ccr-10-1948 1. - clinical cancer research · 2011. 8. 5. · currently in phase ii clinical...

Cancer Therapy: Preclinical

PM02734 (Elisidepsin) Induces Caspase-Independent Cell DeathAssociated with Features of Autophagy, Inhibition of the Akt/mTORSignaling Pathway, and Activation of Death-Associated Protein Kinase

Yi-He Ling1, Miguel Aracil5, Yiyu Zou1, Ziqiang Yuan2, Bo Lu4, Jos�e Jimeno5,Ana Maria Cuervo3, and Roman Perez-Soler1

AbstractPurpose: PM02734 (elisidepsin) is a synthetic marine-derived cyclic peptide of the kahalalide family

currently in phase II clinical development. The mechanisms of cell death induced by PM02734 remain

unknown.

Experimental Design: Human non–small-cell lung cancer (NSCLC) cell lines H322 and A549 were

used to evaluate PM02734-induced cytotoxicity, apoptosis, and autophagy, as well as effects on cell death–

related signaling pathways.

Results: PM02734 at clinically achievable concentrations (0.5–1 mmol/L) was cytotoxic to H322 and

A549 cells but did not cause nuclear fragmentation, PARP cleavage, or caspase activation, suggesting that

classical apoptosis is not its main mechanism of cell death. In contrast, PM02734-induced cell death was

associated with several characteristics of autophagy, including an increase in acidic vesicular organelle

content, levels of GFP-LC3–positive puncta, elevation of the levels of Atg-5/12 and LC3-II, and an

associated compromise of the autophagic flux resulting in increased number of autophagosomes and/or

autolysosomes. Cotreatment with 3-methyladenine (3-MA) and downregulation of Atg-5 gene expression

by siRNA partially inhibited PM02734-induced cell death. PM02734 caused inhibition of Akt/mTOR

signaling pathways and cotreatment with the Akt inhibitor wortmannin or with the mTOR inhibitor

rapamycin led to a significant increase in PM02734-induced cell death. Furthermore, PM02734 caused the

activation of death-associated protein kinase (DAPK) by dephosphorylation at Ser308, and downregula-

tion of DAPK expression with siRNA caused also a partial but significant reduction of PM02734-induced

cell death. In vivo, PM02734 significantly inhibited subcutaneous A549 tumor growth in nude mice

(P < 0.05) in association with induction of autophagy.

Conclusions:Our data indicate that PM02734 causes cell death by a complex mechanism that involves

increased autophagosome content, due for the most part to impairment of autophagic flux, inhibition of

the Akt/mTOR pathway, and activation of DAPK. This unique mechanism of action justifies the continued

development of this agent for the treatment of NSCLC. Clin Cancer Res; 17(16); 1–14. �2011 AACR.

Introduction

PM02734 (elisidepsin; Fig. 1A) is a synthetic marine-derived cyclic peptide belonging to the kahalalide family ofcompounds (1). We and others have previously shown that

PM02734 is a potent cytotoxic agent both in vitro and in vivoin human non–small-cell lung cancer (NSCLC) cell lines(2). PM02734 is currently in phase II clinical developmentwith preliminary evidence of antitumor activity. The cel-lular and molecular mechanisms of PM02734-induced celldeath remain to be elucidated.

Type I programmed cell death (apoptosis) is a highly andgenetically regulated cell suicide response to facilitate nor-mal embryonic development and homeostasis of multi-cellular organisms. Apoptotic cell death is characterizedby cell shrinkage, chromatin condensation, nucleosomalDNA fragmentation, and formation of apoptotic bodies.Activation of the caspase family of cysteine proteases plays acrucial role in the regulation of apoptosis in the initial andexecution stages (3). In addition, apoptosis is an importantmechanism of drug-induced tumor cell killing and sus-ceptibility to apoptosis in tumor cells is an important

Authors' Affiliations: 1Division of Medical Oncology, Departments of2Surgery and 3Developmental and Molecular Biology, Albert EinsteinCollege of Medicine, Bronx, New York; 4Department of Radiation Oncol-ogy, Vanderbilt University, Nashville, Tennessee; and 5Phamamar R & D,Madrid, Spain

Note: Current address for J. Jimeno: Pangaea Biotech, Barcelona, Spain.

Corresponding Author: Roman Perez-Soler, Department of Oncology,Montefiore Medical Center, 111 East 210st Street, Hofheimer 100,Bronx, NY 10467. Phone: 718-920-4001; Fax: 718-798-7474; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-10-1948

�2011 American Association for Cancer Research.

ClinicalCancer

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Cancer Research. on February 11, 2014. © 2011 American Association forclincancerres.aacrjournals.org Downloaded from

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determinant of chemotherapy efficacy (4). Type II pro-grammed cell death, or cell death with autophagy, is char-acterized by the appearance of double- or multiple-membrane structures called autophagosomes, cytoplasmicvesicles that engulf bulk cytoplasm and/or cytoplasmicorganelles such as mitochondria and endoplasmic reticu-lum. The autophagic vesicles and their contents aredegraded by the cellular lysosomal system. Unlike type Iprogrammed cell death, cell death with autophagy is notdependent on the activation of caspases but dependent onthe expression of several tumor suppressor genes involvedboth in the formation of the autophagosomes and theactivation of specific signaling pathways (5). Althoughthe role of autophagy as a cell survival or cell death pathwayis controversial, a number of studies have described celldeath with autophagic features in response to various anti-tumor agents (6–8). The activation of the class I phosphoi-nositide 3-kinase (PI3K)/AKT pathway acts as a cell survivalsignaling pathway. Inhibition of the PI3K/AKT signalingpathway causes cell death associated with apoptosis and/orautophagy (9). The activation of the mTOR signaling path-way, downstream of PI3K/AKT, is implicated in proteinsynthesis, cell-cycle progression, and cell proliferation(10). Interestingly, inhibition of mTOR signaling has beenshown to trigger cell death with autophagy (11), as mTORacts as a repressor of the autophagic pathway.

Death-associated protein kinase (DAPK) is a calcium/calmodulin-regulated serine/threonine kinase associatedwith actin cytoskeleton (12). It plays a role as a positivemediator in response to various apoptotic stimuli such asIFN-g (13), activation of Fas receptors, TNF-a, TGF-b (14),ceramide-, and p53-mediated apoptosis (15), as well as inthe disruption of the extracellular matrix and resulting celldetachment. DAPK appears to function as a sensor thattriggers early apoptotic events (16). In addition, DAPK actsas a tumor suppressor, as loss of DAPK expression second-ary to hypermethylation is involved in tumor formation(17). Recent studies have shown that DAPK is a novelregulator of autophagic signaling in cell death mediated

by estrogen receptor (ER) stress (18). DAPK belongs to afamily of highly related death-associated kinases, whichincludes DRP-1 and ZIP kinases (19). DAPK contains akinase domain, a calmodulin regulatory segment, 8ankyrin repeats, a cytoskeleton binding region, and a deathdomain. The activation of DAPK depends on its depho-sphorylation at serine 308 in the Caþ2/calmodulin regu-latory domain in response to cell death stimuli, resulting inits proapoptotic activation (20). In addition, activation ofDAPK may have a direct role in the regulation of autopha-gic signaling pathways (21).

In this article, we show that PM02734 induces cell deathassociated with autophagic features including an increasein the number of acidic vacuole organelles, due, for themost part, to a remarkable compromise in the clearance ofautophagosomes and/or autolysosomes. PM02734 alsocaused inhibition of AKT/mTOR signaling, and cotreat-ment with the AKT inhibitor wortmannin or with themTOR inhibitor rapamycin enhanced PM02734-inducedcytotoxicity associated with autophagic features. Further-more, PM02734 induced the activation of DAPK anddownregulation of DAPK gene expression by siRNA ledto the attenuation of PM02734-induced cell death.PM02734 exhibited in vivo antitumor activity associatedwith the activation of the autophagic pathway in nudemicebearing human A549 NSCLC xenografts. Overall, theseobservations provide novel insight into the complexmechanism of PM02734-induced cell death that relaysin part in its effects on the autophagic pathway at the levelof steady-state number and clearance of autophagic com-partments. Our findings should help building a rationalbasis for its therapeutic application either alone or incombination with other chemotherapeutic agents.

Materials and Methods

Chemicals and antibodiesPM02734wasmanufacturedbyPharmaMaranddissolved

indimethyl sulfoxide(DMSO)asa stock concentrationof10mmol/L and diluted to the indicated concentrations withculture medium. Polyclonal anti-caspase-8, caspase-9, cas-pase-3, anti-PARP antibody, anti-AKT, anti-phospho-AKT(Ser437) and anti-phospo-AKT (Thr308), anti-mTOR, andanti-phospho-mTOR (Ser2448), anti-S6 ribosomal protein,and anti-phospho-S6 ribosomal protein (Ser235/236), aswell as anti-4E-BP1 antibody and anti-phospho-4E-BP1(Thr37/46) antibodies were purchased from Cell SignalingTechnology. Polyclonal anti-LC3 and anti-Atg-5 antibodieswere obtained from Novus Biologicals. Polyclonal p62/SQSTM1 antibody and monoclonal anti-DAPK and anti-phospho-DAPK (Ser308) antibodies were purchased fromSigma-Aldrich. Other chemicals and reagents were obtainedfrom Sigma-Aldrich, or from Biomol.

Cell lines and cell cultureHuman NSCLC cell lines H322 and A549 were obtained

from American Type Culture Collection. Cell lines weremaintained in RPMI 1640 medium with 10% FBS and

Translational Relevance

The development of novel agents with uniquemechanisms of action is an urgent need for the treat-ment of non–small-cell lung cancer (NSCLC). We hereprovide evidence that PM02734 (elisidepsin) is cyto-toxic against human H322 and A549 NSCLC cell linesand that the mechanism of cell death caused by thisagent is not classic apoptosis but involves features ofautophagy, impaired autophagy clearance, inhibition ofthe Akt/mTOR pathway, and activation of death-associated protein kinase. In addition, we confirm thatPM02734 effectively inhibits A549 tumor growth in vivowithout causing significant toxicity and that such effectis associated with features of autophagy. The uniquemechanism of cell death induced by this agent supportsits continued development for the treatment of NSCLC.

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maintained at 37�C in a humidified atmosphere of 95% airand 5% CO2.

Assay of cell viability and cell deathExponentially growing cells (1 � 105/mL) were plated

in 96-well plates and allowed to attach overnight. Cells

were exposed to various concentrations of PM02734for the indicated times. Cell viability was assessed bythe reduction of tetrazolium bromide (MTT) assay. Forassay of cell death, cells were treated with PM02734at different concentrations for the indicated times.After treatment, cell death was determined by trypan

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Figure 1. PM02734 induces caspase-independent cell death in H322 and A549 cells. A, chemical structure of PM02734. B, left, concentration-dependentcell death induced by PM02734. Cells were treated with various concentrations of PM02734 for 8 hours, and then cell death was assessed by trypanblue exclusion. B, right, time course of PM02734-induced cell death. H322 cells were treated with 0.5 mmol/L PM02734 and A549 cells were treatedwith 1 mmol/L PM02734 for the indicated times. After treatment, cell death was assessed by trypan blue exclusion (left). C, morphologic changes byPM02734 treatment in H322 and A549 cells. H322 cells were treated with 0.5 mmol/L PM02734 and A549 cells were treated with 1 mmol/L PM02734 for8 hours and then stained with 40,6-diamidino-2-phenylindole (DAPI) solution. Morphologic and nuclear changes were assessed by fluorescencemicroscopy. Arrows indicate the condensed nuclei. D, left, effect of PM02734 on the activation of caspases. Cells were treated with 0.5 to 1 mmol/LPM02734 or with same volume of medium as negative control, or 1 mmol/L staurosporine as positive control. After treatment for 8 hours, the activeform of cleaved caspase-8, caspase-9, and caspase-3, and cleavage of PARP protein, were determined by immunoblot analysis using the correspondingantibodies. b-Actin was used as a loading control. D, right, effect of Z-VAD-fmk, a pan-caspase inhibitor, on PM02734-induced cell death in A549 cells. Cellswere treated with 1 mmol/L PM02734 or with 50 mmol/L Z-VAD-fmk alone, or cotreated with both agents, and cotreatment with 1 mmol/L staurosporine as apositive control. After a 24-hour treatment, cell death was determined by trypan blue exclusion. Data represent mean� SD of three independent experiments.*, P < 0.05, compared with staurosporine alone.

PM02734-Induced Dell Death Is Associated with Autophagy

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blue exclusion as described by Scarlatti and colleagues(22).

Caspase activity assayCells were treated with 0.5 to 1 mmol/L PM02734 for 6

hours, and then cell extracts were prepared with extractionbuffer. Ten microliter of cell extracts (10–30 mg of protein)were added into 100 mL of reaction mixture containing 12mmol/L Ac-DEVD-pNA (Biomol) as the substrate for cas-pase-3 in a 96-well microplate. After incubation at roomtemperature for 120 minutes, the amount of p-nitroani-line–derived substrate cleavage by caspase-3 was deter-mined in a microplate reader (Molecular Devices) at405 nm.

Acridine orange staining for autophagy detectionCells were treated with 0.5 to 1 mmol/L PM02734 for the

indicated times. After treatment, cells were washed twicewith PBS solution and then stained with 1 mg/mL acridineorange solution (Invitrogen) at 37�C for 15 minutes. Thelevels of acidic vesicular organelles (AVO) were assessed bycounting the number of bright red fluorescent cells froma total of at least 200 cells with a Nikon fluorescencemicroscope.

