Oncogene (2015), 19

2015 Macmillan Publishers Limited All rights reserved 0950-9232/15

www.nature.com/onc

REVIEW

Cell death by autophagy: emerging molecular mechanisms

and implications for cancer therapy
S Fulda1,2,3 and D Kgel4
Autophagy is a tightly-regulated catabolic process of cellular self-digestion by which cellular components are targeted to lysosomes
for their degradation. Key functions of autophagy are to provide energy and metabolic precursors under conditions of starvation
and to alleviate stress by removal of damaged proteins and organelles, which are deleterious for cell survival. Therefore, autophagy
appears to serve as a pro-survival stress response in most settings. However, the role of autophagy in modulating cell death is
highly dependent on the cellular context and its extent. There is an increasing evidence for cell death by autophagy, in particular in
developmental cell death in lower organisms and in autophagic cancer cell death induced by novel cancer drugs. The deathpromoting and -executing mechanisms involved in the different paradigms of autophagic cell death (ACD) are very diverse and
complex, but a draft scenario of the key molecular targets involved in ACD is beginning to emerge. This review provides an up-todate and comprehensive report on the molecular mechanisms of drug-induced autophagy-dependent cell death and highlights
recent key ndings in this exciting eld of research.
Oncogene advance online publication, 26 January 2015; doi:10.1038/onc.2014.458

INTRODUCTION
Different modes of programmed cell death
Programmed cell death is an evolutionary conserved intrinsic
mechanism enabling damaged and unwanted cells to commit
suicide. This cellular suicide may occur either via apoptosis (type I
cell death)1 or via activation of alternative death programs.2,3
Induction of apoptotic cell death is a major mechanism by which
most chemotherapeutic drugs and radiation kill tumor cells. In the
past couple of years, a tremendous effort has been invested to
develop strategies for triggering cancer cell apoptosis in a targetspecic manner. In addition to apoptosis, an ever increasing
number of studies substantiate the existence of alternative, nonapoptotic forms of programmed cell death,2,4 which may be
exploited for cancer therapy.
Until recently, the nomenclature for different cell death types
was largely based on morphological criteria and has not
been uniformly used and recognized. The emerging knowledge
on the molecular mechanisms of the different forms of
programmed cell death now allows the discrimination into
distinct cell death subroutines, and this classication is continuously rened and further improved.3 Based on the currently
existing knowledge, the Nomenclature Committee on Cell Death
(NCCD), consisting of the leading experts in the eld of cell
death research, has proposed the classication into ve relatively
well-characterized different modes of (programmed) cell
death: (1) extrinsic apoptosis, (2) intrinsic apoptosis, (3) regulated
necrosis, (4) mitotic catastrophe (mitosis), and (5) autophagic cell
death (ACD).3
Apoptosis (type I cell death) is morphologically dened by
cellular and nuclear shrinkage, chromatin condensation (pyknosis),

nuclear fragmentation (karyorrhexis), membrane blebbing and the

formation of apoptotic bodies that are engulfed by neighboring or
specialized cells.5 At the biochemical level, apoptosis is characterized by phosphatidylserine exposure and (in most cases)
activation of effector caspases, the main executors of apoptotic
cell death.1 In extrinsic apoptosis, extracellular death signals act
via specic transmembrane receptors culminating in a caspasedependent, apoptotic type of cell death.3,6 The mitochondria have
a pivotal role in intrinsic apoptosis, which is initiated by a wide
variety of intracellular stress signals/conditions leading to the
activation of the pro-apoptotic BCL-2 family members Bak and
Bak, mitochondrial outer membrane permeabilization, mitochondrial dysfunction and release of pro-apoptotic factors, such as
cytochrome c, apoptosis-inducing factor and Endo G from the
mitochondria into the cytosol.3,6 Depending on the extent of
caspase inhibition (for example, by high overexpression of
Inhibitor of Apoptosis (IAP) family members in tumor cells),
execution of intrinsic apoptosis can occur either in a caspasedependent or -independent fashion.
In contrast to apoptosis, necrotic cell death has been
traditionally characterized as a passive, that is, non-programmed
form, of cell death.4,7 Necrosis is the end result of a bioenergetic
catastrophe resulting from adenosine triphosphate (ATP) depletion, which is incompatible with cell survival and is thought to be
initiated mainly by cellular injury after toxic insults or physical
damage. Morphologically, necrosis is characterized by vacuolization of the cytoplasm, breakdown of the plasma membrane and
inammation around dying cells attributable to the release of
cellular contents and pro-inammatory molecules.7 However, in
addition to passive necrosis, it is now evident that (programmed)
necrosis can also occur in a regulated fashion, for example, after

1
Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany; 2German Cancer Consortium (DKTK), Heidelberg, Germany; 3German Cancer
Research Center (DKFZ), Heidelberg, Germany and 4Experimental Neurosurgery, Center for Neurology and Neurosurgery, Goethe-University Hospital, Frankfurt, Germany.
Correspondence: Professor S Fulda, Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstr. 3a, 60528 Frankfurt, Germany or Professor D Kgel,
Experimental Neurosurgery, Center for Neurology and Neurosurgery, Goethe-University Hospital Frankfurt, Heinrich-Hoffmann Str. 7, 60528 Frankfurt, Germany.
E-mail: simone.fulda@kgu.de or koegel@em.uni-frankfurt.de
Received 10 November 2014; revised 11 December 2014; accepted 12 December 2014

