Teaching the Basics of Reactive Oxygen Species and their Relevance to Cancer Biology: Mitochondrial Reactive Oxygen Species Detection, Redox Signaling, and Targeted Therapies

Reactive oxygen species (ROS) have been implicated in tumorigenesis (tumor initiation, tumor progression, and metastasis). Of the many cellular sources of ROS generation, the mitochondria and the NADPH oxidase family of enzymes are possibly the most prevalent intracellular sources. In this article, we discuss the methodologies to detect mitochondria-derived superoxide and hydrogen peroxide using conventional probes as well as newly developed assays and probes, and the necessity of characterizing the diagnostic marker products with HPLC and LC-MS in order to rigorously identify the oxidizing species. The redox signaling roles of mitochondrial ROS, mitochondrial thiolperoxidases, and transcription factors in response to mitochondria-targeted drugs are highlighted. ROS generation and ROS detoxification in drug-resistant cancer cells and the relationship to metabolic reprogramming are discussed. Understanding the subtle role of ROS in redox signaling and in tumor proliferation, progression, and metastasis as well as the molecular and cellular mechanisms (e.g., autophagy) could help in the development of combination therapies. The paradoxical aspects of antioxidants in cancer treatment are highlighted in relation to the ROS mechanisms in normal and cancer cells. Finally, the potential uses of newly synthesized exomarker probes for in vivo superoxide and hydrogen peroxide detection and the low-temperature electron paramagnetic resonance technique for monitoring oxidant production in tumor tissues are discussed

Large Cross-Effect Dynamic Nuclear Polarisation Enhancements With Kilowatt Inverting Chirped Pulses at 94 GHz

Dynamic nuclear polarisation (DNP) is a process that transfers electron spin polarisation to nuclei by applying resonant microwave radiation, and has been widely used to improve the sensitivity of nuclear magnetic resonance (NMR). Here we demonstrate new levels of performance for static cross-effect proton DNP using high peak power chirped inversion pulses at 94 GHz to create a strong polarisation gradient across the inhomogeneously broadened line of the mono-radical 4-amino TEMPO. Enhancements of up to 340 are achieved at an average power of a few hundred mW, with fast build-up times (3 s). Experiments are performed using a home-built wideband kW pulsed electron paramagnetic resonance (EPR) spectrometer operating at 94 GHz, integrated with an NMR detection system. Simultaneous DNP and EPR characterisation of other mono-radicals and biradicals, as a function of temperature, leads to additional insights into limiting relaxation mechanisms and give further motivation for the development of wideband pulsed amplifiers for DNP at higher frequencies

Large cross-effect dynamic nuclear polarisation enhancements with kilowatt inverting chirped pulses at 94 GHz

This work was supported by UK Research Council EPSRC research grant EP/R13705/1 and Wellcome Trust 099149/Z/12/Z.Dynamic nuclear polarisation (DNP) is a process that transfers electron spin polarisation to nuclei by applying resonant microwave radiation, and has been widely used to improve the sensitivity of nuclear magnetic resonance (NMR). Here we demonstrate new levels of performance for static cross-effect proton DNP using high peak power chirped inversion pulses at 94 GHz to create a strong polarisation gradient across the inhomogeneously broadened line of the mono-radical 4-amino TEMPO. Enhancements of up to 340 are achieved at an average power of a few hundred mW, with fast build-up times (3 s). Experiments are performed using a home-built wideband kW pulsed electron paramagnetic resonance (EPR) spectrometer operating at 94 GHz, integrated with an NMR detection system. Simultaneous DNP and EPR characterisation of other mono-radicals and biradicals, as a function of temperature, leads to additional insights into limiting relaxation mechanisms and give further motivation for the development of wideband pulsed amplifiers for DNP at higher frequencies.Publisher PDFPeer reviewe

Membrane topologies of the PGLa antimicrobial peptide and a transmembrane anchor sequence by Dynamic Nuclear Polarization/ solid-state NMR spectroscopy OPEN

International audienceDynamic Nuclear Polarization (DNP) has been introduced to overcome the sensitivity limitations of nuclear magnetic resonance (NMR) spectroscopy also of supported lipid bilayers. When investigated by solid-state NMR techniques the approach typically involves doping the samples with biradicals and their investigation at cryo-temperatures. Here we investigated the effects of temperature and membrane hydration on the topology of amphipathic and hydrophobic membrane polypeptides. Although the antimicrobial PGLa peptide in dimyristoyl phospholipids is particularly sensitive to topological alterations, the DNP conditions represent well its membrane alignment also found in bacterial lipids at ambient temperature. With a novel membrane-anchored biradical and purpose-built hardware a 17-fold enhancement in NMR signal intensity is obtained by DNP which is one of the best obtained for a truly static matrix-free system. Furthermore, a membrane anchor sequence encompassing 19 hydrophobic amino acid residues was investigated. Although at cryotemperatures the transmembrane domain adjusts it membrane tilt angle by about 10 degrees, the temperature dependence of two-dimensional separated field spectra show that freezing the motions can have beneficial effects for the structural analysis of this sequence

