bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2020‒04‒05
forty-six papers selected by
Kelsey Fisher-Wellman
East Carolina University
  1. A salvage pathway maintains highly functional respiratory complex I.
  2. Fitter Mitochondria Are Associated With Radioresistance in Human Head and Neck SQD9 Cancer Cells.
  3. The MICOS complex, a structural element of mitochondria with versatile functions.
  4. Release of extracellular vesicle miR-494-3p by ARPE-19 cells with impaired mitochondria.
  5. Higher proteotoxic stress rather than mitochondrial damage is involved in higher neurotoxicity of bortezomib compared to carfilzomib.
  6. The short variant of optic atrophy 1 (OPA1) improves cell survival under oxidative stress.
  7. Mitochondrial Respiration in KRAS and BRAF Mutated Colorectal Tumors and Polyps.
  8. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents.
  9. Mitigating effect of paxilline against injury produced by Cd2+ in rat pheochromocytoma PC12 and ascites hepatoma AS-30D cells.
  10. PGRMC1 phosphorylation affects cell shape, motility, glycolysis, mitochondrial form and function, and tumor growth.
  11. Burn-Induced Cardiac Mitochondrial Dysfunction via Interruption of the PDE5A-cGMP-PKG Pathway.
  12. Sennoside A restores colonic barrier function through protecting colon enterocytes from ROS-induced mitochondrial damage in diet-induced obese mice.
  13. Alterations of mitochondrial bioenergetics, dynamics, and morphology support the theory of oxidative damage involvement in autism spectrum disorder.
  14. MitoPlex: A targeted multiple reaction monitoring assay for quantification of a curated set of mitochondrial proteins.
  15. Sex and BNIP3 genotype, rather than acute lipid injection, modulate hepatic mitochondrial function and steatosis risk in mice.
  16. Mitochondrial fission factor (MFF) is a critical regulator of peroxisome maturation.
  17. Mitochondrial-targeted and Near-infrared Fluorescent Probe for Bioimaging and Evaluating Monoamine Oxidase A Activity in Hepatic Fibrosis.
  18. Mitochondrial respiratory inhibition promoted by pyraclostrobin in fungi is also observed in honey bees.
  19. Glucose-Stimulated Insulin Secretion Fundamentally Requires H2O2 Signaling by NADPH Oxidase 4.
  20. Down-regulation of AMPK signaling pathway rescues hearing loss in TFB1 transgenic mice and delays age-related hearing loss.
  21. Comprehensive assessment of mitochondrial respiratory function in freshly isolated nephron segments.
  22. Mitochondrial fragmentation enables localized signaling required for cell repair.
  23. Experimental oxygen concentration influences rates of mitochondrial hydrogen peroxide release from cardiac and skeletal muscle preparations.
  24. Estrogen Signaling Induces Mitochondrial Dysfunction-Associated Autophagy and Senescence in Breast Cancer Cells.
  25. xCT Inhibition Increases Sensitivity to Vorinostat in a ROS-Dependent Manner.
  26. Real-Time Imaging and Simultaneous Quantification of Mitochondrial H2O2 and ATP in Neurons with a Single Two-Photon Fluorescence Lifetime-Based Probe.
  27. Pioglitazone and Deoxyribonucleoside Combination Treatment Increases Mitochondrial Respiratory Capacity in m.3243A>G MELAS Cybrid Cells.
  28. Mitochondrial dysfunction and DNA damage accompany enhanced levels of formaldehyde in cultured primary human fibroblasts.
  29. Acid Sphingomyelinase Downregulation Enhances Mitochondrial Fusion and Promotes Oxidative Metabolism in a Mouse Model of Melanoma.
  30. Pan-Cancer Analysis of Mitochondria Chaperone-Client Co-Expression Reveals Chaperone Functional Partitioning.
  31. Palmatine induces G2/M phase arrest and mitochondrial-associated pathway apoptosis in colon cancer cells by targeting AURKA.
  32. Mitochondria dysfunction and metabolic reprogramming as drivers of idiopathic pulmonary fibrosis.
  33. Metformin enhances the sensitivity of colorectal cancer cells to cisplatin through ROS-mediated PI3K/Akt signaling pathway.
  34. Metformin is distributed to tumor tissue in breast cancer patients in vivo: A 11C-metformin PET/CT study.
  35. Amination of Graphene Oxide Leads to Increased Cytotoxicity in Hepatocellular Carcinoma Cells.
  36. A FRET Based Two-Photon Fluorescent Probe for Visualizing Mitochondrial Thiols of Living Cells and Tissues.
  37. Blocking Parkin/PINK1-mediated mitophagy sensitizes hepatocellular carcinoma cells to sanguinarine-induced mitochondrial apoptosis.
  38. Exercise-Mediated Lowering of Glutamine Availability Suppresses Tumor Growth and Attenuates Muscle Wasting.
  39. Menadione/Ascorbate Induces Overproduction of Mitochondrial Superoxide and Impairs Mitochondrial Function in Cancer: Comparative Study on Cancer and Normal Cells of the Same Origin.
  40. Metformin enhances cytotoxic action of dichloroacetate against Lewis lung carcinoma cells in vitro.
  41. Aurora kinase inhibitor restrains STAT5-activated leukemic cell proliferation by inducing mitochondrial impairment.
  42. Mitochondria at the crossroads of ATM-mediated stress signaling and regulation of reactive oxygen species.
  43. Increased mitochondrial fragmentation in polycystic kidney disease acts as a modifier of disease progression.
  44. Cystine transporter regulation of pentose phosphate pathway dependency and disulfide stress exposes a targetable metabolic vulnerability in cancer.
  45. Redox Modifications of Proteins of the Mitochondrial Fusion and Fission Machinery.
  46. Mitoregulin Controls β-Oxidation in Human and Mouse Adipocytes.
  1. Nat Commun. 2020 Apr 02. 11(1): 1643
    A salvage pathway maintains highly functional respiratory complex I.
    Karolina Szczepanowska, Katharina Senft, Juliana Heidler, Marija Herholz, Alexandra Kukat, Michaela Nicole Höhne, Eduard Hofsetz, Christina Becker, Sophie Kaspar, Heiko Giese, Klaus Zwicker, Sergio Guerrero-Castillo, Linda Baumann, Johanna Kauppila, Anastasia Rumyantseva, Stefan Müller, Christian K Frese, Ulrich Brandt, Jan Riemer, Ilka Wittig, Aleksandra Trifunovic.
      Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.
    DOI:  https://doi.org/10.1038/s41467-020-15467-7
  2. Front Pharmacol. 2020 ;11 263
    Fitter Mitochondria Are Associated With Radioresistance in Human Head and Neck SQD9 Cancer Cells.
    Debora Grasso, Hyllana C D Medeiros, Luca X Zampieri, Vanesa Bol, Pierre Danhier, Marike W van Gisbergen, Caroline Bouzin, Davide Brusa, Vincent Grégoire, Hubert Smeets, Alphons P M Stassen, Ludwig J Dubois, Philippe Lambin, Marie Dutreix, Pierre Sonveaux.
      The clinical management of head and neck squamous cell carcinoma (HNSCC) commonly involves chemoradiotherapy, but recurrences often occur that are associated with radioresistance. Using human SQD9 laryngeal squamous cell carcinoma cancer cells as a model, we aimed to identify metabolic changes associated with acquired radioresistance. In a top-down approach, matched radiosensitive and radioresistant SQD9 cells were generated and metabolically compared, focusing on glycolysis, oxidative phosphorylation (OXPHOS) and ROS production. The cell cycle, clonogenicity, tumor growth in mice and DNA damage-repair were assessed. Mitochondrial DNA (mtDNA) was sequenced. In a bottom-up approach, matched glycolytic and oxidative SQD9 cells were generated using FACS-sorting, and tested for their radiosensitivity/radioresistance. We found that acquired radioresistance is associated with a shift from a glycolytic to a more oxidative metabolism in SQD9 cells. The opposite was also true, as the most oxidative fraction isolated from SQD9 wild-type cells was also more radioresistant than the most glycolytic fraction. However, neither reduced hexokinase expression nor OXPHOS were directly responsible for the radioresistant phenotype. Radiosensitive and radioresistant cells had similar proliferation rates and were equally efficient for ATP production. They were equally sensitive to redox stress and had similar DNA damage repair, but radioresistant cells had an increased number of mitochondria and a higher mtDNA content. Thus, an oxidative switch is associated with but is not responsible for acquired radioresistance in human SQD9 cells. In radioresistant cells, more abundant and fitter mitochondria could help to preserve mitochondrial functions upon irradiation.
    Keywords:  Head and neck cancer; cancer metabolism; mitochondria; oxidative phosphorylation (OXPHOS); radioresistance mechanisms; radiotherapy
    DOI:  https://doi.org/10.3389/fphar.2020.00263
  3. Biol Chem. 2020 Mar 01. pii: /j/bchm.just-accepted/hsz-2020-0103/hsz-2020-0103.xml. [Epub ahead of print]
    The MICOS complex, a structural element of mitochondria with versatile functions.
    Siavash Khosravi, Max E Harner.
      Mitochondria perform a plethora of functions in various cells of different tissues. Their architecture differs remarkably, for instance in neurons versus steroidogenic cells. Furthermore, aberrant mitochondrial architecture results in mitochondrial dysfunction. This indicates strongly that mitochondrial architecture and function are intimately linked. Therefore, a deep knowledge about the determinants of mitochondrial architecture and their function on a molecular level is of utmost importance. In the past decades, various proteins and protein complexes essential for formation of mitochondrial architecture have been identified. Here we will review the current knowledge of the MICOS complex, one of the major structural elements of mitochondria. MICOS is a multi-subunit complex present in the inner mitochondrial membrane. Multiple interaction partners in the inner and outer mitochondrial membrane point to participation in a multitude of important processes, such as generation of mitochondrial architecture, lipid metabolism, and protein import into mitochondria. Since the MICOS complex is highly conserved in form and function throughout evolution, we will highlight the importance of MICOS for mammals. We will emphasize in particular the current knowledge of the association of MICOS with severe human diseases, including Charcot-Marie-Tooth disease type 2, Alzheimer's disease, Parkinson's disease, Frontotemporal Dementia and Amyotrophic Lateral Sclerosis.
    Keywords:  crista formation; mitochondrial architecture; mitochondrial biogenesis; mitochondrial lipid metabolism; mitochondrial protein import; neurodegeneration
    DOI:  https://doi.org/10.1515/hsz-2020-0103
  4. Biochim Biophys Acta Gen Subj. 2020 Mar 30. pii: S0304-4165(20)30110-0. [Epub ahead of print] 129598
    Release of extracellular vesicle miR-494-3p by ARPE-19 cells with impaired mitochondria.
    J Y Ahn, S Datta, E Bandeira, M Cano, E Mallick, U Rai, B Powell, J Tian, K W Witwer, J T Handa, M E Paulaitis.
      BACKGROUND: Mitochondrial function in retinal pigmented epithelial (RPE) cells and extracellular vesicle (EV) formation/release are related through the lysosomal and exocytotic pathways that process and eliminate intracellular material, including mitochondrial fragments. We propose that RPE cells with impaired mitochondria will release EVs containing mitochondrial miRNAs that reflect the diminished capacity of mitochondria within these cells.METHODS: We screened ARPE-19 cells for miRNAs that localize to the mitochondria, exhibit biological activity, and are present in EVs released by both untreated cells and cells treated with rotenone to induce mitochondrial injury. EVs were characterized by vesicle size, size distribution, presence of EV biomarkers: CD81, CD63, and syntenin-1, miRNA cargo, and number concentration of EVs released per cell.
