ConA-binding glycoprotein as a potential serum marker for screening of benign and malignant of bile duct

Chaisiri Wongkham,Arthit Tolek,Nittaya Chamadol,Puangrat Yongvanit,Sopit Wongkham 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

The incidence of cholangiocarcinoma (CCA), a bile duct cancer, is high in the northeastern part of Thailand. At present, there is no known marker that can be used as an early marker for CCA. The surveillance of people at risk of CCA is the effective strategy to detect patients with early CCA, the stage at which surgery for completely cure can be offered. There is accumulated evidence indicated that people with chronic inflammation of bile duct epithelia and peribiliary fibrosis have high risk of CCA. Recently, a high resolution ultrasonography of peri-portal fibrosis was shown to be the acceptable approach to identify people at high risk or early CCA. However, the ultrasonography has to be conducted and determined by an experience radiologist and the operation cannot be offered for people in rural area. In this study, we aimed to find a suitable serum marker that can distinguish people who has benign bilirary diseases (BBD) or CCA from healthy subjects, liver fluke infected persons and patients with other gastro-intestinal cancers. Serum was electrophoresesed in a 12% SDS-polyacrylamide gel electrophoresis and ConA-binding glycoproteins were determined using lectin-biotin/avidin-peroxidase system. ConA blotting of serum from 29 CCA and 23 benign biliary diseases were compared with those from healthy persons (n = 20), active liver fluke infected people (n =10), patients with hepatoma (n =15) and other gastro-intestinal cancers (n =20). A high molecular weight of ConA binding glycoprotein was frequently detected in 85% of serum from CCA and BBD comparing to 24.6% of all serum from the control group. This marker can identify person who had CCA or BBD with a 81.6% sensitivity, 75.4% specificity, 73.3% and 85.9% positive and negative predictive values, respectively. The value of this ConA-specific marker in differentiating people who are at risk of CCA should be re-investigated in a larger cohort including people from the villages that are in the endemic area of liver fluke infection and patients with various pathological conditions.

Transcriptional Profiles of Peripheral Blood Leukocytes Identify Patients with Cholangiocarcinoma and Predict Outcome

Subimerb, Chutima,Wongkham, Chaisiri,Khuntikeo, Narong,Leelayuwat, Chanvit,McGrath, Michael S.,Wongkham, Sopit Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.10

Cholangiocarcinoma (CCA), a slow growing but highly metastatic tumor, is highly prevalent in Northeast Thailand. Specific tests that predict prognosis of CCA remain elusive. The present study was designed to investigate whether peripheral blood leukocyte (PBL) transcriptional profiles might be of use as a prognostic test in CCA patients. Gene expression profiles of PBLs from 9 CCA and 8 healthy subjects were conducted using the Affymetrix HG_U133 Plus 2.0 GeneChip. We indentified informative PBLs gene expression profiles that could reliably distinguish CCA patients from healthy subjects. Of these CCA specific genes, 117 genes were up regulated and 60 were down regulated. The molecular and cellular functions predicted for these CCA specific genes according to the Gene Ontology database indicated differential PBL expression of host immune response and tumor progression genes (EREG, TGF ${\beta}1$, CXCL2, CXCL3, IL-8, and VEGFA). The expression levels of 9 differentially expressed genes were verified in 36 CCA vs 20 healthy subjects. A set of three tumor invasion related genes (PLAU, CTSL and SERPINB2) computed as "prognostic index" was found to be an independent and statistically significant predictor for CCA patient survival. The present study shows that CCA PBLs may serve as disease predictive clinically accessible surrogates for indentifying expressed genes reflective of CCA disease severity.

Hypermethylation of Suppressor of Cytokine Signaling 1 in Hepatocellular Carcinoma Patients

Saelee, Pensri,Chuensumran, Ubol,Wongkham, Sopit,Chariyalertsak, Sunanta,Tiwawech, Danai,Petmitr, Songsak Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.7

Hepatocellular carcinoma (HCC), the most common primary hepatic tumor, is highly prevalent in the Asia-Pacific region, including Thailand. Many genetic and epigenetic alterations in HCC have been elucidated. The aim of this study was to determine whether aberrant methylation of the suppressor of cytokine signaling 1 gene (SOCS1) occurs in HCCs. Methylation specific-PCR assays were performed to identify the methylation status of SOCS1 in 29 tumors and their corresponding normal liver tissues. An abnormal methylation status was detected in 17 (59%), with a higher prevalence of aberrant SOCS1 methylation significantly correlating with HCC treated without chemotherapy (OR=0.04, 95%CI=0.01-0.31; P=0.001). This study suggests that epigenetic aberrant SOCS1 methylation may be a predictive marker for HCC patients.

