Cordycepin suppresses cell proliferation and migration by targeting CLEC2 in human gastric cancer cells via Akt signaling pathway.

PURPOSE: Gastric cancer is a common malignancy worldwide, and is associated with high morbidity and mortality rates. Cordycepin is a 3'-deoxyadenosine drug with significant anti-cancer effects. The aim of this study was to determine the molecular mechanisms underlying cordycepin action on gastric cancer cell proliferation and migration. METHODS: The human gastric cancer cell lines MGC-803 and HGC-27 were treated with different concentrations of cordycepin (25 µM, 50 µM, 100 µM and 5 µM, 25 µM and 50 µM) for 48 h. Cell proliferation was assessed by MTT and colony formation assays, and in vitro migration by the wound healing and transwell assays. In addition, Flow Cytometry was used to detect the cell cycle and apoptosis. RT-PCR and Western blotting were used to evaluate the expression levels of key factors. RESULTS: Cordycepin significantly inhibited gastric cancer cell proliferation and migration in a dose-dependent manner, in addition to inducing apoptosis and arresting the cell cycle at the G2 phase. Mechanistically, cordycepin targeted the PI3K/Akt signaling pathway by significantly altering the expression levels/activation of several key mediators, and upregulated the anti-metastatic factor CLEC2. CONCLUSION: Cordycepin inhibited the proliferation and migration of gastric cancer cells by upregulating CLEC2 via the Akt signaling pathway.

Autophagy may promote carcinoma cell invasion and correlate with poor prognosis in cholangiocarcinoma.

The role of autophagy in cholangiocarcinoma is poorly understood. This study investigated its involvement in cholangiocarcinoma, focusing on carcinoma cell invasion and prognostic significance using cholangiocarcinoma cell lines, CCKS1 and HuCCT1, and human tissues of hilar and extrahepatic cholangiocarcinoma. Nutrient starvation induced the expression of LC3-II and the formation of LC3 puncta in both CCKS1 and HuCCT1, suggesting the occurrence of autophagy. The induction of autophagy was accompanied by the increased expression of an autophagy-related protein, Ambra1, in the cells. Under starvation conditions, the invasive activity of both cells was significantly increased, and a lysosomal inhibitor, chloroquine, attenuated this increased invasive activity. Transforming growth factor-ß1 (TGF-ß1), known as an inducer of epithelial-mesenchymal transition (EMT), increased the invasive activity of both cells, and chloroquine also significantly reduced TGF-ß1-induced cell invasion. Immunohistochemical staining using cholangiocarcinoma tissues showed that the expression of Ambra1 positively correlated with the expression of Snail, one of the major transcriptional factors of EMT. In addition, overexpression of Ambra1 significantly correlated with lymph node metastasis and poor survival rate of the patients. These results suggest that the occurrence of autophagy may be associated with a malignant phenotype and poor prognosis in cholangiocarcinoma, and autophagy is possibly involved in EMT-related cholangiocarcinoma cell invasion.

What are the precursor and early lesions of peripheral intrahepatic cholangiocarcinoma?

Cholangiocarcinoma (CC) is divided into distal, perihilar, and intrahepatic CCs (ICCS), and are further subdivided into large bile duct ICC and peripheral ICC. In distal and perihilar CC and large duct ICC, biliary intraepithelial neoplasm (BilIN) and intraductal papillary neoplasm (IPN) have been proposed as precursor lesions. Peripheral ICC, bile duct adenoma (BDA), biliary adenofibroma (BAF), and von Meyenburg complexes (VMCs) are reportedly followed by development of ICCs. Herein, we surveyed these candidate precursor lesions in the background liver of 37 cases of peripheral ICC and controls (perihilar CC, 34 cases; hepatocellular carcinoma, 34 cases and combined hepatocellular cholangiocarcinoma, 25 cases). In the background liver of peripheral ICC, BDA and BAF were not found, but there were not infrequently foci of BDA-like lesions and atypical bile duct lesions involving small bile ducts (32.4% and 10.8%, resp.). VMCs were equally found in peripheral CCs and also control CCs. In conclusion, BDA, BAF, and VMCs are a possible precursor lesion of a minority of peripheral CCs, and BDA-like lesions and atypical bile duct lesions involving small bile ducts may also be related to the development of peripheral ICC. Further pathologic studies on these lesions are warranted for analysis of development of peripheral ICCs.

Activation of the PI3K/mTOR pathway is involved in cystic proliferation of cholangiocytes of the PCK rat.

The polycystic kidney (PCK) rat is an animal model of Caroli's disease as well as autosomal recessive polycystic kidney disease (ARPKD). The signaling pathways involving the mammalian target of rapamycin (mTOR) are aberrantly activated in ARPKD. This study investigated the effects of inhibitors for the cell signaling pathways including mTOR on cholangiocyte proliferation of the PCK rat. Cultured PCK cholangiocytes were treated with rapamycin and everolimus [inhibitors of mTOR complex 1 (mTOC1)], LY294002 [an inhibitor of phosphatidylinositol 3-kinase (PI3K)] and NVP-BEZ235 (an inhibitor of PI3K and mTORC1/2), and the cell proliferative activity was determined in relation to autophagy and apoptosis. The expression of phosphorylated (p)-mTOR, p-Akt, and PI3K was increased in PCK cholangiocytes compared to normal cholangiocytes. All inhibitors significantly inhibited the cell proliferative activity of PCK cholangiocytes, where NVP-BEZ235 had the most prominent effect. NVP-BEZ235, but not rapamycin and everolimus, further inhibited biliary cyst formation in the three-dimensional cell culture system. Rapamycin and everolimus induced apoptosis in PCK cholangiocytes, whereas NVP-BEZ235 inhibited cholangiocyte apoptosis. Notably, the autophagic response was significantly induced following the treatment with NVP-BEZ235, but not rapamycin and everolimus. Inhibition of autophagy using siRNA against protein-light chain3 and 3-methyladenine significantly increased the cell proliferative activity of PCK cholangiocytes treated with NVP-BEZ235. In vivo, treatment of the PCK rat with NVP-BEZ235 attenuated cystic dilatation of the intrahepatic bile ducts, whereas renal cyst development was unaffected. These results suggest that the aberrant activation of the PI3K/mTOR pathway is involved in cystic proliferation of cholangiocytes of the PCK rat, and inhibition of the pathway can reduce cholangiocyte proliferation via the mechanism involving apoptosis and/or autophagy.

Participation of peribiliary glands in biliary tract pathophysiologies.

AIM: To investigate the roles of peribiliary glands around the bile ducts in the pathophysiology of the biliary tract. METHODS: The expression of fetal pancreatic markers, pancreatic duodenal homeobox factor 1 (PDX1) and hairy and enhancer of split 1 (HES1) and endodermal stem/progenitor (S/P) cell markers [CD44s, chemokine receptor type 4 (CXCR4), SOX9 and epithelial cell adhesion molecule (EpCAM)] were examined immunohistochemically in 32 normal adult livers (autopsy livers) and 22 hepatolithiatic livers (surgically resected livers). The latter was characterized by the proliferation of the peribiliary glands. Immunohistochemistry was performed using formalin-fixed, paraffin-embedded tissue sections after deparaffinization. Although PDX1 and HES1 were expressed in both the nucleus and cytoplasm of epithelial cells, only nuclear staining was evaluated. SOX9 was expressed in the nucleus, while CD44s, CXCR4 and EpCAM were expressed in the cell membranes. The frequency and extent of the expression of these molecules in the lining epithelia and peribiliary glands were evaluated semi-quantitatively based on the percentage of positive cells: 0, 1+ (focal), 2+ (moderate) and 3+ (extensive). RESULTS: In normal livers, PDX1 was infrequently expressed in the lining epithelia, but was frequently expressed in the peribiliary glands. In contrast, HES1 was frequently expressed in the lining epithelia, but its expression in the peribiliary glands was focal, suggesting that the peribiliary glands retain the potential of differentiation toward the pancreas and the lining epithelia exhibit properties to inhibit such differentiation. This unique combination was also seen in hepatolithiatic livers. The expression of endodermal S/P cell markers varied in the peribiliary glands in normal livers: SOX9 and EpCAM were frequently expressed, CD44s infrequently, and CXCR4 almost not at all. The expression of these markers, particularly CD44s and CXCR4, increased in the peribiliary glands and lining epithelia in hepatolithiatic livers. This increased expression of endodermal S/P cell markers may be related to the increased production of intestinal and gastric mucin and also to the biliary neoplasia associated with the gastric and intestinal phenotypes reported in hepatolithiasis. CONCLUSION: The unique expression pattern of PDX1 and HES1 and increased expression of endodermal S/P cell markers in the peribiliary glands may be involved in biliary pathophysiologies.

