title: Retigeric acid B exerts antifungal effect through enhanced reactive oxygen species and decreased cAMP.
abstract: BACKGROUND
  Retigeric acid B (RAB), a triterpene acid isolated from Lobaria kurokawae exerts antifungal effect. The present study was designed to elucidate the underlying mechanisms by which RAB regulates the proliferation and cell death of Candida albicans.
  
  
  METHODS
  We measured the metabolic activity of C. albicans with WST1 Cell Proliferation and Cytotoxicity Assay Kit, analyzed the cell cycle by flow cytometry, visualized the ultrastructure by transmission electron microscopy (TEM) and investigated the apoptosis and necrosis induced by RAB using confocal microscopy. The reactive oxygen species (ROS) accumulation was determined by spectrophotometry, flow cytometry and fluorescent microscopy. The mtΔψ was detected using flow cytometry. And the levels of intracellular cAMP and ATP were measured with cAMP ELISA and ATP Assay Kits, respectively.
  
  
  RESULTS
  The proliferation of the yeasts was blocked in G(2)/M phase by a low dose of RAB treatment and in G(1) phase at high concentration. When cultured in phosphate buffered saline (PBS) deprived of energy source, yeasts displayed the phenotype of death caused by accumulated ROS, mtΔψ hyperpolarization and dramatic decrease in ATP level in the presence of high dose of RAB.
  
  
  CONCLUSIONS
  RAB inhibits the growth of C. albicans by stimulating ROS production and reducing intracellular cAMP. The ROS accumulation, mtΔψ hyperpolarization, ATP depletion and damaged plasma membrane integrity together mediate cell death of C. albicans induced by RAB. Our findings provide a novel molecular mechanism for exploring possible applications of lichen derived metabolites in fighting fungal infection in humans.
authors.affiliation: Department of Natural Product Chemistry, Shandong University, Jinan, People's Republic of China.
authors.forename: Wen-Qiang
authors.initials: WQ
authors.lastname: Chang
authors.forename: Xiu-Zhen
authors.initials: XZ
authors.lastname: Wu
authors.forename: Ai-Xia
authors.initials: AX
authors.lastname: Cheng
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Mei
authors.initials: M
authors.lastname: Ji
authors.forename: Hong-Xiang
authors.initials: HX
authors.lastname: Lou
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001203:Ascomycota / Q000737:chemistry; D003994:Bucladesine / Q000494:pharmacology; D002176:Candida albicans / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D002453:Cell Cycle / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D015394:Molecular Structure; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014315:Triterpenes / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000935:Antifungal Agents; D017382:Reactive Oxygen Species; D014315:Triterpenes; C540615:retigeric acid B; D003994:Bucladesine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP