3330 Research Article

IntroductionMacroautophagy (henceforth referred to as autophagy) is a majorcellular catabolic pathway that consists of a set of complex andhighly regulated processes that lead to the engulfment ofcytoplasmic portions in double-membrane sequestering vesiclescalled autophagosomes. The autophagosomal cargo is subsequentlydelivered to the lysosomal compartment where it is broken downinto its essential constituents and recycled back again to thecytoplasm (Kim and Klionsky, 2000; Mizushima and Klionsky,2007). Activation or repression of autophagy is accompanied by achange in the number of autophagosomes and multivesiculatedlysosomes present in the cells. However, a variation in the numberof these structures does not always reflect an alteration in theexecution of the autophagic pathway. Changes in the autophagicflux, defined as the equilibrium between autophagosome formationand clearance by lysosomes, can also occur from impairment of theability to eliminate autophagic structures from the cytosol throughthe lysosomal compartment (Klionsky et al., 2008).

The main role of autophagy is to provide an alternative energysource during nutrient starvation and certain other adverseconditions in order to ensure cell viability. In addition to enhancedautophagy during starvation, basal levels of autophagy are alsoimportant for intracellular quality control of superfluous anddamaged proteins and organelles (Komatsu et al., 2007a). Besidesthese fundamental roles, autophagy is thought to be involved in thedegradation of intracellular bacteria, antigen presentation, tumoursuppression, cell death and differentiation (Colombo, 2005;Lunemann and Munz, 2008; Maiuri et al., 2008; Orvedahl and

Levine, 2008; Scarlatti et al., 2008). Therefore, it is not surprisingthat alterations in autophagy have been associated with differenthuman pathological conditions (Kundu and Thompson, 2008;Levine and Kroemer, 2008; Mizushima et al., 2008). However,despite the great deal of interest in the regulation of autophagy fortherapeutic purposes, there are only few modulators of theautophagic pathway that show promising pharmacological value(Rubinsztein et al., 2007; Sarkar et al., 2007; Zhang et al., 2007).

Clomipramine (CMI) has been used for over 40 years in thetreatment of patients with psychiatric disorders (Gillman, 2007).It has a long-standing record with good subject tolerance, makingit very attractive to explore further applications for this drug inthe treatment of several medical conditions (Klingenstein et al.,2006; Pilkington et al., 2008). During the course of preliminaryexperiments in an attempt to identify new regulatory activities ofCMI, we observed that treatment with CMI and to an even greaterextent with its active metabolite desmethylclomipramine (DCMI)induced the appearance of autophagy-associated structures in thecytoplasm. Interestingly, this effect occurred at concentrations ofCMI and DCMI that were significantly lower than those previouslyreported to have cytotoxic effects (Daley et al., 2005), thusrevealing a new potential therapeutic role for this group of drugs.These observations prompted us to explore whether DCMI couldalter autophagic flux and, if so, whether this could be exploitedfor novel therapeutic usage. In particular, we focused onaugmenting the sensitivity of transformed cells that exploitautophagy to survive the cytotoxic effects of chemotherapeuticagents.

Alterations in the autophagic pathway are associated with the

onset and progression of various diseases. However, despite the

therapeutic potential for pharmacological modulators of

autophagic flux, few such compounds have been characterised.

Here we show that clomipramine, an FDA-approved drug long

used for the treatment of psychiatric disorders, and its active

metabolite desmethylclomipramine (DCMI) interfere with

autophagic flux. Treating cells with DCMI caused a significant

and specific increase in autophagosomal markers and a

concomitant blockage of the degradation of autophagic cargo.

This observation might be relevant in therapy in which

malignant cells exploit autophagy to survive stress conditions,

rendering them more susceptible to the action of cytotoxic

agents. In accordance, DCMI-mediated obstruction of

autophagic flux increased the cytotoxic effect of

chemotherapeutic agents. Collectively, our studies describe a

new function of DCMI that can be exploited for the treatment

of pathological conditions in which manipulation of autophagic

flux is thought to be beneficial.

Supplementary material available online at

http://jcs.biologists.org/cgi/content/full/122/18/3330/DC1

Key words: LC3, Cell death, Anti-depressant, Doxorubicin,

Chemotherapy, Atg5, p62 (SQSTM1)

Summary

Desmethylclomipramine induces the accumulation ofautophagy markers by blocking autophagic fluxMario Rossi1,*, Eliana Rosa Munarriz1,*, Stefano Bartesaghi1, Marco Milanese2, David Dinsdale1, Maria Azucena Guerra-Martin1, Edward T. W. Bampton1, Paul Glynn1, Giambattista Bonanno2,Richard A. Knight1, Pierluigi Nicotera1 and Gerry Melino1,3,‡

1Medical Research Council, Toxicology Unit, Hodgkin Building, Leicester LE1 9HN, UK2Department of Experimental Medicine, Pharmacology and Toxicology Section, University of Genoa, Viale Cembrano 4, 16148 Genova, Italy3Biochemistry IDI-IRCCS Laboratory, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy*These authors contributed equally to this work‡Author for correspondence (gm89@le.ac.uk)

Accepted 30 June 2009Journal of Cell Science 122, 3330-3339 Published by The Company of Biologists 2009doi:10.1242/jcs.048181

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JCS ePress online publication date 25 August 2009

3331Clomipramine blocks autophagic flux

ResultsDCMI causes an increase in autophagosomal markers in a time-and dose-dependent manner

