sepharose and Liver-Neoplasms

To evaluate the safety of on-line plasma perfusion over protein-A sepharose and the therapeutic advantage of combining plasma perfusion (PP) over protein-A sepharose with 5-fluorouracil (5-FU) chemotherapy in patients with metastatic colorectal carcinoma (MCRC), thirty patients were randomized after surgery of primary CRC to receive a combination of 5-FU and PP over protein-A sepharose (group A), or a combination of 5-FU and PP over sepharose (group B), or 5-FU alone (group C). Bi-weekly on-line PP over 200 ml protein-A sepharose gel (group A) or 200 ml sepharose gel (group B) were performed with a Cobe 2997 blood cell separator for a maximum of 19 treatments per patient. 5-FU was given at 1000 mg/m2/d on days 1-5 of a 4-weekly cycle until progression. PP was well tolerated and no severe or life-threatening toxicity was observed. Mild clinical side-effects consisted of fever and chills (36% in group A, 23% in group B). The most common biological effects of PP over protein-A sepharose were significant drops in IgG (66% of pre-PP values), CH50 and C3 (73% of pre-PP values) and a significant generation of C3a and C5a anaphylatoxins. Tumor response rates were 40% for group A, 0% for group B and 20% for group C. The median survival times tended to be longer in group A (17 months) than in group B (10 months) and in group C (9 months). This is the first randomized trial showing some therapeutic advantage in combining PP over protein-A sepharose with conventional chemotherapy in MCRC.

Topics: Chromatography, Gel; Colorectal Neoplasms; Combined Modality Therapy; Female; Fluorouracil; Humans; Immunosorbent Techniques; Liver Neoplasms; Male; Middle Aged; Perfusion; Plasma; Sepharose; Staphylococcal Protein A

To evaluate the advantage with regard to toxicity, response rate, time to progression and survival of combination chemoimmunotherapy over single-agent chemotherapy in patients with metastatic colorectal carcinoma (CRC), 30 patients were randomized to receive a combination of 5-fluorouracil (5-FU) by continuous i.v. infusion and plasma perfusion (PP) over protein A-Sepharose (group A), or a combination of 5-FU and PP over sepharose (group B) or 5-FU alone (group C). 5-FU was given at 1,000 mg/m2/d on days 1-5 of a 4-weekly cycle until progression. Patients of groups A and B received bi-weekly on-line PPs until disease progression or for a maximum of 19 treatments. PP was well tolerated and no severe or life-threatening toxicity was observed. The response rates were 10% for the group A (1 PR), 0% for the group B and 20% for the group C (1 CR + 1 PR). The times to tumor progression for patients in groups A and C were 22 months, 12 and 11 months, respectively and the median survival times were 17 months, 10 months and 9 months. Although the time to progression and survival tended to be higher in patients treated with protein A. PP, these differences were not statistically significant. This is the first report of a randomized trial showing some therapeutic advantage in combining protein A. PP with 5-FU in CRC patients. Further randomized studies are required to demonstrate the real true value of this chemoimmunotherapeutic approach.

Topics: Adult; Aged; Colorectal Neoplasms; Combined Modality Therapy; Female; Fluorouracil; Humans; Liver Neoplasms; Male; Middle Aged; Plasmapheresis; Randomized Controlled Trials as Topic; Sepharose; Staphylococcal Protein A

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

We combined Michelson-interferometer-based off-axis digital holographic microscopy (DHM) with a common flow cytometry (FCM) arrangement. Utilizing object recognition procedures and holographic autofocusing during the numerical reconstruction of the acquired off-axis holograms, sharply focused quantitative phase images of suspended cells in flow were retrieved without labeling, from which biophysical cellular features of distinct cells, such as cell radius, refractive index and dry mass, can be subsequently retrieved in an automated manner. The performance of the proposed concept was first characterized by investigations on microspheres that were utilized as test standards. Then, we analyzed two types of pancreatic tumor cells with different morphology to further verify the applicability of the proposed method for quantitative live cell imaging. The retrieved biophysical datasets from cells in flow are found in good agreement with results from comparative investigations with previously developed DHM methods under static conditions, which demonstrates the effectiveness and reliability of our approach. Our results contribute to the establishment of DHM in imaging FCM and prospect to broaden the application spectrum of FCM by providing complementary quantitative imaging as well as additional biophysical cell parameters which are not accessible in current high-throughput FCM measurements.

