Information

Could the Warburg effect be used to starve cancer cells in situ?

Could the Warburg effect be used to starve cancer cells in situ?



We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

What is wrong with the following chain of reasoning?

  1. Nearly all cancer cells rely on high rates of glucose uptake (upto 200 times more than normal cells). This is known as the the Warburg effect.
  2. There is only one cell type in the human body that is obligated to use glucose - red blood cells (erythrocytes).
  3. Starve cancer cells of their only energy source by inducing hypoglycaemia in the patient, preserving red blood cells using, for example, erythrocytapheresis or an oxygen-carrying substitute.

I understand that to completely eliminate a tumour such a regime would need to be in place for an extended period, but would like to know why such a treatment isn't feasible in theory?


The human body needs energy (ATP) for even the most basic function. I guess when you decrease glucose levels (which is broken down for ATP), you will be in affect taking away the ability of the body to function normally by depriving it of energy. This is my theory so I would be happy to if someone with more information on the topic could tell me if I am right or wrong.


As a type 1 diabetic for 39 years I can only say that hypoglycaemia is a life threatening state which any of us tries to avoid as much as possible.

  1. Any untreated episode of hypoglycaemia can easily lead to diabetic coma and irreversible brain damage.
  2. When encountering low blood sugar, the organism anyway releases sugar stored in liver just for that case, so prolonged artificially induced hypoglycemia would place a lot (i imagine unsustainable) stress on liver. Not sure, but I suspect it would lead to liver failure.

PH-Responsive Charge-Conversional and Hemolytic Activities of Magnetic Nanocomposite Particles for Cell-Targeted Hyperthermia

Publication History

  • Received 4 December 2017
  • Accepted 16 January 2018
  • Published online 25 January 2018
  • Published in issue 31 January 2018
Article Views
Altmetric
Citations

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.


Introduction

It is now well established that significant heterogeneity exists among human breast cancers. This heterogeneity is observable at every level of examination from the macroscopic to the molecular. Recent large-scale efforts to measure and describe human breast tumor heterogeneity include The Cancer Genome Atlas (TCGA) where a number of high-throughput ‘omic’ technologies were systemically applied to hundreds of primary cancer specimens [1]. Mutation, germ line polymorphisms, DNA copy number, RNA expression, DNA methylation, and protein expression analyses were performed in parallel on a large and carefully curated set of breast cancer specimens to produce the most comprehensive molecular portrait of the disease to date.

One significant metric that was not included in TGCA was an unbiased analysis of tumor metabolism. While metabolic flux cannot be measured in fixed or frozen specimens, steady-state levels of numerous key metabolites may provide insight into these fundamental phenotypic traits. A number of studies in cancer have uncovered relationships between genetic abnormalities and various metabolic reprogramming suggesting that key metabolic process can be altered as a result of specific transformation events [2]-[6]. Relatively nonspecific cancer-related events such as increased proliferation may also underlie some of the inferred/observed metabolic remodeling. Glucose uptake, serine and glutamine auxotrophy, mitochondrial oxidative phosphorylation, and cancer-associated fibroblasts all appear to have roles in defining breast cancer metabolism [7]-[11]. However, it is not clear whether these regulatory relationships can be observed in all or subsets of human tumors.

Breast cancers are broadly categorized as luminal versus basal types possibly derived from different precursor cells or at least different committed lineages [12]-[14]. Within these broad categories, alterations in specific driver genes are believed to produce the heterogeneity observed amongst and within breast tumor subtypes. While the identity and frequency of driver alterations are generally different in basal and luminal cancers, there is still considerable overlap. For example, TP53 mutations are very common in basal tumors and PI3KCA mutations are common in luminal cancer but neither is subtype exclusive. In contrast, MYC (8q24) amplification is common in both types [1]. Each of these genetic drivers has been associated with specific changes in cellular metabolism and therefore may have dominant effects that can be observed across tumor types.

Metabolomic profiling via mass spectrometry or nuclear magnetic resonance (NMR) is now an established approach that has been employed in several studies to analyze primary human breast tissues (normal and cancer) [15],[16]. Building upon transcriptional profiling of breast cancer, there have also been several efforts to integrate steady-state metabolite levels with specific breast cancer subtypes defined by mRNA expression. Expression subtypes are dominated by estrogen receptor and ERBB2 status and thus, metabolic profiling was performed to seek an additional level of information to refine these existing classifications. These analyses identified a subclassification of luminal A-type cancers based on metabolite levels and found higher levels of Warburg-associated metabolites in more aggressive cancer types [9],[17]. A separate study of breast cancer lipidomic identified the association between palmitate-containing phosphatidylcholines with estrogen receptor negative and cancer progression and patient survival [18]. However, none of these studies established associations of particular metabolites or metabolic pathways with specific somatic mutations or expression levels that have been extensively characterized in TCGA.

In order to more fully explore the relationship between genetics, tumor type, and metabolic state, we took advantage of our participation in the breast cancer TCGA to perform joint analyses of metabolomics and genetics in a series of primary cancers. From the current study, we were able to identify several genetic determinants of the metabolic heterogeneity of human breast tumors that confirm and extend prior in vitro and in vivo observations.


Rewiring the System

Cancer as a metabolic disease

Thomas N Seyfried and Laura M Shelton

Biology Department, Boston College, Chestnut Hill, MA 02467, USA

author email corresponding author email

Nutrition & Metabolism 2010, 7:7doi:10.1186/1743-7075-7-7

The electronic version of this article is the complete one and can be found online at: http://www.nutritionandmetabolism.com/content/7/1/7

Received: 15 November 2009
Accepted: 27 January 2010
Published: 27 January 2010
© 2010 Seyfried and Shelton licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Emerging evidence indicates that impaired cellular energy metabolism is the defining characteristic of nearly all cancers regardless of cellular or tissue origin. In contrast to normal cells, which derive most of their usable energy from oxidative phosphorylation, most cancer cells become heavily dependent on substrate level phosphorylation to meet energy demands. Evidence is reviewed supporting a general hypothesis that genomic instability and essentially all hallmarks of cancer, including aerobic glycolysis (Warburg effect), can be linked to impaired mitochondrial function and energy metabolism. A view of cancer as primarily a metabolic disease will impact approaches to cancer management and prevention.

Cancer is a complex disease involving numerous tempo-spatial changes in cell physiology, which ultimately lead to malignant tumors. Abnormal cell growth (neoplasia) is the biological endpoint of the disease. Tumor cell invasion of surrounding tissues and distant organs is the primary cause of morbidity and mortality for most cancer patients. The biological process by which normal cells are transformed into malignant cancer cells has been the subject of a large research effort in the biomedical sciences for many decades. Despite this research effort, cures or long-term management strategies for metastatic cancer are as challenging today as they were 40 years ago when President Richard Nixon declared a war on cancer [1,2].

Confusion surrounds the origin of cancer. Contradictions and paradoxes have plagued the field [3-6]. Without a clear idea on cancer origins, it becomes difficult to formulate a clear strategy for effective management. Although very specific processes underlie malignant transformation, a large number of unspecific influences can initiate the disease including radiation, chemicals, viruses, inflammation, etc. Indeed, it appears that prolonged exposure to almost any provocative agent in the environment can potentially cause cancer [7,8]. That a very specific process could be initiated in very unspecific ways was considered “the oncogenic paradox” by Szent-Gyorgyi [8]. This paradox has remained largely unresolved [7].

In a landmark review, Hanahan and Weinberg suggested that six essential alterations in cell physiology could underlie malignant cell growth [6]. These six alterations were described as the hallmarks of nearly all cancers and included, 1) self-sufficiency in growth signals, 2) insensitivity to growth inhibitory (antigrowth) signals, 3) evasion of programmed cell death (apoptosis), 4) limitless replicative potential, 5) sustained vascularity (angiogenesis), and 6) tissue invasion and metastasis. Genome instability, leading to increased mutability, was considered the essential enabling characteristic for manifesting the six hallmarks [6]. However, the mutation rate for most genes is low making it unlikely that the numerous pathogenic mutations found in cancer cells would occur sporadically within a normal human lifespan [7]. This then created another paradox. If mutations are such rare events, then how is it possible that cancer cells express so many different types and kinds of mutations?

The loss of genomic “caretakers” or “guardians”, involved in sensing and repairing DNA damage, was proposed to explain the increased mutability of tumor cells [7,9]. The loss of these caretaker systems would allow genomic instability thus enabling pre-malignant cells to reach the six essential hallmarks of cancer [6]. It has been difficult, however, to define with certainty the origin of pre-malignancy and the mechanisms by which the caretaker/guardian systems themselves are lost during the emergent malignant state [5,7].

In addition to the six recognized hallmarks of cancer, aerobic glycolysis or the Warburg effect is also a robust metabolic hallmark of most tumors [10-14]. Although no specific gene mutation or chromosomal abnormality is common to all cancers [7,15-17], nearly all cancers express aerobic glycolysis, regardless of their tissue or cellular origin. Aerobic glycolysis in cancer cells involves elevated glucose uptake with lactic acid production in the presence of oxygen. This metabolic phenotype is the basis for tumor imaging using labeled glucose analogues and has become an important diagnostic tool for cancer detection and management [18-20]. Genes for glycolysis are overexpressed in the majority of cancers examined [21,22].

The origin of the Warburg effect in tumor cells has been controversial. The discoverer of this phenomenon, Otto Warburg, initially proposed that aerobic glycolysis was an epiphenomenon of a more fundamental problem in cancer cell physiology, i.e., impaired or damaged respiration [23,24]. An increased glycolytic flux was viewed as an essential compensatory mechanism of energy production in order to maintain the viability of tumor cells. Although aerobic glycolysis and anaerobic glycolysis are similar in that lactic acid is produced under both situations, aerobic glycolysis can arise in tumor cells from damaged respiration whereas anaerobic glycolysis arises from the absence of oxygen. As oxygen will reduce anaerobic glycolysis and lactic acid production in most normal cells (Pasteur effect), the continued production of lactic acid in the presence of oxygen can represent an abnormal Pasteur effect. This is the situation in most tumor cells. Only those body cells able to increase glycolysis during intermittent respiratory damage were considered capable of forming cancers [24]. Cells unable to elevate glycolysis in response to respiratory insults, on the other hand, would perish due to energy failure. Cancer cells would therefore arise from normal body cells through a gradual and irreversible damage to their respiratory capacity. Aerobic glycolysis, arising from damaged respiration, is the single most common phenotype found in cancer.

Based on metabolic data collected from numerous animal and human tumor samples, Warburg proposed with considerable certainty and insight that irreversible damage to respiration was the prime cause of cancer [23-25]. Warburg’s theory, however, was attacked as being too simplistic and not consistent with evidence of apparent normal respiratory function in some tumor cells [26-34]. The theory did not address the role of tumor-associated mutations, the phenomenon of metastasis, nor did it link the molecular mechanisms of uncontrolled cell growth directly to impaired respiration. Indeed, Warburg’s biographer, Hans Krebs, mentioned that Warburg’s idea on the primary cause of cancer, i.e., the replacement of respiration by fermentation (glycolysis), was only a symptom of cancer and not the cause [35]. The primary cause was assumed to be at the level of gene expression. The view of cancer as a metabolic disease was gradually displaced with the view of cancer as a genetic disease. While there is renewed interest in the energy metabolism of cancer cells, it is widely thought that the Warburg effect and the metabolic defects expressed in cancer cells arise primarily from genomic mutability selected during tumor progression [36-39]. Emerging evidence, however, questions the genetic origin of cancer and suggests that cancer is primarily a metabolic disease.

Our goal is to revisit the argument of tumor cell origin and to provide a general hypothesis that genomic mutability and essentially all hallmarks of cancer, including the Warburg effect, can be linked to impaired respiration and energy metabolism. In brief, damage to cellular respiration precedes and underlies the genome instability that accompanies tumor development. Once established, genome instability contributes to further respiratory impairment, genome mutability, and tumor progression. In other words, effects become causes. This hypothesis is based on evidence that nuclear genome integrity is largely dependent on mitochondrial energy homeostasis and that all cells require a constant level of useable energy to maintain viability. While Warburg recognized the centrality of impaired respiration in the origin of cancer, he did not link this phenomenon to what are now recognize as the hallmarks of cancer. We review evidence that make these linkages and expand Warburg’s ideas on how impaired energy metabolism can be exploited for tumor management and prevention.

Energetics of the living cell

In order for cells to remain viable and to perform their genetically programmed functions they must produce usable energy. This energy is commonly stored in ATP and is released during the hydrolysis of the terminal phosphate bond. This is generally referred to as the free energy of ATP hydrolysis [40-42]. The standard energy of ATP hydrolysis under physiological conditions is known as ΔG’ATP and is tightly regulated in all cells between -53 to -60 kJ/mol [43]. Most of this energy is used to power ionic membrane pumps [10,40]. In cells with functional mitochondria, this energy is derived mostly from oxidative phosphorylation where approximately 88% of total cellular energy is produced (about 28/32 total ATP molecules). The other approximate 12% of energy is produced about equally from substrate level phosphorylation through glycolysis in the cytoplasm and through the TCA cycle in the mitochondrial matrix (2 ATP molecules each). Veech and co-workers showed that the ΔG’ATP of cells was empirically formalized and measurable through the energies of ion distributions via the sodium pump and its linked transporters [42]. The energies of ion distributions were explained in terms of the Gibbs-Donnan equilibrium, which was essential for producing electrical, concentration, and pressure work.

A remarkable finding was the similarity of the ΔG’ATP among cells with widely differing resting membrane potentials and mechanisms of energy production. For example, the ΔG’ATP in heart, liver, and erythrocytes was approximately – 56 kJ/mol despite having very different electrical potentials of – 86, – 56, and – 6 mV, respectively [42]. Moreover, energy production in heart and liver, which contain many mitochondria, is largely through respiration, whereas energy production in the erythrocyte, which contains no nucleus or mitochondria, is entirely through glycolysis. Warburg also showed that the total energy production in quiescent kidney and liver cells was remarkably similar to that produced in proliferating cancer cells [24]. Despite the profound differences in resting potentials and in mechanisms of energy production among these disparate cell types, they all require a similar amount of total energy to remain viable.

The constancy of the ΔG’ATP of approximately -56 kJ/mol is fundamental to cellular homeostasis and its relationship to cancer cell energy is pivotal. The maintenance of the ΔG’ATP is the “end point” of both genetic and metabolic processes and any disturbance in this energy level will compromise cell function and viability [40]. Cells can die from either too little or too much energy. Too little energy will lead to cell death by either necrotic or apoptotic mechanisms, whereas over production of ATP, a polyanionic Donnan active material, will disrupt the Gibbs-Donnan equilibrium, alter the function of membrane pumps, and inhibit respiration and viability [42]. Glycolysis or glutaminolysis must increase in cells suffering mitochondrial impairment in order to maintain an adequate ΔG’ATP for viability. This fact was clearly illustrated in showing that total cellular energy production was essentially the same in respiration-normal and respiration-deficient fibroblasts [44].

In addition to its role in replenishing TCA cycle intermediates (anaplerosis), glutamine can also provide energy through stimulation of glycolysis in the cytoplasm and through substrate level phosphorylation in the TCA cycle (glutaminolysis) [45-49]. Energy obtained through substrate level phosphorylation in the TCA cycle can compensate for deficiencies in either glycolysis or oxidative phosphorylation [46,48,50], and can represent a major source of energy for the glutamine-dependent cancers. More energy is produced through substrate level phosphorylation in cancer cells than in normal cells, which produce most of their energy through oxidative phosphorylation. A major difference between normal cells and cancer cells is in the origin of the energy produced rather than in the amount of energy produced since approximately -56 kJ/mol is the amount of energy required for cell survival regardless of whether cells are quiescent or proliferating or are mostly glycolytic or respiratory. It is important to recognize, however, that a prolonged reliance on substrate level phosphorylation for energy production produces genome instability, cellular disorder, and increased entropy, i.e., characteristics of cancer [8,24].

Mitochondrial function in cancer cells

Considerable controversy has surrounded the issue of mitochondrial function in cancer cells [18,29,30,33,34,51-57]. Sidney Weinhouse and Britton Chance initiated much of this controversy through their critical evaluation of the Warburg theory and the role of mitochondrial function [33,34]. Basically, Weinhouse felt that quantitatively and qualitatively normal carbon and electron transport could occur in cancer cells despite the presence of elevated glycolysis [33,34]. Weinhouse assumed that oxygen consumption and CO2 production were indicative of coupled respiration. However, excessive amounts of Donnan active material (ATP) would be produced if elevated glycolysis were expressed together with coupled respiration [42]. Accumulation of Donnan active material will induce cell swelling and produce a physiological state beyond the Gibbs-Donnan equilibrium. The occurrence of up-regulated glycolysis together with normal coupled respiration is incompatible with metabolic homeostasis and cell viability. Chance and Hess also argued against impaired respiration in cancer based on their spectrophotometric studies showing mostly normal electron transfer in ascites tumor cells [58]. These studies, however, failed to assess the level of ATP production as a consequence of normal electron transfer and did not exclude the possibility of elevated ATP production through TCA cycle substrate level phosphorylation. As discussed below, mitochondrial uncoupling can give the false impression of functional respiratory capacity.

Oxygen uptake and CO2 production can occur in mitochondria that are uncoupled and/or dysfunctional [24,59]. While reduced oxygen uptake can be indicative of reduced oxidative phosphorylation, increased oxygen uptake may or may not be indicative of increased oxidative phosphorylation and ATP production [59-62]. Ramanathan and co-workers showed that oxygen consumption was greater, but oxygen dependent (aerobic) ATP synthesis was less in cells with greater tumorigenic potential than in cells with lower tumorigenic potential [61]. These findings are consistent with mitochondrial uncoupling in tumor cells. It was for these types of observations in other systems that Warburg considered the phenomenon of aerobic glycolysis as too capricious to serve as a reliable indicator of respiratory status [24]. Heat production is also greater in poorly differentiated high glycolytic tumor cells than in differentiated low glycolytic cells [63]. Heat production is consistent with mitochondrial uncoupling in these highly tumorigenic cells. Although Burk, Schade, Colowick and others convincingly dispelled the main criticisms of the Warburg theory [55,57,64], citations to the older arguments for normal respiration in cancer cells persist in current discussions of the subject.

Besides glucose, glutamine can also serve as a major energy metabolite for some cancers [65-67]. Glutamine is often present in high concentrations in culture media and serum. Cell viability and growth can be maintained from energy generated through substrate level phosphorylation in the TCA cycle using glutamine as a substrate [47,48]. Energy obtained through this pathway could give the false impression of normal oxidative phosphorylation, as oxygen consumption and CO2 production can arise from glutaminolysis and uncoupled oxidative phosphorylation. Hence, evidence suggesting that mitochondrial function is normal in cancer cells should be considered with caution unless data are provided, which exclude substrate level phosphorylation through glutaminolysis or glycolysis as alternative sources of energy.

Mitochondrial dysfunction in cancer cells

Numerous studies show that tumor mitochondria are structurally and functionally abnormal and incapable of generating normal levels of energy [10,60,61,68-74]. Recent evidence also shows that the in vitro growth environment alters the lipid composition of mitochondrial membranes and electron transport chain function [75]. Moreover, the mitochondrial lipid abnormalities induced from the in vitro growth environment are different from the lipid abnormalities found between normal tissue and tumors that are grown in vivo. It appears that the in vitro growth environment reduces Complex I activity and obscures the boundaries of the Crabtree and the Warburg effects. The Crabtree effect involves the inhibition of respiration by high levels of glucose [76,77], whereas the Warburg effect involves inhibition of respiration from impaired oxidative phosphorylation. While the Crabtree effect is reversible, the Warburg effect is largely irreversible. Similarities in mitochondrial lipids found between lung epidermoid carcinoma and fetal lung cells are also consistent with respiratory defects in tumor cells [78]. The bioenergetic capacity of mitochondria is dependent to a large extent on the content and composition of mitochondrial lipids.

Alterations in mitochondrial membrane lipids and especially the inner membrane enriched lipid, cardiolipin, disrupt the mitochondrial proton motive gradient (ΔΨm) thus inducing protein-independent uncoupling with concomitant reduction in respiratory energy production [41,73,79-82]. Cancer cells contain abnormalities in cardiolipin content or composition, which are associated with electron transport abnormalities [73]. Cardiolipin is the only lipid synthesized almost exclusively in the mitochondria. Proteins of the electron transport chain evolved to function in close association with cardiolipin. Besides altering the function of most electron transport chain complexes including the F1-ATPase, abnormalities in cardiolipin content and composition can also inhibit uptake of ADP through the adenine nucleotide transporter thus altering the efficiency of oxidative phosphorylation [41,79-81,83]. Abnormalities in the content and composition of cardiolipin will also prevent oxidation of the coenzyme Q couple thus producing reactive oxygen species during tumor progression [73,84]. Increased ROS production can impair genome stability, tumor suppressor gene function, and control over cell proliferation [7,85]. Hence, abnormalities in CL can alter cancer cell respiration in numerous ways.

