Page 43 - ebook

Casein kinase 1-induced regulation of autophagy and apoptosis in chloroquine-
mediated adult retinal pigment epithelial-19 cells

1
Haesung Lee , Long Ngo Hoang , Ngoc Buu Tran , Sook-Jeong Lee¹
1
1
1 Department of Bioactive Material Science, Jeonbuk National University, Jeonju, Jeollabuk-do, 54896, Republic of Korea
ABSTRACT 3. D4476 protects ARPE-19 cells from CQ-mediated apoptotic cell death
A B
The antimalarial drug chloroquine (CQ) is a lysosomotropic agent and is currently CQ, 100 μM - + + -
D4476, 10 μM - - + + -tubulin)
used as a potential anticancer agent as well as an autophagy inhibitor, also induces
Bcl-2
retinopathy in which characterized by lysosomotropic alteration. D4476 is a highly Protein expression
Bcl-xL (each protein/
effective, selective ATP-competitive casein kinase 1 (CK1) inhibitor, which may α-tubulin
prevent or enhance apoptosis. In this study, we examined whether D4476 could
attenuate retinopathy induced by CQ in adult human retinal pigment epithelial C
(ARPE-19) cells. CQ-treated ARPE-19 cells resulted in defective lysosomal Veh CQ, 100 μM CQ + D4476 D4476, 10 μM
degradation and caused accumulation of autophagosomes. Furthermore, the
disruption of autophagy initiation and the formation of autolysosome was caused by
PI
Beclin 1 interacting with Bcl-2, which was trapped by CQ. D4476, on the other
hand, mitigated CQ-induced effects, rescuing ARPE-19 cells from CQ-induced
toxicity by modulating the association of Beclin 1 and Bcl-2. As a result, D4476 Annexin-V
regulates autophagy and apoptosis simultaneously by upregulating autophagy flux (A) Western blots and (B) quantitative analysis of Bcl-2 and Bcl-xL in ARPE-19 lysates treated with vehicle, 100
and activating the expression of anti-apoptotic proteins via inhibition of mTOR, μM CQ alone, 10 μM D4476 plus 100 μM CQ, and 10 μM D4476 alone for 6 h. (C) Apoptosis assay using flow
cytometry in ARPE-19 cells. Flow cytometry scatterplots show propidium iodide (PI) versus Annexin-V (Y and X
JNK, and p38 MAPK signals. In conclusion, D4476 appears to be a promising axis, respectively). Cells were treated with vehicle, 100 μM CQ alone, 10 μM D4476 plus 100 μM CQ, and 10 μM
treatment strategy for CQ-mediated retinopathy. D4476 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 (values are relative percentage of total cell population). Data are
means ± SD, n=3; ns, not significant, *P<0.05, **P<0.01 compared with vehicle group.
PURPOSE 4. Effect of D4476 on CQ-induced interaction of Beclin 1 BH3 domain with Bcl-2
and cell proliferation-associated signaling in ARPE-19 cells.
 In order to determine the effect of D4476 on CQ-induced retinopathy ARPE-19 cells and
further elucidate the mechanism of action A CQ, 100 μM - + + - B CQ, 100 μM - + + - C
CQ, 100 μM -
D4476, 10 μM - - + + D4476, 10 μM - - + + D4476, 10 μM - + - + + + - - -
Bcl-2 Bcl-2 Rap, 0.5 μM - - - - +
IP: Bcl-2
Beclin 1 Beclin 1 IB p-mTOR
mTOR
α-tubulin
RESULTS 2.5 CTL p-AMPK
CQ+D4476
1. D4476 protects ARPE-19 cells from CQ-induced retinopathy 2.0 CQ ** AMPK
p-p38
D4476
1.5
p38
A 125 B 125 C Relative protein expression 1.0 * ** * p-JNK
100 * ** 100 *** 0.5 ** ** JNK
% Survival (normalized) 75 50 ** *** 75 # * * ** ** % Survival (normalized) 0.0 Bcl-2 Beclin1 α-tubulin
50
25 25 ARPE-19 cell lysates (A) immunoprecipitated (IP) or (B) immunoblotted (IB) with each antibody. Immunoblots of
anti- Bcl-2 and anti-Beclin 1 antibodies quantified using densitometric analysis in ImageJ. (C) Western blots for p-
0 0 mTOR, mTOR, p-AMPK, AMPK, p-p38, p38, p-JNK, and JNK in APRE-19 cell lysates 6 h after treatment with
0 10 50 75 100 CQ 0 100 100 100 100 100 100 vehicle, 100 μM CQ alone, 10 μM D4476 plus 100 μM CQ, 10 μM D4476 alone, and 0.5 μM Rap alone. Means ±
D4476 0 0 1 2 5 10 20
CQ [ M] SD, n=3; ns, not significant; *P<0.05, **P<0.01 versus vehicle.
