Modulation of Adhesion Proteins Integrin β1 and FAK, and Cytoskeletal Protein Actin by Spermine in MCF-7 Cells

Abstract

Polyamines are essential for cell growth and differentiation; however their precise roles are unclear yet. In the present study, the cytotoxic effect of spermine (spm) on MCF-7 cells was investigated. In the MTT assay of MCF-7 cells treated with spm, cell viability was significantly decreased in a time-and dose-dependent manner. Cell viability measurement was confirmed by trypan blue staining. FACS analysis shows that sub-G1 was increased in a time-and dose-dependent manner too. When the cells were treated with spm, cells started to show morphological changes within 2 hrs. The expression of adhesion proteins (FAK and integrin β1), and cytoskeletal protein (actin) was checked by Western blotting analysis. Integrin β1 levels were slightly decreased, and FAK and actin levels were rapidly decreased with spm treatment. In confocal laser scanning microscopy, the distribution of actin did not change but the expression decreased in a dose-dependent manner with spm treatment. FAK was evenly distributed under the plasma membrane in the untreated control. However, at 10 μM spm FAK seemed to move toward the cell nucleus. Integrin β1, which was mainly found in the focal point of the plasma membrane in the untreated control, dispersed through the entire plasma membrane in spm treatment. The present results indicate that cytotoxic effects of spm are triggered by the disruption of adhesion proteins and cytoskeletal protein. IntroductionPolyamines such as putrescine (put), spermidine (spd), and spermine (spm) are small, flexible molecules, which bind to nucleic acids and proteins and thus affect their conformation and biological activity [25]. Polyamines are important multifunctional cellular components and are considered mediators of cell growth and division. Recently, polyamines have also been known to be implicated in cell death. It appears that polyamines are bivalent regulators of cellular functions, promoting proliferation or cell death depending on the cell type and on environmental signals [12,18]. Among polyamines, spm was most effective in inhibiting cancer cell growth. The cytotoxicity of spm has been suggested by the production of H2O2 and aldehyde(s) from spm in the presence of amine oxidase [1,2].Polyamines are also essential for the normal attachment of cells to the extracellular matrix (ECM), which explains at least some of the reliance of cell migration and the organization of the cytoskeleton on normal polyamine levels [22]. The migration of cells depends on their ability to make and break attachments to the ECM. The ECM controls cells through signals via a family of cell surface receptors called integrins [10]. The malignancy of a tumor is associated with its ability to undergo migration and metastasis. Metastases form after a series of sequential, interlinked steps involving a variety of adhesive interactions between cancer cells and host tissues [7]. The mechanism that plays a role in the cytotoxic properties of polyamine may be related to the down-regulation of integrin-associated signaling. Integrin famil...