RESUMO
The Gi protein-associated A(3) adenosine receptor (A(3) AR) is a member of the adenosine receptor family. Selective agonists at the A(3) AR, such as CF101 and CF102 were found to induce anti-inflammatory and anti-cancer effects. In this study, we examined the differential effect of CF102 in pathological conditions of the liver. The anti-inflammatory protective effect of CF101 was tested in a model of liver inflammation induced by Concanavalin A (Con. A) and the anti-cancer effect of CF102 was examined in vitro and in a xenograft animal model utilizing Hep-3B hepatocellular carcinoma (HCC) cells. The mechanism of action was explored by following the expression levels of key signaling proteins in the inflamed and tumor liver tissues, utilizing Western blot (WB) analysis. In the liver inflammation model, CF102 (100 µg/kg) markedly reduced the secretion of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase in comparison to the vehicle-treated group. Mechanistically, CF102 treatment decreased the expression level of phosphorylated glycogen synthase kinase-3ß, NF-κB, and TNF-α and prevented apoptosis in the liver. This was demonstrated by decreased expression levels of Fas receptor (FasR) and of the pro-apoptotic proteins Bax and Bad in liver tissues. In addition, CF102-induced apoptosis of Hep-3B cells both in vitro and in vivo via de-regulation of the PI3K-NF-κB signaling pathway, resulting in up-regulation of pro-apoptotic proteins. Taken together, CF102 acts as a protective agent in liver inflammation and inhibits HCC tumor growth. These results suggest that CF102 through its differential effect is a potential drug candidate to treat various pathological liver conditions.
Assuntos
Agonistas do Receptor A3 de Adenosina/farmacologia , Adenosina/análogos & derivados , Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Fígado/efeitos dos fármacos , Fígado/patologia , Adenosina/farmacologia , Adenosina/uso terapêutico , Agonistas do Receptor A3 de Adenosina/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Concanavalina A , Hepatite/tratamento farmacológico , Hepatite/patologia , Fígado/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor A3 de Adenosina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: Studies have suggested that rheumatoid arthritis (RA) and osteoarthritis (OA) share common characteristics. The highly selective A(3) adenosine receptor agonist CF101 was recently defined as a potent antiinflammatory agent for the treatment of RA. The purpose of this study was to examine the effects of CF101 on the clinical and pathologic manifestations of OA in an experimental animal model. METHODS: OA was induced in rats by monosodium iodoacetate, and upon disease onset, oral treatment with CF101 (100 microg/kg given twice daily) was initiated. The A(3) adenosine receptor antagonist MRS1220 (100 microg/kg given twice daily) was administered orally, 30 minutes before CF101 treatment. The OA clinical score was monitored by knee diameter measurements and by radiographic analyses. Histologic analyses were performed following staining with hematoxylin and eosin, Safranin O-fast green, or toluidine blue, and histologic changes were scored according to a modified Mankin system. Signaling proteins were assayed by Western blotting; apoptosis was detected via immunohistochemistry and TUNEL analyses. RESULTS: CF101 induced a marked decrease in knee diameter and improved the changes noted on radiographs. Administration of MRS1220 counteracted the effects of CF101. CF101 prevented cartilage damage, osteoclast/osteophyte formation, and bone destruction. In addition, CF101 markedly reduced pannus formation and lymphocyte infiltration. Mechanistically, CF101 induced deregulation of the NF-kappaB signaling pathway, resulting in down-regulation of tumor necrosis factor alpha. Consequently, CF101 induced apoptosis of inflammatory cells that had infiltrated the knee joints; however, it prevented apoptosis of chondrocytes. CONCLUSION: CF101 deregulated the NF-kappaB signaling pathway involved in the pathogenesis of OA. CF101 induced apoptosis of inflammatory cells and acted as a cartilage protective agent, which suggests that it would be a suitable candidate drug for the treatment of OA.
