title: Liposomal temocene (m-THPPo) photodynamic treatment induces cell death by mitochondria-independent apoptosis.
abstract: BACKGROUND
  The cell death pathway activated after photodynamic therapy (PDT) is controlled by a variety of parameters including the chemical structure of the photosensitizer, its subcellular localization, and the photodynamic damage induced. The present study aims to characterize a suitable m-THPPo liposomal formulation, to determine its subcellular localization in HeLa cells and to establish the cell death mechanisms that are activated after photodynamic treatments.
  
  
  METHODS
  Liposomes containing m-THPPo were prepared from a mixture of DPPC and DMPG at a 9:1 molar ratio. In order to procure the best encapsulation efficiency, the m-THPPo/lipid molar ratio was considered. HeLa cells were incubated with liposomal m-THPPo and the subcellular localization of m-THPPo was studied. Several assays such as TUNEL, annexin V/propidium iodide and Hoechst-33258 staining were performed after photodynamic treatments. The apoptotic initiation was assessed by cytochrome c and caspase-2 immunofluorescence.
  
  
  RESULTS
  m-THPPo encapsulated in liposomes showed a decrease of the fluorescence and singlet oxygen quantum yields, compared to those of m-THPPo dissolved in tetrahydrofuran. Liposomal m-THPPo showed colocalization with LysoTracker® and it induced photoinactivation of HeLa cells by an apoptotic mechanism. In apoptotic cells no relocalization of cytochrome c could be detected, but caspase-2 was positive immediately after photosensitizing treatments.
  
  
  CONCLUSIONS
  Photodynamic treatment with liposomal m-THPPo leads to a significant percentage of apoptotic morphology of HeLa cells. The activation of caspase-2, without the relocalization of cytochrome c, indicates a mitochondrial-independent apoptotic mechanism.
  
  
  CONCLUSIONS
  These results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment.
authors.affiliation: Departamento de Biología, Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Jorge
authors.initials: J
authors.lastname: Soriano
authors.forename: María
authors.initials: M
authors.lastname: García-Díaz
authors.forename: Margarita
authors.initials: M
authors.lastname: Mora
authors.forename: Maria Lluïsa
authors.initials: ML
authors.lastname: Sagristá
authors.forename: Santi
authors.initials: S
authors.lastname: Nonell
authors.forename: Angeles
authors.initials: A
authors.lastname: Villanueva
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Stockert
authors.forename: Magdalena
authors.initials: M
authors.lastname: Cañete
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D053143:Caspase 2 / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D020287:In Situ Nick-End Labeling; D008081:Liposomes; D008928:Mitochondria / Q000187:drug effects*; D010778:Photochemotherapy; D011166:Porphyrins / Q000008:administration & dosage / Q000494:pharmacology*
chemical_list: D017304:Annexin A5; D008081:Liposomes; D011166:Porphyrins; C577851:temocene; D045304:Cytochromes c; D053143:Caspase 2
keywords: Apoptosis; DMPG; DMSO; DPPC; H-33258; Hoechst-33258; Liposome; MLVs; PBS; PCS; PDT; PI; PS; Photosensitizer; ROS; TB; THF; Temocene; dimethylsulfoxide; dimyristoylphosphatidylglycerol; dipalmitoylphosphatidylcholine; m-THPPo; m-tetra (hydroxyphenyl) porphycene (temocene); multilamellar vesicles; phosphate buffered saline; photodynamic therapy; photon correlation spectroscopy; photosensitizer; propidium iodide; reactive oxygen species; tetrahydrofuran; toluidine blue