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Photodynamic Therapy (PDT) generates singlet oxygen (1O2) which oxidizes biomolecules in the immediate vicinity of
its formation. The phthalocyanine photosensitizer Pc 4 localizes to mitochondria and endoplasmic reticulum, and the
primary targets of Pc 4-PDT are expected to be lipids and proteins of those membranes. The initial damage then causes
apoptosis in cancer cells via the release of cytochrome c (Cyt-c) from mitochondria into the cytosol, followed by the
activation of caspases. That damage also triggers the induction of autophagy, an attempt by the cells to eliminate
damaged organelles, or when damage is too extensive, to promote cell death. Cyt-c is bound to the cytosolic side of the
mitochondrial inner membrane through association with cardiolipin (CL), a phospholipid containing four unsaturated
fatty acids and thus easily oxidized by 1O2 or by other oxidizing agents. Increasing evidence suggests that oxidation of
CL loosens its association with Cyt-c, and that the peroxidase activity of Cyt-c can oxidize CL. In earlier studies of Cyt-c
in homogeneous medium by MALDI-TOF-MS and LC-ESI-MS, we showed that 1O2 generated by Pc 4-PDT oxidized
histidine, methionine, tryptophan, and unexpectedly phenylalanine but not tyrosine. Most of the oxidation products were
known to be formed by other oxidizing agents, such as hydroxyl radical, superoxide radical anion, and peroxynitrite.
However, two products of histidine were unique to 1O2 and may be useful for reporting the action of 1O2 in cells and
tissues. These products, as well as CL oxidation products, have now been identified in liposomes and mitochondria after
Pc 4-PDT. In mitochondria, the PDT dose-dependent oxidations can be related to specific changes in mitochondrial
function, Bcl-2 photodamage, and Cyt-c release. Thus, the role of PDT-generated 1O2 in oxidizing Cyt-c and CL and the
interplay between protein and lipid targets may be highly relevant to understanding one mechanism for cell killing by
PDT.
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