Serum N-glycome biomarker for monitoring development of DENA-induced hepatocellular carcinoma in rat
© Fang et al. 2010
Received: 9 April 2010
Accepted: 12 August 2010
Published: 12 August 2010
There is a demand for serum markers for the routine assessment of the progression of liver cancer. We previously found that serum N-linked sugar chains are altered in hepatocellular carcinoma (HCC). Here, we studied glycomic alterations during development of HCC in a rat model.
Rat HCC was induced by the hepatocarcinogen, diethylnitrosamine (DENA). N-glycans were profiled using the DSA-FACE technique developed in our laboratory.
In comparison with control rats, DENA rats showed a gradual but significant increase in two glycans (R5a and R5b) in serum total N-glycans during progression of liver cirrhosis and cancer, and a decrease in a biantennary glycan (P5). The log of the ratio of R5a to P1 (NGA2F) and R5b to P1 [log(R5a/P1) and log(R5b/P1)] were significantly (p < 0.0001) elevated in HCC rats, but not in rats with cirrhosis or fibrosis or in control rats. We thus propose a GlycoTest model using the above-mentioned serum glycan markers to monitor the progression of cirrhosis and HCC in the DENA-treated rat model. When DENA-treated rats were subsequently treated with farnesylthiosalicyclic acid, an anticancer drug, progression to HCC was prevented and GlycoTest markers (P5, R5a and R5b) reverted towards non-DENA levels, and the HCC-specific markers, log(R5a/P1) and log(R5b/P1), normalized completely. Conclusions: We found an increase in core-α-1,6-fucosylated glycoproteins in serum and liver of rats with HCC, which demonstrates that fucosylation is altered during progression of HCC. Our GlycoTest model can be used to monitor progression of HCC and to follow up treatment of liver tumors in the DENA rat. This GlycoTest model is particularly important because a rapid non-invasive diagnostic procedure for tumour progression in this rat model would greatly facilitate the search for anticancer drugs.
DNA sequencer-assisted (DSA) and fluorophore-assisted carbohydrate electrophoresis (FACE)
- PNGase F:
peptide N-glycosidase F
bigalacto biantennary glycan
bigalacto core-α-1,6-fucosylated biantennary
Aspergillus oryzae lectin
Coomassie Brilliant blue
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related death in the world . Infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) is the major etiologic factor for HCC . A strong correlation exists between cirrhosis and hepatocarcinogenesis, and most patients with HCC have pre-existing cirrhosis. Chronic hepatitis virus infection commonly leads to fibrosis followed by cirrhosis and finally hepatocellular carcinoma. The diagnosis and certainly the follow-up of liver diseases such as cirrhosis and HCC remains a heavily debated problem [3–6]. Alpha-fetoprotein (AFP) is commonly used as a serum biomarker, but its level is normal in one-third of HCC patients [7, 8]. The gold standard for diagnosis of HCC is histopathological examination of liver biopsy. Thus, a sensitive and specific non-invasive serological marker is needed for the early diagnosis of HCC and for monitoring its treatment.
Abnormal protein glycosylation is associated with malignant transformation of cells. The N-linked sugar chains are altered in various tumors, and certain glycan structures are well-known markers of tumor progression [9–12]. Most serum N-linked glycoproteins are synthesized by the liver or by B-lymphocytes, and any changes in serum total N-glycans could reflect an alteration of liver or B-lymphocyte physiology. Thus, changes in the quantity and type of N-glycans in serum could be utilized for the non-invasive diagnosis of liver diseases. We and others have reported that N-glycan profiles generated by DSA-FACE (DNA Sequencer Assisted - Fluorophore Assisted Carbohydrate Electrophoresis) could be used as a non-invasive marker for HCV-related liver cirrhosis [13, 14], HBV-related fibrosis , and HBV-related HCC [16, 17]. Our previous study also showed that combining serum N-glycan markers with AFP improves the efficacy of diagnosing HBV-related HCC .
Investigations of hepatocarcinogenesis make use of animal models. HCC can be induced in laboratory animals by various chemicals, such as aflatoxin B1, 2-acetylaminofluorene (2-AAF) and diethylnitrosamine (DENA) [19–21]. In this study, we injected rats with DENA, which causes the sequence of fibrosis, cirrhosis and HCC. By monitoring N-glycomic profiles after DENA injection, we identified serum N-glycan biomarkers that can be used to monitor liver fibrosis, cirrhosis and HCC. These serum glycan markers were validated in a rat model in which tumor development is prevented by using an antitumor drug (S-trans-trans-farnesylthiosalicyclic acid; FTS).
Alteration of the Profiles of Serum Protein N-glycans during the development of HCC
Histological diagnosis of liver tissue from rats sacrificed at different time point
Histological diagnosis (no. animals)
Evaluation of serum glycan biomarkers after FTS treatment to prevent HCC
Increased level of core-α-1,6-fucosylation during progression of HCC
The N-glycan structures for the peaks in humans have been described . Overlay of the rat profiles with human serum N-glycan profiles revealed that P1 (NGA2F), P2 (NGA2FB), P5 (NA2) and P6 (NA2F) migrated at the same rates as the corresponding human structures during the CE (Additional file 2, Figure S2). However, peaks R3, R4, R5a and R5b were not found in human serum, which indicates that N-glycosylation is somewhat species-specific. This was confirmed by mass spectrometric glycomics profiling of serum from control and DENA rats. As shown in Additional file 3, Figure S3 the rat serum contains some glycans whose antennae are terminated with alpha galactose moieties. These glycans cannot be biosynthesized in humans [24, 25].
Alteration of the expression level of α1,6-fucosyltransferase (FUT8) in the liver of DENA rats
Injection of DENA in rats led to development of cirrhosis followed by HCC. N-glycan analysis of rat serum showed that levels of N-glycans R5a and R5b were positively correlated with progression of fibrosis and HCC in DENA rats, whereas P1 (NGA2F) and P2 (NGA2FB) first increased with severity of fibrosis and then significantly dropped after development of HCC. Log(R5a/P1) and log(R5b/P1) were tumor specific markers that were significantly elevated in HCC but not in DENA-induced fibrosis and cirrhosis. Moreover, the value of P5 was negatively correlated with fibrosis degree, which makes it a specific marker for progression of liver cirrhosis in DENA rats. We propose that the GlycoTest model involving the above-mentioned serum glycan markers can be used for monitoring the progression of cirrhosis and HCC in the DENA rat model.
We also evaluated the GlycoTest model in DENA rats treated with the anticancer drug, FTS. A previous study demonstrated that administration of DENA leads to formation of numerous whitish HCC nodules over the surface of the liver as well as to the development of cirrhosis, and that FTS treatment prevents both lesions . As expected, the GlycoTest markers for cirrhosis and HCC progression (P5, R5a and R5b) in the FTS treated rats changed towards non-DENA levels. This change correlated with the prevention of progression to cirrhosis and HCC that was confirmed macroscopically and by histology. The GlycoTest for HCC-specific markers, log(R5a/P1) and log(R5b/P1), completely reverted to non-DENA levels in the FTS treated DENA rats, thereby indicating complete prevention of HCC formation. Thus, we demonstrate that our GlycoTest model can be used as a non-invasive biomarker for monitoring progression of cirrhosis and HCC in DENA rats. The GlycoTest model can also be used to screen drug candidates for protective effects against HCC.
