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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 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.
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f3-mmr-12-04-5058: 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.

Mentions: To investigate the effect of ECRG4 overexpression on the proliferative capability of laryngeal cancer cells, cell proliferative capability was examined using the MTT assay and colony formation assay. The results of the MTT assay showed that at day 2, day 3 and day 4, the proliferative capability was severely impaired in the pcDNA3.1-ECRG4 group compared with the pcDNA3.1 group (Fig. 3A; day 2, P<0.05; days 3 and 4, P<0.01). The effect of ECRG4 on the clonogenic capacity of laryngeal cancer cells was further examined using the colony formation assay. The results showed that the colony formation rate in the pcDNA3.1-ECRG4 group was 45.27±7.19%, which was lower than that in the pcDNA3.1 group (83.07±7.51%). The results indicated that ECRG4 significantly reduced the colony formation ability of laryngeal cancer cells (Fig. 3B, P<0.01). This study further investigated the cell-cycle phase distribution in all three groups of cells using flow cytometry. As shown in Fig. 3C, the percentage of cells in the G0/G1 phase was significantly increased in the pcDNA3.1-ECRG4 group compared with that in the pcDNA3.1 group (71.7 vs. 39.41%, P<0.01). By contrast, the percentage of cells in the S phase and G2/M phase were decreased markedly in the pcDNA3.1-ECRG4 group (S phase, 20.14 vs. 44.83%, P<0.01; and G2/M phase, 8.17 vs. 15.76%, P<0.05, respectively). These results indicated that overexpression of ECRG4 inhibited laryngeal cancer cell proliferation and arrested cells in the G0/G1 phase of the cell cycle.


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

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

f3-mmr-12-04-5058: 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.
Mentions: To investigate the effect of ECRG4 overexpression on the proliferative capability of laryngeal cancer cells, cell proliferative capability was examined using the MTT assay and colony formation assay. The results of the MTT assay showed that at day 2, day 3 and day 4, the proliferative capability was severely impaired in the pcDNA3.1-ECRG4 group compared with the pcDNA3.1 group (Fig. 3A; day 2, P<0.05; days 3 and 4, P<0.01). The effect of ECRG4 on the clonogenic capacity of laryngeal cancer cells was further examined using the colony formation assay. The results showed that the colony formation rate in the pcDNA3.1-ECRG4 group was 45.27±7.19%, which was lower than that in the pcDNA3.1 group (83.07±7.51%). The results indicated that ECRG4 significantly reduced the colony formation ability of laryngeal cancer cells (Fig. 3B, P<0.01). This study further investigated the cell-cycle phase distribution in all three groups of cells using flow cytometry. As shown in Fig. 3C, the percentage of cells in the G0/G1 phase was significantly increased in the pcDNA3.1-ECRG4 group compared with that in the pcDNA3.1 group (71.7 vs. 39.41%, P<0.01). By contrast, the percentage of cells in the S phase and G2/M phase were decreased markedly in the pcDNA3.1-ECRG4 group (S phase, 20.14 vs. 44.83%, P<0.01; and G2/M phase, 8.17 vs. 15.76%, P<0.05, respectively). These results indicated that overexpression of ECRG4 inhibited laryngeal cancer cell proliferation and arrested cells in the G0/G1 phase of the cell cycle.

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