bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2020‒03‒01
thirty-one papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Theranostics. 2020 ;10(5): 2141-2157
      Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a malignant disease with a poor prognosis. One prominent aspect of PDAC that contributes to its aggressive behavior is its altered cellular metabolism. The aim of this study was to characterize the oncogenic effects of ubiquinol-cytochrome c reductase core protein I (UQCRC1), a key component of mitochondrial complex III, in PDAC development and to assess its potential as a therapeutic target for PDAC. Experimental Design: The expression of UQCRC1 in human PDAC tissues and p48-Cre/p53Flox/WT/LSL-KrasG12D (KPC) mouse pancreatic intraepithelial neoplasias (PanINs) was determined by immunohistochemistry. The role of UQCRC1 in promoting PDAC growth was evaluated in vitro in PANC-1 and CFPAC-1 cells and in vivo in transplanted mouse models of PDAC. Extracellular flux and RNA-Seq analyses were applied to investigate the mechanism of UQCRC1 in the regulation of mitochondrial metabolism and PDAC cell growth. The therapeutic potential of UQCRC1 in PDAC was assessed by knockdown of UQCRC1 using an RNA interference approach. Results: UQCRC1 expression showed a gradual increase during the progression from PanIN stages to PDAC in KPC mice. Elevated expression of UQCRC1 was observed in 72.3% of PDAC cases and was correlated with poor prognosis of the disease. UQCRC1 promoted PDAC cell growth in both in vitro experiments and in vivo subcutaneous and orthotopic mouse models. UQCRC1 overexpression resulted in increased mitochondrial oxidative phosphorylation (OXPHOS) and ATP production. The overproduced ATP was released into the extracellular space via the pannexin 1 channel and then functioned as an autocrine or paracrine agent to promote cell proliferation through the ATP/P2Y2-RTK/AKT axis. UQCRC1 knockdown or ATP release blockage could effectively inhibit PDAC growth. Conclusion: UQCRC1 has a protumor function and may serve as a potential prognostic marker and therapeutic target for PDAC.
    Keywords:  Extracellular ATP; Mitochondrial Oxidative Phosphorylation; Pancreatic Ductal Adenocarcinoma; UQCRC1
  2. Cell Physiol Biochem. 2020 Feb 27. 54(2): 211-229
      BACKGROUND/AIMS: Mitochondrial ATP synthase, in addition to being involved in ATP synthesis, is involved in permeability transition pore (PTP) formation, which precedes apoptosis in mammalian cells and programmed cell death in yeast. Mutations in genes encoding ATP synthase subunits cause neuromuscular disorders and have been identified in cancer samples. PTP is also involved in pathology. We previously found that in Saccharomyces cerevisiae, two mutations in ATP synthase subunit a (atp6-P163S and atp6-K90E, equivalent to those detected in prostate and thyroid cancer samples, respectively) in the OM45-GFP background affected ROS and calcium homeostasis and delayed yeast PTP (yPTP) induction upon calcium treatment by modulating the dynamics of ATP synthase dimer/oligomer formation. The Om45 protein is a component of the porin complex, which is equivalent to mammalian VDAC. We aimed to investigate yPTP function in atp6-P163S and atp6-K90E mutants lacking the e and g dimerization subunits of ATP synthase.METHODS: Triple mutants with the atp6-P163S or atp6-K90E mutation, the OM45-GFP gene and deletion of the TIM11 gene encoding subunit e were constructed by crossing and tetrad dissection. In spores capable of growing, the original atp6 mutations reverted to wild type, and two compensatory mutations, namely, atp6-C33S-T215C, were selected. The effects of these mutations on cellular physiology, mitochondrial morphology, bioenergetics and permeability transition (PT) were analyzed by fluorescence and electron microscopy, mitochondrial respiration, ATP synthase activity, calcium retention capacity and swelling assays.
    RESULTS: The atp6-C33S-T215C mutations in the OM45-GFP background led to delayed growth at elevated temperature on both fermentative and respiratory media and increased sensitivity to high calcium ions concentration or hydrogen peroxide in the medium. The ATP synthase activity was reduced by approximately 50% and mitochondrial network was hyperfused in these cells grown at elevated temperature. The atp6-C33S-T215C stabilized ATP synthase dimers and restored the yPTP properties in Tim11∆ cells. In OM45-GFP cells, in which Tim11 is present, these mutations increased the fraction of swollen mitochondria by up to 85% vs 60% in the wild type, although the time required for calcium release doubled.
    CONCLUSION: ATP synthase subunit e is essential in the S. cerevisiae atp6-P163S and atp6-K90E mutants. In addition to subunits e and g, subunit a is critical for yPTP induction and conduction. The increased yPTP conduction decrease the S. cerevisiae cell fitness.
    Keywords:  Mitochondria; Permeability transition pore; ATP synthase; ATP6; Subunit a; S. cerevisiae
  3. Biol Chem. 2020 Feb 01. pii: /j/bchm.just-accepted/hsz-2020-0120/hsz-2020-0120.xml. [Epub ahead of print]
      Mitochondria are multifaceted metabolic organelles and adapt dynamically to various developmental transitions and environmental challenges. The metabolic flexibility of mitochondria is provided by alterations in the mitochondrial proteome and is tightly coupled to changes in the shape of mitochondria. Mitochondrial proteases are emerging as important post-translational regulators of mitochondrial plasticity. The i-AAA protease YME1L, an ATP-dependent proteolytic complex in the mitochondrial inner membrane, coordinates mitochondrial biogenesis and dynamics with the metabolic output of mitochondria. mTORC1 dependent lipid signalling drives proteolytic rewiring of mitochondria by YME1L. While the tissue-specific loss of YME1L in mice is associated with heart failure, disturbed eye development and axonal degeneration in the spinal cord, YME1L activity supports growth of pancreatic ductal adenocarcinoma cells. YME1L thus represents a key regulatory protease determining mitochondrial plasticity and metabolic reprogramming and is emerging as a promising therapeutic target.
