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Mitochondria as targets for detection and treatment of cancer

Mitochondria as targets for detection
and treatment of cancer

Josephine S. Modica-Napolitano and Keshav K. Singh

Mitochondria are dynamic intracellular organelles that play a central role in
oxidative metabolism and apoptosis. The recent resurgence of interest in the
study of mitochondria has been fuelled in large part by the recognition that
genetic and/or metabolic alterations in this organelle are causative or
contributing factors in a variety of human diseases including cancer. Several
distinct differences between the mitochondria of normal cells and cancer cells
have already been observed at the genetic, molecular and biochemical levels.
As reviewed in this article, certain of these alterations in mitochondrial structure
and function might prove clinically useful either as markers for the early
detection of cancer or as unique molecular sites against which novel and
selective chemotherapeutic agents might be targeted.

Early studies of differences between the
mitochondria of normal cells and those of
cancer cells focused on the respiratory
deficiencies common to rapidly growing cancer
cells. This led Otto Warburg to propose in 1930
that respiratory deficiency might result in de-
differentiation of cells and hence neoplastic
transformation (Refs 1, 2). Other early studies
suggested that transformation was the result of
submolecular micro-electronic changes involving
errant dismantling and rebuilding of the electron
transport chain during cell division (Ref. 3), and
that mutations in mitochondria might cause
cancers (Ref. 4). More recently, a great deal of
research has been conducted that substantiates,
extends and provides new insight into the role
of mitochondria in cancer (Refs 5, 6, 7, 8). This
review summarises the important aspects of
mitochondrial structure and function, highlights
the observed differences in mitochondria between
normal and cancer cells, and discusses how these
differences might be exploited in the detection and
treatment of cancer.
Mitochondria structure,
function and genome
Historical perspective
Early cytological studies (reviewed in Ref. 9)
indicated the presence of subcellular granules
similar in size and shape to bacteria in a variety
of different cell types. In 1890, Altman postulated
that these granules, which he termed ‘bioblasts’,

Josephine S. Modica-Napolitano
Associate Professor, Department of Biology, Merrimack College, North Andover, MA 01845, USA.
Tel: +1 978 837 5000 x4459; Fax: +1 978 837 5029; E-mail: josephine.modicanapolitano@merrimack.edu

Keshav Singh (corresponding author)

Assistant Professor, Johns Hopkins Oncology Center, Bunting-Blaustein Cancer Research Building,
1650 Orleans St, Baltimore, MD 21231, USA. Tel: +1 410 614 5128; Fax: +1 410 502 7234/7244; E-mail:
singhke@jhmi.edu

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were the basic units of cellular activity.
Interestingly, Altman further speculated that
bioblasts were capable of an independent
existence, yet formed a colonial association with
the cytoplasm of a host cell, and that it was
through this association that the host cell acquired
the properties of life. The term ‘mitochondrion’,
meaning thread-like granule, was first applied to
these subcellular structures by Benda in 1898 .
During the period 1900–1930, most cytologists
recognised the mitochondrion as a well-defined
and ubiquitous organelle, although at that time
there was no agreement about its function. The
identification of mitochondria as centres of energy
metabolism came at the heels of refinements in
cell fractionation techniques during the late 1940s,
which allowed the successful separation of
relatively pure, functionally intact mitochondria
from other cellular components in liver cell
homogenates (Refs 10, 11). By 1949, these
mitochondrial fractions were shown to contain
succinate oxidase and cytochrome oxidase
activities, as well as the enzyme systems required
for fatty acid oxidation and the citric acid cycle
(Refs 12, 13; reviewed in Ref. 9). Today, it is known
that mitochondria produce up to 80% of the
energy needs of a cell and perform a host of
additional cellular functions.
Mitochondrial structure
The mitochondria of different tissues are similar
in their gross morphology (reviewed in Ref. 9). In
electron micrographs of fixed tissue specimens,
mitochondria are most commonly observed as
oval particles, 1–2 µm in length and 0.5–1 µm in
width. These dimensions approximate to those of
the bacterium Escherichia coli. The organelle is
bounded by two membranes. The peripheral, or
outer, membrane encloses the entire contents of
the mitochondrion. The inner membrane has a
much greater surface area and forms a series of
folds or invaginations, called cristae, which project
inward towards the interior space of the organelle.
The total surface area of the inner membrane
varies considerably depending upon the tissue
and type of cell. Since the enzymes involved in
oxidative phosphorylation are located on the
inner mitochondrial membrane, its surface area
and number of cristae are generally correlated
with the degree of metabolic activity exhibited by
a cell. The spatial arrangement of the outer and
inner membranes creates two distinct internal
compartments: the intermembrane space is
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located between the outer and inner membranes;
and the matrix is the space enclosed by the inner
mitochondrial membrane.
By contrast to the static, ‘cigar-shaped’
organelles commonly observed in electron
micrographs, living cells stained with the
lipophilic cation rhodamine 123 (Rh123) and
observed by fluorescence microscopy reveal
mitochondria as a dynamic network of long
filamentous structures, capable of profound
changes in size, form and location (Ref. 14). These
mitochondria can be seen extending, contracting,
fragmenting and even fusing with one another as
they move in three dimensions throughout the
cytoplasm. Interestingly, the treatment of cells
with microtubule-depolymerising agents has been
shown to result in an altered distribution of
mitochondria (Refs 15, 16). This suggests that
mitochondria are associated with and travel along
a molecular ‘highway’ composed of a cytoplasmic
microtubule network.
Mitochondrial function
Mitochondria play a central role in oxidative
metabolism in eukaryotes (reviewed in Ref. 9).
In the catabolism of carbohydrates (Fig. 1a), this
begins with the transport of pyruvate from
the cytosol into the mitochondrion, and its
subsequent oxidative decarboxylation to acetyl
CoA by a soluble, multi-enzyme pyruvate
dehydrogenase complex, which is located in the
mitochondrial matrix. The oxidation of acetyl
CoA is achieved by a cyclic process involving
eight catalytic steps. This process is known as
either the citric acid or the tricarboxylic acid
(TCA) cycle. All but one of the TCA cycle
enzymes are soluble proteins found in the inner
mitochondrial matrix compartment. The single
insoluble enzyme, succinate dehydrogenase, is
tightly bound to the matrix side of the inner
mitochondrial membrane. Each round of the
TCA cycle results in the production of two
molecules of CO2, three molecules of reduced
nicotinamide adenine dinucleotide (NADH), one
molecule of reduced flavin adenine dinucleotide
(FADH2), and one molecule of GTP (the energetic
equivalent of ATP).
The next stage of aerobic metabolism is
oxidative phosphorylation, an energy-generating
process that couples the oxidation of respiratory
substrates (such as the NADH and FADH 2
generated through the TCA cycle) to the synthesis
of ATP. Substrate oxidation involves a series of
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a Carbohydrate metabolism b Fatty acid oxidation c Urea cycle

