bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2020‒07‒05
fifty-two papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Sci Rep. 2020 Jun 29. 10(1): 10304
      For decades, it was not entirely clear why mitochondria develop cristae? The work employing the transmembrane-electrostatic proton localization theory reported here has now provided a clear answer to this fundamental question. Surprisingly, the transmembrane-electrostatically localized proton concentration at a curved mitochondrial crista tip can be significantly higher than that at the relatively flat membrane plane regions where the proton-pumping respiratory supercomplexes are situated. The biological significance for mitochondrial cristae has now, for the first time, been elucidated at a protonic bioenergetics level: 1) The formation of cristae creates more mitochondrial inner membrane surface area and thus more protonic capacitance for transmembrane-electrostatically localized proton energy storage; and 2) The geometric effect of a mitochondrial crista enhances the transmembrane-electrostatically localized proton density to the crista tip where the ATP synthase can readily utilize the localized proton density to drive ATP synthesis.
  2. Redox Biol. 2020 Jun 21. pii: S2213-2317(20)30811-9. [Epub ahead of print]36 101606
      The mitochondrial electron transport chain is a major source of reactive oxygen species (ROS) and is also a target of ROS, with an implied role in the stabilization of hypoxia-inducible factor (HIF) and induction of the AMPK pathway. Here we used varying doses of two agents, Mito-Paraquat and Mito-Metformin, that have been conjugated to cationic triphenylphosphonium (TPP+) moiety to selectively target them to the mitochondrial matrix compartment, thereby resulting in the site-specific generation of ROS within mitochondria. These agents primarily induce superoxide (O2•-) production by acting on complex I. In Raw264.7 macrophages, C2C12 skeletal myocytes, and HCT116 adenocarcinoma cells, we show that mitochondria-targeted oxidants can induce ROS (O2•- and H2O2). In all three cell lines tested, the mitochondria-targeted agents disrupted membrane potential and activated calcineurin and the Cn-dependent retrograde signaling pathway. Hypoxic culture conditions also induced Cn activation and HIF1α activation in a temporally regulated manner, with the former appearing at shorter exposure times. Together, our results indicate that mitochondrial oxidant-induced retrograde signaling is driven by disruption of membrane potential and activation of Ca2+/Cn pathway and is independent of ROS-induced HIF1α or AMPK pathways.
    Keywords:  Calcineurin activation; HIF1α; Hypoxia mediated stress; Macrophage cell line; Mitochondria targeted agents; Mitochondrial ROS; Retrograde signaling
  3. J Membr Biol. 2020 Jul 03.
      Adenosine triphosphate (ATP)-dependent potassium channels openers (KATP) protect skeletal muscle against function impairment through the activation of the mitochondrial KATP channels (mitoKATP). Previous reports suggest that modulators of the mitochondrial KATP channels have additional effects on isolated mitochondria. To determine whether the KATP channel opener nicorandil has non-specific effects that explain its protective effect through the mitochondrial function, chicken muscle mitochondria were isolated, and respiration rate was determined pollarographically. The activity of the electron transport chain (ETC) complexes (I-IV) was measured using a spectrophotometric method. Reactive oxygen species (ROS) levels and lipid peroxidation were assessed using flow cytometry and thiobarbituric acid assay, respectively. Both KATP channel opener nicorandil and KATP channel blocker 5-hydroxydecanoate (5-HD) decreased mitochondrial respiration; nicorandil increased complex III activity and decreased complex IV activity. The effects of nicorandil on complex III were antagonized by 5-HD. Nicorandil increased ROS levels, effect reverted by either 5-HD or the antioxidant N-2-mercaptopropionyl glycine (MPG). None of these drugs affected lipid peroxidation levels. These findings suggest that KATP channel opener nicorandil increases mitochondrial ROS production from complex III. This results by partially blocking electron flow in the complex IV, setting electron carriers in a more reduced state, which is favored by the increase in complex III activity by nicorandil. Overall, our study showed that nicorandil like other mitochondrial KATP channel openers might not act through mitoKATP channel activation.
    Keywords:  Mitochondria; Nicorandil; Reactive oxygen species; Skeletal muscle; mitoKATP channel
  4. Prog Lipid Res. 2020 Jun 24. pii: S0163-7827(20)30030-8. [Epub ahead of print]79 101050
      A growing number of studies describe a connection between glycosphingolipids (GSLs) and glutamine metabolism, glucose metabolism and mitochondrial dysfunction in cancer cells. Since deregulated cell energy metabolism is one of cancer cells hallmarks, investigating this connection is an important step in the development of anti-cancer therapies. GSL species are often aberrantly regulated in human cancers. They cluster in signaling platforms in the plasma membrane and organelle membranes in so called glycosphingolipid enriched microdomains (GEMs), thereby regulating cell signaling pathways. The most important glutamine transporter for epithelial cells, alanine-serine-cysteine transporter 2 (ASCT2) locates in GEMs and is regulated by GEM composition. The accumulation of glucosylceramide and lactosylceramide in mitochondria associated ER membranes (MAMs) leads to increased oxidative phosphorylation. This increases mitochondrial reactive oxygen species (ROS) levels and influences mitochondrial dynamics. Here, we review current knowledge about deregulated GSL species in cancer, GSL influence on glutamine and glucose metabolism. In addition, the role of GSLs in MAMs, oxidative phosphorylation (OXPHOS) and mitochondrial dynamics with a special focus on mechanistic target of rapamycin (mTOR) signaling is discussed. mTOR seems to play a pivotal role in the connection between GSLs and glutamine metabolism as well as in mitochondrial signaling.
    Keywords:  Glucose metabolism; Glutamine metabolism; Mitochondria associated ER membrane; ROS; UGCG; mTOR
  5. Front Physiol. 2020 ;11 677
      Aim: Insulin-resistant skeletal muscle is characterized by metabolic inflexibility with associated alterations in substrate selection, mediated by peroxisome-proliferator activated receptor δ (PPARδ). Although it is established that PPARδ contributes to the alteration of energy metabolism, it is not clear whether it plays a role in mitochondrial fuel competition. While nutrient overload may impair metabolic flexibility by fuel congestion within mitochondria, in absence of obesity defects at a mitochondrial level have not yet been excluded. We sought to determine whether reduced PPARδ content in insulin-resistant rat skeletal muscle of a non-obese rat model of T2DM (Goto-Kakizaki, GK) ameliorate the inhibitory effect of fatty acid (i.e., palmitoylcarnitine) on mitochondrial carbohydrate oxidization (i.e., pyruvate) in muscle fibers.Methods: Bioenergetic function was characterized in oxidative soleus (S) and glycolytic white gastrocnemius (WG) muscles with measurement of respiration rates in permeabilized fibers in the presence of complex I, II, IV, and fatty acid substrates. Mitochondrial content was measured by citrate synthase (CS) and succinate dehydrogenase activity (SDH). Western blot was used to determine protein expression of PPARδ, PDK isoform 2 and 4.
    Results: CS and SDH activity, key markers of mitochondrial content, were reduced by ∼10-30% in diabetic vs. control, and the effect was evident in both oxidative and glycolytic muscles. PPARδ (p < 0.01), PDK2 (p < 0.01), and PDK4 (p = 0.06) protein content was reduced in GK animals compared to Wistar rats (N = 6 per group). Ex vivo respiration rates in permeabilized muscle fibers determined in the presence of complex I, II, IV, and fatty acid substrates, suggested unaltered mitochondrial bioenergetic function in T2DM muscle. Respiration in the presence of pyruvate was higher compared to palmitoylcarnitine in both animal groups and fiber types. Moreover, respiration rates in the presence of both palmitoylcarnitine and pyruvate were reduced by 25 ± 6% (S), 37 ± 6% (WG) and 63 ± 6% (S), 57 ± 8% (WG) compared to pyruvate for both controls and GK, respectively. The inhibitory effect of palmitoylcarnitine on respiration was significantly greater in GK than controls (p < 10-3).
    Conclusion: With competing fuels, the presence of fatty acids diminishes mitochondria ability to utilize carbohydrate derived substrates in insulin-resistant muscle despite reduced PPARδ content.
    Keywords:  bioenergetic; diabetes; fatty acid oxidation; metabolic flexibility; oxidative phosphorylation
  6. Cells. 2020 Jun 27. pii: E1570. [Epub ahead of print]9(7):
      Hürthle cell thyroid carcinoma (HTC) accounts for 3-5% of all thyroid malignancies. Widely invasive HTC is characterized by poor prognosis and limited responsiveness to standard therapy with radioiodine. The molecular landscape of HTC is significantly different from the genetic signature seen in other forms of thyroid cancer. We performed a comprehensive literature review on the association between the molecular features of HTC and cancer metabolism. We searched the Pubmed, Embase, and Medline databases for clinical and translational studies published between 1980 and 2020 in English, coupling "HTC" with the following keywords: "genomic analysis", "mutations", "exome sequencing", "molecular", "mitochondria", "metabolism", "oxidative phosphorylation", "glycolysis", "oxidative stress", "reactive oxygen species", and "oncogenes". HTC is characterized by frequent complex I mitochondrial DNA mutations as early clonal events. This genetic signature is associated with the abundance of malfunctioning mitochondria in cancer cells. HTC relies predominantly on aerobic glycolysis as a source of energy production, as oxidative phosphorylation-related genes are downregulated. The enhanced glucose utilization by HTC is used for diagnostic purposes in the clinical setting for the detection of metastases by fluorodeoxyglucose positron emission tomography (FGD-PET/CT) imaging. A comprehensive metabolomic profiling of HTC in association with its molecular landscape might be necessary for the implementation of tumor-specific therapeutic approaches.
    Keywords:  Hürthle cell; metabolism; mitochondria; oncogenes; oxidative phosphorylation; thyroid cancer
  7. Sci Signal. 2020 Jun 30. pii: eaax6660. [Epub ahead of print]13(638):
      Chaperones in the endoplasmic reticulum (ER) control the flux of Ca2+ ions into mitochondria, thereby increasing or decreasing the energetic output of the oxidative phosphorylation pathway. An example is the abundant ER lectin calnexin, which interacts with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that calnexin stimulated the ATPase activity of SERCA by maintaining its redox state. This function enabled calnexin to control how much ER Ca2+ was available for mitochondria, a key determinant for mitochondrial bioenergetics. Calnexin-deficient cells compensated for the loss of this function by partially shifting energy generation to the glycolytic pathway. These cells also showed closer apposition between the ER and mitochondria. Calnexin therefore controls the cellular energy balance between oxidative phosphorylation and glycolysis.
  8. Nat Metab. 2020 Feb;2(2): 167-178
      The neonatal mammalian heart is capable of regeneration for a brief window of time after birth. However, this regenerative capacity is lost within the first week of life, which coincides with a postnatal shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation, particularly towards fatty-acid utilization. Despite the energy advantage of fatty-acid beta-oxidation, cardiac mitochondria produce elevated rates of reactive oxygen species when utilizing fatty acids, which is thought to play a role in cardiomyocyte cell-cycle arrest through induction of DNA damage and activation of DNA-damage response (DDR) pathway. Here we show that inhibiting fatty-acid utilization promotes cardiomyocyte proliferation in the postnatatal heart. First, neonatal mice fed fatty-acid deficient milk showed prolongation of the postnatal cardiomyocyte proliferative window, however cell cycle arrest eventually ensued. Next, we generated a tamoxifen-inducible cardiomyocyte-specific, pyruvate dehydrogenase kinase 4 (PDK4) knockout mouse model to selectively enhance oxidation of glycolytically derived pyruvate in cardiomyocytes. Conditional PDK4 deletion resulted in an increase in pyruvate dehydrogenase activity and consequently an increase in glucose relative to fatty-acid oxidation. Loss of PDK4 also resulted in decreased cardiomyocyte size, decreased DNA damage and expression of DDR markers and an increase in cardiomyocyte proliferation. Following myocardial infarction, inducible deletion of PDK4 improved left ventricular function and decreased remodelling. Collectively, inhibition of fatty-acid utilization in cardiomyocytes promotes proliferation, and may be a viable target for cardiac regenerative therapies.
  9. Food Funct. 2020 Jul 01.
      Ellagic acid (EA), a natural polyphenol compound that exists in a variety of fruits and vegetables, has been reported to inhibit tumor growth by reducing cell growth, inducing apoptosis, and damaging mitochondria. Recent reports demonstrate that mitochondria regulate cancer cell death through energy metabolism and that different types of cell death coexist in vivo. We showed that EA inhibited lung cancer cell proliferation, markedly decreased ATP levels, decreased the potential of the inner mitochondrial membrane and decreased oxygen consumption in vitro. In addition, EA activated AMP-activated protein kinase (AMPK) and reduced HIF-1α in lung cancer cells. Moreover, the treatment of tumor-bearing mice with EA dramatically inhibited tumor growth, increased p-AMPK and suppressed HIF-1α levels. These findings suggest that EA could be a promising chemotherapeutic agent that targets mitochondrial metabolism in lung cancer.
  10. FASEB J. 2020 Jul 01.
      Dietary restriction and reduced reproduction have been linked to long lifespans in the vast majority of species tested. Although decreased mitochondrial mass and/or function are hallmarks of aging, little is known about the mechanisms by which these organelles contribute to physiological aging or to the effects of lifespan-extending interventions, particularly with respect to oxidative phosphorylation and energy production. Here, we employed the nematode Caenorhabditis elegans to examine the effects of inhibition of germline proliferation and dietary restriction, both of which extend the lifespan of C. elegans, on mitochondrial respiratory activity in whole animals and isolated organelles. We found that oxygen consumption rates and mitochondrial mass were reduced in wild-type (WT) C. elegans subjected to bacterial deprivation (BD) compared with animals fed ad libitum (AL). In contrast, BD decreased the rate of oxygen uptake but not mitochondrial mass in germline-less glp-1(e2144ts) mutants. Interestingly, mitochondria isolated from animals subjected to BD and/or inhibition of germline proliferation showed no differences in complex I-mediated respiratory activity compared to control mitochondria, whereas both interventions enhanced the efficiency with which mitochondria utilized lipids as respiratory substrates. Notably, the combination of BD and inhibition of germline proliferation further increased mitochondrial lipid oxidation compared to either intervention alone. We also detected a striking correlation between lifespan extension in response to BD and/or inhibition of germline proliferation and the capacity of C. elegans to generate ATP from lipids. Our results thus suggest that the ability to oxidize lipids may be determinant in enhanced longevity.
    Keywords:   C. elegans ; dietary restriction; lipid oxidation; longevity; mitochondrial metabolism
  11. Proc Natl Acad Sci U S A. 2020 Jun 29. pii: 201915275. [Epub ahead of print]
      Peroxiredoxins are central to cellular redox homeostasis and signaling. They serve as peroxide scavengers, sensors, signal transducers, and chaperones, depending on conditions and context. Typical 2-Cys peroxiredoxins are known to switch between different oligomeric states, depending on redox state, pH, posttranslational modifications, and other factors. Quaternary states and their changes are closely connected to peroxiredoxin activity and function but so far have been studied, almost exclusively, outside the context of the living cell. Here we introduce the use of homo-FRET (Förster resonance energy transfer between identical fluorophores) fluorescence polarization to monitor dynamic changes in peroxiredoxin quaternary structure inside the crowded environment of living cells. Using the approach, we confirm peroxide- and thioredoxin-related quaternary transitions to take place in cellulo and observe that the relationship between dimer-decamer transitions and intersubunit disulfide bond formation is more complex than previously thought. Furthermore, we demonstrate the use of the approach to compare different peroxiredoxin isoforms and to identify mutations and small molecules affecting the oligomeric state inside cells. Mutagenesis experiments reveal that the dimer-decamer equilibrium is delicately balanced and can be shifted by single-atom structural changes. We show how to use this insight to improve the design of peroxiredoxin-based redox biosensors.
    Keywords:  dimer-decamer equilibrium; fluorescence polarization; homo-FRET; peroxiredoxins; protein oligomerization
  12. J Clin Med. 2020 Jun 24. pii: E1971. [Epub ahead of print]9(6):
      Mitochondria play a crucial role in cell life and death by regulating bioenergetic and biosynthetic pathways. They are able to adapt rapidly to different microenvironmental stressors by accommodating the metabolic and biosynthetic needs of the cell. Mounting evidence places mitochondrial dysfunction at the core of several diseases, notably in the context of pathologies of the cardiovascular and central nervous system. In addition, mutations in some mitochondrial proteins are bona fide cancer drivers. Better understanding of the functions of these multifaceted organelles and their components may finetune our knowledge on the molecular bases of certain diseases and suggest new therapeutic avenues.
    Keywords:  aging; cancer; cardiovascular diseases; infection; inflammation; mitochondria; mitochondrial dysfunction; neurodegenerative diseases
  13. Nat Commun. 2020 Jul 03. 11(1): 3347
      A sharp increase in mitochondrial Ca2+ marks the activation of brown adipose tissue (BAT) thermogenesis, yet the mechanisms preventing Ca2+ deleterious effects are poorly understood. Here, we show that adrenergic stimulation of BAT activates a PKA-dependent mitochondrial Ca2+ extrusion via the mitochondrial Na+/Ca2+ exchanger, NCLX. Adrenergic stimulation of NCLX-null brown adipocytes (BA) induces a profound mitochondrial Ca2+ overload and impaired uncoupled respiration. Core body temperature, PET imaging of glucose uptake and VO2 measurements confirm a thermogenic defect in NCLX-null mice. We show that Ca2+ overload induced by adrenergic stimulation of NCLX-null BAT, triggers the mitochondrial permeability transition pore (mPTP) opening, leading to a remarkable mitochondrial swelling and cell death. Treatment with mPTP inhibitors rescue mitochondrial function and thermogenesis in NCLX-null BAT, while calcium overload persists. Our findings identify a key pathway through which BA evade apoptosis during adrenergic stimulation of uncoupling. NCLX deletion transforms the adrenergic pathway responsible for thermogenesis activation into a death pathway.
