Takaya Shimura

Advantages of endoscopic submucosal dissection over conventional endoscopic mucosal resection

Background:  Endoscopic mucosal resection is an established method for treating intramucosal gastric neoplasms. Conventional endoscopic mucosal resection has predominantly been performed using strip biopsy, but local recurrence sometimes occurs due to such piecemeal resection. Endoscopic submucosal dissection has recently been performed in Japan using new devices such as an insulation‐tip diathermic knife. The efficacy and problems associated with endoscopic submucosal dissection were evaluated by comparison with conventional endoscopic mucosal resection.

Tumor suppressor, AT motif binding factor 1 (ATBF1), translocates to the nucleus with runt domain transcription factor 3 (RUNX3) in response to TGF-β signal transduction

BACKGROUND AND AIMS AT motif binding factor 1 (ATBF1), a homeotic transcription factor, was identified as a tumor suppressor, and loss of heterozygosity at ATBF1 locus occurs frequently in gastric cancers. We previously showed that ATBF1 expression inversely correlated with the malignant character of gastric cancer and that ATBF1 enhanced the promoter activity of p21Waf1/Cip1. We also found that ATBF1 moves between cytoplasm and nucleus, but the precise mechanism of translocation is unknown. In this study, we investigated the mechanism of ATBF1 translocation to the nucleus with the runt domain transcription factor 3 (RUNX3) in cooperation with TGF-beta signal transduction. MATERIALS AND METHODS To analyze the expression of ATBF1 and RUNX3 in gastric cancer cells, we performed immunohistochemistry on 98 resected gastric cancer tissue samples and scored the nuclear staining intensity as grade 0 to grade 5. Co-immunoprecipitation (co-IP) of ATBF1 and RUNX3 was performed. Dual luciferase assays were performed by transfecting ATBF1 and RUNX3 with a p21Waf1/Cip1 reporter vector. To investigate the nuclear translocation of endogenous ATBF1 and RUNX3 in response to TGF-beta signal, we examined the subcellular localization of ATBF1 and RUNX3 in gastric cancer cells treated with recombinant TGF-beta1 using confocal laser scanning microscopy. RESULTS Strong immunohistochemical nuclear staining of ATBF1 was observed in 37 (37.8%) of the gastric cancer tissue samples, and RUNX3 nuclear staining was observed in 15 (15.3%). There was a statistically significant correlation between ATBF1 and RUNX3 nuclear localization (rs=0.433, p<0.001). Co-IP revealed a physical association between ATBF1 and RUNX3. ATBF1 and RUNX3 up-regulated p21Waf1/Cip1 promoter activity synergistically. In SNU16 gastric cancer cells, ATBF1 and RUNX3 were cytoplasmic before TGF-beta1 stimulation, but after 24h of TGF-beta1 stimulation, endogenous ATBF1 and RUNX3 translocated to the nucleus. CONCLUSION ATBF1 associates with RUNX3 and translocates to the nucleus in response to TGF-beta signal transduction and might function in the nucleus as tumor suppressor and transcriptional regulator.

Suppression of proHB-EGF Carboxy-Terminal Fragment Nuclear Translocation: A New Molecular Target Therapy for Gastric Cancer

Purpose: Inactivation of epidermal growth factor (EGF) receptor (EGFR) represents a promising strategy for the development of selective therapies against epithelial cancers and has been extensively studied as a molecular target for cancer therapy. However, little attention has been paid to remnant cell-associated domains created by cleavage of EGFR ligands. The present study focused on recent findings that cleavage of membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF), an EGFR ligand, induces translocation of the carboxyl-terminal fragment (CTF) of HB-EGF from the plasma membrane to the nucleus and regulates cell cycle. Experimental Design: Two gastric cancer cell lines, MKN28 and NUGC4, were used. KB-R7785, an inhibitor of proHB-EGF shedding, was used to suppress HB-EGF-CTF nuclear translocation with cetuximab, which inhibits EGFR phosphorylation. Cell growth was analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay, apoptosis was evaluated by assay of caspase-3 and caspase-7, and cell cycle was investigated by flow cytometry. Results: Immunofluorescence study confirmed that KB-R7785 inhibited HB-EGF-CTF nuclear translocation under conditions of proHB-EGF shedding induction by 12-O-tetradecanoylphorbol-13-acetate in gastric cancer cells. KB-R7785 inhibited cell growth in a dose-dependent manner and high-dose KB-R7785 induced apoptosis. Moreover, KB-R7785 induced cell cycle arrest and increased sub-G1 DNA content. KB-R7785 suppressed cyclin A and c-Myc expression. All effects of KB-R7785 were reinforced by combination with cetuximab. Conclusions: These results suggest that both inhibition of EGFR phosphorylation and inhibition of HB-EGF-CTF nuclear translocation play crucial roles in inhibitory regulation of cancer cell growth. Suppression of HB-EGF-CTF nuclear translocation might offer a new strategy for treating gastric cancer.

