Glycogen Synthase Kinase-3? Mediates Endoplasmic
Reticulum Stress-Induced Lysosomal Apoptosis in Leukemia□
Wei-Ching Huang, Yee-Shin Lin, Chia-Ling Chen, Chi-Yun Wang, Wei-Hsin Chiu, and
Institute of Basic Medical Sciences (W.-C.H., Y.-S.L., C.-Y.W., C.-F.L.), Graduate Institute of Clinical Medicine (W.-C.H.,
C.-Y.W., W.-H.C., C.-F.L.), Center for Gene Regulation and Signal Transduction Research (Y.-S.L.), and Departments of
Microbiology and Immunology (W.-C.H., Y.-S.L., C.-L.C., C.-Y.W., C.-F.L.), and Internal Medicine (W.-H.C.), National Cheng
Kung University Medical College, Tainan, Taiwan
Received October 30, 2008; accepted January 30, 2009
Glycogen synthase kinase (GSK)-3? may modulate endoplasmic
reticulum (ER) stress-induced apoptosis; however, the mecha-
nism remains unclear. Our data showed that human monocytic
not chronic myeloid leukemia K562, cells were susceptible to
apoptosis induced by ER stressor tunicamycin, a protein glyco-
sylation inhibitor. Tunicamycin caused early activation of
caspase-2, -3, -4, and -8, followed by apoptosis, whereas
caspase-9 was slowly activated. Inhibiting caspase-2 reduced
activation of caspase-8 and -3 but had no effect on caspase-4.
Tunicamycin induced apoptosis independently of the mitochon-
drial pathway but caused lysosomal destabilization followed by
lysosomal membrane permeabilization (LMP), cathepsin B reloca-
tion from lysosomes to the cytosol, and caspase-8 and -3 activa-
tion. It is notable that caspase-2 mediated lysosomal destabiliza-
tion. Inhibiting GSK-3? comprehensively reduced lysosomal
apoptosis after caspase-2 inhibition. Unlike U937 and HL-60 cells,
K562 cells showed nonresponsive ER stress and failure of activa-
tion of GSK-3? and caspase-2 in response to tunicamycin. Acti-
vating GSK-3? caused K562 cells to be susceptible to tunicamy-
cin-induced apoptosis. Taken together, we show that GSK-3?
exhibits a mechanism of ER stress-induced lysosomal apoptosis
in leukemia involving caspase-2-induced LMP and cathepsin B
relocation, which result in caspase-8 and -3 activation.
Leukemia is pathologically characterized by abnormal ex-
pansion of hematopoietic progenitor cells. Several drugs have
been developed for therapeutic applications; however, leuke-
mia remains problematic because of relapse and drug resis-
tance. The proapoptotic action of antileukemic agents may
act through the induction of endoplasmic reticulum (ER)
stress (Du et al., 2006; Rahmani et al., 2007). Therefore,
cellular ER is a target for improved, selective anticancer
therapies (Linder and Shoshan, 2005; Boelens et al., 2007).
However, the molecular mechanism of ER stress-triggered
apoptosis in leukemia remains unresolved.
The ER is the site of protein synthesis, modification, and
folding. Multiple insults, such as inhibition of glycosylation,
reduction of disulfide bonds, calcium depletion from the ER
lumen, impairment of protein transport to the Golgi, and
expression of mutated proteins in the ER, can trigger un-
folded protein response (UPR) after ER stress (Wu and Kauf-
man, 2006). These events enhance protein folding and deg-
radation within the ER and down-regulate protein synthesis
until cells have recovered from ER stress. However, pro-
longed ER stress may eventually cause apoptosis, whereas
calcium homeostasis and UPR cannot be restored. In ER
stress-induced apoptosis, several apoptotic signaling path-
ways are demonstrated (Breckenridge et al., 2003). ER
stress-induced CCAAT/enhancer-binding protein homolo-
This work was supported by the National Science Council, Taiwan [Grant
NSC 96-2320-B-006-018-MY3]; and the National Cheng Kung University,
Taiwan [Landmark Project C020].
Article, publication date, and citation information can be found at
S The online version of this article (available at http://jpet.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: ER, endoplasmic reticulum; UPR, unfolded protein response; CHOP, CCAAT/enhancer-binding protein homologous protein;
LMP, lysosomal membrane permeabilization; GSK, glycogen synthase kinase; Z-FA-fmk, benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone;
pepstatin A, Isovalery-Val-Val-Sta-Ala-Sta; SB415286, 3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrol-2,5-dione; LY294002,
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride; DMSO, dimethyl sulfoxide; DAPI, 4,6-diamidino-2-phenylindole; PI, propidium
iodide; AO, acridine orange; Z-VDVAD-fmk, benzyloxycarbonyl-Val-Asp(Ome)-Val-Ala-Asp(Ome)-fluoromethyl ketone; Z-IETD-fmk, benzyloxycar-
bonyl-Ile-Glu(Ome)-Thr-Asp(Ome)-fluoromethyl ketone; Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; GRP, glucose-regu-
lated protein; PBS, phosphate-buffered saline; HIAP2, human inhibitor of apoptosis 2; PP2A, protein phosphatase 2A; 17-AAG, 17-(allylamino)-
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
JPET 329:524–531, 2009
Vol. 329, No. 2
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Supplemental material to this article can be found at:
at ASPET Journals on December 26, 2015
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associated with ER stress-induced GSK-3? activation and apo-
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Our data show that the heat shock protein 90 inhibitor 17-AAG
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and caspase-2 are unable to be activated in K562 cells,
whereas UPR also fails to be induced. Thus, we hypothesize
that these events cause an apoptosis-resistant phenotype in
K562 cells. Compared with the signaling pathways in U937
and HL-60 cells, it became evident that GSK-3? acts as an
apoptosis-promoting target. In accordance, we demonstrated
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implicate the involvement of GSK-3?-regulated lysosomal
pathway in ER stress-induced apoptosis.
We thank Dr. Robert Anderson for critical reading of this manu-
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Address correspondence to: Dr. Chiou-Feng Lin, Institute of Clinical Med-
icine, National Cheng Kung University Medical College, Tainan 701, Taiwan.
ER Stress Induces Lysosomal Apoptosis through GSK-3?
at ASPET Journals on December 26, 2015