This document discusses doxorubicin (Dox), a commonly used chemotherapeutic agent, and its ability to induce autophagy in tumor cells and cardiomyocytes. Recent research has identified several major biomarkers, such as AMPK, p53, and Bcl-2, that are important for Dox-induced apoptosis. In particular, it is Bcl-2's interaction with Beclin-1 that has refocused attention on Dox's ability to induce autophagy. The document suggests that further research into Dox's molecular signaling in neoplastic and normal cells may help redefine how Dox is clinically used and lead to improved cancer management by potentially exploiting autophagy.

1. Doxorubicin-induced death in tumour cells and
cardiomyocytes: is autophagy the key to improving future
clinical outcomes?
Oktay Tacara
and Crispin R. Dassb,c
a
College of Biomedicine and Health, Victoria University, St. Albans, Vic., b
Biosciences Research Precinct and c
School of Pharmacy, Curtin University,
Bentley, WA, Australia
Keywords
autophagy; cardiotoxicity; cancer; cell death;
doxorubicin
Correspondence
Crispin R. Dass, School of Pharmacy, Curtin
University, Bentley 6155, Australia.
E-mail: Crispin.Dass@curtin.edu.au
Received April 1, 2013
Accepted August 20, 2013
doi: 10.1111/jphp.12144
Abstract
Objectives Doxorubicin, a commonly used frontline chemotherapeutic agent for
cancer, is not without side-effects. The original thinking that the drug causes
necrosis in tumours has largely given way to its link with apoptosis over the past
two decades.
Key findings More recently, major biomarkers such as AMPK, p53 and Bcl-2 have
been identified as important to apoptosis induction by doxorubicin. It is Bcl-2 and
its interaction with Beclin-1 that has refocussed research attention on
doxorubicin, albeit this time for its ability to induce autophagy. Autophagy can be
either anticancerous or procancerous however, so it is critical that the reasons for
which cancer cells undergo this type of cell biological event be clearly identified
for future exploitation.
Summary Taking a step back from treating patients with large doses of
doxorubicin, which causes toxicity to the heart amongst other organs, and further
research with this drug’s molecular signalling in not only neoplastic but normal
cells, may indeed redefine the way doxorubicin is used clinically and potentially
lead to better neoplastic disease management.
Introduction
The increasing incidence of cancer poses a great challenge
to global health. Epidemiological data collected by the
American Cancer Society estimates that 1 638 910 new cases
of cancer will be diagnosed by the end of 2012.[1]
Although
the 5-year relative survival rate for all cancers has increased
from 49% (1975–1977) to 67% (2001–2007), currently
marketed drugs are solely treatments, not cures, and have
their own limitations, some of which result in cessation of
further therapy or therapy being performed at suboptimal
doses.[1]
Cancer is characterised as numerous diseases underlined
by uncontrolled growth, producing an array of abnormal
cells that infiltrate healthy tissue. Cancer is caused by inter-
nal factors (inherited genetic mutations, hormone imbal-
ances and immune conditions) and external factors
(radiations, chemicals and smoking). The combination of
these factors can trigger a complex succession of events that
initiate cancer development, often taking over a decade to
become detectable.[2]
Tumours develop clones that are
cytogenetically different from the original cancer cell. These
heterogenous characteristics alter the behaviour of geneti-
cally altered clones and their responses to anticancer treat-
ments.[3]
To rationalise the complexities of cancer, a series of
six biological hallmarks are acquired during the complex
development of a tumour. These hallmarks provide an
understanding of cancer’s diverse framework and biology.
The six hallmarks include sustaining a proliferative signal,
evading growth suppressors, the ability to resist cell
death, replicative immortality, and the abilities to induce
angiogenesis and metastasis.[4]
Cancer chemotherapy, also referred to as antineoplastic
therapy, is clinically used to eradicate neoplastic cells via
chemotherapy, usually in combination with radiotherapy or
surgery. Many factors contribute to the potential success or
failure of chemotherapy agents, a main factor being tumour
cellular sensitivity and diversity. Despite these obstacles,
chemotherapy has been used for treating millions of suffer-
ers worldwide for decades. In many cases though, complete
bs_bs_banner
And Pharmacology
Journal of Pharmacy
Review
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1577–1589 1577

2. elimination fails often because of excessive tumour growth
and late diagnosis. Although complete elimination can
sometimes fail, the drug is used to reduce the number of
neoplastic cells to improve the patient’s chance of survival
and reduce their symptoms. A major problem is that the
tumour cells will usually be in different phases of the cell
cycle, and sometimes, the majority of them paradoxically
are in a phase of decelerated growth. Cells suffer decelerated
growth because of lack of physical space and nutrients as a
result of competition, and this can also occur in larger
tumours.
A chemotherapeutic agent works best when cancer cells
are in the exponential phase of growth, and as a result, the
vast majority of cells will not be affected and show resist-
ance to the drug. Table 1 lists the classes of chemotherapeu-
tic agents currently in clinical usage. The most common
problem with chemotherapeutic agents is that they are not
selective towards cancerous cells, and thus normal healthy
cells are also targeted and eliminated, especially in tissues
where cells divide constantly. Immune cells are also affected,
leaving patients susceptible to viral or bacterial infections.
In this review, we discuss the anticancer effects (Table 2) of
the anticancer agent doxorubicin (Dox), and the relatively
new literature on the link between this drug and autophagy.
This article seminally looks at summarising the various
papers on this link and suggests ways via which autophagy
may be exploited to increase the efficacy attributed to Dox.
Doxorubicin background
The current review will focus on Dox, one of the most com-
monly used chemotherapeutic drugs to date. It functions
primarily by inhibiting topoisomerase I and II, and interca-
lating into the DNA double helix to interfere with its
uncoiling, ultimately inducing cell death.[23]
The degree and
type of cell death caused by Dox varies, often determined by
either the concentration administered or cell line that was
treated. Indeed, Dox has shown great treatment potential
and is regarded as one of the most potent of all 132 Food
and Drug Administration-approved chemotherapeutic
drugs.[24]
Its reputation as a leading chemotherapeutic agent
arises from its ability to combat rapidly dividing cells and
slow disease progression, limited only by its toxicity on
non-cancerous cells in the body.
Chemically, the drug is a non-selective class I
anthracycline, possessing aglyconic and sugar moieties
(Figure 1). The aglycone comprises a tetracyclic ring with
quinine–hydroquinone adjacent groups, methoxy substitu-
ent short side chain followed by the carbonyl group. The
sugar component (also known as daunosamine) is attached
to one of the rings by a glycosidic bond. This comprises a
3-amino-2, 3, 4-trideoxy-L-fucosyl moiety.[25]
Doxorubicin pro-apoptotic
drug action
Dox can treat cancer by effecting various biological cellular
changes, the extent of its effectiveness depending on both
dose and cell line as previously mentioned. Dox acts by
binding to DNA-associated enzymes that intercalate the
base pairs of the DNA’s double helix.[25]
By binding to
multiple molecular targets, such as topoisomerase enzymes
I and II, a range of cytotoxic effects occur in conjunction
with antiproliferation, thus resulting in DNA damage and
the formation of DNA-cleavable complexes.[26]
Table 1 Classes of chemotherapeutic drugs and their activity summaries
Chemotherapeutic agent Function
Alkylating agents Damage DNA to prohibit tumour cells from further dividing
Antimetabolites Interfere with the synthesis of DNA and RNA by substituting for the normal building blocks required
for normal DNA replication and transcription
Antitumour antibiotics (anthracyclines;
example doxorubicin)
Interfere with the enzymes involved in DNA replication and are capable of inflicting their action
regardless of what cell cycle phase the cell is in, though the preference would be in mitotic cells
Topoisomerase inhibitors Interfere with topoisomerase, which is an enzyme responsible for separating the double strands of
DNA, while mitotic inhibitors are natural products that stop mitosis, usually interacting with and
perturbing the microtubule spindle machinery facilitating mitosis
HO HO
OHO O
O
H
OH
HO
OH
OH
OH
NH2
Figure 1 Molecular structure of doxorubicin. Systematic name:
(7S,9S)-7-((2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl)oxy-
6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-
tetracene-5,12-dione.
