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Electron microscopy morphology of the mitochondrial network in human cancer
The International Journal of Biochemistry & Cell Biology 41 (2009) 2062–2068
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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Review
Electron microscopy morphology of the mitochondrial network in human cancer Arismendi-Morillo Gabriel ∗ Laboratorio de Microscopia Electronica-Instituto de Investigaciones Biológicas-Universidad del Zulia, Servicio de Patología-Hospital General del Sur “Dr. Pedro Iturbe”, Maracaibo, Venezuela
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Article history: Available online 13 February 2009 Keywords: Mitochondria Mitochondrial network Cancer Electron microscopy
a b s t r a c t Mitochondria have been implicated in the process of carcinogenesis, which includes alterations of cellular metabolism and cell death pathways. The aim of this review is to describe and analyze the electron microscopy morphology of the mitochondrial network in human cancer. The structural mitochondrial alterations in human tumors are heterogeneous and not specific for any neoplasm. These findings could be representing an altered structural and functional mitochondrial network. The mitochondria in cancer cells, independently of histogenesis, predominantly are seen with lucent-swelling matrix associated with disarrangement and distortion of cristae and partial or total cristolysis and with condensed configuration in minor scale. Mitochondrial changes are associated with mitochondrial-DNA mutations, tumoral microenvironment conditions and mitochondrial fusion–fission disequilibrium. Functionally, the structural alterations suppose the presence of hypoxia-tolerant and hypoxia-sensitive cancer cells. Possibly, hypoxia-tolerant cells are related with mitochondrial condensed appearance and are competent to produce adequate amount of ATP by mitochondrial respiration. Hypoxia-sensitive cells are linked with lucent-swelling and cristolysis mitochondria profile and have an inefficient or null oxidative phosphorylation, which consequently use the glycolytic pathway to generate energy. Additionally, mitochondrial fragmentation is associated with apoptosis; however, alterations in the mitochondrial network are linked with the reduction in sensitivity to apoptosis induces and/or pro-apoptotic conditions. Pharmacological approaches designed to act on both glycolysis and oxidative phosphorylation can be considered as a new approach to selectively kill cancer cells. © 2009 Elsevier Ltd. All rights reserved.
Contents 1.
2. 3. 4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Mitochondrial structure and cell function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Mitochondrial alterations in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1. Which came first, the chicken or the egg? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial electron microscopy features in human tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional implications of mitochondrial pathology in cancer cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potentials applications for treatment of cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction 1.1. Mitochondrial structure and cell function The interest of the scientific community for mitochondria seems to be growing and expanding, since this organelle is involved
∗ Correspondence address: Instituto de Investigaciones Biológicas, Facultad de Medicina, Universidad del Zulia, Postal Code 526, Maracaibo, Venezuela. Tel.: +58 261 7597250; fax: +58 261 7597249. E-mail address: [email protected]. 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.02.002
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not merely in essential pathways of cell metabolisms and energy supply; mitochondria are also the basics in calcium homeostasis, cell-cycle control, development, antiviral responses and cell death. Ultrastructurally, mitochondrion is an organelle constituted by a peripheral and inner membrane. The peripheral membrane encloses the entire contents of the mitochondrion, and internal membrane forms a series of folds, called cristae, which project inward towards the interior space of the organelle. The area between the peripheral and inner membranes is designated as intermembrane space, and the area enclosed by the internal membrane is labeled as mitochondrial matrix. Functionally, the outer membrane includes the apoptosis antagonists and agonists and,
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fission/fusion mitochondrial proteins. The inner membrane contains all the respiratory enzyme complexes and the three electron transporters, necessary for oxidative phosphorylation. In major mammalian tissues, 80–90% of ATP is generated by mitochondria in the process of oxidative phosphorylation (Szewczyk and Wojtczak, 2002; Gilkerson et al., 2003). The mitochondrial matrix contains the enzymatic system of -oxidation and tricarboxylic acid cycle. In the classic electron micrograph, mitochondria appear as solitary and individual organelle. However, recent evidence derived by means of other techniques, supports the idea that mitochondria in living human cells display a large, elongated and branched structures, actually entitled as mitochondrial network (Malka et al., 2004; Benard et al., 2007), extending throughout the cytosol (Amchenkova et al., 1988), and in close contact with the nucleus, the endoplasmic reticulum (Szabadkai et al., 2003), the Golgi complex and the cytoskeleton (Anesti and Scorrano, 2006), and is continually remodeled by both fusion and fission events (Dimmer and Scorrano, 2006; McBride et al., 2006). The mitochondrial morphology is continuously modified by functional requirements to adapt to different cell demands. Mitochondria can exhibit continuous shape changes such as branching, bending and retractions, an increase in the number of cristae or change in their shape and may fuse or increase in size to form giant mitochondria. Mitochondria can be of considerable heterogeneity within a single cell (Collins and Bootmann, 2003). There is accumulating evidence that mitochondrial dynamics are important for many aspects of cellular function (Westermann, 2002). Apparently, the mitochondrial respiration and metabolism may be spatially and temporally regulated by the architecture and positioning of the organelle (McBride et al., 2006). Also, a growing body of evidence suggests that mitochondrial morphology and function is regulated via mostly uncharacterized pathways, by the cytoskeleton (Anesti and Scorrano, 2006). However, the involvement of mitochondrial morphological changes in cell functions is still debated. 1.2. Mitochondrial alterations in cancer 1.2.1. Which came first, the chicken or the egg? The cellular function of mitochondria is reflected in their structure (Westermann, 2002). Consequently, it is logical to consider that if the cancer cell develops abnormal metabolic functions, the mitochondria or mitochondrial network is altered. Defects in mitochondrial function have been suspected to play an important role in the development and progression of cancer (Carew and Huang, 2002; Ohta, 2006). However, it is difficult to know with precision which came first: molecular–structural mitochondrial changes or cancerous transformed cell and microenvironment changes? The answer possibly is both conditions are true and are not exclusive processes. In some cases the primary injury is at molecular–structural level, and in other cases the mitochondrial alterations are derived from the intrinsic conditions of transformed tumoral cell and their microenvironment. There are several facts that address this issue. Some information and interpretations support the first statement, i.e., depletion of mitochondrial DNA in rhoo cells produced functional and morphological changes in mitochondria (Gilkerson et al., 2000; Holmuhamedov et al., 2003). Also, point mutations in mitochondrial DNA are associated with heterogeneous ultrastructural changes of mitochondria (Brantová et al., 2006). The mitochondrial-DNA mutations can initiate a cascade of events leading to a continuous increase in the production of reactive oxygen species (persistent oxidative stress), a condition that probably favors tumor development (Copeland et al., 2002). The mtDNA mutations in tumors may fall into two main classes: (1) severe mutations that inhibit OXPHOS, increase ROS production and promote tumor cell proliferation and (2) milder mutations
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that may permit tumors to adapt to new environments (Brandon et al., 2006). Partial inhibition of oxidative phosphorylation by mitochondrial gene mutations can reduce electron flux through the electron transport chain, increasing mitochondrial reactive oxygen species production, and finally the increased generation of reactive oxygen species mutagenizes nuclear proto-oncogenes and drives nuclear replication, resulting in cancer (Wallace, 2005). Mitochondrial defects have been associated recently with primary hereditary cancers, such as, pheocromocytomas, paragangliomas, leiomyomas and renal cell carcinomas (Eng et al., 2003). Consistent with these facts, mitochondrial dysfunction does appear to be a factor in cancer etiology. However, on the other hand, according to Costello and Franklin (2000), the altered metabolism in cancer cell is probably not the cause of malignancy but, rather, a secondary, albeit essential, adaptation to support malignant activities. In addition, the majority of diverse mtDNA mutations observed in tumors are not important for the process of carcinogenesis or that they play a common oncogenic role (Baysal, 2006). An unequivocal causal link between heritable mitochondrial abnormalities and cancer is provided only by the germ line mutations in the nuclear-encoded genes for succinate dehydrogenase (mitochondrial complex II) and fumarate hydratase (fumarase) (Baysal, 2006). Electron microscopy permits the study of mitochondrial morphology and their overall organization. The aim of this review is to describe and analyze the electron microscopy morphology of the mitochondrial network in human cancer. Probably this represents a contribution to the structural basis of several mitochondrial molecular defects reported in cancer cells that would explain, at least in part, the resistance of malignant solid tumors to conventional chemotherapy.
2. Mitochondrial electron microscopy features in human tumors The observation of mitochondrial ultrastructure by transmission electron microscopy generally shows important differences according to the tissue considered. In cancer, apparently, this consideration is not applicable, since mitochondria exhibit heterogeneous ultrastructural pathology in all kinds of human cancer. Earlier, Menard et al. (1971) reported no major differences in ultrastructure, phosphorylation, rate of substrate oxidation, phosphate ion transport, passive swelling, or cytochrome content between functional mitochondria from normal and neoplastic tissues in mouse mammary gland and mammary adenocarcinoma. Whereas, Gasparre et al. (2007) described an increased mitochondrial mass in five breast carcinoma (mitochondrion-rich tumors). Hackenbrock et al. (1971) presented an orthodox and condensed mitochondrial conformation in ascites tumor cells. Springer (1980) described by means of electron microscopy, the cytoplasmic organelles of 16 human epithelial cell lines derived from normal, nonmalignant tissues of cancerous organs and from colon carcinoma, transitional cell carcinoma of kidney and urethra, carcinosarcoma of breast, pancreatic carcinoma, stomach carcinoma, and metastatic carcinoma. Almost every cell section of the tumor-derived lines had mitochondria showing moderate-to-extensive pleomorphism. Mitochondria displayed outer membrane buckling, cristal disorganization, as well as, mitochondria in which the cristae appear parallel to the long axis of the mitochondrion, grossly altered mitochondrial membranes, matricial myelin figures, matricial vacuoles, and distorted shape. However, in all cases where mitochondrial pleomorphism was observed, normal mitochondria were also visible. In Hürthle cell adenoma of thyroid, an accumulation of numerous irregularly shaped mitochondria, as well as, various degrees of electron density in the matrix of the mitochondria, were reported (Satoh and Yagawa, 1981). Kolosov et al. (1983) described polymorphism
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of mitochondria in endometrioid and serous ovarian adenocarcinomas. Large numbers of very large, swollen and closely packed mitochondria were observed in almost all the tumor cells of malignant papillary cystoadenoma lymphomatosum (Nakashima et al., 1983). Squamous cell carcinoma shows shape, size and structure anomalies of mitochondria (Nicolescu and Eskenasy, 1984). Chordoma exhibits irregularities in mitochondrial sizes and shapes (Sirikulchayanonta and Sriurairatna, 1985). In Warthin’s tumor, mitochondria shows pleomorphism, cup-shaped and concentric ring forms of mitochondria, as well as, aberrant cristae such as the sheaf-like and vesicular types (Kataoka et al., 1991). Human ovarian carcinoma cells revealed major alterations in the cristae structure, for instance, thicker and irregular cristae and absent cristae in many mitochondria (Andrews and Albright, 1992). Salivary duct carcinoma of the parotid gland display a cell type contained numerous mitochondria and other cell with scant organelles (Yoshihara et al., 1994). Bornstein et al. (1996) reported that the ultrastructural characteristics of mitochondria in adrenal cortical adenoma appear to be potential markers for the differentiation of steroid-producing adenomas, since, in aldosterone-producing adrenal cortical adenomas, there was a large number of elongated tubular mitochondria with characteristic bridging of inner membranes, producing a lamellar-type pattern; in cortisol-producing adenomas, mitochondria are large and round with vesicular or tubulovesicular inner membranes surrounded by a characteristic dilated smooth endoplasmic reticulum. Finally, in progesterone-producing adenoma, mitochondria enlarges in a lamellar fashion with bright matrix and reduced number of inner membranes. Clear-cell carcinoma shows swollen or unusually large mitochondria (Kwon et al., 1996). Undifferentiated retinoblastoma showed cytoplasmic scant organelles and mitochondrial swelling following a high degree of hypoxia (Bosun, 1997). In extraskeletal myxoid chondrosarcoma, prominent mitochondria were characterized, whereas, the skeletal myxoid chondrosarcoma revealed inconspicuous organelles (Antonescu et al., 1998). In osteosarcoma cell lines, Gilkerson et al. (2000) reported a reduction in the amount of cristal membranes, often prompting the remaining cristae to adopt a circular appearance in the mitochondrial interior. Many mitochondria–rough endoplasmic reticulum complexes were present in acinar cell carcinoma of the pan˜ creas (Ordónez and Mackay, 2000). Electron microscopy revealed that mitochondria are barely identifiable in hepatocellular carcinoma, and remnants of mitochondria or mitochondrial ghosts were described (Cuezva et al., 2002). Mitochondrial structure of HeLa cells grown in galactose showed a consistent increase in matrix density due to condensation of the mitochondrial matrix and expansion of the cristal spaces (condensed transformation), while during glycolysis, they maintain an orthodox state (Rossignol et al., 2004). In chromophobe renal cell carcinoma, the mitochondria with tubulovesicular cristae fashion and budding from the outer mitochondrial membrane are highly characteristic (Moreno et al., 2005). In mesothelioma with clear cell features marked mitochon˜ drial swelling were seen (Ordonez, 2005). Silent adenoma subtype 3 of the pituitary shows unevenly clustered mitochondria (Horvath et al., 2005). Mitochondria in human leukemia cells are abnormally swollen, with pale matrix and disorganized cristae, these cells were severely deficient in mitochondrial respiration and more active in glucose uptake and lactate accumulation (Xu et al., 2005). Volante et al. (2006) reported that in the majority of cases of nonfunctioning oncocytic endocrine tumors of the pancreas, numerous mitochondria were observed; in addition, nearly 50% of the cases were clinically aggressive. In the case of oncocytic carcinoma in buccal mucosa, numerous dilated mitochondria were described (Sugiyama et al., 2006). In pediatric and adult hepatic embryonal sarcomas dilated mitochondrial and mitochondria–rough endoplasmic reticulum complexes and intracytoplasmic fat droplets were seen
Fig. 1. An area of human astrocytic tumor that shows a micro-vessel (left side) contending condensed mitochondria and neoplastic cells (right side) that exhibits multiple swelling mitochondria of different size, and several degrees of cristae disarrangement and cristolysis. ×12,000.
