RESUMO
The recent demonstration that bone sialoprotein (BSP) is expressed in osteotropic cancers suggests that this bone matrix protein might be implicated in the preferential seed and growth of metastatic cells in bone. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. The exact mechanisms by which BSP may favor bone metastases formation are not clearly established yet. Although BSP expression has been detected in breast, prostate, lung, thyroid, and neuroblastoma primary tumors, no information regarding its expression in metastases is available to date. In this study, we have examined BSP expression in 15 bone and 39 visceral metastatic lesions harvested from 8 breast cancer patients and 7 prostate cancer patients who died of disseminated disease. We were able to retrieve the primary lesions from 5 of the 8 breast cancer patients as well as from all 7 prostate cancer patients. All the primary breast tumor patients and 5 of the 7 primary prostate cancer patients expressed a detectable level of BSP. Bone metastases from all 8 breast cancer patients and from 5 out of 7 prostate cancer patients exhibited detectable levels of the protein. Metastatic cells in close contact with bone trabeculae usually were highly positive for BSP. BSP also was detected in secondary lesions developed at visceral sites including liver, thyroid, lung, and adrenal glands. However, BSP expression was significantly lower in visceral metastases than in skeletal ones (Mann-Whitney test, p < 0.05). Our data represent the first demonstration of an increased expression of BSP in bone metastases compared with nonskeletal metastases in human breast and prostate cancers and add weight to the body of evidence attributing a significant role to this protein in the genesis of bone metastases.
Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Neoplasias da Próstata/patologia , Sialoglicoproteínas/metabolismo , Adulto , Idoso , Feminino , Humanos , Imuno-Histoquímica , Sialoproteína de Ligação à Integrina , Masculino , Pessoa de Meia-IdadeRESUMO
Thromboxane A2 (TXA2) and prostacyclin (PGI2) are two labile products formed from arachidonic acid by the way of cyclooxygenase. An overproduction of thromboxane A2 has been detected in a series of diseases whereby this prostanoid is assumed to contribute to the underlying pathomechanisms by its potent stimulation of platelet aggregation and smooth muscle contraction. This increased TXA2 biosynthesis is frequently accompanied by a stimulation of prostacyclin formation which is one of the most potent inhibitors of platelet aggregation and smooth muscle contraction. Therefore, TXA2 / prostaglandin endoperoxide H2 receptor antagonists, thromboxane synthase inhibitors and drugs which combine both activities have been developed with the aim to suppress the formation and/or the action of thromboxane A2. Since prostacyclin has been demonstrated to counterbalance the pathological effects of TXA2, several PGI2 agonists have also been developed. This review will highlight the evolution and some of the latest findings in the field of prostacyclin and thromboxane A2 modulators mainly those which are under clinical evaluation or marketed.
Assuntos
Epoprostenol/agonistas , Epoprostenol/antagonistas & inibidores , Tromboxano A2/agonistas , Tromboxano A2/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fibrinolíticos/farmacologia , Humanos , Imidazóis/farmacologia , Receptores de Tromboxanos/antagonistas & inibidores , Sulfonamidas/farmacologia , Tromboxano-A Sintase/antagonistas & inibidoresRESUMO
Cyclooxygenase is the key enzyme in the biosynthesis of prostanoids, biologically active substances that are involved in several physiological processes but also in pathological conditions such as inflammation. Since ten years now, it is well known that this enzyme exists under two forms: a constitutive (COX-1) and an inducible form (COX-2). Both enzymes are sensitive to inhibition by conventional nonsteroidal anti-inflammatory drugs (NSAIDs). Observations that COX-1, involved in several homeostatic processes, played a housekeeping role while COX-2 expression was associated with inflammation and other pathologies such as cancer proliferation have led to the development of COX-2 selective inhibitors in order to reduce the classical side-effects, of which gastric irritation is the most common, associated with the use of conventional NSAIDs.