Oktay Tacar and Crispin R. DassDoxorubicin-induced autophagy
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1577–15891578

3. The apoptosis pathway is triggered when the attempt to
repair the breaks in DNA fail, and cellular growth is inhib-
ited by Dox at phases G1 and G2.[27]
Dox is also known to
intercalate itself into the DNA.[28]
The intercalation inhibits
the action of both DNA and RNA polymerases, ultimately
ceasing DNA replication and RNA transcription. This
process occurs as Dox enters the cell through diffusion
using its higher affinity to bind to the proteasome in the
cytoplasm.[24]
A Dox–proteasome complex is formed when
Dox binds to the 20S subunit of the proteosome, whereby it
is then translocated through the nuclear pores into the
nucleus.[29]
In this case, Dox has a higher affinity for the
DNA over the proteasome it is attached to, allowing it to
dissociate itself from the proteasome and bind to the
nuclear DNA. Other Dox actions include free-radical gen-
eration, which causes further DNA damage, inhibits macro-
molecule production, DNA unwinding/separation and
increase in alkylation.[30]
A recent study highlighted the
drug’s ability to intercalate with not only nuclear DNA, but
also mitochondrial DNA.[31]
Furthermore, Dox can affect
the cell membrane directly by binding to plasma proteins
causing enzymatic electron reduction of Dox. This can
cause the formation of highly reactive hydroxyl free radi-
cals, responsible for the dangerous side effects elicited by
the drug’s use.[32]
These mechanisms make Dox a potent
anticancer drug, allowing it to act upon many forms of
cancer in isolation or in combination with other anticancer
drugs – breast, prostate, haematological malignancies, oste-
osarcoma, bile duct neoplasms and cancers of the oesopha-
gus, stomach, uterus, ovary and liver.[33]
Doxorubicin activates adenosine
monophosphate-activated
protein kinase
It is well known that clinical use of Dox can be limited
in most patients because of its life-threatening toxicity.
More importantly is the drug’s tendency to severely
jeopardise cardiac health. Gaining further insight into the
cellular mechanisms involved in these processes will be
Table 2 Molecules influenced by Dox in tumour cell lines
Protein marker Cell line Level of activation References
p53 Myocardial H9c2 Expression ↑ 5–8
Human cervical carcinoma cells Expression ↑ 9
Breast cancer MCF-7 cells Expression ↑ 9
MCF55a cells Expression ↓ 9
Proliferating human umbilical vein endothelial cells (HUVECs) Expression ↑ 10
AMPK Myocardial H9c2 cells Expression ↑ 8
ERK Human cervical carcinoma cells Expression ↑ 9
Breast cancer MCF-7 cells Expression ↑ 9
JNK Jurkat cells Expression ↑ 8,11
Bcl-2 In-vivo adult rat heart Expression – no change 12
Breast cancer MCF-7 cells Expression ↓ 13
MCF55a Expression ↑ 13
Breast cancer cell line MTLn3 Expression ↑ 14
Myeloma cell lines ; 8226, IM-9, U266 Expression ↑ 15
Bax Breast cancer MCF-7 cells Expression ↑ 13
MCF55a cells Expression – o change 9
In-vivo adult rat heart Expression ↓ 12
Caspase-2 Jurkat cells Expression ↑ 11
Caspase-3 Cardiomyocytes Expression ↑↑ 16
Human T-leukemia cells Expression ↑ 17
Human neuroblastoma SKN-SH Expression ↑ 18
Neonate mice in-vivo heart, brain, skeletal muscle Expression ↓ 18
In-vivo adult rat heart Expression ↑ 12
Human ovarian terato-carcinoma (PA-1) Expression ↑ 19
Bovine aortic endothelial cells (BAECs) Expression ↑ 19
Caspase-8 Cardiomyocytes Expression ↑ 16
TLR-2-knockout mice Expression ↓ 16
Caspase-12 Rat heart Expression ↑ 20
NFκB Myeloid leukaemia cell line U937 Expression ↑ 21
Hepatoma cells Expression ↑ 22
AMPK, adenosine monophosphate-activated protein kinase; Bcl-2, B-cell lymphoma 2; ERK, extracellular signal-regulated kinase; NFκB, nuclear
factor kappa B; PA-1, plasminogen activator 1, TLR-2-toll-like receptor-2.
Oktay Tacar and Crispin R. Dass Doxorubicin-induced autophagy
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1577–1589 1579

4. beneficial when optimising Dox treatment. Adenosine
monophosphate-activated protein kinase (AMPK) is a
protein that performs many functions such as acting as an
intracellular energy status sensor, providing a regulatory
role in both cell survival and death when subjected to
pathological stress ((e.g. oxidative stress and endoplasmic
reticulum (ER) stress)).[34–39]
Furthermore, AMPK is known
to mediate apoptotic effects.[40]
Studies on AMPK in rela-
tion to Dox have been extensively undertaken across a wide
range of cell lines, all revealing strikingly similar molecular
pathway involvement. With regard to myocardial toxicity,
numerous studies have shown that Dox induces AMPK
activation in embryonic ventricular myocardial H9c2 rat
cells in response to reactive oxygen species (ROS) accumu-
lation.[8]
Thus, Dox causes the accumulation of ROS, which
in turn activates dependent liver kinase B1 (LKB1), which
provides the upstream signal necessary for AMPK activa-
tion, ultimately leading to p53 phosphorylation at Ser15
and apoptosis (Figure 2). This molecular pathway occurs in
H9c2 cells. In insulin-producing MIN6 cells, AMPK activa-
tion triggers the activation of c-Jun N-terminal kinases
(JNK) to cause apoptosis. Activated JNK isoforms have a
range of pro-apoptotic functions such as phosphorylating
ATF2, c-Jun as well as pro- and anti-apoptotic B-cell lym-
phoma 2 (Bcl-2) family members.[8]
Dox-induced ROS production can occur in various
ways. One reaction involves ‘redox cyclers’ that generate
superoxide when reacted with flavoprotein reductase in the
presence of oxygen. ROS production can also occur through
a Fenton-type reaction by chelating intracellular iron.
This results in the production of highly reactive hydroxyl
radicals.
The exact cascade of molecules involved in JNK stimula-
tion via AMPK is still unknown. The activation of the
AMPK leads to the activation of the JNK pathway.[8]
Furthermore, studies have shown that AMPK induces
apoptosis through JNK stimulation in insulin-producing
β-cells and liver cells.[41,42]
In many cell lines, it can be con-
cluded that Dox induces the production of ROS, which acti-
vates LKB1, in turn activating AMPK, which can then
trigger apoptosis via two pathways. One involves AMPK
activating JNK to trigger apoptosis, although more underly-
ing mechanisms of this process are still relatively unknown.
AMPK can also phosphorylate p53 at the Ser15 region,
causing Bax to migrate to the surface of the mitochondrial
membrane to trigger the traditional mitochondrial
apoptotic cascade.[8,40]
Doxorubicin’s effect on p53
The p53 tumour suppressor protein is a sequence-specific
transcription factor, playing a vital role in regulating the cell
cycle, repairing DNA and angiogenesis. When subjected to
cellular stress, the p53 protein will activate to arrest cell
growth (at G1 or G2) and induce apoptosis in an effort to
prevent cancer as a tumour suppressor.[43]
The protein is
short-lived and is activated by post-transcriptional modifi-
cations such as phosphorylation. Kinases known to phos-
phorylate p53 include extracellular signal-regulated kinase 2
(ERK2), ATR, CK1 and DNA-PK.[9]
The cellular responses
triggered by p53 are influenced by a range of factors includ-
ing the level of stress, cell line and p53 coactivator function.
