An important cause of morbidity and mortality worldwide is the infection by HCV. Progress in understanding HCV biology has remained challenging due to the lack of an efficient cell culture system for virus growth. Establishment of self-replicating full-length HCV genomic replicons from genotypes in cultured cells has provided an important tool for the study of HCV replication mechanisms. This study discusses the system for the HepG2 cell line harboring HCV- genotype-4 replication and examines the expression levels of group of genes in clinical samples obtained from HCC and CH patients. Other studies have reported another systems for HCV replication, the first with HCV GT1 H77 in immortalized human hepatocytes (IHH)  and the other system of HCV GT2 JFH1 in human hepatoma cell line (Huh7) . Kanda et al. suggested that IHH support HCV genome replication and virus assembly by examined HCV core protein-mediated IHH for growth of HCV . Their study described the generation of cell culture-grown HCV from genotype 1a and discuss the concept of HCV replication and assembly of genotype 1a in IHH and speculated that cellular defense mechanisms against HCV infection are attenuated or compromised in IHH . It was reported the HCV production from a HCV-ribozyme construct of genotype 1a (clone H77) in Huh-7 cells with no determination for the virus infectivity . Furthermore, subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. The JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a Huh7, providing a powerful tool for studying the viral life cycle and developing antiviral strategies .
Apoptosis has been demonstrated as an important mechanism for viral clearance. In HCV-infected liver, viral persistence is observed despite enhanced hepatocyte apoptosis ; however, it is not clear whether this apoptotic effect is due to a direct cytopathic effect of the virus, immunological reactions or a contribution of the molecular mechanisms causing liver damage during HCV infection [22, 36]. For understanding the impact of HCV infection on the apoptotic machinery during disease progression, we studied the expression patterns of Bcl-2, Bcl-xL, Bak, Fas, FasL in HCV- genotype-4 infected HepG2 cell line as well as in human tissue samples obtained from patients with HCC and CH as a result of chronic HCV infection. We also analyzed the expression levels of caspases 3, 8 and 9 in tissue culture medium and in HCV infected cells by a colorimetric assay, and viral replication by both RT-PCR and Real-Time PCR for up to 135 days post-infection.
The results of the present study showed that HCV infection disrupted the process of apoptosis through down regulation of Fas and up-regulation of FasL genes expression. However, in tissue samples a higher expression of Fas and FasL genes were detected in CH compared to HCC patients, which explains the presence of severe inflammation in chronic HCV infection and its oncogenic potential. In this regard, previous studies demonstrated that enhanced FasL gene expression induces T-cell apoptosis , which favors viral persistence and indirectly increases the probability of progression to HCC . In addition, the FasL gene exerts proinflammatory activities via IL-1β secretion that is responsible for neutrophils infiltration .
In contrast, other studies [38–40] demonstrated that the ratio of Fas/FasL was significantly lower in HCC than in CH tissue samples or non tumor hepatic tissues. This was attributed to the fact that tumor cells possess more than one safe guard against Fas mediated apoptosis. First, the reduced expression or loss of certain molecules that are involved in the Fas mediated apoptosis pathway such as FADD (Fas-associated protein with death domain), FLICE (FADD like interleukin-1β-coverting enzyme, caspase-8) or FAF (Fas associated factor), or the induction of molecules that would inhibit Fas mediated apoptosis such as FAP (Fas associated phosphatase) . Second, the expression of sFas RNA and FAP-1 may neutralize Fas mediated apoptosis  and third, Fas mutation could be expected. Many investigators suggested that one of the possible mechanisms by which HCV core protein inhibits apoptosis is through a direct binding to downstream domain of FADD and cFLIP leads to viral persistence and cells proliferation . Consequently, it is conceivably possible that the observed decreased apoptosis relative to cell proliferation of infected hepatocytes could be part of the signaling mechanisms in the pathogenesis of HCC .
It has also been reported that the extrinsic (Fas-FasL) pathway plays an important role in liver cell injury directly via HCV infection or indirectly through immune attack of HCV- infected cells with subsequent recruitment and activation of stellate cells and macrophages, resulting in fibrosis and cirrhosis . Also, I was found that during HCV infection, HCV-specific T cells migrate to the liver and recognize viral antigens on the hepatocytes . These immunologically active cells, which are probably induced due to inflammation rather than viral infection, become activated and express FasL that transduces the apoptotic death signal to Fas bearing hepatocytes, resulting in their destruction . Therefore, neither Fas expression nor the degree of liver injury correlates with the intra-hepatic viral load [15, 44]. In such case, the TNF or the IFN-δ might be responsible for the up regulation of Fas expression in infected hepatocytes and FasL in lymphocytes .
