g(m1)-ganglioside and Carcinoma--Hepatocellular

The hepatitis B virus (HBV)-induced chronic liver diseases are serious health threats worldwide. There is evidence to display the alterations of salivary N-linked glycans related to the development of HBV-infected liver diseases. Here, we further investigated the alterations of fucosylated N/O-glycans recognized by LTL in saliva from 120 subjects (30 healthy volunteers (HV), 30 patients with hepatitis B (HB), 30 patients with hepatic cirrhosis (HC), and 30 patients with hepatocellular carcinoma (HCC)) using salivary microarrys and MALDI-TOF/TOF-MS. The results showed that the expression level of fucosylated glycans recognized by LTL was significantly increased in HCC compared with other subjects (P < 0.0001). Besides, the fucosylated glycoproteins were isolated from pooled saliva of HV, HB, HC, and HCC by LTL-magnetic particle conjugates. Then, N/O- glycans were released from the isolated glycoproteins with PNGase F and NaClO, and were identified by MALDI-TOF-MS, respectively. Totally, there were 21/20, 25/18, 29/19, and 28/24 N/O-glycan peaks that were identified and annotated with proposed structures in saliva of HV, HB, HC, and HCC. Among the total, there were 8 N-glycan peaks (e.g., m/z 1905.634, 2158.777 and 2905.036) and 15 O-glycan peaks (e.g., 1177.407, 1308.444 and 1322.444) that only presented in patients with HBV-induced liver diseases. One N-glycan peak (m/z 2205.766) was unique in HC, and 9 O-glycan peaks (e.g., m/z 1157.420, 1163.417 and 1193.402) were unique in HCC. This study could facilitate the discovery of biomarkers for HC and HCC based on precise alterations of fucosylated N/O-glycans in saliva.

Topics: Biomarkers, Tumor; Carcinoma, Hepatocellular; Female; Fibrosis; G(M1) Ganglioside; Hepatitis B virus; Hepatitis, Chronic; Humans; Lectins; Liver Neoplasms; Male; Polysaccharides; Protein Array Analysis; Saliva; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Therefore, developing the early, high-sensitivity diagnostic biomarkers to prevent HCC is urgently needed. Serum a-fetoprotein (AFP), the clinical biomarker in current use, is elevated in only ~60% of patients with HCC; therefore, identification of additional biomarkers is expected to have a significant impact on public health. In this study, we used glycan microarray analysis to explore the potential diagnostic value of several cancer-associated carbohydrate antigens (CACAs) as biomarkers for HCC. We used glycan microarray analysis with 58 different glycan analogs for quantitative comparison of 593 human serum samples (293 HCC samples; 133 chronic hepatitis B virus (HBV) infection samples, 134 chronic hepatitis C virus (HCV) infection samples, and 33 healthy donor samples) to explore the diagnostic possibility of serum antibody changes as biomarkers for HCC. Serum concentrations of anti-disialosyl galactosyl globoside (DSGG), anti-fucosyl GM1 and anti-Gb2 were significantly higher in patients with HCC than in chronic HBV infection individuals not in chronic HCV infection patients. Overall, in our study population, the biomarker candidates DSGG, fucosyl GM1 and Gb2 of CACAs achieved better predictive sensitivity than AFP. We identified potential biomarkers suitable for early detection of HCC. Glycan microarray analysis provides a powerful tool for high-sensitivity and high-throughput detection of serum antibodies against CACAs, which may be valuable serum biomarkers for the early detection of persons at high risk for HCC.

Topics: alpha-Fetoproteins; Antibodies, Viral; Antigens, Neoplasm; Biomarkers, Tumor; Carcinoma, Hepatocellular; Early Diagnosis; Female; G(M1) Ganglioside; Gangliosides; Hepacivirus; Hepatitis B virus; Hepatitis B, Chronic; Hepatitis C, Chronic; Humans; Liver Neoplasms; Male; Polysaccharides; Sensitivity and Specificity; Sex Factors; Tissue Array Analysis

