Fig 1.
Protective effect of protopanaxadiol (PPD) on chloroquine (CQ)-induced adult retinal pigment epithelial (ARPE)-19 cell death and possible autophagy involvement.
Cytotoxicity of (A) various CQ concentrations (0, 10, 50, 75, 100 μM), (B) co-treatment with 100 μM CQ and vehicle or different PPD concentrations (0.5, 1, 2, 5 μM), or (C) 0.5 μM rapamycin (Rap) for 24 h in ARPE-19 cells. Cytotoxicity was assessed using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Mean ± standard deviation (SD), n = 3; *P<0.05, **P<0.01, and #P<0.001 compared to vehicle group.
Fig 2.
Protopanaxadiol (PPD) restored lysosomal pH and activity and chloroquine (CQ)-induced lysosomal alteration in adult retinal pigment epithelial (ARPE)-19 cells.
(A) Colorimetric fluorescence images of ARPE-19 cells stained with pH-sensitive lysosomal dye, Lysosensor Green DND-189 where light green denotes relatively alkaline lysosomal pH. Cells were treated with vehicle, 100 μM CQ, CQ+PPD, and 2 μM PPD for 6 h and stained with 1 μM Lysosensor. Bafilomycin A1 (BA1, 100 nM) and ammonium chloride (NH4Cl, 10 mM) were used as anti-lysosomal experimental controls. (B) Bars denote relative changes in green fluorescence intensity analyzed using “Mean region of interest [ROI]” option in Zen software (n = 3–9). (C) Florescence microscopy images of ARPE-19 cells showing lysosomal intracellular activity. Cells were treated as in (A) with self-quenched substrate. Veh: untreated positive control, BA1: negative control cells. Dark green indicates stronger lysosomal intracellular activity than that shown by light green. (D) Bars show relative changes in green fluorescence intensity analyzed using “Mean ROI” option in Zen software (n = 3–9). (E) Fluorescence-activated cell sorting (FACS) acquisition and analysis of ARPE-19 cells showing lysosomal intracellular activity. Comparison of histograms of flow cytometric analysis (fluorescent channel 1/fluorescein isothiocyanate [FL1/FITC] channel), showing the inhibition of substrate de-quenching following treatment with lysosome-depolarizing agent: unstained (light brown curve), untreated positive control (black curve), CQ (red curve), CQ+PPD (green curve), PPD (crimson curve), and experimental control cells treated with 1× BA1 (blue curve). Scale bar, 20 μm. Mean ± standard deviation (SD), ns; not significant, n = 3; *P<0.05 and #P<0.001 compared to vehicle group.
Fig 3.
Protopanaxadiol (PPD) enhances liberation of adult retinal pigment epithelial (ARPE)-19 cells from chloroquine-induced autophagy congestion.
(A) Phase-contrast photomicrograph of ARPE-19 cells. Large vacuoles in cells treated with CQ for 6 h (black arrows). (B) Confocal images of green fluorescent protein (GFP)-light chain 3 (LC3, green)-transfected cells immunostained with lysosomal membrane-associated protein (LAMP)-2 (red) antibody. Cells treated with vehicle, 100 μM CQ, 2 μM PPD, or 2 μM PPD+100 μM CQ for 6 h. 4’,6-Diamidino-2-phenylindole (blue) counterstained nuclei. Scale bar, 10 μm. Quantification of GFP-LC3-transfected ARPE-19 cells to analyze LC3-positive vacuole (C) size, (D) number (green), and (E) number of LC3-positive (green) vacuoles colocalized (yellow, white arrows) with LAMP-2 (red). (F, H) Western blotting (WB) and (G, I) densitometric analysis of Beclin-1, LC3-II, and p62 in the ARPE-19 cell lysates 6 h after treatment with drugs as in (A). Protein expression levels were compared to levels of α-tubulin and values under blots indicate relative protein expression levels determined by densitometric analysis, unless otherwise specified. Data are means ± standard deviation (SD), n = 3; ns; not significant, *P<0.05, **P<0.01 and #P< 0.001 vs each control.
Fig 4.