Alternatively, the hepatocytes which are likely type II cells in which direct activation of caspase 8 (extrinsic pathway mechanism) is not sufficient to induce apoptosis amplification by a mitochondrial pathway (intrinsic mechanism) are highly required. Accordingly caspase 8 activation causes the proapoptotic cleavage of Bid, which induces cytochrome c release from the mitochondria, which subsequently binds to Apaf-1 and procaspase 9 forming apoptosome complex . In the present study, we assessed the activation of caspases 8, and 9, which represent both death receptor-mediated and the mitochondrial apoptosis pathway and caspase 3 which is an executioner caspase. Our data showed a positive correlation between Fas mediated apoptosis and caspases activation. In HCV infected cells, we observed a loss of caspases after 4 weeks post HCV infection. Some studies provided evidence that monitoring of caspases activation might be helpful as a diagnostic tool to detect the degree of HCV mediated inflammatory liver damage and to evaluate efficacy of HCV therapy [36, 37]. However, it was reported that the extent of caspase activation correlates with the grade of the disease but not with surrogate markers, such as serum transaminases or viral load . This observation indicates that caspase activation is not directly related to HCV mediated damage and suggests the involvement of HCV mediated immune response with Fas triggered hepatocyte apoptosis giving rise to several amplification loops . Similar findings were reported by others, who indicated in their study that the core protein could stimulate caspase-independent apoptosis at later stages of the disease giving relevance to the release of HCV particles from the host cells and to viral spread .
It has been shown that some HCCs are resistant to Fas-mediated apoptosis directly through the expression of HCV proteins or indirectly through up-regulation of Bcl-2 family members . Our data showed that both Bcl-2 and Bcl-xL RNA expression were significantly higher in HCC than in CH and NDT indicating late involvement of those genes in the cascade of HCV-associated hepatocarcinogenesis. We were also able to detect Bcl-2 gene expression in HepG2 cells starting from day 1 post-infection until the end of the experiment, whereas the expression of Bcl-xL was not visible until day 28 when it started to be expressed and its expression was closely associated with the presence of HCV in tumor cells (Table 3) suggesting that Bcl-2 is tumor related whereas Bcl-xL is a viral related. In this context, Bcl-2 was linked to inhibition of apoptosis via interfering with either the recruitment of procaspase 8 to Fas receptors  or by preventing the release of cytochrome C . It has also been shown that the HCV core protein inhibits apoptosis at the mitochondrial level through augmentation of Bcl-xL expression with consequent inhibition of caspase 3 activation . The HCV core protein could induce apoptosis in the Fas death way although this is achieved through the activation of Bax and Bak, both are important mediators of p53 mitochondrial function [5, 36]. Our results showed an increase in Bak-RNA expression at an early stage of HCV infection of HepG2 cells, which is also observed in tissue samples obtained from both CH and HCC patients compared to NDT samples. Our results provided enough evidence that the Bak gene can induce apoptosis in HCC cells even in the presence of high levels of the anti-apoptotic Bcl-2 gene family members, which is in agreement with the findings of others .
The results of gene expression in tissue samples show a significant correlation between Fas expression in HCC cases and the presence of cirrhosis or poorly differentiated tumors. We observed that FasL expression was significantly associated in CH patients with the grade of inflammation and the stage of fibrosis as well as with the presence of severe necro-inflammatory changes. Based on these results we conclude that aberrant expression of Fas and FasL in HCV-infected patients could be considered a marker for increased disease severity with a higher possibility of progression into cirrhosis and/or HCC. Similar results were also reported by others , who indicated that FasL may contribute to malignant transformation of hepatocyte as it was significantly expressed in the peri-cancerous lobules and cirrhotic nodules . Similarly, Bcl-2 expression was significantly associated with poorly-differentiated tumors as well as with the presence of cirrhosis in CH patients. Similar findings were reported previously by some of us . In this study, Bak expression was significantly associated with absence of cirrhosis and well-differentiated tumors, thus Bak gene could be considered a good prognostic marker.
The impact of HCV infection on modulating apoptotic machinery pathway(s) differs during the course of infection, as the disease progresses apoptosis is inhibited leading to cell immortalization and HCC development. HCV infection could exert a direct effect on hepatocytes by inducing Fas-FasL pathway with subsequent inactivation of caspases or indirectly by immune attack on hepatocytes resulting in HCV mediated liver injury, viral persistence and cirrhosis in CH patients with an increasing possibility of hepatocarcinogenesis especially with increasing proliferation rate and acquisition of genetic damage.
Alternatively, HCV infection could induce apoptosis at the early phase of infection followed by modulation of apoptosis by disturbing Fas/FasL. This in turn would cause an inactivation of caspases 3, 8, and 9, up-regulation of Bcl-2 family members, impairment in Bak gene expression and increasing the expression of FasL leading to inhibition of apoptosis in HCV infected patients. This signaling cascade favors cell survival with persistence of HCV infection and enhances the possibility of HCC development. A combination of these effects initiates a circle of hepatocyte damage and repair, which is the hallmark of HCV infection that might progress to HCC. Our study could provide an insight for understanding apoptosis and developing molecular target therapies that could inhibit viral persistence and HCC development. Further studies are still required to clarify the interaction between other HCV proteins in the apoptotic machinery system and the possible involvement of other apoptotic pathways in HCV associated HCC development.