ABSTRACT
BACKGROUND: We analyzed the changes in permeability of endothelial cell layers after photon irradiation, with a focus on the metalloproteases ADAM10 and ADAM17, and on VE-cadherin, components crucial for the integrity of endothelial intercellular junctions, and their roles in the transmigration of cancer cells through endothelial cell monolayers. METHODS: Primary HUVEC were irradiated with 2 or 4 Gy photons at a dose rate of 5 Gy/min. The permeability of an irradiated endothelial monolayer for macromolecules and tumor cells was analyzed in the presence or absence of the ADAM10/17 inhibitors GI254023X and GW280264X. Expression of ADAM10, ADAM17 and VE-Cadherin in endothelial cells was quantified by immunoblotting and qRT. VE-Cadherin was additionally analyzed by immunofluorescence microscopy and ELISA. RESULTS: Ionizing radiation increased the permeability of endothelial monolayers and the transendothelial migration of tumor cells. This was effectively blocked by a selective inhibition (GI254023X) of ADAM10. Irradiation increased both, the expression and activity of ADAM10, which led to increased degradation of VE-cadherin, but also led to higher rates of VE-cadherin internalization. Increased degradation of VE-cadherin was also observed when endothelial monolayers were exposed to tumor-cell conditioned medium, similar to when exposed to recombinant VEGF. CONCLUSIONS: Our results suggest a mechanism of irradiation-induced increased permeability and transendothelial migration of tumor cells based on the activation of ADAM10 and the subsequent change of endothelial permeability through the degradation and internalization of VE-cadherin.
Subject(s)
ADAM10 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Antigens, CD/metabolism , Cadherins/metabolism , Endothelial Cells/radiation effects , Human Umbilical Vein Endothelial Cells/radiation effects , Membrane Proteins/metabolism , Proteolysis/radiation effects , Radiation, Ionizing , Transendothelial and Transepithelial Migration/radiation effects , ADAM10 Protein/antagonists & inhibitors , ADAM10 Protein/genetics , ADAM17 Protein/antagonists & inhibitors , ADAM17 Protein/genetics , ADAM17 Protein/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/genetics , Cell Line, Tumor , Dipeptides/pharmacology , Endothelial Cells/metabolism , Humans , Hydroxamic Acids/pharmacology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Permeability/radiation effects , Radiotherapy/adverse effects , Signal Transduction/radiation effects , Transendothelial and Transepithelial Migration/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
BACKGROUND/AIM: Tumour cells of the profile CD44+/CD24low/- have a high tumorigenic potential. Salinomycin can specifically inhibit the growth of these cells. Herein, we investigated the effects of salinomycin on the viability and migration of triple negative breast cancer cells. MATERIALS AND METHODS: We analysed two cell lines: i) triple-negative MDA-MB 231 breast cancer cells and ii) a cytokeratin 18-transfected, re-differentiated subclone of the MDA-MB 231 cell line. The viability was determined using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, and the migration was determined using 24-h videography. The expression of oestrogen receptor was determined using immunohistochemistry. RESULTS: Salinomycin reduces all migration parameters in MDA-MB 231 cells. A significant correlation was found between increasing salinomycin concentrations and loss of cell viability, which was significantly less noticeable in the transfected control cells. CONCLUSION: With salinomycin there is a specific inhibition of MDA-MB 231 cells. Since MDA-MB 231 has over 90% cells with the profile CD44+/CD24low/-, these might represent a possible point of attack for salinomycin.
