Decreased expression of type Ⅱ tumor suppressor gene RARRES3 in tissues of hepatocellular carcinoma and cholangiocarcinoma

AIM: To analyze the expression of retinoic acid receptor responder 3 (RARRES3) protein in paraffin-embedded tissues of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), and the correlation of RARRES3 production with tumor differentiation. METHODS: Expression of RARRES3 in tissues from 21 CC (10 well-, 7 moderately- and 4 poorly-differentiated) and 32 HCC was determined by immunohistochemistry. RESULTS: Among 21 CC tissues, RARRES3 was detected in 8 (80%) of 10 well-differentiated tumors. Only 2 (18.2%) out of 11 tumors with moderate or poor differentiationshowed positive RARRES3 expression. RARRES3 expression in well-differentiated CC was significantly higher than that in tumors with moderate or poor differentiation (Fisher exact test, P<0.01). Expression of RARRES3 was not different between early (Ⅰ and Ⅱ) and late (Ⅲ and Ⅳ) stages of CC. Among 30 HCC tissues, 17 (56.7%) weakly expressed RARRES3 in HCC cells, and 25 (83.3%) normal tissues adjacent to HCC expressed the protein. RARRES3 expression was significantly decreased in HCC tissues compared to that in adjacent normal tissues (logistic regression analysis, OR = 0.27, 95% Cl (0.11-0.62), P<0.01). CONCLUSION: Expression of RARRES3 is positively correlated to well-differentiated CC, which supports the role of RARRES3 in malignant epithelial differentiation of the tumor. The decrease in RARRES3 expression in tissues of HCC and CC with moderate and poor differentiation suggests that altered RARRES3 expression may play a role in the carcinogenesis of the liver and biliary tract.     

Expression of p53and C—myc genes and its clinical relevance in the hepatocellular carcinomatous and pericarcinomatous tissues

AIM：To investigate the possible roles of p53and C-myc genes in the primary hepatocellular carciogenesis and the relationship between the liver hyperplastic nodule(LHN)and hepatocellular carcinoma(HCC).METHODS:The expression of p53and C-myc genes was detcted immunohist-ochemically in 73and 60cases of HCCand pericarcinomatous tissues,respectively.RESULTS:The positive expression of p53in HCCwas significantly higher than that in pericarcinomatous tissues(P<0.050.In pericarcinomatous tissues,the p53 expression was observed onlyin LHN,but not in liver cirrhosis(LC)and normal liver tissues.The positive expression rate of C-myc in HCC or LHN was significantly higher than that in LCor normal liver tissues(P<0.05and P<0.01).however,no significant difference was found between HCCand LHN(P>0.05).The positive expression rate of p53and C-myc in HCCwas correlated with the histological differentiation,that in the poorly6 differentiated was significantly higher than that in well differentiated samples(P<0.05).CONCLUSION:The overexpression of p53and C-myc genes might play a orle in the carcinogenesis of HCC;And LHN seems a preneoplastic lesion related to hepatocarcinogenesis.No evidence supports that LC contribute directly to the hepatocarcinogenesis.     

Relationship between inducible nitric oxide synthase expression and angiogenesis in primary gallbladder carcinoma tissue

