Objective: This paper applied a transcriptomic approach to investigate the mechanisms of adriamycin(ADR) in treating proliferative vitreoretinopathy(PVR) using ARPE-19 cells. Methods: The growth inhibitory effects of ...Objective: This paper applied a transcriptomic approach to investigate the mechanisms of adriamycin(ADR) in treating proliferative vitreoretinopathy(PVR) using ARPE-19 cells. Methods: The growth inhibitory effects of ADR on ARPE-19 cells were assessed by sulforhodamine B(SRB) assay and propidium iodide(PI) staining using flow cytometry. The differentially expressed genes between ADR-treated ARPE-19 cells and normal ARPE-19 cells and the signaling pathways involved were investigated by microarray analysis. Mitochondrial function was detected by JC-1 staining using flow cytometry and the Bcl-2/Bax protein family. The phosphorylated histone H2 AX(γ-H2 AX), phosphorylated checkpoint kinase 1(p-CHK1), and phosphorylated checkpoint kinase 2(p-CHK2) were assessed to detect DNA damage and repair. Results: ADR could significantly inhibit ARPE-19 cell proliferation and induce caspasedependent apoptosis in vitro. In total, 4479 differentially expressed genes were found, and gene ontology items and the p53 signaling pathway were enriched. A protein–protein interaction analysis indicated that the TP53 protein molecules regulated by ADR were related to DNA damage and oxidative stress. ADR reduced mitochondrial membrane potential and the Bcl-2/Bax ratio. p53-knockdown restored the activation of c-caspase-3 activity induced by ADR by regulating Bax expression, and it inhibited ADR-induced ARPE-19 cell apoptosis. Finally, the levels of the γ-H2 AX, p-CHK1, and p-CHK2 proteins were up-regulated after ADR exposure. Conclusions: The mechanism of ARPE-19 cell death induced by ADR may be caspase-dependent apoptosis, and it may be regulated by the p53-dependent mitochondrial dysfunction, activating the p53 signaling pathway through DNA damage.展开更多
基金Project supported by the Zhejiang Province Key Research and Development Program(No.2015C03042),China
文摘Objective: This paper applied a transcriptomic approach to investigate the mechanisms of adriamycin(ADR) in treating proliferative vitreoretinopathy(PVR) using ARPE-19 cells. Methods: The growth inhibitory effects of ADR on ARPE-19 cells were assessed by sulforhodamine B(SRB) assay and propidium iodide(PI) staining using flow cytometry. The differentially expressed genes between ADR-treated ARPE-19 cells and normal ARPE-19 cells and the signaling pathways involved were investigated by microarray analysis. Mitochondrial function was detected by JC-1 staining using flow cytometry and the Bcl-2/Bax protein family. The phosphorylated histone H2 AX(γ-H2 AX), phosphorylated checkpoint kinase 1(p-CHK1), and phosphorylated checkpoint kinase 2(p-CHK2) were assessed to detect DNA damage and repair. Results: ADR could significantly inhibit ARPE-19 cell proliferation and induce caspasedependent apoptosis in vitro. In total, 4479 differentially expressed genes were found, and gene ontology items and the p53 signaling pathway were enriched. A protein–protein interaction analysis indicated that the TP53 protein molecules regulated by ADR were related to DNA damage and oxidative stress. ADR reduced mitochondrial membrane potential and the Bcl-2/Bax ratio. p53-knockdown restored the activation of c-caspase-3 activity induced by ADR by regulating Bax expression, and it inhibited ADR-induced ARPE-19 cell apoptosis. Finally, the levels of the γ-H2 AX, p-CHK1, and p-CHK2 proteins were up-regulated after ADR exposure. Conclusions: The mechanism of ARPE-19 cell death induced by ADR may be caspase-dependent apoptosis, and it may be regulated by the p53-dependent mitochondrial dysfunction, activating the p53 signaling pathway through DNA damage.
