title: Induction of programmed cell death in ErbB2/HER2-expressing cancer cells by targeted delivery of apoptosis-inducing factor.
abstract: Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with NADH oxidase activity that has a vital function in healthy cells but is also an important mediator of caspase-independent programmed cell death in stressed and damaged cells. Here, we generated a truncated AIF derivative (AIF(Delta100)) that lacks the mitochondrial import signal of the protein. Bacterially expressed AIF(Delta100) was functionally active and induced cell death on microinjection into Vero cells accompanied by clear signs of apoptosis. For specific targeting to tumor cells, AIF(Delta100) was genetically fused to the scFv(FRP5) antibody fragment that recognizes the ErbB2 (HER2) receptor tyrosine kinase frequently overexpressed in many human cancers. Recombinant scFv(FRP5)-AIF(Delta100) (5-AIF(Delta100)) protein and a similar scFv(FRP5)-ETA(252-366)-AIF(Delta100) (5-E-AIF(Delta100)) molecule harboring in addition the nontoxic translocation domain of Pseudomonas exotoxin A as an endosome escape function displayed binding to ErbB2-expressing cells followed by protein internalization and accumulation in intracellular vesicles. In the presence of the endosomolytic reagent chloroquine 5-E-AIF(Delta100) but not the similar 5-AIF(Delta100) protein displayed potent cell killing activity, which was strictly dependent on the expression of ErbB2 on the target cell surface. Our results show that recombinant AIF specifically targeted to human cancer cells and delivered into the cytosol has potent cell killing activity, suggesting this molecule as an effector function suitable for the development of humanized immunotoxin-like molecules.
authors.affiliation: Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, Frankfurt am Main, Germany.
authors.forename: Hayat
authors.initials: H
authors.lastname: Mahmud
authors.forename: Benjamin
authors.initials: B
authors.lastname: Dälken
authors.forename: Winfried S
authors.initials: WS
authors.lastname: Wels
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008845:Microinjections; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection; D014709:Vero Cells
chemical_list: D000906:Antibodies; D051033:Apoptosis Inducing Factor; D005982:Glutathione Transferase; D049452:Green Fluorescent Proteins; D007135:Immunoglobulin Variable Region; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins