A novel mechanism of fluconazole: fungicidal activity through dose-dependent apoptotic responses in Candida albicans

Abstract

Fluconazole (FLC) is a well-known fungistatic agent that inhibits ergosterol biosynthesis. We showed that FLC exhibits dose-dependent fungicidal activity, and investigated the fungicidal mechanism of FLC on Candida albicans. To confirm the relationship between fungicidal activity and the inhibition of ergosterol, we assessed membrane dysfunctions via propidium iodide influx and potassium leakage, as well as morphological change. Interestingly, while membrane disruption was not observed at all tested concentrations of FLC, potassium efflux and cell shrinkage were observed at high dosages of FLC (HDF). Low-dosage FLC (LDF) treatment did not induce significant changes. Next, we examined whether the fungicidal activity of FLC was associated with apoptosis in C. albicans. FLC caused dose-dependent apoptotic responses, including phosphatidylserine externalization and DNA fragmentation. It was also involved in glutathione depletion followed by oxidative damage. In particular, unlike LDF, HDF leads to the disruption of mitochondrial homeostasis, including mitochondrial membrane depolarization and accumulation of calcium and reactive oxygen species. HDF-induced mitochondrial dysfunction promoted the release of cytochrome c from mitochondria to the cytosol, and activated intracellular metacaspase. In conclusion, the dose-dependent fungicidal activity of FLC was due to an apoptotic response in C. albicans.

Synergistic Antifungal Activity of Isoquercitrin: Apoptosis and Membrane Permeabilization Related to Reactive Oxygen Species in Candida albicans

“…Changes in oxidative status by ROS accumulation can cause lipid peroxidation (33). Thus, this study confirmed the involvement of lipid peroxidation after treatment with ISO, AMB, and FLC individually and in combination.…”

“…On the basis of AMB property, we can speculate that ISO enters the cell more easily and generates more ROS through the pore formed by the AMB. In case of FLC, FLC is known to induce ROS accumulation in only high concentration (33). Based on our results, we speculate that ISO accelerates prooxidant activity of FLC followed by ROS accumulation.…”

Lactoferricin B like peptide triggers mitochondrial disruption‐mediated apoptosis by inhibiting respiration under nitric oxide accumulation inCandida albicans

“…Total glutathione levels were determined by incubating the supernatant with 0.01% dithionitrobenzoate for 15 min and reading sample absorbance at 415 nm with a microtiter ELISA reader. Total protein concentrations were determined using the Bradford method, and glutathione levels were evaluated after normalizing to total protein level 22 …”

“…After a 4 hr incubation at 28°C, the cells were stained with JC‐1 in a final concentration of 2.5 μg/ml with warm PBS for 20 min. The intensities of green fluorescence (FL1) and red fluorescence (FL2) were measured using a FACSVerse flow cytometer, and the ratio of FL2 to FL1 was calculated 22 …”

“…Fura‐2AM (Molecular Probes) at a final concentration of 5 μM and Rhod‐2AM (Molecular Probes) at a final concentration of 10 μM were used for the assessment of intracellular Ca 2+ and mitochondrial Ca 2+ , respectively. The fluorescence intensity of each sample, which incubated for 40 min (detection for intracellular Ca 2+ ) or 30 min (detection for mitochondrial Ca 2+ ), was analyzed using a spectrofluorophotometer (Shimadzu RF‐5301PC, Shimadzu, Kyoto, Japan) at the following settings: Ex/Em = 340/510 nm for Fura‐2AM or Ex/Em = 552/581 nm for Rhod‐2AM 22 …”