Cardiolipin abnormalities in cancer cells can arise from any number of unspecific influences to include damage from mutagens and carcinogens, radiation, low level hypoxia, inflammation, ROS, or from inherited mutations that alter mitochondrial energy homeostasis [73]. Considering the dynamic behavior of mitochondria involving regular fusions and fissions [86], abnormalities in mitochondrial lipid composition and especially of cardiolipin could be rapidly disseminated throughout the cellular mitochondrial network and could even be passed along to daughter cells somatically, through cytoplasmic inheritance.

Besides lipidomic evidence supporting the Warburg cancer theory [73], recent studies from Cuezva and colleagues also provide compelling proteomic evidence supporting the theory [21]. Their results showed a drop in the β-F1-ATPase/Hsp60 ratio concurrent with an upregulation of the glyceraldehyde-3-phosphate dehydrogenase potential in most common human tumors [72]. These and other observations indicate that the bioenergetic capacity of tumor cells is largely defective [87-89]. Viewed collectively, the bulk of the experimental evidence indicates that mitochondria structure and function is abnormal in cancer cells. Hence, mitochondrial dysfunction will cause cancer cells to rely more heavily than non-cancer cells on substrate level phosphorylation for energy production in order to maintain membrane pump function and cell viability.

Linking genome instability to mitochondrial dysfunction

Is it genomic instability or is it impaired energy metabolism that is primarily responsible for the origin of cancer? This is more than an academic question, as the answer will impact approaches to cancer management and prevention. Metabolic studies in a variety of human cancers previously showed that that loss of mitochondrial function preceded the appearance of malignancy and aerobic glycolysis [90]. However, the general view over the last 50 years has been that gene mutations and chromosomal abnormalities underlie most aspects of tumor initiation and progression including the Warburg effect and impaired respiratory function. The gene theory of cancer would argue that mitochondrial dysfunction is an effect rather than a cause of cancer, whereas the metabolic impairment theory would argue the reverse. If gene mutations are the primary cause of cancer then the disease can be considered etiologically complicated requiring multiple solutions for management and prevention. This comes from findings that the numbers and types of mutations differ markedly among and within different types of tumors. If, on the other hand, impaired energy metabolism is primarily responsible for cancer, then most cancers can be considered a type of metabolic disease requiring fewer and less complicated solutions.

Although mitochondrial function and oxidative phosphorylation is impaired in tumor cells, it remains unclear how these impairments relate to carcinogenesis and to the large number of somatic mutations and chromosomal abnormalities found in tumors [7,15,91-93]. Most inherited “inborn errors of metabolism” do not specifically compromise mitochondrial function or cause cancer in mammals. There are some exceptions, however, as germ-line mutations in genes encoding proteins of the TCA cycle can increase risk to certain human cancers [94]. For example, risk for paraganglioma involves mutations in the succinate dehydrogenase gene, whereas risk for leiomyomatosis and renal cell carcinoma involves mutations in the fumarate hydratase (fumarase) gene [94-97]. These and similar mutations directly impair mitochondrial energy production leading to increased glycolysis and the Warburg effect [98]. Although rare inherited mutations in the p53 tumor suppressor gene can increase risk for some familial cancers of the Li Fraumeni syndrome [99], most p53 defects found in cancers are not inherited and appear to arise sporadically, as do the vast majority of cancer-associated mutations [6,7,100]. In general, cancer-causing germline mutations are rare and contribute to only about 5-7% of all cancers [5,7]. While germline mutations can cause a few cancers, most cancer mutations are somatic and will contribute more to the progression than to the origin of most cancers.

The cancer mutator phenotype was invoked to explain the large number of somatic mutations found in cancer, but mutations in the p53 caretaker gene are not expressed in all cancers nor does p53 deletion produce cancer in mice suggesting a more complicated involvement of this and other genome guardians in carcinogenesis [7,101-104]. While numerous genetic abnormalities have been described in most human cancers, no specific mutation is reliably diagnostic for any specific type of tumor [7,17,105]. On the other hand, few if any tumors are known, which express normal respiration.

Retrograde response and genomic instability

As an alternative to the genome guardian hypothesis for the origin of somatic mutations, a persistent retrograde response can underlie the genomic instability and mutability of tumor cells. The retrograde (RTG) response is the general term for mitochondrial signaling and involves cellular responses to changes in the functional state of mitochondria [106-110]. Although the RTG response has been most studied in yeast, mitochondrial stress signaling is an analogous response in mammalian cells [110,111]. Expression of multiple nuclear genes controlling energy metabolism is profoundly altered following impairment in mitochondrial energy homeostasis [112,113]. Mitochondrial impairment can arise from abnormalities in mtDNA, the TCA cycle, the electron transport chain, or in the proton motive gradient (ΔΨm) of the inner membrane. Any impairment in mitochondrial energy production can trigger an RTG response. The RTG response evolved in yeast to maintain cell viability following periodic disruption of mitochondrial ATP production [110,114]. This mostly involves an energy transition from oxidative phosphorylation to substrate level phosphorylation. Similar systems are also expressed in mammalian cells [110-113]. Prolonged or continued activation of the retrograde response, however, can have dire consequences on nuclear genome stability and function.

Three main regulatory elements define the RTG response in yeast to include the Rtg2 signaling protein, and the Rtg1/Rtg-3 transcriptional factor complex (both are basic helix-loop-helix-leucine zippers) [110]. Rtg2 contains an N-terminal ATP binding motif that senses changes in mitochondrial ATP production. Rtg2 also regulates the function and cellular localization of the heterodimeric Rtg1/Rtg-3 complex (Figure 1). The RTG response is “off” in healthy cells with normal mitochondrial function. In the off state, the Rtg1/Rtg3 complex is sequestered in the cytoplasm with Rtg1 attached (dimerized) to a highly phosphorylated form of Rtg3 [110]. Besides its role in the cytoplasm as an energy sensor, Rtg2 also functions in the nucleus as a regulator of chromosomal integrity [115].

Figure 1. Activation of the retrograde response (RTG) response in yeast cells. The circled Ps are phosphate groups. SLP, (substrate level phosphorylation). See text for description of the RTG response.

The RTG response is turned “on” following impairment in mitochondrial energy production. In the on state, cytoplasmic Rtg2 disengages the Rtg1/Rtg-3 complex through a dephosphorylation of Rtg3 [110]. The Rtg1 and Rtg3 proteins then individually enter the nucleus where Rtg3 binds to R box sites, Rtg1 reengages Rtg3, and transcription and signaling commences for multiple energy and anti-apoptotic related genes and proteins to include MYC, TOR, p53, Ras, CREB, NFkB, and CHOP [110,112,113,116-118]. The RTG response also involves the participation of multiple negative and positive regulators, which facilitate the bioenergetic transition from respiration to substrate level phosphorylation [110].

The primary role of the RTG response is to coordinate the synthesis of ATP through glycolysis alone or through a combination of glycolysis and glutaminolysis when respiratory function is impaired [110,111]. The RTG response would be essential for maintaining a stable ΔG’ATP for cell viability during periods when respiration is impaired. A prolonged RTG response, however, would leave the nuclear genome vulnerable to instability and mutability [112,117,119]. Mitochondrial dysfunction also increases levels of cytoplasmic calcium, the multi-drug resistance phenotype, production of reactive oxygen species, and abnormalities in iron-sulfur complexes, which together would further accelerate aberrant RTG signaling and genome mutability [85,106,107,110,111,120-122]. Chronic tissue inflammation could further damage mitochondria, which would accelerate these processes [123,124]. Considered collectively, these findings indicate that the integrity of the nuclear genome is dependent to a large extent on the functionality and energy production of the mitochondria.

Similarities between yeast cells and mammalian cells to impaired respiration

Interesting analogies exist between yeast and mammalian cells for the physiological response to impaired respiration [76,112,117,125,126]. Mammalian cells increase expression of hypoxia-inducible factor-1a (HIF-1α) in response to transient hypoxia [127]. HIF-1α is rapidly degraded under normoxic conditions, but becomes stabilized under hypoxia. This is a conserved physiological response that evolved to protect mammalian mitochondria from hypoxic damage and to provide an alternative source of energy to respiration, as HIF-1α induces expression of pyruvate dehydrogenase kinase 1 and most major genes involved with glucose uptake, glycolysis, and lactic acid production [127]. HIF-1α expression is also elevated in most tumor cells whether or not hypoxia is present and could mediate in part aerobic glycolysis [20,28,98,128,129]. Although the mechanisms of HIF-1α stabilization under hypoxic conditions are well defined, the mechanisms by which HIF-1α is stabilized under aerobic or normoxic conditions are less clear [129,130].

HIF-1α is generally unstable in cells under normal aerobic conditions through its interaction with the von Hippel-Lindau (VHL) tumor suppressor protein, which facilitates HIF-1α hydroxylation, ubiquitination, and proteasomal degradation [28]. HIF-1α stabilization under aerobic conditions can be linked to mitochondrial dysfunction through abnormalities in calcium homeostasis, ROS generation, NFkB signaling, accumulation of TCA cycle metabolites (succinate and fumarate), and oncogenic viral infections [131-135]. It is not yet clear if genomic instability can arise through prolonged HIF-1α stabilization under aerobic conditions as would occur during tumor initiation and progression.

Besides HIF-1α function, the human MYC transcription factor also shows homology to the yeast Rtg3 transcription factor [112]. MYC is also a member of the basic, helix-loop-helix leucine zipper family of transcription factors as are RTG1 and RTG3. HIF-1α and MYC also up-regulate many of the same genes for glycolysis [136]. Hence, both HIF-1α and MYC share similarities with components of the yeast RTG system.

Mitochondrial dysfunction and the mutator phenotype

Most human cancer cells display genome instability involving elevated mutation rates, gross chromosomal rearrangements, and alterations in chromosome number [15,17,100,137]. The recent studies of the Singh and the Jazwinski groups provide compelling evidence that mitochondrial dysfunction, operating largely through the RTG response (mitochondrial stress signaling), can underlie the mutator phenotype of tumor cells [71,113,115,117,138]. Chromosomal instability, expression of gene mutations, and the tumorigenic phenotype were significantly greater in human cells with mtDNA depletion than in cells with normal mtDNA. Mitochondrial dysfunction can also down-regulate expression of the apurinic/apyrimidinic endonuclease APE1. This is a redox-sensitive multifunctional endonuclease that regulates DNA transcription and repair [113,139]. APE1 down regulation will increase genomic mutability. Since gene expression is different in different tissues, it is expected that disturbed energy metabolism would produce different kinds of mutations in different types of cancers. Even different tumors within the same cancer type could appear to represent different diseases when evaluated at the genomic level. When evaluated at the metabolic level, however, most cancers and tumors are alike in expressing mitochondrial dysfunction and elevated substrate level phosphorylation. Emerging evidence suggests that mitochondrial dysfunction underlies the mutator phenotype of tumor cells.

Impaired mitochondrial function can induce abnormalities in tumor suppressor genes and oncogenes. For example, impaired mitochondrial function can induce abnormalities in p53 activation, while abnormalities in p53 expression and regulation can further impair mitochondrial function [85,103,113,116,140-143]. The function of the pRB tumor suppressor protein, which controls the cell cycle, is also sensitive to ROS production through the redox state of the cell [144]. Elevated expression of the MYC and Ras oncogenes can be linked to the requirements of substrate level phosphorylation to maintain tumor cell viability. Hence, the numerous gene defects found in various cancers can arise as secondary consequences of mitochondrial dysfunction.

Calcium homeostasis is also dependent on mitochondrial function [110]. It appears that calcium homeostasis is essential for the fidelity of mitosis to include spindle assembly, sister chromosome separation, and cytokinesis [145-150]. Disturbances in cytoplasmic calcium homeostasis, arising as a consequence of mitochondrial dysfunction [111], could contribute to abnormalities in chromosomal segregation during mitosis. These findings suggest that the numerous chromosomal abnormalities found in cancer cells can arise as a consequence of mitochondrial damage.

Recent studies in yeast indicate that damage to the inner mitochondrial membrane potential (ΔΨm) following loss of mtDNA alters the function of several nuclear iron-sulfur-dependent DNA repair enzymes involving the Rad3 helicase, the Pri2 primase, and the Ntg2 glycase [107]. Abnormalities in these DNA repair enzymes contribute to the loss of heterozygosity (LOH) phenotype in specific genes of the affected yeast cells. These findings indicate that LOH, which is commonly observed in many genes of cancer cells [100], can also be linked to mitochondrial dysfunction. Considered collectively, these observations suggest that the bulk of the genetic abnormalities found in cancer cells, ranging from point mutations to gross chromosomal rearrangements, can arise following damage to the structure and function of mitochondria.

Impairment of mitochondrial function can occur following prolonged injury or irritation to tissues including disruption of morphogenetic fields [123,151]. This tumorigenic process could be initiated in the cells of any tissue capable of producing mitochondrial stress signaling following repetitive sub-lethal respiratory damage over prolonged periods. The accumulation of mitochondrial damage over time is what ultimately leads to malignant tumor formation. Acquired abnormalities in mitochondrial function would produce a type of vicious cycle where impaired mitochondrial energy production initiates genome instability and mutability, which then accelerates mitochondrial dysfunction and energy impairment and so on in a cumulative way. An increased dependency on substrate level phosphorylation for survival would follow each round of metabolic and genetic damage thus initiating uncontrolled cell growth and eventual formation of a malignant neoplasm. In other words, the well-documented tumor-associated abnormalities in oncogenes, tumor suppressor genes, and chromosomal imbalances can arise as a consequence of the progressive impairment of mitochondrial function.

Mitochondrial dysfunction following viral infection

Viruses have long been recognized as the cause of some cancers [152]. It is interesting that several cancer-associated viruses localize to, or accumulate in, the mitochondria. Viral alteration of mitochondrial function could potentially disrupt energy metabolism thus altering expression of tumor suppressor genes and oncogenes over time. Viruses that can affect mitochondrial function include the Rous sarcoma virus, Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV), human papilloma virus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), and human T-cell leukemia virus type 1 (HTLV-1) [64,153-155]. Although viral disruption of mitochondrial function will kill most cells through apoptosis following an acute infection, those infected cells that can up-regulate substrate level phosphorylation will survive and potentially produce a neoplasm following chronic infection. Indeed, the hepatitis B × protein (HBx) blocks HIF-1α ubiquitination thus increasing HIF-1α stability and activity in a hypoxia-independent manner [135]. Alterations in calcium homeostasis, ROS production, and expression of NF-kB and HIF-1α are also expected to alter the metabolic state as was previously found for some viral infections [153,154]. It is interesting in this regard that carcinogenesis, whether arising from viral infection or from chemical agent, produces similar impairment in respiratory enzyme activity and mitochondrial function [90]. Thus, viruses can potentially cause cancer through displacement of respiration with substrate level phosphorylation in the infected cells. Alterations in expression of tumor suppressor genes and oncogenes will follow this energy transformation according to the general hypothesis presented here.

Mitochondrial suppression of tumorigenicity

While the mutator phenotype of cancer can be linked to impaired mitochondrial function, normal mitochondrial function can also suppress tumorigenesis. It is well documented that tumorigenicity can be suppressed when cytoplasm from enucleated normal cells is fused with tumor cells to form cybrids, suggesting that normal mitochondria can suppress the tumorigenic phenotype [156-158]. Singh and co-workers provided additional evidence for the role of mitochondria in the suppression of tumorigenicity by showing that exogenous transfer of wild type mitochondria to cells with depleted mitochondria (rho0 cells) could reverse the altered expression of the APE1 multifunctional protein and the tumorigenic phenotype [113]. On the other hand, introduction of mitochondrial mutations can reverse the anti-tumorigenic effect of normal mitochondria in cybrids [159]. It is also well documented that nuclei from cancer cells can be reprogrammed to form normal tissues when transplanted into normal cytoplasm despite the continued presence of the tumor-associated genomic defects in the cells of the derived tissues [160-162]. These findings indicate that nuclear gene mutations alone cannot account for the origin of cancer and further highlight the dynamic role of mitochondria in the epigenetic regulation of carcinogenesis.

It is expected that the presence of normal mitochondria in tumor cells would restore the cellular redox status, turn off the RTG response, and reduce or eliminate the need for glycolysis (Warburg effect) and glutaminolysis to maintain viability. In other words, normal mitochondrial function would facilitate expression of the differentiated state thereby suppressing the tumorigenic or undifferentiated state. This concept can link mitochondrial function to the long-standing controversy on cellular differentiation and tumorigenicity [5,163]. Respiration is required for the emergence and maintenance of differentiation, while loss of respiration leads to glycolysis, dedifferentiation, and unbridled proliferation [8,25]. These observations are consistent with the general hypothesis presented here, that prolonged impairment of mitochondrial energy metabolism underlies carcinogenesis. New studies are necessary to assess the degree to which cellular energy balance is restored in cybrids and in reprogrammed tumor cells.

Linking the acquired capabilities of cancer to impaired energy metabolism

Although the mutator phenotype was considered the essential enabling characteristic for manifesting the six hallmarks of cancer, the pathways by which the acquired capabilities of cancer are linked specifically to impaired energy metabolism remain poorly defined. Kromer and Pouyssegur recently provided an overview on how the hallmarks of cancer could be linked to signaling cascades and to the metabolic reprogramming of cancer cells [164]. As the acquired capabilities of self-sufficiency in growth signals, insensitivity to growth inhibitory (antigrowth) signals, and limitless replicative potential are similar, these capabilities can be grouped and discussed together. The acquired capabilities of evasion of programmed cell death, angiogenesis, and metastasis can be discussed separately.

Growth signaling abnormalities and limitless replicative potential

A central concept in linking abnormalities of growth signaling and replicative potential to impaired energy metabolism is in recognizing that proliferation rather than quiescence is the default state of both microorganisms and metazoans [5,8,165,166]. The cellular default state is the condition under which cells are found when they are freed from any active control. Respiring cells in mature organ systems are quiescent largely because their replicative potential is under negative control through the action of tumor suppressor genes like p53 and the retinoblastoma protein, pRB [144,165]. As p53 function is linked to cellular respiration, prolonged damage to respiration will gradually reduce p53 function thus inactivating the negative control of p53 and of other tumor suppressor genes on cell proliferation.

A persistent impairment in respiratory function will trigger the RTG response, which is necessary for up-regulating the pathways of glycolysis and glutaminolysis in order to maintain the ΔG’ATP for viability. The RTG response will activate MYC, Ras, HIF-1α, Akt, and m-Tor etc, which are required to facilitate and to sustain up-regulation of substrate level phosphorylation [61,110,113,167,168]. In addition to facilitating the uptake and metabolism of alternative energy substrates through substrate level phosphorylation, MYC and Ras further stimulate cell proliferation [136,169,170]. Part of this mechanism also includes inactivation of pRB, the function of which is dependent on mitochondrial activities and the cellular redox state [144]. Disruption of the pRB signaling pathway will contribute to cell proliferation and neoplasia [6]. Hence, the growth signaling abnormalities and limitless replicative potential of tumor cells can be linked directly to the requirements of glycolysis and glutaminolysis and ultimately to impaired respiration.

It is interesting that RTG signaling also underlies replicative life span extension in budding yeast. Yeast longevity is manifested by the number of buds that a mother cell produces before it dies [110]. The greater the loss of mitochondrial function, the greater is the induction of the RTG response, and the greater the longevity (bud production) [108]. As mitochondrial function declines with age, substrate level phosphorylation becomes necessary to compensate for the lost energy from respiration if a cell is to remain alive. A greater reliance on substrate level phosphorylation will induce oncogene expression and unbridled proliferation, which could underlie in part the enhanced longevity in yeast [110,112,119]. When this process occurs in mammalian cells, however, the phenomenon is referred to as neoplasia or “new growth”. We propose that replicative life span extension in yeast and limitless replicative potential in tumor cells can be linked through common bioenergetic mechanisms involving impaired mitochondrial function.

Linking telomerase to mitochondrial function

Emerging evidence indicates that telomerase, a ribonucleoprotein complex, plays a role in tumor progression [171]. Although still somewhat sparse, data suggest that mitochondrial dysfunction could underlie the relocation of telomerase from the mitochondria, where it seems to have a protective role, to the nucleus where it maintains telomere integrity necessary for limitless replicative potential [172-174]. Interestingly, telomerase activity is high during early embryonic development when anaerobic glycolysis and cell proliferation is high, but telomerase expression is suppressed in adult tissues, where cellular energy is derived largely from respiration. Further studies will be necessary to determine how changes in telomerase expression and subcellular localization could be related to mitochondrial dysfunction, elevated substrate level phosphorylation, and to the limitless replication of tumor cells.

Evasion of programmed cell death (apoptosis)

Apoptosis is a coordinated process that initiates cell death following a variety of cellular insults. Damage to mitochondrial energy production is one type of insult that can trigger the apoptotic cascade, which ultimately involves release of mitochondrial cytochrome c, activation of intracellular caspases, and death [6]. In contrast to normal cells, acquired resistance to apoptosis is a hallmark of most types of cancer cells [6]. The evasion of apoptosis is a predictable physiological response of tumor cells that up-regulate substrate level phosphorylation for energy production following respiratory damage during the protracted process of carcinogenesis. Only those cells capable of making the gradual energy transition from respiration to substrate level phosphorylation in response to respiratory damage will be able to evade apoptosis. Cells unable to make this energy transition will die and thus never become tumor cells.

Numerous findings indicate that the genes and signaling pathways needed to up-regulate and sustain substrate level phosphorylation are themselves anti-apoptotic. For example, sustained glycolysis requires participation of mTOR, MYC, Ras, HIF-1α, and the IGF-1/PI3K/Akt signaling pathways [28,110,112,113,128,168]. The up-regulation of these genes and pathways together with inactivation of tumor suppressor genes like p53, which is required to initiate apoptosis, will disengage the apoptotic-signaling cascade thus preventing programmed cell death [142].

Abnormalities in the mitochondrial membrane potential (ΔΨm) can also induce expression of known anti-apoptotic genes (Bcl2 and Ccl-XL) [111]. Tumor cells will continue to evade apoptosis as long as they have access to glucose and glutamine, which are required to maintain substrate level phosphorylation. Glycolytic tumor cells, however, can readily express a robust apoptotic phenotype if their glucose supply is targeted. This was clearly illustrated in experimental brain tumors using calorie restriction [168,175,176]. Hence, the evasion of apoptosis in tumor cells can be linked directly to a dependency on substrate level phosphorylation, which itself is a consequence of impaired respiratory function.

Sustained vascularity (angiogenesis)

Angiogenesis involves neovascularization or the formation of new capillaries from existing blood vessels and is associated with the processes of tissue inflammation, wound healing, and tumorigenesis [123,124,177,178]. Angiogenesis is required for most tumors to grow beyond an approximate size of 0.2-2.0 mm [179]. Vascularity is necessary in order to provide the tumor with essential energy nutrients to include glucose and glutamine, and to remove toxic tumor waste products such as lactic acid and ammonia [49]. In addition to its role in up-regulating glycolysis in response to hypoxia, HIF-1α is also the main transcription factor for vascular endothelial growth factor (VEGF), which stimulates angiogenesis [168,180-182]. HIF-1α is part of the IGF-1/PI3K/Akt signaling pathway that also indirectly influences expression of β FGF, another key angiogenesis growth factor [168,183]. Hence the sustained vascularity of tumors can be linked mechanistically to the metabolic requirements of substrate level phosphorylation necessary for tumor cell survival.

Metastasis is the general term used to describe the spread of cancer cells from the primary tumor to surrounding tissues and to distant organs and is a primary cause of cancer morbidity and mortality [6,184,185]. Metastasis involves a complex series of sequential and interrelated steps. In order to complete the metastatic cascade, cancer cells must detach from the primary tumor, intravasate into the circulation and lymphatic system, evade immune attack, extravasate at a distant capillary bed, and invade and proliferate in distant organs [185-189]. Metastatic cells also establish a microenvironment that facilitates angiogenesis and proliferation, resulting in macroscopic, malignant secondary tumors. A difficulty in better characterizing the molecular mechanisms of metastasis comes in large part from the lack of animal models that manifest all steps of the cascade. Tumor cells that are naturally metastatic should not require intravenous injection in animal models to initiate the metastatic phenotype [190,191]. In vitro models, on the other hand, do not replicate all the steps required for systemic metastasis in vivo. Although the major steps of metastasis are well documented, the process by which metastatic cells arise from within populations of non-metastatic cells of the primary tumor is largely unknown [185,192,193].

Several mechanisms have been advanced to account for the origin of metastasis. The epithelial-mesenchymal transition (EMT) posits that metastatic cells arise from epithelial cells through a step-wise accumulation of gene mutations that eventually transform an epithelial cell into a tumor cell with mesenchymal features [6,100,194-196]. The idea comes from findings that many cancers generally arise in epithelial tissues where abnormalities in cell-cell and cell-matrix interactions occur during tumor progression. Eventually neoplastic cells emerge that appear as mesenchymal cells, which lack cell-cell adhesion and are dysmorphic in shape [185]. These transformed epithelial cells eventually acquire the multiple effector mechanisms of metastasis [185]. Recent studies suggest that ectopic co-expression of only two genes might be all that is necessary to facilitate EMT in some gliomas [197]. Considerable controversy surrounds the EMT hypothesis of metastasis, however, as EMT is not often detected in tumor pathological preparations [198,199].

The macrophage hypothesis of metastasis suggests that metastatic cells arise following fusions of macrophages or bone marrow derived hematopoietic cells with committed tumor cells [193,200,201]. It is well documented that metastatic cancer cells, arising from a variety of tissues, possess numerous properties of macrophages or cells of myeloid lineage including phagocytosis and fusogenicity [190,202-208]. Macrophages and other types of myeloid cells are already genetically programmed to enter and exit tissues. Many of the normal behaviors of macrophages elaborate each step of the metastatic cascade [204]. Fusion of a myeloid cell (macrophage) with a tumor cell could produce a hybrid cell possessing the replicative capacity of the tumor cell and the properties of macrophages including the invasive and inflammatory properties [193,205,209]. As myeloid cells are also part of the immune system, evasion of immune surveillance would be another expected characteristic of metastatic cells derived from macrophage-like cells [205]. Indeed, metastatic melanoma cells can phagocytose live T-cells, which are supposed to kill the tumor cells [210].

Fusions among metastatic myeloid cells at the primary tumor site could, through reprogramming strategies, also produce functional epithelial cells at secondary sites potentially simulating the histological characteristics of the original tissue of origin [200,211,212]. The macrophage fusion hypothesis would also fit with the roles of hematopoietic stem cells in the metastatic niche [208,213]. While the fusion hypothesis is attractive, it would be an exception to the observations showing suppressed tumorigenicity following hybridization between normal cells and tumor cells [163], though some exceptions have been reported [205,206]. However, neither the EMT hypothesis nor the macrophage fusion hypothesis link the origin of metastasis to the Warburg effect or to impaired energy metabolism.

Recent findings of cardiolipin abnormalities in systemic metastatic mouse tumor cells with macrophage properties can link metastasis to impaired respiratory function in these cells [73,190,204]. Most tissues contain resident phagocytes as part of their histoarchitecture or stroma [214]. Tumor associated macrophages (TAM) also become a major cell type in many cancers [215]. While TAM can facilitate the invasive and metastatic properties of tumor cells [213,216], metastatic tumor cells can also express several properties of TAM [190,204].

Damage to the respiratory capacity of resident tissue phagocytes, TAM, or macrophage hybrids would trigger a RTG response, force a reliance on substrate level phosphorylation for energy, and eventually, over time, lead to dysregulated growth and genomic instability as described in the general hypothesis. Metastatic behavior would be an expected outcome following impaired respiratory function in hematopoietic or myeloid-type cells, as macrophages are already mesenchymal cells that embody the capacity to degrade the extracellular matrix, to enter and to exit tissues from the blood stream, to migrate through tissues, and to survive in hypoxic environments. A sampling of human metastatic cancers with properties of macrophage-like cells include brain [204,217-220], breast [221-225], lung [202,225-229], skin [203,205,209,210,230-233], gastric [234], colon [235,236], pancreas [237,238], bladder [239], kidney [240], ovarian [241,242], and muscle [243,244]. It is important to mention that these macrophage properties are expressed in the tumor cells themselves and are not to be confused with similar properties expressed in the non-neoplastic TAM, which are also present in tumors and can facilitate tumor progression [190,213,215,216,245]. Poor prognosis is generally associated with those cancers that display characteristics of macrophages [210,221]. Hence, damage to the respiratory capacity of myeloid or macrophage-like cells would produce “rogue macrophages” leading to cancers with the highest metastatic behavior.

The plethora of the cell surface molecules thought to participate in metastatic tumor cell behavior are also expressed on myeloid cells especially macrophages [185,213]. A robust Warburg effect in human metastatic lesions, detected with combined 18F-fluorodeoxyglucose-positron emission tomography imaging, indicates that metastatic cells have impaired energy metabolism like that of most cancer cells [18,20,246]. Hence, invasion and metastasis can be linked to impaired energy metabolism if this impairment occurs in cells of hematopoietic or myeloid origin.

The path from normal cell physiology to malignant behavior, where all major cancer hallmarks are expressed, is depicted in Figure 2 and is based on the evidence reviewed above. Any unspecific condition that damages a cell’s oxidative phosphorylation, but is not severe enough to induce apoptosis, can potentially initiate the path to a malignant cancer. Some of the many unspecific conditions contributing to carcinogenesis can include inflammation, carcinogens, radiation (ionizing or ultraviolet), intermittent hypoxia, rare germline mutations, viral infections, and disruption of tissue morphogenetic fields. Any of these conditions can damage the structure and function of mitochondria thus activating a specific RTG response in the damaged cell. If the mitochondrial damage persists, the RTG response will persist. Uncorrected mitochondrial damage will require a continuous compensatory energy response involving substrate level phosphorylation in order to maintain the ΔG’ATP of approximately -56 kJ/mol for cell viability. Tumor progression is linked to a greater dependence on substrate level phosphorylation, which eventually becomes irreversible. As the integrity of the nuclear genome is dependent on the efficiency of mitochondrial energy production, the continued impairment of mitochondrial energy production will gradually undermine nuclear genome integrity leading to a mutator phenotype and a plethora of somatic mutations. Activation of oncogenes, inactivation of tumor suppressor genes, and aneuploidy will be the consequence of protracted mitochondrial dysfunction. These gene abnormalities will contribute further to mitochondrial dysfunction while also enhancing those energy pathways needed to up-regulate and sustain substrate level phosphorylation. The greater the dependency on substrate level phosphorylation over time the greater will be the degree of malignancy. Damage to the respiratory capacity of tissue myeloid cells can also produce invasive and metastatic properties according to the macrophage hypothesis of metastasis. This metabolic scenario can account for all major acquired characteristics of cancer to include the Warburg effect.

Figure 2. Linking the hallmarks of cancer to impaired energy metabolism. See text for discussion. SLP and OxPhos represent substrate level phosphorylation and oxidative phosphorylation, respectively. The progressive damage to mitochondria during carcinogenesis is illustrated with a change in shape.
Implications of the hypothesis to cancer management

If cancer is primarily a disease of energy metabolism as reviewed here, then rational approaches to cancer management can be found in therapies that specifically target energy metabolism. Although mitochondrial replacement therapy could in principle restore a more normal energy metabolism and differentiated state to tumor cells, it is unlikely that this therapeutic approach would be available in the foreseeable future. However, numerous studies show that dietary energy restriction is a general metabolic therapy that naturally lowers circulating glucose levels and significantly reduces growth and progression of numerous tumor types to include cancers of the mammary, brain, colon, pancreas, lung, and prostate [10,247-256]. The influence of energy restriction on tumor growth, however, can depend on host background and tumor growth site, as energy restriction is effective in reducing the U87 human glioma when grown orthotopically in the brain of immunodeficient SCID mice [175], but not when grown outside the brain in non-obese diabetic SCID mice [257]. Nevertheless, the bulk of evidence indicates that dietary energy restriction can retard the growth rate of many tumors regardless of the specific genetic defects expressed within the tumor.

Reduced glucose availability will target aerobic glycolysis and the pentose phosphate shunt pathways required for the survival and proliferation of many types of tumor cells. Dietary energy restriction specifically targets the IGF-1/PI3K/Akt/HIF-1α signaling pathway, which underlies several cancer hallmarks to include cell proliferation, evasion of apoptosis, and angiogenesis [168,175,176,250,251,254,258-265]. Calorie restriction also causes a simultaneous down-regulation of multiple genes and metabolic pathways regulating glycolysis [266-268]. This is important, as enhanced glycolysis is required for the rapid growth and survival of many tumor cells [21,22]. In addition, recent findings suggest that a large subset of gliomas have acquired mutations in the TCA cycle gene, isocitrate dehydrogenase (IDH1) [105]. Such mutations are expected to limit the function of the TCA cycle, thus increasing the glycolytic dependence of these tumors. Tumors with these types of mutations could be especially vulnerable to management through dietary energy restriction. Hence, dietary energy or calorie restriction can be considered a broad-spectrum, non-toxic metabolic therapy that inhibits multiple signaling pathways required for progression of malignant tumors regardless of tissue origin.

Besides lowering circulating glucose levels, dietary energy restriction elevates circulating levels of fatty acids and ketone bodies (β-hydroxybutyrate and acetoacetate) [266,269,270]. Fats and especially ketone bodies can replace glucose as a primary metabolic fuel under calorie restriction. This is a conserved physiological adaptation that evolved to spare protein during periods of starvation [271,272]. Many tumors, however, have abnormalities in the genes and enzymes needed to metabolize ketone bodies for energy [273-275]. A transition from carbohydrate to ketones for energy is a simple way to target energy metabolism in glycolysis-dependent tumor cells while enhancing the metabolic efficiency of normal cells [276,277]. The shift from the metabolism of glucose to the metabolism of ketone bodies for energy is due largely to the shift in circulating levels of insulin and glucagon, key hormones that mediate energy metabolism. Insulin, which stimulates glycolysis, is reduced under dietary restriction, while glucagon, which inhibits glycolysis and mobilizes fats, is increased. Glucose reduction not only reduces insulin, but also reduces circulating levels of IGF-1, which is necessary for driving tumor cell metabolism and growth [168,278]. Glucocorticoids, which enhance glucagon action and the stress response, are also elevated under dietary energy restriction [261]. The shift in levels of these metabolic hormones would place greater physiological stress on the tumor cells than on normal cells since the tumor cells lack metabolic flexibility due to accumulated genetic mutations [10,15,277].

Inferences that tumor cells have a growth advantage over normal cells are inconsistent with principles of evolutionary biology [10,277]. Although viewed as a growth advantage, the dysregulated growth of tumor cells is actually an aberrant phenotype. How can tumor cells that express multiple mutations and mitochondrial abnormalities be more “fit” or “advantaged” than normal cells that possess a flexible genome, normal respiratory capacity, and adaptive versatility? The short answer is that they are not. Normal cells can grow much faster than tumor cells during normal wound repair. Metabolism of ketone bodies and fatty acids for energy requires inner mitochondrial membrane integrity and efficient respiration, which tumor cells largely lack [10,273,278]. In contrast to the tumor cells, normal cells evolved to survive extreme shifts in the physiological environment and can readily adapt to fat metabolism when glucose becomes limiting. Glucose transporter expression is higher in mouse brain tumor cells than in neighboring normal cells when circulating glucose levels are high, but the transporter phenotype of these cells becomes reversed under dietary energy restriction [168]. These findings highlight the different responses to energy stress between the metabolically incompetent tumor cells and competent normal cells. Consequently, a shift in energy metabolism from glucose to ketone bodies protects respiratory competent normal cells while targeting the genetically defective and respiratory challenged tumor cells, which depend more heavily on glycolysis than normal cells for survival [10,278,279].

Proof of concept for cancer metabolic therapy was illustrated for the management of malignant astrocytoma in mice, and malignant glioma in children [273,276,280]. Prostate and gastric cancer also appears manageable using low carbohydrate ketogenic diets [252,281,282]. Recent studies show that dietary energy restriction enhances phosphorylation of adenosine monophosphate kinase (AMPK), which induces apoptosis in glycolytic-dependent astrocytoma cells, but protects normal brain cells from death [283]. This further illustrates the differential response of normal cells and tumor cells to energy stress.

A possible concern is how any therapy, which reduces food intake and body weight, can be recommended to individuals who might be losing body weight because of cancer cachexia. Cancer cachexia generally involves anorexia, weight loss, muscle atrophy, and anemia [284,285]. Although some cancer patients could be obese, rapid weight loss from cachexia involving both proteins and fat is a health concern [285]. It is important to recognize that pro-cachexia molecules such as proteolysis-inducing factor are released from the tumor cells into the circulation and contribute to the cachexia phenotype [286,287]. By targeting the glycolytically active tumor cells that produce pro-cachexia molecules, restricted diet therapies can potentially reduce tumor cachexia [278,287]. These therapies could be supplemented with omega-3 fatty acids, which can also reduce the cachexia phenotype [285]. Omega-3 fatty acids from fish oil also have the benefit of maintaining low glucose while elevating ketone levels. Once the tumor becomes managed, individuals can increase caloric consumption to achieve weight gain.

Metabolic therapies involving calorie restriction should be effective in targeting energy-defective cells within a given tumor, and for managing a broad range of glycolysis-dependent tumors. There are no known drugs that can simultaneously target as many tumor-associated signaling pathways as can calorie restriction [168]. Hence, energy restriction can be a cost-effective adjuvant therapy to traditional chemo- or radiation therapies, which are more toxic, costly, and generally less focused in their therapeutic action, than is dietary energy restriction.

In addition to dietary energy restriction, several small molecules that target aerobic glycolysis are under consideration as novel tumor therapeutics to include 2-deoxyglucose, lonidamine, 3-bromopyruvate, imatinib, oxythiamine, and 6-aminonicotinimide among others [129,288-290]. Toxicity can become an issue, however, as some of these compounds target pathways other than glycolysis or nucleotide synthesis and high dosages are sometimes required to achieve efficacy in vivo. A recent study found significant therapeutic synergy in combining low doses of 2-deoxyglucose with a calorie restricted ketogenic diet for managing malignant astrocytoma in mice [291].

It appears that the therapeutic efficacy of anti-glycolytic cancer drugs could be significantly enhanced when combined with dietary energy restriction. The administration of anti-glycolytic drugs together with energy restricted diets, which lower circulating glucose levels while elevating ketone levels, could act as a powerful double “metabolic punch” for the rapid killing of glycolysis dependent tumor cells. This therapeutic approach could open new avenues in cancer drug development, as many drugs that might have minimal therapeutic efficacy or high toxicity when administered alone could become therapeutically relevant and less toxic when combined with energy restricted diets.

Targeting the microenvironment

Some tumors behave as wounds that do not heal [292]. Growth factors and cytokines released by fibroblasts and macrophages, cells programmed to heal wounds, can actually provoke chronic inflammation and tumor progression [213,245]. Part of the wound healing process also involves degradation of the extracellular matrix and enhancement of angiogenesis, which further contribute to tumor progression [180,213]. Dietary energy restriction targets inflammation and the signaling pathways involved with driving tumor angiogenesis [168,258,293]. Indeed, calorie restriction is considered a simple and effective therapy for targeting tumor angiogenesis and inflammation [176,250,279]. As calorie or dietary energy restriction is a systemic therapy that simultaneously targets both the tumor cells as well as the tumor microenvironment, this approach can be effective in retarding tumor progression.

Although dietary energy restriction and anti-glycolytic cancer drugs will have therapeutic efficacy against many tumors that depend largely on glycolysis and glucose for growth, these therapeutic approaches could be less effective against those tumor cells that depend more heavily on glutamine than on glucose for energy [47,65-67]. Glutamine is a major energy metabolite for many tumor cells and especially for cells of hematopoietic or myeloid lineage [47,49,294,295]. This is important as cells of myeloid lineage are considered the origin of many metastatic cancers [17,190,204,221,230]. Moreover, glutamine is necessary for the synthesis of those cytokines involved in cancer cachexia including tumor necrosis factor alpha, (TNF-α) and the interleukins 1 and 6 (IL-1 and -6) [66,284,295,296]. This further indicates a metabolic linkage between metastatic cancer and myeloid cells, e.g., macrophages. It therefore becomes important to also consider glutamine targeting for the metabolic management of metastatic cancer.
Glutamine can be deaminated to glutamate and then metabolized to α-ketoglutarate, a key metabolite of the TCA cycle [49,67]. This occurs either through transamination or through enhanced glutamate dehydrogenase activity depending on the availability of glucose [67]. Besides generating energy through substrate level phosphorylation in the TCA cycle, i.e., transphosphorylation of GTP to ATP, the anapleurotic effect of glutamine can also elevate levels of metabolic substrates, which stimulate glycolysis [49,66]. Glutamine metabolism can be targeted in humans using the glutamine binding drug, phenylacetate, or the glutamine analogue DON (6-Diazo-5-oxo-L-norleucine) [297]. Toxicity, however, can be an issue in attempts to target glutamine metabolism using DON [130,294]. Recent studies suggest that the green tea polyphenol (EGCG) could target glutamine metabolism by inhibiting glutamate dehydrogenase activity under low glucose conditions [67]. This and other glutamine-targeting strategies could be even more effective when combined with energy restricting diets, which lower glucose levels while elevating ketone bodies. Hence, effective non-toxic targeting of both glucose and glutamine metabolism should be a simple therapeutic approach for the global management of most localized and metastatic cancers.

