Small Molecules Destabilize cIAP1 by Activating Auto-ubiquitylation

Abstract

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. 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 anti-apoptotic proteins containing one to three baculoviral IAP repeat (BIR) 2The abbreviations used are: BIRbaculoviral IAP repeatME-BS(–)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-l-leucine methyl esterCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidGSTglutathione S-transferaseE2ubiquitin carrier proteinZ-LLHcarbobenzoxy-l-leucyl-l-leucinalTRAILTNF-related apoptosis-inducing ligandTNFtumor necrosis factorSMAC/DIABLOsecond mitochondria-derived activator of caspase/direct IAP-binding protein with low pI. 2The abbreviations used are: BIRbaculoviral IAP repeatME-BS(–)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-l-leucine methyl esterCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidGSTglutathione S-transferaseE2ubiquitin carrier proteinZ-LLHcarbobenzoxy-l-leucyl-l-leucinalTRAILTNF-related apoptosis-inducing ligandTNFtumor necrosis factorSMAC/DIABLOsecond mitochondria-derived activator of caspase/direct IAP-binding protein with low pI. domains (1Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2278) Google Scholar, 2Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1573) Google Scholar, 3Vaux D.L. Silke J. Nat. Rev. Mol. Cell. Biol. 2005; 6: 287-297Crossref PubMed Scopus (528) Google Scholar), 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 (4Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1716) Google Scholar, 5Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1137) Google Scholar, 6Kasof G.M. Gomes B.C. J. Biol. Chem. 2001; 276: 3238-3246Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 7Vucic D. Stennicke H.R. Pisabarro M.T. Salvesen G.S. Dixit V.M. Curr. Biol. 2000; 10: 1359-1366Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 8Hao Y. Sekine K. Kawabata A. Nakamura H. Ishioka T. Ohata H. Katayama R. Hashimoto C. Zhang X. Noda T. Tsuruo T. Naito M. Nat. Cell Biol. 2004; 6: 849-860Crossref PubMed Scopus (192) Google Scholar, 9Bartke T. Pohl C. Pyrowolakis G. Jentsch S. Mol. Cell. 2004; 14: 801-811Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The BIR domain plays an important role in the interaction with caspases (8Hao Y. Sekine K. Kawabata A. Nakamura H. Ishioka T. Ohata H. Katayama R. Hashimoto C. Zhang X. Noda T. Tsuruo T. Naito M. Nat. Cell Biol. 2004; 6: 849-860Crossref PubMed Scopus (192) Google Scholar, 10Takahashi R. Deveraux Q. Tamm I. Welsh K. Assa-Munt N. Salvesen G.S. Reed J.C. J. Biol. Chem. 1998; 273: 7787-7790Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, 11Srinivasula S.M. Hegde R. Saleh A. Datta P. Shiozaki E. Chai J. Lee R.A. Robbins P.D. Fernandes-Alnemri T. Shi Y. Alnemri E.S. Nature. 2001; 410: 112-116Crossref PubMed Scopus (859) Google Scholar). These IAPs also contain a domain involved in ubiquitin conjugation (RING finger domain or UBC domain), and facilitate proteasomal degradation of caspases and IAPs (8Hao Y. Sekine K. Kawabata A. Nakamura H. Ishioka T. Ohata H. Katayama R. Hashimoto C. Zhang X. Noda T. Tsuruo T. Naito M. Nat. Cell Biol. 2004; 6: 849-860Crossref PubMed Scopus (192) Google Scholar, 12Suzuki Y. Nakabayashi Y. Takahashi R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8662-8667Crossref PubMed Scopus (545) Google Scholar, 13Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (866) Google Scholar). baculoviral IAP repeat (–)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-l-leucine methyl ester 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid glutathione S-transferase ubiquitin carrier protein carbobenzoxy-l-leucyl-l-leucinal TNF-related apoptosis-inducing ligand tumor necrosis factor second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI. baculoviral IAP repeat (–)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-l-leucine methyl ester 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid glutathione S-transferase ubiquitin carrier protein carbobenzoxy-l-leucyl-l-leucinal TNF-related apoptosis-inducing ligand tumor necrosis factor second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI. 