RESUMEN
We recently found that the receptor activator of NF-κB ligand (RANKL)-binding peptide, OP3-4 stimulated the differentiation of both chondrocytes and osteoblasts. OP3-4 is also shown to inhibit cartilage degeneration. To clarify whether the peptide can inhibit cartilage degeneration without stimulating bone formation, we first performed a proliferation assay using C3H10T1/2 (the murine mesenchymal stem cell line), which is the common origin of both chondrocytes and osteoblasts. The RANKL-binding peptides, OP3-4 and W9, promoted cellular proliferation at 24 and 48 h, respectively. Next, we injected both peptides into the intra-articular space of the knee joints of mice with monosodium-iodoacetate (MIA)-induced osteoarthritis to clarify the effects of the peptides on cartilage tissue. Twenty-five nine-week-old male C57BL/6J mice received injections of vehicle, or the same molar amount of W9, OP3-4, or a control peptide (which could not stimulate osteoblast differentiation) on days 7, 14, and 21 after the injection of MIA. The mice were sacrificed on day 28. The histomorphometric analyses revealed that both peptides inhibited the degeneration of cartilage without enhancing bone formation activity. Our data suggest that the stimulation of mesenchymal cell proliferation by the RANKL-binding peptides might lead to the inhibition of cartilage degeneration.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Oligopéptidos/uso terapéutico , Osteoartritis/tratamiento farmacológico , Ligando RANK/metabolismo , Animales , Huesos/efectos de los fármacos , Huesos/patología , Cartílago Articular/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Condrocitos/patología , Humanos , Inyecciones Intraarticulares , Ácido Yodoacético , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones Endogámicos C57BL , Oligopéptidos/farmacología , Osteoartritis/inducido químicamente , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/patologíaRESUMEN
The present study examined the influence of cimetidine on the nephrotoxicity and antitumor effects of cisplatin in vitro and in vivo. When the serum concentration of cimetidine was maintained over 20 µg/ml for 4 h by bolus and continuous intravenous infusion, cimetidine prevented nephrotoxicity of cisplatin without influencing antitumor activity. Cimetidine and the antioxidant N-acetylcysteine (NAC) significantly inhibited the in vitro growth inhibition of cisplatin in cells originating from the kidney, but not in SOSN2 osteosarcoma cells. Cimetidine (1 mM) also did not influence platinum concentration in the cells, regardless of whether the organic cation transporter 2 (OCT2) was expressed. Cisplatin did induce reactive oxygen species (ROS) in the KN41 kidney cell line and cimetidine and NAC significantly reduced ROS production. However, cisplatin did not produce ROS in osteosarcoma cells. From these results, cimetidine clearly inhibits nephrotoxicity induced by cisplatin without any influence on the antitumor activity of cisplatin on osteosarcoma in vitro and in vivo.
