agar and Carcinoma--Hepatocellular

The aim of this study was to characterize the oncogenic function and mechanism of Cathepsin Z (CTSZ) at 20q13.3, a frequently amplified region in hepatocellular carcinoma (HCC). Real-time PCR were used to compare CTSZ expression between paired HCC tumor and non-tumor specimens. CTSZ gene was stably transfected into HCC line QGY-7703 cells and its role in tumorigenicity and cell motility was characterized by soft agar, wound-healing, transwell invasion and cell adhesion assay, and tumor xenograft mouse model. Western blot analysis was used to study expression of proteins associated with epithelial-mesenchymal transition (EMT).Upregulation of CTSZ was detected in 59/137 (43%) of primary HCCs, which was significantly associated with advanced clinical stage (P = 0.000). Functional study found that CTSZ could increase colony formation in soft agar and promote cell motility. Further study found that the metastatic effect of CTSZ was associated with its role in inducing epithelial-mesenchymal transition (EMT) by upregulating mesenchymal markers (fibronectin and vimentin) and downregulating epithelial markers (E-cadherin and α-catenin). In addition, CTSZ could also upregulate proteins associated with extracellular matrix remodeling such as MMP2, MMP3 and MMP9. Taken together, our data suggested that CTSZ was a candidate oncogene within the 20q13 amplicon and it played an important role in HCC metastasis.

Topics: Agar; alpha Catenin; Animals; Cadherins; Carcinoma, Hepatocellular; Cathepsin Z; Cell Adhesion; Cell Movement; Epithelial-Mesenchymal Transition; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Matrix Metalloproteinases; Mice; Mice, SCID; Neoplasm Metastasis; Neoplasm Transplantation; Vimentin; Wound Healing

Hepatitis B x antigen (HBxAg) is a trans-activating protein that contributes to liver cancer, in part, by altering the expression of cellular genes. However, few natural effectors of HBxAg have been identified. Hence, HBxAg positive and negative HepG2 cells were prepared and analyzed by PCR select cDNA subtraction. The results identified elevated vascular endothelial growth factor receptor-3 short form splice variant (VEGFR-3(S)) expression in HBxAg positive compared to negative cells. Normally, VEGFR-3 activates Akt signaling in lymphatic endothelial cells, resulting in lymphangiogenesis. In contrast, the results here show that the expression of VEGFR-3(S) is up-regulated in >75% of HBxAg positive hepatocellular carcinoma (HCC) nodules. VEGFR-3(S) up-regulation correlates with the expression of HBxAg, is associated with decreased survival in tumor bearing patients, and when over-expressed in HepG2 cells, strongly stimulated cell growth in culture, in soft agar, and accelerated tumor formation in a ligand independent manner. VEGFR-3(S) siRNA partially blocked the ability of HBxAg to promote hepatocellular growth. In conclusion, HBxAg may short circuit VEGFR-3(S) signaling in liver cancer. Blocking VEGFR-3(S) signaling may be effective in preventing tumor development and/or prolonging survival in tumor bearing patients.

Topics: Agar; Animals; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Cloning, Molecular; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Viral; Hepatitis B; Hepatitis B virus; Humans; Liver; Liver Neoplasms; Mice; Mice, Nude; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Trans-Activators; Up-Regulation; Vascular Endothelial Growth Factor C; Vascular Endothelial Growth Factor Receptor-3; Viral Regulatory and Accessory Proteins

Hepatocellular carcinoma (HCC) is one of the most common malignancies in Southeast Asia. Although inactivation of pRb2/p130 has been reported in a variety of human cancers, its function in HCC has not been established. In this study we report that loss of expression of pRb2/p130 was detected by immunohistochemistry and western blotting in 15.2% (7 of 46) HCCs examined. High levels of pRb2/p130 expression were found in 84.8% (39 of 46) HCCs studied. Western blot analysis revealed that HCC had 3.5-fold higher pRb2/p130 than adjacent benign liver (ABL) tissues. 71.7% (33 of 46) of HCCs examined exhibited both nuclear and cytoplasmic staining for pRb2/p130. Cytoplasmic staining was found in 93.5% (43 of 46) of ABL tissues. Overproduction of pRb2/p130 in HepG2 cells led to growth suppression, cell cycle arrest in G0/G1, altered cell morphology, inhibition of in vitro colony formation and reduction in tumourigenicity in SCID mice. This demonstration suggests a role of pRb2/p130 as a tumour suppressor protein in HCC and the loss of this protein may lead to the development or progression of HCC. Overexpression of pRb2/p130 in HCC was, therefore, suggested to be a programmed protective response of the organism to uncontrolled proliferation.