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 (14Imoto I. Yang Z.Q. Pimkhaokham A. Tsuda H. Shimada Y. Imamura M. Ohki M. Inazawa J. Cancer Res. 2001; 61: 6629-6634PubMed Google Scholar, 15Imoto I. Tsuda H. Hirasawa A. Miura M. Sakamoto M. Hirohashi S. Inazawa J. Cancer Res. 2002; 62: 4860-4866PubMed Google Scholar, 16Dai Z. Zhu W.G. Morrison C.D. Brena R.M. Smiraglia D.J. Raval A. Wu Y.Z. Rush L.J. Ross P. Molina J.R. Otterson G.A. Plass C. Hum. Mol. Genet. 2003; 12: 791-801Crossref PubMed Scopus (134) Google Scholar, 17Zender L. Spector M.S. Xue W. Flemming P. Cordon-Cardo C. Silke J. Fan S.T. Luk J.M. Wigler M. Hannon G.J. Mu D. Lucito R. Powers S. Lowe S.W. Cell. 2006; 125: 1253-1267Abstract Full Text Full Text PDF PubMed Scopus (907) Google Scholar). 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 (18Tamm I. Kornblau S.M. Segall H. Krajewski S. Welsh K. Kitada S. Scudiero D.A. Tudor G. Qui Y.H. Monks A. Andreeff M. Reed J.C. Clin. Cancer Res. 2000; 6: 1796-1803PubMed Google Scholar). 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 (19Abe F. Matsuda A. Schneider M. Talmadge J.E. Cancer Immunol. Immunother. 1990; 32: 75-80Crossref PubMed Scopus (13) Google Scholar, 20Sasaki S. Fukushima J. Hamajima K. Ishii N. Tsuji T. Xin K.Q. Mohri H. Okuda K. Clin. Exp. Immunol. 1998; 111: 30-35Crossref PubMed Scopus (42) Google Scholar, 21Talmadge J.E. Lenz B.F. Pennington R. Long C. Phillips H. Schneider M. Tribble H. Cancer Res. 1986; 46: 4505-4510PubMed Google Scholar). In a clinical trial with stage I squamous cell lung carcinoma patients, bestatin significantly prolonged survival of these patients (22Ichinose Y. Genka K. Koike T. Kato H. Watanabe Y. Mori T. Iioka S. Sakuma A. Ohta M. J. Natl. Cancer Inst. 2003; 95: 605-610Crossref PubMed Scopus (103) Google Scholar). Bestatin induces apoptosis in human leukemia cells (23Sekine K. Fujii H. Abe F. Leukemia. 1999; 13: 729-734Crossref PubMed Scopus (66) Google Scholar) and augments death ligand-induced apoptosis in human solid tumor cell lines (24Sekine K. Fujii H. Abe F. Nishikawa K. Int. J. Cancer. 2001; 94: 485-491Crossref PubMed Scopus (36) Google Scholar). 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 apoptosis. Mechanistic analysis showed that ME-BS directly interacts with cIAP1, promotes auto-ubiquitylation dependent on its RING domain, and facilitates proteasomal degradation of cIAP1. Destabilization of a particular anti-apoptosis protein by small molecules is a novel strategy for counteracting the pathological overexpression of such proteins known to interfere with cancer treatment. Reagents and Plasmids—ME-BS and analogs of bestatin were synthesized by Nippon Kayaku Co. Ltd. (Tokyo, Japan) as described previously (25Nishizawa R. Saino T. Takita T. Suda H. Aoyagi T. J. Med. Chem. 1977; 20: 510-515Crossref PubMed Scopus (180) Google Scholar). Anti-Fas (CH11, MBL), MG132, and Z-LLH (Peptide Inst.), as well as TRAIL (Chemicon) were purchased commercially. cDNAs encoding human cIAP1, cIAP2, and XIAP (X chromosome-linked inhibitor of apoptosis protein) were amplified by PCR from a Jurkat cDNA library and cloned into a p3xFLAG-CMV-10 expression vector (Sigma). Human Bax cDNA was PCR-amplified and cloned into pcDNA3 vector (Invitrogen). Point mutant constructs were generated using the QuikChange site-directed Mutagenesis kit (Stratagene). All of the constructs generated from PCR products were sequenced. Cell Culture, Drug Treatment, Transfection, and Western Blot Analysis—Human fibrosarcoma HT1080, cervical cancer cell lines (Ca-Ski and SKG-II), and monocytic leukemia THP1 cells were maintained in RPMI 1640 medium (Nissui Co., Ltd., Tokyo, Japan) containing 10% heat-inactivated fetal bovine serum and 100 μg/ml of kanamycin at 37 °C in a humidified atmosphere of 5% CO2. Human squamous cell lung carcinoma EBC-1 and human cervical carcinoma HeLaS3 cells were cultured in Eagle's minimum essential medium (Nissui Co.) containing 10% heat-inactivated fetal bovine serum and 100 μg/ml of kanamycin at 37 °C in a humidified atmosphere of 5% CO2. Proteasome