DOI: 10.14457/tu.the.2015.1563
title: Glycoproteomics analysis of plasma proteins associated with Opisthorchis viverrini infection-induced cholangiocarcinoma in hamster model

description: Cholangiocarcinoma (CCA) is a malignancy arising from bile duct epithelial tissue, which is an important public health problem in several parts of Southeast Asia, particularly in the northeast region of Thailand. Early detection of this type of cancer is still elusive. The aim of the current research was to apply glycoproteomics-based LC-MS/MS to preliminarily investigate the possible potential plasma biomarkers of Opisthorchis viverrini (OV)-associated CCA in OV/dimethylnitrosamine (DMN)-induced CCA hamster model.  Nine Syrian hamsters were divided into 3 groups as follows (n=3 each): normal (healthy control group); OV group; and OV/DMN group (CCA group).  Pooled plasma samples collected from animals in each group at the 6th month post-infection with OV metacercarae were used for glycoproteomics analysis.  Glycoproteins in the pooled sample from each group were initially isolated by concanavilin A (ConA)- and wheat germ agglutinin (WGA)-based affinity chromatography. The expression of glycoproteins isolated by both enrichment methods were determined using LC-MS/MS. The peptide data from LC-MS/MS were matched and normalized by DeCyder MS and proteins were identified using MASCOT software. Finally, bioinformatics tools were applied to predict protein functions and potential biomarkers. The analysis of glycoprotein data was performed using hierarchical clustering analysis (heatmap analysis), Venn diagrams, and STITCH 4 softwares.The glycoproteins isolated by both lectin-based methods were found to be totally different with regard to the number and type of the proteins.  A total of 21 and 24 glycoproteins were isolated from the ConA and WGA-affinity chromatography, respectively. The isolated glycoproteins enriched by both ConA and WGA affinity chromatography were found to be differentially expressed in the pool plasma samples from the OV, OV/DMN (CCA) and control groups. Among the 21 ConA-binding glycoproteins isolated, two proteins, N-myc downstream regulated gene 1 (NDRG1) and fetuin-B (FETUB) were found up-regulated only in the samples from the OV and control groups, but not in the OV/DMN (CCA) groups.  On the other hand, one protein, i.e., NSFL1 cofactor p47 isoform x3 (NSFL1C) was found only in the samples from OV/DMN (CCA) and control groups, but not in the OV group. The remaining 21 proteins were upregulated in the samples from all groups.  With regard to the 24 glycoproteins isolated from WGA-based affinity chromatography, one protein, rho guanine nucleotide exchange factor 6 (ARHGEF6) was found up-regulated only in the plasma sample of the OV group, but not in the OV and control groups.  Furthermore, two proteins, i.e, apolipoprotein B-100 (APOB) and breast cancer type 2 susceptibility protein like protein (BRCA2) were upregulated only in the samples from the OV and CCA group, but not in the control group.  On the other hand, one protein, ankyrin repeat domain-containing protein 42 (ANKRD42) was up-regulated only in the samples of the OV and control groups, but not in the CCA group.  The remaining 21 proteins were upregulated in the samples from all groups.   Results suggest that 7 glycoproteins isolated by the combined ConA- and WGA-based affinity chromatography would be potential biomarkers for CCA.   These potential biomarkers can be exploited as tools for diagnosis, monitoring of disease prognosis and progress of CCA.  Moreover, they also constitute important database of promising chemotherapeutic targets for discovery and development of anti-CCA drugs. Plasma samples with positive expression of APOB, BRCA2 and NSFL1C, with no expression of ARHGEF6, NDRG1 and FETUB are likely suggestive of OV-associated CCA.  On the other hand, plasma samples with positive expression of APOB and BRCA2 and ARHGEF6 are likely suggestive of OV infection.  Nevertheless, this conclusion remains to be confirmed whether this battery test can discriminate OV-associated CCA from other risk factors.  Further investigation into the sensitivity and specificity of this battery test in the human CCA associated with different risk factors is essential for understanding the glycobiology of CCA which could be exploited as as tools for diagnosis, monitoring of disease prognosis and progress of CCA.  Moreover, they also constitute important database of promising chemotherapeutic targets for discovery and development of anti-CCA drugs.