RESUMEN
In this investigation, two cell-permeable synthetic analogs of cAMP, dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP, which are widely used to elevate intracellular cAMP levels under experimental conditions, were investigated for their ability to dose-dependently improve histological and functional outcomes following continuous delivery in two models of incomplete spinal cord injury (SCI). The cAMP analogs were delivered via osmotic minipumps at 1-250 mM through an indwelling cortical cannula or by intrathecal infusion for up to 4 weeks after either a T8 unilateral over-hemisection or a C2-3 dorsolateral quadrant lesion, respectively. In both SCI models, continuous db-cAMP delivery was associated with histopathological changes that included sporadic micro-hemorrhage formation and cavitation, enhanced macrophage infiltration and tissue damage at regions beyond the immediate application site; no deleterious or beneficial effect of agent delivery was observed at the spinal injury site. Furthermore, these changes were accompanied by pronounced behavioral deficits that included an absence of progressive locomotor recovery, increased extensor tone, paralysis, and sensory abnormalities. These deleterious effects were not observed in saline-treated animals, in animals in which the db-cAMP dose did not exceed 1 mM, or in those animals that received a high dose (250 mM) of the alternative cAMP analog, 8-bromo-cAMP. These results demonstrate that, for continuous intraparenchymal or intrathecal administration of cAMP analogs for the study of biological or therapeutic effects within the central nervous system (CNS), consideration of the effective concentration applied as well as the potential toxicity of chemical moieties on the parent molecule and/or their activity needs to be taken into account.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/administración & dosificación , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Bucladesina/administración & dosificación , Bucladesina/uso terapéutico , Traumatismos de la Médula Espinal/tratamiento farmacológico , Animales , Axones/patología , Conducta Animal/fisiología , Corteza Cerebral/metabolismo , Contusiones/tratamiento farmacológico , Relación Dosis-Respuesta a Droga , Femenino , Inmunohistoquímica , Bombas de Infusión Implantables , Inyecciones Espinales , Locomoción/fisiología , Macrófagos/patología , Ratas , Ratas Endogámicas Lew , Médula Espinal/patología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/psicologíaRESUMEN
The study has been designed to investigate the effect of 8-Br-cAMP, an activator of protein kinase A, in hypertension-induced vascular endothelial dysfunction. Rats were uninephroctomized and desoxycortisone acetate (DOCA) (40 mg/kg, s.c.) was administered to rats to produce hypertension (mean arterial blood pressure > 140 mmHg). Vascular endothelial dysfunction was assessed using isolated aortic ring preparation, electron microscopy of thoracic aorta and serum concentration of nitrite/nitrate. The expression of mRNA for p22phox and eNOS was assessed by using reverse transcriptase-polymerase chain reaction. Serum thiobarbituric acid reactive substances concentration and aortic superoxide anion concentration were estimated to assess oxidative stress. 8-Br-cAMP (5 mg/kg, i.p.) or atorvastatin (30 mg/kg, p.o.) prevented hypertension-induced attenuation of acetylcholine-induced endothelium-dependent relaxation, impairment of vascular endothelial lining, decrease in expression of mRNA for endothelial nitric oxide synthase (eNOS), serum nitrite/nitrate concentration and increase in expression of mRNA for p22phox, superoxide anion and serum TBARS. The ameliorative effect of 8-Br-cAMP was prevented by N-nitro-L-arginine methyl ester (25 mg/kg, i.p.) and glibenclamide (30 mg/kg, i.p.). It may be concluded that 8-Br-cAMP may stimulate expression and activity of eNOS and suppress expression of p22phox subunit of NADPH oxidase to reduce oxidative stress and subsequently improve vascular endothelial dysfunction.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Endotelio Vascular/efectos de los fármacos , Hipertensión/tratamiento farmacológico , 8-Bromo Monofosfato de Adenosina Cíclica/administración & dosificación , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Animales , Aorta/efectos de los fármacos , Atorvastatina , Presión Sanguínea/efectos de los fármacos , AMP Cíclico/sangre , Esquema de Medicación , Ácidos Heptanoicos/administración & dosificación , Hipertensión/complicaciones , Masculino , NADPH Oxidasas/metabolismo , Nitratos/sangre , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitritos/sangre , Pirroles/administración & dosificación , Ratas , Ratas Sprague-Dawley , Superóxidos/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/análisis , Vasodilatación/efectos de los fármacosRESUMEN
