**3. Cellular senescence**

#### **3.1. Hepatocytes**

Historical views of liver cell replication supports the physiological properties of the hepatocytes to restore function as response to parenchymal loss [64]. However, massive or unending injury may overcome regenerative processes or may promote a dysfunctional repair process leading to progressive liver fibrosis, development of portal hypertension and eventually liver failure. Senescent status was induced in HepG2 cells by exposure to H<sup>2</sup> O2 . Its consequences and metabolic activity were interrogated [18] and morphological changes were noted with respect to SA-ß-GAL and SAF's expression, cell cycle arrest as well as the upregulation of p53, p21 and p16 genes. Regarding cytokine expression, IL-8 was upregulated while IL-6 was downregulated. Disturbances in glucose and lipid metabolism were evident with upregulation of growth hormone/IGF1 (SOCS2) and glycolysis (PGM2LT). Nonetheless, the downregulation of glucogenolysis and gluconeogenesis (G6PC) were more prominent. The unsaturation of fatty acids was hyperactive (FADS3) with parallel hypo activity of lipoprotein and hepatic lipase activity through the Apo-lipoprotein (APC3) system. APC3 also limits the uptake of chylomicrons by the liver cell. Other fatty acid downregulated proteins included SORL1 (involved in the uptake of LDL), ACSM2B (a medium-chain fatty-acid-CoA ligase) and PHGDH indirectly involved in amino-acid synthesis [18]. In addition, senescent cells secreted a variety of bioactive molecules including proinflammatory cytokines and chemokines that may influence extracellular matrix and the microenvironment but as well modulate the immune response with the promotion of macrophage migration leading to further increase in the inflammatory mielue [65]. Monocyte chemotactic protein (MCP-1) could provide a signal for monocyte recruitment into the liver followed by activation of Kupffer cells with the upregulation of death ligands. The expression of Fas ligand, TNFα, and TNF-related apoptosis inducing ligand (TRAIL) further aggravates lipo-apoptosis [66]. In addition the FFA palmitate increases the expression of TRAIL and abrogation of the TRAIL receptor expression suppresses the inflammation induced by nutrient excess in mice [66].

unknown, most of the monogenic diseases have in common primary alterations in the fat tissue consistent with disturbances of the adipogenesis process or defects in the formation, maintenance and/or regulation of the lipid droplet [63]. Acquired syndromes are seen mainly after HIV therapy with anti-retroviral agents as zidovudine and stavudine (tNRTl's). Agents known to render mitochondrial toxicity with metabolic disturbances similar to the metabolic syndrome seen in obesity. This metabolic adverse effects include premature aging associated with impaired prelamin-A maturation [63]. Lamin-A alterations could produce fragile nuclear envelopes, alter chromatin organization, increase oxidative stress and promote premature senescence at the cellular level. The metabolic disturbances observed in genetic or acquired lipodystrophic syndromes support the hypothesis of a primary fat compartment dysfunction as the source of metabolic disturbances, similar to the ones detected in obesity. Chronic liver disease is associated with an increased translocation of intestinal bacteria contributory to the liver inflammatory response and may promote the development of HCC [12]. Liposaccharide (LPS) produced by Gram (−) bacteria hosted in intestines from obese humans and rodents was associated with the transition of NAFLD to NASH and consequently to its progression to cirrhosis and HCC. LPS is recognized by the Toll like receptor 4 (TLR4) which is expressed upon cell activation on migrating and local macrophages (Kupffer cells). TLR4 is central for the secretion of TNF-ß and IL-6, cytokines present in the chronic inflammatory environment that precedes the detection of malignancy [12]. Further support to the role of LPS was found by interventions such as gut sterilization, removal of LPS or inactivation of TLR4; maneuvers that diminished tumor growth in chronically injured livers [12]. In experimental models, dietary or genetic obesity alterations on the gut microbiota increased levels of metabolites like deoxycholic acid (DCA) that in turn damages DNA. The enterohepatic circulation may further enhance the concentration of such metabolites by both encouraging the senescent-associated secretory phenotype response

