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Science Advances Nov 2020Mitochondria-derived reactive oxygen species (mROS) are required for the survival, proliferation, and metastasis of cancer cells. The mechanism by which mitochondrial...
Mitochondria-derived reactive oxygen species (mROS) are required for the survival, proliferation, and metastasis of cancer cells. The mechanism by which mitochondrial metabolism regulates mROS levels to support cancer cells is not fully understood. To address this, we conducted a metabolism-focused CRISPR-Cas9 genetic screen and uncovered that loss of genes encoding subunits of mitochondrial complex I was deleterious in the presence of the mitochondria-targeted antioxidant mito-vitamin E (MVE). Genetic or pharmacologic inhibition of mitochondrial complex I in combination with the mitochondria-targeted antioxidants, MVE or MitoTEMPO, induced a robust integrated stress response (ISR) and markedly diminished cell survival and proliferation in vitro. This was not observed following inhibition of mitochondrial complex III. Administration of MitoTEMPO in combination with the mitochondrial complex I inhibitor phenformin decreased the leukemic burden in a mouse model of T cell acute lymphoblastic leukemia. Thus, mitochondrial complex I is a dominant metabolic determinant of mROS-dependent cellular fitness.
PubMed: 33148642
DOI: 10.1126/sciadv.abb7272 -
Immunity Jan 2021Several classes of antibiotics have long been known to have beneficial effects that cannot be explained strictly on the basis of their capacity to control the infectious...
Several classes of antibiotics have long been known to have beneficial effects that cannot be explained strictly on the basis of their capacity to control the infectious agent. Here, we report that tetracycline antibiotics, which target the mitoribosome, protected against sepsis without affecting the pathogen load. Mechanistically, we found that mitochondrial inhibition of protein synthesis perturbed the electron transport chain (ETC) decreasing tissue damage in the lung and increasing fatty acid oxidation and glucocorticoid sensitivity in the liver. Using a liver-specific partial and acute deletion of Crif1, a critical mitoribosomal component for protein synthesis, we found that mice were protected against sepsis, an observation that was phenocopied by the transient inhibition of complex I of the ETC by phenformin. Together, we demonstrate that mitoribosome-targeting antibiotics are beneficial beyond their antibacterial activity and that mitochondrial protein synthesis inhibition leading to ETC perturbation is a mechanism for the induction of disease tolerance.
Topics: Animals; Anti-Bacterial Agents; Cell Cycle Proteins; Disease Models, Animal; Doxycycline; Electron Transport; Hep G2 Cells; Humans; Lipid Metabolism; Liver; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Sepsis; Tetracycline
PubMed: 33058782
DOI: 10.1016/j.immuni.2020.09.011 -
Biomolecules Oct 2020Damage to cerebral mitochondria, particularly opening of mitochondrial permeability transition pore (MPTP), is a key mechanism of ischemic brain injury, therefore,...
Damage to cerebral mitochondria, particularly opening of mitochondrial permeability transition pore (MPTP), is a key mechanism of ischemic brain injury, therefore, modulation of MPTP may be a potential target for a neuroprotective strategy in ischemic brain pathologies. The aim of this study was to investigate whether biguanides-metformin and phenformin as well as other inhibitors of Complex I of the mitochondrial electron transfer system may protect against ischemia-induced cell death in brain slice cultures by suppressing MPTP, and whether the effects of these inhibitors depend on the age of animals. Experiments were performed on brain slice cultures prepared from 5-7-day (premature) and 2-3-month old (adult) rat brains. In premature brain slice cultures, simulated ischemia (hypoxia plus deoxyglucose) induced necrosis whereas in adult rat brain slice cultures necrosis was induced by hypoxia alone and was suppressed by deoxyglucose. Phenformin prevented necrosis induced by simulated ischemia in premature and hypoxia-induced-in adult brain slices, whereas metformin was protective in adult brain slices cultures. In premature brain slices, necrosis was also prevented by Complex I inhibitors rotenone and amobarbital and by MPTP inhibitor cyclosporine A. The latter two inhibitors were protective in adult brain slices as well. Short-term exposure of cultured neurons to phenformin, metformin and rotenone prevented ionomycin-induced MPTP opening in intact cells. The data suggest that, depending on the age, phenformin and metformin may protect the brain against ischemic damage possibly by suppressing MPTP via inhibition of mitochondrial Complex I.
Topics: Animals; Brain; Brain Injuries; Electron Transport Complex I; Humans; Male; Metformin; Mitochondria; Mitochondrial Permeability Transition Pore; Mitochondrial Transmembrane Permeability-Driven Necrosis; Necrosis; Neurons; Neuroprotective Agents; Oxidative Phosphorylation; Phenformin; Rats
PubMed: 33019635
DOI: 10.3390/biom10101400 -
Archives of Toxicology Jan 2021Current in vitro genotoxicity tests can produce misleading positive results, indicating an inability to effectively predict a compound's subsequent carcinogenic...
