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

2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês

10.1074/jbc.m207578200

ISSN

1083-351X

Autores

Christina R. Arnt, Mihnea V. Chiorean, Michael P. Heldebrant, Gregory J. Gores, Scott H. Kaufmann,

Tópico(s)

ATP Synthase and ATPases Research

Resumo

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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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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First, overexpression studies demonstrated that XIAP, cIAP1, and cIAP2 diminish apoptosis after treatment with a variety of agents (16Liston P. Roy N. Tamai K. Lefebvre C. Baird S. Cherton-Horvat G. Farahani R. McLean M. Ikeda J.-E. MacKenzie A. Korneluk R.G. Nature. 1996; 379: 349-353Crossref PubMed Scopus (867) Google Scholar, 51Stehlik C. de Martin R. Kumabashiri I. Schmid J.A. Binder B.R. Lipp J. J. Exp. Med. 1998; 188: 211-216Crossref PubMed Scopus (589) Google Scholar, 52Asselin E. Mills G.B. Tsang B.K. Cancer Res. 2001; 61: 1862-1868PubMed Google Scholar, 53Notarbartolo M. Cervello M. Dusonchet L. Cusimano A. D'Alessandro N. Cancer Lett. 2002; 180: 91-101Crossref PubMed Scopus (92) Google Scholar). More recently, studies by MacFarlane et al. (54MacFarlane M. Merrison W. Dinsdale D. Cohen G.M. J. Cell Biol. 2000; 148: 1239-1254Crossref PubMed Scopus (147) Google Scholar) demonstrated that active caspase-3 is sequestered in cytoplasmic aggregates after treatment of tissue culture cells with TRAIL. This sequestration was particularly dramatic in MCF-7 human breast cancer cells transiently transfected with procaspase-3 but was also observed in other cell lines. Studies using MCF-7 cells stably transfected with procaspase-3 not only demonstrated that the activity of cleaved caspase-3 was inhibited in these aggregates but also suggested that XIAP might be present within them (55Kottke T.J. Blajeski A.L. Meng X.W. Svingen P.A. Ruchaud S. Mesner Jr., P.W. Boerner S.A. Samejima K. Henriquez N.V. Chilcote T.J. Lord J. Salmon M. Earnshaw W.C. Kaufmann S.H. J. Biol. Chem. 2002; 277: 804-815Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). These observations prompted us to examine the effect of antagonizing XIAP in this model system as well as other cell lines. In the present study, we demonstrate that the N-terminal sequence of Smac/DIABLO, when delivered into cells by fusion to a peptide carrier, can bind endogenous XIAP and cIAP1, displace caspase-3 from these cytoplasmic aggregates, and enhance cleavage of caspase target polypeptides in situ. These interactions result in increased apoptosis after treatment with a variety of chemotherapeutic agents and enhanced long term antiproliferative effects as assessed by colony forming assays. Reagents were obtained from the following suppliers: paclitaxel and doxorubicin from Sigma; etoposide from Biomol(Plymouth Meeting, PA); TRAIL from R & D Systems (Minneapolis, MN); SN-38 from Amersham Biosciences; gemcitabine from John S. Kovach; Hoescht 33258 from Polyscience (Warrington, PA); ECL-enhanced chemiluminescent reagents from Amersham Biosciences; and thiodiglycol and SuperSignal ULTRATM from Pierce. Peptides were prepared by solid phase synthesis on an ABI 433A Peptide Synthesizer in the Mayo Clinic Protein Chemistry Shared Resource. Biotinylated peptides were synthesized with a pre-biotinylated lysine at the C terminus. Monoclonal antibodies recognizing topoisomerase I and heat shock protein 90 were kindly provided by Y.