RESUMO
In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.
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In prostate carcinogenesis, expression and/or activation of the Transient Receptor Potential Melastatin 8 channel (TRPM8) was shown to block in vitro Prostate Cancer (PCa) cell migration. Because of their localization at the plasma membrane, ion channels, such as TRPM8 and other membrane receptors, are promising pharmacological targets. The aim of this study was thus to use nanocarriers encapsulating a TRPM8 agonist to efficiently activate the channel and therefore arrest PCa cell migration. To achieve this goal, the most efficient TRPM8 agonist, WS12, was encapsulated into Lipid NanoCapsules (LNC). The effect of the nanocarriers on channel activity and cellular physiological processes, such as cell viability and migration, were evaluated in vitro and in vivo. These results provide a proof-of-concept support for using TRPM8 channel-targeting nanotechnologies based on LNC to develop more effective methods inhibiting PCa cell migration in zebrafish xenograft.
Assuntos
Anilidas/farmacologia , Inibição de Migração Celular/efeitos dos fármacos , Mentol/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Canais de Cátion TRPM/agonistas , Anilidas/administração & dosagem , Humanos , Lipídeos/química , Masculino , Mentol/administração & dosagem , Mentol/farmacologia , Nanocápsulas/química , Células PC-3 , Neoplasias da Próstata/metabolismo , Canais de Cátion TRPM/metabolismoRESUMO
Unlike mammals, the CNS of the medicinal leech can regenerate damaged neurites, thus restoring neural functions after lesion. We previously demonstrated that the injured leech nerve cord is able to mount an immune response promoting the regenerative processes. Indeed neurons and microglia express sensing receptors like Hm-TLR1, a leech TLR ortholog, associated with chemokine release in response to a septic challenge or lesion. To gain insights into the TLR signaling pathways involved during these neuroimmune responses, members of the MyD88 family were investigated. In the present study, we report the characterization of Hm-MyD88 and Hm-SARM. The expression of their encoding gene was strongly regulated in leech CNS not only upon immune challenge but also during CNS repair, suggesting their involvement in both processes. This work also showed for the first time that differentiated neurons of the CNS could respond to LPS through a MyD88-dependent signalling pathway, while in mammals, studies describing the direct effect of LPS on neurons and the outcomes of such treatment are scarce and controversial. In the present study, we established that this PAMP induced the relocalization of Hm-MyD88 in isolated neurons.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Hirudo medicinalis/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Sequência de Aminoácidos , Animais , Sistema Nervoso Central/metabolismo , Proteínas do Citoesqueleto/classificação , Proteínas do Citoesqueleto/genética , Humanos , Lipopolissacarídeos/toxicidade , Microglia/metabolismo , Dados de Sequência Molecular , Fator 88 de Diferenciação Mieloide/classificação , Fator 88 de Diferenciação Mieloide/genética , Regeneração Nervosa , Neurônios/metabolismo , Alinhamento de Sequência , Transdução de Sinais/efeitos dos fármacos , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/metabolismoRESUMO
Curcumin, a major active component of turmeric (Curcuma longa, L.), has anticancer effects. In vitro studies suggest that curcumin inhibits cancer cell growth by activating apoptosis, but the mechanism underlying these effects is still unclear. Here, we investigated the mechanisms leading to apoptosis in curcumin-treated cells. Curcumin induced endoplasmic reticulum stress causing calcium release, with a destabilization of the mitochondrial compartment resulting in apoptosis. These events were also associated with lysosomal membrane permeabilization and of caspase-8 activation, mediated by cathepsins and calpains, leading to Bid cleavage. Truncated tBid disrupts mitochondrial homeostasis and enhance apoptosis. We followed the induction of autophagy, marked by the formation of autophagosomes, by staining with acridine orange in cells exposed curcumin. At this concentration, only the early events of apoptosis (initial mitochondrial destabilization with any other manifestations) were detectable. Western blotting demonstrated the conversion of LC3-I to LC3-II (light chain 3), a marker of active autophagosome formation. We also found that the production of reactive oxygen species and formation of autophagosomes following curcumin treatment was almost completely blocked by N-acetylcystein, the mitochondrial specific antioxidants MitoQ10 and SKQ1, the calcium chelators, EGTA-AM or BAPTA-AM, and the mitochondrial calcium uniporter inhibitor, ruthenium red. Curcumin-induced autophagy failed to rescue all cells and most cells underwent type II cell death following the initial autophagic processes. All together, these data imply a fail-secure mechanism regulated by autophagy in the action of curcumin, suggesting a therapeutic potential for curcumin. Offering a novel and effective strategy for the treatment of malignant cells.
