Honokiol induces reactive oxygen species-mediated apoptosis in Candida albicans through mitochondrial dysfunction

PLoS One. 2017 Feb 13;12(2):e0172228. doi: 10.1371/journal.pone.0172228. eCollection 2017.

Abstract

Objective: To investigate the effects of honokiol on induction of reactive oxygen species (ROS), antioxidant defense systems, mitochondrial dysfunction, and apoptosis in Candida albicans.

Methods: To measure ROS accumulation, 2',7'-dichlorofluorescein diacetate fluorescence was used. Lipid peroxidation was assessed using both fluorescence staining and a thiobarbituric acid reactive substances (TBARS) assay. Protein oxidation was determined using dinitrophenylhydrazine derivatization. Antioxidant enzymatic activities were measured using commercially available detection kits. Superoxide dismutase (SOD) genes expression was measured using real time RT-PCR. To assess its antifungal abilities and effectiveness on ROS accumulation, honokiol and the SOD inhibitor N,N'-diethyldithiocarbamate (DDC) were used simultaneously. Mitochondrial dysfunction was assessed by measuring the mitochondrial membrane potential (mtΔψ). Honokiol-induced apoptosis was assessed using an Annexin V-FITC apoptosis detection kit.

Results: ROS, lipid peroxidation, and protein oxidation occurred in a dose-dependent manner in C. albicans after honokiol treatment. Honokiol caused an increase in antioxidant enzymatic activity. In addition, honokiol treatment induced SOD genes expression in C. albicans cells. Moreover, addition of DDC resulted in increased endogenous ROS levels and potentiated the antifungal activity of honokiol. Mitochondrial dysfunction was confirmed by measured changes to mtΔψ. The level of apoptosis increased in a dose-dependent manner after honokiol treatment.

Conclusions: Collectively, these results indicate that honokiol acts as a pro-oxidant in C. albicans. Furthermore, the SOD inhibitor DDC can be used to potentiate the activity of honokiol against C. albicans.

MeSH terms

  • Anti-Infective Agents / pharmacology
  • Apoptosis / drug effects*
  • Biphenyl Compounds / pharmacology*
  • Candida albicans / drug effects*
  • Candida albicans / genetics
  • Candida albicans / metabolism
  • Ditiocarb / pharmacology
  • Dose-Response Relationship, Drug
  • Drug Synergism
  • Gene Expression Regulation, Enzymologic / drug effects
  • Gene Expression Regulation, Fungal / drug effects
  • Lignans / pharmacology*
  • Lipid Peroxidation / drug effects
  • Membrane Potential, Mitochondrial / drug effects
  • Microbial Sensitivity Tests
  • Microscopy, Fluorescence
  • Mitochondria / drug effects*
  • Mitochondria / metabolism
  • Mitochondria / physiology
  • Oxidation-Reduction / drug effects
  • Reactive Oxygen Species / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Superoxide Dismutase / antagonists & inhibitors
  • Superoxide Dismutase / genetics
  • Superoxide Dismutase / metabolism

Substances

  • Anti-Infective Agents
  • Biphenyl Compounds
  • Lignans
  • Reactive Oxygen Species
  • honokiol
  • Ditiocarb
  • Superoxide Dismutase

Grant support

This work was supported by grants from the National Natural Science Foundation of China (NO 81302814) and Jiangsu Province Natural Science Foundation (NO BK20130640; BK20140624).