Inhibition of histone deacetylases (HDACs) leads to hyperacetylation of histones, which in turn regulates transcriptional activation of specific genes through the relaxation of chromatin conformation. Since, the studies with these inhibitors in combination with radiation are lacking, the purpose of the present study was to investigate the effects of three novel HDAC inhibitors: VAD-18 and VAD-20 (Zn2+-chelating, short chain fatty acids) and (S)-11 ((S)-HDAC-42), a second generation inhibitor (an optically active α-branched phenylbutyryl derivative) in prostate cancer cell lines, PC-3, DU145 and LN-3 with ionizing radiation (IR). A decrease in surviving fraction in PC-3 cells with increasing concentration of VAD-18 and VAD-20 was observed. VAD-18 (IC50=0.5 μM) was more cytotoxic than VAD-20 (IC50=7.5 μM). Colony forming assays showed significant radiosensitizing effects with both the inhibitors in PC-3 cells compared to IR alone. IC50 concentrations of (S)-HDAC-42 and SAHA showed that LN-3 cells were most sensitive to both (S)-HDAC-42 and SAHA compared to DU-145 and PC-3 cells. (S)-HDAC-42 (RER=9.8) was able to enhance the radiation effects in PC-3 cells much more effectively than VAD-18 (RER=1.5) and VAD-20 (RER=2.4). A significant transient block in G2/M phase of the cell cycle was observed with either 2 Gy of radiation or drugs alone up to 12 h post-treatment by flow cytometry. Exposure to VAD-18 resulted in an additional delay following irradiation, which was significant, while VAD-20 had a lesser effect. An increase in NFκB activity was observed with either the drugs alone or radiation alone as assessed by EMSA. While, p65 protein was present in the nuclear extract till 6 h post-treatment with either drugs or IR alone (Western blot), it disappeared following the combined treatment implying that the inhibitors sensitize the cells to IR by reducing the levels of IR-induced p65 translocation in the nucleus. In addition, levels of BclXL and Bcl2 reduced following combined treatment while levels of pro-apoptotic protein, Bax increased. VAD-20 in combination with 2 Gy, however reduced the NFκB activity. VAD-20 has more radiosensitizing effects than VAD-18 in PC-3 cells, which could be due to reduced activity of NFκB. These studies demonstrate that radiosensitizing effects of HDAC inhibitors are mediated through cell cycle distribution changes, abrogation of translocation of p65 to the nucleus and inhibition of NFκB activity. Studies are in progress to investigate radiosensitizing effects of (S)-HDAC-42 and SAHA in other prostate cancer cell lines and to further understand the role of NF-κB and IκB in the radiosensitizing effects of HDAC inhibitors. It is hypothesized that radiation in combination with HDAC inhibitors may render effects involving non- epigenetic events such as nucleo-cytoplasmic shuttling. This study was funded by a post-doctoral award from DOD-PCRP. #

[Proc Amer Assoc Cancer Res, Volume 47, 2006]