    RESULTS: We found that miR-494-3p was enriched in ARPE-19 mitochondria. Knockdown of miR-494-3p in ARPE-19 cells decreased ATP production and mitochondrial membrane potential in a dose-dependent manner, and decreased basal oxygen consumption rate and maximal respiratory capacity. Increased number concentrations of EVs released per cell and elevated levels of miR-494-3p in EVs released from ARPE-19 cells treated with rotenone were also measured.
    CONCLUSIONS: ARPE-19 mitochondrial function is regulated by miR-494-3p. Elevated levels of miR-494-3p in EVs released by ARPE-19 cells indicate diminished capacity of the mitochondria within these cells.
    GENERAL SIGNIFICANCE: EV miR-494-3p is a potential biomarker for RPE mitochondrial dysfunction, which plays a central role in non-neovascular age-related macular degeneration, and may be a diagnostic biomarker for monitoring the spread of degeneration to neighboring RPE cells in the retina.
    Keywords:  Age-related macular degeneration; Extracellular vesicles; Mitochondria; Retinal pigment epithelial cells; miR-494-3p; miRNA
    DOI:  https://doi.org/10.1016/j.bbagen.2020.129598
  5. Redox Biol. 2020 Mar 21. pii: S2213-2317(19)31437-5. [Epub ahead of print]32 101502
    Higher proteotoxic stress rather than mitochondrial damage is involved in higher neurotoxicity of bortezomib compared to carfilzomib.
    Ayse Tarbin Jannuzzi, Sema Arslan, Ayse Mine Yilmaz, Gulce Sari, Hande Beklen, Lucía Méndez, Maria Fedorova, Kazim Yalcin Arga, Betul Karademir Yilmaz, Buket Alpertunga.
      Proteasome inhibitors have great success for their therapeutic potential against hematologic malignancies. First generation proteasome inhibitor bortezomib induced peripheral neuropathy is considered as a limiting factor in chemotherapy and its second-generation counterpart carfilzomib is associated with lower rates of neurotoxicity. The mitochondrial toxicity (mitotoxicity) hypothesis arises from studies with animal models of bortezomib induced peripheral neuropathy. However, molecular mechanisms are not fully elucidated and the role of mitotoxicity in bortezomib and carfilzomib induced neurotoxicity has not been investigated comparatively. Herein, we characterized the neurotoxic effects of bortezomib and carfilzomib at the molecular level in human neuronal cells using LC-MS/MS analysis, flow cytometry, RT-qPCR, confocal microscopy and western blotting. We showed that bortezomib and carfilzomib affected the human neuronal proteome differently, and bortezomib caused higher proteotoxic stress via protein oxidation, protein K48-ubiquitination, heat shock protein expression upregulation and reduction of mitochondria membrane potential. Bortezomib and carfilzomib did not affect the gene expression levels related to mitochondrial dynamics (optic atrophy 1; OPA1, mitofusin 1; MFN1, mitofusin 2; MFN2, fission 1; FIS1, dynamin-related protein 1; DRP1) and overall mitophagy rate whereas, PINK1/Parkin mediated mitophagy gene expressions were altered with both drugs. Bortezomib and carfilzomib caused downregulation of the contents of mitochondrial oxidative phosphorylation complexes, voltage-dependent anion channel 1 (VDAC1) and uncoupling protein 2 (UCP2) similarly. Our findings suggest that, both drugs induce mitotoxicity besides proteotoxic stress in human neuronal cells and the higher incidence of neurotoxicity with bortezomib than carfilzomib is not directly related to mitochondrial pathways.
    Keywords:  Bortezomib; Carfilzomib; Mitotoxicity; Neurotoxicity; Peripheral neuropathy
    DOI:  https://doi.org/10.1016/j.redox.2020.101502
  6. J Biol Chem. 2020 Apr 03. pii: jbc.RA119.010983. [Epub ahead of print]
    The short variant of optic atrophy 1 (OPA1) improves cell survival under oxidative stress.
    Hakjoo Lee, Sylvia B Smith, Shey-Shing Sheu, Yisang Yoon.
      Optic atrophy 1 (OPA1) is a dynamin protein that mediates mitochondrial fusion at the inner membrane. OPA1 is also necessary for maintaining the cristae, and thus essential for supporting cellular energetics. OPA1 exists as membrane-anchored long form (L-OPA1) and short form (S-OPA1) that lacks the transmembrane region and is generated by cleavage of L-OPA1. Mitochondrial dysfunction and cellular stresses activate the inner membrane-associated zinc metallopeptidase OMA1 that cleaves L-OPA1, causing S-OPA1 accumulation. The prevailing notion has been that L-OPA1 is the functional form while S-OPA1 is an inactive cleavage product in mammals, and that stress-induced OPA1 cleavage causes mitochondrial fragmentation and sensitizes cells to death. However, S-OPA1 contains all functional domains of dynamin proteins, suggesting that it has a physiological role. Indeed, we recently demonstrated that S-OPA1 can maintain cristae and energetics through its GTPase activity, despite lacking fusion activity. Here, applying oxidant insult that induces OPA1 cleavage, we show that cells unable to generate S-OPA1 are more sensitive to this stress under obligatory respiratory conditions, leading to necrotic death. These findings indicate that L-OPA1 and S-OPA1 differ in maintaining mitochondrial function. Mechanistically, we found that cells that exclusively express L-OPA1 generate more superoxide and are more sensitive to Ca2+-induced mitochondrial permeability transition, suggesting that S-OPA1, and not L-OPA1, protects against cellular stress. Importantly, silencing of OMA1 expression increased oxidant-induced cell death, indicating that stress-induced OPA1 cleavage supports cell survival. Our findings suggest that S-OPA1 generation by OPA1 cleavage is a survival mechanism in stressed cells.
    Keywords:  OPA1; mitochondria; mitochondrial dynamics; mitochondrial permeability transition (MPT); necrosis (necrotic death); oxidative stress; reactive oxygen species (ROS)
    DOI:  https://doi.org/10.1074/jbc.RA119.010983
  7. Cancers (Basel). 2020 Mar 28. pii: E815. [Epub ahead of print]12(4):
    Mitochondrial Respiration in KRAS and BRAF Mutated Colorectal Tumors and Polyps.
    Egle Rebane-Klemm, Laura Truu, Leenu Reinsalu, Marju Puurand, Igor Shevchuk, Vladimir Chekulayev, Natalja Timohhina, Kersti Tepp, Jelena Bogovskaja, Vladimir Afanasjev, Külliki Suurmaa, Vahur Valvere, Tuuli Kaambre.
      This study aimed to characterize the ATP-synthesis by oxidative phosphorylation in colorectal cancer (CRC) and premalignant colon polyps in relation to molecular biomarkers KRAS and BRAF. This prospective study included 48 patients. Resected colorectal polyps and postoperative CRC tissue with adjacent normal tissue (control) were collected. Patients with polyps and CRC were divided into three molecular groups: KRAS mutated, BRAF mutated and KRAS/BRAF wild-type. Mitochondrial respiration in permeabilized tissue samples was observed using high resolution respirometry. ADP-activated respiration rate (Vmax) and an apparent affinity of mitochondria to ADP, which is related to mitochondrial outer membrane (MOM) permeability, were determined. Clear differences were present between molecular groups. KRAS mutated CRC group had lower Vmax values compared to wild-type; however, the Vmax value was higher than in the control group, while MOM permeability did not change. This suggests that KRAS mutation status might be involved in acquiring oxidative phenotype. KRAS mutated polyps had higher Vmax values and elevated MOM permeability as compared to the control. BRAF mutated CRC and polyps had reduced respiration and altered MOM permeability, indicating a glycolytic phenotype. To conclude, prognostic biomarkers KRAS and BRAF are likely related to the metabolic phenotype in CRC and polyps. Assessment of the tumor mitochondrial ATP synthesis could be a potential component of patient risk stratification.
    Keywords:  BRAF; KRAS; colorectal cancer; colorectal polyps; energy metabolism; mitochondria; oxidative phosphorylation
    DOI:  https://doi.org/10.3390/cancers12040815
  8. Nat Rev Mol Cell Biol. 2020 Mar 30.
    Reactive oxygen species (ROS) as pleiotropic physiological signalling agents.
    Helmut Sies, Dean P Jones.
      'Reactive oxygen species' (ROS) is an umbrella term for an array of derivatives of molecular oxygen that occur as a normal attribute of aerobic life. Elevated formation of the different ROS leads to molecular damage, denoted as 'oxidative distress'. Here we focus on ROS at physiological levels and their central role in redox signalling via different post-translational modifications, denoted as 'oxidative eustress'. Two species, hydrogen peroxide (H2O2) and the superoxide anion radical (O2·-), are key redox signalling agents generated under the control of growth factors and cytokines by more than 40 enzymes, prominently including NADPH oxidases and the mitochondrial electron transport chain. At the low physiological levels in the nanomolar range, H2O2 is the major agent signalling through specific protein targets, which engage in metabolic regulation and stress responses to support cellular adaptation to a changing environment and stress. In addition, several other reactive species are involved in redox signalling, for instance nitric oxide, hydrogen sulfide and oxidized lipids. Recent methodological advances permit the assessment of molecular interactions of specific ROS molecules with specific targets in redox signalling pathways. Accordingly, major advances have occurred in understanding the role of these oxidants in physiology and disease, including the nervous, cardiovascular and immune systems, skeletal muscle and metabolic regulation as well as ageing and cancer. In the past, unspecific elimination of ROS by use of low molecular mass antioxidant compounds was not successful in counteracting disease initiation and progression in clinical trials. However, controlling specific ROS-mediated signalling pathways by selective targeting offers a perspective for a future of more refined redox medicine. This includes enzymatic defence systems such as those controlled by the stress-response transcription factors NRF2 and nuclear factor-κB, the role of trace elements such as selenium, the use of redox drugs and the modulation of environmental factors collectively known as the exposome (for example, nutrition, lifestyle and irradiation).
    DOI:  https://doi.org/10.1038/s41580-020-0230-3
  9. Ecotoxicol Environ Saf. 2020 Mar 31. pii: S0147-6513(20)30358-4. [Epub ahead of print]196 110519
    Mitigating effect of paxilline against injury produced by Cd2+ in rat pheochromocytoma PC12 and ascites hepatoma AS-30D cells.
    Elena A Belyaeva, Tatyana V Sokolova.
      On two rat cell lines, pheochromocytoma PC12 and ascites hepatoma AS-30D, and on rat liver mitochondria we studied action of paxilline (lipophilic mycotoxin from fungus Penicillium paxilli which is blocker of large-conductance potassium channels) against harmful effects of Cd(II) - one of the most dangerous toxic metals and environmental pollutants. We investigated an influence of paxilline on cell viability and mitochondrial function in the presence and in the absence of Cd2+. As found, paxilline protected partially from the Cd2+-induced cytotoxicity, namely taken in concentration of 1 μM it decreased the Cd2+-induced cell necrosis in average by 10-14 or 13-23% for AS-30D and PC12 cells, respectively. Nevertheless, paxilline did not affect the Cd2+-induced apoptosis of AS-30D cells. The alleviating concentration of paxilline reduced an intracellular production of reactive oxygen species (ROS) in PC12 cells intoxicated by Cd2+ and enhanced the ROS production in control AS-30D cells; however, it weakly affected mitochondrial membrane potential of the cells in the absence and in the presence of Cd2+. The ameliorative concentration of paxilline decreased the maximal respiration rates of control cells of both types after short-term (3-5 h) treatment with it while the rates reached their control levels after long-term (24-48 h) incubation with the drug. Paxilline was not protective against the Cd2+-induced membrane permeability and respiration rate changes in isolated rat liver mitochondria. As result, the mitochondrial electron transport chain was concluded to contribute in the mitigating effect of paxilline against the Cd2+-produced cell injury.
    Keywords:  Cd(2+)-induced mitochondrial dysfunction-mediated cytotoxicity; Large-conductance potassium channel blocker; Mitochondrial electron transport chain; Paxilline; Protection mechanism(s); Rat liver mitochondria and PC12 and AS-30D cells
    DOI:  https://doi.org/10.1016/j.ecoenv.2020.110519
  10. BMC Mol Cell Biol. 2020 Apr 03. 21(1): 24
    PGRMC1 phosphorylation affects cell shape, motility, glycolysis, mitochondrial form and function, and tumor growth.
    Bashar M Thejer, Partho P Adhikary, Amandeep Kaur, Sarah L Teakel, Ashleigh Van Oosterum, Ishith Seth, Marina Pajic, Katherine M Hannan, Megan Pavy, Perlita Poh, Jalal A Jazayeri, Thiri Zaw, Dana Pascovici, Marina Ludescher, Michael Pawlak, Juan C Cassano, Lynne Turnbull, Mitra Jazayeri, Alexander C James, Craig P Coorey, Tara L Roberts, Simon J Kinder, Ross D Hannan, Ellis Patrick, Mark P Molloy, Elizabeth J New, Tanja N Fehm, Hans Neubauer, Ewa M Goldys, Leslie A Weston, Michael A Cahill.
      BACKGROUND: Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in many cancer cells, where it is associated with detrimental patient outcomes. It contains phosphorylated tyrosines which evolutionarily preceded deuterostome gastrulation and tissue differentiation mechanisms.RESULTS: We demonstrate that manipulating PGRMC1 phosphorylation status in MIA PaCa-2 (MP) cells imposes broad pleiotropic effects. Relative to parental cells over-expressing hemagglutinin-tagged wild-type (WT) PGRMC1-HA, cells expressing a PGRMC1-HA-S57A/S181A double mutant (DM) exhibited reduced levels of proteins involved in energy metabolism and mitochondrial function, and altered glucose metabolism suggesting modulation of the Warburg effect. This was associated with increased PI3K/AKT activity, altered cell shape, actin cytoskeleton, motility, and mitochondrial properties. An S57A/Y180F/S181A triple mutant (TM) indicated the involvement of Y180 in PI3K/AKT activation. Mutation of Y180F strongly attenuated subcutaneous xenograft tumor growth in NOD-SCID gamma mice. Elsewhere we demonstrate altered metabolism, mutation incidence, and epigenetic status in these cells.
    CONCLUSIONS: Altogether, these results indicate that mutational manipulation of PGRMC1 phosphorylation status exerts broad pleiotropic effects relevant to cancer and other cell biology.
    Keywords:  Cytochrome P450; Invasion; Mesenchymal amoeboid transition; Metabolism; Migration; Mitochondria; Proteomics; Tumor biology
    DOI:  https://doi.org/10.1186/s12860-020-00256-3
  11. Int J Mol Sci. 2020 Mar 28. pii: E2350. [Epub ahead of print]21(7):
    Burn-Induced Cardiac Mitochondrial Dysfunction via Interruption of the PDE5A-cGMP-PKG Pathway.
    Jake J Wen, Claire B Cummins, Ravi S Radhakrishnan.
      Burn-induced heart dysfunction is a key factor for patient mortality. However, the molecular mechanisms are not yet fully elucidated. This study sought to understand whether burn-induced heart dysfunction is associated with cardiac mitochondrial dysfunction and interruption of the PDE5A-cGMP-PKG pathway. Sixty percent total body surface area (TBSA) scald burned rats (±sildenafil) were used in this study. A transmission electron microscope (TEM), real-time qPCR, O2K-respirometer, and electron transport chain assays were used to characterized molecular function. Cardiac mitochondrial morphological shapes were disfigured with a decline in mitochondrial number, area, and size, resulting in deficiency of cardiac mitochondrial replication. Burn induced a decrease in all mitDNA encoded genes. State 3 oxygen consumption was significantly decreased. Mitochondrial complex I substrate-energized or complex II substrate-energized and both of respiratory control ratio (RCRs) were decreased after burn. All mitochondrial complex activity except complex II were decreased in the burn group, correlating with decreases in mitochondrial ATP and MnSOD activity. Sildenafil, a inhibitor of the PDE5A-cGMP-PKG pathway, preserved the mitochondrial structure, respiratory chain efficiency and energy status in cardiac tissue. Furthermore, sildenafil treatment significantly restored ADP-conjugated respiration in burned groups. In conclusion, cardiac mitochondrial damage contributes to burn-induced heart dysfunction via the PDE5A-cGMP-PKG pathway.
    Keywords:  burn injury; electron transport chain; mitochondria; oxygen consumption; sildenafil
    DOI:  https://doi.org/10.3390/ijms21072350
  12. Biochem Biophys Res Commun. 2020 Mar 31. pii: S0006-291X(20)30608-2. [Epub ahead of print]
    Sennoside A restores colonic barrier function through protecting colon enterocytes from ROS-induced mitochondrial damage in diet-induced obese mice.
    Li Ma, Xinyu Cao, Xiaotong Ye, Youke Qi, Yawen Zhu, Jianping Ye, Yongning Sun.
      The intestinal barrier dysfunction is closely implicated in low-grade chronic inflammation for insulin resistance in diet-induced obesity (DIO). It is generally believed that degradation of colon enterocytes contributes to intestinal barrier dysfunction in the pathological process of obesity. Sennoside A (SA) is reported to improve metabolic disorders, but the effect and mechanism of SA on colonic barrier function of DIO remains unknown. In this study, SA was found to restore colonic barrier function by protecting the continuity and integrity of colon enterocytes in DIO mice. An increase in mRNA expression of tight junction proteins Occludin, Claudin-2 and ZO-1 provides another mechanism of restoring colonic barrier function in SA-treated group. In the research of mechanism, mitophagy was inhibited by SA via a protection of mitochondrial structure and function in colon. A reduction was found in production of reactive oxygen species (ROS) in the colon, and the benefical effect was attributed to an inhibition of activity in complex I and III with a reduction of protein expression and an increase of Mn-SOD activity. The results indicate that SA can restores colonic barrier function through protecting colon enterocytes from ROS-induced mitochondrial damage in DIO mice.
    Keywords:  Colonic barrier function; Mitochondria damage; Mitophagy; Reactive oxygen species (ROS); Sennoside A
    DOI:  https://doi.org/10.1016/j.bbrc.2020.03.117
  13. FASEB J. 2020 Apr 04.
    Alterations of mitochondrial bioenergetics, dynamics, and morphology support the theory of oxidative damage involvement in autism spectrum disorder.
    Alessandra Pecorelli, Francesca Ferrara, Nicolò Messano, Valeria Cordone, Maria Lucia Schiavone, Franco Cervellati, Brittany Woodby, Carlo Cervellati, Joussef Hayek, Giuseppe Valacchi.
      Autism spectrum disorder (ASD) has been hypothesized to be a result of the interplay between genetic predisposition and increased vulnerability to early environmental insults. Mitochondrial dysfunctions appear also involved in ASD pathophysiology, but the mechanisms by which such alterations develop are not completely understood. Here, we analyzed ASD primary fibroblasts by measuring mitochondrial bioenergetics, ultrastructural and dynamic parameters to investigate the hypothesis that defects in these pathways could be interconnected phenomena responsible or consequence for the redox imbalance observed in ASD. High levels of 4-hydroxynonenal protein adducts together with increased NADPH (nicotinamide adenine dinucleotide phosphateoxidase) activity and mitochondrial superoxide production coupled with a compromised antioxidant response guided by a defective Nuclear Factor Erythroid 2-Related Factor 2 pathway confirmed an unbalanced redox homeostasis in ASD. Moreover, ASD fibroblasts showed overactive mitochondrial bioenergetics associated with atypical morphology and altered expression of mitochondrial electron transport chain complexes and dynamics-regulating factors. We suggest that many of the changes observed in mitochondria could represent compensatory mechanisms by which ASD cells try to adapt to altered energy demand, possibly resulting from a chronic oxinflammatory status.
    Keywords:  4-hydroxynonenal; fission; fusion; mitophagy; nuclear factor erythroid 2-related factor 2
    DOI:  https://doi.org/10.1096/fj.201902677R
  14. J Mol Cell Cardiol. 2020 Mar 28. pii: S0022-2828(20)30076-6. [Epub ahead of print]
    MitoPlex: A targeted multiple reaction monitoring assay for quantification of a curated set of mitochondrial proteins.
    Aleksandr B Stotland, Weston Spivia, Amanda Orosco, Allen M Andres, Roberta A Gottlieb, Jennifer E Van Eyk, Sarah J Parker.
      Mitochondria are the major source of cellular energy (ATP), as well as critical mediators of widespread functions such as cellular redox balance, apoptosis, and metabolic flux. The organelles play an especially important role in the maintenance of cardiac homeostasis; their inability to generate ATP following impairment due to ischemic damage has been directly linked to organ failure. Methods to quantify mitochondrial content are limited to low throughput immunoassays, measurement of mitochondrial DNA, or relative quantification by untargeted mass spectrometry. Here, we present a high throughput, reproducible and quantitative mass spectrometry multiple reaction monitoring based assay of 37 proteins critical to central carbon chain metabolism and overall mitochondrial function termed 'MitoPlex'. We coupled this protein multiplex with a parallel analysis of the central carbon chain metabolites (219 metabolite assay) extracted in tandem from the same sample, be it cells or tissue. In tests of its biological applicability in cells and tissues, "MitoPlex plus metabolites" indicated profound effects of HMG-CoA Reductase inhibition (e.g., statin treatment) on mitochondria of i) differentiating C2C12 skeletal myoblasts, as well as a clear opposite trend of statins to promote mitochondrial protein expression and metabolism in heart and liver, while suppressing mitochondrial protein and ii) aspects of metabolism in the skeletal muscle obtained from C57Bl6 mice. Our results not only reveal new insights into the metabolic effect of statins in skeletal muscle, but present a new high throughput, reliable MS-based tool to study mitochondrial dynamics in both cell culture and in vivo models.
    Keywords:  Metabolomics; Mitochondria; Statins; Targeted mass spectrometry
    DOI:  https://doi.org/10.1016/j.yjmcc.2020.03.011
  15. J Appl Physiol (1985). 2020 Apr 02.
    Sex and BNIP3 genotype, rather than acute lipid injection, modulate hepatic mitochondrial function and steatosis risk in mice.
    Kelly N Z Fuller, Colin S McCoin, Julie Allen, Shelby Bell-Glenn, Devin C Koestler, Gerald W Dorn Ii, John P Thyfault.
      Both lipid oversupply and poor mitochondrial function (low respiration and elevated H2O2 emission) have been implicated in the development of hepatic steatosis and liver injury. Mitophagy, the targeted degradation of low functioning mitochondria, is critical for maintaining mitochondrial quality control. Here, we used intralipid injections combined with acute (4day) and chronic (4-7wk) high-fat diets (HFD) to examine if hepatic mitochondrial respiration would decrease and H202 emission would increase with lipid overload. We tested these effects in male and female wild type (WT) mice and mice null for a critical mediator of mitophagy, BNIP3 (BNIP3 KO) housed at thermoneutral temperatures. Intralipid injection was successful in elevating serum triglycerides and NEFAs but had no impact on hepatic mitochondrial respiratory function or H2O2 emission. However, female mice had greater mitochondrial respiration on the acute HFD, lower H2O2 emission across both HFD durations, and were protected against hepatic steatosis. Unexpectedly, BNIP3 KO animals had greater hepatic mitochondrial respiration, better coupled respiration, and increased electron chain protein content after the 4day HFD compared to WT animals. Altogether, these data suggest that acute lipid overload delivered by a single intralipid bolus does not alter hepatic mitochondrial outcomes, but rather sex and genotype profoundly impact hepatic mitochondrial respiration and H2O2 emission.
    Keywords:  Liver; Metabolism; NAFLD; Reactive Oxygen Species
    DOI:  https://doi.org/10.1152/japplphysiol.00035.2020
  16. Biochim Biophys Acta Mol Cell Res. 2020 Mar 26. pii: S0167-4889(20)30067-7. [Epub ahead of print] 118709
    Mitochondrial fission factor (MFF) is a critical regulator of peroxisome maturation.
    Josiah B Passmore, Ruth E Carmichael, Tina A Schrader, Luis F Godinho, Sacha Ferdinandusse, Celien Lismont, Yunhong Wang, Christian Hacker, Markus Islinger, Marc Fransen, David M Richards, Peter Freisinger, Michael Schrader.
      Peroxisomes are highly dynamic subcellular compartments with important functions in lipid and ROS metabolism. Impaired peroxisomal function can lead to severe metabolic disorders with developmental defects and neurological abnormalities. Recently, a new group of disorders has been identified, characterised by defects in the membrane dynamics and division of peroxisomes rather than by loss of metabolic functions. However, the contribution of impaired peroxisome plasticity to the pathophysiology of those disorders is not well understood. Mitochondrial fission factor (MFF) is a key component of both the peroxisomal and mitochondrial division machinery. Patients with MFF deficiency present with developmental and neurological abnormalities. Peroxisomes (and mitochondria) in patient fibroblasts are highly elongated as a result of impaired organelle division. The majority of studies into MFF-deficiency have focused on mitochondrial dysfunction, but the contribution of peroxisomal alterations to the pathophysiology is largely unknown. Here, we show that MFF deficiency does not cause alterations to overall peroxisomal biochemical function. However, loss of MFF results in reduced import-competency of the peroxisomal compartment and leads to the accumulation of pre-peroxisomal membrane structures. We show that peroxisomes in MFF-deficient cells display alterations in peroxisomal redox state and intra-peroxisomal pH. Removal of elongated peroxisomes through induction of autophagic processes is not impaired. A mathematical model describing key processes involved in peroxisome dynamics sheds further light into the physical processes disturbed in MFF-deficient cells. The consequences of our findings for the pathophysiology of MFF-deficiency and related disorders with impaired peroxisome plasticity are discussed.
    Keywords:  MFF; Mitochondria; Organelle division; PEX14; Peroxisomes; Pexophagy; Redox homeostasis
    DOI:  https://doi.org/10.1016/j.bbamcr.2020.118709
  17. ACS Sens. 2020 Mar 30.
    Mitochondrial-targeted and Near-infrared Fluorescent Probe for Bioimaging and Evaluating Monoamine Oxidase A Activity in Hepatic Fibrosis.
    Zheng-Min Yang, Qing-Yuan Mo, Ji-Man He, Dong-Liang Mo, Jun Li, Hua Chen, Shulin Zhao, Jiang-Ke Qin.
      Monoamine Oxidase A (MAO-A) is a promising diagnostic marker for cancer, depression, parkinson's disease and liver disease. Fluorescence detecting of MAO-A in living animals is of extremely importance for the early diagnosis of related diseases. However, the development of specific and mitochondrial-targeted MAO-A near-infrared (NIR) fluorescence probes is still lacking. Here, we designed and synthesized four NIR fluorescence probes containing dihydroxanthene (DH) skeleton to detect MAO-A in complex biological systems. The specificity of our representative probe of DHMP2 displays a 31-fold fluorescence turn-on in vitro, and can effectively accumulate in mitochondria and specific detect the endogenous MAO-A concentrations in PC-3 and SH-SY5Y cell lines. Furthermore, the probe DHMP2 can be used for the visualization of the endogenous MAO-A activity in zebrafish and tumor-bearing mice. More importantly, it's the first time to detect the MAO-A activity of hepatic fibrosis tissue through the probe DHMP2. The present studies show that the synthesized DHMP2 might be served as a potential tool for monitoring MAO-A activity in vivo and diagnosing related diseases.
    DOI:  https://doi.org/10.1021/acssensors.9b02116
  18. Environ Toxicol Chem. 2020 Apr 02.
    Mitochondrial respiratory inhibition promoted by pyraclostrobin in fungi is also observed in honey bees.
    Daniel Nicodemo, Fábio Erminio Mingatto, David De Jong, Paulo Francisco Veiga Bizerra, Marco Aurélio Tavares, William Cesar Bellini, Eduardo Festozo Vicente, Amanda de Carvalho.
      There is no use restriction associated with bees for many fungicides used in agriculture; however, this does not always mean that these pesticides are harmless for these non-target organisms. We investigated whether the fungicide pyraclostrobin, which acts on fungal mitochondria, also negatively affects honey bee mitochondrial bioenergetics. Honey bees were collected from five hives and anesthetized at 4o C. The thoraces were separated and mitochondria were isolated by grinding, filtering and differential centrifugation. An aliquot of 0.5 mg of mitochondrial proteins was added to 0.5 mL of a standard reaction medium with 4 mM succinate (complex II substrate) plus 50 nM rotenone (complex I inhibitor), and mitochondrial respiration was measured at 30°C using a Clark-type oxygen electrode. Mitochondrial membrane potential was determined spectrofluorimetrically using safranin O as a probe, and ATP synthesis was determined by chemiluminescence. Pyraclostrobin at 0 to 50 μM was tested on the mitochondrial preparations, with three repetitions. Pyraclostrobin inhibited mitochondrial respiration in a dose dependent manner at concentrations of 10 μM and above, demonstrating typical inhibition of oxidative phosphorylation. Pyraclostrobin also promoted a decline in the mitochondrial membrane potential at doses of 5 μM and above and in ATP synthesis at 15 μM and above. We conclude that pyraclostrobin interferes with honey bee mitochondrial function, which is especially critical for the energy-demanding flight activity of foraging bees. This article is protected by copyright. All rights reserved.
    Keywords:   Apis mellifera ; ATP; fungicide; mitochondria; strobilurin; toxicity mechanism
    DOI:  https://doi.org/10.1002/etc.4719
  19. Diabetes. 2020 Apr 03. pii: db191130. [Epub ahead of print]
    Glucose-Stimulated Insulin Secretion Fundamentally Requires H2O2 Signaling by NADPH Oxidase 4.
    Lydie Plecitá-Hlavatá, Martin Jabůrek, Blanka Holendová, Jan Tauber, Vojtěch Pavluch, Zuzana Berková, Monika Cahová, Katrin Schroeder, Ralf P Brandes, Detlef Siemen, Petr Ježek.
      NADPH facilitates glucose-stimulated insulin secretion (GSIS) in pancreatic islet (PI) β-cells by an as yet unknown mechanism. We found NADPH oxidase, isoform-4 (NOX4), to be the major producer of cytosolic H2O2, essential for GSIS, while the increase in ATP/ADP alone was insufficient. The fast GSIS phase was absent in PIs from NOX4-null, β-cell-specific knockout mice (NOX4βKO) (not NOX2KO), and NOX4-silenced or catalase-overexpressing INS-1E cells. Lentiviral NOX4 overexpression or H2O2 rescued GSIS in PIs from NOX4βKO mice. NOX4 silencing suppressed Ca2+ oscillations and the patch-clamped ATP-sensitive potassium channel (KATP) opened more frequently at high glucose. Mitochondrial H2O2, decreasing upon GSIS, provided an alternative redox signaling when 2-oxo-isocaproate or fatty acid oxidation formed superoxide by electron-transport flavoprotein:Q-oxidoreductase. Unlike GSIS, this ceased with mitochondrial antioxidant SkQ1. Both NOX4KO and NOX4βKO strains exhibited impaired glucose tolerance and peripheral insulin resistance. Thus the redox signaling previously suggested to cause β-cell-self-checking - hypothetically induces insulin resistance when absent. In conclusion, ATP plus H2O2 elevations constitute an essential switch-on signal of insulin exocytosis for glucose and branched-chain oxoacids as secretagogues (partly for fatty acids). Redox signaling could be impaired by cytosolic antioxidants, hence those targeting mitochondria should be preferred for clinical applications to treat (pre)diabetes at any stage.
    DOI:  https://doi.org/10.2337/db19-1130
  20. Aging (Albany NY). 2020 Apr 02. 12
    Down-regulation of AMPK signaling pathway rescues hearing loss in TFB1 transgenic mice and delays age-related hearing loss.
    Jingjing Zhao, Gen Li, Xuan Zhao, Xin Lin, Yunge Gao, Nuno Raimundo, Geng-Lin Li, Wei Shang, Hao Wu, Lei Song.
      AMP-activated protein kinase (AMPK) integrates the regulation of cell growth and metabolism. AMPK activation occurs in response to cellular energy decline and mitochondrial dysfunction triggered by reactive oxygen species (ROS). In aged Tg-mtTFB1 mice, a mitochondrial deafness mouse model, hearing loss is accompanied with cochlear pathology including reduced endocochlear potential (EP) and loss of spiral ganglion neurons (SGN), inner hair cell (IHC) synapses and outer hair cells (OHC). Accumulated ROS and increased apoptosis signaling were also detected in cochlear tissues, accompanied by activation of AMPK. To further explore the role of AMPK signaling in the auditory phenotype, we used genetically knocked out AMPKα1 as a rescue to Tg-mtTFB1 mice and observed: improved ABR wave I, EP and IHC function, normal SGNs, IHC synapses morphology and OHC survivals, with decreased ROS, reduced pro-apoptotic signaling (Bax) and increased anti-apoptotic signaling (Bcl-2) in the cochlear tissues, indicating that reduced AMPK attenuated apoptosis via ROS-AMPK-Bcl2 pathway in the cochlea. To conclude, AMPK hyperactivation causes accelerated presbycusis in Tg-mtTFB1 mice by redox imbalance and dysregulation of the apoptosis pathway. The effects of AMPK downregulation on pro-survival function and reduction of oxidative stress indicate AMPK serves as a target to rescue or relieve mitochondrial hearing loss.
    Keywords:  AMPK; NIHL; ROS; apoptosis; mitochondrial deafness
    DOI:  https://doi.org/10.18632/aging.102977
  21. Am J Physiol Renal Physiol. 2020 Mar 30.
    Comprehensive assessment of mitochondrial respiratory function in freshly isolated nephron segments.