Transfection with GFP-LC3 cDNA plasmid or siRNAGFP-LC3 plasmid was a gift from Dr. Mizushima

(Department of Bioregulation and Metabolism, TokyoMetropolitan Institute of Medical Science, Tokyo, Japan).Cells grown at about 70% confluence were transientlytransfected with 1 mg/mL of GFP-LC3 plasmid by usingLipofectamine 2000 (Invitrogen) according to the man-ufacturer’s instructions. The localization of GFP-LC3 intransfected cells was assessed by fluorescence microscopy.For downregulation of Atg-5 and DAPK gene expression,Atg-5 siRNA and DAPK siRNA were purchased from Dhar-macon. Transfections of siRNA were carried out withLipofectamine 2000 according to the manufacturer’sinstructions.

Transmission electron microscopyA549 cells were treated with 1 mmol/L PM02734 or with

the same volume of medium containing 0.1% DMSO as acontrol at 37�C for 8 hours. After treatment, cells werewashed 3 times with PBS, and then fixed with 0.5mL of ice-cold glutaraldehyde (2.5% in 0.1 mol/L cacodylate buffer,pH 7.4) at 4�C overnight. After washing, cells were fixed in1% OsO4 and embedded in polybed resin. The ultrathinsections were doubly stained with uranyl acetate and leadcitrate and analyzed by transmission electron microscopy(JEOL).

Immunoblot analysis and LC3-II flux measurementsCells were scraped from culture dishes, and cell lysates

were prepared with lysis buffer. Immunoblot analysis wasconducted as previously described (23). Autophagic fluxwas calculated from the densitometric analysis of theimmunoblots for LC3, as the ratio of LC3-II levels in cells

in the presence of bafilomycin over untreated cells (LC3flux), or the amount of LC3-II in bafilomycin-treated cellsminus that in untreated cells (LC3 net flux; ref. 24).

Assay of DAPK activityEndogenous DAPK was immunoprecipitated from

PM02734-treated A549 cells at the indicated times andsubjected to an in vitro kinase assay using myosin II reg-ulator light chain (MLC; Sigma-Aldrich) as a substrate asdescribed by Jin and colleagues (25). DAPK activity wasassessed by measurement of phosphorylated MLC byimmunoblot analysis using anti-phos-MLC antibody.

Antitumor activity in vivoExponentially growing A549 cells (2 � 106 cells) were

subcutaneously injected into the flank of nude mice. Sevendays after inoculation mice bearing tumors (volumearound 100 mm3) were divided into 2 groups of 3 animalsand treated with 0.1 mg/kg PM02734 or the same volumeof vehicle as control, via tail vein 3 times per week for 2weeks. Body weight and tumor size were monitored atthe indicated time points. All animal experiments wereconducted in accordance with Institutional Protocol forAnimal Experiments of the Albert Einstein College ofMedicine. After 2 weeks, mice were sacrificed, and tumortissues from control and PM02734-treated mice were har-vested for assay of apoptosis by terminal deoxynucleotidyltransferase–mediated dUTP nick end labeling (TUNEL)staining or for assay of autophagy as measurement ofthe levels of LC3-II by either immunohistochemical stain-ing or by immunoblotting using a polyclonal anti-LC3antibody (NB600-1384; Novus Biologicals).

Statistical analysisAll data are presented as mean � SD. Differences

between groups were analyzed for statistical significanceusing a 2-tailed Student’s t test. P < 0.05 was used as thesignificance level.

Results

PM02734-induced cell death is not associated withactivation of the classic apoptotic pathway in H322and A549 cells

We have previously studied the cytotoxic effect ofPM02734 on a panel of human NSCLC cell lines andobserved that H322 and A549 cell lines are highly sensitiveto PM02734 with IC50 values of 0.5 to 1 mmol/L (2).Initially, we treated H322 and A549 cells with varyingconcentrations of PM02734 for 8 hours or with 0.5mmol/L PM02734 (H322 cells) or 1 mmol/L PM02734(A549 cells) for the indicated times to evaluate drug-induced cell death as measured by trypan blue exclusion.As shown in Figure 1B, PM02734 induces cell death in aconcentration- and time-dependent manner, about 12% to17% cell death can be detected at 4 hours after drugexposure and 37% to 40% at 24 hours. By morphologicassessment, PM02734 induced disruption of membrane

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integrity and nuclear condensation but did not causechromatin fragmentation (Fig. 1C), suggesting that celldeath induced by PM02734 does not have the character-istics of classical apoptosis. To further verify drug-inducedmembrane disruption, we treated A549 cells with varyingconcentrations of PM02734 for 6 hours and then stainedthem with fluorescein isothiocyanate (FITC)-labeledAnnexin V. PM02734 treatment caused FITC-labeledAnnexin V–positive staining cells with exposure of phos-phatidylserine outside the plasma membrane occurring at1 mmol/L and increasing at 10 mmol/L (data not shown).We next explored whether PM02734-induced cell deathwas associated with caspase activation. Immunoblot ana-lysis revealed that PM02734 treatment for 8 hours did notmarkedly induce the formation of the active forms ofcleaved caspase-8, capase-9, and caspase-3, whereas theactive forms of caspase-8 (43 and 41 kDa), caspase-9(39 and 37 kDa), and caspase-3 (28, 19, and 17) wereclearly observed in the tested cell lines following treatmentwith 1 mmol/L staurosporine. Consistently, the cleavage ofPARP, which is a substrate of caspase-3 and a well-knownapoptotic hallmark, was observed only in cells treated withstaurosporine but not in PM02734-treated cells (Fig. 1D,left). Quantitative analysis of caspase-3 activity by colori-metric assay also showed that PM02734 treatment did notalter caspase-3 activity in the tested cell lines, whereasstaurosporine treatment led to an increase in caspase-3activity (data not shown). Moreover, cotreatment with50 mmol/L Z-VAD-fmk, a pan caspase inhibitor, failed toinhibit PM02734-induced A549 cell death but effectivelyreduced staurosporine-induced A549 cell death (Fig. 1D,right). These data suggest that PM02734-induced cell deathis not associated with the caspase-dependent apoptoticsignaling cascade.

PM02734-induced cell death is associated with theactivation of the autophagic signaling pathway inH322 and A549 cellsBecause PM0274-induced cell death appeared to lack the

characteristics of classical apoptotic type I cell death, wesought to determine whether it was associated with changesin the autophagic pathway previously described as char-acteristics of type II cell death.We first determined the effectof PM02734 on the levels of AVOs by staining tested cellswith acridine orange solution as described by Paglin andcolleagues (26). Figure 2A, left, shows acidic vesicle orga-nelles in both tested cell lines following treatment withPM02734 for 8 hours, the cytoplasm fluorescence changingfrom bright green to bright red. Quantitative analysisshowed about 60% to 66% cells with an expansion oftheir AVOs as compared with 15% to 23% in control cells(P < 0.01; Fig. 2A, right). These results support that treat-ment with PM02734 results in enlargement of the endo-somal/lysosomal system in cancer cells. To determine thepossible autophagic nature of the PM02734-induced com-partments, next, we determined the effect of the drug on theintracellular localization of microtubule-associated protein1 light chain 3 (LC3), a specific marker of autophagosomes

