2011 HCV

Research Article

Hepatitis C virus and alcohol: Same mitotic targets butdifferent signaling pathways

Anna Alisi1,2, Monica Ghidinelli1, Alessandro Zerbini3, Gabriele Missale3, Clara Balsano1,4,⇑

1Laboratory of Molecular Virology and Oncology, Fondazione A. Cesalpino, University of Rome, Rome, V. le del Policlinico 155, 00161 Rome, Italy;2Units of Metabolic and Autoimmune Liver Diseases, ‘‘Bambino Gesù’’ Children’s Hospital and Research Institute, P. le S. Onofrio 4, 00165 Rome,

Italy; 3Laboratory of Viral Immunopathology, Azienda Ospedaliero, Universitaria di Parma, Via Gramsci 14, 43100 Parma, Italy; 4

Department of Internal Medicine (M.I.S.P), University of L’Aquila, University of L’Aquila, Piazzale Salvatore Tommasi 1, 67100 L’Aquila, Italy

Background & Aims: Chromosomal aberrations are frequently Introduction
observed in hepatitis C virus (HCV)- and alcohol-related hepato-cellular carcinomas (HCCs). The mechanisms by which chromo-somal aberrations occur during hepatocarcinogenesis are stillunknown. However, these aberrations are considered to be theresult of deregulation of some mitotic proteins, including thealteration of Cyclin B1 and Aurora kinase A expression, andthe phosphorylation of gamma-tubulin. Our study aims at inves-tigating changes in expression of the above mentioned proteinsand related intracellular pathways, in in vitro and in vivo modelsof both HCV- and alcohol- dependent HCCs.Methods: In this study, the molecular defects and the mechanismsinvolved in deregulation of the mitotic machinery were analyzed inhuman hepatoma cells, expressing HCV proteins treated or not withethanol, and in liver tissues from control subjects (n = 10) and patientswith HCV- (n = 10) or alcohol-related (n = 10) HCCs.Results: Expression of Cyclin B1, Aurora kinase A, and tyrosine-phosphorylated gamma-tubulin was analyzed in models repro-ducing HCV infection and ethanol treatment in HCC cells. Inter-estingly, HCV and alcohol increased the expression of Cyclin B,Aurora kinase A, and tyrosine-phosphorylated gamma-tubulinalso in tissues from patients with HCV- or alcohol-related HCCs.In vitro models suggest that HCV requires the expression of PKR(RNA-activated protein kinase), as well as JNK (c-Jun N-terminalkinase) and p38MAPK (p38 mitogen-activated protein kinase)proteins; while, ethanol bypasses all these pathways.Conclusions: Our results support the idea that HCV and alcoholmay promote oncogenesis by acting through the same mitoticproteins, but via different signaling pathways.� 2011 Published by Elsevier B.V. on behalf of the EuropeanAssociation for the Study of the Liver.
Journal of Hepatology 20

Keywords: Aurora kinase A; Cyclin B1; Ethanol; HCC; HCV.Received 4 January 2010; received in revised form 28 July 2010; accepted 15 August2010; available online 22 December 2010⇑ Corresponding author. Address: Fondazione A. Cesalpino, c/o I Clinica Medica,Policlinico Umberto I, Viale del Policlinico n.155, 00161 Rome, Italy. Tel.: +39 649975144; fax: +39 6 49975145.E-mail address: [email protected] (C. Balsano).Abbreviations: HCCs,hepatocellular carcinomas; HBV, hepatitis B virus; HCV, hepatitis C virus; NASH,non-alcoholic steatohepatitis; p38MAPK, p38 mitogen-activated protein kinase;PKR, RNA-activated protein kinase; JNK, c-Jun N-terminal kinase; NF-kB, nuclearfactor-kB; FAK, focal adhesion kinase; PI3K, phosphatydil-inositol-3 kinase; siRNA,small interference RNA.

