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

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Tumor Biol. DOI 10.1007/s13277-013-1137-9

RESEARCH ARTICLE

Increase of exostosin 1 in plasma as a potential biomarker for opisthorchiasis-associated cholangiocarcinoma Jarinya Khoontawad & Nuttanan Hongsrichan & Yaovalux Chamgramol & Porntip Pinlaor & Chaisiri Wongkham & Puangrat Yongvanit & Chawalit Pairojkul & Narong Khuntikeo & Sittiruk Roytrakul & Thidarut Boonmars & Somchai Pinlaor

Received: 16 May 2013 / Accepted: 23 August 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract 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 J. Khoontawad : N. Hongsrichan : T. Boonmars : S. Pinlaor (*) Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected] Y. Chamgramol : C. Pairojkul Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand P. Pinlaor Centre for Research and Development in Medical Diagnostic Laboratory, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand C. Wongkham : P. Yongvanit Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand N. Khuntikeo Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand J. Khoontawad : N. Hongsrichan : Y. Chamgramol : P. Pinlaor : C. Wongkham : P. Yongvanit : C. Pairojkul : N. Khuntikeo : T. Boonmars : S. Pinlaor Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand S. Roytrakul Proteomics Research Laboratory, Genome Institute Biotechnology, Pathum Thani 12120, Thailand

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. Keywords Opisthorchis viverrini . Cholangiocarcinoma . Exostosin 1 . Plasma proteome Abbreviations OV Opisthorchis viverrini CCA Cholangiocarcinoma EXT1 Exostosin 1 ECM Extracellular matrix

Introduction Cholangiocarcinoma (CCA), a bile duct cancer, is highly prevalent where liver flukes, Opisthorchis viverrini and Clonorchis sinensis, are endemic [1]. The incidence of CCA is increasing not only in the liver fluke endemic areas but also worldwide [2].

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In northeast Thailand, CCA is still a major health problem [3] in relation to the high prevalence of O. viverrini infection [4]. CCA is difficult to diagnose, and patients often present late resulting in a high mortality [5]. Detection of some CCA-specific proteins in plasma or other biological fluids may be helpful to discover new diagnostic markers for CCA [6]. O . viverrini infection in combination with nitrosamine administration causes CCA in hamsters [7, 8] with histopathological features similar to human CCA [9]. Nowadays, proteomic analysis has been used to identify tumor-associated biomarkers in the plasma from animals and also from humans [10]. Since plasma is a primary clinical specimen and contains all tissue protein leakage [11], it is a promising specimen to find new markers for the diagnosis and prognosis of CCA [6]. In order to search for the early novel biomarker for CCA, plasma profile of a hamster CCA model was analyzed by proteomic approach. Among upregulated proteins, exostosin 1 (EXT1) was selected for further validation based on the following criteria: (1) having a high ID score, (2) being overexpressed earlier and found at all time points studied, (3) having possible role for carcinogenesis, and (4) never being studied in CCA. Overexpression of EXT1 in both plasma and liver tissues of hamster and human samples was confirmed by western blotting and immunohistochemistry techniques. Moreover, its diagnostic value in the plasma of human CCA was investigated by ELISA.

Materials and methods Parasites Recently caught and dead cyprinid fish, chilled on ice, were obtained from endemic areas in Khon Kaen Province, Thailand. O. viverrini (OV) metacercariae were isolated from naturally infected cyprinid fish by pepsin (Wako, Japan) digestion as described previously [8]. Animals, experimental design, and specimen collection Experiments using the animal model in this study were conducted with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council of Thailand. The Institutional Animal Ethics Committee, Khon Kaen University and the National Research Council of Thailand approved all experimental protocols for use of laboratory animals. All animal samples used in this study were obtained from a previous experiment [8], which was approved by the Animal Ethics Committee of Khon Kaen University, Thailand (AEKKU 17/2552). Seventy male Syrian golden hamsters (Mesocricetus auratus) aged between 4 and 6 weeks were obtained from the Animal Unit, Faculty of Medicine, Khon Kaen University,

