title: Preparation of a claudin-targeting molecule using a C-terminal fragment of Clostridium perfringens enterotoxin.
abstract: Although most malignant tumors are epithelia-derived carcinomas, methods for specific and effective delivery of antitumor agents to carcinomas have not been developed. Recent reports indicate that epithelia overexpress claudin-3 and -4, which are integral membrane proteins of epithelial tight junctions. This suggests that claudins can be targeted for tumor therapy, but there is not currently a method for delivering drugs to claudin-expressing cells. In the present study, we evaluated whether a potent claudin-4-binding C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) would allow targeting to claudin-4-expressing cells. We fused C-CPE to the protein synthesis inhibitory factor (PSIF), which lacks the cell binding domain of Pseudomonas exotoxin. This fusion protein, C-CPE-PSIF, was cytotoxic to MCF-7 human breast cancer cells, which express endogenous claudin-4, but it was not toxic to mouse fibroblast L cells, which lack endogenous claudin-4. The cytotoxicity of C-CPE-PSIF was attenuated by pretreating the MCF-7 cells with C-CPE but not bovine serum albumin. Also, deletion of the claudin-4-binding region of C-CPE reduced the cytotoxicity of C-CPE-PSIF. Finally, we found that C-CPE-PSIF is toxic to L cells expressing claudin-4 but not to normal L cells or cells expressing claudin-1, -2, or -5. These results indicate that use of the C-CPE peptide may provide a novel way to target drugs to claudin-expressing cells.
authors.affiliation: Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Machida-shi, Tokyo 194-8543, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Ebihara
authors.forename: Masuo
authors.initials: M
authors.lastname: Kondoh
authors.forename: Naoki
authors.initials: N
authors.lastname: Hasuike
authors.forename: Motoki
authors.initials: M
authors.lastname: Harada
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Mizuguchi
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Horiguchi
authors.forename: Makiko
authors.initials: M
authors.lastname: Fujii
authors.forename: Yoshiteru
authors.initials: Y
authors.lastname: Watanabe
mesh_terms: D000970:Antineoplastic Agents; D001667:Binding, Competitive / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D062465:Claudin-3; D062506:Claudin-4; D003016:Clostridium perfringens / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems; D004768:Enterotoxins / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D005260:Female; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014343:Trypan Blue / Q000737:chemistry
chemical_list: D000970:Antineoplastic Agents; C568122:CLDN3 protein, human; C568157:CLDN4 protein, human; D062465:Claudin-3; D062506:Claudin-4; C568123:Cldn3 protein, mouse; C568158:Cldn4 protein, mouse; D004768:Enterotoxins; D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C035598:enterotoxin, Clostridium; D007770:L-Lactate Dehydrogenase; D014343:Trypan Blue