In order to investigate whether or not cells treated with DCMIexhibit alterations in the autophagic pathway, we first analysedDCMI-induced changes in the levels and distribution of theautophagosomal marker microtubule-associated protein 1 lightchain 3 (LC3) in a HeLa cell line stably expressing LC3 fused toEGFP (HeLa GFP-LC3). LC3 (and also EGFP-LC3) is processedpost-translationally into LC3-I, which is cytosolic and which is thenconverted to LC3-II, which associates with autophagosomalmembranes. Because LC3-II specifically associates withautophagosomes, the number of LC3-positive vesicles and theamount of LC3-II is a widely accepted approximation of the extentof autophagosomal formation, and represents a specific and sensitivemethod to make inferences about autophagic activity (Klionsky et

al., 2008; Klionsky et al., 2007; Mizushima, 2004). As shown inFig. 1, upon treatment with DCMI we observed a significant increasein the amount of overexpressed and endogenous LC3-II in a dose-dependent (Fig. 1A, upper and middle panel, respectively) and time-dependent (Fig. 1D, upper and middle panel, respectively) manner.This increase in the levels of LC3-II was also accompanied by anincrease in the appearance of punctate LC3 staining in a dose-dependent (Fig. 1B,C) and time-dependent (Fig. 1E,F) manner. Asa positive control for induction of autophagy, incubation of cells innutrient-starvation medium (Fig. 1A-E, St) produced similar effects.To confirm these observations at endogenous levels, we performedsimilar experiments in HeLa parental cells and comparable resultswere obtained (supplementary material Fig. S1). The observedincrease in autophagosomal markers was also confirmed in HeLaGFP-LC3 cells by immunoelectron microscopy using an anti-GFPprimary antibody and a secondary antibody coupled to 10-nm gold

Fig. 1. DCMI increases GFP–LC3-IIaccumulation and the appearance of punctateGFP-LC3 staining in a time- and dose-dependentmanner. (A-F) In all cases, as positive control forinduction of autophagy, cells were incubated for2 hours with EBSS nutrient-starvation medium(St). In the western blot panels, asterisk (*)indicates endogenous or overexpressed LC3-Iand double asterisks (**) indicate endogenous oroverexpressed LC3-II. Equal loading wasverified by anti-GAPDH immunoblotting.(A,B) HeLa cells transfected with GFP-LC3were treated with increasing amounts of DCMIfor a period of 2 hours and then analysed byeither western blotting (A) for overexpressedGFP-LC3 (upper panel) and endogenous LC3(middle panel) or direct GFP fluorescence (B).(A) The GFP–LC3-II to GFP–LC3-I ratio andLC3-II to LC3-I ratio (II:I) are shown below theupper and middle panel, respectively.(C) Statistical analysis of the numbers offluorescent punctate structures per cell in eachtreatment in B. The values reported are means ±s.d.; ***P<0.0001 vs control; **P<0.001. (D-F) HeLa cells transfected with GFP-LC3 weretreated with 10 μM DCMI for the indicated timeperiods and then analysed as in A-C,respectively. (G) HeLa cells transfected withGFP-LC3 were starved, treated with DCMI10 μM for 2 hours and then analysed usingimmunogold electron microscopy with anantibody against GFP. Scale bar: 5 μm. Rightpicture is an inset at higher magnification of thearea of the left picture indicated by an arrow.Scale bar: 1 μm.

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particles. This analysis showed a significant increase in double-membrane structures that were labelled with gold particles forEGFP-LC3 when HeLa GFP-LC3 cells were treated with DCMI(Fig. 1G). Importantly, the changes in autophagosomal markersinduced by DCMI were achieved with concentrations at whichminimal cytotoxic effects were apparent (see section ‘DCMIsensitises cells to cytotoxic agents in an autophagy-dependentmanner’). In addition, we also assessed whether other compoundshaving high structural and therapeutic similarities with DCMI couldproduce similar effects on the autophagic flux. To this end, HeLaGFP-LC3 cells were treated with clomipramine (CMI), imipramine(IMI), nortriptyline (NOR) and doxepin (DOX), and equivalentfindings were obtained (supplementary material Fig. S2).

DCMI interferes with the autophagic flux blocking thedegradation of autophagic cargoThe observed DCMI-dependent increase in the levels ofautophagosomal markers could in principle reflect two differenteffects on the autophagy pathway: either increased autophagosomeformation due to increases in autophagic activity, or reducedturnover of autophagosomes due to impairment in the degradationpathway (Klionsky et al., 2008; Klionsky et al., 2007; Mizushima,2004). Therefore, to establish in which physiological or pathologicalcontext DCMI might have a beneficial therapeutic value, it wasnecessary to thoroughly and unequivocally determine at which levelDCMI affected autophagic flux. To this end we examined the effectof DCMI on LC3-II levels in the presence and absence of the protonATPase inhibitor bafilomycin A1 (known to inhibit lysosomal

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degradation but not autophagosome formation). If DCMI increasedthe levels of autophagy, and consequently augmented the numberof autophagosomes targeted for degradation, treatment with DCMItogether with bafilomycin A1 should result in a considerableincrease of autophagosomal markers. However, when comparedwith cells treated with bafilomycin A1 alone, treatment with DCMIin combination with bafilomycin A1 caused only a slight increasein the endogenous and overexpressed LC3-II levels (Fig. 2A, lanes4 and 5, upper and middle panel) and in the appearance of punctateLC3 staining (Fig. 2B,C). By contrast, upon induction of autophagyin response to nutrient-starvation conditions, addition of eitherbafilomycin A1 or DCMI resulted in a significant and conspicuousincrease in the endogenous and overexpressed LC3-II levels (Fig.2A, lanes 3 and 6, upper and middle panel; Fig. 2D lanes 2, 3, 5and 7, upper and middle panel), and an increased appearance ofpunctate LC3 staining (Fig. 2B,C and Fig. 2E,F, respectively). Asa control for specificity, equivalent experiments were performedsubstituting bafilomycin A1 with chloroquine, another compoundknown to interfere with lysosomal function, and similar results wereobtained (supplementary material Fig. S3). In order to corroborateour results at the endogenous level, parental HeLa cells were treatedwith DCMI and bafilomycin A1, under normal and nutrient-starvation conditions, and comparable results were obtained(supplementary material Fig. S4).

We next investigated whether DCMI also induced theaccumulation of LC3-II and the appearance of punctate LC3staining upon pharmacological induction of autophagy withrapamycin, one of best-characterised drugs that enhances autophagy

Fig. 2. DCMI interferes with the autophagic flux.(A) HeLa cells transfected with GFP-LC3 and leftuntreated (lanes 1-3) or treated with 20 nM bafilomycinA1 (lanes 4-6) were incubated in MEM complete mediumin the absence (Ctrl) or presence of 10 μM DCMI (DCMI)or in EBSS nutrient-starvation medium (St) for 2 hoursand then analysed by western blotting for overexpressedGFP-LC3 (upper panel) and endogenous LC3 (middlepanel). The GFP–LC3-II to GFP–LC3-I ratio and LC3-IIto LC3-I ratio (II:I) are shown below the upper and middlepanel, respectively. (B) Analysis of direct GFPfluorescence of samples shown in A. (C) Statisticalanalysis of the numbers of fluorescent punctate structuresper cell in each treatment in B. The values reported aremeans ± s.d.; ***P<0.0001 vs control; $$$, P<0.0001 vsDCMI plus bafilomycin; +++, P<0.0001 vs starvation andbafilomycin. (D) HeLa cells transfected with GFP-LC3were incubated in MEM complete medium (Ctrl) or inEBSS nutrient-starvation medium (St) for 2 hours in thepresence or absence of increasing amounts of DCMI asindicated and then analysed as in A. (E) Analysis of directGFP fluorescence of samples of shown in D. (F) Statisticalanalysis of the numbers of fluorescent punctate structuresper cell in each treatment in E. The values reported aremeans ± s.d.; ***P<0.0001 vs control; +++, P<0.0001 vsstarvation plus DCMI. In the western blot panels, asterisk(*) indicates endogenous or overexpressed LC3-I anddouble asterisks (**) indicate endogenous oroverexpressed LC3-II. Equal loading was verified by anti-GAPDH immunoblotting.

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3333Clomipramine blocks autophagic flux

by inhibiting mTOR (mammalian target of rapamycin) (Ravikumaret al., 2004). As depicted in Fig. 3, addition of DCMI in combinationwith rapamycin resulted in a significant increase in the levels ofthe autophagosomal markers studied (Fig. 3A, lanes 2 and 3; Fig.2B,C), indicating that the effect of DCMI on the autophagic fluxwas independent of the stimulus used to induce autophagy.

To further confirm whether DCMI inhibited autophagicdegradation, we analysed starvation-induced proteolysis of long-lived proteins that represent a standard and specific end-point assayfor the pathway (Pattingre et al., 2004). Compared with control cells,there was a significant reduction (P<0.0001) in the level ofstarvation-induced degradation in DCMI-treated cells that was

comparable to bafilomycin A1-treated cells (Fig. 4A). In addition,we also observed a DCMI-dependent increase in the steady-statelevels of p62 (also known as SQSTM1) (Fig. 4B, upper panel), aprotein that associates with LC3 and ubiquitin and is degradedthrough the autophagic pathway (Ichimura et al., 2008; Komatsuet al., 2007b; Pattingre et al., 2004).

Taken together, these results suggested that both bafilomycin A1and DCMI stimulated the accumulation of autophagosomal markersby inhibiting the autophagic flux as opposed to an activation of theautophagic pathway, resulting in a significant accumulation ofautophagic cargo components.

DCMI-dependent increase of autophagosomal markersrequires intact autophagic machineryAlthough LC3 is a widespread marker for autophagosomes, recentlyit has been shown that under certain conditions it can easily beincorporated into protein aggregates when autophagy is impaired(Ciechomska and Tolkovsky, 2007; Kochl et al., 2006; Szeto et al.,2006). Therefore, in order to confirm that DCMI-dependent changes

Fig. 3. DCMI interferes with pharmacologically induced autophagic flux.(A) HeLa cells transfected with GFP-LC3 were incubated in MEM completemedium (Ctrl), in EBSS starvation medium (St) or in MEM complete mediumwith 0.2 μM rapamycin for 4 hours in the presence (lanes 1-3) or absence(lanes 4-6) of 10 μM DCMI as indicated and then analysed by either westernblotting for overexpressed GFP-LC3 (upper panel) or endogenous LC3(middle panel). The GFP–LC3-II to GFP-LC3-I ratio and LC3-II to LC3-Iratio (II:I) are shown below the upper and middle panel, respectively. Asterisk(*) indicates endogenous or overexpressed LC3-I and double asterisks (**)indicate endogenous or overexpressed LC3-II. Equal loading was verified byanti-GAPDH immunoblotting. (B) Analysis of direct GFP fluorescence ofsamples show in A. (C) Represent statistical analysis of the numbers offluorescent punctate structures per cell in each treatment in B. The valuesreported are means ± s.d.; ***P<0.0001 vs control; $$$, P<0.0001 vsstarvation plus DCMI; +++, P<0.0001, ++, P<0.001 vs rapamycin plus DCMI.

