Cyclooxygenase-2 is involved in the up-regulation of matrix metalloproteinase-9 in cholangiocarcinoma induced by tumor necrosis factor-alpha.

Matrix metalloproteinase-9 (MMP-9) is an important enzyme in tumor invasion and metastasis in malignant tumors, including cholangiocarcinoma (CC). Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, was recently reported to induce the up-regulation of MMP-9 in cultured CC cells. We examined whether cyclooxygenase-2 (COX-2) and prostaglandin-E2 (PGE2), another endogenous tumor promoter, are involved in the up-regulation of MMP-9 in CC using CC tissue specimens and a CC cell line, HuCCT-1. MMP-9 and COX-2 were immunohistochemically expressed in 58% and 89% of 110 CC cases, respectively; the expression of MMP-9 and COX-2 was correlated (r = 0.32, P = 0.00072). Using zymography, latent MMP-9 was detectable in all cases and active MMP-9 was detected in 24% of cases of the CC specimens. The TNF-alpha/TNF-receptor 1 (TNF-R1) interaction induced MMP-9 production and activation, as well as COX-2 overexpression and PGE2 production, and increased the migration of CC cells. MMP-9 up-regulation was inhibited by COX inhibitors, antagonists of EP2/4 (receptors of PGE2), and COX-1 and COX-2 siRNAs. Inhibitors of both MMP-9 and MMP-9 siRNA treatment abrogated the increase in the migration of CC cells induced by TNF-alpha. In conclusion, we propose a novel signaling pathway of MMP-9 up-regulation in CC cells such that TNF-alpha induces the activation of COX-2 and PGE2 via TNF-R1 followed by the up-regulation of MMP-9 via the PGE2 (EP2/4) receptor.

Cardiopulmonary arrest secondary to compression of the heart owing to esophageal hiatal hernia: a case report.

BACKGROUND: Esophageal hiatal hernia is commonly encountered in clinical practice. We describe a case of cardiac compression caused by an esophageal hiatal hernia that resulted in circulatory failure and cardiac arrest. CASE PRESENTATION: An 82-year-old woman presented to our hospital with vomiting, which progressed to cardiac arrest in the emergency room after computed tomography (CT) imaging. CT revealed gastric herniation into the mediastinum, with marked cardiac compression. Cardiopulmonary resuscitation was performed, and a nasogastric tube was inserted for gastric decompression, which resulted in the return of spontaneous circulation and subsequent hemodynamic stabilization. However, the patient died of aspiration pneumonia 4 days later. CONCLUSION: Gastric decompression can lead to rapid improvements in respiration and circulation in patients with an esophageal hiatal hernia. Nonetheless, to prevent complications, such as those observed in our patient, definitive surgical treatment is warranted.

Expression of matrix metalloproteinase 7 is an unfavorable postoperative prognostic factor in cholangiocarcinoma of the perihilar, hilar, and extrahepatic bile ducts.

Cholangiocarcinoma of the perihilar, hilar, and extrahepatic bile ducts (collectively referred to as the large bile ducts) is an intractable disease, and a papillary phenotype and well-differentiated histologic grade have been proposed as indicators of a favorable prognosis after surgical resection. In this study, we examined the significance of matrix metalloproteinases (MMPs) in cholangiocarcinoma with respect to clinicopathologic features. We subjected 66 surgically resected specimens of cholangiocarcinoma of the large bile ducts to clinicopathologic examination, including postoperative survival, papillary phenotype, and immunohistochemical expression of MMP-2,-7, -9, and membrane type 1 MMP (MT1-MP). Nonneoplastic biliary epithelium did not express these 4 MMPs, whereas cholangiocarcinoma frequently expressed MMP-2 (33.9%), -7 (75.8%), -9 (47.5%), and MT1-MMP (54.5%). In particular, conventional (nonpapillary) cholangiocarcinoma expressed MMP-7 and MT1-MMP more frequently than papillary cholangiocarcinoma. The expression of MMP-7 and MT1-MMP significantly correlated with the nonpapillary phenotype, poorly differentiated histologic grade, perineural invasion, and advanced TNM stage. In contrast, the expression of MMP-2 and -9 was not associated with any of the clinicopathologic features. Univariate analysis of disease-specific survival revealed that a papillary phenotype and expression of MMP-7 were prognostic factors of cholangiocarcinoma, in addition to TNM stage, poorly differentiated histologic grade, perineural invasion, and microscopic margin status. Multivariate analysis showed only TNM stage to be an independent prognostic factor. Expression of MMP-7 in cholangiocarcinoma is an unfavorable postoperative prognostic factor of cholangiocarcinoma arising from the large bile ducts. Underexpression of MMPs in papillary cholangiocarcinoma might be associated with a favorable prognosis.