Traumatic injury of the central nervous system results in formation of a collagenous basement membrane-rich fibrous scar in the lesion centre. Due to accumulation of numerous axon-growth inhibitory molecules the lesion scar is considered a major impediment for axon regeneration. Following transection of the dorsal corticospinal tract (CST) at thoracic level 8 in adult rats, transient suppression of collagenous scarring in the lesion zone by local application of a potent iron chelator and cyclic adenosine monophosphate resulted in the delay of fibrous scarring. Treated animals displayed long-distance growth of CST axons through the lesion area extending for up to 1.5-2 cm into the distal cord. In addition, the treatment showed a strong neuroprotective effect, rescuing cortical motoneurons projecting into the CST that normally die (30%) after thoracic axotomy. Further, anterogradely traced CST axons regenerated through both grey and white matter and developed terminal arborizations in grey matter regions. In contrast to controls, injured animals receiving treatment showed significant functional recovery in the open field, in the horizontal ladder and in CatWalk locomotor tasks. We conclude that the fibrous lesion scar plays a pivotal role as a growth barrier for regenerating axons in adult spinal cord and that a delay in fibrotic scarring by local inhibition of collagen biosynthesis and basement membrane deposition is a promising and unique therapeutic strategy for treating human spinal trauma.
Asunto(s)
Cicatriz/prevención & control , Regeneración Nerviosa/fisiología , Tractos Piramidales/patología , Recuperación de la Función , Corteza Somatosensorial/fisiopatología , Traumatismos de la Médula Espinal/patología , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/uso terapéutico , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Animales , Antígenos/metabolismo , Axones/patología , Axones/fisiología , Conducta Animal , Biotina/análogos & derivados , Biotina/metabolismo , Recuento de Células/métodos , Cicatriz/etiología , Colágeno Tipo IV/metabolismo , Dextranos/metabolismo , Femenino , Compuestos Ferrosos/uso terapéutico , Lateralidad Funcional , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica/métodos , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Proteoglicanos/metabolismo , Ratas , Ratas Wistar , Traumatismos de la Médula Espinal/terapia , Estilbamidinas , Factores de TiempoRESUMEN
Following traumatic injury, the formation of a glial scar and deposition of extracellular matrix (ECM) contributes to the regeneration failure in the adult mammalian central nervous system (CNS). Using a postcommissural fornix transection as a brain lesion model in rat, we have previously shown that the collagenous basement membrane (BM) at the lesion site is a major impediment for axon regeneration. Deposition of BM in this lesion model can be delayed by administration of the iron chelator 2,2'-bipyridine (BPY), an inhibitor of prolyl 4-hydroxylase (PH), a key enzyme of collagen biosynthesis. To examine whether this potential therapeutic approach is transferable to other CNS regions, we have chosen the mechanically lesioned rat spinal cord to investigate the effects of BPY administration on BM formation. Due to the close proximity of the lesion zone to meningeal fibroblasts, a cell-type secreting large amounts of collagen IV, BM deposition was much more extensive in the spinal cord than in the brain lesion. Neither immediate injections nor continuous application of BPY resulted in a detectable reduction of BM formation in the spinal cord. Only a combination of anti-scarring treatments including (i) injection of the more potent PH inhibitor [2,2'-bipyridine]-5,5'-dicarboxylic acid (BPY-DCA), (ii) selective inhibition of fibroblast proliferation and ECM production by 8-Br-cAMP, and (iii) continuous application of BPY-DCA, reduced the lesion-induced BM significantly. The present results clearly demonstrate, that the exclusive application of BPY according to a protocol designed for treatment of brain lesions is not sufficient to reduce BM formation in the lesioned adult rat spinal cord.