Assuntos
Inibidores Enzimáticos/farmacologia , Isoenzimas/antagonistas & inibidores , Prostaglandina-Endoperóxido Sintases/farmacologia , Animais , Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/farmacologia , Ensaios Clínicos como Assunto , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Indução Enzimática , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana , Estrutura Molecular , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/química , Prostaglandinas/fisiologia , Especificidade por SubstratoRESUMO
Cyclooxygenase is the key enzyme in the biosynthesis of prostanoids, biologically active substances involved in several physiological processes and also in pathological conditions such as inflammation. It has been well known for 10 years that this enzyme exists under two forms: a constitutive (COX-1) and an inducible form (COX-2). Both enzymes are sensitive to inhibition by conventional non-steroidal anti-inflammatory drugs (NSAIDs). Observations were made that COX-1 was mainly involved in homeostatic processes, while the COX-2 expression was associated with pathological conditions leading to the development of COX-2 selective inhibitors. Several methods have been reported for the evaluation of the COX-1 and COX-2 inhibitory potency and selectivity of conventional or COX-2 selective NSAIDs. In this study, we evaluated the COXs inhibitory profile of both conventional NSAIDs and COX-2 selective inhibitors using two different in vitro methods: the first test was performed using purified enzymes while the second method consisted of a whole blood assay. The results obtained with reference drugs in these two assays will be discussed and compared in this article.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Inibidores Enzimáticos/farmacologia , Isoenzimas/antagonistas & inibidores , Animais , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Relação Dose-Resposta a Droga , Humanos , Inflamação/metabolismo , Concentração Inibidora 50 , Isoenzimas/sangue , Proteínas de Membrana , Modelos Químicos , Prostaglandina-Endoperóxido Sintases/sangue , OvinosRESUMO
The aim of this work was to evaluate the effects of BM-567 (N-pentyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on both thromboxane A(2) (TXA(2)) receptors (TP) and thromboxane synthase of human platelets. The drug affinity for TP receptors of human washed platelets has been determined. In this test, BM-567 showed a high affinity (IC(50): 1.1+/-0.1nM) for the TP receptors in comparison with BM-531 (IC(50): 7.8+/-0.7nM) and sulotroban (IC(50): 931+/-85nM), two TXA(2) antagonists. We also demonstrated that BM-567 prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) (ED(100): 0.20+/-0.10 microM), U-46619, a stable TXA(2) agonist (1 microM) (ED(50): 0.30+/-0.04 microM) and collagen (1microgram ml(-1)) (% of inhibition: 44.3+/-4.3% at 10 microM) and inhibited the second wave of ADP (2microM). Moreover, when BM-567 was incubated in whole blood from healthy donors, the closure time measured by the Platelet Function analyzer (PFA-100((R))) was significantly prolonged (closure time: 215+/-21s) by using collagen/epinephrine cartridges. Finally, at the concentration of 1 microM, BM-567 completely reduced the TXB(2) production from human platelets stimulated with AA (600 microM). These results indicate that BM-567 is a novel combined TXA(2) receptor antagonist and thromboxane synthase inhibitor characterized by a powerful antiplatelet potency.
Assuntos
Plaquetas/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Receptores de Tromboxano A2 e Prostaglandina H2/antagonistas & inibidores , Compostos de Sulfonilureia/farmacologia , Tromboxano-A Sintase/metabolismo , Plaquetas/fisiologia , Humanos , Tromboxano B2/biossínteseRESUMO
Evidence exists that a large number of tumor cells such as osteosarcoma cells stimulate platelet aggregation, which can be an early step in the metastatic processes of these tumors. Thromboxane A(2) (TXA(2)) is released during platelet aggregation, and it has been suggested that this release may be pathogenic for tumor metastasis for several reasons:Some tumors release large amounts of TXA(2) compared to normal tissue.TXA(2) potentiates tumor growth in culture and increases metastasis in animals.TXA(2) is a potent stimulant of platelet aggregation and causes vascular injuries that may promote implantation of tumor cell-platelet aggregates. If TXA(2) participates in tumor metastasis, it may be hypothesized that TXA(2) inhibitors should decrease tumor metastasis. So, we have evaluated the effects of the original TXA(2) synthase inhibitor and TXA(2) receptor antagonist BM-567 on platelet aggregation induced by osteosarcoma cells using MG-63 tumor cells. Results obtained showed that this drug inhibited both MG-63 tumor-cell-induced platelet aggregation and platelet TXA(2) release following the tumor cell stimulation with IC(50) values of 3.04x10(-7) and 2.51x10(-8)M, respectively.
Assuntos
Plaquetas/efeitos dos fármacos , Neoplasias Ósseas/fisiopatologia , Osteossarcoma/fisiopatologia , Inibidores da Agregação Plaquetária/farmacologia , Receptores de Tromboxano A2 e Prostaglandina H2/antagonistas & inibidores , Compostos de Sulfonilureia/farmacologia , Tromboxano-A Sintase/antagonistas & inibidores , Plaquetas/fisiologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Humanos , Receptores de Tromboxano A2 e Prostaglandina H2/biossínteseRESUMO
In this study we examined the thromboxane A(2)(TXA(2)) receptor antagonist property of BM-531 (N-tert -butyl- N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on platelet function. The drug affinity for human washed platelet TXA(2)receptors labelled with [(3)H]SQ-29,548 has been determined (IC50: 0.0078 microM) and demonstrated to be higher than sulotroban (IC50: 0.93 microM) and SQ-29,548 (IC50: 0.021 microM). The antiaggregatory potency has been confirmed since we demonstrated that BM-531 prevented platelet aggregation in human citrated platelet-rich plasma induced by arachidonic acid (600 microM) (ED100: 0.125 microM), U-46619, a stable TXA(2)agonist (1 microM) (ED50: 0.482 microM) and collagen (1 microg mL(-1)) (% of inhibition: 42.9% at 10 microM) and inhibited the second wave of ADP (2 microM). Moreover, when BM-531 was incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA-100(trade mark)) was significantly prolonged. These results suggest that BM-531 can be regarded as a novel non-carboxylic TXA(2)antagonist with a powerful antiplatelet potency.
Assuntos
Agregação Plaquetária/efeitos dos fármacos , Receptores de Tromboxanos/antagonistas & inibidores , Sulfonamidas/farmacologia , Compostos de Sulfonilureia/farmacologia , Difosfato de Adenosina/farmacologia , Ligação Competitiva , Plaquetas/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes , Relação Dose-Resposta a Droga , Ácidos Graxos Insaturados , Hemostasia/efeitos dos fármacos , Humanos , Hidrazinas/metabolismo , Concentração Inibidora 50 , Estrutura Molecular , Inibidores da Agregação Plaquetária/farmacologia , Sulfonamidas/química , Sulfonamidas/metabolismo , Fatores de Tempo , TorasemidaRESUMO
Torasemide, a new sulfonylurea high ceiling loop diuretic, has been demonstrated to induce a concentration dependent relaxation of canine coronary precontracted with thromboxane A2 (TXA2). With the aim to develop more potent TXA2 receptor antagonists, we investigated a series of torasemide derivatives. This pharmacomodulation led to the discovery of a sulfonyl-cyanoguanidine (BM 144) which presents the same pharmacological profile as sulotroban, a TXA2 receptor antagonist used as reference.
Assuntos
Receptores de Tromboxanos/antagonistas & inibidores , Sulfonamidas/química , Sulfonamidas/farmacologia , Humanos , Técnicas In Vitro , Agregação Plaquetária/efeitos dos fármacos , Sulfonamidas/síntese química , TorasemidaRESUMO
Evidence of the existence of two forms of cyclooxygenases and the clinical relevance of COX-2 inhibition led to the development of COX-2 selective NSAIDs. In order to evaluate this selectivity, we have developed and validated an enzymatic method. The precision and reproducibility of the assay were determined and COX-2 selectivity examined using nimesulide and indometacin.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Indometacina/farmacologia , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Sulfonamidas/farmacologia , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Especificidade por SubstratoRESUMO
OBJECTIVES: To investigate the mechanisms of action underlying the anti-inflammatory action of aceclofenac in vivo, we studied in vitro the effect of aceclofenac and its main metabolite, 4'-hydroxyaceclofenac, in comparison with diclofenac, another metabolite, on cyclooxygenases activity as well as interleukin-1beta, -6 and -8, nitric oxide, and prostaglandin E2 production by human osteoarthritic and normal articular chondrocytes. METHODS: Enzymatically isolated human chondrocytes were cultured for 72 h in the absence or presence of interleukin-1beta (IL-1beta) or lipopolysacharride (LPS) and with or without increased amounts (1 to 30 microM) of