The proteins signalling networks affect both the intrinsic
and extrinsic apoptotic pathways, but are also known to
cause senescence in some cases. Being a major interference
for tumorigenesis, p53 needs to be removed in order for the
tumour to progress. Naturally, the protein is removed or
mutated in approximately 50% of human cancers, with the
remaining cases compromising p53 activity via different
mechanisms.[44]
Doxorubicin-induced apoptosis in normal
and tumour cells
Studies involving p53’s mechanisms involved in Dox-
induced apoptosis in both normal and tumour cells have
shown interesting results. One study evaluated the role of
p53 in Dox-induced apoptosis in normal bovine aortic
endothelial cells (BAECs), adult rat cardiomyocytes and
in tumour cell lines.[19]
Results were mixed, showing
Dox-induced apoptosis is mediated by different signal
transduction pathways. BAECs and tumour cells that were
transiently transfected with p53 luciferase plasmid showed
that Dox-induced activation of p53 was occurring in all cell
lines, yet the response was higher in tumour cells. Despite
the time course being different, p53 was activated after 8 h
of incubation in BAECs, while the tumour cell line showed
that p53 activated during the initial 2 h of incubation, yet
little apoptosis was occurring. Despite p53 being activated
in all cell lines, the lack of apoptosis in the tumour cell line
suggests that Dox-induced apoptosis is independent of p53
activation in endothelial cells while dependent in the
tumour cell. Results were confirmed by inhibiting p53 in
BAECs, which revealed the rate of apoptosis did not recede.
Phosphorylation of p53 via ERK2 activation in
doxorubicin-induced cell death
Studies in both human cervical carcinoma cells and MCF-7
have revealed that phosphorylation of p53 on the Thr55 site
by ERK2 is necessary for Dox-induced p53 activation and
cell death.[9]
p53 can be phosphorylated at different sites by
a range of different kinases to produce specific activity. In
the case of Dox, ERK is involved in this response. Like many
chemotherapeutic drugs that damage DNA, Dox activates
ERK in tumour cells. In order for p53 to be activated, it
must be phosphorylated at specific phosphorylation sites.
Oktay Tacar and Crispin R. DassDoxorubicin-induced autophagy
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1577–15891580

5. Studies have been conducted on the role of the novel
phosphorylation site Thr55 in response to Dox administra-
tion. Results reveal that Dox activates ERK2 in MCF-7
breast cancer cells, which in turn phosphorylates p53 at the
Thr55 region. One theory suggests that DNA damage along
with other cellular stress factors induces the sequential
phosphorylation of p53 on various residues via different
kinases. Phosphorylating and activating different sites may
DOXORUBICIN
Cell membrane
ROS
LK81
AMPKJNK
Bax p53
Bax/Bcl-2
Apaf-1
Cleaved caspase-9
Cleaved caspase-12
Pro/cleaved caspase-3
Pro/cleaved caspase -6, -7
APOPTOSIS
Cytochrome c release
Endoplasmic reticulum
Proteosome
- DNA damage
- Mitochondrial damage
- ER stress
Figure 2 The mitochondrial apoptotic pathway pertinent to doxorubicin. Mitochondrial apoptosis pathway. DNA damage as a result of
doxorubicin (Dox) administration causes the formation of free radicals (ROS), which provides the upstream signal necessary for adenosine
monophosphate-activated protein kinase (AMPK) activation, leading to the phosphorylation of the tumour suppressor p53 at Ser15. p53 can also be
activated if the mitochondria are structurally damaged as a result of drug administration. The activation of p53 triggers the migration of Bax from
the cytosol to the mitochondrial surface where it forms the classic Bcl-2/Bax toggle switch complex. If the ratio of Bcl-2/Bax shifts towards Bcl-2,
apoptosis is avoided. However, if the ratio tilts towards Bax, the apoptosis pathway continues leading to cytochrome c release. The release of
cytochrome c can also occur if the mitochondria are damaged causing a ‘leak’ effect. The released cytochrome c binds with Apaf-1 and
procaspase-9 to form a proteasome, which leads to the sequential cleavage and activation of caspase-9 and caspase-3. Once caspase-3 is cleaved
and activated, the pathway enters the point of no return, resulting in the activation of caspase-6 and -7 and other death substrates to ultimately
induce apoptosis. Furthermore, Dox can also cause stress to the endoplasmic reticulum, which may result in caspase-12 activation leading to
apoptosis.
Oktay Tacar and Crispin R. Dass Doxorubicin-induced autophagy
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1577–1589 1581

6. induce apoptosis and inhibit growth arrest by producing
the effect on different downstream genes. Time-dependent
studies have proven that certain residues are phos-
phorylated before others. For instance, ERK2 activity
phosphorylates p53 at the Thr55 region 6–9 h after Dox
administration.[9]
In contrast, the Ser15 region of p53 was
phosphorylated within minutes of Dox treatment.[45]
However, it is unclear whether or not the phosphorylation
of Ser15 is needed to activate Thr55. ERK’s role in
phosphorylating p53 in response to DNA-damaging drugs
seems to be dependent on the cell line being treated. Previ-
ous studies have shown that ERK induces p53-independent
cell death in neuroblastoma and osteosarcoma cells,
while other studies have shown that treated ovarian cells
cause ERK-mediated p53-independent apoptosis and
cell cycle arrest. Thus, research demonstrates that ERK
phosphorylates p53 at both Ser15 and Thr55 regions. Block-
ing the Thr55 residue prevents p53 from activating, causing
Bcl-2 expression to rise in consequence. This contributes to
apoptosis resistance in breast and cervical carcinoma cells.
When the involvement of the ERK/p53 signal trans-
duction pathway in Dox-induced apoptosis in H9c2 cells
and cardiomyocytes were examined, cell death occurred in a
time-dependent matter.[7]
Initially, ERKs were activated and
translocated from the nucleus where p53 was activated
at the Ser15 region and translocated to the nucleus. The
increase in p53 expression was associated with Bcl-2 levels
decreasing in expression, while Bax was upregulated in
combination with p53. While the Bcl-2/Bax ratio decreased,
levels of p53 upregulated modulator of apoptosis (PUMA)
rose. PUMA has been shown to be induced by p53 and con-
tributes to p53-mediated apoptosis in vivo and in vitro. p53
is thought to bind to Bcl-xL to cause mitochondrial translo-
cation and a conformational alteration of p53 and Bcl-2.
These sequences of events trigger the release of cytochrome
c to form the apoptosome with Apaf-1 and procaspase-9.
The classic apoptosis molecular sequence follows involving
the activation of caspase-9, -3 and poly (ADP-ribose) poly-
merase (PARP) and ultimately cell death via apoptosis. The
exact mechanisms involved in Dox-induced mitochondrial
translocation of both p53 and Bcl-2 have yet to be deter-
mined and require further study.
Weak p53 permits senescence during
doxorubicin-induced cell cycle arrest
p53 is also known to be an inducer of cellular senescence
while also having the ability to suppress it.[46–48]
p53 accom-
plishes suppression of senescence by its ability to inhibit
mammalian target of rapamycin (mTOR), cell growth and
protein synthesis. Its ability to inhibit mTOR depends on
the specific cell line being treated.[49]
Cell lines with low
levels of p53 that are treated with low doses of Dox reveal
that Dox interferes with p53’s ability to suppress mTOR.
