Targeted Inhibition of Rad51 Renders Non–small Cell Lung Cancer Cells More Sensitive to Proton Therapy

Abstract

Radiation therapy using proton beams is an emerging therapy that is being used for cancer treatment due to its superior physical dose distribution, higher RBE and increased effectiveness, leading to greater clinical gains. A recent study has shown a preferential involvement of the homologous recombination (HR) pathway in proton mediated radiosensitization. Rad51, a key protein in the HR pathway of DNA double-strand break repair, is overexpressed in various cancers and plays an essential role in radioresistance. Therefore, this study was designed to evaluate the impact of targeting Rad51on the RBE of protons and photons in NSCLC cell lines. The effects of Rad51 inhibition by genetic (Rad51 siRNA) and pharmacologic (BO2, a small molecule Rad51 inhibitor) approaches on radiosensitization of H1299 and A549 NSCLC cells were determined in vitro. The RBE of protons and photons following siRad51 treatment was compared using clonogenic cell survival assay when protons were delivered with doses of 2, 4 and 6 Gy at the mid-spread out Bragg peak (SOBP) using a reference beam of 15 g/cm2 range and 10 g/cm2modulation. Involvement of the DNA damage response was assessed by western blot analysis for DNA repair proteins and immunofluorescence staining for g-H2AX foci. Rad51 depletion by siRNA sensitized H1299 and A549 cells to protons and photons. However, both H1299 and A549 cell lines were significantly more sensitive to proton irradiation compared to photon. Survival fraction at 2 Gy (SF2) for H1299 cells exposed to proton beam was reduced from (53.7 ± 0.49) % in siScr to (21.8 ± 2.28) % in siRad51-treated group. Similarly, SF2 for A549 cells was reduced from (59.1 ± 1.16) % in siScr to (29.6 ± 4.9) % following siRad51-treatment. Survival enhancement ratios (SER) were calculated at 10% cell survival by dividing proton radiation dose of the siScr to that of siRad51 for that survival. The SER for H1299 cells at 10% survival was 1.9 and for A549 was 1.33, respectively. Similar results were obtained with the Rad51 small molecule inhibitor, BO2. Our results reveal that this enhanced efficacy of protons is associated with increased ATM and Chk1 phosphorylation as well as increased H2AX phosphorylation. Further, Rad51 knockdown led to persistent radiation-induced DNA damage as reflected by γ-H2AX foci staining. Our results demonstrate that Rad51 inhibition significantly increased the radiosensitivity of NSCLC cells, and that this increased radiosensitivity was mediated by the suppression of DNA repair. The effect of siRad51 on radiosensitization to protons was more significant than to photons.