AIM:To explore the relationship between angiogenesis and biological behaviors of primary gallbladder carcinoma (PGBC),the relationship between the expression of inducible nitric oxide synthase (iNOS) and biological behaviors of PGBC and its relationship with the expression of iNOS and angiogenesis of PGBC.METHODS: The expression of iNOS and micro-vessel density (MVD) were assessed by immunohistochemical method and image analysis system in 40 specimens of PGBC and in 8 specimens of normal gallbladder. The immunostaining results and related clinicopathologic materials were analyzed by statistical methods.RESULTS: MVD in PGBC was significantly higher than that in normal gallbladder tissue (46±14 vs 14±6, P<0.05), and was not related with age, gender, tumor size and histological type. MVD of poorly and undifferentiated tumor tissues was higher than that of moderately-differentiated and well-differentiated tumor tissues (52±9 vs43±9 vs33±6, P<0.01).MVD of Nevin IV and V stages was higher than that of NevinI, Ⅱ and Ⅲ stages (52±8 vs 37±13, P<0.01). MVD of cases with lymphatic or liver metastasis was significantly higher than that without liver metastasis (55±6 vs 42±10, P<0.05) or lymphatic metastasis (53±8 vs38±8, P<0.01). The positive level index (PLI) of iNOS in PGBC was 0.435±0.134, and was not related with age, gender, tumor size, histological type,differentiation and clinical stage of PGBC. The PLI of iNOS in cases with lymphatic metastasis was higher than that without lymphatic metastasis (0.573±0.078 vs 0.367±0.064,P<0.01). The PLI of iNOS in cases with liver metastasis was higher than that without liver metastasis (0.533±0.067 vs0.424±0.084, P<0.05). There was a significant correlation between PLI of iNOS and MVD in PGBC (P<0.05).CONCLUSION:Angiogenesis of PGBC is significantly related to the biological behaviors of PGBC. The expression of iNOS is related to the biological behaviors of PGBC. The detection of MVD and the expression of iNOS in PGBC can be used as parameters to determine the degree of malignancy and prognosis.     

Overexpression of p28／gankyrin in human hepatocellular carcinoma and its clinical significance

AIM：To investigate the expression of p28/gankyringene and its role in the carcinogenetic process of human hepatocellular carcinoma(HCC).METHODS:64specimens of HCC and para-carcinoma tissues,22specimens of non-tumor liver tissues(7normal,15cirrhosis),10 specimens of normal human tissues and 5hepatoma cell lines were studied for the expression of p28/gankyrinby Northern blot.The expressionof p28/gankyrin protein was detected immunohistochemically by using the specific polyclonal antibody.RESULTS:Northern blot analysis indicated that the expression of p28/gankyrinmRNA was intensively distributed in brain and heart,weakly in lung,spleen and muscle,undetectable in digestive system including liver,pancreas,stomach,small and large intestines.p28/gankyrinmRNAwas absent in normal liver,weakly detected in liver cirrhosis and in 18of 64para-carcinoma liver tissues.In contrast,the expression of p28/gankyrinmRNA was intensitely detected in all5hepatoma cell lines tested,markedly increased in 57of 64and moderately increased in 5of64HCCsamples.In comparison with liver cirrhosis and para-carcinoma liver tissues.the average expression of p28/gankyrinmRNAin HCCwas increased3.6-(2.901±0.507vs0.805±0.252,P<0.05)and5.2-fold(2.901±0.507vs0.557±0.203,p<0.01),respectively.In addition,p28/gankyrinmRNA expression level was higher in HCCwith portal vein tumor thrombus and microscopic hepatic vein involvement(P=0.021and P=0.047,respectively).Theoverexpression of p28/gankyrin protein in HCCwas targeted in hepatic tumor cells,not in bile duct cells and other interstitial cells.Plays an important role and contributes to the metastasis potential in the process of carcinogenesis.p28/gankyrinmay become a specific biological tissue marker for the pathological diagnosis of HCC.     

Expression and clinical significance of TAp73α, p53, PCNA and apoptosis in hepatocellular carcinoma

