Synthetic Smac/DIABLO Peptides Enhance the Effects of Chemotherapeutic Agents by Binding XIAP and cIAP1 in Situ (2002)

Inhibitor of apoptosis proteins (IAPs) interact with and inhibit caspases-3, -7, and -9. This interaction can be inhibited by Smac/DIABLO, a polypeptide released from mitochondria upon initiation of the apoptotic signaling process. Here we demonstrate that the first 4–8 N-terminal amino acids of Smac/DIABLO fused to theDrosophila antennapaedia penetratin sequence, a carrier peptide, enhance the induction of apoptosis and long term antiproliferative effects of diverse antineoplastic agents including paclitaxel, etoposide, 7-ethyl-10-hydroxycamptothecin (SN-38), and doxorubicin in MCF-7 breast cancer cells. Similar effects were observed in additional breast cancer and immortalized cholangiocyte cell lines. Further analysis demonstrated that the Smac-penetratin fusion peptide crossed the cellular membrane, bound XIAP and cIAP1, displaced caspase-3 from cytoplasmic aggregates, and enhanced drug-induced caspase action in situ. These studies demonstrate that inhibition of IAP proteins can modulate the efficacy of antineoplastic agents. Inhibitor of apoptosis proteins (IAPs) interact with and inhibit caspases-3, -7, and -9. This interaction can be inhibited by Smac/DIABLO, a polypeptide released from mitochondria upon initiation of the apoptotic signaling process. Here we demonstrate that the first 4–8 N-terminal amino acids of Smac/DIABLO fused to theDrosophila antennapaedia penetratin sequence, a carrier peptide, enhance the induction of apoptosis and long term antiproliferative effects of diverse antineoplastic agents including paclitaxel, etoposide, 7-ethyl-10-hydroxycamptothecin (SN-38), and doxorubicin in MCF-7 breast cancer cells. Similar effects were observed in additional breast cancer and immortalized cholangiocyte cell lines. Further analysis demonstrated that the Smac-penetratin fusion peptide crossed the cellular membrane, bound XIAP and cIAP1, displaced caspase-3 from cytoplasmic aggregates, and enhanced drug-induced caspase action in situ. These studies demonstrate that inhibition of IAP proteins can modulate the efficacy of antineoplastic agents. Apoptosis is a distinct form of cell death that is provoked by a variety of stimuli, including cancer chemotherapeutic agents (1Wyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6683) Google Scholar, 2Arends M.J. Wyllie A.H. Int. Rev. Exp. Pathol. 1991; 32: 223-254Crossref PubMed Scopus (1389) Google Scholar). The morphological and biochemical features of apoptosis reflect, at least in part, the activation of a class of cysteine proteases called caspases (3Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4084) Google Scholar, 4Salvesen G.S. Dixit V.M. 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Collectively, cleavages by effector caspases, particularly caspases-3 and -6, result in diminished integrity of the actin and intermediate filament networks within the cell, inhibit protein synthesis, disable the DNA repair machinery, and activate degradative enzymes such as the caspase-activated deoxyribonuclease (9Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2785) Google Scholar). Because of the potentially dire consequences of inadvertent caspase activation, caspase activity within cells is tightly regulated. A family of polypeptides known as IAPs 1The abbreviations used are: IAP, inhibitor of apoptosis protein; BIR, baculovirus inhibitor of apoptosis repeat; cIAP1(2), cellular inhibitor of apoptosis protein 1(2); GFP, green fluorescent protein; PBS, calcium- and magnesium-free Dulbecco's phosphate-buffered saline; RING, really interesting new gene; Smac/DIABLO, second mitochondrial activator of caspases/direct IAP-binding protein with low pI; SN-38, 7-ethyl-10-hydroxycamptothecin; TRAIL, tumor necrosis factor-α-related apoptosis inducing ligand; XIAP, X chromosome-linked IAP 1The abbreviations used are: IAP, inhibitor of apoptosis protein; BIR, baculovirus inhibitor of apoptosis repeat; cIAP1(2), cellular inhibitor of apoptosis protein 1(2); GFP, green fluorescent protein; PBS, calcium- and magnesium-free Dulbecco's phosphate-buffered saline; RING, really interesting new gene; Smac/DIABLO, second mitochondrial activator of caspases/direct IAP-binding protein with low pI; SN-38, 7-ethyl-10-hydroxycamptothecin; TRAIL, tumor necrosis factor-α-related apoptosis inducing ligand; XIAP, X chromosome-linked IAPhas been implicated in this process. 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Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), a of caspase including and and as cells apoptosis and with peptide, of substrates observed that caspase action been enhanced in situ. the enhanced caspase activity in cells were for with peptide followed by of for cells were were in in the of peptide for additional with and for apoptotic A and The with shown in A. were to the of the peptide and peptide in with were from with of and Similar were in MCF-7 cells not that the result in A not to paclitaxel, were doxorubicin and not In each case the to apoptosis in the of with the in A and the peptide at and enhanced the drug-induced apoptosis and and not As in for etoposide, effects of the chemotherapeutic were at and that the fusion peptide be the of cells that apoptosis the