The Effects Of Genistein On The Actin Cytoskeleton Reorganization And Adhesive Ability In Human Breast Cancer Cells

Genistein is an isoflavone present in soybeans, and is a verypotent inhibitor of the protein-tyrosine kinase (PTK) activity of theEGF receptor in vitro. The biological effects of genistein are relatedto cell growth, apoptosis, invasion and metastasis of cancer cells.But the mechanism involved in the inhibition of invasion andmetastasis in cancer cells was unclear. It is well known that complexcommunications through the cell-ECM (extracellular matrix)adhesion initiate the invasion and migration of cancer cell. Actincytoskeleton plays a key role in cell shape and movement. And thereorganization of actin cytoskeleton is involved in the invasion andmigration of cancer cells. There are a number of actin bindingproteins regulating the state and distribution of polymerization offilaments. Hsp27 (heat shock protein 27) is an actin binding protein,in which regulate actin dynamics via p38 mitogen-activated proteinkinase signaling pathway. Recently it is reported that genisteincould inhibit p38 MAPK activation. We hypothesized that genisteinhave a direct effect on the inhibition of the p38 MAPK activation and subsequently inhibit the phosphorylation of Hsp27, leading toloss of cell adhesive ability in human breast cancer cells through thereorganization of actin cytoskeleton. To address this issue, thefollowing studies were designed and performed.In the first study, the MDA-MB-435 cells were treated withdifferent concentrations of genistein (12.5, 25, 50 and 100μmol/L)for 24h. The reorganization of actin cytoskeleton was detected byFITC-phallodin staining and the F-actin contents measured by flowcytometer and western blotting. The adhesive ability of cells toartificial basement membrane (Matrigel) was measured by MTTassay. Compared to the control cells, genistein significantly changedthe redistribution of the F-actin, reduced F-actin contents andreduced the adhesive rate to Matrigel. There results suggest that theless adhesive ability of MDA-MB-435 cells to Matrigel induced bygenistein was related to the reorganization of actin cytoskeleton.In the second study, the MDA-MB-435 cells were treated withdifferent concentrations of genistein (12.5, 25, 50 and 100μmol/L)for 24h. The cell apoptosis was analyzed by flow cytometer. Theexpression of ERK1/2, p38 and Hsp27, phosphorylation of ERK1/2,p38 and Hsp27 was evaluated by western blot analysis. Theexpression of Hsp27 in cytosolic fraction and cytoskeletal fractionwas also detected by by western blotting. After treated by genisteinfor 24h, the cell apoptosis and ERK1/2 activation had no changes inall groups. However, the phosphorylation of p38 and Hsp27 wasattenuated compared to the control cells. Simultaneously Hsp27translocated from the cytosolic fraction to the cytoskeletal fraction. These findings suggest that the reorganization of actin cytoskeletoninduced by genistein was mediated by translocation of Hsp27 incells via p38 signaling pathway.Together with our studies, we conclude that:1. The depolymerization of F-actin induced by genistein maylead to the less adhesive ability of MDA-MB-435 cells to Matrigel.2. The reorganization of actin cytoskeleton induced bygenistein was mediated by translocation of Hsp27 in cells via p38signaling pathway.