Published OnlineFirst July 20, 2012; DOI: 10.1158/0008-5472.CAN-12-0567
Cancer
Research
Molecular and Cellular Pathobiology
Ectopic ATP Synthase Blockade Suppresses Lung
Adenocarcinoma Growth by Activating the Unfolded Protein
Response
Hsin-Yi Chang1, Hsuan-Cheng Huang4,5, Tsui-Chin Huang1,2, Pan-Chyr Yang6,7,8,
Yi-Ching Wang9, and Hsueh-Fen Juan1,2,3
Abstract
Ectopic expression of the mitochondrial F1F0-ATP synthase on the plasma membrane has been reported to
occur in cancer, but whether it exerts a functional role in this setting remains unclear. Here we show that ectopic
ATP synthase and the electron transfer chain exist on the plasma membrane in a punctuated distribution of lung
adenocarcinoma cells, where it is critical to support cancer cell proliferation. Applying ATP synthase inhibitor
citreoviridin induced cell cycle arrest and inhibited proliferation and anchorage-independent growth of lung
cancer cells. Analysis of protein expression profiles after citreoviridin treatment suggested this compound
induced the unfolded protein response (UPR) associated with phosphorylation the translation initiation factor 2a
(eIF2a), triggering cell growth inhibition. Citreoviridin-enhanced eIF2a phosphorylation could be reversed by
siRNA-mediated attenuation of the UPR kinase PKR-like endoplasmic reticulum kinase (PERK) combined with
treatment with the antioxidant N-acetylcysteine, establishing that reactive oxygen species (ROS) boost UPR after
citreoviridin treatment. Thus, a coordinate elevation of UPR and ROS initiates a positive feedback loop that
convergently blocks cell proliferation. Our findings define a molecular function for ectopic ATP synthase at the
plasma membrane in lung cancer cells and they prompt further study of its inhibition as a potential therapeutic
approach. Cancer Res; 72(18); 1–11. 2012 AACR.
Introduction
F1F0-ATP synthase catalyzes the phosphorylation of ADP to
ATP by exploiting a transmembrane proton gradient (1).
Although F1F0-ATP synthase was initially thought to be located
exclusively in the mitochondrial inner membrane, its presence
Authors' Affiliations: 1Institute of Molecular and Cellular Biology, 2Department of Life Science, 3Graduate Institute of Biomedical Electronics and
Bioinformatics, National Taiwan University; 4Institute of Biomedical Informatics, 5Center for Systems and Synthetic Biology, National Yang-Ming
University; 6Department of Internal Medicine, National Taiwan University
Hospital and College of Medicine; 7NTU Center for Genomic Medicine,
National Taiwan University College of Medicine; 8Institute of Biomedical
Sciences, Academia Sinica, Taipei; and 9Department of Pharmacology,
National Cheng Kung University, Tainan, Taiwan
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Authors: Pan-Chyr Yang, Department of Internal Medicine, National Taiwan University Hospital and College of Medicine, No. 7,
Chung-Shan South Road, Taipei 100, Taiwan. Phone: 886-2-23123456 ext.
88671 or 88673; Fax: 886-2-2358-2867; E-mail: [email protected]; YiChing Wang, Department of Pharmacology, College of Medicine, National
Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan. Phone:
886-6-2353535 ext. 5502; Fax: 886-6-2749296; E-mail:
[email protected]; and Hsueh-Fen Juan, Department of Life
Science, Institute of Molecular and Cellular Biology, Graduate Institute of
Biomedical Electronics and Bioinformatics, National Taiwan University, No.
1, Sec. 4, Roosevelt Rd., Taipei 116, Taiwan. Phone: 886-2-3366-4536;
Fax: 886-2-2367-3374; E-mail: [email protected]
doi: 10.1158/0008-5472.CAN-12-0567
2012 American Association for Cancer Research.
has now been described on the outside of the plasma membrane
of highly proliferated cells both normal cells and tumor cells (2–
9). However, these studies reveal ectopic ATP synthase with a
variety functions depending on cell types and relatively little is
known about ectopic ATP synthase in tumor cells.
Ectopic ATP synthase has been shown to have several roles
in normal cells. In endothelial cells, ectopic ATP synthase is the
receptor of angiostatin, an endogenous angiogenesis inhibitor
that blocks neovascularization (2, 10). Angiostatin and the ATP
synthase F1 inhibitor protein IF1 can block ATP synthesis and
hydrolysis by the enzyme and inhibit the proliferation and
migration of cultured endothelial cells (2, 11). In hepatocytes,
ectopic ATP synthase was identified as the receptor for highdensity lipoprotein (HDL) endocytosis, and IF1 significantly
decreases HDL internalization in HepG2 cells, showing the
participation of ectopic ATP synthase in the regulation of
cholesterol homeostasis (5). In keratinocytes, ectopic ATP
synthase mediates the release secretion of ATP into the culture
medium, which plays a crucial role in normal epidermal
homeostasis and wound healing (8). In neural cells, ectopic
ATP synthase has been found to bind to amyloid precursor
protein and amyloid b-peptide, which are involved in the
pathogenesis of Alzheimer's disease (12). During adipogenesis,
ectopic ATP synthase is markedly increased, and may be a
potential target for anti-obesity drugs (4, 7, 13).
