RESUMO
The insecticide Diflubenzuron (DFB), used by many fish farming, when metabolized or degraded produces the extremely toxic compound p-chloroaniline (PCA). Once in the aquatic environment, these compounds can form mixtures and their bioavailability depends on factors such as the presence of soil. The toxic effects of the isolated compounds and their mixtures in the proportions: 75%, 50%, and 25% of PCA were analyzed in tilapia (Oreochromis niloticus) in the presence and absence of soil after 96h. The enzymes catalase (CAT), acid (AcP) and alkaline (AlP) phosphatases and alanine (ALT) and aspartate (AST) aminotransferases of the liver of the tilapia (Oreochromis niloticus) were used as biomarkers. DFB and the mixture containing 75% of this compound did not present high toxicity to fish; however, 25mg/L of PCA alone and 15mg/L of the mixture with 75% of this compound promoted 50% mortality of tilapia (Oreochromis niloticus). In the presence of soil, these toxicity values decreased to 37 and 25mg/L, respectively. Independent of the presence of soil, a synergistic effect was observed when the proportion of PCA was 75% and to the mixture, with 25% PCA was observed the antagonistic effect. Different concentrations of the compounds and their mixtures induced CAT activity independently of the presence of soil. Additionally, increases in phosphatases and transaminases activities were observed. In some cases, the enzymes also had their activities decreased and the dose-dependence effects were not observed. This research showed that the presence of soil influenced the toxicity of the compounds but not altered interaction type among them. Diflubenzuron, p-chloroaniline, and mixtures thereof caused disorders in enzymes important for the health of tilapia (Oreochromis niloticus).
Assuntos
Compostos de Anilina/toxicidade , Diflubenzuron/toxicidade , Inseticidas/toxicidade , Solo , Tilápia/metabolismo , Poluentes Químicos da Água/toxicidade , Animais , Aspartato Aminotransferases/metabolismo , Biomarcadores/metabolismo , Catalase/metabolismo , Ciclídeos/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologiaRESUMO
BACKGROUND: Low-risk patients suffering from prostate cancer (PCa) are currently placed under active surveillance rather than undergoing radical prostatectomy. However, clear parameters for selecting the right patient for each strategy are not available, and new biomarkers and treatment modalities are needed. Low-molecular-weight protein tyrosine phosphatase (LMWPTP) could present such a target. OBJECTIVE: To correlate expression levels of LMWPTP in primary PCa to clinical outcome, and determine the role of LMWPTP in prostate tumor cell biology. DESIGN, SETTING, AND PARTICIPANTS: Acid phosphatase 1, soluble (ACP1) expression was analyzed on microarray data sets, which were subsequently used in Ingenuity Pathway Analysis. Immunohistochemistry was performed on a tissue microarray containing material of 481 PCa patients whose clinicopathologic data were recorded. PCa cell line models were used to investigate the role of LMWPTP in cell proliferation, migration, adhesion, and anoikis resistance. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The association between LMWPTP expression and clinical and pathologic outcomes was calculated using chi-square correlations and multivariable Cox regression analysis. Functional consequences of LMWPTP overexpression or downregulation were determined using migration and adhesion assays, confocal microscopy, Western blotting, and proliferation assays. RESULTS AND LIMITATIONS: LMWPTP expression was significantly increased in human PCa and correlated with earlier recurrence of disease (hazard ratio [HR]:1.99; p<0.001) and reduced patient survival (HR: 1.53; p=0.04). Unbiased Ingenuity analysis comparing cancer and normal prostate suggests migratory propensities in PCa. Indeed, overexpression of LMWPTP increases PCa cell migration, anoikis resistance, and reduces activation of focal adhesion kinase/paxillin, corresponding to decreased adherence. CONCLUSIONS: Overexpression of LMWPTP in PCa confers a malignant phenotype with worse clinical outcome. Prospective follow-up should determine the clinical potential of LMWPTP overexpression. PATIENT SUMMARY: These findings implicate low-molecular-weight protein tyrosine phosphatase as a novel oncogene in prostate cancer and could offer the possibility of using this protein as biomarker or target for treatment of this disease.
