5-Aminolevulinic Acid-mediated Photodynamic Therapy on Hep-2 and MCF-7c3 cells

M. G. ÁLVAREZ, M. S. LACELLI, V. RIVAROLA, A. BATLLE, H. FUKUDA

Buenos Aires

Workshop; 5th International Workshop on Photodynamic Therapy and Photodetection with Porphyrin Precursors; 2006

Introduction: Photodynamic therapy (PDT) is a novel cancer treatment receiving increased attention. PDT is a binary therapy involving the combination of visible light and a photosensitizer. Each component is harmless by itself, but, in combination with molecular oxygen, lead to the generation of singlet oxygen (1O2) and other reactive oxygen species (ROS), oxidative cell damage and cell death. 5-aminolevulinic acid (ALA) induced accumulation of protoporphyrin IX (PpIX) is a useful approach to the early detection and treatment of cancers. oxygen species (ROS), oxidative cell damage and cell death. 5-aminolevulinic acid (ALA) induced accumulation of protoporphyrin IX (PpIX) is a useful approach to the early detection and treatment of cancers. oxygen species (ROS), oxidative cell damage and cell death. 5-aminolevulinic acid (ALA) induced accumulation of protoporphyrin IX (PpIX) is a useful approach to the early detection and treatment of cancers. increased attention. PDT is a binary therapy involving the combination of visible light and a photosensitizer. Each component is harmless by itself, but, in combination with molecular oxygen, lead to the generation of singlet oxygen (1O2) and other reactive oxygen species (ROS), oxidative cell damage and cell death. 5-aminolevulinic acid (ALA) induced accumulation of protoporphyrin IX (PpIX) is a useful approach to the early detection and treatment of cancers. oxygen species (ROS), oxidative cell damage and cell death. 5-aminolevulinic acid (ALA) induced accumulation of protoporphyrin IX (PpIX) is a useful approach to the early detection and treatment of cancers. oxygen species (ROS), oxidative cell damage and cell death. 5-aminolevulinic acid (ALA) induced accumulation of protoporphyrin IX (PpIX) is a useful approach to the early detection and treatment of cancers. increased attention. PDT is a binary therapy involving the combination of visible light and a photosensitizer. Each component is harmless by itself, but, in combination with molecular oxygen, lead to the generation of singlet oxygen (1O2) and other reactive oxygen species (ROS), oxidative cell damage and cell death. 5-aminolevulinic acid (ALA) induced accumulation of protoporphyrin IX (PpIX) is a useful approach to the early detection and treatment of cancers. oxygen species (ROS), oxidative cell damage and cell death. 5-aminolevulinic acid (ALA) induced accumulation of protoporphyrin IX (PpIX) is a useful approach to the early detection and treatment of cancers. oxygen species (ROS), oxidative cell damage and cell death. 5-aminolevulinic acid (ALA) induced accumulation of protoporphyrin IX (PpIX) is a useful approach to the early detection and treatment of cancers. Photodynamic therapy (PDT) is a novel cancer treatment receiving increased attention. PDT is a binary therapy involving the combination of visible light and a photosensitizer. Each component is harmless by itself, but, in combination with molecular oxygen, lead to the generation of singlet oxygen (1O2) and other reactive oxygen species (ROS), oxidative cell damage and cell death. 5-aminolevulinic acid (ALA) induced accumulation of protoporphyrin IX (PpIX) is a useful approach to the early detection and treatment of cancers. oxygen species (ROS), oxidative cell damage and cell death. 5-aminolevulinic acid (ALA) induced accumulation of protoporphyrin IX (PpIX) is a useful approach to the early detection and treatment of cancers. oxygen species (ROS), oxidative cell damage and cell death. 5-aminolevulinic acid (ALA) induced accumulation of protoporphyrin IX (PpIX) is a useful approach to the early detection and treatment of cancers. 1O2) and other reactive oxygen species (ROS), oxidative cell damage and cell death. 5-aminolevulinic acid (ALA) induced accumulation of protoporphyrin IX (PpIX) is a useful approach to the early detection and treatment of cancers. Materials and Methods: Photodynamic effect of ALA was evaluated on human larynx carcinoma (Hep-2) and human breast adenocarcinoma (MCF-7c3) cell lines, the latter stably transfected with human procaspase-3cDNA. The light source used was a Kodak slide projector equipped with a 150 W lamp; light intensity at the treatment site was 60 mW/cm2. Cell survival was evaluated with the MTT assay; necrosis and apoptosis by toluidine blue and Hoechst-33258. toluidine blue and Hoechst-33258. toluidine blue and Hoechst-33258. carcinoma (Hep-2) and human breast adenocarcinoma (MCF-7c3) cell lines, the latter stably transfected with human procaspase-3cDNA. The light source used was a Kodak slide projector equipped with a 150 W lamp; light intensity at the treatment site was 60 mW/cm2. Cell survival was evaluated with the MTT assay; necrosis and apoptosis by toluidine blue and Hoechst-33258. toluidine blue and Hoechst-33258. toluidine blue and Hoechst-33258. carcinoma (Hep-2) and human breast adenocarcinoma (MCF-7c3) cell lines, the latter stably transfected with human procaspase-3cDNA. The light source used was a Kodak slide projector equipped with a 150 W lamp; light intensity at the treatment site was 60 mW/cm2. Cell survival was evaluated with the MTT assay; necrosis and apoptosis by toluidine blue and Hoechst-33258. toluidine blue and Hoechst-33258. toluidine blue and Hoechst-33258. Photodynamic effect of ALA was evaluated on human larynx carcinoma (Hep-2) and human breast adenocarcinoma (MCF-7c3) cell lines, the latter stably transfected with human procaspase-3cDNA. The light source used was a Kodak slide projector equipped with a 150 W lamp; light intensity at the treatment site was 60 mW/cm2. Cell survival was evaluated with the MTT assay; necrosis and apoptosis by toluidine blue and Hoechst-33258. toluidine blue and Hoechst-33258. toluidine blue and Hoechst-33258. 2. Cell survival was evaluated with the MTT assay; necrosis and apoptosis by toluidine blue and Hoechst-33258. Results: PpIX is rapidly accumulated in the cells and tends to a saturation value between 5 and 24 h on both cell lines (3.2 ìg/106 and 8 ìg/106 in Hep-2 cells and MCF-7c3 cells respectively). Cell survival by means of MTT assay shows that in the absence of light ALA does not affect cell viability up to 1mM concentration in both cell lines. In contrast, the combination of ALA with visible light induces a decrease in cell survival that depends on the drug concentration and light dose. Morphologic analysis of MCF-7c3 cells performed with toluidine blue and Hoechst-33258, after incubation with 1mM ALA during 5 h and irradiation with 54 Jcm-2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. respectively). Cell survival by means of MTT assay shows that in the absence of light ALA does not affect cell viability up to 1mM concentration in both cell lines. In contrast, the combination of ALA with visible light induces a decrease in cell survival that depends on the drug concentration and light dose. Morphologic analysis of MCF-7c3 cells performed with toluidine blue and Hoechst-33258, after incubation with 1mM ALA during 5 h and irradiation with 54 Jcm-2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. respectively). Cell survival by means of MTT assay shows that in the absence of light ALA does not affect cell viability up to 1mM concentration in both cell lines. In contrast, the combination of ALA with visible light induces a decrease in cell survival that depends on the drug concentration and light dose. Morphologic analysis of MCF-7c3 cells performed with toluidine blue and Hoechst-33258, after incubation with 1mM ALA during 5 h and irradiation with 54 Jcm-2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. 5 and 24 h on both cell lines (3.2 ìg/106 and 8 ìg/106 in Hep-2 cells and MCF-7c3 cells respectively). Cell survival by means of MTT assay shows that in the absence of light ALA does not affect cell viability up to 1mM concentration in both cell lines. In contrast, the combination of ALA with visible light induces a decrease in cell survival that depends on the drug concentration and light dose. Morphologic analysis of MCF-7c3 cells performed with toluidine blue and Hoechst-33258, after incubation with 1mM ALA during 5 h and irradiation with 54 Jcm-2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. respectively). Cell survival by means of MTT assay shows that in the absence of light ALA does not affect cell viability up to 1mM concentration in both cell lines. In contrast, the combination of ALA with visible light induces a decrease in cell survival that depends on the drug concentration and light dose. Morphologic analysis of MCF-7c3 cells performed with toluidine blue and Hoechst-33258, after incubation with 1mM ALA during 5 h and irradiation with 54 Jcm-2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. respectively). Cell survival by means of MTT assay shows that in the absence of light ALA does not affect cell viability up to 1mM concentration in both cell lines. In contrast, the combination of ALA with visible light induces a decrease in cell survival that depends on the drug concentration and light dose. Morphologic analysis of MCF-7c3 cells performed with toluidine blue and Hoechst-33258, after incubation with 1mM ALA during 5 h and irradiation with 54 Jcm-2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. 