Implications of the hypothesis to cancer prevention
If impaired mitochondrial energy metabolism underlies the origin of most cancers as proposed here, then protecting mitochondria from damage becomes a logical and simple approach for preventing cancer. It is well documented that the incidence of cancer can be significantly reduced by avoiding exposure to those agents or conditions that provoke tissue inflammation such as smoking, alcohol, carcinogenic chemicals, ionizing radiation, obesity etc [1,25,298]. Chronic inflammation, regardless of origin, damages tissue morphogenetic fields that eventually produce neoplastic cells [123,124,166]. Part of this tissue damage will involve injury to the mitochondria in the affected cells. The prevention of inflammation and damage to the tissue microenvironment will go far in reducing the incidence of most cancers. Vaccines against some oncogenic viruses can also reduce the incidence of cancers, as these viruses can damage mitochondria in infected tissues. Hence, simply reducing exposure to cancer risk factors, which produce chronic inflammation and mitochondrial damage, will reduce the incidence of at least 80% of all cancers [1,25]. In principle, there are few chronic diseases more easily preventable than cancer.

In addition to avoiding exposure to established cancer risk factors, the metabolism of ketone bodies protects the mitochondria from inflammation and damaging ROS. ROS production increases naturally with age and damages cellular proteins, lipids, and nucleic acids. Accumulation of ROS decreases the efficiency of mitochondrial energy production. The origin of mitochondrial ROS comes largely from the spontaneous reaction of molecular oxygen (O2) with the semiquinone radical of coenzyme Q, .QH, to generate the superoxide radical O2-. [40,84,299]. Coenzyme Q is a hydrophobic molecule that resides in the inner mitochondrial membrane and is essential for electron transfer. Ketone body metabolism increases the ratio of the oxidized form to the fully reduced form of coenzyme Q (CoQ/CoQH2) [40]. Oxidation of the coenzyme Q couple reduces the amount of the semiquinone radical, thus decreasing superoxide production [84].

Since the cytosolic free NADP+/NADPH concentration couple is in near equilibrium with the glutathione couple, ketone body metabolism will also increase the reduced form of glutathione thus facilitating destruction of hydrogen peroxide [10,84,300]. The reduction of free radicals through ketone body metabolism will therefore reduce tissue inflammation provoked by ROS while enhancing the energy efficiency of mitochondria. Ketone bodies are not only a more efficient metabolic fuel than glucose, but also possess anti-inflammatory potential. Metabolism of ketone bodies for energy will maintain mitochondrial health and efficiency thus reducing the incidence of cancer.

The simplest means of initiating the metabolism of ketone bodies is through dietary energy restriction with adequate nutrition. It is important to emphasize adequate nutrition, as calorie restriction associated with malnutrition can potentially increase cancer incidence [301-303]. Consequently, consumption of foods containing the active groups of respiratory enzymes (iron salts, riboflavin, nicotinamide, and pantothenic acid) could be effective in maintaining health when combined with dietary energy restriction [25]. The lowering of circulating glucose levels through calorie restriction facilitates the uptake and metabolism of ketone bodies for use as an alternative respiratory fuel [84,273,278]. The metabolism of ketone bodies increases succinate dehydrogenase activity while enhancing the overall efficiency of energy production through respiration [304,305]. In essence, dietary energy restriction and ketone body metabolism delays entropy [270]. As cancer is a disease of accelerated entropy [8,25], dietary energy restriction targets the very essence of the disease.

It is well documented that dietary energy restriction can reduce the incidence of both inherited and acquired cancers in experimental animals [256,258,306-309]. Evidence also indicates that dietary energy restriction can reduce the incidence of several human cancers [310,311]. The implementation of periodic dietary energy restriction, which targets multiple cancer provoking factors, can be a simple and cost effective life-style change that is capable of reducing the incidence of cancer. Dietary energy restriction in rodents, however, is comparable to water only therapeutic fasting or to very low caloric diets (500-600 kcal/day) in humans [270]. In light of this fact, it remains to be determined if members of our species are willing or motivated enough to adopt the life style changes necessary to prevent cancer.
Conclusions

Evidence is reviewed supporting a general hypothesis that cancer is primarily a disease of energy metabolism. All of the major hallmarks of the disease can be linked to impaired mitochondrial function. In order to maintain viability, tumor cells gradually transition to substrate level phosphorylation using glucose and glutamine as energy substrates. While cancer causing germline mutations are rare, the abundance of somatic genomic abnormalities found in the majority of cancers can arise as a secondary consequence of mitochondrial dysfunction. Once established, somatic genomic instability can contribute to further mitochondrial defects and to the metabolic inflexibility of the tumor cells. Systemic metastasis is the predicted outcome following protracted mitochondrial damage to cells of myeloid origin. Tumor cells of myeloid origin would naturally embody the capacity to exit and enter tissues. Two major conclusions emerge from the hypothesis first that many cancers can regress if energy intake is restricted and, second, that many cancers can be prevented if energy intake is restricted. Consequently, energy restricted diets combined with drugs targeting glucose and glutamine can provide a rational strategy for the longer-term management and prevention of most cancers.

The authors declare that they have no competing interests.

TNS wrote the manuscript. LMS contributed to the general outline of topic presentation, editorial assistance, and to discussion of key issues. Both authors read and approved the final manuscript.

This work was supported from NIH grants (NS-055195 CA-102135) and from the Boston College Research expense fund. The authors thank Purna Mukherjee, Michael Kiebish, Roberto Flores, Thomas Chiles, Richard Veech, and Jeff Chuang for critical comments. We also thank the students of BI503 (Erin Wolf, Joseph Bravo, Nicholas Buffin, Gregory Della Penna, Robert Hornung, Michelle Levine, Stephen Lo, Brett Pantera, Toan Phan, John Reed, Jeans Santana, and Andrew Syvertsen) for technical assistance and their attempts to disprove the main hypothesis.


Challenges in Analysis of Hydrophilic Metabolites Using Chromatography Coupled with Mass Spectrometry

Hydrophilic metabolites play important roles in cellular energy metabolism, signal transduction, immunity. However, there are challenges in both identification and quantification of the hydrophilic metabolites due to their weak interactions with C18-reversed-phase liquid chromatography (RPLC), leading to poor retention of hydrophilic metabolites on the columns. Many strategies have been put forward to increase the retention behavior of hydrophilic metabolites in the RPLC system. Non-derivatization methods are mainly focused on the development of new chromatographic techniques with different separation mechanisms, such as capillary electrophoresis, ion-pairing RPLC etc. Derivatization methods improve the hydrophobicity of metabolites and can enhance the MS response. This review mainly focused on the illustration of challenges of LCMS in the analysis of hydrophilic metabolomics field, and summarized the non-derivatization and derivatization strategies, with the intention of providing multiple choices for analysis of hydrophilic metabolites.


Energy and Redox Homeostasis in Tumor Cells

Cancer cells display abnormal morphology, chromosomes, and metabolism. This review will focus on the metabolism of tumor cells integrating the available data by way of a functional approach. The first part contains a comprehensive introduction to bioenergetics, mitochondria, and the mechanisms of production and degradation of reactive oxygen species. This will be followed by a discussion on the oxidative metabolism of tumor cells including the morphology, biogenesis, and networking of mitochondria. Tumor cells overexpress proteins that favor fission, such as GTPase dynamin-related protein 1 (Drp1). The interplay between proapoptotic members of the Bcl-2 family that promotes Drp 1-dependent mitochondrial fragmentation and fusogenic antiapoptotic proteins such as Opa-1 will be presented. It will be argued that contrary to the widespread belief that in cancer cells, aerobic glycolysis completely replaces oxidative metabolism, a misrepresentation of Warburg’s original results, mitochondria of tumor cells are fully viable and functional. Cancer cells also carry out oxidative metabolism and generally conform to the orthodox model of ATP production maintaining as well an intact electron transport system. Finally, data will be presented indicating that the key to tumor cell survival in an ROS rich environment depends on the overexpression of antioxidant enzymes and high levels of the nonenzymatic antioxidant scavengers.

1. A Brief Prelude

Every biochemical reaction within living cells involves the transduction of some degree of free energy that is ultimately derived from the oxidation of dietary nutrients. Most of this free energy is made biologically available as reversible phosphorylation reactions involving adenosine triphosphate (ATP) that is continuously being produced and utilized by cells to drive thermodynamically nonspontaneous reactions, such as ion transport, muscle contraction, protein synthesis, and DNA replication. Just as an example of the stupendous biological power, the amount of free energy transduced by our body during light walking is about 3,18 × 10 −3 W/g, which is roughly 16.000 times more than the fusion reactions that take place in the Sun core [1]. It is known that phosphate esterification into ATP can occur by several processes but the best known are the phosphocreatine-ATP shuttle, glycolysis, and oxidative phosphorylation [2]. Oxidative phosphorylation is capable of producing significantly more ATP per mole of substrate than glycolysis in reactions completely dependent on the availability of oxygen. However, the utilization of oxygen by cells, albeit the advantages of oxidative phosphorylation, is not without consequence, since partially reduced oxygen intermediates, the so-called reactive oxygen species (ROS) play key roles in cellular redox homeostasis [3] that may have a role in tumorigenesis.

2. Mitochondria and Bioenergetics

2.1. Fermentation, Pasteur, Warburg, and Crabtree

Fermentation was the first metabolic pathway to be fully known, thanks to the key findings of many researchers such as Louis Pasteur, who defined the biological nature of the process and Eduard Buchner, who showed that cell-free extracts could carry out fermentation. Later, Otto Meyerhoff experimentally demonstrated that a process similar to fermentation occurred in skeletal muscles, although generating a different final product, lactate [4]. He also showed that, in the absence of oxygen, glycogen was converted to lactate and when oxygen was present lactate was converted back to glycogen, establishing the cyclic nature of lactate metabolism in muscles (the lactate shuttle). In the context of cancer, when cells that use glucose as the main substrate to drive ATP synthesis are subjected to hypoxia, as happens to the cells located in the center of the tumor mass, glucose uptake and metabolism increase significantly in order to maintain cellular ATP levels. Since under limited oxygen availability the oxidative phosphorylation machinery is not fully operational, other pathways are recruited in order to supply the energy demand. The reversible nature of increased glucose uptake and metabolism when cells experiment hypoxia is known as the Pasteur Effect. Also relevant is the reversible repressive effect of glucose over respiration, known as the Crabtree effect [5]. Thus, in spite of a functional oxidative phosphorylation machinery, most solid tumors exhibit a reversibly switch of their metabolism towards lactic fermentation, even under normoxia. Thus, contrasting with the Pasteur Effect, the limitation of respiration in the Crabtree effect is not due to oxygen availability, but rather to an acute repressive signaling cascade triggered by glucose over the mitochondrial function. For this reason, sometimes the Crabtree effect is also referred to as Reverse or Inverted Pasteur Effect. However, the molecular mechanisms that underlie the Crabtree effect remain elusive. Finally, the long-term metabolic reprogramming that takes place in many cancer cells and which bears on cancer is known as the Warburg effect [6]. Otto Warburg observed that cancer cells displayed decreased respiration and enhanced lactate production, suggesting that they depended mainly on fermentative metabolism for ATP generation [7]. It is commonly assumed that tumors manifesting the Warburg effect do so because the oxidative phosphorylation machinery is somehow impaired. In this context a growing body of evidence shows in fact that the oxidative phosphorylation is preserved in many cancer cells, as will be discussed in the following section. The point that should be stressed here regarding the main difference between Crabtree and Warburg effects is that in the former the oxidative phosphorylation is rapidly and reversibly downregulated by the repressive effect of glucose, whereas in the latter, there is a long-lasting irreversible effect favoring fermentation due to the increased expression of proteins involved in glucose transport and metabolism [5].

2.2. Mitochondria and the Processes of Energy Transduction

Structurally, mitochondria are organelles enclosed by two very distinct membranes: an outer membrane, moderately selective, and an inner membrane which is protein rich and highly selective. These compartments are structurally and functionally different. The tricarboxylic acid (TCA) cycle enzymes are located within this compartment, whereas the proteins that comprise the electron transport system (ETS) occur in the inner mitochondrial membrane. The redox reactions mediated by different compounds from ubiquinone to iron/copper-sulphur clusters, cytochromes, and finally oxygen reduction to water (respiration) take place at the inner mitochondrial membrane. Recently, an effort to identify the whole set of mitochondrial proteins in different tissues of mice, rat, and human demonstrated that this organelle is composed of almost 1100 different proteins [8].

The ETS is essentially composed of proteins that contain an array of redox centers making up the complexes commonly listed from I to IV [2]. It is important to mention, however, that respiration can be promoted by multiple sites of electrons entry to the ETS in which electrons converge at the ubiquinone reduction (Q-junction) [9]. Importantly, the free energy released during the electrons transport by the ETS complexes is linked to the transport of protons across the inner mitochondrial membrane. Due to its proton impermeable nature, an electrochemical gradient is established [10]. This electrochemical proton gradient has two components one chemical (ΔpH) and the other electrical (Δψ) in nature, which together represent the protonmotive force (pmf). The free energy accumulated in the form of pmf can be converted to chemical energy by means of the complex molecular motor activity of the F1Fo ATP synthase, which allows the return of protons back to the mitochondrial matrix coupled to ATP production [11]. pmf is important not only for ATP synthesis but for many processes such as the control of substrate transport to mitochondrial matrix, respiratory rates [12], calcium homeostasis [13], ROS generation [14] and heat production [15].

2.3. Redox Reactions in the ETS

The first ETS redox centers were described in the nineteenth century by Charles MacMunn. In 1883, he found a peculiar pigment (myohematin) in the muscle of insects, whose light absorption pattern was quite similar to heme. MacMunn proposed the respiratory nature of these pigments and suggested that they were not derived from hemoglobin since they were found in organisms that knowingly did not have it [16]. Decades later the parasitologist David Keilin revisited the problem using an ingenious device, the microspectrophotometer. During his studies, Keilin found the very same four absorption bands identified by MacMunn not only in the fly, but in Bacillus subtilis and in baker’s yeast. Keilin called the ubiquitous colored pigments cytochromes. Eventually, Keilin also determined that light absorption pattern of the four bands changed distinctly when metabolic poisons were administered, or when yeasts were deprived of oxygen. He concluded that the intensity of light absorption bands resulted from the cytochromes reduction. As a result, it became paramount to understand how cytochromes supported respiration.

Besides heme-containing cytochromes, it is known today that many distinct redox centers are involved in the electron transport along the ETS such as the iron/copper-sulphur clusters, the flavin-containing enzymes, and ubiquinone. These compounds differ not only in composition, but also in the number of electrons transported and their redox potentials. An interesting feature of the ETS is the presence of two mobile electron transfers: the nonproteic organic molecule ubiquinone (UQ) and cytochrome c. Although UQ is quite hydrophobic, it is highly mobile and promotes the bridging between complexes I, II, Glycerol-3 phosphate dehydrogenase, and electron transfer flavoprotein-ubiquinone oxidoreductase with complex III [2]. Unlike the cytochromes and iron/copper-sulphur clusters, UQ can be reduced by two electrons, generating the fully reduced form ubiquinol (UQH2). However, during its redox cycle, UQ can be partially reduced, generating an unstable ubisemiquinone (UQ •− ) radical. Cytochrome

is a small heme protein which is loosely bound to the inner mitochondrial membrane and is responsible for the transport of a single electron. Cytochrome also participates as a major inducer of apoptosis, when released by the mitochondria in response to proapoptotic stimuli, such as calcium and oxidative stress conditions [17]. Cytochrome is bound to inner mitochondrial membrane by means of a direct interaction with cardiolipin which can be disrupted when cardiolipin is oxidatively modified in redox imbalance [18].

The ETS complex I, or NADH:ubiquinone oxidoreductase, is considered one of the largest known membrane proteins and can be visualized by electron microscopy, which reveals its characteristic “L” shape [19]. The structure of this huge protein complex was recently elucidated [20]. Complex I activity couples the transfer of two electrons from NADH to ubiquinone, in parallel with the translocation of four protons across the inner mitochondrial membrane. This activity provides about 40% of the proton-motive force generation coupled to mitochondrial ATP synthesis. In mammals, complex I contains 45 subunits resulting in an apparent molecular mass of about 1 MDa and it has been implicated in many human neurodegenerative diseases.

Complex II, also known as succinate dehydrogenase, converts succinate to fumarate, which is the only TCA cycle reaction taking place at the inner mitochondrial membrane. The electrons from succinate oxidation directly contribute to UQ reduction and oxidative phosphorylation as well. The elucidation of complex II structure revealed that the architecture of its redox centers is arranged in a way that prevents ROS production at the FAD site [21, 22]. Complex II contains four subunits, two of which are integral membrane, while the other two face the mitochondrial matrix, which contains covalently bound FAD and three iron-sulphur clusters.

Glycerol 3-phosphate dehydrogenase (G3PDH) has two isoforms, the cytosolic (cG3PDH) and the mitochondrial (mG3PDH). The mG3PDH is bound to the inner membrane facing the mitochondrial intermembrane space [23] and transfer electrons generated from dehydrogenation of G3P to UQ. The activity of this enzyme is closely associated to the oxidation of cytosolic NADH from the glycolytic pathway, regenerating the “pool” of NAD + from glycolysis.

The other component of ETS contributing to electrons entry is the electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), which is an intrinsic membrane protein located in the inner mitochondrial membrane. ETF-QO contains an FAD molecule and a [4Fe4S] cluster. The protein represents the only input site of the ETS for electrons derived from nine flavoprotein acyl-CoA dehydrogenases and two N-methyl dehydrogenases, which are eventually transferred to UQ [24]. Incidentally, of the four distinct ETS electrons entry sites, only complex I contributes to the Δp, as it couples the energy of electrons transport to the proton translocation across inner mitochondrial membrane.

Complex III, also called ubiquinol:cytochrome oxidoreductase, couples the transfer of two electrons from UQH2 to cytochrome generating a proton gradient across the inner mitochondrial membrane. The redox centers involved in complex III activity are the cytochrome b, which has two heme type b (BL and BH), cytochrome

, and the Rieske iron sulphur protein [2Fe-2S] [25]. Complex III is a dimer. Each monomer consists of 11 different polypeptide subunits yielding a total of 240 kDa [25]. The presence of two active sites in complex III is an essential feature for the operation of Q cycle [26]. One of the sites is responsible for UQH2 oxidation and proton translocation to the intermembrane space and is located near the cytoplasmic side of the inner membrane (Qp). The other site is responsible for reducing the UQ, capturing electrons from the inner side of the membrane and is located near the matrix side (Qn). Since UQH2 donates two electrons and the cytochromes are reduced by only one, electron transfer from the UQH2 to complex III is bifurcated. The first step comprises the oxidation of UQH2 at the Qp site of complex III. One of its electrons is transferred to the Rieske iron sulphur protein while the other is transported to heme

. There are two fundamental reasons for the passage of the two electrons from UQH2 to cytochrome and cytochrome . The first, and more obvious, is the fact that UQ is reduced by two electrons and two protons, while cytochrome by a single electron. The other is structural. Because the Rieske center is mobile in complex III and directs the electrons to reduce cytochrome c, eventually it returns back to the Qp site [27]. Thus, at the same time, the Rieske center is close to cytochrome c, and distant from the site receiving the Qp of another UQH2 electron [25]. During the Q cycle, for each pair of electrons, two protons are consumed from the matrix and four protons are released into the intermembrane space, promoting the net transport of two protons.

