Ectopic ATP Synthase Blockade Suppresses Lung ... · Thus, ectopic ATP synthase may be an antigen of tumor cells and involved in the immune response to tumor cells. Treating these

Molecular and Cellular Pathobiology

Ectopic ATP Synthase Blockade Suppresses LungAdenocarcinoma Growth by Activating the Unfolded ProteinResponse

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

AbstractEctopic 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 ectopicATP synthase and the electron transfer chain exist on the plasmamembrane in a punctuated distribution of lungadenocarcinoma cells, where it is critical to support cancer cell proliferation. Applying ATP synthase inhibitorcitreoviridin induced cell cycle arrest and inhibited proliferation and anchorage-independent growth of lungcancer cells. Analysis of protein expression profiles after citreoviridin treatment suggested this compoundinduced the unfolded protein response (UPR) associatedwith phosphorylation the translation initiation factor 2a(eIF2a), triggering cell growth inhibition. Citreoviridin-enhanced eIF2a phosphorylation could be reversed bysiRNA-mediated attenuation of the UPR kinase PKR-like endoplasmic reticulum kinase (PERK) combined withtreatment with the antioxidant N-acetylcysteine, establishing that reactive oxygen species (ROS) boost UPR aftercitreoviridin treatment. Thus, a coordinate elevation of UPR and ROS initiates a positive feedback loop thatconvergently blocks cell proliferation. Our findings define a molecular function for ectopic ATP synthase at theplasma membrane in lung cancer cells and they prompt further study of its inhibition as a potential therapeuticapproach. Cancer Res; 72(18); 4696–706. �2012 AACR.

IntroductionF1F0-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 locatedexclusively in the mitochondrial inner membrane, its presence

has nowbeendescribed on the outsideof the plasma membraneof highly proliferated cells both normal cells and tumor cells (2–9). However, these studies reveal ectopic ATP synthase with avariety functions depending on cell types and relatively little isknown about ectopic ATP synthase in tumor cells.

Ectopic ATP synthase has been shown to have several rolesin normal cells. In endothelial cells, ectopic ATP synthase is thereceptor of angiostatin, an endogenous angiogenesis inhibitorthat blocks neovascularization (2, 10). Angiostatin and the ATPsynthase F1 inhibitor protein IF1 can block ATP synthesis andhydrolysis by the enzyme and inhibit the proliferation andmigration of cultured endothelial cells (2, 11). In hepatocytes,ectopic ATP synthase was identified as the receptor for high-density lipoprotein (HDL) endocytosis, and IF1 significantlydecreases HDL internalization in HepG2 cells, showing theparticipation of ectopic ATP synthase in the regulation ofcholesterol homeostasis (5). In keratinocytes, ectopic ATPsynthasemediates the release secretion of ATP into the culturemedium, which plays a crucial role in normal epidermalhomeostasis and wound healing (8). In neural cells, ectopicATP synthase has been found to bind to amyloid precursorprotein and amyloid b-peptide, which are involved in thepathogenesis of Alzheimer's disease (12). During adipogenesis,ectopic ATP synthase is markedly increased, and may be apotential target for anti-obesity drugs (4, 7, 13).

Although ectopic ATP synthase has been found on theextracellular surface of several different cancer cell types(6, 14–16), unlike in normal cells, its function in tumor cells

Authors' Affiliations: 1Institute ofMolecular andCellular Biology, 2Depart-ment of Life Science, 3Graduate Institute of Biomedical Electronics andBioinformatics, National Taiwan University; 4Institute of Biomedical Infor-matics, 5Center for Systems and Synthetic Biology, National Yang-MingUniversity; 6Department of Internal Medicine, National Taiwan UniversityHospital and College of Medicine; 7NTU Center for Genomic Medicine,National Taiwan University College of Medicine; 8Institute of BiomedicalSciences, Academia Sinica, Taipei; and 9Department of Pharmacology,National Cheng Kung University, Tainan, Taiwan

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Pan-Chyr Yang, Department of Internal Medi-cine, National Taiwan University Hospital and College of Medicine, No. 7,Chung-ShanSouthRoad, Taipei 100, Taiwan. Phone: 886-2-23123456ext.88671 or 88673; Fax: 886-2-2358-2867; E-mail: [email protected]; Yi-Ching Wang, Department of Pharmacology, College of Medicine, NationalChengKungUniversity, No. 1,University Road, Tainan701, Taiwan. Phone:886-6-2353535 ext. 5502; Fax: 886-6-2749296; E-mail:[email protected]; and Hsueh-Fen Juan, Department of LifeScience, Institute of Molecular and Cellular Biology, Graduate Institute ofBiomedical Electronics andBioinformatics,National TaiwanUniversity, 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.

