Drop In ATP Research Articles

A New Quinone-Based Inhibitor of Mitochondrial Complex I in D-Conformation, Producing Invasion Reduction and Sensitization to Venetoclax in Breast Cancer Cells.

Mitochondrial Complex I plays a crucial role in the proliferation, chemoresistance, and metastasis of breast cancer (BC) cells. This highlights it as an attractive target for anti-cancer drugs. Using submitochondrial particles, we identified FRV-1, an ortho-carbonyl quinone, which inhibits NADH:duroquinone activity in D-active conformation and reduces the 3ADP state respiration dependent on Complex I, causing mitochondrial depolarization, ATP drop, increased superoxide levels, and metabolic remodeling towards glycolysis in BC cells. Introducing methyl groups at FRV-1 structure produced analogs that acted as electron acceptors at the Complex I level or increased the inhibitory effect of FCCP-stimulated oxygen consumption rate, which correlated with their redox potential, but increased toxicity on RMF-621 human breast fibroblasts was observed. FRV-1 was inactive in the naphthoquinone oxidoreductase 1 (NOQ1)-positive BC cell line, MCF7, but the sensitivity was recovered by dicoumarol, a NOQ1 inhibitor, suggesting that FRV-1 is a NOQ1 substrate. Importantly, FRV-1 selectively inhibited the proliferation, migration, and invasion of NQO1 negative BC cell, MDA-MB-231, in an OXPHOS- and ROS-dependent manner and sensitized it to the BH3 mimetic drug venetoclax. Overall, FRV-1 is a novel Complex I inhibitor in D-active conformation, blocking possibly the re-activation to A-state, producing selective anti-cancer effects in NQO1-negative BC cell lines.

The αMSH-Dependent PI3K Pathway Supports Energy Metabolism, via Glucose Uptake, in Melanoma Cells.

Stimulation of melanocytes and murine melanoma cells with αMSH plus the PI3K inhibitor LY294002 resulted in ROS increase, oxidative DNA damage, and pigment retention. We performed cellular and molecular biology assays (Western blot, FACS, immunofluorescence analysis, scratch assay) on murine and human melanoma cells. Treatment with αMSH plus LY294002 altered cortical actin architecture. Given that cytoskeleton integrity requires energy, we next evaluated ATP levels and we observed a drop in ATP after exposure to αMSH plus LY294002. To evaluate if the αMSH-activated PI3K pathway could modulate energy metabolism, we focused on glucose uptake by analyzing the expression of the Glut-1 glucose translocator. Compared with cells treated with αMSH alone, those exposed to combined treatment showed a reduction of Glut-1 on the plasma membrane. This metabolic alteration was associated with changes in mitochondrial mass. A significant decrease of the cell migratory potential was also observed. We demonstrated that the αMSH-dependent PI3K pathway acts as a regulator of energy metabolism via glucose uptake, influencing the actin cytoskeleton, which is involved in melanosome release and cell motility. Hence, these results could constitute the basis for innovative therapeutical strategies.

Mitochondrial dysfunction triggers actin polymerization necessary for rapid glycolytic activation.

Mitochondrial damage represents a dramatic change in cellular homeostasis. One rapid response is perimitochondrial actin polymerization, termed acute damage-induced actin (ADA). The consequences of ADA are not understood. In this study, we show evidence suggesting that ADA is linked to rapid glycolytic activation upon mitochondrial damage in multiple cells, including mouse embryonic fibroblasts and effector CD8+ T lymphocytes. ADA-inducing treatments include CCCP, antimycin, rotenone, oligomycin, and hypoxia. The Arp2/3 complex inhibitor CK666 or the mitochondrial sodium-calcium exchanger (NCLX) inhibitor CGP37157 inhibits both ADA and the glycolytic increase within 5 min, supporting ADA's role in glycolytic stimulation. Two situations causing chronic reductions in mitochondrial ATP production, mitochondrial DNA depletion and mutation to the NDUFS4 subunit of complex 1 of the electron transport chain, cause persistent perimitochondrial actin filaments similar to ADA. CK666 treatment causes rapid mitochondrial actin loss and a drop in ATP in NDUFS4 knock-out cells. We propose that ADA is necessary for rapid glycolytic activation upon mitochondrial impairment, to re-establish ATP production.

Acute Sensitivity of V. cholerae to Phosphate Starvation: A Possible Case of Futile Metabolism.

