RESUMO
INTRODUCTION: Recombinant FVIIa (rFVIIa) is an effective treatment for haemophilia through frequent administration. However, the short half-life of rFVIIa decreases its prophylactic ability to reduce bleeding. Carboxy-terminal peptide (CTP)-modified FVIIa (MOD-5014) is a long-acting rFVIIa developed for on-demand treatment of haemophilia using either an intravenous or subcutaneous injection with the aim of less frequent administrations, as well as for prophylactic use. AIM: The comprehensive evaluation of the activity MOD-5014 vs commercially available rhFVIIa, as well as their interaction with cofactors and inhibitors. METHODS: The in vitro characterization included clotting activity, affinity by surface plasmon resonance, cleavage of synthetic substrates, thrombin generation (TG) and rotation thromboelastometry. RESULTS: Reduced specific activity was obtained for MOD-5014 compared to rhFVIIa, while both compounds demonstrated comparable affinity to tissue factor (TF). MOD-5014 showed reduced TG when spiked at a similar concentration as rhFVIIa, suggesting that an increased concentration might be needed in a clinical setting to provide initial haemostatic effect. MOD-5014 demonstrated a slightly lower affinity for binding to activated platelets and slightly lower proteolytic activity on the platelet surface, possibly as the fusion of CTP has the potential to sterically hinder binding to both the platelet membrane and to protein substrates. Both compounds showed a similar dose-dependent stimulatory effect on clot formation, and both showed a similar deactivation pattern following incubation with TF pathway inhibitor (TFPI), antithrombin and heparin. CONCLUSION: The comparable in vitro activity of MOD-5014 and rhFVIIa paves the way for in vivo pharmacology evaluations of MOD-5014 in preparation for clinical studies.
Assuntos
Fator VIIa/química , Fator VIIa/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fator VIIa/administração & dosagem , Fator VIIa/metabolismo , Humanos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Tromboplastina/metabolismoRESUMO
Severe congenital factor VII (FVII) deficiency is a rare bleeding disorder. Prophylaxis with replacement therapy has been suggested to patients, yet the most beneficial dosing regimens and therapy intervals are still to be defined. Due to the lack of evidence-based data, we hereby present our experience with long-term administration and monitoring primary prophylaxis in children with severe FVII deficiency and an extremely high bleeding risk. Four children with familial FVII deficiency, treated by prophylactic recombinant activated factor VII (rFVIIa), 15-30µg/kg/dose, given 2-3 times weekly since infancy, are discussed. Clinical follow up and monitoring laboratory assays, including thrombin generation, measured at various time points after prophylactic rFVIIa administration are presented. Among our treated patients neither FVII activity nor thrombin generation parameters (both already declined 24h post rFVIIa administration) were able to predict the impact of prophylaxis, and could not be used as surrogate markers in order to assess the most beneficial treatment frequency. However, the long clinical follow-up and comprehensive laboratory assessment performed, have shown that early primary prophylaxis as administered in our cohort was safe and effective.
