**3. Mitochondrial alterations in the HCC: the effect of the IFC-305 compound**

Mitochondria are responsible for energy metabolism in eukaryotic cells; they generate ATP through oxidative phosphorylation. In addition, an important part of the ATP synthesis is the donation of electrons by the tricarboxylic acids chain (TCA) to the electron transport chain (ETC), constituted by five complexes (I-V), NADH enters complex I and generates NAD<sup>+</sup> , and complex V forms ATP. Mitochondria regulate the energetic state, the redox state, and the metabolism of the cells, being able to generate the epigenetic intermediaries becoming the main therapeutic target of many kinds of cancer [44].

As a response to stress, the cells acquire a metabolic adaptation, which is an important area of research due to its relationship with different illnesses [45]. In chronic liver diseases like cirrhosis, energetic deficiency and alterations in energy parameters have been demonstrated independently of their etiology [46]. Otto Warburg suggested that mitochondria from tumor cells supply energy through glycolytic flow due to lack of oxygen or genetic-epigenetic alterations that affect oxidative metabolism [47]. Mitochondrial dysfunction is implicated in metabolic reprogramming in HCC. The increased ROS production and the reduced ATP generation may contribute to the HCC malignancy [48]. Metabolic alterations may decrease the levels of acetyl CoA, which also plays an important role as modulator of gene expression [49]. In experimental models, including the CCl4 -induced cirrhosis, mitochondrial dysfunction has been demonstrated because impaired mitochondrial respiration and ATP decreased levels have been observed [50, 51]. A metabolic adaptation in response to the ATP diminished levels is increased glycolysis [51]. A consequence of oxidative stress in chronic liver diseases is the decrease in metabolic flux, which includes alterations in the TCA enzymes, such as isocitrate dehydrogenase (IDH), which can produce oncometabolites when it undergoes mutations [52].

in cell proliferation, survival, morphogenesis, cell motility, and metastasis. This pathway plays a critical role in tissue protection and regeneration. It has been used as a therapeutic agent in fibrosis of different organs. The protective actions of HGF are associated with promotion of cell proliferation, migration, and morphogenesis that would help tissues reorganization [40]. Its protective role is also related to its anti-inflammatory action and its regulation of the cellular redox state, driven by upregulation of the antioxidant enzymes and glutathione reduced (GSH), as well as by repression of two major pro-oxidant systems: NADPH oxidase and/or Cyp2E1 [41]. Nevertheless, the HGF/c-Met pathway in HCC contributes to tumor development by stimulating cell proliferation, invasion, and metastasis [40]. We observed that, in the

[38], which could have a protective role in the regenerative capacity of the liver. On the other hand, in DEN-induced HCC, the IFC305 treatment downregulated HGF and c-Met expression, which contribute to liver tumorigenesis reduction [39]. HGF and c-Met can be potentiated by ROS in hepatoma cells [41, 42]. It was described that, in the sequential model of cirrhosis-HCC with DEN, there are dysfunctional mitochondria and the administration of IFC-305 restored the mitochondrial function and regulated parameters implicated in metabolism, as well as the mitochondrial dynamics modified by DEN intoxication [43]. Therefore, the IFC-305 could be suppressing expression of HGF via the improvement of mitochondrial redox in DEN carcinogenesis. On the other hand, the restoration by IFC-305 treatment of the p53 protein expression


contribute to the restoration of ATP production by OXPHOS and to the decrease of ROS production. However, the exact molecular mechanism by which IFC-305 causes different effects

Mitochondria are responsible for energy metabolism in eukaryotic cells; they generate ATP through oxidative phosphorylation. In addition, an important part of the ATP synthesis is the donation of electrons by the tricarboxylic acids chain (TCA) to the electron transport chain (ETC), constituted by five complexes (I-V), NADH enters complex I and generates NAD<sup>+</sup>

and complex V forms ATP. Mitochondria regulate the energetic state, the redox state, and the metabolism of the cells, being able to generate the epigenetic intermediaries becoming the

As a response to stress, the cells acquire a metabolic adaptation, which is an important area of research due to its relationship with different illnesses [45]. In chronic liver diseases like cirrhosis, energetic deficiency and alterations in energy parameters have been demonstrated independently of their etiology [46]. Otto Warburg suggested that mitochondria from tumor cells supply energy through glycolytic flow due to lack of oxygen or genetic-epigenetic alterations that affect oxidative metabolism [47]. Mitochondrial dysfunction is implicated in metabolic reprogramming in HCC. The increased ROS production and the reduced ATP generation may contribute to the HCC malignancy [48]. Metabolic alterations may decrease the

on hepatocytes proliferation in cirrhosis and HCC requires further clarification.

