Infection, Genetics and Evolution 12 (2012) 236–239

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Infection, Genetics and Evolution

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Review

Dual behavior of HCV Core gene in regulation of apoptosis is importantin progression of HCC

Shah Jahan a,⇑, Usman A. Ashfaq b,⇑, Saba Khaliq a, Baila Samreen a, Nadeem Afzal a

a Department of Immunology, University of Health Sciences Lahore, Pakistanb Department of Bioinformatics and Biotechnology, Government College University, Faisalabad, Pakistan

a r t i c l e i n f o

Article history:Received 8 November 2011Accepted 8 January 2012Available online 15 January 2012

Keywords:HCVHCV Core geneApoptosisCell proliferationHCC

1567-1348/$ - see front matter � 2012 Elsevier B.V. Adoi:10.1016/j.meegid.2012.01.006

⇑ Corresponding authors. Address: Department ofHealth Sciences Lahore, Kheyban-e-Jamiah, Punjab 5+92 03005081072 (S. Jahan), tel.: +92 03314728790 (

E-mail addresses: captainmalik@hotmail.com (S. Jah(U.A. Ashfaq), sabahat711@yahoo.com (S. Khaliq).

a b s t r a c t

Hepatitis C virus (HCV) causes acute and chronic hepatitis which can lead to HCC (Hepatocelluar carci-noma) via oxidative stress, steatosis, insulin resistance, fibrosis and liver cirrhosis. Apoptosis is essentialfor the control and eradication of viral infections. In acute HCV infection, enhanced hepatocyte apoptosisis significant for elimination of viral pathogen. In case of chronic HCV, down regulation of apoptosis andenhanced cell proliferation not only causes HCV infection persistency in the majority of patients. How-ever, the impact of apoptosis in chronic HCV infection is not well understood. It may be harmful by trig-gering liver fibrosis, or essential in interferon (IFN) induced HCV elimination. Regulation of apoptosis inhepatocytes by HCV Core is so important in progression of HCC. This review focuses on the dual characterof HCV Core on regulation of apoptosis and progression of HCC.

� 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2362. Apoptotic pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2373. Caspases and their biological functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2374. Chronic viral hepatitis and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2375. HCV gene regulation and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2386. HCV Core dual role in apoptosis leads to HCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2387. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

1. Introduction

Hepatitis C virus (HCV) causes acute and chronic hepatitis(Hoofnagle, 2002) which can lead to HCC (Hepatocelluar carci-noma) in a significant number of patients via oxidative stress, ste-atosis, insulin resistance, fibrosis and liver cirrhosis (Alter, 1997).HCV is a major health problem with almost 350 million individualschronically HCV infected worldwide (Bartenschlager, 2006)whereas 10% of the Pakistani population is chronically infected

ll rights reserved.

Immunology, University of6400, Lahore, Pakistan. Tel.:U.A. Ashfaq).an), usmancemb@gmail.com

with this viral pathogen (Raja, 2008). Approximately 40–60% ofHCV infected individuals’ leads to chronic liver disease (Khanet al., 2008). Prevalence of HCV associated HCC is higher in Paki-stan as compare to the rest of world (Idrees et al., 2009; Razaet al., 2007).

HCV has a positive-stranded RNA genome of approximately9.5 kb that encodes a large precursor polyprotein of about 3000amino acids. Among the HCV processed proteins, the core proteinof 191 amino acids is essential for nucleo-capsid formation of viralparticle (Choo et al., 1991). Besides structural roles, HCV Core pro-tein has also been reported to modulate gene transcription, cellproliferation, cell death and interference with metabolism leadingto oxidative stress, liver steatosis and eventually HCC (Lin et al.,1994; Moriya et al., 2001; Reed and Rice, 2000). Moreover, HCVCore protein interferes with many biological functions in the host