of apoptosis the that be to cells apoptosis in the of peptide, a of that apoptosis in cells. of peptide in a in apoptosis with this A and Similar were with of in other cell and not fusion peptide apoptosis in a variety of cell lines. breast cancer cells were for with peptide peptide were for with and for additional in in the continued of of peptide and as in to immortalized were for with peptide were for with and with to the interaction of peptide with XIAP and required for the enhanced cells were in with the peptide and the peptide that to bind XIAP and This the of the peptide that the interaction with XIAP is for the of drug-induced Similar effects were observed with SN-38, etoposide, TRAIL, and A and not the first amino acids of Smac/DIABLO are for to XIAP, S.M. R. A. P. Shiozaki E. J. T. Shi Y. Alnemri E.S. Nature. 2001; PubMed Scopus Google the effects of have that N-terminal as long as be required for the of the in apoptosis in the of the of the from to amino acids in the fusion peptide all enhanced apoptosis with peptide, the fusion peptide the most of the three demonstrated that the peptide with XIAP and cIAP1, enhanced caspase activation, and drug-induced apoptosis as demonstrated in as for the peptide not that the effects were cell the effects of the fusion peptide drug-induced apoptosis were in additional cell lines. As in cells were with peptide for of As in the fusion peptide enhanced apoptosis in breast cancer cells the induction of apoptosis by in also enhanced the fusion peptide enhanced the long term effects of chemotherapeutic cells were with paclitaxel, etoposide, for in the of peptide, and for in the of peptide to to the fusion peptide were the apoptotic the of cells that the and of the that form not be by the of the to this the antiproliferative effects of all of the agents were The of that for diminished the in the of peptide the of the the of a of cells to cell of the In addition, the fusion peptide the of of the cells with and of the demonstrated that of cells with a fusion protein 4–8 N-terminal amino acids of Smac/DIABLO the efficacy of a of antineoplastic agents in cancer cell in situ. of that the fusion peptide crossed the and bound to demonstrated that this fusion peptide caspase-3 and enhanced drug-induced caspase the enhanced the induction of apoptosis and the long term antiproliferative effects of agents. These have potentially for the of IAP to modulate antineoplastic the studies were in et D. K. H. S. W.J. Dixit V.M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google that a fusion peptide bind in MCF-7 cells. The of the polypeptide to bind IAPs and drug-induced apoptosis not et R. S. S. D. P. E. C. Wang K. 2002; PubMed Scopus Google that with the amino acids of fused to penetratin apoptosis in cell cells. interaction of the fusion peptide with IAPs not and effects other cell were not As the for and S. M. Nat. 2002; PubMed Scopus Google that the N-terminal amino acids of enhanced apoptosis of cells with TRAIL, various the cellular of the fusion protein not and long term effects were only for of the in a of demonstrated that fusion protein to XIAP and in cell polypeptides as of the fusion studies the of fusion proteins demonstrated that with the displaced caspase-3 from the cytoplasmic that form apoptosis in a variety of cell M. D. G.M. J. Cell Biol. 2000; PubMed Scopus (147) Google Scholar, S. K. J. M. W.C. Kaufmann S.H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). These the first that of active caspase species to IAPs to the of cytoplasmic the studies demonstrated that the fusion peptide not only drug-induced apoptosis in a variety of cell but also the long term antiproliferative effects of a variety of chemotherapeutic agents. A of studies have the that and not the of of the in to result H. J. C. A. G.S. A. A. S.W. Wang X. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). studies have that peptide fused to to peptide has activity S.M. P. T. Z. Alnemri E.S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). These the of to enhance apoptosis in situ. we observed caspase action cells were with in the of peptide Because were in MCF-7 which exhibit of caspase-3 substrates caspase-3 S. K. J. M. W.C. Kaufmann S.H. J. Biol. 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Cell 1998; PubMed Scopus Google the observed apoptotic In the studies that apoptosis observed at with each of the agents not that fusion peptide the of cells the of The peptide also enhanced the effects of that were by themselves A and the that long term effects of the be with this demonstrated that the antiproliferative effects of the agents were enhanced by peptide this peptide antiproliferative effects of In particular, the fusion peptide the of cells to form with SN-38, the active of the and Similar effects were observed with low of is that the peptide not the of understanding of the of action of T. Int. J. 1999; 83: PubMed Scopus Google Scholar), is that this result the of the peptide to enhance of cells that to a cell These the that the of to enhance long term antiproliferative effects of upon the chemotherapeutic and the that is A of studies have that IAP of cells to P. Roy N. Tamai K. Lefebvre C. Baird S. Cherton-Horvat G. Farahani R. McLean M. Ikeda J.-E. MacKenzie A. 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