inhibitors (MG132 and lactacystin) were added to the culture 30 min before the ME-BS treatment. Transfection experiments were carried out with FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. In brief, cells (1 × 105) were seeded in 12-well plates and transfected with a total of 1 μg of plasmid DNA for 18 h. The cells were harvested and lyzed in 25 μl of lysis buffer (10 mm HEPES-KOH, pH 7.4, 2 mm EDTA, 0.1% CHAPS, 5 mm dithiothreitol) containing 1 mm phenylmethylsulfonyl fluoride, and 1:100 (v/v) dilution of aprotinin. Cell lysates containing equal amounts of protein were separated by 4–20% gradient polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Millipore), and Western blotted using appropriate antibodies. Protein bands were detected using Enhanced Chemiluminescence detection (ECL) kits (GE Healthcare). We used the following antibodies for immunoprecipitation and Western blot analysis: anti-cIAP1 and anti-cIAP2 and and that of was generated as described previously Z. H. Naito M. T. A. S. M. H. K. Tsuruo T. 1999; PubMed Scopus Google Scholar). Cell cells were cultured in plates and with drugs for h. cells were as which was by and EBC-1 HeLaS3 cells were with to the cells using a In some cell was by the cells with (24Sekine K. Fujii H. Abe F. Nishikawa K. Int. J. Cancer. 2001; 94: 485-491Crossref PubMed Scopus (36) Google Scholar). activity was using according to the manufacturer's instructions. small to the of cIAP1 and were transfected into for using and the cells were with or TRAIL for h. cells were as The cell lysates were Western blotted to down-regulation of the proteins at activity was as described previously (24Sekine K. Fujii H. Abe F. Nishikawa K. Int. J. Cancer. 2001; 94: 485-491Crossref PubMed Scopus (36) Google Scholar). from cells was at for 30 min with a of bestatin analogs in containing mm and mm and or were added as for and The of from the was with a at and at The of analogs to the activity by was encoding cIAP1, that has that has and XIAP were into plasmid proteins were in cells and using The of the proteins was by and and of the proteins were at In was carried out as described C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). IAPs were with 100 human 2 mm and ubiquitin in μl of buffer mm at pH mm The was by with an equal of by Western blot analysis with anti-cIAP1 was used to the of ME-BS to cIAP1 and cells of a were with cIAP1 or using at 30 μg/ml in mm pH or pH and according to the manufacturer's The was with 1 pH and mm to and and were were at 25 °C with buffer (10 mm pH 7.4, mm mm EDTA, containing 5% as ME-BS was at at a of 30 for 60 and to for an were from the cIAP1 and by with ME-BS Cancer to by cancer cells were with the in the or of of cells apoptosis in the of ME-BS with this apoptosis was in the cells with ME-BS and with cells were to apoptosis by tumor necrosis factor and chemotherapeutic drugs such as and the death apoptosis apoptosis This suggests that ME-BS and by apoptosis by death and DNA at ME-BS is to regulate apoptosis at a of of of cIAP1 by the is a to apoptosis of G. Reed J.C. Nat. Med. 2000; 6: PubMed Scopus Google Scholar, X. Genes Dev. 2001; PubMed Scopus Google Scholar), and IAPs are of (1Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2278) Google Scholar, 2Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1573) Google Scholar, 3Vaux D.L. Silke J. Nat. Rev. Mol. Cell. Biol. 2005; 6: 287-297Crossref PubMed Scopus (528) Google Scholar). we of IAPs in the cells with ME-BS. in Fig. cIAP1 protein was selectively by ME-BS in cancer this was not of The of cIAP1 by ME-BS was by MG132, an inhibitor of Fig. cIAP1, not cIAP2 or was also by ME-BS which was by the inhibitors and not by the inhibitor Z-LLH These results that cIAP1 was selectively by in the cells. the we of cIAP1 in ME-BS cells. cells expressing cIAP1 were by Western blot with Fig. that min a of protein bands that in the gel in the cells with ME-BS and cIAP1 to min of ME-BS ME-BS not such on XIAP Fig. These results suggest that of cIAP1 is ME-BS treatment. the of cIAP1, the cell lysates from cells were with and the were to Western