This may result in phosphorylated p53 either inducing
senescence or failing to suppress it.[50]
Another theory could
be that low doses of Dox cause cell cycle arrest, but does not
induce p53 levels high enough for it to inhibit mTOR and
therefore inhibit senescence. Low levels of phosphorylated
p53 can induce senescence.[50]
In the absence of p53 and
p16, low-dose Dox (50 nM) causes senescence and subse-
quent autophagic induction.[51]
Dox induces cell cycle arrest via several different mecha-
nisms, and many do not involve p53 or inhibition of
mTOR. This is consistent with the notion that low doses of
DNA-damaging agents are needed to cause senescence.[52,53]
When a cell experiences environmental stress or structural
damage, activation of p53 leads to the migration of Bax to
the surface of the mitochondria. Once Bax has migrated to
the surface of the mitochondria, a shift in the Bcl-2/Bax
ratio will occur, determining whether a cell survives or
undergoes apoptotic cell death.
The effect of doxorubicin on the
Bcl-2/Bax pathway
Several studies have focused their attention on the effect
that Dox has on the Bcl-2/Bax complex. The Bcl-2/Bax
complex resides upstream of irreversible cellular damage,
playing a pivotal role in cell survival and death. Bcl-2 is
known for its anti-apoptotic properties that are opposed by
Bax.[54]
It is the ratio of Bcl-2 to Bax that modulates the
cell’s susceptibility to apoptosis.[55]
This crucial pivotal role
makes the complex an ideal target for novel cancer thera-
pies. An imbalance of the Bcl-2/Bax ratio can tilt the scale
towards survival, resulting in cancerous cell lines that are
resistant to cell death stimuli, growth factor withdrawal and
chemotherapeutic drugs such as Dox. A major factor affect-
ing resistance is chromosomal rearrangement, causing the
overproduction of Bcl-2 proteins and thus shifting the Bcl-
2/Bax ratio to increase survival.[55]
By targeting the anti-
apoptotic Bcl-2 protein, it is anticipated that cancer cell
resistance to chemotherapy can be overcome. The ratio of
Bcl-2/Bax can also serve as a predictive marker, allowing
prognosis in patients with carcinomas.[56]
Studies involving Dox-treated breast cancer MCF-7 cells
have shown a decrease in Bcl-2 with increase in Bax.[13]
This
is expected if Dox is to induce apoptosis. When Dox is
administered, increase in ROS stimulates p53 activation,
suggesting that Dox-induced Bcl-2 downregulation is medi-
ated by the p53 pathway. p53 induces a translocation of Bax
to the mitochondria where the ratio shift occurs. As the
level of Bax rises and Bcl-2 is downregulated, cytochrome c
is released from the mitochondria, leading ultimately to
apoptosis. Various studies in M186 and M221 melanoma
cells show similar results where a change in the Bcl-2/Bax
Oktay Tacar and Crispin R. DassDoxorubicin-induced autophagy
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1577–15891582

7. ratio caused apoptosis. To achieve the required threshold in
the Bcl-2/Bax ratio, and thus induce apoptosis, Dox must be
administered at a high concentration or incubated for a
longer period. Indeed, Dox administered at high concentra-
tions results in cellular death via apoptosis, although the
specific type of Dox-induced cell death at lower concentra-
tions is still unknown.
Like many DNA-damaging drugs, Dox may modulate
apoptosis through a variety of other pathways other than
the Bcl-2/Bax complex. Some studies suggest that Dox
employs its effects by stimulating the components within
the Fas/Fas ligand (FasL) extrinsic apoptosis pathway,
although other studies show contradictory results.[57,58]
Thus, drugs do not induce their effects via one universal
pathway, and many factors have to be taken into account. A
rise in cytochrome c levels within the cellular cytosol of
Sprague Dawley rats treated with Dox was noted,[12]
although rather paradoxically, an increase in the Bcl-2/Bax
ratio was noted because of a decrease in the mitochondrial
levels of Bax, which would be protective against apoptosis.
Despite the protective characteristics of this event, it high-
lights the drug’s ability to modulate apoptosis through
other pathways independent of Bcl-2/Bax as previously
mentioned.
Initially, the Bcl-2 protein family was characterised as cell
death regulators. Recent studies have revealed that the Bcl-2
family can also control autophagy.[59]
By pairing with the
autophagy protein Beclin-1 (Becn1), Bcl-2 can inhibit
autophagy by forming the Bcl-2/Becn1 complex.[54]
This
complex works similarly to the Bcl-2/Bax complex in that a
shift in ratio can determine whether or not a cell undergoes
autophagy.
Doxorubicin and autophagy
Autophagy, a cellular process known to provide a survival
advantage to cells experiencing stress or nutrient depriva-
tion has recently been linked cell death.[60]
Hence, apoptosis
is no longer considered the sole means of programmed cell
death via self-elimination. The cell’s decision as to which
death pathway to trigger depends on many scenarios from
the cellular environment to surrounding stimuli. Studies
have revealed that both apoptosis and autophagy share
common molecular regulators. This suggests that they are
not exclusive pathways and that they tend to counteract one
another.[10]
Suffice it to say that studies relating to Dox-
induced apoptosis have been widely studied, yet Dox-
induced autophagy is still to be properly explored.
As abovementioned, apoptosis and autophagy share
common molecular regulatory pathways. The Becn1/Bcl-2
complex is a potential target for Dox-induced autophagy.
Indeed, the cytoprotective function of Bcl-2 arises from its
ability to antagonise Bax and block mitochondrial outer
membrane pores, ultimately preventing apoptosis.61]
In
recent years however, Bcl-2 has been shown to inhibit
autophagy, too, but only when it is present in the ER.[62]
Becn1 is a novel Bcl-2 homology (BH3) domain-only
protein and is localised within cytoplasmic structures such
as the mitochondria and ER, and distributed throughout
the plasma membrane and nucleus.[63]
Becn1 acts as a
phosphorylation substrate for the tumour suppressor
death-associated protein kinase, which phosphorylates
Becn1 to stimulate autophagy. This review will focus pri-
marily on the Becn1/Bcl-2 complex from which Becn1
binds to Bcl-2 through a BH3 domain that mediates
docking to Bcl-2’s BH3-binding groove.
Double knockout Bax-/-
Bak-/-
mouse embryonic fibro-
blasts (MEFs) showed resistance to a range of apoptosis
inducers. When treated with DNA-damaging drugs similar
to Dox, the Bax-/-
Bak-/-
MEFs failed to undergo apoptosis
and instead resulted in substantial autophagy.[64]
It is
known that suppression of apoptosis prompts autophagy
and vice versa. Identifying the regulatory mechanisms
involved in the apoptosis and autophagy relationship is
vital for inhibiting tumour growth and refining chemo-
therapeutic agents. We know that the Bax/Bcl-2 complex
acts as the toggle switch for apoptosis while the Becn1/
Bcl-1 complex acts as the toggle switch for autophagy. One
reason as to why Dox-induced autophagy is yet to be seen
is that Becn1 is deleted in 40–70% of ovarian, prostate,
breast and sporadic tumours. Partial Becn1 silencing has
been shown to aggravate Dox-induced apoptosis in HepG2
cells.[65]
The apoptotic effects of Dox were amplified by
the mitochondria, especially in haepatoma and haepatocyte
cell lines. The mitochondrial membrane potential dissi-
pated far more severely, while in Becn1-/-
cells, the release of
cytochrome c was far more exaggerated when compared
with the control. Thus, although no studies report on Dox
inducing autophagy, there is clearly a relationship between
both apoptosis and autophagy cell death. The results
suggest that Becn1 depletion activates mitochondrial
apoptosis.[65]
The expression levels of Becn1 and Bcl-2 determine
whether a cell undergoing chemotherapy will resist
autophagy or apoptosis.[62]
Low expression of Becn1 has
been linked to poor prognosis and decreases the removal of
damaged organelles via autophagy. The damaged organelles
produce ROS and cellular stress, which already play a major
role in Dox-induced apoptosis. It is possible that Dox
administered at a lower dose in cell lines that are not Becn1-
deficient could induce autophagy, which would remove
damaged organelles, resulting in reduced ROS levels and
cellular stress in an attempt to prevent Dox-induced
apoptosis. Discovery of which pathways are responsible for
Dox-induced autophagy will subsequently lead to develop-
ment of ways to overcome this phenomenon, thereby
Oktay Tacar and Crispin R. Dass Doxorubicin-induced autophagy
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1577–1589 1583

8. possibly leading to better drug activity. One such example is
the use of neuroregulin-1, which modulates the Akt signal-
ling pathway and leads to cardiomyocyte protection against
Dox damage.[66]
As a new therapeutic strategy, autophagy can be targeted
to induce autophagic death or inhibit the protective proper-
ties of autophagy depending on the cell line.[67]
Cell lines
can be resistant to Dox, often because of upregulated anti-
apoptotic Bcl-2 protein members. With many cell lines
being apoptosis defective and drug resistant, chemothera-
peutic agents that evoke autophagic cell death may be more
useful in future oncological therapy. There is plenty of evi-
dence to suggest that cancer cells resistant to apoptosis can
undergo autophagy; therefore, induction of autophagic cell
death may prove to be a novel therapeutic tool.
