ag-213 and daidzein

Genistein, an isoflavone inhibitor of tyrosine-specific protein kinases, was shown to specifically block the 22Na+ influx through voltage-sensitive Na+ channels in cultured rat brain neurons, whereas other tyrosine kinase antagonists such as lavendustin A, compound 5, tyrphostin A47 and an erbstatin analog were inactive at concentrations known to block kinase activity in other neuronal systems. Dose-response curves for genistein indicated a half-maximum effect at 60 microM. Daidzein, an inactive analog of genistein, had a similar inhibitory effect on the 22Na+ influx with a half-maximum effect at 195 microM. The time course of genistein action was rapid, because maximum effect on 22Na+ influx was obtained in less than 20 s at 100 microM. Analysis of Na+ currents by the whole-cell recording technique showed that 20 microM genistein reduced the sodium current and shifted the voltage dependence of both activation and inactivation curves. No competition with [3H]saxitoxin binding was observed, whereas the binding of [3H]batrachotoxinin A 20-alpha-benzoate to rat brain synaptosomal membranes was partially inhibited, which suggested a direct or allosteric interaction with neurotoxin binding site 2. These data taken together clearly indicate that the inhibition of voltage-sensitive sodium channels by genistein is not mediated by tyrosine kinase inhibition.

Topics: Animals; Batrachotoxins; Brain; Caffeic Acids; Cells, Cultured; Enzyme Inhibitors; Estrogens, Non-Steroidal; Fetus; Genistein; Isoflavones; Kinetics; Neurons; Neurotoxins; Phenols; Protein-Tyrosine Kinases; Rats; Saxitoxin; Sodium; Sodium Channel Blockers; Sodium Channels; Tyrphostins

The present study was conducted to examine the effects of two protein tyrosine kinase inhibitors, genistein and tyrphostin 47, on an in vitro model of allergic asthma. Guinea pigs were sensitized with purified IgG raised against ovalbumin (OA). Isolated sensitized bronchial rings contracted in response to OA in a concentration-dependent manner, maximum contraction being achieved at 1 microg/ml. Genistein and tyrphostin 47 concentration-dependently (10-100 microM) inhibited OA-induced anaphylactic contraction of the bronchi, as well as release of histamine and peptidoleukotrienes from chopped lung preparations. Genistein, but not tyrphostin 47, significantly suppressed bronchial contraction to leukotriene D4 at 50 microM and to histamine at 100 microM. Daidzein, an inactive congener of genistein, did not alter OA-induced anaphylactic contraction. However, it slightly reduced bronchial contraction to leukotriene D4 and the OA-stimulated release of peptidoleukotrienes. The inhibitory effects were significantly weaker than those of genistein. Taken together, our results show that tyrphostin 47 inhibited anaphylactic contraction mainly by preventing mast cell degranulation, whereas genistein exerted inhibitory effects partly by blocking mast cell degranulation and partly by attenuating leukotriene D4-induced bronchial contraction. These findings suggest that protein tyrosine kinase inhibitors have a therapeutic potential as mast cell stabilizers in the treatment of allergic diseases such as bronchial asthma.

Topics: Anaphylaxis; Animals; Antigens; Asthma; Bronchoconstriction; Enzyme Inhibitors; Genistein; Guinea Pigs; Histamine Release; In Vitro Techniques; Isoflavones; Leukotriene D4; Nitriles; Phenols; Protein-Tyrosine Kinases; Rabbits; Tyrphostins

We compared the effects of the tyrosine kinase inhibitor genistein, a naturally occurring isoflavone, to those of tyrphostin A25, tyrphostin A47, and herbimycin on avian osteoclasts in vitro. Inactive analogs daidzein and tyrphostin A1 were used to control for nonspecific effects. None of the tyrosine kinase inhibitors inhibited bone attachment. However, bone resorption was inhibited by genistein and herbimycin with ID50s of 3 microM and 0.1 microM, respectively; tyrphostins and daidzein were inactive at concentrations below 30 microM, where nonspecific effects were noted. Genistein and herbimycin thus inhibit osteoclastic activity via a mechanism independent of cellular attachment, and at doses approximating those inhibiting tyrosine kinase autophosphorylation in vitro; the tyrphostins were inactive at meaningful doses. Because tyrosine kinase inhibitors vary widely in activity spectrum, effects of genistein on cellular metabolic processes were compared to herbimycin. Unlike previously reported osteoclast metabolic inhibitors which achieve a measure of selectivity by concentrating on bone, neither genistein nor herbimycin bound significantly to bone. Osteoclastic protein synthesis, measured as incorporation of 3H-leucine, was significantly inhibited at 10 microM genistein, a concentration greater than that inhibiting bone degradation, while herbimycin reduced protein synthesis at 10 nM. These data suggested that genistein may reduce osteoclastic activity at pharmacologically attainable levels, and that toxic potential was lower than that of herbimycin. To test this hypothesis in a mammalian system, bone mass was measured in 200 g ovariectomized rats treated with 44 mumol/day genistein, relative to untreated controls. During 30 d of treatment, weights of treated and control group animals were indistinguishable, indicating no toxicity, but femoral weight in the treated group was 12% greater than controls (P < 0.05). Our data indicate that the isoflavone inhibitor genistein suppresses osteoclastic activity in vitro and in vivo at concentrations consistent with its ID50s on tyrosine kinases, with a low potential for toxicity.

