Zhao-You Tang

p53 mutation in hepatocellular carcinoma after aflatoxin exposure

Mutations of the p53 gene are found in hepatocellular carcinoma (HCC), the most common form of primary liver cancer. Specific mutations might reflect exposure to specific carcinogens and we have screened HCC samples from patients in 14 different countries to determine the frequency of a hotspot mutation at codon 249 of the tumour suppressor p53 gene. We detected mutations in 17% of tumours (12/72) from four countries in south Africa and the southeast coast of Asia. There was no codon 249 mutation in 95 specimens of HCC from other geographical locations including North America, Europe, Middle East, and Japan. Worldwide, the presence of the codon 249 mutation in HCCs correlated with high risk of exposure to aflatoxins and the hepatitis B virus (HBV). Further studies were completed in two groups of HBV-infected patients at different risks of exposure to aflatoxins. 53% of patients (8/15) from Mozambique at high risk of aflatoxin exposure had a tumour with a codon 249 mutation, in contrast with 8% of patients from Transkei (1/12) who were at low risk. HCC is an endemic disease in Mozambique and accounts for up to two thirds of all tumours in men. A codon 249 mutation of the p53 gene identifies an endemic form of HCC strongly associated with dietary aflatoxin intake.

Invasion and metastasis of liver cancer: expression of intercellular adhesion molecule 1.

Abstract Purpose: To study the relationship between intercellular adhesion molecule 1 (ICAM-1) and liver cancer metastasis and to find predicting factors that could indicate the growth and metastasis of liver cancer. Methods: ICAM-1 expression in fresh tissue of normal liver and hepatocellular cancer (HCC) was examined by immunoperoxidase staining. Serum soluble intercellular adhesion molecule 1 (sICAM-1) from patients with a benign HCC tumor, and the expression of ICAM-1 in the orthotopically transplanted LCI-D20 tumor of a nude mouse liver cancer metastasis model, and in human hepatoma, the tumor surrounding tissue and normal liver, was analyzed semiquantitatively by the immuno-dot blot method. Tissue ICAM-1 expression (mRNA level) was detected by Northern blotting. Results: ICAM-1 expression in LD1-20 D metastatic liver cancer had a positive correlation with tumor size and the time after implantation. It increased suddenly as metastasis occurred being 3.03 ± 0.51 before metastasis and 8.24 ± 0.95 after metastasis, P < 0.01, then remained high, appending on the number of sites involved (monosite metastasis 5.48 ± 0.49, multisite metastasis 10.05 ± 1.17, P < 0.05). All six cases of normal liver samples were negative in anti-ICAM-1 immunohistochemical staining, 80.0% (36/45) of the HCC showed some ICAM-1 expression. The rate of positive cells was a little higher in large tumors, tumors with an intact capsule and tumors with metastasis, but there was no significant difference. It was noticed that two cancer emboli also had high ICAM-1 expression. The ICAM-1 concentration in HCC (13.43 ± 0.09) was higher than that in tumor surrounding the liver (5.89 ± 0.17, P < 0.01) and that in normal liver (4.27 ± 0.21, P < 0.01). sICAM-1, like tissue ICAM-1, was higher in HCC patients than in patients (with benign liver tumor and normal controls. Both tissue ICAM-1 and sICAM-1 were higher in the metastasis group than in the group without metastasis (tissue ICAM-1 20.24 ± 0.30 vs 10.23 ± 0.12 P < 0.05; sICAM-1 12.18 ± 0.25 vs 9.77 ± 0.54 P < 0.05). Northern blot analysis revealed that ICAM-1 expression, as indicated by mRNA level, was also higher in HCC and in cancer emboli than in tumor surrounding liver and normal liver. Conclusions: Tissue ICAM-1 and serum sICAM-1 could indicate the stage of HCC, and the potential of hepatoma cells for invasion and metastasis. They may play an important role in the metastasis cascade.

Expression of platelet-derived endothelial cell growth factor and vascular endothelial growth factor in hepatocellular carcinoma and portal vein tumor thrombus.

Purpose: Both platelet-derived endothelial cell growth factor (PD-ECGF) and vascular endothelial growth factor (VEGF) are known to promote the development of new blood vessels, which are fundamental to tumor growth and metastasis. We aimed at evaluating the gene expression of PD-ECGF and VEGF in hepatocellular carcinoma (HCC) and portal vein tumor thrombus (PVTT). Patients and methods: Surgical specimens (28 HCC, 28 nontumorous liver tissues and 18 PVTT) were studied by Northern blot analysis. The levels of PD-ECGF mRNA and VEGF mRNA expression were measured by densitometric scanning of the autoradiographs, and they were normalized to the level of expression of an internal control (glyceraldehyde-phosphate dehydrogenase) mRNA. Results: The expression rates of PD-ECGF mRNA in PVTT, HCC and nontumorous liver tissues were 77.8% (14/18), 67.9% (19/28) and 35.7% (10/28), being 88.9% (16/18), 75.0% (21/28) and 17.9% (5/28) respectively for VEGF mRNA. The expressions of PD-ECGF mRNA and VEGF mRNA were higher in HCC with PVTT than when PVTT was absent (P < 0.05). The PVTT was more often seen in patients with positive expression of both PD-ECGF mRNA and VEGF mRNA in HCC than in patients who were positive for only one of these factors or negative for both (P < 0.05). Conclusion: Both PD-ECGF and VEGF correlated well with the formation of PVTT of HCC.