Although ectopic ATP synthase has been found on the
extracellular surface of several different cancer cell types
(6, 14–16), unlike in normal cells, its function in tumor cells
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is unknown. Expression of the ectopic ATP synthase b subunit
has been reported on Daudi, K562, and RPMI 8226 tumor cells,
and this subunit can be recognized by g/d T lymphocytes
through interaction with the T-cell receptor via apoA-I (9).
Thus, ectopic ATP synthase may be an antigen of tumor cells
and involved in the immune response to tumor cells. Treating
these cancer cells with biological (i.e., antibody or inhibitor of
F1 of ATP synthase, IF1) or chemical synthetic inhibitors of
ectopic ATP synthase markedly inhibits cell growth (15, 17),
thereby highlighting ectopic ATP synthase as a potential and
novel therapeutic target for cancer.
By understanding how inhibitors of ectopic ATP synthase
induce cytotoxicity and by elucidating the molecular mechanisms underlying this process, we aim to improve our understanding of their potential anticancer activity and lay the
foundations for therapeutic application. Specifically, we used
a proteomics approach and constructed a protein–protein
interaction (PPI) network, to investigate the role of tumor ectopic ATP synthase and the effects of its inhibitor citreoviridin,
which exhibits specific inhibition on F1F0 ATP synthase (18, 19).
Materials and Methods
Cell culture
Human A549 lung carcinoma cells and IMR-90 lung fibroblasts were obtained from the American Type Culture Collection. Human lung carcinoma cells CL1-0 were cultured as
previously described (20). Cells were cultured at 37 C and 5%
CO2 in Dulbecco's Modified Eagle's Medium supplemented
with 10% FBS and routinely passaged when 90% to 95% confluent. All the cells were free of mycoplasma as determined by a
PCR-based mycoplasma detection method (MBI Fermentas).
Drug treatment
The ATP synthase inhibitor citreoviridin (Fermentek Biotechnology) was solubilized in dimethyl sulfoxide (DMSO) at
20 mmol/L and diluted in medium at the concentrations
indicated. The control samples were treated with the same
volume of DMSO only (Sigma-Aldrich). All the procedures
including drug preparation and treatment were carried out
in the dark.
Immunofluorescence staining and flow cytometry
Cells were plated onto poly-L-lysine–coated glass coverslips
for 24 hours and fixed in 4% formaldehyde. Immunofluorescence was carried out as described previously (15). Primary
antibodies used to probing NDUFB4, SDHA, UQCRC2, COX5A,
and ATP5B were purchased from Abcam and anti-ATP
synthase complex mouse monoclonal antibody was obtained
from MitoSciences. The secondary antibody used was Alexa
Fluor 488–conjugated goat anti-mouse IgG (Molecular
Probes). All antibody incubations were carried out at room
temperature for 1 hour, after which the samples were washed
three times with PBS. Cell nuclei were stained with 40 -6diamidino-2-phenylindole (DAPI) for 10 minutes, the mitochondria were stained with Mito-ID Red Detection Kit (Enzo
Life Sciences Inc.) and coverslips were mounted with AntiFade
Prolong solution (Molecular Probes). Cells were analyzed
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with a fluorescence microscope or a Leica TCS SP5 spectral
scanning confocal microscope with a Leica HCX PL APO CS
100.0 1.40 OIL objective (Leica Lasertechnik). A stack of
consecutive image planes with vertical distances was taken for
each sample.
For flow cytometric analysis, cells were labeled with ATP5B
and ATP complex antibodies. Cells were washed with PBS
twice, suspended in PBS and then analyzed by FACSCanto
instrument (Becton Dickinson). The fluorescence data were
further analyzed with WinMDI 2.9 software (Scripps Research
Institute, Jupiter, FL).
Extracellular ATP generation assay
The levels of extracellular ATP (eATP) secreted by A549,
CL1-0, and IMR-90 cells were assayed by a bioluminescence
assay kit (Sigma-Aldrich) according to the manual. A total of
2 104 cells were seeded in 24-well plate and allowed to attach
for 16 hours. The cells were refreshed with medium containing
5 mmol/L citreoviridin or DMSO for 30 minutes. Then, after
adding 200 mmol/L ADP for 1 minute, the samples were
centrifuged to eliminate the cells, and the concentration of
ATP in the aliquots was determined according to the user
manual by the bioluminescence assay kit using FlexStation III
(Molecular Devices). Data are expressed in micromoles of ATP
per 1 106 cells on the basis of standards determined for each
independent experiment.
Flow cytometric detection of mitochondrial membrane
potential
To assess the mitochondrial membrane potential (MMP),
after incubation with 5 mmol/L citreoviridin for 48 hours, cells
were incubated with 100 nmol/L DiOC6 for 15 minutes at 37 C.
Then, the cells were washed with PBS twice, suspended in PBS
and then analyzed by FACSCanto instrument (Becton Dickinson). The fluorescence data were further analyzed with
WinMDI 2.9.
Flow cytometric detection of reactive oxygen species
For reactive oxygen species (ROS) detection, cells were
treated with citreoviridin at the IC50 for 6, 12, and 24 hours.
Cells were then washed, trypsinized, and incubated with
1 mmol/L 20 ,70 -dichlorofluorescein diacetate (H2DCFDA,
Molecular Probes) in the dark at 37 C for 30 minutes. Cells
were then washed twice with PBS and analyzed by FACSCanto
instrument (Becton Dickinson) and FlowJo 7.1 (Treestar, Inc.).
Proliferation assay using xCELLigence system and MTS
assays
The xCELLigence System (Roche), an electronic analyzer
with sensor electrodes coated on the tissue culture plate,
provides growth information in real time, which can reflect
the cell behavior immediately once recording after drug treatment. xCELLigence cell index impedance measurements were
done according to the manufacturer's instructions. In brief,
after 30 minutes equilibration in the medium, 5,000 cells were
seeded in 100-mL culture medium to each well of the
E-plate 16, and the attachment, spreading, and proliferation
of the cells were monitored every hour by the xCELLigence
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Targeting Ectopic ATP Synthase in Lung Cancer
system. Approximately 24 hours after seeding, when the cells
were in log phase growth, the cells were exposed to 50 mL of
medium containing the ATP synthase inhibitor citreoviridin or
DMSO only as the control. The final concentration of citreoviridin was 0, 2, 4, and 6 mmol/L. The concentration of DMSO
was 0.1%. All experiments were repeated three times and
examined every hour for 48 hours. The average IC50 was
calculated throughout the 48 hours for each cell lines by
xCELLigence system.
Five thousand cells were plated in 96-well plates and allowed
to adhere overnight. The medium then was discarded, and the
cells were pretreated with chemical chaperones or antioxidants. Chemical chaperones 10 mmol/L tauroursodeoxycholic
acid (TUDCA, Sigma-Aldrich) and 1 mmol/L 4-phenyl butyric
acid (4-PBA, Sigma-Aldrich) were pretreated for 2 hours, and
10 mmol/L antioxidant N-acetyecysteine (NAC, Sigma-Aldrich)
was pretreated for 30 minutes. Citreoviridin was treated for
further 24 hours after chemical chaperones and antioxidant
removed. For extracellular nucleotide and calcium signaling
involvements, 10 mmol/L ATP (Sigma-Aldrich) and 0.1 mmol/L
EGTA (Sigma-Aldrich) were added 5 minutes after citreoviridin
treatment. The concentration of citreoviridin for each cell lines
was used as their IC50. Growth inhibition was measured by
using MTS (Promega Corporation) assay. One hundred percent
viability refers to the MTS value for 0.1% DMSO-treated cells.
Colony formation assay
For anchorage-dependent growth assays, 200 A549 or CL1-0
cells/well were seeded in 6-well plates, incubated with citreoviridin at their IC50 or 0.1% DMSO control for 10 days, fixed with
methanol, and stained with crystal violet. For anchorageindependent growth assays, 500 cells were mixed with 2 mL
low melting point agar (0.35% in DMEM with citreoviridin or
DMSO mentioned above) and overlaid on 0.7% agar (2 mL) in
each well of 6-well plates. The plates were incubated for 14 days,
fixed, and stained. Colonies with a diameter greater than
100 mm were counted.
DNA content analysis
To determinate cell cycle distributions, 1.5 105 A549 or
CL1-0 cells were exposed to citreoviridin at their IC50 or 0.1%
DMSO control in DMEM with 10% FBS for 12 hours or 24 hours.
Cells were washed, trypsinized, collected and fixed in 70% cold
ethanol (–20 C) overnight. Cells were then washed twice with
PBS and resuspended in PBS containing 1 mg/mL RNase A and
incubated at 37 C for 30 minutes and followed by propidium
iodide (PI, 10 mg/mL) staining for 15 minutes. The DNA content
of cells was then analyzed with a FACSCanto instrument
(Becton Dickinson). The percentage of cells in different phases
of the cell cycle was calculated by MultiCycle (DeNovo
software).
Protein extraction
Total protein was extracted from 1 107 cells by 0.5 mL lysis
solution containing 7 M urea (Boehringer), 2 M thiourea (J. T.
Baker), 4% CHAPS (J. T. Baker), and 0.002% bromophenol blue
(Amersco). The mixture was discontinuously sonicated for 2
minutes on ice. The lysates were centrifuged for 30 minutes at
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4 C at 15,000 g. The supernatant was collected, and the
protein concentration was measured by a protein assay kit
(Bio-Rad) according the manual.
Transfection of siRNA
The siRNAs directed against human PKR-like endoplasmic
reticulum kinase (PERK siRNA, pools of three target specific 19
to 25 nt siRNAs) and the nontargeting negative control siRNA
(Control siRNA) were purchased from Santa Cruz Biotechnology Inc. Another independent siRNA targeting PERK and
scrambled control siRNA were obtained from OriGene
(SR306267-3, OriGene). Cancer cells were transfected with the
PERK siRNA or control siRNA with Lipofectamine 2000 (Invitrogen) for 48 hours according to the manufacturer's protocol
and treated with citreoviridin or DMSO for a further 12 hours.
The final concentration of the siRNAs was 10 nmol/L.
Western blotting
The proteins were extracted using lysis buffer as described
previously. Total proteins (20 mg) were separated by PAGE and
blotted onto polyvinyl-difluoride membranes (Millipore). After
blocking with 5% nonfat milk in PBST at room temperature for
30 minutes, membranes were probed with antibodies. Antibodies against BiP, PERK, EroLa, PDI, IRE1a, eIF2a, phosphoeIF2a, and phospho-PERK were purchased from Cell Signaling
Technology Inc. Actin antibody was purchased from Millipore.
All secondary antibodies were obtained from Sigma-Aldrich.
After incubation with primary and secondary antibodies, immunoblots were visualized with the ECL detection kit (Pierce
Biotechnology Inc.) and exposed to Fuji medical X-ray film.
Statistical analysis
All experiments were carried out at least 3 times. Data are
expressed as mean SD. Unpaired 2-tailed t tests were used for
the comparison of two groups. P values < 0.05 were considered
significant.
Results
ATP synthase and the electron transport chain are
expressed on the surface of lung cancer cells
ATP synthase consists of 2 regions, the transmembrane F0
portion and the F1 portion with the ATPase activity. The F1
sector is composed of a3, b3, g, d, and e subunit, where b
subunit provides enzyme activity to convert ADP to ATP as
well as hydrolysis of ATP. To measure the expression levels of
ATP synthase on lung cells, antibodies probed for ATP
synthase b subunit and the ATP synthase complex were
applied to quantitative and qualitative measurements by flow
cytometry (Fig. 1A) and confocal microscopy (Fig. 1B). ATP
synthase b subunit and the whole complex were found to be
expressed on the cell surface of A549 and CL1-0 lung cancer
cells but not on normal fibroblast IMR-90. Without permeablization of the cell, the antibodies were restricted outside the
cell and can only recognize the structures projecting from the
cell. Both the expression of ATP synthase complex and b
subunit are localized on the cell surface not colocalized with
mitochondria staining in A549 and CL1-0 cells (Fig. 1B). With
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Figure 1. Expression of active ATP synthase on the surface of lung cancer cells. A, ectopic ATP synthase is expressed on A549 and CL1-0 lung cancer cells,
but not normal IMR-90 fibroblasts. The expression of ATP synthase b (anti-ATP5B) and the ATP synthase complex (anti-ATP synthase complex) were analyzed
by flow cytometry. B, expression of ectopic ATP synthase was observed by confocal microscopy in A549 and CL1-0 lung cancer cells. DAPI was used
to stain nuclei. b and b0 show the x/z and y/z projections, respectively. C, extracellular ATP concentration was determined after treatment with citreoviridin
(Citreo) or DMSO (vehicle control) for 30 minutes in three cell lines. Asterisks indicate significant differences between the control and the treated group
from three independent experiments (P < 0.01). D, immunocytochemistry of proteins from ETC protein complexes. The ETC complexes were probed by
NUDUFB4, SDHA, UQCRC2, COX5A, and ATP synthase antibodies, followed by Alexa 488 conjugated anti-mouse IgG secondary antibody, and then
counterstained with fluorescent DAPI for DNA. The bottom left squares show the enlarged portion of each panel. Scale bars, 10 mm. E, affinity-enriched
plasma membrane proteins from CL1-0 and A549 cells. Western blot analysis of NDUFB4, SDHA, UQCRC2, COX5A, and ATP5B were component of complex
I, II, III, IV, and V, respectively.
not only the b subunit but the whole catalytic complex located
on the lung cancer cell surface, we suggested that the complete
ectopic ATP synthase may exhibit enzymatic activities. To
further confirm this implication, bioluminescence assay for
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eATP detection was carried out after adding inhibitor and
DMSO control. Relative to control, eATP concentration significantly decreased following treatment with 5 mmol/L citreoviridin for 30 minutes in A549 and CL1-0 cells, but not the
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Targeting Ectopic ATP Synthase in Lung Cancer
ectopic ATP synthase negative cells IMR-90 (Fig. 1C). The remaining level of eATP suggest that there are other ATPase or
ATP-permeable release channels contributing to the homeostasis of eATP, and the inhibitory efficacy of citreoviridin may
be specific for F1F0 ATP synthase.
As the generation of proton gradient is required for ATP
production by ATP synthase, we examined the existence of the
electron transport chain (ETC) on plasma membrane by
immunocytochemistry and purification of plasma membrane
proteins. The distribution pattern of the examined proteins in
nonpermeable cells was punctuated (Fig. 1D), and was quite
different from that in Triton X-100 permeable ones showing
typical mitochondrial pattern (Fig. S1). In addition, the ETC
proteins and the ATP synthase b were presented in the
biotinylated purification of plasma membrane proteins (Fig.
1E and Supplementary Fig. S2). We revealed that not only ATP
synthase but ETC proteins were located on the plasma membrane with a punctuated distribution. Other proteomics studies have also been reviewed for the respiratory chain on cell
surfaces (21), which supports our findings. Taken together,
these results indicate that ectopic ATP synthase and ETC are
localized on the plasma membrane of lung cancer cells and can
generate ATP.
Citreoviridin inhibits the proliferation of lung cancer
cells by inducing G0–G1 phase arrest
To further explore the role of the ectopic ATP synthase on
cell survival of lung cancer cells, we treated both lung cancer
cells and normal lung cells with the ATP synthase inhibitor
citreoviridin and observed their real-time cell growth curves.
