Screening And Identification Of Immunogenic Membrane Antigens In Pancreatic Cancer

BACKGROUNDPancreatic cancer is one of the most lethal human cancers, and its earlydiagnosis is very difficulty. Membrane protein associated with pancreatic cancercarry out many essential cellular functions, and recent studies have shown thatthere are circulating auto-antibodies against tumor antigens in sera of patients.If we can screen and obtain the immunogenic membrane antigens in pancreaticcancer through membrane biology, celelular component proteomics,immuno-proteomics, membrane proteomics, then the candidate membraneantigens might be significantly valuable in serum markers identification,molecular imaging, and targeted immunotherapy of pancreatic cancer.OBJECTIVEIn order to screen and obtain the validate immunogenic membrane antigens inpancreatic cancer.METHODSPancreatic caner cell line SW1990 membrane protein was extracted and put onIEF and separated by 2-DE. One of the three parallel 2-DE gels went forcommassie blue staining while the others underwent immunoblot. Serum IgGwas purified from clinically collected sera of 66 pancreatic cancer patients and24 chronic pancreatitis patients and used as the primary antibodies of theimmunoblot. Positive dots of immunoblot were identified by MALDI-TOF massspectrometry and PMF matching, and then evaluated by bio-informaticsmethods. The candidate membrane antigens were further validated respectivelyin cell lines and tissues by RT-PCR, real time PCR, Western blot, and theirdifferent expression level of gene and protein between pancreatic caner cell lineor pancreatic caner tissue and normal pancreatic tissue were contrastly studied.Using RNA interference to silence the target mRNA of VDAC1, to explore theinterrelationship between VDAC1 and cellular apoptosis, cell growth and proliferation. Membrane protein expression of human pancreatic cancer cell lineSW1990 is successfully analysised.RESULTS1. The immunoblot of SW1990 membrane protein with serum IgG from cancerpatients showed nine positive dots which were not the same as those fromimmunoblot with serum IgG from chronic pancreatitis patients. Six dotswere identified with MALDI and PMF as: VDAC1, VDAC2, VDAC3,Prohibitin, Prohibitin2 and COMT.2. VDAC-1, 2,3 are mitochondria outer membrane proteins which are involvedin substance transport and signal transduction; Prohibitin and Prohibitin2are mitochondrial inner membrane protein, and inhibits DNA synthesis. Ithas a role in regulating proliferation. COMT is associated with thetransferation of a methyl group and the metabolism of endogenoussubstances.3. RT-PCR and real time PCR showed that VDAC1, VDAC2, VDAC3,Prohibitin,Prohibitin2 and COMT were expressed in the pancreatic cancercell line SW1990, AsPc, P3 and/or Panc-1. Western blot showed that VDAC,VDAC1,VDAC2 and COMT were overexpressed in the cell lines comparedwith the normal pancreatic tissue.4. Real time PCR showed that VDAC1, Prohibitin2 were all expressed in theten pancreatic cancer tissue or normal pancreatic tissue(100%). VDAC1 andProhibitin2 were overexpressed of Ca3, 7, 8, 9 and Ca3, 8, 10respectively(40% and 30%) in pancreatic cancer tissue compared with thenormal pancreatic tissue.5. Western blot showed that VDAC1 and Prohibitin2 were significantlyoverexpressed in pancreatic cancer tissue compared with the normalpancreatic tissue (p＜0.05).6. The hVDAC1-expression silencing system is stable and highly specific.Thelevel of hVDAC1 expression was dramatically decreased by 62.5% after transfecting 96hr, indicating the selected shRNA sequence is effective.7. MTT growth curve and Plate colony forming assay showed that, extremelyslowed cell growth and proliferation resulting from suppression of VDAC1expression by siRNA.8. The result of FCM showed that the number of apoptosis cell is obviously increased byVDAC1-RNAi in SW1990 cell line.9. To interfere with the expression of endogenous VDAC1 in transformedSW1990 cells, the RNAi was performed by using the siRNA approach.VDAC1 is related with those apoptosis associated gene bcl-xl,caspase-3,Bax,PARP. The cellular apoptosis is up-regular by silencing endogenous human(h)VDAC1 expression.10. The establishment of Prohibitin2-expression silencing system is effective,the level of Prohibitin2 expression was decreased by 35.8%, which indicatingthe siRNA sequence is highly specific.11. Membrane protein expression of human pancreatic cancer cell line SW1990is successfully analysised.CONCLUSIONS1. VDAC1, VDAC2, VDAC3,Prohibitin, Prohibitin2 and COMT might be thecandidate immunogenic membrane antigens of pancreatic cancer.2. VDAC-1, 2,3 are mitochondria outer membrane proteins which are involvedin substance transport and signal transduction; Prohibitin and Prohibitin2are mitochondrial inner membrane protein, and inhibits DNA synthesis. Ithas a role in regulating proliferation. COMT is associated with thetransferation of a methyl group and the metabolism of endogenoussubstances.3. The gene of VDAC1, VDAC2, VDAC3, Prohibitin,Prohibitin2 and COMTwere expressed in the pancreatic cancer cell line SW1990, AsPc, P3 andPanc-1. VDAC,VDAC1,VDAC2 and COMT were markedly overexpressedin the cell lines compared with the normal pancreatic tissue. 4. The gene of VDAC1, Prohibitin2 were all expressed in the ten pancreaticcancer tissue and normal pancreatic tissue(100%). They were overexpressedin some pancreatic cancer tissue compared with the normal pancreatic tissue.5. The protein of VDAC1, Prohibitin2 were all expressed in the eightpancreatic cancer tissue and normal pancreatic tissue(100%). They wereprominent overexpressed in the most pancreatic cancer tissue(p＜0.05).6. VDAC1 regulated and participated cellular growth and proliferation, whichis positive correlation. VDAC1 is related with cellular apoptosis, which isnegative correlation.7. Membrane protein expression of human pancreatic cancer cell line SW1990is successfully analysised.