Pyruvate Fatty acids Ornithine

Urea

Cytosol
Membrane Soluble
Oxidative Oxidative Fatty + enzymes Urea
Matrix of V in matrix cycle
mito- decarbox decarbox acid V
chondrion -ylase -ylase cycle
β-oxidation
(Mostly)
Acetyl CoA Citrulline
Acetyl CoA
Succinate
dehydrogenase
(inner membrane)
+
Oxidation
V TCA V (Sometimes)
cycle e.g. fasting + Matrix
CO2 or disease enzymes
GTP
NADH Ketones
FADH2
Respiratory enzyme
Oxidative + complexes I-V
phosphorylation (inner membrane) Energy source: brain
heart, etc.
ATP
Adenine nucleotide
+ translocase

Energy use: in cytosol

(and by mitochondrion)

Schematic of key mitochondrial metabolic pathways

Expert Reviews in Molecular Medicine C 2002 Cambridge University Press

Figure 1. Schematic of key mitochondrial metabolic pathways. (a) Carbohydrate metabolism. Pyruvate
produced from glycolysis undergoes oxidative decarboxylation to acetyl CoA, which is then oxidised in an
eight-step process known as the tricarboxylic acid (TCA) cycle. The respiratory substrates NADH and FADH2
generated through the TCA cycle are next oxidised in a process coupled to ATP synthesis. Electrons are
transferred from NADH and FADH2 to oxygen via enzyme complexes located on the inner mitochondrial
membrane. Three of the electron carriers (complexes I, III and IV) are proton pumps, and couple the energy
released by electron transfer to the translocation of protons from the matrix side to the external side of the inner
mitochondrial membrane. Energy stored in the resulting proton gradient (i.e. the proton-motive force) is used
to drive the synthesis of ATP via the mitochondrial enzyme ATP synthetase (complex V). (b) Fatty acid oxidation.
Fatty acids undergo oxidative decarboxylation in the mitochondrial matrix to give acetyl CoA, which is fed into
the TCA cycle, and new acyl CoA molecules that are successively shortened with each round of the cycle.
Under certain conditions (e.g. fasting), acetyl CoA molecules are converted into ketones for use as an alternative
energy source. (c) Urea cycle. Amino acid degradation resulting in excretion of nitrogen as urea occurs partly
in the mitochondrion. The mitochondrion is also essential for several other processes (not shown), including
gluconeogenesis, regulation of cytosolic NAD+, intracellular homeostasis of inorganic ions, and apoptosis
(fig001ksb).
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respiratory enzyme complexes that are located on
the inner mitochondrial membrane and are
capable of accepting and donating electrons in
a specific sequence based on their relative
oxidation–reduction potentials and substrate
specificity. Complex I (NADH-ubiquinone
reductase) transfers electrons from NADH to
the mobile electron carrier ubiquinone, or
coenzyme Q. It is the largest and most labile
of all the respiratory enzyme complexes. In
bovine heart, for example, complex I comprises
at least 41 different protein subunits. Complex
II (succinate-ubiquinone reductase) transfers
reducing equivalents from succinate to
ubiquinone. It comprises four protein subunits,
one of which is the FADH 2-linked TCA cycle
enzyme succinate dehydrogenase. Complex III
(ubiquinone-cytochrome c reductase) is an 11-
subunit respiratory enzyme complex involved in
the transfer of electrons from membrane-bound
ubiquinone to oxidised cytochrome c, another
mobile electron carrier located on the outer surface
of the inner mitochondrial membrane. Complex
IV, or cytochrome c oxidase (COX), is the terminal
electron acceptor. It comprises 13 different protein
subunits and functions in the transfer of electrons
from reduced cytochrome c to molecular oxygen,
to form H2O.
The energy released by the exergonic transfer
of electrons from respiratory substrate to
oxygen is coupled to the translocation of
protons from the matrix side to the external side
of the inner mitochondrial membrane at three
sites: respiratory enzyme complexes I, III and IV.
In intact, well-coupled mitochondria, the inner
membrane is relatively impermeable to the
back flow of these protons. According to the
Chemiosmotic Hypothesis, which was first
proposed by Peter Mitchell in 1961 (and for which
he received the Nobel Prize in Chemistry in 1978),
the energy stored in the resulting proton gradient
(i.e. the proton-motive force) is used to drive the
synthesis of ATP via complex V, the mitochondrial
enzyme ATP synthetase (Ref. 17).
The ATP that is produced by aerobic
metabolism and not used by the mitochondrion
is transported across the inner mitochondrial
membrane in exchange for cytosolic ADP by the
enzyme adenine nucleotide translocase (ANT).
This exchange ensures not only the availabilty of
mitochondrial ADP, which is the principal
control molecule for the rate of oxidative
phosphorylation, but also the availability of
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cytosolic ATP. Oxidative phosphorylation thus
supplies a majority of the cellular energy
produced under aerobic conditions and required
to sustain cell viability and normal cell functions.
Fatty acid oxidation is another important
metabolic activity located in the mitochondria
(Fig. 1b). The beta-oxidation pathway involves
four separate enzymes that are soluble in the
mitochondrial matrix and that function in a
repetitive cycle. With each round of the cycle, a
fatty acid undergoes oxidative decarboxylation to
produce one molecule of acetyl CoA and one
molecule of a new acyl CoA that is two carbons
shorter than the starting fatty acid. The process
continues until the original fatty acid molecule is
completely degraded to acetyl CoA (for example,
the 16-carbon palmitoyl CoA would undergo
seven rounds of beta-oxidation to yield eight
molecules of acetyl CoA). The acetyl CoA
molecules thus generated normally enter into the
TCA cycle where they undergo oxidation to CO2.
However, during conditions of prolonged fasting
and starvation, or in certain metabolic diseases
(e.g. diabetes mellitus), the acetyl CoA molecules
generated by fatty acid oxidation are converted
into ketones (e.g. β-hydroxybutyrate, acetoacetate
and acetone) by enzymes also located in the
mitochondrial matrix. These molecules are then
transported through the blood to other tissues,
such as brain and heart, where they are used as
an alternative energy source to glucose.
In addition to its central role in oxidative
metabolism, the mitochondrion is involved in a
variety of other important cellular functions. For
example, certain enzymes of the urea cycle (Fig.
1c) and gluconeogenesis are located in the
mitochondrial matrix. Mitochondria are involved
in the regeneration of cytosolic NAD+ (required
for the substrate-level phosphorylation step in
glycolysis) and in the intracellular homeostasis of
inorganic ions such as calcium and phosphate. A
wealth of recent studies show that mitochondria
also play an integral role in the cascade of
intracellular events that lead to apoptosis, or
programmed cell death (Refs 18, 19).
Mitochondrial genome
Mammalian cells typically contain 103–104 copies
of mitochondrial DNA (mtDNA). The genome is
a 16.5 kb closed-circular, double-helical molecule
that encodes two rRNAs, 22 tRNAs and 13
polypeptides (reviewed in Ref. 5). Each of these
