Nuevas estrategias para el tratamiento de la leishmaniasis: mecanismo de acción de lípidos antitumorales, interacción hospedero-parásito, y su posible utilidad terapéutica

[ES]A pesar de los pocos conocimientos de la maquinaria de muerte celular en parásitos, se han postulado distintas hipótesis sobre la significancia biológica de la apoptosis en estos microorganismos (Nguewa et al, 2004; Shaha, 2006), aunque está claro que inducir una respuesta apoptótica puede ser una nueva diana terapéutica en el tratamiento de las enfermedades parasitarias. Los denominados lípidos éter sintéticos antitumorales, colectivamente denominados ATLs (de synthetic ¿antitumor lipids¿) constituyen una prometedora familia de compuestos con actividad antitumoral y antiparasitaria, y uno de sus miembros la Miltefosina fue considerado recientemente como el primer tratamiento para el manejo de algunas infecciones por Leishmania. Otro de su miembros la Edelfosina también posee actividad antiparasitaria incluyendo Leishmania y Trypanosoma (Croft et al, 2003). Sin embargo, el tipo de muerte inducida y su mecanismo molecular permanece por ser elucidado. Aunque ciertas evidencias indirectas sugieren que el metabolismo lipídico (Lux et al, 2000) y la mitocondria (Santa-Rita et al, 2004, 2006) pueden estar implicados. La edelfosina, que se ha considerado como el prototipo standard de ATLs induce apoptosis de forma selectiva en células tumorales (Mollinedo et al 1997), a través de un mecanismo de acción que implica los dominios de membrana ¿lípid rafts¿ (Gajate & Mollinedo, 2001; Gajate et al, 2004). ). En tumores sólidos parece actuar vía un mecanismo diferente que implica la activación de la apoptosis a través del retículo endoplásmico (Nieto-Miguel et al, 2006; 2007). A pesar de que este proceso de apoptosis mediada por ATLs se inicie a través de distintos mecanismos, la mitocondria juega un papel fundamental en la inducción de apoptosis por edelfosina (Gajate et al, 2000; Vrablic et al, 2001) por lo cual pretendemos extrapolar este proceso de apoptosis mediada por los lipid rafts y la mitocondria a la terapia antiparasitaria. Estos estudios se han extendido con éxito en nuestro grupo en levaduras (Zaremberg et al., 2005) Tambien llevaremos a cabo estudios in vitro e in vivo, necesarios para determinar la posible utilidad clínica de la edelfosina como antiparasitario. Además, en este trabajo se evalúo la actividad antiparasitaria de otra molécula lipídica antitumoral adicional, la espisulosina (ES-285), estudiándose sus mecanismos de acción. Adicionalmente se estudian los factores del hospedero que el parásito aprovecha para establecer la infección y así encontrar nuevos blancos terapéuticos que sean de utilidad para una terapia combinada

Data in support of dyslipidemia-associated alterations in B cell subpopulations frequency and phenotype during experimental atherosclerosis

ABSTARCT: Cardiovascular diseases are the most common cause of death in the world, atherosclerosis being its main underlying disease. Information about the role of B cells during atherosclerotic process is scarce, but both proatherogenic and atheroprotective properties have been described in the immunopathology of this disease. Frequency and phenotype of B cell subpopulations were studied in wild type and apolipoprotein-E-deficient (apoE / ) mice fed or not with high-fat diet (HFD), by flow cytometry. Here, we provide the information about the materials, methods, analysis and additional information related to our study published in Atherosclerosi

Mitochondria and lipid raft-located FOF1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine.

Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated.Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast.The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis

Edelfosine induces breakage of kinetoplast DNA prior to nuclear DNA breakdown, and accumulates in mitochondria in <i>Leishmania</i> parasites and cancer cells.

<p>(<b>A</b>) <i>L</i>. <i>panamensis</i> promastigotes were untreated (Control) or treated with 10 μM edelfosine (EDLF) for 6 and 9 h, and then analyzed by confocal microscopy for propidium iodide (PI) staining and TUNEL assay. The positions of the nucleus (N) and kinetoplast (K) are indicated by arrows. Merging of PI and TUNEL panels (Merge) shows the DNA-containing organelles with DNA disruption in yellow. The corresponding differential interference contrast (DIC) images were included in the Merge panels to highlight parasite morphology and facilitate kinetoplast identification. (<b>B</b>) <i>L</i>. <i>panamensis</i> promastigotes and (<b>C</b>) HeLa cancer cells were incubated with 10 μM PTE-ET (blue fluorescence) for 1 h, 100 nM MitoTracker (red fluorescence) for 20 min to localize mitochondria, and then analyzed by fluorescence microscopy. Areas of colocalization between mitochondria and PTE-ET in merge panels are purple. The corresponding differential interference contrast (DIC) images are also shown. Images are representative of three independent experiments. Bar, 20 μm.</p

F_OF₁-ATPase recruitment into rafts in the antileishmanial activity of edelfosine and oligomycin inhibitory effect on cytotoxicity.