Each year, 550,000 new patients are diagnosed with hepatocellu-lar carcinoma (HCC) worldwide, thus, at present, liver cancer isconsidered the fifth most frequent neoplasm and, because of itspoor prognosis, the third leading cause of cancer death [1]. Oneof the most important clinical challenges associated with chronichepatitis rises from the evidence that a considerable number ofpatients develop end-stage liver disease and HCC. HCC develop-ment and progression may depend on several etiologic factors,including hepatitis B virus (HBV), hepatitis C virus (HCV), alcoholabuse, and non-alcoholic steatohepatitis (NASH) [2,3]. HCV andalcohol consumption often coexist and may act synergistically,leading to a more rapid progression of liver disease and increas-ing the risk of HCC [4–6]. Accordingly, several studies from differ-ent geographic areas demonstrate that the risk for developingHCC is higher in patients with HCV infection and alcohol abusethan in patients with hepatitis C alone [6–8].

During HCC development, unrespectable to its etiologic ori-gin, hepatic cells, committed to proliferate, may accumulatemutations in key cell cycle genes or bear epigenetic changes,microsatellite instability, and several chromosomal aberrations,including aneuploidy [9,10]. Chromosomal aberrations are fre-quently discovered in dysplastic nodules of cirrhotic liversand are often present in HCC, suggesting the occurrence ofchromosomal defects at the early stages of hepatocarcinogene-sis [9–11].

Although the mechanisms by which chromosomal aberrationsarise during hepatocarcinogenesis are still unknown, it is knownthat deregulation of some mitotic events, usually found in othertumors, occurs. Alteration of expression and/or activity of mitoticregulatory components (cyclin B1, Aurora kinase A, etc.), as wellas deregulation of continuous cycles of microtubule polymeriza-tion/depolymerization and phosphorylation of structural proteins(i.e. tubulins), radically influence the control of centrosome mat-uration and separation, bipolar spindle assembly, chromosomealignment and segregation, and cytokinesis in human cells, lead-ing to aneuploidy [12–14]. Cyclin B1 was found over-expressed inHCC with advanced stage, portal invasion, and intrahepaticmetastasis [15]. Recently, the role of aurora kinase A in HCChas moved into the focus of preclinical research. In fact, the up-regulation of the Aurora-A gene in HCC nodules compared withnontumorous liver tissues has been demonstrated [16].

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JOURNAL OF HEPATOLOGY
Moreover, modifications of beta-tubulin isotypes were found inliver cancer [17]. The close association between mitotic defectsand hepatocarcinogenesis is also supported by in vivo and vitrostudies demonstrating that both HCV and ethanol are able tointerfere with the regular control of mitosis in hepatocarcinomacell lines [18–20]. HCV is able to induce an accumulation of cellsin G2/M phase and, concomitantly, an increase of cyclin B1/cdk1complex nuclear translocation, through its effects on p38 mito-gen-activated protein kinase (p38MAPK) and RNA-activated pro-tein kinase (PKR), both molecules implicated in the control of G2/M phase progression [18,19]. Animal and human studies suggestthat alcohol may be involved in initiation, promotion, and pro-gression of HCC, through the activation of various molecularmechanisms including oxidative stress, changes in DNA methyla-tion, immunosuppression, and genetic susceptibility [21,22].Interestingly, it has been reported that HepG2 cells stablyexpressing alcohol dehydrogenase showed 6-fold increase inthe percentage of cells in G2/M phase after ethanol exposure.The impairment in cell-cycle progression was due to the accumu-lation of the phosphorylated inactive form of Cdk1 [20,23].
Unfortunately, regarding the pathogenesis of HCV-associatedHCC, it still remains controversial whether the virus plays a director an indirect role, and how alcohol operates in the accelerationof HCC development [24,25].

We postulate that HCV, acting in synergism with alcoholabuse, might promote mitotic aberrations through the activa-tion/inhibition of different intracellular pathways; therefore, inthis study we analyzed the changes in expression of some impor-tant mitotic regulators in HCC tissues derived from alcoholics orHCV infected patients.

Although several mitotic regulators have been implicated incancer development and progression, we chose to study cyclinB1 and aurora kinase A as they form a network of interactionsregulating the onset of mitosis, the centrosome biogenesis andmicrotubule nucleation, and the cytokinesis. Moreover, gamma-tubulin is a little known common partner of these two proteins[26,27].