Case of telangiectatic/inflammatory hepatocellular adenoma arising in a patient with primary sclerosing cholangitis.

Hepatocellular adenomas (HCA) have been recently identified as a heterogeneous group, differing based on genotypic as well as morphological characteristics. HCA are most frequently found in women on oral contraception. A type of HCA, inflammatory HCA, is also known as telangiectatic HCA and was previously referred to as telangiectatic focal nodular hyperplasia. We present the first case of HCA arising from the liver with primary sclerosing cholangitis (PSC). This case is a 30-year-old man with a past medical history of PSC, ulcerative colitis and diabetes mellitus. A routine ultrasonography for PSC detected the gradually enlarged intrahepatic mass. Liver biopsy could reveal the diagnosis of telangiectatic/ inflammatory HCA by morphological and immunohistochemical analyses. Partial hepatectomy was performed and the resected liver was pathologically diagnosed as the telangiectatic/inflammatory HCA arising in PSC. This is the first case report of such an association and here we review the current developments and published work of this rare tumor and the association with an activated inflammatory related tumorogenic pathway and PSC.

Significance of immunoglobulin G4 (IgG4)-positive cells in extrahepatic cholangiocarcinoma: molecular mechanism of IgG4 reaction in cancer tissue.

UNLABELLED: IgG4 reactions consisting of marked infiltration by immunoglobulin G4 (IgG4)-positive plasma cells in affected organs is found in cancer patients as well as patients with IgG4-related diseases. Notably, extrahepatic cholangiocarcinomas accompanying marked IgG4 reactions clinicopathologically mimic IgG4-related sclerosing cholangitis. The regulatory cytokine interleukin (IL)-10 is thought to induce the differentiation of IgG4-positive cells. In this study, to clarify the mechanism of the IgG4 reaction in extrahepatic cholangiocarcinoma, we investigated nonprofessional antigen-presenting cells (APCs) generating IL-10-producing regulatory T cells (anergy T cells) and Foxp3-positive regulatory cells producing IL-10. Immunohistochemistry targeting IgG4, HLA-DR, CD80, CD86, and Foxp3 was performed using 54 cholangiocarcinoma specimens from 24 patients with gallbladder cancer, 22 patients with common bile duct cancer, and eight patients with cancer of the Papilla of Vater. Moreover, a molecular analysis of Foxp3 and IL-10 was performed using a cultured human cholangiocarcinoma cell line. Consequently, 43% of the cholangiocarcinomas were found to be abundant in IgG4. Those expressing HLA-DR but lacking costimulatory molecules (CD80 and CD86) and those expressing Foxp3 detected by an antibody recognizing the N terminus accounted for 54% and 39% of cases, respectively. Moreover, the number of IgG4-positive cells was larger in these cases than in other groups. In cultured cells, the presence of a splicing variant of Foxp3 messenger RNA and the expression of IL-10 were demonstrated. CONCLUSION: Extrahepatic cholangiocarcinoma is often accompanied by significant infiltration of IgG4-positive cells. Cholangiocarcinoma cells could play the role of nonprofessional APCs and Foxp3-positive regulatory cells, inducing IgG4 reactions via the production of IL-10 indirectly and directly, respectively.