Assuntos
Biomarcadores Tumorais/metabolismo , Movimento Celular , Neoplasias da Próstata/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Idoso , Anoikis , Biomarcadores Tumorais/genética , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Distribuição de Qui-Quadrado , Quinase 1 de Adesão Focal/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Peso Molecular , Análise Multivariada , Metástase Neoplásica , Recidiva Local de Neoplasia , Seleção de Pacientes , Paxilina/metabolismo , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Prostatectomia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas/genética , Medição de Risco , Fatores de Risco , Fatores de Tempo , Análise Serial de Tecidos , Transfecção , Regulação para Cima , Conduta ExpectanteRESUMO
Phoneutria nigriventer spider venom (PNV) contains Ca(2+), K(+) and Na(+) channel-acting peptides that affect neurotransmitter release and causes excitotoxicity in PNS and CNS. It has been demonstrated that PNV causes blood-brain barrier (BBB) breakdown of hippocampal microvessels time-dependently through enhanced microtubule-mediated vesicular transport. Herein, it is hypothesized that PNV can cause BBB breakdown in the hippocampus and cerebellum time-dependently through other molecular mechanisms. The BBB integrity was assessed through the analysis of expression of Poly-glycoprotein (P-gp) efflux transporter protein, laminin from basement membrane and endothelial tight junctional and adhesion junctional (TJ/AJ) proteins. Phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A) expression, which are known to have a role in the phosphorylation of junctional proteins and BBB opening, were also investigated. Astrocytes P-gp activity was determined by flow cytometry. The study demonstrated temporary decreased expression of laminin, TJ and AJ proteins (ZO1//occludin//claudin-5//beta-catenin) and P-gp (more prominently in hippocampus), which was completely or partially resolved between 2 and 5 h (and more quickly for cerebellum). PNV inhibited P-gp activity in astrocytes. PP2A phosphorylation, which inhibits the enzyme activity, was increased in both regions (15-45 min); however the phosphorylation level returned to baseline after 2 h. In conclusion, PNV disrupts paracellular transport in the BBB and possesses substrates for the active P-gp efflux transporter located in the BBB complex. Further studies into cellular mechanisms of astrocyte/endothelial interactions, using PNV as tool, may identify how astrocytes regulate the BBB, a characteristic that may be useful for the temporary opening of the BBB.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Junção Neuromuscular/metabolismo , Neurotoxinas/farmacologia , Venenos de Aranha/farmacologia , Aranhas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Western Blotting , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Imunofluorescência , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Laminina/biossíntese , Masculino , Proteínas do Tecido Nervoso/genética , Junção Neuromuscular/efeitos dos fármacos , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/fisiopatologia , Fosforilação , Ratos , Ratos WistarRESUMO
The flavonoid naringin is a polyphenolic compound that naturally occurs in citrus. Patients with cancer generally present features of malnutrition and cachexia. Levels of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) are raised in patients with cancer. This study was designed to analyze the in vivo effect of naringin in the therapeutic treatment of rats bearing Walker 256 carcinosarcoma (W256). Rats were treated intraperitoneally with different doses of naringin (10, 25 and 35 mg/kg), for 50 days. At 25 mg/kg, naringin inhibited tumor growth by ~75%. With this treatment, TNF-α and IL-6 levels decreased (p<0.05) in comparison with the control. In addition, two rats presented complete tumor regression. Inhibition of tumor growth, survival increase and the reduction of TNF-α and IL-6 levels in rats bearing W256 treated with naringin strongly suggest that this compound has potential as an anticarcinogenic drug.
Assuntos
Carcinoma 256 de Walker/tratamento farmacológico , Carcinoma 256 de Walker/metabolismo , Flavanonas/uso terapêutico , Interleucina-6/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Caquexia/tratamento farmacológico , Caquexia/metabolismo , Carcinoma 256 de Walker/mortalidade , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Ratos , Ratos Wistar , Taxa de SobrevidaRESUMO
Several biomaterials have been widely used in bone regeneration in both orthopedic and oral surgeries. However, it is poorly understood how these biomaterials alter osteoblast phenotype. It prompted us to examine the involvement of signaling proteins during preosteoblast adhesion (attachment), proliferation, and differentiation on natural hydroxyapatite (HA) from bovine bone. Our results indicated that natural HA is able to promote osteoblast adhesion, proliferation, and differentiation. The osteoblast/HA interaction requires phosphorylation of tyrosine residues of focal adhesion kinase, Src, and Paxillin upon integrin activation, which culminates in the control of cofilin phosphorylation (at serine 03) via rac-1 activation. In part, these signaling pathways are responsible for actin-rearrangement, responsible to adapt cell-shape on HA particles. In regarding to osteoblast differentiation, we showed that natural HA favored extracellular matrix remodeling by stimulating matrix metalloproteinase activities and alkaline phosphatase activity. Overall, this study demonstrates that osteoblast response toward bovine bone HA is initially mediated by activation of focal adhesion components, culminating on actin-rearrangement executed by cofilin activation via rac-1. Moreover, bovine bone HA provided an excellent microenvironment for osteoblast activity, since adhesion to differentiation.
Assuntos
Durapatita/farmacologia , Osteoblastos/citologia , Células 3T3 , Actinas/química , Fosfatase Alcalina/metabolismo , Animais , Bovinos , Adesão Celular , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Cofilina 1/química , Colágeno/química , Gelatina/química , Camundongos , Osteoblastos/metabolismo , Fosforilação , Transdução de SinaisRESUMO
Quercetin, a flavonoid abundantly present in fruit, vegetables, wine and tea, has revealed several properties such as antioxidant, antiproliferative and anticancer. Cachexia is a poorly understood syndrome present in already compromised cancer patients, decreasing the quality of life and increasing mortality. Many studies have been performed in an attempt to discover an effective treatment for cachexia, but none of the tested therapies has fulfilled expectations. The objective of the present study was to analyze the effect of quercetin in the therapeutic treatment of cachexia and reversion of tumor growth in rats bearing Walker 256 carcinosarcoma (W256). Rats bearing W256 were treated daily with I.P. quercetin injections, at different doses (10, 15, 25 and 35 mg/kg). The results show that 10 mg/kg quercetin inhibited tumor growth by about 50% (ED(50)) when compared with controls (CTR). Moreover, two animals of this group presented complete tumor regression. Matrix metalloproteinase-2 (MMP-2) activity and vascular endothelial growth factor (VEGF) expression decreased in rats bearing W256 treated with 10 mg/kg quercetin when compared with CTR. Thus, the inhibition of tumor growth, survival increase, decrease of MMP-2 and VEGF levels and reduction of cachexia in animals treated with quercetin strongly support the anticancer function of this flavonoid.
Assuntos
Antineoplásicos/uso terapêutico , Caquexia/prevenção & controle , Carcinoma 256 de Walker/tratamento farmacológico , Quercetina/uso terapêutico , Animais , Caquexia/etiologia , Carcinoma 256 de Walker/complicações , Fígado/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Ratos , Ratos Wistar , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important regulatory mechanism in eukaryotes. Therefore, the goal of this study is to assess osteoblast adhesion through a molecular prism under a peptide array technology, revealing essential signaling proteins governing adhesion-related events. First, we showed that there are main morphological changes on osteoblast shape during adhesion up to 3 h. Second, besides classical proteins activated upon integrin activation, our results showed a novel network involving signaling proteins such as Rap1A, PKA, PKC, and GSK3beta during osteoblast adhesion on polystyrene. Third, these proteins were grouped in different signaling cascades including focal adhesion establishment, cytoskeleton rearrangement, and cell-cycle arrest. We have thus provided evidence that a global phosphorylation screening is able to yield a systems-oriented look at osteoblast adhesion, providing new insights for understanding of bone formation and improvement of cell-substratum interactions. Altogether, these statements are necessary means for further intervention and development of new approaches for the progress of tissue engineering.
