Chutima Talabnin

Overexpression of microRNA-21 regulating PDCD4 during tumorigenesis of liver fluke-associated cholangiocarcinoma contributes to tumor growth and metastasis.

MicroRNA, an endogenous noncoding RNA modulating gene expression, is a key molecule that by its dysregulation plays roles in inflammatory-driven carcinogenesis. This study aimed to investigate the role of oncomiR miR-21 and its target, the programmed cell death 4 (PDCD4) in tumor growth and metastasis of the liver fluke Opisthorchis viverrini-associated cholangiocarcinoma (CCA). The expression levels of miR-21 and PDCD4 were analyzed using the TaqMan miRNA expression assay and immunohistochemistry in liver tissues of both O. viverrini plus N-nitrosodimethylamine (NDMA)-treated hamsters and human CCA samples (n = 23 cases). The functional assay for miR-21 was performed in CCA cell lines by the anti-miR-21 and pre-miR-21 transfection procedures. The peak of miR-21 levels were reached at 2 (hyperplastic lesions) and 6 (CCA) months of the O. viverrini plus NDMA-induced group and had a reverse response with its target PDCD4 proteins. In human CCA, miR-21 was overexpressed in tumor tissues when compared with nontumor tissues (P = 0.0034) and had a negative correlation with PDCD4 protein (P = 0.026). It was also found that high expression of miR-21 was significantly correlated with shorter survival (P < 0.05) and lymph node metastasis (P = 0.037) of CCA patients. Transient transfection of pre-miR-21 reduced the PDCD4 level and resulted in an increase of M213 CCA cell growth and wound-induced migration ability. These results indicated that miR-21 plays a role in the carcinogenesis and metastasis of O. viverrini-associated CCA by suppressing the function of PDCD4. Modulation of aberrantly expressed miR-21 may be a useful strategy to inhibit tumor cell phenotypes or improve response to chemotherapy.

Piperlongumine induces G2/M phase arrest and apoptosis in cholangiocarcinoma cells through the ROS-JNK-ERK signaling pathway

Cholangiocarcinoma (CCA) is an aggressive, metastatic bile duct cancer. CCA is difficult to diagnose, and responds poorly to current radio- and chemo-therapy. Piperlongumine (PL) is a naturally-occurring small molecule selectively toxic to cancer cells by targeting reactive oxygen species (ROS). In this study, we demonstrated the potential anticancer activity of PL in CCA. PL markedly induced death in CCA cell lines in a dose- and time-dependent manner through the activation of caspase-3 and PARP. PL also stimulated ROS accumulation in CCA. Co-exposure of PL with the ROS scavenger N-acetyl-l-cysteine or GSH completely blocked PL-induced apoptosis in CCA cell lines. Increased p21 via the p53-independent pathway in PL-treated CCA cells led to G2/M phase arrest and cell apoptosis. In addition, the study showed that PL trigger CCA cell lines death through JNK-ERK activation. Furthermore, the different antioxidant capacity of CCA cell lines also indicates the susceptibility of the cells to PL treatment. Our findings reveal that PL exhibits anti-tumor activity and has potential to be used as a chemotherapeutic agent against CCA.

Activation of Vimentin is Critical to Promote a Metastatic Potential of Cholangiocarcinoma Cells.

Cholangiocarcinoma (CCA) is a highly metastatic tumor, and the majority of patients with CCA have a short survival time because there are no available effective treatments. Hence, a better understanding regarding CCA metastasis may provide an opportunity to improve the strategies for treatment. A comparison study between the highly metastatic cells and their parental cells is an approach to uncover the molecular mechanisms underlying the metastatic process. In the present study, a lung metastatic CCA cell line, KKU-214L5, was established by the in vivo selection of the tail vein-injected mouse model. KKU-214L5 cells possessed mesenchymal spindle-like morphology with higher migration and invasion abilities in vitro than the parental cells (KKU-214). KKU-214L5 also exhibited extremely aggressive lung colonization in the tail vein-injected metastatic model. Epithelial-mesenchymal transition (EMT) was clearly observed in KKU-214L5 cells. Significant downregulation of epithelial markers (ZO-1 and claudin-1), with unique upregulation of E-cadherin and mesenchymal markers (vimentin, β-catenin, and slug), was observed in KKU-214L5. Increasing MMP-2 and MMP-9 activities and CD147 expression reflected the high invasion activity in KKU-214L5 cells. Suppression of vimentin using siRNA significantly decreased the migration and invasion capabilities of KKU-214L5 to almost the basal levels of the parental cells without any change on the expression levels of other EMT markers and the activities of MMPs. These results suggest that vimentin activation is essential to potentiate the metastatic characters of CCA cells, and suppression of vimentin expression could be a potential strategy to improve the treatment of CCA, a highly metastatic cancer.

