Electron Transfer Chain Complexes Research Articles

AMPK phosphorylation of FNIP1 (S220) controls mitochondrial function and muscle fuel utilization during exercise.

Exercise-induced activation of adenosine monophosphate-activated protein kinase (AMPK) and substrate phosphorylation modulate the metabolic capacity of mitochondria in skeletal muscle. However, the key effector(s) of AMPK and the regulatory mechanisms remain unclear. Here, we showed that AMPK phosphorylation of the folliculin interacting protein 1 (FNIP1) serine-220 (S220) controls mitochondrial function and muscle fuel utilization during exercise. Loss of FNIP1 in skeletal muscle resulted in increased mitochondrial content and augmented metabolic capacity, leading to enhanced exercise endurance in mice. Using skeletal muscle-specific nonphosphorylatable FNIP1 (S220A) and phosphomimic (S220D) transgenic mouse models as well as biochemical analysis in primary skeletal muscle cells, we demonstrated that exercise-induced FNIP1 (S220) phosphorylation by AMPK in muscle regulates mitochondrial electron transfer chain complex assembly, fuel utilization, and exercise performance without affecting mechanistic target of rapamycin complex 1-transcription factor EB signaling. Therefore, FNIP1 is a multifunctional AMPK effector for mitochondrial adaptation to exercise, implicating a mechanism for exercise tolerance in health and disease.

Oksipösedanin kaynaklı antikanser aktivitenin in siliko ve in vitro değerlendirilmesi: Mitotoksisite?

Aim: This study aims to evaluate the alterations in Oxypeucedanin (OXY)-mediated anticancer activity in different media. Second aim is to predict the affinity of OXY to electron transfer chain (ETC) complexes. Materials and Methods: MTT and LDH leakage assays were performed with OXY. Molecular docking studies were also conducted to predict the affinity of OXY to ETC complexes. Results: 250 µM OXY reduced viability in glucose media. ≥50 µM OXY decreased viability in galactose media. ≥50 µM OXY increased membrane disruption in galactose media. Molecular docking studies also showed that OXY might possess the capacity to bind to the inhibition sites of Complex I and IV. Conclusion: Galactose-conditioned media exacerbated the OXY-mediated cytotoxicity. Preliminary results suggested that mitotoxicity might take part in anticancer activity. Furthermore, OXY might cause ETC dysfunctions due to selective inhibition of Complex I and IV.

Targeting Mitochondrial Oxidative Stress: Potential Neuroprotective Therapy for Spinal Cord Injury.

Spinal cord injury (SCI) is a serious central nervous system (CNS) injury disease related to hypoxia-ischemia and inflammation. It is characterized by excessive reactive oxygen species (ROS) production, oxidative damage to nerve cells, and mitochondrial dysfunction. Mitochondria serve as the primary cellular origin of ROS, wherein the electron transfer chain complexes within oxidative phosphorylation frequently encounter electron leakage. These leaked electrons react with molecular oxygen, engendering the production of ROS, which culminates in the occurrence of oxidative stress. Oxidative stress is one of the common forms of secondary injury after SCI. Mitochondrial oxidative stress can lead to impaired mitochondrial function and disrupt cellular signal transduction pathways. Hence, restoring mitochondrial electron transport chain (ETC), reducing ROS production and enhancing mitochondrial function may be potential strategies for the treatment of SCI. This article focuses on the pathophysiological role of mitochondrial oxidative stress in SCI and evaluates in detail the neuroprotective effects of various mitochondrial-targeted antioxidant therapies in SCI, including both drug and non-drug therapy. The objective is to provide valuable insights and serve as a valuable reference for future research in the field of SCI.

Mouse embryonic stem cells embody organismal-level cold resistance

During hibernation, some mammals show low body temperatures (<10°C). Tissues from hibernators exhibit cold resistance even when the animal is not hibernating. Mice can also enter hypothermic fasting-induced torpor (FIT), but the cold resistance of FIT has never been related to their tissues. Here, we show that an inbred mouse STM2 exhibits lower body temperature during FIT than C57BL/6J or MYS/Mz. Thus, STM2 resists the cold more than other strains. Analysis of strain-specific mouse embryonic stem (ES) cells shows that STM2 ES cells are more cold-resistant than others and rely on the oxidative phosphorylation (OXPHOS) pathway but respire independently of the electron transfer chain complex I in the cold. We also found that the liver of STM2 uses OXPHOS more in cold than other strains. This study demonstrates that an organismal phenotype associated with torpor can be effectively studied in an invitro setup using mouse cells.