aceclofenac or metabolites. The production of different cytokines was measured by Enzyme Amplified Sensitivity Immunoassays (EASIA). Prostaglandin E2 was quantified by a specific radioimmunoassay. Nitrite and nitrate concentrations in the culture supernatants were determined by spectrophotometric method based upon the Griess reaction. Cyclooxygenase-2, inducible NO synthase and IL-1beta gene expression were quantified by reverse transcription of mRNA followed by real time and quantitative polymerase chain reaction. Finally, cyclooxygenase inhibitory potency of the drugs was also tested in both a cell-free system using purified ovine cyclooxygenase-1 and -2 (COX-1 and COX-2) and at a cellular level using human whole blood assay. RESULTS: We have demonstrated that aceclofenac, 4'-hydroxyaceclofenac and diclofenac significantly decreased interleukin-6 production at concentrations ranged among 1 to 30 microM and fully blocked prostaglandin E2 synthesis by IL-1beta- or LPS-stimulated human chondrocytes. Aceclofenac and diclofenac had no effect on interleukin-8 production while 4'-hydroxyaceclofenac slightly decreased this parameter at the highest dose (30 microM). Aceclofenac was without effect on IL-1beta- or LPS-stimulated nitric oxide production. At 30 microM, 4'-hydroxyaceclofenac inhibited both IL-1beta or LPS-stimulated nitric oxide production while diclofenac inhibited only the LPS-stimulated production. Finally, at 30 microM, the three drugs significantly decreased IL-1beta mRNA. In the whole blood test, aceclofenac and 4'-hydroxyaceclofenac weakly inhibited COX-1 with IC50 values superior to 100 microM, but decreased by 50% COX-2 activity at the concentration of 0.77 and 36 microM, respectively. Diclofenac strongly inhibited both COX-1 and COX-2 with IC50 values of 0.6 and 0.04 microM, respectively. On the other hand, aceclofenac and diclofenac weakly inhibited purified ovine cyclooxygenases with IC50 values superior to 100 microM, whereas 4'-hydroxyaceclofenac was without effect. CONCLUSIONS: These results suggest that aceclofenac actions are multifactorial and that metabolites could contribute to its anti-inflammatory actions.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Condrócitos/metabolismo , Diclofenaco/análogos & derivados , Diclofenaco/farmacologia , Mediadores da Inflamação/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Condrócitos/efeitos dos fármacos , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Citocinas/metabolismo , DNA/biossíntese , DNA/genética , Dinoprostona/metabolismo , Humanos , Técnicas In Vitro , Interleucina-1/biossíntese , Interleucina-1/genética , Isoenzimas/metabolismo , Proteínas de Membrana , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Prostaglandina-Endoperóxido Sintases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BM-531 (N-tert-butyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, is a novel noncarboxylic thromboxane receptor antagonist and thromboxane synthase inhibitor. Indeed, its affinity for human washed platelet TXA2 receptors labeled with [3H]SQ-29548 (IC50 = 0.0078 microM) is higher than sulotroban (IC50 = 0.93 microM) and SQ-29548 (IC50 = 0.021 microM). Moreover, BM-531 is characterized by a potent antiaggregatory property. Indeed, on one hand, in human citrated platelet-rich plasma BM-531 prevents platelet aggregation induced by arachidonic acid (600 microM) (ED100 = 0.125 microM), U-46619, a stable TXA2 agonist (1 microM) (ED50 = 0.482 microM) or collagen (1 microgram/mL) (percentage of inhibition: 42.9% at 10 microM) and inhibits the second wave of ADP (2 microM)-induced aggregation. On the other hand, when BM-531 is incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA-100) is significantly prolonged. In addition, at the concentrations of 10 and 1 microM, BM-531 totally prevents the production of TXB2 by human platelets activated by arachidonic acid. Finally, at 10 microM, BM-531 significantly prevents rat fundus contractions induced by U-46619 but not by prostacyclin. These results suggest that BM-531, which is devoid of the diuretic property of torasemide, can be regarded as a promising antiplatelet agent.