Hirokazu Fukui

Candidate markers for stem and early progenitor cells, Musashi‐1 and Hes1, are expressed in crypt base columnar cells of mouse small intestine

Musashi‐1, a neural RNA‐binding protein, is important for maintaining neural stem cells. Both Musashi‐1 and Hes1, a transcriptional factor regulated by Musashi‐1, are expressed in the small intestine. Here we show that Musashi‐1 is present in a few epithelial cells just above the Paneth cells in the small intestinal crypt, the putative position of stem cells, whereas Hes1 is expressed in lower crypt cells just above the Paneth cells, including Musashi‐1‐positive cells. Musashi‐1 and Hes1 were not expressed in Paneth cells. Notably, Musashi‐1 and Hes1 were coexpressed in the crypt base columnar cells located between the Paneth cells. These findings suggest that not only the cells just above Paneth cells but also the crypt base columnar cells between the Paneth cells have stem cell characteristics.

STAT3 is constitutively activated and supports cell survival in association with survivin expression in gastric cancer cells

Signal transduction and activator of transcription 3(STAT3) signaling is constitutively activated in various tumors, and is involved in cell survival and proliferation during oncogenesis. There are few reports, however, on the role of STAT3 signaling in gastric cancer. The aim of the present study was to clarify the role of STAT3 signaling in apoptosis and cellular proliferation in gastric cancer. Here we reported that STAT3 was constitutively activated in various human gastric cancer cells and its inhibition by ectopic dominant-negative STAT3 or Janus kinase inhibitor, tyrphostin AG490, induced apoptosis. Furthermore, STAT3 inhibition markedly decreased survivin expression, and forced expression of survivin rescued AGS cells from apoptosis induced by STAT3 inhibition. Although some reports demonstrated that the PI3K/Akt pathway regulates survivin expression, inhibition of the PI3K/Akt pathway did not affect survivin expression in AGS and MKN1 cells. Finally, activated form of STAT3, Tyr-705 phospho-stat3, was found in the nucleus of cancer cells in 11 of 40 (27.5%) human gastric cancer specimens. These findings suggest that constitutively activated STAT3 signaling supports gastric cancer cell survival in association with survivin expression.

Regenerating gene protein may mediate gastric mucosal proliferation induced by hypergastrinemia in rats.

BACKGROUND & AIMS Regenerating gene (Reg) has been isolated from rat regenerating pancreatic islets, and Reg protein is mitogenic to islet cells. We have recently shown that Reg gene and Reg protein are expressed in gastric enterochromaffin-like (ECL) cells. This study aimed to clarify whether gastrin enhances Reg protein production in ECL cells and whether Reg protein is mitogenic to gastric mucosal cells. METHODS Reg gene expression in response to acute and chronic hypergastrinemia was investigated in rats. Immunohistochemical studies, Northern blotting, and in situ hybridization were performed to investigate the expression of Reg protein and Reg gene. The direct effect of gastrin on Reg gene expression was investigated using isolated ECL cells, and the trophic effect of Reg protein on cultured gastric epithelial cells was assessed by [3H]thymidine uptake. RESULTS Both chronic hypergastrinemia and short-term gastrin administration stimulated Reg gene expression and Reg protein production in fundic mucosa. Reg gene expression was also augmented in isolated ECL cells after incubation with rat gastrin. Reg protein was mitogenic to cultured rat gastric epithelial cells. CONCLUSIONS Gastrin stimulates the production of Reg protein in gastric ECL cells, which may be involved in the gastrin-induced gastric mucosal cell growth.

Growth arrest‐specific gene 6 and Axl signaling enhances gastric cancer cell survival via Akt pathway

Activation of tyrosine kinases is an important factor during cancer development. Axl, one of the receptor tyrosine kinases, binds to the specific ligand growth arrest‐specific gene 6 (Gas6), which encodes a vitamin K‐dependent γ‐carboxyglutamyl protein. Although many receptor tyrosine kinases and their ligands are involved in gastric carcinogenesis, whether Gas6‐Axl signaling is involved in gastric carcinogenesis has not been elucidated. The aim of this study was to investigate the expression of Gas6 and Axl in gastric cancer and also their roles during gastric carcinogenesis. mRNA and protein of Gas6 and Axl were highly expressed in a substantial proportion of human gastric cancer tissue and cell lines, and Gas6 expression was significantly associated with lymph node metastasis. With recombinant Gas6 and a decoy‐receptor of Axl in vitro, we demonstrated that Gas6‐Axl signaling pathway enhanced cellular survival and invasion and suppressed apoptosis via Akt pathway. Our results suggests that Gas6‐Axl signaling plays a role during gastric carcinogenesis, and that targeting Gas6‐Axl signaling could be a novel therapeutic for gastric cancer.

