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|Title:||The Mechanisms of Cellular Sensitivity to Photofrin-mediatd photodynamic therapy.|
|Advisor:||Rainbow, A. J.|
|Abstract:||<p>Photodynamic therapy (PDT) is a cancer treatment in which a photosensitizing drug is retained by tumor cells and activated by visible light to create toxic photoproducts including singlet oxygen and other free radicals. Photofrinmediated PDT is being applied in many clinics throughout the world. Although PDT is a promising treatment for many types of tumors, the response of cells to PDT varies between different cell types. The mechanisms of cell response to PDT are not fully understood. Therefore, an improved understanding of mechanisms of cell response to PDT may lead to novel strategies for increasing the cellular response to PDT, which has the potential to improve significantly the cure rate of cancers. Although there is evidence that the p53 tumor suppressor plays a role in tumor response to the chemotherapy and radiation therapy, its role in the response of human cells to PDT is less clear. In the present study, Li-Fraumeni syndrome (LFS) skin fibroblast cells that express only mutant p53 and normal human skin fibroblasts (NHF) strains expressing wild-type p53 were used to elucidate the role of p53 in the cell response to Photofrin-mediated PDT. NHF cells were found to have a higher sensitivity to Photofrin-mediated PDT compared to LFS cells. Reintroduction of wild-type p53 reduced the viability of LFS cells following PDT. These data suggest a role for p53 in the response of human cells to PDT. Although the treatment of PDT resulted in an increased level of p53 proteins in NHF cells, no apoptosis or any alteration in the cell cycle was found. Whereas LFS cells displayed a prolonged accumulation of cells in G2/M phase and the cells underwent apoptosis. Thus, the loss of p53 may result in a prolonged G2/M arrest that contributes to the photoresistance of LFS cells. In order to see the possible role of p53 in the cellular response to Nile Blue A (NBA), a secondary photosensitizer-mediated PDT, the clonogenic survival assay was used to compare the viability of LFS cells to NHF cells following NBA-mediated PDT. We found an extreme sensitivity of NHF cells compared to LFS and several tumour cells for treatment with NBA alone. In addition, Nile Blue A was found to be unable to produce a significant photo-cytotoxic effect on human cells using NBA concentrations which have relatively low toxicity for normal human fibroblasts. Since the LFS cells and the human tumour cells examined have some alteration in the expression of p53, these data suggest that p53 contributes to the high sensitivity of NHF cells to Nile Blue A. PDT eradicates tumor cells through the intracellular generation of reactive oxygen species (ROS). ROS is able to trigger several cellular events including cell apoptosis and several signaling pathways. It has been suggested that mitogen-activated protein kinases (MAPKs) playa crucial role in controlling cell proliferation, differentiation and cell survival. However, the activation of MAPKs by Photofrinmediated PDT and its role in the cell response to PDT are lacking. We report here that Photofrin-mediated PDT rapidly activated MAPKs including extracellular signal-regulated kinase 1 and 2(ERK1/2), c-jun NH2-terminal kinase 1(JNK1) and p38 MAPK in both LFS and NHF cells. The more PDT resistant LFS cells exhibited a sustained activation of MAPKs while the activation of MAPKS in the PDT sensitive NHF cells was transient. Blocking of the sustained activation of ERK1/2 pathway in LFS cells led to a reduction in cell viability following PDT. In contrast, blocking of prolonged p38 activation in LFS cells has no effect on the cell sensitivity to PDT. These data suggest that sustained ERK1/2 activation protects cells from cell killing by Photofrin-mediated PDT. However, the p38 pathway alone does not playa major role in the sensitivity of LFS cells to Photofrin-mediated PDT. The role of JNK1 activation remains to be clarified. Several studies have suggested the importance of raf-1 in the regulation of ERK1/2 activity, as well as the importance of MAPK phosphatase 1 (MKP-1) in the negative regulation of ERK1/2 activity. In the present study, it was found that Photofrin-mediated PDT increased expression of MKP-1 at both mRNA and protein levels. The levels of MKP-1 were inversely correlated with the kinetics of ERK1/2 activity following PDT. In addition, PDT resulted in a rapid reduction of the raf-1 expression. These data demonstrated a role of MKP-1 in the regulation of PDT-induced ERK1/2 activation, and suggested that PDT-induced ERK1/2 activity was raf-1 independent.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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