Topics: Agar; Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cell Division; Cell Line; Cytoplasm; Disease Progression; DNA, Complementary; Electrophoresis, Agar Gel; Flow Cytometry; G1 Phase; Humans; Immunohistochemistry; Liver Neoplasms; Mice; Mice, SCID; Neoplasm Transplantation; Neoplasms; Precipitin Tests; Protein Biosynthesis; Proteins; Resting Phase, Cell Cycle; Retinoblastoma-Like Protein p130; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transfection; Tumor Cells, Cultured

The central role of T cell activation in hepatocellular injury has been well documented. In this article, we provide evidence suggesting that T cells may also play a protective role in liver disease by releasing interleukin-22 (IL-22), a recently identified T cell-derived cytokine whose biological significance is unclear. IL-22 messenger RNA and protein expression are significantly elevated in T cell-mediated hepatitis induced by concanavalin A (ConA) but are less extensively elevated in the carbon tetrachloride-induced liver injury model. Activated CD3(+) T cells are likely responsible for the production of IL-22 in the liver after injection of ConA. The IL-22 receptor is normally expressed at high levels by hepatocytes and further induced after ConA injection. IL-22 blockade with a neutralizing antibody reduces signal transducer and activator of transcription factor 3 (STAT3) activation and worsens liver injury in T cell-mediated hepatitis, whereas injection of recombinant IL-22 attenuates such injury. In vitro treatment with recombinant IL-22 or overexpression of IL-22 promotes cell growth and survival in human hepatocellular carcinoma HepG2 cells. Stable overexpression of IL-22 in HepG2 cells constitutively activates STAT3 and induces expression of a variety of antiapoptotic (e.g., Bcl-2, Bcl-xL, Mcl-1) and mitogenic (e.g., c-myc, cyclin D1, Rb2, CDK4) proteins. Blocking STAT3 activation abolishes the antiapoptotic and mitogenic actions of IL-22 in hepatic cells. In conclusion, the T cell-derived cytokine IL-22 is a survival factor for hepatocytes; this suggests that T cell activation may also prevent and repair liver injury by releasing hepatoprotective cytokine IL-22 in addition to its previously documented central role in hepatocellular injury.

Topics: Agar; Animals; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Cell Survival; Chemical and Drug Induced Liver Injury; Concanavalin A; DNA-Binding Proteins; Hepatitis; Hepatocytes; Humans; Interleukin-22; Interleukins; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Receptors, Interleukin; STAT3 Transcription Factor; T-Lymphocytes; Trans-Activators

Mad protein has been shown as an antagonist of c-Myc protein in some cell lines. The effect of Mad protein to the malignant phenotype of human hepatoma BEL-7404 cell line was investigated experimentally. An eukarryotic vector pCDNA III containing full ORF fragment of mad cDNA was transfected into targeted cells. Under G418 selection, stable Mad-overexpressed cells were cloned. Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells. DNA synthesis, cell proliferation and anchorage-independent growth in soft-agar of the mad-transfected cells were partially inhibited in comparison to control cells. Flow Cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase, resulting in the retardation of cell proliferation. RT-PCR detected a marked inhibition of the expression of cdc25A, an important regulator gene of G0/G1 to S phase in cell cycle. It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-7404-M1 cells in the absence of serume. Thus, Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL-7404 cells.

Topics: Agar; Apoptosis; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Carcinoma, Hepatocellular; Cell Division; DNA; DNA-Binding Proteins; Down-Regulation; Genetic Vectors; Humans; Repressor Proteins; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We have shown previously that approximately 1 in 10,000 primary hepatocytes isolated from untreated rats undergo clonal growth in soft agar in vitro in response to the synergistic action of nafenopin, a peroxisome proliferator (PP) and epidermal growth factor (EGF), a naturally occurring liver growth regulator. Here, we demonstrate that prior treatment of the animals with the genotoxic hepatocarcinogen diethylnitrosamine (DEN) caused a dose-dependent increase in soft agar colony numbers formed in vitro. These data suggest that the colony assay may offer a method of detecting in vitro hepatocytes transformed in vivo by DEN. It is known that rats treated with DEN develop enzyme altered foci prior to the development of tumours. The majority of these foci express high levels of gamma-glutamyl transpeptidase (GGT). However, foci promoted by PPs do not show this increased enzyme activity. In the present study, the colonies we have generated in vitro mimicked this pattern since the majority (approximately 80%) of the spontaneous colonies expressed GGT whereas colonies promoted by the synergistic action of nafenopin and EGF were mainly (75%) GGT negative. The proportion of colonies positive for GGT were similar using either hepatocytes isolated from control or from DEN-initiated rats. Further studies are required to assess if the hepatocytes selected for clonal expansion by this EGF/nafenopin regime reflect the presumed pre-neoplastic cells induced by genotoxin in vivo and associated with an increased propensity to cancer.

