Seminars in Cancer Biology 19 (2009) 5766

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Seminars in Cancer Biology

journal homepage: www.elsevier.com/locate/semcancer

Review

Mitochondria as targets for cancer chemotherapy

Vladimir Gogvadze , Sten Orrenius, Boris Zhivotovsky
Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, Stockholm, SE-171 77, Sweden

a r t i c l e
Keywords:
Cell death
Tumor
Mitochondria
Therapy
Warburg effect

i n f o

a b s t r a c t
Heterogeneity of tumors dictates an individual approach to anticancer treatment. Despite their variability,
almost all cancer cells demonstrate enhanced uptake and utilization of glucose, a phenomenon known
as the Warburg effect, whereas mitochondrial activity in tumor cells is suppressed. Considering the key
role of mitochondria in cell death, it appears that resistance of most tumors towards treatment can be,
at least in part, explained by mitochondrial silencing in cancer cells. This review is devoted to the role
of mitochondria in cell death, and describes how targeting of mitochondria can make tumor cells more
susceptible to anticancer treatment.
2008 Elsevier Ltd. All rights reserved.

1. Introduction
In their millennium review Hanahan and Weinberg described
six essential alterations in cell physiology that collectively might
dictate malignant growth: self-sufciency in growth signals,
insensitivity to growth-inhibitory (antigrowth) signals, limitless
replicative potential, sustained angiogenesis, tissue invasion and
metastasis, and evasion of programmed cell death (apoptosis)
[1]. Accumulating evidence now suggests that, in addition to
these alterations, another feature of tumor cells, i.e. their dependence on glycolysis for ATP generation, might be added to this
list.
In 1926 Otto Warburg found that cancer cells produce most of
their ATP via glycolysis, also in the presence of oxygen [2]. At rst
sight, this nding contradicted the Pasteur effect named after Louis
Pasteur, who found that yeast cease fermentation when exposed
to oxygen. Extensive glycolytic production of ATP was found to be
a prerogative of most cancer cells, and this observation was conrmed in various laboratories. A comparison of multiple cancer cell
lines revealed that they rely on glycolysis for ATP production to
different extents, and typically, the more glycolytic tumor cells
were shown to be the most aggressive ones [3]. Warburg originally
suggested that aerobic glycolysis in tumors might reect defects in
mitochondrial respiration: The aerobic glycolysis of the tumor cell
is derived in any case from a disturbance of the respiration. As a
rule, the respiration of the tumor cell is small, but in recent years
tumor cells with a large respiration have also been found. [2]. He
assumed that such aerobic glycolysis was a universal property of
malignant cells and suggested that cancer is caused by impaired
mitochondrial metabolism.

Corresponding author. Tel.: +46 8 5248 7515; fax: +46 8 32 90 41.

E-mail address: Vladimir.Gogvadze@ki.se (V. Gogvadze).
1044-579X/$ see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.semcancer.2008.11.007

However, subsequent studies revealed that tumor mitochondria

could function normally with regards to the oxidation of respiratory substrates and maintenance of P/O and respiratory control
ratios [4]. Warburgs hypothesis was questioned by Sidney Weinhouse who wrote: no substantial evidence has been found that
would indicate a respiratory defect, either in the machinery of electron transport or in the coupling of respiration with ATP formation,
or in the unique presence or absence of mitochondrial enzymes
or cofactors involved in electron transport [5]. However, whether
mitochondrial respiration is low or not, there is now general agreement that cancer cells do exhibit high rates of glycolysisaerobic
or anaerobic. In fact, the extensive glucose utilization by malignant cells is widely used nowadays for the visualization of tumors
by positron emission tomography, emphasizing the importance of
Warburgs original observation.
The fact that mitochondria play a key role in the regulation of
cell death pathways makes them an attractive target for cancer
chemotherapy. In fact, targeting of mitochondria as a tool for tumor
cell elimination was proposed by Otto Warburg in his early publications. He hypothesized that malignant cells can be eliminated
through the inhibition of mitochondrial oxidative phosphorylation
(for example by moderate doses of ionizing radiation), which would
reduce the activity of these organelles below a threshold level critical for cell survival. In contrast, mitochondria in normal cells would
still be able to produce ATP and to survive.
However, mitochondrial contribution to cellular physiology is
not restricted to ATP production for metabolic demands. Mitochondria also produce reactive oxygen species (ROS), which participate
in physiological cell signaling, but which might cause cell damage if produced excessively. Further, mitochondria are involved in
the maintenance of intracellular Ca2+ homeostasis; compromising
their capability to do so might lead to irreparable cell deterioration.
Of note, perturbation of mitochondrial ROS production or Ca2+ buffering capacity might also affect the mitochondrial regulation of

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the cell death machinery. Hence, all these mitochondrial functions

are of critical importance for the growth and survival of tumor cells.
2. What makes mitochondria in cancer cells so special?
One of the main characteristics of tumor cells is their fast proliferation. Due to the high growth rate, tumor tissue easily becomes
hypoxic because of the inability of the local vasculature to supply an
adequate amount of oxygen. Similar conditions are often lethal to
non-malignant cells due to hypoxida-induced, p53-mediated apoptosis [6], but tumor cells can successfully escape hypoxia-mediated
death due to mutations, or lowered expression, of p53 [7].
The inability of mitochondria to provide enough ATP for cell
survival under hypoxic conditions, results in upregulation of the
glycolytic pathway. This occurs by induction of hypoxia-inducible
factor 1 (HIF-1), which not only stimulates key steps of glycolysis, but also regulates genes that control angiogenesis, as well as
cell survival and invasion [8]. The role of HIF-1 is not restricted
to upregulation of the enzymes stimulating glucose utilization.
Recent ndings demonstrate that, in addition, HIF-1 suppresses
mitochondrial function in tumor cells, suggesting that it modulates
the reciprocal relationship between glycolysis and oxidative phosphorylation. Normally, the switch between glycolysis and oxidative
phosphorylation is controlled by the relative activities of two

