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

Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps

Reactive oxygen species are by-products of aerobic metabolism involved in the onset and evolution of various pathological conditions. Among them, the superoxide radical is of special interest as the origin of several damaging species such as H2O2, hydroxyl radical, or peroxynitrite (ONOO-). Spin trapping coupled with ESR is a method of choice to characterize these species in chemical and biological systems and the metabolic stability of the spin adducts derived from reaction of superoxide and hydroxyl radicals with nitrones is the main limit to the in vivo application of the method. Recently, new cyclic nitrones bearing a triphenylphosphonium or permethylated β-cyclodextrin moiety have been synthesized and their spin adducts demonstrated increased stability in buffer. In this article, we studied the stability of the superoxide adducts of four new cyclic nitrones in the presence of liver subcellular fractions and biologically relevant reductants using an original setup combining a stopped-flow device and an ESR spectrometer. The kinetics of disappearance of the spin adducts were analyzed using an appropriate simulation program. Our results highlight the interest of the new spin trapping agents CD-DEPMPO and CD-DIPPMPO for specific detection of superoxide with high stability of the superoxide adducts in the presence of liver microsomes. © 2013 Elsevier Inc. All rights reserved

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

A global phylogeny of butterflies reveals their evolutionary history, ancestral hosts and biogeographic origins

Butterflies are a diverse and charismatic insect group that are thought to have evolved with plants and dispersed throughout the world in response to key geological events. However, these hypotheses have not been extensively tested because a comprehensive phylogenetic framework and datasets for butterfly larval hosts and global distributions are lacking. We sequenced 391 genes from nearly 2,300 butterfly species, sampled from 90 countries and 28 specimen collections, to reconstruct a new phylogenomic tree of butterflies representing 92% of all genera. Our phylogeny has strong support for nearly all nodes and demonstrates that at least 36 butterfly tribes require reclassification. Divergence time analyses imply an origin similar to 100 million years ago for butterflies and indicate that all but one family were present before the K/Pg extinction event. We aggregated larval host datasets and global distribution records and found that butterflies are likely to have first fed on Fabaceae and originated in what is now the Americas. Soon after the Cretaceous Thermal Maximum, butterflies crossed Beringia and diversified in the Palaeotropics. Our results also reveal that most butterfly species are specialists that feed on only one larval host plant family. However, generalist butterflies that consume two or more plant families usually feed on closely related plants

Novel genetic loci associated with hippocampal volume

The hippocampal formation is a brain structure integrally involved in episodic memory, spatial navigation, cognition and stress responsiveness. Structural abnormalities in hippocampal volume and shape are found in several common neuropsychiatric disorders. To identify the genetic underpinnings of hippocampal structure here we perform a genome-wide association study (GWAS) of 33,536 individuals and discover six independent loci significantly associated with hippocampal volume, four of them novel. Of the novel loci, three lie within genes (ASTN2, DPP4 and MAST4) and one is found 200 kb upstream of SHH. A hippocampal subfield analysis shows that a locus within the MSRB3 gene shows evidence of a localized effect along the dentate gyrus, subiculum, CA1 and fissure. Further, we show that genetic variants associated with decreased hippocampal volume are also associated with increased risk for Alzheimer's disease (rg =-0.155). Our findings suggest novel biological pathways through which human genetic variation influences hippocampal volume and risk for neuropsychiatric illness

Novel genetic loci underlying human intracranial volume identified through genome-wide association

Intracranial volume reflects the maximally attained brain size during development, and remains stable with loss of tissue in late life. It is highly heritable, but the underlying genes remain largely undetermined. In a genome-wide association study of 32,438 adults, we discovered five novel loci for intracranial volume and confirmed two known signals. Four of the loci are also associated with adult human stature, but these remained associated with intracranial volume after adjusting for height. We found a high genetic correlation with child head circumference (ρgenetic=0.748), which indicated a similar genetic background and allowed for the identification of four additional loci through meta-analysis (Ncombined = 37,345). Variants for intracranial volume were also related to childhood and adult cognitive function, Parkinson’s disease, and enriched near genes involved in growth pathways including PI3K–AKT signaling. These findings identify biological underpinnings of intracranial volume and provide genetic support for theories on brain reserve and brain overgrowth

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Antiproliferative effects of mitochondria-targeted N-acetylcysteine and analogs in cancer cells

Abstract N-acetylcysteine (NAC) has been used as an antioxidant drug in tumor cells and preclinical mice tumor xenografts, and it improves adaptive immunotherapy in melanoma. NAC is not readily bioavailable and is used in high concentrations. The effects of NAC have been attributed to its antioxidant and redox signaling role in mitochondria. New thiol-containing molecules targeted to mitochondria are needed. Here, mitochondria-targeted NAC with a 10-carbon alkyl side chain attached to a triphenylphosphonium group (Mito10-NAC) that is functionally similar to NAC was synthesized and studied. Mito10-NAC has a free sulfhydryl group and is more hydrophobic than NAC. Mito10-NAC is nearly 2000-fold more effective than NAC in inhibiting several cancer cells, including pancreatic cancer cells. Methylation of NAC and Mito10-NAC also inhibited cancer cell proliferation. Mito10-NAC inhibits mitochondrial complex I-induced respiration and, in combination with monocarboxylate transporter 1 inhibitor, synergistically decreased pancreatic cancer cell proliferation. Results suggest that the antiproliferative effects of NAC and Mito10-NAC are unlikely to be related to their antioxidant mechanism (i.e., scavenging of reactive oxygen species) or to the sulfhydryl group-dependent redox modulatory effects