AIM: To study the prognostic role of TAp73α, p53,proliferating cell nuclear antigen (PCNA) and apoptosis in patients with hepatocellular carcinoma (HCC) after surgical tumor ablation.METHODS: Forty-seven human resected HCC tissues and 42 adjacent non-cancerous tissues were studied with 10 normal liver tissues as control group. TAp73α, p53, and PCNA were detected with Elivision immunohistochemistry.Terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick-end labeling (TUNEL) method was used to detect the apoptosis cells. All clinical and pathological materials were analyzed by SPSS10.0statistical package.RESULTS: TAp73α overexpressed in HCC tissues (36.2%)when compared with adjacent non-cancerous tissues(2.38%, P<0.005) and normal liver tissues (0, P<0.01).Mutant type p53 (rot-p53) overexpressed in HCC tissues(38.3%) when contracted with adjacent non-cancerous tissues (16.7%, P<0.05) and normal liver tissues (0,P<0.01). Proliferation index (PI) level in HCC tissues was significantly higher than that in adjacent non-cancerous tissues (30.34%±4.46% vs27.88%±5.89%, t, P= 0.028).Apoptosis index (AI) level in HCC tissues was higher than that in adjacent non-cancerous tissues (8.62%±2.28%vs7.38%±2.61%, t, P = 0.019). Expression of TAp73α was associated with lymph node metastasis and rot-p53,with r = 0.407 and 0.265, respectively. Expression of rot-p53 was associated with Edmondson‘s stage and AFP,with r = 0.295 and -0.357, respectively. In Kaplan-Meier univariant analysis, TAp73α, AFP, TNM stage, portal vein invasion, liver membrane invasion and HBsAg correlated with prognosis (log rank, P= 0.039, 0.012, 0.002, 0.000,0.014, 0.007, respectively). Multivariant Cox regression analysis showed that TAp73α, AFP, TNM stage, portalve in invasion, liver membrane invasion and age were independent factors of prognosis.CONCLUSION: These results suggest that TAp73α can be used as a prognostic indicator of patients with HCC undergoing surgical tumor ablation. AFP, TNM, portal vein invasion, liver membrane invasion and age also have a potency of predicting the prognosis of HCC.     

Expression of TIMP-1 and TIMP-2 in rats with hepatic fibrosis

AIM: To investigate the location and expression of TIMP-1 and TIMP-2 in the liver of normal and experimental hepatic fibrosis in rats. METHODS: The rat models of experimental immunity hepatic fibrosis (n=20) were prepared by the means of immunologic attacking with human serum albumin (HSA),and normal rats (n=10) served as control group. Both immunohistochemistry and in situ hybridization methods were respectively used to detect the TIMP-1 and TIMP-2 mRNA and related antigens in liver. The liver tissue was detected to find out the gene expression of TIMP-1 and TIMP-2 with RT-PCR. RESULTS: The TIMP-1 and TIMP-2 related antigens in livers of experimental group were expressed in myofibroblasts and fibroblasts (TIMP-1: 482±65 vs 60±20; TIMP-2:336±48 vs 50±19, P<0.001). This was the most obvious in portal area and fibrous septum. The positive signals were located in cytoplasm, not in nucleus. Such distribution and location were confirmed bysitu hybridization (TIMP-1/β-actin: 1.86±0.47 vs 0.36±0.08; TIMP-2/β-actin: 1.06±0.22 vs 0.36±0.08,P<0.001). The expression of TIMP-1 and TIMP-2 was seen in the liver of normal rats, but the expression level was very low. However, the expression of TIMP-1 and TIMP-2 in the liver of experimental group was obviously high. CONCLUSION: In the process of hepatic fibrosis, fibroblasts and myofibroblasts are the major cells that express TIMPs.The more serious the hepatic fibrosis is in the injured liver,the higher the level of TIMP-1 and TIMP-2 gene expression.     

Expression of transforming growth factor-alpha and hepatitis B surface antigen in human hepatocellular carcinoma tissues and its significance

AIM:To evaluate the expression of transforming growthfactor-alpha (TGF-α) and hepatitis B surface antigen (HBsAg) in human hepatocellular carcinoma (HCC) tissues and its significance.METHODS:Seventy specimens of HCC tissues were detected by immunohistochemical method. Five specimens of normal human liver tissues were used as control.RESULTS: The TGF-o~ positive expression rates in HCC and its surrounding tissues were 74.3%(52/70) and 88.1%(52/59), respectively. TGF-α positive granules were mainly in the cytoplasm and fewer existed on the karyotheca. The TGF-α positive expressing rate in well differentiated HCC was significantly higher than that in moderately and poorly differentiated HCC (P<0.05).The TGF-α positive expression also was observed in intrahepatic bile ducts (part of those were hyperplastic ducts).The HBsAg positive expression rates in HCC and its surrounding tissues were 21.4%(15/70) and 79.7%(47/59), respectively.HBsAg positive granules were in the cytoplasm, inclusion and on the karyotheca.There was a prominent positive correlation between TGF-α and HBsAg expression in HCC surrounding tissues (P<0.05,γ=0.34). TGF-α was usually existed with HBsAg in regenerated and/or dysplastic liver cells.In the five normal liver tissues, TGF-α and HBsAg were not detectable in hepatocytes and bile ducts.CONCLUSION:Hepatitis B virus infection is dosely related with hepatocarcinogenesis.The overexpression of TGF-α in the liver seems to be associated with the regeneration of hepatocytes injured by HBsAg.The continued expression of TGF-α might lead to dysplasia of liver cells and development of HCC. Furthermore, TGF-α might play a role in morphogenesis and regeneration of intrahepatic bile ducts.     

Levels of plasma des-γ-carboxy protein C and prothrombin in patients with liver diseases

AIM: To study the plasma des-γ-carboxy protein C activity, antigen and prothrombin levels in patients with liver diseases and their clinical significance. METHODS: Plasma protein C activity (PC:C) was detected by chromogenic assay and antigen (PC:Ag) and des-γ-carboxy protein C (DCPC) were detected by ELISA. Total prothrombin and unabsorbed prothrombin in plasma were detected by ecarin chromogenic assay. RESULTS: Compared with the control, the levels of PC:C and PC:Ag in patients with hepatocellular carcinoma (HCC) and liver cirrhosis (LC) were lower (PCC: 104.65±23.0%,62.50±24.89%, 56.75±20.14%, PC:Ag: 5.31±1.63 μg/mL, 2.28±1.15 μg/mL, 2.43±0.79 μg/mL, P<0.05). The levels of PC:Ag in patients with acute viral hepatitis (AVH) also was lower (2.98±0.91 μg/mL, P<0.01), but PC:C was close to the control (93.76±30.49%, P>0.05). The levels of DCPC in patients with HCC were remarkably higher (0.69±0.29 μg/mL,1.18±0.63 μg/mL, 0.45±0.21 μg/mL, P<0.05) and its averagewas up to 50% of total PC:Ag. But those of DCPC in patients with AVH were not significantly different from the control. The levels of total prothrombin were lower in patients with LC, but higher in patients with HCC. The levels of unabsorbed prothrombin were predominantly higher than those of other groups. CONCLUSION: PC:C and PC:Ag in patients with liverdiseases (except PC:C in AVH) were lower. The total prothrombin was lower in patients with LC. The higher level of unabsorbed prothrombin may be used as a scanning marker for HCC. DCPC may be used as a complementary marker in the diagnosis of HCC.     

原发性肝细胞癌及癌旁组织中环氧合酶-2的表达及其意义

目的：研究人原发性肝细胞癌（HCC）组织和癌旁非瘤组织中的环氧合酶－2（COX－2）蛋白及基因表达情况,方法：采用免疫组织化法和原位分子杂交法,研究27对原发性肝细胞癌及癌旁非肿瘤组织,5例正常肝组织中COX－2的蛋白和基因表达,结果：高分化HCC中COX－2蛋白表达显著高于中分化和低分化HCC（P分别＜0．05）以及癌旁组织和正常组织（P分别＜0．01）,癌旁组织的COX－2表达显著高于正常组织（P＜0．05）,癌旁组织,中分化和低分化HCC之间COX－2的表达强度差异无显著性（P＞0．05）,在COX－2蛋白阳性的肝癌细胞和癌旁肝细胞胸胞胞质中可见到COX－2mRNA呈阳性表达,结论：COX－2的过度表达可能参与了高分子HCC的致癌过程。     

Expression of cyclooxygenase-2 in human hepatocellular carcinoma: relevance to tumor dedifferentiation

