Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface.

• MeSH
• diferenciální skenovací kalorimetrie MeSH
• fosforylcholin analogy a deriváty   metabolismus   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie s uhlíkem 13C MeSH
• mitochondriální membrány metabolismus   MeSH
• oxidace-redukce MeSH
• permeabilita MeSH
• protein X asociovaný s bcl-2 metabolismus   MeSH
• protonová magnetická rezonanční spektroskopie MeSH
• unilamelární lipozómy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mitocans are drugs selectively killing cancer cells by destabilizing mitochondria and many induce apoptosis via generation of reactive oxygen species (ROS). However, the molecular events by which ROS production leads to apoptosis has not been clearly defined. In this study with the mitocan alpha-tocopheryl succinate (alpha-TOS) the role of the Bcl-2 family proteins in the mechanism of malignant cell apoptosis has been determined. Exposure of several different cancer cell lines to alpha-TOS increased expression of the Noxa protein, but none of the other proteins of the Bcl-2 family, an event that was independent of the cellular p53 status. alpha-TOS caused a profound conformational change in the pro-apoptotic protein, Bak, involving oligomerization in all cell types, and this also applied to the Bax protein, but only in non-small cell lung cancer cells. Immunoprecipitation studies indicated that alpha-TOS activates the two BH1-3 proteins, Bak or Bax, to form high molecular weight complexes in the mitochondria. RNAi knockdown revealed that Noxa and Bak are required for alpha-TOS-induced apoptosis, and the role of Bak was confirmed using Bak- and/or Bax-deficient cells. We conclude that the major events induced by alpha-TOS in cancer cells downstream of ROS production leading to mitochondrial apoptosis involve the Noxa-Bak axis. It is proposed that this represents a common mechanism for mitochondrial destabilization activated by a variety of mitocans that induce accumulation of ROS in the early phases of apoptosis.

• MeSH
• alfa-tokoferol chemie   toxicita   MeSH
• apoptóza MeSH
• Jurkat buňky MeSH
• lidé MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondrie účinky léků   MeSH
• nádorový supresorový protein p53 genetika   MeSH
• protein Bak chemie   genetika   metabolismus   MeSH
• protinádorové látky chemie   toxicita   MeSH
• protoonkogenní proteiny c-bcl-2 metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• transportní proteiny mitochondriální membrány metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Apoptóza je typ programované buněčné smrti (typ I), který je nezbytný pro správný vývoj organizmu a tkáňovou homeostázu. Její průběh může být určen dvěma signálními drahami – vnější (receptorovou) dráhou řízenou receptory smrti a vnitřní (mitochondriální) apoptotickou dráhou, kde klíčovou roli plní mitochondrie. Mitochondrie jsou důležité buněčné organely s nepostradatelnými funkcemi pro život buňky, jako je např. tvorba energie ve formě molekul ATP (adenosintrifosfátu). Mitochondriální buněčná smrt je charakteristická změnou transmembránového potenciálu a permeabilizací vnější mitochondriální membrány. Mitochondrie jsou elektronegativní organely a depolarizace mitochondriální membrány je důležitá pro uvolnění proapoptotických signálů. Narušená regulace mitochondriální buněčné smrti se může podílet na patogenezi různých onemocnění, včetně rakoviny. Mitochondrie jsou také zdrojem reaktivních kyslíkových radikálů, iontů Ca2+ a proteinů ovlivňujících procesy iniciace a progrese nádorů nezávisle na indukci apoptózy. Současné studie se zaměřují na výzkum mitochondriálního membránového potenciálu a kyslíkových radikálů, které modulují různé signální dráhy uvnitř buňky a vymezení jejich významu v kancerogenezi, případně v léčbě onkologických pacientů. Monitorování apoptotických markerů, jako je stav mitochondriálního membránového potenciálu a určení hladiny reaktivních kyslíkových radikálů ve vzorcích nádorových pacientů, má prediktivní hodnotu pro výstup léčebných protokolů.

