Mesenchymal Stem Cells Increase Drug Tolerance of A431 Cells Only in 3D Spheroids, Not in 2D Co-Cultures.

Mesenchymal stem cells (MSCs) are an integral part of the tumor microenvironment (TME); however, their role is somewhat controversial: conflicting reports suggest that, depending on the stage of tumor development, MSCs can either support or suppress tumor growth and spread. Additionally, the influence of MSCs on drug resistance is also ambiguous. Previously, we showed that, despite MSCs proliferating significantly more slowly than cancer cells, there are chemotherapeutic drugs which proved to be similarly toxic to both cell types. Here we established 2D co-cultures and 3D co-culture spheroids from different ratios of GFP-expressing, adipose tissue-derived MSCs and A431 epidermoid carcinoma cells tagged with mCherry to investigate the effect of MSCs on cancer cell growth, survival, and drug sensitivity. We examined the cytokine secretion profile of mono- and co-cultures, explored the inner structure of the spheroids, applied MSC-(nutlin-3) and cancer cell-targeting (cisplatin) treatments separately, monitored the response with live-cell imaging and identified a new, double-fluorescent cell type emerging from these cultures. In 2D co-cultures, no effect on proliferation or drug sensitivity was observed, regardless of the changes in cytokine secretion induced by the co-culture. Conversely, 3D spheroids developed a unique internal structure consisting of MSCs, which significantly improved cancer cell survival and resilience to treatment, suggesting that physical proximity and cell-cell connections are required for MSCs to considerably affect nearby cancer cells. Our results shed light on MSC-cancer cell interactions and could help design new, better treatment options for tumors.

Connexons Coupling to Gap Junction Channel: Potential Role for Extracellular Protein Stabilization Centers.

Connexin (Cx) proteins establish intercellular gap junction channels (Cx GJCs) through coupling of two apposed hexameric Cx hemichannels (Cx HCs, connexons). Pre- and post-GJ interfaces consist of extracellular EL1 and EL2 loops, each with three conserved cysteines. Previously, we reported that known peptide inhibitors, mimicking a variety of Cx43 sequences, appear non-selective when binding to homomeric Cx43 vs. Cx36 GJC homology model subtypes. In pursuit of finding potentially Cx subtype-specific inhibitors of connexon-connexon coupling, we aimed at to understand better how the GJ interface is formed. Here we report on the discovery of Cx GJC subtype-specific protein stabilization centers (SCs) featuring GJ interface architecture. First, the Cx43 GJC homology model, embedded in two opposed membrane bilayers, has been devised. Next, we endorsed the fluctuation dynamics of SCs of the interface domain of Cx43 GJC by applying standard molecular dynamics under open and closed cystine disulfide bond (CS-SC) preconditions. The simulations confirmed the major role of the unique trans-GJ SC pattern comprising conserved (55N, 56T) and non-conserved (57Q) residues of the apposed EL1 loops in the stabilization of the GJC complex. Importantly, clusters of SC patterns residing close to the GJ interface domain appear to orient the interface formation via the numerous SCs between EL1 and EL2. These include central 54CS-S198C or 61CS-S192C contacts with residues 53R, 54C, 55N, 197D, 199F or 64V, 191P, respectively. In addition, we revealed that GJC interface formation is favoured when the psi dihedral angle of the nearby 193P residue is stable around 180° and the interface SCs disappear when this angle moves to the 0° to -45° range. The potential of the association of non-conserved residues with SC motifs in connexon-connexon coupling makes the development of Cx subtype-specific inhibitors viable.

Molecular Plasticity of the Nucleus Accumbens Revisited-Astrocytic Waves Shall Rise.

Part of the ventral striatal division, the nucleus accumbens (NAc) drives the circuit activity of an entire macrosystem about reward like a "flagship," signaling and leading diverse conducts. Accordingly, NAc neurons feature complex inhibitory phenotypes that assemble to process circuit inputs and generate outputs by exploiting specific arrays of opposite and/or parallel neurotransmitters, neuromodulatory peptides. The resulting complex combinations enable versatile yet specific forms of accumbal circuit plasticity, including maladaptive behaviors. Although reward signaling and behavior are elaborately linked to neuronal circuit activities, it is plausible to propose whether these neuronal ensembles and synaptic islands can be directly controlled by astrocytes, a powerful modulator of neuronal activity. Pioneering studies showed that astrocytes in the NAc sense citrate cycle metabolites and/or ATP and may induce recurrent activation. We argue that the astrocytic calcium, GABA, and Glu signaling and altered sodium and chloride dynamics fundamentally shape metaplasticity by providing active regulatory roles in the synapse- and network-level flexibility of the NAc.

Effects of nucleosides on glia - neuron interactions open up new vistas in the development of more effective antiepileptic drugs.

One-third of epileptic patients are drug refractory due to the limited efficacy of antiepileptic therapy. Thus, there is an immense need to find more effective, safer and well-tolerated antiepileptic drugs. A great deal of results suggests that adenosine (Ado), guanosine (Guo), inosine (Ino) or uridine (Urd) are endogenous antiepileptogenic modulators. Furthermore, nucleosides and their derivatives may be safe and effective potential drugs in the treatment of epilepsy. Conversely, nucleosidergic modulatory system implying nucleoside levels, metabolism, receptors and transporters may also be involved in seizure pathomechanisms. Application of Ado receptor agonists as well as antagonists, elevation of nucleoside levels (e.g., by nucleoside metabolism inhibitors, and Adoreleasing implants) or utilization of non-Ado nucleosides may also turn to be useful approaches to decrease epileptic activity. However, all drugs exerting their effects on the nucleosidergic modulatory system may affect the fine regulation of glia-neuron interactions that are intimately governed by various nucleosidergic processes. Perturbation of the complex, bidirectional communication between neurons and astrocytes through these nucleosidergic modulatory mechanisms may lead to pathological changes in the central nervous system (CNS) and therefore may cause significant side effects. Thus, a deeper understanding of the nucleosidergic modulatory control over glia-neuron interactions is essential in order to develop more effective and safe nucleoside-based antiepileptic drugs. In this review article we focus on the role of Ado and Urd in glia-neuron interactions, placing emphasis on their implications for the treatment of epilepsy.

