Hepatocellular carcinoma (HCC)-related mortality is high because early detection modalities are hampered by inaccuracy, expense and inherent procedural risks. Thus there is an urgent need for minimally invasive, highly specific and sensitive biomarkers that enable early disease detection when therapeutic intervention remains practical. Successful therapeutic intervention is predicated on the ability to detect the cancer early. Similar unmet medical needs abound in most fields of medicine and require novel methodological approaches. Proteomic profiling of body fluids presents a sensitive diagnostic tool for early cancer detection. Here we describe such a strategy of comparative proteomics to identify potential serum-based biomarkers to distinguish high-risk chronic hepatitis C virus infected patients from HCC patients. In order to compensate for the extraordinary dynamic range in serum proteins, enrichment methods that compress the dynamic range without surrendering proteome complexity can help minimize the problems associated with many depletion methods. The enriched serum can be resolved using 2D-difference in-gel electrophoresis and the spots showing statistically significant changes selected for identification by liquid chromatography-tandem mass spectrometry. Subsequent quantitative verification and validation of these candidate biomarkers represent an obligatory and rate-limiting process that is greatly enabled by selected reaction monitoring (SRM). SRM is a tandem mass spectrometry method suitable for identification and quantitation of target peptides within complex mixtures independent on peptide-specific antibodies. Ultimately, multiplexed SRM and dynamic multiple reaction monitoring can be utilized for the simultaneous analysis of a biomarker panel derived from support vector machine learning approaches, which allows monitoring a specific disease state such as early HCC. Overall, this approach yields high probability biomarkers for clinical validation in large patient cohorts and represents a strategy extensible to many diseases.

• Hepatocellular carcinoma
• Biomarkers
• Early detection
• Selected reaction monitoring
• Targeted proteomics

• R01 ES026874 NIEHS NIH HHS

AIM: To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy.

METHODS: Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis (2DE). The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching.

RESULTS: We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein I, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.

CONCLUSION: Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

• Human hepatocarcinoma cell line SMMC-7721
• Protein identification
• Functional analysis
• Heat-shock protein
• Tumorigenesis

• Biomarkers, Tumor/metabolism
• Carcinoma, Hepatocellular/metabolism
• Cell Line, Tumor
• Humans
• Liver Neoplasms/metabolism
• Proteomics

AIM: To analyze the proteome of hepatocellular carcinoma cell lines, MHCC97-H (high metastasis) and MHCC97-L (low metastasis), with different metastasis potentials by two-dimentional liquid phase chromatographic fractionation followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS).

METHODS: Cell lysates of MHCC97-H and MHCC97-L were fractionated according to PI using chromatofocusing with analytical columns in the first dimension, followed by separation of the proteins in each PI fraction using nonporous reversed-phase high performance liquid chromatography (HPLC). A two-dimensional map of the protein content for each cell line based upon pI versus hydrophobicity as detected by UV absorption was generated and a differential display map indicating the presence of up- or down-regulated proteins was exhibited using ProteoVue and DltaVue software. The different proteins were digested by trypsin and MALDI-TOF/MS was performed.

RESULTS: Image analysis of two-dimensional map revealed 72 differentially expressed proteins between MHCC97-H and MHCC97-L cells, and 9 protein peaks (including M2 pyruvate kinase, α-subunit of ATP synthase, heat shock protein 60, Toll-like receptor 9, flavoprotein oxygenase, pro-calreticulin, manganese superoxide dismutase, nm23-H1, and G-protein-coupled receptor kinase 5) were further identified by tryptic digestion, peptide mass fingerprinting and mass spectrometry. Four proteins were up-regulated while 5 were down-regulated in MHCC97-H cells.

CONCLUSION: The identified differentially expressed proteins may play important roles in the metastasis of hepatocellular carcinoma.

• Proteome
• Hepatocellular carcinoma
• Metastasis
• Two-dimentional liquid phase chromatographic fractionation
• Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

AIM: To establish the two-dimensional gel electrophoresis (2-DE) profiles of indomethacin (IN)-treated human colon cancer cell line HCT116, and to provide a new way to study its anti-tumor molecular mechanism through analyzing a variety of protein maps.

METHODS: Two-DE profiles of HCT116 were established in IN-treated and untreated groups. Total proteins were separated by immobilized pH gradient-based 2-DE. The gels were stained by silver, scanned by ImageScanner, and analyzed with Image Master software.

RESULTS: Clear background, well-resolved and reproducible 2-DE patterns of HCT116 cells were acquired in IN-treated and untreated group. The average deviation of spot position was 0.896±0.177 mm in IEF direction and 1.106±0.289 mm in SDS-PAGE direction respectively. In IN-treated group, 1169±36 spots were detected and 1061±32 spots were matched, the average matching rate was 90.6% in three gels. In untreated group, 1256±50 spots were detected and 1168±46 spots were matched, the average matching rate was 93.0% in three gels. Forty-five differential protein spots were displayed between IN-treated and untreated groups. Of which, 34 protein spots decreased and 9 showed higher expression in IN-treated group, and only two protein spots showed an expression in untreated cells.

CONCLUSION: Two-DE profiles of IN-treated and untreated HCT116 cells were established. Apparent 45 different protein spots were detected in IN-treated and untreated HCT116 cells. The analysis on differential protein spots may serve as a new way to study the molecule mechanism of IN-treated colon cancer.

• Gel electrophoresis
• Indomethacin
• Colon cancer

• Antineoplastic Agents/pharmacology
• Cell Division/drug effects
• Cell Line, Tumor
• Colonic Neoplasms/chemistry
• Colonic Neoplasms/drug therapy
• Electrophoresis, Gel, Two-Dimensional/methods
• Electrophoresis, Gel, Two-Dimensional/standards
• Humans
• Indomethacin/pharmacology
• Proteomics/methods
• Reproducibility of Results

AIM: To study the differential expression of proteins in normal and cancerous gastric tissues, and further identify new molecular markers for diagnosis and prognosis of gastric carcinoma, as well as develop new therapeutic targets of the disease.

METHODS: Matched pairs of tissues from 6 gastric cancer patients were analyzed for their two-dimensional electrophoresis (2DE) profiles. Soluble fraction proteins from human normal and cancerous gastric tissue were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG, pH3-10) strips, and by 125 g/L sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension with silver nitrate staining. Protein differential expression was analyzed by use of image analysis software to find out candidates for gastric cancer-associated proteins.

RESULTS: Nine protein spots overexpressed in tumor tissues as compared with noncancerous regions. In the next step, 9 tumor-specific spots were cut off from Coomassie Brilliant Blue staining gels, digested in gel with L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-trypsin. Protein identification was done by peptide mass fingerprinting with matrix assisted laser desorption/ ionization-time of flight-mass spectrometry (MALDI-TOF-MS). In total, 5 tumor-specific protein spots corresponding to 5 different polypeptide chains were identified, including annexin V, carbonic anhydrase, prohibitin, fibrin beta and fibrinogen fragment D. Among these 5 spots, the potential significance of the differential expressions is discussed.

CONCLUSION: Differential expression analysis of proteomes may be useful for the development of new molecular markers for diagnosis and prognosis of gastric carcinoma.

• Biomarkers, Tumor/analysis
• Carcinoma/chemistry
• Electrophoresis, Gel, Two-Dimensional
• Humans
• Neoplasm Proteins/analysis
• Reproducibility of Results
• Stomach Neoplasms/chemistry

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