Assuntos
Adenosina/análogos & derivados , Anti-Inflamatórios/uso terapêutico , Cartilagem Articular/patologia , Inflamação/tratamento farmacológico , Osteoartrite/tratamento farmacológico , Osteoartrite/patologia , Adenosina/efeitos adversos , Adenosina/farmacologia , Adenosina/uso terapêutico , Antagonistas do Receptor A3 de Adenosina , Animais , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Modelos Animais de Doenças , Inflamação/metabolismo , Inflamação/patologia , Iodoacetatos/efeitos adversos , Masculino , NF-kappa B/metabolismo , Osteoartrite/induzido quimicamente , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The study of the A(3) adenosine receptor (A(3)AR) represents a rapidly growing and intense area of research in the adenosine field. The present chapter will provide an overview of the expression patterns, molecular pharmacology and functional role of this A(3)AR subtype under pathophysiological conditions. Through studies utilizing selective A(3)AR agonists and antagonists, or A(3)AR knockout mice, it is now clear that this receptor plays a critical role in the modulation of ischemic diseases as well as in inflammatory and autoimmune pathologies. Therefore, the potential therapeutic use of agonists and antagonists will also be described. The discussion will principally address the use of such compounds in the treatment of brain and heart ischemia, asthma, sepsis and glaucoma. The final part concentrates on the molecular basis of A(3)ARs in autoimmune diseases such as rheumatoid arthritis, and includes a description of clinical trials with the selective agonist CF101. Based on this chapter, it is evident that continued research to discover agonists and antagonists for the A(3)AR subtype is warranted.
Assuntos
Receptor A3 de Adenosina/fisiologia , Animais , Doenças Autoimunes/etiologia , Isquemia Encefálica/etiologia , Humanos , Inflamação/etiologia , Isquemia Miocárdica/etiologia , Receptor A3 de Adenosina/efeitos dos fármacos , Receptor A3 de Adenosina/genética , Transdução de SinaisRESUMO
The A(1), A(2A), A(2B) and A(3) G-protein-coupled cell surface adenosine receptors (ARs) are found to be upregulated in various tumor cells. Activation of the receptors by specific ligands, agonists or antagonists, modulates tumor growth via a range of signaling pathways. The A(1)AR was found to play a role in preventing the development of glioblastomas. This antitumor effect of the A(1)AR is mediated via tumor-associated microglial cells. Activation of the A(2A)AR results in inhibition of the immune response to tumors via suppression of T regulatory cell function and inhibition of natural killer cell cytotoxicity and tumor-specific CD4+/CD8+ activity. Therefore, it is suggested that pharmacological inhibition of A(2A)AR activation by specific antagonists may enhance immunotherapeutics in cancer therapy. Activation of the A(2B)AR plays a role in the development of tumors via upregulation of the expression levels of angiogenic factors in microvascular endothelial cells. In contrast, it was evident that activation of A(2B)AR results in inhibition of ERK1/2 phosphorylation and MAP kinase activity, which are involved in tumor cell growth signals. Finally, A(3)AR was found to be highly expressed in tumor cells and tissues while low expression levels were noted in normal cells or adjacent tissue. Receptor expression in the tumor tissues was directly correlated to disease severity. The high receptor expression in the tumors was attributed to overexpression of NF-kappaB, known to act as an A(3)AR transcription factor. Interestingly, high A(3)AR expression levels were found in peripheral blood mononuclear cells (PBMCs) derived from tumor-bearing animals and cancer patients, reflecting receptor status in the tumors. A(3)AR agonists were found to induce tumor growth inhibition, both in vitro and in vivo, via modulation of the Wnt and the NF-kappaB signaling pathways. Taken together, A(3)ARs that are abundantly expressed in tumor cells may be targeted by specific A(3)AR agonists, leading to tumor growth inhibition. The unique characteristics of these A(3)AR agonists make them attractive as drug candidates.
Assuntos
Neoplasias/etiologia , Receptores Purinérgicos P1/fisiologia , Antagonistas do Receptor A2 de Adenosina , Antagonistas do Receptor A3 de Adenosina , Animais , Antineoplásicos/farmacologia , Humanos , Imunoterapia , NF-kappa B/fisiologia , Neoplasias/imunologia , Receptor A1 de Adenosina/fisiologia , Receptor A2A de Adenosina/fisiologia , Receptor A2B de Adenosina/fisiologia , Receptor A3 de Adenosina/fisiologia , Transdução de Sinais , Proteínas Wnt/fisiologiaRESUMO
The Gi protein associated A(3) adenosine receptor (A(3)AR) was recently defined as a novel anti-inflammatory target. The aim of this study was to look at A(3)AR expression levels in peripheral blood mononuclear cells (PBMCs) of patients with autoimmune inflammatory diseases and to explore transcription factors involved receptor expression. Over-expression of A(3)AR was found in PBMCs derived from patients with rheumatoid arthritis (RA), psoriasis and Crohn's disease compared with PBMCs from healthy subjects. Bioinformatics analysis demonstrated the presence of DNA binding sites for nuclear factor-kappaB (NF-kappaB) and cyclic AMP-responsive element binding protein (CREB) in the A(3)AR gene promoter. Up-regulation of NF-kappaB and CREB was found in the PBMCs from patients with RA, psoriasis and Crohn's disease. The PI3K-PKB/Akt signaling pathway, known to regulate both the NF-kappaB and CREB, was also up-regulated in the patients' PBMCs. Taken together, NF-kappaB and CREB are involved with the over-expression of A(3)AR in patients with autoimmune inflammatory diseases. The receptor may be considered as a specific target to combat inflammation.