blot analysis with to the cIAP1 The bands with the which the cIAP1, were in the cells with and in the cells with ME-BS and was with was not by ME-BS Fig. In addition, which has and domains C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), was in by which was by an for and Fig. of and XIAP were not by ME-BS Fig. these results that ME-BS induces of cIAP1. cIAP1 a RING domain at the that is involved in the auto-ubiquitylation of cIAP1 Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (866) Google Scholar). study the role of the RING domain in the of cIAP1, a was in a in the RING The level of the RING mutant cIAP1 not in the cells that the RING domain plays an essential role in auto-ubiquitylation of cIAP1 by ME-BS. ME-BS induces of cIAP1 by its RING domain and facilitates the proteasomal degradation of cIAP1. has an activity against as which was to be involved in apoptosis (24Sekine K. Fujii H. Abe F. Nishikawa K. Int. J. Cancer. 2001; 94: 485-491Crossref PubMed Scopus (36) Google Scholar). study the and of aminopeptidase in cIAP1 a of bestatin analogs were for to cIAP1, and and The of the analogs and for the are in Fig. We that analogs of bestatin with a such as and cIAP1 and the apoptosis by in cells Bestatin with a acid and with an a activity for cIAP1 and apoptosis. showed ME-BS cIAP1, bestatin 30 for with the to cIAP1 and to apoptosis results were in and cervical cancers cIAP1 by genetic amplification Fig. These results suggest that the apoptosis sensitization by these is to cIAP1 In to the cIAP1 down-regulation and apoptosis was in the of and cIAP1 that the aminopeptidase was not involved in the cIAP1 down-regulation or apoptosis sensitization by ME-BS. the to cIAP1, we on ME-BS in this study because of the activity to cIAP1, the and the of the We the of cIAP1 destabilization by of ME-BS Fig. 1 the cIAP1 level was maintained for was maintained for with 30 ME-BS. of ME-BS showed activity to and EBC-1 cells to Fig. We cIAP1 down-regulation is for the apoptosis sensitization by ME-BS. of apoptosis in which was by of and cIAP1. ME-BS the apoptosis by cIAP1 not the by the mutant In addition, small down-regulation of cIAP1 the cells to apoptosis These results that the down-regulation of cIAP1 at in for the apoptosis. ME-BS with BIR3 of cIAP1 not cIAP2, a of cIAP1 acid study the of destabilization of cIAP1 in we a of molecules containing of cIAP1 and cIAP2 which the domains from cIAP1 and the domains from cIAP2, well to which the domains from cIAP1 and the domains from cIAP2, in which the BIR3 domain of cIAP1 was with the BIR3 domain from cIAP2, was not by ME-BS in which the BIR3 domain of cIAP2 was by that of cIAP1, was by ME-BS Fig. These results that the BIR3 domain of cIAP1 plays a role in We cIAP1 with a in a in the BIR the BIR3 mutant was not by and were which suggests a role of the BIR3 domain in destabilization of cIAP1. We the interaction of ME-BS with cIAP1 by ME-BS directly to cIAP1 in this The in the and of ME-BS the cIAP1 are which has a activity to cIAP1, and the analogs and the were of that these molecules not or with cIAP1 with ME-BS. We by of analysis the interaction of ME-BS with and with as a the was a was with ME-BS to not to Bestatin a to the the at 100 from the showed that analogs and with BIR3 bestatin and the analogs and The interaction of glutathione with and with as a These results that ME-BS directly interacts with the BIR3 domain of cIAP1. IAPs are a family of anti-apoptotic proteins that are frequently overexpressed in cancer cells (1Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2278) Google Scholar, 2Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1573) Google Scholar, 3Vaux D.L. Silke J. Nat. Rev. Mol. Cell. Biol. 2005; 6: 287-297Crossref PubMed Scopus (528) Google Scholar). The anti-apoptotic activity of IAPs are by that is from into in to C. Fang M. Y. L. X. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A.M. M. Silke J. D.L. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The IAP-binding at the of plays a role in the of IAP and a of IAP that the interaction of and IAPs L. R.M. H. X. Science. 