Topics: Algorithms; Cell Line, Tumor; Female; Flow Cytometry; Holography; Humans; Image Processing, Computer-Assisted; Interferometry; Liver Neoplasms; Microscopy, Phase-Contrast; Microspheres; Middle Aged; Pancreatic Neoplasms; Refractometry; 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

Anchorage-dependent cells including hepatocytes, the main functional cellular constituent comprising liver tissue, require a substrate for cell adhesion when cultured outside their native tissue. The challenge with hepatocyte culture is that material substrates and designs supporting hepatocyte attachment, phenotype, and function are not readily available. Our laboratory previously published that type I collagen found in the liver extracellular matrix supports hepatocyte culture. We hypothesized that micropatterned agarose with a coating of collagen covalently bound to the surface would facilitate hepatocyte adhesion and phenotype. To test this hypothesis, primary canine hepatocytes and neoplastic human HepG2 hepatocellular carcinoma cells were cultured on these substrates. Hepatocyte adhesion was dependent on the cell type and also the micropattern design. Viable normal and neoplastic hepatocytes attached to the microchannel troughs rather than on the ridges. In contrast, hepatocyte adhesion on the microcircular patterns was similar to control agarose as cells did not sense differences in surface topology on these substrates. Neoplastic cells exhibited a distinct difference in growth behavior following 7 days in culture on the microchannel patterns, exhibiting aberrant proliferation relative to normal hepatocytes which did not proliferate. Our results suggest that patterned microchannel agarose may be useful to evaluate hepatoprotective and noxious agents.

Topics: Animals; Cell Adhesion; Cell Shape; Cells, Cultured; Collagen; Dogs; Hep G2 Cells; Hepatocytes; Humans; Inflammation; Liver Neoplasms; Microtechnology; Oxidative Stress; Sepharose

RF ablation uses RF current to heat and kill cancer applied via an electrode inserted under image guidance. Tumor has about half the electrical resistivity of normal tissue below 20 kHz, but similar resistivity above 500 kHz. We placed normal porcine liver tissue in contact with agar gel having similar resistivity as tumor within 20-450 kHz. A needle electrode was placed with half of the electrically active tip in each layer. We performed ablation with electric current applied for 12 min at 30 W, either at 20 or 450 kHz (n = 7 each), while measuring temperature via thermocouples 4 and 8 mm from the electrode. Mathematical heat-transfer models were created of an equivalent configuration and temperature profile determined at both frequencies. At 8-mm distance, at 450 kHz, tumor gel phantom and normal tissue obtained similar temperatures (57.5 ± 1.4 versus 58.7 ± 2.5 (°)C); at 20 kHz, tumor phantom obtained significantly higher temperatures than normal tissue (65.6 ± 2.0 versus 57.2 ± 5.6 (°)C, p < 0.01). Computer models confirm these results, and show the ablation zone diameter to be larger within the tumor phantom at 20 kHz compared to 450 kHz. Heating at low RFs may thus allow targeted heating of tumor tissue and reduced heating of normal tissue.

Topics: Ablation Techniques; Animals; Cell Survival; Computer Simulation; Electric Conductivity; Hot Temperature; Hyperthermia, Induced; Liver; Liver Neoplasms; Models, Biological; Phantoms, Imaging; Radiofrequency Therapy; Sepharose; Swine

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

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

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

An isoelectric focusing technique in agarose gel is presented which is suitable for alkaline phosphatases from both serum and tissue sources. An anomaly in the literature about isoelectric focusing of serum alkaline phosphatase from liver origin is discussed and a possible explanation is proposed. The presented technique is used to demonstrate some differences in behaviour of serum liver and bone isoenzymes towards neuraminidase treatment.

Topics: Alkaline Phosphatase; Humans; Isoelectric Focusing; Isoenzymes; Liver; Liver Neoplasms; Neuraminidase; Sepharose

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

The isoenzymes of canine alkaline phosphatase have been separated by isoelectric focusing on agarose gels. Alkaline phosphatase from normal tissue extracts gave heterogeneous focusing patterns with the following isoelectric points: liver pH 4.3, bone pH 4.0 to 4.9, kidney pH 4.4 to 4.7 and intestine pH 3.6 to 4.6. Plasma alkaline phosphatase isoenzymes were examined in 123 dogs with activities at least twice the normal maximum. The isoenzymes from these plasma samples had multiple bands at pH 4.3 to 4.6 but in 67 per cent of all cases, the predominant isoenzyme, named isoenzyme A, was a single band of isoelectric point pH 3.5. Isoenzyme A occurred in all dogs with diabetes mellitus or Cushing's syndrome and in 93 per cent of animals receiving steroid therapy. Increased activities of the pH 4.3 to 4.6 isoenzymes were more likely to occur in cases of hepatic degeneration.

Topics: Alkaline Phosphatase; Animals; Dog Diseases; Dogs; Female; Hot Temperature; Hydrogen-Ion Concentration; Isoelectric Focusing; Isoelectric Point; Isoenzymes; Liver Neoplasms; Male; Sepharose

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

Topics: Adenocarcinoma; Animals; Binding Sites, Antibody; Carcinoembryonic Antigen; Chromatography, Affinity; Colonic Neoplasms; Goats; Humans; Immunoelectrophoresis; Immunoglobulin G; Iodine Radioisotopes; Liver Neoplasms; Neoplasm Metastasis; Radioimmunoassay; Sepharose

Topics: Adenocarcinoma; Aluminum Hydroxide; Animals; Animals, Newborn; Antibody Formation; Carcinoembryonic Antigen; Colonic Neoplasms; Goats; Horses; Immune Sera; Immune Tolerance; Immunization, Secondary; Immunodiffusion; Immunoelectrophoresis; Liver Neoplasms; Neoplasm Metastasis; Rabbits; Radioimmunoassay; Sepharose