Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients: A proof-of-concept study.

Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients. Data showed decreased glycolytic activity in red blood cells of 2/3 of patients with lower-risk MDS. These results highlight a potential effect of the PK activator in this setting.

INTEGRATE: Model-based multi-omics data integration to characterize multi-level metabolic regulation.

Metabolism is directly and indirectly fine-tuned by a complex web of interacting regulatory mechanisms that fall into two major classes. On the one hand, the expression level of the catalyzing enzyme sets the maximal theoretical flux level (i.e., the net rate of the reaction) for each enzyme-controlled reaction. On the other hand, metabolic regulation controls the metabolic flux through the interactions of metabolites (substrates, cofactors, allosteric modulators) with the responsible enzyme. High-throughput data, such as metabolomics and transcriptomics data, if analyzed separately, do not accurately characterize the hierarchical regulation of metabolism outlined above. They must be integrated to disassemble the interdependence between different regulatory layers controlling metabolism. To this aim, we propose INTEGRATE, a computational pipeline that integrates metabolomics and transcriptomics data, using constraint-based stoichiometric metabolic models as a scaffold. We compute differential reaction expression from transcriptomics data and use constraint-based modeling to predict if the differential expression of metabolic enzymes directly originates differences in metabolic fluxes. In parallel, we use metabolomics to predict how differences in substrate availability translate into differences in metabolic fluxes. We discriminate fluxes regulated at the metabolic and/or gene expression level by intersecting these two output datasets. We demonstrate the pipeline using a set of immortalized normal and cancer breast cell lines. In a clinical setting, knowing the regulatory level at which a given metabolic reaction is controlled will be valuable to inform targeted, truly personalized therapies in cancer patients.

A metabolic core model elucidates how enhanced utilization of glucose and glutamine, with enhanced glutamine-dependent lactate production, promotes cancer cell growth: The WarburQ effect.

Cancer cells share several metabolic traits, including aerobic production of lactate from glucose (Warburg effect), extensive glutamine utilization and impaired mitochondrial electron flow. It is still unclear how these metabolic rearrangements, which may involve different molecular events in different cells, contribute to a selective advantage for cancer cell proliferation. To ascertain which metabolic pathways are used to convert glucose and glutamine to balanced energy and biomass production, we performed systematic constraint-based simulations of a model of human central metabolism. Sampling of the feasible flux space allowed us to obtain a large number of randomly mutated cells simulated at different glutamine and glucose uptake rates. We observed that, in the limited subset of proliferating cells, most displayed fermentation of glucose to lactate in the presence of oxygen. At high utilization rates of glutamine, oxidative utilization of glucose was decreased, while the production of lactate from glutamine was enhanced. This emergent phenotype was observed only when the available carbon exceeded the amount that could be fully oxidized by the available oxygen. Under the latter conditions, standard Flux Balance Analysis indicated that: this metabolic pattern is optimal to maximize biomass and ATP production; it requires the activity of a branched TCA cycle, in which glutamine-dependent reductive carboxylation cooperates to the production of lipids and proteins; it is sustained by a variety of redox-controlled metabolic reactions. In a K-ras transformed cell line we experimentally assessed glutamine-induced metabolic changes. We validated computational results through an extension of Flux Balance Analysis that allows prediction of metabolite variations. Taken together these findings offer new understanding of the logic of the metabolic reprogramming that underlies cancer cell growth.

A Novel Strategy for Glioblastoma Treatment by Natural Bioactive Molecules Showed a Highly Effective Anti-Cancer Potential.

Glioblastoma (GBM) is a severe form of brain tumor that has a high fatality rate. It grows aggressively and most of the time results in resistance to traditional treatments like chemo- and radiotherapy and surgery. Biodiversity, beyond representing a big resource for human well-being, provides several natural compounds that have shown great potential as anticancer drugs. Many of them are being extensively researched and significantly slow GBM progression by reducing the proliferation rate, migration, and inflammation and also by modulating oxidative stress. Here, the use of some natural compounds, such as Allium lusitanicum, Succisa pratensis, and Dianthus superbus, was explored to tackle GBM; they showed their impact on cell number reduction, which was partially given by cell cycle quiescence. Furthermore, a reduced cell migration ability was reported, accomplished by morphological cytoskeleton changes, which even highlighted a mesenchymal-epithelial transition. Furthermore, metabolic studies showed an induced cell oxidative stress modulation and a massive metabolic rearrangement. Therefore, a new therapeutic option was suggested to overcome the limitations of conventional treatments and thereby improve patient outcomes.

Fusion-fission-mitophagy cycling and metabolic reprogramming coordinate nerve growth factor (NGF)-dependent neuronal differentiation.

Neuronal differentiation is regulated by nerve growth factor (NGF) and other neurotrophins. We explored the impact of NGF on mitochondrial dynamics and metabolism through time-lapse imaging, metabolomics profiling, and computer modeling studies. We show that NGF may direct differentiation by stimulating fission, thereby causing selective mitochondrial network fragmentation and mitophagy, ultimately leading to increased mitochondrial quality and respiration. Then, we reconstructed the dynamic fusion-fission-mitophagy cycling of mitochondria in a computer model, integrating these processes into a single network mechanism. Both the computational model and the simulations are able to reproduce the proposed mechanism in terms of mitochondrial dynamics, levels of reactive oxygen species (ROS), mitophagy, and mitochondrial quality, thus providing a computational tool for the interpretation of the experimental data and for future studies aiming to detail further the action of NGF on mitochondrial processes. We also show that changes in these mitochondrial processes are intertwined with a metabolic function of NGF in differentiation: NGF directs a profound metabolic rearrangement involving glycolysis, TCA cycle, and the pentose phosphate pathway, altering the redox balance. This metabolic rewiring may ensure: (a) supply of both energy and building blocks for the anabolic processes needed for morphological reorganization, as well as (b) redox homeostasis.

Plastic leachate-induced toxicity during sea urchin embryonic development: Insights into the molecular pathways affected by PVC.

Microplastics are now polluting all seas and, while studies have found numerous negative interactions between plastic pollution and marine animals, the effects on embryonic development are poorly understood. A potentially important source of developmental ecotoxicity comes from chemicals leached from plastic particles to the marine environment. Here we investigate the effects of leachates from new and beach-collected pellets on the embryonic and larval development of the sea urchin Strongylocentrotus purpuratus and demonstrate that exposure of developing embryos to these leachates elicits severe, consistent and treatment-specific developmental abnormalities including radialisation of the embryo and malformation of the skeleton, neural and immune cells. Using a multi-omics approach we define the developmental pathways disturbed upon exposure to PVC leachates and provide a mechanistic view that pinpoints cellular redox stress and energy production as drivers of phenotypic abnormalities following exposure to PVC leachates. Analysis of leachates identified high concentrations of zinc that are the likely cause of these observed defects. Our findings point to clear and specific detrimental effects of marine plastic pollution on the development of echinoderms, demonstrating that chemicals leached from plastic particles into sea water can produce strong developmental abnormalities via specific pathways, and therefore have the potential to impact on a wide range of organisms.

Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth.

Oncogenes such as K-ras mediate cellular and metabolic transformation during tumorigenesis. To analyze K-Ras-dependent metabolic alterations, we employed ¹³C metabolic flux analysis (MFA), non-targeted tracer fate detection (NTFD) of ¹5N-labeled glutamine, and transcriptomic profiling in mouse fibroblast and human carcinoma cell lines. Stable isotope-labeled glucose and glutamine tracers and computational determination of intracellular fluxes indicated that cells expressing oncogenic K-Ras exhibited enhanced glycolytic activity, decreased oxidative flux through the tricarboxylic acid (TCA) cycle, and increased utilization of glutamine for anabolic synthesis. Surprisingly, a non-canonical labeling of TCA cycle-associated metabolites was detected in both transformed cell lines. Transcriptional profiling detected elevated expression of several genes associated with glycolysis, glutamine metabolism, and nucleotide biosynthesis upon transformation with oncogenic K-Ras. Chemical perturbation of enzymes along these pathways further supports the decoupling of glycolysis and TCA metabolism, with glutamine supplying increased carbon to drive the TCA cycle. These results provide evidence for a role of oncogenic K-Ras in the metabolic reprogramming of cancer cells.

