ABSTRACT
BACKGROUND/AIMS: Neogenin-1 (Neo1) has been reported to be involved in diverse physiology and pathology functions, including cell proliferation, differentiation and migration. The present study aimed to explore the functional role of neogenin-1 (Neo1) in gastric cancer (GC), together with underlying mechanisms. METHODS: Neo1 expression was analyzed by qRT-PCR and Western blot analysis in both human GC cell lines and normal gastric epithelial cell line. Neo1 was respectively overexpressed or silenced by transfection with pcDNA3.1 or siRNA, and then the cells were incubated with or without different concentrations of cisplatin, transforming growth factor (TGF)-ß1, and/or inhibitors of Rac-1 and PI3K. Thereafter, cell viability, invasion, and adhesion were measured by CCK-8, wound healing and adhesion assays, respectively. The expression levels of key factors involved in epithelial mesenchymal transition (EMT) and the PI3K/AKT pathway were analyzed by Western blot analysis. RESULTS: The results showed that the Neo1 level was significantly increased in GC cell lines, with the highest level in SGC-7901 cells. Overexpression of Neo1 significantly reduced the GC cell sensitivity to cisplatin and increased the cell viability, motility and adhesion ability, and while silencing of Neo1 showed contrary results. Moreover, overexpression of Neo1 dramatically downregulated the E-Cadherin level and upregulated the levels of N-Cadherin and Vimentin. In addition, the data revealed that Neo1 positively regulated the expression of Zinc finger E-box-binding homeobox 1 (ZEB1) by activating the Rac1/PI3K/AKT pathway. CONCLUSIONS: Neo1 could promote cell proliferation, motility, and adhesion by up-regulation of ZEB1 via activating the Rac1/PI3K/AKT pathway in GC cells.
Subject(s)
Cell Proliferation , Nerve Tissue Proteins/metabolism , Receptors, Cell Surface/metabolism , Signal Transduction , Zinc Finger E-box-Binding Homeobox 1/metabolism , Antineoplastic Agents/pharmacology , Cadherins/genetics , Cadherins/metabolism , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cisplatin/pharmacology , Epithelial-Mesenchymal Transition/drug effects , Humans , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cell Surface/genetics , Signal Transduction/drug effects , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Up-Regulation/drug effects , Vimentin/genetics , Vimentin/metabolism , Zinc Finger E-box-Binding Homeobox 1/genetics , rac1 GTP-Binding Protein/metabolismABSTRACT
AIMS: The alteration of ROS level is frequently observed in the course of morphine addiction, and ROS is proverbially involved in this process. This study aims to explore the relationship among morphine addiction, reactive oxygen species (ROS) and expression of µ-opioid receptor (MOR) in differentiated SH-SY5Y cells. MAIN METHODS: SH-SY5Y cells were induced to differentiation by treatment with retinoic acid (RA); the activity of lactate dehydrogenase (LDH) and the nitro blue tetrazolium (NBT) reduction were assessed by spectrophotometry. Intracellular reactive oxygen species (ROS) was measured with the 2,7-dichlorofluorescin diacetate (DCFH-DA) assay. Cellular cAMP was determined by using a competitive protein binding kit. The mRNA expression of µ-opioid receptor (MOR) was evaluated by qRT-PCR. KEY FINDINGS: Morphine-induced ROS are generated in a concentration- and time-dependent manner and inhibited by naloxone. Exogenous oxidants increase the level of ROS and aggravate morphine addiction, while the exogenous antioxidants efficiently reverse these effects. Morphine decreases the mRNA level of MOR in a concentration-dependent manner. And the mRNA level of MOR is remarkably reduced in the presence of exogenous oxidants and effectively promoted by antioxidants. SIGNIFICANCE: This study indicates that ROS can affect morphine addiction through involving MOR. Treatment with ROS scavenging can serve as a medical therapy for morphine addiction.
