Histone Deacetylase Inhibitors Radiosensitize Human Melanoma Cells by Suppressing DNA Repair Activity

Abstract

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.

Modulation of cellular radiation responses by histone deacetylase inhibitors

“…It is established that the g-H2AX form of the histone has an important role in recruiting repair factors to nuclear foci following induction of DSBs (Paull et al, 2000;Celeste et al, 2003;Olive and Banath, 2004). Recent studies have shown that incubation of cells with HDAC inhibitors including MS-275 and sodium butyrate, results in prolonged expression of induced g-H2AX foci (Camphausen et al, 2004a;Munshi et al, 2005). These findings indicate that HDAC inhibitor-mediated radiosensitization is associated with a decrease in the repair of DSBs.…”

“…For example, it has been shown that the radiation-induced increase in Rad51 and DNA-PKcs expression is markedly attenuated by pretreatment of a human prostate cancer cell-line with SAHA (Chinnaiyan et al, 2005). Similarly, another study has demonstrated that sodium butyrate decreases the expression of critical DNA repair proteins Ku70, Ku86 and DNA-PKcs in two melanoma cell-lines (Munshi et al, 2005).…”

“…Classical molecules such as sodium butyrate (Arundel et al, 1985;Arundel and Leith, 1987;Leith, 1988;Chung et al, 2000;Munshi et al, 2005), phenyl butyrate (Munshi et al, 2005), tributyrin (Munshi et al, 2005) (Zhang et al, 2004a) and numerous hydroxamic acid analogues such as M344 (Zhang et al, 2004b;Jung et al, 2005), have been investigated for their radiation sensitizing properties in vitro. The common theme is a synergistic effect following treatment of cells with HDAC inhibitors and subsequent irradiation.…”

“…However, it has been shown that HDAC inhibitors can modulate the sensitivity of cells to ionizing radiation even at relatively low concentrations, that are sufficient to alter histone acetylation status but are nontoxic and do not induce cell-cycle arrest. The ability of HDAC inhibitors to modulate chromatin architecture, to interact with critical signal transduction proteins involved in DNA damage response pathways and to regulate transcription, particularly of key genes involved in the DNA DSB repair pathway, have been proposed to account for the synergistic effect (Camphausen et al, 2004a, b;Kim et al, 2004;Zhang et al, 2004a;Jung et al, 2005;Munshi et al, 2005).…”

Cutaneous T‐cell lymphoma: 2011 update on diagnosis, risk‐stratification, and management

“…The 18 human HDACs may be classified as either zinc-or NAD 1 -dependent and further subclassified into class I (HDAC1, 2, 3, and 8), class II (HDAC4, 5, 6, 7, 9, and 10), class III (including NAD 1 -dependent sirtuins), and class IV (HDAC11) HDACs. As HDACs regulate a wide variety of processes involved in carcinogenesis, multiple mechanisms may explain the clinical activity of HDAC inhibitors [249,250], including altered gene expression of cell-cycle and apoptotic regulatory proteins [251][252][253][254][255], acetylation of nonhistone proteins regulating cell growth and survival [256][257][258][259], angiogenesis [260,261], aggresome formation [262], and DNA repair [263]. In addition, HDAC inhibitors may have important effects on the tumor microenvironment via reactive oxygen species [264,265], enhanced antigen presentation [266] and downregulation of immunomodulatory cytokines, like IL-10 [267].…”