Characterization of events associated with apoptosis/anoikis induced by snake venom metalloproteinase BaP1 on human endothelial cells.
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, IkappaBalpha, an inhibitor of NFkappaB, 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.
Díaz
Cecilia
C
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. cdiaz@cariari.ucr.ac.cr
Valverde
Lorena
L
Brenes
Oscar
O
Rucavado
Alexandra
A
Gutiérrez
José María
JM
D023102:Anoikis / Q000187:drug effects*; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012910:Snake Venoms / Q000201:enzymology*; D013876:Thiophenes / Q000494:pharmacology; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494826:BAX protein, human; C494811:BCL2L1 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D012910:Snake Venoms; D013876:Thiophenes; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D010649:Phenylalanine; C080985:batimastat; D045726:Metalloproteases; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases