Significant correlation between expression level of HSP gp96 and progression of hepatitis B virus induced diseases

AIM: Gp96, also known as Grp94, is a member of heat shock protein (HSP) family and binds repertoires of peptides thereof eliciting peptide-specific T cell immune responses. It predominantly locates inside the endoplasmic reticulum (ER) with some cell surface expression in certain cancerous cells. Previous studies have shown that gp96 expression level was up-regulated in tumor cells, including hepatocellular carcinoma (HCC). However, relationship between the extent of gp96 expression and disease progression especially HBV-induced chronic infection, cirrhosis and hepatocellular carcinoma, has not been addressed before. As primary HCC can be induced and progressed from chronic hepatitis B virus (HBV) infection and HBV-induced cirrhosis, we designed an immunohistochemical experiment to test the correlation between gp96 expression level and HBV-induced disease progression, from chronic HBV infection, cirrhosis to HCC. METHODS: We chose liver samples from different patients of hepatitis B virus induced diseases, including chronic hepatitis B (77 patients), cirrhosis (27 patients) and primary HCC (30 patients), to test the expression level of gp96 in different affected groups. Formalin-fixed, and paraffin-embedded liver tissues taken from these patients were immuno-stained by using an anti-gp96 monoclonal antibody for the expression level of gp96 protein in the sections. In addition, Western blotting of whole cell lysates derived from established human embryonic liver cell lines and several human HCC cell lines (Huh7, HepG2, SSMC-7721) was compared with the expression of gp96. RESULTS: We found that the extent of elevated gp96 expression was significantly correlated with the disease progression, and was the highest in HCC patients, lowest in chronic HBV infection and was that of the cirrhosis in the middle. CONCLUSION: Increased expression of gp96 might be used as a diagnostic or prognostic bio-marker for the HBV infection and HBV-induced diseases.     

Quantification of hepatitis B virus covalently closed circular DNA in patients with hepatocellular carcinoma

BACKGROUND/AIMS: This study aimed to measure the intrahepatic total hepatitis B virus (HBV) DNA and covalently closed circular DNA (cccDNA) levels in tumor and non-tumor tissues in hepatocellular carcinoma (HCC) patients. METHODS: Intrahepatic total HBV DNA and cccDNA were measured in 25 HCC patients (21 hepatitis B surface antigen [HBsAg]-positive and 4 HBsAg-negative) by the Invader assay. RESULTS: A low level of intrahepatic HBV DNA was detectable in all HBsAg-negative patients. For HBsAg-positive patients, the intrahepatic total HBV DNA levels in the tumor and non-tumor tissues were comparable (P=0.903). However, the tumor tissues had significantly higher levels of cccDNA (0.35 vs. 0.16 copies/cell, P=0.030) and higher proportion of intrahepatic HBV DNA in the form of cccDNA (100% vs. 84%, P=0.004) than the non-tumor tissues. Seventeen out of 21 (81%) tumor tissues had intrahepatic HBV DNA solely in cccDNA form. Analysis of HBV mRNA expression indicated that HBV replication appeared to be lower in the tumor tissues than the non-tumor tissues. CONCLUSIONS: Compared to the non-tumor tissues, the levels of HBV replication in the tumor tissues appeared to be lower, and cccDNA was the predominant form of HBV DNA in the tumor tissues.     

Prognostic Value of HLA-G in Malignant Liver and Pancreas Lesions

Background: Human leukocyte antigen (HLA)-G is a nonclassical HLA class I molecule with modulatory effects on NK and T cells. Because HLA-G expression is frequently detected in different solid tumors, it may be involved in tumor immune evasion. Objective: This study was designed to elucidate the prognostic value of HLA-G in hepatocellular carcinoma (HCC) and pancreatic adenocarcinoma (PADC). The influence of hepatitis B virus (HBV) infection on HLA-G expression was also evaluated in patients with HCC. Methods: HLA-G expression was investigated in tumor tissues from patients with HCC (n=74) or PADC (n=42) with immunohistochemical techniques. The presence of HBV genome was also examined in HCC tumor tissues by PCR. Results: HLA-G expression was detected in 66% of PADC and in 31% of HCC samples. In contrast to HCC, HLA-G overexpression was associated with advanced stages and grades in PADC. HBV genome was detected in 31% of HCC samples but we found no correlation between HLA-G expression and the presence of HBV genome in these tumors. Conclusion: Our findings showed that HLA-G overexpression in tumor tissue correlated with poor prognosis in PADC. HLA-G expression is apparently affected by the patient’s genetic background and other epigenetic factors rather than by HBV infection.     