Assuntos
Deficiência do Fator VII/prevenção & controle , Fator VIIa/uso terapêutico , Criança , Pré-Escolar , Deficiência do Fator VII/sangue , Deficiência do Fator VII/complicações , Deficiência do Fator VII/metabolismo , Fator VIIa/administração & dosagem , Feminino , Hemorragia/sangue , Hemorragia/etiologia , Hemorragia/metabolismo , Hemorragia/prevenção & controle , Humanos , Masculino , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Trombina/metabolismoRESUMO
Treatment of haemophilia A patients with inhibitors is challenging, and may require individually tailored regimens. Whereas low titre inhibitor patients may respond to high doses of factor VIII (FVIII), high-responding inhibitor patients render replacement therapy ineffective and often require application of bypassing agents. Thrombin generation (TG) assays may be used to monitor haemostasis and/or predict patients' response to bypass agents. In this study we defined by TG, the potential contribution of FVIII to recombinant activated factor VII (rFVIIa)-induced haemostasis in inhibitor plasma. Based upon results, prospectively designed individual regimens of coadministration of rFVIIa and FVIII were applied. Plasma samples from 14 haemophilia patients with inhibitors (including high titre inhibitors) were tested. The response to increasing concentrations of FVIII, rFVIIa or both was assayed by TG. Eight patients, chosen following consent and at physician's discretion, comprised the combined FVIII-rFVIIa therapy clinical study cohort. Combined spiking with FVIII/rFVIIa improved TG induced by rFVIIa alone in all inhibitor plasmas. Combined rFVIIa and FVIII therapy was applied during bleeding or immune tolerance to eight patients, for a total of 393 episodes. Following a single combined dose, 90% haemostasis was documented and neither thrombosis nor any complications evolved. During study period decline of inhibitor levels and bleeding frequency were noted. Pre-analytical studies enabled us to prospectively tailor individual therapy regimens. We confirmed for the first time that the in vitro advantage of combining FVIII and rFVIIa, indeed accounts for improved haemostasis and may safely be applied to inhibitor patients.
Assuntos
Fator VIII/administração & dosagem , Fator VIIa/administração & dosagem , Hemofilia A/tratamento farmacológico , Trombina/biossíntese , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Fator VIII/imunologia , Hemofilia A/sangue , Hemofilia A/imunologia , Hemostasia/efeitos dos fármacos , Humanos , Lactente , Isoanticorpos/biossíntese , Isoanticorpos/sangue , Isoanticorpos/imunologia , Pessoa de Meia-Idade , Projetos Piloto , Proteínas Recombinantes/administração & dosagem , Resultado do Tratamento , Adulto JovemRESUMO
Patients with severe haemophilia A and an inhibitor may become refractory to FEIBA and/or recombinant factor VIIa (rFVIIa). Sequential therapy with both products has been reported in such patients. In this pilot study, we examined the safety and efficacy of combined rFVIIa and FEIBA therapy in patients with haemophilia A and inhibitors during bleeding episodes. We also tried to evaluate whether thrombin generation (TG), by various mixtures of these agents, can serve as a guide for tailoring therapy. TG was measured in plasma taken from eight haemophilia A patients. Increasing concentrations of rFVIIa, FEIBA or both were added ex vivo to the plasmas, and TG was induced by recalcification. Since low concentrations of rFVIIa and FEIBA had either an additive or a synergistic effect in all patients, the lowest combination, yielding TG comparable or lower than TG achieved with either FEIBA 100 U kg(-1) or rFVIIa 160 microg kg(-1) alone, was selected for the treatment of bleeding episodes. Five patients with a high titre of an inhibitor (8-1300 BU), including one previously refractory to infusions of rFVIIa at doses up to 400 microg kg(-1) X4 daily, were treated with combinations of 30-70 microg kg(-1) rFVIIa and 20-30 U kg(-1) FEIBA during a total number of 400 bleeding episodes with excellent haemostatic effect. No adverse events and no DIC were observed following these infusions. Concomitant infusion of low-dose rFVIIa and low-dose FEIBA, seems to be safe, efficacious and economical in patients refractory to rFVIIa and probably other haemophilia A patients with an inhibitor.