Multi-nutrient starvation profiles are emerging as an essential feature of human pathogen studies. In the present work, we attempted to understand why V. cholerae cannot survive in the growth media that includes carbon and nitrogen but lacks phosphate. However, it can survive starvation in artificial seawater without all three major nutrients (carbon, nitrogen, and phosphate). V. cholerae survival was critically affected below ten mM of phosphate content in the media. Interestingly, the survival of the bacteria in low phosphate conditions improved when cultured under sub-optimal growth conditions, sub-optimal C: N ratio, non-metabolizable nutrient sources, and activation of stringent response. The phosphate starved cells had low ATP levels and high NADH levels; A quick drop in ATP, coupled with impaired redox potential, may lead to cell death. We also observed the acute sensitivity to phosphate limitation among the other members of the genus Vibrio. Among those, V. vulnificus and V. cholerae showed the highest sensitivity (< 0.1%). Among the members of γ-proteobacteria, E. coli and C. sakazaki and S. Typhimurium showed the lowest sensitivity (10%), and the A. hydrophila and V. harvey showing intermediate (1 - 2%) survival in low phosphate condition. The presence of carbon and nitrogen in the media over-weigh the information about the lack of phosphate that would cause continued but futile metabolism. The current study shows a general lack of coordination between carbon, nitrogen, and phosphate metabolism in V. cholerae.

Evidence of a Synergistic Cross-Talk between the B Cell Receptor (BCR) and Nicotinamide Phosphoribosyl Transferase (NAMPT) in Richter's Syndrome Patient-Derived Xenograft Models: Therapeutic Implications

Background: A rare complication of Chronic Lymphocytic Leukemia (CLL), Richter's Syndrome (RS) represents the transformation of a pre-existing CLL into a Diffuse Large B-cell Lymphoma (DLBCL), generally associated with poor prognosis. Current therapeutic approaches are limited and do not significantly reduce disease progression. For these reasons there is intense investigation to identify potentially druggable molecular circuits, opening the way to innovative combination therapies.Among the several oncogenic signalling pathways that may contribute to disease progression, the B Cell Receptor (BCR) is a main driver and an actionable target. We previously showed that BCR ligation in CLL cells increases expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage NAD pathway starting from nicotinamide, a finding in line with the notion of oncogenic-driven metabolic reprogramming. In this context, increased NAMPT expression leading to heightened NAD+ levels could sustain proliferation through the modulation of the activity of several intracellular enzymes, including sirtuins (NAD-dependent deacetylases).Aim: This work explores the connections between BCR signalling and the NAMPT/NAD/sirtuin axis in RS cells, asking the question of whether BCR and NAMPT can be simultaneously targeted.Methods: RS-PDX cells were freshly purified from tumor masses grown in mice and immediately used for short ex-vivo experiments. For in vivo experiments, NSG mice were injected subcutaneously with RS-PDX cells, which were left to engraft until a palpable mass was evident. Mice were then randomized to receive duvelisib, OT82 or a combination of the two for two consecutive weeks. Control mice were similarly treated with vehicle only. Mice were under a niacin-free diet.Results: By using our 4 RS patient-derived xenografts (PDX) models, we invariably observed high levels of NAMPT expression. High levels of NAMPT expression were also observed in 15 primary RS lymph node biopsies analyzed by RNA sequencing. BCR engagement through αIgM polyclonal antibodies significantly up-regulated NAMPT expression, as determined by qRT-PCR and protein analysis, with a concomitant increase in intracellular NAD+ levels. We then asked whether RS cells are sensitive to NAMPT inhibition, alone or in combination with drugs that target the BCR pathway. As most RS patients would likely have been treated during the preceding CLL phase with a BTK inhibitor, possibly developing resistance, we turned to PI3K inhibitors, which are less commonly used for CLL therapy. As NAMPT inhibitors (NAMPTi) we used both FK866 and OT-82, which are validated small molecules. Results indicated that the combination of the dual PI3K-δ/γ inhibitor duvelisib, with either FK866 or OT-82 induces dramatic apoptosis in all 4 models tested, as confirmed by annexinV/PI staining, by caspase 3 activation and by a significant drop in ATP and NAD+ levels. Importantly, two RS-PDX models (RS9737 and RS1316) were fully resistant to NAMPTi used alone, likely due to high levels of nicotinate phosphoribosyltransferase (NAPRT), which is the rate-limiting enzyme in the NAD salvage pathway that starts from nicotinic acid. However, addition of duvelisib, which was mildly effective when used alone, was followed by marked apoptosis even in these two models.Molecular dissection of the pathway showed that the combination of duvelisib and NAMPTi was followed by complete inhibition of the PI3K pathway, which was only partially blocked by duvelisib alone, even at high doses. The connection between NAMPT and PI3K is represented by cytoplasmic sirtuins, particularly SIRT2, which activate AKT through de-acetylation. Immunoprecipitation and two-dimensional gel electrophoresis showed that in the presence of NAMPTi, the amount of acetylated, i.e., inactive, AKT increased considerably. Consistently, treatment of RS-PDX mice with a combination of duvelisib and OT-82 was followed by significantly higher responses and longer animal survival.Conclusions: These results highlight a crosstalk between BCR signalling and NAMPT/sirtuin axis in RS models, showing the increased efficacy of the dual targeting (i.e., PI3K-δ/γ and NAMPT), and supporting this novel and promising therapeutic strategy for the treatment of RS patients. DisclosuresDeaglio: Heidelberg Pharma: Research Funding; Astra Zeneca: Research Funding.

Depletion of cardiolipin induces major changes in energy metabolism in Trypanosoma brucei bloodstream forms.

The mitochondrial inner membrane glycerophospholipid cardiolipin (CL) associates with mitochondrial proteins to regulate their activities and facilitate protein complex and supercomplex formation. Loss of CL leads to destabilized respiratory complexes and mitochondrial dysfunction. The role of CL in an organism lacking a conventional electron transport chain (ETC) has not been elucidated. Trypanosoma brucei bloodstream forms use an unconventional ETC composed of glycerol-3-phosphate dehydrogenase and alternative oxidase (AOX), while the mitochondrial membrane potential (ΔΨm) is generated by the hydrolytic action of the Fo F1 -ATP synthase (aka Fo F1 -ATPase). We now report that the inducible depletion of cardiolipin synthase (TbCls) is essential for survival of T brucei bloodstream forms. Loss of CL caused a rapid drop in ATP levels and a decline in the ΔΨm. Unbiased proteomic analyses revealed a reduction in the levels of many mitochondrial proteins, most notably of Fo F1 -ATPase subunits and AOX, resulting in a strong decline of glycerol-3-phosphate-stimulated oxygen consumption. The changes in cellular respiration preceded the observed decrease in Fo F1 -ATPase stability, suggesting that the AOX-mediated ETC is the first pathway responding to the decline in CL. Select proteins and pathways involved in glucose and amino acid metabolism were upregulated to counteract the CL depletion-induced drop in cellular ATP.

Imatinib and Dasatinib Provoke Mitochondrial Dysfunction Leading to Oxidative Stress in C2C12 Myotubes and Human RD Cells

Tyrosine kinase inhibitors (TKIs) can cause skeletal muscle toxicity in patients, but the underlying mechanisms are mostly unclear. The goal of the current study was to better characterize the role of mitochondria in TKI-associated myotoxicity. We exposed C2C12 murine myoblasts and myotubes as well as human rhabdomyosarcoma cells (RD cells) for 24 h to imatinib (1–100 µM), erlotinib (1–20 µM), and dasatinib (0.001–100 µM). In C2C12 myoblasts, imatinib was membrane toxic at 50 µM and depleted the cellular ATP pool at 20 µM. In C2C12 myotubes exposed to imatinib, ATP depletion started at 50 µM whereas membrane toxicity was not detectable. In myoblasts and myotubes exposed to dasatinib, membrane toxicity started at 0.5 µM and 2 µM, respectively, and the ATP drop was visible at 0.1 µM and 0.2 µM, respectively. When RD cells were exposed to imatinib, ATP depletion started at 20 µM whereas membrane toxicity was not detectable. Dasatinib was membrane toxic at 20 µM and depleted the cellular ATP pool already at 0.5 µM. Erlotinib was not toxic in both cell models. Imatinib (20 µM) and dasatinib (1 µM) reduced complex I activity in both cell models. Moreover, the mitochondrial membrane potential (Δψm) was dissipated for both TKIs in myotubes. In RD cells, the Δψm was reduced only by dasatinib. Both TKIs increased mitochondrial superoxide accumulation and decreased the mitochondrial copy number in both cell lines. In consequence, they increased protein expression of superoxide dismutase (SOD) 2 and thioredoxin 2 and cleavage of caspase 3, indicating apoptosis in C2C12 myotubes. Moreover, in both cell models, the mRNA expression of Sod1 and Sod2 increased when RD cells were exposed to dasatinib. Furthermore, dasatinib increased the mRNA expression of atrogin-1 and murf-1, which are important transcription factors involved in muscle atrophy. The mRNA expression of atrogin-1 increased also in RD cells exposed to imatinib. In conclusion, imatinib and dasatinib are mitochondrial toxicants in mouse C2C12 myotubes and human RD cells. Mitochondrial superoxide accumulation induced by these two TKIs is due to the inhibition of complex I and is probably related to impaired mitochondrial and myocyte proliferation.

