bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2020‒03‒15
27 papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Nat Commun. 2020 Mar 11. 11(1): 1312
      The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.
  2. Exp Neurol. 2020 Mar 09. pii: S0014-4886(20)30113-8. [Epub ahead of print] 113282
      Cell-based respirometers such as the Seahorse Extracellular Flux Analyzer are valuable tools to assess the functionality of mitochondria within adherent neurons, as well as other cell types. The Mito Stress Test is the most frequently employed protocol of drug additions to evaluate mitochondrial bioenergetic function. Sequential exposure of cells to an ATP synthase inhibitor such as oligomycin and an uncoupler such as FCCP cause changes in oxygen consumption rate that allow estimation of the cellular efficiency and capacity for mitochondrial ATP synthesis. While a useful first step in assessing whether an experimental treatment or genetic manipulation affects mitochondrial energetics, the Mito Stress Test does not identify specific sites of altered respiratory chain function. This article discusses limitations of the Mito Stress Test, proposes a refined protocol for comparing cell populations that requires independent drug titrations at multiple cell densities, and describes a stepwise series of respirometry-based assays that "map" locations of electron transport deficiency. These include strategies to test for cytochrome c release, to probe the functionality of specific electron transport chain complexes within intact or permeabilized cells, and to measure NADH oxidation by the linked activity of Complexes I, III, and IV. To illustrate utility, we show that although UK5099 and ABT-737 each decrease the spare respiratory capacity of cortical neurons, the stepwise assays reveal different underlying mechanisms consistent with their established drug targets: deficient Complex I substrate supply induced by the mitochondrial pyruvate carrier inhibitor UK5099 and cytochrome c release induced by the anti-apoptotic BCL-2 family protein inhibitor ABT-737.
    Keywords:  ABT-737; BCL-2; BH3; Bioenergetics; Cytochrome c; Oxygen; Pyruvate; Respiration; Seahorse; UK5099
  3. Oncotarget. 2020 Feb 25. 11(8): 801-812
      SLC25A32 is a member of the solute carrier 25 family of mitochondrial transporters. SLC25A32 transports tetrahydrofolate (THF) as well as FAD into mitochondria and regulates mitochondrial one-carbon metabolism and redox balance. While it is known that cancer cells require one-carbon and FAD-dependent mitochondrial metabolism to sustain cell proliferation, the role of SLC25A32 in cancer cell growth remains unexplored. Our results indicate that the SLC25A32 gene is highly amplified in different tumors and that amplification correlates with increased mRNA expression and reduced patients´ survival. siRNA-mediated knock-down and CRISPR-mediated knock-out of SLC25A32 in cancer cells of different origins, resulted in the identification of cell lines sensitive and resistant to SLC25A32 inhibition. Mechanistically, tracing of deuterated serine revealed that SLC25A32 knock-down does not affect the mitochondrial/cytosolic folate flux as measured by Liquid Chromatography coupled Mass Spectrometry (LC-MS). Instead, SLC25A32 inhibition results in a respiratory chain dysfunction at the FAD-dependent complex II enzyme, induction of Reactive Oxygen Species (ROS) and depletion of reduced glutathione (GSH), which impairs cancer cell proliferation. Moreover, buthionine sulfoximine (BSO) treatment further sensitizes cells to ROS-mediated inhibition of cell proliferation upon SLC25A32 knock-down. Treatment of cells with the FAD precursor riboflavin and with GSH rescues cancer cell proliferation upon SLC25A32 down-regulation. Our results indicate that the reduction of mitochondrial FAD concentrations by targeting SLC25A32 has potential clinical applications as a single agent or in combination with approved cancer drugs that lead to increased oxidative stress and reduced tumor growth.
    Keywords:  FAD; ROS; metabolism; mitochondria; transporter
  4. Cells. 2020 Mar 05. pii: E628. [Epub ahead of print]9(3):
      Triple-negative breast cancer (TNBC) stands out for its aggressiveness and accelerated rate of proliferation. Evidence shows that exercise may exert antitumorigenic effects, but the biochemical mechanisms underlying them remain unclear. Our objective was to evaluate the ability of exercise to modulate tumor growth and energy metabolism in an experimental TNBC model. Female BALB/c mice were sedentary or trained for 12 weeks and inoculated with 1 × 104 4T1 cells in the eighth week. Analyzes of macronutrient oxidation, mitochondrial respiration, and expression of genes related to cell metabolism were performed. The results showed that the trained group had a smaller tumor mass and the mitochondria in the tumors presented lower respiratory rates in the state of maximum electron transport capacity. Additionally, the tumors of the exercised group showed a higher expression of genes related to tumor suppressors, while the genes linked with cellular growth were similar between groups. Furthermore, the training modulated the corporal macronutrient oxidation to almost exclusive carbohydrate oxidation, while the sedentary condition metabolized both carbohydrate and lipids. Therefore, the exercise reduced tumor growth, with an impact on mitochondrial and macronutrient metabolism. Our results shed light on the understanding of the antitumorigenic effects of physical exercise, particularly regarding the metabolic transformations in TNBC.