Topics: Animals; Benzoquinones; Bone Resorption; Caffeic Acids; Cells, Cultured; Chickens; Enzyme Inhibitors; Female; Femur; Genistein; Isoflavones; Lactams, Macrocyclic; Nitriles; Osteoclasts; Ovariectomy; Protein Biosynthesis; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Tyrphostins

1. Islets from normal mice were used to test the acute effects of genistein, a potent tyrosine kinase inhibitor, on stimulus-secretion coupling in pancreatic beta-cells. 2. Genistein produced a concentration-dependent (10-100 microM), reversible, increase of insulin release. This effect was marginal on basal release or in the presence of non-metabolized secretagogues, and much larger in the presence of glucose or other nutrients. The increase in insulin release caused by 100 microM genistein was abolished by adrenaline or omission of extracellular Ca2+. It was not accompanied by any rise of cyclic AMP, inositol phosphate or adenine nucleotide levels. 3. Although genistein slightly inhibited ATP-sensitive K+ channels, as shown by 86Rb efflux and patch-clamp experiments, this effect could not explain the action of the drug on insulin release because the latter persisted when ATP-sensitive K+ channels were all blocked by maximally effective concentrations of glucose and tolbutamide. Genistein was also effective when ATP-sensitive K+ channels were opened by diazoxide and the beta-cell membrane depolarized by 30 mM K, but ineffective in the presence of diazoxide and normal extracellular K. 4. Genistein paradoxically decreased Ca2+ influx in beta-cells, as shown by the inhibition of glucose-induced electrical activity, by the inhibition of Ca2+ currents (perforated patches) and by the lowering of cytosolic [Ca2+]i (fura-2 technique). Genistein thus increases insulin release in spite of a lowering of [Ca2+]i in beta-cells. 5. Daidzein, an analogue of genistein reported not to affect tyrosine kinases, was slightly less potent than genistein on K+ and Ca2+ channels, but increased insulin secretion in a similar way. Three other tyrosine kinase inhibitors, tyrphostin A47, herbimycin A and an analogue of erbstatin variably affected insulin secretion.6. Genistein exerts a number of heretofore unrecognized effects. The unusual mechanisms, by which genistein increases insulin release in spite of a decrease in beta-cell [Ca2+]i and without activating known signalling pathways, do not seem to result from an inhibition of tyrosine kinases.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Analysis of Variance; Animals; Benzoquinones; Calcium; Catechols; Cyclic AMP; Cytosol; Dose-Response Relationship, Drug; Epinephrine; Female; Genistein; Hydroquinones; Inositol Phosphates; Insulin; Insulin Secretion; Islets of Langerhans; Isoflavones; Lactams, Macrocyclic; Mice; Nitriles; Patch-Clamp Techniques; Potassium Channels; Protein-Tyrosine Kinases; Quinones; Rifabutin; Rubidium; Tyrphostins

In addition to a constitutive cyclo-oxygenase (Cox-1), human endothelial cells also possess an inducible cyclo-oxygenase (Cox-2) which plays an important role in the regulation of the synthesis of prostacyclin (prostaglandin I2). Cox-2 is regulated and expressed in large quantities upon activation of the cells by inducers such as phorbol myristate acetate (PMA), an activator of protein kinase C (PKC), or interleukin-1 alpha. We have investigated the involvement of protein tyrosine kinases in Cox-2 expression by human endothelial cells upon activation by these inducers. PMA or interleukin-1 alpha provoke an increase in the phosphorylation of substrates of 110 and 120 kDa and additional phosphorylations for a broad band of multiple substrates in the 70 kDa range. This stimulation was accompanied by the induction of Cox-2 protein, detectable after stimulation for 1 h, which is consistent with an increase in activity reflected by prostacyclin synthesis; no variation in the expression of Cox-1 could be observed. Three distinct inhibitors of protein tyrosine kinases, genistein, herbimycin or AG-213, reduced tyrosine phosphorylation of cell substrates, consistently with their pharmacological effects. Under these conditions, there was selective reduction of Cox-2 expression without modification of Cox-1. Regulation of Cox-2 induction is also dependent on the activation of PKC since Ro 31-8220 or PKC depletion by PMA prevented its induction. Our results suggest that within the time-frame of our experiments these effects on kinases are specific for Cox-2 rather than Cox-1.

Topics: Benzoquinones; Blotting, Western; Catechols; Cells, Cultured; Endothelium, Vascular; Enzyme Induction; Enzyme Inhibitors; Genistein; Humans; Indoles; Interleukin-1; Isoenzymes; Isoflavones; Kinetics; Lactams, Macrocyclic; Nitriles; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Recombinant Proteins; Rifabutin; Tyrphostins; Umbilical Veins

We examined the involvement of tyrosine kinase activity in the bradykinin (BK)-mediated signal transduction process. Immunoblots with anti-phosphotyrosine antibodies following BK stimulation of human fibroblasts showed tyrosine phosphorylation of specific proteins that could be inhibited by the tyrosine kinase inhibitors genistein and tyrphostin. Image analysis data from individual cells stimulated by BK in the presence of genistein and tyrphostin showed that these inhibitors block the plateau phase but not the rapid transient phase of the BK-induced calcium response. That the loss of the plateau phase was due to blockage of calcium entry rather than stimulation of calcium pump activity was confirmed by examining the influx of Ba2+ following BK stimulation. The Ca2+ imaging results were confirmed by 45Ca2+ uptake measurements and extended to another tyrosine kinase inhibitor (methyl 2,5-dihydroxycinnamate), which was found to interfere with the fura-2 signal and therefore could not be used in imaging experiments. The fact that three structurally distinct inhibitors of tyrosine kinase activity inhibited BK-stimulated calcium influx, while an inactive analogue of genistein (daidzein) did not, strongly suggests the involvement of tyrosine kinases in the regulation of a BK-induced calcium entry pathway. To our knowledge, this is the first report of tyrosine kinase involvement in the regulation of calcium entry following activation of a receptor that lacks endogenous tyrosine kinase activity and is known to be coupled to phosphatidylinositol turnover. We found that calcium entry in HSWP (human foreskin fibroblast) cells can also be stimulated by emptying the intracellular calcium stores with thapsigargin. Genistein also inhibits the plateau phase of the thapsigargin-induced calcium response while leaving the transient phase intact. This suggests that the Ca2+ influx pathway induced by depletion of intracellular calcium stores with thapsigargin also may be regulated via a tyrosine kinase pathway.

Topics: Barium; Bradykinin; Calcium; Calcium-Transporting ATPases; Catechols; Cells, Cultured; Fibroblasts; Fura-2; Genistein; Humans; Immunoblotting; Isoflavones; Kinetics; Male; Nitriles; Phosphotyrosine; Protein-Tyrosine Kinases; Skin; Terpenes; Thapsigargin; Tyrosine; Tyrphostins

The effect of the isoflavones, genistein, daidzein, and biochanin A on the growth of the LNCaP and DU-145 human prostate cancer cell lines has been examined. Genistein and biochanin A, but not daidzein, inhibit both serum and EGF-stimulated growth of LNCaP and DU-145 cells (IC50 values from 8.0 to 27 micrograms/ml for serum and 4.3 to 15 micrograms/ml for EGF), but have no significant effect of the EGF receptor tyrosine autophosphorylation. In contrast, tyrphostin 25, a specific EGF receptor tyrosine kinase inhibitor, inhibits EGF-stimulated growth and EGF receptor tyrosine autophosphorylation in these whole cells, but does not inhibit serum-stimulated growth. These data suggest that the mechanism of action of genistein and biochanin A does not depend on inhibition of EGF receptor tyrosine autophosphorylation, but on a more distal event in the EGF receptor-mediated signal transduction cascade.

Topics: Anticarcinogenic Agents; Catechols; Cell Division; Epidermal Growth Factor; ErbB Receptors; Estrogens, Non-Steroidal; Genistein; Humans; Isoflavones; Male; Nitriles; Phosphorylation; Prostatic Neoplasms; Protein-Tyrosine Kinases; Tumor Cells, Cultured; Tyrosine; Tyrphostins