Asunto(s)
2,2'-Dipiridil/farmacología , Membrana Basal/efectos de los fármacos , Cicatriz/prevención & control , Colágeno Tipo IV/antagonistas & inhibidores , Indicadores y Reactivos/farmacología , Regeneración Nerviosa/efectos de los fármacos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Médula Espinal/efectos de los fármacos , 2,2'-Dipiridil/uso terapéutico , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Animales , Membrana Basal/metabolismo , Membrana Basal/patología , Quelantes/farmacología , Quelantes/uso terapéutico , Cicatriz/etiología , Cicatriz/fisiopatología , Colágeno Tipo IV/biosíntesis , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Femenino , Gliosis/etiología , Gliosis/fisiopatología , Gliosis/prevención & control , Hierro/metabolismo , Masculino , Regeneración Nerviosa/fisiología , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/metabolismo , Ratas , Ratas Wistar , Médula Espinal/fisiopatología , Médula Espinal/cirugía , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/fisiopatología , Resultado del TratamientoRESUMEN
From a series of preclinical studies and animal experiments, we have been able to demonstrate that DNA vaccines are a promising tool in strategies for protecting hosts from a variety of infectious diseases. Since the promoter activity of the human cytomegalovirus immediate-early promoter/ enhancer (CMV promoter) is known to be responsive to an elevation in the level of intracellular cAMP, we hypothesized that use of cAMP analogue (8-Bromo adenosine 3'5'-cyclic monophosphate, 8 Br-cAMP) would increase the level of transgene expression supported by the CMV, and enhance the ability of DNA vaccines to evoke an immune response against the transgene product in vivo. To evaluate this hypothesis, immune responses against HIV-1 envelope protein, gp160, an immunogenic HIV-1 component expressed under the control of the CMV promoter, were evaluated in BALB/c mice with or without stimulation by 8 Br-cAMP. DNA vaccine with 8 Br-cAMP was intramuscularly (i.m.) or intranasally (i.n.) administered to BALB/c mice twice on days 0 and 14. Regardless of which route was used, the combination increased the serum IgG antibody (Ab) titer, HIV-1-specific cytotoxic T lymphocyte (CTL) activity and the delayed-type hypersensitivity (DTH) response, compared with the effect of using the vaccine alone. When administered via the i.n. route, the combination also remarkably increased the titer of secretory IgA (sIgA). Moreover, it induced increased production of interferon-gamma with reduction in IL-4 synthesis, and decreased the ratio of serum IgG1/IgG2a. However, these enhancements were not observed when 8 Br-cAMP was coadministered with peptide vaccine or protein antigen. These data suggest that 8 Br-cAMP is able to enhance both humoral and cellular immune responses induced by the DNA vaccine. The induction of T helper type 1 (Th1) immunity against HIV-1 was also enhanced by coadministration of 8 Br-cAMP. A CAT assay study demonstrated that the adjuvant effect of 8 Br-cAMP may be due to the activation of the CMV promoter in the DNA vaccine. The virus challenge experiment in a mouse influenza model also proved our hypothesis.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Terapia Genética/métodos , Proteínas gp160 de Envoltorio del VIH/genética , Hipersensibilidad Tardía/tratamiento farmacológico , Linfocitos T Citotóxicos/efectos de los fármacos , Vacunas de ADN/uso terapéutico , Administración Intranasal , Animales , Terapia Combinada , Citomegalovirus/genética , Relación Dosis-Respuesta a Droga , Vectores Genéticos/administración & dosificación , Hipersensibilidad Tardía/inmunología , Inmunoglobulina G/análisis , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Regiones Promotoras Genéticas , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The cyclic AMP (cAMP)-dependent protein kinase regulatory subunit RI is overexpressed in cancer cells. 