Mitochondrion-mediated apoptosis induced by Photofrin-PDT

摘要

Apoptosis is an important cellular event that plays a key role in pathogeny and therapy of many diseases. The mechanisms of the initiation and regulation of PDT-induced apoptosis are complex. Some PDT-associated apoptosis pathways involved plasma membrane death receptors, mitochondria, lysosomes and endoplasmic reticulum (ER). In order to determine the apoptosis pathway induced by Photofrin-PDT, we used fluorescence resonance energy transfer (FRET) technique and probe SCAT3 to monitor the dynamics of caspase-3 activation after PDT treatment and also measured caspase-8 activity. With laser scanning confocal microscopy, we found that Photofrin were localized primarily in mitochondria, the primary targets of Photofrin-PDT. Formation of mitochondrial reactive oxygen species (ROS) was detected within minutes after PDT treatment. This was followed by mitochondrial membrane potential (ΔΨm), cytochrome c release, caspase-9 activity, caspase-3 activity and apoptosis. After PDT treatment, caspase-3 was activated rapidly while caspase-8 remained inactivated. Our results indicated that PDT-induced apoptosis was initiated from mitochondria pathway and independent of caspase-8 activation. The activation of caspase-3 by PDT started 20 minutes after treatment and completed in about 15 minutes. PDT-induced apoptosis is directly initiated from mitochondria pathway and not involved in the death receptors-dependent pathway. Our results demonstrated that FRET could be an effective tool to determine PDT-induced apoptosis and other cell death mechanism.