Counteracting Colon Cancer by Inhibiting Mitochondrial Respiration and Glycolysis with a Selective PKCδ Activator.

Metabolic reprogramming is a central hub in tumor development and progression. Therefore, several efforts have been developed to find improved therapeutic approaches targeting cancer cell metabolism. Recently, we identified the 7α-acetoxy-6β-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz) as a PKCδ-selective activator with potent anti-proliferative activity in colon cancer by stimulating a PKCδ-dependent mitochondrial apoptotic pathway. Herein, we investigated whether the antitumor activity of Roy-Bz, in colon cancer, could be related to glucose metabolism interference. The results showed that Roy-Bz decreased the mitochondrial respiration in human colon HCT116 cancer cells, by reducing electron transfer chain complexes I/III. Consistently, this effect was associated with downregulation of the mitochondrial markers cytochrome c oxidase subunit 4 (COX4), voltage-dependent anion channel (VDAC) and mitochondrial import receptor subunit TOM20 homolog (TOM20), and upregulation of synthesis of cytochrome c oxidase 2 (SCO2). Roy-Bz also dropped glycolysis, decreasing the expression of critical glycolytic markers directly implicated in glucose metabolism such as glucose transporter 1 (GLUT1), hexokinase 2 (HK2) and monocarboxylate transporter 4 (MCT4), and increasing TP53-induced glycolysis and apoptosis regulator (TIGAR) protein levels. These results were further corroborated in tumor xenografts of colon cancer. Altogether, using a PKCδ-selective activator, this work evidenced a potential dual role of PKCδ in tumor cell metabolism, resulting from the inhibition of both mitochondrial respiration and glycolysis. Additionally, it reinforces the antitumor therapeutic potential of Roy-Bz in colon cancer by targeting glucose metabolism.

SIRT5-related desuccinylation modification of AIFM1 protects against compression-induced intervertebral disc degeneration by regulating mitochondrial homeostasis

Mitochondrial dysfunction plays a major role in the development of intervertebral disc degeneration (IDD). Sirtuin 5 (SIRT5) participates in the maintenance of mitochondrial homeostasis through its desuccinylase activity. However, it is still unclear whether succinylation or SIRT5 is involved in the impairment of mitochondria and development of IDD induced by excessive mechanical stress. Our 4D label-free quantitative proteomic results showed decreased expression of the desuccinylase SIRT5 in rat nucleus pulposus (NP) tissues under mechanical loading. Overexpression of Sirt5 effectively alleviated, whereas knockdown of Sirt5 aggravated, the apoptosis and dysfunction of NP cells under mechanical stress, consistent with the more severe IDD phenotype of Sirt5 KO mice than wild-type mice that underwent lumbar spine instability (LSI) surgery. Moreover, immunoprecipitation-coupled mass spectrometry (IP-MS) results suggested that AIFM1 was a downstream target of SIRT5, which was verified by a Co-IP assay. We further demonstrated that reduced SIRT5 expression resulted in the increased succinylation of AIFM1, which in turn abolished the interaction between AIFM1 and CHCHD4 and thus led to the reduced electron transfer chain (ETC) complex subunits in NP cells. Reduced ETC complex subunits resulted in mitochondrial dysfunction and the subsequent occurrence of IDD under mechanical stress. Finally, we validated the efficacy of treatments targeting disrupted mitochondrial protein importation by upregulating SIRT5 expression or methylene blue (MB) administration in the compression-induced rat IDD model. In conclusion, our study provides new insights into the occurrence and development of IDD and offers promising therapeutic approaches for IDD.

