bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2021‒01‒03
forty-three papers selected by
Kelsey Fisher-Wellman
East Carolina University


  1. Mol Cell. 2020 Dec 22. pii: S1097-2765(20)30904-7. [Epub ahead of print]
    Luengo A, Li Z, Gui DY, Sullivan LB, Zagorulya M, Do BT, Ferreira R, Naamati A, Ali A, Lewis CA, Thomas CJ, Spranger S, Matheson NJ, Vander Heiden MG.
      Aerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is associated with proliferation across many organisms and conditions. To better understand that association, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the NAD+/NADH ratio. This change in NAD+/NADH is caused by increased mitochondrial membrane potential that impairs mitochondrial electron transport and NAD+ regeneration. Uncoupling respiration from ATP synthesis or increasing ATP hydrolysis restores NAD+/NADH homeostasis and proliferation even when glucose oxidation is increased. These data suggest that when demand for NAD+ to support oxidation reactions exceeds the rate of ATP turnover in cells, NAD+ regeneration by mitochondrial respiration becomes constrained, promoting fermentation, despite available oxygen. This argues that cells engage in aerobic glycolysis when the demand for NAD+ is in excess of the demand for ATP.
    Keywords:  Aerobic Glycolysis; Cell Metabolism; Fermentation; NAD+; PDK; Warburg Effect
    DOI:  https://doi.org/10.1016/j.molcel.2020.12.012
  2. Cancer Discov. 2020 Dec 18. pii: CD-20-1065. [Epub ahead of print]
    Fresquet V, Garcia-Barchino MJ, Larrayoz MJ, Celay J, Vicente C, Fernandez-Galilea M, Larrayoz MJ, Calasanz MJ, Panizo C, Junza A, Han J, Prior C, Fortes P, Pio R, Oyarzabal J, Martínez-Baztán Á, Paiva B, Moreno-Aliaga MJ, Odero MD, Agirre X, Yanes O, Prósper F, Martinez-Climent JA.
      During millions of years, endogenous retroelements have remained transcriptionally silent within mammalian genomes by epigenetic mechanisms. Modern anti-cancer therapies targeting the epigenetic machinery awaken retroelement expression, inducing anti-viral responses that eliminate tumors through mechanisms not completely understood. Here we find that massive binding of epigenetically-activated retroelements by RIG-I and MDA5 viral sensors promotes ATP hydrolysis and depletes intracellular energy, driving tumor killing independently of immune signaling. Energy depletion boosts compensatory ATP production by switching glycolysis to mitochondrial oxidative phosphorylation, thereby reversing the Warburg effect. However, hyperfunctional succinate dehydrogenase in mitochondrial electron transport chain generates excessive oxidative stress that unleashes RIP1-mediated necroptosis. To maintain ATP generation, hyperactive mitochondrial membrane blocks intrinsic apoptosis by increasing BCL2 dependency. Accordingly, drugs targeting BCL2-family proteins and epigenetic inhibitors yield synergistic responses in multiple cancer types. Thus, epigenetic therapy kills cancer cells by rewiring mitochondrial metabolism upon retroelement activation, which primes mitochondria to apoptosis by BH3-mimetics.
    DOI:  https://doi.org/10.1158/2159-8290.CD-20-1065
  3. Cell Rep. 2020 Dec 29. pii: S2211-1247(20)31551-5. [Epub ahead of print]33(13): 108562
    Patananan AN, Sercel AJ, Wu TH, Ahsan FM, Torres A, Kennedy SAL, Vandiver A, Collier AJ, Mehrabi A, Van Lew J, Zakin L, Rodriguez N, Sixto M, Tadros W, Lazar A, Sieling PA, Nguyen TL, Dawson ER, Braas D, Golovato J, Cisneros L, Vaske C, Plath K, Rabizadeh S, Niazi KR, Chiou PY, Teitell MA.
      Generating mammalian cells with desired mitochondrial DNA (mtDNA) sequences is enabling for studies of mitochondria, disease modeling, and potential regenerative therapies. MitoPunch, a high-throughput mitochondrial transfer device, produces cells with specific mtDNA-nuclear DNA (nDNA) combinations by transferring isolated mitochondria from mouse or human cells into primary or immortal mtDNA-deficient (ρ0) cells. Stable isolated mitochondrial recipient (SIMR) cells isolated in restrictive media permanently retain donor mtDNA and reacquire respiration. However, SIMR fibroblasts maintain a ρ0-like cell metabolome and transcriptome despite growth in restrictive media. We reprogrammed non-immortal SIMR fibroblasts into induced pluripotent stem cells (iPSCs) with subsequent differentiation into diverse functional cell types, including mesenchymal stem cells (MSCs), adipocytes, osteoblasts, and chondrocytes. Remarkably, after reprogramming and differentiation, SIMR fibroblasts molecularly and phenotypically resemble unmanipulated control fibroblasts carried through the same protocol. Thus, our MitoPunch "pipeline" enables the production of SIMR cells with unique mtDNA-nDNA combinations for additional studies and applications in multiple cell types.
    Keywords:  cell engineering; differentiation, MitoPunch, mitochondrial transplantation, mitochondrial replacement, mitonuclear communication, isolated mitochondria; mitochondrial transfer; mtDNA; reprogramming
    DOI:  https://doi.org/10.1016/j.celrep.2020.108562
  4. Front Cell Dev Biol. 2020 ;8 603292
    Mohammadalipour A, Dumbali SP, Wenzel PL.
      Mesenchymal stromal cell (MSC) metabolism plays a crucial role in the surrounding microenvironment in both normal physiology and pathological conditions. While MSCs predominantly utilize glycolysis in their native hypoxic niche within the bone marrow, new evidence reveals the importance of upregulation in mitochondrial activity in MSC function and differentiation. Mitochondria and mitochondrial regulators such as sirtuins play key roles in MSC homeostasis and differentiation into mature lineages of the bone and hematopoietic niche, including osteoblasts and adipocytes. The metabolic state of MSCs represents a fine balance between the intrinsic needs of the cellular state and constraints imposed by extrinsic conditions. In the context of injury and inflammation, MSCs respond to reactive oxygen species (ROS) and damage-associated molecular patterns (DAMPs), such as damaged mitochondria and mitochondrial products, by donation of their mitochondria to injured cells. Through intercellular mitochondria trafficking, modulation of ROS, and modification of nutrient utilization, endogenous MSCs and MSC therapies are believed to exert protective effects by regulation of cellular metabolism in injured tissues. Similarly, these same mechanisms can be hijacked in malignancy whereby transfer of mitochondria and/or mitochondrial DNA (mtDNA) to cancer cells increases mitochondrial content and enhances oxidative phosphorylation (OXPHOS) to favor proliferation and invasion. The role of MSCs in tumor initiation, growth, and resistance to treatment is debated, but their ability to modify cancer cell metabolism and the metabolic environment suggests that MSCs are centrally poised to alter malignancy. In this review, we describe emerging evidence for adaptations in MSC bioenergetics that orchestrate developmental fate decisions and contribute to cancer progression. We discuss evidence and potential strategies for therapeutic targeting of MSC mitochondria in regenerative medicine and tissue repair. Lastly, we highlight recent progress in understanding the contribution of MSCs to metabolic reprogramming of malignancies and how these alterations can promote immunosuppression and chemoresistance. Better understanding the role of metabolic reprogramming by MSCs in tissue repair and cancer progression promises to broaden treatment options in regenerative medicine and clinical oncology.
    Keywords:  MSC differentiation; cancer metabolism; hematological malignancy; mesenchymal stromal cells (MSCs); metabolic reprogramming; mitochondrial biogenesis; mitochondrial dynamics; mitochondrial transfer
    DOI:  https://doi.org/10.3389/fcell.2020.603292
  5. Biochim Biophys Acta Mol Basis Dis. 2020 Dec 29. pii: S0925-4439(20)30410-5. [Epub ahead of print] 166062
    Schirris TJJ, Rossell S, de Haas R, Frambach SJCM, Hoogstraten CA, Renkema GH, Beyrath JD, Willems PHGM, Huynen MA, Smeitink JAM, Russel FGM, Notebaart RA.
      The majority of cellular energy is produced by the mitochondrial oxidative phosphorylation (OXPHOS) system. Failure of the first OXPHOS enzyme complex, NADH:ubiquinone oxidoreductase or complex I (CI), is associated with multiple signs and symptoms presenting at variable ages of onset. There is no approved drug treatment yet to slow or reverse the progression of CI-deficient disorders. Here, we present a comprehensive human metabolic network model of genetically characterized CI-deficient patient-derived fibroblasts. Model calculations predicted that increased cholesterol production, export, and utilization can counterbalance the surplus of reducing equivalents in patient-derived fibroblasts, as these pathways consume considerable amounts of NAD(P)H. We show that fibrates attenuated increased NAD(P)H levels and improved CI-deficient fibroblast growth by stimulating the production of cholesterol via enhancement of its cellular efflux. In CI-deficient (Ndufs4-/-) mice, fibrate treatment resulted in prolonged survival and improved motor function, which was accompanied by an increased cholesterol efflux from peritoneal macrophages. Our results shine a new light on the use of compensatory biological pathways in mitochondrial dysfunction which may lead to novel therapeutic interventions for mitochondrial diseases for which currently no cure exists.