Increased levels of fucosylation have been reported in cancer, such as increased core-fucosylation of AFP in HCC [27, 28], and increased fucosylation of haptoglobin in ovarian, lung, breast and pancreatic cancer [29, 30]. Strong expression of α-1,6-FucT was observed in 3'-MeDAB-induced rat hepatomas and some rat hepatoma cell lines . As recently reviewed by Miyoshi et al. , fucosylation is one of the most important types of sugar modification on glycoproteins in cancer. In agreement, our lectin blots showed increased fucosylation of N-glycans on total proteins in serum and liver in cirrhosis and HCC in the DENA rats. The total amounts of serum core-fucosylated structures were increased also during progression of HCC, partly due to alteration of the core fucosylated N-glycans of R5a, R5b, P1 (NGA2F), P2 (NGA2FB) and P 6 (NA2F). As a consequence, P5 (NA2), which is a precursor for core-fucosylation of biantennary glycan, was significantly decreased. N-glycans are synthesized in the ER and Golgi by the sequential addition of saccharides, such as fucose, by the corresponding glycosyltransferases [33, 34]. Changes in the N-glycan profile in serum could be related to changes in the expression levels of glycosyltransferases in hepatocytes . Basically, core-fucosylation is regulated by core-fucosyltransferase (FUT) and the GDP-L-fucose synthetic pathway. Based on sugar donor specificities, FUT transfers the substrate of GDP-L-fucose to the reducing terminal GlcNAc of the core structure of an asparagine-linked oligosaccharide. GDP-L-fucose is synthesized through a de novo pathway, a dominant pathway that involves conversion of cellular GDP-D-mannose to GDP-L-fucose by GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (FX). Noda K et al.  described the relationship between elevated FX expression, increased production of GDP-L-fucose, and high levels of fucosylation in human hepatocellular carcinoma and hepatoma cell lines. Alpha-1,6-fucosyltransferase (FUT8) is the only fucosyltransferase involved in core-fucosylation in mice . However, we did not detect increased expression of Fut8 gene in DENA rats, which might indicate that another yet unknown alpha-1,6-fucosyltransferase is involved. The mechanism behind the alteration of fucosylation during HCC development is not fully understood. It has been reported that overexpression of α-1,6-FucT in hepatoma cells suppressed intrahepatic metastasis after splenic injection in athymic mice, partly by a decrease in the adhesion of core fucosylated α5β1 integrin to fibronectin . Recently, the function of core-fucosylation was associated with EGFR. The EGF-induced phosphorylation of EGFR is substantially blocked in Fut8 KO cells, and the binding of EGF to its receptor (EGFR) requires core fucosylation of N-glycans . Diverse lines of evidence indicate that activation of the TGF-α/EGFR pathway contributes to HCC formation. It has been reported that the levels of TGF-α and EGFR mRNA increase at different stages of cirrhosis and HCC in the DENA rat model, and that blocking EGFR activity prevents development of HCC in DENA rats . Increased core-fucosylation in HCC might be associated with enhanced core-fucosylation of EGFR, and thus with activation of EGF-induced phosphorylation of the EGFR pathway.
We demonstrate that serum N-glycan markers can be used to monitor progression of HCC and to evaluate drugs in the DENA rat model. This GlycoTest model is particularly important because a rapid non-invasive diagnostic procedure for tumour progression in this rat model would greatly facilitate the search for anticancer drugs. Although the molecular mechanism by which N-glycosylation is altered in liver disease remains unsolved, our study confirms that abnormal fucosylation is important in HCC development, and that fucosylated glycoproteins could be exploited effectively for diagnosis of HCC in the DENA rat model.
Experiment design 1
Male Sprague-Dawley rats weighing 180-220 g were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. and housed in cages under standard animal laboratory conditions in the experimental animal center of the Second Military Medical University. The rats (n = 60) were intraperitoneally injected with DENA at 20 mg/kg body weight three times a week for 14 weeks. The rats in the control group (n = 24) were injected with an equivalent volume of physiological saline at the same times. All animals were kept in a temperature controlled environment with a 12-h light-dark cycle and had free access to food and water throughout the study. To monitor tumor development, the rats were sacrificed at different times during and after DENA administration. Blood samples were collected for serum N-glycan analysis and several biochemical liver function tests. Liver tissues were fixed in 10% formalin for histological examination or frozen in liquid nitrogen and stored at -80°C for molecular biological study. All animal care and experimentation conformed to the guidelines established by the Second Military Medical University.
Experiment design 2
It has been reported that trans-farnesylthiosalicyclic acid (FTS) prevents formation of DENA-induced HCC in rats . Therefore, FTS-treated animals could be used as a validation group for HCC prevention. In brief, male Wistar rats (body weight 180-190 g) received weekly intraperitoneal injections of DENA at 50 mg/kg body weight for 16 weeks (DENA group). FTS was administered intraperitoneally at 5 mg/kg body weight three times a week starting six weeks after DENA induction (DENA-FTS group). The animals were sacrificed one week after the last DENA injection.
