Abstract
Background: Hepatocellular carcinoma (HCC) is one of the most lethal diseases, and one of the major causes of death in Japan. Our previous research of proteomics for cancerous and paired non-cancerous tissues from patients with HCC with hepatitis C virus infection (HCV-HCC) by means of the combination of two-dimensional gel electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) reported that four of numerous spots of weaker intensity in cancerous tissues than in paired non-cancerous tissues were identified as four isoforms of liver type aldolase (aldolase B). In the present study, two-dimensional (2-D) Western blot analysis demonstrated a significantly lower expression of four isoforms of aldolase B in cancerous than in non-cancerous tissues. Conclusion: Our finding of differences of expression aldolase B isoforms between cancerous and paired non-cancerous tissues for HCV-HCC may be useful for shedding light on some behaviors of aldolase B during hepatocellular carcinogenesis.
Hepatocellular carcinoma (HCC) is one of the world's most lethal diseases (1). Chronic infection with hepatitis C virus (HCV) is the major cause of the rising incidence of HCC in many countries (1, 2). Eighty percent of deaths from HCC are related to HCV infection in Japan (3).
In recent years, with the development of study of the proteome in HCV-related HCC, there has been a great progress in diagnosis and pathogenesis of HCV-HCC. Heat-shock protein 70 family members were identified as biomarkers and play important roles in the pathogenesis of HCV-HCC (5). Proteomic profiling demonstrated that some metabolic enzymes were decreased in cancerous tissues from patients with HCV-HCC compared to paired non-cancerous tissues, such as enoyl-CoA hydratase, aldolase B and arginase 1 (6). Plenty of biomarkers that showed differential expression between cancerous and non-cancerous tissues were found in HCC, by proteomic technologies, and were classified by following HCC with distinct causes (7). Our recent proteomic study of HCVHCC tissues showed eight proteins to be down-regulated in HCC tissues from over 50% of the patients, identified as aldolase B, tropomyosin beta-chain, ketohexokinase, enoyl-CoA hydratase, albumin, smoothelin, ferritin light chain, and arginase 1. In particular, aldolase B was identified as four spots on the two-dimensional electrophoresis (2-DE) gel.
In the present study, we investigated these four spots which were identified as aldolase B by means of 2-D Western blot analysis with anti-aldolase B antibody.
Materials and Methods
Sample preparation. Twenty-two pairs of cancerous and paired non-cancerous liver tissues were obtained from patients with HCV-HCC who had undergone surgical hepatectomy at the Department of Surgery II at Yamaguchi University Hospital. None of the patients received any preoperative therapy. Written informed consent was obtained from all patients prior to surgery. The study protocol was approved by the Institutional Review Board for Human Use of the Yamaguchi University School of Medicine. Specimens were resuspended and homogenized in lysis buffer (1% NP-40, 1mM sodium vanadate, 1 mM PMSF, 10 mM NaF, 10 mM EDTA, 50 mM Tris, 165 mM NaCl, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) on ice (8). Suspensions were incubated for 2 h at 4°C, and centrifuged at 15 000 × g for 30 min at 4°C, then the supernatants were extracted and stored at −80°C until use.
2-DE. Eighty micrograms of protein was used for each electrophoretic run. Sample was mixed with 200 μl of rehydration buffer [8 M urea, 2% 3-(3-cholamidopropyl) dimethylammonio-1-propanesulfonate (CHAPS), 0.01% bromophenol blue, 1.2% Destreak reagent (GE Healthcare, Bukinghamshire, UK) and 0.5% IPG buffer (GE Healthcare). Rehydration was performed in an IPGphor 3 Isolelectric Focusing (IEF) unit (GE Healthcare) on 11 cm, immobilized pH 3-10 linear gradient IPG strips (Bio-Rad, Hercules, CA, USA) at 50 μA/strip. IEF was performed as following conditions: rehydration for 10 h; 0 to 500 V for 4 h; 500 to 1000 V for 1 h; 1000 to 8000 V for 4 h; 8000 V for 20 min; and the final phase of 500 V from 20000 to 30000 Vh. The strips were subsequently equilibrated in equilibration buffer 1 [6 M urea, 0.5 M Tris-HCl pH 8.8, 30% glycerol, 2% sodium lauryl sulfate (SDS), 2% 2-mercaptoethanol (2-ME)] for 10 min and then continued in equilibration buffer 2 (6 M urea, 0.5 M Tris-HCl pH 8.8, 30% glycerol, 2% SDS, 2.5% iodoacetoamide) for another 10 min. The strips were then transferred onto gels and run at 200 V (9).
Image analysis and spot picking. The SDS-PAGE gels were incubated with solution containing 40% ethanol and 10% acetic acid for 2.5 h. The gels were then stained with a fluorescent gel staining, Flamingo™ Fluorescent Gel Stain (Bio-Rad) overnight (10). The gels were scanned by using a ProEXPRESS 2 D Proteomic Imaging System (PerkinElmer, Waltham, MA, USA) and then analyzed by using Progenesis Samespots software (Nonlinear, Newcastle, upon Tyne, UK). After image analysis, the gels were stained with See Pico™ (Benebiosis, Seoul, Korea) overnight (11). Protein spots were picked out from each gel and immersed in 70 μl ultra-pure water.
