Interleukin-6 decreases senescence and increases telomerase activity in malignant human cholangiocytes

BACKGROUND/AIMS: Cellular senescence results in irreversible growth arrest. In malignant cells, senescence is prevented by maintenance of chromosomal length by telomerase activity. Telomerase activity is increased in malignant, but not in normal cholangiocytes. Interleukin-6 (IL-6) is an autocrine promoter of cholangiocarcinoma growth. Our aims were to assess the relationship between IL-6 activated p38 mitogen-activated protein kinase (MAPK) pathways and senescence in malignant cholangiocytes. METHODS: Cell senescence and telomerase activity was assessed in Mz-ChA-1 malignant human cholangiocytes. The effect of inhibitors of p38 MAPK and telomerase activity on cell proliferation was assessed, and the interaction between these inhibitors was quantitated by median effects analysis. RESULTS: Mz-ChA-1 cells rapidly underwent senescence during repeated passaging. IL-6 increased telomerase activity and decreased cellular senescence during repeated passaging. However, basal telomerase activity was increased by inhibition of p38 MAPK. Inhibition of telomerase activity decreased IL-6 induced proliferation and had a synergistic effect with p38 MAPK inhibitors. Thus, IL-6 increases telomerase activity independent of p38 MAPK signaling and maintenance of telomerase activity promotes cholangiocarcinoma growth. CONCLUSION: Enhanced telomerase activity in response to IL-6 stimulation can prevent cellular senescence and thereby contribute to cholangiocarcinoma growth. Inhibition of telomerase activity may therefore be therapeutically useful in biliary tract malignancies.     

Targeting cholangiocarcinoma

Cholangiocarcinoma (CCA) represents a diverse group of epithelial cancers associated with the biliary tract, and can best be stratified anatomically into intrahepatic (iCCA), perihilar (pCCA) and distal (dCCA) subsets. Molecular profiling has identified genetic aberrations associated with these anatomic subsets. For example, IDH catalytic site mutations and constitutively active FGFR2 fusion genes are predominantly identified in iCCA, whereas KRAS mutations and PRKACB fusions genes are identified in pCCA and dCCA. Clinical trials targeting these specific driver mutations are in progress. However, The Tumor Genome Atlas (TCGA) marker analysis of CCA also highlights the tremendous molecular heterogeneity of this cancer rendering comprehensive employment of targeted therapies challenging. CCA also display a rich tumor microenvironment which may be easier to target. For example, targeting cancer associated fibroblasts for apoptosis with BH3-mimetics and/or and reversing T-cell exhaustion with immune check point inhibitors may help aid in the treatment of this otherwise devastating malignancy. Combinatorial therapy attacking the tumor microenvironment plus targeted therapy may help advance treatment for CCA. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

Autophagy in intra-hepatic cholangiocarcinoma

Intra-hepatic cholangiocarcinoma (IHCC) is a primary cancer of the liver that shares histological features with the hepatic bile ducts from which it is thought to arise. The incidence of this disease is increasing, possibly related to increased inflammatory liver diseases such as viral hepatitis and steatohepatitis (commonly called “fatty liver”), induced by obesity, diabetes, and other metabolic derangements. Its prognosis is generally poor with early metastasis and presently there are limited effective treatments. A basic understanding of the disease has long been hampered by limited tumor cell lines and good model systems. Similarly, such limitations have obstructed new therapeutic inroads.     

Autophagy in intra-hepatic cholangiocarcinoma

Intra-hepatic cholangiocarcinoma (IHCC) is a primary cancer of the liver that shares histological features with the hepatic bile ducts from which it is thought to arise. The incidence of this disease is increasing, possibly related to increased inflammatory liver diseases such as viral hepatitis and steatohepatitis (commonly called “fatty liver”), induced by obesity, diabetes, and other metabolic derangements. Its prognosis is generally poor with early metastasis and presently there are limited effective treatments. A basic understanding of the disease has long been hampered by limited tumor cell lines and good model systems. Similarly, such limitations have obstructed new therapeutic inroads.     

Cholangiocytes in the pathogenesis of primary sclerosing cholangitis and development of cholangiocarcinoma