Assuntos
Osteoblastos/citologia , Osteoblastos/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Transdução de Sinais , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Motivos de Aminoácidos , Animais , Adesão Celular , Linhagem Celular , Proliferação de Células , Forma Celular , Citoesqueleto/metabolismo , Adesões Focais/enzimologia , Camundongos , Osteoblastos/enzimologia , Fosfoproteínas/química , Fosfotransferases/metabolismo , Análise Serial de Proteínas , Reprodutibilidade dos Testes , Serina/metabolismo , Fatores de TempoRESUMO
The presence of copper in water environment may have detrimental effects on aquatic organisms, including algae, where different enzymatic systems can be affected. Algae acid phosphatase plays important roles in metabolic processes such as decomposition of organic phosphate, autophagic digestive process, recycling cellular materials and zygote formation during reproduction. This work describes an in vitro activation effect of copper on the acid phosphatase of the green algae Pseudokirchneriella subcapitata (formely Selenastrum capricornutum) under preincubation condition. Apparent Michaelis constant values of 1.21 and 0.37 mM, and activation energy values of 26.8 and 13.6 kJ mol(-1) were determined in the absence and in the presence of 0.2 mM Cu(2+), respectively. The dissociation constant value for Cu(2+) binding to the enzyme was determined to be 22.04 microM. The decrease of the apparent Michaelis constant (Km) and activation energy values in the presence of Cu(2+) correlates well with its activating effect on the acid phosphatase activity. This propriety could be used as a sensitive bioindicator for copper in environmental samples.
Assuntos
Fosfatase Ácida/antagonistas & inibidores , Clorófitas/enzimologia , Sulfato de Cobre/farmacologia , Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Clorófitas/crescimento & desenvolvimento , Sulfato de Cobre/química , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Cinética , Relação Estrutura-AtividadeRESUMO
Acid phosphatases play important roles in algae metabolism such availability/recycling of inorganic phosphate and autophagic digestive processes. Chemicals released into the environment from agricultural activities and through industrial and urban wastes, may impair algae enzyme activity. The aim of this work was to evaluate the in vitro activation/inhibition effect of ten metals, commonly present as contaminants in soil and water, on the acid phospahatase extracted from the green algae Pseudokirchneriella subcapitata. Results demonstrated that Hg, Al, Mo, Pb, Se, and Cd inhibited the enzyme activity in 56.3, 54.5, 30.6, 25.5, 23.1 and 11.5 per cent respectively. This corresponds to the maximum percentage of effect attained at the metal concentration tested (0.02-2.0 mM). On the other hand, Cu, Zn, Ni and Cr exhibited an increment on phosphatase activity equal to 95.5, 87.6, 87.6, 77.6 and 42.8 per cent, respectively. Kinetic parameters values were calculated for the metals that showed hisghest effects. Thus, Ki ( inhibition constant) and Kd (dissociation constant) values equal to 0.0400 and 0.0016 mM were determined for Hg and Cu, respectively. A non-competitive inhibition mechanism was attributed to the former. Results improved the understanding of the basic events of the impact of metals at biochemical lebels in primary producers organisms.
Assuntos
Enzimas , Fitoplâncton , Poluentes Ambientais , Poluição Ambiental , Efeitos da Contaminação da Água , Poluição da ÁguaRESUMO
Pre-osteoblast adhesion attracts increasing interest in both medicine and dentistry. However, how this physiological event alters osteoblast phenotype is poorly understood. We therefore attempted to address this question by investigating key biochemical mechanism that governs pre-osteoblast adhesion on polystyrene surface. Importantly, we found that cofilin activity was strongly modulated by PP2A (Ser/Thr phosphatase), while cell-cycle was arrested. Accordingly, we observed that the profile of cofilin phosphorylation (at Ser03) was similar to phospho-PP2A (at Tyr307). Also, it is plausible to suggest during pre-osteoblast adhesion that PP2A phosphorylation at Y307 was executed by phospho-Src (Y416). In addition, it was observed that MAPKp38, but not MAPK-erk, played a key role on pre-osteoblast adhesion by phosphorylating MAPKAPK-2 and ATF-2 (also called CRE-BP1). Also, the up-modulation of RhoA reported here suggests its involvement at the beginning of osteoblast attachment, while Akt remained active during all periods. Altogether, our results clearly showed that osteoblast adhesion is under an intricate network of signaling molecules, which are responsible to guide their interaction with substrate mainly via cytoskeleton rearrangement.
