Bryostatin 1 enhances paclitaxel-induced mitochondrial dysfunction and apoptosis in human leukemia cells (U937) ectopically expressing Bcl-x_L

Abstract

The effects of the protein kinase C (PKC) activator and down-regulator bryostatin 1 were examined with respect to paclitaxel-induced apoptosis and antiproliferative activity in human myeloid leukemia cells (U937) displaying enforced expression of the anti-apoptotic protein Bcl-x_L. Overexpression of Bcl-x_L blocked various aspects of paclitaxel-mediated apoptosis, including caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), loss of mitochondrial membrane potential (Δ Ψ_m), and release of cytochrome c. However, subsequent (but not prior) exposure of paclitaxel-treated U937/Bcl-x_L cells (500 nM; 6 h) to bryostatin 1 (10 nM; 15 h) restored the extent of apoptosis, caspase activation, and mitochondrial damage to levels approximating those in paclitaxel-treated empty-vector control cells (U937/Neo). Potentiation of paclitaxel-induced apoptosis by bryostatin 1 in U937/Bcl-x_L cells occurred primarily in the G₂M cell population, and was associated with alterations in Bcl-x_L gel mobility and a reduction in paclitaxel-mediated stimulation of CDK1 activity. Enhancement of paclitaxel-induced apoptosis by bryostatin 1 in Bcl-x_L overexpressors was accompanied by a corresponding reduction in clonogenic potential. In contrast to its effects on apoptosis, bryostatin 1 failed to restore paclitaxel-mediated increases in free Bax levels in U937/Bcl-x_L cells. Lastly, the actions of bryostatin 1 were mimicked by a pharmacologic inhibitor of the MEK1/MAP kinase pathway (PD98059), but not by SB203580, an inhibitor of p 38 MAP kinase. Moreover, sequential exposure of both U937/Neo or/Bcl-x_L cells to paclitaxel followed by bryostatin 1 or PD98059 was associated with a net reduction in MAP kinase activity. Collectively, these findings indicate that protection against paclitaxel-mediated mitochondrial dysfunction and apoptosis in human U937 leukemia cells conferred by Bcl-x_L overexpression can be substantially overcome by bryostatin 1 and possibly other agents that interrupt the MAP kinase signal transduction pathway.