Characterization of the effect of pomegranate crude extract, and its post-harvesting preservation procedures, on redox tone, cellular growth and metabolic profile of MDA-MB-231 cell line.

BACKGROUND: Pomegranate is known for its beneficial properties due to its high content in antioxidants and might constitute a natural option for preventing and treatment of different pathologies including cancer. Since mitochondria are involved in tumorigenesis through ROS production and modulation of oxidative metabolism, we investigated the biological effects of pomegranate on cellular redox state, proliferation and metabolism in the breast cancer cell line MDA-MB-231 (MDA). METHODS: MDA were treated for 24 h with graded concentration of filtered Pomegranate juice (PJ) and tested for metabolic Flux Analysis with XFe96 Extracellular Flux Analyzer, for proliferation using the xCELLigence System Real-Time Cell Analyzer and for intracellular ROS content by Confocal Microscopy Imaging. RESULTS: Cells-treatment with freshly prepared pomegranate juice (PJ) resulted in a significant reduction of the intracellular ROS content already at the lower concentration of PJ tested. Additionally, it enhanced mitochondria respiration, and decreased glycolysis at high concentrations, inhibiting at the same time cell proliferation. As pomegranate is a seasonal fruit, assessment of optimum storage conditions preserving its bio-active properties was investigated. Our results indicated that storage conditions under controlled atmosphere for 30 days was able to enhance mitochondrial respiration at the same extent than freshly extracted PJ. Conversely, freezing procedure, though retaining the antioxidant and cell-growth inhibitory property, elicited an opposite effect on the metabolic profile as compared with fresh extract. CONCLUSION: Overall, the results of our study, on the one hand, confirms the preventive/therapeutic potential of PJ, as well as of its post-harvested processing, for cancer management. On the other hand, it highlights the intrinsic difficulties in attaining mechanistic insights when a multiplicity of effects is elicited by a crude mixture of bio-active compounds.

Myoglobin expression by alternative transcript in different mesenchymal stem cells compartments.

BACKGROUND: The metabolic phenotype of stem cells is increasingly recognized as a hallmark of their pluripotency with mitochondrial and oxygen-related metabolism playing a not completely defined role in this context. In a previous study, we reported the ectopic expression of myoglobin (MB) in bone marrow-derived hematopoietic stem/progenitor cells. Here, we have extended the analysis to mesenchymal stem cells (MSCs) isolated from different tissues. METHODS: MSCs were isolated from human placental membrane, mammary adipose tissue and dental pulp and subjected to RT-PCR, Western blotting and mass spectrometry to investigate the expression of MB. A combination of metabolic flux analysis and cyto-imaging was used to profile the metabolic phenotype and the mitochondria dynamics in the different MSCs. RESULTS: As for the hematopoietic stem/progenitor cells, the expression of Mb was largely driven by an alternative transcript with the protein occurring both in the monomer and in the dimer forms as confirmed by mass spectrometry analysis. Comparing the metabolic fluxes between neonatal placental membrane-derived and adult mammary adipose tissue-derived MSCs, we showed a significantly more active bioenergetics profile in the former that correlated with a larger co-localization of myoglobin with the mitochondrial compartment. Differences in the structure of the mitochondrial network as well as in the expression of factors controlling the organelle dynamics were also observed between neonatal and adult mesenchymal stem cells. Finally, the expression of myoglobin was found to be strongly reduced following osteogenic differentiation of dental pulp-derived MSCs, while it was upregulated following reprogramming of human fibroblasts to induce pluripotent stem cells. CONCLUSIONS: Ectopic expression of myoglobin in tissues other than muscle raises the question of understanding its function therein. Properties in addition to the canonical oxygen storage/delivery have been uncovered. Finding of Mb expressed via an alternative gene transcript in the context of different stem cells with metabolic phenotypes, its loss during differentiation and recovery in iPSCs suggest a hitherto unappreciated role of Mb in controlling the balance between aerobic metabolism and pluripotency. Understanding how Mb contributes through modulation of the mitochondrial physiology to the stem cell biology paves the way to novel perspectives in regenerative medicine as well as in cancer stem cell therapy.