Autophagy and cell death

S Fulda and D Kgel

2
Autophagy and ACD
Autophagy is a cellular stress response and a quality control
mechanism that in general acts in a pro-survival manner. Different
forms of autophagy can be discriminated, including macroautophagy (hereafter simply denoted as autophagy), microautophagy
and chaperone-mediated autophagy. During autophagy, which
serves to regulate the turnover of long-lived proteins and
damaged organelles, these cellular constituents are engulfed in
double-membrane-containing vesicles called autophagosomes
(Figure 1).6,8,9 Their vesicular content is subsequently digested
by lysosomal proteases after fusion of autophagosomes with
lysosomes.8,9 Autophagy is a complex, multistep process that is
genetically regulated by the ~ 30 autophagy-related genes
(ATG) discovered hitherto in mammals. In addition, autophagy
is subject to posttranscriptional regulation, for example, by
microRNAs, as miR-101 has been shown to suppress autophagy

alkylating DNA damage, during excitotoxicity and after ligation of

death receptors.3 Regulated necrotic cell death often involves
activation of the kinases receptor-interacting protein 1 (RIP1) and
RIP3 and can be blocked by the specic RIP1 inhibitor necrostatin1. The term necroptosis is restricted to these necrostatin-1inhibitable forms of necrotic cell death.3,4 Mechanistically, death
execution via necroptosis is currently not well understood but
may involve energy failure, oxidative stress and lysosomal
membrane permeabilization.
The term mitotic catastrophe is usually used for cell death
induced by aberrant mitosis and executed either during mitosis or
in the subsequent interphase (this type of cell death is sometimes
also called mitoptosis).3 However, mitotic catastrophe can exhibit
features of apoptosis or necrosis, and the concept that mitotic
catastrophe truly represents a distinct mode of cell death is
currently not generally accepted.

stress conditions
cancer drugs
(Resveratrol, APO866, TMZ, THC, HDACs)

autophagy
induction

Vps34
inhibitors

lysosome/
endosome

mTOR

autolysosome

Beclin-1
depletion

autophagic
cell death

autophagic
initiation
membrane

fusion
vesicle
elongation

flux

cargo
degradation

autophagosome

vesicle
nucleation

Atg5

LC3-II
BECN1
core complex

chloroquine
BafA1
Betulinic acid B10

Atg5/Atg7
depletion

Vps34

Bcl-2/xL
Mcl-1

BECN1

complex
formation

membrane
recruitment

Atg5
FADD

complex
disruption

Vps34

autophagosome

BECN1

BH3 mimetics

RIP1

RIP3

Bcl-2/xL
Mcl-1

necroptosis
Figure 1. Different stages of autophagy and denition of ACD. Autophagy can be induced by multiple stimuli, including metabolic stress,
organelle dysfunction, protein aggregation and several cancer drugs, many of which target the central autophagy regulator mTOR. The
different stages of this process are tightly regulated by the core autophagy proteins encoded by ATGs. Autophagosome biogenesis starts
with the formation of an initiation membrane that can be derived from the ER and several other cellular membrane sources. Vesicle nucleation
is promoted by a large macromolecular complex containing the lipid kinase Vps34 (BECN1 core complex). BECN1 (ATG6) serves to activate
Vps34 leading to formation of PtdIns3P, which is required for this stage of the autophagic pathway. Vesicle elongation is regulated
by two ubiquitin-like conjugation systems involving several ATG proteins: (1) a large protein complex containing ATG5 (and ATG12/ATG16)
and (2) ATG7/ATG3-driven attachment of phosphatidylethanolamine to LC3-I, leading to the generation of LC3-II, which is inserted into
the autophagosomal membrane. Following vesicle closure, mature autophagosomes fuse with lysosomes or endosomes to autolysosomes in
which the autophagosomal content is digested by lysosomal proteases. Excessive activation of the autophagy pathway can lead
to an autophagy-dependent cell death in several paradigms. The term ACD should be exclusively limited to cases of cell death that are
mediated and not simply accompanied by autophagy. Therefore, only cases where inhibition of the autophagic pathway suppresses cell
death can be considered as true ACD. The effects of autophagy inhibition on cell death can be experimentally addressed at different
stages of autophagy, either by Vps34 inhibitors or by knockout/knockdown of core autophagic modulators, such as ATG5, ATG7 or BECN1.
It is currently controversially discussed whether the term ACD should be reserved only for cases in which the nal cell death process is
mediated by an enhanced autophagic ux rather than by alternative forms of cell death, such as necroptosis. In light of this controversy
and the cytotoxicity of drugs inhibiting the autophagic ux from autophagosomes to lysosomes (chloroquine, Balomycin A1),
interference at this stage of the autophagy pathway is currently not a generally accepted approach to analyze ACD. BH3 mimetics
have been implicated in several paradigms of ACD. They are capable to induce the release of BECN1 from its inhibitory interaction with
BCL-2/BCL-xL and have been shown to recruit necrosome components to the autophagosomal membrane, thereby inducing ACD (for details,
please refer to the main text).
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Autophagy and cell death

