onse to AZD1152 treatment. The results presented here have confirmed our hypothesis that AZD1152 treatment of human derived PC3 and DU145 MLN8237 Aurora Kinase inhibitor prostate cancer cells results in increased sensitivity to radiation. One of the further primary goals of these investigations was to maximize the radiosensitizing effects of AZD1152 for these androgen insensitive prostate cancer cell lines. Because G2/M and polyploid cells predominately contain double stranded DNA, we sought to determine the treatment conditions with AZD1152 that result in the greatest proportion of G2/M phase and polyploid cells. Our experiments showed that AZD1152 induced inhibition of AURKB is both dose and time dependent and that 60 nM AZD1152 for 48 h resulted in the largest increase in polyploid and G2/M phase cells in both PC3 and DU145 cells.
These conditions were subsequently used to investigate the effects of radiation on DNA damage and cell survival. To better characterize the temporal effects of radiation and AURKB inhibition on PC3 and DU145 cells, we quantified DNA damage at two times. The first, at 30 Roscovitine 186692-46-6 min postirradiation, reflects the initial susceptibility of these cells to radiation induced DNA damage. The Niermann et al. Page 6 Radiat Res. Author manuscript, available in PMC 2012 April 1. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript second, at 6 h postirradiation, when compared longitudinally to the first time, is indicative of the extent of DNA repair. DNA repair begins soon after irradiation. γ H2AX foci peak within an hour, and focus half lives average between 2 and 4 h .
More damage was induced by radiation in both treated and control cells, though it was more sustained in AZD1152 treated populations. PC3 cells, which exhibited an increase in both G2/M phase and polyploid cells, sustained more damage than DU145 cells, in which polyploid cells predominated. Also of note, PC3 cells lack p53 while DU145 cells express p53 mutations. These data are thus consistent with previous observations that p53 deficient cells have a longer H2AX half life . Individual cells that are incapable of repairing DNA breaks will eventually undergo cell death . Thus either an increase in DNA damage or a delay in DNA repair or both may result in increased radiosensitization. This was borne out in the radiation survival data .
Greater cytotoxicity was exhibited by PC3 cells treated with AZD1152 compared to control , with a drug enhancement ratio of 1.53 at a surviving fraction of 0.1 . In comparison, DU145 cells, which were previously shown to be composed primarily of polyploid cells after AZD1152 treatment, also showed enhanced radiosensitization, with a drug enhancement ratio of 1.71 at a surviving fraction of 0.4 . Although it is possible that factors other than DNA damage may play a role in radiosensitization, these data indicate that polyploid cells may be more susceptible to radiation induced cell death. AURKB is highly expressed in hormone refractory prostate cancer in patients and in DU145 and PC3 cells . Inhibition of AURKB using siRNA technology was associated with inhibition of growth of prostate cancer xenografts .
Additionally, concomitant use of siRNAs against AURKB and EGFR resulted in further suppression of tumor growth. These results demonstrate the value of targeting several pathways and using multiple modalities to achieve optimal response to therapy. Radiotherapy is an essential treatment modality for prostate cancer and is frequently used with hormone therapy in managing locally advanced cases. Resistance of prostate cancer to the current available treatments, including hormone therapy, surgery and radiation therapy, is a significant clinical problem that affects the survival of patients, and the development of new treatment strategies is therefore critical for improving patient outcome. Our data indicate a potential role for AZD1152 induced AURKB inhibition in the treatment of prostate cancer with radiation therapy. AZD1152 in