Here, we identify the intracellular mechanism(s) potentiallyinvolved in mitotic aberrations, by the use of a cell-based in vitromodel.

Materials and methods

Cells, plasmids, and polyclones

Human hepatocarcinoma cell lines (HepG2 and Huh7) were grown in Dulbecco’smodified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum(FBS), 100 U/ml penicillin, and 100 lg/ml streptomycin at 37 �C in a 5% CO2 incu-bator. Cells were grown to 70–80% confluence in 100-mm dishes and then trans-fected, using Lipofectamin 2000 reagent (Invitrogen, Carlsbad, CA, USA),according to the manufacturer’s protocols, with two different plasmids: an emptyvector (pcDNA3-FLAG) or a unique construct encoding for entire HCV products(p34-9,7-HCVwt 1a) (gently provided by Dott. La Monica Nicola, IRBM-Pomezia,Italy) [28].

Control polyclones (with the empty vector) and polyclones stably expressingall HCV proteins were obtained by 21 days geneticin-selection of transfected cells.Polyclones were cryo-preserved at �80 �C and used at the occurrence.

Polyclones selected after transfection with the empty vector were used asinternal experimental controls: preliminary experiments were performed toexclude the presence of substantial differences in comparison with non-transfec-ted cells (see Supplementary Fig. 1).

For all reported experiments, polyclonal cells were alternatively synchronizedin G1/S (by 2 mM thymidine) or G2/M transition (by 200 ng/ml nocodazole). SeeSupplementary Table 1 for FACS analysis.


Mitotic index

The mitotic index was calculated for G1/S-synchronized polyclones using doublethymidine block [29]. After block, G1 cells were released to progress through thecell cycle over the next 15 h, and then treated or not with ethanol. Mitotic indexwas calculated by DAPI (40–60-diamino-2-phenylindole) staining, as alreadydescribed [18]. After staining, the slides were extensively washed and mountedin Vectashield (Vector Laboratories Inc., Burlingame, CA, USA), before examination.

The mitotic index was expressed as the mean (%) of positive cells with respectto the total cell population per field, for a total of 20 fields. Cells were counted byfluorescence microscope (Nikon Eclipse E600 microscope, Nikon, Italy).

Western blotting

For Western blot analysis, equal amounts of proteins from tissue or cell extractsobtained after lysis in Ripa buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% NonidetP-40, 5 mM EDTA, SDS 0,1%, 2 mM phenylmethilsulfonyl fluoride, 1 g/ml aproti-nin, 1 g/ml of leupeptin and phosphatase inhibitors), were electrophoresed onSDS–PAGE. Proteins were transferred to PVDF membrane (Millipore, Marlbor-ough, MA, USA) and treated with specific primary antibodies overnight at 4 �C.Filters were then washed four times with PBS-Tween 20 and newly incubatedwith peroxidase-coupled secondary antibodies for 1 h at RT. After incubation,the blots were visualized by ECL (Amersham Pharmacia Biotechnology, Freiburg,Germany).

Immunoprecipitation

For immunoprecipitation, 0.1 mg of total lysate was incubated with 0.1 lg ofanti-phosphotyrosine antibody (Santa Cruz) overnight at 4 �C. Samples were thenincubated with protein A agarose (Amersham Pharmacia) for 1 h at 4 �C, washedthree times with Ripa buffer, and resuspended in 10 ll of SDS-sample buffer. Eachsample was then electrophoresed on SDS–PAGE, and Western blot to revealgamma-tubulin was performed as described above.

Antibodies

The following antibodies were used: anti-core protein monoclonal antibody(Affinity Bioreagents, Denver, CO, USA); anti-NS5A mouse monoclonal antibody,anti-actin goat polyclonal antibody, anti-cyclinB1 mouse monoclonal antibodyand anti-PKR rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz,CA, USA); anti-phosphotyrosin mouse monoclonal antibodies (Upstate Biotech-nology, Lake Placid, NY, USA); anti-aurora A rabbit polyclonal antibody, andanti-gamma-tubulin rabbit polyclonal antibody (GeneTex Inc., San. Antonio, TX,USA); peroxidase-conjugated goat anti-rabbit, goat anti-mouse, and rabbit anti-goat IgG (Santa Cruz).

Inhibitors treatment

Cells were pre-treated for 30 min with 10 lM SP600125 or SB203580 (Sigma–Aldrich, Milano, Italy) to inhibit the activity of c-Jun N-terminal kinase (JNK)and p38MAPK, respectively.

siRNA transfection

A cocktail of siRNA directed against several regions of PKR (siPKR) was designedby New England Biolabs (Beverly, MA, USA) and transfected using Lipofectamin2000 reagent (Invitrogen), according to the manufacturer’s protocols. Dosingexperiments showed that optimal silencing was achieved using 10 nM PKR siRNA(see Supplementary Fig. 2). Cells were also transfected with siRNA double target-ing GFP gene (Quiagen, Germantown, MD, USA), as a negative control.

Patients and liver samples

The study was performed on 60 archived liver tissues obtained from 10 patientswith HCV-related HCC, 10 patients with alcohol-associated HCC, and control non-cirrhotic subjects obtained from 10 surgically treated patients during the courseof routine clinical care at the Surgical Departments, University of ‘‘La Sapienza‘‘Rome’’ and at the Azienda Ospedaliero-Universitaria di Parma. At the time ofsurgical resection the tumor area was separated by the surrounding tissue. Then,part of the resected sample was fixed in formalin and embedded in paraffin forhistological diagnosis, another part of all tissues were snap frozen in liquid nitro-

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Table 1. Mitotic index values in HepG2 polyclones. p value refers to each condition vs. control.

C HCVA

Research Article

gen and then stored at –80 �C for molecular analysis. This study was approved bythe local ethical committee and all samples were obtained with the patient’sinformed consent.

Statistical analysis

Results are expressed as mean ± standard deviation (SD) of four samples fromat least three independent experiments. In particular, we performed an ANOVAamong groups for repeated measures, followed by Bonferroni’s correction. pvalue < 0.05/n (where n is the number of comparisons) was considered to indi-cate a statistically significant difference vs control. ⁄p <0.001, ⁄⁄p <0.01,⁄⁄⁄p <0.05.

CC + EtOHHCVHCV + EtOH

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Fig. 1. HCV and ethanol affect the expression of cyclin B1, and Aurora kinaseA in HepG2 cells. (A) Expression levels of cyclin B1 and Aurora kinase A in thecontrol and HCV polyclones at 0, 2, 6, and 24 h after release from G2/M blocking.Beta-actin is present as the control of equal loading. (B) Expression levels of cyclinB1 and Aurora kinase A in the control and HCV polyclones, treated or not withethanol (EtOH), were analyzed 24 h after nocodazole release from G2/M blocking.To demonstrate equal loading, membranes were re-probed with the beta-actinantibody. Immunoblots are representative of at least three independent exper-iments. (C) Semiquantitative densitometric data are reported in the histograms asmean values of four independent experiments ± SD (bars). p <0.001; ⁄⁄p <0.01versus control polyclones.

Results

HCV and ethanol impair mitosis in HCC cells

Since both HepG2 and Huh7 polyclones showed similar results, forbrevity we reported only the results of experiments performed onHepG2 cells. Here, we investigated whether HCV and ethanol altermitosis in cell-based models of HCV infection and alcohol abuse.Control and HCV polyclones, established as described in Materialsand methods, were firstly treated with a 25 mM minimum toxicconcentration of ethanol (see Supplementary Fig. 3), or PBS alonefor 24 h, and then synchronized in G1/S phase with double thymi-dine block. The mitotic index was evaluated with DAPI staining,after 0, 24, and 48 h from synchronization. As reported in Table1, the accumulation of cells in M phase reached the maximal valueat 48 h in polyclones expressing HCV proteins. Ethanol treatmentinduced a statistically significant increase of M phase cells in thecontrol, but not in HCV polyclones.

