Background Cholangiocarcinoma (CCA) is a fatal cancer of the bile duct epithelial cell lining. The misdiagnosis of CCA and other biliary diseases may occur due to the similarity of clinical manifestations and blood tests resulting in inappropriate or delayed treatment. Thus, an accurate and less-invasive method for differentiating CCA from other biliary diseases is inevitable. Methods We quantified methylation of OPCML , HOXA9 , and HOXD9 in serum cell-free DNA (cfDNA) of CCA patients and other biliary diseases using methylation-sensitive high-resolution melting (MS-HRM). Their potency as differential biomarkers between CCA and other biliary diseases was also evaluated by using receiver operating characteristic (ROC) curves. Results The significant difference of methylation levels of OPCML and HOXD9 was observed in serum cfDNA of CCA compared to other biliary diseases. Assessment of serum cfDNA methylation of OPCML and HOXD9 as differential biomarkers of CCA and other biliary diseases showed the area under curve (AUC) of 0.850 (0.759–0.941) for OPCML which sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were 80.00%, 90.00%, 88.88%, 81.81%, and 85.00%, respectively. The AUC of HOXD9 was 0.789 (0.686–0.892) with sensitivity, specificity, PPV, NPV, and accuracy of 67.50%, 90.00%, 87.09%, 73.46%, and 78.75%, respectively. The combined marker between OPCML and HOXD9 showed sensitivity, specificity, PPV, and NPV of 62.50%, 100%, 100%, and 72.72%, respectively, which may be helpful to prevent a misdiagnosis between CCA and other biliary diseases. Conclusions Our findings suggest the application of serum cfDNA methylation of OPCML and HOXD9 for differential diagnosis of CCA and other biliary diseases due to its less invasiveness and clinically practical method which may benefit the patients by preventing the misdiagnosis of CCA and avoiding unnecessary surgical intervention. Electronic supplementary material The online version of this article (10.1186/s13148-019-0634-0) contains supplementary material, which is available to authorized users.

Diagnosis of cholangiocarcinoma (CCA) is difficult when patients do not show jaundice. The aim of this study was to examine the feasibility of using the total serum bile acid (TSBA) level as an aid for the diagnosis of CCA in patients without jaundice. For this purpose, TSBA of the following groups were measured using a Beckman Synchron CX4 clinical chemistry analyzer: 60 cases of CCA with total serum bilirubin ≤2 mg/dL (low total bilirubin group, LTB); 32 cases of CCA with total serum bilirubin >2 mg/dL (high total bilirubin group, HTB); and 115 healthy controls. Liver function parameters such as serum cholesterol, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were also examined. The results showed that the TSBA of both LTB and HTB groups of the CCA patients were significantly higher than that of the healthy controls. Also, significant correlation was observed between TSBA and total bilirubin levels in the HTB group of CCA patients. However, no such correlation was seen in the LTB group. The cut-off value of TSBA was determined for the LTB group of CCA patients using the receiver operating characteristic curve analysis, and it was 6.05 µmol/L with the sensitivity and specificity of 46.7% and 84.4%, respectively. In addition, the ALP level was correlated well with the TSBA level and ALP in HTB group was significantly higher than that of LTB group. Moreover, the combination of high TSBA and high ALP levels gave higher specificity up to 97.4%. TSBA might be useful for the diagnosis of CCA patients without jaundice.

The use of gold nanoparticles (AuNP) has been established in nanocarriers, diagnostics, and biosensors. Access to the targeted sites of these nanomaterials could directly involve the first line of defense, the innate immune system. Charges of nanomaterials play a critical role in a number of aspects such as stabilization, cellular uptake, modulation, and function of cells. Interactions and modulations of the charged nanomaterials against the innate immune system may occur even at very low concentration. To understand the effects of charges on monocyte behavior, in this study, the positively and negatively charged AuNP (AuNP+ve and AuNP-ve) of the similar size and shape on cytotoxicity, recognition, cellular behavior, and function were evaluated in vitro using U937 human monocyte cells as an innate immunity model. Both types of AuNP at various concentrations (0−5 nM) exhibited low toxicity. In addition, the cellular internalization of the AuNP+ve and AuNP-ve, as determined by TEM, occurred by different mechanisms, and the internalization had no effect on cellular destruction, as implied by the low levels of %LDH. Interestingly, the AuNP+ve recognition and internalization seemingly entered cells through receptor dependence and strongly affected cellular response to express both pro-inflammatory (IL-1β) and anti-inflammatory (TGF-β) cytokines, while the AuNP-ve stimulated TNF-α expression. Nevertheless, the AuNP-treated cells maintained normal function when exposed to planktonic bacteria. Thus, these results indicated that one part of the immune system interacted with different surface-charged AuNP, suggesting appropiate immunomodulation in biomedicine.

