BACKGROUND: Hypoxia is a common feature of many solid tumors and causes radiotherapy and immunotherapy resistance. Pharmacological inhibition of oxidative phosphorylation (OXPHOS) has emerged as a therapeutic strategy to reduce hypoxia. However, the OXPHOS inhibitors tested in clinical trials caused only moderate responses in hypoxia alleviation or trials were terminated due to dose-limiting toxicities. To improve the therapeutic benefit, FDA approved OXPHOS inhibitors (e.g. atovaquone) were conjugated to triphenylphosphonium (TPP+) to preferentially target cancer cell's mitochondria. In this study, we evaluated the hypoxia reducing effects of several mitochondria-targeted OXPHOS inhibitors and compared them to non-mitochondria-targeted OXPHOS inhibitors using newly developed spheroid models for diffusion-limited hypoxia. METHODS: B16OVA murine melanoma cells and MC38 murine colon cancer cells expressing a HIF-Responsive Element (HRE)-induced Green Fluorescent Protein (GFP) with an oxygen-dependent degradation domain (HRE-eGFP-ODD) were generated to assess diffusion-limited hypoxia dynamics in spheroids. Spheroids were treated with IACS-010759, atovaquone, metformin, tamoxifen or with mitochondria-targeted atovaquone (Mito-ATO), PEGylated mitochondria-targeted atovaquone (Mito-PEG-ATO) or mitochondria-targeted tamoxifen (MitoTam). Hypoxia dynamics were followed and quantified over time using the IncuCyte Zoom Live Cell-Imaging system. RESULTS: Hypoxic cores developed in B16OVA.HRE and MC38.HRE spheroids within 24 h hours after seeding. Treatment with IACS-010759, metformin, atovaquone, Mito-PEG-ATO and MitoTam showed a dose-dependent reduction of hypoxia in both B16OVA.HRE and MC38.HRE spheroids. Mito-ATO only alleviated hypoxia in MC38.HRE spheroids while tamoxifen was not able to reduce hypoxia in any of the spheroid models. The mitochondria-targeted OXPHOS inhibitors demonstrated stronger anti-hypoxic effects compared to the non-mito-targeted OXPHOS inhibitors. CONCLUSIONS: We successfully developed a high-throughput spheroid model in which hypoxia dynamics can be quantified over time. Using this model, we showed that the mitochondria-targeted OXPHOS inhibitors Mito-ATO, Mito-PEG-ATO and MitoTam reduce hypoxia in tumor cells in a dose-dependent manner, potentially sensitizing hypoxic tumor cells for radiotherapy.

• Publikační typ
• časopisecké články MeSH

BACKGROUND: Fast adaptation of glycolytic and mitochondrial energy pathways to changes in the tumour microenvironment is a hallmark of cancer. Purely glycolytic ρ0 tumour cells do not form primary tumours unless they acquire healthy mitochondria from their micro-environment. Here we explored the effects of severely compromised respiration on the metastatic capability of 4T1 mouse breast cancer cells. METHODS: 4T1 cell lines with different levels of respiratory capacity were generated; the Seahorse extracellular flux analyser was used to evaluate oxygen consumption rates, fluorescent confocal microscopy to assess the number of SYBR gold-stained mitochondrial DNA nucleoids, and the presence of the ATP5B protein in the cytoplasm and fluorescent in situ nuclear hybridization was used to establish ploidy. MinION nanopore RNA sequence analysis was used to compare mitochondrial DNA transcription between cell lines. Orthotopic injection was used to determine the ability of cells to metastasize to the lungs of female Balb/c mice. RESULTS: OXPHOS-deficient ATP5B-KO3.1 cells did not generate primary tumours. Severely OXPHOS compromised ρ0D5 cells generated both primary tumours and lung metastases. Cells generated from lung metastasis of both OXPHOS-competent and OXPHOS-compromised cells formed primary tumours but no metastases when re-injected into mice. OXPHOS-compromised cells significantly increased their mtDNA content, but this did not result in increased OXPHOS capacity, which was not due to decreased mtDNA transcription. Gene set enrichment analysis suggests that certain cells derived from lung metastases downregulate their epithelial-to-mesenchymal related pathways. CONCLUSION: In summary, OXPHOS is required for tumorigenesis in this orthotopic mouse breast cancer model but even very low levels of OXPHOS are sufficient to generate both primary tumours and lung metastases.

