Kouichiro Kitagawa

Berberine Sulfate Inhibits Tumor-Promoting Activity of Teleocidin in Two-Stage Carcinogenesis on Mouse Skin

Berberine sulfate, an isoquinoline alkaloid isolated from Hydrastis canadensis L., inhibited the effects of the tumor promoters 12-O-tetradecanoylphorbol-13-acetate and teleocidin, such as increased 32Pi-incorporation into phospholipids of cell membrane and hexose transport. Berberine sulfate also markedly suppressed the promoting effect of teleocidin on skin tumor formation in mice initiated with 7,12-dimethylbenz[a]anthracene.

Effect of protein kinase C activation and Ca2+ mobilization on hexose transport in Swiss 3T3 cells

Down-modulation of Ca2+-activated, phospholipid-dependent protein kinase (protein binase C), which was accomplished by pretreatment with phorbol-12,13-dibutyrate for 24 h, resulted in the loss of a phorbol ester-induced stimulation of hexose transport activity in Swiss 3T3 cells. In these cells, however, platelet-derived growth factor as well as Ca2+ ionophore A23187 were still able to induce stimulation of hexose transport activity accompanied by the elevation of intracellular free Ca2+ concentration. Since chelation of extracellular Ca2+ inhibited this stimulation, inflow of extracellular Ca2+ into cytoplasm seemed to be essential for the stimulatory effect of platelet-derived growth factor and A23187 on hexose transport. Epidermal growth factor and insulin also stimulated hexose transport activity regardless of the absence of protein kinase C. However, in the case of epidermal growth factor, intracellular Ca2+, but not extracellular Ca2+, was found to be necessary for the stimulation. On the other hand, insulin stimulated the hexose transport independent of both intra- and extracellular Ca2+.

Tumor promoter-stimulated translocation of glucose transport system in mouse embryo fibroblast Swiss 3T3 cell

Assay for D-glucose-inhibitable 3H-cytochalasin B-binding was carried out to elucidate the action mechanism of the tumor promoter-induced enhancement of glucose transport activity in Swiss 3T3 cells. Incubation of the cells with 12-0-tetradecanoylphorbol-13-acetate (TPA) increased the amount of D-glucose-inhibitable cytochalasin B-binding sites in plasma membrane from 13.5 to 40.1 pmol/mg protein. On the other hand, TPA treatment resulted in the decrease of binding sites in microsomal membrane from 68.9 to 34.1 pmol/mg protein. The tumor promoter-induced translocation of hexose transport system from microsomal membrane to plasma membrane was inhibited by the treatment with 2,4-dinitrophenol before the addition of TPA but was not affected by the treatment with cycloheximide. By removal of the promoter from its receptor, the stimulatory effect of the promoter on the translocation of hexose transport system was decreased. The analysis by electrophoresis demonstrated that among the affinity labeled hexose transporter components of Mr 48,000 and Mr 55,000, the former was responsible for the TPA-induced increase in hexose transport activity in plasma membrane.

N-(6-Phenylhexyl)-5-chloro-1-naphthalenesulfonamide is one of a new class of activators for Ca2+-activated, phospholipid-dependent protein kinase

N-(6-Phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9) activated Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). SC-9 acted as a substitute for phosphatidylserine, which is one of the endogenous factors in activating protein kinase C. SC-9 was also effective in regulating the physiological functions at the whole-cell level. For example, SC-9 stimulated hexose transport activity in mouse fibroblasts, a protein kinase C-regulated cellular function. Thus, SC-9 may be useful to study the molecular basis of the regulation of protein kinase C activity, and the biological significance of this enzyme.

Effect of tunicamycin on hexose transport in mouse embryo fibroblast Swiss 3T3 cells.

The role of glycosylation of the carrier in the transporting activity was investigated in Swiss 3T3 cells. Inhibition of protein glycosylation by tunicamycin resulted in the decrease of hexose uptake in a dose- and time-dependent manner without a cytotoxic effect. From kinetic analysis, a decrease in the number or availability of hexose carriers in the plasma membrane was suggested. This was in good correlation with the decrease in the amount of photoaffinity cytochalasin B binding in the plasma membrane by the treatment with tunicamycin. The rate of phorbol 12,13-dibutyrate-induced translocation of the hexose carrier from microsomal to plasma membrane was reduced in tunicamycin-treated cells, which may be correlated with the decrease in the number of the completely glycosylated carrier translocatable from the microsomal membrane. In both tunicamycin-treated and untreated cells, the stimulation of hexose transport by phorbol 12,13-dibutyrate was abolished by the removal of phorbol 12,13-dibutyrate, and upon its readdition the stimulation recovered to the same degree as before the removal. Thus, the recycling of the functionally mature hexose carrier appeared not to be affected by the treatment with tunicamycin. These results suggested that complete glycosylation of the carrier may be necessary for the translocation of the carrier from microsomal to plasma membrane to accomplish its function on the cell surface.

Analysis of hexose transport in untransformed and sarcoma virus-transformed mouse 3T3 cells by photoaffinity binding of cytochalasin B

The effect of simian virus 40 transformation on the hexose transport system in mouse embryo fibroblast Swiss 3T3 cells was examined. The concentration of hexose transporters was estimated by measuring D-glucose-inhibitable cytochalasin B binding. The binding of cytochalasin B to the plasma membranes of simian virus 40-transformed mouse 3T3 cells (SV3T3 cells) was significantly greater than that of 3T3 cells. On the other hand, cytochalasin B binding to the microsomal membranes of SV3T3 cells was decreased, and the total amount of binding to plasma and microsomal membranes was not significantly changed in both cell lines. The electrophoretic analysis demonstrated that both hexose-transporter components of Mr 46 000 and Mr 58 000 affinity labeled were responsible for an increase in the hexose transport by viral transformation. These results suggested that the higher hexose-transport activity of transformed cells is caused by a redistribution of transporter from intracellular membranes to plasma membranes.

Inhibition of the Specific Binding of 12-O-Tetradecanoylphorbol-13-Acetate to Mouse Epidermal Membrane Fractions by Glycyrrhetic Acid

Glycyrrhetic acid inhibited the specific binding of 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse epidermal membrane fractions in a dose- and time-dependent manner. Glycyrrhizic acid, a glycoside of glycyrrhetic acid, has no inhibitory effect on TPA binding. The results of kinetic analysis suggested that glycyrrhetic acid directly binds to the TPA receptor, resulting in the competitive inhibition of the binding of TPA to its receptor without affecting the number of binding sites (Ki = 2.2 X 10(-4) M). The inhibitory effect of glycyrrhetic acid on TPA binding to the membrane receptor may play a role in its antitumor-promoting activity in vivo.

Ca2+-dependent stimulation of 3-O-methylglucose transport in mouse fibroblast Swiss 3T3 cells induced by phorbol-12,13-dibutyrate

Binding of phorbol-12,13-dibutyrate (PDBu), a tumor promoter, to quiescent Swiss 3T3 cells increased the number of hexose carriers, resulting in stimulation of membrane transport of 3-O-methylglucose (3MeGlu) in a Ca2+-dependent fashion. Extracellular Ca2+ was necessary to initiate the binding of PDBu to its receptor, and intracellular Ca2+ was required to maintain it. The loss of PDBu-binding, caused by elimination of Ca2+, was accompanied by a loss of stimulation of hexose transport. These results indicated that Ca2+-dependent, continuous binding of PDBu to its receptor was essential to induce the stimulation of hexose transport.

Possible involvement of protein kinase C in the stimulation of amino acid transport by phorbol ester, platelet-derived growth factor and A23187 in Swiss 3T3 cells

Stimulation of amino acid transport induced by phorbol-12, 13-dibutyrate, platelet-derived growth factor or A23187 was not observed in cells lacking protein kinase C. On the other hand, stimulation of transport by epidermal growth factor or insulin was not affected. These results suggested that the stimulation of amino acid transport is mediated by at least two separate pathways.

Ca2+-dependent translocation of hexose carrier in mouse fibroblast Swiss 3T3 cells

Ca2+-induced translocation of hexose carriers from microsomal membrane to plasma membrane was demonstrated in saponin-permeabilized Swiss 3T3 cells by a specific D-glucose-inhibitable cytochalasin B-binding assay. The number of hexose carriers in the plasma membrane and the hexose transport activity in intact cells were also compared. The incubation of permeabilized cells with 10 microM Ca2+ at 37 degrees C rapidly increased the number of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane from 13 to 40 pmol/mg protein and concomitantly decreased that in the microsomal membrane from 66 to 36 pmol/mg protein, each with a half-time of approx. 2 min. Furthermore, when Ca2+-stimulated cells were exposed to 50 microM EGTA, the effect of Ca2+ on the translocation of D-glucose-inhibitable cytochalasin B-binding sites was reversed with a half-time of approx. 5 min. The concentration of Ca2+ required for the half-maximal effect was approx 500 nM. The magnitude of the stimulatory effect of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane closely correlated with the magnitude of stimulatory action of Ca2+ on 3-O-methylglucose transport in the intact cells. These results suggest that Ca2+ regulates the activity of hexose transport across the plasma membrane through a rapid and reversible translocation of hexose carrier between microsomal and plasma membranes of mouse fibroblast Swiss 3T3 cells.