**1. Introduction**

Hepatocellular carcinoma (HCC) represents 80% of the primary liver cancer and, in minor proportion, bile duct cancer and angiosarcoma of the blood vessels in the liver, but all of them have a poor prognosis. HCC is a major cause of cancer-related deaths globally. The incidence of HCC is increasing and has been rising in the last few decades [1]. The HCC is a complex pathology associated in 80–90% with chronic liver diseases like cirrhosis of diverse etiologies. Cirrhosis is a chronic degenerative disease of the hepatic parenchyma characterized by an inflammation process that leads to liver fibrogenesis. This process induces the loss of liver architecture and a diminution of functional parenchyma, which over time changes the environment of the cells resulting in chromosomal instability. The cause of cirrhosis transformation into HCC is not well known, but chromosomal instability could be an important factor for HCC generation in cirrhotic patients. The main problem of this pathology is the lack of early detection, recurrence of tumors following resection [2], and there are no effective therapies. To understand this complex pathology, it is convenient to have some knowledge of the structure and functions of the liver. Therapeutic options for HCC are very limited, and the incidence is very similar to the death rate per year. Only in the early stage of the disease, there are some approved therapies such as tumor ablation, surgical resection, and liver transplantation, but in advanced stages, when most patients are diagnosed, these treatments are not recommended. There is an average of 5-year survival below 20% with these therapies [3]. In intermediate and advanced stage-HCC, the approved options are transcatheter arterial chemoembolization (TACE) and the multi-kinase inhibitor, sorafenib. TACE therapy could extend survival to 2 years [3]. Sorafenib extends survival of patients with advanced stage disease for only 3 months, and this medication causes considerable adverse effects and offers no symptom palliation [4]. There are other several clinical trial efforts focused on therapies involving multiple signaling pathways, most commonly related to tyrosine-kinase growth factor receptors, but they have inferior survival benefits and several adverse effects. Immunotherapy has demonstrated some efficacy, but, in general, molecular characterization to find effective treatments of HCC is needed.

When they are activated, they play an essential role in the synthesis and degradation of the extracellular matrix (ECM) proteins and fibrogenic cytokines, like hepatocyte growth factor (HGF), insulin growth factor (IGR), transforming growth factor-β (TGF-β), and, consequently, induce cirrhosis. Biliary epithelial cells participate in the formation of bile; they are transported to the bile ducts or Canals of Hering. These cells have the potential to become oval cells [7]. The cell-free hepatic tissue represents 20% of the liver volume and constitutes the ECM located in the Disse space. The ECM contains structural proteins like collagen of different types, glycoproteins, fibronectin, tenascin, laminin, entactin, and perlecan. Their function is to maintain the hepatic architecture and the organization of the entire organ. Hepatocytes contribute with 80–90% of the synthesis of liver collagen, which is degraded by metalloproteinases (MMPs) [12]. The liver has multiple functions needed for its own metabolism and for other organs; it participates intensely in the intermediary metabolism that occurs mainly in hepatocytes and is connected with the nutrients of the diet, reaching from the portal circulation, that is, in carbohydrates, proteins, and lipid metabolism. The liver also generates purines and pyrimidines for its own use and their distribution to other tissues in the form of adenosine, inosine, and hypoxanthine [13]. It also synthetizes and secretes plasma proteins and participates in the

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Previously, we have demonstrated that adenosine is a metabolic modulator of glucose and lipids in the liver and adipose tissue [14]. This molecule also modulates in vivo the energy charge in the liver [15]. The nucleoside adenosine is a substance with multiphysiological effects in different tissues, the central nervous system, and cardiovascular system; it is responsible for the modulation of the immune response and acts as metabolic regulator. Its action could be autocrine, paracrine, and endocrine; its metabolism is very active with a high turnover and a very short half-live. Adenosine presents circadian variations in the rat, which correlated with energetic homeostasis of the cell, modulation of membrane structure and function, cell proliferation, and genetic expression by regulating physiological methylation [16]. Exogenous adenosine administration to normal rats showed some pharmacological effects, like increased ATP levels simultaneous to a decrease in ADP and AMP, resulting in an increase of the energy charge of the liver [14]. Also, in the liver of fasted rats, adenosine induces an enhancement of glycogen synthesis [16] and an inhibition of fatty acid oxidation by inhibiting the extramitochondrial acyl CoA synthase and decreasing the plasma ketone bodies [17] These findings allowed us to demonstrate in vivo the Atkinson hypothesis of metabolism regulation by energy charge [18].

