title: Targeted induction of apoptosis in glioblastoma multiforme cells by an MRP3-specific TRAIL fusion protein in vitro.
abstract: Single-chain Fv fragments (scFvs) consist of the variable heavy-chain (VH) and variable light-chain (VL) domains, which are the smallest immunoglobulin fragments containing the whole antigen-binding site. Human soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) proves to acquire a potent pro-apoptotic activity only after selective binding to a predefined tumor cell surface antigen and has no off-target effects towards normal cells. Glioblastoma multiforme (GBM) is the most frequent and aggressive type of brain tumor and overexpresses human multidrug resistance protein 3 (MRP3). In this study, we designed a novel fusion protein, termed scFvM58-sTRAIL, in which the MRP3-specific scFv antibody M58 was genetically fused to the N-terminus of human soluble TRAIL (sTRAIL). The recombinant scFvM58-sTRAIL fusion protein, expressed in Escherichia coli, was purified by chromatography and tested for cytotoxicity. scFvM58-sTRAIL showed a significant apoptosis-inducing activity towards MRP3-positive GBM cells in vitro. The pro-apoptotic activity of scFvM58-sTRAIL towards GBM cells was strongly inhibited in the presence of the parental scFvM58 antibody, suggesting that cytotoxic activity is MRP3-restricted. In a control experiment with MRP3-negative Jurkat cells, scFvM58-sTRAIL did not induce apparent apoptosis. In addition, through target antigen-restricted binding, scFvM58-sTRAIL was capable of activating not only TRAIL-R1 but also TRAIL-R2. In conclusion, our results suggest that fusion protein scFvM58-sTRAIL with specificity for MRP3 is a highly selective therapeutic agent and may provide an alternative therapy for human GBM.
authors.affiliation: Department of Biochemistry and Molecular Biology, Second Military Medical University, Xiangyin Road No. 800, Shanghai, 200433, China.
authors.forename: Liang-Hua
authors.initials: LH
authors.lastname: Wang
authors.forename: Chang-Wei
authors.initials: CW
authors.lastname: Ni
authors.forename: Yong-Zhong
authors.initials: YZ
authors.lastname: Lin
authors.forename: Lin
authors.initials: L
authors.lastname: Yin
authors.forename: Chang-Bin
authors.initials: CB
authors.lastname: Jiang
authors.forename: Cui-Ting
authors.initials: CT
authors.lastname: Lv
authors.forename: Yuan
authors.initials: Y
authors.lastname: Le
authors.forename: Yue
authors.initials: Y
authors.lastname: Lang
authors.forename: Chen-Yang
authors.initials: CY
authors.lastname: Zhao
authors.forename: Kang
authors.initials: K
authors.lastname: Yang
authors.forename: Bing-Hua
authors.initials: BH
authors.lastname: Jiao
authors.forename: Jian
authors.initials: J
authors.lastname: Yin
mesh_terms: D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology; D017209:Apoptosis / Q000235:genetics; D001932:Brain Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D006801:Humans; D019169:Jurkat Cells; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000627:therapeutic use; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use
chemical_list: D000954:Antigens, Surface; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; C114462:multidrug resistance-associated protein 3