**6.3. Mitochondrial network and mitochondria-associated membranes abnormalities in human astrocytomas**

Regards to the mitochondrial network, lucent-swelling mitochondria with disarrangement and distortion of cristae and partial or total cristolysis is predominant in the astrocytoma cells. In a minor proportion of astrocytoma cells, the presence of mitochondria with dense matrix displayed in closed groups exists [7–10].

Considerable variations in MAM ultrastructure is observed in the glioma tissue with respect to density, length, and width of the interfacing ER and mitochondrial membranes (**Figure 3**). In some astrocytoma cells, the MAM displayed a network or "work station" Functional and Therapeutic Implications of Mitochondrial Network and Mitochondria… http://dx.doi.org/10.5772/intechopen.77224 19

**6.1. Mitochondrial network**

18 Glioma - Contemporary Diagnostic and Therapeutic Approaches

both fusion and fission events [19].

**6.2. Mitochondria-associated membranes**

cell demands or microenvironment stimuli [2, 21].

displayed in closed groups exists [7–10].

**in human astrocytomas**

Ultrastructurally, mitochondrion is an organelle constituted by a peripheral and inner membrane. The peripheral membrane encloses the entire contents of the mitochondrion, and internal membrane forms a series of folds, called cristae, which project inward toward the interior space of the organelle. The area between the peripheral and inner membranes is designated as intermembrane space, and the area enclosed by the internal membrane is labeled as a mitochondrial matrix. Functionally, the outer membrane includes the apoptosis antagonists and agonists and fission/fusion mitochondrial proteins. The inner membrane contains all the respiratory enzyme complexes and the three electron transporters, necessary for oxidative phosphorylation. In major mammalian tissues, 80–90% of ATP is generated by mitochondria in the process of oxidative phosphorylation [17, 18]. The mitochondrial matrix contains the enzymatic system of β-oxidation and tricarboxylic acid cycle. Mitochondria in living human cells display large, elongated and branched structures, actually entitled as mitochondrial network, extending throughout the cytosol and in close contact with the nucleus, the endoplasmic reticulum, the Golgi complex and the cytoskeleton, and is continually remodeled by

In some cell types, mitochondria exist as single and randomly dispersed organelles; in other cells, mitochondria may also exit as dynamic networks that often changes shape and subcellular distribution. Depending on the cell type, mitochondria localized in different site-specific

MAM is a membranous and protein structure (inter-membranous structure) composed by three pieces: (1) endoplasmic reticulum membrane; (2) mitochondrial membrane (outer mitochondrial membrane); and (3) tethers (proteins). Consequently, it displays biological mem-

To date, MAM is considered as a fundamental cellular structure tightly regulated and with multifaceted roles that include Ca2+ signaling, lipid synthesis and exchange, metabolic control, and others. MAM formation might depend on several factors relating to differences in

Regards to the mitochondrial network, lucent-swelling mitochondria with disarrangement and distortion of cristae and partial or total cristolysis is predominant in the astrocytoma cells. In a minor proportion of astrocytoma cells, the presence of mitochondria with dense matrix

Considerable variations in MAM ultrastructure is observed in the glioma tissue with respect to density, length, and width of the interfacing ER and mitochondrial membranes (**Figure 3**). In some astrocytoma cells, the MAM displayed a network or "work station"

**6.3. Mitochondrial network and mitochondria-associated membranes abnormalities** 

regions of a cell may display dissimilar morphology and biochemical properties [20].

branous processes such as molecules trafficking and signaling events.

**Figure 3.** (A–C) Glioblastoma cells displays variable organization of endoplasmic reticulum membrane associated with mitochondria (circles, ellipses, and arrows). M/m denotes mitochondria; er: endoplasmic reticulum profiles. N: cellular nucleus. Lucent-swelling mitochondria with disarrangement and distortion of cristae, and partial or total cristolysis, are seen.

(an area with high density of MAM and predicted the functional activity). Close or direct association (mitochondria-endoplasmic reticulum interface <30 nm) and detached or disrupted (>30 nm) associations is present. The shortest span of MAM was 96 nm, and the longest was 652 nm [10].

In the ultrastructural perspective, we identified two remarkable cell types: (1) poorly differentiated glioma stem cells and (2) well-differentiated glioma cells. The first one exhibits a poorly developed mitochondrial network and scarce MAM (named by us "MAM-deficient cells"). The second contains a well-developed MN and numerous MAM (named by us "MAMenriched cells"). MAM displayed a network or "work station" in some well-differentiated glioma cells [10] (**Figure 4**).

