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ROCK inhibition activates MCF-7 cells.

Yang S, Kim HM - PLoS ONE (2014)

Bottom Line: Rho-associated kinase (ROCK) inhibition disrupted cell junction, promoted cell proliferation and migration / invasion in both two-dimensional and three-dimensional substrates.The disintegration of cell junction upon ROCK inhibition, coupled with the loss of E-cadherin and b-catenin from the cell membrane, was associated with the activation of Rac1 upon ROCK inhibition.However, the activation of MCF-7 cells upon ROCK inhibition was not associated with the up-regulation of typical EMT markers, such as snail and slug.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for the Study of Molecular Biointerfaces, Department of Oral Histology and Developmental Biology, Program of Cell and Developmental Biology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Republic of Korea.

ABSTRACT
Dormant carcinoma cancer cells showing epithelial characteristics can be activated to dissipate into the surrounding tissue or organs through epithelial-mesenchymal transition (EMT). However, the molecular details underlying the activation of dormant cancer cells have been less explored. In this study, we examined the molecular pathway to activate dormant breast cancer cells. Rho-associated kinase (ROCK) inhibition disrupted cell junction, promoted cell proliferation and migration / invasion in both two-dimensional and three-dimensional substrates. The disintegration of cell junction upon ROCK inhibition, coupled with the loss of E-cadherin and b-catenin from the cell membrane, was associated with the activation of Rac1 upon ROCK inhibition. Migration / invasion also increased upon ROCK inhibition. However, the activation of MCF-7 cells upon ROCK inhibition was not associated with the up-regulation of typical EMT markers, such as snail and slug. Based on these results, we suggest the potential risk for dormant cancer cells to dissipate through non-typical EMT when ROCK activity is down-regulated.

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Adhesion strength has influence on ROCK activity and various cell activities in a dose dependent manner.A. To compare the relative levels of ROCK activation, depending on the amount of precoated FN, P-MLC expression levels were assessed through immunoblotting analysis using an anti-P-MLC antibody. B. The cells were cultured for three days on the hydrophobic culture dishes precoated with indicated amounts of fibronectin. Cell number was analyzed using CCK-8 kit. The data are expressed as the means ± SD. n = 3 dishes. The p-value was less than 0.05 for comparisons between any two groups. *, p<0.05. C. The cells seeded on the Transwell chambers precoated with the indicated amounts of fibronectin (FN) for the migration assay were incubated for 48 hrs. The cells migrated into the lower chamber were manually counted after staining cells with hematoxylin. The data are expressed as the mean ± SD. n = 3. The p-value was less than 0.05 for comparisons between any two groups. *, p<0.05. D. MCF-7 Cells were cultured for two days on hydrophobic culture dishes precoated with the indicated amounts of fibronectin (FN). The substrates were blocked with albumin (5% v/v) after coating with fibronectin. The localized patterns of F-actin and E-cadherin were examined using FITC-phalloidin and Cy3-E-cadherin through confocal laser microscopy. E. MCF-7 Cells were cultured for two days on hydrophobic culture dishes precoated with the indicated amounts of fibronectin (FN). The substrates were blocked with albumin (5% v/v) after coating with fibronectin. Cell morphology was examined using phase-contrast microscopy.
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pone-0088489-g007: Adhesion strength has influence on ROCK activity and various cell activities in a dose dependent manner.A. To compare the relative levels of ROCK activation, depending on the amount of precoated FN, P-MLC expression levels were assessed through immunoblotting analysis using an anti-P-MLC antibody. B. The cells were cultured for three days on the hydrophobic culture dishes precoated with indicated amounts of fibronectin. Cell number was analyzed using CCK-8 kit. The data are expressed as the means ± SD. n = 3 dishes. The p-value was less than 0.05 for comparisons between any two groups. *, p<0.05. C. The cells seeded on the Transwell chambers precoated with the indicated amounts of fibronectin (FN) for the migration assay were incubated for 48 hrs. The cells migrated into the lower chamber were manually counted after staining cells with hematoxylin. The data are expressed as the mean ± SD. n = 3. The p-value was less than 0.05 for comparisons between any two groups. *, p<0.05. D. MCF-7 Cells were cultured for two days on hydrophobic culture dishes precoated with the indicated amounts of fibronectin (FN). The substrates were blocked with albumin (5% v/v) after coating with fibronectin. The localized patterns of F-actin and E-cadherin were examined using FITC-phalloidin and Cy3-E-cadherin through confocal laser microscopy. E. MCF-7 Cells were cultured for two days on hydrophobic culture dishes precoated with the indicated amounts of fibronectin (FN). The substrates were blocked with albumin (5% v/v) after coating with fibronectin. Cell morphology was examined using phase-contrast microscopy.

Mentions: Next we explored the biological significance of the lowered ROCK activity in association with the activation of MCF-7 cells. In general, adhesion signaling is poor in dormant cancer cells [13], and weak adhesion signaling up-regulates RhoA-ROCK signaling [10], [11]. Thus, the ROCK activity in dormant MCF-7 cells was monitored on hydrophobic substrates precoated with various amounts of fibronectin to show a dependency on the strength of the adhesion signaling through an examination of the phosphorylated myosin light chain (p-MLC) [7]. Consistent with our hypothesis that ROCK activity was associated with the degree of adhesion signaling, the expression of p-MLC in MCF-7 cells, which reflects ROCK activity, decreased with increased adhesion signaling, and ROCK inhibition decreased the p-MLC expression in MCF-7 cells [Fig. 7A]. The MCF-7 cells presented the same phenotypes as shown for ROCK inhibition on the substrates precoated with fibronectin. Cell proliferation and migration were up-regulated on the substrates precoated with fibronectin in a dose dependent manner [Fig. 7B and C]. Furthermore, the E-cadherin expression at the cell-cell junction decreased on the fibronectin-coated substrates in a dose dependent manner [Fig. 7D], and the MCF-7 cells dissipated on the substrates precoated with a higher amount of fibronectin [Fig. 7D and E]. Based on these results, we can establish a hypothesis that the activation of MCF-7 cells may be associated with the ROCK activity down-regulated on the substrates with a higher adhesion strength. Further study is required to confirm this hypothesis.


ROCK inhibition activates MCF-7 cells.

Yang S, Kim HM - PLoS ONE (2014)

Adhesion strength has influence on ROCK activity and various cell activities in a dose dependent manner.A. To compare the relative levels of ROCK activation, depending on the amount of precoated FN, P-MLC expression levels were assessed through immunoblotting analysis using an anti-P-MLC antibody. B. The cells were cultured for three days on the hydrophobic culture dishes precoated with indicated amounts of fibronectin. Cell number was analyzed using CCK-8 kit. The data are expressed as the means ± SD. n = 3 dishes. The p-value was less than 0.05 for comparisons between any two groups. *, p<0.05. C. The cells seeded on the Transwell chambers precoated with the indicated amounts of fibronectin (FN) for the migration assay were incubated for 48 hrs. The cells migrated into the lower chamber were manually counted after staining cells with hematoxylin. The data are expressed as the mean ± SD. n = 3. The p-value was less than 0.05 for comparisons between any two groups. *, p<0.05. D. MCF-7 Cells were cultured for two days on hydrophobic culture dishes precoated with the indicated amounts of fibronectin (FN). The substrates were blocked with albumin (5% v/v) after coating with fibronectin. The localized patterns of F-actin and E-cadherin were examined using FITC-phalloidin and Cy3-E-cadherin through confocal laser microscopy. E. MCF-7 Cells were cultured for two days on hydrophobic culture dishes precoated with the indicated amounts of fibronectin (FN). The substrates were blocked with albumin (5% v/v) after coating with fibronectin. Cell morphology was examined using phase-contrast microscopy.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921164&req=5

