Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of ouabain on migration of A549 cells. (a) In vitro migration assay was performed twice in triplicate using a 48-well Boyden chamber with a gelatin-coated polycarbonate membrane. DMEM containing either 0.1% FBS or 10% FBS was added into the bottom chamber and cells were loaded into the upper chamber and incubated at 37°C for 6 h. The cells on the upper side of the membrane were removed, and the cells on the bottom of the filter membrane were stained with Diff-Quick solution. (b) The numbers of migrated cells were counted under a light microscope. The data are presented as mean ± standard deviation (*P < 0.05; **P < 0.01; ***P < 0.001).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4383155&req=5

fig4: Effect of ouabain on migration of A549 cells. (a) In vitro migration assay was performed twice in triplicate using a 48-well Boyden chamber with a gelatin-coated polycarbonate membrane. DMEM containing either 0.1% FBS or 10% FBS was added into the bottom chamber and cells were loaded into the upper chamber and incubated at 37°C for 6 h. The cells on the upper side of the membrane were removed, and the cells on the bottom of the filter membrane were stained with Diff-Quick solution. (b) The numbers of migrated cells were counted under a light microscope. The data are presented as mean ± standard deviation (*P < 0.05; **P < 0.01; ***P < 0.001).

Mentions: Human A549 cells have been reported to be highly metastatic, and cardiac glycosides have been reported to inhibit the migration of cancer cells by the specific inhibition of the Na+/K+-ATPase α1 subunit [13, 14]. Therefore, we examined the effect of ouabain on the expression of Na+/K+-ATPase subunits and the migration of A549 cells using the Boyden chamber analysis. As shown in Figure 3(c), ouabain treatment for 1 day of A549 cells strongly inhibited the expression levels of Na+/K+-ATPase α1 and β1. Furthermore, it decreased the migration of A549 cells in a dose-dependent manner even when it was exposed to A549 cells for 4 h before migration (Figures 4(a) and 4(b)).


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)

Effect of ouabain on migration of A549 cells. (a) In vitro migration assay was performed twice in triplicate using a 48-well Boyden chamber with a gelatin-coated polycarbonate membrane. DMEM containing either 0.1% FBS or 10% FBS was added into the bottom chamber and cells were loaded into the upper chamber and incubated at 37°C for 6 h. The cells on the upper side of the membrane were removed, and the cells on the bottom of the filter membrane were stained with Diff-Quick solution. (b) The numbers of migrated cells were counted under a light microscope. The data are presented as mean ± standard deviation (*P < 0.05; **P < 0.01; ***P < 0.001).
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Effect of ouabain on migration of A549 cells. (a) In vitro migration assay was performed twice in triplicate using a 48-well Boyden chamber with a gelatin-coated polycarbonate membrane. DMEM containing either 0.1% FBS or 10% FBS was added into the bottom chamber and cells were loaded into the upper chamber and incubated at 37°C for 6 h. The cells on the upper side of the membrane were removed, and the cells on the bottom of the filter membrane were stained with Diff-Quick solution. (b) The numbers of migrated cells were counted under a light microscope. The data are presented as mean ± standard deviation (*P < 0.05; **P < 0.01; ***P < 0.001).
Mentions: Human A549 cells have been reported to be highly metastatic, and cardiac glycosides have been reported to inhibit the migration of cancer cells by the specific inhibition of the Na+/K+-ATPase α1 subunit [13, 14]. Therefore, we examined the effect of ouabain on the expression of Na+/K+-ATPase subunits and the migration of A549 cells using the Boyden chamber analysis. As shown in Figure 3(c), ouabain treatment for 1 day of A549 cells strongly inhibited the expression levels of Na+/K+-ATPase α1 and β1. Furthermore, it decreased the migration of A549 cells in a dose-dependent manner even when it was exposed to A549 cells for 4 h before migration (Figures 4(a) and 4(b)).

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