Polystyrene micro and nano-particles induce metabolic rewiring in normal human colon cells: A risk factor for human health.

Polystyrene is a thermoplastic polymer widely used in commercial products. Like all plastics, polystyrene can be degraded into microplastic and nanoplastic particles and ingested via food chain contamination. Although the ecological impact due to plastic contamination is well known, there are no studies indicating a carcinogenic potential of polystyrene microplastics (MPs) and nanoplastics (NPs). Here, we evaluated the effects of the MPs and NPs on normal human intestinal CCD-18Co cells. Our results show that internalization of NPs and MPs induces metabolic changes under both acute and chronic exposure by inducing oxidative stress, increasing glycolysis via lactate to sustain energy metabolism and glutamine metabolism to sustain anabolic processes. We also show that this decoupling of nutrients mirrors the effect of the potent carcinogenic agent azoxymethane and HCT15 colon cancer cells, carrying out the typical strategy of cancer cells to optimize nutrients utilization and allowing metabolic adaptation to environmental stress conditions. Taken together our data provide new evidence that chronic NPs and MPs exposure could act as cancer risk factor for human health.

Production of recombinant human xCT (SLC7A11) and reconstitution in proteoliposomes for functional studies.

The plasma membrane transporter xCT belongs to the SLC7 family and has the physiological role of mediating the exchange of glutamate and cystine across the cell plasma membrane, being crucial for redox control. The xCT protein forms a heterodimer with the ancillary protein CD98. Over the years, xCT became a hot pharmacological target due to the documented over-expression in virtually all human cancers, which rely on cystine availability for their progression. Notwithstanding, several unknown aspects of xCT biology still exist that require a suitable single protein experimental model, to be addressed. To this aim, the recombinant host Escherichia coli has been exploited to over-express the human isoform of xCT. In this widely used and low-cost system, the optimization for growth and protein production has been achieved by acting on the metabolic needs of the bacterial strains. Then, the His-tagged protein has been purified by Ni2+-chelating chromatography and reconstituted in proteoliposomes for transport activity assays. The expressed protein was in a folded/active state allowing functional and kinetic characterization. Interestingly, the features of the recombinant protein meet those of the native one extracted from intact cells, further confirming the suitability of E. coli as a host for the expression of human proteins. This study opens perspectives for elucidating other molecular aspects of xCT, as well as for studying the interaction with endogenous and exogenous compounds, relevant to human health.

Fundamental Role of Pentose Phosphate Pathway within the Endoplasmic Reticulum in Glutamine Addiction of Triple-Negative Breast Cancer Cells.

Cancer utilization of large glutamine equivalents contributes to diverging glucose-6-P flux toward the pentose phosphate shunt (PPP) to feed the building blocks and the antioxidant responses of rapidly proliferating cells. In addition to the well-acknowledged cytosolic pathway, cancer cells also run a largely independent PPP, triggered by hexose-6P-dehydrogenase within the endoplasmic reticulum (ER), whose activity is mandatory for the integrity of ER-mitochondria networking. To verify whether this reticular metabolism is dependent on glutamine levels, we complemented the metabolomic characterization of intermediates of the glucose metabolism and tricarboxylic acid cycle with the estimation of proliferating activity, energy metabolism, redox damage, and mitochondrial function in two breast cancer cell lines. ER-PPP activity and its determinants were estimated by the ER accumulation of glucose analogs. Glutamine shortage decreased the proliferation rate despite increased ATP and NADH levels. It depleted NADPH reductive power and increased malondialdehyde content despite a marked increase in glucose-6P-dehydrogenase. This paradox was explained by the deceleration of ER-PPP favored by the decrease in hexose-6P-dehydrogenase expression coupled with the opposite response of its competitor enzyme glucose-6P-phosphatase. The decreased ER-PPP activity eventually hampered mitochondrial function and calcium exchanges. These data configure the ER-PPP as a powerful, unrecognized regulator of cancer cell metabolism and proliferation.

Gold nanoparticles coated with polyvinylpyrrolidone and sea urchin extracellular molecules induce transient immune activation.

We report that the immunogenicity of colloidal gold nanoparticles coated with polyvinylpyrrolidone (PVP-AuNPs) in a model organism, the sea urchin Paracentrotus lividus, can function as a proxy for humans for in vitro immunological studies. To profile the immune recognition and interaction from exposure to PVP-AuNPs (1 and 10 µg mL-1), we applied an extensive nano-scale approach, including particle physicochemical characterisation involving immunology, cellular biology, and metabolomics. The interaction between PVP-AuNPs and soluble proteins of the sea urchin physiological coelomic fluid (blood equivalent) results in the formation of a protein "corona" surrounding the NPs from three major proteins that influence the hydrodynamic size and colloidal stability of the particle. At the lower concentration of PVP-AuNPs, the P. lividus phagocytes show a broad metabolic plasticity based on the biosynthesis of metabolites mediating inflammation and phagocytosis. At the higher concentration of PVP-AuNPs, phagocytes activate an immunological response involving Toll-like receptor 4 (TLR4) signalling pathway at 24 hours of exposure. These results emphasise that exposure to PVP-AuNPs drives inflammatory signalling by the phagocytes and the resolution at both the low and high concentrations of the PVP-AuNPs and provides more details regarding the immunogenicity of these NPs.

Transcriptomics and Metabolomics Integration Reveals Redox-Dependent Metabolic Rewiring in Breast Cancer Cells.

Rewiring glucose metabolism toward aerobic glycolysis provides cancer cells with a rapid generation of pyruvate, ATP, and NADH, while pyruvate oxidation to lactate guarantees refueling of oxidized NAD+ to sustain glycolysis. CtPB2, an NADH-dependent transcriptional co-regulator, has been proposed to work as an NADH sensor, linking metabolism to epigenetic transcriptional reprogramming. By integrating metabolomics and transcriptomics in a triple-negative human breast cancer cell line, we show that genetic and pharmacological down-regulation of CtBP2 strongly reduces cell proliferation by modulating the redox balance, nucleotide synthesis, ROS generation, and scavenging. Our data highlight the critical role of NADH in controlling the oncogene-dependent crosstalk between metabolism and the epigenetically mediated transcriptional program that sustains energetic and anabolic demands in cancer cells.

Systems metabolomics: from metabolomic snapshots to design principles.

Metabolomics is a rapidly expanding technology that finds increasing application in a variety of fields, form metabolic disorders to cancer, from nutrition and wellness to design and optimization of cell factories. The integration of metabolic snapshots with metabolic fluxes, physiological readouts, metabolic models, and knowledge-informed Artificial Intelligence tools, is required to obtain a system-level understanding of metabolism. The emerging power of multi-omic approaches and the development of integrated experimental and computational tools, able to dissect metabolic features at cellular and subcellular resolution, provide unprecedented opportunities for understanding design principles of metabolic (dis)regulation and for the development of precision therapies in multifactorial diseases, such as cancer and neurodegenerative diseases.

Methionine Supplementation Affects Metabolism and Reduces Tumor Aggressiveness in Liver Cancer Cells.

Liver cancer is one of the most common cancer worldwide with a high mortality. Methionine is an essential amino acid required for normal development and cell growth, is mainly metabolized in the liver, and its role as an anti-cancer supplement is still controversial. Here, we evaluate the effects of methionine supplementation in liver cancer cells. An integrative proteomic and metabolomic analysis indicates a rewiring of the central carbon metabolism, with an upregulation of the tricarboxylic acid (TCA) cycle and mitochondrial adenosine triphosphate (ATP) production in the presence of high methionine and AMP-activated protein kinase (AMPK) inhibition. Methionine supplementation also reduces growth rate in liver cancer cells and induces the activation of both the AMPK and mTOR pathways. Interestingly, in high methionine concentration, inhibition of AMPK strongly impairs cell growth, cell migration, and colony formation, indicating the main role of AMPK in the control of liver cancer phenotypes. Therefore, regulation of methionine in the diet combined with AMPK inhibition could reduce liver cancer progression.