(Agaram et al., 2006). Kummoona (2007) described degenerated mitochondria in jaw lymphomas. In human pancreatic cancer cell line treated with oleandrin shows mitochondrial condensation and translocation to a perinuclear position accompanied by vacuoles (Newman et al., 2007). Oncocytic thyroid tumors are composed of large cells rich in closely packed, swollen mitochondria (Gasparre et al., 2007). In contrast, cultured cells from oncocytic lesions retained only a few large mitochondria with frequently observed secondary lysosomal structures. They conclude that disruptive mutations in complex I subunits are markers of thyroid oncocytic tumors. In fibrillary astrocytomas, anaplastic astrocytoma, and glioblastoma multiforme, mitochondrial swelling-associated disarrangement of cristae and partial or total cristolysis were the most constant ultrastructural findings. Also, mitochondria show variability in the abnormalities in number, size and shape, included in the same specimen, as well as, the degree of severity of internal ultrastructural mitochondrial changes. Mitochondria with dense matrix were seen. The mitochondria were localized predominantly in cellular bodies, close to nuclear membrane and rough endoplasmic reticulum. Lipid droplets between or near to the mitochondria were seen. On the contrary, in cell processes the presence of mitochondria was inconspicuous. The “normal” rod-shaped mitochondria with clearly defined and closely apposed outer membranes and intact cristae organized perpendicular to the long axis of the mitochondrion were observed occasionally. In pilocytic astrocytomas as well as in undifferentiated neoplastic cells particularly, in addition to swelling mitochondria, mitochondria with increased thickness and remarkably electron dense cristae was seen. The presence of enlarged mitochondria was characterized. In a few mitochondria folds of inner mitochondrial membranes were seen. Matricial condensation and existence of vacuoles, as well as scarce mitochondria with onion-like structure were also seen. Mitochondrial fusion–fission phenomena and presence of amorphous matricial densities were categorized (Arismendi-Morillo and Castellano-Ramirez, 2008). Chatterjee et al. (2008) reported decrease in mitochondria amount in hairy cell leukemia after treatment with cladribine. Undoubtedly, the structural mitochondrial alterations in human tumors are heterogeneous and not specific for any neoplasm (Fig. 1). In my opinion, these findings are associated with several aspects: first, a possible reason is the frequent presence of mutations and instability of mitochondrial DNA in cancer. The deletion of mitochondrial DNA produces functional and morphological changes in mitochondria (Gilkerson et al., 2000). Human mitochondrial DNA encodes 13 polypeptide components of the respiratory chain and, therefore, in rhoo cells, the oxidative phosphorylation machinery is incompletely assembled (Logan, 2006). This fact has a dramatic
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Fig. 2. Two swelling mitochondria inside of human astrocytoma cell that exhibits electron-lucent matrix and total and partial cristolysis. ×40,000.
effect on the internal structure of the mitochondria: the cristal membranes are greatly reduced and disorganized, yet the inner boundary membrane remains visibly unaltered (Gilkerson et al., 2000) (see Fig. 2). Mutations in mitochondrial DNA have been identified in breast, ovarian, colorectal, gastric, hepatic, esophageal, pancreatic, renal, prostate, brain, thyroid, bladder, head and neck, lung, hematological cancers (Carew and Huang, 2002). The presence of mitochondrial-DNA mutations in cancer cells is consistent with the intrinsic susceptibility of mitochondrial DNA to damage and constitutive oxidative stress (Carew and Huang, 2002); second, one of the major conceptual advances in oncology over the last decade has been the appreciation that all major aspects of cancer biology are influenced by the tumor microenvironment (Melillo and Semenza, 2006). The tumoral microenvironment is characterized by hypoxia and acidosis (Brown and Giaccia, 1998). The mitochondria are affected by variations in the energy substrates and cell physiological state. Consequentially, the variations in intra-tumoral conditions would be an added factor. The presence of hypoxic cells in solid tumors has long been recognized. In the tumors are present a state of inadequate oxygen and nutrients supply to tumoral cells, especially those that are distant from functional blood vessels (Minchinton and Tannock, 2006). A gradient of decreasing tumor cells proliferation forms with increasing distance from tumor blood vessel, in parallel with decreasing nutrient and oxygen concentration (Minchinton and Tannock, 2006). The mitochondrial swelling with distortion of the cristae is associated with hypoxic–ischemic conditions (Steinbach et al., 2003) (see Fig. 2). Therefore, is possible that the most important and conspicuous mitochondrial changes exist within deeper layers of cells; third, mitochondrial shape, size, and number are controlled by the dynamically opposing processes of fission and fusion (Logan, 2006), apparently, mitochondrial fission is predominant in tumoral cells (Arismendi-Morillo and Castellano-Ramirez, 2008), since mitochondrial fusion in mammalian cells requires an intact mitochondrial inner membrane potential (Mattenberger et al., 2003). Mitochondrial fragmentation is the result of excessive fission, and small, punctuate mitochondria can be derived from mitochondrial fission (Bereiter-Hahn et al., 2008). 3. Functional implications of mitochondrial pathology in cancer cell Mitochondria have major roles in bioenergetics and vital signaling of the mammalian cell. This organelle has been implicated in the process of carcinogenesis, which includes alterations of cellular metabolism and cell death pathways. Indeed, the structural mitochondrial alterations in human tumors are heterogeneous and not specific for any neoplasm. On the other hand, in almost all cases
Fig. 3. (A) Several undifferentiated human astrocytic tumoral cells that show several condensed mitochondria characterized by remarkably electron dense mitochondrial matrix and cristae. ×20,000. (B) Higher magnification of a condensed mitochondrion. ×70,000.