The redox state of the cell in different compartments, calculated by the NAD<sup>+</sup>

tetrachloride (CCl4

and NADH nicotinamide adenine dinucleotide, oxidized and reduced) system, has been shown to be a key point in the control of metabolism [19]. Adenosine administration induces mitochondrial oxidation and promotes the oxidized state in the cytosol and mitochondria in the presence of fatty acid oxidation inhibition, which is induced by the nucleoside. It has been reported that adenosine modulates vasodilatation and vasoconstriction in the hepatic vessels controlling blood flow from the hepatic artery [20]. All these results observed in normal animals led us to test the effects of the nucleoside in several models of acute hepatotoxicity: one induced with ethanol [21], the second with cycloheximide, and the third with carbon

similar response generating a fatty liver that was prevented by adenosine [21–23]. In this way, the nucleoside, through different mechanisms, protects the liver against acute toxicity.

). Although the toxic mechanism of each one is different, they yielded a

/NADH (NAD+

biotransformation of endogenous and exogenous compounds.

The liver is the largest internal and heterogeneous organ in the body constituted by different kinds of cells like hepatocytes, endothelial cells, cells of the bile duct, Kupffer cells, hepatic stellate cells (HSC), oval cells and pit cells [5]. The liver is an organ highly irrigated by the portal venous system and blood is distributed by the hepatic sinusoids and the hepatic artery [6]. About 80% of the liver cells are hepatocytes, and are epithelial cells that form cords with high metabolic activity and contain a complete set of organelles: mitochondria, peroxisomes, lysosomes, Golgi complex and a well-organized cytoskeleton [7]. The space between cords of hepatocytes and the endothelium is called the space of Disse. Endothelial cells constitute the wall of the hepatic sinusoids and are separated from the parenchymal cells by the space of Disse. They possess pores or fenestrae that permit the exchange of fluids [8]. These cells show endocytic activity and secrete several mediators such as interleukin-1 (IL-1), interleukin-6 (IL-6), interferon, and nitric oxide as paracrine modulators. Kupffer cells are the fixed macrophages of the liver that can migrate along sinusoids. Their main function is an immunomodulatory one [9]. Pit cells are intrahepatic leucocytes with natural killer cell activity [10] and exert a cytotoxic activity toward tumor and virus-infected cells [11]. HSC, also known as lipocytes, fat storing cells, perisinusoidal cells, and vitamin A storing cells, are quiescent in normal conditions. When they are activated, they play an essential role in the synthesis and degradation of the extracellular matrix (ECM) proteins and fibrogenic cytokines, like hepatocyte growth factor (HGF), insulin growth factor (IGR), transforming growth factor-β (TGF-β), and, consequently, induce cirrhosis. Biliary epithelial cells participate in the formation of bile; they are transported to the bile ducts or Canals of Hering. These cells have the potential to become oval cells [7]. The cell-free hepatic tissue represents 20% of the liver volume and constitutes the ECM located in the Disse space. The ECM contains structural proteins like collagen of different types, glycoproteins, fibronectin, tenascin, laminin, entactin, and perlecan. Their function is to maintain the hepatic architecture and the organization of the entire organ. Hepatocytes contribute with 80–90% of the synthesis of liver collagen, which is degraded by metalloproteinases (MMPs) [12]. The liver has multiple functions needed for its own metabolism and for other organs; it participates intensely in the intermediary metabolism that occurs mainly in hepatocytes and is connected with the nutrients of the diet, reaching from the portal circulation, that is, in carbohydrates, proteins, and lipid metabolism. The liver also generates purines and pyrimidines for its own use and their distribution to other tissues in the form of adenosine, inosine, and hypoxanthine [13]. It also synthetizes and secretes plasma proteins and participates in the biotransformation of endogenous and exogenous compounds.

**1. Introduction**

140 Liver Cancer

Hepatocellular carcinoma (HCC) represents 80% of the primary liver cancer and, in minor proportion, bile duct cancer and angiosarcoma of the blood vessels in the liver, but all of them have a poor prognosis. HCC is a major cause of cancer-related deaths globally. The incidence of HCC is increasing and has been rising in the last few decades [1]. The HCC is a complex pathology associated in 80–90% with chronic liver diseases like cirrhosis of diverse etiologies. Cirrhosis is a chronic degenerative disease of the hepatic parenchyma characterized by an inflammation process that leads to liver fibrogenesis. This process induces the loss of liver architecture and a diminution of functional parenchyma, which over time changes the environment of the cells resulting in chromosomal instability. The cause of cirrhosis transformation into HCC is not well known, but chromosomal instability could be an important factor for HCC generation in cirrhotic patients. The main problem of this pathology is the lack of early detection, recurrence of tumors following resection [2], and there are no effective therapies. To understand this complex pathology, it is convenient to have some knowledge of the structure and functions of the liver. Therapeutic options for HCC are very limited, and the incidence is very similar to the death rate per year. Only in the early stage of the disease, there are some approved therapies such as tumor ablation, surgical resection, and liver transplantation, but in advanced stages, when most patients are diagnosed, these treatments are not recommended. There is an average of 5-year survival below 20% with these therapies [3]. In intermediate and advanced stage-HCC, the approved options are transcatheter arterial chemoembolization (TACE) and the multi-kinase inhibitor, sorafenib. TACE therapy could extend survival to 2 years [3]. Sorafenib extends survival of patients with advanced stage disease for only 3 months, and this medication causes considerable adverse effects and offers no symptom palliation [4]. There are other several clinical trial efforts focused on therapies involving multiple signaling pathways, most commonly related to tyrosine-kinase growth factor receptors, but they have inferior survival benefits and several adverse effects. Immunotherapy has demonstrated some efficacy, but, in

general, molecular characterization to find effective treatments of HCC is needed.