Considering these results, we analyzed the expression or theactivity of some molecules specifically involved in the control ofmitosis, such as cyclin B1, Aurora A kinase, and gamma-tubulin.For this purpose, control and HCV polyclones, treated or not with eth-anol, were synchronized in G2/M phase with nocodazole and, after24 h, total proteins were extracted to perform Western blotting.

In polyclones expressing HCV proteins, the expression ofcyclin B1 and Aurora kinase A was up-regulated at all time points(Fig. 1A). Thus, we investigated the effect of ethanol on the samemitotic regulatory molecules. As shown in Fig. 1B, ethanol treat-ment caused a further increase in cyclin B1 and Aurora kinase Aprotein expression levels, as compared to HCV expressing cells. Asemiquantitative densitometric analysis of immunoblots wasperformed (Fig. 1C).

Moreover, we analyzed, in cell-based models, the pattern oftyrosine-phosphorylation of gamma-tubulin at G2/M transition.As shown in Fig. 2A, HCV was able to induce a significant andsustained increase in tyrosine-phosphorylation of gamma-tubu-lin. Noteworthy, ethanol further increased gamma-tubulin phos-phorylation levels compared to HCV expressing cells (Fig. 2B andC). Newly, the ethanol effects were additive to those observed inuntreated HCV polyclones (Figs. 1C and 2C).

958 Journal of Hepatology 201

Expression of mitotic regulatory molecules in HCC tissues

To verify the in vivo relevance of results obtained in in vitro mod-els, we analyzed the expression profile of cyclin B1 and Aurora Akinase, and the phosphorylation rate of gamma-tubulin in livertissues derived from 30 subjects: 10 with HCV-related HCC, 10with alcohol-associated HCC, and 10 with normal liver. In partic-

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P-γ-Tubulin

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Fig. 2. HCV and ethanol alter tyrosine-phosphorylation of gamma-tubulin inHCC cells. (A) Tyrosine-phosphorylation levels of gamma-tubulin in the controland HCV polyclones at 0, 2, 6, and 24 h after release from blocking in G2/M withnocodazole. (B) Tyrosine-phosphorylation levels of gamma-tubulin in the controland HCV polyclones treated or not with ethanol (EtOH) for 24 h after release fromblocking in G2/M. Panels P (upper) show the pellets containing the tyrosine-phosphorylated form of gamma-tubulin; while panels S (lower) show the beta-actin levels in supernatants as loading controls. Immunoblots are representativeof at least three independent experiments. (C) Semiquantitative densitometricdata are reported in the histograms as mean values of four independentexperiments ± SD (bars). p <0.001 versus control polyclones.

A

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β-Actin

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Aurora A

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Et-OH Et-OHC HCV HCV

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HCC sHCC

HCC sHCC

Fig. 3. Expression levels of cyclin B1, Aurora kinase A, and phosphorylatedgamma-tubulin in liver tissues. (A) Cyclin B1 and Aurora kinase A proteinexpression levels in liver tissue of control subjects (C), and in HCC (HCC) andsurrounding tissue (sHCC) of patients with hepatocarcinoma associated with HCVinfection (HCV) or alcohol abuse (EtOH). Beta-actin is present as the control ofequal loading of proteins. (B) Levels of gamma-tubulin expressed in pelletsresulting from the immunoprecipitation of tyrosine-phosphorylated proteins inthe controls and HCV or ethanol-related HCC and surrounding tissues. Panels P(upper) show the pellets containing tyrosine-phosphorylated form of gamma-tubulin; while panels S (lower) show the beta-actin levels in supernatants asloading controls. The images shown are representative of at least threeindependent experiments.


ular, the expression levels of cyclin B1 and Aurora kinase A wereanalyzed in control normal livers, in HCC tissues (HCC), and insurrounding tumor tissues (sHCC). As shown in Fig. 3A, cyclinB1 and Aurora kinase A were significantly over-expressed inHCC tissues (lanes 2 and 3). We also examined the expressionof the tyrosine-phosphorylated form of gamma-tubulin. Asreported in Fig. 3B, the level of the tyrosine-phosphorylated formof gamma-tubulin is up-regulated in HCC (lanes 2 and 3) and insHCC (lanes 4 and 5) and seems to be higher in alcohol-relatedcompared to HCV-related HCC. Quantitative data, obtained bydensitometric analysis of immunoblotting of the studied mole-cules, is shown in Table 2. In the same table we reported clinic-o-pathological findings of examined liver tissues.

