Histone deacetylase inhibitors radiosensitize human melanoma cells by suppressing DNA repair activity.
OBJECTIVE
Histone deacetylase (HDAC) inhibitors have emerged recently as promising anticancer agents. They arrest cells in the cell cycle and induce differentiation and cell death. The antitumor activity of HDAC inhibitors has been linked to their ability to induce gene expression through acetylation of histone and nonhistone proteins. However, it has recently been suggested that HDAC inhibitors may also enhance the activity of other cancer therapeutics, including radiotherapy. The purpose of this study was to evaluate the ability of HDAC inhibitors to radiosensitize human melanoma cells in vitro.


METHODS
A panel of HDAC inhibitors that included sodium butyrate (NaB), phenylbutyrate, tributyrin, and trichostatin A were tested for their ability to radiosensitize two human melanoma cell lines (A375 and MeWo) using clonogenic cell survival assays. Apoptosis and DNA repair were measured by standard assays.


RESULTS
NaB induced hyperacetylation of histone H4 in the two melanoma cell lines and the normal human fibroblasts. NaB radiosensitized both the A375 and MeWo melanoma cell lines, substantially reducing the surviving fraction at 2 Gy (SF2), whereas it had no effect on the normal human fibroblasts. The other HDAC inhibitors, phenylbutyrate, tributyrin, and trichostatin A had significant radiosensitizing effects on both melanoma cell lines tested. NaB modestly enhanced radiation-induced apoptosis that did not correlate with survival but did correlate with functional impairment of DNA repair as determined based on the host cell reactivation assay. Moreover, NaB significantly reduced the expression of the repair-related genes Ku70 and Ku86 and DNA-dependent protein kinase catalytic subunit in melanoma cells at the protein and mRNA levels. Normal human fibroblasts showed no change in DNA repair capacity or levels of DNA repair proteins following NaB treatment. We also examined gamma-H2AX phosphorylation as a marker of radiation response to NaB and observed that compared with controls, gamma-H2AX foci persisted long after ionizing exposure in the NaB-treated cells.


CONCLUSIONS
HDAC inhibitors radiosensitize human tumor cells by affecting their ability to repair the DNA damage induced by ionizing radiation and that gamma-H2AX phosphorylation can be used as a predictive marker of radioresponse.
Department of Experimental Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA. amunshi@mdanderson.org
Anupama
A
Munshi
John F
JF
Kurland
Takashi
T
Nishikawa
Toshimitsu
T
Tanaka
Marvette L
ML
Hobbs
Susan L
SL
Tucker
Sheikh
S
Ismail
Craig
C
Stevens
Raymond E
RE
Meyn
D000107:Acetylation / Q000187:drug effects; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000528:radiation effects; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D000072200:Ku Autoantigen; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D014280:Triglycerides / Q000494:pharmacology
D034961:Antigens, Nuclear; D002087:Butyrates; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C468783:H2AX protein, human; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D014280:Triglycerides; C012589:trichostatin A; D006655:Histone Deacetylases; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; C005830:tributyrin