High glucose-ROS conditions enhance the progression in cholangiocarcinoma via upregulation of MAN2A2 and CHD8.

Diabetes is a major risk factor in the development and progression of several cancers including cholangiocarcinoma (CCA). However, the molecular mechanism by which hyperglycemia potentiates progression of CCA is not clearly understood. Here, we showed that a high glucose condition significantly increased reactive oxygen species (ROS) production and promoted aggressive phenotypes of CCA cells, including proliferation and migration activities. Mannosidase alpha class 2a member 2 (MAN2A2), was upregulated at both mRNA and protein levels in a high glucose- and ROS-dependent manner. In addition, cell proliferation and migration were significantly reduced by MAN2A2 knockdown. Based on our proteome and in silico analyses, we further found that chromodomain helicase DNA-binding protein 8 (CHD8) was induced by ROS signaling and regulated MAN2A2 expression. Overexpression of CHD8 increased MAN2A2 expression, while CHD8 knockdown dramatically reduced proliferation and migration as well as MAN2A2 expression in CCA cells. Moreover, both MAN2A2 and CHD8 were highly expressed with positive correlation in CCA tumor tissues. Collectively, these data suggested that high glucose conditions promote CCA progression through ROS-mediated upregulation of MAN2A2 and CHD8. Thus, glucose metabolism is a promising therapeutic target to control tumor progression in patients with CCA and diabetes.

Homophilic Interaction of CD147 Promotes IL-6-Mediated Cholangiocarcinoma Invasion via the NF-κB-Dependent Pathway.

Cholangiocarcinoma (CCA), an aggressive cancer of bile ducts, is a well-known chronic inflammation-related disease. The major impediment in CCA treatment is limited treatment options for advanced disease; hence, an alternative is urgently required. The role of CD147 on cytokine production has been observed in inflammation-related diseases, but not in CCA. Therefore, this study was focused on CD147-promoting proinflammatory cytokine production and functions. Proinflammatory cytokine profiles were compared between CD147 expressing CCA cells and CD147 knockout cells (CD147 KO). Three cytokines, namely interleukin (IL)-6, IL-8, and granulocyte-monocyte colony-stimulating factor (GM-CSF), were dramatically diminished in CD147 KO clones. The involvement of the CD147-related cytokines in CCA invasion was established. CD147-promoted IL-6, IL-8, and GM-CSF secretions were regulated by NF-κB nuclear translocation, Akt activation, and p38 phosphorylation. CD147-fostering IL-6 production was dependent on soluble CD147, CD147 homophilic interaction, and NF-κB function. The overexpression of specific genes in CCA tissues compared to normal counterparts emphasized the clinical importance of these molecules. Altogether, CD147-potentiated proinflammatory cytokine production leading to CCA cell invasion is shown for the first time in the current study. This suggests that modulation of CD147-related inflammation might be a promising choice for advanced CCA treatment.

A Novel Serum Glycobiomarker for Diagnosis and Prognosis of Cholangiocarcinoma Detected by Butea monosperma Agglutinin.

Plant lectins are widely used in medical glycosciences and glycotechnology. Many lectin-based techniques have been applied for the detection of disease-associated glycans and glycoconjugates. In this study, Butea monosperma agglutinin (BMA), a lectin purified from seeds of the medicinal plant Butea monosperma, was used for the detection of cholangiocarcinoma (CCA)-associated glycans. Expression of BMA-binding N-acetyl galactosamine/galactose (GalNAc/Gal)-associated glycan (BMAG) in CCA tissues was determined using BMA lectin histochemistry; the results showed that BMAG was undetectable in normal bile ducts and drastically increased in preneoplastic bile ducts and CCA. The study in hamsters showed that an increase of BMAG was associated with carcinogenesis of CCA. Using an in-house double BMA sandwich enzyme-linked lectin assay, BMAG was highly detected in the sera of CCA patients. The level of serum BMAG in CCA patients (N = 83) was significantly higher than non-CCA controls (N = 287) and it was applicable for diagnosis of CCA with 55.4% sensitivity, 81.9% specificity, and 76.0% accuracy. A high level of serum BMAG (≥82.5 AU/mL) was associated with unfavorable survival of CCA patients; this information suggested the potential of serum BMAG as a poor prognostic indicator of CCA. In summary, BMAG was aberrantly expressed in preneoplastic bile ducts and CCA, it was also highly detected in patient serum which potentially used as a marker for diagnosis and prognostic prediction of CCA.

