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Narintorn Rattanata,1 Sompong Klaynongsruang,1 Chanvit Leelayuwat,2 Temduang Limpaiboon,2 Aroonlug Lulitanond,2 Patcharee Boonsiri,3 Sirinart Chio-Srichan,4 Siriwat Soontaranon,4 Supagorn Rugmai,4 Jureerut Daduang2 1Department of Biochemistry, Faculty of Science, 2Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, 3Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, 4Synchrotron Light Research Institute (Public Organization), Nakhon Ratchasima, Thailand Abstract: Foodborne pathogens, including Plesiomonas shigelloides and Shigella flexneri B, are the major cause of diarrheal endemics worldwide. Antibiotic drug resistance is increasing. Therefore, bioactive compounds with antibacterial activity, such as gallic acid (GA), are needed. Gold nanoparticles (AuNPs) are used as drug delivery agents. This study aimed to conjugate and characterize AuNP–GA and to evaluate the antibacterial activity. AuNP was conjugated with GA, and the core–shell structures were characterized by small-angle X-ray scattering and transmission electron microscopy. Antibacterial activity of AuNP–GA against P. shigelloides and S. flexneri B was evaluated by well diffusion method. AuNP–GA bactericidal mechanism was elucidated by Fourier transform infrared microspectroscopic analysis. The results of small-angle X-ray scattering showed that AuNP–GA conjugation was successful. Antibacterial activity of GA against both bacteria was improved by conjugation with AuNP because the minimum inhibitory concentration value of AuNP–GA was significantly decreased (P

The nucleotide sequence of cosmid B1790, carrying the Rif-Str regions of the Mycobacterium leprae chromosome, has been determined. Twelve open reading frames were identified in the 36716bp sequence, representing 40% of the coding capacity. Five ribosomal proteins, two elongation factors and the β and β' subunits of RNA polymerase have been characterized and two novel genes were found. One of these encodes a member of the so-called ABC family of ATP-binding proteins while the other appears to encode an enzyme involved in repairing genomic lesions caused by free radicals. This finding may well be significant as M. leprae, an intracellular pathogen, lives within macrophages. [ABSTRACT FROM AUTHOR]

Colorectal carcinoma (CRC) is one of the most common malignancies, though there are no effective therapeutic regimens at present. This study aimed to investigate the inhibitory effects of mannooligosaccharides extracted from coconut meal (CMOSs) on the proliferation and migration of human colorectal cancer HCT116 cells in vitro . The results showed that CMOSs exhibited significant inhibitory activity against HCT116 cell proliferation in a concentration-dependent manner with less cytotoxic effects on the Vero normal cells. CMOSs displayed the ability to increase the activation of caspase-8, -9, and -3/7, as well as the generation of reactive oxygen species (ROS). Moreover, CMOSs suppressed HCT116 cell migration in vitro . Interestingly, treatment of human microvascular endothelial cells (HMVECs) with CMOSs resulted in the inhibition of cell proliferation, cell migration, and capillary-like tube formation, suggesting its anti-vascular angiogenesis. In summary, the results of this study indicate that CMOSs could be a valuable therapeutic candidate for CRC treatment., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

A limited self-healing ability of injured articular cartilage results in osteoarthritis and a joint dysfunction afterward. Cartilage tissue engineering is a promising approach to increase the treatment efficiency. Moreover, host response to implanted biomaterial has been increasingly concerned. Thus, this study aimed to establish three-dimensional (3D) scaffold that could support cartilage tissue engineering and reduce inflammatory. The various ratios of silk fibroin (SF), gelatin (G), chondroitin sulfate (C), hyaluronic acid (H), and aloe vera (A) were used to fabricate 3D scaffolds by lyophilization, designated as SF, SF-A, SF-gelatin/chondroitin sulfate/hyaluronic acid (GCH)-A-411, and SF-GCH-A-111. The physical and biological characteristics of the scaffolds were investigated. All scaffolds possessed interconnected porous structures, which the highest pore size of 209 μm was found in SF and SF-GCH-A-411 scaffolds. Moreover, high porosity, high water uptake, and good mechanical strength were observed in the SF-GCH-A-411 scaffold. The SF, SF-A, and SF-GCH-A-411 scaffolds could retain their structures up to 21 days, while SF-GCH-A-111 was rapidly degraded. The proliferation of human bone marrow mesenchymal stem cells (BM-MSCs) was significantly higher in SF-A and SF-GCH-A-411 than in the SF scaffold. Besides, the SF-A and SF-GCH-A-411 revealed significantly lower expression of pro-inflammatory cytokine, interleukin-1 beta than the SF scaffold, suggesting the beneficial role of aloe vera in anti-inflammatory effect. Furthermore, the SF-GCH-A-411 scaffold could support chondrogenic differentiation of BM-MSCs. In conclusion, based on its superior physical and biological characteristics that support chondrogenesis of BM-MSCs, the SF-GCH-A-411 scaffold is recommended for cartilage tissue engineering., (© 2023 Wiley Periodicals LLC.)