8-Chloro-cAMP (8-Cl-cAMP) is an RII site-specific analogue that down-regulates RI and inhibits the growth of a wide range of cancer cells in vitro and in vivo. We performed a Phase I trial of 8-Cl-cAMP in 32 patients with malignancies that were refractory to standard treatments. 8-Cl-cAMP was initially given in a 1-month cycle by constant infusion at 0.005 mg/kg/h for 21 days, followed by 1 week of rest. The dose was escalated to 0.045 mg/kg/h, but hypercalcemia became the dose-limiting toxicity. The length of drug administration was, therefore, reduced to 5 days per week for the first 3 weeks of the cycle, but it was not possible to increase the drug dose without producing hypercalcemia. Hence, the length of drug administration was reduced to 3 days per week for the first 3 weeks of the cycle. The maximum tolerated dose for this regimen was 0.15 mg/kg/h, and the dose-limiting toxicities were reversible hypercalcemia and hepatotoxicity. Stable disease for > or =4 months was observed in two patients treated at > or =0.045 mg/kg. cAMP-dependent protein kinase is involved in hormone- and cytokine-mediated signaling, and so representative hormone, cytokine, and peripheral lymphocyte subsets were measured. The drug had a parathyroid hormone-like effect on calcium homeostasis and significantly increased circulating luteinizing hormone and 17-hydoxyprogesterone levels (P < 0.02 and P < 0.0006, respectively). We conclude that 8-Cl-cAMP is well tolerated without attendant myelotoxicity, and in this study, it was associated with biological effects. In Phase II studies, a dose of 0.11 mg/kg/h for 3 days per week would be appropriate.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , 8-Bromo Monofosfato de Adenosina Cíclica/efectos adversos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacocinética , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Adulto , Anciano , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Citocinas/metabolismo , Femenino , Hormonas/metabolismo , Humanos , Hipercalcemia/inducido químicamente , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/metabolismo , Masculino , Persona de Mediana Edad , Náusea/inducido químicamente , Neoplasias/metabolismo , Resultado del Tratamiento , Vómitos/inducido químicamenteRESUMEN
We have evaluated the antiproliferative effect of a novel mixed backbone antisense oligonucleotide generated against the 5'-coding region of the human CRIPTO mRNA in GEO human colon cancer cells. We have also evaluated the effects of this anti-CRIPTO antisense oligonucleotide in combination with a chimeric anti-human epidermal growth factor receptor (EGFR) monoclonal antibody (MAb C225) and with 8-Cl-cAMP, a cAMP analog that specifically inhibits type I protein kinase A (PKAI), since a functional EGFR-driven autocrine pathway is operative and PKAI is overexpessed in GEO colon cancer cells. Treatment with a single agent at low doses determined a 15-35% growth inhibition. A synergistic antiproliferative effect was observed when combinations of two agents were used with a co-operativity quotient ranging between 1.5 and 2.2. Furthermore, the combined treatment with all three drugs caused an almost complete suppression of the ability of GEO cells to form colonies in soft agar. We next evaluated whether any combination of 8-Cl-cAMP, the anti-CRIPTO antisense oligonucleotide and MAb C225 could induce programmed cell death in GEO cells. Treatment with each agent alone at all doses tested did not cause DNA fragmentation. The treatment with any combination of two agents was not able to induce apoptosis. In contrast, treatment with all three compounds determined an approximately three-fold increase in DNA fragmentation. In conclusion, the combination of selective antineoplastic agents directed against different but related key signal tranduction pathways efficiently inhibits cell growth and causes apoptosis in human colorectal cancer cells.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Apoptosis , Neoplasias del Colon , Factor de Crecimiento Epidérmico , Glicoproteínas de Membrana , Proteínas de Neoplasias/genética , Oligonucleótidos Antisentido/uso terapéutico , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Antineoplásicos/uso terapéutico , División Celular/efectos de los fármacos , División Celular/genética , Cetuximab , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Sinergismo Farmacológico , Proteínas Ligadas a GPI , Marcación de Gen , Sustancias de Crecimiento/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular , Oligonucleótidos Antisentido/genética , ARN Mensajero/genética , Células Tumorales CultivadasRESUMEN
X-adrenoleukodystrophy (X-ALD) is an inherited fatty acid metabolic disorder with secondary manifestation of neuroinflammatory disease process. We report that compounds (forskolin, 8-bromo cAMP, and rolipram) that increase cAMP and activate protein kinase A (PKA) were found to stimulate the peroxisomal beta-oxidation of lignoceric acid (C24:0) whereas compounds (H-89 and myristoylated PKI) that decrease cAMP and PKA activity inhibited the peroxisomal beta-oxidation of lignoceric acid in cultured skin fibroblasts from X-ALD patients. Consistent with the stimulation of beta-oxidation of lignoceric acid, activators of PKA normalized the level of very long chain fatty acids (VLCFA) in X-ALD cultured skin fibroblasts. This normalization of VLCFA in X-ALD cells with forskolin, 8-Br cAMP or with rolipram, an inhibitor of cAMP phosphodiesterase, was realized independent of expression of mRNA or protein of the ALD gene, suggesting that cAMP derivatives can correct the metabolic defect in X-ALD fibroblasts without involving the candidate gene for the disease. Because astrocytes and microglia in demyelinating lesions of X-ALD brain express proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), we examined the effect of cAMP derivatives or rolipram on lipopolysaccharide-stimulated rat primary astrocytes and microglia and found that cAMP derivatives and rolipram inhibited the induction of TNF-alpha and IL-1beta in both astrocytes and microglia. The ability of cAMP derivatives and rolipram to block the induction of TNF-alpha and IL-1beta in astrocytes and microglia and to normalize the fatty acid pathogen in skin fibroblasts of x-adrenoleukodystrophy (X-ALD) clearly identify cAMP analogs or rolipram as candidates for potential therapy for X-ALD patients.
Asunto(s)
Adrenoleucodistrofia/tratamiento farmacológico , AMP Cíclico/metabolismo , Citocinas/biosíntesis , Ácidos Grasos/sangre , Sulfonamidas , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Adrenoleucodistrofia/genética , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Colforsina/análogos & derivados , Colforsina/uso terapéutico , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Ligamiento Genético , Interleucina-1/biosíntesis , Isoquinolinas/uso terapéutico , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Oxidación-Reducción , Inhibidores de Proteínas Quinasas , Pirrolidinonas/uso terapéutico , Ratas , Rolipram , Factor de Necrosis Tumoral alfa/biosíntesis , Cromosoma XRESUMEN
Rats were bilaterally implanted with indwelling cannulae in the CA1 region of the dorsal hippocampus, the entorrhinal cortex or the posterior parietal cortex. After recovery from surgery, they were trained in a one-trial step-down inhibitory avoidance task using a 0.3 mA footshock. The animals received i.p. 15 min before training either saline (1 ml/kg) or midazolam (1 mg/kg). Three hours after training they received, through the cannulae, infusions of saline, norepinephrine (0.3 microg/side), SKF38393 (7.5 microg/side), or 8-Br-cAMP (1.25 microg/side) into the brain regions mentioned. Animals were tested for retention 24 h after the training session. Midazolam produced anterograde amnesia, and the post-training treatments (with the exception of SKF38393 given into the entorrhinal cortex) caused retrograde memory facilitation. The amnestic effect of midazolam and the facilitatory effect of the treatments given into the brain cancelled each other out. Therefore, the mechanisms triggered by midazolam can interact with others in areas involved in memory processing several hours after their onset.