Assuntos
Artrite Reumatoide/metabolismo , Doença de Crohn/metabolismo , Psoríase/metabolismo , Receptor A3 de Adenosina/biossíntese , Adulto , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Humanos , Quinase I-kappa B/metabolismo , Leucócitos Mononucleares/metabolismo , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/fisiologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Receptor A3 de Adenosina/genética , Proteínas Supressoras de Tumor , Regulação para CimaRESUMO
The A3 adenosine receptor (A(3)AR) is highly expressed in tumors and was suggested as a target for cancer treatment. In this study, we show that A(3)AR is highly expressed in tumor tissues and in peripheral blood mononuclear cells (PBMCs) derived from patients with HCC, as well as from HCC tumor-bearing rats. The high expression level of the receptor was directly correlated to overexpression of NF-kappaB, known as a transcription factor of A(3)AR. CF102, a synthetic highly selective agonist to A(3)AR induced a marked dose response inhibition of tumor growth in N1S1 HCC tumor rats, via de-regulation of the NF-kappaB and the Wnt signal transduction pathways, resulting in apoptosis of tumor cells. Taken together, A(3)AR is highly expressed in tumors and PBMCs of HCC patients and tumor-bearing rats. CF102 induced apoptosis and tumor growth inhibition. These data suggest A(3)AR as a novel targeted therapy to treat HCC.
Assuntos
Adenosina/análogos & derivados , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , NF-kappa B/efeitos dos fármacos , Proteínas Wnt/efeitos dos fármacos , Adenosina/farmacologia , Agonistas do Receptor A3 de Adenosina , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Carcinoma Hepatocelular/metabolismo , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor A3 de Adenosina/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/metabolismoRESUMO
The A(3) adenosine receptor (A(3)AR) is over-expressed in inflammatory cells and was defined as a target to combat inflammation. Synthetic agonists to this receptor, such as IB-MECA and Cl-IB-MECA, exert an anti-inflammatory effect in experimental animal models of adjuvant- and collagen-induced arthritis. In this study we present a novel A(3)AR agonist, CF502, with high affinity and selectivity at the human A(3)AR. CF502 induced a dose dependent inhibitory effect on the proliferation of fibroblast-like synoviocytes (FLS) via de-regulation of the nuclear factor-kappa B (NF-kappaB) signaling pathway. Furthermore, CF502 markedly suppressed the clinical and pathological manifestations of adjuvant-induced arthritis (AIA) in a rat experimental model when given orally at a low dose (100 microg/kg). As is typical of other G-protein coupled receptors, the A(3)AR expression level was down-regulated shortly after treatment with agonist CF502 in paw and in peripheral blood mononuclear cells (PBMCs) derived from treated AIA animals. Subsequently, a decrease in the expression levels of protein kinase B/Akt (PKB/Akt), IkappaB kinase (IKK), I kappa B (IkappaB), NF-kappaB and tumor necrosis factor-alpha (TNF-alpha) took place. In addition, the expression levels of glycogen synthase kinase-3 beta (GSK-3beta), beta-catenin, and poly(ADP-ribose)polymerase (PARP), known to control the level and activity of NF-kappaB, were down-regulated upon treatment with CF502. Taken together, CF502 inhibits FLS growth and the inflammatory manifestations of arthritis, supporting the development of A(3)AR agonists for the treatment of rheumatoid arthritis.
Assuntos
Agonistas do Receptor A3 de Adenosina , Adenosina/análogos & derivados , Artrite Reumatoide/tratamento farmacológico , NF-kappa B/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Membrana Sinovial/patologia , Adenosina/farmacologia , Adenosina/uso terapêutico , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Artrite/metabolismo , Artrite/patologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Modelos Animais de Doenças , Humanos , Inflamação/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Membrana Sinovial/metabolismoRESUMO
The A3 adenosine receptor (A3AR) is highly expressed in various human solid tumor cells whereas low expression is found in the adjacent normal tissues. Activation of the A3AR with synthetic highly selective agonists, such as IB-MECA, Cl-IB-MECA or LJ529, induces tumor growth inhibition of melanoma, lymphoma, breast, hepatoma, prostate and colon carcinoma cells both in vitro and in vivo. Two molecular events take place upon receptor activation and include: a. receptor internalization and subsequent degradation, followed by decreased receptor mRNA and protein expression level. b. modulation of down-stream signal transduction pathways, including those related to Wnt and NF-κB. Subsequently, the levels of cyclin D1 and c-Myc are decreased leading to tumor growth inhibition. IB-MECA synergizes with chemotherapeutic agents to yield an additive anti-tumor effect and protects against myelotoxicity induced by chemotherapy. Taken together, A3AR agonists may be suggested as a new family of orally bioavailable compounds to be developed as potent inhibitors of malignant diseases.
RESUMO
OBJECTIVES: The anti-inflammatory effect of adenosine is partially mediated via the A3 adenosine receptor (A3AR), a Gi protein associated cell surface receptor. The highly selective A3AR agonist, IB-MECA was earlier shown to prevent the clinical and pathological manifestations of arthritis in experimental animal models of collagen and adjuvant induced arthritis (AIA). In this study we tested the effect of IB-MECA on the prevention of bone resorption in AIA rats and looked at the molecular mechanism of action. METHODS: Rats with AIA were treated orally twice daily with IB-MECA starting upon onset of disease and the clinical score was evaluated every other day. At study termination the foot, knee and hip region of both vehicle and IB-MECA treated animals were subjected to histomorphometric analysis. Western blot analysis was carried out on paw protein extracts. RESULTS: IB-MECA ameliorated the clinical manifestations of the disease and reduced pannus and fibrosis formation, attenuated cartilage and bone destruction and decreased the number of osteoclasts. In cell protein extracts derived from paw of AIA rats, A3AR was highly expressed in comparison to naïve animals. In paw extracts derived from IB-MECA treated AIA rats, down-regulation of the A3AR protein expression level was noted. PI3K, PKB/Akt, IKK, NF-kappaB, TNF-alpha and RANKL were down-regulated whereas caspase 3 was up-regulated. CONCLUSION: IB-MECA, a small highly bioavailable molecule, induces modulation of proteins which control survival and apoptosis resulting in the amelioration of the inflammatory process and the preservation of bone mass in AIA rats.
Assuntos
Agonistas do Receptor A3 de Adenosina , Adenosina/análogos & derivados , Artrite Experimental/tratamento farmacológico , Conservadores da Densidade Óssea/uso terapêutico , Reabsorção Óssea/prevenção & controle , Adenosina/uso terapêutico , Administração Oral , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Feminino , Fibrose/induzido quimicamente , Fibrose/patologia , Membro Posterior/efeitos dos fármacos , Membro Posterior/metabolismo , Membro Posterior/patologia , Articulações/efeitos dos fármacos , Articulações/metabolismo , Articulações/patologia , Ratos , Ratos Endogâmicos Lew , Receptor A3 de Adenosina/metabolismoRESUMO
OBJECTIVES: To assess safety, tolerability, pharmacokinetics and hemodynamic effects of oral CF 101, an A3 adenosine receptor (A3AR) agonist, in healthy men. METHODS: One single and 1 repeated dose, parallel-group, ascending dose, double-blind and placebo-controlled study in normal volunteers. In the single dose study, n = 15 subjects received 1, 5 or 10 mg oral CF101; in each group 1 subject received placebo, the remainder active CF101. In the repeat-dose study, n = 28 subjects received repeated 12-hourly oral doses of CF 101 (2, 3, 4 or 5 mg) for 7 days, in each group 2 subjects received placebo, the remainder active CF101. TEST MATERIALS: Single-dose study: CF101 in 30% Cremophor RH40. Multiple-dose sudy: CF101 