    Allison McCrimmon, Mark Domondon, Regina Sultanova, Daria V Ilatovskaya, Krisztian Stadler.
      Changes in mitochondrial function are central to many forms of kidney disease including acute injury, diabetic nephropathy, hypertension and chronic kidney diseases. As such, there is an increasing need for reliable and fast methods for assessing mitochondrial respiratory function in renal cells. Despite being indispensable for many mechanistic studies, cultured cells or isolated mitochondria, however, often do not recapitulate in vivo or close-to-in vivo situations. Cultured and/or immortalized cells often change their bioenergetic profile and phenotype compared to in vivo or ex vivo situations, and isolated mitochondria are simply removed from their cellular milieu. This is especially important for extremely complex organs such as the kidney. Here we report the development and validation of a new approach for rapid assessment of mitochondrial oxygen consumption on freshly isolated glomeruli or proximal tubular (PT) fragments using the Agilent SeaHorse XFe24 and XF96 Extracellular Flux Analyzers. We validated the technique in several healthy and diseased rodent models - the C57BL/6J mouse, the diabetic db/db mouse and their matching db/+ control and the Dahl salt sensitive rat. We compared the data to respiration from isolated mitochondria. The method can be adapted and used for rapid assessment of mitochondrial oxygen consumption from any rodent model of the investigator's choice. The isolation methods presented here ensure viable and functional PT fragments and glomeruli, with preserved cellular environment for studying mitochondrial function within the context of their surroundings and interactions.
    Keywords:  bioenergetics; glomeruli; mitochondria; proximal tubules
    DOI:  https://doi.org/10.1152/ajprenal.00031.2020
  22. J Cell Biol. 2020 May 04. pii: e201909154. [Epub ahead of print]219(5):
    Mitochondrial fragmentation enables localized signaling required for cell repair.
    Adam Horn, Shreya Raavicharla, Sonna Shah, Dan Cox, Jyoti K Jaiswal.
      Plasma membrane injury can cause lethal influx of calcium, but cells survive by mounting a polarized repair response targeted to the wound site. Mitochondrial signaling within seconds after injury enables this response. However, as mitochondria are distributed throughout the cell in an interconnected network, it is unclear how they generate a spatially restricted signal to repair the plasma membrane wound. Here we show that calcium influx and Drp1-mediated, rapid mitochondrial fission at the injury site help polarize the repair response. Fission of injury-proximal mitochondria allows for greater amplitude and duration of calcium increase in these mitochondria, allowing them to generate local redox signaling required for plasma membrane repair. Drp1 knockout cells and patient cells lacking the Drp1 adaptor protein MiD49 fail to undergo injury-triggered mitochondrial fission, preventing polarized mitochondrial calcium increase and plasma membrane repair. Although mitochondrial fission is considered to be an indicator of cell damage and death, our findings identify that mitochondrial fission generates localized signaling required for cell survival.
    DOI:  https://doi.org/10.1083/jcb.201909154
  23. Am J Physiol Regul Integr Comp Physiol. 2020 Apr 01.
    Experimental oxygen concentration influences rates of mitochondrial hydrogen peroxide release from cardiac and skeletal muscle preparations.
    Lance C Li Puma, Michael Hedges, Joseph M Heckman, Alissa B Mathias, Madison R Engstrom, Abigail B Brown, Adam J Chicco.
      Mitochondria utilize the majority of oxygen (O2) consumed by aerobic organisms as the final electron acceptor for oxidative phosphorylation (OXPHOS), but also to generate reactive oxygen species (mtROS) that participate in cell signaling, physiological hormesis and disease pathogenesis. Simultaneous monitoring of mtROS production and oxygen consumption (JO2) from tissue mitochondrial preparations is an attractive investigative approach, but introduces dynamic changes in media O2 concentration ([O2]) that can confound experimental results and interpretation. We utilized high-resolution fluoro-respirometry to evaluate JO2 and hydrogen peroxide release (JH2O2) from isolated mitochondria (Mt), permeabilized fibers (Pf), and tissue homogenates (Hm) prepared from murine heart and skeletal muscle across a range of experimental [O2]s typically encountered during respirometry protocols (400-50 µM). Results demonstrate notable variations in JH2O2 across tissues and sample preparations during non-phosphorylating (LEAK) and OXPHOS-linked respiration states at 250 µM [O2], but a linear decline in JH2O2 of 5-15% per 50 µM decrease in chamber [O2] in all samples. JO2 was generally stable in Mt and Hm across [O2]s above 50 µM, but tended to decline below 250 µM in Pf, leading to wide variations in assayed rates of JH2O2/O2 across chamber [O2]s and sample preparations. Development of chemical background fluorescence from the H2O2 probe (Amplex Red) was also O2-sensitive, emphasizing relevant calibration considerations. These studies highlight the importance of monitoring and reporting the chamber [O2] at which JO2 and JH2O2 are recorded during fluoro-respirometry experiments, and provide a basis for selecting sample preparations for studies addressing the role mtROS in physiology and disease.
    Keywords:  bioenergetics; mitochondria; oxidative stress; reactive oxygen species; respirometry
    DOI:  https://doi.org/10.1152/ajpregu.00227.2019
  24. Biology (Basel). 2020 Apr 01. pii: E68. [Epub ahead of print]9(4):
    Estrogen Signaling Induces Mitochondrial Dysfunction-Associated Autophagy and Senescence in Breast Cancer Cells.
    Khuloud Bajbouj, Jasmin Shafarin, Jalal Taneera, Mawieh Hamad.
      Previous work has shown that although estrogen (E2) disrupts cellular iron metabolism and induces oxidative stress in breast and ovarian cancer cells, it fails to induce apoptosis. However, E2 treatment was reported to enhance the apoptotic effects of doxorubicin in cancer cells. This suggests that E2 can precipitate anti-growth effects that render cancer cells more susceptible to chemotherapy. To investigate such anti-growth non-apoptotic, effects of E2 in cancer cells, MDA-MB-231 and MCF-7 cells were evaluated for the expression of key autophagy and senescence markers and for mitochondrial damage following E2 treatment. Treated cells experienced mitochondrial membrane depolarization along with increased expression of LC3-I/II, Pink1 and LAMP2, increased LC3-II accumulation and increased lysosomal and mitochondrial accumulation and flattening. E2-treated MCF-7 cells also showed reduced P53 and pRb780 expression and increased Rb and P21 expression. Increased expression of the autophagy markers ATG3 and Beclin1 along with increased levels of β-galactosidase activity and IL-6 production were evident in E2-treated MCF-7 cells. These findings suggest that E2 precipitates a form of mitochondrial damage that leads to cell senescence and autophagy in breast cancer cells.
    Keywords:  Estrogen; MCF-7; MDA-MB-231; autophagy; mitochondria; senescence
    DOI:  https://doi.org/10.3390/biology9040068
  25. Cancers (Basel). 2020 Mar 30. pii: E827. [Epub ahead of print]12(4):
    xCT Inhibition Increases Sensitivity to Vorinostat in a ROS-Dependent Manner.
    Keiko Miyamoto, Motoki Watanabe, Shogen Boku, Mamiko Sukeno, Mie Morita, Haruhito Kondo, Koichi Sakaguchi, Tetsuya Taguchi, And Toshiyuki Sakai.
      As histone deacetylase inhibitors (HDACIs) have limited efficacy against solid tumors, we investigated whether and how oxidative stress is involved in sensitivity to HDACIs to develop a novel therapeutic option of HDACIs treatment. We first tested whether a reduction of the antioxidant glutathione (GSH) by glutamine deprivation affects sensitivity to a commercially available HDACI vorinostat and reactive oxygen species (ROS) accumulation. Next we investigated the relationship between a glutamate-cystine transporter xCT and the efficacy of vorinostat using siRNA of xCT and bioinformatic analyses. Finally, we verified the combinatory effects of vorinostat and the xCT inhibitor salazosulfapyridine (SASP) on ROS accumulation, cell death induction, and colony formation. Glutamine deprivation increased vorinostat-mediated cell death with ROS accumulation. Genetic ablation of xCT improved the efficacy of vorinostat, consistent with the results of public data analyses demonstrating that xCT expressions positively correlate with insensitivity to HDACIs in many types of cancer cell lines. Vorinostat caused ROS accumulation when combined with SASP, possibly resulting in synergistic ferroptosis. Our study provides a novel mechanistic insight into the mechanism underlying sensitivity to HDACIs involving xCT, suggesting xCT to be a promising predictive marker of HDACIs and rationalizing combinatory therapy of HDACIs with xCT inhibitors to induce ferroptosis.
    Keywords:  HDAC inhibitor; ROS; ferroptosis; glutathione; salazosulfapyridine; vorinostat; xCT
    DOI:  https://doi.org/10.3390/cancers12040827
  26. J Am Chem Soc. 2020 Apr 01.
    Real-Time Imaging and Simultaneous Quantification of Mitochondrial H2O2 and ATP in Neurons with a Single Two-Photon Fluorescence Lifetime-Based Probe.
    Zhou Wu, Mengmeng Liu, Zhichao Liu, Yang Tian.
      Mitochondrial oxidative stress and energy metabolism are vital biological events and involved in various physiological and pathological processes, like apoptosis and necrosis. However, it remains unclear that how the dynamic pattern of mitochondrial hydrogen peroxide (H2O2) and adenosine-5'-triphosphate (ATP) changes in these events, and more importantly how they affect each other. Herein, we developed a single two-photon fluorescence lifetime-based probe (TFP), which offered real-time imaging and simultaneous determination of mitochondrial H2O2 and ATP changes in two well-separated fluorescence channels without spectral crosstalk. The fluorescence lifetime of TFP exhibited good responses and selectivity in the detection range of 0.4-10 μM H2O2 and 0.5-15 mM ATP, taking of the advantages of accuracy and quantitative ability of fluorescence lifetime imaging. Using this useful probe, we studied the relationship between H2O2 and ATP in mitochondria as well as visualized the dynamic level changes of mitochondrial H2O2 and ATP induced by superoxide anion (O2•-). It was discovered that O2•- stimulation in a short period of time (8 min) temporarily change the levels of H2O2 and ATP in mitochondria, and neurons were capable of recovering to the initial state in short time. However, the increasing time up to 50 min of O2•- stimulation led to permanent oxidative damage and energy deficiency. Meanwhile, it was first found that exogenous stimulation of O2•- and H2O2 exhibited different impacts on the levels of mitochondrial H2O2 and ATP, in which O2•- demonstrated more serious and negative consequences. As a matter of fact, this work has not only provided a general molecular design methodology for multiple species imaging, but also has discovered oxidative stress-induced intracellular functions related to H2O2 and ATP in mitochondria based on this developed TFP probe.
    DOI:  https://doi.org/10.1021/jacs.0c00771
  27. Int J Mol Sci. 2020 Mar 20. pii: E2139. [Epub ahead of print]21(6):
    Pioglitazone and Deoxyribonucleoside Combination Treatment Increases Mitochondrial Respiratory Capacity in m.3243A>G MELAS Cybrid Cells.
    Harrison J Burgin, M Isabel G Lopez Sanchez, Craig M Smith, Ian A Trounce, Matthew McKenzie.
