Glucose Deprivation Conditions Research Articles

CBMT-27. ABERRANT AMINO ACID METABOLISM EXPOSE NOVEL FUNCTIONAL VULNERABILITIES IN GLIOBLASTOMA

Abstract Despite advances in molecularly characterizing glioblastoma, metabolic alterations driving its aggressive phenotype are only beginning to be recognized. Integrative cross-platform analyses coupling global metabolomic and gene expression profiling identified aberrant amino acid (AA) metabolism as a central node in glioblastoma. This metabolic phenotype was recapitulated in preclinical models and through a series of investigations designed to determine the biologic consequence of individual AA, we identified branched chain AA (BCAA) and glutamine as the only indispensable AA in glioblastoma, serving as the sole source of nucleotide pools and glutathione, respectively. Although molecularly and/or chemically perturbing these pathways resulted in cytotoxicity in glioblastoma, normal astrocytes demonstrated a similar response, suggesting therapeutic limitations in targeting these core metabolic pathways in cancer. As the glioblastoma microenvironment typically represents a nutrient-deprived state, we went on to determine the capacity of these cells to adapt to AA restricted conditions by only providing these cells with the above-identified indispensable AA. Intriguingly, glioblastoma cells had the unique ability to revert a state of metabolic dormancy. In addition to triggering a reversible proliferative arrest, this dormant phenotype displayed a near-complete shutdown of glycolysis that allowed these cells to adapt and maintain survival in glucose-deprived conditions and elicited profound resistance to ionizing radiation. Studies designed to systemically understand molecular underpinnings driving this unique metabolically dormant state uncovered a functional reliance upon mTOR/p21 signaling to maintain proliferative arrest in nutrient unfavorable conditions. Consistent with these findings, p21 expression was differentially expressed in the perinecrotic core of glioblastoma when compared to the peripheral edge in patient samples. Targeting this novel functional vulnerability through p21 inhibition, thereby, ‘forcing’ proliferation of these cells in nutrient unfavorable conditions, led to robust cytotoxicity specific to dormant cells and enhanced radiation response. Targeting functional vulnerabilities in otherwise therapeutically resistant cells represents a promising clinical strategy in glioblastoma.

Targeting Glycolysis with Epigallocatechin-3-Gallate Enhances the Efficacy of Chemotherapeutics in Pancreatic Cancer Cells and Xenografts.

Pancreatic cancer is a complex disease, in need of new therapeutic approaches. In this study, we explored the effect and mechanism of action of epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, alone and in combination with current chemotherapeutics on pancreatic cancer cell growth, focusing on glycolysis metabolism. Moreover, we investigated whether EGCG’s effect is dependent on its ability to induce reactive oxygen species (ROS). EGCG reduced pancreatic cancer cell growth in a concentration-dependent manner and the growth inhibition effect was further enhanced under glucose deprivation conditions. Mechanistically, EGCG induced ROS levels concentration-dependently. EGCG affected glycolysis by suppressing the extracellular acidification rate through the reduction of the activity and levels of the glycolytic enzymes phosphofructokinase and pyruvate kinase. Cotreatment with catalase abrogated EGCG’s effect on phosphofructokinase and pyruvate kinase. Furthermore, EGCG sensitized gemcitabine to inhibit pancreatic cancer cell growth in vitro and in vivo. EGCG and gemcitabine, given alone, reduced pancreatic tumor xenograft growth by 40% and 52%, respectively, whereas the EGCG/gemcitabine combination reduced tumor growth by 67%. EGCG enhanced gemcitabine’s effect on apoptosis, cell proliferation, cell cycle and further suppressed phosphofructokinase and pyruvate kinase levels. In conclusion, EGCG is a strong combination partner of gemcitabine reducing pancreatic cancer cell growth by suppressing glycolysis.

