The redox state of cytochrome C modulates resistance to methotrexate in human MCF7 breast cancer cells

Background: Methotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells. Results: Cell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexateinduced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells. Conclusions: We conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells.

Natural abundance of stable isotopes (15N and 13C) and isotopic fractionation of sea bream muscle as markers of food and culture conditions and its relationship with metabolic and proteomic analyses

Els isòtops estables com a traçadors de la cadena alimentària, s'han utilitzat per caracteritzar la relació entre els consumidors i els seus aliments, ja que el fraccionament isotòpic implica una discriminació en contra de certs isòtops. Però les anàlisis d'isòtops estables (SIA), també es poden dur a terme en peixos cultivats amb dietes artificials, com la orada (Sparus aurata), la especie más cultivada en el Mediterráneo. Canvis en l'abundància natural d'isòtops estables (13C i 15N) en els teixits i les seves reserves poden reflectir els canvis en l'ús i reciclatge dels nutrients ja que els enzims catabòlics implicats en els processos de descarboxilació i desaminació mostren una preferència pels isòtops més lleugers. Per tant, aquestes anàlisis ens poden proporcionar informació útil sobre l'estat nutricional i metabòlic dels peixos. L'objectiu d'aquest projecte va ser determinar la capacitat dels isòtops estables per ser utilitzats com a marcadors potencials de la capacitat de creixement i condicions de cria de l'orada. En aquest sentit, les anàlisis d'isòtops estables s'han combinat amb altres metabòlics (activitats citocrom-c-oxidasa, COX, i citrat sintasa, CS) i els paràmetres de creixement (ARN/ADN). El conjunt de resultats obtinguts en els diferents estudis realitzats en aquest projecte demostra que el SIA, en combinació amb altres paràmetres metabòlics, pot servir com una eina eficaç per discriminar els peixos amb millor potencial de creixement, així com a marcador sensible de l'estat nutricional i d'engreix. D'altra banda, la combinació de l'anàlisi d'isòtops estables amb les eines emergents, com ara tècniques de proteòmica (2D-PAGE), ens proporciona nous coneixements sobre els canvis metabòlics que ocorren en els músculs dels peixos durant l‟increment del creixement muscular induït per l'exercici.

Defective TNF-alpha-mediated hepatocellular apoptosis and liver damage in acidic sphingomyelinase knockout mice

This study addressed the contribution of acidic sphingomyelinase (ASMase) in TNF-alpha-mediated hepatocellular apoptosis. Cultured hepatocytes depleted of mitochondrial glutathione (mGSH) became sensitive to TNF-alpha, undergoing a time-dependent apoptotic cell death preceded by mitochondrial membrane depolarization, cytochrome c release, and caspase activation. Cyclosporin A treatment rescued mGSH-depleted hepatocytes from TNF-alpha-induced cell death. In contrast, mGSH-depleted hepatocytes deficient in ASMase were resistant to TNF-alpha-mediated cell death but sensitive to exogenous ASMase. Furthermore, although in vivo administration of TNF-alpha or LPS to galactosamine-pretreated ASMase(+/+) mice caused liver damage, ASMase(-/-) mice exhibited minimal hepatocellular injury. To analyze the requirement of ASMase, we assessed the effect of glucosylceramide synthetase inhibition on TNF-alpha-mediated apoptosis. This approach, which blunted glycosphingolipid generation by TNF-alpha, protected mGSH-depleted ASMase(+/+) hepatocytes from TNF-alpha despite enhancement of TNF-alpha-stimulated ceramide formation. To further test the involvement of glycosphingolipids, we focused on ganglioside GD3 (GD3) because of its emerging role in apoptosis through interaction with mitochondria. Analysis of the cellular redistribution of GD3 by laser scanning confocal microscopy revealed the targeting of GD3 to mitochondria in ASMase(+/+) but not in ASMase(-/-) hepatocytes. However, treatment of ASMase(-/-) hepatocytes with exogenous ASMase induced the colocalization of GD3 and mitochondria. Thus, ASMase contributes to TNF-alpha-induced hepatocellular apoptosis by promoting the mitochondrial targeting of glycosphingolipids.

