A preliminary study of the effect of ECRG4 overexpression on the proliferation and apoptosis of human laryngeal cancer cells and the underlying mechanisms

Abstract

Human esophageal cancer‑related gene 4 (ECRG4) is a potential tumor suppressor gene isolated from human esophageal epithelial cells. Studies have shown that ECRG4 effectively inhibits the proliferation of tumor cells and induces apoptosis. However, the role of ECRG4 in laryngeal cancer has not yet been clearly defined. In this study, a human laryngeal cancer cell line stably overexpressing ECRG4 was established. The effect of ECRG4 on the proliferation and apoptosis of laryngeal cancer cells and the associated mechanisms were investigated. The Hep‑2 human laryngeal carcinoma cell line exhibited a low basal level of ECRG4 expression and was selected for the present study. The eukaryotic expression plasmid pcDNA3.1‑ECRG4 was constructed and introduced into Hep‑2 cells by transfection reagents. Western blot analysis, reverse transcription-quantitative polymerase chain reaction and immunofluorescence staining confirmed high‑level expression of ECRG4. The 3‑(4, 5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay and colony formation assay showed that ECRG4 overexpression suppressed the proliferative capacity of laryngeal cancer cells in vitro. Cell cycle analysis showed that ECRG4 induced cell cycle arrest at the G0/G1 phase. Flow cytometric analysis and Hoechst staining demonstrated that overexpression of ECRG4 significantly induced apoptosis. Western blot analysis confirmed that Bcl‑2‑associated X protein, cleaved‑caspase‑3 and cleaved‑poly (ADP‑ribose) polymerase were upregulated in the apoptotic process, whereas B‑cell lymphoma 2 was downregulated. In conclusion, overexpression of ECRG4 inhibited laryngeal cancer cell proliferation and induced cancer cell apoptosis. Therefore, ECRG4 exhibits potential as an effective target in gene therapy for laryngeal cancer.

Figure 1

Selection of cell lines. The expression levels of ECRG4 in Hep-2 and LSC-1 human laryngeal cancer cell lines were examined by western blot analysis. Grayscale analysis was performed using β-actin as the internal control. Experimental data are expressed as the mean ± standard deviation. ^*P<0.05, compared with Hep-2 cells. ECRG4, human esophageal cancer-related gene 4.

Figure 2

Establishment of a cell line stably overexpressing ECRG4. (A) Western blot analysis of ECRG4 protein expression. Grayscale analysis was conducted using β-actin as the internal control. (B) Reverse transcription-quantitative polymerase chain reaction analysis of ECRG4 mRNA expression. (C) Immunofluorescence examination of ECRG4 distribution. ECRG4 expression was visible under a fluorescence microscope (red). The nuclei were stained blue. Representative results of several replica experiments are shown in the figure (magnification, x600). Experimental data are expressed as the mean ± standard deviation. ^**P<0.01, compared with the pcDNA3.1 group. ECRG4, human esophageal cancer-related gene 4.

Figure 3

Overexpression of ECRG4 inhibited the proliferation of laryngeal cancer cells. (A) Examination of the proliferative capacity of laryngeal cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cells from the three experimental groups were seeded into 96-well plates. Five replica wells were set up for each experimental group. The OD₄₉₀ values were measured at days 0, 1, 2, 3 and 4 after cell inoculation. (B) Determination of the clonogenic capacity of laryngeal cancer cells using colony formation assay. Cells from the experimental groups were seeded into Petri dishes. After day 14 of culture, the cells were fixed and the colony formation rates were calculated under a microscope. (C) Analysis of the cell cycle by flow cytometric analysis. Experimental data are presented as the mean ± standard deviation. ^*P<0.05, and ^**P<0.01, compared with the pcDNA3.1 group. ECRG4, human esophageal cancer-related gene 4.

Figure 4

Overexpression of ECRG4 induced apoptosis in laryngeal cancer cells through regulation of the expression of apoptosis-related factors. (A) Examination of apoptosis by flow cytometric analysis. The figure shows the representative results of several repeated experiments. (B) Examination of apoptosis by Hoechst staining. Apoptotic cells exhibited intensive chromatin condensation and dense, strongly Hoechst stained nuclei (magnification, ×400). (C) Examination of the expression of Bax, Bcl-2, cleaved-caspase-3 and cleaved-PARP by western blot analysis. Grayscale analysis was performed using β-actin as the internal control. Experimental data are expressed as the mean ± standard deviation. Compared with the pcDNA3.1 group, ^**P<0.01. ECRG4, human esophageal cancer-related gene 4; Bax, Bcl-2-associated X protein; Bcl-2, anti-B-cell lymphoma 2; PARP, poly ADP-ribose polymerase.