Restoration of cell volume in the continued presence of osmotic stimuli is essential, particularly in hepatocytes, which swell upon nutrient uptake. Responses to swelling involve the Ca2+-dependent activation of K+ channels, which promote fluid efflux to drive volume recovery; however, the channels involved in hepatocellular volume regulation have not been identified. We found that hypotonic exposure of HTC hepatoma cells evoked the opening of 50 pS K+-permeable channels, consistent with intermediate conductance (IK) channels. We isolated from rat liver and HTC cells a cDNA with sequence identity to the coding region of IK1. Swelling-activated currents were inhibited by transfection with a dominant interfering IK1 mutant. The IK channel blockers clotrimazole and TRAM-34 inhibited whole cell swelling-activated K+ currents and volume recovery. To determine whether IK1 underwent volume-sensitive localization, we expressed a green fluorescent protein fusion of IK1 in HTC cells. The localization of IK1 was suggestive of distribution in lipid rafts. Consistent with this, there was a time-dependent increase in colocalization between IK1 and the lipid raft ganglioside GM1 on the plasma membrane, which subsequently decreased with volume recovery. Pharmacological disruption of lipid rafts altered the plasma membrane distribution of IK1 and inhibited volume recovery after hypotonic exposure. Collectively, these findings support the hypothesis that IK1 regulates compensatory responses to hepatocellular swelling and suggest that regulation of cell volume involves coordination of signaling from lipid rafts with IK1 function.

Topics: Animals; Calcium; Carcinoma, Hepatocellular; Cell Membrane; G(M1) Ganglioside; Green Fluorescent Proteins; Hepatocytes; Intermediate-Conductance Calcium-Activated Potassium Channels; Liver; Male; Membrane Microdomains; Rats; Rats, Sprague-Dawley; Signal Transduction; Transfection

Hypoxia-inducible factor 1 alpha (HIF-1alpha), a component of HIF-1, is expressed in human tumors and renders cells able to survive and grow under hypoxic (low-oxygen) conditions. YC-1, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole, an agent developed for circulatory disorders that inhibits platelet aggregation and vascular contraction, inhibits HIF-1 activity in vitro. We tested whether YC-1 inhibits HIF-1 and tumor growth in vivo.. Hep3B hepatoma, NCI-H87 stomach carcinoma, Caki-1 renal carcinoma, SiHa cervical carcinoma, and SK-N-MC neuroblastoma cells were grown as xenografts in immunodeficient mice (69 mice total). After the tumors were 100-150 mm(3), mice received daily intraperitoneal injections of vehicle or YC-1 (30 microg/g) for 2 weeks. HIF-1 alpha protein levels and vascularity in tumors were assessed by immunohistochemistry, and the expression of HIF-1-inducible genes (vascular endothelial growth factor, aldolase, and enolase) was assessed by reverse transcription-polymerase chain reaction. All statistical tests were two-sided.. Compared with tumors from vehicle-treated mice, tumors from YC-1-treated mice were statistically significantly smaller (P<.01 for all comparisons), expressed lower levels of HIF-1 alpha (P<.01 for all comparisons), were less vascularized (P<.01 for all comparisons), and expressed lower levels of HIF-1-inducible genes, regardless of tumor type.. The inhibition of HIF-1 alpha activity in tumors from YC-1-treated mice is associated with blocked angiogenesis and an inhibition of tumor growth. YC-1 has the potential to become the first antiangiogenic anticancer agent to target HIF-1 alpha.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Carcinoma; Carcinoma, Hepatocellular; Cell Hypoxia; Culture Media, Conditioned; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Female; G(M1) Ganglioside; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoblotting; Indazoles; Intercellular Signaling Peptides and Proteins; Kidney Neoplasms; Killer Cells, Natural; Liver Neoplasms; Lymphokines; Male; Mice; Mice, SCID; Neoplasms; Neovascularization, Pathologic; Neuroblastoma; Platelet Endothelial Cell Adhesion Molecule-1; Precipitin Tests; Rats; Reverse Transcriptase Polymerase Chain Reaction; Stomach Neoplasms; Transcription Factors; Transplantation, Heterologous; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

A GDP-fucose:GM1 alpha1-->2 fucosyltransferase (FucT) is induced during early stages of chemical hepatocarcinogenesis in parenchymal cells of Fischer 344 rats fed a diet supplemented with 0.03% N-2-acetylaminofluorene (AAF). This enzyme is undetectable in normal rat liver tissues but is highly expressed in many rat hepatoma cell lines, including rat hepatoma H35 cells. Enzymatic properties and acceptor specificity of native rat hepatoma H35 cell alpha1-->2FucT, expressed recombinant full-length H35 cell alpha1-->2FucT, and a truncated form missing the first 27 amino acid residues from the N-terminus, comprising the cytoplasmic and transmembrane domains of the enzyme, were studied. The results indicate that the recombinant full-length enzyme has a specific activity over 80-fold higher than the truncated enzyme. Both the native and recombinant full-length enzymes display significant activity in the absence of detergent or phospholipid and optimal activity in the presence of Triton CF-54 detergent. The truncated enzyme is optimally activated by CHAPSO, showing little activity in its absence. These findings are in agreement with previous studies demonstrating a requirement of a lipidic environment for optimal activity with this enzyme and suggest that the N-terminal transmembrane domain is important either in the maintenance of an active conformation or in allowing efficient interaction with acceptor glycolipids. Both the full-length and truncated enzymes transfer fucose not only to GM1 and asialo-GM1 (Gg4) but also to galactosyl globoside (Gb5) as well. Weak or undetectable transfer to lacto- and neolacto-series acceptors was observed, demonstrating a strong preference for terminal Galbeta1-->3GalNAc- structures. The structures of two reaction products generated by expressed recombinant full-length alpha1-->2FucT, which are known to be important tumor-associated antigens (fucosyl-GM1 and fucosyl-Gb5), were unambiguously confirmed by 1H-NMR spectral analysis.

Topics: Amino Acid Sequence; Animals; Antigens, Tumor-Associated, Carbohydrate; Base Sequence; Carbohydrate Sequence; Carcinoma, Hepatocellular; COS Cells; Detergents; Enzyme Activation; Fucosyltransferases; G(M1) Ganglioside; Galactoside 2-alpha-L-fucosyltransferase; Glycosphingolipids; Humans; Liver Neoplasms, Experimental; Male; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments; Phospholipids; Rats; Rats, Inbred F344; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Deletion; Stage-Specific Embryonic Antigens; Substrate Specificity; Transfection; Tumor Cells, Cultured

We have studied the neutral glycolipid composition of spontaneous hepatomas in LEC female rats. Neutral lipid fractions were isolated and purified by column chromatographies on DEAE-Toyopearl 650(M) and Iatrobeads. The neutral glycolipid fraction contained 3.2 to 4.4 micrograms lipid-bound glucose (Glc) per mg protein, and consisted of isogloboside (iso-Gb4, 50.8% of total neutral glycolipids) and IV3Gal, IV2Fuc, GgOse4Cer (asialo-BGM1, 13.5%) as the major neutral glycolipids and Gb3 and iso-Gb3 (9.2%), GlcCer (7.2%), LacCer (6.1%) as the other species. The structure of iso-Gb4 was elucidated by gas-liquid chromatography (GLC), permethylation study, liquid secondary ion (LSI) mass spectrometry, and nuclear Overhauser enhancement spectroscopy (NOESY) and that for asialo-BGM1 by GLC, LSI mass spectrometry, and high-performance thin-layer chromatography (HPTLC)-overlay method using anti-asialo-BGM1 antibody. Isogloboside and asialo-BGM1 which are found in negligible amounts in normal liver tissues may represent excellent markers for studying tumor metastasis and cellular adhesion.

Topics: ABO Blood-Group System; Animals; Biomarkers, Tumor; Carbohydrate Sequence; Carcinoma, Hepatocellular; Epitopes; Female; G(M1) Ganglioside; Globosides; Glycolipids; Liver Neoplasms; Male; Molecular Sequence Data; Rats

The use of lymphokine activated killer (LAK) cells for adoptive transfer therapy has been reported from number of clinical trials. To our knowledge, however, there has been no report concerning the cell cycle progression of LAK cells. Thus, for the present study we have attempted to examine the LAK cell cycle either before or after transfer. In vitro and in vivo analyses of LAK cells labeled with bromodeoxyuridine (BrdU) were carried out using two-parameter flow cytometries of their nuclear staining using fluorescein isothiocyanate(FITC)-conjugated anti-BrdU antibody and propidium iodide (PI). The in vitro growth of BrdU-positive cells showed the cells to divide once in the S phase, continue to the G2-M phase and return to the G0-G1 phase with a similar pattern after 48 or 72 h culture. They formed a definite subpopulation and were of phenotypes, thy-1.2 (+), Lyt-1.1 (-), Lyt-2.1 (+), L3T4 (-) and AGM1 (+). The percentage of BrdU-positive cells decreased significantly (P < 0.05) when treated with complement plus anti-thy-1.2, anti-Lyt-2.1 or anti-AGM1 anti-bodies, demonstrating BrdU-labeled LAK cells to have the same phenotype as control LAK cells. As in vitro, the in vivo cell cycle of LAK cells 48 and 72 h after transfer had a similar pattern, and the LAK cells also continued on to the S phase after the first division. The in vivo growth of the LAK cells treated with interleukin-2 (IL-2) was promoted 24 and 48 h after transfer. In conclusion, the present study showed LAK cells to be capable of dividing following transfer and to keep their own phenotypic characteristics; also, that treatment with IL-2 may promote their division. Adoptive transfer therapy seems to be a viable therapeutic method.

