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Inhibition of cell motility by troglitazone in human ovarian carcinoma cell line.

Yang YC, Ho TC, Chen SL, Lai HY, Wu JY, Tsao YP - BMC Cancer (2007)

Bottom Line: The effects of TGZ on phosphorylation of FAK, PTEN, Akt were assessed by immunoblotting using phospho-specific antibodies.These results indicate that TGZ can suppress cultured ES-2 cells migration.Our data suggest that the anti-migration potential of TGZ involves in regulations of FAK and PTEN activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Mackay Medicine, Nursing and Management College, Taipei, Taiwan. eugene@ms2.mmh.org.tw

ABSTRACT

Background: Troglitazone (TGZ) is a potential anticancer agent. Little is known about the effect of this agent on cancer cell migration.

Methods: Human ovarian carcinoma cell line, ES-2 cells were treated with various concentrations of TGZ. Cell migration was evaluated by wound-healing and Boyden chamber transwell experiments. PPARgamma expression was blocked by PPARgamma small interfering RNA. The effects of TGZ on phosphorylation of FAK, PTEN, Akt were assessed by immunoblotting using phospho-specific antibodies. The cellular distribution of paxillin, vinculin, stress fiber and PTEN was assessed by immunocytochemistry.

Results: TGZ dose- and time-dependently impaired cell migration through a PPARgamma independent manner. TGZ treatment impaired cell spreading, stress fiber formation, tyrosine phosphorylation of focal adhesion kinase (FAK), and focal adhesion assembly in cells grown on fibronectin substratum. TGZ also dose- and time-dependently suppressed FAK autophosphorylation and phosphorylation of the C-terminal of PTEN (a phosphatase). At concentration higher than 10 muM, TGZ caused accumulation of PTEN in plasma membrane, a sign of PTEN activation.

Conclusion: These results indicate that TGZ can suppress cultured ES-2 cells migration. Our data suggest that the anti-migration potential of TGZ involves in regulations of FAK and PTEN activity.

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Related in: MedlinePlus

TGZ inhibits stress fiber and focal adhesion formation. (A) Effect of TGZ on the distribution of paxillin and stress fibers. ES-2 cells were plated on FN (10 μg/mL)-coated coverslips and incubated in 10% FBS medium with 0.1% DMSO or 20 μM TGZ for 4 h. Cells were then double stained with rhodamine-labeled phalloidin and antibodies to paxillin (FITC). These results are representative of triplicate experiments. (B) Time-course study of the tyrosine phosphorylation level of focal adhesion kinase (FAK) in TGZ-treated ES-2 cells. ES-2 cells were treated with 20 μM TGZ for different intervals. The cell homogenates were immunoprecipitated from 1 mg of total cellular protein by anti-FAK antibody and subjected to Western blot analysis with anti-phosphotyrosine antibody (p-FAK) or anti-FAK antibody (FAK). The cell homogenates were also analyzed for β-actin levels by Western blotting as indicated. Immunoblot results are from a representative experiment performed in triplicate with β-actin as loading control. Symbol (*) indicates cells that were treated with DMSO for 8 h.
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Figure 2: TGZ inhibits stress fiber and focal adhesion formation. (A) Effect of TGZ on the distribution of paxillin and stress fibers. ES-2 cells were plated on FN (10 μg/mL)-coated coverslips and incubated in 10% FBS medium with 0.1% DMSO or 20 μM TGZ for 4 h. Cells were then double stained with rhodamine-labeled phalloidin and antibodies to paxillin (FITC). These results are representative of triplicate experiments. (B) Time-course study of the tyrosine phosphorylation level of focal adhesion kinase (FAK) in TGZ-treated ES-2 cells. ES-2 cells were treated with 20 μM TGZ for different intervals. The cell homogenates were immunoprecipitated from 1 mg of total cellular protein by anti-FAK antibody and subjected to Western blot analysis with anti-phosphotyrosine antibody (p-FAK) or anti-FAK antibody (FAK). The cell homogenates were also analyzed for β-actin levels by Western blotting as indicated. Immunoblot results are from a representative experiment performed in triplicate with β-actin as loading control. Symbol (*) indicates cells that were treated with DMSO for 8 h.

Mentions: We next examined the effect of TGZ on focal adhesions (FAs) formation because its impairment has been shown to reduce cell migration [6]. After four hours of adhesion to fibronectin (FN)-coated surfaces, ES-2 cells formed numerous FAs, which were stained by anti-paxillin. In cells incubated with 20 μM TGZ, FAs were substantially reduced (Figure 2A). Similar inhibitory effect of TGZ was observed when FAs formation was identified by anti-vinculin antibody (data not shown). Reduction in the number of FAs was accompanied by an overall decrease in actin stress fiber (Figure 2A). It is highly possible that TGZ treatment decreases the transport or maintenance of cytoskeletal proteins in FAs, such as paxillin and vinculin, thereby reducing the number of complexes available for the formation of strong focal points and actin bundling.


Inhibition of cell motility by troglitazone in human ovarian carcinoma cell line.

Yang YC, Ho TC, Chen SL, Lai HY, Wu JY, Tsao YP - BMC Cancer (2007)

TGZ inhibits stress fiber and focal adhesion formation. (A) Effect of TGZ on the distribution of paxillin and stress fibers. ES-2 cells were plated on FN (10 μg/mL)-coated coverslips and incubated in 10% FBS medium with 0.1% DMSO or 20 μM TGZ for 4 h. Cells were then double stained with rhodamine-labeled phalloidin and antibodies to paxillin (FITC). These results are representative of triplicate experiments. (B) Time-course study of the tyrosine phosphorylation level of focal adhesion kinase (FAK) in TGZ-treated ES-2 cells. ES-2 cells were treated with 20 μM TGZ for different intervals. The cell homogenates were immunoprecipitated from 1 mg of total cellular protein by anti-FAK antibody and subjected to Western blot analysis with anti-phosphotyrosine antibody (p-FAK) or anti-FAK antibody (FAK). The cell homogenates were also analyzed for β-actin levels by Western blotting as indicated. Immunoblot results are from a representative experiment performed in triplicate with β-actin as loading control. Symbol (*) indicates cells that were treated with DMSO for 8 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2233635&req=5

Figure 2: TGZ inhibits stress fiber and focal adhesion formation. (A) Effect of TGZ on the distribution of paxillin and stress fibers. ES-2 cells were plated on FN (10 μg/mL)-coated coverslips and incubated in 10% FBS medium with 0.1% DMSO or 20 μM TGZ for 4 h. Cells were then double stained with rhodamine-labeled phalloidin and antibodies to paxillin (FITC). These results are representative of triplicate experiments. (B) Time-course study of the tyrosine phosphorylation level of focal adhesion kinase (FAK) in TGZ-treated ES-2 cells. ES-2 cells were treated with 20 μM TGZ for different intervals. The cell homogenates were immunoprecipitated from 1 mg of total cellular protein by anti-FAK antibody and subjected to Western blot analysis with anti-phosphotyrosine antibody (p-FAK) or anti-FAK antibody (FAK). The cell homogenates were also analyzed for β-actin levels by Western blotting as indicated. Immunoblot results are from a representative experiment performed in triplicate with β-actin as loading control. Symbol (*) indicates cells that were treated with DMSO for 8 h.
Mentions: We next examined the effect of TGZ on focal adhesions (FAs) formation because its impairment has been shown to reduce cell migration [6]. After four hours of adhesion to fibronectin (FN)-coated surfaces, ES-2 cells formed numerous FAs, which were stained by anti-paxillin. In cells incubated with 20 μM TGZ, FAs were substantially reduced (Figure 2A). Similar inhibitory effect of TGZ was observed when FAs formation was identified by anti-vinculin antibody (data not shown). Reduction in the number of FAs was accompanied by an overall decrease in actin stress fiber (Figure 2A). It is highly possible that TGZ treatment decreases the transport or maintenance of cytoskeletal proteins in FAs, such as paxillin and vinculin, thereby reducing the number of complexes available for the formation of strong focal points and actin bundling.

Bottom Line: The effects of TGZ on phosphorylation of FAK, PTEN, Akt were assessed by immunoblotting using phospho-specific antibodies.These results indicate that TGZ can suppress cultured ES-2 cells migration.Our data suggest that the anti-migration potential of TGZ involves in regulations of FAK and PTEN activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Mackay Medicine, Nursing and Management College, Taipei, Taiwan. eugene@ms2.mmh.org.tw

ABSTRACT

Background: Troglitazone (TGZ) is a potential anticancer agent. Little is known about the effect of this agent on cancer cell migration.

Methods: Human ovarian carcinoma cell line, ES-2 cells were treated with various concentrations of TGZ. Cell migration was evaluated by wound-healing and Boyden chamber transwell experiments. PPARgamma expression was blocked by PPARgamma small interfering RNA. The effects of TGZ on phosphorylation of FAK, PTEN, Akt were assessed by immunoblotting using phospho-specific antibodies. The cellular distribution of paxillin, vinculin, stress fiber and PTEN was assessed by immunocytochemistry.

Results: TGZ dose- and time-dependently impaired cell migration through a PPARgamma independent manner. TGZ treatment impaired cell spreading, stress fiber formation, tyrosine phosphorylation of focal adhesion kinase (FAK), and focal adhesion assembly in cells grown on fibronectin substratum. TGZ also dose- and time-dependently suppressed FAK autophosphorylation and phosphorylation of the C-terminal of PTEN (a phosphatase). At concentration higher than 10 muM, TGZ caused accumulation of PTEN in plasma membrane, a sign of PTEN activation.

Conclusion: These results indicate that TGZ can suppress cultured ES-2 cells migration. Our data suggest that the anti-migration potential of TGZ involves in regulations of FAK and PTEN activity.

Show MeSH
Related in: MedlinePlus