Elevated activity of N -acetylglucosaminyltransferase V in human hepatocellular carcinoma

Abstract Cell-surface glycoproteins are regarded as candidates for involvement in the spread of tumor cells. N-linked β1-6 branched oligosaccharides may contribute directly to the malignant or metastatic phenotypes of tumor cells. Increased β1-6 branching has been associated with an increased level of N-acetylglucosaminyltransferase V (GlcNAc transferase V), the glycosyltransferase that initiates the β1-6 branching. In this report, 33 pathologically verified hepatocellular carcinoma (HCC) specimens, six non-cancerous tissues surrounding HCC and five normal liver specimens have been studied. We have quantified N-linked β1-6 branched oligosaccharides indirectly by measuring GlcNac transferase V activity. The average GlcNac transferase V activities in hepatocellular carcinoma (HCC), noncancerous tissues surrounding HCC and normal liver tissues were 324.2 ± 269.8, 84.8 ± 20.7 and 7.0 ± 6.2 pmol product h−1 mg protein−1 (P < 0.05) respectively. In addition, the activity was correlated with the TNM classification of HCC. The average activities of GlcNAc transferase V in stages T1, T2–3 and T4 were 77.6 ± 57.8, 369.0 ± 294.7 and 329.9 ± 205.9 pmol product h−1 mg protein h−1 respectively (P < 0.05), showing that the activity of the enzyme in advanced HCC was higher than that in early HCC. Our preliminary results indicated that GlcNAc transferase V activity increased in human HCC and was correlated with its progression.

Improved Survival with Resectionafter Transcatheter ArterialChemoembolization (TACE) for Unresectable Hepatocellular Carcinoma

Aim: This retrospective study was undertaken to analyze the outcome of hepatic resection in hepatocellular carcinomas (HCCs) that shrunk after transcatheter hepatic arterial chemoembolization (TACE) in 65 patients with unresectable HCCs between June 1987 and September 1996. Materials and Methods: Among these 65 patients, the median diameter of the tumor was 9.9 cm (5.6–20.0) prior to the first TACE, after 1–6 times of TACE (median 3) the median tumor diameter reduced to 3.7 cm (1.9–12.5) prior to resection. The duration between the last TACE treatment and sequential resection varied from 1 to 9 months (median 2.5). Serum α-fetoprotein (AFP) levels were abnormal in 39 out of the 65 patients. In AFP producing HCCs, the AFP level returned to normal (≤20 µg/l) in 14 out of 39 patients (35.9%). Hepatic segmentectomy, multiple hepatic segmentectomy or partial hepatic resection were performed in 61 patients, right hemihepatectomy in 1, left trisegmentectomy in 2, and left hemihepatectomy in 1. Results: Tumor necrosis ranged from 40 to 100% and pathologically and complete tumor necrosis occurred in 11 patients (16.9%). Of 14 patients with AFP levels decreased to normal, 10 still had microscopic living tumor foci. The 1-, 3- and 5-year survival rates of the 65 patients were 80.0, 65.0 and 56.0% respectively. Conclusion: TACE treatment can provide a chance of tumor resection for those patients with initially judged unresectable HCCs, and liver resection should be performed when the tumor has shrunk to be resectable, even when the AFP level has returned to normal.

High-intensity focused ultrasound in the treatment of experimental liver tumour.

This project aimed to determine the adequacy and accuracy of high-intensity focused ultrasound (HIFU) for ablating experimental liver tumour, and to assess imaging methods for monitoring the therapeutic results. The rabbit liver pseudotumour model was established by injection of Freunds complete adjuvant into the liver; the animals then received HIFU therapy via laparotomy at the focal point of the beam (1.1 MHz, 500 W/cm2, 20 s). The rabbits were sacrificed at scheduled times after treatment and liver tumours were examined histologically. Sequential imaging of the liver tumour was performed before and after HIFU treatment. HIFU accurately destroyed the rabbit liver tumour and induced coagulation necrosis 24 h later. Sonographic imaging studies revealed that characteristic changes occurred. A hyperechoic mass turned to a hypoechoic lesion with no Doppler signal, and a high echogenic rim appeared 24 h after HIFU treatment, correlating well with the pathological changes of a sonoablated lesion. These results verify that HIFU has the power to ablate liver tumour quite adequately and accurately, and that sonography is useful for monitoring sonoablated liver tumour.

Expression of the integrinα5 subunit and its mediated cell adhesion in hepatocellular carcinoma

Tumor invasion and metastasis are complex processes, requiring the ability of tumor cells to interact with proteins of the extracellular matrix through cell-adhesion molecules on the cell surface. Integrins are heterodimeric membrane glycoproteins, consisting of α and β subunits, which enable cells to recognize adhesive substrates in the extracellular matrix. The roles of the integrinα5β1 in tumor invasion are highlighted by finding that some tumor cells have lost or reducedα5β1 expression. It therefore functions as a negative signaling regulator. Expression ofα5β1 and its mediation of cell adhesion in hepatocellular carcinoma (HCC) have not been elucidated. In surgical specimens of HCC we found, by immunohistochemistry and Northern blot analysis, that theα5-positive rates in cancerous tissues were lower than the corresponding rates in non-cancerous tissues. Reduced expression of the integrinα5 occurred more frequently in HCC with more malignant phenotypes, such as poor differentiation, large size (more than 10-cm in diameter), absence of capsule and high invasion. Reverse transcription/polymerase chain reaction, a more sensitive assay, was used to detect theα5 mRNA level in LCID20, a highly metastatic model of human HCC, and LCID35, a low-metastasis model. The results showed that integrinα5 was negative in the former and positive in the latter. Cell adhesion assays showed the maximal percentage inhibition of anti-α5 mAb on SMMC 7721 cell adhesion to fibronectin to be 68.9±4.9% at the saturation concentrations of each antibody (200 μg/ml). If anti-α5 mAb was combined with anti-β1 mAb, the inhibition was 74.1±11.1%. It is concluded that reduced expression of the integrinα5 subunit is correlated with more malignant phenotypes of human HCC. Any change in the adhesion of hepatocellular carcinoma cells to fibronectin is mainly dependent upon the function of the integrinα5β1.

Mutations on Free and Integrated Hepatitis B Virus DNA in a Hepatocellular Carcinoma: Footprints of Homologous Recombination

Hepatitis B virus nucleotide sequences derived from a hepatocellular carcinoma with free and multiply integrated viral DNAs were determined. Based on a comparison within the X-gene region, cloned free viral DNA previously had been attributed to two distinct groups of preC minus genomes. The comparison of the complete sequence identified one of the genome equivalents as a recombinant between members of these groups. Four different integrated viral DNA elements were cloned and analysed. Similarity to either one of two DNAs representing the two groups of free viral DNA on one hand and the presence of certain mutations only on integrated DNA on the other hand, allowed to recognize distinct segments within the integrants. The data suggest a contribution of different but related genotypes to contiguous stretches of integrated viral DNA via homologous recombination. On this basis an evolutionary relationship between free and integrated DNAs of the preC and the preC minus genotype could be recognized when short sequence segments were compared. The observed coexistence on a given integrated DNA of segments homologous to free viral DNA and of segments homologous to another integrated DNA is consistent with (1) a long lasting association of individual genotypes with dividing cells and (2) multiple integration events being the result of a series of steps not separated by a long time span.

Inhibitory effect of the angiogenesis inhibitor TNP-470 on tumor growth and metastasis in nude mice bearing human hepatocellular carcinoma.

The antitumor and anti-metastatic effects of a potent angiogenesis inhibitor,O-(chloroacetyl-carbamoyl)fumagillol (TNP-470), was investigated in a highly metastatic model of human hepatocellular carcinoma—LCI-D20. Small pieces of LCI-D20 tumor tissue were implanted subcutaneously into the right axillary region of 24 nude mice; the mice were then randomized into two groups. To one group, TNP-470 30 mg/kg was given as a subcutaneous injection every other day from day 1 to day 15 and the mice were sacrificed on day 26. An antitumor effect of TNP-470 was clearly demonstrated by tumor weight (0.97±0.34 g compared to 2.04±0.34 g,P<0.001) and α-Fetoprotein value (93±59 μg/L compared to 769±282 μg/L,P<0.001). There was also an anti-metastatic effect of TNP-470. Lung metastases developed in only 1 of 12 mice in the treated group, while they developed in 6 of mice of the control group. No severe side-effect of TNP-470 was found in this study. In vitro study revealed that the purified hepatoma cells were insensitive to TNP-470 (the 50% inhibitory concentration was 43 μg/ml). These results suggest that the angiogenesis inhibitor TNP-470 has both strong antitumor and anti-metastatic effects on a human hepatocellular carcinoma model in nude mice.

Experimental models of hepatocellular carcinoma: developments and evolution

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. The biological mechanisms of hepatocarcinogenesis and progression are poorly understood. Experimental models of HCC provide valuable tools to evaluate the risk factors, new treatment modalities and biologic characteristics. Under the constant evolution in model design and technology development, new experimental models continue to emerge, including spontaneous models, induced models, viral models, transplantable models, and genetically engineered models. These models are used as tools to investigate basic biological mechanisms of growth and differentiation, oncogene function, and as systems to test new diagnostic and therapeutic approaches. Each model has its own advantages and disadvantages. The progress in HCC model construction and studies are summarized in this review.