Liposomal temocene (m-THPPo) photodynamic treatment induces cell death by mitochondria-independent apoptosis |
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Authors: | Jorge Soriano María García-Díaz Margarita Mora Maria Lluïsa Sagristá Santi Nonell Angeles Villanueva Juan Carlos Stockert Magdalena Cañete |
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Institution: | 1. Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Darwin 2, 28049 Cantoblanco, Madrid, Spain;2. Grup d''Enginyeria Molecular, IQS School of Engineering, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain;3. Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 643, 08028 Barcelona, Spain |
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Abstract: | BackgroundThe 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.MethodsLiposomes 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.Resultsm-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.ConclusionsPhotodynamic 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.General significanceThese results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment. |
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Keywords: | DMPG dimyristoylphosphatidylglycerol DMSO dimethylsulfoxide DPPC dipalmitoylphosphatidylcholine Hoechst-33258 H-33258 m-THPPo m-tetra (hydroxyphenyl) porphycene (temocene) MLVs multilamellar vesicles PDT photodynamic therapy PI propidium iodide PS photosensitizer ROS reactive oxygen species TB toluidine blue THF tetrahydrofuran PCS photon correlation spectroscopy PBS phosphate buffered saline |
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