Abstract
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.
Highlights
Glioblastoma is the most common and aggressive type of human brain tumor
We checked the effects of Ag+ application on intracellular Ca2+ level in U251 glioblastoma cells
All the cells were responsive to 10 μM of silver ion application and after washout the intracellular Ca2+ levels dropped to resting levels (Fig. 1A)
Summary
Glioblastoma is the most common and aggressive type of human brain tumor. Its poor prognosis largely derives from its diffused invasiveness into normal brain tissues which precludes successful surgical resection[1,2]. Transporters and ion channels can regulate these mechanisms through two different ways: by changing the concentration of internal Ca2+ and by modifying the volume of the cell, a process closely related to a two-dimensional migration[6]. In the presence of defects of the plasma membrane, the transitory pressure increase causes a cytosol flow and bleb protrusion, caused by the interaction with the underlying cytoskeleton. During this phase, actin undetectably on bleb surface only becomes evident when cytosol inflation is slow. Depending on the nature of the inducing agent, its concentration or cell line model, blebbing can become not transient, but can continuously grow until it produces extracellular vesicles[10]. We studied the properties of ionic currents associated to the treatment with silver ions and the involvement of the intracellular Ca2+ homeostasis
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