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is unknown. Expression of the ectopic ATP synthase b subunithas been reported on Daudi, K562, and RPMI 8226 tumor cells,and this subunit can be recognized by g/d T lymphocytesthrough interaction with the T-cell receptor via apoA-I (9).Thus, ectopic ATP synthase may be an antigen of tumor cellsand involved in the immune response to tumor cells. Treatingthese cancer cells with biological (i.e., antibody or inhibitor ofF1 of ATP synthase, IF1) or chemical synthetic inhibitors ofectopic ATP synthase markedly inhibits cell growth (15, 17),thereby highlighting ectopic ATP synthase as a potential andnovel therapeutic target for cancer.By understanding how inhibitors of ectopic ATP synthase

induce cytotoxicity and by elucidating the molecular mechan-isms underlying this process, we aim to improve our under-standing of their potential anticancer activity and lay thefoundations for therapeutic application. Specifically, we useda proteomics approach and constructed a protein–proteininteraction (PPI) network, to investigate the role of tumor ecto-pic ATP synthase and the effects of its inhibitor citreoviridin,which exhibits specific inhibition on F1F0 ATP synthase (18, 19).

Materials and MethodsCell cultureHuman A549 lung carcinoma cells and IMR-90 lung fibro-

blasts were obtained from the American Type Culture Collec-tion. Human lung carcinoma cells CL1-0 were cultured aspreviously described (20). Cells were cultured at 37�C and 5%CO2 in Dulbecco's Modified Eagle's Medium supplementedwith 10% FBS and routinely passaged when 90% to 95% con-fluent. All the cells were free of mycoplasma as determined by aPCR-based mycoplasma detection method (MBI Fermentas).

Drug treatmentThe ATP synthase inhibitor citreoviridin (Fermentek Bio-

technology) was solubilized in dimethyl sulfoxide (DMSO) at20 mmol/L and diluted in medium at the concentrationsindicated. The control samples were treated with the samevolume of DMSO only (Sigma-Aldrich). All the proceduresincluding drug preparation and treatment were carried outin the dark.

Immunofluorescence staining and flow cytometryCells were plated onto poly-L-lysine–coated glass coverslips

for 24 hours and fixed in 4% formaldehyde. Immunofluores-cence was carried out as described previously (15). Primaryantibodies used to probing NDUFB4, SDHA, UQCRC2, COX5A,and ATP5B were purchased from Abcam and anti-ATPsynthase complex mouse monoclonal antibody was obtainedfrom MitoSciences. The secondary antibody used was AlexaFluor 488–conjugated goat anti-mouse IgG (MolecularProbes). All antibody incubations were carried out at roomtemperature for 1 hour, after which the samples were washedthree times with PBS. Cell nuclei were stained with 40-6-diamidino-2-phenylindole (DAPI) for 10 minutes, the mito-chondria were stained with Mito-ID Red Detection Kit (EnzoLife Sciences Inc.) and coverslips were mounted with AntiFadeProlong solution (Molecular Probes). Cells were analyzed

with a fluorescence microscope or a Leica TCS SP5 spectralscanning confocal microscope with a Leica HCX PL APO CS100.0 � 1.40 OIL objective (Leica Lasertechnik). A stack ofconsecutive image planes with vertical distances was taken foreach sample.

For flow cytometric analysis, cells were labeled with ATP5Band ATP complex antibodies. Cells were washed with PBStwice, suspended in PBS and then analyzed by FACSCantoinstrument (Becton Dickinson). The fluorescence data werefurther analyzed with WinMDI 2.9 software (Scripps ResearchInstitute, Jupiter, FL).

Extracellular ATP generation assayThe levels of extracellular ATP (eATP) secreted by A549,

CL1-0, and IMR-90 cells were assayed by a bioluminescenceassay kit (Sigma-Aldrich) according to the manual. A total of2� 104 cells were seeded in 24-well plate and allowed to attachfor 16 hours. The cells were refreshed with medium containing5 mmol/L citreoviridin or DMSO for 30 minutes. Then, afteradding 200 mmol/L ADP for 1 minute, the samples werecentrifuged to eliminate the cells, and the concentration ofATP in the aliquots was determined according to the usermanual by the bioluminescence assay kit using FlexStation III(Molecular Devices). Data are expressed in micromoles of ATPper 1� 106 cells on the basis of standards determined for eachindependent experiment.

Flow cytometric detection of mitochondrial membranepotential

To assess the mitochondrial membrane potential (MMP),after incubation with 5 mmol/L citreoviridin for 48 hours, cellswere incubated with 100 nmol/L DiOC6 for 15minutes at 37�C.Then, the cells were washed with PBS twice, suspended in PBSand then analyzed by FACSCanto instrument (Becton Dick-inson). The fluorescence data were further analyzed withWinMDI 2.9.

