Tsutsumi S

Galectin-3, a Novel Binding Partner of β-Catenin

Galectin-3 (gal-3), a pleiotrophic protein, is an important regulator of tumor metastasis, which like β-catenin shuttles between the nucleus and the cytosol in a phosphorylation-dependent manner. We report herein that β-catenin stimulation of cyclin D1 and c-myc expression is gal-3 dependent. Gal-3 binds to β-catenin/Tcf complex, colocalizes with β-catenin in the nucleus, and induces the transcriptional activity of Tcf-4 as determined by the TOP/FOPFLASH reporter system. We have identified the β-catenin–gal-3–binding sequences, which are in the NH2 and COOH termini of the proteins encompassing amino acid residues 1 to 131 and 143 to 250, respectively. These data indicate that gal-3 is a novel binding partner for β-catenin involved in the regulation of Wnt/β-catenin signaling pathway.

E/N-cadherin switch mediates cancer progression via TGF- β -induced epithelial-to-mesenchymal transition in extrahepatic cholangiocarcinoma

Background:Epithelial-to-mesenchymal transition (EMT) is a fundamental process governing not only morphogenesis in multicellular organisms, but also cancer progression. During EMT, epithelial cadherin (E-cadherin) is downregulated while neural cadherin (N-cadherin) is upregulated, referred to as ‘cadherin switch’. This study aimed to investigate whether cadherin switch promotes cancer progression in cholangiocarcinoma (CC).Methods:CC cell lines were examined for migration, invasion, and morphological changes with typical EMT-induced model using recombinant TGF-β1. The changes in E-cadherin and N-cadherin expression were investigated during EMT. We also examined E-cadherin and N-cadherin expression in resected specimens from extrahepatic CC patients (n=38), and the associations with clinicopathological factors and survival rates.Results:TGF-β1 treatment activated cell migration, invasion, and fibroblastic morphological changes, especially in extrahepatic CC HuCCT-1 cells. These changes occurred with E-cadherin downregulation and N-cadherin upregulation, that is, cadherin switch. Patients with low E-cadherin expression had a significantly lower survival rate than patients with high E-cadherin expression (P=0.0059). Patients with decreasing E-cadherin and increasing N-cadherin expression had a significantly lower survival rate than patients with increasing E-cadherin and decreasing N-cadherin expression (P=0.017).Conclusion:Cadherin switch promotes cancer progression via TGF-β-induced EMT in extrahepatic CC, suggesting a target for elucidating the mechanisms of invasion and metastasis in extrahepatic CC.

MicroRNA-7 expression in colorectal cancer is associated with poor prognosis and regulates cetuximab sensitivity via EGFR regulation

MicroRNA-7 (miR-7) has been reported to be a tumor suppressor in all malignancies including colorectal cancer (CRC). However, its significance for CRC clinical outcomes has not yet been explored. The potential for miR-7 to act as a tumor suppressor by coordinately regulating the epidermal growth factor receptor (EGFR) signaling pathway at several levels was examined. We investigated the tumor inhibitory effect of miR-7 in CRC, with particular focus on the relationship between miR-7 and the EGFR pathway. Quantitative reverse transcription-PCR was used to evaluate miR-7 expression in 105 CRC cases to determine the clinicopathologic significance of this miRNA. The regulation of EGFR by miR-7 was examined with miR-7 precursor-transfected cells. Furthermore, we investigated whether miR-7 suppresses proliferation of CRC cells in combination with cetuximab, a monoclonal antibody against EGFR. Multivariate analysis indicated that low miR-7 expression was an independent prognostic factor for poor survival (P = 0.0430). In vitro assays showed that EGFR and RAF-1 are direct targets of miR-7, which potently suppressed the proliferation of CRC cells, and, interestingly, that the growth inhibitory effect of each of these was enhanced by cetuximab. miR-7 is a meaningful prognostic marker. Furthermore, these data indicate that miR-7 precursor, alone or in combination with cetuximab, may be useful in therapy against CRC.

Circulating soluble Fas ligand in patients with gastric carcinoma.