The O-GalNAcylating enzyme GALNT5 mediates carcinogenesis and progression of cholangiocarcinoma via activation of AKT/ERK signaling.

Mucin type O-glycosylation is a posttranslational modification of membrane and secretory proteins. Transferring of N-acetylgalactosamine, the first sugar of O-glycosylation, is catalyzed by one of the 20 isoforms of polypeptide N-acetylgalactosaminyltransferases (GALNTs). In this study, Vicia villosa lectin (VVL), a lectin that recognizes O-GalNAcylated glycans, was used to detect VVL-binding glycans (VBGs) in cholangiocarcinoma (CCA). The elevation of VBGs in tumor tissues of the liver fluke associated with CCA from hamsters and patients was noted. VBGs were detected in hyperplastic/dysplastic bile ducts and CCA but not in normal biliary epithelia and hepatocytes, indicating the association of VBGs with CCA development and progression. GALNT5 was shown to be the major isoform found in human CCA cell lines with high VBG expression. Suppression of GALNT5 expression using siRNA significantly reduced VBG expression, signifying the connection of GALNT5 and VBGs observed. Knocked-down GALNT5 expression considerably inhibited proliferation, migration and invasion of CCA cells. Increased expression of GALNT5 using pcDNA3.1-GALNT5 expression vector induced invasive phenotypes in CCA cells with low GALNT5 expression. Increasing of claudin-1 and decreasing of slug and vimentin expression together with inactivation of Akt/Erk signaling were noted in GALNT5 knocked-down cells. These observations were reversed in GALNT5 over-expressing cells. GALNT5-modulated progression of CCA cells was shown to be, in part, via GALNT5-mediated autocrine/paracrine factors that stimulated activations of Akt/Erk signaling and the epithelial to mesenchymal transition process. GALNT5 and its O-GalNAcylated products may have important roles in promoting progression of CCA and could possibly be novel targets for treatment of metastatic CCA.

Differential Protein Expression Marks the Transition From Infection With Opisthorchis viverrini to Cholangiocarcinoma.

Parts of Southeast Asia have the highest incidence of intrahepatic cholangiocarcinoma (CCA) in the world because of infection by the liver fluke Opisthorchis viverrini (Ov). Ov-associated CCA is the culmination of chronic Ov-infection, with the persistent production of the growth factors and cytokines associated with persistent inflammation, which can endure for years in Ov-infected individuals prior to transitioning to CCA. Isobaric labeling and tandem mass spectrometry of liver tissue from a hamster model of CCA was used to compare protein expression profiles from inflammed tissue (Ovinfected but not cancerous) versus cancerous tissue (Ov-induced CCA). Immunohistochemistry and immunoblotting were used to verify dysregulated proteins in the animal model and in human tissue. We identified 154 dysregulated proteins that marked the transition from Ov-infection to Ov-induced CCA, i.e. proteins dysregulated during carcinogenesis but not Ov-infection. The verification of dysregulated proteins in resected liver tissue from humans with Ov-associated CCA showed the numerous parallels in protein dysregulation between human and animal models of Ov-induced CCA. To identify potential circulating markers for CCA, dysregulated proteins were compared with proteins isolated from exosomes secreted by a human CCA cell line (KKU055) and 27 proteins were identified as dysregulated in CCA and present in exosomes. These data form the basis of potential diagnostic biomarkers for human Ov-associated CCA. The profile of protein dysregulation observed during chronic Ovinfection and then in Ov-induced CCA provides insight into the etiology of an infection-induced inflammation-related cancer.

Increase of MAL-II Binding Alpha2,3-Sialylated Glycan Is Associated with 5-FU Resistance and Short Survival of Cholangiocarcinoma Patients.