Safety Evaluation of TiO2 Nanoparticle-Based Sunscreen UV Filters on the Development and the Immunological State of the Sea Urchin Paracentrotus lividus.

Sunscreens are emulsions of water and oil that contain filters capable of protecting against the detrimental effects of ultraviolet radiation (UV). The widespread use of cosmetic products based on nanoparticulate UV filters has increased concerns regarding their safety and compatibility with both the environment and human health. In the present work, we evaluated the effects of titanium dioxide nanoparticle (TiO2 NP)-based UV filters with three different surface coatings on the development and immunity of the sea urchin, Paracentrotus lividus. A wide range of NP concentrations was analyzed, corresponding to different levels of dilution starting from the original cosmetic dispersion. Variations in surface coating, concentration, particle shape, and pre-dispersant medium (i.e., water or oil) influenced the embryonic development without producing a relevant developmental impairment. The most common embryonic abnormalities were related to the skeletal growth and the presence of a few cells, which were presumably involved in the particle uptake. Adult P. lividus immune cells exposed to silica-coated TiO2 NP-based filters showed a broad metabolic plasticity based on the biosynthesis of metabolites that mediate inflammation, phagocytosis, and antioxidant response. The results presented here highlight the biosafety of the TiO2 NP-based UV filters toward sea urchin, and the importance of developing safer-by-design sunscreens.

Titanium dioxide nanoparticles temporarily influence the sea urchin immunological state suppressing inflammatory-relate gene transcription and boosting antioxidant metabolic activity.

Titanium dioxide nanoparticles (TiO2NPs) are revolutionizing biomedicine due to their potential application as diagnostic and therapeutic agents. However, the TiO2NP immune-compatibility remains an open issue, even for ethical reasons. In this work, we investigated the immunomodulatory effects of TiO2NPs in an emergent proxy to human non-mammalian model for in vitro basic and translational immunology: the sea urchin Paracentrotus lividus. To highlight on the new insights into the evolutionarily conserved intracellular signaling and metabolism pathways involved in immune-TiO2NP recognition/interaction we applied a wide-ranging approach, including electron microscopy, biochemistry, transcriptomics and metabolomics. Findings highlight that TiO2NPs interact with immune cells suppressing the expression of genes encoding for proteins involved in immune response and apoptosis (e.g. NF-κB, FGFR2, JUN, MAPK14, FAS, VEGFR, Casp8), and boosting the immune cell antioxidant metabolic activity (e.g. pentose phosphate, cysteine-methionine, glycine-serine metabolism pathways). TiO2NP uptake was circumscribed to phagosomes/phagolysosomes, depicting harmless vesicular internalization. Our findings underlined that under TiO2NP-exposure sea urchin innate immune system is able to control inflammatory signaling, excite antioxidant metabolic activity and acquire immunological tolerance, providing a new level of understanding of the TiO2NP immune-compatibility that could be useful for the development in Nano medicines.

Two high-rate pentose-phosphate pathways in cancer cells.

The relevant role of pentose phosphate pathway (PPP) in cancer metabolic reprogramming has been usually outlined by studying glucose-6-phosphate dehydrogenase (G6PD). However, recent evidence suggests an unexpected role for a less characterized PPP, triggered by hexose-6-phosphate dehydrogenase (H6PD) within the endoplasmic reticulum (ER). Studying H6PD biological role in breast and lung cancer, here we show that gene silencing of this reticular enzyme decreases cell content of PPP intermediates and D-ribose, to a similar extent as G6PD silencing. Decrease in overall NADPH content and increase in cell oxidative status are also comparable. Finally, either gene silencing impairs at a similar degree cell proliferating activity. This unexpected response occurs despite the absence of any cross-interference between the expression of both G6PD and H6PD. Thus, overall cancer PPP reflects the contribution of two different pathways located in the cytosol and ER, respectively. Disregarding the reticular pathway might hamper our comprehension of PPP role in cancer cell biology.

Disruption of redox homeostasis for combinatorial drug efficacy in K-Ras tumors as revealed by metabolic connectivity profiling.

