Human NT2 Research Articles

PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

The Gαq/phospholipase C-β (PLCβ) signaling system mediates calcium responses to a variety of hormones and neurotransmitters. Recent studies suggest that PLCβ1 expression plays a role in the differentiation of two types of cultured neuronal cells (PC12 and SK-N-SH) through a mechanism independent of Gαq. Here, we show that, similar to that observed in PC12 and SK-N-SH cells, PLCβ1 expression increases when human NT2 cells are induced to differentiate either through cytosine-β-D-arabinofuranoside or retinoic acid. Preventing this increase, abolishes differentiation, and down-regulating PLCβ1 in rat primary astrocytes causes cells to adapt an undifferentiated morphology. Surprisingly, transfecting PLCβ1 into undifferentiated PC12 or NT2 cells induces differentiation without the need for differentiating agents. Studies to uncover the underlying mechanism focused on the transcription factor early growth response 1 (Egr-1) which mediates PLCβ1 expression early in differentiation. Over-expressing PLCβ1 in HEK293 cells enhances Egr-1 expression and induces morphological changes. We show that increased levels of cytosolic PLCβ1 in undifferentiated PC12 cells disrupts the association between Egr-1 and its cytosolic binding partner (Tar RNA binding protein), promoting relocalization of Egr-1 to the nucleus, which promotes transcription of proteins needed for differentiation. These studies show a novel mechanism through which differentiation can be modulated.

Synthesis and Biological Evaluation of Novel Benzylidene Thiazolo Pyrimidin-3(5H)-One Derivatives

Starting compound 1 was synthesized according to reference. 1 Benzylidene thiazole pyrimidin-3(5H)-ones were synthesized reactions of 1 with bromoacetic acid and various aryl-aldehydes in the same vessel via one-step, unlike studies in the literature. Quantum chemical parameters and full geometry optimizations for all compounds were computed using DFT based on B3LYP. Cytotoxic action potential of synthesized compounds was evaluated using trypan blue dye exclusion and MTT assays in different cell lines including adenocarcinoma alveolar basal epithelial-like adherent A549 cells, the colon adenocarcinoma HT-29 cells, prostate adenocarcinoma DU-145 cells, and diploid ARPE-19 retinal pigment epithelial cells. Embryotoxicity and genotoxicity assessments were performed on pluripotent human embryonal carcinoma NT2 and human lymphocyte cells, respectively. Compound A1 exhibited good anticancer activity on A549 and DU-145 cell lines, and the compounds including A3, 4, 6, and 9 induced cytotoxicity on A549 cells. The compounds A1-10 also showed a good biosafety profile at relatively lower concentrations.

Toxicity of Glycyl-l-Prolyl-l-Glutamate Pseudotripeptides: Cytotoxic, Oxidative, Genotoxic, and Embryotoxic Perspectives.

The tripeptide H-Gly-Pro-Glu-OH (GPE) and its analogs began to take much interest from scientists for developing effective novel molecules in the treatment of several disorders including Alzheimer's disease, Parkinson's disease, and stroke. The peptidomimetics of GPEs exerted significant biological properties involving anti-inflammatory, antiapoptotic, and anticancer properties. The assessments of their hematological toxicity potentials are critically required for their possible usage in further preclinical and clinical trials against a wide range of pathological conditions. However, there is so limited information on the safety profiling of GPE and its analogs on human blood tissue from cytotoxic, oxidative, and genotoxic perspectives. And, their embryotoxicity potentials were not investigated yet. Therefore, in this study, measurements of mitochondrial viability (using MTT assay) and lactate dehydrogenase (LDH) release as well as total antioxidant capacity (TAC) assays were performed on cultured human whole blood cells after treatment with GPE and its three novel peptidomimetics for 72 h. Sister chromatid exchange (SCE), micronucleus (MN), and 8-oxo-2-deoxyguanosine (8-OH-dG) assays were performed for determining the genotoxic damage potentials. In addition, the nuclear division index (NDI) was figured out for revealing their cytostatic potentials. Embryotoxicity assessments were performed on cultured human pluripotent NT2 embryonal carcinoma cells by MTT and LDH assays. The present results from cytotoxicity, oxidative, genotoxicity, and embryotoxicity testing clearly propounded that GPEs had good biosafety profiles and were trouble-free from the toxicological point of view. Noncytotoxic, antioxidative, nongenotoxic, noncytostatic, and nonembryotoxic features of GPE analogs are worthwhile exploring further and may exert high potentials for improving the development of novel disease-modifying agents.

Effects of Tolerance-Induced Preconditioning on Mitochondrial Biogenesis in Undifferentiated and Differentiated Neuronal Cells.

Mitochondrial biogenesis occurs in response to chronic stresses as an adaptation to the increased energy demands and often renders cells more refractive to subsequent injuries which is referred to as preconditioning. This phenomenon is observed in several non-neuronal cell types, but it is not yet fully established in neurons, although it is fundamentally important for neuroprotection and could be exploited for therapeutic purposes. This study was designed to examine whether the preconditioning treatment with hypoxia or nitric oxide could trigger biogenesis in undifferentiated and differentiated neuronal cells (rat PC12 and human NT2 cells) as well as in primary mouse cortical neurons. The results showed that both preconditioning paradigms induced mitochondrial biogenesis in undifferentiated cell lines, as indicated by an increase of mitochondrial mass (measured by flow cytometry of NAO fluorescence) and increased expression of genes required for mitochondrial biogenesis (Nrf1, Nrf2, Tfam, Nfκb1) and function (Cox3, Hk1). All these changes translated into an increase in the organelle copy number from an average of 20-40 to 40-60 mitochondria per cell. The preconditioning treatments also rendered the cells significantly less sensitive to the subsequent oxidative stress challenge brought about by oxygen/glucose deprivation, consistent with their improved cellular energy status. Mitochondrial biogenesis was abolished when preconditioning treatments were performed in the presence of antioxidants (vitamin E or CoQ10), indicating clearly that ROS-signaling pathway(s) played a critical role in the induction of this phenomenon in undifferentiated cells. However, mitochondrial biogenesis could not be re-initiated by preconditioning treatments in any of the post-mitotic neuronal cells tested, i.e., neither rat PC12 cells differentiated with NGF, human NT2 cells differentiated with retinoic acid nor mouse primary cortical neurons. Instead, differentiated neurons had a much higher organelle copy number per cell than their undifferentiated counterparts (100-130 mitochondria per neuron vs. 20-40 in proliferating cells), and this feature was not altered by preconditioning. Our study demonstrates that mitochondrial biogenesis occurred during the differentiation process resulting in more beneficial energy status and improved tolerance to oxidative stress in neurons, putting in doubt whether additional enhancement of this phenomenon could be achieved and successfully exploited as a way for better neuroprotection.

Molecular and Functional Characterization of Caveolae in Mixed Cultures of Human NT-2 Neurons and Astrocytes

Caveolae are plasma membrane invaginations that are enriched in cholesterol-binding proteins called caveolins. The presence of caveolae and caveolins in mixed cultures of human neurons and glia has not been investigated. Here, we sought to determine the presence of caveolae and caveolins in human NTera-2 (NT2/D1) cells, differentiated with retinoic acid into neuron-like (NT2/N) and astrocyte-like (NT2/A) cells. We found that while caveolin-3 mRNA levels remained relatively constant, caveolin-1 and -2 levels were upregulated in NT2/A and downregulated in NT2/N. No caveolin-1 immunoreactivity was detected in NT2/N. Electron microscopy revealed numerous flask-shaped invaginations (~86–102 nm in diameter) in the plasma membrane of NT2/A and NT2/N cells, while only few were detected in NT2/D1 cells. Immunoelectron microscopy localized caveolin-1 gold particles in the flask-shaped structures on plasmalemma and cytoplasmic vesicles of NT2/A cells. Furthermore, NT2/A endocytosed Alexa 488 conjugated-cholera toxin B subunit (CTX-B) through a caveolae- and clathrin-dependent pathway, whereas NT2/N endocytosed CTX-B through a caveolae-independent pathway. We have established that while NT2/A expressed functional caveolae, the molecular identity of the plasma membrane invaginations in NT2/N is unknown. The expression of caveolin proteins was differentially regulated in these cells. Taken together, our findings support the usefulness of the human NT2 model system to study the role of caveolins in neuron–glia communication, and their involvement in brain health and disease.

