Normal Human Liver Cell Line Research Articles

Effect of the extracts from Glycyrrhiza uralensis Fisch on the growth characteristics of human cell lines: Anti-tumor and immune activation activities.

Immune modulating activity of ethanol extracts from Glycyrrhiza uralensis Fisch was investigated by conserving growth characteristics of several human cell lines. All of the samples did not show severe cytotoxicity on normal human liver cell line, WRL-68, showing less than 25% inhibition of cell growth. The crude extract and its fractionized samples (F1 and F3) inhibited the growth of human hepatoma, Hep3B, down to ca. 70% of normal cell growth in adding 1.0 g l(-1) of fraction F3. The result of anticancer experiments was well matched to the results of antimutagenicity using Chinese Hamster Lung cell lines(CHL V79). In adding 1.0 g l(-1) of fraction F1, the growth of human B cell was enhanced, up to 60% of control growth. The secretion of two kinds of cytokines, Interleukin-6 and Tumor Necrosis Factor-alpha from human B cells was also enhanced in adding the crude extract, but not the standards such as Glycyrrhizin (GL) or 18,beta-glycyrrhetinic acid (GM). It was found that both of the apoptosis and differentiation were more accelerated in supplementing the crude extract and fraction F1 than in adding the standards. A spot was found only in the crude extract and fractions, not standards by Thin Layer Chromatography(TLC) analysis. It tells that there must be another unknown component in crude and/or fraction F1 as a possible candidate of immune modulators. This component seems to be a derivative of a monomer, GM since its R(f) was close to the monomer. It was also interesting that glycyrrhizin, a major component in G. uralensis Fisch was biologically activated by first being hydrolyzed by an enzyme.

Identification of differentially expressed proteins between human hepatoma and normal liver cell lines by two-dimensional electrophoresis and liquid chromatography-ion trap mass spectrometry.

In the previous study, the proteomes of the human hepatoma cell line BEL-7404 and the normal human liver cell line L-02 were separated by high resolution two-dimensional electrophoresis (2-DE). Image analysis revealed that 99 protein spots showed quantitative and qualitative variations that were significant (P < 0.01) and reproducible. Here we report the identification results of some of these protein spots. Protein spots excised from 2-D gels were subjected to in-gel digestion with trypsin, and the resulting peptides were measured by microbore high performance liquid chromatography - ion trap - mass spectrometry (LC-IT-MS) to obtain the tandem mass (MS/MS) spectra. Twelve protein spots were identified with high confidence using SEQUEST with uninterpreted MS/MS raw data. Besides inosine-5'-monophosphate dehydrogenase 2, heat shock 27 kDa protein, calreticulin and calmodulin, whose expression was elevated in hepatoma cells, glutathione-S-transferase P was identified from hepatoma cells in which its level was 18-fold higher compared to human liver cells. Two spots were identified as the homologs of reticulocalbin for the first time in hepatoma cells and their expression increased compared to liver cells. However, tubulin beta-1 chain and natural killer cell enhancing factor B were downregulated in hepatoma cells. A tumor suppressing serpin, maspin precursor, was identified from one spot whose quantity was much higher in the normal liver cell line. More interestingly, epidermal fatty acid-binding protein (E-FABP) and fatty acid-binding protein, adipocyte-type (A-FABP), were detected in liver cells but not in hepatoma cells. The functional implication of the identified proteins was discussed.

Two-dimensional gel electrophoresis analysis of the proteomes expressed in the human hepatoma cell line BEL-7404 and normal liver cell line L-02

Proteome analysis technology has been used extensively in conducting discovery research of biology and has become one of the most essential technologies in functional genomics. The proteomes of the human hepatoma cell line BEL-7404 and the normal human liver cell line L-02 have been separated by high resolution two-dimensional gel electrophoresis (2-DE) with immobilized pH gradient isoelectric focusing (IPG-IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension (IPG-DALT). The resulting images have been analyzed using 2-D analysis software. Quantitative analysis reveals that 7 protein spots are detected only in hepatoma BEL-7404 cells, 14 only in L-02 cells, and 78 protein spots show significant fluctuation in quantity in both cell lines (P< 0.01). These protein spots have been displayed on a proteome differential expression map. Analysis for the reproducibility of 2-DE indicates that the positional variability in the IEF dimension is 0.73 mm, while the variability in the SDS-PAGE dimension is 0.44 mm, and the quantitative variability is 17.6%–19.2%. These results suggest that the reproducibility of 2-DE has been suitable for the study of differential expression of proteomes. Proteome differential expression maps can be useful tools for disease diagnosis, drug-target validation analysis and biological process elucidation.

In vitro inhibition of human malignant brain tumour cell line proliferation by anti-urokinase-type plasminogen activator monoclonal antibodies.

A brain tumour-associated marker, urokinase (UK), was investigated using rabbit anti-UK polyclonal and murine anti-UK monoclonal antibodies, which were prepared by immunization with low molecular weight UK (LMW-UK) and high molecular weight urokinase (HMW-UK) synthetic peptide respectively. The polyclonal antibody cross-reacted with both LMW-UK and HMW-UK, whereas the murine MAbs were specific for HMW-UK. These immunological probes were used to study urokinase in glioma extracts, tissues, sera and cell lines that had been prepared from primary cultures of freshly dissected gliomas. Radioimmunoassays showed that glioma extracts had much higher level (5- to 44-fold) of UK than normal human brain extracts. This result was confirmed by immunoblotting of electrophoresis gels of glioma and human brain extracts. Immunohistochemical study using anti-UK MAb demonstrated much higher levels of UK in glioma tissue than normal brain tissue. Immunohistochemical study using anti-UK MAbs localized UK on the cell surface of glioma cells. Anti-UK MAbs inhibited the proliferation of AA cell lines and GB cell lines (50% to > 90%) and exerted minor effects (< or = 20%) on normal human liver, intestine and lymphocyte cell lines. Taken together, these results suggest that anti-UK MAbs may have therapeutic potential for human gliomas and cancer metastasis.

In vitro efficacy of anti-glial fibrillary acidic protein monoclonal antibodies against human malignant glioma cell lines.

Our studies have confirmed the presence of large concentrations of various intermediate filament proteins (IFPs) in glioma tissue compared to normal brain. This avenue of research was extended to assess the anti‐proliferative activity of anti‐intermediate filament protein monoclonal antibodies (anti‐IFP mAbs)against human glioma cells. In this study, anti‐proliferative activity of glial fibrillary acidic protein monoclonal antibodies (anti‐GFAP mAbs) has been tested in vitro, using glioma cell lines prepared and established from freshly resected brain tumors. One anaplastic astrocytoma (AA), two glioblastoma multiforme (GB1 and GB2) cell lines and three anti‐GFAP mAbs (Bi2C4, B12B4 and B6C6, all IgG15, kappa) were used. Immunofiuorescence study indicated the ability of anti‐GFAP mAbs to recognize the cell surface of glioma cells and the inhibition study showed that mAb B12B4 inhibited the proliferation of GB1, (96%), GB2(85%)and AA(93%) at a concentration of 3.2x 10−10M. mAb B12C4 inhibited the proliferation of GB1 (95%), GB2 (86%) and A A (91%) at a concentration of 3.26 X10‐10M and mAb B6C5 inhibited the proliferation of GB1 (75%), GB2 (75%) and AA (91%) at a concentration of 2.074 X10 −19M. Thymidine release assay demonstrated the cytolytic activities of anti‐GFAP mAbs towards these glioma cell lines, and this observation was confirmed by dye exclusion, which indicated the lysis of glioma cells after anti‐GFAP mAbs treatment. Anti‐GFAP mAbs had little effect (<120%) on normal human lymphocyte, liver and intestine cell lines. These results look promising for radioimaging and immunotherapy of human gliomas.

Identification of chemopreventive agents by screening for induction of glutathione-S-transferase as a biomarker

For selection and identification of potential chemopreventive agents, a biochemical assay using induction of a phase II enzyme, glutathione-S-transferase (GST) in a liver cell culture is described. A normal human liver cell line (Chang liver cells) was selected as the candidate cell line for induction of GST (liver tissues are abundant in GST) by a known chemopreventive agent, oltipraz. Exponentially growing cells plated for 24 hours are exposed to various doses of a chemopreventive agent for an additional 24 hours, homogenized by sonication, and the homogenate is assayed for GST in a modified microplate enzyme assay using CDNB (1-chloro-2,4-dinitrobenzene) as a substrate. A concurrent protein assay is performed to determine the specific enzyme activity. As the assay is modified from a spectrophotometric assay to a microplate assay, it is reliable, sensitive and fast, a significant number of test agents can be screened in a short time.

Induction of NADPH-quinone reductase as a screening assay for potential chemopreventive agents

An in vitro biochemical assay using induction of a phase II enzyme, NADPH-quinone reductase (NADPH-QR) for selection and identification of potential chemopreventive agents is described. A normal human liver cell line (Chang liver cells) or a rodent rat liver cell line (buffalo rat liver cells) was selected as the candidate cell line for induction of QR by a known chemopreventive agent, genistein. The choice of a liver cell line is based on the fact that liver being a major metabolic organ, phase II enzymes are commonly elaborated in liver tissues and is therefore appropriate for enzyme induction studies.