(27), in the tested cell lines. Initially, we transiently trans-fected cells with GFP-LC3 plasmid for 36 hours and thentreated them with PM02734, or with the same volume ofmedium containing 0.1% DMSO as a control, for anadditional 8 hours. The cellular localization of GFP-LC3protein was assessed by fluorescencemicroscopy. As shownin Figure 2B, left, representative fluorescence micrographsshow a punctate pattern of GFP-LC3 in PM02734-treatedcells in contrast to the diffuse pattern in control cells.Quantitative analysis revealed a significant increase in cellswith GFP-LC3 punctate pattern in PM02734-treated cells ascompared with control cells (P < 0.01), that is, �38%versus �20% in H322 cells and �36% versus �17% inA549 cells, respectively (right). To further verify an increasein autophagic markers induced by PM02734, we assessedthe effect of PM02734 on the levels of Atg-5/12, a proteincomplex formed by activation of autophagy (28), and onthe conversion of the cytoplasmic form of LC3-I protein(18 kDa) to the preautophagosomal and autophagosomalmembrane–bound form of LC3-II (16 kDa). As shown inFigure 2C, left, the immunoblot analysis revealed thatPM02734 treatment led to a time-dependent increase inthe levels of conjugated Atg-5 and LC3-II proteins in bothtested cell lines. Quantitative analysis indicated thatPM02734 treatment led to an increase in Atg-5/12 andLC3-II levels as early as 4 hours after drug exposure andgradually increasing thereafter (Fig. 2C, right). To deter-mine whether PM02734-induced accumulation of LC3-II iscaused by enhanced formation of autophagic vacuoles or toblockade of autophagic vacuole clearance, we treated cellswith PM02734 in the absence or presence of the inhibitorof lysosomal proteolysis bafilomycin A1 (BFA; ref. 24). Asshown in Figure 2D (top), LC3-II levels were increased inPM02734-treated cells compared with that in control cellsboth in the presence and in the absence of bafilomycin.However, measurement of the increase in LC3-II afteraddition of bafilomycin (which reflects the amount ofautophagosomes degraded in lysosomes) revealed thatdifferences between PM02734-treated cells and untreatedcells were no longer evident (Fig. 2D). These results supportthat the increase in the amount of autophagosomes was inlarge part resulting from their compromised clearance bylysosomes. Reduced autophagosome clearance was alsosupported when following the degradation of p62/SQSTM1, a polyubiquitin-binding protein that is degradedby autophagy (29, 30). Although levels of p62 were mark-edly reduced in H322 and A549 cells treated withPM02734, the difference was not due to increased degrada-tion as the lower p62 content in treated cells was stillevident upon bafilomycin blockade of lysosomal degrada-tion (Fig. 2D); however, the fact that even after treatmentof PM02734, we found a slight but consistent increase inp62 levels in bafilomycin-treated cells supports that somelevel of autophagosome/lysosome fusion still occurs inthese cells. These data suggest that PM02734 may lead toincrease content of cellular autophagosomes (increasedAtg-5/12 levels) by preventing their clearance (reducedautophagic flux; Fig. 2D, bottom). Finally, we confirmed





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the increased content of autophagic vacuoles by transmis-sion electron microscopy in A549 cells following treatmentwith 1 mmol/L PM02734, or with the same volume ofmedium containing 0.1% DMSO as a control, for 8 hours.As shown in Figure 2E, treatment with PM02734 caused theaccumulation of autophagic vacuoles, which exhibitedautophagosome and/or autolysosomal characteristics

(double andmultilayer membranes containing identifiablemitochondrial and cytoplasmic components), whereasonly a few vacuoles were observed in control cells. Overall,these results suggest that PM02734-induced cell death mayresult, at least in part, from the abnormal accumulation ofautophagic compartments, which has been shown to betoxic for cells in different cellular settings (31).

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Figure 2. PM02734 induces changes in autophagy in H322 and A549 cells. A, PM02734 induces an expansion of the autophagic/lysosomal compartment.Cells were treated with 0.5 to 1 mmol/L PM02734 or with the same volume of medium as control for 8 hours. Following treatment, cells were stainedwith 1 mg/mL acridine orange at 37�C for 15 minutes. A representative slide under fluorescence microscopy shows PM02734-induced AVO displaying redbright fluorescence staining (left). Quantitative analysis of cells with red bright fluorescence staining was analyzed by counting at least 200 cells using afluorescence microscope (right). Each column represents the mean � SD of 3 independent experiments. **, P < 0.01, compared with control. B, effectof PM02734 on intracellular localization of GFP-LC3 protein. Cells were transiently transfected with GFP-LC3 plasmid for 36 hours, and then treated with0.5 to 1 mmol/L PM02734 for 8 hours. After treatment, PM02734-induced autophagy manifesting as dots of GFP-LC3 was assessed by fluorescencemicroscopy (left). Quantitatively, percentage of cells with dots of GFP-LC3 in control or PM02734-treated cells were assessed by counting at least 100 cellsunder fluorescence microscopy (right). Each column represents mean � SD of 3 independent experiments. **, P < 0.01, compared with control. C, effectof PM02734 on the expression of Atg-5/12 and LC3-II in human NSCLC cell lines. H322 cells were treated with 0.5 mmol/L PM02734 and A549 cellswere treated with 1 mmol/L PM02734 for the indicated times. After treatment, cell extracts were prepared for immunoblot analysis. The levels of Atg-5/12 andLC3-I and LC3-II were detected using the corresponding antibodies. b-Actin was used as a loading control (left). The amount of Atg-5/12, LC3-I, andLC3-II was quantified by a laser densitometer. After normalization to b-actin, the relative levels of Atg-5 and LC3-II were compared with those at time 0 as avalue of one. Data represent mean � SD of 3 independent experiments (right). D, effect of PM02734 on autophagic flux. Cells were treated with PM02734 asdescribed above in the absence and presence of 2.5 nmol/L BFA. After treatment, cell extracts were prepared for detection of LC3-II, and p62/SQSTM1 byimmunoblot analysis using the corresponding antibodies. b-Actin was used as sample loading control (top). Quantitative analysis of LC3 flux and net LC3flux were conducted as described in Materials and Methods. Each column represents mean of 2 independent experiments (bottom). E, ultrastructuralevidence of increased autophagosome content induced by PM02734 in A549 cells. Cells were treated with 1 mmol/L PM02734 for 8 hours. After treatment,cells were harvested and the ultrastructural changes were detected by electron microscopy as described in Materials and Methods. Arrows indicate theformation of autophagosomes and autolysosomes in PM02734-treated cells. MW, molecular weight.

