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Inactivation of Src-to-ezrin pathway: a possible mechanism in the ouabain-mediated inhibition of A549 cell migration.

Shin HK, Ryu BJ, Choi SW, Kim SH, Lee K - Biomed Res Int (2015)

Bottom Line: Employing proteomic techniques, we found 7 proteins downregulated by ouabain in A549 including p-ezrin, a protein associated with pulmonary cancer metastasis in a dose-dependent manner.In addition, when the relative phosphorylation levels of 39 intracellular proteins were compared between control and ouabain-treated A549 cells, p-Src (Y416) was also found to be downregulated by ouabain.The inhibitory effect of ouabain and Src inhibitor PP2 on the migration of A549 cells was confirmed by Boyden chamber assay.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea.

ABSTRACT
Ouabain, a cardiac glycoside found in plants, is primarily used in the treatment of congestive heart failure and arrhythmia because of its ability to inhibit Na(+)/K(+)-ATPase pump. Recently ouabain has been shown to exert anticancer effects but the underlying mechanism is not clear. Here, we explored the molecular mechanism by which ouabain exerts anticancer effects in human lung adenocarcinoma. Employing proteomic techniques, we found 7 proteins downregulated by ouabain in A549 including p-ezrin, a protein associated with pulmonary cancer metastasis in a dose-dependent manner. In addition, when the relative phosphorylation levels of 39 intracellular proteins were compared between control and ouabain-treated A549 cells, p-Src (Y416) was also found to be downregulated by ouabain. Furthermore, western blot revealed the ouabain-mediated downregulation of p-FAK (Y925), p-paxillin (Y118), p130CAS, and Na(+)/K(+)-ATPase subunits that have been shown to be involved in the migration of cancer cells. The inhibitory effect of ouabain and Src inhibitor PP2 on the migration of A549 cells was confirmed by Boyden chamber assay. Anticancer effects of ouabain in A549 cells appear to be related to its ability to regulate and inactivate Src-to-ezrin signaling, and proteins involved in focal adhesion such as Src, FAK, and p130CAS axis are proposed here.

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Proteome profiler array analysis of phosphokinase and validation. (a) Effects of ouabain on the expression and phosphorylation of ezrin were evaluated by Western blot analysis. Actin was used as an internal control. (b) For phosphokinase array study, 300 μg of proteins obtained from A549 cells (5 × 105 cells in a 60 mm2 dish) treated with vehicle (DMSO) or ouabain octahydrate for 24 h in the membranes was probed. (c) Effects of ouabain on the expression and/or phosphorylation of Src, FAK, p130CAS, paxillin, and Na+/K+-ATPase subunits were evaluated by Western blot analysis.
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fig3: Proteome profiler array analysis of phosphokinase and validation. (a) Effects of ouabain on the expression and phosphorylation of ezrin were evaluated by Western blot analysis. Actin was used as an internal control. (b) For phosphokinase array study, 300 μg of proteins obtained from A549 cells (5 × 105 cells in a 60 mm2 dish) treated with vehicle (DMSO) or ouabain octahydrate for 24 h in the membranes was probed. (c) Effects of ouabain on the expression and/or phosphorylation of Src, FAK, p130CAS, paxillin, and Na+/K+-ATPase subunits were evaluated by Western blot analysis.

Mentions: Based on the PMF, the estimated pI and molecular weight by 2-DE map, the circle-indicated protein in the 2-DE gel was identified as ezrin. These characteristics are listed in Table 1. Ouabain-induced decrease in the ezrin signal in 2-DE gel was further differentiated by Western blot analysis. As shown in Figure 3(a), ouabain dose-dependently decreased the level of phosphoezrin (Y353), but not that of total ezrin (Figure 3(a)). Further, we carried out phosphokinase array analysis to investigate molecular pathways that potentially contribute to ouabain-mediated cell death. We found that p-Src (Y416) was downregulated by ouabain in 39 intracellular proteins in the control and ouabain-treated A549 cells (Figure 3(b)). Ouabain-mediated decrease of p-Src (Y416) was also confirmed by Western blot analysis (Figure 3(c)).