In-gel digestion. The See Pico™ dye was removed by washing three times in 60% methanol, 0.05 M ammonium bicarbonate, and 0.005 M DL-dithiothreitol (DTT) for 15 min. The sample in the gel piece was reduced twice in 50% methanol, 0.05 M ammonium bicarbonate, and 0.005 M DTT for 10 min. The gel pieces were dehydrated twice in 100% acetonitrile (ACN) for 30 min and incubated with an in-gel digestion reagent containing 10 μg/ml sequencing-grade-modified trypsin (Promega, Madison, WI, USA) in 30% ACN, 0.05 M ammonium bicarbonate, and 0.005 M DTT at 30°C overnight. The samples were lyophilized overnight with the use of Labconco Lyph-lock 1L Model 77400 (Labconco, Kansas, MO, USA) (12).
LC-MS/MS. The lyophilized samples were dissolved in 30 μl formic acid and centrifuged at 15,000 × g for 5 min. Peptide sequencing of identified protein spots was performed by using an Agilent 1100 LC-MSD Trap XCT (Agilent Technologies, Palo Alto, CA, USA). Fifteen microliters of each sample was injected and separated on a Zorbax 300SB-C18 column (75 μm, 150 mm; Agilent Technologies). The Agilent 1100 capillary pump worked with the following conditions: solvent A, 0.1% formic acid; solvent B, ACN in 0.1% formic acid. Column flow, 0.3 μl/min; primary flow 300 μl/min. Gradient, 0-5 min 2% B, 60 min 60% B. Stop time: 60 min. Proteins were identified in an Agilent Spectrum Mill MS proteomics workbench against the Swiss-Prot protein database search engine (http://kr.expasy.org/sprot/) and MASCOT MS/MS Ions Search engine (http//www.matrixscience.com/search_form_select.html). Standard for induction of candidate proteins were set as follows: filter by protein score >10.0, and filter peptide by score >8. The Spectrum Mill workbench searched MS/MS spectra using an MS/MS ion search (13, 14).
2-D Western blot analysis. Four pairs of samples were separated on 2-DE gels and then transfer onto polyvinylidene fluoride (PVDF) membranes (Immobilon; Millipore, Bedford, MA, USA) at 90 mA for 78 min. The membranes were blocked overnigh with Tris-buffered saline (TBS) containing 5% milk at 4°C (8). Membranes were incubated with the primary antibody against aldolase B (Anti-aldolase B goat polyclonal antibody 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were incubated with the secondary antibody conjugated with horseradish peroxidase (1:10,000) for 1 h at room temperature after washing three times with TBS containing Tween-20 and once with TBS. Membranes were then treated with a chemiluminescent reagent (ImmunoStar Long Detection, Wako, Osaka, Japan) and imaged using an Image Reader LAS-1000 Pro (Fujifilm Corporation, Tokyo, Japan).
Results
Detection of protein spots on 2-DE gels. Twenty-two pairs of cancerous and paired non-cancerous tissues from patients with HCV-HCC were analyzed by 2-DE. Hundreds of protein spots were visualized on 2-DE gels. Four spots of down-regulated proteins (spots 1-4) were revealed on 2-DE gels of cancerous and paired non-cancerous tissues with a >1.5-fold difference in intensity (Figure 1).
Identification of proteins by LC-MS/MS. The four spots were digested and analyzed by MS. Each spot provided a good spectrum of amino acids by analysis with LC-MS/MS. Aldolase B isoforms (spots 1-4) showing underexpression in cancerous tissues compared to paired non-cancerous tissues on 2-DE gels were identified and listed in Table I. MS and MS/MS spectra of trypsin-digested spot 2 are shown in Figure 2.
2-D Western blot analysis of aldolase B isoforms. Four pairs of cancerous and paired non-cancerous tissues from patients with HCV-HCC were analyzed by 2-D Western blot analysis with specific anti-aldolase B antibody. The spots were confirmed as four aldolase B isoforms (Figure 3A). The differential intensities of protein spots were quantified (Figure 3B) and the four proteins were found to be significantly down-regulated (p<0.05) in cancerous tissues compared to non-cancerous tissues.
