ABSTRACT
Cigarette smoking has been shown to be the most significant risk factor for lung cancer. Recent studies have also indicated that RNA-binding motif protein 5 (RBM5) can modulate apoptosis and suppress tumor growth. The present study focused on the role of RBM5 in the regulation of cigarette smoke extract (CSE)-induced transformation of bronchial epithelial cells into the cancerous phenotype and its mechanism of action. Herein, we exposed normal BEAS-2B cells for 8 days to varying concentrations of CSE or dimethylsulfoxide (DMSO), followed by a recovery period of 2 weeks. Next, the RBM5 protein was overexpressed in these transformed BEAS-2B cells though lentiviral infection. Later, the morphological changes, cell proliferation, cell cycle, apoptosis, invasion and migration were assessed. In addition, we analyzed the role of RBM5 in xenograft growth. The expression of RBM5 along with the genes related to cell cycle regulation, apoptosis and invasion were also examined. Finally, our results revealed that BEAS-2B cells exposed to 100 µg/ml CSE acquired phenotypic changes and formed tumors in nude mice, indicative of their cancerous transformation and had reduced RBM5 expression. Subsequent overexpression of RBM5 in these cells significantly inhibited their proliferation, induced G1/S arrest, triggered apoptosis and inhibited their invasion and migration, including xenograft growth. Thus, we established an in vitro model of CSE-induced cancerous transformation and concluded that RBM5 overexpression inhibited the growth of these transformed cells through cell cycle arrest and induction of apoptosis. Therefore, our study suggests the importance of RBM5 in the pathogenesis of smoking-related cancer.
Subject(s)
Cell Cycle Checkpoints/drug effects , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Transformation, Neoplastic/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Smoking/adverse effects , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Animals , Apoptosis , Cell Line , Cell Movement , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Transformation, Neoplastic/pathology , Down-Regulation , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, NudeABSTRACT
Lung cancer is the leading cause of cancer death in the world. Schizandrin B (Sch B) is one of the main dibenzocyclooctadiene lignans present in the fruit of Schisandra chinensis (Schisandraceae). Sch B has multiple functions against cancer. The aim of this study was to determine the effect of Sch B on the proliferation, cell cycling, apoptosis and invasion of lung adenocarcinoma A549 cells by MTT, flow cytometry, wound healing and transwell invasion assays. Treatment with Sch B inhibited the proliferation of A549 cells in a dose-dependent manner. Sch B induced cell cycle arrest at G0/G1 phase by down-regulating the expression of cyclin D1, cyclin-dependent kinase (CDK)4, and CDK6, but up-regulating p53 and p21 expression in A549 cells. Furthermore, Sch B triggered A549 cell apoptosis by increasing Bax, cleaved caspase-3, 9, Cyto C, but decreasing Bcl-2 and PCNA expression. In addition, Sch B inhibited the invasion and migration of A549 cells by down-regulating the expressions of HIF-1, VEGF, MMP-9 and MMP-2. Therefore, Sch B has potent anti-tumor activity and may be a promising traditional Chinese medicine for human lung carcinoma.
ABSTRACT
Cigarette smoking is closely associated with various respiratory diseases. Oxidants and carcinogens in cigarettes are reported to induce various airway epithelial injuries. However, the underlying mechanisms remain unclear. The aims of the present study were to determine the involvement of RNA-binding motif protein 5 (RBM5) and Wnt/ß-catenin signaling in cigarette smoke-induced alveolar epithelial injury, as well as the interaction between both. A549 cells were treated with cigarette smoke extract (CSE). The MTT assay was used to assess the effects of CSE on cell viability. The levels of RBM5 and Wnt/ß-catenin/GSK3ß were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. A luciferase assay was used to assess the activity of ß-catenin/T-cell factor (TCF) signaling. The results revealed that CSE inhibited A549 cell viability in both a dose- and time-dependent manner. Cytosolic and nuclear ß-catenin levels were significantly increased following CSE treatment, compared with those in the control cells (P<0.05). The luciferase activity in CSE-exposed cells transfected with the TCF luciferase reporter wild-type plasmid (pGL3-OT) was significantly greater than that in cells without CSE exposure (33,167±3,085 vs. 19,978±1,916, respectively, P<0.05). Both the mRNA and protein levels of RBM5 in the CSE-treated cells were significantly reduced compared to the levels in the controls (all P<0.05). The overexpression of RBM5 inhibited Wnt/ß-catenin signaling in the A549 cells, while silencing of RBM5 enhanced Wnt/ß-catenin signaling. The ß-catenin/TCF signaling inhibitor ICG-001 had no apparent effect on the RBM5 levels. Downregulation of RBM5 and activation of Wnt/ß-catenin signaling are involved in CSE-induced alveolar epithelial injury. RBM5 acts as an upstream molecule that negatively regulates the activity of Wnt/ß-catenin signaling.
