Modulation of radiation response by histone deacetylase inhibition.

PURPOSE: Histone deacetylase (HDAC) inhibitors, which modulate chromatin structure and gene expression, represent a class of anticancer agents that hold particular potential as radiation sensitizers. In this study, we examine the capacity of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) to modulate radiation response in human tumor cell lines and explore potential mechanisms underlying these interactions.

METHODS AND MATERIALS: Cell proliferation: Exponentially growing tumor cells were incubated in medium containing 0-10 microM of SAHA for 72 h. Cells were fixed/stained with crystal violet to estimate cell viability. Apoptosis: Caspase activity was analyzed by fluorescence spectroscopy using a fluorescein labeled pan-caspase inhibitor. Cells were harvested after 48 h of exposure to SAHA (1.0 microM), radiation (6 Gy), or the combination. Whole cell lysates were evaluated for poly(ADP-ribose) polymerase (PARP) cleavage by western blot analysis. Radiation survival: Cells were exposed to varying doses of radiation +/- 3 days pretreatment with SAHA (0.75-1.0 microM). After incubation intervals of 14-21 days, colonies were stained with crystal violet and manually counted. Immunocytochemistry: Cells were grown and treated in chamber slides. At specified times after treatment with SAHA, cells were fixed in paraformaldehyde, permeabilized in methanol, and probed with primary and secondary antibody solutions. Slides were analyzed using an epifluorescent microscope.

RESULTS: SAHA induced a dose-dependent inhibition of proliferation in human prostate (DU145) and glioma (U373vIII) cancer cell lines. Exposure to SAHA enhanced radiation-induced apoptosis as measured by caspase activity (p < 0.05) and PARP cleavage. The impact of SAHA on radiation response was further characterized using clonogenic survival analysis, which demonstrated that treatment with SAHA reduced tumor survival after radiation exposure. We identified several oncoproteins and DNA damage repair proteins (epidermal growth factor receptor, AKT, DNA-PK, and Rad51) that show differential expression after exposure to SAHA. These proteins may contribute to mechanistic synergy between HDAC inhibition and radiation response.

CONCLUSION: These preclinical results suggest that treatment with the HDAC inhibitor SAHA can enhance radiation-induced cytotoxicity in human prostate and glioma cells. We are examining the capacity of HDAC inhibitors to modulate radiation response and tumor control in animal xenograft model systems to strengthen the rationale for future clinical trial exploration.