angiogenin and Carcinoma--Hepatocellular

Angiogenin (ANG) is known to alter multiple cell behaviors by directly targeting downstream targets, but its role in hepatocellular carcinoma (HCC) remains to be elucidated. The expression of ANG in HCC cell lines was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The effects of ANG expression on cell proliferation, cell migration, and hallmarks of epithelial-mesenchymal transition (EMT) process were also investigated. The relationship between ANG and high mobility group AT-hook 2 (HMGA2) was evaluated. ANG expression was increased in HCC cell lines. Downregulating of ANG inhibits proliferation, migration, and EMT of HCC cells. The direct regulation of ANG on HMGA2 was verified by luciferase activity reporter assay and western blot assay. Furthermore, overexpression of HMGA2 reversed the inhibitory effects of ANG downregulation on HCC cell behaviors. Our results illustrated the mechanism that ANG promote the EMT of HCC through targeting HMGA2.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Epithelial-Mesenchymal Transition; HMGA2 Protein; Humans; Liver Neoplasms; Ribonuclease, Pancreatic; RNA, Small Interfering; Transfection; Up-Regulation

Patients with hepatitis B virus (HBV) infection are at a high risk of developing hepatocellular carcinoma (HCC). In this study, we aim to investigate the roles of HBV on angiogenin (ANG), as well as the effects on cell proliferation in presence of ANG down-regulation.. Serum ANG was determined by ELISA. The expression of ANG mRNA and protein in HCC cell lines with or without HBV/HBx were determined. Western blot and ELISA were conducted to determine the effects of HBV/HBx on IL-6 expression. The role of IL-6 on ANG was evaluated by IL-6 recombinant protein or IL-6 neutralizing antibody. Immunofluorescence staining was used to detect the nuclear translocation of ANG. MTT was performed to evaluate the relative inhibition ratio.. In vivo experiments showed elevation of serum ANG in patients infected with HBV. In vitro experiments showed HBV and HBx contributed to the transcription and translation of ANG. ANG expression showed increase after IL-6 stimulation, and ANG protein decreased in the presence of IL-6 blocking with its antibody. HBV promoted nuclear translocation of ANG. Inhibiting ANG expression or blocking of nuclear transfer of ANG attenuated the 45S rRNA synthesis and cell proliferation.. HBV and HBx protein can increase the level of ANG through IL-6. HBV and HBx contributed to the nuclear translocation of ANG. Cell proliferation was inhibited after inhibiting the expression or nuclear transfer of ANG.

Topics: Adult; Antibodies; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Female; Hep G2 Cells; Hepatitis B; Hepatitis B virus; Humans; Interleukin-6; Liver Neoplasms; Male; Middle Aged; Ribonuclease, Pancreatic; RNA Interference; RNA, Ribosomal; Trans-Activators; Up-Regulation; Viral Regulatory and Accessory Proteins

Persistent infections with hepatitis B virus (HBV) or hepatitis C virus (HCV) account for the majority of cases of hepatic cirrhosis and hepatocellular carcinoma (HCC) worldwide. Small, non-coding RNAs play important roles in virus-host interactions. We used high throughput sequencing to conduct an unbiased profiling of small (14-40 nts) RNAs in liver from Japanese subjects with advanced hepatitis B or C and hepatocellular carcinoma (HCC). Small RNAs derived from tRNAs, specifically 30-35 nucleotide-long 5' tRNA-halves (5' tRHs), were abundant in non-malignant liver and significantly increased in humans and chimpanzees with chronic viral hepatitis. 5' tRH abundance exceeded microRNA abundance in most infected non-cancerous tissues. In contrast, in matched cancer tissue, 5' tRH abundance was reduced, and relative abundance of individual 5' tRHs was altered. In hepatitis B-associated HCC, 5' tRH abundance correlated with expression of the tRNA-cleaving ribonuclease, angiogenin. These results demonstrate that tRHs are the most abundant small RNAs in chronically infected liver and that their abundance is altered in liver cancer.

