Research On Mechemism Of Photodynamic Threapy-Induced Apoptosis

Apoptosis is a very important cellular event that plays a key role in pathogeny and therapy of many diseases. Photodynamic therapy (PDT) is a new cancer treatment modality and attracts many countries' attention. In response to photodynamic therapy (PDT), apoptosis has been found to be a prominent form of cell death for many cells in culture. The mechanisms of the initiation and regulation of apoptosis induced by PDT are complex and diverse. So we investigated the status and progress of the PDT induced apoptosis, we used FRET technique and a specific chemiluminescence probe (FCLA) to study PDT-induced apoptosis. The results are primary but significative, suggesting that they are feasible methods for studying the mechanism of PDT-induced apoptosis.In this paper, the base information of PDT and the mechanism of ablating tumor are introduced firstly, and the characteristicsand the regulation of apoptosis are given in details. In addition, the principle and the application of FRET technique are described.According to the former research, we have done the following work.In the first experimental study, we studied TNF-a and PDT induced caspase-3 activation with fluorescence resonance energy transfer (FRET) technique. A recombinant caspase-3 substrate, SCAT3, was used as the FRET probe. FRET fluorescence images were collected after PDT or TNF-a induced apoptosis. By analyzing the dynamic changes of FRET fluorescence, the results indicate that the caspase-3 activation started immediately after the PDT treatment. In contrast, FRET disruption caused by caspase-3 activation started at 3 hours after TNF-a treatment, due to different signaling pathway. The results have proofed, for the first time, that FRET is a sensitive technique that can be used to investigate PDT-induced activation of caspase-3 in real-time and in single cells. By choosing appropriate recombinant substrates as FRET probes, it is likely that FRET technique will provides a new real-time means to study the mechanism of PDT at single cell level.In the second experimental study, to determine the apoptosis pathway induced by Photofrin-PDT, we used the FRET probe SCAT3 to monitor the dynamics of caspase-3 activation after PDT treatment and the caspase-8 activity was measured. With laser-scanning confocal microscopy, we found that Photofrin can localize in mitochondria. The mitochondria are the primary targets of Photofrin-PDT. After PDT treatment, caspase-3 was activated rapidly while caspase-8 was not activated. The results indicated that PDT-induced apoptosis was initiated from mitochondria pathway and independent on caspase-8 activation. As a parallel study to confirm and compare apoptosis initiated by the death receptor pathway, the activation of caspase-3 was induced by TNF-a. TNF-α acts by binding to its receptors and recruiting components of death-inducing complexes that directly activate caspase-8. Compared with PDT-induced apoptosis, the onset of caspase-3 activation was delayed 3 hours and the caspase-3 activation was required a significantly longer time. The differential kinetics of the FRET probe cleavage in cells with TNF-α or PDT suggests that the different dynamics of caspase-3 activation are induced by these two-apoptotic stimuli. The results have proofed that the initiation and process of caspase-3 activation are different in response to different apoptosis pathways, PDT-induced apoptosis is directly initiated from mitochondria pathway and not involved in the death receptors-dependent pathway and the apoptosis progress initiation from the death receptors pathway is slower than that from the mitochondria pathway.In the third experiment study, we reported a preliminary result of morphological study on permeating efficiency and localization of FCLA and hematoporphyrin derivative (HpD) through cellular membrane. In the experiment, we found that both HpD and FCLA could permeate through cellular membrane and localize to cytoplasm. Although the molecular weight of HpD is close to FCLA's, the permeating efficiency of HpD through membrane is obviously differe...