RESUMO
BACKGROUND: The main mechanism underlying cancer dissemination is the epithelial to mesenchymal transition (EMT). This process is orchestrated by cytokines like TGFß, involving "non-canonical" AKT- or STAT3-driven pathways. Recently, the alteration of copper homeostasis seems involved in the onset and progression of cancer. METHODS: We expose different breast cancer cell lines, including two triple negative (TNBC) ones, an HER2 enriched and one cell line representative of the Luminal A molecular subtype, to short- or long-term copper-chelation by triethylenetetramine (TRIEN). We analyse changes in the expression of EMT markers (E-cadherin, fibronectin, vimentin and αSMA), in the levels and activity of extracellular matrix components (LOXL2, fibronectin and MMP2/9) and of copper homeostasis markers by Western blot analyses, immunofluorescence, enzyme activity assays and RT-qPCR. Boyden Chamber and wound healing assays revealed the impact of copper chelation on cell migration. Additionally, we explored whether perturbation of copper homeostasis affects EMT prompted by TGFß. Metabolomic and lipidomic analyses were applied to search the effects of copper chelation on the metabolism of breast cancer cells. Finally, bioinformatics analysis of data on breast cancer patients obtained from different databases was employed to correlate changes in kinases and copper markers with patients' survival. RESULTS: Remarkably, only HER2 negative breast cancer cells differently responded to short- or long-term exposure to TRIEN, initially becoming more aggressive but, upon prolonged exposure, retrieving epithelial features, reducing their invasiveness. This phenomenon may be related to the different impact of the short and prolonged activation of the AKT kinase and to the repression of STAT3 signalling. Bioinformatics analyses confirmed the positive correlation of breast cancer patients' survival with AKT activation and up-regulation of CCS. Eventually, metabolomics studies demonstrate a prevalence of glycolysis over mitochondrial energetic metabolism and of lipidome changes in TNBC cells upon TRIEN treatment. CONCLUSIONS: We provide evidence of a pivotal role of copper in AKT-driven EMT activation, acting independently of HER2 in TNBC cells and via a profound change in their metabolism. Our results support the use of copper-chelators as an adjuvant therapeutic strategy for TNBC.
Assuntos
Transição Epitelial-Mesenquimal , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/metabolismo , Fibronectinas/metabolismo , Fibronectinas/farmacologia , Fibronectinas/uso terapêutico , Cobre/farmacologia , Cobre/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Disponibilidade Biológica , Trientina/farmacologia , Trientina/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular , Fator de Crescimento Transformador beta/metabolismo , Aminoácido Oxirredutases/metabolismo , Aminoácido Oxirredutases/farmacologia , Aminoácido Oxirredutases/uso terapêuticoRESUMO
Aging-related sarcopenia is currently the most common sarcopenia. The main manifestations are skeletal muscle atrophy, replacement of muscle fibers with fat and fibrous tissue. Excessive fibrosis can impair muscle regeneration and function. Lysyl oxidase-like 2 (LOXL2) has previously been reported to be involved in the development of various tissue fibrosis. Here, we investigated the effects of LOXL2 inhibitor on D-galactose (D-gal)-induced skeletal muscle fibroblast cells and mice. Our molecular and physiological studies show that treatment with LOXL2 inhibitor can alleviate senescence, fibrosis, and increased production of reactive oxygen species in fibroblasts caused by D-gal. These effects are related to the inhibition of the TGF-ß1/p38 MAPK pathway. Furthermore, in vivo, mice treatment with LOXL2 inhibitor reduced D-gal-induced skeletal muscle fibrosis, partially enhanced skeletal muscle mass and strength and reduced redox balance disorder. Taken together, these data indicate the possibility of using LOXL2 inhibitors to prevent aging-related sarcopenia, especially with significant fibrosis.