S. Jahan et al. / Infection, Genetics and Evolution 12 (2012) 236–239 237

cell, such as cellular growth, malignant transformation and apop-tosis which could be involved in HCV-related liver diseases. Inter-estingly, constitutive expression of HCV Core protein induced HCCin transgenic mice and the expression level of HCV Core protein inthe liver of these mice was similar to chronic HCV patients (Moriyaet al., 1998). Regulation of apoptosis is important predictor in dis-ease progression of hepatitis and HCC progression. HCV causes li-ver damage through the induction of apoptotic pathways.Primarily, host immune responses induce apoptosis and to someextent HCV viral proteins are involved in regulation of apoptosisin chronic hepatitis. Enhanced apoptosis of hepatocytes has beendescribed during chronic HCV infection and it correlated with thedegree of liver damage (Bantel et al., 2001b).

Overall data regarding the roles of different HCV proteins is con-troversial and different viral protein’s pro- or anti-apoptotic effectsdepend on the experimental system used. Further, HCV is geneti-cally highly variable and exists as quasispecies in the patient. Dif-ferent pro- and anti-apoptotic effects of HCV Core protein from anindividual patient (Pavio and Lai, 2003) suggested special proper-ties of different quasispecies proteins. The differences in these pro-teins may explain in part the diverse effects of viral proteins onapoptosis. Studies about the contribution of genotypes or quasi-species to apoptosis are largely missing. Our recent studies demon-strated that core of different genotype of HCV differentiallyregulate cellular genes involved in cell survival, angiogenesis andit ultimately leads to HCC. Therefore, this review is an attempt todiscuss the role of HCV Core gene in regulation of apoptosis whichleads to HCC.

2. Apoptotic pathway

Activation of apoptosis takes place externally via interaction ofcell-surface receptors or intracellular through activation of mito-chondrial proteins. These pathways converge into a common apop-totic pathway at the point at which caspase enzyme cascade isactivated. At this juncture, intracellular components are degradedand DNA-degrading enzymes are activated. Ultimately, cells arebroken down into small apoptotic bodies that are disposed of byphagocytes (Bantel and Schulze-Osthoff, 2003). The intrinsic path-way is triggered by extra- or intra-cellular signals, including DNAdamage, oxidative stress, growth factor deprivation and viral pro-teins (Guicciardi and Gores, 2010). These stress signals activatepro-apoptotic members of Bcl-2 family such as Bax, Bak and Bid,thereby increases permeability of outer mitochondrial membrane.As a result of stress signal, cytochrome c released from mitochon-dria, binds to apoptotic protease activating factor-1 (Apaf-1) toform a complex in the presence of ATP, which is known as apopto-some. Procaspase-9 is recruited to apoptosome and is convertedinto active caspase-9 through oligomerisation, resulting in activa-tion of caspase-3, -6 and -7, and subsequently cell death (Porterand Jänicke, 1999). The extrinsic pathway is initiated at the plasmamembrane where death ligand interactions are responsible for therecruitment of adaptor proteins such as Fas-associated death do-main protein (FADD) and caspase-8 and -10 that result in the for-mation of death-inducing signal complex (DISC). Activation ofcaspase-8 and -10 in turn activate caspase-3, causing cell death(Porter and Jänicke, 1999).

3. Caspases and their biological functions

In mammals, the function and regulation of Caspase in celldeath is complex, nonetheless, caspase-2, 8, 9 and-10 are thoughtto be the initiator caspase, whereas caspase-3, and to a lesser ex-tent caspase-6 and -7, serve as effecter caspase (Muzio et al.,1996; Varfolomeev et al., 1998). Caspase-8 is the key initiator of