      The lack of effective treatments for mitochondrial disease has seen the development of new approaches, including those that aim to stimulate mitochondrial biogenesis to boost ATP generation above a critical disease threshold. Here, we examine the effects of the peroxisome proliferator-activated receptor γ (PPARγ) activator pioglitazone (PioG), in combination with deoxyribonucleosides (dNs), on mitochondrial biogenesis in cybrid cells containing >90% of the m.3243A>G mutation associated with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). PioG + dNs combination treatment increased mtDNA copy number and mitochondrial mass in both control (CON) and m.3243A>G (MUT) cybrids, with no adverse effects on cell proliferation. PioG + dNs also increased mtDNA-encoded transcripts in CON cybrids, but had the opposite effect in MUT cybrids, reducing the already elevated transcript levels. Steady-state levels of mature oxidative phosphorylation (OXPHOS) protein complexes were increased by PioG + dNs treatment in CON cybrids, but were unchanged in MUT cybrids. However, treatment was able to significantly increase maximal mitochondrial oxygen consumption rates and cell respiratory control ratios in both CON and MUT cybrids. Overall, these findings highlight the ability of PioG + dNs to improve mitochondrial respiratory function in cybrid cells containing the m.3243A>G MELAS mutation, as well as their potential for development into novel therapies to treat mitochondrial disease.
    Keywords:  MELAS; OXPHOS; cybrid; deoxyribonucleosides; mitochondrial biogenesis; mitochondrial disease; oxidative phosphorylation; pioglitazone
    DOI:  https://doi.org/10.3390/ijms21062139
  28. Sci Rep. 2020 Mar 27. 10(1): 5575
    Mitochondrial dysfunction and DNA damage accompany enhanced levels of formaldehyde in cultured primary human fibroblasts.
    Cristina A Nadalutti, Donna F Stefanick, Ming-Lang Zhao, Julie K Horton, Rajendra Prasad, Ashley M Brooks, Jack D Griffith, Samuel H Wilson.
      Formaldehyde (FA) is a simple biological aldehyde that is produced inside cells by several processes such as demethylation of DNA and proteins, amino acid metabolism, lipid peroxidation and one carbon metabolism (1-C). Although accumulation of excess FA in cells is known to be cytotoxic, it is unknown if an increase in FA level might be associated with mitochondrial dysfunction. We choose to use primary human fibroblasts cells in culture (foreskin, FSK) as a physiological model to gain insight into whether an increase in the level of FA might affect cellular physiology, especially with regard to the mitochondrial compartment. FSK cells were exposed to increasing concentrations of FA, and different cellular parameters were studied. Elevation in intracellular FA level was achieved and was found to be cytotoxic by virtue of both apoptosis and necrosis and was accompanied by both G2/M arrest and reduction in the time spent in S phase. A gene expression assessment by microarray analysis revealed FA affected FSK cells by altering expression of many genes including genes involved in mitochondrial function and electron transport. We were surprised to observe increased DNA double-strand breaks (DSBs) in mitochondria after exposure to FA, as revealed by accumulation of γH2A.X and 53BP1 at mitochondrial DNA foci. This was associated with mitochondrial structural rearrangements, loss of mitochondrial membrane potential and activation of mitophagy. Collectively, these results indicate that an increase in the cellular level of FA can trigger mitochondrial DNA double-strand breaks and dysfunction.
    DOI:  https://doi.org/10.1038/s41598-020-61477-2
  29. Cells. 2020 Mar 31. pii: E848. [Epub ahead of print]9(4):
    Acid Sphingomyelinase Downregulation Enhances Mitochondrial Fusion and Promotes Oxidative Metabolism in a Mouse Model of Melanoma.
    Marco Coazzoli, Alessandra Napoli, Paulina Roux-Biejat, Clara De Palma, Claudia Moscheni, Elisabetta Catalani, Silvia Zecchini, Vincenzo Conte, Matteo Giovarelli, Sonia Caccia, Patrizia Procacci, Davide Cervia, Emilio Clementi, Cristiana Perrotta.
      Melanoma is the most severe type of skin cancer. Its unique and heterogeneous metabolism, relying on both glycolysis and oxidative phosphorylation, allows it to adapt to disparate conditions. Mitochondrial function is strictly interconnected with mitochondrial dynamics and both are fundamental in tumour progression and metastasis. The malignant phenotype of melanoma is also regulated by the expression levels of the enzyme acid sphingomyelinase (A-SMase). By modulating at transcriptional level A-SMase in the melanoma cell line B16-F1 cells, we assessed the effect of enzyme downregulation on mitochondrial dynamics and function. Our results demonstrate that A-SMase influences mitochondrial morphology by affecting the expression of mitofusin 1 and OPA1. The enhanced expression of the two mitochondrial fusion proteins, observed when A-SMase is expressed at low levels, correlates with the increase of mitochondrial function via the stimulation of the genes PGC-1alpha and TFAM, two genes that preside over mitochondrial biogenesis. Thus, the reduction of A-SMase expression, observed in malignant melanomas, may determine their metastatic behaviour through the stimulation of mitochondrial fusion, activity and biogenesis, conferring a metabolic advantage to melanoma cells.
    Keywords:  acid sphingomyelinase; melanoma; mitochondrial dynamics; mitochondrial function
    DOI:  https://doi.org/10.3390/cells9040848
  30. Cancers (Basel). 2020 Mar 30. pii: E825. [Epub ahead of print]12(4):
    Pan-Cancer Analysis of Mitochondria Chaperone-Client Co-Expression Reveals Chaperone Functional Partitioning.
    Geut Galai, Hila Ben-David, Liron Levin, Martin F Orth, Thomas G P Grünewald, Shai Pilosof, Shimon Berstein, Barak Rotblat.
      Metabolic reprogramming is a hallmark of cancer. Such reprogramming entails the up-regulation of the expression of specific mitochondrial proteins, thus increasing the burden on the mitochondrial protein quality control. However, very little is known about the specificity of interactions between mitochondrial chaperones and their clients, or to what extent the mitochondrial chaperone-client co-expression is coordinated. We hypothesized that a physical interaction between a chaperone and its client in mitochondria ought to be manifested in the co-expression pattern of both transcripts. Using The Cancer Genome Atlas (TCGA) gene expression data from 13 tumor entities, we constructed the mitochondrial chaperone-client co-expression network. We determined that the network is comprised of three distinct modules, each populated with unique chaperone-clients co-expression pairs belonging to distinct functional groups. Surprisingly, chaperonins HSPD1 and HSPE1, which are known to comprise a functional complex, each occupied a different module: HSPD1 co-expressed with tricarboxylic acid cycle cycle enzymes, while HSPE1 co-expressed with proteins involved in oxidative phosphorylation. Importantly, we found that the genes in each module were enriched for discrete transcription factor binding sites, suggesting the mechanism for the coordinated co-expression. We propose that our mitochondrial chaperone-client interactome can facilitate the identification of chaperones supporting specific mitochondrial pathways and bring forth a fundamental principle in metabolic adaptation.
    Keywords:  bioinformatics analysis; cancer; chaperone; co-expression; mitochondria
    DOI:  https://doi.org/10.3390/cancers12040825
  31. Biochem Pharmacol. 2020 Mar 26. pii: S0006-2952(20)30161-1. [Epub ahead of print]175 113933
    Palmatine induces G2/M phase arrest and mitochondrial-associated pathway apoptosis in colon cancer cells by targeting AURKA.
    Xiaojiang Liu, Yaru Zhang, Siqi Wu, Minmin Xu, Youfeng Shen, Min Yu, Jinhua Fan, Sijia Wei, Chaohang Xu, Lu Huang, Han Zhao, Xuegang Li, Xiaoli Ye.
      Studies have shown that palmatine (PAL) has anti-cancer effects. However, the activity and potential mechanisms of PAL against colorectal cancer remain elusive. The results showed that PAL significantly inhibited the proliferation of colon cancer cells in vitro and in vivo without significant effect on non-tumorigenic colon cells. Target prediction and clinical sample database analysis suggested that PAL may contribute to colon cancer cells phase arrest and apoptosis by targeting aurora kinase A (AURKA). Inhibition and overexpression of AURKA proved that PAL induces G2/M phase arrest and apoptosis in colon cancer cells by targeting AURKA. Moreover, PAL promoted intracellular Reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (ΔΨm). PAL reduced the levels of AURKA, Bcl-xl and Bcl2 proteins, and promoted the expression of pro-apoptotic proteins P53, P73, Caspase3 and Caspase9, as well as the increase of cytochrome c (cyt. c) in cell lysates in vitro and in vivo. Together, our study confirmed that PAL induced G2/M phase arrest and mitochondrial-associated pathway apoptosis in colon cancer cells by targeting AURKA. PAL may provide a novel solution for the treatment of colon cancer by serving as a new AURKA inhibitor.
    Keywords:  AURKA; Apoptosis; Colon cancer; Palmatine (PAL)
    DOI:  https://doi.org/10.1016/j.bcp.2020.113933
  32. Redox Biol. 2020 Mar 19. pii: S2213-2317(20)30178-6. [Epub ahead of print] 101509
    Mitochondria dysfunction and metabolic reprogramming as drivers of idiopathic pulmonary fibrosis.
    Marta Bueno, Jazmin Calyeca, Mauricio Rojas, Ana L Mora.
      Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown etiology. It is characterized by deposition of extracellular matrix proteins, like collagen and fibronectin in the lung interstitium leading to respiratory failure. Our understanding of the pathobiology underlying IPF is still incomplete; however, it is accepted that aging is a major risk factor in the disease while growing evidence suggests that the mitochondria plays an important role in the initiation and progression of pulmonary fibrosis. Mitochondria dysfunction and metabolic reprogramming had been identified in different IPF lung cells (alveolar epithelial cells, fibroblasts, and macrophages) promoting low resilience and increasing susceptibility to activation of profibrotic responses. Here we summarize changes in mitochondrial numbers, biogenesis, turnover and associated metabolic adaptations that promote disrepair and fibrosis in the lung. Finally, we highlight new possible therapeutic approaches focused on ameliorate mitochondrial dysfunction.
    Keywords:  Aging; Epithelial cells; Fibroblast; Fibrosis; Macrophage; Mitochondrial dysfunction
    DOI:  https://doi.org/10.1016/j.redox.2020.101509
  33. Gene. 2020 Mar 25. pii: S0378-1119(20)30292-4. [Epub ahead of print] 144623
    Metformin enhances the sensitivity of colorectal cancer cells to cisplatin through ROS-mediated PI3K/Akt signaling pathway.
    Pei Zhang, Surong Zhao, Xingyue Lu, Zongfen Shi, Hao Liu, Bing Zhu.
      Metformin and cisplatin have been widely studied as antitumor agents. However, the effect of metformin combined with cisplatin has not been investigated in colorectal cancer (CRC) cells. This study was aimed to explore the effect of metformin or/and cisplatin on cell viability, apoptosis, and the related signaling pathways in CRC SW480 and SW620 cells. We found that metformin or cisplatin inhibited cell viability of SW480 and SW620 cells in a concentration- and time-dependent manner. Furthermore, metformin combined with cisplatin obviously inhibited cell viability, decreased colony formation, induced apoptosis, mediated cleavage of caspase-9, caspase-3 and PARP, activated mitochondrial membrane potential, downregulated Mcl-1 and Bcl-2 expression, upregulated Bak and Bax expression, and increased reactive oxygen species (ROS) production, compared to the individual agent in SW480 and SW620 cells, which were attenuated by N-acetyl-L-cysteine (NAC), a ROS scavenger. Moreover, NAC could recover the downregulation of p-PI3K and p-Akt treated with combination of metformin and cisplatin, which subsequently activated the PI3K/Akt signaling pathway. Taken together, our results demonstrated that metformin enhanced the sensitivity of CRC cells to cisplatin through ROS-mediated PI3K/Akt signaling pathway.