Effect of glucose deprivation on the expression of genes encoding glucocorticoid receptor and some related factors in ERN1-knockdown U87 glioma cells

The aim of the present study was to examine the effect of glucose deprivation on the expression of genes encoded glucocorticoid receptor (NR3C1) and some related proteins (NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme 1) for evaluation of their possible significance in the control of glioma growth through endoplasmic reticulum stress signaling mediated by IRE1 and glucose deprivation. The expression of NR3C1, NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3 genes in U87 glioma cells transfected by empty vector pcDNA3.1 (control cells) and cells without ERN1 signaling enzyme function (transfected by dnERN1) under glucose deprivation was studied by real time quantitative polymerase chain reaction. It was shown that the expression level of NR3C2, AHR, SGK1, SGK3, and NNT genes was up-regulated in control U87 glioma cells under glucose deprivation condition in comparison with the control cells growing with glucose. At the same time, the expression of NRIP1 gene is down-regulated in these glioma cells under glucose deprivation, but NR3C1 and ARHGAP35 genes was resistant to this experimental condition. We also showed that inhibition of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose deprivation condition. Thus, effect of glucose deprivation on the expression level of NR3C2, AHR, and SGK1 genes was significantly stronger in ERN1 knockdown U87 glioma cells since the expression of NNT gene was resistant to glucose deprivation condition. Moreover, the inhibition of ERN1 enzymatic activities in U87 glioma cells led to up-regulation of ARHGAP35 gene expression and significant down-regulation of the expression of SGK3 gene in response to glucose deprivation condition. Results of this study demonstrated that glucose deprivation did not change the expression level of NR3C1 gene but it significantly affected the expression of NR3C2, AHR, NRIP, SGK1, SGK3, and NNT genes in vector-transfected U87 glioma cells in gene specific manner and possibly contributed to the control of glioma growth since the expression of most studied genes in glucose deprivation condition was significantly dependent on the functional activity of IRE1 signaling enzyme.

Refinements of LC-MS/MS Spectral Counting Statistics Improve Quantification of Low Abundance Proteins

Mass spectrometry-based spectral count has been a common choice of label-free proteome quantification due to the simplicity for the sample preparation and data generation. The discriminatory nature of spectral count in the MS data-dependent acquisition, however, inherently introduces the spectral count variation for low-abundance proteins in multiplicative LC-MS/MS analysis, which hampers sensitive proteome quantification. As many low-abundance proteins play important roles in cellular processes, deducing low-abundance proteins in a quantitatively reliable manner greatly expands the depth of biological insights. Here, we implemented the Moment Adjusted Imputation error model in the spectral count refinement as a post PLGEM-STN for improving sensitivity for quantitation of low-abundance proteins by reducing spectral count variability. The statistical framework, automated spectral count refinement by integrating the two statistical tools, was tested with LC-MS/MS datasets of MDA-MB468 breast cancer cells grown under normal and glucose deprivation conditions. We identified about 30% more quantifiable proteins that were found to be low-abundance proteins, which were initially filtered out by the PLGEM-STN analysis. This newly developed statistical framework provides a reliable abundance measurement of low-abundance proteins in the spectral count-based label-free proteome quantification and enabled us to detect low-abundance proteins that could be functionally important in cellular processes.

Abstract 4745: Reversal by inhibiting XBP1 activation of glucose deprivation-induced tolerance to multiple anticancer therapies

Abstract Tumor microenvironment is harsh condition including low pH, hypoxia and nutrient deprivation. Cancer cells can switch their phenotypes and characteristics dependent on the microenvironment. Most previous researches have focused mainly on the effect of hypoxia on cancer cells plasticity and resistance to anticancer therapies. Despite the accumulation of emerging evidences, the effect of nutrient deficiency to multiple anticancer therapies is still unclear.In this study, human lung adenocarcinoma cells (A549), triple-negative breast cancer (MDA-MB-231), hepatocellular carcinoma cells (HepG2), pancreas adenocarcinoma cells (AsPC-1) were cultured in the medium containing low concentrations of glucose. Low sensitivity to cisplatin or taxol was observed in the cancer cells maintained for 28 weeks or so under the glucose-deprived condition. In addition, the multidrug tolerant cells were also insensitive to cytotoxic T cell (CTL)-based immunotherapy and had increased migration ability compared with control group under normal glucose condition. Reversion to normal glucose condition only for short term period (at least 4 days) made the multidrug tolerant cells susceptible to cisplatin-induced apoptotic cell death. In the unfolded protein response, BiP-XBP1 signaling was increased but PERK signaling was decreased in response to long-term glucose deprivation. Sensitivity to cisplatin treatment and CTL-based immunotherapy was restored by inhibiting XBP1 splicing in glucose deprivation-induced multidrug tolerant cells in various types of cancer.This study demonstrated that glucose deprivation-induced tolerance to multiple anti-cancer therapies is transient and reversible, and requires activation of IRE1-XBP1 pathway.This research was supported by the National Research Foundation of Korea (2018R1D1A1B07045297, 2016R1D1A1A02936945 and 2017R1D1A1B03036008). Citation Format: Mini Jeong, Serin Jo, Serkin Park, Jeongwon Sohn, Yun Gyu Park. Reversal by inhibiting XBP1 activation of glucose deprivation-induced tolerance to multiple anticancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4745.

