The Research Of Claudin-3 In Prostate Cancer As A Potential Target For Diagnostic And Therapeutic Strategy

Prostate cancer is a most common malignant carcinoma and main reason for death in male in the world. Mortality attributable to prostate cancer is the second factor causing of cancer-related death in men of United States. The failure rate of local therapy for early stage disease combined with the limited duration of effective palliative management with hormonal manipulation for advanced metastatic disease beg the development of newer therapeutic agents and strategies. Recent advances in the use of combination chemotherapy for advanced hormone-refractory prostate cancer are encouraging but remain inadequate. Epithelial-derived tumors account for 90% of all malignant tumors, and their resistance to chemotherapy is a major clinical problem. Recent progress in combinatorial chemistry, proteomics, and genomics research has further advanced the development of effective drugs against carcinomas, but, for clinical application, it is also essential to develop selective and efficient drug delivery systems for these novel drugs.Tight junctions (TJs), which are points of intercellular contact and interaction, are characteristic and complex structures in the epithelia. TJs play a critical role in forming a barrier between apical and basal sides of the cell, and they are present on the lateral side of the cell where they mediate intercellular interactions. Loss of polarity is a typical feature of transformation in epithelial cells.Furthermore, abnormal localization of membrane proteins, including TJ components, adherence junction proteins, and apical and basal proteins, is observed during carcinogenesis. These findings indicate that abnormally localized membrane proteins may be useful for targeting drugs to carcinoma cells.Claudin is an approximately 23KDa transmembrane protein found in the TJs, and it plays a pivotal role in the barrier function of the TJ. There are more than 20 members of claudin family including Claudin-3 and Claudin-4, and they are expressed in a tissue-specific manner. Interestingly, the overexpression of claudins is frequently observed in the epithelium of ovarian cancer, hepatocellular carcinoma, malignant pancreatic cancer, and prostate cancer. Therefore, claudins are promising candidates for the targeting of anticancer drugs to carcinoma cells.Clostridium perfringens enterotoxin (CPE) is a single polypeptide with a molecular mass of 35 KDa that causes food poisoning associated with most human food-borne illnesses. CPE is made up of two functionally distinct domains: an approximately 22-KDa N-terminal domain that mediates cytotoxicity, and an approximately 13KDa C-terminal domain (C-CPE) that mediates binding allowing partial insertion of CPE into the target cell membrane with resultant initiation of massive small molecule permeability changes, osmotic cell ballooning, and lysis. However, only claudin-3 and 4 in the family of claudin proteins are the receptors for CPE to bind it and mediate toxin-dependent cytolysis. These findings suggest that CPE could be used for the targeting of claudins on epithelial carcinoma cells. Indeed, CPE has been successfully used to treat human ovarian and pancreatic cancers, both of which express high levels of claudin-3 or -4. The present study was designed to determine whether claudin-3 is expressed in malignant epithelial cells of prostate cancer to serve as a potential target for a CPE-mediated therapeutic strategy.Methods: The expressions of Claudin-3 protein in prostate cancer and benign hyperplasia of prostate were detected by immunohistochemistry and Western blot, and its contribution on pathological stage of tumor were analysed. The stand Clostridium perfringens 64615 which could produce Clostridium perfringens enterotoxin was incubated and identified. Then the full-length gene of cpe which was extracted and amplified from the whole-length DNA of the Cl. Perfringens was correctly inserted into the vector pET-28a by restriction endonuclease digestion with NdeI plus XhoI. The protein CPE was expressed after IPTG induction, then it was isolated and purified from the total proteins of E. coli BL21 transformed by the recombinant prokaryotic plasmid pET-28a-cpe depending on Ni-NTA affinity chromatography. The human prostate cancer cell line 22RV1 and PC-3 were cultured separately and detected by fluorescein stain. Changes of the cultured human prostate cancer cell line 22RV1 were observed after CPE was added into the 96 well plate. The Nude mice Balb/c were subcutaneously inoculated human prostate cancer cell line 22RV1. The mice of different groups were injected different dose of CPE in the local position of the inoculated tumor. The volume of the mice tumor were recorded and the pathological changes were observed by HE staining and immunohistochemistry.Results:1. Claudin-3 was expressed on the cell membrane of prostate inequablely. Compared with those sections of benign hyperplasia of prostate, the expression of Claudin-3 was significantly increased in the cases of prostate cancer by immunohistochemistry and Western blot.2. The stand Clostridium perfringens 64615 which could produce Clostridium perfringens enterotoxin was revived,incubated and identified. The full-length gene of cpe which was extracted and amplified from the whole-length DNA of the Cl. Perfringens was correctly inserted into the vector pET-28a by restriction endonuclease digestion with NdeI plus XhoI. The recombinant prokaryotic plasmid pET-28a-cpe was confirmed by partial nucleotide sequencing and restriction endonuclease digestion.3. The reconstructed protein was expressed after IPTG induction and isolated and purified from the total proteins of E. coli BL21. By induction of IPTG, the E. coli BL21 transformed by the recombinant prokaryotic plasmid pET-28a-cpe. Ni-NTA affinity chromatography was used for the Purification. The purity of the protein CPE was detected with SDS-PAGE, which was confirmed to have a purity of more than 90%.4. The human prostate cancer cell line 22RV1 could highly expressed claudin-3 detected by fluorescein stain compared with the human prostate cancer cell line PC-3. Cell line 22RV1 was inhibited by CPE mediating toxin-dependent cytolysis after CPE was added into the 96 well plate.5. The Nude mice Balb/c were subcutaneously inoculated human prostate cancer cell line 22RV1 successfully. After injecting CPE in the local position of the inoculated tumor, the volume of the mice tumor was reduced and the cellular necrosis of the inoculated tumor was observed by HE staining and immunohistochemistry.Conclusions: Claudin-3 protein is significantly highly expressed in prostate cancer. The protein CPE was successfully expressed in E. coli BL21 and purified by gene engineering. CPE could cause biological effect and mediate toxin-dependent cytolysis in cultured cells in vitro and in inoculated tumor of the Nude mice. Claudin-3 protein may become a new marker in the diagnose and treatment of prostate cancer. It is may be possible that CPE would be used as a new therapeutic strategy on the reasearch of clinic treatment for tumor in future.