The Role of Mitochondrial NADPH-Dependent Isocitrate Dehydrogenase in Cancer Cells

Isocitrate dehydrogenase 2 (IDH2) is located in the mitochondrial matrix. IDH2 acts in the forward Krebs cycle as an NADP+-consuming enzyme, providing NADPH for maintenance of the reduced glutathione and peroxiredoxin systems and for self-maintenance by reactivation of cystine-inactivated IDH2 by glutaredoxin 2. In highly respiring cells, the resulting NAD+ accumulation then induces sirtuin-3-mediated activating IDH2 deacetylation, thus increasing its protective function. Reductive carboxylation of 2-oxoglutarate by IDH2 (in the reverse Krebs cycle direction), which consumes NADPH, may follow glutaminolysis of glutamine to 2-oxoglutarate in cancer cells. When the reverse aconitase reaction and citrate efflux are added, this overall “anoxic” glutaminolysis mode may help highly malignant tumors survive aglycemia during hypoxia. Intermittent glycolysis would hypothetically be required to provide ATP. When oxidative phosphorylation is dormant, this mode causes substantial oxidative stress. Arg172 mutants of human IDH2—frequently found with similar mutants of cytosolic IDH1 in grade 2 and 3 gliomas, secondary glioblastomas, and acute myeloid leukemia—catalyze reductive carboxylation of 2-oxoglutarate and reduction to D-2-hydroxyglutarate, which strengthens the neoplastic phenotype by competitive inhibition of histone demethylation and 5-methylcytosine hydroxylation, leading to genome-wide histone and DNA methylation alternations. D-2-hydroxyglutarate also interferes with proline hydroxylation and thus may stabilize hypoxia-induced factor α

Nová tvář buněčné bioenergetiky

Cancer cells generally present abnormal bioenergetic properties including an elevated glucose uptake, a high glycolysis and a poorly efficient oxidative phosphorylation system. However, the determinants of cancer cells metabolic reprogramming remain unknown. The main question in this project was how environmental conditions in vivo can influence functioning of mitochondrial OXPHOS, because details of mitochondrial bioenergetics of cancer cells are poorly documented. We have combined two conditions, namely glucose and oxygen deprivation, to measure their potential interaction. We examined the impact of glucose deprivation and oxygen deprivation on cell survival, overall bioenergetics and OXPHOS protein expression. As a model, we have chosen a human breast carcinoma (HTB-126) and appropriate control (HTB-125) cultured cells, as large fraction of breast malignancies exhibit hypoxic tumor regions with low oxygen concentrations and poor glucose delivery. The results demonstrate that glucose presence or absence largely influence functioning of mitochochondrial oxidative phosphorylation. The level of mitochondrial respiration capacity is regulated by glucose; by Crabtree effect, by energy substrate channeling towards anabolic pathways that support cell growth and by mitochondrial biogenesis pathways. Both oxygen...Rakovinné buňky jsou charakteristické netypickými bioenergetickými vlastnostmi, jako je zvýšený metabolizmus glukózy a neefektivní oxidativní fosforylace. Přesný mechanizmus této metabolické proměny není doposud znám. Cílem tohoto projektu bylo ujasnit, jak prostředí tumoru ovlivňuje fungování mitochondriální oxidativní fosforylace. Zaměřovali jsme se na dvě základní podmínky, tj. deprivace glukózy a kyslíku, a analyzovali růst buněk, bioenergetické vlastnosti a expresi proteinů oxidativní fosforylace. Jako experimentální model jsme použili rakovinné buňky tumoru mléčné žlázy (HTB-126), které jsme porovnávali s příslušnou kontrolní linií (HTB-125), protože prostředí karcinomu prsu bývá často hypoxické a aglykemické. Výsledky této práce jasně ukazují závislost mitochondriální bioenergetiky na přítomnosti glukózy u rakovinných buněk. Mitochondriální respirace je regulována glukózou na několika úrovních, jako je Crabtree efekt, odvádění energetického substrátu z mitochondrií k podpoře buněčného růstu a proliferace a na úrovni biogeneze mitochondrií. Fungování oxidativní fosforylace je jasně ovlivněna přítomností glukózy i kyslíku. Součástí přizpůsobení se hypoxii je potlačení oxidativní fosforylace přítomností glukózy mnohem více, než v normoxii. Naše data ukazují, že energetické vlastnosti rakovinných buněk...Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

