Biology contributions
Modulation of radiation response by histone deacetylase inhibition

https://doi.org/10.1016/j.ijrobp.2004.12.088Get rights and content

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 μM 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 μM), 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 μM). 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.

Introduction

Histone deacetylase (HDAC) inhibitors represent a class of anticancer agents that may prove effective as radiation sensitizers. HDAC inhibitors serve to regulate chromatin architecture through modification of histone tails. Histone proteins organize DNA into nucleosomes that are regular repeating structures of chromatin. The acetylation status of histones influences chromatin structure, which in turn serves to regulate gene expression. Two classes of enzymes can affect the acetylation of histones—histone acetyltransferases (HATs) and HDACs. Alterations in baseline HAT or HDAC activity have been identified in several human cancers. HDAC inhibition de-represses transcriptional activity of a set of genes that are “preprogrammed” to be responsive to changes in histone acetylation status (1, 2). HDAC inhibitors therefore promote hyperacetylation of histones, which in turn leads to chromatin relaxation and selective expression of genes that promote tumor suppression.

The rationale for combining HDAC inhibitors with radiation in cancer therapy can be considered at several levels. The combination of HDAC inhibitors with radiation may provide general therapeutic advantage because of the differential toxicity associated with each treatment modality. Further, underlying mechanisms of action for HDAC inhibitors suggest the potential for synergy with radiation deriving from physical interaction between HDAC inhibitors and chromatin architecture as well as differential expression of genes regulated by histone acetylation.

The present study explores the potential therapeutic value of combining the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) with radiation. Prostate and glioma cell lines are selected for study in light of the central role of radiation for these cancers, and the need to identify new methods to enhance local tumor control. The data confirm that treatment with SAHA reduces clonogenic survival and enhances radiation-induced apoptosis in human prostate and glioma cancer cell lines. Furthermore, potential cellular mechanisms underlying the enhanced antitumor activity are examined. Preliminary data suggest that exposure to SAHA induces differential regulation of genes involved in promitogenic and survival signaling and DNA damage repair.

Section snippets

Chemicals

Cell culture media was obtained from Life Technologies Inc. (Gaithersburg, MD). Primary antibodies against epidermal growth factor receptor (EGFR), poly(ADP-ribose) polymerase (PARP), and AKT were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); pAKT was obtained from Cell Signaling Technologies (Beverly, MA); Rad51 and DNA-PK were obtained from Neomarker (Freemont, CA); and α-tubulin was obtained from Oncogene Research Products (Cambridge, MA). ECL+ chemiluminescence detection

SAHA inhibits cell proliferation and clonogenic capacity

We examined the antiproliferative effects of SAHA in prostate (DU145) and glioma (U373vIII) cancer cell lines. As shown in Fig. 1a, cellular proliferation was inhibited in a dose-dependent manner after a 3-day exposure to SAHA. Based on known action mechanisms of HDAC inhibitors, which involve gene regulation and biochemical differentiation (3), we hypothesized that maximal growth inhibitory effects of SAHA might be achieved after longer evaluation. We therefore examined the potential of

Discussion

Several promising HDAC inhibitors are progressing through clinical trials (2, 13, 14). HDAC inhibitor mechanisms of action suggest potential for enhancing radiation response at several levels. These include physical modifications of chromatin structure (and thus altered potential for radiation damage) and differential oncoprotein regulation after histone acetylation. This study demonstrates the capacity of the HDAC inhibitor SAHA to modulate radiation response by enhancing radiation-induced

Conclusion

The current data demonstrate the capacity of the HDAC inhibitor SAHA to enhance radiation response in prostate and glioma cells. Mechanisms of interaction may include direct interactions with chromatin structure, abrogation of key DNA damage repair processes, and attenuation of pro-survival signals which confer radiation resistance. Studies are in progress to characterize the capacity of SAHA to modulate radiation response and tumor eradication in vivo. Such studies may provide a scientific

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