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Extracellular vesicles (EV) within the cellular secretome are emerging as modulators of pathological processes involved in tumor growth through their ability to transfer donor‐derived RNA into recipient cells. While the effects of tumor and stromal cell EVs within the tumor microenvironment have been studied, less is known about the contributions of normal, nontransformed cells. We examined the impact of EVs within the cellular secretome from nonmalignant cells on transformed cell growth and behavior in cholangiocarcinoma cells. These effects were enhanced in the presence of the pro‐fibrogenic mediator TGF‐β. We identified miR‐195 as a TGF‐β responsive miRNA in normal cells that can be transferred via EV to tumor cells and regulate cell growth, invasion, and migration. The effects of miR‐195 involve modulation of the epithelial–mesenchymal transition through direct effects on the transcription factor Snail. These studies provide in vitro and in vivo evidence for the impact of normal cellular secretome on transformed cell growth, show the importance of EV RNA transfer, and identify mechanisms of EV‐mediated transfer of miRNA as a contributor to tumor development, which may provide new therapeutic opportunities for targeting human cholangiocarcinoma., Cholangiocyte cell secretome can include extracellular vesicles (EV) containing tumor‐suppressive noncoding RNA such as miR‐195 and contribute to microenvironmental modulation of cholangiocarcinoma growth. miR‐195 can suppress the epithelial–mesenchymal transition and inhibit tumor invasion, migration, and growth.

Creative Commons Attribution 3.0 License, Early-staged cholangiocarcinoma (CCA) is difficult to diagnose due to its high potential for invasion and metastasis. Epithelial-mesenchymal transition (EMT) is induced by transforming growth factor-β (TGF-β) in a process thought to be important for invasion and metastasis in several cancers, including CCA. Although microRNAs (miRNAs) have been implicated in the pathogenesis of several malignancies, their roles to CCA are not clearly understood. Some miRNAs were reported to be included in extracellular vesicles (EVs) and transferred from their donor cells to other cells, modulating recipient cell behaviors. In this study, the involvement and functional roles of EV-contained miRNAs during EMT in human CCA were determined. Expression profiling identified a subset of miRNAs that were reduced by TGF-β in CCA cells. Among these, miR-30e was highly downregulated by TGF-β and predicted to target Snail, which is an EMT-inducible transcription factor. MiR-30e overexpression suppressed cell invasion and migration via inhibiting EMT, whereas miR-30e inhibition promoted EMT, cell invasion and migration. Moreover, miR-30e was enriched in EVs derived from CCA cells after miR-30e overexpression, and miR-30e intercellular transfer through EVs suppressed EMT, cell invasion and migration in recipient CCA cells. Together, our results suggest that EV-mediated miR-30e transfer could inhibit EMT via directly targeting Snail, which subsequently suppresses CCA cell invasion and migration. These findings provide several new insights into regulatory mechanisms of tumor invasion and metastasis in human CCA.

application/pdf, Fucoidan is a complex of sulfated polysaccharides derived from non-mammalian origin such as marine brown algae and induces cytokine expression. We investigated the effect of fucoidan on concanavalin A (Con A)-induced liver injury in mice. Liver injury was induced by an intravenous injection of Con A (18.5 mg/kg). Various doses of fucoidan (1-30 mg/kg) were intravenously administered 30 min before Con A injection. The plasma alanine aminotransferase (ALT) and several cytokines levels were determined, and hepatic histological changes were also assessed. The effect of fucoidan administration by itself on induction of interleukin (IL)-10 in plasma and liver tissue was investigated. Con A administration induced an elevation of plasma ALT level, and fucoidan administration dose-dependently prevented the Con A-induced elevation of plasma ALT. Con A administration increased plasma TNF-alpha and IFN-gamma levels, and fucoidan pretreatment significantly inhibited these alterations and increased plasma IL-10 level. The inhibitory effect of fucoidan on Con A-induced liver injury and production of proinflammatory cytokines were reversed by anti-mouse IL-10 antibody pretreatment. Fucoidan induced the IL-10 production in plasma and liver tissue. These findings suggest that fucoidan prevents Con A-induced liver injury by mediating the endogenous IL-10 production and the inhibition of proinflammatory cytokine in mice. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Elsevier , Nakamura, K; Okada, M; Yoneda, M; Takamoto, S; Nakade, Y; Tamori, K; Aso, K; Makino, I, JOURNAL OF HEPATOLOGY, 35(2), 2001, 217-224. author, Background/Aims: Macrophage inflammatory protein-2 (MIP-2), one of CXC chemokines is involved in the recruitment of neutrophils in several tissue injury. In this study, we investigated a role of MIP-2 in concanavalin A (Con A)-induced liver injury in mice. Methods:Liver injury was induced by intravenous injection of Con A (15 mg/kg) and plasma alanine aminotransferase (ALT), MIP-2 levels were determined and histological assessment of the liver was performed. Anti-mouse MIP-2 antibody was intravenously administered 30 min before Con A injection. Results: Plasma ALT level significantly elevated and reached a maximum at 8 h after Con A injection. Plasma MIP-2 level was also elevated and reached peak value at 2 h after Con A injection. The elevated ALT level by Con A injection was significantly inhibited by MIP-2 antibody. The elevated plasma MIP-2 level after Con A injection was significantly reduced by TNF-a antibody, and MIP-2 was induced in plasma after recombinant TNF-a injection. Hepatic necrosis and infiltration of neutrophils were observed after Con A injection, and these histological changes were attenuated by MIP-2 antibody. Conclusion: These findings suggest that Con A induces TNF-a release, and this TNF-a stimulates MIP-2 induction, at least partially contributing to the liver injury mediated through the recruitment of neutrophils.