polypeptides is a highly hydrophobic subunit of
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one of four respiratory enzyme complexes with Leber’s hereditary optic neuropathy and
localised to the inner mitochondrial membrane. familial mitochondrial encephalomyopathy
They include seven subunits of respiratory (Refs 27, 28, 29). Since then, there has been a
enzyme complex I, one subunit of complex III, steady growth in the list of diseases associated
three subunits of complex IV, and two subunits with mitochondrial dysfunction arising from
of complex V. All other mitochondrial proteins, mtDNA mutations (Table 1).
including those involved in the replication, Although mtDNA represents less than 1% of
transcription and translation of mtDNA, are total cellular DNA, its gene products are essential
encoded by nuclear genes and are targeted to for normal cell function. Unlike nuclear DNA,
the mitochondrion by a specific transport mammalian mtDNA contains no introns, has no
system (Ref. 20). In humans and other mammals, protective histones and is exposed to deleterious
mitochondrial genes display a maternal
inheritance (i.e. are inherited from the female Table 1. Mitochondrial diseasesa
parent). This is probably because the number of (tab001ksb)
mtDNA copies in the egg is typically 103-fold
greater than that in the sperm (Ref. 21). Tissue/organ
Alternatively, paternal genomes and organelles affected Clinical condition
might be preferentially degraded in the zygote Blood Pearson’s syndrome
(Refs 22, 23).
Although the mitochondrial and nuclear Brain Seizures
genomes are physically distinct, the high degree Myoclonus
of functional interdependence between them is Ataxia
Stroke
suggestive of a host–parasite relationship. The Demetia
‘endosymbiont’ theory proposes that early in Migraine
the evolution of the eukaryote, a primitive proto-
eukaryote cell that was incapable of aerobic Colon Pseudo-obstruction
respiration served as host to a eubacterium
Eye Optic neuropathy
with the unique capacity for oxidative Ophthalmoplegia
metabolism (Ref. 24). During the early stages of Retinopathy
this endosymbiotic association, the eubacterium
retained its genetic autonomy. In time, however, Heart Conduction disorder
most of its genetic material was transferred to the Wolff–Parkinson–White
syndrome
nuclear genome of the host. The resulting Cardiomyopathy
mitochondrion retained only those few (i.e. 13)
genes encoding polypeptides that are essential to Inner ear Sensorineural hearing loss
aerobic ATP production yet have a hydrophobicity
that precludes nuclear synthesis and cytoplasmic Kidney Fanconi’s syndrome
Glomerulopathy
transport to mitochondria. It is of interest to
recall Altman’s perceptive characterisation of Liver Hepatopathy
mitochondrial function, and his suggestion of a
colonial association between the newly discovered Skeletal muscle Myopathy
‘bioblasts’ and the host cell within which they Neuropathy
reside.
a
Data in the table are derived from Ref. 38.
Mitochondria and disease Mitochondrial diseases can arise from mutations in
The first mitochondrial disease was described nuclear DNA or in mitochondrial DNA. Deficits in
by Rolf Luft in 1962 (Ref. 25), a year prior to ATP production might result from altered functions
of proteins involved directly in oxidative
the discovery of mtDNA (Ref. 26). At the phosphorylation or involved in communication
genetic level, mitochondrial disease was not between the nucleus and mitochondria, and might
definitively described until 1988 with the have deleterious effects on several organ systems.
identification of mtDNA mutations in patients Many mitochondrial diseases are so new that they
with mitochondrial myopathies, as well as the have not yet been mentioned in the medical
textbooks or in the medical literature.
molecular genetic characterisation of patients
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reactive oxygen species generated by oxidative
phosphorylation. In addition, replication of
mtDNA might be error prone (Refs 8, 30, 31, 32,
33, 34). The accumulation of mutations in mtDNA
is approximately tenfold greater than that in
nuclear DNA (Refs 35, 36).
Inherited disorders of mitochondria produce
childhood and adult diseases with a variety of
clinical symptoms (Ref. 37). Mitochondrial
dysfunction has been found frequently as the
basis of developmental defects. It is estimated that
of the 4 million children born each year in the
USA, up to 4000 develop diseases related to
mitochondrial dysfunction (Refs 5, 30). Congenital
mitochondrial diseases such as Kearns–Sayre/
chronic progressive external opthalmoplegia and
Leber’s hereditary optic neuropathy are either
maternally inherited or are derived from a
founder mutation early in embryogenesis. Since
mitochondria perform a variety of different
functions in different tissues, since the proportion
of normal to mutated mtDNA can vary, and since
tissues have different aerobic dependencies, each
mtDNA mutation can produce a wide spectrum
of phenotypes that have proven to be extremely
perplexing to scientists and physicians.
Mitochondrial dysfunction is also increasingly
recognised as an important cause of adult human
pathology (reviewed in Ref. 5). Dysfunctional
mitochondria are found in diverse adult-onset
diseases, including diabetes, cardiomyopathy,
infertility, migraine, blindness, deafness, kidney
and liver diseases, and stroke. The accumulation
of somatic mutations in mtDNA has been
suggested to play a causative or contributing
role in aging, in age-related neurodegenerative
disorders such as Parkinson’s, Alzheimer’s and
Huntington’s disease, and in cancer (see
below). Other adult-onset pathologies might
result from the biochemical toxicity or mtDNA
damage caused by various drugs, including those
used against human immunodeficiency virus
(HIV) (Ref. 38), or by other endogenous or
environmental agents (Refs 30, 39). Although
human mitochondrial diseases are often multi-
system disorders, constitutively highly oxidative
tissues such as myocardium, brain and kidney,
as well as episodically oxidative tissues such
as skeletal muscle, are especially vulnerable to
mtDNA damage (Refs 35, 36). To date, over
100 point mutations and 200 deletions and
rearrangements have been shown to be associated
with mitochondrial disease and new mutations
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Mitochondria as targets for detection and treatment of cancer
are being described every year (Ref. 37). Thus,
mitochondria play a central role in many diseases
that can affect any organ, at any age.
Mitochondria and cancer
Phenotypic differences in tumour
mitochondria
Cancer cells have an altered metabolism that
includes: a higher rate of glycolysis (Ref. 39), an
increased rate of glucose transport (Ref. 40),
increased gluconeogenesis (Ref. 41), reduced
pyruvate oxidation and increased lactic acid
production (Ref. 42), increased glutaminolytic
activity (Ref. 43), reduced fatty acid oxidation (Ref.
44), increased glycerol and fatty acid turnover
(Ref. 45), modified amino acid metabolism (Ref.
46), and increased pentose phosphate pathway
activity (Ref. 47).
Mitochondria are involved either directly or
indirectly in many aspects of altered metabolism
