Pancreatic Cancer Serum Differential Proteomics

Background: Pancreatic carcinoma has become one of the most commonmalignant tumors in the digestive tract and has the lowest survival ratecompared to any other solid cancer. During the last 2 decades, the incidence ofpancreatic carcinoma in the world is rising. However, the 5-year survival ofpatients with pancreatic carcinoma has not improved significantly. Currently,there are lack of markers for early detection and targets for therapy. Theprocess of cancer initiation, progression, and formation of metastasis are notsufficiently understood. The proteomic approach has offered manyopportunities for identifying new tumor markers and therapeutic targets, aswell as well understanding pathogenesis of the disease.Objectives: 1. Using the two-dimensional gel electrophoresis (2-DE)technical to analysis the serum proteome of pancreatic carcinoma patients,gastric cancer patients, benign pancreatic disease patients, and healthyvolunteers. The technology of mass spectrometry and the methods ofbioinformatics were combined to identify the differentially expressed proteinsin pancreatic carcinoma. 2. Two newly screened proteins (cyclin-Ⅰand RabGDP dissociation inhibitorβ) were validated to find possible candidatebiomarkers for early diagnosis of pancreatic carcinoma and to gain new insightinto the pathological mechanisms of tumor formation and development.Methods: 1.Serum samples from 16 pancreatic carcinoma patients, 16gastric carcinoma patients, 16 benign pancreatic disease patients, and 16 healthy volunteers were obtained. Serum samples of the same group werepooled, and then purified by albumin and immunoglobulin G (IgG) depletion.After concentrated and desalted, pooled serum samples were separated by2-DE. Protein spots of pancreatic carcinoma pooling group was compared withthat of other three pooling groups by 2-DE analysis software, respectively. Thecommon differential protein spots which were up-expressed in sera frompancreatic carcinoma patients were selected and analysised by matrix-assistedlaser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS). Peptide mass fingerprints (PMFs) obtained by theMALDI-TOF-MS analysis were used to search SWISS-PROT database byusing Mascot software. 2.Western blot and immunohistochemical analysiswere used to validate cyclin-Ⅰand Rab GDP dissociation inhibitorβ(Rab GDIβ). Sera from 12 pancreatic carcinoma patients, 12 benign pancreatic diseasepatients were used for Western blot analysis of cyclin-Ⅰ. Sera from 18pancreatic carcinoma patients, 18 benign pancreatic disease patients, 18healthy volunteers and 10 calculus or benign stricture of bile duct patients, andpancreatic juice from 10 pancreatic carcinoma patients, 10 calculus or benignstricture of bile duct patients were used for Western blot analysis of Rab GDIβ.Expression of cyclin-I and Rab GDIβwere also observed in an additional 36pancreatic carcinoma, 9 benign pancreatic disease, and one healthy humanpancreas by immunohistochemistry.Results: 1.2-D maps with a high reproducibility were obtained. Nine common differential protein spots which were up-expressed in sera frompancreatic carcinoma patients were identified by MALDI-TOF-MS. Twospots were cyclin-Ⅰand other four were Rab GDIβ,α-1-antitrypsin precursor,haptoglobin precursor, serotransferrin precursor, respectively. 2. Western blotanalyses showed that four of twelve carcinomas, and five of twelve benignpancreatic disease sera had high levels of cyclin-Ⅰexpression. However,almost all of the twelve carcinomas with cyclin-Ⅰexpression evinced eitherprocessed protein bands or degraded protein fragments, which were absent incontrol sera. Western blot analyses showed the detection of Rab GDIβin100％sera and 80％pancreatic juice from pancreatic carcinoma patients, 50％benign pancreatic disease sera, and 30％sera and none of pancreatic juicefrom calculus or benign stricture of bile duct patients, and in none of normalsera. Immunohistochemistry analyses showed cyclin-I was observed in themajority of pancreatic carcinoma cells, the minority of benign adjacent ductalepithelial cells (P＜0.05), and in none of benign pancreatic disease andhealthy human pancreatic tissue. The positive immunolabeling of Rab GDIβwas observed in pancreatic carcinoma cells and benign ductal epithelial cellsand could not be observed in healthy human pancreatic. tissue.Conclusions: The combination of 2-DE and mass spectrometry (MS)strategy is an ideal platform and powerful approach for differential proteomicanalysis of human sera. Our results of comparative serum proteomics analysismight provide clues for further elucidating the role of these proteins in pancreatic carcinoma carcinogenesis and progression, as well as developingpotential associated biomarkers.