We describe a patient with an eating disorder and hyperamylasemia originating from the salivary glands, who developed pancreatitis with a huge pancreatic pseudocyst. A 40-year-old woman was referred for the treatment of an eating disorder that had persisted for 9 years. She was admitted with abdominal pain, diarrhea, and nausea. She had bilateral parotid enlargement with marked elevation of total serum amylase level (3288 IU/l; normal range, 60-220) and an isolated increase of salivary isoamylase activity. After her symptoms resolved, oral intake of food was commenced. She subsequently complained of abdominal pain; this was associated with a slight elevation of serum pancreatic isoamylase and lipase levels, and a huge pancreatic pseudocyst was detected. Percutaneous drainage of the pseudocyst was successful. Endoscopic retrograde cholangiopancreatography demonstrated irregularity of the pancreatic duct. Based on these findings, the final diagnosis was parotid enlargement and acute exacerbation of chronic pancreatitis associated with a pancreatic pseudocyst in a patient with an eating disorder.

Angiotensin II type 1 receptor blockers (ARBs) have been reported to attenuate hepatic fibrosis in non-alcoholic steatohepatitis (NASH). However, it is uncertain whether ARBs prevent hepatocarcinogenesis in NASH even after hepatic fibrosis has developed. Male Wistar rats were fed with a choline-deficient, l-amino acid-defined (CDAA) diet for 24 weeks, and then fed with the CDAA diet with telmisartan (2 mg/kg/day), a novel ARB, or vehicle for another 24 weeks. The liver histology and the expression of genes and proteins related to angiogenesis were investigated. The 24-week CDAA diet induced liver cirrhosis. The 48-week CDAA diet exacerbated liver cirrhosis, and developed hepatocellular carcinoma (HCC) in 54.6 % of the rats concurrently with increases of hypoxia-inducible factor-1α (HIF-1α) protein and vascular endothelial growth factor (VEGF) mRNA, which are potent angiogenic factors in the liver. Telmisartan inhibited hepatic fibrosis and preneoplastic lesions and prevented the development of HCC along with inducing decreases in HIF-1α protein and VEGF mRNA. These data indicated that telmisartan may prevent hepatocarcinogenesis through the inhibition of hepatic angiogenesis even after liver cirrhosis has been established.

The extracellular matrix component hyaluronan (HA) modulates the production of various cytokines and chemokines by activated inflammatory cells. In this study, we investigated whether exogenous administration of HA influences T-cell-mediated liver injury and cytokine production.Liver injury was induced by administration of concanavalin A (Con A) or D-galactosamine/lipopolysaccharide (GalN/LPS), and 0.05%-0.35% (v/v) HA (MW 250, 470, 780, 900, and 1200 kDa) was administered intravenously 18 h before Con A or GalN/LPS injection. Plasma ALT level was determined enzymatically and plasma cytokine levels were determined by ELISA.The elevated plasma levels of ALT at 8 h after Con A and at 7 h after GalN/LPS injection were significantly decreased by pretreatment with high molecular weight HAs (780, 900, and 1200 kDa) but not low molecular weight HAs (250 and 470 kDa). High molecular weight HA (900 kDa) significantly reduced plasma tumor necrosis factor-alpha, interferon gamma, macrophage inflammatory protein 2, and interleukin 4 levels after Con A injection. However, this inhibitory effect on plasma cytokines was not observed with low molecular weight HA (250 kDa) pretreatment.The present results suggest that high molecular weight but not low molecular weight HA prevents liver injury by reducing proinflammatory cytokines in a T-cell-mediated liver injury model. The extracellular matrix component hyaluronan (HA) modulates the production of various cytokines and chemokines by activated inflammatory cells. In this study, we investigated whether exogenous administration of HA influences T-cell-mediated liver injury and cytokine production.

Elsevier , Nakamura, K. ; Ito, T. ; Yoneda, M. ; Takamoto, S. ; Nakade, Y. ; Okamoto, S. ; Okada, M. ; Yokohama, S. ; Aso, K. ; Makino, I., JOURNAL OF HEPATOLOGY, 36(6), 2002, 766-773. author, Background/Aims: Recently, we have reported that macrophage inflammatory protein-2 (MIP-2) plays a pivotal role in concanavalin A (Con A)-induced liver injury. In this study, we investigated the effect of antithrombin III (AT-III) on liver damage, and production of MIP-2 and prostacyclin in this model. Methods: Liver injury was induced by intravenous injection of Con A (15 mg/kg) and AT-III was administered (50, 250 and 500 units/kg, iv) 30 min before Con A injection. Plasma levels of alanine aminotransferase (ALT), MIP-2 and 6-keto-prostaglandin F1a (6k-PG-F1a), stable metabolite of prostaglandin I2 (prostacyclin), were determined. Results: The elevated plasma ALT levels 8, 16, 24 h after Con A injection were inhibited by AT-III pretreatment. The elevated plasma MIP-2 levels were significantly inhibited by AT-III pretreatment compared with vehicle treatment. The inhibitory effect of AT-III on plasma ALT and MIP-2 in Con A-induced liver injury was attenuated by indomethacin (5 mg/kg, ip). Plasma concentration of 6k-PG-F1a at 2 h after AT-III injection was significantly elevated compared with baseline and vehicle pretreatment. Conclusion: These findings suggest that AT-III prevents Con A-induced liver injury through an inhibition of MIP-2 release and a production of prostacyclin.