Fig. 4. DCMI blocks autophagic-cargo degradation. (A) The rate ofdegradation of 14C-labelled valine long-lived proteins was measured in HeLacells transfected with GFP-LC3 incubated either in MEM complete medium(Control) or EBBS starvation medium (Starv) as described under Materialsand Methods. Experiments were performed in the presence or absence of10 μM DCMI or 20 nM bafilomycin A1. The values reported are means ± s.d.of three independent experiments in triplicate; ***P<0.0001, ** P<0.001 vscontrol; +++P<0.0001 vs starvation; N.S., not significant. (B) HeLa cellstransfected with GFP-LC3 were treated as indicated: (Ctrl) control, (St) EBBSstarvation medium, (Baf) 20 nM bafilomycin A1, (DCMI) 10 μM DCMI, orthe combination indicated and then subjected to western blotting analysis ofendogenous p62 (upper panel), overexpressed GFP-LC3 (second panel) andendogenous LC3 (third panel). The ratio of treatment vs control of p62 isshown below the upper panel. The GFP–LC3-II and GFP–LC3-I ratio andLC3-II to LC3-I ratio (II:I) are shown below second and third panel,respectively. Asterisk (*) indicates endogenous or overexpressed LC3-I anddouble asterisks (**) indicate endogenous or overexpressed LC3-II. Equalloading was verified by anti-GAPDH immunoblotting.

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in LC3-II and punctate LC3 staining were autophagy-dependent,we next set out to investigate the effect of DCMI on autophagosomalmarkers in an autophagy-deficient cellular model. To this end weused immortalised mouse embryonic fibroblasts (MEFs) obtainedfrom Atg5-deficient mice (Atg5–/–) (Bampton et al., 2005). The Atg5protein plays a key role in the autophagic cascade, and its presenceand proper conjugation with the ubiquitin-like molecule Atg12 arespecifically required for the formation of autophagosomes. Atg5–/–

MEFs were transfected with either a plasmid vector expressing Atg5cDNA or with empty vector and single clones were selected. Inempty-vector-reconstituted Atg5–/– MEFs (pCDNA MEFs),treatment with DCMI or bafilomycin A1, under control or nutrient-deprivation conditions, failed to induce the appearance of LC3puncta (Fig. 5A) or accumulation of LC3-II (Fig. 5B, middle panel,lanes 3-6). As a control, to confirm impairment of the autophagiccascade in the pCDNA MEFs, formation of Atg5-Atg12 conjugationwas examined. To this end the samples were analysed by westernblotting using an anti-Atg5 antibody and the appearance of animmunoreactive band at around 50 kDa (representing the combinedmolecular weight of Atg5 and Atg12) was evaluated. As expected,we did not detect any anti-Atg5 signal in the pCDNA MEFs treatedwith the different stimuli indicated (Fig. 5D, upper panel, lanes 1-7). On the contrary, when we reintroduced Atg5 in the Atg5–/– MEFs(ATG5 MEFs) we observed a DCMI-dependent and bafilomycin-A1-dependent accumulation of autophagosomal markers both in

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control and nutrient-starvation conditions; this accumulation wascomparable with our previous experimental results (Fig. 5C,D; Fig.5E, middle panel, lanes 3-6). In all cases, we observed the presenceof the Atg5-Atg12 conjugated form, indicating a normal progressof the autophagic process (Fig. 5E, upper panel, lanes 1-7). Werepeated similar experiments using a mixed cell population obtainedfrom reconstituted Atg5–/– MEFs with the same vectors used forthe selection of the stable clones (supplementary material Fig. S5)and verified that the results observed were not specific for theparticular stable clones used. As an additional control we alsotransfected wild-type MEFs with the empty vector (pCDNA),obtaining equivalent results (supplementary material Fig. S6).

To further confirm that the DCMI-induced changes inautophagosomal markers were dependent on autophagic activity,we compared the levels of GFP–LC3-II and appearance of punctuateGFP-LC3 staining in HeLa cells transfected with either wild-typeLC3 (GFP-LC3 WT) or a mutant form of LC3 (GFP-LC3G120A)with a single amino acid substitution (Gly120Ala) in its C-terminus.This particular LC3 mutant cannot undergo proteolytic processingand consequently its conjugation to phosphatidylethanolamine andtargeting to the autophagosomal membrane is impaired (Szeto etal., 2006). As shown in Fig. 6, treatment with DCMI in normal ornutrient-starvation medium only induced the appearance of GFP-LC3 puncta in cells transfected with GFP-LC3 WT (Fig. 6A,B),but not in cells transfected with GFP-LC3G120A (Fig. 6C). In

Fig. 5. DCMI-dependent increase of LC3-II and punctateLC3 staining requires intact autophagic machinery.(A,C) Atg5–/– MEFs were stably transfected with either anempty vector (pCDNA; A) or reconstituted with a plasmidexpressing ATG5 (ATG5 clone #9; C). Cells fromindividual clones for each transfection were incubatedeither in DMEM complete medium (Control) or EBBSstarvation medium (Starv) in the presence or absence of10 μM DCMI or 20 nM bafilomycin A1 (Baf) or differentcombinations as indicated. Indirect immunofluorescencefor endogenous LC3 was assessed as described inMaterials and Methods. (D) Statistical analysis of thenumbers of fluorescent punctate structures per cell in eachtreatment in B. The values reported are means ± s.d.;***P<0.0001 vs control; +++, P<0.0001 vs starvationplus DCMI; N.S., not significant. (B,E) Western blotanalysis for Atg5 (upper panel) and endogenous LC3(middle panel) of samples described in A and B,respectively. The ratio of treatment vs control of Atg5immunoreactive band is shown below the upper panel.The LC3-II to LC3-I ratio (II:I) is shown below themiddle panel. Asterisk (*) indicates endogenous oroverexpressed LC3-I and double asterisks (**) indicateendogenous or overexpressed LC3-II. Equal loading wasverified by anti-GAPDH immunoblotting.

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3335Clomipramine blocks autophagic flux

agreement, we only detected an increase in the levels of theGFP–LC3-II band in cells transfected with GFP-LC3 WT, but notGFP-LC3G120A under the same experimental conditions (Fig. 6D,upper panel, compare lanes 1-4 and 5-8). Finally, we also measuredthe levels of endogenous LC3-II in the different conditions testedto assess whether overexpression of GFP-LC3G120A affectedautophagy-dependent processing of LC3. As observed in Fig. 6D,the changes in the levels of endogenous LC3-II were comparablein cells transfected with either GFP-LC3 WT or GFP-LC3G120A (Fig.6D, middle panel), confirming the existence of normal autophagy-dependent LC3 processing in both cases.

Taken together, these observations confirmed that theaccumulation of autophagosomal markers induced by DCMIresulted from inhibition of autophagic flux and not from aninduction of non-specific protein aggregation.

DCMI sensitises cells to cytotoxic agents in an autophagy-dependent mannerFinally, we decided to explore potential therapeutic applications forthe inhibitory action of DCMI on autophagic flux, particularly inthe context of cancer therapy. In fact, although our understandingof the role of autophagy in tumour biology is at an early stage,increasing evidence indicates that autophagy might protect cancercells against the effect of cytotoxic compounds and allow continuedsurvival of transformed cells (Kondo et al., 2005; Levine, 2007;Mathew et al., 2007). Genetic or pharmacological inhibition ofautophagy has already been shown to enhance the anti-tumourefficacy of different chemotherapeutic agents in the treatment oftumour cells that have become chemo-resistant (Amaravadi et al.,2007; Carew et al., 2007; Dang, 2008; Degtyarev et al., 2008;Maclean et al., 2008). In addition, it has been demonstrated recentlythat the beneficial effect of blocking autophagy in order to increasethe cytotoxic effect of chemotherapeutic agents depends on boththe mechanism of cellular injury and the compensatory changes inother forms of autophagy (Wang et al., 2008). This highlights thediversity of response of different tumour cells to chemotherapeutictreatments and the necessity to expand our arsenal of potentialcompounds in order to develop tailored treatment for specific typesof cancer.

In this regard, we investigated whether DCMI could potentiatethe cytotoxic effect of doxorubicin, a DNA-damaging agentwidely used in cancer therapy, known to synergise with inhibitorsof autophagy (Lambert et al., 2008; Meschini et al., 2008; Munoz-Gamez et al., 2009). To this end we treated HeLa cells for 24hours with both compounds, either alone or in combination.Whereas DCMI had minimal effect on cell survival, it stronglysensitised the cells to the toxic effects of doxorubicin as assessedby both estimation of the number of surviving cells (ATP levels)and cells undergoing cell death that externalisedphosphatidylserine (PS) but did not lose plasma-membraneintegrity (single annexin-V positive) (Fig. 7A,B, respectively). Inorder to verify that, in these experimental conditions, DCMI wasexerting the inhibitory effect on the autophagic flux reported inthe previous sections, we analysed the levels of endogenous LC3and p62. As observed in Fig. 7C, treatment with DCMI alone orin combination with doxorubicin induced accumulation of p62and LC3-II, confirming a DCMI-dependent impairment of theautophagic flux.

We next investigated whether enhancement of doxorubicincytotoxicity by DCMI was a result of the inhibitory effect of DCMIon the autophagic pathway. For this purpose we used thereconstituted autophagy-deficient Atg5–/– MEFs described before.Treatment of ATG5 MEFs with doxorubicin together with DCMIfor 24 hours significantly reduced cell number and increased celldeath (Fig. 7D,E, respectively, diagonally striped bars,). Bycontrast, pCDNA MEFs were more sensitive to the cytotoxic effectof doxorubicin alone and this level of toxicity was not significantlyincreased by addition of DCMI (Fig. 7D,E, solid white bars),suggesting that the potentiating effect of DCMI was mainly dueto an inhibition of the autophagy pathway. In agreement,combination indices (CIs) (Chou and Talalay, 1984), as determinedusing the CalcuSyn software (Biosoft), showed synergisticcytotoxicity between doxorubicin and DCMI in ATG5reconstituted MEFs but not in pCDNA reconstituted MEFs(supplementary material Fig. S7). As described above, only inATG5 MEFs, but not in pCDNA MEFs, did we observe a DCMI-dependent increase in the protein levels of both LC3-II and p62

Fig. 6. A single point mutation that blocks LC3 activation abrogates theDCMI-dependent increase of GFP–LC3-II and punctate GFP-LC3 staining.(A,C) Parental HeLa cells were transiently transfected with either wild-typeLC3 (GFP-LC3 WT; A) or with the EGFP-LC3 mutant version G120A (GFP-LC3G120A; C). At 24 hours post-transfection, cells were treated for 2 hours asindicated: (Control) DMEM complete medium, (Starv) EBSS nutrient-starvation medium and (DCMI) 10 μM of DCMI. Direct GFP fluorescencewas then analysed. (B) Statistical analysis of the numbers of fluorescentpunctate structures per cell in each treatment in A. The values reported aremeans ± s.d.; ***P<0.0001 vs control; +++, P<0.0001, ++, P>0.001 vsstarvation plus DCMI. (D) Western blot analysis for overexpressed GFP-LC3(upper panel) and endogenous LC3 (middle panel) of samples described in Aand C. Asterisk (*) indicates endogenous or overexpressed LC3-I and doubleasterisks (**) indicate endogenous or overexpressed LC3-II. Equal loadingwas verified by anti-GAPDH immunoblotting.

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(Fig. 7F,G). Interestingly, the vast majority of the cells treatedwith doxorubicin alone or in combination with DCMI thatexternalised PS were negative for DNA staining with 7-aminoactinomycin (7-ADD), indicating that plasma-membraneintegrity was not compromised (compare Fig. 7 and supplementarymaterial Fig. S8). This suggested that apoptosis, rather thannecrosis, was the major mechanism of cell death induced by thedifferent treatments in both HeLa cells and MEFs.

To complement the analysis of the combination of DCMI anddoxorubicin, we performed a colony-formation assay and estimatedthe number of surviving cells using a standard cell-viability assay(MTT reduction). As shown in Fig. 8, although DCMI alone hadvirtually no effect on cell survival, we observed a very strongreduction in the number of colonies when ATG5 MEFs were treatedwith DCMI in combination with doxorubicin (Fig. 8A,B), even ata concentration that was one order of magnitude lower than that

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used in previous experiments. Interestingly, the concentration ofDCMI used in this experiment was very close to previouslyreported steady-state DCMI plasma levels of patients treated withCMI (Della Corte et al., 1979; Nielsen et al., 1992; Vandel et al.,1982). In agreement with our previous results, pCDNA MEFs weremore sensitive to doxorubicin alone and the potentiating effect ofDCMI on doxorubicin cytotoxicity in this cell line was very modestcompared with ATG5 MEFs (Fig. 8A,B). As a control, we alsomeasured the levels of endogenous LC3-II in replica experimentsand observed an increase in this autophagosomal marker after 10days of treatment with 1 μM DCMI (Fig. 8C,D). This resultconfirmed that, in this experimental setup, DCMI also interferedwith autophagosomal degradation.

Taken together, these results strongly indicated that DCMIpotentiates doxorubicin toxicity and this potentiation requires theinhibitory action of DCMI on autophagic flux.

Fig. 7. DCMI increases doxorubicin cytotoxicity inan autophagy-dependent manner. (A,B) HeLa cellswere treated with the indicated concentrations ofdoxorubicin and DCMI, alone or in combination.After 24 hours, cytotoxicity was assessed by eitherquantisation of the ATP present (indicating number ofviable cells; A) or by PS externalisation usingannexin-V–FITC and 7-ADD for DNA staining (B).In B, only the single-positive annexin-V cells areplotted. The values reported are means ± s.d. of threeindependent experiments in triplicate; ***P<0.0001;+++, P<0.0001; ++, P<0.001 vs doxorubicin.(C) Western blot analysis of endogenous p62 (upperpanel) and endogenous LC3 (middle panel). The ratioof treatment vs control p62, and the LC3-II to LC3-Iratio (II:I) are shown below the upper and middlepanel, respectively. (D,E) To investigate whether theeffect observed was autophagy-dependent, pCDNAMEFs (white bars) or ATG5 MEFs (striped bars)were incubated for 24 hours with the indicatedconcentrations of doxorubicin and DCMI, alone or incombination. Cytotoxicity was evaluated by eitherquantisation of the ATP present (indicating number ofviable cells; D) or by PS externalisation usingannexin-V–FITC and 7-ADD for DNA staining (E).In E, only the single-positive annexin-V cells areplotted. The values reported are means ±s.d. of threeindependent experiments in triplicate; ***P<0.0001;**P<0.001; *P<0.05 ATG5 MEFs vs pCDNA MEFs.(F,G) Western blot analysis for endogenous Atg5(upper panel), endogenous p62 (second panel) andendogenous LC3 (third panel). The ratios of treatmentvs control of Atg5 immunoreactive band and of p62are shown below the upper and second panel,respectively. The LC3-II to LC3-I ratio (II:I) is shownbelow the third panel. Asterisk (*) indicatesendogenous LC3-I and double asterisks (**) indicateendogenous LC3-II. Equal loading was verified byanti-GAPDH immunoblotting.

