15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and Leukemia--Erythroblastic--Acute

Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 microM, diosgenin induced megakaryocytic differentiation, in contrast to 40 microM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Cattle; Cell Differentiation; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Diosgenin; Fractionation, Field Flow; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Erythroblastic, Acute; Megakaryocytes; Microsomes; Platelet Membrane Glycoprotein IIb; Ploidies; Thromboxane-A Synthase

The human leukaemic cell line K562 is a pluripotent stem cell with the potential to mature along a megakaryocytic or erythroid line. In these cells, thrombin and U46619 (9,11-dideoxy-9 alpha, 11 alpha-methanoepoxy prostaglandin F2 alpha), a thromboxane A2 analogue, increased intracellular Ca2+ in a rapid and concentration-dependent manner. The peak transient observed with both thrombin and U46619 was preserved upon stimulation in the absence of extracellular calcium and blunted with phorbol myristate acetate, suggestive of activation of phospholipase C. Short-term treatment with leupeptin abolished the calcium response to thrombin, but did not alter that to U46619. Both pertussis toxin (PT) and DMSO pretreatment inhibited thrombin- but not U46619-stimulated intracellular calcium elevation, indicating that these agonists signal through different G-proteins. Western blot analysis of crude membranes from K562 cells revealed the presence of G12 alpha and G13 alpha; the other known PT-substrates, Gi1 alpha and G0 alpha, were not detected. Consistent with this observation, ADP-ribosylation experiments revealed the presence of two PT substrates which co-migrated with human erythrocyte G12 alpha and G13 alpha. An antibody raised against Gq/11 alpha, a subfamily of G-protein alpha subunits unmodified by PT, specifically recognized 42 kDa protein(s) in K562 cells. PCR amplification of reverse-transcribed K562 RNA followed by DNA sequencing showed that these cells express messages for both Gq alpha and G11 alpha. Treatment of K562 cells with DMSO reduced the levels of thrombin receptor mRNA, without simultaneous changes in the expression of G12 alpha and G13 alpha. We have thus identified Ca(2+)-mobilizing agonists and related G-proteins in K562 cells, together with changes induced by DMSO in this signalling pathway.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amino Acid Sequence; Base Sequence; Blotting, Northern; Blotting, Western; Calcium; Dimethyl Sulfoxide; DNA Probes; Gene Expression; GTP-Binding Proteins; Humans; Lanthanum; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Nickel; Polymerase Chain Reaction; Prostaglandin Endoperoxides, Synthetic; Sequence Homology, Nucleic Acid; Signal Transduction; Thrombin; Thromboxane A2; Tumor Cells, Cultured

Thromboxane A2 (TxA2), a platelet aggregator and vasoconstrictor, has been implicated as a potential mediator of cardiovascular diseases. Abuse of androgenic steroids has been associated with thrombotic cardiovascular diseases. Human erythroleukemia (HEL) cells, a megakaryocyte-like cell line, express functional TxA2/prostaglandin H2 (PGH2) receptors with characteristics similar to those seen in platelets. This study characterized testosterone regulation of HEL cell TxA2/PGH2 receptors. TxA2/PGH2 receptor affinity (Kd) and density (Bmax) were determined via equilibrium binding experiments using the radiolabeled TxA2 mimetic (1S-[1 alpha,2 beta(5Z),3 alpha(1E,3R*),4 alpha])-7-(3-[3-hydroxy-4-(4'- iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]heptan-2-yl)-5-he ptenoic acid (125I-labeled BOP). Testosterone (200 nM) but not estradiol increased Bmax from 108 +/- 9 fmol/mg protein to 157 +/- 9 fmol/mg protein (n = 7 experiments; P < 0.01) without any significant change in Kd. Testosterone had no significant effect on alpha 2-adrenergic receptor density. The maximum increase in intracellular free calcium induced by the TxA2 agonists I-BOP or U-46619 was significantly (P < 0.005) greater in testosterone-treated cells compared with controls. Hydroxyflutamide (1 microM), an androgen-receptor antagonist, completely blocked the effect of testosterone (P < 0.01). Dihydrotestosterone, the active metabolite of testosterone, also increased Bmax in a concentration-dependent manner and was more potent than testosterone. The effect of testosterone to increase Bmax was significantly (P < 0.01) inhibited by coincubation with cycloheximide (0.1 microgram/ml) or actinomycin D (10 ng/ml). These results indicate that androgenic steroids regulate the expression of functional TxA2/PGH2 receptors in HEL cells. These findings may have relevance to cardiovascular disease.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 5-alpha Reductase Inhibitors; Azasteroids; Calcium; Cell Line; Cycloheximide; Dactinomycin; Dihydrotestosterone; Estradiol; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Testosterone; Thromboxane A2; Tumor Cells, Cultured; Vasoconstrictor Agents

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Binding, Competitive; Blood Platelets; Bridged Bicyclo Compounds; Cell Membrane; Fatty Acids, Monounsaturated; Humans; Leukemia, Erythroblastic, Acute; Neoplasm Proteins; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Tetradecanoylphorbol Acetate; Thromboxane A2; Tumor Cells, Cultured