Involvement of CD95-independent caspase 8 activation in arsenic trioxide-induced apoptosis

Arsenic trioxide (As2O3)-treatment is effective in acute promyelocytic leuk emia (APL) patients with t(15;17). Clinically achievable concentrations of As2O3 induce apoptosis in NB4, an APL cell line, in vitro. Here, to study t he mechanism of As2O3-induced apoptosis, we established an As2O3-resistant subline, NB4/As. Growth of NB4/As was inhibited by 50% after 2 day-treatmen t (IC50) at 1.6 mu M As2O3, whereas IC50 of NB4 was 0.3 mu M. Degradation o f PML-RAR alpha and change of the PML-subcellular localization were similar ly induced by As2O3 in NB4 and NB4/As, suggesting that their contribution t o apoptosis is small. Treatment with 1 mu M As2O3 induced the activation of caspase 3 as well as a loss of mitochondrial transmembrane potential (Delt a Psi m) in NB4 but not in NB4/As. Caspase 8 and Bid were also activated by As2O3 in NB4 but not in NB4/As. In NB4, an inhibitor of caspase 8 blocked not only the activation of caspase 3 but also the loss of Delta Psi m. Neit her cell line expressed CD95/Fas, and agonistic anti-Fas antibody (CH-11) f ailed to cause apoptosis. Neither antagonistic anti-CD95/Fas antibody nor a nti-fas ligand antibodies influenced the As2O3-induced apoptosis. NB4/As ha d a higher concentration of intracellular glutathione (GSH) than NB4 (96 vs 32 nmol/mg). Reduction of the GSH level by buthionine sulfoxide (BSO) comp letely restored the sensitivity to As2O3 in NB4/As. Furthermore, caspase ac tivation and the loss of Delta Psi m were recovered by combination treatmen t with BSO. These findings suggest that the As2O3 treatment activates caspa se 8 in a CD95-independent but GSH concentration-dependent manner. In combi nation with BSO, As2O3 might be applied to therapy of leukemia/cancers whic h are insensitive to the clinically achievable concentrations of As2O3.