<title>ABSTRACT</title>
Proton beam therapy (PBT) combined with chemotherapy, such as cis-diamminedichloroplatinum (II) (CDDP) and 5-fluorouracil (5-FU), has been employed as an alternative approach to improve clinical outcomes. PBT has been reported to be effective against esophageal cancer. However, apart from 5-FU and CDDP, almost no other drug has been tested in combined chemotherapy with PBT. Therefore, we investigated the effects of a poly (ADP-ribose) polymerase inhibitor on enhancing proton beam effects using esophageal cancer cell lines that exhibit resistance to radiation and CDDP. Esophageal squamous cell carcinoma cell lines OE-21 and KYSE-450 were exposed to the drugs for 1 h prior to irradiation. The cell survival curve was obtained using a clonogenic assay and the sensitizing effect ratio (SER) was calculated. The clonogenic assay was used to compare the effect of multi-fractioned irradiation between 8 Gy/1 fraction (fr) and 8 Gy/4 fr. γH2AX, Rad51, BRCA1, BRCA2 and 53BP1 foci were detected via immunofluorescence. Olaparib exhibited an SER of 1.5–1.7 on PBT. The same sensitizing effect was exhibited in multi-fractioned irradiation, and the combined use increased the expression of double-strand breaks and homologous recombination-related genes in an additive manner. Such additive effects were not observed on non-homologous end joining-related genes. We demonstrated that olaparib has a high sensitizing effect on PBT in platinum- and radiation-resistant esophageal cancer cells. Our results suggest a potential clinical application of olaparib-proton irradiation (PT) against platinum- and radiation-resistant esophageal cancer.