PKR is involved in HCV-related mitotic defects

Then, we investigated which intracellular mechanism(s) might beresponsible for the interesting results we obtained both in vivoand in vitro.

As already reported, PKR is one mediator of HCV core proteineffects on G2/M progression [18,19]. Thus, here, we firstlyevaluated whether PKR could be involved in the HCV-related


deregulation of cyclin B1, Aurora kinase A, and tyrosine-phosphorylated gamma-tubulin. To this aim, HCV and controlpolyclones were transfected with a mix of small interference(si)RNAs (10 nM) against PKR, or alternatively, with a siRNAagainst GFP as the control. Three hours later, we treated the con-trol cells with 25 mM ethanol or an equal amount of PBS for 24 h.G2/M synchronized cells were collected immediately afterrelease and at 24 h.

Silencing of PKR abrogated all mitotic HCV-related effects,while it was unable to revert the ethanol-dependent alterationsof the mitotic molecules (Fig. 4A). Again, in HCV expressing cells,gamma-tubulin tyrosine phosphorylation was affected by PKRsilencing (Fig. 4B). Densitometric analysis was performed andreported as fold changes in protein levels compared to the controlconsidered as 1 after normalization against beta-actin (Fig. 4C).

Our results clearly indicate that PKR is involved in HCV- butnot in ethanol-dependent mitotic protein deregulation. The rele-vance of PKR in HCV-dependent effects was also reinforced by theanalysis of the mitotic index in HCV polyclones in the presence ofsiPKR (see Supplementary Fig. 4).

The effect produced on mitotic proteins by PKR silencing inHCV polyclones stimulated with ethanol was comparable to thatobserved in untreated HCV polyclones (data not shown).

Emodin counteracts the ethanol-dependent mitotic effects

To analyze the mechanisms by which ethanol induces mitoticderegulation, we treated polyclonal cells with several differentdrugs inhibiting important signal transduction intracellular path-ways. In particular, we used SP600125 (10 lM) to inhibit JNK andSB203580 (10 lM) to specifically inhibit p38MAPK, two impor-tant intracellular pathways activated by ethanol treatment. Asreported in Fig. 5A and B (lanes 7–9), both inhibitors were inef-

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Table 2. Clinicopathological findings and quantitative values obtained from densitometric analysis of proteins analyzed in 30 liver samples.

⁄p <0.001, ⁄⁄p <0.01, ⁄⁄⁄p <0.05 vs. control samples without tumor. HCC: HCC tissue; sHCC surrounding HCC tissue.

Research Article

fective on ethanol-induced mitotic biological effects. On the con-trary, JNK and p38MAPK inhibitors were able to equally revertHCV-dependent mitotic alterations in untreated (Fig. 5A and B,lanes 4–6) and in ethanol-treated HCV polyclones (data notshown).

In the light of the difficulties encountered in searching possi-ble intracellular pathways responsible for molecular changes inethanol-treated cells, we decided to avoid selective inhibitorspreferring a compound with a broad spectrum of biological activ-ities. We checked the effects of a new active bio-molecule derivedby the rhizome of Rheum palmatum L., known as Emodin. Emodinwas chosen because it is a drug described to interfere with multi-ple signaling pathways, including NF-kB (nuclear factor-kB), FAK(focal adhesion kinase), and PI3K (phosphatydil-inositol-3kinase) intracellular signaling [30–32]. Interestingly, Emodin(40 lM) was able to completely revert alcohol effects on tyro-sine-phosphorylation of gamma-tubulin (Fig. 5C); while cyclinB1 and Aurora kinase A expression remained unchanged(Fig. 5D). These last data, confirmed by densitometric analysis(Fig. 5E), together with the analysis of the mitotic index in etha-nol treated control polyclones in the presence of Emodin (seeSupplementary Fig. 5), demonstrated that Emodin only partiallyreverts the ethanol-dependent mitotic deregulation.