The loaded Ferula assa-foetida seed essential oil in Solid lipid nanoparticles (FSEO-SLN) as the strong apoptosis inducer agents in human NTERA-2 embryocarcinoma cells

ABSTRACT In the current study,Ferula assa foetida seed oil (FS) (FSEO) was loaded into Solid lipid nanoparticles (SLN) to analyze its apoptotic and anti-angiogenic potential, respectively. The FSEO-loaded SLN (FSEO-SLN) was synthesized and characterized by TEM, DLS, FTIR, and Zeta potential analysis. The cytotoxic, apoptotic, and anti-angiogenic activities of the FSEO-SLN were evaluated by studying the NT2 survival rate, cell cycle phases, and apoptotic gene expression profile (TNF-α, P21, and Cas3). Finally, the impact of FSEO-SLN on the length and number of the CAM blood vessels were analyzed. The FSEO-SLN nanoparticles exhibited a significant cell-growth suppressive impact on the human NT-2 cancer stem cell line. They induced apoptotic death by up-regulating the expression of TNF-α, P21, Cas3 genes. The FSEO-SLN remarkably suppressed angiogenesis in CAM tissue by decreasing the length and number of its blood vessels. Therefore, it has the potential to be studied as an efficient anti-cancer agent.

Developmental Neurotoxicity of Fipronil and Rotenone on a Human Neuronal In Vitro Test System

Pesticide exposure during in utero and early postnatal development can cause a wide range of neurological defects. However, relatively few insecticides have been recognized as developmental neurotoxicants, so far. Recently, discovery of the insecticide, fipronil, in chicken eggs has raised public concern. The status of fipronil as a potential developmental neurotoxicant is still under debate. Whereas several in vivo and in vitro studies suggest specific toxicity, other in vitro studies could not confirm this concern. Here, we tested fipronil and its main metabolic product, fipronil sulfone both at concentrations between 1.98 and 62.5 µM, alongside with the established developmental neurotoxicant, rotenone (0.004–10 µM) in vitro on the human neuronal precursor cell line NT2. We found that rotenone impaired all three tested DNT endpoints, neurite outgrowth, neuronal differentiation, and precursor cell migration in a dose-dependent manner and clearly separable from general cytotoxicity in the nanomolar range. Fipronil and fipronil sulfone specifically inhibited cell migration and neuronal differentiation, but not neurite outgrowth in the micromolar range. The rho-kinase inhibitor Y-27632 counteracted inhibition of migration for all three compounds (EC50 between 12 and 50 µM). The antioxidant, n-acetyl cysteine, could ameliorate the inhibitory effects of fipronil on all three tested endpoints (EC 50 between 84 and 164 µM), indicating the involvement of oxidative stress. Fipronil sulfone had a stronger effect than fipronil, confirming the importance to test metabolic products alongside original pesticides. We conclude that in vitro fipronil and fipronil sulfone display specific developmental neurotoxicity on developing human model neurons.

Cultured Cell Line Models of Neuronal Differentiation: NT2, PC12, and SK-N-MC.

The lack of a convenient, easily maintained, and inexpensive in vitro human neuronal model to study neurodegenerative diseases prompted us to develop a rapid, 1-h differentiated neuronal cell model based on human NT2 cells and C3 transferase. Here, we describe the rapid differentiation of human neuronal NT2 cells, and the differentiation, transduction, and transfection of human SK-N-MC cells and rat PC12 cells to obtain cells with the morphology of differentiated neurons that can express exogenous genes of interest at high level.

The t-PA –7351C>T enhancer polymorphism decreases Sp1 and Sp3 protein binding affinity and transcriptional responsiveness to retinoic acid

AbstractWe have previously identified a common polymorphism at the tissue-type plasminogen activator (t-PA) locus (–7351C>T), located within a GC-box in the retinoic acid (RA) and steroid hormone responsive t-PA enhancer. The aim of the present study was to functionally characterize this t-PA variant. Electrophoretic mobility shift assays (EMSAs) using crude nuclear extracts from human endothelial, HeLa, and NT2 neuronal cells revealed a 10-fold greater protein binding affinity to the wild-type C allele compared with the mutant T allele variant. Sp1 and Sp3 were identified as the GC-box binding proteins. Luciferase reporter assays showed that the C allele generated higher transcriptional activity after induction by RA, compared with the T allele variant. Further EMSAs showed that RA treatment enhanced Sp1/Sp3 binding to the GC-box. Formation of the Sp1/Sp3 containing complex was inhibited by anti–RA receptor (RAR) antibodies, suggesting that Sp1/Sp3 and RAR interact. The t-PA –7351C>T polymorphism is therefore functional at the level of transcription. The reduced binding affinity of Sp1/Sp3 to the T allele could explain our earlier observations of a reduced t-PA release and an increased risk of myocardial infarction in individuals carrying this allele.