Asunto(s)
Amnesia/prevención & control , Corteza Entorrinal/efectos de los fármacos , Hipocampo/efectos de los fármacos , Memoria/efectos de los fármacos , Midazolam/farmacología , Lóbulo Parietal/efectos de los fármacos , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/uso terapéutico , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Amnesia/inducido químicamente , Animales , Agonistas de Dopamina/farmacología , Agonistas de Dopamina/uso terapéutico , Vías de Administración de Medicamentos , Interacciones Farmacológicas , Corteza Entorrinal/fisiología , Hipocampo/fisiología , Masculino , Lóbulo Parietal/fisiología , Prótesis e Implantes , Ratas , Ratas WistarRESUMEN
The treatment of cancer patients with conventional chemotherapy is sometimes associated with severe systemic toxicity and only a minimal survival benefit. Because of this, new less toxic and more efficacious treatments have been sought. 8-Chloro-cAMP (8-Cl-cAMP) is one of a new generation of anticancer drugs that act at the level of signal transduction. In preclinical models, 8-Cl-cAMP modulates protein kinase A (PKA) leading to growth inhibition and increased differentiation of cancer cells. 8-Cl-cAMP was given to 16 patients with advanced cancer as an infusion via an indwelling subclavian venous catheter. We showed that 8-Cl-cAMP had a parathyroid hormone-like effect leading to increased synthesis of renal 1,25-dihydroxyvitamin D [up to 14 times the baseline value, median 3.6 times; P = 0.00001 (Student's paired t test)]. This produced the dose-limiting toxicity of reversible hypercalcemia that could not be controlled by the administration of either pamidronate or dexamethasone. The treatment was otherwise well tolerated, and other cAMP-dependent pathways (cortisol and TSH) were not affected, emphasizing the marked differences between organs in their sensitivity to this cAMP analog. Our results have shown that 8-Cl-cAMP is biologically active, and it is feasible that if the hypercalcemia can be controlled, then this drug may have a role as a single agent, or as a short infusion between cycles of chemotherapy.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Antineoplásicos/efectos adversos , AMP Cíclico/análogos & derivados , Hipercalcemia/inducido químicamente , Neoplasias/metabolismo , Vitamina D/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/administración & dosificación , 8-Bromo Monofosfato de Adenosina Cíclica/efectos adversos , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Adolescente , Adulto , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Relación Dosis-Respuesta a Droga , Estudios de Factibilidad , Humanos , Hormona Paratiroidea/sangre , Vitamina D/biosíntesisRESUMEN
8-Chloro-cyclic AMP (8-Cl-cAMP) is a cAMP analogue that specifically down-regulates type I protein kinase A, a signaling protein directly involved in cell proliferation and neoplastic transformation, and that causes growth inhibition in a variety of human cancer cell types. In this report, we have investigated the effects of 8-Cl-cAMP on the expression of several growth factors in human colon (GEO and LS174T) and breast (MDA-MB468) cancer cell lines. 8-Cl-cAMP treatment caused in the three cancer cell lines a significant dose- and time-dependent inhibition in the expression of various endogenous autocrine growth factors, such as transforming growth factor alpha, amphiregulin, and CRIPTO, and of two angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor, at both the mRNA and protein levels. Furthermore, 8-Cl-cAMP treatment markedly inhibited the ability of all three cell lines to invade a basement membrane matrix in a chemoinvasion assay. Finally, 8-Cl-cAMP-induced inhibition of GEO tumor growth in nude mice was accompanied by a significant suppression of transforming growth factor alpha, amphiregulin, CRIPTO, basic fibroblast growth factor, and vascular endothelial growth factor production by the tumor cells, and of neoangiogenesis, as detected by factor VIII staining of host blood cells. These results demonstrate that 8-Cl-cAMP is a novel anticancer drug that inhibits the production of various autocrine and paracrine tumor growth factors that are important in sustaining autonomous local growth and facilitate invasion and metastasis.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Antineoplásicos/toxicidad , Neoplasias del Colon/tratamiento farmacológico , Factores de Crecimiento Endotelial/genética , Factor de Crecimiento Epidérmico , Factor 2 de Crecimiento de Fibroblastos/genética , Péptidos y Proteínas de Señalización Intercelular , Linfocinas/genética , Glicoproteínas de Membrana , Neovascularización Patológica/prevención & control , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , 8-Bromo Monofosfato de Adenosina Cíclica/toxicidad , Anfirregulina , Animales , Antineoplásicos/uso terapéutico , Neoplasias de la Mama , División Celular/efectos de los fármacos , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Neoplasias Colorrectales , Familia de Proteínas EGF , Factores de Crecimiento Endotelial/biosíntesis , Femenino , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Proteínas Ligadas a GPI , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Sustancias de Crecimiento/biosíntesis , Sustancias de Crecimiento/genética , Humanos , Linfocinas/biosíntesis , Ratones , Ratones Desnudos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Factor de Crecimiento Transformador alfa/biosíntesis , Factor de Crecimiento Transformador alfa/genética , Trasplante Heterólogo , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Significant