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice.

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol's critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

Abstract 374: Direct measurements of cellular ATP levels in tumor cell lines using real-time, quantitative live-cell analysis

Abstract Metabolic reprogramming is a hallmark of tumor cells, owing to the increased energetic and biosynthetic demands associated with maintaining high rates of proliferation. Therapeutic strategies targeting unique, critical metabolic pathways of tumor cells are under investigation and in clinical trials. Standard approaches to monitoring drug induced metabolic perturbations are limited to endpoint assays that provide population-based measurements and limited kinetic information. We developed a genetically encoded ATP sensor to enable direct, automated analysis of cellular ATP levels using IncuCyte® S3. In this study, our live cell imaging approach was utilized to evaluate the effect of cancer therapeutics on ATP levels in tumor cell lines, focusing primarily on compounds that target metabolic vulnerabilities. Cell lines stably expressing a genetically encoded, fluorescent ATP sensor or a control (non-ATP binding) sensor were generated. ATP levels were monitored and analyzed using an IncuCyte® S3 equipped with a specialized filter set and data acquisition module. Cellular ATP levels were measured over the course of hours to days following compound treatment. Reductions in ATP levels could be observed within an hour of administration, highlighting the sensitivity and temporal resolution of the IncuCyte® S3 ATP sensor assay. Transient reductions in ATP followed by recovery, which would have been missed by typical endpoint assays, were noted using our live cell analysis approach. Sustained decreases in ATP were associated with enhanced antiproliferative efficacy compared to conditions under which recovery of ATP levels were observed. For example, triple-negative breast cancer (TNBC) cell lines have been shown to be more dependent on activity of glutaminase 1 (GLS1), which catalyzes the first step in utilization of glutamine to fuel mitochondrial metabolism, than their receptor-positive counterparts. Using our live cell analysis approach, we observed a rapid drop in ATP upon glutamine deprivation or inhibition of GLS1 by CB-839 in the TNBC cell line MDA-MB-231. ATP levels remained below that of vehicle-treated cells for the duration of the three-day time course. In contrast, the estrogen receptor-positive cell line MCF-7 displayed a more modest decrease in ATP and full recovery within 48 hours. Quantification of phase confluence confirmed that CB-839 had a stronger antiproliferative effect in MDA-MB-231 cells compared to MCF-7 cells. Visualization of morphology in tandem with automated ATP sensor analysis provide additional insight into the heterogeneity of cellular responses to compound addition. Overall, these data highlight the ability of IncuCyte® S3 to provide direct, kinetic measurement of ATP by live-cell analysis. Citation Format: Cicely L. Schramm, Grigory S. Filonov, Michael L. Bowe, Yong X. Chen, Laura A. Skerlos, Dyke P. McEwen, Daniel M. Appledorn. Direct measurements of cellular ATP levels in tumor cell lines using real-time, quantitative live-cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 374.

Abstract 3513: Curcumin induces a fatal energetic impairment by inhibiting ATP-synthase activity and decreasing ATP generation and oxygen consumption in in vitro and in vivo tumor models