BACKGROUND AND OBJECTIVES: Sialylation plays important roles in tumor progression. Our present study aimed to demonstrate the alteration of sialylation and its role in cholangiocarcinoma (CCA). MATERIALS AND METHODS: The α2,3- and α2,6-sialylation in CCA tissue was analyzed by lectin-histochemistry using Maackia amurensis lectin-II (MAL-II) and Sambucus nigra agglutinin (SNA). CCA cell lines were treated with the pan-sialylation inhibitor 3Fax-peracetyl-Neu5Ac (3F-Sia) followed by proliferation and chemosensitivity assays. RESULTS: MAL-II binding α2,3-Sialylated Glycan (MAL-SG) and SNA binding α2,6-Sialylated Glycan (SNA-SG) were both elevated in CCA compared with hyperplastic/dysplastic (HP/DP) and normal bile ducts (NBD). The positive staining for MAL-SG or SNA-SG were found in 82% (61/74) of the CCA cases. Higher expression of MAL-SG in CCA was associated with shorter survival of the patients. The median survival of patients with high and low MAL-SG were 167 and 308 days, respectively, with overall survival of 233 days, suggesting the involvement of MAL-SG in CCA progression. MAL-SG expression of CCA cell lines was markedly decreased after treatment with 3F-Sia for 48 to 72 h. While proliferation of CCA cells were not affected by 3F-Sia treatment, their susceptibility to 5-fluorouracil (5-FU) was significantly enhanced. These results suggest that sialylation is involved in the development of 5-FU resistance and the sialylation inhibitor 3F-Sia can be used as a chemosensitizer for CCA. CONCLUSIONS: Sialylation is critically involved in the development of chemoresistance of CCA, and sialylation inhibitors may be used as a chemosensitizer in CCA treatment.

Artesunate and chloroquine induce cytotoxic activity on cholangiocarcinoma cells via different cell death mechanisms.

Chemotherapy for cholangiocarcinoma (CCA) is not quite successful. In this study, we revisited the possibility of artesunate (ART) and chloroquine (CQ), the antimalarial drugs, as therapeutic agents against CCA. The possible mechanisms of these drugs to exert cytotoxicity on CCA cells were also explored. The effects of ART and CQ on proliferation and death patterns of two CCA cell lines, KKU-214 and its highly metastatic subtype KKU-214L5, were examined using water soluble tetrazolium (WST) assay and time-lapse photometry, respectively. To differentiate and verify the death patterns between necrosis and apoptosis, lactate dehydrogenase (LDH) release, and caspase 3 activity were measured. CellROXTM green reagent staining method was used to assess reactive oxygen species (ROS) production in ART- and CQ-treated cells. ART and CQ significantly inhibited proliferation of CCA cells. Both drugs kill malarial parasites via similar mechanism depending on ROS formation, however, ART induced necrotic cell death and CQ induced apoptotic cell death in CCA cells. ART induced LDH release, whereas CQ activated caspase 3, confirming induction of necrotic and apoptotic cell deaths by ART and CQ, respectively. ART treatment induced higher ROS production than CQ. ART and CQ induce CCA cells death via different death pathways. ART should be suitable for necrosis-sensitive CCA, whereas CQ is more suitable for apoptosis-sensitive CCA.

Immuno-proteomic analysis of Trichinella spiralis, T. pseudospiralis, and T. papuae extracts recognized by human T. spiralis-infected sera.

The present study explored potentially immunogenic proteins of the encapsulated (Trichinella spiralis) and non-encapsulated (T. pseudospiralis, T. papuae) species within the genus Trichinella. The somatic muscle larval extracts of each species were subjected to immunoblotting analysis using human T. spiralis-infected serum samples. Fifteen reactive bands of all three species were selected for further protein identification by liquid chromatography-tandem mass spectrometry, and their possible functions were ascertained using the gene ontology. Our findings showed immunogenic protein patterns with molecular mass in the range of 33-67 kDa. Proteomic and bioinformatic analysis revealed a wide variety of functions of 17 identified proteins, which are associated with catalytic, binding, and structural activities. Most proteins were involved in cellular and metabolic processes that contribute in the invasion of host tissues and the larval molting processes. The parasite proteins were identified as actin-5C, serine protease, deoxyribonuclease-2, and intermediate filament protein ifa-1. This information may lead to alternative tools for selection of potential diagnostic protein markers or aid in the design of vaccine candidates for prevention and control of Trichinella infection.