The electron present at the cytochrome is then transferred to the terminal ETS complex IV, also known as cytochrome oxidase. The mammalian complex IV is composed of 13 subunits and contains several redox centers such as two hemes, one cytochrome

, and two copper centers, the CuA and CuB centers. In fact, the site of oxygen reduction to water is composed of a binuclear center which contains cytochrome and CuB. Complex IV catalyzes the transfer of four electrons from four reduced cytochrome to oxygen, completely reducing it to two water molecules [28]. Oxygen reduction involves a complex redox cycle in which CuA and CuB centers, as well as the heme a, heme and a tyrosine residue participate. Firstly, the oxygen molecule binds to the enzyme complex at the heme -CuB binuclear center on its fully reduced state in the following redox configuration: heme

. In fact, molecular oxygen binds to the binuclear center at the heme site and then the bonds between oxygen atoms are disrupted in such a way that one of the oxygen atoms remains bound to heme site and the other one, to the CuB center. During this step, two electrons are transferred from the heme to the oxygen atom bound, adopting the heme

oxidation state (ferryl). The other oxygen atom is reduced by means of transfer of two electrons, one originating from center, which becomes , and the other one from the tyrosine 244 residue, which is cross-linked to histidine 240 where the CuB is bound. In the next step, the tyrosine is regenerated with electrons from one reduced cytochrome molecule which is transferred via heme . An additional electron is transferred from one reduced cytochrome molecule, through heme a, to the heme , which converts to its +3 redox state. Complete enzyme regeneration is achieved by further delivery of two electrons, from two reduced cytochrome molecules, through heme a, to the active site, restoring the heme and redox configuration and allowing each of the two oxygen atoms originally in oxygen to dissociate as water. Interestingly, the very same site where oxygen binds to the cytochrome oxidase, the heme at the binuclear center, is also able to bind other ligands such as cyanide (CN-) carbon monoxide (CO) and nitric oxide (NO), all of which are respiration inhibitors [2].

During the sequential redox reactions of electrons transport along the ETS to their final acceptor, molecular oxygen, a significant part of the energy is conserved as protons are transported from the mitochondrial matrix to the intermembrane space, generating the Δp, specifically at the complexes I, III, and IV. As proton transport is thermodynamically unfavorable, coupling the energy released by the electrons transport at complexes I, III, and IV overcomes the energy barrier.

2.4. The Chemiosmotic-Dependent ATP Synthesis

The ETS-dependent proton transport across the inner mitochondrial membrane allows the Δp formation and represents the ultimate energy source by which mitochondrial ATP is synthesized [10]. One of the first proposals was based on the idea that electrons passage through the ETS would release a finite amount of energy that would be trapped in the form of a phosphorylated intermediate “X” which would then transfer its “high energy phosphate” to ADP by specific enzymes [29]. Initial efforts to identify intermediate “X,” which for a while was thought to be phosphohistidine, failed.

Then, a revolutionary idea was proposed by Mitchell in 1961 [30]. Mitchell’s hypothesis was based on the concept known as vectorial metabolism, which stated that substrates could be transported across a membrane by an enzyme with a particular orientation against a chemical and electrical gradient. This could only be achieved if it was coupled to another thermodynamically favorable reaction such as ATP hydrolysis. Mitchell’s chemioosmotic hypothesis explained mitochondrial ATP synthesis, which in several ways resembles the fuel cell-type, and set the basis for a concept hinging on the coupling of electron transfer to ADP phosphorylation. The supramolecular organization of the enzymes at the inner mitochondrial membrane is an essential component to be considered in this proposal and Mitchell referred to it as anisotropy. In essence, the anisotropic enzymes would act as molecular charge splitters across the inner mitochondrial membrane, in which the redox reactions at the ETS complexes resulted in the separation of protons and hydroxyl anions to each side of membrane, creating the so-called protonmotive force (Δp). As respiration proceeded, the resulting increase in the Δp would drive the separation of protons and hydroxyl anions at the active center of the ATPase, allowing ADP and inorganic phosphate dehydration and ATP formation. In addition, this ingenious proposal offered explanations for many other observations such as (i) the effect of uncouplers, (ii) the regulation of redox state of the ETS components by the magnitude of the electrochemical potential, (iii) the photochemical phosphorylation in chloroplasts, and (iv) the swelling and shrinkage effects on mitochondria associated to the changes in the electrochemical potential. This hypothesis was extensively validated experimentally, being eventually consolidated in 1967 after Mitchell’s answers to the criticisms raised by Slater [10]. Although the respiration coupled to ATP synthesis was mechanistically demonstrated, it did not explain how the ATP molecules were produced by the ATPase using the energy accumulated in the form of Δp.

The fundamental basis of the mechanism by which the F1Fo ATP synthase complex produces ATP at the expenses of the Δp was made possible by the research conducted in the Laboratories of Efraim Racker, John E. Walker, Paul D. Boyer and many others. In fact, a mitochondrial ATPase activity was directly involved in the mechanism by which ADP phosphorylation is coupled to electron transport [31]. This complex was first observed by electron microscopy in the 1960s [32]. Two distinct subcomplexes were seen: one associated to the inner mitochondrial membrane (Fo) and the other facing towards the matrix (F1). Purification of the whole F1Fo ATP synthase was achieved as well as and the characterization of both F1 and Fo activities [33]. In 1973, Paul Boyer observed that the exchange of labeled oxygen between inorganic phosphate and water was not blocked by uncouplers of oxidative phosphorylation, but was inhibited by oligomycin (a compound that blocks F1Fo ATP synthase activity). Interpretation of these results led to the suggestion that ATP might be formed at the catalytic site of this complex. Thus, a significant part of the energy transduced by oxidative phosphorylation is utilized to drive the release of preformed ATP from the enzyme [11]. Later, in 1977, Boyer advanced this concept by proposing an alternating site model for oxidative phosphorylation, in which ATP is formed at one site of the enzyme site, but is transitorily tightly bound. This ATP was not released from enzyme unless ADP and Pi bound a second enzyme site and the ATPase complex became energized [34]. Therefore, net ATP formation by oxidative phosphorylation occurs by a cooperative mechanism involving alternate conformational changes in the β subunits of F1, promoted by the passage of protons through the Fo site of this complex [35].

The passage of protons from the intermembrane space to the mitochondrial matrix mediated by the Fo site would drive a rotational movement of the whole Fo which, in turn, would transfer the rotation movement to the γ subunit and then to the β subunits at the F1 site. As the interactions between the γ subunit and each one of the three β subunits are unique, γ subunit rotation induces a specific conformation in each of the β subunits (open, loose, and tight). When the β subunit adopts an open conformation, ATP is released from the enzyme and the active site becomes empty, while the neighboring β subunit adopts a loose conformation, binding ADP, and inorganic phosphate. Finally, the third β subunit is in the closed conformation, expelling water from its active site, allowing the thermodynamically spontaneous ATP synthesis.

2.5. Mitochondrial Redox Metabolism

Since a long time, it was known that oxygen played essential biological functions ranging from biomolecule modification to cellular respiration. However, life arose long before oxygen could accumulate in the atmosphere in order to be utilized by cytochrome oxidase. In fact, evidence indicates that organisms in the primitive Earth had simpler metabolic pathways that were not able to fully utilize the energy contained in nutrients. Also, the process of respiration seems to have emerged before the occurrence of significant amounts of oxygen in the atmosphere, as a result of photosynthetic activity. Evidence supporting this interpretation was derived from microorganisms that utilize electron acceptors other than oxygen. Examples are iron, sulfate, vanadium, and even uranium. Along Earth’s evolutionary history, organisms that were able to use the sunlight as an energy source to allow water oxidation coupled to molecular oxygen production had a clear advantage. From the energy perspective, oxygen utilization allows a more efficient use of the energy stored in the nutrients through the process of oxidative phosphorylation. Thus, organisms lacking oxygen transport and storage systems, relying simply on its diffusion to the inner parts of its body, exhibited a growth rate that strongly limited by oxygen availability. This idea seems to offer an excellent explanation for the large number of giant fossil records aged approximately 300 million years that lived when atmospheric oxygen levels reached about 35%.

Oxygen is not an inert gas and its toxicity was firstly reported by Paul Bert way back in 1878. He showed that oxygen was toxic to a number of invertebrates as well as fungi, germinating seeds, birds, and even other higher animals. In the central nervous system of mammals, oxygen toxicity was referred to as the “Paul Bert effect.” The mechanisms underlying cellular oxygen toxicity were further studied by Rebecca Gerschman in 1954 who proposed that oxygen potentiated cell death induced by X-ray irradiation [36]. The conclusion was that oxygen and ionizing radiation share mechanisms that possibly involved the formation of “oxidizing free radicals.” A free radical is defined as any atom or molecule that has at least one unpaired electron in an orbital [37]. The term ROS is used to designate not only oxygen-derived free radicals, but also nonradical oxygen species that are capable to generate highly reactive oxygen radicals, such as hydroxyl radical [37]. Because free radicals have unpaired electrons, they tend to achieve stability by donating or removing electrons from adjacent biomolecules such as sugars, lipids, and proteins, resulting in their structural modification. The accumulation of altered or damaged biomolecules by free radicals is associated to a multitude of functional changes in cells, such as apoptosis, mutations, inhibition of enzyme activities, and oxidative stress [37].

Much of the biomedical interest regarding the ROS are due their potential role in the pathogenesis of many diseases and also in aging. In this regard, the seminal work of Harman in 1956, established the well-known “Free-radical theory of aging,” which stated that aging is a result of chronic oxidative modification of biomolecules and structures within the cells that ultimately culminate in death [38]. According to Harman, the cellular free radicals would probably arise by reactions involving molecular oxygen as a result of dehydrogenase activity. Later in 1969, McCord and Fridovich made a central contribution by establishing a link between biology and free-radical chemistry. A copper-containing enzyme, previously identified by Keilin in 1939 as hemocuprein, was found to have a key activity of dismutating superoxide radicals into oxygen and hydrogen peroxide in bovine erythrocytes [39].

Most of the oxygen consumed by the cell is completely reduced to H2O by cytochrome oxidase. However, a small portion of this is partially reduced by mitochondria, generating ROS. Complexes I, II, and III of ETS are sources of ROS. In fact, the ETS is not only capable of generating free radicals such as the superoxide radical (O2 •− ), but also ROS such as hydrogen peroxide (H2O2). The first demonstration of mitochondrial ROS formation was made in 1966 by Jensen, who showed that succinate and NADH were able to support hydrogen peroxide formation and this was strongly potentiated by the metabolic poison antimycin A [40]. In the cell, ROS production usually occurs during electron transport along ETS, however, some pathophysiological conditions can increase mitochondrial ROS production because they reduce the activity of ETS or decrease ADP content in mitochondria leading to an increase in the magnitude of the membrane potential, a condition associated with increased ROS “leakage” [41]. Further developments have shown that not only the oxygen pressure, but also the magnitude of the membrane potential strongly affected mitochondrial ROS generation [42]. However, mitochondria are not the only cellular source of ROS. NADPH oxidases, peroxisomes, and endoplasmic reticulum also represent important sites of ROS production.

The imbalance between ROS generation and removal might lead to the so-called oxidative stress. The antioxidant defenses found in biological systems to avoid oxidative stress may be divided into preventive (inhibition of ROS generation), scavengers (suppression of unpaired electrons), and repair (repair of molecules damaged by ROS). The UCPs and hexokinase bound to mitochondria act as preventive antioxidant systems, because both mechanisms aim to reduce membrane potential, promoting mitochondrial depolarization. Cancer cells as well are characterized among other features, by a high expression of hexokinase bound to mitochondria through the VDAC protein. Originally, it was thought that the VDAC bound hexokinase had an exclusive role in the maintenance of the high glycolytic flux typical of cancer cells, but similarly to the mouse brain cells, hexokinase of tumor cells may also maintain the redox balance through an ADP recycling mechanism [43, 44]. The remaining two groups of scavengers are enzymatic (enzymes like superoxide dismutase, catalase and glutathione peroxidase) and nonenzymatic (α-tocopherol, thioredoxin). Examples of the repair antioxidant defenses are the poly (ADP ribose) polymerase (PARP) and aldehyde dehydrogenase.

3. ROS and Cancer

In the light of Otto Warburg’s original observations concerning aerobic glycolysis, it became pertinent to ask whether the mitochondria of tumor cells were functional or not. This question is by no means easily answered and even after 85 years into the post-Warburg era, the issue remains conflicting, or at best riddled with misconceptions. One example of this situation is the oft repeated puzzlement about the cancer cell’s selection of the less energy efficient anaerobic glycolysis over the more ATP-rich tricarborxylic acid cycle in conjunction with oxidative phosphorylation. Actually, what cancer cells frequently do is to combine the best of the two pathways in order to sustain the intense proliferation as well as the metastasis that accompanies the transformed state. The glycolytic pathway does not just generate ATP and lactate as the end product. Many of its intermediate metabolites are recognizably anaplerotic in nature as is the case of 3-phosphoglycerate which acts as a precursor to serine, glycine, and cysteine synthesis, all of which are essential for the anabolic condition seen in tumorigenesis. Indeed, it has been recently shown that in human breast cancer, one of the genes that consistently displays a gain in copy number is phosphoglycerate dehydrogenase a result that highlights the association between glucose uptake and aminoacid synthesis [45]. Besides, it must be remembered that glycolysis is a much faster process in terms of ATP synthesis, so that when not hindered by limited amounts of glucose, tumor cells are not badly off. When dealing with the term functionality with regards to mitochondria it must be taken into account that the organelles are a hub to many essential cellular processes. Whilst mitochondria of tumor cells may perform some functions as well as those from normal cells others may be deficient and thus there is a need to specify which of these bear on cancer causally. This question will be addressed below with emphasis on those pathways that are at the same time anomalous and, therefore, amenable to interference by specific inhibitors, especially those connected to the bioenergetics scenario associated to cell transformation.

Functionality of the mitochondria has been addressed in several ways. Many papers dealing with the properties of the organelle, particularly when comparing tumor and normal cells, approach the question in a rather loose and sectored manner. For example, it is common to refer to the physiological status of the mitochondria in terms of its morphology, its respiratory function including ATP synthesis, its role as regulators of intracellular Ca 2+ homeostasis, or as essential elements in the processes of cell proliferation and apoptosis. Frequently in the literature, the mitochondria are considered functional or not on the basis of analysis that may not be entirely informative of the status of the organelle. For example, in some cases, conclusions about the integrity of mitochondria, or the cell as a whole, are based solely on the ability of certain enzymes to reduce tetrazolium salts and on the assumption that if the enzymes are active, then the organelle and the cells are also viable. Clearly, then, it becomes important to consider what weight should one attribute to these individual parameters and whether they could represent bona fide markers regarding the general health of the mitochondria and sufficient to classify them as dysfunctional. Perhaps a better description for the supposedly aberrant behavior of the organelles within the context of malignant transformation would be “deviant.” The questions that naturally follow this initial discussion are deviant mitochondria are the cause or consequence of cancer? If mitochondria are in fact key elements in cell transformation, which alterations predispose cells to cancer?

3.1. Morphology

It is not simple to define what is the normal morphology of mitochondria, mainly because they exhibit considerable plasticity and change their shape radically even when functioning within normal cells. Mitochondria may display transient shape changes as a result of energy demand, that is, oxidative phosphorylation (OXPHOS). A condensed appearance has been associated to mitochondria actively undergoing OXPHOS, whereas the orthodox conformation reflected diminished oxygen consumption. Since in many types of cancer cells the mitochondria have been shown to respire normally, even though with less intensity than glycolysis, it may be difficult to observe morphologies that diverge significantly from the condensed appearance. In other words, the cancer-related morphology of mitochondria may be merely a reflection of the occurrence, or not of the Warburg effect (see below).

Morphology of the organelle refers also to the dynamic processes of fission and fusion with one another and with other organelles that mitochondria undergo in parallel with the cell cycle. Mitochondrial fusion and elongation produces a branched tubular network spreading throughout the cytosol that characterizes what is generally known as mitochondrial networking. Although the mechanism of mitochondria fission, fusion and elongation, is not yet fully understood, some of the key players in this process have been identified and it was the analysis of these components that suggested to a certain degree the distinction between normal and tumor cells. In normal cells, mitochondrial fission occurs in synchrony with cell division. As the cells enter mitosis, mitochondria too begin to fragment, an event which is largely regulated by a GTPase dynamin-related protein 1 (Drp 1), a major component of the fission apparatus. The fission machinery also requires the presence of hFis 1 which is integrated in the outer membrane of the mitochondria. It is thought that interaction between hFis 1 and Drp 1 alters the conformation of the latter leading to the formation of a constricting ring around the mitochondria which ultimately produces fragmentation [46, 47]. In turn, Drp 1 activity itself depends on posttranslational modifications, namely, phosphorylation catalyzed by Cdk 1/cyclin. In addition, the half-life of Drp 1can be modified. It is enhanced by sumoylation by SUMO1 which protects Drp 1 from degradation via proteasome and decreased by deSUMOylation mediated by the protease SenP5. Incidentally, these reactions illustrate quite well how mitochondrial fission responds to elements that normally control the cell cycle and thus becomes synchronous with cytokinesis. Exception should be made to tissues in which cells do not proliferate, such as in muscle. Although the dynamin-related GTPases are the core components of the mitochondrial fission mechanism, recent data have implicated other proteins as upstream regulators of the Drp 1/hFis 1complex and hence of mitochondrial networking [48, 49].

Upon completion of cytokinesis, mitochondria reconnect again through fusion, a complex event that involves the merging of the double lipid membrane of the mitochondria. This is mediated by profusion proteins located on the surfaces of the inner and outer membranes, the complex formed between Mgm1p and the optic atrophy protein OPA1 together with mitofusins (Mfn1 and Mfn2), respectively. The mitofusins also play a role in tethering the mitochondria to other organelles such as the endoplasmic reticulum [50]. This type of interaction does affect Ca 2+ signaling and exemplifies how mitochondrial networking could have far reaching effects on metabolism as well. By the same token, loss of Mfn2 is known to affect the expression of the subunits that make up the respiratory complexes leading to reduced cellular oxygen consumption. Independently of direct actions of mitofusins on mitochondrial tethering, Mfn2 also regulates the ERK/MAPK signaling pathway a feature that can have a direct bearing on tumorigenesis as it will be discussed ahead [51].

So what is the consensus regarding mitochondria morphology when comparing normal and tumor cells? Within the context of mitochondrial networking, it has been proposed that mitochondria of normal cells spend most of their time in a fragmented mode (postfission state) before they fuse again [52, 53]. Along those lines it has been reported that tumor cells also display a higher frequency of fragmented mitochondria [54], which would indicate the prevalence of fissional events in those organelles. A situation that could be considered analogous would be apoptosis. Reports have shown that when cells are subjected to overexpression of proapototic members of the Bcl-2 family, they exhibit a higher rate of Drp-1-dependent mitochondrial fragmentation and that over expression of fusogenic proteins such as Opa-1 (promoting mitochondrial fusion) protects from apoptosis [55, 56]. Our own results confirm this interpretation indirectly. The mitochondria of H460 cells treated with sodium butyrate, which inhibited cell proliferation, were shown to be more elongated than controls. This was accompanied by a higher expression of Mfn1 suggesting that mitochondrial fusion could be associated to lower rates of cell proliferation [57]. Taken together, the data indicate that except for those cases in which oxidative stress directly induces mitochondrial fission [58], the balance between mitochondrial fusion and fission might depend primarily on the proliferative (or apoptotic) status of the cells. Along those lines, it would be interesting to compare the variations of mitochondrial morphology in different types of synchronized cultures of tumor cells versus that of the normal cell counterparts. In vivo, however, there would be experimental complications since it is known that cell doubling time and mitotic indexes are highly heterogeneous even within tumors and also when different types of cancer are compared. In conclusion, so far the morphology of mitochondria cannot be unequivocally associated to cell transformation. The structural mitochondrial alterations observed in many types of tumors cannot be ascribed to any specific neoplasm and this question remains largely unresolved.

3.2. Are Mitochondria of Tumor Cells Dysfunctional?

Papers that address the intermediary metabolism of tumor cells typically begin the discussion by quoting the Warburg effect and usually mention that glycolysis replaces mitochondrial oxidative phosphorylation as the principal source of cellular ATP. Frequently, these opening remarks are followed by statements that indicate that the high glycolytic flux adaptation occurs because the mitochondria of tumor cells are dysfunctional or partially disabled. However, the aerobic glycolysis described by Warburg did not imply that the mitochondria were dysfunctional since he himself acknowledged that tumor cells continued to consume oxygen at levels comparable to those of normal cells. In other words, what Warburg really noted was that tumor cells did not exhibit the Pasteur effect, that is, glycolysis in tumor cells persisted even in the presence of oxygen.

The longstanding notion that mitochondria are somehow defective presumably derives from the fact that researchers approach the problem in a nonholistic fashion and frequently assume that if one set of results obtained from tumor cells significantly differs from the normal cell counterparts, other downstream events, even if not investigated, will vary as well. Some metabolic modifications have indeed been detected that when considered individually justified the belief that the mitochondria of cancer cells were somehow impaired. These include the preference for particular respiratory substrates, rates of electron transport, and the activities of enzymes involved in oxidative phosphorylation [59]. Albeit those early reports, data have accumulated to the effect that recently, there has been a shift in opinion. There is now a tendency to accept that mitochondria of cancer cells rarely present defects and seem to retain their capacity to carry out oxidative phosphorylation and consume oxygen with levels comparable to those of normal cells, much as Warburg himself had stated [60]. According to this view, the enhanced lactate production observed in tumor cells does not necessarily imply the cessation of mitochondrial activity, as confirmed by several experiments in which oxygen consumption of cancer cells was evaluated. Such results also indicate some degree of independence between cytoplasmic glycolysis and mitochondrial metabolism acquired by the tumor cells which may explain the absence of the Pasteur effect in cancer cells through the loss of a regulatory interface. In this respect, it was shown that the cytotoxic effect of 2-deoxyglucose (2-DG) on A549 lung cancer cells depended on inactivation of mitochondria in p53 -/- cells. The observation that the Warburg effect was only evidenced in cells in which mitochondria were impaired supported the idea of a functional link between glycolysis and the oxidative reactions of the organelle [61]. Furthermore, the often quoted idea that in cancer cells the mitochondria stop respiring in order to save carbon skeletons for the biosynthesis of other biomolecules required for rapid growth is not compatible with the observation that tumors actually excrete high amounts of lactate [62]. That is not to say that the macromolecules and lipids found in the intramitochondrial milieu do not display alterations that while observable may not significantly compromise mitochondrial respiratory function. The enhanced production of reactive species of oxygen (ROS) and reactive nitrogen species (RNS) associated to cancer cells (discussed in Section 3.3) can definitely cause damage to proteins, DNA, RNA, and membranes. However, the mutations produced in mtDNA are not necessarily synonymous with tumorigenesis. It is known that germline mutations of mitochondrial DNA can cause diseases that affect children and adults ranging from mitochondrial myopathies to retinitis pigmentosa and possibly even to autism, but not cancer. In an analogous manner, sporadic mutations that may result from oxidative damage due to elevated ROS in the mitochondria are suggested by many to be the culprits of tumorigenesis. This has been difficult to demonstrate, however, mainly because there is no strong evidence showing that the mtDNA mutations are driver mutations. Also, the majority of somatic mutations found in mitochondrial DNA are not harmful to the cells and populational surveys showed that the so called specific cancer mutations (varying from 30–100%) are frequently found in mitochondrial DNA of individuals with no history of cancer. Hence, it is thought that ROS-induced mtDNA mutations may actually occur as a consequence of metabolic reconfiguration of cancer cells. Nevertheless, papers abound that correlate the mitochondrial mutations to several types of cancer [63].

How to resolve this quandary? There is a very tight connection between the nuclear and mitochondrial genomes coordinating the expression of exons encodings different subunits of proteins making up the electron transport chain. Thus, it would be desirable to separate the individual contributions of nuclear and mitochondrial DNA mutations to the cancer phenotype. After the advent of the technique of cytoplasmic hybrids (cybrids), or transmitochondrial cybrids, it became possible to repopulate cells from which mitochondria had been depleted, with exogenous mitochondria present in enucleated cells. In this manner, the information obtained from the fused cells, for example, cancer cells, should highlight which events could be assigned to the mitochondria alone [64]. Results obtained with this experimental approach have demonstrated that many of the respiratory alterations ascribed to mitochondria of cancer cells (and other pathologies) could be reproduced in the “transplanted” cells. For example, Imanishi and collaborators [65] were able to show that mitochondrial respiration defects observed in human breast cancer cells caused by mtDNA mutations were responsible for the expression of high metastatic potential in recipient cells. Interestingly, these experiments with cybrids demonstrated that the mutated mtDNA affected metastasis, but not cell transformation. Results that confirmed that the increase in metastatic potential was acquired were also obtained in a mouse model in which the transferred mtDNA had a mutation in the NADH dehydrogenase subunit 6, a deficiency that augmented the production of ROS. In these experiments, the metastatic potential of the cybrids was enhanced and use of a scavenger such as N-Acetyl cysteine was able to counteract it [66]. Correlations exist showing that ROS and RNS are able to inhibit the process of anoikis, that is, the occurrence of apoptosis as a result of loss of cell adhesion. In normal cells, anoikis prevents detached cells from colonizing different tissues. According to this, the oxidative stress generated by ROS and RNS overproduction in tumor cells would favor metastasis by means of activation of prosurvival signaling pathways that in turn would inhibit anoikis and in this manner boost the progression of cancer [67]. It would be very interesting if it could be demonstrated that alterations in the mtDNA alone and by extension the higher production of ROS could be responsible for metastasis. Other parameters such as diminished cellular oxygen consumption and ATP synthesis observed in human breast cancer cells could also be successfully passed onto the cybrids and thus were firmly interpreted as derived from mitochondria [68]. Diminished respiratory rates, except when connected to damage or reversal of the electron transport chain, however, would generate less ROS a result which would go against the grain according to the canonic view of ROS-induced tumorigenesis.

Although with the aid of the cybrid technology, the role of mitochondria in tumorigenesis could be better appreciated, caution should still be exercised to avoid misinterpreting, or overstating certain selected parameters. Some of those have limited information. For instance, when studying mitochondrial respiratory control, values for the P/O ratio, a very popular analysis, do not really define whether mitochondria are functional or not since on its own they reflect proton leak as a result in fluctuations of states 3 and 4 of respiration. Others, as in the case of experiments using isolated mitochondria, may result from manifestations that occur independently of the regulatory grid that normally control the organelles. Thus, experiments generating data based on morphology (see Section 3.1) as well as evaluation of reactions to specific stressors have to be interpreted with reserve because they abrogate the innate responses connected to HIF-1α and hypoxia. In addition, when using intact cells to investigate mitochondrial function, researchers have to make use of detergents such as digitonin in order to allow access of cell-impermeant substrates to the organelle. This experimental resource may produce artifacts due to damage to the mitochondria outer membrane resulting in the release of components such as cytochrome c that in turn may trigger cellular responses independently of the bioenergetics analysis being carried out. In a recent review, those questions have been clearly and carefully dissected pointing out the pros and cons of each approach [69].

The importance of mitochondria as fully functional organelles in cancer cells has been strengthened by considering the recently proposed hypothesis that metabolically they actually conform to the orthodox model of ATP production via the regular set of mitochondrial oxidative reactions, like the TCA cycle, oxidative phosphorylation, and the anaplerotic glutamine utilization pathway. According to this empirically based hypothesis, cancer cells are not considered as rogue cells that become immortalized and manage to live independently of other tissues. Reports have described a lactate shuttle that is formed between stromal cells and the cancer cells, in which the former predominantly glycolytic, feed the latter, the oxidative tumor cells that utilize mitochondria to their full capacity. This hypothesis maintains that aerobic glycolysis, the hallmark of many tumors, is actually carried out by the cancer associated fibroblasts rather than the cancer cells themselves [70, 71]. With the metabolic symbiosis thus established, the stromal fibroblasts would undergo autophagy and mitophagy and as a result secrete and supply lactate to the cancer cells. In turn the fibroblasts would profit from the available mitochondria in the latter. This phenomenon was termed the “reverse Warburg effect” and the autophagy/mitophagy occurring in the stromal fibroblasts would be induced by oxidative stress triggered by the cancer cells. The proponents of this model went as far as mentioning that, in some cases, the Warburg effect might in fact represent an in vitro artifact. Although a similar metabolic symbiosis seems to occur in normal cells, it remains to be demonstrated whether the reverse Warburg effect could be extrapolated to other types of cancer [72]. At any rate lactate fueled respiration, a feature of metabolic symbiosis, has been demonstrated in tumor cells in mice, thus strengthening the idea of the interdependence that exists between normal and tumor cells [73].

In conclusion, it can be safely stated that the mitochondria of tumor cells are functional and that they may have a significant role in the maintenance of proliferation and metastasis.

3.3. ROS and RNS Production in Tumor Cells

This section tries to appraise the role of ROS and RNS in cancer formation. At the same time, it introduces the question whether the oxidative stress signature of cancer cells can indeed be a prime target for therapy. ROS and RNS can be regarded as decidedly toxic to the cells by considering many of their direct or indirect effects on biomolecule targets. However, toxicity, or for that matter the adjuvant role in carcinogenesis that ROS and RNS have appeared to depend ultimately on their final concentrations at a given instant and on the duration of the stimulus. In consequence, ROS and RNS levels depend on the regulation of the pathways that generate them as well as those that degrade them (scavengers). When ROS and RNS concentrations are within a certain range of the concentration gradient, “physiological” steady-state levels of cellular ROS and RNS perform the housekeeping coordination of metabolic and genetic processes. As such H2O2, for example, stimulates cell proliferation by acting as modulators of various transcription factors that in turn influence several important cellular processes. Among the transcription factors, NF-

B, Nrf2, p53, HIF-1α, and STAT3 could be mentioned. The mechanism whereby ROS modulate transcription factors involves the reversible oxidation of cysteine residues of proteins belonging to signaling pathways such as protein tyrosine kinases and phosphatases, lipid phosphatases, proteases, and signaling effectors. Exposure of proteins to the oxidative environment generates dityrosine residues that can be considered as oxidation markers. Apart from its effect on proliferation, H2O2 also mediates cell differentiation and migration. As opposed to the physiological role of ROS in normal cells, the oxidative stress is characterized by situations in which the levels of molecular oxygen or its ROS derivatives increase above the threshold of normality and produce widespread irreversible oxidation of aminoacids, polydesaturation of fatty acids, and mutations on DNA and RNA. Mutations in nucleic acids occur by formation of C5-OH and C6-OH adducts of thymine and cytosine or similarly with purines, or by reaction with the sugar moiety of the polymer leading in some cases to single or double-strand breaks, intrastrand cross-links, and protein-DNA cross-links [74–76]. This destabilizing effect of ROS on DNA can promote genomic instability which as a consequence may predispose the cells to malignant transformation. In this context, cancer cells that are able to survive in a supposedly hostile microenvironment of high ROS and RNS could be regarded as a subpopulation that were selected in terms of their peculiar metabolic adaptations. This view, however, is not without controversy. Before discussing the possible mechanisms of adaptation, it is relevant to inquire whether there is a consensus concerning the ROS associated etiology of cancer.

What then, if there is a pattern, is the ROS/RNS phenotype of cancer cells? Are ROS and RNS able to act as stimulants of cell transformation cells by subverting the normal control network through a sustained oxidative environment, or do they act directly producing harmful modifications on lipids, proteins and RNA/DNA? Or both? The earlier work of Szatrowski and Nathan [77] showed that relatively large amounts of hydrogen peroxide, comparable only to polymorphonuclear leukocytes and monocytes, were produced by a number of tumors. Since then, the list has grown and many reports indicate that cancer cells produce massive amounts of ROS to levels that exceed the capacity of the antioxidant enzymes that are normally in charge of ROS detoxification. Adding to this, there is evidence showing that excessive ROS production causes the progressive inactivation of the antioxidant enzymatic systems, a condition that favors the maintenance of high concentration of ROS and the induction of a chronic oxidative state. Results published by several groups indicate that it is this situation that sets the scene for transformation. According to this hypothesis, the maintenance of parameters such as cell proliferation within the boundaries of normalcy would depend primarily on the redox balance established between ROS production and the activity of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, glutathione reductase, catalase, and thioredoxin as well as the nonenzymatic antioxidants scavengers glutathione and vitamins C and D.

In contrast, increased levels of ROS, that may result from a failure of the antioxidant system rather than from overproduction, have been associated to chronic degenerative conditions such as cancer, aging, diabetes, and cardiovascular diseases [78, 79]. The site of the oxidative stress has been narrowed down to mitochondria and was covered extensively in a recent review [80] that highlights the prooxidant role of the altered organelles as protagonists of tumorigenesis. Corroborating this view, the high levels of ROS associated to the pathophysiology of a wide variety of cancers have prompted clinicians to examine the amount of dityrosine residues in proteins exposed to an oxidative environment. Proteins that are highly susceptible to oxidative attack have in fact been termed as advanced oxidation protein products (AOPP) and currently serve as markers for monitoring cancer patients [81]. Numerous other reports have generalized the notion that cancer cells are more challenged by oxidative stress than normal cells [82]. Among the types of cancer that have been linked to increased oxidative stress are bladder, brain, breast, cervical, gastric, liver, lung, melanoma, multiple myeloma, leukemia, lymphoma, ovarian, pancreatic, and prostrate [83]. In a number of reports, it has been found that genes involved in oncogenic pathways and those linked to tumor suppression are frequently mutated in transformed cells all of them sharing the common feature of increased amounts of ROS [84–88]. Finally, the hypothesis that ROS and RNS are early effectors of tumorigenesis is in agreement with data revolving around the inflammatory reactions. A hegemonic view on the etiology of cancer states that when an inflammatory condition lasts long enough it behaves as a prodrome to cancer. The normal course of an inflammatory reaction produced by a number of different agents including bacteria, carcinogens, and radiation progresses to a stage in which mast cells and leukocytes are mobilized to the sites of lesion. These cells produce what could be described as respiratory bursts that in turn contribute towards the increase in local production of ROS. The respiratory bursts are actually amplified several fold by other inflammatory cells attracted to the site of inflammation by chemokines and cytokines released by the former cells, so that the oxidative stress actually propagates to neighboring cells. In this context, it is known that the extent of tumor associated macrophage infiltrates correlates well with the prognosis of certain types of cancer. If inflammation is not reversed, it could then create a vicious circle whose outcome is chronic ROS-induced oxidative stress and ultimately, cell transformation. The occurrence of inflammatory reactions with the participation of cell infiltrates in premalignant senescent hepatocytes has also been demonstrated in inflammation-based mouse models of hepatocellular carcinoma [89].

One question could be raised concerning the increase in ROS as an oncogenic factor. Why, in view of the known roles of high ROS in inducing senescence and apoptosis, cancer cells do not undergo apoptosis? Presumably in these transformed cells the pathways connected to senescence and apoptosis are somehow blocked. One proposed mechanism for this inactivation involves p38 MAPK pathway. In normal cells, it is known that elevated ROS induces apoptosis via the p38α MAPK. In contrast, human cell lines in which p38α is inactivated are refractory to ROS-induced apoptosis which suggests that deficiencies in this pathway, as well as those which involve p53 (often mutated in most cancers) allow cancer cells to remain viable in the presence of high ROS [90]. The question whether cancer cells survive in a hostile ROS environment as a result of enhanced activity of antioxidant enzymes and compounds must also be considered.

Many papers attest to the fact that tumor cells are usually well adapted to high levels of ROS and that their viability is only possible due to enhancement of antioxidant activity [60]. However, it should be borne in mind that tumor cells, which occur in a tumor in different stages of transformation, should exhibit heterogeneous metabolic profiles. This would be due to oncogenic gain-of-function and/or loss of tumor suppressors. These alterations may generate a mosaic of metabolic patterns resulting from differentially expressed enzymes which in the same tumor would reveal increased ROS levels and downregulated antioxidant systems. Such a scenario is compatible with the model of waves of gene expression proposed by Smolková and collaborators [5].

As alluded some findings cannot be generalized to all types of tumors and to all situations. Some points that require further discussion are listed below.

(a) Source of ROS: scientists do not agree as to the source of ROS. Many reports state flatly that mitochondria are the main producers of ROS and highlight that organelle as the site where the tumorigenic process begins [91]. Others call attention to the fact that there are alternative and perhaps more important intra- and extracellular ROS generating reactions that include the endoplasmic reticulum, peroxisomes, the cytosol, plasma membrane, and the extracellular space [92]. Among the most important nonmitochondrial sources of ROS is the NADPH oxidase family of enzymes. These are bound to the plasma membranes and to the membranes of phagosomes. Of the seven oxidases that comprise the NADPH family, Nox1, Nox2, and Nox4 are expressed by several types of cancer cells, including colon, prostate, gliomas, melanomas, pancreatic adenocarcinomas, renal carcinomas, and ovarian. Other incidental nonmitochondrial sources of ROS include those that are generated by external factors such as carcinogens and radiation that are known effectors of inflammatory reactions that usually precede cell transformation (see below).

In the context of cancer, the NADPH oxidases should be highlighted not only because of their quantitative contributions to ROS formation, but also as promoters of tumor-induced angiogenesis, an important process adjuvant to tumor growth and metastasis [93]. Supporting the role of NADPH derived superoxides as promoters of tumor induced angiogenesis, experiments have shown that antioxidants such as vitamins C and E reduced the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR-2) in mice [94]. Likewise, in vivo experiments demonstrated that the scavenger NAC was able to inhibit sarcoma-induced angiogenesis, thus strengthening the ROS-induced tumorigenesis model [95].

(b) Are ROS tumorigenic? There is a growing consensus as to the role of ROS in tumorigenesis. In many works, ROS are clearly singled out as the main tumorigenic factors [96, 97]. Some reports, however, indicate that ROS are harmful and tumor cells are only able to survive thanks to efficient or exacerbated antioxidant defense systems [98, 99]. Put another way, mitochondria of tumor cells, which as mentioned above are believed to be fully functional and which according to many are the main producers of ROS, play the simultaneous role of providing energy to the cells, and also of introduce the tenuously controlled oxidative stress, the sword of Damocles as it were [70]. Interestingly, ROS production seems to be much higher in mitochondria in which damage to the respiratory chain occurs, or mitochondria that are actively undergoing reverse electron transport, than those that are normally producing ATP [100]. This situation illustrates then how the processes of autophagy and mitophagy are beneficial to the cells as removers of potential sources of cell transforming ROS [53]. Adding support to the toxic effect of ROS to cancer cells, it is worth mentioning that both, radiotherapy and chemotherapy, normally employed to eradicate cancer cells do so by producing ROS-mediated oxidative stress [97]. Although it is known that ROS have multiple downstream effects, what is emerging as an explanation to conciliate all the conflicting data is that the overall outcome of the ROS-triggered activations/inhibitions depends primarily on their final concentration. In order to even out the data in the literature that show a wide variation in ROS concentrations produced by different types of tumor cells, it would be important to show whether the observed oscillations are actually due to the activities of antioxidant enzymes or the presence of higher levels of free radical scavengers in these cells. This seems to be the case of cancer stem cells that appear to have lower level of ROS than normal cells [101]. Likewise, hematopoietic systems display low levels of ROS although the progenitor cells myeloid produce high levels of ROS. If the hypothesis of antioxidants as the regulators of ROS and hence as pace makers of tumorigenesis proves to be right, the management of cancer should afford many strategies of interference based not only on the use of antioxidants, but on inhibitors acting on ROS downstream signaling pathways. Distinction should be made among the ROS, however. Nitric oxide (NO), for example, has been shown to have antiproliferative effects in both, normal and tumor cells [102, 103], and it has been demonstrated that superexpression of nitric oxide synthase (iNOS) caused inhibition of proliferation of pancreatic tumor cells. When considering the effects of ROS on transformed cells, another issue has to be taken into account which involves the plasticity of tumor cells regarding exposure to ROS. Results of experiments in which tumor cells in culture were incubated in the presence of increasing amounts of ROS showed that they responded by displaying an enhanced tolerance to the oxidative stress. Results obtained by Onul et al. [104] evidenced further that A549 lung cancer cells adapted to long-term high levels of hydrogen peroxide grew better in culture than the parental cell line and were more resistant to the chemotherapeutic agent Doxorubicin. Interestingly, those adapted cells definitely favored a more anaerobic metabolic profile suggesting that the survival strategy adopted might be independent of mitochondria. The same standing applies for high concentrations of nitric oxide (NO). In breast tumor cell lines, high concentrations of NO were shown to induce a phenotypic change [105]. Thus, it may well be that it is the metabolic adaptation of tumour-initiating cells that could dictate the development of cancer rather than HIF activation or other signaling pathways that have been known to affect solid tumor growth. Whether the cells adapted to high ROS would be resistant to antioxidant therapy remains to be investigated. In conclusion, the individual contributions of different cell compartments to the final ROS concentration within the cells may predispose them to transformation, mainly in those cases when the antioxidant systems are not effective in counteracting the oxidative stress. Excess of ROS is also harmful to cancer cells.