dose-related inhibition of growth of HT29 human colorectal cancer xenografts and ZR-75-1 breast cancer xenografts in immune-suppressed mice was induced by the cyclic AMP analogue, 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cyclic AMP) when given by alzet mini-pumps over a 7-day period at doses of either 50 or 100 mg/kg/day. Levels and types of cyclic AMP binding proteins were measured by ligand binding and photoaffinity labelling, respectively, in tumours harvested at the end of the treatment period. Compared with levels in tumours from control animals, values of tumour cyclic AMP binding proteins from treated animals were significantly reduced. These effects were associated with an apparent modulation of the types of cyclic AMP binding proteins, 8-Cl-cyclic AMP-treated xenografts displaying a reduced ratio of RI/RII isoforms compared with untreated control tumours.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/patología , Neoplasias del Colon/patología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , División Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Terapia de Inmunosupresión , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Tumorales CultivadasRESUMEN
BACKGROUND: Gastrin regulates growth of human colon cancer cells by activation of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA). Gastrin and 8-Br-cAMP, a membrane-permeable cAMP analog, inhibit growth of HCT116 cells; both stimulate growth of LoVo cells. This dual effect on growth may be explained by relative amounts of the regulatory subunit (RI alpha or RII beta) of PKA within the cancer cells. Antisense oligodeoxynucleotides (ASO) to either RI alpha or RII beta inhibit protein translation of the target mRNA by sequence-specific binding; subsequently, cellular PKA content and the cAMP-mediated growth may be altered. We determined whether ASO to either the RI alpha or RII beta subunit altered the cAMP-mediated growth of HCT116 and LoVo human colon cancer cells. METHODS: HCT116 cells were treated with RII beta ASO (15 mumol/L, 4 days) and then treated with 8-Br-cAMP (25 mumol/L); tritiated thymidine incorporation was measured after 24 hours, and the cell number was determined on alternate days. Protein and mRNA levels of the RII beta subunit were determined by Western and Northern blotting, respectively. Similar studies with an ASO against the RI alpha subunit were performed on LoVo cells. RESULTS: RII beta ASO reversed the cAMP-mediated inhibition of growth of HCT116 cells, and RII beta ASO decreased the protein level of the RII beta subunit. RII beta ASO did not alter the basal growth of HCT116 cells. RI alpha ASO reversed the cAMP-mediated stimulation of growth of LoVo cells. CONCLUSIONS: The regulatory subunits of PKA are potential targets to alter growth of human colon cancer cells. Gene therapy directed to alter specific steps in signal transduction pathways may provide new therapeutic strategies.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Neoplasias del Colon/terapia , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Terapia Genética , Oligonucleótidos Antisentido/uso terapéutico , Secuencia de Bases , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Humanos , Datos de Secuencia Molecular , ARN Mensajero/análisis , Células Tumorales CultivadasRESUMEN
The effect of a new anticancer drug, 8-chloroadenosine 3',5'-monophosphate (8-Cl-cAMP-), a site selective cAMP analog, that inhibits growth of cancer cells in vitro, was examined in L5178Y (LY) murine lymphoma cells. Two LY sublines were used, grown in full Fisher's medium: LY-R, radiation resistant and LY-S, radiation sensitive. The latter was also adapted to grow in simplified medium. In the full medium conversion of 8-Cl-cAMP to 8-chloroadenosine presumably was the case of cytotoxicity. In the simplified medium this conversion was limited and the cytotoxic effect much less pronounced. Cytotoxicity was equal in LY-R and LY-S cells and it was not related to changes in the cell cycle distribution; the latter were observed in LY-S, but not in LY-R cells. There was no interaction of the drug with x-rays in LY cells grown either in full or simplified medium.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Antineoplásicos/uso terapéutico , Factores Inmunológicos/uso terapéutico , Linfoma/tratamiento farmacológico , Linfoma/radioterapia , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , División Celular/efectos de los fármacos , División Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Terapia Combinada , Ensayos de Selección de Medicamentos Antitumorales , Linfoma/patología , Ratones , Células Tumorales CultivadasRESUMEN
A site-selective analogue of the cyclic adenosine monophosphate 8-chloro-adenosine-3',5'-cyclophosphate was studied for its effects on the growth of transplanted murine melanoma B-16. When the agent was given to the mice, a substantial effect on the growth of the tumor was produced by a number of factors, which included the route of administration, concentration of the agent, the time and duration of therapy. Intraperitoneal injections of the agent in a dose of 20 mg/kg/day which were made during three consecutive days, beginning from day 5 after tumor transplantation caused a 58% decrease in tumor growth as compared to the controls. An examination of tumour biopsy specimen revealed that after a course of the injections there was a significant suppression of the activity of cAMP-dependent protein kinase, type I, and a drastic increase in that of cAMP-dependent protein kinase, type II.
