Trichostatin A enhances radiosensitivity and radiation-induced DNA damage of esophageal cancer cells

Abstract

Background: Trichostatin A (TSA) is emerging as a potential component of anticancer therapy. In this study, we aimed to identify the radiosensitizing effects of TSA in esophageal squamous carcinoma cell lines and identify the genomic alteration of histone acetylation associated with TSA treatment.

Methods: EC109 and KYSE450 cells were pretreated with TSA (0.1 µM) for 12 hours prior to irradiation, and the cell viability, flow cytometry, and comet assays were performed to analyze cell growth, cell apoptosis, and DNA damage, respectively. Chromatin immunoprecipitation sequencing (ChIP-Seq) was performed to identify the acetylation sites of histone H3 lysine 9 (H3K9), which was altered by TSA.

Results: Our data showed that TSA could sensitize esophageal cancer cells to radiation by inducing cell cycle arrest and increasing cell apoptosis. DNA damage induced by radiation was enhanced by TSA treatment. In addition, a total of 105 differential peak-related genes were found to be associated with TSA treatment, which was identified using ChIP-Seq with specific antibodies against acetylated histone H3K9.

Conclusions: Our data suggest that pretreatment with TSA can enhance ionizing radiation-induced DNA damage of esophageal cancer cells, which was associated with the altered histone modification of whole genome. TSA has potential implications for clinical use in increasing the anticancer efficacy of radiation.

Keywords: DNA damage; Trichostatin A (TSA); esophageal cancer cells; ionizing radiation.

Figure 1

Radiosensitivity of esophageal squamous cell carcinoma was enhanced by TSA. (A) EC109 and KYSE450 cells were pretreated with TSA for 12 h, and then irradiated with 5 Gy. Cell viability was determined using Cell Counting Kit-8 assay. (B) EC109 and KYSE450 cells were seeded in 6-well plates and treated with TSA, and then irradiated with or without 5 Gy. Long-term survival was detected using clonogenic assay. N, untreated control. *, P<0.05; **, P<0.05 vs. N; ^#, P<0.05 vs. 5 Gy or TSA. TSA, trichostatin A; N, untreated control.

Figure 2

TSA promoted mitotic G2 gap 2 (G2/M) arrest induced by radiation. EC109 (A) and KYSE450 (B) cells were pretreated with TSA, followed by 5-Gy irradiation. Then cell cycle distributions were analyzed using flow cytometry. TSA, trichostatin A; N, untreated control.

Figure 3

Apoptotic cell death induced by radiation was enhanced by TSA. (A) EC109 and KYSE450 cells were pretreated with TSA, followed by 5-Gy irradiation. Apoptotic cells were detected using staining with Annexin V-FITC Apoptosis Detection Kit and propidine iodide. (B) Protein samples from treated EC109 and KYSE450 cells were detected by western blot. Glyceraldehyde-3-phosphate dehydrogenase protein expression was measured as a control for equal loading. Fold changes of protein level were calculated by normalizing that of untreated control. *, P<0.05; **, P<0.01 vs. N; ^#, P<0.05 vs. 5 Gy or TSA. TSA, trichostatin A; N, untreated control.

Figure 4

Radiation-induced DNA damage was enhanced using TSA in esophageal cancer cells. EC109 cells were pretreated with TSA, followed by 5-Gy irradiation. Then DNA damage was detected using immunofluorescent staining of γ-H2AX (A) and single-cell gel electrophoresis (B,C). a: untreated control; b: TSA; c: 5 Gy; d: TSA + 5 Gy. *, P<0.05; **, P<0.01 vs. N; ^#, P<0.05 vs. 5 Gy or TSA. TSA, trichostatin A; N, untreated control.

Figure 5

Acetylated histone H3K9 related DNA fragments were detected using ChIP-sequencing. EC109 cells were pretreated with TSA for 12 h, then immunoprecipitation with an antibody against acetylated histone H3K9 was carried out to enrich for acetylated histone H3-bound DNA and subsequent sequencing. (A) The distribution of peaks in the genome was analyzed both in control EC109 or TSA-treated EC109 cells. (B) The number of differential chromatin immunoprecipitation (ChIP peaks) (EC109-TSA vs. EC109-N) on different chromosomes. (C) Gene Ontology annotation of differential peak-related genes in terms of biological process, cellular component, and molecular function. TSA, trichostatin A.