    Keywords:  Lipin1; YME1L; cancer; i-AAA protease; mTORC1; mitochondria; mitochondrial plasticity; mitochondrial proteases
  4. Int J Mol Sci. 2020 Feb 25. pii: E1555. [Epub ahead of print]21(5):
      : Mitochondrial respiratory chain supercomplexes (RCS), particularly, the respirasome, which contains complexes I, III, and IV, have been suggested to participate in facilitating electron transport, reducing the production of reactive oxygen species (ROS), and maintaining the structural integrity of individual electron transport chain (ETC) complexes. Disassembly of the RCS has been observed in Barth syndrome, neurodegenerative and cardiovascular diseases, diabetes mellitus, and aging. However, the physiological role of RCS in high energy-demanding tissues such as the heart remains unknown. This study elucidates the relationship between RCS assembly and cardiac function. Adult male Sprague Dawley rats underwent Langendorff retrograde perfusion in the presence and absence of ethanol, isopropanol, or rotenone (an ETC complex I inhibitor). We found that ethanol had no effects on cardiac function, whereas rotenone reduced heart contractility, which was not recovered when rotenone was excluded from the perfusion medium. Blue native polyacrylamide gel electrophoresis revealed significant reductions of respirasome levels in ethanol- or rotenone-treated groups compared to the control group. In addition, rotenone significantly increased while ethanol had no effect on mitochondrial ROS production. In isolated intact mitochondria in vitro, ethanol did not affect respirasome assembly; however, acetaldehyde, a byproduct of ethanol metabolism, induced dissociation of respirasome. Isopropanol, a secondary alcohol which was used as an alternative compound, had effects similar to ethanol on heart function, respirasome levels, and ROS production. In conclusion, ethanol and isopropanol reduced respirasome levels without any noticeable effect on cardiac parameters, and cardiac function is not susceptible to moderate reductions of RCS.
    Keywords:  ethanol; heart; mitochondria; respirasome; respiratory chain supercomplexes
  5. J Biol Chem. 2020 Feb 24. pii: jbc.RA119.011902. [Epub ahead of print]
      Mitochondrial oxidative phosphorylation (OXPHOS) and cellular workload are tightly balanced by the key cellular regulator, calcium (Ca2+). Current models assume that cytosolic Ca2+ regulates workload and that mitochondrial Ca2+ uptake precedes activation of matrix dehydrogenases, thereby matching OXPHOS substrate supply to ATP demand. Surprisingly, knock-out (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice results in only minimal phenotypic changes and does not alter OXPHOS. This implies that adaptive activation of mitochondrial dehydrogenases by intramitochondrial Ca2+ cannot be the exclusive mechanism for OXPHOS control. We hypothesized that cytosolic Ca2+, but not mitochondrial matrix Ca2+, may adapt OXPHOS to workload by adjusting the rate of pyruvate supply from the cytosol to the mitochondria. Here, we studied the role of malate aspartate shuttle (MAS)-dependent substrate supply on OXPHOS responses to changing Ca2+ concentrations in isolated brain and heart mitochondria, synaptosomes, fibroblasts, and thymocytes from wild-type (WT) and MCU KO mice, and the isolated working rat heart. Our results indicate that extramitochondrial Ca2+ controls up to 85% of maximal pyruvate-driven OXPHOS rates, mediated by the activity of the complete MAS, and that intramitochondrial Ca2+ accounts for the remaining 15%. Of note, the complete MAS as applied here, included besides its classical NADH oxidation reaction the generation of cytosolic pyruvate. Part of this largely neglected mechanism has previously been described as the "mitochondrial gas pedal". Its implementation into OXPHOS control models integrates seemingly contradictory results and warrants a critical reappraisal of metabolic control mechanisms in health and disease.
    Keywords:  bioenergetics; calcium; malate-aspartate shuttle; mitochondria; mitochondrial calcium uniporter; mouse; respiratory chain
  6. Oxid Med Cell Longev. 2020 ;2020 8956504
      Benzalkonium chloride (BAC) is currently the most commonly used antimicrobial preservative in ophthalmic solutions, nasal sprays, and cosmetics. However, a large number of clinical and experimental investigations showed that the topical administration of BAC-containing eye drops could cause a variety of ocular surface changes, from ocular discomfort to potential risk for future glaucoma surgery. BAC-containing albuterol may increase the risk of albuterol-related systemic adverse effects. BAC, commonly present in personal care products, in cosmetic products can induce irritation and dose-dependent changes in the cell morphology. The cationic nature of BAC (it is a quaternary ammonium) suggests that one of the major targets of BAC in the cell may be mitochondria, the only intracellular compartment charged negatively. However, the influence of BAC on mitochondria has not been clearly understood. Here, the effects of BAC on energy parameters of rat liver mitochondria as well as on yeast cells were examined. BAC, being a "weaker" uncoupler, potently inhibited respiration in state 3, diminished the mitochondrial membrane potential, caused opening of the Ca2+/Pi-dependent pore, blocked ATP synthesis, and promoted H2O2 production by mitochondria. BAC triggered oxidative stress and mitochondrial fragmentation in yeast cells. BAC-induced oxidative stress in mitochondria and yeast cells was almost totally prevented by the mitochondria-targeted antioxidant SkQ1; the protective effect of SkQ1 on mitochondrial fragmentation was only partial. Collectively, these data showed that BAC acts adversely on cell bioenergetics (especially on ATP synthesis) and mitochondrial dynamics and that its prooxidant effect can be partially prevented by the mitochondria-targeted antioxidant SkQ1.
  7. Biosci Trends. 2020 Feb 25.
      The roots of Angelica dahurica have long been used as a traditional medicine in Korea to treat various diseases such as toothache and cold. In this study, we investigated the effect of ethanol extract from the roots of this plant on metastatic melanoma, a highly aggressive skin cancer, in B16F10 melanoma cells and B16F10 cell inoculated-C57BL/6 mice. Our results showed that the ethanol extracts of Angelicae dahuricae Radix (EEAD) suppressed cell growth and induced apoptotic cell death in B16F10 cells. EEAD also activated the mitochondria-mediated intrinsic apoptosis pathway, with decreased mitochondrial membrane potential, and increased production of intracellular reactive oxygen species and ration of Bax/Bcl-2 expression. Furthermore, EEAD reduced the migration, invasion, and colony formation of B16F10 cells through the reduced expression and activity of matrix metalloproteinase (MMP)-2 and -9. In addition, in vivo results demonstrated that oral administration of EEAD inhibited lactate dehydrogenase activity, hepatotoxicity, and nephrotoxicity without weight loss in B16F10 cell inoculated-mice. Importantly, EEAD was able to markedly suppress lung hypertrophy, the incidence of B16F10 cells lung metastasis, and the expression of tumor necrosis factor-alpha in lung tissue. Taken together, our findings suggest that EEAD may be useful for managing metastasis and growth of malignant cancers, including melanoma.
    Keywords:  Angelica dahurica; B16F10 cells; apoptosis; invasion; lung metastasis
  8. Nat Commun. 2020 Feb 26. 11(1): 1050
      Organisms respond to tissue damage through the upregulation of protective responses which restore tissue structure and metabolic function. Mitochondria are key sources of intracellular oxidative metabolic signals that maintain cellular homeostasis. Here we report that tissue and cellular wounding triggers rapid and reversible mitochondrial fragmentation. Elevated mitochondrial fragmentation either in fzo-1 fusion-defective mutants or after acute drug treatment accelerates actin-based wound closure. Wounding triggered mitochondrial fragmentation is independent of the GTPase DRP-1 but acts via the mitochondrial Rho GTPase MIRO-1 and cytosolic Ca2+. The fragmented mitochondria and accelerated wound closure of fzo-1 mutants are dependent on MIRO-1 function. Genetic and transcriptomic analyzes show that enhanced mitochondrial fragmentation accelerates wound closure via the upregulation of mtROS and Cytochrome P450. Our results reveal how mitochondrial dynamics respond to cellular and tissue injury and promote tissue repair.
  9. Int J Biochem Cell Biol. 2020 Feb 22. pii: S1357-2725(20)30049-2. [Epub ahead of print] 105732
      Cisplatin is one of the most widely used anticancer agents for patients with tongue squamous cell carcinoma (TSCC), but its efficacy is limited by chemoresistance. Accumulated evidence has demonstrated that reactive oxygen species (ROS) plays a critical role in multiple tumor chemotherapy resistance. In the present study, we aimed to investigate the role of ROS in cisplatin resistance of TSCC and explore its underlying molecular mechanism in vitro. Our results showed that pre-treatment with ROS scavenger N-acetylcysteine reduced cisplatin-induced cytotoxicity, autophagy, and apoptosis in TSCC cells. Down-regulation of intracellular ROS attenuated apoptosis and autophagy of TSCC cisplatin-resistant CAL27/CDDP cells by reversing the inhibition of p38MAPK/mTOR pathway. Taken together, these findings suggest that down-regulation of intracellular ROS reduces the cytotoxicity of cisplatin by inhibiting apoptosis and autophagy in TSCC cells involving p38MAPK/mTOR mediated pathway. Low intracellular ROS levels may be one of the main mechanisms of cisplatin resistance in TSCC.
    Keywords:  Apoptosis; Autophagy; Chemoresistance; Cisplatin; ROS
  10. Sci Rep. 2020 Feb 27. 10(1): 3603
      The mitochondrial mutator mouse is a well-established model of premature aging. In addition to accelerated aging, these mice develop hypertrophic cardiomyopathy at ~13 months of age, presumably due to overt mitochondrial dysfunction. Despite evidence of bioenergetic disruption within heart mitochondria, there is little information about the underlying changes to the mitochondrial proteome that either directly underly or predict respiratory insufficiency in mutator mice. Herein, nLC-MS/MS was used to interrogate the mitochondria-enriched proteome of heart and skeletal muscle of aged mutator mice. The mitochondrial proteome from heart tissue was then correlated with respiratory conductance data to identify protein biomarkers of respiratory insufficiency. The majority of downregulated proteins in mutator mitochondria were subunits of respiratory complexes I and IV, including both nuclear and mitochondrial-encoded proteins. Interestingly, the mitochondrial-encoded complex V subunits, were unchanged or upregulated in mutator mitochondria, suggesting a robustness to mtDNA mutation. Finally, the proteins most strongly correlated with respiratory conductance were PPM1K, NDUFB11, and C15orf61. These results suggest that mitochondrial mutator mice undergo a specific loss of mitochondrial complexes I and IV that limit their respiratory function independent of an upregulation of complex V. Additionally, the role of PPM1K in responding to mitochondrial stress warrants further exploration.