Previously, we suggest that the MAM could be involved in the invasive properties of glioma cells. Human glioma cell invadopodia show mitochondria with a dense matrix condensed configuration, indicating an active state. The mitochondria were frequently in close contact with an extended smooth endoplasmic reticulum displaying an endoplasmic reticulum subfraction associated with mitochondria MAM. Fluorescent microscopy confirmed that D54 and U251 glioma cells growing in vitro also contained filopodia with mitochondria (**Figure 5**). The U251 glioma cells' filopodia that penetrated through 1.2-μm pores of transwell chambers also contained mitochondria, suggesting that the mitochondria are actively involved in the invasion process [9].

In the vascular microenvironment components of gliomas, the mitochondrial network exhibit similar changes to describe in tumoral cells. The mitochondria display mainly two patterns: (1) swelling associated with disarrangement of cristae and partial or total cristolysis and (2) condensed configuration [8].

**Figure 4.** (A) Glioma like-stem cell exhibited, adjacent to nuclei, an endoplasmic reticulum an endoplasmic reticulum profile, and a small amount of electron-dense mitochondrion displayed a "MAM network" (black rectangle) with six direct interorganellar close associations with small span (white rectangles). (B) Well-differentiated tumor cell displays electron-lucent mitochondrion (m) in close association with multiple endoplasmic reticulum profiles establishing multiple MAM (rectangle) conforming a huge "MAM network". Similar fashion is observed in three cellular processes (arrows); es: denotes extracellular space.

### **6.4. Functional and therapeutics implications**

In the case of astrocytomas, the dense mitochondria could be capable of producing energy by oxidative phosphorylation, and lucent-swelling mitochondria with disarrangement and distortion of cristae and partial or total cristolysis are incapable of generating energy by oxidative phosphorylation. Possibly, the astrocytoma cells that hold dense mitochondria are able to generate sufficient ATP concentration by oxidative phosphorylation. In contrast, the astrocytoma cells that contain lucent swelling mitochondria with disarrangement and distortion of cristae and partial or total cristolysis are incompetent to produce an adequate amount of ATP by mitochondrial respiration. These findings suggest that the majority of astrocytoma cells are incompetent to produce an adequate amount of energy by means of oxidative phosphorylation [7–9]. The glycolytic inhibition and inhibition or down-regulation of mitochondrial respiration would be a potential tool for future therapeutic strategies in cases of human astrocytic tumors.

increased tumorigenicity, stem cell-ness, and invasiveness of invasive glioblastoma cells [23]. Molina et al. [23] reported that the glioma cells with high Akt activation actively invaded the surrounding parenchyma along blood vessels and with matter tracts. In human astrocytomas, the co-option vessel shows invadopodia with mitochondria that display dense matrix condensed configuration [9]. This finding possibly represents the ultrastructural basis of the molecular process expressed above, which permits the invasiveness of glioblastoma cells. On another hand, the PI3K-Rac and PI3K-3/Akt pathways are involved in the production of ROS that accumulates at the membrane ruffles [24], ROS production stimulates cytoskeletal reorganization required for a migratory response. Migrating glioma cells show activation of the PI3K/Akt pathway, and PI3K inhibitors have been tested experimentally, resulting in a decrease in migration [25]. Therefore, Inhibition of mitochondrial ROS generation may repre-

**Figure 5.** (A) Two glioblastoma multiforme cells exhibit several invadopodia that contain mitochondria with dense matrix condensed configuration (arrows). The cytosol shows multiple mitochondria with similar morphologies and physically adjacent to distended endoplasmic reticulum MAM. EM: denotes extracellular matrix. (B and C) Glioma cells filopodias (f). M denotes: mitochondria; \* designates: dilated endoplasmic reticulum cystern; arrows indicate: filiform projections. (D) Under fluorescent microscopy, U251 glioma cells stained with MitoTracker Red (label the mitochondria). The mitochondria are in the filopodia (circle and arrow). Green: actin filaments. Blue: nuclei. (personal communication and courtesy from Martin R. Jadus, Diagnostic & Molecular Health Care Group, Veterans Affairs Medical Center, Long Beach, California, USA, Neuro-Oncology Program, Chao Comprehensive Cancer, University of California–Irvine, Orange, California, and USA; and Pathology and Laboratory Medicine, Med. Sci. I, University of California, Irvine,

Functional and Therapeutic Implications of Mitochondrial Network and Mitochondria…

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21

The degree of development of MN and quantity of MAM could be linked to the functional or metabolic state of the different tumor cells found in human astrocytic tumors. Then, the welldifferentiated glioma cells (or "MAM-enriched cells") could be more active in these processes than the poorly differentiated glioma stem cells (or "MAM deficient cells") [10]. A recent study

sent another important therapeutic target to most gliomas.