Topics: Animals; Base Sequence; Carcinoma, Hepatocellular; Hepatitis B, Chronic; Hepatitis C, Chronic; High-Throughput Nucleotide Sequencing; Humans; Immunoprecipitation; Liver; Liver Neoplasms; MicroRNAs; Molecular Sequence Data; Pan troglodytes; Real-Time Polymerase Chain Reaction; Ribonuclease, Pancreatic; RNA, Transfer; Sequence Analysis, RNA

Hepatocellular carcinoma (HCC) frequently develops in a pro-inflammatory and pro-fibrogenic environment with hepatic stellate cells (HSCs) remodeling the extracellular matrix composition. Molecules secreted by liver tumors contributing to HSC activation and peritumoral stromal transformation remain to be fully identified. Here we show that conditioned medium from HCC cell lines, Hep3B and HepG2, induced primary mouse HSCs transdifferentiation, characterized by profibrotic properties and collagen modification, with similar results seen in the human HSC cell line LX2. Moreover, tumor growth was enhanced by coinjection of HepG2/LX2 cells in a xenograft murine model, supporting a HCC-HSC crosstalk in liver tumor progression. Protein microarray secretome analyses revealed angiogenin as the most robust and selective protein released by HCC compared to LX2 secreted molecules. In fact, recombinant angiogenin induced in vitro HSC activation requiring its nuclear translocation and rRNA transcriptional stimulation. Moreover, angiogenin antagonism by blocking antibodies or angiogenin inhibitor neomycin decreased in vitro HSC activation by conditioned media or recombinant angiogenin. Finally, neomycin administration reduced tumor growth of HepG2-LX2 cells coinjected in mice. In conclusion, angiogenin secretion by HCCs favors tumor development by inducing HSC activation and ECM remodeling. These findings indicate that targeting angiogenin signaling may be of potential relevance in HCC management.

Topics: Animals; Carcinoma, Hepatocellular; Hep G2 Cells; Hepatic Stellate Cells; Heterografts; Humans; Liver Neoplasms, Experimental; Mice; Neoplasm Proteins; Neoplasm Transplantation; Ribonuclease, Pancreatic

Staphylococcal nuclease domain-containing 1 (SND1) is a multifunctional protein that is overexpressed in multiple cancers, including hepatocellular carcinoma (HCC). Stable overexpression of SND1 in Hep3B cells expressing a low level of SND1 augments, whereas stable knockdown of SND1 in QGY-7703 cells expressing a high level of SND1 inhibits establishment of xenografts in nude mice, indicating that SND1 promotes an aggressive tumorigenic phenotype. In this study we analyzed the role of SND1 in regulating tumor angiogenesis, a hallmark of cancer. Conditioned medium from Hep3B-SND1 cells stably overexpressing SND1 augmented, whereas that from QGY-SND1si cells stably overexpressing SND1 siRNA significantly inhibited angiogenesis, as analyzed by a chicken chorioallantoic membrane assay and a human umbilical vein endothelial cell differentiation assay. We unraveled a linear pathway in which SND1-induced activation of NF-κB resulted in induction of miR-221 and subsequent induction of angiogenic factors Angiogenin and CXCL16. Inhibition of either of these components resulted in significant inhibition of SND1-induced angiogenesis, thus highlighting the importance of this molecular cascade in regulating SND1 function. Because SND1 regulates NF-κB and miR-221, two important determinants of HCC controlling the aggressive phenotype, SND1 inhibition might be an effective strategy to counteract this fatal malady.

Topics: Carcinoma, Hepatocellular; Cell Nucleus; Chemokine CXCL16; Chemokines, CXC; Endonucleases; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Liver Neoplasms; MicroRNAs; Neoplasm Metastasis; Neovascularization, Pathologic; NF-kappa B; Nuclear Proteins; Receptors, Scavenger; Ribonuclease, Pancreatic; RNA, Small Interfering

