sepharose and Carcinoma--Hepatocellular

Folate ingestion below and above the physiologic dose has been shown to play a tumorigenic role in certain cancers. Also, excessive folate supplementation after establishment of pre-established lesions led to an advancement in the growth of a few tumors. However, such information has not yet been achieved in the case of HCC. In our study, HepG2 cells were administered with three different concentrations of folic acid i.e. folic acid normal (FN) (2.27 µM), folic acid deficient (FD) (no folic acid), folic acid oversupplementation (FO) (100 µM) for 10 days. Intracellular folate levels were assayed by Elecsys Folate III kit based method. The migratory and invasive abilities were estimated by transwell migration and matrigel invasion methods respectively. FACS was done to evaluate cell viability and apoptosis. Agarose-coated plates were used to access cancer stem cells (CSCs) number. Quantitative RT-PCR and western blotting approaches were used for gene and protein expression of certain tumor suppressor genes (TSGs), respectively. FD cells depicted increased migration, invasion, apoptosis, necrosis and decreased cell viability, CSCs. On the other hand, FO cells showed increased migration, invasion, cell viability and number of CSCs and decreased apoptosis and necrosis. TSGs revealed diminished expression with both FA modulations with respect to FN cells. Thus, FA deficiency as well as abundance enhanced the HCC progression by adapting different mechanisms.

Topics: Carcinogenesis; Carcinoma, Hepatocellular; Folic Acid; Hep G2 Cells; Humans; Liver Neoplasms; Necrosis; Sepharose

The present study aims to create and characterise a cell-embedding tissue-mimicking material (TMM) that has thermal and acoustic properties similar to liver tissue, in order to enable study and optimisation of protocols for ultrasound-induced hyperthermia and drug delivery.. An agarose-based, cell-embedding TMM was iteratively developed and characterised. The acoustic properties (attenuation coefficient, speed of sound and cavitation threshold) and thermal response of the material were compared with those of fresh degassed liver tissue over a range of acoustic pressures and frequencies. A luminescence intensity assay was used to evaluate viability of HuH-7 cells in the material. The efficacy of ultrasound-mediated chemotherapeutic treatment in the material was tested by localised activation of low temperature thermally sensitive liposomes. Drug activation was measured by fluorescence microscopy.. Similar acoustic properties (attenuation coefficient, speed of sound) to liver tissue were achieved over the therapeutically relevant frequency range of 1-4 MHz and similar thermal response was achieved for acoustic pressures up to 4.8 MPa peak to peak (ppk) at 1.1 MHz. Above 4.8 MPa ppk cavitation enhanced heating occurred in the TMM. Drug release from low-temperature-sensitive liposomes was achieved with 4.4 MPa ppk 6-s exposures at 1.1 MHz and cell compatibility of the material was confirmed.. A platform for in vitro work for activation of thermally sensitive liposomes using high intensity focused ultrasound (HIFU)-induced hyperthermia was established. The TMM presents similar acoustic properties and thermal response to liver tissue over a broad range of ultrasound exposure conditions.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Doxorubicin; Drug Delivery Systems; Gels; High-Intensity Focused Ultrasound Ablation; Humans; Liposomes; Liver; Liver Neoplasms; Phantoms, Imaging; Sepharose; Tissue Embedding

A dimeric 64-kDa hemagglutinin was isolated with a high yield from dried Phaseolus vulgaris cultivar "French bean number 35" seeds using a chromatographic protocol that involved Blue-Sepharose, Q-Sepharose, and Superdex 75. The yield was exceptionally high (1.1g hemagglutinin per 100g seed), which is around 10-85 times higher than other Phaseolus cultivars. Its N-terminal sequence resembled those of other Phaseolus hemagglutinins. The hemagglutinating activity of the hemagglutinin was stable in the pH range 6-8, and in the temperature range 0 degrees C-50 degrees C. It inhibited HIV-1 reverse transcriptase with an IC50 of 2microM. It suppressed mycelial growth in Valsa mali with an IC50 of 10microM. It inhibited proliferation of hepatoma HepG2 cells and breast cancer MCF-7 cells with an IC50 of 100 and 2microM, respectively. It had no antiproliferative effect on normal embryonic liver WRL68 cells.