  11. Oxid Med Cell Longev. 2020 ;2020 3631272
      A new mitochondria-targeted probe MitoCLox was designed as a starting compound for a series of probes sensitive to cardiolipin (CL) peroxidation. Fluorescence microscopy reported selective accumulation of MitoCLox in mitochondria of diverse living cell cultures and its oxidation under stress conditions, particularly those known to cause a selective cardiolipin oxidation. Ratiometric fluorescence measurements using flow cytometry showed a remarkable dependence of the MitoCLox dynamic range on the oxidation of the sample. Specifically, MitoCLox oxidation was induced by low doses of hydrogen peroxide or organic hydroperoxide. The mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decyltriphenyl-phosphonium (SkQ1), which was shown earlier to selectively protect cardiolipin from oxidation, prevented hydrogen peroxide-induced MitoCLox oxidation in the cells. Concurrent tracing of MitoCLox oxidation and membrane potential changes in response to hydrogen peroxide addition showed that the oxidation of MitoCLox started without a delay and was complete during the first hour, whereas the membrane potential started to decay after 40 minutes of incubation. Hence, MitoCLox could be used for splitting the cell response to oxidative stress into separate steps. Application of MitoCLox revealed heterogeneity of the mitochondrial population; in living endothelial cells, a fraction of small, rounded mitochondria with an increased level of lipid peroxidation were detected near the nucleus. In addition, the MitoCLox staining revealed a specific fraction of cells with an increased level of oxidized lipids also in the culture of human myoblasts. The fraction of such cells increased in high-density cultures. These specific conditions correspond to the initiation of spontaneous myogenesis in vitro, which indicates that oxidation may precede the onset of myogenic differentiation. These data point to a possible participation of oxidized CL in cell signalling and differentiation.
  12. Onco Targets Ther. 2020 ;13 1187-1198
      Purpose: To explore the regulatory effect of HMGB1 upon hypoxia-induced mitochondrial biogenesis in pancreatic cancer PANC1/CFPAC1 cells.Methods: After a down-regulation of HMGB1 expression by lentivirus-mediated RNAi, the effect of knocking down HMGB1 on hypoxia-induced mitochondrial biogenesis was examined. NRF-1/TFAM expression, mtDNA copy number, ATP content and mitochondrial number/morphology in hypoxia-treated pancreatic cancer cells were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, mtDNA and ATP assay kits and electron microscopy, respectively. Cell proliferation was measured by MTS assay. And protein and acetylation levels of PGC-1α and SIRT1 activity were detected by Western blot, immunoprecipitation (IP) and SIRT1 activity kit.
    Results: Hypoxia enhanced the expressions of NRF-1/TFAM, boosted mtDNA copy number and ATP content and increased the number of mitochondria in pancreatic cancer cells while induction was suppressed by a knockdown of HMGB1. Knocking down HMGB1 expression lowered hypoxia-induced PGC-1α/SIRT1 expression and activity, phosphorylation of AMPK. PGC-1α over-expression by a plasmid transfection failed to boost mtDNA copy number or ATP content in HMGB1-knockdown cells. A knockdown of HMGB1 attenuated hypoxia with AICAR (an AMPK activator)-induced expression of NRF-1, TFAM, PGC-1α, SIRT1 and the proteins of complexes Ⅰ& Ⅲ and reduced the acetylation level of PGC-1α/SIRT1 activity. Additionally, SRT1720 (a SIRT1 activator)-induced elevation in SIRT1 activity boosted hypoxia-induced PGC-1α deacetylation, except in HMGB1-knockdown cells.
    Conclusion: As a novel regulator of mitochondrial biogenesis via AMPK/SIRT1 pathway under hypoxia, HMGB1 may become a potential drug target for therapeutic interventions in pancreatic cancer.
    Keywords:  AMPK/SIRT1 pathway; HMGB1; PGC-1α; mitochondrial biogenesis; pancreatic cancer
  13. Onco Targets Ther. 2019 ;12 11715-11727
      Purpose: Cutaneous squamous cell carcinoma (cSCC) is the second most common form of skin cancer and its incidence continues to rise yearly. Photodynamic therapy (PDT) is a non-invasive form of cancer therapy, which utilizes the combined action of a photosensitizer, light, and oxygen molecules to selectively cause cellular damage to tumor cells. Vitamin K3 (VitK3) has been shown to induce apoptosis and inhibit the growth of tumor cells in humans. The purpose of this study was to determine the effect of VitK3 and ultraviolet radiation B (UVB) on oxidative damage, proliferation and apoptosis of A431 cells.Methods: CCK-8 assay was used to detect cell proliferation; Hoechst staining, TUNEL assay and flow cytometry analysis were used to detect apoptosis. Western Blot was perfomed to measure the expression of apoptosis-related proteins. Flow cytometry analysis was employed to detect the reactive oxygen species (ROS) levels and mitochondrial membrane potential. Finally, the role of VitK3 in combination with UVB on the proliferation and apoptosis of A431 cells was investigated using mice xenograft models.
    Results: We found that the co-treatment of VitK3 combined with UVB more significantly inhibited the growth and proliferation of A431 cells than either VitK3 or UVB alone. Hoechst 33258 staining and flow cytometry analysis revealed that apoptosis was more pronounced in the VitK3-UVB group compared to the VitK3 and UVB groups. Moreover, flow cytometry analysis showed that ROS and the depolarization of the mitochondrial membrane potential were higher in all the co-treatment groups compared to the control, VitK3, and UVB groups. The VitK3-UVB group exhibited a significantly lower tumor growth rate in mouse xenograft models.
    Conclusion: This study reveals that VitK3 combined with UVB inhibits the growth and induces apoptosis of A431 cells in vitro and suppresses tumor growth and promotes apoptosis of cSCC in vivo.