5 and 24 h on both cell lines (3.2 ìg/106 and 8 ìg/106 in Hep-2 cells and MCF-7c3 cells respectively). Cell survival by means of MTT assay shows that in the absence of light ALA does not affect cell viability up to 1mM concentration in both cell lines. In contrast, the combination of ALA with visible light induces a decrease in cell survival that depends on the drug concentration and light dose. Morphologic analysis of MCF-7c3 cells performed with toluidine blue and Hoechst-33258, after incubation with 1mM ALA during 5 h and irradiation with 54 Jcm-2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. respectively). Cell survival by means of MTT assay shows that in the absence of light ALA does not affect cell viability up to 1mM concentration in both cell lines. In contrast, the combination of ALA with visible light induces a decrease in cell survival that depends on the drug concentration and light dose. Morphologic analysis of MCF-7c3 cells performed with toluidine blue and Hoechst-33258, after incubation with 1mM ALA during 5 h and irradiation with 54 Jcm-2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. respectively). Cell survival by means of MTT assay shows that in the absence of light ALA does not affect cell viability up to 1mM concentration in both cell lines. In contrast, the combination of ALA with visible light induces a decrease in cell survival that depends on the drug concentration and light dose. Morphologic analysis of MCF-7c3 cells performed with toluidine blue and Hoechst-33258, after incubation with 1mM ALA during 5 h and irradiation with 54 Jcm-2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. PpIX is rapidly accumulated in the cells and tends to a saturation value between 5 and 24 h on both cell lines (3.2 ìg/106 and 8 ìg/106 in Hep-2 cells and MCF-7c3 cells respectively). Cell survival by means of MTT assay shows that in the absence of light ALA does not affect cell viability up to 1mM concentration in both cell lines. In contrast, the combination of ALA with visible light induces a decrease in cell survival that depends on the drug concentration and light dose. Morphologic analysis of MCF-7c3 cells performed with toluidine blue and Hoechst-33258, after incubation with 1mM ALA during 5 h and irradiation with 54 Jcm-2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. respectively). Cell survival by means of MTT assay shows that in the absence of light ALA does not affect cell viability up to 1mM concentration in both cell lines. In contrast, the combination of ALA with visible light induces a decrease in cell survival that depends on the drug concentration and light dose. Morphologic analysis of MCF-7c3 cells performed with toluidine blue and Hoechst-33258, after incubation with 1mM ALA during 5 h and irradiation with 54 Jcm-2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. respectively). Cell survival by means of MTT assay shows that in the absence of light ALA does not affect cell viability up to 1mM concentration in both cell lines. In contrast, the combination of ALA with visible light induces a decrease in cell survival that depends on the drug concentration and light dose. Morphologic analysis of MCF-7c3 cells performed with toluidine blue and Hoechst-33258, after incubation with 1mM ALA during 5 h and irradiation with 54 Jcm-2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. ìg/106 and 8 ìg/106 in Hep-2 cells and MCF-7c3 cells respectively). Cell survival by means of MTT assay shows that in the absence of light ALA does not affect cell viability up to 1mM concentration in both cell lines. In contrast, the combination of ALA with visible light induces a decrease in cell survival that depends on the drug concentration and light dose. Morphologic analysis of MCF-7c3 cells performed with toluidine blue and Hoechst-33258, after incubation with 1mM ALA during 5 h and irradiation with 54 Jcm-2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. -2 light doses, revealed the typical morphological changes of necrosis 24 h post-PDT. Instead, the same conditions applied to the Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. Conclusions: The different mechanisms of cell death can be explained by the difference in the pattern of PpIX accumulation: in Hep-2 cells fluorescence is confined into the lysosome, while in MCF-7c3 cells, in the nuclei. These results also confirm that the cell response to PDT is dependent on both the light dose and the cell type. ___ in the pattern of PpIX accumulation: in Hep-2 cells fluorescence is confined into the lysosome, while in MCF-7c3 cells, in the nuclei. These results also confirm that the cell response to PDT is dependent on both the light dose and the cell type. ___ in the pattern of PpIX accumulation: in Hep-2 cells fluorescence is confined into the lysosome, while in MCF-7c3 cells, in the nuclei. These results also confirm that the cell response to PDT is dependent on both the light dose and the cell type. ___ The different mechanisms of cell death can be explained by the difference in the pattern of PpIX accumulation: in Hep-2 cells fluorescence is confined into the lysosome, while in MCF-7c3 cells, in the nuclei. These results also confirm that the cell response to PDT is dependent on both the light dose and the cell type. ___ ______________________________________________________________________________________