Background/Aim: Cholangiocarcinoma (CCA) is an aggressive cancer for which standard treatments are still ineffective. This study demonstrated the antiproliferative and anti-metastatic activity of metformin, an anti-diabetic drug, in CCA cells. Materials and Methods: Cell proliferation, migration/invasion and anoikis resistance were determined. The underlying mechanisms were identified using western blotting and immunocytofluorescence. Results: Metformin significantly suppressed proliferation of CCA cells in a dose- and time-dependent manner, regardless of glucose present in the medium. A low dose of metformin significantly increased anoikis and inhibited migration/ invasion of CCA cells that was in concert with the decrease of vimentin, matrix metalloproteinase (MMP)-2 and -7. Activation of 5’ adenosine monophosphate-activated protein kinase (AMPK) by phosphorylation together with suppression of nuclear translocation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-ĸB) were the underlying mechanisms for these effects. Conclusion: Metformin is a potent antiproliferative and anti-metastatic agent against human CCA cells. These findings encourage the repurposing of metformin in clinical trials to improve CCA treatment.

Background: Cyclophilin A (CypA) expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of CypA in liver fluke-associated cholangiocarcinoma (CCA) are not presently known. In this study, we investigated the expression of CypA in CCA tumor tissues and CCA cell lines as well as regulation mechanisms of CypA in tumor growth using CCA cell lines. Methods: CypA expression was determined by real time RT-PCR, Western blot or immunohistochemistry. CypA silence or overexpression in CCA cells was achieved using gene delivery techniques. Cell proliferation was assessed using MTS assay or Ki-67 staining. The effect of silencing CypA on CCA tumor growth was determined in nude mice. The effect of CypA knockdown on ERK1/2 activation was assessed by Western blot. Results: CypA was upregulated in 68% of CCA tumor tissues. Silencing CypA significantly suppressed cell proliferation in several CCA cell lines. Likewise, inhibition of CypA peptidyl-prolyl cis-trans isomerase (PPIase) activity using cyclosporin A (CsA) decreased cell proliferation. In contrast, overexpression of CypA resulted in 30% to 35% increases in proliferation of CCA cell lines. Interestingly, neither silence nor overexpression of CypA affected cell proliferation of a non-tumor human cholangiocyte cell line, MMNK1. Suppression of CypA expression attenuated ERK1/2 activity in CCA M139 cells by using both transient and stable knockdown methods. In the in vivo study, there was a 43% reduction in weight of tumors derived from CypA-silenced CCA cell lines compared with control vector CCA tumors in mice; these tumors with stable CypA silencing showed a reduced cell proliferation. Conclusions: CypA is upregulated in majority of CCA patients' tissues and confers a significant growth advantage in CCA cells. Suppression of CypA expression decreases proliferation of CCA cell lines in vitro and reduces tumor growth in the nude mouse model. Inhibition of CypA activity also reduces CCA cell proliferation. The ERK1/2 pathway may be involved in the CypA-mediated CCA cell proliferation. Thus, CypA may represent an important new therapeutic target for liver fluke-associated CCA.

Cholangiocarcinoma (CCA) is a deadly malignant tumor of the liver. It is a significant health problem in Thailand. The critical obstacles of CCA diagnosis and treatment are the high heterogeneity of disease and considerable resistance to treatment. Recent multi-omics studies revealed the promising targets for CCA treatment; however, limited models for drug discovery are available. This study aimed to develop a patient-derived xenograft (PDX) model as well as PDX-derived cell lines of CCA for future drug screening. From a total of 16 CCA frozen tissues, 75% (eight intrahepatic and four extrahepatic subtypes) were successfully grown and subpassaged in Balb/c Rag-2^-/-/Jak3^-/- mice. A shorter duration of PDX growth was observed during F0 to F2 transplantation; concomitantly, increased Oct-3/4 and Sox2 were evidenced in 50% and 33%, respectively, of serial PDXs. Only four cell lines were established. The cell lines exhibited either bile duct (KKK-D049 and KKK-D068) or combined hepatobiliary origin (KKK-D131 and KKK-D138). These cell lines acquired high transplantation efficiency in both subcutaneous (100%) and intrasplenic (88%) transplantation models. The subcutaneously transplanted xenograft retained the histological architecture as in the patient tissues. Our models of CCA PDX and PDX-derived cell lines would be a useful platform for CCA precision medicine.

