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Characterization of events associated with apoptosis/anoikis induced by snake venom metalloproteinase BaP1 on human endothelial cells
Author(s) -
Díaz Cecilia,
Valverde Lorena,
Brenes Oscar,
Rucavado Alexandra,
Gutiérrez José María
Publication year - 2004
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.20322
Subject(s) - anoikis , apoptosis , dna fragmentation , tunel assay , matrix metalloproteinase , biology , programmed cell death , terminal deoxynucleotidyl transferase , microbiology and biotechnology , metalloproteinase , snake venom , caspase , cancer research , venom , biochemistry
Human endothelial EA.hy926 cells were incubated with BaP1, a hemorrhagic metalloproteinase purified from Bothrops asper snake venom. Since the first hour of incubation with the proteinase, cells started showing DNA fragmentation, detected by a terminal deoxynucleotidyl transferase‐mediated dUDP nick‐end labeling (TUNEL)‐based photometric enzyme‐linked immunosorbent assay (ELISA). At later times, DNA fragments were predominantly located outside the cells, evidencing plasma membrane rupture. DNA fragmentation was completely abolished by Batimastat, a potent inhibitor of metalloproteinase enzymatic activity. Apoptosis induced by BaP1 on endothelial cells was independent of two Bcl‐2 family members (anti‐apototic Bcl‐xL and pro‐apoptotic Bax), that did not show any changes in their expression during a 24 h‐treatment period. Interestingly, IκBα, an inhibitor of NFκB, decreased after 24 h of treatment, suggesting further activation of the transcription factor. When some elements of the apoptotic extrinsic pathway were assessed, it was observed that procaspase‐8 completely disappeared after 24 h of treatment with BaP1, probably indicating its activation by a death receptor, whereas caspase‐8 inhibitor, cellular FLICE‐inhibitory protein (cFLIP L ), increased its expression since the first hours of BaP1 incubation. In conclusion, treatment of human endothelial cells with BaP1 induces apoptosis/anoikis, independently of Bcl‐2 family members Bax and Bcl‐xL and associated with caspase‐8 activation and cFLIP L up‐regulation. Apoptosis was completely dependent on BaP1 enzymatic activity. Similarities between this and other endothelial cell anoikis‐related systems suggest that BaP1 and other snake venom metalloproteinases may be useful experimental tools in the study of death‐related events that occur when adherent cells loose contact with extracellular matrix. © 2004 Wiley‐Liss, Inc.