California, USA).

Mitochondria are present at the invadopodia and their apparent function appears linked with the ROS generation and subsequent activation of several pathways essentials for glioma invasiveness. Mitochondria are a major source of ROS, which occurs mainly at complexes I and III of the respiratory chain. In cancer cells, mitochondria can generate ROS and redox signals, specifically via an increase in the NAD+ /NADH ratio [22]. H<sup>2</sup> O2 induces Akt (protein kinase B) activation, and their pathway is redox regulated. Akt activation correlated with the Functional and Therapeutic Implications of Mitochondrial Network and Mitochondria… http://dx.doi.org/10.5772/intechopen.77224 21

**Figure 5.** (A) Two glioblastoma multiforme cells exhibit several invadopodia that contain mitochondria with dense matrix condensed configuration (arrows). The cytosol shows multiple mitochondria with similar morphologies and physically adjacent to distended endoplasmic reticulum MAM. EM: denotes extracellular matrix. (B and C) Glioma cells filopodias (f). M denotes: mitochondria; \* designates: dilated endoplasmic reticulum cystern; arrows indicate: filiform projections. (D) Under fluorescent microscopy, U251 glioma cells stained with MitoTracker Red (label the mitochondria). The mitochondria are in the filopodia (circle and arrow). Green: actin filaments. Blue: nuclei. (personal communication and courtesy from Martin R. Jadus, Diagnostic & Molecular Health Care Group, Veterans Affairs Medical Center, Long Beach, California, USA, Neuro-Oncology Program, Chao Comprehensive Cancer, University of California–Irvine, Orange, California, and USA; and Pathology and Laboratory Medicine, Med. Sci. I, University of California, Irvine, California, USA).

**6.4. Functional and therapeutics implications**

20 Glioma - Contemporary Diagnostic and Therapeutic Approaches

(arrows); es: denotes extracellular space.

signals, specifically via an increase in the NAD+

In the case of astrocytomas, the dense mitochondria could be capable of producing energy by oxidative phosphorylation, and lucent-swelling mitochondria with disarrangement and distortion of cristae and partial or total cristolysis are incapable of generating energy by oxidative phosphorylation. Possibly, the astrocytoma cells that hold dense mitochondria are able to generate sufficient ATP concentration by oxidative phosphorylation. In contrast, the astrocytoma cells that contain lucent swelling mitochondria with disarrangement and distortion of cristae and partial or total cristolysis are incompetent to produce an adequate amount of ATP by mitochondrial respiration. These findings suggest that the majority of astrocytoma cells are incompetent to produce an adequate amount of energy by means of oxidative phosphorylation [7–9]. The glycolytic inhibition and inhibition or down-regulation of mitochondrial respiration would be a potential tool for future therapeutic strategies in cases of human astrocytic tumors. Mitochondria are present at the invadopodia and their apparent function appears linked with the ROS generation and subsequent activation of several pathways essentials for glioma invasiveness. Mitochondria are a major source of ROS, which occurs mainly at complexes I and III of the respiratory chain. In cancer cells, mitochondria can generate ROS and redox

**Figure 4.** (A) Glioma like-stem cell exhibited, adjacent to nuclei, an endoplasmic reticulum an endoplasmic reticulum profile, and a small amount of electron-dense mitochondrion displayed a "MAM network" (black rectangle) with six direct interorganellar close associations with small span (white rectangles). (B) Well-differentiated tumor cell displays electron-lucent mitochondrion (m) in close association with multiple endoplasmic reticulum profiles establishing multiple MAM (rectangle) conforming a huge "MAM network". Similar fashion is observed in three cellular processes

kinase B) activation, and their pathway is redox regulated. Akt activation correlated with the

/NADH ratio [22]. H<sup>2</sup>

O2

induces Akt (protein

increased tumorigenicity, stem cell-ness, and invasiveness of invasive glioblastoma cells [23]. Molina et al. [23] reported that the glioma cells with high Akt activation actively invaded the surrounding parenchyma along blood vessels and with matter tracts. In human astrocytomas, the co-option vessel shows invadopodia with mitochondria that display dense matrix condensed configuration [9]. This finding possibly represents the ultrastructural basis of the molecular process expressed above, which permits the invasiveness of glioblastoma cells. On another hand, the PI3K-Rac and PI3K-3/Akt pathways are involved in the production of ROS that accumulates at the membrane ruffles [24], ROS production stimulates cytoskeletal reorganization required for a migratory response. Migrating glioma cells show activation of the PI3K/Akt pathway, and PI3K inhibitors have been tested experimentally, resulting in a decrease in migration [25]. Therefore, Inhibition of mitochondrial ROS generation may represent another important therapeutic target to most gliomas.

The degree of development of MN and quantity of MAM could be linked to the functional or metabolic state of the different tumor cells found in human astrocytic tumors. Then, the welldifferentiated glioma cells (or "MAM-enriched cells") could be more active in these processes than the poorly differentiated glioma stem cells (or "MAM deficient cells") [10]. A recent study showed that glioma stem cells are less glycolytic than differentiated glioma cells, consuming lower levels of glucose, and producing lower amounts of lactate while maintaining higher ATP levels compared with their differentiated progeny [26]. Another study, by means of transmission electron microscopy, analysis revealed that the number of mitochondria with distinct cristae and electron-dense matrices increased significantly in the non-stem differentiated glioma cells when compared to their undifferentiated glioma stem cells. The final conclusion was that glioma stem cells prefer a relatively higher glucose metabolism, which implies that they utilize different mitochondrial biosynthesis and metabolic pathways when compared to differentiated glioma cells [27]. Other research established that glioma stem cells displayed diminished endoplasmic reticulum-mitochondria contacts compared to glioma differentiated cells. Forced endoplasmic reticulum-mitochondria contacts in glioma stem cells increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. The final conclusion was that endoplasmic reticulum-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells [28].

has been marginal [43]; added benefits from Temozolomide [44] and bevacizumab [45] are modest, and patient overall survival remains poor. Increasing recognition of the metabolic peculiarities of cancer has prompted investigations of nutritional strategies targeting glycemic modulation in cancer treatment, predominantly through the use of high-fat and low-carbohydrate diets (ketogenic diets; KDs), but also caloric restriction (CR), intermittent fasting (IF), and other combinatorial dietary protocols [46, 47]. All of these strategies induce a physiological state of systemic ketosis that metabolically compensates for the therapeutic reduction of carbohydrate intake and a concurrent decrease in blood glucose levels [47]. Both glycemic reduction and systemic ketosis are established key metabolic correlates of these nutritional strategies and are thought to mediate their therapeutic efficacy [35, 47]. The reduced availability of glucose as an energy substrate has been shown to selectively starve glioma cells both in vitro and in vivo [48–54]. Glioma cells are metabolically maladapted to utilize ketone bodies [48, 52, 55]. Unlike highly selective pharmacological blocking agents, KMT might produce a global dampening of insulin-related signaling with potentially more efficacy and less side effects [56]. On a functional level, several preclinical studies could demonstrate that ketogenic metabolic therapy (in particular, KD treatment, and/or CR) induces a metabolic shift in malignant brain tissue toward a proapoptotic, antiangiogenic, anti-invasive, and anti-inflammatory state accompanied by a marked reduction in tumor growth in vivo [57]. According to the current literature, ketogenic metabolic therapy is a safety and feasible alternative for malignant glioma. Cumulative clinical trials suggest that ketogenic metabolic therapy is emerging as a potential therapeutic option and might be combinable with existing anti-neoplastic treatments for malignant glioma [57]. Recently, a press-pulse therapeutic strategy for cancer management was presented [58]. The press-pulse therapeutic strategy for cancer management is illustrated with calorie-restricted ketogenic diets used together with drugs and procedures that create both chronic and intermittent acute stress on tumor cell energy metabolism, while protecting and enhancing the energy metabolism of normal cells. Optimization of dosing, timing, and scheduling of the presspulse therapeutic strategy will facilitate the eradication of tumor cells with minimal patient toxicity. This therapeutic strategy can be used as a framework for the design of clinical trials

Functional and Therapeutic Implications of Mitochondrial Network and Mitochondria…

http://dx.doi.org/10.5772/intechopen.77224

23

There is a great need to develop new therapies for gliomas. The ultrastructural findings observed in MN and MAM in the human gliomas indicate that: (1) The majority of glioma cells are incompetent to produce adequate amount of energy by means of oxidative phosphorylation and compensatory increases in glycolytic ATP production and (2) The variability of the ultrastructural aspects of MAM observed on astrocytic tumors suggests a dynamic regulation of the interorganellar junction that can be modified by functional requirements needed to adapt to different cell demands. These findings possibly represent the ultrastructural basis of the metabolic processes of glioma cells. MAM-resident mTORC2 controls the MAM integrity and mitochondrial functions, and mTORC2 can promote growth and chemotherapy resistance in cancer cells as well as tumor metabolism including glycolysis, glutaminolysis,

for the non-toxic management of most cancers [58].