enzymes, pyruvate dehydrogenase (PDH) and lactate dehydrogenase (LDH) (Fig. 1). The activity of PDH is regulated by pyruvate
dehydrogenase kinase 1 (PDK1). HIF-1 was shown to induce PDK1
and thereby inactivate PDH and, as a result, to suppress the Krebs
cycle and mitochondrial respiration [9]. In addition, HIF-1 was
found to stimulate the expression of lactate dehydrogenase A,
which facilitates the conversion of pyruvate to lactate [10]. As
a result, mitochondrial contribution to ATP synthesis declines,
although the mitochondria might remain functionally intact.
However, it should be noticed that a relatively small mitochondrial contribution to ATP production can be observed in a variety
of fast-growing cells, which are not necessarily malignant [11]. An
early indication of the inhibition of mitochondrial respiration by
stimulated glycolysis, a phenomenon known as the Crabtree effect
(reviewed in [12]), was observed in cells with approximately equal
glycolytic and respiratory capacities for ATP synthesis. Thus, the
Crabtree effect is absent in thymocytes in the resting state, whereas
thymocytes in the proliferative state demonstrate a signicantly
higher rate of glycolysis and a strong glucose-induced inhibition
of oxygen consumption [13]. Several mechanisms have been proposed to explain the Crabtree effect in tumor cells. According to one
of them, glucose induces the release of Ca2+ from the endoplasmic
reticulum, leading to enhanced uptake of Ca2+ by mitochondria and
subsequent inhibition of oxidative phosphorylation by an increased

Fig. 1. Pathways of mitochondrial silencing and therapeutic stimulation of tumor cell death. Mutation of p53 in tumors decreases production of Synthesis of Cytochrome c
Oxidase 2 (SCO2), a copper transporter critical for the synthesis of the cytochrome oxidase complex in the mitochondrial respiratory chain, causing suppression of respiration
and, hence, mitochondrial ATP production. HIF-1 can induce pyruvate dehydrogenase kinase (PDK), thereby, inactivating pyruvate dehydrogenase (PDH) and stimulate the
expression of lactate dehydrogenase A, causing suppression of the Krebs cycle and mitochondrial respiration. Inhibition of PDK by dichloroacetate can shift cellular metabolism
from glycolysis to glucose oxidation. The permeabilization of the OMM engages pro-apoptotic members of the Bcl-2 family proteins (square A). In many tumors anti-apoptotic
members of the Bcl-2 family sequester pro-apoptotic members preventing OMM permeabilization. A small molecule, ABT-737, can disrupt interaction between Bcl-2 and Bax
allowing Bax oligomerization and pore formation in the OMM. Stimulation of mitochondrial activity facilitates pore opening followed by mitochondrial swelling and OMM
permeabilization (square B).

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association of the inhibitory subunit of the ATP synthase [14]. This

explanation would be in agreement with the observed elevation of
the Ca2+ content in tumor mitochondria.
In addition to modulation of metabolic pathways, mitochondrial silencing in cancer cells might also result from suppression
of key mitochondrial components. Thus, one of the hallmarks of
most human carcinomas is the down-regulation of the catalytic
subunit of the mitochondrial H+ -ATP synthase (beta-F1-ATPase).
As a result, cells start to consume glucose by aerobic glycolysis. A
similar increase in the rate of aerobic glycolysis can be achieved by
incubation of cancer cells with oligomycin, an inhibitor of mitochondrial ATP synthase [15]. These ndings demonstrate that a
switch to glycolysis might also result from suppression of mitochondrial oxidative phosphorylation.
Another characteristic feature of tumor cells is an elevated level
of ROS [16]. They are generated continuously in cells and play
important roles in a variety of cellular processes. ROS stimulate
cell proliferation and cellular migration. The mitochondria are the
most important intracellular source of ROS. Also in non-malignant
cells under physiological conditions, about 2% of the mitochondrially consumed oxygen is converted into the superoxide anion
radical, the precursor of most ROS. Under physiological conditions
the level of ROS is controlled by cellular antioxidant enzymes in conjunction with water- and lipid-soluble antioxidants. Uncontrolled
production of ROS can cause cell damage by oxidation of macromolecules, including DNA, which in turn might lead to genetic
lesions, tumorigenicity and tumor progression [17]. Detailed analysis of mitochondrial DNA revealed mutations in seven out of ten
human colorectal cancer cell lines. The majority of mutations were
transitions at purines, consistent with ROS-related derivation [18].
These mutations may contribute to abnormal metabolic and apoptotic processes in cancer.
Cellular hypoxia and reoxygenation are two essential elements of ischemia-reperfusion injury. Massive production of ROS
is observed during reoxygenation, but the oxidant level might also
be elevated in the reduced state that characterizes cellular hypoxia,
apparently due to electron leakage from mitochondrial respiratory
complexes [19]. Another reason for the enhanced ROS production in
cancer cells could be the hyperpolarized mitochondria. It is known
that the formation of ROS intensies when the mitochondrial membrane potential increases [20]. Many tumor cells demonstrate an
elevated mitochondrial membrane potential due to its decreased
utilization for ATP synthesis, and this could be one reason for the
elevated ROS production in cancer cells. Indeed, overexpression of
UCP2 in HCT116 human colon cancer cells, and the subsequent dissipation of the mitochondrial membrane potential, hinders ROS
accumulation and apoptosis after exposure to chemotherapeutic
agents [21].
Many antitumor agents, such as vinblastine, cisplatin, mitomycin C, doxorubicin, camptothecin, inostamycin, neocarzinostatin
and several others, exhibit antitumor activity via ROS-mediated
cell killing. Normally, antioxidant defense systems can cope with
increased ROS production, but it is well known that several antioxidant enzymes, including catalase and SOD, are down-regulated
in most solid tumors [22]. In contrast, heme oxygenase-1, a major
antioxidant enzyme in tumors, is highly upregulated. This provides
a therapeutic rationale for attempts to eliminate cancer cells via
stimulation of ROS production, which might spare non-malignant
cells with better antioxidant defense. In fact, Kong and Lillehei
[23] speculated that many chemotherapeutic agents, as well as
ionizing radiation, exert their lethal effect on cancer cells by the
production of free radicals, and that the elevated ROS level in
tumor cells might exhaust the capacity of SOD and other adaptive
antioxidant defenses. Indeed, modulation of cellular redox balance
through pharmacological stimulation of ROS production and/or
depletion of protective reducing metabolites e.g., glutathione [GSH]