Non-adenosine nucleoside inosine, guanosine and uridine as promising antiepileptic drugs: a summary of current literature.

Adenosine (Ado) and some non-adenosine (non-Ado) nucleosides including inosine (Ino), guanosine (Guo) and uridine (Urd) are modulatory molecules in the central nervous system (CNS), regulating different physiological and pathophysiological processes in the brain such as sleep and epilepsy. Indeed, different drugs effective on adenosinergic system (e.g., Ado metabolism inhibitors, agonists and antagonists of Ado receptors) are being used in drug development for the treatment of epileptic disorders. Although (i) endogenous Ino, Guo and Urd showed anticonvulsant/antiepileptic effects (e.g., in quinolinic acid - induced seizures and in different epilepsy models such as hippocampal kindling models), and (ii) there is a need to generate new and more effective antiepileptic drugs for the treatment of drug-resistant epilepsies, our knowledge about antiepileptic influence of non-Ado nucleosides is far from complete. Thus, in this review article, we give a short summary of anticonvulsant/antiepileptic effects and mechanisms evoked by Ino, Guo, and Urd. Finally, we discuss some non-Ado nucleoside derivatives and their structures, which may be candidates as potential antiepileptic agents.

Calcium signals in the nucleus accumbens: activation of astrocytes by ATP and succinate.

BACKGROUND: Accumulating evidence suggests that glial signalling is activated by different brain functions. However, knowledge regarding molecular mechanisms of activation or their relation to neuronal activity is limited. The purpose of the present study is to identify the characteristics of ATP-evoked glial signalling in the brain reward area, the nucleus accumbens (NAc), and thereby to explore the action of citric acid cycle intermediate succinate (SUC). RESULTS: We described the burst-like propagation of Ca2+ transients evoked by ATP in acute NAc slices from rat brain. Co-localization of the ATP-evoked Ca2+ signalling with immunoreactivities of the astroglia-specific gap junction forming channel protein connexin43 (Cx43) and the glial fibrillary acidic protein (GFAP) indicated that the responsive cells were a subpopulation of Cx43 and GFAP immunoreactive astrocytes. The ATP-evoked Ca2+ transients were present under the blockade of neuronal activity, but were inhibited by Ca2+ store depletion and antagonism of the G protein coupled purinergic P2Y1 receptor subtype-specific antagonist MRS2179. Similarly, Ca2+ transients evoked by the P2Y1 receptor subtype-specific agonist 2-(Methylthio)adenosine 5'-diphosphate were also blocked by MRS2179. These characteristics implied that intercellular Ca2+ signalling originated from the release of Ca2+ from internal stores, triggered by the activation of P2Y1 receptors. Inhibition by the gap junction blockers carbenoxolone and flufenamic acid and by an antibody raised against the gating-associated segment of Cx43 suggested that intercellular Ca2+ signalling proceeded through gap junctions. We demonstrated for the first time that extracellular SUC also evoked Ca2+ transients (EC50 = 50-60 µM) in about 15% of the ATP-responsive NAc astrocytes. By contrast to glial cells, electrophysiologically identified NAc neurons surrounded by ATP-responsive astrocytes were not activated simultaneously. CONCLUSIONS: We concluded, therefore, that ATP- and SUC-sensitive Ca2+ transients appear to represent a signalling layer independent of NAc neurons. This previously unrecognised glial action of SUC, a major cellular energy metabolite, may play a role in linking metabolism to Ca2+ signalling in astrocytic networks under physiological and pathological conditions such as exercise and metabolic diseases.

Characterisation of an uridine-specific binding site in rat cerebrocortical homogenates.

Parameters of [3H]uridine binding to synaptic membranes isolated from rat brain cortex (K(D)=71+/-4 nM, B(max)=1.37+/-0.13 pmol/mg protein) were obtained. Pyrimidine and purine analogues displayed different rank order of potency in displacement of specifically bound [3H]uridine (uridine>5-F-uridine>5-Br-uridine approximately adenosine>>5-ethyl-uridine approximately suramin>theophylline) and in the inhibition of [14C]uridine uptake (adenosine>uridine>5-Br-uridine approximately 5-F-uridine approximately 5-ethyl-uridine) into purified cerebrocortical synaptosomes. Furthermore, the effective ligand concentration for the inhibition of [14C]uridine uptake was about two order of magnitude higher than that for the displacement of specifically bound [3H]uridine. Adenosine evoked the transmembrane Na(+) ion influx, whereas uridine the transmembrane Ca(2+) ion influx much more effectively. Also, uridine was shown to increase free intracellular Ca(2+) ion levels in hippocampal slices by measuring Calcium-Green fluorescence. Uridine analogues were found to be ineffective in displacing radioligands that were bound to various glutamate and adenosine-recognition and modulatory-binding sites, however, increased [35S]GTPgammaS binding to membranes isolated from the rat cerebral cortex. These findings provide evidence for a rather specific, G-protein-coupled site of excitatory action for uridine in the brain.