Background/aim: Cartilage tissue engineering has been popularly applied in the treatment of articular cartilage defect because it is more effective in generating functional engineered cartilage than traditional methods. Although the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) is well established, it is often accompanied by undesired hypertrophy. Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a crucial mediator in the ion channel pathway which is known to be involved in chondrogenic hypertrophy. Therefore, this study aimed to reduce the hypertrophy of BM-MSCs by inhibiting CaMKII activation., Materials and Methods: BM-MSCs were cultured in three-dimensional (3D) scaffold under chondrogenic induction with and without CaMKII inhibitor, KN-93. After cultivation, markers of chondrogenesis and hypertrophy were investigated., Results: KN-93 at a concentration of 2.0 μM had no effect on the viability of BM-MSCs, while the activation of CaMKII was suppressed. A long period of KN-93 treatment significantly up-regulated the expression of SRY-box transcription factor 9 and aggrecan on day 28 compared to untreated BM-MSCs. Furthermore, KN-93 treatment significantly down-regulated the expression of RUNX family transcription factor 2 and collagen type X alpha 1 chain on days 21 and 28. Immunohistochemistry showed increased expression of aggrecan and type II collagen while the expression of type X collagen was reduced., Conclusion: A CaMKII inhibitor, KN-93 is able to enhance chondrogenesis of BM-MSCs and suppress chondrogenic hypertrophy, suggesting its potential applicability in cartilage tissue engineering., (Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Cholangiocarcinoma (CCA) is a rare malignancy of the biliary tract with extremely poor clinical outcomes due to a lack of effective therapies to improve disease management. The emerging green synthesis of gold nanoparticles (AuNPs) has extensively provided their use in biomedical applications. In this study, we developed AuNPs via reducing gold salts with apigenin (4',5,7-trihydroxyflavone). The synthesized apigenin-conjugated AuNPs (api-AuNPs) were physicochemically characterized by various techniques before evaluation their biological and functional inhibition in a CCA cell line, KKU-M055. The mean size of api-AuNPs was 90.34 ± 22.82 nm with zeta potential of -36 ± 0.55. The half-maximal inhibitory concentration (IC 50 , 0.8 mg/mL) of api-AuNPs on cell proliferation of KKU-M055 was 1.9-fold less than that of an immortalized human cholangiocyte cell line, MMNK1 (IC 50 , 1.5 mg/mL). Moreover, api-AuNPs induced cell apoptosis via the up-regulation of Bax , Bid , and Caspase 3 , and down-regulation of Bcl2 , leading to elevated caspase 3/7, 8, 9 activities and reactive oxygen species (ROS) production. The api-AuNPs significantly inhibited the migration of KKU-M055 cells and suppressed the proliferation, migration, and in vitro tube formation of vascular endothelial cells. Collectively, our findings indicate the dual abilities of api-AuNPs that potentially inhibit cancer cell growth and motility as well as endothelial cell-mediated angiogenesis, which may offer a novel therapeutic avenue to treat CCA patients effectively., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Cholangiocarcinoma (CCA), a malignancy of the biliary epithelium, can arise at any point in the biliary system. We previously reported that CIAPIN1 is detectable in the sera and that its overexpression was associated with poor prognosis and metastasis of CCA patients. In this study, we investigated further its expression in CCA tissues, biological functions, and related signaling pathways in CCA cells. First, we examined CIAPIN1 expression in CCA tissues of 39 CCA patients using immunohistochemistry (IHC). Then, CIAPIN1-related proteins expressed in CCA cells were identified using RNA interference (siRNA) and liquid chromatography-mass spectrometry (LC-MS/MS). To predict the functions and signaling pathways of CIAPIN1 in CCA cells, the identified proteins were analyzed using bioinformatics tools. Then, to validate the biological functions of CIAPIN1 in the CCA cell line, transwell migration/invasion assays were used. CIAPIN1 was overexpressed in CCA tissues compared with adjacent noncancerous tissues. Its overexpression was correlated with lymph node metastasis. Bioinformatic analyses predicted that CIAPIN1 is connected to the TGF-β/SMADs signaling pathway via nitric oxide synthase 1 (NOS1) and is involved in the metastasis of CCA cells. In fact, cell migration and invasion activities of the KKU-100 CCA cell line were significantly suppressed by CIAPIN1 gene silencing. Our results unravel its novel function and potential signaling pathway in metastasis of CCA cells. CIAPIN1 can be a poor prognostic factor and can be a promising target molecule for CCA chemotherapy.