BACKGROUND: Rewiring of metabolism induced by oncogenic K-Ras in cancer cells involves both glucose and glutamine utilization sustaining enhanced, unrestricted growth. The development of effective anti-cancer treatments targeting metabolism may be facilitated by the identification and rational combinatorial targeting of metabolic pathways. METHODS: We performed mass spectrometric metabolomics analysis in vitro and in vivo experiments to evaluate the efficacy of drugs and identify metabolic connectivity. RESULTS: We show that K-Ras-mutant lung and colon cancer cells exhibit a distinct metabolic rewiring, the latter being more dependent on respiration. Combined treatment with the glutaminase inhibitor CB-839 and the PI3K/aldolase inhibitor NVP-BKM120 more consistently reduces cell growth of tumor xenografts. Maximal growth inhibition correlates with the disruption of redox homeostasis, involving loss of reduced glutathione regeneration, redox cofactors, and a decreased connectivity among metabolites primarily involved in nucleic acid metabolism. CONCLUSIONS: Our findings open the way to develop metabolic connectivity profiling as a tool for a selective strategy of combined drug repositioning in precision oncology.

Towards a systems biology approach to mammalian cell cycle: modeling the entrance into S phase of quiescent fibroblasts after serum stimulation.

BACKGROUND: The cell cycle is a complex process that allows eukaryotic cells to replicate chromosomal DNA and partition it into two daughter cells. A relevant regulatory step is in the G0/G1 phase, a point called the restriction (R) point where intracellular and extracellular signals are monitored and integrated.Subcellular localization of cell cycle proteins is increasingly recognized as a major factor that regulates cell cycle transitions. Nevertheless, current mathematical models of the G1/S networks of mammalian cells do not consider this aspect. Hence, there is a need for a computational model that incorporates this regulatory aspect that has a relevant role in cancer, since altered localization of key cell cycle players, notably of inhibitors of cyclin-dependent kinases, has been reported to occur in neoplastic cells and to be linked to cancer aggressiveness. RESULTS: The network of the model components involved in the G1 to S transition process was identified through a literature and web-based data mining and the corresponding wiring diagram of the G1 to S transition drawn with Cell Designer notation. The model has been implemented in Mathematica using Ordinary Differential Equations. Time-courses of level and of sub-cellular localization of key cell cycle players in mouse fibroblasts re-entering the cell cycle after serum starvation/re-feeding have been used to constrain network design and parameter determination. The model allows to recapitulate events from growth factor stimulation to the onset of S phase. The R point estimated by simulation is consistent with the R point experimentally determined. CONCLUSION: The major element of novelty of our model of the G1 to S transition is the explicit modeling of cytoplasmic/nuclear shuttling of cyclins, cyclin-dependent kinases, their inhibitor and complexes. Sensitivity analysis of the network performance newly reveals that the biological effect brought about by Cki overexpression is strictly dependent on whether the Cki is promoting nuclear translocation of cyclin/Cdk containing complexes.

An Intricate Connection between Alternative Splicing and Phenotypic Plasticity in Development and Cancer.

During tumor progression, hypoxia, nutrient deprivation or changes in the extracellular environment (i.e., induced by anti-cancer drugs) elicit adaptive responses in cancer cells. Cellular plasticity increases the chance that tumor cells may survive in a challenging microenvironment, acquire new mechanisms of resistance to conventional drugs, and spread to distant sites. Re-activation of stem pathways appears as a significant cause of cellular plasticity because it promotes the acquisition of stem-like properties through a profound phenotypic reprogramming of cancer cells. In addition, it is a major contributor to tumor heterogeneity, depending on the coexistence of phenotypically distinct subpopulations in the same tumor bulk. Several cellular mechanisms may drive this fundamental change, in particular, high-throughput sequencing technologies revealed a key role for alternative splicing (AS). Effectively, AS is one of the most important pre-mRNA processes that increases the diversity of transcriptome and proteome in a tissue- and development-dependent manner. Moreover, defective AS has been associated with several human diseases. However, its role in cancer cell plasticity and tumor heterogeneity remains unclear. Therefore, unravelling the intricate relationship between AS and the maintenance of a stem-like phenotype may explain molecular mechanisms underlying cancer cell plasticity and improve cancer diagnosis and treatment.