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.     

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.     

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.     

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 SPSS10.0 statistical 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 (mt-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% vs 27.88%±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% vs 7.38%±2.61%, t, P=0.019). Expression of TAp73a was associated with lymph node metastasis and mt-p53, with r=0.407 and 0.265, respectively. Expression of mt-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, portal vein 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.     

Significance of cyclooxygenase—2 expression in human primary hepatocellular carcinoma

AIM：To clarife the significance of cyclooxygenase-2(COX-2)expression in human primary hepatcellular carcinoma(HCC)and adjacent nontumorous tissues.METHODS;TheCOX-2protein and mRNA were investigated in 27HCC tissues with adjacent nontumorous tissues,and 5histologically normal liver tissues,using immunohistochemistry and in situ hybridization.RESULTS:The well-differentiated HCC expressed COX-2protein(5.68&#177;1.19)more strongly than moderated HCC(3.43&#177;1.98)and poor differentiated HCC(3.33&#177;1.50)(P&lt;0.05 respectively),adjacent nontumorous tissues(4.93&#177;1.05)and normal live tissues(3.20&#177;1.92)(P&lt;0.01 respectively);More intensive staining of COX-2in adjacent nontumorous tissues was observed than that in normal liver tissues(P&lt;0.05).There was no significant difference among adjacent nontumorous tissues,moderately differentiated HCC and poorly differentiated HCC(P&gt;0.05).The expression of COX-2mRNA was observed in the cytoplasm of the cells of HCC and of gtthe hepatocytes in adjacent nontumorous tissues in which COX-2 protein was positive.CONCLUSION:The overexpression of COX-2 in well-differentiated HCsuggets that COX-2 may play a role in the early stages of hepatocarcinogensis.     

HLA classⅠexpression in primary hepatocellular carcinoma

AIM：To investigate whetherCTL vaccine therapy is suitable for primary hepatocellular carcinoma(HCC)from the viewpoint of HLA classIantigens expression.METHODS:The immunocytochemistry,image analysis,flow cytometry,and labeled streptavidin bioti(LSAB)method of immunohistochemistry were applied respectively to study 4HCCcell lines(e.g.Alexander,HepG2,SMMC-7721,andQGY-7703)cultured in vitro and 6frozen tissue specimens of HCC.RESULTS:The positive control cell line Raji had very strong positive staining,Most mitotic and nonmitotic cells of the 4HCCcell lines had various intensity of HLAclassⅠantigens expression.The negative control cell K562 and the control slides of all the cell lines had no positive staining,In the 6HCCspecimens immunohistochemically studied,histological normal hepatocytes had no or very weak positive staining and the liver sinus had very strong positive staining.Most HCCcells in the sections from the 6HCCspecimens had strong positive HLAclassⅠantigens staining.The positive staining was located in the cytoplasm,the perinuclear area,and at the cell membrane of the liver cancer cells.Flow cytometry also revealed that Raji and those 4HCCcell lines had strong HLAclassⅠantigens expression.which was confirmed quantitatively by the image analysis.It showed that the objective grayscale values of Raji and those 4HCCcell lines were significantly different from that of K562(Raji114.04&#177;10.94,Alexander165.97&#177;5.35,HepG2167.02&#177;12.60,QGY-7703161.46&#177;7.13,SMMC-7721 165.93&#177;5.21,K562244.89&#177;4.60,P&lt;0.01).Significant differences were also found ebtween Raji and the 4HCCcell lines.CONCLUSION:HCC cells express HLA classI antigens strongly,Fromthis point of view.the active specific immunotherapy of CTL vaccine is suitable and practicable for HCC.     