• Publikační typ
• časopisecké články MeSH

• Publikační typ
• abstrakt z konference MeSH

Common inbred strains of the laboratory rat can be divided into four major mitochondrial DNA (mtDNA) haplotype groups represented by the BN, F344, LEW, and SHR strains. In the current study, we investigated the metabolic and hemodynamic effects of the SHR vs. F344 mtDNA by comparing the SHR vs. SHR-mt(F344) conplastic strains that are genetically identical except for their mitochondrial genomes. Altogether 13 amino acid substitutions in protein coding genes, seven single nucleotide polymorphisms in tRNA genes, and 12 single nucleotide changes in rRNA genes were detected in F344 mtDNA compared with SHR mtDNA. Analysis of oxidative phosphorylation system (OXPHOS) in heart left ventricles (LV), muscle, and liver revealed reduced activity and content of several respiratory chain complexes in SHR-mt(F344) conplastic rats compared with the SHR strain. Lower function of OXPHOS in LV of conplastic rats was associated with significantly increased relative ventricular mass and reduced fractional shortening that was independent of blood pressure. In addition, conplastic rats exhibited reduced sensitivity of skeletal muscles to insulin action and impaired glucose tolerance. These results provide evidence that inherited alterations in mitochondrial genome, in the absence of variation in the nuclear genome and other confounding factors, predispose to insulin resistance, cardiac hypertrophy and systolic dysfunction.

• MeSH
• adeninnukleotidy metabolismus   MeSH
• elektrokardiografie MeSH
• fenotyp MeSH
• funkce levé komory srdeční účinky léků   MeSH
• genová dávka MeSH
• glukosa metabolismus   MeSH
• glukózový toleranční test MeSH
• haplotypy genetika   MeSH
• inzulin farmakologie   MeSH
• inzulinová rezistence genetika   MeSH
• kardiomegalie genetika   patofyziologie   MeSH
• krevní tlak účinky léků   MeSH
• metabolismus lipidů účinky léků   MeSH
• mitochondriální DNA genetika   MeSH
• mitochondriální geny MeSH
• molekulární sekvence - údaje MeSH
• oxidativní fosforylace * účinky léků   MeSH
• potkani inbrední F344 MeSH
• potkani inbrední SHR MeSH
• RNA transferová genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• systola * účinky léků   MeSH
• transport elektronů účinky léků   MeSH
• velikost orgánu účinky léků   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Navzdory velkému úsilí je řada typů nádorových onemocnění téměř či zcela neléčitelná s tím, že resekce je u určitého počtu pacientů jedinou možností. Proto je zapořebí nový, účinnější způsob léčby. Nedávno jsme zjistili, že mitochondriální respirace je zcela nutná pro proces tvorby a růstu nádoru rakovinnými buňkami. V kontextu tvorby a růstu nádorů nesouvisí respirace s tvorbou ATP, nýbrž je potřebná pro efektivní tvorbu pyrimidinů kvůli své roli v redoxním cyklování ubichinonu, přičemž tento koenzym je pořebný pro průběh důležitého kroku de novo biosyntézy pyrimidinů. Proto navrhujeme cílení na respiraci jako potenciálně velmi účinný způsob léčby nádorových onemocnění, neboť nádorové buňky nemohou mutovat svoje geny důležité pro udržení respirace. Naše nová hypotéza říká, že různé typy nádorů mají různou prahovou hladinu respirace, která je potřebná pro jejich proliferaci. Tomuto fenoménu říkáme OXPHOS adikce a navrhujeme ji jako nový, účinný přístup k léčbě nádorů.; In spite of concerted effort, a number of types of cancer are very hard to cure or are beyond any treatment with resection being the only option in a limited number of patients. Therefore, new and efficient therapeutic approach is needed. We have recently discovered that mitochondrial respiration is essential for cancer cells to form and progress tumours. In the context of tumour formation/progression, respiration is unrelated to ATP provision; rather, it is required for efficient pyrimidine synthesis due to the role of respiration in ubiquinone cycling, the coenzyme being essential for the rate-limiting step in pyrimidine de novo biosynthetic pathway. Therefore, we propose that targeting respiration is potentially a very efficient way to treat cancer, since cancer cells will not mutate their genes necessary to maintain respiration. Our hypothesis is that different types of cancer have different requirement for 'respiration threshold' needed for their proliferation. We refer to this phenomenon as 'OXPHOS Addiction' and propose it as a novel approach to efficient tumour therapy.

