Autonomous Oscillatory Mitochondrial Respiratory Activity: Results of a Systematic Analysis Show Heterogeneity in Different In Vitro-Synchronized Cancer Cells.

Circadian oscillations of several physiological and behavioral processes are an established process in all the organisms anticipating the geophysical changes recurring during the day. The time-keeping mechanism is controlled by a transcription translation feedback loop involving a set of well-characterized transcription factors. The synchronization of cells, controlled at the organismal level by a brain central clock, can be mimicked in vitro, pointing to the notion that all the cells are endowed with an autonomous time-keeping system. Metabolism undergoes circadian control, including the mitochondrial terminal catabolic pathways, culminating under aerobic conditions in the electron transfer to oxygen through the respiratory chain coupled to the ATP synthesis according to the oxidative phosphorylation chemiosmotic mechanism. In this study, we expanded upon previous isolated observations by utilizing multiple cell types, employing various synchronization protocols and different methodologies to measure mitochondrial oxygen consumption rates under conditions simulating various metabolic stressors. The results obtained clearly demonstrate that mitochondrial respiratory activity undergoes rhythmic oscillations in all tested cell types, regardless of their individual respiratory proficiency, indicating a phenomenon that can be generalized. However, notably, while primary cell types exhibited similar rhythmic respiratory profiles, cancer-derived cell lines displayed highly heterogeneous rhythmic changes. This observation confirms on the one hand the dysregulation of the circadian control of the oxidative metabolism observed in cancer, likely contributing to its development, and on the other hand underscores the necessity of personalized chronotherapy, which necessitates a detailed characterization of the cancer chronotype.

The Relationship between Clock Genes, Sirtuin 1, and Mitochondrial Activity in Head and Neck Squamous Cell Cancer: Effects of Melatonin Treatment.

The circadian clock is a regulatory system, with a periodicity of approximately 24 h, which generates rhythmic changes in many physiological processes, including mitochondrial activity. Increasing evidence links chronodisruption with aberrant functionality in clock gene expression, resulting in multiple diseases such as cancer. Melatonin, whose production and secretion oscillates according to the light-dark cycle, is the principal regulator of clock gene expression. In addition, the oncostatic effects of melatonin correlate with an increase in mitochondrial activity. However, the direct links between circadian clock gene expression, mitochondrial activity, and the antiproliferative effects of melatonin in cancers, including head and neck squamous cell carcinoma (HNSCC), remain largely unknown. In this study, we analyzed the effects of melatonin on HNSCC cell lines (Cal-27 and SCC9), which were treated with 500 and 1000 µM melatonin. We found that the antiproliferative effect of melatonin is not mediated by the Bmal1 clock gene. Additionally, high doses of melatonin were observed to result in resynchronization of oscillatory circadian rhythm genes (Per2 and Sirt1). Surprisingly, the resynchronizing effect of melatonin on Per2 and Sirt1 did not produce alterations in the oscillation of mitochondrial respiratory activity. These results increase our understanding of the possible antiproliferative mechanisms in melatonin in the treatment of head and neck squamous cell carcinoma and suggest that its antiproliferative effects are independent of clock genes but are directly related to mitochondrial activity.

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.

The Histone Variant MacroH2A1 Impacts Circadian Gene Expression and Cell Phenotype in an In Vitro Model of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. A foremost risk factor for HCC is obesity/metabolic syndrome-related non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), which is prompted by remarkable changes in transcription patterns of genes enriching metabolic, immune/inflammatory, and circadian pathways. Epigenetic mechanisms play a role in NAFLD-associated HCC, and macroH2A1, a variant of histone H2A, is involved in the pathogenesis modulating the expression of oncogenes and/or tumor suppressor genes and interacting with SIRT1, which crucially impacts the circadian clock circuitry. Hence, we aimed to appraise if and how macroH2A1 regulated the expression patterns of circadian genes in the setting of NAFLD-associated HCC. We took advantage of an in vitro model of liver cancer represented by HepG2 (human hepatocarcinoma) cells stably knocked down for macroH2A1 and conducted whole transcriptome profiling and deep phenotyping analysis. We found up-regulation of PER1 along with several deregulated circadian genes, enriching several important pathways and functions related to cancer onset and progression, such as epithelial-to-mesenchymal transition, cell cycle deregulation, and DNA damage. PER1 silencing partially mitigated the malignant phenotype induced by the loss of macroH2A1 in HCC cells. In conclusion, our findings suggest a modulatory role for the core circadian protein PER1 in liver carcinogenesis in the context of a lack of the macroH2A1 epigenetic and transcriptional landscape.

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.

High mobilization of CD133+/CD34+ cells expressing HIF-1α and SDF-1α in septic abdominal surgical patients.

