Modulation of radiation response by histone deacetylase inhibition.
OBJECTIVE
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
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.


CONCLUSIONS
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.
Chinnaiyan
Prakash
P
Department of Human Oncology, University of Wisconsin School of Medicine and Comprehensive Cancer Center, Madison, WI 53792-0600, USA.
Vallabhaneni
Geetha
G
Armstrong
Eric
E
Huang
Shyh-Min
SM
Harari
Paul M
PM
D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004354:Drug Screening Assays, Antitumor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism; D005910:Glioma / Q000201:enzymology / Q000473:pathology / Q000532:radiotherapy*; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008297:Male; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000473:pathology / Q000532:radiotherapy*; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011836:Radiation Tolerance / Q000187:drug effects; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D000077337:Vorinostat
D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D011518:Proto-Oncogene Proteins; D011838:Radiation-Sensitizing Agents; D000077337:Vorinostat; D011065:Poly(ADP-ribose) Polymerases; D066246:ErbB Receptors; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020169:Caspases