in 0.5% methylcellulose suspension. Both studies: the corresponding vehicles were used as placebos. Galenicals were prepared remotely from the clinical study site to ensure double-blind nature of the study. RESULTS TOLERABILITY: Single doses up to 5 mg CF101 were safe and well-tolerated. However, the single dose of 10 mg CF101 was associated with flushing, tachycardia, nausea and vomiting, which were viewed as dose-limiting in normal volunteers. Single doses of CF101 (as well as the first of the multiple doses) were associated with increases in heart rate (8 - 24 beats/min after 5 mg and 18 - 55 beats/min after 10 mg). Multiple doses up to 4 mg 12-hourly for 7 days were safe and well-tolerated. However, the 5 mg multiple-dose group reported headache, drowsiness, hot flushes and dizziness on standing; this declined with dosing duration and was not dose-limiting in this study. Adverse events were commonest near t(max). RESULTS PHARMACOKINETICS: For oral CF101, the t(max) was always 1 - 2 h post-dose and t 1/2 about 9 h, in both the single- and multiple-dose studies. For a single 5 mg dose (mean +/- SD) C(max) = 81.6 +/- 23.6 ng/ml in the single dose study, and 63.6 +/- 22.0 ng/ml after the first of the multiple doses; AUC if was 904.0 +/- 221.9 ng.h/ml and 596.1 +/- 196.6 ng.h/ml for the 2 studies, respectively. After 7 days of multiple dosing there was little change, and AUC(0-24h) = 601.0 +/- 163.6 ng.h/ml. These pharmacokinetic parameters were linearly proportional to dose in the other treatment groups. RESULTS PHARMACODYNAMICS: Increases in heart rate were related to plasma concentration and evident only in the upper range of concentrations observed. There were no changes on ECG monitoring beyond sinus tachycardia, and, in particular, no evidence of PR prolongation in any subject (n = 43). In comparison with single doses, this response was almost absent after 7 days of dosing. Leucocytosis (increases up to about 1.5 x 10(9)/l after 5 and 10 mg) was similarly transient and reversible after multiple dosing. CONCLUSIONS: Single oral doses up to 5 mg CF101 and repeated doses up to 4 mg 12-hourly for 7 days were safe and well-tolerated. Multiple-dose CF101 pharmacokinetics were unchanged and predictable from single-dose estimates, and were linearly proportional to dose. Increases in heart rate and neutrophil count were reversible during multiple dosing and were not dose-limiting in the repeat dose study. CF101 warrants further study for its efficacy in treating human disease.
Assuntos
Agonistas do Receptor A3 de Adenosina , Adenosina/análogos & derivados , Adenosina/farmacocinética , Adenosina/administração & dosagem , Adenosina/efeitos adversos , Administração Oral , Adulto , Área Sob a Curva , Relação Dose-Resposta a Droga , Método Duplo-Cego , Tolerância a Medicamentos , Meia-Vida , Frequência Cardíaca/efeitos dos fármacos , Humanos , Contagem de Leucócitos , Masculino , Neutrófilos/metabolismoRESUMO
Adenosine is a purine nucleoside that acts as a regulatory molecule by binding to specific G-protein-coupled A1, A(2A), A(2B), and A3 cell surface receptors. We have recently demonstrated that adenosine inhibits tumour cell growth and concomitantly stimulates bone marrow cell proliferation via activation of the A3 adenosine receptor (A3AR). In the present study, we show that a synthetic agonist to the A3AR, CF101, at the low nanomolar concentration range, inhibits HCT-116 human colon carcinoma cell growth. This effect was reversed by the selective A3AR antagonist MRS1523, demonstrating the specificity of the response. CF101 (given orally) was efficacious in inhibiting the development of primary tumours in xenograft and syngeneic models in which mice were inoculated subcutaneously with human HCT-116 or murine CT-26 colon carcinoma cells, respectively. Moreover, CF101 suppressed (50%, P<0.01) colon cancer liver metastases in syngeneic mice inoculated to the spleen with CT-26 cells. The mechanism of action entailed upregulation of interleukin-12 production in the CF101-treated groups and potentiation of NK cell activity. In the HCT-116 xenograft model in which a combined therapy of CF101 and 5-fluorouracyl (5-FU) was examined, an additive antitumour effect was demonstrated. Moreover, CF101 prevented the 5-FU-induced myelotoxicity, resulting in normal values of white blood cell and neutrophil counts. We conclude that the A3AR agonist CF101, a small orally bioavailable molecule, exerts systemic anticancer, antimetastatic, and myeloprotective effects in colon carcinoma-bearing mice, and may serve as an adjuvant treatment to enhance the chemotherapeutic index and prevent myelotoxicity.
