Hidenobu Kamohara

Glypican-3, overexpressed specifically in human hepatocellular carcinoma, is a novel tumor marker

With the global pandemic of hepatitis B and C infections, the incidence of Hepatocellular carcinoma (HCC) is rapidly increasing world wide. We identified glypican-3 (GPC3), a novel oncofetal gene over-expressed specifically in human HCC, as based on data of cDNA microarrays. As GPC3 is a GPI-anchored membrane protein and could be secreted, we attempted to detect secreted GPC3 protein in sera from HCC patients using Western blotting and ELISA. GPC3 protein was positive in sera of 40.0% (16/40) of HCC patients, and negative in sera from subjects with liver cirrhosis (LC) (0/13), chronic hepatitis (CH) (0/34), and healthy donors (0/60). All subjects were Japanese. Although 12 of 40 HCC patients were negative for both alpha-fetoprotein (AFP) and PIVKA-II well known tumor markers of HCC, four of these were GPC3-positive in the sera. We also observed vanishing GPC3 protein in the sera of three patients after the surgical treatment for HCC. On the other hand, immunohistochemical analysis revealed that HCC expressed GPC3 protein in all 14 HCC patients tested. In conclusion, GPC3, as defined in this study was shown to be a useful tumor marker for cancer-diagnosis for large numbers of patients with HCC.

MicroRNA-21 Regulates the Proliferation and Invasion in Esophageal Squamous Cell Carcinoma

Purpose: MicroRNAs are ∼22 nucleotide noncoding RNA molecules that posttranscriptionally regulate gene expression. The aim of this study was (a) to determine a role of microRNA-21 in esophageal squamous cell carcinoma and (b) to elucidate the regulation of the programmed cell death 4 (PDCD4) gene by microRNA-21. Experimental Design: MicroRNA-21 expression was investigated in 20 matched normal esophageal epitheliums and esophageal squamous cell carcinomas and seven esophageal squamous cell carcinoma cell lines (TE6, TE8, TE10, TE11, TE12, TE14, KYSE30) by TaqMan quantitative real-time PCR and in situ hybridization. To evaluate the role of microRNA-21, cell proliferation and invasion were analyzed with anti–microRNA-21–transfected cells. In addition, the regulation of PDCD4 by microRNA-21 was elucidated to identify the mechanisms of this regulation. Results: Of 20 paired samples, 18 cancer tissues overexpressed microRNA-21 in comparison with matched normal epitheliums. Specifically, patients with lymph node metastasis or venous invasion showed significantly high expression of microRNA-21. In situ hybridization for microRNA-21 showed strong positive staining in paraffin-embedded esophageal squamous cell carcinoma tissues. All seven esophageal squamous cell carcinoma cell lines also overexpressed microRNA-21, and anti–microRNA-21–transfected cells showed significant reduction in cellular proliferation and invasion. The PDCD4 protein levels in esophageal squamous cell carcinoma cells have an inverse correlation with microRNA-21 expression. Anti–microRNA-21–transfected cells increased PDCD4 protein expression without changing the PDCD4 mRNA level and increased a luciferase-reporter activity containing the PDCD4-3′ untranslated region construct. Conclusions: MicroRNA-21 targets PDCD4 at the posttranscriptional level and regulates cell proliferation and invasion in esophageal squamous cell carcinoma. It may serve as a novel therapeutic target in esophageal squamous cell carcinoma.

Clinical impact of serum exosomal microRNA‐21 as a clinical biomarker in human esophageal squamous cell carcinoma

Exosomes are 40‐nm to 100‐nm membrane vesicles that are secreted by various cells, and they play a major role in cell‐cell communication. The objective of this study was to clarify the significance of the levels of microRNA in exosomes extracted from the sera of patients with esophageal squamous cell cancer (ESCC).

