The inhibitors of apoptosis proteins (IAP) are important intrinsic cellular inhibitors of apoptosis, highly expressed in cancer cells and play an important role in resistance to apoptosis induced by cancer therapy. Since IAP blocks apoptosis at the down-stream effector phase, a point where multiple signaling pathways converge, IAP is a promising molecular target for discovery of novel anticancer drugs aiming at overcoming apoptosis-resistance. We first discovered that Embelin, a natural compound from the Japanese Ardisia herb, is a small molecule inhibitor that binds to the XIAP BIR3 domain with an affinity similar to that of the natural Smac peptide. Our NMR analysis conclusively confirmed that Embelin interacts with several crucial residues in the XIAP BIR3 domain with which Smac and caspsase-9 bind. Embelin potently inhibits cell growth, induces apoptosis and activates caspase-9 in prostate cancer cells with high levels of XIAP, but has a minimal effect on normal prostate epithelial and fibroblast cells with low levels of XIAP. Embelin potentiated Cisplatin- and docetaxel-induced apoptosis in human prostate cancer PC-3 cells that have high levels of XIAP and cIAP-1. Embelin effectively overcame the protective effect of XIAP and enhanced etoposide-induced apoptosis in Jurkat cells stably transfected with XIAP, but not in Jurkat cells transfected with control vector. In Jurkat cells stably transfected with a BIR1-2-deleted version of XIAP which retains a functional BIR3 domain, Embelin completely overcame the apoptosis-resistance rendered by the IAP, indicating that BIR3 domain in XIAP is involved in Embelin-induced apoptosis. In nude mouse xenograft models of human prostate cancer PC-3 and DU-145, oral administration of Embelin at 50mg/kg daily for 3 weeks significantly inhibited the growth of established xenograft tumors, with no obvious toxicity to the animals. We have also synthesized a series of more potent IAP inhibitors that bind to XIAP BIR3 domain 20-30-times more potent than Smac peptide. Using NMR methods, we conclusively show that these IAP inhibitors bind to the pocket in the XIAP BIR3 domain where Smac binds. Biotin-labeled compound pull-down assay showed that XIAP, cIAP-1 and cIAP-2 proteins can be pulled down by the IAP-inhibitors. Our IAP inhibitors potently enhanced chemotherapy-induced apoptosis in PC-3 cells and sensitized PC-3 cells to ionizing irradiation up to 10-fold in a clonogenic assay. Studies with Jurkat cells transfected with XIAP or XIAP-BIR3 further confirm that BIR3 domain in XIAP is indeed the molecular target of the IAP inhibitors in apoptosis-potentiation. Taken together, our results demonstrate that the small molecule IAP inhibitors can overcome apoptosis-resistance in cancer cells with high levels of IAP proteins. Modulating IAP family of proteins holds promise as a potential novel molecular therapy for human prostate cancer.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]