title: The effect of direct translocation across endosomes on the cytotoxicity of the recombinant protein e23sFv-Fdt-casp6 to HER2 positive gastric cancer cells.
abstract: HER2-positive cancers represent a class of malignancies with high metastasis and poor prognosis. We previously generated the e23sFv-PEA II-casp6 recombinant, which contains an anti-HER2 single-chain antibody (e23sFv), a Pseudomonas exotoxin A translocation domain (PEA II), and a constitutively active caspase-6 (casp6), and demonstrated its potent selective anti-tumor activities. In this study, we generated a smaller-sized recombinant e23sFv-Fdt-casp6, in which the PEA II domain was replaced with the furin cleavage sequence from diphtheria toxin (Fdt), and explored its translocation pathway and specific killing mechanism. We found that e23sFv-Fdt-casp6 proteins, following their receptor-mediated endocytosis in HER2-positive gastric cancer cells, underwent furin-mediated cleavage in endosome and engaged in direct translocation of the released C-terminal fragment (active caspase-6) instead of via the trans-Golgi and the endoplasmic reticulum (ER) pathway. The active caspase-6 cleaved its well-documented substrate, Lamin A, and subsequently triggered the apoptosis of cancer cells. The e23sFv-Fdt-casp6 proteins produced from genetically modified cells showed a selective cytotoxicity to cultured HER2-positive gastric cancer cells. Similar to the results of our previous research on e23sFv-PEA II-casp6, the delivery of liposome-encapsulated e23sFv-Fdt-casp6 constructs in tumor-adjacent muscles also inhibited tumor growth and prolonged animal survival in a nude mouse xenograft tumor model. Moreover, e23sFv-Fdt-casp6 proteins were also cytotoxic to trastuzumab-resistant gastric cancer cells characterized by downregulated HER2 expression. Accordingly, e23sFv-Fdt-casp6 recombinant provides a promising therapeutic alternative for HER2-positive and trastuzumab-resistant gastric cancers.
authors.affiliation: State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
authors.forename: Jun-Lin
authors.initials: JL
authors.lastname: Ren
authors.forename: Yan-Ling
authors.initials: YL
authors.lastname: Meng
authors.forename: Bin
authors.initials: B
authors.lastname: Hu
authors.forename: Lin-Tao
authors.initials: LT
authors.lastname: Jia
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: Yan-Ming
authors.initials: YM
authors.lastname: Xu
authors.forename: Qiao-Sheng
authors.initials: QS
authors.lastname: Xie
authors.forename: Ying-Qi
authors.initials: YQ
authors.lastname: Zhang
authors.forename: Bo-Quan
authors.initials: BQ
authors.lastname: Jin
authors.forename: Si-Yi
authors.initials: SY
authors.lastname: Chen
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.forename: An-Gang
authors.initials: AG
authors.lastname: Yang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D053178:Caspase 6 / Q000235:genetics / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism; D004167:Diphtheria Toxin / Q000235:genetics / Q000627:therapeutic use*; D011992:Endosomes / Q000378:metabolism*; D045683:Furin / Q000378:metabolism; D006801:Humans; D034904:Lamin Type A / Q000378:metabolism; D008081:Liposomes; D051379:Mice; D008819:Mice, Nude; D021381:Protein Transport; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000627:therapeutic use*; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000378:metabolism
chemical_list: D000906:Antibodies; D053178:Caspase 6; D004167:Diphtheria Toxin; D045683:Furin; D034904:Lamin Type A; D008081:Liposomes; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins