Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

Background -- N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a synthetic retinoid with potent pro-apoptotic activity against several types of cancer, but little is known regarding mechanisms leading to chemoresistance. Ceramide and, more recently, other sphingolipid species (e.g., dihydroceramide and dihydrosphingosine) have been implicated in 4-HPR-mediated tumor cell death. Because sphingolipid metabolism has been reported to be altered in drug-resistant tumor cells, we studied the implication of sphingolipids in acquired resistance to 4-HPR based on an acute lymphoblastic leukemia model. Methods -- CCRF-CEM cell lines resistant to 4-HPR were obtained by gradual selection. Endogenous sphingolipid profiles and in situ enzymatic activities were determined by LC/MS, and resistance to 4-HPR or to alternative treatments was measured using the XTT viability assay and annexin V-FITC/propidium iodide labeling. Results -- No major crossresistance was observed against other antitumoral compounds (i.e. paclitaxel, cisplatin, doxorubicin hydrochloride) or agents (i.e. ultra violet C, hydrogen peroxide) also described as sphingolipid modulators. CCRF-CEM cell lines resistant to 4-HPR exhibited a distinctive endogenous sphingolipid profile that correlated with inhibition of dihydroceramide desaturase. Cells maintained acquired resistance to 4-HPR after the removal of 4-HPR though the sphingolipid profile returned to control levels. On the other hand, combined treatment with sphingosine kinase inhibitors (unnatural (dihydro)sphingosines ((dh)Sph)) and glucosylceramide synthase inhibitor (PPMP) in the presence or absence of 4-HPR increased cellular (dh)Sph (but not ceramide) levels and were highly toxic for both parental and resistant cells. Conclusions -- In the leukemia model, acquired resistance to 4-HPR is selective and persists in the absence of sphingolipid profile alteration. Therapeutically, the data demonstrate that alternative sphingolipid-modulating antitumoral strategies are suitable for both 4-HPR-resistant and sensitive leukemia cells. Thus, whereas sphingolipids may not be critical for maintaining resistance to 4-HPR, manipulation of cytotoxic sphingolipids should be considered a viable approach for overcoming resistance.

Development of catalytic methane reforming systems integrating hydrogen selective PdCu membrane modules

En la presente tesis doctoral se ha estudiado la integración del proceso de producción de hidrógeno con su purificación mediante el empleo de membranas selectivas de hidrógeno. La producción de hidrógeno se realiza empleando catalizadores no convencionales de níquel soportado sobre magnesia y alúmina en un reactor catalítico. Se analiza la actividad de los catalizadores y la producción de hidrógeno mediante distintos procesos con metano como son la oxidación parcial catalítica (OPC), OPC húmeda y reformadoLa purificación de hidrógeno se realiza en un módulo provisto de una membrana selectiva de hidrógeno de PdCu depositado en un soporte poroso cerámico. Una vez optimizada su preparación mediante deposición no electrolítica se caracterizan. Para ello se determina su permeabilidad a distintas temperaturas y realizando ciclos térmicos en atmósferas inerte y de hidrógeno, que puede fragilizar el metal. Una vez preparados los catalizadores y las membranas se integran los dos sistemas y se determinan los parámetros de operación óptimos como la presión de la línea de alimentación y el caudal de gas de arrastre en el módulo de membrana. Ambos parámetros se optimizan para lograr la máxima recuperación de hidrógeno en el módulo de membrana. Por últimos se realizan ensayos completos de producción y purificación, que permiten observar el rendimiento del sistema y también el efecto que los compuestos de la mezcla compleja alimentada a las membranas tienen en su comportamiento. Para concluir la integración de procesos se realizan ensayos añadiendo azufre de forma que el sistema sea más similar al proceso real. Esto permite también analizar el efecto del azufre tanto en los catalizadores como en las membranas.

Apaf-1 Inhibitors Protect from Unwanted Cell Death in In Vivo Models of Kidney Ischemia and Chemotherapy Induced Ototoxicity

Background: Excessive apoptosis induces unwanted cell death and promotes pathological conditions. Drug discovery efforts aimed at decreasing apoptotic damage initially targeted the inhibition of effector caspases. Although such inhibitors were effective, safety problems led to slow pharmacological development. Therefore, apoptosis inhibition is still considered an unmet medical need. Methodology and Principal Findings: The interaction between Apaf-1 and the inhibitors was confirmed by NMR. Target specificity was evaluated in cellular models by siRNa based approaches. Cell recovery was confirmed by MTT, clonogenicity and flow cytometry assays. The efficiency of the compounds as antiapoptotic agents was tested in cellular and in vivo models of protection upon cisplatin induced ototoxicity in a zebrafish model and from hypoxia and reperfusion kidney damage in a rat model of hot ischemia. Conclusions: Apaf-1 inhibitors decreased Cytc release and apoptosome-mediated activation of procaspase-9 preventing cell and tissue damage in ex vivo experiments and in vivo animal models of apoptotic damage. Our results provide evidence that Apaf-1 pharmacological inhibition has therapeutic potential for the treatment of apoptosis-related diseases.

Vanadyl arsenates as catalysts for selective oxidation of organic sulfides and alkenes

artículo científico (postprint)