Assuntos
Citoesqueleto/metabolismo , Osteoblastos/citologia , Transdução de Sinais , Animais , Adesão Celular , Ciclo Celular , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Modelos Biológicos , Osteoblastos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Treonina/genética , Treonina/metabolismo , Tirosina/genética , Tirosina/metabolismoRESUMO
Sewage sludge applied to soils as a fertilizer often contains metals and linear alkylbenzene sulphonate (LAS) as contaminants. These pollutants can be transported to the aquatic environment where they can alter the phosphatase activity in living organisms. The acid phosphatase of algae plays important roles in metabolism such as decomposing organic phosphate into free phosphate and autophagic digestive processes. The order of in vitro inhibition of Pseudokirchneriella subcapitata acid phosphatase at the highest concentration tested was LAS > Hg2+ = Al3+ > Se4+ = Pb2+ > Cd2+. A non-competitive inhibition mechanism was obtained for Hg2+ (Ki = 0.040 mM) and a competitive inhibition for LAS (Ki = 0.007 mM). In vivo studies with treated algae cultures showed that the inhibition of specific activity was observed in algae exposed during 7 days, in contrast to short term (24 h) treatments with both these chemicals. Our results suggest that the inhibition parameters in vitro did not markedly differ between the two chemicals. On the other hand, in vivo evaluations showed strong differences between both pollutants regarding the concentration values and the degree of response.
Assuntos
Fosfatase Ácida/antagonistas & inibidores , Proteínas de Algas/antagonistas & inibidores , Ácidos Alcanossulfônicos/química , Eucariotos/enzimologia , Metais Pesados/química , Poluentes Químicos da Água/química , Fosfatase Ácida/isolamento & purificação , Proteínas de Algas/isolamento & purificação , Ácidos Alcanossulfônicos/farmacologia , Eucariotos/efeitos dos fármacos , Concentração Inibidora 50 , Metais Pesados/farmacologia , Poluentes Químicos da Água/farmacologiaRESUMO
Pseudokirchneriella subcapitata is a unicellular green algae widely distributed in freshwater and soils. Due to its cosmopolitan characteristic, its use is recommended by national and international protocols in ecotoxicity studies. The alteration of phosphatase activities by agriculture pollutants like heavy metals has been extensively used as a biomarker in risk assessment and biomonitoring. In this study, we compared the extraction of acid phosphatase from P. subcapitata by different procedures and we studied the stability, substrates specificity, kinetics and the effect of Hg2+ in the crude extract. The freezing and thawing technique associated with probe sonication was the most suitable method of extraction. The enzyme was stable when frozen at -20ºC for at least six months, showed an optimum pH of 5 and a Km value of 0.27 mM for p-nitrophenylphosphate (pNPP) as substrate. Some natural organic substrates were cleaved by a similar extent as the synthetic substrate pNPP. Short term exposure (24 hours) to Hg2+ had little effect but inhibition of the specific activity was observed after 7 days with EC50 (concentration of Hg2+ that promotes 50% decrease of specific activity) value of 12.63 M Hg2+.
Pseudokirchneriella subcapitata é uma alga verde unicelular amplamente distribuída em corpos d´agua e solos. Devido a sua natureza cosmopolita, seu uso é recomendado por protocolos nacionais e internacionais na realização de estudos de ecotoxicidade. A alteração da atividade de fosfatases por agentes poluentes de origem agrícola, como metais pesados, tem sido largamente usada como um biomarcador na avaliação de risco e biomonitoramento. No presente trabalho foi comparada a extração da fosfatase ácida de P. subcapitata por diferentes métodos e estudada a sua estabilidade, especificidade por substratos, cinética e efeito do Hg2+ no extrato bruto. O congelamento e descongelamento, associado com ultrassom, foi o método que proporcionou maior rendimento de extração. A enzima, praticamente estável por armazenamento a -20ºC, durante aproximadamente seis meses, demonstrou uma atividade ótima em pH 5 e um valor de Km para o p-nitrofenilfostato (pNPP) de 0,27 mM. Alguns substratos naturais foram hidrolisados com uma intensidade semelhante à do substrato sintético pNPP. Diferentemente dos estudos de exposição a curto prazo (24 horas), observou-se inibição da atividade específica nas culturas expostas durante 7 dias, com um valor de CE50 (concentração de Hg2+ que promove 50% de decréscimo da atividade específica) equivalente a 12,63 M Hg2+.
