Clinical investigation: biology contribution
Susceptibility and radiosensitization of human glioblastoma cells to trichostatin A, a histone deacetylase inhibitor

https://doi.org/10.1016/j.ijrobp.2004.03.001Get rights and content

Abstract

Purpose

Histone deacetylase inhibitors (HDAC-Is) show in vitro and in vivo antitumor activity in various types of cancer cells and are being studied in clinical trials. However, studies addressing the combination of HDAC-I and radiation are lacking. The purpose of this study was to assess the effect of trichostatin A (TSA), an HDAC-I, on the radiosensitivity of U373MG and U87MG (human glioblastoma) cell lines.

Methods and materials

Intrinsic TSA toxicity was determined by measuring survival in exponentially growing cells treated with 0–200 nM TSA for 0–24 h. To assay the radiosensitizing effect of TSA, cells were exposed to 0–200 nM TSA for 18 h before irradiation, and radiation survival curves were obtained. Radiation survival of TSA-treated cells was determined by clonogenic assay.

Results

The human glioblastoma cells showed a dose-dependent reduction in survival and radiosensitization with TSA treatment in the range of 50–200 nM. Exposure to 200 nM TSA resulted in reduced survival of both cell lines, and survival was further reduced with time. Exposure of these cells to TSA before irradiation led to dose-dependent radiosensitization.

Conclusions

These results suggest that HDAC-Is may be a useful adjunct in the treatment of glioblastoma and merit further investigation. Given the limited efficacy of standard treatments for patients afflicted with glioblastoma, the results reported here provide support for clinical trials integrating HDAC-I with radiation therapy.

Introduction

The organization of chromatin is crucial for the regulation of gene expression. Chromatin is composed of nucleosomes, which consist of 146 bp DNA tightly wrapped around an octameric histone core. The core histone octamers consist of two H2A, two H2B, two H3, and two H4 histones, which are subject to a variety of enzyme-catalyzed posttranslational modifications. Concerning these modifications, the acetylation status of core histones is associated with transcriptional regulation and is determined by two classes of enzymes: histone acetyl transferases (HATs) and histone deacetylases (HDACs) (1). The acetylation of histones is thought to loosen the histone-DNA contact by neutralizing the positively charged lysine residues in the core histones. This loosening leads to a more open DNA conformation, allowing transcription factors and transcription apparatus to gain access to DNA and express specific genes (2). Nonhistone proteins, such as p53, have been known to serve as substrates of HAT and HDAC in vitro and in vivo, and the acetylation status of cellular proteins is thought to modulate the activity of target proteins 3, 4. In addition, aberrant acetylation has been reported to be associated with carcinogenesis in multiple organs (5).

So far, several HDAC-Is have been discovered or synthesized (6). HDAC-Is, in addition to deacetylase activity, show various biologic effects—for example, morphologic change (7), transcriptional change 8, 9, cell differentiation (10), cell cycle arrest 11, 12, antiangiogenesis (13), and apoptosis 12, 14, 15, 16. In addition, HDAC-Is have in vitro and in vivo antitumor activity against transformed cells of various histologic origins 17, 18, 19, 20, 21, 22, 23, 24. Several HDAC-Is have been synthesized and tested for toxicity and antitumor effect in clinical trials 25, 26, 27, 28, 29, 30. Several Phase I/II clinical trials using HDAC-Is are under way.

Although research on HDAC-Is has progressed to the clinical trial stage, studies on the interaction between HDAC-Is and ionizing radiation are lacking. Recently, two studies reported the in vitro radiosensitizing effect of HDAC-Is in human nasopharyngeal (31) and colon carcinoma cells (32).

In this study, we investigated the effect of trichostatin A (TSA), the most potent HDAC-I discovered so far, on the radiosensitivity of human glioblastoma cells, and found that treatment with TSA at nanomolar concentrations sensitizes human glioblastoma cells to radiation-induced cellular lethality.

Section snippets

Cell culture

The U373MG and U87MG, human glioblastoma cell lines, were purchased from the Korean Cell Line Bank (33). Cells were grown as attached monolayers in 25-cm2 flasks in RPMI 1640 media (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) and 12.5 μg/mL gentamicin (Gibco). Cells were incubated at the exponential growth phase in humidified 5% CO2/95% air atmosphere at 37°C.

Assay of intrinsic TSA cytotoxicity

Cells were trypsinized from exponentially growing monolayer cultures, and 2 × 105 cells per 25-cm2 flask

Intrinsic TSA cytotoxicity

The clonogenicities of U373MG and U87MG cells were not significantly influenced by treating cells with 50–100 nM TSA for up to 24 h (Fig. 1). However, exposure to 200 nM TSA showed significant cytotoxicity in two cell lines. The cytotoxicity of 200 nM TSA showed time-dependence. We chose to test the radiosensitizing effect of TSA at 50, 100, and 200 nM, because significant intrinsic toxicity was expected at TSA concentration of 200 nM or higher.

TSA effect on radiosensitivity

SF2s of untreated U373MG and U87MG were 0.694 ±

Discussion

Histone deacetylase inhibitors have diverse biologic effects, such as transcriptional change, differentiation induction, cell cycle arrest, and apoptosis, and are known to inhibit the growth of various transformed cells. To date, several HDAC-Is have been discovered, and TSA has the most potent deacetylase activity among these. The low bioavailability of TSA has led to the development of several synthetic HDAC-Is 6, 19, 21, 34, 35. Some of the synthetic HDAC-Is selectively inhibit specific

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