in cancer cells (Ref. 48) and several notable
differences between the mitochondria of normal
versus transformed cells have been discovered
(reviewed in Refs 49, 50 and 51). For example,
various tumour cell lines exhibit differences in
the number, size and shape of their mitochondria
relative to normal controls. The mitochondria of
rapidly growing tumours tend to be fewer in
number, smaller and have fewer cristae than
mitochondria from slowly growing tumours;
the latter are larger and have characteristics
more closely resembling those of normal cells.
Interestingly, the usually benign oncocytoma of
thyroid, salivary gland, kidney, parathyroid
and breast is characterised by the presence of
cells containing abnormally large numbers of
mitochondria, and high levels of oxidative
enzymes (Ref. 52). The ultrastructural features
of mitochondria in these cells show similarities
with mitochondrial encephalomyopathies, where
mitochondria are found as large aggregates and
display a variety of morphological alterations.
MtDNA mutations are also commonly found in
oncocytic tumours (Ref. 52).
Alterations in the molecular composition of the
inner membranes of tumour mitochondria have
also been noted (Refs 53, 54, 55, 56, 57, 58).
Polypeptide profiles of normal liver versus
hepatoma mitochondria demonstrate differences
in the appearance and/or relative abundance of
several protein subunits. One major band that is
deficient or absent in several tumours studied has
a mobility near or equal to the B subunit of the F1-
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ATPase (approximately 57 kDa). Other bands that
are present in tumour mitochondria appear to be
deficient or absent in control mitochondria. In
addition, analysis of the inner membrane lipid
composition of various tumour mitochondria has
indicated elevated levels of cholesterol, varying
total phospholipid content, and/or changes in the
amount of individual phospholipids relative to
normal controls.
Many differences in the mitochondria of
normal versus transformed cells have also been
noted with regard to: (1) the preference for
substrates oxidised; (2) the magnitude of the
acceptor control ratio; (3) the rates of electron and
anion transport; (4) the capacity to accumulate and
retain calcium; (5) the amounts and forms of DNA;
and (6) the rates of protein synthesis and organelle
turnover. However, there is apparently no
universal metabolic alteration that is common
to all tumours. For example, although the
pathogenesis of prostate cancer involves the
mitochondrial metabolic transformation of citrate-
producing cells to citrate-oxidising cells, this
metabolic abnormality is not reported in other
cancers (Ref. 59). Additionally, it is important to
note that the altered metabolism in cancer cells is
probably not the cause of malignancy but, rather,
a secondary, albeit essential, adaptation to support
malignant activities (Ref. 59).
Rh123 uptake
In the early 1980s, Chen and colleagues discovered
an interesting phenotype that was found to be
common to nearly all types of carcinoma tested
(Refs 14, 60, 61). It was observed that the lipophilic
cation Rh123 could serve as a highly specific
vital stain for mitochondria, providing low-
background, high-resolution fluorescent images
of the organelle in a variety of cell types (Fig. 2).
It was further observed that relative to the
mitochondria of normal epithelial cells, the
mitochondria of carcinoma cells displayed an
increased uptake and prolonged retention of
Rh123, and that this phenomenon correlated with
a selective cytotoxicity for carcinoma cells in vitro
and in vivo (Refs 62, 63, 64). For example, whereas
Rh123 was shown to have a minimal effect on the
clonal growth of those cell types that display little
uptake and short retention of the dye (e.g. primary
cultures of normal mouse bladder epithelial cells;
and CCL-34 and BSC-1, the non-tumourigenic
dog and monkey kidney epithelial cell lines,
respectively), it markedly inhibited the clonal
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Mitochondria of neuron revealed by
staining with a rhodamine 123
derivative
Expert Reviews in Molecular Medicine
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Figure 2. Mitochondria of neuron revealed by
staining with a rhodamine 123 derivative. Neurons
were stained with tetramethylrhodamine, ethyl
ester, perchlorate (TMRE). TMRE accumulates in
mitochondria and emits high fluorescence. The cell
images were recorded with a digital camera and
processed to generate high-resolution images. The
image is courtesy of Dr Gary E. Gibson, Weill Medical
College of Cornell University, Burke Medical
Research Institute, White Plains, NY 10605, USA.
Magnification, ~X760 (fig002ksb).
growth of those cultured carcinomas cell lines that
display high uptake and prolonged retention of
Rh123 (e.g. MB49, the transformed mouse
epithelial cell line; and MCF-7 and HUT, the
human breast and lung carcinoma cell lines,
respectively). In addition, at a constant exposure
of 10 µg/ml Rh123, greater than 50% cell death
occurred within seven days in 9/9 of the
carcinoma cell types tested, whereas 6/6 control
epithelial cell types remained unaffected.
Standard chemotherapeutic agents such as
arabinosyl cytosine (Ara-c) and methotrexate
exhibit no such selectivity for carcinoma cells. In
vivo, Rh123 was shown to prolong the survival
of mice implanted with Ehrlich ascites tumour
or MB49 mouse bladder carcinoma cells as
much as 260%, although the extent of survival
prolongation was highly dependent on the dose
and schedule of administration of the dye. As
expected, Rh123 did not significantly prolong the
survival rate of mice implanted with tumours
of cell types shown to be short retainers of the
dye (e.g. L1210 and P388 leukaemias, and B6
melanoma).
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Increased membrane potential in
carcinoma cells
It became apparent from early studies that two
chemical properties of Rh123 were important in
promoting its uptake into mitochondria. The first
is the lipophilicity of Rh123, which allows the
compound to penetrate the hydrophobic barriers
of the plasma and mitochondrial membranes; and
the second is its electrical charge – rhodamines
that are positively charged at physiological pH
stain mitochondria specifically, whereas the
neutral rhodamines do not (Ref. 14). Initially, there
was much indirect evidence to suggest that Rh123
uptake occurs as a function of the magnitude of
the mitochondrial membrane potential. For
example, the addition of ionophores that dissipate
the mitochondrial electrical gradient (such as
valinomycin or dinitrophenol), or respiratory
inhibitors that prevent the establishment of the
electrical gradient (such as cyanide, antimycin or
rotenone), were demonstrated to diminish
mitochondrial-specific fluorescence in cells pre-
stained with the dye (Ref. 61). Later, experimental
manipulation of the membrane potential in
isolated mitochondria and concurrent
measurement of the amount of Rh123 associated
with the organelle definitively established that
Rh123 is concentrated by cells and into
mitochondria in response to negative-inside
transmembrane potentials (Ref. 65). Furthermore,