-C. Cheng (Yale University of Medicine, New Haven, CT) and David Toft (Mayo Clinic, Rochester, MN), respectively. Polyclonal sera raised against lamin A and lamin B1 were described previously (56Kaufmann S.H. J. Biol. Chem. 1989; 264: 13946-13955Abstract Full Text PDF PubMed Google Scholar). The remaining antibodies were purchased from the following sources: peroxidase-coupled affinity-purified secondary antibodies from Kirkegaard & Perry (Gaithersburg, MD); monoclonal antibodies to β-catenin and gelsolin from BD Biosciences; goat anti-actin from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-procaspase-6 from Upstate Biotechnology, Inc. (Lake Placid, NY); rabbit anti-Smac from Biomol; and rabbit antisera against XIAP, cIAP1, and cIAP2 from R & D Systems. MCF-7, T47D, and MDA-MB-468 (American Type Culture Collection, Manassas, VA) were cultured as instructed by the supplier. MCF-7/wt1 cells were stably transfected with procaspase-3 cDNA as recently described (55Kottke T.J. Blajeski A.L. Meng X.W. Svingen P.A. Ruchaud S. Mesner Jr., P.W. Boerner S.A. Samejima K. Henriquez N.V. Chilcote T.J. Lord J. Salmon M. Earnshaw W.C. Kaufmann S.H. J. Biol. Chem. 2002; 277: 804-815Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Mouse immortalized cholangiocytes (NMC) were kindly provided by N. F. LaRusso (57Mano Y. Ishii M. Kisara N. Kobayashi Y. Ueno Y. Kobayashi K. Hamada H. Toyota T. Lab. Invest. 1998; 78: 1467-1468PubMed Google Scholar) and cultured in Dulbecco's modified essential medium containing 10% heat-inactivated fetal bovine serum, 100 nm insulin, 50 μg/ml gentamycin, 100 μg/ml streptomycin, and 100 units/ml penicillin G. To assess the long term antiproliferative effects of the Smac/penetratin peptide in concert with chemotherapeutic agents, multiple aliquots containing 750 MCF-7/wt1 cells were plated in 35-mm plates and allowed to adhere for 16–18 h. Triplicate samples were then treated with varying drug concentrations for 24 h in the absence or presence of Smac/penetratin peptide, washed, and incubated in drug-free medium in the continued presence or absence of Smac/penetratin peptide. After 14 days to allow colony formation, plates were washed, stained with Coomassie Blue, and manually counted. After cells were treated for 3 h (58Schwarze S.R., Ho, A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2160) Google Scholar) with the Smac/penetratin peptide, drug was added at a concentration determined previously to induce apoptosis in 25–50% of cells 48–72 h after drug addition. At the predetermined time point, adherent cells were released from tissue culture plates with trypsin-EDTA or cell scrapers, combined with the non-adherent cells, sedimented at 500 × g for 5 min, fixed in methanol/acetic acid (3:1 v/v), stained with 1 μg/ml Hoescht 33258 in 50% glycerol containing 0.1 m Tris-HCl (pH 7.4 at 21 °C), and examined by fluorescence microscopy. A minimum of 500 cells/treatment were scored for apoptotic changes (chromatin condensation and nuclear fragmentation) as illustrated previously (59Kottke T.J. Blajeski A.L. Martins L.M. Mesner Jr., P.W. Davidson N.E. Earnshaw W.C. Armstrong D.K. Kaufmann S.H. J. Biol. Chem. 1999; 274: 15927-15936Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Samples were photographed using a Zeiss Axioplan microscope equipped with an N.A. 1.40 63× objective, a 365-nm excitation filter, and a 420-nm emission filter. Alternatively, for some experiments cells were stained with 5 μg/ml 4′,6-diamidino-2-phenylindole dihydrochloride for 3 h and examined by fluorescence microscopy using excitation and emission filters of 380 and 430 nm, respectively. Parental MCF-7 cells were transfected with 10 μg of plasmid encoding the C163S mutant of procaspase-3 fused in-frame with enhanced GFP at its C terminus in the plasmid pEGFP-N1 (Clontech) as described previously (55Kottke T.J. Blajeski A.L. Meng X.W. Svingen P.A. Ruchaud S. Mesner Jr., P.W. Boerner S.A. Samejima K. Henriquez N.V. Chilcote T.J. Lord J. Salmon M. Earnshaw W.C. Kaufmann S.H. J. Biol. Chem. 2002; 277: 804-815Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Briefly, log phase cells were trypsinized, washed twice in PBS, resuspended in the buffer described by van den Hoff et al. (60van den Hoff M.J. Christoffels V.M. Labruyere W.T. Moorman A.F. Lamers W.H. Methods Mol. Biol. 1995; 48: 185-197PubMed Google Scholar),incubated for 5 min with plasmid or empty vector, and permeabilized using a T820 square wave electroporator (BTX, San Diego, CA) delivering a 240-V pulse for 10 ms. After return to tissue culture plates for 24 h, cells were treated with diluent or 100 nmpaclitaxel for 24 h in the absence or presence of the indicated peptide, washed, and incubated in drug-free medium in the continued absence