  14. J Cell Biol. 2020 Aug 03. pii: e202003131. [Epub ahead of print]219(8):
      Mitochondria, so much more than just being energy factories, also have the capacity to synthesize macromolecules including phospholipids, particularly cardiolipin (CL) and phosphatidylethanolamine (PE). Phospholipids are vital constituents of mitochondrial membranes, impacting the plethora of functions performed by this organelle. Hence, the orchestrated movement of phospholipids to and from the mitochondrion is essential for cellular integrity. In this review, we capture recent advances in the field of mitochondrial phospholipid biosynthesis and trafficking, highlighting the significance of interorganellar communication, intramitochondrial contact sites, and lipid transfer proteins in maintaining membrane homeostasis. We then discuss the physiological functions of CL and PE, specifically how they associate with protein complexes in mitochondrial membranes to support bioenergetics and maintain mitochondrial architecture.
  15. Nat Commun. 2020 Jul 03. 11(1): 3326
      Tumour cells adapt to nutrient deprivation in vivo, yet strategies targeting the nutrient poor microenvironment remain unexplored. In melanoma, tumour cells often experience low glutamine levels, which promote cell dedifferentiation. Here, we show that dietary glutamine supplementation significantly inhibits melanoma tumour growth, prolongs survival in a transgenic melanoma mouse model, and increases sensitivity to a BRAF inhibitor. Metabolomic analysis reveals that dietary uptake of glutamine effectively increases the concentration of glutamine in tumours and its downstream metabolite, αKG, without increasing biosynthetic intermediates necessary for cell proliferation. Mechanistically, we find that glutamine supplementation uniformly alters the transcriptome in tumours. Our data further demonstrate that increase in intra-tumoural αKG concentration drives hypomethylation of H3K4me3, thereby suppressing epigenetically-activated oncogenic pathways in melanoma. Therefore, our findings provide evidence that glutamine supplementation can serve as a potential dietary intervention to block melanoma tumour growth and sensitize tumours to targeted therapy via epigenetic reprogramming.
  16. J Biochem Mol Toxicol. 2020 Jul 03. e22544
      Breast cancer is one of the leading causes of death in cancer categories, followed by lung, colorectal, and ovarian among the female gender across the world. 10H-3,6-diazaphenothiazine (PTZ) is a thiazine derivative compound that exhibits many pharmacological activities. Herein, we proceed to investigate the pharmacological activities of PTZ toward breast cancer MCF-7 cells as a representative in vitro breast cancer cell model. The PTZ exhibited a proliferation inhibition (IC50  = 0.895 µM) toward MCF-7 cells. Further, cell cycle analysis illustrated that the S-phase checkpoint was activated to achieve proliferation inhibition. In vitro cytotoxicity test on three normal cell lines (HEK293 normal kidney cells, MCF-10A normal breast cells, and H9C2 normal heart cells) demonstrated that PTZ was more potent toward cancer cells. Increase in the levels of reactive oxygen species results in polarization of mitochondrial membrane potential (ΔΨm), together with suppression of mitochondrial thioredoxin reductase enzymatic activity suggested that PTZ induced oxidative damages toward mitochondria and contributed to improved drug efficacy toward treatment. The RT2 PCR Profiler Array (human apoptosis pathways) proved that PTZ induced cell death via mitochondria-dependent and cell death receptor-dependent pathways, through a series of modulation of caspases, and the respective morphology of apoptosis was observed. Mechanistic studies of apoptosis suggested that PTZ inhibited AKT1 pathways resulting in enhanced drug efficacy despite it preventing invasion of cancer cells. These results showed the effectiveness of PTZ in initiation of apoptosis, programmed cell death, toward highly chemoresistant MCF-7 cells, thus suggesting its potential as a chemotherapeutic drug.
    Keywords:  10H-3,6-Diazaphenothiazine; AKT1 pathways; apoptosis; breast cancer; mitochondrial thioredoxin reductase
  17. Breast Cancer Res Treat. 2020 Jun 29.
      PURPOSE: Mitochondrial unfolding protein are abundant in breast cancer cells, but the mechanism by which breast cancer cells resist apoptosis is still not fully elucidated. In this study, we explored the role of mitochondrial unfolded protein response (mtUPR)-related proteins in four types of breast cancer tissues.METHODS: Mitochondrial fractions were taken from four breast cancer tissues (luminal A, luminal B, Her2 -overexpression, and TNBC) and the expression of mitochondrial polyubiquitinated proteins was observed by western blot and ELISA. In addition, the expression of hsp10, hsp60, and clpp in mitochondria was observed by western blot in breast cancer tissues and adjacent tissues, and confirmed by ELISA. The expression levels of hsp10 and hsp60 were correlated with clinicopathological parameters in 114 breast cancer patients.
    RESULTS: We found an increase in the performance of mitochondrial polyubiquitinated proteins in breast cancer tissues of luminal A, luminal B, Her2-overexpression, and TNBC. The mitochondrial hsp10, hsp60, and clpp are abundantly expressed in breast cancer tissues rather than adjacent noncancerous tissues. The expression levels of mitochondrial hsp10 and hsp60 were highest in histological grade 3 breast cancer tissues. Additionally, mitochondria with high hsp60 expression were more present in Her2-positive tumors.
    CONCLUSIONS: We observed that mtUPR was specifically activated in breast cancer tissues but inactivated in normal mammary tissue. MtUPR had also exhibited a particular increase in Her2-overexpression tumors but not in ER- or PR-positive tumors. Taken together, we suggested that mtUPR may act as a potential candidate for developing novel Her2-overexpression breast cancer therapy.
    Keywords:  Breast cancer‧mitochondrial unfolded protein response; Her2-overexpression; mtUPR
  18. Int J Biochem Cell Biol. 2020 Jun 25. pii: S1357-2725(20)30113-8. [Epub ahead of print] 105796
      pH gradient reversal refers to intracellular alkalization and extracellular acidification commonly seen in malignant tumors. To meet their high anabolic demand, cancer cells rewire their glucose metabolism from oxidative phosphorylation to lactate fermentation, which results in the excessive generation of protons. To avoid lethal cytosolic acidification, lactate-fermenting cancer cells activate multiple acid removal pathways leading to the acidification of the extracellular space. This acidification is often further intensified by the defective capacity of the disorganized tumor vasculature to dilute protons away from the cancer tissue. The cancer-specific proton equilibrium with highly alkaline cytosol and acidic extracellular space is emerging as a fundamental driving force for cancer growth. Here, we discuss how cancer cells establish and maintain reversed pH gradient, how pH gradient reversal fuels cancer progression, and how these mechanisms can be targeted in cancer therapy.
    Keywords:  cancer; cellbiology; glycolysis; lysosome; pH gradient reversal
  19. Oxid Med Cell Longev. 2020 ;2020 5304768
      Circadian clocks regulate metabolic processes in a tissue-specific manner, which deteriorates during aging. Skeletal muscle is the largest metabolic organ in our body, and our previous studies highlight a key role of circadian regulation of skeletal muscle mitochondria in healthy aging. However, a possible circadian regulation of cardiolipin (CL), the signature lipid class in the mitochondrial inner membrane, remains largely unclear. Here, we show that CL levels oscillate during the diurnal cycle in C2C12 myotubes. Disruption of the Ror genes, encoding the ROR nuclear receptors in the secondary loop of the circadian oscillator, in C2C12 cells was found to dampen core circadian gene expression. Importantly, several genes involved in CL synthesis, including Taz and Ptpmt1, displayed rhythmic expression which was disrupted or diminished in Ror-deficient C2C12 cells. In vivo studies using skeletal muscle tissues collected from young and aged mice showed diverse effects of the clock and aging on the oscillatory expression of CL genes, and CL levels in skeletal muscle were enhanced in aged mice relative to young mice. Finally, consistent with a regulatory role of RORs, Nobiletin, a natural agonist of RORs, was found to partially restore transcripts levels of CL synthesis genes in aged muscle under a dietary challenge condition. Together, these observations highlight a rhythmic CL synthesis in skeletal muscle that is dependent on RORs and modifiable by age and diet.
  20. Cancer Discov. 2020 Jun 30. pii: CD-20-0226. [Epub ahead of print]
      Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor metabolite and is the currency of metabolic transactions critical for cell survival. Depending on tissue context and genotype, cancer cells have unique dependencies on NAD+ metabolic pathways. Poly(ADP-ribose) polymerases (PARPs) catalyze oligomerization of NAD+ monomers into poly(ADP-ribose) (PAR) chains during cellular response to alkylating chemotherapeutics, including procarbazine or temozolomide. Here, we find that, in endogenous IDH1 mutant tumor models, alkylator-induced cytotoxicity is markedly augmented by pharmacologic inhibition or genetic knockout of the PAR breakdown enzyme poly(ADP-ribose) glycohydrolase (PARG). Both in vitro and in vivo, we observe that concurrent alkylator and PARG inhibition depletes freely available NAD+ by preventing PAR breakdown, resulting in NAD+ sequestration and collapse of metabolic homeostasis. This effect reversed with NAD+ rescue supplementation, confirming the mechanistic basis of cytotoxicity. Thus, alkylating chemotherapy exposes a genotype-specific metabolic weakness in tumor cells that can be exploited by PARG inactivation.
  21. Mol Cancer Res. 2020 Jul 01. pii: molcanres.0339.2020. [Epub ahead of print]
      O-GlcNAc transferase (OGT) is a nutrient-sensitive glycosyltransferase that is overexpressed in prostate cancer, the most common cancer in males. We recently developed specific and potent inhibitor targeting this enzyme, and here we report a synthetic lethality screen using this compound. Our screen identified pan-cyclin-dependent kinase (CDK) inhibitor AT7519 as lethal in combination with OGT inhibition. Follow-up chemical and genetic approaches identified CDK9 as the major target for synthetic lethality with OGT inhibition in prostate cancer cells. OGT expression is regulated through retention of the fourth intron in the gene and CDK9 inhibition blunted this regulatory mechanism. CDK9 phosphorylates carboxy-terminal domain (CTD) of RNA Polymerase II (RNA Pol II) to promote transcription elongation. We show that OGT inhibition augments effects of CDK9 inhibitors on CTD phosphorylation and general transcription. Finally, the combined inhibition of both OGT and CDK9 blocked growth of organoids derived from metastatic prostate cancer patients but had minimal effects on normal prostate spheroids. We report a novel synthetic lethal interaction between inhibitors of OGT and CDK9 that specifically kills prostate cancer cells but not normal cells. Our study highlights the potential of combining OGT inhibitors with other treatments to exploit cancer specific vulnerabilities. Implications: The primary contribution of OGT to cell proliferation is unknown, and in this study we use a compound screen to indicate that OGT and CDK9 collaborate to sustain a cancer cell specific pro-proliferative program. A better understanding of how OGT and CDK9 cross-talk will refine our understanding of this novel synthetic lethal interaction.
  22. J Am Osteopath Assoc. 2020 Jul 01. 120(7): 446-455
      CONTEXT: Patients with diabetes are susceptible to heart failure. Defective mitochondria can cause cardiac damage. Mitochondrial autophagy or mitophagy is a quality control mechanism that eliminates dysfunctional mitochondria through lysosome degradation. Mitophagy is essential for maintaining a pool of healthy mitochondria for normal cardiac function. However, the effect of diabetes on the functional status of cardiac mitophagy remains unclear.OBJECTIVE: To determine and compare cardiac mitophagy flux between diabetic and nondiabetic mice.
    METHODS: Using a novel dual fluorescent mitophagy reporter termed mt-Rosella, we labeled and traced mitochondrial fragments that are sequestered by the autophagosome and delivered to and degraded in the lysosome.
    RESULTS: Mitophagic activity was reduced in high-glucose-treated cardiomyocytes and in the heart tissue of type 1 diabetic mice.
    CONCLUSIONS: Mitophagy was impaired in the heart of diabetic mice, suggesting that restoring or accelerating mitophagy flux may be a useful strategy to reduce cardiac injury caused by diabetes.
  23. Cell Death Differ. 2020 Jul 01.
      FOXP3+ regulatory T (Treg) cells are essential for maintaining immunological tolerance. Given their importance in immune-related diseases, cancer and obesity, there is increasing interest in targeting the Treg cell compartment therapeutically. New pharmacological inhibitors that specifically target the prosurvival protein MCL-1 may provide this opportunity, as Treg cells are particularly reliant upon this protein. However, there are two distinct isoforms of MCL-1; one located at the outer mitochondrial membrane (OMM) that is required to antagonize apoptosis, and another at the inner mitochondrial membrane (IMM) that is reported to maintain IMM structure and metabolism via ATP production during oxidative phosphorylation. We set out to elucidate the relative importance of these distinct biological functions of MCL-1 in Treg cells to assess whether MCL-1 inhibition might impact upon the metabolism of cells able to resist apoptosis. Conditional deletion of Mcl1 in FOXP3+ Treg cells resulted in a lethal multiorgan autoimmunity due to the depletion of the Treg cell compartment. This striking phenotype was completely rescued by concomitant deletion of the apoptotic effector proteins BAK and BAX, indicating that apoptosis plays a pivotal role in the homeostasis of Treg cells. Notably, MCL-1-deficient Treg cells rescued from apoptosis displayed normal metabolic capacity. Moreover, pharmacological inhibition of MCL-1 in Treg cells resistant to apoptosis did not perturb their metabolic function. We conclude that Treg cells require MCL-1 only to antagonize apoptosis and not for metabolism. Therefore, MCL-1 inhibition could be used to manipulate Treg cell survival for clinical benefit without affecting the metabolic fitness of cells resisting apoptosis.
  24. Nutrients. 2020 Jun 29. pii: E1930. [Epub ahead of print]12(7):
      β-hydroxybutyrate is the main ketone body generated by the liver under starvation. Under these conditions, it can sustain ATP levels by its oxidation in mitochondria. As mitochondria can modify its shape and function under different nutritional challenges, we study the chronic effects of β-hydroxybutyrate supplementation on mitochondrial morphology and function, and its relation to exercise capacity. Male C57BL/6 mice were supplemented with β-hydroxybutyrate mineral salt (3.2%) or control (CT, NaCl/KCl) for six weeks and submitted to a weekly exercise performance test. We found an increase in distance, maximal speed, and time to exhaustion at two weeks of supplementation. Fatty acid metabolism and OXPHOS subunit proteins declined at two weeks in soleus but not in tibialis anterior muscles. Oxygen consumption rate on permeabilized fibers indicated a decrease in the presence of pyruvate in the short-term treatment. Both the tibialis anterior and soleus showed decreased levels of Mitofusin 2, while electron microscopy assessment revealed a significant reduction in mitochondrial cristae shape in the tibialis anterior, while a reduction in the mitochondrial number was observed only in soleus. These results suggest that short, but not long-term, β‑hydroxybutyrate supplementation increases exercise capacity, associated with modifications in mitochondrial morphology and function in mouse skeletal muscle.
    Keywords:  endurance; ketone bodies; mitochondrial morphology; skeletal muscle; β-hydroxybutyrate
  25. Front Oncol. 2020 ;10 963
      Lung squamous cell carcinoma (LUSC) is one of the leading causes of tumor-driven deaths in the world. To date, studies on the tumor heterogeneity of LUSC at genomic level have only revealed limited therapeutic benefits. Therefore, system-wide research of LUSC at proteomic level may further improve precision medicine strategies on individual demands. To this end, we performed proteomic and phosphoproteomic study for LUSC samples of 25 Chinese patients. From our results, two subgroups (Cluster I and II) based on proteomic data were identified, which were associated with distinct molecular characteristics and clinicopathologic features. Combined with phosphoproteomic data, our result showed that spliceosome pathway was enriched in Cluster I, while focal adhesion pathway, immune-related pathways and Ras signaling pathway were enriched in Cluster II. In addition, we found that lymph node metastasis (LNM) was associated with our proteomic subgroups and cell cycle pathway was enriched in patients with LNM. Further analysis showed that MCM2, a DNA replication licensing factor involved in cell cycle pathway, was highly expressed in patients with poor prognosis, which was further proved by immunohistochemistry (IHC) analysis. In summary, our study provided a resource of the proteomic and phosphoproteomic features of LUSC in Chinese patients.
    Keywords:  cell cycle; clustering; lung squamous cell carcinoma; lymph node metastasis; phosphoproteomics; proteomics
  26. J Control Release. 2020 Jun 26. pii: S0168-3659(20)30344-8. [Epub ahead of print]
      Primary tumor metastasis remains to be a tough obstacle for clinical breast cancer treatment. Since evidences have shown that mitochondria play a crucial role in tumor metastasis, we designed a mitochondrial targeted drug delivery system (P-D-R8MTS) based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers to simultaneously inhibit breast cancer progression and metastasis. A novel mitochondrial targeted hybrid peptide R8MTS, which consists of a cell penetrating peptide octaarginine (R8) and a mitochondrial targeting sequence ALD5MTS, was used as targeting ligand and attached to doxorubicin (DOX) as model drug (DOX-R8MTS). After entering into the tumor cells, DOX-R8MTS was pH-responsibly released from HPMA copolymer backbone in acidic lysosome and efficiently targeted to mitochondria, resulting in enhanced reactive oxygen species (ROS) generation and apoptosis initiation. By destroying mitochondria, P-D-R8MTS not only inhibited cell proliferation but also suppressed migration and invasion of breast cancer 4T1 and MDA-MB-231 cells in vitro. Moreover, P-D-R8MTS exhibited superior inhibition of tumor growth and showed no apparent lung metastatic nodules on 4T1-bearing mice in vivo, which was partially via down-regulation of typical proteins associated with tumor metastasis and invasion: matrix metalloproteinases-2 (MMP-2), vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β). Collectively, our work provided a prospectively potential strategy for metastatic cancer treatment through mitochondrial targeted drug delivery.
    Keywords:  Breast cancer; Hybrid peptide; Metastatic cancer treatment; Mitochondrial targeting; Tumor metastasis
  27. Onco Targets Ther. 2020 ;13 5419-5428
      Background: Noscapine is an opium alkaloid that has recently been shown to potentiate anti-cancer therapeutic effects by inducing apoptosis in various malignant cells without any detectable toxicity. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear.Materials and Methods: In this study, we explored the anti-cancer activity of noscapine in 5-fluorouracil (5-FU)-resistant human colon cancer cell lines HT29/5-FU and LoVo/5-FU and investigated the possible underlying mechanism. The apoptosis and mitochondrial morphology of cells were detected by TUNEL assay and transmission electron microscopy (TEM). The mitochondrial membrane potential (MMP) was determined using JC-1. The mitochondrial permeability transition pore (mPTP) opening was detected by the calcein-AM/cobalt assay. The levels of glucose, lactic, and ATP in cells were evaluated by ELISA kits. Relative protein expression levels were detected by Western blot.