Primary sclerosing cholangitis (PSC) is an idiopathic cholangiopathy strongly associated with inflammatory bowel disease (IBD) and characterized by cholestasis, chronic immune infiltration and progressive fibrosis of the intrahepatic and extrahepatic bile ducts. PSC confers a high risk of cholangiocarcinoma (CCA) with PSC-CCA representing the leading cause of PSC-associated mortality. PSC-CCA is derived from cholangiocytes and associated progenitor cells – a heterogeneous group of dynamic epithelial cells lining the biliary tree that modulate the composition and volume of bile production by the liver. Infection, inflammation and cholestasis can trigger cholangiocyte activation leading to an increased expression of adhesion and antigen-presenting molecules as well as the release of various inflammatory and fibrogenic mediators. As a result, activated cholangiocytes engage in a myriad of cellular processes, including hepatocellular proliferation, apoptosis, angiogenesis and fibrosis. Cholangiocytes can also regulate the recruitment of immune cells, mesenchymal cells, and endothelial cells that participate in tissue repair and destruction in settings of persistent inflammation. In PSC, the role of cholangiocytes and the mechanisms governing their transformation to PSC-CCA are unclear however localization of disease suggests that cholangiocytes are a key target and potential regulator of hepatobiliary immunity, fibrogenesis and tumorigenesis. Herein, we summarize mechanisms of cholangiocyte activation in PSC and highlight new insights into disease pathways that may contribute to the development of PSC-CCA. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

Synthesis of 14-deoxy-11,12-didehydroandrographolide analogues as potential cytotoxic agents for cholangiocarcinoma

A series of 14-deoxy-11,12-didehydroandrographolide analogues were synthesized from naturally occurring andrographolide and their cytotoxicity evaluated against nine cancer cell lines including cholangiocarcinoma. Analogues 5a and 5b exhibited the most potent cytotoxicity with ED₅₀s of 3.37 and 3.08?μM on KKU-M213 cell lines and 2.93 and 3.27?μM on KKU-100 cell lines, respectively. Selective cytotoxicity on cholangiocarcinoma cell lines identified in this study highlight the importance of structural modification in the development of drugs for this cancer.     

Expression of oxysterol binding protein isoforms in opisthorchiasis-associated cholangiocarcinoma: A potential molecular marker for tumor metastasis

Oxysterols are oxygenated derivatives of cholesterol generated by enzymatic reactions mediated by cytochrome P450 family enzymes or by inflammation-associated non-enzymatic reactions. Oxysterol binding proteins (OSBPs) are cytosolic high affinity receptors for oxysterols. We previously found that OSBPL-8 is upregulated in liver fluke (Opisthorchis viverrini)-induced hamster cholangiocarcinoma (CCA). Our aims were to determine the expression patterns of OSBP isoforms in human CCA tissues and to evaluate whether OSBPs could be used as molecular markers for the identification of blood-borne CCA metastasis. Expression levels of OSBP1, OSBP2, OSBPL-7 and OSBPL-8 in CCA tissues were detected using qRT-PCR and immunohistochemistry. Expression of OSBPs at mRNA level in the blood of CCA patients was also investigated. We confirmed increased expression of OSBPL-8 in O. viverrini -induced hamster CCA tissues. Moreover, increased expression of OSBP1, OSBP2, OSBPL-7 and OSBPL-8 was seen in human CCA tissues. Notably, a significant increased level of OSBPL-7 mRNA was observed in tumor compared to non-tumor liver tissue. Immunohistochemistry supported the mRNA results, in that OSBPL-7 protein was over-expressed in cancer cells and hepatocytes but not in normal biliary cells and surrounding inflammatory cells. Interestingly, in our preliminary results, significantly higher levels of OSBP2 and OSBPL-7 mRNA were seen in blood samples from CCA patients than in healthy controls. These results suggest that OSBP2 and OSBPL-7 might serve as molecular markers for the identification of CCA metastasis in the bloodstream.     

胆道肿瘤动物模型建立的研究现状

胆管癌是一种侵袭性很强的胆道恶性肿瘤,全世界的发病率和死亡率逐步增加。建立合适的胆管癌动物模型对胆管癌的早期诊断和治疗具有重要意义。本文就胆道肿瘤动物模型建立方法的现状进行综述。     

Secreted Opisthorchis viverrini glutathione S-transferase regulates cell proliferation through AKT and ERK pathways in cholangiocarcinoma

Opisthorchis viverrini can develop mitogenic substances into the excretory/secretory product (ESP) that may play an important role in promoting the genesis of cholangiocarcinoma (CCA). In the present study, glutathione S-transferase (GST) is identified as being secreted into Ov-ESP and acting as one of the parasitic mitogens. Its proliferative effect and possible mechanism were explored and its association with the tumor development is proposed. Ov-ESP was concentrated and purified by gel filtration chromatography. SDS-PAGE, 2-DE, and LC-MS/MS identified GST predominantly expressed in the proliferative ESP fraction. The recombinant OvGST (rOvGST) was produced by wheat germ cell-free expression and confirmed by an MTS assay to have a proliferative function on NIH-3T3 murine fibroblasts and MMNK1 non-tumorigenic human bile duct epithelial cells in a dose dependent manner with different optimal doses. The cell surface binding of rOvGST was confirmed in vitro and the activation of both pAKT and pERK was revealed as the mechanism of OvGST-mediated cell proliferation. With support from the observation of secreted OvGST on the biliary cells surrounding the parasites, it is suggested that OvGST can promote cell proliferation that consequently may accelerate the genesis of CCA.     

