DNA damage response in prostate cancer cells by proton microbeam irradiation

Background: In cancer radiotherapy, microbeam is an advanced and effective tool in investigating radiobiology. Currently, evidence to support the radiobiology of proton beam radiotherapy for prostate cancer is limited. This study aimed to investigate the DNA damage response of proton microbeam irradiation in prostate cancer. Methods: Single-particle irradiation system to cells (SPICE) was used to perform the proton microbeam radiation-induced DNA damage response. The SPICE can deliver defined number of protons (3.4 MeV) to the cell nucleus. Different quantities of protons were irradiated to observe differential dose responses in prostate cancer cells. A total of 500 protons or defined proton doses were applied to PC-3 cell nucleus to investigate the kinetics of DNA double-strand breaks (DSB) repair after different time intervals; between 1 and 24 h post-irradiation. Subsequently, immunofluorescent staining of gamma-H2AX was performed to detect DSB, and images were captured by immunofluorescence microscopy. Finally, gamma-H2AX fluorescence intensity in each nucleus was quantified with Image J software. Results: Proton microbeam radiation-induced DSB were dependent on proton dose applied. After irradiated with 500 protons, relative expression levels of gamma-H2AX were time dependent during DSB repair process. The gamma-H2AX fluorescence intensity was maximum at 1 h post-irradiation. However, a gradual decrease was observed from 4 to 24 h. Conclusions: Microbeam is a valuable tool for the exploration of DSB response. The findings of the present study show that microbeam irradiation targeted the nucleus with precision. This study is the first to reveal that immune-stained gamma-H2AX assay with proton microbeam irradiation could predict DSB repair kinetics in PC-3 cells.