Citreoviridin inhibited lung cancer cell proliferation in a dosedependent manner but did not have an effect on the growth of
normal human IMR-90 fibroblasts (Fig. 2A). According to the
results, cell proliferation and attachment was inhibited 4 to 6
hours after treatment with citreoviridin. The average half
maximal inhibitory concentrations (IC50) for 48 hours treatment were 1.5, 4.65, and more than 6 mmol/L for A549, CL1-0,
and IMR-90 cells, respectively. The variation of IC50 may be
due to the difference of the amount of ectopic ATP synthase in
each cell line. We assumed that the more expression of ectopic
ATP synthase, the higher concentration of citreoviridin was
needed to reach the inhibitory threshold. To distinguish the
effects of citreoviridin on cell proliferation, whether the mitochondria function is inhibited may need further to be explored.
To determine the effects of the inhibitor treatment on the
mitochondrial electron transfer chain, we measured the MMP
by DiOC6 in both CL1-0 and A549 cells (Fig. 2B and C).
Comparing to paraformaldehyde caused depletion of MMP
(Gate M1), cells treated with citreoviridin for 48 hours were
maintained in the population of M2 where retained the MMP
as the DMSO treated control. The results suggest that citreoviridin only inhibit the activity of ectopic, and not mitochondrial, ATP synthase.
Next, we examined the means by which the inhibition of
ectopic ATP synthase inhibited lung cancer cell proliferation.
Cell-cycle analysis by flow cytometry indicated that
Figure 2. Citreoviridin does not affect mitochondrial membrane potential (MMP), but selectively inhibits proliferation of lung cancer cells. A, cell proliferation
was monitored by the xCELLigence RTCA system. The cell index was normalized to the time when the drug was added. Growth was measured for 48 hours.
The average IC50 of 48 hours was calculated by the RTCA system to be 1.5, 4.65, and more than 6 mmol/L for A549, CL1-0 and IMR-90 cells, respectively. B and
C, citreoviridin does not affect the MMP. Cells were stained with DiOC6 and monitored by fluorescence microscopy (B) and flow cytometry (C). Scale
bars, 50 mm. Positive CTL, cells were treated with 4% paraformaldehyde for 15 minutes as the positive control. Cells with altered MMP were gated by M1 and
M2 for depolarized and polarized MMP, respectively. The proportion of M1 and M2 gated cells is plotted.
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citreoviridin increased the percentage of cells in the G0–G1
phase in both CL1-0 and A549 lung cells within 12 hours and
markedly at 24 hours after treatment (Fig. 3A and B). This
suggests that citreoviridin may inhibit cell proliferation
through the inhibition of cell cycle progression at a specific
phase.
Citreoviridin reduces anchorage-independent growth of
lung cancer cells
To determine whether citreoviridin may inhibit anchoragedependent or anchorage-independent growth by blocking
oncogenes that are required for cell survival and/or growth
signals normally provided to adherent nontransformed cells by
the extracellular matrix via integrins, we investigated anchorage-dependent growth by colony formation on tissue culture
plates and anchorage-independent growth by soft agar assay at
their IC50 to both cell lines. We found that citreoviridin
inhibited anchorage-dependent growth of both CL1-0 and
A549 cells after treatment of 2 weeks (Fig. 3C). Regarding
anchorage-independent growth, cells treated with IC50 citreo-
viridin formed significantly fewer and smaller colonies in the
soft agar than cells treated with DMSO alone (Fig. 3D). These
results suggest that the inhibition of ectopic ATP synthase is
involved in the attenuation of anchorage-independent growth,
a feature of malignant transformation of lung cancer cells.
Proteomic analysis identifies changes in CL1-0 cells with
citreoviridin treatment
To investigate the effects of citreoviridin on protein expression, comprehensive time-course protein expression profiles
were analyzed by proteomic analysis (Supplementary Fig. S3).
Performing 2DE, the amounts of protein spots were quantified
using ImageMaster and proteins were identified using mass
spectrometry (see Table S1 for the differentially expressed
proteins). We also analyzed the PPI network of the identified
differentially expressed proteins. In total, 30 of 49 MS-identified
proteins were mapped to the PPI network (P < 0.005). Gene
ontology (GO) functional enrichment analysis of the dataset
indicated that protein folding (8 proteins), negative regulation
of ubiquitin-protein ligase activity involved in mitotic cell cycle
Figure 3. Citreoviridin affects cell-cycle distribution and reduces colony formation in lung cancer cells. A, CL1-0 and B, A549 cells were treated with IC50
citreoviridin, 4.65 and 1.5 mmol/L, respectively. Cells treated with citreoviridin (Citreo) or DMSO for 12 and 24 hours were harvested and analyzed for
cell-cycle distribution by PI staining. The percentage of cells in each phase was calculated by ModFit LT 3.0. Asterisks indicate significant differences between
the control and treated group from three independent experiments (P < 0.01). C, anchorage-dependent growth of CL1-0 and A549 was estimated after
14 days treatment with IC50 citreoviridin (Citreo). D, anchorage-independent growth of CL1-0 and A549 cells was estimated in 0.35% soft agar
after 14 days treatment of IC50 citreoviridin. The AIG colonies were observed using microscopy or a high-resolution scanner (top left). Scale bars,
100 mm. The numbers of colonies were counted. Asterisks indicate significant differences between the control and treated group from three independent
experiments (P < 0.01).
