With the goal of gaining insight into the signaling pathways involved, we examined the activation of caspases 3, 8 and Dizocilpine GluR Chemicals 9, in addition to the effect of caspase inhibitors. Because no caspase 9 activation or mitochondrial cytochrome c release to cytosol was found, the mitochondrial pathway did not contribute notably to the apoptotic process. More over, death receptor mediated apoptosis was recommended by the translocation of Fas associated death domain to the cell membrane together with caspase 8 activation. The same susceptibility was shown by human peripheral lymphocytes either stimulated with phytohemagglutinin or not, to viability decrease induced by these trypsin inhibitors. G. dubium seeds were by hand gathered from trees growing in Misiones, Argentina and were kindly given by Dr. Teresa Arg?elles y Andr?s, from the Universidad Forestal of Misiones. P. dubium trypsin inhibitor was isolated as described before by affinity chromatography on a Organism agarose column. All PDTI preparations were checked for endotoxin contamination by LAL check, Gel clot Pyrotel, and the last endotoxin content of PDTI found in this study was b0. 2 endotoxin models /mg of protein. Soybean trypsin inhibitor. trypsinagarose, RPMI medium, HEPES buffer, penicillin, streptomycin, glutamine, RNase A, RNase T, propidium iodide, staurosporine, phytohemagglutinin, bovine serum albumin and rabbit antiactin antibody were obtained from Sigma Chemical Co.. High glucose Dulbeccos altered Eagle medium was from Gibco. Camptothecin was from Fluka, mouse anti human FADD and mouse anti human cytochrome c monoclonal antibodies were obtained from BD Pharmingen, goat anti mouse IgG and anti rabbit IgG coupled to horseradish peroxidase were from Santa Cruz Biotechnology, Inc. General caspase inhibitor was from Calbiochem and caspase 8 inhibitor and caspase 9 inhibitor were obtained from natural product libraries Santa Cruz Biotechnology, Inc. The human Jurkat severe T cell leukemia cell line was grown in RPMI 1640 supplemented with 10% heatinactivated fetal bovine serum. 50 mM HEPES 50 ug/ml streptomycin, 50 U/ml penicillin, buffer and 2 mM L glutamine at 37 C in a humidified atmosphere of five hundred CO2. The human primary hepatocellular carcinoma cell line, HepG2 and human cervical adenocarcinoma cell line, HeLa were cultured in high glucose Dulbeccos modified Eagle medium supplemented with 10% warmth inactivated, 50 U/ml penicillin and 50 ug/ml streptomycin at 37 C in a atmosphere of 5% CO2. For the experiments, cells were plated 24 h ahead of the treatments to permit adherence. Human blood was mixed with heparin sulfate, obtained from healthier donors and diluted with phosphate saline buffer.