ABSTRACT
Triple negative breast cancer (TNBC) is known for its heterogeneous nature and aggressive onset. The unresponsiveness to hormone therapies and immunotherapy and the toxicity of chemotherapeutics account for the limited treatment options for TNBC. Ion channels have emerged as possible therapeutic candidates for cancer therapy, but little is known about how ligand gated ion channels, specifically, GABA type A ligand-gated ion channel receptors (GABAAR), affect cancer pathogenesis. Our results show that the GABAA ß3 subunit is expressed at higher levels in TNBC cell lines than non-tumorigenic cells, therefore contributing to the idea that limiting the GABAAR via knockdown of the GABAA ß3 subunit is a potential strategy for decreasing the proliferation and migration of TNBC cells. We employed pharmacological and genetic approaches to investigate the role of the GABAA ß3 subunit in TNBC proliferation, migration, and cell cycle progression. The results suggest that pharmacological antagonism or genetic knockdown of GABAA ß3 subunit decreases TNBC proliferation and migration. In addition, GABAA ß3 subunit knockdown causes cell cycle arrest in TNBC cell lines via decreased cyclin D1 and increased p21 expression. Our findings suggest that membrane bound GABAA receptors containing the ß3 subunit can be further developed as a potential novel target for the treatment of TNBC.
Subject(s)
Cell Cycle , Cell Movement , Receptors, GABA-A , Triple Negative Breast Neoplasms , Female , Humans , Cell Cycle/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cyclin D1/metabolism , Cyclin D1/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p21/genetics , Receptors, GABA-A/metabolism , Receptors, GABA-A/genetics , Triple Negative Breast Neoplasms/pathology , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/geneticsABSTRACT
Mounting evidence indicates that ethanol (EtOH) exposure activates neuroimmune signaling. Alterations in pro-inflammatory cytokines after acute and chronic EtOH exposure have been heavily investigated. In contrast, little is known about the regulation of neurotransmission and/or modulation by anti-inflammatory cytokines in the brain after an acute EtOH exposure. Recent evidence suggests that interleukin-10 (IL-10), an anti-inflammatory cytokine, is upregulated during withdrawal from chronic EtOH exposure. In the present study, we show that IL-10 is increased early (1 h) after a single intoxicating dose of EtOH (5 g/kg, intragastric) in Sprague Dawley rats. We also show that IL-10 rapidly regulates GABAergic transmission in dentate gyrus neurons. In brain slice recordings, IL-10 application dose-dependently decreases miniature inhibitory postsynaptic current (mIPSC) area and frequency, and decreases the magnitude of the picrotoxin sensitive tonic current (Itonic), indicating both pre- and postsynaptic mechanisms. A PI3K inhibitor LY294002 (but not the negative control LY303511) ablated the inhibitory effects of IL-10 on mIPSC area and Itonic, but not on mIPSC frequency, indicating the involvement of PI3K in postsynaptic effects of IL-10 on GABAergic transmission. Lastly, we also identify a novel neurobehavioral regulation of EtOH sensitivity by IL-10, whereby IL-10 attenuates acute EtOH-induced hypnosis. These results suggest that EtOH causes an early release of IL-10 in the brain, which may contribute to neuronal hyperexcitability as well as disturbed sleep seen after binge exposure to EtOH. These results also identify IL-10 signaling as a potential therapeutic target in alcohol-use disorders and other CNS disorders where GABAergic transmission is altered.