Assuntos
Fatores de Coagulação Sanguínea/administração & dosagem , Fator VIIa/administração & dosagem , Hemartrose/tratamento farmacológico , Hemofilia A/tratamento farmacológico , Trombina/administração & dosagem , Adolescente , Adulto , Inibidores dos Fatores de Coagulação Sanguínea/administração & dosagem , Inibidores dos Fatores de Coagulação Sanguínea/economia , Fatores de Coagulação Sanguínea/economia , Análise Custo-Benefício , Relação Dose-Resposta a Droga , Esquema de Medicação , Quimioterapia Combinada , Fator VIIa/economia , Hemartrose/economia , Hemofilia A/economia , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/economia , Trombina/economia , Adulto JovemRESUMO
Factor VIII inhibitor bypass activity (FEIBA) and recombinant factor VIIa (rFVIIa) are the common bypassing agents for treating haemophilia A or haemophilia B patients who developed an inhibitor to factor VIII or IX, respectively. As these preparations differ in their composition and mode of action, combined therapy, either sequential or simultaneous has recently been used for achieving haemostasis during bleeding episodes in patients who became refractory to FEIBA or rFVIIa when each was given alone. In this in vitro study, we show by a sensitive assay of thrombin generation that phospholipids present in FEIBA and other procoagulants contribute to FEIBA's activity and that exogenous phospholipids are essential for the activity of rFVIIa. We also demonstrate that the combination of FEIBA and rFVIIa has a marked synergistic effect on thrombin generation in plasma of a haemophilia A patient with a high titre of an inhibitor. It is conceivable that simultaneous administration of small doses of FEIBA and rFVIIa may be beneficial in treating haemophilia A patients, with an inhibitor to FVIII, who are resistant to conventional therapy.
Assuntos
Fatores de Coagulação Sanguínea/farmacologia , Fator VIIa/farmacologia , Hemofilia A/sangue , Trombina/biossíntese , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Sinergismo Farmacológico , Humanos , Técnicas In Vitro , Proteínas Recombinantes/farmacologia , Trombina/efeitos dos fármacosRESUMO
BACKGROUND: Recombinant factor VIIa (rFVIIa) used for the treatment of hemophilia A or B patients with an inhibitor is hemostatically effective because it induces thrombin generation (TG), despite grossly impaired FVIII- and FIX-dependent amplification of FX activation. Tissue factor (TF) and or activated platelets were shown to be essential for the rFVIIa activity. OBJECTIVE: To evaluate the relative effects of TF and phospholipids on rFVIIa-induced TG in FVIII-, FIX- and FXI-deficient plasmas. METHODS: Phospholipids had an independent effect that was augmented by TF. The contribution of blood-borne TF in FVIII-, FIX- and FXI-deficient plasma to rFVIIa-induced TG was demonstrated by removing microparticles and use of anti-TF antibodies. RESULTS: At increasing concentrations of rFVIIa, the dependence of rFVIIa-induced TG on TF declined, but the presence of phospholipids was essential. rFVIIa was also shown to activate purified FIX and FX in the presence of phospholipids and absence of TF. rFVIIa-induced TG was dramatically augmented in FVIII- or FIX-deficient plasma in which the level of FVIII or FIX was increased to 1 or 2 U dL(-1). CONCLUSIONS: The data indicate that rFVIIa-induced TG is affected by TF, phospholipids, rFVIIa concentration, and the presence of FVIII and FIX.
Assuntos
Transtornos de Proteínas de Coagulação/metabolismo , Fator VII/farmacologia , Trombina/biossíntese , Plaquetas/metabolismo , Plaquetas/fisiologia , Transtornos de Proteínas de Coagulação/tratamento farmacológico , Relação Dose-Resposta a Droga , Fator IX/metabolismo , Fator IX/farmacologia , Fator VIIa , Fator X/metabolismo , Fator X/farmacologia , Deficiência do Fator XI/tratamento farmacológico , Deficiência do Fator XI/metabolismo , Hemofilia A/tratamento farmacológico , Hemofilia A/metabolismo , Hemofilia B/tratamento farmacológico , Hemofilia B/metabolismo , Humanos , Cinética , Fosfolipídeos/farmacologia , Plasma/metabolismo , Proteínas Recombinantes/farmacologia , Trombina/efeitos dos fármacos , Tromboplastina/farmacologia , Tromboplastina/fisiologiaRESUMO