Mitofusin-2 Down-Regulation Predicts Progression of Non-Muscle Invasive Bladder Cancer.

Identification of markers predicting disease outcome is a major clinical issue for non-muscle invasive bladder cancer (NMIBC). The present study aimed to determine the role of the mitochondrial proteins Mitofusin-2 (Mfn2) and caseinolytic protease P (ClpP) in predicting the outcome of NMIBC. The study population consisted of patients scheduled for transurethral resection of bladder tumor upon the clinical diagnosis of bladder cancer (BC). Samples of the main bladder tumor and healthy-looking bladder wall from patients classified as NMIBC were tested for Mfn2 and ClpP. The expression levels of these proteins were correlated to disease recurrence, progression. Mfn2 and ClpP expression levels were significantly higher in lesional than in non-lesional tissue. Low-risk NMIBC had significantly higher Mfn2 expression levels and significantly lower ClpP expression levels than high-risk NMIBC; there were no differences in non-lesional levels of the two proteins. Lesional Mfn2 expression levels were significantly lower in patients who progressed whereas ClpP levels had no impact on any survival outcome. Multivariable analysis adjusting for the EORTC scores showed that Mfn2 downregulation was significantly associated with disease progression. In conclusion, Mfn2 and ClpP proteins were found to be overexpressed in BC as compared to non-lesional bladder tissue and Mfn2 expression predicted disease progression.

How Cells Communicate with Each Other in the Tumor Microenvironment: Suggestions to Design Novel Therapeutic Strategies in Cancer Disease.

Connexin- and pannexin (Panx)-formed hemichannels (HCs) and gap junctions (GJs) operate an interaction with the extracellular matrix and GJ intercellular communication (GJIC), and on account of this they are involved in cancer onset and progression towards invasiveness and metastatization. When we deal with cancer, it is not correct to omit the immune system, as well as neglecting its role in resisting or succumbing to formation and progression of incipient neoplasia until the formation of micrometastasis, nevertheless what really occurs in the tumor microenvironment (TME), which are the main players and which are the tumor or body allies, is still unclear. The goal of this article is to discuss how the pivotal players act, which can enhance or contrast cancer progression during two important process: "Activating Invasion and Metastasis" and the "Avoiding Immune Destruction", with a particular emphasis on the interplay among GJIC, Panx-HCs, and the purinergic system in the TME without disregarding the inflammasome and cytokines thereof derived. In particular, the complex and contrasting roles of Panx1/P2X7R signalosome in tumor facilitation and/or inhibition is discussed in regard to the early/late phases of the carcinogenesis. Finally, considering this complex interplay in the TME between cancer cells, stromal cells, immune cells, and focusing on their means of communication, we should be capable of revealing harmful messages that help the cancer growth and transform them in body allies, thus designing novel therapeutic strategies to fight cancer in a personalized manner.

Unveiling Metabolic Vulnerability and Plasticity of Human Osteosarcoma Stem and Differentiated Cells to Improve Cancer Therapy.

Defining the metabolic phenotypes of cancer-initiating cells or cancer stem cells and of their differentiated counterparts might provide fundamental knowledge for improving or developing more effective therapies. In this context we extensively characterized the metabolic profiles of two osteosarcoma-derived cell lines, the 3AB-OS cancer stem cells and the parental MG-63 cells. To this aim Seahorse methodology-based metabolic flux analysis under a variety of conditions complemented with real time monitoring of cell growth by impedentiometric technique and confocal imaging were carried out. The results attained by selective substrate deprivation or metabolic pathway inhibition clearly show reliance of 3AB-OS on glycolysis and of MG-63 on glutamine oxidation. Treatment of the osteosarcoma cell lines with cisplatin resulted in additive inhibitory effects in MG-63 cells depleted of glutamine whereas it antagonized under selective withdrawal of glucose in 3AB-OS cells thereby manifesting a paradoxical pro-survival, cell-cycle arrest in S phase and antioxidant outcome. All together the results of this study highlight that the efficacy of specific metabolite starvation combined with chemotherapeutic drugs depends on the cancer compartment and suggest cautions in using it as a generalizable curative strategy.

