Mechanisms Inolved in Spontaneous and Viscum album Agglutinin-I-Induced Human Neutrophil Apoptosis: Viscum album Agglutinin-I Accelerates the Loss of Antiapoptotic Mcl-1 Expression and the Degradatio of Cytoskeletal Paxillin and Vimentin Proteins Via

摘要

Viscum album agglutinin-I (VAA-I) is a plant lectin that possesses interesting potential therapeutic properties and immunomodu-latory activities. We have recently found that VAA-I is a potent inducer of human neutophil apoptosis, but the mechanism(s) involved require further elucidation. In this study, we found that VAA-I alters mitochondrial transmembrane potential and increases intracellular levels of reactive oxygen species (ROS). Despite these observations, treatment with the mitochondrial stabilizer, bongkrekic acid, or with catalase, known to degrade H_2O_2, fails to reverse VAA-I-induced apoptosis. Moreover, VAA-I was found to induce apoptosis in PLB-985 cells deficient in gp91~(phox), indicating that the lectin acts via an ROS-independent mechanism. Pretreatment of neutrophils with brefeldin a, an inhibitor of vesicular transport, was found to reverse VAA-I-induced apoptosis. Protein expression of Mcl-1 was decreased by VAA-I. The role of caspases in the degradation of cytoskeletal proteins during both spontaneous and VAA-I-induced neutrophil apoptosis was also investigated. Paxillin and vimentin were markedly degraded by VAA-I when compare with neutrophils that undergo spontaneous apoptosis, but not vinculin or #alpha#- and #beta#-tubulin. Caspases were involved in cytoskeletal protein degradation because preincubation with the pan-caspase inhibitor N-benzyloxy-carbonyl-V-A-D-O-methylfluoromethyl ketone was found to reverse protein cleavage. We conclude that VAA-I needs to be internalized to mediate apoptosis and tat its activity is not dependent on a cell surface receptor-mediated pathway. Also, we conclude that VAA-I induces apoptosis by ROS-independent and Mcl-1-dependent mechanisms and that caspases are involved in cytoskeletal protein degradation in both spontaneous and VAA-I-induced neutrophil apoptosis.