Discussion

HCCs are associated with high incidence of genetic alterations,which increases during the carcinogenic process. Persistent infec-tion with HCV has been considered a major risk for the develop-ment of HCC, as well as heavy alcohol abuse, which has beenlinked with earlier progression to HCC in chronic hepatitis C


patients [25]. However, molecular mechanisms inducing this syn-ergism of action are still controversial. Oxidative stress andderegulation of cellular gene expression, controlling cell-cycleprogression, seem to be dominant mechanisms for the synergicaction of alcohol and HCV [33].

HCV infection, as well as ethanol, impairs cell-cycle progres-sion leading to a G2/M arrest in liver cells [18,20]. Interest-ingly, over-expression of mitotic molecules, such as cyclin B1and Aurora kinase A, was found in human HCCs; however, nostudies analyzed differences in the expression of these proteinsin relation to possible different etiologies of HCCs [34,16,35].We decided to study the expression and the activity of threemolecules involved in the control of mitosis: two with func-tional roles (i.e. cyclin B1 and Aurora kinase A), and anotherwith structural importance (i.e. gamma-tubulin). Cyclin B1and gamma-tubulin play different roles during mitosis: the firstregulates entry/exit from M phase while the second is impor-tant for centrosome maturation. Aurora kinase A is a commonpartner for these two proteins: it physically interacts withcyclin B1 enhancing its stability and disrupting cytokinesis, fur-thermore it recruits gamma-tubulin, and other centrosomalproteins to promote centrosome maturation and microtubulenucleation ability [36,37].

Our results provide novel evidence that the expression of allHCV proteins, alone and even more in association with ethanol,may induce mitotic defects in HCC cells. Accordingly, bothHCV- and ethanol-related HCCs, analyzed by us, are characterizedby a deregulation of cyclin B1, Aurora kinase A, and tyrosine-phosphorylation of gamma-tubulin. The finding that eitherHCV or ethanol may enhance the expression of some mitoticmolecules is interesting, but even more fascinating is their effecton tyrosine-phosphorylation of gamma-tubulin.

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CC + siPKRHCV

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Fig. 4. Role of PKR in the HCV-related mitotic effects. (A) Protein expressionlevels of PKR, cyclin B1 and Aurora kinase A, analyzed 24 h after release from G2/M blocking, in control and HCV polyclones exposed or not to the ethanol,transfected or not transfected with siPKR (10 nM) or with siGFP (10 nM) as thecontrol. Beta-actin is present as the control of equal protein loading. (B) Tyrosine-phosphorylation levels of gamma-tubulin 24 h after release from G2/M blocking,in the control and HCV polyclones exposed or not to ethanol, and transfected ornot with siPKR (10 nM) or siGFP (10 nM) as the control. Panels P (upper) show thepellets containing tyrosine-phosphorylated form of gamma-tubulin; while panelsS (lower) show the beta-actin levels in supernatants as loading controls.Immunoblots are representative of at least three independent experiments. (C)Densitometric analysis reported as fold changes in protein levels ± SD (bars)respect to the control considered as 1 after normalization against beta-actin.⁄p <0.001 versus control polyclones.

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Fig. 5. Role of Emodin in ethanol-related mitotic effects. (A) Protein expressionlevels of cyclin B1 and Aurora kinase A observed 24 h after release from G2/Mblocking, in HCV polyclones and in control polyclones exposed or not to ethanol,treated or not with SP600125 (10 M) and SB203580 (10 M). (B) Protein expressionlevels of tyrosine-phosphorylated gamma-tubulin, observed 24 h after releasefrom G2/M blocking, in HCV polyclones and in control polyclones exposed or notto ethanol, treated or not with SP600125 (10 M) and SB203580 (10 M). (C) Proteinexpression levels of tyrosine-phosphorylated gamma-tubulin, 24 h after releasefrom G2/M blocking, in control polyclones exposed or not to ethanol, and treatedor not with Emodin (40 M). (D) Protein expression levels of Aurora A and cyclinB1, in the control polyclones exposed or not to ethanol and treated or not withEmodin. Beta-actin is reported as the control of equal loading. Immunoblots arerepresentative of at least three independent experiments. (E) Densitometricanalysis reported as fold changes in protein levels compared to the controlconsidered as 1 after normalization against beta-actin. ⁄p <0.001 versus controlpolyclones.