    Keywords:  Leigh syndrome; NAD(P)H; Ndufs4(-/-) mice; cholesterol biosynthesis; complex I deficiency; metabolic network modelling
    DOI:  https://doi.org/10.1016/j.bbadis.2020.166062
  6. Int J Mol Sci. 2020 Dec 28. pii: E198. [Epub ahead of print]22(1):
    Wang W, Liparulo I, Rizzardi N, Bolignano P, Calonghi N, Bergamini C, Fato R.
      Mitochondrial dysfunction plays a significant role in the metabolic flexibility of cancer cells. This study aimed to investigate the metabolic alterations due to Coenzyme Q depletion in MCF-7 cells.METHOD: The Coenzyme Q depletion was induced by competitively inhibiting with 4-nitrobenzoate the coq2 enzyme, which catalyzes one of the final reactions in the biosynthetic pathway of CoQ. The bioenergetic and metabolic characteristics of control and coenzyme Q depleted cells were investigated using polarographic and spectroscopic assays. The effect of CoQ depletion on cell growth was analyzed in different metabolic conditions.
    RESULTS: we showed that cancer cells could cope from energetic and oxidative stress due to mitochondrial dysfunction by reshaping their metabolism. In CoQ depleted cells, the glycolysis was upregulated together with increased glucose consumption, overexpression of GLUT1 and GLUT3, as well as activation of pyruvate kinase (PK). Moreover, the lactate secretion rate was reduced, suggesting that the pyruvate flux was redirected, toward anabolic pathways. Finally, we found a different expression pattern in enzymes involved in glutamine metabolism, and TCA cycle in CoQ depleted cells in comparison to controls.
    CONCLUSION: This work elucidated the metabolic alterations in CoQ-depleted cells and provided an insightful understanding of cancer metabolism targeting.
    Keywords:  bioenergetics; cancer metabolism targeting; coenzyme Q; glutamine metabolism; glycolysis; metabolic reprogramming; mitochondrial dysfunction; spheroids
    DOI:  https://doi.org/10.3390/ijms22010198
  7. Pharmaceutics. 2020 Dec 23. pii: E10. [Epub ahead of print]13(1):
    Zavadskis S, Weidinger A, Hanetseder D, Banerjee A, Schneider C, Wolbank S, Marolt Presen D, Kozlov AV.
      Reactive oxygen species (ROS) have recently been recognized as important signal transducers, particularly regulating proliferation and differentiation of cells. Diphenyleneiodonium (DPI) is known as an inhibitor of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) and is also affecting mitochondrial function. The aim of this study was to investigate the effect of DPI on ROS metabolism and mitochondrial function in human amniotic membrane mesenchymal stromal cells (hAMSCs), human bone marrow mesenchymal stromal cells (hBMSCs), hBMSCs induced into osteoblast-like cells, and osteosarcoma cell line MG-63. Our data suggested a combination of a membrane potential sensitive fluorescent dye, tetramethylrhodamine methyl ester (TMRM), and a ROS-sensitive dye, CM-H2DCFDA, combined with a pretreatment with mitochondria-targeted ROS scavenger MitoTEMPO as a good tool to examine effects of DPI. We observed critical differences in ROS metabolism between hAMSCs, hBMSCs, osteoblast-like cells, and MG-63 cells, which were linked to energy metabolism. In cell types using predominantly glycolysis as the energy source, such as hAMSCs, DPI predominantly interacted with NOX, and it was not toxic for the cells. In hBMSCs, the ROS turnover was influenced by NOX activity rather than by the mitochondria. In cells with aerobic metabolism, such as MG 63, the mitochondria became an additional target for DPI, and these cells were prone to the toxic effects of DPI. In summary, our data suggest that undifferentiated cells rather than differentiated parenchymal cells should be considered as potential targets for DPI.
    Keywords:  NADPH-oxidase; differentiation; diphenyleneiodonium; mitochondria; proliferation; reactive oxygen species
    DOI:  https://doi.org/10.3390/pharmaceutics13010010
  8. Free Radic Biol Med. 2020 Dec 28. pii: S0891-5849(20)31897-9. [Epub ahead of print]
    Okoye CN, Stevens D, Kamunde C.
      Oxygen (O2) deprivation and metals are common environmental stressors and their exposure to aquatic organisms can induce oxidative stress by disrupting cellular reactive oxygen species (ROS) homeostasis. Mitochondria are a major source of ROS in the cell wherein a dozen sites located on enzymes of the electron transport system (ETS) and substrate oxidation produce superoxide anion radicals (O2˙¯) or hydrogen peroxide (H2O2). Sites located on ETS enzymes can generate ROS by forward electron transfer (FET) and reverse electron transfer (RET) reactions; however, knowledge of how exogenous stressors modulate site-specific ROS production is limited. We investigated the effects of anoxia-reoxygenation and cadmium (Cd) on H2O2 emission in fish liver mitochondria oxidizing glutamate-malate, succinate or palmitoylcarnitine-malate. We find that anoxia-reoxygenation attenuates H2O2 emission while the effect of Cd depends on the substrate, with monotonic responses for glutamate-malate and palmitoylcarnitine-malate, and a biphasic response for succinate. Anoxia-reoxygenation exerts a substrate-dependent inhibition of mitochondrial respiration which is more severe with palmitoylcarnitine-malate compared with succinate or glutamate-malate. Additionally, specific mitochondrial ROS-emitting sites were sequestered using blockers of electron transfer and the effects of anoxia-reoxygenation and Cd on H2O2 emission were evaluated. Here, we find that site-specific H2O2 emission capacities depend on the substrate and the direction of electron flow. Moreover, anoxia-reoxygenation alters site-specific H2O2 emission rates during succinate and glutamate-malate oxidation whereas Cd imposes monotonic or biphasic H2O2 emission responses depending on the substrate and site. Contrary to our expectation, anoxia-reoxygenation blunts the effect of Cd. These results suggest that the effect of exogenous stressors on mitochondrial oxidant production is governed by their impact on energy conversion reactions and mitochondrial redox poise. Moreover, direct increased ROS production seemingly does not explain the increased adverse effects associated with combined exposure of aquatic organisms to Cd and low dissolved oxygen levels.
    Keywords:  anoxia-reoxygenation; cadmium; mitochondria; reactive oxygen species; respiration
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2020.12.234
  9. Cell Metab. 2020 Dec 08. pii: S1550-4131(20)30655-0. [Epub ahead of print]
    Zhang GF, Jensen MV, Gray SM, El K, Wang Y, Lu D, Becker TC, Campbell JE, Newgard CB.
      Metabolic fuels regulate insulin secretion by generating second messengers that drive insulin granule exocytosis, but the biochemical pathways involved are incompletely understood. Here we demonstrate that stimulation of rat insulinoma cells or primary rat islets with glucose or glutamine + 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (Gln + BCH) induces reductive, "counter-clockwise" tricarboxylic acid (TCA) cycle flux of glutamine to citrate. Molecular or pharmacologic suppression of isocitrate dehydrogenase-2 (IDH2), which catalyzes reductive carboxylation of 2-ketoglutarate to isocitrate, results in impairment of glucose- and Gln + BCH-stimulated reductive TCA cycle flux, lowering of NADPH levels, and inhibition of insulin secretion. Pharmacologic suppression of IDH2 also inhibits insulin secretion in living mice. Reductive TCA cycle flux has been proposed as a mechanism for generation of biomass in cancer cells. Here we demonstrate that reductive TCA cycle flux also produces stimulus-secretion coupling factors that regulate insulin secretion, including in non-dividing cells.
    Keywords:  NADPH; anaplerosis; insulin secretion; isocitrate dehydrogenase-2; metabolic flux; pancreatic islet β cells; reductive TCA cycle; stable isotopes
    DOI:  https://doi.org/10.1016/j.cmet.2020.11.020
  10. Biochim Biophys Acta Mol Cell Res. 2020 Dec 23. pii: S0167-4889(20)30300-1. [Epub ahead of print] 118942
    Bahri H, Buratto J, Rojo M, Dompierre JP, Salin B, Blancard C, Cuvellier S, Rose M, Elgaaied ABA, Tetaud E, di Rago JP, Devin A, Duvezin-Caubet S.
      Mitochondrial ATP-synthesis is catalyzed by a F1Fo-ATP synthase, an enzyme of dual genetic origin enriched at the edge of cristae where it plays a key role in their structure/stability. The enzyme's biogenesis remains poorly understood, both from a mechanistic and a compartmentalization point of view. The present study provides novel molecular insights into this process through investigations on a human protein called TMEM70 with an unclear role in the assembly of ATP synthase. A recent study has revealed the existence of physical interactions between TMEM70 and the subunit c (Su.c), a protein present in 8 identical copies forming a transmembrane oligomeric ring (c-ring) within the ATP synthase proton translocating domain (Fo). Herein we analyzed the ATP-synthase assembly in cells lacking TMEM70, mitochondrial DNA or F1 subunits and observe a direct correlation between TMEM70 and Su.c levels, regardless of the status of other ATP synthase subunits or of mitochondrial bioenergetics. Immunoprecipitation, two-dimensional blue-native/SDS-PAGE, and pulse-chase experiments reveal that TMEM70 forms large oligomers that interact with Su.c not yet incorporated into ATP synthase complexes. Moreover, discrete TMEM70-Su.c complexes with increasing Su.c contents can be detected, suggesting a role for TMEM70 oligomers in the gradual assembly of the c-ring. Furthermore, we demonstrate using expansion super-resolution microscopy the specific localization of TMEM70 at the inner cristae membrane, distinct from the MICOS component MIC60. Taken together, our results show that TMEM70 oligomers provide a scaffold for c-ring assembly and that mammalian ATP synthase is assembled within inner cristae membranes.
    Keywords:  ATP synthase; cristae; human mitochondria; membrane protein; organelle biogenesis; protein complex
    DOI:  https://doi.org/10.1016/j.bbamcr.2020.118942
  11. Mol Metab. 2020 Dec 22. pii: S2212-8778(20)30228-3. [Epub ahead of print] 101154
    Shannon CE, Ragavan M, Palavicini JP, Fourcaudot M, Bakewell T, Valdez IA, Ayala I, Jin ES, Madesh M, Han X, Merritt ME, Norton L.
      BACKGROUND: Insulin resistance and altered hepatic mitochondrial function are central features of type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD), but the etiological role of these processes in disease progression remains unclear.OBJECTIVES: & Approach We investigated the molecular links between insulin resistance, mitochondrial remodeling, and hepatic lipid accumulation in a rodent model of T2D/NAFLD.
    RESULTS: Livers from obese, insulin resistant mice displayed augmented mitochondrial content and increased TCA cycle and pyruvate dehydrogenase (PDH) activities. Insulin sensitization with the thiazolidinedione pioglitazone mitigated pyruvate-driven TCA cycle activity and PDH activation via both allosteric (intracellular pyruvate availability) and covalent (PDK4 and PDP2) mechanisms that were dependent on PPARγ activity in isolated primary hepatocytes. Improved mitochondrial function following pioglitazone treatment was entirely dissociated from changes in hepatic triglycerides, diacylglycerides or fatty acids. Instead, we highlight a role for the mitochondrial phospholipid cardiolipin, which underwent pathological remodeling in livers from obese mice that was reversed by insulin sensitization.
    CONCLUSIONS: Our findings identify targetable mitochondrial features of T2D and NAFLD and highlight the benefit of insulin sensitization in managing the clinical burden of obesity-associated disease.
    DOI:  https://doi.org/10.1016/j.molmet.2020.101154
  12. Comp Biochem Physiol B Biochem Mol Biol. 2020 Dec 23. pii: S1096-4959(20)30146-9. [Epub ahead of print] 110552
    Else PL.
      This is the story of a series of reductionist studies that started with an attempt to explain what underpins the high-level of aerobic metabolism in mammals (i.e. associated with the evolution of endothermy) and almost forty years later had led to investigations into the role of membrane lipids in determining metabolism. Initial studies showed that the increase in aerobic metabolism in mammals was driven by a combination of increases in mitochondrial volume and membrane densities, organ size and changes in the molecular activity of enzymes. The increase in the capacity to produce energy was matched by an increase in energy use, notably driven by increases in H+, Na+ and K+ fluxes. In the case of increased Na+ flux, it was found this was matched by increases in Na+-dependent metabolism at the tissue level and increases in enzyme activity at a cellular level but not by an increase in the number of sodium pumps. To maintain Na+ gradient across cell membranes, increased Na+ flux is not controlled by an increase in sodium pump number but rather by an increase in sodium pump molecular activity (i.e. an increase the substrate turnover rate of each sodium pump) in tissues of endotherms. This increase in molecular activity is coupled to an increase in the level of highly unsaturated polyunsaturated fatty acids (PUFA) in membranes, a mechanism similar to that used by ectotherms to ameliorate decreasing activities of metabolic processes in the cold. Determination of how changes in membrane fatty acid composition can change the activities of proteins in membranes will be the next step in this story.
    Keywords:  Body mass; Ectotherm; Endotherm; Fatty acid; Lipid; Mitochondria; Na(+) flux; Organ size; Phospholipid; Protein concentration; Proton; Sodium pump
    DOI:  https://doi.org/10.1016/j.cbpb.2020.110552
  13. PLoS One. 2020 ;15(12): e0244499
    Iaubasarova IR, Khailova LS, Firsov AM, Grivennikova VG, Kirsanov RS, Korshunova GA, Kotova EA, Antonenko YN.
      The synthesis of a mitochondria-targeted derivative of the classical mitochondrial uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP) by alkoxy substitution of CCCP with n-decyl(triphenyl)phosphonium cation yielded mitoCCCP, which was able to inhibit the uncoupling action of CCCP, tyrphostin A9 and niclosamide on rat liver mitochondria, but not that of 2,4-dinitrophenol, at a concentration of 1-2 μM. MitoCCCP did not uncouple mitochondria by itself at these concentrations, although it exhibited uncoupling action at tens of micromolar concentrations. Thus, mitoCCCP appeared to be a more effective mitochondrial recoupler than 6-ketocholestanol. Both mitoCCCP and 6-ketocholestanol did not inhibit the protonophoric activity of CCCP in artificial bilayer lipid membranes, which might compromise the simple proton-shuttling mechanism of the uncoupling activity on mitochondria.
    DOI:  https://doi.org/10.1371/journal.pone.0244499
  14. Cell Metab. 2020 Dec 17. pii: S1550-4131(20)30662-8. [Epub ahead of print]
    Kang YP, Mockabee-Macias A, Jiang C, Falzone A, Prieto-Farigua N, Stone E, Harris IS, DeNicola GM.
      Cysteine is required for maintaining cellular redox homeostasis in both normal and transformed cells. Deprivation of cysteine induces the iron-dependent form of cell death known as ferroptosis; however, the metabolic consequences of cysteine starvation beyond impairment of glutathione synthesis are poorly characterized. Here, we find that cystine starvation of non-small-cell lung cancer cell lines induces an unexpected accumulation of γ-glutamyl-peptides, which are produced due to a non-canonical activity of glutamate-cysteine ligase catalytic subunit (GCLC). This activity is enriched in cell lines with high levels of NRF2, a key transcriptional regulator of GCLC, but is also inducible in healthy murine tissues following cysteine limitation. γ-glutamyl-peptide synthesis limits the accumulation of glutamate, thereby protecting against ferroptosis. These results indicate that GCLC has a glutathione-independent, non-canonical role in the protection against ferroptosis by maintaining glutamate homeostasis under cystine starvation.
    Keywords:  GCLC; NRF2; cysteine; cystine; ferroptosis; glutamate; γ-glutamyl
    DOI:  https://doi.org/10.1016/j.cmet.2020.12.007
  15. PLoS One. 2020 ;15(12): e0244540
    Kloepping KC, Kraus AS, Hedlund DK, Gnade CM, Wagner BA, McCormick ML, Fath MA, Seol D, Lim TH, Buettner GR, Goswami PC, Pigge FC, Spitz DR, Schultz MK.
      Despite dramatic improvements in outcomes arising from the introduction of targeted therapies and immunotherapies, metastatic melanoma is a highly resistant form of cancer with 5 year survival rates of <35%. Drug resistance is frequently reported to be associated with changes in oxidative metabolism that lead to malignancy that is non-responsive to current treatments. The current report demonstrates that triphenylphosphonium(TPP)-based lipophilic cations can be utilized to induce cytotoxicity in pre-clinical models of malignant melanoma by disrupting mitochondrial metabolism. In vitro experiments demonstrated that TPP-derivatives modified with aliphatic side chains accumulated in melanoma cell mitochondria; disrupted mitochondrial metabolism; led to increases in steady-state levels of reactive oxygen species; decreased total glutathione; increased the fraction of glutathione disulfide; and caused cell killing by a thiol-dependent process that could be rescued by N-acetylcysteine. Furthermore, TPP-derivative-induced melanoma toxicity was enhanced by glutathione depletion (using buthionine sulfoximine) as well as inhibition of thioredoxin reductase (using auranofin). In addition, there was a structure-activity relationship between the aliphatic side-chain length of TPP-derivatives (5-16 carbons), where longer carbon chains increased melanoma cell metabolic disruption and cell killing. In vivo bio-distribution experiments showed that intratumoral administration of a C14-TPP-derivative (12-carbon aliphatic chain), using a slow-release thermosensitive hydrogel as a delivery vehicle, localized the drug at the melanoma tumor site. There, it was observed to persist and decrease the growth rate of melanoma tumors. These results demonstrate that TPP-derivatives selectively induce thiol-dependent metabolic oxidative stress and cell killing in malignant melanoma and support the hypothesis that a hydrogel-based TPP-derivative delivery system could represent a therapeutic drug-delivery strategy for melanoma.
    DOI:  https://doi.org/10.1371/journal.pone.0244540
  16. Cell Metab. 2020 Dec 17. pii: S1550-4131(20)30660-4. [Epub ahead of print]
    Jeong S, Savino AM, Chirayil R, Barin E, Cheng Y, Park SM, Schurer A, Mullarky E, Cantley LC, Kharas MG, Keshari KR.
