RESUMO
Psoralidin has been reported to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production, but the mechanisms of the action remain unclear. Thus, the impact of psoralidin on signaling pathways known to be implicated in NO synthesis was explored in LPS-activated RAW264.7 macrophages by using RT-PCR and Western blotting. Consistent with NO inhibition, psoralidin suppressed LPS-induced expression of inducible NO synthase (iNOS) by abolishing IκB kinase (IKK) phosphorylation, IκB degradation and nuclear factor κB (NF-κB) nuclear translocation without effecting mitogen-activated protein kinases (MAPKs) phosphorylation. Exposure to wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression, suggesting activation of such a signal pathway might also be phosphoinositide-3-kinase (PI3K) dependent. By using Src inhibitor PP2, Janus kinase 2 (JAK-2) inhibitor AG490, Bruton's tyrosine kinase (Btk) inhibitor LFM-A13 and spleen tyrosine kinase (Syk) inhibitor piceatannol, the results showed that piceatannol clearly repressed NO production more potently than the other inhibitors. Furthermore, piceatannol significantly repressed LPS-induced PI3K/Akt phosphorylation and the downstream IKK/IκB activation, suggesting that Syk is an upstream key regulator in the activation of PI3K/Akt-mediated signaling. In fact, transfection with siRNA targeting Syk obviously reduced iNOS expression. Interestingly, LPS-induced phosphorylations of Syk and PI3K-p85 were both significantly blunted by psoralidin treatment. The present results show that interfering with Syk-mediated PI3K phosphorylation might contribute to the NO inhibitory effect of psoralidin via blocking IKK/IκB signaling propagation in LPS-stimulated RAW 264.7 macrophages.
Assuntos
Benzofuranos/farmacologia , Cumarínicos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Quinase SykRESUMO
In the field of osteoporosis, there has been growing interest in anabolic agents that enhance bone formation. Here, we examined the effects of betulinic acid on cell proliferation, differentiation, and mineralization of MC3T3-E1 osteoblasts. Then, the impact of betulinic acid on signaling pathways known to be implicated in osteoblastogenesis was explored. Betulinic acid (1-20 microM) markedly increased alkaline phosphatase (ALP) activity and calcium nodule formation, although without a notable effect on cell proliferation. Stimulation with betulinic acid not only increased the osteopontin level and osteocalcin mRNA expression but also upregulated the osteoprotegerin (OPG)/RANKL ratio. Noggin, but not ICI 182780, significantly repressed betulinic acid-induced ALP activity, suggesting a possible action of betulinic acid through the bone morphogenetic protein (BMP) pathway. This was strengthened by the induction of BMP-2 expression, increases in Smad1/5/8 phosphorylation, and Runx2 expression. Furthermore, betulinic acid increased the nuclear level of the active form beta-catenin. These results suggested that betulinic acid has the potential to enhance osteoblastogenesis probably through the activation of BMP/Smad/Runx2 and beta-catenin signal pathways. Furthermore, upregulation of the OPG/RANKL ratio to repress bone catabolism may also indirectly contribute to the bone anabolic effect of betulinic acid.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoblastos/citologia , Transdução de Sinais/efeitos dos fármacos , Triterpenos/farmacologia , beta Catenina/metabolismo , Animais , Proteína Morfogenética Óssea 2/genética , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Triterpenos Pentacíclicos , beta Catenina/genética , Ácido BetulínicoRESUMO
Apoptosis is a common mode of cell death after exposure of tumor cells to radiation and/or chemotherapy. The factors that determine the rate of induction of apoptosis are generally related to the functioning of cell cycle checkpoints. In the present study, we investigated the involvement of several genes in cell cycle redistribution and induction of apoptosis in U937 cells after low and high doses of radiation. Activation of CDC2 was observed after both low and high doses of radiation in U937 cells that underwent apoptosis. Expression of CDK2, CDC2 and cyclin A was induced rapidly in the process of radiation-induced apoptosis. In addition, we investigated the use of a clinically relevant dose of radiation to promote As2O3-induced apoptosis in U937 cells. We found that combining radiation and As2O3 may be a new and more effective means of cancer treatment.