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Basic fibroblast growth factor (bFGF), which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Its expression is essential for the progression and metastasis of HCC. This study aims to investigate the effects of bFGF on the expression of angiogenin, another growth factor, which plays an important role in tumor angiogenesis, and on cell proliferation in H7402 human hepatoma cells. The bFGF sense cDNA or antisense cDNA was stably transfected into H7402 cells. Genomic DNA PCR analysis demonstrated that human bFGF sense cDNA or antisense cDNA was inserted into the genome. Furthermore, the expression of bFGF and angiogenin was examined by RT-PCR and Western blot assays. MTT and colony formation assays were employed to determine cell proliferation. Stable bFGF over-expressing and under-expressing transfectants were successfully established. Expression of angiogenin was decreased in the over-expressing bFGF cells (sense transfectants) and was increased in the under-expressing bFGF cells (antisense transfectants). Cell proliferation increased in the bFGF sense transfectants and decreased in the bFGF antisense transfectants. These results demonstrated that the endogenous bFGF may not only negatively regulate the angiogenin expression but also contribute to the overall cell proliferation in H7402 human hepatoma cells. This study may be helpful in finding a potential therapeutic approach to HCC.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; DNA; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Genome, Human; Humans; Liver Neoplasms; Ribonuclease, Pancreatic; Transgenes

Physiological angiogenesis occurs during liver regeneration, leading to the formation of new functional sinusoids. Pathological angiogenesis occurs in hepatocellular carcinoma (HCC). We aimed to evaluate the expression of angiogenic factors in hepatitis C virus (HCV)-HCC tissues and the utility of angiogenesis soluble factors as noninvasive markers of HCC and tumor growth.. Thirty-eight HCV-HCC tumors with 10 corresponding nontumor cirrhotic tissues, as well as 42 independent HCV cirrhotic and 6 normal liver tissues were studied using high-density oligonucleotide arrays. Human angiogenesis microarray was used for the protein detection of EGF, TIMP-1, TIMP-2, HGF, angiopn-1, angiopn-2, VEGF-A, IP-10, PDGF, KGF, angiogenin, VEGF-D, ICAM-1, and FGF in plasma samples from 40 patients (30 HCCs and 10 HCV cirrhosis).. From the gene expression analysis of the HCV-HCC tumors compared to normal livers, we found an important number of genes related to angiogenesis differentially expressed (alpha=0.01), including VEGF, PDGF, AGPTL2, ANG, EGFL6, EGFR, angiopn-1, angiopn-2, ICAM2, TIMP-2, among others. Moreover, angiogenic genes were also differentially expressed when HCV-HCC samples were compared to HCV cirrhotic tissues (alpha=0.01; VEGF, EGFL3, EGFR, VEGFB, among others). Ten out of 14 angiogenic proteins analyzed were statistically differentially expressed between HCV cirrhosis and HCV-HCC groups (TIMP-1, TIMP-2, HGF, angiopn-1, angiopn-2, VEGF-A, IP-10, PDGF, KGF, and FGF; P<0.05). In addition, we observed that angiopn-2 was the most significant predictor (area under the curve: 0.83).. Differentially expressed angiogenesis genes were observed between HCV patients with and without HCC. Soluble angiogenic factors might be useful for monitoring high-risk HCV patients.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Female; Fibroblast Growth Factors; Hepatitis C; Humans; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neovascularization, Physiologic; Oligonucleotide Array Sequence Analysis; Platelet-Derived Growth Factor; Ribonuclease, Pancreatic; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Vascular Endothelial Growth Factor A; Waiting Lists

Hepatocellular carcinoma (HCC) is a hypervascular malignancy. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and angiogenin (ANG) are important angiogenic factors of neoangiogenesis. This study investigated the predictive value of serum VEGF, bFGF, and ANG in tumor recurrence, disease-free survival (DFS), and overall survival (OS) in HCC patients.. Preoperative serum VEGF, bFGF, and ANG were measured in 98 patients with resectable HCC and in 15 healthy controls. The median follow-up time was 43 months.. Preoperative serum VEGF was increased in patients with resectable HCC compared with healthy controls (P <.05). Increased serum VEGF was correlated with tumor recurrence (P =.001). Univariate analysis showed that serum VEGF, tumor-node-metastasis stage, tumor size and number, macroscopic portal vein invasion, and microscopic vascular invasion were correlated with OS and DFS. Serum bFGF and ANG were not associated with survival. Multivariate analysis showed that serum VEGF was the most significant predictor of DFS (relative risk, 2.35; 95% confidence interval, 1.26-4.39; P =.007) and OS (relative risk, 3.44; 95% confidence interval, 1.81-6.57; P <.001) in HCC patients after surgical resection.. Preoperative serum VEGF is a significant independent predictor of tumor recurrence, DFS, and OS in patients with resectable HCC.