Cyclooxygenase (COX) is a key enzyme in the synthesis of prostanoids. Two isoforms of this enzyme have been identified: COX-1 and COX-2. Recent studies have suggested that COX-2, but not COX-1, may play a role in colorectal tumorigenesis. In the present study, we investigated the expression of COX-2 as well as COX-1 in human hepatocellular carcinoma (HCC) tissues using immunohistochemistry and immunoblotting. Forty-four surgically resected HCC tissues with adjacent nontumorous livers (NTs), involving 17 cases of chronic viral hepatitis and 27 cases of cirrhosis, and 7 surgically resected, histologically normal liver tissues were used. The well-differentiated HCC expressed COX-2 more frequently and strongly than less-differentiated HCC or hepatocytes of NTs. Less-differentiated HCCs expressed less COX-2 than hepatocytes of NTs, which showed scattered, strong COX-2 expression. Histologically normal liver was weakly positive for COX-2. The expression of COX-1 was weaker than that of COX-2 in hepatic neoplastic and non-neoplastic parenchymal cells. An enhanced expression of COX-1 was not observed in well-differentiated HCCs. Immunoblotting also confirmed up-regulation of COX-2, but not COX-1, in well-differentiated HCCs. The present study is the first to demonstrate a high expression of COX-2 in well-differentiated HCC and a low expression in advanced HCC, in contrast to its continuous expression during colorectal carcinogenesis. These findings suggested that COX-2 may play a role in the early stages of hepatocarcinogenesis, but not in the advanced stages, and may consequently be related to HCC dedifferentiation.     

Pathomorphological study on location and distribution of Kupffer cells in hepatocellular carcinoma

AIM: To clarify the location and distribution of Kupffer cells in hepatocellular carcinoma (HCC), and to investigate their role in hepatocarcinogenesis.METHODS: Kupffer cells were immunohistochemically stained by streptavadin-peroxidase conjugated method (S-P). The numbers of Kupffer cells in cancerous, para-cancerous and adjacent normal liver tissues of 48 HCCs were comparatively examined.RESULTS: The mean number of Kupffer cells in cancerous,para-cancerous and adjacent normal liver tissues was 12.7±6.8, 18.1±8.2 and 18.9±7.9 respectively. The number of Kuppfer cells in cancerous tissues was significantly lower than that in para-cancerous tissues (t=2.423, P＜0.05) and adjacent normal liver tissues (t=2.52t, P＜0.05). As tumor size increased, the number of Kupffer cells in cancerous tissues significantly decreased (F=4.61, P＜0.05). Moreover,there was also a significant difference in the number of Kupffer cells among well-differentiated, moderately-differentiated and poorly-differentiated cases(F＝4.49, P＜0.05).CONCLUSION: This study suggests that decrease of Kupffer cells in HCCs may play an important role in the carcinogenesis of HCC, the number of Kupffer cells in HCC is closely related to the size and differentiation grade of the tumor.     

Expression of microsomal prostaglandin E synthase-1 in human hepatocelluar carcinoma.

BACKGROUND/AIMS: The objective of this study was to evaluate the expression of microsomal prostaglandin E synthase-1 (mPGES-1) in hepatocellular carcinoma (HCC) tissues. METHODS: Forty surgically resected HCC tissues with adjacent non-tumorous liver tissues and 14 surgically resected, histologically normal liver tissues were used. The immunohistochemical expressions of the mPGES-1 protein in these HCC tissues and normal control livers were analysed. mPGES-1 mRNA expression was also analysed by the real-time polymerase chain reaction method using the same tissues. RESULTS: Microsomal prostaglandin E synthase-1 was not expressed in hepatocytes but instead in vascular endothelial cells and bile duct epithelial cells in normal liver tissues. The mPGES-1 expression in HCC tissues was significantly greater than its expression in the non-tumorous tissues. All types of HCC expressed more mPGES-1 than normal or hepatitis livers, and the levels of mPGES-1 expression in poorly differentiated HCC were similar to the levels in well-differentiated HCC. The mPGES-1 mRNA expression paralleled its protein expression in these tumorous and non-tumorous tissues. CONCLUSIONS: The present study is the first to demonstrate a high expression of mPGES-1 in well-differentiated HCC as well as in poorly differentiated HCC. These findings suggest that mPGES-1 may play a role in the advanced as well as early stage of hepatocarcinogenesis.     