or presence of peptide for 24 h. Adherent and nonadherent cells were then examined on a Zeiss LSM 510 laser scanning confocal microscope. Recently published results (55Kottke T.J. Blajeski A.L. Meng X.W. Svingen P.A. Ruchaud S. Mesner Jr., P.W. Boerner S.A. Samejima K. Henriquez N.V. Chilcote T.J. Lord J. Salmon M. Earnshaw W.C. Kaufmann S.H. J. Biol. Chem. 2002; 277: 804-815Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) have demonstrated that the localization of caspase-3/GFP in MCF-7 cells closely parallels the localization of caspase-3 in MCF-7/wt1 cells. Cells were incubated with biotinylated Smac/penetratin peptide for 30 min. At the completion of the incubation, cells were released by brief trypsinization, pelleted for 5 min at 500 rpm, and washed twice with ice-cold PBS. All further steps were performed at 4 °C. Cells were lysed by incubation for 20 min in lysis buffer consisting of 30 mm Tris-HCl (pH 7.5 at 4 °C), 1% (w/v) Triton X-100, 150 mm NaCl, 1% (w/v) thiodiglycol, 1 mm Na3VO4, 100 mm NaF, 20 nm microcystin, 1 mmα-phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml pepstatin. After lysis, samples were centrifuged at 14,000 ×g for 15 min. Smac/penetratin fusion peptide was recovered from the supernatant by incubation for 3 h with 40 μl of streptavidin-agarose beads (Amersham Biosciences). The resulting complexes were washed 5 times in lysis buffer and recovered from the beads by heating for 20 min at 70 °C in SDS sample buffer. Polypeptides were separated by one-dimensional SDS-PAGE on a 5–15% acrylamide gradient gel and probed as described below. Treated cells were pelleted at 500 ×g for 5 min at 4 °C, rinsed twice with ice-cold PBS, and lysed in buffer consisting of 6 m guanidine hydrochloride, 250 mm Tris-HCl (pH 8.5 at 21 °C), 10 mmEDTA, 1 mm phenylmethylsulfonyl fluoride, and 1% (v/v) β-mercaptoethanol (61Kaufmann S.H. McLaughlin S.J. Kastan M.B. Liu L.F. Karp J.E. Burke P.J. Cancer Res. 1991; 51: 3534-3543PubMed Google Scholar). After preparation for electrophoresis as described previously (61Kaufmann S.H. McLaughlin S.J. Kastan M.B. Liu L.F. Karp J.E. Burke P.J. Cancer Res. 1991; 51: 3534-3543PubMed Google Scholar), aliquots containing 50 μg of total cellular protein (estimated by the bicinchoninic acid method (62Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18349) Google Scholar)) were separated by one-dimensional SDS-PAGE on a 5–15% acrylamide gradient gel. Polypeptides were transferred to nitrocellulose, probed with antibodies, and visualized by enhanced chemiluminescence using techniques described previously (63Kaufmann S.H. Anal. Biochem. 2001; 296: 283-286Crossref PubMed Scopus (52) Google Scholar). Data shown are representative of the indicated number of replicate experiments. Differences in paired groups were analyzed using a χ2 pairwise analysis withp values indicated in the text. All statistical analysis was done in collaboration with the Department of Statistics, Mayo Clinic and Foundation (Rochester, MN). Smac/DIABLO has been shown to interact with a number of IAPs in vitro and in vivo. Based on structural analyses, which showed that the N-terminal four amino acids of Smac/DIABLO are critical for binding to XIAP (39Liu Z. Sun C. Olejniczak E.T. Meadows R.P. Betz S.F. Oost T. Herrmann J., Wu, J.C. Fesik S.W. Nature. 2000; 408: 1004-1008Crossref PubMed Scopus (538) Google Scholar), we hypothesized that 4–8 amino acids might be sufficient to mediate the effects of Smac/DIABLO in situ. To test this hypothesis, we synthesized peptides (Fig. 1 A) containing 4–8 of the N-terminal amino acids of Smac/DIABLO fused to the penetratin sequence of the Drosophila transcription factor antennapaedia. This peptide carrier was chosen because it is well characterized and has little toxicity when applied to cells in the absence of cargo (64Prochaintz A. Curr. Opin. Neurobiol. 1996; 6: 629-634Crossref PubMed Scopus (192) Google Scholar, 65Derossi D. Chassaing G. Prochaintz A. Trends Cell Biol. 1998; 8: 84-87Abstract Full Text PDF PubMed Scopus (644) Google Scholar, 66Thoren P.E.G. Persson D. Karlsson M. Norden B. FEBS Lett. 2000; 482: 265-268Crossref PubMed Scopus (207) Google Scholar). Because the N-terminal alanine of Smac/DIABLO has been reported previously (39Liu Z. Sun C. Olejniczak E.T. Meadows R.P. Betz S.F. Oost T. Herrmann J., Wu, J.C. Fesik S.W. Nature. 