MicroRNA-200b Regulates Cell Proliferation, Invasion, and Migration by Directly Targeting ZEB2 in Gastric Carcinoma

BackgroundThe microRNA-200 (miR-200) family has been reported to induce epithelial differentiation and suppress epithelial–mesenchymal transition (EMT) by inhibiting translation of zinc finger E-box-binding homeobox (ZEB) 1 and 2 mRNAs in several types of cancers. This study aimed to clarify the role of miR-200b in regulating EMT and promoting cellular proliferation, invasion, and migration in gastric cancer.MethodsThe relationships among the expression levels of miR-200b, ZEB1 and ZEB2, and E-cadherin mRNAs were analyzed by quantitative real-time reverse transcription–polymerase chain reaction in frozen tissue samples from 40 gastric cancer patients who underwent gastrectomy from 2008 to 2010. The effects of miR-200b on EMT in gastric cancer cells in vitro were also analyzed.ResultsDiffuse histologic type, depth of tumor, tumor size, lymph node metastasis, and lymphatic invasion were significantly higher in the low-miR-200b expression group compared with the high expression group. There was a strong correlation between the levels of miR-200b, and ZEB2 and E-cadherin mRNAs in gastric cancer patients. Upregulation of miR-200b in gastric cancer cells changed the cell morphology from fibroblast- to epithelial-like, associated with localization of E-cadherin to the plasma membrane. ZEB2 mRNA levels fell, while E-cadherin expression levels increased in gastric cells overexpressing miR-200b, associated with significantly reduced cellular proliferation, and inhibition of cellular migration and invasion.ConclusionsmiR-200b regulates ZEB2 expression and thus controls metastasis in gastric cancer.

Group II phospholipase A2 is increased in peritoneal and pleural effusions in patients with various types of cancer

Serum levels of group II phospholipase A2 (PLA2) have been reported to be associated with stage of disease in cancer patients. These levels are also related to the malignant potential in tissues, and are an important prognostic factor. We radioimmunoassayed group II PLA2 levels in pleural and peritoneal effusions from patients with various cancers. We also investigated the production of group II PLA2 in cells in effusions from cancer patients by Northern blotting, immunocytochemistry and in situ hybridization. Immunoreactive group II PLA2 levels were significantly higher in effusions from 47 patients with various cancers, compared with those in sera and cirrhotic ascites. There was no significant correlation between group II PLA2 levels in effusions and those in sera. Group II PLA2 mRNA was expressed at a high level in cells from effusions, by Northern blot analysis, but not in those cells from blood. The localization of group II PLA2 protein and mRNA was intense in carcinoma cells and CD68‐positive macrophages, determined by immunocytochemistry and in situ hybridization. In addition, IL‐6 and IL‐8 levels were significantly higher in effusions, in comparison with those in sera from patients, suggesting that cancer cells and macrophages produce group II PLA2 by IL‐6. These group II PLA2 levels are apparently significantly increased in effusions, and the carcinoma cells and macrophages produce group II PLA2, as noted in effusions from patients with various cancers. Int. J. Cancer 74:245‐250, 1997.

Overexpression of microRNA-223 regulates the ubiquitin ligase FBXW7 in oesophageal squamous cell carcinoma

Background:F-box and WD repeat domain-containing 7 (FBXW7) is a cell cycle regulatory gene whose protein product ubiquitinates positive cell cycle regulators such as c-Myc, cyclin E, and c-Jun, thereby acting as a tumour-suppressor gene. This study focused on microRNA-223 (miR-223), which is a candidate regulator of FBXW7 mRNA. The aim of this study was to clarify the clinical significance of miR-223 and FBXW7 in oesophageal squamous cell carcinoma (ESCC) patients, and to elucidate the mechanism by which FBXW7 is regulated by miR-223.Methods:The expression levels of miR-223 and the expression of FBXW7 protein was examined using 109 resected specimens to determine the clinicopathological significance. We also investigated the role of miR-223 in the regulation of FBXW7 expression in ESCC cell lines in an in vitro analysis.Results:We found that miR-223 expression was significantly higher in cancerous tissues than in the corresponding normal tissues. There was a significant inverse relationship between the expression levels of miR-223 and FBXW7 protein. Moreover, patients with high miR-223 expression demonstrated a significantly poorer prognosis than those with low expression. On the basis of a series of gain-of-function and loss-of-function studies in vitro, we identified FBXW7 as a functional downstream target of miR-223.Conclusion:Our present study indicates that high expression of miR-223 had a significant adverse impact on the survival of ESCC patients through repression of the function of FBXW7.

Serum microRNA-21 is a novel biomarker in patients with esophageal squamous cell carcinoma

It was recently revealed that microRNAs (miRNAs) can stably exist in serum and may affect the pathogenesis of several diseases. However, there are few reports that have demonstrated the significance of miRNAs in the serum of patients with esophageal squamous cell carcinoma (ESCC). Thus, the aims of this study were to clarify the status of miRNA‐21 in serum of ESCC patients and to reveal the usefulness of this molecule as a biomarker.