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Effects of 3-MA and downregulation of Atg-5expression on PM02734-induced autophagic celldeath in H322 and A549 cellsTo confirm the contribution of the increase in the

autophagic compartment to PM02734-induced cell death,we analyzed the effect of 3-methyladenine (3-MA), a well-known inhibitor of autophagosome formation (32). First,we cotreated H322 and A549 cells with 2 mmol/L 3-MAand 0.5 to 1 mmol/L PM02734 for 8 hours, and thenharvested cell pellets to determine the amount of the

autophagic marker LC3-II. As expected, PM02734 treat-ment resulted in increased levels of LC3-II in H322 andA549 cells. 3-MA treatment led to a significant decrease inPM02734-induced LC3-II expression in the 2 cell lines(Fig. 3A and B, top and middle). In addition, we testedwhether cotreatment with 3-MA could affect PM02734-induced cell death. As shown in Figure 3A and B, bottom,cotreatment with 2 mmol/L 3-MA resulted in a significantbut partial inhibition of PM02734-induced cell death, asmeasured byMTT assay, in H322 and A549 cell lines. Given

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Figure 3. Effects of 3-MA and downregulation of Atg-5 expression by siRNA on PM02734-induced cell death in H322 and A549 cells. A and B, effect of 3-MAon PM02734-induced autophagic cell death. Cells were treated with 0.5 to 1 mmol/L PM02734 or with 2 mmol/L 3-MA alone, or a combination of bothagents or with the same volume of medium as control for 8 hours. After treatment, cells were harvested and cell extracts were prepared forimmunoblot analysis. The levels of LC3-II as an autophagy marker were monitored by immunoblot analysis using a polyclonal anti-LC3 antibody. b-Actin wasused as a loading control (top). The fold increase of LC3-II was analyzed by comparison with that in control which was assigned a value of one. Each columnrepresents mean � SD of 3 independent experiments. *, P < 0.05 and **, P < 0.01, compared with control or PM02734 alone (middle). Cell viability wasdetermined by MTT assay in cells treated with various concentrations of PM02734 in the absence or presence of 2 mmol/L of 3-MA for 24 hours.Data represent mean � SD of 3 independent experiments. *, P < 0.05, compared with PM02734 alone (bottom). C and D, effect of downregulation of Atg-5expression on PM02734-induced cell viability. Cells were transiently transfected with mock siRNA or with Atg-5 siRNA for 24 hours, and then treated withvarious concentrations of PM02734 for 24 hours. After treatment, cell viability was determined by MTT assay. The expression of Atg-5 in nontransfection,mock, and Atg-5 siRNA transfection was detected by immunoblot analysis. b-Actin was used as a loading control. Each point represents mean � SD of 3independent experiments. **, P < 0.01, compared with nontransfection.





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that Atg-5 protein is essential for induction of autophagy(33), we next determined whether the downregulation ofAtg-5 gene expression by transfection with Atg-5 siRNAcould affect PM02734-induced cell death. As shown inFigure 3C and D, downregulation of Atg-5 gene expressionproduced a significant although partial attenuation ofPM02734-induced cell death as measured by MTT assayin H322 and A549 cells. The fact that both chemical andgenetic blockage of autophagosome formation reduced celldeath confirmed that the observed accumulation of autop-hagosomes in cells treated with this drug contributes atleast, in part, to its cellular toxicity.

Role of Akt/mTOR signaling pathways in PM02734-induced cell death in H322 and A549 cells

Activation of the Akt signaling pathway has been shownto be involved in cell survival through phosphorylation atThr308 by PDK1 or at Ser473 by mTOR kinase (34).mTOR, one of downstream targets of Akt, plays a centralrole in the regulation of protein synthesis by phosphoryla-tion of p70 S6 kinase and 4E-BP1 (35). Recent investiga-tions have showed that the inhibition of this signalingpathway is linked to the triggering of autophagy (36). Thus,we sought to test whether PM02734 could induce autop-hagy by inhibition of the Akt/mTOR signaling pathways. Asshown in Figure 4A, PM02734 treatment caused a markedreduction in Akt phosphorylation at Ser473 and Thr308,along with the inhibition of phosphorylation of mTOR atSer2448, as early as 4 hours after exposure and increasingthereafter. In parallel, PM02734 inhibited the phosphor-ylation of S6 ribosomal protein and 4E-BP1, which are thedownstream targets in response to Akt/mTOR signaling. Ofinterest, 4E-BP1 appeared to be more susceptible toPM02734 as strong inhibition of 4E-BP1 phosphorylationwas already observed at 2 hours of drug exposure. To furtherexplore the role of Akt/mTOR signaling inhibition in theregulation of PM02734-induced cell death, we cotreatedH322 and A549 cells with PM02734 and the Akt inhibitorwortmannin or the mTOR inhibitor rapamycin for 24hours. Following treatment, cell viability was determinedby MTT assay, and the LC3-II levels were assayed by immu-noblotting. As shown in Figure 4B, either cotreatment withwortmannin or with rapamycin resulted in a significantenhancement of cell death as compared with PM02734alone (P < 0.05–0.01). Although wortmannin has beenshown to inhibit PI3K class III and compromise autopha-gosome formation, in a similarmanner topreviously shownfor 3MA, this did not seem to be the case in these cells in theconditions of our assay. In fact, the immunoblot analysisrevealed an increased amount of LC3-II in these cells whentreated with wortmannin almost comparable to the oneinduced by rapamycin. Addition of PM02734 furtherincreased LC3-II levels in cells treated with either wortman-nin or rapamycin, further supporting that the effect ofPM02734 was not only at the level of autophagosomeformation but also on autophagosome clearance (Fig. 4C).

To further test whether cotreatment with Akt and mTORinhibitors could affect PM02734-induced autophagic flux,

we also determined the intracellular levels of p62 protein.As shown in Fig. 4D, PM02734 markedly suppressed p62expression as compared with that in control cells. Treat-ment with Akt inhibitor wortmannin alone led to a remark-able accumulation of p62 protein; however, treatment withmTOR inhibitor rapamycin caused an effective decrease inp62 protein. Of interest, cotreatment with PM02734resulted in a significant suppression of p62 levels as com-pared with wortmannin or rapamycin alone (P < 0.01).These results suggest that the inactivation of Akt/mTORpathway by PM02734 may be involved in the regulation ofdrug-induced autophagy and cell death.