Caroli's Disease: Current Knowledge of Its Biliary Pathogenesis Obtained from an Orthologous Rat Model.

Caroli's disease belongs to a group of hepatic fibropolycystic diseases and is a hepatic manifestation of autosomal recessive polycystic kidney disease (ARPKD). It is a congenital disorder characterized by segmental saccular dilatations of the large intrahepatic bile duct and is frequently associated with congenital hepatic fibrosis (CHF). The most viable theory explaining its pathogenesis suggests that it is related to ductal plate malformation. The development of the polycystic kidney (PCK) rat, an orthologous rodent model of Caroli's disease with CHF as well as ARPKD, has allowed the molecular pathogenesis of the disease and the therapeutic options for its treatment to be examined. The relevance of the findings of studies using PCK rats and/or the cholangiocyte cell line derived from them to the pathogenesis of human Caroli's disease is currently being analyzed. Fibrocystin/polyductin, the gene product responsible for ARPKD, is normally localized to primary cilia, and defects in the fibrocystin from primary cilia are observed in PCK cholangiocytes. Ciliopathies involving PCK cholangiocytes (cholangiociliopathies) appear to be associated with decreased intracellular calcium levels and increased cAMP concentrations, causing cholangiocyte hyperproliferation, abnormal cell matrix interactions, and altered fluid secretion, which ultimately result in bile duct dilatation. This article reviews the current knowledge about the pathogenesis of Caroli's disease with CHF, particularly focusing on studies of the mechanism responsible for the biliary dysgenesis observed in PCK rats.

Biliary infection may exacerbate biliary cystogenesis through the induction of VEGF in cholangiocytes of the polycystic kidney (PCK) rat.

Cholangitis arising from biliary infection dominates the prognosis in Caroli's disease. To clarify the influences of bacterial infection on the biliary cystogenesis, in vivo and in vitro studies were performed using the polycystic kidney (PCK) rat as an animal model of Caroli's disease. Cholangitis became a frequent histological finding in aged PCK rats, and neovascularization around the bile ducts also increased in aged PCK rats. Immunohistochemistry revealed that expression of vascular endothelial growth factor (VEGF) was increased in PCK rat biliary epithelium. In vitro, PCK cholangiocytes overexpressed VEGF, and the supernatant of cultured PCK cholangiocytes significantly increased the proliferative activity, migration, and tube formation of cultured rat vascular endothelial cells. Stimulation with lipopolysaccharide (LPS) further induced VEGF expression in PCK cholangiocytes, which might be mediated by signaling pathways involving phosphatidylinositol 3-kinase (PI3K)-Akt and c-Jun N-terminal kinase (JNK). Both LPS and VEGF increased cell proliferative activity in PCK cholangiocytes, and siRNA against VEGF significantly reduced LPS-induced cell proliferation. Thus, LPS-induced overexpression of VEGF in the biliary epithelium may lead to hypervascularity around the bile ducts; concurrently, LPS and VEGF act as cell proliferation factors for cholangiocytes. Biliary infection may thus exacerbate biliary cystogenesis in PCK rats.

Monocyte chemoattractant protein-1 derived from biliary innate immunity contributes to hepatic fibrogenesis.