Flow cytometric detection of reactive oxygen speciesFor 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 with1 mmol/L 20,70-dichlorofluorescein diacetate (H2DCFDA,Molecular Probes) in the dark at 37�C for 30 minutes. Cellswere then washed twice with PBS and analyzed by FACSCantoinstrument (Becton Dickinson) and FlowJo 7.1 (Treestar, Inc.).

Proliferation assay using xCELLigence system and MTSassays

The xCELLigence System (Roche), an electronic analyzerwith sensor electrodes coated on the tissue culture plate,provides growth information in real time, which can reflectthe cell behavior immediately once recording after drug treat-ment. xCELLigence cell index impedance measurements weredone according to the manufacturer's instructions. In brief,after 30 minutes equilibration in the medium, 5,000 cells wereseeded in 100-mL culture medium to each well of theE-plate 16, and the attachment, spreading, and proliferationof the cells were monitored every hour by the xCELLigence

Targeting Ectopic ATP Synthase in Lung Cancer

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system. Approximately 24 hours after seeding, when the cellswere in log phase growth, the cells were exposed to 50 mL ofmedium containing the ATP synthase inhibitor citreoviridin orDMSO only as the control. The final concentration of citreo-viridin was 0, 2, 4, and 6 mmol/L. The concentration of DMSOwas 0.1%. All experiments were repeated three times andexamined every hour for 48 hours. The average IC50 wascalculated throughout the 48 hours for each cell lines byxCELLigence system.

Five thousand cells were plated in 96-well plates and allowedto adhere overnight. The medium then was discarded, and thecells were pretreated with chemical chaperones or antioxi-dants. Chemical chaperones 10 mmol/L tauroursodeoxycholicacid (TUDCA, Sigma-Aldrich) and 1 mmol/L 4-phenyl butyricacid (4-PBA, Sigma-Aldrich) were pretreated for 2 hours, and10mmol/L antioxidantN-acetyecysteine (NAC, Sigma-Aldrich)was pretreated for 30 minutes. Citreoviridin was treated forfurther 24 hours after chemical chaperones and antioxidantremoved. For extracellular nucleotide and calcium signalinginvolvements, 10 mmol/L ATP (Sigma-Aldrich) and 0.1mmol/LEGTA (Sigma-Aldrich)were added 5minutes after citreoviridintreatment. The concentration of citreoviridin for each cell lineswas used as their IC50. Growth inhibition was measured byusingMTS (PromegaCorporation) assay. One hundred percentviability refers to the MTS value for 0.1% DMSO-treated cells.

Colony formation assayFor anchorage-dependent growth assays, 200 A549 or CL1-0

cells/well were seeded in 6-well plates, incubated with citreo-viridin at their IC50 or 0.1%DMSOcontrol for 10 days,fixedwithmethanol, and stained with crystal violet. For anchorage-independent growth assays, 500 cells were mixed with 2 mLlow melting point agar (0.35% in DMEM with citreoviridin orDMSO mentioned above) and overlaid on 0.7% agar (2 mL) ineachwell of 6-well plates. Theplateswere incubated for 14days,fixed, and stained. Colonies with a diameter greater than100 mm were counted.

DNA content analysisTo 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 DMEMwith 10%FBS for 12 hours or 24 hours.Cells were washed, trypsinized, collected and fixed in 70% coldethanol (–20�C) overnight. Cells were then washed twice withPBS and resuspended in PBS containing 1mg/mL RNase A andincubated at 37�C for 30 minutes and followed by propidiumiodide (PI, 10mg/mL) staining for 15minutes. TheDNA contentof cells was then analyzed with a FACSCanto instrument(Becton Dickinson). The percentage of cells in different phasesof the cell cycle was calculated by MultiCycle (DeNovosoftware).

Protein extractionTotal protein was extracted from 1� 107 cells by 0.5mL 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 2minutes on ice. The lysates were centrifuged for 30 minutes at

4�C at 15,000 � g. The supernatant was collected, and theprotein concentration was measured by a protein assay kit(Bio-Rad) according the manual.

Transfection of siRNAThe siRNAs directed against human PKR-like endoplasmic

reticulum kinase (PERK siRNA, pools of three target specific 19to 25 nt siRNAs) and the nontargeting negative control siRNA(Control siRNA) were purchased from Santa Cruz Biotechnol-ogy Inc. Another independent siRNA targeting PERK andscrambled control siRNA were obtained from OriGene(SR306267-3, OriGene). Cancer cells were transfected with thePERK siRNA or control siRNA with Lipofectamine 2000 (Invi-trogen) for 48 hours according to the manufacturer's protocoland treated with citreoviridin or DMSO for a further 12 hours.The final concentration of the siRNAs was 10 nmol/L.

