Studies of mechanisms of oxidative stress have shown that it activates signaling cascades (including MAPK pathway), which can seriously influence regulation of cell growth and transformation processes [84
]. Particularly, MAP kinases may be involved in pathogenesis of some diseases associated with oxidative stress.
It is known that the oxidative stress status has a key role in HCC development and progression.
The most important reactive oxygen species (ROS) derived by molecular oxygen include free oxygen radicals [e.g., superoxide (O2•-), hydroxyl radical (OH.), nitric oxide (NO.) radicals] as well as nonradical ROS [e.g., hydrogen peroxide (H2O2), organic hydroperoxides, and hypochloride].
A low level of ROS is indispensable in several physiologic processes of the cell including proliferation, apoptosis, cell cycle arrest, cell senescence, etc. [85
]. However, an increased level of ROS causes oxidative stress and creates a potentially toxic environment to the cells. In normal physiologic condition, a balance between ROS generation and oxidative defences exists in a cell. A significant role is played by endogenous antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) that act on O2
•- and H2
, respectively, and glutathione peroxidase (Gpx1) that uses glutathione as co-substrate. Despite the basal production of radicals is hampered by the anti-oxidant defences, the generation of ROS is amplified in response to various environmental perturbations.
This stressful condition is known to play a major role in cancer development mainly by enhancing DNA damage and by modifying some key cellular processes, such as DNA damage caused primarily by hydroxyl radicals [64
], cell proliferation, apoptosis, and cell motility cascades by superoxide radicals and hydrogen peroxides playing an important role in cancer development.
Although extensive or limited damage may trigger cell death, many cells can tolerate and repair the occasional hit from ROS. In the Fruehauf model [86
], when the balance tips further in favour of ROS, programmed cell death becomes a near certainty.
Excessive ROS, which the cellular enzymes cannot neutralize, alters the chemical environment within the mitochondria; in fact, the pore protein that forms a channel through the mitochondrial membranes becomes jammed in the open position, allowing cytochrome c to escape into the cytoplasm thus triggering programmed cell death.
The increase of ROS is associated with the increase of the inducible mitochondrial manganese SOD (MnSOD) expression. Elevated serum MnSOD levels have been found in patients with HCC [87
] and relatively high values of the enzyme have also been observed in patients with chronic hepatitis and liver cirrhosis. Therefore, it could be hypothesized that during induction of the malignant process in cirrhotic liver, the increase in MnSOD activity can already occur in the precancerous phase.
In cancer biology, NO can be involved either in promotion or in prevention of tumour occurrence dependently from tumour microenvironment, NO concentration and time of exposure [88
]. NO is a product of endothelial cells that binds and activates the guanylate cyclase, which catalyzes the conversion of GTP to the second messenger molecule cyclic GMP (cGMP). Concentrations of NO ranging between 1 and 30 nM produce high levels of cGMP promoting angiogenesis and proliferation of endothelial cells. In these conditions, ERK phosphorylation stimulates the proliferation of endothelial cells. Concentrations of NO ranging between 30 and 100 nM correspond to an increase of proliferative and anti-apoptotic AKT and ERK-dependent pathways in tumour cells [89
]. This range of concentrations seems to protect tumour cells from apoptosis and enhance angiogenic effects. In these conditions, the molecules activated by NO can be considered as factors correlated to poor prognosis events. On the other hand, higher NO levels (> 300 nM) promote apoptosis and are responsible for anti-tumour activity. NO levels are influenced also by ROS and, specifically, by superoxide anions that can attenuate the NO-mediated pathway. In fact, superoxide anions and ROS, through the scavenging of NO, can lower NO levels favouring its tumour-promoting activity [92
]. Accordingly, tumours have high levels of ROS and low levels of SOD.
Similarly to oxidative stress, the expression of nitrosative stress supports the de-regulated synthesis or overproduction of NO and NO-derived products and its toxic physiological consequences [93
]. The main source of NO in the mammals is the enzymatic oxidation of L
-arginine by NO synthases [94
]. As ROS, NO may limit oxidative damage by acting as a chain-breaking radical scavenger or may cause damage and kill cells by mechanisms that include inhibition of protein [95
] and DNA [96
] synthesis, downregulation of antioxidative enzymes [97
] and depletion of intracellular GSH [98
]. Nitrosative insult may occur in vivo
also in pathologies associated with inflammatory processes, neurotoxicity and ischaemia [99
NO is able to reduce oxidative injury via several mechanisms. NO reacts with peroxy and oxy radicals generated during the process of lipid peroxidation. The reactions between NO and these ROS can terminate lipid peroxidation and protect tissues from ROS-induced injuries [100
]. Through the Fenton reaction, hydrogen peroxide oxidizes iron (II) and the process generates an extremely reactive intermediate (the hydroxyl radical) which then carries out oxidations of different substrates [H2
+ OH- + hydroxyl radical (·OH)]. NO prevents hydroxyl radical formation by blocking the predominant iron catalyst in the Fenton reaction. In fact, NO reacts with iron and forms an iron-nitrosyl complex, inhibiting iron's catalytic functions in the Fenton reaction [101
Treatment of rat hepatocytes with NO induces resistance to H2
-induced cell death by induction of the rate-limiting antioxidant enzyme, heme oxygenase (HO-1) [102
]. In addition, NO prevents the induction of some ROS-induced genes during tissue injury such as early growth response-1 (EGR-1), which activates a number of adhesion molecules and accelerates oxidative tissue injuries [103
Regulatory events and their alterations depend on the magnitude and duration of the change in ROS or RNS concentration. ROS and RNS normally occur in living tissues at relatively low steady-state levels. The increase in superoxide or NO production leads to a temporary imbalance that forms the basis of redox regulation. The persistent production of abnormally large amounts of ROS or RNS, however, may lead to persistent changes in signal transduction and gene expression, which, in turn, may give rise to pathological conditions [104
3.1 Stress and HCC
Oxidative stress has emerged as a key player in both development and progression of many pathological conditions, including HCV- and HBV-induced liver diseases.
ER stress is a homeostatic mechanism, that regulates cellular metabolism and protein synthesis in response to perturbations in protein folding and biosynthesis [105
]. Moderate ER stress modulates protein synthesis initiation and causes a reduction in cell growth, whereas extreme or prolonged ER stress leads to apoptosis mediated by the activation of the ER-associated caspase 12 [106
Signaling from ER susceptible to stress is closely related to cell metabolism and intracellular redox status [107
]. Changes in cell metabolism can cause an increase of mutation processes including stimulation of cell proliferation and apoptosis [84
Studies of mechanisms of oxidative stress have shown that the latter activates signaling cascades (including MAP kinase pathway), which can seriously influence regulation of cell growth and transformation processes [84
] and may be involved in pathogenesis of some diseases associated with oxidative stress.
Oxidative stress also activates hepatic stellate cells that represent the main connective tissue cells in the liver, involved in formation of extracellular matrix and required for normal growth and differentiation of cells during liver damage. In this case, the stellate cells divide in response to various cytokines, growth factors, and chemokines produced by the damaged liver. Chronic activation of stellate cells in response to oxidative stress induced by viral replication may contribute to fibrogenesis and increase proliferation of hepatocytes chronically infected with HBV and HCV that, together with activation of MAP kinases, may induce HCC [108
The nuclear transcription factor-κB (NF-κB) is the major stress-inducible antiapoptotic transcription factor. NF-κB activation is associated with cancer, and it has been found to be strongly activated in many types of cancer, including HCC [108
Moreover, markers of acute intracellular oxidative stress were found elevated in patients with chronic HCV [109
] with accumulation of DNA adduct 8-hydroxydeoxyguanosine [110
]. Transgenic mice expressing HCV core protein show an increased accumulation of ROS that correlates with HCC development [111
The increased generation of ROS and RNS, together with the decreased antioxidant defense, promotes the development and progression of hepatic and extrahepatic complications of HCV infection.