Antitumor activity of C5 against HL60 and M14 cell lines.
1 × 106 cells/mL were exposed to increasing concentrations (25-150 μg/mL) C5 for 18 hours, and cell death was determined by the MTT assay (HL60) or crystal violet assay (M14), as described in “Materials and methods.” Data shown are mean ± SD of 3 independent experiments performed in triplicate. (B) Determination of caspase 3 activation by a fluorometric assay designed to detect cleavage of tetrapeptide substrate DEVD-AFC. HL60 cells (1 × 106) were treated with 50-150 μg/mL C5 for 18 hours, and lysates were analyzed for DEVDase (caspase 3) activity, as described in “Materials and methods.” Data shown are mean ± SD of 3 independent experiments and are expressed asx increase in activity over the untreated HL60 cells. C2 (100 μg/mL), a potent inducer of caspase 3 activity, was used for the sake of comparison. The dotted line represents the activity obtained in lysates from untreated HL60 cells. (C) HL60 cells were exposed to C5 (150 μg/mL) or C2 (100 μg/mL) for 12 hours in the presence or absence of DEVD (1 μmol/L), permeabilized, incubated with phycoerythrin-conjugated antiactive caspase 3 antibody, and analyzed by flow cytometry, as described in “Materials and methods.” At least 10 000 events were analyzed for caspase 3 processing.