Identification of antigenic proteins in Strongyloides stercoralis by proteomic analysis.

Strongyloides stercoralis is an intestinal helminth that infects people worldwide. Hyperinfection or disseminated human strongyloidiasis can involve vital organs, leading to lethal outcomes. We analyzed immunoproteomics of antigenic spots, derived from S. stercoralis third-stage larvae and reacted with human strongyloidiasis sera, by two-dimensional gel electrophoresis and immunoblotting. Of 26 excised antigenic spots analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry, 20 proteins were identified. Most proteins were associated with enzymes involved in the metabolic process, energy generation, and oxidation-reduction. The proteins relate to promotion of worm development, cell division, cell signaling and transportation, and regulation of muscular contraction. Identification of antigenic proteins shows promise in helping to discover potential diagnostic protein markers or vaccine candidates for S. stercoralis infection.

High expression of apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) in human cholangiocarcinoma.

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.

Inhibition of carbonic anhydrase potentiates bevacizumab treatment in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a unique liver cancer subtype with an increasing incidence globally. The lack of specific symptoms and definite diagnostic markers results in a delayed diagnosis and disease progression. Systemic chemotherapy is commonly selected for advanced CCA even though its advantages remain unknown. Targeted therapy, especially anti-vascular endothelial growth factor (VEGF) therapy, is promising for CCA; however, improvements in the therapeutic regimen are necessary to overcome subsequent resistance. We demonstrated VEGF expression was higher in CCA cell lines than in other liver cancer cells. Secreted VEGFs played roles in the induction of peri- and intra-tumoral vascularization. VEGF neutralization by bevacizumab effectively reduced tumor growth, mainly through the suppression of angiogenesis; however, increases in the expression of hypoxia-inducible factor 1α (HIF1α) and HIF1α-responsive genes (such as VEGF, VEGFR1, VEGFR2, carbonic anhydrase (CA) IX and CAXII) indicated the potential for subsequent therapeutic resistance. Supplementation with a carbonic anhydrase inhibitor, acetazolamide, enhanced the anti-CCA effects of bevacizumab. Anti-angiogenesis and anti-proliferation were observed with the combination treatment. These results suggested a novel treatment strategy to overcome anti-angiogenesis resistance and the importance of "induced essentiality" in the treatment of CCA.

Thymosin ß10 as a predictive biomarker of response to 5-fluorouracil chemotherapy in cholangiocarcinoma.

UNLABELLED:  Introduction and aim. 5-Fluorouracil (5-FU) is the most commonly used chemotherapeutic drug in the treatment of cholangiocarcinoma (CCA). Since development of drug resistance to 5-FU in CCA patients is the primary cause of treatment failure, a better understanding of the mechanism of drug resistance of this cancer is essential to improve the efficacy of 5-FU in CCA therapy. MATERIAL AND METHODS: A 5-FU resistant CCA cell line (M214-5FUR) for a comparative chemo-resistance study was established. Real time RT-PCR was used to determine gene expression levels. Cell cytotoxicity was measured by the MTT assay. Protein expression levels were detected by the immunofluorescene method. RESULTS: It was found that 5-FU resistance was associated with the overexpression of T?10 in CCA cell lines. 5-FU treatment at various concentrations induced the expressions of T?10 and ABC transporters (ABCB1, ABCG2 ABCA3) in two CCA cell lines, KKU-M055 and KKU-M214. M214-5FUR, a 5-FU-resistant cell line, exhibited a 5-FU resistant phenotype with a 16-fold extremely high expression of T?10 and ABC transporters, as compared to the parental cells, KKU-M214. siRNA targeted to T?10 significantly reduced expression of ABC transporters tested in the M214-5FUR cells (P < 0.05). CONCLUSIONS: The present novel findingsof T?10 connected with drug resistance as shown in this study provides a new insight for the therapeutic value of T?10 as a predictive biomarker of 5-FU chemoresistance. Inhibiting T?10 may be a valuable adjunct for suppression of ABC transporters and sensitizing chemotherapy treatment, especially 5-FU in CCA patients.

Suppression of trophoblast cell surface antigen 2 enhances proliferation and migration in liver fluke-associated cholangiocarcinoma.

BACKGROUND AND AIM: Trophoblast cell surface antigen 2 (TROP2) or tumor-associated calcium signal transducer 2 (TACSTD2) is a 36-kDa type I transmembrane glycoprotein and exerts dual functions as an oncogene and tumor suppressor in cancer cells. In this study, we investigated the expression and functions of TROP2 in liver fluke-associated cholangiocarcinoma (CCA). MATERIAL AND METHODS: TROP2 expression in 85 CCA tissues was detected by using immunohistochemistry. The methylation status of TROP2 promoter was studied in 15 matched pairs of normal and CCA formalin fixed paraffin embedded (FFPE) tissues using the bisulfite genomic sequencing (BGS) method. The functions of TROP2 on cancer cell behavior were investigated using siRNA in CCA cell lines. Proliferation, migration and invasion assays were performed. A PCR array was used to evaluate the impact of TROP2 knockdown on the gene expression profiles. RESULTS: TROP2 was highly expressed in all normal bile duct epithelia, but significantly down-regulated in CCA cells. Sixty percent of CCA revealed promoter hypermethylation compared to the corresponding adjacent normal tissues. TROP2 knockdown significantly enhanced the proliferation and migration in CCA cell lines, and altered the expressions of MARCK, EMP1 and FILIP1L. CONCLUSION: We provide new evidence that TROP2 is epigenetically down-regulated and operates as a negative regulator of cell proliferation and migration in liver fluke-associated CCA.

Strongyloides stercoralis diagnostic polypeptides for human strongyloidiasis and their proteomic analysis.

Human strongyloidiasis is a deleterious gastrointestinal disease mainly caused by Strongyloides stercoralis infection. Strongyloides stercoralis is a soil-transmitted helminthiasis that is distributed around the globe. Although definitive diagnosis is carried out through the detection of parasite objects in human stool samples, the development of reliable immunological assays is an important alternative approach for supportive diagnosis. We characterized the two sensitive and specific bands of S. stercoralis filariform larvae that reacted with human strongyloidiasis sera based on immunoblot analysis. Serum samples obtained from strongyloidiasis patients showed a sensitivity of 90 and 80 % at the approximate molecular mass of 26 and 29-kDa polypeptide bands, respectively. The reactive specificity of the 26-kDa band was 76.5 % while for the 29-kDa band was 92.2 %. Proteomic analysis identified the 26-kDa band protein was 14-3-3 protein zeta, while the 29-kDa band protein was ADP/ATP translocase 4. The results provided a basic framework for further studies regarding the potential of the S. stercoralis recombinant antigen to become a leading to diagnostic tool.

Secreted cyclophilin A mediates G1/S phase transition of cholangiocarcinoma cells via CD147/ERK1/2 pathway.