death receptor-mediated apoptosis (Fuentes-Prior and Salvesen,2004). In response to the activation of death receptors of TNF fam-ily, caspase-8 is recruited to the DISC via binding to the adaptorprotein FADD (Muzio et al., 1996). This recruitment results in cas-pase-8 activation and cell death (Varfolomeev et al., 1998). Cas-pase-3 is activated following cleavage by caspase-8 which occursexternally via ligation of cell-surface receptors or intracellularthrough the activation of mitochondrial proteins (Porter andJänicke, 1999) and it is the main downstream effecter caspase thatcleaves the majority of cellular substrates in apoptotic cells.Derangements in apoptosis of liver cells are mechanisticallyimportant in the pathogenesis of end stage liver disease. Vulnera-ble hepatocytes can undergo apoptosis via an extrinsic, deathreceptor-mediated pathway, or alternatively intracellular stresscan activate intrinsic pathway of apoptosis. If this killing processworks adequately, it is beneficial for the organism and may leadto elimination of virus-infected cells, however deregulation of thissystem might result in liver damage. Persistent apoptosis is a fea-ture of chronic liver diseases, and massive apoptosis is a feature ofacute liver diseases (Malhi and Gores, 2008). Caspase-3, -8, -9, -10,(proteases involved in apoptosis signaling cascade) Caspase-3, -9,p53 are the main downstream effecters activated by caspase 8,10 essential factors of the extrinsic cell death pathway (Bantelet al., 2001a). Cyto and p53 interaction is important in apoptosis(Bantel et al., 2001b).

Different HCV genotypes regulate apoptotic pathway differen-tially. Very few studies about HCV genotypes have been under-taken and further studies are needed to expose the ambiguity ofdiverse responses of different genotypes. In mammals, regulationof caspase in cell death is complex, however, caspase-2, -8, -9and -10 are initiator caspase whereas caspase 3, 6 and 7, serve aseffecter caspases (Muzio et al., 1996; Varfolomeev et al., 1998).Caspase-8 as the key initiator of death receptor-mediated apopto-sis (Fuentes-Prior and Salvesen, 2004). Cyto C is associated withcaspase-9 (Otsuka et al., 2000) in intrinsic apoptosis pathwayand P53 tumor suppressor gene is an important marker in apopto-sis (Chou et al., 2005; Otsuka et al., 2000). Expression of caspasefamily is higher in HCV infection, and enhanced hepatocyte apop-tosis occurs through the intrinsic apoptosis pathway via mitochon-dria (Calabrese et al., 2000; Fischer et al., 2007). Studies of caspasesare better alternative for investigation of apoptosis in HCV infec-tion as caspase activation occurs earlier than DNA cleavage (Muzioet al., 1996; Varfolomeev et al., 1998).

4. Chronic viral hepatitis and apoptosis

HCV influences apoptosis mediated by both external and inter-nal pathways. Apoptosis of hepatocytes occur in chronic HCV pa-tients (Bantel and Schulze-Osthoff, 2003). Bantel and colleaguesidentified a caspase cleavage epitope of cytokeratin-18 (CK-18);major intermediate filament in hepatocytes of HCV-infected pa-tients. Levels of CK-18 cleavage products in serum and liver biopsyspecimens were increased in HCV infected patients compared withuninfected controls, indicating that caspase activation occurs inHCV-infected patients. Hepatocytes are subject to high rate of cellinjury and destruction due to continuous intrahepatic inflamma-tion by chronic HCV infection. Apoptosis play a role in HCV-medi-ated liver damage through the induction of apoptotic pathways,primarily as a result of host immune responses. Enhanced apopto-sis of hepatocytes has been described during chronic HCV infectionand correlates with the degree of liver damage (Bantel et al.,2001a). An increase in caspase activation has also been demon-strated in HCV-infected liver (Bantel et al., 2001b). Previous studieshave shown that caspase-3-knockout mice were largely resistant

238 S. Jahan et al. / Infection, Genetics and Evolution 12 (2012) 236–239

to liver damage and suppression of hepatic injury by using caspaseinhibitors (Rouquet et al., 1996).

