Increasing the efficiency of photodynamic tumor therapy by controlling the cell death mode via the intracellular energy homeostasis

Photodynamic tumour therapy (PDT: photosensitizers detect and destroy selectively tumour tissue upon activation with visible light) as a rather new approach to cancer treatment is successfully applied since many years and permitted by now in several countries for use on different kinds of tumours. As in other tumour therapies, cell death via apoptosis can be induced by reduced sensitizer and/or light doses, which is expected to increase the efficiency and reduce side effects such as skin photosensitivity. In contrast to this, necrosis stimulates the immune system, which is an important component in tumour eradication. PDT is proved to be very efficient possibly by providing both cell death modes. It is therefore of high interest to explore and control the mechanisms leading to cell death. Mitochondria seem to be regulators of apoptosis and the intracellular ATP level might determine the mode of cell death; both types can be investigated best by exploring the network of their regulation via their common source, the energy pool. The condition of the energy homeostasis could be a prerequisite for the decision of the cell to survive, to start apoptotic processes or to die by necrosis. The aim of the present research project is therefore to investigate the energy homeostasis for the pathways leading to PDT-induced tumour cell death, and to control the processes in order to enhance the efficiency and safety of PDT. Both cell death modes should finally appear in an optimized ratio. Manipulating the "death" programs should be possible by monitoring signalling pathways indirectly via the overlaying energy pattern. The energy source behind the programs is the ATP-pool. ATP production can be strongly influenced by reactive oxygen intermediates (ROI) as generated in PDT. Especially the endogenous photosensitizer protoporphyrin IX (PpIX), which is induced in mitochondria, might influence the ATP-pool and the energy distribution via R0I. This in turn could initiate signal transduction with messages for up- or down-regulation of cell proliferation and for cell death induction. The main part of the project is to measure the energy metabolism (distribution, homeostasis and energy dynamics by continuous recording) in single cells with the focus on mitochondria. Detection and evaluation of the metabolic response (ATP, ADP, AMP = energy charge and Ca2+) of transformed mouse fibroblasts (L929) will be carried out with a Low Light Imaging system by chemoluminescence and with low intensity, highly time-resolved fluorescence analysis. The cells will be stably transfected by those genes, whose gene products (Luciferase, Acquorin) catalyze the luminescence reaction. The energy metabolism will be recorded following photodynamic treatment with 5-aminolevulinic acid-induced endogenous PpIX and under different control conditions. The questions to be answered are: Could the different states of the cells, the two modes of cell death and the different types of apoptosis be discriminated or even influenced by the energy charge? The generation of ROI by PDT will be studied in L929 and normal mouse fibroblasts (3T3) with regard to intracellular structures and sensitizer distribution, and correlated to the energy metabolism. It should be investigated, how far the different states of the cells and the two modes of cell death are influenced or even caused by R01. Apoptosis/necrosis detection will. be carried out by at least three independent test methods. The mitochondrial membrane potential and cytochrome c release will be measured in order to detect their role in photodynamically induced cell death. Finally, factors influencing the cell death processes should be detected and used for further manipulation towards an optimized ratio of apoptosis/necrosis according to the results of this project and to our immunological investigations. This should lead to a clinically applicable PDT protocol, which is able to improve the therapeutical success. Beside that, answers to important questions concerning the energy metabolism of apoptotic cells could be given.

Summary: Apoptotic cells depend on a functional cellular energy supply until late stages in active cell death; this is maintained by glycolytic ATP-production. Necrotic cells loose ATP rapidly and completely. Thus cellular energetics is one of the parameters determining the cell death mode after photodynamic therapy and could, if altered, lead to a desired necrosis/ apoptosis ratio optimal for complete tumor eradication by stimulation of the immune system. Abstract: Photodynamic therapy represents a promising method for the treatment of several malignant and non- malignant diseases. It is carried out as a two-step protocol comprising the preferential accumulation of photosensitizing molecules in the target cells and the subsequent irradiation of the loaded cells with visible or near- infrared light. Thereby reactive oxygen species are being formed, which oxidize cellular components and cause cell death. PDT can trigger both, apoptosis and necrosis in target cells. Apoptosis is, in contrast to passive necrosis, an active, controlled and energy-requiring process and several steps in the apoptotic cascade have been shown to depend on ATP. The project aimed at the examination of the energetic status of cells following treatment with photodynamic therapy. This is of importance since cellular energetics determines the mode of cell death, the latter influencing at the level of patient treatment the response of the immune system and thus the overall efficiency of the therapy. Our data revealed some interesting details about the energetics of apoptotic cells after AlPcS4 -PDT: 1) Clear apoptosis induction could be found in A431 cells when AlPcS4 was used as a photosensitizer. We found an apoptotic window at light fluences ranging from 3 to 5 J.cm-2 , independently identified by several assays. Lower fluences caused repair and survival of treated cells, higher fluences necrosis. 2) It could be shown that - in clear contrast to the literature - apoptotic cells maintain high levels of intracellular ATP until late steps of the apoptotic program. On the contrary necrosis leads to a rapid and complete drop of intracellular ATP. The kinetics of the intracellular ATP-level provides deep insight in the detailed composition of PDT-treated samples, comprising survival, apoptosis and necrosis. 3) The mitochondrial membrane potential (), a chemi-osmotic gradient over the inner mitochondrial membrane, which is generated by oxidative phosphorylation in these organelles, is the main source of ATP under normal conditions. By employing the potential-sensitive dye JC-1 and FACS analysis we could show that decreases early, but not completely in apoptosis. Necrotic cells show a rapid and complete loss of the . 4) The loss in is compensated by glycolysis to maintain high ATP-levels required for the energy-dependent steps in the apoptotic cascade. We could show apoptosis after AlPcS4 -PDT to inevitably depend on the availability of glucose. Neither caspase activation, nor nuclear fragmentation or morphological changes could be observed after PDT-treatment, when cells were deprived of glucose. Lack of glucose causes cells to shift from apoptosis to necrosis. All knowledge gained leads to a deep understanding of cellular energetics of apoptosis after PDT. The impact of metabolic manipulation on the effectiveness of anticancer-treatments in general has been discussed recently by several authors. Our results provide some experimental evidence for these ideas and should be expanded to clinical treatment protocols, which, by manipulating cellular energetics, shall induce a desired apoptosis/ necrosis ratio most favorable for tumor therapy.