Proteomic analysis of differentially expressed proteins in hepatitis B virus-related hepatocellular carcinoma tissues

Background

Hepatocellular carcinoma (HCC), a major cause of cancer death in China, is preceded by chronic hepatitis and liver cirrhosis (LC). Although hepatitis B virus (HBV) has been regarded as a clear etiology of human hepatocarcinogenesis, the mechanism is still needs to be further clarified. In this study, we used a proteomic approach to identify the differential expression protein profiles between HCC and the adjacent non-tumorous liver tissues.

Methods

Eighteen cases of HBV-related HCC including 12 cases of LC-developed HCC and 6 cases of chronic hepatitis B (CHB)-developed HCC were analyzed by two-dimensional electrophoresis (2-DE) combined with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and the results were compared to those of paired adjacent non-tumorous liver tissues.

Results

A total of 17 differentially expressed proteins with diverse biological functions were identified. Among these, 10 proteins were up-regulated, whereas the other 7 proteins were down-regulated in cancerous tissues. Two proteins, c-Jun N-terminal kinase 2 and ADP/ATP carrier protein were found to be up-regulated only in CHB-developed HCC tissues. Insulin-like growth factor binding protein 2 and Rho-GTPase-activating protein 4 were down-regulated in LC-developed and CHB-developed HCC tissues, respectively. Although 11 out of these 17 proteins have been already described by previous studies, or are already known to be involved in hepatocarcinogenesis, this study revealed 6 new proteins differentially expressed in HBV-related HCC.

Conclusion

These findings elucidate that there are common features between CHB-developed HCC and LC-developed HCC. The identified proteins are valuable for studying the hepatocarcinogenesis, and may be potential diagnostic markers or therapeutic targets for HBV-related HCC.     

The Rho kinase inhibitor fasudil is involved in p53-mediated apoptosis in human hepatocellular carcinoma cells

Purpose

Rho kinase is an important factor in tumor progression. We demonstrated that Rho kinase-associated coil-containing protein kinase (ROCK) is expressed in hepatic tissues in hepatocellular carcinoma (HCC) and confirmed its roles in cell survival in HCC cells using the ROCK inhibitor, fasudil.

Methods

ROCK protein levels were estimated in hepatic tissues with HCC compared with healthy liver tissues or hepatic hemangioma tissues using immunohistochemistry. Furthermore, HepG2 and Huh7 cells were cultured with ROCK inhibitor, fasudil for 24?h in vitro. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay, and apoptotic cells were detected by cell death ELISA. The expression apoptosis-related proteins were analyzed using Western blotting.

Results

Fasudil significantly decreased cell proliferation and induced apoptosis mediated by increases in p53, Bax, caspase-9, and caspase-3 in HepG2 and Huh7 cells. The induction of apoptosis was inhibited in HCC cells precultured with p53 decoy oligodeoxynucleotide.

Conclusion

These results suggest that ROCK inhibits the p53-mediated apoptosis pathway in HCC. Fasudil may thus be a beneficial approach to HCC therapy.     

Hepatitis B Virus-Associated Hepatocellular Carcinoma from India: Role of Viral Genotype and Mutations in CTNNB1 (Beta-Catenin) and TP53 Genes

Purpose

Chronic hepatitis B virus (HBV) infection is the major risk factor for hepatocellular carcinoma (HCC) in India. Studies from other countries have linked HBV genotype C to a higher risk for HCC. This study was carried out to determine the association between genotype and HCC and also the frequency of mutations in CTNNB1 (beta-catenin) and TP53 genes in HBV-related HCC.

Methods

Formalin-fixed paraffin-embedded (FFPE) tissues from 20 (15 autopsy, five resected specimens) cases of HBV-associated HCC were examined. Viral genotype was determined by sequencing portions of the HBV S gene using four overlapping PCR amplicons. Exon 3 of CTNNB1 and exon 7 of TP53 were sequenced.

Results

HBV genotyping was possible in 14 of 20 cases; genotype D was most common (n?=?11) followed by C (n?=?2) and A (n?=?1). CTNNB1 mutations were noted in two of 15 amplifiable cases while two of 10 specimens showed TP53 mutations.

Conclusions

HBV genotype can be ascertained from FFPE sections by sequencing multiple overlapping fragments to avoid the limitation of fragmented DNA. Genotype D was the common genotype in HBV-associated HCC. The very low frequency of TP53 mutation suggests low levels of aflatoxin B₁ exposure. The beta-catenin pathway appears not to be significantly involved in HBV-related HCC in India. However, further larger studies are required to confirm these findings.     

Risk Profile of Hepatocellular Carcinoma Reveals Dichotomy among US Veterans

Background

Hepatocellular carcinoma (HCC) is traditionally associated with chronic liver injury resulting from hepatitis B virus (HBV) and hepatitis C virus (HCV) infection or excessive consumption of alcohol. In addition, recent evidence links HCC to diabetes.

Aims

Since these risk factors are prevalent among US veterans, we analyzedcontribution of various etiologies toHCC incidence in this population.

Methods

Clinicopathological correlates of 150 male US veterans diagnosed with HCC between 2001 and 2010 were analyzed and compared to frequency-matched (2:1) non-cancer controls in a single center.

Results

HCC was associated with cirrhosis (odds ratio [OR], 250.84; 95 % confidence interval [CI], 86.92–723.88; p?<?0.0001), chronic hepatitis B (OR, 34.30 95 % CI, 1.97–598.47; p?=?0.015), chronic hepatitis C (OR, 6.84; 95 % CI, 3.89–12.04; p?<?0.0001), alcohol use (OR, 6.76; 95 % CI, 4.35–10.52; p?<?0.0001), and smoking (OR, 1.83; 95 % CI, 1.23–2.89; p?=?0.009), but surprisingly not with diabetes. Only in a subgroup of HCC patients with no “traditional” risk factors did diabetes become a strong independent predictor of HCC when compared to HCC patients with at least one such risk factor (OR, 10.69; 95 % CI, 1.88–60.63, p?=?0.007). This subgroup was further distinguished by older age, increased prevalence of hypertension, nonsmoking, and a trend to develop noncirrhotic HCC.

Conclusions

While HCC in US veterans is overwhelmingly linked to cirrhosis due to “traditional” risk factors, it also occurs with a separate clinical profile characterized by diabetes and no evidence of cirrhosis, suggesting distinct mechanisms of hepatocarcinogenesis and needs for surveillance.     

Microvesicle microRNA profiles and functional roles between chronic hepatitis B and hepatocellular carcinoma

Objectives

Hepatocellular carcinoma (HCC) is an inflammation-related malignancy and chronic hepatitis B (CHB) predisposes to HCC. Microvesicles (MVs) transfer various bioactive molecules including microRNA (miRNA) between cells and exert biological functions. The purpose of this study was to detect CHB-MV and HCC-MV miRNAs and analyze the expression profiles and functional roles between CHB and HCC.

Methods

We examined MV miRNA profiles of CHB and HCC using miRNA microarrays. TargetScan, PicTar and miRanda were exerted to predict target genes regulating these differentially expressed miRNAs.

Results

A total of 272 and 242 aberrant fluctuation miRNAs were identified in CHB-MVs and HCC-MVs, respectively. Among them, there were 53 miRNAs co-expressing both in CHB-MVs and HCC-MVs. These miRNAs affected cellular apoptosis, proliferation and molecular signaling pathways. Among them, 25 co-expressed MV miRNAs targeted 21 inflammatory factors and these miRNAs may be a tight linkage between CHB and HCC. Interestingly, there were 14 co-expressed MV miRNAs targeting 17 oncogenes and 7 miRNAs targeting 9 tumor suppressors in the study. In addition, MVs were enriched with maladjusted miRNAs regulating zinc finger proteins and chromosome open reading frame. Those MV miRNAs may play roles in CHB developing to HCC.

Conclusions

We demonstrated that CHB and HCC displayed aberrantly co-expressed MV miRNA profiles for the first time, which may have a link between CHB and HCC. Those MV miRNAs may serve as early biomarkers for HCC and may aid to promote CHB developing to HCC.     

Comparison of Cancer Care for Hepatocellular Carcinoma at Two Tertiary-Care Referral Centers from High and Low Endemic Regions for Viral Hepatitis

Background

Risk factors for hepatocellular carcinoma (HCC) have geographic variability but differences in care have not been described. We reviewed the presentation, management, and outcomes of HCC patients from two tertiary-referral centers in Central Saudi Arabia and Atlantic Canada during 1997–2002.

Methods

Data were extracted from health records of 96 Saudi and 80 Canadian consecutive patients with HCC.

Results

Mean age (±SEM) of the two groups were similar (64?+?1 and 65?+?1 years) with 93% versus 75% males amongst Canadian and Saudi patients, respectively. In Canada, underlying disease was alcohol-related cirrhosis (45%), cryptogenic cirrhosis (26%), or hepatitis C (13%). For Saudis, HCC cases were attributed to hepatitis C (47%), cryptogenic cirrhosis (27%), and hepatitis B (21%). At initial presentation, Saudi patients had more vascular invasion and distant metastases while Canadians had more advanced liver disease. The tumor-specific prognostic classifications were comparable. Due to center-specific expertise or preference, symptomatic treatment was more common amongst Saudi patients (83% versus 42%) while more Canadians underwent local palliative interventions (52% versus 12%). Frequency of potentially curative therapies including resection and transplantation were similar at both centers. There was no difference in overall median survival (14 versus 10 months) amongst Canadian and Saudi patients.

Conclusions

This study validates divergence in HCC presentation between low and high endemic regions for viral hepatitis. In addition, for the first time, differences in cancer care of HCC are documented.     

Hepatocellular Carcinoma in Non-cirrhotic Liver Without Evidence of Iron Overload in a Patient with Primary Hemochromatosis. Review

Background

We report a case of a 70-year-old male with hepatocellular carcinoma (HCC) with a history of hemochromatosis but with no evidence of cirrhosis or iron overload and with a history of exposure to atomic bomb radiation. It is very rare to see hepatocellular carcinoma in the absence of evidence of liver injury.

Methods

We did an extensive review of current English medical literature through Pubmed from 1980 to 2009 and found 14 case reports of patient with hepatocellular cancer in absence of cirrhosis. The details of these cases were reanalyzed as reported and documented for review.

Results

There are 14 previous case reports of HCC developing in hemochromatosis in absence of cirrhosis but ten of them had evidence of iron overload in the non-tumorous livers. Our case is the fifth case of Hepatocellular cancer in hemochromatosis in absence of cirrhosis and iron overload.

Conclusion

Hepatocellular carcinoma is a very rare in absence of cirrhosis but patient with other risk factors like hemochromatosis, viral infections, radiation, and toxin exposure should be monitored closely for any sign and symptoms suggestive of malignancy.     

Effects of Ginkgo biloba extract on expression of biomarkers during aflatoxin B1-induced hepatocarcinogenesis in Wistar rats

Objective

The aim of this study was to study the effect of Ginkgo biloba extract (EGb761) on metabolism of aflatoxin B₁ (AFB₁) in Wistar rats.

Methods

Seventy one Wistar rats were assigned at random to groups A, B and C. Rats in groups A, B were injected with AFB₁ (intraperitoneal, 100–200 μg/kg body weight, 1–3 times/week). Group C was normal control. Rats in group B were fed in food with EGb761, while rats in groups A, C were given normal food. Blood samples were collected and liver biopsies were performed on the 14th, 28th and 42nd week. All the rats were sacrificed on the 64th week. The incidence of hepatocarcinoma was investigated. The hepatic phase I drug-metabolizing enzyme Cytochrome-P450 (CYP450) and phase II metabolizing enzyme glutathione S-transferase (GST) were analyzed with spectrometry. Serum AFB₁-lysine adduct levels were assessed with high performance liquid chromatography (HPLC). The expression of 8-hydroxydeoxyguanosine (8-OHdG) was measured with immunohistochemistry.

Results

The incidence of hepatocellular carcinoma (HCC) in group B was significantly lower than that in group A (26.92% vs 76.00%, P < 0.001). No HCC developed in group C. EGb761 showed no effects on the activities of CYP450 and GST in rat liver tissues. The level of AFB1-lysine adduct reached the peak (4356.01 pg/mg albumin) at the 14th week in group A. EGb761 significantly inhibited the formation of AFB1-lysine adduct in serum by 13.07% at the 14th week (P = 0.033), and 73.63% at the 42nd week (P = 0.002). The expression of 8-OHdG protein in rat liver tissues in group B was significantly lower than that in group A at the 28th, 42nd, and 64th week (P < 0.05).

