The Regulating Effects And Mechanisms Of CD40 Signal On The Immunoediting Of Human Gastric Cancer

Objective: To investigate the expression of CD40 in gastric cancer, and to explore the correlation of CD40 with the outcome of the patients. Methods: The expression of CD40 was examined by immunohistochemistry in human gastric cancer tissues (73 cases) and adjacent normal gastric tissues (51 cases). CD40mRNA was detected by RT-PCR in 11 gastric cancer and 11 adjacent normal gastric tissues. The patients were followed up of 60 months.The correlation of CD40 expression and the overall survival time were evaluated by Kaplan-Meier chart and Log-rank test . Cox model was used for multivariate analysis.Results: The immunoreactivity of CD40 increased in 35/73 gastric cancer tissues (47.9%) as compared to the tumor-free tissues (3.9%), respectively(P=0.0063). CD40mRNA were detected in 72.7% gastric cancer tissues, while not in all tumor-free tissues. After 5 years follow up,the survival rate in CD40-negative patients was 57.9%(22/38), and in CD40-positive patients was 5.7%(2/35), P=0.0022. The median survival time of patients with negative, positive, and strong CD40 expression was 56, 29, and 11 months,respectively(P=0.0085). Cox regression analysis suggested that CD40, lymph node metastasis, and TNM stage were independent factors affecting the prognosis of gastric cancer patients. Conclusion: Normal and malignant gastric tissues are found to have its own unique CD40 expression patterns. CD40 may play a role in metastatic spread of gastric cancer and its expression may be used as an important survival predictor in human gastric cancer. Objective: To screen and identify tumor immunoedting-related signal transduction pathway on gene expression profile of gastric cancer AGS cells obtained before and after activation of CD40 signaling by bioinformatics analysis and molecular biology techniques. And to construct gene silencing vector as the specific blocker of the relevant signaling pathways. Methods: Gastric cancer immunoediting associated signal transduction pathways were screened on DNA microarray of differentially expressed genes before and after activation of CD40 signaling with Hierarchical Cluster analysis and were verified using Real-time PCR and Western blot. The PI3K siRNA template DNA sequence for short hairpin RNA(shRNA) was designed and synthesized. The annealed siRNA template was inserted into Psilencer1.0-U6 plasmid. The recombinant plasmid (Psilencer1.0-U6-PI3K-siRNA) was transformed into DH5αstrain and identified by restrictive enzyme digestion and sequence analysis. The effect of the recombinant plasmid on the PI3K expression of human gastric cancer AGS cells was detected by RT-PCR and Western blot. Results: Bioinformatics analysis revealed 414 differentially expressed genes, including 209 up-regulated genes, 205 down-regulated genes. PI3K and JNK/SAPK signaling pathway related genes were significantly upregulated and their upregulation was validated by real-time PCR and Western blot. It was confirmed by restrictive enzyme digestion and sequence analysis that the recombinant plasmid was cloned and the aim sequence was obtained. The PI3K expression of AGS cells was inhibited at mRNA and protein levels 72 hours after transfected with Psilencer1.0-U6-PI3K-siRNA. Conclusion: The activation of CD40 signaling in AGS cell can activate a number of different signal transduction pathways. The aberrant activation of PI3K and JNK/SAPK signaling pathway might be a major molecular mechanism of the two-way biological behavior of gastric cancer cells regulated by CD40 signaling. PI3K/Akt signaling pathway can increase the expression of survivin and VEGF and may promote gastric cancer invasion, metastasis and immunoediting. Targeting PI3K siRNA gene silencing vector could specifically inhibit the PI3K expression.Objective: To investigate the effect of silencing PI3K by small interfering RNA ( siRNA ) on CD40 signaling-induced tumor growth, invasion and related proteins expression of gastric cancer. Methods: In vitro, plasmids of siRNA for PI3K gene ( Psilencer1. 0-U6-PI3K-siRNA) were constructed and stably transfected into AGS cells. The cells were divided into 4 groups: A. sCD40L+AGS cells transfected with Psilencer1.0-U6-PI3K-siRNA;B. sCD40L+AGS cells; C. NS+AGS cells transfected with Psilencer1.0-U6-PI3K-siRNA; D. NS+AGS cells. Cell viability were tested by MTT. Cell cycle and apoptosis rate were detected by flow cytometry. The cells were subjected to wound closure assay and transwell assay to determine the migration of AGS cells and those after stable transfection. The expression of PI3K, survivin, VEGF and Fas were evaluated by Western blot. Results: In vitro, cell cycle arrest and apoptosis rate of group A was significantly increased than other groups, but the invasive ability decreased(P < 0. 01~0.05). The expression of PI3K, VEGF and survivin of group A were significantly reduced than other groups, but Fas expression enhanced (P<0. 01~0.05). Conclusion: CD40 induce the invasion and metastasis of gastric cancer by activating PI3K signaling. The molecular mechanisms may involve upregulation of VEGF, Survivin and downregulation of Fas. Silencing PI3K can increase the specific antitumor effect induced by CD40 signaling in human gastric cancer. Objective: To investigate the effect of sCD40L combined with PI3K siRNA on tumor growth and microenvironment of a nude mice model bearing human gastric cance. Methods: In vivo, 24 nude mice were randomly divided into four groups, and 5×106 cells per mouse was subcutaneously injected on the right HS abdomen of mice:Ⅰ. sCD40L+ AGS cells transfected with Psilencer1.0-U6-PI3K-siRNA;Ⅱ.sCD40L+ AGScells;Ⅲ.NS+ AGS cell stransfected with Psilencer1.0-U6-PI3K-siRNA;Ⅳ. NS+AGS cells. All mice were sacrificed 42 days later. The volume and weight of tumor were measured and the rates of tumor inhibition were evaluated. The microvessel density (MVD) of tumor tissue was measured. Apoptotic cells were detected by TUNEL. The expression of PI3K, survivin, VEGF and Fas were evaluated by Western blot. Results: In vivo, the volume of tumors were significantly different between groupⅠand other groups(P<0.05). The rates of tumor apoptosis of groupⅠwas significantly increased than other groups, but MVD decreased(P <0.01). The expression of PI3K, VEGF and survivin of groupⅠwere significantly reduced than other groups, but Fas expression enhanced (P<0. 01~0.05). Conclusion: sCD40L combined with PI3K siRNA significantly can inhibit tumor growth, angiogenesis and promote apoptosis in a nude mice model of gastric cancer. Silencing PI3K may increase the specific antitumor effect induced by CD40 signaling in human gastric cancer.Objectives: To extract, purify Astragalus polysaccharides(APS) and to analyze its chemical structure. To explore anti-tumor effects and immune modulating activities of APS in rats with gastric cancer and its impacts on CD40-PI3K signaling pathway.Methods: APS was extracted from natural Astragalus by NaOH method and purified by thin-layer chromatography (TLC) and Sephadex G-100 chromatography. HPLC and IR methods were used for a qualitative and quantitative determination of from polysaccharides of Astragalus. The Wistar rats were randomly divided into five groups. Except for normal control group, the other four groups were injected Arac and radiated with 60CO, then transplanted with AGS gastric cancer cells to form gastric cancer rat models. Three groups of which were treated separately by APS low dose, moderate dose and high dose. The normal control group and the model control group were gived NS. Ten days later, blood Ig A, Ig M, Ig G and IL-2 levels were measured with ELISA kit. Spleen lymphoproliferation rate and natural killer (NK) cells activity was measured by biochemical analysis. Peripheral blood T cell subsets were detected by flow cytometry. Tumor growth were observed and the inhibition rate were calculated in each group. The expression of CD40 and PI3K proteins were evaluated by Western blot. Results: APS is an a-(1-4)-d-glucan with a-(1-6)-linked branches attached to the O-6 of branch points. Bioactivity tests showed that APS is active for spleen lymphocytes proliferation and NK cells in dose-dependent manner. APS significantly increased serum IgA, IgG, IgM levels and CD4+, CD4+/CD8+ in dose-dependent manner. APS showed significant anti-tumor effects. After APS treatment, CD40 expression were upregulaed while PI3K expression were downregulaed. Conclusions: APS presents significant immune modulating activity and anti-tumor effect. The treatment effect of APS in rats with gastric cancer may be associated with regulating CD40-PI3K signal way. These results together support the popular use of APS in the treatment of gastric cancer diseases.