normal mitochondria are present. In the first place, the heterogeneous ultrastructural pathology could be representing an altered mitochondrial network. This finding possibly implies a strong inhibition of mitochondrial energy production (Benard et al., 2007). In addition, mitochondrial swelling with partial or total cristolysis suggests that the ability of neoplastic cells to generate ATP by mitochondrial oxidative phosphorylation would be diminished. Early, Warburg (1956) initially posited that mitochondrial oxidative phosphorylation was defective in tumor cells, and that this initial insult led to gradual and compensatory increases in glycolytic ATP production as the central event of the cell transformation. An altered oxidative phosphorylation is one of the determinants that underlie the abnormal aerobic glycolysis of the cancer cell (Lopez-Rios et al., 2007). It has been a long-held belief that this glycolytic phenotype is due to cancer-specific defects in mitochondrial oxidative phosphorylation (Bui and Thompson, 2006). The mitochondria in cancer cells, independently of histogenesis, are seen with dense matrix or condensed configuration (Fig. 3) and with lucent-swelling matrix associated with disarrangement and distortion of cristae and partial or total cristolysis (Fig. 4). The mitochondrial dense appearance is a functional form; hence produces energy by oxidative phosphorylation (Hackenbrock et al., 1971; Ikrenyi et al., 1976; Rossignol et al., 2004). Possibly, cells with dense mitochondria are hypoxia-tolerant, therefore, are able to generate sufficient ATP by oxidative phosphorylation. In contrast, the mitochondria with lucent-swelling matrix associated with disarrangement and distortion of cristae and partial or total cristolysis,
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Fig. 4. (A) Sector of a human astrocytoma cell which demonstrates swelling mitochondria that exhibit partial or total cristolysis. ×15,000. (B) Higher magnification of a mitochondrion with electron-lucent matrix, subtotal cristolysis and the presence of amorphous matricial densities. ×50,000.
are hypoxia-sensitive cells, and subsequently, are incompetent to produce adequate amount of ATP by mitochondrial respiration. Mitochondria in cancer cells are often relatively resistant to the induction of mitochondrial membrane permeabilization, which is the rate-limiting step of the intrinsic pathway of apoptosis (Rustin and Kroemer, 2007). The resistance of cancer mitochondria against apoptosis-associated membrane permeabilization and the altered contribution of these organelles to metabolism are closely related (Kroemer and Pouyssegur, 2008). One of the steps in apoptosis is the mitochondrial fragmentation. Recent reports have described dramatic alterations in mitochondrial morphology during the early stages of apoptotic cell death, a fragmentation of the mitochondrial network and the remodeling of the cristae and the proteins associated with the regulation of apoptosis have been shown to affect mitochondrial ultrastructure (Karbowski and Youle, 2003). Fragmentation of the mitochondrial network appears to occur in situations where the mitochondrial inner membrane potential is abnormal (Benard et al., 2007) and in response to oxidative phosphorylation impairment (Ishihara et al., 2006). However, fragmentation of mitochondria per se does not evoke apoptosis (Bereiter-Hahn et al., 2008). Cuezva et al. (2002) point out that cancer cells with low bioenergetic index would be prone to more resistance to apoptosis in response to oxidative stress.
Fig. 5. (A) Mitochondria in human astrocytic tumor that exhibits an onion-like structure composed of five concentric layers of double leaflets of inner mitochondrial membrane. ×50,000. (B) Mitochondria in human astrocytoma cell that displays an onion-like structure constituted by three concentric layers of double leaflets of inner mitochondrial membrane, note the dilated mitochondrial matrix occupied by lipoidic-like material. ×34,000.
Finally, apparently in cancer cells the presence of mitochondria that display a few concentric layers of double leaflets of inner mitochondrial membrane contained inside a continuous envelope of outer membrane (onion ring-like structure) (see Fig. 5) is very scarce. This mitochondrial morphology is linked with the loss of subunit e or g of the mitochondrial ATP synthase in yeast cells (Paumard et al., 2002; Arselin et al., 2004). The ATP synthaseassociated subunits e and g are indispensable for the biogenesis of the mitochondrial cristae (Paumard et al., 2002). The full loss of subunit e or g decreased the mitochondrial ATPase activity by 50% (Arselin et al., 2004); however, it is not sufficient to affect growth by oxidative phosphorylation (Mukhopadhyay et al., 1994). Possibly, in the case of the cancer cells, the contribution to energy metabolism of this rare mitochondrial conformation is not substantial, due to the amount and their apparently diminished energy production because of defective oxidative phosphorylation. 4. Potentials applications for treatment of cancer Cancer-associated mitochondrial alterations and bioenergetics may be taken advantage of for the development of antineoplastic drugs (Kroemer, 2006). An approach would target glycolysis and/or revert the Warburg phenomenon. The glycolytic inhibitors are particularly effective against cancer cells with mitochondrial defects or under hypoxic conditions, which are frequently associated with
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cellular resistance to conventional anticancer treatments and radiation therapy (Pelicano et al., 2006; Chen et al., 2007). Inhibition of glycolysis effectively kills colon cancer cells and lymphoma cells in a hypoxic environment; in addition, depletion of ATP by glycolytic inhibition also potentially induces apoptosis in multidrug-resistant cells (Xu et al., 2005). In accordance with Noble and Dietrich (2002), tumors cannot even be treated as a homogeneous mass of identical cells. This statement is very important because a neoplasm is constituted by hypoxia-tolerant and hypoxia-sensitive cells. Griguer et al. (2005) described oxidative phosphorylation-dependent cells and glycolytic-dependent cells in human and mouse malignant gliomas cell lines. Therefore, the glycolysis inhibition would be effective in hypoxia-sensitive cells. While, the hypoxia-tolerant cells potentially represent a sensible target to inhibition or down-regulation of mitochondrial respiration, given that mitochondrial insult or failure can rapidly lead to the inhibition of cell survival and proliferation (Dorward et al., 1997; Dias and Bailly, 2005). These considerations possibly imply plasticity of tumor metabolism. Fantin et al. (2006) reported that most tumor cells have a substantial reserve capacity to produce ATP by oxidative phosphorylation when glycolysis is suppressed. On the other hand, recent findings suggest that forcing cancer cells into mitochondrial metabolism efficiently suppresses cancer growth, and that impaired mitochondrial respiration may even have a role in metastatic process (Ristow, 2006). In view of these facts, a mixed therapy emerged as the most appropriate one. Indeed, pharmacological approaches designed to act on both glycolysis and oxidative phosphorylation can be considered as a new approach to selectively kill cancer cells (Griguer et al., 2007; Arismendi-Morillo and Castellano-Ramirez, 2008).
5. Conclusions Mitochondria exhibit heterogeneous and not specific ultrastructural pathology in all kinds of human cancer. These are associated with the frequent presence of mutations and instability of mitochondrial DNA in cancer since the deletion of mitochondrial DNA produces functional and morphological changes in mitochondria; variations within intra-tumoral conditions related with the energy substrates and cancer cell physiological state; and with mitochondrial fragmentation as a result of excessive fission. The heterogeneous ultrastructural pathology could be representing an altered mitochondrial network that possibly implies that almost all cancer cells have a strong impediment for obtaining ATP through mitochondrial respiration and, therefore generate energy by the glycolytic pathway, and consequently exist hypoxia-tolerant and hypoxia-sensitive cancer cells. A mixed therapy emerged as the most appropriate one. Finally, tumors cannot even be treated as a homogeneous mass of identical cells, and for these reasons correspondingly designed pharmacological strategies to act on both glycolysis and oxidative phosphorylation can be considered as a new tactic to selectively kill cancer cells.