Assuntos
Galactose , Sarcopenia , Aminoácido Oxirredutases/metabolismo , Aminoácido Oxirredutases/farmacologia , Animais , Fibrose , Galactose/farmacologia , Camundongos , Músculo Esquelético/metabolismo , Proteína-Lisina 6-Oxidase/farmacologia , Sarcopenia/induzido quimicamente , Sarcopenia/tratamento farmacológico , Sarcopenia/patologiaAssuntos
Aminoácido Oxirredutases/farmacologia , Antifibróticos/farmacologia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/normas , Aminoácido Oxirredutases/análise , Aminoácido Oxirredutases/uso terapêutico , Antifibróticos/análise , Antifibróticos/uso terapêutico , Técnicas Biossensoriais/métodos , Fibrose/tratamento farmacológico , Fibrose/prevenção & controle , HumanosRESUMO
A novel express method is developed to determine activity of antitumor enzyme L-lysine-α-oxidase obtained by culturing Trichoderma harzianum Rifai F-180 fungus. The carcinogenic reagent ortho-dianisidine-hydrochloride was replaced in the reaction medium with environmentally friendly reagents of the chromogenic mixture that included tetramethylbenzidine. This method improved precision and sensitivity of ELISA by 10 and 40 times, respectively. In addition, it could detect activity of L-lysine-α-oxidase not only in the producer strains with a pronounced activity of this enzyme, but also in the strains where this activity has not been previously determined.
Assuntos
Aminoácido Oxirredutases/isolamento & purificação , Aminoácido Oxirredutases/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Hypocreales/enzimologia , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Colorimetria/métodos , Meios de Cultura/química , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Hypocreales/química , Temperatura , Fatores de TempoRESUMO
Knee meniscus injury is frequent, resulting in over 1 million surgeries annually in the United States and Europe. Because of the near-avascularity of this fibrocartilaginous tissue and its intrinsic lack of healing, tissue engineering has been proposed as a solution for meniscus repair and replacement. This study describes an approach employing bioactive stimuli to enhance both extracellular matrix content and organization of neomenisci toward augmenting their mechanical properties. Self-assembled fibrocartilages were treated with TGF-ß1, chondroitinase ABC, and lysyl oxidase-like 2 (collectively termed TCL) in addition to lysophosphatidic acid (LPA). TCL + LPA treatment synergistically improved circumferential tensile stiffness and strength, significantly enhanced collagen and pyridinoline crosslink content per dry weight, and achieved tensile anisotropy (circumferential/radial) values of neomenisci close to 4. This study utilizes a combination of bioactive stimuli for use in tissue engineering studies, providing a promising path toward deploying these neomenisci as functional repair and replacement tissues. STATEMENT OF SIGNIFICANCE: This study utilizes a scaffold-free approach, which strays from the tissue engineering paradigm of using scaffolds with cells and bioactive factors to engineer neotissue. While self-assembled neomenisci have attained compressive properties akin to native tissue, tensile properties still require improvement before being able to deploy engineered neomenisci as functional tissue repair or replacement options. In order to augment tensile properties, this study utilized bioactive factors known to augment matrix content in combination with a soluble factor that enhances matrix organization and anisotropy via cell traction forces. Using a bioactive factor to enhance matrix organization mitigates the need for bioreactors used to apply mechanical stimuli or scaffolds to induce proper fiber alignment.
Assuntos
Matriz Extracelular/metabolismo , Fibrocartilagem/metabolismo , Menisco/metabolismo , Engenharia Tecidual/métodos , Aminoácido Oxirredutases/farmacologia , Animais , Bovinos , Condrócitos/metabolismo , Condroitina ABC Liase/farmacologia , Módulo de Elasticidade , Matriz Extracelular/efeitos dos fármacos , Fibrocartilagem/efeitos dos fármacos , Humanos , Lisofosfolipídeos/farmacologia , Teste de Materiais , Menisco/efeitos dos fármacos , Resistência à Tração , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Tissue engineers rely on expensive, time-consuming, and destructive techniques to monitor the composition, microstructure, and function of engineered tissue equivalents. A non-destructive solution to monitor tissue quality and maturation would greatly reduce costs and accelerate the development of tissue-engineered products. The objectives of this study were to (a) determine whether matrix stabilization with exogenous lysyl oxidase-like protein-2 (LOXL2) with recombinant hyaluronan and proteoglycan link protein-1 (LINK) would result in increased compressive and tensile properties in self-assembled articular cartilage constructs, (b) evaluate whether label-free, non-destructive fluorescence lifetime imaging (FLIm) could be used to infer changes in both biochemical composition and biomechanical properties, (c) form quantitative relationships between destructive and non-destructive measurements to determine whether the strength of these correlations is sufficient to replace destructive testing methods, and (d) determine whether support vector machine (SVM) learning can predict LOXL2-induced collagen crosslinking. The combination of exogenous LOXL2 and LINK proteins created a synergistic 4.9-fold increase in collagen crosslinking density and an 8.3-fold increase in tensile strength as compared with control (CTL). Compressive relaxation modulus was increased 5.9-fold with addition of LOXL2 and 3.4-fold with combined treatments over CTL. FLIm parameters had strong and significant correlations with tensile properties (R2 = 0.82; p < 0.001) and compressive properties (R2 = 0.59; p < 0.001). SVM learning based on FLIm-derived parameters was capable of automating tissue maturation assessment with a discriminant ability of 98.4%. These results showed marked improvements in mechanical properties with matrix stabilization and suggest that FLIm-based tools have great potential for the non-destructive assessment of tissue-engineered cartilage.