    Keywords:  OXPHOS; TNBC; mitochondria; training
  5. Antioxidants (Basel). 2020 Mar 05. pii: E215. [Epub ahead of print]9(3):
      Oocytes are postulated to repress the proton pumps (e.g., complex IV) and ATP synthase to safeguard mitochondrial DNA homoplasmy by curtailing superoxide production. Whether the ATP synthase is inhibited is, however, unknown. Here we show that: oligomycin sensitive ATP synthase activity is significantly greater (~170 vs. 20 nmol/min-1/mg-1) in testes compared to oocytes in Xenopus laevis (X. laevis). Since ATP synthase activity is redox regulated, we explored a regulatory role for reversible thiol oxidation. If a protein thiol inhibits the ATP synthase, then constituent subunits must be reversibly oxidised. Catalyst-free trans-cyclooctene 6-methyltetrazine (TCO-Tz) immunocapture coupled to redox affinity blotting reveals several subunits in F1 (e.g., ATP-α-F1) and Fo (e.g., subunit c) are reversibly oxidised. Catalyst-free TCO-Tz Click PEGylation reveals significant (~60%) reversible ATP-α-F1 oxidation at two evolutionary conserved cysteine residues (C244 and C294) in oocytes. TCO-Tz Click PEGylation reveals ~20% of the total thiols in the ATP synthase are substantially oxidised. Chemically reversing thiol oxidation significantly increased oligomycin sensitive ATP synthase activity from ~12 to 100 nmol/min-1/mg-1 in oocytes. We conclude that reversible thiol oxidation inhibits the mitochondrial ATP synthase in X. laevis oocytes.
    Keywords:  ATP synthase; Xenopus laevis; click chemistry; mitochondria; oocyte; redox signaling; thiol
  6. Int J Cancer. 2020 Mar 13.
      Copy number gains and increased expression levels of cellular Inhibitor of Apoptosis protein (cIAP)1 and cIAP2 have been identified in primary diffuse large B-cell lymphoma (DLBCL) tissues. Smac (second mitochondria-derived activator of caspases) mimetics were designed to antagonize IAP proteins. However, since their effect as single agents is limited, combination treatment represents a strategy for their clinical development. Therefore, we investigated the Smac mimetic BV6 in combination with proteasome inhibitors and analyzed the molecular mechanisms of action. We discovered that BV6 treatment sensitizes DLBCL cells to proteasome inhibition. We show a synergistic decrease in cell viability and induction of apoptosis by BV6/Carfilzomib (CFZ) treatment, which was confirmed by calculation of combination index (CI) and Bliss score. BV6 and CFZ acted together to trigger activation of BAX and BAK, which facilitated cell death, as knockdown of BAX and BAK significantly reduced BV6/CFZ-mediated cell death. Activation of BAX and BAK was accompanied by loss of mitochondrial membrane potential (MMP) and activation of caspases. Pre-treatment with the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) rescued BV6/CFZ-induced cell death, confirming caspase dependency. Treatment with CFZ alone or in combination with BV6 caused accumulation of NOXA, which was required for cell death, as gene silencing by siRNA or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated NOXA inactivation inhibited BV6/CFZ-induced cell death. Together, these experiments indicate that BV6 and CFZ cooperatively induce apoptotic cell death via the mitochondrial pathway. These findings emphasize the role of Smac mimetics for sensitizing DLBCL cells to proteasome inhibition with important implications for further (pre)clinical studies. This article is protected by copyright. All rights reserved.
    Keywords:  Diffuse large B-cell lymphoma; NOXA; Smac mimetics; intrinsic apoptosis; proteasome inhibitor
  7. FASEB Bioadv. 2020 Mar;2(3): 188-202
      Pseudomonas aeruginosa is a Gram-negative bacterium of the proteobacteria class, and one of the most common causes of nosocomial infections. For example, it causes chronic pneumonia in cystic fibrosis patients. Patient sputum contains 2-heptyl-4-hydroxyquinoline N-oxide [HQNO] and Pseudomonas quorum sensing molecules such as the Pseudomonas quinolone signal [PQS]. It is known that HQNO inhibits the enzyme activity of mitochondrial and bacterial complex III at the Qi (quinone reduction) site, but the target of PQS is not known. In this work we have shown that PQS has a negative effect on mitochondrial respiration in HeLa and A549 cells. It specifically inhibits the complex I of the respiratory chain. In vitro analyses showed a partially competitive inhibition with respect to ubiquinone at the IQ site. In competing studies with Rotenone, PQS suppressed the ROS-promoting effect of Rotenone, which is typical for a B-type inhibitor. Prolonged incubation with PQS also had an effect on the activity of complex III.