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3337Clomipramine blocks autophagic flux

DiscussionTricyclic antidepressants such as CMI and DCMI are FDA-approved compounds that have been in clinical use for many years(Gillman, 2007), and a wealth of information is already availableon their bioavailability, toxicity and dosing. Therefore, their use inan alternative setting would avoid one of the major time- and effort-consuming issues in drug discovery, namely gaining FDA approvalfor a new chemical entity (O’Connor and Roth, 2005).

In relation to the ideas outlined above, our observations describea new function for this group of drugs as autophagy inhibitors thatmight have therapeutic implications outside the field of depressiveillness. In particular, we show that DCMI-dependent blockage ofautophagy enhances the efficacy of doxorubicin, a genotoxic

chemotherapeutic agent used in the treatment of a wide variety ofcancers. Importantly, we also provide compelling evidence showingthat this action of DCMI occurs at doses comparable with thoseused in anti-depressive therapy, underscoring its potential fortherapeutic use.

Taken together, these observations provide a platform for futurestudies to explore the combination of antidepressants with cytotoxicagents for the treatment of chemo-refractory malignant disease inwhich autophagy plays an important role in the chemo-resistance.

Materials and MethodsCell cultureHeLa cell lines were routinely cultured in MEM medium supplemented with 10%foetal calf serum (FCS). SV40-immortalised Atg5+/+ (wild type) and Atg5–/–MEFswere maintained in DMEM supplemented with 10% FCS. Autophagy was inducedby different methods: with the addition of 0.2 μM rapamycin for 4 hours and byamino acid starvation. For the latter, cells were washed twice with Earl’s balancedsalt solution (EBSS) and incubated in EBSS for the indicated periods. HeLa cellsstably expressing EGFP-LC3 and immortalized Atg5–/– MEFs were a gift from AvivaTolkovsky (University of Cambridge, Cambridge, UK).

Transfection and cell-line selectionSV40-immortalised and Atg5–/– MEFs were transfected with Atg5 cDNA in a pCDNA-hygro vector (or with vector alone) using a Calcium Phosphate Transfection kit(Invitrogen). Cells stabling carrying the plasmids indicated above were selected usingincreasing concentrations of hygromycin over 3 weeks and single clones picked andexpanded. pCDNA-hygro-Atg5 plasmid was a gift from Gerry Cohen (MRCToxicology Unit, Leicester, UK). HeLa cells were transfected with pEGFP-LC3 WTor pEGFP-LC3G120A mutant using the same method.

Western blottingHeLa cell lines were treated with MEM containing 10% FCS or starvation mediumwith either DCMI, 20 nM bafilomycin A1 (Sigma) or 10 μM chloroquine (Sigma).Cells were lysed in RIPA buffer (150 mM NaCl; 10 mM Tris-HCl, pH 7.2; 0.1%Triton X-100; 1% sodium deoxycholate; 5 mM EDTA), sonicated and finally clarifiedby centrifugation (15,700 g for 10 minutes). Total protein (30 μg) was loaded andresolved on 10% SDS-PAGE gels. Proteins were transferred to nitrocellulose andprobed with anti-GFP antibody (1:2000, Invitrogen). To confirm that EGFP-LC3conversion matched that of endogenous LC3, the same protein extracts were run ona separate 13% SDS-PAGE gel and the blot was probed with anti-LC3 antibody(1:1000, Sigma). To detect endogenous p62, 15 μg of total cell protein were run on10% SDS-PAGE gels and blots were probed with anti-p62 antibody (1:3000,Biomol). To detect overexpressed Atg5, blots were probed with anti-Atg5 antibody(1:500, Abcam). Equal protein loading was confirmed by blotting with anti-GAPDHantibody (1:10,000, Santa Cruz Biotechnology). All blots were subsequently probedwith peroxidase-conjugated secondary antibodies and immunoreactive proteins wererevealed using the ECL Plus detection system (Amersham). Western blot results werequantified by densitometric analysis using GeneTools software (Syngene).

ImmunocytochemistryCells cultured under the same conditions as those used for western blot experimentswere grown on coverslips: at the same time points, these cells were fixed in 4%paraformaldehyde for 10-15 minutes, washed twice in PBS and stained with 1 μg/mlHoechst 33342, rinsed a further three times and mounted in ProLong Gold anti-fadereagent (Invitrogen). For indirect immunofluorescence, cells were fixed in 4%paraformaldehyde for 10-15 minutes and post-fixed for 5 minutes in methanol at–20°C. Coverslips were incubated overnight at 4°C with anti-LC3 antibody (1:750)diluted in blocking solution (1% gelatin; 2.5% goat serum; 0.5% Triton X-100; 100mM Na2HPO4; 200 mM NaCl). After three rinses in washing buffer (20 mMNa2HPO4; 500 mM NaCl), coverslips were incubated for 30 minutes with anti-rabbitAlexa-Fluor-488-conjugated antibodies (0.5 μg/ml in blocking buffer). Cells werecounterstained with 1 μg/ml Hoechst 33342, rinsed a further three times with washingbuffer and mounted in ProLong Gold anti-fade reagent (Invitrogen). All imaging wasperformed on a Zeiss LMS510 confocal microscope using a 63� objective.Quantitative analysis of the numbers of LC3 fluorescent punctate structures per cellwas determined using ImproVision Volocity software.