Abstract. Cholangiocarcinoma (CCA) is a malignant transformation of biliary epithelial cells. It is a slow growing tumor, but is also highly metastatic with a poor prognosis. Bile acids are known to transactivate the epidermal growth factor receptor (EGFR) in cholangiocytes and induce cyclooxygenase-2 expression. The protein expression profiles of bile acid-treated CCA cells were studied using a proteomic approach. To elucidate the possible mechanisms involved in the bile acid-mediated enhancement of CCA cell migration, the effects of six bile acids, including cholic, deoxycholic, taurocholic, taurodeoxycholic, glycocholic and glycodeoxycholic acid, on the migration of CCA cells were examined in vitro using wound healing assays. Subsequently, the possible proteins involved in enhanced CCA cell migration were investigated using a proteomic approach. Changes to the protein expression profiles of CCA cells following bile acid treatment was examined using two-dimensional electrophoresis and mass spectrometry. The results demonstrated that cholic and deoxycholic acid significantly enhanced the migration of CCA cells, compared with the treated MMNK-1 control cells. CCA cells had 77 overexpressed protein spots following cholic acid treatment, and 50 protein spots following deoxycholic acid treatment, compared with the treated MMNK-1 control cells. Liquid chromatography tandem-mass spectrometry analysis revealed that coiled-coil domain containing 25 (CCDC25) was significantly overexpressed in cholic acid-treated CCA cells compared with in cholic acid-treated control cells. When the expression levels of CCDC25 were investigated using western blot analysis, CCDC25 was demonstrated to be highly expressed in CCA tissues, but not in the adjacent non-cancerous tissue samples. The identified proteins were further analyzed for protein-chemical interactions using STITCH version 3.1 software. CCDC25 protein was identified to be associated with Son of sevenless homolog 1 and growth factor receptor-bound protein 2, which are involved in EGFR signaling. The results of the present study demonstrated that following cholic acid treatment, CCDC25 is overexpressed in CCA cells, which is associated with significantly enhanced cell migration. This suggests that CCDC25 is a potential therapeutic target for the treatment of patients with CCA. IntroductionCholangiocarcinoma (CCA) is a malignant transformation of biliary epithelial cells. It is a slow growing tumor, but it is also highly metastatic with a poor prognosis (1). The highest incidence of this cancer has been identified in Asia, particularly in Northeast Thailand (2). In the majority of cases of CCA, the etiology is unknown. Chronic inflammation and cellular injury within bile ducts, together with the partial obstruction of bile flow result in high-risk conditions for CCA development (3). The tumor not only may develop mechanisms to survive in the toxic environment of bile, but may also utilize bile components to promote cell growth, survival and invasion (4-6). Bile acids are known...

Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium which is caused by liver fluke infection. The clinical symptoms of CCA were revealed as the disease progresses to advanced stage. Thus, specific diagnostic biomarkers are important for this fatal disease. We applied methylation-sensitive high-resolution melting (MS-HRM) to quantify DNA methylation levels of opioid binding protein/cell adhesion molecule-like gene (OPCML) and Secreted frizzled-related protein 1 (SFRP1) in 73 primary CCA and 10 adjacent normal tissues and evaluated the sensitivity, specificity, and accuracy of the assay. The median methylation level of OPCML in CCA was 38.7 % (ranged from 0 to 82.2 %) and of SFRP1 was 31.5 % (ranged from 0 to 86.2 %). Methylation cutoff values of OPCML and SFRP1 derived from adjacent normal tissue were 6.90 and 10.44 %, respectively. With these cutoff values, the area under curve (AUC) of OPCML was 0.932 (95 % CI 0.878-0.986) and of SFRP1 was 0.951 (95 % CI 0.905-0.996). The sensitivity, specificity, and accuracy of OPCML were 89.04, 100, and 90.36 %, respectively, and of SFRP1 were 83.56, 100, and 85.54 %, respectively. In conclusion, the DNA methylation levels of OPCML and SFRP1 could be potential biomarkers for diagnosis of CCA with high specificity, sensitivity, and accuracy, in particular for biopsy specimens. Further validation in noninvasive samples such as serum or plasma is warranted for clinical applicability, especially as early diagnostic biomarkers.