Inactivation of Src-to-ezrin pathway: a possible mechanism in the ouabain-mediated inhibition of A549 cell migration.

Shin HK, Ryu BJ, Choi SW, Kim SH, Lee K - Biomed Res Int (2015)

Proteome profiler array analysis of phosphokinase and validation. (a) Effects of ouabain on the expression and phosphorylation of ezrin were evaluated by Western blot analysis. Actin was used as an internal control. (b) For phosphokinase array study, 300 μg of proteins obtained from A549 cells (5 × 105 cells in a 60 mm2 dish) treated with vehicle (DMSO) or ouabain octahydrate for 24 h in the membranes was probed. (c) Effects of ouabain on the expression and/or phosphorylation of Src, FAK, p130CAS, paxillin, and Na+/K+-ATPase subunits were evaluated by Western blot analysis.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4383155&req=5

fig3: Proteome profiler array analysis of phosphokinase and validation. (a) Effects of ouabain on the expression and phosphorylation of ezrin were evaluated by Western blot analysis. Actin was used as an internal control. (b) For phosphokinase array study, 300 μg of proteins obtained from A549 cells (5 × 105 cells in a 60 mm2 dish) treated with vehicle (DMSO) or ouabain octahydrate for 24 h in the membranes was probed. (c) Effects of ouabain on the expression and/or phosphorylation of Src, FAK, p130CAS, paxillin, and Na+/K+-ATPase subunits were evaluated by Western blot analysis.
Mentions: Based on the PMF, the estimated pI and molecular weight by 2-DE map, the circle-indicated protein in the 2-DE gel was identified as ezrin. These characteristics are listed in Table 1. Ouabain-induced decrease in the ezrin signal in 2-DE gel was further differentiated by Western blot analysis. As shown in Figure 3(a), ouabain dose-dependently decreased the level of phosphoezrin (Y353), but not that of total ezrin (Figure 3(a)). Further, we carried out phosphokinase array analysis to investigate molecular pathways that potentially contribute to ouabain-mediated cell death. We found that p-Src (Y416) was downregulated by ouabain in 39 intracellular proteins in the control and ouabain-treated A549 cells (Figure 3(b)). Ouabain-mediated decrease of p-Src (Y416) was also confirmed by Western blot analysis (Figure 3(c)).

Bottom Line: Employing proteomic techniques, we found 7 proteins downregulated by ouabain in A549 including p-ezrin, a protein associated with pulmonary cancer metastasis in a dose-dependent manner.In addition, when the relative phosphorylation levels of 39 intracellular proteins were compared between control and ouabain-treated A549 cells, p-Src (Y416) was also found to be downregulated by ouabain.The inhibitory effect of ouabain and Src inhibitor PP2 on the migration of A549 cells was confirmed by Boyden chamber assay.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea.

ABSTRACT
Ouabain, a cardiac glycoside found in plants, is primarily used in the treatment of congestive heart failure and arrhythmia because of its ability to inhibit Na(+)/K(+)-ATPase pump. Recently ouabain has been shown to exert anticancer effects but the underlying mechanism is not clear. Here, we explored the molecular mechanism by which ouabain exerts anticancer effects in human lung adenocarcinoma. Employing proteomic techniques, we found 7 proteins downregulated by ouabain in A549 including p-ezrin, a protein associated with pulmonary cancer metastasis in a dose-dependent manner. In addition, when the relative phosphorylation levels of 39 intracellular proteins were compared between control and ouabain-treated A549 cells, p-Src (Y416) was also found to be downregulated by ouabain. Furthermore, western blot revealed the ouabain-mediated downregulation of p-FAK (Y925), p-paxillin (Y118), p130CAS, and Na(+)/K(+)-ATPase subunits that have been shown to be involved in the migration of cancer cells. The inhibitory effect of ouabain and Src inhibitor PP2 on the migration of A549 cells was confirmed by Boyden chamber assay. Anticancer effects of ouabain in A549 cells appear to be related to its ability to regulate and inactivate Src-to-ezrin signaling, and proteins involved in focal adhesion such as Src, FAK, and p130CAS axis are proposed here.

Show MeSH
Related in: MedlinePlus