Electron microscopy and immunoelectron microscopyCells were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4)at 4°C overnight and post-fixed with 1% osmium tetroxide/1% potassium ferrocyanidefor 1 hour at room temperature. After fixation, cells were stained en bloc with 5%aqueous uranyl acetate overnight at room temperature, dehydrated, and embedded inTaab epoxy resin (Taab Laboratories Equipment). Duplicate samples were embeddedin LR-white resin (Agar Scientific) and labelled with immunogold using a modificationof the published technique (Zaccheo et al., 2004). Ultra-thin sections were stained

Fig. 8. DCMI potentiates the inhibitory effect of doxorubicin on colonyformation. (A) Reconstituted ATG5 MEFs or pCDNA MEFs were incubatedwith 1 μM of DCMI and 0.05 μM of doxorubicin, alone or in combination.After 24 hours, drug-free medium was applied to the samples and, 10 dayslater, colonies were fixed in methanol, stained with Crystal Violet, washedwith water and photographed. (B) In order to estimate the number of cellsremaining in the plates after the different treatments, replica samples werestained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) for 1hour, then lysed in DMSO and absorbance was read at 570 nm using astandard plate reader. The values reported are means ± s.d. of threeindependent experiments in triplicate; ***P<0.0001 Doxo vs Doxo+DCMI.(C,D) Western blot analysis for endogenous Atg5 (upper panel) andendogenous LC3 (middle panel). The ratio of treatment vs control of the Atg5immunoreactive band is shown below the upper panel. The LC3-II to LC3-Iratio (II:I) is shown below middle panel. Asterisk (*) indicates endogenousLC3-I and double asterisks (**) indicate endogenous LC3-II. Equal loadingwas verified by anti-GAPDH immunoblotting.

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3338 Journal of Cell Science 122 (18)

with lead citrate/uranyl acetate and examined in a Zeiss 902A or a Jeol 100-CXIIelectron microscope.

Degradation of long-lived proteinsAs previously described (Pattingre et al., 2004), HeLa cells stably expressing EGFP-LC3 were incubated for 24 hours with 0.25 μCi/ml L-[U-14C]valine (Amersham).Cells were rinsed three times with PBS to remove unincorporated radioisotope andthen chased in fresh complete medium containing 10 mM unlabelled valine for 1hour to allow degradation of short-lived proteins. After this, cells were pretreatedwith complete medium supplemented with 10 mM valine, bafilomycin A1 or DCMIfor 30 minutes. Cells were rinsed three times in EBSS and incubated for 2 hours witheither complete media or EBSS supplemented with 10 mM HEPES, 10 mM valinewith bafilomycin A1 or DCMI. Cells were then scraped and lysed in RIPA buffer asdescribed above. Using trichloroacetic acid (TCA), proteins were precipitated fromboth the incubation media and the cell lysates. Proteolysis was assessed as the TCA-soluble radioactivity divided by TCA-insoluble radioactivity.

Cell-death and cell-proliferation assaysHeLa cells and immortalised MEFs were grown in 96-well plates and treated withdoxorubicin (Sigma) and DCMI alone or in combination. After 24 hours, cellproliferation was evaluated using the Cell-Titer 96 Assay (Promega) according tothe manufacturer’s instructions. To assess cell death, cell lines were grown in 60-cmdishes and were treated with doxorubicin and DCMI alone or in combination. After24 hours, cells were collected and stained with annexin-V–FITC and 7-ADD (BDBiosciences) in annexin-binding buffer (10 mM HEPES; 150 mM NaCl; 5 mM KCl;1 mM MgCl; 1.8 mM CaCl2) to detect PS externalisation. Samples were processedwith the FACScan cytofluorimeter and analysed with the Cellquest software (bothBD Biosciences).

Drug interaction and CITo calculate CIs, ATG5 MEFs and pCDNA MEFs were treated with drug-free medium(control), or the indicated concentration of DCMI and doxorubicin alone or incombination. After 24 hours, cells were collected and stained with annexin-V–FITCand 7-ADD as described before to detect PS externalisation. Drug interaction wasassessed using CalcuSyn software version 2.1 (Biosoft), which performs multipledrug dose-effect calculations using the median effects method described by Chouand Talalay (Chou and Talalay, 1984), to determine the CI of the combined treatmentof doxorubicin and DCMI. A CI value of <0.9 indicates synergy, and CI of >0.9 and<1.2 indicates additivity.

Colony-formation assay and MTT assaysA total of 25,000 MEFs were plated in 35-mm-diameter plates and incubated withdifferent concentrations of DCMI, doxorubicin alone or in combination. After 24hours, drug-free medium was applied to the samples. After 10 days, colonies werefixed in methanol, stained with crystal violet, washed with water, counted andphotographed. In order to evaluate the long-term effect of the different treatments oncell viability, an equivalent set of samples were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) for 1 hour, then lysed in DMSO and absorbancewas read at 570 nm using a standard plate reader.

Statistical analysisThe statistical significance of differences was determined by one-way or two-wayanalysis of variance (ANOVA) followed by multiple comparison tests (Bonferroni’stest). A significance level of 95% (P<0.05) was accepted.

The authors would like to thank Judy McWilliam and Tim Smith forthe preparation of samples for electron microscopy. The work wassupported by grants from the Medical Research Council, TelethonAIRC, EU (LSGBH-2005-019067-Epistem), Philip Morris USA Inc.,FIRB-RBNE01KJHT_004 (Marino), MIUR, MinSan, ISS ‘ProgramItalia-USA’ N526D5 and ACC12, Italian Human ProteomeNet:(RBRN07BMCT_007) to G.M. Deposited in PMC for release after 6months.

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