59

and NADPH), or suppression of antioxidant enzymes, such as SOD

[24], can lead to oxidative stress, destabilization of mitochondria
and induction of apoptosis [25].
3. Mitochondriakey participants in cell death
Multiple forms of cell death might be triggered in tumors
in order to kill malignant cellsapoptosis, necrosis, autophagy.
Apoptosis is an evolutionarily conserved and genetically regulated
process of critical importance for embryonic development and
maintenance of tissue homeostasis in the adult organism, which
also plays a signicant role in tumor cell biology [26]. Apoptotic
cell death is characterized by a host of morphological and biochemical features, including permeabilization of the outer mitochondrial
membrane (OMM) and the release of pro-apoptotic proteins [27].
Apoptosis might be involved in both the spontaneous elimination
of potentially malignant cells and in therapeutically induced tumor
regression. Hence, defects in the apoptotic death programs may
contribute to tumorigenesis and resistance to treatment.
For a long time, necrosis was regarded as a form of non-regulated
cell death. It is characterized by cell and organelle swelling, disruption of the plasma membrane, chromatin digestion and DNA
hydrolysis and, nally, cell lysis. A characteristic feature of necrosis is a drastic drop in ATP level due to mitochondrial collapse and,
as a result thereof, perturbation of intracellular Ca2+ homeostasis.
However, recent evidence suggests that in some instances necrosis
can be regulated. There are many examples of necrosis occurring
during embryogenesis, tissue renewal, and as part of the immune
response. Both necrosis and apoptosis can also be triggered by internal as well as external stimuli (e.g., cytokines, ischemia/reperfusion,
heat, irradiation, pathogens) in the same cell population. Moreover,
both processes might involve activation of similar signaling pathways, such as death receptors, kinase cascades, and mitochondrial
and/or lysosomal permeabilization, and modulation of these pathways can cause a switch between apoptosis and necrosis (reviewed
in [28]). Apoptosis and necrosis can often be initiated by the same
type of insult, although with different doses or intensities; and in
some cases anti-apoptotic mechanisms, such as Bcl-2/Bcl-XL proteins or heat shock proteins, are similarly effective in protecting
from both forms of cell death. One form of programmed necrosis is
a caspase-independent programmed cell death with necrotic morphology [29], which has been reported to be dependent on ROS
[30], and which may have benecial therapeutic consequences in
treating both apoptosis-resistant tumors and degenerating adult
neurons [31].
Autophagy is a regulated lysosomal pathway involved in the
degradation and recycling of long-lived proteins and organelles
within cells. Although the mechanisms of autophagy-induced cell
death remain unclear, some evidence indicates mitochondrial
involvement also in this process [32]. In particular, it appears
that mitochondrial permeability transition (see below) and permeabilization of the OMM are responsible for stimulation of
mitochondrial autophagy, a process that may be important for the
removal of damaged mitochondria [33].
Autophagy can be a highly efcient inducer of cell death by
excessive self-digestion, for instance in apoptosis-decient cells
stimulated by radiation [34]. The role of autophagy in tumor progression and/or elimination is, however, still unclear. Autophagy
can lead to either cell survival or cell death [35]. In cancer cells with
defects in the apoptosis program, autophagy allows prolonged survival and can also limit tumor necrosis and inammation, as well as
mitigate genome damage in response to metabolic stress [36]. On
the other hand, defects in autophagy are associated with increased
tumorigenesis [37], although the mechanism behind this effect has
not been determined yet. Despite controversies with regards to the
role of autophagy in carcinogenesis, this mode of cell death can

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be envisioned as a spare (reserve) mechanism of cell elimination,

when other mechanisms are unsuccessful. Indeed, inhibition of caspases was shown to stimulate autophagy and enhanced the effect of
radiotherapy in a mouse model of lung cancer [38]. Further, embryonic broblasts from Bax/Bak double knockout mice, which are
resistant to apoptosis, underwent non-apoptotic cell death associated with autophagosome/autolysosome formation, when treated
with etoposide or staurosporine [39].
Recently it has been shown that starvation-stimulated
autophagy was dependent on formation of ROS, specically
H2 O2 . A target for H2 O2 was the cysteine protease HsAtg4, particularly, a cysteine residue located near the HsAtg4 catalytic site [40].
Hence, it is tempting to speculate that mitochondrial targeting and
stimulation of ROS production might be a factor contributing to
autophagy initiation.
Different alterations of mitochondrial activity such as the release
of apoptogenic proteins from the intermembrane space, loss of
mitochondrial membrane potential, stimulation of ROS production,
inhibition of mitochondrial respiratory complexes, and decreases
in ATP synthesis have been shown to be involved in, and possibly
responsible for, the different manifestations of cell death (reviewed
in [41]). Thus mitochondrial destabilization, could be a promising
step in stimulating tumor cell death.
3.1. Mechanisms of OMM permeabilization
Investigations of mitochondrial participation in apoptosis
revealed that their release of certain proteins (particularly
cytochrome c) from the intermembrane space is a point of no
return in many models of apoptotic cell death. Once released,
cytochrome c triggers the formation of the apoptosome complex in
the cytosol and subsequent activation of the caspase cascade, which
is primarily responsible for the cleavage of cellular proteins leading
to the biochemical and morphological characteristics of apoptosis.
Hence, permeabilization of the OMM is a critical step in apoptosis
initiation/propagation.
The permeabilization of the OMM engages pro-apoptotic members of the Bcl-2 family of proteins. The rst indication that genes
and proteins, which play a role in tumorigenesis, might be involved
in the negative regulation of cell death came from a study on
the Bcl-2 protein that is overexpressed as a result of a chromosomal translocation in B cell lymphomas [42]. Overexpression of
this protein was shown to inhibit cell death induced by different
stimuli, such as IL-3 deprivation, chemotherapeutic agents, and
heat shock (reviewed in [43]). In an early publication, the Bcl-2
protein was shown to be localized to the mitochondria, although
the precise mechanism of its protective action was unclear at that
time [44]. Accumulation of Bcl-2 in mitochondria triggered a set of
metabolic changes leading to the stabilization of this organelle. Cells
overexpressing Bcl-2 demonstrated higher mitochondrial membrane potential than wild-type cells, which was thought to be
a reason of their enhanced resistance towards TNF cytotoxicity
[45].
Today, more than thirty members of the Bcl-2 family and related
proteins have been identied. They can be divided into three subgroups: Bcl-2-like survival factors, Bax-like, and BH3-only death
factors [46]. There is accumulating evidence suggesting that antiapoptotic members of the Bcl-2 family act as oncogenes [47]. Thus,
transgenic overexpression of Bcl-XL induced lymphomagenesis, or
development of pancreatic -cell tumors, and overexpression of
Mcl-1 resulted in B-cell lymphomas. Bcl-w, which is expressed in
almost all murine myeloid cell lines analyzed and in a wide range of
tissues, is frequently overexpressed in colorectal adenocarcinomas
and appears to play a role in the progression from adenoma to adenocarcinoma in the colorectal epithelium. Bcl-w is also expressed
in a majority of inltrative gastric adenocarcinomas.