Current in vitro genotoxicity tests can produce misleading positive results, indicating an inability to effectively predict a compound's subsequent carcinogenic potential in vivo. Such oversensitivity can incur unnecessary in vivo tests to further investigate positive in vitro results, supporting the need to improve in vitro tests to better inform risk assessment. It is increasingly acknowledged that more informative in vitro tests using multiple endpoints may support the correct identification of carcinogenic potential. The present study, therefore, employed a holistic, multiple-endpoint approach using low doses of selected carcinogens and non-carcinogens (0.001-770 µM) to assess whether these chemicals caused perturbations in molecular and cellular endpoints relating to the Hallmarks of Cancer. Endpoints included micronucleus induction, alterations in gene expression, cell cycle dynamics, cell morphology and bioenergetics in the human lymphoblastoid cell line TK6. Carcinogens ochratoxin A and oestradiol produced greater Integrated Signature of Carcinogenicity scores for the combined endpoints than the "misleading" in vitro positive compounds, quercetin, 2,4-dichlorophenol and quinacrine dihydrochloride and toxic non-carcinogens, caffeine, cycloheximide and phenformin HCl. This study provides compelling evidence that carcinogens can successfully be distinguished from non-carcinogens using a holistic in vitro test system. Avoidance of misleading in vitro outcomes could lead to the reduction and replacement of animals in carcinogenicity testing.
Topics: Carcinogenicity Tests; Carcinogens; Cell Cycle Checkpoints; Cell Line; Cell Shape; Endpoint Determination; Energy Metabolism; Gene Expression Regulation; Humans; Micronuclei, Chromosome-Defective; Micronucleus Tests; Phosphorylation; Research Design; Risk Assessment; Tumor Suppressor Protein p53
PubMed: 32910239
DOI: 10.1007/s00204-020-02902-3 -
Toxicology Research Jul 2020Medicines are usually prescribed for repeated use over shorter or longer times. Unfortunately, repeated-dose animal toxicity studies do not correlate well with...
Medicines are usually prescribed for repeated use over shorter or longer times. Unfortunately, repeated-dose animal toxicity studies do not correlate well with observations in man. As emphasized by the '3Rs' and the desire to phase-out animal research, models are needed. One potential approach uses clinostat-cultured 3D HepG2-C3A liver-mimetic spheroids. They take 18 days to recover physiological functionality and reach a metabolic equilibrium, which is thereafter stable for a year. Acute and chronic repeated-dose studies of six drugs (amiodarone, diclofenac, metformin, phenformin, paracetamol and valproic acid) suggest that spheroids are more predictive of human toxicity than either 2D-cultured HepG2 cells or primary human hepatocytes. Repeated non-lethal treatment results in a clear response and return to equilibrium. Mitochondrial toxic compounds can be identified using a galactose-based medium. Some drugs induced a protective (or stress) response that intensifies after the second treatment. This 3D spheroid model is inexpensive, highly reproducible and well-suited for the determination of repeated-dose toxicity of compounds (naturally or chemically synthesized).
PubMed: 32905230
DOI: 10.1093/toxres/tfaa033 -
Cells Sep 2020The greatest challenge in cancer therapy is posed by drug-resistant recurrence following treatment. Anticancer chemotherapy is largely focused on targeting the rapid...
The greatest challenge in cancer therapy is posed by drug-resistant recurrence following treatment. Anticancer chemotherapy is largely focused on targeting the rapid proliferation and biosynthesis of cancer cells. This strategy has the potential to trigger autophagy, enabling cancer cell survival through the recycling of molecules and energy essential for biosynthesis, leading to drug resistance. Autophagy recycling contributes amino acids and ATP to restore mTOR complex 1 (mTORC1) activity, which leads to cell survival. However, autophagy with mTORC1 activation can be stalled by reducing the ATP level. We have previously shown that cytosolic NADH production supported by aldehyde dehydrogenase (ALDH) is critical for supplying ATP through oxidative phosphorylation (OxPhos) in cancer cell mitochondria. Inhibitors of the mitochondrial complex I of the OxPhos electron transfer chain and ALDH significantly reduce the ATP level selectively in cancer cells, terminating autophagy triggered by anticancer drug treatment. With the aim of overcoming drug resistance, we investigated combining the inhibition of mitochondrial complex I, using phenformin, and ALDH, using gossypol, with anticancer drug treatment. Here, we show that OxPhos targeting combined with anticancer drugs acts synergistically to enhance the anticancer effect in mouse xenograft models of various cancers, which suggests a potential therapeutic approach for drug-resistant cancer.