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Review
Electron microscopy morphology of the mitochondrial network in human cancer Arismendi-Morillo Gabriel ∗ Laboratorio de Microscopia Electronica-Instituto de Investigaciones Biológicas-Universidad del Zulia, Servicio de Patología-Hospital General del Sur “Dr. Pedro Iturbe”, Maracaibo, Venezuela
a r t i c l e
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Article history: Available online 13 February 2009 Keywords: Mitochondria Mitochondrial network Cancer Electron microscopy
a b s t r a c t Mitochondria have been implicated in the process of carcinogenesis, which includes alterations of cellular metabolism and cell death pathways. The aim of this review is to describe and analyze the electron microscopy morphology of the mitochondrial network in human cancer. The structural mitochondrial alterations in human tumors are heterogeneous and not specific for any neoplasm. These findings could be representing an altered structural and functional mitochondrial network. The mitochondria in cancer cells, independently of histogenesis, predominantly are seen with lucent-swelling matrix associated with disarrangement and distortion of cristae and partial or total cristolysis and with condensed configuration in minor scale. Mitochondrial changes are associated with mitochondrial-DNA mutations, tumoral microenvironment conditions and mitochondrial fusion–fission disequilibrium. Functionally, the structural alterations suppose the presence of hypoxia-tolerant and hypoxia-sensitive cancer cells. Possibly, hypoxia-tolerant cells are related with mitochondrial condensed appearance and are competent to produce adequate amount of ATP by mitochondrial respiration. Hypoxia-sensitive cells are linked with lucent-swelling and cristolysis mitochondria profile and have an inefficient or null oxidative phosphorylation, which consequently use the glycolytic pathway to generate energy. Additionally, mitochondrial fragmentation is associated with apoptosis; however, alterations in the mitochondrial network are linked with the reduction in sensitivity to apoptosis induces and/or pro-apoptotic conditions. Pharmacological approaches designed to act on both glycolysis and oxidative phosphorylation can be considered as a new approach to selectively kill cancer cells. © 2009 Elsevier Ltd. All rights reserved.
Contents 1.
2. 3. 4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Mitochondrial structure and cell function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Mitochondrial alterations in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1. Which came first, the chicken or the egg? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial electron microscopy features in human tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional implications of mitochondrial pathology in cancer cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potentials applications for treatment of cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction 1.1. Mitochondrial structure and cell function The interest of the scientific community for mitochondria seems to be growing and expanding, since this organelle is involved
∗ Correspondence address: Instituto de Investigaciones Biológicas, Facultad de Medicina, Universidad del Zulia, Postal Code 526, Maracaibo, Venezuela. Tel.: +58 261 7597250; fax: +58 261 7597249. E-mail address: [email protected]. 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.02.002
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not merely in essential pathways of cell metabolisms and energy supply; mitochondria are also the basics in calcium homeostasis, cell-cycle control, development, antiviral responses and cell death. Ultrastructurally, mitochondrion is an organelle constituted by a peripheral and inner membrane. The peripheral membrane encloses the entire contents of the mitochondrion, and internal membrane forms a series of folds, called cristae, which project inward towards the interior space of the organelle. The area between the peripheral and inner membranes is designated as intermembrane space, and the area enclosed by the internal membrane is labeled as mitochondrial matrix. Functionally, the outer membrane includes the apoptosis antagonists and agonists and,
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fission/fusion mitochondrial proteins. The inner membrane contains all the respiratory enzyme complexes and the three electron transporters, necessary for oxidative phosphorylation. In major mammalian tissues, 80–90% of ATP is generated by mitochondria in the process of oxidative phosphorylation (Szewczyk and Wojtczak, 2002; Gilkerson et al., 2003). The mitochondrial matrix contains the enzymatic system of -oxidation and tricarboxylic acid cycle. In the classic electron micrograph, mitochondria appear as solitary and individual organelle. However, recent evidence derived by means of other techniques, supports the idea that mitochondria in living human cells display a large, elongated and branched structures, actually entitled as mitochondrial network (Malka et al., 2004; Benard et al., 2007), extending throughout the cytosol (Amchenkova et al., 1988), and in close contact with the nucleus, the endoplasmic reticulum (Szabadkai et al., 2003), the Golgi complex and the cytoskeleton (Anesti and Scorrano, 2006), and is continually remodeled by both fusion and fission events (Dimmer and Scorrano, 2006; McBride et al., 2006). The mitochondrial morphology is continuously modified by functional requirements to adapt to different cell demands. Mitochondria can exhibit continuous shape changes such as branching, bending and retractions, an increase in the number of cristae or change in their shape and may fuse or increase in size to form giant mitochondria. Mitochondria can be of considerable heterogeneity within a single cell (Collins and Bootmann, 2003). There is accumulating evidence that mitochondrial dynamics are important for many aspects of cellular function (Westermann, 2002). Apparently, the mitochondrial respiration and metabolism may be spatially and temporally regulated by the architecture and positioning of the organelle (McBride et al., 2006). Also, a growing body of evidence suggests that mitochondrial morphology and function is regulated via mostly uncharacterized pathways, by the cytoskeleton (Anesti and Scorrano, 2006). However, the involvement of mitochondrial morphological changes in cell functions is still debated. 1.2. Mitochondrial alterations in cancer 1.2.1. Which came first, the chicken or the egg? The cellular function of mitochondria is reflected in their structure (Westermann, 2002). Consequently, it is logical to consider that if the cancer cell develops abnormal metabolic functions, the mitochondria or mitochondrial network is altered. Defects in mitochondrial function have been suspected to play an important role in the development and progression of cancer (Carew and Huang, 2002; Ohta, 2006). However, it is difficult to know with precision which came first: molecular–structural mitochondrial changes or cancerous transformed cell and microenvironment changes? The answer possibly is both conditions are true and are not exclusive processes. In some cases the primary injury is at molecular–structural level, and in other cases the mitochondrial alterations are derived from the intrinsic conditions of transformed tumoral cell and their microenvironment. There are several facts that address this issue. Some information and interpretations support the first statement, i.e., depletion of mitochondrial DNA in rhoo cells produced functional and morphological changes in mitochondria (Gilkerson et al., 2000; Holmuhamedov et al., 2003). Also, point mutations in mitochondrial DNA are associated with heterogeneous ultrastructural changes of mitochondria (Brantová et al., 2006). The mitochondrial-DNA mutations can initiate a cascade of events leading to a continuous increase in the production of reactive oxygen species (persistent oxidative stress), a condition that probably favors tumor development (Copeland et al., 2002). The mtDNA mutations in tumors may fall into two main classes: (1) severe mutations that inhibit OXPHOS, increase ROS production and promote tumor cell proliferation and (2) milder mutations
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that may permit tumors to adapt to new environments (Brandon et al., 2006). Partial inhibition of oxidative phosphorylation by mitochondrial gene mutations can reduce electron flux through the electron transport chain, increasing mitochondrial reactive oxygen species production, and finally the increased generation of reactive oxygen species mutagenizes nuclear proto-oncogenes and drives nuclear replication, resulting in cancer (Wallace, 2005). Mitochondrial defects have been associated recently with primary hereditary cancers, such as, pheocromocytomas, paragangliomas, leiomyomas and renal cell carcinomas (Eng et al., 2003). Consistent with these facts, mitochondrial dysfunction does appear to be a factor in cancer etiology. However, on the other hand, according to Costello and Franklin (2000), the altered metabolism in cancer cell is probably not the cause of malignancy but, rather, a secondary, albeit essential, adaptation to support malignant activities. In addition, the majority of diverse mtDNA mutations observed in tumors are not important for the process of carcinogenesis or that they play a common oncogenic role (Baysal, 2006). An unequivocal causal link between heritable mitochondrial abnormalities and cancer is provided only by the germ line mutations in the nuclear-encoded genes for succinate dehydrogenase (mitochondrial complex II) and fumarate hydratase (fumarase) (Baysal, 2006). Electron microscopy permits the study of mitochondrial morphology and their overall organization. The aim of this review is to describe and analyze the electron microscopy morphology of the mitochondrial network in human cancer. Probably this represents a contribution to the structural basis of several mitochondrial molecular defects reported in cancer cells that would explain, at least in part, the resistance of malignant solid tumors to conventional chemotherapy.
2. Mitochondrial electron microscopy features in human tumors The observation of mitochondrial ultrastructure by transmission electron microscopy generally shows important differences according to the tissue considered. In cancer, apparently, this consideration is not applicable, since mitochondria exhibit heterogeneous ultrastructural pathology in all kinds of human cancer. Earlier, Menard et al. (1971) reported no major differences in ultrastructure, phosphorylation, rate of substrate oxidation, phosphate ion transport, passive swelling, or cytochrome content between functional mitochondria from normal and neoplastic tissues in mouse mammary gland and mammary adenocarcinoma. Whereas, Gasparre et al. (2007) described an increased mitochondrial mass in five breast carcinoma (mitochondrion-rich tumors). Hackenbrock et al. (1971) presented an orthodox and condensed mitochondrial conformation in ascites tumor cells. Springer (1980) described by means of electron microscopy, the cytoplasmic organelles of 16 human epithelial cell lines derived from normal, nonmalignant tissues of cancerous organs and from colon carcinoma, transitional cell carcinoma of kidney and urethra, carcinosarcoma of breast, pancreatic carcinoma, stomach carcinoma, and metastatic carcinoma. Almost every cell section of the tumor-derived lines had mitochondria showing moderate-to-extensive pleomorphism. Mitochondria displayed outer membrane buckling, cristal disorganization, as well as, mitochondria in which the cristae appear parallel to the long axis of the mitochondrion, grossly altered mitochondrial membranes, matricial myelin figures, matricial vacuoles, and distorted shape. However, in all cases where mitochondrial pleomorphism was observed, normal mitochondria were also visible. In Hürthle cell adenoma of thyroid, an accumulation of numerous irregularly shaped mitochondria, as well as, various degrees of electron density in the matrix of the mitochondria, were reported (Satoh and Yagawa, 1981). Kolosov et al. (1983) described polymorphism
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of mitochondria in endometrioid and serous ovarian adenocarcinomas. Large numbers of very large, swollen and closely packed mitochondria were observed in almost all the tumor cells of malignant papillary cystoadenoma lymphomatosum (Nakashima et al., 1983). Squamous cell carcinoma shows shape, size and structure anomalies of mitochondria (Nicolescu and Eskenasy, 1984). Chordoma exhibits irregularities in mitochondrial sizes and shapes (Sirikulchayanonta and Sriurairatna, 1985). In Warthin’s tumor, mitochondria shows pleomorphism, cup-shaped and concentric ring forms of mitochondria, as well as, aberrant cristae such as the sheaf-like and vesicular types (Kataoka et al., 1991). Human ovarian carcinoma cells revealed major alterations in the cristae structure, for instance, thicker and irregular cristae and absent cristae in many mitochondria (Andrews and Albright, 1992). Salivary duct carcinoma of the parotid gland display a cell type contained numerous mitochondria and other cell with scant organelles (Yoshihara et al., 1994). Bornstein et al. (1996) reported that the ultrastructural characteristics of mitochondria in adrenal cortical adenoma appear to be potential markers for the differentiation of steroid-producing adenomas, since, in aldosterone-producing adrenal cortical adenomas, there was a large number of elongated tubular mitochondria with characteristic bridging of inner membranes, producing a lamellar-type pattern; in cortisol-producing adenomas, mitochondria are large and round with vesicular or tubulovesicular inner membranes surrounded by a characteristic dilated smooth endoplasmic reticulum. Finally, in progesterone-producing adenoma, mitochondria enlarges in a lamellar fashion with bright matrix and reduced number of inner membranes. Clear-cell carcinoma shows swollen or unusually large mitochondria (Kwon et al., 1996). Undifferentiated retinoblastoma showed cytoplasmic scant organelles and mitochondrial swelling following a high degree of hypoxia (Bosun, 1997). In extraskeletal myxoid chondrosarcoma, prominent mitochondria were characterized, whereas, the skeletal myxoid chondrosarcoma revealed inconspicuous organelles (Antonescu et al., 1998). In osteosarcoma cell lines, Gilkerson et al. (2000) reported a reduction in the amount of cristal membranes, often prompting the remaining cristae to adopt a circular appearance in the mitochondrial interior. Many mitochondria–rough endoplasmic reticulum complexes were present in acinar cell carcinoma of the pan˜ creas (Ordónez and Mackay, 2000). Electron microscopy revealed that mitochondria are barely identifiable in hepatocellular carcinoma, and remnants of mitochondria or mitochondrial ghosts were described (Cuezva et al., 2002). Mitochondrial structure of HeLa cells grown in galactose showed a consistent increase in matrix density due to condensation of the mitochondrial matrix and expansion of the cristal spaces (condensed transformation), while during glycolysis, they maintain an orthodox state (Rossignol et al., 2004). In chromophobe renal cell carcinoma, the mitochondria with tubulovesicular cristae fashion and budding from the outer mitochondrial membrane are highly characteristic (Moreno et al., 2005). In mesothelioma with clear cell features marked mitochon˜ drial swelling were seen (Ordonez, 2005). Silent adenoma subtype 3 of the pituitary shows unevenly clustered mitochondria (Horvath et al., 2005). Mitochondria in human leukemia cells are abnormally swollen, with pale matrix and disorganized cristae, these cells were severely deficient in mitochondrial respiration and more active in glucose uptake and lactate accumulation (Xu et al., 2005). Volante et al. (2006) reported that in the majority of cases of nonfunctioning oncocytic endocrine tumors of the pancreas, numerous mitochondria were observed; in addition, nearly 50% of the cases were clinically aggressive. In the case of oncocytic carcinoma in buccal mucosa, numerous dilated mitochondria were described (Sugiyama et al., 2006). In pediatric and adult hepatic embryonal sarcomas dilated mitochondrial and mitochondria–rough endoplasmic reticulum complexes and intracytoplasmic fat droplets were seen
Fig. 1. An area of human astrocytic tumor that shows a micro-vessel (left side) contending condensed mitochondria and neoplastic cells (right side) that exhibits multiple swelling mitochondria of different size, and several degrees of cristae disarrangement and cristolysis. ×12,000.
(Agaram et al., 2006). Kummoona (2007) described degenerated mitochondria in jaw lymphomas. In human pancreatic cancer cell line treated with oleandrin shows mitochondrial condensation and translocation to a perinuclear position accompanied by vacuoles (Newman et al., 2007). Oncocytic thyroid tumors are composed of large cells rich in closely packed, swollen mitochondria (Gasparre et al., 2007). In contrast, cultured cells from oncocytic lesions retained only a few large mitochondria with frequently observed secondary lysosomal structures. They conclude that disruptive mutations in complex I subunits are markers of thyroid oncocytic tumors. In fibrillary astrocytomas, anaplastic astrocytoma, and glioblastoma multiforme, mitochondrial swelling-associated disarrangement of cristae and partial or total cristolysis were the most constant ultrastructural findings. Also, mitochondria show variability in the abnormalities in number, size and shape, included in the same specimen, as well as, the degree of severity of internal ultrastructural mitochondrial changes. Mitochondria with dense matrix were seen. The mitochondria were localized predominantly in cellular bodies, close to nuclear membrane and rough endoplasmic reticulum. Lipid droplets between or near to the mitochondria were seen. On the contrary, in cell processes the presence of mitochondria was inconspicuous. The “normal” rod-shaped mitochondria with clearly defined and closely apposed outer membranes and intact cristae organized perpendicular to the long axis of the mitochondrion were observed occasionally. In pilocytic astrocytomas as well as in undifferentiated neoplastic cells particularly, in addition to swelling mitochondria, mitochondria with increased thickness and remarkably electron dense cristae was seen. The presence of enlarged mitochondria was characterized. In a few mitochondria folds of inner mitochondrial membranes were seen. Matricial condensation and existence of vacuoles, as well as scarce mitochondria with onion-like structure were also seen. Mitochondrial fusion–fission phenomena and presence of amorphous matricial densities were categorized (Arismendi-Morillo and Castellano-Ramirez, 2008). Chatterjee et al. (2008) reported decrease in mitochondria amount in hairy cell leukemia after treatment with cladribine. Undoubtedly, the structural mitochondrial alterations in human tumors are heterogeneous and not specific for any neoplasm (Fig. 1). In my opinion, these findings are associated with several aspects: first, a possible reason is the frequent presence of mutations and instability of mitochondrial DNA in cancer. The deletion of mitochondrial DNA produces functional and morphological changes in mitochondria (Gilkerson et al., 2000). Human mitochondrial DNA encodes 13 polypeptide components of the respiratory chain and, therefore, in rhoo cells, the oxidative phosphorylation machinery is incompletely assembled (Logan, 2006). This fact has a dramatic
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Fig. 2. Two swelling mitochondria inside of human astrocytoma cell that exhibits electron-lucent matrix and total and partial cristolysis. ×40,000.
effect on the internal structure of the mitochondria: the cristal membranes are greatly reduced and disorganized, yet the inner boundary membrane remains visibly unaltered (Gilkerson et al., 2000) (see Fig. 2). Mutations in mitochondrial DNA have been identified in breast, ovarian, colorectal, gastric, hepatic, esophageal, pancreatic, renal, prostate, brain, thyroid, bladder, head and neck, lung, hematological cancers (Carew and Huang, 2002). The presence of mitochondrial-DNA mutations in cancer cells is consistent with the intrinsic susceptibility of mitochondrial DNA to damage and constitutive oxidative stress (Carew and Huang, 2002); second, one of the major conceptual advances in oncology over the last decade has been the appreciation that all major aspects of cancer biology are influenced by the tumor microenvironment (Melillo and Semenza, 2006). The tumoral microenvironment is characterized by hypoxia and acidosis (Brown and Giaccia, 1998). The mitochondria are affected by variations in the energy substrates and cell physiological state. Consequentially, the variations in intra-tumoral conditions would be an added factor. The presence of hypoxic cells in solid tumors has long been recognized. In the tumors are present a state of inadequate oxygen and nutrients supply to tumoral cells, especially those that are distant from functional blood vessels (Minchinton and Tannock, 2006). A gradient of decreasing tumor cells proliferation forms with increasing distance from tumor blood vessel, in parallel with decreasing nutrient and oxygen concentration (Minchinton and Tannock, 2006). The mitochondrial swelling with distortion of the cristae is associated with hypoxic–ischemic conditions (Steinbach et al., 2003) (see Fig. 2). Therefore, is possible that the most important and conspicuous mitochondrial changes exist within deeper layers of cells; third, mitochondrial shape, size, and number are controlled by the dynamically opposing processes of fission and fusion (Logan, 2006), apparently, mitochondrial fission is predominant in tumoral cells (Arismendi-Morillo and Castellano-Ramirez, 2008), since mitochondrial fusion in mammalian cells requires an intact mitochondrial inner membrane potential (Mattenberger et al., 2003). Mitochondrial fragmentation is the result of excessive fission, and small, punctuate mitochondria can be derived from mitochondrial fission (Bereiter-Hahn et al., 2008). 3. Functional implications of mitochondrial pathology in cancer cell Mitochondria have major roles in bioenergetics and vital signaling of the mammalian cell. This organelle has been implicated in the process of carcinogenesis, which includes alterations of cellular metabolism and cell death pathways. Indeed, the structural mitochondrial alterations in human tumors are heterogeneous and not specific for any neoplasm. On the other hand, in almost all cases
Fig. 3. (A) Several undifferentiated human astrocytic tumoral cells that show several condensed mitochondria characterized by remarkably electron dense mitochondrial matrix and cristae. ×20,000. (B) Higher magnification of a condensed mitochondrion. ×70,000.