    Results: We verified that PTEN was involved in noscapine-induced apoptosis in HT29/5-FU and LoVo/5-FU cells. Noscapine greatly increased mitochondrial damage by altering mitochondrial morphology, inducing mitochondrial membrane potential depolarization, and enabling mitochondrial permeability transition pore opening in HT29/5-FU and LoVo/5-FU cells. In addition, noscapine inhibited the Warburg effect by decreasing the levels of glucose, lactic acid, and ATP and inhibiting the protein expression of glucose transporter 1, lactate dehydrogenase-B, hexokinase 2, and pyruvate kinase M2 in HT29/5-FU and LoVo/5-FU cells. However, PTEN interference counteracted the effect of noscapine on mitochondrial damage and the Warburg effect in HT29/5-FU and LoVo/5-FU cells by decreasing the activation of PI3K/mTOR signaling.
    Conclusion: These results indicated that noscapine induced the apoptosis of HT29/5-FU and LoVo/5-FU human colon cancer cells by regulating mitochondria damage and the Warburg effect via PTEN, and the process is closely related to the PI3K/mTOR signaling pathway.
    Keywords:  PI3K/mTOR; PTEN; colon cancer; mitochondrial damage; noscapine
  28. Anticancer Res. 2020 Jul;40(7): 3819-3830
      BACKGROUND: Picrasma quassioides (PQ) is a traditional Asian herbal medicine with anti-tumor properties that can inhibit the viability of HepG2 liver cancer cells. H-Ras is often mutated in liver cancer, however, the effect of PQ treatment on H-Ras mutated liver cancer is unclear. This study aimed to investigate the role of PQ on ROS accumulation and mitochondrial dysfunction in H-ras mutated HepG2 (HepG2G12V) cells.MATERIALS AND METHODS: PQ ethanol extract-induced HepG2G12V apoptosis was analyzed by the MTT assay, fluorescence microscopy, flow cytometry and western blotting.
    RESULTS: PQ treatment affected cell migration and colony formation in HepG2G12V cells. Cleaved-caspase-3, cleaved-caspase-9 and BCL2 associated agonist of cell death (BAD) expression levels were increased, while the levels of B-cell lymphoma-extra large (Bcl-xL) were decreased with PQ treatment. PQ treatment led to a reduction of H-Ras expression levels in liver cancer cells, thus reducing their abnormal proliferation. Furthermore, it led to increased expression levels of Peroxiredoxin VI, which regulates the redox signal in cells.
    CONCLUSION: Taken together these results provide a new functional significance for the role of PQ in treating HepG2G12V liver cancer.
    Keywords:  Liver cancer; Picrasma quassioides; ROS; mitochondria
  29. Sci Rep. 2020 Jun 30. 10(1): 10665
      Mitochondrial lysine acetylation regulates several metabolic pathways in cardiac cells. The current study investigated whether GCN5L1-mediated lysine acetylation regulates cardiac mitochondrial metabolic proteins in response to a high fat diet (HFD). GCN5L1 cardiac-specific knockout (cKO) mice showed significantly reduced mitochondrial protein acetylation following a HFD relative to wildtype (WT) mice. GCN5L1 cKO mice did not display any decrease in ex vivo cardiac workload in response to a HFD. In contrast, ex vivo cardiac function in HFD-fed WT mice dropped ~ 50% relative to low fat diet (LFD) fed controls. The acetylation status of electron transport chain Complex I protein NDUFB8 was significantly increased in WT mice fed a HFD, but remained unchanged in GCN5L1 cKO mice relative to LFD controls. Finally, we observed that inhibitory acetylation of superoxide dismutase 2 (SOD2) at K122 was increased in WT (but not cKO mice) on a HFD. This correlated with significantly increased cardiac lipid peroxidation in HFD-fed WT mice relative to GCN5L1 cKO animals under the same conditions. We conclude that increased GCN5L1 expression in response to a HFD promotes increased lysine acetylation, and that this may play a role in the development of reactive oxygen species (ROS) damage caused by nutrient excess.
  30. J Immunol. 2020 Jun 29. pii: ji1900820. [Epub ahead of print]
      NF-κB is a reduction-oxidation-sensitive transcription factor that plays a key role in regulating the immune response. In these studies, we intended to investigate the role of mitochondrial-derived reactive oxygen species in regulating NF-κB activation by studying transgenic mice that overexpress mitochondrial-targeted human catalase (mCAT). We treated wild-type (WT) and mCAT mice with intratracheal instillation of Escherichia coli LPS and found that mCAT mice had exaggerated NF-κB activation in the lungs, increased neutrophilic alveolitis, and greater lung inflammation/injury compared with WT mice. Additional studies using bone marrow chimeras revealed that this hyperinflammatory phenotype was mediated by immune/inflammatory cells. Mechanistic studies using bone marrow-derived macrophages (BMDMs) showed that LPS treatment induced a sustained increase in NF-κB activation and expression of NF-κB-dependent inflammatory mediators in mCAT BMDMs compared with WT BMDMs. Further investigations showed that cytoplasmic, but not mitochondrial, hydrogen peroxide levels were reduced in LPS-treated mCAT BMDMs. However, mCAT macrophages exhibited increased glycolytic and oxidative metabolism, coupled with increased ATP production and an increased intracellular NADH/NAD+ ratio compared with BMDMs from WT mice. Treatment of BMDMs with lactate increased the intracellular NADH/NAD+ ratio and upregulated NF-κB activation after LPS treatment, whereas treatment with a potent inhibitor of the mitochondrial pyruvate carrier (UK5099) decreased the NADH/NAD+ ratio and reduced NF-κB activation. Taken together, these findings point to an increased availability of reducing equivalents in the form of NADH as an important mechanism by which metabolic activity modulates inflammatory signaling through the NF-κB pathway.
  31. Curr Pharm Des. 2020 Jun 27.
      BACKGROUND: Copper (Cu) is a transition metal active in Fenton redox cycling from reduced Cu+ and H2O2, to oxidized Cu2+ and the hydroxyl radical (∙OH) highly reactive oxygen species (ROS). At homeostatic Cu levels, ROS promote cell proliferation, migration, angiogenesis, and wound repair. To limit ROS toxicity, cells use Cu-dependent chaperone proteins, Cu-binding ceruloplasmin, and Cu-modulated enzymes like superoxide dismutases (SOD) like SOD1 and SOD3 to scavenge excess superoxide anion which favours Cu+ reduction, and mitochondrial cytochrome c oxidase, important in aerobic energy production. Because Cu helps drive tumor cell proliferation by promoting growth factorindependent receptor tyrosine kinase signaling, and Cu-dependent MEK1 involved in oncogenic BRAFV600E signaling, further augmenting bioavailable Cu may promote ROS overproduction, cancer progression and eventually tumor cell death. For these reason, the following clinically approved copper chelators are being repurposed as anti-cancer agents: a) ammonium tetrathiomolybdate (TTM) used to treat Wilson's disease (copper overload) and Menkes disease (copper deficiency); b) Disulfiram (DSF), used against alcoholism, since it inhibits Aldehyde Dehydrogenase (ALDH1), enzyme important in ethanol detoxification, and a key target against cancer stem cells. Moreover, TTM and DSF are also relevant in cancer clinical trials, because they increase uptake of both Cu and Platinum (Pt)-containing anti-cancer drugs, since Pt and Cu share the same CTR1 copper transporter.PURPOSE: The majority of reports on Cu chelators dealt separately with either TTM, DSF or others. Here, we compare in parallel, the anti-cancer efficacy of low doses of TTM and DSF, asking whether they can be synergistic or antagonistic. The relevance of their unequal ROS inducing abilities, and their different behavior as ionophores is also addressed.
    SIGNIFICANCE: The potential of Cu chelators as repurposed anti-cancer drugs, should be greater in patients with higher endogenous Cu levels. Since Platinum and Cu share uptake receptors, the synergism by drugs containing these metals should not be under-estimated. The potential of disulfiram or its metabolically active Cu-containing form, to inhibit ALDH1-positive tumor cells is therapeutically very important.
    Keywords:  Cu chelation; Cu-MEK activation; disulfiram; oxaliplatin; tetrathiomolybdate
  32. Sci Rep. 2020 Jun 30. 10(1): 10618
      Mitochondria are dynamic organelles that undergo fusion and fission in response to various physiological and stress stimuli, which play key roles in diverse mitochondrial functions such as energy metabolism, intracellular signaling, and apoptosis. OPA1, a mitochondrial dynamin-like GTPase, is responsible for the inner membrane fusion of mitochondria, and the function of OPA1 is regulated by proteolytic cleavage in response to various metabolic stresses. Growing evidences highlighted the importance of mitochondrial adaptation in response to metabolic stimuli. Here, we demonstrated the role of p32/C1QBP in mitochondrial morphology by regulating OMA1-dependent proteolytic processing of OPA1. Genetic ablation of p32/C1QBP activates OMA1, cleaves OPA1, and leads mitochondrial fragmentation and swelling. The loss of p32/C1QBP decreased mitochondrial respiration and lipid utilization, sensitized cells to mitochondrial stress, and triggered a metabolic shift from oxidative phosphorylation to glycolysis, which were correlated with apoptosis in cancer cells and the inhibition of 3D-spheroid formation. These results suggest a unique regulation of cell physiology by mitochondria and provide a basis for a new therapeutic strategy for cancer.
  33. iScience. 2020 Jun 10. pii: S2589-0042(20)30443-0. [Epub ahead of print]23(7): 101257
      Immune escape contributes to viral persistence, yet little is known about human polyomaviruses. BK-polyomavirus (BKPyV) asymptomatically infects 90% of humans but causes premature allograft failure in kidney transplant patients. Despite virus-specific T cells and neutralizing antibodies, BKPyV persists in kidneys and evades immune control as evidenced by urinary shedding in immunocompetent individuals. Here, we report that BKPyV disrupts the mitochondrial network and membrane potential when expressing the 66aa-long agnoprotein during late replication. Agnoprotein is necessary and sufficient, using its amino-terminal and central domain for mitochondrial targeting and network disruption, respectively. Agnoprotein impairs nuclear IRF3-translocation, interferon-beta expression, and promotes p62/SQSTM1-mitophagy. Agnoprotein-mutant viruses unable to disrupt mitochondria show reduced replication and increased interferon-beta expression but can be rescued by type-I interferon blockade, TBK1-inhibition, or CoCl2-treatment. Mitochondrial fragmentation and p62/SQSTM1-autophagy occur in allograft biopsies of kidney transplant patients with BKPyV nephropathy. JCPyV and SV40 infection similarly disrupt mitochondrial networks, indicating a conserved mechanism facilitating polyomavirus persistence and post-transplant disease.
    Keywords:  Biological Sciences; Cell Biology; Immunology; Virology
  34. Onco Targets Ther. 2020 ;13 5375-5386
      Background: Carboplatin is a platinum-based chemotherapeutic drug that is commonly used as a treatment for ovarian cancer. However, high doses and repeated use of carboplatin usually reduce the sensitivity of cancer cells to the drug. There is an urgent need to develop strategies to increase the sensitivity of ovarian cancer cells to carboplatin.Materials and Methods: Quantitative reverse-transcriptase real-time PCR was used to detect miR-124-3p.1 levels in ovarian cancer tissues and cell lines. Transfection with miR-124-3p.1 and caveolin-1 (CAV1) was used for gain-of-function experiments. Western blot and immunoprecipitation assays were performed to evaluate the expression and function of CAV1, AKT, Bad, and Bcl-xl. Flow cytometry analysis was used to measure the apoptosis rates of SKOV3 and A2780 cells.
    Results: Expression levels of miR-124-3p.1 were decreased in ovarian cancer tissues and cell lines. Furthermore, overexpression of miR-124-3p.1 enhanced carboplatin-induced apoptotic cell death of ovarian cancer cell lines. Regarding the mechanism of this effect, we showed that CAV1 was the target of miR-124-3p.1 in ovarian cancer. Overexpression of miR-124-3p.1 suppressed the expression of CAV1, thereby reducing the activation of AKT and phosphorylation of Bad. As a result, the function of Bcl-xl was inhibited and carboplatin-induced mitochondrial apoptosis was enhanced.
    Conclusion: miR-124-3p.1 sensitizes carboplatin-induced mitochondrial apoptosis through suppression of CAV1 in ovarian cancer. Increasing miR-124-3p.1 expression may represent a novel strategy to improve carboplatin sensitivity in ovarian cancer.
    Keywords:  CAV1; carboplatin; miR-124-3p.1; ovarian cancer
  35. Cancers (Basel). 2020 Jun 26. pii: E1703. [Epub ahead of print]12(6):
      The monocarboxylate transporter 1 (MCT1) is a key element in tumor cell metabolism and inhibition of MCT1 with AZD3965 is undergoing clinical trials. We aimed to investigate nutrient fluxes associated with MCT1 inhibition by AZD3965 to identify possible biomarkers of drug action. We synthesized an 18F-labeled lactate analogue, [18F]-S-fluorolactate ([18F]-S-FL), that was used alongside [18F]fluorodeoxyglucose ([18F]FDG), and 13C-labeled glucose and lactate, to investigate the modulation of metabolism with AZD3965 in diffuse large B-cell lymphoma models in NOD/SCID mice. Comparative analysis of glucose and lactate-based probes showed a preference for glycolytic metabolism in vitro, whereas in vivo, both glucose and lactate were used as metabolic fuel. While intratumoral L-[1-13C]lactate and [18F]-S-FL were unchanged or lower at early (5 or 30 min) timepoints, these variables were higher compared to vehicle controls at 4 h following treatment with AZD3965, which indicates that inhibition of MCT1-mediated lactate import is reversed over time. Nonetheless, AZD3965 treatment impaired DLBCL tumor growth in mice. This was hypothesized to be a consequence of metabolic strain, as AZD3965 treatment showed a reduction in glycolytic intermediates and inhibition of the TCA cycle likely due to downregulated PDH activity. Glucose ([18F]FDG and D-[13C6]glucose) and lactate-based probes ([18F]-S-FL and L-[1-13C]lactate) can be successfully used as biomarkers for AZD3965 treatment.
    Keywords:  AZD3965; cancer metabolism; diffuse large B-cell lymphoma; glycolysis; lactate; metabolic flux; monocarboxylate transporter 1; positron emission tomography (PET)
  36. Arch Toxicol. 2020 Jun 30.
      Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.
    Keywords:  ECAR; ETC; HepG2; MMP; Mitochondria; RPTEC/TERT1; Seahorse
  37. Cells. 2020 Jun 28. pii: E1572. [Epub ahead of print]9(7):
      Pancreatic ductal adenocarcinoma (PDAC) is typically characterized by high chemoresistance and metastatic spread, features mainly attributable to cancer stem cells (CSCs). It is of central interest the characterization of CSCs and, in particular, the study of their metabolic features in order to selectively identify their peculiarities for an efficient therapeutic approach. In this study, CSCs have been obtained by culturing different PDAC cell lines with a specific growth medium. Cells were characterized for the typical stem/mesenchymal properties at short-, medium-, and long-term culture. Metabolomics, proteomics, analysis of oxygen consumption rate in live cells, and the effect of the inhibition of lactate transporter on cell proliferation have been performed to delineate the metabolism of CSCs. We show that gradually de-differentiated pancreatic cancer cells progressively increase the expression of both stem and epithelial-to-mesenchymal transition markers, shift their metabolism from a glycolytic to an oxidative one, and lastly gain a quiescent state. These quiescent stem cells are characterized by high chemo-resistance, clonogenic ability, and metastatic potential. Re-differentiation reverts these features, re-activating their proliferative capacity and glycolytic metabolism, which generally correlates with high aggressiveness. These observations add an important piece of knowledge to the comprehension of the biology of CSCs, whose metabolic plasticity could be exploited for the generation of promising and selective therapeutic approaches for PDAC patients.
    Keywords:  cancer metabolism; cancer stem cells; metabolic plasticity; pancreatic ductal adenocarcinoma; quiescence
  38. Circulation. 2020 Jun 29.
      Background: Increased fatty acid oxidation (FAO) has long been considered a culprit in the development of obesity/diabetes induced cardiomyopathy. However, enhancing cardiac FAO by removing the inhibitory mechanism of long-chain fatty acids transport into mitochondria via deletion of acetyl-CoA carboxylase 2 (ACC2) does not cause cardiomyopathy in non-obese mice, suggesting that high FAO is distinct from cardiac lipotoxicity. We hypothesize that cardiac pathology associated obesity is attributable to the imbalance of fatty acid supply and oxidation. Thus, we here seek to determine whether further increasing FAO by inducing ACC2 deletion prevents obesity induced cardiomyopathy, and if so, to elucidate the underlying mechanisms. Methods: We induced high FAO in adult mouse hearts by cardiac-specific deletion of ACC2 using a tamoxifen inducible model (ACC2 iKO). Control (Con) and ACC2 iKO mice were subjected to high fat diet (HFD) feeding for 24 weeks to induce obesity. Cardiac function, mitochondria function and mitophagy activity were examined. Results: Despite both Con and ACC2 iKO mice exhibiting similar obese phenotype, increasing FAO oxidation by deletion of ACC2 prevented HFD induced cardiac dysfunction, pathological remodeling as well as mitochondria dysfunction. Similarly, increasing FAO by knock down of ACC2 prevented palmitate induced mitochondria dysfunction and cardiomyocyte death in vitro. Furthermore, HFD suppressed mitophagy activity and caused damaged mitochondria to accumulate in the heart, which was partially attenuated in ACC2 iKO heart. Mechanistically, ACC2 iKO prevented HFD induced downregulation of parkin. During stimulation for mitophagy, mitochondria localized parkin was severely reduced in Con HFD-fed mouse heart, which was partially restored in ACC2 iKO HFD-fed mice. Conclusions: These data show that increasing cardiac FAO alone does not cause cardiac dysfunction but protect against cardiomyopathy in chronically obese mice. The beneficial effect of enhancing cardiac FAO in HFD induced obesity is mediated, in part, by maintenance of mitochondria function through regulating parkin mediated mitophagy. Our findings also suggest that targeting the parkin dependent mitophagy pathway could be an effective strategy against the development of obesity induced cardiomyopathy.