表面增强的激光解析电离飞行时间质谱技术在肝细胞癌血清学诊断中的应用

目的:检测HBV感染携带者与肝细胞癌(HCC)患者血清蛋白质的差异性表达,以发现HCC的肿瘤诊断标志物.方法:用表面增强的激光解析电离飞行时间质谱(SELDI-TOF-MS)技术检测27例HCC患者,27例HBV感染携带者,25例健康对照血清中的蛋白质谱,并用Biomarker Patterns System 5．0软件分析,建立诊断模型．结果:检测HCC患者与HBV感染携带者。正常对照与HCC患者,正常对照与HBV感染携带者的差异性蛋白分子,据此构建分类模型,得到的灵敏度和特异度分别为93％,85％; 96％,96％;84％,89％．其中相对分子质量为8141 Da的蛋白峰在HCC组明显高于HBV感染组(p<10-5);相对分子质量为3448 Da的蛋白峰在HCC及HBV感染携带组表达均较正常组显著增高(P<10-5),而在HCC-HBV组无明显差异(P>0．05),提示其可能为HBV感染的一种标志蛋白;相对分子质量为777) Da的蛋白峰在3组中均有差异性表达．结论:SELDI蛋白芯片技术检测血清蛋白质谱法诊断HCC具有较高的灵敏度和特异度,操作简单快捷,临床应用前景广阔,为HCC诊断提供了新的血清学方法．     

乙型肝炎病毒复制上调肝癌组织TGF-β1和IGF-Ⅱ的表达

目的分析肝癌组织HBV复制与转化生长因子（TGF）-β1和胰岛素样生长因子（IGF）-Ⅱ表达的关系。方法以自身对照法收集人肝细胞癌（HCC）组织及癌旁组织，以生物素标记的HBV—DNA探针检测肝癌组织中HBV—DNA，采用免疫组织化学方法检测组织中TGF-β1和IGF-Ⅱ的表达，分析TGF-β1和IGF-Ⅱ表达与HBV复制的临床病理学关系。结果肝癌组织中TGF-β1和IGF-Ⅱ均呈较高表达，其阳性率均为83．3％，肝癌组明显高于癌旁组（P〈0．01）。癌灶组TGF-β1和IGF-Ⅱ阳性表达与肿瘤分化程度显著相关（P〈0．05）；而与肿瘤直径、数目无关（P〉0．05）；TGF-β1和IGF-Ⅱ表达与HBV复制显著相关，且HBV—DNA阳性组显著高于HBV—DNA阴性组。结论肝癌组织中TG-β1和IGF—II过度表达，且与HBV复制和肝癌的分化程度有关。     

Expression of heat shock proteins （HSP27, HSP60, HSP70, HSP90,GRP78, GRP94） in hepatitis B virus-related hepatocellular carcinomas and dysplastic nodules

AIM: Expression of heat shock proteins (HSPs) is frequently up-regulated in hepatocellular carcinoma (HCC), which evolves from dysplastic nodule (DN) and early HCC to advanced HCC. However, little is known about the differential expression of HSPs in multistep hepatocarcinogenesis. It was the purpose of this study to monitor the expression of HSPs in multistep hepatocarcinogenesis and to evaluate their prognostic significance in hepatitis B virus (HBV)-related HCC. METHODS: Thirty-eight HCC and 19 DN samples were obtained from 52 hepatitis B surface antigen-positive Korean patients. Immunohistochemical and dot immunoblot analyses of HSP27, HSP60, HSP70, HSP90, glucose regulated protein (GRP)78, and GRP94 were performed and their expression at different stages of HCC development was statistically analyzed. RESULTS: Expression of HSP27, HSP70, HSP90, GRP78, and GRP94 increased along with the stepwise progression of hepatocarcinogenesis. Strong correlation was found only in GRP78 (Spearman's r= 0.802). There was a positive correlation between the expressions of GRP78, GRP94, HSP90, or HSP70 and prognostic factors of HCC. Specifically, the expression of GRP78, GRP94, or HSP90 was associated significantly with vascular invasion and intrahepatic metastasis. CONCLUSION: The expressions of HSPs are commonly up-regulated in HBV-related HCCs and GRP78 might play an important role in the stepwise progression of HBV-related hepatocarcinogenesis. GRP78, GRP94, and HSP90 may be important prognostic markers of HBV-related HCC, strongly suggesting vascular invasion and intrahepatic metastasis.     