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The growing number of studies suggested that inhibition of autophagy enhances the efficacy of Akt kinase inhibitors in cancer therapy. Here, we provide evidence that ML-9, a widely used inhibitor of Akt kinase, myosin light-chain kinase (MLCK) and stromal interaction molecule 1 (STIM1), represents the 'two-in-one' compound that stimulates autophagosome formation (by downregulating Akt/mammalian target of rapamycin (mTOR) pathway) and inhibits their degradation (by acting like a lysosomotropic agent and increasing lysosomal pH). We show that ML-9 as a monotherapy effectively induces prostate cancer cell death associated with the accumulation of autophagic vacuoles. Further, ML-9 enhances the anticancer activity of docetaxel, suggesting its potential application as an adjuvant to existing anticancer chemotherapy. Altogether, our results revealed the complex effect of ML-9 on autophagy and indentified ML-9 as an attractive tool for targeting autophagy in cancer therapy through dual inhibition of both the Akt pathway and the autophagy.
Assuntos
Autofagia/efeitos dos fármacos , Azepinas/farmacologia , Lisossomos/efeitos dos fármacos , Neoplasias da Próstata/patologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Regulação para Baixo/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Lisossomos/ultraestrutura , Masculino , Modelos Biológicos , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/ultraestrutura , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Bacillus strains are often isolated from biofilms in the food industries. Previous works have demonstrated that sporulation could occur in biofilms, suggesting that biofilms would be a significant source of food contamination with spores. In this study, we investigated the properties of mono-species and mixed Bacillus biofilms and the ability of Bacillus strains to sporulate inside biofilms. Bacillus strains were able to form mono-species biofilms on stainless steel coupons, with up to 90% spores after a 48 h-incubation. These spores were highly resistant to cleaning but were easily transferred to agar, mimicking the cross-contamination of food, thereby suggesting that biofilms would be of particular concern due to a potential for Bacillus spore food contamination. This hypothesis was strengthened by the fact that Bacillus strains were able to form mixed biofilms with resident strains and that sporulation still occurred easily in these complex structures.
Assuntos
Bacillus/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Contaminação de Equipamentos , Equipamentos e Provisões/microbiologia , Manipulação de Alimentos/instrumentação , Esporos Bacterianos/crescimento & desenvolvimento , Bacillus/efeitos dos fármacos , Bacillus/fisiologia , Biofilmes/efeitos dos fármacos , Desinfetantes/farmacologia , Esporos Bacterianos/efeitos dos fármacos , Aço Inoxidável/análiseRESUMO
This study was designed to evaluate the respective roles of mechanical and chemical effects on the removal of Bacillus spores during cleaning-in-place. This analysis was performed on 12 strains belonging to the Bacillus cereus group (B. cereus, Bacillus anthracis, Bacillus thuringiensis) or to less related Bacillus species (Bacillus pumilus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus subtilis). Adherent spores were subjected to rinsing-in-place (mechanical action) and cleaning-in-place (mechanical and chemical actions) procedures, the latter involving NaOH 0.5% at 60°C. Results revealed that mechanical action alone only removed between 53 and 89% of the attached spores at a shear stress of 500 Pa. This resistance to shear was not related to spore surface properties. Conversely, in the presence of NaOH at a shear stress of 4 Pa, spores were readily detached, with between 80 and 99% of the adherent spores detached during CIP and the chemical action greatly depended on the strain. This finding suggests that chemical action plays the major role during CIP, whose efficacy is significantly governed by the spore surface chemistry.
Assuntos
Bacillus/fisiologia , Aderência Bacteriana , Conservação de Alimentos/métodos , Esporos Bacterianos/química , Esporos Bacterianos/fisiologia , Bacillus/química , Bacillus/classificação , Bacillus/crescimento & desenvolvimento , Esporos Bacterianos/classificação , Esporos Bacterianos/crescimento & desenvolvimentoRESUMO
This study was designed to evaluate how conditions encountered by spores during cleaning-in-place (CIP) procedures affected their surface properties, their viability and ability to contaminate materials. Spores from five Bacillus cereus strains were treated with NaOH at high temperature. Results revealed that high temperatures (exceeding 60 degrees C) and NaOH concentrations (over 0.5%) were required to significantly decrease spore viability (3-5log decrease). In these conditions, modifications were also clearly observed by microscopy to various surface structures of spores (appendages, exosporium, and especially to the hair-like nap) but also to their coat. Therefore, the ability of culturable spores to adhere decreased for the majority of strains tested. We then demonstrated that spores in suspension in NaOH could adhere to surfaces of a CIP rig and that the contamination level was controlled by flow pattern. Consequently, re-adhesion along the processing line might occur during CIP procedures and this phenomenon must be taken into account when defining cleaning strategies.