Assuntos
Cartilagem Articular/fisiologia , Matriz Extracelular/metabolismo , Aminoácido Oxirredutases/farmacologia , Animais , Fenômenos Biomecânicos , Cartilagem Articular/efeitos dos fármacos , Bovinos , Colágeno/metabolismo , Força Compressiva , Reagentes de Ligações Cruzadas/química , Matriz Extracelular/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Humanos , Proteoglicanas/farmacologia , Máquina de Vetores de Suporte , Resistência à Tração , Engenharia Tecidual , Alicerces Teciduais/químicaRESUMO
Strategies to overcome the limited availability of human articular chondrocytes and their tendency to dedifferentiate during expansion are required to advance their clinical use and to engineer functional cartilage on par with native articular cartilage. This work sought to determine whether a biochemical factor (transforming growth factor-ß1 [T]), a biophysical agent (chondroitinase-ABC [C]), and a collagen crosslinking enzyme (lysyl oxidase-like 2 [L]) are efficacious in forming three-dimensional human neocartilage from expanded human articular chondrocytes. Among the treatment regimens, the combination of the three stimuli (TCL treatment) led to the most robust glycosaminoglycan content, total collagen content, and type II collagen production. In particular, TCL treatment synergistically increased tensile stiffness and strength of human neocartilage by 3.5-fold and 3-fold, respectively, over controls. Applied to two additional donors, the beneficial effects of TCL treatment appear to be donor independent; tensile stiffness and strength were increased by up to 8.5-fold and 3-fold, respectively, over controls. The maturation of human neocartilage in response to TCL treatment was examined following 5 and 8 weeks of culture, demonstrating maintenance or further enhancement of functional properties. The present study identifies a novel strategy for engineering human articular cartilage using serially passaged chondrocytes.
Assuntos
Aminoácido Oxirredutases/farmacologia , Cartilagem/metabolismo , Condrócitos/metabolismo , Condroitina ABC Liase/farmacologia , Engenharia Tecidual , Fator de Crescimento Transformador beta1/farmacologia , Adulto , Cartilagem/citologia , Condrócitos/citologia , Humanos , Masculino , Resistência à TraçãoRESUMO
Studies of the effects of Trichoderma harzianum Rifai F-180 culture fluid concentrate containing L-lysine-α-oxidase antitumor enzyme produced by the fungus and the homogenous enzyme, on ultrahazardous bacterium Acidovorax citrulli demonstrated the antibacterial activity of the concentrate. Trichoderma harzianum Rifai F-180 producing L-lysine-α-oxidase was cultured in a technological device at G. K. Skryabin Institute of Biochemistry and. Physiology of Microorganisms, Russian Academy of Sciences. Activity of L-lysine-α-oxidase in the resulted culture fluid concentrate was 0.54 U/ml, activity of the homogenous enzyme was 50 U/mg.
Assuntos
Aminoácido Oxirredutases/farmacologia , Comamonadaceae/efeitos dos fármacos , Proteínas Fúngicas/farmacologia , Fungicidas Industriais/farmacologia , Trichoderma/química , Aminoácido Oxirredutases/isolamento & purificação , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Comamonadaceae/crescimento & desenvolvimento , Comamonadaceae/isolamento & purificação , Comamonadaceae/patogenicidade , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Reposicionamento de Medicamentos , Proteínas Fúngicas/isolamento & purificação , Fungicidas Industriais/isolamento & purificação , Plantas/microbiologia , Trichoderma/crescimento & desenvolvimentoRESUMO
We studied the effects of a concentrate of metabolites of Trichoderma harzianum Rifai F-180, an active producer of L-lysine-α-oxidase, and homogenous enzyme on a highly virulent bacteria Erwinia amylovora. The producer of antitumor and antiviral Trichoderma enzyme L-lysine-α-oxidase was cultured on a processing system of G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (Pushchino). Activity of L-lysine-α-oxidase in the prepared concentrate of metabolic products of the producer was 5.4 U/ml, and activity of the homogenous enzyme was 50 U/ml. Antibacterial activity of the enzyme was shown in our experiments.
Assuntos
Aminoácido Oxirredutases/farmacologia , Antibacterianos/farmacologia , Proteínas Fúngicas/farmacologia , Trichoderma/química , Aminoácido Oxirredutases/isolamento & purificação , Antibacterianos/isolamento & purificação , Meios de Cultura/química , Erwinia amylovora/efeitos dos fármacos , Erwinia amylovora/crescimento & desenvolvimento , Fermentação , Proteínas Fúngicas/isolamento & purificação , Testes de Sensibilidade Microbiana , Trichoderma/enzimologiaRESUMO
A producing strain of an anti-tumor and antiviral enzyme L-lysine-α-oxidase from Trichoderma was cultured using a technological device of G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (Pushchino). L-lysine-α-oxidase activity in the obtained metabolite concentrate was 5.4 U/ml. We studied the effects of the concentrate of active L-lysine-α-oxidase producer on the highly infectious Tobacco ringspot virus and revealed anti-viral activity of it when enzyme concentration was at least 1.0 U/ml.
Assuntos
Aminoácido Oxirredutases/farmacologia , Antivirais/farmacologia , Proteínas Fúngicas/farmacologia , Nepovirus/efeitos dos fármacos , RNA Viral/antagonistas & inibidores , Trichoderma/enzimologia , Aminoácido Oxirredutases/biossíntese , Aminoácido Oxirredutases/isolamento & purificação , Antivirais/isolamento & purificação , Antivirais/metabolismo , Balsaminaceae/virologia , Meios de Cultura/química , Fermentação , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Nepovirus/genética , Nepovirus/crescimento & desenvolvimento , RNA Viral/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trichoderma/química , Trichoderma/crescimento & desenvolvimentoRESUMO
The antitumor effect of L-lysine-α-oxidase from the culture fluid of Trichoderma harzianum Rifai F-180 was investigated for the first time. The in vitro studies revealed its high activity on a model of the forest-spring encephalitis virus and no activity against the Sindbis, Western Nile, Tyaginya and Dhori viruses.
Assuntos
Aminoácido Oxirredutases/farmacologia , Antineoplásicos/farmacologia , Antivirais/farmacologia , Vírus da Encefalite Transmitidos por Carrapatos/efeitos dos fármacos , Proteínas Fúngicas/farmacologia , Trichoderma/química , Aminoácido Oxirredutases/isolamento & purificação , Animais , Antineoplásicos/isolamento & purificação , Antivirais/isolamento & purificação , Chlorocebus aethiops , Reposicionamento de Medicamentos , Vírus da Encefalite Transmitidos por Carrapatos/crescimento & desenvolvimento , Proteínas Fúngicas/isolamento & purificação , Humanos , Camundongos , Orthobunyavirus/crescimento & desenvolvimento , Sindbis virus/crescimento & desenvolvimento , Suínos , Thogotovirus/crescimento & desenvolvimento , Trichoderma/enzimologia , Células Vero , Vírus do Nilo Ocidental/crescimento & desenvolvimentoRESUMO
BACKGROUND: Virus replication strongly depends on host metabolic machinery and essential cellular factors, in particular, on amino acid profiles. Amino acids play an important role in the pathogenesis of all virus-related infections both as basic substrates for protein synthesis and as regulators in many metabolic pathways, including gene expression. The inhibitory effects of deficiency or excess of these essential elements on virus replication are widely appreciated. Although the same interrelationship between host cellular factors and HIV have been recognized for a long time, the effects of amino acids on HIV-1 RNA replication dynamic is not yet well documented. Our aim was to determine in this pilot study the direct effect of L-lysine amino acid on HIV-1 RNA replication in vitro in HIV-infected