Topics: Agar; Animals; Carcinogenicity Tests; Carcinoma, Hepatocellular; Cells, Cultured; Contact Inhibition; Diethylnitrosamine; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; gamma-Glutamyltransferase; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Male; Nafenopin; Rats; Rats, Wistar; Tumor Stem Cell Assay

To characterize epidermal growth factor-related transforming growth factors in the urine of patients with hepatocellular carcinoma, gel filtration with Bio-Gel P-30 was performed in seven hepatocellular carcinoma patients and seven sex-matched and age-matched healthy controls. Distinct profiles of soft agar growth assay in the hepatocellular carcinoma patients and the normal controls were seen. Three peaks (A, B and C) in the urine were examined. Peak C in most hepatocellular carcinoma patients was higher than that in healthy controls. Similar profiles were detected with epidermal growth factor radioreceptor assay and cellular DNA synthesis assay. This result might indicate that transforming growth factors with low molecular weight were found in the urine of hepatocellular carcinoma patients. An exceptional HCC patient had an additional peak (A') that corresponded to the high molecular weight protein. We concluded that there were transforming growth factors with functional activity in the urine of patients with hepatocellular carcinoma.

Topics: Adult; Agar; Aged; Carcinoma, Hepatocellular; Cell Division; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Liver Neoplasms; Middle Aged; Radioligand Assay; Transforming Growth Factors; Tumor Cells, Cultured

Epithelial cultures established from adult rat liver and from rat hepatomas induced in vivo with aromatic amine carcinogens have been compared by light and electron microscopy and by growth properties in liquid medium and in agar. The morphology and growth patterns of all of these cultures indicate that they have characteristics of epithelial rather than fibroblast cells. The criteria generally used to score for transformation of fibroblasts were not satisfactory for distinguishing normal epithelial cells from hepatoma cells in culture. Growth in agar, however, provides a simple and objective method of scoring for transformed epithelial cells, because only the tumorigenic cells grow in agar. Since none of the normal cultures had hydrocortisone-inducible tryosine aminotransferase, we lack definitive evidence that they are derived from liver parenchymal cells. The outstanding feature in the ultrastructure of the hepatoma cells in culture was the presence of type A and C viral particles. Whereas five hepatoma cultures and a spontaneously transformed normal liver cell line were positive for these particles, five independently isolated cell cultures from normal adult rat liver were negative. Evidence is presented that the viral particles seen in hepatoma cultures are due to activation of latent viruses rather than to in vitro contamination. The possible significance of these particles in hepatocarcinogenesis is discussed.

Topics: Agar; Animals; Carcinogens; Carcinoma, Hepatocellular; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Epithelium; Liver; Liver Neoplasms; Microscopy, Electron; Neoplasms, Experimental; Oncogenic Viruses; Rats; Retroviridae

Topics: Agar; Animals; Carbon Isotopes; Carcinoma, Hepatocellular; Electrophoresis; Gels; Glycine; Isoenzymes; Liver; Liver Neoplasms; Mice; Mice, Inbred C3H; Neoplasms, Experimental; Proteins; Pyridoxal Phosphate; Rats; Serine; Spectrophotometry; Tetrahydrofolates; Transferases

Topics: Agar; Alloys; Aluminum; Animals; Arsenic; Carcinoma, Hepatocellular; Cell Adhesion; Cell Division; Cell Line; Densitometry; Ethylmaleimide; Glass; Iodoacetates; Kinetics; Liver Neoplasms; Mercuribenzoates; Mercury; Polyethylenes; Polymers; Polytetrafluoroethylene; Rats; Silicones

Topics: Agar; Animals; Anti-Bacterial Agents; Antineoplastic Agents; Ascites; Carcinoma, Hepatocellular; Clone Cells; Culture Techniques; Diffusion; Liver Neoplasms; Mercaptopurine; Methods; Mitomycins; Neoplasms, Experimental; Nitrogen Mustard Compounds; Rats; Thiotepa

Topics: Agar; Animals; Carcinoma; Carcinoma, Hepatocellular; Chemical Precipitation; Cricetinae; Electrophoresis; gamma-Globulins; In Vitro Techniques; Kidney; Liver Neoplasms; Neoplasm Proteins; Neoplasms, Experimental; Proteins; Quaternary Ammonium Compounds; Rats; Tissue Extracts

Topics: Agar; Animals; Antigens; Carcinoma, Hepatocellular; Cytoplasm; Liver; Liver Neoplasms; Rats