    Keywords:  Cardiomyopathy; Fatty acid oxidation; Mitophagy
  39. Cancers (Basel). 2020 Jun 29. pii: E1731. [Epub ahead of print]12(7):
      Colorectal cancer (CRC) is among the most frequent cancer entities worldwide. Multiple factors are causally associated with CRC development, such as genetic and epigenetic alterations, inflammatory bowel disease, lifestyle and dietary factors. During malignant transformation, the cellular energy metabolism is reprogrammed in order to promote cancer cell growth and proliferation. In this review, we first describe the main alterations of the energy metabolism found in CRC, revealing the critical impact of oncogenic signaling and driver mutations in key metabolic enzymes. Then, the central role of mitochondria and the tricarboxylic acid (TCA) cycle in this process is highlighted, also considering the metabolic crosstalk between tumor and stromal cells in the tumor microenvironment. The identified cancer-specific metabolic transformations provided new therapeutic targets for the development of small molecule inhibitors. Promising agents are in clinical trials and are directed against enzymes of the TCA cycle, including isocitrate dehydrogenase, pyruvate dehydrogenase kinase, pyruvate dehydrogenase complex (PDC) and α-ketoglutarate dehydrogenase (KGDH). Finally, we focus on the α-lipoic acid derivative CPI-613, an inhibitor of both PDC and KGDH, and delineate its anti-tumor effects for targeted therapy.
    Keywords:  CPI-613; TCA cycle; cancer metabolism; colorectal cancer; mitochondria; oncogenic signaling; targeted therapy; tumor microenvironment
  40. Trends Cancer. 2020 Jul;pii: S2405-8033(20)30084-4. [Epub ahead of print]6(7): 593-604
      Over the past decade, it has become apparent that metabolic reprogramming is a key event in tumor progression. The tumor microenvironment (TME) is a source of metabolites for tumor cells. Lipid-filled mature adipocytes are frequently found in proximity to invasive human tumors and release free fatty acids (FFAs) through lipolysis. These FFAs are taken up by tumor cells and used to promote tumor progression by mechanisms that include mitochondrial fatty acid oxidation (FAO). This review discusses recent advances in our understanding of this metabolic symbiosis between adipocytes and cancer cells and underlines the differences in this metabolic crosstalk between the various types of cancer and their localization.
    Keywords:  adipocyte; cancer metabolism; fatty acids; metabolic crosstalk
  41. Onco Targets Ther. 2020 ;13 5241-5255
      Background: The transfer of whole mitochondria has been demonstrated to be beneficial for treating breast cancer because it induces apoptosis and drug sensitivity; however, in vivo evidence of this benefit remains scant. The present study compared the transplantation of mitochondria with instinctive (Mito) and membrane-fused morphologies induced by Pep-1 conjugation (P-Mito) using a mouse model of triple-negative breast cancers.Materials and Methods: Mice with advanced severe immunodeficiency received orthotopic implantation of MDA-MB-231 human breast cancer cells followed by transplants of 5-bromo-2'-deoxyuridine (BrdU)-labeled Mito or P-Mito (200 μg [10 μg/μL]) through intratumoral injection at multiple points once a week for 4 weeks.
    Results: After 1 month of consecutive treatment, 8.2% and 14.2% of the BrdU-labeled mitochondria were preserved in tumors of the Mito and P-Mito groups, respectively. Both Pep-1 and P-Mito treatments reduced tumor weight (21.7% ± 2.43% vs 40.6% ± 2.28%) and led to marked inhibition of Ki67 staining and angiogenesis. However, only the P-Mito group exhibited obvious necrosis and DNA fragmentation accompanied by an altered tumor microenvironment, which included reduced oxidative stress and size of cancer-associated fibroblast populations and enhanced immune cell infiltration. Transmission electron microscopy images further revealed an elongated network of perinuclear mitochondria fused with a few peripheral mitochondria in the nonnecrotic area in the P-Mito group as well as increases in mitochondrial fusion proteins and parkin compared with mitochondrial fission proteins.
    Conclusion: In this study, the results of mitochondrial transplantation emphasized that the facilitation of mitochondrial fusion is a critical regulator in breast cancer therapy.
    Keywords:  MDA-MB-231; Pep-1; animal model of breast cancer; mitochondrial fusion; mitochondrial transplantation; tumor growth
  42. J Biol Chem. 2020 Jul 01. pii: jbc.AC120.014189. [Epub ahead of print]
      The transcription factor aryl hydrocarbon receptor (AHR) drives the expression of genes involved in detoxification pathways in cells exposed to pollutants and other small molecules. Moreover, AHR supports transcriptional programs that promote ribosome biogenesis and protein synthesis in cells stimulated to proliferate by the oncoprotein MYC. Thus, AHR is necessary for the proliferation of MYC-overexpressing cells. To define metabolic pathways in which AHR cooperates with MYC in supporting cell growth, here we used LC-MS-based metabolomics to examine the metabolome of MYC-expressing cells uponAHR knockdown. We found that AHR knockdown reduced lactate, S-lactoyl-glutathione,N-acetyl-L-alanine, 2-hydroxyglutarate, and uridine-5-monophosphate (UMP) levels. Using our previously obtained RNA-seq data, we found that AHR mediates the expression of the UMP-generating enzymes dihydroorotate dehydrogenase (quinone) (DHODH) and uridine monophosphate synthetase (UMPS), as well as lactate dehydrogenase A (LDHA), establishing a mechanism by which AHR regulates lactate and UMP production in MYC-overexpressing cells. AHR knockdown in glioblastoma cells also reduced the expression of LDHA (and lactate), DHODH,and UMPS, but did not affect UMP levels, likely due to compensatory mechanisms in these cells. Our results indicate that AHR contributes to the regulation of metabolic pathways necessary for the proliferation of transformed cells.
    Keywords:  Myc (c-Myc); aryl hydrocarbon receptor (AhR) (AHR); cancer; gene regulation; glioblastoma; glycolysis; metabolism; metabolomics; oncogene; pyrimidine
  43. Front Oncol. 2020 ;10 915
      Background: The aldehyde dehydrogenase 1 family member A3 (ALDH1A3) is a key enzyme associated with a variety of metabolic processes, including glucose metabolism. We recently uncovered that glucose metabolism played an essential role in promoting metastasis of pancreatic ductal adenocarcinoma (PDAC). As ALDH1A3 labels an aggressive subtype of PDAC, we hypothesized that ALDH1A3 functionally promoted PDAC metastasis via its metabolic effect on glucose metabolism. Methods: Expression of ALDH1A3 was detected in human PDAC tissues by immunohistochemistry. ALDH1A3 was knocked down or overexpressed in PDAC cells by either shRNA or overexpression vector. The functional roles of ALDH1A3 were characterized in vitro and in vivo. Transcriptional profiling via RNA-sequencing was used to explore the possible underlying molecular mechanisms. Glucose uptake, extracellular lactate, and ATP production were measured to access the metabolic influence of ALDH1A3 on PDAC cells. Results: ALDH1A3 was associated with poor prognosis in PDAC patients. Functionally, ALDH1A3 promoted PDAC metastasis in vitro and in vivo. Further studies revealed that ALDH1A3 activated PI3K/AKT/mTOR signaling pathway and its downstream target-PPARγ (peroxisome proliferator-activated receptor gamma). This led to increase the expression of HK2 (hexokinase 2), which subsequently enhanced the glycolysis in PDAC cells. Additionally, the pharmacological inhibition of PPARγ activity in ALDH1A3-positive cells impaired glycolytic genes expression, PI3K/AKT/mTOR activity and cellular glycolysis. Conclusions: ALDH1A3 promotes PDAC metastasis via its metabolic influence on glucose metabolism. PPARγ and its downstream PI3K/AKT/mTOR signaling pathway maybe involved in this process.
    Keywords:  ALDH1A3; HK2; PPARγ; glycolysis; pancreatic ductal adenocarcinoma; tumor metastasis
  44. ACS Sens. 2020 Jul 02.
      Remarkable variation between cell-free and cellular measurements of enzyme activity triggered the unmet need for developing tools for monitoring enzyme activity in living cells. Such tools will advance our understanding of the biological functions of enzymes and their potential impact on drug discovery. We report in this study a universal assay for monitoring ATP-generating enzymes in living cells using a self-assembled Tb3+ complex probe. Modulation of the rheological properties of cell culture media enabled shifting the lifetime of the Tb3+ complex in the presence of ATP from micro-to-millisecond range. Based on the response of the Tb3+ complex to ATP, cellular assays for 5 ATP-generating enzymes were developed. Remarkably, assessment of the activity of these enzymes in living cells is made possible for the first time. The pyruvate kinase M2 (PKM2) assay has been optimized for high-throughput screening (HTS) and further implemented in the identification of novel scaffolds as PKM2 inhibitors.