Khon Kaen, Thailand. Animals were divided into four groups: a group without treatment (control group), a group infected with 50 O . viverrini metacercariae (OV group), a group given N -nitrosodimethylamine (NDMA) (NDMA group), and a group treated with the combination of O . viverrini infection and given NDMA (OV + NDMA group). NDMA was dissolved in water and fed to the hamsters ad libitum for 2 months and then withdrawn thereafter. Animals (n =5 for each subgroup) were sacrificed at 21 days posttreatment and then every month up to 6 months. Hamsters were euthanized with an overdose of diethyl ether, and blood was collected from the heart. EDTA–blood was centrifuged at 3,000×g for 10 min at 4 °C and the plasma was collected and stored at −80 °C until analysis. The hilar region and the adjacent areas of the liver were dissected and were placed in 10 % buffered formalin and used to evaluate histopathological changes [8]. Patients and specimen collection Plasma and liver tissues of CCA patients and controls were obtained from the BioBank Collection in the Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. A written informed consent was obtained from 91 volunteers consisting of healthy individuals (n =11), patients with benign biliary diseases (BBD, i.e., chronic cholangitis, cholelithiasis, and cholestasis, n =12), and patients with CCA (n =68). All the CCA patients underwent liver resection in the Srinagarind Hospital, Khon Kaen University, Thailand, during 1999–2010. All patients possessed antibodies to O. viverrini crude somatic antigens as determined by ELISA. Normal hepatobiliary tracts were confirmed by ultrasonography and were selected as controls based on age (average 52.09±2.63) and sex matching. Ten milliliters of peripheral blood was obtained by aseptic venipuncture, collected in heparin-containing tubes, and centrifuged at 3,000×g for 10 min at 4 °C. The plasma was separated and stored at −80 °C until analysis. Tissue microarrays were constructed from the archival paraffin-embedded tissue samples of 68 intrahepatic CCA patients and the liver tissues of six normal healthy volunteers. Diagnosis of CCA patients was evaluated by clinical data, imaging analysis, tumor markers, and pathology. The study protocol using human materials was approved by the Human Research Ethics Committee, Khon Kaen University, Thailand (HE551407). 1-D PAGE gel, trypsin digestion, and LC-MS/MS-based proteomics approach Proteomics approach by 1-D PAGE gel, trypsin digestion, and LC-MS/MS was performed as described previously [12]. In brief, an equal amount of plasma (n =5) was pooled separately

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for each subgroup. Pooled plasma (50 μg protein) was fractionated by SDS-PAGE using both 7.5 and 12.5 % gels (Fig. 1). To recover all possible target proteins in the plasma and to exclude the most prominent band of albumin, the gels above the albumin band at 7.5 % and below the albumin band at 12.5 % were sliced. After reduction and alkylation, the gel pieces were digested overnight in 10 ng/μl trypsin in 50 % acetonitrile (ACN)/10 mM ammonium bicarbonate. The Fig. 1 SDS-PAGE fractionation of pooled plasma samples from normal and OV+NDMA groups. Fifty micrograms of pooled plasma protein obtained from five animals at each time point was fractionated on 7.5 and 12.5 % acrylamide gels (8×13 cm), and protein bands were visualized with silver staining. Each gel lane was sliced and then subjected to in-gel digestion prior to LC-MS/ MS analysis. For the consistency of results, the assay was done as three independent experiments. Molecular weights of protein marker bands are indicated in kilodalton (kDa) on the right of the images. OV Opisthorchis viverrini, NDMA Nnitrosodimethylamine, OV + NDMA combined OV infection and NDMA administration, D days, M months

peptides were extracted three times with 50 % ACN and 0.1 % formic acid, dried in a vacuum centrifuge, and kept at −80 °C for further MS analysis. Peptide solutions were analyzed using an HCTultra PTM Discovery System (Bruker Daltonics Ltd., Cambridge, UK) coupled to an UltiMate 3000 LC System (Dionex Ltd., Surrey, UK) and a nano-column (PepSwift monolithic column 100 μm i.d. × 50 mm). Peptides were analyzed by DeCyder MS

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Differential Analysis software (DeCyderMS, GE Healthcare, Piscataway, NJ, USA). For protein quantitation and identification, DeCyder MS Differential Analysis software (DeCyderMS, GE Healthcare) was used. The analyzed mass spectrometry data from DeCyderMS were searched against the NCBI database using the Mascot search engine (Matrix Science, London, UK). The protein expression ratio at each time point was analyzed by comparing the protein intensity values between the OV+NDMA

group and the normal hamster group at each time point. To reduce the number of candidate proteins for searching for the potential diagnostic markers for CCA, only the proteins with at least two peptides from MASCOT with an ID score for the protein more than 15 and overexpression of at least three times across the time points were listed. The Multi Experiment Viewer (MeV, version 4.6.1) software was used to show maps of protein levels corresponding to the expression patterns in Table 1.

Table 1 Overexpression of 37 proteins in the plasma of the hamster CCA Protein name

Biological process

Database ID no.