Abstract 5259: Fatty acid oxidation represents a metabolic vulnerability in glioblastoma in nutrient-deprived conditions

Abstract Glioblastoma represents an aggressive, primary brain tumor with limited treatment options. We performed an integrative, cross-platform analysis coupling global metabolomics and gene expression profiling in >100 patient-derived gliomas to begin to understand the diverse metabolic programs driving the aggressive phenotype of this malignancy. Alterations in fatty acid β-oxidation (FAO) emerged as a key metabolic node differentiating glioblastoma from low-grade astrocytoma, as demonstrated by an accumulation of acylcarnitines. Metabolic heterogeneity was observed within glioblastoma that could further define tumors as FAO ‘high’ and ‘low’. Integrative analyses identified these metabolic subtypes to be enriched with mesenchymal (MES) and proneural (PN) glioblastoma subtypes, respectively. These findings were metabolomically and functionally recapitulated in molecular subtype-specific preclinical models. Analysis of gene expression profiles from these lines uncovered an orchestrated transcriptional program designed to promote fatty acid uptake, activation, and mitochondrial oxidation. Studies designed to determine the biologic consequence of enhanced FAO in glioblastoma only identified a role in glucose-deprived conditions, where it served as a vital, alternate source for ATP synthesis. Based on these findings, we tested the hypothesis that dual targeting of glycolysis and FAO would elicit energetic stress-mediated cell death in glioblastoma. Accordingly, the glycolysis inhibitor 2DG and the FAO inhibitor etomoxir only demonstrated anti-proliferative activity in MES glioblastoma cell lines when used as single agents in vitro, while the combination resulted in robust, necroptosis-mediated cell death. Synergistic anti-tumor activity was observed following combined glycolysis and FAO inhibition when extended in vivo in an orthotopic model. Collectively, our findings suggest FAO provides metabolic plasticity in MES glioblastoma, allowing these cells to adapt to nutrient-deprived microenvironments. Combinatorial strategies designed to inhibit glycolysis and FAO represents an attractive therapeutic approach in glioblastoma. Citation Format: Shiva Kant, Antony Dayalan, Pravin Kesarwani, Prakash Chinnaiyan. Fatty acid oxidation represents a metabolic vulnerability in glioblastoma in nutrient-deprived conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5259.

LIMS1 Promotes Pancreatic Cancer Cell Survival under Oxygen-Glucose Deprivation Conditions by Enhancing HIF1A Protein Translation.

Oxygen and glucose deprivation is a common feature of the solid tumor. Regulatory network underlying the adaptation of cancer cells to the harsh microenvironment remains unclear. We determined the mechanistic role of LIM and senescent cell antigen-like-containing domain protein 1 (LIMS1) in cancer cell survival under oxygen-glucose deprivation conditions. The expression level of LIMS1 was determined by IHC staining and analyzing the mRNA expression profiles from The Cancer Genome Atlas of three human solid tumors. Roles of LIMS1 in cancer cell metabolism and growth were determined by molecular and cell biology methods. A jetPEI nanocarrier was used as the vehicle for anti-LIMS1 siRNAs in mouse models of cancer therapeutics. LIMS1 expression was drastically elevated in pancreatic ductal adenocarcinoma (PDAC). High LIMS1 level was associated with advanced TNM stage and poor prognosis of patients with tumor. Increased LIMS1 expression was pivotal for tumor cells to survive in the oxygen-glucose deprivation conditions. Mechanistically, LIMS1 enhanced GLUT1 expression and membrane translocation, which facilitated tumor cell adaptation to the glucose deprivation stress. Furthermore, LIMS1 promoted HIF1A protein translation by activating AKT/mTOR signaling, while hypoxia-inducible factor 1 (HIF1) transactivated LIMS1 transcription, thus forming a positive feedback loop in PDAC cell adaptation to oxygen deprivation stress. Inhibition of LIMS1 with jetPEI nanocarrier-delivered anti-LIMS1 siRNAs significantly increased cell death and suppressed tumor growth. LIMS1 promotes pancreatic cancer cell survival under oxygen-glucose deprivation conditions by activating AKT/mTOR signaling and enhancing HIF1A protein translation. LIMS1 is crucial for tumor adaptation to oxygen-glucose deprivation conditions and is a promising therapeutic target for cancer treatment.

