| Abstract |
The dynamic state of actin cytoskeleton is strongly correlated with the expression of malignant phenotype. Indeed, the actin polymerization dynamics may characterize malignant transformation, since quantitative determinations of the sensitive dynamic equilibrium of G- to F-actin in various malignant cell types revealed important modifications of the actin cytoskeleton. In particular these modifications include decreased F- and total actin content, increased G/total actin ratio and impaired microfilament organization and stability. Several extracellular signals, including hormones, neuropeptides and growth factors, were shown to modify actin polymerization and microfilament reorganization in malignant cells, indicating a potentially important role of actin dynamics in regulating cell phenotype during malignant transformation. The cellular models used in the present study were the non-malignant (MCF12A) and the malignant (MCF7) human breast epithelial cell lines. Triton soluble insoluble experiments and DNase I inhition assay, showed differential actin polymerization state in these cell lines. In particular G/total actin ratio is signiffiantly increased in MCF7 compared to MCF12A cells. Stress fibers are evenly distributed in the cytoplasm of non malignant mammary cells, while actin microfilaments are localized mainly in the periphery of the cell creating a ring attached to the plasma membrane. The mechanisms through which opioids regulate the activity of malignant breast epithelial cells are currently unknown. In the present study we report the differential actin cytoskeleton reorganization induced by opioids in malignant (MCF7) and non-malignant (MCF12A) breast epithelial cells. Exposure of MCF7 cells to the opioid agonist αs1 casomorphin induced important actin assembly and reorganization, casomorphin inhibites cell motility to the substratum compaired to control cells, while in MCF12A cells this effect is elimineted. including the formation of filopodia and lamellipodia. In contrary, incubation of MCF12A cells with αs1 casomorphin revealed a partial but transient disassembly of actin microfilaments. Immunoprecipitation and immunoblot analysis showed rapid phosphorylation of the focal adhesion kinase (FAK) and vinculin in opioid treated MCF7 cells. Moreover, FAK associates with phosphatidylinositol-3 (PI-3 kinase), the latter being subsequently phosphorylated and activated. In addition, a substantial activation of the small GTPase Rac1 was observed. Pre-treatment of MCF7 cell with the specific PI 3-kinase inhibitor wortmannin abolished both, the activation of Rac 1 and the actin reorganization, while the opioid induced phosphorylation of FAK and vinculin remained unaffected. Interestingly, in opioid treated MCF12A cells this signaling cascade remained inactive, while we identified rapid phosphorylation of the actin regulating protein villin. Finally, wound healing assay showed that opioids affect differentially cell motility in each cell line. Incubation of MCF7 cells with αs1 Actin polymerimerization induced by opioids was observed also in melanoma A375 cell line. Additionally FAK kinase, vinculin and PI-3 kinase are phoshorylated after opioid treatment, suggesting that the same pathway is also activated in other cancer cells beside epithelial mammary cells. These data suggest a distinct, opioid induced, signaling pathway activated in malignant breast epithelial cells, leading to important actin reorganization. These findings may indicate a potential antineoplastic role of opiates, based on the activation of differential signaling mechanisms.
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Μελέτη των μεταβολών της δυναμικής ισορροπίας της ακτίνης στη βιολογία του καρκίνου