• MeSH
• dihydroorotátdehydrogenasa škodlivé účinky   MeSH
• lidé MeSH
• mitochondriální DNA MeSH
• mitochondrie patologie   MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• nádory etiologie   terapie   MeSH
• oncogene addiction MeSH
• oxidativní fosforylace MeSH
• protinádorové látky MeSH
• pyrimidiny MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• onkologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Mitochondrial oxidative phosphorylation (OXPHOS) generates ATP, but OXPHOS also supports biosynthesis during proliferation. In contrast, the role of OXPHOS during quiescence, beyond ATP production, is not well understood. Using mouse models of inducible OXPHOS deficiency in all cell types or specifically in the vascular endothelium that negligibly relies on OXPHOS-derived ATP, we show that selectively during quiescence OXPHOS provides oxidative stress resistance by supporting macroautophagy/autophagy. Mechanistically, OXPHOS constitutively generates low levels of endogenous ROS that induce autophagy via attenuation of ATG4B activity, which provides protection from ROS insult. Physiologically, the OXPHOS-autophagy system (i) protects healthy tissue from toxicity of ROS-based anticancer therapy, and (ii) provides ROS resistance in the endothelium, ameliorating systemic LPS-induced inflammation as well as inflammatory bowel disease. Hence, cells acquired mitochondria during evolution to profit from oxidative metabolism, but also built in an autophagy-based ROS-induced protective mechanism to guard against oxidative stress associated with OXPHOS function during quiescence.Abbreviations: AMPK: AMP-activated protein kinase; AOX: alternative oxidase; Baf A: bafilomycin A1; CI, respiratory complexes I; DCF-DA: 2',7'-dichlordihydrofluorescein diacetate; DHE: dihydroethidium; DSS: dextran sodium sulfate; ΔΨmi: mitochondrial inner membrane potential; EdU: 5-ethynyl-2'-deoxyuridine; ETC: electron transport chain; FA: formaldehyde; HUVEC; human umbilical cord endothelial cells; IBD: inflammatory bowel disease; LC3B: microtubule associated protein 1 light chain 3 beta; LPS: lipopolysaccharide; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; mtDNA: mitochondrial DNA; NAC: N-acetyl cysteine; OXPHOS: oxidative phosphorylation; PCs: proliferating cells; PE: phosphatidylethanolamine; PEITC: phenethyl isothiocyanate; QCs: quiescent cells; ROS: reactive oxygen species; PLA2: phospholipase A2, WB: western blot.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• autofagie * MeSH
• cystein metabolismus   MeSH
• dextrany metabolismus   MeSH
• dýchání MeSH
• endoteliální buňky metabolismus   MeSH
• fibroblasty metabolismus   MeSH
• formaldehyd metabolismus   MeSH
• fosfatidylethanolaminy metabolismus   MeSH
• idiopatické střevní záněty * metabolismus   MeSH
• isothiokyanatany MeSH
• lidé MeSH
• lipopolysacharidy metabolismus   MeSH
• mitochondriální DNA metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• mTORC1 metabolismus   MeSH
• myši MeSH
• proteinkinasy aktivované AMP metabolismus   MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• sirolimus MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Mitochondria and oxidative phosphorylation (OXPHOS) are emerging as intriguing targets for the efficient elimination of cancer cells. The specificity of this approach is aided by the capacity of non-proliferating non-cancerous cells to withstand oxidative insult induced by OXPHOS inhibition. Recently we discovered that mitochondrial targeting can also be employed to eliminate senescent cells, where it breaks the interplay between OXPHOS and ATP transporters that appear important for the maintenance of mitochondrial morphology and viability in the senescent setting. Hence, mitochondria/OXPHOS directed pharmacological interventions show promise in several clinically-relevant scenarios that call for selective removal of cancer and senescent cells.

• MeSH
• adenosindifosfát metabolismus   MeSH
• adenosintrifosfát metabolismus   MeSH
• biologický transport MeSH
• buněčná smrt MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• nádory metabolismus   patologie   MeSH
• oxidativní fosforylace MeSH
• proliferace buněk MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• stárnutí buněk * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Cancer cell bioenergetics, maintaining mixed aerobic glycolysis (Warburg phenotype) and oxidative phosphorylation (OXPHOS), is not fully elucidated. Hypoxia-dependent OXPHOS suppression determines aerobic glycolysis. To elucidate further details, we studied hypoxic adaptation (up to 72 h at 5 % oxygen) of hepatocellular carcinoma HepG2 cells. The key regulatory component, hypoxia-inducible factor (HIF)-1α (HIF-1α) was stabilized at 5 h in 5 % oxygen for all three studied regimens, i.e. in glycolytic cells at 5 mM or 25 mM glucose, or in aglycemic (OXPHOS) cells when glucose was replaced by galactose. However, the conventional HIF-mediated suppression of respiration was prevented at aglycemia, which correlated with a high proportion of unphosphorylated pyruvate dehydrogenase (PDH) at 5 % oxygen. Such a modified HIF response in OXPHOS cells, termed as a non-canonical one, contrasted to conventional respiration suppression down to 45 % or 43 %, observed in hypoxia-adapted glycolytic cells at 5 mM or 25 mM glucose, respectively. These hypoxic glycolytic cells had normally highly phosphorylated PDH and most likely utilized pyruvate by aminotransferase reaction of glutaminolysis to feed at least suppressed respiration. Also, glycolytic cells were rather resistant towards the staurosporine-induced apoptosis, whereas aglycemic (OXPHOS) HepG2 cells exhibited much higher susceptibility. We conclude that aglycemia modulates the hypoxic HIF signaling toward a non-canonical response that is unable to carry out complete PDH phosphorylation, allowing a high pyruvate input for OXPHOS from the elevated glycolysis, which together with ongoing glutaminolysis maintain a virtually unchanged respiration. Similar OXPHOS revival may explain distinct tumor sensitivity to chemotherapy and other pharmacological interventions.