2004; PubMed Scopus Google Scholar, C. H. S.W. A. T. H. S.W. J. Med. Chem. 2004; PubMed Scopus Google Scholar, H. Z. Yang L. Y. Krajewski K. S. J. Med. Chem. 2004; PubMed Scopus Google Scholar, K. L. E. D. K. Chem. Biol. 2006; PubMed Scopus Google Scholar). These IAP the interaction IAPs and a small of which the IAP-binding and apoptosis. IAP that were also to apoptosis in cancer cells Welsh K. C. Z. M. Kitada S. G. Scudiero D. E. Salvesen G. A. J.M. R.A. Reed J.C. Cancer Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We described in this a novel of cIAP1 destabilization of cIAP1 by its analysis revealed the BIR3 domain of cIAP1 plays a role in destabilization and ME-BS directly interacts with the BIR3 domain in We that ME-BS with the BIR3 domain induces a in cIAP1 that interacts with the BIR3 domain of cIAP1 C. Fang M. Y. L. X. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A.M. M. Silke J. D.L. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), we a role of in destabilization of cIAP1. the of to cIAP1 was not by ME-BS. of in ubiquitin (8Hao Y. Sekine K. Kawabata A. Nakamura H. Ishioka T. Ohata H. Katayama R. Hashimoto C. Zhang X. Noda T. Tsuruo T. Naito M. Nat. Cell Biol. 2004; 6: 849-860Crossref PubMed Scopus (192) Google Scholar, A.M. D. J. Duckett C.S. P. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) not the destabilization of cIAP1. the cIAP1 mutant was by not to Fig. These results suggest that is not for the destabilization of cIAP1. The of cIAP1 apoptosis is not the of the cIAP1 to directly caspases Salvesen G.S. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). is that of IAP such as and the of the proteins to of the role of cIAP1 in tumor necrosis factor because cIAP1 interacts with proteins and in the of the anti-apoptotic of cIAP1 was not cIAP1 is overexpressed in certain cancers because of the genetic amplification of cIAP1 and the cIAP1 overexpression is involved in the resistance to therapy. a study that of the cIAP1 in results in L. D.A. T. Ashwell J.D. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar), cIAP1 could be a promising target for cancer therapy. ME-BS is a of bestatin that has immunomodulatory activity (19Abe F. Matsuda A. Schneider M. Talmadge J.E. Cancer Immunol. Immunother. 1990; 32: 75-80Crossref PubMed Scopus (13) Google Scholar, 20Sasaki S. Fukushima J. Hamajima K. Ishii N. Tsuji T. Xin K.Q. Mohri H. Okuda K. Clin. Exp. Immunol. 1998; 111: 30-35Crossref PubMed Scopus (42) Google Scholar, 21Talmadge J.E. Lenz B.F. Pennington R. Long C. Phillips H. Schneider M. Tribble H. Cancer Res. 1986; 46: 4505-4510PubMed Google Scholar) and is approved in Japan to treat patients with adult acute nonlymphatic clinical trial with patients stage I squamous cell lung carcinoma has showed bestatin as a significantly prolonged survival of these patients (22Ichinose Y. Genka K. Koike T. Kato H. Watanabe Y. Mori T. Iioka S. Sakuma A. Ohta M. J. Natl. Cancer Inst. 2003; 95: 605-610Crossref PubMed Scopus (103) Google Scholar). bestatin has a on cIAP1, is to that the of bestatin on the down-regulation of cIAP1 is involved in the prolonged survival reported in the lung cancer as we to in a with human tumor cIAP1, therapeutic by the of or in with study is to the role of cIAP1 down-regulation in the survival by bestatin treatment. In we in this that selective destabilization of cIAP1 is with a class of small by its the of the IAP is also of to develop a small to IAPs containing RING finger and UBC such as and the overexpression of which has reported in cancers G.M. Gomes B.C. J. Biol. Chem. 2001; 276: 3238-3246Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 7Vucic D. Stennicke H.R. Pisabarro M.T. Salvesen G.S. Dixit V.M. Curr. Biol. 2000; 10: 1359-1366Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, Z. Naito M. S. T. T. Tsuruo T. Res. 1999; PubMed Scopus Google Scholar, P. S.W. J.M. G. 2001; 12: Full Text PDF PubMed Scopus Google Scholar, M. Krajewski S. S. X. L. A. A. D. G.J. Reed J.C. Clin. Cancer Res. 2003; Google Scholar). We J. Inazawa and Y. Shimada for cell lines with cIAP1 We also T. for in We H. Y. A. A. Y. and N. for with