RESUMO
Coprisin is a 43-mer defensin-like peptide from the dung beetle, Copris tripartitus. In this study, we tested its minimum inhibitory concentration and performed combination assays to confirm the antibacterial susceptibility of coprisin and synergistic effects with antibiotics. The synergistic effects were evaluated by testing the effects of coprisin in combination with ampicillin, vancomycin, and chloramphenicol. The results showed that coprisin possessed antibacterial properties and had synergistic activities with the antibiotics. To understand the synergistic mechanism(s), we conducted hydroxyl radical assays. Coprisin alone and in combination with antibiotics generated hydroxyl radicals, which are highly reactive oxygen forms and the major property of bactericidal agents. Furthermore, the antibiofilm effect of coprisin alone and in combination with antibiotics was investigated. Biofilm formation is the source of many relentless and chronic bacterial infections. The results indicated that coprisin alone and in combination with antibiotics also had antibiofilm activity. Therefore, we conclude that coprisin has the potential to be used as a combinatorial therapeutic agent for the treatment of infectious diseases caused by bacteria.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Sinergismo Farmacológico , Proteínas de Insetos/farmacologia , Bactérias/química , Fenômenos Fisiológicos Bacterianos , Radical Hidroxila/análise , Testes de Sensibilidade MicrobianaRESUMO
Amentoflavone was isolated from an ethyl acetate extract of the whole plant of Selaginella tamariscina. It is a traditional herb for the therapy of chronic trachitis and exhibits some anti-tumor activity. Previously, we confirmed the antifungal effects of amentoflavone. The objective of this study was to investigate the antifungal mechanism(s) of amentoflavone, such as mitochondria-mediated apoptotic cell death. The cells that were treated with amentoflavone exhibited a series of cellular changes that were consistent with apoptosis: externalization of phosphatidylserine, DNA and nuclear fragmentation, accumulation of intracellular reactive oxygen species (ROS) and hydroxyl radicals, and activation of metacaspase. In addition, diagnostic markers of apoptosis, including the reduction of mitochondrial inner-membrane potential and the release of cytochrome c from mitochondria, were observed. These phenomena are important changes in mitochondria-mediated apoptosis. Furthermore, the effect of thiourea as hydroxyl radical scavenger on amentoflavone-induced apoptosis was evaluated. A hydroxyl radical is a more active ROS species. Mitochondrial dysfunction was inhibited, which was indicated by decreased levels of intracellular hydroxyl radicals. Taken together, our results present the first evidence that amentoflavone induces apoptosis in C. albicans cells and is associated with the mitochondrial dysfunction. Besides, amentoflavone-induced hydroxyl radicals may play a significant role in mitochondria-mediated apoptosis.
Assuntos
Antifúngicos/farmacologia , Apoptose/efeitos dos fármacos , Biflavonoides/farmacologia , Candida albicans/citologia , Candida albicans/efeitos dos fármacos , Extratos Vegetais/farmacologia , Selaginellaceae/química , Candida albicans/metabolismo , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Lariciresinol is an enterolignan precursor isolated from the herb Sambucus williamsii, a folk medicinal plant used for its therapeutic properties. In this study, the antifungal properties and mode of action of lariciresinol were investigated. Lariciresinol displays potent antifungal properties against several human pathogenic fungal strains without hemolytic effects on human erythrocytes. To understand the antifungal mechanism of action of lariciresinol, the membrane interactions of lariciresinol were examined. Fluorescence analysis using the membrane probe 3,3'-diethylthio-dicarbocyanine iodide (DiSC(3)-5) and 1,6-diphenyl-1,3,5-hexatriene (DPH), as well as a flow cytometric analysis with propidium iodide (PI), a membrane-impermeable dye, indicated that lariciresinol was associated with lipid bilayers and induced membrane permeabilization. Therefore, the present study suggests that lariciresinol possesses fungicidal activities by disrupting the fungal plasma membrane and therapeutic potential as a novel antifungal agent for the treatment of fungal infectious diseases in humans.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Furanos/farmacologia , Lignanas/farmacologia , Sambucus/química , Antifúngicos/química , Antifúngicos/isolamento & purificação , Benzotiazóis/química , Carbocianinas/química , Células Cultivadas , Difenilexatrieno/química , Eritrócitos/efeitos dos fármacos , Citometria de Fluxo , Corantes Fluorescentes/química , Furanos/química , Furanos/isolamento & purificação , Hemólise , Hemolíticos/química , Hemolíticos/isolamento & purificação , Hemolíticos/farmacologia , Humanos , Lignanas/química , Lignanas/isolamento & purificação , Propídio/químicaRESUMO
Cruciferous vegetables contain glucobrassicin which, during metabolism, yields indole-3-carbinol (I3C). The aim of this study was to find whether indole-3-carbinol caused apoptosis and its mechanism in Candida albicans. We found that treatment of Candida albicans with indole-3-carbinol significantly increased the reactive oxygen species and hydroxyl radical accumulation. The hydroxyl radical is one of the most active components of oxygen, and it is the end product of an oxidative damage cellular death pathway. We investigated the general phenotypes of apoptosis and then investigated whether there were other distinct markers of apoptosis. Furthermore, the effects of thiourea as a hydroxyl radical scavenger and protective effect of trehalose, which is the result of the fungal immune system, was also assured. This study indicates that indole-3-carbinol has apoptosis effects, including a production of hydroxyl radicals, cytochrome c release and activation of metacaspase. Both hydroxyl radicals and metacaspases triggered apoptosis in Candida albicans.
