Radiosensitisation of glioblastoma cells using a histone deacetylase inhibitor (SAHA) comparing carbon ions with X-rays

Short running title: HDAC inhibition with low and high LET radiation Abstract Purpose: Prognosis for patients with glioblastoma (GBM) remains poor, and new treatments are needed. Here we use a combination of two novel treatment modalities, carbon ions and a histone deacetylase inhibitor (HDACi). We compare these to conventional X-rays, measuring the increased effectiveness of carbon ions as well as radiosensitisation using HDACi. Materials and methods: Suberoylanilide hydroxamic acid (SAHA) was used at a non-toxic concentration of 0.5 µM in combination with 85 keV μm-1 carbon ions, and 250 kVp X-rays for comparison. Effects were assayed using clonogenic survival, γH2AX foci repair kinetics and measuring chromatin decondensation. Results: Dose toxicity curves show that human GBM LN18 cells were more sensitive to SAHA compared to U251 cells at higher doses, but there was little effect at low doses. When combined with radiation, clonogenic assays show that the Sensitiser Enhancement Ratio with carbon ions at 50% survival (SER 50) was about 1.2 and 1.5 for LN18 and U251 respectively, but was similar for X-rays at about 1.3. The repair half-life of γH2AX foci was slower for cells treated with SAHA and was most noticeable in U251 cells treated with carbon ions where after 24 hr, more than double the number of foci remained in comparison to the untreated cells. Hoechst fluorescent dye incorporation into the nucleus showed significant chromatin decondensation and density homogenisation with SAHA treatment for both cell lines. Conclusions: Our results suggest a vital role of histone deacetylases (HDAC) in the modulation of DNA damage response and support the use of SAHA for the treatment of GBM through the combination with heavy ion therapy.