Relevance of death receptors in nervous system: role in the pathogenesis of neurodegenerative diseases and targets for therapy

L’apoptosi és un procés fisiològic que controla el nombre de cèl·lules en organismes superiors. L’apoptosi està estrictament regulada i s’ha vist que està implicada en la patogènesi d’algunes malalties del sistema nerviós. En aquest sentit, un excés de mort cel·lular contribueix a les malalties neurodegenerati- ves, mentre que, el seu dèficit és una de les raons del desenvolupament de tumors. El punt principal de regulació del procés apoptòtic és l’activació de les caspases, cisteïna-proteases que tenen especificitat pels residus aspàrtic. Les caspases es poden activar per dos mecanismes principals: (1) alliberament de citocrom C dels mitocondris alterats al citoplasma i (2) l’activació dels receptors de la membrana anomenats receptors de mort (DR, de l’anglès death receptor). Aquests receptors s’han caracteritzat extensament en el sistema immunitari, mentre que en el sistema nerviós les seves funcions són encara desconegudes. El present article se centra en el paper dels DR en la patogènesi de malalties neurodegeneratives i suggereix el seu potencial des del punt de vista terapèutic. També es descriuen diverses molècules intracel·lulars caracteritzades per la seva habilitat en la modulació dels DR. Entre elles, presentem dues noves proteïnes – lifeguard i FAIM – que s’expressen específicament al sistema nerviós.

Natural or naturalized? phylogeography suggests that the abundant sea urchin arbacia lixula Is a recent Colonizer of the Mediterranean

We present the global phylogeography of the black sea urchin Arbacia lixula, an amphi-Atlantic echinoid with potential to strongly impact shallow rocky ecosystems. Sequences of the mitochondrial cytochrome c oxidase gene of 604 specimens from 24 localities were obtained, covering most of the distribution area of the species, including the Mediterranean and both shores of the Atlantic. Genetic diversity measures, phylogeographic patterns, demographic parameters and population differentiation were analysed. We found high haplotype diversity but relatively low nucleotide diversity, with 176 haplotypes grouped within three haplogroups: one is shared between Eastern Atlantic (including Mediterranean) and Brazilian populations, the second is found in Eastern Atlantic and the Mediterranean and the third is exclusively from Brazil. Significant genetic differentiation was found between Brazilian, Eastern Atlantic and Mediterranean regions, but no differentiation was found among Mediterranean sub-basins or among Eastern Atlantic sub-regions. The star-shaped topology of the haplotype network and the unimodal mismatch distributions of Mediterranean and Eastern Atlantic samples suggest that these populations have suffered very recent demographic expansions. These expansions could be dated 94-205 kya in the Mediterranean, and 31-67 kya in the Eastern Atlantic. In contrast, Brazilian populations did not show any signature of population expansion. Our results indicate that all populations of A. lixula constitute a single species. The Brazilian populations probably diverged from an Eastern Atlantic stock. The present-day genetic structure of the species in Eastern Atlantic and the Mediterranean is shaped by very recent demographic processes. Our results support the view (backed by the lack of fossil record) that A. lixula is a recent thermophilous colonizer which spread throughout the Mediterranean during a warm period of the Pleistocene, probably during the last interglacial. Implications for the possible future impact of A. lixula on shallow Mediterranean ecosystems in the context of global warming trends must be considered.

Long-term treatment with insulin induces apoptosis in brown adipocytes: role of oxidative stress

Trying to define the precise role played by insulin regulating the survival of brown adipocytes, we have used rat fetal brown adipocytes maintained in primary culture. The effect of insulin on apoptosis and the mechanisms involved were assessed. Different from the known effects of insulin as a survival factor, we have found that long-term treatment (72 h) with insulin induces apoptosis in rat fetal brown adipocytes. This process is dependent on the phosphatidylinositol 3-kinase/mammalian target of rapamycin/p70 S6 kinase pathway. Short-term treatment with the conditioned medium from brown adipocytes treated with insulin for 72 h mimicked the apoptotic effect of insulin. During the process, caspase 8 activation, Bid cleavage, cytochrome c release, and activation of caspases 9 and 3 are sequentially produced. Treatment with the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (Z-VAD), prevents activation of this apoptotic cascade. The antioxidants, ascorbic acid and superoxide dismutase, also impair this process of apoptosis. Moreover, generation of reactive oxygen species (ROS), probably through reduced nicotinamide adenine dinucleotide phosphate oxidases, and a late decrease in reduced glutathione content are produced. According to this, antioxidants prevent caspase 8 activation and Bid cleavage, suggesting that ROS production is an important event mediating this process of apoptosis. However, the participation of uncoupling protein-1, -2, and -3 regulating ROS is unclear because their levels remain unchanged upon insulin treatment for 72 h. Our data suggest that the prolonged hyperinsulinemia might cause insulin resistance through the loss of brown adipose tissue.