Background/aim: Cholangiocarcinoma (CCA) is a stem cell-based cancer. The in vivo tumor microenvironment is not present in two-dimensional (2D) cultures, which is one of the limitations in cancer stem cell (CSC) research. Thus, we aimed to establish three-dimensional (3D) culture mimicking extracellular matrix (ECM) that could serve as a niche for CSC enrichment in CCA., Materials and Methods: Silk fibroin-gelatin/hyaluronic acid/heparan sulfate (SF-GHHs) scaffolds were fabricated by lyophilization in various ratios and compared to silk fibroin (SF) scaffold. The physical and biological characteristics of the scaffolds were investigated., Results: The SF-GHHs 1:2 scaffold with pore size of 350±102 μm harbored optimal porosity, good water uptake, and stable beta-sheet that supported the increase in KKU-213A cell proliferation and aggregation. The CSC and the epithelial-mesenchymal transition (EMT) markers were significantly upregulated in this scaffold compared to 2D. Moreover, drug sensitivity against cisplatin and gemcitabine in 3D culture was significantly higher than that in 2D culture., Conclusion: The SF-GHHs 1:2 scaffold could simulate ECM that may serve as a CSC niche of CCA, and reinforce stemness and EMT properties, suggesting its suitability for 3D CCA model, which supports CSC and new targeting drug research in CCA., (Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Cholangiocarcinoma (CCA) is a type of liver cancer originating from bile duct epithelium which has an unfavorable prognosis. Therefore, novel prognostic markers and effective therapeutic regimens are required. Opioid-binding protein/cell adhesion molecule-like (OPCML) is a tumor-suppressor protein that suppresses CCA cell proliferation via AXL receptor tyrosine kinase/signal transducer and activator of transcription 3 (AXL/STAT3) inactivation. However, this association in clinical samples remains unknown. We aimed to determine OPCML and AXL expression and investigate their association with clinicopathological features in patients with CCA. In addition, we also addressed whether OPCML enhanced the sensitivity of CCA cells to AXL inhibitor R428 in vitro., Materials and Methods: The expression of OPCML and AXL was determined by immunohistochemistry in 90 CCA tissue samples. The study of CCA cell line sensitivity to R428 was performed by cell viability assay., Results: The expression of OPCML was significantly lower while AXL expression was substantially higher in CCA than in adjacent normal tissue (p<0.001). Furthermore, high AXL expression was significantly associated with lymph node metastasis (p=0.035). Interestingly, patients with combined low OPCML/high AXL expression had significantly shorter overall survival (p=0.007). OPCML enhanced the effect of AXL inhibitor R428 in AXL-expressing CCA cell lines., Conclusion: Combined expression of OPCML and AXL shows potential value as a prognostic marker and OPCML as an agent enhancing the effect of R428 may contribute to better prognosis for patients with CCA., (Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: We previously demonstrated that a mitochondrial protein, apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) is over-expressed in cholangiocarcinoma (CCA) and its serum levels can be a prognostic biomarker for CCA. To elucidate the functional roles of AIFM3 in CCA progression, we aimed to determine the signaling pathways of AIFM3 in CCA., Materials and Methods: AIFM3 gene in CCA cells was silenced and AIFM3-related proteins were identified using mass spectrometry and bioinformatics tools. The relationships between AIFM3 and 441 related proteins were explored. To validate the functions of AIFM3, transwell migration/invasion assays were used., Results: Bioinformatic analyses predicted that AIFM3 interacts with formin-like protein 3 (FMNL3) and is involved in tumor cell motilities. Online database analysis revealed higher AIFM3 mRNA expression levels in CCA, particularly with lymph node metastasis. After AIFM3 gene silencing, CCA cell migration/invasion was significantly decreased (p<0.001). Furthermore, AIFM3 expression levels were significantly associated with lymph node metastasis (p=0.0009) and shorter survival time (p=0.020)., Conclusion: The AIFM3 signaling pathway is mediated via FMNL3 and involved in metastasis, suggesting that AIFM3 might be a molecular target to prevent CCA metastasis., (Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Opioid-binding protein/cell adhesion molecule-like (OPCML) plays a crucial role in the suppression of tumor progression in several cancer types. Nevertheless, the association between OPCML functions and cholangiocarcinoma (CCA) progression remains unknown. We aimed to investigate biological functions of OPCML and related signaling pathways in CCA cell lines., Materials and Methods: Methylation status and ectopic expression of OPCML were determined in CCA cell lines using methylation-specific polymerase chain reaction and pcDNA3.1+/C-(K)DYK-OPCML, respectively. Cell proliferation, migration and invasion were investigated., Results: OPCML was found to be epigenetically silenced by DNA methylation. Ectopic expression of OPCML inhibited CCA proliferation by inducing apoptosis via AXL receptor tyrosine kinase/signal transducer and activator of transcription 3 (AXL/STAT3) inactivation. It also suppressed cell migration and invasion via down-regulation of Rho GTPases, ras homolog family member A (RHOA), Rac family small GTPase 1 (RAC1) and cell division cycle 42 (CDC42)., Conclusion: We are the first to unravel the antitumor effects and the related signaling pathways of OPCML in CCA. The loss of OPCML expression due to promoter hypermethylation can cause a decrease in cell death but increase in cell migration and invasion, which may at least in part contribute to CCA progression., (Copyright© 2021, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The mortality rate of cholangiocarcinoma (CCA) is high since there is a lack of a non-invasive technique to accurately detect tumors at the early stage. CCA biomarkers are consistently needed for various purposes including screening, early diagnosis, prognosis and follow-up. Herein, using bioinformatic analysis of our mitochondrial proteome database of CCA tissues, we identified cytokine-induced apoptosis inhibitor 1 (CIAPIN1) as a potential prognostic biomarker for CCA. CIAPIN1 levels in the sera of 159 CCA patients and 93 healthy controls (HC) were measured using a dot blot assay. The median level ± quartile deviation of CIAPIN1 level in the sera of CCA patient group was 0.5144 ± 0.34 µg/µL, which was significantly higher than 0.2427 ± 0.09 µg/µL of the HC group ( p < 0.0001). In CCA patients, higher serum CIAPIN1 level was significantly associated with lymph node metastasis ( p = 0.024) and shorter overall survival time ( p = 0.001, Kaplan-Meier test). Cox regression analysis showed that the serum CIAPIN1 level can be an independent prognostic indicator for the survival of CCA patients. Moreover, for the prediction of CCA prognosis, CIAPIN1 is superior to CEA, CA19-9 and ALP. In conclusion, CIAPIN1 can be a serum biomarker candidate for the poor prognosis of CCA.

