Cancer Res Treat.  2013 Dec;45(4):334-342.

Sequence-Dependent Radiosensitization of Histone Deacetylase Inhibitors Trichostatin A and SK-7041

• Kim JH
• Kim IH
• Shin JH
• Kim HJ
• Kim IA

Abstract

PURPOSE
This preclinical study is to determine whether the capacity of histone deacetylase (HDAC) inhibitors to enhance radiation response depends on temporal sequences of HDAC inhibition and irradiation.
MATERIALS AND METHODS
The effects of HDAC inhibitors trichostatin A (TSA) and SK-7041 on radiosensitivity in human lung cancer cells were examined using a clonogenic assay, exposing cells to HDAC inhibitors in various sequences of HDAC inhibition and radiation. We performed Western blot of acetylated histone H3 and flow cytometry to analyze cell cycle phase distribution.
RESULTS
TSA and SK-7041 augmented radiation cell lethality in an exposure time-dependent manner when delivered before irradiation. The impact of TSA and SK-7041 on radiosensitivity rapidly diminished when HDAC inhibition was delayed after irradiation. Radiation induced the acetylation of histone H3 in cells exposed to TSA, while irradiation alone had no effect on the expression of acetylated histone H3 in TSA-naive cells. Preirradiation exposure to TSA abrogated radiation-induced G2/M-phase arrest. When delivered after irradiation, TSA had no effect on the peak of radiation-induced G2/M-phase arrest.
CONCLUSION
TSA and SK-7041 enhances radiosensitivity only when delivered before irradiation. Unless proven otherwise, it seems prudent to apply scheduling including preirradiation HDAC inhibition so that maximal radiosensitization is obtained.

Keyword

Histone deacetylase inhibitors; Radiation-sensitizing agents; Preclinical drug evaluation

MeSH Terms

Acetylation
Blotting, Western
Cell Cycle
Drug Evaluation, Preclinical
Flow Cytometry
Histone Deacetylase Inhibitors*
Histone Deacetylases*
Histones*
Humans
Lung Neoplasms
Radiation Tolerance
Radiation-Sensitizing Agents
Histone Deacetylase Inhibitors
Histone Deacetylases
Histones
Radiation-Sensitizing Agents

Figure

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  Fig. 1 Plating efficacy and surviving fraction of A549 cells treated with histone deacetylase inhibitors (HDACI). Cells were exposed to trichostatin A (TSA) of 200 nM or SK-7041 of 200 nM for various times. HDAC, histone deacetylase.

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  Fig. 2 The effect of histone deacetylase (HDAC) inhibition on tumor cell radiosensitivity. (A) A549 cells were treated for 3 to 24 hours with trichostatin A (TSA; 200 nM) and irradiated with graded doses of X-ray. After irradiation (IR), cells were grown in HDAC inhibitor-free media till colony formation was determined 14-21 days afterwards and survival curves were generated. Values represent the mean±SD from at least triplicate experiments. (B) As described previously (A), cells were treated with SK-7041 (200 nM) and irradiated. (C) Sensitizer enhancement ratioof HDAC inhibition prior to IR. Values represent the mean±SE from triplicate experiments.

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  Fig. 3 The effect of postirradiation histone deacetylase (HDAC) inhibitors (HDACI) on tumor cell radiosensitivity. (A) Schema of HDACI treatment and irradiation (IR). A549 cells were treated with HDACIs prior to or after IR with graded doses of X-ray. Cells were exposed to 200 nM trichostatin A (TSA) or 200 nM SK-7041 for 18 hours (represented by open horizontal bars). For postirradiation treatment, cells were subjected to HDACI immediately, at 3 hours, 6 hours, and 12 hours after IR. Drugs were washed off immediately after intended exposure time and cells were grown in HDACI-free media thereafter. (B, C) Clonogenic survival of A549 cells treated with TSA and SK-7041. Values represent the mean±SD from at least triplicate experiments. (D) Sensitizer enhancement ratio of preirradiation and postirradiation HDAC inhibition calculated from previous data (B, C). Values represent the mean±SE from triplicate experiments.

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  Fig. 4 Immunoblots for acetyl histone H3 (Ac-H3) from lysates from trichostatin A (TSA)-treated cells following irradiation. A549 cells were treated with 200 nM TSA for 18 hours prior to or immediately after irradiation (IR) with 8 Gy X-ray.

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  Fig. 5 Cell cycle profiles after histone deacetylase inhibition and irradiation (IR). (A) As previously described (Fig. 3A), A549 cells were treated with 200 nM trichostatin A (TSA) for 18 hours prior to or after IR with 8 Gy X-ray. For postirradiation treatment, cells were exposed to TSA immediately following (IR → TSA) or at 12 hours (IR → 12 hours → TSA) after IR. Cells were collected, fixed, stained with propidium iodide, and analyzed via fluorescence-activated cell sorting for DNA content at 18 hours after IR except for cells in the group IR → 12 hours → TSA, where cells were fixed after TSA treatment was completed (that is, 30 hours after IR). At least 10×104 cells were counted for each sample. (B) Proportion of cells in G2/M phase DNA content. As described previously (A), cells were treated with TSA and irradiated. Then, cells were serially collected, fixed and stained at specified times and analyzed for DNA content. Values represent the proportion of cells with G2/M-phase DNA content. Cells were irradiated to 8 Gy X-ray at time zero on the horizontal axis. Thus, cells were exposed to TSA from -18 hours to 0 hour and from 0 hour to 18 hours for preirradiation treatment and postirradiation treatment, respectively.

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