Cancer-specific cytotoxicity of pyridinium-based ionic liquids by regulating hypoxia-inducible factor-1α-centric cancer metabolism

Owing to their unique properties and biological activities, ionic liquids (ILs) have attracted research interest in pharmaceutics and medicine. Hypoxia-inducible factor (HIF)− 1α is an attractive cancer drug target involved in cancer malignancy in the hypoxic tumor microenvironment. Herein, we report the inhibitory activity of ILs on the HIF-1α pathway and their mechanism of action. Substitution of a dimethylamino group on pyridinium reduced hypoxia-induced HIF-1α activation. It selectively inhibited the viability of the human colon cancer cell line HCT116, compared to that of the normal fibroblast cell line WI-38. These activities were enhanced by increasing the alkyl chain length in the pyridinium. Under hypoxic conditions, dimethylaminopyridinium reduced the accumulation of HIF-1α and its target genes without affecting the HIF1A mRNA level in cancer cells. It suppressed the oxygen consumption rate and ATP production by directly inhibiting electron transfer chain complex I, which led to enhanced intracellular oxygen content and oxygen-dependent degradation of HIF-1α under hypoxia. These results indicate that dimethylaminopyridinium suppresses the mitochondria and HIF-1α-dependent glucose metabolic pathway in hypoxic cancer cells. This study provides insights into the anticancer activity of pyridinium-based ILs through the regulation of cancer metabolism, making them promising candidates for cancer treatment.

Ginsenoside Rh2 stimulates the production of mitochondrial reactive oxygen species and induces apoptosis of cervical cancer cells by inhibiting mitochondrial electron transfer chain complex.

Ginsenoside Rh2 (G-Rh2) is a monomeric compound that extracted from ginseng and possesses anti-cancer activities both in vitro and in vivo. Previously, we reported that G-Rh2 induces apoptosis in HeLa cervical cancer cells and that the process was related to reactive oxygen species (ROS) accumulation and mitochondrial dysfunction. However, the upstream mechanisms of G-Rh2, along with its cellular targets, remain to be elucidated. In the present study, the Cell Counting Kit-8 assay, flow cytometry and Hoechst staining revealed that G-Rh2 significantly inhibited cell viability and induced apoptosis of cervical cancer cells. However, G-Rh2 was demonstrated to be non-toxic to End1/e6e7 cells. JC-1, rhodamine 123 staining, oxidative phosphorylation and glycolysis capacity assays demonstrated that G-Rh2 exposure caused an immediate decrease in mitochondrial transmembrane potential due to its inhibition of mitochondrial oxidative phosphorylation, as well as glycolysis, both of which reduced cellular ATP production. Western blotting and electron transport chain (ETC) activity assays revealed that G-Rh2 significantly inhibited the activity of ETC complexes I, III and V. Overexpression of ETC complex III partially significantly restored mitochondrial ROS and inhibited the apoptosis of cervical cancer cells induced by G-Rh2. The predicted results of binding energy in molecular docking, confirmed that G-Rh2 was highly likely to induce mitochondrial ROS production and promote cell apoptosis by targeting the ETC complex, especially for ETC complex III. Taken together, the present results revealed the potential anti-cervical cancer activity of G-Rh2 and provide direct evidence for the contribution of impaired ETC complex activity to cervical cancer cell death.

Protective effects of lycopene on mitochondrial oxidative injury and dysfunction in the liver of aflatoxin B1-exposed broilers