Assuntos
Bacillus cereus/fisiologia , Contaminação de Equipamentos , Temperatura Alta , Saneamento/métodos , Hidróxido de Sódio/farmacologia , Aderência Bacteriana , Qualidade de Produtos para o Consumidor , Microbiologia de Alimentos , Indústria de Processamento de Alimentos/normas , Viabilidade Microbiana , Esporos Bacterianos/crescimento & desenvolvimentoRESUMO
Accumulating data point to K(+) channels as relevant players in controlling cell cycle progression and proliferation of human cancer cells, including prostate cancer (PCa) cells. However, the mechanism(s) by which K(+) channels control PCa cell proliferation remain illusive. In this study, using the techniques of molecular biology, biochemistry, electrophysiology and calcium imaging, we studied the expression and functionality of intermediate-conductance calcium-activated potassium channels (IK(Ca1)) in human PCa as well as their involvement in cell proliferation. We showed that IK(Ca1) mRNA and protein were preferentially expressed in human PCa tissues, and inhibition of the IK(Ca1) potassium channel suppressed PCa cell proliferation. The activation of IK(Ca1) hyperpolarizes membrane potential and, by promoting the driving force for calcium, induces calcium entry through TRPV6, a cation channel of the TRP (Transient Receptor Potential) family. Thus, the overexpression of the IK(Ca1) channel is likely to promote carcinogenesis in human prostate tissue.
Assuntos
Cálcio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/fisiologia , Neoplasias da Próstata/patologia , Benzimidazóis/farmacologia , Canais de Cálcio/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/análise , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27 , Fase G1 , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/análise , Peptídeos e Proteínas de Sinalização Intracelular/análise , Masculino , Potenciais da Membrana , Neoplasias da Próstata/metabolismo , RNA Mensageiro/análise , Proteínas S100/análise , Canais de Cátion TRPV/fisiologia , Proteína Supressora de Tumor p53/fisiologiaRESUMO
This study was designed to evaluate the occurrence of Bacillus cereus spores with a damaged exosporium and the consequences of such damages on spore adhesion. The analysis of nine strains sporulated under optimal conditions (Spo8-agar, 30 degrees C) revealed that damaged exosporia were systematically found in one strain (B. cereus D17) and occasionally in two others (B. cereus ATCC 14579T and B. cereus D6). The prevalence of spores with damaged exosporia increased when sporulation occurred under less favorable conditions (Spo8-broth or high temperature); for example, more than 50% of the B. cereus ATCC 14579T spores were damaged when sporulation occurred at 40 degrees C on Spo8-agar or at 30 degrees C in Spo8-broth. Furthermore, when subjected to shear stresses by circulation of spore suspensions through a peristaltic pump, the exosporium of a significant amount of spores became partially or totally shorn off (for example, 40% of the B. cereus ATCC 14579T spores). The ability of damaged spores to adhere to inert surfaces and to resist cleaning under shear stress was significantly affected when compared with intact spores, resulting in a decreased number of adhering spores (P < or = 0.004) and enhanced resistance to cleaning (P < or = 0.008). This study provides evidence that, under various conditions, the exosporium of B. cereus spores can be partly or wholly damaged, thereby affecting the ability of spores to contaminate the surfaces of food processing lines. The presence of spores devoid of exosporium will be of importance in determining the risk associated with B. cereus spores adherent to food processing line surfaces.
