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Inhibition of Calcium-Activated Chloride Channel ANO1/TMEM16A Suppresses Tumor Growth and Invasion in Human Lung Cancer.

Jia L, Liu W, Guan L, Lu M, Wang K - PLoS ONE (2015)

Bottom Line: Knocking-down ANO1 by small hairpin RNAs inhibited proliferation, migration and invasion of GLC82 and NCI-H520 cancel cells evaluated by CCK-8, would-healing, transwell and 3D soft agar assays.ANO1 protein is overexpressed in 77.3% cases of human lung adenocarcinoma tissues detected by immunohistochemistry.Furthermore, the tumor growth in nude mice implanted with GLC82 cells was significantly suppressed by ANO1 silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Pharmacology, State Key Laboratory of Natural and Biomimetic Drugs, Peking University School of Pharmaceutical Sciences, Beijing 100191, China.

ABSTRACT
Lung cancer or pulmonary carcinoma is primarily derived from epithelial cells that are thin and line on the alveolar surfaces of the lung for gas exchange. ANO1/TMEM16A, initially identified from airway epithelial cells, is a member of Ca2+-activated Cl- channels (CaCCs) that function to regulate epithelial secretion and cell volume for maintenance of ion and tissue homeostasis. ANO1/TMEM16A has recently been shown to be highly expressed in several epithelium originated carcinomas. However, the role of ANO1 in lung cancer remains unknown. In this study, we show that inhibition of calcium-activated chloride channel ANO1/TMEM16A suppresses tumor growth and invasion in human lung cancer. ANO1 is upregulated in different human lung cancer cell lines. Knocking-down ANO1 by small hairpin RNAs inhibited proliferation, migration and invasion of GLC82 and NCI-H520 cancel cells evaluated by CCK-8, would-healing, transwell and 3D soft agar assays. ANO1 protein is overexpressed in 77.3% cases of human lung adenocarcinoma tissues detected by immunohistochemistry. Furthermore, the tumor growth in nude mice implanted with GLC82 cells was significantly suppressed by ANO1 silencing. Taken together, our findings provide evidence that ANO1 overexpression contributes to tumor growth and invasion of lung cancer; and suppressing ANO1 overexpression may have therapeutic potential in lung cancer therapy.

No MeSH data available.


Related in: MedlinePlus

Upregulation of ANO1 expression in human lung cancer cell lines.Top panel: representative western blot images of ANO1 expression in different lung cell lines. Bottom panel: statistical analysis bar chart (n = 3) of ANO1 relative levels in NCI-H520, GLC82, Calu-3, A549 and H1299 cell lines (compared with normal 2BS cells). The expression of ANO1 was normalized to the expression level of β-Actin. ANO1 is significantly overexpressed in GLC82, Calu-3 and H1299 cell lines. Statistical significance by ANOVA is given as *p< 0.05; **p< 0.01 and ***p< 0.001. All data are shown as mean ± s.e.m.
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pone.0136584.g002: Upregulation of ANO1 expression in human lung cancer cell lines.Top panel: representative western blot images of ANO1 expression in different lung cell lines. Bottom panel: statistical analysis bar chart (n = 3) of ANO1 relative levels in NCI-H520, GLC82, Calu-3, A549 and H1299 cell lines (compared with normal 2BS cells). The expression of ANO1 was normalized to the expression level of β-Actin. ANO1 is significantly overexpressed in GLC82, Calu-3 and H1299 cell lines. Statistical significance by ANOVA is given as *p< 0.05; **p< 0.01 and ***p< 0.001. All data are shown as mean ± s.e.m.

Mentions: Using immunohistochemistry, our initial finding revealed that ANO1 was overexpressed in human lung cancer tissues especially in adenocarcinoma. To confirm the immunohistochemical finding and determine whether ANO1 is also upregulated in different pathological types of lung cancer cell lines, we selected and tested 6 different cell lines that include the 2BS cells for normal human lung cell line, GLC82 and Calu-3 cells for adenocarcinoma, NCI-H520 cells for squamous cell carcinoma, and A549 and H1299 cells for unconfirmed type of lung cancer (Fig 2). Proteins extracted from 2BS, NCI-H520, GLC82, Calu-3, A549 or H1299 cells were separated by gel electrophoresis under denaturing conditions and probed with ANO1 specific antibody. As shown in the lower panel of Fig 2, quantitative analysis of ANO1 protein expression level showed an elevation about 2.8-fold in NCI-H520, 6.9-fold in GLC82, 6.3-fold in Calu-3, 3.1-fold in A549 and 8.0-fold in H1299, as compared with normal lung 2BS cells. The increase in GLC82, Calu-3 and H1299 cells was statistically significant, but not for NCI-H520 and A549 cells (P = 0.149 and P = 0.238, respectively) although there was a trend of increase in ANO1 expression. This result is consistent with the immunohistochemical analysis of lung cancer tissues obtained from patients (Table 1), further confirming that ANO1 expression is higher in lung adenocarcinoma GLC 82 cells than squamous carcinoma HCI-H520 cells. The GLC82 and NCI-H520 cells that had different expression levels of ANO1 proteins were selected for further investigations.