This study was conducted to investigate the effects of lycopene (LYC) on mitochondrial oxidative injury and dysfunction in the liver of aflatoxin B1 (AFB1)-exposed broilers. A total of 192 healthy 1-day-old male broilers were randomly divided into 3 groups with 8 replicates of 8 birds each. Birds in the 3 groups were fed basal diet (control), basal diet with 100 µg/kg AFB1, and basal diet with 100 µg/kg AFB1 and 200 mg/kg LYC, respectively. The experiment lasted 42 d. The results showed that AFB1 decreased average daily body weight gain (ADG), average daily feed intake, and gain to feed ratio (G :F) compared to the control group, the LYC supplementation increased ADG and G/F compared to AFB1 group (P < 0.05). Broilers in the AFB1 group had lower mitochondrial glutathione (mGSH) concentration and glutathione peroxidase (GSH-Px), manganese superoxide dismutase (MnSOD), and thioredoxin reductase activities, and higher hydrogen peroxide (H2O2) and reactive oxygen species (ROS) concentrations than the control group (P < 0.05). The LYC increased mGSH concentration and GSH-Px and MnSOD activities, and decreased H2O2 and ROS concentrations compared to AFB1 group (P < 0.05). Broilers fed the AFB1 diet showed increased mitochondrial swelling and decreased adenosine triphosphate concentration than the control group, and LYC had opposite effects (P < 0.05). The AFB1 decreased the activities of mitochondrial electron transfer chain (ETC) complexes I, II, III, and V, downregulated the mRNA expression levels of hepatic MnSOD, thioredoxin 2, thioredoxin reductase, peroxiredoxin-3, peroxisome proliferator-activated receptor γ coactivator 1α, nuclear respiratory factor 1, and mitochondrial transcription factor A compared with the control group (P < 0.05), and LYC increased activities of mitochondrial ETC complexes III and V, and upregulated mRNA expression levels of these genes in comparison to AFB1 group (P < 0.05). In conclusion, the LYC protected broilers from AFB1-induced liver mitochondrial oxidative injury and dysfunction by stimulating mitochondrial antioxidant capacity and maintaining mitochondrial biogenesis.

The Energy Status of Astrocytes Is the Achilles' Heel of eIF2B-Leukodystrophy.

Translation initiation factor 2B (eIF2B) is a master regulator of global protein synthesis in all cell types. The mild genetic Eif2b5(R132H) mutation causes a slight reduction in eIF2B enzymatic activity which leads to abnormal composition of mitochondrial electron transfer chain complexes and impaired oxidative phosphorylation. Previous work using primary fibroblasts isolated from Eif2b5(R132H/R132H) mice revealed that owing to increased mitochondrial biogenesis they exhibit normal cellular ATP level. In contrast to fibroblasts, here we show that primary astrocytes isolated from Eif2b5(R132H/R132H) mice are unable to compensate for their metabolic impairment and exhibit chronic state of low ATP level regardless of extensive adaptation efforts. Mutant astrocytes are hypersensitive to oxidative stress and to further energy stress. Moreover, they show migration deficit upon exposure to glucose starvation. The mutation in Eif2b5 prompts reactive oxygen species (ROS)-mediated inferior ability to stimulate the AMP-activated protein kinase (AMPK) axis, due to a requirement to increase the mammalian target of rapamycin complex-1 (mTORC1) signalling in order to enable oxidative glycolysis and generation of specific subclass of ROS-regulating proteins, similar to cancer cells. The data disclose the robust impact of eIF2B on metabolic and redox homeostasis programs in astrocytes and point at their hyper-sensitivity to mutated eIF2B. Thereby, it illuminates the central involvement of astrocytes in Vanishing White Matter Disease (VWMD), a genetic neurodegenerative leukodystrophy caused by homozygous hypomorphic mutations in genes encoding any of the 5 subunits of eIF2B.

Disrupting Mitochondrial Electron Transfer Chain Complex I Decreases Immune Checkpoints in Murine and Human Acute Myeloid Leukemic Cells.

Simple SummaryDespite all of the advancements made in recent years in the treatment of acute myeloid leukemia (AML), long-term survival is achieved by only 30–40% of AML patients. Thus, new therapeutic strategies are strongly needed. This work confirms the increase in oxidative phosphorylation upon cytarabine resistance in AML murine cells, reinforcing the interest of targeting it. In addition, it identifies a new role of the first complex of the electron transport chain (ETCI) in the regulation of the immune checkpoints PD-L1 and CD39 in murine and human leukemic cells. Thus, this work opens the door to the evaluation of ETCI inhibitors in combination with immunotherapy.Oxidative metabolism is crucial for leukemic stem cell (LSC) function and drug resistance in acute myeloid leukemia (AML). Mitochondrial metabolism also affects the immune system and therefore the anti-tumor response. The modulation of oxidative phosphorylation (OxPHOS) has emerged as a promising approach to improve the therapy outcome for AML patients. However, the effect of mitochondrial inhibitors on the immune compartment in the context of AML is yet to be explored. Immune checkpoints such as ectonucleotidase CD39 and programmed dead ligand 1 (PD-L1) have been reported to be expressed in AML and linked to chemo-resistance and a poor prognosis. In the present study, we first demonstrated that a novel selective electron transfer chain complex (ETC) I inhibitor, EVT-701, decreased the OxPHOS metabolism of murine and human cytarabine (AraC)-resistant leukemic cell lines. Furthermore, we showed that while AraC induced an immune response regulation by increasing CD39 expression and by reinforcing the interferon-γ/PD-L1 axis, EVT-701 reduced CD39 and PD-L1 expression in vitro in a panel of both murine and human AML cell lines, especially upon AraC treatment. Altogether, this work uncovers a non-canonical function of ETCI in controlling CD39 and PD-L1 immune checkpoints, thereby improving the anti-tumor response in AML.

