Electrical signals elicited by integrin interaction with ECM components and their role in neurite outgrowth were studied in two clones (N1 and N7) isolated from 41A3 murine neuroblastoma cell line. Although the two clones similarly adhered to fibronectin (FN) and vitronectin (VN), this adhesion induced neurite outgrowth in N1 but not in N7 cells. Patch clamp recordings in whole cell configuration showed that, upon adhesion to FN or VN but not to platelet factor 4 (PF4), N1 cells undergo a marked (approximately equal to 20 mV) hyperpolarization of the resting potential (Vrest) that occurred within the first 20 min after cell contact with ECM, and persisted for approximately 1 h before reverting to the time zero values. This hyperpolarization was totally absent in N7 cells. A detailed analysis of the molecular mechanisms involved in N1 and N7 cell adhesion to ECM substrata was performed by using antibodies raised against the FN receptor and synthetic peptides variously competing with the FN or VN binding to integrin receptor (GRGDSP and GRGESP). Antibodies, as well as GRGDSP, abolished adhesion of N1 and N7 clones to FN and VN, revealing a similar implication of integrins in the adhesion of these clones to the ECM proteins. However, these anti-adhesive treatments, while ineffective on Vrest of N7 cells, abolished in N1 cells the FN- or VN-induced hyperpolarization and neurite outgrowth, that appeared therefore strictly associated and integrin-mediated phenomena. The nature of this association was deepened through a comparative analysis of the integrin profiles and the ion channels of N1 and N7 cells. The integrin immunoprecipitation profile resulted very similarly in the two clones, with only minor differences concerning the alpha V containing complexes. Both clones possessed Ca2+ and K+ delayed rectifier (KDR) channels, while only N1 cells were endowed with inward rectifier K+ (KIR) channels. The latter governed the Vrest, and, unlike KDR channels, were blocked by Ba2+ and Cs+. By moving patched cells in contact with FN-coated beads, it was shown that KIR channel activation was responsible for the FN-mediated hyperpolarization of Vrest. Treatment with Pertuxis toxin (PTX) abolished this hyperpolarization and neurite outgrowth, indicating that a G protein is interposed between integrins and KIR channels and that the activation of these channels is required for neuritogenesis. In fact, the block of KIR channels by Cs+ abolished both hyperpolarization and neurite outgrowth, provided that the cation was supplied during the first two hours after N1 cell contact with FN.(ABSTRACT TRUNCATED AT 400 WORDS)

Thymoquinone (TQ) is the main active ingredient of Nigella sativa essential oil, with remarkable anti-neoplastic activities with anti-invasive and anti-migratory abilities on a variety of cancer cell lines. However, its poor water solubility, high instability in aqueous solution and pharmacokinetic drawbacks limits its use in therapy. Soluplus^® and Solutol^® HS15 were employed as amphiphilic polymers for developing polymeric micelles (SSM). Chemical and physical characterization studies of micelles are reported, in terms of size, homogeneity, zeta potential, critical micelle concentration (CMC), cloud point, encapsulation efficiency (EE%), load capacity (DL), in vitro release, and stability. This study reports for the first time the anti-migratory activity of TQ and TQ loaded in SSM (TQ-SSM) in the SH-SY5Y human neuroblastoma cell line. The inhibitory effect was assessed by the wound-healing assay and compared with that of the unformulated TQ. The optimal TQ-SSM were provided with small size (56.71 ± 1.41 nm) and spherical shape at ratio of 1:4 (Soluplus:Solutol HS15), thus increasing the solubility of about 10-fold in water. The entrapment efficiency and drug loading were 92.4 ± 1.6% and 4.68 ± 0.12, respectively, and the colloidal dispersion are stable during storage for a period of 40 days. The TQ-SSM were also lyophilized to obtain a more workable product and with increased stability. In vitro release study indicated a prolonged release of TQ. In conclusion, the formulation of TQ into SSM allows a bio-enhancement of TQ anti-migration activity, suggesting that TQ-SSM is a better candidate than unformulated TQ to inhibit human SH-SY5Y neuroblastoma cell migration.

Posidonia oceanica (L.) Delile is a marine plant with interesting biological properties potentially ascribed to the synergistic combination of bioactive compounds. Our previously described extract, obtained from the leaves of P. oceanica, showed the ability to impair HT1080 cell migration by targeting both expression and activity of gelatinases. Commonly, the lack of knowledge about the mechanism of action of phytocomplexes may be an obstacle regarding their therapeutic use and development. The aim of this study was to gain insight into the molecular signaling through which such bioactive compounds impact on malignant cell migration and gelatinolytic activity. The increase in autophagic vacuoles detected by confocal microscopy suggested an enhancement of autophagy in a time and dose dependent manner. This autophagy activation was further confirmed by monitoring pivotal markers of autophagy signaling as well as by evidencing an increase in IGF-1R accumulation on cell membranes. Taken together, our results confirm that the P. oceanica phytocomplex is a promising reservoir of potent and cell safe molecules able to defend against malignancies and other diseases in which gelatinases play a major role in progression. In conclusion, the attractive properties of this phytocomplex may be of industrial interest in regard to the development of novel health-promoting and pharmacological products for the treatment or prevention of several diseases.