RESUMO
Pseudokirchneriella subcapitata is a unicellular green algae widely distributed in freshwater and soils. Due to its cosmopolitan characteristic, its use is recommended by national and international protocols in ecotoxicity studies. The alteration of phosphatase activities by agriculture pollutants like heavy metals has been extensively used as a biomarker in risk assessment and biomonitoring. In this study, we compared the extraction of acid phosphatase from P. subcapitata by different procedures and we studied the stability, substrates specificity, kinetics and the effect of Hg2+ in the crude extract. The freezing and thawing technique associated with probe sonication was the most suitable method of extraction. The enzyme was stable when frozen at -20ºC for at least six months, showed an optimum pH of 5 and a Km value of 0.27 mM for p-nitrophenylphosphate (pNPP) as substrate. Some natural organic substrates were cleaved by a similar extent as the synthetic substrate pNPP. Short term exposure (24 hours) to Hg2+ had little effect but inhibition of the specific activity was observed after 7 days with EC50 (concentration of Hg2+ that promotes 50% decrease of specific activity) value of 12.63 M Hg2+.
Pseudokirchneriella subcapitata é uma alga verde unicelular amplamente distribuída em corpos d´agua e solos. Devido a sua natureza cosmopolita, seu uso é recomendado por protocolos nacionais e internacionais na realização de estudos de ecotoxicidade. A alteração da atividade de fosfatases por agentes poluentes de origem agrícola, como metais pesados, tem sido largamente usada como um biomarcador na avaliação de risco e biomonitoramento. No presente trabalho foi comparada a extração da fosfatase ácida de P. subcapitata por diferentes métodos e estudada a sua estabilidade, especificidade por substratos, cinética e efeito do Hg2+ no extrato bruto. O congelamento e descongelamento, associado com ultrassom, foi o método que proporcionou maior rendimento de extração. A enzima, praticamente estável por armazenamento a -20ºC, durante aproximadamente seis meses, demonstrou uma atividade ótima em pH 5 e um valor de Km para o p-nitrofenilfostato (pNPP) de 0,27 mM. Alguns substratos naturais foram hidrolisados com uma intensidade semelhante à do substrato sintético pNPP. Diferentemente dos estudos de exposição a curto prazo (24 horas), observou-se inibição da atividade específica nas culturas expostas durante 7 dias, com um valor de CE50 (concentração de Hg2+ que promove 50% de decréscimo da atividade específica) equivalente a 12,63 M Hg2+.
RESUMO
Ferruginol, a bioactive compound isolated from a Chilean tree (Podocarpaceae), attracts attention as a consequence of its pharmacological properties, which include anti-fungal, anti-bacterial, cardioprotective, anti-oxidative, anti-plasmodial and anti-ulcerogenic actions. Nevertheless, the molecular basis for these actions remains only partly understood and hence we investigated the effects of ferruginol on androgen-independent human prostate cancer cells (PC3), a known model for solid tumor cells with an exceptional resistance to therapy. The results show that ferruginol induces PC3 cell death via activation of caspases as well as apoptosis-inducing factor (AIF) as confirmed by its translocation into the nucleus. In order to clarify the biochemical mechanism responsible for the anti-tumor activity of ferruginol, we analyzed a set of molecular mediators involved in tumor cell survival, progression and aggressiveness. Ferruginol was able to trigger inhibition/downregulation of Ras/PI3K, STAT 3/5, protein tyrosine phosphatase and protein kinases related to cell cycle regulation. Importantly, the toxic effect of ferruginol was dramatically impeded in a more reducing environment, which indicates that at least in part, the anti-tumoral activity of ferruginol might be related to redox status modulation. This study supports further examination of ferruginol as a potential agent for both the prevention and treatment of prostate cancer.