Role of activation of DAPK in PM02734-induced celldeath in A549 cells

Recent studies have shown that the activation of DAPK isassociated with the triggering of types I and II cell death (5,19, 37). Thus, we wanted to test whether PM02734-induced cell death could be associated with the activationof DAPK in A549 cells. Initially, we tested the effect ofPM02734 on DAPK expression and found that PM02734treatment caused the degradation of full-size DAPK (160kDa) accompanied by increased levels of its correspondingcleaved fragments (100 and 60 kDa) in a concentration-and time-dependent manner (Fig. 5A). Although the sig-nificance of DAPK degradation remains largely unknown,several studies have shown that the generation of proteo-lytic DAPK fragments (100 or 60 kDa) is involved in theinitiation of the apoptotic and autophagic cascades (20,38). Furthermore, recent investigations have shown thatthe activation of DAPK depends on its dephosphorylationat Ser308 (20). We therefore determined the effect ofPM02734 on DAPK phosphorylation at Ser308 by immu-noblot analysis using the corresponding antibody. Ofinterest, PM02734 treatment strongly induced the depho-sphorylation of DAPK at Ser308 in a concentration- andtime-dependent manner. As shown in Figure 5B, left,1 mmol/L PM02734 led to about 80% of DAPK depho-sphorylation at Ser308 in A549 cells. Time course studiesshowed about 65% DAPK dephosphorylation as early as 1hour after PM02734exposure and about 80%DAPKdepho-sphorylation during the 4- to 24-hour period after drugexposure (Fig. 5B, right). To further confirm that PM02734-induced DAPK dephosphorylation was correlated with theenhancement of DAPK activity, we used MLC as a substrateto determine the effect of PM02734onDAPK activity in vitrousing immunoprecipitated endogenous DAPK as describedin Materials and Methods. As shown in Figure 5C, DAPKactivity was increased by about 150% as early as 1 hour ofdrug exposure and reached its maximum (280% to 250%)after 2 to 4 hours of exposure, declining to basal levels after24-hour exposure. Moreover, we determined the effect ofdownregulation of DAPK expression on PM02734-inducedcell death. As shown in Figure 5D, downregulation ofDAPK expression by siRNA resulted in a pronounced andsignificant attenuation of PM02734-induced cell growthinhibition as measured by MTT assay. In addition, down-regulation of DAPK by siRNA transfection also resulted in a

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significant although incomplete attenuation of PM02734-induced increase in LC3-II levels in A549 cells (Fig. 5E).These results suggest a relationship between PM02734-induced activation of DAPK and autophagy.

PM02734 inhibition of human NSCLC A549 xenograftsis associated with autophagosome accumulationTo investigate the antitumor efficacy of PM02734 in vivo,

we conducted a dose escalation toxicity trial in mice andfound that an intravenous dose of 0.1 mg/kg was well

tolerated. We then established a xenograft model of humanA549 NSCLC cells in nude mice. Exponentially growingA549 cells (2 � 106 cells) were subcutaneously inoculatedin the flank of nude mice. Seven days after inoculation,when tumor volume is about 100 mm3, mice were ran-domly assigned to control (vehicle) and PM02734 treat-ment groups. Mice bearing established A549 tumors weregiven intravenous injections of 0.1 mg/kg PM02734 or thesame volume of vehicle 3 times per week for 2 weeks. Asshown in Figure 6A, top, PM02734 caused about 50%

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Figure 4. Role of inhibition of Akt/mTOR-related signaling pathways in PM02734-induced cell death in H322 and A549 cells. A, effect of PM02734on the phosphorylation of Akt/mTOR signaling pathways. H322 cells were treated with 0.5 mmol/L PM02734 and A549 cells were treated with 1 mmol/LPM02734 for the indicated times. After treatment, cells were harvested and cell extracts were prepared for immunoblot analysis. The levels of total Akt andmTOR, and phospho-Akt, and phospho-mTOR, total S6 ribosomal protein (RP) and 4E-BPl, as well as phospho-S6 RP and phospho-4E-BP1 weredetected by immunoblot analysis using the corresponding antibodies. b-Actin was used as a loading control. B, effects of cotreatment with wortmannin andrapamycin on PM02734-induced cell death. Cells were treated with 0.5 to 1 mmol/L PM02734 or 10 mmol/L wortmannin (wort), an AKT inhibitor, or 1 mmol/Lrapamycin (rap), an mTOR inhibitor, alone or in combination with PM02734 for 24 hours. After treatment, cell viability was determined by MTT assay. Eachcolumn represents the mean � SD of 3 independent experiments. *, P < 0.05; **, P < 0.01, compared with PM02734 alone. C, effects of cotreatment withwortmannin and rapamycin on PM02734-induced increase of LC3 protein levels. Cells were treated with PM02734, wortmannin, or rapamycin alone,or with combinations of PM02734 and either other agent as described above. After treatment, cells were harvested and cell extracts were prepared forimmunoblot analysis. The levels of LC3-II were detected by immunoblot using a polyclonal anti-LC3 antibody. b-Actin was used as a loading control (top). Afternormalization to b-actin, relative levels of LC3-II were analyzed by comparison with control which was given the value of 1. Each column represents mean� SDof 3 independent experiments. *, P < 0.05 and **, P < 0.01 (bottom). D, effects of PM02734, wortmannin, rapamycin alone, or combined treatment onintracellular p62 levels. After treatment as described above, cells were harvested and cell lysates were prepared for detection of p62 protein by immunoblotusing a polyclonal anti-p62 antibody. b-Actin was used as a loading control (top). After normalization with b-actin, the relative levels of p62were comparedwithcontrol which was given the value of 1. Each column represents the mean � SD of 3 independent experiments. **, P < 0.01 (bottom).





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Figure 5. Role of the activation of DAPK in PM02734-induced cell death in A549 cells. A, PM02734 induces the degradation of DAPK in aconcentration- and time-dependent manner. A549 cells were treated with various concentrations of PM02734 for 8 hours or with 1 mmol/L PM02734for the indicated times. Following treatment, cells were harvested and cell extracts were prepared for immunoblot analysis. The full size of DAPK and itscleaved fragments were detected by immunoblot with a monoclonal anti-DAPK antibody (left). b-Actin was used as a loading control. The relative levels of full-size and cleaved DAPK were analyzed in comparison with control or exposure time 0 (right). Each column represents the mean � SD of 3 independentexperiments. *, P < 0.05; **, P < 0.01, compared with control or with time 0. B, PM02734 induces DAPK dephosphorylation at Ser308 in a concentration- andtime-dependent manner. Cells were treated with PM02734 as described above. After treatment, cell extracts were prepared for assay of DAPKphosphorylation at Ser308 by immunoblot using a monoclonal anti-phospho-DAPK antibody. b-Actin was used as a loading control. The relative levelof phospho-DAPK at Ser308 was analyzed in comparison with control or with exposure time 0. Each column represents the mean � SD of 3 independentexperiments. **, P < 0.01, compared with control or with time 0. C, effect of PM02734 on DAPK activity. A549 cells were treated with 1 mmol/L PM02734 for theindicated times. Following treatment, endogenous DAPK was immunoprecipitated, and DAPK activity was assessed by measurement of MLCphosphorylation as described in Materials and Methods. Each point represents the mean of 2 independent experiments. D, effect of downregulation of DAPKgene expression by siRNA on PM02734-induced cell viability. A549 cells were transiently transfected with mock or DAPK siRNA or nontransfection for 24hours. After transfection, cells were treated with various concentrations of PM02734 for 24 hours, and cell viability was determined by MTT assay.The expression of DAPK was monitored by immunoblot using anti-DAPK antibody, b-actin was used as a loading control (right). Each point represents themean � SD of 3 independent experiments. **, P < 0.01, compared with nontransfection. E, effect of downregulation of DAPK on PM02734-inducedLC3-II expression. siRNA-transfected or controlled A549 cells were treated with 1 mmol/L PM02734 or with the same volume of medium as control for8 hours. After treatment, cell extracts were prepared for assay of LC3 expression by immunoblot using a polyclonal anti-LC3 antibody. b-Actin was used as aloading control. The relative LC3-II levels were analyzed in comparison with control which was assigned a value of 1. Each column represents meanþ SD of 3independent experiments. *, P < 0.05, compared with control cells.