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(7 proteins), and mRNA processing (5 proteins) were the top
three cellular functions that are altered by citreoviridin treatment (Fig. 4).
Citreoviridin induces the unfolded protein response
Further analysis of the GO terms revealed that protein
folding was the process most affected by the citreoviridin
treatment (Fig. 4). Disruption of ER homeostasis leads to the
accumulation of unfolded proteins. The ER has developed an
adaptive mechanism known as the unfolded protein response
(UPR) to cope with altered protein folding. Accumulation of
unfolded proteins in the ER induces the dissociation of the
chaperone protein BiP from an ER transmembrane sensor
known as PKR-like ER-localized eIF2a kinase (PERK) and
inosital-requiring 1a (IRE1a). The dimerization and phosphorylation of PERK and IRE1a subsequently activate the UPR.
Activated PERK phosphorylates eIF2a, which in turn inhibits
the assembly of translational machinery and thereby represses
protein synthesis, which reduces the workload of the ER and
can reduce protein accumulation in the ER. Expression of the
ER stress markers in CL1-0 and A549 lung cancer cells was
investigated by western blotting (Fig. 5A and B). Citreoviridin
treatment increased the expression of chaperone proteins,
including BiP, and protein disulfide isomerase (PDI). Oxidizing
proteins, such as IRE1a, were also increased. The most immediate response to ER stress is transient attenuation of mRNA
translation by increased phosphorylation of eIF2a. Accordingly, although the protein levels of eIF2a remain constant
following citreoviridin treatment, the data showed that eIF2a
was phosphorylated following treatment with the inhibitor, in
both cell lines tested (Fig. 5C). We observed a corresponding
increase in the expression of PERK following citreoviridin
treatment (Fig. 5B). These results suggested that the growth
attenuation of citreoviridin-treated cells may be due to the
induction of the UPR and the inhibition of protein synthesis.
To confirm that PERK was involved in eIF2a phosphorylation in citreoviridin treated cells, we carried out PERK knockdown in both cell lines using RNA interference for 48 hours, and
then treated them with citreoviridin for 12 hours. The phosphorylation and expression levels of eIF2a were analyzed by
western blotting and normalized to the expression of actin
(Fig. 5D). The phosphorylation of eIF2a was abolished after
citreoviridin treatment in the PERK siRNA cells, whereas the
total eIF2a levels were unaffected. These data suggest that the
phosphorylation of eIF2a is mediated by PERK.
Citreoviridin induces ROS dependent UPR
Studies have indicated cross-talk between ER stress and
oxidative stress (22, 23). Our proteomics data also showed that
glutathione S-transferase Mu 3 and glutathione S-transferase P,
enzymes participating in detoxification by conjugating
reduced glutathione to electrophilic substrates were upregulated upon citreoviridin treatment. To verify whether citreoviridin caused ROS accumulation, H2DCFDA was used to
measure the level of endogenous ROS. The geometric mean
of fluorescence intensity was measured by flow cytometric
analysis (Fig. 6A). The results indicated that ROS levels were
elevated by citreoviridin in a time-dependent manner. To see
whether the citreoviridin induced UPR was ROS dependent,
free radical scavenger NAC was used. Treating with citreoviridin, the phosphorylation level of eIF2a was reduced (Fig. 6B)
and the cell viability was recovered (Fig. 6C) upon NAC
pretreatment, indicating citreoviridin induced UPR was ROS
dependent.
Figure 4. The PPI network of MS-identified proteins (round node) and their common interacting partners (triangle node). The interactions are indicated by gray
lines using the IntAct PPI database as the reference dataset. Modules in color represent enrichment of GO terms (right). Note that the top enriched function is
protein folding.
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Figure 5. Citreoviridin induces the UPR and phosphorylation of eIF2a. Proteins involved in the UPR were examined by immunoblotting of IC50 citreoviridintreated (A) CL1-0 and (B) A549 cells. C, phosphorylation of eIF2a was detected in both cell lines with citreoviridin treatment at their IC50 concentrations. The
value of each band was normalized to its actin control and the time point 0 hour. D, CL1-0 and A549 cells were transfected with siRNA targeting PERK or a
scrambled control. The siRNA knockdown was carried out independently with two individual systems, which were obtained from Santa Cruz Biotechnology
(siRNA-1) and OriGene (siRNA-2). Knockdown was assessed by Western blot analysis. Phosphorylation of eIF2a was determined and normalized to the level
of total eIF2a. The knockdown of PERK was validated in protein expression level. Actin was used as the internal control.
Citreoviridin-induced UPR does not activate the
apoptotic cascade
Misfolded or unassembled proteins retained in the ER are
degraded by ER-associated degradation (ERAD) through the
ubiquitin/26S proteosome-dependent pathway (24) or autophagy (25). If stressed cells fail to cope with the UPR, cells
undergo cell death, mainly via apoptosis (26). However, we did
not observe apoptotic cell death by annexin V/PI staining in
citreoviridin-treated CL1-0 and A549 cells (Supplementary Fig.
S4), indicating the citreoviridin-induced inhibition on cell
proliferation was caused by restriction of cell cycle progression
but not cell death. Recent studies have highlight that p53
selectively transactivate quite different responses, ranging
from cell cycle arrest to cell death and senescence (27–29).
