title: [Target-specific cytotoxic activity of recombinant fusion toxin C-CPE-ETA' against CLDN-3,4-overexpressing ovarian cancer cells].
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  Quantitive RT-PCR results showed there existed high levels of CLDN-3 and CLDN-4 in ovarian cancer cells, CAOV3, OVCAR3 and SKOV3. Moreover, high expressions of CLDN-3 and CLDN-4 were observed in 90.0% (18/20) and 60.0% (12/20) of ovarian cancer tissues, with an expression level 10-fold higher than that in the normal ovarian tissue. A 58 000 recombinant protein C-CPE-ETA' was demonstrated by Western blot and Coomassie blue staining. Purified and recombinant C-CPE-ETA' was bound with high affinity to CLDN-3,4-overexpressing ovarian cancer cells, CAOV3, OVCAR3 and SKOV3 cells. C-CPE-ETA' was strongly proapoptotic and cytotoxic towards the CLDN-3,4-overexpressing ovarian cancer cells. The concentration of IC(50) was 7.364 ng/ml for CAOV3 cells, 8.110 ng/ml for OVCAR3 cells and 22.340 ng/ml for SKOV3 cells, respectively. However, control CLDN-3,4-deficient cell line HUVEC was not susceptible to the recombinant C-CPE-ETA' at a concentration up to 10 µg/ml.
  
  
  CONCLUSIONS
  The C-CPE-ETA' protein exhibits remarkably specific cytotoxicity for CLDN-3,4-overexpressing ovarian cancer cells. Its therapeutic potential warrants further development for ovarian cancer molecular targeted therapy.
authors.affiliation: Department of Obstetrics and Gynecology, Affiliated Hospital of Qingdao University Medical Collage, Qingdao 266003, China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Yao
authors.forename: Qing-Mei
authors.initials: QM
authors.lastname: Zheng
authors.forename: Jun-Feng
authors.initials: JF
authors.lastname: Wen
authors.forename: Teng
authors.initials: T
authors.lastname: Lü
authors.forename: Ming-Qian
authors.initials: MQ
authors.lastname: Wei
authors.forename: Shu-Zhen
authors.initials: SZ
authors.lastname: Dai
mesh_terms: D036002:ADP Ribose Transferases / Q000378:metabolism* / Q000502:physiology; D017209:Apoptosis; D001427:Bacterial Toxins / Q000378:metabolism*; D045744:Cell Line, Tumor; D062465:Claudin-3; D062506:Claudin-4; D057167:Claudins / Q000235:genetics / Q000378:metabolism*; D004768:Enterotoxins / Q000378:metabolism* / Q000502:physiology; D005098:Exotoxins / Q000378:metabolism* / Q000502:physiology; D005260:Female; D006801:Humans; D000922:Immunotoxins / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000502:physiology; D037521:Virulence Factors / Q000378:metabolism* / Q000502:physiology; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D062465:Claudin-3; D062506:Claudin-4; D057167:Claudins; D004768:Enterotoxins; D005098:Exotoxins; D000922:Immunotoxins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C568122:CLDN3 protein, human; C568157:CLDN4 protein, human; C035598:enterotoxin, Clostridium