Glucose Deprivation Conditions Research Articles

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.

Mi R -15a/15b Cluster Modulates Survival of Mesenchymal Stem Cells to Improve Its Therapeutic Efficacy of Myocardial Infarction.

BackgroundThe poor viability of transplanted mesenchymal stem cells (MSCs) hampers their therapeutic efficacy for ischemic heart disease. MicroRNAs are involved in regulation of MSC survival and function. The present study was designed to investigate the molecular effects of miR‐15a/15b on MSC survival, focusing on the role of vascular endothelial growth factor receptor 2.Methods and ResultsWe first harvested donor luc(Luciferase)‐MSCs (5×105) isolated from the luciferase transgenic mice with FVB background. Luc‐MSCs were transfected with miR‐15a/15b mimics or inhibitors and cultured under oxygen glucose deprivation condition for 12 hours to mimics the harsh microenvironment in infarcted heart; they were subjected to MTT (3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide?Thiazolyl Blue Tetrazolium Bromide) assay, bioluminescence imaging, quantitative reverse transcription–polymerase chain reaction, transferase‐mediated deoxyuridine triphosphate–digoxigenin nick‐end labeling assay, and flow cytometry. Furthermore, the levels of vascular endothelial growth factor receptor 2, protein kinase B, p(Phosphorylate)‐protein kinase B, Bcl‐2, Bax, and caspase‐3 proteins were available by Western blotting assay. In vivo, acute myocardial infarction was induced in 24 mice by coronary ligation, with subsequent receipt of Luc‐MSCs, Luc‐MSCs+miR‐15a/15b inhibitors, or PBS treatment. The therapeutic procedure and treatment effects were tracked and assessed using bioluminescence imaging and echocardiographic measurement. Next, ex vivo imaging and immunohistochemistry were conducted to verify the distribution of MSCs. We demonstrated that miR‐15a/15b targeted vascular endothelial growth factor receptor 2 to modulate MSC survival, possibly via phosphatidylinositol 3‐kinase/protein kinase B signaling pathway, which was proved by bioluminescence imaging, immunohistochemistry analysis, and echocardiographic measurement.ConclusionsLuc‐MSCs could be followed dynamically in vitro and in vivo by bioluminescence imaging, and the role of miR‐15a/b could be inferred from the loss of signals from luc‐MSCs. This finding may have practical clinical implications in miR‐15a/15b–modified MSC transplantation in treating myocardial infarction.

Targeted Inhibitory Effect of Nasopharyngeal Carcinoma Cells by Hre2.Grp78 Chimeric Promoter Regulating Fusion Gene TK/VP3.

Objective:To construct plasmids with Hre2.Grp78 chimeric promoter regulating fusion gene TK/VP3 and elaborate the effects of overexpressed TK/VP3 on nasopharyngeal carcinoma cells.Methods:Four plasmids were constructed, including pcDNA3.1-CMV-TK/VP3, pcDNA3.1-Hre2.TK/VP3, pcDNA3.1-Grp78.TK/VP3, and pcDNA3.1-Hre2.Grp78.TK/VP3. The human nasopharyngeal carcinoma cell line HNE1 cells were transfected with the 4 plasmids, respectively. Cell viabilities were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was conducted using flow cytometry analysis. The expression of TK, VP3, Grp78, and hypoxia-inducible factor 1α and apoptosis-related proteins was determined by real-time quantitative polymerase chain reaction and Western blotting.Results:The recombinant plasmids that could steadily overexpress TK and VP3 were successfully constructed. Expression of TK and VP3 in cells transfected with pcDNA3.1-Hre2.TK/VP3 and pcDNA3.1-Grp78.TK/VP3 was significantly higher than pcDNA3.1-CMV-TK/VP3, and expression in cells transfected with pcDNA3.1-Hre2.Grp78.TK/VP3 was the highest. Under glucose deprivation or hypoxia condition, Grp78 or hypoxia-inducible factor 1α was overexpressed so that expression of TK and VP3 was significantly upregulated, which could further inhibit cell proliferation and enhance cell apoptosis.Conclusion:We successfully constructed 4 plasmids with Hre2.Grp78 chimeric promoter regulating fusion gene TK/VP3, which could significantly inhibit the proliferation as well as enhance the apoptosis of nasopharyngeal carcinoma cells under glucose deprivation or hypoxia condition.

Activation of AMPK by metformin promotes renal cancer cell proliferation under glucose deprivation through its interaction with PKM2.

Metformin, a common therapeutics for type 2 diabetics, was recently demonstrated to possess antitumor activity in various cancer types. However, its therapy effect in renal cell carcinoma (RCC) still remains controversial. In this study, we found that metformin treatment in RCC cells lead to activation of AMPK, which suppressed the cell proliferation under normal condition, but enhanced cell proliferation under glucose deprivation (GD) condition. Depletion of AMPK by siRNA abolished the proliferation effect of MF under GD condition. Mechanistic investigations revealed that the effect of AMPK on cell proliferation under GD condition is dependent on its nuclear translocation. Moreover, the nuclear AMPK recruits PKM2 and β-Catenin to form a complex, which promotes the transcription of cell proliferation related genes, including CCND1 and c-Myc. Furthermore, depletion of PKM2 or β-Catenin abrogated the proliferative effects of metformin under GD condition. And inhibition of PKM2 also re-sensitized the A498 xenograft in response to metformin treatment. Together, our results suggested that combined of AMPK activation and PKM2 depletion or inhibition can achieve better therapeutic effect for RCC patients.

GPx1 is involved in the induction of protective autophagy in pancreatic cancer cells in response to glucose deprivation

Given the dense stroma and poor vascularization, access to nutrients is limited in the microenvironment of pancreatic ductal adenocarcinoma (PDA). PDA cells can efficiently recycle various metabolic substrates through the activation of different rescuing pathways, including the autophagy pathway. However, the precise roles of autophagy in cancer metabolism are not yet fully understood. In the present study, we first monitored the effect of glucose deprivation on autophagy and on the expression of glutathione peroxidase-1 (GPx1) in PDA cells under the glucose-free environment. Glucose starvation induced progressive autophagy activation in PDA cells via the activation of ROS/AMPK signaling. GPx1 degradation caused by glucose deprivation led to further ROS-dependent autophagy activation. Both GPx1 overexpression and autophagy inhibition sensitized cells to starvation-induced cell death through the activation of caspase-dependent apoptosis. Moreover, GPx1 may regulate glycolysis inhibition in PDA cells under glucose-deprived conditions. In summary, this study increases our understanding of the role of GPx1 in the induction of protective autophagy in PDA cells under extreme glucose starvation and may provide new therapeutic targets or innovative therapies.