<p>(<b>A</b>) <i>L</i>. <i>panamensis</i> promastigotes untreated (Control) and treated with 10 μM edelfosine for 9 h were lysed in 1% Triton X-100 and subjected to discontinuous sucrose density gradient centrifugation. Individual fractions were electrophoresed, and location of GM1 was determined. (<b>B</b>) Proteins from lipid rafts of untreated control and edelfosine-treated <i>L</i>. <i>panamensis</i> promastigotes were subjected to two-dimensional gel electrophoresis followed by MALDI-TOF analysis. Mitochondrial F_OF₁-ATP synthase β subunit is indicated by an arrow. (<b>C</b>) Mass spectrum of the tryptic peptides of the F_OF₁-ATP synthase β subunit spot. Mass values (m/z) and putative amino acid position assignments are indicated above peaks. (<i>Inset</i>) Peptide coverage map of <i>Leishmania</i> F_OF₁-ATP synthase β subunit; the peptides used for identification are highlighted in bold characters and underlined. (<b>D</b>) <i>L</i>. <i>panamensis</i> were untreated (Control) or preincubated with 1 μM oligomycin for 1 h and then incubated in the absence or presence of 10 μM edelfosine for 9 h, and ΔΨ_m disruption (Low ΔΨ_m) and DNA breakdown (hypodiploids) were evaluated. Data shown are means ± SD or representative of three independent experiments. (*) <i>P</i><0.05. (**) <i>P</i><0.01.</p

Schematic model of mitochondria involvement in the killing activity of edelfosine against <i>Leishmania</i> parasites and tumor cells.

<p>This is a schematic diagram to portray one currently plausible mechanism of how edelfosine induces cell death in <i>Leishmania</i> parasites and tumor cells through its main mitochondrial localization in both biological systems. Protection of mitochondria by Bcl-X_L ectopic expression restrains cell death. See text for details.</p

Inhibition of apoptosis-like cell death by ectopic expression of Bcl-X_L in <i>L</i>. <i>infantum</i> promastigotes and HeLa tumor cells.

<p>Inhibition of apoptosis-like cell death by ectopic expression of Bcl-X_L in <i>L</i>. <i>infantum</i> promastigotes and HeLa tumor cells.</p

Edelfosine resistance of <i>atp7</i>Δ mutant in <i>Saccharomyces cerevisiae</i> yeast.

<p>Growth curves of wild-type (BY4741) (<b>A</b>), <i>ATP7</i> knock-out mutant (<i>atp7</i>Δ) (<b>B</b>) and the mutant strain harboring the corresponding cognate gene (<i>atp7</i>Δ+pRS416-<i>ATP7</i>) (<b>C</b>) in SDC medium containing different concentrations of edelfosine. The cultures were carried out in duplicate and in at least three independent experiments. Data shown are mean values of three independent experiments. SD values were less than 10% of the mean values.</p

Edelfosine is taken up by macrophages and inhibits macrophage-derived inflammatory mediators.

<p>(<b>A</b>) Incorporation of edelfosine (EDLF) in mouse bone marrow-derived macrophages (BMM) and mouse RAW 309 Cr.1 tumor macrophage cell line. 10⁶ cells were incubated with 10 μM edelfosine (containing 0.05 μCi [³H]edelfosine) for the indicated times to measure drug uptake. (<b>B</b>) Edelfosine is cytotoxic for transformed macrophages but spares BMM. 2 x 10⁶ cells were incubated for 24 h in the absence or presence of the indicated concentrations of edelfosine (EDLF), and cytotoxicity was determined by the WST-1 reduction method. (<b>C</b>) Edelfosine inhibits superoxide anion generation in BMM. Superoxide anion was measured as lucigenin-dependent chemiluminescence (relative light units, RLU) in untreated control (C) or edelfosine (EDLF)-treated BMM that were incubated with medium alone or zymosan to induce the respiratory burst. (<b>D-F</b>) BMM from edelfosine-fed mice show a decreased generation of inflammatory mediators. BMM from untreated control mice (C) and from mice given orally edelfosine (EDLF) for two weeks were analyzed for their capacity to generate zymosan-induced superoxide anion (<b>D</b>), LPS-induced nitric oxide (<b>E</b>), and IL-12+IL-18-induced IFN-γ (<b>F</b>). Cells incubated with medium alone were run in parallel as a negative control of each assay. Data shown are means ± SD of five independent determinations. Asterisks indicate values that are significantly different from those of control mice (comparison between the black histograms of control and edelfosine-treated groups) at <i>P</i><0.05 (*) and <i>P</i><0.01 (**).</p