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. 2012 Nov;78(2):138-43.
doi: 10.1016/j.lungcan.2012.08.003. Epub 2012 Aug 25.

Epigenetic therapy with 3-deazaneplanocin A, an inhibitor of the histone methyltransferase EZH2, inhibits growth of non-small cell lung cancer cells

Junko Kikuchi  1 , Taichi Takashina, Ichiro Kinoshita, Eiki Kikuchi, Yasushi Shimizu, Jun Sakakibara-Konishi, Satoshi Oizumi, Victor E Marquez, Masaharu Nishimura, Hirotoshi Dosaka-Akita
Affiliations

Epigenetic therapy with 3-deazaneplanocin A, an inhibitor of the histone methyltransferase EZH2, inhibits growth of non-small cell lung cancer cells

Junko Kikuchi et al. Lung Cancer. 2012 Nov.

Abstract

EZH2 (enhancer of zeste homolog 2) is the catalytic subunit of PRC2 (polycomb repressive complex 2), which mediates histone methyltransferase activity and functions as transcriptional repressor involved in gene silencing. EZH2 is involved in malignant transformation and biological aggressiveness of several human malignancies. We previously demonstrated that non-small cell lung cancers (NSCLCs) also overexpress EZH2 and that high expression of EZH2 correlates with poor prognosis. Growing evidence indicates that EZH2 may be an appropriate therapeutic target in malignancies, including NSCLCs. Recently, an S-adenosyl-l-homocysteine hydrolase inhibitor, 3-Deazaneplanocin A (DZNep), has been shown to deplete and inhibit EZH2. The aim of this study was to determine the effect of DZNep in NSCLC cells. Knockdown of EZH2 by small-interfering RNA (siRNA) resulted in decreased growth of four NSCLC cell lines. MTT assays demonstrated that DZNep treatment resulted in dose-dependent inhibition of proliferation in the NSCLC cell lines with a half maximal inhibitory concentration (IC50) ranging from 0.08 to 0.24 μM. Immortalized but non-cancerous bronchial epithelial and fibroblast cell lines were less sensitive to DZNep than the NSCLC cell lines. Soft agarose assays demonstrated that anchorage-independent growth was also reduced in all three NSCLC cell lines that were evaluated using this assay. Flow cytometry analysis demonstrated that DZNep induced apoptosis and G1 cell cycle arrest in NSCLC cells, which was partially associated with cyclin A decrease and p27(Kip1) accumulation. DZNep depleted cellular levels of EZH2 and inhibited the associated histone H3 lysine 27 trimethylation. These results indicated that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach to treatment of NSCLCs.

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Figures

Fig. 1
Fig. 1
Effects of EZH2 knockdown on cell proliferation in NSCLC cell lines. NCI-H1299 (H1299), NCI-H1975 (H1975), A549, and PC-3 cells were transfected with EZH2 siRNA or a negative control siRNA every 72 h. After 6 days, cell proliferation was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are means ± SD of quadruplet samples in one of three independent experiments. Similar results were obtained from all three independent experiments. *p < 0.05 and *p < 0.01 versus cells treated with negative control siRNA by unpaired, two-sided Student’s t-test. Cell lysates were collected 72 hrs after transfection with EZH2 or a negative control siRNA; lysates were subjected to western blot analysis. EZH2; transfected with EZH2 siRNA, NC; transfected with negative control siRNA.
Fig. 2
Fig. 2
Inhibition of anchorage-independent growth by DZNep. Representative data from one of three independent experiments is shown. Data are means ± SD of triplicate samples. Similar results were obtained from all three independent experiments. * p < 0.05 and ** p < 0.01 between indicated groups by one-way ANOVA with Tukey’s multiple comparison test.
Fig. 3
Fig. 3
Effect of DZNep on cell cycle in NSCLC cells. Cells were transfected with indicated doses of DZNep. After 72 hrs, the percentage of cells in each cell cycle phase was measured using a FACS flow cytometer and ModFitLT software. Representative data from one of three independent experiments is shown. Similar results were obtained in all three independent experiments.
Fig. 4
Fig. 4
Cell apoptosis analysis of NSCLC cells using flow cytometry with Annexin V-FITC and PI staining. Cells were treated with indicated doses of DZNep. After 72 hrs, the percentage of cells in the apoptotic fraction was measured using a FACS flow cytometer. Data are means ± SD of triplicate samples from one of three independent experiments. Similar results were obtained in all three independent experiments. * p < 0.05 and ** p < 0.01 between indicated groups by one-way ANOVA with Tukey’s multiple comparison test.
Fig. 5
Fig. 5
Western blot analysis of NSCLC cells. Cell lysates were collected 72 hrs after the indicated doses of DZNep were administered; lysates were then subjected to western blot analysis. Representative western blots of EZH2, SUZ12, EED, trimethylation of lysine 27 on histon H3 (H3K27me3), cyclin A, p27 Kip1, and actin are shown.
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