Genetic diversity and species richness patterns in Baetidae (Ephemeroptera) in the Montseny Mountain range (North-East Iberian Peninsula)

This study aimed to describe patterns of diversity of Baetidae (Ephemeroptera) at the ommunity and population levels within the Montseny Mountain range (North-East Iberian Peninsula). We studied both the distribution of 4 species of baetids in 20 sites among three catchments along the altitudinal gradient (350-1700 masl); and the genetic diversity of the mtDNA cytochrome c oxidase subunit I (cox1) gene of the two common species Baetis alpinus and Baetis rhodani. We found a gradual replacement of the dominant species along the altitudinal gradient. Baetis alpinus inhabited sites at high-altitudes, and this species was replaced by B. rhodani when the altitude decreased. Baetis melanonyx and Alainites muticus attained low abundance at all river sections, and no clear altitudinal trend appeared. Our hypothesis at the population level was that genetic structuring is associated with geographic distance and limited by drainage boundaries among the three studied catchments because of the short-time dispersion of adults. Unexpectedly, analyses of molecular variance (AMOVA) and isolation-bydistance (IBD) showed genetic diversity was unstructured by distance for both species, which may be explained by the relatively short spatial scale studied and small topographic barriers among the three catchments. The Generalized Mixed Yule-Coalescent (GMYC) model showed that B. rhodani had two differentiated genetic lineages that co-occurred in all sites. Overall, diversity of baetids was structured at the community level along the altitudinal gradient, whereas it was unstructured at the population level within the Montseny Mountain range.

Genetic diversity and species richness patterns in Baetidae (Ephemeroptera) in the Montseny Mountain range (North-East Iberian Peninsula)

This study aimed to describe patterns of diversity of Baetidae (Ephemeroptera) at the ommunity and population levels within the Montseny Mountain range (North-East Iberian Peninsula). We studied both the distribution of 4 species of baetids in 20 sites among three catchments along the altitudinal gradient (350-1700 masl); and the genetic diversity of the mtDNA cytochrome c oxidase subunit I (cox1) gene of the two common species Baetis alpinus and Baetis rhodani. We found a gradual replacement of the dominant species along the altitudinal gradient. Baetis alpinus inhabited sites at high-altitudes, and this species was replaced by B. rhodani when the altitude decreased. Baetis melanonyx and Alainites muticus attained low abundance at all river sections, and no clear altitudinal trend appeared. Our hypothesis at the population level was that genetic structuring is associated with geographic distance and limited by drainage boundaries among the three studied catchments because of the short-time dispersion of adults. Unexpectedly, analyses of molecular variance (AMOVA) and isolation-bydistance (IBD) showed genetic diversity was unstructured by distance for both species, which may be explained by the relatively short spatial scale studied and small topographic barriers among the three catchments. The Generalized Mixed Yule-Coalescent (GMYC) model showed that B. rhodani had two differentiated genetic lineages that co-occurred in all sites. Overall, diversity of baetids was structured at the community level along the altitudinal gradient, whereas it was unstructured at the population level within the Montseny Mountain range.

Contribution of cytochrome P450 and ABCB1 genetic variability on methadone pharmacokinetics, dose requirements, and response

Although the efficacy of methadone maintenance treatment (MMT) in opioid dependence disorder has been well established, the influence of methadone pharmacokinetics in dose requirement and clinical outcome remains controversial. The aim of this study is to analyze methadone dosage in responder and nonresponder patients considering pharmacogenetic and pharmacokinetic factors that may contribute to dosage adequacy. Opioid dependence patients (meeting Diagnostic and Statistical Manual of Mental Disorders, [4th Edition] criteria) from a MMT community program were recruited. Patients were clinically assessed and blood samples were obtained to determine plasma concentrations of (R,S)-, (R) and (S)- methadone and to study allelic variants of genes encoding CYP3A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, and P-glycoprotein. Responders and nonresponders were defined by illicit opioid consumption detected in random urinalysis. The final sample consisted in 105 opioid dependent patients of Caucasian origin. Responder patients received higher doses of methadone and have been included into treatment for a longer period. No differences were found in terms of genotype frequencies between groups. Only CYP2D6 metabolizing phenotype differences were found in outcome status, methadone dose requirements, and plasma concentrations, being higher in the ultrarapid metabolizers. No other differences were found between phenotype and responder status, methadone dose requirements, neither in methadone plasma concentrations. Pharmacokinetic factors could explain some but not all differences in MMT outcome and methadone dose requirements.