Introduction: Double strand breaks are amongst the most critical lesions caused by ionizing radiation that can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). PARP1, Ku70 and DNA-PKcs play an important role in the in NHEJ whereas BRCA2 and Rad51 are essential for the HR. Hyperthermia temporary inactivates of BRCA2 hereby interfering with the HR and Cisplatin disrupts the NHEJ. Poly-[ADP-ribose]-polymerase-1, which plays important role in SSB repair, NHEJ and backup-NHEJ is blocked by the PARP1-inhibitor. Our aim is to further enhance the effectiveness of cisplatin and PARP1-inhibition in combination with radiotherapy and/or hyperthermia.
Methods: Cervical carcinoma cells (SiHa) were treated at different temperatures (39-45˚C), gamma-irradiation doses (0-8Gy), cDDP and PARP1-inhibitor (NU1025). Clonogenic assays were performed to measure survival and γH2AX staining was used to visualize DSBs. To elucidate mechanisms of action expression levels of different DNA repair proteins BRCA2, Ku70, Rad51 and DNA-PKcs were investigated after 42.5˚C (1 h) using western blot.
Results: Combined treatment of hyperthermia and radiation resulted in increased number of γH2AX foci as compared to radiation alone. Hyperthermia when combined with cDDP & PARP1-inhibitor and radiation & PARP1 inhibitor significantly decreased cell survival. Western blot demonstrated a decreased expression of BRCA2 protein at 30 min after hyperthermia treatment.
Discussion/Conclusion: Adding PARP1-inhibitor significantly improves the effectiveness of combined hyperthermia-radiotherapy and combined hyperthermia-cDDP treatment on cervical carcinoma cells. Hyperthermia affects DNA-DSB repair as is indicated by increased γH2AX foci numbers and decreased BRCA2 expression.
Financial support from The Dutch Cancer Foundation (UVA 2008-4019 and UVA 2012-5540)