Nancy L. Oleinick

Photodynamic therapy (PDT) is dependent on the uptake of a photosensitizing dye, often a porphyrin-related macrocycle, by the tumor or other abnormal tissue that is to be treated, the subsequent irradiation of the tumor with visible light of an appropriate wavelength matched to the absorption spectrum of the dye, and molecular oxygen to generate reactive oxygen intermediates. The initial oxidative reactions lead to damage to organelles in which the dye is bound, culminating in cell death and destruction of the tumor or abnormal tissue. Apoptosis is a common mechanism of cell death after PDT both in vitro and in vivo. PDT also triggers the activation of several signal transduction pathways in the treated cells; some of these are stress responses aimed at cell protection, while others are likely to contribute to the cell death process. The photosensitizers of greatest interest in PDT bind to various cytoplasmic membranes but are not found in the nucleus and do not bind to DNA. Nevertheless, some DNA damage is produced that can lead to mutagenesis, the extent of which is dependent on the photosensitizer, the cellular repair properties and the target gene. Thus, in spite of generating some responses common to ionizing radiation and other oxidative stresses, PDT is unique in the subcellular localization of damage, the combination of signaling pathways that are activated, and rapid kinetics of the induction of cell death processes.

The mode of cell death of two strains of mouse lymphoma L5178Y cells was studied following photodynamic therapy (PDT) sensitized by chloroaluminum phthalocyanine. Strains LY-R and LY-S differ in their relative sensitivities to UVC radiation, X-radiation, and PDT; both responded to PDT by undergoing apoptosis. The DNA was degraded into fragments with lengths which are multiples of approximately 180-190 base pairs (i.e., oligonucleosome size), a biochemical marker of apoptosis. The DNA fragmentation was dose and time dependent which indicates this response to be an enzymic process related to cell killing. Cycloheximide, a protein synthesis inhibitor, and actinomycin D, an RNA synthesis inhibitor, enhanced the endonucleolytic DNA fragmentation. Transmission electron microscopy revealed chromatin condensation around the periphery of the nucleus, which is also characteristic of apoptosis. The induction of apoptosis in L5178Y cells by PDT was rapid, with marked degradation of DNA occurring in as little as 30 min. The rapidity of the response to PDT suggests that cellular damage produced by PDT can directly activate endonucleolysis and chromatin condensation, thereby by-passing many of the early steps in the signal transduction program which are acted upon by other agents causing apoptosis.

Photodynamic therapy (PDT), a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in human epidermoid carcinoma A431 cells. However, the precise mechanism of PDT-induced apoptosis is not well characterized. To dissect the pathways of PDT-induced apoptosis, we investigated the involvement of mitochondrial damage by examining a second generation photosensitizer, the silicon phthalocyanine 4 (Pc 4). By using laser-scanning confocal microscopy, we found that Pc 4 localized to cytosolic membranes primarily, but not exclusively, in mitochondria. Formation of mitochondrial reactive oxygen species (ROS) was detected within minutes when cells were exposed to Pc 4 and 670-675 nm light. This was followed by mitochondrial inner membrane permeabilization, depolarization and swelling, cytochrome c release, and apoptotic death. Desferrioxamine prevented mitochondrial ROS production and the events thereafter. Cyclosporin A plus trifluoperazine, blockers of the mitochondrial permeability transition, inhibited mitochondrial inner membrane permeabilization and depolarization without affecting mitochondrial ROS generation. These data indicate that the mitochondrial ROS are critical in initiating mitochondrial inner membrane permeabilization, which leads to mitochondrial swelling, cytochrome c release to the cytosol, and apoptotic death during PDT with Pc 4.