Hepatitis B virus,alpha-fetoprotein synthesis,and hepatocellular carcinoma in Zaire

ABSTRACT— We investigated the epidemiology of hepatocellular carcinoma (HCC) in Zaire, and evaluated the association between exposure to hepatitis B virus (HBV) and the development of HCC. Two hundred and twenty-three consecutive cases of HCC diagnosed over 19 years (1966–1985) were reviewed. HCC represented 8.32% of all carcinomas and 5.56% of all cancers. Frequency was higher in males (75.7%) than in females (24.3%); a sex ratio of 3/1. The majority (82.1%o) of patients were aged 14 to 55 years with a peak occurrence in the fourth decade (28.6%). The mean age in males (41.27 ± 17.5 years) and females (37.40 ± 15.16 years) was significantly different (p<0.02). Sera from 40 patients and 68 age and sex-matched controls were analyzed for markers of HBV infection: patients and controls had comparable rates of exposure (96%) vs 72.1%, respectively). However, patients had significantly higher HBsAg carrier rates (56.7% vs 7.35%; p < 0.001), and lower anti-HBsAg seroconversion rates (25% vs 63.2%, p < 0.05). Using immunohistochemical analysis, the livers of patients were evaluated for HBsAg and HBcAg. These HBV antigens were more frequent in non-tumorous hepatocytes (53.3% vs 23.3%, respectively) than in HCC cells (13.3% vs 3.3%). Serum alpha-fetoprotein (AFP) was abnormal (>20 ng/ml) in 90%) of patients. The geometric mean (GM) AFP was 7273.8 ng/ml. AFP levels were significantly higher in HBsAg-positive HCC cases (GM: 19 322.6 ng/ml; 95% confidence interval (CI): [3639.2, 102 565.2]) than in antigen negative cases (GM: 1939.5 ng/ml; 95%) CI: [182.8, 19952.6]), but did not correlate with HBV replication. Immunohistochemical detection of AFP revealed a similar correlation between AFP and HBsAg. Neither AFP level nor HBsAg production correlated with cellular atypia or tumor grade.     

Hepatitis B virus, alpha-fetoprotein synthesis, and hepatocellular carcinoma in Zaire.

We investigated the epidemiology of hepatocellular carcinoma (HCC) in Zaire, and evaluated the association between exposure to hepatitis B virus (HBV) and the development of HCC. Two hundred and twenty-three consecutive cases of HCC diagnosed over 19 years (1966-1985) were reviewed. HCC represented 8.32% of all carcinomas and 5.56% of all cancers. Frequency was higher in males (75.7%) than in females (24.3%); a sex ratio of 3/1. The majority (82.1%) of patients were aged 14 to 55 years with a peak occurrence in the fourth decade (28.6%). The mean age in males (41.27 +/- 17.5 years) and females (37.40 +/- 15.16 years) was significantly different (p < 0.02). Sera from 40 patients and 68 age and sex-matched controls were analyzed for markers of HBV infection: patients and controls had comparable rates of exposure (96% vs 72.1%, respectively). However, patients had significantly higher HBsAg carrier rates (56.7% vs 7.35%; p < 0.001), and lower anti-HBsAg seroconversion rates (25% vs 63.2%, p < 0.05). Using immunohistochemical analysis, the livers of patients were evaluated for HBsAg and HBcAg. These HBV antigens were more frequent in non-tumourous hepatocytes (53.3% vs 23.3%, respectively) than in HCC cells (13.3% vs 3.3%). Serum alpha-fetoprotein (AFP) was abnormal (> 20 ng/ml) in 90% of patients. The geometric mean (GM) AFP was 7273.8 ng/ml. AFP levels were significantly higher in HBsAg-positive HCC cases (GM: 19,322.6 ng/ml; 95% confidence interval (CI): [3639.2, 102,565.2]) than in antigen negative cases (GM: 1939.5 ng/ml; 95% CI: [182.8, 19,952.6]), but did not correlate with HBV replication. Immunohistochemical detection of AFP revealed a similar correlation between AFP and HBsAg. Neither AFP level nor HBsAg production correlated with cellular atypia or tumor grade.     

