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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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RhoA and ROCK activities regulate E-cadherin and b-catenin in MCF-7 cells.A. The localized patterns of E-cadherin and b-catenin were examined using specific antibodies through confocal laser microscopy. MCF-7 cells were cultured for 24 hrs in the absence or presence of Y-27632 (20 µM) to inhibit ROCK activity. B. The activity of RhoA, a major upstream regulating molecule of ROCK, was regulated to examine the role of RhoA-ROCK in regulating the membrane localization of E-cadherin and b-catenin after transfecting MCF-7 cells with constitutively active RhoA (EGFP-RhoA-Q63L, EGFP-RhoA CA) or dominant negative RhoA (EGFP-RhoA-T19N, EGFP-RhoA DN). E-cadherin and b-catenin were analyzed after staining the fixed cells with specific antibodies under confocal laser microscopy. The cells were also stained with DAPI to localize the nuclei. C. Membrane fraction level of E-cadherin was analyzed through immunoblotting using specific antibodies after treating MCF-7 cells with Y-27632 (20 µM) to inhibit ROCK activity for 24 hrs. An antibody to flotillin-1 was used to confirm the equal load of the membrane fraction. D. The level of E-cadherin in the membrane fraction was examined through immunoblotting using specific antibodies after transfecting MCF-7 cells with the pCDNA3-EGFP plasmid (Empty) or pCDNA3-EGFP-RhoA-T19N plasmid (RhoA DN) to inhibit RhoA-ROCK. An antibody to flotillin-1 was used to confirm the equal load of the membrane fraction. E. The total expression of E-cadherin or b-catenin was determined after treating MCF-7 cells with the indicated concentration of Y-27632 for 24 hrs through immunoblotting using the specific antibodies. F. Total expression level of E-cadherin was determined after transfecting cells with control si-RNA (scramble), si-RNA targeting ROCK1 (si-ROCK1) or ROCK2 (si–ROCK2) for 72 hrs through immunoblotting.
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pone-0088489-g003: RhoA and ROCK activities regulate E-cadherin and b-catenin in MCF-7 cells.A. The localized patterns of E-cadherin and b-catenin were examined using specific antibodies through confocal laser microscopy. MCF-7 cells were cultured for 24 hrs in the absence or presence of Y-27632 (20 µM) to inhibit ROCK activity. B. The activity of RhoA, a major upstream regulating molecule of ROCK, was regulated to examine the role of RhoA-ROCK in regulating the membrane localization of E-cadherin and b-catenin after transfecting MCF-7 cells with constitutively active RhoA (EGFP-RhoA-Q63L, EGFP-RhoA CA) or dominant negative RhoA (EGFP-RhoA-T19N, EGFP-RhoA DN). E-cadherin and b-catenin were analyzed after staining the fixed cells with specific antibodies under confocal laser microscopy. The cells were also stained with DAPI to localize the nuclei. C. Membrane fraction level of E-cadherin was analyzed through immunoblotting using specific antibodies after treating MCF-7 cells with Y-27632 (20 µM) to inhibit ROCK activity for 24 hrs. An antibody to flotillin-1 was used to confirm the equal load of the membrane fraction. D. The level of E-cadherin in the membrane fraction was examined through immunoblotting using specific antibodies after transfecting MCF-7 cells with the pCDNA3-EGFP plasmid (Empty) or pCDNA3-EGFP-RhoA-T19N plasmid (RhoA DN) to inhibit RhoA-ROCK. An antibody to flotillin-1 was used to confirm the equal load of the membrane fraction. E. The total expression of E-cadherin or b-catenin was determined after treating MCF-7 cells with the indicated concentration of Y-27632 for 24 hrs through immunoblotting using the specific antibodies. F. Total expression level of E-cadherin was determined after transfecting cells with control si-RNA (scramble), si-RNA targeting ROCK1 (si-ROCK1) or ROCK2 (si–ROCK2) for 72 hrs through immunoblotting.

Mentions: Epithelial cells bind together through the homophilic binding of E-cadherins, which form an intracellular molecular complex with several molecules, such as b-catenin and actin filaments [20], [21]. Thus, the loss of E-cadherin and b-catenin from the cell membrane was examined upon cell separation through ROCK inhibition using confocal microscopy. ROCK inhibition clearly resulted in the loss of the E-cadherin expression from the cell membrane [Fig. 3A]. The loss of E-cadherin and b-catenin, associated with ROCK inhibition from the cell membrane, were further confirmed through the constitutive overexpression of active RhoA (RhoA-CA) to activate ROCK or the dominant negative RhoA (RhoA-DN) to inhibit ROCK. MCF-7 cells overexpressing RhoA-DN induced the loss of E-cadherin and b-catenin from the cell membrane in immunolocalization assays [Fig. 3B]. However, MCF-7 cells overexpressing RhoA-CA presented strong E-cadherin and b-catenin expression at the cell membrane. In the Western blotting analysis, the E-cadherin membrane fraction was reduced after ROCK inhibition or the overexpression of RhoA-DN [Fig. 3C and D]. In addition, the total expression of E-cadherin and b-catenin was also down-regulated upon ROCK inhibition [Fig. 3E]. These results suggest that the disruption of cell junction upon ROCK inhibition might be associated with the loss of E-cadherin and b-catenin from the cell membrane.


ROCK inhibition activates MCF-7 cells.