pone-0088489-g007: Adhesion strength has influence on ROCK activity and various cell activities in a dose dependent manner.A. To compare the relative levels of ROCK activation, depending on the amount of precoated FN, P-MLC expression levels were assessed through immunoblotting analysis using an anti-P-MLC antibody. B. The cells were cultured for three days on the hydrophobic culture dishes precoated with indicated amounts of fibronectin. Cell number was analyzed using CCK-8 kit. The data are expressed as the means ± SD. n = 3 dishes. The p-value was less than 0.05 for comparisons between any two groups. *, p<0.05. C. The cells seeded on the Transwell chambers precoated with the indicated amounts of fibronectin (FN) for the migration assay were incubated for 48 hrs. The cells migrated into the lower chamber were manually counted after staining cells with hematoxylin. The data are expressed as the mean ± SD. n = 3. The p-value was less than 0.05 for comparisons between any two groups. *, p<0.05. D. MCF-7 Cells were cultured for two days on hydrophobic culture dishes precoated with the indicated amounts of fibronectin (FN). The substrates were blocked with albumin (5% v/v) after coating with fibronectin. The localized patterns of F-actin and E-cadherin were examined using FITC-phalloidin and Cy3-E-cadherin through confocal laser microscopy. E. MCF-7 Cells were cultured for two days on hydrophobic culture dishes precoated with the indicated amounts of fibronectin (FN). The substrates were blocked with albumin (5% v/v) after coating with fibronectin. Cell morphology was examined using phase-contrast microscopy.
Mentions: Next we explored the biological significance of the lowered ROCK activity in association with the activation of MCF-7 cells. In general, adhesion signaling is poor in dormant cancer cells [13], and weak adhesion signaling up-regulates RhoA-ROCK signaling [10], [11]. Thus, the ROCK activity in dormant MCF-7 cells was monitored on hydrophobic substrates precoated with various amounts of fibronectin to show a dependency on the strength of the adhesion signaling through an examination of the phosphorylated myosin light chain (p-MLC) [7]. Consistent with our hypothesis that ROCK activity was associated with the degree of adhesion signaling, the expression of p-MLC in MCF-7 cells, which reflects ROCK activity, decreased with increased adhesion signaling, and ROCK inhibition decreased the p-MLC expression in MCF-7 cells [Fig. 7A]. The MCF-7 cells presented the same phenotypes as shown for ROCK inhibition on the substrates precoated with fibronectin. Cell proliferation and migration were up-regulated on the substrates precoated with fibronectin in a dose dependent manner [Fig. 7B and C]. Furthermore, the E-cadherin expression at the cell-cell junction decreased on the fibronectin-coated substrates in a dose dependent manner [Fig. 7D], and the MCF-7 cells dissipated on the substrates precoated with a higher amount of fibronectin [Fig. 7D and E]. Based on these results, we can establish a hypothesis that the activation of MCF-7 cells may be associated with the ROCK activity down-regulated on the substrates with a higher adhesion strength. Further study is required to confirm this hypothesis.

Bottom Line: Rho-associated kinase (ROCK) inhibition disrupted cell junction, promoted cell proliferation and migration / invasion in both two-dimensional and three-dimensional substrates.The disintegration of cell junction upon ROCK inhibition, coupled with the loss of E-cadherin and b-catenin from the cell membrane, was associated with the activation of Rac1 upon ROCK inhibition.However, the activation of MCF-7 cells upon ROCK inhibition was not associated with the up-regulation of typical EMT markers, such as snail and slug.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for the Study of Molecular Biointerfaces, Department of Oral Histology and Developmental Biology, Program of Cell and Developmental Biology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Republic of Korea.

ABSTRACT
Dormant carcinoma cancer cells showing epithelial characteristics can be activated to dissipate into the surrounding tissue or organs through epithelial-mesenchymal transition (EMT). However, the molecular details underlying the activation of dormant cancer cells have been less explored. In this study, we examined the molecular pathway to activate dormant breast cancer cells. Rho-associated kinase (ROCK) inhibition disrupted cell junction, promoted cell proliferation and migration / invasion in both two-dimensional and three-dimensional substrates. The disintegration of cell junction upon ROCK inhibition, coupled with the loss of E-cadherin and b-catenin from the cell membrane, was associated with the activation of Rac1 upon ROCK inhibition. Migration / invasion also increased upon ROCK inhibition. However, the activation of MCF-7 cells upon ROCK inhibition was not associated with the up-regulation of typical EMT markers, such as snail and slug. Based on these results, we suggest the potential risk for dormant cancer cells to dissipate through non-typical EMT when ROCK activity is down-regulated.

Show MeSH
Related in: MedlinePlus