Western blottingThe proteins were extracted using lysis buffer as described

previously. Total proteins (20 mg) were separated by PAGE andblotted onto polyvinyl-difluoridemembranes (Millipore). Afterblocking with 5% nonfat milk in PBST at room temperature for30 minutes, membranes were probed with antibodies. Anti-bodies against BiP, PERK, EroLa, PDI, IRE1a, eIF2a, phospho-eIF2a, and phospho-PERKwere purchased from Cell SignalingTechnology Inc. Actin antibody was purchased fromMillipore.All secondary antibodies were obtained from Sigma-Aldrich.After incubation with primary and secondary antibodies, im-munoblots were visualized with the ECL detection kit (PierceBiotechnology Inc.) and exposed to Fuji medical X-ray film.

Statistical analysisAll experiments were carried out at least 3 times. Data are

expressed asmean� SD. Unpaired 2-tailed t tests were used forthe comparison of two groups. P values < 0.05 were consideredsignificant.

ResultsATP synthase and the electron transport chain areexpressed on the surface of lung cancer cells

ATP synthase consists of 2 regions, the transmembrane F0portion and the F1 portion with the ATPase activity. The F1sector is composed of a3, b3, g , d, and e subunit, where bsubunit provides enzyme activity to convert ADP to ATP aswell as hydrolysis of ATP. To measure the expression levels ofATP synthase on lung cells, antibodies probed for ATPsynthase b subunit and the ATP synthase complex wereapplied to quantitative and qualitative measurements by flowcytometry (Fig. 1A) and confocal microscopy (Fig. 1B). ATPsynthase b subunit and the whole complex were found to beexpressed on the cell surface of A549 and CL1-0 lung cancercells but not on normal fibroblast IMR-90. Without permea-blization of the cell, the antibodies were restricted outside thecell and can only recognize the structures projecting from thecell. Both the expression of ATP synthase complex and bsubunit are localized on the cell surface not colocalized withmitochondria staining in A549 and CL1-0 cells (Fig. 1B). With

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not only the b subunit but the whole catalytic complex locatedon the lung cancer cell surface, we suggested that the completeectopic ATP synthase may exhibit enzymatic activities. Tofurther confirm this implication, bioluminescence assay for

eATP detection was carried out after adding inhibitor andDMSO control. Relative to control, eATP concentration signif-icantly decreased following treatment with 5 mmol/L citreo-viridin for 30 minutes in A549 and CL1-0 cells, but not the

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-90fibroblasts. The expression ofATPsynthaseb (anti-ATP5B) and theATPsynthase complex (anti-ATPsynthasecomplex)were analyzedby flow cytometry. B, expression of ectopic ATP synthase was observed by confocal microscopy in A549 and CL1-0 lung cancer cells. DAPI was usedto 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 groupfrom three independent experiments (P < 0.01). D, immunocytochemistry of proteins from ETC protein complexes. The ETC complexes were probed byNUDUFB4, SDHA, UQCRC2, COX5A, and ATP synthase antibodies, followed by Alexa 488 conjugated anti-mouse IgG secondary antibody, and thencounterstained with fluorescent DAPI for DNA. The bottom left squares show the enlarged portion of each panel. Scale bars, 10 mm. E, affinity-enrichedplasmamembraneproteins fromCL1-0 andA549 cells.Western blot analysis of NDUFB4, SDHA,UQCRC2,COX5A, andATP5Bwere component of complexI, II, III, IV, and V, respectively.






ectopic ATP synthase negative cells IMR-90 (Fig. 1C). The re-maining level of eATP suggest that there are other ATPase orATP-permeable release channels contributing to the homeo-stasis of eATP, and the inhibitory efficacy of citreoviridin maybe specific for F1F0 ATP synthase.

As the generation of proton gradient is required for ATPproduction by ATP synthase, we examined the existence of theelectron transport chain (ETC) on plasma membrane byimmunocytochemistry and purification of plasma membraneproteins. The distribution pattern of the examined proteins innonpermeable cells was punctuated (Fig. 1D), and was quitedifferent from that in Triton X-100 permeable ones showingtypical mitochondrial pattern (Fig. S1). In addition, the ETCproteins and the ATP synthase b were presented in thebiotinylated purification of plasma membrane proteins (Fig.1E and Supplementary Fig. S2). We revealed that not only ATPsynthase but ETC proteins were located on the plasma mem-brane with a punctuated distribution. Other proteomics stud-ies have also been reviewed for the respiratory chain on cellsurfaces (21), which supports our findings. Taken together,these results indicate that ectopic ATP synthase and ETC arelocalized on the plasmamembrane of lung cancer cells and cangenerate ATP.