Nuclear expression of phosphorylated EGFR is associated with poor prognosis of patients with esophageal squamous cell carcinoma.

Objectives: Although it has been reported that epidermal growth factor receptor (EGFR) is able to translocate from the plasma membrane to the nucleus, the pathophysiological role of this translocation in tumorigenicity is still unclear. In the present study, to elucidate the pathophysiological significance of EGFR translocation, we investigated the expression not only of conventional EGFR but also its phosphorylated form (pEGFR), focusing on its cellular localization in esophageal cancer tissues. Methods: Fifty-two specimens of esophageal squamous cell carcinoma (SCC) obtained by surgery were examined immunohistochemically for their EGFR and pEGFR immunostaining patterns. The relationships between clinicopathological parameters and EGFR or pEGFR immunostaining patterns were then analyzed. Results: In 37 (71.2%) of the 52 esophageal SCCs, EGFR immunoreactivity was clearly localized at the plasma membrane of the cancer cells, whereas pEGFR immunoreactivity was clearly localized in the nucleus in 19 (36.5%) cases. Nuclear expression of pEGFR significantly correlated with TNM stage and lymph node metastasis, and moreover was associated with a poor outcome of esophageal SCC. Conclusions: Nuclear translocalization of pEGFR is associated with an increase in the malignant potential of esophageal SCC and may affect prognosis in patients with esophageal SCC.

Possible role of REG Iα protein in ulcerative colitis and colitic cancer

Background and aims: Although regenerating gene (REG) Iα protein may be involved in the inflammation and carcinogenesis in the gastrointestinal tract, its pathophysiological role in ulcerative colitis (UC) and the resulting colitic cancer remains unclear. We investigated expression of the REG Iα gene and its protein in UC and colitic cancer tissues. We examined whether cytokines are responsible for REG Iα gene expression and whether REG Iα protein has a trophic and/or an antiapoptotic effect on colon cancer cells. Methods: Expression of REG Iα mRNA and its gene product in UC tissues was analysed by real time reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. The effects of cytokines on REG Iα promoter activity were examined in LoVo cells by luciferase reporter assay. The effects of REG Iα protein on growth and H2O2 induced apoptosis were examined in LoVo cells by MTT and TUNEL assays, respectively. Results: REG Iα protein was strongly expressed in inflamed epithelium and in dysplasias and cancerous lesions in UC tissues. The level of REG Iα mRNA expression in UC tissues correlated significantly with severity of inflammation and disease duration. REG Iα promoter activity was enhanced by stimulation with interferon γ or interleukin 6. REG Iα protein promoted cell growth and conferred resistance to H2O2 induced apoptosis in LoVo cells. REG Iα protein promoted Akt phosphorylation and enhanced Bcl-xL and Bcl-2 expression in LoVo cells. Conclusions: The REG Iα gene is inducible by cytokines and its gene product may function as a mitogenic and/or an antiapoptotic factor in the UC-colitic cancer sequence.

Involvement of the IL-22/REG Iα axis in ulcerative colitis

The Regenerating gene (REG) Iα protein, a trophic and/or anti-apoptotic factor, is important in the pathophysiology of gastrointestinal inflammation. Interleukin (IL)-22 is a recently identified cytokine that is suggested to have pivotal roles in inflammatory bowel diseases. We therefore investigated the involvement of the IL-22/REG Iα axis and examined the mechanism of regulation of REG Iα expression by IL-22 stimulation in ulcerative colitis (UC) mucosa. Expression of IL-22, IL-22 receptor 1 (IL-22R1), and REG Iα in UC mucosa was analyzed by real-time RT-PCR and immunohistochemistry. The effects of IL-22 on REG Iα protein expression were examined using a small-interfering RNA for STAT3, an MAPK inhibitor or a PI3K inhibitor. The element responsible for IL-22-induced REG Iα promoter activation was determined by a promoter deletion and electrophoretic mobility shift assay. The expression of IL-22 was enhanced in infiltrating inflammatory cells, and that of IL-22R1 and REG Iα was concurrently enhanced in the inflamed epithelium in UC mucosa. The levels of REG Iα and IL-22 mRNA expression were strongly correlated, and the distributions of REG Iα- and IL-22R1-positive epithelial cells were very similar. IL-22 simulation enhanced the expression of REG Iα protein through STAT3 tyrosine phosphorylation in colon cancer cells. The IL-22-responsive element was located between −142 and −134 in the REG Iα promoter region. REG Iα protein may have a pathophysiological role as a biological mediator for immune cell-derived IL-22 in the UC mucosa.

