Molecular Cancer Therapeutics 1343 Sequence and helicity requirements for the proapoptotic activity of Bax BH3 peptides Sanjeev Shangary,1 Christopher L. Oliver,2 PC-3 cells. Our results define 15 amino acids as the minimal Tommy S. Tillman,3 Michael Cascio,3 and length required for Bax BH3 peptide biological activity and Daniel E. Johnson1,4 show that amino acids COOH terminal to the BH3 core sequence are less critical than those located NH terminal 1 2 3 2 Departments of Medicine, Otolaryngology, Molecular Genetics to the core. In addition, circular dichroism spectroscopy and Biochemistry, and 4Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania revealed that high a-helical content generally correlated with, but was not sufficient for, peptide activity. Taken together, these studies provide a basis for future optimiza- Abstract tion of Bax BH3 peptide as a therapeutic anticancer agent. [Mol Cancer Ther 2004;3(11):1343–53] Overexpression of the antiapoptotic proteins Bcl-2 and Bcl-XL is commonly observed in human malignancies and contributes to chemotherapy and radiation resistance. Bcl- Introduction 2 and Bcl-X inhibit apoptosis by binding to proapoptotic L Apoptosis is a genetically defined form of cell suicide that proteins such as Bax, thereby preventing chemotherapy- is critically important for tissue homeostasis. The Bcl-2 induced or radiation-induced release of cytochrome c from protein family, which consists of both proapoptotic and mitochondria and subsequent activation of the caspase antiapoptotic members, acts to regulate apoptosis via protease cascade. Efforts to inhibit Bcl-2 or Bcl-XL governance of the ‘‘intrinsic’’ pathway of cell death. The function in tumor cells have focused on developing agents intrinsic or mitochondrial-mediated pathway is character- to inhibit the interactions of these proteins with proapo- ized by mitochondrial release of apoptogenic factors ptotic proteins. Peptides derived from the BH3 domains of including cytochrome c, Smac/DIABLO, and apoptosis- proapoptotic proteins have been shown to disrupt the inducing factor following treatment of cells with death interactions of Bcl-2 and Bcl-X with key binding partners L stimuli such as chemotherapy drugs or ionizing radiation in cell-free reactions and to promote cellular apoptosis. (1–5). The release of cytochrome c and Smac/DIABLO However, less is known about the targets of BH3 peptides leads to subsequent activation of the caspase protease in intact cells as well as the sequence, length, and cascade, which promotes the internal destruction of the cell conformational requirements for peptide biological activ- (1, 2, 4–7). Antiapoptotic Bcl-2 family members, including ity. In this report, we show that cell-permeable Bax BH3 Bcl-2 and Bcl-XL, inhibit apoptosis by preventing the peptides physically disrupt Bax/Bcl-2 heterodimerization in release of apoptogenic proteins into the cytosol (1–3), intact cells and that this disruption correlates with peptide- whereas proapoptotic proteins, including Bax and Bak, induced cell death. A point-mutant, control peptide that promote such release (8–10). In view of their ability to failed to disrupt intracellular Bax/Bcl-2 interactions also prevent apoptosis, it is not surprising that Bcl-2 and Bcl-X failed to promote apoptosis. To determine important L are commonly overexpressed in human malignancies (11– sequence, length, and structural requirements for peptide 14). Moreover, in several forms of cancer, Bcl-2 or Bcl-XL activity, we generated and systematically analyzed the overexpression has been shown to correlate with chemo- biological activities of 17 Bax BH3 peptide variants. therapy and radiation resistance as well as with poor Peptides were quantitatively examined for their ability clinical prognosis (11–14). These observations highlight the to inhibit Bax/Bcl-2 and Bax/Bcl-XL heterodimerization need to develop effective agents that specifically target Bcl- in vitro and to promote cytochrome c release from 2 or Bcl-X in the tumor cells. mitochondria isolated from Jurkat, HL-60, U937, and L Antisense molecules directed against mRNA for Bcl-2 or Bcl-XL have been used to down-regulate the expression of these proteins in cell culture and in vivo (15–17). In Received 7/13/04; revised 8/18/04; accepted 8/27/04. particular, G3139, an 18-nucleotide antisense molecule, has Grant support: Leukemia and Lymphoma Society of America Translational been shown to down-regulate Bcl-2 expression in tumor Research Award 6456 and NIH grant RO1 CA86980 (D.E. Johnson). cell lines (17) and has exhibited anticancer activity in The costs of publication of this article were defrayed in part by the clinical trials (13, 16, 18, 19). More recently, several agents payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to have been developed that target Bcl-2 or Bcl-XL function. indicate this fact. The application of a variety of techniques, including Requests for reprints: Daniel E. Johnson, Division of Hematology/ fluorescence polarization binding assays, nuclear magnetic Oncology, University of Pittsburgh, and University of Pittsburgh Cancer resonance shift assays, computer modeling, and cell-based Institute, Room 2.18c, Hillman Cancer Center, 5117 Center Avenue, Pittsburgh, PA 15213. Phone: 412-623-3245; Fax: 412-623-7768. cytotoxicity assays, has led to the identification of several E-mail: [email protected] small organic molecules that bind and/or inhibit Bcl-2 and C Copyright 2004 American Association for Cancer Research. Bcl-XL, including HA-14-1 (20), antimycin A3 (21), BH3Is Mol Cancer Ther 2004;3(11). November 2004 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2004 American Association for Cancer Research. 1344 Structural Requirements of Bax BH3 Peptide (22), gossypol (23), and compound 6 (ref. 24; small organic Materials and Methods molecule inhibitors reviewed in refs. 13, 25, 26). In addition, Cell Lines and Reagents increased understanding of the three-dimensional struc- Vector-transfected Jurkat T leukemic cells and Jurkat cells tures of Bcl-2 family members, and the interactions that overexpressing Bcl-2 were generated as described previous- occur between these proteins, has led to the design of ly (47). Jurkat cells overexpressing Bcl-XL were a kind gift of peptide-based agents to inhibit Bcl-2 and Bcl-XL function Dr. Craig Thompson (University of Pennsylvania, Phila- (27–29). delphia, PA). Transfected Jurkat cell lines were maintained Structural and functional studies have revealed that in RPMI 1640 (Life Technologies, Gaithersburg, MD) protein-protein interactions among Bcl-2 family members containing 10% fetal bovine serum, 2 mmol/L L-glutamine, play a key role in the ability of these proteins to regulate 1% penicillin/streptomycin, 0.2% fungizone, and 0.5 mg/ apoptosis. Following treatment of cells with agents that j mL G418 in a humidified atmosphere of 5% CO2 at 37 C. activate the intrinsic pathway, Bax and/or Bak homooligo- HL-60 and U937 cells were cultured in RPMI 1640 and PC-3 merize in the mitochondrial outer membrane and facilitate prostate cancer cells were maintained in F-12K medium the release of apoptogenic factors (10, 30–32). Antiapop- (Mediatech, Inc., Herndon, VA) supplemented with 10% totic Bcl-2 and Bcl-XL can inhibit this process by hetero- fetal bovine serum, 2 mmol/L L-glutamine, 1% penicillin/ dimerizing with the Bax or Bak proteins (33–37). streptomycin, and 0.2% fungizone. Mutational analyses have determined that the BH3 domain Polyclonal anti-Bax antibody (N20) was obtained from of proapoptotic proteins is necessary and sufficient for Santa Cruz Biotechnology (Santa Cruz, CA), monoclonal heterodimerization with antiapoptotic proteins (38–40). anti-Bcl-2 antibody (clone 124) was from DAKO (Carpin- Thus, it has been reasoned that peptides based on the BH3 teria, CA), polyclonal anti-caspase-3 was from Cell Signal- domains of proapoptotic proteins may have the capacity to ing Technology (Beverly, MA), anti-tubulin (clone DM1A) disrupt physical interactions between proapoptotic and was from Sigma Chemical Co. (St. Louis, MO), and anti– antiapoptotic Bcl-2 family members and thereby promote cytochrome c oxidase IV (clone 2OE8) was from Molecular apoptosis in Bcl-2-overexpressing or Bcl-XL-overexpressing Probes (Eugene, OR). Monoclonal antibodies recognizing cells. poly(ADP-ribose) polymerase (clone 4C10-5) and cyto- Previous studies have shown that peptides derived chrome c (clone 7H8.2C12) were purchased from PharMin- from the BH3 domains of Bak (19-mer), Bid (20-mer), Bad gen (San Diego, CA). (21-mer), and Bax (20-mer) are capable of inducing Peptide Synthesis apoptosis in a variety of cell line models (41–44). Peptides were synthesized on a Pioneer peptide synthe- Peptide-induced apoptosis was associated with activation sizer (Applied Biosystems, Foster City, CA) using a of the intrinsic cell death pathway and release of fluoren-9-ylmethoxycarbonyl synthesis protocol (Peptide cytochrome c from the mitochondria (41, 43, 45). The Synthesis Facility, Molecular Medicine Institute, University ability of Bax, Bid, and Bim BH3 peptides to provoke of Pittsburgh). Synthesis was done by stepwise addition of cytochrome c release from purified mitochondria suggests activated amino acids to the solid support (polyethylene that the peptides directly target Bcl-2 family members glycol-polystyrene resin) starting from the COOH termi- present in the outer mitochondrial membrane