it was determined that the mitochondrial
membrane potential of carcinoma cells is
approximately 60 mV higher than that of control
epithelial cells (Ref. 65). Since Rh123 distributes
across the inner mitochondrial membrane in
accordance with the Nernst equation (Ref. 66), this
difference alone is sufficient to account for a
tenfold greater accumulation of the compound in
carcinoma versus control epithelial mitochondria.
In whole cells, however, the plasma membrane
potential pre-concentrates Rh123 relative to the
external medium, thus affecting the cytoplasmic
concentration of Rh123 and the amount of dye
available for mitochondrial uptake. The higher
plasma membrane potential observed in some
carcinoma cells versus control epithelial cell types
therefore further contributes to increased Rh123
accumulation in carcinoma mitochondria (Ref.
67). Finally, Rh123 was found to exhibit a
concentration-dependent toxicity in mitochondria
by inhibition of ATP synthetase (Refs 68, 69). Since
mitochondria are the primary sites of ATP
synthesis in cells undergoing aerobic metabolism,
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selective mitochondrial toxicity in carcinoma cells
resulting from enhanced uptake and retention of
Rh123 provided the basis for the selective anti-
carcinoma activity displayed by this compound.
Delocalised lipophilic cations
Rh123 provided a prototype for a new class of
anti-cancer agents that exploit the difference in
mitochondrial membrane potential between
normal epithelial and carcinoma cells to achieve
a selective mitochondrial toxicity and consequent
selective cytotoxicity for carcinoma cells. In the
past few years, several members of this class of
compounds, known collectively as delocalised
lipophilic cations (DLCs), have exhibited at least
some degree of efficacy in carcinoma cell killing
in vitro and/or in vivo (Refs 70, 71, 72, 73, 74, 75).
For example, dequalinium chloride (DECA) has
demonstrated 100-fold greater inhibition of the
clonal growth of carcinoma versus control
epithelial cells in culture, anti-carcinoma activity
in human colon adenocarcinoma cells injected
subcutaneously in nude mice, and significant
regression of tumours in rats carrying in situ
mammary adenocarcinomas induced by
dimethyl-bezanthracene (Refs 71, 72). The
thiopyrylium AA-1 was shown to prolong the
survival of mice implanted with either mouse
bladder carcinoma, human melanoma, and
human ovarian carcinoma cell lines, achieving
treated:control ratios as high as 450% (Ref. 74).
The rhodacyanine MKT-077 also appears
particularly promising (Refs 73, 76, 77). In recent
studies, MKT-077 demonstrated significant
growth-inhibitory activity against a variety of
keratin-positive human cancer cell lines, as
measured by clonogenic assays and growth
inhibition of cultured cells. In vivo, MKT-077
demonstrated significant anti-tumour activity in
nude mice implanted with either the human
melanoma LOX, the human renal carcinoma A498,
or the human prostate carcinoma DU145, all of
which are highly refractory to a variety of
traditional therapies. As the first DLC with a
favourable pharmacological and toxicological
profile in preclinical studies, MKT-077 has
undergone Phase I clinical trials, approved by the
US Food and Drug Administration, for the
treatment of carcinoma.
Mechanism of action of DLCs
It is of interest to note that, although all DLCs are
taken up into mitochondria by a common
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mechanism (i.e. in response to negative-inside
transmembrane potentials), the mechanism of
mitochondrial toxicity exhibited by these
compounds is quite varied. For example, among
the DLCs that display a concentration-dependent
toxicity to mitochondria, Rh123 and AA-1 inhibit
mitochondrial ATP synthesis at the level of F0F1-
ATPase activity (Refs 68, 74), whereas DECA and
certain DLC thiacarbocyanines interfere with
NADH-ubiquinone reductase activity (Refs 70,
72). In addition, the selective cytotoxicity to
carcinoma cells exhibited by MKT-077 in vitro
and in vivo has been attributed to a selective
inhibition of mitochondrial respiration in cancer
cells, most probably as a result of a general
perturbation of mitochondrial membranes and
consequent inhibition of the activity of
membrane-bound enzymes (Ref. 76). The selective
cytotoxicity might also be a consequence of a mild
to moderate degradative effect on mtDNA, but
not nuclear DNA, of various carcinoma cell types
(Ref. 76).
It is of further interest to note that, although
increased membrane potential is necessary to
achieve selective cytotoxicity by DLCs, it alone is
not sufficient. If this were the case then cardiac
muscle cells, which have also been shown to
exhibit a high mitochondrial membrane potential
(Ref. 78), would be susceptible to the cytotoxic
effects of these compounds. Yet significant cardiac
toxicity has not been observed following in vivo
administration of either MKT-077 or DECA. This
suggests the sensitivity of any particular cell
type to the effects of DLCs might depend on
different cytoplasmic characteristics, such as those
involving the kinetics of uptake and retention of
the compound, or on properties inherent to the
mitochondria, such as differential sensitivity of
the target molecule against which the DLC exerts
its cytotoxic effect.
DLCs and photochemotherapy
Some research groups have explored the use of
certain DLCs in photochemotherapy (PCT), an
investigational cancer treatment involving
light activation of a photoreactive drug, or
photosensitiser, that is selectively taken up or
retained by malignant cells (Refs 79, 80, 81, 82).
There has been considerable interest recently in
PCT as a form of treatment for neoplasms of the
skin, lung, breast, bladder, brain or any other
tissue accessible to light transmitted either
through the body surface or internally via fibre
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Mitochondria as targets for detection and treatment of cancer
optic endoscopes. Cationic photosensitisers are
particularly promising as potential PCT agents.
Like other DLCs, these compounds are
concentrated by cells into mitochondria in
response to negative-inside transmembrane
potentials, and are thus selectively accumulated
in the mitochondria of carcinoma cells. In response
to localised photoirradiation, the photosensitiser
can be converted to a more reactive and highly
toxic species, thus enhancing the selective toxicity
to carcinoma cells and providing a means of
highly specific tumour cell killing without injury
to normal cells.
Several cationic photosensitisers have shown
promise for use in PCT. For example, selective
phototoxicity of carcinomas in vitro and in vivo
has been observed for a series of triarylmethane
derivatives (Ref. 83), and for 2-ethyl-1,3-dioxylene
kryptocyanine (EDKC) (Ref. 77). Both Rh123 and
the chalcogenapyrylium dye 8b have been
evaluated as photosensitisers for the
photochemotherapy of malignant gliomas (Refs
84, 85, 86). As is the case for the non-
photosensitising DLCs, the mitochondrion has
been implicated as an important, perhaps primary,
subcellular site of damage by these and several
other cationic photosensitisers (Refs 79, 87, 88, 89,