3.4. Different Tumors, Different Biochemistries

Attempts to build a grand unifying model of metabolic reprograming in cancer cells that would make biochemical sense are perhaps premature. As it became clear from the preceding discussion, the available data do not always fit into coherent mechanisms and so far ideas do not converge to a single stratagem to combat cancer cells. Apart from the controversies, experimental difficulties, and conflicting interpretations, there are inherent differences that have to be considered before one draws a standard biochemical profile analysis of different tumors. Firstly, normal tissues have individual metabolic rates that would certainly influence the type of metabolism occurring in the cognate transformed cells. Slow-twitch and fast-twitch muscle tissue, for example, obtain ATP preferentially from different pathways. The same applies for several other tissues in which the metabolic diversity reflects the presence of specific isoforms of enzymes, as occurs in liver and brain tissue. It is plausible then to imagine situations in which as a function of distinct metabolic rates, variable levels of ROS would be produced that could cause different types of local lesions. Secondly, individuals are themselves different when considering their biochemical buildup. The emerging field of pharmacogenomics recognizes these differences and the trend is now, taking advantage on available high throughput technology, to carry out individual genome and transcriptome analysis for patients undergoing long term chemotherapy. However, biochemical diversity seems to transcend genes. Population studies have suggested that the majority of cancers is in fact of the sporadic type and hence would reflect the life style of individuals more than their genetic background. So pharmacogenomics must be complemented by “pharmacometabolomics” which could be adapted to the individual needs according to the redox profile of the tumors under treatment. ROS-based treatment would thus select combinations of prooxidant and antioxidants agents that would best counteract the anomalous oxidative stress generated, preferably at the transformative stage. In other words, the prooxidant, and antioxidant medication would be tissue tailored. That the antioxidant, system renders itself as a target for chemotherapy was eloquently shown by Raj and collaborators [99] using a murine model. In this work, cancer cells from different types of tumors, but not normal cells, were effectively killed by piperlongumine, a small molecule derived from the plant Piper longum. In addition, the authors were able to demonstrate that the cytotoxic effect of piperlongumine was achieved through interference with the antioxidant systems of the tumor cells. Such an approach has the added advantage that inhibitors targeting antioxidant systems could also be prescribed on a preventive setting.

References

  1. G. Schatz, “The tragic matter,” FEBS Letters, vol. 536, no. 1𠄳, pp. 1–2, 2003. View at: Google Scholar
  2. D. G. Nicholls and S. J. Ferguson, Bioenergetics, Academic Press, London, UK, 3rd edition, 2002.
  3. A. J. Kowaltowski, N. C. de Souza-Pinto, R. F. Castilho, and A. E. Vercesi, “Mitochondria and reactive oxygen species,” Free Radical Biology and Medicine, vol. 47, no. 4, pp. 333–343, 2009. View at: Publisher Site | Google Scholar
  4. N. Kresge, R. D. Simoni, and R. L. Hill, “Otto Fritz Meyerhof and the elucidation of the glycolytic pathway,” The Journal of Biological Chemistry, vol. 280, no. 4, p. e3, 2005. View at: Google Scholar
  5. K. Smolkova, L. Plecita-Hlavata, N. Bellance, G. Benard, R. Rossignol, and P. Jezek, “Waves of gene regulation suppress and then restore oxidative phosphorylation in cancer cells,” The International Journal of Biochemistry & Cell Biology, vol. 43, no. 7, pp. 950–968, 2011. View at: Google Scholar
  6. R. Diaz-Ruiz, M. Rigoulet, and A. Devin, “The Warburg and Crabtree effects: on the origin of cancer cell energy metabolism and of yeast glucose repression,” Biochimica et Biophysica Acta, vol. 1807, no. 6, pp. 568–576, 2011. View at: Publisher Site | Google Scholar
  7. W. H. Koppenol, P. L. Bounds, and C. V. Dang, “Otto Warburg's contributions to current concepts of cancer metabolism,” Nature Reviews Cancer, vol. 11, no. 5, pp. 325–337, 2011. View at: Publisher Site | Google Scholar
  8. D. J. Pagliarini, S. E. Calvo, B. Chang et al., “A mitochondrial protein compendium elucidates complex I disease biology,” Cell, vol. 134, no. 1, pp. 112–123, 2008. View at: Publisher Site | Google Scholar
  9. E. Gnaiger, “Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology,” International Journal of Biochemistry and Cell Biology, vol. 41, no. 10, pp. 1837–1845, 2009. View at: Publisher Site | Google Scholar
  10. P. Mitchell and J. Moyle, “Chemiosmotic hypothesis of oxidative phosphorylation,” Nature, vol. 213, no. 5072, pp. 137–139, 1967. View at: Publisher Site | Google Scholar
  11. P. D. Boyer, R. L. Cross, and W. Momsen, “A new concept for energy coupling in oxidative phosphorylation based on a molecular explanation of the oxygen exchange reactions,” Proceedings of the National Academy of Sciences of the United States of America, vol. 70, no. 10, pp. 2837–2839, 1973. View at: Google Scholar
  12. B. Chance and G. R. Williams, “Respiratory enzymes in oxidative phosphorylation. I. Kinetics of oxygen utilization,” The Journal of Biological Chemistry, vol. 217, no. 1, pp. 383–393, 1955. View at: Google Scholar
  13. H. Rottenberg and A. Scarpa, “Calcium uptake and membrane potential in mitochondria,” Biochemistry, vol. 13, no. 23, pp. 4811–4817, 1974. View at: Google Scholar
  14. P. K. Jensen, “Antimycin-insensitive oxidation of succinate and reduced nicotinamide-adenine dinucleotide in electron-transport particles II. Steroid effects,” Biochim Biophys Acta, vol. 122, no. 2, pp. 167–174, 1966. View at: Google Scholar
  15. D. G. Nicholls, “Brown adipose tissue mitochondria,” Biochimica et Biophysica Acta, vol. 549, no. 1, pp. 1–29, 1979. View at: Google Scholar
  16. C. A. Macmunn, “An address on some of the applications of the spectroscope to medicine,” BMJ, vol. 1, no. 1566, pp. 3–9, 1891. View at: Publisher Site | Google Scholar
  17. J. C. Yang and G. A. Cortopassi, “Induction of the mitochondrial permeability transition causes release of the apoptogenic factor cytochrome C,” Free Radical Biology and Medicine, vol. 24, no. 4, pp. 624–631, 1998. View at: Publisher Site | Google Scholar
  18. Y. Shidoji, K. Hayashi, S. Komura, N. Ohishi, and K. Yagi, “Loss of molecular interaction between cytochrome c and cardiolipin due to lipid peroxidation,” Biochemical and Biophysical Research Communications, vol. 264, no. 2, pp. 343–347, 1999. View at: Publisher Site | Google Scholar
  19. N. Grigorieff, “Structure of the respiratory NADH:ubiquinone oxidoreductase (complex I),” Current Opinion in Structural Biology, vol. 9, no. 4, pp. 476–483, 1999. View at: Publisher Site | Google Scholar
  20. R. G. Efremov, R. Baradaran, and L. A. Sazanov, “The architecture of respiratory complex I,” Nature, vol. 465, no. 7297, pp. 441–445, 2010. View at: Publisher Site | Google Scholar
  21. V. Yankovskaya, R. Horsefield, S. Törnroth et al., “Architecture of succinate dehydrogenase and reactive oxygen species generation,” Science, vol. 299, no. 5607, pp. 700–704, 2003. View at: Publisher Site | Google Scholar
  22. M. D. Brand, “The sites and topology of mitochondrial superoxide production,” Experimental Gerontology, vol. 45, no. 7-8, pp. 466–472, 2010. View at: Publisher Site | Google Scholar
  23. M. Klingenberg, “Localization of the glycerol-phosphate dehydrogenase in the outer phase of the mitochondrial inner membrane,” European Journal of Biochemistry, vol. 13, no. 2, pp. 247–252, 1970. View at: Google Scholar
  24. J. Zhang, F. E. Frerman, and J. J. P. Kim, “Structure of electron transfer flavoprotein-ubiquinone oxidoreductase and electron transfer to the mitochondrial ubiquinone pool,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 44, pp. 16212–16217, 2006. View at: Publisher Site | Google Scholar
  25. S. Iwata, J. W. Lee, K. Okada et al., “Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex,” Science, vol. 281, no. 5373, pp. 64–71, 1998. View at: Publisher Site | Google Scholar
  26. M. Saraste, “Oxidative phosphorylation at the fin de siecle,” Science, vol. 283, no. 5407, pp. 1488–1493, 1999. View at: Publisher Site | Google Scholar
  27. Z. Zhang, L. Huang, V. M. Shulmeister et al., “Electron transfer by domain movement in cytochrome bc1,” Nature, vol. 392, no. 6677, pp. 677–684, 1998. View at: Publisher Site | Google Scholar
  28. T. Tsukihara, H. Aoyama, E. Yamashita et al., “The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 Å,” Science, vol. 272, no. 5265, pp. 1136–1144, 1996. View at: Google Scholar
  29. E. C. Slater, “Mechanism of phosphorylation in the respiratory chain,” Nature, vol. 172, no. 4387, pp. 975–978, 1953. View at: Publisher Site | Google Scholar
  30. P. Mitchell, “Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism,” Nature, vol. 191, no. 4784, pp. 144–148, 1961. View at: Publisher Site | Google Scholar
  31. H. S. Penefsky, M. E. Pullman, A. Datta, and E. Racker, “Partial resolution of the enzymes catalyzing oxidative phosphorylation. II. Participation of a soluble adenosine tolphosphatase in oxidative phosphorylation,” The Journal of Biological Chemistry, vol. 235, pp. 3330–3336, 1960. View at: Google Scholar
  32. H. Fernández-Morán, T. Oda, P. V. Blair, and D. E. Green, “A macromolecular repeating unit of mitochondrial structure and function. correlated electron microscopic and biochemical studies of isolated mitochondria and submitochondrial particles of beef heart muscle,” The Journal of Cell Biology, vol. 22, no. 1, pp. 63–100, 1964. View at: Google Scholar
  33. R. Serrano, B. I. Kanner, and E. Racker, “Purification and properties of the proton translocating adenosine triphosphatase complex of bovine heart mitochondria,” Journal of Biological Chemistry, vol. 251, no. 8, pp. 2453–2461, 1976. View at: Google Scholar
  34. C. Kayalar, J. Rosing, and P. D. Boyer, “An alternating site sequence for oxidative phosphorylation suggested by measurement of substrate binding patterns and exchange reaction inhibitions,” Journal of Biological Chemistry, vol. 252, no. 8, pp. 2486–2491, 1977. View at: Google Scholar
  35. P. D. Boyer, “The ATP synthase𠅊 splendid molecular machine,” Annual Review of Biochemistry, vol. 66, pp. 717–749, 1997. View at: Publisher Site | Google Scholar
  36. R. Gerschman, D. L. Gilbert, S. W. Nye, P. Dwyer, and W. O. Fenn, “Oxygen poisoning and X-irradiation: a mechanism in common,” Science, vol. 119, no. 3097, pp. 623–626, 1954. View at: Google Scholar
  37. B. Halliwell and J. M. C. Gutteridge, Free Radicals in Biology and Medicine, Oxford University Press, Oxford, UK, 2007.
  38. D. Harman, “Aging: a theory based on free radical and radiation chemistry,” Journal of gerontology, vol. 11, no. 3, pp. 298–300, 1956. View at: Google Scholar
  39. J. M. McCord and I. Fridovich, “Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein),” Journal of Biological Chemistry, vol. 244, no. 22, pp. 6049–6055, 1969. View at: Google Scholar
  40. P. K. Jensen, “Antimycin-insensitive oxidation of succinate and reduced nicotinamide-adenine dinucleotide in electron-transport particles I. pH dependency and hydrogen peroxide formation,” BBA - Enzymology and Biological Oxidation, vol. 122, no. 2, pp. 157–166, 1966. View at: Google Scholar
  41. S. S. Korshunov, V. P. Skulachev, and A. A. Starkov, “High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria,” FEBS Letters, vol. 416, no. 1, pp. 15–18, 1997. View at: Publisher Site | Google Scholar
  42. A. Boveris and B. Chance, “The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen,” Biochemical Journal, vol. 134, no. 3, pp. 707–716, 1973. View at: Google Scholar
  43. W. S. Da-Silva, A. Gómez-Puyou, M. T. De Gómez-Puyou et al., “Mitochondrial bound hexokinase activity as a preventive antioxidant defense. Steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria,” Journal of Biological Chemistry, vol. 279, no. 38, pp. 39846–39855, 2004. View at: Publisher Site | Google Scholar
  44. P. L. Pedersen, S. Mathupala, A. Rempel, J. F. Geschwind, and Y. H. Ko, “Mitochondrial bound type II hexokinase: a key player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention,” Biochimica et Biophysica Acta, vol. 1555, no. 1𠄳, pp. 14–20, 2002. View at: Publisher Site | Google Scholar
  45. R. Possemato, K. M. Marks, Y. D. Shaul et al., “Functional genomics reveal that the serine synthesis pathway is essential in breast cancer,” Nature, vol. 476, no. 7360, pp. 346–350, 2011. View at: Google Scholar
  46. D. I. James, P. A. Parone, Y. Mattenberger, and J. C. Martinou, “hFis1, a novel component of the mammalian mitochondrial fission machinery,” Journal of Biological Chemistry, vol. 278, no. 38, pp. 36373–36379, 2003. View at: Publisher Site | Google Scholar
  47. S. Grandemange, S. Herzig, and J. C. Martinou, “Mitochondrial dynamics and cancer,” Seminars in Cancer Biology, vol. 19, no. 1, pp. 50–56, 2009. View at: Publisher Site | Google Scholar
  48. Y. Lu, S. G. Rolland, and B. Conradt, “A molecular switch that governs mitochondrial fusion and fission mediated by the BCL2-like protein CED-9 of Caenorhabditis elegans,” Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 41, pp. E813–E822, 2011. View at: Google Scholar
  49. D. F. Kashatus, K. H. Lim, D. C. Brady, N. L. K. Pershing, A. D. Cox, and C. M. Counter, “RALA and RALBP1 regulate mitochondrial fission atmitosis,” Nature Cell Biology, vol. 13, no. 9, pp. 1108–1115, 2011. View at: Publisher Site | Google Scholar
  50. O. M. De Brito and L. Scorrano, “Mitofusin 2 tethers endoplasmic reticulum to mitochondria,” Nature, vol. 456, no. 7222, pp. 605–610, 2008. View at: Publisher Site | Google Scholar
  51. K. H. Chen, X. Guo, D. Ma et al., “Dysregulation of HSG triggers vascular proliferative disorders,” Nature Cell Biology, vol. 6, no. 9, pp. 872–883, 2004. View at: Publisher Site | Google Scholar
  52. G. Twig, A. Elorza, A. J. A. Molina et al., “Fission and selective fusion govern mitochondrial segregation and elimination by autophagy,” EMBO Journal, vol. 27, no. 2, pp. 433–446, 2008. View at: Publisher Site | Google Scholar
  53. G. Twig, B. Hyde, and O. S. Shirihai, “Mitochondrial fusion, fission and autophagy as a quality control axis: the bioenergetic view,” Biochimica et Biophysica Acta, vol. 1777, no. 9, pp. 1092–1097, 2008. View at: Publisher Site | Google Scholar
  54. V. G. Arciuch, M. E. Elguero, J. J. Poderoso, and M. C., Carreras, “Mitochondrial regulation of cell cycle and proliferation,” Antioxid Redox Signal, vol. 16, no. 10, pp. 1150–1180, 2012. View at: Publisher Site | Google Scholar
  55. A. Jourdain and J. C. Martinou, “Mitochondrial outer-membrane permeabilization and remodelling in apoptosis,” International Journal of Biochemistry and Cell Biology, vol. 41, no. 10, pp. 1884–1889, 2009. View at: Publisher Site | Google Scholar
  56. S. G. Rolland and B. Conradt, “New role of the BCL2 family of proteins in the regulation of mitochondrial dynamics,” Current Opinion in Cell Biology, vol. 22, no. 6, pp. 852–858, 2010. View at: Publisher Site | Google Scholar
  57. N. D. Amoອo, M. F. Rodrigues, P. Pezzuto et al., “Energy metabolism in H460 lung cancer cells: effects of histone deacetylase inhibitors,” PLoS ONE, vol. 6, no. 7, Article ID e22264, 2011. View at: Publisher Site | Google Scholar
  58. N. Gregersen and P. Bross, “Protein misfolding and cellular stress: an overview,” Methods in Molecular Biology, vol. 648, pp. 3–23, 2010. View at: Google Scholar
  59. J. S. Modica-Napolitano and K. K. Singh, “Mitochondrial dysfunction in cancer,” Mitochondrion, vol. 4, no. 5-6, pp. 755–762, 2004. View at: Publisher Site | Google Scholar
  60. R. A. Cairns, I. S. Harris, and T. W. Mak, “Regulation of cancer cell metabolism,” Nature Reviews Cancer, vol. 11, no. 2, pp. 85–95, 2011. View at: Publisher Site | Google Scholar
  61. C. Sinthupibulyakit, W. Ittarat, W. H. S. Clair, and D. K. S. Clair, “p53 protects lung cancer cells against metabolic stress,” International Journal of Oncology, vol. 37, no. 6, pp. 1575–1581, 2010. View at: Publisher Site | Google Scholar
  62. F. Sotgia, U. E. Martinez-Outschoorn, S. Pavlides, A. Howell, R. G. Pestell, and M. P. Lisanti, “Understanding the Warburg effect and the prognostic value of stromal caveolin-1 as a marker of a lethal tumor microenvironment,” Breast Cancer Research, vol. 13, no. 4, p. 213, 2011. View at: Google Scholar
  63. B. A. Kaipparettu, Y. Ma, and L. J. C. Wong, “Functional effects of cancer mitochondria on energy metabolism and tumorigenesis: utility of transmitochondrial cybrids,” Annals of the New York Academy of Sciences, vol. 1201, pp. 137–146, 2010. View at: Publisher Site | Google Scholar
  64. M. P. King and G. Attardi, “Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation,” Science, vol. 246, no. 4929, pp. 500–503, 1989. View at: Google Scholar
  65. H. Imanishi, K. Hattori, R. Wada et al., “Mitochondrial DNA mutations regulate metastasis of human breast cancer cells,” PLoS One, vol. 6, no. 8, Article ID e23401, 2011. View at: Google Scholar
  66. K. Ishikawa, K. Takenaga, M. Akimoto et al., “ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis,” Science, vol. 320, no. 5876, pp. 661–664, 2008. View at: Publisher Site | Google Scholar
  67. E. Giannoni, M. Parri, and P. Chiarugi, “EMT and oxidative stress: a bidirectional interplay affecting tumor malignancy,” Antioxidants and Redox Signaling, vol. 16, no. 11, pp. 1248–1263, 2012. View at: Publisher Site | Google Scholar
  68. Y. Ma, R. K. Bai, R. Trieu, and L. J. C. Wong, “Mitochondrial dysfunction in human breast cancer cells and their transmitochondrial cybrids,” Biochimica et Biophysica Acta, vol. 1797, no. 1, pp. 29–37, 2010. View at: Publisher Site | Google Scholar
  69. M. D. Brand and D. G. Nicholls, “Assessing mitochondrial dysfunction in cells,” Biochemical Journal, vol. 435, no. 2, pp. 297–312, 2011. View at: Publisher Site | Google Scholar
  70. D. Whitaker-Menezes, U. E. Martinez-Outschoorn, N. Flomenberg et al., “Hyperactivation of oxidative mitochondrial metabolism in epithelial cancer cells in situ: visualizing the therapeutic effects of metformin in tumor tissue,” Cell Cycle, vol. 10, no. 23, pp. 4047–4064, 2011. View at: Google Scholar
  71. M. G. V. Heiden, L. C. Cantley, and C. B. Thompson, “Understanding the warburg effect: the metabolic requirements of cell proliferation,” Science, vol. 324, no. 5930, pp. 1029–1033, 2009. View at: Publisher Site | Google Scholar
  72. E. C. Nakajima and B. Van Houten, “Metabolic symbiosis in cancer: refocusing the Warburg lens,” Molecular Carcinogenesis. In press. View at: Publisher Site | Google Scholar
  73. P. Sonveaux, F. Végran, T. Schroeder et al., “Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice,” Journal of Clinical Investigation, vol. 118, no. 12, pp. 3930–3942, 2008. View at: Publisher Site | Google Scholar
  74. Y. Wang, “Bulky DNA lesions induced by reactive oxygen species,” Chemical Research in Toxicology, vol. 21, no. 2, pp. 276–281, 2008. View at: Publisher Site | Google Scholar
  75. M. Dizdaroglu, P. Jaruga, M. Birincioglu, and H. Rodriguez, “Free radical-induced damage to DNA: mechanisms and measurement,” Free Radical Biology and Medicine, vol. 32, no. 11, pp. 1102–1115, 2002. View at: Publisher Site | Google Scholar
  76. M. E. Goetz and A. Luch, “Reactive species: a cell damaging rout assisting to chemical carcinogens,” Cancer Letters, vol. 266, no. 1, pp. 73–83, 2008. View at: Publisher Site | Google Scholar
  77. T. P. Szatrowski and C. F. Nathan, “Production of large amounts of hydrogen peroxide by human tumor cells,” Cancer Research, vol. 51, no. 3, pp. 794–798, 1991. View at: Google Scholar
  78. R. M. Balliet, C. Capparelli, C. Guido et al., “Mitochondrial oxidative stress in cancer-associated fibroblasts drives lactate production, promoting breast cancer tumor growth: understanding the aging and cancer connection,” Cell Cycle, vol. 10, no. 23, pp. 4065–4073, 2011. View at: Google Scholar
  79. S. H. Park, O. Ozden, H. Jiang et al., “Sirt3, mitochondrial ROS, ageing, and carcinogenesis,” International Journal of Molecular Sciences, vol. 12, no. 9, pp. 6226–6239, 2011. View at: Google Scholar
  80. S. J. Ralph, S. Rodríguez-Enríquez, J. Neuzil, E. Saavedra, and R. Moreno-Sánchez, “The causes of cancer revisited: "Mitochondrial malignancy" and ROS-induced oncogenic transformation - Why mitochondria are targets for cancer therapy,” Molecular Aspects of Medicine, vol. 31, no. 2, pp. 145–170, 2010. View at: Publisher Site | Google Scholar
  81. F. L. Zhou, W. G. Zhang, Y. C. Wei et al., “Involvement of oxidative stress in the relapse of acute myeloid leukemia,” Journal of Biological Chemistry, vol. 285, no. 20, pp. 15010–15015, 2010. View at: Publisher Site | Google Scholar
  82. F. Weinberg, R. Hamanaka, W. W. Wheaton et al., “Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity,” Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 19, pp. 8788–8793, 2010. View at: Publisher Site | Google Scholar
  83. S. Reuter, S. C. Gupta, M. M. Chaturvedi, and B. B. Aggarwal, “Oxidative stress, inflammation, and cancer: how are they linked?” Free Radical Biology and Medicine, vol. 49, no. 11, pp. 1603–1616, 2010. View at: Publisher Site | Google Scholar
  84. V. Nogueira, Y. Park, C. C. Chen et al., “Akt determines replicative senescence and oxidative or oncogenic premature senescence and sensitizes cells to oxidative apoptosis,” Cancer Cell, vol. 14, no. 6, pp. 458–470, 2008. View at: Publisher Site | Google Scholar
  85. A. A. Sablina, A. V. Budanov, G. V. Ilyinskaya, L. S. Agapova, J. E. Kravchenko, and P. M. Chumakov, “The antioxidant function of the p53 tumor suppressor,” Nature Medicine, vol. 11, no. 12, pp. 1306–1313, 2005. View at: Publisher Site | Google Scholar
  86. O. Vafa, M. Wade, S. Kern et al., “c-Myc can induce DNA damage, increase reactive oxygen species, and mitigate p53 function: a mechanism for oncogene-induced genetic instability,” Molecular Cell, vol. 9, no. 5, pp. 1031–1044, 2002. View at: Publisher Site | Google Scholar
  87. W. Hu, C. Zhang, R. Wu, Y. Sun, A. Levine, and Z. Feng, “Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant function,” Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 16, pp. 7455–7460, 2010. View at: Publisher Site | Google Scholar
  88. K. Bensaad, E. C. Cheung, and K. H. Vousden, “Modulation of intracellular ROS levels by TIGAR controls autophagy,” EMBO Journal, vol. 28, no. 19, pp. 3015–3026, 2009. View at: Publisher Site | Google Scholar
  89. T. W. Kang, T. Yevsa, N. Woller et al., “Senescence surveillance of pre-malignant hepatocytes limits liver cancer development,” Nature, vol. 479, no. 7374, pp. 547–551, 2011. View at: Google Scholar
  90. I. Dolado and A. R. Nebreda, “AKT and oxidative stress team up to kill cancer cells,” Cancer Cell, vol. 14, no. 6, pp. 427–429, 2008. View at: Publisher Site | Google Scholar
  91. Z. Y. Li, Y. Yang, M. Ming, and B. Liu, “Mitochondrial ROS generation for regulation of autophagic pathways in cancer,” Biochemical and Biophysical Research Communications, vol. 414, no. 1, pp. 5–8, 2011. View at: Google Scholar
  92. G. C. Brown and V. Borutaite, “There is no evidence that mitochondria are the main source of reactive oxygen species in mammalian cells,” Mitochondrion, vol. 12, no. 1, pp. 1–4, 2012. View at: Publisher Site | Google Scholar
  93. S. Coso, I. Harrison, C. B. Harrison et al., “NADPH oxidases as regulators of tumor angiogenesis: Current and emerging concepts,” Antioxidants and Redox Signaling, vol. 16, no. 11, pp. 1229–1247, 2012. View at: Publisher Site | Google Scholar
  94. B. Nespereira, M. Perez-Ilzarbe, P. Fernandez, A. M. Fuentes, J. A. Paramo, and J. A. Rodriguez, “Vitamins C and E downregulate vascular VEGF and VEGFR-2 expression in apolipoprotein-E-deficient mice,” Atherosclerosis, vol. 171, no. 1, pp. 67–73, 2003. View at: Google Scholar
  95. T. Cai, G. Fassina, M. Morini et al., “N-acetylcysteine inhibits endothelial cell invasion and angiogenesis,” Laboratory Investigation, vol. 79, no. 9, pp. 1151–1159, 1999. View at: Google Scholar
  96. P. M. Scarbrough, K. A. Mapuskar, D. M. Mattson, D. Gius, W. H. Watson, and D. R. Spitz, “Simultaneous inhibition of glutathione- and thioredoxin-dependent metabolism is necessary to potentiate 17AAG-induced cancer cell killing via oxidative stress,” Free Radical Biology and Medicine, vol. 52, no. 2, pp. 436–443, 2012. View at: Google Scholar
  97. S. C. Gupta, D. Hevia, S. Patchva, B. Park, W. Koh, and B. B. Aggarwal, “Upsides and downsides of reactive oxygen species for cancer: the roles of reactive oxygen species in tumorigenesis, prevention, and therapy,” Antioxidants and Redox Signaling, vol. 16, no. 11, pp. 1295–1322, 2012. View at: Publisher Site | Google Scholar
  98. N. M. Gruning and M. Ralser, “Cancer: sacrifice for survival,” Nature, vol. 480, no. 7376, pp. 190–191, 2011. View at: Google Scholar
  99. L. Raj, T. Ide, A. U. Gurkar et al., “Selective killing of cancer cells by a small molecule targeting the stress response to ROS,” Nature, vol. 475, no. 7355, pp. 231–234, 2011. View at: Publisher Site | Google Scholar
  100. M. P. Murphy, “How mitochondria produce reactive oxygen species,” Biochemical Journal, vol. 417, no. 1, pp. 1–13, 2009. View at: Publisher Site | Google Scholar
  101. M. Diehn, R. W. Cho, N. A. Lobo et al., “Association of reactive oxygen species levels and radioresistance in cancer stem cells,” Nature, vol. 458, no. 7239, pp. 780–783, 2009. View at: Publisher Site | Google Scholar
  102. A. Villalobo, “Nitric oxide and cell proliferation,” FEBS Journal, vol. 273, no. 11, pp. 2329–2344, 2006. View at: Publisher Site | Google Scholar
  103. Q. I. Lu, F. L. Jourd'Heuil, and D. Jourd'Heuil, “Redox control of G1/S cell cycle regulators during nitric oxide-mediated cell cycle arrest,” Journal of Cellular Physiology, vol. 212, no. 3, pp. 827–839, 2007. View at: Publisher Site | Google Scholar
  104. A. Onul, K. M. Elseth, and H. De Vitto, “Long-term adaptation of the human lung tumor cell line A549 to increasing concentrations of hydrogen peroxide,” Tumor Biology, vol. 33, no. 3, pp. 739–748, 2012. View at: Publisher Site | Google Scholar
  105. B. J. Vesper, K. M. Elseth, G. Tarjan, G. K. Haines, and J. A. Radosevich, “Long-term adaptation of breast tumor cell lines to high concentrations of nitric oxide,” Tumor Biology, vol. 31, no. 4, pp. 267–275, 2010. View at: Publisher Site | Google Scholar

Copyright

Copyright © 2012 Marcus Fernandes de Oliveira et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Conclusion

Immunometabolism is among the critical features that define the intimate relationship between host and the Mtb pathogen a clear understanding of these interactions will be essential for limiting the progression of the TB. Metabolic reprogramming from OXPHOS to glycolysis in Mtb infection results in the upregulated expression of numerous pro-inflammatory cytokines and antimicrobial effector molecules. Further investigation will be needed in order to understand more fully the relationship between Mtb and host metabolism. How and when Mtb exploit the host metabolism is not clearly understood at this time clarification will be critical in order to identify the most appropriate candidates for HDT. Among those currently under consideration is Mtb-mediated modulation of glucose and/or lipid metabolism. Glucose metabolism might be targeted at the early stage, which would ultimately provide a boost to the Warburg effect. Thus, more efficient elimination of Mtb bacteria by contrast, targeting glucose metabolism at a later stage may result in a much needed- alleviation of hyperinflammation. A better understanding of metabolic reprogramming in TB will provide further insights toward novel therapeutic strategies.


MTORC1 and Mitochondrial Regulation by miRNAs in Cancer

The expression of a large number of oncogenes and tumor suppressor genes is regulated by miRNAs, which altered expression, is currently though as a hallmark of cancer. miRNAs or microRNAs are small non-coding RNAs (21� nt), that regulate gene expression by targeting mRNAs for degradation or suppressing translation (76). In cancer, miRNAs are divided into two categories, oncogenic miRNAs and tumor suppressor miRNAs, which are up regulated and down regulated during tumorigenesis (77). According to its role as a master regulator of cell growth, mTORC1 activity is modulated by different extracellular signals and intracellular mechanisms, interestingly it has been shown that some miRNAs can also regulate the mTORC1 activity directly or through targeting upstream regulators such as PI3K/Akt pathway. For instance, miR-451 is upregulated in glioma compared with control brain tissue furthermore decreased miR-451 expression was associated to a suppressed tumor cell proliferation via CAB39/AMPK/mTOR pathway in two glioma cell lines (78). Furthermore, over expression of miR-405 promoted caspase-3/-9 and Bax protein expression, and suppressed cyclin D1 protein expression and the PI3K/Akt/mTOR pathway inhibiting cell proliferation and promoting cell apoptosis in gastric cancer-derived cells (78). On the other hand evidence shown that mTORC1 regulates miRNAs biogenesis and given the broad function of miRNAs in cancer development, it is possible that a significant portion of mTORC1 function, may be through its ability to control miRNA biogenesis. It was shown that chronic treatment with rapamycin leads to significant alterations in miRNA profiles and these changes correlate with resistance to rapamycin. The miRNAs associated to rapamycin resistance were miR-370, miR-17-92 and its related miR-106a-92, and miR-106b-25 clusters, which have been shown to have oncogenic properties in several types of cancer (79). Ye and collaborators (2015) report that mTORC1 activation downregulates miRNA biogenesis through upregulation of Mdm2, which is a necessary and sufficient E3 ligase for ubiquitinylation of Drosha an essential RNase dedicated to processing pri-miRNA in response to the cellular environment (80). On the other hand it was shown that mTORC1 in TSC2 deficient cells, promotes the miRNA biogenesis through of GSK3β regulation. mTORC1 induces the activity of the microprocessor, a nuclear complex that includes the nuclease Drosha and its partner DGCR8, this complex cleaves the stem loop of pri-miRNA to form premiRNA via the nuclease activity of Drosha (81).

On the other hand it was reported that several miRNAs targeting several mRNAs of nuclear-encoded mitochondrial proteins, integrating miRNAs into the landscape of translational regulation of mitochondrial functions such as TCA cycle, production of ROS and glutamine metabolism and mitochondrial fission process (82). miR-125a is frequently downregulated in several human cancer such as ovarian, non small-cell lung and gastric cancer and colorectal cancer (83�) Moreover low expression of miR-125a is associated with increased tumor diameter, high Ki67 expression and poor overall survival of patients with gastric carcinoma (86) Additionally miR-125a deficiency enhances agiogenic processes through metabolic reprogramming of endothelial cells (87). Interestingly it was demonstrated that miR-125a is decreased in pancreatic cancer cells (PANC-1), accompanied by an increase in the contents of mitofusin 2 (MFN2) an important regulator of mitochondrial fission. Interestingly reintroduction of miR-125a triggered mitochondrial fission via downregulation of MFN2. Excessive mitochondrial fission contributes to activation of mitochondria-dependent apoptosis and impairs cellular migration via induction of F-actin degradation (88).

miRNAs are encoded in the nuclear genome and exported to the cytosol where they perform most of their functions, however, the expression of miRNAs within the mitochondrion has been recently demonstrated, which can be either mitochondrial encoded or transcribed within the nucleus and subsequently localized to mitochondria, this miRNAs are termed as mitomiRs (89). MitomiRs are likely to contribute to some post-transcriptional regulation of gene expression related to the mitochondrial functions (90). Interestingly mitomiRs have been shown to play a very important role in chemotherapy resistance through the regulation of metabolic changes. For instance, it was demonstrated that mito-miR-2392 regulates the cisplatin resistance by reprogramming the balance between OXPHOS and glycolysis in tongue squamous cell carcinoma (TSCC) cells. Furthermore, in a retrospective analysis of TSCC patient tumor revealed a significant association of miR2392 and the expression of mitochondrial gene with chemosensitivity and overall survival (91).

Although several cancer processes are regulated by miRNAs, there is a lacking of investigation aimed to determine the role of the mitomiRs and mTORC1 regulation either, in metabolic responses to therapy as well as mitochondrial functions, representing an open opportunity for future research.


Could the Warburg effect be used to starve cancer cells in situ? - Biology

Academia.edu no longer supports Internet Explorer.

To browse Academia.edu and the wider internet faster and more securely, please take a few seconds to upgrade your browser.

mitochondrial bioenergetics researcher

400 pA/μM) and an improved limit of detection (

40 nM). Finally, they were applied to the analysis within an oxygraphy chamber of mitochondrial production of H2O2. This first study paves the way for the detection of several biomarkers on an integrated microchip and therefore for multi-parametric monitoring of mitochondrial metabolism in physiological and pathological conditions.


DISCUSSION

Our data suggest that growth factors regulate cellular growth and survival by modulating the ability to take up a range of extracellular molecules, including glucose, amino acids, cholesterol, and iron. The loss of transporters for each of these molecules would have important consequences for cellular homeostasis, and their coordinated down-regulation would present an almost insurmountable hurdle to continued cell growth. Loss of amino acid transport along with the decrease in glucose uptake would result in a shortage of bioenergetic metabolites, forcing the cell to break down its constituent macromolecules to sustain bioenergetics. Cholesterol is an important component of cellular membranes. Although cells are capable of synthesizing cholesterol, loss of access to external sources would decrease the available supply and increase the energy drain on the cell as it is forced to synthesize all the cholesterol required. Decreased iron uptake would have important consequences for the activity of enzymes such as ribonucleotide reductase, which turns over rapidly and requires a continuous supply of iron to maintain its activity (Cazzolaet al., 1990). In addition, cellular iron deficiency results in oxidative damage and loss of function in mitochondria, and treatment of cells with iron chelators decreases the activity of tricarbolic acid cycle enzymes, increases the NAD/NADH ratio, decreases oxygen consumption, and increases lactate production, indicating that iron plays a critical role in metabolic homeostasis (Oexle et al., 1999 Walter et al., 2001).

The Akt kinase is a component of the growth factor signal transduction cascade regulating cell growth and survival. The kinase activity of mTOR may be modulated by Akt activity (Scott et al., 1998Sekulic et al., 2000), and several studies suggest that the transforming effects of Akt involve stimulation of mTOR (Aoki et al., 2001 Neshat et al., 2001 Podsypanina et al., 2001). Our studies indicate that Akt-mediated increases in cell size and survival result from increased surface expression of nutrient transporters. Furthermore, the ability of Akt activity to increase cell size, cell survival, and surface expression of nutrient transporters depends on the activity of mTOR. The TOR kinase is highly conserved from yeast to humans, and parallels can be drawn between the functions of yeast and mammalian TOR. In yeast, TOR coordinates the cellular response to extracellular nutrient levels and allows yeast cells to respond adaptively to changes in the extracellular environment by modulating nutrient transporter expression (Schmidt et al., 1998 Beck et al., 1999). Our results raise the possibility that in mammalian cells, mTOR functions in the growth factor signal transduction pathway to maintain cellular access to extracellular nutrients. Rapamycin treatment in yeast produces a response equivalent to starvation (Schmelzle and Hall, 2000 Raughtet al., 2001), and it may be that growth factor withdrawal induces a similar state of pseudostarvation in part by limiting mTOR activity. The data presented herein show that mTOR regulates the trafficking of nutrient transporters in myrAkt-expressing cells. Consistent with the low toxicity of rapamycin in normal cells (Millset al., 2001 Neshat et al., 2001), rapamycin had limited effects on control cells growing in the presence of IL3. Growth factor signaling probably supports nutrient transporter expression through multiple, redundant pathways and only the mTOR pathway would be rapamycin sensitive. In contrast, activated Akt seems to increase transporter expression solely through an mTOR-dependent pathway as rapamycin treatment ablates the effects of Akt on transporter surface expression. These results are also consistent with the observations that, although the maintenance of muscle mass is not dependent on the Akt, the activities of Akt and mTOR are critical for load-induced hypertrophy and recovery of mass after atrophy (Bodine et al., 2001).

If access to extracellular molecules is an important mechanism of growth factor control over cell survival and proliferation then it follows that cancer cells have developed mechanisms to supply themselves with these molecules in spite of the lack of growth factor support. It is well established that tumors and cell lines have enhanced rates of glucose uptake. Tumor cells are also avid consumers of amino acids and take up nitrogen from host proteins as well as from the diet (Medina, 2001). In fact, overexpression of 4F2hc in 3T3 cells results in transformation and renders the cells tumorigenic in nude mice (Hara et al., 1999, 2000). Rapidly growing and dividing cells must constantly make new membrane, and inhibition of cholesterol synthesis by chemical inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase can cause apoptosis in ∼50% of tumor cell lines tested, whereas normal human fibroblast lines remain unaffected (Dimitroulakos et al., 2001). Interfering antibodies to the transferrin receptor can arrest tumor cell growth at the G1/S interface, and iron chelators have shown some efficacy as antineoplastic agents (Cazzola et al., 1990). Furthermore, we have shown that when cells are growth factor withdrawn and Akt-mediated elevated transporter expression is prevented by rapamycin treatment, cell survival and Δψm decline, suggesting that nutrient acquisition has a regulatory role in cellular apoptosis. Rapamycin treatment decreases colony formation by myrPI3K and myrAkt transformed fibroblasts and has been shown to have activity against PTEN-deficient tumors (Hidalgo and Rowinsky, 2000 Aoki et al., 2001 Neshat et al., 2001 Podsypanina et al., 2001). Our results further support the evaluation of rapamycin and its analogs as cancer therapy agents in cells with an activated PI3K signaling pathway.

Growth factor withdrawal is known to decrease the rate of protein synthesis, a process that consumes a large fraction of cellular energy. An alternate explanation for decreased nutrient uptake during IL3 withdrawal is that cellular demand for nutrients is reduced due to arrested translation and decreased ATP utilization. This could account for the observed decline in amino acid and glucose uptake upon growth factor withdrawal, but not for the decline in the mitochondrial potential. A decrease in the rate of translation and a commensurate decline in ATP consumption should result in an increase rather than a decrease in the mitochondrial membrane potential. The fact that Δψm declines in the face of reduced energy demand suggests that cells are unable to meet even this lower energy requirement. The cellular atrophy observed upon growth factor withdrawal is also consistent with a catabolic state in which cells degrade their constituent proteins for energy due to a negative cellular energy balance.

The observed decrease in nutrient transporter protein expression after growth factor withdrawal also does not result solely from a decrease in protein translation. When IL3 is withdrawn from control cells or from rapamycin-treated, myrAkt-expressing cells, the 4F2hc staining pattern changes from a surface to a lysosomal pattern. Loss of this protein due to the interruption of its translation would be reflected as decreased surface staining rather than a shift to a lysosomal localization. Our data suggest that these nutrient transporter proteins are actively targeted for lysosomal degradation upon IL3 withdrawal and that preventing transporter down-regulation may be an important step in tumorigenesis. Several recent reviews have highlighted the possible involvement of proteins regulating endocytosis in some human cancers (Floyd and De Camilli, 1998 Di Fiore and Gill, 1999). It will be interesting to determine whether the antiproliferative effects of rapamycin on PTEN-deficient tumor cells results from a decrease in mTOR-dependent surface expression of nutrient transporters, thereby limiting cellular access to extracellular nutrients.


Watch the video: Three must-dos to cure cancer. Timothy Cripe. TEDxColumbus (August 2022).