Recent reports have suggested that 5-aminolevulinic acid (5-ALA), which is a precursor to protoporphyrin IX (PpIX), leads to selective accumulation of PpIX in tumor cells and acts as a radiation sensitizer in vitro and in vivo in mouse models of melanoma, glioma, and colon cancer. In this study, we investigated the effect of PpIX under X-ray irradiation through ROS generation and DNA damage. ROS generation by the interaction between PpIX and X-ray was evaluated by two kinds of probes, 3′-(p-aminophenyl) fluorescein (APF) for hydroxyl radical (•OH) detection and dihydroethidium (DHE) for superoxide (O2•-). •OH showed an increase, regardless of the dissolved oxygen. Meanwhile, the increase in O2•- was proportional to the dissolved oxygen. Strand breaks (SBs) of DNA molecule were evaluated by gel electrophoresis, and the enhancement of SBs was observed by PpIX treatment. We also studied the effect of PpIX for DNA damage in cells by X-ray irradiation using a B16 melanoma culture. X-ray irradiation induced γH2AX, DNA double-strand breaks (DSBs) in the context of chromatin, and affected cell survival. Since PpIX can enhance ROS generation even in a hypoxic state and induce DNA damage, combined radiotherapy treatment with 5-ALA is expected to improve therapeutic efficacy for radioresistant tumors.
X-ray responsivity resulting in the generation of reactive oxygen species (ROS) was investigated in 9600 organic compounds that were selected by considering their structural diversity. We focused on superoxides that were primarily detected using dihydroethidium (DHE) and hydroxyl radicals, that were identified fluorometrically using 3’-(p-aminophenyl) Fluorescein (APF). Many organic compounds were discovered that responded to the DHE and/or APF assay using X-ray irradiation. These results suggest that some of these organic compounds emit either superoxides or hydroxyl radicals whereas others emit both under the influence of X-ray irradiation. The response of the derivatives of a hit compound with a partial change in the structure was also investigated. The products produced from DHE by X-ray irradiation were identified by HPLC to confirm the integrity of the process. Although, the reactions were suppressed by the superoxide dismutase (SOD), not only 2-hydroxyethidium (2-OH-E+), but also ethidium (E+) were detected. The results suggest that apart from a direct reaction, an indirect reaction may occur between DHE and the superoxides. Although X-ray responsiveness could not be inferred due to the molecular complexity of the investigated compounds, delineation of these reactions will facilitate the development of the next generation of radiosensitizers.
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