**7. Conclusions**

The length of the interface is changing under different biochemical conditions [29, 30]. Apparently, the execution of the physiological programs is dependent on the length of the MAM, since the structural plasticity of the MAM cleft accompanies changes in cell metabolism [29]. Changing the thickness of MAM would impact on the activity of several enzymes of the Krebs cycle and on the strength of the IP3R Ca2+ signaling pathway [30]. Furthermore, the variability of the ultrastructural aspects observed on astrocytic tumors suggests a dynamic regulation of the interorganellar junction that can be modified by functional requirements needed to adapt to different cell demands. Solid and glycolytic tumor tissue is frequently characterized by a loss of normal MAM architecture and formation [6]. Today, altered Ca2+ signaling at the MAM is recognized as a hallmark of cancer cells that shifts their metabolism to glycolysis and increases their resistance to cell death [31]. MAM-resident mTORC2 controls the MAM integrity and mitochondrial functions [4, 32] and is the core of MAM signaling hub that controls growth and metabolism. Recent studies suggest that mTORC2 can promote glioblastoma growth and chemotherapy resistance in cancer cells as well as controlling genome stability and tumor metabolism including glycolysis, glutaminolysis, lipogenesis, and nucleotide and reactive oxygen species metabolism [33]. Glucose is required to activate mTORC2 and promote tumor growth [33] by means an auto-activation loop of mTORC2, rendering glioblastoma resistant to EGFR, PI3K, or AKT-targeted therapies. Then, if sufficient nutrients are present, glioblastoma cells maintain mTORC2 signaling to drive cell proliferation, and survival [33, 34]. mTOCR2 markedly increases glycolysis in glioblastoma [33]. Consequently, replacement of fermentable fuels like glucose and glutamine with nonfermentable fuels like ketone bodies becomes a logical approach to management [35, 36]. The dietary intervention prevents glioma cells accessing their preferred fuel source, i.e., glucose [37–40], and consequently, the signal transduction of mTORC2, cell proliferation and survival are diminished [35]. Therefore, impairments in glucose availability can be devastating for glioma survival [26].

The current standard of care for glioblastoma patients consists of maximal safe resection, followed by radiotherapy, and concurrent chemotherapy with Temozolomide [15, 41, 42]. Despite substantial clinical research efforts over the past decades, therapeutic progress has been marginal [43]; added benefits from Temozolomide [44] and bevacizumab [45] are modest, and patient overall survival remains poor. Increasing recognition of the metabolic peculiarities of cancer has prompted investigations of nutritional strategies targeting glycemic modulation in cancer treatment, predominantly through the use of high-fat and low-carbohydrate diets (ketogenic diets; KDs), but also caloric restriction (CR), intermittent fasting (IF), and other combinatorial dietary protocols [46, 47]. All of these strategies induce a physiological state of systemic ketosis that metabolically compensates for the therapeutic reduction of carbohydrate intake and a concurrent decrease in blood glucose levels [47]. Both glycemic reduction and systemic ketosis are established key metabolic correlates of these nutritional strategies and are thought to mediate their therapeutic efficacy [35, 47]. The reduced availability of glucose as an energy substrate has been shown to selectively starve glioma cells both in vitro and in vivo [48–54]. Glioma cells are metabolically maladapted to utilize ketone bodies [48, 52, 55]. Unlike highly selective pharmacological blocking agents, KMT might produce a global dampening of insulin-related signaling with potentially more efficacy and less side effects [56]. On a functional level, several preclinical studies could demonstrate that ketogenic metabolic therapy (in particular, KD treatment, and/or CR) induces a metabolic shift in malignant brain tissue toward a proapoptotic, antiangiogenic, anti-invasive, and anti-inflammatory state accompanied by a marked reduction in tumor growth in vivo [57]. According to the current literature, ketogenic metabolic therapy is a safety and feasible alternative for malignant glioma. Cumulative clinical trials suggest that ketogenic metabolic therapy is emerging as a potential therapeutic option and might be combinable with existing anti-neoplastic treatments for malignant glioma [57]. Recently, a press-pulse therapeutic strategy for cancer management was presented [58]. The press-pulse therapeutic strategy for cancer management is illustrated with calorie-restricted ketogenic diets used together with drugs and procedures that create both chronic and intermittent acute stress on tumor cell energy metabolism, while protecting and enhancing the energy metabolism of normal cells. Optimization of dosing, timing, and scheduling of the presspulse therapeutic strategy will facilitate the eradication of tumor cells with minimal patient toxicity. This therapeutic strategy can be used as a framework for the design of clinical trials for the non-toxic management of most cancers [58].
