25 February 2008 Contribution of mitochondria and lysosomes to photodynamic therapy-induced death in cancer cells
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Abstract
In photodynamic therapy (PDT), visible light activates a photosensitizing drug added to a tissue, resulting in singlet oxygen formation and cell death. Employing confocal microscopy, we previously found that the phthalocyanine Pc 4 localized primarily to mitochondrial membranes in various cancer cell lines, resulting in mitochondrial reactive oxygen species (ROS) production, followed by inner membrane permeabilization (mitochondrial permeability transition) with mitochondrial depolarization and swelling, which in turn led to cytochrome c release and apoptotic death. Recently, derivatives of Pc 4 with OH groups added to one of the axial ligands were synthesized. These derivatives appeared to be taken up more avidly by cells and caused more cytotoxicity than the parent compound Pc 4. Using organelle-specific fluorophores, we found that one of these derivatives, Pc 181, accumulated into lysosomes and that PDT with Pc 181 caused rapid disintegration of lysosomes. We hypothesized that chelatable iron released from lysosomes during PDT contributes to mitochondrial damage and subsequent cell death. We monitored cytosolic Fe2+ concentrations after PDT with calcein. Fe2+ binds to calcein causing quenching of calcein fluorescence. After bafilomycin, an inhibitor of the vacuolar proton-translocating ATPase, calcein fluorescence became quenched, an effect prevented by starch desferal s-DFO, an iron chelator that enters cells by endocytosis. After Pc 181-PDT, cytosolic calcein fluorescence also decreased, indicating increased chelatable Fe2+ in the cytosol, and apoptosis occurred. s-DFO decreased Pc 181-PDT-induced apoptosis as measured by a decrease of caspase-3 activation. In isolated mitochondria preparations, Fe2+ induced mitochondrial swelling, which was prevented by Ru360, an inhibitor of the mitochondrial Ca2+ uniporter. The data support a hypothesis of oxidative injury in which Pc 181-PDT disintegrates lysosomes and releases constituents that synergistically promote mitochondrial permeabilization and apoptotic signaling. One important constituent seems to be Fe2+ that is taken up by mitochondria through the Ca2+ uniporter to promote mitochondrial ROS-dependent chain reactions. Lysosomal proteases may also directly promote apoptotic signaling, e.g., through cleavage/activation of the pro-apoptotic protein Bid.ÿÿÿ
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Anna-Liisa Nieminen, Kashif Azizuddin, Ping Zhang, Malcolm E. Kenney, Peter Pediaditakis, John J. Lemasters, Nancy L. Oleinick, "Contribution of mitochondria and lysosomes to photodynamic therapy-induced death in cancer cells", Proc. SPIE 6845, Optical Methods for Tumor Treatment and Detection: Mechanisms and Techniques in Photodynamic Therapy XVII, 684506 (25 February 2008); doi: 10.1117/12.767356; https://doi.org/10.1117/12.767356
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