Resveratrol helps recovery from fatty liver and protects against hepatocellular carcinoma induced by hepatitis B virus X protein in a mouse model

Resveratrol is a natural polyphenol that has beneficial effects across species and various disease models. Here, we investigate whether resveratrol is effective against hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) using HBV X protein (HBx) transgenic mice. We found that resveratrol (30 mg/kg/d) has a therapeutic effect on HBx-induced fatty liver and the early stages of liver damage. Resveratrol decreased intracellular reactive oxygen species and transiently stimulated hepatocyte proliferation. Interestingly, resveratrol inhibited LXRα and downregulated the expression of the lipogenic genes, Srebp1-c and PPARγ. The decrease in Srebp1-c seems to further downregulate the expression of its target genes, Acc and Fas. In addition, resveratrol stimulated the activity of Ampk and SirT1. Thus, resveratrol has a pleiotropic effect on HBx transgenic mice in terms of the downregulation of lipogenesis, the promotion of transient liver regeneration, and the stimulation of antioxidant activity. Furthermore, at the later precancerous stages, resveratrol delayed HBx-mediated hepatocarcinogenesis and reduced HCC incidence from 80% to 15%, a 5.3-fold reduction. Resveratrol should be considered as a potential chemopreventive agent for HBV-associated HCC.     

Downregulation of E-cadherin by hepatitis B virus X antigen in hepatocellullar carcinoma

Hepatitis B virus (HBV)-encoded X antigen (HBxAg) contributes to the development of hepatocellular carcinoma (HCC). A frequent characteristic of HCC is reduced or absent expression of the cell adhesion protein, E-cadherin, although it is not known whether HBxAg plays a role. To address this, the levels of E-cadherin were determined in HBxAg-positive and -negative HepG2 cells in culture, and in tumor and surrounding nontumor liver from a panel of HBV carriers. The results showed an inverse relationship between HBxAg and E-cadherin expression both in tissue culture and in vivo. In HBxAg-positive cells, E-cadherin was suppressed at both the mRNA and protein levels. This was associated with hypermethylation of the E-cadherin promoter. Depressed E-cadherin correlated with HBxAg trans-activation function, as did the migration of HepG2 cells in vitro. Decreased expression of E-cadherin was also associated with the accumulation of beta-catenin in the cytoplasm and/or nuclei in tissues and cell lines, which is characteristic of activated beta-catenin. Additional work showed that HBxAg-activated beta-catenin. Together, these results suggest that the HBxAg is associated with decreased expression of E-cadherin, accumulation of beta-catenin in the cytoplasm and nucleus, and increased cell migration, which may contribute importantly to hepatocarcinogenesis.     

Identification and characterization of a prevalent hepatitis B virus X protein mutant in Taiwanese patients with hepatocellular carcinoma

The aim of this study was to investigate whether there was a particular hepatitis B virus (HBV) X protein (HBx) mutant associated with Taiwanese patients with hepatocellular carcinoma (HCC). Initially, the entire coding region of HBx gene from the serum samples of 14 Taiwanese patients were sequenced. A novel mutant, HBx-A31, was preferentially found in patients with HCC. Sera from 67 patients with HCC and 100 patients with chronic hepatitis B were thus subjected for codon 31 analysis using a dual amplification created restriction site method. HBx-A31 was detected more frequently in patients with HCC (52% versus 12%; P<0.001) and in patients with liver cirrhosis (44% versus 6%; P<0.001). Site directed mutagenesis experiment revealed that HBx-A31 was less effective in transactivating HBV enhancer I-X promoter complex, less efficient in supporting HBV replication, and less potent in enhancing TNF-alpha induced increment of CPP32/caspase 3 activities in HepG2 cells. In conclusion, a prevalent HBx mutant was identified in Taiwanese patients with hepatocellular carcinoma. Development of this mutant might represent a strategy of the virus to escape immune surveillance and thus contribute to the process of multiple-step hepatocarcinogenesis.     

乙肝病毒X蛋白促进肝细胞癌侵袭转移相关分子的作用机制

乙肝病毒X蛋白(HBx)是乙型肝炎病毒编码的病毒蛋白,在介导肝细胞癌(HCC)转移中起重要作用.新近研究发现,HBx可通过介导肿瘤转移相关分子,如基质金属蛋白酶(MMP)、血管内皮生长因子(VEGF)及尿激酶纤溶酶原激活因子(uPA)等表达,进而活化转移相关信号传导通路,促进HCC转移.     

乙型肝炎病毒X蛋白与肝癌研究进展

X蛋白(HBx)被认为是乙型肝炎病毒(HBV)致原发性肝癌(HCC)的重要物质,具体机制不明,己成为研究的热点之一。近年来对HBx致肝癌机制的研究显示,HBx是一种多功能的病毒蛋白,有转录调控作用,对肝细胞内许多癌基因和/或抑癌基因的表达有直接或间接影响,可影响细胞的生存、增殖和凋亡,调节转录因子活性,促进细胞的侵袭和转移等,在肝癌形成中起重要作用。     

Functional analysis of transactivation by mutants of hepatitis B virus X gene in human hepatocellular carcinoma

The X protein of the hepatitis B virus transactivates various cellular and viral promoters and enhancers. In this study, wild-type and mutants of the X gene, including the point mutations at codons 130 (AAG-->ATG, lysin-->methionine) and 131 (GTC-->ATC, valine-->isoleucine), commonly found in patients with human hepatocellular carcinoma (HCC), or deletions encompassing the same region, were cloned and inserted into expression vectors. Functional analysis of the mutants of the X gene was performed on the long terminal repeat of the Rous sarcoma virus in a transient transfection assay. A transactivating function was observed in the vector containing point mutations at codons 130 and 131 at the same level as that of wild-type X gene. Two constructs, each containing a different type of 8-nucleotide deletion mutant (codons 128-130 or 130-132,) dramatically lost their transactivating function. These findings suggest that the transactivating function is not necessarily associated with the development of HCC, and that not only transactivation by the X gene but also the mutation-enhanced oncogenic potential of the gene products could contribute to hepatocarcinogenesis.     

The hepatitis B virus X protein promotes hepatocellular carcinoma metastasis by upregulation of matrix metalloproteinases

The hepatitis B virus (HBV) is a major cause of human hepatocellular carcinoma (HCC) which has a very high mortality rate due to high incidence of metastasis. It is unknown whether HBV contributes to HCC metastasis. In this report, we present clinical data obtained from HCC patients indicating that the expression of hepatitis B virus X protein (HBx) in HCC is associated with an increased expression of membrane-type 1 matrix metalloproteinase (MT1-MMP), and matrix metalloproteinase-2(MMP-2), which correlates with a poor prognosis. We further demonstrate experimentally that HBx upregulates MT1-MMP, which in turn induces MMP-2. Significantly, HBx-mediated MMP activation is associated with a marked increase of cell migration, as revealed by both wound-healing and transwell migration assays, suggesting that HBx may facilitate tumor cell invasion by upregulation of MMPs and subsequent destruction of the extracellular matrix. Together, our results support a model in which HBx contributes to HCC metastasis by upregulation of MMPs.     

Hepatitis B virus induced hepatocellular carcinoma

A number of risk factors appear to play a role in Hepatocellularcinoma (HCC), HBV infection being one of the most important. Chronic inflammation and cytokines are key determinants in the development of fibrosis and liver cell proliferation. HBV DNA integration and/or expression of HBV proteins may have a direct effect on cellular functions. Occult hepatitis B virus infection is characterized by persistence of HBV DNA in hepatitis B surface antigen-negative individuals. There are evidences that occult HBV is a risk factor for the development of HCC and that the potential mechanisms whereby overt HBV might induce tumour formation are mostly maintained.     

X protein mutations in hepatitis B virus DNA predict postoperative survival in hepatocellular carcinoma

Hepatitis B virus (HBV) DNA is prone to mutations because of the proofreading deficiencies of HBV polymerase. The postoperative prognostic value of HBV mutations in HBV X protein (HBx) gene was assessed in HBV associated hepatocellular carcinoma (HCC) patients. The HBx gene was amplified and sequenced, the HBV mutations was identified according to NCBI database (http://www.ncbi.nlm.nih.gov/genome/5536). The relationship between the HBV mutations and HCC survival was compared. Survival curves were generated using the Kaplan–Meier method, and comparisons between the curves were made using the log-rank test. Multivariate survival analysis was performed using a Cox proportional hazards model. After adjusting for clinical characteristics, the following eight mutational sites were identified as statistically significant independent predictors of HCC survival: 1383, 1461, 1485, 1544, 1613, 1653, 1719, and 1753. In addition, the following four mutational sites were identified for their association with survival at a border-line significance level: 1527, 1637, 1674, and 1762/1764. A total of 12 mutations in HBx gene region were identified as independent predictors of postoperative survival in HCC patients. The analysis of HBV DNA mutations may help identify patient subgroups with poor prognosis and may help refine therapeutic decisions regarding HCC patients.     

Integration of hepatitis B virus DNA in hepatocellular carcinoma

Integration of hepatitis B virus (HBV) DNA into genomic DNA was investigated in 34 livers bearing hepatocellular carcinoma (HCC) by Southern blot hybridization using 32P-labeled, cloned and purified HBV DNA as a probe. Rehybridization of nitrocellulose paper with a probe containing only the cloning vector was performed after dehybridization to avoid possible false-positive results. Integrated HBV DNA was detected in all 9 hepatitis B surface antigen (HBsAg)-seropositive cases and 3 out of 25 (12%) HBsAg seronegative cases. The hybridization patterns of viral DNA were the same among several cancer nodules in two HCC cases with multiple liver tumors, indicating unicentric hepatocarcinogenesis in these two cases. These results, obtained with avoidance of false-positive results, showed that only a minority of HBsAg-seronegative HCC cases in Japan had demonstrable HBV DNA in the tumors studied by the Southern blot hybridization technique.     

Does hepatitis B virus cause hepatocellular carcinoma?

Evaluation of the hypothesis of an association between hepatitis and hepatocellular carcinoma (HCC), first suspected on pathologic grounds, was made easier by the discovery of hepatitis B surface antigen. Population correlation and analytical epidemiologic studies established that there is a strong and specific association between hepatitis B virus (HBV) and HCC. The association is restricted to chronically active forms of HBV infection and is universally present, equally strong in the USA and Europe as in Africa and Asia. The causal nature of the association between HBV and HCC appears indisputable, even though the pathogenesis of HBV-related HCC has not been established. There is no evidence to support any of the non-causal interpretations, i.e., that a third factor causes both persistence of HBV and HCC, or that HCC increases susceptibility to the HBV carrier state. Since about 200 million people are hepatitis B surface antigen carriers, HBV would appear to be second only to cigarette smoking as the most important known human carcinogen. However, a substantial proportion of HCC cases throughout the world show no evidence of active HBV infection; for those cases other causal agents must be invoked. Aflatoxin may be an important etiologic factor in Africa and Asia, whereas in Europe and the USA cigarette smoking and alcohol drinking are serious and numerically important suspects for hepatocellular carcinogenicity. Several other factors, including natural and synthetic chemical carcinogens, infectious agents and steroid hormones are also suspected, but the supporting evidence is weak and the numerical importance of the factors probably limited.     

肝细胞癌患者B和C基因型乙型肝炎病毒X蛋白的变异特点

背景与目的：X蛋白是乙型肝炎病毒(hepatitis B virus,HBV)最重要的致病因子之一,它在HBV相关性肝细胞癌(hepatocellular carcinoma,HCC)的发生发展中起着重要作用。目前已知B、C基因型HBV相关性HCC的临床及病理表现不尽相同,但目前尚不清楚这种差别是否与B、C基因型HBV X基因之间的差别有关。因此本实验拟研究B、C基因型间HBV X蛋白氨基酸差异及其在HCC患者中的变异及特点,初步探讨其与HCC发生、发展的关系。方法：PCR扩增22份乙型肝炎病毒表面抗原(HBsAg)阳性HCC患者血清HBV X基因,克隆、测序并以Vector NTI6．0软件分析其基因型。以DNAMAN软件对标准HBV及HCC来源的HBV X蛋白进行氨基酸同源分析。结果：检测的22个HBV X基因片段均属于B或C基因型。B、C基因型HBV X蛋白之间存在14个氨基酸的差异,HCC患者来源的B、C型HBV X蛋白存在4个氨基酸的共有变异,C型HBV X蛋白尚有6个型特异性变异。这些差异或变异氨基酸均位于X蛋白B、T细胞表位或反式激活区及调节区内。结论：B、C基因型HBV X蛋白之间存在氨基酸差异,且在HCC中发生基因型特异性变异,这些差异或变异氨基酸可能导致X蛋白免疫学功能及反式激活功能的不同。     

Decreased expression of Bid in human hepatocellular carcinoma is related to hepatitis B virus X protein

As a mitochondrial membrane death ligand, Bid oligomerises Bak to release cytochrome C and its deficiency renders hepatocytes resistant to apoptosis induced by Fas. The Bid level in hepatocellular carcinoma (HCC) is unknown. In this report, we examined the expression of Bid protein and mRNA in HCC cancerous tissues and their corresponding non-cancerous ones. The effect of the hepatitis B x protein (HBx) on the expression of Bid was also evaluated by transfecting hepatoma cells with the HBx gene. The results showed that the expression of Bid was significantly lower in cancerous tissues than that in their corresponding non-cancerous tissues. Immunohistochemical study revealed that Bid molecule was mainly localised in hepato-cytoplasm. Some nuclei were also positive for Bid antigen though to a lesser degree. In vitro experiments demonstrated that the expression of Bid in cells transfected with HBx was significantly lower than that in the cells without HBx transfection. This finding suggests that HBx may play a causative role in the reduction of Bid expression in HCC. This in vitro result is, to some degree, supported by clinical data that all the HCC examined are positive for hepatitis B virus (HBV). We conclude from this data that the expression of Bid in HCC is significantly decreased and the reduction of Bid may result from a mechanism associated with HBx, a major hepatocarcinogenic product from HBV. The imbalance of increased anti-apoptosis and decreased pro-apoptosis seen in HCC is a critical mechanism leading to the uncontrolled growth of tumour cells. Therefore, this study suggests that a deficiency in the expression of Bid may contribute to the development of such an imbalance in HCC.     

Maternal transmission of hepatitis B virus in childhood hepatocellular carcinoma

Fifty-one children, aged between 3 and 16 years, were diagnosed to have hepatocellular carcinoma (HCC) in the last 15 years. Serum hepatitis B surface antigen (HBsAg) was positive in all the latter 34 HCC children checked by radioimmunoassay, and was positive in five of the 17 earlier HCC children studied by double immunodiffusion method. Serum HBsAg was studied in 31 mothers of the latter 33 HCC children, including two pairs of affected siblings and was positive in 29 (94%). The positive rate was much higher than that (50%) of the mothers of control HBsAg carrier children (P less than 0.001). The serum HBsAg positive rate was also higher in the siblings of HCC children than that of the control group. On the contrary, the serum HBsAg positive rate was not different between the fathers of HCC children and that of the control group. Our observation demonstrated that transmission of hepatitis B virus from the mothers during the perinatal period or early childhood is the most important mode of hepatitis B virus infection in HCC children in Taiwan.     

乙型肝炎病毒X蛋白氨基端变异与肝癌发生的相关性分析

目的:探讨乙型肝炎病毒X蛋白(hepatitis B virus X protein, HBx)氨基端1～90位氨基酸(amino acid, AA)变异与肝细胞癌发生之间的关系.方法:采用PCR扩增产物直接测序的方法,对48例肝癌组织、159例肝癌和144例慢性肝炎患者血清中HBV X基因进行序列分析.通过病例-对照研究HBx变异与肝癌发生之间的关系.结果: 肝癌组织中HBx氨基端变异集中于1～49 AA区域,突变率为4.1%; 50～90 AA为保守区,突变率仅为0.46%. 血清检测结果显示,HBx氨基端突变在肝癌患者中的发生率显著高于肝炎患者(2.2% vs 1.8%,P<0.05); HBx第36位Ala/Thr/Pro→Ser的突变,在肝癌患者中的发生率为10.1%,显著高于肝炎患者中的2.1%[P<0.01,比数比(odds ratio,OR)=5.259, 95%可信区间为1.499～18.444)].A/T/P36S突变仅发生于C基因型病毒.结论:HBx 氨基端突变的累积可能与肝癌的发生有关.HBx A/T/P36S突变能增加C基因型HBV感染者发生肝癌的危险度.     

Childhood primary hepatocellular carcinoma and hepatitis B virus infection

In the 7-year period between 1980 and 1987, six cases of childhood primary hepatocellular carcinoma (PHC) were confirmed histologically in our institution. Hepatitis B surface antigen (HBsAg) seropositivity was confirmed in five of the cases, and tissue HBsAg was shown in four of these using the Shikata's orcein stain. An associated maternal HBsAg seropositivity was shown in two of the seropositive children. The youngest seropositive patient who developed PHC was 7 years old. The mother of this patient was also seropositive. These observations support a causal relation between childhood Hepatitis B virus infection and PHC. The importance of vertical or perinatal transmission of HBV in the causation of childhood PHC and the prophylactic role of childhood vaccination is emphasized. Attention is also drawn to the relative short malignant transformation time seen in some of these patients.