Abstract Curcumin has been reported to inhibit inflammation, tumor growth, angiogenesis and metastasis by decreasing cell growth and by inducing apoptosis mainly through the inhibition of nuclear factor kappa-B (NFkB), a master regulator of inflammation. Recent reports also indicate potential metabolic effects of the polyphenol and we therefore analyzed whether and how it affects the energy metabolism of tumor cells. We show that curcumin inhibits the activity of ATP-synthase in isolated mitochondrial membranes leading to a dramatic drop of ATP and a reduction of oxygen consumption in in vitro in several murine tumor cell lines (CT26 colon cancer, B16 melanoma, L1210 lymphocytic leukemia, 4T1 breast cancer) and in vivo in syngeneic tumor models. The effects of curcumin on ATP-synthase are independent of the inhibition of nuclear factor kB (NFkB) since the IkB Kinase inhibitor, SC-514, inhibits the expression of the NFkB target gene, BCL2, but does not affect the activity of the ATP-synthase. The activities of the glucose metabolism enzymes hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase are only slightly affected in a cell type specific manner. The energy impairment translates into decreased tumor cell viability. Apoptosis is induced by promoting the generation of reactive oxygen species and malondialdehyde (MDA), a marker of lipid oxidation. Tumor autophagy is induced by curcumin at least in part due to the activation of the AMP-activated protein kinase (AMPK). These activities translate into a significant delay of in vivo tumor growth likely due to a reduction of tumor angiogenesis since we observe reduced number and size of tumor vessels in vivo. These results establish the ATP-synthase, a central enzyme of the cellular energy metabolism, as a target of the anti-tumoral polyphenol leading to inhibition of cancer cell growth and a general reprogramming of tumor metabolism. Citation Format: Ulrich Pfeffer, Giovanna Bianchi, Silvia Ravera, Chiara Traverso, Adriana Amaro, Francesca Piaggio, Laura Emionite, Tiziana Bacchetti, Lizzia Raffaghello. Curcumin induces a fatal energetic impairment by inhibiting ATP-synthase activity and decreasing ATP generation and oxygen consumption in in vitro and in vivo tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3513.

Curcumin induces a fatal energetic impairment in tumor cells in vitro and in vivo by inhibiting ATP-synthase activity.

Curcumin has been reported to inhibit inflammation, tumor growth, angiogenesis and metastasis by decreasing cell growth and by inducing apoptosis mainly through the inhibition of nuclear factor kappa-B (NFκB), a master regulator of inflammation. Recent reports also indicate potential metabolic effects of the polyphenol, therefore we analyzed whether and how it affects the energy metabolism of tumor cells. We show that curcumin (10 µM) inhibits the activity of ATP synthase in isolated mitochondrial membranes leading to a dramatic drop of ATP and a reduction of oxygen consumption in in vitro and in vivo tumor models. The effects of curcumin on ATP synthase are independent of the inhibition of NFκB since the IκB Kinase inhibitor, SC-514, does not affect ATP synthase. The activities of the glycolytic enzymes hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase are only slightly affected in a cell type-specific manner. The energy impairment translates into decreased tumor cell viability. Moreover, curcumin induces apoptosis by promoting the generation of reactive oxygen species (ROS) and malondialdehyde (MDA), a marker of lipid oxidation, and autophagy, at least in part due to the activation of the AMP-activated protein kinase (AMPK). According to the in vitro anti-tumor effect, curcumin (30 mg/kg body weight) significantly delayed in vivo cancer growth likely due to an energy impairment but also through the reduction of tumor angiogenesis. These results establish the ATP synthase, a central enzyme of the cellular energy metabolism, as a target of the antitumoral polyphenol leading to inhibition of cancer cell growth and a general reprogramming of tumor metabolism.

Metabolic adjustment upon repetitive substrate perturbations using dynamic 13C-tracing in yeast

BackgroundNatural and industrial environments are dynamic with respect to substrate availability and other conditions like temperature and pH. Especially, metabolism is strongly affected by changes in the extracellular space. Here we study the dynamic flux of central carbon metabolism and storage carbohydrate metabolism under dynamic feast/famine conditions in Saccharomyces cerevisiae.ResultsThe metabolic flux reacts fast and sensitive to cyclic perturbations in substrate availability. Compared to well-documented stimulus–response experiments using substrate pulses, different metabolic responses are observed. Especially, cells experiencing cyclic perturbations do not show a drop in ATP with the addition of glucose, but an immediate increase in energy charge. Although a high glycolytic flux of up to 5.4 mmol gDW−1 h−1 is observed, no overflow metabolites are detected. From famine to feast the glucose uptake rate increased from 170 to 4788 μmol gDW−1 h−1 in 24 s. Intracellularly, even more drastic changes were observed. Especially, the T6P synthesis rate increased more than 100-fold upon glucose addition. This response indicates that the storage metabolism is very sensitive to changes in glycolytic flux and counterbalances these rapid changes by diverting flux into large pools to prevent substrate accelerated death and potentially refill the central metabolism when substrates become scarce. Using 13C-tracer we found a dilution in the labeling of extracellular glucose, G6P, T6P and other metabolites, indicating an influx of unlabeled carbon. It is shown that glycogen and trehalose degradation via different routes could explain these observations. Based on the 13C labeling in average 15% of the carbon inflow is recycled via trehalose and glycogen. This average fraction is comparable to the steady-state turnover, but changes significantly during the cycle, indicating the relevance for dynamic regulation of the metabolic flux.ConclusionsComparable to electric energy grids, metabolism seems to use storage units to buffer peaks and keep reserves to maintain a robust function. During the applied fast feast/famine conditions about 15% of the metabolized carbon were recycled in storage metabolism. Additionally, the resources were distributed different to steady-state conditions. Most remarkably is a fivefold increased flux towards PPP that generated a reversed flux of transaldolase and the F6P-producing transketolase reactions. Combined with slight changes in the biomass composition, the yield decrease of 5% can be explained.