Topics: Aldehyde Dehydrogenase; Animals; Antineoplastic Agents; Autophagy; Drug Resistance, Neoplasm; Drug Synergism; Electron Transport Complex I; Gossypol; HT29 Cells; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Neoplasms; Oxidative Phosphorylation; Phenformin; Xenograft Model Antitumor Assays
PubMed: 32883024
DOI: 10.3390/cells9092013 -
The Laryngoscope Jul 2021Iatrogenic laryngotracheal stenosis (iLTS) is the pathological narrowing of the glottis, subglottis, and/or trachea due to scar tissue. Patients with type 2 diabetes...
OBJECTIVES
Iatrogenic laryngotracheal stenosis (iLTS) is the pathological narrowing of the glottis, subglottis, and/or trachea due to scar tissue. Patients with type 2 diabetes mellitus (T2DM) are over 8 times more likely to develop iLTS and represent 26% to 53% of all iLTS patients. In this investigation, we compared iLTS scar-derived fibroblasts in patients with and without T2DM.
STUDY DESIGN
Controlled ex vivo study.
METHODS
iLTS scar fibroblasts were isolated and cultured from subglottic scar biopsies in iLTS patients diagnosed with or without type 2 diabetes (non-T2DM). Fibroblast proliferation, fibrosis-related gene expression, and metabolic utilization of oxidative phosphorylation (OXPHOS) and glycolysis were assessed. Contractility was measured using a collagen-based assay. Metabolically targeted drugs (metformin, phenformin, amobarbital) were tested, and changes in fibrosis-related gene expression, collagen protein, and contractility were evaluated.
RESULTS
Compared to non-T2DM, T2DM iLTS scar fibroblasts had increased α-smooth muscle actin (αSMA) expression (8.2× increased, P = .020), increased contractility (mean 71.4 ± 4.3% vs. 51.7 ± 16% Δ area × 90 minute , P = .016), and reduced proliferation (1.9× reduction at 5 days, P < .01). Collagen 1 (COL1) protein was significantly higher in the T2DM group (mean 2.06 ± 0.19 vs. 0.74 ±.44 COL1/total protein [pg/μg], P = .036). T2DM iLTS scar fibroblasts had increased measures of OXPHOS, including basal respiration (mean 86.7 vs. 31.5 pmol/minute/10 μg protein, P = .016) and adenosine triphosphate (ATP) generation (mean 97.5 vs. 25.7 pmol/minute/10 μg protein, P = .047) compared to non-T2DM fibroblasts. Amobarbital reduced cellular contractility; decreased collagen protein; and decreased expression of αSMA, COL1, and fibronectin. Metformin and phenformin did not significantly affect fibrosis-related gene expression.
CONCLUSION
T2DM iLTS scar fibroblasts demonstrate a myofibroblast phenotype and greater contractility compared to non-T2DM. Their bioenergetic preference for OXPHOS drives their increased contractility, which is selectively targeted by amobarbital.
LEVEL OF EVIDENCE
NA Laryngoscope, 131:1570-1577, 2021.
Topics: Adult; Aged; Amobarbital; Biopsy; Case-Control Studies; Cell Proliferation; Cells, Cultured; Cicatrix; Constriction, Pathologic; Diabetes Mellitus, Type 2; Energy Metabolism; Female; Glottis; Glycolysis; Humans; Hypoglycemic Agents; Iatrogenic Disease; Intubation, Intratracheal; Laryngostenosis; Male; Metformin; Middle Aged; Muscle Contraction; Myofibroblasts; Oxidative Phosphorylation; Phenformin; Primary Cell Culture; Trachea; Tracheal Stenosis; Tracheostomy; Young Adult
PubMed: 32857885
DOI: 10.1002/lary.29026 -
The Journal of Investigative Dermatology Jan 2021Phenformin is a drug in the biguanide class that was previously used to treat type 2 diabetes. We have reported the antitumor activities of phenformin to enhance the...
Phenformin is a drug in the biguanide class that was previously used to treat type 2 diabetes. We have reported the antitumor activities of phenformin to enhance the efficacy of BRAF-MAPK kinase-extracellular signal-regulated kinase pathway inhibition and to inhibit myeloid-derived suppressor cells in various melanoma models. Here we demonstrate that phenformin suppresses tumor growth and promotes keratinocyte differentiation in the 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate two-stage skin carcinogenesis mouse model. Moreover, phenformin enhances the suspension-induced differentiation of mouse and human keratinocytes. Mechanistically, phenformin induces the nuclear translocation of NFATc1 in keratinocytes in an AMPK-dependent manner. Pharmacologic or genetic inhibition of calcineurin and NFAT signaling reverses the effects of phenformin on keratinocyte differentiation. Taken together, our study reveals an antitumor activity of phenformin to promote keratinocyte differentiation that warrants future translational efforts to repurpose phenformin for the treatment of cutaneous squamous cell carcinomas.