Assuntos
Adenosina/farmacologia , Carcinoma/secundário , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Neoplasias Hepáticas/secundário , Agonistas do Receptor Purinérgico P1 , Adenosina/análogos & derivados , Administração Oral , Animais , Disponibilidade Biológica , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Adenosine, a purine nucleoside, acts as a regulatory molecule, by binding to specific G-protein-coupled A(1), A(2A), A(2B), and A(3) cell surface receptors. We have recently demonstrated that adenosine induces a differential effect on tumor and normal cells. While inhibiting in vitro tumor cell growth, it stimulates bone marrow cell proliferation. This dual activity was mediated through the A3 adenosine receptor. This study showed that a synthetic agonist to the A3 adenosine receptor, 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyl-uronamide (Cl-IB-MECA), at nanomolar concentrations, inhibited tumor cell growth through a cytostatic pathway, i.e., induced an increase number of cells in the G0/G1 phase of the cell cycle and decreased the telomeric signal. Interestingly, Cl-IB-MECA stimulates murine bone marrow cell proliferation through the induction of granulocyte-colony-stimulating factor. Oral administration of Cl-IB-MECA to melanoma-bearing mice suppressed the development of melanoma lung metastases (60.8 +/- 6.5% inhibition). In combination with cyclophosphamide, a synergistic anti-tumor effect was achieved (78.5 +/- 9.1% inhibition). Furthermore, Cl-IB-MECA prevented the cyclophosphamide-induced myelotoxic effects by increasing the number of white blood cells and the percentage of neutrophils, demonstrating its efficacy as a chemoprotective agent. We conclude that A3 adenosine receptor agonist, Cl-IB-MECA, exhibits systemic anticancer and chemoprotective effects.
Assuntos
Neoplasias/prevenção & controle , Neoplasias/terapia , Receptores Purinérgicos P1/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Administração Oral , Animais , Antineoplásicos Alquilantes/farmacologia , Células da Medula Óssea/metabolismo , Ciclo Celular , Divisão Celular , Ciclofosfamida/farmacologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Ligação Proteica , Receptor A3 de Adenosina , Telômero/metabolismo , Células Tumorais CultivadasRESUMO
Tumor metastases are extremely rare in striated muscles. Lately, we have found that muscle cell conditioned medium (MCM) inhibits the proliferation of various tumor cells while maintaining the growth of normal murine bone marrow cells. This dual activity was confirmed in vivo when the MCM was administered orally, i.e., it inhibited the development of tumor growth in mice and prevented the myelotoxic effects of chemotherapy. Adenosine was found to be one of the active components of MCM, inhibiting tumor cell growth while maintaining bone marrow cell proliferation in vitro. Adenosine is known to act as an important regulatory molecule through its binding to specific G-protein-associated A1, A(2a), A(2b) and A3 cell surface receptors. In distinction from MCM, adenosine did not suppress tumor development in mice and was not active as a chemoprotective agent when administered orally or intravenously. Thus, the in vivo activity of MCM could not be attributed to adenosine. In this study, MCM from which adenosine was enzymatically removed still retained its dual activity that was also found to be mediated through the A3 adenosine receptor (A3AR). This result led to the conclusion that natural agonists to A3AR were responsible for the activity of MCM. We further tested synthetic agonist to the A3AR and demonstrated that it possessed the same in vitro and in vivo activity profile as MCM. Taken together, muscle cells, in addition to adenosine, secrete natural agonists to A3AR. These agonists are stable nondegradable molecules and may contribute to the systemic anticancer and chemoprotective activity exerted by MCM. This group of molecules may account for the rarity of tumor metastases in muscle.
Assuntos
Neoplasias Musculares/metabolismo , Músculos/metabolismo , Agonistas do Receptor Purinérgico P1 , Adenosina/metabolismo , Animais , Antineoplásicos/farmacologia , Células da Medula Óssea/metabolismo , Divisão Celular , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta a Droga , Humanos , Camundongos , Modelos Químicos , Metástase Neoplásica , Antagonistas de Receptores Purinérgicos P1 , Receptor A3 de Adenosina , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Adenosine is an ubiquitous nucleoside present in all body cells. It is released from metabolically active or stressed cells and subsequently acts as a regulatory molecule through binding to specific A1, A2A, A2B and A3 cell surface receptors. The synthesis of agonists and antagonists to the adenosine receptors and their cloning enabled the exploration of their physiological functions. As nearly all cells express specific adenosine receptors, adenosine serves as a physiological regulator and acts as a cardioprotector, neuroprotector, chemoprotector, and as an immunomodulator. At the cellular level, activation of the receptors by adenosine initiates signal transduction mechanisms through G-protein associated receptors. Adenosine's unique characteristic is to differentially modulate normal and transformed cell growth, depending upon its extracellular concentration, the expression of adenosine cell surface receptors, and the physiological state of the target cell. Stimulation of cell proliferation following incubation with adenosine has been demonstrated in a variety of normal cells in the range of low micromolar concentrations, including mesangial and thymocyte cells, Swiss mouse 3T3 fibroblasts, and bone marrow cells. Induction of apoptosis in tumor or normal cells was shown at higher adenosine concentrations (>100 microM) such as in leukemia HL-60, lymphoma U-937, A431 epidermoid cells, and GH3 tumor pituitary cell lines. It was further noted that the A3 adenosine receptor (A3AR) plays a key role in the inhibitory and stimulatory growth activities of adenosine. Modulation of the A3AR was found to affect cell growth either positively or negatively depending on the concentration of the agonist, similar to the effect described for adenosine. At nanomolar concentrations, the A3AR agonists possess dual activity, i.e., antiproliferative activity toward tumor cells and stimulatory effect on bone marrow cells. In vivo, these agonists exerted anti-cancer effects, and when given in combination with chemotherapy, they enhanced the chemotherapeutic index and acted as chemoprotective agents. Taken together, activation of the A3AR, by minute concentrations of its natural ligand or synthetic agonists, may serve as a new approach for cancer therapy.
