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To date, it is well-established that mitochondrial dysfunction does not only play a vital role in cancer but also in other pathological conditions such as neurodegenerative diseases and inflammation. An important tool for the analysis of cellular metabolism is the application of stable isotope labeled substrates, which allow for the tracing of atoms throughout metabolic networks. While such analyses yield very detailed information about intracellular fluxes, the determination of compartment specific fluxes is far more challenging. Most approaches for the deconvolution of compartmented metabolism use computational models whereas experimental methods are rare. Here, we developed an experimental setup based on selective permeabilization of the cytosolic membrane that allows for the administration of stable isotope labeled substrates directly to mitochondria. We demonstrate how this approach can be used to infer metabolic changes in mitochondria induced by either chemical or genetic perturbations and give an outlook on its potential applications. [ABSTRACT FROM AUTHOR]

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment. [ABSTRACT FROM AUTHOR]

Cancer cells generally rely mostly on glycolysis rather than oxidative phosphorylation (OXPHOS) for ATP production. In fact, they are particularly sensitive to glycolysis inhibition and glucose depletion. On the other hand mitochondrial dysfunctions, involved in the onset of the Warburg effect, are sometimes also associated with the resistance to apoptosis that characterizes cancer cells. Therefore, combined treatments targeting both glycolysis and mitochondria function, exploiting peculiar tumor features, might be lethal for cancer cells. In this study, we show that glucose deprivation and mitochondrial Complex I inhibitors synergize in inducing cancer cell death. In particular, our results reveal that low doses of Complex I inhibitors, ineffective on immortalized cells and in high glucose growth, become specifically cytotoxic on cancer cells deprived of glucose. Importantly, the cytotoxic effect of the inhibitors on cancer cells is strongly enhanced by forskolin, a PKA pathway activator, that we have previously shown to stimulate OXPHOS. Taken together, we demonstrate that induction in cancer cells of a switch from a glycolytic to a more respirative metabolism, obtained by glucose depletion or mitochondrial activity stimulation, strongly increases their sensitivity to low doses of mitochondrial Complex I inhibitors. Our findings might be a valuable approach to eradicate cancer cells. [ABSTRACT FROM AUTHOR]

Synthesis and in vitro testing of a prodrug designed for the controlled delivery of the anticancer drug camptothecin within pancreatic cancer cells are reported. Our study reveals a non-conventional pharmacokinetic release characterized by an exponential pattern before reaching the half-life (t1/2) and a linear pattern thereafter. The release mechanism was triggered either by hydrolytic enzymes and/or by the acid microenvironment of cancer cells. [ABSTRACT FROM AUTHOR]

Metabolism and mitochondrial biology have gained a prominent role as determinants of stem cell fate and function. In the context of regenerative medicine, innovative parameters predictive of therapeutic efficacy could be drawn from the association of metabolic or mitochondrial parameters to different degrees of stemness and differentiation potentials. Herein, this possibility was addressed in human mesenchymal stromal/stem cells (hMSC) previously shown to differ in lifespan and telomere length. First, these hMSC were shown to possess significantly distinct proliferation rate, senescence status and differentiation capacity. More potential hMSC were associated to higher mitochondrial (mt) DNA copy number and lower mtDNA methylation. In addition, they showed higher expression levels of oxidative phosphorylation subunits. Consistently, they exhibited higher coupled oxygen consumption rate and lower transcription of glycolysis-related genes, glucose consumption and lactate production. All these data pointed at oxidative phosphorylation-based central metabolism as a feature of higher stemness-associated hMSC phenotypes. Consistently, reduction of mitochondrial activity by complex I and III inhibitors in higher stemness-associated hMSC triggered senescence. Finally, functionally higher stemness-associated hMSC showed metabolic plasticity when challenged by glucose or glutamine shortage, which mimic bioenergetics switches that hMSC must undergo after transplantation or during self-renewal and differentiation. Altogether, these results hint at metabolic and mitochondrial parameters that could be implemented to identify stem cells endowed with superior growth and differentiation potential. [ABSTRACT FROM AUTHOR]

