Apollon (also known as BRUCE or BIRC6) is a large protein containing baculoviral-IAP-repeat (BIR) and ubiquitin-conjugating enzyme (UBC) domains at the amino- and carboxy termini, respectively. Apollon inhibits apoptosis, but its molecular and physiological function remains unclear. Here we report that Apollon binds to, ubiquitinates and facilitates proteasomal degradation of SMAC and caspase-9, which both contain IAP-binding motifs. Targeted disruption of Apollon in mice caused embryonic and neonatal lethality. Notably, SMAC induced apoptosis in Apollon-deficient cells, but not in Apollon-expressing cells. Furthermore, the IAP-binding motif of SMAC was required to induce apoptosis in Apollon-deficient cells. These results suggest that Apollon has an essential function in preventing SMAC-induced apoptosis.
Overexpression of an anti-apoptotic protein cIAP1 caused by its genetic amplification was reported in certain cancers, such as hepatocellular carcinoma, esophageal squamous cell carcinoma, cervical cancer, and lung cancer, which confers resistance to chemotherapy and radiotherapy. Here we report cIAP1 to be selectively down-regulated by a class of small molecules ((؊)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-L-leucine methyl ester (ME-BS)), resulting in a sensitization of cancer cells to apoptosis. ME-BS directly interacts with the BIR3 domain of cIAP1, promotes auto-ubiquitylation dependent on its RING domain, and facilitates proteasomal degradation of cIAP1. Other IAPs such as XIAP and cIAP2 were not affected by ME-BS. These results suggest targeted destabilization of cIAP1 by small molecules as a novel method to treat cancers expressing cIAP1, which interferes with treatment. Manipulation of the intrinsic ubiquitinligase activity could be a novel strategy to develop small molecules for therapeutic purposes.IAPs (inhibitor of apoptosis proteins) are a family of antiapoptotic proteins containing one to three baculoviral IAP repeat (BIR) 2 domains (1-3), some of which are frequently overexpressed in malignant cells. Certain IAPs such as XIAP/ hILP/BIRC4, cIAP1/MIHB/hiap-2/BIRC2, cIAP2/MIHC/hiap-1/BIRC3, ML-IAP/Livin/BIRC7 and Apollon/BRUCE/BIRC6 directly interact with and regulate caspases (4 -9). The BIR domain plays an important role in the interaction with caspases (8, 10, 11). These IAPs also contain a domain involved in ubiquitin conjugation (RING finger domain or UBC domain), and facilitate proteasomal degradation of caspases and IAPs (8, 12, 13). cIAP1 (cellular inhibitor of apoptosis protein 1) is overexpressed in human cancers such as esophageal squamous cell carcinoma, hepatocellular carcinoma, cervical cancer, and lung cancer, because of its genetic amplification and is regarded as an oncogene (14 -17). cIAP1 overexpression in cervical cancers correlates with resistance to radiotherapy. In addition, a comparative study of the expression of IAP family proteins and the sensitivity to chemotherapeutic drugs in 60 cell lines revealed the level of cIAP1 significantly correlates with resistance against anti-cancer drugs such as carboplatin, cisplatin, etoposide, and cytosine arabinoside (18). This evidence suggests cIAP1 to be a promising target for cancer therapy.Bestatin, an inhibitor of aminopeptidase N, has an immunomodulatory activity and is approved in Japan to treat patients with adult acute nonlymphatic leukemia (19 -21). In a clinical trial with stage I squamous cell lung carcinoma patients, bestatin significantly prolonged survival of these patients (22). Bestatin induces apoptosis in human leukemia cells (23) and augments death ligand-induced apoptosis in human solid tumor cell lines (24). In this paper, we describe selective down-regulation of cIAP1 by esterified analogs of bestatin represented by bestatin-methyl ester (ME-BS) (see Fig. 1A), resulting in a sensitization of cancer cells to ...
We investigated the growth inhibitory activity of bestatin, an inhibitor of aminopeptidase N (CD13), on six human leukemic cell lines. Proliferation of all the cell lines except KG1 was inhibited by bestatin. P39/TSU, HL60 and U937 were highly sensitive, with 50% growth inhibitory concentrations (IC 50 ) close to the maximum serum concentration when bestatin was orally administered at 30 mg in clinical application. All cell lines except for K562 highly expressed CD13, but a clear correlation between the sensitivity to bestatin and expression of CD13 was not observed. Other aminopeptidase inhibitors such as amastatin A, arphamenine B and WM15 antibody showed no growth inhibitory effects. To confirm the growth inhibitory effects of bestatin, we quantitatively examined DNA fragmentation in five bestatin-sensitive cell lines. Bestatin dose-dependently induced DNA fragmentation in those cell lines. In case of U937, bestatin induced DNA fragmentation quantitatively and DNA ladder and enhanced caspase-3 activity. Furthermore, the growth inhibition by bestatin was reduced by the caspase inhibitor Z-Asp-CH 2 -DCB. These results suggested that bestatin exhibits direct antileukemic effects against human leukemic cell lines through the induction of apoptosis.
We previously reported that the aminopeptidase inhibitor bestatin induced apoptosis in several human leukemia cell lines. The present study was performed to examine whether bestatin can also induce apoptosis in solid tumor cell lines. Bestatin alone exhibited neither direct growth inhibition nor induction of apoptosis in the tumor cell lines examined. However, it significantly augmented the growth-inhibitory effect and induction of apoptosis by agonistic anti-Fas antibody (CH11). The augmentation by bestatin was also observed with other death ligands including tumor necrosis factor-␣ (TNF-␣) in EBC-1 cells, a cell line sensitive to these death ligands. However, the HeLa S3 cell line, which is insensitive to TNF-␣, showed no growth inhibition even by combination treatment. Bestatin methyl ester, a more cell-permeable derivative of bestatin with similar inhibitory activity to cytosolic neutral aminopeptidase, potentiated cell growth inhibition of CH11 more efficiently than bestatin. Other cytosolic neutral aminopeptidase inhibitors such as actinonin and puromycin also augmented cell growth suppression by CH11, while an enantiomer of bestatin lacking aminopeptidase inhibitory action did not increase the growth-inhibitory effects of CH11. The combination of 10 g/ml of bestatin with CH11 promoted processing of capase 3 to the active form p17 and efflux of mitochondrial cytochrome c into the cytosol more quickly and more intensely than CH11 alone. Inhibition of aminopeptidase was not involved in dATP-and cytochrome c-dependent caspase 3-activation in a cell-free system. Bestatin significantly augmented activation of caspase 8, which is upstream of cytochrome c efflux in the apoptosis cascade. These results suggested that intracellular neutral aminopeptidase might play an important role in Fas-or TNF-␣-induced solid tumor cell apoptosis.
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