90, 91). Again, the mechanisms of mitochondrial
toxicity exhibited by these compounds have been
shown to vary from an inhibition of NADH-
ubiquinone reductase (e.g. in the case of EDKC,
and the triarylmethane derivative VB-BO) to a
non-specific perturbation of mitochondrial
function most probably resulting from membrane
damage induced by singlet oxygen (e.g. in the case
of certain chalcogenapyrylium dyes). More
recently, photoactivation has been shown to
enhance the mitochondrial toxicity of MKT-077,
with evidence for the involvement of lipid
peroxidation in this process. These results have
positive implications for the use of MKT-077 in
PCT.
Future directions for research
involving DLCs
Although the use of DLCs as anti-cancer agents
has shown promise, as yet there is no real
understanding of the biochemical basis for the
increased mitochondrial membrane potential in
carcinoma cells. Consequently, the choice for the
design or selection of potentially therapeutic
lipophilic cations has been based almost solely
on physical properties (i.e. lipid solubilty,
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delocalisation of positive charge, etc.), and
preliminary screening for the selective cytotoxicity
of these compounds has been empirical. Although
there is sufficient evidence to support the idea that
a therapeutic mechanism based on differences
in mitochondrial membrane potential might
be exploited for the treatment of cancers, a
knowledge of the specific biochemical alterations
that account for increased mitochondrial
membrane potential in these cells would
undoubtedly lead to a more rational approach to
the choice of highly selective DLCs for clinical use.
To this end, a comprehensive, comparative
biochemical analysis of mitochondria isolated
from several control and carcinoma cell lines that
display differences in mitochondrial membrane
potential is currently under way (J.S. Modica-
Napolitano, unpublished). This type of study
will contribute to an understanding of the
phenomenon of increased uptake of DLCs by
carcinoma mitochondria and might also reveal
molecular differences between the mitochondria
of normal epithelial and carcinoma cells against
which novel, selective and site-specific DLCs
could be targeted. The information obtained will
be used in the rational design of a more efficacious
form of DLC that exhibits a dual selectivity for
carcinoma cells based on both differential
accumulation and selective action against a
unique molecular target.
Molecular basis for increased membrane
potential in carcinoma cells
It is logical to assume that the observed differences
in the magnitude of the mitochondrial membrane
potential between normal epithelial and
carcinoma cells might arise from differences in
the structure and function of one or more of
those organelle components that serve to create
and/or maintain the electrical gradient.
Possibilities include differences in mitochondrial
respiratory enzyme complexes, electron carriers,
ATP synthetase, ANT and membrane lipid
structure. Differences that affect electron transfer
activity, or proton translocation, utilisation or
conductance, are also candidates for the molecular
basis for increased mitochondrial membrane
potential in carcinoma cells.
Interestingly, some differences of these types
are already known to exist between the
mitochondria of normal and malignant cells.
For example, under certain assay conditions,
mitochondria isolated from hepatomas of varying
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Mitochondria as targets for detection and treatment of cancer
growth rates and degrees of differentiation
display a decreased capacity for uncoupler-
stimulated ATP hydrolysis relative to that
found in normal liver (Ref. 92). In addition,
mitochondria isolated from biopsies of human
hepatocellular carcinoma have decreased rates
of respiration-linked ATP synthesis and a
reduced phosphorylative capacity compared with
normal human liver (Refs 93, 94). Furthermore,
the measured maximal velocity for ATPase
activity in submitochondrial particles isolated
from hepatocellular carcinoma is considerably
lower than that in normal liver. It has been
suggested that these alterations in enzyme
function might be associated with a decrease
in immunodetectable levels of the B subunit
of the F1 component of mitochondrial ATPase
and/or with overexpression of the ATPase
inhibitor protein (IF1) in tumour mitochondria
(Refs 93, 94, 95).
COX activity
A comparison of COX activities in cultured
carcinoma versus normal epithelial cells suggests
a possible correlation between membrane
potential and the activity of this enzyme.
Measurements of COX activity in samples from
the total cellular homogenate and the
mitochondrial subfraction from the cultured
human carcinoma cell lines MCF-7 (breast), T47D
(breast) and DU-145 (prostate) demonstrate
significantly lower specific activities of the
enzyme compared with that measured in the
normal monkey kidney epithelial cell line CV-1
(Ref. 96). Similar decreases in COX activity were
found when comparing the specific activity of
the enzyme in biopsies of human colonic
adenocarcinoma versus normal colon mucosa
(Ref. 97), and in cultured rat HC252 hepatoma
cells versus non-neoplastic liver (Ref. 98).
It is not clear whether the decrease in specific
activity of COX in cancer cells can be explained
by alterations in the level of gene expression.
For example, in one study involving human
colonic biopsies, the mean level of expression of
mitochondrially encoded COX subunit III was
found to be lower in carcinoma versus normal
mucosa samples (Ref. 99). Cultured HT29 colon
carcinoma cells also exhibited low levels of the
COX III transcript; however, expression of COX
III returned to higher (normal) levels when the
cells were induced to differentiate by exposure
to sodium butyrate. By contrast, increased
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levels of RNA transcripts of the nuclear-encoded
subunit COX IV and mitochondrially encoded
COX subunits I and II have been observed in
Zajdela hepatoma as compared with normal liver
(Ref. 100).
Alternatively, and perhaps more interestingly,
the decreased specific activity of COX in tumour
mitochondria might reflect a kinetic difference
caused by genetic mutations that alter the
structure and function of the enzyme. These same
structural changes might be hypothesised to affect
the proton-pumping capacity of the enzyme,
and thus the magnitude of the mitochondrial
membrane potential.
Mitochondrial permeability transition
pore activity
The magnitude of the mitochondrial membrane
potential might also be dependent upon
membrane permeability as regulated generally
by the membrane lipid–protein structure, or more
specifically by the mitochondrial permeability
transition pore (MPTP). This is a multi-protein
structure formed at contact sites between the inner
and outer mitochondrial membranes. It is a
voltage-dependent, cyclosporin-A-sensitive,
high-conductance inner membrane channel, the
opening of which transiently depolarises the
mitochondrial membrane potential. Although
the complete physiological role of the MPTP is
unknown at this time, it is probably involved
in the early apoptotic changes that affect
