Itraconazole (ITC), a well-known fungistatic agent, has potent fungicidal activity against Candida albicans. However, its mechanism of fungicidal activity has not been elucidated yet, and we aimed to identify the mechanism of ITC against C. albicans. ITC caused cell shrinkage via potassium leakage through the ion channel. Since shrunken cells could indicate apoptosis, we investigated apoptotic features. Annexin V-FITC and TUNEL assays indicated that fungicidal activity of ITC was involved in apoptosis. Subsequently, we confirmed an intracellular factor that could cause apoptosis. ITC treatment caused reactive oxygen species (ROS) accumulation. To confirm whether ROS is related with ITC-triggered cell death, cell viability was examined using the ROS scavenger N-acetylcysteine (NAC). NAC pretreatment recovered ITC-induced cell death, indicating that antifungal activity of ITC is associated with ROS, which is also confirmed by impaired glutathione-related antioxidant system and oxidized intracellular lipids. Moreover, ITC-induced mitochondrial dysfunction, in turn, triggered cytochrome c release and metacaspase activation, leading to apoptosis. Unlike the only ITC-treatment group, cells with NAC pretreatment did not show significant damage to mitochondria, and attenuated apoptotic features. Therefore, our results suggest that ITC induces apoptosis as fungicidal mechanism, and intracellular ROS is major factor to trigger the apoptosis by ITC in C. albicans.

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.

Phytol, isolated from Aster yomena, is widely distributed as a constituent of chlorophyll. In the present study, we confirmed the antibacterial activity of phytol and its mechanism inducing oxidative cell death in Pseudomonas aeruginosa. In phytol-treated cells, elevated level of intracellular reactive oxygen species (ROS) and transient NADH depletion were observed. These results demonstrated that phytol induced ROS accumulation and that the electron transport chain was involved in increase of ROS. Due to this ROS generation, the imbalance developed between intracellular ROS and the antioxidant defense system, leading to decrease of reduced glutathione (GSH). Moreover, severe DNA damage was shown after treatment with phytol. DNA electrophoresis and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were conducted with pretreatment with the antioxidant N-acetylcysteine (NAC) to evaluate the cause of DNA damage. In NAC-pretreated cells, alleviated damage was confirmed and it supports that phytol induces oxidative stress-mediated DNA damage. In conclusion, phytol exerts the antibacterial property via inducing oxidative stress response in P. aeruginosa.

No abstract

No abstract