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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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Related in: MedlinePlus

ROCK inhibition of MCF-7 cells increases cell migration and invasion through Rac1 activation.A. MCF-7 cells were cultured with the indicated concentration of Y-27632 for three days. The cell proliferation rate was subsequently measured through CCK-8 assay. The data are expressed as the mean ± SD. n = 3 dishes. The p-value was less than 0.05 for comparisons between un-treated and treatment groups (Y-27632) upon Krusal-Wallis test. *, p<0.05. B. MCF-7 cells were cultured with Y-27632 (20 µM) in the absence or presence of Rac1 inhibitor (15 or 25 µM) for three days. The cell proliferation rate was subsequently measured through CCK-8 assay. The data are expressed as the mean ± SD. n = 3 dishes. The p-value was less than 0.05 for comparisons between any two groups except comparison between the group of Y-27632 with 25 mM Rac1 inhibitor and the group of 15 mM Rac1 inhibitor upon one-way analysis of variance. *, p<0.05. C. The cells seeded on the Transwell chambers for the migration assay were incubated for 30 hrs in the absence or presence of Y-27632 (20 µM) to inhibit ROCK activity (upper panel). The cells seeded on the Transwell chambers covered with Matrigel for the invasion assay were cultured for 30 hrs in the absence or presence of Y-27632 (20 µM) to inhibit ROCK activity. 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 chambers. *, P<0.05 compared between groups treated with or without Y-27632. D. The cells transfected with control si-RNA (scramble), si-RNA targeting ROCK1 (si-ROCK1) or ROCK2 (si-ROCK2) were seeded on the Transwell chambers for migration assay and incubated for 48 hrs. The cells migrated onto the lower chamber were manually counted after staining cells with hematoxylin. The data are expressed as the mean ± SD. The p-value was less than 0.05 for comparisons between scramble group and transfected groups. *, p<0.05. E. The cells seeded on the Transwell chambers for migration assay were incubated with Y-27632 (20 µM) and the indicated concentration (10, 25 50 mM) of Rac1 inhibitor for 18 hrs. The cells migrated into the lower chamber were stained with hematoxylin. 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 Y-27632 group without Rac1 inhibition and any Y-27632 group with Rac1 inhibition. *, p<0.05. F. Cells transfected with control siRNA (scramble) or siRNA targeting PTEN (si-PTEN) were incubated for 2 days. Subsequently, the cells were replated into the Transwell chambers for migration assay. The cells migrated into the lower chamber for 24 hrs were stained with hematoxylin (upper panel). PTEN expression was analyzed through immunoblotting using specific antibodies against PTEN. G. The cells seeded in Transwell chambers for the migration assay were incubated with the indicated concentration of bpV (PTEN inhibitor; 1, 3, 5 µM) for 24 hrs. Cells migrated into the lower chamber were stained with hematoxylin. H. The cells were seeded in the Transwell chambers for the migration assay and incubated with ROCK inhibitors (Y-27632; 20 µM) and/or PI3-K inhibitors (LY-294002; 10 µM) for 24 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. There was no statistical significance between ROCK inhibition groups (Y-27632) with / without PI3K inhibition (LY-294002).
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pone-0088489-g005: ROCK inhibition of MCF-7 cells increases cell migration and invasion through Rac1 activation.A. MCF-7 cells were cultured with the indicated concentration of Y-27632 for three days. The cell proliferation rate was subsequently measured through CCK-8 assay. The data are expressed as the mean ± SD. n = 3 dishes. The p-value was less than 0.05 for comparisons between un-treated and treatment groups (Y-27632) upon Krusal-Wallis test. *, p<0.05. B. MCF-7 cells were cultured with Y-27632 (20 µM) in the absence or presence of Rac1 inhibitor (15 or 25 µM) for three days. The cell proliferation rate was subsequently measured through CCK-8 assay. The data are expressed as the mean ± SD. n = 3 dishes. The p-value was less than 0.05 for comparisons between any two groups except comparison between the group of Y-27632 with 25 mM Rac1 inhibitor and the group of 15 mM Rac1 inhibitor upon one-way analysis of variance. *, p<0.05. C. The cells seeded on the Transwell chambers for the migration assay were incubated for 30 hrs in the absence or presence of Y-27632 (20 µM) to inhibit ROCK activity (upper panel). The cells seeded on the Transwell chambers covered with Matrigel for the invasion assay were cultured for 30 hrs in the absence or presence of Y-27632 (20 µM) to inhibit ROCK activity. 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 chambers. *, P<0.05 compared between groups treated with or without Y-27632. D. The cells transfected with control si-RNA (scramble), si-RNA targeting ROCK1 (si-ROCK1) or ROCK2 (si-ROCK2) were seeded on the Transwell chambers for migration assay and incubated for 48 hrs. The cells migrated onto the lower chamber were manually counted after staining cells with hematoxylin. The data are expressed as the mean ± SD. The p-value was less than 0.05 for comparisons between scramble group and transfected groups. *, p<0.05. E. The cells seeded on the Transwell chambers for migration assay were incubated with Y-27632 (20 µM) and the indicated concentration (10, 25 50 mM) of Rac1 inhibitor for 18 hrs. The cells migrated into the lower chamber were stained with hematoxylin. 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 Y-27632 group without Rac1 inhibition and any Y-27632 group with Rac1 inhibition. *, p<0.05. F. Cells transfected with control siRNA (scramble) or siRNA targeting PTEN (si-PTEN) were incubated for 2 days. Subsequently, the cells were replated into the Transwell chambers for migration assay. The cells migrated into the lower chamber for 24 hrs were stained with hematoxylin (upper panel). PTEN expression was analyzed through immunoblotting using specific antibodies against PTEN. G. The cells seeded in Transwell chambers for the migration assay were incubated with the indicated concentration of bpV (PTEN inhibitor; 1, 3, 5 µM) for 24 hrs. Cells migrated into the lower chamber were stained with hematoxylin. H. The cells were seeded in the Transwell chambers for the migration assay and incubated with ROCK inhibitors (Y-27632; 20 µM) and/or PI3-K inhibitors (LY-294002; 10 µM) for 24 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. There was no statistical significance between ROCK inhibition groups (Y-27632) with / without PI3K inhibition (LY-294002).