The liver tissues were fixed with 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). Sirius red stain was used to detect fibrosis. Liver fibrosis was staged histologically by an experienced hepatopathologist using Scheuer's classification, which uses a scale from F0 to F4: F0, no fibrosis; F1, enlarged fibrotic portal tracts; F2, periportal or portal-portal septa, but intact architecture; F3, fibrosis with architectural distortion, but no obvious cirrhosis; F4, cirrhosis [39, 40].
Routine biochemical tests on sera, including total bilirubin (TBil), alanine aminotransferase activity (ALT), aspartate aminotransferase activity (AST) and γ-glutamyltransferase (GGT), were done using the standard methods (Hitachi 7600 Analyzer, Hitachi, Japan Wako Diagnostics reagents, Wako Pure Chemical Industries Ltd., Japan).
N-glycan profiling using DSA-FACE
N-glycan profiles were generated from 2 μl serum samples by DSA-FACE technology by using a capillary electrophoresis (CE)-based ABI3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) as previously described [16, 41]. Total N-linked glycans were released from serum proteins by digestion with peptide N-glycosidase F (PNGase F) (New England Biolabs, MA, USA), which cleaves most common mammalian N-linked oligosaccharides at the N-glycosidic linkage. N-glycans with reducing ends were derivatized with APTS (Molecular Probes, Eugene, OR, USA) and directly separated using the CE-based DNA sequencer ABI 3130. Because most serum glycans have a terminal sialic acid, which contains a negative charge, removal of sialic acid with hydrolyzing exoglycosidase (a sialidase from Arthrobacter ureafaciens) (Roche, Indianapolis, IN, USA) makes the glycan profile much clearer. The heights of the peaks were measured by GeneMapper version 3.7 software (Applied Biosystems), and the height of each peak was converted to a percent of the total of all peaks.
Structure analysis of N-glycans using exoglycosidase digestion
One microliter of APTS-labeled desialylated N-glycan obtained from the DSA-FACE procedure was digested overnight with various mixtures of exoglycosidases in 10 mM NH4Ac (pH 5.0) at 37°C. The products were then subjected to electrophoresis on an ABI 3130. The exoglycosidases were α-1,3/4-Fucosidase (almond meal) (Glyko, Novato, CA, USA), which cleaves non-reducing terminal α-1,3/4-linked fucose, and α-1,6-Fucosidase (bovine kidney) (Glyko, Novato, CA, USA), which cleaves non-reducing terminal α-1,6-linked fucose.
MS and MS/MS Analyses of Permethylated Glycans
Approximately 1 μL of rat serum sample was desialylated with hydrolyzing exoglycosidase (a sialidase from Arthrobacter ureafaciens) (Roche, Indianapolis, IN, USA). And then desialylated serum was subjected to reduction, carboxymethylation, and tryptic digestion: the sample was reduced in 1 ml of 50 mm Tris-HCl buffer, pH 8.5, containing 2 mg/ml dithiothreitol. Reduction was performed at 37°C in a water bath for 1 h. Carboxymethylation was carried out by the addition of iodoacetic acid (5-fold molar excess over dithiothreitol), and the reaction was allowed to proceed at room temperature in the dark for 1.5 h. Carboxymethylation was terminated by dialysis against 4 × 4.5 liters of 50 mm ammonium bicarbonate, pH 8.5, at 4°C for 48 h. After dialysis, the sample was lyophilized. The reduced carboxymethylated proteins were then digested with N-p-tosyl-l-phenylalanine chloromethyl ketone-pretreated bovine pancreas trypsin (EC 220.127.116.11; Sigma) for 16 h at 37°C in 50 mm ammonium bicarbonate buffer, pH 8.4. The products were purified by C18 Sep-Pak® (Waters) as described previously .
Peptide N-glycosidase F digestion of the tryptic glycopeptides was carried out in 50 mM ammonium bicarbonate, pH 8.5, for 20 h at 37°C with 5 units of enzyme (Roche Applied Science, U.K.). The released N-glycans were purified from O-glycopeptides and peptides by chromatography on a Sep-Pak C18 cartridge (Waters Corp., Milford, MA) and subsequently methylated using the sodium hydroxide permethylation procedure as described previously .