Various cytokines are involved in carcinogenesis and tumor progression. Some tumor cells produce cytokines by themselves. Using secretome analysis, a high expression of APEX-1 was found in cholangiocarcinoma (CCA) cell lines. During this secretome analysis, it was found that CCA cell lines overexpressed some cytokines and related molecules, including interleukin 25 (IL-25). In the present study, we first performed precise secretome analysis on cytokines and related molecules in CCA cell lines and identified that IL-25 was overexpressed in CCA cell lines. Then, using immunohistochemical methods, we investigated the expression of IL-25 in the cancer tissues from 20 CCA patients in Northeast Thailand. Correlation between IL-25 expression levels and patients' clinical parameters were analyzed. The results showed that IL-25 expression was significantly (P<0.0001) higher in cancerous tissues than in the normal bile ducts and in the adjacent tissues. Overexpression of IL-25 protein in CCA tissue was confirmed using western blot analysis. Moreover, IL-25 expression in cancerous tissues was significantly (P<0.0015) higher in CCA patients with metastasis than in CCA patients without metastasis. Survival analysis revealed that a high expression of IL-25 was correlated with shorter survival time of CCA patients (P=0.0260). Aberrant expression of IL-25 in CCA tissue was associated with tumor metastasis and poor prognosis, suggesting that IL-25 is a potential prognostic biomarker. Biological roles of IL-25 in CCA genesis and progression should be explored in future., (Copyright: © Kaewsarabhumi et al.)