**3. Mitochondrial alterations in the HCC: the effect of the IFC-305** 

main therapeutic target of many kinds of cancer [44].

, the hepatoprotector IFC-305 incremented HGF expression

,

cirrhotic liver induced by CCl4

in CCl4

144 Liver Cancer

**compound**

The redox state can be represented by the NAD+ /NADH ratio, which is regulated by the ETC. Several enzymes depend on NAD+ like sirtuin-1 (Sirt-1), a member of deacetylases, and poly (ADP-ribose) polymerase-1 (PARP-1). A Sirt-1 substrate is the peroxisome proliferatoractivated receptor gamma co-activator 1-alpha (PGC-1α), which is upregulated in HCC and is responsible for orchestrating mitochondrial biogenesis, favoring accumulation of defective mitochondria [44]. On the other hand, PARP-1 modulates the transcription and DNA repair; however, in HCC, it is upregulated and is considered a hallmark of cancer [53]. The over-regulation of both enzymes in HCC may deplete the NAD+ that can be related to loss of mitochondrial membrane potential (ψm) and mitochondrial dysfunction [54]. Alterations in ψm induce the process of mitochondrial dynamics as a repair response to possible damage to this organelle. Mitochondrial dynamics depends on two mechanisms: fission and fusion; the first one is caused by various types of stress and requires protein activity such as Drp-1, on the other hand, fusion requires the recovery of ψm and proteins such as mitofusin 1 and 2 (MFN 1 and 2) [44]. Mitochondrial fusion promotes cristae formation and normal mitochondria phenotype [55]. Morphological alterations in mitochondria determined through electronic microscopy in various models of hepatic fibrosis have been described a long time ago [56, 57].

Previously, it has been discussed some of the effects of adenosine (base molecule of IFC-305), which include increase in energy parameters and regulation of the redox state. Considering this background and what has been described regarding the metabolic and mitochondrial changes in chronic liver damage, such as cirrhosis and HCC, it was decided to evaluate whether IFC-305 had any mitochondrial effect in the sequential model of cirrhosis-HCC.

In the sequential model of cirrhosis-HCC, decreased mitochondrial respiration, determined through oxygen consumption, and a decreased ψm were found, which reflected in a diminished ATP synthesis. In fact, the dimeric form (active form) of the F1F0 complex of ATPase is lost [43].

On the other hand, alterations in the mitochondrial redox state were observed, determined through the ratio of the levels of β-hydroxybutyrate/acetoacetate (NAD<sup>+</sup> /NADH). The activity of NAD-dependent enzymes was also affected, such is the case of IDH and PARP-1; this alteration induced a metabolic adaptation because increased levels of lactate were observed suggesting an increase in aerobic glycolysis [43].

It is known that the mitochondrion is capable of responding to several insults of stress through the activity of various nuclear-encoded proteins like PGC-1α and Sirt-1. However, the over-regulation of these proteins has been associated with the accumulation of dysfunctional mitochondria, as described above. In the model previously described, these proteins were found increased. Dysfunctional mitochondria have been related to their morphology, and we know that morphology is closely linked to dynamism. The ratio of Drp-1/MFN-2, proteins that regulate the mitochondrial dynamics, was increased favoring the fragmented form of mitochondria as verified through electron microscopy [43].

two different pharmaceutical formulations, finding a decrease in the mitochondrial generation of ROS and a decrease in the dissipation of the mitochondrial membrane potential [59]. Moreover, two of the most studied hepatoprotective compounds until now are resveratrol and N-acetylcysteine (NAC) [60]. On the one hand, resveratrol inhibits the formation of hepatocyte nodules in the DEN-induced HCC model plus phenobarbital administration; moreover, it is capable of modulating mitochondrial biogenesis [61]. On the other hand, NAC blocked phosphorylation of β-catenin, JNK, and c-jun activation, avoiding the development of liver damage in HCC transaldolase-deficient mice, a limiting enzyme for the non-oxidative branch of the pentose phosphate pathway, which is, at least in part, responsible for HCC generation [62]; furthermore, NAC stabilizes the mitochondrial membrane potential regulating mitochondrial dynamics [61].

Interaction of Mitochondrial and Epigenetic Regulation in Hepatocellular Carcinoma

http://dx.doi.org/10.5772/intechopen.79923

147

**4. Interaction of mitochondria and epigenetics in HCC: An overview**

lated by mitochondrial intermediates, like acetyl-CoA, ATP, NAD+

The epigenome can be altered not only by environmental factors, such as exposure to exogenous chemicals [63] but also by changes in the levels of endogenous cofactors and metabolites [64, 65]. The exact correlation between nucleus and mitochondrion allows for the maintenance of mitochondrial structure and function. On the one hand, the nuclear gene expression is regu-

which are the link between the epigenome and calorie availability [47, 66]. In addition to the production of epigenetic substrates, mitochondria may be modified in their DNA (mtDNA). Some mitochondrial genes have been reported as hypermethylated in HCC; for example, mitochondrial ribosomal protein S12 (*Mrps12*), mitochondria-localized glutamic acid-rich protein *(Mgrap*), and transmembrane protein 70 (*Tmem70*) genes [67, 68]. On the other hand, the disruption of the step in the methylation of 5-mC to 5-hmC in the mitochondrial genome leads to the alteration of several OXPHOS genes, such as: NADH dehydrogenase (ubiquinone) 1 subunit C2 (NDUFC2), NADH dehydrogenase (ubiquinone) flavoprotein 1 (NDUFV1), NADH: ubiquinone oxidoreductase subunit S6 (NDUFS6) from complex 1. These modifications, added to the mitochondrial damage by oxidative stress, can favor the loss of ETC function. In addition to that, it has been reported that the mitochondrial genome damage can affect the expression of nuclear genes [69–71]. Moreover, there is a deregulation of hepatic one carbon, and TCA cycle, therefore it driving the aberrant epigenetics changes [72–74]. The main consequence of depressing the TCA cycle is the reduced availability of α-ketoglutarate, leading to a decrease in the activity of α-ketoglutarate-dependent proteins, which are responsible for the hydroxyl-

ation of many substrates in the cell that are important in epigenomic control [74].

genes and large chromosomal instability [77, 78].

and HCC have been confirmed [79, 80].

Tumor cell metabolism can be linked to epigenetic changes during carcinogenesis; recent research has focused on epigenetic studies in relation to metabolic pathways [75, 76]. HCC is a heterogeneous disease affected by various lifestyles and environmental factors. Epigenetic alterations are frequently caused by these factors and contribute to hepatocarcinogenesis. During HCC development, different alterations in global DNA methylation have been described; for example, global hypomethylation leads to aberrant overexpression of onco-

In cirrhosis and HCC, distinct patterns of aberrant DNA methylation associated with cirrhosis

, and s-adenosylmethionine,

Important findings were observed with the IFC-305 treatment as described in **Table 1** [43].

Uncoupled mitochondria depicted lower ATP synthesis due to the altered ψm and complex I activity. Previously, it has been demonstrated that complex I is sensitive to DEN toxicity, as NAD+ linked respiration is inhibited [58]. Recovery of these parameters with IFC-305 treatment was observed, including the activity of NAD+ -dependent IDH. The PARP-1 activity inhibition probably favored the NAD+ availability and contributed to the maintenance of the redox state. Mitochondrial function preservation and restoration allowed the normalization of the metabolism observed by lactate levels diminution.

