Fasudil Action Analysis in Cell Migration of Tumour Cell Line MDA‐MB 231

The ability of cancer cells to undergo invasion and migration is a prerequisite for tumor metastasis. Rho, a Ras‐related small GTPase, and the Rho‐associated coiled coil–containing protein kinases (Rho kinases, ROCK1 and ROCK2), are key regulators of focal adhesion, actomyosin contraction, and thus cell motility. The concept of Rho Kinase inhibition as an antimetastatic therapy for cancer can now be clinically explored. Therefore, it is of great interest to investigate whether Fasudil (Rho kinase inhibitor) affects early events in human breast carcinoma (MDA‐MB‐231) cell line migration and show the signaling pathways involved in cell migration as well as cell cytoskeleton changes and thus would have potential for the treatment of human breast cancer. For the study, the cell viability was evaluated by MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐difenltetrazolium), and fasudil has been used at concentrations 0.1, 1, 10, 50 and 100 μM. The migration was evaluated using wound healing assay in which it is a scraping is made in the cell monolayer and subject to the closing of the shaved area by the migration of the cells for 24 hours. Morphological analysis was carried out by phase‐contrast micrography, and analysed using Image J software. Actin structures can be visualized by fluorescent phalloidins. Immunofluorescence assays were performed with anti‐vimentin antibody. ROCK‐activity was evaluated by immunofluorescence. Statistical analyses were performed by ANOVA with Newman‐Keuls‐post‐test (*p<0.05). The results showed the the cell viability of the MDA‐MB 231 cell line was significantly reduced only at the concentration of 100 μm of Fasudil when incubated for 24 and 48 hours. The effect of the treatment with 10 μm of Fasudil decreased cell migration 39.4% when compared to control. Through the morphological analysis, treated cells with 50 μm presented area, height and width reduced by 50%, fusiform and protrusions membrane with filopodia, different from control cells that had sprawling, with cytoplasm extended and forming lamelipodios ( Figure 1). The staining for F‐actin showed thet the cell body became elongated less spread with a narrowing of the cell body shape compared to control. In the immunofluorescence assay of vimentin, a predominantly perinuclear marking was observed with less pronounced marking in the cytoplasm of treated cells with Fasudil 10 μm when compared to the control. It is concluded partially that the treatment with 10 μm does not reduce the viability of MDA‐MB 231 cells, reduces the cell migration, alters cell morphology and interferes with the formation of F‐actin, which is essential for cell movement during migration. These results add to a better understanding of the process of cell migration and contribute to the discovery of drugs that act to reduce tumour metastasis processes. Support or Funding Information Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 1Morphological analysis was carried out by phase‐contrast micrography, and analyzed using Image J software. A (control), B (Fasudil). Treated cells with 50 μm for 24 hours presented area, height and width reduced by 50%, fusiform and protrusions membrane with filopodia, different from control cells that had sprawling, with cytoplasm extended and forming lamelipodios