BCL6 degradation caused by the interaction with the C-terminus of pro-HB-EGF induces cyclin D2 expression in gastric cancers

BCL6 is a transcriptional repressor that has important functions in lymphocyte differentiation and lymphomagenesis, but there have been no reports of BCL6 expression in gastric cancers. In the present study, we investigated the BCL6 function in gastric cancers. Treatment with TPA resulted in BCL6 degradation and cyclin D2 upregulation. This phenomenon was inhibited by the suppression of the nuclear translocation of HB-EGF-CTF (C-terminal fragment of pro-HB-EGF). The HB-EGF-CTF nuclear translocation leads to the interaction of BCL6 with HB-EGF-CTF and the nuclear export of BCL6, and after that BCL6 degradation was mediated by ubiquitin/proteasome pathway. Real-time RT–PCR and siRNA targeting BCL6 revealed that BCL6 suppresses cyclin D2 expression. Our data indicate that BCL6 interacts with nuclear-translocated HB-EGF-CTF and that the nuclear export and degradation of BCL6 induces cyclin D2 upregulation. We performed immunohistochemical analyses of BCL6, HB-EGF and cyclin D2 in human gastric cancers. The inverse correlation between BCL6 and cyclin D2 was also found in HB-EGF-positive human gastric cancers. BCL6 degradation caused by the HB-EGF-CTF also might induce cyclin D2 expression in human gastric cancers. Inhibition of HB-EGF-CTF nuclear translocation and maintenance of BCL6 function are important for the regulation of gastric cancer progression.

Clinical features of interstitial lung disease induced by standard chemotherapy (FOLFOX or FOLFIRI) for colorectal cancer

BACKGROUND Chemotherapy-induced interstitial lung disease (ILD) in colorectal cancer (CRC) patients is rarely reported, but its clinical features remain to be clarified. PATIENTS AND METHODS Using a computerized database, we retrospectively identified patients who developed ILD from 734 patients with CRC treated with infusional 5-fluorouracil, leucovorin and oxaliplatin (FOLFOX) or infusional 5-fluorouracil, leucovorin and irinotecan (FOLFIRI) from April 2005 to December 2008 at the National Cancer Center Hospital East. RESULTS Of 734 patients, 11 patients developed ILD (1.5%) and 4 of those patients died (0.54%). Of the 11 patients, 10 showed pulmonary shadows other than lung metastases before chemotherapy. ILD developed during FOLFOX in six patients, at 137 days after completion of FOLFOX in one patient, during oxaliplatin interruption of FOLFOX in one patient and during FOLFIRI in the remaining three patients. FOLFOX had been administered at some point for all ILD patients, with a median of 10 cycles (range 2-17 cycles) and a median dose of administered oxaliplatin of 850 mg/m(2) (range 170-1445 mg/m(2)). CONCLUSIONS ILD following FOLFOX or FOLFIRI is an uncommon but life-threatening complication. Care must be taken regarding the onset of ILD, not only during but also after chemotherapy for CRC.

Incidence of gastrointestinal bleeding in patients with cardiovascular disease: buffered aspirin versus enteric-coated aspirin

Abstract Objective. Aspirin-induced enteropathy is increasing, but whether the type of aspirin affects the gastrointestinal (GI) bleeding, especially small intestine, is unclear. The incidence of GI bleeding for buffered aspirin and enteric-coated aspirin was evaluated in patients receiving long-term low-dose aspirin (LDA) for cardiovascular (CV) diseases. Methods. This retrospective cohort study assessed overt GI bleeding, decreased hemoglobin levels suspecting small bowel blood loss, and CV death in patients taking LDA for more than 1 year (LDA group) and in patients not taking LDA (control group). The LDA group was divided into two subgroups, patients taking either buffered aspirin (buffered subgroup) or enteric-coated aspirin (enteric subgroup), and their outcomes were compared. Results. A total of 1402 patients (LDA group 701, control group 701; median follow-up duration 1778 ± 747 days) were assessed. The incidences of overt GI bleeding and decreased hemoglobin were 3.9% and 1.4% in LDA group, respectively, significantly higher than the control group (p < 0.01; p < 0.01). In the LDA group, 3% died during the follow-up period. Ten (3.7%) in the buffered subgroup (n = 267) and 17 (3.9%) in the enteric subgroup (n = 434) developed GI bleeding (p = 0.92). One (0.3%) in the buffered subgroup and nine (2%) in the enteric subgroup developed decreased hemoglobin (p = 0.06, log-rank test). Conclusions. The type of aspirin does not affect the incidence of overt GI bleeding and decreased hemoglobin, but enteric-coated aspirin may be associated with an increased incidence of decreased hemoglobin.

Blockage of angiotensin II type 1 receptor regulates TNF-α-induced MAdCAM-1 expression via inhibition of NF-κB translocation to the nucleus and ameliorates colitis

Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) is an important target in the treatment of inflammatory bowel disease (IBD). Recently, treatment of IBD with an antibody to alpha4beta7-integrin, a ligand for MAdCAM-1, has been an intense focus of research. Our aim was to clarify the mechanism by which MAdCAM-1 is regulated via angiotensin II type 1 receptor (AT1R), and to verify if AT1R might be a novel target for IBD treatment. The role of AT1R in the expression of MAdCAM-1 in SVEC (a murine high endothelial venule cell) and MJC-1 (a mouse colonic endothelial cell) was examined following cytokine stimulation. We further evaluated the effect of AT1R on the pathogenesis of immune-mediated colitis using AT1R-deficient (AT1R-/-) mice and a selective AT1R blocker. AT1R blocker significantly suppressed MAdCAM-1 expression induced by TNF-alpha, but did not inhibit phosphorylation of p38 MAPK or of IkappaB that modulate MAdCAM-1 expression. However, NF-kappaB translocation into the nucleus was inhibited by these treatments. In a murine colitis model induced by dextran sulfate sodium, the degree of colitis, judged by body weight loss, histological damage, and the disease activity index, was much milder in AT1R-/- than in wild-type mice. The expression of MAdCAM-1 was also significantly lower in AT1R-/- than in wild-type mice. These results suggest that AT1R regulates the expression of MAdCAM-1 under colonic inflammatory conditions through regulation of the translocation of NF-kappaB into the nucleus. Furthermore, inhibition of AT1R ameliorates colitis in a mouse colitis model. Therefore, AT1R might be one of new therapeutic target of IBD via regulation of MAdCAM-1.

Role of ES cell-expressed Ras (ERas) in tumorigenicity of gastric cancer.

ERas, a unique member of the Ras family, was initially found only in embryonic stem (ES) cells, where it plays a crucial role in the transformation of transplanted ES cells to teratomas. ERas is involved in ES cell survival, and unlike other Ras family members, is constitutively active without any mutations. The aim of this study was to investigate the expression and role of ERas in human gastric cancer. To test whether ERas played a significant role in human cancer cells, we examined its expression and function in gastric cancer. ERas was expressed in gastric cancer cell lines at different levels. Induction of ERas expression activated the phosphatidylinositol 3 kinase (PI3K)/Akt axis and then enhanced anchorage-independent growth and ERas knockdown by siRNA suppressed cell invasion. Immunohistochemical analyses revealed that ERas was expressed in 38.7% (55/142) of human gastric carcinoma tissues, and its expression was significantly associated with metastasis to the liver (P < 0.0001) and lymph nodes (P < 0.05). ERas up-regulated transcription regulatory factors including ZFHX1A, ZFHX1B, and TCF3, which repress E-cadherin. These data suggest that ERas is activated in a significant population of gastric cancer, where it may play a crucial role in gastric cancer cell survival and metastases to liver via down-regulation of E-cadherin.

Involvement of oxidative stress and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in inflammatory bowel disease.

The pathophysiology of inflammatory bowel disease involves excessive immune effects of inflammatory cells against gut microbes. In genetically predisposed individuals, these effects are considered to contribute to the initiation and perpetuation of mucosal injury. Oxidative stress is a fundamental tissue-destructive mechanisms that can occur due to the reactive oxygen species and reactive nitrogen metabolites which are released in abundance from numerous inflammatory cells that have extravasated from lymphatics and blood vessels to the lamina propria. This extravasation is mediated by interactions between adhesion molecules including mucosal addressin cell adhesion molecule-1 and vascular cell adhesion molecule-1 on the surface of lymphocytes or neutrophils and their ligands on endothelial cells. Thus, reactive oxygen species and adhesion molecules play an important role in the development of inflammatory bowel disease. The present review focuses on the involvement of oxidative stress and adhesion molecules, in particular mucosal addressin cell adhesion molecule-1, in inflammatory bowel disease.

TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

BACKGROUND AND AIMS Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation. MATERIALS AND METHODS The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. RESULT TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGFβ enhanced gastric cancer cell growth and ADAM inhibitors suppressed this effect. EGFR phosphorylation, HB-EGF-CTF nuclear translocation, and cell growth were suppressed in ADAM17 knockdown cells. CONCLUSION HB-EGF-CTF nuclear translocation and EGFR transactivation from proHB-EGF shedding mediated by ADAM17 activated by TGFβ might be an important pathway of gastric cancer cell proliferation by TGFβ.