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

Jia J, Dai S, Sun X, Sang Y, Xu Z, Zhang J, Cui X, Song J, Guo X - Mol Med Rep (2015)

Bottom Line: Studies have shown that ECRG4 effectively inhibits the proliferation of tumor cells and induces apoptosis.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology‑Head and Neck Surgery, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China.

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.

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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.
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f4-mmr-12-04-5058: 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.

Mentions: Cell apoptosis was measured by flow cytometry and fluorescence microscopy using Annexin V/PI and Hoechst staining, as well as the analysis of the expression of apoptosis-related factors. The results of flow cytometric analysis showed that the apoptotic rate was significantly elevated in the pcDNA3.1-ECRG4 group compared with the pcDNA3.1 group (19.37±0.67 vs. 0.66±0.09%; Fig. 4A, P<0.01). Hoechst staining showed that compared with cells in the parental group and the pcDNA3.1 group, cells in the pcDNA3.1-ECRG4 group exhibited significantly increased chromatin condensation and more densely stained nuclei (Fig. 4B). To determine whether ECRG4-induced apoptosis of laryngeal cancer cells affected the expression of apoptosis-related factors, western blot analysis was performed to examine the expression of Bax, Bcl-2, cleaved-caspase-3 and cleaved-PARP. The results showed that the expression levels of cleaved-PARP, cleaved-caspase-3 and Bax were significantly elevated in cells from the pcDNA3.1-ECRG4 group compared with that from the pcDNA3.1 group (Fig. 4C, P<0.01). By contrast, the expression level of Bcl-2 was markedly decreased (P<0.01). These results demonstrated that overexpression of ECRG4 significantly induced apoptosis in laryngeal cancer cells.


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

Jia J, Dai S, Sun X, Sang Y, Xu Z, Zhang J, Cui X, Song J, Guo X - Mol Med Rep (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581775&req=5

f4-mmr-12-04-5058: 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.
Mentions: Cell apoptosis was measured by flow cytometry and fluorescence microscopy using Annexin V/PI and Hoechst staining, as well as the analysis of the expression of apoptosis-related factors. The results of flow cytometric analysis showed that the apoptotic rate was significantly elevated in the pcDNA3.1-ECRG4 group compared with the pcDNA3.1 group (19.37±0.67 vs. 0.66±0.09%; Fig. 4A, P<0.01). Hoechst staining showed that compared with cells in the parental group and the pcDNA3.1 group, cells in the pcDNA3.1-ECRG4 group exhibited significantly increased chromatin condensation and more densely stained nuclei (Fig. 4B). To determine whether ECRG4-induced apoptosis of laryngeal cancer cells affected the expression of apoptosis-related factors, western blot analysis was performed to examine the expression of Bax, Bcl-2, cleaved-caspase-3 and cleaved-PARP. The results showed that the expression levels of cleaved-PARP, cleaved-caspase-3 and Bax were significantly elevated in cells from the pcDNA3.1-ECRG4 group compared with that from the pcDNA3.1 group (Fig. 4C, P<0.01). By contrast, the expression level of Bcl-2 was markedly decreased (P<0.01). These results demonstrated that overexpression of ECRG4 significantly induced apoptosis in laryngeal cancer cells.

Bottom Line: Studies have shown that ECRG4 effectively inhibits the proliferation of tumor cells and induces apoptosis.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology‑Head and Neck Surgery, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China.

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.

Show MeSH
Related in: MedlinePlus