Nová tvář buněčné bioenergetiky

Rakovinné buňky jsou charakteristické netypickými bioenergetickými vlastnostmi, jako je zvýšený metabolizmus glukózy a neefektivní oxidativní fosforylace. Přesný mechanizmus této metabolické proměny není doposud znám. Cílem tohoto projektu bylo ujasnit, jak prostředí tumoru ovlivňuje fungování mitochondriální oxidativní fosforylace. Zaměřovali jsme se na dvě základní podmínky, tj. deprivace glukózy a kyslíku, a analyzovali růst buněk, bioenergetické vlastnosti a expresi proteinů oxidativní fosforylace. Jako experimentální model jsme použili rakovinné buňky tumoru mléčné žlázy (HTB-126), které jsme porovnávali s příslušnou kontrolní linií (HTB-125), protože prostředí karcinomu prsu bývá často hypoxické a aglykemické. Výsledky této práce jasně ukazují závislost mitochondriální bioenergetiky na přítomnosti glukózy u rakovinných buněk. Mitochondriální respirace je regulována glukózou na několika úrovních, jako je Crabtree efekt, odvádění energetického substrátu z mitochondrií k podpoře buněčného růstu a proliferace a na úrovni biogeneze mitochondrií. Fungování oxidativní fosforylace je jasně ovlivněna přítomností glukózy i kyslíku. Součástí přizpůsobení se hypoxii je potlačení oxidativní fosforylace přítomností glukózy mnohem více, než v normoxii. Naše data ukazují, že energetické vlastnosti rakovinných buněk...Cancer cells generally present abnormal bioenergetic properties including an elevated glucose uptake, a high glycolysis and a poorly efficient oxidative phosphorylation system. However, the determinants of cancer cells metabolic reprogramming remain unknown. The main question in this project was how environmental conditions in vivo can influence functioning of mitochondrial OXPHOS, because details of mitochondrial bioenergetics of cancer cells is poorly documented. We have combined two conditions, namely glucose and oxygen deprivation, to measure their potential interaction. We examined the impact of glucose deprivation and oxygen deprivation on cell survival, overall bioenergetics and OXPHOS protein expression. As a model, we have chosen a human breast carcinoma (HTB-126) and appropriate control (HTB-125) cultured cells, as large fraction of breast malignancies exhibit hypoxic tumor regions with low oxygen concentrations and poor glucose delivery. (...) Apoptosis is a natural, genetically controlled process of cell elimination. The mechanisms of its activation and regulation is a fundamental scientific question and growing body of evidence reveal further molecular pathways of apoptotic machinery. The well-known caspase activation cascade along with pro- and anti-apoptotic members of BCL family is the basic...1. lékařská fakultaFirst Faculty of Medicin

Non-canonical Bioenergetics of the Cell

Cancer cells generally present abnormal bioenergetic properties including an elevated glucose uptake, a high glycolysis and a poorly efficient oxidative phosphorylation system. However, the determinants of cancer cells metabolic reprogramming remain unknown. The main question in this project was how environmental conditions in vivo can influence functioning of mitochondrial OXPHOS, because details of mitochondrial bioenergetics of cancer cells is poorly documented. We have combined two conditions, namely glucose and oxygen deprivation, to measure their potential interaction. We examined the impact of glucose deprivation and oxygen deprivation on cell survival, overall bioenergetics and OXPHOS protein expression. As a model, we have chosen a human breast carcinoma (HTB-126) and appropriate control (HTB-125) cultured cells, as large fraction of breast malignancies exhibit hypoxic tumor regions with low oxygen concentrations and poor glucose delivery. (...) Apoptosis is a natural, genetically controlled process of cell elimination. The mechanisms of its activation and regulation is a fundamental scientific question and growing body of evidence reveal further molecular pathways of apoptotic machinery. The well-known caspase activation cascade along with pro- and anti-apoptotic members of BCL family is the basic..

Non-canonical Bioenergetics of the Cell

Cancer cells generally present abnormal bioenergetic properties including an elevated glucose uptake, a high glycolysis and a poorly efficient oxidative phosphorylation system. However, the determinants of cancer cells metabolic reprogramming remain unknown. The main question in this project was how environmental conditions in vivo can influence functioning of mitochondrial OXPHOS, because details of mitochondrial bioenergetics of cancer cells is poorly documented. We have combined two conditions, namely glucose and oxygen deprivation, to measure their potential interaction. We examined the impact of glucose deprivation and oxygen deprivation on cell survival, overall bioenergetics and OXPHOS protein expression. As a model, we have chosen a human breast carcinoma (HTB-126) and appropriate control (HTB-125) cultured cells, as large fraction of breast malignancies exhibit hypoxic tumor regions with low oxygen concentrations and poor glucose delivery. (...) Apoptosis is a natural, genetically controlled process of cell elimination. The mechanisms of its activation and regulation is a fundamental scientific question and growing body of evidence reveal further molecular pathways of apoptotic machinery. The well-known caspase activation cascade along with pro- and anti-apoptotic members of BCL family is the basic..