The Fas/Fas ligand (FasL) system is involved in cancer cell death induced by the immune system. Most of the tumors may escape the host immune attack by imitating themselves as immune‐privileged sites by overexpressing FasL. FasL is synthesized as a membrane‐bound protein that can be cleaved to the soluble isoform (sFasL). The objectives of this work were to determine whether the serum concentrations of sFasL in patients with gastric carcinoma were correlated with clinicopathologic features and survival rates.

Animal model of para-aortic lymph node metastasis

The purpose of this study was to establish a model of experimental lymph node metastasis by intra-rectal implantation of human cancer cells in nude mice. Four types of human cancer cell lines (TE-1, MKN-45, HT-29, and MIAPaca-2) were investigated. Tumor cells suspended in Matrigel were injected into the submucosal layer of the rectum. All cancer cell lines produced locally aggressive rectal tumors and, subsequently, para-aortic lymph node metastasis. We were unable to produce other distant metastases in the dying state in such locations as the liver, spleen, lung, and peritoneum. However, using this method, we were able to evaluate the effect of the anti-cancer agent uracil/tegafur (UFT) on primary tumor growth and lymph node metastasis. Oral intake of UFT significantly suppressed implanted tumor volume and inhibited lymph node metastasis. We expect that the process of lymph node metastasis shown in this model will be studied as an experimental model of lymph node metastasis simulating human cancers.

Significance of contracted cholecystitis lesions as high risk for gallbladder carcinogenesis.

A precancerous change has been identified incidentally in resected specimens from patients who have undergone cholecystectomy. We focused on chronic cholecystitis, showing a thick and sclerotic wall caused by recurrent inflammation, e.g. contracted cholecystitis, and examined the malignant potential of these lesions. We studied 88 patients who had undergone cholecystectomy. Contracted cholecystitis was diagnosed, using our criteria, in 28 of these cases. Ordinary chronic cholecystitis was diagnosed in 50 cases and gallbladder carcinoma in ten cases. We examined the expression of p53, Ki-67, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) immunohistochemically. Severe dysplasia or carcinoma in situ in a very small portion of the specimen was identified with hematoxylin-eosin staining in four cases (14.3%) of contracted cholecystitis. These specimens revealed a positive expression of not only p53, but also Ki-67, iNOS, and COX-2. Statistical significance was shown among the three disease groups in terms of the incidence of p53 overexpression, respectively (P<0.05). The results of this study suggest that contracted cholecystitis could be an early change leading to carcinogenesis.

Loss of standard type of CD44 expression in invaded area as a good indicator of lymph-node metastasis in colorectal carcinoma

PURPOSE: Recent advances have made possible the treatment of small invasive colorectal cancer by means of polypectomy or endoscopic mucosal resection. CD44 expression in cancer cells was identified as an indicator of lymph-node metastasis, which could be evaluated in specimens removed by colonoscopy. METHODS: The correlation between lymph-node metastasis and the expression of standard-type CD44 in cancer cells was examined immunohistologically using the invaded cancer cells of 61 tissue samples of superficially invasive colorectal cancer. We defined the above as invasive cancer restricted within the colorectal wall. Of the 61 samples, 31 had submucosal invasion and 30 had muscular invasion. RESULTS: Standard-type CD44 expression in the area of invasion in cases with lymph-node metastasis was remarkably down-regulated. In 43 cases with no lymph-node metastasis, 36 (83.7 percent) of patients had CD44 expression in invaded cells, whereas only two of 18 cases (11.1 percent) with lymph-node metastasis had expression of standard-type CD44 in the same area (P<0.0001). A total of 69.6 percent (16/23) of patients with loss of standard-type CD44 expression in invaded sites were found to have positive metastasis in the lymph nodes. These results suggest that standard-type CD44 in invasive colon cancer cells could suppress metastasis to the regional lymph nodes. CONCLUSION: In cases of invasive colorectal cancer, the loss of standard-type CD44 expression in the invaded area is a sensitive marker for metastasis to the lymph nodes. Further investigation with larger patient groups is required to clarify the reliability of loss of standard-type CD44 expression as an indicator for additional surgery after endoscopic resection of submucosal invasive colorectal carcinoma.

Double endoscopic intraluminal operation for upper digestive tract diseases: proposal of a novel procedure.

Background and Objective:Endoscopic treatment of digestive tract diseases, such as early esophageal and gastric neoplasia, has become increasingly popular in recent years as an alternative to surgical procedures in the hope of providing an improved quality of life for these patients. However, one of the limitations of a conventional endoscopic mucosal resection, such as an aspiration mucosectomy and a strip biopsy, has been the size of the lesions to be resected. Both an aspiration mucosectomy and strip biopsy are useful variants for removing flat lesions measuring less than 20 mm in maximal diameter. To overcome such limitations, we devised a double endoscopic intraluminal operation (DEILO), which enables us to resect mucosal lesions by using 2 fine endoscopes and monopolar shears. Methods:DEILO was performed on patients with esophageal and gastric lesions measuring up to 40 mm in diameter. This novel technique is characterized by the use of 2 endoscopes (one for lifting the lesion and the other for cutting the lesions) inserted into the esophagus or stomach through an overtube. A mucosal resection is then performed by dissecting the mucosal margin with newly developed monopolar shears, thereby separating the mucosa from the submucosa. Results:A total of 25 lesions in the esophagus (8 lesions) and stomach (17 lesions) were resected by DEILO. The sizes of the esophageal lesions ranged from 8 to 40 mm in diameter (mean, 21.1 mm) whereas gastric lesions ranged from 8 to 30 mm (mean, 13.3 mm) in diameter, and histopathologic examinations revealed the resection margin to be clear and without any tumor. No complications or instances of recurrence were observed in this series. Conclusions:DEILO is considered to be feasible for the mucosal resection of esophageal and gastric lesions measuring more than 10 mm in diameter without submucosal invasion, whereas conventional endoscopic mucosal resection is indicated for such lesions measuring less than 10 mm in size.

Clinical significance of soluble form of HLA class I molecule in Japanese patients with pancreatic cancer

In recent studies a soluble form of human leukocyte antigen class I (sHLA-I) has been found in blood, urine, ascitic fluid, and various other tissues. Research has been focused on the role of sHLA-I in the induction of immunotolerance in organ transplantation. To examine the role of sHLA-I in the immune system of patients with malignancy, we examined serum sHLA-I levels in patients with pancreatic, biliary, hepatic malignancy, and other diseases. We examined sHLA-I levels in the sera of patients with pancreatic cancer (n = 19), benign biliary disease and chronic pancreatitis (n = 20), hepatocellular carcinoma (n = 51), gallbladder cancer (n = 6), cholangiocellular carcinoma (n = 6), and in normal controls (n = 22), using enzyme-linked immunosorbent assay (ELISA). In patients with pancreatic cancer we also analyzed the relationship between sHLA-I and CA19-9, and the specificity and sensitivity of sHLA-I. When patients with acute or chronic hepatitis were excluded from analysis, the mean sHLA-I level in patients with pancreatic cancer was significantly higher than that of normal controls (p < 0.01) and patients with benign disease (p < 0.01), hepatocellular carcinoma (p < 0.01), gallbladder cancer (p < 0.05), and cholangiocarcinoma (p < 0.05). We determined a serum sHLA-I cutoff level for normal controls of 2000 ng/ml; serum levels of sHLA-I were higher than the cutoff in ten patients with pancreatic cancer, and serum levels of CA19-9 were lower than 37 IU/l in 9 of 14 patients; sensitivity and specificity were 88.2% and 85.5%, respectively. Serum levels of sHLA-I in pancreatic cancer patients were higher than in the other diseases, although we found that pancreatic cancer cell lines did not produce the sHLA-I. The evaluation of serum sHLA-I levels could have clinical significance in pancreatic cancer.

Association of DNase I phenotype 2 with colorectal carcinoma risk in Japanese populations

We have already demonstrated a significant association of deoxyribonuclease I (DNase I; EC3.1.21.1) polymorphism with liver disease and gastric carcinoma. The aim of this study was to determine whether DNase I polymorphism is closely related to the incident of colorectal carcinoma. We have found a close statistical association between colorectal carcinoma and a high frequency of DNase I phenotype 2 in Japanese populations. However, there was no significant difference in the phenotype distribution between a group of patients with benign diseases and controls. These findings suggest that DNase I phenotype 2 may be potentially useful for identifying patients who are at risk of harboring or developing colorectal carcinoma.