Hilar Cholangiocarcinoma Tissues Research Articles

Bsep expression\xa0in hilar cholangiocarcinoma of rat model

Develop a rat model of hilar cholangiocarcinoma for detecting bile salt export pump (Bsep) expression in hilar cholangiocarcinoma tissues, in order to provide a new therapeutic target for the gene therapy of hilar cholangiocarcinoma. Sixty male Wistar rats (body weight, 190 ± 8 g) were randomly divided into three groups (the experimental group, the control group and the sham operation group, n = 20 each) as follows: The three groups were fed a standard diet, the experimental group was injected by cholangiocarcinoma QBC939 cell suspension along the hilar bile duct into the bile duct bifurcation with microsyringe, the control group was injected by normal saline, the sham operation group did not inject anything. Every day assess the rats’ mental state, diet, and motion by using Basso–Beattie–Bresnahan and combined behavioral score. At 4 weeks, one rat of the experimental group was sacrificed after it was administered anesthesia, and we recorded changes in hilar bile duct size, texture, and form. This procedure was repeated at 6 weeks. After 6 weeks, hilar cholangiocarcinoma developed only in the experimental group, thereby establishing an experimental model for studying QBC939-induced hilar cholangiocarcinoma. Tumor formation was confirmed by pathological examination, and hilar bile duct tissues were harvested from both the groups. A real-time polymerase chain reaction assay and an immunohistochemical assay were used to analyze the expression of Bsep in hilar bile duct tissues of each group. From the second week, the rats in experimental group began to eat less, and their body mass decreased compared with control group and sham operation group. After 6 weeks, we detected hilar cholangiocarcinoma in the hilar bile duct tissues of 18 rats (90%) in the experimental group. In the experimental group with hilar cholangiocarcinoma, we found that the levels of total cholesterol, total bilirubin, and direct bilirubin were higher compared with those in the control group and sham operation group. Simultaneously, muddy stones emerged from the bile ducts of rats in the experimental group. The Bsep/Gapdh mRNA ratio in hilar cholangiocarcinoma, control group and sham operation group differed markedly. Light microscopy revealed a granular pattern of Bsep protein expression which reacted with the anti-Bsep antibody. Each section was randomly divided into six regions, with 80 cells were observed in every region. Sections with > 10% positive cells were designated positive, Sections with < 10% positive cells were designated negative. Each group included 4800 cells. In the experimental group, 1200 cells (25%) were positive, in the control group, 3648 cells (76%) were positive and in the sham operation group 3598 cells (75%) were positive, and this difference was statistically significant. Bsep expression significantly decreased in hilar cholangiocarcinoma of rats than those in control group and sham operation group, suggesting that drugs targeting Bsep are a new strategy for hilar cholangiocarcinoma.