Assuntos
Antifúngicos/farmacologia , Apoptose/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Indóis/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Anexina A5/metabolismo , Antifúngicos/química , Biomarcadores/metabolismo , Candida albicans/citologia , Candida albicans/metabolismo , Citocromos c/análise , Avaliação Pré-Clínica de Medicamentos , Radicais Livres/análise , Glucosinolatos/química , Marcação In Situ das Extremidades Cortadas , Indóis/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Terapia de Alvo Molecular , Espécies Reativas de Oxigênio/análise , Tioureia/farmacologiaRESUMO
In this study, antibacterial effects of (+)-Medioresinol isolated from stem bark of Sambucus williamsii and its synergistic activities in combination with antibiotics such as ampicillin, cefotaxime, and chloramphenicol were tested by antibacterial susceptibility testing and checkerboard assay. (+)-Medioresinol possessed antibacterial effects against antibiotics-susceptible- or antibiotics-resistant strains. Most of combinations between (+)-Medioresinol and each antibiotic showed synergistic interaction (fractional inhibitory concentration index ≤ 0.5) against bacterial strains including antibiotics-resistant Pseudomonas aeruginosa. Furthermore, the antibiofilm effect of (+)-Medioresinol alone or in combination with each antibiotic was investigated. The results indicated that not only (+)-Medioresinol but also its combination with each antibiotic had antibiofilm activities. It concludes that (+)-Medioresinol has potential as a therapeutic agent and adjuvant for treatment of bacterial infection.
Assuntos
Antibacterianos/administração & dosagem , Biofilmes/crescimento & desenvolvimento , Lignanas/administração & dosagem , Lignanas/isolamento & purificação , Pseudomonas aeruginosa/fisiologia , Sambucus/química , Biofilmes/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Pseudomonas aeruginosa/efeitos dos fármacosRESUMO
Silver nanoparticles have been shown to be detrimental to fungal cells although the mechanism(s) of action have not been clearly established. In this study, we used Candida albicans cells to show that silver nanoparticles exert their antifungal effect through apoptosis. Many studies have shown that the accumulation of reactive oxygen species induces and regulates the induction of apoptosis. Furthermore, hydroxyl radicals are considered an important component of cell death. Therefore, we assumed that hydroxyl radicals were related to apoptosis and the effect of thiourea as a hydroxyl radical scavenger was investigated. We measured the production of reactive oxygen species and investigated whether silver nanoparticles induced the accumulation of hydroxyl radicals. A reduction in the mitochondrial membrane potential shown by flow cytometry analysis and the release of cytochrome c from mitochondria were also verified. In addition, the apoptotic effects of silver nanoparticles were detected by fluorescence microscopy using other confirmed diagnostic markers of yeast apoptosis including phosphatidylserine externalization, DNA and nuclear fragmentation, and the activation of metacaspases. Cells exposed to silver nanoparticles showed increased reactive oxygen species and hydroxyl radical production. All other phenomena of mitochondrial dysfunction and apoptotic features also appeared. The results indicate that silver nanoparticles possess antifungal effects with apoptotic features and we suggest that the hydroxyl radicals generated by silver nanoparticles have a significant role in mitochondrial dysfunctional apoptosis.