    Keywords:  PI3K/Akt signaling pathway; apoptosis; cisplatin; colorectal cancer; metformin; reactive oxygen species
    DOI:  https://doi.org/10.1016/j.gene.2020.144623
  34. Breast Cancer Res Treat. 2020 Apr 02.
    Metformin is distributed to tumor tissue in breast cancer patients in vivo: A 11C-metformin PET/CT study.
    Elias Immanuel Ordell Sundelin, Nidal Al-Suliman, Pernille Vahl, Mikkel Vendelbo, Ole Lajord Munk, Steen Jakobsen, Steen Bønløkke Pedersen, Jørgen Frøkiær, Lars C Gormsen, Niels Jessen.
      PURPOSE: Epidemiological studies and randomized clinical trials suggest that the antidiabetic drug, metformin, may have anti-neoplastic effects. The mechanism that mediates these beneficial effects has been suggested to involve direct action on cancer cells, but this will require distribution of metformin in tumor tissue. The present study was designed to investigate metformin distribution in vivo in breast and liver tissue in breast cancer patients.METHODS: Seven patients recently diagnosed with ductal carcinoma were recruited. Using PET/CT, tissue distribution of metformin was determined in vivo for 90 min after injection of a carbon-11-labeled metformin tracer. After surgery, tumor tissue was investigated for gene expression levels of metformin transporter proteins.
    RESULTS: Tumor tissue displayed a distinct uptake of metformin compared to normal breast tissue AUC0-90 min (75.4 ± 5.5 vs 42.3 ± 6.3) g/ml*min (p = 0.01). Maximal concentration in tumor was at 1 min where it reached approximately 30% of the activity in the liver. The metformin transporter protein with the highest gene expression in tumor tissue was multidrug and toxin extrusion 1 (MATE 1) followed by plasma membrane monoamine transporter (PMAT).
    CONCLUSION: This study confirms that metformin is transported into tumor tissue in women with breast cancer. This finding support that metformin may have direct anti-neoplastic effects on tumor cells in breast cancer patients. However, distribution of metformin in tumor tissue is markedly lower than in liver, an established metformin target tissue.
    Keywords:  Antidiabetic treatment; Ductal carcinoma; Oncology; Organic cation transporters
    DOI:  https://doi.org/10.1007/s10549-020-05621-6
  35. Int J Mol Sci. 2020 Mar 31. pii: E2427. [Epub ahead of print]21(7):
    Amination of Graphene Oxide Leads to Increased Cytotoxicity in Hepatocellular Carcinoma Cells.
    Milena Georgieva, Bela Vasileva, Giorgio Speranza, Dayong Wang, Kalin Stoyanov, Milena Draganova-Filipova, Plamen Zagorchev, Victoria Sarafian, George Miloshev, Natalia Krasteva.
      Clinically, there is an urgent need to identify new therapeutic strategies for selectively treating cancer cells. One of the directions in this research is the development of biocompatible therapeutics that selectively target cancer cells. Here, we show that novel aminated graphene oxide (haGO-NH2) nanoparticles demonstrate increased toxicity towards human hepatocellular cancer cells compared to pristine graphene oxide(GO). The applied novel strategy for amination leads to a decrease in the size of haGO-NH2 and their zeta potential, thus, assuring easier penetration through the cell membrane. After characterization of the biological activities of pristine and aminated GO, we have demonstrated strong cytotoxicity of haGO-NH2 toward hepatic cancer cells - HepG2 cell line, in a dose-dependent manner. We have presented evidence that the cytotoxic effects of haGO-NH2 on hepatic cancer cells were due to cell membrane damage, mitochondrial dysfunction and increased reactive oxygen species (ROS) production. Intrinsically, our current study provides new rationale for exploiting aminated graphene oxide as an anticancer therapeutic.
    Keywords:  GO; HepG2; cytotoxicity; genotoxicity; haGO-NH2; hydroxylamine; nanoparticle functionalization
    DOI:  https://doi.org/10.3390/ijms21072427
  36. Sensors (Basel). 2020 Mar 21. pii: E1746. [Epub ahead of print]20(6):
    A FRET Based Two-Photon Fluorescent Probe for Visualizing Mitochondrial Thiols of Living Cells and Tissues.
    Zhengkun Liu, Qianqian Wang, Hao Wang, Wenting Su, Shouliang Dong.
      Glutathione (GSH) is the main component of the mitochondrial thiol pool and plays key roles in the biological processes. Many evidences have suggested that cysteine and homocysteine also exist in mitochondria and are interrelated with GSH in biological systems. The fluctuation of the levels of mitochondrial thiols has been linked to many diseases and cells' dysfunction. Therefore, the monitoring of mitochondrial thiol status is of great significance for clinical studies. We report here a novel fluorescence resonance energy transfer based two-photon probe MT-1 for mitochondrial thiols detection. MT-1 was constructed by integrating the naphthalimide moiety (donor) and rhodamine B (accepter and targeting group) through a newly designed linker. MT-1 shows a fast response, high selectivity, and sensitivity to thiols, as well as a low limit of detection. The two-photon property of MT-1 allows the direct visualization of thiols in live cells and tissues by two-photon microscopy. MT-1 can serve as an effective tool to unravel the diverse biological functions of mitochondrial thiols in living systems.
    Keywords:  FRET; fluorescent probe; mitochondrial thiols; two-photon
    DOI:  https://doi.org/10.3390/s20061746
  37. Toxicol In Vitro. 2020 Mar 28. pii: S0887-2333(20)30157-0. [Epub ahead of print] 104840
    Blocking Parkin/PINK1-mediated mitophagy sensitizes hepatocellular carcinoma cells to sanguinarine-induced mitochondrial apoptosis.
    Qi Su, Jingjing Wang, Feng Liu, Yanmin Zhang.
      Hepatocellular carcinoma (HCC) remains a major clinical challenge. Although mitophagy is implicated in hepatocarcinogenesis, novel therapeutic options targeting mitophagy for HCC treatment still await further studies. Here, we demonstrate that sanguinarine induces cell death in HCC cell line MHCC-97H through the mitochondrial apoptosis pathway. Sanguinarine triggers mitochondrial dysfunction and PTEN-induced putative kinase 1 (PINK1)/Parkin upregulation and recruitment to mitochondria. Elevated levels of p62 and LC3-II/I ratios suggest that sanguinarine is both an inducer of autophagy and a blocker of autolysosome formation, which is further confirmed by LC3-II conversion levels in presence of autophagy and mitophagy inhibitors, as well as an autophagy activator. In addition, blocking autophagy promotes sanguinarine-induced cell death, indicating mitophagy plays a cytoprotective role in sanguinarine-treated cells. Our findings suggest that blocking mitophagy may contribute to sanguinarine-induced mitochondrial apoptosis through the prevention of damaged mitochondrial clearance.
    Keywords:  Apoptosis; Hepatocellular carcinoma; Mitophagy; Sanguinarine
    DOI:  https://doi.org/10.1016/j.tiv.2020.104840
  38. iScience. 2020 Mar 12. pii: S2589-0042(20)30162-0. [Epub ahead of print]23(4): 100978
    Exercise-Mediated Lowering of Glutamine Availability Suppresses Tumor Growth and Attenuates Muscle Wasting.
    Katrine S Pedersen, Francesco Gatto, Bo Zerahn, Jens Nielsen, Bente K Pedersen, Pernille Hojman, Julie Gehl.
      Glutamine is a central nutrient for many cancers, contributing to the generation of building blocks and energy-promoting signaling necessary for neoplastic proliferation. In this study, we hypothesized that lowering systemic glutamine levels by exercise may starve tumors, thereby contributing to the inhibitory effect of exercise on tumor growth. We demonstrate that limiting glutamine availability, either pharmacologically or physiologically by voluntary wheel running, significantly attenuated the growth of two syngeneic murine tumor models of breast cancer and lung cancer, respectively, and decreased markers of atrophic signaling in muscles from tumor-bearing mice. In continuation, wheel running completely abolished tumor-induced loss of weight and lean body mass, independently of the effect of wheel running on tumor growth. Moreover, wheel running abolished tumor-induced upregulation of muscular glutamine transporters and myostatin signaling. In conclusion, our data suggest that voluntary wheel running preserves muscle mass by counteracting muscular glutamine release and tumor-induced atrophic signaling.
    Keywords:  Cancer; Physiology; Specialized Functions of Cells
    DOI:  https://doi.org/10.1016/j.isci.2020.100978
  39. Anticancer Res. 2020 Apr;40(4): 1963-1972
    Menadione/Ascorbate Induces Overproduction of Mitochondrial Superoxide and Impairs Mitochondrial Function in Cancer: Comparative Study on Cancer and Normal Cells of the Same Origin.
    Severina Semkova, Zhivko Zhelev, Thomas Miller, Kimihiko Sugaya, Ichio Aoki, Tatsuya Higashi, Rumiana Bakalova.
      BACKGROUND/AIM: The menadione/ascorbate (M/A) combination has attracted attention due to the unusual ability of pro-vitamin/vitamin combination to kill cancer cells without affecting the viability of normal cells. The aim of this study was to elucidate the role of M/A in targeting cancerous mitochondria.MATERIALS AND METHODS: Several cancer and normal cell lines of the same origin were used. Cells were treated with different concentrations of M/A for 24 h. The cell viability, mitochondrial superoxide, mitochondrial membrane potential, and succinate were analyzed using conventional analytical tests.
    RESULTS: M/A exhibited a highly specific suppression on cancer cell growth and viability, without adversely affecting the viability of normal cells at concentrations attainable by oral or parenteral administration in vivo. This effect was accompanied by: (i) an extremely high production of mitochondrial superoxide in cancer cells, but not in normal cells; (ii) a significant dose-dependent depolarization of mitochondrial membrane and depletion of oncometabolite succinate in cancer cells.
    CONCLUSION: The anticancer effect of M/A is related to the induction of severe mitochondrial oxidative stress in cancer cells only. Thus, M/A has a potential to increase the sensitivity and vulnerability of cancer cells to conventional anticancer therapy and immune system.
    Keywords:  Menadione; ascorbate; cancer; mitochondria; succinate; superoxide
    DOI:  https://doi.org/10.21873/anticanres.14151
  40. Exp Oncol. 2020 03;42(1): 35-39
    Metformin enhances cytotoxic action of dichloroacetate against Lewis lung carcinoma cells in vitro.
    D L Kolesnik, O N Pyaskovskaya, O Gorbach, G I Solyanik.
      Tumor cell metabolism is considered one of the hallmarks of cancer. This concept is exploited in the development of new ways of anticancer therapy based on the use of substances capable of changing drastically bioenergetic metabolism of tumor cells. Among them, sodium dichloroace-tate (DCA), an inhibitor of pyruvate dehydrogenase kinase, and metformin (MTF), an antidiabetic hypoglycemic drug, an inhibitor of the mitochondrial respiratory chain (complex I), both have been long used in clinical non-oncological practice, and presently are considered promising candidates in oncology.AIM: To study the capability of MTF to enhance the antitumor action of DCA against Lewis lung carcinoma cells in vitro.
    MATERIALS AND METHODS: LLC/R9, a low metastatic variant of Lewis lung carcinoma cells, was used. Effects of 30 mM DCA in combination with 2 mM MTF on cell survival, cell cycle distribution, apoptosis, mitochondrial potential, intracellular ATP level, glucose consumption, and lactate production rates were determined in vitro.
    RESULTS: MTF was shown to enhance the cytotoxic/cytostatic action of DCA against LLC/R9 cells in vitro. Treatment of LLC/R9 cells with 30 mM DCA in combination with 2 mM MTF resulted in a 39% decrease in the number of viable cells (p < 0.05), a 2.8-fold increase of the number of dead cells (p < 0.05), a near 2-fold decrease in the proportion of cells at the S-phase (p < 0.05), a 4-fold increase in the apoptosis (p < 0.05) and significant reduction (p < 0.05) of the mitochondrial membrane potential of tumor cells as compared to corresponding values in control. DCA alone reduced glucose consumption and lactate production rates by more than 26% (p < 0.05) and 34% (p < 0.05), respectively, whereas MTF counteracted these effects. Nevertheless, in the cells treated with both DCA and DCA in combination with MTF, the intracellular adenosine triphosphate increased by 33-35% compared with that in the control (p < 0.05).
    CONCLUSION: MTF enhanced the cytotoxic/cytostatic action of DCA against LLC/R9 cells in vitro, which points on their possible synergistic antitumor action in vivo.
  41. J Cell Physiol. 2020 Apr 02.
    Aurora kinase inhibitor restrains STAT5-activated leukemic cell proliferation by inducing mitochondrial impairment.
    Jin-Xing Wang, Ling Zhang, Ze-Wei Huang, Xue-Ning Zhang, Yan-Yan Jiang, Fang-Jie Liu, Liang Long, Man-Jie Xue, Gui Lu, Quentin Liu, Zi-Jie Long.
      Current chemotherapy regimens on acute myeloid leukemia (AML) still have some drawbacks, such as intolerance and drug resistance, which calls need for the development of targeted therapy. Signal transducer and activator of transcription 5 (STAT5) is often overexpressed or abnormally activated in leukemia and involved in cell self-renewal, proliferation, and stress adaptation. Overexpressed Aurora A (AURKA) is associated with poor prognosis in tumors, and inhibitors against AURKA are already in clinical trials. However, it has rarely been reported whether AURKA inhibitors restrain STAT5-activated leukemia cells. In this study, we constructed STAT5 constitutively activated (cS5) cells and found that STAT5 promoted cell proliferation and colony formation. Moreover, cS5 cells showed elevated reactive oxygen species (ROS) and adenosine triphosphate (ATP) levels, which indicated higher mitochondrial metabolism in cS5 cells. A novel AURKA inhibitor AKI604 was synthesized and showed significant inhibitory effects to the proliferation and colony formation in both STAT5 constitutively activated and nonactivated AML cells. AKI604 induced mitochondrial impairment, leading to the disruption of mitochondrial membrane potential and the elevation of ROS as well as cellular calcium (Ca2+ ) levels. AKI604 could also decline basal oxygen consumption rate and ATP biosynthesis, indicating the damage of oxidative phosphorylation. Furthermore, AKI604 exhibited significant antitumor effect in the HL-60 cS5 xenograft model of the BALB/c nude mice without an obvious influence on mice body weight and other healthy indicators. This study suggested that AKI604 was a potential strategy to overcome STAT5-induced leukemic proliferation in AML treatment by inducing mitochondrial impairment.
    Keywords:  AML; AURKA inhibitor; STAT5; mitochondrial impairment; targeted therapy
    DOI:  https://doi.org/10.1002/jcp.29680
  42. Redox Biol. 2020 Mar 21. pii: S2213-2317(20)30203-2. [Epub ahead of print]32 101511
    Mitochondria at the crossroads of ATM-mediated stress signaling and regulation of reactive oxygen species.
    Ji-Hoon Lee, Tanya T Paull.
      The Ataxia-telangiectasia mutated (ATM) kinase responds to DNA double-strand breaks and other forms of cellular stress, including reactive oxygen species (ROS). Recent work in the field has uncovered links between mitochondrial ROS and ATM activation, suggesting that ATM acts as a sensor for mitochondrial derived ROS and regulates ROS accumulation in cells through this pathway. In addition, characterization of cells from Ataxia-telangiectasia patients as well as ATM-deficient mice and cell models suggest a role for ATM in modulating mitochondrial gene expression and function. Here we review ROS responses related to ATM function, recent evidence for ATM roles in mitochondrial maintenance and turnover, and the relationship between ATM and regulation of protein homeostasis.
    Keywords:  Ataxia; DNA repair; Mitochondria; Reactive oxygen species
    DOI:  https://doi.org/10.1016/j.redox.2020.101511
  43. FASEB J. 2020 Apr 02.
    Increased mitochondrial fragmentation in polycystic kidney disease acts as a modifier of disease progression.
    Laura Cassina, Marco Chiaravalli, Alessandra Boletta.
      Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic disorder, characterized by bilateral renal cyst formation. Multiple pathways are de-regulated in cystic epithelia offering good opportunities for therapy. Others and we have previously reported that metabolic reprogramming, including alterations of the TCA cycle, are prominent features of ADPKD. Several lines of evidence suggest that mitochondrial impairment might be responsible for the metabolic alterations. Here, we performed morphologic and morphometric evaluation of mitochondria by TEM in an orthologous mouse model of PKD caused by mutations in the Pkd1 gene (Ksp-Cre;Pkd1flox/- ). Furthermore, we measured mitochondrial respiration by COX and SDH enzymatic activity in situ. We found several alterations including reduced mitochondrial mass, altered structure and fragmentation of the mitochondrial network in cystic epithelia of Ksp-Cre;Pkd1flox/- mice. At the molecular level, we found reduced expression of the pro-fusion proteins OPA1 and MFN1 and up-regulation of the pro-fission protein DRP1. Importantly, administration of Mdivi-1, which interferes with DRP1 rescuing mitochondrial fragmentation, significantly reduced kidney/body weight, cyst formation, and improved renal function in Ksp-Cre;Pkd1flox/- mice. Our data indicate that impaired mitochondrial structure and function play a role in disease progression, and that their improvement can significantly modify the course of the disease.
    Keywords:  Mdivi-1; cellular respiration; mitochondrial fragmentation; polycystic kidney disease
    DOI:  https://doi.org/10.1096/fj.201901739RR
  44. Nat Cell Biol. 2020 Mar 30.
    Cystine transporter regulation of pentose phosphate pathway dependency and disulfide stress exposes a targetable metabolic vulnerability in cancer.
    Xiaoguang Liu, Kellen Olszewski, Yilei Zhang, Esther W Lim, Jiejun Shi, Xiaoshan Zhang, Jie Zhang, Hyemin Lee, Pranavi Koppula, Guang Lei, Li Zhuang, M James You, Bingliang Fang, Wei Li, Christian M Metallo, Masha V Poyurovsky, Boyi Gan.
      SLC7A11-mediated cystine uptake is critical for maintaining redox balance and cell survival. Here we show that this comes at a significant cost for cancer cells with high levels of SLC7A11. Actively importing cystine is potentially toxic due to its low solubility, forcing cancer cells with high levels of SLC7A11 (SLC7A11high) to constitutively reduce cystine to the more soluble cysteine. This presents a significant drain on the cellular NADPH pool and renders such cells dependent on the pentose phosphate pathway. Limiting glucose supply to SLC7A11high cancer cells results in marked accumulation of intracellular cystine, redox system collapse and rapid cell death, which can be rescued by treatments that prevent disulfide accumulation. We further show that inhibitors of glucose transporters selectively kill SLC7A11high cancer cells and suppress SLC7A11high tumour growth. Our results identify a coupling between SLC7A11-associated cystine metabolism and the pentose phosphate pathway, and uncover an accompanying metabolic vulnerability for therapeutic targeting in SLC7A11high cancers.
    DOI:  https://doi.org/10.1038/s41556-020-0496-x
  45. Cells. 2020 Mar 27. pii: E815. [Epub ahead of print]9(4):
    Redox Modifications of Proteins of the Mitochondrial Fusion and Fission Machinery.
    Christina Wolf, Víctor López Del Amo, Sabine Arndt, Diones Bueno, Stefan Tenzer, Eva-Maria Hanschmann, Carsten Berndt, Axel Methner.
      Mitochondrial fusion and fission tailors the mitochondrial shape to changes in cellular homeostasis. Players of this process are the mitofusins, which regulate fusion of the outer mitochondrial membrane, and the fission protein DRP1. Upon specific stimuli, DRP1 translocates to the mitochondria, where it interacts with its receptors FIS1, MFF, and MID49/51. Another fission factor of clinical relevance is GDAP1. Here, we identify and discuss cysteine residues of these proteins that are conserved in phylogenetically distant organisms and which represent potential sites of posttranslational redox modifications. We reveal that worms and flies possess only a single mitofusin, which in vertebrates diverged into MFN1 and MFN2. All mitofusins contain four conserved cysteines in addition to cysteine 684 in MFN2, a site involved in mitochondrial hyperfusion. DRP1 and FIS1 are also evolutionarily conserved but only DRP1 contains four conserved cysteine residues besides cysteine 644, a specific site of nitrosylation. MFF and MID49/51 are only present in the vertebrate lineage. GDAP1 is missing in the nematode genome and contains no conserved cysteine residues. Our analysis suggests that the function of the evolutionarily oldest proteins of the mitochondrial fusion and fission machinery, the mitofusins and DRP1 but not FIS1, might be altered by redox modifications.
    Keywords:  fission; fusion; metabolism; mitochondria; redox; thiol switch
    DOI:  https://doi.org/10.3390/cells9040815
  46. Stem Cell Reports. 2020 Apr 01. pii: S2213-6711(20)30070-9. [Epub ahead of print]
    Mitoregulin Controls β-Oxidation in Human and Mouse Adipocytes.
    Max Friesen, Curtis R Warren, Haojie Yu, Takafumi Toyohara, Qiurong Ding, Mary H C Florido, Carolyn Sayre, Benjamin D Pope, Loyal A Goff, John L Rinn, Chad A Cowan.
      We previously discovered in mouse adipocytes an lncRNA (the homolog of human LINC00116) regulating adipogenesis that contains a highly conserved coding region. Here, we show human protein expression of a peptide within LINC00116, and demonstrate that this peptide modulates triglyceride clearance in human adipocytes by regulating lipolysis and mitochondrial β-oxidation. This gene has previously been identified as mitoregulin (MTLN). We conclude that MTLN has a regulatory role in adipocyte metabolism as demonstrated by systemic lipid phenotypes in knockout mice. We also assert its adipocyte-autonomous phenotypes in both isolated murine adipocytes as well as human stem cell-derived adipocytes. MTLN directly interacts with the β subunit of the mitochondrial trifunctional protein, an enzyme critical in the β-oxidation of long-chain fatty acids. Our human and murine models contend that MTLN could be an avenue for further therapeutic research, albeit not without caveats, for example, by promoting white adipocyte triglyceride clearance in obese subjects.
    Keywords:  MTLN; adipocyte; human stem cells; metabolic disease; metabolism; mitochondrial metabolism
    DOI:  https://doi.org/10.1016/j.stemcr.2020.03.002
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