5. HCV gene regulation and apoptosis

Viral proteins interfere with cellular apoptotic signaling path-way and block key cellular elements of host cell. Until recently,due to lack of HCV tissue culture system, studies about the impactof HCV infection on hepatocyte apoptosis could not be done. Over-all, the data regarding the role of different HCV proteins are contro-versial and ascribe to a given viral protein pro- and anti-apoptoticeffects, depending on the experimental system used. Further; HCVis genetically highly variable and exists as quasispecies in a givenpatient. Different pro- and anti-apoptotic effects of the HCV Coreprotein from an individual patient have been described (Pavioand Lai, 2003) suggesting special properties of different quasispe-cies proteins. These protein differences may explain in part the dif-ferent effects of viral proteins on apoptosis. Studies of thecontribution of genotypes or quasispecies to the effects on apopto-sis are largely missing. Further, experiments designed to study theimpact of HCV infection on hepatocyte apoptosis must also con-sider the interactions between the different HCV proteins.

6. HCV Core dual role in apoptosis leads to HCC

HCV Core has pro- and anti-apoptotic effects in death ligand-mediated hepatocyte. Core-dependent inhibition of TNF-a-(Rayet al., 1998) and CD95 ligand-induced apoptosis has been de-scribed in hepatoma cell line (Ruggieri et al., 1997). HCV Core pro-tein inhibits CD95 ligand-mediated apoptosis by prevention ofcytochrome C released from mitochondria and consecutive activa-tion of caspase-9, -3 and -7 (Machida et al., 2001). Further, directbinding to the downstream death domain of FADD and c-FLIPshowed anti-apoptotic effects (Saito et al., 2006). Recently, inhibi-tion of TGF-b-pathway by direct interaction of Core protein withthe DNA-binding domain of Smad3 has been demonstrated (Pavioand Lai, 2003). Several studies confirmed binding of HCV Core pro-tein to p53, either inhibiting or activating p53 following with anti-or pro-apoptotic effects (Fig. 1) (Kao et al., 2004; Otsuka et al.,2000) and HCV counteracts p53 growth suppression through acti-vation of MAPK and PI3K/Akt signaling cascades which enhancescell growth and proliferation. In some studies apoptosis was inhib-ited in hepatoma through Core-dependent phosphorylation and

Fig. 1. Dual role of HCV Core gene in regulation of apoptosis and progression tohepatocellular carcinoma.

activation of STAT3 that induced anti-apoptotic bcl-XL (Otsukaet al., 2002).

There are increasing evidences suggesting that liver cell damagein chronic HCV infection is mediated by apoptosis (Bantel et al.,2001b). However, the comparative contribution of apoptosis aswell as functional role of caspases and their association in liverdamage is largely unknown. For virtually all HCV proteins, pro-and anti-apoptotic effects have been described, especially for theCore and E2 protein (Bantel and Schulze-Osthoff, 2003). To date,it is not known which HCV protein affects apoptosis in vivo andwhether the infectious virions act as pro- or anti-apoptotic agent.With the availability of HCV tissue culture system, effect of HCVinfection and different HCV proteins on apoptosis and of corre-sponding signaling cascades. HCV has a high mutation rate and ex-ists as genetically heterogeneous quasispecies in individualpatients due to lack of proofreading function of RNA-dependentRNA-polymerase (NS5B) (Bartenschlager, 2006; Dustin and Rice,2007; Pavio and Lai, 2003). Various genotypes differ geneticallyfrom one another by at least 30%, and different subtypes within agenotype by more than 20%. This genetic heterogeneity makes itdifficult to compare apoptotic pathways obtained from differentHCV genotypes. In general, apoptosis is central to viral clearance.In HCV-infected liver, however, despite enhanced hepatocyteapoptosis, viral persistence is observed. Consistent with the‘‘dual-signal’’ hypothesis postulating coupling of proliferationpathways with those of cell death, These studies support hypothe-sis that HCV Core protein triggers two mechanisms: and an anti-apoptotic PI(3)K/Akt pathway and increases in cell proliferationfact, Bataller et al., recently demonstrated that expression of HCVCore protein in hepatic stellate cells increased cell proliferationin Ras/ERK and PI(3)K/Akt-dependent manner. Recently, it hasbeen reported that PI(3)K/Akt pathway was active in human livertumors and in cultured hepatoma cells, and this activation causeddephosphorylation of C/EBPa on Ser 193, leading to proliferation ofcells. In agreement with these results, a number of previous inves-tigations indicated that inhibition of Akt in many hepatoma celllines leads to growth arrest. Although the mechanisms explainingactivation of Akt in hepatoma cells are still unclear, we demon-strated that expression of HCV Core 3a protein resulted in muchhigher Akt activity and increased cell proliferation which mightlead to HCC (Jahan et al., 2011).

