RESUMEN
Thrombin, which plays a crucial role in hemostasis, is also implicated in cancer progression. In the present study, the effects of the thrombintargeting recombinant tyrosinesulfated madanin1 on cancer cell behavior and signaling pathways compared with madanin1 wildtype (WT) were investigated. Recombinant madanin1 2 sulfation (madanin1 2S) and madanin1 WT proteins were generated using Escherichia coli. SKOV3 and MDAMB231 cells were treated with purified recombinant proteins with or without thrombin stimulation. Migration and invasion of cells were analyzed by wound healing assay and Transwell assay, respectively. Thrombin markedly increased cell migration and invasion in both SKOV3 and MDAMB231 cells, which were significantly suppressed by madanin1 2S (P<0.05). Madanin1 2S also significantly suppressed thrombininduced expression of phosphorylated (p)Akt and pextracellular signalregulated kinase in both cell lines (P<0.05), whereas madanin1 WT had no effect on the expression levels of these proteins in MDAMB231 cells. Furthermore, madanin1 2S significantly reversed the effects of thrombin on Ecadherin, Ncadherin and vimentin expression in MDAMB231 cells (P<0.05), whereas madanin1 WT did not show any effect. In conclusion, madanin1 2S suppressed the migration and invasion of cancer cells more effectively than madanin1 WT. It is hypothesized that inhibiting thrombin via the sulfated form of madanin1 may be a potential candidate for enhanced cancer therapy; however, further in vivo validation is required.
Asunto(s)
Movimiento Celular , Proteínas Recombinantes , Trombina , Humanos , Cadherinas/metabolismo , Cadherinas/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Trombina/antagonistas & inhibidores , Trombina/farmacología , Tirosina/metabolismo , Tirosina/farmacologíaRESUMEN
BACKGROUND AND OBJECTIVES: Chemokines have various biological functions and potential roles in the development or progression of neuroinflammatory diseases. However, the specific pathogenic roles of chemokines in the major cause for vision loss among the elderly, the leading cause of blindness in older individuals, remain elusive. Chemokines interact with their receptors expressed in the endothelium and on leukocytes. The sulfation of tyrosine residues in chemokine receptors increases the strength of ligand-receptor interaction and modulates signaling. Therefore, in the present study, we aimed to construct a human recombinant sulfated CXCR3 peptide trap (hCXCR3-S2) and mouse recombinant sulfated CXCR3 peptide trap (mCXCR3-S2) to demonstrate in vivo effects in preventing choroidal neovascularization (CNV) and chemotaxis. MATERIALS AND METHODS: We generated expression vectors for mCXCR3-S2 and hCXCR3-S2 with GST domains and their respective cDNA sequences. Following overexpression in E. coli BL21 (DE3), we purified the fusion proteins from cell lysates using affinity chromatography. First, the impact of hCXCR3-S2 was validated in vitro. Subsequently, the in vivo efficacy of mCXCR3-S2 was investigated using a laser-induced CNV mouse model, a mouse model of neovascular age-related macular degeneration (AMD). RESULTS: hCXCR3-S2 inhibited the migration and invasion of two human cancer cell lines. Intravitreal injection of mCXCR3-S2 attenuated CNV and macrophage recruitment in neovascular lesions of mouse models. These in vitro and in vivo effects were significantly stronger with CXCR3-S2 than with wild-type CXCR3 peptides. CONCLUSION: These findings demonstrate that the sulfated form of the CXCR3 peptide trap is a valuable tool that could be supplemented with antivascular endothelial growth factors in AMD treatment.