2-(4-Methoxyphenyl)Ethyl-2-Acetamido-2-Deoxy-β-D-Pyranoside Exerts a Neuroprotective Effect through Regulation of Energy Homeostasis and O-GlcNAcylation.

Dysfunction of energy metabolism exerts a central role in triggering neuron death following cerebral ischemia. Neuronal energy metabolism is highly dependent on glucose. O-GlcNAcylation, a post-translational modification, is a novel pro-survival pathway that modulates glucose homeostasis in ischemic stroke. Here, we explored whether activation O-GlcNAcylation and maintaining energy homeostasis mediated the neuroprotective effect of 2-(4-methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside, a synthetic salidroside analog (named SalA-4g) which was previously developed in our laboratory. For in vivo analyses, SalA-4g improved the outcome after transient middle cerebral artery occlusion (MCAO). 18F-FDG PET/MRI indicated that SalA-4g accelerated the recovery of energy metabolism in the ipsilateral hippocampus in MCAO rats. In vitro analyses showed that glucose uptake was markedly increased, and O-GlcNAcylation was also activated by SalA-4g in hippocampal neurons under both normal and oxygen glucose deprivation (OGD) conditions. Moreover, SalA-4g exerted obvious neuroprotective effects in hippocampal neurons against moderate OGD injury. Our study indicates that boosting a pro-survival pathway-GlcNAcylation-and regulating energy homeostasis are important biochemical mechanisms responsible for SalA-4g neuroprotection.

Metabolic stress in cancer cells induces immune escape through a PI3K-dependent blockade of IFN\u03b3 receptor signaling

BackgroundT-cell mediated immunotherapy brought clinical success for many cancer patients. Nonetheless, downregulation of MHC class I antigen presentation, frequently occurring in solid cancers, limits the efficacy of these therapies. Unraveling the mechanisms underlying this type of immune escape is therefore of great importance. We here investigated the immunological effects of metabolic stress in cancer cells as a result of nutrient deprivation.MethodsTC1 and B16F10 tumor cell lines were cultured under oxygen- and glucose-deprivation conditions that mimicked the tumor microenvironment of solid tumors. Presentation of peptide antigens by MHC class I molecules was measured by flow cytometry and via activation of tumor-specific CD8 T cell clones. The proficiency of the IFNy-STAT1 pathway was investigated by Western blots on phosphorylated proteins, transfection of constitutive active STAT1 constructs and qPCR of downstream targets. Kinase inhibitors for PI3K were used to examine its role in IFNy receptor signal transduction.ResultsCombination of oxygen- and glucose-deprivation resulted in decreased presentation of MHC class I antigens on cancer cells, even in the presence of the stimulatory cytokine IFNy. This unresponsiveness to IFNy was the result of failure to phosphorylate the signal transducer STAT1. Forced expression of constitutive active STAT1 fully rescued the MHC class I presentation. Furthermore, oxygen- and glucose-deprivation increased PI3K activity in tumor cells. Pharmacological inhibition of this pathway not only restored signal transduction through IFNy-STAT1 but also improved MHC class I presentation. Importantly, PI3K inhibitors also rendered tumor cells sensitive for recognition by CD8 T cells in culture conditions of metabolic stress.ConclusionsThese data revealed a strong impact of metabolic stress on the presentation of tumor antigens by MHC class I and suggest that this type of tumor escape takes place at hypoxic areas even during times of active T cell immunity and IFNy release.

A Novel Role of VEGFC in Cerebral Ischemia With Lung Injury.