Assuntos
Antifúngicos/farmacologia , Apoptose/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Radical Hidroxila/farmacologia , Nanopartículas Metálicas/química , Prata/farmacologia , Antifúngicos/química , Candida albicans/citologia , Candida albicans/metabolismo , Caspases/metabolismo , Citocromos c/metabolismo , Dano ao DNA , Ativação Enzimática/efeitos dos fármacos , Radical Hidroxila/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Prata/químicaRESUMO
Silver nanoparticles (nano-Ags), which have well-known antimicrobial properties, are used extensively in various medical and general applications. In this study, the combination effects between nano-Ags and the conventional antibiotics ampicillin, chloramphenicol and kanamycin against various pathogenic bacteria were investigated. The MIC and fractional inhibitory concentration index (FICI) were determined to confirm antibacterial susceptibility and synergistic effects. The results showed that nano-Ags possessed antibacterial effects and synergistic activities. The antibiofilm activities of nano-Ags alone or in combination with antibiotics were also investigated. Formation of biofilm is associated with resistance to antimicrobial agents and chronic bacterial infections. The results indicated that nano-Ags also had antibiofilm activities. To understand these effects of nano-Ags, an ATPase inhibitor assay, permeability assay and hydroxyl radical assay were conducted. The antibacterial activity of nano-Ags was influenced by ATP-associated metabolism rather than by the permeability of the outer membrane. Additionally, nano-Ags generated hydroxyl radicals, a highly reactive oxygen species induced by bactericidal agents. It was concluded that nano-Ags have potential as a combination therapeutic agent for the treatment of infectious diseases by bacteria.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Nanopartículas Metálicas , Prata/farmacologia , Sinergismo Farmacológico , Enterococcus faecium/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Streptococcus mutans/efeitos dos fármacosRESUMO
Antimicrobial peptides (AMPs) exert antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and viruses by various mechanisms. AMPs commonly possess particular characteristics by harboring cationic and amphipathic structures and binding to cell membranes, resulting in the leakage of essential cell contents by forming pores or disturbing lipid organization. These membrane disruptive mechanisms of AMPs are possible to explain according to the various structure forming pores in the membrane. Some AMPs inhibit DNA and/or RNA synthesis as well as apoptosis induction by reactive oxygen species (ROS) accumulation and mitochondrial dysfunction. Specifically, mitochondria play a major role in the apoptotic pathway. During apoptosis induced by AMPs, cells undergo cytochrome c release, caspase activation, phosphatidylserine externalization, plasma or mitochondrial membrane depolarization, DNA and nuclei damage, cell shrinkage, apoptotic body formation, and membrane blebbing. Even AMPs, which have been reported to exert membrane-active mechanisms, induce apoptosis in yeast. These phenomena were also discovered in tumor cells treated with AMPs. The apoptosis mechanism of AMPs is available for various therapeutics such as antibiotics for antibiotic-resistant pathogens that resist to the membrane active mechanism, and antitumor agents with selectivity to tumor cells.
Assuntos
Anti-Infecciosos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias/fisiopatologia , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Infecções Bacterianas/fisiopatologia , Humanos , Neoplasias/tratamento farmacológicoRESUMO
Coprisin is a 43-mer defensin-like peptide from the dung beetle, Copris tripartitus. Here, we investigated the induction of apoptosis by coprisin in Candida albicans cells. Coprisin exerted antifungal and fungicidal activity without any hemolytic effect. Confocal microscopy indicated that coprisin accumulated in the nucleus of cells. The membrane studies, 1,6-diphenyl-1,3,5-hexatriene, calcein-leakage, and giant unilamellar vesicle assays, confirmed that coprisin did not disrupt the fungal plasma membrane at all. Moreover, the activity of coprisin was energy- and salt-dependent. Next, we investigated whether coprisin induced apoptosis in C. albicans. Annexin V-FITC staining and TUNEL assay showed that coprisin was involved with both the early and the late stages of apoptosis. Coprisin also increased the intracellular reactive oxygen species level, and hydroxyl radicals were included at high levels among the species. The effect of thiourea as a hydroxyl radical scavenger further confirmed the existence of the hydroxyl radicals. Furthermore, coprisin induced mitochondrial membrane potential dysfunction, cytochrome c release, and activation of metacaspases. In summary, this study suggests that coprisin could be a model molecule for a large family of novel antimicrobial peptides possessing apoptotic activity.
Assuntos
Antifúngicos/farmacologia , Candida albicans/fisiologia , Proteínas de Insetos/farmacologia , Antifúngicos/síntese química , Apoptose/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Células Cultivadas , Defensinas/imunologia , Metabolismo Energético/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Imunidade , Proteínas de Insetos/síntese química , Tioureia/farmacologiaRESUMO
The phytochemical (+)-Medioresinol, a furofuran type lignan identification and isolation on the stem bark of Sambucus williamsii, which is a folk medicinal plant used in traditional medicine. (+)-Medioresinol is known to possess a lesishmanicidal activity and cardiovascular disease risk reduction but its antifungal effects have not yet been identified. In this study, to confirm (+)-Medioresinol's antifungal properties and mode of action, we observed morphological and physiological change in Candida albicans. In cells exposed to (+)-Medioresinol, arrested the cell cycle and intracellular reactive oxygen species (ROS) which is a major cause of apoptosis were increased. The increase of ROS induced oxidative stress and the mitochondria dysfunction which causes release of pro-apoptotic factors. We investigated a series of characteristic cellular changes of apoptosis by using various apoptosis detection methods. We report here for the first time that (+)-Medioresinol has effects on mitochondria and induced the accumulation of ROS in C. albicans cells. We demonstrated that one of the important features of apoptosis, mitochondrial membrane depolarization is caused by ROS. Substantially, we investigated the release of cytochrome c, which is one of the factors of metacaspase activity. We also show that the effects of (+)-Medioresinol are mediated at an early stage in apoptosis acting on the plasma membrane phosphatidylserine externalization. In addition, (+)-Medioresinol induced apoptotic morphological changes, showing the reduced cell size (low FSC) and enhanced intracellular density (high SSC). In late stage of confirmation of diagnostic markers in yeast apoptosis include the effects of nucleus morphological change, DNA fragmentation and condensation by influence of oxidative stress. These apoptotic phenomena represent that oxidative stress and mitochondria dysfunctions by inducing the phytochemical (+)-Medioresinol must be an important factors of the apoptotic process in C. albicans. These results support the elucidation of the underlying antifungal mechanisms of (+)-Medioresinol.