Apoptosis is type I programmed cell death, a process that is essential for development and tissue homeostasis. It is a prevalent form of cell death and it proceeds via two signaling pathways – external (receptor pathway) triggered by death receptors and intrinsic (mitochondrial) apoptotic pathway with major involvement of mitochondria. Mitochondria are important cellular organelles producing energy stored in molecules of adenosine triphosphate that are essential for cell survival. The mitochondrial cell death is characterized by permeabilization of the mitochondrial outer membrane and dissipation of the transmembrane potential. Mitochondria are electronegative organelles and depolarization of the mitochondrial membrane is important for the release of proapoptotic signals. Aberrant control of the mitochondrial cell death might contribute to several diseases including cancer. Mitochondria are also a source of reactive oxygen species, Ca2+ ions and other proteins that affect processes important for the initiation and progression of tumors independently of apoptosis. Current studies focus on research of mitochondrial membrane potential and reactive oxygen species modulating various signaling pathways within the cell, their importance in carcinogenesis, and in treatment of oncological patients. Monitoring of the apoptotic markers, such as the mitochondrial membrane potential (MMP), and the level of reactive oxygen species in samples of oncological patients has a predictive value for the output of treatment protocols. Key words: mitochondria – flow cytometry – apoptosis – free radicals – mitochondrial membrane potential This work was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101) and IntegRECAMO CZ.1.07/2.3.00/20.0097). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 13. 1. 2014 Accepted: 11. 4. 2014

• MeSH
• apoptóza * fyziologie   MeSH
• buněčná smrt fyziologie   MeSH
• ethidium analogy a deriváty   diagnostické užití   MeSH
• fluoresceiny diagnostické užití   MeSH
• fluorescenční barviva diagnostické užití   MeSH
• lidé MeSH
• membránový potenciál mitochondrií * MeSH
• mitochondriální membrány MeSH
• mitochondrie MeSH
• průtoková cytometrie * metody   MeSH
• reaktivní formy kyslíku * analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Mitochondrial membrane potential (Deltapsi(m)) plays important roles in the normal function of cells and in pathobiochemical situations. The application of ion-selective electrodes for the measurement of Deltapsi(m) is important for studying normal biological reactions and pathways and mitochondrial diseases. We constructed and optimized a computerized device for real-time monitoring of the Deltapsi(m), which included modification of tetraphenylphosphonium (TPP(+))-selective membrane that improved reproducibility of the TPP(+)-selective electrode. Application of MATLAB software increased the sensitivity of the system. We tested our improved device for membrane potential measurements of isolated mitochondria (in absolute scale of millivolts). In addition, we assessed relative changes of Deltapsi(m) (as changes in TPP(+) concentration) of digitonin-permeabilized cells (hepatocytes, control transmitochondrial cybrids, HeLa G and BSC-40) after addition of substrates, inhibitors, and uncoupler of respiratory chain. Our system can be successfully used for studies of many aspects of the regulation of mitochondrial bioenergetics and as a diagnostic tool for mitochondrial oxidative phosphorylation disorders.

• MeSH
• elektrofyziologie metody   přístrojové vybavení   MeSH
• financování organizované MeSH
• indikátory a reagencie chemie   MeSH
• iontově selektivní elektrody MeSH
• jaterní mitochondrie metabolismus   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• membránové potenciály fyziologie   MeSH
• mitochondriální membrány metabolismus   MeSH
• nádorové buňky kultivované MeSH
• oniové sloučeniny chemie   MeSH
• organofosforové sloučeniny chemie   MeSH
• periferní zařízení počítače MeSH
• počítačové systémy MeSH
• potkani Wistar MeSH
• reprodukovatelnost výsledků MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• hodnotící studie MeSH