BACKGROUND: The control of endothelial progenitor cells (CD133+/CD34+ EPCs) migrating from bone marrow to peripheral blood is not completely understood. Emerging evidence suggests that stromal cell-derived factor-1α (SDF-1α) mediates egression of EPCs from bone marrow, while the hypoxia inducible factor (HIF) transcriptional system regulates SDF-1α expression. Our study aimed to investigate the time course of circulating CD133+/CD34+ EPCs and its correlation with the expression of HIF-1α protein and SDF-1α in postoperative laparoscopic abdominal septic patients. METHODS: Postoperative patients were divided in control (C group) and septic group (S group) operated immediately after the diagnosis of sepsis/septic shock. Blood samples were collected at baseline (0), 1, 3 and 7 postoperative days for CD133+/CD34+ EPCs count expressing or not the HIF-1α and SDF-1α analysis. RESULTS: Thirty-two patients in S group and 39 in C group were analyzed. In C group CD133+/CD34+ EPCs count remained stable throughout the study period, increasing on day 7 (173 [0-421] /µl vs baseline: P = 0.04; vs day 1: P = 0.002). In S group CD133+/CD34+ EPCs count levels were higher on day 3 (vs day 1: P = 0.006 and day 7: P = 0.026). HIF-1α expressing CD133+/CD34+ EPCs count decreased on day 1 as compared with the other days in C group (day 0 vs 1: P = 0.003, days 3 and 7 vs 1: P = 0.008), while it was 321 [0-1418] /µl on day 3 (vs day 1; P = 0.004), and 400 [0-587] /µl on day 7 in S group. SDF-1α levels were higher not only on baseline but also on postoperative day 1 in S vs C group (219 [124-337] pg/ml vs 35 [27-325] pg/ml, respectively; P = 0.01). CONCLUSION: Our results indicate that sepsis in abdominal laparoscopic patients might constitute an additional trigger of the EPCs mobilization as compared with non-septic surgical patients. A larger mobilization of CD133+/CD34+ EPCs, preceded by enhanced plasmatic SDF-1α, occurs in septic surgical patients regardless of HIF-1α expression therein. TRIAL REGISTRATION: ClinicalTrials.gov no. NCT02589535 . Registered 28 October 2015.

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.

The Circadian Clock Regulates Metabolic Phenotype Rewiring Via HKDC1 and Modulates Tumor Progression and Drug Response in Colorectal Cancer.

An endogenous molecular clockwork drives various cellular pathways including metabolism and the cell cycle. Its dysregulation is able to prompt pathological phenotypes including cancer. Besides dramatic metabolic alterations, cancer cells display severe changes in the clock phenotype with likely consequences in tumor progression and treatment response. In this study, we use a comprehensive systems-driven approach to investigate the effect of clock disruption on metabolic pathways and its impact on drug response in a cellular model of colon cancer progression. We identified distinctive time-related transcriptomic and metabolic features of a primary tumor and its metastatic counterpart. A mapping of the expression data to a comprehensive genome-scale reconstruction of human metabolism allowed for the in-depth functional characterization of 24 h-oscillating transcripts and pointed to a clock-driven metabolic reprogramming in tumorigenesis. In particular, we identified a set of five clock-regulated glycolysis genes, ALDH3A2, ALDOC, HKDC1, PCK2, and PDHB with differential temporal expression patterns. These findings were validated in organoids and in primary fibroblasts isolated from normal colon and colon adenocarcinoma from the same patient. We further identified a reciprocal connection of HKDC1 to the clock in the primary tumor, which is lost in the metastatic cells. Interestingly, a disruption of the core-clock gene BMAL1 impacts on HKDC1 and leads to a time-dependent rewiring of metabolism, namely an increase in glycolytic activity, as well as changes in treatment response. This work provides novel evidence regarding the complex interplay between the circadian clock and metabolic alterations in carcinogenesis and identifies new connections between both systems with pivotal roles in cancer progression and response to therapy.

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.

Clock-genes and mitochondrial respiratory activity: Evidence of a reciprocal interplay.

In the past few years mounting evidences have highlighted the tight correlation between circadian rhythms and metabolism. Although at the organismal level the central timekeeper is constituted by the hypothalamic suprachiasmatic nuclei practically all the peripheral tissues are equipped with autonomous oscillators made up by common molecular clockworks represented by circuits of gene expression that are organized in interconnected positive and negative feed-back loops. In this study we exploited a well-established in vitro synchronization model to investigate specifically the linkage between clock gene expression and the mitochondrial oxidative phosphorylation (OxPhos). Here we show that synchronized cells exhibit an autonomous ultradian mitochondrial respiratory activity which is abrogated by silencing the master clock gene ARNTL/BMAL1. Surprisingly, pharmacological inhibition of the mitochondrial OxPhos system resulted in dramatic deregulation of the rhythmic clock-gene expression and a similar result was attained with mtDNA depleted cells (Rho0). Our findings provide a novel level of complexity in the interlocked feedback loop controlling the interplay between cellular bioenergetics and the molecular clockwork. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Clock genes-dependent acetylation of complex I sets rhythmic activity of mitochondrial OxPhos.