The liver is the largest internal and heterogeneous organ in the body constituted by different kinds of cells like hepatocytes, endothelial cells, cells of the bile duct, Kupffer cells, hepatic stellate cells (HSC), oval cells and pit cells [5]. The liver is an organ highly irrigated by the portal venous system and blood is distributed by the hepatic sinusoids and the hepatic artery [6]. About 80% of the liver cells are hepatocytes, and are epithelial cells that form cords with high metabolic activity and contain a complete set of organelles: mitochondria, peroxisomes, lysosomes, Golgi complex and a well-organized cytoskeleton [7]. The space between cords of hepatocytes and the endothelium is called the space of Disse. Endothelial cells constitute the wall of the hepatic sinusoids and are separated from the parenchymal cells by the space of Disse. They possess pores or fenestrae that permit the exchange of fluids [8]. These cells show endocytic activity and secrete several mediators such as interleukin-1 (IL-1), interleukin-6 (IL-6), interferon, and nitric oxide as paracrine modulators. Kupffer cells are the fixed macrophages of the liver that can migrate along sinusoids. Their main function is an immunomodulatory one [9]. Pit cells are intrahepatic leucocytes with natural killer cell activity [10] and exert a cytotoxic activity toward tumor and virus-infected cells [11]. HSC, also known as lipocytes, fat storing cells, perisinusoidal cells, and vitamin A storing cells, are quiescent in normal conditions. Previously, we have demonstrated that adenosine is a metabolic modulator of glucose and lipids in the liver and adipose tissue [14]. This molecule also modulates in vivo the energy charge in the liver [15]. The nucleoside adenosine is a substance with multiphysiological effects in different tissues, the central nervous system, and cardiovascular system; it is responsible for the modulation of the immune response and acts as metabolic regulator. Its action could be autocrine, paracrine, and endocrine; its metabolism is very active with a high turnover and a very short half-live. Adenosine presents circadian variations in the rat, which correlated with energetic homeostasis of the cell, modulation of membrane structure and function, cell proliferation, and genetic expression by regulating physiological methylation [16]. Exogenous adenosine administration to normal rats showed some pharmacological effects, like increased ATP levels simultaneous to a decrease in ADP and AMP, resulting in an increase of the energy charge of the liver [14]. Also, in the liver of fasted rats, adenosine induces an enhancement of glycogen synthesis [16] and an inhibition of fatty acid oxidation by inhibiting the extramitochondrial acyl CoA synthase and decreasing the plasma ketone bodies [17] These findings allowed us to demonstrate in vivo the Atkinson hypothesis of metabolism regulation by energy charge [18].

The redox state of the cell in different compartments, calculated by the NAD<sup>+</sup> /NADH (NAD+ and NADH nicotinamide adenine dinucleotide, oxidized and reduced) system, has been shown to be a key point in the control of metabolism [19]. Adenosine administration induces mitochondrial oxidation and promotes the oxidized state in the cytosol and mitochondria in the presence of fatty acid oxidation inhibition, which is induced by the nucleoside. It has been reported that adenosine modulates vasodilatation and vasoconstriction in the hepatic vessels controlling blood flow from the hepatic artery [20]. All these results observed in normal animals led us to test the effects of the nucleoside in several models of acute hepatotoxicity: one induced with ethanol [21], the second with cycloheximide, and the third with carbon tetrachloride (CCl4 ). Although the toxic mechanism of each one is different, they yielded a similar response generating a fatty liver that was prevented by adenosine [21–23]. In this way, the nucleoside, through different mechanisms, protects the liver against acute toxicity.

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 cirrhosis after 10 weeks of CCl4 administration. Five weeks after suspension of the toxin, animals 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 adenosine as a potential therapeutic agent in the treatment of chronic hepatic disease.

mass and architecture. This process is regulated by cell cycle proteins, cytokines, growth fac-

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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

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 contrib-

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

uting to restoring p53 protein expression to control levels [39].


is impaired, and instead a net deposition of fibrillar collagen is predominant [33].

tors, and matrix remodeling [32].

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 the CCl4 induced cirrhosis. The aspartate of adenosine, named IFC-305, showed interesting 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 modification of immunity genes which where explored in the CCl<sup>4</sup> model; it was found that the IFC-305 compound reduced inflammatory cytokines and increased the anti-inflammatory ones like IL-10, supporting the modulation of the macrophage phenotypes M1 and M2 [31].