7. Conclusion

The role of apoptosis in HCV infection is not well defined. Kinet-ics and extent of hepatocytes apoptosis as well as the pro- andanti-apoptotic mechanisms involved remain unclear. DifferentHCV genotypes and quasispecies may induce different effects,and most studies employed none physiologically over expressedviral proteins. In HCV infected patients, by comparison, only verylow quantities of HCV proteins were detectable, and balancedexpression of these proteins might be essential. Therefore, the re-sults obtained to date have to be interpreted with great cautious.The available HCV tissue culture systems as well as future in vivomodel systems may give answers to these questions, and may helpto clarify the interference of HCV with apoptotic pathways and itsrole in the pathogenesis of HCV infection and clearance.

Several molecular interactions between Core protein and cellu-lar genes of the apoptotic machinery have been found (Bantel andSchulze-Osthoff, 2003). Other studies showed Core induced apop-tosis through mitochondrial cytochrome C release and indirectactivation of baxTRAIL-induced apoptosis in hepatoma cells seemsenhanced by Core-dependent bid-cleavage (Chou et al., 2005). Ta-ken together, it remains unclear whether HCV Core protein inhibitsor induces death receptor-mediated apoptosis of hepatocytes. This

S. Jahan et al. / Infection, Genetics and Evolution 12 (2012) 236–239 239

molecular mechanism of anti-apoptotic model by HCV Core pro-tein might play a critical part of hepatocyte growth regulationand development of human HCC mediated by chronic HCVinfection.

Recently, we showed an increased cell proliferation in HCV 3aCore transfected liver cells to understand the molecular mecha-nisms of HCV Core protein, and identified that HCV Core proteinactivates Cox-2 and VEGF (Jahan et al., 2011) Akt acts as an impor-tant signal mediator, which regulates cell survival and proliferation(Nishizono et al., 1993; Niwa et al., 1991). Activation of pAkt causescell growth and proliferation. Despite several studies, mechanismsinvolved in HCV-associated pathogenesis are not completelyunderstood; it may vary as a function of viral genotype (Nishizonoet al., 1993). The mechanisms of cell proliferation and transforma-tion are intrinsically linked to the process of apoptosis. The Ras/phosphatidylinositol-3-kinase (PI3 K)/Akt pathway is critical inanti-apoptotic signaling (Downward, 2004). In this pathway, ser-ine-threonine kinase Akt directly phosphorylates many substrates,including Bad, caspase-9, CREB, Forkhead and GSK-3, and is criticalfor cell survival in response to growth factor stimulation. In our re-cent study, we examined the activation of the PI(3)K/Akt pathwayin cells expressing HCV Core 3a protein, and identified that HCVCore protein activated Akt through Ras/PI(3)K pathway. Our resultsindicated that HCV Core protein promoted activated anti-apoptoticsignaling pathway including Akt and IKK, thus linking anti-apopto-tic signaling with transcription machinery. This molecular mecha-nism of anti-apoptotic model by HCV Core protein might play acritical role in hepatocyte growth regulation and development ofhuman HCC mediated by chronic HCV infection. HCV Core proteineffects on Caspase signaling, we investigated the expression levelsof molecules downstream caspase signaling including Caspaces (3,8, 9, and 10), Cytochrom C and P53 which are important in apop-totic pathway. We have shown increased cell growth and reducedexpression of host genes involved in apoptosis was significantlydecreased in liver cells expressing HCV Core 3a protein as com-pared to HCV Core 1a (Jahan et al., 2011).

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