Permeabilization of the OMM during apoptosis was shown

to require the oligomeric form of Bax or Bak (Fig. 1, square A).
Oligomerization of Bax can result from its binding to the truncated form of the BH3-only, pro-apoptotic protein Bid (tBid), which
is cleaved by caspase-8 and several other death proteases. Cells
decient in both Bax and Bak, but not cells lacking only one of
these proteins, have been found to be resistant to tBid-induced
cytochrome c release and apoptosis. Moreover, Bax- and Bakdecient cells are also resistant to a variety of other apoptotic
stimuli that act through the mitochondrial pathway [48]. Thus, activation of a multidomain, pro-apoptotic Bcl-2 family member, Bax
or Bak, appears to be a principal gateway to mitochondrial release
of pro-apoptotic proteins required for cell death in response to
diverse stimuli. Anti-apoptotic proteins, e.g., Bcl-2, Bcl-XL , Mcl-1,
and Bcl-w, interact with the pro-apoptotic proteins, Bax and Bak, to
prevent their oligomerization. Hence, a critical factor that regulates
cellular apoptosis susceptibility is the balance between pro- and
anti-apoptotic proteins. It has been shown for many tumors that
the mitochondrial apoptosis pathway is suppressed due to a disproportion between anti- and pro-apoptotic Bcl-2 family proteins,
in favor of the former [49]. Thus, overexpression of Bcl-2 and BclXL in tumor cells, in particular neuroblastomas, contributes to the
drug resistance characteristic of high-risk neuroblastomas. Disturbance of the balance between anti- and pro-apoptotic Bcl-2 family
members in favor of the latter, can proceed by mechanisms involving BH3-only proteins that bind to and occupy the anti-apoptotic
proteins, thereby liberating Bax and Bak.
Another mechanism that may be responsible for OMM permeabilization is induction of mitochondrial permeability transition
(MPT). This phenomenon was described some thirty years ago by
Haworth and Hunter [50], who found that Ca2+ uptake by mitochondria stimulates drastic changes in mitochondrial morphology
and functional activity due to the opening of a non-specic pore
in the mitochondrial inner membrane, commonly known as the
MPT pore. Pore opening is facilitated by inorganic phosphate, oxidation of pyridine nucleotides, ATP depletion, low pH, and ROS [51].
Traditionally, the MPT pore has been regarded as a multimeric complex, composed of the voltage dependent anion channel (VDAC)
located in the OMM, the adenine nucleotide translocase (ANT), an
integral protein of the inner mitochondrial membrane (IMM), and
a matrix protein, cyclophilin D (CyP-D). In addition, other proteins
may bind to the pore complex, particularly kinases (e.g., hexokinase,
creatine kinase) [52]. The pore is thought to form at contact sites
between the mitochondrial inner and outer membranes. However,
it should be noted that the proteins responsible for pore formation
have not been conclusively identied, and that there is recent evidence that the inorganic phosphate transporter of the IMM may
play an important role in pore formation [53].
Opening of Ca2+ -dependent, non-specic pores in the IMM is
followed by the inux of water and ions into the matrix causing mitochondrial swelling, rupture of the OMM, and the release
of intermembrane space proteins, including cytochrome c (Fig. 1,
square B). Mitochondria in tumor cells are, however, characterized
by a certain resistance towards Ca2+ -induced MPT. Thus, comparison of Ca2+ accumulation by mitochondria from liver and hepatoma
revealed that hepatoma mitochondria were able to accumulate
from two to ve times more Ca2+ than rat liver mitochondria before
the permeability transition was induced. This difference might have
been due to higher expression of the Bcl-2 protein in hepatoma cells
than in hepatocytes, since mitochondria from hepatoma had substantially larger amounts of Bcl-2 protein than liver mitochondria
[54], although the mechanism(s) linking Bcl-2 and MPT is unclear.
MPT was shown to be a key event in necrotic as well as in
some experimental models of apoptotic cell death. If permeability
transition and subsequent uncoupling of mitochondria occur in a
large subpopulation of the organelles, this would lead to destructive