Topics: Antifungal Agents; Antineoplastic Agents, Phytogenic; Ascomycota; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Female; Hemagglutinins; Hep G2 Cells; Hepatocytes; HIV Reverse Transcriptase; HIV-1; Humans; Inhibitory Concentration 50; Liver Neoplasms; Mycelium; Phaseolus; Plant Extracts; Plant Lectins; Reverse Transcriptase Inhibitors; Seeds; Sepharose

A dimeric 70-kDa chymotrypsin inhibitor with substantial N-terminal sequence homology to serine protease inhibitors was isolated from Acacia confusa seeds. The chymotrypsin inhibitor was purified using a protocol that entailed ion exchange chromatography on Q-Sepharose, SP-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. The chymotrypsin inhibitor was unadsorbed on both Q-Sepharose and SP-Sepharose. Its chymotrypsin inhibitory activity was stable from pH 3 to 10 and from 0 to 50 degrees C. It exerted antiproliferative activity toward breast cancer MCF-7 cells with an IC(50) of 10.7+/-4.2 microM. It inhibited HIV-1 reverse transcriptase with an IC(50) of 8+/-1.5 microM. It was devoid of antifungal activity toward a variety of fungal species. The distinctive features of the chymotrypsin inhibitor included dimeric nature, a high molecular mass, lack of trypsin inhibitory activity, highly potent HIV-1 reverse transcriptase inhibitory activity, specific antitumor activity and relatively high pH-stability.

Topics: Acacia; Adsorption; Amino Acid Sequence; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chromatography; Chymotrypsin; Dimerization; Dose-Response Relationship, Drug; Female; Fungi; Hep G2 Cells; HIV Reverse Transcriptase; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Molecular Structure; Phytotherapy; Plant Proteins; Reverse Transcriptase Inhibitors; Seeds; Sepharose; Sequence Homology; Temperature

Whereas hepatocytes secrete the major human plasma high density lipoproteins (HDL)-protein, apo A-I, as lipid-free and lipidated species, the biogenic itineraries of apo A-II and apo E are unknown. Human plasma and HepG2 cell-derived apo A-II and apo E occur as monomers, homodimers and heterodimers. Dimerization of apo A-II, which is more lipophilic than apo A-I, is catalyzed by lipid surfaces. Thus, we hypothesized that lipidation of intracellular and secreted apo A-II exceeds that of apo A-I, and once lipidated, apo A-II dimerizes. Fractionation of HepG2 cell lysate and media by size exclusion chromatography showed that intracellular apo A-II and apo E are fully lipidated and occur on nascent HDL and VLDL respectively, while only 45% of intracellular apo A-I is lipidated. Secreted apo A-II and apo E occur on small HDL and on LDL and large HDL respectively. HDL particles containing both apo A-II and apo A-I form only after secretion from both HepG2 and Huh7 hepatoma cells. Apo A-II dimerizes intracellularly while intracellular apo E is monomeric but after secretion associates with HDL and subsequently dimerizes. Thus, HDL apolipoproteins A-I, A-II and E have distinct intracellular and post-secretory pathways of hepatic lipidation and dimerization in the process of HDL formation. These early forms of HDL are expected to follow different apolipoprotein-specific pathways through plasma remodeling and reverse cholesterol transport.

Topics: Apolipoprotein A-I; Apolipoprotein A-II; Apolipoproteins E; Carcinoma, Hepatocellular; Chromatography, Gel; Hep G2 Cells; Humans; Intracellular Space; Lipoproteins, HDL; Liver Neoplasms; Models, Biological; Protein Binding; Protein Multimerization; Sepharose; Time Factors; Ultracentrifugation

We recently developed a sensitive method using biotin-N-maleimide (biotin-NM) as a probe to positively identify oxidized mitochondrial proteins. In this study, biotin-NM was used to identify oxidized cytosolic proteins in alcohol-fed mouse livers. Alcohol treatment for 6 wk elevated the levels of CYP2E1 and nitrotyrosine, a marker of oxidative stress. Markedly increased levels of oxidized proteins were detected in alcohol-fed mouse livers compared to pair-fed controls. The biotin-NM-labeled oxidized proteins from alcohol-exposed mouse livers were subsequently purified with streptavidin-agarose and resolved on 2-DE. More than 90 silver-stained protein spots that displayed differential intensities on 2-D gels were identified by MS. Peptide sequence analysis revealed that many enzymes or proteins involved in stress response, chaperone activity, intermediary metabolism, and antioxidant defense systems such as peroxiredoxin were oxidized after alcohol treatment. Smaller fragments of many proteins were repeatedly detected only in alcohol-fed mice, indicating that many oxidized proteins after alcohol exposure were degraded. Immunoblot results showed that the level of oxidized peroxiredoxin (inactivated) was markedly increased in the alcohol-exposed mouse livers and ethanol-sensitive hepatoma cells compared to the corresponding controls. Our results may explain the underlying mechanism for cellular dysfunction and increased susceptibility to other toxic agents following alcohol-mediated oxidative stress.

