Photodynamic therapy (PDT) is a new and promising cancer treatment modality that is being investigated worldwide. The long term objective of this research is to investigate the fundamental mechanisms of its mode of action at the biochemical and cellular level and hence find ways to enhance the efficacy of PDT. We hypothesize that an environment that enhances free radical lipid peroxidation processes will lead to increased effectiveness of PDT. The working hypothesis of this proposal is that photodynamic therapy with a hematoporphyrin derivative preparation such as Photofrin (PDT/HPD) produces singlet oxygen in cell membranes, both tumor cell membranes and endothelial cell membranes of the tumor vascular system. This singlet oxygen reacts with unsaturated lipids and cholesterol, yielding hydroperoxides, which can serve as initiators of free radical lipid peroxidation chain reactions, disrupting membrane function. We believe that the microvascular endothelial cell is a key target for these peroxidative processes. We further hypothesize that an environment that will enhance the 'dark' free radical lipid peroxidation reactions after the delivery of light will result in enhanced endothelial cell damage and death. The endothelial cell appears to be particularly sensitive to free radical-mediated oxidative stress. Therefore, in all experiments we will compare results obtained with microvascular endothelial cells to those from vascular epithelial cells and two tumor cell lines. L1210 murine leukemia and MCF-7 human breast carcinoma. The working hypothesis of this project will be tested with the following Specific Aims: I. Determine if pro-oxidants, such as iron, or ascorbate, or iron with ascorbate, will enhance the phototoxicity of PDT/HPD to cells, and conversely, determine if small-molecule antioxidants such as vitamin E, lazaroids, and BHT will provide protection against peroxidative phototoxicity. II. Determine if membrane-derived free radicals are produced in intact, functioning cells by PDT/HPD, and if pro-oxidants will enhance the free radical peroxidation processes, then determine if radical production correlates with phototoxicity. III. Determine if making cell membranes more oxidizable by increasing the proportion of polyunsaturated fatty acids will increase the phototoxicity of PDT/HPD. IV. We will use molecular biology techniques to modify the antioxidant enzyme profile of cells and determine if this will alter their sensitivity to PDT/HPD. V. Study in detail the fundamental chemical reactions that may be key to PDT/HPD phototoxicity.
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