AIMS: Monocyte chemoattractant protein-1 (MCP-1) is a major chemotactic factor for hepatic stellate cells (HSCs) associated with hepatic fibrosis. In this study, among several fibrogenetic factors derived from biliary epithelial cells (BECs), MCP-1 produced by the biliary innate immune system was found to be most critical in the histogenesis of hepatic fibrogenesis. METHODS: Using cultured human BECs, the expression of five fibrogenetic factors including MCP-1 on stimulation with Toll-like receptor ligands, inflammatory cytokines or bile acids was examined. Moreover, in situ detection of MCP-1 and α-smooth muscle actin proteins was performed using sections from normal and diseased livers by immunohistochemistry. RESULTS: All fibrogenetic factors were detected in BECs, but only MCP-1 expression was upregulated, by all the Toll-like receptor ligands, IL-1ß, and tumour necrosis factor-alpha. Proliferating bile ductules in interface areas expressed MCP-1 in diseased livers accompanying α-smooth muscle actin-positive activated HSCs. CONCLUSIONS: Bile ductules proliferate in various hepatobiliary diseases, and its significance is still unknown. This study demonstrated that BECs in bile ductules could produce MCP-1, particularly, via biliary innate immunity, suggesting that MCP-1 derived from BECs plays an important role in the recruitment of HSCs to interface areas and the activation of HSCs resulting in the progression of periportal fibrosis.

Significance of periductal Langerhans cells and biliary epithelial cell-derived macrophage inflammatory protein-3α in the pathogenesis of primary biliary cirrhosis.

BACKGROUND/AIMS: To clarify the primary biliary cirrhosis (PBC)-specific antigen-presenting mechanism, we examined the distribution and phenotypic characteristics of infiltrating dendritic cells (DCs) with respect to bile ducts and the mechanism of migration in terms of the periductal cytokine milieu and biliary innate immunity. METHODS AND RESULTS: Immunohistochemistry using liver sections from patients with PBC and controls revealed that blood dendritic cell antigen (BDCA)-2(+) plasmacytoid DCs were found mainly in the portal tracts in PBC and the controls, but their distribution was not related to bile ducts. BDCA-1(+) and CD19(-) myeloid DCs were also found in portal tracts in PBC and the controls and, in particular, Langerin+Langerhans cells (LCs) were dominantly scattered around or within biliary epithelial layers of the damaged bile ducts in PBC. Moreover, experiments with cultured human biliary epithelial cells (BECs) showed that an LC-attracting chemokine, macrophage inflammatory protein-3α, was produced by BECs in the response to cytokines [interleukin (IL)-1ß, tumour necrosis factor-α and IL-17] and pathogen-associated molecular patterns. CONCLUSIONS: LCs existing around or within biliary epithelial layers are important as periductal antigen-presenting cells in PBC and the migration of LCs into bile ducts is closely associated with the periductal cytokine milieu and biliary innate immunity in PBC.

A fatal case of progressive steatohepatitis, possibly chemotherapy-associated steatohepatitis related to gemcitabine.

We report the case of an 80-year-old female suffering from pancreatic cancer who developed severe non-alcoholic steatohepatitis (NASH) resulting in fatal hepatic failure after anti-cancer chemotherapy with gemcitabine. Hepatic encephalopathy appeared 1 year after the chemotherapy, and the patient developed progressive liver failure and eventually died. Radiological examination showed severe fatty liver. Histopathological examination of a liver needle necropsy showed almost panlobular macrovesicular fatty change. Ballooning degeneration and necrosis of hepatocytes accompanying neutrophil infiltration, Mallory bodies, and a few bile plugs were found in zone 3. Marked perivenular and pericellular/perisinusoidal fibrosis and extensive bridging fibrosis were also found. Together, these findings indicated steatohepatitis at a precirrhotic stage. Because the patient had no history of drinking in excess, we made a diagnosis of NASH, in particular, chemotherapy-associated steatohepatitis (CASH). Gemcitabine is a pyrimidine nucleoside antimetabolite with anti-cancer activity. A few reports have mentioned fatal hepatotoxicity caused by gemcitabine, but, to our knowledge, this is the first report of steatohepatitis, possibly associated with gemcitabine. Physicians treating patients with this drug should be aware of the possibility of steatohepatitis.