Signal transducers and activators of transcription 3 activation is involved in nuclear accumulation of β-catenin in colorectal cancer

Nuclear accumulation of beta-catenin is a key event for the development of colorectal cancer. Little is known, however, about the mechanisms underlying translocation of beta-catenin from the cytoplasm or the membrane to the nucleus. The present study examined whether signal transducers and activators of transcription 3 (STAT3) activation is involved in the nuclear accumulation of beta-catenin in colorectal cancer cells. Of the 90 primary colorectal cancer tissues, 40 (44.4%) were positive for nuclear staining of p-STAT3 and 63 (70.0%) were positive for nuclear staining of beta-catenin. The nuclear staining of both p-STAT3 and beta-catenin were observed predominantly in the periphery of the cancer tissues. Importantly, of the 40 tumors with p-STAT3 nuclear staining, 37 (92.5%) were also positive for nuclear beta-catenin staining and there was a significant correlation between p-STAT3 and beta-catenin nuclear staining (P < 0.01). Coexpression of nuclear p-STAT3 and beta-catenin was associated with lower patient survival (P < 0.01). In an in vitro study using a human colon cancer cell line, SW480, inhibition of STAT3 by dominant negative STAT3 or the Janus kinase inhibitor, AG490, induced translocation of beta-catenin from the nucleus to the cytoplasm or membrane. Luciferase assays revealed that STAT3 inhibition resulted in significant suppression of beta-catenin/T-cell factor transcription in association with significant inhibition of cell proliferation (P < 0.05). These findings suggest that in colorectal cancer, STAT3 activation is involved in the nuclear accumulation of beta-catenin, resulting in poor patient survival.

Relationship between severity and symptoms of reflux oesophagitis in elderly patients in Japan

Since information concerning reflux oesophagitis in the elderly is limited, particularly in Japan, the severity and symptomatic profiles of reflux oesophagitis in elderly patients were investigated. One hundred and nineteen patients with reflux oesophagitis found among 2278 endoscopy cases between 1993 and 1996 were investigated in this study. The patients were divided into two groups, elderly and non‐elderly. The severity of reflux oesophagitis was estimated by the Los Angeles classification. The presence or absence of typical symptoms (heartburn and regurgitation) was determined by interview. Reflux oesophagitis was not only more frequently found in the elderly group, but was more severe than in the non‐elderly. Although the degree of manifestation of typical symptoms was similar between the elderly and the non‐elderly with high‐grade oesophagitis, the elderly patients with mild reflux oesophagitis were less symptomatic than the non‐elderly. Mild reflux oesophagitis in the elderly may be missed due to its rarity of typical reflux symptoms and a substantial number of elderly persons might have subclinical reflux oesophagitis.

Colonic expression of heme oxygenase-1 is associated with a better long-term survival in patients with colorectal cancer

Objective. Heme oxygenase-1 (HO-1) has emerged as a crucial mediator of mucosal defense in the gastrointestinal tract. Its metabolic pathway products, biliverdin/bilirubin and carbon monoxide, can reduce oxidative stress and inflammation, and promote resistance to apoptosis. The role of HO-1 in gastrointestinal malignancies, however, remains to be elucidated. The purpose of this study was to analyze HO-1 expression in human colon adenoma and cancer samples. Material and methods. Fifty-five paraffin-embedded surgical specimens of colorectal cancer and 19 colonic adenoma samples were stained immunhistochemically for HO-1 expression using an anti-HO-1 monoclonal antibody. HO-1 expression was evaluated independently by two different investigators and subsequently correlated to clinical data and patients’ life expectancy. Results. Focal HO-1 expression could be documented in 41.8% (23/55) of patients with colorectal cancer. HO-1 expression in colonic adenoma was detectable in 36.8% (7/19) of cases. The rate of lymphatic tumor invasion was significantly lower in colorectal cancer samples expressing HO-1 (p=0.048). Additionally, fewer lymph node metastases were found in colorectal cancer samples with HO-1 expression, but these differences did not reach statistical significance. Mean observation period was 65.87±3.96 months. Kaplan-Meier analysis showed a significantly better survival for colorectal cancer patients with colonic HO-1 expression (p=0.018). Conclusions. This study demonstrates that colonic HO-1 may be a prognostic marker of colorectal-cancer outcome.