Mentions: For the activation of dormant cancer cells, cell proliferation, migration, and invasion should also be up-regulated concomitant with the disruption of cell junctions, which potentiates the dissipation of dormant cancer cells into surrounding tissues or contributes to the enlargement of the cancer mass. Cell proliferation was also accompanied by ROCK inhibition [Fig. 1B and Fig. 5A]. To examine the involvement of Rac1 up-regulation in the cell proliferation promoted by ROCK inhibition, the Rac1 activity of MCF-7 cells was inhibited by treating cells with a Rac1-specific inhibitor in concomittant with ROCK inhibition. Rac1 inhibition down-regulated the increased cell proliferation by ROCK inhibition in a dose-dependent manner [Fig. 5B]. These results indicate that Rac1 activation upon ROCK inhibition may also be involved in promoting the cell proliferation of MCF-7 cells.


ROCK inhibition activates MCF-7 cells.

Yang S, Kim HM - PLoS ONE (2014)

ROCK inhibition of MCF-7 cells increases cell migration and invasion through Rac1 activation.A. MCF-7 cells were cultured with the indicated concentration of Y-27632 for three days. The cell proliferation rate was subsequently measured through CCK-8 assay. The data are expressed as the mean ± SD. n = 3 dishes. The p-value was less than 0.05 for comparisons between un-treated and treatment groups (Y-27632) upon Krusal-Wallis test. *, p<0.05. B. MCF-7 cells were cultured with Y-27632 (20 µM) in the absence or presence of Rac1 inhibitor (15 or 25 µM) for three days. The cell proliferation rate was subsequently measured through CCK-8 assay. The data are expressed as the mean ± SD. n = 3 dishes. The p-value was less than 0.05 for comparisons between any two groups except comparison between the group of Y-27632 with 25 mM Rac1 inhibitor and the group of 15 mM Rac1 inhibitor upon one-way analysis of variance. *, p<0.05. C. The cells seeded on the Transwell chambers for the migration assay were incubated for 30 hrs in the absence or presence of Y-27632 (20 µM) to inhibit ROCK activity (upper panel). The cells seeded on the Transwell chambers covered with Matrigel for the invasion assay were cultured for 30 hrs in the absence or presence of Y-27632 (20 µM) to inhibit ROCK activity. 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 chambers. *, P<0.05 compared between groups treated with or without Y-27632. D. The cells transfected with control si-RNA (scramble), si-RNA targeting ROCK1 (si-ROCK1) or ROCK2 (si-ROCK2) were seeded on the Transwell chambers for migration assay and incubated for 48 hrs. The cells migrated onto the lower chamber were manually counted after staining cells with hematoxylin. The data are expressed as the mean ± SD. The p-value was less than 0.05 for comparisons between scramble group and transfected groups. *, p<0.05. E. The cells seeded on the Transwell chambers for migration assay were incubated with Y-27632 (20 µM) and the indicated concentration (10, 25 50 mM) of Rac1 inhibitor for 18 hrs. The cells migrated into the lower chamber were stained with hematoxylin. 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 Y-27632 group without Rac1 inhibition and any Y-27632 group with Rac1 inhibition. *, p<0.05. F. Cells transfected with control siRNA (scramble) or siRNA targeting PTEN (si-PTEN) were incubated for 2 days. Subsequently, the cells were replated into the Transwell chambers for migration assay. The cells migrated into the lower chamber for 24 hrs were stained with hematoxylin (upper panel). PTEN expression was analyzed through immunoblotting using specific antibodies against PTEN. G. The cells seeded in Transwell chambers for the migration assay were incubated with the indicated concentration of bpV (PTEN inhibitor; 1, 3, 5 µM) for 24 hrs. Cells migrated into the lower chamber were stained with hematoxylin. H. The cells were seeded in the Transwell chambers for the migration assay and incubated with ROCK inhibitors (Y-27632; 20 µM) and/or PI3-K inhibitors (LY-294002; 10 µM) for 24 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. There was no statistical significance between ROCK inhibition groups (Y-27632) with / without PI3K inhibition (LY-294002).