INTRODUCTION: Bleeding symptoms in severe thrombocytopenia range from mild to severe. The aim of this in vitro study was to improve blood clotting and protect against fibrinolysis in reconstituted severe thrombocytopenia blood. METHODS: Thrombocytopenia [(16 ± 4) × 10(6) /mL] was created by high-speed centrifugation of normal blood with subsequent mixing plasma with packed cells. The blood samples were subjected to clotting by CaCl2 and tissue factor and to fibrinolysis by the addition of tissue plasminogen activator. Blood was spiked with fibrinogen, activated prothrombin complex concentrate (FEIBA), thrombin activatable fibrinolysis inhibitor (TAFI), or their combinations. To mimic the situation that may occur in patients subjected to massive transfusion of plasma substitutes, blood was diluted by 40% of TRIS/saline buffer. Clotting time (CT), α-Angle, maximum clot firmness (MCF), and lysis onset time (LOT) were evaluated using rotation thromboelastometry. RESULTS: Spiking thrombocytopenia blood with FEIBA led to reduction of CT. Fibrinogen and FEIBA enhanced α-Angle and MCF both in the absence and in the presence of tPA. LOT values were prolonged by TAFI and to less extent by FEIBA. Dilution of thrombocytopenia blood was followed by reduction of α-Angle and MCF compared to nondiluted blood which partly reversed by either fibrinogen or FEIBA being higher using fibrinogen and FEIBA together. Clot strength was enhanced, and fibrinolysis was inhibited by TAFI. CONCLUSION: The results of this study suggest that combined spiking of blood with fibrinogen and FEIBA may be enough to correct the clot formation disorder in severe thrombocytopenia, whereas in thrombocytopenia and blood dilution, additive inhibition of fibrinolysis may be needed.
Assuntos
Fatores de Coagulação Sanguínea/farmacologia , Testes de Coagulação Sanguínea/métodos , Coagulação Sanguínea/efeitos dos fármacos , Carboxipeptidase B2/farmacologia , Fibrinogênio/farmacologia , Testes de Coagulação Sanguínea/instrumentação , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Cloreto de Cálcio/farmacologia , Humanos , Modelos Biológicos , Cultura Primária de Células , Rotação , Índice de Gravidade de Doença , Tromboelastografia/instrumentação , Trombocitopenia/sangue , Trombocitopenia/patologia , Ativador de Plasminogênio Tecidual/farmacologiaRESUMO
Comparison of LCC2, the E(2)-independent, tamoxifen-resistant subline of the MCF-7 human breast cancer cell line with its parent line, disclosed that it is more resistant to growth inhibition and apoptosis induction by a variety of agents acting by diverse mechanisms. Thus, LCC2 cells can serve as a useful in-vitro model for the study of the molecular mechanisms of this resistance. It was found that bcl-2 protein and mRNA were elevated and that bax protein and mRNA were reduced in LCC2 compared with MCF-7 cells. Incubation of both lines in the presence of bcl-2 antisense caused growth inhibition and reduced bcl-2 protein levels only in MCF-7 cells, suggesting the involvement of bcl-2 in the regulation of normal growth of breast cancer cells. Increased bcl-2 expression in breast cancer cells may correlate with their resistance to growth inhibitory agents. Bcl-2 is a useful target for enhancing the effects of growth inhibitory agents.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Inibidores do Crescimento/farmacologia , Neoplasias Hormônio-Dependentes/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos , Estradiol/fisiologia , Moduladores de Receptor Estrogênico/farmacologia , Humanos , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/patologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/biossíntese , Tamoxifeno/farmacologia , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
