Masuko Kobori

Flavonoids Inhibit Cell Growth and Induce Apoptosis in B16 Melanoma 4A5 Cells

We investigated the growth inhibitory activity of several flavonoids, including apigenin, luteolin, kaempherol, quercetin, butein, isoliquiritigenin, naringenin, genistein, and daizein against B16 mouse melanoma 4A5 cells. Isoliquiritigenin and butein, belonging to the chalcone group, markedly suppressed the growth of B16 melanoma cells and induced cell death. The other flavonoids tested showed little growth inhibitory activity and scarcely caused cell death. In cells treated with isoliquiritigenin or butein, condensation of nuclei and fragmentation of nuclear DNA, which are typical phenomena of apoptosis, were observed by Hoechst 33258 staining and by agarose gel electrophoresis of DNA. Flowcytometric analysis showed that isoliquiritigenin and butein increased the proportion of hypodiploid cells in the population of B16 melanoma cells. These results demonstrate that isoliquiritigenin and butein inhibit cell proliferation and induce apoptosis in B16 melanoma cells. Extracellular glucose decreased the proportion of hypodiploid cells that appeared as a result of isoliquiritigenin treatment. p53 was not detected in cells treated with either of these chalcones, however, protein of the Bcl-2 family were detected. The level of expression of Bax in cells treated with either of these chalcones was markedly elevated and the level of Bcl-XL decreased slightly. Isoliquiritigenin did not affect Bcl-2 expression, but butein down-regulated Bcl-2 expression. From these results, it seems that the pathway by which the chalcones induce apoptosis may be independent of p53 and dependent on proteins of the Bcl-2 family. It was supposed that isoliquiritigenin induces apoptosis in B16 cells by a mechanism involving inhibition of glucose transmembrane transport and promotion of Bax expression. On the other hand, it was suggested that butein induces apoptosis via down-regulation of Bcl-2 expression and promotion of Bax expression. This mechanism differs from the isoliquiritigenin induction pathway.

Phloretin-induced apoptosis in B16 melanoma 4A5 cells by inhibition of glucose transmembrane transport

Phloretin, a naturally occurring dihydrochalcone, is known to inhibit tumor cell growth in vitro and in vivo. To clarify the anti-tumor effects of phloretin, its apoptosis-inducing effects in B16 melanoma 4A5 cells were examined. Phloretin induced the internucleosomal DNA fragmentation typical of apoptosis in B16 melanoma cells. The addition of extracellular glucose remarkably inhibited the phloretin-induced apoptosis in the cells. When apoptosis was strongly induced in the B16 cells by phloretin, protein kinase C activity was inhibited in the cells. Our results suggest that phloretin induced apoptosis in B16 melanoma 4A5 cells mainly through the inhibition of glucose transmembrane transport. Inhibition of protein kinase C activity by phloretin probably promotes the ratio of apoptotic cells in the cells.

Stimulation of cell growth in the U-937 human myeloid cell line by honey royal jelly protein

Royal jelly was fractionated by ion-exchange chromatography and a protein (DIII protein) that had growth stimulating activity to the U-937 human myeloid cell line was obtained. The molecular weight of the DIII protein was 58 kDa on SDS-PAGE. The growth stimulating activity of the DIII protein was shown to be relatively heat and pH stable.

Growth stimulation with honey royal jelly DIII protein of human lymphocytic cell lines in a serum-free medium

The growth stimulating effects of a royal jelly protein (DIII protein) were studied. The DIII protein stimulated the growth of five human lymphocytic cell lines in serum-free conditions. Cell cycle analysis showed that U-937 cells cultured with the DIII protein did not arrest to the G1 phase. Furthermore, a binding assay using europium-labeled DIII protein showed U-937 cells had a large number of low affinity receptors on the cell surface.

Effect of some constituents of chicken egg yolk lipoprotein on the growth and IgM production of human-human hybridoma cells and other human-derived cells

Chicken egg yolk lipoprotein (YLP) was partially fractionated into some constituents, and the effect of constituents of YLP were examined on the growth and immunoglobulin (IgM and IgG) secretion of a HB4C5 human-human hybridoma cell line cultured in serum-free medium. Among the fractions, YP-1 and YP-2 fractions (LDL-rich fractions) were found to enhance the growth and IgM secretion of HB4C5 cells. The promoting activity was found in the commercial LDL. The lipid fraction in YP-2 fraction conjugated with 2-maltosyl-a-cyclodextrin was found to enhance the growth and IgM secretion of HB4C5 cells. Livetin-rich YP-3 and YP-4 fractions had no significant promoting activity. Commercial γ-livetin and phosphatidyl choline possessed no growth-promoting activity. Phosphatidyl choline enhanced the IgM secretion of HB4C5 cells.

Establishment of a mouse hybrid-hybridoma secreting bispecific antibodies to bovine lactoferrin and horseradish peroxidase

A mouse hybridoma HS@03A secreting anti-horseradish peroxidase (HRPO) monoclonal antibodies (IgG1) was established. A HAT sensitive clone of HS@03A was obtained by culturing the hybridoma cells in a 6-thioguanine supplemented medium. The resulting clone 03AR10-2 was fused with a 5-bromo-2′-deoxyuridine resistant (HAT sensitive) clone of a mouse hybridoma HB8852 secreting anti-bovine lactoferrin (bLF) antibodies. Hybrid-hybridomas secreting bispecific antibodies were selected and a hybrid-hybridoma clone HH1-4-3 was established. The bispecific antibodies secreted by the hybrid-hybridoma HH1-4-3 were found to be useful for the analysis of bLF by competitive ELISA.

Protein-free culture of a human-human hybridoma HB4C5

A human-human hybridoma HB4C5 secreting human IgM class monoclonal antibody was examined for possible adaptation to a protein-free medium. HB4C5 could be cultured for more than two months in a protein-free medium without any significant changes in antibody production.

Analysis of increased leukocyte ratio induced by tumor necrosis factor-α using several inhibitors in the undisturbed microcirculation of the rat mesentery

Increased leukocyte ratio induced by intraperitoneal injection of TNF-alpha was analyzed in undisturbed rat mesentery venules, a long-term experimental model. TNF-alpha induced a biphasic increase in PMNL ratio in the mesentery venules; in particular, there was a large increase in PMNL ratio 3 h after injection of TNF-alpha. Anti-P and anti-L-selectin compounds, fucoidin and dextran sulfate, inhibited the PMNL ratio by about 40%. Dexamethasone completely inhibited the increased ratio. These results suggest that all selectins may be involved in the increased ratio, i.e. rolling and adhesion reaction.

Preparation of a Bispecific Antibody to Ovomucoid and Horseradish Peroxidase

We prepared a bispecific antibody reactive to ovomucoid (OM) and horseradish peroxidase (HRPO) by a chemical recombination method. Hybridomas, HS@03A (anti HRPO) and HS@07A (anti OM), were cultured in a CELLMAX artificial capillary system with MPS module in a serum free medium. Purified antibodies (IgGl) were digested with immobilized ficin and resulting F(ab’)2 fragments were reduced with 2-aminoethanethiol hydrochloride followed by treatment with Ellman’s reagent to form stable Fab’-TNB-derivatives. Anti-HRPO Fab’-TNB was reduced again, reacted with anti-OM Fab’-TNB and we obtained a bispecific F(ab’)2 antibody. The bispecific antibody gave a good standard curve in a competitive ELISA at OM concentrations of 0.1 to 100 μ/ml.

Some Properties of Bispecific Antibodies to Bovine Lactoferrin and Horseradish Peroxidase Secreted by a Hybrid-Hybridoma HH1-4-3

A mouse hybrid-hybridoma HH1-4-3 secreting IgGl class bispecific antibodies to bovine lactoferrin (bLF) and horseradish peroxidase (HRPO) was established. Using the supernatants as bispecific antibodies, a competitive ELISA was carried out to measure bLF concentration. The competitive ELISA gave a good standard curve at bLF concentrations of 2 to 100 μg/ml. However, the competitive ELISA using the HH1-4-3 culture supernatant was not sensitive enough to measure bLF concentration in biological fluids. To improve the sensitivity of the competitive ELISA, we fractionated bispecific antibodies by antigen affinity column chromatography. The competitive ELISA for bLF using the affinity purified bispecific antibodies through HRPO-immobilized column chromatography showed a good standard curve at bLF concentrations of 10 ng/ml to 100 μg/ml.