    Keywords:  depolarization; oxidative; photodynamic; photosensitizer
  14. Cancers (Basel). 2020 Feb 20. pii: E493. [Epub ahead of print]12(2):
      Mitochondrial transcription factor A (TFAM) is required for mitochondrial DNA replication and transcription, which are essential for mitochondrial biogenesis. Previous studies reported that depleting mitochondrial functions by genetic deletion of TFAM impaired autophagic activities. However, the underlying mechanisms remain largely unknown. In the current study, we identified that knockdown of TFAM repressed the synthesis of autophagy bio-marker LC3-II in tumor cells and decreased the expression of phosphatidyl-serine decarboxylase (PISD). Besides, downregulation of PISD with siRNA reduced the level of LC3-II, indicating that depletion of TFAM retarded autophagy via inhibiting PISD expression. Furthermore, it was found that the tumor repressor p53 could stimulate the transcription and expression of PISD by binding the PISD enhancer. Additionally, the protein stability and transcriptional activity of p53 in TFAM knockdown tumor cells was attenuated, and this was associated with decreased acetylation, especially the acetylation of lysine 382 of p53. Finally, we identified that TFAM knockdown increased the NAD+/NADH ratio in tumor cells. This led to the upregulation of Sirtuin1 (SIRT1), a NAD-dependent protein deacetylase, to deacetylate p53 and attenuated its transcriptional activation on PISD. In summary, our study discovered a new mechanism regarding disturbed autophagy in tumor cells with mitochondrial dysfunction due to the depletion of TFAM.
    Keywords:  LC3-II; PISD; TFAM; autophagy; p53; p53 acetylation
  15. Int J Mol Sci. 2020 Feb 21. pii: E1468. [Epub ahead of print]21(4):
      The voltage-dependent anion-selective channels (VDACs), which are also known as eukaryotic porins, are pore-forming proteins, which allow for the passage of ions and small molecules across the outer mitochondrial membrane (OMM). They are involved in complex interactions that regulate organelle and cellular metabolism. We have recently reported the post-translational modifications (PTMs) of the three VDAC isoforms purified from rat liver mitochondria (rVDACs), showing, for the first time, the over-oxidation of the cysteine residues as an exclusive feature of VDACs. Noteworthy, this peculiar PTM is not detectable in other integral membrane mitochondrial proteins, as defined by their elution at low salt concentration by a hydroxyapatite column. In this study, the association of tryptic and chymotryptic proteolysis with UHPLC/High Resolution nESI-MS/MS, allowed for us to extend the investigation to the human VDACs. The over-oxidation of the cysteine residues, essentially irreversible in cell conditions, was as also certained in VDAC isoforms from human cells. In human VDAC2 and 3 isoforms the permanently reduced state of a cluster of close cysteines indicates the possibility that disulfide bridges are formed in the proteins. Importantly, the detailed oxidative PTMs that are found in human VDACs confirm and sustain our previous findings in rat tissues, claiming for a predictable characterization that has to be conveyed in the functional role of VDAC proteins within the cell. Data are available via ProteomeXchange with identifier PXD017482.
    Keywords:  Cysteine over-oxidation; Orbitrap Fusion Tribrid; hydroxyapatite; mitochondria; mitochondrial intermembrane space; outer mitochondrial membrane; post-translational modification
  16. Curr Opin Cell Biol. 2020 Feb 24. pii: S0955-0674(20)30007-7. [Epub ahead of print]63 162-173
      The lipids that make up biological membranes tend to be the forgotten molecules of cell biology. The paucity of data on these important entities likely reflects the difficulties of studying and understanding their biological roles, rather than revealing a lack of importance. Indeed, the lipid composition of biological membranes has a profound impact on a diverse array of cellular processes. The focus of this review is on the effects of different lipid classes on the function of mitochondria, particularly bioenergetics, in health and disease.
  17. Aging Cell. 2020 Feb 27. e13127
      Age-related changes at the cellular level include the dysregulation of metabolic and signaling pathways. Analyses of blood leukocytes have revealed a set of alterations that collectively lower their ability to fight infections and resolve inflammation later in life. We studied the transcriptomic, epigenetic, and metabolomic profiles of monocytes extracted from younger adults and individuals over the age of 65 years to map major age-dependent changes in their cellular physiology. We found that the monocytes from older persons displayed a decrease in the expression of ribosomal and mitochondrial protein genes and exhibited hypomethylation at the HLA class I locus. Additionally, we found elevated gene expression associated with cell motility, including the CX3CR1 and ARID5B genes, which have been associated with the development of atherosclerosis. Furthermore, the downregulation of two genes, PLA2G4B and ALOX15B, which belong to the arachidonic acid metabolism pathway involved in phosphatidylcholine conversion to anti-inflammatory lipoxins, correlated with increased phosphatidylcholine content in monocytes from older individuals. We found age-related changes in monocyte metabolic fitness, including reduced mitochondrial function and increased glycose consumption without the capacity to upregulate it during increased metabolic needs, and signs of increased oxidative stress and DNA damage. In conclusion, our results complement existing findings and elucidate the metabolic alterations that occur in monocytes during aging.
    Keywords:  DNA methylation; aging; glucose metabolism; monocytes; phosphatidylcholines; transcriptome
  18. Antioxidants (Basel). 2020 Feb 19. pii: E171. [Epub ahead of print]9(2):
      Obesity and diabetes are independent risk factors for cardiovascular diseases, and they are associated with the development of a specific cardiomyopathy with elevated myocardial oxygen consumption (MVO2) and impaired cardiac efficiency. Although the pathophysiology of this cardiomyopathy is multifactorial and complex, reactive oxygen species (ROS) may play an important role. One of the major ROS-generating enzymes in the cardiomyocytes is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), and many potential systemic activators of NOX2 are elevated in obesity and diabetes. We hypothesized that NOX2 activity would influence cardiac energetics and/or the progression of ventricular dysfunction following obesity. Myocardial ROS content and mechanoenergetics were measured in the hearts from diet-induced-obese wild type (DIOWT) and global NOK2 knock-out mice (DIOKO) and in diet-induced obese C57BL/6J mice given normal water (DIO) or water supplemented with the NOX2-inhibitor apocynin (DIOAPO). Mitochondrial function and ROS production were also assessed in DIO and DIOAPO mice. This study demonstrated that ablation and pharmacological inhibition of NOX2 both improved mechanical efficiency and reduced MVO2 for non-mechanical cardiac work. Mitochondrial ROS production was also reduced following NOX2 inhibition, while cardiac mitochondrial function was not markedly altered by apocynin-treatment. Therefore, these results indicate a link between obesity-induced myocardial oxygen wasting, NOX2 activation, and mitochondrial ROS.