Overexpression of PDGFA and its receptor during carcinogenesis of Opisthorchis viverrini-associated cholangiocarcinoma

Cholangiocarcinoma (CCA) is a crucial health problem in northeastern part of Thailand, which is caused by a combination of Opisthorchis viverrini infection and nitrosamine. A better understanding of its molecular mechanism is an important step to discover and develop the new diagnostics and therapies for CCA. To reveal the involvement of potential genes in the development of CCA, the present study investigated the expression kinetics of platelet-derived growth factor alpha (Pdgfa) and its receptor (Pdgfra) during the tumorigenesis of CCA induced by O. viverrini infection with quantitative RT-PCR, and confirmed the expression with immunohistological staining. The results showed that in the hamster model of opisthorchiasis-associated CCA, the expression of Pdgfa was increased after infection plus N-nitrosodimethylamine (NDMA) administration, reached its peak at 2 months post infection, and remained at the high level until 6 months. Similarly, the expression of Pdgfra was increased time-dependently. The positive immunostaining for PDGFA proteins was observed in the cytoplasm of epithelial tumor cells of hamster CCA. Moreover, the analysis of the expression of these genes in 10 cases of human opisthorchiasis-associated CCA showed that Pdgfa was overexpressed in 80%, and Pdgfra was overexpressed in 40% cases (> 3.0 folds, compared with the expressions of adjacent normal tissues). This result suggests that PDGFA is likely involved in the tumorigenesis of opisthorchiasis-associated CCA, and may be a promising candidate biomarker for diagnosis and treatment strategies of CCA.     

Inhibition of the AKT pathway in cholangiocarcinoma by MK2206 reduces cellular viability via induction of apoptosis

Introduction

Cholangiocarcinoma (CCA) is an aggressive disease with limited effective treatment options. The PI3K/Akt/mTOR pathway represents an attractive therapeutic target due to its frequent dysregulation in CCA. MK2206, an allosteric Akt inhibitor, has been shown to reduce cellular proliferation in other cancers. We hypothesized that MK2206 mediated inhibition of Akt would impact CCA cellular viability.

Study methods

Post treatment with MK2206 (0-2 μM), cellular viability was assessed in two human CCA cell lines—CCLP-1 and SG231—using an MTT assay. Lysates from the MK2206 treated CCA cells were then examined for apoptotic marker expression levels using Western blot analysis. Additionally, the effect on cellular proliferation of MK2206 treatment on survivin depleted cells was determined.

Results

CCLP-1 and SG231 viability was significantly reduced at MK2206 concentrations of 0.5, 1, and 2 μM by approximately 44%, 53%, and 64% (CCLP-1; p = 0.01) and 32%, 32%, and 42% (SG231; p < 0.00005) respectively. Western analysis revealed a decrease in AKT^Ser473, while AKT^Thr308 expression was unchanged. In addition, cleaved PARP as well as survivin expression increased while pro-caspase 3 and 9 levels decreased with treatment. Depletion of survivin in CCLP-1 cells resulted in apoptosis as evidenced by increased cleaved PARP. In addition, survivin siRNA further enhanced the antitumor activity of MK2206.

Conclusions

This study demonstrates that by blocking phosphorylation of Akt at serine473, CCA cellular growth is reduced. The growth suppression appears to be mediated via apoptosis. Importantly, combination of survivin siRNA transfection and MK2206 treatment significantly decreased cell viability.     

Macrophage-capping protein as a tissue biomarker for prediction of response to gemcitabine treatment and prognosis in cholangiocarcinoma

Cholangiocarcinoma is one of the deadliest malignancies worldwide. Recent studies reported that treatment with gemcitabine was effective in prolonging survival. However, as the treatment only benefited a limited subset of patients, selection of patients before treatment is required. To discover biomarkers predictive of the response to gemcitabine treatment in cholangiocarcinoma, we examined the proteome of three types of material resource; ten cell lines, nine xenografts and nine surgically resected primary tumors from patients who exhibited different response to gemcitabine treatment. Two-dimensional difference gel electrophoresis generated quantitative protein expression profiles including 3571 protein spots. We detected 172 protein spots with significant correlation with response to gemcitabine treatment. All proteins corresponding to these 172 protein spots were identified by mass spectrometry. We found that the macrophage-capping protein (CapG) was associated with response to gemcitabin treatment in all three types of material source. Immunohistochemical validation in an additional set of 196 cholangiocarcinoma cases revealed that CapG expression was associated with lymphatic invasion status and overall survival. Multivariate analysis showed that CapG protein expression was an independent prognostic factor for overall survival. In conclusion, CapG was identified as a novel candidate biomarker to predict response to gemcitabine treatment and survival in cholangiocarcinoma.     