Background: Thymosin β10 (Tβ10) expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of Tβ10 in liver fluke-associated cholangiocarcinoma (CCA) are not fully understood. In this study, we investigated the expression of Tβ10 in CCA tumor tissues and cell lines as well as molecular mechanisms of Tβ10 in tumor metastasis of CCA cell lines.Methods: Tβ10 expression was determined by real time RT-PCR or immunocytochemistry. Tβ10 silence or overexpression in CCA cells was achieved using gene delivery techniques. Cell migration was assessed using modified Boyden chamber and wound healing assay. The effect of silencing Tβ10 on CCA tumor metastasis was determined in nude mice. Phosphorylation of ERK1/2 and the expression of EGR1, Snail and matrix metalloproteinases (MMPs) were studied.Results: Ten pairs of CCA tissues (primary and metastatic tumors) and 5 CCA cell lines were studied. With real time RT-PCR and immunostaining analysis, Tβ10 was highly expressed in primary tumors of CCA; while it was relatively low in the metastatic tumors. Five CCA cell lines showed differential expression levels of Tβ10. Silence of Tβ10 significantly increased cell migration, invasion and wound healing of CCA cellsin vitro; reversely, overexpression of Tβ10 reduced cell migration compared with control cells (P<0.05). In addition, silence of Tβ10 in CCA cells increased liver metastasis in a nude mouse model of CCA implantation into the spleen. Furthermore, silence of Tβ10 activated ERK1/2 and increased the expression of Snail and MMPs in CCA cell lines. Ras-GTPase inhibitor, FPT inhibitor III, effectively blocked Tβ10 silence-associated ERK1/2 activation, Snail expression and cell migration.Conclusions: Low expression of Tβ10 is associated with metastatic phenotype of CCAin vitroandin vivo, which may be mediated by the activation of Ras, ERK1/2 and upregulation of Snail and MMPs. This study suggests a new molecular pathway of CCA pathogenesis and a novel strategy to treat or prevent CCA metastasis.

Cholangiocarcinoma (CCA) is difficult to diagnose at an early stage and most tumors are detected at late stage where surgery or other therapy is ineffective. Many advanced techniques are applied to diagnose CCA; however, most are expensive and have varying degrees of accuracy. A less invasive and simpler procedure such as serum markers would be of substantial clinical benefit for diagnosis, monitoring, and predicting outcome for CCA patients. Recent advances in “Omics” technologies offer remarkable opportunities for establishment of biomarker‐related to diseases. In this review, the potential biomarkers obtained from proteomics and glycomic studies are evaluated. Several protein markers were discovered from patient specimen, using two dimensional‐differential gel electrophoresis couple with liquid chromatography tandem mass spectrometry (2D‐DIGE/LC‐MS‐MS), matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF‐MS), surface enhanced laser desorption/ionization (SELDI)‐TOF‐MS and capillary electrophoresis (CE)‐MS, etc. Newly reported CCA‐associated glyco‐biomarkers were identified using lectin‐assisted, monoclonal antibody‐assisted or specific‐target strategies. The combination between carbohydrate binding‐lectin and core protein‐binding mAb significantly increased the values for detection of the glyco‐biomarkers for CCA. Searching for specific and sensitive molecular markers to be used for population screening is worth being evaluated. This could lead to earlier diagnosis and improve outcome. Further investigation of those biomarker functions is also of value in order to better understand the tumor biology and use them as targets for future therapeutic agents.