Non-canonical bioenergetics of the cell

Cancer cells generally present abnormal bioenergetic properties including an elevated glucose uptake, a high glycolysis and a poorly efficient oxidative phosphorylation system. However, the determinants of cancer cells metabolic reprogramming remain unknown. The main question in this project was how environmental conditions in vivo can influence functioning of mitochondrial OXPHOS, because details of mitochondrial bioenergetics of cancer cells are poorly documented. We have combined two conditions, namely glucose and oxygen deprivation, to measure their potential interaction. We examined the impact of glucose deprivation and oxygen deprivation on cell survival, overall bioenergetics and OXPHOS protein expression. As a model, we have chosen a human breast carcinoma (HTB-126) and appropriate control (HTB-125) cultured cells, as large fraction of breast malignancies exhibit hypoxic tumor regions with low oxygen concentrations and poor glucose delivery. The results demonstrate that glucose presence or absence largely influence functioning of mitochochondrial oxidative phosphorylation. The level of mitochondrial respiration capacity is regulated by glucose; by Crabtree effect, by energy substrate channeling towards anabolic pathways that support cell growth and by mitochondrial biogenesis pathways. Both oxygen..

Silica-coated upconversion lanthanide nanoparticles: The effect of crystal design on morphology, structure and optical properties

NaYF4:Yb3+/Er3+ nanoparticles were synthesized by thermal decomposition of lanthanide trifluoroacetates using oleylamine (OM) as both solvent and surface binding ligand. The effect of reaction temperature and time on the properties of the particles was investigated. The nanoparticles were characterized by transmission electron microscopy (TEM), electron diffraction (ED), energy dispersive spectroscopy (EDX), dynamic light scattering (DLS), thermogravimetric analysis (TGA), elemental analysis and X-ray diffraction (XRD) to determine morphology, size, polydispersity, crystal structure and elemental composition of the nanocrystals. TEM microscopy revealed that the morphology of the nanoparticles could be fine-tuned by modifying of the synthetic conditions. A cubic-to-hexagonal phase transition of the NaYF4:Yb3+/Er3+ nanoparticles at temperatures above 300 °C was confirmed by both ED and XRD. Upconversion luminescence under excitation at 980 nm was observed in the luminescence spectra of OM–NaYF4:Yb3+/Er3+ nanoparticles. Finally, the OM–NaYF4:Yb3+/Er3+ nanoparticles were coated with a silica shell to enable further functionalization and increase biocompatibility and stability in aqueous media, preventing particle aggregation

Antioxidant Role and Cardiolipin Remodeling by Redox-Activated Mitochondrial Ca²⁺-Independent Phospholipase A₂γ in the Brain

Mitochondrial Ca2+-independent phospholipase A2γ (iPLA2γ/PNPLA8) was previously shown to be directly activated by H2O2 and release free fatty acids (FAs) for FA-dependent H+ transport mediated by the adenine nucleotide translocase (ANT) or uncoupling protein 2 (UCP2). The resulting mild mitochondrial uncoupling and consequent partial attenuation of mitochondrial superoxide production lead to an antioxidant effect. However, the antioxidant role of iPLA2γ in the brain is not completely understood. Here, using wild-type and iPLA2γ-KO mice, we demonstrate the ability of tert-butylhydroperoxide (TBHP) to activate iPLA2γ in isolated brain mitochondria, with consequent liberation of FAs and lysophospholipids. The liberated FA caused an increase in respiratory rate, which was fully inhibited by carboxyatractyloside (CATR), a specific inhibitor of ANT. Employing detailed lipidomic analysis, we also demonstrate a typical cleavage pattern for TBHP-activated iPLA2γ, reflecting cleavage of glycerophospholipids from both sn-1 and sn-2 positions releasing saturated FAs, monoenoic FAs, and predominant polyunsaturated FAs. The acute antioxidant role of iPLA2γ-released FAs is supported by monitoring both intramitochondrial superoxide and extramitochondrial H2O2 release. We also show that iPLA2γ-KO mice were more sensitive to stimulation by pro-inflammatory lipopolysaccharide, as reflected by the concomitant increase in protein carbonyls in the brain and pro-inflammatory IL-6 release in the serum. These data support the antioxidant and anti-inflammatory role of iPLA2γ in vivo. Our data also reveal a substantial decrease of several high molecular weight cardiolipin (CL) species and accumulation of low molecular weight CL species in brain mitochondria of iPLA2γ-KO mice. Collectively, our results support a key role of iPLA2γ in the remodeling of lower molecular weight immature cardiolipins with predominantly saturated acyl chains to high molecular weight mature cardiolipins with highly unsaturated PUFA acyl chains, typical for the brain