RESUMEN
Sun or therapy-related ultraviolet B (UVB) irradiation induces different cell death modalities such as apoptosis, necrosis/necroptosis and autophagy. Understanding of mechanisms implicated in regulation and execution of cell death program is imperative for prevention and treatment of skin diseases. An essential component of death-inducing complex is Fas-associated protein with death domain (FADD), involved in conduction of death signals of different death modalities. The purpose of this study was to enlighten the role of FADD in the selection of cell death mode after narrow-band UVB (NB-UVB) irradiation using specific cell death inhibitors (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (zVAD-fmk), Necrostatin-1 and 3-Methyladenine) and FADD-deficient (FADD-/-) mouse embryonic fibroblasts (MEFs) and their wild type (wt) counterparts. The results imply that lack of FADD sensitized MEFs to induction of receptor-interacting protein 1 (RIPK1)-dependent apoptosis by the generation of reactive oxygen species (ROS), but without activation of the proteins p53, Bax and Bcl-2 as well as without the enrolment of calpain-2. Autophagy was established as a contributing factor to NB-UVB-induced death execution. By contrast, wt cells triggered intrinsic apoptotic pathway that was resistant to the inhibition by zVAD-fmk and Necrostatin-1 pointing to the mechanism overcoming the cell survival. These findings support the role of FADD in prevention of autophagy-dependent apoptosis.
Asunto(s)
Apoptosis/efectos de la radiación , Autofagia/efectos de la radiación , Proteína de Dominio de Muerte Asociada a Fas/deficiencia , Fibroblastos/citología , Fibroblastos/efectos de la radiación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Rayos Ultravioleta , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Daño del ADN , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Imidazoles/farmacología , Indoles/farmacología , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Reactive oxygen species (ROS) generated after exposure to ionizing radiation and toxic peptides, in mitochondrial metabolism and during aging contribute to damage of cell's structural and functional components and can lead to diseases. Monomers and small oligomers of amyloid beta (Aß) peptide, players in Alzheimer's disease, are recently suggested to be involved in damaging of neurons, instead of extracellular Aß plaques. We demonstrate that externally applied disaggregated Aß1-42 peptide interacts preferentially with acidic compartments (lysosomes). We compared standard cell cultivation (21% O2) to more physiological cell cultivation (5% O2). Cells did not exhibit a dramatic increase in ROS and change in glutathione level upon 4 µM Aß peptide treatment, whereas exposure to 2 Gy X-rays increased ROS and changed glutathione level and ATP concentration. The occurrence of the 4977 bp deletion in mtDNA and significant protein carbonylation were specific effects of IR and more pronounced at 21% O2. An increase in cell death after Aß peptide treatment or irradiation was unexpectedly restored to the control level or below when both were combined, particularly at 5% O2. Therefore, Aß peptide at low concentration can trigger neuroprotective mechanisms in cells exposed to radiation. Oxygen concentration is an important modulator of cellular responses to stress.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Estrés Oxidativo/fisiología , Oxígeno/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos beta-Amiloides/administración & dosificación , Péptidos beta-Amiloides/farmacocinética , Animales , Línea Celular Tumoral , Humanos , Inflamación/metabolismo , Ratones , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/farmacocinéticaRESUMEN
Cytotoxic effects, including oxidative stress, of low linear energy transfer (LET)-ionizing radiation are often underestimated and studies of their mechanisms using cell culture models are widely conducted with cells cultivated at atmospheric oxygen that does not match its physiological levels in body tissues. Also, cell differentiation status plays a role in the outcome of experiments. We compared effects of 2 Gy X-ray irradiation on the physiology and mitochondrial proteome of nondifferentiated and human neuroblastoma (SH-SY5Y) cells treated with retinoic acid cultivated at 21% and 5% O2. Irradiation did not affect the amount of subunits of OxPhos complexes and other non-OxPhos mitochondrial proteins, except for heat shock protein 70, which was increased depending on oxygen level and differentiation status. These two factors were proven to modulate mitochondrial membrane potential and the bioenergetic status of cells. We suggest, moreover, that oxygen plays a role in the differentiation of human SH-SY5Y cells.
Asunto(s)
Diferenciación Celular/efectos de la radiación , Proteínas Mitocondriales/metabolismo , Neuroblastoma/patología , Oxígeno/farmacología , Proteoma/efectos de los fármacos , Proteoma/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Electroforesis en Gel Bidimensional , Humanos , Immunoblotting , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de la radiación , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Neuroblastoma/metabolismo , Rayos XRESUMEN
Fusion proteins combining oligomeric assemblies of a genetically obtained single-chain (sc) variant of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with antibodies directed against tumor-associated antigens represent a promising strategy to overcome the limited therapeutic activity of conventional soluble TRAIL. To further improve the scTRAIL module in order to obtain a robust, thermostable molecule of high activity, we performed a comprehensive analysis of the minimal TNF homology domain (THD) and optimized linkers between the 3 TRAIL subunits constituting a scTRAIL. Through a stepwise mutagenesis of the N- and C-terminal region and the joining linker sequences, we generated bioactive scTRAIL molecules comprising a covalent linkage of the C-terminal Val280 and the N-terminal position 122 by only 2 amino acid residues in combination with conservative exchanges at positions 122 and 279. The increased thermal stability and solubility of such optimized scTRAIL molecules translated into increased bioactivity in the diabody-scTRAIL (Db-scTRAIL) format, exemplified here for an epidermal growth factor receptor-specific Db-scTRAIL. Additional modifications within the diabody linkers resulted in a fusion protein exerting high, target-dependent apoptosis induction in tumor cell lines in vitro and potent antitumor activity in vivo. Our results illustrate that protein engineering of scTRAIL and associated peptide linkers provides a promising strategy to develop antibody-scTRAIL fusion proteins as effective antitumor therapeutics.