Historical views of liver cell replication supports the physiological properties of the hepatocytes to restore function as response to parenchymal loss [64]. However, massive or unending injury may overcome regenerative processes or may promote a dysfunctional repair process leading to progressive liver fibrosis, development of portal hypertension and eventually liver fail-

metabolic activity were interrogated [18] and morphological changes were noted with respect to SA-ß-GAL and SAF's expression, cell cycle arrest as well as the upregulation of p53, p21 and p16 genes. Regarding cytokine expression, IL-8 was upregulated while IL-6 was downregulated. Disturbances in glucose and lipid metabolism were evident with upregulation of growth hormone/IGF1 (SOCS2) and glycolysis (PGM2LT). Nonetheless, the downregulation of glucogenolysis and gluconeogenesis (G6PC) were more prominent. The unsaturation of fatty acids was hyperactive (FADS3) with parallel hypo activity of lipoprotein and hepatic lipase activity through the Apo-lipoprotein (APC3) system. APC3 also limits the uptake of chylomicrons by the liver

O2

. Its consequences and

ure. Senescent status was induced in HepG2 cells by exposure to H<sup>2</sup>

and favoring a tumor-promoting environment.

82 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

**3. Cellular senescence**

**3.1. Hepatocytes**

Prior assumptions on cellular senescence determined that cell cycle arrest was a mechanism to protect the cell towards tumorigenesis. Nevertheless, it has been shown that the cell in cycle arrest can produce pro-inflammatory mediators, the senescence-associated secretory phenotype that promotes tumor growth [67]. During chronic liver disease, senescent machinery becomes "hijacked" perhaps triggering proliferation and transformation of hepatocytes, thus, promoting metabolic adaptation which may enhance tumor grafting and growth [68, 69]. The above metabolic paths could at least in part, be mediated by the over expression of the phosphatase and tensin homolog (PTEN) described in T-leukemia but later shown in liver tumors to inhibit the pentose phosphatase pathway (PPP) by binding to glucose-6-phophodiesterase (G6PD). With no active G6DP dimer, cells favor glycolysis with the production of lactate even in the presence of oxygen [70].

Aging and senescent liver cells have different genetic paths that may converge to similar metabolic traits. Aging liver cells have a proliferative response after injury associated with the repression of C/EBPα, Farnesoid X Receptor (FXR), telomere reverse transcriptase (TERT), and a decrease in the Wnt signaling pathway [71, 72]. A physiological Wnt signaling pathway involves a soluble ligand that binds to the Frizzled receptor (Fzd) and the LRP5/6 co-receptor on the plasma membrane; this interaction activates the cytoplasmic Disheveled protein which inhibits the ß-catenin (Ctnnb1) destruction complex (APC, GSK3ß, and Axin) by preventing Ctnnb1 phosphorylation and its subsequent destruction. Stable ß-catenin (intact Wnt signaling) translocates to the nucleus to form a complex with Lef and Tcf transcription factors that target genes as c-Myc and Cyclin D1. In cell culture and a mice model of HCC, tumor growth was ablated by the suppression of N-Myc downregulated gene 1 (NDRG1) expression; it promoted HCC cells to go into cell arrest [73]. The induction of senescence on malignant cells was accomplished by upregulation of the tumor suppressor genes p53, p21 and p16 in addition to decreased phosphorylated Rb. Senescent liver cells response to injury included transcription of Nf-kB, Myb, Nkx2-1, Nr5a2 and Ep300 factors; proteins known to be involved in inflammation, cell differentiation, lipid metabolism and chromatin remodeling. In addition, the chronic inflammatory phenotype of senescent cells induces telomere dysfunction and accelerates liver cell aging [74]. Thus, decreased physiological cell signaling that occurs with aging plus stress induced cell senescence may add to the lipid toxic microenvironment by promoting a vicious circle that overrules redundant mechanisms that prevent uncontrolled cell division. Mechanisms that imply an apoptosis "switch" from a pro-apoptotic to an anti-apoptotic status. Nonetheless, it is no clear the role of mitochondrial Bcl-2 proteins family and their expression may determine cellular fate [75].