Yang S, Kim HM - PLoS ONE (2014)

RhoA and ROCK activities regulate E-cadherin and b-catenin in MCF-7 cells.A. The localized patterns of E-cadherin and b-catenin were examined using specific antibodies through confocal laser microscopy. MCF-7 cells were cultured for 24 hrs in the absence or presence of Y-27632 (20 µM) to inhibit ROCK activity. B. The activity of RhoA, a major upstream regulating molecule of ROCK, was regulated to examine the role of RhoA-ROCK in regulating the membrane localization of E-cadherin and b-catenin after transfecting MCF-7 cells with constitutively active RhoA (EGFP-RhoA-Q63L, EGFP-RhoA CA) or dominant negative RhoA (EGFP-RhoA-T19N, EGFP-RhoA DN). E-cadherin and b-catenin were analyzed after staining the fixed cells with specific antibodies under confocal laser microscopy. The cells were also stained with DAPI to localize the nuclei. C. Membrane fraction level of E-cadherin was analyzed through immunoblotting using specific antibodies after treating MCF-7 cells with Y-27632 (20 µM) to inhibit ROCK activity for 24 hrs. An antibody to flotillin-1 was used to confirm the equal load of the membrane fraction. D. The level of E-cadherin in the membrane fraction was examined through immunoblotting using specific antibodies after transfecting MCF-7 cells with the pCDNA3-EGFP plasmid (Empty) or pCDNA3-EGFP-RhoA-T19N plasmid (RhoA DN) to inhibit RhoA-ROCK. An antibody to flotillin-1 was used to confirm the equal load of the membrane fraction. E. The total expression of E-cadherin or b-catenin was determined after treating MCF-7 cells with the indicated concentration of Y-27632 for 24 hrs through immunoblotting using the specific antibodies. F. Total expression level of E-cadherin was determined after transfecting cells with control si-RNA (scramble), si-RNA targeting ROCK1 (si-ROCK1) or ROCK2 (si–ROCK2) for 72 hrs through immunoblotting.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088489-g003: RhoA and ROCK activities regulate E-cadherin and b-catenin in MCF-7 cells.A. The localized patterns of E-cadherin and b-catenin were examined using specific antibodies through confocal laser microscopy. MCF-7 cells were cultured for 24 hrs in the absence or presence of Y-27632 (20 µM) to inhibit ROCK activity. B. The activity of RhoA, a major upstream regulating molecule of ROCK, was regulated to examine the role of RhoA-ROCK in regulating the membrane localization of E-cadherin and b-catenin after transfecting MCF-7 cells with constitutively active RhoA (EGFP-RhoA-Q63L, EGFP-RhoA CA) or dominant negative RhoA (EGFP-RhoA-T19N, EGFP-RhoA DN). E-cadherin and b-catenin were analyzed after staining the fixed cells with specific antibodies under confocal laser microscopy. The cells were also stained with DAPI to localize the nuclei. C. Membrane fraction level of E-cadherin was analyzed through immunoblotting using specific antibodies after treating MCF-7 cells with Y-27632 (20 µM) to inhibit ROCK activity for 24 hrs. An antibody to flotillin-1 was used to confirm the equal load of the membrane fraction. D. The level of E-cadherin in the membrane fraction was examined through immunoblotting using specific antibodies after transfecting MCF-7 cells with the pCDNA3-EGFP plasmid (Empty) or pCDNA3-EGFP-RhoA-T19N plasmid (RhoA DN) to inhibit RhoA-ROCK. An antibody to flotillin-1 was used to confirm the equal load of the membrane fraction. E. The total expression of E-cadherin or b-catenin was determined after treating MCF-7 cells with the indicated concentration of Y-27632 for 24 hrs through immunoblotting using the specific antibodies. F. Total expression level of E-cadherin was determined after transfecting cells with control si-RNA (scramble), si-RNA targeting ROCK1 (si-ROCK1) or ROCK2 (si–ROCK2) for 72 hrs through immunoblotting.
Mentions: Epithelial cells bind together through the homophilic binding of E-cadherins, which form an intracellular molecular complex with several molecules, such as b-catenin and actin filaments [20], [21]. Thus, the loss of E-cadherin and b-catenin from the cell membrane was examined upon cell separation through ROCK inhibition using confocal microscopy. ROCK inhibition clearly resulted in the loss of the E-cadherin expression from the cell membrane [Fig. 3A]. The loss of E-cadherin and b-catenin, associated with ROCK inhibition from the cell membrane, were further confirmed through the constitutive overexpression of active RhoA (RhoA-CA) to activate ROCK or the dominant negative RhoA (RhoA-DN) to inhibit ROCK. MCF-7 cells overexpressing RhoA-DN induced the loss of E-cadherin and b-catenin from the cell membrane in immunolocalization assays [Fig. 3B]. However, MCF-7 cells overexpressing RhoA-CA presented strong E-cadherin and b-catenin expression at the cell membrane. In the Western blotting analysis, the E-cadherin membrane fraction was reduced after ROCK inhibition or the overexpression of RhoA-DN [Fig. 3C and D]. In addition, the total expression of E-cadherin and b-catenin was also down-regulated upon ROCK inhibition [Fig. 3E]. These results suggest that the disruption of cell junction upon ROCK inhibition might be associated with the loss of E-cadherin and b-catenin from the cell membrane.

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