The effect of doxorubicin on the
caspase family
It has been shown that caspases can cleave Becn1, destroy-
ing its pro-autophagic function.[68]
Once Becn1 is cleaved,
the C-terminal fragment is formed with a newly acquired
function to amplify mitochondrial-mediated apoptosis. The
BH3 domain of Becn1 showed no pro-apoptotic activity
and remained within the N-terminal fragment. Caspases
are cysteine proteases that cleave a number of different
substrates within the cytoplasm and nucleus to mediate
apoptosis.[69]
The caspase family are not only responsible for
many of the morphological features of apoptosis, but they
also play a vital role in regulatory events such as axon
pruning in neural functions.[70]
For decades, it has been well
established that caspases are required for successful comple-
tion of apoptosis. For this reason, the effect of Dox on these
particular proteolytic enzymes is of great interest. There are
numerous reports of Dox-induced apoptosis occuring
via caspase activation, with only a few cases expressing
Dox-induced apoptosis occurs via caspase-independent
pathways.[71]
Studies have shown that caspase activation
can occur through four pathways; mitochondria-mediated
pathway, receptor-mediated pathway, ER-mediated pathway
and the granzyme B-mediated pathway.[72]
Of these four
pathways, Dox-induced apoptosis via caspase activation is
best understood in the context of mitochondria-mediated
and receptor-mediated pathways.
The caspase family can be separated into two groups;
the initiator caspases-1, -8, -9 and -10, and executioner
caspases-3 and -7.[73]
Many studies into Dox-induced car-
diomyopathy consider Dox to be intimately implicated in
caspase activation via the mitochondrial pathway. Dox-
induced apoptosis in cardiomyocytes resulted in an
increased activity of caspase-3 but not of caspase-8 activity,
a caspase that plays a vital role in receptor-mediated
apoptosis.[16]
It has been well documented that Dox generates ROS,
with oxygen species thought to play a major role in Dox-
induced toxicity. As ROS impairs the mitochondria, the
mitochondrial membrane potential collapses, opening the
transition pore through which cytochrome c is released in
abundance. This in turn cleaves and activates caspase-9,
which interacts with Apaf-1, leading to the sequential cleav-
age and activation of caspase-3.[12]
Once caspase-3 is acti-
vated, the sequence hits the point of no return, leading to
the downstream activation of caspase-6 and -7, and ulti-
mately to full-blown apoptosis.
Other studies also support the notion of Dox-inducing
apoptosis via cytochrome c release and caspase-3 activation
in cardiomyopathy.[74]
Studies conducted in human T-cell
leukemia has also revealed that Dox-induced apoptosis
occurs through a Fas-independent pathway with high levels
of caspase-3 activity.[17]
Although Dox-induced apoptosis
has been documented to occur via both the mitochondrial
pathway and Fas/FasL receptor pathway, the vast majority
revolve around the mitochondrial pathway. Furthermore, it
is speculated that caspase-3 deficiency in tumour cells may
contribute to chemotherapeutic resistance to Dox.[75]
While the roles of caspases-3, -6, -7, -8 and -9 are clearly
defined in the apoptotic pathways, the role of caspase-2 in
apoptosis is still unclear, and this is also the case for its
involvement in Dox-induced apoptosis. The poorly defined
role of caspase-2 in apoptosis stems from its enigmatic
status and the inability of researchers to formally define it as
an initiator or executioner caspase.[73]
However, despite
having features from both caspase families, the substrate
has more in common with the executioner proteins than
initiator caspases. Recent studies indicate that caspase-2
may have a role in suppressing tumorigenesis, making it a
potential safeguard against tumour development.[76,77]
The
involvement of caspase-2 in Dox-induced apoptosis has
been studied in various cell lines such as mouse oocytes and
Jurkat cells, but its direct involvement in stress-induced
apoptosis specifically in Dox-induced apoptosis is largely
unknown.[11]
General activation of caspase-2 in specific
stressful scenarios may not be enough to trigger cellular
apoptosis, but may affect the cell in other ways such as cell
cycle arrest.
The activation platform for caspase-2 is the protein
complex known as the PIDDosome comprising of the
proteins PIDD, RAIDD and caspase-2. The PIDDosome
recruites procaspase-2 once cellular stress has occurred (e.g.
DNA damage). Procaspase-2 is then cleaved and activated,
binding to Bid to induce MOMP and eventually apoptosis
through caspase-9 mediated activation[78]
(Figure 3). Alter-
native (non-caspase-3) pathways have been reported for
caspase-2,[79,80]
and it is intriguing whether such can also be
discovered for Dox, particularly at doses not usually used
clinically – that is, at low doses.
Oktay Tacar and Crispin R. DassDoxorubicin-induced autophagy
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1577–15891584

9. An alternative pathway involving caspase-2 may exist,
where Dox requires the sequential activation of caspase-2,
protein kinase Cδ (PKCδ) and c-Jun NH2-terminal kinase
to induce apoptosis.[11]
Dox-induced apoptosis in Jurkat
cells occurs via caspase-2-dependent cleavage and down-
stream PKCδ activation, which activates Bak and
cytochrome c release. The nature of Dox-induced cell death
via caspase-2-dependent pathways appears to be cell spe-
cific, as in U266 cells that were treated under the exact same
conditions; Dox did not require activation of caspase-2 to
induce apoptosis. The activation of tumour suppressor
caspase-2 does not appear frequently in Dox-treated cell
lines because it is often defective or absent in certain
cancers, often contributing to drug resistance. Typically,
caspase-2 activates and translocates Bid to the mitochon-
dria to release cytochrome c and thus induce apoptosis;
however, this was not the case. Treating Bid-/- cells with
Dox revealed that Bid did not play a regulatory role in Dox-
induced apoptosis.[81]
These results further highlight the
alternative pathway activated by Dox in activating caspase-2
and thereby inducing apoptosis.
Because the caspase family plays such a prominent role
in chemotherapeutic tumour suppression, inhibition of
apoptosis naturally results in chemoresistance. In contrast,
Dox-induced DNA damage
PIDD
PIDDosome
procaspase-2
Cleaved
caspase-2
RAIDD
Cleaved caspase-9
Pro/cleaved caspase-3
Pro/cleaved caspase -6, -7
APOPTOSIS
Cytochrome c
Figure 3 The caspase-2 activation pathway pertinent to doxorubicin. Caspase-2 is proposedly activated by the PIDDosome. The PIDDosome
recruits procaspase-2 in response to cellular stress (e.g. Dox-induced DNA damage), causing the cleavage and activation of caspase-2, which acti-
vates Bid. Bid-induced mitochondrial outer membrane pores (MOMPs), which trigger cleavage and activation of caspase-9 on the pathway to
fullblown apoptosis.
Oktay Tacar and Crispin R. Dass Doxorubicin-induced autophagy
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1577–1589 1585

10. recent studies have revealed that caspase inhibitors channel
cells down an alternative form of cell growth suppression
known as senescence.[18]
Over the last few decades, studies
on Dox have revealed the drug’s ability to induce at least
three types of cell growth suppression – necrosis, apoptosis
and senescence. Although autophagy can theoretically occur
via the Becn1/Bcl-2 toggle switch complex, further study is
needed. Almost the same can be said for Dox-induced
senescence, which is a relatively new process.
Neuroblastoma cells (SKN-SH) treated with Dox at
medium (10−6
m) and high (10−5
m) concentrations undergo
apoptosis, and lowering the drug concentration to 10−7
m
revealed molecular characteristics of senescence followed by
delayed death. The elevated expression levels of senescence-
associated β-Gal, p21/WAF1 and caspase-3 provide further
evidence of senescence occurring. Senescence is used by
cells to exit cell cycle activation by various oncogenes, in
essence acting as a tumour suppression program. There are
still many unknown molecular factors that contribute to
senescence as well as autophagy.[18]
Dox-induced apoptosis
has been well documented for decades, but senescence and
autophagy are two cellular events that have not been
explored to their full potential and may provide vital clues
to better cancer therapy in the future.
Doxorubicin-induced cardiomyocyte
cell death
As previously mentioned, the administration of Dox is
limited by its cytotoxic effect on virtually all cell types. The
most common form of Dox-induced toxicity involves
cardiomyocytes. Acute cardiac toxicity (cardiotoxicity)
typically occurs during the course of high dose administra-
tion and is characterised by acute heart failure and
tachyarrhythmia. Furthermore, chronic toxicity in cardiac
cells is dose dependent. Eventually, both levels of toxicity
have the ability to contribute to cardiomyopathy, dysfunc-
tion and ultimately heart failure.[82]
Many studies attribute ROS to be a major contributor to
Dox-induced cellular stress in cardiomyocytes. The chemi-
cal composition of Dox has the tendency to generate ROS
by being converted into an unstable semi-quinine inter-
mediate that favours ROS production by endothelial nitric
oxide synthase.[83]
Once Dox has caused cellular stress (e.g.
DNA damage), numerous molecular mechanisms initiate in
a specific cascade to bring upon cell death. The type of cell
death caused by Dox varies by cell line and dose. However,
studies have shown that cardiomyocytes are susceptible to
all four forms of Dox-induced cell death (apoptosis,
autopsy, necrosis and senescence).
Previous studies provide evidence of Dox increasing ROS
while also disrupting calcium homeostasis in the cytosol. By
stimulating the release of calcium from the sarcoplasmic
reticulum, ROS increases intracellular calcium levels. The
rise of intracellular calcium occurs when the sarcoplasmic
reticulum’s ryanodine receptors open, which causes the
production of ROS to occur via calcium-sensitive ROS-
generating enzymes. In cardiomyocytes, the mitochondria
are situated in close proximity to the calcium-releasing sites
of the sarcoplasmic reticulum, allowing the mitochondria to
accumulate high levels of calcium.[82]
Once the level of
calcium surpasses the threshold, mitochondrial membrane
potential is lost while the mitochondria swells and the outer
membrane bursts. Cytochrome c is released from the rup-
tured membrane triggering apoptosis via the intrinsic
pathway.
Dox-induced apoptosis in cardiomyocytes also correlates
with increased activation of p53. As previously mentioned,
Dox can directly activate ERK1/2, which phosphorylates
and activates p53. p53 can also be activated when Dox
increases ROS levels, which cause DNA lesions. The DNA
damage would trigger p53 activation, leading to down-
stream proteins being activated such as Bax and thus initiat-
ing apoptosis.[82]
When p53 in H9c2 rat cardiomyocytes was
inhibited with pifithrin-α,[7]
Dox-induced apoptosis was
inhibited. Studies in p53 -/- mice confirm this.[84,85]
The process of autophagy typically occurs in the myocar-
dium. The process is known to be a ‘double-edged sword’
because of its ability to maintain homeostasis by removing
damaged organelles and acting as a prosurvival pathway,
while at the same time, overamplified autophagy causes cell
death.[33,86]
Both apoptosis and autophagy have the ability to
crosstalk through the Bcl-2 family proteins, although the
crosstalk at a molecular level as a result of Dox administra-
tion is still largely unknown.
ROS has the ability to trigger autophagy by increasing
intracellular calcium levels, while p53 also plays a role by
activating AMP kinase. Typically, ROS, AMP kinase and p53
contribute to apoptosis, and this is also the case in H9c2
cells. Studies have shown that when ROS-dependent LKB1
is activated, it provides the upstream signal necessary for
AMP kinase activation, leading to Dox induced H9c2
apoptosis. In contrast to this, Dox-generated ROS increases
calcium levels and activates calmodulin-dependent kinase
as well as AMP kinase activation, ultimately inducing
autophagy in H9c2.[87]
It is known that the type of cell death occurring is Dox
dependent. In this case, the mitochondria act as the cross-
road for all four types of cell death. It is thought that cellu-
lar stress increases with the dose administered. With
increasing stress, apoptosis occurs in most cell types
because of the cytochrome c released from the mitochon-
dria. Extreme stress implemented on cells via high levels of
Dox causes necrosis. This is because the intracellular supply
of ATP becomes exhausted as a result of all mitochondria
undergoing permeability transition. Autophagy occurring
Oktay Tacar and Crispin R. DassDoxorubicin-induced autophagy
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1577–15891586

11. in cardiomyocytes, however, has yet to be seen and requires
further research. It could simply be that cardiomyocytes
are protected or die depending on the dose of Dox, and
autophagy is integral to this decision. Bcl-2 is one protein
that has to be further studied in this regard.
Declaration
Conflict of interest
The authors state that there is no conflict of interest in
writing and submission of this article.
Acknowledgements
The authors acknowledge the support of a Medical Research
and Technology in Victoria grant to CRD.
References
1. ACS – American Cancer Society. Facts
and Figures. Philadelphia, PA: ACS,
2012.
2. Hahn WC, Weinberg RA. Rules for
making human tumor cells. N Engl J
Med 2002; 347: 1593–1603.
3. Croce CM. Oncogenes and cancer. N
Engl J Med 2008; 358: 502–511.
4. Hanahan D, Weinberg RA. Hallmarks
of cancer: the next generation. Cell
2011; 144: 646–674.
5. L’ecuyer T et al. DNA damage is an
early event in doxorubicin-induced
cardiac myocyte death. Am J Physiol
Heart Circ Physiol 2006; 291: H1273–
1280.
6. Liu X et al. Pifithrin-alpha protects
against doxorubicin-induced apopto-
sis and acute cardiotoxicity in mice.
Am J Physiol Heart Circ Physiol 2004;
286: H933–H939.
7. Liu J et al. ERKs/p53 signal
transduction pathway is involved in
doxorubicin-induced apoptosis in
H9c2 cells and cardiomyocytes. Am J
Physiol Heart Circ Physiol 2008; 295:
H1956–1965.
8. Chen MB et al. Activation of AMP-
activated protein kinase contributes to
doxorubicin-induced cell death and
apoptosis in cultured myocardial
H9c2 cells. Cell Biochem Biophys 2011;
60: 311–322.
9. Yeh PY et al. Phosphorylation of p53
on Thr55 by ERK2 is necessary for
doxorubicin-induced p53 activation
and cell death. Oncogene 2004; 23:
3580–3588.
10. Lorenzo E et al. Doxorubicin induces
apoptosis and CD95 gene expres-
sion in human primary endothelial
cells through a p53-dependent mecha-
nism. J Biol Chem 2002; 277: 10883–
10892.
11. Panaretakis T et al. Doxorubicin
requires the sequential activation of
caspase-2, protein kinase Cdelta, and
c-Jun NH2-terminal kinase to induce
apoptosis. Mol Biol Cell 2005; 16:
3821–3831.
12. Childs AC et al. Doxorubicin treat-
ment in vivo causes cytochrome C
release and cardiomyocyte apoptosis,
as well as increased mitochondrial
efficiency, superoxide dismutase activ-
ity, and Bcl-2 : Bax ratio. Cancer Res
2002; 62: 4592–4598.
13. Leung LK, Wang TT. Differential
effects of chemotherapeutic agents on
the Bcl-2/Bax apoptosis pathway in
human breast cancer cell line MCF-7.
Breast Cancer Res Treat 1999; 55:
73–83.
14. Huigsloot M et al. Differential regula-
tion of doxorubicin-induced mito-
chondrial dysfunction and apoptosis
by Bcl-2 in mammary adenocarci-
noma (MTLn3) cells. J Biol Chem
2002; 277: 35869–35879.
15. Tu Y et al. Upregulated expression of
BCL-2 in multiple myeloma cells
induced by exposure to doxorubicin,
etoposide, and hydrogen peroxide.
Blood 1996; 88: 1805–1812.
16. Ueno M et al. Doxorubicin induces
apoptosis by activation of caspase-3
in cultured cardiomyocytes in vitro
and rat cardiac ventricles in vivo.
J Pharmacol Sci 2006; 101: 151–158.
17. Gamen S et al. Doxorubicin-induced
apoptosis in human T-cell leukemia is
mediated by caspase-3 activation in a
Fas-independent way. FEBS Lett 1997;
417: 360–364.
18. Rebbaa A et al. Caspase inhibition
switches doxorubicin-induced apop-
tosis to senescence. Oncogene 2003; 22:
2805–2811.
19. Wang S et al. Doxorubicin induces
apoptosis in normal and tumor cells
via distinctly different mechanisms.
intermediacy of H(2)O(2)- and p53-
dependent pathways. J Biol Chem
2004; 279: 25535–25543.
20. Jang YM et al. Doxorubicin treatment
in vivo activates caspase-12 mediated
cardiac apoptosis in both male and
female rats. FEBS Lett 2004; 577: 483–
490.
21. Maestre N et al. Cell surface-directed
interaction of anthracyclines leads to
cytotoxicity and nuclear factor kappaB
activation but not apoptosis signaling.
Cancer Res 2001; 61: 2558–2561.
22. Tietze MK et al. IkappaBalpha gene
therapy in tumor necrosis factor-
alpha- and chemotherapy-mediated
apoptosis of hepatocellular carcino-
mas. Cancer Gene Ther 2000; 7: 1315–
1323.
23. Box VG. The intercalation of DNA
double helices with doxorubicin and
nogalamycin. J Mol Graph Model 2007;
26: 14–19.
24. Carvalho C et al. Doxorubicin: the
good, the bad and the ugly effect. Curr
Med Chem 2009; 16: 3267–3285.
25. Hilmer SN et al. The hepatic
pharmacokinetics of doxorubicin and
liposomal doxorubicin. Drug Metab
Dispos 2004; 32: 794–799.
26. Buchholz TA et al. Global gene expres-
sion changes during neoadjuvant
chemotherapy for human breast
cancer. Cancer J 2002; 8: 461–468.
27. Lal S et al. Pharmacogenetics of target
genes across doxorubicin disposition
Oktay Tacar and Crispin R. Dass Doxorubicin-induced autophagy
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1577–1589 1587

12. pathway: a review. Curr Drug Metab
2010; 11: 115–128.
28. Tan ML et al. Doxorubicin delivery
systems based on chitosan for cancer
therapy. J Pharm Pharmacol 2009; 61:
131–142.
29. Kiyomiya K et al. Mechanism of spe-
cific nuclear transport of adriamycin:
the mode of nuclear translocation
of adriamycin-proteasome complex.
Cancer Res 2001; 61: 2467–2471.
30. Minotti G et al. Anthracyclines:
molecular advances and pharma-
cologic developments in antitumor
activity and cardiotoxicity. Pharmacol
Rev 2004; 56: 185–229.
31. Ashley N, Poulton J. Mitochondrial
DNA is a direct target of anti-cancer
anthracycline drugs. Biochem Biophys
Res Commun 2009; 378: 450–455.
32. Cardoso S et al. Doxorubicin increases
the susceptibility of brain mitochon-
dria to Ca(2+)-induced permeability
transition and oxidative damage.
Free Radic Biol Med 2008; 45: 1395–
1402.
33. Tacar O et al. Doxorubicin: an update
on anticancer molecular action, toxic-
ity and novel drug delivery systems.
J Pharm Pharmacol 2012; 65: 157–170.
34. Kim YM et al. Involvement of AMPK
signaling cascade in capsaicin-induced
apoptosis of HT-29 colon cancer cells.
Ann N Y Acad Sci 2007; 1095: 496–
503.
35. Kim YD et al. Compound K induces
apoptosis via CAMK-IV/AMPK path-
ways in HT-29 colon cancer cells. J
Agric Food Chem 2009; 57: 10573–
10578.
36. Guo D et al. The AMPK agonist
AICAR inhibits the growth of
EGFRvIII-expressing glioblastomas by
inhibiting lipogenesis. Proc Natl Acad
Sci U S A 2009; 106: 12932–12937.
37. Jones RG et al. AMP-activated protein
kinase induces a p53-dependent meta-
bolic checkpoint. Mol Cell 2005; 18:
283–293.
38. Pan W et al. AMPK mediates
curcumin-induced cell death in
CaOV3 ovarian cancer cells. Oncol Rep
2008; 20: 1553–1559.
39. Cao C et al. AMP-activated protein
kinase contributes to UV- and H2O2-
induced apoptosis in human skin
keratinocytes. J Biol Chem 2008; 283:
28897–28908.
40. Okoshi R et al. Activation of AMP-
activated protein kinase induces p53-
dependent apoptotic cell death in
response to energetic stress. J Biol
Chem 2008; 283: 3979–3987.
41. Kefas BA et al. AMP-activated protein
kinase can induce apoptosis of
insulin-producing MIN6 cells through
stimulation of c-Jun-N-terminal
kinase. J Mol Endocrinol 2003; 30:
151–161.
42. Meisse D et al. Sustained activation of
AMP-activated protein kinase induces
c-Jun N-terminal kinase activation
and apoptosis in liver cells. FEBS Lett
2002; 526: 38–42.
43. Lee TK et al. Doxorubicin-induced
apoptosis and chemosensitivity in
hepatoma cell lines. Cancer Chemother
Pharmacol 2002; 49: 78–86.
44. Haupt S et al. Apoptosis – the p53
network. J Cell Sci 2003; 116: 4077–
4085.
45. Appella E, Anderson CW. Post-
translational modifications and acti-
vation of p53 by genotoxic stresses.
Eur J Biochem 2001; 268: 2764–2772.
46. Levine AJ, Oren M. The first 30 years
of p53: growing ever more complex.
Nat Rev Cancer 2009; 9: 749–758.
47. Vousden KH, Prives C. Blinded by the
light: the growing complexity of p53.
Cell 2009; 137: 413–431.
48. Brown CJ et al. Awakening guardian
angels: drugging the p53 pathway. Nat
Rev Cancer 2009; 9: 862–873.
49. Demidenko ZN et al. Paradoxical sup-
pression of cellular senescence by p53.
Proc Natl Acad Sci U S A 2010; 107:
9660–9664.
50. Leontieva OV et al. Weak p53 permits
senescence during cell cycle arrest. Cell
Cycle 2010; 9: 4323–4327.
51. Yang MY et al. Induction of cellular
senescence by doxorubicin is associ-
ated with upregulated miR-375 and
induction of autophagy in K562 cells.
PLoS ONE 2012; 7: e37205. doi:
10.1371/journal.pone.0037205.
52. Vigneron A et al. Src inhibits
adriamycin-induced senescence and
G2 checkpoint arrest by blocking the
induction of p21waf1. Cancer Res
2005; 65: 8927–8935.
53. Xue C et al. p53 determines multidrug
sensitivity of childhood neuroblas-
toma. Cancer Res 2007; 67: 10351–
10360.
54. Levine B et al. Bcl-2 family members:
dual regulators of apoptosis and
autophagy. Autophagy 2008; 4: 600–
606.
55. Kang MH, Reynolds CP. Bcl-2
inhibitors: targeting mitochondrial
apoptotic pathways in cancer therapy.
Clin Cancer Res 2009; 15: 1126–1132.
56. Yoshino T et al. Bcl-2 expression as
a predictive marker of hormone-
refractory prostate cancer treated with
taxane-based chemotherapy. Clin
Cancer Res 2006; 12: 6116–6124.
57. Liu WH, Chang LS. Fas/FasL-
dependent and -independent activa-
tion of caspase-8 in doxorubicin-
treated human breast cancer MCF-7
cells: ADAM10 down-regulation acti-
vates Fas/FasL signaling pathway. Int J
Biochem Cell Biol 2011; 43: 1708–
1719.
58. Yamaoka M et al. Apoptosis in rat
cardiac myocytes induced by Fas
ligand: priming for Fas-mediated
apoptosis with doxorubicin. J Mol Cell
Cardiol 2000; 32: 881–889.
59. Pattingre S et al. Bcl-2 antiapoptotic
proteins inhibit Beclin 1-dependent
autophagy. Cell 2005; 122: 927–939.
60. Eisenberg-Lerner A et al. Life and
death partners: apoptosis, autophagy
and the cross-talk between them. Cell
Death Differ 2009; 16: 966–975.
61. Maiuri MC et al. Self-eating and self-
killing: crosstalk between autophagy
and apoptosis. Nat Rev Mol Cell Biol
2007; 8: 741–752.
62. Maiuri MC et al. Crosstalk between
apoptosis and autophagy within the
Beclin 1 interactome. EMBO J 2010;
29: 515–516.
63. Wang J. Beclin 1 bridges auto-
phagy, apoptosis and differentiation.
Autophagy 2008; 4: 947–948.
64. Shimizu S et al. Role of Bcl-2 family
proteins in a non-apoptotic pro-
grammed cell death dependent on
autophagy genes. Nat Cell Biol 2004a;
6: 1221–1228.
Oktay Tacar and Crispin R. DassDoxorubicin-induced autophagy
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1577–15891588

13. 65. Daniel F et al. Partial Beclin 1 silenc-
ing aggravates doxorubicin- and Fas-
induced apoptosis in HepG2 cells.
World J Gastroenterol 2006; 12: 2895–
2900.
66. Dalby KN et al. Targeting the pro-
death and prosurvival functions of
autophagy as novel therapeutic strat-
egies in cancer. Autophagy 2010; 6:
322–329.
67. Wirawan E et al. Caspase-mediated
cleavage of Beclin-1 inactivates Beclin-
1-induced autophagy and enhances
apoptosis by promoting the release of
proapoptotic factors from mitochon-
dria. Cell Death Dis 2010; 1. doi:
10.1038/cddis.2009.16.
68. An T et al. Neuregulin-1 attenuates
doxorubicin-induced autophagy in
neonatal rat cardiomyocytes. J Cardio-
vasc Pharmacol 2013; 62: 130–137.
69. Fulda S, Debatin KM. Extrinsic versus
intrinsic apoptosis pathways in anti-
cancer chemotherapy. Oncogene 2006;
25: 4798–4811.
70. Hyman BT, Yuan J. Apoptotic and
non-apoptotic roles of caspases in
neuronal physiology and pathophysi-
ology. Nat Rev Neurosci 2012; 13: 395–
406.
71. Youn HJ et al. Induction of caspase-
independent apoptosis in H9c2
cardiomyocytes by adriamycin treat-
ment. Mol Cell Biochem 2005; 270:
13–19.
72. Fernando P, Megeney LA. Is caspase-
dependent apoptosis only cell differ-
entiation taken to the extreme? FASEB
J 2007; 21: 8–17.
73. Bouchier-Hayes L. The role of
caspase-2 in stress-induced apoptosis.
J Cell Mol Med 2010; 14: 1212–
1224.
74. Narula J et al. Apoptosis in heart
failure: release of cytochrome c from
mitochondria and activation of
caspase-3 in human cardiomyopathy.
Proc Natl Acad Sci U S A 1999; 96:
8144–8149.
75. Yang XH et al. Reconstitution of
caspase 3 sensitizes MCF-7 breast
cancer cells to doxorubicin- and
etoposide-induced apoptosis. Cancer
Res 2001; 61: 348–354.
76. Ho LH et al. A tumor suppressor
function for caspase-2. Proc Natl Acad
Sci U S A 2009; 106: 5336–5341.
77. Dass CR. Use of caspase-2 as a
biomarker for osteosarcoma. J Mol
Biomarkers Diag 2011; 2: 29.
78. Tinel A, Tschopp J. The PIDDosome, a
protein complex implicated in activa-
tion of caspase-2 in response to
genotoxic stress. Science 2004; 304:
843–846.
79. Kim SH, Dass CR. Induction of
caspase-2 activation by a DNA
enzyme evokes tumor cell apoptosis.
DNA Cell Biol 2012; 31: 1–7.
80. Dass CR et al. A c-jun DNAzyme
which is a potent inducer of caspase-2
activation. Oligonucleotides 2010; 20:
137–146.
81. Panaretakis T et al. Activation of Bak,
Bax, and BH3-only proteins in the
apoptotic response to doxorubicin. J
Biol Chem 2002; 277: 44317–44326.
82. Zhang YW et al. Cardiomyocyte
death in doxorubicin-induced cardio-
toxicity. Arch Immunol Ther Exp
(Warsz) 2009; 57: 435–445.
83. Neilan TG et al. Disruption of nitric
oxide synthase 3 protects against the
cardiac injury, dysfunction, and mor-
tality induced by doxorubicin. Circula-
tion 2007; 116: 506–514.
84. Shizukuda Y et al. Targeted disrup-
tion of p53 attenuates doxorubicin-
induced cardiac toxicity in mice. Mol
Cell Biochem 2007; 273: 25–32.
85. Shimizu S et al. Role of Bcl-2 family
proteins in a non-apoptotic pro-
grammed cell death dependent on
autophagy genes. Nat Cell Biol 2004b;
6: 1221–1228.
86. Gustafsson AB, Gottlieb RA.
Autophagy in ischemic heart disease.
Circ Res 2009; 104: 150–158.
87. Hoyer-Hansen M et al. Control
of macroautophagy by calcium,
calmodulin-dependent kinase kinase-
beta, and Bcl-2. Mol Cell 2007; 25:
193–205.
Oktay Tacar and Crispin R. Dass Doxorubicin-induced autophagy
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 1577–1589 1589