Protective effect of grape seed extract and orlistat co-treatment against stroke: Effect on oxidative stress and energy failure

Ischemic stroke is a major health concern and a leading cause of mortality worldwide. Oxidative stress is an early event in the course of stroke inducing neuro-inflammation and cell death. Grape seed extract (GSE) is a natural phytochemical mixture exhibiting antioxidant, anti-inflammatory and neuroprotective properties. Orlistat (ORL) is an anti-obesity agent and a gastro-intestinal lipase inhibitor which showed recently beneficial effects on brain lipotoxicity. Recent studies reported the increase of lipase activity upon stroke which led us to investigate the neuroprotective effect of ORL on rat brain I/R injury as well as the putative synergism with GSE. I/R insult infarcted the brain parenchyma as assessed by TTC staining, induced an oxidative stress as revealed by increased lipoperoxidation along with alteration of antioxidant enzymes activities which was corrected using the cotreatment of ORL + GSE. I/R also disturbed the main metabolic pathways involved in brain fueling as glycolysis, neoglucogenesis, glycogenolysis, TCA cycle and electron transfer chain (ETC) complexes. These disturbances were also corrected with the cotreatment ORL + GSE which maintained energetic activities near to the control level. I/R also disrupted transition metals distribution, along with associated enzymes as tyrosinase, LDH or glutamine synthetase activities and induced hippocampal inflammation as revealed by glycogen depletion from dentate gyrus area along with depressed anti-inflammatory IL1β cytokine and increased pro-inflammatory CD68 antigen. Interestingly almost all I/R-induced disturbances were corrected either partially upon ORL and GSE on their own and the best neuroprotection was obtained in the presence of both drugs (ORL + GSE) enabling robust neuroprotection of the sub granular zone within hippocampal dentate gyrus area.

ZFHX3 knockdown dysregulates mitochondrial adaptations to tachypacing in atrial myocytes through enhanced oxidative stress and calcium overload.

To investigate the role of zinc finger homeobox 3 gene (ZFHX3) in tachypacing-induced mitochondrial dysfunction and explore its molecular mechanisms and potential as a therapeutic target in atrial fibrillation (AF). Through a bioluminescent assay, a patch clamp, confocal fluorescence and fluorescence microscopy, microplate enzyme activity assays and Western blotting, we studied ATP and ADP production, mitochondrial electron transfer chain complex activities, ATP-sensitive potassium channels (IKATP ), mitochondrial oxidative stress, Ca2+ content, and protein expression in control and ZFHX3 knockdown (KD) HL-1 cells subjected to 1 and 5-Hz pacing for 24hours. Compared with 1-Hz pacing, 5-Hz pacing increased ATP and ADP production, IKATP , phosphorylated adenosine monophosphate-activated protein kinase and inositol 1,4,5-triphosphate (IP3 ) receptor (IP3 R) protein expression. Tachypacing induced mitochondrial oxidative stress and Ca2+ overload in both cell types. Furthermore, under 1- and 5-Hz pacing, ZFHX3 KD cells showed higher IKATP , ATP and ADP production, mitochondrial oxidative stress and Ca2+ content than control cells. Under 5-Hz pacing, 2-aminoethoxydiphenyl borate (2-APB; 3μmol/L, an IP3 R inhibitor) and MitoTEMPO (10µmol/L, a mitochondria-targeted antioxidant) reduced ADP and increased ATP production in both cell types; however, only 2-APB significantly reduced mitochondrial Ca2+ overload in control cells. Under 5-Hz pacing, mitochondrial oxidative stress was significantly reduced by both MitoTEMPO and 2-APB and only by 2-APB in control and ZFHX3 KD cells respectively. ZFHX3 KD cells modulate mitochondrial adaptations to tachypacing in HL-1 cardiomyocytes through Ca2+ overload, oxidative stress and metabolic disorder. Targeting IP3 R signalling or oxidative stress could reduce AF.

Multiwalled carbon nanotubes inhibit cell migration and invasion by destroying actin cytoskeleton via mitochondrial dysfunction in ovarian cancer cells.

Objective: This study aimed to investigate the effects of multiwalled carbon nanotubes (MWCNTs) on cytotoxicity and tumor metastasis in ovarian cancer cells, and further explored its mechanism.Results: MWCNTs significantly inhibited cell viability and the clone number, increased the cell number of S phage, promoted cell apoptosis, as well as suppressed cell migration and invasion, and damaged the structure of actin cytoskeleton in a dose-dependent manner in SKOV3. Moreover, MWCNTs treatment obviously damaged the structure of actin cytoskeleton of SKOV3, and inhibited the activities of mitochondrial electron transfer chain complexes I-V.Conclusions: MWCNTs might influence the assembly of actin cytoskeleton by disrupting mitochondrial function, thereby inhibiting migration and invasion of SKOV3.Methods: The characterization of MWCNTs was analyzed by UV visible light absorption spectroscopy and transmission electron microscopy. SKOV3 cells were exposed to different doses of MWCNTs. Then, in vitro cytotoxicity of MWCNTs was evaluated by MTT assay, colony-forming assay, cell cycle, and cell apoptosis assay. Moreover, the effects of MWCNTs on cell migration and invasion as well as actin cytoskeleton were explored in SKOV3 cells. Furthermore, the mitochondrial membrane potential and the activities of mitochondrial electron transfer chain complexes I-V were measured.

Short Term Metformin Intervention Inhibits IL-6 and IL-1β Secretion and Prevents Acute Respiratory Distress Syndrome-Implications for COVID-19

Acute respiratory distress syndrome (ARDS) is a leading cause of COVID-19 death. Long-term metformin usage decreases COVID-19 mortality through an obscure mechanism unrelated to glycemic control. It is unclear whether and how short-term metformin-based intervention can block acute inflammation. We show that metformin inhibits IL-6 and IL-1β secretion from LPS or SARS-CoV-2 Spike protein primed macrophages undergoing NLRP3 inflammasome activation, a key to ARDS initiation. Reduced IL-6 production correlates with blunted NFAT and C/EBPβ/NFIL-6 activation, whereas inhibition of IL-1β secretion reflects interference with NLRP3 inflammasome activation. By targeting electron transfer chain complex 1, but independently of AMPK and mitophagy, metformin inhibits ATP-dependent mitochondrial DNA synthesis and blocks generation of oxidized mitochondrial DNA, a trigger of inflammasome assembly. Correspondingly, short-term metformin treatment abrogates LPS-induced ARDS, lung macrophage recruitment, damage and mortality. We suggest that metformin, a cheap and safe drug, can be used to prevent ARDS onset in COVID-19 patients.Funding: The UCSD Tissue Technology Shared Resource supported by a National Cancer Institute Cancer Center Support Grant (CCSG P30CA23100). M.A. was supported by NIH (R01AI072726). E.S-L. was supported by NIAMS (K01AR077111). Research was supported by NIH grant awards to M.K. and M.A. (U54CA260591), and M.K. (R01A1043477, P42ES010337), who is an American Cancer Research Society Professor and holds the Ben and Wanda Hildyard Chair for Mitochondrial and Metabolic Diseases. Conflict of Interest: M.K. is a founder of Elgia Pharmaceuticals and receives research support from Gossamer Bio and Jansen Pharmaceuticals. All other authors declare no competing interests.Ethical Approval: All mouse studies were conducted in accordance with UCSD and NIH guidelines and regulations for the housing and treatment of laboratory animals using protocols approved by the UCSD Institutional Animal Care and Use Committee.

Glioma cells are resistant to inflammation‑induced alterations of mitochondrial dynamics.

Accumulating evidence suggests that inflammation is present in solid tumors. However, it is poorly understood whether inflammation exists in glioma and how it affects the metabolic signature of glioma. By analyzing immunohistochemical data and gene expression data downloaded from bioinformatic datasets, the present study revealed an accumulation of inflammatory cells in glioma, activation of microglia, upregulation of proinflammatory factors (including IL-6, IL-8, hypoxia-inducible factor-1α, STAT3, NF-κB1 and NF-κB2), destruction of mitochondrial structure and altered expression levels of electron transfer chain complexes and metabolic enzymes. By monitoring glioma cells following proinflammatory stimulation, the current study observed a remodeling of their mitochondrial network via mitochondrial fission. More than half of the mitochondria presented ring-shaped or spherical morphologies. Transmission electron microscopic analyses revealed mitochondrial swelling with partial or total cristolysis. Furthermore, proinflammatory stimuli resulted in increased generation of reactive oxygen species, decreased mitochondrial membrane potential and reprogrammed metabolism. The defective mitochondria were not eliminated via mitophagy. However, cell viability was not affected, and apoptosis was decreased in glioma cells after proinflammatory stimuli. Overall, the present findings suggested that inflammation may be present in glioma and that glioma cells may be resistant to inflammation-induced mitochondrial dysfunction.

Melatonin Improves Mitochondrial Dynamics and Function in the Kidney of Zücker Diabetic Fatty Rats.

Obesity and associated diabetes (diabesity) impair kidney mitochondrial dynamics by augmenting fission and diminishing fusion, which results in mitochondrial and renal dysfunction. Based on available evidence, the antioxidant activities of melatonin may improve impaired renal mitochondrial function in obese diabetic animals by restoring the imbalanced dynamics through inhibiting fission and promoting fusion. Male Zücker diabetic fatty (ZDF) rats and lean littermates (ZL) were orally treated either with melatonin (10 mg/kg BW/day) (M-ZDF and M-ZL) or vehicle (C-ZDF and C-ZL) for 17 weeks. Kidney function was evaluated by measurement of total urine volume, proteinuria, creatinine clearance, and assessment of kidney mitochondrial dynamics and function. C-ZDF exhibited impaired dynamics and function of kidney mitochondria in comparison to C-ZL. Melatonin improved nephropathy of ZDF rats and modulated their mitochondrial dynamics by reducing expression of Drp1 fission marker and increasing that of fusion markers, Mfn2 and Opa1. Furthermore, melatonin ameliorated mitochondrial dysfunction by increasing respiratory control index and electron transfer chain complex IV activity. In addition, it lowered mitochondrial oxidative status. Our findings show that melatonin supplementation improves nephropathy likely via modulation of the mitochondrial fission/fusion balance and function in ZDF rats.

Abstract 1459: Selective killing of SMARCA4-deficient gynecologic cancers by mitochondria oxidative phosphorylation inhibitors

Abstract Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) and dedifferentiated carcinoma of the ovary or endometrium are rare but highly aggressive types of gynecologic cancers. We and others have recently demonstrated that genetic inactivation of SMARCA4, one of the two ATPases of the SWI/SNF chromatin remodeling complex, along with protein loss is the only recurrent somatic mutation of SCCOHT. Furthermore, SMARCA4 loss appears as a key event in the development of a subset of dedifferentiated carcinoma of the ovary, endometrium or other organs. These SMARCA4-deficient dedifferentiated tumors and SCCOHT usually lose the expression of SMARCA2, the alternative ATPase of the SWI/SNF complex. Through mining the publicly available genome-wide CRISPR screen database from the Broad Institute DepMap Project for about 500 cell lines, we identified that SMARCA4/A2-dual deficient ovarian cell lines are selectively sensitive to the genetic ablation of multiple components of mitochondria electron transfer chain (ETC). Using seahorse metabolism assays, we revealed that SCCOHT cells have reduced glycolysis in comparison to other ovarian cancer cells and utilize mitochondria respiration for energy production regardless of the availability of glucose. Accordingly, both SCCOHT and SMARCA4-deficient dedifferentiated ovarian carcinoma cell lines are remarkably more sensitive to several inhibitors of ETC complex I, and tigecycline, a selective inhibitor of the mitochondria ribosomal translation, than other ovarian cancer cells, such as SMARCA4-intact ovarian high-grade serous carcinoma cells and clear cell ovarian carcinoma cells regardless of the expression level of ARID1A, a SWI/SNF complex subunit frequently lost in clear cell ovarian carcinoma. Re-expression of SMARCA4 increased the expression of SLC2A1, encoding the glucose transporter GLUT1, and decreased the sensitivity of SCCOHT cells to ETC complex I inhibitors, suggesting that SMARCA4 loss may reduce the transport of glucose leading to reduced glycolysis and increased reliance on mitochondria respiration, which is currently under investigation. Therefore, our data suggest that selective targeting mitochondria respiratory complex function can be an effective strategy for SCCOHT and other SMARCA4-deficient dedifferentiated cancers of gynecologic tract or other organs. Citation Format: Yemin Wang, Dionzie Ong, Shary Yuting Chen, Eunice Li, Krystal Orlando, Elizabeth Raupach, Jennifer Ji, Bernard Weissman, Patrick Pirrotte, David Humtsman. Selective killing of SMARCA4-deficient gynecologic cancers by mitochondria oxidative phosphorylation inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1459.

