Proceedings Article | 25 February 2008
KEYWORDS: Photodynamic therapy, Luminescence, Cell death, Iron, Cancer, Confocal microscopy, Oxygen, Proteins, Injuries, Calcium
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
