title: Mitochondria are a primary target of hypericin phototoxicity: synergy of intracellular calcium mobilisation in cell killing.
abstract: Hypericin, a naturally occurring anthraquinone synthesised by hypericum, upon light activation exhibits photodynamic cytotoxicity attributed mainly to the production of reactive oxygen species. This study aimed to elucidate the primary subcellular targets and mechanistic aspects of hypericin photosensitization in human prostate carcinoma cells. Depletion of intracellular glutathione (>85%) via inhibition of gamma-glutamyl-cysteine synthase had no effect on hypericin (5 microM) phototoxicity, thus precluding any direct oxidative involvement of H2O2. There was no change in intracellular SOD activity immediately after hypericin irradiation (1.5-5 J cm(-2)). Evaluation of the lysosomal enzyme hexosaminidase activity showed: (a) 60% cell loss 22 h following irradiation (1.5 J cm(-2)) and (b) a steady rate of lysosomal leakage to the cytosol (25%), at the same time and irradiation. However, lysosomal damage appears to be a slower process compared to the rapid loss of mitochondrial function, as reflected from parallel tetrazolium to formazan assays. The activity of cytosolic and mitochondrial aconitase, an enzyme exquisitely sensitive to oxidation, revealed a dose correlated loss of activity in the mitochondria immediately following hypericin photoactivation. The use of ionomycin, which modulates both internal Ca2+ stores and external Ca2+ transport during hypericin photosensitization, profoundly enhanced photocytotoxicity. Our data supports a direct mitochondrial hypericin phototoxicity that does not involve glutathione/H2O2 homeostasis. Further a potential synergistic treatment combining mitochondrial targeting of photosensitisers and Ca2+ mobilisation was identified.
authors.affiliation: Department of Medicine, The Rayne Institute, 5 University Street, University College London, London WC1E 6JJ, UK. t.theodossiou@ucl.ac.uk
authors.forename: Theodossis A
authors.initials: TA
authors.lastname: Theodossiou
authors.forename: Alberto
authors.initials: A
authors.lastname: Noronha-Dutra
authors.forename: John S
authors.initials: JS
authors.lastname: Hothersall
mesh_terms: D000154:Aconitate Hydratase / Q000378:metabolism; D000873:Anthracenes; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D019069:Cell Respiration / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008027:Light; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D010569:Perylene / Q000031:analogs & derivatives* / Q000494:pharmacology; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D000873:Anthracenes; D011838:Radiation-Sensitizing Agents; D017382:Reactive Oxygen Species; D010569:Perylene; C004965:hypericin; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D000154:Aconitate Hydratase; D005978:Glutathione; D002118:Calcium