NOS2 inhibitor 1400W Induces Autophagic Flux and Influences Extracellular Vesicle Profile in Human Glioblastoma U87MG Cell Line

Abstract

The relevance of nitric oxide synthase 2 (NOS2) as a prognostic factor in Glioblastoma Multiforme (GBM) malignancy is emerging. We analyzed the effect of NOS2 inhibitor 1400W on the autophagic flux and extracellular vesicle (EV) secretion in U87MG glioma cells. The effects of glioma stem cells (GSC)-derived EVs on adherent U87MG were evaluated. Cell proliferation and migration were examined while using Cell Counting Kit-8 assay (CCK-8) and scratch wound healing assay. Cell cycle profile and apoptosis were analyzed by flow cytometry. Autophagy-associated acidic vesicular organelles were detected and quantified by acridine orange staining. The number and size of EVs were assessed by nanoparticle tracking analysis. EV ultrastructure was verified by transmission electron microscopy (TEM). WB was used to analyze protein expression and acid sphingomyelinase was determined through ceramide levels. 1400W induced autophagy and EV secretion in both adherent U87MG and GSCs. EVs secreted by 1400W-treated GSC, but not those from untreated cells, were able to inhibit adherent U87MG cell growth and migration while also inducing a relevant level of autophagy. The hypothesis of NOS2 expression as GBM profile marker or interesting therapeutic target is supported by our findings. Autophagy and EV release following treatment with the NOS2 inhibitor could represent useful elements to better understand the complex biomolecular frame of GBM.

Targeting iNOS As a Valuable Strategy for the Therapy of Glioma

“…Recently, it was reported that 1400 W significantly reduces the proliferation, migration, colony formation, and neurosphere generation ability in U‐87 MG and T98G glioma cell lines, as well as in the human glioma primary cells. This study sustains the emerging relevance of iNOS as a prognostic factor of glioma malignancy and recurrence, supporting the potential therapeutic value of safer iNOS inhibitors for a better management of this cancer …”

Trauma Coagulopathy and Its Outcomes

“…Unlike other pathologies [24,[33][34][35][36][37][38][39][40][41][42][43][44][45][46][47], the molecular mechanism underlying the pathophysiology of acquired coagulopathy is still not well known, although studies have reported that a few bioumoral parameters (pH, lactates, and BE) are significantly more compromised in trauma coagulopathy patients [24,32,48]. Because ours was not a pathophysiological study, we do not know whether this correlation stems from the fact that MT results more easily in coagulopathy or whether trauma coagulopathy increases the severity of the trauma itself or whether there is a mutual influence.…”

Extracellular Vesicle-Mediated Communication between the Glioblastoma and Its Microenvironment

“…Therefore, it is important to take into account that GSCs are targets for EVs released within the GBM microenvironment, and, at the same time, GSCs release EVs which can induce protumorigenic effects on GBM cells (Figure 2). For instance, Palumbo et al [74] have shown that EVs released by GSCs exert a stimulatory effect on cell growth and migration of the adherent GBM cell line U87MG. GSC-derived EVs decorated with the cell adhesion molecule L1CAM are also able to induce motility, proliferation and invasiveness of different types of GBM cell lines [75].…”