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tumor growth inhibition (P < 0.05) as compared withcontrol animals. No body weight loss was observed indrug-treated mice compared with control mice (Fig. 6A,bottom). To investigate the mechanism of PM02734-induced tumor growth inhibition in vivo, mice were sacri-ficed and tumor tissues collected 1 day after the lastPM02734 dose. Apoptosis was assessed by TUNEL stainingand autophagy by LC3 expression by immunohistochem-ical staining and immunoblot analysis. Figure 6B showsno differences in the fraction of TUNEL-positive stainingcells between PM02734-treated tumors and controltumors. In contrast, the expression of total cytoplasmicLC3 detected by immunohistochemical staining wasmarkedly elevated in PM02734 treated tumors comparedwith control tumors (Fig. 6C). Consistently, the immuno-blot analysis revealed that LC3-II levels were higher inPM02734-treated tumors than in control tumors, whereasno differences were observed between PM02734 treatmentand control tumors in Bcl-2 and Bax levels (Fig. 6D). All

these data suggest that autophagosome accumulation isassociated with PM02734-induced tumor growth inhibi-tion in vivo.

Discussion

Our results indicate that PM02734-induced cell death isassociated with features of autophagy including an increasein cellular content of AVOs and compromised autophagicclearance. The role of autophagy in cancer therapeutics isstill controversial. Autophagy has been found to play a roleas a cell survival mechanism by which cells clear damagedcytoplasmic proteins and organelles through lysosomaldegradation and survivemetabolic stress (39). On the otherhand, autophagy has been also found to contribute to typeII programmed cell death in response to hypoxia, che-motherapeutic agents, virus infection, and toxins (40).Our results suggest that the observed abnormal increaseof the autophagic compartment can only partially explain

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Figure 6. PM02734 inhibits tumor growth and induces autophagy in A549 xenografts in vivo. A, top, PM02734 inhibits the growth of A549 xenograftsin vivo. Exponentially growing A549 cells (2 � 106 cells) were subcutaneously inoculated into nude mice. When the tumor growth reached about 100 mm2

in volume on day 7, nude mice were randomly divided into 2 groups (n ¼ 3), and were treated either with 0.1 mg/kg PM02734 intravenously or the samevolume of vehicle as control 3 times/wk for 2 weeks. Tumor growth was determined 3 times each week. Each point represents the mean � SD of 3 animals.*, P < 0.05, compared with control. A, bottom, effect of PM02734 on animal body weight in vivo. Each point represents the mean of 3 animals. B,effect of PM02734 on induction of apoptosis assessed by TUNEL reaction. After treatment with 0.1mg/kg PM02734 or vehicle for 2 weeks, paraffin-embeddedtumors were labeled with FITC-dUTP for TUNEL assay. Nuclei were stained with propidium iodide. C, representative micrograph of immunohistochemicalstaining with anti-LC3 antibody in PM02734-treated and control tumors. Tumor-bearing animals were treated with PM02734 and vehicle as describedabove, and the paraffin-embedded tumor samples were subjected to immunohistochemical staining with a polyclonal anti-LC3 antibody. D, effect of PM02734on the expression of LC3-II, Bcl-2, and Bax. After treatment with 0.1 mg/kg PM02734 or with vehicle for 2 weeks as described above, tumor samples wereharvested, and proteins were prepared and subjected to immunoblot analysis using the corresponding antibodies. b-Actin was used as a loading control (left).Quantitative analysis of detected proteins was analyzed with a laser densitometer. After normalization to b-actin, the relative levels of LC3-II, Bcl-2, and BAX inPM02734-treated tumors were compared with vehicle-treated tumors which were given a value of 1. Data represent mean � SD of 3 independentexperiments. *, P < 0.05 (right).





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the cytotoxic effect of PM02734, as cotreatment with 3-MA,a well-known autophagy inhibitor, or Agt-5 siRNA, anessential component for autophagosome formation, hadonly a partial protective effect on PM02734-induced celldeath in H322 and A549 cells. It is possible that thePM02734-induced increase in autophagosome contentcould be, in part, related to induction of autophagythrough its inhibitory effect on the Akt/mTOR signalingpathway and activation of DAPK. The accumulation ofautophagic vacuoles induced by PM02734 was confirmedin vivo in A549 xenografts. This study is the first to showthat PM02734-induced antitumor effect is, in part,mediated by an autophagic mechanism.

Apoptosis is an active form of cell death characterized bycell shrinking, cytoplasmic condensation, DNA ladderdegradation, and nuclear fragmentation resulting in theformation of apoptotic bodies. In contrast, type II pro-grammed cell death is characterized by the presence ofvisible autophagic cytoplasmic vacuoles, mitochondrialdilation, enlargement of the endoplasmic reticulum andthe Golgi apparatus, and nuclear condensation withoutDNA laddering (41). Apoptosis and cell death with autop-hagy are not mutually exclusive and can coincide in vivo incertain tissues and in cell culture (42). In addition, apop-tosis and autophagy may share certain mechanisms andalterations such as loss of mitochondrial permeability andmembrane potential (43). The fact that PM02734-inducedcell death could only be attenuated partially with autop-hagy inhibitors suggests that other mechanisms of celldeath are involved in PM02734-induced caspase-indepen-dent cell death. In a previous work, we and others haveshown that kahalalide F, a parent compound of PM02734,induces necrotic cell death (type III programmed celldeath) linked to damage of the endoplasmic reticulum,Golgi apparatus, and the activation of lysosomes, as well asloss of the mitochondrial membrane potential and releaseof lactate dehydrogenase (LDH) from cytoplasm (44).Preliminary studies also show that PM02734 leads to arapid depletion of intracellular ATP and loss of mitochon-drial membrane potential in H322 and A549 cells (data notshown), suggesting that activation of necrotic signalingpathways may also be involved in PM02374-induced celldeath.

