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. 2010 Aug 12:9:215.
doi: 10.1186/1476-4598-9-215.

Serum N-glycome biomarker for monitoring development of DENA-induced hepatocellular carcinoma in rat

Meng Fang  1 , Sylviane Dewaele, Yun-Peng Zhao, Peter Stärkel, Valerie Vanhooren, Yue-Ming Chen, Xin Ji, Ming Luo, Bao-Mu Sun, Yves Horsmans, Anne Dell, Stuart M Haslam, Paola Grassi, Claude Libert, Chun-Fang Gao, Cuiying Chitty Chen
Affiliations

Serum N-glycome biomarker for monitoring development of DENA-induced hepatocellular carcinoma in rat

Meng Fang et al. Mol Cancer. .

Abstract

Background: 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.

Results: 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-alpha-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.

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Figures

Figure 1
Figure 1
Body weight and biochemical tests at different time points after DENA administration (weeks). (A) Body weight (g); (B) serum total bilirubin (TBil, μmol/L); (C) serum alanine aminotransferase (ALT, IU/L); (D) serum aspartate aminotransferase (AST, IU/L); and (E) serum γ-glutamyltransferase (GGT, IU/L). All the values are expressed as mean ± SD on the vertical axis. "Open square" represents the control group and "slash square" represents the DENA group. Asterisks indicate statistically significant differences between the groups (* p < 0.05, **p < 0.01 and ***p < 0.001).
Figure 2
Figure 2
Representative serum desialylated N-glycan profiles in DENA rats. (A) Liver of untreated control rat (left) and liver of DENA treated rat showing multifocal HCC nodules. (B) The four panels (from top to bottom) are typical serum N-glycan fingerprints from control, fibrotic, cirrhotic and HCC rats. Eight major glycan peaks were detected in rat serum, and their patterns in the four rat groups showed considerable differences. The vertical axis represents the glycan values of the peaks as percent relative fluorescence level. The X-axis represents the retention time of N-glycans.
Figure 3
Figure 3
N-glycan values that differ significantly between control, fibrotic, cirrhotic and HCC groups. The vertical axis represents the glycan values of GycoTest markers R5a, R5b, log(R5a/P1), log(R5b/P1) and P5. Error bars represent 95% confidence intervals for means. Statistical significance of differences between groups is indicated by the p value.
Figure 4
Figure 4
A scheme for using the GlycoTest model to monitor progression of HCC in DENA rats. The GlyoTest model includes the following steps: (1) DENA administration, (2) blood sampling during progression of HCC, (3) serum N-glycan profiling using DSA-FACE, and (4) analysis of GlycoTest markers. The GlycoTest markers are as follows: a decrease in P5 indicates fibrosis progression; an increase in R5a and R5b indicates HCC progression; and an elevation of log(R5a/P1) and log(5Rb/P1) points to development of HCC.
Figure 5
Figure 5
Desialylated N-glycan profiles of serum proteins from DENA rats treated with FTS. (A) Liver histology after 16 weeks of treatment with DENA or DENA plus FTS (DENA-FTS). Sirius staining shows that FTS treatment prevented development of cirrhosis. (B) The three groups of rats were the untreated control (top), DENA plus FTS (DENA-FTS) (middle), and DENA alone (bottom). The vertical axis represents the glycan values of the peaks as percent relative fluorescence level. The horizontal axis represents the retention time of N-glycans.
Figure 6
Figure 6
The GlycoTest markers in DENA rats treated with FTS. The levels of GlycoTest markers changed towards non-DENA values after FTS treatment. Error bars represent 95% confidence intervals for means. Statistical significance of differences between groups is indicated by the p value.
Figure 7
Figure 7
Exoglycosidase sequencing of N-glycans from rat serum glycoproteins. The upper panel shows separation of desialylated rat serum N-glycans: desialylated N-glycans digested with bovine kidney α-1,6-fucosidase (middle panel) and desialylated N-glycans digested with almond meal α-1,3-fucosidase (lower panel). Peaks P1, P2, R5a, R5b and P6 contain α-1,6-fucosylated structures. The arrows indicate the changes in the glycan peaks due to glycosidase digestion that are outlined in the dashed squares. The structures of the N-glycan peaks are shown below the panels. P1 is an asialo, agalacto, core-α-1,6-fucosylated biantennary glycan (NGA2F). P2 is an asialo, agalacto, core-α-1,6-fucosylated bisected biantennary (NGA2FB). P5 is an asialo, bigalacto, biantennary glycan (NA2). P6 is an asialo, bigalacto, core-α-1,6-fucosylated biantennary (NA2F). The symbols used in the structural formulas are the following: "black square" stands for N-acetylglucosamine (GlcNAc); "open circle" stands for galactose; "grey triangle" stands for α-1,6-linked fucose; "grey circle" stands for mannose.
Figure 8
Figure 8
Total serum core-fucose residues measured by DSA-FACE. Error bars represent 95% confidence intervals for means. Statistical significance of differences between groups is indicated by the p value.
Figure 9
Figure 9
SDS-PAGE of proteins from serum and liver and western blots probed with AOL. The upper panel shows a western AOL blot of serum proteins (left) and a gel stained with Coomassie Blue (CBB) of the same serum proteins (right). The lower panel shows a western blot of liver proteins probed with AOL and β-actin. The lanes are as follows: 1-2: controls; 3-4: cirrhosis; 5-6: HCC. 2+F, 4+F and 6+F are the proteins in lanes 2, 4 and 6 digested with PNGase F and used as negative controls for AOL binding. The data were reproducible in three independent experiments.
Figure 10
Figure 10
The relative gene expression level of α-1,6-fucosyltransferase (FUT8) in the liver as measured by Q-PCR. The horizontal axis represents the experimental groups: control (n = 6), fibrosis (n = 6), cirrhosis (n = 6) and HCC (n = 6). The vertical axis indicates the expression level of FUT8 relative to the mean of two housekeeping genes. Statistical significance of differences between groups is indicated by asterisks: *** p < 0.001.
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