Topics: Carcinoma, Hepatocellular; Endothelial Growth Factors; Female; Fibroblast Growth Factor 2; Humans; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Lymphokines; Male; Middle Aged; Multivariate Analysis; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Portal Vein; Prognosis; Ribonuclease, Pancreatic; Survival Analysis; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Neovascularization is known to be one of the major characteristics of human hepatocellular carcinoma (HCC). Angiogenin (ANG), originally discovered in a human colon cancer cell line, is a liver-derived polypeptide that shows strong angiogenic activity in vivo. However, the role of ANG on the development of HCC remains unknown. The present study was designed to examine the implication of ANG in the neovascularization of human HCC.. Forty-one HCC patients who had undergone conventional celiac angiography were used in this study. ANG protein expression and microvessel density (MVD) in HCC specimens obtained by liver biopsy or surgical resection were examined by immunohistochemistry, and the levels were quantified by the KS-400 image analyzing system. ANG mRNA expression in liver tissues was evaluated by in situ hybridization. Serum ANG concentrations were measured by an ELISA. Survival rates were calculated using the Kaplan-Meier method.. Immunohistochemistry and in situ hybridization showed greater increments of ANG protein expression and mRNA expression, respectively, in HCC tissues than in the surrounding nontumorous tissues. MVD within tumorous tissues increased according to dedifferentiation of the histological grade of HCC, showing a significant correlation (r = 0.877, P = 0.0009) with ANG expression levels. Mean +/- SD serum ANG levels of healthy subjects and chronic hepatitis (CH) patients were 362.3 +/- 84.1 ng/ml and 331.9 +/- 133.8 ng/ml, respectively, with no significant difference. Serum ANG levels of liver cirrhosis patients (242.4 +/- 126.9 ng/ml) were lower than those of healthy subjects or CH patients and decreased as the fibrosis grade advanced. In HCC patients, despite the cirrhotic background, serum ANG levels increased as the tumor vascularity increased (197.8 +/- 64.9 ng/ml for hypovascular, 326.7 +/- 148.6 ng/ml for hypervascular, and 405.0 +/- 121.3 ng/ml for very hypervascular), in good accordance with histological grading, and significantly decreased (P = 0.015) after successful treatment with transcatheter arterial embolization or percutaneous ethanol injection. HCC patients were conventionally divided into two groups according to the serum level of ANG, those with values higher than the mean level (332.9 +/- 143.8 ng/ml) and those with values lower than the mean,; the 5-year survival rate of the latter group was determined to be significantly higher than that in the former group.. These results suggest that ANG is one of the neovascularization defining factors of HCC. Thus, measuring serum ANG may assist in monitoring the disease, and targeting ANG may provide a new strategy for treating advanced HCC.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunohistochemistry; In Situ Hybridization; Liver; Liver Neoplasms; Male; Middle Aged; Neovascularization, Pathologic; Ribonuclease, Pancreatic; RNA, Messenger; Time Factors

The polypeptide ligand angiogenin, a potent inducer of angiogenesis, localizes in the nucleus/nucleolus subsequent to endocytosis by relevant cell types. This study examines the kinetic properties of the nucleolar targeting signal (NTS) of angiogenin (IMRRRGL(35)) at the single cell level. We show that the NTS is sufficient to target green fluorescent protein (GFP), but not beta-galactosidase, to the nucleolus of rat hepatoma cells. Mutation of Arg(33) to Ala within the NTS abolishes targeting activity. Nuclear/nucleolar import conferred by the NTS of angiogenin is reduced by cytosolic factors as well as ATP and is independent of importins and Ran. The NTS also confers the ability to bind to nuclear/nucleolar components which is inhibited by ATP hydrolysis; nonhydrolysable GTP analogs prevent nuclear accumulation in the absence of an intact nuclear envelope through an apparent cytoplasmic retention mechanism. Since the lectin wheat germ agglutinin does not inhibit transport, we postulate a mechanism for angiogenin nuclear/nucleolar import involving passive diffusion of angiogenin through the nuclear pore and NTS-mediated nuclear/nucleolar retention, and with cytoplasmic retention modulating the process. This pathway is clearly distinct from that of conventional signal-mediated nuclear protein import.