Expression of transforming growth factor-α 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 itssignificance.METHODS:Seventy specimens of HCC tissues weredetected by immunohistochemical method.Five specimensof normal human liver tissues were used as control.RESULTS:The TGF-α positive expression rates in HCCand its surrounding tissues were 74.3%(52/70) and88.1%(52/59),respectively.TGF-α positive granules weremainly in the cytoplasm and fewer existed on thekaryotheca.The TGF-α positive expressing rate in welldifferentiated HCC was significantly higher than that inmoderately and poorly differentiated HCC (P<0.05).TheTGF-α positive expression also was observed in intrahepaticbile ducts (part of those were hyperplastic ducts).TheHBsAg positive expression rates in HCC and its surroundingtissues were 21.4%(15/70) and 79.7%(47/59),respectively.HBsAg positive granules were in the cytoplasm,inclusionand on the karyotheca.There was a prominent positivecorrelation between TGF-a and HBsAg expression in HCCsurrounding tissues (P<0.05,γ=0.34).TGF-α was usuallyexisted with HBsAg in regenerated and/or dysplastic livercells.In the five normal liver tissues,TGF-α and HBsAgwere not detectable in hepatocytes and bile ducts.CONCLUSION:Hepatitis B virus infection is closely relatedwith hepatocarcinogenesis.The overexpression of TGF-αin the liver seems to be associated with the regenerationof hepatocytes injured by HBsAg.The continued expressionof TGF-α might lead to dysplasia of liver cells anddevelopment of HCC.Furthermore,TGF-α might play arole in morphogenesis and regeneration of intrahepaticbile ducts.     

Expression of E-selectin, integrin β1 and immunoglobulin superfamily member in human gastric carcinoma cells and its clinicopathologic significance

AIM: To study the expression levels of E- selectin, integrin beta1 and immunoglobulin superfamily member-intercellular adhesion molecule-1 (ICAM-1) in human gastric carcinoma cells, and to explore the relationship between these three kinds of cell adhesion molecules and gastric carcinoma. METHODS: The serum contents of E-selectin, integrin beta1 and ICAM-1 were detected by enzyme-linked immunosorbent assay (ELISA), in 47 healthy individuals (control group) and in 57 patients with gastric carcinoma (gastric carcinoma group) respectively prior to operation and 7 d after operation.RESULTS: The serum E-selectin, ECAM-1 and integrin beta1 were found to be expressed in both control and gastric carcinoma groups. However, they were highly expressed in patients with gastric carcinoma patients before operation or with unresectable tumours. The expression levels of ICAM-1 and integrin beta1 were significantly higher in gastric carcinoma patients than in controls (P < 0.01). A comparison of the E-selectin levels between the two groups showed statistically insignificant difference (P = 0.64). In addition, the expression levels were all decreased substantially in the postoperative patients subjected to radical resection of the tumours, indicating that the high level expressions of these compounds might be the important factor for predicting the prognosis of these patients. CONCLUSION: Serum E-selectin, ICAM-1 and integrin beta1 expression levels are probably related to the metastasis and relapse of gastric cancer.     

Clinicopathological and prognostic implications of endoglin （CD105） expression in hepatocellular carcinoma and its adjacent non-tumorous liver

AIM: The expression pattern of endoglin (CD105) in hepatocellular carcinoma (HCC) has not been reported so far. We hypothesized that CD105 could differentially highlight a subset of microvessels in HCC, and intratumoral microvessel density (IMVD) by CD105 immunostaining (IMVD-CD105) could provide better prognostic information than IMVD by CD34 immunostaining (IMVD-CD34). METHODS: Paraffin blocks of tumor and adjacent non-tumorous liver tissues from 86 patients who underwent curative resection of HCC were used for this study. Serial sections were stained for CD105 and CD34, respectively, to highlight the microvessels. IMVD was counted according to a standard protocol. RESULTS: In the HCC tissues, CD105 was either negatively or positively stained only in a subset of microvessels. In contrast, CD34 showed positive and more extensive microvessel staining in all cases examined. However, in the adjacent non-tumorous liver sections, CD105 showed a diffuse pattern of microvessel staining in 20 of 86 cases, while CD34 showed negative or only focal staining of the sinusoids around portal area. Correlation with clinicopathological data demonstrated that lower scores of IMVD-CD105 were found in larger sized tumors [mean 41.4/0.74 mm2 (>5 cm tumor) vs 65.9/0.74 mm2 (≤5 cm tumor), P= 0.043] and more aggressive tumors, as indicated by venous infiltration [36.8/0.74 mm2 (present) vs 64.2/0.74 mm2 (absent), P = 0.020], microsatellite nodules [35.1/0.74 mm2 (present) vs 65.9/0.74 mm2 (absent), P= 0.012], and advanced TNM tumor stage [38.8/0.74 mm2 (stage 3 or 4) vs 68.3/0.74 mm2 (stage 1 or 2), P= 0.014]. No prognostic significance was observed when median values were used as cut-off points using either IMVD-CD105 or IMVD-CD34. However, the presence of the diffuse pattern of CD105 expression in the adjacent non-tumorous liver tissues predicted a poorer disease-free survival (median 8.6 vs 21.5 mo, P= 0.026). CONCLUSION: Our data demonstrate that a lower IMVD-CD105 is associated with larger and more aggressive tumors. In this study, IMVD-CD105 did not provide significant prognostic information. However, active angiogenesis as highlighted by diffuse CD105 staining of the microvessels in the adjacent non-tumorous liver tissues is predictive of early recurrence.     