Asunto(s)
Acetilcisteína/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Cimetidina/uso terapéutico , Cisplatino/efectos adversos , Riñón/efectos de los fármacos , Osteosarcoma/tratamiento farmacológico , Fármacos Renales/uso terapéutico , Acetilcisteína/farmacología , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Neoplasias Óseas/metabolismo , Línea Celular , Línea Celular Tumoral , Cimetidina/farmacología , Cisplatino/uso terapéutico , Infusiones Intravenosas , Masculino , Proteínas de Transporte de Catión Orgánico/metabolismo , Osteosarcoma/metabolismo , Platino (Metal)/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Fármacos Renales/farmacologíaAsunto(s)
Huesos/metabolismo , Portadores de Fármacos/metabolismo , Sistemas de Liberación de Medicamentos , Oligopéptidos , Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Secuencia de Aminoácidos , Huesos/citología , Portadores de Fármacos/química , Estradiol/química , Estradiol/uso terapéutico , Humanos , Sialoproteína de Unión a Integrina , Datos de Secuencia Molecular , Estructura Molecular , Oligopéptidos/química , Oligopéptidos/metabolismo , Osteomielitis/tratamiento farmacológico , Osteopontina/genética , Osteopontina/metabolismo , Osteoporosis/tratamiento farmacológico , Quinolonas/química , Quinolonas/uso terapéutico , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismoRESUMEN
To assess the combination effect of calcitonin and the phosphodiesterase 4 inhibitor rolipram on osteoclastogenesis, adherent cell-depleted bone marrow cells from mouse tibia and femur (ACD-BMCs), which were cultured in the presence of 25 ng/ml colony-stimulating factor 1 (CSF-1) and 100 ng/ml soluble receptor activator of NF-kappaB ligand (sRANKL), were utilized. Calcitonin inhibited formation of tartrate-resistant acid phosphatase-positive multinucleated cells, as mature osteoclasts, by 70% even at 20 pM, whereas rolipram (10 microM) scarcely affected osteoclast formation; in contrast, the combination of both agents led to significant inhibition of multinucleation and pit formation ability of osteoclasts. The combined administration of calcitonin and rolipram attenuated calcitonin receptor mRNA expression in comparison to treatment with either agent alone, whereas expression of RANK and CSF-1 receptor mRNAs was unchanged. Alone, these agents scarcely elevated intracellular cyclic AMP (cAMP) concentration; however, combination treatment with both agents significantly increased cAMP concentration in osteoclast progenitors and osteoclasts. The combination effect was abolished by H-89, an inhibitor of protein kinase A. It appears that rolipram inhibited hydrolysis of cAMP formed by calcitonin in cells and potentiated the inhibitory effect of calcitonin on osteoclastogenesis. The escape phenomenon following calcitonin treatment may also be prevented by concomitant treatment with the phosphodiesterase 4 inhibitor.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Calcitonina/farmacología , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Rolipram/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Animales , Calcitonina/agonistas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Ratones , ARN MensajeroRESUMEN
UNLABELLED: The mechanism of osteoblast formation by a novel PDE4 inhibitor, XT-611, was studied in the in vitro bone marrow culture system. The compound potentiated the osteoblast differentiation through accumulation of cyclic AMP after autocrine stimulation of EP4 receptor by PGE2 in pro-osteoblastic cells. INTRODUCTION: We previously reported that inhibitors of phosphodiesterase (PDE)4 isoenzyme increase osteoblast formation in an in vitro bone marrow culture system and inhibit bone loss in animal osteoporosis models. Here we investigated the mechanism of the effect of a novel PDE4 inhibitor, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (XT-611), on osteoblast formation in the in vitro bone marrow culture system. MATERIALS AND METHODS: Rodent bone marrow cells were cultured in the presence of 0.2 mM ascorbic acid phosphate ester, 1 mM beta-glycerophosphate, and 10 nM dexamethasone for 10 days. Drug treatments were done for 24 h on day 3 of culture. RESULTS: PDE4 inhibitors, including XT-611, but not PDE3 and PDE5 inhibitors, increased mineralized nodule formation in rat and mouse bone marrow cell cultures. During culture of the bone marrow cells, prostaglandin E2 (PGE2) production increased with a peak on day 4, but the increase was completely inhibited by indomethacin, an unselective cyclo-oxygenase (COX) inhibitor. Spontaneous and XT-611-induced mineralized-nodule formation was also inhibited by indomethacin and COX-2 inhibitors, in a similar potential. Alkaline phosphatase-positive nodule formation in the absence or presence of XT-611 was inhibited by an antagonist of EP4 receptor, AH23848B, and synergistically potentiated by 11-deoxy-PGE1, but it was not influenced by other EP antagonists and agonists examined. The expression of PDE4 and EP4 mRNAs was observed in bone marrow cells. The effect of XT-611 was also confirmed to involve an increase of cyclic AMP and the cyclic AMP-dependent protein kinase pathway. CONCLUSION: These results suggest that PGE2 stimulates differentiation of osteoblast progenitor cells through the EP4 receptor in an autocrine manner, and the PDE4 inhibitor potentiates the differentiation by inhibiting hydrolysis of cyclic AMP in the cells.