| Object:Gastric cancer is a global disease that threaten human health.Even after standardized treatment,the 5-year survival rate of advanced gastric cancer is still less than 30%.Target therapy is a hot point for gastric cancer individual treatment.To find a safe and high-efficient target is a key for target therapy.In this study,we try to use the protein pathway array technique(PPA)and meta-analysis for the screening of target proteins and genes in the advance gastric cancer.We also study the effect of ABT-737 on gastric cancer cells.Alpha-fetoprotein(AFP)producing gastric cancer is considered as a rare subtype of gastric cancer.Compared with AFP non-producing gastric cancer,our study and other previous studies showed that AFP producing gastric cancer is more aggressive and prone to liver metastasis.In this study,we try to use PPA to find important proteins in AFP positive gastric cancer.Method:The first part of this study is to screen the differential expressed proteins and genes for advanced gastric cancer.The screening of differential protein isbased on Protein Pathway array(PPA)technology of proteomics research methods,using high-throughput Protein electrophoresis and fast Protein Analysis.In this step,we compared 77 tumor-normal tissue pairs of advanced gastric cancer cases.For the screening the differentially expressed proteins,we selected 286 kinds of antibody.The second step for this part,we collected and selected 11 datasets of advanced gastric cancer Microarray data from GEO databases.Totally,455 cases of tumor-normal paired samples were analyzed by using pairing Meta data Analysis Package.The third step was the cross-analysis of differential expressed protein and gene expression data.Finally,the Ingenuity Pathway Analysis(IPA)Analysis software was used to analyze the cross-analysis results.The important Pathway signaling in advanced gastric cancer was listed.The second part studies by using the Protein Pathway Array,11 of out of286 proteins tested were found to be differentially expressed between AFP producing(n=32)and AFP non-producing(n=45)gastric adenocarcinoma tissues.Kaplan-Meier survival and log-rank analyses were used for survival analysis.In order to determine the risk factors associated with relapse-free survival(RFS)and overall survival(OS),univariate and multivariate analyses were performed by using Cox regression method and Wald tests to assess the significance.For all statistical analysis,a p value less than 0.05(P<0.05)was considered significant.In the third part of this study,we studied the effect of ABT-737 on gastric cancer cell viability through Cell Titer-Glo?.To determine whether the growth inhibition of gastric cancer cells by ABT-737 was associated with cell cycle arrest or apoptosis,flow cytometry were performed.We determine the effect of ABT-737 on cell invasion ability through Trasnwell and on expression of Bcl-2 in cell lines through Western-blot.Results:Out of 286 proteins studied,119 were detected in all 77 cases,and 24 of119 proteins were found to be differentially expressed between the two groups based on the paired t test and paired SAM analysis(P<0.05 and q<5%).Among these 24 proteins,19 were up-regulated in tumor samples,5 proteins were down-regulated in in tumor samples.By using pairing Meta data package analysis,a total of 3430 genes were selected to be differentially expressed genes(P.adj<0.01),2918 were up-regulated in tumor samples and 512 were down-regulated.After cross-analysis of those differentially expressed genes and proteins,a total of 14 protein is consistent with the trend of gene expression,13 up-regulated in tumor samples,: FEN 1 [FEN1],PCNA(PC10)(PCNA),cdk2(M2)(cdk2),annexin A1 [ANXA1],Maspin(C-20)[SERPINB5],Hsp90(AC88)[HSP90AA1],the Bcl-2-2(C)[BCL2],p38 lightning beta(A-12)[MAPK11],NFk B p50 [NFKB1],HER2(Erb B2)/Erb B2,Caspase 1 p10(M-20)[CASP1],XIAP(48 / h ILP)(XIAP),CTLA 4(H-126)/CTLA4,1 down-regulated: Glutamine Synthetase [GLUL].IPA analysis software results showed that these proteins and corresponding genes are involved in tumor growth,progression,and metastasis of tumor apoptosis,proliferation.The top five genes in those functions were BCL2,ERBB2,ANXA1,CASP1,MAPK11.The main involved pathways were p53 signaling,apoptosis signaling,GADD45 signaling,cyclins and cell cycle regulation,etc.In the second part of the experiment,we found the high level expression of XIAP and IGF-Ir? in gastric adenocarcinoma tissues was independent factors for poor prognosis in AFP producing gastric adenocarcinoma patients.A risk model based on the XIAP and IGF-Ir? expression levels can separate.AFP producing gastric adenocarcinoma patients into 2 subgroups and each subgroup had a distinct set of signaling pathways involved.In the third part of the experiment,we confirmed by Western-blot that the Bcl-2 were highlly expressed in the gastric cancer samples.The cell experiment results suggested ABT-737,as a kind of the Bcl-2 protein inhibitors,can inhibit the Bcl-2 protein expression and promote apoptosis in both AGS and BCG823 gastric cancer cell lines.Italso had obvious inhibitory effect on cell proliferation,and invasion.Those inhibition effects were concentration dependent.Conclusion: Multiple genes,proteins and pathways may involve in the occurrence and development of gastric cancer.We pioneered the cross-analysisof the results from the PPA technology for protein expression and GEO database meta-analysis of gene expression.The selected differentially expressed proteins and corresponding genes in advanced gastric cancer may be as potential targets for targeted therapy.The ABT-737 had obvious inhibitory effect on cell proliferation,invasion and promoted the apoptosis of the cells.The effects were concentration dependent.The effects of ABT-737 in gastric cancer cells showed it's potent to be a target for individual therapy for advanced gastric cancer.AFP producing gastric adenocarcinoma is a heterogeneous cancer with different clinical outcomes,biological behaviors and underlying molecular alterations. |
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