Target-specific cytotoxic activity of recombinant fusion toxin C-CPE-ETA' against CLDN-3,4-overexpressing ovarian cancer cells

• Published in: Zhōnghuá zhŏngliú zázhì Vol. 32; no. 12; p. 897
• Main Authors: Yao, Qin, Zheng, Qing-Mei, Wen, Jun-Feng, Lü, Teng, Wei, Ming-Qian, Dai, Shu-Zhen
• Format: Journal Article
• Language: Chinese
• Published: China 01.12.2010
• Subjects: ADP Ribose Transferases - metabolism; ADP Ribose Transferases - physiology; Apoptosis; Bacterial Toxins - metabolism; Cell Line, Tumor; Claudin-3; Claudin-4; Claudins - genetics; Claudins - metabolism; Enterotoxins - metabolism; Enterotoxins - physiology; Exotoxins - metabolism; Exotoxins - physiology; Female; Humans; Immunotoxins - metabolism; Ovarian Neoplasms - metabolism; Ovarian Neoplasms - pathology; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - physiology; RNA, Messenger - metabolism; Virulence Factors - metabolism; Virulence Factors - physiology
• ISSN: 0253-3766

The aim of this study was to explore the possibility of creating a toxin, C-CPE-ETA', by fusing C-terminal high affinity binding domain of CPE (C-CPE) with a truncated form of Pseudomonas aeruginosa exotoxin A (ETA') and to examine whether C-CPE-ETA' could specifically target CLDN-3, 4 molecule and the targeted toxin was cytotoxic against CLDN-3,4-overexpressing ovarian cancer. CLDN-3 and CLDN-4 expressions were analyzed at the mRNA level in three ovarian cancer cell lines and epithelial ovarian cancer tissues from 20 patients. After transforming an expression plasmid of C-CPE-ETA' into E. coli BL21 (DE3) plysS strain, the recombinant protein was purified using His-Bind resin chromatography column and analyzed by Western blot and Coomassie blue staining. The specific binding, proapoptotic and cytolytic activities were evaluated by flow cytometry, fluorescence microscopy with the JC-1 probe and MTT assay in CLDN-3,4-overexpressing ovarian cancer cells. Quantitive RT-PCR results showed there existed high levels