RESUMO
The antiproliferative effects of 15-LOX (15-lipoxygenase) metabolites of arachidonic acid {(15S)-HPETE [(15S)-hydroperoxyeicosatetraenoic acid] and (15S)-HETE [(15S)-hydroxyeicosatetraenoic acid]} and the mechanism(s) involved were studied in the human T-cell leukaemia cell line Jurkat. (15S)-HPETE, the hydroperoxy metabolite of 15-LOX, inhibited the growth of Jurkat cells 3 h after exposure and with an IC(50) value of 10 microM. The hydroxy metabolite of 15-LOX, (15S)-HETE, on the other hand, inhibited the growth of Jurkat cells after 6 h of exposure and with an IC(50) value of 40 microM. The cells exposed to 10 microM (15S)-HPETE for 3 h or to 40 microM (15S)-HETE for 6 h showed increased expression of Fas ligand and FADD (Fas-associated death domain), caspase 8 activation, Bid (BH3-interacting domain death agonist) cleavage, decrease in mitochondrial membrane potential, cytochrome c release, caspase 3 activation, PARP-1 [poly(ADP-ribose) polymerase-1] cleavage and DNA fragmentation, suggesting the involvement of both extrinsic and intrinsic death pathways. Further studies on ROS (reactive oxygen species) generation revealed the involvement of NADPH oxidase. In conclusion, the present study indicates that NADPH oxidase-induced ROS generation activates the Fas-mediated death pathway.
Assuntos
Proteína Ligante Fas/metabolismo , Leucotrienos/química , Leucotrienos/farmacologia , Peróxidos Lipídicos/química , Peróxidos Lipídicos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Humanos , Células Jurkat , Potencial da Membrana Mitocondrial/efeitos dos fármacos , NADPH Oxidases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estereoisomerismo , Receptor fas/metabolismoRESUMO
Umbilical cord blood (UCB) is gaining more prominence in recent times as a source of non-embryonic multipotent stem cells. Global annual human birth rate (100 million) presents UCB as the largest non-controversial stem cell source, with an added advantage of naive immune status. Cord blood stem cells are routinely utilized in stem cell transplantation in leukemia patients and carry huge potential to treat other human diseases with less concern of rejection. Because UCB contains low number of stem cells, their use is associated with significant delays in engraftment of neutrophils and platelets. Development of reliable methods for isolation and expansion of cord blood stem cells is critical for consequent clinical application. The focus of this chapter is to review the methods currently used by different research groups and to recommend an isolation protocol that yields optimal number of UCB stem cells.