Synergistic cellular effects including mitochondrial destabilization, autophagy and apoptosis following low-level exposure to a mixture of lipophilic persistent organic pollutants

Humans are exposed to multiple exogenous environmental pollutants. Many of these compounds are parts of mixtures that can exacerbate harmful effects of the individual mixture components. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is primarily produced via industrial processes including incineration and the manufacture of herbicides. Both endosulfan and TCDD are persistent organic pollutants which elicit cytotoxic effects by inducing reactive oxygen species generation. Sublethal concentrations of mixtures of TCDD and endosulfan increase oxidative stress, as well as mitochondrial homeostasis disruption, which is preceded by a calcium rise and, in fine, induce cell death. TCDD+Endosulfan elicit a complex signaling sequence involving reticulum endoplasmic destalilization which leads to Ca2+ rise, superoxide anion production, ATP drop and late NADP(H) depletion associated with a mitochondrial induced apoptosis concomitant early autophagic processes. The ROS scavenger, N-acetyl-cysteine, blocks both the mixture-induced autophagy and death. Calcium chelators act similarly and mitochondrially targeted anti-oxidants also abrogate these effects. Inhibition of the autophagic fluxes with 3-methyladenine, increases mixture-induced cell death. These findings show that subchronic doses of pollutants may act synergistically. They also reveal that the onset of autophagy might serve as a protective mechanism against ROS-triggered cytotoxic effects of a cocktail of pollutants in Caco-2 cells and increase their tumorigenicity.

Differential effects of energy deprivation on intracellular sodium homeostasis in neurons and astrocytes.

The maintenance of a low intracellular sodium concentration by the Na+ /K+ -ATPase (NKA) is critical for brain function. In both neurons and glial cells, NKA activity is required to counteract changes in the sodium gradient due to opening of voltage- and ligand-gated channels and/or activation of sodium-dependent secondary active transporters. Because NKA consumes about 50% of cellular ATP, sodium homeostasis is strictly dependent on an intact cellular energy metabolism. Despite the high energetic costs of electrical signaling, neurons do not contain significant energy stores themselves, but rely on a close metabolic interaction with surrounding astrocytes. A disruption of energy supply as observed during focal ischemia causes a rapid drop in ATP in both neurons and astrocytes. There is accumulating evidence that dysregulation of intracellular sodium is an inherent consequence of a reduction in cellular ATP, triggering secondary failure of extra- and intracellular homeostasis of other ions -in particular potassium, calcium, and protons- and thereby promoting excitotoxicity. The characteristics, cellular mechanisms and direct consequences of harmful sodium influx, however, differ between neurons and astrocytes. Moreover, recent work has shown that an intact astrocyte metabolism and sodium homeostasis are critical to maintain the sodium homeostasis of surrounding neurons as well as their capacity to recover from imposed sodium influx. Understanding the mechanisms of sodium increases upon metabolic failure and the differential responses of neurons and glial cells as well as their metabolic interactions will be critical to fully unravel the events causing cellular malfunction, failure and cell death following energy depletion. © 2017 Wiley Periodicals, Inc.

Antiretrovirals, Methamphetamine, and HIV-1 Envelope Protein gp120 Compromise Neuronal Energy Homeostasis in Association with Various Degrees of Synaptic and Neuritic Damage.

HIV-1 infection frequently causes HIV-associated neurocognitive disorders (HAND) despite combination antiretroviral therapy (cART). Evidence is accumulating that components of cART can themselves be neurotoxic upon long-term exposure. In addition, abuse of psychostimulants, such as methamphetamine, seems to aggravate HAND and compromise antiretroviral therapy. However, the combined effect of virus and recreational and therapeutic drugs on the brain is poorly understood. Therefore, we exposed mixed neuronal-glial cerebrocortical cells to antiretrovirals (ARVs) (zidovudine [AZT], nevirapine [NVP], saquinavir [SQV], and 118-D-24) of four different pharmacological categories and to methamphetamine and, in some experiments, the HIV-1 gp120 protein for 24 h and 7 days. Subsequently, we assessed neuronal injury by fluorescence microscopy, using specific markers for neuronal dendrites and presynaptic terminals. We also analyzed the disturbance of neuronal ATP levels and assessed the involvement of autophagy by using immunofluorescence and Western blotting. ARVs caused alterations of neurites and presynaptic terminals primarily during the 7-day incubation and depending on the specific compounds and their combinations with and without methamphetamine. Similarly, the loss of neuronal ATP was context specific for each of the drugs or combinations thereof, with and without methamphetamine or viral gp120. Loss of ATP was associated with activation of AMP-activated protein kinase (AMPK) and autophagy, which, however, failed to restore normal levels of neuronal ATP. In contrast, boosting autophagy with rapamycin prevented the long-term drop of ATP during exposure to cART in combination with methamphetamine or gp120. Our findings indicate that the overall positive effect of cART on HIV infection is accompanied by detectable neurotoxicity, which in turn may be aggravated by methamphetamine.

On-target effect of FK866, a nicotinamide phosphoribosyl transferase inhibitor, by apoptosis-mediated death in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) remains incurable despite advances in therapy. In this study, we characterize the effect of nicotinamide phosphoribosyltransferase (NAMPT) inhibition by FK866 in primary CLL cells from patients with various clinical prognostic markers. CLL cells were treated with FK866 to assess viability by Annexin V/PI staining. Functional analysis of FK866 included time- and concentration-dependent evaluation of cellular NAD, ATP, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and apoptotic signaling. Chemosensitization potential by FK866 to fludarabine was also assessed. Prognostic markers were correlated with drug response. FK866 induced CLL cell death by depleting cellular NAD content by day 1, followed by a drop in ATP on day 2. We observed loss of MMP, ROS increase, and induction of apoptotic signaling at day 3. On-target activity of FK866 was confirmed by NAD-mediated rescue of NAD and ATP loss, apoptotic signaling, and viability. The response to FK866 was independent of most prognostic markers. Higher doses were required with short lymphocyte doubling time and positive CD38 status, whereas CLL cells resistant to fludarabine in vitro and from patients with del17p13.1 were equally sensitive to FK866. FK866 did not upregulate the p53-target p21, nor did the p53 activator Nutlin improve FK866-mediated cell death. Furthermore, fludarabine and FK866 were synergistic at clinically relevant concentrations. NAMPT inhibition by FK866 may be a potential treatment for CLL, including patients with del17p13.1 or other high-risk features. FK866 may complement standard agents to enhance their efficacy and/or allow dose reduction for improved tolerability.

Knockdown of malic enzyme 2 suppresses lung tumor growth, induces differentiation and impacts PI3K/AKT signaling.

Mitochondrial malic enzyme 2 (ME2) catalyzes the oxidative decarboxylation of malate to yield CO2 and pyruvate, with concomitant reduction of dinucleotide cofactor NAD+ or NADP+. We find that ME2 is highly expressed in many solid tumors. In the A549 non-small cell lung cancer (NSCLC) cell line, ME2 depletion inhibits cell proliferation and induces cell death and differentiation, accompanied by increased reactive oxygen species (ROS) and NADP+/NADPH ratio, a drop in ATP, and increased sensitivity to cisplatin. ME2 knockdown impacts phosphoinositide-dependent protein kinase 1 (PDK1) and phosphatase and tensin homolog (PTEN) expression, leading to AKT inhibition. Depletion of ME2 leads to malate accumulation and pyruvate decrease, and exogenous cell permeable dimethyl-malate (DMM) mimics the ME2 knockdown phenotype. Both ME2 knockdown and DMM treatment reduce A549 cell growth in vivo. Collectively, our data suggest that ME2 is a potential target for cancer therapy.