S Fulda and D Kgel

by downregulation of STMN1, RAB5A and ATG4D.10 The process of

autophagy begins with the formation of a nascent initiation
membrane, which serves as a prestage of autophagosomes.
Several ATG proteins (that is, ATG5, ATG7, ATG10 and ATG12) have
been implicated in autophagosome formation. The mammalian
ortholog of yeast ATG8 was originally identied as the light chain
3 (LC3) of microtubule-associated proteins. LC3 exists in two
forms, that is, LC3-I and its lipidated derivative LC3-II, which are
localized in the cytosol (LC3-I) or in autophagosomal membranes
(LC3-II), respectively.11 BECN1 (ATG6) is a core element of cellular
autophagy and a component of the class III phosphatidylinositol
3-kinase complex required both for formation and transport of
autophagosomes. BECN1 is a tumor-suppressor gene monoallelically deleted in several types of cancer, including ovarian, breast
and prostate cancer.12 In addition, the BECN1-binding protein
ultraviolet irradiation resistance-associated gene (UVRAG) has
been implicated as a tumor-suppressor gene that is monoallelically mutated at high frequency in human colon cancers.13 Bif-1
(also known as Endophilin B1) is another BECN1-interacting
protein that can act as a tumor suppressor, as knockout of Bif-1
has been reported to increase the development of spontaneous
tumors in mice.14 Furthermore, Ambra1 has been demonstrated to
interact with the BECN1 complex and deciency of Ambra1 in
mouse embryos has been shown to result in uncontrolled cell
proliferation,15 suggesting that Ambra1 has some tumorsuppressive properties. The quality-control function of autophagy,
especially in the context of genomic integrity, is correlated to its
role in cancer development: autophagy can act as a tumorsuppressor mechanism, as impaired autophagy, that is, the lack of
proper removal of toxic protein aggregates and organelles such as
dysfunctional mitochondria, has been shown to promote oxidative
stress, DNA lesions and genomic instability.16
The net effect of autophagy on cell death is highly contextual,
and both cytoprotective and cytotoxic functions of autophagy
have been reported. Autophagy comprises a primordial prosurvival stress response, for example, under conditions of nutrient
deprivation where it serves to ensure energy balance. In addition,
there is now substantial evidence showing that enforced overactivation of autophagy can lead to ACD (type II cell death)
(Figure 1), that is, cellular self-digestion via the autophagosomal
lysosomal pathway beyond the point of allowing cell
survival.3,4,8,16,17 It was proposed that the apparent dichotomy
between pro-survival and pro-death autophagy may be causally
related to the extent and duration of autophagy, indicating that
this dual function may represent a threshold effect of autophagy,
as also observed for other stress responses such as the
endoplasmatic reticulum (ER) stress response or activation of
p53.17
A number of studies suggest that (at least in some instances) it
is not simply overactivation of unspecic bulk autophagy but
rather the selective removal of autophagy substrates that
promotes cell death. Oxidative stress is a key feature observed
in many paradigms of autophagy-dependent cell death,18,19 and
the autophagy-mediated selective degradation of the endogenous reactive oxygen species (ROS) scavenger catalase has been
shown to induce ACD.20 In an analogous manner, inhibition of
selective autophagy by targeting the autophagy receptors
p62/SQSTM1 and BNIP3 was proposed to cause ACD.21,22 Further
molecular mechanisms possibly underlying the dual function of
autophagy in promoting either cell death or cell survival are
currently being addressed.
The exact denition of ACD has been controversially discussed
in the eld. It was suggested that many of the older, descriptive
cases of the so-called ACD actually reect death accompanied by
the block of the autophagic ux rather than death mediated by
autophagy.6 According to the criteria proposed by the NCCD, the
term autophagic cell death should be exclusively used from a
functional perspective and limited to cases of cell death that are
2015 Macmillan Publishers Limited

3
mediated by autophagy and can be suppressed by the inhibition
of the autophagic pathway (either by Vps34 inhibitors or by
knockout/knockdown of core autophagic modulators, such as
ATG5, ATG12 or BECN1) (Figure 1).23 Some authors have argued
for even more stringent criteria and have proposed to use the
term ACD only in cases where the nal cell death process is
mediated by an enhanced autophagic ux rather than by
apoptosis or necroptosis.6,24 In contrast to the authentic cases of
ACD as dened by the NCCD, cases of cell death that simply
exhibit markers of autophagy, such as an increase in autophagosomes, the lipidation of LC3 or an increased degradation of the
autophagic substrate p62, but cannot be suppressed by autophagy inhibition, should not be classied as ACD. Probably, the
most convincing examples of bona de ACD have so far been
observed in developmental cell death in lower model organisms,
including Caenorhabditis elegans, Drosophila melanogaster and
Dyctiostelium discoideum.6,2527 In addition, there are an ever
increasing number of studies demonstrating true ACD in
mammalian cells. Nevertheless, given the fact that autophagy
represents a double-edge sword with both tumor-suppressive and
-promoting properties, it remains to be determined whether or
not engagement of autophagy even under conditions of ACD may
elicit a more complex cellular response beyond modulation of cell
death. Some recent examples supporting the mechanistic concept
of ACD are outlined below.
The oncogene H-RAS was shown to trigger upregulation of the
BH3-only proteins Noxa and BECN1 and a caspase-independent
ACD, thereby limiting clonogenic survival.28 In this context, ACD
may represent a safeguard mechanism to limit the oncogenic
potential of deregulated RAS signals.28
Recently, an autophagy-dependent type of cell death termed
autosis was described.29 In this study, a cell-permeable autophagy-inducing peptide, that is, Tat-BECN1, was shown to induce
autophagy and, importantly, cell death in a dose-dependent
manner.29 Autotic cell death was also observed in starved cells
in vitro and in hippocampal rat neurons during cerebral ischemia
in vivo.29 This type of cell death was blocked by pharmacological
or genetic inhibition of autophagy, antagonists of the ion pump
Na+/K+-ATPase or genetic knockdown of the Na+/K+-ATPase 1
subunit, whereas inhibition of apoptosis or necroptosis provided
no protection.29 The results of this study also suggest that high
amounts of the BH3-only protein BECN1 are sufcient to trigger
ACD in the absence of other cellular stress conditions. In a similar
manner, overexpression of the BH3-only protein apolipoprotein L1
was previously shown to trigger ACD.30
In another recent study, caspase-10 was identied in an RNAi
screen to be required for suppression of an intrinsic form of ACD
in multiple myeloma.31 Caspase-10 was demonstrated to be
essential for dampening intrinsic autophagy by cleaving the
BCL-2-interactor and potent inducer of autophagy BCLAF1,
thereby preventing overactivation of autophagy to avoid ACD.31
Furthermore, the orphan nuclear receptor TR3 was shown to
promote ACD in melanoma cells.32 In this study, the TR3-targeting
compound 1-(3,4,5-trihydroxyphenyl)nonan-1-one was used to
trigger TR3 translocation to the mitochondrial inner membrane
through Tom40 and Tom70 channel proteins, dissipation of the
mitochondrial membrane potential and induction of autophagy
associated with excessive mitochondria clearance and irreversible
cell death. These ndings underscore the notion that selective
mitophagy, which typically acts in a cytoprotective manner, may
reach a certain threshold level at which it will turn into a deathpromoting process.
Despite the notion that induction of autophagy in response to
anticancer treatments often represents a cytoprotective mechanism of cells trying to cope with stress,33 overall there is now an
increasing and solid evidence for the existence of true ACD. Given
the fact that disrupted autophagy as well as excess autophagy can
have detrimental consequences on cell viability, abrogation as
Oncogene (2015), 1 9

Autophagy and cell death

S Fulda and D Kgel

4
well as overactivation of the autophagy pathway may both
represent relevant strategies for cancer therapy. Accordingly,
induction of autophagy-dependent cell death by pro-autophagic
drugs has emerged as a novel concept to sensitize cancer cells to
therapy or to directly kill them with the aim of exploiting caspaseindependent programmed cell death pathways for the development of novel cancer therapies. Recent relevant examples for
ACD induced by cancer drugs are outlined in the following
paragraphs (Table 1).
ACD INDUCED BY CANCER DRUGS
Gossypol
Antiapoptotic BCL-2 family members can form a complex with
BECN1/ATG63436 and formation/dissociation of this complex may
have an important role in modulating autophagy in tumor cells.
BCL-2 and BCL-xL sequester BECN1 via binding to its BH3 domain
and prevent it from forming a multiprotein complex essential for
vesicle nucleation during the early steps of the autophagic
process, thereby inhibiting autophagy. Consequently, BH3
mimetics are capable to activate both apoptosis and
autophagy.37 Gossypol is a natural polyphenolic compound and
BH3 mimetic derived from cotton seeds, which was initially
identied as an antifertility agent in China during the 1950s and
shown to possess cell death-promoting effects in various in vivo

Table 1.

and in vitro models.3841 Gossypol acts as a pan-BCL-2 inhibitor

and can inactivate BCL-2, BCL-xL, MCL-1 and BCL-w. There are two
enantiomers of gossypol, (+)-gossypol and ( )-gossypol (also
called AT-101), the latter being more potent as an inhibitor of
tumor growth. In cancer cells with an intact apoptotic machinery,
( )-gossypol has been reported to induce apoptotic cell
death.3942 In contrast, cell death triggered by gossypol largely
seems to depend on induction of ACD in apoptosis-decient
prostate cancer and malignant glioma cells.43,44
In glioma cells, ( )-gossypol was shown to trigger translocation
of LC3 to autophagosomes, lysosomal activity and a late
cytochrome c release, but cell death occurred in the absence of
overt lysosomal damage and effector caspase activation.43
( )-Gossypol and the alkylating agent temozolomide (TMZ)
cooperated to activate ACD in O6-methylguanine-DNA-methyltransferase-negative glioma cells, and lentiviral knockdown of
BECN1 or ATG5 in different glioma cell lines strongly diminished
cell death induced by ( )-gossypol and combined treatment with
TMZ, indicating that autophagy contributes to this type of cell
death.43 In contrast, stable knockdown of the endogenous
autophagy inhibitor mammalian target of rapamycin (mTOR)
increased ACD.43
In line with these ndings, ( )-gossypol has been reported to
cause caspase-independent, non-apoptotic cell death in malignant peripheral nerve sheath tumor cells, which could be blocked

Examples of autophagic cell death-inducing anticancer drugs

Substances

Cancer types

Autophagy inhibition used

References

BH3 mimetics
Obatoclax
Obatoclax
Obatoclax
Obatoclax
Obatoclax
Obatoclax
Obatoclax
Obatoclax
Gossypol
Gossypol
Gossypol

ALL
RMS
ALL
Colon carcinoma
Breast carcinoma
Pancreatic carcinoma
AML
B-cell lymphoma
Prostate carcinoma
GBM
Peripheral nerve sheath tumor

BECN1 and ATG7 siRNA, 3-MA

ATG5 shRNA, ATG7 siRNA, BafA1
BECN1 and ATG5 siRNA, 3-MA
BECN1 and ATG5 siRNA,
BECN1 siRNA, 3-MA
BECN1 siRNA
CQ
BECN1 siRNA
BECN1 and ATG5 siRNA, 3-MA
BECN1 and ATG5 siRNA, BafA1
3-MA

Natural products
Betulinic acid derivative B10
Betulinic acid
Resveratrol
Resveratrol

GBM
Cervical carcinoma
Breast carcinoma
CML

BECN1 and ATG5, ATG7 siRNA

ATG5 siRNA, ATG5 / MEFs
BECN1 and hVP34, ATG7 siRNA
ATG5, p62 and LC3 siRNA

63

HDACs
SAHA
SAHA
SAHA, OSU-HDAC 42

Chondrosarcoma
Cervical carcinoma
HCC

3-MA
BECN1 and ATG7 siRNA
ATG5 siRNA, 3-MA

68

Chemotherapeutics
TMZ
TMZ

GBM
GBM

3-MA
BECN1 and ATG5 siRNA

71

Cannabinoids
THC
THC, OWH-015

GBM
HCC

ULK1, ATG5 and Ambra-1 siRNA, ATG5 / MEFs

ATG5 siRNA, 3-MA

74

Others
Lapatinib
APO866

HCC
Leukemia, lymphoma cells

BECN1 and ATG5, ATG7 shRNA, 3-MA

ATG5 and ATG7 siRNA, 3-MA

50
51
52
53
55
56
57
58
44,76
43
45

64
65
66

69
70

43

75

54
19,67

Abbreviations: AML = acute myeloid leukemia; BafA1 = Balomycin A1; CML = chronic myeloid leukemia; CQ = chloroquine; GBM = glioblastoma;
HCC = hepatocellular carcinoma; HDAC = histone deacetylase; 3-MA = 3-methyladenine; MEF = mouse embryonic broblast; RMS = rhabdomyosarcoma;
SAHA = suberoylanilide hydroxamic acid; shRNA = short hairpin RNA; siRNA = small interfering RNA; THC = tetrahydrocannabinol; TMZ = temozolomide.