Topics: Animals; Calcineurin; Cell Differentiation; Humans; Hypoglycemic Agents; Keratinocytes; Melanoma; Mice; Neoplasms, Experimental; Nitrofurans; Phenformin; Signal Transduction; Skin; Skin Neoplasms
PubMed: 32619504
DOI: 10.1016/j.jid.2020.05.114 -
Journal of Biomolecular Structure &... Sep 2021SARS-CoV-2 or Coronavirus disease 19 (COVID-19) is a rapidly spreading, highly contagious, and sometimes fatal disease for which drug discovery and vaccine development...
SARS-CoV-2 or Coronavirus disease 19 (COVID-19) is a rapidly spreading, highly contagious, and sometimes fatal disease for which drug discovery and vaccine development are critical. SARS-CoV-2 papain-like protease (PL) was used to virtually screen 1697 clinical FDA-approved drugs. Among the top results expected to bind with SARS-CoV-2 PL strongly were three cell protectives and antioxidants (NAD+, quercitrin, and oxiglutatione), three antivirals (ritonavir, moroxydine, and zanamivir), two antimicrobials (doripenem and sulfaguanidine), two anticancer drugs, three benzimidazole anthelmintics, one antacid (famotidine), three anti-hypertensive ACE receptor blockers (candesartan, losartan, and valsartan) and other miscellaneous systemically or topically acting drugs. The binding patterns of these drugs were superior to the previously identified SARS CoV PL inhibitor, 6-mercaptopurine (6-MP), suggesting a potential for repurposing these drugs to treat COVID-19. The objective of drug repurposing is the rapid relocation of safe and approved drugs by bypassing the lengthy pharmacokinetic, toxicity, and preclinical phases. The ten drugs with the highest estimated docking scores with favorable pharmacokinetics were subjected to molecular dynamics (MD) simulations followed by molecular mechanics/generalized Born surface area (MM/GBSA) binding energy calculations. Phenformin, quercetin, and ritonavir all demonstrated prospective binding affinities for COVID-19 PL over 50 ns MD simulations, with binding energy values of -56.6, -40.9, and -37.6 kcal/mol, respectively. Energetic and structural analyses showed phenformin was more stable than quercetin and ritonavir. The list of the drugs provided herein constitutes a primer for clinical application in COVID-19 patients and guidance for further antiviral studies.Communicated by Ramaswamy H. Sarma.
Topics: Anthelmintics; Anti-Bacterial Agents; Antioxidants; Antiviral Agents; COVID-19; Drug Repositioning; Humans; Molecular Docking Simulation; Papain; Peptide Hydrolases; Prospective Studies; SARS-CoV-2
PubMed: 32597315
DOI: 10.1080/07391102.2020.1784291 -
Neoplasia (New York, N.Y.) Aug 2020Using a mini-library of 1062 lentiviral shRNAs targeting 40 nuclear hormone receptors and 70 of their co-regulators, we searched for potential therapeutic targets that...
Using a mini-library of 1062 lentiviral shRNAs targeting 40 nuclear hormone receptors and 70 of their co-regulators, we searched for potential therapeutic targets that would be important during in vivo tumor growth using a parallel in vitro and in vivo shRNA screening strategy in the non-small cell lung cancer (NSCLC) line NCI-H1819. We identified 21 genes essential for in vitro growth, and nine genes specifically required for tumor survival in vivo, but not in vitro: NCOR2, FOXA1, HDAC1, RXRA, RORB, RARB, MTA2, ETV4, and NR1H2. We focused on FOXA1, since it lies within the most frequently amplified genomic region in lung adenocarcinomas. We found that 14q-amplification in NSCLC cell lines was a biomarker for FOXA1 dependency for both in vivo xenograft growth and colony formation, but not mass culture growth in vitro. FOXA1 knockdown identified genes involved in electron transport among the most differentially regulated, indicating FOXA1 loss may lead to a decrease in cellular respiration. In support of this, FOXA1 amplification was correlated with increased sensitivity to the complex I inhibitor phenformin. Integrative ChipSeq analyses reveal that FOXA1 functions in this genetic context may be at least partially independent of NKX2-1. Our findings are consistent with a neomorphic function for amplified FOXA1, driving an oncogenic transcriptional program. These data provide new insight into the functional consequences of FOXA1 amplification in lung adenocarcinomas, and identify new transcriptional networks for exploration of therapeutic vulnerabilities in this patient population.
Topics: Adenocarcinoma of Lung; Animals; Apoptosis; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Genome-Wide Association Study; Genomics; Hepatocyte Nuclear Factor 3-alpha; Humans; Insulin-Like Growth Factor Binding Protein 3; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Receptors, Cytoplasmic and Nuclear; Thrombospondin 1; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 32512502
DOI: 10.1016/j.neo.2020.04.005