Cellular events that follows are the activation/repression of factors involved in cell proliferation. In the liver cell, the known transcriptional shift includes activation of FOXO3, FOXII, E2F1, c-jun, C/EBPß, Myb, USF and neutralization of inhibitors of cell proliferation such as Rb family and C/EBP family of proteins [76]. In C/EBP-S193A mice, failure to stop liver regeneration after surgery correlated with the epigenetic repression of C/EBPß, p53, FXR, SIRT1, PGC1α and TERT. The repression was performed by a protein formed by C/EBPß-HDCAC1 complex which also inhibit the promoters of enzymes for glucose synthesis PEPCK and G6P [76]. The response of cell cycle engaged hepatocytes and cell cycle arrested hepatocytes (senescent cell) to injury is different and it may awake an unregulated cell growth on quiescent stem liver cells [76, 77]. Oval shaped liver cells may differentiate into cholangiocytes with a distinct metabolism and perhaps pathway towards malignancy [15, 16]. Although cholangiocytes are metabolically very active cells involved in the secretion and resorption of water and soluble bile components, they are not directly involved in the metabolism and/or regulation of biliary lipid species (cholesterol, bile acids and phosphatidyl-choline vesicles) [78, 79].

since 90% of the HCC's flourish in a highly progressive fibrotic ECM, the question raises if it is the changes on the microenvironment that further promotes metabolic transformation with an "apoptotic switch" and tumor development. Interestingly, progressive liver fibrogenic ECM becomes enriched with vascular growth factor (VGF) receptor promoting angiogenesis, paving

The different components of the ECM, cellular and non-cellular interact directly and indirectly with malignant cells therefore changing the phenotype of the evolving cells that in turn produces feedback signals to further adapt the microenvironment to the needs of the malignant cell. The link between the actin cytoskeleton and the microenvironment provides an input of intracellular contractile forces capable of regulating signaling pathways fundamental to the definition of cell phenotype, mechanism that constitute the ECM "out-side-in" code to the cell. In response, the anchored cells expressed adhesions molecules and secreted proteins that signals HSC and other ECM regulators increasing anchoring sites in response to the "in-side-out" signaling [67]. Therefore, the metabolic transformation of the already stressed parenchymal cells help to choose a path different to senescence and necrosis but to a path of unregulated regeneration, thus escaping apoptosis. A path that needs an ECM differentiation

SEC's are specialized endothelial cells that lie flat in the liver sinusoids along and in direct contact with the hepatocytes. Through their membranes and specialized pores or fenestra passes high concentrations of metabolites, proteins and other blood compounds, traffic which is regulated by the size of the fenestra. SEC's play a critical role in immune-activation, rolling of T cells, macrophages and PMN migration. Liver sinusoidal endothelial cells may be affected with age and obesity. SEC from old individuals have impaired and reduced expression of VEGF likely due to impaired nuclear transport of P-STST3 and P-CREB transcription factors [82, 83]. In a rodent model of sepsis, endothelial nitrogen oxide synthase (eNOS) deficient mice and aging mice had the same mortality and mitochondrial dysfunction upon the isolation of SEC mitochondrion [84]. In obesity and during early fibrogenesis, SEC lose their fenestra, decreasing the exchange of metabolites and increase the secretion of several basement membrane components (type IV collagen, perlecan, entactin and laminin) [64]. Authors concluded that an endothelial base-line dysfunction in the aging animal is manifested by a weakened antioxidant response and inappropriate energy production from mitochondrial dysfunction due to a tipped-balance of the SEC oxi-redox systems when exposed to additional stress. This is seen in the obese towards a state of energy depletion and cellular death, apoptosis or activation of a pro-coagulant/pro-fibrogenic phase. The

potential effects on mitochondrial function, a pro-fibrogenesis state and the promotion of insulin resistance status. Changes exaggerated in obesity, implying obesity may promote accelerated SEC aging processes. Interestingly, endothelial cellular senescence was inhibited in vitro and in the rodent by the activation of the liver x receptor (LXR), a nuclear receptor involved in the control of hepatic lipid and cholesterol metabolism [85]. Furthermore, LXR has been shown to play an important role in glucose metabolism, cytokine production

supply for HCC expansion [67].

Cellular Senescence and Their Role in Liver Metabolism in Health and Disease...

http://dx.doi.org/10.5772/intechopen.71659

85

delivery and availability to liver cells with its

the way for the much needed arterial high O<sup>2</sup>

**3.3. Sinusoidal endothelial cells (SEC)**

changes of SEC's with aging may limit O<sup>2</sup>

and anti-inflammatory response.

to assure cell survival in a non-efficient energy redox status.
