Cholangiocarcinoma (CCA) is a tumor arising from cholangiocytes lining the bile ducts. Vascular invasion and lymph node metastasis are important prognostic factors for disease staging as well as clinical therapeutic decisions for CCA patients. In the present study, we applied CCA sera proteomic analysis to identify a potential biomarker for prognosis of CCA patients. Then, using bioinformatics tools, we identified angiopoietin-like protein 4 (ANGPTL4) which expressed highest signal intensity among candidate proteins in proteomic analysis of CCA sera. Expression of ANGPTL4 in CCA tissues was determined using immunohistochemistry. The results showed that ANGPTL4 was stained at higher level in CCA cells when compared with normal cholangiocytes. The high expression of ANGPTL4 was associated with lymph node metastasis and advanced tumor stage (p = 0.013 and p = 0.031, respectively). Furthermore, serum ANGPTL4 levels in CCA and healthy control (HC) were analyzed using a dot blot assay. And it was found that ANGPTL4 level was significantly higher in CCA than HC group (p < 0.0001). ROC curve analysis revealed that serum ANGPTL4 level was effectively distinguished CCA from healthy patients (cutoff = 0.2697 arbitrary unit (AU), 80.0% sensitivity, 72.7% specificity, AUC = 0.825, p < 0.0001). Serum ANGPTL4 level was associated with vascular invasion and lymph node metastasis (p = 0.0004 and p = 0.006), so that it differentiated CCA with vascular invasion from CCA without vascular invasion (cutoff = 0.5526 AU, 64.9% sensitivity, 92.9% specificity, AUC = 0.751, p = 0.006) and it corresponded to CCA with/without lymph node metastasis (cutoff = 0.5399 AU, 71.4% sensitivity, 70.8% specificity, AUC = 0.691, p = 0.01) by ROC analysis. Serum ANGPTL4 levels showed superior predictive efficiency compared with CA 19-9 and CEA for vascular invasion and lymph node metastasis. In addition, serum ANGPTL4 level was an independent predictive indicator by multivariate regression analysis. In conclusion, serum ANGPTL4 could be a novel prognostic biomarker for prediction of vascular invasion and lymph node metastasis of CCA patients.

Pyrvinium pamoate (PP), an FDA-approved anthelmintic drug, has been validated as a highly potent anti-cancer agent and patented recently as a potential chemotherapeutic drug for various cancers. The aims of this study were, therefore, to investigate the ability of PP in anti-proliferative activity and focused on the lipid profiles revealing the alteration of specific lipid species in the liver fluke Opisthorchis viverrini (Ov)-associated cholangiocarcinoma (CCA) cells. PP inhibited CCA cell viability through suppressing mitochondrial membrane potential (MMP) and ATP productions, leading to apoptotic cell death. Liquid chromatography-mass spectrometry combined with chemometrics was performed to investigate lipid alteration during PP-induced apoptosis. The lipidomic analyses showed the altered lipid signatures of CCA cell types including S-acetyldihydrolipoamide, methylselenopyruvate, and triglycerides that were increased in PP-treated CCA cells. In contrast, the levels of sphinganine and phosphatidylinositol were lower in the PP-treated group compared with its counterpart. The orthogonal partial-least squares regression analysis revealed that PP-induced MMP dysfunction, leading to remarkably reduced ATP level, was significantly associated with triglyceride (TG) accumulation observed in PP-treated CCA cells. Our findings indicate that PP could suppress the MMP function, which causes inhibition of CCA cell viability through lipid production, resulting in apoptotic induction in CCA cells. These findings provide an anti-cancer mechanism of PP under apoptotic induction ability that may serve as the alternative approach for CCA treatment.