The effects of glucose and lipids in steatotic and non‐steatotic livers in conditions of partial hepatectomy under ischaemia‐reperfusion

Steatosis is a risk factor in partial hepatectomy (PH) under ischaemia-reperfusion (I/R), which is commonly applied in clinical practice to reduce bleeding. Nutritional support strategies, as well as the role of peripheral adipose tissue as energy source for liver regeneration, remain poorly investigated. To investigate whether the administration of either glucose or a lipid emulsion could protect steatotic and non-steatotic livers against damage and regenerative failure in an experimental model of PH under I/R. The relevance of peripheral adipose tissue in liver regeneration following surgery is studied. Steatotic and non-steatotic rat livers were subjected to surgery and the effects of either glucose or lipid treatment on damage and regeneration, and part of the underlying mechanisms, were investigated. In non-steatotic livers, treatment with lipids or glucose provided the same protection against damage, regeneration failure and ATP drop. Adipose tissue was not required to regenerate non-steatotic livers. In the presence of hepatic steatosis, lipid treatment, but not glucose, protected against damage and regenerative failure by induction of cell cycle, maintenance of ATP levels and elevation of sphingosine-1-phosphate/ceramide ratio and phospholipid levels. Peripheral adipose tissue was required for regenerating the steatotic liver but it was not used as an energy source. Lipid treatment in non-steatotic livers provides the same protection as that afforded by glucose in conditions of PH under I/R, whereas the treatment with lipids is preferable to reduce the injurious effects of liver surgery in the presence of steatosis.

Reactive oxygen species generated in different compartments induce cell death, survival, or senescence

Although reactive oxygen species (ROS) are well-established mediators of oxidative damage and cell demise, the mechanisms by which they trigger specific cell death modalities and the temporal/spatial requirements underlying this phenomenon are largely unknown. Yet, it is well established that most anticancer therapies depend on ROS production for efficient tumor eradication. Using several non-small-cell lung cancer cell lines, we have dissected how the site of ROS production and accumulation in various cell compartments affect cell fate. We demonstrate that high levels of exogenously generated H2O2 induce extensive DNA damage, ATP depletion, and severe cytotoxicity. Although these effects were independent of caspase activity, they could—at least in part—be prevented by RIP1 kinase inhibition. In contrast, low levels of exogenously produced H2O2 triggered a modest drop in ATP level, delayed toxicity, G2/M arrest, and cell senescence. Mitochondrially produced H2O2 induced a reversible ATP drop without affecting cell viability. Instead, the cells accumulated in the G1/S phase of the cell cycle and became senescent. Concomitant inhibition of glycolysis was found to markedly sensitize cells to death in the presence of otherwise nontoxic concentrations of H2O2, presumably by the inhibition of ATP-restoring mechanisms. Combined, our data provide evidence that ROS might dictate different cellular consequences depending on their overall concentration at steady-state levels and on their site of generation.

Oxidative stress modulates mitochondrial failure and cyclophilin D function in X-linked adrenoleukodystrophy

A common process associated with oxidative stress and severe mitochondrial impairment is the opening of the mitochondrial permeability transition pore, as described in many neurodegenerative diseases. Thus, inhibition of mitochondrial permeability transition pore opening represents a potential target for inhibiting mitochondrial-driven cell death. Among the mitochondrial permeability transition pore components, cyclophilin D is the most studied and has been found increased under pathological conditions. Here, we have used in vitro and in vivo models of X-linked adrenoleukodystrophy to investigate the relationship between the mitochondrial permeability transition pore opening and redox homeostasis. X-linked adrenoleukodystrophy is a neurodegenerative condition caused by loss of function of the peroxisomal ABCD1 transporter, in which oxidative stress plays a pivotal role. In this study, we provide evidence of impaired mitochondrial metabolism in a peroxisomal disease, as fibroblasts in patients with X-linked adrenoleukodystrophy cannot survive when forced to rely on mitochondrial energy production, i.e. on incubation in galactose. Oxidative stress induced under galactose conditions leads to mitochondrial damage in the form of mitochondrial inner membrane potential dissipation, ATP drop and necrotic cell death, together with increased levels of oxidative modifications in cyclophilin D protein. Moreover, we show increased expression levels of cyclophilin D in the affected zones of brains in patients with adrenomyeloneuropathy, in spinal cord of a mouse model of X-linked adrenoleukodystrophy (Abcd1-null mice) and in fibroblasts from patients with X-linked adrenoleukodystrophy. Notably, treatment with antioxidants rescues mitochondrial damage markers in fibroblasts from patients with X-linked adrenoleukodystrophy, including cyclophilin D oxidative modifications, and reverses cyclophilin D induction in vitro and in vivo. These findings provide mechanistic insight into the beneficial effects of antioxidants in neurodegenerative and non-neurodegenerative cyclophilin D-dependent disorders.