Topics: Animals; Bacterial Proteins; Biomarkers, Tumor; Biotin; Carcinoma, Hepatocellular; Central Nervous System Depressants; Computational Biology; Cytochrome P-450 CYP2E1; Cytosol; Electrophoresis, Gel, Two-Dimensional; Ethanol; Liver; Liver Extracts; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Oxidation-Reduction; Peroxidases; Peroxiredoxins; Sepharose; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tyrosine

Heavy alcohol consumption can damage various cells and organs partly through production of reactive oxygen species (ROS) and mitochondrial dysfunction. Treatment with antioxidants can significantly reduce the degree of damage. Despite well established roles of ROS in alcohol-induced cell injury, the proteins that are selectively oxidized by ROS are poorly characterized. We hypothesized that certain cysteinyl residues of target proteins are oxidized by ROS upon alcohol exposure, and these modified proteins may play roles in mitochondrial dysfunction. A targeted proteomics approach utilizing biotin-N-maleimide (biotin-NM) as a specific probe to label oxidized cysteinyl residues was employed to investigate which mitochondrial proteins are modified during and after alcohol exposure. Human hepatoma HepG2 cells with transduced CYP2E1 (E47 cells) were used as a model to generate ROS through CYP2E1-mediated ethanol metabolism. Following exposure to 100 mM ethanol for 4 and 8 h, the biotin-NM-labeled oxidized proteins were purified with agarose coupled to either streptavidin or monoclonal antibody against biotin. The purified proteins were resolved by two-dimensional gel electrophoresis and protein spots that displayed differential abundances were excised from the gel, in-gel digested with trypsin and analyzed for identity utilizing either matrix-assisted laser desorption-time of flight mass spectrometry or microcapillary reversed-phase liquid chromatography-tandem mass spectrometry. The results demonstrate that heat shock protein 60, protein disulfide isomerase, mitochondrial aldehyde dehydrogenases, prohibitin, and other proteins were oxidized after alcohol exposure. The identity of some of the proteins purified with streptavidin-agarose was also confirmed by immunoblot analyses using the specific antibody to each target protein. This method was also used to identify oxidized mitochondrial proteins in the alcohol-fed mouse liver. These results suggest that exposure to ethanol causes oxidation of various mitochondrial proteins that may negatively affect their function and contribute to alcohol-induced mitochondrial dysfunction and cellular injury.

Topics: Alcohol Drinking; Animals; Bacterial Proteins; Biotin; Carcinoma, Hepatocellular; Cysteine; Electrophoresis, Gel, Two-Dimensional; Ethanol; Humans; Liver; Mice; Mitochondria; Mitochondrial Proteins; Oxidation-Reduction; Sepharose; Sequence Analysis, Protein

To purify hsp70 protein and observe its anti-tumor effect on mouse H22 hepatoma.. Cytosolic hsp70 protein of heat treated H22 cells was purified successively by chromatographic procedures, including DEAE 52-cellulose, ConA-sepharose 4B and ADP-argarose chromatography. The molecular weight and the identity of the purified hsp70 protein were confirmed by SDS-PAGE and Western blot. Tumorigenicity of H22 hepatoma was examined in purified hsp70-treated C3H mice.. A sharp stained protein band with a molecular weight of about 70 kD was obtained and shown to be hsp70 as confirmed by Western blot. In C3H mice challenged with H22 hepatoma, tumor developed in 6 of 6 mice while in hsp70 pretreated mice, none of the 6 H22 challenged mice developed tumor. Nevertheless, C57BL/6 mice pretreated with hsp70 or RPMI 1640 all developed tumor upon challenge with EL-4 lymphoma.. The results suggest that the immunoprotective effect of hsp70 protein is due not to hsp70 per se, but rather to the specific antigenic peptides it carries.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Disease Models, Animal; HSP70 Heat-Shock Proteins; Liver Neoplasms, Experimental; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Neoplasm Transplantation; Sepharose; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Recent studies have revealed binding of mitochondrial enoyl-CoA isomerase (ECI) to S-hexylglutathione-Sepharose, an affinity matrix used for purification of glutathione transferases (GSTs), and the enzyme has been suggested to be identical with the Alpha class form of GST with a subunit molecular mass of about 30 kDa. In the present study, S-hexylglutathione-binding proteins of human hepatocellular carcinomas were characterized to examine their identity. Supernatant fractions of carcinoma and surrounding tissues were applied to an affinity column, and bound fractions were resolved into three proteins with subunit molecular masses/pI values of 33 kDa/7.0, 30 kDa/5.8 and 29 kDa/5.8 in addition to the well-characterized four GST subunits, A1, A2, M1 and P1, by two-dimensional gel electrophoresis. The proteins were further purified by chromatofocusing at pH 7.4-4.0. The 30 and 29 kDa proteins were eluted at pH 4.9 and by 1 M NaCl respectively, and could be clearly separated from each other. The 29 kDa protein exhibited a low but significant activity towards 1-chloro-2,4-dinitrobenzene (4.25 micromol/min per mg of protein) and reacted with anti-(GST A1-2) antibody, suggesting that it is a member of the GST Alpha class. The 30 kDa protein did not react with anti-GST antibodies and was identified as ECI by immunoblotting and N-terminal-amino-acid-sequencing analyses. The results thus indicated that the Alpha class GST form composed of the 29 kDa subunits and ECI are two different proteins. The 33 kDa protein was eluted from the chromatofocusing column at pH 7.0 and did not react with either anti-GST antibodies or antibodies against mitochondrial enzymes involved in the beta-oxidation of fatty acids. However, it exhibited a carbonyl reductase activity with menadione and ubiquinone, and amino acid sequences of its peptides cleaved by Staphylococcus aureus V8 proteinase were consistent with those reported for the enzyme. Thus this protein binding to S-hexylglutathione-Sepharose was identified as carbonyl reductase.

