Evaluation of Histone Deacetylase Inhibitors as Radiosensitizers for Proton and Light Ion Radiotherapy.

Alicia M Johnson,Paul F Wilson,Paula V Bennett,James H Jardine,Deborah J Keszenman,Anthony Hoang,Katherine Z Sanidad

doi:10.3389/fonc.2021.735940

Abstract

Significant opportunities remain for pharmacologically enhancing the clinical effectiveness of proton and carbon ion-based radiotherapies to achieve both tumor cell radiosensitization and normal tissue radioprotection. We investigated whether pretreatment with the hydroxamate-based histone deacetylase inhibitors (HDACi) SAHA (vorinostat), M344, and PTACH impacts radiation-induced DNA double-strand break (DSB) induction and repair, cell killing, and transformation (acquisition of anchorage-independent growth in soft agar) in human normal and tumor cell lines following gamma ray and light ion irradiation. Treatment of normal NFF28 primary fibroblasts and U2OS osteosarcoma, A549 lung carcinoma, and U87MG glioma cells with 5–10 µM HDACi concentrations 18 h prior to cesium-137 gamma irradiation resulted in radiosensitization measured by clonogenic survival assays and increased levels of colocalized gamma-H2AX/53BP1 foci induction. We similarly tested these HDACi following irradiation with 200 MeV protons, 290 MeV/n carbon ions, and 350 MeV/n oxygen ions delivered in the Bragg plateau region. Unlike uniform gamma ray radiosensitization, effects of HDACi pretreatment were unexpectedly cell type and ion species-dependent with C-12 and O-16 ion irradiations showing enhanced G0/G1-phase fibroblast survival (radioprotection) and in some cases reduced or absent tumor cell radiosensitization. DSB-associated foci levels were similar for proton-irradiated DMSO control and SAHA-treated fibroblast cultures, while lower levels of induced foci were observed in SAHA-pretreated C-12 ion-irradiated fibroblasts. Fibroblast transformation frequencies measured for all radiation types were generally LET-dependent and lowest following proton irradiation; however, both gamma and proton exposures showed hyperlinear transformation induction at low doses (≤25 cGy). HDACi pretreatments led to overall lower transformation frequencies at low doses for all radiation types except O-16 ions but generally led to higher transformation frequencies at higher doses (>50 cGy). The results of these in vitro studies cast doubt on the clinical efficacy of using HDACi as radiosensitizers for light ion-based hadron radiotherapy given the mixed results on their radiosensitization effectiveness and related possibility of increased second cancer induction.

Highlights

Despite the improved dose distributions and increased relative biological effectiveness (RBE) afforded by accelerated proton and carbon ion-based hadron radiotherapies [1], significant opportunities remain for enhancing their clinical effectiveness through pharmacological means to achieve tumor cell radiosensitization and normal tissue radioprotection
Survival curves for NFF28, A549, U2OS, and U87MG cells irradiated with 0.5–6 Gy g-rays are shown in Figure 1 for cultures pretreated with Suberoylanilide hydroxamic acid (SAHA) and Supplementary Figures 2, 3 for cultures pretreated with M344 and S-[6-(4-phenyl-2-thiazolylcarbamoyl)hexyl] thioisobutyrate (PTACH), respectively
Results of g-H2AX/53BP1 foci analyses conducted in quiescent G0/G1-phase cultures of NFF28 fibroblasts pretreated for 18 h with 10 μM SAHA and subsequently irradiated with 5–25 cGy doses of cesium-137 g-rays, 200 MeV protons, and 290 MeV/n C-12 ions are shown in Figures 8A–C, respectively, reported as numbers of mean (± SEM) IR-induced foci per unit dose

Summary

Introduction

Despite the improved dose distributions and increased relative biological effectiveness (RBE) afforded by accelerated proton and carbon ion-based hadron radiotherapies [1], significant opportunities remain for enhancing their clinical effectiveness through pharmacological means to achieve tumor cell radiosensitization and normal tissue radioprotection. Identifying effective radiosensitizers that synergistically enhance charged particle-induced tumor cell killing would allow for lower doses per fraction to be used, thereby reducing normal tissue exposures. Charged particles have been shown to induce higher relative frequencies of closely localized DNA double-strand breaks (DSBs) and clustered DNA damages along their tracks compared to low linear energy transfer (LET) photon radiations [2, 3]. This feature underlies their higher RBE for cell killing in vitro and tumor control in vivo [4]. Exposures to charged particles, including low LET protons, are consistently associated with higher relative induction of DSBassociated foci, prematurely condensed chromosomal breaks, and simple and complex chromosomal aberrations measured in vitro post-irradiation compared to X- and g-rays [9, 12,13,14,15]

Methods

Results

Conclusion