Tumor necrosis factor-alpha induces the aberrant expression of mucus core protein-2 in non-neoplastic biliary epithelial cells via the upregulation of CDX2 in chronic cholangitis.

AIM: Chronic cholangitis, such as hepatolithiasis, is frequently associated with goblet cell metaplasia and the aberrant expression of mucus core protein-2 (MUC2). In this study, we clarified the role of inflammatory cytokines in the expression of MUC2 in lining biliary epithelial cells (BEC) in chronic cholangitis with an emphasis on CDX2, an intestine-specific transcription factor. METHODS: We used human hepatolithiatic livers and polycystic kidney (PCK) rats, an animal model of Caroli's disease, and cultured BEC from PCK rats. As a control, extrahepatic biliary obstruction and histologically normal livers and Crj:CD rats were used. RESULTS: Immunohistochemically, tumor necrosis factor-alpha (TNF-alpha) was expressed in periductal inflammatory cells and BEC of the affected intrahepatic bile ducts with an aberrant expression of MUC2 and CDX2 in hepatolithiasis and the PCK rats. In cultured BEC, TNF-alpha, interleukin (IL)-1beta, IL-6, and interferon-gamma induced the expression of CDX2 mRNA, though only TNF-alpha additionally induced the expression of MUC2 mRNA. The expression of CDX2 mRNA and the MUC2 protein induced in BEC by TNF-alpha were abolished by pretreatment of nuclear factor-kappaB inhibitors. CONCLUSION: The aberrant expression of CDX2 and MUC2 in the affected bile ducts showing goblet cell metaplasia was closely associated with TNF-alpha expressed in periductal infiltrating inflammatory cells and BEC. TNF-alpha induced the expression of CDX2 and MUC2 in cultured BEC. Taken together, it seems likely that TNF-alpha plays a role in MUC2 expression via CDX2 upregulation in the bile ducts with chronic cholangitis and goblet cell metaplasia.

Frequent p16ink4a inactivation is an early and frequent event of intraductal papillary neoplasm of the liver arising in hepatolithiasis.

Intraductal papillary neoplasm of the liver (IPNL) is a precursor lesion of intrahepatic cholangiocarcinoma (ICC) arising in hepatolithiasis. In this study, 98 foci of IPNL identified in 39 surgically resected hepatolithiatic livers were investigated for expression of p16INK4a, cyclin D1, p21WAF1/CIP1, p53, mouse double-minute 2 (MDM2), and pRb. In addition, methylation-specific polymerase chain reaction (MSP) for p16 INK4a promoter region was performed in these foci. Nonneoplastic bile ducts from 11 hepatolithiatic livers, 5 histologically normal livers, and 9 cases of nonpapillary conventional ICC were used as controls. Decreased expression of p16INK4A was seen in IPNL group 1 with mild dysplasia and continued along the progression of IPNL to ICC. The expression of cyclin D1, p21WAF1/CIP1,and pRb gradually increased along the progression of IPNL to ICC and became significantly high in IPNL of group 3 (carcinoma in situ). The expression of p53 and MDM2 was increased in IPNL group 3 and group 4 with evident invasive carcinoma. MSP revealed that 54.6% of 44 IPNL foci harbored p16INK4a promoter hypermethylation, and such foci were significantly correlated with decreased expression of p16INK4a protein. Ki-67 labeling index exhibited a stepwise increase from IPNL group 1 to group 4. We conclude that p16INK4a inactivation, due mainly to its promoter hypermethylation, is a frequent and early event of IPNL and may be responsible for genetic and epigenetic alterations of other cell cycle regulators in IPNL.

Expression of trefoil factor family 1, 2, and 3 peptide is augmented in hepatolithiasis.

Trefoil factor family (TFF) 1, 2, and 3 is a mucin-associated protein involved in the maintenance of mucosal barrier and restitution of lining epithelial cells. In this study, the expression of TFF1, 2, and 3 and MUC mucins were examined immunohistochemically in hepatolithiasis and control livers. The expression of TFF1, 2, and 3 were augmented markedly in biliary mucosa in hepatolithiasis in coordinate with gel-forming mucin. TFF3 was detected in hepatic bile samples of hepatolithiasis. Augmented expression and secretion of TFF in biliary mucosa in hepatolithiasis may play a role in lithogenesis together with gel-forming mucin in addition to mucosal defense and repair.