    Keywords:  HyPer‐3; IQ site inhibition; Pseudomonas aeruginosa; Pseudomonas quinolone signal (PQS); ROS; Respiratory complex I; mitochondria
  8. Free Radic Biol Med. 2020 Mar 09. pii: S0891-5849(20)30007-1. [Epub ahead of print]
      Both oxidative stress (OS) and reductive stress (RS) are the two extreme facets of redox imbalances that can have deleterious effects on sperm function. However, there is a lack of information on the physiological range of oxidation-reduction potential (ORP). The aim of this study was to investigate effect of OS and RS on sperm functions and associated molecular changes in normal spermatozoa in order to establish physiological range of ORP. In current study, total and progressive motility remained unchanged in spermatozoa exposed to ORP values 0.33 and 0.72 mV/106 sperm/mL. However, a significant (P < 0.05) declines in total and progressive motility were observed at ORP values 1.48, 2.75, -11.24, -9.76 and -9.35 mV/106 sperm/mL. Sperm vitality also decreased significantly (P < 0.0001) at 2.75, -11.24 and -9.76 mV/106 sperm/mL. Spermatozoa exposed to ORP levels 2.75 and -11.24 mV/106 sperm/mL showed significant (P < 0.01) decrease in mitochondrial membrane potential. Intracellular reactive oxygen species (iROS) production increased (P < 0.05) in spermatozoa exposed to ORP levels 1.48 and 2.75 mV/106 sperm/mL, while iROS decreased (P < 0.05) at ORP levels -9.76 and -11.24 mV/106 sperm/mL. No significant change in sperm DNA fragmentation was noted in sperm exposed to OS/RS and the values were below reference range (<19.25%). Western blot analysis revealed decreased expression of CV-ATPA, CIII-UQCRC2, CIV-MTCO1 proteins at 60 and 120 min (P < 0.05) in both OS and RS conditions. This is the first study to report physiological range of ORP (between -9.76 and 1.48 mV/106 sperm/mL) and to elucidate role of altered expression of oxidative phosphorylation (OXPHOS) complexes proteins in mediating deleterious effects of oxidative and reductive condition on sperm functions. A decreased expression of OXPHOS proteins and associated mitochondrial dysfunction contributes to decreased sperm motility and vitality under oxidative and reductive stress.
    Keywords:  Human spermatozoa; Oxidation-reduction potential; Oxidative stress; Reactive oxygen species; Reductive stress
  9. J Neuroinfect Dis. 2018 ;pii: 283. [Epub ahead of print]9(4):
      Objective: Mitochondrial dysfunction is known to be implicated in stroke, but the complex mechanisms of stroke have led to few stroke therapies. The present study to disrupted mitochondrial oxidative phosphorylation through a known electron transport chain (ETC) uncoupler, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP). Analyzing the resulting neurological deficits as well as infarct volume could help determine the role of mitochondria in stroke outcome and determine whether uncoupling the ETC could potentially be a strategy for new stroke therapies. The objective of this study was to determine the effects of uncoupling electron flow on mitochondrial oxidative phosphorylation and stroke infarction.Methods: Cerebral endovascular cells (CECs) were treated with various concentrations of FCCP, and bioenergetics were measured. For the stroke mouse model, FCCP (1 mg/kg, i.p) or vehicle was administered followed by 1-hour transient middle cerebral artery occlusion (tMCAO). Infarct volume was measured after a 23-hour reperfusion, and triphenyl tetrazolium chloride (TTC) staining was used to assess infarct volume.
    Results: FCCP significantly decreased basal respiration, ATP turnover, maximal respiration, and spare capacity when the concentration of FCCP was greater than 1000 nM. The mice pretreated with FCCP had a significantly increased infarct volume within the cortex, striatum, and total hemisphere. Mice receiving FCCP had a significantly increased neurological deficit score compared to the vehicle.
    Conclusions: FCCP compromised mitochondrial oxidative phosphorylation in CECs in a dose-dependent manner. Uncoupling the electron transport chain with FCCP prior to tMCAO exacerbated stroke infarction in mice.
    Keywords:  Blood-Brain Barrier (BBB); Carbonyl Cyanide-4 (trifluoromethoxy) Phenylhydrazone (FCCP); Cerebral Endovascular Cells (CECs); Electron Transport Chain (ETC); Ischemia; Transient Middle Cerebral Artery Occlusion (tMCAO); Triphenyl Tetrazolium Chloride (TTC)
  10. Cell Death Dis. 2020 Mar 12. 11(3): 181
      Fatty acids are the most major substrate source for adult cardiac energy generation. Prohibitin 2 (PHB2), a highly conserved protein located in mitochondrial inner membrane, plays key roles in cellular energy metabolic homeostasis. However, its functions in regulating cardiac fatty acid metabolism have remained largely unknown. Our study demonstrates that cardiac-specific knockout of Phb2 leads to accumulation of lipid droplets and causes heart failure. Mechanistically, ablation of PHB2 impairs cardiac fatty acid oxidation (FAO) through downregulating carnitine palmitoyltransferase1b (CPT1b), a rate-limiting enzyme of cardiac mitochondrial FAO. Moreover, overexpression of CPT1b alleviates impaired FAO in PHB2-deficient cardiomyocytes. Thus, our study provides direct evidence for the link between PHB2 and cardiac fatty acid metabolism. Our study points out that PHB2 is a potential FAO regulator in cardiac mitochondrial inner membrane, as well as the connection between PHB2 and CPT1b and their relationships to cardiac pathology especially to cardiac fatty acid metabolic disorder.
  11. J Oncol. 2020 ;2020 2314693
      Gliomas are the most common primary tumors of the central nervous system (CNS) in the adult. Previous data showed that estrogen affects cancer cells, but its effect is cell-type-dependent and controversial. The present study aimed to analyze the effects of estradiol (E2, 5 nM) in human glioblastoma multiforme U87-MG cells and how it may impact on cell proliferation and mitochondrial fitness. We monitored cell proliferation by xCELLigence technology and mitochondrial fitness by assessing the expression of genes involved in mitochondrial biogenesis (PGC1α, SIRT1, and TFAM), oxidative phosphorylation (ND4, Cytb, COX-II, COX IV, NDUFA6, and ATP synthase), and dynamics (OPA1, MNF2, MNF1, and FIS1). Finally, we evaluated Nrf2 nuclear translocation by immunocytochemical analysis. Our results showed that E2 resulted in a significant increase in cell proliferation, with a significant increase in the expression of genes involved in various mechanisms of mitochondrial fitness. Finally, E2 treatment resulted in a significant increase of Nrf2 nuclear translocation with a significant increase in the expression of one of its target genes (i.e., heme oxygenase-1). Our results suggest that E2 promotes proliferation in glioblastoma cells and regulate the expression of genes involved in mitochondrial fitness and chemoresistance pathway.
  12. J Cancer Res Clin Oncol. 2020 Mar 09.
      OBJECTIVE: Increasing evidence has revealed that mechanical stress and elevated mechanical signals promote malignant tumor transformation and metastasis. This study aimed to explore the function of the mechanically activated ion-channel Piezo1 in the colon cancer metastasis and its potential regulatory mechanism.METHODS: First, we examined the expression levels of Piezo1 and mitochondrial calcium uniporter (MCU) both in colon cancer tissues and assessed the prognostic value of Piezo1 and MCU in a colon cancer cohort (n = 110). Second, functional assays were performed to investigate the effects of Piezo1 and MCU on colon cancer cell migration, invasion, and mitochondrial membrane potential. Third, we analyzed the expression of Piezo1, MCU, and HIF-1α by overexpressing/silencing each other's expression.
    RESULTS: We found that Piezo1 was up-regulated and MCU was down-regulated in colon cancer tissues. Piezo1 and MCU were both correlated with poor prognosis of patients with colon cancer. Overexpressing Piezo1 and silencing MCU could promote colon cancer cell migration and metastasis, reduce mitochondrial membrane potential, and promote each other's expression. We also found that HIF-1α was up-regulated in colon cancer tissues. Additionally, silencing Piezo1 inhibited the expression of HIF-1α and VEGF, which was contrary to MCU silencing. Intriguingly, Piezo1-overexpressing cells did not regain their migration behaviors when HIF-1α expression was inhibited, which was accompanied with the re-expression of MCU and VEGF.
    CONCLUSION: In our study, Piezo1 is involved in colon cancer cell metastasis. Furthermore, our findings indicated a possible Piezo1-MCU-HIF-1α-VEGF axis, which still need further exploration.
    Keywords:  Colon cancer; HIF-1α; Metastasis; Mitochondrial calcium uniporter; Piezo1
  13. Sci Rep. 2020 Mar 10. 10(1): 4401
      Mitochondrial dysfunctions belong amongst the most common metabolic diseases but the signalling networks that lead to the manifestation of a disease phenotype are often not well understood. We identified the subunits of respiratory complex I, III and IV as mediators of major signalling changes during Drosophila wing disc development. Their downregulation in larval wing disc leads to robust stimulation of TOR activity, which in turn orchestrates a complex downstream signalling network. Specifically, after downregulation of the complex I subunit ND-49 (mammalian NDUFS2), TOR activates JNK to induce cell death and ROS production essential for the stimulation of compensatory apoptosis-induced proliferation within the tissue. Additionally, TOR upregulates Notch and JAK/STAT signalling and it directs glycolytic switch of the target tissue. Our results highlight the central role of TOR signalling in mediating the complex response to mitochondrial respiratory dysfunction and they provide a rationale why the disease symptoms associated with respiratory dysfunctions are often alleviated by mTOR inhibitors.
  14. Cell Signal. 2020 Mar 09. pii: S0898-6568(20)30075-9. [Epub ahead of print] 109598
      Recent evidence has demonstrated that the signal transducer and activator of transcription 3 (STAT3) gene are abnormally active in glioblastoma multiforme (GBM), and this change is crucial for the tumor survival and chemotherapy-resistant. Certain preclinical pharmacology studies have focused on STAT3 phosphorylation and homodimerization, and have developed a class of salicylic acid-based inhibitors, which blocks the nuclear translocation-dependent canonical STAT3 signaling. In the present study, we demonstrated that the salicylic acid-based compound SH-4-54 was quite toxic to temozolomide (TMZ)-resistant GBM cells and could trigger apoptosis in these cells via enhancing mitochondrial translocation-dependent non-canonical STAT3 pathway. We demonstrated that incubation of TMZ-resistant GBM cells with SH-4-54 led to mitochondrial STAT3 (mitoSTAT3) activation and respiratory dysfunction reflected by disrupted (or suppressed) activities of oxidative phosphorylation complexes and oxygen consumption rate. Mechanistically, we proved that SH-4-54 could increase mitoSTAT3 transmembrane import via GRIM-19 and reinforce the association between mitoSTAT3 and mitochondrial transcription factor A (TFAM), indicating that SH-4-54 could facilitate the binding of mitoSTAT3 to mitochondria DNA (mtDNA) and negatively regulate mitochondrial-encoded genes, thus leading to the abnormal oxidation respiratory. Lastly, using GRIM-19 knockout cell line and subcutaneous xenotransplanted tumor model, we elaborately showed the enrichment of SH-4-54 in mitochondria by LC-MS/MS analysis. In conclusion, our data demonstrate thatthe salicylic acid-based compound SH-4-54 is quite effective in killing TMZ-resistant GBM cells and this cytotoxicity is attributed tomitoSTAT3 activation.
    Keywords:  Glioblastoma; Mitochondrial translocation; Respiratory chain dysfunction; SH-4-54; STAT3
  15. Redox Biol. 2020 Feb 07. pii: S2213-2317(19)31460-0. [Epub ahead of print] 101450
      Reactive Oxygen Species (ROS) are essential cellular messengers required for cellular homeostasis and regulate the lifespan of several animal species. The main site of ROS production is the mitochondrion, and within it, respiratory complex I (CI) is the main ROS generator. ROS produced by CI trigger several physiological responses that are essential for the survival of neurons, cardiomyocytes and macrophages. Here, we show that CI produces ROS when electrons flow in either the forward (Forward Electron Transport, FET) or reverse direction (Reverse Electron Transport, RET). We demonstrate that ROS production via RET (ROS-RET) is activated under thermal stress conditions and that interruption of ROS-RET production, through ectopic expression of the alternative oxidase AOX, attenuates the activation of pro-survival pathways in response to stress. Accordingly, we find that both suppressing ROS-RET signalling or decreasing levels of mitochondrial H2O2 by overexpressing mitochondrial catalase (mtCAT), reduces survival dramatically in flies under stress. Our results uncover a specific ROS signalling pathway where hydrogen peroxide (H2O2) generated by CI via RET is required to activate adaptive mechanisms, maximising survival under stress conditions.
    Keywords:  AOX; Alternative oxidase; Complex I; Heat stress; Reactive oxygen species; Reverse electron transport
  16. Proc Natl Acad Sci U S A. 2020 Mar 09. pii: 201916414. [Epub ahead of print]
      The mitochondria of various tissues from mice, naked mole rats (NMRs), and bats possess two mechanistically similar systems to prevent the generation of mitochondrial reactive oxygen species (mROS): hexokinases I and II and creatine kinase bound to mitochondrial membranes. Both systems operate in a manner such that one of the kinase substrates (mitochondrial ATP) is electrophoretically transported by the ATP/ADP antiporter to the catalytic site of bound hexokinase or bound creatine kinase without ATP dilution in the cytosol. One of the kinase reaction products, ADP, is transported back to the mitochondrial matrix via the antiporter, again through an electrophoretic process without cytosol dilution. The system in question continuously supports H+-ATP synthase with ADP until glucose or creatine is available. Under these conditions, the membrane potential, ∆ψ, is maintained at a lower than maximal level (i.e., mild depolarization of mitochondria). This ∆ψ decrease is sufficient to completely inhibit mROS generation. In 2.5-y-old mice, mild depolarization disappears in the skeletal muscles, diaphragm, heart, spleen, and brain and partially in the lung and kidney. This age-dependent decrease in the levels of bound kinases is not observed in NMRs and bats for many years. As a result, ROS-mediated protein damage, which is substantial during the aging of short-lived mice, is stabilized at low levels during the aging of long-lived NMRs and bats. It is suggested that this mitochondrial mild depolarization is a crucial component of the mitochondrial anti-aging system.
    Keywords:  aging; antioxidant; mild depolarization; mitochondria; naked mole rat
  17. Antioxidants (Basel). 2020 Mar 10. pii: E226. [Epub ahead of print]9(3):
      According to the free radical theory of aging, reactive oxygen species (ROS) have been proposed to be a major cause of aging for a long time. Meanwhile, it became clear that ROS have diverse functions in a healthy organism. They act as second messengers, and as transient inhibitors of phosphatases and others. In fact, their detrimental role is highly dependent on the context of their production. NADPH oxidases (Nox) have been discovered as a controllable source of ROS. NoxO1 enables constitutive ROS formation by Nox1 by acting as a constitutively active cytosolic subunit of the complex. We previously found that both Nox1 and NoxO1 were highly expressed in the colon, and that NoxO1-/- deficiency reduces colon health. We hypothesized that a healthy colon potentially contributes to longevity and NoxO1 deficiency would reduce lifetime, at least in mouse. In contrast, here we provide evidence that the knockout of NoxO1 results in an elongated life expectancy of mice. No better endothelial function, nor an improved expression of genes related to longevity, such as Sirt1, were found, and therefore may not serve as an explanation for a longer life in NoxO1 deficiency. Rather minor systemic differences, such as lower body weight occur. As a potential reason for longer life, we suggest better DNA repair capacity in NoxO1 deficient mice. Although final fatal DNA damage appears similar between wildtype and NoxO1 knockout animals, we identified less intermediate DNA damage in colon cells of NoxO1-/- mice, while the number of cells with intact DNA is elevated in NoxO1-/- colons. We conclude that NoxO1 deficiency prolongs lifetime of mice, which correlates with less intermediate and potentially fixable DNA damage at least in colon cells.
