| Abstract |
Διατμηματικό, συνεργαζόμενα τμήματα Βιολογίας και Ιατρικής. Opioids are known for their antiproliferative properties against cancer cells. Also, it has been shown that actin filaments are prefferentially depolymerized in cancer cells as compared to higher levels of actin polymerization in their normal counterparts, suggesting that the dynamic state of actin correlates with the expression of the malignant phenotype. The aim of our study was to investigate whether opioids might have other antineoplastic properties that are releted to the modulation of actin polymerization and stabilization. We used as model for opioid action on actin organization the Cys 374 mutated human β-actin in the opposum kidney cells and the kidney cancer cell line Caki-2. Triton fractionation and immunoblot analysis showed that exposure of the mutated OK cells to the opioids αs1-casomorphin and ethylketocyclazocine and TNF-α for 30 min, resulted in a rapid and substantial actin microfilament reorganisation, as it was documented by a significant increase of the G/Total actin ratio. Exposure of Caki-2 cells to the opioid αs1-casomorphin resulted in a transient increase of the G/F actin ratio, after 15 min, with return to the normal levels after further incubation, whereas the opioid ethylketocyclazocine constantly decreased the G/F actin ratio of Caki-2 cells, for at least 2h. In order to explore the signal transduction pathway of opioid action in Caki-2 cells, we first investigated whether opioids induse tyrosine phosphorylation of specific actin relating proteins. Immunoprecipitation and immunoblot analysis with monoclonal phosphotyrosine antibody showed that the opioid agonist ethylketokyclazocine strongly affects only the phosphorylation of two proteins, the Focal Adhesion Kinase (FAK) and Paxillin in Caki-2 cells. Interestingly, in Caki-2 cells these proteins were not phosphorylated significantly after exposure to the opioid αs1-casomorphin. We further examined a possible relationship between PI-3 kinase activation and Fak and Paxillin phosphorylation. For this we performed the above mentioned experiment in the presence of Wortmannin, a specific PI-3 kinase inhibitor. Our experiment demonstrated that Wortmannin affected tyrosine phosphorylation of both focal adhesion proteins, suggesting that FAK and Paxillin phsphorylation pathway is depedent on PI-3 kinase activation. Our findings demonstrate that two different opioids induce opposite modifications in the dynamics of actin polymerization in malignant Caki-2 cells and tyrosine phosphorylation of FAK and paxillin, after exposure to ethylketocyclazocine is depedent on the activation of PI3-Kinase. These results may be important for understanding the mechanism of opioids and their role as potential agent against the cancer cells.
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Επίδραση οπιοειδών στους κυτταρικούς κλώνους που εκφράζουν διαφορετικές μεταλλάξεις της ακτίνης και στη νεοπλασματική σειρά Caki-2