      A significant increase in dietary fructose consumption has been implicated as a potential driver of cancer. Metabolic adaptation of cancer cells to utilize fructose confers advantages for their malignant growth, but compelling therapeutic targets have not been identified. Here, we show that fructose metabolism of leukemic cells can be inhibited by targeting the de novo serine synthesis pathway (SSP). Leukemic cells, unlike their normal counterparts, become significantly dependent on the SSP in fructose-rich conditions as compared to glucose-rich conditions. This metabolic program is mediated by the ratio of redox cofactors, NAD+/NADH, and the increased SSP flux is beneficial for generating alpha-ketoglutarate from glutamine, which allows leukemic cells to proliferate even in the absence of glucose. Inhibition of PHGDH, a rate-limiting enzyme in the SSP, dramatically reduces leukemia engraftment in mice in the presence of high fructose, confirming the essential role of the SSP in the metabolic plasticity of leukemic cells.
    Keywords:  in vivo isotope tracing; metabolic flux; redox; serine synthesis pathway
    DOI:  https://doi.org/10.1016/j.cmet.2020.12.005
  17. J Am Chem Soc. 2020 Dec 30. 142(52): 21758-21766
    Röpke M, Saura P, Riepl D, Pöverlein MC, Kaila VRI.
      The respiratory complex I is a gigantic (1 MDa) redox-driven proton pump that reduces the ubiquinone pool and generates proton motive force to power ATP synthesis in mitochondria. Despite resolved molecular structures and biochemical characterization of the enzyme from multiple organisms, its long-range (∼300 Å) proton-coupled electron transfer (PCET) mechanism remains unsolved. We employ here microsecond molecular dynamics simulations to probe the dynamics of the mammalian complex I in combination with hybrid quantum/classical (QM/MM) free energy calculations to explore how proton pumping reactions are triggered within its 200 Å wide membrane domain. Our simulations predict extensive hydration dynamics of the antiporter-like subunits in complex I that enable lateral proton transfer reactions on a microsecond time scale. We further show how the coupling between conserved ion pairs and charged residues modulate the proton transfer dynamics, and how transmembrane helices and gating residues control the hydration process. Our findings suggest that the mammalian complex I pumps protons by tightly linked conformational and electrostatic coupling principles.
    DOI:  https://doi.org/10.1021/jacs.0c09209
  18. STAR Protoc. 2020 Dec 18. 1(3): 100160
    Liao PC, Yang EJ, Pon LA.
      The redox state of mitochondria is one indicator of the functional state of the organelles. Mitochondria are also the primary endogenous source of reactive oxygen species (ROS). Therefore, the redox state of the organelles also reflects their function in ROS production. Here, we provide step-by-step protocols for live-cell imaging and quantification of mitochondrial redox state using the genetically encoded fluorescent biosensor, mitochondria-targeted redox sensing GFP (mito-roGFP), and mitochondrial ROS using the membrane-permeant small molecule dihydroethidium (DHE) in budding yeast cells. For complete details on the use and execution of this protocol, please refer to Liao et al. (2020c).
    DOI:  https://doi.org/10.1016/j.xpro.2020.100160
  19. Biomed Pharmacother. 2020 Dec 24. pii: S0753-3322(20)31332-9. [Epub ahead of print]134 111139
    Benot-Dominguez R, Tupone MG, Castelli V, d'Angelo M, Benedetti E, Quintiliani M, Cinque B, Forte IM, Cifone MG, Ippoliti R, Barboni B, Giordano A, Cimini A.
      Breast and ovarian cancers are the leading and fifth reason for tumor death among females, respectively. Recently, many studies demonstrated antiproliferative activities of natural aliments in cancer. In this study, we investigated the antitumor potential of Olive Leaf Extract (OLE) in triple-negative breast and ovarian cancer cells. A HPLC/DAD analysis on OLE has been performed to assess the total polyphenolics and other secondary metabolites content. HCEpiC, MDA-MB-231, and OVCAR-3 cell lines were used. MTS, Cytofluorimetric, Western Blot analysis were performed to analyze cell viability, cell proliferation, apoptosis, and oxidative stress. Fluorimetric and IncuCyte® analyses were carried out to evaluate apoptosis and mitochondrial function. We confirmed that OLE, containing a quantity of oleuropein of 87 % of the total extract, shows anti-proliferative and pro-apoptotic activity on MDA-MB-231 cells. For the first time, our results indicate that OLE inhibits OVCAR-3 cell viability inducing cell cycle arrest, and it also increases apoptotic cell death up-regulating the protein level of cleaved-PARP and caspase 9. Moreover, our data show that OLE treatment causes a significant decrease in mitochondrial functionality, paralleled by a reduction of mitochondrial membrane potential. Interestingly, OLE increased the level of intracellular and mitochondrial reactive oxygen species (ROS) together with a decreased activity of ROS scavenging enzymes, confirming oxidative stress in both models. Our data demonstrate that mitochondrial ROS generation represented the primary mechanism of OLE antitumor activity, as pretreatment with antioxidant N-acetylcysteine prevented OLE-induced cell cycle arrest and apoptosis.
    Keywords:  Mediterranean diet; Mitochondria; OLE; Ovarian cancer; ROS; TNBC
    DOI:  https://doi.org/10.1016/j.biopha.2020.111139
  20. IUBMB Life. 2020 Dec 25.
    Lechuga-Vieco AV, Justo-Méndez R, Enríquez JA.
      The oxidative phosphorylation (OXPHOS) system is the only structure in animal cells with components encoded by two genomes, maternally transmitted mitochondrial DNA (mtDNA), and biparentally transmitted nuclear DNA (nDNA). MtDNA-encoded genes have to physically assemble with their counterparts encoded in the nucleus to build together the functional respiratory complexes. Therefore, structural and functional matching requirements between the protein subunits of these molecular complexes are rigorous. The crosstalk between nDNA and mtDNA needs to overcome some challenges, as the nuclear-encoded factors have to be imported into the mitochondria in a correct quantity and match the high number of organelles and genomes per mitochondria that encode and synthesize their own components locally. The cell is able to sense the mito-nuclear match through changes in the activity of the OXPHOS system, modulation of the mitochondrial biogenesis, or reactive oxygen species production. This implies that a complex signaling cascade should optimize OXPHOS performance to the cellular-specific requirements, which will depend on cell type, environmental conditions, and life stage. Therefore, the mitochondria would function as a cellular metabolic information hub integrating critical information that would feedback the nucleus for it to respond accordingly. Here, we review the current understanding of the complex interaction between mtDNA and nDNA.
    Keywords:  cytoplasmic communication; intergenomic coadaptation; mito-nuclear interactions; mitochondrial DNA; mitochondrial haplotypes; nucleo-mitochondrial mismatch; respiratory complexes and supercomplexes; retrograde responses
    DOI:  https://doi.org/10.1002/iub.2434
  21. Free Radic Biol Med. 2020 Dec 24. pii: S0891-5849(20)31685-3. [Epub ahead of print]163 306-313
    Sokolovski SG, Rafailov EU, Abramov AY, Angelova PR.
      Oxygen, in form of reactive oxygen species (ROS), has been shown to participate in oxidative stress, one of the major triggers for pathology, but also is a main contributor to physiological processes. Recently, it was found that 1267 nm irradiation can produce singlet oxygen without photosensitizers. We used this phenomenon to study the effect of laser-generated singlet oxygen on one of the major oxygen-dependent processes, mitochondrial energy metabolism. We have found that laser-induced generation of 1O2 in neurons and astrocytes led to the increase of mitochondrial membrane potential, activation of NADH- and FADH-dependent respiration, and importantly, increased the rate of maximal respiration in isolated mitochondria. The activation of mitochondrial respiration stimulated production of ATP in these cells. Thus, we found that the singlet oxygen generated by 1267 nm laser pulse works as an activator of mitochondrial respiration and ATP production in the brain.
    Keywords:  1267 nm; Brain; Energy metabolism; Mitochondrial respiration; Singlet oxygen
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2020.12.022
  22. Cancer Discov. 2020 Dec 28. pii: CD-20-0387. [Epub ahead of print]
    Li J, Duran MA, Dhanota N, Chatila WK, Bettigole SE, Kwon J, Sriram RK, Humphries MP, Salto-Tellez M, James JA, Hanna MG, Melms JC, Vallabhaneni S, Litchfield K, Usaite I, Biswas D, Bareja R, Li HW, Martin ML, Dorsaint P, Cavallo JA, Li P, Pauli C, Gottesdiener L, DiPardo BJ, Hollmann TJ, Merghoub T, Wen HY, Reis-Filho JS, Riaz N, Su SM, Kalbasi A, Vasan N, Powell SN, Wolchok JD, Elemento O, Swanton C, Shoushtari AN, Parkes EE, Izar B, Bakhoum SF.
      Cytosolic DNA is characteristic of chromosomally unstable metastatic cancer cells, resulting in constitutive activation of the cGAS-STING innate immune pathway. How tumors co-opt inflammatory signaling while evading immune surveillance remains unknown. Here we show that the ectonucleotidase ENPP1 promotes metastasis by selectively degrading extracellular cGAMP, an immune stimulatory metabolite whose breakdown products include the immune suppressor, adenosine. ENPP1 loss suppresses metastasis, restores tumor immune infiltration, and potentiates response to immune checkpoint blockade in a manner dependent on tumor cGAS and host STING. Conversely, overexpression of wildtype ENPP1, but not an enzymatically weakened mutant, promotes migration and metastasis, in part, through the generation of extracellular adenosine, and renders otherwise sensitive tumors completely resistant to immunotherapy. In human cancers, ENPP1 expression correlates with reduced immune cell infiltration, increased metastasis, and resistance to anti-PD1/PD-L1 treatment. Thus, cGAMP hydrolysis by ENPP1 enables chromosomally unstable tumors to transmute cGAS activation into an immune suppressive pathway.