Subject(s)
Brain/drug effects , Central Nervous System Depressants/pharmacology , Ethanol/pharmacology , Interleukin-10/metabolism , Synaptic Transmission/drug effects , gamma-Aminobutyric Acid/metabolism , Animals , Binge Drinking/metabolism , Brain/metabolism , Cells, Cultured , Disease Models, Animal , Dose-Response Relationship, Drug , Hypnotics and Sedatives/pharmacology , Inhibitory Postsynaptic Potentials/drug effects , Inhibitory Postsynaptic Potentials/physiology , Male , Miniature Postsynaptic Potentials/drug effects , Miniature Postsynaptic Potentials/physiology , Neurons/drug effects , Neurons/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Rats, Sprague-Dawley , Receptors, GABA-A/metabolism , Sleep/drug effects , Sleep/physiology , Synaptic Transmission/physiology , Tissue Culture TechniquesABSTRACT
The neurotransmitter GABA mediates the majority of rapid inhibition in the CNS. Inhibition can occur via the conventional mechanism, the transient activation of subsynaptic GABAA receptors (GABAA-Rs), or via continuous activation of high-affinity receptors by low concentrations of ambient GABA, leading to "tonic" inhibition that can control levels of excitability and network activity. The GABAA-R alpha4 subunit is expressed at high levels in the dentate gyrus and thalamus and is suspected to contribute to extrasynaptic GABAA-R-mediated tonic inhibition. Mice were engineered to lack the alpha4 subunit by targeted disruption of the Gabra4 gene. alpha4 Subunit knockout mice are viable, breed normally, and are superficially indistinguishable from WT mice. In electrophysiological recordings, these mice show a lack of tonic inhibition in dentate granule cells and thalamic relay neurons. Behaviorally, knockout mice are insensitive to the ataxic, sedative, and analgesic effects of the novel hypnotic drug, gaboxadol. These data demonstrate that tonic inhibition in dentate granule cells and thalamic relay neurons is mediated by extrasynaptic GABAA-Rs containing the alpha4 subunit and that gaboxadol achieves its effects via the activation of this GABAA-R subtype.
Subject(s)
Dentate Gyrus/metabolism , Isoxazoles/pharmacology , Receptors, GABA-A/physiology , Thalamus/metabolism , Animals , Dentate Gyrus/drug effects , GABA-A Receptor Agonists , Mice , Mice, Knockout , Receptors, GABA-A/deficiency , Receptors, GABA-A/genetics , Thalamus/drug effectsABSTRACT
Xenopus laevis oocytes are used extensively in the study of ion channel coupled receptors. Efficient use of oocytes for ion channel characterization requires a system that is inherently stable, reproducible, minimizes drug volumes, and maximizes oocyte longevity. We have constructed a vertical flow oocyte recording chamber to address the aforesaid issues, where the oocyte is placed in a funnel-shaped chamber and perfused from the bottom of the funnel. The vertical rather than horizontal flow of perfusate results in an unusually stable environment for oocyte recording. Two-electrode voltage clamp recordings from a single oocyte are acquired easily and routinely over several hours while maintaining stable baseline currents and reproducible response profiles. Chamber characteristics were tested using a serotonin ligand-gated ion channel receptor (5-HT3R). Data obtained from this system corresponds well with published data. To further test the stability and reliability of this perfusion chamber, we constructed an automated oocyte perfusion system utilizing a commonly available HPLC autosampler. We were able to obtain dose-response curves for various 5-HT3AR ligands using the automated perfusion system with minimal user intervention. Such a system can easily satisfy need for automated oocyte electrophysiology in academic settings, especially small to medium sized laboratories.
Subject(s)
Automation/instrumentation , Diffusion Chambers, Culture/instrumentation , Drug Evaluation, Preclinical/methods , Electrophysiology , Oocytes/drug effects , Animals , Automation/methods , Computer-Aided Design , Diffusion Chambers, Culture/methods , Dose-Response Relationship, Drug , Electric Conductivity , Female , Humans , Membrane Potentials/drug effects , Membrane Potentials/physiology , Microinjections/methods , Oocytes/physiology , Patch-Clamp Techniques , Perfusion/methods , RNA, Complementary/biosynthesis , Receptors, Serotonin, 5-HT3/drug effects , Receptors, Serotonin, 5-HT3/genetics , Receptors, Serotonin, 5-HT3/metabolism , Serotonin/pharmacology , Xenopus laevisABSTRACT
The design, synthesis, and use of lerisetron-based molecular probes to investigate the 5-HT(3)R binding site are described. A SAR study, which involved distance and electronic parameter modifications of lerisetron's N-benzyl group, resulted in the discovery of a partial agonist.