    Keywords:  NADPH oxidase; NoxO1; longevity; mice
  18. Obesity (Silver Spring). 2020 Mar 13.
      OBJECTIVE: This study aimed to test the hypothesis that young adults with obesity and cold-activated brown adipose tissue (BAT) are less likely to have metabolic dysfunction (dyslipidemia, insulin resistance, and hypertension) than those without cold-activated BAT. Previous studies have noted a potentially protective effect of BAT and higher adiponectin/leptin ratios, but they have acknowledged that the clinical implications of these findings remain uncertain.METHODS: Twenty-one females and twenty-three males with obesity (BMI ≥ 30 kg/m2 ) underwent a 2-hour cooling protocol before 18 F-fluorodeoxyglucose (18 F-FDG)-positron emission tomography/x-ray computed tomography scan to determine the prevalence, volume, and 18 F-FDG uptake of cold-activated BAT.
    RESULTS: Cold-activated BAT was identified in 43% of participants (11 female, 8 male); females had greater 18 F-FDG uptake. Those with cold-activated BAT had a lesser degree of metabolic dysfunction. Cold-activated BAT volume correlated with triglycerides (inversely) and adiponectin (concordantly). Body-mass-adjusted cold-activated BAT activity correlated with high-density lipoprotein cholesterol (concordantly). Males with cold-activated BAT had lower leptin and higher adiponectin/leptin ratio.
    CONCLUSIONS: A high prevalence of cold-activated BAT was found in the study participants. BAT could be important in decreasing metabolic dysfunction among young adults with obesity, making it a potential target for treating metabolically unhealthy obesity.
  19. FASEB J. 2020 Mar 12.
      Mitochondrial dysfunction is the leading cause of reactive oxygen species (ROS) burst and apoptosis in hepatic ischemia/reperfusion (I/R) injury. Ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE) is a hepatotargeted agent that exerts hepatoprotective roles by regulating lipid metabolism. Our previous studies have shown that UDCA-LPE improves hepatic I/R injury by inhibiting apoptosis and inflammation. However, the role of UDCA-LPE in lipid metabolism and mitochondrial function in hepatic I/R remains unknown. In the present study, we investigated the role of UDCA-LPE in hepatic I/R by focusing on the interface of phospholipid metabolism and mitochondrial homeostasis. Livers from 28-week-old mice, primary hepatocytes and HepG2 cells were subjected to in vivo and in vitro I/R, respectively. Analyses of oxidative stress, imaging, ATP generation, genetics, and lipidomics showed that I/R was associated with mitochondrial dysfunction and a reduction in phospholipids. UDCA-LPE alleviated mitochondria-dependent oxidative stress and apoptosis and prevented the decrease of phospholipid levels. Our study found that cytosolic phospholipase A2 (cPLA2 ), a phospholipase that is activated during I/R, hydrolyzed mitochondrial membrane phospholipids and led to mitochondria-mediated oxidative stress and apoptosis. UDCA-LPE inhibited the interaction between cPLA2 and mitochondria and reduced phospholipid hydrolysis-mediated injury. UDCA-LPE might regulate the crosstalk between the phospholipid metabolism and the mitochondria, restore mitochondrial function and ameliorate I/R injury.
    Keywords:  cPLA2; defect mitochondria; mitochondrial membrane; oxidative stress; phospholipid metabolism disorders; reperfusion injury
  20. Mult Scler Relat Disord. 2020 Feb 25. pii: S2211-0348(20)30101-2. [Epub ahead of print]41 102025
      BACKGROUND: Multiple sclerosis (MS) is a multifactorial disease of the central nervous system in young adults. Mitochondrial respiration provides fuel necessary for cellular function and is especially important in cells with large energy demand including neurons. Various studies suggest that the pathogenesis of MS may be associated with mitochondrial dysfunction.METHODS: We examined 145 volunteers including 62 MS patients and healthy controls. MS patients were divided into two groups according to their disease severity: those with mild disability (EDSS=0-3.0) and those with moderate-severe MS (EDSS=3.5-8). After signing an informed consent, blood was taken and was separated to platelets and lymphocytes. Mitochondria activity was monitored as mitochondrial transmembrane potential following staining with JC1 dye in platelets and lymphocytes utilizing flow cytometry.
    RESULTS: We examined mitochondria activity as JC1 values from all separated lymphocyte samples and found significantly higher levels of mitochondrial activity in lymphocytes separated from healthy controls vs. MS patients (mean of 87.9% vs. 75.6%, p = 0.001). Significant differences in mitochondrial activity were also found when comparing means of groups divided according to MS disease severity. Interestingly, there were no significant differences in mitochondrial activity between patients treated with diverse medications or untreated patients. Mitochondrial activity was also examined in platelets, but no significant differences were found between groups.
    CONCLUSIONS: Results obtained here show that mitochondrial activity was significantly lower in MS patients in comparison to healthy controls. In addition, there was a significant difference in mitochondrial activity depending on MS degree of disability. These initial findings in a peripheral examination hold potential for new diagnostic biomarkers to be considered in the future.
