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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 ([email protected])
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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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
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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
Journal of Cell Science 122 (18)
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
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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
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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
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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
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3336
(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
Journal of Cell Science 122 (18)
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
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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
-
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 and
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