Mitochondria are crucial compartments of eukaryotic cells because they function as the cellular power plant and play a central role in the early stages of programmed cell death (apoptosis). To avoid undesired cell death, this apoptotic pathway is tightly regulated by members of the Bcl-2 protein family, which interact on the external surface of the mitochondria, i.e., the mitochondrial outer membrane (MOM), and modulate its permeability to apoptotic factors, controlling their release into the cytosol. A growing body of evidence suggests that the MOM lipids play active roles in this permeabilization process. In particular, oxidized phospholipids (OxPls) formed under intracellular stress seem to directly induce apoptotic activity at the MOM. Here we show that the process of MOM pore formation is sensitive to the type of OxPls species that are generated. We created MOM-mimicking liposome systems, which resemble the cellular situation before apoptosis and upon triggering of oxidative stress conditions. These vesicles were studied using (31)P solid-state magic-angle-spinning nuclear magnetic resonance spectroscopy and differential scanning calorimetry, together with dye leakage assays. Direct polarization and cross-polarization nuclear magnetic resonance experiments enabled us to probe the heterogeneity of these membranes and their associated molecular dynamics. The addition of apoptotic Bax protein to OxPls-containing vesicles drastically changed the membranes' dynamic behavior, almost completely negating the previously observed effect of temperature on the lipids' molecular dynamics and inducing an ordering effect that led to more cooperative membrane melting. Our results support the hypothesis that the mitochondrion-specific lipid cardiolipin functions as a first contact site for Bax during its translocation to the MOM in the onset of apoptosis. In addition, dye leakage assays revealed that different OxPls species in the MOM-mimicking vesicles can have opposing effects on Bax pore formation.

• MeSH
• apoptóza fyziologie   MeSH
• diferenciální skenovací kalorimetrie MeSH
• Escherichia coli MeSH
• fluorescenční barviva MeSH
• fosfolipidy metabolismus   MeSH
• kardiolipiny metabolismus   MeSH
• lidé MeSH
• lipidové dvojvrstvy chemie   MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• oxidace-redukce MeSH
• oxidační stres fyziologie   MeSH
• permeabilita buněčné membrány MeSH
• protein X asociovaný s bcl-2 metabolismus   MeSH
• teplota MeSH
• unilamelární lipozómy chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The Bcl-2 protein family comprises both pro- and antiapoptotic members that control the permeabilization of the mitochondrial outer membrane, a crucial step in the modulation of apoptosis. Recent research has demonstrated that the carboxyl-terminal transmembrane domain (TMD) of some Bcl-2 protein family members can modulate apoptosis; however, the transmembrane interactome of the antiapoptotic protein Mcl-1 remains largely unexplored. Here, we demonstrate that the Mcl-1 TMD forms homooligomers in the mitochondrial membrane, competes with full-length Mcl-1 protein with regards to its antiapoptotic function, and induces cell death in a Bok-dependent manner. While the Bok TMD oligomers locate preferentially to the endoplasmic reticulum (ER), heterooligomerization between the TMDs of Mcl-1 and Bok predominantly takes place at the mitochondrial membrane. Strikingly, the coexpression of Mcl-1 and Bok TMDs produces an increase in ER mitochondrial-associated membranes, suggesting an active role of Mcl-1 in the induced mitochondrial targeting of Bok. Finally, the introduction of Mcl-1 TMD somatic mutations detected in cancer patients alters the TMD interaction pattern to provide the Mcl-1 protein with enhanced antiapoptotic activity, thereby highlighting the clinical relevance of Mcl-1 TMD interactions.