Age-Dependent Decrease in Requirement for Glycolysis in Hematopoietic Stem Cells

Metabolic process governs cell cycle and differentiation status of hematopoietic stem cells (HSCs) through regulating the balance between oxidative phosphorylation and glycolysis. Cell cycle and self-renewal capacity dynamically changes during developmental and aging process; in early stage of life, HSCs are highly cycling and self-renew, whereas adult HSCs exhibit quiescent in cell-cycle, and gradually decline in its function as they age. How functional alteration of HSCs is coupled with metabolic program is an important issue for understanding the lifecourse of HSCs. Phosphoglycerate mutase 1 (Pgam1) is an enzyme catalyzing conversion of 3-phosphoglycerate into 2-phosphoglycerate. By deleting Pgam1, ATP production through glycolysis was abrogated while pentose-phosphate pathway (PPP) was spared, allowing us to examine dependence on glycolytic energy production without affecting nucleotide and NADPH production through PPP. Following inducible Pgam1 deletion in mice at various ages, the number of HSCs was measured. At an early life stage (5-week old), Pgam1 deletion resulted in decreased number of HSCs, suggesting an essential role of glycolysis for HSC maintenance. Notably, the number of HSCs in Pgam1-deleted mice restored in an age-dependent manner, with 2-year old mice exhibiting almost comparable number of HSCs with wild type mice. By contrast, upon HSC transplantation, the repopulation capacity of Pgam1-deficient HSCs from any stages of life was uniformly abrogated, indicating that glycolytic energy production is essential for stressed condition in which HSCs are forced to proliferate. Reverse transplantation of wild type HSCs into Pgam1-floxed recipients followed by conditional deletion in non-hematopoietic cells showed no phenotypes in hematopoietic lineages, suggesting a cell-autonomous requirement of Pgam1 for blood cells.To further determine the relevance of glycolysis with maintenance/proliferation balance, we performed in vitro culture utilizing newly-developed quiescence-maintaining condition with or without glucose. The number of HSCs under the quiescence-maintaining condition was irrelevant to the presence or absence of glucose, while cell number under the proliferating condition was markedly decreased in glucose-deprived condition. Furthermore, lymphoid-primed multipotential progenitors (LMPP) with marker phenotype of Flt3+ linage markers- Sca-1+ c-Kit+ cells, which show high cycling status in vivo, were highly dependent on glucose for survival in vitro. Collectively, proliferating cells require glucose irrespective to its cell type, while quiescent HSCs are less dependent on glycolytic energy production in line with age-dependent decrease of requirement for glycolysis in vivo.ATP concentration directly reflects ATP production/consumption rate inside cells. We utilized ATP-biosensor (GO-ATeam2)-transgenic mice to monitor ATP concentration in live HSCs under various metabolic environments in vitro. HSCs cultured in the proliferating condition exhibited gradual decrease in ATP concentration during 7-day culture, whereas in the quiescence-maintaining condition ATP was kept high. In the steady state, cycling multipotent progenitors (MPPs) exhibited lower ATP concentration than quiescent HSCs. These results suggest that quiescent state is characterized by lower ATP production/consumption rate than the proliferating cells. We then compared ATP concentration in young and old HSCs using the same system. The decrease of ATP under the proliferating condition was less prominent in old HSCs, while ATP concentration was comparable in the quiescence-maintaining condition. The result indicates the lower ATP production/consumption rate in old HSCs, presumably explaining lower dependence on glycolytic energy production in the steady state hematopoiesis.Altogether, we concluded that the dependence on glycolysis-driven ATP production derived from glucose decreased with age, underlying mechanism of which is explained by lower ATP consumption rate in old HSCs. DisclosuresOkamoto:Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co.,Ltd.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.:: Research Funding; Nippon Shinyaku Co., Ltd: Research Funding; Shionogi & Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Asahi Kasei Pharma Corp.:: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Teijin Pharma Limited: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

Infiltrative and drug-resistant slow-cycling cells support metabolic heterogeneity in glioblastoma.

Metabolic reprogramming has been described in rapidly growing tumors, which are thought to mostly contain fast-cycling cells (FCCs) that have impaired mitochondrial function and rely on aerobic glycolysis. Here, we characterize the metabolic landscape of glioblastoma (GBM) and explore metabolic specificities as targetable vulnerabilities. Our studies highlight the metabolic heterogeneity in GBM, in which FCCs harness aerobic glycolysis, and slow-cycling cells (SCCs) preferentially utilize mitochondrial oxidative phosphorylation for their functions. SCCs display enhanced invasion and chemoresistance, suggesting their important role in tumor recurrence. SCCs also demonstrate increased lipid contents that are specifically metabolized under glucose-deprived conditions. Fatty acid transport in SCCs is targetable by pharmacological inhibition or genomic deletion of FABP7, both of which sensitize SCCs to metabolic stress. Furthermore, FABP7 inhibition, whether alone or in combination with glycolysis inhibition, leads to overall increased survival. Our studies reveal the existence of GBM cell subpopulations with distinct metabolic requirements and suggest that FABP7 is central to lipid metabolism in SCCs and that targeting FABP7-related metabolic pathways is a viable therapeutic strategy.

Glucose Deprivation Enhances the Antiproliferative Effects of Oral Hypoglycemic Biguanides in Different Molecular Subtypes of Breast Cancer: an in Vitro Study.

Extensive in vitro studies have been conducted to evaluate the anticancer activity of oral hypoglycemic agents. Many of these studies experienced detrimental limitations, since they were conducted on cancer cells commonly grown in culture media consisting of extremely high concentrations of growth factors and glucose. The present study was aimed at exploring the antiproliferative effects of the commonly studied metformin and the less frequently reported phenformin oral hypoglycemic agents on different molecular subtypes of breast cancer under rich glucose and glucose deprived conditions. Our results indicate that under glucose deprived conditions, which better reflect the factual glucose-starved solid tumors in vivo, biguanides exert more antiproliferative activities against the three molecular subtypes of breast cancer cell lines examined in this study. In addition, the observed antiproliferative activities of biguanides appear to be mediated by apoptosis induction in breast cancer cells. This induction is significantly augmented under glucose deprived conditions.