胆道微生态的研究进展

人体微生态是微生物与宿主在长期相互作用过程中形成的特殊生态系统,在人体的健康与疾病中起着重要作用。目前对消化系统微生态的研究主要集中在肠道,而胆道系统可能存在独特的微生态体系,它们与胆道系统的健康和疾病之间的关系尚不明确,研究人员探讨了胆道的微生物群与胆囊结石、胆总管结石、Oddi括约肌受损、胆总管结石复发、原发性硬化性胆管炎和胆道肿瘤等肝胆疾病的发生发展、诊断和治疗的关系。本文从健康人群、肝移植供肝者的胆道及以上疾病着手,对胆道微生态的相关研究和进展作一综述。

     

胆道梗阻合并胆道感染的病原学特征和危险因素及与肝脏损伤的关系研究

目的:探讨胆道梗阻合并胆道感染的病原学特征和危险因素,并分析胆道感染与肝脏损伤的关系。方法:回顾性分析250例胆道梗阻患者的临床资料,分析胆道梗阻合并胆道感染的病原菌分布和主要病原菌的耐药性,分析胆道梗阻合并胆道感染的危险因素,比较各组的肝功能指标[天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、谷氨酰转移酶(GGT)、直接胆红素(DBIL)]和肝纤维化指标[层黏蛋白(LN)、透明质酸(HA)、Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(Ⅳ-C)]。结果:250例胆道梗阻中共有132例合并胆道感染,感染率为52.80%,共分离出病原菌150株,以革兰阴性菌和革兰阳性菌为主。粪肠球菌对红霉素的耐药率最高,屎肠球菌对林可霉素的耐药率最高,均对万古霉素的耐药率最低;大肠埃希菌和肺炎克雷伯菌均对氨苄西林的耐药率最高,对妥布霉素的耐药率最低。年龄≥60岁、有胆道手术史、肝功能Child-Pugh评分≥11分是胆道梗阻合并胆道感染的危险因素(P<0.05)。胆道感染组的AST、ALT、GGT、DBIL、LN、HA、PCⅢ、Ⅳ-C水平高于无胆道感染组和对照组,且无胆道感染组高于对照组(P<0.05)。结论:胆道梗阻患者胆道感染的发生率较高,并且胆道感染会进一步加重胆道梗阻患者的肝脏损伤,临床应根据其病原学特征和危险因素做好相应的防治工作。     

新型近红外荧光探针MHI85在胆道系统成像的实验研究

目的:观察一种新型近红外荧光探针MHI85在器官中的成像特点,寻找特异性的器官成像荧光探针,为手术提供帮助。方法:用海洋光学测量系统检测近红外荧光探针MHI85的吸光度和荧光强度,分析其光学特点。随后将近红外荧光探针MHI85注射到CD-1小鼠体内,4小时后观察小鼠体内腹腔、胆囊和胆管、离体小鼠腹部脏器的近红外荧光成像情况。并测量离体脏器的信号背景比(SBR)。结果:近红外荧光探针MHI85最大吸收峰值和荧光峰值分别在690 nm和713 nm,说明其发光谱在700 nm左右,且成像稳定。利用小动物活体成像系统发现,近红外荧光探针MHI85在小鼠胆囊、胆囊管、左右肝管、肝总管可见明显荧光信号。心、肺、肝、胰、脾、肾、十二指肠、小肠均无荧光信号,而胆囊中可见明显的荧光信号。离体脏器SBR结果显示,胆囊的SBR明显高于其他脏器。结论:近红外荧光分子探针MHI85对胆囊及胆道系统具有良好的靶向性,且成像清晰、定位准确。     

684例胆道感染患者胆汁病原菌分布及耐药性分析

目的研究胆道感染患者胆汁病原菌的分布及其耐药性,为临床合理使用抗菌药物提供依据。方法对684例胆道感染患者胆汁进行培养,应用Micro Scan Walk Away40细菌鉴定及药敏分析系统对分离菌进行鉴定及药敏测定,用WHONET 5.6软件进行数据分析。结果从684例患者胆汁中分离出315株病原菌,革兰阴性杆菌占65.1%,革兰阳性球菌占34.3%,真菌占0.6%。革兰阴性杆菌对亚胺培南、美罗培南、头孢哌酮/舒巴坦、哌拉西林/他唑巴坦、左氧氟沙星、头孢吡肟、庆大霉素耐药率较低。肠球菌属、葡萄球菌属对喹诺酮类耐药率较低,未发现耐万古霉素、利奈唑胺的革兰阳性球菌。结论胆道感染仍以革兰阴性杆菌为主,病原菌分布广泛,耐药情况较严重,治疗使用抗生素需要进行病原菌检测及耐药性分析。