• MeSH
• apoptóza fyziologie   MeSH
• buněčná smrt fyziologie   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• HeLa buňky MeSH
• lidé MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• protein MCL-1 metabolismus   MeSH
• proteinové domény MeSH
• protoonkogenní proteiny c-bcl-2 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

CONTEXT: Acetaminophen (APAP) hepatotoxicity is often studied in primary cultures of hepatocytes of various species, but there are only few works comparing interspecies differences in susceptibility of hepatocytes to APAP in vitro. OBJECTIVES: The aim of our work was to compare hepatotoxicity of APAP in rat and mouse hepatocytes in primary cultures. MATERIALS AND METHODS: Hepatocytes isolated from male Wistar rats and C57Bl/6J mice were exposed to APAP for up to 24 h. We determined lactate dehydrogenase (LDH) activity in culture medium, activity of cellular dehydrogenases (WST-1) and activity of caspases 3 in cell lysate as markers of cell damage/death. We assessed content of intracellular reduced glutathione, production of reactive oxygen species (ROS) and malondialdehyde (MDA). Respiration of digitonin-permeabilized hepatocytes was measured by high resolution respirometry and mitochondrial membrane potential (MMP) was visualized (JC-1). RESULTS: APAP from concentrations of 2.5 and 0.75 mmol/L induced a decrease in viability of rat (p < 0.001) and mouse (p < 0.001) hepatocytes (WST-1), respectively. In contrast to rat hepatocytes, there was no activation of caspase-3 in mouse hepatocytes after APAP treatment. Earlier damage to plasma membrane and faster depletion of reduced glutathione were detected in mouse hepatocytes. Mouse hepatocytes showed increased glutamate + malate-driven respiration in state 4 and higher susceptibility of the outer mitochondrial membrane (OMM) to APAP-induced injury. CONCLUSION: APAP displayed dose-dependent toxicity in hepatocytes of both species. Mouse hepatocytes in primary culture however had approximately three-fold higher susceptibility to the toxic effect of APAP when compared to rat hepatocytes.

• MeSH
• biologické markery metabolismus   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• druhová specificita MeSH
• glutathion metabolismus   MeSH
• hepatocyty cytologie   účinky léků   metabolismus   MeSH
• jaterní mitochondrie účinky léků   enzymologie   metabolismus   MeSH
• kultivované buňky MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• mitochondriální membrány účinky léků   metabolismus   MeSH
• myši inbrední C57BL MeSH
• neopioidní analgetika škodlivé účinky   MeSH
• oxidace-redukce MeSH
• oxidační stres účinky léků   MeSH
• paracetamol škodlivé účinky   MeSH
• peroxidace lipidů účinky léků   MeSH
• potkani Wistar MeSH
• reaktivní formy kyslíku agonisté   metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Epigallocatechin-3-gallate (EGCG) is the main compound of green tea with well-described antioxidant, anti-inflammatory, and tumor-suppressing properties. However, EGCG at high doses was reported to cause liver injury. In this study, we evaluated the effect of EGCG on primary culture of rat hepatocytes and on rat liver mitochondria in permeabilized hepatocytes. The 24-hour incubation with EGCG in concentrations of 10 μmol/L and higher led to signs of cellular injury and to a decrease in hepatocyte functions. The effect of EGCG on the formation of reactive oxygen species (ROS) was biphasic. While low doses of EGCG decreased ROS production, the highest tested dose induced a significant increase in ROS formation. Furthermore, we observed a decline in mitochondrial membrane potential in cells exposed to EGCG when compared to control cells. In permeabilized hepatocytes, EGCG caused damage of the outer mitochondrial membrane and an uncoupling of oxidative phosphorylation. EGCG in concentrations lower than 10 μmol/L was recognized as safe for hepatocytes in vitro.