Mitochondrial calcium drives clock gene-dependent activation of pyruvate dehydrogenase and of oxidative phosphorylation.

Regulation of metabolism is emerging as a major output of circadian clock circuitry in mammals. Accordingly, mitochondrial oxidative metabolism undergoes both in vivo and in vitro daily oscillatory activities. In a previous study we showed that both glycolysis and mitochondrial oxygen consumption display a similar time-resolved rhythmic activity in synchronized HepG2 cell cultures, which translates in overall bioenergetic changes as here documented by measurement of the ATP level. Treatment of synchronized cells with specific metabolic inhibitors unveiled pyruvate as a major source of reducing equivalents to the respiratory chain with its oxidation driven by the rhythmic (de)phosphorylation of pyruvate dehydrogenase. Further investigation enabled to causally link the autonomous cadenced mitochondrial respiration to a synchronous increase of the mitochondrial Ca2+. The rhythmic change of the mitochondrial respiration was dampened by inhibitors of the mitochondrial Ca2+ uniporter as well as of the ryanodine receptor Ca2+ channel or the ADPR cyclase, indicating that the mitochondrial Ca2+ influx originated from the ER store, likely at contact sites with the mitochondrial compartment. Notably, blockage of the mitochondrial Ca2+ influx resulted in deregulation of the expression of canonical clock genes such as BMALl1, CLOCK, NR1D1. All together our findings unveil a hitherto unexplored function of Ca2+-mediated signaling in time keeping the mitochondrial metabolism and in its feed-back modulation of the circadian clockwork.

Loss of Function of the Gene Encoding the Histone Methyltransferase KMT2D Leads to Deregulation of Mitochondrial Respiration.

KMT2D encodes a methyltransferase responsible for histone 3 lysine 4 (H3K4) mono-/di-methylation, an epigenetic mark correlated with active transcription. Here, we tested the hypothesis that KMT2D pathogenic loss-of-function variants, which causes the Kabuki syndrome type 1, could affect the mitochondrial metabolic profile. By using Seahorse technology, we showed a significant reduction of the mitochondrial oxygen consumption rate as well as a reduction of the glycolytic flux in both Kmt2d knockout MEFs and skin fibroblasts of Kabuki patients harboring heterozygous KMT2D pathogenic variants. Mass-spectrometry analysis of intermediate metabolites confirmed alterations in the glycolytic and TCA cycle pathways. The observed metabolic phenotype was accompanied by a significant increase in the production of reactive oxygen species. Measurements of the specific activities of the mitochondrial respiratory chain complexes revealed significant inhibition of CI (NADH dehydrogenase) and CIV (cytochrome c oxidase); this result was further supported by a decrease in the protein content of both complexes. Finally, we unveiled an impaired oxidation of glucose and larger reliance on long-chain fatty acids oxidation. Altogether, our findings clearly indicate a rewiring of the mitochondrial metabolic phenotype in the KMT2D-null or loss-of-function context that might contribute to the development of Kabuki disease, and represents metabolic reprogramming as a potential new therapeutic approach.

Nandrolone induces a stem cell-like phenotype in human hepatocarcinoma-derived cell line inhibiting mitochondrial respiratory activity.

Nandrolone is a testosterone analogue with anabolic properties commonly abused worldwide, recently utilized also as therapeutic agent in chronic diseases, cancer included. Here we investigated the impact of nandrolone on the metabolic phenotype in HepG2 cell line. The results attained show that pharmacological dosage of nandrolone, slowing cell growth, repressed mitochondrial respiration, inhibited the respiratory chain complexes I and III and enhanced mitochondrial reactive oxygen species (ROS) production. Intriguingly, nandrolone caused a significant increase of stemness-markers in both 2D and 3D cultures, which resulted to be CxIII-ROS dependent. Notably, nandrolone negatively affected differentiation both in healthy hematopoietic and mesenchymal stem cells. Finally, nandrolone administration in mice confirmed the up-regulation of stemness-markers in liver, spleen and kidney. Our observations show, for the first time, that chronic administration of nandrolone, favoring maintenance of stem cells in different tissues would represent a precondition that, in addition to multiple hits, might enhance risk of carcinogenesis raising warnings about its abuse and therapeutic utilization.

A joint molecular networking study of a Smenospongia sponge and a cyanobacterial bloom revealed new antiproliferative chlorinated polyketides.

The bloom-forming cyanobacteria Trichodesmium sp. have been recently shown to produce some of the chlorinated peptides/polyketides previously isolated from the marine sponge Smenospongia aurea. A comparative analysis of extracts from S. aurea and Trichodesmium sp. was performed using tandem mass spectrometry-based molecular networking. The analysis, specifically targeted to chlorinated metabolites, showed that many of them are common to the two organisms, but also that some general differences exist between the two metabolomes. Following this analysis, six new chlorinated metabolites were isolated and their structures elucidated: four polyketides, smenolactones A-D (1-4) from S. aurea, and two new conulothiazole analogues, isoconulothiazole B (5) and conulothiazole C (6) from Trichodesmium sp. The absolute configuration of smenolactone C (3) was determined by taking advantage of the conformational rigidity of open 1,3-disubstituted alkyl chains. The antiproliferative activity of smenolactones was evaluated on three tumor cell lines, and they were active at low-micromolar or sub-micromolar concentrations.

In Vitro Antiproliferative Evaluation of Synthetic Meroterpenes Inspired by Marine Natural Products.

Several marine natural linear prenylquinones/hydroquinones have been identified as anticancer and antimutagenic agents. Structure-activity relationship studies on natural compounds and their synthetic analogs demonstrated that these effects depend on the length of the prenyl side chain and on the type and position of the substituent groups in the quinone moiety. Aiming to broaden the knowledge of the underlying mechanism of the antiproliferative effect of these prenylated compounds, herein we report the synthesis of two quinones 4 and 5 and of their corresponding dioxothiazine fused quinones 6 and 7 inspired to the marine natural product aplidinone A (1), a geranylquinone featuring the 1,1-dioxo-1,4-thiazine ring isolated from the ascidian Aplidium conicum. The potential effects on viability and proliferation in three different human cancer cell lines, breast adenocarcinoma (MCF-7), pancreas adenocarcinoma (Bx-PC3) and bone osteosarcoma (MG-63), were investigated. The methoxylated geranylquinone 5 exerted the highest antiproliferative effect exhibiting a comparable toxicity in all three cell lines analyzed. Interestingly, a deeper investigation has highlighted a cytostatic effect of quinone 5 referable to a G0/G1 cell-cycle arrest in BxPC-3 cells after 24 h treatment.

Dichloroacetate (DCA) and Cancer: An Overview towards Clinical Applications.

An extensive body of literature describes anticancer property of dichloroacetate (DCA), but its effective clinical administration in cancer therapy is still limited to clinical trials. The occurrence of side effects such as neurotoxicity as well as the suspicion of DCA carcinogenicity still restricts the clinical use of DCA. However, in the last years, the number of reports supporting DCA employment against cancer increased also because of the great interest in targeting metabolism of tumour cells. Dissecting DCA mechanism of action helped to understand the bases of its selective efficacy against cancer cells. A successful coadministration of DCA with conventional chemotherapy, radiotherapy, other drugs, or natural compounds has been tested in several cancer models. New drug delivery systems and multiaction compounds containing DCA and other drugs seem to ameliorate bioavailability and appear more efficient thanks to a synergistic action of multiple agents. The spread of reports supporting the efficiency of DCA in cancer therapy has prompted additional studies that let to find other potential molecular targets of DCA. Interestingly, DCA could significantly affect cancer stem cell fraction and contribute to cancer eradication. Collectively, these findings provide a strong rationale towards novel clinical translational studies of DCA in cancer therapy.

Gap Junction Intercellular Communication in the Carcinogenesis Hallmarks: Is This a Phenomenon or Epiphenomenon?

If occupational tumors are excluded, cancer causes are largely unknown. Therefore, it appeared useful to work out a theory explaining the complexity of this disease. More than fifty years ago the first demonstration that cells communicate with each other by exchanging ions or small molecules through the participation of connexins (Cxs) forming Gap Junctions (GJs) occurred. Then the involvement of GJ Intercellular Communication (GJIC) in numerous physiological cellular functions, especially in proliferation control, was proven and accounts for the growing attention elicited in the field of carcinogenesis. The aim of the present paper is to verify and discuss the role of Cxs, GJs, and GJIC in cancer hallmarks, pointing on the different involved mechanisms in the context of the multi-step theory of carcinogenesis. Functional GJIC acts both as a tumor suppressor and as a tumor enhancer in the metastatic stage. On the contrary, lost or non-functional GJs allow the uncontrolled proliferation of stem/progenitor initiated cells. Thus, GJIC plays a key role in many biological phenomena or epiphenomena related to cancer. Depending on this complexity, GJIC can be considered a tumor suppressor in controlling cell proliferation or a cancer ally, with possible preventive or therapeutic implications in both cases.

Deferasirox drives ROS-mediated differentiation and induces interferon-stimulated gene expression in human healthy haematopoietic stem/progenitor cells and in leukemia cells.

BACKGROUND: Administration of the iron chelator deferasirox (DFX) in transfusion-dependent patients occasionally results in haematopoiesis recovery by a mechanism remaining elusive. This study aimed to investigate at a molecular level a general mechanism underlying DFX beneficial effects on haematopoiesis, both in healthy and pathological conditions. METHODS: Human healthy haematopoietic stem/progenitor cells (HS/PCs) and three leukemia cell lines were treated with DFX. N-Acetyl cysteine (NAC) and fludarabine were added as antioxidant and STAT1 inhibitor, respectively. In vitro colony-forming assays were assessed both in healthy and in leukemia cells. Intracellular and mitochondrial reactive oxygen species (ROS) as well as mitochondrial content were assessed by cytofluorimetric and confocal microscopy analysis; mtDNA was assessed by qRT-PCR. Differentiation markers were monitored by cytofluorimetric analysis. Gene expression analysis (GEA) was performed on healthy HS/PCs, and differently expressed genes were validated in healthy and leukemia cells by qRT-PCR. STAT1 expression and phosphorylation were assessed by Western blotting. Data were compared by an unpaired Student t test or one-way ANOVA. RESULTS: DFX, at clinically relevant concentrations, increased the clonogenic capacity of healthy human CD34+ HS/PCs to form erythroid colonies. Extension of this analysis to human-derived leukemia cell lines Kasumi-1, K562 and HL60 confirmed DFX capacity to upregulate the expression of specific markers of haematopoietic commitment. Notably, the abovementioned DFX-induced effects are all prevented by the antioxidant NAC and accompanied with overproduction of mitochondria-generated reactive oxygen species (ROS) and increase of mitochondrial content and mtDNA copy number. GEA unveiled upregulation of genes linked to interferon (IFN) signalling and tracked back to hyper-phosphorylation of STAT1. Treatment of leukemic cell lines with NAC prevented the DFX-mediated phosphorylation of STAT1 as well as the expression of the IFN-stimulated genes. However, STAT1 inhibition by fludarabine was not sufficient to affect differentiation processes in leukemic cell lines. CONCLUSIONS: These findings suggest a significant involvement of redox signalling as a major regulator of multiple DFX-orchestrated events promoting differentiation in healthy and tumour cells. The understanding of molecular mechanisms underlying the haematological response by DFX would enable to predict patient's ability to respond to the drug, to extend treatment to other patients or to anticipate the treatment, regardless of the iron overload.

Dichloroacetate Affects Mitochondrial Function and Stemness-Associated Properties in Pancreatic Cancer Cell Lines.