Cerebral ischemia (CI) is a severe brain injury resulting in a variety of motor impairments combined with secondary injury in remote organs, especially the lung. This condition occurs due to insufficient blood supply to the brain during infancy. However, it has a molecular linkage that needs to be thoroughly covered. Here, we report on the role of vascular endothelial growth factor C (VEGFC) in lung injury induced by CI. The middle cerebral artery occlusion (MCAO) was depended to establish the animal model of CI. Rats were used and brain ischemia was confirmed through TTC staining. Serum was used for protein chip analysis to study the proteomic interaction. Immunohistochemistry analyses were used to quantify and locate the VEGFC in the lung and brain. The role of VEGFC was detected by siVEGFC technology in SY5Y, HUCEV, and A549 cell lines, under normal and oxygen glucose deprivation (OGD) conditions in vitro. As a result, the TTC staining demonstrated that the model of brain ischemia was successfully established, and MPO experiments reported that lung damage was induced in MCAO rats. VEGFC levels were up-regulated in serum. On the other hand, immunohistochemistry showed that VEGFC increased significantly in the cytoplasm of neurons, the endothelium of small trachea and the lung cells of CI animals. On a functional level, siVEGFC effectively inhibited the proliferation of SY5Y cells and decreased the viability of HUVEC cells in normal cell lines. But under OGD conditions, siVEGFC decreased the growth of HUVEC and increased the viability of A549 cells, while no effect was noticed on SYSY cells. Therefore, we confirmed the different role of VEGFC played in neurons and lung cells in cerebral ischemia-reperfusion injury. These findings may contribute to the understanding the molecular linkage of brain ischemia and lung injury, which therefore provides a new idea for the therapeutic approach to cerebral ischemia-reperfusion.

Lactic Acid Promotes Cell Survival by Blocking Autophagy of B16F10 Mouse Melanoma Cells under Glucose Deprivation and Hypoxic Conditions.

In solid tumors, cancer cells are exposed to microenvironment stress, including hypoxia and insufficient nutrients. An acidic microenvironment in tumors is facilitated by the increase in synthesis of lactic acid; this is known as Warburg effect. We previously showed that B16F10 melanoma cells were induced autophagic cell death by glucose-deprivation stress, and lactic acid suppressed the cell death through the inhibition of autophagy. In this study, effects of lactic acid on cell death of B16F10 cells under hypoxic and glucose-depleted double stress conditions were investigated. The double stress promoted autophagic cell death earlier than glucose-depleted stress alone. Lactic acid repressed the double stress-induced cell death by inhibiting autophagy. These results suggest that lactic acid serves for cell survival under microenvironmental stress conditions in B16F10 melanoma cells.

A chemical toolbox for the study of bromodomains and epigenetic signaling

Bromodomains (BRDs) are conserved protein interaction modules which recognize (read) acetyl-lysine modifications, however their role(s) in regulating cellular states and their potential as targets for the development of targeted treatment strategies is poorly understood. Here we present a set of 25 chemical probes, selective small molecule inhibitors, covering 29 human bromodomain targets. We comprehensively evaluate the selectivity of this probe-set using BROMOscan and demonstrate the utility of the set identifying roles of BRDs in cellular processes and potential translational applications. For instance, we discovered crosstalk between histone acetylation and the glycolytic pathway resulting in a vulnerability of breast cancer cell lines under conditions of glucose deprivation or GLUT1 inhibition to inhibition of BRPF2/3 BRDs. This chemical probe-set will serve as a resource for future applications in the discovery of new physiological roles of bromodomain proteins in normal and disease states, and as a toolset for bromodomain target validation.

Inhibition of miRNA-27b enhances neurogenesis via AMPK activation in a mouse ischemic stroke model.

Stroke is a leading cause of death and disability, but treatment options remain limited. Recent studies have suggested that cerebral ischemia‐induced neurogenesis plays a vital role in post‐stroke repair. Overactivation of AMP‐activated protein kinase (AMPK), a master sensor of energy balance, has been reported to exacerbate neuron apoptosis, but the role of chronic AMPK stimulus in post‐stroke recovery remains unclear. MicroRNAs have emerged as regulators of neurogenesis and have been reported to be involved in neurological function. In this study, we verified that miR‐27b directly targets AMPK and inhibits AMPK expression. In cultured neural stem cells, miR‐27b inhibitor improved proliferation and differentiation via the AMPK signaling pathway, but did not have an obvious effect on cell viability under oxygen and glucose deprivation conditions. In a mouse middle cerebral artery occlusion model, administration of miR‐27b inhibitor significantly enhanced behavioral function recovery and spatial memory. Up‐regulation of neurogenesis was observed both in the subventricular zone and in the hippocampal dentate gyrus. Collectively, our data suggest that miR‐27b inhibition promotes recovery after ischemic stroke by regulating AMPK activity. These findings may facilitate the development of novel therapeutic strategies for stroke.