Evaluating the effect of siRNA on SOX2OT expression in the human neuron-committed teratocarcinoma NT2 cell line.

Non-coding RNA elongated (lncRNAs) have recently attracted as molecules that regulate gene expression of the pluripotent properties (pluripotency) of stem cells. Recently our colleagues examined the role of one of these RNAs called SOX2OT in esophageal squamous cell carcinoma, and found a concomitant increase in its expression with some regulatory genes of cell proliferation. In the present study, using the design of suitable primers from SOX2OT gene, we investigated the effect of siRNA on expression of SOX2OT.

Chemical hypoxia in human pluripotent NT2 stem cell-derived neurons: Effect of hydroxamic acid and benzamide-based epigenetic drugs

Hypoxia-induced oxidative stress contributes to neuronal damage leading to many neurodegenerative disorders. Hypoxia promotes many downstream effectors such as hypoxia-inducible factor-1α (HIF-1α) in order to restore respiratory homeostasis due to low oxygen availability and increased ROS. Use of histone deacetylase (HDAC) inhibitors may modulate hypoxia-induced neuronal cell damage. In this study, we used two chemically diverse HDAC inhibitors to investigate their effect on hypoxia-exposed neuronal cells. Human pluripotent NT-2 stem cell-derived neuronal differentiated cells were exposed to CoCl2 pre-treatment for 6h to induce hypoxia, prior to supplementation of HDAC inhibitor (SAHA or MGCD0103). Treatment with HDAC inhibitor improved cell viability in hypoxia-induced neuronal cells. The increased HIF1α expression in hypoxia-induced neuronal cells was blunted by these HDAC inhibitors with a concomitant decrease in ROS production. CoCl2 treatment caused an increase in IL-1β, which was significantly inhibited by these HDAC inhibitors. Furthermore, apoptosis induced in these CoCl2 treated neuronal cells was mitigated by SAHA as well MGCD0103 suggesting that these HDAC inhibitors are capable of reducing cellular toxicity, inflammation and apoptosis, and thus, could be beneficial as therapeutic molecules for many neuropathological conditions.

Human tryptophanyl-tRNA synthetase is an IFN-γ-inducible entry factor for Enterovirus.

Enterovirus A71 (EV-A71) receptors that have been identified to date cannot fully explain the pathogenesis of EV-A71, which is an important global cause of hand, foot, and mouth disease and life-threatening encephalitis. We identified an IFN-γ-inducible EV-A71 cellular entry factor, human tryptophanyl-tRNA synthetase (hWARS), using genome-wide RNAi library screening. The importance of hWARS in mediating virus entry and infectivity was confirmed by virus attachment, in vitro pulldown, antibody/antigen blocking, and CRISPR/Cas9-mediated deletion. Hyperexpression and plasma membrane translocation of hWARS were observed in IFN-γ-treated semipermissive (human neuronal NT2) and cDNA-transfected nonpermissive (mouse fibroblast L929) cells, resulting in their sensitization to EV-A71 infection. Our hWARS-transduced mouse infection model showed pathological changes similar to those seen in patients with severe EV-A71 infection. Expression of hWARS is also required for productive infection by other human enteroviruses, including the clinically important coxsackievirus A16 (CV-A16) and EV-D68. This is the first report to our knowledge on the discovery of an entry factor, hWARS, that can be induced by IFN-γ for EV-A71 infection. Given that we detected high levels of IFN-γ in patients with severe EV-A71 infection, our findings extend the knowledge of the pathogenicity of EV-A71 in relation to entry factor expression upon IFN-γ stimulation and the therapeutic options for treating severe EV-A71-associated complications.

The Superiority of Sucrose Cushion Centrifugation to Ultrafiltration and PEGylation in Generating High-Titer Lentivirus Particles and Transducing Stem Cells with Enhanced Efficiency

Viral gene delivery is hailed as a great milestone in gene-based therapeutic approaches. The human immunodeficiency virus-derived lentiviral vectors (LVs) are advantageous in infecting both dividing and non-dividing cells leading to continuous expression of transgenes. A variety of protocols are available for concentration of LVs. We primarily generated our internal ribosome entry site (IRES)-based LVs. Virus titration and transduction efficiency were compared between various strategies that included sucrose cushion centrifugation (SCC), protein column ultrafiltration and polyethylene glycol precipitation. Among these approaches, SCC resulted in concentration of high-titer EGFP-expressing lentivirus (1.4±0.3×109TU/ml) with the lowest protein impurities. Further, we examined transduction strengths of our three methods on two challenging stem cells. Both human NT2 and mouse bone marrow-derived mesenchymal stem cells demonstrated high transduction using SCC of 65±2.8 and 49±0.8%, respectively. Finally, lentivirus particles harboring IRES-based transfer vectors of specific genes, concentrated by SCC, integrated into host genome. Taken together, development of cost-effective and efficient concentration strategies such as our SCC method is yet highly demanded to broaden the horizons of lentivirus application in clinical and translational research.

