Inhibition of poly (ADP-ribose) polymerase-1 (PARP-1) to increase radiosensitivity of human pancreatic cancer (PAC) cell lines proficient in homology-directed repair (HDR).

Abstract

204 Background: Radiation-induced single strand DNA breaks (SSBs) are primarily repaired by base excision repair, of which PARP1 is a key component. Unrepaired SSBs may cause collapse of stalled replication forks during DNA replication generating DNA double-strand breaks (DSBs). PARP inhibition has been shown to potentiate the effect of radiotherapy (RT) in a variety of human cancers in vitro and in vivo. We investigated the ability of the PARP inhibitor olaparib to radiosensitize BRCA1/2 wild-type human PAC cell lines and quantified repair capability of an induced DSB by HDR using the I-SceI-induced DSB DR-GFP assay. Methods: MiaPaCa -2 and ASPC-1 human PAC cells lines were exposed to RT administered in a single fraction by 157-gammacell irradiator at doses of 2, 4, 6 and 8 Gy in the presence or absence of olaparib at a dose of 500nM (4 hours pre and 20 hours post RT). Plates were incubated at 37C for 10-14 days and stained with Giemsa. Clonogenic survival assays following olaparib continuous exposure at concentrations up to 10 µM for 10-14 days were also performed. HDR of an induced DNA DSB was assessed in ASPC1 and MiaPaCa cell lines by co-transfection with an I-SceI expression vector and the DR-GFP reporter plasmid(Pierce et al., 1999), and in BRCA2 deficient CAPAN-1 cells harboring a stably integrated chromosomal DR-GFP gene (Moynahan et al. 2001). Results: Linear quadratic models were fitted to the data sets to generate survival curves. The half maximal inhibitory concentration (IC 50) of RT was 2Gy for both cell lines. The sensitivity enhancement ratio (SER) at 25% survival was 1.44 and 1.53 for MiaPaCa-2 and ASPC-1 cell lines respectively. Both cell lines were resistant to treatment with single agent olaparib to dose of 10µM and were proficient in HDR of DNA DSBs when compared to the BRCA2-deficient Capan-1 cell line. Conclusions: Inhibition of PARP increases radiosensitivity in human PAC cell lines proficient in HDR of DNA DSBs and resistant to treatment with single agent PARP inhibitor. Therapeutic strategies incorporating PARP inhibition and RT offer potential to improve clinical outcomes for patients presenting with localized sporadic and BRCA mutated PAC.