RESUMO
It was found in our previous study that berberine (BBR) and fluconazole (FLC) used concomitantly exhibited a synergism against FLC-resistant Candida albicans in vitro. The aim of the present study was to clarify how BBR and FLC worked synergistically and the underlying mechanism. Antifungal time-kill curves indicated that the synergistic effect of the two drugs was BBR dose dependent rather than FLC dose dependent. In addition, we found that BBR accumulated in C. albicans cells, especially in the nucleus, and resulted in cell cycle arrest and significant change in the transcription of cell cycle-related genes. Besides BBR, other DNA intercalators, including methylene blue, sanguinarine, and acridine orange, were all found to synergize with FLC against FLC-resistant C. albicans. Detection of intracellular BBR accumulation by fluorescence measurement showed that FLC played a role in increasing intracellular BBR concentration, probably due to its effect in disrupting the fungal cell membrane. Similar to the case with FLC, other antifungal agents acting on the cell membrane were able to synergize with BBR. Interestingly, we found that the efflux of intracellular BBR was FLC independent but strongly glucose dependent and associated with the drug efflux pump Cdr2p. These results suggest that BBR plays a major antifungal role in the synergism of FLC and BBR, while FLC plays a role in increasing the intracellular BBR concentration.
Assuntos
Antifúngicos/farmacologia , Berberina/farmacologia , Candida albicans/efeitos dos fármacos , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Laranja de Acridina/farmacologia , Benzofenantridinas/farmacologia , Transporte Biológico , Candida albicans/genética , Candida albicans/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Relação Dose-Resposta a Droga , Farmacorresistência Fúngica/genética , Sinergismo Farmacológico , Proteínas Fúngicas/metabolismo , Substâncias Intercalantes/farmacologia , Isoquinolinas/farmacologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Azul de Metileno/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
Candida albicans is the most common opportunistic fungal pathogen and its apoptosis is inducible by environmental stress. Based on our previous finding that transcription factor Cap1p was involved in baicalein-induced apoptosis, the present study aimed to further clarify the role of Cap1p in apoptosis by observing the impact of CAP1 deletion on cell fate. It was found that apoptotic stimulation with amphotericin B, acetic acid and hydrogen peroxide increased the number of apoptotic and necrotic cells, caspase activity and the accumulation of reactive oxygen species, whereas it decreased the mitochondrial membrane potential and intracellular ATP level in the cap1Δ/Δ mutant. The cell fate was, at least partly, caused by glutathione depletion and attenuation of the expression of the glutathione reductase gene in the cap1Δ/Δ mutant. Collectively, our data suggest that Cap1p participated in the apoptosis of C. albicans by regulating the expression of the glutathione reductase gene and glutathione content.
Assuntos
Apoptose/fisiologia , Candida albicans/citologia , Candida albicans/metabolismo , Proteínas de Ciclo Celular/fisiologia , Proteínas Fúngicas/fisiologia , Fatores de Transcrição/fisiologia , Anfotericina B/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Fatores de Transcrição de Zíper de Leucina Básica , Candida albicans/genética , Proteínas de Ciclo Celular/genética , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos , Glutationa/metabolismo , Glutationa Redutase/genética , Glutationa Redutase/metabolismoRESUMO
1. The aim of the present study was to investigate the effects of ascorbic acid (AA) on the antifungal activity of fluconazole (FCZ) in a systemic murine candidiasis model as well as in vitro. 2. The murine model was established by infusion of Candida albicans via the tail vein. Control mice received no further treatment. Other groups of mice were injected with FCZ (0.5 mg/kg, i.p.) and then treated or not with 50 or 500 mg/kg AA intragastrically (i.g.) or i.p. In all groups, FCZ was administered i.p. 2 h after fungal inoculation, whereas AA was administered 6 h after fungal inoculation. Survival rate, kidney fungal burden and renal pathological changes were evaluated. 3. The in vitro effects of AA (5, 1 and 0.2 mmol/L) on the growth of various Candida strains in the presence of FCZ (0.125-64 microg/mL) were also investigated. The in vitro effects of two anti-oxidants, namely N-acetylcysteine (NAC; 5, 1 and 0.2 mmol/L) and reduced glutathione (GSH; 5, 1 and 0.2 mmol/L), on FCZ activity were evaluated to determine the mechanism of action of AA. 4. Intragastric administration of AA (50 or 500 mg/kg) significantly decreased the antifungal effect of 0.5 mg/kg FCZ. Although i.p. administration of AA (50 or 500 mg/kg) had no significant effect on the survival of mice, it dose-dependently inhibited the activity of FCZ, with significant inhibition observed with 500 mg/kg AA. 5. In vitro, AA decreased the activity of FCZ against various Candida strains. Both NAC and GSH dose-dependently decreased the activity of FCZ. 6. The results of the present study indicate that AA inhibits the antifungal activity of FCZ, suggesting that the two should not be used together clinically for the treatment of candidiasis.