Oncogene (2015), 1 9

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Autophagy and cell death

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5
with 3-methyladenine (3-MA) and involved intracellular iron
chelation and hypoxia-inducible factor-1-induced expression of
the
BH3-only protein BNIP3.45 Similarly, BNIP3 was found to be
involved in autophagy-related cell death induced by combined
treatment of pancreatic adenocarcinoma cells with ( )-gossypol
and BRD4770, a small-molecule inhibitor of the histone methyltransferase G9a.46 These observations suggest that the BH3-only
protein and mitophagy-regulator BNIP3 may have an important
function in ACD,47 although it should be noted that the latter
study failed to address the question as to whether or not
autophagy does indeed contribute to this particular case of cell
death. In addition to the release of BECN1 from BCL-2/BCL-xL and
activation of BNIP3, oxidative stress is presumably involved in the
prominent pro-autophagic and death-promoting effects of
( )-gossypol. Indeed, ( )-gossypol and its derivative apogossypolone have previously been shown to be potent inductors of
oxidative stress in cancer cells.38,48
As indicated above, the effects of ( )-gossypol-induced
autophagy on cell death appear to be highly dependent on the
cellular context as also demonstrated in prostate cancer and
breast carcinoma cells. In androgen-independent prostate cancer
cells expressing high levels of BCL-2 and resistant to apoptosis,
( )-gossypol also preferentially induced ACD, which could
partially be blocked by knockdown of ATG5 and BECN1.44 In
contrast, apoptosis was preferentially induced in cells with low
BCL-2 expression.44 In this study, ( )-gossypol was shown to
induce BECN1- and ATG5-dependent autophagy via releasing
BECN1 from BCL-2 and BCL-xL at the ER, thus triggering the
autophagic cascade.44 In addition, oral administration of ( )-gossypol signicantly inhibited the growth of androgen-independent
prostate cancer xenografts, suggesting a potential relevance of
ACD for the treatment of human hormone-refractory prostate
cancer with BCL-2 overexpression.44 In contrast to these observations, ( )-gossypol inhibited the interaction between BECN1 and
BCL-2 to induce BECN1-dependent autophagy, which was
followed by the execution of apoptotic cell death in apoptosiscompetent MCF-7 human breast adenocarcinoma cells.49 Knockdown of Vps34 and ATG5 reduced ( )-gossypol-induced autophagy, and in this particular case, ( )-gossypol-mediated apoptotic
cell death was potentiated by treatment with or by small
interfering RNAs against core autophagy genes (Vps34, BECN1
and ATG5),49 suggesting a cytoprotective function of ( )-gossypol-induced autophagy in this apoptosis-procient cell model.
Obatoclax
There are a number of studies implicating autophagy as a
cytotoxic mechanism in obatoclax-induced cell death. In childhood acute lymphoblastic leukemia (ALL), subcytotoxic concentrations of obatoclax have been reported to overcome
glucocorticoid resistance by inducing ACD.50 Induction of
autophagy has been associated with disruption of the interaction
of BECN1 with MCL-1 as well as suppression of mTOR activity.50 In
line with the known suppressive function of mTOR on autophagy
induction, the mTOR inhibitor rapamycin acted in concert with
dexamethasone to induce ACD in this model.50 Concomitant
knockdown experiments showing that silencing of BECN1 or ATG7
rescued obatoclax-mediated sensitization to glucocorticoids in
both cell viability and clonogenic assays conrmed that stimulation of autophagy was critical for the ability of obatoclax to
overcome glucocorticoid resistance.50 The authors went on to
demonstrate that RIP1 as well as cylindromatosis (CYLD) were
required for the execution of cell death upon treatment with
obatoclax and glucocorticoids, whereas both RIP1 and CYLD were
dispensable for the initiation of autophagy in this context.50 As
RIP1 and CYLD are known as critical regulatory proteins in
necroptotic signaling, this study provided a genetic link between
2015 Macmillan Publishers Limited

ACD and necroptosis, although the underlying molecular mechanisms have remained elusive at that point.
Obatoclax subsequently was shown to stimulate the interaction
of components of autophagosomal membranes such as
ATG5 with proteins of the necrosome complex such as RIP1 in
an ATG5-dependent fashion, as ATG5 silencing inhibited this
interaction.51 The requirement of autophagy for cell death
induction was demonstrated by genetic silencing, as depletion
of ATG5 or ATG7 inhibited obatoclax-mediated autophagosome
formation and cell death.51 Data showing that genetic or
pharmacological ablation of RIP1 inhibited obatoclax-induced cell
death conrmed that RIP1 is a crucial mediator of cell death upon
treatment with obatoclax, while RIP1 turned out to be dispensable
for obatoclax-stimulated autophagosome formation.51
Furthermore, Heidari et al.52 reported that obatoclax is able to
bypass glucocorticoid resistance in ALL via the induction of
autophagy in addition to triggering apoptosis. Treatment of ALL
cells with obatoclax resulted in a rapid LC3 conversion and
degradation of p62 protein, both used as markers of autophagy.52
Knockdown of ATG5 prevented obatoclax-stimulated autophagy
as well as cell death,52 underlining that autophagy represents a
cell death mechanism in this context. By comparison, silencing of
BECN1 failed to block obatoclax-stimulated autophagy
induction,52 pointing to a BECN1-independent mode of autophagy. Similarly, pharmacological inhibition by 3-MA did not affect
obatoclax-mediated autophagy.52
In colon and breast carcinoma cells, obatoclax has been
reported to enhance cell death induced by lapatinib,53 a smallmolecule tyrosine kinase inhibitor targeting epidermal growth
factor receptors and Her2/Neu, which is capable to trigger ACD
either alone54 or in combination with other drugs. Obatoclaxmediated sensitization to lapatinib was associated with LC3
conversion and suppression of AKT/mTOR signaling as indicated
by reduced phosphorylation of AKT, mTOR and S6K1.53 Knockdown of BECN1 or, alternatively, of ATG5 protected against
obatoclax-/lapatinib-induced cytotoxicity,53 underscoring that the
induction of autophagy was necessary for combination treatmentmediated antitumor activity. Similar to obatoclax, knockdown of
MCL-1 or BCL-xL increased LC3 vesiculation and cell death by
lapatinib.53 Also, Tang et al.55 reported that obatoclax acts in
concert with lapatinib to trigger LC3 conversion and cytotoxic
autophagy in breast carcinoma cells, as genetic inhibition of
autophagy by knockdown of BECN1 or pharmacological inhibition
by using 3-MA signicantly reduced cell death induced by the
combination therapy. Mechanistic studies showed that obatoclax-/
lapatinib-mediated autophagy was associated with Noxamediated displacement of BECN1 from MCL-1 as well as inhibition
of mTOR signaling.55
Furthermore, obatoclax has been described to enhance the
induction of cell death upon treatment with histone deacetylase
(HDAC) inhibitors such as vorinostat or sodium valproate together
with the multikinase inhibitor sorafenib via autophagy.56 This
obatoclax-mediated sensitization to HDAC inhibitor-/sorafenibtriggered cell death was accompanied by LC3 vesiculation and
inhibited by knockdown of BECN1, consistent with an autophagic
form of cell death.56 Also, in acute myeloid leukemia obatoclax
was shown to exert antileukemic activity in concert with HDAC
inhibitors, that is, vorinostat and MGCD0103.57 This synergistic
antileukemic activity by obatoclax and HDAC inhibitors was
associated with the induction of both autophagy and apoptosis.57
The authors conclusion that autophagy contributes to the
synergistic antileukemic effects by the combination treatment is,
however, only based on experiments using chloroquine to inhibit
autophagy but lacks genetic evidence showing that autophagy
represents a cell death mechanism under these conditions.
Therefore, the question as to whether or not autophagy indeed
mediates cell death during obatoclax/HDAC inhibitor combination
treatment remains to be claried.
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S Fulda and D Kgel

6
In B-cell lymphoma, obatoclax was reported to stimulate LC3
conversion and ACD in a caspase-independent manner that was
signicantly inhibited by BECN1 knockdown.58
Importantly, the conclusion that autophagy represents a
cytotoxic process contributing to obatoclax-induced cell death is
based, in most studies, on genetic evidence showing that
knockdown of essential autophagy genes such as BECN1, ATG5
or ATG7 rescue cell death upon treatment with obatoclax.
However, there are also some studies concluding that obatoclax
triggers cell death via autophagy that are solely based on
experiments using pharmacological inhibitors of autophagy such
as chloroquine or 3-MA, which lack absolute specicity and may
also affect additional processes besides autophagy. This calls
for some caution as far as the functional impact of
autophagy induction in the context of obatoclaxs cytotoxicity is
concerned.
In addition to the pro-death function of autophagy in the
course of obatoclax-induced cell death, autophagy has also been
implicated as a cytoprotective or bystander mechanism in
response to treatment with obatoclax in some studies.5961 In
lung carcinoma cells, obatoclax-induced autophagy as documented by LC3 processing has been reported to depend on ATG7 but
not on BECN1.61 However, ATG7 was found to be dispensable for
obatoclax-induced cell death in this model,61 implying that the
induction of autophagy by obatoclax was not required for
obatoclax-mediated cytotoxicity. In esophageal carcinoma and
osteosarcoma cells, treatment with obatoclax caused conversion
of LC3 and ultrastructural changes consistent with the formation
of autophagosomes.59 Based on experiments showing that the
cytotoxicity of obatoclax was signicantly reduced by addition of
chloroquine or 3-MA, the authors concluded that obatoclaxinduced autophagy exerts cytoprotective functions under these
conditions.59 In breast carcinoma cells, BECN1 turned out to be
required for obatoclax-stimulated autophagosome formation but
not for obatoclax-induced cell death.60 In addition to engaging
autophagosome formation in this model, obatoclax was found to
block autophagic degradation of vesicular cargo by attenuating
cathepsin activity.60
Natural products
In addition to gossypol, other natural products have been
reported to engage cell death pathways via autophagy. For
example, the plant-derivative betulinic acid, a pentacyclic
triterpenoid derived from white birch trees,62 has been implicated
in modulating autophagy-mediated cell death. The semisynthetic
glycosylated derivative of betulinic acid B10 has been shown both
to trigger autophagy and to abrogate the autophagic ux in
glioblastoma cells, thereby switching autophagy into a cytotoxic
process.63 This B10-induced cell death was found to be associated
with destabilization of lysosomes and release of lysosomal
enzymes into the cytoplasm.63 Consistently, the cathepsin
inhibitor Ca074Me signicantly decreased B10-induced cell death,
further supporting that the release of lysosomal enzymes
contributes to B10-triggered cell death.63 In line with the notion
that destabilization of lysosomes can convert autophagy into a
detrimental pathway once autophagy has been initiated, inhibition of lysosomal enzyme activity was shown to protect against
B10-mediated cytotoxicity.63 Genetic studies showing that silencing of core autophagy genes, including BECN1, ATG5 or ATG7,
rescues cell death upon exposure to B10 supported the conclusion
that autophagy represents a cell death mechanism in the course
of B10 treatment.63 By comparison, induction of autophagy by the
parental compound betulinic acid has been implied as a
cytoprotective response, as cervical carcinoma cells with knockdown of ATG5 or mouse embryonic broblasts lacking ATG5 or
ATG7 exhibited increased sensitivity to betulinic acid.64 It will
therefore be interesting to explore whether different derivatives of
Oncogene (2015), 1 9

betulinic acid vary in their ability to modulate the autophagic

pathway.
Resveratrol represents another naturally occurring compound
that has been reported to trigger autophagy-mediated cell death
in cancer cells. Interestingly, a non-canonical, BECN1-independent
pathway of autophagy induction was documented upon treatment with resveratrol in human breast carcinoma.65 This
conclusion is based on experiments using BECN1 and Vps34 small
interfering RNAs that failed to inhibit resveratrol-stimulated
autophagy and cell death, whereas knockdown of ATG7 protected
breast carcinoma cells against resveratrol-mediated autophagy
and cell death.65 Engagement of the autophagic program by
resveratrol was shown to involve resveratrol-imposed inhibition of
AKT/mTOR signaling as well as conversion of LC3-I to LC3-II.65 In
chronic myeloid leukemia, resveratrol has been demonstrated to
promote ACD via c-Jun N-terminal kinase-mediated p62/SQSTM1
expression as well as activation of adenosine monophosphateactivated kinase, which in turn inhibited mTOR signaling.66
Knockdown experiments showing that silencing of key autophagy
components such as ATG5, LC3 or p62 protected against
resveratrol-triggered loss of cell viability conrmed that autophagy serves as a cytotoxic process in this model.66
APO866
There is evidence showing that cancer cells have a higher
nicotinamide adenine dinucleotide (NAD+) turnover rate than
non-transformed cells, suggesting that this biosynthetic pathway
represents an attractive target for cancer treatment.67 Recently, it
was reported that APO866, an inhibitor of nicotinamide phosphoribosyltransferase, the rate-limiting enzyme involved in NAD
(+) synthesis, induced an autophagy-dependent type of cell death
in leukemia and lymphoma cells,19,67 although both studies differ
with regard to the potential role of apoptosis in APO866-induced
cell demise. In the study by Cea et al.,67 the authors observed
APO866-dependent induction of autophagy and cell death, but
the pan-caspase inhibitor zVAD-fmk as well as specic inhibitors of
caspase-3 and -9 failed to protect from APO866-induced cell
death. In contrast, the pharmacological autophagy inhibitors
wortmannin, LY294002, 3-MA and chloroquine all reduced
APO866-induced cell death. However, genetic ablation of autophagy genes was not performed in this study. In the study by
Ginet et al.,19 the authors demonstrated that APO866 induces
autophagosome formation and SQSTM1/p62 degradation in
parallel to caspase-3 activation. Interestingly, APO866 treatment
evoked depletion of the ROS scavenger catalase, thus leading to
enhanced ROS levels and cell death. Inhibition of autophagy by
knockdown of ATG5 and ATG7 abrogated catalase degradation,
ROS production, caspase activation and cell death after APO866
treatment, which could be rescued by addition of exogenous
catalase.
HDAC INHIBITORS (HDACIS)
As aberrant regulation of epigenetic control of gene expression,
including histone acetylation, represents a hallmark of human
cancer, small-molecule inhibitors of HDACs are considered as
promising cancer therapeutics. Among their various cellular
effects, HDACIs have been reported to engage autophagy that
has also been linked to the induction of cell death. For example,
the HDACI suberoylanilide hydroxamic acid (SAHA) was described
to trigger autophagy-associated cell death in chondrosarcoma
cells.68 Cell death upon treatment with SAHA was accompanied by
increased lipidation of LC3 as well as ultrastructural changes
consistent with autophagosome formation.68 Furthermore, the
addition of 3-MA signicantly restored cell viability upon
treatment with SAHA, leading the authors to the conclusion that
autophagy-associated cell death is involved in the antitumor
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Autophagy and cell death

S Fulda and D Kgel

activity of SAHA against chondrosarcoma cells.68 However, the

question as to whether or not autophagy represents a cytotoxic
mechanism in this contest remains to be answered, as genetic
evidence supporting this conclusion is currently lacking.
Independent studies similarly reported that SAHA stimulates
autophagy-associated cell death in cancer cells. Induction of
autophagy by SAHA in HeLa cervical carcinoma cells was
associated with the inhibition of mTOR and accompanied by
typical ultrastructural features consistent with autophagy as well
as biochemical evidence such as conversion of LC3-I to LC3-II.69
Although induction of autophagy was shown to depend on key
genetic components of autophagy signaling, including BECN1 and
ATG7,69 the functional relevance of autophagy in SAHA-induced
cell death remains to be determined. In hepatocellular carcinoma,
HDACIs, including SAHA and OSU-HDAC-42, were described to
trigger ACD based on both pharmacological as well as genetic
evidence demonstrating that the addition of 3-MA or knockdown
of ATG5 reduced SAHA-mediated cytotoxicity.70 In parallel,
treatment with SAHA caused morphological changes typical for
autophagy, that is, autophagosome formation as well as lipidation
of LC3-I to generate LC3-II, and was accompanied by downregulation of p62, consistent with increased autophagic ux.70
Engagement of autophagy by HDACIs might involve inhibition of
mTOR signaling, as SAHA and OSU-HDAC-42 resulted in downregulation of AKT/mTOR signaling activity.70
TEMOZOLOMIDE
Furthermore, the DNA-alkylating agent TMZ, which causes
formation of O6-methylguanine in DNA that mispairs with thymine
during the following cycle of DNA replication and is routinely used
in the treatment of malignant glioma, was shown to stimulate
autophagy associated with the recruitment of LC3 to autophagosomal membranes.71 The role of autophagy in TMZ-induced
glioma cell death is currently controversial. On one side, early
interference with autophagy by 3-MA inhibited both autophagosome formation as well as TMZ-mediated cytotoxicity,71 arguing
for a pro-death role of autophagy in TMZ-triggered cell death. In
line with these observations, cell death induced by single agent
treatment with TMZ or by TMZ in combination with ( )-gossypol
was attenuated by early interference with autophagy via lentiviral
depletion of ATG5 and BECN1.43 On the other side, the study by
Kanzawa et al.71 also demonstrated that interference with
autophagy at a later stage using the vacuolar type H+ (ATPase)
inhibitor Balomycin A1 increased rather than reduced TMZinduced cell death. These data suggest that concomitant
induction of autophagy and disruption of the autophagic ux
enhances TMZ-triggered cytotoxicity, possibly through lysosomal
and mitochondrial membrane permeabilization and a shift
towards apoptotic cell death.71 To complicate matters even
further, it was also proposed that TMZ stimulates autophagy as
a cytoprotective response in glioblastoma that contributes to
chemoresistance.72 This notion is supported by experimental data
demonstrating that (1) TMZ can stimulate an autophagydependent surge of ATP that limits non-apoptotic glioma cell
death and that (2) genetic ablation of BECN1 reduced colony
formation after TMZ treatment in glioma cells.72 The opposing
effects of autophagy described in these studies are difcult to
explain but may be related to the different genetic backgrounds
of the cell models used72 and/or to dose-dependent changes in
the predominant DNA lesions induced by TMZ (O6-methylguanine
lesions versus N-alkylations).73 The precise role of autophagy and
the relevance of autophagy-dependent mechanisms for the
modulation of TMZ-induced glioma cell death await further
clarication.
2015 Macmillan Publishers Limited

