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Several inhibitors of BCL2 proteins have been identified that induce apoptosis in a variety of tumor cells, indicating their potential in cancer therapy. We investigated the specificity of six putative BCL2 inhibitors (obatoclax, gossypol, apogossypol, EM20-25, chelerythrine and ABT-737). Using cells deficient either for Bax/Bak or caspase-9, we found that only ABT-737 specifically targeted BCL2 proteins and induced apoptosis by activation of caspase-9, as only ABT-737 induced apoptosis was completely inhibited in cells deficient for Bax/Bak or caspase-9. Our data show that only ABT-737 is a specific BCL2 inhibitor and all other compounds investigated were not specific for BCL2 proteins. Furthermore, investigations of the effects of these compounds in primary chronic lymphocytic leukemic cells showed that all compounds induced certain biochemical hallmarks of apoptosis, such as release of cytochrome c and caspase cleavage. However, they all caused strikingly different ultrastructural changes. ABT-737 induced all the characteristic ultrastructural changes of apoptosis together with early rupture of the outer mitochondrial membrane, whereas obatoclax, chlelerythrine and gossypol induced pronounced mitochondrial swelling with formation of phospholipid inclusions. Therefore, we conclude that biochemical measurements used earlier to define apoptosis like mitochondrial release of cytochrome c and caspase cleavage, are insufficient to distinguish between classic apoptosis and other forms of cell death.

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Apoptosis in megakaryocytes results in the formation of platelets. The role of apoptotic pathways in platelet turnover and in the apoptotic-like changes seen after platelet activation is poorly understood. ABT-263 (Navitoclax), a specific inhibitor of antiapoptotic BCL2 proteins, which is currently being evaluated in clinical trials for the treatment of leukemia and other malignancies, induces a doselimiting thrombocytopenia. In this study, the relationship between BCL2/BCL-X L inhibition, apoptosis, and platelet activa- IntroductionAll nucleated cells in multicellular organisms are genetically programmed to undergo apoptosis to remove unnecessary or damaged cells from the whole organism. This program has been recognized as the central mechanism of platelet production from megakaryocytes. 1 However, the role of apoptosis in anuclear, mature platelets is less well characterized, with apoptotic-like changes seen in both aging platelets and in the formation of procoagulant microparticles after agonist stimulation.Two main pathways lead to the execution of apoptosis: the extrinsic and the intrinsic (or mitochondrial) pathways. Both converge into the activation of caspases, which are proteases that cleave Ͼ 500 cellular targets and induce typical morphologic changes associated with apoptosis in nucleated cells. A critical step in the intrinsic pathway is the loss of mitochondrial membrane potential (MMP) and the release of cytochrome c into cytosol, where it triggers the activation of caspase-9. Therefore, the release of cytochrome c from mitochondria needs to be tightly regulated: a function that is fulfilled by the BCL2 protein family, which consists of proapoptotic and antiapoptotic members that promote or block the release of cytochrome c, respectively. 2,3 The proapoptotic family members BAX and BAK play an essential role in directly mediating the release of cytochrome c by forming a pore in the outer mitochondrial membrane. Antiapoptotic BCL2 proteins, including BCL2, BCL-X L , BCL-w, MCL1, and BCL2A1, prevent the activation of BAX and BAK. Besides their function in regulating mitochondrial cytochrome c release, BCL2 proteins have also been implicated in the regulation of intracellular calcium homeostasis at the endoplasmic reticulum (ER), possibly by interacting with inositol triphosphate receptors. 4,5 Because of their key role, the antiapoptotic BCL2 proteins are attractive targets for anticancer therapy, with several small molecule inhibitors currently in preclinical testing or early clinical trials. 6,7 Among these, the most promising and specific inhibitors are ABT-263 (Navitoclax) and 9 ABT-737 shows promising antitumor activity in animal models of leukemia and lymphoma. A related compound, ABT-263, is metabolically more stable and currently in phase 1 and 2 clinical trials for leukemia and other malignancies. 10 Both compounds have often been regarded as interchangeable because they bind with high affinity to BCL2, BCL-X L , and BCL-w but do not inhibit MCL1 or BCL2A1. 11 Early results from the clinica...