    Keywords:  NADPH-oxidase; ROS; cardiac efficiency; metabolism; myocardial oxygen consumption; obesity
  19. Exp Ther Med. 2020 Mar;19(3): 2179-2187
      The aim of the current study was to investigate luteolin-induced apoptosis and the molecular mechanisms underlying it in HT29 cells. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess the cytotoxicity of luteolin on HT29 cells, and a dichloro-dihydro-fluorescein diacetate assay was used to measure cellular levels of reactive oxygen species (ROS). The effects of luteolin on the mitochondrial membrane potential were also evaluated. Bax and Bcl-2 mRNA expression were determined using reverse transcription-quantitative PCR. Additionally, western blot analysis was performed to assess changes in cytochrome c and caspase-3 protein expression. Localization of nuclear factor erythroid 2-related factor 2 (Nrf2) in the nucleus was also assessed using immunofluorescence. Luteolin exhibited cytotoxicity on HT29 cells in a time- and concentration-dependent manner. Additionally, ROS production was indicated to be increased and ROS scavenging was decreased, which resulted in a significant increase in the levels of ROS in the cells. The mitochondrial membrane potential was indicated to decrease following luteolin treatment. At the molecular level, luteolin significantly increased the mRNA expression of Bax and the protein expression of cytochrome c, caspase-3, p47phox and p22phox. The results revealed that luteolin decreased Bcl-2 protein expression and inhibited the nuclear localization of Nrf2. In conclusion, the current study indicated that luteolin inhibited HT29 cell proliferation and induced apoptosis via the mitochondrial pathway.
    Keywords:  HT29 cells; luteolin; mitochondrial apoptosis; nuclear factor erythroid 2-related factor 2/antioxidant responsive element
  20. Sci Rep. 2020 Feb 26. 10(1): 3528
      MitoBlue is a fluorescent bisamidine that can be used to easily monitor the changes in mitochondrial degradation processes in different cells and cellular conditions. MitoBlue staining pattern is exceptional among mitochondrial dyes and recombinant fluorescent probes, allowing the dynamic study of mitochondrial recycling in a variety of situations in living cells. MitoBlue is a unique tool for the study of these processes that will allow the detailed characterization of communication between mitochondria and lysosomes.
  21. Anal Chem. 2020 Feb 26.
      Real-time imaging and quantification of adenosine triphosphate (ATP) dynamics in living cells are important for understanding relationship between energy metabolism and cell functions. However, few synthetic fluorescent probes have been reported to tackle this challenge due to lack of accurate fluorescence readout and suitable response concentration. In this work, we developed a ratiometric fluorescent probe (Rh6G-ACFPN) for quantitatively monitoring the fluctuation of mitochondrial ATP in living cells. Rh6G-ACFPN selectively and reversibly responds to ATP with an ideal dissociation constant (Kd) of 4.72 mM (3-10 mM: the range of mitochondrial ATP concentrations). Live-cell imaging allows us to directly monitor the dynamic changes of mitochondrial ATP in high temporal resolution. Moreover, for the first time, mitochondrial ATP in normal and cancer cells lines was successfully quantified and discriminated. These results demonstrate the versatility of Rh6G-ACFPN as a useful imaging tool to elucidate the function of mitochondrial ATP in living cells.
  22. Nat Commun. 2020 Feb 26. 11(1): 1048
      The membrane deforming dynamin family members MxA and MxB are large GTPases that convey resistance to a variety of infectious viruses. During viral infection, Mx proteins are known to show markedly increased expression via an interferon-responsive promoter to associate with nuclear pores. In this study we report that MxB is an inner mitochondrial membrane GTPase that plays an important role in the morphology and function of this organelle. Expression of mutant MxB or siRNA knockdown of MxB leads to fragmented mitochondria with disrupted inner membranes that are unable to maintain a proton gradient, while expelling their nucleoid-based genome into the cytoplasm. These findings implicate a dynamin family member in mitochondrial-based changes frequently observed during an interferon-based, anti-viral response.
  23. J Natl Cancer Inst. 2020 Feb 25. pii: djaa024. [Epub ahead of print]
      BACKGROUND: Neuroblastoma is a childhood malignancy that arises from the developing sympathetic nervous system. Although mitochondrial dysfunctions have been implicated in the pathophysiology of neuroblastoma, the role of mitochondrial DNA (mtDNA) has not been extensively investigated.METHODS: 2,404 Caucasian children diagnosed with neuroblastoma and 9,310 ancestry-matched controls were recruited at the Children's Hospital of Philadelphia. The mtDNA haplogroups were identified from SNP array data of two independent cohorts. We conducted a case-control study to explore potential associations of mtDNA haplogroups with the susceptibility of neuroblastoma. The genetic effect of neuroblastoma was measured by odds ratios of mitochondrial haplogroups. All tests were two-sided.
    RESULTS: Haplogroup K was statistically significantly associated with reduced risk of neuroblastoma in the discovery cohort consisting 1,474 cases and 5,699 controls (odds ratio 0.72, 95%CI 0.57-0.90, P = 0.005). The association was replicated in an independent cohort (odds ratio 0.69, 95%CI 0.53-0.92, P = 0.01) of 930 cases and 3,611 controls. Pooled analysis was performed by combing the two data sets. The association remained highly statistically significant after correction for multiple testing (odds ratio 0.71, 95%CI 0.59-0.84, P = 1.96 ✕ 10-4, Pcorrected = 0.002). Further analysis focusing on neuroblastoma subtypes indicated haplogroup K was more associated with high-risk neuroblastoma (odds ratio 0.57, 95%CI 0.43-0.76, P = 1.46 ✕ 10-4) than low-risk and intermediate-risk neuroblastoma.
    CONCLUSIONS: Haplogroup K is an independent genetic factor associated with reduced risk of developing neuroblastoma in European descents. These findings provide new insights into the genetic basis of neuroblastoma, implicating mitochondrial DNA encoded proteins in the etiology of neuroblastoma.
  24. Biochem Biophys Res Commun. 2020 Feb 19. pii: S0006-291X(20)30320-X. [Epub ahead of print]
      Berberine (BBR), a natural isoquinoline alkaloid, has been shown to be a promising therapeutic agent for colorectal cancer (CRC), but the molecular mechanism remains unclear. Here, we used mass spectrometry-based label-free proteomics to explore the potential targets of BBR in CRC cells. Comprehensive proteomic profiles demonstrated that of 8051 identified proteins, 503 and 277 differentially expressed proteins (DEPs) were screened out of CACO2 and LOVO cells, respectively. 83 DEPs were overlapped and most of these were down-regulated. A pathway enrichment analysis pinpointed mitochondrial translation, respiratory electron transport and the citric acid (TCA) cycle as biological effectors. The data of proteomics was subsequently confirmed by citrate synthase (CS), Tu translation elongation factor (TUFM), pentatricopeptide repeat domain 3 (PTCD3) and mitochondrial ribosomal protein L48 (MRPL 48) protein measurement. CS protein expression in CRC cells and tissues was higher than it was in normal specimens. Additionally, forcible downregulation of CS led to remarkable cell proliferation inhibition. Taken together, we concluded that the anticancer effects of BBR are attributable to mitochondrial protein synthesis, TCA and respiratory electron transport inhibition and that CS might be a useful therapeutic target in CRC treatment.
    Keywords:  Berberine; Colorectal cancer; Label-free; Mechanism; Proteomics
  25. Aging (Albany NY). 2020 Feb 23. 12
      Numerous genetic manipulations that extend lifespan in mice have been discovered over the past two decades, the most robust of which has arguably been the down regulation of growth hormone (GH) signaling. However, while decreased GH signaling has been associated with improved health and lifespan, many of the underlying physiological changes and molecular mechanisms associated with GH signaling have yet to be elucidated. To this end, we have completed the first transcriptomic and metabolomic study on long-lived growth hormone releasing hormone knockout (GHRH-KO) and wild-type mice in brown adipose tissue (transcriptomics) and blood serum (metabolomics). We find that GHRH-KO mice have increased transcript levels of mitochondrial and amino acid genes with decreased levels of extracellular matrix genes. Concurrently, mitochondrial metabolites are differentially regulated in GHRH-KO. Furthermore, we find a strong signal of genotype-by-sex interactions, suggesting the sexes have differing physiological responses to GH deficiency. Overall, our results point towards a strong influence of mitochondrial metabolism in GHRH-KO mice which potentially is tightly intertwined with their extended lifespan phenotype.
    Keywords:  aging; growth hormone; metabolite; mouse; transcriptomics
  26. Cell. 2020 Feb 25. pii: S0092-8674(20)30156-2. [Epub ahead of print]
      Mammalian tissues engage in specialized physiology that is regulated through reversible modification of protein cysteine residues by reactive oxygen species (ROS). ROS regulate a myriad of biological processes, but the protein targets of ROS modification that drive tissue-specific physiology in vivo are largely unknown. Here, we develop Oximouse, a comprehensive and quantitative mapping of the mouse cysteine redox proteome in vivo. We use Oximouse to establish several paradigms of physiological redox signaling. We define and validate cysteine redox networks within each tissue that are tissue selective and underlie tissue-specific biology. We describe a common mechanism for encoding cysteine redox sensitivity by electrostatic gating. Moreover, we comprehensively identify redox-modified disease networks that remodel in aged mice, establishing a systemic molecular basis for the long-standing proposed links between redox dysregulation and tissue aging. We provide the Oximouse compendium as a framework for understanding mechanisms of redox regulation in physiology and aging.
    Keywords:  ROS; aging; cysteine; proteomics; reactive oxygen species
  27. Aging Cell. 2020 Feb 28. e13123
      Growth hormone receptor knockout (GHRKO) mice are remarkably long-lived and have improved glucose homeostasis along with altered energy metabolism which manifests through decreased respiratory quotient (RQ) and increased oxygen consumption (VO2 ). Short-term exposure of these animals to increased environmental temperature (eT) at 30°C can normalize their VO2 and RQ. We hypothesized that increased heat loss in the diminutive GHRKO mice housed at 23°C and the consequent metabolic adjustments to meet the increased energy demand for thermogenesis may promote extension of longevity, and preventing these adjustments by chronic exposure to increased eT will reduce or eliminate their longevity advantage. To test these hypotheses, GHRKO mice were housed at increased eT (30°C) since weaning. Here, we report that contrasting with the effects of short-term exposure of adult GHRKO mice to 30°C, transferring juvenile GHRKO mice to chronic housing at 30°C did not normalize the examined parameters of energy metabolism and glucose homeostasis. Moreover, despite decreased expression levels of thermogenic genes in brown adipose tissue (BAT) and elevated core body temperature, the lifespan of male GHRKO mice was not reduced, while the lifespan of female GHRKO mice was increased, along with improved glucose homeostasis. The results indicate that GHRKO mice have intrinsic features that help maintain their delayed, healthy aging, and extended longevity at both 23°C and 30°C.
