Kiyohito Kato

The antidiabetic drug metformin inhibits gastric cancer cell proliferation in vitro and in vivo

Recent studies suggest that metformin, which is commonly used as an oral anti-hyperglycemic agent of the biguanide family, may reduce cancer risk and improve prognosis, but the mechanisms by which metformin affects various cancers, including gastric cancer, remains unknown. The goal of the present study was to evaluate the effects of metformin on human gastric cancer cell proliferation in vitro and in vivo and to study microRNAs (miRNA) associated with antitumor effect of metformin. We used MKN1, MKN45, and MKN74 human gastric cancer cell lines to study the effects of metformin on human gastric cancer cells. Athymic nude mice bearing xenograft tumors were treated with or without metformin. Tumor growth was recorded after 4 weeks, and the expression of cell-cycle-related proteins was determined. In addition, we used miRNA array tips to explore the differences among miRNAs in MKN74 cells bearing xenograft tumors treated with or without metformin in vitro and in vivo. Metformin inhibited the proliferation of MKN1, MKN45, and MKN74 in vitro. Metformin blocked the cell cycle in G0–G1 in vitro and in vivo. This blockade was accompanied by a strong decrease of G1 cyclins, especially in cyclin D1, cyclin-dependent kinase (Cdk) 4, Cdk6 and by a decrease in retinoblastoma protein (Rb) phosphorylation. In addition, metformin reduced the phosphorylation of epidermal growth factor receptor and insulin-like growth factor-1 receptor in vitro and in vivo. The miRNA expression was markedly altered with the treatment of metformin in vitro and in vivo. Various miRNAs altered by metformin also may contribute to tumor growth in vitro and in vivo. Mol Cancer Ther; 11(3); 549–60. ©2012 AACR.

Effect of the anti-diabetic drug metformin in hepatocellular carcinoma in vitro and in vivo.

Metformin is a commonly used oral anti-hyperglycemic agent of the biguanide family. Recent studies suggest that metformin may reduce cancer risk and improve prognosis. However, the antitumor mechanism of metformin in several types of cancers, including hepatocellular carcinoma (HCC), has not been elucidated. The goal of the present study was to evaluate the effects of metformin on HCC cell proliferation in vitro and in vivo, and to study microRNAs (miRNAs) associated with the antitumor effect of metformin in vitro. We used the cell lines Alex, HLE and Huh7, and normal hepatocytes to study the effects of metformin on human HCC cells. In an in vivo study, athymic nude mice bearing xenograft tumors were treated with metformin or left untreated. Tumor growth was recorded after 4 weeks, and the expression of cell cycle-related proteins was determined. Metformin inhibited the proliferation of Alex, HLE and Huh7 cells in vitro and in vivo. Metformin blocked the cell cycle in G0/G1 in vitro and in vivo. This blockade was accompanied by a strong decrease of G1 cyclins, especially cyclin D1, cyclin E and cyclin-dependent kinase 4 (Cdk4). In addition, microRNA (miRNA) expression was markedly altered by the treatment with metformin in vitro and in vivo. In addition, various miRNAs induced by metformin also may contribute to the suppression of tumor growth. Our results demonstrate that metformin inhibits the growth of HCC, possibly by inducing G1 cell cycle arrest through the alteration of microRNAs.

Identification and Characterization of Wolframin, the Product of the Wolfram Syndrome Gene (WFS1), as a Novel Calmodulin-Binding Protein

To search for calmodulin (CaM) targets, we performed affinity chromatography purification of a rat brain extract using CaM fused with GST as the affinity ligand. Proteomic analysis was then carried out to identify CaM-binding proteins. In addition to identifying 36 known CaM-binding proteins, including CaM kinases, calcineurin, nNOS, the IP(3) receptor, and Ca(2+)-ATPase, we identified an ER transmembrane protein, wolframin [the product of the Wolfram syndrome gene (WFS1)] as interacting. A CaM overlay and an immunoprecipitation assay revealed that wolframin is capable of binding the Ca(2+)/CaM complex in vitro and in transfected cells. Surface plasmon resonance analysis and zero-length cross-linking showed that the N-terminal cytoplasmic domain (residues 2-285) of wolframin binds to an equimolar unit of CaM in a Ca(2+)-dependent manner with a K(D) for CaM of 0.15 muM. Various truncation and deletion mutants showed that the Ca(2+)/CaM binding region in wolframin is located from Glu90 to Trp186. Furthermore, we demonstrated that three mutations (Ala127Thr, Ala134Thr, and Arg178Pro) associated with Wolfram syndrome completely abolished CaM binding of wolframin. This observation may indicate that CaM binding is important for wolframin function and that impairment of this interaction by mutation contributes to the pathology seen in Wolfram syndrome.

Human MicroRNAs Originated from Two Periods at Accelerated Rates in Mammalian Evolution

MicroRNAs (miRNAs) are short, noncoding RNAs that modulate genes posttranscriptionally. Frequent gains and losses of miRNA genes have been reported to occur during evolution. However, little is known systematically about the periods of evolutionary origin of the present miRNA gene repertoire of an extant mammalian species. Thus, in this study, we estimated the evolutionary periods during which each of 1,433 present human miRNA genes originated within 15 periods, from human to platypus–human common ancestral branch and a class “conserved beyond theria,” primarily using multiple genome alignments of 38 species, plus the pairwise genome alignments of five species. The results showed two peak periods in which the human miRNA genes originated at significantly accelerated rates. The most accelerated rate appeared in the period of the initial phase of hominoid lineage, and the second appeared shortly before Laurasiatherian divergence. Approximately 53% of the present human miRNA genes have originated within the simian lineage to human. In particular, approximately 28% originated within the hominoid lineage. The early phase of placental mammal radiation comprises approximately 28%, while no more than 15% of human miRNAs have been conserved beyond placental mammals. We also clearly showed a general trend, in which the miRNA expression level decreases as the miRNA becomes younger. Intriguingly, amid this decreasing trend of expression, we found one significant rise in the expression level that corresponded to the initial phase of the hominoid lineage, suggesting that increased functional acquisitions of miRNAs originated at this particular period.

Antitumor effect of metformin in esophageal cancer: In vitro study

Recent studies suggest that metformin, which is a member of the biguanide family and commonly used as an oral anti-hyperglycemic agent, may reduce cancer risk and improve prognosis of numerous types of cancer. However, the mechanisms underlying the antitumor effect of metformin on esophageal cancer remain unknown. The goal of the present study was to evaluate the effects of metformin on the proliferation of human ESCC in vitro, and to study changes in the expression profile of microRNAs (miRNAs), since miRNAs have previously been associated with the antitumor effects of metformin in other human cancers. The human ESCC cell lines T.T, KYSE30 and KYSE70 were used to study the effects of metformin on human ESCC in vitro. In addition, we used miRNA array tips to explore the differences between miRNAs in KYSE30 cells with and without metformin treatment. Metformin inhibited the proliferation of T.T, KYSE30 and KYSE70 cells in vitro. Metformin blocked the cell cycle in G0/G1 in vitro. This blockade was accompanied by a strong decrease of G1 cyclins, especially cyclin D1, as well as decreases in cyclin-dependent kinase (Cdk)4, Cdk6 and phosphorylated retinoblastoma protein (Rb). In addition, the expression of miRNAs was markedly altered with the treatment of metformin in vitro. Metformin inhibited the growth of three ESCC cell lines, and this inhibition may have involved reductions in cyclin D1, Cdk4 and Cdk6.

Factors predicting survival and pathological subtype in patients with ampullary adenocarcinoma

Carcinoma of the ampulla of Vater is uncommon. This study aimed to clarify predictors of survival for ampullary adenocarcinoma and to identify characteristics of its two major pathological subtypes.

Antidiabetic drug metformin inhibits esophageal adenocarcinoma cell proliferation in vitro and in vivo.

Esophageal carcinoma is the eighth most common cancer worldwide and the sixth leading cause of cancer-related deaths, with one of the worst prognoses of any form of cancer. Treatment with the anti-diabetic drug metformin has been associated with reduced cancer incidence in patients with type 2 diabetes. This study therefore evaluated the effects of metformin on the proliferation, in vitro and in vivo, of human esophageal adenocarcinoma cells, as well as the microRNAs associated with the antitumor effects of metformin. Metformin inhibited the proliferation of the esophageal adenocarcinoma cell lines OE19, OE33, SK-GT4 and OACM 5.1C, blocking the G0 to G1 transition in the cell cycle. This was accompanied by strong reductions in G1 cyclins, especially cyclin D1, cyclin-dependent kinase (Cdk)4, and Cdk6, and decreases in retinoblastoma protein phosphorylation. In addition, metformin reduced the phosphorylation of epidermal growth factor receptor and insulin-like growth factor and insulin-like growth factor-1 receptor, as well as angiogenesis-related proteins, such as vascular endothelial growth factor, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2. Metformin also markedly altered microRNA expression. Treatment with metformin of athymic nude mice bearing xenograft tumors reduced tumor proliferation. These findings suggest that metformin may have clinical use in the treatment of esophageal adenocarcinoma.

Galectin-9 suppresses cholangiocarcinoma cell proliferation by inducing apoptosis but not cell cycle arrest

Cholangiocarcinoma is the most common biliary malignancy and the second most common hepatic malignancy after hepatocellular carcinoma (HCC). Galectin-9 (Gal-9) is a tandem-repeat-type galectin that has recently been shown to exert antiproliferative effects on cancer cells. Therefore, the present study evaluated the effects of Gal-9 on the proliferation of human cholangiocarcinoma cells in vitro as well as the microRNAs (miRNAs) associated with the antitumor effects of Gal-9. Gal-9 suppressed the proliferation of cholangiocarcinoma cell lines in vitro and the growth of human cholangiocarcinoma cell xenografts in nude mice. Our data further revealed that Gal-9 increased caspase‑cleaved keratin 18 (CCK18) levels, and the expression of cytochrome c increased in Gal-9-treated cholangiocarcinoma cell lines. These data suggested that Gal-9 induced cholangiocarcinoma cell apoptosis via the intrinsic apoptosis pathway mediated by caspase-dependent or -independent pathways. In addition, Gal-9 reduced the phosphorylation of the epidermal growth factor receptor (EGFR), insulin-like growth factor and insulin-like growth factor-1 receptor (IGF-1R), hepatocyte growth factor receptor and fibroblast growth factor receptor 3 (FGFR3). These findings suggest that Gal-9 can be a candidate of therapeutic target in the treatment of cholangiocarcinoma.

Prediction of invasion depth for submucosal differentiated gastric cancer by magnifying endoscopy with narrow-band imaging

The usefulness of determining gastric cancer invasion depth by magnifying endoscopy with narrow-band imaging (NBI-ME) has not been established. The objective of our study was to retrospectively compare NBI-ME images of differentiated submucosal (SM) 1 cancer with those of SM2 to identify the indicators of invasion depth for SM2 gastric cancer. Fifteen patients with SM1 differentiated gastric cancer and 20 with SM2 removed by endoscopic submucosal resection (ESD) were included. NBI-ME images matching the invasion depth of pathological specimens were examined to define the following three findings as diagnostic indicators of SM2: non-structure, scattery vessels and multi-caliber vessels. The relationship between indicators and invasion depth and between indicator score and invasion depth was examined in 27 patients (SM1/SM2: 11/16) with depressed-type gastric cancer (D-GC) and in 8 (SM1/SM2: 4/4) with protruding-type gastric cancer (P-GC). Diagnostic accuracy for invasion depth determined by four endoscopists using regular endoscopic images was compared with that determined by the same endoscopists using NBI-ME. In D-GC, all three indicators were significantly more frequent in SM2 than in SM1 (p<0.05). All D-GC with ≥2 points were SM2, demonstrating a significant difference in score distribution between SM1 and SM2 (p<0.05). In D-GC, diagnostic accuracy by NBI-ME was higher than that by regular endoscopy by all 4 endoscopists (p<0.05). NBI-ME findings of non-structure, scattery vessels and multi-caliber vessels can possibly serve as indicators of SM2 invasion in differentiated D-GC. Scoring of the three indicators was significant.

Molecular Biologic Approach to the Diagnosis of Pancreatic Carcinoma Using Specimens Obtained by EUS-Guided Fine Needle Aspiration

We review the utility of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), a rapid, safe, cost-effective, and accurate diagnostic modality for evaluating pancreatic tumors. EUS-FNA is currently used for the diagnosis and staging of pancreatic tumors. The sensitivity of EUS-FNA for pancreatic malignancy ranges from 75% to 94%, and its specificity approaches 100% in most studies. However, EUS-FNA has some limitations in the diagnosis of well-differentiated or early-stage cancers. Recent evidence suggests that molecular biological analysis using specimens obtained by EUS-FNA improves diagnostic sensitivity and specificity, especially in borderline cytological cases. It was also reported that additional information regarding patient response to chemotherapy, surgical resectability, time to metastasis, and overall survival was acquired from the genetic analysis of specimens obtained by EUS-FNA. Other studies have revealed that the analysis of KRAS, MUC, p53, p16, S100P, SMAD4, and microRNAs is helpful in making the diagnosis of pancreatic carcinoma. In this paper, we describe the present state of genetic diagnostic techniques for use with EUS-FNA samples in pancreatic diseases. We also discuss the role of molecular biological analyses for the diagnosis of pancreatic carcinoma.