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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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Effects of Src inhibitor, PP2, on cell migration and on the phosphorylation of ezrin and paxillin. (a) In vitro migration assay was performed twice in triplicate using a 48-well Boyden chamber with a gelatin-coated polycarbonate membrane. Serially diluted PP2 was added into the bottom chamber and cells were loaded into the upper chamber. Following incubation 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). (c) Src inhibitor, PP2, was treated for indicated time and then Western blot analysis was performed as described in Materials and Methods.
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fig5: Effects of Src inhibitor, PP2, on cell migration and on the phosphorylation of ezrin and paxillin. (a) In vitro migration assay was performed twice in triplicate using a 48-well Boyden chamber with a gelatin-coated polycarbonate membrane. Serially diluted PP2 was added into the bottom chamber and cells were loaded into the upper chamber. Following incubation 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). (c) Src inhibitor, PP2, was treated for indicated time and then Western blot analysis was performed as described in Materials and Methods.

Mentions: The involvement of Src in the antimigration activity of ouabain was further confirmed by the pharmacologic inhibition study. Src inhibitor, PP2, also exhibited significant inhibition effect in the migration of A549 cells from the top to the bottom chamber at 6 h, in a dose-dependent fashion when several doses of PP2 (3, 10, and 30 μM) were administered in the bottom chamber (Figures 5(a) and 5(b)). Additionally, A549 cells were treated with PP2 for 30 min and after 24 h the phosphorylation of signaling molecules including Src, ezrin, and paxillin was assessed by Western blotting. As shown in Figure 5(c), PP2-induced inhibition of Src resulted in reduced phosphorylation of ezrin and paxillin. These results suggest that ouabain exerts its antimigration effect by inactivating ezrin and paxillin via Src inhibition.


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)

Effects of Src inhibitor, PP2, on cell migration and on the phosphorylation of ezrin and paxillin. (a) In vitro migration assay was performed twice in triplicate using a 48-well Boyden chamber with a gelatin-coated polycarbonate membrane. Serially diluted PP2 was added into the bottom chamber and cells were loaded into the upper chamber. Following incubation 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). (c) Src inhibitor, PP2, was treated for indicated time and then Western blot analysis was performed as described in Materials and Methods.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4383155&req=5

fig5: Effects of Src inhibitor, PP2, on cell migration and on the phosphorylation of ezrin and paxillin. (a) In vitro migration assay was performed twice in triplicate using a 48-well Boyden chamber with a gelatin-coated polycarbonate membrane. Serially diluted PP2 was added into the bottom chamber and cells were loaded into the upper chamber. Following incubation 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). (c) Src inhibitor, PP2, was treated for indicated time and then Western blot analysis was performed as described in Materials and Methods.
Mentions: The involvement of Src in the antimigration activity of ouabain was further confirmed by the pharmacologic inhibition study. Src inhibitor, PP2, also exhibited significant inhibition effect in the migration of A549 cells from the top to the bottom chamber at 6 h, in a dose-dependent fashion when several doses of PP2 (3, 10, and 30 μM) were administered in the bottom chamber (Figures 5(a) and 5(b)). Additionally, A549 cells were treated with PP2 for 30 min and after 24 h the phosphorylation of signaling molecules including Src, ezrin, and paxillin was assessed by Western blotting. As shown in Figure 5(c), PP2-induced inhibition of Src resulted in reduced phosphorylation of ezrin and paxillin. These results suggest that ouabain exerts its antimigration effect by inactivating ezrin and paxillin via Src inhibition.

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