Assuntos
Abietanos/toxicidade , Antineoplásicos/toxicidade , Neoplasias da Próstata/metabolismo , Transdução de Sinais/efeitos dos fármacos , Abietanos/química , Androgênios , Antineoplásicos/química , Fator de Indução de Apoptose/metabolismo , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Quinase I-kappa B/metabolismo , Masculino , Oxirredução , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição STAT/metabolismoRESUMO
Flavonoids, polyphenolic phytochemicals, are ubiquitous in plants and are commonly present in the human diet. They may exert diverse beneficial effects, including antioxidant and anticarcinogenic activities. The present study was designed to evaluate three biomolecules that play important roles in the apoptotic process: mitogen-activated protein kinases, protein phosphatases and NFkappaB, using HL60 cells treated with fisetin as an experimental model. Our results demonstrated that cells treated with fisetin presented high expression of NFkappaB, activation of MAPK p38 and an increase of phosphoprotein levels; inhibition of enzymes involved in redox status maintenance were also observed. Our findings reinforce the hypothesis that fisetin is likely to exert beneficial and/or toxic actions on cells not through its potential as antioxidant but rather through its modulation of protein kinase and phosphatase signaling cascades. Additionally, our results also indicate that the cellular effects of fisetin will ultimately depend on the cell type and on the extent to which they associate with the cells, either by interactions at the membrane or by uptake into the cytosol.
Assuntos
Flavonoides/metabolismo , Sistema de Sinalização das MAP Quinases , NF-kappa B/biossíntese , Fosfoproteínas/química , Antioxidantes/química , Antioxidantes/metabolismo , Citosol/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Flavonóis , Células HL-60 , Humanos , Linfócitos/metabolismo , Modelos Químicos , Estresse Oxidativo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Acid phosphatase plays important roles in algae metabolism such as availability and recycling of inorganic phosphate, autophagic digestive processes and fertilization. Chemicals released into the environment from agriculture activities may impair algae phosphatase activity. The aim of this work was to evaluate the in vitro effect of twenty-four organic compounds and six metals used as pesticides, or present as contaminants in sewage sludge, on the acid phosphatase activity extracted from Pseudokirchneriella subcapitata. Results demonstrated that only the linear surfactant alkyl benzenesulphonate (LAS) and the heavy metals Hg(2+), Al(3+) and Cu(2+) markedly altered (50%) the enzyme activity. Join action inhibition studies indicated that Hg(2+) was more potent inhibitor than Al(3+) or LAS, and that the Hg(2+)+Al(3+) and Hg(2+)+LAS mixtures have, respectively, additive and slight antagonism effects. Copper, which demonstrated an activator effect when preincubated with the enzyme, behaved as a slight antagonist for the inhibitor effect of Hg(2+).
Assuntos
Fosfatase Ácida/metabolismo , Agroquímicos/toxicidade , Clorófitas/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Agroquímicos/química , Clorófitas/enzimologia , Clorófitas/crescimento & desenvolvimento , Sinergismo Farmacológico , Poluentes Ambientais/química , Cadeia AlimentarRESUMO
Among the structurally related flavonoids tested on the bovine kidney low molecular weight protein tyrosine phosphatase (LMrPTP) activity, quercetin activated by about 2.6-fold the p-nitrophenyl-phosphate (p-NPP)-directed reaction, in contrast to morin that acted as a competitive inhibitor, with Ki values of 87, 73 and 50 microM for p-NPP, FMN, and tyrosine-phosphate, respectively. Other related flavonoids, such as rutin, kaempferol, catechin, narigin, phloretin and taxifolin did not significantly affect the LMrPTP activity. The positions of the hydroxyl groups in the structures of the flavonoids were important for their distinct effects on LMrPTP activity. The hydroxyl groups at C3' and C4' and the presence of a double bond at C2 and C3 were essential for the activating effect of quercetin. The absence of the 3'-OH (kaempferol), absence of the double bond (taxifolin) and the presence of the sugar rutinose at the 3-OH (rutin) suppressed the effect of quercetin. The C2'- and C4'-hydroxyl groups, the presence of the double bond, and a C4-ketone group were important requirements for the inhibitory effects of morin.
Assuntos
Inibidores Enzimáticos/farmacologia , Flavonoides/química , Rim/enzimologia , Proteínas Tirosina Fosfatases/química , Animais , Bovinos , Química Farmacêutica , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/química , Flavonóis/química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Modelos Químicos , Peso Molecular , Quercetina/análogos & derivados , Quercetina/químicaRESUMO
Reversible phosphorylation of tyrosine residues is a key regulatory mechanism for numerous cellular events. Protein tyrosine kinases and protein tyrosine phosphatases (PTPs) have a pivotal role in regulating both normal cell physiology and pathophysiology. Accordingly, deregulated activity of both protein tyrosine kinases and PTPs is involved in the development of numerous congenitically inherited and acquired human diseases, prompting obvious pharmaceutical and academic research interest. The development of compound libraries with higher selective PTP inhibitory activity has been bolstered by the realization that many natural products have such activity and thus are interesting biologically lead compounds, which properties are widely exploited. In addition, more rational approaches have focused on the incorporation of phosphotyrosine mimetics into specific peptide templates (peptidomimetic backbones). Additional factors furthering discovery as well as therapeutic application of new bioactive molecules are the integration of functional genomics, cell biology, structural biology, drug design, molecular screening and chemical diversity. Together, all these factors will lead to new avenues to treat clinical disease based on PTP inhibition.
Assuntos
Produtos Biológicos/química , Inibidores Enzimáticos/química , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Animais , Produtos Biológicos/uso terapêutico , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/uso terapêutico , Humanos , Estrutura Molecular , Proteínas Tirosina Fosfatases/metabolismoRESUMO
Besides having a pivotal biological function as a component of coenzymes, riboflavin appears a promissing antitumoral agent, but the underlying molecular mechanism remains unclear. In this work, we demonstrate that irradiated riboflavin, when applied at microM concentrations, induces an orderly sequence of signaling events finally leading to leukemia cell death. The molecular mechanism involved is dependent on the activation of caspase 8 caused by overexpression of Fas and FasL and also on mitochondrial amplification mechanisms, involving the stimulation of ceramide production by sphingomyelinase and ceramide synthase. The activation of this cascade led to an inhibition of mitogen activated protein kinases: JNK, MEK and ERK and survival mediators (PKB and IAP1), upregulation of the proapoptotic Bcl2 member Bax and downregulation of cell cycle progression regulators. Importantly, induction of apoptosis by irradiated riboflavin was leukaemia cell specific, as normal human lymphocytes did not respond to the compound with cell death. Our data indicate that riboflavin selectively activates Fas cascade and also constitutes a death receptor-engaged drug without harmful side effects in normal cells, bolstering the case for using this compound as a novel avenue for combating cancerous disease.
Assuntos
Morte Celular/efeitos dos fármacos , Leucemia/patologia , Riboflavina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Luz , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Riboflavina/efeitos da radiação , Riboflavina/uso terapêuticoRESUMO
OBJECTIVE: Tetrahydroxyquinone is a molecule best known as a primitive anticataract drug but is also a highly redox active molecule that can take part in a redox cycle with semiquinone radicals, leading to the formation of reactive oxygen species (ROS). Its potential as an anticancer drug has not been investigated. METHODS: The effects of tetrahydroxyquinone on HL60 leukemia cells are investigated using fluorescein-activated cell sorting-dependent detection of phosphatidylserine exposure combined with 7-amino-actinomycin D exclusion, via Western blotting using phosphospecific antibodies, and by transfection of constitutively active protein kinase B. RESULTS: We observe that in HL60 leukemia cells tetrahydroxyquinone causes ROS production followed by apoptosis through the mitochondrial pathway, whereas cellular physiology of normal human blood leukocytes was not affected by tetrahydroxyquinone. The antileukemic effect of tetrahydroxyquinone is accompanied by reduced activity of various antiapoptotic survival molecules including the protein kinase B pathway. Importantly, transfection of protein kinase B into HL60 cells and thus artificially increasing protein kinase B activity inhibits tetrahydroxyquinone-dependent cytotoxicity. CONCLUSION: Tetrahydroxyquinone provokes cytotoxic effects on leukemia cells by reduced protein kinase B-dependent survival signaling followed by apoptosis through the mitochondrial pathway. Thus, tetrahydroxyquinone may be representative of a novel class of chemotherapeutic drugs, inducing apoptosis in cancer cells through diminished survival signaling possibly as a consequence of ROS generation.