Prognosis of cholangiocarcinoma (CCA) patients is absolutely poor. Since improvement of prognosis and/or response to treatment by personalized and precision treatments requires earlier and precise diagnostic markers, discovery of prognostic markers attracts more attention. Apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) is highly expressed in several cancers including CCA. The present study investigated whether the serum AIFM3 level can be used as a potential marker for CCA prognosis. For this purpose, we first determined secretory protein nature of AIFM3 using bioinformatic tools. The results show that although AIFM3 lacks signal peptide, it can be secreted into plasma/serum via an unconventional pathway. Then, the AIFM3 levels in the sera of 141 CCA patients and 70 healthy controls (HC) were measured using a semi-quantitative dot blot assay. The results show that the AIFM3 level in the sera of CCA group was significantly higher than that of HC. When correlation between serum AIFM3 levels and the clinicopathological parameters of CCA patients were examined, serum AIFM3 levels correlated significantly with lymph node metastasis, age, and the patients' overall survival (OS). Higher AIFM3 levels were significantly associated with shorter OS, and only AIFM3 was an independent prognostic marker for CCA. In conclusion, AIFM3 can be used as a prognostic marker for CCA.

Background: Cholangiocarcinoma (CCA) is the second most common primary hepatobiliary cancer. These patients have meager prognosis and short-term survival. Precise assessment of glomerular filtration rate is a fundamental aspect of clinical care in cancer patients. Cystatin C has been proposed to be superior to creatinine, a well-known marker of renal function. This study aimed to evaluate cystatin C as a marker of GFR calculation in CCA patients. Materials and Methods: One hundred thirty serum samples from CCA patients and 32 from controls were included in this study. Serum cystatin C was measured using immunoturbidity assay. Estimated glomerular filtration rate was calculated by three equations established by chronic kidney disease epidemiology collaboration (based on creatinine and/or cystatin C). Results: Serum cystatin C in CCA patients was higher than that of controls (p=0.0002). Cystatin C was positively correlated with BUN in CCA group (p=0.019). eGFR based on cystatin C and based on both cystatin C and creatinine in CCA was low with significantly different from those of control (p<0.001). Although there was no difference in eGFR using three equations in control, creatinine based eGFR was high with significantly different from eGFR based on cystatin C and on both creatinine and cystatin C in CCA (P=0.000). Proportion in each eGFR stage by three equations showed a high sensitivity with significantly different in CCA (p<0.05). Conclusion: There was a high sensitivity of cys C with significant difference between creatinine and/or cystatin C based eGFR in CCA patients. It should be taken into consideration of mild changes in eGFR by cystatin C which is important in managing drug dosage for CCA patients., (Copyright : © International Journal of Hematology-Oncology and Stem Cell Research & Tehran University of Medical Sciences.)

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., (Copyright: © Chanakankun et al.)

Biliary tract cancers (BTCs) currently have no approved targeted therapies. Although genomic profiling of primary BTCs has identified multiple potential drug targets, accurate models are needed for their evaluation. Genomic profiling of 22 BTC cell lines revealed they harbor similar mutational signatures, recurrently mutated genes, and genomic alterations to primary tumors. Transcriptomic profiling identified two major subtypes, enriched for epithelial and mesenchymal genes, which were also evident in patient-derived organoids and primary tumors. Interrogating these models revealed multiple mechanisms of MAPK signaling activation in BTC, including co-occurrence of low-activity BRAF and MEK mutations with receptor tyrosine kinase overexpression. Finally, BTC cell lines with altered ERBB2 or FGFRs were exquisitely sensitive to specific targeted agents, whereas surprisingly, IDH1-mutant lines did not respond to IDH1 inhibitors in vitro. These findings establish BTC cell lines as robust models of primary disease, reveal specific molecular disease subsets, and highlight specific molecular vulnerabilities in these cancers., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Cholangiocarcinoma (CCA), although is an uncommon liver cancer originating from bile duct epithelial cells, is one of the top 10 most fatal cancers. Chemoresistance is an unmet need always found in CCA patients. Tumor microenvironment conditions such as hypoxia, nutrient starvation and acidic extracellular pH play critical roles in chemoresistance and cancer progression. However, the effect of acidic extracellular pH on cellular response and chemoresistance in CCA has not been studied. Methods: Human CCA cell lines (KKU-M213, KKU-M055 and KKU-100) were cultured under acidic (pH 6.5) or non-acidic (pH 7.4) condition and were used for gene expression, doubling time and cytotoxicity assay. Results: The acidic extracellular pH (pH 6.5) significantly increased doubling times of CCA cell lines compared with non-acidic condition (pH 7.4). Interestingly, extracellular acid condition induced gemcitabine resistance in CCA cell lines. We showed that Octamer-binding transcription factor 4 (Oct4) was upregulated in these cell lines under extracellular acid condition. Conclusion: Our findings demonstrate that CCA cells can adapt to survive in acidic environment after which chemoresistance has been developed. Oct4 may be a key transcriptional regulator which mediates chemoresistance in response to acidic extracellular pH.

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.

Background: Cholangiocarcinoma (CCA) is a fatal liver cancer arising from bile duct epithelium. Polycomb repressive complex 2 (PRC2) is a histone methyltransferase enzyme that catalyzes trimethylation of histone H3 on lysine 27, resulting transcriptional gene silencing. The key components of PRC2 are EZH2, SUZ12 and EED, which EZH2 is a catalytic subunit. The defect of individual PRC2 components has been shown to enhance carcinogenesis and cancer progression. The aim of this study was to determine the expression of individual PRC2 components and evaluate its association with clinicopathological data in CCA patients., Methods: The expression of PRC2 components including EZH2, SUZ12 and EED was determined by immunohistochemistry in 40 CCA tissue samples., Results: The expression of EZH2 and SUZ12 in CCA tissue was significantly higher than that in adjacent non-cancerous tissue (P < 0.001). The high cytoplasmic EZH2 expression was significantly associated with short overall survival in CCA (P = 0.030). Interestingly, a combined high nuclear and cytoplasmic expression of EZH2 was found to be a worse prognostic marker for overall survival (P = 0.015). Moreover, combined high expression of EZH2 and SUZ12/EED was also associated with short overall survival (P < 0.05)., Conclusions: Our findings suggest that overexpression of the PRC2 key components especially EZH2 in both nucleus and cytoplasm can be potentially used as a prognostic marker for CCA., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Pyruvate dehydrogenase kinase (PDK) is a Ser/Thr kinase that inactivates mitochondrial pyruvate dehydrogenase and serves a key role in aerobic glycolysis, which is a hallmark of cancer cells. The present study determined the PDK expression in cholangiocarcinoma (CCA) tissues and sera to evaluate their applicability as a biomarker for CCA. Using proteomic analysis, PDK was revealed to be the most overexpressed mitochondrial protein in CCA tissues. Then, the expression of PDK isoforms in CCA tissues was examined in 15 CCA cases by immunohistochemistry. The PDK3 isoform levels in the sera were measured using a dot blot assay for 39 patients with CCA, 20 patients with benign biliary disease and 19 healthy volunteers. The results revealed a 27-fold overexpression of PDK3 in cancerous tissues when compared with adjacent non-cancerous tissues. The immunohistochemical results demonstrated that the PDK1, 2 and 3, but not the PDK4, isoforms were overexpressed in cancerous tissues. When the PDK3 levels in the sera were examined, they were significantly higher in CCA when compared with the BBD and healthy groups. The specificity and sensitivity of PDK3 as a marker for CCA were 97.5 and 33.0%, respectively, and high PDK3 levels in the sera were correlated with a short survival time for CCA. In conclusion, PDK3 can be used as a diagnostic/prognostic marker for CCA.

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.

Persistent infection with human papillomavirus (HPV) type 16 and 18 is known to be a major risk factor for cervical cancer. Increased prevalence of co-infection with these high-risk types has been observed in pre-cancerous and cancerous tissues. The determination of physical status and copy numbers of viruses is therefore useful in clinical settings. A simple multiplex quantitative polymerase chain reaction (qPCR) for HPV16/HPV18 co-infection in one tube reaction was established in the present study using TaqMan ® -based PCR for E2 and E6 viral DNA. The detection range was up to 10 6 copies with 100% specificity and high precision (CV of cycle time <0.5%). The analytical accuracy and robustness were verified by competitive assay using an unequal mixture of HPV16/HPV18 DNA. No significant effect was demonstrated when compared with the simplex qPCR. The detection of physical status was evaluated in cervical samples, including 5 pre-cancerous and 15 cancerous samples. No significant difference was observed between simplex and multiplex qPCR (P=0.372). In conclusion, the developed multiplex qPCR method successfully demonstrated the viral status of the common HPV types in one tube. This assay will facilitate viral assessment and monitoring of cervical cancer associated with HPV16 and HPV18 co-infection.