    Keywords:  GHRKO; growth hormone receptor; lifespan; metabolism; temperature; thermogenesis
  28. Aging (Albany NY). 2020 Feb 25. 12
      Healthy aging depends on a complex gene-environment network that is ultimately reflected in the expression of different proteins. We aimed to perform a comparative analysis of the plasma proteome of healthy centenarians (n=9, 5 women, age range 100-103 years) with a notably preserved ambulatory capacity (as a paradigm of 'successful' aging), and control individuals who died from a major age-related disease before the expected life expectancy (n=9, 5 women, age range: 67-81 years), and while having impaired ambulatory capacity (as a paradigm of 'unsuccessful' aging). We found that the expression of 49 proteins and 86 pathways differed between the two groups. Overall, healthy centenarians presented with distinct expression of proteins/pathways that reflect a healthy immune function, including a lower pro-inflammatory status (less 'inflammaging' and autoimmunity) and a preserved humoral immune response (increased B cell-mediated immune response). Compared with controls, healthy centenarians also presented with a higher expression of proteins involved in angiogenesis and related to enhanced intercellular junctions, as well as a lower expression of proteins involved in cardiovascular abnormalities. The identification of these proteins/pathways might provide new insights into the biological mechanisms underlying the paradigm of healthy aging.
    Keywords:  elderly; healthy aging; immune system; proteomics; senescence
  29. Biochem Pharmacol. 2020 Feb 20. pii: S0006-2952(20)30097-6. [Epub ahead of print] 113869
      The mitochondrial chaperone TRAP1 has been involved in several mitochondrial functions, and modulation of its expression/activity has been suggested to play a role in the metabolic reprogramming distinctive of cancer cells. TRAP1 posttranslational modifications, i.e. phosphorylation, can modify its capability to bind to different client proteins and modulate its oncogenic activity. Recently, it has been also demonstrated that TRAP1 is S-nitrosylated at Cys501, a redox modification associated with its degradation via the proteasome. Here we report molecular dynamics simulations of TRAP1, together with analysis of long-range structural communication, providing a model according to which Cys501 S-nitrosylation induces conformational changes to distal sites in the structure of the protein. The modification is also predicted to alter open and closing motions for the chaperone function. By means of colorimetric assays and site directed mutagenesis aimed at generating C501S variant, we also experimentally confirmed that selective S-nitrosylation of Cys501 decreases ATPase activity of recombinant TRAP1. Coherently, C501S mutant was more active and conferred protection to cell death induced by staurosporine. Overall, our results provide the first in silico, in vitro and cellular evidence of the relevance of Cys501 S-nitrosylation in TRAP1 biology.
  30. Int J Colorectal Dis. 2020 Feb 25.
      PURPOSE: To evaluate the effect of metformin as a treatment for the mortality of colorectal cancer (CRC) patients with type 2 diabetes mellitus (T2DM).METHODS: We searched Medline, PubMed, EMBASE, Clinical (, and the Cochrane Collaboration Library from inception to November 2019. To analyze the relationship between metformin and the overall mortality, specific mortality, and sex differences in CRC patients with T2DM, hazard ratios (HRs) with 95% confidence intervals (CIs) were used. Egger's test and Begg's test were used to assess publication bias.
    RESULTS: We included 8 cohort studies in our meta-analysis. CRC patients with T2DM treated with metformin had a lower overall mortality than CRC patients with T2DM who did not receive metformin (HR = 0.80, 95% CI 0.67-0.95). There was no significant difference in CRC-specific mortality between CRC patients with T2DM who used metformin and those who did not (HR = 0.84, 95% CI 0.65-1.08). However, females had a lower CRC-specific mortality among CRC patients with T2DM than males (HR = 0.63, 95% CI 0.41-0.97).
    CONCLUSION: Metformin reduced the overall mortality of CRC patients with T2DM. Moreover, female CRC patients with T2DM using metformin had lower CRC-specific mortality than male CRC patients with T2DM.
    Keywords:  Colorectal cancer; Meta-analysis; Metformin; Mortality; Sex difference; T2DM
  31. World J Oncol. 2020 Feb;11(1): 23-32
      Background: The present study aimed to review the use of hypoglycemic drugs and clinicopathological data in breast cancer patients with type 2 diabetes mellitus (T2DM), and to investigate the effect of metformin on the clinicopathological features of breast cancer in patient with T2DM.Methods: Eighty-nine patients with breast cancer hospitalized in the Second Affiliated Hospital of Xi'an Jiaotong University from January 2012 to December 2014 were included. Thirty-three patients were on metformin (metformin group) and 56 patients were on control group. Streptavidin-peroxidase (SP) method was used to quantify protein expression of molecular markers (estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2)), molecular markers of proliferation (Ki-67 and epidermal growth factor receptor (EGFR)) and epithelial-mesenchymal transition (EMT) molecular markers (matrix metalloproteinase-2 (MMP-2), E-cadherin and downstream N-cadherin). Fluorescence in situ hybridization was used to detect HER-2 (+ and ++).
    Results: The rate of lymph node metastasis and the level of Ki-67/MMP-2 in the metformin group were significantly lower than those in the control group (P < 0.05). The ratio of luminal pattern in metformin group was higher than that in the control group (P < 0.05). However, there were no differences in the parameters of age, duration of diabetes, body mass index, tumor size, histological grade of cancer and clinical pathological features between the two groups. No significant difference was observed in the expressions of ER, PR, HER-2, EGFR, E-cadherin, N-cadherin and the recurrence rate between two groups.
    Conclusions: Metformin is associated with luminal breast cancer and can inhibit breast cancer invasion and metastasis in some cases. It may be associated with EMT and is beneficial to the prognosis of breast cancer.
    Keywords:  Breast cancer; Epithelial-mesenchymal transition; Metformin; Molecular typing; Type 2 diabetes mellitus