RESUMO
Disruption of the finely tuned osteoblast-osteoclast balance is the underlying basis of several inflammatory bone diseases, such as osteomyelitis, osteoporosis, and septic arthritis. Prolonged and unrestrained exposure to inflammatory environment results in reduction of bone mineral density by downregulating osteoblast differentiation. Earlier studies from our laboratory have identified that Anacardic acid (AA), a constituent of Cashew nut shell liquid that is used widely in traditional medicine, has potential inhibitory effect on gelatinases (MMP2 and MMP9) which are over-expressed in numerous inflammatory conditions (Omanakuttan et al. in Mol Pharmacol, 2012 and Nambiar et al. in Exp Cell Res, 2016). The study demonstrated for the first time that AA promotes osteoblast differentiation in lipopolysaccharide-treated osteosarcoma cells (MG63) by upregulating specific markers, like osteocalcin, receptor activator of NF-κB ligand, and alkaline phosphatase. Furthermore, expression of the negative regulators, such as nuclear factor-κB, matrix metalloproteinases (MMPs), namely MMP13, and MMP1, along with several inflammatory markers, such as Interleukin-1ß and Nod-like receptor protein 3 were downregulated by AA. Taken together, AA expounds as a novel template for development of potential pharmacological therapeutics for inflammatory bone diseases.
Assuntos
Ácidos Anacárdicos/farmacologia , Doenças Ósseas/tratamento farmacológico , Inflamassomos/antagonistas & inibidores , Osteoblastos/efeitos dos fármacos , Osteocalcina/agonistas , Ligante RANK/agonistas , Doenças Ósseas/metabolismo , Doenças Ósseas/patologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Inflamassomos/metabolismo , NF-kappa B/antagonistas & inibidores , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Ligante RANK/metabolismoRESUMO
Earlier studies from our laboratory have identified Anacardic acid (AA) as a potent inhibitor of gelatinases (MMP-2 and 9), which are over-expressed in a wide variety of cancers (Omanakuttan et al., 2012). Disruption of the finely tuned matrix metalloproteinase (MMP) activator/inhibitor balance plays a decisive role in determining the fate of the cell. The present study demonstrates for the first time, that in addition to regulating the expression as well as activity of gelatinases, AA also inhibits the expression of its endogenous activators like MMP-14 and Extracellular Matrix MetalloProteinase Inducer (EMMPRIN) and induces the expression of its endogenous inhibitor, REversion-inducing Cysteine-rich protein with Kazal motifs (RECK). In addition to modulating gelatinases, AA also inhibits the expression of various components of the Epidermal Growth Factor (EGF) pathway like EGF, Protein Kinase B (Akt) and Mitogen-activated protein kinases (MAPK). Furthermore, AA also activates the expression of Sprouty 2 (Spry2), a negative regulator of EGF pathway, and silencing Spry2 results in up-regulation of expression of gelatinases as well as MMP-14. The present study thus elucidates a novel mechanism of action of AA and provides a strong basis for utilizing this molecule as a template for cancer therapeutics.
Assuntos
Ácidos Anacárdicos/farmacologia , Basigina/metabolismo , Proteínas Ligadas por GPI/metabolismo , Gelatinases/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Gelatinases/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Modelos Biológicos , Invasividade Neoplásica , Transdução de Sinais/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
An investigation on the properties of an alkaline protease secreted by Bacillus circulans BM15 strain isolated from a mangrove sediment sample was carried out in order to characterize the enzyme and to test its potency as a detergent additive. The protease was purified to apparent homogeneity by ammonium sulphate precipitation and was a 30-kDa protease as shown by SDS-PAGE and its proteolytic activity was detected by casein zymography. It had optimum activity at pH 7, was stable at alkaline pH range (7 to 11), had optimum temperature of activity 40°C and was stable up to a temperature of 55°C after incubation for one hour. Hg(2+), Zn(2+), Co(2+), and Cu(2+)completely inhibited the enzyme activity, while Ca(2+), Mg(2+), K(+) and Fe(3+) were enhancing the same. The serine protease inhibitor PMSF and metal chelator EDTA inhibited the activity of this protease while the classic metalloprotease inhibitor 1, 10 phenanthroline did not show inhibition. The enzyme was stable in SDS, Triton-X-100 and H(2) O(2) as well as in various commercial detergents after incubation for one hour. The extracellular production of the enzyme, the pH and temperature stability and stability in presence of oxidants, surfactants and commercial detergents suggest its possible use as a detergent additive.