RESUMO
Earlier studies showed that the oxidant menadione (MD) induces apoptosis in certain cells and also has anticancer effects. Most of these studies emphasized the role of the mitochondria in this process. However, the engagement of other organelles is less known. Particularly, the role of lysosomes and their proteolytic system, which participates in apoptotic cell death, is still unclear. The aim of this study was to investigate the role of lysosomal cathepsins on molecular signaling in MD-induced apoptosis in U937 cells. MD treatment induced translocation of cysteine cathepsins B, C, and S, and aspartic cathepsin D. Once in the cytosol, some cathepsins cleaved the proapoptotic molecule, Bid, in a process that was completely prevented by E64d, a general inhibitor of cysteine cathepsins, and partially prevented by the pancaspase inhibitor, z-VAD-fmk. Upon loss of the mitochondrial membrane potential, apoptosome activation led to caspase-9 processing, activation of caspase-3-like caspases, and poly (ADP-ribose) polymerase cleavage. Notably, the endogenous protein inhibitor, stefin B, was degraded by cathepsin D and caspases. This process was prevented by z-VAD-fmk, and partially by pepstatin A-penetratin. These findings suggest that the cleaved Bid protein acts as an amplifier of apoptotic signaling through mitochondria, thus enhancing the activity of cysteine cathepsins following stefin B degradation.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Cistatina B/genética , Regulação Neoplásica da Expressão Gênica , Lisossomos/efeitos dos fármacos , Vitamina K 3/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/genética , Apoptossomas/efeitos dos fármacos , Apoptossomas/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Catepsina B/metabolismo , Catepsina C/antagonistas & inibidores , Catepsina C/genética , Catepsina C/metabolismo , Catepsina D/antagonistas & inibidores , Catepsina D/genética , Catepsina D/metabolismo , Catepsinas/antagonistas & inibidores , Catepsinas/genética , Catepsinas/metabolismo , Cistatina B/metabolismo , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Lisossomos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Pepstatinas/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , Transdução de Sinais , Células U937RESUMO
Lysosomal cathepsins were previously found to be involved in tumor necrosis factor-α (TNFα)-induced apoptosis. However, there are opposing views regarding their role as either initiators or amplifiers of the signaling cascade as well as the order of molecular events during this process. In this study, we investigated the role of cathepsin D (catD) in TNFα/cycloheximide-induced apoptosis in U937 human monocytic cells. TNFα-induced apoptosis proceeds through caspase-8 activation, processing of the pro-apoptotic molecule Bid, mitochondrial membrane permeabilization, and caspase-3 activation. The translocation of lysosomal catD into the cytosol was a late event, suggesting that lysosomal membrane permeabilization and the release of cathepsins are not required for the induction of apoptosis, but rather amplifies the process through the generation of reactive oxygen species. For the first time, we show that apoptosis is accompanied by degradation of the cysteine cathepsin inhibitor stefin B (StfB). CatD did not exhibit a crucial role in this step. However, this degradation was partially prevented through pre-incubation with the antioxidant N-acetyl cysteine, although it did not prevent apoptosis and its progression. These results suggest that the degradation of StfB, as a response to TNFα, could induce a cell death amplification effect as a result of progressive damage to lysosomes during TNFα treatment. J. Cell. Biochem. 118: 4813-4820, 2017. © 2017 Wiley Periodicals, Inc.