Autophagy related protein 9A increase in hepatitis B virus-associated hepatocellular carcinoma and the role in apoptosis

The majority of hepatocellular carcinoma (HCC) cases are associated with the hepatitis B virus (HBV) infection. Autophagy related protein 9A (ATG9A) is a transmembrane protein required for autophagosome formation. In order to investigate the role of ATG9A in HBV-associated HCC, ATG9A protein expression was determined in tumor liver tissues and compared with adjacent nontumor tissues from HCC patients with or without HBV infection. In HBV-associated HCC tissues, ATG9A protein level was increased in tumor liver tissues, but not in cases of non-HBV HCC. Our findings suggested that ATG9A might be involved in HBV and cancer cell survival. Therefore, we aimed to analyze the function of ATG9A in HBV replication using RNA interference to evaluate the HBV DNA level using real-time PCR. In the present study, there were no significant differences between shATG9A-transfected HepG2.2.15 cells and the mock control. However, we found that silencing ATG9A affected apoptosis in HepG2.2.15 and HepG2 cell lines. Our results indicated that ATG9A might be partly involved in the survival of HCC. Thus, the inhibition of ATG9A together with other targets might be a potential drug target for HCC treatment.     

肝细胞癌热休克蛋白70过表达与自发性癌细胞凋亡

目的探讨肝细胞癌（hepatocellularcarcinoma,HCC)热休克蛋白70（heatshockproteins70,HSP70）的表达及其与自发性癌细胞凋亡的关系。方法采用免疫组化SP法和原位末端转移酶标记法（TUNEL）检测68例HCC手术标本中HSP70表达和细胞凋亡指数,以癌旁肝组织作为对照。结果HCC和癌旁组织中HSP70阳性率分别为71%和28%,过表达分别为53%和21%,两组之间差异有非常显著意义（P＜0.01),HSP70阳性率和凋亡指数有正相关关系（r＝0.8765,P＜0.05),两者都随HCC组织学分级增高而增加。结论HCC组织中存在HSP70过表达和自发性癌细胞凋亡,两者正相关性机理尚不明了,可能是HCC生物学恶性行为的标志。     

Overexpression of HBxAg in hepatocellular carcinoma and its relationship with Fas／FasL system

AIM: To study the expression and serum level of HBxAg,Fas and FasL in tissues of HCC patients, and to assess the relationship between HBxAg and Fas/FasL system.METHODS: Tissues from 50 patients with HCC were tested for the expression of HBxAg, Fas and FasL by S-P immunohistochemistry. Serum levels of sFas/sFasL and HBsAg/HBeAg were measured by ELISA assay. HBV X gene was detected by PCR in serum and confirmed by automatic sequencing. Fifty cases of liver cirrhosis and 30 normal controls were involved in serum analysis.RESULTS: The expression of HBxAg, Fas and FasL in carcinoma tissues was 96 %, 84 % and 98 %, respectively.Staining of HBxAg, Fas and FasL was observed predominately in cytoplasms, no significant difference was found in intensity between HBxAg, Fas and FasL (P＞0.05). HBxAg, Fas and FasL might express in the same area of carcinoma tissues and this co-expression could be found in most patients with HCC. The mean levels of sFas in serum from HCC, cirrhosis and normal controls were 762.29±391.56 μg@ L-1 835.36±407.33 μg@L-1 and 238.27±135.29 μg@L-1. The mean levels of sFasL in serum from HCC, cirrhosis and normal controls were 156.36±9.61iμg@ L-1, 173.63±18.74 μg@L-1 and 121.96±7.83 μg@ L-1.Statistical analysis showed that both sFas and sFasL in HCC and cirrhosis patients were significantly higher than those in normal controls (P＜0.01). Serum HBV X gene was found in 32 % of HCC patients and ,46 % of cirrhotic patients.There was no significant relationship between serum level of sFas/sFasL and serum X gene detection (P＞0.05). Eight percent of HCC patients with negative HBsAg and HBeAg in serum might have X gene in serum and HBxAg expression in carcinoma tissues.CONCLUSION: Our data suggest that HBxAg and Fas/FasL system plays an important role in the development of human HCC. Expression of HBxAg can leads to expression of Fas/FasL system which and reverse apoptosis of hepatocellular carcinoma induced by FasL.     

HBV cccDNA的水平及临床因素对肝细胞癌术后预后的判断价值

目的:研究HBV cccDNA水平及临床因素对肝细胞癌术后预后的影响.方法:回顾性分析2003-2006年我院收治60例术后病理证实原发性肝癌患者,采用荧光定量PCR检测乙型肝炎病毒(hepatitis B virus,HBV)共价闭合环状DNA(cccDNA)和HBV DNA;55例获得完整随访,选择血清HBV DNA、肝组织cccDNA及临床、病理特征等指标分析其对无瘤生存率、总体生存率的影响.采用Kaplan-Meier法计算无瘤生存率、总体生存率,Log-rank检验比较组间差异,多因素分析采用Cox回归模型.结果:血清cccDNA仅有1例阳性(1/35),肝癌组织cccDNA阳性率20.0%(11/55),肝癌组织cccDNA与血清HBV DNA之间存在相关性(r=0.364;P=0.006).全组1、3、5年总体生存率为73%、51%和38%,无瘤生存率为63%、29%和19%;多因素分析结果表明,肿块数目(P=0.011)、血管侵犯(P=0.001)是影响术后总体生存率的独立危险因素;癌组织cccDNA水平(P=0.007)、生长方式(P=0.002)是影响术后无瘤生存率的独立危险因素.结论:单发肿瘤、无血管侵犯的患者术后总体生存率较高.癌组织cccDNA水平<3log10copies/μg、肝癌膨胀性生长的患者术后无瘤生存率较高.     