Assuntos
Adenosina/fisiologia , Neoplasias/patologia , Receptores Purinérgicos P1/fisiologia , Animais , Divisão Celular/fisiologia , Humanos , Receptor A3 de Adenosina , Valores de Referência , Transdução de Sinais/fisiologiaRESUMO
In this study, we demonstrated several mechanisms exploring the inhibitory effect of low-dose adenosine on lymphoma cell growth. Adenosine, a purine nucleoside present in plasma and other extracellular fluids, acts as a regulatory molecule, by binding to G-protein associated cell-surface receptors, A1, A2 and A3. Recently we showed that low-dose adenosine released by muscle cells, inhibits tumour cell growth and thus attributes to the rarity of muscle metastases. In the present work, a cytostatic effect of adenosine on the proliferation of the Nb2-11C rat lymphoma cell line was demonstrated. This effect was mediated through the induction of cell cycle arrest in the G0/G1 phase and by decreasing the telomeric signal in these cells. Adenosine was found to exert its antiproliferative effect mainly through binding to its A3 receptor. The cytostatic anticancer activity, mediated through the A3 adenosine receptor, turns it into a potential target for the development of anticancer therapies.
Assuntos
Adenosina/fisiologia , Linfoma/patologia , Receptores Purinérgicos P1/fisiologia , Animais , Apoptose/fisiologia , Divisão Celular/fisiologia , DNA de Neoplasias/análise , Antagonistas de Receptores Purinérgicos P1 , Ratos , Receptor A3 de Adenosina , Telômero/química , Células Tumorais CultivadasRESUMO
Adenosine, a ubiquitous nucleoside, is released into the extracellular environment from metabolically active or stressed cells. It binds to cells through specific A1, A(2A), A(2B), and A3 G-protein-associated cell-surface receptors, thus acting as a signal-transduction molecule by regulating the levels of adenylyl cyclase and phospholipase C. In this study, we showed that adenosine stimulates the proliferation of murine bone marrow cells in vitro. Pharmacological studies, using antagonists to the adenosine receptors, revealed that this activity was mediated through the binding of adenosine to its A1 and A3 receptors. This result was further corroborated by showing that the two selective A1 and A3 receptor agonists, N-cyclopentyladenosine (CPA) and 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-be ta-D-ribofuranuronamide (IB-MECA) respectively, induced bone marrow cell proliferation in a manner similar to adenosine. Adenosine's interaction with its A1 and A3 receptors induced G-CSF production, which led to its stimulatory effect on bone marrow cells. These results were confirmed in vivo when we demonstrated that low-dose adenosine (0.25 mg/kg) acted as a chemoprotective agent. When administered after chemotherapy, it restored the number of leukocytes and neutrophils to normal levels, compared with the decline in these parameters after chemotherapy alone. It is suggested that low-dose adenosine, already in clinical use, may also be applied as a chemoprotective agent.
