Chem. knockdown of Bid. However, knockdown of X chromosome-linked IAP (XIAP) or antagonism of XIAP allows FasL or DR5 agonist antibody to induce activation of effector caspases efficiently Cobimetinib (racemate) without the need for mitochondrial amplification of the apoptotic signal and thus rescues the effect of Bid knockdown in these cells. Introduction Apoptosis, or programmed cell death, is a genetically regulated process with critical roles in development and homeostasis in metazoans (1). Deficient apoptosis leads to the absence of normal cell death and contributes to the development and progression of human cancers (2). Apoptotic cell death can be initiated through the engagement of cell surface proapoptotic receptors by their specific ligands or by changes in internal cellular integrity (3, 4). Both of these pathways Cobimetinib (racemate) converge at the activation of caspases, cysteine-dependent aspartyl-specific proteases that comprise the effector arm of apoptotic cell death (5, 6). The intrinsic or mitochondrial pathway is initiated by developmental cues or cellular stress signals. These signals activate Bcl-2 homology 3 (BH3)3 proteins, leading to neutralization of the antiapoptotic proteins, such as Bcl-2, Bcl-xL, or Mcl-1, activation of proapoptotic proteins Bax and Bak, and subsequent disruption of mitochondrial membrane potential (7). The resulting release of cytochrome from the mitochondria into the cytoplasm leads to Apaf-1-mediated caspase-9 activation and consequent activation of effector caspases-3 and -7 and culminates in cell death. The extrinsic apoptotic pathway is triggered when proapoptotic receptors such as Fas or death receptor 5 (DR5) are engaged by their respective ligands, resulting in recruitment of the adaptor protein FADD and the apical caspases 8- or -10 (3). Incorporation of these caspases into the receptor-associated death-inducing signaling complex causes their autoactivation and leads to ensuing activation of effector caspases-3 and -7. In most cell types (type II cells), amplification of extrinsic pathway signaling through caspase-8-mediated activation of the BH3-only protein Bid is critical for efficient execution of apoptosis (8, 9); in type I cells direct activation of effector caspases by caspase-8 is sufficient. Bid plays an important role in a number of cellular pathways including regulation of Fas- and TNFR1-mediated hepatocellular injury (9,C13). In addition to stimulation by their respective ligands, proapoptotic receptors can be engaged by agonistic antibodies (14). DR5 agonist antibody (“type”:”entrez-protein”,”attrs”:”text”:”PRO95780″,”term_id”:”1357785992″,”term_text”:”PRO95780″PRO95780) binds DR5 tightly and selectively, triggering apoptosis in various types of cancer cells and inhibiting tumor xenograft growth (15, 16). IAP proteins represent the ultimate line of defense against Cobimetinib (racemate) cellular suicide by regulating caspase activity and preventing caspase activation (17). c-IAP1 and c-IAP2 are components of TNF receptor (TNFR) complexes where they modulate apoptotic signaling and caspase-8 activation (18,C20). X chromosome-linked IAP (XIAP) is the only true endogenous inhibitor of caspases because other IAP proteins exhibit weak binding to and inhibition of caspases (21). XIAP inhibits caspases-3 and -7 Cobimetinib (racemate) using the linker region between its baculoviral IAP-repeat (BIR) domain 1 (BIR1) and BIR2 as well as the BIR2 domain, whereas inhibition of caspase-9 relies on the binding of the BIR3 domain to an N-terminal IAP-binding motif of partially Rabbit polyclonal to AGAP processed caspase-9 (21, 22). Caspase inhibition by XIAP is blocked by second mitochondrial activator of caspases (SMAC) (23, 24). During induction of apoptosis, SMAC undergoes proteolyic processing, resulting in its release from mitochondria into the cytoplasm where it can bind to and antagonize the BIR2 and BIR3 domains of XIAP via an exposed IAP-binding motif (23, 24). IAP-mediated inhibition of cell death and promotion of survival signaling pathways are important for tumor maintenance and therapeutic resistance to anticancer agents. These properties distinguish IAP proteins as attractive targets for anticancer therapeutic intervention (25). Efforts to identify small molecule antagonists of IAPs have led to the discovery of a number of IAP antagonistic compounds that possess antiapoptotic activity both and (26, 27). These IAP antagonists induce cell death that depends on TNF signaling, stimulation of NF-B pathways, and caspase activation (28,C31). Here, we demonstrate that combination of IAP antagonists and proapoptotic receptor agonists (PARAs), namely Fas ligand (FasL) or DR5 agonist antibody, synergistically activates apoptosis in cancer cells and inhibits tumor growth test. Tumor Xenograft Study All procedures involving animals were performed in accordance with the guidelines of the Genentech Institutional Animal Care and Use Committee. To generate tumors, 100 l of a single-cell suspension containing 5 106 MDA-MB-231-X1.1 cells was injected subcutaneously into the right thoracic flanks of female C.B-17 mice (Charles River Laboratories, Hollister, CA).