5-Aminolevulinic acid strongly enhances delayed intracellular production of reactive oxygen species (ROS) generated by ionizing irradiation: quantitative analyses and visualization of intracellular ROS production in glioma cells in vitro.

Postoperative adjuvant radiotherapy has important roles in multimodal treatment for highly aggressive malignant gliomas. Previously, we demonstrated that multi-dose ionizing irradiation with repetitive administration of 5-aminolevulinic acid (5-ALA) enhanced the host antitumor response and strongly inhibited tumor growth in experimental glioma. However, the mechanism of the radiosensitizing effect of 5-ALA is not known. Ionizing irradiation not only causes reactive oxygen species (ROS) formation initially by water radiolysis but also induces delayed production of mitochondrial ROS for mediating the long-lasting effects of ionizing irradiation on tumor cells. 5-ALA leads to high accumulation of protoporphyrin IX (PpIX) in the mitochondria of tumor cells, yet can also improve dysfunction of the mitochondrial respiratory chain in tumor cells. Here, we assessed the effect of 5-ALA-induced PpIX synthesis and delayed production of intracellular ROS after ionizing irradiation with 5-ALA in glioma cells in vitro. Temporal changes in intracellular 5-ALA-induced PpIX synthesis after ionizing irradiation in glioma cell lines were evaluated using flow cytometry (FCM). Then, the effect of 5-ALA on delayed production of intracellular ROS 12 h after ionizing irradiation in glioma cells was evaluated by FCM and confocal laser scanning microscopy. Ionizing irradiation had no effect on 5-ALA-induced PpIX synthesis in glioma cells. Delayed intracellular production of ROS was significantly higher than that just after ionizing irradiation, but 5-ALA pretreatment strongly enhanced the delayed intracellular production of ROS, mainly in the cytoplasm of glioma cells. This 5-ALA-induced increase in the delayed production of ROS tended to be higher in the case of 5-ALA treatment before rather than after ionizing irradiation. These results suggest that 5-ALA can affect tumor cells under ionizing irradiation, and greatly increase secondary intracellular production of ROS long after ionizing irradiation, thereby causing a radiosensitizing effect in glioma cells.