Citreoviridin inhibits the proliferation of lung cancercells by inducing G0–G1 phase arrest

To further explore the role of the ectopic ATP synthase oncell survival of lung cancer cells, we treated both lung cancer

cells and normal lung cells with the ATP synthase inhibitorcitreoviridin and observed their real-time cell growth curves.Citreoviridin inhibited lung cancer cell proliferation in a dose-dependent manner but did not have an effect on the growth ofnormal human IMR-90 fibroblasts (Fig. 2A). According to theresults, cell proliferation and attachment was inhibited 4 to 6hours after treatment with citreoviridin. The average halfmaximal inhibitory concentrations (IC50) for 48 hours treat-ment 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 bedue to the difference of the amount of ectopic ATP synthase ineach cell line. We assumed that the more expression of ectopicATP synthase, the higher concentration of citreoviridin wasneeded to reach the inhibitory threshold. To distinguish theeffects of citreoviridin on cell proliferation, whether the mito-chondria function is inhibitedmay need further to be explored.To determine the effects of the inhibitor treatment on themitochondrial electron transfer chain, we measured the MMPby 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 weremaintained in the population of M2 where retained the MMPas the DMSO treated control. The results suggest that citreo-viridin only inhibit the activity of ectopic, and not mitochon-drial, ATP synthase.

Next, we examined the means by which the inhibition ofectopic 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 proliferationwas 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 48hourswas calculatedby theRTCAsystem tobe1.5, 4.65, andmore than6mmol/L forA549,CL1-0 and IMR-90cells, respectively. B andC, citreoviridin does not affect the MMP. Cells were stained with DiOC6 and monitored by fluorescence microscopy (B) and flow cytometry (C). Scalebars, 50 mm. Positive CTL, cells were treated with 4%paraformaldehyde for 15minutes as the positive control. Cells with alteredMMPwere gated byM1 andM2 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 andmarkedly at 24 hours after treatment (Fig. 3A and B). Thissuggests that citreoviridin may inhibit cell proliferationthrough the inhibition of cell cycle progression at a specificphase.

Citreoviridin reduces anchorage-independent growth oflung cancer cellsTo determine whether citreoviridin may inhibit anchorage-

dependent or anchorage-independent growth by blockingoncogenes that are required for cell survival and/or growthsignals normally provided to adherent nontransformed cells bythe extracellular matrix via integrins, we investigated anchor-age-dependent growth by colony formation on tissue cultureplates and anchorage-independent growth by soft agar assay attheir IC50 to both cell lines. We found that citreoviridininhibited anchorage-dependent growth of both CL1-0 andA549 cells after treatment of 2 weeks (Fig. 3C). Regardinganchorage-independent growth, cells treated with IC50 citreo-

viridin formed significantly fewer and smaller colonies in thesoft agar than cells treated with DMSO alone (Fig. 3D). Theseresults suggest that the inhibition of ectopic ATP synthase isinvolved in the attenuation of anchorage-independent growth,a feature of malignant transformation of lung cancer cells.

Proteomic analysis identifies changes in CL1-0 cells withcitreoviridin treatment

To investigate the effects of citreoviridin on protein expres-sion, comprehensive time-course protein expression profileswere analyzed by proteomic analysis (Supplementary Fig. S3).Performing 2DE, the amounts of protein spots were quantifiedusing ImageMaster and proteins were identified using massspectrometry (see Table S1 for the differentially expressedproteins). We also analyzed the PPI network of the identifieddifferentially expressed proteins. In total, 30 of 49MS-identifiedproteins were mapped to the PPI network (P < 0.005). Geneontology (GO) functional enrichment analysis of the datasetindicated that protein folding (8 proteins), negative regulationof ubiquitin-protein ligase activity involved inmitotic 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 forcell-cycle distribution byPI staining. Thepercentage of cells in eachphasewas calculated byModFit LT 3.0. Asterisks indicate significant differences betweenthe control and treated group from three independent experiments (P < 0.01). C, anchorage-dependent growth of CL1-0 and A549 was estimated after14 days treatment with IC50 citreoviridin (Citreo). D, anchorage-independent growth of CL1-0 and A549 cells was estimated in 0.35% soft agarafter 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 independentexperiments (P < 0.01).






(7 proteins), and mRNA processing (5 proteins) were the topthree cellular functions that are altered by citreoviridin treat-ment (Fig. 4).