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consequences for the cell. Indeed, under such circumstances the

mitochondria would start to actively hydrolyze cytosolic ATP
(uncoupling-stimulated ATPase activity), and the drop in cellular ATP content would cause an impairment of energy-dependent
ion transport with perturbation of intracellular Ca2+ homeostasis. Subsequent activation of various catabolic enzymes (proteases,
phospholipases, etc.) might lead to necrotic cell death. Conversely,
pore opening in only a minor fraction of mitochondria would leave
the majority of the organelles functionally intact, but it could still
lead to the release of enough cytochrome c to trigger apoptosome
formation and apoptotic cell death, although cellular ATP content
and calcium homeostasis would not change signicantly.
Recently, a new mode of OMM permeabilization was found
to occur through the opening of mitochondrial apoptosis-induced
channels (MAC), large enough to allow passing of intermembrane
space proteins, in particular cytochrome c, into the cytosol [55].
The permeability of MAC can be regulated by Bcl-2 family proteins.
Thus, depletion of Bax, which is an essential constituent of MAC in
some systems, signicantly diminished MAC activity, whereas overexpression of Bcl-2 blocked the formation of MAC and cytochrome
c release [56]. In contrast to MPT induction, opening of MAC does
not affect the barrier properties of the IMM.
3.2. ROS facilitate OMM permeabilization
Both Bax/Bak- and MPT-dependent release of cytochrome c is
facilitated by ROS. Cytochrome c is bound to the outer surface of
the IMM by both electrostatic and hydrophobic interactions with
the unique mitochondrial phospholipid, cardiolipin. Stimulation of
ROS generation and dissociation of cytochrome c from oxidized
cardiolipin are critical early steps in cytochrome c release into
the cytosol through pores formed by Bax/Bak [57]. This mechanism might provide a reasonable explanation for the anti-apoptotic
effects reported for multiple mitochondrial antioxidant enzymes
[58]. ROS formation is also a key factor that facilitates MPT pore
opening. In fact, it has been proposed that mitochondria can regulate the release of proteins inducing apoptosis through excessive
ROS generation and self-directed induction of MPT.
4. Targeting mitochondriaa tool for tumor cell
elimination
4.1. Inhibition of glycolysis in tumor cells
Since tumor cells rely on the glycolytic pathway of ATP production, much more than non-malignant cells, exploitation of the
Warburg effect could represent an approach to overcome some of
the current limitations of radio- and chemotherapies. Thus, drugs
able to perturb cancer cell metabolism, specically at the level of
glycolysis, might display benecial therapeutic effects in cancer.
The antitumor effect of these glycolytic inhibitors is based predominantly on ATP depletion, but also involves diminution of multi-drug
resistance and reduced hypoxia-linked tumor cell resistance [59].
Indeed, in a variety of cancer cells inhibition of glycolysis, using a
non-metabolizable glucose analogue, 2-deoxyglucose (2-DG) [60]
or 3-bromopyruvate [61], caused a marked decrease in ATP level,
especially in clones where mitochondrial ATP supply was compromised. ATP depletion led also to rapid dephosphorylation of
the pro-apoptotic protein Bad, migration of Bax to the mitochondria, permeabilization of the OMM and subsequent massive cell
death [62]. Similar to the effects of inhibition of key steps in the
glycolytic pathway, suppression of glucose transport might also
sensitize tumor cells to anticancer agents. Thus, a glucose transporter inhibitor, phloretin, was reported to markedly enhance the
anticancer effects of daunorubicin [63]. Combining inhibitors of glycolysis with conventional chemotherapeutic drugs might provide a

61

novel therapeutic strategy to overcome drug resistance in hypoxia.

Presently, several glycolytic inhibitors are in preclinical and clinical development. Thus, the cardiac drug, digitoxin, was shown to
inhibit growth and induce apoptosis in cancer cells at concentrations commonly found in the plasma of cardiac patients treated
with this drug [64]. A key mechanism by which this natural product
selectively targets cancer cells is inhibition of glycolysis.
4.2. Neutralization of anti-apoptotic Bcl-2 family proteins
Another therapeutic approach targeting mitochondria involves
activation of their pro-apoptotic potential. This can be accomplished in different ways. As mentioned above, Bcl-XL and Bcl-2 are
overexpressed in many cancers and contribute to tumor initiation,
progression and resistance to therapy. Localization of these proteins
in the OMM makes this membrane stable towards permeabilization
and attenuates the release of cytochrome c and other pro-apoptotic
proteins upon treatment with anticancer agents. Since a majority of
human tumors are defective in the p53 pathway, or overexpress a
Bcl-2 homologue [65], investigators have focused on nding potential anticancer drugs with properties similar to the BH3-domain, i.e.
neutralizing binding of anti-apoptotic proteins to Bax or Bak and
facilitating pore formation in the OMM [66].
Recently, ABT-737, a small-molecular inhibitor of the antiapoptotic proteins Bcl-2, Bcl-XL and Bcl-w, was synthesized and
found to have a binding afnity that was two to three orders of
magnitude higher than that of any previously reported compound.
It has been demonstrated that ABT-737 can disrupt intracellular
Bcl-2 family proteinprotein interaction (Fig. 1). Mechanistic studies revealed that ABT-737 does not directly initiate the apoptotic
process, but enhances the effects of death signals and displays synergistic cytotoxicity with chemotherapeutics and radiation [67].
ABT-737 was found to augment TRAIL-induced cell killing by releasing Bim and Bak from their binding sites and to enhance Bax
conformational changes induced by TRAIL in human pancreatic
cancer cells [68]. Further, ABT-737 stimulated the activity of vincristine, L-ASP, and dexamethasone in lymphoblastic leukemia in
vitro and in vivo [69]. In addition, ABT-737 was shown to induce
apoptosis in chronic myeloid leukemia cells with diverse drugresistance mechanisms [70], lymphomas, small-cell lung carcinoma
lines, as well as in primary patient-derived cells and in animal models [71]. ABT-737 was also shown to improve survival and initiate
regression of established tumors in a high percentage of tumorbearing mice [67].
In addition to the disruption of pro- and anti-apoptotic Bcl-2
protein interaction, another mechanism of ABT-737 action has been
described recently. ABT-737 was shown to induce both OMM and
IMM permeabilization, resulting in mitochondrial matrix swelling
and rupture of the OMM, thereby permitting the rapid efux of
cytochrome c from the mitochondrial intermembrane space [71].
This observation can be viewed as additional support for a relationship between Bcl-2 family proteins and MPT induction [72].
ABT-737 and a related orally active derivative, ABT-263, bind with
high afnity to Bcl-2, Bcl-XL and Bcl-w, and both promise to be
useful tools for mechanistic studies. ABT-263 is in early clinical trials in lymphomas, small-cell lung cancer and chronic lymphocytic
leukemia (CLL) [73].
A novel BH3-mimetic, 072RB, was described recently [74]. The
authors demonstrated mitochondrial localization of this compound, followed by cell death in various cultured leukemic cells,
as well as in cells derived from acute myeloid leukemia (AML)
patients. No signicant cytotoxic effect was observed when 072RB
was administered to cultures of peripheral blood mononuclear
cells, either resting or PHA-stimulated, or to bone marrow cells from
healthy donors. Intravenous administration of 072RB to xenografts
of human AML cells in NOD/SCID mice caused a signicant delay