Topics: Aged; Carbon-Carbon Double Bond Isomerases; Carcinoma, Hepatocellular; Chromatography, Affinity; Dodecenoyl-CoA Isomerase; Glutathione; Glutathione Transferase; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Proteins; Protein Binding; Sepharose

Topics: Adult; Aged; Arthritis, Rheumatoid; Carcinoma, Hepatocellular; Cell Line; Chromatography, Affinity; Concanavalin A; Female; Humans; Liver Neoplasms; Male; Orosomucoid; Protein Binding; Sepharose; Tumor Cells, Cultured

Topics: Animals; Carcinoma, Hepatocellular; Cells, Cultured; Chromatography, Affinity; gamma-Glutamyltransferase; Humans; Liver; Liver Neoplasms; Liver Neoplasms, Experimental; Neuraminidase; Phytohemagglutinins; Rats; Sepharose; Species Specificity

We used affinity chromatography on concanavalin A Sepharose to study the serum alpha-fetoprotein of 10 patients with histologically proven germ-cell tumors and 12 patients with primary liver cancer. Less than 50% of the fetoprotein from germ-cell tumors bound to concanavalin A, as compared with more than 80% of the alpha-fetoprotein from primary liver cancers.

Topics: Adult; Aged; alpha-Fetoproteins; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Female; Humans; Liver Neoplasms; Male; Mesonephroma; Middle Aged; Ovarian Neoplasms; Sepharose; Testicular Neoplasms

Topics: Animals; Antibodies, Neoplasm; Carcinoma, Hepatocellular; Complement System Proteins; Concanavalin A; Cytotoxicity, Immunologic; Guinea Pigs; Immunoglobulin G; Immunoglobulin M; Liver Neoplasms; Neoplasms, Experimental; Rabbits; Sepharose; Staphylococcus aureus

tRNAAsp from rabbit liver, rat liver and rat ascites hepatoma was readily isolated by concanavalin A-Sepharose (Con A-Sepharose) affinity column chromatography. tRNATyr from these sources was extensively purified by Ricinus communis lectin-Sepharose column chromatography. These results, together with the chromatographic behaviour of four tRNAs (tRNATyr, tRNAHis, tRNAAsn and tRNAAsp) on acetylated DBAE-cellulose column chromatography suggested that tRNAAsp contains a Q nucleoside species having a mannose moiety while tRNATyr contains Q nucleoside with galactose. The sugars attached in 4-position of cyclopentene diol in the Q molecule are therefore not present at random in the four tRNAs, but present only in each specific tRNA. This is the first case which shows that plant agglutinin interacts with nucleic Acid as well as polysaccharide and glycoproteins.

Topics: Animals; Aspartic Acid; Carcinoma, Hepatocellular; Chromatography, Affinity; Concanavalin A; Liver; Liver Neoplasms; Neoplasms, Experimental; Rabbits; Rats; RNA, Transfer; Sepharose; Tyrosine

Topics: Animals; Binding Sites; Carcinoma, Hepatocellular; Cell Line; Dactinomycin; Dexamethasone; Enzyme Induction; Freeze Drying; Insulin; Kinetics; Liver Neoplasms; Macromolecular Substances; Polysaccharides; Protein Binding; Rats; Sepharose; Swine; Time Factors; Tyrosine Transaminase