engleski

Integrin αvβ3 mediated drug resistance in human tongue squamous carcinoma cells

Integrins act as mechanosensors, adhesion molecules and signal transduction platforms. Integrin-mediated interactions with the extracellular matrix are required for cell attachment, cytoskeletal organization, migration, proliferation, differentiation and survival. Expression of integrin αvβ3 is increased in a variety of tumors in which it promotes tumor growth, metastasis and survival. The aim of this study was to investigate the influence of de novo expression of integrin αvβ3 in human tongue squamous carcinoma cells Cal27 on the sensitivity to antitumor drugs, cell adhesion and migration, as well as the involved signaling pathways. Integrin β3-stably transfected cell clones (integrin β3-clones) were isolated by the stable transfection of Cal27 cells with the plasmid containing the integrin subunit β3 gene. They differ from the control Cal27 cells in de novo expression of integrin αvβ3, the increased expression of integrin αvβ5 and the increased adhesion to vitronectin and fibronectin, which correlates with the amount of expression of integrin αvβ3 and/or αvβ5. Using MTT assay and plating efficiency assay we showed that integrin β3-clones are resistant to antitumor drugs cisplatin, doxorubicin, mitomycin C, etoposide and 5-fluorouracil, as compared to Cal27 cells. Integrin β5 and αv-specific silencing using siRNA demonstrated that de novo expression of integrin αvβ3 in integrin β3-clones, and not the increased expression of integrin αvβ5, is responsible for the resistance phenomenon. Western blot analysis has shown that the expression of FAK and pFAK (Y397) was similar in integrin β3-clones and Cal27 control cells. The absence of involvement of focal adhesion kinase (FAK) was additionally confirmed by the expression of a dominant-interfering mutant form of FAK, termed FRNK using a replication-defective adenovirus. Integrin β3-clones have a reduced amount of Src and pSrc (Y418), an increased level of integrin linked kinase ILK, and a reduced amount of pILK (T173). The involvement of ILK in integrin β3-mediated antitumor drug resistance was excluded by silencing ILK using siRNA in integrin β3-clones. Conversely, in Cal27 cells the silencing of Src expression using siRNA, as well as the inhibition of Src (Y418) phosphorylation by dasatinib, led to the resistance to all above-mentioned antitumor drugs. We showed that in Cal27 cells integrin αvβ5 transmits a signal to pSrc(Y418), while neither of integrins αv signal through pSrc(Y418) in integrin β3-clones. Integrin β3-clones showed reduced migration ability, very likely due to the reduced amount of pSrc(Y418). Although the observed upregulation of ILK in the integrin β3-stably transfected clone is not responsible for drug resistance, a signal transmitted through ILK increases the expression of interleukin 6. Our findings could have clinical importance in terms of identifying integrin αvβ3 as a drug resistance biomarker in tumor cells, as well as a tumor therapy target molecule. Our results also show that drugs that inhibit the phosphorylation of Src(Y418), which have been identified as molecules for the treatment of many cancers, cannot, in some cases, be used simultaneously with antitumor drugs which act through the DNA molecule, as they may reduce the therapeutic effect

integrin αvβ3 ; integrin αvβ5 ; integrin mediated drug resistance ; pSrc (Y418) ; ILK ; migration

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