人宫颈癌基因蛋白在人肝细癌中的表达及其临床意义

人宫颈癌基因(HCCR)是新近确定的一种癌基因,定位于人染色体12q,编码HCCR-1和HCCR-2两种蛋白。HCCR不仅在宫颈癌组织中表达,而且在人其他肿瘤如白血病、淋巴瘤、乳腺癌、肾癌、胃癌、结肠癌、肝癌和子宫癌中均有过度表达,可作为肝癌及乳腺癌诊断的标志物。本研究通过制备HCCR蛋白多克隆抗体,检测人肝细胞癌及肝癌细胞株中HCCR的表达,旨在了解HCCR的表达与肝细胞癌临床病理特征之间的关系。     

人肝癌组织中原癌基因Pokemon的表达及其临床意义

目的 探讨肝癌组织中Pokemon基因的表达,并分析其与肿瘤的大小、分化程度、TNM分期等临床资料的相关性.方法 收集30例原发性肝癌患者的肿瘤及其癌旁组织,用逆转录酶聚合链反应(RT-PCR)及免疫印迹(Western blot)的方法分别从核酸及蛋白水平检测标本中Pokemon基因的表达,并对其与患者的性别、肿瘤大小、淋巴结转移、肿瘤分化程度、肿瘤的TNM分期等临床资料进行统计分析.结果 RT-PCR检测肝癌和癌旁组织mRNA表达的相对强度分别为0.71±0.03、0.17±0.01.Western blot检测蛋白水平的相对表达强度分别为10.64±0.92、1.06±0.18,两者比较有统计学意义(P<0.05),肝癌组织中Pokemon的表达与肿瘤的大小有关,而与患者的年龄、肿瘤的分化程度、淋巴结转移及肿瘤TNM分期等无关.结论 Pokemon的表达上调在肝癌的增殖中可能发挥重要作用.     

Overexpression of P-glycoprotein in hepatocellular carcinoma and its clinical implication

INTRODUCTION Most advanced hepatocellular garcinoma (HCC) is insensitive to most anticancer drugs which might be related to the high frequency of expression of the multidrug resistance-1 ( MDR1 ) gene[1] and its product, P-glycoprotein (P-gp)[2]. P-gp expression may also be concerned with tumor progression and differentiation[3].     

p27、Cyclin A在肝细胞癌中的表达及其意义

细胞周期调节因子p27及Cyclin A蛋白水平升高或基因改变与多种肿瘤的发生有关。用免疫组织化学SABC法，联合检测45例肝细胞癌(HCC)组织及30例癌旁肝组织中p27及Cyclin A的表达，以探讨其与HCC发生、发展及侵袭转移的关系。     

Expression of p28GANK and its correlation with RB in human hepatocellular carcinoma.

BACKGROUND: Aberrance of retinoblastoma protein (RB) signal pathway is known to play an important role in the carcinogenesis of human hepatocellular carcinoma (HCC). p28GANK, originally purified from human 26S proteasome as a non-ATPase subunit, was recently found in HCC and shown to interact with RB. The aim of this study was to investigate the expression profile of p28GANK and its correlation with RB in HCC. METHODS: The expression of p28GANK was evaluated in 55 surgically resected HCCs by immunohistochemistry (IHC), and the associations were explored between p28GANK level and clinicopathologic features as well as tumor suppressor RB. Western blotting was performed to determine p28GANK expression level in 12 HCCs. Immunofluorescence stainings of p28GANK and RB in U2-OS cells were examined by confocal microscopy. RESULTS: Positive p28GANK cytoplasmic staining was recognized in 55 HCCs. Nuclear positive occurrence of p28(GANK) in HCCs was more frequent than paracancerous hepatic tissues (P < 0.05). The overexpression probability of p28GANK was inversely associated with Edmonson's grade: overexpression occurred in nine out of 11 (81.8%), 22 out of 35 (62.9%) and two out of nine (22.2%) in I-II, III and IV graded cases, respectively (P = 0.004). Total cellular expression of p28GANK had curvilinear correlation with the nuclear expression of RB (r = 0.475, P = 0.019), while the nuclear expression of p28GANK had not. Western blot analysis showed that up-regulation of p28GANK expression was found in nine out of 12 HCCs compared with paracancerous liver tissues. Exogenously expressed p28GANK colocalized with RB in cytoplasm of U2-OS cells. CONCLUSIONS: These results confirm the role of p28GANK as a highly expressed oncoprotein in HCC by in situ examination. Its overexpression correlates with the differentiation status of HCC. The whole cellular p28GANK activation, not nuclear portion only, influences the alteration of RB. Underlying nuclear translocation of p28GANK may contribute to the counteraction against RB through a feed back loop. These data provide new evidence for p28GANK to be used as a promising drug target of a therapeutic agent against HCC.     

原发性肝细胞癌组织Smad4、p21蛋白的表达及其临床意义

目的 探讨原发性肝细胞癌组织Smad4和p21蛋白表达及其相关性.方法 采用免疫组化法检测60例原发性肝细胞癌组织及20例癌旁组织中Smad4、p21蛋白的表达,同时结合临床资料进行分析.结果 癌及癌旁组织中Smad4蛋白阳性表达率分别为36.7%、80.0%,p21蛋白阳性表达率分别为41.7%、70.0%,两种组织Smad4和p21蛋白表达差异均有统计学意义(P＜0.01,＜0.05).癌组织中Smad4蛋白表达与组织分级、肿瘤转移有关(P＜0.05).Smad4与p21蛋白的表达呈正相关(r=0.69,P＜0,01).结论 Smad4及p21蛋白在原发性肝细胞癌组织中表达均显著下降,并与肝癌生物学行为关系密切.     

Overexpression of DNA methyltransferase 1 and its biological significance in primary hepatocellular carcinoma

AIM： To explore the relationship between DNA methyltransferase 1 （DNMT1） and hepatitis B virus （HBV）-related hepatocellular carcinoma （HCC） and its biological significance in primary HCC. METHODS： We carried out an immunohistochemical examination of DNMT1 in both HCC and paired nonneoplastic liver tissues from Chinese subjects. DNMT1 mRNA was further examined in HCC cell lines by real-time PCR. We inhibited DNMT1 using siRNA and detected the effect of depletion of DNMT1 on cell proliferation ability and cell apoptosis in the HCC celt line SMMC-7721. RESULTS： DNMT1 protein expression was increased in HCCs compared to histologically normal nonneoplastic liver tissues and the incidence of DNMT1 immunoreactivity in HCCs correlated significantly with poor tumor differentiation （P = 0.014）. There were more cases with DNMT1 overexpression in HCC with HBV （42.85%） than in HCC without HBV （28.57%）. However, no significant difference in DNMT1 expression was found in HBV-positive and HBV-negative cases in the Chinese HCC group. There was a trend that DNMT1 RNA expression increased more in HCC cell lines than in pericarcinoma cell lines and normal liver cell lines. In addition, we inhibited DNMT1 using siRNA in the SMMC-7721 HCC cell line and found depletion of DNMT1 suppressed cells growth independent of expression of proliferating cell nuclear antigen （PCNA）, even in HCC cell lines where DNMT1 was stably decreased. CONCLUSION： The findings implied that DNMT1 plays a key role in HBV-retated hepatocellular tumorigenesis. Depletion of DNMT1 mediates growth suppression in SMMC-7721 cells.     

死亡相关蛋白激酶在肝细胞癌中的表达及其临床意义

目的研究死亡相关蛋白激酶(death associated protein kinase,DAPK)在原发性肝细胞癌(HCC)组织中的表达及其与HCC临床病理特征的关系。方法应用免疫组织化学SP法检测DAPK在50例HCC组织及其癌旁组织和5例正常肝脏组织中的表达。结果 DAPK在HCC组织中的阳性率为36%,明显低于癌旁组织62%及正常肝组织100%(P<0.05);DAPK低表达与HCC组织分化程度、淋巴结转移有关(P<0.05)。结论 DAPK表达下调在HCC的发生发展中可能起重要作用。