Although the detailed regulating mechanism remains unclear,
we postulate that the p53 regulated cell cycle/apoptosis decision may be involved in the citreoviridin-induced pathway.
To further confirm this possibility, we treated the ectopic
ATP synthase expressed p53 null cell line H1299 with citreoviridin ranged from 0 to 8 mmol/L (Supplementary Fig. S5).
The results showed citreoviridin only inhibited 12.5% of cell
proliferation at the highest concentration, indicating citreoviridin induced inhibition on cell growth is p53 mediated.
Discussion
In the past decade, ectopic ATP synthase has been shown to
involve a variety of functions in lipid metabolism, immune
recognition, and invasiveness of tumors (3, 6, 9, 16), regulation
of intracellular pH (14, 30), differentiation (13), control of
proliferation and cell death (3, 10, 15). Ectopic ATP synthase
has been shown to localize on the cell membrane of different
cancer cell types. Here, we show that the ATP synthase
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Cancer Res; 72(18) September 15, 2012
complex and ETC are localized on the membrane of lung
cancer cells. In an attempt to shed light on the cellular
processes affected by the action of this complex, and to provide
further insights into the mechanistic action of the ATP
synthase inhibitor citreoviridin, we show that the inhibition
of ectopic ATP synthase is associated with the inhibition of
lung cancer cell growth and the activation of UPR. By disrupting the homeostasis of the ER, citreoviridin could specifically
target ectopic ATP synthase-expressing cancer cells and effectively inhibit growth with limited side effects on normal cells.
Cancer cells can pose numerous microenvironmental challenges to surrounding tissues, such as through hypoxia, nutrient limitation, oxidative stress, metabolic dysregulation, or low
pH. In turn, these stresses can promote the activation of
specific signaling pathways, sometimes from the ER via the
accumulation of misfolded proteins in the lumen (31). To
account for this severe microenvironment and support proliferation, tumor cells have a higher capacity for rapid protein
synthesis and degradation than normal cells (32–35). Inhibition of the ERAD pathway by proteasomal inhibitors (36) or
protein folding by PDI inhibitors (37) induces the UPR and
cytotoxicity in tumor cells. This implies that the homeostasis of
ER capacity is critical in tumor progression and recurrence (38,
39).
But how could citreoviridin activate UPR? There are several
possible mechanisms. First, it may be due to the inhibition of
eATP formation by citreoviridin. Because we have now shown
that lung cancer ectopic ATP synthase generates ATP (Fig. 1C),
it is reasonable to speculate that citreoviridin disturbs the
homeostasis of extracellular nucleotides, which may have
further effects on cell signaling. For example, eATP can activate
plasma membrane-localized ATP-gated ion channel (P2X)
receptors and G protein-coupled (P2Y) receptors in an
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Targeting Ectopic ATP Synthase in Lung Cancer
Figure 6. Citreoviridin induces ROS accumulation and chemicals confer citreoviridin induced growth inhibition. A, A549 and CL1-0 cells were treated with
citreoviridin (Citreo) at their IC50 for indicated times. DMSO was used as the vehicle control. The ROS scavenger NAC was administered to cells at 5 and 10
mmol/L for 30 minutes. After washing, cells were detached, and the total ROS were detected by the H2DCFDA. The signal of the fluorescence was measured
by flow cytometry and the geometric mean was analyzed by FlowJo7.1. Experiments were repeated three times independently. Asterisks indicate significant
differences between the control and treated group (P < 0.05). B, the phosphorylation level of eIF2a was determined in citreoviridin (IC50) treated or combined
with 10 mmol/L antioxidant NAC pretreated group. Phosphorylation and total level of eIF2a was normalized to each actin and the DMSO treated control. C,
attempted rescue of ectopic ATP synthase reverse the citreoviridin-induced growth inhibition. ATP and the calcium chelator EGTA were administered 5
minutes later after citreoviridin (Citreo) treatment at their IC50 concentrations, and DMSO was used as the vehicle control. Cells were pretreated with the ROS
inhibitor NAC for 30 minutes and chemical chaperon 4-PBA or TUDCA for 2 hours. The cell viability was measured by the MTS assay and normalized to the
DMSO control treatment. The relative cell viability after 24-hour treatment from three independent experiments is shown. Asterisks indicate significant
differences from the DMSO control group (P < 0.05).
autocrine or paracrine manner (40). Furthermore, ATP is
degraded rapidly to ADP and 50 -AMP, the latter of which is
subsequently converted by ectopic 50 -nucleotidases into adenosine, which acts as an agonist of the P1 receptor (41). Although
extracellular nucleotides and nucleosides are important for
growth and death signal transduction (40, 42), whether the
disruption of P1 and P2 receptor signaling by the inhibition of
ectopic ATP synthase activity participates in the regulation of
growth or other effects remains to be seen. Yang and colleagues
showed a delicate proteomics study of the human ABCC1
interacting proteome and revealed ATP synthase a binds to
ABCC1 in plasma membranes and may cooperate to regulate
eATP level and purinergic signaling cascade (43), supporting
the regulation of eATP by ectopic ATP synthase.
Second, citreoviridin-induced acidosis may activate the
UPR. Treatment with citreoviridin for 48 hours decreased the
pH of the culture medium from 7.0 to 6.7 and 7.1 to 6.8 for CL1-0
and A549 cells, respectively. The acidosis of culture medium
may induce ER stress and cause cytotoxicity (44). Ectopic ATP
synthase may act as an intracellular pH regulator because of its
role in proton transport (12, 14, 45), and the inhibition of
ectopic ATP synthase is enhanced under acidic conditions (14).
The inhibition of ectopic ATP synthase by citreoviridin may
disrupt homeostasis of intracellular pH and cause extracellular
acidosis, thus triggering the UPR by increasing inhibitory
efficacy of citreoviridin.
www.aacrjournals.org
Third, the induction of ROS forms a positive feedback loop to
enhance ER stress. Our results showed that citreoviridin
induces ROS production (Fig. 6A). Raj and colleagues reported
that piperlongumine selectively kills cancer cells by increasing
the level of ROS, but this was dependent on the cancer
genotype (46). Furthermore, a mitochondrial mutation in
MTATP6, one of the subunit of ATP synthase, which results
in an elevated level of cytosolic ROS and MMP has been shown
(47), suggesting the dysfunction of ATP synthase may contribute to impaired oxidative phosphorylation and ROS production. As there is the whole respiratory chain in the plasma
membrane that generates partial eATP in lung cancer cells
and the MMP was not affected by citreoviridin, we assumed
that the citreoviridin-induced production of ROS may be due
to the impairment of oxidative phosphrylation in the plasma
membrane but not mitochondria. The active excessive ROS
oxidize proteins and ultimately results in protein damage.
Generation of ROS directly or indirectly affects ER homeostasis
and protein folding by calcium signaling, thus results in ER
stress and vise versa (48). Our results therefore suggest that
citreoviridin-induced ROS elevation may contribute to the
selective inhibition of growth in cancer cells.
Fourth, besides those arguments listed above, the aberrant
regulation of intracellular calcium from cancer cell activity
may also induce ER stress. Once the newly synthesized nascent
proteins extrude from polysomes into the lumen, they undergo
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Chang et al.
extensive modifications, including glycosylation and disulfide
bond formation. The ER contains a pool of calcium-dependent
molecular chaperone proteins (49), such as Grp94, calnexin,
and calreticulin, which assist in protein folding, disulfide bond
formation, and N-linked glycosylation. The depletion of ER
luminal calcium and increased cytosolic calcium cause dysfunction of the molecular chaperones and leads to the UPR
(50).
The addition of ATP, the antioxidant NAC, chemical chaperones, or the calcium chelator EGTA rescued citreoviridininduced growth inhibition (Fig. 6C), suggesting that regulation
of the citreoviridin-induced inhibition of cell proliferation is
complex, and many mechanisms in addition to UPR activation
are involved (Supplementary Fig. S6). It would therefore be
interesting to investigate whether pathways enriched in the
PPI network could have a synergic/addictive effect with UPR in
citreoviridin-induced growth inhibition. Here, we provided
evidence that in combination of only dose of 1 mmol/L citreoviridin with 10 nmol/L 26S proteosome inhibitor bortezomib
caused significantly decreasing in cell viability when comparing to single agent treatment (Fig. S7), implying the possibility
of synergic/addictive therapy for citreoviridin and proteosome
inhibitors.
This study provides the first evidence that the inhibition of
ectopic ATP synthase induces the UPR, which disrupts the
balance between life and death in lung cancer cells and highlights the therapeutic potential of ectopic ATP synthase inhibition in cancer cells. Further investigations of the ectopic ATP
synthase PPI network, including the active and nonactive state
downstream signaling, will be crucial for a more comprehensive understanding of its function in the cell membrane of
tumor cells.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: H.-Y. Chang, H.-C. Huang, T.-C. Huang, P.-C. Yang, H.F. Juan
Development of methodology: H.-Y. Chang, T.-C. Huang, P.-C. Yang, H.-F. Juan
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): H.-Y. Chang, T.-C. Huang, H.-F. Juan
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): H.-Y. Chang, H.-C. Huang, T.-C. Huang, P.-C. Yang, H.F. Juan
Writing, review, and/or revision of the manuscript: H.-Y. Chang, T.-C.
Huang, P.-C. Yang, Y.-C. Wang, H.-F. Juan
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T.-C. Huang, H.-F. Juan
Study supervision: P.-C. Yang, Y.-C. Wang, H.-F. Juan
Grant Support
This work was supported by National Research Program for Genomic
Medicine (NSC 100-3112-B-002-011), National Science Council of Taiwan (NSC
97-2311-B-002-010-MY3 and NSC 99-2621-B-002-005-MY3), National Taiwan
University Cutting-Edge Steering Research Project (10R70602C3) and the National Health Research Institutes, Taiwan (NHRI-EX98-9819PI).
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received February 16, 2012; revised July 11, 2012; accepted July 12, 2012;
published OnlineFirst July 20, 2012.
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Ectopic ATP Synthase Blockade Suppresses Lung
Adenocarcinoma Growth by Activating the Unfolded Protein
Response
Hsin-Yi Chang, Hsuan-Cheng Huang, Tsui-Chin Huang, et al.
Cancer Res Published OnlineFirst July 20, 2012.
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