Expression and significance of L-FABP in hilar cholangiocarcinoma

Objective To explore the expression of liver fatty acid binding protein (L-FABP) in tissues of hilar cholangiocarcinoma and the relationship between expression of L-FABP and clinicopathological factors and prognosis of the patients. Methods The retrospective case-control study was conducted. The clinicopathological data of 132 patients with hilar cholangiocarcinoma who were admitted to the Navy General Hospital between January 2003 and January 2013 were collected. The expression of L-FABP in tumor tissues and adjacent tissues of hilar cholangiocarcinoma and normal bile duct tissues were respectively detected by immunohistochemistry. Observation indicators: (1) expression of L-FABP by immunohistochemistry; (2) relationship between clinicopathological factors of patients and expression of L-FABP in tumor tissues; (3) follow-up and survival situations; (4) prognostic analysis of patients after radical resection of hilar cholangiocarcinoma. Follow-up using outpatient examination and telephone interview was performed to detect postoperative overall survival time up to June 2017. Count data were described as percentage and compared using the chi-square test. The survival time was calculated by the Kaplan-Meier method. Measurement data with skewed distribution were described as M (range). The univariate analysis and multivariate analysis were respectively done using the nonparametric test and COX regression model. Results (1) Expression of L-FABP by immunohistochemistry: the positive expressions of L-FABP were located in the cytoplasm. The low, moderate and high expression rates of L-FABP in tumor tissues were respectively 11.36%(15/132), 71.97%(95/132) and 16.67%(22/132), and positive-staining cells showed platy and / or diffuse distribution; the low, moderate and high expression rates of L-FABP in adjacent tissues of hilar cholangiocarcinoma were respectively 77.27%(102/132), 7.58%(10/132) and 15.15%(20/132), and positive-staining cells showed scattered or platy distribution, with a weaker staining intensity compared with tumor tissues; there was no positive expression in normal bile duct tissues. There was a statistically significant difference in expressions of L-FABP among tumor tissues and adjacent tissues of hilar cholangiocarcinoma and normal bile duct tissues (χ2=5.423, P 5 cm , with a statistically significant difference (χ2=10.171, P< 0.05). (3) Follow-up and survival situations: 132 patients were followed up for 5-90 months, with a median time of 33 months. During the follow-up, postoperative overall median survival time of 132 patients was 31 months. (4) Prognostic analysis of patients after radical resection of hilar cholangiocarcinoma: results of univariate analysis showed that tumor differentiation, lymph node metastasis and expressions of L-FABP in tumor tissues were related factors affecting prognosis of patients after radical resection of hilar cholangiocarcinoma (Z=1.845, 3.156, 1.243, P<0.05). Results of multivariate analysis showed that tumor differentiation, lymph node metastasis and expressions of L-FABP in tumor tissues were independent factors affecting prognosis of patients after radical resection of hilar cholangiocarcinoma (odds ratio=0.431, 1.806, 3.692, 95% confidence interval: 0.292-0.693, 0.974-2.973, 1.875-11.364, P<0.05). Conclusions The high expression of L-FABP in tumor tissues is significantly correlated with the tumor diameter. Tumor differentiation, lymph node metastasis and expressions of L-FABP in tumor tissues are independent factors affecting prognosis of patients after radical resection of hilar cholangiocarcinoma. Key words: Hilar cholangiocarcinoma; Liver fatty acid binding protein; Expressions; Radical resection; Prognosis

Elevated AQP1 Expression Is Associated With Unfavorable Oncologic Outcome in Patients With Hilar Cholangiocarcinoma.

Hilar cholangiocarcinomas are malignant tumors with a poor prognosis. An early prediction of prognosis for patients may help us determine treatment strategies. Aquaporin 1 is a cell membrane channel involved in water transport, cell motility, and proliferation. Increasing evidences showed that aquaporin 1 played a role in tumor prognosis and diagnosis. The purpose of this study is to evaluate the role of aquaporin 1 in hilar cholangiocarcinoma. Here, we analyzed messenger RNA expression data of genes function as bile secretion in a data set of 169 samples using the R2 bioinformatic platform ( http://r2.amc.nl ). Quantitative polymerase chain reaction was performed to verify the gene expression in 17 hilar cholangiocarcinoma samples. Immunohistochemistry was also performed in a series of specimens from 62 hilar cholangiocarcinoma tissues, and its clinical significance was assessed by clinical correlation and Kaplan-Meier analyses. All data were analyzed using the R2 web application, aquaporin 1 was selected for further analysis. The significant expression variation of aquaporin 1 among 17 cases with cholangiocarcinoma was also found using quantitative polymerase chain reaction. The expression level of aquaporin 1 protein significantly correlated with tumor-node-metastasis stage ( P = .002) and overall survival time ( P = .010). Higher aquaporin 1 expression indicated poor prognostic outcomes ( P <.05, log-rank test). Multivariate analysis also showed strong aquaporin 1 protein expression was an independent adverse prognosticator in hilar cholangiocarcinoma ( P = .002). This study highlighted the prognostic value of aquaporin 1 in hilar cholangiocarcinoma. Strong aquaporin 1 expression predicts poor survival, regardless of pathological features. Immunohistochemical detection of aquaporin 1, as a prognostic marker, may contribute to predicting clinical outcome for patients with hilar cholangiocarcinoma.

PKM2 regulates neural invasion of and predicts poor prognosis for human hilar cholangiocarcinoma.

BackgroundThe therapeutic and prognostic value of the glycolytic enzymes hexokinase, phosphofructokinase, and pyruvate kinase (PK) has been implicated in a variety of cancers, while their roles in treatment of and prognosis for hilar cholangiocarcinoma (HC) remain unclear. In this study, we determined the expression of PKM2 in and its impact on biology and clinical outcome of human HC.MethodsThe regulation and function of PKM2 in HC pathogenesis was evaluated using human tissues, molecular and cell biology, and animal models, and its prognostic significance was determined according to its impact on patient survival.ResultsWe found that expression of hexokinase 1 and the M2 splice isoform of PK (PKM2) was upregulated in HC tissues and that this expression correlated with tumor recurrence and outcome. PKM2 expression was increased in HC cases with chronic cholangitis as demonstrated by isobaric tags for relative and absolute quantification. High PKM2 expression was highly correlated with high syndecan 2 (SDC2) expression and neural invasion. PKM2 downregulation led to a decrease in SDC2 expression. Treatment with metformin markedly suppressed PKM2 and SDC2 expression at both the transcriptional and posttranscriptional levels and inhibited HC cell proliferation and tumor growth.ConclusionsPKM2 regulates neural invasion of HC cells at least in part via regulation of SDC2. Inhibition of PKM2 and SDC2 expression contributes to the therapeutic effect of metformin on HC. Therefore, PKM2 is an independent prognostic factor and potential therapeutic target for human HC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0462-6) contains supplementary material, which is available to authorized users.

Downregulation of the expression of HDGF attenuates malignant biological behaviors of hilar cholangiocarcinoma cells.

Hepatoma-derived growth factor (HDGF) has been reported to be a potential predictive and prognostic marker for several types of cancer and important in malignant biological behaviors. However, its role in human hilar cholangiocarcinoma remains to be elucidated. Our previous study demonstrated that high expression levels of HDGF in hilar cholangiocarcinoma tissues correlates with tumor progression and patient outcome. The present study aimed to elucidate the detailed functions of the HDGF protein. This was performed by downregulating the protein expression of HDGF in the FRH0201 hilar cholangiocarcinoma cell line by RNA interference (RNAi) invitro, and revealed that downregulation of the HDGF protein significantly inhibited the malignant biological behavior of the FRH0201 cells. In addition, further investigation revealed that downregulation of the protein expression of HDGF significantly decreased the secretion of vascular endothelial growth factor, which may be the mechanism partially responsible for the inhibition of malignant biological behaviors. These findings demonstrated that HDGF is important in promoting malignant biological behaviors, including proliferation, migration and invasion of hilar cholangiocarcinoma FRH0201 cells. Inhibition of the expression of HDGF downregulated the malignant biological behaviors, suggesting that downregulation of the protein expression of HDGF by RNAi may be a novel therapeutic approach to inhibit the progression of hilar cholangiocarcinoma.

Expression and clinical significance of PIWIL2 in hilar cholangiocarcinoma tissues and cell lines.

