Comparative transcriptomic analysis reveals adriamycin-induced apoptosis via p53 signaling pathway in retinal pigment epithelial cells.
- Author:
Yu-Chen LIN
1
;
Ze-Ren SHEN
1
;
Xiao-Hui SONG
1
;
Xin LIU
1
;
Ke YAO
1
Author Information
- Publication Type:Journal Article
- Keywords: Adriamycin; Proliferative vitreoretinopathy; Retinal pigment epithelial; p53; Apoptosis
- MeSH: Apoptosis; Caspases/metabolism*; Cell Proliferation; Cell Survival/drug effects*; Doxorubicin/pharmacology*; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation; Humans; Membrane Potential, Mitochondrial; Oligonucleotide Array Sequence Analysis; Oxidative Stress/drug effects*; Phosphorylation; Propidium/chemistry*; RNA, Small Interfering/metabolism*; Retinal Pigment Epithelium/metabolism*; Rhodamines/chemistry*; Signal Transduction/drug effects*; Transcriptome; Tumor Suppressor Protein p53/metabolism*; Vitreoretinopathy, Proliferative/drug therapy*
- From: Journal of Zhejiang University. Science. B 2018;19(12):895-909
- CountryChina
- Language:English
-
Abstract:
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 H2AX (γ-H2AX), 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 caspase-dependent 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-H2AX, 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.
