Antimyeloma activity of NK012, a micelle-forming macromolecular prodrug of SN-38, in an orthotopic model.

NK012 is a micelle-forming macromolecular prodrug of 7-ethyl-10-hydroxy camptothecin (SN-38), an active metabolite of irinotecan. It is accumulated and retained in tumor tissues and gradually releases SN-38 in an enzyme-independent manner. NK012 was previously demonstrated to have stronger antitumor activity than irinotecan in a broad range of human solid-tumor xenograft models. In our study, we used an orthotopic multiple myeloma (MM) model created by injecting CD138-positive U266B1, a myeloma cell line that produces human IgE lambda light chain (monoclonal protein, M protein), into immunodeficient NOD/Shi-scid, IL-2Rγc (null) mice. This model shows typical bone marrow infiltration by the human myeloma cells. We evaluated the antimyeloma activity of intravenously administered NK012 in this model and showed that it suppressed the M protein concentration in the plasma and proliferation of myeloma cells in the bone marrow in a dose-dependent manner. NK012 suppressed the progression of hind-leg paralysis and prolonged the survival time of the mice compared to the untreated control group. In combination with bortezomib (BTZ), NK012 increased the median survival time compared to that with BTZ alone. In conclusion, these results suggest that NK012 is a potential candidate for use-alone and in combination-in the treatment of MM in humans.

Degradation-promoters of cellular inhibitor of apoptosis protein 1 based on bestatin and actinonin.

A series of hybrid compounds of bestatin (1) and actinonin (3), which promote degradation of cellular inhibitor of apoptosis protein 1 (cIAP1), were designed and synthesized. Structure-activity relationship studies indicated that absolute configuration, hydrophobicity at the alpha-position of the internal amide carbonyl group, and the presence of a small substituent at the alpha-position of the ester group are important factors for the expression of potent cIAP1 degradation-promoting activity. HAB-5A (30b) showed the most potent activity (IC(50)=0.53 microM) among the compounds prepared.

Small molecules destabilize cIAP1 by activating auto-ubiquitylation.

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 ubiquitin-ligase activity could be a novel strategy to develop small molecules for therapeutic purposes.

A derivative of aminopeptidase inhibitor (BE15) has a dual inhibitory effect of invasion and motility on tumor and endothelial cells.

Bestatin is an inhibitor of aminopeptidase N (APN)/CD13 and aminopeptidase B. In our previous report, bestatin inhibited the tumor cell invasion and the angiogenesis induced by the inoculation of B16-BL6 melanoma cells into mice and capillary formation on human umbilical vein endothelial cells (HUVECs) in vitro. The results show that the enzymatic activity of APN is deeply involved in tumor invasion and angiogenesis. We investigated the effect of three bestatin derivatives on A375 human melanoma cells and in vitro. All the derivatives inhibited the activity of APN, but BE15 was most effective and controlled the migration of A375 cells and HUVECs and capillary formation of HUVECs. Furthermore, the bestatin derivatives had an inhibitory effect not only on aminopeptidase activity but also on cell motility. Compared with bestatin and the other derivatives, BE15 had a marked inhibitory effect on the formation of capillary structure by HUVECs in vitro. These results suggest that new anti-metastatic and anti-angiogenic agents, which have a dual inhibitory effect on the degradation of the extra cellular matrix and cell motility, may be developed from bestatin.

Aminopeptidase N (APN/CD13) is selectively expressed in vascular endothelial cells and plays multiple roles in angiogenesis.

Proteolytic enzyme-mediated degradation of the extracellular matrix (ECM) is crucial for the formation of both tumor metastasis and angiogenesis. Recently, several reports have suggested that aminopeptidases are involved in this process, but precisely how is largely unknown. We found here that aminopeptidase N (APN/CD13) was selectively expressed in vascular endothelial cells including human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC), and was not detectable in a majority of normal cells and tumor cell lines we examined. RNA interference (RNAi) of APN resulted in the inhibition of capillary tube formation of HUVEC on Matrigel. APN siRNA suppressed the migration of HUVEC through a fibronectin-coated Transwell membrane, and reduced the cellular adhesion to Matrigel and various adhesion molecules including type IV collagen, type I collagen and fibronectin. These findings suggest that APN is a multifunctional protein with important roles in vascular endothelial morphogenesis during angiogenesis.

HtrA2 cleaves Apollon and induces cell death by IAP-binding motif in Apollon-deficient cells.

Apollon/BRUCE is a giant IAP protein that has BIR and UBC domains in its amino- and carboxy-terminals, respectively. Apollon binds and ubiquitylates SMAC/DIABLO and caspase9, and regulates apoptosis by facilitating proteasomal degradation of these proteins. Apollon overexpression inhibits apoptosis, while its downregulation sensitizes cells to apoptosis, suggesting that Apollon level is important for apoptosis regulation. Here we show that HtrA2/Omi catalytically cleaves Apollon with its serine protease activity. Conversely, Apollon ubiquitylates and facilitates proteasomal degradation of HtrA2 that binds to Apollon through IAP-binding motif. Thus, Apollon and HtrA2 mutually downregulate each other. Expression of catalytically active, but not inactive, HtrA2 induced apoptosis in Apollon-expressing cells. In Apollon-deficient cells, however, expression of catalytically inactive HtrA2 mutant with IAP-binding motif also induced apoptosis. These results indicate that HtrA2 induces apoptosis in two different mechanisms, one with serine protease domain and the other with IAP-binding motif, in Apollon-deficient cells.

Apollon ubiquitinates SMAC and caspase-9, and has an essential cytoprotection function.

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.