Study on vascular shutdown effects of carcinoma blood vessels and cytotoxic effects of carcinoma cells induced by photochemical reaction.

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Photodynamic therapy (PDT) is a clinically effective cancer treatment which is based on cytotoxicity of reactive oxygen species (ROS) generated by an interaction between photosensitizer and light. PDT induces vascular shutdown (VSD) effects based on thrombus formation realized by administration of photosensitizing agent. This treatment results in a sequence of photochemical processes to produce cytotoxic ROSS which bring irreversible photo damage to tissue cells. ^1O_2 and O_2^-strongly activated adhesive function of platelet and leukocyte, and also induced endothelial cell retraction resulted in increase in sub endothelial area. Activation of both platelet and endothelial cells also released adhesion molecules that followed strong cell interaction indispensable for the platelet aggregation and thrombus formation. The platelet adhesion and thrombus formation in the microvessels accelerated under photodynamic reaction. For visualization of flow behavior of erythrocytes, leukocytes and p … More latelets, fluorescent dye probes were used and the flow images were observed under fluorescent microscope. Blood oxygen pressure was also measured by using phosphorescence quenching of Pd porphyrin with oxygen molecules. In order to measure erythrocyte velocity and to evaluate the degree of platelet adhesion and leukocyte sticking, high-speed CCD camera and image processing system were used and made possible to evaluate the contribution of ROSS to the VSD and pharmacological effect on the VSD. ROSs induced protein transformation of cytoplasm in the endothelial cells, especially actin filament polymerisation was observed that affected on increase in subendothelial area. These phenomena accelerated activation of platelets and leukocytes which progressively adhered on the endothelial cells and subendothelial area. On the other hand, cytotoxic effect of PDT on tumor cells, mainly apoptosis, was induced by ROSs attributed to photochemical reaction. Ca^<2+> is one of the most famous second messenger in cell function, especially, the cytoplasmic concentration of free calcium ions[Ca^<2+>]i has important role in apoptosis. In this study, we examined the role of Ca^<2+> in PDT-induced apoptosis of human lung small carcinoma (Ms-1) and human leukemia cell line (HL-60 cells). We performed the imaging of Ca^<2+> and monitoring Ca^<2+> concentration in apoptosis induced by PDT. With intracellular calcium indicator Fluo3-AM, increase of [Ca^<2+>]i was observed for 6 hours after PDT and was inhibited by extracellular Ca^<2+> chelator BAPTA. Agarose gel electrophresis revealed that PDT-induced apoptosis was inhibited by BAPTA. PDT induced loss of mitochondrial membrane potential and increase of Ca^<2+> in mitochondria. As a result, we have revealed that increase of [Ca^<2+>]i is induced by transcellular calcium influx from extracellular fluid to intracellular fluid, and induce apoptosis in Ms-1 and HL-60 cells. These results indicate that Ca^<2+> plays an important role in apoptosis induced by PDT. Less