Cyclophilin A (CypA) was shown to be upregulated in human cholangiocarcinoma (CCA) tissues. Suppression of intracellular CypA (inCypA) significantly reduces cell proliferation in vitro and tumor growth in nude mice. In the present study, the effect and potential mechanism of secreted CypA (sCypA) on cell proliferation of CCA cell lines were further investigated. CCA cells were treated with sCypA-containing conditioned media (CM) or with purified recombinant human CypA (rhCypA). Cell proliferation, cell cycle, ERK1/2, p38 MAPK, NF-κB, and STAT3 activities were examined by MTS assay, flow cytometry, and Western blot. sCypA was detected in CM from MMNK1 (an immortalized human cholangiocyte cell line) and six CCA cell lines. The sCypA levels corresponded to the inCypA levels indicating the intracellular origin of sCypA. Both sCypA-containing CM and rhCypA significantly increased proliferation of CCA cells. CD147 depletion by shRNA-knockdown or neutralizing with a CD147-monoclonal antibody significantly reduced sCypA-, and rhCypA-mediated cell proliferation. Upon rhCypA treatment, ERK1/2 was rapidly phosphorylated; whereas neutralizing CD147 inhibited ERK1/2 phosphorylation. Cell cycle analysis showed a significant increase in S phase and decrease in G1 population in rhCypA-treated cells. The expression levels of cyclin D1 and phosphorylated-retinoblastoma protein in the rhCypA-treated cells were increased compared with those in the non-treated control cells. p38 MAPK pathway was shown to be suppressed in siCypA-treated cells. In summary, CypA is secreted from CCA cells and enhances cell proliferation in an autocrine/paracrine manner, at least via direct binding with CD147, which may activate the ERK1/2 and p38 MAPK signaling pathways.

Proteomic analysis identification of antigenic proteins in Gnathostoma spinigerum larvae.

Gnathostoma spinigerum is the causative agent of human gnathostomiasis. The advanced third stage larva (AL3) of this nematode can migrate into the subcutaneous tissues, including vital organs, often producing severe pathological effects. This study performed immuno-proteomic analysis of antigenic spots, derived from G. spinigerum advanced third stage larva (GSAL3) and recognized by human gnathostomiasis sera, using two-dimensional (2-DE) gel electrophoresis based-liquid chromatography/tandem mass spectrometry (LC/MS-MS), and followed by the aid of a database search. The crude GSAL3 extract was fractionated using IPG strips (pH 3-11NL) and followed by SDS-PAGE in the second dimension. Each gel was stained with colloidal Coomassie blue or was electro-transferred onto a nitrocellulose membrane and probed with gnathostomiasis human sera by immunoblotting. Individual Coomassie-stained protein spots corresponding to the antigenic spots recognized by immunoblotting were excised and processed using LC/MS-MS. Of the 93 antigenic spots excised, 87 were identified by LC/MS-MS. Twenty-seven protein types were found, the most abundant being Ascaris suum37. Six spots showed good quality spectra, but could not be identified. This appears to be the first attempt to characterize antigenic proteins from GSAL3 using a proteomic approach. Immuno-proteomics shows promise to assist the search for candidate proteins for diagnosis and vaccine/drug design and may provide better understand of the host-parasite relationship in human gnathostomiasis.

Cytokine/chemokine secretion and proteomic identification of upregulated annexin A1 from peripheral blood mononuclear cells cocultured with the liver fluke Opisthorchis viverrini.

We investigated the cytokine/chemokine secretions and alteration of protein expression from peripheral blood mononuclear cells (PBMCs) cocultured with adult liver flukes (Opisthorchis viverrini) for 6 to 24 h. PBMC-derived proteins were identified by two-dimensional electrophoresis and mass spectrometry, and the cytokines/chemokines in the supernatant were assessed using a cytokine array. Exposure to O. viverrini induced increases in secretion of proinflammatory cytokines, costimulating protein, adhesion molecules, and chemotactic chemokines relative to untreated controls. In contrast, secretion of the CD40 ligand, interleukin 16, and macrophage inflammatory protein 1ß decreased. Proteomic analysis revealed that expression of 48 proteins was significantly altered in PBMCs stimulated with O. viverrini. Annexin A1 (ANXA1) was selected for further study, and immunoblotting showed upregulation of ANXA1 expression in PBMCs after 12 and 24 h coculture with liver flukes. In an in vivo study, transcription and translation of ANXA1 significantly increased in livers of hamsters infected with O. viverrini at 21 days and from 3 months onwards compared to normal controls. Interestingly, immunohistochemistry revealed that ANXA1 was present not only in the cytoplasm of inflammatory cells but also in the cytoplasm of cholangiocytes, which are in close contact with the parasite and its excretory/secretory products in the biliary system. Expression of ANXA1 increased with time concomitant with bile duct enlargement, bile duct formation, and epithelial cell proliferation. In conclusion, several cytokines/chemokines secreted by PBMCs and upregulation of ANXA1 in PBMCs and biliary epithelial cells might have a role in host defense against O. viverrini infection and tissue resolution of inflammation.

Increase of exostosin 1 in plasma as a potential biomarker for opisthorchiasis-associated cholangiocarcinoma.

A proteomic-based approach was used to search for potential markers in the plasma of hamsters in which cholangiocarcinoma (CCA) was induced by Opisthorchis viverrini infection and N-nitrosodimethylamine treatment. The plasma proteins of CCA-induced hamsters were resolved by 1-D PAGE, digested by trypsin, and analyzed by LC-MS/MS. From the criteria of protein ID scores >15 and an overexpression of at least three times across all time points, 37 proteins were selected. These overexpressed proteins largely consisted of signal transduction, structural, transport, and transcriptional proteins in the order. Among the most frequently upregulated proteins, exostosin 1 (EXT1) was selected for further validation. By western blot analysis, the EXT1 expression level in the plasma of hamster CCA was significantly higher than that of controls at 1 month and thereafter. Immunohistochemistry revealed that EXT1 was expressed at vascular walls and fibroblasts at 21 days (before tumor onset) and at 2 months (early CCA) posttreatment. Its expression was also observed in bile duct cancer cells during tumor progression at 6 months posttreatment. In the human CCA tissue microarray, EXT1 immunoreactivity was found not only in vascular walls and fibroblasts but also in bile duct cancer cells and was positive in 89.7 % (61/68) of the cases. By ELISA and immunoblotting, plasma EXT1 level was significantly higher in human CCA compared to healthy controls. In conclusion, these results suggest that increased expression of EXT1 level in the plasma might be involved in CCA genesis and might be a potential biomarker of CCA.

CA-S27: a novel Lewis a associated carbohydrate epitope is diagnostic and prognostic for cholangiocarcinoma.

Early and specific diagnosis is critical for treatment of cholangiocarcinoma (CCA). In this study, a carbohydrate antigen-S27 (CA-S27) monoclonal antibody (mAb) was established using pooled CCA tissue-extract as immunogen. The epitope recognized by CA-S27-mAb was a new Lewis-a (Le(a)) associated modification of MUC5AC mucin. A Soybean agglutinin/CA-S27-mAb sandwich ELISA to determine CA-S27 in serum was successfully developed. High level of CA-S27 was detected in serum of CCA patients and could differentiate CCA patients from those of gastro-intestinal cancers, hepatomas, benign hepatobiliary diseases and healthy subjects with high sensitivity (87.5%) and high negative predictive value (90.4%). The level of serum CA-S27 was dramatically reduced after tumor removal, indicating tumor origin of CA-S27. Patients with high serum CA-S27 had significantly shorter survivals than those with low serum CA-S27 regardless of serum MUC5AC levels. Fucosyltransferase-III (FUT3) was shown to be a regulator of CA-S27 expression. Suppression of CA-S27 expression with siRNA-FUT3 or neutralization with CA-S27 mAb significantly reduced growth, adhesion, invasion and migration potentials of CCA cells in vitro. In summary, we demonstrate that serum CA-S27, a novel carbohydrate antigen, has potential as diagnostic and prognostic markers for CCA patients. CA-S27 involves in promoting cell growth, adhesion, migration and invasion of CCA cells.

Roles and mechanisms of ß-thymosins in cell migration and cancer metastasis: an update.

ß-thymosins, including thymosin ß4 (Tß4), Tß10, and Tß15, are a family of highly conserved 5 kDa peptides. They are involved not only in normal cell migration, but also in tumor metastasis. However, the molecular mechanisms of ß-thymosins to regulate cell migration and other functions are not fully understood. Recently, this important area is under active investigation worldwide. Many new discoveries have been made from molecular biology and cell culture models as well as animal models and human diseases. This timely review provides the most updated information about functional roles and molecular mechanisms of ß-thymosins in normal tissues and disease conditions.