Motivation The elucidation of dysfunctional cellular processes that can induce the onset of a disease is a challenging issue from both the experimental and computational perspectives. Here we introduce a novel computational method based on the coupling between fuzzy logic modeling and a global optimization algorithm, whose aims are to (1) predict the emergent dynamical behaviors of highly heterogeneous systems in unperturbed and perturbed conditions, regardless of the availability of quantitative parameters, and (2) determine a minimal set of system components whose perturbation can lead to a desired system response, therefore facilitating the design of a more appropriate experimental strategy. Results We applied this method to investigate what drives K-ras-induced cancer cells, displaying the typical Warburg effect, to death or survival upon progressive glucose depletion. The optimization analysis allowed to identify new combinations of stimuli that maximize pro-apoptotic processes. Namely, our results provide different evidences of an important protective role for protein kinase A in cancer cells under several cellular stress conditions mimicking tumor behavior. The predictive power of this method could facilitate the assessment of the response of other complex heterogeneous systems to drugs or mutations in fields as medicine and pharmacology, therefore paving the way for the development of novel therapeutic treatments. Availability and implementation The source code of FUMOSO is available under the GPL 2.0 license on GitHub at the following URL: https://github.com/aresio/FUMOSO Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

A library of GlcNAc 6‐ or 1‐phosphate analogues was designed, and each compound was evaluated computationally through docking studies for its binding affinity to AGM1/PGM3. The compounds with the highest binding affinity, as ranked through a docking score, were synthesised and screened for their ability to inhibit the production of UDP‐GlcNAc. A glycofused oxazoline analogue showed good inhibition, and gave significant results in vitro. [ABSTRACT FROM AUTHOR]

To assess the evidence on SARS-CoV2 infection and Covid-19 in relation to deficiency and supplementation of vitamin D, we conducted a systematic review up to April 2021. We summarised data from 38 eligible studies, which presented risk estimates for at least one endpoint, including two RCT and 27 cohort-studies: 205565 patients with information on 25OHD status and 2022 taking vitamin D supplementation with a total of 1197 admitted to the ICU or who needed invasive mechanical ventilation or intubation and hospital stay, and more than 910 Covid-19 deaths. Primary outcomes were severity and mortality and the main aim was to evaluate the association with vitamin D supplementation. Random effects models showed that supplementation was associated with a significant lower risk of both Covid-19 severe disease (SRR 0.38, 95% CI 0.20–0.72, 6 studies) and mortality (SRR 0.35, 95% CI 0.17–0.70, 8 studies). There were no statistically significant dose differences between studies: summary estimates with regular doses remain statistically significant, suggesting that higher doses are not necessary. For patients on vitamin D supplementation, a greater reduction in mortality risk emerged in older individuals and at higher latitudes. Regarding the quality of studies, assessed using the New Castle-Ottawa quality scale, the analysis revealed in most cases no statistically significant differences between low, medium or high quality studies. We found significant associations of vitamin D supplementation with Covid-19, encompassing risks of disease worsening and mortality, especially in seasons characterized by 25OHD deficiency and with not severe patients. Dedicated randomized clinical studies are encouraged to confirm these results. [ABSTRACT FROM AUTHOR]

