Serum Cea-negative Colorectal Cancer Patients With Proteomics Study

Background: Colorectal cancer (CRC) is one of the third most common cancer and remains the leading cause of cancer-related mortality worldwide. Therefore,pay more attention to biological characteristics and mechanism of CRC.High-resolution two-dimensional polyacrylamide gel electrophoresis(2-D PAGE) and followed by protein identification using MS,has been used to study a wide variety of cancer biological systems,and has been an important technique for biomarker discovery.Resently, more and more attention has been pay on human sera proteome because of the central role sera plays in clinical diagnostics. Protein concentrations in sera are tightly controlled to balance their physiological functions in areas such as immunity, small molecule transport ,inflammation, and lipid metabolism.Lack of function and out-of-balance concentrations of sera proteins can cause or result from disease processes.To identify biomarkers that improve the diagnosis and lead to possible therapeutic targets in CEA negative CRC patients, we used proteomic approach to compare serum protein expression patterns of CEA negative CRC patients, CEA positive CRC patients with healthy individuals to search for protein biomarkers that may be detected in serum of CEA negative CRC patients. Nowadays, only a few proteins from serum have been described as biomarkers in CRC: carcinoembryonic antigen (CEA), CA19-9 and CA125.Among these biomarkers, CEA is a commonly used as tumor marker and is currently available to assist with monitoring treatment for CRC. However, CEA is not specific for CRC and is not expressed at all colorectal tumors, about one-third of patients with CRC have normal levels. As such, CEA test is not an ideal screening test or detection for CRC, especially for CEA negative CRC patients. Therefore, new and better serum biomarkers of colorectal cancer are needed.Aim: In the current study, to identify potential serum markers for CEA negative colorectal cancer, we used a proteomics approach that combined 2-DE analyses and MALDI-TOF-MS/MS, which represents a promising approach for the identification of useful tumor biomarkers, to examine protein alterations in the serum of CEA negative CRC patients and CEA positive CRC patients compared to healthy controls.Methods: Pooled sera from 11 CEA negative CRC patients, 11 CEA positive CRC patients and 11 healthy individuals as controls were collected, Albumin and IgG removal were carried out and serum total proteins were extracted. Two-dimensional gel electrophoresis (2-DE) was used to isolate the total proteins; colloidal coomassie blue G-250 stain was used to visualize the protein spots. The differential protein spots were identified by MALDI-TOF-MS/MS. The biological information of the proteins was analysis.Western blot were used to validate two of these differentially expressed protein in an independent series of 96 serum samples.Results: 2-DE serum protein profiles were obtained from the patient suffering from colorectal cancers and healthy controls in each group.From optimized 2-DE gel images of the above groups.13 protein spots with more than 2-fold different between the three groups were selected and were identified by MALDI-TOF-MS/MS. Compared with healthy controls, The spots that were down-regulated in CEA negative cancer patients were: sficolin 2 isoform a precursor, ficolin 3; apolipoprotein L1; inter-alpha-trypsin inhibitor heavy chain H4; transthyretin (TTR), one spot was up–regulated and was identified as Ig lambda light chain.Compared with healthy controls, the spots that were down-regulated in CEA positive cancer patients were : ficolin 2 isoform a precursor; ficolin 3; apolipoprotein L1; inter-alpha-trypsin inhibitor heavy chain H4; transthyretin (TTR),the spots that up- regulated were: Apolipoprotein E; haptoglobin. Compared with healthy controls, CEA positive cancer patients, one spot was up–regulated and: Ig lambda light chain. Two differential expression proteins , apolipoprotein L1and haptoglobin were confirmed by western blot.Conclusion: 2-DE-based serum proteome analysis may be useful in the screening of serum biomarkers for CRC cancer. Some proteins of differential expression in CEA negative CRC patients, positive CRC patients and normal subject group have been foud,and the biological function of these proteins were analysid.Apolipoprotein L1 and haptoglobin may be potential biomarkers in the diagnosis of CEA negative CRC patients.