Project description:Although therapy responsiveness to therapy in Burkitt lymphoma (BL) is high, relapsed disease and and chemoresistance remain a clinical challenge, and complete mechanisms underlying BL chemoresistance and how it can be circumvented is yet to be fully elucidated. In this study we present data showing that chymotrypsin-like serine proteases inhibitor Nα-tosyl-L-phenylalanine chloromethylketone (TPCK) and specific NF-κB inhibitor Bay-11 7082 can induce caspase-independent apoptosis in chemoresistant BL cells. We also demonstrate that both TPCK and Bay-11 7082-treatment leads to decreased NF-κB nuclear activity and that this is associated with sensitization of chemoresistant Burkitt lymphoma cells. Furthermore we investigated global transcriptional changes induced by Bay-11 7082 and TPCK in the DG-75 and Raji cell lines, respectively, by microarray analysis using Illumina BeadChips. TPCK-treatment of Raji and DG-75 cells resulted in 59 and 21 differently expressed genes, respectively, while Bay-11-treated Raji and DG-75 cells displayed 1403 and 8 differently expressed genes, respectively. Gene Ontology (GO) categorization confirmed enrichment of multiple GOs in Bay 11-treated Raji and DG-75 cells. Fifty percent of the 61 categories in Raji cells were categories sorting under Biological Processes and represented mostly increased gene expression. In DG-75 cells Bay-11 7082 induced significant gene ontology enrichment in only two categories, where the increased/decreased ratio was 1:1. Further unsupervised and supervised bioinformatics processing by Ingenuity Pathway Analysis indicated significant networks in response to TPCK and Bay 11 respectively, including association to NF-κB. Bay-11 7082 demonstrated deregulated NF-κB related members of receptor mediated cell death signaling, i.e TRAF2 and TRADD, as well as deregulated members of the NF-κB signaling pathway from the cytoplasmic compartment, i.e RELB, in Raji cells. Comparably NF-κB network analysis of Raji- and DG-75 cells treated with Bay-11 7082 and Raji cells treated with TPCK demonstrated deregulation of NF-κB target genes CD69 and IL8. These data indicates that NF-kB may play a role in overcoming chemoresistance in BL cells with defective classical apoptosis signaling. NF-κB network analysis of Raji- and DG-75 cells treated with Bay-11 7082 and Raji cells treated with TPCK

Project description:Although therapy responsiveness to therapy in Burkitt lymphoma (BL) is high, relapsed disease and and chemoresistance remain a clinical challenge, and complete mechanisms underlying BL chemoresistance and how it can be circumvented is yet to be fully elucidated. In this study we present data showing that chymotrypsin-like serine proteases inhibitor Nα-tosyl-L-phenylalanine chloromethylketone (TPCK) and specific NF-κB inhibitor Bay-11 7082 can induce caspase-independent apoptosis in chemoresistant BL cells. We also demonstrate that both TPCK and Bay-11 7082-treatment leads to decreased NF-κB nuclear activity and that this is associated with sensitization of chemoresistant Burkitt lymphoma cells. Furthermore we investigated global transcriptional changes induced by Bay-11 7082 and TPCK in the DG-75 and Raji cell lines, respectively, by microarray analysis using Illumina BeadChips. TPCK-treatment of Raji and DG-75 cells resulted in 59 and 21 differently expressed genes, respectively, while Bay-11-treated Raji and DG-75 cells displayed 1403 and 8 differently expressed genes, respectively. Gene Ontology (GO) categorization confirmed enrichment of multiple GOs in Bay 11-treated Raji and DG-75 cells. Fifty percent of the 61 categories in Raji cells were categories sorting under Biological Processes and represented mostly increased gene expression. In DG-75 cells Bay-11 7082 induced significant gene ontology enrichment in only two categories, where the increased/decreased ratio was 1:1. Further unsupervised and supervised bioinformatics processing by Ingenuity Pathway Analysis indicated significant networks in response to TPCK and Bay 11 respectively, including association to NF-κB. Bay-11 7082 demonstrated deregulated NF-κB related members of receptor mediated cell death signaling, i.e TRAF2 and TRADD, as well as deregulated members of the NF-κB signaling pathway from the cytoplasmic compartment, i.e RELB, in Raji cells. Comparably NF-κB network analysis of Raji- and DG-75 cells treated with Bay-11 7082 and Raji cells treated with TPCK demonstrated deregulation of NF-κB target genes CD69 and IL8. These data indicates that NF-kB may play a role in overcoming chemoresistance in BL cells with defective classical apoptosis signaling.