Inhibition of EGFR signaling with Spautin-1 represents a novel therapeutics for prostate cancer

BackgroundProstate cancer (PCa) remains a challenge worldwide. Due to the development of castration-resistance, traditional first-line androgen deprivation therapy (ADT) became powerlessness. Epidermal growth factor receptor (EGFR) is a well characterized therapeutic target to treat colorectal carcinoma and non-small cell lung cancer. Increasing studies have unraveled the significance of EGFR and its downstream signaling in the progression of castration-resistant PCa.MethodMTS, colony formation and Edu staining assays were used to analyze the cell proliferation of PCa cells. Flow cytometry was used to analyze PCa cell cycle distribution and cell apoptosis. Western blot was used to measure the expression of key proteins associated with cell cycle progression, apoptosis and EGFR signaling pathways. Transfection of exogenous small interfering RNA (siRNA) or plasmid was used to intervene specific gene expression. Nude mouse model was employed to test the in vivo effect of Spautin-1.ResultsThe current study reveals that Spautin-1, a known inhibitor of ubiquitin-specific peptidase 10 (USP10) and USP13, inhibits EGFR phosphorylation and the activation of its downstream signaling. Inhibition of EGFR signaling induced by Spautin-1 leads to cell cycle arrest and apoptosis of PCa in a USP10/USP13 independent manner. The application of Spautin-1 reduces the expression of glucose transporter 1 (Glut1) and dramatically induces cell death under glucose deprivation condition. In vivo experiments show a potent anti-tumor effect of Spautin-1 alone and in combination with Enzalutamide.ConclusionThis study demonstrates the therapeutic potential of EGFR signaling inhibition by the use of Spautin-1 for PCa treatment.

Intake of psyllium seed husk reduces white matter damage in a rat model of chronic cerebral hypoperfusion

Vascular dementia (VaD) develops through a pre-VaD step during which blood vessels narrow due to atherosclerosis attributed to risk factors, including hyperlipidemia. This is followed by a VaD progression step during which inadequate blood supply results in white matter damage and consequent cognitive impairment. Furthermore, administration of arabinoxylan attenuated white matter damage in a rat model of VaD. Thus, we hypothesized that consumption of psyllium seed husk (PSH), containing a high level of arabinoxylan (~60%), could inhibit the VaD progression step. To test this hypothesis, rats were supplemented with PSH at various dosages for 33 days in a model of bilateral common carotid artery occlusion. PSH supplementation decreased astrocytic and microglial activation in the optic tract (opt) and, consequently, attenuated white matter damage in the opt. Attenuation of white matter damage resulted in improvement of pupillary light reflex, an indicator reflecting intactness of the opt. In addition, PSH treatment improved survival of glial cells cultured under hypoxic and glucose-deprived conditions by inhibiting both apoptosis and autophagy. These findings indicate that PSH consumption can inhibit the VaD progression step through a decrease of white matter damage. Therefore, these results support our hypothesis that PSH consumption prevents VaD due to the high arabinoxylan content in the rat model. PSH consumption has already been shown to reduce risk factors, thereby inhibiting the pre-VaD step. Consequently, PSH consumption can contribute to the prevention of VaD by inhibiting both the pre-VaD and VaD progression steps. In conclusion, our rat study suggests that PSH might be a candidate to explore its use in clinical studies to reduce VaD.

