Caspase-8-mediated BID Cleavage and Release of Mitochondrial Cytochrome cduringNω-Hydroxy-l-arginine-induced Apoptosis in MDA-MB-468 Cells

We have previously reported thatNω-hydroxy-l-arginine (NOHA), a stable intermediate product formed during the conversion ofl-arginine to nitric oxide, induced apoptosis in MDA-MB-468 cells, and this action was antagonized in the presence ofl-ornithine. We also reported that apoptosis induced by NOHA in this cell line could not be explained on the basis of a reduction of intracellular polyamines. In the current study, we investigated other potential mechanism(s) by which NOHA may have induced apoptosis in this cell line. We observed that NOHA initially activated caspase-8 and induced cleavage of BH3interacting domain. This was followed by release of cytochrome cand subsequently, activation of downstream caspases-9 and -3 to cleave poly(ADP-ribose) polymerase. We also observed that NOHA induced a rapid and persistent hyperpolarization of the mitochondrial membrane potential rather than depolarization indicating that the release of cytochrome cby NOHA was by a mechanism independent of the mitochondrial transition pore. Exogenous l-ornithine did not inhibit NOHA-induced caspase-8 activation and cleavage of BH3interacting domain but acted at the mitochondrial level and inhibited the NOHA-induced cytochrome crelease and apoptosis.