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Photodynamic therapy (PDT) is a proven clinical intervention for many dermatological conditions, including non-melanoma skin cancer. Amino-laevulinic acid- (ALA-) based PDT relies on three components: photosensitiser accumulation in targeted cells, photo-irradiation, and the presence of O2. Irradiation excites the photosensitiser – protoporphyrin IX (PpIX) – resulting in intracellular reactive oxygen species (ROS) formation and cancer cell death. Cell killing by PDT may be increased by raising O2 availability. One way to increase tissue O2 in patients is the use of hyperbaric O2 (HBO), in combination with PDT.
To test the potential efficacy of this treatment strategy, changes to skin O2 concentration were modelled in vitro by increasing the O2 from a baseline concentration of 2.0% (physioxic) to an HBO-achievable concentration of 18.6% O2, prior to photo-irradiation of the cells. It was necessary to ensure that the baseline cells were phenotypically adapted to 2.0% O2, before comparing the effects of photodynamically-induced cell killing at 2.0% and 18.6% O2. Additional experiments were carried out, to determine the effects of adjunctive treatments, using exogenous nitric oxide donors and small molecule nitroxides, on the efficacy of photodynamic cell killing.
A431 epidermoid carcinoma cells were cultured in flasks under 2.0% or 18.6% O2 for 48 hours, to reflect physioxia and hyperoxia for this cell type. A concentration-response curve was established using the pro-drug ALA or its methyl ester, methyl-laevulinic acid (MAL).
Spectrofluorometry was performed to measure PpIX levels. Cells were incubated for 24-h intervals, up to 96 h, before treatment with the pro-drug. This was to establish the optimal duration of long-term incubation under physioxia. Having carried out this preliminary work, A431 cells were incubated for 48 h under physioxia or hyperoxia, to adapt the cells before carrying out photodynamic treatment. Cell death was measured via flow cytometry using annexin V-FITC and propidium iodide. The presence of nitrated proteins, widely regarded as a post-translational proxy for oxidative stress, was assessed using western blots. Chemiluminescence was undertaken to indirectly quantify, the levels of NO released.
Optimal loading of cells with ALA (0.5 mM) and MAL (1.0 mM) was deduced from the concentration-response curves. There was no significant change in the levels of cell death after 48 h pre-adaptation to physioxia, compared to longer time-points. Rates of necrosis and late apoptosis in cells pre-adapted, and subsequently photo-irradiated, under 2.0% O2 were almost double that of cells adapted and treated under 18.6% O2. When the NO donor was added to cells which had been adapted to physioxia and then photodynamically irradiated under physioxia, there was an even more pronounced increase in cellular late apoptosis (n=4, P<0.001), an effect that diminished when photodynamic irradiation was performed under 18.6% O2. Using western blots, protein nitration was detected in bands between 35 and 40 kDa. During the 96-h adaptation experiment, the generation of 3-nitrotyrosine after photodynamic irradiation was greater in cells cultured under hyperoxia during both the adaptation and photodynamic irradiation phases. A trio of low-molecular weight bands (between 15-20 kDa) was observed in all samples, but the bands’ fluorescence intensity was stronger in cells cultured under hyperoxia, compared to those cells pre-adapted and photo-irradiated under physioxia (n=2). Putative assignments were made in respect of the proteins responsible for tyrosine nitration in western blots. The mitochondria-targeted nitroxide antioxidant (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO), elicited a reduction in cell death following hyperoxia pre-adaptation and photodynamic treatment. Yet, mitoTEMPO elicited an increase in cell death in cells pre-adapted (and irradiated) under physioxic conditions.
48 h was an appropriate duration to adapt A431 cells ‘long-term’ to physioxia before photodynamic treatment. After this pre-adaptation, there was an increase in the killing of photodynamically-treated cells in the presence of an NO donor (spermine NONOate, sperNO) under physioxia, compared to photodynamically treated cells in the presence of an NO donor, under 18.6% O2 (after adaptation to physioxia) (n= 4; p< 0.001). ALA-loaded cells, which had been adapted to physioxia before photo-irradiation, showed an increase in levels of cell death when sperNO was added in combination with photo-irradiation.
Overall, this work has set out to establish an appropriate cell culture model, using physiologically relevant O2 concentrations to adapt A431, before carrying out photodynamic irradiation. The results presented in this thesis show that long-term pre-adaptation to physioxia leads to different cellular responses to photodynamic irradiation, compared to simply maintaining conventional incubation conditions throughout an experiment. When sperNO was added to cells that had been adapted to physioxia and also photo-irradiated under physioxia, there was an even more pronounced increase in overall cell death. This was reduced significantly when photodynamic irradiation was performed under hyperoxia. |
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