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of leukemic cell growth with no evidence of toxicity to normal tissue. Thus, BH3 mimetics can be used as anticancer agents, both in
mono- and combinatorial therapy.
Anti-apoptotic properties of Bcl-2 can be converted into proapoptotic effects. The unstructured loop of Bcl-2, which links the
BH3- and BH4-domains, might represent an important target for
conversion of Bcl-2 into a pro-apoptotic molecule. Thus, nuclear
receptor Nur77 was shown to convert Bcl-2 into a killer molecule by
binding this loop [75]. Recently, a Nur77-derived Bcl-2-converting
peptide with nine amino acids (NuBCP-9) was identied, and shown
to induce apoptosis of cancer cells in vitro and in animals. NuBCP9s acts as a molecular switch to dislodge the Bcl-2 BH4-domain,
exposing its BH3-domain, which in turn blocks the activity of antiapoptotic Bcl-XL [76].
Similar effects were achieved using non-peptide inhibitors
of Bcl-2. Thus, HA14-1 (ethyl 2-amino-6-bromo-4-(1-cyano2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate) was the rst
small molecular Bcl-2 inhibitor shown to induce apoptosis in several tumor cell lines [77], and antimycin A, a mitochondrial complex
III inhibitor, which binds Bcl-2 and Bcl-XL [78]. Other pan-Bcl-2
inhibitors, GX15-003 and GX15-070 (obatoclax), induced apoptosis in B cells from nine of the eleven CLL samples studied [79].
GX15-070 was shown to promote the release of cytochrome c from
isolated leukemia cell mitochondria. Apoptosis induced by this
agent is preceded by the release of Bak from Mcl-1, liberation of Bim
from both Bcl-2 and Mcl-1, and the formation of an active Bak/Bax
complex. Apoptosis was diminished, but not fully prevented, in the
absence of Bak/Bax or Bim, suggesting that obatoclax has additional
targets that contribute to its cytotoxicity [80].
4.3. Induction of MPT
As mentioned above, overexpression of anti-apoptotic Bcl-2
family proteins in tumors might inhibit OMM permeabilization by
Bax/Bak-mediated pore formation. If so, induction of MPT might
overcome the resistance of OMM to permeabilization due to overexpression of antiapoptotic Bcl-2 family proteins. The anticancer
effect of multiple conventional treatments (e.g., ionizing radiation,
etoposide and arsenates) is based on their ability to stimulate production of ROSkey factors facilitating MPT induction. Anticancer
drug-induced MPT may thus result from ROS-mediated modication of components of the MPT pore. For example, at higher
doses the chemotherapeutic agent, arsenic trioxide, was found to
cause oxidative modication of thiol groups in ANT and subsequent release of cytochrome c through MPT induction. However,
at clinically achievable concentrations the same drug stimulated
cytochrome c release and apoptosis through a Bax/Bak-dependent
mechanism [81] [82]. Distinct pathways of cytochrome c release
were also shown for the DNA-damaging anticancer drug, etoposide.
This drug was found to stimulate MPT induction and cytochrome c
release [83], but at low, therapeutic doses it also affected mitochondria through activation of caspase-2. Apparently, both pathways
might be relevant for its clinical effects.
Another component of the MPT pore is VDAC. Interaction of
VDAC with hexokinase II, a key glycolytic enzyme that is usually
upregulated in tumor cells (see the review by Pedersen in this
issue), not only facilitates glucose phosphorylation at the expense
of ATP produced by mitochondria, but also keeps VDAC in the
open state, which counteracts OMM permeabilization. Another
consequence of hexokinaseVDAC interaction is that it prevents
binding of pro-apoptotic proteins to VDAC and thereby the induction of apoptosis [84]. A variety of compounds stimulate cell
death via interaction with VDAC. Thus, avicins, pro-apoptotic, antiinammatory molecules with antioxidant effects both in vitro and
in vivo, perturb mitochondrial functions and initiate apoptosis in
tumor cells. Biophysical studies using lipid bilayers, revealed that