• MeSH
• čaj chemie   metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• hepatocyty cytologie   účinky léků   metabolismus   MeSH
• jaterní mitochondrie účinky léků   metabolismus   MeSH
• kaspasa 3 metabolismus   MeSH
• katechin analogy a deriváty   toxicita   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• oxidativní fosforylace účinky léků   MeSH
• potkani Wistar MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• TNF-alfa metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Východiska: Nejčastější pánevní gynekologickou malignitou je endometriální karcinom, kterému obvykle předchází hyperplazie děložní sliznice. Hypoteticky estrogeny inhibují především apoptotické proteiny, zatímco progestiny inhibují silněji anti-apoptotické proteiny než apoptotické. Pod vlivem neoponovaných estrogenů převáží anti-apoptotické faktory, je stimulována endometriální proliferace, která je spojena s vyšším rizikem mutací a atypických změn vyúsťujících v rozvoj endometriálního karcinomu. Apoptóza jeden z nejdůležitějších a nezbytných mechanismů udržujících tkáňovou homeostázu. Apoptóza v endometriu kromě jiného zahrnuje tři skupiny proteinů rodiny Bcl-2: anti-apoptotické proteiny (např. Bcl-2, Bcl-xl), pro-apoptotické proteiny se třemi (např. Bax, Bak) nebo jednou Bcl-2 homologní (BH) doménou (např. Bad, Bid). Tzv. “BH3-only” proteiny, Bad a Bid spouštějí apoptotickou kaskádu oligomerizací proteinů Bak a Bax, které permeabilizují zevní mitochondriální membránu. Na rozdíl od Bidu, který je přímým spouštěčem apoptózy, Bad spouští apoptózu nepřímo. Bad vazbou na anti-apoptotické proteiny rodiny Bcl-2 snižuje práh, při kterém je apoptóza indukována. Jejich vzájemné protichůdné působení nebo synergismus v lidském endometriu však zatím nebyly popsány. Soubor a metoda: U 75 pacientek byly stanoveny hodnoty Bid a Bad pomocí SDS-PAGE a Western blotu se specifickými protilátkami s cílem analyzovat množství Bid a Bad v normálním (NE), hyperplastickém (HE) a kancerózním endometriu (CE). Výsledky: Hladina proteinu Bid v kancerózním endometriu dosahovala pouze 47 % hladiny pozorované v NE a 50 % hodnot v HE. Naopak hladina Bad proteinu v hyperplastickém endometriu dosahovala pouze 40 % hladiny detekované v NE, resp. 36 % hodnot v CE. Závěr: Trendy exprese proteinů Bid a Bad jsou zjevně opačné v HE a CE. Předpokládáme, že buňky hyperplastického endometria si uchovávají dostatečný apoptotický program. Naopak kancerózní buňky vykazují sníženou hladinu přímého apoptotického spouštěče Bid a porušenou apoptózu a endometrium postižené adenokarcinomem jeví snahu zachránit defektní apoptotický program alespoň nepřímo, snížením apoptotického prahu cestou vyšších hladin Bad, který vyvazuje anti-apoptotický protein Bcl-2.

Background: The most frequent pelvic gynaecological malignancy is endometrial cancer, who is commonly proceeded by endometrial hyperplasia. Hypothetically, estrogens inhibit predominantly apoptotic proteins, whilst progestins inhibit more strongly anti-apoptotic proteins than apoptotic ones. Under the influence of unopposed estrogenes, anti-apoptotic factors overbalance, endometrial proliferation is stimulated which is associated with higher risk of mutations and atypical changes resulting in development of endometrial cancer. Apoptosis is one of the most important and necessary mechanism maintaining tissue homeostasis. Endometrial apoptosis involves among others three groups of proteins of the Bcl-2 family: anti-apoptotic proteins (e. g. Bcl-2, Bcl-xL), pro-apoptotic proteins with three (e. g. Bax, Bak) or one (e. g. Bad, Bid) Bcl-2 homologous (BH) domains. So-called BH3-only proteins, Bad and Bid trigger the apoptotic cascade with the oligomerization of Bak and Bax protein, which permeabilize the outer mitochondrial membrane. Unlike Bid, which is the direct apoptotic trigger, Bad triggers apoptosis indirectly. Bad lowers the threshold at which apoptosis is induced, by binding anti-apoptotic Bcl-2 family proteins. However, their mutual counterbalance or synergism in the human endometrium have not been reported yet. Patients and Method: In total of 75 patients, the levels of Bid and Bad were assessed using SDS-PAGE and Western blotting with specific antibodies, with the aim to analyse amount of Bid and Bad proteins in normal (NE), hyperplastic (HE) and cancerous (CE) endometrium. Results: Bid level in CE reached only 47 % and 50 % of this observed in NE and HE. Conversely, Bad level in HE reached only 40 % and 36 % of this observed in NE and CE, respectively. Conclusion: Trends of Bid and Bad protein levels are obviously opposite in HE and CE. We suggest that cells in HE maintain sufficient apoptotic program. On the contrary, cancerous cells (CE) show decreased level of direct apoptotic trigger protein Bid and impaired apoptosis and endometrium affected with adenocarcinoma make efforts to save the disrupted apoptosis program at least indirectly, by lower apoptotic threshold via higher Bad levels sequestrating anti-apoptotic protein Bcl-2.