Anti‐edematous effect of neurolysin in ischemic stroke

During ischemic stroke brain injury occurs due to oxygen and nutrient deficiency, which initiates secondary injury cascades, including intracellular sodium and calcium accumulation, release of neurotoxic dopamine and glutamate, ion gradient dissipation, accumulation of reactive oxygen species, mitochondrial dysfunction, and excitotoxicity. All of these pathophysiologic processes contribute to cellular and vasogenic edema, which can be a life‐threatening effect of stroke and the primary reason for patient mortality after stroke.Neuropeptides similar to neurotransmitters play a significant role in different signaling pathways. They act on the cellular surface after releasing into the extracellular spaces and their functions are terminated by endogenous neuropeptidases. Neurolysin (Nln), a zinc metallopeptidase, has been reported to be up‐regulated during stroke and plays a crucial role in the improvement of stroke outcomes. Functional importance of this enzyme in post‐stroke brain repair is due to its capability in processing a diverse group of neuropeptides, such as substance P, bradykinin and neurotensin, which all have distinct functions in the development of inflammation, excitotoxicity, and cell death after stroke. The objective of our present study was to investigate the role of Nln through the deactivation of the above neuropeptide substrates and subsequent reduction of brain edema after a model of ischemic stroke.For an ex‐vivo model of ischemia, an acute brain slice model has been used. Briefly, 5–7 month old CD1 male mouse brain was extracted and 400 μm slices were exposed to 1 hour of oxygen glucose deprivation (OGD) condition followed by reperfusion (R) for 2 hours in the presence and absence of Nln substrates, i.e., substance P, bradykinin and neurotensin. To study the role of Nln, Nln substrates were co‐incubated with Nln during OGD/R. We observed that the 21% water content in slices increased when exposed to OGD/R compared to normoxia which further increased in the presence of Nln substrates during reperfusion, suggesting the role of Nln substrate in edema formation. Interestingly, the presence of rNln attenuated the enhanced edema formation by Nln substrates. These results indicated the role of Nln in reducing brain edema during ischemic stroke. Furthermore, to confirm that the effect of Nln is through deactivation of neuropeptides, we confirmed the hydrolysis of neuropeptides in the presence of Nln by using the LC‐MS/MS method.These results imply that cellular edema formation after stroke can be worsened by common Nln substrates (substance P, bradykinin, and neurotensin), which all have well‐documented neurovascular effects that exacerbate brain edema formation, and treatment with rNln can significantly attenuate cerebral swelling. This study is instrumental in explaining the possible role of Nln substrates and their deleterious effect in edema formation during a stroke and further studies are needed to study the positive role of Nln activation in stroke recovery and reduction of brain edema.Support or Funding InformationThis research was supported by NIH R01 NS106879 to VK and TA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Myo‐inositol in Ischemic Stroke Outcome in a Preclinical Tobacco Smoke Exposed Mouse Model

Tobacco smoking is a preventable risk factor for ischemic stroke in both males and females and contributes up to 14% of all stroke deaths. It has been reported that an increase in brain edema after ischemic stroke worsens long‐term clinical outcome, such as seen in tobacco smokers. Myo‐inositol (MI) is a major organic “non‐perturbing” osmolyte that promotes cell membrane stability and also a precursor for phosphoinositide signaling pathway. Our previous work reported that MI attenuated brain infarct and edema ratio in an ischemic stroke model coupled to preclinical diabetes and suggests that MI and MI transporters provide neuroprotection during/following stroke both in non‐diabetic and diabetic conditions through protection from brain edema. Since our lab has shown that tobacco smoke exposure worsens stroke edema similarly to diabetic conditions, the aim of the present study was to assess the neuroprotective effects of MI associated with ischemic mice exposed to tobacco smoke. In this study, we also investigated the role of MI transporters during stroke and post‐stroke outcomes especially related to the role of astrocytes cells, which are the most abundant glial cells in the brain and play an important role in post‐stroke reorganization, and indirectly protecting neuronal metabolic support. Primary mouse astrocytes were incubated with tobacco smoke extract (TSE) overnight prior for in vitro studies. Toxicity of TSE was measured using MTT assay after incubating astrocytes with TSE 6hr and 24hr. Therapeutic efficacy of MI was evaluated in a transient‐middle cerebral artery occlusion (tMCAO) mouse model after smoking 3R4F research cigarettes for 10 days. Uptake studies showed an increase of MI uptake increased with longer incubation of TSE in normoxic conditions. Additionally, it was also observed that uptake of MI increased during Oxygen Glucose Deprivation(OGD) conditions with prior exposure of TSE. These results are in support with our previous reports, where we found an increased expression of MI transports, Sodium Myo‐inositol Transporter 1 (SMIT1) and Sodium Dependent Glucose Transporter 6 (SGLT6) during OGD exposure. Preliminary animal studies using middle cerebral artery occlusion mouse model for in vivo stroke studies, demonstrated that post‐stroke administration of MI attenuated infarction size and edema ratios in tobacco smoke exposed animals compared to non‐tobacco smoked ischemic animals and also improved motor function in behavioral tests. Taken together with previous work, this study provides an insight into how MI may play a neuroprotective role in ischemic stroke injury and could significantly improve stroke outcome, especially in tobacco smoke populations. Further studies are required to understand the neuroprotective mechanisms of MI in ischemic stroke injury in reducing brain edema.Support or Funding InformationR01NS076012 and R01DA029121This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of O-Glcnacylated Proteins in Trypanosoma cruzi.