  45. Anal Chem. 2020 Jun 29.
      The ratio between the cytosolic concentrations of monocarboxylates lactate and pyruvate is a direct readout of the balance between glycolysis and mitochondrial oxidative metabolism. Current approaches to detect these metabolites do not allow detection of the lactate/pyruvate ratio in a single readout with high spatial/temporal resolution in living systems. Using a FRET-based screening strategy, we found that the orphan transcriptional factor LutR from Bacillus licheniformis is an endogenous sensor of the lactate/pyruvate ratio, suitable to be used as a binding moiety to develop a lactate/pyruvate ratio FRET-based genetically encoded indicator, Lapronic. The sensitivity of the indicator to lactate and pyruvate was characterized through changes in fluorescence FRET ratio and validated with isothermal titration calorimetry. Lapronic was insensitive to physiological pH and temperature and did not respond to structurally related molecules like acetate and β-hydroxybutyrate or cofactors NAD+ and NADH. Lapronic was expressed in HEK293 cells showing a homogeneous cytosolic localization and was also targeted to the mitochondrial matrix. A calibration protocol was designed to quantitatively assess the lactate/pyruvate ratio in intact mammalian cells. Purified protein from Escherichia coli showed a robust stability over time and found suitable for lactate/pyruvate ratio detection in biological samples. We envision that Lapronic will be of practical interest for basic and applied research.
  46. Trends Cancer. 2020 Jul;pii: S2405-8033(20)30106-0. [Epub ahead of print]6(7): 536-537
      Mounting evidence indicates that vitamins C and D are linked to tumor growth, but the relevance of vitamin B6 remains uncertain. In a recent study, Chen et al. demonstrate that pyridoxal kinase promotes vitamin B6 phosphorylation, producing the active form pyridoxal 5'-phosphate, which regulates two key metabolic enzymes required for acute myeloid leukemia (AML) cell growth.
  47. Sci Rep. 2020 Jul 02. 10(1): 10821
      Mitochondrial heteroplasmy, which fundamentally means intracellular heterogeneity of mitochondrial DNA (mtDNA), has been measured in a group of cells, regardless of intercellular heterogeneity. Ordinal methods for mitochondrial heteroplasmy cannot discriminate between an intercellular homogenic population composed of cells with similar intracellular heterogeneity for mtDNA and an intercellular heterogenic population composed of cells with different rates of mutated mtDNA. A high-throughput method to determine mitochondrial heteroplasmy in a single cell was developed by using droplet digital PCR with TaqMan polymerase in this study. This technique revealed that there are three different cell populations of cultured fibroblasts derived from patients with mitochondrial disease carrying a mutation in the mtDNA; cells with homoplasmy of either mutated or healthy mtDNA; and cells mixed with mutated and healthy mtDNA. The presence of intercellular heterogeneity, even in uniformed cultured fibroblasts, suggests that heterogeneity should exist among different kinds of cells. The diagnosis of intercellular heterogeneity with respect to mitochondrial heteroplasmy by this methodology could provide novel insight into developing a treatment strategy for mitochondrial diseases.
  48. Cancers (Basel). 2020 Jun 24. pii: E1670. [Epub ahead of print]12(6):
      Background: Cisplatin (CDDP) is commonly utilized in the treatment of advanced solid tumors including head and neck squamous cell carcinoma (HNSCC). Cisplatin response remains highly variable among individual tumors and development of cisplatin resistance is common. We hypothesized that development of cisplatin resistance is partially driven by metabolic reprogramming. Methods: Using a pre-clinical HNSCC model and an integrated approach to steady state metabolomics, metabolic flux and gene expression data we characterized the interaction between cisplatin resistance and metabolic reprogramming. Results: Cisplatin toxicity in HNSCC was driven by generation of intra-cellular oxidative stress. This was validated by demonstrating that acquisition of cisplatin resistance generates cross-resistance to ferroptosis agonists despite the fact that cisplatin itself does not trigger ferroptosis. Acquisition of cisplatin resistance dysregulated the expression of genes involved in amino acid, fatty acid metabolism and central carbon catabolic pathways, enhanced glucose catabolism and serine synthesis. Acute cisplatin exposure increased intra-tumoral levels of S-methyl-5-thiadenosine (MTA) precursors and metabotoxins indicative of generalized oxidative stress. Conclusions: Acquisition of cisplatin resistance is linked to metabolic recovery from oxidative stress. Although this portends poor effectiveness for directed metabolic targeting, it supports the potential for biomarker development of cisplatin effectiveness using an integrated approach.
    Keywords:  amino acid; cisplatin; fatty acid; ferroptosis; head and neck cancer; oxidative stress
  49. Proc Natl Acad Sci U S A. 2020 Jun 29. pii: 202000640. [Epub ahead of print]
      Calcium uptake by the mitochondrial calcium uniporter coordinates cytosolic signaling events with mitochondrial bioenergetics. During the past decade all protein components of the mitochondrial calcium uniporter have been identified, including MCU, the pore-forming subunit. However, the specific lipid requirements, if any, for the function and formation of this channel complex are currently not known. Here we utilize yeast, which lacks the mitochondrial calcium uniporter, as a model system to address this problem. We use heterologous expression to functionally reconstitute human uniporter machinery both in wild-type yeast as well as in mutants defective in the biosynthesis of phosphatidylethanolamine, phosphatidylcholine, or cardiolipin (CL). We uncover a specific requirement of CL for in vivo reconstituted MCU stability and activity. The CL requirement of MCU is evolutionarily conserved with loss of CL triggering rapid turnover of MCU homologs and impaired calcium transport. Furthermore, we observe reduced abundance and activity of endogenous MCU in mammalian cellular models of Barth syndrome, which is characterized by a partial loss of CL. MCU abundance is also decreased in the cardiac tissue of Barth syndrome patients. Our work raises the hypothesis that impaired mitochondrial calcium transport contributes to the pathogenesis of Barth syndrome, and more generally, showcases the utility of yeast phospholipid mutants in dissecting the phospholipid requirements of ion channel complexes.
    Keywords:  Barth syndrome; EMRE; cardiolipin; mitochondrial calcium uniporter (MCU); uniplex
  50. Cell Stem Cell. 2020 Jun 24. pii: S1934-5909(20)30272-1. [Epub ahead of print]
      Self-renewing embryonic stem cells (ESCs) respond to environmental cues by exiting pluripotency or entering a quiescent state. The molecular basis underlying this fate choice remains unclear. Here, we show that histone acetyltransferase MOF plays a critical role in this process through directly activating fatty acid oxidation (FAO) in the ground-state ESCs. We further show that the ground-state ESCs particularly rely on elevated FAO for oxidative phosphorylation (OXPHOS) and energy production. Mof deletion or FAO inhibition induces bona fide quiescent ground-state ESCs with an intact core pluripotency network and transcriptome signatures akin to the diapaused epiblasts in vivo. Mechanistically, MOF/FAO inhibition acts through reducing mitochondrial respiration (i.e., OXPHOS), which in turn triggers reversible pluripotent quiescence specifically in the ground-state ESCs. The inhibition of FAO/OXPHOS also induces quiescence in naive human ESCs. Our study suggests a general function of the MOF/FAO/OXPHOS axis in regulating cell fate determination in stem cells.
    Keywords:  FAO; MOF; cell fate decision; embryo development; epigenetics; quiescence; self-renewal; stem cell metabolism
  51. Sci Rep. 2020 Jun 30. 10(1): 10659
      In human blood, oxygen is mainly transported by red blood cells. Accordingly, the dissolved oxygen level in plasma is expected to be limited, although it has not been quantified yet. Here, by developing dedicated methods and tools, we determined that human plasma pO2 = 8.4 mmHg (1.1% O2). Oxygen solubility in plasma was believed to be similar to water. Here we reveal that plasma has an additional ascorbate-dependent oxygen-reduction activity. Plasma experimental oxygenation oxidizes ascorbate (49.5 μM in fresh plasma vs < 2 μM in oxidized plasma) and abolishes this capacity, which is restored by ascorbate supplementation. We confirmed these results in vivo, showing that the plasma pO2 is significantly higher in ascorbate-deficient guinea pigs (Ascorbateplasma < 2 μM), compared to control (Ascorbateplasma > 15 μM). Plasma low oxygen level preserves the integrity of oxidation-sensitive components such as ubiquinol. Circulating leucocytes are well adapted to these conditions, since the abundance of their mitochondrial network is limited. These results shed a new light on the importance of oxygen exposure on leucocyte biological study, in regards with the reducing conditions they encounter in vivo; but also, on the manipulation of blood products to improve their integrity and potentially improve transfusions' efficacy.
  52. Mitochondrial DNA A DNA Mapp Seq Anal. 2020 Jul 01. 1-5
      Leukemia is the most common childhood malignancy and studies had been carried out with promising revelations in its diagnosis and prognosis. However, majority of the studies are focused on nuclear alterations, while mitochondrial mutations are not well studied. Although there are studies of mitochondrial mutations in the adult leukemias, it does not represent the same for childhood malignancy. This is the first scientific report on the mtDNA mutational pattern of pediatric leukemic cases from a endogamous tribal population in Northeast India. ATP6 involved in the Complex V was found to be more altered with respect to the Non-synonymous variants. mtDNA variations in the non-coding region (D-Loop - g.152 T>C) and in the coding region (MT-ND2, g.4824 A>G, p.T119A) showed a maternal inheritance which could reveal a genetic predisposition with lower penetrance. D-Loop variant (g.152 T>C) could be a diagnostic marker in accordance with previous report but is in contrast to pertaining only in AML - M3 subtype rather was found across in myeloid malignancies.
    Keywords:  ATP6; D-loop; Mt-DNA; complex V; pediatric leukemia; tribal population