avicins target and close VDAC. Closure of VDAC would lead to an

overall lowering of the cell energy metabolism, subsequently pushing these cells towards the apoptotic pathway by permeabilization
of the OMM and release of cytochrome c [85]. This observation,
however, is in contrast with the report that HK-I, which binds to
VDAC and causes its closure, inhibits cytochrome c release and
protects against apoptotic cell death [86]. Apparently, the role of
VDAC in apoptotic cell death is still obscure and needs to be further
investigated.
4.4. Modulation of mitochondrial respiratory chain activity
Various attempts have been performed to stimulate tumor cell
death by modulation of mitochondrial respiratory chain activity.
Thus, inhibitors of the mitochondrial respiratory chain, rotenone
or antimycin A, and an inhibitor of mitochondrial ATP synthase,
oligomycin, were shown to induce apoptotic cell death in cultured human lymphoblastoid and other mammalian cells within
1218 h [87]. No signs of apoptosis were detected in cells lacking mitochondrial DNA, indicating that cell death was a result of a
direct effect on mitochondrial respiratory chain activity or on mitochondrial ATP synthase. Inhibitors of the mitochondrial respiratory
chain were also shown to enhance cell susceptibility towards other
apoptotic stimuli. Thus, Fas-mediated HeLa cell death occurred at
lower concentrations of Fas in cells pretreated with respiratory
inhibitors [88]. Cell death induced by inhibitors of the mitochondrial respiratory chain is often mediated by ROS. Thus, benzyl
isothiocyanate targets the mitochondrial respiratory chain to trigger ROS-dependent apoptosis in human breast cancer cells [89]. The
benzyl isothiocyanate-induced ROS production and apoptosis were
signicantly inhibited by overexpression of catalase and Cu,Znsuperoxide dismutase, as well as by pharmacological inhibition of
the mitochondrial respiratory chain. Similarly, cells lacking mitochondrial DNA were resistant to benzyl isothiocyanate-mediated
ROS generation and apoptosis.
Inhibition of the mitochondrial respiratory chain can induce
other forms of cell death as well. Thus, inhibitors of complex I
(rotenone) and complex II (TTFA) caused autophagic cell death,
in the transformed cell line HEK 293 and in cancer cell lines U87
and HeLa [90]. Cell death was markedly suppressed by blocking
the expression of autophagic genes (beclin 1 and ATG5) by siRNAs,
or by the autophagy inhibitor, 3-methyladenine. Further, overexpression of SOD2 in HeLa cells also attenuated cell death. These
results indicate that inhibitors of respiratory chain complexes I and
II can selectively induce autophagic cell death via a ROS-mediated
mechanism.
In 1982 Prasad and Edwards-Prasad reported for the rst time
that -tocopheryl succinate (-TOS), a redox-silent analogue of
vitamin E induces morphological changes and growth inhibition in
mouse melanoma cells in culture [91]. -TOS was shown to inhibit
the proliferation of virus-transformed RECC-UTC4-1 (C4-1) lymphoblastoid cells in a dose-dependent manner, to block the cells
in the G2/M cell cycle phase, and to trigger cellular apoptosis [92].
Subsequent studies revealed that -TOS selectively killed malignant cells at concentrations that were non-toxic to normal cells and
tissues [93], and regardless of whether the cells contained mutations in key tumor suppressor genes, such as p53 [94]. Further, the
lethal effect of -TOS was shown to involve destabilization of mitochondria with unleashing of their apoptogenic potential, resulting
in suppression of tumor growth. There are several explanations for
the anticancer effect of -TOS. Thus, it has been reported to interact
with complex II of the mitochondrial respiratory chain [95], to stimulate cell death via ROS production [96], and to induce cytochrome
c release and caspase activation as a result of translocation of Bax
from the cytosol to the mitochondria [97]. Thus, -TOS might be a
promising candidate for mitochondria-directed antitumor strategy.

V. Gogvadze et al. / Seminars in Cancer Biology 19 (2009) 5766

A novel approach to mitochondria-targeted anticancer therapy

was demonstrated recently based on different levels of mitochondrial cholesterol in tumor cells as compared to non-malignant cells.
Cancer cells exhibit enhanced mitochondrial cholesterol, which
supposedly contributes to chemotherapy resistance in hepatocellular carcinoma. Indeed, isolated mitochondria from hepatocellular
carcinoma with increased cholesterol levels were resistant to mitochondrial membrane permeabilization and release of cytochrome c
or Smac/DIABLO in response to various apoptotic stimuli, including
Bax [98]. Accordingly, tumor cell susceptibility to chemotherapy
was enhanced upon cholesterol depletion by inhibition of HMGCoA reductase, or squalene synthase, which catalyzes the rst
committed step in cholesterol biosynthesis [98]. Apparently, mitochondrial cholesterol contributes to chemotherapy resistance by
increasing membrane order and lessening its susceptibility to permeabilization. Lowering of mitochondrial cholesterol level might
therefore be useful as a potential therapeutic approach in cancer.
4.5. Stimulation of mitochondrial activity in tumor cells
Silencing of mitochondria, a characteristic feature of most tumor
cells, might thus be one reason for their evasion of cell death. This is
not only because apoptosis is dependent on mitochondrial energy
metabolism, but also because respiring mitochondria produce ROS,
which enhance the size of the releasable pool of cytochrome c and
sensitize mitochondria towards MPT. Hence, stimulation of mitochondrial activity and shifting energy production from glycolysis
to oxidative phosphorylation might be expected to increase the
apoptotic potential of mitochondria. Several possibilities can be
anticipated.
In tumor cells, pyruvate, the end product of glycolysis, is converted into lactate by PDH, which, as discussed above, can be
inhibited through phosphorylation by PDK (Fig. 1). Experiments
performed recently clearly demonstrate that inhibition of PDK by
dichloroacetate can shift cellular metabolism from glycolysis to glucose oxidation. As a result of such treatment, mitochondrial H2 O2
production increased, whereas mitochondrial membrane potential
decreased in cancer cells, but not in normal cells [99]. This reversed
the suppressed mitochondrial apoptosis pathway in cancer and
resulted in inhibition of tumor growth both in vitro and in vivo
[100].
It has been reported that overexpression of frataxin, a protein
associated with Friedreich ataxia an inherited neurodegenerative disorder stimulated oxidative metabolism and enhanced
mitochondrial membrane potential and ATP content in several
colon cancer cell lines [101]. Frataxin was shown to promote
mitochondrial oxidative metabolism, apparently via stimulation
of the synthesis of Fe/S clusters in mitochondria. In addition to
stimulation of mitochondrial activity, frataxin inhibited colony formation and markedly suppressed tumor formation when injected
into nude mice. These results are in accordance with those
reported above, demonstrating that stimulation of mitochondrial
metabolism in cancer cells can efciently suppress tumor growth.
Apparently, the mechanism by which stimulation of mitochondrial functional activity might increase apoptosis susceptibility
depends on cell type as well as the mode of cell death induction.
5. Involvement of p53 in mitochondria-mediated cell death
The p53 protein plays a major role in the maintenance of
genome stability in mammalian cells. Mutations of p53 occur in
a majority of tumors and are often responsible for tumor resistance
to chemotherapeutic agents. Examination of transcripts induced
by p53 expression before the onset of apoptosis revealed that

