title: Clostridium perfringens enterotoxin as a potential drug for intravesical treatment of bladder cancer.
abstract: The current intravesical treatment of bladder cancer (BC) is limited to a few chemotherapeutics that show imperfect effectiveness and are associated with some serious complications. Thus, there is an urgent need for alternative therapies, especially for patients with high-risk non-muscle invasive (NMIBC). Clostridium perfringens enterotoxin (CPE), cytolytic protein binds to its receptors: claudin 3 and 4 that are expressed in epithelial cells. This binding is followed by rapid cell death. Claudin 4 is present in several epithelial tissue including bladder urothelium and its expression is elevated in some forms of BC. In addition to directly targeting BC cells, binding of CPE to claudins increases urothelium permeability that creates conditions for better accession of the tumor. Therefore, we evaluated CPE as a candidate for intravesical treatment of BC using a cellular model. We examined cytotoxicity of CPE against BC cells lines and 3D cultures of cells derived from surgical samples. To better elucidate cellular mechanisms, activated by CPE and to consider the use of CPE non-toxic fragment (C-CPE) for combination treatment with other drugs we synthesized C-CPE, compared its cytotoxic activity with CPE and examined claudin 4 expression and intracellular localization after C-CPE treatment. CPE induced cell death after 1 h in low aggressive RT4 cells, in moderately aggressive 5637 cells and in the primary 3D cultures of BC cells derived from NMIBC. Conversely, non-transformed urothelial cells and cells derived from highly aggressive tumor (T24) survived this treatment. The reason for this resistance to CPE might be the lower expression of CLDNs or their inaccessibility for CPE in these cells. C-CPE treatment for 48 h did not affect cell viability in tested cells, but declined expression of CLDN4 in RT4 cells. C-CPE increased sensitivity of RT4 cells to Mitommycin C and Dasatinib. To better understand mechanisms of this effect we examined expression and phosphorylation status of EphA2 and Src after C-CPE treatment and found changes in expression and phosphorylated status of these regulatory molecules. These observations show that after additional preclinical studies CPE and C-CPE in combinations with other drugs can be considered as a potential modalities for intravesical treatment of BC because of its ability to effectively destroy BC cells expressing claudin 4 and low toxicity against normal urothelium.
authors: Department of Medicine, School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.
authors: Theodore G
authors: TG
authors: Gabig
authors: Department of Urology, School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.
authors: Wayne C
authors: WC
authors: Waltzer
authors: Department of Urology, School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.
authors: Terry
authors: T
authors: Whyard
authors: Department of Urology, School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA. Electronic address: victor.romanov@stonybrookmedicine.edu.
authors: Victor
authors: V
authors: Romanov
mesh_terms: D000283:Administration, Intravesical; D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D062506:Claudin-4 / Q000235:genetics* / Q000378:metabolism; D003016:Clostridium perfringens / Q000737:chemistry*; D000069439:Dasatinib / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D004768:Enterotoxins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016685:Mitomycin / Q000494:pharmacology; D008954:Models, Biological; D058990:Molecular Targeted Therapy; D011485:Protein Binding; D036104:Receptor, EphA2 / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D018874:Spheroids, Cellular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D019459:Urothelium / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; C568157:CLDN4 protein, human; D062506:Claudin-4; D004768:Enterotoxins; D011994:Recombinant Proteins; C035598:enterotoxin, Clostridium; D016685:Mitomycin; D036104:Receptor, EphA2; D019061:src-Family Kinases; D000069439:Dasatinib
keywords: Bladder cancer; Claudin 4; Clostridium perfringens enterotoxin