Conclusion

The main mechanism underlying the effect of EGb761 in blocking hepatocarcinogenesis induced by AFB1 may not be fully attributable to its influence on the activity of liver phase I and phase II metabolizing enzymes. EGb761 inhibits the production of AFB₁-lysine adducts, decreases the expression of 8-OHdG protein, and finally alleviates the DNA oxidative injury, which may be one of the mechanisms for the effects of EGb761 in inhibiting or delaying AFB1-induced hepatocarcinogenesis.     

Overexpression of retinoic acid-induced protein 3 predicts poor prognosis for hepatocellular carcinoma

Aim

To investigate clinical significance of retinoic acid-induced protein 3 (RAI3) in hepatocellular carcinoma (HCC).

Methods

Expression of RAI3 at both mRNA and protein levels in tumor, para-tumor and normal liver tissues was detected in 106 HCC patients by real-time quantitative RT-PCR, Western blot and immunohistochemistry. Then, the correlation of RAI3 expression with clinicopathological characteristics and survivals of HCC patients was analyzed.

Results

Our data first found that RAI3 mRNA and protein expression were both significantly higher in HCC than in para-tumor (both P < 0.001) and normal liver tissues (both P < 0.001). The correlation analysis showed a positive correlation between RAI3 mRNA level and RAI3 protein level in HCC tissues (r = 0.8, P < 0.001). Immunohistochemistry data also revealed that overexpression of RAI3 was present in 73.6 % (78/106) of HCC tissues. In addition, high RAI3 protein expression was correlated with advanced TNM stage (P = 0.001), high serum AFP (P = 0.008), vascular invasion (P = 0.01) and tumor recurrence (P = 0.008). Moreover, HCC patients with overexpression of RAI3 had significantly shorter overall (P = 0.01) and disease-free survival (P = 0.01). Furthermore, multivariate analysis showed that overexpression of RAI3 was an independent prognostic factor for both overall (P = 0.02) and disease-free survival (P = 0.03) in HCC.

Conclusion

Our data for the first time provide a basis for the concept that overexpression of RAI3 may contribute to the malignant progression of HCC and predict poor prognosis for patients with this deadly disease after curative hepatectomy. RAI3 might be an important marker for tumor progression and prognosis, as well as a potential therapeutic target of HCC.     

Expression and prognostic value of Ars2 in hepatocellular carcinoma

Background

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China. Arsenic resistance protein 2 (Asr2) was reported to be important for microRNA (miR) biogenesis, and its depletion could reduce the levels of several miRs, including miR-21, which is over-expressed in HCC. We hypothesized that Ars2 is also overexpressed in HCC and may be involved in the biological properties of HCC.

Methods

Ars2 immunolabeling was evaluated in 132 HCCs. Ars2 immunolabeling, Ars2 qRT-PCR and miR-21 were evaluated in 20 HCCs and in paired normal tissues. Ars2 shRNA was transfected into SMCC-7721 and HepG2 HCC cells. The cell proliferation and expression of Ars2 and miR-21 were subsequently evaluated.

Results

Ars2 was expressed primarily in the nucleus of HCC cells. The expression of Ars2 was statistically correlated with the loss of HCC differentiation and pathological stage. The survival rates of patients with low Ars2 expression in HCC were statistically higher than patients with overexpressed Ars2 in HCC. Ars2 and miR-21 were more highly expressed in HCC specimens than normal tissues, and they were also correlated. The knockdown of Ars2 in HCC cells inhibited miR-21 expression and cell proliferation.

Conclusions

Ars2 is overexpressed in HCC and may have prognostic value; it might play an important role in HCC proliferation and miR-21 expression.     

Restoration of klotho expression induces apoptosis and autophagy in hepatocellular carcinoma cells

Purpose

Klotho has been identified as a tumor suppressor in several human malignancies including hepatocellular carcinoma (HCC). However, the signaling pathways involved in the tumor suppressive role of klotho in HCC have not been reported. Here, we investigated the role of klotho in HCC cell proliferation, apoptosis, autophagy, and invasion, as well as its associated signal transduction pathways.