Characterization of the binding of a glycosylated serine protease from Euphorbia cf. lactea latex to human fibrinogen

In this study, the binding of a glycosylated serine protease (EuP‐82) with human fibrinogen was investigated by isothermal titration calorimetry (ITC). ITC analysis indicated that the binding of EuP‐82 to fibrinogen in the conditions with or without the activator (Ca2+) was an exothermic reaction (dominant negative enthalpy), which tended to be driven by hydrogen bonding and van der Waals interactions. In contrast, the binding of fibrinogen−EuP‐82 in the condition with the inhibitor (Zn2+) was an unfavorable endothermic reaction. EuP‐82 could not inhibit the platelet activity in citrated whole blood via the ADP–receptor pathways (mainly, P2Y1 and P2Y12), but it could enhance the platelet aggregation. The ITC together with whole blood platelet aggregation suggested that EuP‐82 provided multiple fibrinogen‐binding sites that were not related to the arginine‐glycine‐aspartate (RGD) and the dodecapeptide sequences of fibrinogen. In addition, EuP‐82 had neither thrombin‐like activity nor anticoagulant activity. The SR‐FTIR spectra revealed that EuP‐82 was a glycoprotein. Deglycosylation of EuP‐82 did not affect its proteolytic activity. Moreover, EuP‐82 did not exhibit any toxicity to the living cells (NIH‐3T3). This study supports that EuP‐82 may be useful for wound‐healing material through stabilizing the clot via the platelet induction for the first process.

Ring finger protein 43 expression is associated with genetic alteration status and poor prognosis among patients with intrahepatic cholangiocarcinoma

Ring finger E3 ligases have roles in processes central to maintenance of genomic integrity and cellular homeostasis. Many ring finger E3 ligases are implicated in malignancy. Ring finger protein 43 (RNF43) is a ring finger E3 ligase that negatively regulates the Wnt/β-catenin signaling pathway. RNF43 is frequently mutated in several types of malignancy, including intrahepatic cholangiocarcinoma (ICC). The significance of its expression in ICC has not, however, been reported. We determined RNF43 expression and identified RNF43 polymorphisms in ICC tissues. We also investigated the correlation between RNF43 expression and RNF43 mutation status, RNF43 polymorphisms, clinicopathological features, and prognosis of ICC patients. RNF43 reduced expression in ICC, and the reduction of RNF43 messenger RNA expression was significantly correlated with the presence of rs2257205 and RNF43 somatic mutations, confirming that all RNF43 somatic mutations in ICC are inactivating. Overall survival was worst in patients with down-regulation of RNF43. Univariate and multivariate analyses revealed that RNF43 expression was an independent prognostic factor. There was no statistically significant association between RNF43 messenger RNA and protein expression nor any clinicopathological features or RNF43 polymorphisms. The results imply that RNF43 is down-regulated in ICC and may play a crucial role during development of ICC.

Differential Expression of O-glycoprotein Glycans in Cholangiocarcinoma Cell Lines.

Protein glycosylation is the most common posttranslational modification in mammalian cells. Aberrant protein glycosylation has been reported in various diseases, including cancer. We identified and quantified the glycan structures of O-linked glycoprotein from cholangiocarcinoma (CCA) cell lines from different histological types and compared their profiles by nanospray ionization-linear ion trap mass spectrometry (NSI-MSn). Five human CCA cell lines, K100, M055, M139, M213 and M214 were characterized. The results showed that the O-linked glycans of the CCA cell lines comprised tri- to hexa-saccharides with terminal galactose and sialic acids: NeuAc1Gal1GalNAc1, Gal2GlcNAc1GalNAc1, NeuAc2Gal1GalNAc1 NeuAc1Gal2GlcNAc1GalNAc1 and NeuAc2Gal2GlcNAc1GalNAc1 All five CCA cell lines showed a similar glycan pattern, but with differences in their quantities. NeuAc1Gal1GalNAc1 proved to be the most abundant structure in poorly differentiated adenocarcinoma (K100; 57.1%), moderately differentiated adenocarcinoma (M055; 42.6%) and squamous cell carcinoma (M139; 43.0%), while moderately to poorly differentiated adenocarcinoma (M214; 40.1%) and adenosquamous cell carcinoma (M213; 34.7%) appeared dominated by NeuAc2Gal1GalNAc1. These results demonstrate differential expression of the O-linked glycans in the different histological types of CCA. All five CCA cell lines have abundant terminal sialic acid (NeuAc) O-linked glycans, suggesting an important role for sialic acid in cancer cells. Our structural analyses of glycans may provide important information regarding physiology of disease-related glycoproteins in CCA.

Increased expression of the high‑mannose M6N2 and NeuAc3H3N3M3N2F tri‑antennary N‑glycans in cholangiocarcinoma

Changes in protein glycosylation have been reported in various types of cancer, including cholangiocarcinoma (CCA). Nanospray ionization-linear ion trap mass spectrometry (NSI-MSn) was used in the present study to determine the comparative structural glycomics of the N-linked glycans in the serum of patients with CCA compared with healthy controls. A total of 5 high-mannose and 4 complex N-linked glycans were detected. Mannose7-N-acetyl-glucosamine2 was the most abundant structure among the high-mannose types (control 12.12±2.54 vs. CCA 9.27±2.66%), whereas NeuAc2H2N2M3N2 predominated the complex types (control 61.17±2.55 vs. CCA 64.68±4.23%). The expression of 3 different N-glycans differed significantly between the CCA cases and controls. These included mannose6-N-acetyl-glucosamine2 (P=0.044), mannose9-N-acetyl-glucosamine2 (Ρ=0.030) and NeuAc3H3N3M3N2F (Ρ=0.002). These three glycan structures may therefore be associated with tumor progression in CCA and may be useful for its diagnosis.