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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

Inhibition of cell proliferation by ANO1 knockdown.(A) RNAi knockdown of ANO1 protein expression in ANO1 expressing cells was shown by Western blot images. The membrane proteins extracted from GLC82 and NCI-H520 cells on the 3rd day after transfection of different ANO1 shRNAs (shRNA1, shRNA2 and shRNA3) were immunoblotted with ANO1 antibody. ANO1 shRNA1 showed the most effective inhibition of ANO1 expression, compared with the other two shRNAs and the scrambled shRNA. (B) GLC82 or NCI-H520 cell proliferation was assessed by CCK8 assay based on the extracellular decreasing of WST8 by NADH produced in mitochondria. Using a microplate reader, the number of cells was quantified by the measurement of absorbance at 450 nm. Transfection of ANO1 shRNA1 resulted in significant inhibition of GLC82 and NCI-H529 cell viability in time-dependent manner as compared with cells treated with scrambled shRNA (n = 6). (C) Cell proliferation of carcinoma cells was assessed by the colony formation assay in culture in the presence of ANO1 shRNA1. Quantitative analysis of ANO1 silencing group showed less colony genesis, compared with scrambled shRNA group (n = 3). Representative images are showed below bar charts. (D) RNAi of ANO1 inhibits the clonogenicity of GLC82 cells in soft agar (n = 3). NCI-H520 failed to form colonies in soft agar. Statistical significance by Student’s t-test is indicated as *p< 0.05; **p< 0.01 and ***p< 0.001. Data are expressed as mean ± s.e.m.
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pone.0136584.g003: Inhibition of cell proliferation by ANO1 knockdown.(A) RNAi knockdown of ANO1 protein expression in ANO1 expressing cells was shown by Western blot images. The membrane proteins extracted from GLC82 and NCI-H520 cells on the 3rd day after transfection of different ANO1 shRNAs (shRNA1, shRNA2 and shRNA3) were immunoblotted with ANO1 antibody. ANO1 shRNA1 showed the most effective inhibition of ANO1 expression, compared with the other two shRNAs and the scrambled shRNA. (B) GLC82 or NCI-H520 cell proliferation was assessed by CCK8 assay based on the extracellular decreasing of WST8 by NADH produced in mitochondria. Using a microplate reader, the number of cells was quantified by the measurement of absorbance at 450 nm. Transfection of ANO1 shRNA1 resulted in significant inhibition of GLC82 and NCI-H529 cell viability in time-dependent manner as compared with cells treated with scrambled shRNA (n = 6). (C) Cell proliferation of carcinoma cells was assessed by the colony formation assay in culture in the presence of ANO1 shRNA1. Quantitative analysis of ANO1 silencing group showed less colony genesis, compared with scrambled shRNA group (n = 3). Representative images are showed below bar charts. (D) RNAi of ANO1 inhibits the clonogenicity of GLC82 cells in soft agar (n = 3). NCI-H520 failed to form colonies in soft agar. Statistical significance by Student’s t-test is indicated as *p< 0.05; **p< 0.01 and ***p< 0.001. Data are expressed as mean ± s.e.m.

Mentions: To evaluate the biological role of ANO1 in lung cancer cell proliferation, we used shRNAs to knockdown the expression of ANO1 in different cell lines. The effectiveness of three shRNAs was evaluated by Western blot in GLC82 and NCI-H520 lung cancer cells. Compared with transfection of scrambled shRNA, ANO1 shRNA1 transfection was most effective in silencing endogenous expression of ANO1 proteins in both GLC82 and NCI-H520 cells (Fig 3A), and thus it was used for the rest of experiments.


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)

Inhibition of cell proliferation by ANO1 knockdown.(A) RNAi knockdown of ANO1 protein expression in ANO1 expressing cells was shown by Western blot images. The membrane proteins extracted from GLC82 and NCI-H520 cells on the 3rd day after transfection of different ANO1 shRNAs (shRNA1, shRNA2 and shRNA3) were immunoblotted with ANO1 antibody. ANO1 shRNA1 showed the most effective inhibition of ANO1 expression, compared with the other two shRNAs and the scrambled shRNA. (B) GLC82 or NCI-H520 cell proliferation was assessed by CCK8 assay based on the extracellular decreasing of WST8 by NADH produced in mitochondria. Using a microplate reader, the number of cells was quantified by the measurement of absorbance at 450 nm. Transfection of ANO1 shRNA1 resulted in significant inhibition of GLC82 and NCI-H529 cell viability in time-dependent manner as compared with cells treated with scrambled shRNA (n = 6). (C) Cell proliferation of carcinoma cells was assessed by the colony formation assay in culture in the presence of ANO1 shRNA1. Quantitative analysis of ANO1 silencing group showed less colony genesis, compared with scrambled shRNA group (n = 3). Representative images are showed below bar charts. (D) RNAi of ANO1 inhibits the clonogenicity of GLC82 cells in soft agar (n = 3). NCI-H520 failed to form colonies in soft agar. Statistical significance by Student’s t-test is indicated as *p< 0.05; **p< 0.01 and ***p< 0.001. Data are expressed as mean ± s.e.m.
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getmorefigures.php?uid=PMC4549304&req=5

pone.0136584.g003: Inhibition of cell proliferation by ANO1 knockdown.(A) RNAi knockdown of ANO1 protein expression in ANO1 expressing cells was shown by Western blot images. The membrane proteins extracted from GLC82 and NCI-H520 cells on the 3rd day after transfection of different ANO1 shRNAs (shRNA1, shRNA2 and shRNA3) were immunoblotted with ANO1 antibody. ANO1 shRNA1 showed the most effective inhibition of ANO1 expression, compared with the other two shRNAs and the scrambled shRNA. (B) GLC82 or NCI-H520 cell proliferation was assessed by CCK8 assay based on the extracellular decreasing of WST8 by NADH produced in mitochondria. Using a microplate reader, the number of cells was quantified by the measurement of absorbance at 450 nm. Transfection of ANO1 shRNA1 resulted in significant inhibition of GLC82 and NCI-H529 cell viability in time-dependent manner as compared with cells treated with scrambled shRNA (n = 6). (C) Cell proliferation of carcinoma cells was assessed by the colony formation assay in culture in the presence of ANO1 shRNA1. Quantitative analysis of ANO1 silencing group showed less colony genesis, compared with scrambled shRNA group (n = 3). Representative images are showed below bar charts. (D) RNAi of ANO1 inhibits the clonogenicity of GLC82 cells in soft agar (n = 3). NCI-H520 failed to form colonies in soft agar. Statistical significance by Student’s t-test is indicated as *p< 0.05; **p< 0.01 and ***p< 0.001. Data are expressed as mean ± s.e.m.
Mentions: To evaluate the biological role of ANO1 in lung cancer cell proliferation, we used shRNAs to knockdown the expression of ANO1 in different cell lines. The effectiveness of three shRNAs was evaluated by Western blot in GLC82 and NCI-H520 lung cancer cells. Compared with transfection of scrambled shRNA, ANO1 shRNA1 transfection was most effective in silencing endogenous expression of ANO1 proteins in both GLC82 and NCI-H520 cells (Fig 3A), and thus it was used for the rest of experiments.

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