Abstract
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.
Highlights
Radiotherapy (RT) is one of the major therapies for cancer [1,2]
The reaction mixture was prepared in a final volume of 100 μL in a 96-well black plate with the following reagents at indicated final concentrations: 5 μM protoporphyrin IX (PpIX), 5 μM Aminophenyl fluorescein (APF), 0.25% dimethyl sulfoxide (DMSO), and PBS buffer
The cell suspension was diluted 10-fold, and 100 μL was plated on Luria Bertani Agar medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agarose) supplemented with 20 μg/mL ampicillin or tetracycline
Summary
The total clinical radiation dose is limited to a threshold value to avoid causing any damage to normal cells [3,4] To overcome this issue, a series of studies on the combination treatment of 5-aminolevulinic acid (5-ALA) and ionizing radiation have been conducted by some research groups using mouse models of melanoma, glioma, and colon cancer [5,6,7,8,9,10,11,12,13,14,15]. It is known that 5-ALA administration results in the accumulation of protoporphyrin IX (PpIX) in cancer cells by inhibiting the conversion PpIX to heme [16,17]. It has ahlsaos ablesoenbereenporerpteodrtethdatthtaht ethme majoajrorddamamaaggeettooccancerr cceelllssbbyyXX-r-aryaystesmtesmfsrofmroRmORSOanSdatnhdattthhaetsethese ROSRaOreSraerseproenspsiobnlseibfloerfDorNDANAbrberaekasks[2[72]7.].IInntthhiis stuuddyy,, wweeaannaalylyzezdedthtehreorleoloef oRfORSOgeSngeeranterdaftreodmfrom
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