    Keywords:  JC1; Lymphocytes; Medication; Mitochondria; Multiple sclerosis
  21. Front Genet. 2020 ;11 24
      Mitochondrial complex I deficiency is associated with a diverse range of clinical phenotypes and can arise due to either mitochondrial DNA (mtDNA) or nuclear gene defects. We investigated two adult patients who exhibited non-syndromic neurological features and evidence of isolated mitochondrial complex I deficiency in skeletal muscle biopsies. The first presented with indolent myopathy, progressive since age 17, while the second developed deafness around age 20 and other relapsing-remitting neurological symptoms since. A novel, likely de novo, frameshift variant in MT-ND6 (m.14512_14513del) and a novel maternally-inherited transversion mutation in MT-ND1 were identified, respectively. Skewed tissue segregation of mutant heteroplasmy level was observed; the mutant heteroplasmy levels of both variants were greater than 70% in muscle homogenate, however, in blood the MT-ND6 variant was undetectable while the mutant heteroplasmy level of the MT-ND1 variant was low (12%). Assessment of complex I assembly by Blue-Native PAGE demonstrated a decrease in fully assembled complex I in the muscle of both cases. SDS-PAGE and immunoblotting showed decreased levels of mtDNA-encoded ND1 and several nuclear encoded complex I subunits in both cases, consistent with functional pathogenic consequences of the identified variants. Pathogenicity of the m.14512_14513del was further corroborated by single-fiber segregation studies.
    Keywords:  deafness; mitochondrial DNA; muscle biopsy; myopathy; tissue segregation
  22. Cancers (Basel). 2020 Mar 11. pii: E650. [Epub ahead of print]12(3):
      Mitochondrial bioenergetics profiling, a measure of oxygen consumption rates, correlates with prognostic markers and can be used to assess response to therapy in chronic lymphocytic leukemia (CLL) cells. In this study, we measured mitochondrial respiration rates in primary CLL cells using respirometry to evaluate mitochondrial function. We found significant increases in mitochondrial respiration rates in CLL versus control B lymphocytes. We also observed amongst CLL patients that advanced age, female sex, zeta-chain-associated protein of 70 kD (ZAP-70+), cluster of differentiation 38 (CD38+), and elevated β2-microglobulin (β2-M) predicted increased maximal respiration rates. ZAP-70+ CLL cells exhibited significantly higher bioenergetics than B lymphocytes or ZAP-70- CLL cells and were more sensitive to the uncoupler, carbonyl cyanide-p-trifluoro-methoxyphenylhydrazone (FCCP). Univariable and multivariable linear regression analysis demonstrated that ZAP-70+ predicted increased maximal respiration. ZAP-70+ is a surrogate for B cell receptor (BCR) activation and can be targeted by ibrutinib, which is a clinically approved Bruton's tyrosine kinase (BTK) inhibitor. Therefore, we evaluated the oxygen consumption rates (OCR) of CLL cells and plasma chemokine (C-C motif) ligands 3 and 4 (CCL3/CCL4) levels from ibrutinib-treated patients and demonstrated decreased OCR similar to control B lymphocytes, suggesting that ibrutinib treatment resets the mitochondrial bioenergetics, while diminished CCL3/CCL4 levels indicate the down regulation of the BCR signaling pathway in CLL. Our data support evaluation of mitochondrial respiration as a preclinical tool for the response assessment of CLL cells.
    Keywords:  B cell receptor; CCL3/CCL4; Oroboros oxygraph; ZAP-70; chronic lymphocytic leukemia; ibrutinib; mitochondria; oxygen consumption rates
  23. Proc Natl Acad Sci U S A. 2020 Mar 10. pii: 201910141. [Epub ahead of print]
      Oxidative phosphorylation, the primary source of cellular energy in eukaryotes, requires gene products encoded in both the nuclear and mitochondrial genomes. As a result, functional integration between the genomes is essential for efficient adenosine triphosphate (ATP) generation. Although within populations this integration is presumably maintained by coevolution, the importance of mitonuclear coevolution in key biological processes such as speciation and mitochondrial disease has been questioned. In this study, we crossed populations of the intertidal copepod Tigriopus californicus to disrupt putatively coevolved mitonuclear genotypes in reciprocal F2 hybrids. We utilized interindividual variation in developmental rate among these hybrids as a proxy for fitness to assess the strength of selection imposed on the nuclear genome by alternate mitochondrial genotypes. Developmental rate varied among hybrid individuals, and in vitro ATP synthesis rates of mitochondria isolated from high-fitness hybrids were approximately two-fold greater than those of mitochondria isolated from low-fitness individuals. We then used Pool-seq to compare nuclear allele frequencies for high- or low-fitness hybrids. Significant biases for maternal alleles were detected on 5 (of 12) chromosomes in high-fitness individuals of both reciprocal crosses, whereas maternal biases were largely absent in low-fitness individuals. Therefore, the most fit hybrids were those with nuclear alleles that matched their mitochondrial genotype on these chromosomes, suggesting that mitonuclear effects underlie individual-level variation in developmental rate and that intergenomic compatibility is critical for high fitness. We conclude that mitonuclear interactions can have profound impacts on both physiological performance and the evolutionary trajectory of the nuclear genome.
    Keywords:  coevolution; copepod; incompatibilities; intergenomic; mitonuclear
  24. Eur Biophys J. 2020 Mar;49(2): 193-205
      Quinidine is an antiarrhythmic drug commonly used for the treatment of cardiac ailments. It affects oxidative phosphorylation, calcium uptake, and ion channels of mitochondria. We have investigated the interaction of Quinidine and mitochondrial voltage-dependent anion channel (VDAC). VDAC was purified from neuronal tissue of Wistar rats and in vitro bilayer electrophysiology experiments were performed on it. 50-mM Quinidine treatment on VDAC leads to a sudden drop in its conductance. The dose of Quinidine leading to a half-maximal current through a single-channel VDAC was calculated using Quinidine at different concentrations. In silico molecular docking studies using Autodock-4.2 software indicate interaction between Quinidine and VDAC. Docking results demonstrate the interaction of Quinidine and VDAC on its Glutamic acid residue (Glu-206 of VDAC). Fluorescence spectroscopy results on Quinidine and Glutamic acid interaction show an increase in the intensity and wavelength of Quinidine fluorescence, whereas no interaction between Quinidine and Cysteine was observed. This further supports the Glutamic acid and Quinidine interaction. In conclusion, we report Quinidine partially blocks VDAC due to the interaction of Glutamic acid and Quinidine in the channel pore.
    Keywords:  Bilayer electrophysiology; Bilayer membrane (BLM); Docking; Fluorescence spectroscopy; Quinidine; Voltage-dependent anion channel (VDAC)
  25. FASEB J. 2020 Mar 10.
      Oxidative stress and hypoxia are two opposite microenvironments involved in HCC metastasis. Thioredoxin (TXN) and hypoxia-inducible factor 2α (HIF-2α) are typical proteins involved in these two different microenvironments, respectively. How these two factors interact to influence the fate on tumor cells remains unknown. Hypoxia facilitated HCC cells withstood oxidative stress and eventually promoted HCC cells metastasis, in which TXN and HIF-2α were mostly involved. Upregulation of TXN/HIF-2α correlated with poor HCC prognosis and promoted HCC metastasis both in vitro and in vivo. Epithelial-mesenchymal transition (EMT) process was involved in TXN/HIF-2α-enhanced invasiveness of HCC cells. Additionally, the stability and activity of HIF-2α were precisely regulated by TXN via SUMOylation and acetylation, which contributed to HCC metastasis. Our data revealed that the redox protein TXN and HIF-2α are both associated with HCC metastasis, and the fine regulation of TXN on HIF-2α contributes essentially during the process of metastasis. Our study provides new insight into the interaction mechanism between hypoxia and oxidative stress and implies potential therapeutic benefits by targeting both TXN and HIF-2α in the treatment of HCC metastasis.
    Keywords:  HIF-2α; hepatocellular carcinoma; metastasis; thioredoxin
  26. Front Oncol. 2020 ;10 196
      Exercise is recommended for the healthy population as it increases fitness and prevents diseases. Moreover, exercise is also applied as an adjunct therapy for patients with various chronic diseases including cancer. Childhood cancer is a rare, heterogeneous disease that differs from adult cancer. Improved therapeutic strategies have increased childhood cancer survival rates to above 80% in developed countries. Although this is higher than the average adult cancer survival rate of about 50%, therapy results often in substantial long-term side effects in childhood cancer survivors. Exercise in adult cancer patients has many beneficial effects and may slow down tumor progression and improve survival in some cancer types, suggesting that exercise may influence cancer cell behavior. In contrast to adults, there is not much data on general effects of exercise in children. Whilst it seems possible that exercise might delay cancer progression or improve survival in children as well, there is no reliable data yet to support this hypothesis. Depending on the type of cancer, animal studies of adult cancer types show that the exercise-induced increase of the catecholamines epinephrine and norepinephrine, have suppressive as well as promoting effects on cancer cells. The diverse effects of exercise in adult cancer patients require investigating whether these results can be achieved in children with cancer.
    Keywords:  adjunct therapy; cancer; childhood cancer; exercise training; molecular mechanisms; tumor
  27. Mol Carcinog. 2020 Mar 09.
      Colorectal cancer (CRC) is a common malignancy. Many reports have implicated aberrant mitochondrial activity in the progression of CRC, with particular emphasis on the dysregulation of redox signaling and oxidative stress. In this study, we focused on manganese superoxide dismutase (MnSOD/SOD2), a key antioxidant enzyme, which maintains intracellular redox homeostasis. Current literature presents conflicting mechanisms for how SOD2 influences tumorigenesis and tumor progression. Here, we explored the role of SOD2 in CRC specifically. We found high levels of SOD2 expression in CRC tissues. We carried out a series of experiments to determine whether knockdown of SOD2 expression in CRC cell lines would reverse features of tumorigenesis. We found that reduced SOD2 expression decreased cell proliferation, migration, and invasion activity in CRC cells. Results from an additional series of experiments on mitochondrial function implicated a dual role for SOD2 in promoting CRC progression. First, proper level of SOD2 helped CRC cells maintain mitochondrial function by disposal of superoxide (O2 .- ). Second, over-expression of SOD2 induced H2 O2 -mediated tumorigenesis by upregulating AMPK and glycolysis. Our results indicate that SOD2 may promote the occurrence and development of CRC by regulating the energy metabolism mediated by AMPK signaling pathways.
    Keywords:  colorectal cancer (CRC); glycolysis; manganese superoxide dismutase (MnSOD/SOD2); redox