normal mitochondria are present. In the first place, the heterogeneous ultrastructural pathology could be representing an altered mitochondrial network. This finding possibly implies a strong inhibition of mitochondrial energy production (Benard et al., 2007). In addition, mitochondrial swelling with partial or total cristolysis suggests that the ability of neoplastic cells to generate ATP by mitochondrial oxidative phosphorylation would be diminished. Early, Warburg (1956) initially posited that mitochondrial oxidative phosphorylation was defective in tumor cells, and that this initial insult led to gradual and compensatory increases in glycolytic ATP production as the central event of the cell transformation. An altered oxidative phosphorylation is one of the determinants that underlie the abnormal aerobic glycolysis of the cancer cell (Lopez-Rios et al., 2007). It has been a long-held belief that this glycolytic phenotype is due to cancer-specific defects in mitochondrial oxidative phosphorylation (Bui and Thompson, 2006). The mitochondria in cancer cells, independently of histogenesis, are seen with dense matrix or condensed configuration (Fig. 3) and with lucent-swelling matrix associated with disarrangement and distortion of cristae and partial or total cristolysis (Fig. 4). The mitochondrial dense appearance is a functional form; hence produces energy by oxidative phosphorylation (Hackenbrock et al., 1971; Ikrenyi et al., 1976; Rossignol et al., 2004). Possibly, cells with dense mitochondria are hypoxia-tolerant, therefore, are able to generate sufficient ATP by oxidative phosphorylation. In contrast, the mitochondria with lucent-swelling matrix associated with disarrangement and distortion of cristae and partial or total cristolysis,
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Fig. 4. (A) Sector of a human astrocytoma cell which demonstrates swelling mitochondria that exhibit partial or total cristolysis. ×15,000. (B) Higher magnification of a mitochondrion with electron-lucent matrix, subtotal cristolysis and the presence of amorphous matricial densities. ×50,000.
are hypoxia-sensitive cells, and subsequently, are incompetent to produce adequate amount of ATP by mitochondrial respiration. Mitochondria in cancer cells are often relatively resistant to the induction of mitochondrial membrane permeabilization, which is the rate-limiting step of the intrinsic pathway of apoptosis (Rustin and Kroemer, 2007). The resistance of cancer mitochondria against apoptosis-associated membrane permeabilization and the altered contribution of these organelles to metabolism are closely related (Kroemer and Pouyssegur, 2008). One of the steps in apoptosis is the mitochondrial fragmentation. Recent reports have described dramatic alterations in mitochondrial morphology during the early stages of apoptotic cell death, a fragmentation of the mitochondrial network and the remodeling of the cristae and the proteins associated with the regulation of apoptosis have been shown to affect mitochondrial ultrastructure (Karbowski and Youle, 2003). Fragmentation of the mitochondrial network appears to occur in situations where the mitochondrial inner membrane potential is abnormal (Benard et al., 2007) and in response to oxidative phosphorylation impairment (Ishihara et al., 2006). However, fragmentation of mitochondria per se does not evoke apoptosis (Bereiter-Hahn et al., 2008). Cuezva et al. (2002) point out that cancer cells with low bioenergetic index would be prone to more resistance to apoptosis in response to oxidative stress.
Fig. 5. (A) Mitochondria in human astrocytic tumor that exhibits an onion-like structure composed of five concentric layers of double leaflets of inner mitochondrial membrane. ×50,000. (B) Mitochondria in human astrocytoma cell that displays an onion-like structure constituted by three concentric layers of double leaflets of inner mitochondrial membrane, note the dilated mitochondrial matrix occupied by lipoidic-like material. ×34,000.
Finally, apparently in cancer cells the presence of mitochondria that display a few concentric layers of double leaflets of inner mitochondrial membrane contained inside a continuous envelope of outer membrane (onion ring-like structure) (see Fig. 5) is very scarce. This mitochondrial morphology is linked with the loss of subunit e or g of the mitochondrial ATP synthase in yeast cells (Paumard et al., 2002; Arselin et al., 2004). The ATP synthaseassociated subunits e and g are indispensable for the biogenesis of the mitochondrial cristae (Paumard et al., 2002). The full loss of subunit e or g decreased the mitochondrial ATPase activity by 50% (Arselin et al., 2004); however, it is not sufficient to affect growth by oxidative phosphorylation (Mukhopadhyay et al., 1994). Possibly, in the case of the cancer cells, the contribution to energy metabolism of this rare mitochondrial conformation is not substantial, due to the amount and their apparently diminished energy production because of defective oxidative phosphorylation. 4. Potentials applications for treatment of cancer Cancer-associated mitochondrial alterations and bioenergetics may be taken advantage of for the development of antineoplastic drugs (Kroemer, 2006). An approach would target glycolysis and/or revert the Warburg phenomenon. The glycolytic inhibitors are particularly effective against cancer cells with mitochondrial defects or under hypoxic conditions, which are frequently associated with
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cellular resistance to conventional anticancer treatments and radiation therapy (Pelicano et al., 2006; Chen et al., 2007). Inhibition of glycolysis effectively kills colon cancer cells and lymphoma cells in a hypoxic environment; in addition, depletion of ATP by glycolytic inhibition also potentially induces apoptosis in multidrug-resistant cells (Xu et al., 2005). In accordance with Noble and Dietrich (2002), tumors cannot even be treated as a homogeneous mass of identical cells. This statement is very important because a neoplasm is constituted by hypoxia-tolerant and hypoxia-sensitive cells. Griguer et al. (2005) described oxidative phosphorylation-dependent cells and glycolytic-dependent cells in human and mouse malignant gliomas cell lines. Therefore, the glycolysis inhibition would be effective in hypoxia-sensitive cells. While, the hypoxia-tolerant cells potentially represent a sensible target to inhibition or down-regulation of mitochondrial respiration, given that mitochondrial insult or failure can rapidly lead to the inhibition of cell survival and proliferation (Dorward et al., 1997; Dias and Bailly, 2005). These considerations possibly imply plasticity of tumor metabolism. Fantin et al. (2006) reported that most tumor cells have a substantial reserve capacity to produce ATP by oxidative phosphorylation when glycolysis is suppressed. On the other hand, recent findings suggest that forcing cancer cells into mitochondrial metabolism efficiently suppresses cancer growth, and that impaired mitochondrial respiration may even have a role in metastatic process (Ristow, 2006). In view of these facts, a mixed therapy emerged as the most appropriate one. Indeed, pharmacological approaches designed to act on both glycolysis and oxidative phosphorylation can be considered as a new approach to selectively kill cancer cells (Griguer et al., 2007; Arismendi-Morillo and Castellano-Ramirez, 2008).
5. Conclusions Mitochondria exhibit heterogeneous and not specific ultrastructural pathology in all kinds of human cancer. These are associated with the frequent presence of mutations and instability of mitochondrial DNA in cancer since the deletion of mitochondrial DNA produces functional and morphological changes in mitochondria; variations within intra-tumoral conditions related with the energy substrates and cancer cell physiological state; and with mitochondrial fragmentation as a result of excessive fission. The heterogeneous ultrastructural pathology could be representing an altered mitochondrial network that possibly implies that almost all cancer cells have a strong impediment for obtaining ATP through mitochondrial respiration and, therefore generate energy by the glycolytic pathway, and consequently exist hypoxia-tolerant and hypoxia-sensitive cancer cells. A mixed therapy emerged as the most appropriate one. Finally, tumors cannot even be treated as a homogeneous mass of identical cells, and for these reasons correspondingly designed pharmacological strategies to act on both glycolysis and oxidative phosphorylation can be considered as a new tactic to selectively kill cancer cells.
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