On the other hand, the decreased Sirt-1 and PGC-1α in the groups treated with IFC-305 suggested that abnormal mitochondrial accumulation was inhibited. In fact, mitochondrial dynamics regulation was induced by IFC-305. These results demonstrated mitochondrial impairment through functional, metabolic, and dynamic alterations in HCC, and the hepatoprotector IFC-305 helps to repair them, being a tumor suppressive mechanism.

These findings support the mitochondrial role in the establishment of HCC and the interplay with the nuclear genome as targets in the design of new therapeutic strategies for the HCC treatment. In this regard, the IFC-305 supports that idea and emerges as a new possible HCC therapy through mitochondrial regulation.

According to the above, there is a growing interest to find pharmacological strategies to block the effects of mitochondrial dysfunction in HCC. Regarding this, in the model of HCC induced with DEN, a study was conducted to determine the mitochondrial effects of ginkgolide B in


**Table 1.** Effects of IFC-305 administration in mitochondria in the sequential model of cirrhosis-HCC.

two different pharmaceutical formulations, finding a decrease in the mitochondrial generation of ROS and a decrease in the dissipation of the mitochondrial membrane potential [59]. Moreover, two of the most studied hepatoprotective compounds until now are resveratrol and N-acetylcysteine (NAC) [60]. On the one hand, resveratrol inhibits the formation of hepatocyte nodules in the DEN-induced HCC model plus phenobarbital administration; moreover, it is capable of modulating mitochondrial biogenesis [61]. On the other hand, NAC blocked phosphorylation of β-catenin, JNK, and c-jun activation, avoiding the development of liver damage in HCC transaldolase-deficient mice, a limiting enzyme for the non-oxidative branch of the pentose phosphate pathway, which is, at least in part, responsible for HCC generation [62]; furthermore, NAC stabilizes the mitochondrial membrane potential regulating mitochondrial dynamics [61].

and we know that morphology is closely linked to dynamism. The ratio of Drp-1/MFN-2, proteins that regulate the mitochondrial dynamics, was increased favoring the fragmented

Important findings were observed with the IFC-305 treatment as described in **Table 1** [43].

Uncoupled mitochondria depicted lower ATP synthesis due to the altered ψm and complex I activity. Previously, it has been demonstrated that complex I is sensitive to DEN toxicity, as

redox state. Mitochondrial function preservation and restoration allowed the normalization

On the other hand, the decreased Sirt-1 and PGC-1α in the groups treated with IFC-305 suggested that abnormal mitochondrial accumulation was inhibited. In fact, mitochondrial dynamics regulation was induced by IFC-305. These results demonstrated mitochondrial impairment through functional, metabolic, and dynamic alterations in HCC, and the hepato-

These findings support the mitochondrial role in the establishment of HCC and the interplay with the nuclear genome as targets in the design of new therapeutic strategies for the HCC treatment. In this regard, the IFC-305 supports that idea and emerges as a new possible HCC

According to the above, there is a growing interest to find pharmacological strategies to block the effects of mitochondrial dysfunction in HCC. Regarding this, in the model of HCC induced with DEN, a study was conducted to determine the mitochondrial effects of ginkgolide B in

protector IFC-305 helps to repair them, being a tumor suppressive mechanism.

• mitochondrial respiration

• normal cellular redox state

Metabolic • Recovered the normal mitochondrial redox state • recovered the IDH activity • reduced lactate production

• Sirt-3 increment

• mitochondrial membrane potential • dimeric form of the F1F0 ATPase subunit

• diminished increased PARP-1 activity Dynamics Avoided the accumulation of dysfunctional mitochondria through: • down-regulation of PGC-1α and Sirt-1 • diminution of DRP-1/MFN-2 ratio

**Table 1.** Effects of IFC-305 administration in mitochondria in the sequential model of cirrhosis-HCC.

• ATP synthesis

linked respiration is inhibited [58]. Recovery of these parameters with IFC-305 treat-


availability and contributed to the maintenance of the

form of mitochondria as verified through electron microscopy [43].

ment was observed, including the activity of NAD+

of the metabolism observed by lactate levels diminution.

inhibition probably favored the NAD+

therapy through mitochondrial regulation.

Function Maintained and recovered:

**Mitochondrial parameter Effect**

NAD+

146 Liver Cancer