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pone-0088489-g005: ROCK inhibition of MCF-7 cells increases cell migration and invasion through Rac1 activation.A. MCF-7 cells were cultured with the indicated concentration of Y-27632 for three days. The cell proliferation rate was subsequently measured through CCK-8 assay. The data are expressed as the mean ± SD. n = 3 dishes. The p-value was less than 0.05 for comparisons between un-treated and treatment groups (Y-27632) upon Krusal-Wallis test. *, p<0.05. B. MCF-7 cells were cultured with Y-27632 (20 µM) in the absence or presence of Rac1 inhibitor (15 or 25 µM) for three days. The cell proliferation rate was subsequently measured through CCK-8 assay. The data are expressed as the mean ± SD. n = 3 dishes. The p-value was less than 0.05 for comparisons between any two groups except comparison between the group of Y-27632 with 25 mM Rac1 inhibitor and the group of 15 mM Rac1 inhibitor upon one-way analysis of variance. *, p<0.05. C. The cells seeded on the Transwell chambers for the migration assay were incubated for 30 hrs in the absence or presence of Y-27632 (20 µM) to inhibit ROCK activity (upper panel). The cells seeded on the Transwell chambers covered with Matrigel for the invasion assay were cultured for 30 hrs in the absence or presence of Y-27632 (20 µM) to inhibit ROCK activity. 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 chambers. *, P<0.05 compared between groups treated with or without Y-27632. D. The cells transfected with control si-RNA (scramble), si-RNA targeting ROCK1 (si-ROCK1) or ROCK2 (si-ROCK2) were seeded on the Transwell chambers for migration assay and incubated for 48 hrs. The cells migrated onto the lower chamber were manually counted after staining cells with hematoxylin. The data are expressed as the mean ± SD. The p-value was less than 0.05 for comparisons between scramble group and transfected groups. *, p<0.05. E. The cells seeded on the Transwell chambers for migration assay were incubated with Y-27632 (20 µM) and the indicated concentration (10, 25 50 mM) of Rac1 inhibitor for 18 hrs. The cells migrated into the lower chamber were stained with hematoxylin. 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 Y-27632 group without Rac1 inhibition and any Y-27632 group with Rac1 inhibition. *, p<0.05. F. Cells transfected with control siRNA (scramble) or siRNA targeting PTEN (si-PTEN) were incubated for 2 days. Subsequently, the cells were replated into the Transwell chambers for migration assay. The cells migrated into the lower chamber for 24 hrs were stained with hematoxylin (upper panel). PTEN expression was analyzed through immunoblotting using specific antibodies against PTEN. G. The cells seeded in Transwell chambers for the migration assay were incubated with the indicated concentration of bpV (PTEN inhibitor; 1, 3, 5 µM) for 24 hrs. Cells migrated into the lower chamber were stained with hematoxylin. H. The cells were seeded in the Transwell chambers for the migration assay and incubated with ROCK inhibitors (Y-27632; 20 µM) and/or PI3-K inhibitors (LY-294002; 10 µM) for 24 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. There was no statistical significance between ROCK inhibition groups (Y-27632) with / without PI3K inhibition (LY-294002).
Mentions: For the activation of dormant cancer cells, cell proliferation, migration, and invasion should also be up-regulated concomitant with the disruption of cell junctions, which potentiates the dissipation of dormant cancer cells into surrounding tissues or contributes to the enlargement of the cancer mass. Cell proliferation was also accompanied by ROCK inhibition [Fig. 1B and Fig. 5A]. To examine the involvement of Rac1 up-regulation in the cell proliferation promoted by ROCK inhibition, the Rac1 activity of MCF-7 cells was inhibited by treating cells with a Rac1-specific inhibitor in concomittant with ROCK inhibition. Rac1 inhibition down-regulated the increased cell proliferation by ROCK inhibition in a dose-dependent manner [Fig. 5B]. These results indicate that Rac1 activation upon ROCK inhibition may also be involved in promoting the cell proliferation of MCF-7 cells.

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