[ M]
Cytotoxicity of ARPE-19 cells treated with (A) varying concentrations of CQ alone, (B) 100 μM CQ with vehicle
or different D4476 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<0.001 compared to vehicle; *P<0.05, **P<0.01,
and ***P<0.001, compared to CQ alone. 5. Schematic representation of D4476 effects on crosstalk between autophagy
and apoptosis in CQ-treated ARPE-19 cells.
2. D4476 attenuates CQ-induced autophagy defects in ARPE-19 cells D4476 CQ D4476
JNK p38 MAPK
Phase contrast GFP-LC3 LAMP-2 DAPI Merge
A B mTORC1
Veh C CQ, 100 μM - + + - -
D4476, 10 μM - - + + -
Rap, 0.5 μM - - - - + Beclin1 BH3 Bcl-2
Beclin 1
p62 LC3-II Bcl-2
p62
CQ LC3
Autophagy Apoptosis
α-tubulin
D
CQ
Relative expression
+ -tubulin)
D4476 Cell Survival Cell Death
D4476 (each protein/ CONCLUSIONS
10 μm 10 μm
1. We found that D4476 prevented ARPE-19 cells from CQ-mediated cytotoxicity.
(A) Phase-contrast photomicrograph and (B) confocal images of GFP-LC3 (green)-transfected cells after 2. D4476 protected retinal cells from CQ-induced autophagy inhibition.
immunostaining with LAMP-2 (red) antibody. Cells were treated with vehicle, 100 μM CQ alone, 10 μM
D4476 alone, or 10 μM D4476 plus 100 μM CQ for 6 h. DAPI (blue) was used to counterstain nuclei. Scale 3. D4476 rescued ARPE-19 cells from CQ-induced apoptotic death.
bar, 10 μm. Quantification of LC3- positive vacuole. (C) Western blots and (D) quantitative analysis of 4. D4476 effects were caused by disruption of CQ-induced interaction between
Beclin 1, p62, and light chain 3 (LC3) expression in ARPE-19 cell lysates 6 h after treatment with vehicle,
100 μM CQ alone, 10 μM D4476 plus 100 μM CQ, 10 μM D4476 alone, and 0.5 μM rapamycin (Rap) Beclin 1 and Bcl-2.
[alone, with vehicle, or with 100 nM BA1]. Data are means ± SD, n=3; ns, not significant; *P<0.05,
**P<0.01 and ***P<0.001 versus vehicle.
REFERENCES
ACKNOWLEDGEMENT 1. Carrino M et al. Prosurvival autophagy is regulated by protein kinase CK1 alpha in
multiple myeloma. Cell Death Discov. 2019;5:98.
This work was supported by the Basic Science Research Program through the National 2. Cheong JK et al. Casein kinase 1 alpha-dependent feedback loop controls autophagy
Research Foundation of Korea (NRF) funded by the Ministry of Education in RAS-driven cancers. J Clin Invest. 373 2015;125:1401-1418.
(No.2016R1D1A1B04934383) and by the NRF grant funded by the Korea government 3. Gaynes BI et al. Retinal toxicity of chloroquine hydrochloride administered by
(MSIT) (No.2021R1A2C1005980). intraperitoneal injection. J Appl Toxicol. 2008;28:895-900.