The pathways of reactive oxygen species (ROS)-mediated apoptosis induction, of Bax activation and the sensitization of tumor cells for TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis are still largely elusive.,Here, sensitization of melanoma cells for TRAIL by the PI3-kinase inhibitor wortmannin correlated to the activation of mitochondrial apoptosis pathways.,Apoptosis was dependent on Bax and abrogated by Bcl-2 overexpression.,The synergistic enhancement was explained by Bax activation through wortmannin, which tightly correlated to the characteristic Bax phosphorylation patterns.,Thus, wortmannin resulted in early reduction of the Bax-inactivating phosphorylation at serine-184, whereas the Bax-activating phosphorylation at threonine-167 was enhanced.,Proving the responsibility of the pathway, comparable effects were obtained with an Akt inhibitor (MK-2206); while suppressed phosphorylation of serine-184 may be attributed to reduced Akt activity itself, the causes of enhanced threonine-167 phosphorylation were addressed here.,Characteristically, production of ROS was seen early in response to wortmannin and MK-2206.,Providing the link between ROS and Bax, we show that abrogated ROS production by α-tocopherol or by NADPH oxidase 4 (NOX4) siRNA suppressed apoptosis and Bax activation.,This correlated with reduced Bax phosphorylation at threonine-167.,The data unraveled a mechanism by which NOX4-dependent ROS production controls apoptosis via Bax phosphorylation.,The pathway may be considered for proapoptotic, anticancer strategies.
Bim is known to be critical in killing of melanoma cells by inhibition of the RAF/MEK/ERK pathway.,However, the potential role of the most potent apoptosis-inducing isoform of Bim, BimS, remains largely unappreciated.,Here, we show that inhibition of the mutant B-RAFV600E triggers preferential splicing to produce BimS, which is particularly important in induction of apoptosis in B-RAFV600E melanoma cells.,Although the specific B-RAFV600E inhibitor PLX4720 upregulates all three major isoforms of Bim, BimEL, BimL, and BimS, at the protein and mRNA levels in B-RAFV600E melanoma cells, the increase in the ratios of BimS mRNA to BimEL and BimL mRNA indicates that it favours BimS splicing.,Consistently, enforced expression of B-RAFV600E in wild-type B-RAF melanoma cells and melanocytes inhibits BimS expression.,The splicing factor SRp55 appears necessary for the increase in BimS splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of BimS and apoptosis induced by PLX4720.,The PLX4720-induced, SRp55-mediated increase in BimS splicing is also mirrored in freshly isolated B-RAFV600E melanoma cells.,These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAFV600E inhibitors.