Targeting metabolism represents a possible successful approach to treat cancer. Dichloroacetate (DCA) is a drug known to divert metabolism from anaerobic glycolysis to mitochondrial oxidative phosphorylation by stimulation of PDH. In this study, we investigated the response of two pancreatic cancer cell lines to DCA, in two-dimensional and three-dimension cell cultures, as well as in a mouse model. PANC-1 and BXPC-3 treated with DCA showed a marked decrease in cell proliferation and migration which did not correlate with enhanced apoptosis indicating a cytostatic rather than a cytotoxic effect. Despite PDH activation, DCA treatment resulted in reduced mitochondrial oxygen consumption without affecting glycolysis. Moreover, DCA caused enhancement of ROS production, mtDNA, and of the mitophagy-marker LC3B-II in both cell lines but reduced mitochondrial fusion markers only in BXPC-3. Notably, DCA downregulated the expression of the cancer stem cells markers CD24/CD44/EPCAM only in PANC-1 but inhibited spheroid formation/viability in both cell lines. In a xenograft pancreatic cancer mouse-model DCA treatment resulted in retarding cancer progression. Collectively, our results clearly indicate that the efficacy of DCA in inhibiting cancer growth mechanistically depends on the cell phenotype and on multiple off-target pathways. In this context, the novelty that DCA might affect the cancer stem cell compartment is therapeutically relevant.

Studying Werner syndrome to elucidate mechanisms and therapeutics of human aging and age-related diseases.

Aging is a natural and unavoidable part of life. However, aging is also the primary driver of the dominant human diseases, such as cardiovascular disease, cancer, and neurodegenerative diseases, including Alzheimer's disease. Unraveling the sophisticated molecular mechanisms of the human aging process may provide novel strategies to extend 'healthy aging' and the cure of human aging-related diseases. Werner syndrome (WS), is a heritable human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. As a classical premature aging disease, etiological exploration of WS can shed light on the mechanisms of normal human aging and facilitate the development of interventional strategies to improve healthspan. Here, we summarize the latest progress of the molecular understandings of WRN protein, highlight the advantages of using different WS model systems, including Caenorhabditis elegans, Drosophila melanogaster and induced pluripotent stem cell (iPSC) systems. Further studies on WS will propel drug development for WS patients, and possibly also for normal age-related diseases.

Clogging the Ubiquitin-Proteasome Machinery with Marine Natural Products: Last Decade Update.

The ubiquitin-proteasome pathway (UPP) is the central protein degradation system in eukaryotic cells, playing a key role in homeostasis maintenance, through proteolysis of regulatory and misfolded (potentially harmful) proteins. As cancer cells produce proteins inducing cell proliferation and inhibiting cell death pathways, UPP inhibition has been exploited as an anticancer strategy to shift the balance between protein synthesis and degradation towards cell death. Over the last few years, marine invertebrates and microorganisms have shown to be an unexhaustive factory of secondary metabolites targeting the UPP. These chemically intriguing compounds can inspire clinical development of novel antitumor drugs to cope with the incessant outbreak of side effects and resistance mechanisms induced by currently approved proteasome inhibitors (e.g., bortezomib). In this review, we report about (a) the role of the UPP in anticancer therapy, (b) chemical and biological properties of UPP inhibitors from marine sources discovered in the last decade, (c) high-throughput screening techniques for mining natural UPP inhibitors in organic extracts. Moreover, we will tell about the fascinating story of salinosporamide A, the first marine natural product to access clinical trials as a proteasome inhibitor for cancer treatment.

Targeting Endoplasmic Reticulum and/or Mitochondrial Ca2+ Fluxes as Therapeutic Strategy for HCV Infection.

Chronic hepatitis C is characterized by metabolic disorders and by a microenvironment in the liver dominated by oxidative stress, inflammation and regeneration processes that can in the long term lead to liver cirrhosis and hepatocellular carcinoma. Several lines of evidence suggest that mitochondrial dysfunctions play a central role in these processes. However, how these dysfunctions are induced by the virus and whether they play a role in disease progression and neoplastic transformation remains to be determined. Most in vitro studies performed so far have shown that several of the hepatitis C virus (HCV) proteins also localize to mitochondria, but the consequences of these interactions on mitochondrial functions remain contradictory and need to be confirmed in the context of productively replicating virus and physiologically relevant in vitro and in vivo model systems. In the past decade we have been proposing a temporal sequence of events in the HCV-infected cell whereby the primary alteration is localized at the mitochondria-associated ER membranes and causes release of Ca2+ from the ER, followed by uptake into mitochondria. This ensues successive mitochondrial dysfunction leading to the generation of reactive oxygen and nitrogen species and a progressive metabolic adaptive response consisting in decreased oxidative phosphorylation and enhanced aerobic glycolysis and lipogenesis. Here we resume the major results provided by our group in the context of HCV-mediated alterations of the cellular inter-compartmental calcium flux homeostasis and present new evidence suggesting targeting of ER and/or mitochondrial calcium transporters as a novel therapeutic strategy.

Para-hydroxyphenylpyruvate inhibits the pro-inflammatory stimulation of macrophage preventing LPS-mediated nitro-oxidative unbalance and immunometabolic shift.

Targeting metabolism is emerging as a promising therapeutic strategy for modulation of the immune response in human diseases. In the presented study we used the lipopolysaccharide (LPS)-mediated activation of RAW 264.7 macrophage-like cell line as a model to investigate changes in the metabolic phenotype and to test the effect of p-hydroxyphenylpyruvate (pHPP) on it. pHPP is an intermediate of the PHE/TYR catabolic pathway, selected as analogue of the ethyl pyruvate (EP), which proved to exhibit antioxidant and anti-inflammatory activities. The results obtained show that LPS-priming of RAW 264.7 cell line to the activated M1 state resulted in up-regulation of the inducible nitric oxide synthase (iNOS) expression and consequently of NO production and in release of the pro-inflammatory cytokine IL-6. All these effects were prevented dose dependently by mM concentrations of pHPP more efficiently than EP. Respirometric and metabolic flux analysis of LPS-treated RAW 264.7 cells unveiled a marked metabolic shift consisting in downregulation of the mitochondrial oxidative phosphorylation and upregulation of aerobic glycolysis respectively. The observed respiratory failure in LPS-treated cells was accompanied with inhibition of the respiratory chain complexes I and IV and enhanced production of reactive oxygen species. Inhibition of the respiratory activity was also observed following incubation of human neonatal fibroblasts (NHDF-neo) with sera from septic patients. pHPP prevented all the observed metabolic alteration caused by LPS on RAW 264.7 or by septic sera on NHDF-neo. Moreover, we provide evidence that pHPP is an efficient reductant of cytochrome c. On the basis of the presented results a working model, linking pathogen-associated molecular patterns (PAMPs)-mediated immune response to mitochondrial oxidative metabolism, is put forward along with suggestions for its therapeutic control.

Rewiring carbohydrate catabolism differentially affects survival of pancreatic cancer cell lines with diverse metabolic profiles.

An increasing body of evidence suggests that targeting cellular metabolism represents a promising effective approach to treat pancreatic cancer, overcome chemoresistance and ameliorate patient's prognosis and survival. In this study, following whole-genome expression analysis, we selected two pancreatic cancer cell lines, PANC-1 and BXPC-3, hallmarked by distinct metabolic profiles with specific concern to carbohydrate metabolism. Functional comparative analysis showed that BXPC-3 displayed a marked deficit of the mitochondrial respiratory and oxidative phosphorylation activity and a higher production of reactive oxygen species and a reduced NAD+/NADH ratio, indicating their bioenergetic reliance on glycolysis and a different redox homeostasis as compared to PANC-1. Both cell lines were challenged to rewire their metabolism by substituting glucose with galactose as carbon source, a condition inhibiting the glycolytic flux and fostering full oxidation of the sugar carbons. The obtained data strikingly show that the mitochondrial respiration-impaired-BXPC-3 cell line was unable to sustain the metabolic adaptation required by glucose deprivation/substitution, thereby resulting in a G2\M cell cycle shift, unbalance of the redox homeostasis, apoptosis induction. Conversely, the mitochondrial respiration-competent-PANC-1 cell line did not show clear evidence of cell sufferance. Our findings provide a strong rationale to candidate metabolism as a promising target for cancer therapy. Defining the metabolic features at time of pancreatic cancer diagnosis and likely of other tumors, appears to be crucial to predict the responsiveness to therapeutic approaches or coadjuvant interventions affecting metabolism.