RESUMO
Essential oils and their main components, monoterpenes, have been proven to be important alternatives for the control of pathogenic and spoiling microorganisms, but the mode of action of these compounds is poorly understood. This work aimed to determine the mode of action of citral and geraniol on the model yeast Saccharomyces cerevisiae using a flow cytometry approach. Exponentially growing yeast cells were treated with different concentrations of citral and geraniol for 3 h, and evaluated for cell wall susceptibility to glucanase, membrane integrity, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential, and metacaspase activity. Results provide strong evidence that citral and geraniol acute fungicidal activity against Saccharomyces cells involves the loss of membrane and cell wall integrity resulting in a dose-dependent apoptotic/necrotic cell death. However, yeast cells that escape this first cell membrane disruption, particularly evident on sub-lethal concentration, die by metacaspase-mediated apoptosis induced by the accumulation of intracellular ROS. The deleted mutant on the yca1 gene showed high tolerance to citral and geraniol.
Assuntos
Monoterpenos Acíclicos/farmacologia , Caspases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Apoptose , Membrana Celular/metabolismo , Farmacorresistência Fúngica , Citometria de Fluxo , Deleção de Genes , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genéticaRESUMO
Dithianon is a broad-spectrum anthraquinone fungicide used to control several diseases of grapes, apples, and other fruits and vegetables. Its mode of action is described as multi-site and associated to thiol-reactivity. As other fungicides can affect non-phytopathogenic organisms as yeasts and bacteria, with impact on microbial population, diversity, and fermentation processes. In this context, we study the effect of dithianon on the model organism and fermentative yeast Saccharomyces cerevisiae in order to elucidate the mechanisms involved in yeast cell death., and explain its interference on wine fermentation kinetics. Thus for, we analyzed cellular protein and non-protein thiols, membrane and cell wall integrity, reactive oxygen species accumulation, mitochondrial membrane potential, and phosphatidylserine externalization. The results showed that when exponentially aerobic growing cells of S. cerevisiae are submitted to acute dithianon treatment they loss cell wall and membrane integrity, dying by necrosis, and this behavior is associated to a depletion of reduced proteic and non-proteic thiol groups. We also detected an important increase of cellular reactive oxygen species (ROS) associated to mitochondrial membrane potential modifications on dithianon treated cells. ROS accumulation was not associated to apoptotic cell death, but can be responsible for intracellular damages. Moreover, necrotic cell death induced by dithianon explains its effect on the kinetics of wine fermentations.
Assuntos
Antraquinonas/farmacologia , Morte Celular/efeitos dos fármacos , Fungicidas Industriais/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fermentação , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Necrose , Fosfatidilserinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Compostos de Sulfidrila/metabolismo , Vitis/metabolismo , VinhoRESUMO
Cumulative evidence from research studies has shown that the shiitake culinary-medicinal mushroom, Lentinus edodes, is an excellent source of natural antitumor agents and is capable of inhibiting cancer cell growth. However, the cell signaling pathway that leads tumor cells to apoptosis is not well understood because many chemical compounds may be acting. This study investigated the chemopreventive effects of an L. edodes aqueous extract on human HEp-2 epithelial larynx carcinoma cells and normal human MRC-5 lung fibroblasts by identifying proliferative and apoptotic pathways. The chemical characterization of the dry powder was assessed by high-performance liquid chromatography. Antiproliferative and proapoptotic effects induced by the extract were evaluated by assessing proliferative markers, cell sorting through flow cytometry, and expression levels of apoptotic proteins with Western blotting. The results suggest that inhibition of cell proliferation was more prominent in HEp-2 than in MRC-5 cells. Cell death analysis showed the appearance of cell populations in the sub-G1 phase, with late apoptotic signal increased in a dose-dependent manner. In addition, the aqueous extract induced depolarization of mitochondria, activating the generation of intracellular reactive oxygen species in HEp-2 cells. These observations suggest that L. edodes extract may exert a chemopreventive effect, regulating mitotic induction of apoptogenic signals. These findings highlight the mushroom's pharmacological potential in cancer treatment.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cogumelos Shiitake/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ácido Gálico/análise , Humanos , Concentração Inibidora 50 , Laringe/citologia , Laringe/patologia , Lentinano/farmacologia , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitose/efeitos dos fármacos , Fenóis/análise , Fenóis/química , Espécies Reativas de Oxigênio/metabolismo , Água/químicaRESUMO
Captan is one of the most widely used broad-spectrum fungicide applied to control several early and late diseases of grapes, apples, and other fruits and vegetables, and as other phthalimide fungicides is defined as a multisite compound with thiol-reactivity. Captan can affect non-target organisms as yeasts, modifying microbial populations and fermentation processes. In this study, we asked whether Captan thiol-reactivity and other mechanisms are involved in acute Captan-induced cell death on aerobic growing Saccharomyces cerevisiae. Thus for, we analyze cellular protein and non-protein thiols, cell membrane integrity, reactive oxygen species accumulation, phosphatidylserine externalization, and apoptotic mutants behavior. The results showed that when submitted to acute Captan treatment most cells lost their membrane integrity and died by necrosis due to Captan reaction with thiols. However, part of the cells, even maintaining their membrane integrity, lost their culture ability. These cells showed an apoptotic behavior that may be the result of non-protein thiol depletion and consequent increase of reactive oxygen species (ROS). ROS accumulation triggers a metacaspase-dependent apoptotic cascade, as shown by the higher viability of the yca1-deleted mutant. Together, necrosis and apoptosis are responsible for the high mortality detected after acute Captan treatment of aerobically growing cells of S. cerevisiae.