Poster Session : Biotechnology ; 5` SAGE transcriptome of adult Liver fluke (opisthorchis viverrini)

( Nopporn Chutiwitoonchai ),( Chaisiri Wongkham ),( Sopit Wongkham ),( Yen Shen ),( Huajun Zheng ),( Hui Xiong ),( Guoping Zhao ) 한국생화학분자생물학회 (구 한국생화학회) 2007 생화학분자생물학회 춘계학술발표논문집 Vol.2007 No.-

Serum Cathepsin B to Cystatin C Ratio as a Potential Marker for the Diagnosis of Cholangiocarcinoma

Monsouvanh, Ammala,Proungvitaya, Tanakorn,Limpaiboon, Temduang,Wongkham, Chaisiri,Wongkham, Sopit,Luvira, Vor,Proungvitaya, Siriporn Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.21

Cholangiocarcinoma (CCA) is a cancer of the bile duct epithelial cells. The highest incidence rate of CCA with a poor prognosis and poor response to chemotherapy is found in Southeast Asian countries, especially in northeastern Thailand and Lao PDR. Cathepsin B is a lysosomal cysteine protease which is regulated by cysteine proteinase inhibitors such as cystatin C. Elevation of cathepsin B levels in biological fluid has been observed in patients with inflammatory diseases and many cancers. We aimed to investigate the serum cathepsin B and cystatin C levels of CCA patients to evaluate the feasibility of using cathepsin B and cystatin C as markers for the diagnosis of CCA. Fifty-six sera from CCA patients, 17 with benign biliary diseases (BBD) and 13 from controls were collected and the cathepsin B and cystatin C levels were determined. In addition, cathepsin B expression was investigated immunohistochemically for 9 matched-pairs of cancerous and adjacent tissues of CCA patients. Serum cathepsin B, but not cystatin C, was significantly higher in CCA and BBD patient groups compared to that in the control group. Consistently, all cancerous tissues strongly expressed cathepsin B while adjacent tissues were negative in 7 out of 9 cases. In contrast, serum cystatin C levels were comparable between CCA and control groups, although serum cystatin C levels in the BBD group was higher than that in the control or CCA groups. When the serum cathepsin B to cystatin C ratio was calculated, that of the CCA group was significantly higher than that of the control group, and, although statistically not significant, the ratio of CCA group showed a trend to be higher than that of the BBD group. Thus, the cathepsin B to cystatin C ratio might be used as an alternative marker for aiding diagnosis of CCA.

Total Serum Bile Acid as a Potential Marker for the Diagnosis of Cholangiocarcinoma without Jaundice

Sombattheera, Sutthikan,Proungvitaya, Tanakorn,Limpaiboon, Temduang,Wongkham, Sopit,Wongkham, Chaisiri,Luvira, Vor,Proungvitaya, Siriporn Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.4

Diagnosis of cholangiocarcinoma (CCA) is difficult when patients do not show jaundice. The aim of this study was to examine the feasibility of using the total serum bile acid (TSBA) level as an aid for the diagnosis of CCA in patients without jaundice. For this purpose, TSBA of the following groups were measured using a Beckman Synchron CX4 clinical chemistry analyzer: 60 cases of CCA with total serum bilirubin ${\leq}2mg/dL$ (low total bilirubin group, LTB); 32 cases of CCA with total serum bilirubin >2 mg/dL (high total bilirubin group, HTB); and 115 healthy controls. Liver function parameters such as serum cholesterol, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were also examined. The results showed that the TSBA of both LTB and HTB groups of the CCA patients were significantly higher than that of the healthy controls. Also, significant correlation was observed between TSBA and total bilirubin levels in the HTB group of CCA patients. However, no such correlation was seen in the LTB group. The cut-off value of TSBA was determined for the LTB group of CCA patients using the receiver operating characteristic curve analysis, and it was $6.05{\mu}mol/L$ with the sensitivity and specificity of 46.7% and 84.4%, respectively. In addition, the ALP level was correlated well with the TSBA level and ALP in HTB group was significantly higher than that of LTB group. Moreover, the combination of high TSBA and high ALP levels gave higher specificity up to 97.4%. TSBA might be useful for the diagnosis of CCA patients without jaundice.

