f AURKB, is known to induce TW-37 Bcl-2 inhibitor cell cycle arrest, yielding G2/M phase cells or polyploidy . Previous studies have linked G2/M phase cells with increased radiosensitization in adenocarcinoma and colon carcinoma cell lines . Because AURKB inhibition results in increased levels of cellular polyploidy , inhibition of AURKB results in increased susceptibility to apoptosis . This provides a strong rationale that other treatments administered concurrently with AURKB inhibitors, including radiation therapy, could be quite effective in increasing treatment efficacy. Among the various types of prostate cancer cell lines that have been established for preclinical testing, both PC3 and DU145 human derived prostate cancer cells lines are notable for their relative insensitivity to androgen treatment, owing to their lack of the intracellular androgen receptor .
These cell lines model an important population of patients who have prostate cancer that is resistant or refractory to hormone ablation therapy. The effects of AZD1152 on prostate cancer have not been studied previously, and it is unknown whether the AURKB inhibitor Cyclopamine 4449-51-8 AZD1152 increases the sensitivity of androgen resistant human prostate cancer cells to radiation treatment. Herein we examined the effects of AZD1152 on cell cycle distribution, DNA damage and radiosensitivity of PC3 and DU145 prostate cancer cells. We tested the hypothesis that AZD1152 increases the radiosensitivity of androgen insensitive PC3 and DU145 human prostate cancer cells.
If AZD1152 or other AURKB inhibitors could be demonstrated to increase the therapeutic index for androgen resistant prostate cancer, this would have a significant clinical impact. MATERIALS AND METHODS Cell Culture and Reagents PC 3 and DU145 cells were cultured in RPMI 1640 medium containing 10 % fetal bovine serum and 1% penicillin/streptomycin. All cells were incubated at 37°C in 95% air/5% CO2. AZD1152 was obtained from AstraZeneca . Western Immunoblotting Cells were treated with various concentrations of AZD1152. They were collected at various times and then washed with ice cold PBS twice before the addition of lysis buffer including protease inhibitor cocktail and phosphatase inhibitor cocktail I . Protein concentration was quantified by the Bio Rad method. Equal amounts of protein were loaded into each well and separated by 12.5% or 15% SDS PAGE gel, followed by transfer onto PVDF membranes .
Membranes were blocked with 5% nonfat dry milk in PBST for 1 h at room temperature. The blots were then incubated with Niermann et al. Page 2 Radiat Res. Author manuscript, available in PMC 2012 April 1. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript anti phosphohistone H3 , anti Aurora B , and anti Actin for 1 h at 4°C. Goat anti rabbit IgG secondary was incubated for 45 min at room temperature. Western blots were developed using the chemiluminescence detection system according to the manufacturer,s protocol and autoradiography. Cell Cycle Analysis Cells were seeded in 10 cm2 dishes 24 h before AZD1152 treatment and then treated with various doses of AZD1152 for 48 h. The cells were then collected by trypsinization, fixed with 70% ethanol, and stored overnight at �?0°C.
Cells were then collected by centrifugation, resuspended in 1 ml of PBS with 100 μl of 200 g/ml DNase free RNase A, and incubated at 37°C for 30 min. Propidium iodide was then added, and the cells were incubated at room temperature for 5 min. The number of cells in each phase of the cell cycle was determined and calculated as a percentage of the total cell population. Clonogenic Assay Cells were treated with AZD1152 or DMSO . Cells were irradiated with 0 to 6 Gy at a dose rate of 1.8 Gy/min using a 137Cs irradiator . After irradiation, the medium was changed and cells were incubated at 37°C for 8 days. Cells were then fixed for 30 min with 70% methanol and stained for 30 min with 1% methylene blue in water. After staining, colonies were counted