MALDI-TOF data were acquired on a Voyager-DE STR mass spectrometer (Applied Biosystems, Foster City, CA) in the reflectron mode with delayed extraction. Permethylated samples were dissolved in 10 μl of 70% (v/v) aqueous methanol, and 1 μl of the dissolved sample was premixed with 1 μl of matrix (20 mg/ml 2,5-dihydroxybenzoic acid in 80% (v/v) aqueous methanol), spotted onto a target plate, and dried under vacuum.
Further MS/MS analyses of peaks observed in the MS spectra were carried out using a 4800 MALDI-TOF/TOF (Applied Biosystems) mass spectrometer in positive ion mode. Signals selected for MS/MS were (M + Na)+ molecular ions.
The collision energy was set to 1 kV, and argon was used as collision gas. Samples were dissolved in 10 μL of methanol, and 1 μL was mixed at a 1:1 ratio (v/v) with 2,5-dihydroxybenzoic acid (20 mg/mL in 70% methanol in water) as matrix.
The MS and MS/MS data were processed using Data Explorer 4.9 Software (Applied Biosystems, U.K.). The mass spectra were baseline corrected (default settings) and noise filtered (with correction factor of 0.7), and then converted to ASCII format. The processed spectra were then subjected to manual assignment and annotation with the aid of a glycobioinformatics tool known as GlycoWorkBench .
Peak picking was done manually, and proposed assignments for the selected peaks were based on molecular mass composition of the 12C isotope together with knowledge of the biosynthetic pathways. Sequence ambiguities occurring in proposed structures were resolved by data obtained from MS/MS experiments.
Western lectin blot
A total of 25 μg serum proteins or 50 μg proteins extracted from frozen liver tissues were separated by electrophoresis in 10% sodium dodecyl sulfate-polyacrylamide gel. The proteins were then transferred to a nitrocellulose membrane for lectin blot analysis. The membranes were blocked overnight at 4°C with 3% bovine serum albumin (BSA) in Tris-buffered saline (140 mM NaCl, 10 mM Tris-HCl, TBS) and then incubated for 1 h at room temperature with 5 μg/ml of biotinylated Aspergillus oryzae L-fucose-specific lectin (AOL) (Funakoshi Co., Ltd., Tokyo, Japan) in TBST buffer (TBS containing 0.05% Tween 20). After four washes of 5 min each with TBST, the membranes were incubated with 1/1000 diluted horseradish peroxidase-conjugated Streptavidin (R&D Systems, Minneapolis, MN, USA) for 1 h at room temperature. The membranes were washed four times again with TBST and developed with an ECL system (Amersham Biosciences Inc., Piscataway, NJ, USA).
RNA extraction and real-time RT-PCR
RNA was extracted from frozen liver tissues with Qiagen RNeasy mini kit according to the manufacturer's instructions (QIAGEN GmbH, Hilden, Germany). The purity and concentration of RNA were determined by a Nano Drop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). cDNA was synthesized starting from 2 μg of total RNA by using iScript cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, California, USA).
PCR primer pairs used in the present study
Rattus Norvegicus Gene
Forward Primer (5'-3')
Reverse Primer (5'-3')
Amplicon Length (bp)
All quantitative variables are presented as mean ± SD. The unpaired t-test for comparison of two groups was used, and Welch's correction was applied if the assumption of equal variance was not accepted. For comparison of more than two groups, one-way ANOVA was performed. All reported p-values are two-tailed, and p-values < 0.05 are considered statistically significant. Statistical analysis was performed with SPSS 15.0 software (SPSS, Chicago, IL, USA). The coefficients of variation (CVs) for peak intensity were averaged in Microsoft Excel.
We thank Dr. Amin Bredan for editing the manuscript. This work was supported by research grant from Ghent University (BOF No. 01106205), Flanders-China Bilateral project (011S605 and BL/02/C42), Natural Science Foundation of China (NSFC No. 30971345, 30571774), Science and Technology Commission of Shanghai (No. 064119522, 09XD1405800), Astra-Zeneca Belgium, Roche Belgium and the Fondation St. Luc Belgium. The mass spectrometry work was supported by the Biotechnology and Biological Sciences Research Council (BBF0083091) and Marie Curie Initial Training Network, EuroglycoArrays Project, part of the FP7 People Programme.
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