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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

In vivo suppression of xenograft tumor growth by silencing ANO1.Normal GLC82 cells or stable GLC82 cells expressing ANO1 shRNA 1, ANO1 shRNA 2 and scrambled shRNA were inoculated hypodermically into the right forelimbs of 5-week-old female nude mice (1X107 cells per mouse). (A) Western analysis of knockdown efficiency for wild type GLC82 cells and GLC82 cells stable expressing scrambled shRNA, ANO1 shRNA 1 and ANO1 shRNA 2. (B) Representative picture of nude mice carrying GLC82 implanted tumors in four groups: blank group (n = 7), scrambled shRNA group (n = 8), ANO1 shRNA1 group (n = 8) and ANO1 shRNA2 group (n = 8). (C) Tumor size was measured every 2–4 days by Vernier caliper during its development since the 7th day after injection. The growth of tumors expressing ANO1 shRNA1 and ANO1 shRNA2 was significantly inhibited in a time dependent manner, compared with the scrambled shRNA group. (D) Representative picture of tumors picked from mice on the 12th day of generation. (E) Tumors were weighed and analyzed after surgical removal. Comparing with scrambled shRNA tumors, ANO1 shRNAs tumors were lighter. Statistical significance (ANOVA) is shown as *p< 0.05; **p< 0.01 and ***p< 0.001. Data are expressed as mean ± s.e.m. (F) Representative images of immunohistochemical staining of ANO1 expression in xenograft tumor tissues. (G) Analysis of ANO1 expression in xenograft tumor tissues was conducted by scoring from 0 to 3 according to the intensity and area of the staining.
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pone.0136584.g006: In vivo suppression of xenograft tumor growth by silencing ANO1.Normal GLC82 cells or stable GLC82 cells expressing ANO1 shRNA 1, ANO1 shRNA 2 and scrambled shRNA were inoculated hypodermically into the right forelimbs of 5-week-old female nude mice (1X107 cells per mouse). (A) Western analysis of knockdown efficiency for wild type GLC82 cells and GLC82 cells stable expressing scrambled shRNA, ANO1 shRNA 1 and ANO1 shRNA 2. (B) Representative picture of nude mice carrying GLC82 implanted tumors in four groups: blank group (n = 7), scrambled shRNA group (n = 8), ANO1 shRNA1 group (n = 8) and ANO1 shRNA2 group (n = 8). (C) Tumor size was measured every 2–4 days by Vernier caliper during its development since the 7th day after injection. The growth of tumors expressing ANO1 shRNA1 and ANO1 shRNA2 was significantly inhibited in a time dependent manner, compared with the scrambled shRNA group. (D) Representative picture of tumors picked from mice on the 12th day of generation. (E) Tumors were weighed and analyzed after surgical removal. Comparing with scrambled shRNA tumors, ANO1 shRNAs tumors were lighter. Statistical significance (ANOVA) is shown as *p< 0.05; **p< 0.01 and ***p< 0.001. Data are expressed as mean ± s.e.m. (F) Representative images of immunohistochemical staining of ANO1 expression in xenograft tumor tissues. (G) Analysis of ANO1 expression in xenograft tumor tissues was conducted by scoring from 0 to 3 according to the intensity and area of the staining.

Mentions: To investigate the effect of ANO1 knockdown on growth of xenograft tumor in vivo, three groups of GLC82 cells were individually transfected with ANO1 shRNA1, ANO1 shRNA2 and scrambled shRNAs, and their knockdown efficiency was confirmed by western blot before injection for formation of tumor xenograft in mice (Fig 6A). We injected 107 GLC82 cells per mouse into right forelimbs of 5-week old nude mice in four groups for formation of a tumor xenograft. The size of tumors was first measured 7 days after the injection (as day 0). As shown in Fig 6B and 6C, ANO1 silencing resulted in a significant reduction of tumor growth by 68.8% in shRNA1 group and 42.1% in shRNA2 group, respectively, as compared with the scrambled group at the observation of day 12. At day 12, the tumors were removed from mice and weighted. A remarkable reduction of tumor weight in ANO1 shRNA groups was observed (Fig 6D and 6E). As compared with the control scrambled shRNA, the average tumor weight of ANO1 shRNA1 and shRNA2 groups was about 38.7% and 70.1%, respectively (Fig 6D and 6E). Further immunohistochemical staining of ANO1 in xenograft tumor confirmed that the reduction of ANO1 protein expression levels was consistent with the tumor volume in the groups treated by ANO1 shRNAs with different potency and in the control group (Fig 6F and 6G). These results indicate that ANO1 silencing not only inhibits the migration and invasion of lung cancer cells but also significantly suppresses tumor growth.


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)

In vivo suppression of xenograft tumor growth by silencing ANO1.Normal GLC82 cells or stable GLC82 cells expressing ANO1 shRNA 1, ANO1 shRNA 2 and scrambled shRNA were inoculated hypodermically into the right forelimbs of 5-week-old female nude mice (1X107 cells per mouse). (A) Western analysis of knockdown efficiency for wild type GLC82 cells and GLC82 cells stable expressing scrambled shRNA, ANO1 shRNA 1 and ANO1 shRNA 2. (B) Representative picture of nude mice carrying GLC82 implanted tumors in four groups: blank group (n = 7), scrambled shRNA group (n = 8), ANO1 shRNA1 group (n = 8) and ANO1 shRNA2 group (n = 8). (C) Tumor size was measured every 2–4 days by Vernier caliper during its development since the 7th day after injection. The growth of tumors expressing ANO1 shRNA1 and ANO1 shRNA2 was significantly inhibited in a time dependent manner, compared with the scrambled shRNA group. (D) Representative picture of tumors picked from mice on the 12th day of generation. (E) Tumors were weighed and analyzed after surgical removal. Comparing with scrambled shRNA tumors, ANO1 shRNAs tumors were lighter. Statistical significance (ANOVA) is shown as *p< 0.05; **p< 0.01 and ***p< 0.001. Data are expressed as mean ± s.e.m. (F) Representative images of immunohistochemical staining of ANO1 expression in xenograft tumor tissues. (G) Analysis of ANO1 expression in xenograft tumor tissues was conducted by scoring from 0 to 3 according to the intensity and area of the staining.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0136584.g006: In vivo suppression of xenograft tumor growth by silencing ANO1.Normal GLC82 cells or stable GLC82 cells expressing ANO1 shRNA 1, ANO1 shRNA 2 and scrambled shRNA were inoculated hypodermically into the right forelimbs of 5-week-old female nude mice (1X107 cells per mouse). (A) Western analysis of knockdown efficiency for wild type GLC82 cells and GLC82 cells stable expressing scrambled shRNA, ANO1 shRNA 1 and ANO1 shRNA 2. (B) Representative picture of nude mice carrying GLC82 implanted tumors in four groups: blank group (n = 7), scrambled shRNA group (n = 8), ANO1 shRNA1 group (n = 8) and ANO1 shRNA2 group (n = 8). (C) Tumor size was measured every 2–4 days by Vernier caliper during its development since the 7th day after injection. The growth of tumors expressing ANO1 shRNA1 and ANO1 shRNA2 was significantly inhibited in a time dependent manner, compared with the scrambled shRNA group. (D) Representative picture of tumors picked from mice on the 12th day of generation. (E) Tumors were weighed and analyzed after surgical removal. Comparing with scrambled shRNA tumors, ANO1 shRNAs tumors were lighter. Statistical significance (ANOVA) is shown as *p< 0.05; **p< 0.01 and ***p< 0.001. Data are expressed as mean ± s.e.m. (F) Representative images of immunohistochemical staining of ANO1 expression in xenograft tumor tissues. (G) Analysis of ANO1 expression in xenograft tumor tissues was conducted by scoring from 0 to 3 according to the intensity and area of the staining.
Mentions: To investigate the effect of ANO1 knockdown on growth of xenograft tumor in vivo, three groups of GLC82 cells were individually transfected with ANO1 shRNA1, ANO1 shRNA2 and scrambled shRNAs, and their knockdown efficiency was confirmed by western blot before injection for formation of tumor xenograft in mice (Fig 6A). We injected 107 GLC82 cells per mouse into right forelimbs of 5-week old nude mice in four groups for formation of a tumor xenograft. The size of tumors was first measured 7 days after the injection (as day 0). As shown in Fig 6B and 6C, ANO1 silencing resulted in a significant reduction of tumor growth by 68.8% in shRNA1 group and 42.1% in shRNA2 group, respectively, as compared with the scrambled group at the observation of day 12. At day 12, the tumors were removed from mice and weighted. A remarkable reduction of tumor weight in ANO1 shRNA groups was observed (Fig 6D and 6E). As compared with the control scrambled shRNA, the average tumor weight of ANO1 shRNA1 and shRNA2 groups was about 38.7% and 70.1%, respectively (Fig 6D and 6E). Further immunohistochemical staining of ANO1 in xenograft tumor confirmed that the reduction of ANO1 protein expression levels was consistent with the tumor volume in the groups treated by ANO1 shRNAs with different potency and in the control group (Fig 6F and 6G). These results indicate that ANO1 silencing not only inhibits the migration and invasion of lung cancer cells but also significantly suppresses tumor growth.

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