patients. METHODS: A total of 100 HIV-1-infected males without highly active antiretroviral therapy (HAART) were monitored in our center. The patients were in stage A of the disease according to the 1993 Centers for Disease Control (CDC) classification system for HIV-infection. Patients with HIV were enrolled in one stage (A) of the disease with the average amount CD4 lymphocytes in the range of 200-300 cells/µL at the time of sample acquisition. For evaluation of the effects of essential L-lysine amino acid on HIV-1 RNA replication level, we used a model of amino acid-excess system in vitro following incubation of plasma samples for 24 h at 25 °C. Quantitative HIV-1 RNA assay was performed using (RT-PCR) reverse-transcriptase polymerase chain reaction (Rotor-Gene Q, QIAGEN, Germany). RESULTS: The mean HIV-1 RNA levels were significantly higher in the enriched peripheral blood mononuclear cells plasma samples HIV-infected subjects after 24 h incubation at 25 °C temperature than in the plasma samples the same patients studied on the date of blood tests (p < 0.0001). The number of HIV-1 RNA copies increased in 1.5 times. We observed that in plasma of the same HIV-infected patients after adding L-lysine and following incubation in vitro, viral load increased significantly in comparison with standard samples (p < 0.0001). The increased viral load was found in 100/92 (92%) of HIV-infected subjects. The average number of HIV-1 RNA copies in samples had increased by 4.0 times. However, we found no difference in HIV-1 RNA levels after replacement of L-lysine for L-arginine in comparison samples in the same HIV-infected patients. It is obvious that the addition of L-arginine does not increase viral replication in vitro as L-lysine amino acid supplement does. Additionally, no increase in viral load was determined after adding L-lysine and non toxic doses of its inhibitor (L-lysine alpha-oxidase) in plasma samples. CONCLUSIONS: The results show that L-lysine amino acid excess is characterized by significant increased of HIV-1 RNA copies in enriched peripheral blood mononuclear cells plasma samples of HIV-infected patients. There was evidence for an association between L-lysine supplementation and HIV-1 RNA replication and the level changes of this host essential nutritional element play a key role in the synthesis of the virus proteins and in transcription initiation of the retrovirus life cycle. High intake of L-lysine amino acid may increase the risk of high viral load, subsequent acceleration of immunosuppression and HIV progression. Overall results demonstrate that the simple L-lysine-related model in vitro can be widely used for practical purposes to evaluate HIV-1 RNA replication dynamic, disease prognosis and new approaches in treatment of the patients with human immunodeficiency virus. Although the impact mechanism of L-lysine amino acid on the viral load in the pathogenesis of HIV-infection is at present conjectural and requires further development, the results highlight an interesting target in antiviral therapy, and this statement remains to be proved in further research and clinical trials.
Assuntos
HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Lisina/farmacologia , RNA Viral/biossíntese , Replicação Viral/efeitos dos fármacos , Adulto , Aminoácido Oxirredutases/farmacologia , Arginina/farmacologia , Infecções por HIV/sangue , HIV-1/genética , Humanos , Leucócitos Mononucleares/virologia , Masculino , Projetos Piloto , RNA Viral/sangue , Adulto JovemRESUMO
L-Lysine α-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L-lysine to form α-keto-ε-aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 Å resolution revealed that the overall structure is similar to that of snake venom L-amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX.
Assuntos
Aminoácido Oxirredutases/genética , Trichoderma/enzimologia , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/farmacologia , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Sequência de Bases , Clonagem Molecular , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Homologia de Sequência de Aminoácidos , Streptomyces lividans/genéticaRESUMO
Synergism effects of cisplatin and L-lysine-alpha-oxidase (LO), while sequential (no interval) administration of drugs depends on the tumor model and duration of treatment. Synergism is identified at intraperitoneal daily (during 3 days) administration of cisplatin to experimental animals in single doses of 1.5 or 3.0 mg/kg and intravenously 5-fold after 48 h administration of LO and also administered intravenously in cumulative doses of 300-600 E / kg discretely, the first dose--doubled. Synergism of cisplatin and LO is showed by significant (p < 0.05) therapeutic gain against cisplatin at such indicators as increased survival of mice with P388 tumor and increased inhibition of primary tumor melanoma B16.
Assuntos
Aminoácido Oxirredutases/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cisplatino/farmacologia , Leucemia P388/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Aminoácido Oxirredutases/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Cisplatino/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Análise de Sobrevida , Resultado do TratamentoRESUMO
L-Lysine α-oxidase (LO) from a novel Trichoderma strain: Trichoderma cf. aureoviride Rifai shows favorable biochemical and kinetic properties (Km for L-lysine of 17.9 µmol/l, optimum pH 8.0, high stability) and significant antiproliferative activity both in vitro and in vivo. The molecular weight of LO was determined to be 115-116 kDa; the active dimer consists of two identical 57-58 kDa subunits. LO shows considerable cytotoxicity against the following tumor cell lines: K562, LS174T, HT29, SCOV3, PC3, and MCF7, with the inhibition concentration (IC50) ranging from 3.0×10 to 7.8×10 U/ml (3.2×10 to 8.2×10 mg/ml). Two human colon cancer xenografts HCT116 and LS174T and breast adenocarcinoma T47D implanted subcutaneously into Balb/c nude mice showed high sensitivity to LO with a T/C of 12, 37, and 36%, respectively (P<0.05). The antitumor efficacy of LO was observed in the absence of pronounced morbidity or toxicity in vivo. Taken together, these data suggest that LO may be considered as an effective anticancer agent for the treatment of solid tumors in vivo. This study presents promising data on the possible application of LO in clinical oncology for patients with colorectal cancer.
Assuntos
Aminoácido Oxirredutases/metabolismo , Aminoácido Oxirredutases/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacologia , Neoplasias/tratamento farmacológico , Trichoderma/enzimologia , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estabilidade Enzimática , Feminino , Células HT29 , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Células K562 , Cinética , Lisina/metabolismo , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Peso Molecular , Neoplasias/patologia , Multimerização Proteica , Especificidade por Substrato , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Bone morphogenetic protein-2 (BMP2) is among the most popular anabolic agents and substantially increase bone volume related to enhanced osteoblast differentiation. Here we demonstrate a remarkable deterioration in the nanomechanical properties of mineralized tissue induced from osteoblasts solely by the function of BMP2. Mineralized tissue of primary osteoblasts cultured with BMP2 shows molecular features of both bone and cartilage, but depletion of lysyl oxidase family members leads to poor nanomechanical properties of the mineralized tissue. Lysyl oxidase like-2 supplementation reinforces the inferior mineralized tissue induced from osteoblasts by BMP2 through intermolecular cross-linking of type II or type X collagen-rich extracellular matrix. This may also mimic a consolidation of bone fracture gaps, despite the fact that the distribution of the bone properties in such microenvironments has been poorly elucidated. These findings confirm the importance of testing newly induced bone down to the microscale and nanoscale in bone tissue engineering. FROM THE CLINICAL EDITOR: Bone morphogenetic protein-2 is known to substantially increase bone volume related to enhanced osteoblast differentiation; however, this team of investigators report a remarkable deterioration in the nanomechanical properties of mineralized tissue induced from osteoblasts solely by the function of BMP2.
Assuntos
Aminoácido Oxirredutases/farmacologia , Proteína Morfogenética Óssea 2/farmacologia , Fenômenos Mecânicos/efeitos dos fármacos , Nanopartículas/química , Osteogênese/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Animais , Calcificação Fisiológica/efeitos dos fármacos , Células Cultivadas , Perfilação da Expressão Gênica , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Proteínas Recombinantes/farmacologia , Análise Espectral RamanRESUMO
Adult human dental pulp stem cells (hDPSCs) are a unique population of precursor cells those are isolated from postnatal dental pulp and have the ability to differentiate into a variety of cell types utilized for the formation of a reparative dentin-like complex. Using LC-MS/MS proteomics approaches, we identified the proteins secreted from the differentiating hDPSCs in mineralization media. Lysyl oxidase-like 2 (LOXL2) was identified as a protein that was down-regulated in the hDPSCs that differentiate into odontoblast-like cells. The role of LOXL2 has not been studied in dental pulp stem cells. LOXL2 mRNA levels were reduced in differentiating hDPSCs, whereas the levels of other LOX family members including LOX, LOXL1, LOXL3, and LOXL4, are increased. The protein expression and secretion levels of LOXL2 were also decreased during odontogenic differentiation. Recombinant LOXL2 protein treatment to hDPSCs resulted in a dose-dependent decrease in the early differentiation and the mineralization accompanying with the lower levels of odontogenic markers such as DSPP, DMP-1 and ALP. These results suggest that LOXL2 has a negative effect on the differentiation of hDPSCs and blocking LOXL2 can promote the hDPSC differentiation to odontoblasts.
Assuntos
Células-Tronco Adultas/fisiologia , Aminoácido Oxirredutases/metabolismo , Diferenciação Celular/genética , Polpa Dentária/fisiologia , Odontogênese/genética , Células-Tronco Adultas/efeitos dos fármacos , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/farmacologia , Biomarcadores/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL27/metabolismo , Polpa Dentária/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Odontogênese/efeitos dos fármacos , Fosfoproteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Sialoglicoproteínas/metabolismo , Nicho de Células-TroncoRESUMO
The effect of L-lysine-alpha-oxidase from Trichoderma harzianum Rifai on the functional activity of T-lymphocytes was investigated. It was shown that in a dose of 35 units/kg administered parentally the enzyme had no suppressive effect on the T-lymphocyte functional activity. An inhibitory effect of L-lysine-a-oxidase on some indices of the macrophages functional activity was observed. L-Lyzine-alpha-oxidase had a selective lymphotropic action and showed no mytostatic activity, which is in favour of the enzyme vs. other antitumor agents.
Assuntos
Aminoácido Oxirredutases/farmacologia , Antineoplásicos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Trichoderma/enzimologia , 5'-Nucleotidase/metabolismo , Aminoácido Oxirredutases/isolamento & purificação , Animais , Antineoplásicos/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Imunomodulação , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos CBARESUMO
The effect of L-lysine-alpha-oxidase from a representative of the Trichoderma genus on the humoral immune response to protein antigens was studied. It was shown that repeated fife-fold intravenous administrations of L-lysine-alpha-oxidase in a dose of 35 U/kg had no depressive action on the humoral immunity. The enzyme had no suppressive effect on the delayed type hypersensitivity to xenogenous erythrocytes. The use of L-lysine-alpha-oxidase in a therapeutic dose or in a twice as higher dose had no reliable effect on the leukocyte migration capacity vs. the control.
Assuntos
Aminoácido Oxirredutases/farmacologia , Formação de Anticorpos/efeitos dos fármacos , Antineoplásicos/farmacologia , Imunidade Humoral/imunologia , Trichoderma/enzimologia , Animais , Anticorpos/sangue , Anticorpos/imunologia , Asparaginase/imunologia , Movimento Celular/imunologia , Avaliação Pré-Clínica de Medicamentos , Eritrócitos/imunologia , Escherichia coli/enzimologia , Imunização , Leucócitos/fisiologia , Camundongos , Camundongos Endogâmicos CBA , Ovalbumina/imunologia , OvinosRESUMO
A new l-amino acid oxidase (LAAO) was isolated from the Central Asian cobra Naja naja oxiana venom by size exclusion, ion exchange and hydrophobic chromatography. The N-terminal sequence and the internal peptide sequences share high similarity with other snake venom l-amino acid oxidases, especially with those isolated from elapid venoms. The enzyme is stable at low temperatures (-20 degrees C, -70 degrees C) and loses its activity by heating at 70 degrees C. Specific substrates for the isolated protein are l-phenylalanine, l-tryptophan, l-methionine and l-leucine. The enzyme has antibacterial activity inhibiting the growth of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. N. naja oxiana LAAO dose-dependently inhibited ADP- or collagen-induced platelet aggregation with IC(50) of 0.094 microM and 0.036 microM, respectively. The antibacterial and anti-aggregating activity was abolished by catalase.