Purpose: 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. Experimental Design: 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 g-H2AX phosphorylation as a marker of radiation response to NaB and observed that compared with controls, g-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 g-H2AX phosphorylation can be used as a predictive marker of radioresponse.

Vorinostat (suberoylanilide hydroxamic acid) is the prototype of a family of hybrid polar compounds that can induce growth arrest in transformed cells and shows promise for the treatment of cancer. Vorinostat specifically binds to and inhibits the activity of histone deacetylases resulting in acetylation of nucleosomal histones and an activation of gene transcription. Because histone deacetylases modulate chromatin structure and gene expression, both of which can influence radioresponse, this study was designed to examine the capacity of Vorinostat to influence radiation response in human tumor cells and investigate the mechanism underlying these interactions. Vorinostat induced hyperacetylation of histone H4 in a dose-dependent manner. We tested its ability to radiosensitize three human tumor cell lines (A375, MeWo, and A549) using clonogenic cell survival assays. Clonogenic cell survival assay showed that Vorinostat significantly radiosensitized all three tumor cell lines, substantially reducing the surviving fraction at 2 Gy. We examined potential mechanisms that may contribute to the enhanced radiation response induced by Vorinostat. Vorinostat and radiation alone did not induce apoptosis in the melanoma cell line. However, enhanced apoptosis was observed when cells were exposed to both Vorinostat and radiation, suggesting that Vorinostat renders tumor cells more susceptible to radiation-induced apoptosis. Results from DNA damage repair analysis in cultured A375 cells showed that Vorinostat had a strong inhibitory effect on the nonhomologous end joining pathway after radiation. A detailed examination of the involvement of the DNA repair pathway following Vorinostat treatment showed that Vorinostat reduced the expression of the repair-related genes Ku70, Ku80, and Rad50 in A375 cells as detected by Western blot analysis. We also examined ;-H2AX phosphorylation as a predictive marker of radiotherapy response to Vorinostat and observed that the combination of Vorinostat and radiation caused a prolongation of expression of DNA repair proteins such as ;-H2AX. Overall, we conclude that Vorinostat enhances tumor radioresponse by multiple mechanisms that may involve antiproliferative growth inhibition and effects on DNA repair after exposure to radiation.

Purpose: Overexpression of the epidermal growth factor receptor (EGFR) promotes unregulated growth, inhibits apoptosis, and likely contributes to clinical radiation resistance of non^small cell lung cancer (NSCLC). Molecular blockade of EGFR signaling is an attractive therapeutic strategy for enhancing the cytotoxic effects of radiotherapy that is currently under investigation in preclinical and clinical studies. In the present study, we have investigated the mechanism by which gefitinib, a selective EGFR tyrosine kinase inhibitor, restores the radiosensitivity of NSCLC cells. Experimental Design: Two NSCLC cell lines, A549 and H1299, were treated with 1 Amol/L gefitinib for 24 h before irradiation and then tested for clonogenic survival and capacity for repairing DNA double strand breaks (DSB). Four different repair assays were used: host cell reactivation, detection of g-H2AX and pNBS1 repair foci using immunofluorescence microscopy, the neutral comet assay, and pulsed-field gel electrophoresis. Results: In clonogenic survival experiments, gefitinib had significant radiosensitizing effects on both cell lines. Results from all four DNA damage repair analyses in cultured A549 and H1299 cells showed that gefitinib had a strong inhibitory effect on the repair of DSBs after ionizing radiation.The presence of DSBs was especially prolonged during the first 2 h of repair compared with controls. Immunoblot analysis of selected repair proteins indicated that pNBS1 activation was prolonged by gefitinib correlating with its effect on pNBS1-labeled repair foci. Conclusions: Overall, we conclude that gefitinib enhances the radioresponse of NSCLC cells by suppressing cellular DNA repair capacity, thereby prolonging the presence of radiation-induced DSBs.

No abstract