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Ginsenoside protopanaxadiol-induced changes in autophagy and apoptosis in
chloroquine-induced adult retinal pigment epithelial-19 cells

1
1
1
Haesung Lee , Long Ngo Hoang , Ngoc Buu Tran , Sook-Jeong Lee*¹
1 Department of Bioactive Material Science, Jeonbuk National University, Jeonju, Jeollabuk-do, 54896, Republic of Korea

ABSTRACT 3. Inhibitory effect of PPD on CQ-induced apoptosis and ROS formation in ARPE-19 cells
A B
Ginsenosides are the major active component found in ginseng (Panax ginseng). CQ, 100 μM - + + -
PPD, 2 μM - - + + -tubulin)
Among the ginsenosides, (20S)-Protopanaxadiol (PPD) ginsenosides Rg3, Rh2
Bcl-2
have been demonstrated for their hepatoprotection, tumor suppression and Bcl-XL Relative expression
diabetes resistance. Chloroquine (CQ) is an antimalarial drug known to inhibit α-tubulin (each protein/
autophagy flux by impairing autophagosome–lysosome fusion, frequently causes
C Veh CQ, 100 μM
serious eye and vision problems. In this study, we determined whether the PPD
reduces CQ-induced retinopathy by restoring lysosomotropic abnormalities in
D 40 E
human adult retinal pigment epithelial (ARPE)-19 cells. The accumulation of 50 #
30 # 40
autophagosomes with fusion defects in lysosomes in CQ-treated ARPE-19 cells, 30 **
PI CQ + PPD PPD, 2 μM 20 Population (%) (apotototic + necrotic) 20
which trapped Beclin-1 with B-cell lymphoma 2 (Bcl-2), interfered with autophagy 10 ** ns 10 ns
initiation and autophagosome development. PPD alleviated the CQ-induced toxicity 0 0
CQ - + + - CQ - + + -
by modulating the interaction between Beclin-1 and Bcl-2, and this effect was PPD - - + + PPD - - + +
mediated by the 5' adenosine monophosphate-activated protein kinase-mammalian
Annexin-V
target of rapamycin signal axis. The results indicate that autophagy and apoptosis
(A) Western blots and (B) quantitative analysis of Bcl-2 and Bcl-XL in ARPE-19 lysates treated with
were simultaneously controlled by PPD via the upregulation of autophagy flux and 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 apoptotic cells in ARPE-19 cells treated with different drug
increase of anti-apoptotic protein expression. These findings suggest that PPD may 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.
be a promising treatment strategy for CQ-mediated retinopathy.
4. Regulatory effect of PPD on crosstalk between apoptosis and autophagy via
PURPOSE interference with BH3-domain-associated interaction between Beclin-1 and Bcl-2
A
Ø In order to determine whether the PPD reduces CQ-induced retinopathy by restoring CQ, 100 μM - + + - C CQ, 100 μM - + + - E
PPD, 2 μM - - + + PPD, 2 μM - - + + Rap, 0.5 μM + - - - -
lysosomotropic abnormalities in human adult retinal pigment epithelial (ARPE)-19 cells. CQ, 100 μM - - + + -
Bcl-2 Bcl-2
IP: Bcl-2 PPD, 2 μM - - - + +
Beclin 1 Beclin 1 IB
p-AMPK
α-tubulin
B 2.0 CTL CQ+PPD D AMPK
RESULTS CQ PPD 2.0 CTL CQ+PPD p-mTOR
PPD
#
CQ
mTOR
1.5
ns 1.5 #
ns p-JNK
ns
1. Protective effect of PPD on CQ-induced ARPE-19 cell death and possible 1.0 ** ns 1.0 ns ns ns JNK
autophagy involvement ** ns p-p38
0.5 0.5
p38
A B C 0.0 0.0 α-tubulin
Bcl-2 Beclin1 Bcl-2 Beclin1
ARPE-19 cell lysates (A, B) immunoprecipitated (IP) or (C, D) immunoblotted (IB) with antibodies. (E) Western
blots of p-AMPK, AMPK, p- mTOR, mTOR, p-JNK, JNK, p-p38 MAPK (p-p38), and p38 MAPK (p38)
expression in ARPE-19 cells lysates 6h 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 ± SD, n=3; ns, *P<0.05, **P<0.01, #P<0.001 vs each
control.
5. Schematic representation of effect of PPD on crosstalk between autophagy and
apoptosis in CQ-treated ARPE-19 cells
Cytotoxicity of ARPE-19 cells treated with (A) varying concentrations of CQ alone, (B) 100 μM CQ with
vehicle or different PPD concentrations, (C) 100 μM CQ with vehicle or 1 μM Rap for 24h. Cytotoxicity
#
assessed using MTT assay. Mean ± standard deviation [SD], n=3, *P<0.05, **P<0.01, P<0.001,
compared to CQ alone.
2. Effect of PPD on liberation of ARPE-19 cells from CQ-induced autophagy
congestion
PPD inhibited CQ-induced increase of mTORC1 activity, but activated AMPK activity, which altered CQ-
mediated interaction between Beclin-1 and Bcl-2 via BH3 domain. PPD may act critically on gateway of
autophagy and apoptosis intersection in CQ-treated ARPE-19 toxicity, restoring normal cellular homeostasis.
CONCLUSIONS
1. Autophagy and apoptosis were simultaneously controlled by PPD via the upregulation of
autophagy flux and increased anti-apoptotic protein expression.
2. PPD may be a promising treatment strategy for CQ-mediated retinopathy.
(A) Phase-contrast photomicrograph of ARPE-19 cells. Large vacuoles in cells treated with CQ for 6 h
(black arrows). (B) Confocal images of GFP-LC3 (green)-transfected cells immunostained with LAMP-2
(red) antibody. Cells treated with vehicle, 100 μM CQ, 2 μM PPD, or 2 μM PPD+100 μM CQ for 6 h. DAPI REFERENCES
(blue) counterstained nuclei. Scale bar, 10 μm. (C) Western blotting and and (D) densitometric analysis of
Beclin-1, LC3, and p62 from the lysates of ARPE-19 cells exposed to different drug combinations for 6hr.
1 Gaynes BI et al. Retinal toxicity of chloroquine hydrochloride administered by
intraperitoneal injection. J Appl Toxicol 2008 Oct; 28(7): 895-900.
ACKNOWLEDGEMENT http://doi.org/10.1002/jat.1353.
2. Kang R et al. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ
This work was supported by the Basic Science Research Program through the National 2011 Apr; 18(4): 571-80. http://doi.org/10.1038/cdd.2010.191.
Research Foundation of Korea (NRF) funded by the Ministry of Education 3. Attele AS et al. Ginseng pharmacology: multiple constituents and multiple actions.
Biochem Pharmacol 1999 Dec 1; 58(11):1685-93. http://doi.org/10.1016/s0006-
(No.2016R1D1A1B04934383) and by the NRF grant funded by the Korea government
2952(99)00212-9.
(MSIT) (No.2021R1A2C1005980).