Abstract A107: S 78454/PCI-24781, a novel HDAC inhibitor, targets the DNA damage response leading to radiosensitization of both normoxic and hypoxic NSCLC cell lines.
Authors: Rivera Sofia , Leteur Celine , Menard Jean , Lelievre Helene , Bescond Anne Jacquet , Berthier Laurence Kraus , Depil Stephane , Deutsch Eric 
Published: June 2012

Introduction: Histone deacetylases inhibitors (HDACi) are promising anti-cancer agents. Recent data showing synergy of HDACi with ionizing radiation (IR) and other DNA damaging agents have suggested that HDACi may act, in part, by inhibiting DNA repair. Tumor hypoxia is a negative prognostic marker for patients undergoing IR. Hypoxic tumor cells are less radiosensitive than well-oxygenated ones and this is correlated with a lower rate of radioinduced DNA double strand breaks (DSB) under hypoxia. The purpose of this study was to determine the effect and mechanism of action of S 78454 (also called PCI-24781), a novel HDACi, in combination to IR under normoxia and hypoxia in NSCLC cells.

Material and Methods: We tested the effectiveness of combining IR and S 78454 on the human NSCLC cell line H460 and A549 using clonogenic survival assays. H460 and A549 cells were treated with vehicle control or respectively 0.1 M or 0.4μM of S 78454 for 24 hours under normoxia (21% oxygen) or hypoxia (0,1% oxygen), irradiated to 0–6Gy with a 137Cs source, and incubated for 24 hours. Cells were then incubated without S 78454 for 12 days and colonies were stained and counted. Plating efficiency and surviving fractions were calculated relative to those of unirradiated cells. Flow cytometry was performed for apoptosis analysis. γH2AX foci were assessed using immunofluorescence staining assays. Histone3 acetylated, γH2AX and Rad51 protein levels were evaluated by western blotting.

Results: Pretreating H460 and A549 cells with S 78454 respectively for 24 hours did synergistically enhance cell killing after IR compared with untreated irradiated control cells irrespective of hypoxia. Under normoxia, the mean surviving fraction at 2Gy (SF2) of cells untreated by S 78454 was 0.85 and 1 for H460 and A549 cells respectively, whereas the SF2 for H460 and A549 cells treated with S 78454 was 0.73 and 0.64 respectively. Under hypoxia, the mean SF2 of cells untreated by S 78454 was 0.63 and 0.72 for H460 and A549 cells respectively, whereas the SF2 for H460 and A549 cells treated with S 78454 was 0.36 and 0.56 respectively. A dose effect relationship was observed with increased radiosensitization when increasing S 78454 doses. S 78454 alone and in combination with IR (4Gy) enhanced DNA damage as evidenced by increased expression of H2AX which increased from 4 hours to at least 24 hours after treatment. The combination delayed replication recovery and caused a moderate induction of apoptosis as shown by increased sub-G1 population and Z-VAD/annexineV assay in H460 and A549 cells. Western blot analysis showed that after 24h of treatment by S 78454 levels of Rad51 decreased in both cell lines under hypoxia. IR (4Gy) slightly increased Rad51 expression under hypoxia in both cell lines from 1 hour after IR. In 24 hours pretreated hypoxic cells Rad51 expression decreased 1 hour after IR while levels of Rad51 remained stable in normoxic cells.

Conclusions: Our results indicate that inhibition of HDACs by S 78454 increases sensitivity of both normoxic and hypoxic NSCLC cells to IR and suggest that decreased DSB repair might play a key role even under hypoxia. Our data suggest that impaired DSB repair does not necessarily correlate with Rad51 diminution underscoring the fact that other DNA repair proteins might account for S 78454 induced radiosentization.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A107.