RESUMO
Bcl-2-associated athanogene 1 (Bag-1) is a multifunctional and antiapoptotic protein that binds to the antiapoptosis regulator Bcl-2 and promotes cell survival. To investigate the protective function of Bag-1, we examined the effects of Bag-1L, one isoform of Bag-1, in an in vitro cell culture model of lung ischemia-reperfusion injury (LIRI) generated by treatment of A549 cells with hypoxia/reoxygenation. Overexpression of full-length Bag-1L increased the viability of A549 cells and reduced cell apoptosis in response to 6 h of hypoxia/reoxygenation treatment. Furthermore, Bag-1L overexpression enhanced the heat shock protein 70 (HSP70) and Bcl-2 protein levels, increased the phosphorylation of AKT, decreased Bax and cleaved caspase-3 levels, and was able to overcome cell cycle arrest. These effects were not observed in A549 cells overexpressing a truncated form of Bag-1L lacking the "Bag domain," denoted Bag-1Lâ³C. The "Bag domain" is the C-terminal 47 amino acids. Taken together, the results of this study suggest that Bag-1L overexpression can protect against oxidative stress and apoptosis in an in vitro LIRI model, with a dependence on the Bag domain.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/farmacologia , Pulmão/metabolismo , Substâncias Protetoras/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Fatores de Transcrição/farmacologia , Células A549 , Adenoviridae , Ciclo Celular , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Hipóxia , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Transcrição/genéticaRESUMO
PURPOSE: B-cell lymphoma-2 (Bcl-2) associated athanogene 1 (Bag-1) is a multifunctional protein, and Bag -1 overexpression is associated with progression, metastasis, and drug resistance in lung cancer. This study assessed the effects of Bag-1 siRNA on sensitization of cisplatin on non-small cell lung cancer (NSCLC) cells. MATERIAL AND METHODS: NSCLC A549 cell line was transfected with Bag-1 or negative control siRNA and then treated with cisplatin for cell viability, CCK-8, LDH, and flow cytometry assays. The Ca2+ levels were analyzed using Fluo-3/AM fluorescence staining, and the protein levels were assessed using Western blot analysis. RESULTS: Bag-1 siRNA significantly knocked down Bag-1 expression and inhibited cell invasion versus the negative control siRNA, while Bag-1 silence sensitized cisplatin to induce A549 cells to apoptosis by induction of cell cycle G1 arrest. At protein level, Bag-1 silence reduced the expression ratio of Bcl-2 to Bcl-2 associated X protein (Bax), downregulated activity of the PI3K/AKT and mitogen-activated protein kinase (MAPK) pathways, and potently upregulated the calcium signaling-mediated pathway. CONCLUSION: This study demonstrated that Bag-1 silencing sensitized A549 to cisplatin to enhance A549 cell apoptosis by modified multiple gene pathways. Further study will evaluate the usefulness of Bag-1 siRNA as a potential targeting therapy for NSCLC.
RESUMO
BACKGROUND: Transplant vasculopathy is a major cause of chronic rejection of transplanted organs. In the present study, we examined the effects of CX-5461, a novel selective inhibitor of RNA polymerase I, on development of transplant vasculopathy using a modified model of rat aortic transplantation. METHODS: The thoracic aortas from Fischer rats were transplanted into the abdominal cavity of Lewis rats. CX-5461 was mixed in pluronic gel and administered via perivascular release. RESULTS: Treatment with CX-5461 mitigated the development of neointimal hyperplasia and vascular inflammation. This effect was likely to be attributable in part to inhibition of macrophage-dependent innate immunity reactions. Specifically, CX-5461 exhibited potent inhibitory effects on macrophage migration and lipopolysaccharide-induced activation. Treatment with CX-5461 also prevented macrophage differentiation and maturation from primary bone marrow cells. In macrophages, CX-5461 did not alter the total amount of p53 protein, but significantly increased p53 phosphorylation, which was involved in regulating cytokine-stimulated macrophage proliferation. CONCLUSIONS: In conclusion, our results suggest that pharmacological inhibition of RNA polymerase I may be a novel strategy to treat transplantation-induced arterial remodeling.