Rho/ROCK Contributes To Hypoxia-induced Actin Cytoskeleton Reorganization In Breast Cancer MDA-MB-435 Cells

Background: Hypoxia could play a significant role in tumor progression, as associated with increased cell migration. The family of Rho GTPases (Rho, Rac, and Cdc42) plays crucial role in the regulation of cytoskeletal dynamics that underlie changes in cell motility and adhesion during migration. Rho-associated kinase (ROCK) is a major effector of Rho. Upon activation by Rho, ROCK results in an increase in cell motility.Objective: To explore the implication of cytoskeletal rearrangements in the cell adhesion and motility in hypoxic breast cancer MDA-MB-435 cells and relative contribution of Rho/ROCK signaling to actin polymerization or depolymerization.Methods: MDA-MB-435 cells were treated with or without Rho-kinase inhibitor, Y-27632 for 1h and subsequently exposed to hypoxia for 16h. The reorganization of actin cytoskeleton was detected by FITC-Phallodin staining and the F-actin contents in cytosolic fraction and cytoskeletal fraction using western blotting method. The adhesive ability of cells to artificial basement membrane (Matrigel) was measured by MTT assay. The cell motility was measured by wound healing migration assay.Results: Compared to the control cells, motility of MDA-MB-435 cells was significantly enhanced and adhesive ability was significantly decreased after exposed to hypoxia for 16h, accompanied by a rearrangement of stress fibers and increased contents of F-actin. All of these actions were abolished by the pretreatment of Y-27632.Conclusion: The reduced adhesive ability and increased cell motility in the hypoxic breast cancer MDA-MB-435 cells were positively correlated with the promote actin polymerization. These actions were largely dependent on the Rho/ ROCK signaling activity.