Photogenerated Radical in Phenylglyoxylic Acid for in Vivo Hyperpolarized 13C MR with Photosensitive Metabolic Substrates

Whether for 13C magnetic resonance studies in chemistry, biochemistry or biomedicine, hyperpolarization methods based on dynamic nuclear polarization (DNP) have become ubiquitous. DNP requires a source of unpaired electrons, which are commonly added to the sample to be hyperpolarized in the form of stable free radicals. Once polarized, the presence of these radicals is unwanted. These radicals can be replaced by nonpersistent radicals created by photo-irradiation of pyruvic acid (PA), which are annihilated upon dissolution or thermalization in the solid state. However, since PA is readily metabolized by most cells, its presence may be undesirable for some metabolic studies. In addition, some 13C substrates are photo-sensitive and, therefore, may degrade during photo-generation of PA radical, which requires ultraviolet (UV) light. We show here that photoirradiation of phenylglyoxylic acid (PhGA) using visible light produces a non-persistent radical that, in principle, can be used to hyperpolarize any molecule. We compare radical yields in samples containing PA and PhGA upon photo-irradiation with broadband and narrowband UV-visible light sources. To demonstrate the suitability of PhGA as a radical precursor for DNP, we polarized the gluconeogenic probe 13C-dihydroxyacetone, which is UV-sensitive, using a commercial 3.35 T DNP polarizer and then injected this into a mouse and followed its metabolism in vivo.This work is part of a project that has received funding from the European Union’s Horizon 2020 European Research Council (ERC Consolidator Grant) under grant agreement no. 682574 (ASSIMILES). Funding was also received from a Cancer Research UK Programme grant (17242) and from the CRUK-EPSRC Imaging Centre in Cambridge and Manchester (16465). F.K. and S.P. received funding from the European Union’s Horizon 2020 Research and Innovation Program under Marie Sklodowska-Curie grant agreement no. 642773 (EUROPOL). A. Capozzi received funding from the European Union’s Horizon 2020 Research and Innovation Program under Marie Sklodowska-Curie grant agreement no. 713683 (COFUNDfellowsDTU)

Terapias selectivas contra cáncer hepático y de seno, dirigidas a la bioenergética mitocondrial