Basic fibroblast growth factor (bFGF) is a classical mitogen in fibroblasts and endothelial cells. Our previous studies have demonstrated that bFGF inhibits the growth of MCF-7 human breast cancer cells. The aim of the present study was to examine the effect of bFGF on cis-diamminedichloroplatinum(cisplatin)-induced cytotoxicity in MCF-7 breast cancer cells as compared to normal endothelial cells. MCF-7/NCF cells transduced with a vector expressing the bFGF gene and overexpressing its product, and MCF-7/N2 cells transduced with the backbone vector were incubated with a combination of bFGF and cisplatin for 5 days; results were compared with those obtained with bovine aortic endothelial cells. Cell proliferation was assessed with the sulforhodamine B colorimetric cytotoxicity assay. Apoptosis was quantitatively determined by flow-cytometric analysis for DNA damage and the apoptotic death assay for DNA fragmentation, and qualitatively by electron microscopy. Reverse transcriptase/polymerase chain reaction analysis and an enzyme immunoassay were used to determine the mRNA and protein level, respectively, of the anti-apoptotic bcl-2 gene product. We found that bFGF enhanced cisplatin-induced cytotoxicity in MCF-7 breast cancer sublines. bFGF enhanced proliferation of normal endothelial cells and did not increase cisplatin-induced cytotoxicity. This effect was accompanied by down-regulation of the anti-apoptotic protooncogene bcl-2 and the enhancement of cisplatin-induced apoptosis. We suggest that the improved understanding of the role of bFGF in the differential modulation of the response of breast cancer and normal endothelial cells to chemotherapy may enable active intervention to alter the therapeutic ratio favorably in breast cancer patients.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Cisplatino/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias da Mama/química , Neoplasias da Mama/patologia , Separação Celular , Cisplatino/uso terapêutico , DNA de Neoplasias/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Humanos , Microscopia Eletrônica , Proteínas Proto-Oncogênicas c-bcl-2/análise , RNA Neoplásico/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
We report here on a novel mechanism involved in epidermal growth factor (EGF) action, which shows that EGF rapidly stimulates binding of the glycolytic enzymes, phosphofructokinase (EC 2.7.1.11), and aldolase (EC 4.1.2.13) to muscle cytoskeleton. This effect was demonstrated both in vivo, in the tibialis anterior muscle from rats injected with EGF, and in vitro, in the isolated rat diaphragm muscle incubated with EGF. The increase in cytoskeleton-bound glycolytic enzymes induced by EGF was prevented, in both the in vivo and in vitro experiments, by treatment with the calmodulin antagonists trifluoperazine or CGS 9343B (a potent and selective inhibitor of calmodulin activity), which strongly suggests that Ca2+ and calmodulin are involved in this effect of EGF. Our previous findings have revealed that insulin or Ca2+ exert a similar rapid stimulation of cytoskeletal glycolysis, which is also calmodulin mediated. We now hypothesize that this may be a general mechanism of signal transduction in the cell, involving Ca(2+)-mobilizing hormones and growth factors, and supplying local ATP, in the vicinity of cytoskeleton-membrane, which is required for the rapid cytoskeletal-membrane rearrangements upon membrane-induced events.
Assuntos
Calmodulina/antagonistas & inibidores , Citoesqueleto/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Glicólise/efeitos dos fármacos , Músculos/enzimologia , Animais , Benzimidazóis/farmacologia , Cálcio/metabolismo , Citoesqueleto/efeitos dos fármacos , Fator de Crescimento Epidérmico/antagonistas & inibidores , Frutose-Bifosfato Aldolase/metabolismo , Técnicas In Vitro , Músculos/efeitos dos fármacos , Fosfofrutoquinase-1/metabolismo , Ligação Proteica , Ratos , Músculos Respiratórios/efeitos dos fármacos , Músculos Respiratórios/enzimologia , Trifluoperazina/farmacologiaRESUMO
1. We show here that treatment of diaphragm muscle with 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, abolished the stimulatory action of insulin on binding of the glycolytic enzymes, phosphofructokinase (PFK) and aldolase, to muscle cytoskeleton. This effect was demonstrated with low concentration of DNP, which caused only a small decrease in ATP and did not affect the basic levels of cytoskeleton-bound glycolytic enzymes. 2. Higher concentrations of DNP, which induced a drastic decline in ATP content, caused a decrease in cytoskeleton-bound glycolytic enzymes and damage to myofibrils. 3. These results suggest that mitochondrial ATP is required for both the preservation of the basal levels of cytoskeleton-bound glycolytic enzymes and cell structure, as well as for the expression of the stimulatory action of insulin on glycolytic enzymes' binding to muscle cytoskeleton.