Citreoviridin induces the unfolded protein responseFurther analysis of the GO terms revealed that protein

folding was the process most affected by the citreoviridintreatment (Fig. 4). Disruption of ER homeostasis leads to theaccumulation of unfolded proteins. The ER has developed anadaptive mechanism known as the unfolded protein response(UPR) to cope with altered protein folding. Accumulation ofunfolded proteins in the ER induces the dissociation of thechaperone protein BiP from an ER transmembrane sensorknown as PKR-like ER-localized eIF2a kinase (PERK) andinosital-requiring 1a (IRE1a). The dimerization and phosphor-ylation of PERK and IRE1a subsequently activate the UPR.Activated PERK phosphorylates eIF2a, which in turn inhibitsthe assembly of translational machinery and thereby repressesprotein synthesis, which reduces the workload of the ER andcan reduce protein accumulation in the ER. Expression of theER stress markers in CL1-0 and A549 lung cancer cells wasinvestigated by western blotting (Fig. 5A and B). Citreoviridintreatment increased the expression of chaperone proteins,including BiP, and protein disulfide isomerase (PDI). Oxidizingproteins, such as IRE1a, were also increased. The most imme-diate response to ER stress is transient attenuation of mRNAtranslation by increased phosphorylation of eIF2a. According-ly, although the protein levels of eIF2a remain constantfollowing citreoviridin treatment, the data showed that eIF2awas phosphorylated following treatment with the inhibitor, inboth cell lines tested (Fig. 5C). We observed a correspondingincrease in the expression of PERK following citreoviridin

treatment (Fig. 5B). These results suggested that the growthattenuation of citreoviridin-treated cells may be due to theinduction of the UPR and the inhibition of protein synthesis.

To confirm that PERK was involved in eIF2a phosphoryla-tion in citreoviridin treated cells, we carried out PERK knock-down in both cell lines using RNA interference for 48 hours, andthen treated them with citreoviridin for 12 hours. The phos-phorylation and expression levels of eIF2a were analyzed bywestern blotting and normalized to the expression of actin(Fig. 5D). The phosphorylation of eIF2a was abolished aftercitreoviridin treatment in the PERK siRNA cells, whereas thetotal eIF2a levels were unaffected. These data suggest that thephosphorylation of eIF2a is mediated by PERK.

Citreoviridin induces ROS dependent UPRStudies have indicated cross-talk between ER stress and

oxidative stress (22, 23). Our proteomics data also showed thatglutathione S-transferaseMu3 andglutathione S-transferase P,enzymes participating in detoxification by conjugatingreduced glutathione to electrophilic substrates were upregu-lated upon citreoviridin treatment. To verify whether citreo-viridin caused ROS accumulation, H2DCFDA was used tomeasure the level of endogenous ROS. The geometric meanof fluorescence intensity was measured by flow cytometricanalysis (Fig. 6A). The results indicated that ROS levels wereelevated by citreoviridin in a time-dependent manner. To seewhether the citreoviridin induced UPR was ROS dependent,free radical scavenger NAC was used. Treating with citreovir-idin, the phosphorylation level of eIF2a was reduced (Fig. 6B)and the cell viability was recovered (Fig. 6C) upon NACpretreatment, indicating citreoviridin induced UPR was ROSdependent.

Figure 4. The PPI network of MS-identified proteins (round node) and their common interacting partners (triangle node). The interactions are indicated by graylines using the IntAct PPI database as the reference dataset. Modules in color represent enrichment of GO terms (right). Note that the top enriched function isprotein folding.

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Citreoviridin-induced UPR does not activate theapoptotic cascadeMisfolded or unassembled proteins retained in the ER are

degraded by ER-associated degradation (ERAD) through theubiquitin/26S proteosome-dependent pathway (24) or autop-hagy (25). If stressed cells fail to cope with the UPR, cellsundergo cell death, mainly via apoptosis (26). However, we didnot observe apoptotic cell death by annexin V/PI staining incitreoviridin-treated CL1-0 and A549 cells (Supplementary Fig.S4), indicating the citreoviridin-induced inhibition on cellproliferationwas caused by restriction of cell cycle progressionbut not cell death. Recent studies have highlight that p53selectively transactivate quite different responses, rangingfrom 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 deci-sion may be involved in the citreoviridin-induced pathway.To further confirm this possibility, we treated the ectopicATP synthase expressed p53 null cell line H1299 with citreo-viridin ranged from 0 to 8 mmol/L (Supplementary Fig. S5).The results showed citreoviridin only inhibited 12.5% of cellproliferation at the highest concentration, indicating citreo-viridin induced inhibition on cell growth is p53 mediated.