ALCAM is a Novel Cytoplasmic Membrane Protein in TNF-α Stimulated Invasive Cholangiocarcinoma Cells

Adisakwattana, Poom,Suwandittakul, Nantana,Petmitr, Songsak,Wongkham, Sopit,Sangvanich, Polkit,Reamtong, Onrapak Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.9

Background: Cholangiocarcinoma (CCA), or bile duct cancer, is incurable with a high mortality rate due to a lack of effective early diagnosis and treatment. Identifying cytoplasmic membrane proteins of invasive CCA that facilitate cancer progression would contribute toward the development of novel tumor markers and effective chemotherapy. Materials and Methods: An invasive CCA cell line (KKU-100) was stimulated using TNF-${\alpha}$ and then biotinylated and purified for mass spectrometry analysis. Novel proteins expressed were selected and their mRNAs expression levels were determined by real-time RT-PCR. In addition, the expression of ALCAM was selected for further observation by Western blot analysis, immunofluorescent imaging, and antibody neutralization assay. Results: After comparing the proteomics profile of TNF-${\alpha}$ induced invasive with non-treated control cells, over-expression of seven novel proteins was observed in the cytoplasmic membrane of TNF-${\alpha}$ stimulated CCA cells. Among these, ALCAM is a novel candidate which showed significant higher mRNA- and protein levels. Immunofluorescent assay also supported that ALCAM was expressed on the cell membrane of the cancer, with increasing intensity associated with TNF-${\alpha}$. Conclusions: This study indicated that ALCAM may be a novel protein candidate expressed on cytoplasmic membranes of invasive CCA cells that could be used as a biomarker for development of diagnosis, prognosis, and drug or antibody-based targeted therapies in the future.

Novel Mutations in Cholangiocarcinoma with Low Frequencies Revealed by Whole Mitochondrial Genome Sequencing

Muisuk, Kanha,Silsirivanit, Atit,Imtawil, Kanokwan,Bunthot, Suphawadee,Pukhem, Ake,Pairojkul, Chawalit,Wongkham, Sopit,Wongkham, Chaisiri Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.5

Background: Mitochondrial DNA (mtDNA) mutations have been shown to be associated with cancer. This study explored whether mtDNA mutations enhance cholangiocarcinoma (CCA) development in individuals. Materials and Methods: The whole mitochondrial genome sequences of 25 CCA patient tissues were determined and compared to those of white blood cells from the corresponding individuals and 12 healthy controls. The mitochondrial genome was amplified using primers from Mitoseq and compared with the Cambridge Reference Sequence. Results: A total of 161 mutations were identified in CCA tissues and the corresponding white blood cells, indicating germline origins. Sixty-five (40%) were new. Nine mutations, representing those most frequently observed in CCA were tested on the larger cohort of 60 CCA patients and 55 controls. Similar occurrence frequencies were observed in both groups. Conclusions: While the correspondence between the cancer and mitochondrial genome mutation was low, it is of interest to explore the functions of the missense mutations in a larger cohort, given the possibility of targeting mitochondria for cancer markers and therapy in the future.

Differential Expression of O-glycoprotein Glycans in Cholangiocarcinoma Cell Lines

Talabnin, Krajang,Talabnin, Chutima,Ishihara, Mayumi,Azadi, Parastoo,Wongkham, Sopit,Sripa, Banchob Asian Pacific Journal of Cancer Prevention 2016 Asian Pacific journal of cancer prevention Vol.17 No.2

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-$MS^n$). 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 $NeuA_{c2}Gal_1GalNA_{c1}$. 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.

Soluble CD30: A Possible Serum Tumor Marker for Primary Effusion Lymphoma

Michai, Manthana,Goto, Hiroki,Hattori, Shinichiro,Vaeteewoottacharn, Kulthida,Wongkham, Chaisiri,Wongkham, Sopit,Okada, Seiji Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.10

Background: The serum level of soluble CD30 (sCD30) is known to be increased with several lymphomas and to correlate with prognosis. Primary effusion lymphoma (PEL) is a highly aggressive malignant lymphoma with poor prognosis, but the existence and significance of sCD30 in PEL have not yet been investigated in detail. Objectives: Since the membrane type of CD30 is frequently expressed on the surface of PEL cells, we compared the expression of the membrane type of CD30 and the production of sCD30 among PEL cell lines as well as other lymphomas. Methods: The expression of surface CD30 in various lymphoma cell lines was analyzed with flow cytometry ans sCD30 was quantified by ELISA. Results: Both surface and sCD30 were detected on PEL cell lines as well as on Hodgkin's lymphoma and adult T-cell leukemia/lymphoma cell lines. Surface CD30 and sCD30 levels of each cell lines correlated with each other. Conclusion: The serum level of sCD30 appear to be a useful biological tumor marker for the diagnosis and management of CD30-positive PEL.