CANNABINOIDS
Moreover, the main active component of cannabinoids, that is,
tetrahydrocannabinol (THC), has been demonstrated to trigger
autophagy-mediated cell death in different cancer entities,
including glioblastoma and hepatocellular carcinoma.74,75 The
functional requirement of autophagy to mediate THC-induced
antitumor activity has been established both in vitro and in vivo.
Accordingly, genetic silencing of autophagy-related genes,
including ULK1, ATG5 and Ambra-1, in glioblastoma cells as well
as knockout of ATG5 in broblasts rescued THC-imposed
cytotoxicity.74 Also in vivo using a tumor xenograft model based
on transformed mouse embryonic broblasts procient or
decient in ATG5, the induction of autophagy has been shown
to be critical for THC-imposed antitumor activity.74 In mechanistic
terms, the authors demonstrated that THC stimulates accumulation of ceramide and phosphorylation of eukaryotic translation
initiation factor 2 alpha, which engages an ER stress response that
leads to upregulation of ER stress-related proteins, including CHOP
and tribbles homologue 3 (TRB3).74 In turn, TRB3 facilitates
autophagy via inhibition of AKT/mTOR signaling.74 Interestingly,
autophagy was shown to be upstream of apoptosis that involved
loss of mitochondrial membrane potential and production of
ROS.74 Accordingly, THC-induced features of apoptotic cell death,
including Annexin-V positivity, caspase-3 activation and loss of
mitochondrial membrane potential, were attenuated in cells
decient in key autophagy genes.74 Vice versa, BAX/BAK double
knockout mouse embryonic broblasts exhibited similar conversion of LC3-I to LC3-II compared with wild-type cells, conrming
that stimulation of autophagy occurs upstream of apoptosis in
THC-induced cancer cell death.74 It is interesting to note that THCstimulated autophagy-mediated cell death has been documented
in different types of cancer cells but not in non-transformed
astrocytes,74 pointing to a potential therapeutic window that
could be exploited for cancer therapy.
In hepatocellular carcinoma, the cannabinoid receptor
2-selective agonist JWH-015 was reported to trigger autophagymediated cell death in addition to THC.75 In this study, the
engagement of the autophagic program by cannabinoids was
shown to involve activation of adenosine monophosphateactivated kinase via calmodulin-activated kinase beta in addition
to the induction of TRB3 and subsequent inhibition of AKT/mTOR
signaling.75 Genetic or pharmacological inhibition of autophagy
by ATG5 silencing or addition of 3-MA rescued the THC- or
JWH-015-imposed suppression of tumor growth of subcutaneous
hepatocellular carcinoma xenografts in vivo, emphasizing that
activation of autophagy was required for the antitumor activity of
THC and JWH-015.75
OUTLOOK
Autophagy has been shown to exert dual functions in human
cancers, that is, both as tumor suppressor and as tumor promoter.
As many anticancer drugs can engage autophagy, a better
understanding of the molecular mechanisms that regulate ACD
can pave the avenue for rational exploitation of this cellular
program for therapeutic purposes. There are now a number of
examples showing that the induction of ACD indeed represents a
crucial event for the drugs antitumor activity. This opens new
perspectives for the development of novel therapeutic strategies
and drug discovery. In addition, engagement of ACD may offer
new options to overcome treatment resistance, as autophagy has
been reported to serve as a backup mechanism with important
implications to bypass resistance especially in apoptosis-refractory
tumors. However, the molecular determinants that are responsible
for turning autophagy into a death process are currently still
poorly understood. As autophagy is a double-edged sword at the
interface of cell survival and cell death, an improved
Oncogene (2015), 1 9

Autophagy and cell death

S Fulda and D Kgel

8
understanding of the underlying signaling pathways that regulate
the impact of autophagy on cell death versus survival decisions
will be critical for the further exploitation of autophagy as a
strategy for cancer therapy.
ABBREVIATIONS
THC, tetrahydrocannabinol; TMZ, temozolomide.
CONFLICT OF INTEREST
The authors declare no conict of interest.

ACKNOWLEDGEMENTS
The expert secretarial assistance of C Hugenberg is greatly appreciated. This work has
been partly supported by a grant from the BMBF (to SF).

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