Assuntos
Bacillus cereus/fisiologia , Contaminação de Equipamentos , Contaminação de Alimentos/análise , Indústria de Processamento de Alimentos/normas , Medição de Risco , Bacillus cereus/ultraestrutura , Aderência Bacteriana , Contagem de Colônia Microbiana , Microbiologia de Alimentos , Esporos Bacterianos/fisiologia , Esporos Bacterianos/ultraestrutura , Temperatura , Fatores de TempoRESUMO
Cardiolipin (CL) is a mitochondria-specific phospholipid synthesized by CL synthase (CLS). We describe here a human gene for CLS and its analysis via RNAi knockdown on apoptotic progression. Although mitochondrial membrane potential is unchanged in cells containing only 25% of the normal amount of CL, free cytochrome c (cyt. c) is detected in the intermembrane space and the mitochondria exhibit signs of reorganized cristae. However, the release of cyt. c from the mitochondria still requires apoptotic stimulation. Increased sensitivity to apoptotic signals and accelerated rates of apoptosis are observed in CL-deficient cells, followed by elevated levels of secondary necrosis. Apoptosis is thought to progress via binding of truncated Bid (tBid) to mitochondrial CL, followed by CL oxidation which results in cyt. c release. The exaggerated and accelerated apoptosis observed in CL-deficient cells is matched by an accelerated reduction in membrane potential and increased cyt. c release, but not by decreased tBid binding. This study suggests that the CL/cyt. c relationship is important in apoptotic progression and that regulating CL oxidation or/and deacylation could represent a possible therapeutic target.
Assuntos
Cardiolipinas/metabolismo , Citocromos c/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Apoptose , Cardiolipinas/fisiologia , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Proteínas de Membrana/genética , Camundongos , Células NIH 3T3 , Interferência de RNA , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
TRPM8 (melastatine-related transient receptor potential member 8), a member of the transient receptor potential (TRP) superfamily of cation channels, has been shown to be a calcium-channel protein. TRPM8 mRNA has also been shown to be overexpressed in prostate cancer and is considered to play an important role in prostate physiology. This study was designed to determine the androgen-regulation mechanisms for TRPM8 mRNA expression and to identify the phenotype of TRPM8-expressing cells in the human prostate. Our findings show that trpm8 gene expression requires a functional androgen receptor. Furthermore, this article argues strongly in favour of the fact that the trpm8 gene is a primary androgen-responsive gene. Single-cell reverse transcriptase PCR and immunohistochemical experiments also showed that the trpm8 gene was mainly expressed in the apical secretory epithelial cells of the human prostate and trpm8 down-regulation occurred during the loss of the apical differentiated phenotype of the primary cultured human prostate epithelial cells. The androgen-regulated trpm8 expression mechanisms are important in understanding the progression of prostate cancer to androgen-independence. These findings may contribute to design a strategy to predict prostate cancer status from the TRPM8 mRNA level. Furthermore, as the TRPM8 channel is localized in human prostate cells, it will be interesting to understand its physiological function in the normal prostate and its potential role in prostate cancer development.
Assuntos
Regulação Neoplásica da Expressão Gênica , Canais Iônicos/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/fisiologia , 5-alfa-Di-Hidroprogesterona/metabolismo , 5-alfa-Di-Hidroprogesterona/farmacologia , Androgênios/metabolismo , Células Epiteliais/química , Células Epiteliais/metabolismo , Humanos , Canais Iônicos/metabolismo , Masculino , Miócitos de Músculo Liso/química , Miócitos de Músculo Liso/metabolismo , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas/genética , Próstata/citologia , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Elementos de Resposta , Canais de Cátion TRPM , Células Tumorais CultivadasRESUMO
Prostate smooth muscle cells predominantly express alpha1-adrenoceptors (alpha1-AR). alpha1-AR antagonists induce prostate smooth muscle relaxation and therefore they are useful therapeutic compounds for the treatment of benign prostatic hyperplasia symptoms. However, the Ca(2+) entry pathways associated with the activation of alpha1-AR in the prostate have yet to be elucidated. In many cell types, mammalian homologues of transient receptor potential (TRP) genes, first identified in Drosophila, encode TRPC (canonical TRP) proteins. They function as receptor-operated channels (ROCs) which are involved in various physiological processes such as contraction, proliferation, apoptosis, and differentiation. To date, the expression and function of TRPC channels have not been studied in prostate smooth muscle. In fura-2 loaded PS1 (a prostate smooth muscle cell line) which express endogenous alpha1A-ARs, alpha-agonists epinephrine (EPI), and phenylephrine (PHE) induced Ca(2+) influx which depended on the extracellular Ca(2+) and PLC activation but was independent of PKC activation. Thus, we have tested two membrane-permeable analogues of diacylglycerol (DAG), oleoyl-acyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG). They initiated Ca(2+) influx whose properties were similar to those induced by the alpha-agonists. Sensitivity to 2-aminoethyl diphenylborate (2-APB), SKF-96365 and flufenamate implies that Ca(2+)-permeable channels mediated both alpha-agonist- and OAG-evoked Ca(2+) influx. Following the sarcoplasmic reticulum (SR) Ca(2+) store depletion by thapsigargin (Tg), a SERCA inhibitor, OAG and PHE were both still able to activate Ca(2+) influx. However, OAG failed to enhance Ca(2+) influx when added in the presence of an alpha-agonist. RT-PCR and Western blotting performed on PS1 cells revealed the presence of mRNAs and the corresponding TRPC3 and TRPC6 proteins. Experiments using an antisense strategy showed that both alpha-agonist- and OAG-induced Ca(2+) influx required TRPC3 and TRPC6, whereas the Tg-activated ("capacitative") Ca(2+) entry involved only TRPC3 encoded protein. It may be thus concluded that PS1 cells express TRPC3 and TRPC6 proteins which function as receptor- and store-operated Ca(2+) entry pathways.
