Chronic infection with HCV is a major health problem affecting more than 170 million individuals in the world. Although the initial infection is largely asymptomatic, prolonged infection carries a high risk of chronic hepatitis, cirrhosis, and HCC. The mechanism of HCV related hepatocarcinogenesis remains to be clarified [5
]. PKR is an integral part of human innate immune response mechanism, which is the cell's first line of defence against viral infection [24
]. The molecular mechanisms that regulate PKR function in normally dividing cells are largely unknown. PKR is implicated in controlling HCV replication and mediating interferon- induced antiviral state against HCV replication [20
]. Some viruses including hepatitis viruses can evolve various devices to down-regulate PKR and overcome the host defence mechanism against virus replication [29
Our results showed that hepatitis C viraemia was much higher in HCC cases but no significance was detected. Others reported a significant increase in viral load after the development of HCC denoting the involvement of HCV in the process of hepatocarcinogenesis [2
]. On the contrary, Hung observed that HCV replication persists in HCC cells at a lower level than non-malignant cells as they reported significantly lower viraemia in HCC patients [23
]. We correlated fibrosis and HAI in all patients with HCV-4 and we got significant results in which patients with chronic HCV-4 related HCC showed higher stage of fibrosis and higher HAI when compared to chronic hepatitis patients without HCC; this may be explained by advanced viral C infection with severe tissue insult. A positive correlation was found between AST but not ALT and the degree of inflammation in chronic hepatitis patients. These results disagree with others who found that ALT was significantly correlated with HAI while, AST was correlated with stage of fibrosis [30
]. We obtained a significant negative correlation between viral load and HAI in both HCV-4 chronic hepatitis cases and HCV-4 related HCC cases in which the increase in score of HAI is associated with decrease in the viral load. This correlation was not previously discussed in literature. Consistent with previous reports, no correlation was found between serum HCV-RNA levels and stage of fibrosis in HCV-4 patients [31
]. Thus we can deduce that serum HCV-RNA level does not determine the degree of hepatic injury precisely and liver biopsy is mandatory to evaluate the extent of liver damage. In contrast a correlation was suggested by other authors between serum HCV-RNA levels and the fibrosis stage in HCV patients without cirrhosis [32
HCV participates in the oncogenic mechanisms leading to the development of HCC via inhibiting PKR functions on cell growth control and host apoptotic program [11
]. Although, PKR is generally thought to be regulated solely by dsRNA, an intermediate in replication of many viruses including HCV, recent studies have proven the existence of other PKR activation pathways which involve cellular activators that are independent of dsRNA, such as growth factors, lipopolysaccharide, Ca2+
and PKR-activating protein [15
]. Thus HCV core protein may induce a shift of PKR activity from its classical pathway by targeting other substrates [14
]. Chen and his colleagues found that PKR level was much reduced in HCC than that in non-tumour tissues [34
]. This is not surprising, as PKR is considered a tumour suppressor in some cancer cells. Those cells with low PKR are resistant to apoptosis, thus promoting the growth of tumour. However, in the present study, there was a significantly higher PKR gene expression in HCC tissues and non-tumorous tissues adjacent to malignancy than in tissues of chronic liver disease without a nearby malignancy. Comparing gene expression in chronic liver disease without HCC to those with adjacent malignancy may indicate that higher PKR expression is acquired in premalignant state and during the process of cancer development or even it may be a step in HCV induced hepatocarcinogenesis. This is the first time that PKR-RNA is quantified in HCV-4 related tumour and non-tumour tissues, giving an explanation for the difference from those results obtained by Chen as their study of PKR was immunohistochemical expression, thus lacking a quantitative measurement. Moreover, in our study we have reached a sensitive and specific cut-off for PKR level which can be used in segregation of HCV patients into low risk and high risk groups for tumour development. Some authors support our findings as they suggested that PKR was not acting as a classical tumour suppressor protein but rather as a potential growth stimulus [35
]. Also other studies showed that expression of PKR was lower in liver of HCC with HBV than in HCV infection [26
]. These findings may reflect the difference in viral impact on PKR expression and suggest that PKR might have a tumour promoting action in some cancer cells.
The PKR activation pathway may be inhibited as a survival benefit for the cancer cell. Increased PKR expression has been documented in cancer cells, but its activity was in-fact decreased; therefore we can add that increased PKR expression is a finding but this PKR is non-functioning. This may be attributed to mutation in PKR gene induced by the virus or epigenetics changes whether caused by methylation or phosphorylation. It should however be noted that increased PKR expression was recently described in breast cancer-derived cell lines [25
]. Defective programmed cell death mechanisms contribute significantly to the origin and progression of cancer. These tumours show high levels of PKR, suggesting that the ability of PKR to inhibit cell proliferation is impaired in some way [27
]. Thus loss of PKR function may constitute one step in the pathway to tumorigenesis. Our observations imply that liver cell proliferation observed during liver carcinogenesis is associated with the selection of viral genomes whose core products activate PKR. The specific association of HCV core protein expressed in HCC tissues with PKR leads to the increased activity of the kinase. PKR activation is not sufficient to cause cell death because of the strong anti-apoptotic signalling in HCV infected cells [16
]. Our work showed no significant difference in expression of PKR among patients with well differentiated tumours and those with poor or moderate differentiation, this is in concordance with other study [34
]. While a previous report showed that the expression of PKR was increased significantly in well-differentiated HCC related HBV infection, compared with that in poorly differentiated HCC [36
]. These different results from ours may be due to the method used for PKR measurement. A TaqMan RT-PCR used in the present study is a quantitative method while the immunohistochemical staining method used in the previous report is not. Also the impact of HCV on PKR may be different from that of HBV.
Further studies showed that normal tissues tend to have lower PKR levels than their neoplastic counterparts [18
]. This was in parallel with our data as we found a significant increase in PKR in HCV-4 related HCC cases when compared with normal subjects. There was no correlation between PKR gene expression in liver tissue and age, ALT, AST, bilirubin and AFP. So, PKR was found to be an independent prognostic factor indicating the important biological significance of this gene in the HCC disease process. In a study on peripheral adenocarcinoma of the lung, PKR showed a prognostic impact in which patients with high PKR expression had significantly shorter survival periods than those with low PKR expression [37
]. This may be of great help if estimated in HCC patients. We also found that higher PKR gene expression was associated with higher scores of fibrosis and HAI in both HCV-4 and HCV-4 related HCC cases. No other thesis evaluated this relationship.
We detected a significant negative correlation between PKR gene expression and serum viral load in HCV-4 and HCV-4 related HCC cases, in which HCV copy number was significantly decreased by increasing PKR gene expression, these data are compatible with those obtained by others [35
]. It appears that HCV protein expression is directly dependent on PKR expression. PKR is antiviral towards HCV thus the over-expression of PKR significantly suppressed HCV levels. From these correlative data, we suggest that PKR is potentially functional in decreasing viral replication. Other authors demonstrated that the expressed endogenous PKR plays a role in controlling the initiation of HCV infection but does not affect HCV replication in infected cells in which persistent viral replication is fully established [5
]. Once activated by these low concentrations of dsRNA, PKR cannot be inhibited by subsequent addition of high concentrations of dsRNA [16
]. Our findings therefore suggest that the activation of PKR may reduce HCV protein expression and the elimination of infected cells, thus favouring viral persistence.