Plumbagin induces RPE cell cycle arrest and apoptosis via p38 MARK and PI3K/AKT/mTOR signaling pathways in PVR

Abstract

Background: This study aimed to explore the effects of plumbagin (PLB) on ARPE-19 cells and underlying mechanism.

Methods: Cultured ARPE-19 cells were treated with various concentrations (0, 5, 15, and 25 μM) of PLB for 24 h or with 15 μM PLB for 12, 24 and 48 h. Then cell viability was evaluated by MTT assay and DAPI staining, while apoptosis and cell cycle progression of ARPE cells were assessed by flow cytometric analysis. Furthermore, the level of main regulatory proteins was examinated by Western boltting and the expression of relative mRNA was tested by Real-Time PCR.

Results: PLB exhibited potent inducing effects on cell cycle arrest at G2/M phase and apoptosis of ARPE cells via the modulation of Bcl-2 family regulators in a concentration- and time-dependent manner. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways contributing to the anti-proliferative activities in ARPE cells.

Conclusions: This is the first report to show that PLB could inhibit the proliferation of RPE cells through down-regulation of modulatory signaling pathways. The results open new avenues for the use of PLB in prevention and treatment of proliferative vitreoretinopathy.

Keywords: Plumbagin; Proliferation; RPE.

Fig. 1

Effects of plumbagin on the viability of ARPE cells. Notes: (a) ARPE cells were treated with indicated concentrations of plumbagin for 24 h and cell survival was measured by MTT assay. The results are expressed as percentage of control; (b) The morphological characteristic of cells and nuclears were analyzed with phase-contrast image (Scale bar 50 μm) and DAPI staining (Scale bar 20 μm). Arrows indicated apoptotic morphological changes (e.g., chromatin condensation, shrinkage and nuclear fragmentation). Figures were selected as representative data from three independent experiments

Fig. 2

Plumbagin enhanced apoptosis of ARPE cells. The apoptosis of cells was analyzed by Annexin V-FITC/PI assay. Data were represented the mean ± SD of three individual experiments. (*P < 0.05 by one-way ANOVA.). Notes: (a) Flow cytometric plots show cells in the live, early apoptosis, and late apoptosis stages when the cells were treated with PLB at 0, 5, 15 and 25 μM for 24 h; (b) Bar graphs show the percentage of specific cell populations (live, early apoptosis, or late apoptosis) and total apoptosis cell population when the cells were treated with PLB at 0, 5, 15, and 25 μM for 24 h; (c) Flow cytometric plots show cells in the live, early apoptosis, and late apoptosis stages when the cells were treated with 15 μM PLB at 0, 12, 24,and 48 h; (d) Bar graphs show the percentage of specific cell populations (live, early apoptosis, or late apoptosis) and total apoptosis cell population when the cells were treated with 15 μM PLB at 0, 12, 24, and 48 h

Fig. 3

Plumbagin induces G2/M in ARPE cells. The population of cells was analyzed by flow cytometry. Data represented the mean ± SD of three individual experiments. (* P < 0.05 by one-way ANOVA.). Notes: (a) Flow cytometric histograms show the cell cycle distribution when the cells were treated with PLB at 0, 5, 15 and 25 μM for 24 h; (b) Bar graphs show the cell cycle distribution (G0/G1、S and G2/M phase) when the cells were treated with PLB at 0, 5, 15, and 25 μM for 24 h. c Flow cytometric histograms show the cell cycle distribution when the cells were treated with 15 μM PLB at 0, 12, 24, and 48 h; (d) Bar graphs show the cell cycle distribution (G0/G1、S and G2/M phase) when the cells were treated with 15 μM PLB at 0, 12, 24, and 48 h

Fig. 4

Effect of plumbagin on the expression of apoptosis-related proteins in ARPE cells. (*:P < 0.05 by one-way ANOVA,n = 3). Notes: (a) The expression levels of Bax, Bak, Bcl-2 and Bcl-xl determined by Western blotting assay when ARPE cells were treated with plumbagin at 0,5,15 and 25 μM. β-actin was used as the internal control. b Bar graphs show the expression levels of Bax and Bak when ARPE cells were treated with plumbagin at 0,5,15 and 25 μM for 24 h. c Bar graphs show the expression levels of Bcl-2 and Bcl-xl when ARPE cells were treated with plumbagin at 0,5,15 and 25 μM for 24 h. d Bar graphs show the expression levels of Bax and Bak when ARPE cells were treated with plumbagin at 15 μM for 0, 12, 24 and 48 h. e Bar graphs show the expression levels of Bcl-2 and Bcl-xl when ARPE cells were treated with plumbagin at 15 μM for 0, 12, 24 and 48 h. f The expression levels of Bax, Cytochrome C, Caspase-3 and Caspase-8 determined by ELISA when ARPE cells were treated with plumbagin at 0,5,15 and 25 μM for 48 h

Fig. 5

Effect of plumbagin on the different signal pathways in ARPE cells. (*:P < 0.05 by one-way ANOVA, n = 3). Notes: (a) The relative mRNA expression levels of p38 MARK, PI3K, β-catenin and Notch-1 determined by QPCR when ARPE cells were treated with plumbagin at 0,5,15 and 25 μM. β-actin was used as the internal control; (b) The relative mRNA expression levels of p38 MARK and PI3K determined by QPCR when ARPE cells were treated with plumbagin at 15 μM for 0, 12, 24 and 48 h. β-actin was used as the internal control; (c) The protein levels of p-p38 MARK, p-PI3K, β-catenin and Notch-1 determined by Western blotting assay. GAPDH was used as the internal control; (d) The relative protein levels of p-p38 MARK, p-PI3K, β-catenin and Notch-1 when ARPE cells were treated with plumbagin at 0,5,15 and 25 μM; (e) The relative protein levels of p-p38 MARK, p-PI3K, β-catenin and Notch-1 when ARPE cells were treated with plumbagin at 15 μM for 0, 12, 24 and 48 h