The objective of this study was to explore the relationship between PIWI-like protein 2 (PIWIL2) and clinicopathological charac-teristics and prognosis after radical resection. To accomplish this, we analyzed PIWIL2 expression in hilar cholangiocarcinoma tissues and cell lines. PIWIL2 expression was detected by immunohistochemistry in 41 hilar cholangiocarcinoma samples and 10 control tissues. Western blotting and immunocytofluorescence were used to investigate PIWIL2 expression in the cholangiocarcinoma cell line QBC939 and the bile duct epithelial cell line HIBEpic. Univariate and multivariate surviv-al analyses were performed using the Kaplan-Meier method for hilar cholangiocarcinoma patients who underwent radical resection. PIWIL2 expression was significantly higher in the hilar cholangiocarcinoma tissues and QBC939 cells than in control tissues and HIBEpic cells, respectively (P < 0.05). Poorly and moderately differentiated cholan-giocarcinoma tissues had significantly higher PIWIL2 expression than well-differentiated tissues (P < 0.05). Univariate analysis demonstrated that high PIWIL2 expression was associated with shorter survival time after radical resection (P < 0.05). Multivariate analysis showed that PI-WIL2 expression was an independent prognostic factor after radical re-section of hilar cholangiocarcinoma (P < 0.05). PIWIL2 expression was also associated with tumor-node-metastasis stage and differentiation. PIWIL2 was an independent prognostic factor after radical resection of hilar cholangiocarcinoma.

Overexpression of Sig1R is closely associated with tumor progression and poor outcome in patients with hilar cholangiocarcinoma.

Nonopioid Sigma1 receptor (Sig1R), which regulates various metabolism functions, has been implicated in cancers; yet, its role in hilar cholangiocarcinoma remains unclear. In the present study, we examined Sig1R expression in hilar cholangiocarcinoma (HC) tissues and explored its possible clinical values. Tissue microarray blocks containing 92 HC tissues and matched non-cancerous bile duct tissues were examined immunohistochemically for expression of Sig1R protein. Overexpression of Sig1R was found in 43 (46.7%) of the 92 primary tumor tissues. Overexpression of Sig1R was significantly associated with poor/undifferentiation (P = 0.011), tumor invasion (P = 0.001), lymph node metastasis (P = 0.047), and advanced disease stage (P = 0.024) of HC patients. Kaplan-Meier analysis showed that patients overexpressing Sig1R had an earlier recurrence and worse overall survival than those not overexpressing Sig1R. Cox regression analysis revealed that Sig1R was an independent factor to predict HC recurrence and prognosis of HC patients. Our results suggest that Sig1R is frequently activated in human HC tissue and overexpression of Sig1R might serve as a predictor for tumor recurrence and a prognostic biomarker for HC patients.

Expression and clinical significance of PIWIL2 in hilar cholangiocarcinoma tissues and in cell lines

Objective To explore the relationship between PIWIL2 and clinicopathological characteristics and prognosis after radical resection by detecting the expression of PIWIL2 in hilar cholangiocarcinoma tissues and in cell lines.Methods The expression of PIWIL2 was detected by immunohistochemistry in 41 hilar cholangiocarcinomas and 10 control tissues,Western-blot and immunocytofluorescent were used to investigate the PIWIL2 expression in cholangiocarcinoma cell lines QBC939 and bile duct epithelial cell lines HIBEpic.Univariate and multivariate analysis of survival was performed using the Kaplan-Meier curve in hilar cholangiocarcinoma patients who bad underwent radical resection.Result The expression of PIWIL2 in hilar cholangiocarcinoma tissues and QBC939 cells were significantly higher than the control tissues and HIBEpic cells (P ＜ 0.05).The expression of PIWIL2 in poor and moderate differentiated cholangiocarcinoma was significantly higher than the well differentiated group (P ＜ 0.05).Univariate analysis showed that high expression of PIWIL2 was associated with short-survival time after radical resection (P ＜ 0.05).Multivariate analysis showed that the expression of PIWIL2 was an independent prognostic factor after radical resection of hilar cholangiocarcinoma (P ＜ 0.05).Conclusions The expression of PIWIL2 was associated with TNM stage and differentiation; PIWIL2 was an independent factor for prognosis after radical resection of hilar cholangiocarcinoma. Key words: PIWI protein, PIWIL2 ; Hilar cholangiocarcinoma; Prognosis ; Risk factors

Down-Regulation of Gab1 Inhibits Cell Proliferation and Migration in Hilar Cholangiocarcinoma

Hilar cholangiocarcinoma is a highly aggressive malignancy originating from the hilar biliary duct epithelium. Due to few effective comprehensive treatments, the prognosis of hilar cholangiocarcinoma is poor. In this study, immunohistochemistry was first used to detect and analyze the expression of Gab1, VEGFR-2, and MMP-9 in hilar cholangiocarcinoma solid tumors and the relationships to the clinical pathological features. Furthermore, Gab1 and VEGFR-2 siRNA were used to interfere the hilar cholangiocarcinoma cell line ICBD-1 and then detect the PI3K/Akt signaling pathway, MMP-9 levels and malignant biological behaviors of tumor cells. The data showed that 1. Gab1, VEGFR-2, and MMP-9 were highly expressed and positively correlated with each other in hilar cholangiocarcinoma tissues, which were related to lymph node metastasis and differentiation. 2. After Gab1 or VEGFR-2 siRNA interference, PI3K/Akt pathway activity and MMP-9 levels were decreased in ICBD-1 cells. At the same time, cell proliferation decreased, cell cycle arrested in G1 phase, apoptosis increased and invasion decreased. These results suggest that the expression of Gab1, VEGFR-2, and MMP-9 are significantly related to the malignant biological behavior of hilar cholangiocarcinoma. Gab1 regulates growth, apoptosis and invasion through the VEGFR-2/Gab1/PI3K/Akt signaling pathway in hilar cholangiocarcinoma cells and influences the invasion of tumor cells via MMP-9.

Prognostic significance of snail expression in hilar cholangiocarcinoma

Many patients with hilar cholangiocarcinoma (HC) have a poor prognosis. Snail, a transcription factor and E-cadherin repressor, is a novel prognostic factor in many cancers. The aim of this study was to evaluate the relationship between snail and E-cadherin protein expression and the prognostic significance of snail expression in HC. We examined the protein expression of snail and E-cadherin in HC tissues from 47 patients (22 males and 25 females, mean age 61.2 years) using immunohistochemistry and RT-PCR. Proliferation rate was also evaluated in the same cases by the MIB1 index. High, low and negative snail protein expression was recorded in 18 (38%), 17 (36%), and 12 (26%) cases, respectively, and 40.4% (19/47) cases showed reduced E-cadherin protein expression in HC samples. No significant correlation was found between snail and E-cadherin protein expression levels (P = 0.056). No significant correlation was found between snail protein expression levels and gender, age, tumor grade, vascular or perineural invasion, nodal metastasis and invasion, or proliferative index. Cancer samples with positive snail protein expression were associated with poor survival compared with the negative expresser groups. Kaplan-Meier curves comparing different snail protein expression levels to survival showed highly significant separation (P < 0.0001, log-rank test). With multivariate analysis, only snail protein expression among all parameters was found to influence survival (P = 0.0003). We suggest that snail expression levels can predict poor survival regardless of pathological features and tumor proliferation. Immunohistochemical detection of snail protein expression levels in routine sections may provide the first biological prognostic marker.

Expression and clinical significance of FXYD3 protein in hilar cholangiocarcinoma

Objective To investigate the expression of FXYD3 protein in hilar cholangiocarcinoma and the relationship of FXYD3 protein expression with the biological characteristics of hilar cholangiocarcinoma cells.Methods Strept avidin-biotin complex (SABC) immunohistochemical method was used to detect the expression of FXYD3 protein in 43 hilar cholangiocarcinoma and paraneoplastic tissues respectively.The relationship of FXYD3 protein expression with the biological characteristics of hilar cholangiocarcinoma cells was analyzed.Results The positive expression rate of FXYD3 protein in hilar cholangiocarcinoma was 67.44％ (29/43),significantly higher than that of 32.56％ ( 14/43 ) in paraneoplastic tissue (P＜ 0.01 ).FXYD3 protein expression was significantly correlated with the histological classification (P ＜ 0.01 ),but not with patient' s age,gender,TNM stage and casbohydrate antigen19-9 (CA19-9) expression (P ＞ 0.05).Conclusion FXYD3 protein expression was significantly correlated with the histological classification,suggesting it may be a very critical factor in primary cell transformation and progress of hilar cholangiocarcinoma. Key words: Hilar cholangiocarcinoma; FXYD3 protein; Biology