Physiology of living beings show circadian rhythms entrained by a central timekeeper present in the hypothalamic suprachiasmatic nuclei. Nevertheless, virtually all peripheral tissues hold autonomous molecular oscillators constituted essentially by circuits of gene expression that are organized in negative and positive feed-back loops. Accumulating evidence reveals that cell metabolism is rhythmically controlled by cell-intrinsic molecular clocks and the specific pathways involved are being elucidated. Here, we show that in vitro-synchronized cultured cells exhibit BMAL1-dependent oscillation in mitochondrial respiratory activity, which occurs irrespective of the cell type tested, the protocol of synchronization used and the carbon source in the medium. We demonstrate that the rhythmic respiratory activity is associated to oscillation in cellular NAD content and clock-genes-dependent expression of NAMPT and Sirtuins 1/3 and is traceable back to the reversible acetylation of a single subunit of the mitochondrial respiratory chain Complex I. Our findings provide evidence for a new interlocked transcriptional-enzymatic feedback loop controlling the molecular interplay between cellular bioenergetics and the molecular clockwork.

Hepatitis C virus-linked mitochondrial dysfunction promotes hypoxia-inducible factor 1 alpha-mediated glycolytic adaptation.

Hepatitis C virus (HCV) infection induces a state of oxidative stress by affecting mitochondrial-respiratory-chain activity. By using cell lines inducibly expressing different HCV constructs, we showed previously that viral-protein expression leads to severe impairment of mitochondrial oxidative phosphorylation and to major reliance on nonoxidative glucose metabolism. However, the bioenergetic competence of the induced cells was not compromised, indicating an efficient prosurvival adaptive response. Here, we show that HCV protein expression activates hypoxia-inducible factor 1 (HIF-1) by normoxic stabilization of its alpha subunit. In consequence, expression of HIF-controlled genes, including those coding for glycolytic enzymes, was significantly upregulated. Similar expression of HIF-controlled genes was observed in cell lines inducibly expressing subgenomic HCV constructs encoding either structural or nonstructural viral proteins. Stabilization and transcriptional activation of HIF-1alpha was confirmed in Huh-7.5 cells harboring cell culture-derived infectious HCV and in liver biopsy specimens from patients with chronic hepatitis C. The HCV-related HIF-1alpha stabilization was insensitive to antioxidant treatment. Mimicking an impairment of mitochondrial oxidative phosphorylation by treatment of inducible cell lines with oligomycin resulted in stabilization of HIF-1alpha. Similar results were obtained by treatment with pyruvate, indicating that accumulation of intermediate metabolites is sufficient to stabilize HIF-1alpha. These observations provide new insights into the pathogenesis of chronic hepatitis C and, possibly, the HCV-related development of hepatocellular carcinoma.

Characterization of mitochondrial and extra-mitochondrial oxygen consuming reactions in human hematopoietic stem cells. Novel evidence of the occurrence of NAD(P)H oxidase activity.

This study was aimed to characterize the mitochondrial and extra-mitochondrial oxygen consuming reactions in human CD34+ hematopoietic stem cells. Cell samples were collected by apheresis following pre-conditioning by granulocyte colony-stimulating factor and isolated by anti-CD34 positive immunoselection. Polarographic analysis of the CN-sensitive endogenous cell respiration revealed a low mitochondrial oxygen consumption rate. Differential absorbance spectrometry on whole cell lysate and two-dimensional blue native-PAGE analysis of mitoplast proteins confirmed a low amount of mitochondrial respiratory chain complexes thus qualifying the hematopoietic stem cell as a poor oxidative phosphorylating cell type. Confocal microscopy imaging showed, however, that the intracellular content of mitochondria was not homogeneously distributed in the CD34+ hematopoietic stem cell sample displaying a clear inverse correlation of their density with the expression of the CD34 commitment marker. About half of the endogenous oxygen consumption was extra-mitochondrial and completely inhibitable by enzymatic scavengers of reactive oxygen species and by diphenylene iodinium. By spectral analysis, flow cytometry, reverse transcriptase-PCR, immunocytochemistry, and immunoprecipitation it was shown that the extra-mitochondrial oxygen consumption was contributed by the NOX2 and NOX4 isoforms of the O2-*. producer plasma membrane NAD(P)H oxidase with low constitutive activity. A model is proposed suggesting for the NAD(P)H oxidase a role of O2 sensor and/or ROS source serving as redox messengers in the activation of intracellular signaling pathways leading (or contributing) to mitochondriogenesis, cell survival, and differentiation in hematopoietic stem cells.