Roles of NTE protein and encoding gene in development and neurodevelopmental toxicity

Neuropathy Target Esterase (NTE) is a membrane protein codified by gene PNPLA6. NTE was initially discovered as a target of the so-called organophosphorus-induced delayed polyneuropathy triggered by the inhibition of the NTE-associated esterase center by neuropathic organophosphorus compounds (OPs). The physiological role of NTE might be related to membrane lipid homeostasis and seems to be involved in adult organisms in maintaining nervous system integrity. However, NTE is also involved in cell differentiation and embryonic development. NTE is expressed in embryonic and adult stem cells, and the silencing of Pnpla6 by interference RNA in D3 mouse cells causes significant alterations in several genetic pathways related to respiratory tube and nervous system formation, and in vasculogenesis and angiogenesis. The silencing of gene PNPLA6 in human NT2 cells at the beginning of neurodifferentiation causes severe phenotypic alterations in neuron-like differentiated cells; e.g. reduced electrical activity and the virtual disappearance of markers of neural tissue, synapsis and glia. These phenotypic effects were not reproduced when NTE esterase activity was inhibited by neuropathic OP mipafox instead of being silenced at the genetic level. Neuropathic OP chlorpyrifos seems able to induce neurodevelopmental alterations in animals. However, the effects of chlorpyrifos in the expression of biomarker genes of differentiation in D3 cells differ considerably from the effects induced by Pnpla6 silencing. In conclusion, available information suggests that PNPLA6 and/or the NTE protein play a role in early neurodifferentiation stages, although this role is not dependent upon the esterase NTE center. Therefore, impairments caused by OPs, such as chlorpyrifos, on neurodevelopment are not due to inhibition of NTE esterase enzymatic activity.

Regulation of Microglial Phagocytosis by RhoA/ROCK-Inhibiting Drugs.

Inflammation within the central nervous system (CNS) is a major component of many neurodegenerative diseases. The underlying mechanisms of neuronal loss are not fully understood, but the activation of CNS resident phagocytic microglia seems to be a significant element contributing to neurodegeneration. At the onset of inflammation, high levels of microglial phagocytosis may serve as an essential prerequisite for creating a favorable environment for neuronal regeneration. However, the excessive and long-lasting activation of microglia and the augmented engulfment of neurons have been suggested to eventually govern widespread neurodegeneration. Here, we investigated in a functional assay of acute inflammation how the small GTPase RhoA and its main target the Rho kinase (ROCK) influence microglial phagocytosis of neuronal debris. Using BV-2 microglia and human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA activation and microglial phagocytosis of neuronal cell fragments. Inhibition of the downstream effector ROCK with the small-molecule agents Y-27632 and Fasudil reduces the engulfment of neuronal debris and attenuates the production of the inflammatory mediator nitric oxide during stimulation with lipopolysaccharide. Our results support a therapeutic potential for RhoA/ROCK-inhibiting agents as an effective treatment of excessive inflammation and the resulting progression of microglia-mediated neurodegeneration in the CNS.

Carbon Monoxide Releasing Molecule-A1 (CORM-A1) Improves Neurogenesis: Increase of Neuronal Differentiation Yield by Preventing Cell Death.

Cerebral ischemia and neurodegenerative diseases lead to impairment or death of neurons in the central nervous system. Stem cell based therapies are promising strategies currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase (HO) activity. Administration of CO at low concentrations produces several beneficial effects in distinct tissues, namely anti-apoptotic and anti-inflammatory. Herein the CO role on modulation of neuronal differentiation was assessed. Three different models with increasing complexity were used: human neuroblastoma SH-S5Y5 cell line, human teratocarcinoma NT2 cell line and organotypic hippocampal slice cultures (OHSC). Cell lines were differentiated into post-mitotic neurons by treatment with retinoic acid (RA) supplemented with CO-releasing molecule A1 (CORM-A1). CORM-A1 positively modulated neuronal differentiation, since it increased final neuronal production and enhanced the expression of specific neuronal genes: Nestin, Tuj1 and MAP2. Furthermore, during neuronal differentiation process, there was an increase in proliferative cell number (ki67 mRNA expressing cells) and a decrease in cell death (lower propidium iodide (PI) uptake, limitation of caspase-3 activation and higher Bcl-2 expressing cells). CO supplementation did not increase the expression of RA receptors. In the case of SH-S5Y5 model, small amounts of reactive oxygen species (ROS) generation emerges as important signaling molecules during CO-promoted neuronal differentiation. CO’s improvement of neuronal differentiation yield was validated using OHSC as ex vivo model. CORM-A1 treatment of OHSC promoted higher levels of cells expressing the neuronal marker Tuj1. Still, CORM-A1 increased cell proliferation assessed by ki67 expression and also prevented cell death, which was followed by increased Bcl-2 expression, decreased levels of active caspase-3 and PI uptake. Likewise, ROS signaling emerged as key factors in CO’s increasing number of differentiated neurons in OHSC. In conclusion, CO’s increasing number of differentiated neurons is a novel biological role disclosed herein. CO improves neuronal yield due to its capacity to reduce cell death, promoting an increase in proliferative population. However, one cannot disregard a direct CO’s effect on specific cellular processes of neuronal differentiation. Further studies are needed to evaluate how CO can potentially modulate cell mechanisms involved in neuronal differentiation. In summary, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies.

Nitric oxide regulates antagonistically phagocytic and neurite outgrowth inhibiting capacities of microglia.

Traumatic injury or the pathogenesis of some neurological disorders is accompanied by inflammatory cellular mechanisms, mainly resulting from the activation of central nervous system (CNS) resident microglia. Under inflammatory conditions, microglia up-regulate the inducible isoform of NOS (iNOS), leading to the production of high concentrations of the radical molecule nitric oxide (NO). At the onset of inflammation, high levels of microglial-derived NO may serve as a cellular defense mechanism helping to clear the damaged tissue and combat infection of the CNS by invading pathogens. However, the excessive overproduction of NO by activated microglia has been suggested to govern the inflammation-mediated neuronal loss causing eventually complete neurodegeneration. Here, we investigated how NO influences phagocytosis of neuronal debris by BV-2 microglia, and how neurite outgrowth of human NT2 model neurons is affected by microglial-derived NO. The presence of NO greatly increased microglial phagocytic capacity in a model of acute inflammation comprising lipopolysaccharide (LPS)-activated microglia and apoptotic neurons. Chemical manipulations suggested that NO up-regulates phagocytosis independently of the sGC/cGMP pathway. Using a transwell system, we showed that reactive microglia inhibit neurite outgrowth of human neurons via the generation of large amounts of NO over effective distances in the millimeter range. Application of a NOS blocker prevented the LPS-induced NO production, totally reversed the inhibitory effect of microglia on neurite outgrowth, but reduced the engulfment of neuronal debris. Our results indicate that a rather simple notion of treating excessive inflammation in the CNS by NO synthesis blocking agents has to consider functionally antagonistic microglial cell responses during pharmaceutic therapy.

Abstract 4215: Small molecule compounds that destabilize stem cell factors

Abstract Cancer stem cells (CSCs) are thought to be present in malignant tumors at various proportions, and their phenotype can drift over time. Based on the current view, this elusive population of cancer cells is likely responsible for distant metastases, drug resistance and recurrence. However, the process by which such cell population is generated and maintained in a tumor mass has been unclear. Based on our previous study (PNAS Vol. 110: 6097-102 2013) and others, this process may involve the elevated expression of stem cell factors (SOX2, OCT4, NANOG, LIN28, KLF4, MYC, MYCN and those with equivalent functions). If so, it is conceivable that destabilization of these proteins would facilitate the eradication of CSC compartments in tumor mass. We have previously identified several small molecule compounds that confer the anti-growth effect on the stem cell-like neuroblastoma cells via screening of the Prestwick Chemical Library®, containing 1200 FDA-approved small molecules and of other selected experimental small molecules by using MTS assays. These compounds were structurally and biologically diverse molecules, including Alexidine (Alex), Benzethonium (Benz), Ketoconazole (Keto), Flubendazole (Flu), Auranofin (Aura), Nifuroxazide (Nif), Itraconazole (Itac) and JQ1. In this study, we investigated the effects of these selected small molecules on the expression of stem cell factors using the human teratocarcinoma cell lines NT2 and NCCIT as model systems, because these cells retain the expression of the majority of stem cell factors. The teratocarcinoma cell lines were treated with the small molecules at 0.5 to 10 μM for 24 hours and subjected to Western blot analysis. The expression of stem cell factors was down-regulated in NT2 cells by the several small molecules tested. The expression of SOX2 was down-regulated by Benz (5 μM), Flu (10 μM), Nif (10 μM), Aura (1 μM) and JQ1 (5 μM); the expression of OCT4 was down-regulated by Benz, Flu, Nif, Alex and JQ1; the NANOG expression was down-regulated by the all compounds tested; the expression of LIN28 was down-regulated by Benz, Flu and Aura; the expression of MYC/MYCN was down-regulated by Benz, Flu, Nif, Alex, Keto and JQ1. Interestingly, Auranofin treatment augmented the expression of MYC/MYCN, although the compound was growth suppressive to NT2 cells. Unlike the stem cell factors, the expression of LIN28B, which is the homologue of LIN28, was not affected by any of the compounds tested. A similar observation was made for NCCIT cells, but the effect of the compounds on the expression of stem cell factors appeared to be weaker. Collectively, the data suggest that small molecules identified via the process described could become useful lead chemicals for developing drug-like compounds that can target the most malignant stem cell-like cancer cells. Citation Format: Naohiko Ikegaki, Sarah Lomahan, Xao Tang. Small molecule compounds that destabilize stem cell factors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4215. doi:10.1158/1538-7445.AM2015-4215

Exogenous carbon monoxide improves neuronal differentiation: a near-death experience

Cerebral ischemic injuries and neurodegenerative disorders lead to death or impairment of neurons in the central nervous system. Application of stem cell based therapies, namely stimulation of endogenous neurogenesis or cell transplantation, are promising strategies and currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase. Although there is no published data reporting CO as a factor involved in stem cell differentiation, several evidences support this hypothesis. This gasotransmitter induces mitochondrial biogenesis, which is also broadly described to be involved in metabolic shifts during neuronal differentiation process. Likewise, CO-induced pathways can occur via generation of small amounts of ROS, which are also important signaling molecules in neuronal differentiation. The CO effect on modulation of neuronal differentiation is assessed in three different models with increasing complexity: human neuroblastome SH-S5Y5 cell line, human teratocarcinome NT2 cell line and hippocampal organotypic slice cultures (HOSC). CO does increase the final yield of post-mitotic neurons. During neuronal differentiation, CO promotes an increase on precursor cell proliferation and in parallel CO inhibits cell death. Furthermore, cell mitochondrial population is increased by CORM-A1 supplementation. Further work is needed for assessing the mechanisms underlying CO effects in neuronal differentiation, namely by targeting modulation of cellular metabolic pathways, redox alterations and autophagy related pathways. In conclusion, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies.

Enhanced neurite outgrowth of human model (NT2) neurons by small-molecule inhibitors of Rho/ROCK signaling.

Axonal injury in the adult human central nervous system often results in loss of sensation and motor functions. Promoting regeneration of severed axons requires the inactivation of growth inhibitory influences from the tissue environment and stimulation of the neuron intrinsic growth potential. Especially glial cell derived factors, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, and myelin in general, prevent axon regeneration. Most of the glial growth inhibiting factors converge onto the Rho/ROCK signaling pathway in neurons. Although conditions in the injured nervous system are clearly different from those during neurite outgrowth in vitro, here we use a chemical approach to manipulate Rho/ROCK signalling with small-molecule agents to encourage neurite outgrowth in cell culture. The development of therapeutic treatments requires drug testing not only on neurons of experimental animals, but also on human neurons. Using human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA (Ras homolog gene family, member A GTPase) activation and promotes neurite growth. Inhibition of the downstream effector Rho kinase by the drug Y-27632 results in a strong increase in neurite outgrowth. Conversely, activation of the Rho pathway by lysophosphatidic acid results in growth cone collapse and eventually to neurite retraction. Finally, we show that blocking of Rho kinase, but not RhoA results in an increase in neurons bearing neurites. Due to its anti-inflammatory and neurite growth promoting action, the use of a pharmacological treatment of damaged neural tissue with Ibuprofen should be explored.