The activated caspase 9 leads to the activation of downstream eector caspase, such as caspase 3, which cleaves cell death to be executed by a number of cellular proteins. It has recently been suggested that in receptor mediated apoptosis, Bid, activated by caspase 8, is translocated to the mitochondria and induces the release of cytochrome c, Capecitabine 154361-50-9 whereas in chemical induced apoptosis, cytochrome c release is caspaseindependent and is not mediated by cleavage of Bid. Bax is a proapoptotic members of the Bcl 2 family that resides in the cytosol and translocates to mitochondria upon induction of apoptosis. Recently, Bax has been shown to induce caspase activation and cytochrome c release in vivo and in vitro. In contrast, BclxL and antiapoptotic Bcl 2 could block cytochrome c release in cells under-going apoptosis. The antiapoptotic Bcl 2 family live on the outer mitochondrial membrane and can prevent apoptosis by many things including homo or heterodimerization with other family members, maintenance of normal mitochondrial membrane resulting in the prevention of cytochrome c release and subsequent caspase activation. Recent studies show that Bcl xL abolishes apoptosis, caspase 3 action, and release of cytochrome c induced by ceramide. Currently, it’s still Lymph node not yet determined how ceramide acts on mitochondria. Within this statement, we examined pathways downstream of ceramide, with specific emphasis on the capability of Bax to induce the release of cytochrome c and apoptosis, and we considered the associations between caspase activation and mitochondrial dysfunction. By using a speci?c bax antisense oligonucleotide, we show the crucial functional part of Bax in ceramide induced apoptosis. We demonstrate that antisense bax inhibits cytochrome c release, poly polymerase cleavage and cell death. In-addition, ceramide induces translocation of Bax to mitochondria and escalates the ratio of Bax to Bcl xL. Our findings Enzalutamide supplier suggest that Bax plays an essential role in managing the apoptotic process upstream of cytochrome c release induced by ceramide. C6 ceramide was obtained from Sigma. Lipofectamine was obtained from Life Technologies. Fetal bovine serum was from Gibco BRL, ECL kit from Amersham Pharmacia, caspase 3, 8, 9 substrates from Biomol, and Hoechst 33258 from Molecular Probes. Antibody to cytochrome c was from Pharmingen. Antibodies to Bcl 2, Bax, BclxL and HRP conjugated secondary antibody were from Santa Cruz Biotechnology. zVAD fmk was from Enzyme System Products. HL 60 cells were washed with serum free RPMI. Ceramide, zVADfmk o-r vehicle was diluted in to serum free RPMI in the indicated concentrations. DNA fragmentation and cell viability were analyzed as described previously.