Histone deacetylase 6 inhibitor ACY1215 ameliorates mitochondrial dynamic and function injury in hepatocytes by activating AMPK signaling pathway in acute liver failure mice.

Acute liver failure (ALF) is often accompanied by dynamic and functional disorders of mitochondria in hepatocytes. The histone deacetylase 6 inhibitor Rocilinostat (ACY1215) has a hepatoprotective effect. However, its protective effect on mitochondria of hepatocytes and its related mechanisms in ALF remain unknown. The purpose of the present study was to elucidate the protective effect of ACY1215 on mitochondrial of hepatocytes in ALF by regulating AMPK signaling pathway. LPS and D-Gal were used to induce ALF model in C57BL/6 mice. D-Gal and TNF-α were applied in L02 cells as model group. ACY1215 was administered to the mice or culture cells before the model' s establishment as ACY1215 group. The normal group in mice and L02 cells was not given any drug intervention. ACY1215 improves liver histological and functional changes in ALF model mice. Compared with normal group, the expression of p-AMPK and p-ACC proteins was decreased in model group. ACY1215 activated the AMPK signaling pathway with an increase of p-AMPK and p-ACC proteins level in model group. ACY1215 treatment decreased levels of mitochondrial fission proteins DRP1 and FIS1, and enhanced levels of mitochondrial fusion proteins MFN1, MFN2 and OPA1 in models. MtDNA copies in model group was decreased compared with normal group, but ACY1215 elevated the mtDNA copies in models. Mitochondrial respiratory electron transfer chain Complex I-III and citrate synthase (CS) activities in model group were decreased compared with normal group, but ACY1215 treatment enhanced these activities in model group. ACY1215 protects against dynamic disorders and dysfunction of mitochondria in hepatocytes in ALF by activating AMPK signaling pathway.

New screening system using Twist1 promoter activity identifies dihydrorotenone as a potent drug targeting cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells in tumor microenvironments. These cells strongly support tumor progression and are considered to be potent therapeutic targets. Therefore, drugs targeting CAFs have been developed, but most of them have failed in clinical trials. The discovery of additional drugs to inactivate or eliminate CAFs is thus essential. In this study, we developed a high-throughput screening system to find anti-CAF drugs using reporter cells that express Twist1 promoter-GFP. This screening system uses the activity of the Twist1 promoter as an indicator of CAF activation because Twist1 is known to be a central player in CAF activation. Using this screening system, we found that dihydrorotenone (DHR), an inhibitor of electron transfer chain complex 1 in mitochondria, can effectively deactivate CAFs. DHR-treated CAFs exhibited reduced expression of CAF-enriched markers, decreased capability of collagen gel contraction, and impaired ability to engage in tumor-promoting activities, such as facilitating the proliferation and colonization of cancer cells. Furthermore, conditioned media from DHR-treated CAFs attenuated tumor progression in mice grafted with MNK28 cells. In conclusion, DHR can be considered as a candidate drug targeting CAFs.