cellular matrices in glioma cells. Nestin may serve as a novel candidate for molecular-targeted therapy for gliomas, including glioblastomas.