Assuntos
Cálcio/metabolismo , Canais Iônicos/metabolismo , Músculo Liso/citologia , Próstata/citologia , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Linhagem Celular , Diglicerídeos/farmacologia , Expressão Gênica , Receptores de Inositol 1,4,5-Trifosfato , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/genética , Masculino , RNA Mensageiro/análise , Ratos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Canais de Cátion TRPC , Canal de Cátion TRPC6RESUMO
Inhibitors of histone deacetylase (HDAC) are considered as potential anticancer agents. We have previously demonstrated that an inhibitor of HDAC, sodium butyrate (NaB), induces apoptosis of breast cancer cells in a P53-independent and P21(waf1)-dependent manner. In this study, we showed that tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), and anti-Fas agonist antibody potentiated NaB-induced growth inhibition through synergistic induction of apoptosis in breast cancer cell lines (MCF-7, T47-D, and BT-20). In MCF-7 cells, NaB increased the expression of death receptors; NaB alone or in combination with TNF-alpha, TRAIL, and anti-Fas agonist antibody increased the levels of Bid, tBid, and that of cytosolic cytochrome c. Synergistic induction of apoptosis was strongly inhibited by dominant-negative Fas-associated death domain (FADD) and inhibitors of caspases-8 and -9, indicating that potentiation of apoptosis involved key elements of death receptors' signaling pathways. Moreover, cotreatment of NaB and ligands of death receptors up-regulated the levels of P21(waf1) and that of proliferating cell nuclear antigen (PCNA) associated with P21(waf1). Transient transfections of p21(waf1) antisense or p21(waf1) deficient for its interaction with PCNA abolished synergistic induction of apoptosis. This suggested that potentiation of apoptosis by cotreatments required P21(waf1) and its interaction with PCNA. Since breast tumors contain rarely p21 mutations, our results may open interesting prospects in the fight against breast cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Carcinoma/tratamento farmacológico , Ciclinas/efeitos dos fármacos , Receptor fas/efeitos dos fármacos , Anticorpos/farmacologia , Anticorpos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Apoptose/genética , Proteínas Reguladoras de Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Butiratos/farmacologia , Butiratos/uso terapêutico , Carcinoma/genética , Carcinoma/metabolismo , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Ciclinas/metabolismo , Citocromos c/efeitos dos fármacos , Citocromos c/metabolismo , Sinergismo Farmacológico , Proteína de Domínio de Morte Associada a Fas , Feminino , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Humanos , Glicoproteínas de Membrana/farmacologia , Glicoproteínas de Membrana/uso terapêutico , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/uso terapêutico , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Receptor fas/imunologia , Receptor fas/metabolismoRESUMO
beta-1,2-Oligomannosides (beta-1,2-Man) derived from Candida albicans mannan have been shown to act as adhesins and to induce protective antibodies. We used monoclonal antibodies specific for beta-1,2-Man in electron, confocal, and fluorescence microscopy to study the surface expression of beta-1,2-Man epitopes. These monoclonal antibodies were also used for Western blotting of cell surface extracts to study the nature of the molecules expressing the beta-Man epitopes. Evidence was obtained for the contribution of a glycolipid, phospholipomannan (PLM), to the complex expression of beta-1,2-Man epitopes at the cell wall surfaces of yeasts grown on solid media. PLM was present in intercellular matrixes of colonies grown on agar and was detected as a contaminant in mannan batches prepared by conventional methods.