Significance: Histone deacetylases (HDACs) activity and cell metabolism are considered important targets for cancer therapy, as both are deregulated and associated with the onset and maintenance of tumors. Recent Advances: Besides the classical function of HDACs as HDAC enzymes controlling the transcription, it is becoming increasingly evident that these proteins are involved in the regulation of several other cellular processes by their ability to deacetylate hundreds of proteins with different functions in both the cytoplasm and the nucleus. Importantly, recent high-throughput studies have identified as important target proteins several enzymes involved in different metabolic pathways. Conversely, it has been also shown that metabolic intermediates may control HDACs activity. Consequently, the acetylation/deacetylation of metabolic enzymes and the ability of metabolic intermediates to modulate HDACs may represent a cross-talk connecting cell metabolism, transcription, and other HDACs-controlled processes in physiological and pathological conditions. Critical Issues: Since metabolic alterations and HDACs deregulation are important cancer hallmarks, disclosing connections among them may improve our understanding on cancer mechanisms and reveal novel therapeutic protocols against this disease. Future Directions: High-throughput metabolic studies performed by using more sophisticated technologies applied to the available models of conditional deletion of HDACs in cell lines or in mice will fill the gap in the current understanding and open directions for future research. Antioxid. Redox Signal. 23, 30-50. [ABSTRACT FROM AUTHOR]

Alterations in glucose and glutamine utilizing pathways and in fatty acid metabolism are currently considered the most significant and prevalent metabolic changes observed in almost all types of tumors. Glucose, glutamine and fatty acids are the substrates for the hexosamine biosynthetic pathway (HBP). This metabolic pathway generates the “sensing molecule” UDP-N-Acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is the substrate for the enzymes involved in protein N- and O-glycosylation, two important post-translational modifications (PTMs) identified in several proteins localized in the extracellular space, on the cell membrane and in the cytoplasm, nucleus and mitochondria. Since protein glycosylation controls several key aspects of cell physiology, aberrant protein glycosylation has been associated with different human diseases, including cancer. Here we review recent evidence indicating the tight association between the HBP flux and cell metabolism, with particular emphasis on the post-transcriptional and transcriptional mechanisms regulated by the HBP that may cause the metabolic rewiring observed in cancer. We describe the implications of both protein O- and N-glycosylation in cancer cell metabolism and bioenergetics; focusing our attention on the effect of these PTMs on nutrient transport and on the transcriptional regulation and function of cancer-specific metabolic pathways. [ABSTRACT FROM AUTHOR]

Macrophages undergo extensive metabolic reprogramming during classical pro-inflammatory polarization (M1-like). The accumulation of itaconate has been recognized as both a consequence and mediator of the inflammatory response. In this study we first examined the specific functions of itaconate inside fractionated mitochondria. We show that M1 macrophages produce itaconate de novo via aconitase decarboxylase 1 (ACOD1) inside mitochondria. The carbon for this reaction is not only supplied by oxidative TCA cycling, but also through the reductive carboxylation of α-ketoglutarate by isocitrate dehydrogenase (IDH). While macrophages are capable of sustaining a certain degree of itaconate production during hypoxia by augmenting the activity of IDH-dependent reductive carboxylation, we demonstrate that sufficient itaconate synthesis requires a balance of reductive and oxidative TCA cycle metabolism in mouse macrophages. In comparison, human macrophages increase itaconate accumulation under hypoxic conditions by augmenting reductive carboxylation activity. We further demonstrated that itaconate attenuates reductive carboxylation at IDH2, restricting its own production and the accumulation of the immunomodulatory metabolites citrate and 2-hydroxyglutarate. In line with this, reductive carboxylation is enhanced in ACOD1-depleted macrophages. Mechanistically, the inhibition of IDH2 by itaconate is linked to the alteration of the mitochondrial NADP+/NADPH ratio and competitive succinate dehydrogenase inhibition. Taken together, our findings extend the current model of TCA cycle reprogramming during pro-inflammatory macrophage activation and identified novel regulatory properties of itaconate. [Display omitted] • Fractionated mitochondria produce itaconate de novo. • Itaconate is produced via IDH-dependent reductive carboxylation. • Itaconate synthesis requires bidirectional TCA cycle metabolism • Reductive carboxylation at IDH2 is readily inhibited by itaconate. • Divergent reprogramming of TCA cycle fluxes in different macrophage types. [ABSTRACT FROM AUTHOR]