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8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Antineoplásicos/uso terapéutico , Melanoma Experimental/tratamiento farmacológico , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Animales , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Factores de TiempoAsunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , AMP Cíclico/fisiología , Neoplasias/patología , Receptores de AMP Cíclico/fisiología , Transducción de Señal , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Línea Celular , Células , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Femenino , Humanos , Sustancias Macromoleculares , Metástasis de la Neoplasia , Oligonucleótidos Antisentido/farmacología , Proteínas Quinasas/genética , ARN Mensajero/genéticaRESUMEN
8-Chloroadenosine 3',5'-monophosphate has been reported to inhibit growth of various mammalian cell lines at micromolar concentrations. We have used Chinese hamster ovary cell lines with mutated cyclic AMP-dependent protein kinase or altered cyclic nucleotide metabolism to show that a metabolite, 8-chloroadenosine, is formed in the medium and is the active inhibitor of cell growth in Chinese hamster ovary cells. Adding adenosine deaminase to the Chinese hamster ovary cell growth media removes the inhibition of cell growth attributed to 8-chloroadenosine 3',5'-monophosphate. Adenosine deaminase or dipyridamole also protects Molt-4 lymphoblasts from the growth-inhibitory effects of 8-chloroadenosine 3',5'-monophosphate.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Adenosina/metabolismo , Antineoplásicos/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapéutico , Adenosina Desaminasa/farmacología , Animales , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , División Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Proteínas Quinasas/metabolismoRESUMEN
Site-selective cyclic AMP (cAMP) analogues inhibit growth and induce changes in morphology in a spectrum of human cancer cell lines (D. Katsaros et al., FEBS Lett., 223:97, 1987). The cellular events underlying such effects of cAMP analogues include differential regulation of type I versus type II cAMP-dependent protein kinase isozymes (S. Ally et al., Proc. Natl. Acad. Sci. USA, 85: 6319, 1988). Infusion (i.p.) of 8-Cl-cAMP, the most potent site-selective cAMP analogue, for 7 days produced regression of LX-1 lung carcinoma in athymic mice in a dose-dependent manner. The tumor regression correlated with the changing levels of cAMP receptor proteins, RI alpha and RII beta, the regulatory subunits of cAMP-dependent protein kinase type I and type II, respectively. By photoaffinity labeling with 8-N3-[32P]cAMP and immunoblotting with a monospecific anti-RII antibody, RI alpha (Mr 49,000) and RII beta (Mr 51,000) were identified in the untreated control tumors. 8-Cl-cAMP treatment induced a rapid increase of both RI alpha and RII beta in tumor cytosols and translocation (within 1 h) of only RII beta from the cytosol to the nucleus. RII beta in both cytosols and nuclei remained elevated during 8-Cl-cAMP treatment, whereas RI alpha in the cytosols gradually decreased with time of treatment after its initial transient increase. Northern blot analyses demonstrated that the RII beta mRNA level increased within 6 h of 8-Cl-cAMP treatment and remained elevated during treatment, whereas the RI alpha mRNA level decreased to below that of the untreated control tumor level after its transient increase during 1-6 h of treatment. 8-Cl-cAMP treatment also caused a sharp decrease in both N-ras and c-myc mRNA levels. These results suggest that the fundamental basis for the antineoplastic activity of 8-Cl-cAMP may reside in the restoration of normal gene regulation in neoplasms in which cAMP receptor proteins play a role.