    DOI:  https://doi.org/10.1158/2159-8290.CD-20-0387
  23. Front Cell Dev Biol. 2020 ;8 592348
    Papa L, Djedaini M, Martin TC, Zangui M, Beaumont KG, Sebra R, Parsons R, Schaniel C, Hoffman R.
      Ex vivo expansion strategies of human hematopoietic stem cell (HSC) grafts with suboptimal stem cell dose have emerged as promising strategies for improving outcomes of HSC transplantation in patients with hematological malignancies. While exposure of HSCs to ex vivo cultures expands the number of phenotypically identifiable HSCs, it frequently alters the transcriptomic and metabolic profiles, therefore, compromising their long-term (LT) hematopoietic reconstitution capacity. Within the heterogeneous pool of expanded HSCs, the precise phenotypic, transcriptomic and metabolic profile and thus, the identity of HSCs that confer LT repopulation potential remains poorly described. Utilizing valproic acid (VPA) in ex vivo cultures of umbilical cord blood (UCB)-CD34+ cells, we demonstrate that expanded HSCs phenotypically marked by expression of the stem cell markers CD34, CD90 and EPCR (CD201) are highly enriched for LT-HSCs. Furthermore, we report that low mitochondrial membrane potential, and, hence, mitochondrial activity distinguishes LT-HSCs within the expanded pool of phenotypically defined HSCs. Remarkably, such reduced mitochondrial activity is restricted to cells with the highest expression levels of CD34, CD90 and EPCR phenotypic markers. Together, our findings reveal that high expression of CD34, CD90 and EPCR in conjunction with low mitochondrial activity is critical for identification of functional LT-HSCs generated within ex vivo expansion cultures.
    Keywords:  CD90; EPCR; ex vivo expansion; functional fitness; mitochondrial membrane potential; phenotype; valproic acid
    DOI:  https://doi.org/10.3389/fcell.2020.592348
  24. Proc Natl Acad Sci U S A. 2021 Jan 05. pii: e2015632118. [Epub ahead of print]118(1):
    Dagvadorj J, Mikulska-Ruminska K, Tumurkhuu G, Ratsimandresy RA, Carriere J, Andres AM, Marek-Iannucci S, Song Y, Chen S, Lane M, Dorfleutner A, Gottlieb RA, Stehlik C, Cassel S, Sutterwala FS, Bahar I, Crother TR, Arditi M.
      The balance between NLRP3 inflammasome activation and mitophagy is essential for homeostasis and cellular health, but this relationship remains poorly understood. Here we found that interleukin-1α (IL-1α)-deficient macrophages have reduced caspase-1 activity and diminished IL-1β release, concurrent with reduced mitochondrial damage, suggesting a role for IL-1α in regulating this balance. LPS priming of macrophages induced pro-IL-1α translocation to mitochondria, where it directly interacted with mitochondrial cardiolipin (CL). Computational modeling revealed a likely CL binding motif in pro-IL-1α, similar to that found in LC3b. Thus, binding of pro-IL-1α to CL in activated macrophages may interrupt CL-LC3b-dependent mitophagy, leading to enhanced Nlrp3 inflammasome activation and more robust IL-1β production. Mutation of pro-IL-1α residues predicted to be involved in CL binding resulted in reduced pro-IL-1α-CL interaction, a reduction in NLRP3 inflammasome activity, and increased mitophagy. These data identify a function for pro-IL-1α in regulating mitophagy and the potency of NLRP3 inflammasome activation.
    Keywords:  IL-1α; autophagy; cardiolipin; inflammasome; mitochondria
    DOI:  https://doi.org/10.1073/pnas.2015632118
  25. Front Physiol. 2020 ;11 604069
    Kobayashi S, Zhao F, Zhang Z, Kobayashi T, Huang Y, Shi B, Wu W, Liang Q.
      Hyperglycemia-induced mitochondrial dysfunction plays a key role in the pathogenesis of diabetic cardiomyopathy. Injured mitochondrial segments are separated by mitochondrial fission and eliminated by autophagic sequestration and subsequent degradation in the lysosome, a process termed mitophagy. However, it remains poorly understood how high glucose affects the activities of, and the relationship between, mitochondrial fission and mitophagy in cardiomyocytes. In this study, we determined the functional roles of mitochondrial fission and mitophagy in hyperglycemia-induced cardiomyocyte injury. High glucose (30 mM, HG) reduced mitochondrial connectivity and particle size and increased mitochondrial number in neonatal rat ventricular cardiomyocytes, suggesting an enhanced mitochondrial fragmentation. SiRNA knockdown of the pro-fission factor dynamin-related protein 1 (DRP1) restored mitochondrial size but did not affect HG toxicity, and Mdivi-1, a DRP1 inhibitor, even increased HG-induced cardiomyocyte injury, as shown by superoxide production, mitochondrial membrane potential and cell death. However, DRP1 overexpression triggered mitochondrial fragmentation and mitigated HG-induced cardiomyocyte injury, suggesting that the increased mitochondrial fission is beneficial, rather than detrimental, to cardiomyocytes cultured under HG conditions. This is in contrast to the prevailing hypothesis that mitochondrial fragmentation mediates or contributes to HG cardiotoxicity. Meanwhile, HG reduced mitophagy flux as determined by the difference in the levels of mitochondria-associated LC3-II or the numbers of mitophagy foci indicated by the novel dual fluorescent reporter mt-Rosella in the absence and presence of the lysosomal inhibitors. The ability of HG to induce mitochondrial fragmentation and inhibit mitophagy was reproduced in adult mouse cardiomyocytes. Overexpression of Parkin, a positive regulator of mitophagy, or treatment with CCCP, a mitochondrial uncoupler, induced mitophagy and attenuated HG-induced cardiomyocyte death, while Parkin knockdown had opposite effects, suggesting an essential role of mitophagy in cardiomyocyte survival under HG conditions. Strikingly, Parkin overexpression increased mitochondrial fragmentation, while DRP1 overexpression accelerated mitophagy flux, demonstrating a reciprocal activation loop that controls mitochondrial fission and mitophagy. Thus, strategies that promote the mutual positive interaction between mitochondrial fission and mitophagy while simultaneously maintain their levels within the physiological range would be expected to improve mitochondrial health, alleviating hyperglycemic cardiotoxicity.
    Keywords:  DRP1; Parkin; cardiomyocytes; cell death; diabetes; hyperglycemia; mitochondrial fission; mitophagy
    DOI:  https://doi.org/10.3389/fphys.2020.604069
  26. Cancers (Basel). 2020 Dec 22. pii: E15. [Epub ahead of print]13(1):
    Nanni S, Aiello A, Salis C, Re A, Cencioni C, Bacci L, Pierconti F, Pinto F, Ripoli C, Ostano P, Baroni S, Lazzarino G, Tavazzi B, Pugliese D, Bassi P, Grassi C, Panunzi S, Chiorino G, Pontecorvi A, Gaetano C, Farsetti A.
      The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes growth and progression in prostate cancer (PCa); however, little is known about its possible impact in PCa metabolism. The aim of this work has been the assessment of the metabolic reprogramming associated with MALAT1 silencing in human PCa cells and in an ex vivo model of organotypic slice cultures (OSCs). Cultured cells and OSCs derived from primary tumors were transfected with MALAT1 specific gapmers. Cell growth and survival, gene profiling, and evaluation of targeted metabolites and metabolic enzymes were assessed. Computational analysis was made considering expression changes occurring in metabolic markers following MALAT1 targeting in cultured OSCs. MALAT1 silencing reduced expression of some metabolic enzymes, including malic enzyme 3, pyruvate dehydrogenase kinases 1 and 3, and choline kinase A. Consequently, PCa metabolism switched toward a glycolytic phenotype characterized by increased lactate production paralleled by growth arrest and cell death. Conversely, the function of mitochondrial succinate dehydrogenase and the expression of oxidative phosphorylation enzymes were markedly reduced. A similar effect was observed in OSCs. Based on this, a predictive algorithm was developed aimed to predict tumor recurrence in a subset of patients. MALAT1 targeting by gapmer delivery restored normal metabolic energy pathway in PCa cells and OSCs.
    Keywords:  MALAT1; biomarkers; long non-coding RNA; metabolic reprogramming; metabolism; precision medicine; predictive model; prostate cancer; transcription
    DOI:  https://doi.org/10.3390/cancers13010015
  27. Free Radic Biol Med. 2020 Dec 19. pii: S0891-5849(20)31682-8. [Epub ahead of print]163 243-254
    Pagacz J, Broniec A, Wolska M, Osyczka A, Borek A.