Immunohistochemical analysis of the progression of flat and papillary preneoplastic lesions in intrahepatic cholangiocarcinogenesis in hepatolithiasis.

BACKGROUND: Two types of precursor lesions, flat-type 'biliary intraepithelial neoplasia (BilIN)' and papillary-type 'intraductal papillary neoplasm of the bile duct (IPNB)', are proposed in the tumorigenesis of intrahepatic cholangiocarcinoma (ICC) in hepatolithiasis. METHODS: In this study, the participation of cancer-related molecules in the progression of these two precursor lesions was examined, using 64 hepatolithiatic livers with BilIN lesions (45 livers) and IPNB lesions (19 livers) and 10 hepatolithiatic livers without neoplastic lesions as a control. The expression of E-cadherin, beta-catenin, matrix metalloproteinase-7 (MMP-7), membrane type 1-MMP (MT1-MMP), cyclin D1 and c-myc was immunohistochemically examined. RESULTS: The membranous expression of beta-catenin decreased along with the progression in both BilIN and IPNB lineages. Membranous expression of E-cadherin was significantly decreased in invasive ICC with BilIN and IPNB in comparison with non-invasive counterparts. MMP-7 and MT1-MMP were commonly expressed in invasive ICC with BilIN (100%), while non-invasive lesions (BilIN-1, -2, -3) and the IPNB lineage were only occasionally and weakly positive for these molecules. Cyclin D1 and c-myc, target molecules of Wnt signalling, were frequently positive in the IPNB lineage (65 and 54% respectively), and interestingly nuclear beta-catenin staining, reflecting activation of Wnt signalling, was observed only in the IPNB lineage (22%) (P<0.05). CONCLUSIONS: Decreased membranous expression of beta-catenin and E-cadherin is an early event in the tumorigenesis of both BilIN and IPNB lineages. The expression of MMP-7 and MT1-MMP was closely associated with invasive growth of the BilIN lineage. The Wnt signalling pathway may play an important role in the tumorigenesis of the IPNB lineage.

Possible regulation of migration of intrahepatic cholangiocarcinoma cells by interaction of CXCR4 expressed in carcinoma cells with tumor necrosis factor-alpha and stromal-derived factor-1 released in stroma.

Intrahepatic cholangiocarcinoma (ICC) is highly fatal because of early invasion, widespread metastasis, and lack of an effective therapy. We examined roles of CXCR4 and its ligand, stromal cell-derived factor (SDF)-1, in migration of ICC with respect to tumor-stromal interaction by using two ICC cell lines, a fibroblast cell line (WI-38), and 28 human ICC tissues. The two ICC cell lines expressed CXCR4 mRNA and protein, and WI-38 fibroblasts expressed SDF-1 mRNA and protein. Migration of cultured ICC cells in Matrigel was induced by co-culture with WI-38 fibroblasts and by incubation with SDF-1. Anti-SDF-1 antibody suppressed migration, demonstrating that SDF-1 released from WI-38 fibroblasts was responsible for this migration. Tumor necrosis factor (TNF)-alpha pretreatment of ICC cells up-regulated CXCR4 mRNA and protein expression in a concentration-dependent manner. Administration of SDF-1 and TNF-alpha increased synergistically ICC cell migration, which was suppressed by the CXCR4 antagonist AMD3100. In ICC tissue, TNF-alpha was mainly expressed in infiltrated macrophages, CXCR4 in ICC cells, and SDF-1 in stromal fibroblasts. In conclusion, the interaction of SDF-1 released from fibroblasts and CXCR4 expressed on ICC cells may be actively involved in ICC migration, and TNF-alpha may enhance ICC cell migration by increasing CXCR4 expression. CXCR4 could be a therapeutic target to prevent ICC invasion.

Local balance of transforming growth factor-beta1 secreted from cholangiocarcinoma cells and stromal-derived factor-1 secreted from stromal fibroblasts is a factor involved in invasion of cholangiocarcinoma.