Originally an anthropozoonosis in the Americas, Chagas disease has spread from its previous borders through migration. It is caused by the protozoan Trypanosoma cruzi. Differences in disease severity have been attributed to a natural pleomorphism in T. cruzi. Several post-translational modifications (PTMs) have been studied in T. cruzi, but to date no work has focused on O-GlcNAcylation, a highly conserved monosaccharide-PTM of serine and threonine residues mainly found in nucleus, cytoplasm, and mitochondrion proteins. O-GlcNAcylation is thought to regulate protein function analogously to protein phosphorylation; indeed, crosstalk between both PTMs allows the cell to regulate its functions in response to nutrient levels and stress. Herein, we demonstrate O-GlcNAcylation in T. cruzi epimastigotes by three methods: by using specific antibodies against the modification in lysates and whole parasites, by click chemistry labeling, and by proteomics. In total, 1,271 putative O-GlcNAcylated proteins and six modification sequences were identified by mass spectrometry (data available via ProteomeXchange, ID PXD010285). Most of these proteins have structural and metabolic functions that are essential for parasite survival and evolution. Furthermore, O-GlcNAcylation pattern variations were observed by antibody detection under glucose deprivation and heat stress conditions, supporting their possible role in the adaptive response. Given the numerous biological processes in which O-GlcNAcylated proteins participate, its identification in T. cruzi proteins opens a new research field in the biology of Trypanosomatids, improve our understanding of infection processes and may allow us to identify new therapeutic targets.

Structure-Activity Relationship of Biakamide, Selective Growth Inhibitors under Nutrient-Starved Condition from Marine Sponge.

The tumor microenvironment is considered as one of the important targets for anticancer drug discovery. In particular, nutrient deficiency may be observed in tumor microenvironment; biakamides A-D (1-4) isolated from marine sponge Petrosaspongia sp. as growth inhibitors against cancer cells adapted to glucose-deprived conditions have potential as new drugs and tools for elucidating adaptation mechanisms to these conditions. In this paper, we investigated structure-activity relationship (SAR) of biakamide to create easily accessible analog and gain insights about participation of the substructures to growth-inhibitory activity toward development of anticancer drug. This work revealed that 14,15-dinor-biakamide C (5), which is easily accessible, has similar activity to natural biakamide C (3). In addition, detailed SAR study showed the terminal acyl chain is important for interacting with target molecule and amide part including thiazole ring has acceptability to convert structures without losing activity.

Effects of 2-(2-Chlorophenyl)ethylbiguanide on ERAD Component Expression in HT-29 Cells Under a Serum- and Glucose-Deprived Condition.

We recently characterized the cytotoxic action of a novel phenformin derivative, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on HT-29 cells under a serum- and glucose-deprived condition and found that 2-Cl-Phen attenuated ATF4 and GRP78, typical downstream targets of the unfolded protein response (UPR), together with c-Myc protein expression in a transcriptional and posttranscriptional manner. In the current study, we focused on the expression of ER-associated protein degradation (ERAD) components after treatment with 2-Cl-Phen under a serum- and glucose-deprived condition. Among nine ER-localizing factors regulating protein quality control within the ER, the amounts of Herp, GRP78, GRP94, and OS9 proteins were significantly downregulated by treatment with 2-Cl-Phen. In particular, replacement of the culture medium with the serum- and glucose-deprived medium induced the expression of Herp protein at the early phase. This increase in Herp protein was accompanied by an increase in its mRNA, and its induction was significantly dampened by 2-Cl-Phen. However, cotreatment with a proteasome inhibitor, MG132, restored Herp expression only to a limited extent. Taken together, these results show that 2-Cl-Phen changed the expression of several ERAD components, especially by transcriptional inhibition of Herp induction by 2-Cl-Phen when it occurred at an early phase, and this finding provides new insights into understanding the mechanisms of 2-Cl-Phen-mediated cytotoxicity.