Topics: Animals; Bromodeoxyuridine; Carcinoma, Hepatocellular; Cell Cycle; Complement System Proteins; Cytotoxicity, Immunologic; Flow Cytometry; Fluorescent Antibody Technique; G(M1) Ganglioside; Immunotherapy, Adoptive; Interleukin-2; Isoantibodies; Killer Cells, Lymphokine-Activated; Male; Mice; Mice, Inbred C3H; Mice, Inbred Strains; Specific Pathogen-Free Organisms; Time Factors; Tumor Cells, Cultured

A 76-yr-old male patient with carcinomatous neuropathy associated with hepatic cell carcinoma, whose initial symptom was deep sensory disturbance followed by muscle weakness is described. The onset was subacute, followed by slow progression. Sural nerve biopsy, as well as electrophysiological examinations, revealed severe axonal degeneration without any evidence of demyelination. The autopsy findings were similar to findings described in the literature on carcinomatous neuropathy. Although carcinomatous neuropathy is usually associated with lung cancer, this report describes an association with hepatic cell carcinoma. The patient also had motor nerve involvement with positive serum anti-GM1 ganglioside antibody which decreased after immunosuppressant therapy in parallel with recovery of muscle weakness. The anti-GM1 ganglioside antibody may be involved in the pathogenesis of motor disturbance in the present case.

Topics: Aged; Antibodies; Carcinoma, Hepatocellular; G(M1) Ganglioside; Humans; Immunosuppressive Agents; Liver Neoplasms; Male; Nerve Degeneration; Neural Conduction; Neurons, Afferent; Peripheral Nervous System Diseases

Hyperplastic nodules and hepatocellular carcinomas were induced in livers of rats by a low-protein diet containing 0.05% of the carcinogen N-2-fluorenylacetamide. Ganglioside amounts and composition were determined for histologically different hepatocellular carcinomas and compared with those for control livers, hyperplastic nodules, and liver tissue surrounding hepatomas and nodules as well as those for livers of fetal, newborn, 1-week-old, weanling, and adult Sprague-Dawley rats. Ganglioside sialic acid levels were elevated above those of normal adult liver in all liver tissues following the carcinogen treatment regimen. Livers of fetal and newborn rats contained nearly twice the amount of ganglioside sialic acid on a protein or DNA basis as did livers of adult rats. Analyses of individual nodules and hepatomas revealed two populations of tumors in which the levels of ganglioside sialic acid were 2.3 and 3.8 times normal. Ganglioside sialic acid content was at hepatoma levels in small nodules. Individual gangliosides were evenly distributed between products of the monosialoganglioside and disialoganglioside pathways in normal liver with a ratio of [N-acetylneuraminic acid (sialic acid)] (NAN)-galactose (Gal)-N-acetylgalactosamine (GalNAc)-(NAN)-Gal-glucose (Glc)-ceramide (Cer) (GD1a) to Gal-GalNAc-(NAN)2-Gal-Glc-Cer (GD1b) of about one. In contrast, the monosialogangliosides predominated in liver tissues following administration of the carcinogen. Increased levels of specific monosialogangliosides were present in nodules, in liver of carcinogen-treated animals prior to the appearance of tumors, and in the liver tissues surrounding nodules and hepatomas. In single hepatomas, ganglioside patterns correlated with tumorigenicity. A well-differentiated hepatoma had a normal complement of most gangliosides but was deficient in trisialogangliosides. In a poorly diferentiated but well-circumscribed hepatoma, the relative levels of all higher gangliosides were reduced. The monosialoganglioside Gal-GalNAc-(NAN)-Gal-Glc-Cer (GM1) accounted for 80% of the total ganglioside in a poorly circumscribed and poorly differentiated hepatoma. The ganglioside pattern of fetal livers most closely resembled that of a poorly differentiated hepatoma. During the first week post natum, levels of all higher monosialogangliosides and disialogangliosides declined, but the decline was most pronounced for gangliosides GM1 and GD1a. The ratio of GM1 + GD1a to GD1b + NAN-Gal-GalNAc-(NAN)

Topics: 2-Acetylaminofluorene; Animals; Animals, Newborn; Carcinoma, Hepatocellular; Fluorenes; G(M1) Ganglioside; G(M2) Ganglioside; G(M3) Ganglioside; Gangliosides; Hyperplasia; Liver; Liver Neoplasms; Neoplasms, Experimental; Precancerous Conditions; Rats; Sialic Acids