Abnormal beta-catenin gene expression with invasiveness of primary hepatocellular carcinoma in China

AIM: To study the abnormal expression of beta-catenin gene and its relationship ith invasiveness of primary hepatocellular carcinoma among Chinese people. METHODS: Thirty-four hepatocellular carcinoma (HCC) specimens and adjacent para-cancerous tissues, 4 normal liver tissues were immunohistochemically stained to study subcellular distribution of beta-catenin. Semiquantitive analysis of expression of beta-catenin gene exon 3 mRNA was examined by RT-PCR and in situ hybridization. The relationship between expressions of both beta-catenin protein, mRNA and clinicopathological characteristics of HCC was also analyzed. RESULTS: Immuno-histochemistry showed that all normal liver tissues and para-cancerous tissues examined displayed membranous type staining for beta-catenin protein, occasionally with weak expression in the cytoplasm. While 21 cases (61.8%) of HCC examined showed accumulated type in cytoplasms or nuclei. The accumulated type Labling Index (LI) of cancer tissue and para-cancerous tissue was (59.9 +/- 26.3) and (18.3 +/- 9.7) respectively (P<0.01). Higher accumulated type LI was closely related with invasiveness of HCC. Results of RT-PCR showed the beta-catenin gene exon 3 mRNA Expression Index (EI) of 34 HCCs was higher than that of para-cancerous tissue and normal liver tissue. Using in situ hybridization, the signal corresponding to beta-catenin gene exon 3 mRNA was particularly strong in cytoplasm of HCC when compared with those of para-cancerous and normal liver tissues. Over expression of beta-catenin exon 3 was also found to be correlated with high metastatic potential of HCC. CONCLUSION: Abnormal expression of beta-catenin gene may contribute importantly to the invasiveness of HCC among Chinese people.     

Tissue microarray for high-throughput analysis of gene expression profiles in hepatocellular carcinoma

AIM: To study the expression profiles of HBsAg, HBcAg, p21WAF1/CIP1 (p21), Rb genes in hepatocellular carcinoma (HCC) and to investigate their roles in the hepatocar-cinogenesis. METHODS: HCC tissue microarray containing 120-min tissues of 40 HCC cases was constructed. HBsAg, HBcAg, p21 and Rb proteins were immunohistochemically stained by streptavidin-peroxidase conjugated method (S-P). The expression loss of these genes in cancerous, para-cancerous tissues and adjacent normal liver tissues of 40 HCCs were comparatively examined. RESULTS: The positive rate of HBsAg expression in cancerous tissues of 40 HCCs was 7.5%, which was lower than that in para-cancerous and adjacent normal liver tissues (X2=12.774, P<0.01; X2=18.442, P<0.01). The positive rate of HBcAg expression in cancerous tissues of 40 HCCs was 20.0%, which was also lower than that in para-cancerous and adjacent normal liver tissues (X2=9.482, P<0.01; X2=14.645, P<0.01). p21 protein deletion rate in cancerous tissues of 40 HCCs was 27.5%, which was higher than that in para-cancerous and adjacent normal liver tissues (X2=7.439, P<0.01; X2=11.174, P<0.01). p21 protein deletion correlated remarkably with the pathological grade of HCC (X2=0.072, P<0.05). Rb protein deletion rate in cancerous tissues of 40 HCCs was 42.5%, which was also higher than that in para-cancerous and adjacent normal liver tissues (X2=10.551, P<0.01; X2=18.353, P<0.01). Rb protein deletion rate did not correlate remarkably with tumor size or pathological grade of HCC (X2=0.014, P>0.05; X2=0.017, P>0.05). CONCLUSION: Expression deletion of HBsAg, HBcAg, p21 and Rb proteins in HCCs may play important roles in the carcinogenesis of HCC. Tissue microarray is an effective high-throughput technique platform for cancer research.