Cross-taking Of Cellular Signalling Network In Gastric Cancer Cell Targeted Inhibition Of NOTCH Signaling Pathway

AimsNOTCH signaling plays an important role in cell proliferation, differentiation and apoptosis. A growing body of research and clinical evidence are in support of NOTCH’s oncogenic or tumor suppressive role in a wide variety of cancers. It, therefore, places Notch signaling as a potential target for cancer therapeutics. Our previous study found that abnormal expression of the NOTCH signaling pathway in gastric cancer. The aim of this study was to investigate the possibility of NOTCH signaling pathway as a potential therapeutic target in gastric cancer, and explain the molecular mechanism which inhibition of Notch pathway induces apoptosis in gastric cancer cells. It would provide a theoretical basis for the treatment of gastric cancer.MethodsGastric epithelial cell line GES-1was co-cultured with H. pylori for24h. The signaling pathways activated by H. pylori were identified by RT2Profiler PCR Array-Human Signal Transduction Pathway Finder microarray. Total RNA and protein were isolated from GES-1incubation with different strains of H. pylori and were cocultured for24h. The activated signaling pathway verified by qPCR and Western Blot. Gastric cancer lines (MKN-45, SGC-7901, BGC-823) will be treated with or without GSI, Notch receptor siRNA and ligands siRNA. Cell cycle and apoptosis were detected by Annexin-V-FITC/PI and flow cytometry. Cross-talking between NOTCH signaling pathway and other signaling pathways analyzed by RT2Profiler PCR Array-Human Signal Transduction Pathway Finder microarray. The molecular mechanism which inhibition of NOTCH pathway induces apoptosis in gastric cancer cells analyzed by Human Apoptosis Antibody Array.ResultsThere are22different expression genes between GSI with and without H. pylori11637by RT2Profiler PCR Array-Human Signal Transduction Pathway Finder microarray. These genes were associated with cell proliferation, cell cycle and transcriptional regulation. DAVID Pathway analysis showed that the H. pylori infection mainly affects Cyclins and Cell Cycle Regulation and NOTCH Signaling Pathway. The expression levels of NOTCH ligand DLL4were significantly increased by H. pylori11637and SSI. H. pylori inhibited cell proliferation through the regulation of cell cycle-related proteins.Gastric cancer cell lines (BGC-823, MKN-45, SGC-7901) were treated with or without20μmol/L GSI for24h. The expression levels of mRNA and protein of NOTCH1, NOTCH2and HES1were inhibited significantly. Inhibitor induced cell-cycle arrest in G1phase, inhibited cell proliferation and induced apoptosis. NOTCH2siRNA and DLL4siRNA significantly inhibited the expression of the corresponding protein levels and decreased significantly HES1. siRNA group did not affect gastric cancer cell cycle. Both GSI group and siRNA group induced significantly gastric cancer cells apoptosis. NOTCH signaling pathway closely related to WNT signaling pathway, P53signaling pathway and hypoxia signaling pathways by analysis of Array-Human Signal Transduction Pathway Finder in group GSI, NOTCH2siRNA and DLL4siRNA treatment gastric cancer cell MKN-45. Human Apoptosis Antibody Array was used to identify differentially expressed protein related apoptosis in the control and GSI, NOTCH2siRNA and DLL4siRNA treatment. Our results showed that a number of apoptotic signaling proteins were modulated following treatment. Some proteins related mitochondrial pathway were elevated in GSI group. Tumor necrosis factor-a and its receptors were significantly higher in all treatment group as compared with control.ConclusionNOTCH signaling can be used as a potential target for the treatment of gastric cancer. There are similar effect in inhibitors, NOTCH receptor siRNA and ligand siRNA. Inhibition of NOTCH signaling pathway could reduce gastric cancer cell proliferation and induce apoptosis, through regulating WNT signaling pathway, P53signaling pathway, and hypoxia signaling pathways. siRNA induced cell apoptosis via death receptor-mediated pathway. GSI induced cell apoptosis via both the mitochondrial and death receptor-mediated pathway.