Project description:[Aim] EBVaGC accounts for about 10% of gastric cancer and has a better prognosis than EBVnGC, but its molecular mechanism is still unclear. We investigated the role of EBV-miR-BART1-3p in the occurrence and development of EBVaGC by applying bioinformatics methods, high-throughput sequencing, in vitro experiments, target gene screening and verification, differential gene protein interaction network and gene regulatory network construction. [Materials and Methods] The data set GSE51575 was normalized, and the outlier samples were deleted. Finally, gene expression profiles of 11 EBVaGC and 14 EBVnGC tumor tissues and non-tumor control tissues were obtained. Differential gene function analysis was performed, and molecular regulatory network was constructed. The mRNA expression levels of SNU-719 (EBV-positive gastric cancer cell line), AGS, AGS-NC (EBV-negative gastric cancer cell line) and AGS-OE (lentivirus-transfected miR-BART1-3p overexpression cell line) were detected by high-throughput next-generation sequencing technology, and differential gene expression characteristics were analyzed. To investigate the effect of miR-BART1-3p overexpression on mRNA expression level of gastric cancer cells and construct the protein interaction network of differential genes and the regulatory network of miR-BART1-3p gene. DIANA-MR micro-T, miRnada, miRDB, TarBase v.8, and ViRBase online databases were used to predict the target genes of miR-BART1-3p and GO enrichment analysis of target gene function was performed. Real-time fluorescence quantitative PCR was used to detect the expression levels of miR-BART1-3p and target genes in SNU719, AGS-OE and AGS gastric cancer cells. The effects of overexpression of EBV-miR-BART1-3p on proliferation, apoptosis, invasion, and migration of gastric cancer cells were investigated by CCK-8, cell cloning, cell scratch and Transwell. [Results] By analyzing the gene expression profile of the GSE51575 dataset, we found that EBVaGC up-regulated the expression of immune cell membrane protein receptors and chemokine family, and was involved in inducing tumor immunity, suggesting a better prognosis. EBVnGC significantly up-regulated the expression of AURK, BUB1B, CCNA2 and CDC20, suggesting the risk of malignant progression and recurrence. High-throughput next-generation sequencing of SNU719 and AGS to detect differences in gene expression revealed that EBVaGC cells (SNU719) up-regulated the expression of genes that inhibit malignant progression as well as potential tumor immunotherapy targets. These results are consistent with those found in the GSE51575 dataset. Fifteen miR-BART1-3p target genes, including FAM168A, TET3, HBP1, ZBTB5, ATXN7L3, KMT5C, SPARC and BMF, were predicted and screened by online tools. After the overexpression of miR-BART1-3p in AGC(EBVnGC) cells, the proliferation, migration, and invasion ability were significantly inhibited, and the signaling pathway promoting tumor progression was also limited in activation. [Conclusions] The overexpression of miR-BART1-3p may inhibit the proliferation and invasion and migration of gastric cancer cells by inhibiting the activation of STAT3/ pSTAT3 and PI3K/AKT signaling pathways and may become a target for the treatment of EBVaGC, which is worthy of further study.

Project description:Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T-cells into regulatory T-cells in vitro. The marker CD69 is a target of canonical NF-κB signaling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3+ T-cells were activated and cultured in the presence or absence of MSCs. CD4+ cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 and a siRNA against RELB were used to explore the differential roles of canonical and non-canonical NF-κB signaling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69+ cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69+ cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF-κB signaling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signaling on the 3rd day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signaling. In order to study the molecular basis by which Multipotent Mesenchymal Stromal/Stem Cells (MSC) exert their immune regulatory function, immunomagnetically purified CD3+ T-cells from the peripheral blood of 3 individuals were activated and cultured in the presence or absence of MSCs. Following 5 days, CD4+ and CD8+ T-cells were further immunomagnetically selected and their gene expression profiles were obtained by microarrays and compared. Paired samples from 3 individuals were used for this analysis.

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EBV-induced CXCL8 upregulation promotes vasculogenic mimicry in gastric carcinoma via NF-κB signaling

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