Methods

Restoration of klotho gene expression was established by delivering a klotho gene expression vector into the human HCC cell lines HepG2 and MHCC-97-H. Cell viability was measured using a cell counting (CCK-8) assay and apoptosis was analyzed through flow cytometry. Autophagy was measured via LC3-I and LC3-II protein expression levels and tumor cell invasion was assessed using a Matrigel invasion chamber assay. Expression and phosphorylation of several apoptosis and survival related proteins were assessed using Western blot assays.

Results

Exogenous klotho gene expression significantly inhibited HCC cell proliferation, induced HCC cell apoptosis, increased LC3-I and LC3-II protein expression in HCC cells, and decreased migration of HCC cells in a Matrigel invasion chamber assay. Exogenous klotho gene expression also down-regulated the phosphorylation levels of the IGF-1 receptor, and the downstream Akt, ERK, and p70S6K proteins. Both apoptosis and autophagy inhibitors decreased klotho-induced apoptosis and autophagy.

Conclusion

Klotho is a tumor suppressor that, through the regulation of IGF-1R phosphorylation and subsequent activation of downstream Akt-p70S6K and ERK signaling, regulates HCC tumor cell proliferation, apoptosis, autophagy and invasion.     

An expression signature of phenotypic resistance to hepatocellular carcinoma identified by cross-species gene expression analysis

Background and aims

Hepatocarcinogenesis is under polygenic control. We analyzed gene expression patterns of dysplastic liver nodules (DNs) and hepatocellular carcinomas (HCCs) chemically-induced in F344 and BN rats, respectively susceptible and resistant to hepatocarcinogenesis.

Methods

Expression profiles were performed by microarray and validated by quantitative RT-PCR and Western blot.

Results

Cluster analysis revealed two distinctive gene expression patterns, the first of which included normal liver of both strains and BN nodules, and the second one F344 nodules and HCC of both strains. We identified a signature predicting DN and HCC progression, characterized by highest expression of oncosuppressors Csmd1, Dmbt1, Dusp1, and Gnmt, in DNs, and Bhmt, Dmbt1, Dusp1, Gadd45g, Gnmt, Napsa, Pp2ca, and Ptpn13 in HCCs of resistant rats. Integrated gene expression data revealed highest expression of proliferation-related CTGF, c-MYC, and PCNA, and lowest expression of BHMT, DMBT1, DUSP1, GADD45g, and GNMT, in more aggressive rat and human HCC. BHMT, DUSP1, and GADD45g expression predicted patients?? survival.

Conclusions

Our results disclose, for the first time, a major role of oncosuppressor genes as effectors of genetic resistance to hepatocarcinogenesis. Comparative functional genomic analysis allowed discovering an evolutionarily conserved gene expression signature discriminating HCC with different propensity to progression in rat and human.     

Clonal state of human hepatocellular carcinoma and non-tumorous hepatocytes

We determined the clonal state in specimens of hepatocellular carcinoma (HCC) and non-tumorous hepatocytes from the integration mode of hepatitis B virus (HBV) DNA. The integration mode of HBV DNA in several parts of the tumors and non-tumorous regions of the same liver, as well as in metastatic tumors, was examined using the Southern blot analysis. In 13 of the 14 cases of HCC, the liver tumors, including metastatic tumors in lymph nodes and the lungs, were monoclonal. In one case, a different HCC clone was found in one part of the liver tumor. The integration of HBV DNA was also observed in non-tumorous tissues in 38 of the 78 cases (49%) of chronic hepatitis with and without HCC; in 16 cases of chronic hepatitis in which HBV DNA was integrated, several clones of the hepatocytes that had HBV DNA integrated into their chromosomal DNA and had proliferated clonally were found in non-tumorous tissues. These clones were different from the tumor clone of the same liver. Thus HCCs were usually monoclonal. The development of different tumor clones appeared to be unusual, but the nontumorous hepatocytes could have proliferated clonally from different multicentric clones before carcinogenesis. The clonal growth of the non-tumorous hepatocytes suggests that the integration of HBV DNA plays an important role in hepatocarcinogenesis.     