Assuntos
Adenosina/farmacologia , Células da Medula Óssea/fisiologia , Fator Estimulador de Colônias de Granulócitos/biossíntese , Receptores Purinérgicos P1/fisiologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Ciclofosfamida/toxicidade , Fator Estimulador de Colônias de Granulócitos/sangue , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Antagonistas de Receptores Purinérgicos P1 , Quinazolinas/farmacologia , Receptor A3 de Adenosina , Teobromina/análogos & derivados , Teobromina/farmacologia , Triazóis/farmacologia , Xantinas/farmacologiaRESUMO
Tumor metastases are extremely rare in striated muscles. This is surprising given the fact that this tissue constitutes 60% of body weight. The present study focuses on small molecules produced and secreted by muscle cells which possess anti-cancer activity in vivo. Recently we have shown that a low molecular weight fraction (< 1000 Dalton) of skeletal muscle cell conditioned medium (muscle factor-MF), markedly inhibits the proliferation of carcinoma, sarcoma or melanoma cell lines in vitro. The MF exerts a cytostatic effect on tumor cell growth and arrests the cells in the G0/G1 of the cell cycle. However, normal cell proliferation, such as bone marrow and fibroblasts, was stimulated following incubation with MF. In this study, the effect of orally administered MF on melanoma and sarcoma growth was examined in mice. The administration of MF to mice inoculated intravenously with melanoma (B16-F10) or sarcoma (MCA-105) cells, resulted in a statistically significant inhibition of metastatic lung foci. In a different model, melanoma was induced in the foot pad and after development of a local lesion, the leg was amputated. A prolonged survival time was observed in the MF treated groups. Since the MF stimulated bone marrow cell proliferation in vitro, we decided to test its efficacy as an inhibitor of the myelotoxic effect exerted by chemotherapy, in vivo. MF, administered after chemotherapy, restored the number of white blood cells and yielded an increased percentage of neutrophils compared with the decline in these parameters after administration of chemotherapy alone. Thus, it is indicated that MF exerted a systemic anti tumor and chemoprotective effect when given orally. It can be concluded that it is bioavailable and is not biodegradable in the digestive system. MF may be considered as a potential therapy for the prevention of tumor spread.
Assuntos
Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Proteínas Musculares/administração & dosagem , Sarcoma Experimental/patologia , Administração Oral , Animais , Antineoplásicos/efeitos adversos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/farmacologia , Sarcoma Experimental/tratamento farmacológicoRESUMO
In this study, we investigated the basis of the resistance of muscles to tumor metastases. We found that a low molecular weight fraction (Mr <3000) of skeletal muscle cell-conditioned medium (MCM) markedly inhibits the proliferation of carcinoma, sarcoma, or melanoma cell lines in vitro. The MCM exerts a cytostatic effect on tumor cell growth and arrests the cells in G0/G1 of the cell cycle. However, normal cell proliferation of cells such as bone marrow cells or fibroblasts was found to be refractory to the influence of the MCM. A reduction in melanoma growth was observed in mice treated with the MCM. Adenosine was identified as one of the active components in the MCM by using high-performance liquid chromatography separations, mass spectra, and nuclear magnetic resonance analyses. At a concentration of 4 microM, equal to that found in the MCM, adenosine inhibits the proliferation of tumor cell lines (Nb2 lymphoma, K-562 leukemia, and LNCaP prostate carcinoma cells) while stimulating the proliferation of normal murine bone marrow cells. By similar methods, additional inhibitory components were detected in the MCM in a molecular mass range of 600-800 Da. The ability of adenosine and other low molecular weight components to specifically inhibit tumor cell growth in vitro and in vivo may account for the resistance of muscle to tumor metastases.
Assuntos
Adenosina/farmacologia , Divisão Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Músculos/química , Neoplasias/prevenção & controle , Animais , Ciclo Celular/efeitos dos fármacos , Humanos , Camundongos , Ratos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Following the clinical observations that tumor metastases are extremely rare in striated muscles we defined recently a low molecular weight factor which is released by muscle cells (muscle factor, MF) and possesses specific anti-proliferative activity against tumor cells. we demonstrate that peripheral blood mononuclear cells constitutively release low molecular weight factor (LMF) similar to the MF which is capable of inhibiting in vitro the proliferation of carcinoma, melanoma, leukemia and lymphoma cell lines. The proliferation of normal cells such as bone marrow or fibroblasts was not inhibited but slightly stimulated following incubation with the LMF. Biochemical purification of this factor by several HPLC steps revealed that the inhibitory activity against tumor cells was concentrated within two definitive peaks. The LMF affects tumor cell growth by arresting them in the G0/G1 of the cell cycle and its activity is species and tumor non-specific. In vivo studies in melanoma- bearing mice revealed that the LMF inhibited melanoma growth when given either intraperitoneally or orally. Mononuclear cells from cancer patients with different malignancies (non-Hodgkin lymphoma, malignant melanoma, colon carcinoma and carcinoma of the rectum) secreted lower level of LMF in comparison to healthy subjects. The capability of the LMF to inhibit tumor cell growth and promote normal cell proliferation combined with its bioavailability in vivo may lead to its potential therapeutic and diagnostic use.