      Cytochrome bc1, also known as mitochondrial complex III, is considered to be one of the important producers of reactive oxygen species (ROS) in living organisms. Under physiological conditions, a certain level of ROS produced by mitochondrial electron transport chain (ETC) might be beneficial and take part in cellular signaling. However, elevated levels of ROS might exhibit negative effects, resulting in cellular damage. It is well known that inhibiting the electron flow within mitochondrial complex III leads to high production of ROS. However, superoxide production by cytochrome bc1 in a non-inhibited system remained controversial. Here, we propose a novel method for ROS detection in ETC hybrid system in solution comprising bacterial cytochrome bc1 and mitochondrial complex IV. We clearly show that non-inhibited cytochrome bc1 generates ROS and that adaptive and pathogenic mitochondrial mutations suppress and enhance ROS production, respectively. We also noted that cytochrome bc1 produces ROS in a rate-dependent manner and that the mechanism of ROS generation changes according to the rate of operation of the enzyme. This dependency has not yet been reported, but seems to be crucial when discussing ROS signaling originating from mitochondria.
    Keywords:  Amplex red; Cytochrome bc(1); Mitochondrial mutations; Reactive oxygen species; Superoxide anion radical; aa(3) oxidase
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2020.12.019
  28. Nat Rev Mol Cell Biol. 2020 Dec 22.
    Covarrubias AJ, Perrone R, Grozio A, Verdin E.
      Nicotinamide adenine dinucleotide (NAD+) is a coenzyme for redox reactions, making it central to energy metabolism. NAD+ is also an essential cofactor for non-redox NAD+-dependent enzymes, including sirtuins, CD38 and poly(ADP-ribose) polymerases. NAD+ can directly and indirectly influence many key cellular functions, including metabolic pathways, DNA repair, chromatin remodelling, cellular senescence and immune cell function. These cellular processes and functions are critical for maintaining tissue and metabolic homeostasis and for healthy ageing. Remarkably, ageing is accompanied by a gradual decline in tissue and cellular NAD+ levels in multiple model organisms, including rodents and humans. This decline in NAD+ levels is linked causally to numerous ageing-associated diseases, including cognitive decline, cancer, metabolic disease, sarcopenia and frailty. Many of these ageing-associated diseases can be slowed down and even reversed by restoring NAD+ levels. Therefore, targeting NAD+ metabolism has emerged as a potential therapeutic approach to ameliorate ageing-related disease, and extend the human healthspan and lifespan. However, much remains to be learnt about how NAD+ influences human health and ageing biology. This includes a deeper understanding of the molecular mechanisms that regulate NAD+ levels, how to effectively restore NAD+ levels during ageing, whether doing so is safe and whether NAD+ repletion will have beneficial effects in ageing humans.
    DOI:  https://doi.org/10.1038/s41580-020-00313-x
  29. Free Radic Biol Med. 2020 Dec 23. pii: S0891-5849(20)31675-0. [Epub ahead of print]163 196-209
    Ghanem A, Melzer AM, Zaal E, Neises L, Baltissen D, Matar O, Glennemeier-Marke H, Almouhanna F, Theobald J, Abu El Maaty MA, Berkers C, Wölfl S.
      The idea to use megadoses of ascorbate (vitamin C) for cancer treatment has recently been revived. Despite clear efficacy in animal experimentation, our understanding of the cellular and molecular mechanisms of this treatment is still limited and suggests a combined oxidative and metabolic mechanism behind the selective cytotoxicity of ascorbate towards cancerous cells. To gain more insight into the cellular effects of high doses of ascorbate, we performed a detailed analysis of metabolic changes and cell survival of both luminal and basal-like breast cancer cells treated with ascorbate and revealed a distinctive metabolic shift virtually reversing the Warburg effect and triggering a severe disruption of redox homeostasis. High doses of ascorbate were cytotoxic against MCF7 and MDA-MB231 cells representing luminal and basal-like breast cancer phenotypes. Cell death was dependent on ascorbate-induced oxidative stress and accumulation of ROS, DNA damage, and depletion of essential intracellular co-factors including NAD+/NADH, associated with a multifaceted metabolic rewiring. This included a sharp disruption of glycolysis at the triose phosphate level, a rapid drop in ATP levels, and redirection of metabolites toward lipid droplet accumulation and increased metabolites and enzymatic activity in the pentose phosphate pathway (PPP). High doses of ascorbate also inhibited the TCA cycle and increased oxygen consumption. Together the severe disruptions of the intracellular metabolic homeostasis on multiple levels "redox crisis and energetic catastrophe" consequently trigger a rapid irreversible cell death.
    Keywords:  Ascorboic acid; Breast cancer; Cancer; Cancer metabolism; Metabolic rewiring; Oxidative burst; Oxidative stress; Peroxide; Redox; Reversing warburg effect; Targetting cancer metabolism; Vitamin C
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2020.12.012
  30. Int J Mol Sci. 2020 Dec 28. pii: E204. [Epub ahead of print]22(1):
    Olivas-Aguirre M, Torres-López L, Pottosin I, Dobrovinskaya O.
      Anticancer activity of different phenols is documented, but underlying mechanisms remain elusive. Recently, we have shown that cannabidiol kills the cells of acute lymphoblastic leukemia (ALL) by a direct interaction with mitochondria, with their consequent dysfunction. In the present study, cytotoxic effects of several phenolic compounds against human the T-ALL cell line Jurkat were tested by means of resazurin-based metabolic assay. To unravel underlying mechanisms, mitochondrial membrane potential (∆Ψm) and [Ca2+]m measurements were undertaken, and reactive oxygen species generation and cell death were evaluated by flow cytometry. Three out of eight tested phenolics, cannabidiol, curcumin and quercetin, which displayed a significant cytotoxic effect, also dissipated the ∆Ψm and induced a significant [Ca2+]m increase, whereas inefficient phenols did not. Dissipation of the ∆Ψm by cannabidiol was prevented by cyclosporine A and reverted by Ru360, inhibitors of the permeation transition pore and mitochondrial Ca2+ uniporter, respectively. Ru360 prevented the phenol-induced [Ca2+]m rise, but neither cyclosporine A nor Ru360 affected the curcumin- and quercetin-induced ∆Ψm depolarization. Ru360 impeded the curcumin- and cannabidiol-induced cell death. Thus, all three phenols exert their antileukemic activity via mitochondrial Ca2+ overload, whereas curcumin and quercetin suppress the metabolism of leukemic cells by direct mitochondrial uncoupling.
    Keywords:  acute lymphoblastic leukemia; cannabidiol; curcumin; cytotoxicity; mitochondria; quercetin
    DOI:  https://doi.org/10.3390/ijms22010204
  31. Int J Cancer. 2020 Dec 24.
    Mitra D, Vega-Rubin-de-Celis S, Royla N, Bernhardt S, Wilhelm H, Tarade N, Poschet G, Buettner M, Binenbaum I, Borgoni S, Vetter M, Kantelhardt EJ, Thomssen C, Chatziioannou A, Hell R, Kempa S, Müller-Decker K, Wiemann S.
      Uncontrolled proliferation and altered metabolic reprogramming are hallmarks of cancer. Active glycolysis and glutaminolysis are characteristic features of these hallmarks and required for tumorigenesis. A fine balance between cancer metabolism and autophagy is a prerequisite of homeostasis within cancer cells. Here we show that glutamate pyruvate transaminase 2 (GPT2), which serves as a pivot between glycolysis and glutaminolysis, is highly upregulated in aggressive breast cancers, particularly the triple negative breast cancer (TNBC) subtype. Abrogation of this enzyme results in decreased TCA cycle intermediates, which promotes the rewiring of glucose carbon atoms and alterations in nutrient levels. Concordantly, loss of GPT2 results in an impairment of mechanistic target of rapamycin complex 1 (mTORC1) activity as well as the induction of autophagy. Furthermore, in vivo xenografts studies have shown that autophagy induction correlates with decreased tumor growth and that markers of induced autophagy correlate with low GPT2 levels in patient samples. Taken together, these findings indicate that cancer cells have a close network between metabolic and nutrient sensing pathways necessary to sustain tumorigenesis, and that aminotransferase reactions play an important role in maintaining this balance.
    Keywords:  Autophagy; Breast Cancer; Cancer metabolism; GPT2; mTORC1
    DOI:  https://doi.org/10.1002/ijc.33456
  32. Mitochondrion. 2020 Dec 27. pii: S1567-7249(20)30231-2. [Epub ahead of print]
    Adlimoghaddam A, Albensi BC.
      Hyperammonemia is very toxic to the brain, leading to inflammation, disruption of brain cellular energy metabolism and cognitive function. However, the underlying mechanism(s) for these impairments is still not fully understood. This study investigated the effects of ammonia in hippocampal astroglia derived from C57BL/6 mice. Parameters measured included oxygen consumption rates (OCR), ATP, cytochrome c oxidase (COX) activity, alterations in oxidative phosphorylation (OXPHOS), nuclear factor kappa B (NF-κB) subunits, key regulators of mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator1-alpha (PGC-1α), calcium/calmodulin-dependent protein kinase II (CaMKII), cAMP-response element binding protein (CREB), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), early growth response (Egr) factor family of proteins, and mitochondrial transcription factor A (TFAM). Ammonia was found to decrease mitochondrial numbers, potentially through a CaMKII-CREB-PGC1α-Nrf2 pathway in astroglia. Ammonia did not alter the levels of Egrs and TFAM in astroglia. Ammonia decreased OCR, ATP, COX, and OXPHOS levels in astroglia. To assess whether energy metabolism is reduced by ammonia through NF-κB associated pathways, astroglia were treated with ammonia alone or with NF-κB inhibitors such as Bay11-7082 or SN50. Mitochondrial OCR levels were reduced in the presence of NF-κB inhibitors; however co-treatment of NF-κB inhibitors and ammonia reversed mitochondrial deficits. Further, ammonia increased translocation of the NF-κB subunit into the nucleus of astroglia that correlates with an increased expression of NF-κB. These findings suggest that the NF-κB signaling pathway is putatively involved in ammonia-induced changes in bioenergetics in astroglia. Such research has critical implications for the treatment of disorders in which brain bioenergetics is compromised.