Topics: Active Transport, Cell Nucleus; Adenosine Triphosphate; Amino Acid Motifs; Animals; beta-Galactosidase; Carcinoma, Hepatocellular; Cell Nucleolus; Cell Nucleus; Cholic Acids; Cytosol; Detergents; Genes, Reporter; Green Fluorescent Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Liver Neoplasms, Experimental; Luminescent Proteins; Mutagenesis, Site-Directed; Protein Binding; ran GTP-Binding Protein; Rats; Recombinant Fusion Proteins; Ribonuclease, Pancreatic; Tumor Cells, Cultured; Wheat Germ Agglutinins

Angiogenin is a potent inducer of neovascularization in vivo. However, like other angiogenic molecules, its specific physiologic roles and mechanisms regulating its expression remain to be elucidated. Angiogenin is a liver-derived component of normal serum whose concentration can increase in various disease states. This suggests that it might participate in the acute-phase response. In an initial study we showed that angiogenin protein and mRNA levels transiently increased in mice following an acute inflammatory stimulus. We now report that IL-6, a major inducer of acute-phase proteins, stimulates the synthesis and secretion of angiogenin protein in human HepG2 cells within 24 hr following treatment, an effect enhanced by dexamethasone. IL-6 also increases the amount of angiogenin mRNA without altering its half-life. This increase, suppressible by cycloheximide, peaks at 12 hr following stimulation and returns to basal levels by 48 hr. IL-1 alone slightly decreases the basal production of angiogenin protein and mRNA, but essentially abolishes the response to IL-6 in the absence or presence of dexamethasone. This antagonistic effect by IL-1 on IL-6 activity is not a result of changes in mRNA stability nor is it dependent on new protein synthesis. Thus, the combined effects of IL-6, IL-1, glucocorticoids, and perhaps other related factors may specifically control angiogenin expression. Since angiogenin is regulated in a manner similar to that of acute phase proteins both in vitro and in vivo, it may play a role in the host response to injury.

Topics: Acute-Phase Proteins; Carcinoma, Hepatocellular; Cytokines; Fibrinogen; Gene Expression Regulation; Humans; Interleukin-1; Interleukin-6; Proteins; Ribonuclease, Pancreatic; RNA, Messenger; Time Factors; Tumor Cells, Cultured

Using digoxigenin-labelled, synthetic oligonucleotide probe cocktails of angiogenin and bFGF genes, the expression of the two genes was observed by in situ hybridization in ten colonic adenocarcinomas, seven gastric adenocarcinomas, and four hepatocellular carcinomas. The angiogenin gene was expressed in eight of the ten cases of colonic adenocarcinoma and in all of the four cases where dysplastic glands were found. Angiogenin expression was evident in four of the seven cases of gastric adenocarcinoma. bFGF expression was detected in only five of the seven gastric carcinoma cases. The mRNAs for angiogenin and bFGF were mainly cytoplasmic in distribution and were only occasionally seen in the nuclei of the positive cells. Neither the angiogenin gene mRNA nor the bFGF mRNA was expressed in the four cases of hepatocellular carcinoma. It is postulated that the angiogenin gene may play an important role in angiogenesis in colonic adenocarcinomas; in gastric cancers, both angiogenin and bFGF were involved in this process. For hepatocellular carcinomas, neither angiogenin nor bFGF production appeared to be related to angiogenesis.

Topics: Adenocarcinoma; Angiogenesis Inducing Agents; Carcinoma, Hepatocellular; Colonic Neoplasms; Fibroblast Growth Factor 2; Gastrointestinal Neoplasms; Humans; In Situ Hybridization; Liver Neoplasms; Neoplasm Proteins; Neovascularization, Pathologic; Proteins; Ribonuclease, Pancreatic; RNA, Messenger; Stomach Neoplasms