Prognosis of cholangiocarcinoma (CCA) patients is absolutely poor in spite of extensive efforts for the development of chemotherapy. Mitochondrial proteins play key roles in carcinogenesis of various cancers. Therefore, mitochondria are considered as the target organelles for chemotherapy of several cancers including CCA. The purpose of this study is to identify potential candidate proteins for chemotherapy using mitochondrial proteome analysis for CCA tissues. A shotgun proteomic approach using SDS-PAGE coupled with LC-MS/MS was applied to compare the expression of mitochondrial proteins in CCA and the adjacent non-cancerous tissues. Using the proteomic analysis for the pooled mitochondrial proteins purified from three each of papillary and non-papillary types of CCA and their adjacent tissues, 281 proteins were identified as mitochondrial proteins, and 105 of them have significantly different expression levels compared with the corresponding counterparts. Among the 105 proteins, apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) was a unique protein commonly over-expressed in both papillary and non-papillary types of CCA tissues but not in the adjacent non-cancerous tissues. In conclusion, AIFM3 was aberrantly expressed only in the mitochondria of CCA tissues. This finding suggests that AIFM3 could be a potential target molecule for CCA chemotherapy.

Coiled-coil domain containing 25 (CCDC25) was previously reported to be upregulated in cholangiocarcinoma (CCA) tissues compared with adjacent normal tissues. The present study investigated whether serum CCDC25 level may be used as a potential marker for the diagnosis of CCA. Bioinformatics tools were used to reveal that CCDC25 is secreted into plasma/serum via a non-conventional pathway, which secretes proteins independently from the endoplasmic reticulum/golgi complex, but is yet to be fully elucidated. Subsequently, the CCDC25 levels in the sera of patients with CCA (n=141), patients with benign biliary disease (BBD; n=53) and healthy controls (HC; n=72) were measured using a quantitative dot blot assay based on the standard curve created using recombinant CCDC25 protein. The results demonstrated that the serum CCDC25 level in the CCA group (0.28±0.06 ng/µl) was significantly higher compared with that in the BBD (0.15±0.03 ng/µl) or HC (0.0017±0.0008 ng/µl) groups. Serum CCDC25 level provided an improved resolution (P=0.0001) compared with carcinoembryonic antigen (P=0.098) or carbohydrate antigen 19-9 (P=0.271) for the differential diagnosis between BBD and CCA. Receiver operating characteristic curve analysis revealed high sensitivity and specificity of serum CCDC25 level to differentiate between patients with CCA and HC (93.0 and 100%, respectively), and also to differentiate between patients with CCA and patients with BBD (75.0 and 84.0%, respectively). CCDC25 expression was further investigated in 23 CCA tissues, and CCDC25 expression in cancer tissues was moderately correlated with the serum CCDC25 level (r 2 =0.52, P=0.01). Among patients with CCA, serum CCDC25 level was significantly higher in patients with non-metastatic CCA compared with patients with metastatic CCA. Correspondingly, a higher serum CCDC25 level was associated with a longer overall survival time in patients with CCA. In conclusion, serum CCDC25 level may be a promising screening and diagnostic marker for the differential diagnosis of CCA.

Diagnostic and/or prognostic biomarkers for cholangiocarcinoma (CCA) are still insufficient with poor prognosis of patients. To discover a new CCA biomarker, we constructed our secretome database of three CCA cell lines and one control cholangiocyte cell line using GeLC-MS/MS. We selected candidate proteins by five bioinformatics tools for secretome analysis. The inclusion criteria were as follows: having predicted signal peptide or being predicted as non-classically secreted protein; together with having no transmembrane helix and being previously detected in plasma and having the highest number of signal peptide cleavage sites. Eventually, apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) was selected for further analysis. To validate APEX1 as a bio-marker for CCA, serum APEX1 levels of 80, 39, and 40 samples collected from CCA, benign biliary diseases (BBD), and healthy control groups, respectively, were measured using dot blot analysis. The results showed that serum APEX1 level in CCA group was significantly higher than that in BBD or healthy control group. Among CCA patients, serum APEX1 level was significantly higher in patients having metastasis than in those without metastasis. The higher level of serum APEX1 was correlated with the shorter survival time of the patients. Serum APEX1 level might be a diagnostic and prognostic biomarker for CCA.