title: Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency.
abstract: BACKGROUND
  Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosomal/lysosomal compartment. The latter step remains a particular challenge. Claudins 3 and 4 are tight junction proteins that are over-expressed in many types of tumors. This study utilized the C-terminal 30 amino acid fragment of C. perfringens enterotoxin (CPE), which binds to claudins 3 and 4, to deliver a toxin in the form of recombinant gelonin (rGel) to the cytoplasm of the human ovarian carcinoma cell line 2008.
  
  
  RESULTS
  CPE was fused to rGel at its N-terminal end via a flexible G4S linker. This CPE-G4S-rGel molecule was internalized into vesicles from which location it produced little cytotoxicity. To enhance release from the endosomal/lysosomal compartment a poly-arginine sequence (R9) was introduced between the CPE and the rGel. CPE-R9-rGel was 10-fold more cytotoxic but selectivity for claudin-expressing cells was lost. The addition of a poly-glutamic acid sequence (E9) through a G4S linker to R9-rGel (E9-G4S-R9-rGel) largely neutralized the non-selective cell membrane penetrating activity of the R9 motif. However, introduction of CPE to the E9-G4S-R9-rGel fusion protein (CPE-E9-G4S-R9-rGel) further reduced its cytotoxic effect. Treatment with the endosomolytic reagent chloroquine increased the cytotoxicity of CPE-E9-G4S-R9-rGel. Several types of linkers susceptible to cleavage by furin and endosomal cathepsin B were tested for their ability to enhance R9-rGel release but none of these modifications further enhanced the cytotoxicity of CPE-E9-G4S-R9-rGel.
  
  
  CONCLUSIONS
  We conclude that while a claudin-3 and -4 ligand serves to deliver rGel into 2008 cells the delivered molecules were entrapped in intracellular vesicles. Incorporation of R9 non-specifically increased rGel cytotoxicity and this effect could be masked by inclusion of an E9 sequence. However, the putative protease cleavable sequences tested were inadequate for release of R9-rGel from CPE-E9-G4S-R9-rGel.
authors.affiliation: Department of Medicine and Rebecca and John Moores UCSD Cancer Center, University of California-San Diego, 3855 Health Sciences Drive, La Jolla, CA 92093-0819, USA.
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Yuan
authors.forename: Xinjian
authors.initials: X
authors.lastname: Lin
authors.forename: Gerald
authors.initials: G
authors.lastname: Manorek
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Howell
mesh_terms: D045744:Cell Line, Tumor; D062465:Claudin-3; D062506:Claudin-4; D004768:Enterotoxins / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D005260:Female; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism*; D058990:Molecular Targeted Therapy / Q000379:methods; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D054789:Ribosome Inactivating Proteins, Type 1 / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics
chemical_list: C568122:CLDN3 protein, human; C568157:CLDN4 protein, human; D062465:Claudin-3; D062506:Claudin-4; D004768:Enterotoxins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C035598:enterotoxin, Clostridium; C025674:GEL protein, Gelonium multiflorum