    Keywords:  Hyperammonemia; and astroglia; bioenergetics; biogenesis; mitochondria; oxidative phosphorylation
    DOI:  https://doi.org/10.1016/j.mito.2020.12.008
  33. Haematologica. 2020 Dec 23. Online ahead of print
    Carter BZ, Mak PY, Tao W, Warmoes M, Lorenzi PL, Mak D, Ruvolo V, Tan L, Cidado J, Drew L, Andreeff M.
      MCL-1 and BCL-2 are both frequently overexpressed in acute myeloid leukemia and critical for the survival of acute myeloid leukemia cells and acute myeloid leukemia stem cells. MCL-1 is a key factor in venetoclax resistance. Using genetic and pharmacological approaches, we discovered that MCL-1 regulates leukemia cell bioenergetics and carbohydrate metabolisms, including the TCA cycle, glycolysis and pentose phosphate pathway and modulates cell adhesion proteins and leukemia-stromal interactions. Inhibition of MCL-1 sensitizes to BCL-2 inhibition in acute myeloid leukemia cells and acute myeloid leukemia stem/progenitor cells, including those with intrinsic and acquired resistance to venetoclax through cooperative release of pro-apoptotic BIM, BAX, and BAK from binding to anti-apoptotic BCL-2 proteins and inhibition of cell metabolism and key stromal microenvironmental mechanisms. The combined inhibition of MCL-1 by MCL-1 inhibitor AZD5991 or CDK9 inhibitor AZD4573 and BCL-2 by venetoclax greatly extended survival of mice bearing patient-derived xenografts established from an acute myeloid leukemia patient who acquired resistance to venetoclax/decitabine. These results demonstrate that co-targeting MCL-1 and BCL-2 improves the efficacy of and overcomes preexisting and acquired resistance to BCL-2 inhibition. Activation of metabolomic pathways and leukemia-stroma interactions are newly discovered functions of MCL-1 in acute myeloid leukemia, which are independent from canonical regulation of apoptosis by MCL-1. Our data provide new mechanisms of synergy and rationale for co-targeting MCL-1 and BCL-2 clinically in patients with acute myeloid leukemia and potentially other cancers.
    DOI:  https://doi.org/10.3324/haematol.2020.260331
  34. Mol Ther Nucleic Acids. 2021 Mar 05. 23 232-243
    Guo S, Zhou K, Yuan Q, Su L, Liu Y, Ji X, Gu X, Guo X, Xing J.
      Next-generation sequencing technology has been commonly applied to detect mitochondrial DNA (mtDNA) mutations, which are reported to be strongly associated with cancers. However, several key challenges still exist regarding bioinformatics analysis of mtDNA sequencing data that greatly affect the detection accuracy of mtDNA mutations. Here we comprehensively evaluated several key analysis procedures in three different sample types. We found that a trimming procedure was essential for improving mtDNA mapping performance in plasma but not tissue samples. Mapping with a revised Cambridge reference sequence and human genome 19 reference was strongly suggested for mtDNA mutation detection in plasma samples because of the extreme abundance of nuclear DNA of mitochondrial origin. Moreover, our results showed that a setting of 3 mismatches was most appropriate for mtDNA mutation calling. Importantly, we revealed the presence of a negative logarithmic relationship between mtDNA site sequencing depth and minimum detectable mutation frequency and built an innovative and efficient filtering strategy to increase the accuracy and sensitivity of mutation detection. Finally, we verified that higher sequencing depth was required for a PCR-based compared with a capture-based enrichment strategy. We established an innovative data analysis strategy that is of great significance for improving the accuracy of mtDNA mutation detection for different types of tumor samples.
    Keywords:  bioinformatics; mtDNA mutation; next-generation sequencing
    DOI:  https://doi.org/10.1016/j.omtn.2020.11.002
  35. Biomed Pharmacother. 2021 Jan;pii: S0753-3322(20)31250-6. [Epub ahead of print]133 111058
    Datta S, Sears T, Cortopassi G, Woolard K, Angelastro JM.
      Glioblastoma Multiforme (GBM) tumors contain a small population of glioma stem-like cells (GSCs) among the various differentiated GBM cells (d-GCs). GSCs drive tumor recurrence, and resistance to Temozolomide (TMZ), the standard of care (SoC) for GBM chemotherapy. In order to investigate a potential link between GSC specific mitochondria function and SoC resistance, two patient-derived GSC lines were evaluated for differences in their mitochondrial metabolism. In both the lines, GSCs had significantly lower mitochondrial -content, and -function compared to d-GCs. In vitro, the standard mitochondrial-specific inhibitors oligomycin A, antimycin A, and rotenone selectively inhibited GSC proliferation to a greater extent than d-GCs and human primary astrocytes. These findings indicate that mitochondrial inhibition can be a potential GSC-targeted therapeutic strategy in GBM with minimal off-target toxicity. Mechanistically the standard mitochondrial inhibitors elicit their GSC-selective cytotoxic effects through the induction of apoptosis or autophagy pathways. We tested for GSC proliferation in the presence of 3 safe FDA-approved drugs--trifluoperazine, mitoxantrone, and pyrvinium pamoate, all of which are also known mitochondrial-targeting agents. The SoC GBM therapeutic TMZ did not trigger cytotoxicity in glioma stem cells, even at 100 μM concentration. By contrast, trifluoperazine, mitoxantrone, and pyrvinium pamoate exerted antiproliferative effects in GSCs about 30-50 fold more effectively than temozolomide. Thus, we hereby demonstrate that FDA-approved mitochondrial inhibitors induce GSC-selective cytotoxicity, and targeting mitochondrial function could present a potential therapeutic option for GBM treatment.
    Keywords:  Antimycin A (CID: 6604296); Cancer stem cells; Chemotherapy; Drug repurposing; Glioblastoma multiforme; Mitochondria; Mitoxantrone (CID:4212); Oligomycin A (CID: 5281899); Pyrvinium pamoate (CID:54680693); Rotenone (CID:6758); Therapeutics; Trifluoperazine (CID:5566)
    DOI:  https://doi.org/10.1016/j.biopha.2020.111058
  36. Biology (Basel). 2020 Dec 18. pii: E479. [Epub ahead of print]9(12):
    Moro L.
      Mitochondria are highly dynamic and regulated organelles implicated in a variety of important functions in the cell, including energy production, fatty acid metabolism, iron homeostasis, programmed cell death, and cell signaling. Changes in mitochondrial metabolism, signaling and dynamics are hallmarks of cancer. Understanding whether these modifications are associated with alterations of the mitochondrial proteome is particularly relevant from a translational point of view because it may contribute to better understanding the molecular bases of cancer development and progression and may provide new potential prognostic and diagnostic biomarkers as well as novel molecular targets for anti-cancer treatment. Making an inventory of the mitochondrial proteins has been particularly challenging given that there is no unique consensus targeting sequence that directs protein import into mitochondria, some proteins are present at very low levels, while other proteins are expressed only in some cell types, in a particular developmental stage or under specific stress conditions. This review aims at providing the state-of-the-art on methodologies used to characterize the mitochondrial proteome in tumors and highlighting the biological relevance of changes in expression and delocalization of proteins in and out the mitochondria in cancer biology.
    Keywords:  cancer; immunofluorescence; interactome; mass spectrometry; mitochondria; mitochondrial proteome
    DOI:  https://doi.org/10.3390/biology9120479
  37. Breast Cancer. 2020 Dec 24.
    Shafei MA, Flemban A, Daly C, Kendrick P, White P, Dean S, Qualtrough D, Conway ME.
      BACKGROUND: Biological characterisation of breast cancer subtypes is essential as it informs treatment regimens especially as different subtypes have distinct locoregional patterns. This is related to metabolic phenotype, where altered cellular metabolism is a fundamental adaptation of cancer cells during rapid proliferation. In this context, the metabolism of the essential branched-chain amino acids (BCAAs), catalysed by the human branched-chain aminotransferase proteins (hBCAT), offers multiple benefits for tumour growth. Upregulation of the cytosolic isoform of hBCAT (hBCATc), regulated by c-Myc, has been demonstrated to increase cell migration, tumour aggressiveness and proliferation in gliomas, ovarian and colorectal cancer but the importance of the mitochondrial isoform, hBCATm has not been fully investigated.METHODS: Using immunohistochemistry, the expression profile of metabolic proteins (hBCAT, IDH) was assessed between breast cancer subtypes, HER2 + , luminal A, luminal B and TNBC. Correlations between the percentage and the intensity of protein expression/co-expression with clinical parameters, such as hormone receptor status, tumour stage, lymph-node metastasis and survival, were determined.
    RESULTS: We show that hBCATc expression was found to be significantly associated with the more aggressive HER2 + and luminal B subtypes, whilst hBCATm and IDH1 associated with luminal A subtype. This was concomitant with better prognosis indicating a differential metabolic reliance between these two subtypes, in which enhanced expression of IDH1 may replenish the α-ketoglutarate pool in cells with increased hBCATm expression.
    CONCLUSION: The cytosolic isoform of BCAT is associated with tumours that express HER2 receptors, whereas the mitochondrial isoform is highly expressed in tumours that are ER + , indicating that the BCAT proteins are regulated through different signalling pathways, which may lead to the identification of novel targets for therapeutic applications targeting dysregulated cancer metabolism.
    Keywords:  BCAT; Breast cancer; HER2 + ; IDH; Luminal A
    DOI:  https://doi.org/10.1007/s12282-020-01197-7
  38. Biochim Biophys Acta Bioenerg. 2020 Dec 13. pii: S0005-2728(20)30205-X. [Epub ahead of print]1862(3): 148355
    Jarman OD, Biner O, Hirst J.
      F1FO-ATP synthase is a crucial metabolic enzyme that uses the proton motive force from respiration to regenerate ATP. For maximum thermodynamic efficiency ATP synthesis should be fully reversible, but the enzyme from Paracoccus denitrificans catalyzes ATP hydrolysis at far lower rates than it catalyzes ATP synthesis, an effect often attributed to its unique ζ subunit. Recently, we showed that deleting ζ increases hydrolysis only marginally, indicating that other common inhibitory mechanisms such as inhibition by the C-terminal domain of the ε subunit (ε-CTD) or Mg-ADP may be more important. Here, we created mutants lacking the ε-CTD, and double mutants lacking both the ε-CTD and ζ subunit. No substantial activation of ATP hydrolysis was observed in any of these strains. Instead, hydrolysis in even the double mutant strains could only be activated by oxyanions, the detergent lauryldimethylamine oxide, or a proton motive force, which are all considered to release Mg-ADP inhibition. Our results establish that P. denitrificans ATP synthase is regulated by a combination of the ε and ζ subunits and Mg-ADP inhibition.
    Keywords:  ATP hydrolysis; F(1)F(O)-ATP synthase; Mg-ADP inhibition; P. denitrificans; ε subunit; ζ subunit
    DOI:  https://doi.org/10.1016/j.bbabio.2020.148355
  39. Mol Cell Proteomics. 2020 Dec 28. pii: mcp.RA120.002301. [Epub ahead of print]
    Swovick K, Firsanov D, Welle KA, Hryhorenko J, Wise JP, George C, Sformo TL, Seluanov A, Gorbunova V, Ghaemmaghami S.
      Cells continually degrade and replace damaged proteins. However, the high energetic demand of protein turnover generates reactive oxygen species (ROS) that compromise the long-term health of the proteome. Thus, the relationship between aging, protein turnover and energetic demand remains unclear. Here, we used a proteomic approach to measure rates of protein turnover within primary fibroblasts isolated from a number of species with diverse lifespans including the longest-lived mammal, the bowhead whale. We show that organismal lifespan is negatively correlated with turnover rates of highly abundant proteins. In comparison to mice, cells from long-lived naked mole rats have slower rates of protein turnover, lower levels of ATP production and reduced ROS levels. Despite having slower rates of protein turnover, naked mole rat cells tolerate protein misfolding stress more effectively than mouse cells. We suggest that in lieu of rapid constitutive turnover, long-lived species may have evolved more energetically efficient mechanisms for selective detection and clearance of damaged proteins.
    Keywords:  Aging; Energy metabolism; Protein Degradation*; Protein Turnover*; Proteostasis; SILAC
    DOI:  https://doi.org/10.1074/mcp.RA120.002301
  40. Aging Cell. 2020 Dec 23. e13283
    Lazo S, Noren Hooten N, Green J, Eitan E, Mode NA, Liu QR, Zonderman AB, Ezike N, Mattson MP, Ghosh P, Evans MK.
      The mitochondrial free radical theory of aging suggests that accumulating oxidative damage to mitochondria and mitochondrial DNA (mtDNA) plays a central role in aging. Circulating cell-free mtDNA (ccf-mtDNA) isolated from blood may be a biomarker of disease. Extracellular vesicles (EVs) are small (30-400 nm), lipid-bound vesicles capable of shuttling proteins, nucleic acids, and lipids as part of intercellular communication systems. Here, we report that a portion of ccf-mtDNA in plasma is encapsulated in EVs. To address whether EV mtDNA levels change with human age, we analyzed mtDNA in EVs from individuals aged 30-64 years cross-sectionally and longitudinally. EV mtDNA levels decreased with age. Furthermore, the maximal mitochondrial respiration of cultured cells was differentially affected by EVs from old and young donors. Our results suggest that plasma mtDNA is present in EVs, that the level of EV-derived mtDNA is associated with age, and that EVs affect mitochondrial energetics in an EV age-dependent manner.
    Keywords:  aging; biomarker; circulating cell-free mitochondrial DNA; exosomes; extracellular vesicles; intercellular communication; microvesicles; mitochondrial DNA
    DOI:  https://doi.org/10.1111/acel.13283
  41. Front Oncol. 2020 ;10 559568
    Zhang H, Guo W, Zhang F, Li R, Zhou Y, Shao F, Feng X, Tan F, Wang J, Gao S, Gao Y, He J.
      Abnormal metabolism is one of the hallmarks of cancer cells. Monoacylglycerol lipase (MGLL), a key enzyme in lipid metabolism, has emerged as an important regulator of tumor progression. In this study, we aimed to characterize the role of MGLL in the development of lung adenocarcinoma (LUAD). To this end, we used tissue microarrays to evaluate the expression of MGLL in LUAD tissue and assessed whether the levels of this protein are correlated with clinicopathological characteristics of LUAD. We found that the expression of MGLL is higher in LUAD samples than that in adjacent non-tumor tissues. In addition, elevated MGLL expression was found to be associated with advanced tumor progression and poor prognosis in LUAD patients. Functional studies further demonstrated that stable short hairpin RNA (shRNA)-mediated knockdown of MGLL inhibits tumor proliferation and metastasis, both in vitro and in vivo, and mechanistically, our data indicate that MGLL regulates Cyclin D1 and Cyclin B1 in LUAD cells. Moreover, we found that knockdown of MGLL suppresses the expression of matrix metalloproteinase 14 (MMP14) in A549 and H322 cells, and in clinical samples, expression of MMP14 is significantly correlated with MGLL expression. Taken together, our results indicate that MGLL plays an oncogenic role in LUAD progression and metastasis and may serve as a potential biomarker for disease prognosis and as a target for the development of personalized therapies.
    Keywords:  lung adenocarcinoma; matrix metalloproteinase 14; metastasis; monoacylglycerol lipase; prognosis; proliferation
    DOI:  https://doi.org/10.3389/fonc.2020.559568
  42. Front Oncol. 2020 ;10 583217
    Atas E, Oberhuber M, Kenner L.
      A metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis-known as the Warburg effect-is characteristic for many cancers. It gives the cancer cells a survival advantage in the hypoxic tumor microenvironment and protects them from cytotoxic effects of oxidative damage and apoptosis. The main regulators of this metabolic shift are the pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase (PDK) isoforms 1-4. PDK is known to be overexpressed in several cancers and is associated with bad prognosis and therapy resistance. Whereas the expression of PDK1-3 is tissue specific, PDK4 expression is dependent on the energetic state of the whole organism. In contrast to other PDK isoforms, not only oncogenic, but also tumor suppressive functions of PDK4 have been reported. In tumors that profit from high OXPHOS and high de novo fatty acid synthesis, PDK4 can have a protective effect. This is the case for prostate cancer, the most common cancer in men, and makes PDK4 an interesting therapeutic target. While most work is focused on PDK in tumors characterized by high glycolytic activity, little research is devoted to those cases where PDK4 acts protective and is therefore highly needed.
    Keywords:  Warburg effect; aerobic glycolysis; cancer metabolism; oxidative phosphorylation; prostate cancer; pyruvate dehydrogenase kinase; therapy resistance; tricarboxylic acid cycle
    DOI:  https://doi.org/10.3389/fonc.2020.583217
  43. Biotechnol Lett. 2021 Jan 01.
    Yi H, Shen X, Wang H, Luo S, Guo W, Chen P, Hu L, Liang L, Gong Y, Xiao X, Liu J.
      OBJECTIVE: Two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF mass spectrometry were performed to compare the proteomic alterations of lycorine-treated and control cells to further investigate the anti-multiple myeloma (MM) mechanisms of lycorine.RESULTS: Mass spectrometry results showed that after lycorine treatment of MM cells, 42% of the differentially expressed proteins had subcellular localization, mainly, on mitochondria. Voltage-dependent anion-selective channel protein 2 (VDAC2), the most abundant protein in the outer mitochondrial membrane, was up-regulated after treatment with lycorine and was subsequently verified by western blot analysis. Further studies on mitochondria found that lycorine was able to increase abnormal mitochondria and increase mitochondrial membrane potential.
    CONCLUSIONS: Lycorine can achieve the effect of resisting multiple myeloma by acting on VDAC2 and causing mitochondrial abnormalities.
    Keywords:  2-DE; Abnormal mitochondria; Lycorine; MALDI-TOF/TOF mass spectrometry; Multiple myeloma; VDAC2
    DOI:  https://doi.org/10.1007/s10529-020-03053-2