• MeSH
• buněčné kultury MeSH
• buňky Hep G2 MeSH
• faktor 1 indukovatelný hypoxií - podjednotka alfa metabolismus   MeSH
• hypoxie buňky MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• oxidativní fosforylace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Human hepatocellular carcinoma HepG2 cells are forced to oxidative phosphorylation (OXPHOS), when cultured in aglycemic conditions at galactose and glutamine. These Oxphos cells represent a prototype of cancer cell bioenergetics with mixed aerobic glycolysis and OXPHOS. We aimed to determine fractions of (i) glutaminolytic pathway involving aminotransferase reaction supplying 2-oxoglutarate (2OG) to the Krebs cycle vs. (ii) active segment of the Krebs cycle with aconitase and isocitrate dehydrogenase-3 (ACO-IDH3), which is typically inactive in cancer cells due to the citrate export from mitochondria. At normoxia, Oxphos cell respiration was decreased down to ~15 and ~10% by the aminotransferase inhibitor aminooxyacetate (AOA) or with AOA plus the glutamate-dehydrogenase inhibitor bithionol, respectively. Phosphorylating to non-phosphorylating respiration ratios dropped from >6.5 to 1.9 with AOA and to zero with AOA plus bithionol. Thus, normoxic Oxphos HepG2 cells rely predominantly on glutaminolysis. Addition of membrane-permeant dimethyl-2-oxoglutarate (dm2OG) to inhibited cells instantly partially restored respiration, evidencing the lack of 2OG-dehydrogenase substrate upon aminotransferase inhibition. Surprisingly, after 72 hr of 5% O2 hypoxia, the AOA (bithionol) inhibition ceased and respiration was completely restored. Thus in aglycemic HepG2 cells, the hypoxia-induced factor (HIF) upregulation of glycolytic enzymes enabled acceleration of glycolysis pathway, preceded by galactolysis (Leloir pathway), redirecting pyruvate via still incompletely blocked pyruvate dehydrogenase toward the ACO-IDH3. Glycolytic flux upregulation at hypoxia was evidently matched by a higher activity of the Leloir pathway in Oxphos cells. Hypoxic Oxphos cells increased 2-fold the NADPH oxidase activity, whereas hypoxic glycolytic cells decreased it. Oxphos cells and glycolytic cells at 5 mM glucose decreased their reduced glutathione fraction. In contrast to aglycemic cells, glycolytic HepG2 cells decreased their respiration at hypoxia despite the dm2OG presence, i.e., even at unlimited respiratory substrate availability for 72 hr at 5% O2, exhibiting the canonical HIF-mediated adaptation. Nevertheless, their ATP content was much higher with dm2OG as compared to its absence during hypoxic adaptation. Thus, the metabolic plasticity of cancer cells is illustrated under conditions frequently established for solid tumors in vivo, such as aglycemia plus hypoxia. Consequently, a wide acceptance of the irreversible and exclusive Warburg phenotype in cancer cells is incorrect.

• Publikační typ
• časopisecké články MeSH

Mitochondriální medicína je jedna z nejrychlejších rozvíjejících se interdisciplinárních oblastí medicíny, je součástí klinické a experimentální medicíny, která přispívá k objasnění patobiochemických mechanismů různých nemocí na úrovni mitochondrií. Monografie poskytuje současné poznatky o mitochondriálních nemocech z hlediska klinických, metabolických, patologických a genetických informací, důležitých pro určení diagnózy a cílenou terapii mitochondriálních nemocí. (Zadní strana knihy. Kráceno)

• MeSH
• karnitin MeSH
• kyseliny mastné omega-6 MeSH
• lékové transportní systémy MeSH
• minerální vody MeSH
• mitochondriální nemoci diagnóza   farmakoterapie   MeSH
• mitochondrie fyziologie   imunologie   patologie   MeSH
• omega-3 mastné kyseliny MeSH
• ubichinon MeSH
• vitaminy MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biochemie
• patologie
• NLK Publikační typ
• kolektivní monografie