An emerging body of evidence indicates that the Akt/mTOR pathway plays a crucial role in the regulation ofboth apoptosis and autophagy (36). We present here dataindicating that PM02734 markedly inhibits Akt phosphor-ylation at Ser473 and at Thr308, as well as mTOR phos-phorylation at Ser2448. In addition, we observed thatdownregulation of mTOR protein by PM02734 mayexplain at least in part mTOR dephosphorylation. Timecourse studies indicated that the inhibition of the Akt/mTOR signaling pathway occurred as early as 4 hours afterPM02734 exposure, suggesting that the inhibition of Akt/mTOR is an early event in the drug-induced cell deathprocess. As expected, phosphorylation of S6 ribosomalprotein and 4E-BP-1, downstream targets of Akt/mTORpathway, were inhibited by PM02734. However, we found

that PM02734 caused degradation and dephosphorylationof 4E-BP-1, a translation factor for protein synthesis and adownstream element of the Akt/mTOR axis 2 hours afterPM02734 exposure, suggesting that inactivation of the S6ribosomal protein/4E-BP1 pathway preceded Akt/mTORinhibition. Although the pharmacologic significance ofPM02734-induced downregulation and inactivation ofAkt/mTOR downstream events is unknown, these findingssuggest that the effect of PM02734 on this pathway couldbe 2-fold; in one hand, it may induce autophagosomeformation, which associated with the observed compro-mise in clearance would lead to cellular clogging, andin addition, it may directly or indirectly affect proteinbiosynthesis, which could further lead to suppression ofcell proliferation and cell growth. Wortmannin has beenshown to be an autophagy inhibitor via inhibition of classIII PI3K (45); however, its effect over class I PI3K wouldresult in mTOR inhibition. In this work, we found thatwortmannin markedly induces accumulation of the intra-cellular levels of LC3-II in H322 and A549 cells, indicatingthat this compoundmay have a preferential effect on class IPI3K in these cells, as it did not seem to impact theconversion of LC3-I into LC3-II, or could be interferingwith autophagosome clearance by lysosomes. Recent inves-tigations have shown that autophagy inhibition at an earlystage could trigger cell survival signaling pathways, pre-venting death due to stress; whereas, inhibition of autop-hagy at a late stage could cause accelerated cell death underautophagy-inducing conditions via the activation of apop-totic signaling (46). Further studies are required to deter-mine whether a combined effect of PM02734 activatingearly autophagy steps and compromising late autophagicstages may be behind its marked negative effect on cellviability. Interestingly, we here present data that cotreat-ment with 3-MA, which is an early autophagy inhibitor(45), led to the attenuation of PM02734-induced celldeath, whereas cotreatment with wortmannin, whichmay inhibit autophagy at a late stage as discussed above,caused a significant enhancement of PM02734-inducedcytotoxicity in 2 tested cell lines. Consistent with ourfindings, Shingu and colleagues recently reported thatinhibition of autophagy by 3-MA attenuated imatinib-induced cell death, but inhibition of autophagy at a latestagewith bafilomycin A1 enhanced drug-induced cytotoxi-city in human malignant glioma cells (47). In addition, wealso observed that cotreatment with the mTOR inhibitorrapamycin and PM02734 resulted in an additive effect oncell death associated with an increase in autophagosomecontent as shown by increase in LC3-II, suggesting that theinhibition of mTOR signaling may play a role in PM02734-induced autophagy. Overall, the role of the PI3K/Akt/mTOR axis in modulating PM02734-induced cell deathappears highly complex and deserves further investigation.

DAPK, Ca2þ/calmodulin-regulated serine/threoninekinases, has a wide range of functions in the regulationof cell growth and cell death. It functions as a tumorsuppressor, is involved in p53-mediated checkpointcontrol resulting in inhibition of oncogene-induced cell

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transformation, and is also an antimetastasis factor (12).Recent studies have shown that DAPK acts as a positivemediator, regulating different types of cell death signalingpathways including apoptosis and autophagy (18, 21). Ofnote, data presented in this work indicate that PM02734induces the cleavage of full-size DAPK (160 kDa) into 2small fragments (100 and 60 kDa) in a dose- and time-dependent manner. The biological role of such cleavage isunclear, although Shamloo and colleagues (38) havereported that the cleavage of full-size DAPK resulting in100 and 60 kDa fragments was correlated with the induc-tion of cell death in damaged rat brain by ischemic injury,suggesting that these cleaved fragments may represent theactivated form of DAPK. Moreover, a number of investiga-tions have showed that the activation of DAPK depends onits dephosphorylation at Ser308 (21). Consistently, we alsofound that PM02734 at a low concentration (1 mmol/L)induces DAPK dephosphorylation at Ser308 as early as 1hour after exposure, and an increase in DAPK dephosphor-ylation over time. Accordingly, DAPK activity was inducedafter PM02734 exposure for 1 hour, peaked at 4 to 8 hoursand declined thereafter, suggesting that the activation ofDAPK may be an initiator of cell death. Of interest, down-regulation of DAPK expression by siRNA caused a signifi-cant attenuation of PM02734-induced cytotoxicity and alsoblocked drug-induced LC3-II accumulation, suggestingthat the activation of DAPK may contribute toPM02734-induced cell death with autophagic features.Recently, Harrison and colleagues reported that DAPKcan interact with microtubule-associated protein 1B, whichwas proven to interact with autophagosomal protein Atg-8(LC3), resulting in the stimulation of DAPK-dependentmembrane blebbing and autophagy (48). In addition,Zalckvar and colleagues showed that DAPK phosphorylatesbeclin 1, an essential protein for autophagy at threonine119, resulting in the dissociation of beclin 1 from Bcl-XLand induction of autophagy (49). In addition, we hereobserved that downregulation of DAPK by siRNA causesgreater extent of blockade of PM02734-induced cell deaththan direct inhibition of autophagy by 3-MA treatment orby Atg-5 downregulation, suggesting that other lethal cas-

pase-independent pathways induced by DAPK such asactivation of programmed necrosis may be involved inPM02734-induced cell death. Thus, the cellular and mole-cular mechanism by which PM02734 induces DAPK acti-vation and cell death associated with autophagic featuresdeserves to be studied further. Consistent with the in vitroresults, we found that PM02374 has antitumor activity invivo and that the extent of LC3-II, an autophagic marker,was elevated in PM02734-treated xenograft tumor tissuesas measured by immunohistochemical staining and immu-noblot analysis. However, TUNEL assay and the levels ofBcl-2 and Bax did not show evidence of apoptosis afterPM02734 treatment.

In summary, we have shown that PM02734 induces celldeath in human NSCLC cell lines both in vitro and in vivoby a complex mechanism that appears to involve accu-mulation of autophagosomes, mainly by compromise ofautophagosome clearance along with inhibition of theAkt/mTOR signaling pathway and activation of DAPK.Consistent with in vitro results, PM02734 inhibits tumorgrowth accompanied by marked accumulation of autop-hagosomes in vivo in A549 xenografts. These observationsrepresent a potentially first important step toward under-standing the mechanism of lethality of this structurallyunique agent and may be of help in rationally integratingPM02734 with other agents for the treatment of NSCLCpatients.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported in part by NIH grants CA84119 and CA96515and by PharmaMar R & D.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 29, 2010; revised May 20, 2011; accepted June 1, 2011;published OnlineFirst June 20, 2011.

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Published OnlineFirst June 20, 2011.Clin Cancer Res Yi-He Ling, Miguel Aracil, Yiyu Zou, et al. Death-Associated Protein Kinasethe Akt/mTOR Signaling Pathway, and Activation ofDeath Associated with Features of Autophagy, Inhibition of PM02734 (Elisidepsin) Induces Caspase-Independent Cell

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