**3. Mitochondrial dehydrogenases and cancer**

appears to occur through the reversible glutathionylation of the E2-lipoic acid domain of the dehydrogenase [14], thereby protecting the E2 domain, and the catalytic site of the dehydrogenase, from damaging oxidative stress. As the flux of electrons through the electron chain decreases, so does the production of free radicals, optimizing the conditions for the mitochon-

*Isocitrate Dehydrogenase* (1.1.1.42 and 1.1.1.41): This enzyme catalyzes the oxidative decarbox-

isoform of this enzyme, IDH3, is a heterotetramer composed by two alpha, one beta, and one gamma subunits (2α1β1γ) Two other isoforms (IDH1 and IDH2) of the enzyme are known in humans. They catalyze the same reaction in the cytosol and in peroxisomes converting

Owing to the large negative free energy change involved in the reaction, the step catalyzed by this dehydrogenase represents one of the irreversible reactions in the citric acid cycle. Hence, tight regulation is required to avoid unnecessary depletion of isocitrate. The reaction is pro-

which both bind specific active sites on the IDH structure, and inhibited by ATP levels [15] *Succinate dehydrogenase* (1.3.5.1): This enzyme participates in two key cycles within the mitochondrion. It is part of the respiratory complex II (whereby it is also defined as succinatecoenzyme Q reductase) and as such is key in controlling the electron flux through the electron transport chain [16]. In addition, it participates in the citric acid cycle, in which it catalyzes the

Structurally, the succinate dehydrogenase is composed of 2 hydrophilic and 2 hydrophobic subunits. The two hydrophilic subunits possess a covalently attached flavin adenine dinucleotide (FAD) cofactor and the succinate binding site (subunit SdhA) and three iron–sulfur clusters (SdhB). The subunit SdhA provides the binding site for the oxidation of succinate. The two hydrophobic subunits SdhC and SdhD act as membrane anchors. Human mitochondria contain 2 distinct SdhA isoforms (FpI and FpII), which form a membrane bound cytochrome b complex with 1 heme b group and 1 ubiquinone binding site. More specifically, the ubiquinone binding site is located in an enclosure formed by SdhB, and the two hydrophobic subunits SdhC and SdhD. These two subunits also present one cardiolipin and one phospha-

Functionally, the succinate-binding site in SdhA and the ubiquinone-binding site in the pocket formed by the three other subunits are connected by a chain of redox centers that include FAD and the three iron–sulfur clusters [17]. Due to its localization, the succinate dehydrogenase participates to both the citric acid cycle and the respiratory chain, as indicated previously. Electron removed from succinate during its oxidation to fumarate are transferred through the iron–sulfur clusters present in SdhB to the ubiquinone molecule, and ultimately to the two hydrophobic subunits anchoring the complex to the mitochondrial membrane. The acceptance of the electrons by the ubiquinone occurs in a two-step process whereby, after the first electron transfer, the ubiquinone is converted to a semiquinone radical species. The transfer of the second electron fully converts this intermediate to ubiquinol [18]. Hence, SdhA acts as an intermediate in the enzymatic activity of the succinate dehydrogenase complex in that:

oxidation of succinate to fumarate with the reduction of ubiquinone to ubiquinol.

in the citric acid cycle. The reaction

to NADH. The mitochondrial

and Mg2+ (or Mn2+),

drial detox systems to scavenger these toxic agents.

to NADPH in the process.

128 Secondary Metabolites - Sources and Applications

tidylethanolamine molecule attached [17].

NADP<sup>+</sup>

ylation of isocitrate to generate a-ketoglutarate and CO<sup>2</sup>

is a two-step process, supported by the conversion of NAD<sup>+</sup>

moted by substrate availability and the presence of cofactors such as NAD<sup>+</sup>

Cancer cells are characterized by increased glycolytic ATP production as a result of decreased mitochondria effectiveness. Inhibition (or decreased activity) of the citric acid cycle is considered to be one of the main causes forcing the cells to generate ATP through anaerobic glycolysis [21]. This reprogramming results from oncogene activation or inhibition of tumor suppressors [21]. Consistent with this observation, inhibition of glycolysis by dichloroacetate, a pyruvate inhibitor, shifts cell metabolism back to oxidative phosphorylation, at least to a certain extent [22]. This metabolic reprogramming is considered to be necessary to meet the needs of the rapid proliferative rate exhibited by cancer cells. Two key bioenergetics parameters resulting from the mentioned metabolic reprogramming are anaerobic glycolysis, with associated extracellular acidification, and mitochondrial respiration.

*Mitochondrial ROS and Cancer*: Reactive oxygen species (ROS) are by-products of mitochondrial electron transport chain, generated by the incomplete reduction of oxygen as electrons flow from one complex to the next. Under physiological conditions, 1–2% of the molecular oxygen utilized by the mitochondria is converted to ROS [31]. Reactive oxygen species are highly reactive molecules, which act as oxidants removing electrons from DNA, proteins, and lipids. While ROS can be generated at the level of the plasma membrane (NADPH oxidase) and the endoplasmic reticulum of cell abundant in cytochrome activity (e.g. hepatocytes), the main intracellular source of ROS in most cells is indeed represented by mitochondria. In

gests that it can cross the mitochondrial membrane and diffuse into the cytoplasm through VDAC and other not-yet identified mitochondrial channels [32]. Either in the mitochondria

any surrounding macromolecule, including proteins, lipids, nucleic acids, and carbohydrates [33]. Interaction of ROS with surrounding macromolecules may result in damage of biological membrane, enzyme inactivation, and genotoxicity. High levels of ROS can induce apoptosis and cell death whereas low levels of ROS can act as signaling molecules, and either path can

Cellular DNA and mitochondrial DNA are both highly susceptible to ROS-induced damage. This susceptibility is more pronounced for mitochondrial DNA, due to the close proximity to the electron transport chain where ROS are generated, the absence of protective histones, and the limited DNA repair capability. As a result, the mutation rate of mitochondrial DNA is two orders of magnitude higher than that of nuclear DNA. Consistent with this notion, mutations in mitochondrial DNA - in particular mutations of oxidative phosphorylation enzymes - have been observed in many neoplastic cells (see previous section), in which they result in oxidative phosphorylation dysfunction, increased ROS formation, and energetic shift towards

The role of ROs as signaling molecules in cell proliferation, differentiation, migration, metastatic colonization, and gene transcription is now fairly well recognized. The ability of ROS to act as signaling molecules depends on the presence of redox-sensitive proteins that operate as 'ROS sensors'. In most cases, the sensing ability lies in the reversible oxidation of sulfhy-

O2

that particular protein. For the most part, this inhibition is transient in that the cell possesses scavenging mechanisms (e.g. glutathione) to reduce the sulfhydryl groups back to the original

The levels of ROS produced by tumor cells are usually higher than those present in normal cells, and they results in DNA damage but also in increased tumorigenesis and metastasis via direct activation of signaling pathways [35]. Two signaling pathways activated by ROS are MAP Kinase and phosphoinositide 3-kinase, which both control cell survival and proliferation. The

−

Haber-Weiss reaction), generating the more reactive but short-lived OH−

is the main byproduct of oxidative phosphorylation, and acts as precursor of

O2

− to H<sup>2</sup> O2

cannot diffuse across biological membrane. However, evidence sug-

is highly reactive towards cysteine residues, and this reactivity is

) and hydroxyl radical (OH−

Physiology and Pathology of Mitochondrial Dehydrogenases

http://dx.doi.org/10.5772/intechopen.76403

and iron or other transition metals (Fenton reaction or

or other ROS, resulting in the inhibition of

, which can now diffuse freely

, which interacts with

). Due to

131

particular, O<sup>2</sup>

−

its negative charge, O<sup>2</sup>

across membranes. H<sup>2</sup>

anaerobic glycolysis.

other ROS products such as hydrogen peroxide (H<sup>2</sup>

or in the cytoplasm superoxide dismutases convert O<sup>2</sup>

−

O2

further enhanced in the presence of O<sup>2</sup>

results in tumor initiation and progression.

dryl groups in specific cysteine residues by H<sup>2</sup>

state, thus restoring the protein's activity or signaling properties [34].

Altered functioning of any of the mentioned dehydrogenases has been observed in different cancer cells, and specific correlations have been observed and documented. For example, glutamate dehydrogenase can be considered as biomarkers for cancer cell growth [23]. Similarly, defects in isocitrate dehydrogenase [24], a-ketoglutarate dehydrogenase [25] succinate dehydrogenase [26], and pyruvate dehydrogenase [27] have all been reported in a variety of cancer cells, and associated with the metabolic reprogramming these cells undergo. The picture that emerges is that many cancer cells are hypoxic and therefore metabolize lactate to pyruvate to generate ATP [28]. The occurrence of hypoxia results in the activation of hypoxia-inducible factor 1 (HIF-1) heterodimeric DNA-binding complex and pro-neoplastic genes in tumor cells. HIF-1α is continuously synthesized and degraded, with a half-life of ~6 min under normoxic conditions. Under hypoxia conditions, however, the rate of HIF-1α degradation decreases significantly as a result of prolyl hydroxylation and proteosomal degradation suppression by accumulating succinate and increasing ROS formation and enzyme modification. As a result, HIF-1 heterodimer accumulates, and translocated to the nucleus of the cells where it activates genes responsible for increased glucose uptake (mostly GLUT1) and lactate production. At the same time, the increase in succinate levels further depresses mitochondrial respiration, with increased routing of O<sup>2</sup> towards ROS formation. In addition to upregulating the glucose transporter, HIF-1 promotes the expression of various glycolytic enzymes as well as PDK-1, the kinase that phosphorylates and inactivates the pyruvate dehydrogenase subunit E1. Inactivation of this dehydrogenase prevents pyruvate entry into the TCA cycle, thus downregulating mitochondria respiration [29].

Another pathway involved in modulating mitochondria activity and responsiveness in cancer cells is the one tapping onto peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC1α). This pathway has been observed to be overexpressed in some subsets of melanomas in which it activates mitochondrial oxidative phosphorylation and TCA-cycle specific genes for the metabolic needs of the cancer cells [30]. In addition, PGC1α promotes the expression of genes involved in *de novo* lipogenesis and in the pentose phosphate pathway, increasing NADPH production for fatty acid synthesis purposes [30]. While genes involved in oxidative phosphorylation and TCA cycle are located in mitochondria, those involved in the pentose phosphate pathway and fatty acid synthesis are located in the cytoplasm of the cell. How exactly Acyl-CoA generated within the mitochondrion through aerobic glycolysis (pyruvate dehydrogenase) is utilized to enhance fatty acid synthesis is not completely understood. Experimental evidence would suggest that PGC1α may do so by inducing the expression of genes responsible for converting citrate back to oxaloacetate and Acyl-CoA [30]. Irrespective of the precise mechanisms involved in the metabolic reprogramming of cancer cells, PGC1α-positive cancer cells are particular sensitive to pharmacological (e.g. metformin) or chemical (e.g. rotenone or FCCP) inhibitors of oxidative phosphorylation, in stark contrast to PGC1α-negative cancer cells, which are more sensitive to anti-glycolytic agents (e.g. 2-deoxyglucose, or 2-DG).

*Mitochondrial ROS and Cancer*: Reactive oxygen species (ROS) are by-products of mitochondrial electron transport chain, generated by the incomplete reduction of oxygen as electrons flow from one complex to the next. Under physiological conditions, 1–2% of the molecular oxygen utilized by the mitochondria is converted to ROS [31]. Reactive oxygen species are highly reactive molecules, which act as oxidants removing electrons from DNA, proteins, and lipids. While ROS can be generated at the level of the plasma membrane (NADPH oxidase) and the endoplasmic reticulum of cell abundant in cytochrome activity (e.g. hepatocytes), the main intracellular source of ROS in most cells is indeed represented by mitochondria. In particular, O<sup>2</sup> − is the main byproduct of oxidative phosphorylation, and acts as precursor of other ROS products such as hydrogen peroxide (H<sup>2</sup> O2 ) and hydroxyl radical (OH− ). Due to its negative charge, O<sup>2</sup> − cannot diffuse across biological membrane. However, evidence suggests that it can cross the mitochondrial membrane and diffuse into the cytoplasm through VDAC and other not-yet identified mitochondrial channels [32]. Either in the mitochondria or in the cytoplasm superoxide dismutases convert O<sup>2</sup> − to H<sup>2</sup> O2 , which can now diffuse freely across membranes. H<sup>2</sup> O2 is highly reactive towards cysteine residues, and this reactivity is further enhanced in the presence of O<sup>2</sup> − and iron or other transition metals (Fenton reaction or Haber-Weiss reaction), generating the more reactive but short-lived OH− , which interacts with any surrounding macromolecule, including proteins, lipids, nucleic acids, and carbohydrates [33]. Interaction of ROS with surrounding macromolecules may result in damage of biological membrane, enzyme inactivation, and genotoxicity. High levels of ROS can induce apoptosis and cell death whereas low levels of ROS can act as signaling molecules, and either path can results in tumor initiation and progression.

a pyruvate inhibitor, shifts cell metabolism back to oxidative phosphorylation, at least to a certain extent [22]. This metabolic reprogramming is considered to be necessary to meet the needs of the rapid proliferative rate exhibited by cancer cells. Two key bioenergetics parameters resulting from the mentioned metabolic reprogramming are anaerobic glycolysis, with

Altered functioning of any of the mentioned dehydrogenases has been observed in different cancer cells, and specific correlations have been observed and documented. For example, glutamate dehydrogenase can be considered as biomarkers for cancer cell growth [23]. Similarly, defects in isocitrate dehydrogenase [24], a-ketoglutarate dehydrogenase [25] succinate dehydrogenase [26], and pyruvate dehydrogenase [27] have all been reported in a variety of cancer cells, and associated with the metabolic reprogramming these cells undergo. The picture that emerges is that many cancer cells are hypoxic and therefore metabolize lactate to pyruvate to generate ATP [28]. The occurrence of hypoxia results in the activation of hypoxia-inducible factor 1 (HIF-1) heterodimeric DNA-binding complex and pro-neoplastic genes in tumor cells. HIF-1α is continuously synthesized and degraded, with a half-life of ~6 min under normoxic conditions. Under hypoxia conditions, however, the rate of HIF-1α degradation decreases significantly as a result of prolyl hydroxylation and proteosomal degradation suppression by accumulating succinate and increasing ROS formation and enzyme modification. As a result, HIF-1 heterodimer accumulates, and translocated to the nucleus of the cells where it activates genes responsible for increased glucose uptake (mostly GLUT1) and lactate production. At the same time, the increase in succinate levels further depresses mitochondrial respiration,

transporter, HIF-1 promotes the expression of various glycolytic enzymes as well as PDK-1, the kinase that phosphorylates and inactivates the pyruvate dehydrogenase subunit E1. Inactivation of this dehydrogenase prevents pyruvate entry into the TCA cycle, thus down-

Another pathway involved in modulating mitochondria activity and responsiveness in cancer cells is the one tapping onto peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC1α). This pathway has been observed to be overexpressed in some subsets of melanomas in which it activates mitochondrial oxidative phosphorylation and TCA-cycle specific genes for the metabolic needs of the cancer cells [30]. In addition, PGC1α promotes the expression of genes involved in *de novo* lipogenesis and in the pentose phosphate pathway, increasing NADPH production for fatty acid synthesis purposes [30]. While genes involved in oxidative phosphorylation and TCA cycle are located in mitochondria, those involved in the pentose phosphate pathway and fatty acid synthesis are located in the cytoplasm of the cell. How exactly Acyl-CoA generated within the mitochondrion through aerobic glycolysis (pyruvate dehydrogenase) is utilized to enhance fatty acid synthesis is not completely understood. Experimental evidence would suggest that PGC1α may do so by inducing the expression of genes responsible for converting citrate back to oxaloacetate and Acyl-CoA [30]. Irrespective of the precise mechanisms involved in the metabolic reprogramming of cancer cells, PGC1α-positive cancer cells are particular sensitive to pharmacological (e.g. metformin) or chemical (e.g. rotenone or FCCP) inhibitors of oxidative phosphorylation, in stark contrast to PGC1α-negative cancer cells, which

are more sensitive to anti-glycolytic agents (e.g. 2-deoxyglucose, or 2-DG).

towards ROS formation. In addition to upregulating the glucose

associated extracellular acidification, and mitochondrial respiration.

with increased routing of O<sup>2</sup>

130 Secondary Metabolites - Sources and Applications

regulating mitochondria respiration [29].

Cellular DNA and mitochondrial DNA are both highly susceptible to ROS-induced damage. This susceptibility is more pronounced for mitochondrial DNA, due to the close proximity to the electron transport chain where ROS are generated, the absence of protective histones, and the limited DNA repair capability. As a result, the mutation rate of mitochondrial DNA is two orders of magnitude higher than that of nuclear DNA. Consistent with this notion, mutations in mitochondrial DNA - in particular mutations of oxidative phosphorylation enzymes - have been observed in many neoplastic cells (see previous section), in which they result in oxidative phosphorylation dysfunction, increased ROS formation, and energetic shift towards anaerobic glycolysis.

The role of ROs as signaling molecules in cell proliferation, differentiation, migration, metastatic colonization, and gene transcription is now fairly well recognized. The ability of ROS to act as signaling molecules depends on the presence of redox-sensitive proteins that operate as 'ROS sensors'. In most cases, the sensing ability lies in the reversible oxidation of sulfhydryl groups in specific cysteine residues by H<sup>2</sup> O2 or other ROS, resulting in the inhibition of that particular protein. For the most part, this inhibition is transient in that the cell possesses scavenging mechanisms (e.g. glutathione) to reduce the sulfhydryl groups back to the original state, thus restoring the protein's activity or signaling properties [34].

The levels of ROS produced by tumor cells are usually higher than those present in normal cells, and they results in DNA damage but also in increased tumorigenesis and metastasis via direct activation of signaling pathways [35]. Two signaling pathways activated by ROS are MAP Kinase and phosphoinositide 3-kinase, which both control cell survival and proliferation. The upregulation of these pathways increases the expression of oncogenes and proteins involved in metastasis (e.g. matrix metalloproteinases) and in epithelial to mesenchymal transition [36]. Of note, oncogene activation has been reported to enhance mitochondrial ROS production, and ROS generation appears to be required for oncogene-mediated cell transformation [37].

within the mitochondrion of streptozotocin-treated rats. As mentioned in the previous section, the consequences of this dysfunction are three-fold: 1) the inability of the electron transport chain to operate properly is compromised, resulting in decreased ATP production; 2) the production of ROS is enhanced, further damaging macromolecules within the mitochondrion and its surroundings, and 3) forcing the cardiac myocytes to depend on glycolysis to produce ATP. Interestingly, these changes were observed predominantly in cardiac and liver tissue but not in the kidney of the diabetic animals [49], rising the intriguing question of which protec-

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http://dx.doi.org/10.5772/intechopen.76403

133

The term obesity refers to a condition in which the amount of fat tissue in the body is increased to an excessive degree (i.e. more than 25% in body weight in men and more than 30% in women). The incidence of obesity or just overweight has been on the rising for the last several decades, and currently affects anywhere between 35 and 45% of the population, in developed and developing countries [50]. Obesity is the sixth most important risk factor, and the number of affected individuals has reached 2.1 billion worldwide, including 10% of all children. The main complications include particular forms of cancer (e.g. colon and breast cancer, in particular), type 2 diabetes mellitus, cardiovascular pathologies including stroke, and musculoskeletal dysfunctions, with a prohibitive price tag in terms of healthcare costs, morbidity and mortality [51]. Several reviews have addressed the changes in mitochondria functioning in obesity, and we refer to them for an in-depth understanding of the changes occurring in the organelle [52–53]. The emerging picture indicates that cardiac cells, adipocytes, skeletal muscle cells, beta-cells, liver cells and others are affected to a varying degree by lipotoxicity [54], which also affects mitochondria operation. Whether the dysfunction strictly depends on the abnormal presence of fatty acid in the cells and the biological membrane of the organelle, or other not-yet identified factors remains undefined. The most common mitochondrial dysfunctions associated with obesity have been identified in an abnormally low number of mitochondria with altered morphology, decreased expression of the F1-ATPase subunit, with consequent low ATP generation [53], higher expression of the

tive mechanism(s) may operate in the latter organ under diabetic conditions.

uncoupling protein 2 (UCP2) [52] with associated increased production of O<sup>2</sup>

ponents within the cell nucleus and defective import into already altered organelles.

**6. Conclusions**

and apoptosis [52]. At the same time, the abnormal presence of fatty acids and ceramide within the cell activate specific signaling that further impair the tissue response to insulin [53]. Due to the decline in number and the altered morphology of mitochondria, an unanswered question remains as to whether these modifications are secondary to intrinsic defects in mitochondrial dehydrogenases that link electron transport chain to TCA cycle and/or to proper ATP synthesis, or whether defects in mitochondria enzymes are consequence of defective synthesis of key com-

The picture we have attempted to draw in the present review moves from the important role specific mitochondrial dehydrogenases play in cell metabolism. Under conditions in which metabolic reprogramming occurs (i.e. cancer, diabetes, obesity), cells move away from efficient

−

, ROS in general,

**5. Mitochondrial dysfunction in obesity**