Discussion
Fructose-1,6-(bis)phosphase aldolase exists as three isozymes with different tissue distributions: aldolase A (muscle and red blood cell), aldolase B (liver, kidney and small intestine), and aldolase C (brain and neuronal tissues) (15). Aldolase B catalyzes the reversible cleavage of fructose-1,6-(bis) phosphate (FBP) and fructose-1-phosphate (F1P) to dihydroxyacetone phosphate and either glyceraldehyde-3-phosphate or glyceraldehyde, respectively (16). Not only that, but aldolase B is also involved in the two opposite metabolic pathways, glycolysis and gluconeogenesis (17). The mRNA of aldolase B was down-regulated in HCC patients and its protein expression was shown to be down-regulated in cancerous tissue compared with surrounding non-cancerous tissues in HCV-HCC (6, 18). However, aldolase A was up-regulated in hepatoma cell lines and cancerous tissues from patients with HCC compared to surrounding non-cancerous tissues (19, 20). Because aldolase A seems to be customized for a glycolytic role, while aldolase B may function mainly in glyconeogenesis (17), this kind of displacement between aldolase A and B in cancerous tissues in HCC may be involved in the imbalance of glycometabolism in patients' liver tissues (21, 22), leading to tumor progression for HCC.
Down-regulated expression of four proteins in HCC. Four protein spots (numbered 1-4) were found to be differentially expressed on 2-DE gels of proteins from samples of cancerous and paired non-cancerous tissues.
Identification of the protein spots in cancerous tissues.
In this study, aldolase B was identified as existing in four isoforms in liver tissues from patients with HCV-HCC, and 2-D Western blotting demonstrated that protein expression of the four isoforms of aldolase B was significantly down-regulated in cancerous compared to paired non-cancerous tissues. However, the relationships between functional mechanisms and post-translational modifications (PTMs) of aldolase B isoforms in liver tissue remain unclear. The cognate aldolase B mRNA encodes a 364 amino acid protein with a molecular weight of 39.3 kDa (23). It has strictly conserved residues in the active site consisting of Asp33, Arg42, Lys107, Lys146, Glu187, Ser271, Arg303 and Lys229 (24). Arg303 and Arg42 residues are involved in one of the two alternate C6-binding sites (25). Lys146, a residue required for carbon–carbon bond cleavage, is able to perturb the pKa values of important residues in catalysis, such as the Schiff-base Lys229 and general acid/base catalyst Asp33, towards neutrality (24, 26). The subtle changes in position of Lys146 may alter the reactivity of these essential residues (27). Lys146 and Glu187 are directly bound to the product of the catalytic reaction (DHAP) (28). Presumably, aldolase B isoforms in liver tissue may be associated with the PTMs of these functional positions. Despite that less than 22% of a full length sequence of aldolase B was analyzed by using an MS system (shown in Table I), no interesting differences of PTMs among four isoforms of aldolase B were found. Therefore, in order to understand the effects of aldolase B isoforms on occurrence and development of HCV-HCC, the key PTMs among them should be identified in further study. Accordingly, it is necessary to improve quantity and purity of protein samples for analysis by LC-MS/MS. In addition, aldolase not only plays an essential role in glycometabolism, but also interacts with macromolecules unrelated to the glycometabolic pathway, such as F-actin (29), cell surface adhesins (30), RNA (31), tubulin (32) and liver cytoskeleton (33). Therefore aldolase B isoforms might be involved in tumor progression in a glycometabolism-independent manner.
MS and MS/MS spectra of trypsin-digested spot 2. A: LC-MS spectra of trypsin-digested of spot 2; aldolase B precursor ion m/z is 622.4. B and C: LC-MS/MS spectrum of a precursor ion with m/z 622.4 marked by a diamond in (A). The MS/MS spectrum identifies the partial tryptic peptide [R]ALQASALAAWGGK[A] from aldolase B processed with a spectrum Mill workbench.
Conclusion
Proteomic technology, such as 2-DE and MS analysis, has been widely employed in the research of discovery of biomarkers in diseases, especially in cancer. Differences of expression of aldolase B isoforms between cancerous and paired non-cancerous tissues were identified in HCV-HCC by those of technologies in current study. Simultaneously, four isoforms of aldolase B were identified and quantified by performing 2-D Western blotting and statistical analysis. These findings may be useful for shedding light on some behaviors of aldolase B during hepatocellular carcinogenesis. In order to assess the function of aldolase B in HCV-HCC, we will focus on finding the key PTMs among four isoforms of aldolase B in further study.
2-D Western blot analysis of aldolase B. A: Four pairs of cancerous and paired non-cancerous tissues from patients with HCV-HCC underwent 2-D Western blot analysis. Using specific anti-aldolase B antibody, the spots were confirmed as four aldolase B isoforms. Each spot number is the same as those shown in Figure 1A. B: The differential intensities of protein spots in non-cancerous tissues (N) and cancerous tissues (C) in HCC were quantified by Student's t-test (n=4, p<0.05 for each spot). The error bars represent standard errors of the means.
Acknowledgements
This work was supported in part by Grants-in-Aid from the Ministry of Health, Labour and Welfare of Japan (No. H20-Bio-005 to Kazuyuki Nakamura).
- Received June 8, 2011.
- Revision received July 13, 2011.
- Accepted July 14, 2011.
- Copyright © 2011 The Author(s). Published by the International Institute of Anticancer Research.
