Recombinant Cytotoxin C-CPE-ETA' Effectively Kills Claudin-4-Expressing Cancer Cells

Abstract

Claudin-4 is a natural receptor for Clostridium perfringens enterotoxin (CPE). It has recently been found to be overexpressed on the plasma membrane of a range of cancer cells, such as ovarian, breast, colon and prostate cancer. Hence, we hypothesised that a CPE-based cytotoxin may be of potential use in targeted cytolysis of claudin-4 positive cancers. We created such a novel toxin, C-CPEETA', by fusing C-terminal high affinity binding domain of CPE (C-CPE) with a truncated form of Pseudomonas aeruginosa exotoxin A (ETA'), aiming to examine whether C-CPE will specifically target claudin-4 molecule and the targeted toxin is cytotoxic. After cloning into an expression plasmid and transforming it into a bacterial strain, a 58 kDa recombinant protein was evident by Western blot and Coomassie blue staining. Flow cytometric analysis and immune assays were performed using recombinant C-CPEETA' in a range of claudin-4 positive and negative cancer cell lines. The proapoptotic and cytolytic activities were evaluated using JC-1 staining and MTT assay. Purified and recombinant C-CPE-ETA' bound with high affinity to claudin- 4-positive cancer cells, including breast cancer MCF-7, colon cancer SW480, HT29, prostate cancer DU145, PC3 and ovarian cancer CAOV3. Interestingly, after initial binding, C-CPE-ETA' was sequentially internalized into claudin-4- positive cells within 60 mins, which resulted in significant apoptotic cell death. This apoptotic effects increased when the incubation was further extended to 24h. The IC50 of recombinant C-CPE-ETA' ranged from 0.7 ng/ml to 50 ng/ml in claudin-4-positive cancer cells. However, control claudin-4-negative cell lines, Hela and HUVEC, were not susceptible to the recombinant C-CPE-ETA' at concentrations up to 10姯ml, suggesting the strict claudin-4 selectivity. Further study in a MCF-7 subcutaneous xerograft tumour model system showed that intratumoural administration of recombinant C-CPE-ETA' significantly inhibited tumour growth. Our results suggest that the C-CPE-ETA' exhibits remarkably specific cytotoxicity. Its therapeutic potentials for claudin-4-postitive cancer warrants further development.