DiscussionIn the past decade, ectopic ATP synthase has been shown to

involve a variety of functions in lipid metabolism, immunerecognition, and invasiveness of tumors (3, 6, 9, 16), regulationof intracellular pH (14, 30), differentiation (13), control ofproliferation and cell death (3, 10, 15). Ectopic ATP synthasehas been shown to localize on the cell membrane of differentcancer cell types. Here, we show that the ATP synthase

complex and ETC are localized on the membrane of lungcancer cells. In an attempt to shed light on the cellularprocesses affected by the action of this complex, and to providefurther insights into the mechanistic action of the ATPsynthase inhibitor citreoviridin, we show that the inhibitionof ectopic ATP synthase is associated with the inhibition oflung cancer cell growth and the activation of UPR. By disrupt-ing the homeostasis of the ER, citreoviridin could specificallytarget ectopic ATP synthase-expressing cancer cells and effec-tively inhibit growth with limited side effects on normal cells.

Cancer cells can pose numerous microenvironmental chal-lenges to surrounding tissues, such as through hypoxia, nutri-ent limitation, oxidative stress, metabolic dysregulation, or lowpH. In turn, these stresses can promote the activation ofspecific signaling pathways, sometimes from the ER via theaccumulation of misfolded proteins in the lumen (31). Toaccount for this severe microenvironment and support pro-liferation, tumor cells have a higher capacity for rapid proteinsynthesis and degradation than normal cells (32–35). Inhibi-tion of the ERAD pathway by proteasomal inhibitors (36) orprotein folding by PDI inhibitors (37) induces the UPR andcytotoxicity in tumor cells. This implies that the homeostasis ofER capacity is critical in tumor progression and recurrence (38,39).

But how could citreoviridin activate UPR? There are severalpossible mechanisms. First, it may be due to the inhibition ofeATP formation by citreoviridin. Because we have now shownthat lung cancer ectopic ATP synthase generates ATP (Fig. 1C),it is reasonable to speculate that citreoviridin disturbs thehomeostasis of extracellular nucleotides, which may havefurther effects on cell signaling. For example, eATP can activateplasma membrane-localized ATP-gated ion channel (P2X)receptors and G protein-coupled (P2Y) receptors in an

Figure 5. Citreoviridin induces the UPR and phosphorylation of eIF2a. Proteins involved in the UPR were examined by immunoblotting of IC50 citreoviridin-treated (A) CL1-0 and (B) A549 cells. C, phosphorylation of eIF2awas detected in both cell lines with citreoviridin treatment at their IC50 concentrations. Thevalue 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 ascrambled 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 byWestern blot analysis. Phosphorylation of eIF2awas determined and normalized to the levelof total eIF2a. The knockdown of PERK was validated in protein expression level. Actin was used as the internal control.






autocrine or paracrine manner (40). Furthermore, ATP isdegraded rapidly to ADP and 50-AMP, the latter of which issubsequently converted by ectopic 50-nucleotidases into aden-osine, which acts as an agonist of the P1 receptor (41). Althoughextracellular nucleotides and nucleosides are important forgrowth and death signal transduction (40, 42), whether thedisruption of P1 and P2 receptor signaling by the inhibition ofectopic ATP synthase activity participates in the regulation ofgrowth or other effects remains to be seen. Yang and colleaguesshowed a delicate proteomics study of the human ABCC1interacting proteome and revealed ATP synthase a binds toABCC1 in plasma membranes and may cooperate to regulateeATP level and purinergic signaling cascade (43), supportingthe regulation of eATP by ectopic ATP synthase.

Second, citreoviridin-induced acidosis may activate theUPR. Treatment with citreoviridin for 48 hours decreased thepHof the culturemedium from7.0 to 6.7 and 7.1 to 6.8 for CL1-0and A549 cells, respectively. The acidosis of culture mediummay induce ER stress and cause cytotoxicity (44). Ectopic ATPsynthasemay act as an intracellular pH regulator because of itsrole in proton transport (12, 14, 45), and the inhibition ofectopic ATP synthase is enhanced under acidic conditions (14).The inhibition of ectopic ATP synthase by citreoviridin maydisrupt homeostasis of intracellular pH and cause extracellularacidosis, thus triggering the UPR by increasing inhibitoryefficacy of citreoviridin.

Third, the induction of ROS forms a positive feedback loop toenhance ER stress. Our results showed that citreoviridininduces ROS production (Fig. 6A). Raj and colleagues reportedthat piperlongumine selectively kills cancer cells by increasingthe level of ROS, but this was dependent on the cancergenotype (46). Furthermore, a mitochondrial mutation inMTATP6, one of the subunit of ATP synthase, which resultsin an elevated level of cytosolic ROS andMMP has been shown(47), suggesting the dysfunction of ATP synthase may contrib-ute to impaired oxidative phosphorylation and ROS produc-tion. As there is the whole respiratory chain in the plasmamembrane that generates partial eATP in lung cancer cellsand the MMP was not affected by citreoviridin, we assumedthat the citreoviridin-induced production of ROS may be dueto the impairment of oxidative phosphrylation in the plasmamembrane but not mitochondria. The active excessive ROSoxidize proteins and ultimately results in protein damage.Generation of ROS directly or indirectly affects ER homeostasisand protein folding by calcium signaling, thus results in ERstress and vise versa (48). Our results therefore suggest thatcitreoviridin-induced ROS elevation may contribute to theselective inhibition of growth in cancer cells.

