Abstract
Carcinogenesis is often accompanied by dysfunctional tight junction (TJs), resulting in the loss of cellular polarity. Claudin, a tetra-transmembrane protein, plays a pivotal role in the barrier and fence functions of TJs. Claudin-4 is deregulated in various cancers, including breast, prostate, ovarian, and gastric cancer. Claudin-4 may be a promising target molecule for tumor therapy, but the claudin-targeting strategy has never been fully developed. In the present study, we prepared a claudin-4-targeting molecule by fusion of the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) with the protein synthesis inhibitory factor (PSIF) derived from Pseudomonas aeruginosa exotoxin. PSIF was not cytotoxic to claudin-4-expressing cells, whereas C-CPE-PSIF was cytotoxic. Cells that express claudin-1, -2, and -5 were less sensitive to C-CPE-PSIF. Pretreatment of the cells with C-CPE attenuated C-CPE-PSIF-induced cytotoxicity, and mutation of C-CPE in the claudin-4-binding residues attenuated the cytotoxicity of C-CPE-PSIF. TJ-undeveloped cells were more sensitive to C-CPE-PSIF than TJ-developed cells. It is noteworthy that polarized epithelial cells are sensitive to C-CPE-PSIF applied to the basal side, whereas the cells were less sensitive to C-CPE-PSIF applied to the apical side. Intratumoral injection of C-CPE-PSIF reduced tumor growth. This is the first report to indicate that a claudin-4-targeting strategy may be a promising method to overcome the malignant tumors.
- TJ, tight junction
- C-CPE, C-terminal fragment of Clostridium perfringens enterotoxin from 194 to 319 amino acids
- PSIF, protein synthesis inhibitory factor derived from P. aeruginosa exotoxin
- CPE, Clostridium perfringens enterotoxin
- PE, P. aeruginosa exotoxin
- C-CPE-PSIF, C-terminal fragment of Clostridium perfringens enterotoxin-fused protein synthesis inhibitory factor derived from P. aeruginosas exotoxin
- PAGE, polyacrylamide gel electrophoresis
- BV, budded baculovirus
- Ab, antibody
- TER, transepithelial electric resistance
- FBS, fetal bovine serum
- MCS, multiple cloning sites
- PCR, polymerase chain reaction
- PBS, phosphate-buffered saline
- BSA, bovine serum albumin
- TBS, Tris-buffered saline
- LDH, lactate dehydrogenase
- aa, amino acid.
Footnotes
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This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan; a Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan; Takeda Science Foundation; a Suzuken Memorial Foundation; and a Grant from Kansai Biomedical Cluster project in Saito, which is promoted by the Knowledge Cluster Initiative of the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- Received June 8, 2009.
- Accepted July 28, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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