Assuntos
Citoesqueleto/enzimologia , Frutose-Bifosfato Aldolase/metabolismo , Insulina/farmacologia , Mitocôndrias/metabolismo , Músculos/enzimologia , Fosforilação Oxidativa , Fosfofrutoquinase-1/metabolismo , 2,4-Dinitrofenol , Trifosfato de Adenosina/metabolismo , Animais , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Dinitrofenóis/farmacologia , Glicólise , Técnicas In Vitro , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Músculos/efeitos dos fármacos , Músculos/ultraestrutura , Fosforilação Oxidativa/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , RatosRESUMO
Glycolytic enzymes are known to be controlled by reversible binding to cytoskeleton. Our previous experiments have shown that insulin, epidermal growth factor (EGF), and Ca2+ induce a rapid and transient stimulation of binding of glycolytic enzymes to muscle cytoskeleton. We show here that platelet-derived growth factor (PDGF) exerts a similar action. Incubation of rat diaphragm muscle in the presence of PDGF resulted in rapid and transient stimulation of binding of phosphofructokinase (EC 2.7.11) and aldolase (EC 4.1.2.13) to muscle cytoskeleton. The increase in cytoskeleton-bound glycolytic enzymes induced by PDGF was prevented by treatment with the calmodulin antagonists trifluoperazine or CGS 9343B (a potent and selective inhibitor of calmodulin activity), which strongly suggests that Ca(2+)-calmodulin is involved in this effect of PDGF. Similarly, we previously found that stimulation of cytoskeleton-bound glycolytic enzymes exerted by insulin, EGF, or Ca2+, was also calmodulin mediated. The present and previous results suggest that the rapid, Ca(2+)-calmodulin-mediated increase in cytoskeleton-bound glycolytic enzymes, may be a general mechanism in the cell, in signal transduction of insulin, growth factors, and other Ca(2+)-mobilizing hormones. The accelerated cytoskeletal glycolysis will provide local ATP, which is required for the rapid cytoskeletal-membrane rearrangements following binding of growth factor or hormone to its receptor.
Assuntos
Calmodulina/antagonistas & inibidores , Citoesqueleto/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Músculo Liso/metabolismo , Fosfofrutoquinase-1/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Cálcio/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Técnicas In Vitro , Insulina/farmacologia , Músculo Liso/ultraestrutura , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Ligação Proteica , Ratos , Estimulação Química , Fatores de TempoRESUMO
We show here that long-term streptozotocin diabetes affects differently the intracellular distribution of phosphofructokinase (PFK), the rate-limiting enzyme of glycolysis, in tibialis anterior and gastrocnemius muscles. Diabetes, which causes ultrastructural damage in both muscle fibers, induced a decrease in PFK binding to cytoskeleton in gastrocnemius muscle but not in the tibialis anterior muscle. However, the allosteric activity of cytoskeleton-bound and soluble PFK was reduced in both kinds of muscles, most probably due to the decrease in the level of glucose 1,6-bisphosphate, the potent allosteric activator of the enzyme. Levels of fructose 2,6-bisphosphate remained unchanged. A change in the allosteric properties of the cytoskeleton-bound PFK was found only in the diabetic tibialis anterior muscle; in contrast to normal muscle, where only the soluble but not the bound enzyme responded to allosteric effectors, in the diabetic tibialis anterior muscle, the bound enzyme exhibited allosteric properties similar to the soluble enzyme. The reduction in both cytosolic and cytoskeletal PFK, and, thereby, glycolysis in these two kinds of muscles, which results most probably from the reported high pathological intracellular Ca2+ concentration, may contribute to muscle damage in diabetes.