63

only 14 out of 7202 transcripts were markedly increased in p53expressing cells. Interestingly, many of these genes were genes
that are responsible for encoding proteins, which generate ROS
or respond to oxidative stress [102]. The authors presume that
apparently different cells have different capacities to cope with
oxidative stress and cells with a low capacity end up in apoptosis. Recent observations have revealed that p53, in addition to its
role as a central regulator of the cellular stress response, can modulate the balance between the glycolytic pathway and mitochondrial
ATP synthesis by oxidative phosphorylation. Mitochondrial impairment in p53-decient human cancer cells has been observed earlier.
In particular, the activity of mitochondrial cytochrome c oxidase
(COX) was found to be signicantly diminished in cells lacking p53
[103]. This decrease in COX activity was attributed to decreased
protein level of the COXII subunit encoded by the mitochondrial
genome and was not due to mutation in the mitochondrial COXII
gene. Recently, it has been found that the p53 protein regulates
the production of Synthesis of Cytochrome c Oxidase 2, a copper transporter critical for synthesis of the COX complex in the
mitochondria and thereby for mitochondrial ATP production [104]
(Fig. 1).
Among the targets of p53 is the TP53-induced glycolysis and
apoptosis regulator, TIGAR. Expression of TIGAR lowered fructose2,6-bisphosphate levels in cells, resulting in inhibition of glycolysis,
while stimulating NADPH generation via the pentose phosphate
shunt [105]. Keeping glucose away from energy production might
be important for the synthesis of nucleotides that are essential for
the repair of DNA lesions. Interestingly, one of the p53 target genes,
p53R2, is involved in regulation of the nucleotide pool within damaged cells [106]. On the other hand, TIGAR-mediated stimulation of
the pentose shunt results in increased GSH levels that are important for ROS scavenging. These functions of TIGAR correlated with
the ability to protect cells from ROS-associated apoptosis. Accordingly, knockdown of endogenous TIGAR expression sensitized cells
to p53-mediated death. In this context, it should be noted that
NADPH accumulation might block the p53-dependent activation of
caspase-2 in response to DNA damage mediated by the p53 target
PIDD [107]. Thus, it seems that the anti-apoptotic effect of TIGAR
might be realized via several pathways, including regulation of DNA
repair, inhibition of caspase-2 activation, and/or GSH-mediated ROS
scavenging. Additional work is required to establish whether these
pathways operate simultaneously or separately, and under which
circumstances.
The role of p53 is not restricted to its transcriptional activity. Hence, under stress conditions, including oxidative stress, a
fraction of cellular p53 (2%) was shown to migrate to mitochondria and initiate apoptosis [108]. Once in the mitochondria, p53
can bind to, and inhibit, MnSOD, enhancing ROS formation and
thereby promoting cell death [109]. Thus, restoration of the normal
function of mutated p53 represents an attractive tool for cancer
cell elimination. Such attempts to restore the activity of mutant
p53 have recently been performed using the low-molecular-weight
compound PRIMA-1(MET), which was shown to induce apoptosis in human tumor cells and inhibit tumor xenograft growth in
vivo [110]. It has been demonstrated that PRIMA-1(MET) induced
mitochondria-mediated apoptosis through activation of caspase2 with subsequent cytochrome c release and further activation of
downstream caspase-9 and caspase-3 [111]. Inhibition of caspase2 by a selective inhibitor and/or siRNA treatment, prevented
cytochrome c release and caused a signicant reduction in PRIMA1(MET)-induced cell death. In addition, PRIMA-1 can stimulate cell
death via restoration of p53 DNA binding activity to promoters of
pro-apoptotic genes, such as Bax and PUMA [112]. Knockdown of
p53 protein in breast cancer MCF-7 cells, using siRNA, followed by
PRIMA-1 treatment resulted in a decline in the expression of the
Bax and PUMA proteins.

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V. Gogvadze et al. / Seminars in Cancer Biology 19 (2009) 5766

6. Concluding remarks
Selective stimulation of cell death in tumor cells remains a primary strategic aim in cancer therapy. In spite of the heterogeneity
of tumors, which dictates an individual approach to anticancer
treatment, almost all tumor cells demonstrate enhanced uptake
and utilization of glucose, a phenomenon known as the Warburg
effect. One of the consequences of such a shift towards glycolytic
ATP production is enhanced mitochondrial stability and resistance
to apoptosis induction caused by the upregulation of pro-survival
proteins from the Bcl-2 family observed in a variety of tumors, as
well as suppression of mitochondrial activity. The success of an anticancer attack must be based on the concerted modulation of cellular
energy metabolism, mitochondrial stability, and other mechanisms
responsible for the resistance of tumor cells to death stimuli. In
this regard, mitochondrial targeting appears to be a promising
tool for promotion of tumor cell death. Stimulation of mitochondrial activity not only restores cellular energy metabolism to what
is characteristic for non-malignant cells but also promotes mitochondrial ROS production, which might sensitize mitochondria and
increase the susceptibility of the tumor cells to death-inducing
stimuli. The intimate interplay between apoptotic regulators and
mitochondrial energy metabolism should therefore be considered
during the search for novel anticancer drugs.
Conict of interest
No competing interest.

[15]

[16]
[17]
[18]

[19]

[20]

[21]

[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]

Acknowledgements
[31]

Work in the authors laboratory was supported by grants from

the Swedish and Stockholm Cancer Societies, Swedish Childhood
Cancer Foundation, The Swedish Research Council, the EC-FP-6
(Oncodeath and Chemores) and EC-FP-7 (APO-SYS). We apologize
to authors whose primary references could not be cited due to space
limitations.

[32]
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