Silver ions promote blebs growth in U251 glioblastoma cell by activating nonselective cationic currents

Glioblastoma (GBM) is the most common and aggressive human brain cancer with low prognosis and therefore the discovery of new anticancer agents is needful. Sulfydryl reagents, such as silver, have been shown to induce membrane vesiculation in several cellular models through a mechanism that has not been yet completely clarified. Using U251 glioblastoma cells, we observed that silver induced irreversible bleb formation of the plasma membrane. This morphological event was anticipated by an increase of intracellular Ca²⁺ associated to extracellular Ca²⁺ influx. Accordingly, using patch-clamp whole cell recording during silver ion application, inward current/s (IAg) at -90 mV were detected and cells were permeable to Ca²⁺ and monovalent ions such as Na⁺. IAg activation and the intracellular Ca²⁺ increase promoted by silver ions (Ag⁺) were prevented by co-application of 20 µM cysteine and 300 µM DIDS (4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid), suggesting a critical role of thiol groups in the biological effects of silver ions. IAg was partially inhibited by 1 mM Gd³⁺, an unspecific inhibitor of cationic currents. Cysteine, Gd³⁺ and extracellular free Ca²⁺ solution completely abolished blebbing formation promoted by Ag⁺. Furthermore, extracellular Na⁺ ion replacement with TEA or an increase of extracellular tonicity by sucrose (100 mM) reduced both size and growth of membrane blebbing. Our data suggest that Ag⁺ promotes the formation necrotic blebs as consequence of the increase of intracellular Ca²⁺ and intracellular hydrostatic pressure associated to the activation of cationic currents. Since silver-induced blebs were less evident in benign glial human Müller MIO-M1 cells, silver compounds could represent new adjuvant to anticancer agents to improve GBM therapies.