Overcoming resistance to treatment is an essential issue in many cancers including glioblastoma (GBM), the deadliest primary tumor of the central nervous system. As dependence on iron is a key feature of tumor cells, using chelators to reduce iron represents an opportunity to improve conventional GBM therapies. The aim of the present study was, therefore, to investigate the cytostatic and cytotoxic impact of the new iron chelator deferasirox (DFX) on human GBM cells in well-defined clinical situations represented by radiation therapy and mild-hypoxia. Under experimental normoxic condition (21 % O2), deferasirox (DFX) used at 10 μM for 3 days reduced proliferation, led cell cycle arrest in S and G2-M phases and induced cytotoxicity and apoptosis in U251 and U87 GBM cells. The abolition of the antineoplastic DFX effects when cells were co-treated with ferric ammonium sulfate supports the hypothesis that its effects result from its ability to chelate iron. As radiotherapy is the main treatment for GBM, the combination of DFX and X-ray beam irradiation was also investigated. Irradiation at a dose of 16 Gy repressed proliferation, cytotoxicity and apoptosis, but only in U251 cells, while no synergy with DFX was observed in either cell line. Importantly, when the same experiment was conducted in mild-hypoxic conditions (3 % O2), the antiproliferative and cytotoxic effects of DFX were abolished, and its ability to deplete iron was also impaired. Taken together, these in vitro results could raise the question of the benefit of using iron chelators in their native forms under the hypoxic conditions often encountered in solid tumors such as GBM. Developing new chemistry or a new drug delivery system that would keep DFX active in hypoxic cells may be the next step toward their application.

Neonicotinoid insecticides are nicotine-derived molecules which exert acute neurotoxic effects over the insect central nervous system by activating nicotinic acetylcholine receptors (nAChRs). However, these receptors are also present in the mammalian central and peripheral nervous system, where the effects of neonicotinoids are faintly known. In mammals, cholinergic synapses are crucial for the control of vascular tone, blood pressure and skeletal muscle contraction. We therefore hypothesized that neonicotinoids could affect cholinergic networks in mammals and sought to highlight functional consequences of acute intoxication in rats with sub-lethal concentrations of the highly used acetamiprid (ACE) and clothianidin (CLO). In this view, we characterized their electrophysiological effects on rat $\alpha$3$\beta$4 nAChRs, knowing that it is predominantly expressed in ganglia of the vegetative nervous system and the adrenal medulla, which initiates catecholamine secretion. Both molecules exhibited a weak agonist effect on $\alpha$3$\beta$4 receptors. Accordingly, their influence on epinephrine secretion from rat adrenal glands was also weak at 100 μM, but it was stronger at 500 μM. Challenging ACE or CLO together with nicotine (NIC) ended up with paradoxical effects on secretion. In addition, we measured the rat arterial blood pressure (ABP) in vivo by arterial catheterization. As expected, NIC induced a significant increase in ABP. ACE and CLO did not affect the ABP in the same conditions. However, simultaneous exposure of rats to both NIC and ACE/CLO promoted an increase of ABP and induced a biphasic response. Modeling the interaction of ACE or CLO on $\alpha$3$\beta$4 nAChR is consistent with a binding site located in the agonist pocket of the receptor. We present a transversal experimental approach of mammal intoxication with neonicotinoids at different scales, including in vitro, ex vivo, in vivo and in silico. It paves the way of the acute and chronic toxicity for this class of insecticides on mammalian organisms.

