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Expert Reviews in Molecular Medicine: http://www.expertreviews.org/
Accession information: (02)00445-3h.htm (shortcode: txt001ksb); 11 April 2002
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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.
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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’, 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
mm in length and 0.5–1 mm
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 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 FADH2 generated through the TCA cycle) to the synthesis of ATP. Substrate oxidation involves a series of 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 FADH2-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 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. b-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 one of four respiratory
enzyme complexes localised to the inner mitochondrial membrane. They include
seven subunits of respiratory enzyme complex I, one subunit of complex III,
three subunits of complex IV, and two subunits of complex V. All other mitochondrial
proteins, including those involved in the replication, transcription and translation
of mtDNA, are encoded by nuclear genes and are targeted to the mitochondrion
by a specific transport system (Ref. 20). In humans and other
mammals, mitochondrial genes display a maternal inheritance (i.e. are inherited
from the female parent). This is probably because the number of mtDNA copies
in the egg is typically 103-fold greater than that in the sperm (Ref. 21).
Alternatively, paternal genomes and organelles might be preferentially degraded
in the zygote (Refs 22, 23).
Although the mitochondrial and nuclear genomes are physically distinct, the high degree of functional interdependence between them is suggestive of a host–parasite relationship. The ‘endosymbiont’ theory proposes that early in the evolution of the eukaryote, a primitive proto-eukaryote cell that was incapable of aerobic respiration served as host to a eubacterium with the unique capacity for oxidative metabolism (Ref. 24). During the early stages of this endosymbiotic association, the eubacterium retained its genetic autonomy. In time, however, most of its genetic material was transferred to the nuclear genome of the host. The resulting mitochondrion retained only those few (i.e. 13) genes encoding polypeptides that are essential to aerobic ATP production yet have a hydrophobicity that precludes nuclear synthesis and cytoplasmic transport to mitochondria. It is of interest to recall Altman’s perceptive characterisation of mitochondrial function, and his suggestion of a colonial association between the newly discovered ‘bioblasts’ and the host cell within which they reside.
Mitochondria
and disease
The first mitochondrial
disease was described by Rolf Luft in 1962 (Ref. 25), a year
prior to the discovery of mtDNA (Ref. 26). At the genetic
level, mitochondrial disease was not definitively described until 1988 with
the identification of mtDNA mutations in patients with mitochondrial myopathies,
as well as the molecular genetic characterisation of patients with Leber’s
hereditary optic neuropathy and familial mitochondrial encephalomyopathy (Refs
27, 28, 29). Since then,
there has been a steady growth in the list of diseases associated with mitochondrial
dysfunction arising from mtDNA mutations (Table 1).
Although mtDNA represents less than 1% of total cellular DNA, its gene products are essential for normal cell function. Unlike nuclear DNA, mammalian mtDNA contains no introns, has no protective histones and is exposed to deleterious 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 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-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 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 mg/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).
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, 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.
Mechanism of action
of DLCs
It is of interest to note that, although all DLCs are taken up into mitochondria
by a common 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 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, 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 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 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 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 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 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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| Further
reading, resources and contacts Wallace, D.C. (1997) Mitochondrial DNA in aging and disease. Sci Am 277, 40-47, PubMed 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, The Mitochondria Research Society website provides basic information and useful links on mitochonrial disease for scientists, clinicians and patients. Website providing introduction to mitochondrial DNA for students (‘The fire within: the unfolding story of human mtDNA’):
The Mitomap database lists polymorphisms and mutations of human mitochondrial DNA. Keshav K. Singh home page: |
|
Features associated with this article
Figures Figure 2. Mitochondria of neuron revealed by staining with a rhodamine
123 derivative Tables Table 2. Mitochondrial
DNA mutation in cancers |
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