Adult chondrocytes are surrounded by a protein- and glycosaminoglycan-rich extracellular matrix and are subjected to dynamic mechanical compression during daily activities. The extracellular matrix and mechanical stimuli play an important role in chondrocyte biosynthesis and homeostasis. In this study, we aimed to develop scaffold and compressive loading conditions that mimic the native cartilage micro-environment and enable enhanced chondrogenesis for tissue engineering applications. Towards this aim, we fabricated porous scaffolds based on silk fibroin (SF) and SF with gelatin/chondroitin sulfate/hyaluronate (SF-GCH), seeded the scaffolds with either human bone marrow mesenchymal stromal cells (BM-MSCs) or chondrocytes, and evaluated their performance with and without dynamic compression. Human chondrocytes derived from osteoarthritic joints and BM-MSCs were seeded in scaffolds, precultured for 1 week, and subjected to compression with 10% dynamic strain at 1 Hz, 1 hr/day for 2 weeks. When dynamic compression was applied, chondrocytes significantly increased expression of aggrecan (ACAN) and collagen X (COL10A1) up to fivefold higher than free-swelling controls. In addition, dynamic compression dramatically improved the chondrogenesis and chondrocyte biosynthesis cultured in both SF and SF-GCH scaffolds evidenced by glycosaminoglycan (GAG) content, GAG/DNA ratio, and immunostaining of collagen type II and aggrecan. However, both chondrocytes and BM-MSCs cultured in SF-GCH scaffolds under dynamic compression showed higher GAG content and compressive modulus than those in SF scaffolds. In conclusion, the micro-environment provided by SF-GCH scaffolds and dynamic compression enhances chondrocyte biosynthesis and matrix accumulation, indicating their potential for cartilage tissue engineering applications., (Copyright © 2018 John Wiley & Sons, Ltd.)

Cervical cancer (CxCa) is a major health problem globally and is associated with the presence of human papillomavirus infection. Cisplatin (CDDP) is a platinum-based chemotherapeutic agent. Owing to its side effects and drug-resistance, novel anticancer agents with lower toxicity, including caffeic acid (CFC), are of interest. However, the effects of CDDP and CFC in combination are, to the best of our knowledge, uninvestigated. The present study investigated the effectiveness of CDDP and CFC in combination and its mechanism of action on four human cervical cancer cell lines, which were compared with the Chlorocebus sabaeus normal kidney Vero cell line. Cell viability was evaluated using a sulforhodamine B assay. Caspase-Glo assay kits, measuring the activity of caspases-3, -7, -8 and -9, were used to detect caspase activation in HeLa and CaSki cell lines in response to CDDP and CFC in combination. The results revealed that CDDP and CFC alone reduced the proliferation of HeLa, CaSki, SiHa and C33A cell lines. Treatment with CFC exhibited no significant cytotoxicity towards Vero cells. In addition, CDDP-CFC significantly inhibited cell growth of HeLa and CaSki cell lines. In HeLa and CaSki cell lines, a combination index <1 for CDDP and CFC indicated the synergistic growth inhibition; the combination of the two also significantly increased expression of caspase-3, -7 and -9. In conclusion, CFC may be a candidate anticancer agent that, when use in combination, may increase the therapeutic efficacy of CDDP.

Vasculogenic mimicry (VM) is an alternative microvascular system which tumour cells orchestrate, independent of endothelial cell-mediated angiogenesis. VM develops tumour vascular networks that correlate with tumour growth, metastasis, and short survival time of patients with a number of cancers. However, little is known regarding VM in the vascularisation of cervical cancer. Chitinase 3 like 1 (CHI3L1) has been previously reported to display the ability to induce angiogenesis in cervical cancer. Here, we explored a pathological role of CHI3L1 in tumour cell-mediated vascularisation. Sixty-six samples of cervical cancer were collected to examine CHI3L1 expression and VM formation using immunohistochemistry and CD34-periodic acid-Schiff (PAS) dual staining. CHI3L1 expression was significantly correlated with formation of tumour cell-associated vascular channels in the absence of endothelial cells (p=0.031). Interestingly, tumour samples lacking VM were positively correlated with non-metastasis (p=0.035). Patients with VM positive tumours tended to have decreased overall survival (OS) compared to those with VM negative samples (43.9 versus 64.6 months, p=0.079). In addition, recombinant CHI3L1 enhanced cervical cancer cell lines to form tube-like structures, supporting the notion that CHI3L1 mediates VM in cervical cancer. Our present data reveal the crucial role of CHI3L1 in the formation of VM, which may contribute to tumour aggressiveness. Therefore, targeting CHI3L1 may be a valuable strategy for the reduction of cervical cancer vascularisation and metastasis., (Copyright © 2018 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Cholangiocarcinoma (CCA) is the most common primary liver cancer in Northeastern Thailand where liver fluke infection is highly endemic. Although aberrant DNA methylation in CCA has been reported by several investigators, little is known regarding the associations between them. In the present study, the results obtained from our previously published methylation array were analyzed and 10 candidate genes involved in DNA repair [protein phosphatase 4 catalytic subunit (PPP4C)], apoptosis [runt related transcription factor 3 (RUNX3), interferon regulatory factor 4 (IRF4), ubiquitin C‑terminal hydrolase L1 (UCHL1) and tumor protein p53 inducible protein 3 (TP53I3)], cell proliferation [cyclin D2 (CCND2) and Ras association domain family member 1 (RASSF1)], drug metabolism [aldehyde dehydrogenase 1 family member A3 (ALDH1A3) and solute carrier family 29 member 1 (SLC29A1)] and angiogenesis [human immunodeficiency virus‑1 tat interactive protein 2 (HTATIP2)] were selected for quantification of their methylation levels in 54 CCA and 19 adjacent normal tissues using methylation‑sensitive high‑resolution melting. The associations between the methylation status of the individual genes and clinical parameters were statistically analyzed. High methylation levels were observed in UCHL1, IRF4, CCND2, HTATIP2 and TP53I3. The median methylation level of UCHL1 was 57.3% (range, 3.15 to 88.7%) and HTATIP2 was 13.6% (range, 7.5 to 36.7%). By contrast, low methylation of HTATIP2 and UCHL1 was identified in adjacent normal tissues. The methylation status of HTATIP2 and UCHL1 was associated with patients' overall survival. CCA patients with high methylation of HTATIP2 and low methylation of UCHL1 exhibited longer overall survival. In addition, multivariate Cox regression analysis demonstrated that UCHL1 methylation was an independent factor for CCA with hazard ratio of 1.81 (95% confidence interval, 1.01‑3.25) in high methylation group. The combination of HTATIP2 and UCHL1 methylation status strongly supported their potential predictive biomarker in which patients with CCA who had high methylation of HTATIP2 and low methylation of UCHL1 showed longer overall survival than those with low HTATIP2 methylation and high UCHL1 methylation. In conclusion, the present study revealed the value of aberrant DNA methylation of HTATIP2 and UCHL1, which may serve as a potential predictive biomarker for CCA.