Fourth, besides those arguments listed above, the aberrantregulation of intracellular calcium from cancer cell activitymay also induce ER stress. Once the newly synthesized nascentproteins extrude from polysomes into the lumen, they undergo

Figure 6. Citreoviridin induces ROS accumulation and chemicals confer citreoviridin induced growth inhibition. A, A549 and CL1-0 cells were treated withcitreoviridin (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 10mmol/L for 30 minutes. After washing, cells were detached, and the total ROSwere detected by the H2DCFDA. The signal of the fluorescence wasmeasuredby flow cytometry and the geometric mean was analyzed by FlowJo7.1. Experiments were repeated three times independently. Asterisks indicate significantdifferences between the control and treated group (P < 0.05). B, the phosphorylation level of eIF2awas determined in citreoviridin (IC50) treated or combinedwith 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 5minutes later after citreoviridin (Citreo) treatment at their IC50 concentrations, and DMSOwas used as the vehicle control. Cells were pretreated with the ROSinhibitor 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 theDMSO control treatment. The relative cell viability after 24-hour treatment from three independent experiments is shown. Asterisks indicate significantdifferences from the DMSO control group (P < 0.05).

Chang et al.





extensive modifications, including glycosylation and disulfidebond formation. The ER contains a pool of calcium-dependentmolecular chaperone proteins (49), such as Grp94, calnexin,and calreticulin, which assist in protein folding, disulfide bondformation, and N-linked glycosylation. The depletion of ERluminal calcium and increased cytosolic calcium cause dys-function of the molecular chaperones and leads to the UPR(50).The addition of ATP, the antioxidant NAC, chemical cha-

perones, or the calcium chelator EGTA rescued citreoviridin-induced growth inhibition (Fig. 6C), suggesting that regulationof the citreoviridin-induced inhibition of cell proliferation iscomplex, and many mechanisms in addition to UPR activationare involved (Supplementary Fig. S6). It would therefore beinteresting to investigate whether pathways enriched in thePPI network could have a synergic/addictive effect with UPR incitreoviridin-induced growth inhibition. Here, we providedevidence that in combination of only dose of 1 mmol/L citreo-viridin with 10 nmol/L 26S proteosome inhibitor bortezomibcaused significantly decreasing in cell viability when compar-ing to single agent treatment (Fig. S7), implying the possibilityof synergic/addictive therapy for citreoviridin and proteosomeinhibitors.This study provides the first evidence that the inhibition of

ectopic ATP synthase induces the UPR, which disrupts thebalance between life and death in lung cancer cells and high-lights the therapeutic potential of ectopic ATP synthase inhi-bition in cancer cells. Further investigations of the ectopic ATPsynthase PPI network, including the active and nonactive state

downstream signaling, will be crucial for a more comprehen-sive understanding of its function in the cell membrane oftumor cells.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: H.-Y. Chang, H.-C. Huang, T.-C. Huang, P.-C. Yang, H.-F. JuanDevelopment ofmethodology:H.-Y. Chang, T.-C. Huang, P.-C. Yang, H.-F. JuanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H.-Y. Chang, T.-C. Huang, H.-F. JuanAnalysis 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. JuanWriting, review, and/or revision of the manuscript: H.-Y. Chang, T.-C.Huang, P.-C. Yang, Y.-C. Wang, H.-F. JuanAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): T.-C. Huang, H.-F. JuanStudy supervision: P.-C. Yang, Y.-C. Wang, H.-F. Juan

Grant SupportThis work was supported by National Research Program for Genomic

Medicine (NSC 100-3112-B-002-011), National Science Council of Taiwan (NSC97-2311-B-002-010-MY3 and NSC 99-2621-B-002-005-MY3), National TaiwanUniversity Cutting-Edge SteeringResearch Project (10R70602C3) and theNation-al Health Research Institutes, Taiwan (NHRI-EX98-9819PI).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance 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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2012;72:4696-4706. Published OnlineFirst July 20, 2012.Cancer Res Hsin-Yi Chang, Hsuan-Cheng Huang, Tsui-Chin Huang, et al. ResponseAdenocarcinoma Growth by Activating the Unfolded Protein Ectopic ATP Synthase Blockade Suppresses Lung

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