Interleukin-17A affects extracellular vesicles release and cargo in human keratinocytes.

Psoriasis is a chronic inflammatory systemic disease caused by deregulation of the interleukin-23/-17 axis that allows the activation of Th17 lymphocytes and the reprogramming of keratinocytes proliferative response, thereby inducing the secretion of cyto-/chemokines and antimicrobial peptides. Beside cell-to-cell contacts and release of cytokines, hormones and second messengers, cells communicate each other through the release of extracellular vesicles containing DNA, RNA, microRNAs and proteins. It has been reported the alteration of extracellular vesicles trafficking in several diseases, but there is scarce evidence of the involvement of extracellular vesicles trafficking in the pathogenesis of psoriasis. The main goal of the study was to characterize the release, the cargo content and the capacity to transfer bioactive molecules of extracellular vesicles produced by keratinocytes following recombinant IL-17A treatment if compared to untreated keratinocytes. A combined approach of standard ultracentrifugation, RNA isolation and real-time RT-PCR techniques was used to characterize extracellular vesicles cargo. Flow cytometry was used to quantitatively and qualitatively analyse extracellular vesicles and to evaluate cell-to-cell extracellular vesicles transfer. We report that the treatment of human keratinocytes with IL-17A significantly modifies the extracellular vesicles cargo and release. Vesicles from IL-17A-treated cells display a specific pattern of mRNA which is undid by IL-17A neutralization. Extracellular vesicles are taken up by acceptor cells irrespective of their content but only those derived from IL-17A-treated cells enable recipient cells to express psoriasis-associated mRNA. The results imply a role of extracellular vesicles in amplifying the pro-inflammatory cascade induced in keratinocyte by pro-psoriatic cytokines.

BK channels blockage inhibits hypoxia-induced migration and chemoresistance to cisplatin in human glioblastoma cells.

Glioblastoma (GBM) cells express large-conductance, calcium-activated potassium (BK) channels, whose activity is important for several critical aspects of the tumor, such as migration/invasion and cell death. GBMs are also characterized by a heavy hypoxic microenvironment that exacerbates tumor aggressiveness. Since hypoxia modulates the activity of BK channels in many tissues, we hypothesized that a hypoxia-induced modulation of these channels may contribute to the hypoxia-induced GBM aggressiveness. In U87-MG cells, hypoxia induced a functional upregulation of BK channel activity, without interfering with their plasma membrane expression. Wound healing and transwell migration assays showed that hypoxia increased the migratory ability of U87-MG cells, an effect that could be prevented by BK channel inhibition. Toxicological experiments showed that hypoxia was able to induce chemoresistance to cisplatin in U87-MG cells and that the inhibition of BK channels prevented the hypoxia-induced chemoresistance. Clonogenic assays showed that BK channels are also used to increase the clonogenic ability of U87-MG GBM cells in presence, but not in absence, of cisplatin. BK channels were also found to be essential for the hypoxia-induced de-differentiation of GBM cells. Finally, using immunohistochemical analysis, we highlighted the presence of BK channels in hypoxic areas of human GBM tissues, suggesting that our findings may have physiopathological relevance in vivo. In conclusion, our data show that BK channels promote several aspects of the aggressive potential of GBM cells induced by hypoxia, such as migration and chemoresistance to cisplatin, suggesting it as a potential therapeutic target in the treatment of GBM.

Overexpression of Large-Conductance Calcium-Activated Potassium Channels in Human Glioblastoma Stem-Like Cells and Their Role in Cell Migration.

Glioblastomas (GBMs) are brain tumors characterized by diffuse invasion of cancer cells into the healthy brain parenchyma, and establishment of secondary foci. GBM cells abundantly express large-conductance, calcium-activated potassium (BK) channels that are thought to promote cell invasion. Recent evidence suggests that the GBM high invasive potential mainly originates from a pool of stem-like cells, but the expression and function of BK channels in this cell subpopulation have not been studied. We investigated the expression of BK channels in GBM stem-like cells using electrophysiological and immunochemical techniques, and assessed their involvement in the migratory process of this important cell subpopulation. In U87-MG cells, BK channel expression and function were markedly upregulated by growth conditions that enriched the culture in GBM stem-like cells (U87-NS). Cytofluorimetric analysis further confirmed the appearance of a cell subpopulation that co-expressed high levels of BK channels and CD133, as well as other stem cell markers. A similar association was also found in cells derived from freshly resected GBM biopsies. Finally, transwell migration tests showed that U87-NS cells migration was much more sensitive to BK channel block than U87-MG cells. Our data show that BK channels are highly expressed in GBM stem-like cells, and participate to their high migratory activity. J. Cell. Physiol. 232: 2478-2488, 2017. © 2016 Wiley Periodicals, Inc.