• MeSH
• apoptóza * fyziologie   genetika   MeSH
• časové faktory MeSH
• dospělí MeSH
• endometrium fyziologie   metabolismus   MeSH
• hyperplazie endometria * genetika   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• membránové proteiny fyziologie   genetika   metabolismus   MeSH
• nádory endometria * genetika   patologie   MeSH
• odběr biologického vzorku MeSH
• protein Bad genetika   metabolismus   MeSH
• protein Bid genetika   metabolismus   MeSH
• senioři MeSH
• western blotting metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH

Mitochondria play a pivotal role in apoptosis: permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic proteins from the intermembrane space of mitochondria are regarded as the key event in apoptosis induction. Here we demonstrate how non-toxic doses of the mitochondrial Complex II inhibitor thenoyltrifluoroacetone (TTFA), which specifically inhibits the ubiquinone-binding site of succinate dehydrogenase (SDH), synergistically stimulated cell death, induced by harmless doses of cisplatin in a panel of chemoresistant neuroblastoma cell lines. Apoptotic cell death was confirmed by cytochrome c release from the mitochondria, cleavage of poly ADP-ribose polymerase, processing of caspase-3, which is an important executive enzyme in apoptosis, and caspase-3-like activity. Methyl malonate, an inhibitor of the SDHA subunit partially reversed apoptosis stimulated by TTFA in SK-N-BE(2) neuroblastoma cells (NB), indicating that sensitization requires oxidation of succinate. In contrast, in IMR-32 NB cells, the same concentrations of TTFA markedly suppressed cisplatin-induced apoptosis. Comparison of oxygen consumption in cisplatin-resistant SK-N-BE(2) and cisplatin-sensitive IMR-32 cells clearly demonstrated impaired Complex II activity in IMR-32 cells. We also found that in SK-N-BE(2) cells co-treatment with cisplatin and TTFA markedly stimulated formation of reactive oxygen species (ROS), whereas in IMR cells, cisplatin-mediated ROS production was attenuated by TTFA, which explains apoptosis suppression in these cells. Thus, functionally active SDH is a prerequisite for the ROS-mediated sensitization to treatment by TTFA.

• MeSH
• apoptóza účinky léků   MeSH
• chemorezistence MeSH
• cílená molekulární terapie * MeSH
• cisplatina farmakologie   MeSH
• kyselina jantarová metabolismus   MeSH
• lidé MeSH
• mitochondrie účinky léků   enzymologie   MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny antagonisté a inhibitory   MeSH
• neuroblastom patologie   MeSH
• oxidace-redukce MeSH
• protinádorové látky farmakologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• respirační komplex II antagonisté a inhibitory   MeSH
• screeningové testy protinádorových léčiv MeSH
• spotřeba kyslíku účinky léků   MeSH
• superoxidy metabolismus   MeSH
• synergismus léků MeSH
• thenoyltrifluoraceton farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH