Figure 6. NGR-peptide-1-induced cell death does not depend upon Bcl-2 family proteins and caspases' activity.

(A and B) U937 cells were cultured for in the absence or presence of 50 μM NGR-peptides for 10 min or 100 nM flavopiridol for 14 h (positive control) or diluted DMSO (control for flavopiridol). After which lysates were western blotted with antibodies against (A) the pro-apoptotic proteins Bak and Bax (active proteins) and Bid, (B) the anti-apoptotic proteins Bcl-2 and Mcl-1, and (A, B) actin. One of three representative experiments is shown. (C) U937 cells were cultured for in the absence or presence of 50 μM NGR-peptides for 10 min and 14 h, or 100 nM flavopiridol for 14 h. Caspase-3,-8 and -9 activities were determined using the substrates DEVD-pNA, IETD-pNA and LEHD-pNA, respectively. Release of pNA was measured at 405 nm. Data are expressed as a fold-increase relative to the corresponding untreated samples (baseline values for caspase-8, caspase-9, and caspase-3 activity at 10 min were 17 ± 2, 4 ± 1, and 8 ± 2 pmol/60 min/mg protein at 37°C, respectively; baseline values for caspase-8, caspase-9, and caspase-3 activity at 14 h were 21 ± 2, 6 ± 2, and 12 ± 2 pmol/60 min/mg protein at 37°C, respectively). Data are mean ± SD of three independent determinations. (D) Cells were incubated with 50 μM NGR-peptides for 15 min or 100 nM flavopiridol for 14 h, after 30 min of pretreatment with 50 μM ZVAD-fmk (a broad-spectrum caspase inhibitor). The proportion (%) of dead cells (L2 + L3 gates) is shown in the box. No effect was observed with ZVAD-fmk alone. One of three representative experiments is shown.