Assuntos
Candida albicans/metabolismo , Glicolipídeos/metabolismo , Oligossacarídeos/biossíntese , Membrana Celular/metabolismo , Parede Celular , Epitopos de Linfócito B/biossíntese , Epitopos de Linfócito B/imunologia , Mananas/isolamento & purificação , Mercaptoetanol , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Microscopia Imunoeletrônica/métodos , Oligossacarídeos/imunologiaRESUMO
Human mature erythrocytes have been considered as unable to undergo programmed cell death (PCD), due to their lack of mitochondria, nucleus and other organelles, and to the finding that they survive two conditions that induce PCD in vitro in all human nucleated cells, treatment with staurosporine and serum deprivation. Here we report that mature erythrocytes can undergo a rapid self-destruction process sharing several features with apoptosis, including cell shrinkage, plasma membrane microvesiculation, phosphatidylserine externalization, and leading to erythrocyte disintegration, or, in the presence of macrophages, to macrophage ingestion of dying erythrocytes. This regulated form of PCD was induced by Ca(2+) influx, and prevented by cysteine protease inhibitors that allowed erythrocyte survival in vitro and in vivo. The cysteine proteinases involved seem not to be caspases, since (i) proforms of caspase 3, while present in erythrocytes, were not activated during erythrocyte death; (ii) cytochrome c, a critical component of the apoptosome, was lacking; and (iii) cell-free assays did not detect activated effectors of nuclear apoptosis in dying erythrocytes. Our findings provide the first identification that a death program can operate in the absence of mitochondria. They indicate that mature erythrocytes share with all other mammalian cell types the capacity to self-destruct in response to environmental signals, and imply that erythrocyte survival may be modulated by therapeutic intervention.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Eritrócitos/fisiologia , Mitocôndrias/fisiologia , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Caspase 3 , Caspases/metabolismo , Caspases/farmacologia , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Eritrócitos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leupeptinas/metabolismo , Leupeptinas/farmacologia , Ativação de Macrófagos/imunologia , Camundongos , Modelos Biológicos , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologiaRESUMO
The present study demonstrates for the first time that intracellular calcium-ATPases and calcium pool content are closely associated with prostate cancer LNCaP cell growth. Cell growth was modulated by changing the amount of epidermal growth factor, serum, and androgene in culture media. Using the microspectrofluorimetric method with Fura-2 and Mag Fura-2 as probes, we show that in these cells, the growth rate is correlated with intracellular calcium pool content. Indeed, an increased growth rate is correlated with an increase in the calcium pool filling state, whereas growth-inhibited cells show a reduced calcium pool load. Using Western blotting and immunocytochemistry, we show that endoplasmic reticulum calcium pump expression is closely linked to LNCaP cell growth, and are a common target of physiological stimuli that control cell growth. Moreover, we clearly demonstrate that inhibition of these pumps, using thapsigargin, inhibits LNCaP cell growth and prevents growth factor from stimulating cell proliferation. Our results thus provide evidence for the essential role of functional endoplasmic reticulum calcium pumps and calcium pool in control of prostate cancer LNCaP cell growth, raising the prospect of new targets for the treatment of prostate cancer.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/enzimologia , Neoplasias da Próstata/metabolismo , Retículo Sarcoplasmático/enzimologia , Western Blotting , ATPases Transportadoras de Cálcio/biossíntese , Divisão Celular , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/antagonistas & inibidores , Corantes Fluorescentes/farmacologia , Fura-2/farmacologia , Humanos , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Masculino , Microscopia de Fluorescência , Microssomos/metabolismo , Ligação Proteica , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Espectrofotometria , Tapsigargina/farmacologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
We have previously shown that overexpressing cRel, a transcription factor of the Rel/NF-kappa B family, concomitantly inhibits proliferation of HeLa cells and makes them resistant against TNF alpha-induced apoptosis. Both effects rely on the upregulation of the manganese superoxide dismutase (MnSOD), a mitochondrial enzyme that converts O(2)(*-) in H(2)O(2). Here we describe additional alterations induced by cRel, namely mitochondrial clustering and accumulation of dense dark granules near the nucleus. These changes preferentially occur in cells that display a sustained cRel expression in the nucleus and that are cell-cycle arrested. As the cell-cycle arrest, these changes are reproduced by directly overexpressing MnSOD or by treating cells with H(2)O(2), suggesting they are due to MnSOD induction and ensuing H(2)O(2) accumulation. We propose that mitochondria cluster because they are damaged by the H(2)O(2) they overproduce. They would then be autophagocytosed and degraded in secondary lysosomes. In support of this scenario, we documented the occurrence of oxidative damage and the presence of lysosomes in the area of mitochondrial clustering. In addition, we identified the dense dark granules as lipofuscin, based on their autofluorescence. Lipofuscin could directly originate from the mitochondrial degradation products that would aggregate and become indigestible because of the presence of H(2)O(2) in the secondary lysosomes. Altogether, our findings show that cRel overexpression in HeLa cells creates, via the induction of MnSOD, an oxidative injury that culminates in mitochondrial degeneration, proliferation blockage, and resistance against TNF alpha-induced apoptosis.
Assuntos
Núcleo Celular/fisiologia , Células HeLa/citologia , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/fisiologia , Proteínas Proto-Oncogênicas c-rel/metabolismo , Superóxido Dismutase/metabolismo , Regulação para Cima/genética , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Células HeLa/metabolismo , Humanos , Lipofuscina/metabolismo , Oxirredução , Proteínas Proto-Oncogênicas c-rel/genética , Superóxido Dismutase/genética , Regulação para Cima/fisiologiaRESUMO
The malaria parasite, Plasmodium falciparum, synthesises and exports several proteins inducing morphological and biochemical modifications of erythrocytes during the erythrocytic cycle. The protein trafficking machinery of the parasite is similar to that of other eukaryotic cells in several ways. However, some unusual features are also observed. The secretion of various polypeptides was inhibited when P. falciparum-infected erythrocytes were incubated with Brefeldin A. Immunoelectron microscopy studies revealed substantial morphological changes in the endoplasmic reticulum following exposure of parasitised erythrocytes to the drug. Immunofluorescence studies of Brefeldin A-treated parasites suggest that polypeptide sorting to different intracellular destinations begins at the endoplasmic reticulum. The parasite also secretes polypeptides by a Brefeldin A-insensitive route that bypasses the classical endoplasmic reticulum-Golgi complex pathway.
Assuntos
Plasmodium falciparum/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Proteínas de Protozoários/metabolismo , Animais , Brefeldina A/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/fisiologia , Eritrócitos/parasitologia , Imunofluorescência , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/fisiologia , Humanos , Malária Falciparum/parasitologia , Microscopia Imunoeletrônica , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/fisiologia , Plasmodium falciparum/ultraestrutura , Sinais Direcionadores de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Proteínas de Protozoários/fisiologiaRESUMO
A putative glutathione peroxidase gene (Swiss-Prot accession number Z 68200) of Plasmodium falciparum, the causative agent of tropical malaria, was expressed in Escherichia coli and purified to electrophoretic homogeneity. Like phospholipid hydroperoxide glutathione peroxidase of mammals, it proved to be monomeric. It was active with H(2)O(2) and organic hydroperoxides but, unlike phospholipid hydroperoxide glutathione peroxidase, not with phosphatidylcholine hydroperoxide. With glutathione peroxidases it shares the ping-pong mechanism with infinite V(max) and K(m) when analyzed with GSH as substrate. As a homologue with selenocysteine replaced by cysteine, its reactions with hydroperoxides and GSH are 3 orders of magnitude slower than those of the selenoperoxidases. Unexpectedly, the plasmodial enzyme proved to react faster with thioredoxins than with GSH and most efficiently with thioredoxin of P. falciparum (Swiss-Prot accession number 202664). It is therefore reclassified as thioredoxin peroxidase. With plasmodial thioredoxin, the enzyme also displays ping-pong kinetics, yet with a limiting K(m) of 10 microm and a k(1)' of 0.55 s(-)1. The apparent k(1)' for oxidation with cumene, t-butyl, and hydrogen peroxides are 2.0 x 10(4) m(-1) s(-1), 3.3 x 10(3) m(-1) s(-1), and 2.5 x 10(3) m (-1) s(-1), respectively. k(2)' for reduction by autologous thioredoxin is 5.4 x 10(4) m(-1) s(-1) (21.2 m(-1) s(-1) for GSH). The newly discovered enzymatic function of the plasmodial gene product suggests a reconsideration of its presumed role in parasitic antioxidant defense.