Studying On The Expression Of IDO1、IDO2in Gallbladder Carcinoma Cells And The Role Of Immune Tolerance

Primary carcinoma of the gallbladder (PCG) is a common malignant tumors ofthe biliary system, ranking fifth to sixth of digestive tract tumors.PCG patients oftenconcealed with gallbladder stones and other diseases, early diagnosis rate was only9.1%.It`s often at advanced stage when diagnosised, losting opportunity to surgery.With the rapid development of tumor molecular biology and tumor immunology, andother related disciplines,tumor immune therapy has become the new cancer treatmentfollowing surgery, chemotherapy and radiotherapy three conventional therapy. Inorder to play the best efficacy of tumor immunotherapy. People must have a clear andcomprehensive understanding of tumor immune tolerance mechanisms. Studies haveshown that indoleamine2,3-dioxygenase1-mediated tumor immune tolerance hasbecome a hotspots. IDO1were over expressed in a variety of tumor tissues,whichInhibited of lymphocyte proliferation by reducing the concentration of tryptophan inthe tumor microenvironment.Recent study found a protein that has similar structurewith IDO1called indoleamine2,3-dioxygenase2(IDO2). Speculated that IDO2alsoplay an important role in the tumor formation of immune tolerance.How IDO1andIDO2express in gallbladder carcinoma? What role in the formation of immunetolerance respectively? There were no reports in the literature.In this study,we investigate the expression of indoleamine2,3-dioxygenase1,(IDO1) andtranscription factor Forkhead box P3（FOXP3）in gallbladder adenocarcinoma and theexpression of IDO1、IDO2in gallbladder carcinoma cells and the role of immunetolerance.Paper I The expression and clinical significance ofIDO1and FOXP3in gallbladder carcinomaIDO1is a cell containing enzyme of the heme can catalyze oxidative cleavage ofthe indole ring in tryptophan molecules. that tryptophan catabolism along theKynurenine pathway. IDO1play an important role in tumor immune tolerance. Whenthe tumor antigen stimulate macrophages and dendritic cells, they produce IDO1tochange the local concentration of the microenvironment of tryptophan and itsmetabolites to inhibit tumor antigen-specific clonal proliferation of T cells. Whenstimulated with IFN-gamma,tumor cells produced IDO1to avoid the attack of thebody’s immune system. FOXP3has been known as specific markers of regulatory Tcells (regulatory T-cells, Tregs). Tregs can autocrine TGF-beta, IL-10inhibitscytokine. To inhibit of antigen-specific CD4+T cells, cytotoxic CD8+T cells andnatural killer T cell function. IDO1and Tregs exists regulation circuits. Interactioncan promote each other’s expression,and were abundantly expressed in the tumorsurrounding lymph nodes and draining lymph nodes. So we suppose that IDO1andFOXP3also have a high expression in gallbladder carcinomaObjectiveTo investigate the expression of IDO1and FOXP3in gallbladder adenocarcinoma and toevaluate the relationship between the expression of IDO1and FOXP3protein and theclinicopathology, and lymph node metastasis of gallbladder carcinoma.MethodsExpression of IDO1and FOXP3in51cases of primary gallbladder cancer，90casesof chronic cholecystitis,20cases of normal gallbladder tissues were detected withimmunohistochemical staining Results1. The positive expression rate of IDO1and FOXP3in gallbladder carcinoma was72.5%(37/51) and60.8%(31/51)respectively, in normal gallbladder mucous was5%(1/20) and20.0%(4/20) respectively, in cholecystitis and gallstone was11.1%(10/90) and32.2%(29/90) respectively, and there is significant differencebetween the three groups in IDO1and FOXP3expression values(P<0.01, P<0.01,respectively).2. There is significant difference between Nevin stage group, lymph node metastasisgroup and gallbladder stone in IDO1and FOXP3expression value. In gallbladdercarcinoma.However, there is no significant difference between sex group, agegroup in IDO1and FOXP3expression value (P>0.05, respectively)．3. In gallbladder carcinoma，the expression of IDO1had a positive correlation withthat of FOXP3expression in lymphocyte.(r=0.406，P=0.003)Conclusion4. IDO1and FOXP3may be play synergistic effect involved in the formation ofgallbladder carcinoma immune tolerance The results provide a reference forclinical use of immunotherapy to treat gallbladder carcinoma． PAPER II Studying on the expression of IDO1、IDO2in gallbladder carcinoma cells and the role ofimmune toleranceIDO1is a cell containing enzyme of the heme can catalyze oxidative cleavage ofthe indole ring in tryptophan molecules. that tryptophan catabolism along theKynurenine pathway. IDO1play an important role in tumor immune tolerance.Therefore, the high expression of IDO1is one of the important factor in immunetolerance. Recent studies have found a substances that was similar as IDO1in gene sequence, molecular structure and biological activity.It was called IDO2, In order todistinguish with IDO, previous studies of IDO is now known as IDO1.ObjectiveTo investigate expression of indoleamine2,3-dioxygenase1,(IDO1)、indoleamine2,3-dioxygenase2,(IDO2) in gallbladder carcinoma cells and the function in immunetoleranceMethods1. The gallbladder carcinoma cell line SGC-996was treated by interferon-gamma(IFNgamma) and the levels of IDO1and IDO2expression was analyzed bywestern blot and immunofluorescence.2. RNA interference the expression of IDO1or IDO2in cell line SGC-996.Theinterference efficiency was analyzed by Western blotting.3. The kynurenine in medium from which was interfered IDO1or IDO2of SGC-996respectively was analysed by reverse phase high-performance liquidchromatography (HPLC).4. PBLs were cultured in SGC-996conditioned medium derived from IFNγ-treatedSGC-996cells with IDO1siRNA or IDO2siRNA for a further24h. Cell death wasdetected by flow cytometric analysis, using annexin V and propidium iodide asindicators.Results1. IDO1expression was acutely induced in SGC-996by low dose interferon-gamma(IFNgamma).IDO2expression was constant expression regardless of the presenceof IFNgamma.2. Confocal microscopy in the detection IDO1, Without IFN-γ, IDO1content isvery low, IFN-gamma stimulation, IDO1expression increased and IDO1locatedin the cytoplasm. But IDO2constant expression, not affected by IFN-gamma,located in the cytoplasm and nucleus.3. The concentration of kynurenine analysed by HPLC in medium which in SGC-996by IDO1siRNA was significant decreased.But concentration of kynurenine inSGC-996by IDO2siRNA was no significant difference. 4. The percent of cell death of PBLs which were cultured in SGC-996conditionedmedium derived from IFNγ-treated SGC-996cells for24h. The negativegroup,IDO1silence group, the IDO2silent group mortality was0.8%,1.1%,1.9%.In the positive control group the late apoptotic and dead cells increased,respectively,15.4%,12.4%,13.6%. The difference of IDO1silent group, IDO2silent group between the positive control group was statistically significant.ConclusionIDO1play a major role in the IDO enzyme catalysis.To Inhibit the activity of IDO1orIDO2in tumor cells may decrease the mortality of lymphocytes. The results providea reference for the research of IDO1and IDO2role in the formation of immunetolerance.