**2. Hepatocytes proliferation in cirrhosis and cancer, modulation by IFC-305**

The liver is an organ with regenerative capacity. Partial hepatectomy or diverse stimuli promote proliferation of parenchymal and non-parenchymal cells in order to recover the liver mass and architecture. This process is regulated by cell cycle proteins, cytokines, growth factors, and matrix remodeling [32].

Continuous acute hepatotoxicity results in chronic liver injury with subsequent cirrhosis, with accumulation of ECM proteins, mainly collagen type I [24], accompanied by a deficient degradation of deposited collagen [25]. These conditions will induce a change in liver architecture with loss of its function. This is a complex process, for which no effective treatment has been developed yet. To study the effects of adenosine in this process, a model of cirrhosis induced in rats with CCl<sup>4</sup> was developed, in which two conditions were tested: prevention during cirrhosis development and reversion once it is already established [26, 27]. The simultaneous administration of adenosine partially blocked the stimulated collagen synthesis induced by the hepatotoxin, maintained high levels of hepatic collagenase activity, resulting in 50% diminution of fibrosis [26]. The effect of the nucleoside was clearly observed also in the reversion model; it was tested in well-established

were treated with saline or adenosine, the saline group increased the cirrhotic characteristics but the group of animals treated with the nucleoside revealed blocked fibrogenesis, increased collagen degradation and normalized collagen types ratio, promoted hepatocyte proliferation, accelerated normalization of liver function, and decreased oxidative stress. These results suggest

The transfer of an interesting research finding to a clinical setting is complicated, but in collaboration with Dr. Francisco Hernández Luis from the National Autonomous University of Mexico's School of Chemistry, we prepared several adenosine derivatives that were tested in

results [28]; beneficial effects in structure and functional recovery were obtained with a fourfold lower dose of this adenosine derivative because it has a longer half-life. The hepatoprotective mechanism of IFC-305 on fibrogenesis was investigated by means of DNA microarrays analysis [29], showing that the expression of 413 differential genes deregulated in cirrhosis tended to be normalized by IFC-305 treatment. Fibrogenic genes, such as TGF-β, collagen type I, fibronectin I, increased their expression in cirrhotic groups, and IFC-305 diminished their expression supporting the antifibrogenic action of the compound. These results highly suggest a diminution of chromosomal instability. With the increased understanding in chromatin organization of the eukaryote genome at genetic and epigenetic levels and remembering the previously commented role of adenosine on physiological methylations, a possible epigenetic mechanism of the IFC-305 could participate in the obtained results. Global changes in DNA methylation, 5-hydroxymethylation and histone H4 acetylation were decreased in cirrhosis and after the IFC-305 treatment the normal values were recuperated. In contrast, the promoter of *Col1a1* gene is hypomethylated in cirrhosis but gains DNA methylation upon treatment with IFC-305, correlating with a decrease of Col1a1 transcript and protein level, showing that the treatment restores globally and specifically epigenetic modifications [30]. The microarray analysis also showed

IFC-305 compound reduced inflammatory cytokines and increased the anti-inflammatory ones

The liver is an organ with regenerative capacity. Partial hepatectomy or diverse stimuli promote proliferation of parenchymal and non-parenchymal cells in order to recover the liver

like IL-10, supporting the modulation of the macrophage phenotypes M1 and M2 [31].

**2. Hepatocytes proliferation in cirrhosis and cancer, modulation by IFC-305**

induced cirrhosis. The aspartate of adenosine, named IFC-305, showed interesting

adenosine as a potential therapeutic agent in the treatment of chronic hepatic disease.

modification of immunity genes which where explored in the CCl<sup>4</sup>

administration. Five weeks after suspension of the toxin, animals

model; it was found that the

cirrhosis after 10 weeks of CCl4

the CCl4

142 Liver Cancer

In acute liver injury, there is a classic wound healing process in which inflammation triggers scar formation that is subsequently resolved to enable regeneration of the damaged hepatic parenchyma. However, when there is a chronic liver injury, the normal regenerative process is impaired, and instead a net deposition of fibrillar collagen is predominant [33].