Cholangiocarcinoma (CCA) is a cancer of biliary epithelial cell origin, which is prevalent in northeastern Thailand. The majority of patients with CCA are diagnosed at the advanced-stage of the disease. Although the early detection and diagnosis of CCA is critical to improve the prognosis of patients, there are presently no specific tumor markers for CCA. A previous study demonstrated that the total serum bile acid (TSBA) levels of patients with CCA are significantly increased, compared with those of healthy controls. In addition, although statistically insignificant, the TSBA levels in the sera of patients with CCA tended to be increased, as compared with the sera of patients with benign biliary disease (BBD). In the present study, the high performance liquid chromatographic (HPLC) patterns of bile acid composition were compared in the sera of patients with CCA, patients with BBD and normal controls. The results revealed that serum bile acid patterns in patients with CCA varied, compared with those in patients with BBD and normal controls. As hypothesized, glycocholic acid (GCA) levels in the sera of patients with CCA and BBD were high, compared with those in healthy controls. In addition, GCA levels in the sera of patients with CCA tended to be higher, as compared with patients with BBD; however, this result was not statistically significant. Therefore, determination of the bile acid patterns and GCA levels in sera using HPLC is feasible, and may aid the diagnosis of CCA.

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.

Cholangiocarcinoma (CCA), a cancer of bile duct epithelium, is a major health problem in Thailand especially in the northeast. Overall treatment outcomes have not shown much improvement because the disease is usually detected at an advanced stage and often shows chemotherapeutic resistance. High-throughput Fourier Transform Infrared (FTIR) microspectroscopy can be used for cell classification and has the potential to diagnose cancer and possibly predict chemo-response. This study was aimed to differentiate gemcitabine-sensitive and gemcitabine-resistant induction in two CCA cell lines (KKU-M139 and KKU-M214) and xenograft tissues using synchrotron-FTIR microspectroscopy. Partial Least Squares Discriminant Analysis (PLS-DA) could discriminate between chemo-sensitive and chemo-resistant cells in the FTIR fingerprint spectral region (1800-1000 cm -1 ) with more than 90% sensitivity and specificity. The chemo-resistant and chemo-sensitive phenotypes were different in protein (amide I, amide II), lipids (carbonyl group and CH 3 deformation) and phosphodiester from nucleic acids. Additionally, spectra from xenograft tissues showed similar results to the cell line study with marked differences between chemo-resistant and chemo-sensitive CCA tissues, and PLS-DA could discriminate the chemotherapeutic response with 98% sensitivity and specificity. This is the first study to demonstrate the use of FTIR microspectroscopy to assess chemo-response both in vitro and in vivo., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.