Assuntos
Diabetes Mellitus Experimental/complicações , Frutosedifosfatos/análise , Glucose-6-Fosfato/análogos & derivados , Glucofosfatos/análise , Músculo Esquelético/química , Músculo Esquelético/enzimologia , Fosfofrutoquinase-1/metabolismo , Animais , Citoesqueleto/enzimologia , Citosol/enzimologia , Diabetes Mellitus Experimental/enzimologia , Frutosedifosfatos/metabolismo , Glucofosfatos/metabolismo , Insulina/deficiência , Masculino , Microscopia Eletrônica , Músculo Esquelético/ultraestrutura , Ratos , Ratos Endogâmicos , Estreptozocina/farmacologia , Fatores de TempoRESUMO
1. We report here on a novel action of insulin which shows that the hormone stimulates binding of phosphofructokinase (PFK) and aldolase to muscle cytoskeleton. 2. This effect was demonstrated both in vivo, by injection of insulin, in the tibialis anterior and gastrocnemius muscles, as well as in vitro, in the isolated rat diaphragm muscle incubated with insulin. 3. Insulin exerted this effect at physiologic range of concentrations and very rapidly (about 50% stimulation of binding occurred within 1 min). 4. The possible physiological significance of this rapid action of insulin, is to provide local ATP, generated by the accelerated cytoskeletal glycolysis, for other rapidly insulin-stimulated membrane-cytoskeleton processes.
Assuntos
Citoesqueleto/enzimologia , Frutose-Bifosfato Aldolase/metabolismo , Insulina/fisiologia , Músculos/enzimologia , Fosfofrutoquinase-1/metabolismo , Animais , Bovinos , Cinética , Ligação Proteica , RatosRESUMO
Insulin was shown in our previous experiments to induce an increase in binding of glycolytic enzymes to muscle cytoskeleton. We show here the same stimulatory effect of insulin in C-6 glial cells in culture. In these cells, like in muscle, a short time of incubation with insulin (1-10 min) induced an increase in cytoskeleton bound phosphofructokinase and aldolase. This stimulatory effect of insulin could be prevented by treatment with calmodulin antagonists trifluoperazine, thioridazine or CGS 9343 B (a potent and selective inhibitor of calmodulin activity), which strongly suggests that calmodulin is involved in this action of insulin. Our previous experiments have shown that growth factors and Ca(2+) also induce a rapid, calmodulin-mediated stimulation of binding of glycolytic enzymes to cytoskeleton. The present and previous results suggest that the rapid binding of glycolytic enzymes to cytoskeleton, may be a general mechanism, in different cells, in signal transduction of insulin, growth factors and other Ca(2+) -mobilizing hormones. The accelerated cytoskeletal glycolysis will supply local ATP, which is required for the rapid cytoskeletal-membrane rearrangements following the binding of hormone to its receptor.
RESUMO
Recent studies from our laboratory have revealed that basic fibroblast growth factor (bFGF) selectively inhibits the proliferation of human MCF-7 breast cancer cells. It has also been shown to enhance cis-platinum-induced apoptosis, decrease levels of the anti-apoptotic gene product bcl-2, and increase levels of the cyclin-dependent protein kinase inhibitor p21/WAF1/Cip1. Transforming growth factor beta-1 (TGFbeta1), a cell growth regulator has been found to have an inhibitory effect on breast cancer cells. The aim of the present study was to evaluate the possible role of TGFbeta1 in the antiproliferative effects of bFGF in MCF-7 breast cancer cells. We found that exogenous, as well as endogenous (overexpressed) bFGF increased TGFbeta1 mRNA expression in the cells and enhanced the secretion of TGFbeta1 into culture medium. However, exogenous addition of TGFbeta1 neither led to a decrease in bcl-2 nor induced an increase in the levels of p21/WAF1/Cip1 and neutralizing antibodies to TGFbeta1, did not reverse bFGF-induced G1 arrest northe increase in p21/WAF1/Cip1 level. In contrast, antisense oligonucleotides to TGFbeta1 abrogated the antiproliferative effects and inhibited the induction of p21/WAF1/Cip1 by bFGF in MCF-7 cells. These data suggest that the anti-proliferative effects of bFGF in human MCF-7 breast cancer cells are mediated by endogenous TGFbeta1, while exogenous TGFbeta1 does not mimic all the effects of bFGF on these breast cancer cells. These findings provide an important basis for further investigations into the autocrine and paracrine processes that control the growth of breast cancer cells.