Tetrodotoxin (TTX) poisoning through the consumption of contaminated fish leads to lethal symptoms, including severe hypotension. This TTX-induced hypotension is likely due to the downfall of peripheral arterial resistance through direct or indirect effects on adrenergic signaling. TTX is a high-affinity blocker of voltage-gated Na⁺ (Na_V) channels. In arteries, Na_V channels are expressed in sympathetic nerve endings, both in the intima and media. In this present work, we aimed to decipher the role of Na_V channels in vascular tone using TTX. We first characterized the expression of Na_V channels in the aorta, a model of conduction arteries, and in mesenteric arteries (MA), a model of resistance arteries, in C57Bl/6J mice, by Western blot, immunochemistry, and absolute RT-qPCR. Our data showed that these channels are expressed in both endothelium and media of aorta and MA, in which scn2a and scn1b were the most abundant transcripts, suggesting that murine vascular Na_V channels consist of Na_V1.2 channel subtype with Na_Vβ1 auxiliary subunit. Using myography, we showed that TTX (1 µM) induced complete vasorelaxation in MA in the presence of veratridine and cocktails of antagonists (prazosin and atropine with or without suramin) that suppressed the effects of neurotransmitter release. In addition, TTX (1 µM) strongly potentiated the flow-mediated dilation response of isolated MA. Altogether, our data showed that TTX blocks Na_V channels in resistance arteries and consecutively decreases vascular tone. This could explain the drop in total peripheral resistance observed during mammal tetrodotoxications.

Thanks to the crosstalk between Na⁺ and Ca²⁺ channels, Na⁺ and Ca²⁺ homeostasis interplay in so-called excitable cells enables the generation of action potential in response to electrical stimulation. Here, we investigated the impact of persistent activation of voltage-gated Na⁺ (Na_V) channels by neurotoxins, such as veratridine (VTD), on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a model of excitable cells, the rat pituitary GH3b6 cells, in order to identify the molecular actors involved in Na⁺-Ca²⁺ homeostasis crosstalk. By combining RT-qPCR, immunoblotting, immunocytochemistry, and patch-clamp techniques, we showed that GH3b6 cells predominantly express the Na_V1.3 channel subtype, which likely endorses their voltage-activated Na⁺ currents. Notably, these Na⁺ currents were blocked by ICA-121431 and activated by the β-scorpion toxin Tf2, two selective Na_V1.3 channel ligands. Using Fura-2, we showed that VTD induced a [Ca²⁺]_i increase. This effect was suppressed by the selective Na_V channel blocker tetrodotoxin, as well by the selective L-type Ca_V channel (LTCC) blocker nifedipine. We also evidenced that crobenetine, a Na_V channel blocker, abolished VTD-induced [Ca²⁺]_i elevation, while it had no effects on LTCC. Altogether, our findings highlight a crosstalk between Na_V and LTCC in GH3b6 cells, providing a new insight into the mode of action of neurotoxins.

Voltage-gated Na⁺ (Na_V) channels are significant therapeutic targets for the treatment of cardiac and neurological disorders, thus promoting the search for novel Na_V channel ligands. With the objective of discovering new blockers of Na_V channel ligands, we screened an In-House vegetal alkaloid library using fluorescence cell-based assays. We screened 62 isoquinoline alkaloids (IA) for their ability to decrease the FRET signal of voltage sensor probes (VSP), which were induced by the activation of Na_V channels with batrachotoxin (BTX) in GH3b6 cells. This led to the selection of five IA: liriodenine, oxostephanine, thalmiculine, protopine, and bebeerine, inhibiting the BTX-induced VSP signal with micromolar IC₅₀. These five alkaloids were then assayed using the Na⁺ fluorescent probe ANG-2 and the patch-clamp technique. Only oxostephanine and liriodenine were able to inhibit the BTX-induced ANG-2 signal in HEK293-hNa_V1.3 cells. Indeed, liriodenine and oxostephanine decreased the effects of BTX on Na⁺ currents elicited by the hNa_V1.3 channel, suggesting that conformation change induced by BTX binding could induce a bias in fluorescent assays. However, among the five IA selected in the VSP assay, only bebeerine exhibited strong inhibitory effects against Na⁺ currents elicited by the hNav1.2 and hNav1.6 channels, with IC₅₀ values below 10 µM. So far, bebeerine is the first BBIQ to have been reported to block Na_V channels, with promising therapeutical applications.