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Resistance to apoptosis is a hallmark of pancreatic cancer, a leading cause of cancer deaths. Therefore, novel strategies are required to target apoptosis resistance. Here, we report that the combination of X-linked inhibitor of apoptosis (XIAP) inhibition and tumor necrosis factor-related apoptosisinducing ligand (TRAIL) is an effective approach to trigger apoptosis despite Bcl-2 overexpression and to suppress pancreatic cancer growth in vitro and in vivo. Knockdown of XIAP by RNA interference cooperates with TRAIL to induce caspase activation, loss of mitochondrial membrane potential, cytochrome c release, and apoptosis in pancreatic carcinoma cells. Loss of mitochondrial membrane potential and cytochrome c release are extensively inhibited by a broad range or caspase-3 selective caspase inhibitor and by RNAi-mediated silencing of caspase-3, indicating that XIAP inhibition enhances TRAIL-induced mitochondrial damage in a caspase-3-dependent manner. XIAP inhibition combined with TRAIL even breaks Bcl-2-imposed resistance by converting type II cells that depend on the mitochondrial contribution to the death receptor pathway to type I cells in which TRAILinduced activation of caspase-3 and caspase-9 and apoptosis proceeds irrespective of high Bcl-2 levels. Most importantly, XIAP inhibition potentiates TRAIL-induced antitumor activity in two preclinical models of pancreatic cancer in vivo. In the chicken chorioallantoic membrane model, XIAP inhibition significantly enhances TRAIL-mediated apoptosis and suppression of tumor growth. In a tumor regression model in xenograft-bearing mice, XIAP inhibition acts in concert with TRAIL to cause even regression of established pancreatic carcinoma. Thus, this combination of XIAP inhibition plus TRAIL is a promising strategy to overcome apoptosis resistance of pancreatic cancer that warrants further investigation. [Cancer Res 2008;68(19):7956-65]

Evasion of apoptosis is a characteristic feature of pancreatic cancer, a prototypic cancer that is refractory to current treatment approaches. Hence, there is an urgent need to design rational strategies that counter apoptosis resistance. To explore X-linked inhibitor of apoptosis (XIAP) as a therapeutic target in pancreatic cancer, we analyzed the expression of XIAP in pancreatic tumor samples and evaluated the effect of small molecule XIAP inhibitors alone and in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against pancreatic carcinoma in vitro and in vivo. Here, we report that XIAP is highly expressed in pancreatic adenocarcinoma samples compared with normal pancreatic ducts. Small molecule XIAP inhibitors synergize with TRAIL to induce apoptosis and to inhibit long-term clonogenic survival of pancreatic carcinoma cells. In contrast, they do not reverse the lack of toxicity of TRAIL on nonmalignant cells in vitro or normal tissues in vivo, pointing to a therapeutic index. Most importantly, XIAP inhibitors cooperate with TRAIL to trigger apoptosis and suppress pancreatic carcinoma growth in vivo in two preclinical models, i.e., the chorioallantoic membrane model and a mouse xenograft model. Parallel immunohistochemical analysis of tumor tissue under therapy reveals that the XIAP inhibitor acts in concert with TRAIL to cause caspase-3 activation and apoptosis. In conclusion, our findings provide, for the first time, evidence in vivo that XIAP inhibitors prime pancreatic carcinoma cells for TRAIL-induced apoptosis and potentiate the antitumor activity of TRAIL against established pancreatic carcinoma. These findings build the rationale for further (pre)clinical development of XIAP inhibitors and TRAIL against pancreatic cancer. [Cancer Res 2009;69(6):2425-34]