mitochondrial membrane permeability. Several
differences have been found when comparing the
MPTP in normal versus malignant cells.
One of the key structural components of the
MPTP complex is ANT. The primary role of ANT
is to facilitate the one-for-one exchange of ATP
(out) for ADP (in) across the inner mitochondrial
membrane. More recently, it has been suggested
that ANT also acts as a non-specific pore that
renders the mitochondrial inner membrane
permeable to solutes less than 1.5 kDa in size (Ref.
101). Interestingly, the adenine nucleotide
exchange function of ANT is known to be
decreased in certain hepatoma versus normal liver
mitochondria (Refs 102, 103, 104). In addition, the
sensitivity of this enzyme to bongkrekic acid, an
inhibitor of both adenine nucleotide exchange and
formation of the MPTP is also decreased in
hepatoma versus normal liver (Refs 104, 105, 106).
Furthermore, high transcript levels for ANT2, the
gene encoding one of three isoforms of the
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Mitochondria as targets for detection and treatment of cancer
translocase, have been observed in several de-
differentiated, proliferating, renal tumour cell
types, whereas expression of ANT2 is usually
repressed in quiescent cells (Refs 107, 108).
Additional known or putative MPTP
components also exhibit alterations in gene
expression between normal and cancer cells.
Among those genes overexpressed in cancer cells
are the anti-apoptotic oncogenes encoding Bcl-2
and Bcl-XL, which have a direct inhibitory effect
on pore opening, and genes encoding the
peripheral benzodiazepin receptor (PBR), the
PBR-associated protein Prax-1, and mitochondrial
creatine kinase (Refs 109, 110, 111, 112, 113, 114,
115, 116). Conversely, the expression of BAX, a pro-
apoptotic, inner mitochondrial membrane protein
that facilitates pore opening, has been shown to
be reduced in some cancer cell lines (Refs 117, 118).
However, whether these changes in gene
expression contribute to steady-state differences
in membrane permeability between normal
epithelial and carcinoma cells has yet to be
determined.
Mitochondrial DNA mutations in
carcinoma cells
Mitochondrial dysfunction is one of the most
profound features of cancer cells. Consistently,
mutations in mtDNA have been reported in a
variety of cancers. These include ovarian, thyroid,
salivary, kidney, liver, lung, colon, gastric, brain,
bladder, head and neck, and breast cancers, and
leukaemia (see Table 2 for references). The types
of mutations observed in mtDNA range from
point mutations, to deletions and duplications.
Most tumours contain homoplasmic (100% pure)
mutant mtDNA because of the clonal nature of
cancers (Refs 5, 6, 119).
The abundance and homoplasmic nature of
mitochondria make mtDNA an attractive
molecular marker of cancer. Indeed, mutant
mtDNA in tumour cells is reported to be 220 times
as abundant as a mutated nuclear marker (Ref.
119). Furthermore, a recent study of mtDNA from
patients with bladder, head and neck, and lung
cancers reported that mutated mtDNA is readily
detectable in urine, blood and saliva samples
from these patients (Ref. 119). Thus, mtDNA
mutation might prove to be an extremely useful
biomarker for the detection of many cancers.
Ongoing research on DNA repair genes involved
in maintaining the genetic integrity of the
mitochondrial genome combined with further
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Table 2. Mitochondrial DNA mutation in
cancersa (tab002ksb)
Cancer Refs
Leukaemia 123, 124, 125, 126, 127
Ovary 128
Thyroid 129, 130
Salivary 131, 132
Kidney 133, 134, 135
Liver 136, 137
Lung 138
Colon 6, 139
Gastric 140
Brain 141
Goiter 52
Breast 142
a
Point mutations, deletions or duplications of
mitochondrial DNA mutations are found in a wide
range of cancers.
analysis of the nature of mtDNA mutations will
greatly aid progress in this area.
Mitochondrial dysfunction also has important
implications in cancer therapy. This has been
demonstrated by measuring the cell survival
of a cervical tumour cell line (with parental
mitochondrial function, i.e. Rho + ) and its
derivative isogenic cell line that completely lacked
mtDNA (with dysfunctional mitochondria, i.e.
Rho0) after exposure to a variety of anti-cancer
agents (Ref. 119). It was found that mitochondrial
dysfunction leads to increased cell survival
after exposure to cancer therapeutic agents
such as adriamycin and porphyrin-catalysed
phototoxicity. By contrast, no measurable
difference was found in the cell survival of the
Rho+ and Rho0 cells to high doses of ionising
radiation. These results underscore the
importance of the mitochondrial genome in
development of cancer therapeutic drugs.
Concluding remarks
and clinical implications
The many distinct differences in mitochondrial
structure and function between normal cells and
cancer cells offer a unique potential for the clinical
use of mitochondria as markers for the early
detection of cancer. Additionally, these differences
offer the possibility for the design and synthesis
of effective anti-cancer agents that deliver potent
mitochondrial inhibitors to selectively kill tumour
cells (reviewed in Ref. 120). As summarised
previously, one current chemotherapeutic strategy
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utilises lipophilic cations that accumulate
selectively in carcinoma cells in response to
increased mitochondrial membrane potential.
An alternative strategy employs mitochondrial
protein-import machinery to deliver
macromolecules to mitochondria. For example, a
mitochondrial signal sequence has been used to
direct green fluorescent protein to mitochondria,
which allows the visualisation of mitochondria
within living cells (Ref. 121). Interestingly, certain
short peptides readily penetrate the mitochondrial
membrane and become toxic when internalised
into the targeted cells by disruption of
mitochondrial membranes (Ref. 122). Another
chemotherapeutic strategy employs specific
interaction of drugs with certain mitochondrial
proteins (Ref. 101).
Traditional chemotherapies, aimed at DNA
replication in actively dividing cells, have
achieved only limited success in the treatment of
cancer largely because of their lack of specificity
for cells of tumourigenic origin. It is important,
therefore, to search for novel cellular targets
that are sufficiently different between normal cells
and cancer cells so as to provide a basis for
selective cytotoxicity. As this review suggests, the
mitochondrion is one such target.
Acknowledgements and funding
We thank the members of our laboratories for their
contributions to this article. Our research has been
supported by grants from the National Institutes
of Health (RO1-097714, P50 CA88843, P20
CA86346), an American Heart Association
Scientist Development Award (9939223N) to
K.K.S., and a National Institutes of Health grant
(R15 Ca78323-01S1) to J.S.M-N. We also thank Dr
June R. Aprille, Tufts University, Medford, MA,
USA and Lene J. Rasmussen, Roskilde University,
Roskilde, Denmark for their helpful critique of this
article.
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Mitochondria as targets for detection and treatment of cancer

Further reading, resources and contacts

Wallace, D.C. (1997) Mitochondrial DNA in aging and disease. Sci Am 277, 40-47, PubMed ID: 97388606

Singh, K.K. (1998) Mitochondrial DNA Mutation in Aging, Disease and Cancer, Springer, New York

Scheffler, I.E. (1999) Mitochondria, Wiley-Liss, New York

Naviaux, R.K. (1997) Spectrum of Mitochondrial Diseases, Exceptional Parent, http://

biochemgen.ucsd.edu/mmdc/ep-toc.htm

The Mitochondria Research Society website provides basic information and useful links on mitochonrial
disease for scientists, clinicians and patients.

http://www.mitoresearch.org

Website providing introduction to mitochondrial DNA for students (‘The fire within: the unfolding story of
human mtDNA’):

http://biocrs.biomed.brown.edu/books/essays/mitochondrialDNA.html

The Mitomap database lists polymorphisms and mutations of human mitochondrial DNA.

http://www.gen.emory.edu/mitomap.html

Keshav K. Singh home page:

http://www.jhmi.edu/~kksingh

Features associated with this article

Figures
Figure 1. Schematic of key mitochondrial metabolic pathways (fig001ksb).
Figure 2. Mitochondria of neuron revealed by staining with a rhodamine 123 derivative (fig002ksb).

Tables
Table 1. Mitochondrial diseases (tab001ksb).
Table 2. Mitochondrial DNA mutation in cancers (tab002ksb).

Citation details for this article

Josephine S. Modica-Napolitano and Keshav K. Singh (2002) Mitochondria as targets for detection and
treatment of cancer. Exp. Rev. Mol. Med. 11 April, http://www-ermm.cbcu.cam.ac.uk/02004453h.htm

19
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