Taken together, our findings may imply that not only pro-found changes in gene expression but also early phosphorylationevents may contribute to hepatocyte transformation opening anew attractive research field in molecular hepatocarcinogenesis,which surely requires further investigations. Furthermore, thestudy of a larger sample of tissues, including HCC and othersources, may not only strengthen our results but also identifysome possible correlations between the expression of these pro-teins and the degree of tumor transformation.

Moreover, here, we reinforce our previous findings demon-strating that HCV proteins modulate mitotic molecules via PKR[18]. In particular, here we find that the HCV-dependent altera-tions of cyclin B1, Aurora kinase A, and tyrosine-phosphorylation


of gamma-tubulin are mediated by a mechanism stronglydependent on PKR, as well as on p38MAPK and JNK pathways.Interestingly, PKR is a relevant mediator of p38MAPK and JNKactivity in several conditions. Goh et al. demonstrated that PKRmediates the activation of p38MAPK and JNK by specific proin-flammatory stress stimuli, such as: interleukin-1beta, lypopoly-saccharide, TNF (tumor necrosis factor)-alpha, etc. [38]. Inmouse fibroblasts, PKR influences TNF-alpha signaling positively

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Research Article
regulating JNK and negatively regulating p38MAPK [39]. Morerecently, it has been reported that the depletion of PKR impairsp38 and JNK phosphorylation induced by either the E3L deletionmutant of vaccinia virus or double-stranded RNA [40]. Based onour previous data demonstrating that HCV core expression leadsto deregulation of the mitotic checkpoint via a p38/PKR-depen-dent pathway [19], we hypothesize that also JNK might be adownstream effector of PKR in the HCV-dependent mitoticeffects. On the other hand, our results demonstrated that ethanoltreatment modifies the expression of the same mitotic moleculestargeted by HCV virus, but in a PKR, JNK, and p38MAPK-indepen-dent way. These data suggest that other signaling molecules maybe involved in the ethanol-dependent mitotic effect. Interest-ingly, Emodin a novel anticancer drug that interferes with theactivity of multiple signaling pathways including NF-kB, FAK,and PI3K, completely reverts the ethanol-associated over-expres-sion and up-regulation of the tyrosine-phosphorylated form ofgamma-tubulin [30–32]. Further investigations are required toanalyze molecular pathways involved in the ethanol-dependenteffects on cyclin B1 and Aurora kinase A. In addition, Emodin,which has a well documented hepatoprotective effect [41,42],has been recently reported as capable to inhibit hepatoma cellgrowth affecting genes potentially associated with liver tumorprogression, including cyclins [43]. These findings make this nat-ural agent a potential candidate to improve hepatocarcinomatreatment.
In conclusion, our study demonstrates, for the first time, thatHCV proteins and alcohol synergistically alter the mitotic appara-tus, using different intracellular pathways; furthermore, we haveidentified new molecular mechanisms associated with HCV- andalcohol-dependent mitotic abnormalities. Our findings provideimportant new insights into HCV- and alcohol-associated hepato-carcinogenesis furnishing a good starting point to develop inno-vative combined therapeutic strategies [44,45]. However,specific multiple effectors and downstream signal cascades haveto be deeply investigated and the possible correlations amongthese signaling molecules and the stage and grade of HCC are stillunclear.

Financial support

For this work Dr. Anna Alisi was supported by a fellowship fromItalian Association for the Study of the Liver: AISF.

Conflict of interest

The authors who have taken part in this study declared that theydo not have anything to disclose regarding funding or conflict ofinterest with respect to this manuscript.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jhep.2010.08.016.

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