Tumor-stromal interactions are important for the progression of malignant tumors. The purpose of the present study was to examine interactions of cholangiocarcinoma (CC) cells and stromal fibroblasts with respect to stromal-derived factor-1 (SDF-1) and transforming growth factor (TGF)-beta1. Two cell lines of CC (HuCCT-1 and CCKS-1) and WI-38 fibroblast cell line were used for cell culture, and 12 CC tissue specimens for immunohistochemical studies. Invasion of CC cells was increased significantly by the supernatant from fibroblast cultures, but not by the supernatant from fibroblasts cocultured with CC cells. Expression of SDF-1 in cultured fibroblasts was downregulated by TGF-beta1 treatment, and coculture with CC cells and anti-TGF-beta1 neutralizing antibody restored the decreased SDF-1 expression, suggesting that TGF-beta1 secreted from CC cells might have reduced the expression of SDF-1 by fibroblasts and might have reduced the increased invasion of CC cells induced by the supernatant from fibroblasts. Immunohistochemical expression of TGF-beta1 in CC cells was focal or negative and that of SDF-1 was evident in stromal fibroblasts at the invasive front of CC. In conclusion, local mutual influence of TGF-beta1 secreted from carcinoma cells and SDF-1 expressed by stromal fibroblasts may be involved in invasion of CC cells.

Lipopolysaccharide activates nuclear factor-kappaB through toll-like receptors and related molecules in cultured biliary epithelial cells.

To clarify the innate immunity of the intrahepatic biliary tree, we examined expression of Toll-like receptors and intracellular signalings in biliary epithelial cells in response to bacterial components by using cultured biliary epithelial cells (murine biliary cells and human cholangiocarcinoma cell lines). The expression of Toll-like receptors in cultured cells was examined by reverse transcription and PCR and immunohistochemistry. Intracellular signalings after Toll-like receptors activation by lipopolysaccharide was examined by analysis of nuclear factor (NF)-kappaB activation and inhibition studies using inhibitors for NF-kappaB and mitogen-activated protein kinase and blocking antibody. The mRNAs of Toll-like receptors 2, 3, 4, and 5, and related molecules (MD-2, MyD88, and CD14) were detected, and their proteins were expressed in cultured cells. Lipopolysaccharide was shown to bind to the cell surface of cultured cells. Lipopolysaccharide treatment induced the production of TNF-alpha, and nuclear translocation of NF-kappaB and increased NF-kappaB-DNA binding in cultured cells. This induction of TNF-alpha was partially inhibited by anti-Toll-like receptor 4 antibody. The nuclear translocation and increased binding of NF-kappaB by lipopolysaccharide were blocked by addition of MG132, an inhibitor of NF-kappaB. In conclusion, lipopolysaccharide appears to form a receptor complex of CD14, Toll-like receptor 4, MD-2, and MyD88 in cultured biliary epithelial cells and seems to regulate activation of NF-kappaB and synthesis of TNF-alpha. The recognition of pathogen-associated molecular patterns using Toll-like receptors and related molecules in biliary epithelial cells, which is demonstrated in this in vitro study, may participate in an immunopathology of the intrahepatic biliary tree in vivo.

Aberrant expression of CDX2 is closely related to the intestinal metaplasia and MUC2 expression in intraductal papillary neoplasm of the liver in hepatolithiasis.

Intraductal papillary neoplasia of the liver (IPNL) frequently presents gastrointestinal metaplasia with aberrant expression of MUC2 and MUC5AC and oversecretion of mucin into the ductal lumen. In this study, the involvement of CDX2, a homeodomain protein involved in the regulation of intestinal development and differentiation, in the expression of MUC2 was examined in mucinous intrahepatic cholangiocarcinoma (ICC) (n=7) and IPNL with hepatolithiasis (n=19) with comparison to conventional ICC (n=11), and intraductal papillary mucinous tumor and invasive ductal carcinoma of the pancreas (n=9 and 11, respectively). A total of 33 cases of hepatolithiasis, extrahepatic biliary obstruction and normal livers were used as the control. Immunohistochemically, both MUC2 and MUC5AC were frequently expressed in mucinous ICC and IPNL, while expression of MUC2 was not seen in conventional ICC. The nuclear expression of CDX2 was closely associated with the expression of MUC2 in mucinous ICC and IPNL. This intimate association of MUC2 and CDX2 was confirmed by double immunostaining. The cytoplasmic CDX2 expression was frequent in the mucinous and the conventional ICC and pancreatic carcinoma, irrespective of MUC2 and MUC5AC expression. CDX2 mRNA was detected in neoplastic cells showing cytoplasmic as well as nuclear expression of CDX2 by reverse transcriptase-polymerase chain reaction. One IPMT expressed MUC2 associated with nuclear CDX2 expression, while the other IPMT and conventional pancreatic carcinoma expressed MUC5AC only. Aberrant expression of CDX2 is closely related to the overexpression of MUC2 in mucinous ICC and IPNL associated with hepatolithiasia, suggesting its role in intestinal differentiation and its association with carcinogenesis in these tumors.