title: Eradication of chemotherapy-resistant CD44+ human ovarian cancer stem cells in mice by intraperitoneal administration of Clostridium perfringens enterotoxin.
abstract: BACKGROUND
  Emerging evidence has suggested that the capability to sustain tumor formation, growth, and chemotherapy resistance in ovarian as well as other human malignancies exclusively resides in a small proportion of tumor cells termed cancer stem cells. During the characterization of CD44(+) ovarian cancer stem cells, we found a high expression of the genes encoding for claudin-4. Because this tight junction protein is the natural high-affinity receptor for Clostridium perfringens enterotoxin (CPE), we have extensively investigated the sensitivity of ovarian cancer stem cells to CPE treatment in vitro and in vivo.
  
  
  METHODS
  Real-time polymerase chain reaction and flow cytometry were used to evaluate claudin-3/-4 expression in ovarian cancer stem cells. Small interfering RNA knockdown experiments and MTS assays were used to evaluate CPE-induced cytotoxicity against ovarian cancer stem cell lines in vitro. C.B-17/SCID mice harboring ovarian cancer stem cell xenografts were used to evaluate CPE therapeutic activity in vivo.
  
  
  RESULTS
  CD44(+) ovarian cancer stem cells expressed claudin-4 gene at significantly higher levels than matched autologous CD44(-) ovarian cancer cells, and regardless of their higher resistance to chemotherapeutic agents died within 1 hour after exposure to 1.0 μg/mL of CPE in vitro. Conversely, small-interfering RNA-mediated knockdown of claudin-3/-4 expression in CD44(+) cancer stem cells significantly protected cancer stem cells from CPE-induced cytotoxicity. Importantly, multiple intraperitoneal administrations of sublethal doses of CPE in mice harboring xenografts of chemotherapy-resistant CD44(+) ovarian cancer stem cells had a significant inhibitory effect on tumor progression leading to the cure and/or long-term survival of all treated animals (ie, 100% reduction in tumor burden in 50% of treated mice; P < .0001).
  
  
  CONCLUSIONS
  CPE may represent an unconventional, potentially highly effective strategy to eradicate chemotherapy-resistant cancer stem cells.
authors.affiliation: Division of Gynecologic Oncology, Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale School of Medicine, New Haven, Connecticut 06520-8063, USA. alessandro.santin@yale.edu
authors.forename: Francesca
authors.initials: F
authors.lastname: Casagrande
authors.forename: Emiliano
authors.initials: E
authors.lastname: Cocco
authors.forename: Stefania
authors.initials: S
authors.lastname: Bellone
authors.forename: Christine E
authors.initials: CE
authors.lastname: Richter
authors.forename: Marta
authors.initials: M
authors.lastname: Bellone
authors.forename: Paola
authors.initials: P
authors.lastname: Todeschini
authors.forename: Eric
authors.initials: E
authors.lastname: Siegel
authors.forename: Joyce
authors.initials: J
authors.lastname: Varughese
authors.forename: Dan
authors.initials: D
authors.lastname: Arin-Silasi
authors.forename: Masoud
authors.initials: M
authors.lastname: Azodi
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Rutherford
authors.forename: Sergio
authors.initials: S
authors.lastname: Pecorelli
authors.forename: Peter E
authors.initials: PE
authors.lastname: Schwartz
authors.forename: Alessandro D
authors.initials: AD
authors.lastname: Santin
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D000077216:Carcinoma, Ovarian Epithelial; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D062465:Claudin-3; D057167:Claudins / Q000235:genetics / Q000378:metabolism; D003016:Clostridium perfringens / Q000737:chemistry* / Q000378:metabolism; D004768:Enterotoxins / Q000096:biosynthesis / Q000493:pharmacokinetics / Q000494:pharmacology*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis*; D007274:Injections, Intraperitoneal; D051379:Mice; D016513:Mice, SCID; D008875:Middle Aged; D009375:Neoplasms, Glandular and Epithelial / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D060888:Real-Time Polymerase Chain Reaction; D014709:Vero Cells; D023041:Xenograft Model Antitumor Assays
chemical_list: C568122:CLDN3 protein, human; D062465:Claudin-3; D057167:Claudins; C568123:Cldn3 protein, mouse; D004768:Enterotoxins; D018960:Hyaluronan Receptors; C035598:enterotoxin, Clostridium