Aims: Vorinostat (suberoylanilide hydroxamic acid; SAHA) is a histone deacetylase inhibitor (HDACi) approved in the clinics for the treatment of T-cell lymphoma and with the potential to be effective also in breast cancer. We investigated the responsiveness to SAHA in human breast primary tumors and cancer cell lines. Results: We observed a differential response to drug treatment in both human breast primary tumors and cancer cell lines. Gene expression analysis of the breast cancer cell lines revealed that genes involved in cell adhesion and redox pathways, especially glutathione metabolism, were differentially expressed in the cell lines resistant to SAHA compared with the sensitive ones, indicating their possible association with drug resistance mechanisms. Notably, such an association was also observed in breast primary tumors. Indeed, addition of buthionine sulfoximine (BSO), a compound capable of depleting cellular glutathione, significantly enhanced the cytotoxicity of SAHA in both breast cancer cell lines and primary breast tumors. Innovation: We identify and validate transcriptional differences in genes involved in redox pathways, which include potential predictive markers of sensitivity to SAHA. Conclusion: In breast cancer, it could be relevant to evaluate the expression of antioxidant genes that may favor tumor resistance as a factor to consider for potential clinical application and treatment with epigenetic drugs (HDACis). Antioxid. Redox Signal. 23, 15-29. [ABSTRACT FROM AUTHOR]

Environmental contributions to cancer development are widely accepted, but only a fraction of all pertinent exposures have probably been identified. Traditional toxicological approaches to the problem have largely focused on the effects of individual agents at singular endpoints. As such, they have incompletely addressed both the pro-carcinogenic contributions of environmentally relevant low-dose chemical mixtures and the fact that exposures can influence multiple cancer associated endpoints over varying timescales. Of these endpoints, dysregulated metabolism is one of the most common and recognizable features of cancer, but its specific roles in exposure-associated cancer development remain poorly understood. Most studies have focused on discrete aspects of cancer metabolism and have incompletely considered both its dynamic integrated nature and the complex controlling influences of substrate availability, external trophic signals and environmental conditions. Emerging high throughput approaches to environmental risk assessment also do not directly address the metabolic causes or consequences of changes in gene expression. As such, there is a compelling need to establish common or complementary frameworks for further exploration that experimentally and conceptually consider the gestalt of cancer metabolism and its causal relationships to both carcinogenesis and the development of other cancer hallmarks. A literature review to identify environmentally relevant exposures unambiguously linked to both cancer development and dysregulated metabolism suggests major gaps in our understanding of exposure-associated carcinogenesis and metabolic reprogramming. Although limited evidence exists to support primary causal roles for metabolism in carcinogenesis, the universality of altered cancer metabolism underscores its fundamental biological importance, and multiple pleiomorphic, even dichotomous, roles for metabolism in promoting, antagonizing or otherwise enabling the development and selection of cancer are suggested. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death and the search for a resolutive therapy is still a challenge. Since KRAS is commonly mutated in PDAC and is one of the main drivers of PDAC progression, its inhibition should be a key strategy for treatment, especially considering the recent development of specific KRAS inhibitors. Nevertheless, the effects of KRAS inhibition can be increased through the co-inhibition of other nodes important for cancer development. One of them could be the hexosamine biosynthetic pathway (HBP), whose enhancement is considered fundamental for PDAC. Here, we demonstrate that PDAC cells expressing oncogenic KRAS, owing to an increase in the HBP flux, become strongly reliant on HBP for both proliferation and survival. In particular, upon treatment with two different compounds, 2-deoxyglucose and FR054, inhibiting both HBP and protein N-glycosylation, these cells undergo apoptosis significantly more than PDAC cells expressing wild-type KRAS. Importantly, we also show that the combined treatment between FR054 and the pan-RAS inhibitor BI-2852 has an additive negative effect on cell proliferation and survival by means of the suppression of both Akt activity and cyclin D1 expression. Thus, co-inhibition of HBP and oncogenic RAS may represent a novel therapy for PDAC patients. [ABSTRACT FROM AUTHOR]