HIV-1 viral protein R downregulates Ebp1 and stabilizes p53 in glioblastoma U87MG cells

Purpose

HIV-1 viral protein R (Vpr) inhibits cell growth and induces apoptosis in a wide range of cancers. However, the mechanism by which Vpr induces cell cycle arrest and apoptosis in GBM cell lines is unclear. The present work was taken to detect the proteins interacted with Vpr in U87MG cells.

Methods

We analyzed the differential expression of proteins between glioblastoma cell U87MG treated with Ad-Vpr and untreated by 2-DE. We used antibody array analysis to analyze the common molecules in the apoptosis of U87MG induced by Vpr.

Results

We analyzed the differential expression of proteins between U87MG cell treated with Ad-Vpr and untreated, and found that proteins related to DNA damage repair or different apoptosis pathways were involved in the G₂ arrest and apoptosis mediated by Vpr. In addition, proliferation-associated protein 2G4 (PA2G4), also known as Ebp1, was down-regulated and p53 was up-regulated in U87MG cells treated with Ad-Vpr.

Conclusions

Our data suggest that Vpr may inhibit Ebp1 to stabilize p53, which in turn leads to G₂ arrest and apoptosis in U87MG cells.     

Radiologic Complete Response with Sirolimus and Sorafenib in a Hepatocellular Carcinoma Patient who Relapsed After Orthotopic Liver Transplantation

Background

No standard therapies have been established for the treatment of recurrent hepatocellular carcinoma (HCC) after liver transplantation.

Discussion

Sirolimus is a mTOR inhibitor which has been used as an immunosuppressive medication in patients who are at high risk of tumor reoccurrence after liver transplantation. Sorafenib is a multikinase inhibitor approved for the treatment of advanced HCC. However the role of sorafenib in patients with HCC reoccurrence after liver transplantation is unclear.

Results

Combination of sirolimus and sorafenib appears to have synergistic effect when treating HCC in preclinical settings. We report a case of a post-liver transplant patient treated with sorafenib and sirolimus for hepatic HCC recurrence who exhibited complete radiologic response after 5 months of therapy.     

The emerging role of histone lysine demethylases in prostate cancer

Background

We had previously demonstrated overexpression of fibroblast growth factor receptor-4 (FGFR4) in hepatocellular carcinoma (HCC). However, additional molecular mechanisms resulting in amplified FGFR4 signaling in HCC remain under-studied. Here, we studied the mechanistic role of its co-receptor klotho-beta (KLB) in driving elevated FGFR4 activity in HCC progression.

Results

Quantitative real-time PCR analysis identified frequent elevation of KLB gene expression in HCC tumors relative to matched non-tumor tissue, with a more than two-fold increase correlating with development of multiple tumors in patients. KLB-silencing in Huh7 cells decreased cell proliferation and suppressed FGFR4 downstream signaling. While transient repression of KLB-FGFR4 signaling decreased protein expression of alpha-fetoprotein (AFP), a HCC diagnostic marker, prolonged inhibition enriched for resistant HCC cells exhibiting increased liver stemness.

Conclusions

Elevated KLB expression in HCC tissues provides further credence to the oncogenic role of increased FGFR4 signaling in HCC progression and represents a novel biomarker to identify additional patients amenable to anti-FGFR4 therapy. The restricted tissue expression profile of KLB, together with the anti-proliferative effect observed with KLB-silencing, also qualifies it as a specific and potent therapeutic target for HCC patients. The enrichment of a liver stem cell-like population in response to extended KLB-FGFR4 repression necessitates further investigation to target the development of drug resistance.     

Non-Viral Causes of Hepatocellular Carcinoma

Introduction

Hepatocellular carcinoma (HCC) is a major cause of cancer worldwide. The vast majority of cases occur in individuals with a chronic HBV or HCV infection. In addition, a number of metabolic diseases of the liver are associated with the development of HCC.

Pathophysiologic Mechanisms

The mechanisms responsible for the progression of the metabolic liver disease and HCC differ from those associated with viral liver disease.

Conclusions

The purpose of this report is to describe the mechanisms responsible for the disease progression and HCC in case of metabolic liver disease. A secondary goal is to identify the frequency of HCC development in the disorders described.