ID score

NP

Ankyrin repeat domain-containing protein 50 Myosin-Id Exostosin-like 1 (homology to exostosin 1) Rho GTPase-activating protein 25 Multidrug resistance associated protein 3 NADPH oxidase activator 1

Signal transduction Signal transduction Signal transduction Signal transduction Signal transduction Signal transduction

gi|123295151 gi|51100974 gi|20138356 gi|83582811 gi|30984103 gi|74759404

23.8 19.5 19.1 19.0 18.8 18.6

2 19 2 3 4 5

Disabled homolog 2, mitogen-responsive phosphoprotein RhoGEF and PH domain containing 6 Inversin Aggrecanase 1 Fibronectin Kinesin-like protein KIF18A Fascin 3 Plectin isoform 1b A-kinase anchor protein 11 Contactin associated protein 1 Protocadherin 24 Collagen alpha-1(VII) chain Protocadherin beta 15 Apolipoprotein A-I Syntaxin 11 Sodium/glucose cotransporter 5 Anoctamin 8 Syntaxin 18

Signal transduction Signal transduction Signal transduction Signal transduction Structural Structural Structural Structural Structural Structural Structural Structural Structural Transport Transport Transport Transport Transport

gi|149634255 gi|148689626 gi|73949740 gi|17432929 gi|2497975 gi|148612831 gi|73975872 gi|1477646 gi|73989321 gi|148671950 gi|194219569 gi|2326168 gi|119895631 gi|62899898 gi|71043714 gi|59709496 gi|50511029 gi|119602835

17.9 16.1 16.1 15.9 46.0 20.7 16.2 15.6 15.3 20.9 18.9 18.9 17.8 42.6 19.7 17.0 15.9 15.7

2 2 5 2 4 8 2 13 2 2 4 7 2 3 2 2 3 2

T-complex protein 10a Protein kinase/ribonuclease IRE1 beta Zinc finger protein ZNF223 Poly [ADP-ribose] polymerase 1 (PARP-1) MHC class I antigen Interferon-gamma receptor beta chain T-cell receptor beta chain Fibrinogen gamma chain PEST proteolytic signal-containing nuclear protein Huntingtin interacting protein HIP3RP 60S ribosomal protein L29 Glycosyltransferase Heat shock protein 90

Transcription Transcription Transcription Transcription Immune response Immune response Immune response Blood coagulation Cell cycle Apoptosis Translation Metabolism Stress response

gi|227484 gi|12407081 gi|6118383 gi|130782 gi|32306917 gi|114684351 gi|9844142 gi|222985 gi|71480098 gi|242987962 gi|27671155 gi|146284146 gi|31982980

28.7 22.5 17.0 15.1 21.3 17.1 16.5 40.6 23.7 22.3 20.5 17.0 17.0

15 6 8 20 2 2 20 20 6 14 2 22 6

These proteins were selected by an ID score of >15 and increased expression of at least three times across time points in comparison with the control group NP number of peptides matches

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Molecular functions and biological processes of the identified proteins were assigned according to the Uniprot database (http:// www.uniprot.org). The candidate protein was chosen for further immunoblot and immunohistochemical analyses. Western blot analysis Plasma protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, USA). Ten micrograms of plasma protein was separated on a 12 % SDS-PAGE gel and transferred to a polyvinylidenedifluoride membrane (PVDF, Amersham Bioscience, Piscataway, NJ, USA) for 2 h at 60 V. The membrane was incubated overnight at 4 °C with goat polyclonal anti-exostosin 1 (EXT1) antibody (1:500, Santa Cruz, Heidelberg, Germany) diluted in 2 % nonfat dried milk/1× phosphate buffered saline Tween-20 (PBST). Subsequently, the membrane was incubated with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:3,000, GE Healthcare) diluted in 2 % nonfat dried milk/1× PBST. The immunoreactive material was visualized by enhanced chemiluminescence using the ECL Western blotting Detection Reagent (GE Healthcare). The ImageQuant TL software v2005 (1.1.0.1) (Nonlinear Dynamics, Durham, NC, USA) was used for quantitative analysis of each band. Then, the relative band intensity of the OV+NDMA group was compared to that of the normal control group for each time point. Histopathological study, tissue microarray, and immunohistochemistry Hematoxylin and eosin (H & E) staining, tissue microarray of 68 human CCA cases, and immunohistochemistry (IHC) were performed as described previously [8]. The staining density and intensity in hamster tissue was scored as described previously [13]. In brief, the intensity of protein expression was graded as follows: 0, no staining; 1+, mild; 2+, moderate; and 3+, strong. Only moderate and strong staining were considered positive. The staining density was quantified as the percentage of cells stained positively in tissue as follows: 0, no staining; 1, positive staining in 50 %. Intensity score was multiplied with the density score to yield an overall score of 0–9 for each specimen. For tissue microarray of 68 human CCA, cases were scored by the percentage of positive area as follows: negative (
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