Radiosensitization by PARP inhibition to proton beam irradiation in cancer cells

https://doi.org/10.1016/j.bbrc.2016.07.062Get rights and content

Highlights

  • Effective radiosensitizers for particle radiation therapy have not been reported.

  • PARP inhibitor treatment radiosensitized after proton beam irradiation.

  • The sensitization at Bragg peak was greater than that at entrance region.

  • DSB induction and G2/M arrest is involved in the sensitization mechanism.

Abstract

The poly(ADP-ribose) polymerase (PARP)-1 regulates DNA damage responses and promotes base excision repair. PARP inhibitors have been shown to enhance the cytotoxicity of ionizing radiation in various cancer cells and animal models. We have demonstrated that the PARP inhibitor (PARPi) AZD2281 is also an effective radiosensitizer for carbon-ion radiation; thus, we speculated that the PARPi could be applied to a wide therapeutic range of linear energy transfer (LET) radiation as a radiosensitizer. Institutes for biological experiments using proton beam are limited worldwide. This study was performed as a cooperative research at heavy ion medical accelerator in Chiba (HIMAC) in National Institute of Radiological Sciences. HIMAC can generate various ion beams; this enabled us to compare the radiosensitization effect of the PARPi on cells subjected to proton and carbon-ion beams from the same beam line. After physical optimization of proton beam irradiation, the radiosensitization effect of the PARPi was assessed in the human lung cancer cell line, A549, and the pancreatic cancer cell line, MIA PaCa-2. The effect of the PARPi, AZD2281, on radiosensitization to Bragg peak was more significant than that to entrance region. The PARPi increased the number of phosphorylated H2AX (γ-H2AX) foci and enhanced G2/M arrest after proton beam irradiation. This result supports our hypothesis that a PARPi could be applied to a wide therapeutic range of LET radiation by blocking the DNA repair response.

Introduction

Radiation therapy using accelerated particles such as proton and carbon-ion beams is used as cancer treatment for various organs and good clinical outcomes are being reported [1]. Because of a physical characteristic called Bragg peak (BP), accelerated charged particle radiation therapies can supply a proper dose distribution with fewer gates compared to photon radiation therapy. In addition to this advantage, proton beam radiation has an almost equal biological effect to photon radiation [2], making its clinical application easier as an alternative to photon therapy since therapeutic efficiency and adverse events could be predicted. There have been several reports that showed the radiosensitization effect on the carbon-ion beam [3], [4], [5]; however, effective radiosensitizers for particle radiation therapy are not currently available.

Targeting enzyme molecules that are important in DNA repair pathways with small molecules has recently been considered an attractive strategy for cancer therapy [6]. Poly(ADP-ribose) polymerase (PARP)-1 is one of the essential proteins involved in base excision repair (BER) and promotes DNA repair by modifying key proteins [7]. PARP inhibition causes synthetic lethality in cells harboring mutations in BRCA1/2, which encode for important proteins in the homologous recombination (HR) pathway or in HR-deficient cancer cells. Recently, AZD2281, a PARP inhibitor (PARPi), has been approved by the FDA and the European Commission as an anti-cancer drug for serous ovarian cancer with BRCA1/2 mutations [8]. PARP inhibition also enhances the cytotoxicity of ionizing radiation in various cancer cells and animal models [9], [10], [11]. We have demonstrated that this PARPi is an effective radiosensitizer for carbon-ion irradiation, as well as γ-ray irradiation [3]. The underlying mechanism includes the induction of S phase arrest through an increased DSB levels and a local delay in DNA double strand break (DSB) processing. From this result, we speculated that the PARPi could be applied to a wide therapeutic range of linear energy transfer (LET) radiation as a radiosensitizer. In this study, we investigated the effect of PARP inhibition on the responses to proton beam irradiation.

Section snippets

Chemicals and antibodies

The PARPi AZD2281 (olaparib) was obtained from Selleck Chemicals (Houston, TX, USA) and was dissolved in dimethyl sulfoxide (DMSO). The anti-γ-H2AX (Ser-139) antibody was purchased from EMD Millipore (Billerica, MA, USA). Anti-phosphorylated p53 (Ser-15) and anti-histone H3 antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-phosphorylated histone H3 (Ser-10) antibody was purchased from Abcam (Cambridge, UK). Anti-β-actin was purchased from Sigma-Aldrich (St. Louis,

Sensitization effect on cancer cell survival by treatment with the PARPi AZD2281

To assess the radiosensitization effect of AZD2281 on different LET proton beams, BP and ER beam irradiation were used for clonogenic survival assays. Because proton therapy is already applied clinically for lung and pancreatic cancer treatments, we used A549 (human lung cancer) and MIA PaCa-2 (human pancreatic cancer) cell lines in this study [13], [14].

Fig. 2 shows the dose-response curves of A549 (A, B) and MIA PaCa-2 (C, D) cells irradiated with proton beams, with either ER (A, C) or BP (B,

Discussion

In the present study, we demonstrated that the PARPi AZD2281 is an effective radiosensitizer for proton beam irradiation. The underlying mechanism of radiosensitization to proton beam irradiation by the PARPi is suggested to be caused by a delay in DDR and DSB processing.

The effect of the PARPi on the cell cycle progression of A549 cells was different from the effect in MIA PaCa-2 cells. In the A549 cell line, where the p53 gene is unmutated [15], p53 induces a prolonged G1 phase arrest with

Acknowledgements

This study was one of the Research Projects with Heavy Ions at NIRS-HIMAC (12J394). We appreciate the help and suggestions provided by the HIMAC support team, Dr. Akira Fujimori at the NIRS, Dr. Keisuke Sasai at Juntendo University Faculty of Medicine and Dr. Hitoshi Nakagama at the National Cancer Center. This work was supported by a grant-in-aid for Young Scientists (B) of Scientific Research (KAKEN), Japan (grant no. 30626669) and MEXT KAKENHI (15K14416).

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      Thus, three studies investigated the effect of PARP inhibition on the response of high-LET irradiation and showed that in vitro enhancement ratio ranged from 1.4 to 2.5, depending on LET and PARPi concentrations (Hirai et al., 2016, 2012; Wéra et al., 2019). Authors implied that PARPi sensitized cells with high-LET irradiation by enhancing the number of sub-lethal oxidative clustered DNA damage, thus leading to a delay in DNA damage repair (Hirai et al., 2016). Recently, one study showed that a RAD51 inhibitor enhanced radiosensitization after protontherapy (Wéra et al., 2019).

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