EZH2 silencing with RNAi enhances irradiation-induced inhibition of human lung cancer growth in vitro and in vivo

Abstract

Non-small cell lung cancer (NSCLC) has a high mortality rate and poor prognosis. The aim of the present study was to silence EZH2 and explore the antitumor effect of small interfering RNA (siRNA)-EZH2 in combination with radiotherapy, which is a main treatment for NSCLC. The results showed that irradiation in the presence of siRNA-EZH2 arrested A549 cells in the G(0) and G(1) phases, delayed cell cycle progression and effectively inhibited cell proliferation, compared with cells that received radiotherapy alone. The combined therapy enhanced the percentage of apoptotic A549 cells in vitro and reduced the tumor size, in addition to increasing the survival rate in tumor xenograft experiments. This study demonstrates the antitumor activity of ionizing radiation therapy in combination with siRNA-EZH2 in NSCLC, both in vitro and in vivo, as well as providing a scientific rationale for targeting EZH2 to enhance the sensitivity of cancer to radiotherapy in NSCLC patients.

Figure 1

EZH2 protein expression was analyzed by western blotting. Lanes show the control group, cells treated with γ-ray irradiation at ~2 Gy/min and cells treated with irradiation in the presence of siRNA-EZH2. The corresponding β-actin levels are shown as a loading control. siRNA, small interfering RNA.

Figure 2

Effect of combination therapy on the growth of the A549 and HTB-56 human lung cancer cell lines in vitro. The cells were treated with γ-ray irradiation at ~2 Gy/min in the presence and absence of siRNA-EZH2. Data show the standard deviations of the mean. Each experiment was performed in triplicate. siRNA, small interfering RNA.

Figure 3

Effect of combination therapy on the cell cycle in A549 and HTB-56 cells. Representative profiles of an analysis of cell cycle kinetics are shown. The cells were treated with γ-ray irradiation at ~2 Gy/min in the absence and presence of siRNA-EZH2. Cell cycle analysis was performed 2 days after treatment. siRNA, small interfering RNA.

Figure 4

Effect of combination therapy on apoptosis in A549 and HTB-56 cells. The cells were evaluated by flow cytometry following treatment. The cells were treated with γ-ray irradiation at ~2 Gy/min alone and treated with irradiation in the presence of siRNA-EZH2. Cell apoptosis analysis was performed 2 days after transfection with siRNA-EZH2. siRNA, small interfering RNA; PI, propidium iodide.

Figure 5

Therapeutic effect of irradiation combined with siRNA-EZH2 on subcutaneous human glioma tumor xenografts. Tumor volume was calculated at the indicated times as shown on the right. Tumors were measured using calipers and the statistical significance of tumor volume changes was calculated using the Student’s t-test. Each time point represents the mean tumor volume for each group (n=10). The survival fraction following treatment is shown on the left. Irradiation in the presence of siRNA-EZH2 enhanced tumor growth inhibition and led to longer survival times than treatment with irradiation alone. siRNA, small interfering RNA.