Cirrhosis is characterized by a decrease in hepatocyte proliferation, in part, because liver cells have a limited regenerative capacity restricted by telomere length. After several rounds of replication, telomeres reach a critically short length that induces cell cycle arrest, senescence, and apoptosis of hepatocytes. Telomere shortening also activates DNA repair pathways leading to chromosomal fusions and instability [34]. During cirrhosis-activated HSC, inflammatory cells secrete proliferative and angiogenic cytokines that contribute to a proliferative condition milieu, including: HGF, vascular endothelial growth factor (VEGF), and IL-6 [33]. This proliferative milieu could stimulate the proliferation of altered hepatocytes carrying mutations of cell cycle checkpoint genes or could select genetically altered clones, promoting HCC [34].

Among the principal cell cycle checkpoints that are generally altered in HCC are the tumor suppressor p53 and Rb proteins. p53 is implicated in cell cycle control, DNA repair, apoptosis, and regulates different metabolic pathways [35, 36]. p53 is frequently mutated in HCC (28–50%) and core proteins from hepatitis B and C viruses can repress p53 activity [36]. The pRB protein is implicated in the progression from G1 into S phase. The Rb pathway is disrupted in more than 80% of human HCC [34]. Gankyrin binds Mdm2 promoting proteasomal degradation of p53 and pRb. Both gankyrin and Mdm2 proteins are frequently overexpressed in human HCC [34, 35]. p53 is also implicated in the stimulation of ATP production by oxidative phosphorylation (OXPHOS). p53 also decreases glycolysis and cellular reactive oxygen species (ROS) production by inducing a protein called TP53-induced glycolysis and apoptosis regulator (TIGAR). TIGAR blocks glycolysis by degrading fructose-2,6-bisphosphate. This inhibition redirects glucose-6-phosphate into the pentose phosphate pathway, which increases NADPH production increasing the antioxidant defenses. The inactivation of p53 should decrease OXPHOS and increase glycolysis and ROS production in cancer cells [37].

It has been demonstrated that IFC-305 is able to stimulate hepatocytes proliferation in CCl4 induced cirrhotic liver through the upregulation of proliferating cell nuclear antigen (PCNA), HGF, and p53, with an increase in energy and preservation of mitochondrial function [38].

On the other hand, in a sequential model of cirrhosis-HCC induced by diethylnirosamine (DEN), IFC-305 caused a tumor reduction, and this protective effect was associated with decreased cell proliferation in the HCC stage. This effect was associated with a decreased expression of PCNA, thymidylate synthase, HGF and its receptor c-Met, and the induction of the cell cycle inhibitor p27. IFC-305 also induced a diminution of gankyrin expression contributing to restoring p53 protein expression to control levels [39].

How could the same compound IFC-305 have opposing effects on proliferation in normal versus transformed hepatocytes? These could be mediated partly by a differential expression of the HGF-c-Met pathway driven by IFC-305 treatment, and the dual role of HGF/c-Met in cirrhosis and liver tumorigenesis. HGF expression is restricted to cells of mesenchymal origin, whereas the receptor c-Met is expressed in epithelial and endothelial cells. HGF is implicated 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 cirrhotic liver induced by CCl4 , the hepatoprotector IFC-305 incremented HGF expression [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 in CCl4 -induced cirrhosis and in DEN-induced carcinogenesis, among other effects, could 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 on hepatocytes proliferation in cirrhosis and HCC requires further clarification.

levels of acetyl CoA, which also plays an important role as modulator of gene expression [49].

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].

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

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

ity 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

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,

through the ratio of the levels of β-hydroxybutyrate/acetoacetate (NAD<sup>+</sup>

suggesting an increase in aerobic glycolysis [43].


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

145

Interaction of Mitochondrial and Epigenetic Regulation in Hepatocellular Carcinoma

/NADH ratio, which is regulated by the

that can be related to loss of

/NADH). The activ-

like sirtuin-1 (Sirt-1), a member of deacetylases, and

In experimental models, including the CCl4

The redox state can be represented by the NAD+

over-regulation of both enzymes in HCC may deplete the NAD+

ETC. Several enzymes depend on NAD+