2000; 408: 1004-1008Crossref PubMed Scopus (538) Google Scholar) to be critical for the Smac/XIAP interaction, a peptide containing an alanine-to-methionine mutation (A1M peptide, Fig. 1 A) was synthesized as a negative control. To determine whether a Smac/penetratin fusion peptide could successfully interact with IAPs, we treated two cell lines, MCF-7/wt1 and MDA-MB-468, with a biotinylated version of the 8-mer peptide. After a 30-min incubation, cells were washed and lysed in neutral detergent. Polypeptides bound to the Smac/penetratin fusion peptide were recovered with streptavidin-agarose, subjected to SDS-PAGE, and analyzed by immunoblotting. This analysis demonstrated that the Smac/penetratin fusion peptide interacted with XIAP and cIAP1 in both cell lines tested (Fig. 1 B). Reactions run without peptide as well as those run with the A1M mutant peptide served as negative controls. Previous studies have suggested that cleaved caspase-3 is sequestered in cytoplasmic aggregates (54MacFarlane M. Merrison W. Dinsdale D. Cohen G.M. J. Cell Biol. 2000; 148: 1239-1254Crossref PubMed Scopus (147) Google Scholar, 55Kottke T.J. Blajeski A.L. Meng X.W. Svingen P.A. Ruchaud S. Mesner Jr., P.W. Boerner S.A. Samejima K. Henriquez N.V. Chilcote T.J. Lord J. Salmon M. Earnshaw W.C. Kaufmann S.H. J. Biol. Chem. 2002; 277: 804-815Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) and inactivated (55Kottke T.J. Blajeski A.L. Meng X.W. Svingen P.A. Ruchaud S. Mesner Jr., P.W. Boerner S.A. Samejima K. Henriquez N.V. Chilcote T.J. Lord J. Salmon M. Earnshaw W.C. Kaufmann S.H. J. Biol. Chem. 2002; 277: 804-815Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) in a variety of epithelial cell lines. This effect is particularly prominent in MCF-7 cells transfected either transiently or stably with procaspase-3 (54MacFarlane M. Merrison W. Dinsdale D. Cohen G.M. J. Cell Biol. 2000; 148: 1239-1254Crossref PubMed Scopus (147) Google Scholar,55Kottke T.J. Blajeski A.L. Meng X.W. Svingen P.A. Ruchaud S. Mesner Jr., P.W. Boerner S.A. Samejima K. Henriquez N.V. Chilcote T.J. Lord J. Salmon M. Earnshaw W.C. Kaufmann S.H. J. Biol. Chem. 2002; 277: 804-815Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Fractionation studies indicated that XIAP was present in the same insoluble cell fraction (55Kottke T.J. Blajeski A.L. Meng X.W. Svingen P.A. Ruchaud S. Mesner Jr., P.W. Boerner S.A. Samejima K. Henriquez N.V. Chilcote T.J. Lord J. Salmon M. Earnshaw W.C. Kaufmann S.H. J. Biol. Chem. 2002; 277: 804-815Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), raising the possibility that an interaction between XIAP and cleaved caspase-3 might contribute to the observed aggregation. Because release of Smac/DIABLO from mitochondria would be expected to disrupt caspase-3/XIAP interactions, we examined Smac expression and release from mitochondria in MCF-7 cells. Immunoblotting revealed that Smac was readily detectable in each of the 10 human breast cancer cell lines examined (Fig. 2 A and data not shown). When cells were harvested 48–72 h after the initiation of a 24-h paclitaxel treatment, Smac was readily detectable in cytosol of floating (apoptotic) MDA-MB-468 cells (Fig. 2 B, lane 2). In contrast, Smac was not released to the cytosol in apoptotic MCF-7 cells (Fig. 2 B, lane 4), possibly reflecting the recently reported dependence of Smac release on caspase activity (67Adrain C. Creagh E.M. Martin S.J. EMBO J. 2001; 20: 6627-6636Crossref PubMed Scopus (351) Google Scholar). To determine whether this paucity of Smac release might contribute to caspase-3 aggregation in MCF-7 cells, MCF-7 cells transfected with a procaspase-3/GFP fusion construct were treated with paclitaxel in the absence or presence of 80 μg/ml Smac/penetratin peptide. When transfected cells were treated with either diluent or Smac/penetratin peptide alone, none of the cells became apopt

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