Protopanaxadiol (PPD) protected adult retinal pigment epithelial (APRE)-19 cells from chloroquine-mediated reactive oxygen species (ROS) formation and apoptosis.
(A) Western blots and (B) quantitative analysis of Bcl-2 and Bcl-xL in ARPE-19 lysates treated with vehicle, 100 μM CQ, 2 μM PPD+100 μM CQ, and 2 μM PPD for 6 h. (C) Apoptosis assay using flow cytometry and (D, E) quantitative analysis of apoptosis of ARPE-19 cells treated with different drug combinations for 24 h. Bottom left and right show normal and early apoptotic cells, respectively; top right and left show late apoptotic and necrotic cells, respectively. (F) Fluorescence images and (G) relative intensity of DCF-positive fluorescence in ARPE-19 cells. Cells treated with different drug combinations for 24 h were stained with H2DCFDA for 1 h. Staurosporine (0.1 μM) was positive control for ROS measurement. Means ± standard deviation (SD), ns, *P<0.05, **P<0.01, and #P< 0.001 vs each control.
Fig 5.
Protopanaxadiol (PPD)-induced alteration on chloroquine-mediated interaction of Beclin-1 BH3 domain with Bcl-2 and cell proliferation associated signaling in adult retinal pigment epithelial (ARPE)-19 cells.
ARPE-19 cell lysates (A, B) immunoprecipitated (IP) or (C, D) immunoblotted (IB) with antibodies. Cells were treated with vehicle, 100 μM CQ, 2 μM PPD+100 μM CQ, and 2 μM PPD for 6 h, followed by IP and IB. Data are means ± standard deviation (SD), n = 3; ns, *P<0.05, **P<0.01, and #P<0.001 vs each control.
Fig 6.
AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) signals are associated with cytoprotective effect of protopanaxadiol (PPD) in chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells.
(A) Western blotting (WB) and (B) quantitative analysis of phosphorylated (p)-AMPK, AMPK, p-mTOR, mTOR, p-c-Jun N-terminal kinase (JNK), JNK, p-p38 mitogen-activated protein kinase (MAPK, p-p38), and p38 MAPK (p38) expression in ARPE-19 cells 6 h after treatment with vehicle, 100 μM CQ, 2 μM PPD+100 μM CQ, 2 μM PPD, and 0.5 μM rapamycin (Rap). Data are means ± standard deviation (SD), n = 3; ns, *P<0.05, **P<0.01, and #P<0.001 vs each control.
Fig 7.
Modulation of AMP-activated protein kinase (AMPK)-induced alteration of protopanaxadiol (PPD) effects on chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells.
(A) Relative survival rate of ARPE-19 cells treated with vehicle, 100 μM CQ, CQ+PPD, and 2 μM PPD with vehicle, 10 μM AICAR, or 500 nM Compound C for 24 h. Cytotoxicity was assessed using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (B, C) Western blotting (WB) and quantitative analysis of phosphorylated (p)-AMPK, AMPK, light chain 3 (LC3) I/II, p62, and B-cell lymphoma 2 (Bcl-2). Cells were treated with various drug combinations as indicate in (A) for 6 h. Bars denote relative changes in each protein. Data are means ± standard deviation (SD), n = 3; ns, *P<0.05, **P<0.01, and #P<0.001 vs control vehicle.
Fig 8.
Schematic representation of effect of protopanaxadiol (PPD) on crosstalk between autophagy and apoptosis in chloroquine-treated adult retinal pigment epithelial (ARPE)-19 cells.
PPD activated AMP-activated protein kinase (AMPK) activity but inhibited CQ-induced increase of mechanistic target of rapamycin (mTORC) activity and anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) expression. This action altered CQ-mediated interactions between Beclin-1 and Bcl-2 via Bcl-2 homology region 3 (BH3) domain, to activate Beclin-1. Beclin-1, LC3, and p62 are associated with autophagosome formation and autophagy flux. PPD may act critically on gateway of autophagy and apoptosis intersection in mediating CQ-treated ARPE-19 cell toxicity, restoring normal cellular homeostasis.