转化生长因子bⅠ型受体表达与肝癌预后的关系

目的研究转化生长因子βⅠ型受体在人肝癌组织中的表达,探讨其临床意义。方法 以＾１２５Ｉ－ＴＧＦ－β１为配体的放射性配体结合分析法,检测了ＴβＲⅠ在３０例肝癌组织及癌周肝组织和４例正常对照肝组织中的表达。分析了肝细胞癌组织中ＴβＲⅠ相对表达率与肝癌的一些临床病量资料的关系。结果 肝癌组织ＴβＲⅠ表达较癌周肝组织及正常肝组织明显降低,后两者间ＴβＲⅠ表达一致。     

树Qu实验性肝癌发生过程p53基因的变化

目的探讨由人乙型肝炎病毒(HBV)和黄曲霉毒素B1(AFB1)诱发的树鼩肝细胞癌变过程,p53基因的表达及变化.方法将树鼩分为四组A组HBV+AFB1,B组只感染HBV;C组只摄入AFB1;D组作空白对照.定期肝活检,用免疫组织化学、分子生物学等技术对实验树鼩肝及肿瘤组织进行检测.结果 (1)接受HBV及AFB1双因素的A组,肝细胞癌(HCC)发生率(66.7%)明显高于只接受HBV的B组或AFB1的C组(30%),而且HCC的平均发生时间也明显早于C组,(120.0±16.6)周与(153.3±5.8)周,t=3.336,P＜0.01.(2)在第75周前各组动物肝均未检出突变的p53蛋白.(3)105周时,A组p53蛋白表达率为78.6%,B组为60%,C组为71.4%,D组为10%(x2≥5.03,P＜0.05).在A、C组检出p53基因异常带.(4)树鼩肝癌p53基因突变点分别位于2 7 5、7 8及1 3密码子;其野生型p 5 3基因的核苷酸及氨基酸序列与人的p 5 3基因的核苷酸及氨基酸序列的同源性分别为91.7%、93.4%.结论再次证实HBV和AFB1有协同致肝癌作用;突变的p53蛋白出现于肝细胞发生癌变之前,p 5 3基因的突变促进了肝癌的发生和演进.HBV可能协同AFB1致p 5 3基因突变.     

Transarterial chemo-lipiodolization can reactivate hepatitis B virus replication in patients with hepatocellular carcinoma

BACKGROUND/AIMS: Reactivation of hepatitis B virus (HBV) replication is a well-known complication in cancer patients receiving chemotherapy. The aims of this study were to determine the incidence of HBV reactivation in hepatocellular carcinoma (HCC) patients undergoing transarterial chemo-lipiodolization, and to clarify factors contributing to HBV reactivation. METHODS: From April 2001 to September 2002, 146 HBsAg positive patients newly diagnosed as HCC were enrolled in the study. Among these, 83 patients underwent transarterial chemo-lipiodolization using epirubicin and/or cisplatin, and 63 received other treatments. RESULTS: In total, HBV reactivation occurred in 30 (20.5%) patients (28 with chemo-lipiodolization and 2 with other treatments), and of the 30 patients, 19 (13.0%) (18 with chemo-lipiodolization and 1 with other treatments) developed hepatitis. Chemo-lipiodolization was significantly correlated with a higher incidence of hepatitis attributed to HBV reactivation than other treatments (21.7% vs. 1.6%, P<0.001), irrespective of HBeAg or HBV DNA. Among 83 patients undergoing chemo-lipiodolization, HBV reactivation occurred in 28 (33.7%) patients, and HBeAg seropositivity was the only independent predictor of HBV reactivation (P=0.013). Three (10.7%) of them died of hepatic decompensation resulting from HBV reactivation. CONCLUSIONS: Transarterial chemo-lipiodolization can reactivate HBV, and HBeAg-positive HCC patients receiving chemo-lipiodolization should be closely monitored for HBV reactivation.