Inhibition of Calcium-Activated Chloride Channel ANO1/TMEM16A Suppresses Tumor Growth and Invasion in Human Lung Cancer.

Jia L, Liu W, Guan L, Lu M, Wang K - PLoS ONE (2015)

Upregulation of ANO1 expression in human lung cancer cell lines.Top panel: representative western blot images of ANO1 expression in different lung cell lines. Bottom panel: statistical analysis bar chart (n = 3) of ANO1 relative levels in NCI-H520, GLC82, Calu-3, A549 and H1299 cell lines (compared with normal 2BS cells). The expression of ANO1 was normalized to the expression level of β-Actin. ANO1 is significantly overexpressed in GLC82, Calu-3 and H1299 cell lines. Statistical significance by ANOVA is given as *p< 0.05; **p< 0.01 and ***p< 0.001. All data are shown as mean ± s.e.m.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549304&req=5

pone.0136584.g002: Upregulation of ANO1 expression in human lung cancer cell lines.Top panel: representative western blot images of ANO1 expression in different lung cell lines. Bottom panel: statistical analysis bar chart (n = 3) of ANO1 relative levels in NCI-H520, GLC82, Calu-3, A549 and H1299 cell lines (compared with normal 2BS cells). The expression of ANO1 was normalized to the expression level of β-Actin. ANO1 is significantly overexpressed in GLC82, Calu-3 and H1299 cell lines. Statistical significance by ANOVA is given as *p< 0.05; **p< 0.01 and ***p< 0.001. All data are shown as mean ± s.e.m.
Mentions: Using immunohistochemistry, our initial finding revealed that ANO1 was overexpressed in human lung cancer tissues especially in adenocarcinoma. To confirm the immunohistochemical finding and determine whether ANO1 is also upregulated in different pathological types of lung cancer cell lines, we selected and tested 6 different cell lines that include the 2BS cells for normal human lung cell line, GLC82 and Calu-3 cells for adenocarcinoma, NCI-H520 cells for squamous cell carcinoma, and A549 and H1299 cells for unconfirmed type of lung cancer (Fig 2). Proteins extracted from 2BS, NCI-H520, GLC82, Calu-3, A549 or H1299 cells were separated by gel electrophoresis under denaturing conditions and probed with ANO1 specific antibody. As shown in the lower panel of Fig 2, quantitative analysis of ANO1 protein expression level showed an elevation about 2.8-fold in NCI-H520, 6.9-fold in GLC82, 6.3-fold in Calu-3, 3.1-fold in A549 and 8.0-fold in H1299, as compared with normal lung 2BS cells. The increase in GLC82, Calu-3 and H1299 cells was statistically significant, but not for NCI-H520 and A549 cells (P = 0.149 and P = 0.238, respectively) although there was a trend of increase in ANO1 expression. This result is consistent with the immunohistochemical analysis of lung cancer tissues obtained from patients (Table 1), further confirming that ANO1 expression is higher in lung adenocarcinoma GLC 82 cells than squamous carcinoma HCI-H520 cells. The GLC82 and NCI-H520 cells that had different expression levels of ANO1 proteins were selected for further investigations.

Bottom Line: Knocking-down ANO1 by small hairpin RNAs inhibited proliferation, migration and invasion of GLC82 and NCI-H520 cancel cells evaluated by CCK-8, would-healing, transwell and 3D soft agar assays.ANO1 protein is overexpressed in 77.3% cases of human lung adenocarcinoma tissues detected by immunohistochemistry.Furthermore, the tumor growth in nude mice implanted with GLC82 cells was significantly suppressed by ANO1 silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Pharmacology, State Key Laboratory of Natural and Biomimetic Drugs, Peking University School of Pharmaceutical Sciences, Beijing 100191, China.

ABSTRACT
Lung cancer or pulmonary carcinoma is primarily derived from epithelial cells that are thin and line on the alveolar surfaces of the lung for gas exchange. ANO1/TMEM16A, initially identified from airway epithelial cells, is a member of Ca2+-activated Cl- channels (CaCCs) that function to regulate epithelial secretion and cell volume for maintenance of ion and tissue homeostasis. ANO1/TMEM16A has recently been shown to be highly expressed in several epithelium originated carcinomas. However, the role of ANO1 in lung cancer remains unknown. In this study, we show that inhibition of calcium-activated chloride channel ANO1/TMEM16A suppresses tumor growth and invasion in human lung cancer. ANO1 is upregulated in different human lung cancer cell lines. Knocking-down ANO1 by small hairpin RNAs inhibited proliferation, migration and invasion of GLC82 and NCI-H520 cancel cells evaluated by CCK-8, would-healing, transwell and 3D soft agar assays. ANO1 protein is overexpressed in 77.3% cases of human lung adenocarcinoma tissues detected by immunohistochemistry. Furthermore, the tumor growth in nude mice implanted with GLC82 cells was significantly suppressed by ANO1 silencing. Taken together, our findings provide evidence that ANO1 overexpression contributes to tumor growth and invasion of lung cancer; and suppressing ANO1 overexpression may have therapeutic potential in lung cancer therapy.

No MeSH data available.


Related in: MedlinePlus