El Cáncer Hepático o Carcinoma Hepatocelular (HCC) y el Cáncer de Seno Triple Negativo (por sus siglas en inglés TNBC, Triple Negative Breast Cancer), son problemas de salud pública en el mundo, por su difícil tratamiento y alta resistencia a la quimioterapia. En el caso del HCC, dado la aparición tardía de los síntomas y signos de la enfermedad, rara vez se diagnóstica a tiempo, siendo fatal dentro de los 3 a 6 meses siguientes a su diagnóstico. TNBC representa aproximadamente el 15-20% de todos los casos de cáncer de mama, y generalmente se considera como el más severo subgrupo, dado que no expresa los genes para los receptores de estrógenos, progesterona y HER2 (por sus siglas en inglés, Human Epidermal Growth Factor Receptor 2), por lo tanto, no responde a la hormonoterapia (como tamoxifeno o inhibidores de la aromatasa) ni a las terapias dirigidas a los receptores de HER2, como Herceptin (nombre genérico: trastuzumab), conllevando un mayor riesgo de recaída y una tasa de mortalidad más alta en comparación con otros subtipos de cáncer de mama.En la actualidad, los fármacos utilizados para el tratamiento del cáncer, como la doxorubicina, son agentes citostáticos que causan arresto celular, y que inducen apoptosis por aumento de niveles de Bax y p38 MAPK, mediados por la inhibición de Akt. Desafortunadamente, la Doxorrubicina, a pesar de sus propiedades contra el cáncer, induce miocardiopatía severa, aparentemente por la inhibición de la Citocromo C oxidasa Subunidad Vb y por aumentar la producción de ROS. Otros enfoques incluyen inhibidores de la vía MAPK kinasa como el Nexavar® (Sorafenib), e inhibidores Tirosin-Kinasa con actividad contra los receptores del factor de crecimiento epidérmico 1 y 2, como el Tacerva® (Erlotinib). Sin embargo, ninguno de ellos son eficaces, no sólo porque no son capaces de inhibir totalmente la proliferación tumoral, sino también porque afectan a las células normales conduciendo en la mayoría de los casos a insuficiencia renal aguda y muerte.Se conoce que tanto el HCC y el TNBC se caracterizan por la disfunción mitocondrial, la glicólisis elevada, el aumentó en el metabolismo glutaminólisis, la producción de lactato y la generación de especies reactivas de oxígeno (ROS). Actualmente, está ampliamente aceptado que el metabolismo mitocondrial está normalmente reprogramado para permitir el crecimiento de células de cáncer y la proliferación. Por ejemplo, la fosforilación oxidativa mitocondrial en el cáncer es esencial para satisfacer la creciente demanda de la biosíntesis de metabolitos necesarios para la proliferación de células tumorales sin restricciones. Del mismo modo, los niveles alterados de ciertos subproductos metabólicos de las mitocondrias, tales como ROS, han sido implicados en la iniciación del tumor y mantenimiento, así como son esenciales para tumorigenecidad mediada por Kras.Las terapias selectivas dirigidas a la afectación de la bioenergética mitocondrial de HCC y TNBC, se convierten en una estrategia potencial con resultados prometedores.Recientemente hemos sintetizado dos compuestos dirigidos a la mitocondria MTA (Mitochondria Target Agent), MitoSG1 y Mitometformina, cuyos compuestos parenterales son SG1 y Metformina respectivamente, son conjugado a un catión de alkyl triphenylphosphonium. Dado las diferencias del potencial de membrana de las células tumorales, estos compuestos tienen una acumulación selectiva permitiendo su acción directa sobre la mitocondria de las células de HCC y TNBC.En nuestros resultados preliminares encontramos que MitoSG1 inhibe el crecimiento de la línea celular de HCC HepG2, a concentraciones de 2.5µM a las 24 horas de tratamiento. MitoSG1 ejerce efectos citotóxicos con una IC50 de 3.6µM, con intervalos entre 2.28µM y 5.9µM. Adicionalmente MitoSG1 genera una afectación del potencial de membrana a las 24 horas de tratamiento, evidenciando una inducción temprana a apoptosis vía mitocondrial. Por su parte la Mitometformina presentó sobre la línea celular HepG2 una IC50 de 358.7µM, una concentración mayor que la requerida por MitoSG1, concluyendo en HCC que la línea celular es más susceptible al desequilibro del estado REDOX ocasionado por MitoSG1, que a la posible afectación causada por la activación de AMPK o inhibición de la Glicerofosfato Deshidrogenasa, ocasionada por Mitometformina.MitoSG1 genera efectos citotóxicos en las líneas celulares Triple Negativas de seno MDA MB231 y MDA MB468, con IC50 que oscilan entren 1 y 2µM del compuesto, así como una afectación del potencial de membrana, indicándonos que la mitocondria es su blanco de acción.MitoSG1 presentó efectos sinérgicos con Erlotinib y Doxorubicina en la línea Celular HepG2, así como un efecto aditivo con Sorafenib. En el caso de MDA MB231, MitoSG1 potencia los efectos citotóxicos  de  Doxorubicina, sugiriéndonos su potencial uso como coadyuvante del tratamiento.Aspectos éticos.En el desarrollo de esta investigación no se incluyeron estudios en humano o animales, por lo que su desarrollo no implicó ningún tipo de riesgo y por ende, no le aplican las consideraciones de las Resolución 8430 de 1993. Los experimentos fueron realizados en líneas celulares comerciales

Enhanced Intersystem Crossing and Transient Electron Spin Polarization in a Photoexcited Pentacene-Trityl Radical

Identifying and characterizing systems that generate well-defined states with large electron spin polarization is of high interest for applications in molecular spintronics, high-energy physics and magnetic resonance spectroscopy. The generation of electron spin polarization on free-radical substituents tethered to pentacene derivatives has recently gained a great deal of interest for its applications in molecular electronics. After photoexcitation of the chromophore, pentacene-radical derivatives can rapidly form spin-polarized triplet excited states through enhanced intersystem crossing. Under the right conditions, the triplet spin polarization, arising from mS-selective intersystem crossing rates, can be transferred to the tethered stable radical. The efficiency of this spin polarization transfer depends on many factors: local magnetic and electric fields, excited state energetics, molecular geometry, and spin-spin coupling. Here we present transient electron paramagnetic resonance (EPR) measurements on three pentacene derivatives tethered to Finland trityl, BDPA or TEMPO radicals to explore the influence of the nature of the radical on the spin polarization transfer. We observe efficient polarization transfer between the pentacene excited triplet and the trityl radical, but do not observe the same for the BDPA and TEMPO derivatives. The polarization transfer behavior in the pentacene-trityl system is also investigated in different glassy matrices and is found to depend markedly on the solvent used. The EPR results are rationalized with the help of femtosecond and nanosecond transient absorption measurements, yielding complementary information on the excited-state dynamics of the three pentacene derivatives. Notably, we observe a two orders of magnitude difference in the timescale of triplet formation between the pentacene-trityl system and the pentacene systems tethered with the BDPA and TEMPO radicals