Leukemia inhibitory factor induces apoptosis and proliferation of human carcinoma cells through different oncogene pathways

Leukemia inhibitory factor (LIF) affects the growth of carcinoma cells, and we thus analyzed its underlying mechanisms. Carcinoma cells constitutively express LIF mRNA, and 23 lines (92.0%) and all (100%) of 25 lines express LIF receptor mRNAs of LIFRβ and gp130, respectively. Exogenous addition of LIF promoted significant cell proliferation in 4 lines (MCF‐7, ZR‐75‐1, Hs‐700T and Panc‐1) and suppressed cell growth in 3 lines (AZ‐521, GBK‐1 and HT‐29). LIF significantly induced an immediate early response of genes c‐fos and junB 3 hr after stimulation, but not of c‐jun during the process of proliferation of MCF‐7 and Hs‐700T cells, with maximum levels at 30–60 min. The cell‐cycle‐related gene cyclin E was also induced in MCF‐7 and Hs‐700T cells, whereas cyclinA, cdk2, c‐myc, c‐myb and p53 mRNAs were not induced. On the other hand, LIF inhibited growth and increased the rate of cell death of AZ‐521 and GBK‐1 cells. LIF increased the number of TUNEL‐positive cells in AZ‐521 cells and DNA fragmentation in AZ‐521 and GBK‐1 cells. LIF induced apoptosis related genes c‐myc and ICE during suppression of cell growth, but p53, p21, c‐fos, cyclin A and cyclin E were not induced. Our results suggest that LIF is linked to cell proliferation and apoptosis in some human carcinoma cell lines. It is considered that this is related to differences in signal transduction and induction of oncogenes. Int. J. Cancer 72:687–695, 1997.

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3,5-dione) Blocks the Chemotaxis of Neutrophils by Inhibiting Signal Transduction through IL-8 Receptors

We investigated the impact of curcumin on neutrophils. Chemotactic activity via human recombinant IL-8 (hrIL-8) was significantly inhibited by curcumin. Curcumin reduced calcium ion flow induced by internalization of the IL-8 receptor. We analyzed flow cytometry to evaluate the status of the IL-8 receptor after curcumin treatment. The change in the distribution of receptors intracellularly and on the cell surface suggested that curcumin may affect the receptor trafficking pathway intracellulary. Rab11 is a low molecular weight G protein associated with the CXCR recycling pathway. Following curcumin treatment, immunoprecipitation studies showed that the IL-8 receptor was associated with larger amounts of active Rab11 than that in control cells. These data suggest that curcumin induces the stacking of the Rab11 vesicle complex with CXCR1 and CXCR2 in the endocytic pathway. The mechanism for antiinflammatory response by curcumin may involve unique regulation of the Rab11 trafficking molecule in recycling of IL-8 receptors.

Surgical trauma induces group II phospholipase A2 production by neutrophils at a local site after surgery.

OBJECTIVES Group II phospholipase A2 (PLA2) regulates eicosanoids and platelet activating factor (PAF) production and plays an important role in regulating critical mediators in inflammatory diseases such as trauma, sepsis and multiple organ failure. To elucidate the local effect of surgical trauma, we investigated the production of group II PLA2 at a local site after surgery. DESIGN AND METHODS We utilized a radioimmunoassay to measure group II PLA2 levels in peritoneal exudates from the operative field and blood in patients who underwent gastrectomy. We also investigated the production of group II PLA2 in cells from peritoneal exudates by Northern blotting and immunocytochemistry. RESULTS Immunoreactive group II PLA2 levels were significantly increased from 3 h after surgery and peaked at 12 h peritoneal exudates. However, serum group II PLA2 levels peaked at 24-48 h and decreased gradually after surgery, findings similar to levels of postoperative serum C-reactive protein (CRP). There was no significant correlation between group II PLA2 levels in peritoneal exudates and those in blood. Group II PLA2 mRNA was expressed at high level in cells from peritoneal exudates, by Northern blot analysis, but not those from blood. The localization of group II PLA2 protein was intense in neutrophils, as determined by immunocytochemistry. No group II PLA2 expression was observed in corresponding peripheral blood cells. CONCLUSIONS After surgery, group II PLA2 is increased in peritoneal exudates prior to elevation in the blood circulation and is produced by neutrophils recruited and activated at a local site. Group II PLA2 produced in peritoneal exudates by neutrophils has an important role in the physiological and pathological states at a local site, after surgery.