Malignant transformation of the cultured human normal biliary tract epithelial cells induced by hepatitis C virus core protein

High level expression of hepatitis C virus core protein (HCV-C) was detected in hilar cholangiocarcinoma tissues in our previous studies. This protein played an important role in the process of cancer cell inversion and proliferation, by some direct and indirect effects on certain genes. Based on this observation, we investigated the effect of HCV-C on human normal biliary epithelial (hBE) cell transformation and tumor development. Plasmid pHCV-C encoding the gene of HCV core protein was constructed and transfected into hBE cells. The expression and the biological effect of HCV-C in HCV-C gene-modified hBE cells were determined in vitro and in vivo. The clone formation rates of hBE cells transfected with pHCV-C, pcdna3.1 and mock-transfected cells were 36, 2.5 and 1.5%, respectively. Tumor developed in 7 of 7 nude mice after incubated with pHCV-C transfected hBE cells, while no tumor appeared in mice injected with pcdna3.1- and mock-transfected hBE cells. To investigate the possible mechanism of malignant transformation, we further studied the telomerase activity and human telomerase reverse transcriptase (hTERT) expression in pHCV-C transfected hBE cells. The elevated expression of hTERT was confirmed by RT-PCR, immunocytochemistry (ICC) and Western blot analysis, which in turn elevated the telomerase activity, confirmed by TRAP-ELISA. These results indicated that HCV-C protein could participate in malignant transformation of human normal biliary epithelial cells and induce cholangiocarcinoma tumorigenesis, and the activation of telomerase was one of the possible mechanisms.

Expression of RECK gene and MMP-9 in hilar cholangiocarcinoma and its clinical significance

In order to study the expression of transformation suppressor gene RECK and MMP-9 in hilar cholangiocarcinomas and its clinical significance, and explore the roles of RECK gene in metastasis and invasion of hilar cholangiocarcinoma, the expression levels of RECK, and MMP-9 mRNA were detected by using reverse transcription-polymerase reaction in 42 paraffin-embedded samples of hilar cholangiocarcinomas and 10 samples of benign bile duct diseases. The results showed that in hilar cholangiocarcinoma tissues, the expression of RECK gene was 0.235 +/- 0.062, significantly lower than in normal bile duct tissues (0.533 +/- 0.024, P < 0.05). In hilar cholangiocarcinoma tissues, the expression of MMP-9 (0.528 +/- 0.039) was significantly higher than in the normal tissues (0.311 +/- 0.032, P < 0.05). The expression of RECK gene was closely related to the intrahepatic and surrounding organs invasion (P < 0.05). It was concluded that RECK gene could inhibit the expression of MMP-9 in hilar cholangiocarcinomas and closely correlated with the biological behaviors. The abnormal expression of RECK gene might be one of the molecular mechanisms of hilar cholangiocarcinoma metastasis.

Fas counterattack in cholangiocarcinoma: a mechanism for immune evasion in human hilar cholangiocarcinomas.

To investigate FasL expression in hilar cholangiocarcinoma tissues and cultured cholangiocarcinoma cells, and to assess its ability to induce apoptosis. We studied the expression of FasL by human hilar cholangiocaroinomas tissues by immunohistochemistry, and the QBC939 cholangiocarcinoma cell line by RT-PCR, immunohistochemistry, and Western Blot. TUNEL and flow cytometry were used to detect apoptotic cells. Prevalent expression of FasL was detected in 39 resected hilar cholangiocarcinoma tissues. TUNEL staining disclosed a high level of cell death among lymphocytes infiltrating FasL positive areas of tumor. FasL mRNA and protein expressions in cholangiocarcinoma cells could induce Jurkat cells. Hilar cholangiocarcinomas may elude immunological surveillance by inducing, via Fas/FasL system, the apoptosis of activated lymphocytes.